Toll-like receptor agonists and their use

Abstract

A Toll-like receptor agonist is a class C CpG-ODN capable of effectively activating TLR9, wherein its nucleotide sequence includes one of the nucleotide sequences shown in SEQ ID NOs: 1-4, either modified or unmodified. The Toll-like receptor agonist has a significant inhibitory effect on tumor growth. The provided lipid-encapsulated Toll-like receptor agonist can inhibit tumor growth, prevent tumor recurrence, and improve the efficiency of immune activation, either alone or in combination with other therapeutic agents, exhibiting improved performance compared to free Toll-like receptor agonists.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to application CN202311008396.X filed on 10 August 2023, application CN202410324833.7 filed on 20 March 2024, and application CN202410567565.1 filed on 9 May 2024. These prior applications are incorporated together as part of this application.

[0002] Technical field This disclosure relates to the biomedical field, and more specifically, to Toll-like receptor agonists. [Background technology]

[0003] Toll-like receptors (TLRs) are highly conserved pattern recognition receptors found in various cell types and play a crucial role in the surveillance of microbial pathogens by innate immune cells. In humans, ten TLRs have already been identified, expressed in T cells, B cells, APCs, and various non-immune cells such as epithelial and endothelial cells. TLRs are localized to two distinct regions of the cell: the cell membrane and the intracellular body. TLR1, 2, 5, and 6 are located on the cell membrane, TLR3, 7, 8, and 9 are expressed in the intracellular body, and TLR4 is present in both regions. In the tumor microenvironment, TLRs are expressed in immune cells and tumor cells, and when activated, they inhibit the function of Treg cells and CD8 + This can enhance T cell survival, proliferation, and cytokine release.

[0004] To date, much research has been conducted on treating tumors using TLR agonists. Since TLR9 activation leads to the activation of dendritic cells, T cells, and B cells, TLR9 agonists have great potential in the field of oncology by inducing interferon-α secretion and promoting the migration of T cells to the tumor microenvironment. TLR9 agonists are mainly unmethylated bacterial CpG DNA, and it has also been demonstrated that artificially synthesized CpG oligonucleotides (CpG-ODNs) can effectively activate TLR9. CpG-ODNs are generally classified into class A, class B, and class C. Class A CpG-ODNs have a palindromic sequence containing CpG dinucleotides as their core, with poly G termini at both ends, and a phosphodiester bond skeleton that is partially thiolated. The palindromic sequence and poly G form a higher-order structure that can activate plasmacytoid dendritic cells and induce the production of large amounts of type I interferon, while exhibiting weak activity against B cells. Class B CpG-ODN is a fully thiolated linear CpG-ODN that has very strong immunostimulatory activity against B cells but cannot activate plasmacytoid dendritic cells. Class C CpG-ODN is a fully thiolated CpG-ODN that can form dimers due to its palindromic sequence and possesses the activity of both Class A and Class B CpG-ODN, activating not only plasmacytoid dendritic cells but also B cells. CpG-ODN is used as an adjuvant component in vaccines to enhance the immune response, and currently, some products, such as the hepatitis B vaccine HEPLISAV-B, are approved for sale. In addition to vaccines, TLR9 agonists can also be used to treat inflammatory diseases and infections, but all of these are currently in the clinical stage.

[0005] TLR9 agonists hold great potential in treating tumors, but several challenges remain in developing them as antitumor drugs: 1) Systemic administration of TLR9 agonists causes systemic inflammation and significant side effects, and because only small amounts of the drug enter the tumor microenvironment, the efficacy is poor. 2) Some researchers have reduced the dosage to some extent by using intratumoral injection, but the drug diffuses quickly, and very little of the drug remains in the tumor. 3) TLR9 is an intracellular receptor, and only small amounts of nude CpG-ODN can enter the cell and function.

[0006] Currently, improvements to TLR9 agonists primarily focus on two areas: preventing the rapid degradation of oligonucleotides and enhancing endocytosis. Mologen Ag's product, Lefitolimod, extends the half-life of oligonucleotides by modifying linear oligonucleotides into dumbbell-shaped oligonucleotides. However, it did not yield favorable results or clear efficacy in phase 2 and phase 3 clinical trials for advanced colorectal cancer after monotherapy and small cell lung cancer after monotherapy, and therefore there are currently no ongoing oncological clinical trials. Checkmate's intratumoral injection product, CMP-001, is a class A CpG TLR9 agonist that significantly extends the half-life by preventing the degradation of oligonucleotides by nucleases through encapsulation of virus-like particles. However, delivering oligonucleotides through virus-like particle encapsulation is a complex process and has high manufacturing costs. [Overview of the project] [Problems that the invention aims to solve]

[0007] The present disclosure provides Toll-like receptor agonists having an antitumor effect, and further provides Toll-like receptor agonists encapsulated with lipids or multi-active ingredients in which a Toll-like receptor agonist is combined with other therapeutic agents. The Toll-like receptor agonist encapsulated with the lipid component provided by the present disclosure or the multi-active ingredient in which a Toll-like receptor agonist is combined with other therapeutic agents has a significant broad-spectrum antitumor effect, can prevent tumor recurrence, and can prevent tumor metastasis. The Toll-like receptor agonist encapsulated with the lipid component provided by the present disclosure or the multi-active ingredient in which a Toll-like receptor agonist is combined with other therapeutic agents has prolonged retention in tumors compared with the free Toll-like receptor agonist, has an immunostimulatory effect, can upregulate the expression of cytokines, and after systemic administration, has a broad-spectrum antitumor effect, an effect of preventing tumor recurrence, and an effect of preventing tumor metastasis. Moreover, the survival period of the test animals is effectively prolonged, and it has good safety and tolerance. The Toll-like receptor agonist encapsulated with the lipid component provided by the present disclosure or the multi-active ingredient in which a Toll-like receptor agonist is combined with other therapeutic agents has fewer side effects than the free Toll-like receptor agonist, and good drug efficacy is expected in suppressing human tumors.

Means for Solving the Problems

[0008] In a first aspect of the present disclosure, there is provided a Toll-like receptor agonist comprising any one of the following: (a) any one of the nucleotides represented by SEQ ID NOs: 1 to 4, which may be modified or unmodified; (b) a functional variant of any one of the nucleotides represented by SEQ ID NOs: 1 to 4, which may be modified or unmodified; and (c) any one of pharmaceutically acceptable salts of the nucleotides described in (a) or (b), wherein all or part of the nucleotides are ribonucleotides or all or part of the nucleotides are deoxyribonucleotides.

[0009] In a second aspect of the present disclosure, there is provided an active ingredient encapsulated by a lipid component, which is an active ingredient containing a Toll-like receptor agonist provided by the first aspect of the present disclosure. In some embodiments, the lipid component includes DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG.

[0010] In a third aspect of the present disclosure, step (a) of preparing the Toll-like receptor agonist provided by the first aspect of the present disclosure as an acidic solution, step (b) of preparing each component of the lipid component as a lipid ethanol solution, step (c) of mixing and reacting the solutions prepared in steps (a) and (b) above to obtain a primary nano-lipid particle solution, optionally, repeating steps (a) to (c) above to obtain more primary nano-lipid particle solutions, step (d), step (e) of removing ethanol in the primary nano-lipid particle solution above, adjusting the concentration and pH of the active ingredient, and then obtaining a nano-lipid particle suspension, optionally, step (f) of uniformly mixing another therapeutic agent and the nano-lipid particle suspension above to obtain a multi-active ingredient liposome is provided, and a method for producing an active ingredient encapsulated by a lipid component is provided.

[0011] In a fourth aspect of the present disclosure, there is provided a composition comprising an active ingredient encapsulated by a lipid component provided by the second aspect of the present disclosure and another therapeutic agent.

[0012] In a fifth aspect of the present disclosure, there is provided a delivery system comprising an active ingredient and a lipid component, wherein the active ingredient comprises a Toll-like receptor agonist provided by the first aspect of the present disclosure.

[0013] A sixth aspect of this disclosure provides a pharmaceutical composition comprising a Toll-like receptor agonist provided in the first aspect of this disclosure, an active ingredient encapsulated with a lipid component provided in the second aspect, a composition provided in the fourth aspect, or a delivery system provided in the fifth aspect, and a pharmaceutically acceptable carrier or excipient.

[0014] A seventh aspect of this disclosure provides a vaccine adjuvant or vaccine characterized by comprising a Toll-like receptor agonist provided in the first aspect of this disclosure, an active ingredient encapsulated with a lipid component provided in the second aspect, a composition provided in the fourth aspect, a delivery system provided in the fifth aspect, or a pharmaceutical composition provided in the sixth aspect.

[0015] In the eighth aspect of this disclosure, (a) Use in the manufacture of drugs to prevent the development of tumors (b) Use in the manufacture of drugs for the treatment of tumors (c) Use in the manufacture of drugs to inhibit tumor growth (d) Use in the manufacture of drugs to prevent metastasis of tumor cells (e) Use in the manufacture of drugs to prevent tumor recurrence (f) Use in the manufacture of vaccines (g) Use in the manufacture of drugs for the treatment or prevention of inflammatory diseases or infections (h) Use in the manufacture of immunoactivating drugs (i) Use in the manufacture of drugs for upregulating cytokine expression (j) Use to upregulate cytokine expression for purposes other than therapeutic use The use of a Toll-like receptor agonist in the first aspect of the Disclosure, an active ingredient encapsulated with a lipid component in the second aspect, a composition in the fourth aspect, a delivery system in the fifth aspect, or a pharmaceutical composition in the sixth aspect is provided in any one of the above paragraphs (a) to (j).

[0016] In the ninth aspect of this disclosure, (a) Methods to prevent the development of tumors (b) Methods of treating tumors (c) Methods to suppress tumor growth (d) Methods to prevent metastasis of tumor cells (e) Methods to prevent tumor recurrence (f) Methods for treating or preventing inflammatory diseases or infections (g) Methods to activate the immune system (h) Methods for upregulating cytokine expression Any of the above methods (a) to (h) A method is provided, characterized by administering to a subject in need a therapeutically effective amount of a Toll-like receptor agonist according to the first aspect of this disclosure, an active ingredient encapsulated with a lipid component according to the second aspect, a composition according to the fourth aspect, a delivery system according to the fifth aspect, or a pharmaceutical composition according to the sixth aspect.

[0017] In the tenth aspect of this disclosure, Step (a) of preparing a Toll-like receptor agonist according to a first aspect of this disclosure as a Toll-like receptor agonist buffer with a buffer, (b) a step of preparing the lipid components as a lipid ethanol solution with anhydrous ethanol, Step (c) involves mixing the lipid ethanol solution and the Toll-like receptor agonist buffer so that the mass ratio of the lipid component to the Toll-like receptor agonist is (8-12):1. A method for manufacturing a delivery system is provided, characterized by comprising the step (d) of removing ethanol, diluting, and adjusting the pH to 6.8 to 7.2. [Brief explanation of the drawing]

[0018] [Figure 1] This is a curve showing the time course of tumor volume during intratumoral administration to MC38 tumor-bearing mice when administered physiological saline or different CpG-ODNs, namely T0009-1, T0009-2, T0009-3, and T0009-4, in Example 1. [Figure 2]This is a time-course curve of tumor volume in MC38 tumor-bearing mice when Qtolimod (T0009-1 Qtolimod, T0009-2 Qtolimod, T0009-3 Qtolimod, T0009-4 Qtolimod) prepared with physiological saline or different CpG-ODNs was administered in Example 8. 'it' represents an intratumoral injection, and 'q3d×5' represents a total of 5 injections administered every 3 days. [Figure 3] This is a time-course curve of the percentage change in body weight (%) during intratumoral administration to MC38 tumor-bearing mice when Qtolimod prepared with physiological saline or a different CpG-ODN was administered in Example 8. 'it' represents intratumoral administration, and 'q3d×5' represents a total of 5 injections, administered once every 3 days. [Figure 4] This is a time-course curve of tumor volume in B16F10 tumor-bearing mice when Qtolimod prepared with physiological saline or different CpG-ODNs was administered in Example 9. [Figure 5] This is a time-course curve of the percentage change in body weight (%) during administration to B16F10 tumor-bearing mice when Qtolimod prepared with physiological saline or a different CpG-ODN was administered in Example 9. [Figure 6] This is a time-course curve of survival rate in B16F10 tumor-bearing mice when Qtolimod prepared with physiological saline or a different CpG-ODN was administered in Example 9. [Figure 7] The graph shows the time course of tumor volume in MC38 tumor-bearing mice when administered physiological saline, T0009-1 Qtolimod, Tritolimod, or Doxotolimod in Example 10 (the time of the first tumor measurement was the day before the first administration, which is day -1). [Figure 8] This is a time-course curve of the percentage change in body weight (%) during administration to MC38 tumor-bearing mice when physiological saline, T0009-1 Qtolimod, Tritolimod, or Doxotolimod were administered in Example 10. [Figure 9]This is a time-course curve of tumor volume in B16F10 tumor-bearing mice during tail vein administration when Tritolimod or physiological saline was administered in Example 11. [Figure 10] This is a time-course curve of survival rate in B16F10 tumor-bearing mice during tail vein administration when Tritolimod or physiological saline was administered in Example 11. [Figure 11] This is a time-course curve of the percentage change in body weight (%) during intratumoral administration to MC38 tumor-bearing mice when administered physiological saline, lipid solution (QTsome group), or T0009-1 Qtolimod at doses of 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, or 2 mg / kg in Example 12 (the time of the first tumor measurement was the day before the first administration, which is day -1). [Figure 12] The graph shows the time course of tumor volume in MC38 tumor-bearing mice during intratumoral administration when T0009-1 Qtolimod was administered at physiological saline, lipid solution (QTsome group), 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, or 2 mg / kg in Example 12 (the time of the first tumor measurement was the day before the first administration, which is day -1). [Figure 13A] In Example 12, a control group was again established on day 36, and MC38 cells were subcutaneously transplanted into the left shoulder of mice in the control group and each Qtolimod group. The graph shows the time course of tumor volume in tumor-bearing mice (no further administration was given to any of the groups). [Figure 13B] In Example 12, a control group was again established on day 36. MC38 cells were subcutaneously transplanted into the left shoulder of mice in the control group and each Qtolimod group. The tumor volume of the left shoulder of the tumor-bearing mice at the experimental endpoint was then measured (no further administration was given to any of the groups). [Figure 14A]This image shows in vivo imaging of drugs at different time points between intratumoral administration of T0009-1 Qtolimod and free T0009-1 to MC38 tumor-bearing mice in Example 13. Free T0009-1 was administered to the mouse on the left, and T0009-1 Qtolimod was administered to the mouse on the right. The colored areas on the mice correspond to the main distribution areas of the drug; yellow indicates high fluorescence intensity, red indicates moderate fluorescence intensity, and black indicates low fluorescence intensity. [Figure 14B] This is a bar graph of fluorescence intensity imaging of drugs at different time points during intratumoral administration to MC38 tumor-bearing mice when T0009-1 Qtolimod and free T0009-1 were administered in Example 13. [Figure 15A] The images show the CD11b immunohistochemical image (left) and the percentage of CD11b-positive area (right) of mouse tumor sections after administration of physiological saline or T0009-1 Qtolimod in Example 14. In the immunohistochemical image, brown areas indicate CD11b positivity. [Figure 15B] The images show the CD8 immunohistochemical image (left) and the percentage of CD8-positive T cells relative to the total number of tumor cells (right) of mouse tumor sections after administration of physiological saline or T0009-1 Qtolimod in Example 14. [Figure 15C] The images show the F4 / 80 immunohistochemical image (left) and the percentage of F4 / 80 positive area (right) of tumor sections from tumor-bearing mice after administration of physiological saline or T0009-1 Qtolimod in Example 14. In the immunohistochemical image, brown areas indicate F4 / 80 positivity. [Figure 16A] The concentrations of IL-12, IL-10, and IFN-γ in serum samples and tumor samples of tumor-bearing mice after administration of physiological saline, lipid solution (QTsome group), or T0009-1 Qtolimod in Example 14 are shown. [Figure 16B]The concentrations of IL-1β, IL-6, and TNF-α in serum samples and tumor samples of tumor-bearing mice after administration of physiological saline, lipid solution (QTsome group), or T0009-1 Qtolimod in Example 14 are shown. [Figure 17] This shows the time-course changes in body weight of female and male mice when administered subcutaneously with physiological saline (solvent control group), lipid solution (QTsome group), free T0009-1 (API group), or T0009-1 Qtolimod (Qtolimod group) in Example 15. [Figure 18] These are the survival curves for each group of animals during the administration period in Example 16. [Figure 19] This shows the trend in body weight change for each group of animals during the administration period in Example 16. [Figure 20] This shows the trend in subcutaneous tumor volume changes in each group of animals during the administration period in Example 16. [Figure 21] This shows the change in the relative tumor growth rate (T / C) value of animals in each group during the administration period in Example 16. [Figure 22] This is the weight of the subcutaneous tumors in each group of animals in Example 16. [Figure 23] This shows the distribution of metastatic tumor nodules in each tissue of the animals in Example 16. [Figure 24] This is the number of metastatic tumor nodules in the lung tissue of the animals in each group in Example 16. [Figure 25] This is the total number of metastatic tumor nodules in each group of animals in Example 16. [Figure 26] This shows the trend in the volume change of subcutaneous tumors in each group of animals during the administration period in Example 17. [Figure 27] This shows the trend in body weight change for each group of animals during the administration period in Example 17. [Figure 28A] This shows the trend in subcutaneous tumor volume changes in animals in each of the EMT6 groups during the administration period in Example 18. [Figure 28B] This shows the trend in body weight change in each EMT6 group of animals during the administration period in Example 18. [Figure 28C]This shows the trend in subcutaneous tumor volume changes in each EMT6-HPV18 group of animals during the administration period in Example 18. [Figure 28D] This shows the trend in body weight changes in each EMT6-HPV18 group of animals during the administration period in Example 18. [Figure 29A] This shows the trend in subcutaneous tumor volume changes in each K7M2 group of animals during the administration period in Example 19. [Figure 29B] This shows the trend in body weight change in each K7M2 group of animals during the administration period in Example 19. [Figure 29C] This shows the survival status of the animals in each group during the observation period after administration in Example 19. [Figure 30-1] This figure shows the measurement of immune cells in tumor tissue at different time points after different numbers of intratumoral administrations to tumor-bearing mice in Example 20. There are samples from three mice at each time point, and one dot in the figure represents one sample. [Figure 30-2] This figure shows the measurement of immune cells in tumor tissue at different time points after different numbers of intratumoral administrations to tumor-bearing mice in Example 20. There are samples from three mice at each time point, and one dot in the figure represents one sample. [Figure 30-3] This figure shows the measurement of immune cells in tumor tissue at different time points after different numbers of intratumoral administrations to tumor-bearing mice in Example 20. There are samples from three mice at each time point, and one dot in the figure represents one sample. [Figure 30-4] This figure shows the measurement of immune cells in tumor tissue at different time points after different numbers of intratumoral administrations to tumor-bearing mice in Example 20. There are samples from three mice at each time point, and one dot in the figure represents one sample. [Figure 30-5] This figure shows the measurement of immune cells in tumor tissue at different time points after different numbers of intratumoral administrations to tumor-bearing mice in Example 20. There are samples from three mice at each time point, and one dot in the figure represents one sample. [Figure 30-6]This figure shows the measurement of immune cells in tumor tissue at different time points after different numbers of intratumoral administrations to tumor-bearing mice in Example 20. There are samples from three mice at each time point, and one dot in the figure represents one sample. [Figure 31-1] This figure shows the measurement of immune cells in spleen tissue at different time points after different doses of intratumoral administration to tumor-bearing mice in Example 20. There are samples from three mice at each time point, and one dot in the figure represents one sample. [Figure 31-2] This figure shows the measurement of immune cells in spleen tissue at different time points after different doses of intratumoral administration to tumor-bearing mice in Example 20. There are samples from three mice at each time point, and one dot in the figure represents one sample. [Figure 31-3] This figure shows the measurement of immune cells in spleen tissue at different time points after different doses of intratumoral administration to tumor-bearing mice in Example 20. There are samples from three mice at each time point, and one dot in the figure represents one sample. [Figure 31-4] This figure shows the measurement of immune cells in spleen tissue at different time points after different doses of intratumoral administration to tumor-bearing mice in Example 20. There are samples from three mice at each time point, and one dot in the figure represents one sample. [Figure 31-5] This figure shows the measurement of immune cells in spleen tissue at different time points after different doses of intratumoral administration to tumor-bearing mice in Example 20. There are samples from three mice at each time point, and one dot in the figure represents one sample. [Figure 31-6] This figure shows the measurement of immune cells in spleen tissue at different time points after different doses of intratumoral administration to tumor-bearing mice in Example 20. There are samples from three mice at each time point, and one dot in the figure represents one sample. [Figure 32] This is a proteomic analysis of mouse and human PBMC cells after in vitro stimulation in Example 21. [Figure 33] This is an anatomical pathological image of a mouse with bladder intraepithelial carcinoma after administration in Example 22. [Modes for carrying out the invention]

[0019] Unless otherwise specified, terms used herein have the ordinary meanings understood by those skilled in the art. Those skilled in the art can interpret each numerical parameter, depending on the properties and effects sought by this application, by the number of significant figures and the usual rounding methods, or in a manner understandable to those skilled in the art. Generally, the nomenclature and organic chemical, medicinal chemical, or biological experimental procedures used herein are well known and commonly used in the art. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meanings understood by those skilled in the art. If a term has multiple definitions, unless otherwise specified, the definition in this section shall prevail.

[0020] Unless otherwise specified, all numerical values ​​used in this specification and in the claims, such as content, concentration, proportion, weight, particle size, percentage, and technical effect, should be understood to be modified in all cases by the term "approximately" or "about." Therefore, unless otherwise stated, the numerical parameters listed in the following descriptions and in the claims below are approximations. "Approximately" or "about" can be understood to mean within a range of ±10%, ±20%, ±30%, ±40%, or ±50% of the given numerical value.

[0021] As used herein, the expression "A and / or B" includes three cases: (1) A, (2) B, and (3) A and B. The expression "A, B and / or C" includes seven cases: (1) A, (2) B, (3) C, (4) A and B, (5) A and C, (6) B and C, and (7) A, B and C. The meanings of similar expressions are determined accordingly.

[0022] As used herein, the terms "contains" and "include" indicate that elements other than those listed are not excluded.

[0023] (definition) As used herein, the term “pharmaceutically acceptable salt” includes acid addition salts and base addition salts. Suitable acid addition salts are produced from acids that produce non-toxic salts. Examples include acetates, adipines, aspartates, benzoates, benzenesulfons, bicarbonates / carbonates, bisulfates / sulfates, borates, dl-camphor sulfons, citrates, cyclohexylamine sulfons, ethanedisulfons, formates, trans-2-butene diates, glucoheptons, glucons, glucurons, hexafluorophosphates, 2-(4-hydroxybenzyl)benzoates, hydrochlorides / chlorides, hydrobroms / bromides, and hydroiodides / iodides. This includes, but is not limited to, substances, 2-hydroxyethanesulfonates, lactates, malates, cis-2-butene diates, malons, methanesulfons, methylsulfates, naphthoates, 2-naphthalenesulfons, nicotinates, nitrates, orotates, oxalates, hexadecanates, phosphates / hydrogen phosphates / dihydrogen phosphates, L-pyroglutamates, saccharates, stearates, salicylates, tannates, tartrates, toluenesulfonates, and trifluoroacetates. Suitable base addition salts are produced from bases that produce non-toxic salts. Examples include, but are not limited to, aluminum salts, arginine salts, calcium salts, choline salts, diethylamine salts, diethanolamine salts, glycine salts, lysine salts, magnesium salts, meglumine salts, ethanolamine salts, potassium salts, sodium salts, trometamol salts, and zinc salts. Hemi salts of acids and bases, such as hemisulfates and hemicalcium salts, can also be produced. For an overview of appropriate salts, see Handbook of Pharmaceutical Salts: Properties, Selection and Use by Stahl and Wermuth (Wiley-VCH, 2002).

[0024] As used herein, the term “functional variant” means a Toll-like receptor agonist having substantial or significant sequence matching or similarity with a parent Toll-like receptor agonist, CpG ODN, wherein the functional variant retains at least 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the biological activity of the parent Toll-like receptor agonist.

[0025] (Toll-like receptor agonists) This disclosure provides a Toll-like receptor agonist comprising (a) any one of the modified or unmodified nucleotides represented by SEQ ID NOs: 1 to 4, (b) a functional variant of any one of the modified or unmodified nucleotides represented by SEQ ID NOs: 1 to 4, and (c) any pharmaceutically acceptable salt of the nucleotide described in (a) or (b), wherein the nucleotide is all or partly a ribonucleotide, or all or partly a deoxyribonucleotide.

[0026] In some embodiments, modifications include one or more of the following: replacement of all or some P=O bonds between two adjacent nucleotides in a sequence with P=S bonds; replacement of all or some deoxyribonucleosides in a sequence with ribonucleosides and replacement of all or some ribonucleosides in a sequence with deoxyribonucleosides; replacement of the 2'-OH group of a ribonucleoside or deoxyribonucleoside with a methoxy group, a methoxyethyl group or a halogen; and formation of locked nucleic acids or (S)-cEt-BNA by crosslinking of the second and fourth carbon atoms of the nucleotide. In some embodiments, the halogen is selected from fluorine, chlorine, bromine, and iodine.

[0027] (Active ingredients encapsulated by lipids) This disclosure provides an active ingredient encapsulated by a lipid component, characterized in that it is an active ingredient containing a Toll-like receptor agonist provided herein.

[0028] In some embodiments, the mass ratio of the lipid component to the Toll-like receptor agonist provided herein is (8-12):1, preferably 10:1.

[0029] In some embodiments, the lipid component comprises one or more of cationic polymers, nanolipid particles (LNPs), cationic liposomes, QTsomes, lipopolyplexes, microparticles, microspheres, and nanoemulsions. QTsomes are lipid nanoparticles in which quaternary and trivalent cationic phospholipids coexist. In some embodiments, the disclosure employs QTsome lipid nanotechnology to encapsulate CpG-ODNs and introduce a combination of cationic lipids that can be ionized under specific conditions, thereby leveraging the liposome's sensitivity to the pH of the external environment to maximize oligonucleotide loading and promote intracellular delivery of CpG-ODNs.

[0030] In some embodiments, the lipid component includes one or more of the following: cationic lipids, ionizable lipids, neutral lipids, and PEGylated lipids.

[0031] Cationic lipids include DOTAP (CAS No. 132172-61-3, (2,3-Dioleoyloxy-propyl)-trimethylammonium-chloride), DOTMA (CAS No. 104872-42-6), DDBA, DMRIE, DOTIM, SAINT, DC-Chol (DC cholesterol, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride), BGTC, CTAP, DOSPA, DORIE, DODAB, DOIC, DMEPC, DOGS, DIMRI, DC-6-14, and CLIP. 1. This includes, but is not limited to, what is disclosed in DORIE, DOSPA, CLIP6, CLIP9, and U.S. Patent No. 5,049,386, International Publication Nos. WO91 / 16024, WO97 / 019675, WO2005 / 121348, WO2009 / 086558 and WO2011 / 13636.

[0032] Ionizable lipids include tertiary amines and their derivatives, pyrrolidines and their derivatives, piperazines and their derivatives, piperidines and their derivatives, for example, DODMA (1,2-dioleyloxy-3-dimethylaminopropane, CAS number 104162-47-2), ALC-0159, A066(Z016)(1- (2,3-bis(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)propyl)pyrrolidine, 1-(2,3-bis(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)propyl)pyrrolidine), L-319 (CAS No. 1351586-50-9), DODAP, C12-200, 5A2-SC8, 306Oi10, Moderna Lipid 5, Acuitas A9, ALC-0315, SM-102 (also known as HUO, Moderna Lipid H; 1-octylnonyl 5 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoate, 1-octylnonyl 5 8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoate)), DLin-MC3-DMA ((6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate, (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl This includes, but is not limited to, 4-(dimethylamino)butanoate, DLin-K-DMA, DLin-KC2-DMA, DLin-KC3-DMA, DLin-KC4-DMA, DLinDMA, other DLinDMA derivatives, other DLin-K-DMA derivatives, and lipid-like 5A2-SC8.

[0033] In some embodiments, the neutral lipids include, but are not limited to, 1,2-bishexadecanoyl-sn-glycero-3-phosphorylcholine (DPPC), dioleoyl L-α-phosphatidylethanolamine (DOPE, CAS No. 4004-05-1), 1,2-bis-(9Z-octadecenoyl)-sn-glycero-3-phosphorylcholine (DOPC, CAS No. 4235-95-4), 1-hexadecane 2-(cis-9-octadecenoyl)-sn-glycero-3-phosphorylcholine (POPC), egg yolk lecithin (EPC), distearoylphosphatidylcholine (DSPC, CAS No. 816-94-4), and cholesterol. In some embodiments, the neutral lipids include neutral phospholipids, phosphatidylcholine and / or phosphatidylethanolamine, for example, distearoyl phosphatidylcholine (DSPC), egg yolk lecithin (EPC), soy lecithin, hydrogenated soy lecithin (HSPC), dioleoyl phosphatidylethanolamine (DOPE), dilauroyl phosphatidylcholine (DLPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), 1,2-dioleoyl phosphatidyl This includes, but is not limited to, phosphorus (DOPC), diarachidonoylphosphatidylcholine (DAPC), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), 1-palmitoyl-2-oleoylethanolamine (POPE), and 1-hexadecane 2-(cis-9-octadecenoyl)-sn-glycero-3-phosphorylcholine (POPC).

[0034] In some embodiments, the pegylated lipids include, but are not limited to, PEG-docosylacetamide, PEG-myristoyl diglyceride, PEG-diacylglycerol, PEG-dialkyloxypropyl, PEG-phospholipids, PEG-ceramide, DMG-PEG, PEG-DSPE, and DSG-PEG. In some embodiments, the pegylated lipids can be selected from at least one of DSG-PEG and DMG-PEG.

[0035] In some embodiments, the lipid component comprises DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG. In some preferred embodiments, the lipid component comprises DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG, and the molar ratio of DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG is (1-30):(5-50):(5-40):(15-60):(0-10). In some more preferred embodiments, the lipid component comprises DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG, and the molar ratio of DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG is 15:25:20:38.5:1.5.

[0036] In some embodiments, the active ingredient encapsulated with a lipid component comprises a Toll-like receptor agonist provided by this disclosure and another therapeutic agent. In some preferred embodiments, the molar ratio of the other therapeutic agent to the Toll-like receptor agonist provided by this disclosure is (0.1 to 5):1, preferably 1:2.

[0037] In some embodiments, the other therapeutic agent is selected from one or more antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the other therapeutic agent is selected from one or more polyinosinate-polycytidylic acid (Poly(I:C)), STING agonists, imiquimod, reximod, sorafenib, sunitinib, erlotinib, doxorubicin, paclitaxel, gemcitabine, and radiotherapy. In some preferred embodiments, the other therapeutic agent is one or more of reximod and doxorubicin.

[0038] In some embodiments, the antibodies include, but are not limited to, antibodies having antitumor effects. Antitumor-containing antibodies include, but are not limited to, antibodies targeting PD-1, such as nivolumab, pembrolizumab, cemiprimab, tripalimab, cintilimab, and camrelizumab; antibodies targeting PD-L1, such as atezolizumab, avelumab, and durvalumab; and antibodies targeting CTLA4, such as ipilimumab and tremelimumab. In some embodiments, the Toll-like receptor agonists, CpG-ODNs, provided herein are antitumor-containing antibody drugs that, when used in combination with, for example, antibody drugs targeting PD-1 or PD-L1, can effectively enhance the responsiveness of immune checkpoint inhibitors or overcome resistance to immune checkpoint inhibitors, which is more advantageous in further expanding the patient population for PD-1 or PD-L1 inhibitors, improving efficacy, and expanding indications.

[0039] In some embodiments, the antibodies include, but are not limited to, antitumor-associated antigen (TAA) antibodies. TAAs include, but are not limited to, GPC, CEA, immature laminin receptor, TAG-72, HPV E6, HPV E7, EGFR, Ep-CAM, EphA3, Her2, Her3, ROR2, PSMA, STEAP1, FGFR2, TROP2, B7-H3, B7-H4, B7-H6, FOLR1, BAGE family, CAGE family, GAGE ​​family, MAGE family, SAGE family, XAGE family, SSX-2, fibronectin, MART-2, PDL-1, VEGFR, CLAUDIN, and the like.

[0040] In some embodiments, the chemotherapy agents include, but are not limited to, doxorubicin, paclitaxel, gemcitabine, and radiotherapy.

[0041] In some embodiments, small molecule drugs include, but are not limited to, pattern recognition receptor agonists other than the Toll-like receptor agonists provided herein, anthracycline compounds, camptothecin compounds, immunogenic cell death (ICD) inducers, tyrosine kinase inhibitors, paclitaxel compounds, Bruton's tyrosine kinase (BTK) inhibitors, PI3K inhibitors, HDAC inhibitors, ERK inhibitors, MAPK inhibitors, PD-1 / PD-L1 inhibitors, CTLA-4 inhibitors, TIGIT inhibitors, TIM3 inhibitors, EGFR inhibitors, VEGF inhibitors, PARP inhibitors, Her2, LAG-3, and TNFR2 inhibitors.

[0042] In some embodiments, the anthracycline compound includes, but is not limited to, doxorubicin, epirubicin, pirarubicin, daunorubicin, acralubicin, idarubicin, and amrubicin.

[0043] In some embodiments, camptothecin compounds include, but are not limited to, topotecan, irinotecan, verotecan, exatecan (DX-8951), lutotecan, sinotecan, rubitecan (9-NC), 9-aminocamptothecin (9-AC), gimatecan, karenitecin, and DB-67.

[0044] In some embodiments, the tyrosine kinase inhibitor is sorafenib, sunitinib, gefitinib, erlotinib, lapatinib, afatinib, dacomitinib, vandetanib, neratinib, peritinib (EKB-569), canertinib (CI-1033), osimertinib, rociletinib (CO-1686, Clovis Oncology), olmutinib (HM61713, Beijing Hanmi Pharmaceutical Co., Ltd.), naquotinib (ASP8273, Astellas Pharma), tesevatinib (XL647 / KD019, Kadmon This includes, but is not limited to, nazartinib (EGF816, Novartis) and PF-06747775 (Pfizer), among others.

[0045] In some embodiments, the immunogenic cell death inducer includes, but is not limited to, DNA damaging agents, bleomycin, and the like.

[0046] In some embodiments, the paclitaxel-based compound includes, but is not limited to, paclitaxel, docetaxel, and the like.

[0047] Pattern recognition receptor agonists other than Toll-like receptor agonists provided in this disclosure include, but are not limited to, Toll-like receptors, RIG-I-like receptors, Nod-like receptors, AIM2-like receptors, and type C lectin receptors, as well as cGas and other intraplasmic DNA sensor agonists.

[0048] In some embodiments, resiquimod (a TLR7 / 8 agonist, also known as R848) and the CpG-ODNs provided herein can be encapsulated together to obtain a dual active ingredient liposome (Tritolimod), which can further enhance the immune response in the tumor microenvironment and effectively improve the therapeutic effect on tumors.

[0049] In some embodiments, the molar ratio of resiquimod to CpG-ODN is (0.1 to 5):1, preferably 1:2.

[0050] In some embodiments, the chemotherapy drug doxorubicin hydrochloride (DOX) and the CpG-ODNs provided herein can be encapsulated together to obtain a dual active ingredient liposome (Doxotolimod), thereby enabling doxorubicin to promote the release of tumor antigens, thereby activating CD8 by the CpG-ODNs. + This can enhance the recognition and killing of tumor cells by T cells, thereby effectively improving the effectiveness of tumor treatment.

[0051] In some embodiments, the molar ratio of doxorubicin to CpG-ODNs is (0.1 to 5):1, preferably 1:2.

[0052] (Delivery system) This disclosure provides a delivery system comprising an active ingredient and a lipid component, wherein the active ingredient comprises a Toll-like receptor agonist provided by this disclosure.

[0053] In some embodiments, the mass ratio of the lipid component to the Toll-like receptor agonist provided herein is (8-12):1, preferably 10:1.

[0054] In some embodiments, the lipid component includes one or more of cationic polymers, nanolipid particles (LNPs), cationic liposomes, QTsomes, lipopolyplexes, microparticles, microspheres, and nanoemulsions.

[0055] In some embodiments, the lipid component includes one or more of cationic lipids, ionizable lipids, neutral lipids, and PEGylated lipids. In some embodiments, the PEGylated lipid can be selected from at least one of DSG-PEG and DMG-PEG.

[0056] In some embodiments, the lipid component comprises DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG. In some preferred embodiments, the lipid component comprises DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG, and the molar ratio of DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG is (1-30):(5-50):(5-40):(15-60):(0-10). In some more preferred embodiments, the lipid component comprises DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG, and the molar ratio of DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG is 15:25:20:38.5:1.5.

[0057] In some embodiments, the active ingredient in the delivery system comprises a Toll-like receptor agonist provided in this disclosure and another therapeutic agent. In some preferred embodiments, the molar ratio of the other therapeutic agent to the Toll-like receptor agonist provided in this disclosure is (0.1 to 5):1, preferably 1:2.

[0058] In some embodiments, the other therapeutic agent is selected from one or more antibodies, chemotherapeutic agents, and small molecule drugs. In some embodiments, the other therapeutic agent is selected from one or more polyinosinate-polycytidylic acid (Poly(I:C)), STING agonists, imiquimod, reximod, sorafenib, sunitinib, erlotinib, doxorubicin, paclitaxel, gemcitabine, and radiotherapy. In some preferred embodiments, the other therapeutic agent is one or more of reximod and doxorubicin.

[0059] In some embodiments, resiquimod (a TLR7 / 8 agonist, also known as R848) and the CpG-ODNs provided herein can be encapsulated together to obtain a dual active ingredient liposome (Tritolimod), which can further enhance the immune response in the tumor microenvironment and effectively improve the therapeutic effect on tumors.

[0060] In some embodiments, the molar ratio of resiquimod to CpG-ODN is (0.1 to 5):1, preferably 1:2.

[0061] In some embodiments, the chemotherapy drug doxorubicin hydrochloride (DOX) and the CpG-ODNs provided herein can be encapsulated together to obtain a dual active ingredient liposome (Doxotolimod), thereby enabling doxorubicin to promote the release of tumor antigens, thereby activating CD8 by the CpG-ODNs. + This can enhance the recognition and killing of tumor cells by T cells, thereby effectively improving the effectiveness of tumor treatment.

[0062] In some embodiments, the molar ratio of doxorubicin to CpG-ODNs is (0.1 to 5):1, preferably 1:2.

[0063] The delivery system having a specific lipid composition provided by this disclosure can provide a stable coating to CpG-ODNs, protect them from degradation by endogenous enzymatic systems, improve the bioavailability of CpG-ODNs, extend the half-life of oligonucleotides, and improve drug particle size and cell adsorption, thereby achieving the objective of improving drug retention within tumors. When the delivery system provided by this disclosure is used in combination with intratumoral administration, it becomes possible to effectively improve drug retention within tumors, efficiently deliver drugs to tumor-infiltrating immune cells, enhance the immune response of tumor tissue, and improve the efficacy of tumor therapy.

[0064] The delivery system provided in this disclosure targets dendritic cells, macrophages, and CD3 cells at the tumor site. + T cells, CD8 + It can effectively enhance the infiltration of immune cells such as T cells and NK cells, and promote the release of factors such as interleukins, interferons, chemokines, and granzymes, thereby realizing tumor immunotherapy. Conventional alum adjuvants, squalene, and QS21 are microemulsions, and they have local retention and sustained release properties. They can induce antigen-specific immunity and have good safety, but they cannot induce a strong type I immune response. Conventional CpG adjuvants are appropriate for CD8 + While conventional CpG adjuvants can induce a T-cell response, they can rapidly spread throughout the body, causing adverse side effects and interfering with treatment. Furthermore, in conventional CpG adjuvants, the adjuvant separates from the antigen, inducing nonspecific autoimmunity. The delivery system provided in this disclosure induces an appropriate type I immune response without inducing systemic cytokines and also has the advantage of inducing antigen-specific immunity compared to conventional adjuvants.

[0065] The delivery systems comprising CpG-ODNs provided herein can effectively inhibit tumor growth and can be combined with one or more pharmaceutically acceptable excipients to manufacture drugs for use in treating tumors.

[0066] The delivery system comprising CpG-ODNs provided herein can effectively prevent the metastasis of tumor cells to other tissues and organs, or prevent tumor recurrence.

[0067] (Manufacturing method) This disclosure is, Step (a) of preparing a Toll-like receptor agonist, CpG-ODNs, provided in a first aspect of this disclosure, as an acidic solution, (b) a step of preparing each component of the lipid as a lipid ethanol solution, Step (c) involves mixing and reacting the solutions prepared in steps (a) and (b) above to obtain a primary solution of nanolipid particles, Selectively repeat the above steps (a) to (c) to obtain a larger amount of nanolipid particle primary solution (d), Step (e) removes ethanol from the above nanolipid particle primary solution, adjusts the concentration and pH of the active ingredient, and then obtains a nanolipid particle suspension. The present invention provides a method for producing an active ingredient encapsulated by a lipid component, characterized by comprising the step (f) of selectively mixing another therapeutic agent and the above-mentioned nanolipid particle suspension uniformly to obtain a multi-active ingredient liposome.

[0068] In some embodiments, when using the above method to produce a single active ingredient encapsulated with a lipid component, step (f) is an optional step.

[0069] In some embodiments, step (f) is performed when producing a multi-active ingredient encapsulated with a lipid component using the method described above. In some embodiments, the other therapeutic agent used in step (f) is preferably reximod or doxorubicin.

[0070] This disclosure further provides a method for manufacturing a delivery system, comprising the steps of: preparing a Toll-like receptor agonist as a Toll-like receptor agonist buffer with a buffer; preparing a lipid component as a lipid ethanol solution with anhydrous ethanol; and mixing the lipid ethanol solution and the Toll-like receptor agonist buffer so that the mass ratio of the lipid component to CpG-ODN is (8-12):1, removing the ethanol, diluting, and adjusting the pH to 6.8-7.2 to complete the process.

[0071] (Composition, pharmaceutical composition) This disclosure provides a composition characterized by comprising an active ingredient encapsulated by a lipid component provided herein and another therapeutic agent. In some embodiments, the other therapeutic agent is selected from one or more antibodies, chemotherapeutic agents and small molecule drugs. In some embodiments, the other therapeutic agent is selected from one or more polyinosinate-polycytidylic acid (poly IC), STING agonists, imiquimod, reximod, sorafenib, sunitinib, erlotinib, doxorubicin, paclitaxel, gemcitabine, and radiotherapy. In some preferred embodiments, the other therapeutic agent is selected from one or more reximod and doxorubicin.

[0072] This disclosure provides a pharmaceutical composition characterized by comprising a Toll-like receptor agonist, an active ingredient encapsulated with a lipid component, a composition or delivery system provided herein, and a pharmaceutically acceptable carrier or excipient.

[0073] As used herein, the term “pharmaceutical composition” refers to a mixture of a Toll-like receptor agonist, an active ingredient encapsulated with a lipid component, a composition, or a delivery system provided herein, and other chemical components, such as carriers, stabilizers, diluents, dispersants, suspenders, thickeners, and / or excipients. Pharmaceutical compositions are designed to facilitate the administration of the Toll-like receptor agonist, the active ingredient encapsulated with a lipid component, the composition, or the delivery system to a living organism. In the art, there are many methods of administration of pharmaceutical compositions, including, but not limited to, intratumoral injection, intravenous injection, subcutaneous injection, intramuscular injection, arterial injection, intraperitoneal injection, and intracentral nervous system injection, for example, intrathecal injection, intravesical instillation, interventional therapy, oral administration, transdermal administration, transpulmonary administration, intraocular administration, and topical administration.

[0074] As described herein, the pharmaceutical composition can be formulated into dosage forms suitable for administration to a subject via a desired route of administration. The dosage forms include, but are not limited to, tablets, capsules, caplets, pills, tablets, powders, syrups, elixirs, suspensions, solutions, emulsions, transdermal patches, suppositories, inhalants, creams, ointments, washes, pastes, sprays, lyophilized preparations, injections, and gels.

[0075] The term “pharmaceutically acceptable carrier” includes, for example, pharmaceutically acceptable materials, compositions, or carriers relating to liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials that carry or transport, or carry, to a subject, an active ingredient, composition, or delivery system encapsulated by the Toll-like receptor agonist or lipid component of the Disclosure, and deliver to a subject to perform its predetermined function. Each salt or carrier must be “acceptable” in terms of compatibility with the other components of the formulation and must not harm the subject. Examples of materials that can be used as pharmaceutically acceptable carriers include sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth gum powder; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; and polyols, such as glycerol and sorbitol. Examples include tall, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffers; diluents; granulators; lubricants; binders; disintegrants; wetting agents; emulsifiers; colorants; release agents; coating agents; sweeteners; flavoring agents; preservatives; antioxidants; plasticizers; gelling agents; thickeners; curing agents; molding additives; suspending agents; surfactants; humectants; carriers; stabilizers; and other non-toxic and suitable substances used in pharmaceutical formulations, or any combination thereof.

[0076] (Vaccine, vaccine adjuvant) The CpG-ODNs provided by this disclosure can also be used in the vaccine and vaccine adjuvant fields to enhance the immune response and improve antibody titers. In some embodiments, CpG-ODNs can be used as the main component to manufacture vaccine adjuvants, and the CpG-ODNs can be present in the vaccine formulation as lipid-bound, directly encapsulated, or as free particles in the vaccine formulation.

[0077] This disclosure provides a vaccine adjuvant or vaccine characterized by comprising a Toll-like receptor agonist, an active ingredient encapsulated with a lipid component, a composition, a delivery system, or a pharmaceutical composition provided herein.

[0078] In some embodiments, the vaccine or vaccine adjuvant described above is a vaccine or vaccine adjuvant against KRAS, RSV, TERT, TP53, hepatitis viruses, neoantigens, etc.

[0079] (Usage and Instructions) This disclosure is, (a) Use in the manufacture of drugs to prevent the development of tumors (b) Use in the manufacture of drugs for the treatment of tumors (c) Use in the manufacture of drugs to inhibit tumor growth (d) Use in the manufacture of drugs to prevent metastasis of tumor cells (e) Use in the manufacture of drugs to prevent tumor recurrence (f) Use in the manufacture of vaccines (g) Use in the manufacture of drugs for the treatment or prevention of inflammatory diseases or infections (h) Use in the manufacture of immunoactivating drugs (i) Use in the manufacture of drugs for upregulating cytokine expression (j) Use to upregulate cytokine expression for purposes other than therapeutic use The present disclosure provides the use of any one of the above-mentioned Toll-like receptor agonists, active ingredients encapsulated with lipid components, compositions, delivery systems, or pharmaceutical compositions.

[0080] In some embodiments, immune activation includes, but is not limited to, activation of the B cell pathway, activation of the T cell pathway, activation of the myeloid-derived lymphocyte pathway, and activation of the lymphoid-derived lymphocyte pathway. In some embodiments, immune activation involves macrophages, plasmacytoid dendritic cells (pDCs), B cells, T cells, and CD3 cells. + T cells, CD8 + This includes, but is not limited to, the activation of immune cells such as T cells, NK cells, and granulocytes.

[0081] In some embodiments, the drug can be administered by many methods, including but not limited to intratumoral injection, intravenous injection, subcutaneous injection, intramuscular injection, arterial injection, intraperitoneal injection, and central nervous system injection, for example, intrathecal injection, intravesical instillation, interventional therapy, oral administration, transdermal administration, transpulmonary administration, intraocular administration, and local administration.

[0082] In some embodiments, the tumor is selected from solid tumors or tumors to which drugs can be injected, and is, for example, at least one of head and neck cancer, esophageal cancer, oral cancer, nasopharyngeal cancer, thyroid cancer, lung cancer, gastric cancer, liver cancer, pancreatic cancer, splenic cancer, non-small cell lung cancer, colorectal cancer, colon cancer, intestinal cancer, kidney cancer, brain tumor, glioma, bladder cancer, prostate cancer, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, uterine cancer, bone cancer, osteosarcoma, osteochondroma, liposarcoma, neuroblastoma, synovial sarcoma, astrocytoma, glioblastoma pleomorphic, anaplastic astrocytoma, peritoneal cancer, soft tissue sarcoma, sarcoma, rhabdomyosarcoma, end-stage myxoid disease, melanoma, skin cancer, for example, non-melanoma skin cancer.

[0083] Inflammatory diseases include, but are not limited to, delayed-type allergic reactions, allergic diseases, systemic lupus erythematosus, graft-versus-host disease, vasculitis due to hepatitis C, type 1 diabetes, type 2 diabetes, multiple sclerosis, rheumatoid arthritis, alopecia areata, atherosclerosis, psoriasis, organ transplant rejection, dryness syndrome, Behçet's disease, spontaneous abortion, atopic diseases, asthma, and inflammatory bowel disease.

[0084] Infections include, but are not limited to, fungal infections, bacterial infections, mycoplasma or chlamydia infections, and viral infections such as viral hepatitis.

[0085] This disclosure further states that (a) Methods to prevent the development of tumors (b) Methods of treating tumors (c) Methods to suppress tumor growth (d) Methods to prevent metastasis of tumor cells (e) Methods to prevent tumor recurrence (f) Methods for treating or preventing inflammatory diseases or infections (g) Methods to activate the immune system (h) Methods for upregulating cytokine expression Any of the above methods (a) to (h) The present invention provides a method characterized by administering a therapeutically effective amount of the Toll-like receptor agonist, active ingredient, composition, delivery system, or pharmaceutical composition encapsulated with a lipid component, to a subject with a need.

[0086] In some preferred embodiments, to prevent tumor development, treat tumors, inhibit tumor growth, prevent metastasis of tumor cells, and prevent tumor recurrence, subjects with a need are administered the Toll-like receptor agonist or active ingredient encapsulated with a lipid component of the present disclosure at a dose of at least 0.1 mg / kg (mpk) of body weight, for example, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, or 8 mg / kg of body weight. In some preferred embodiments, to prevent tumor development, treat tumors, inhibit tumor growth, prevent metastasis of tumor cells, and prevent tumor recurrence, the Toll-like receptor agonist or active ingredient encapsulated with a lipid component of the Disclosure is administered to a subject in need at least once a week, for a total of at least three times, for example, once every three days for a total of four times, once every three days for a total of five times, once a week for a total of three times, once a week for a total of four times, or once a week for a total of five times. In some preferred embodiments, the subject in need is a mouse. Those skilled in the art, knowing the above dosages and frequencies, can convert the dosages and frequencies for a particular animal, e.g., a mouse, to the dosages and frequencies for another animal, e.g., a human, by referring to the usual technical means of the art.

[0087] In some embodiments, the tumor is selected from solid tumors or tumors to which drugs can be injected, and is, for example, at least one of head and neck cancer, esophageal cancer, oral cancer, nasopharyngeal cancer, thyroid cancer, lung cancer, gastric cancer, liver cancer, pancreatic cancer, splenic cancer, non-small cell lung cancer, colorectal cancer, colon cancer, intestinal cancer, kidney cancer, brain tumor, glioma, bladder cancer, prostate cancer, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, uterine cancer, bone cancer, osteosarcoma, osteochondroma, liposarcoma, neuroblastoma, synovial sarcoma, astrocytoma, glioblastoma pleomorphic, anaplastic astrocytoma, peritoneal cancer, soft tissue sarcoma, sarcoma, rhabdomyosarcoma, end-stage myxoid disease, melanoma, skin cancer, for example, non-melanoma skin cancer.

[0088] Inflammatory diseases include, but are not limited to, delayed-type allergic reactions, allergic diseases, systemic lupus erythematosus, graft-versus-host disease, vasculitis due to hepatitis C, type 1 diabetes, type 2 diabetes, multiple sclerosis, rheumatoid arthritis, alopecia areata, atherosclerosis, psoriasis, organ transplant rejection, dryness syndrome, Behçet's disease, spontaneous abortion, atopic diseases, asthma, and inflammatory bowel disease.

[0089] Infections include, but are not limited to, fungal infections, bacterial infections, mycoplasma or chlamydia infections, and viral infections such as viral hepatitis.

[0090] As used herein, the term “subject” includes animals such as vertebrates, preferably mammals, such as dogs, cats, pigs, cattle, goats, horses, rabbits, rodents (e.g., mice, rats, or guinea pigs), or primates (e.g., monkeys, gorillas, chimpanzees, or humans).

[0091] As used herein, the term “treatment” means alleviating or improving a disease or disorder (i.e., delaying or preventing the progression of the disease or at least one clinical symptom), or reducing or improving at least one physical parameter or biomarker associated with the disease or disorder.

[0092] As used herein, the term “therapeutic dose” means the amount that provides benefit or alleviates disease compared to a corresponding subject who has not received that amount, and is also, in reasonable medical judgment, sufficiently low to avoid serious side effects. The therapeutic dose of the Toll-like receptor agonist, lipid-encapsulated active ingredient, composition, delivery system, or pharmaceutical composition described herein varies depending on factors such as the selected Toll-like receptor agonist, lipid-encapsulated active ingredient, composition, delivery system, or pharmaceutical composition; route of administration; severity of the disease being treated; age, body type, weight, and physical condition of the patient being treated; patient's medical history; duration of treatment; characteristics of parallel treatments; and desired therapeutic effect, but can be determined by ordinary means by those skilled in the art.

[0093] As used herein, the term “prevention” includes prevention of the onset or recurrence of the disease in individuals who are susceptible to the disease but have not yet been diagnosed with the disease.

[0094] This disclosure further states that (a) Prevention of tumor development (b) Treatment of tumors (c) Suppression of tumor growth (d) Prevention of tumor cell metastasis (e) Prevention of tumor recurrence (f) Treatment or prevention of inflammatory diseases or infections (g) Immune activation (h) Upregulation of cytokine expression The present disclosure provides a Toll-like receptor agonist, an active ingredient encapsulated with a lipid component, a composition, a delivery system, or a pharmaceutical composition for use in any of the above uses (a) to (h).

[0095] The various embodiments described above relating to the Toll-like receptor agonists, lipid-encapsulated active ingredients, and delivery systems of this disclosure are also suitable for the pharmaceutical compositions, compositions, uses, and methods of this disclosure (unless they are inconsistent), and various embodiments combining these are also considered part of this disclosure. [Examples]

[0096] The following description of exemplary embodiments of the present application, with reference to the drawings, includes various details of the embodiments for ease of understanding. These are illustrative only and should be understood as not intended to limit the scope of protection of the present application. The scope of protection of the present application is limited only to the claims. Therefore, those skilled in the art should understand that various changes and modifications can be made to the embodiments described herein without departing the scope of the present application. Furthermore, for clarity and conciseness, descriptions of well-known functions and structures have been omitted in the following description.

[0097] Unless otherwise specified, the experimental methods in the following examples are all conventional methods and should be carried out in accordance with the techniques or conditions described in the literature in the art, or in accordance with the product specifications. Unless otherwise specified, commercially available materials and reagents can be used in the following examples.

[0098] Example 1: Effect of CpG-ODN on the growth of colon cancer tumors in mice The experimental drugs were the four CpG-ODNs (T0009-1 to T0009-4) listed in Table 1, and in the sequences shown in Table 1, all phosphodiester bonds were thiolated. [Table 1]

[0099] The experimental drugs described above were diluted to 1 μg / μL using physiological saline (0.9% sodium chloride aqueous solution).

[0100] 1. Experimental Procedure (1) 50 seven-week-old C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., equal numbers of males and females) were reared for one week to allow them to adapt, and then their weight was measured. (2) The mouse colon cancer MC38 cell line (Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP60825) was revived and subcultured. When the cell state was good, MC38 cells (seeding amount was 10 6 cells) were transplanted subcutaneously into the back of the mice, and the changes in the body weight of the mice and the size of the tumors were recorded. (3) When the tumor reached approximately 100 mm 3 , the mice were grouped. Mice with tumors that were too large or too small were removed, and the same number of mice as at the time of incorporation was maintained. They were randomly divided into 5 groups using the complete randomization grouping method, denoted as group T0009-1, group T0009-2, group T0009-3, group T0009-4, and the physiological saline group, with 7 mice in each group. (4) On day 0, 25 μL of T0009-1, T0009-2, T0009-3, T0009-4, or physiological saline (0.9% sodium chloride aqueous solution) was administered intratumorally to each mouse at a dosage of 1 mg / kg body weight (1 mg / kg), once every 3 days, for a total of 5 injections. The first injection was on day 0. (5) The growth and proliferation status of the colon tumors in the subcutaneous back of the tumor-bearing mice in the administration group and the physiological saline control group were observed and recorded daily, including the measurement of the mouse body weight and tumor volume, and the creation of the tumor volume change curve and body weight change curve. (6) On day 18, the mice were sacrificed, the tumor masses were excised, the volume of the tumor masses was measured, the net weight of the tumor masses was weighed, and based on the relative tumor volume (RTV) of each group of mice on day 18 at the time of grouping, the tumor growth inhibition rate (TGI) was calculated. The calculation formula for TGI (%) was TGI (%) = [1 - RTV (administration group) / RTV (control group)] × 100%.

[0101] 2. Experimental results As shown in Figure 1, each treatment group showed a very significant tumor-suppressing effect, with T0009-1 being the most effective. The calculated TGI% for T0009-1 to T0009-4 were 73.72%, 56.12%, 58.41%, and 65.37%, respectively, with T0009-1 showing the best tumor-suppressing effect. No mice died in any of the treatment groups from the start of administration until 18 days later.

[0102] Examples 2-5: Production of lipid nanoparticles (Qtolimod) containing encapsulated CpG-ODNs The CpG-ODNs used in Examples 2-5 were T0009-1, T0009-2, T0009-3, and T0009-4, respectively, and the CpG-ODN Qtolimods produced in Examples 2-5 were T0009-1 Qtolimod, T0009-2 Qtolimod, T0009-3 Qtolimod, and T0009-4 Qtolimod, respectively.

[0103] A method for manufacturing Qtolimod containing encapsulated CpG-ODN includes the following steps. (1) CpG-ODN was obtained, and a CpG-ODN sodium acetate solution was prepared using sodium acetate buffer (pH 5.0) with a pH of 5.0, a CpG-ODN concentration of 0.25 mg / mL, and a sodium acetate concentration of 25 mM. (2) DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG were obtained, and a lipid ethanol solution with a total lipid concentration of 7.5 mg / mL was prepared using anhydrous ethanol, in which the molar ratio of DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG was 15:25:20:38.5:1.5. (3) After preheating 0.75 mL of the CpG-ODN sodium acetate solution prepared in step (1) and 0.25 mL of the lipid ethanol solution prepared in step (2) in a 25°C water bath, the lipid ethanol solution was poured into the CpG-ODN sodium acetate solution and incubated for 1 minute with magnetic stirring. Then, the solution was diluted fourfold with sodium acetate buffer at pH 5.0 and concentration 25 mM to obtain a Qtolimod primary solution in which CpG-ODN was encapsulated by QTsome. Steps (1) to (3) above can be repeated, and by combining them, a larger amount of primary solution of nanolipid particles (LNPs) in which CpG-ODN was encapsulated can be obtained. (4) Using an ultracentrifuge filter with a fractional molecular weight cutoff of 100 kD, the solution was ultrafiltered at 2000 g to remove ethanol from the LNP primary solution, adjusted to an appropriate nucleic acid concentration with 25 mM Tris buffer, and the pH was adjusted to 7.0 with 0.1 M sodium hydroxide solution to obtain a CpG-ODN Qtolimod suspension, which is an LNP suspension in which different CpG-ODNs are encapsulated. The lipid solution used in the QTsome group, which serves as a control in in vivo and in vitro experiments, is a lipid solution. The lipid solution used in the QTsome group and the lipid solution used in Example 14 were obtained by directly ultrafiltration the lipid ethanol solution described in (2) to remove ethanol and adjust the pH, without going through the step of mixing with CpG-ODN described in (3).

[0104] Example 6: Production of a dual-active ingredient liposome (Tritolimod) containing resiquimod and CpG-ODN encapsulated in a single molecule. The LNP suspension (T0009-1 Qtolimod suspension) containing T0009-1, prepared in Example 2, and resiquimod (Resiquimod, Selleck, catalog number S8133) were uniformly mixed so that the molar ratio of CpG-ODN to resiquimod was 2:1, thereby forming a dual active ingredient liposome (Tritolimod). Resiquimod has a high logP value and can spontaneously disperse inside the LNP.

[0105] Example 7: Production of a dual-active ingredient liposome (Doxotolimod) containing encapsulated doxorubicin (DOX) and CpG-ODN. The LNP suspension containing T0009-1, prepared in Example 2 (T0009-1 Qtolimod suspension), and DOX were uniformly mixed so that the molar ratio of CpG-ODN to DOX (Selleck, catalog number S1208) was 2:1, thereby forming a dual active ingredient liposome (Doxotolimod). DOX can be remotely supported and enter the interior of the LNP by binding to CpG and / or the captured complex (e.g., acetate).

[0106] Example 8: Effect of Qtolimod produced with different CpG-ODNs on the growth of colon cancer tumors in mice The experimental drug was Qtolimod prepared in Examples 2-5. The formulation was pre-dispensed, stored at -20°C, and reconstituted on the day of use.

[0107] 1. Experimental Procedure (1) 50 seven-week-old C57BL / 6 mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., equal numbers of males and females) were reared for one week to allow them to adapt, and then their weight was measured. (2) Resurrect the mouse colon cancer MC38 cell line (Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP60825), subculture it, and when the cell condition is good, inoculate the MC38 cells (inoculation amount is 10 6 The cells were transplanted subcutaneously into the backs of mice, and changes in the mice's body weight and tumor size were recorded. (3) The tumor is approximately 100 mm 3At this point, the mice were divided into groups. Mice with tumor volumes that were too large or too small were removed, maintaining the same number of mice as when they were first enrolled. Using a fully randomized grouping method, they were randomly divided into five groups, which were named T0009-1 Qtolimod group, T0009-2 Qtolimod group, T0009-3 Qtolimod group, T0009-4 Qtolimod group, and the saline group, with seven mice per group. (4) On day 0, each mouse was administered 100 μL of T0009-1 Qtolimod, T0009-2 Qtolimod, T0009-3 Qtolimod, T0009-4 Qtolimod, or physiological saline (0.9% sodium chloride aqueous solution) intratumorally (in some cases, injections could be administered at two sites). The dose was 1 mg / kg, administered once every three days for a total of five injections, with the first injection given on day 0. (5) Observe and record the growth and proliferation of subcutaneous colon tumors on the backs of tumor-bearing mice in the treatment group and the saline control group daily, measure the body weight and tumor volume, and create tumor volume change curves and body weight change curves. (6) On day 30, the mice were killed, the tumor mass was removed, the volume of the tumor mass was measured, the net weight of the tumor mass was measured, and the tumor growth inhibition rate was calculated.

[0108] 2. Experimental Results As can be seen in Figures 2 and 3, a clear drug effect became apparent in each treatment group on day 8 of the experiment, and on day 30, the tumor volume of the mice in the saline control group reached the humane endpoint (2500 mm²). 3 The TGI% reached a certain level, and all groups treated with Qtolimod showed a very significant tumor suppression effect. The calculated TGI% for the T0009-1 to T0009-4 Qtolimod groups were 84.42%, 85.522%, 89.152%, and 85.802%, respectively. In all treatment groups, no mice died from the start of administration until they were killed on day 30.

[0109] Throughout the study, there were no apparent abnormalities in body weight or clinical condition in either the saline control group or the treatment group of mice, indicating that the animals tolerated the investigational drug well under the conditions of this tumor model.

[0110] In short, T0009-1 Qtolimod, T0009-2 Qtolimod, T0009-3 Qtolimod, and T0009-4 Qtolimod demonstrated a clear inhibitory effect on transplanted tumor growth in mice, and the animals showed good tolerance to the drugs. There were no significant differences among the treatment groups in terms of improved survival time, tumor suppression, and clinical symptoms in tumor-bearing animals.

[0111] Example 9: Effect of Qtolimod produced with different CpG-ODNs on the growth of melanoma in mouse skin. The experimental drug was Qtolimod prepared in Examples 2-5. The formulation was pre-dispensed, stored at -20°C, and reconstituted on the day of use.

[0112] 1. Experimental Procedure (1) Fifty 7-week-old C57BL / 6 mice were housed for one week to allow them to adapt, and then their weight was measured.

[0113] (2) The conditions for vaccination were as shown in Table 2. [Table 2]

[0114] (3) After vaccination, measure the tumor volume and body weight once a week, and the average tumor volume is approximately 40-60 mm. 3When the mice reached a certain tumor volume, they were divided into groups. Mice with excessively large or small tumor volumes were removed, maintaining the same number of mice as when they were first enrolled. These mice were then randomly divided into five groups using a fully randomized grouping method, and were labeled T0009-1 Qtolimod group, T0009-2 Qtolimod group, T0009-3 Qtolimod group, T0009-4 Qtolimod group, and the saline group, with 8 mice per group. After grouping, administration was started immediately, or saline (0.9% sodium chloride aqueous solution) was administered. 50 μL was injected intratumorically into each mouse (it) (in some cases, injections could be given at two sites), with a dose of 1 mg / kg. Injections were given once every 3 days for a total of 5 times, with the first injection on day 0.

[0115] (4) Once administration was started, the body weight and tumor volume of the mice were measured three times a week. 3 When the animals reached a certain threshold, they were euthanized and considered dead. The median survival time (days, MST, Medium survival time) for each group was calculated, survival curves were created, and statistical comparisons were performed.

[0116] 2. Experimental Results As can be seen in Figures 4 to 6, on the 7th day of the experiment, clear drug effects were observed in all treatment groups, and among them, the T0009-1 Qtolimod group showed a good tumor-suppressing effect.

[0117] Analysis of the median survival time of mice in each group revealed that the median survival time for animals in the saline control group was 9 days, while the median survival times for animals in the T0009-1 to T0009-4 Qtolimod groups were 16 days, 12 days, 11 days, and 12 days, respectively. Calculations showed that the TGI% for the T0009-1 Qtolimod group was 75%, which was significantly higher than the saline control group (P<0.01 in all cases), and the T0009-1 Qtolimod group had the longest survival time among all the treatment groups.

[0118] Throughout the study, there were no apparent abnormalities in body weight or clinical condition in either the saline control group or the treatment group of mice, indicating that the animals tolerated the investigational drug well under the conditions of this tumor model.

[0119] In summary, T0009-1 Qtolimod, T0009-2 Qtolimod, T0009-3 Qtolimod, and T0009-4 Qtolimod showed a clear inhibitory effect on transplanted tumor growth in mice, and the animals had good tolerance to the drugs. T0009-1 Qtolimod was the best in terms of improving the survival time of tumor-bearing animals, tumor suppression, and clinical symptoms.

[0120] Example 10: Effect of dual-active-component liposomes on the growth of colon cancer tumors in mice The experimental drugs were T0009-1 Qtolimod prepared in Example 2, the dual active ingredient liposome (Tritolimod) prepared in Example 6, and the dual active ingredient liposome (Doxotolimod) prepared in Example 7. The formulations were pre-dispensed, stored at -20°C, and reconstituted on the day of use.

[0121] 1. Experimental Procedure The experimental procedure was the same as in Example 8.

[0122] 2. Experimental Results As can be seen in Figures 7 and 8, a clear drug effect became apparent in each treatment group on day 6 of the experiment, and by day 12, a very significant (P<0.001) tumor-suppressing effect was observed in all three groups: the Qtolimod group (administered with T0009-1 Qtolimod), the Tritolimod group (administered with Tritolimod), and the Doxotolimod group (administered with Doxotolimod). Calculated TGI% for the Qtolimod, Tritolimod, and Doxotolimod groups were 90.97%, 94.09%, and 91.03%, respectively. Throughout the study, there were no apparent abnormalities in body weight or clinical condition in the saline control group and the treatment groups, indicating that the animals tolerated the investigational drug well under the conditions of this tumor model.

[0123] Example 11: Effects of systemic administration of dual-active ingredient liposomes (Tritolimod) on the growth of melanoma in mouse skin. The experimental drug was a dual-active-component liposome (Tritolimod) prepared in Example 6. The formulation was pre-dispensed, stored at -20°C, and reconstituted on the day of use.

[0124] 1. Experimental Procedure This example is similar to Example 9, except that this example uses tail vein injection as the administration method.

[0125] 2. Experimental Results As shown in Figures 9 and 10, systemic administration of tritolimod effectively suppressed tumor growth in tumor-bearing mice. Calculations showed that the TGI in the treatment group reached 80%, a significant difference compared to the saline control group (P<0.001), and the survival time of the mice in the treatment group was effectively extended.

[0126] Example 12: Study on dose-dependent antitumor effect and recurrence prevention effect In this example, intratumoral administration was employed to investigate the effect of T0009-1 Qtolimod on tumor growth. The experimental drug was the lipid nanoparticle T0009-1 Qtolimod prepared in Example 2. The formulation was pre-dispensed, stored at -20°C, and reconstituted on the day of use.

[0127] 1. Experimental Procedure (1) Resuscitate MC38 cells, culture them in vitro, and prepare 5 × 10⁶ cells. 6 We obtained individual cells. (2) Thirty-six seven-week-old female C57BL / 6 mice were reared for one week to allow them to adapt, and then their weight was measured. (3) Resurrect the mouse colon cancer MC38 cell line, subculture it, and when the cell condition is good, inoculate the MC38 cells (inoculation amount is 10 6 We transplanted (100 cells) subcutaneously into the right shoulder of a mouse and recorded changes in the mouse's body weight and tumor size. (4) The tumor is approximately 100 mm 3 At this point, the mice were divided into groups. Mice with tumor volumes that were too large or too small were removed, maintaining the same number of mice as when they were first enrolled. These mice were then randomly divided into six groups using a fully randomized grouping method, and were labeled as follows: QTsome group, T0009-1 Qtolimod (0.5 mg / kg) group, T0009-1 Qtolimod (1 mg / kg) group, T0009-1 Qtolimod (1.5 mg / kg) group, T0009-1 Qtolimod (2 mg / kg) group, and physiological saline control group. Each group consisted of six female mice. The QTsome group was administered the lipid solution described in step (4) of Example 2, while the control group was administered a 0.9% sodium chloride aqueous solution. (5) On day 0, each mouse was administered intratumorally with 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, or 2 mg / kg of T0009-1 Qtolimod, lipid solution, or physiological saline (0.9% sodium chloride aqueous solution) in 50 μL doses (in some cases, injections could be administered at two sites), once every 3 days for a total of 5 injections, with the first injection given on day 0. (6) The growth and proliferation of subcutaneous colon tumors in the right shoulder of tumor-bearing mice in the treatment group and the control group were observed and recorded daily, and the mouse body weight and tumor volume were measured, along with the calculation of the tumor growth suppression rate and the creation of tumor volume change curves (the results are shown in Figures 11 and 12). (7) To determine whether this drug has an effect in suppressing tumor recurrence, on day 36, MC38 cells were subcutaneously transplanted into the left shoulder of mice in the T0009-1 Qtolimod (0.5 mg / kg, i.e., 0.5 mpk) group, the T0009-1 Qtolimod (1 mpk) group, the T0009-1 Qtolimod (1.5 mpk) group, and the T0009-1 Qtolimod (2 mpk) group (inoculation amount: 10 6 (individual cells). Since the animals in the saline control group and the QTsome group died, a new control group was established in this study. The newly established control group consisted of six female C57BL / 6 mice of the same age as the mice in each of the T0009-1 Qtolimod groups described above. The same number of MC38 cells were inoculated into the same site on the left side of the mice in the newly established control group, but the six female mice were not administered the cells. After inoculating all mice with tumors, the growth of the tumors in them was monitored, and tumor growth curves were created (the results are shown in Figures 13A and 13B).

[0128] 2. Experimental Results In in vivo efficacy experiments using female C57BL / 6 mouse homology models inoculated with MC38 mouse colorectal cancer cells, a clear therapeutic effect was observed in each Qtolimod group on day 6 of the experiment. Calculations showed that the TGI% for each Qtolimod administration group was greater than 63%. On day 26, the tumor volume of the mice in the saline control group reached the humane endpoint, while the other Qtolimod administration groups (0.5mpk, 1mpk, 1.5mpk, and 2mpk) all showed very significant tumor suppression effects, with no statistically significant differences. Calculations showed that the TGI% for the Qtolimod administration groups (0.5mpk, 1mpk, 1.5mpk, and 2mpk) were 96.20%, 92.45%, 97.73%, and 98.82%, respectively. Monitoring of tumor changes in each mouse revealed that on day 56, half of the mice in the 0.5mpk group died due to a recurrence of the tumor on their right shoulder, while one-third of the mice showed continuous reduction of the tumor on the right side. In the 1.0mpk group, one-third of the mice died due to a recurrence of the tumor on their right shoulder, while the remaining two-thirds showed continuous reduction of the tumor on the right side. In the 1.5mpk and 2.0mpk groups, no mice died due to tumor exacerbation. In the 1.5mpk group, five mice had complete elimination of the tumor on their right shoulder, and in the 2mpk group, all six mice had complete elimination of the tumor on their right shoulder. The above data clearly demonstrate a dose-dependent relationship between animal survival rate and tumor resolution. In a study simulating tumor recurrence, with the exception of some mice in the 0.5mpk and 1mpk groups that died due to the growth of the tumor on the right side, in almost all treatment groups, the tumor on the left side of the mice grew slowly before gradually disappearing. Mice treated for the tumor on the right side developed tumor-specific immunomemory cells (especially CD8) that could recognize and eliminate the same type of tumor cells in other parts of the body. + This demonstrated the generation of T cells.

[0129] Example 13: Comparison of intratumoral content of Qtolimod and free CpG ODN injected intratumor. The test drugs used in this experiment were T0009-1 Qtolimod and free T0009-1, prepared using the method described in Example 2. In this experiment, intratumoral administration was employed to examine the drug's retention within tumor tissue. Both T0009-1 (CpG ODN) used in T0009-1 Qtolimod and free T0009-1 were labeled with cy5. The preparations were pre-dispensed, stored at -20°C, and reconstituted on the day of use.

[0130] 1. Experimental Procedure (1) Resuscitate MC38 cells, culture them in vitro, and prepare 5 × 10⁶ cells. 6 We obtained individual cells. (2) Two 7-week-old female C57BL / 6 mice were reared for one week to allow them to adapt, and then their weight was measured. (3) Resurrect the mouse colon cancer MC38 cell line, subculture it, and when the cell condition is good, inoculate the MC38 cells (inoculation amount is 10 6 We transplanted (100 cells) subcutaneously into the right shoulder of a mouse and recorded changes in the mouse's body weight and tumor size. (4) The tumor is approximately 150 mm 3 When the mice reached a certain stage, they were divided into a T0009-1 Qtolimod group and a free T0009-1 group, and intratumoral administration (1 mg / kg) was performed. (5) Bioimaging (excitation 630 nm, emission 699 nm) was performed using Tanon (ABL-X) at 30 minutes, 3 hours, day 1, and day 3 after administration, and the imaging results are shown in Figure 14A.

[0131] 2. Experimental Results After labeling with cy5, the retention of free T0009-1 and T0009-1 Qtolimod within tumors was compared. As can be seen in Figures 14A and 14B, the content of free T0009-1 in tumor tissue decreased rapidly after intratumoral administration. It decreased to 50% in 3 hours and only 8% remained after 72 hours. On the other hand, the content of T0009-1 Qtolimod in tumor tissue after intratumoral administration showed almost no decrease after 3 hours, then decreased by 20% after 24 hours, and approximately 50% of the drug still remained in the tumor tissue after 72 hours. These results indicate that CpG ODN encapsulated with QTsome can significantly improve drug retention in tumor tissue and significantly reduce the proportion entering the systemic circulation. This not only reduces systemic toxicity and improves drug safety, but also ensures drug exposure in tumor tissue, achieving a sustained-release effect and significantly improving the efficacy of tumor treatment.

[0132] Example 14: Study on the immune activation mechanism of the test drug The test drug used in this example was T0009-1 Qtolimod from Example 2, and the control group used physiological saline or the lipid solution described in step (4) of Example 2. The procedure for the animal experiment was the same as in Example 8.

[0133] I. Immunohistochemistry Experiment After all administrations were completed, tumors were isolated from the mice and immunohistochemical experiments were performed according to the following procedure. 1. Sample Processing Samples were taken from the received tumor-bearing samples, trimmed, and cut in half along the widest surface of the tissue from the needle insertion point for the experiment. The mouse tissue was then dehydrated using a Leica tissue processor (Leica HistoCore PEGASUS).

[0134] 2. Labeling of tumor-bearing tissue with F4 / 80, CD8 alpha, and CD11b by immunohistochemical staining. (1) In preparation for immunohistochemical staining, the dehydrated tissue was embedded in paraffin, and tissue paraffin sections (4 μm sections, 5 sections per tissue) were prepared. (2) Immunohistochemical staining was performed on paraffin sections of mouse tumor tissue using F4 / 80 (CST, 70076T), CD8 alpha (abcam, ab209775), and CD11b (abcam, ab133357) as primary antibodies. The primary antibody was added dropwise to the processed tissue sample sections. After incubation at room temperature for 1 hour, the samples were placed in 0.1% Triton TBS and washed three times for 5 minutes each while shaking in a shaker. A goat anti-rabbit secondary antibody (Peroxidase AffiniPure Goat Anti-Rabbit IgG(H+L), Jacksonimmuno, 111-035-003, diluted 1:1000 in PBS) was used, incubated at room temperature for 30 minutes, placed in 0.1% Triton TBS, and washed three times for 5 minutes each while shaking in a shaker. (3) Inducing color development of the substrate. Approximately 200 μL of DAB color developer (Fuzhou Maixin Medical Laborator Co., Ltd., DAB-1031, DAB kit (20×)) was added dropwise, and the substrate was allowed to develop color at room temperature for 5 minutes. The reaction was then immediately stopped by immersing it in tap water. (4) Double stain the cell nuclei and mount the dehydrated sections. After double staining the cell nuclei with hematoxylin for 1 minute, the sections were dehydrated with gradient ethanol and xylene, and then manually mounted using neutral resin. (5) All sections were scanned and digitized (using an Olympus VS200), and the tissue necrosis rate was analyzed for any immunohistochemical (IHC) stained scan image using the digital quantitative analysis software QuPath (semi-automatic labeling of necrotic tissue in areas without cell nuclei). QuPath was used to statistically analyze the labeled F4 / 80, CD8 alpha, and CD11b positive signals (brown).

[0135] 3. Data Analysis Using GraphPad prism, the mean ± standard error (Mean ± Sem) was calculated, and a t-test was used for significance testing. A statistically significant difference between the two groups was considered to exist if P < 0.05. The results are shown in Figures 15A to 15C.

[0136] II. The expression levels of cytokines IL-12, IL-10, IFN-γ, TNF-α, IL-6, and IL-1β were measured in mouse blood and tumor tissue samples after administration using ELISA. 1. Sample Processing After administering T0009-1 Qtolimod and completing the administration of physiological saline or lipid solution, blood was collected from the hearts of mice. The blood was allowed to stand at room temperature for 1 hour, and then serum samples were collected by running the blood at 5500 rpm for 20 minutes. Tumors were removed from the mice and trimmed using scissors to a size of approximately 1 mm. 3 After cutting, 2 mL of PBS was added, and the mixture was vortexed at 2000 rpm for 2 minutes. The supernatant was collected as a tumor sample.

[0137] 2. ELISA measurement The ELISA measurement kit was provided by QuanLab.CUSABIO, and the experimental procedure was as follows: (1) Transfer each reagent to room temperature (18-25°C) and allow to equilibrate for at least 30 minutes, preparing the reagents according to the kit instructions. A blank well (S0), two standard wells (parallel), and a sample well were prepared and loaded. 100 μL of the standard or sample (serum sample, tumor sample) from the kit was added to each well, an equal volume of sample diluent was added to the blank well (S0), the plates were gently shaken to mix uniformly, the plates were sealed, and incubated at 37°C for 2 hours. (2) Discard the liquid, shake to dry, and there is no need to wash. (3) Add 100 μL of biotin-labeled antibody working solution to each well, cover with a new plate seal, and incubate at 37°C for 1 hour. (4) Discard the liquid in the wells, shake to dry, and wash the plate three times. Immerse the wells in 200 μL for 2 minutes each time, shake to dry. (5) Add 100 μL of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate seal, and incubate at 37°C for 1 hour. (6) Discard the liquid in the wells, shake to dry, and wash the plate five times. Immerse each well in 200 μL for 2 minutes, shake to dry. (7) Add 90 μL of substrate solution to each well sequentially and allow to develop color at 37°C in the dark for 15 to 30 minutes. (8) 50 μL of stop solution was added sequentially to each well to stop the reaction. (9) Within 5 minutes after the reaction, the optical density (OD value) of each well was sequentially measured at a wavelength of 450 nm using a microplate reader.

[0138] 3. Data Processing A curve was created by subtracting the values ​​in the S0 well from the standard and sample values, and the values ​​for parallel wells were calculated using the average. After creating a standard curve using the relevant software, the measured OD values ​​were substituted into the standard curve equation to calculate the sample concentration. The results are shown in Figures 16A and 16B.

[0139] II. Experimental Results Immunohistochemical results showed that macrophages and CD8 cells at tumor sites were more numerous in the treatment group compared to the saline control group. + The infiltration of T cells was significantly increased, indicating the effects of direct tumor killing, removal of tumor tissue fragments, and activation of adaptive immunity through antigen presentation. Infiltration of CD11b-positive cells (monocytes, granulocytes, activated lymphocytes, NK cells, dendritic cells) was also increased, playing an important role in inducing efficient and specific cellular immune responses to tumor-associated antigens and mobilizing immune cells. Cytokine measurements showed that IL-12, IL-10, IFN-γ, and TNF-α were significantly upregulated in tumor tissue in the Qtolimod-treated group compared to the saline and LNP control groups, while there were no significant changes in IL-1β and IL-6, indicating that their expression levels in the blood were all below the detection limit. IL-12 is expressed by NK cells and CD8 + It promotes T cell proliferation and cytotoxic activity, induces Th1 differentiation and IFN-γ secretion, and promotes antigen presentation. IL-10 is produced by antigen-presenting cells and CD8 +It promotes T cell activation and, by activating macrophages, suppresses the expression of inflammatory cytokines such as TNF-α, IL-6, and IL-1. IFN-γ is mainly produced by T cells, NK cells, and NK T cells, and exerts its killing effect by inducing apoptosis or promoting non-apoptotic cell death, thereby indirectly causing tumor cells to respond to an apoptotic immune response.

[0140] Example 15: Study on the impact of delivery systems on safety To study the effect of the QTsome delivery system on the safety of the aforementioned CpG ODN, this experiment included a solvent control group, a QTsome group, an active ingredient (API) group, and a Qtolimod group. The solvent control group used physiological saline (0.9% sodium chloride aqueous solution), the QTsome group used the lipid solution described in step (4) of Example 2, the API group used free T0009-1, and the Qtolimod group used T0009-1 Qtolimod. The survival and body weight changes of the mice after administration were observed.

[0141] 1. Experimental Procedure (1) Each group consisted of 6 CD1(ICR) mice weighing 20-30g and aged 6-8 weeks (Beijing Vital River Laboratory Animal Technology Co., Ltd., equal number of males and females). (2) On days 1, 8, 15, 22, and 29, physiological saline, lipid solution, and free T0009-1 or T0009-1 Qtolimod were administered subcutaneously (SC) once a week (QW), for a total of 5 doses over a 29-day period. The dose was 20 mg / kg, and the lipid content of the QTsome group was the same as that of the Qtolimod group. The first subcutaneous injection was administered on day 1.

[0142] 2. Experimental Results As shown in Figure 17, during the experiment, the mice other than those in the API group tolerated the drug well and did not show significant weight loss. Mice administered with free T0009-1 experienced weight loss after administration, and on day 12, one male and two females, representing 50% of the total, died. After dissection of the non-dead mice in the API group, organs were weighed and observed. It was found that the kidneys were noticeably lighter in color and slightly lighter in weight, while the liver and spleen were noticeably enlarged. The liver weight of the males was 2.5 times that of the solvent control group, and the spleen weight was 5.4 times that of the solvent control group. The liver weight of the females was 3 times that of the solvent control group, and the spleen weight was 6.8 times that of the solvent control group. At the endpoint (after completion of 5 doses), dissection of mice in the Qtolimod group revealed that male liver weight was 1.7 times greater than that of the solvent control group, and spleen weight was 2.9 times greater. Similarly, female liver weight was 1.7 times greater than that of the solvent control group, and spleen weight was 3.4 times greater. These experimental results demonstrate that the QTsome delivery system can effectively improve the safety of this product.

[0143] Example 16: Efficacy of the investigational drug in a mouse melanoma B16-Luc metastatic tumor model. In this experiment, the investigational drug T0009-1 Qtolimod was produced by encapsulating T0009-1 using lipid nanoparticles, employing the methods described in Examples 2 to 5 above. Subcutaneous tumor and systemic metastatic tumor mouse models were established by transplanting mouse melanoma B16-Luc cells into the subcutaneous tissue and tail vein of each mouse, and the therapeutic effects of the investigational drug on the melanoma B16-Luc allogeneic subcutaneous transplant tumor model and the systemic metastatic tumor model were evaluated.

[0144] 1. Construction of subcutaneous tumor and metastatic tumor models Mouse melanoma B16-Luc cells with a base area of ​​75 cm² 2The cells were dispensed into multiple culture flasks and cultured using the standard method, i.e., in DMEM medium containing 10% fetal bovine serum, in a 37°C, 5% CO2 incubator. When the cells were in the logarithmic growth phase, they were digested with 0.25% trypsin. After the cells were detached, digestion was stopped and the cell suspension was collected. After centrifugation, the supernatant was removed, and the cells were washed twice with PBS to prepare a cell suspension, which was then adjusted to the desired cell concentration for animal transplantation. Using 1:1 sterile PBS buffer and Matrigel, 1 × 10⁶ cells were extracted. 7 The cells were resuspended until the concentration reached 1 / mL, and then subcutaneously transplanted at 0.1 mL / mice into the left axilla of C57BL / 6J mice to construct a subcutaneous tumor mouse model. Simultaneously, 5 × 10⁶ cells were hydrated in sterile PBS buffer. 6 The cells were resuspended until the concentration reached cells / mL, and then injected into C57BL / 6J mice at a dose of 0.2 mL / mice via tail vein injection to construct a mouse model of systemic metastatic tumors.

[0145] 2. Experiments on subcutaneous tumors and systemic metastatic tumors The T0009-1 Qtolimod preparation was pre-dispensed, stored at -20°C, and reconstituted on the day of use. Subcutaneous tumor volume was 68-105 mm². 3 When the tumor reached a certain stage, tumor-bearing mice with well-developed tumors that did not rupture were selected. Mice with tumors that were too large or too small were removed, maintaining the same number of mice as the initial group. These mice were then randomly divided into two groups using a fully randomized grouping method, designated as the T0009-1 Qtolimod 1.5 mg / kg group and the physiological saline control group, with 16 mice per group. The T0009-1 Qtolimod 1.5 mg / kg body weight group was administered to the mice, while the control group was administered a 0.9% sodium chloride aqueous solution. The day of group division was designated as Day 1 (D1). Specifically, 2.5 mL of the drug / kg body weight was administered every three days, on days 1, 4, 7, and 10, for a total of four doses. During the administration period, subcutaneous intratumoral injection was used, and the same amount of saline solution was administered to mice in the saline control group.

[0146] The experimental results showed that on days 8, 9, 10, and 11 after administration, one animal died in each of the saline control groups, while no animals died in the T0009-1 Qtolimod 1.5 mg / kg group. Figure 18 shows the survival status of the animals in each group. In the T0009-1 Qtolimod 1.5 mg / kg group, the health status, circumstances of death, mental state, behavior, fecal characteristics, and other abnormalities of all animals were observed daily throughout the entire period, including before administration, during administration, and before euthanasia, and no obvious abnormalities were found.

[0147] Table 3 and Figure 19 show the changes in animal body weight during the administration period. There was no significant difference in body weight between the groups before the first administration (at the time of group assignment) (P>0.05). On days 3, 5, 9, and the experimental endpoint (day 11) after administration, the T0009-1 Qtolimod 1.5 mg / kg group showed a significant decrease in body weight compared to the saline control group (Day 3: P<0.05, Day 5: P<0.05, Day 9: P<0.001, Experimental endpoint: P<0.001). [Table 3]

[0148] Table 4 and Figure 20 show the volume changes of subcutaneous tumors during the administration period. There was no significant difference in subcutaneous tumor volume between the animal groups before the first administration (at the time of group assignment) (P>0.05). On days 5, 7, and 9 after administration, and at the experimental endpoint, the volume of subcutaneous tumors in the T0009-1 Qtolimod 1.5 mg / kg group was significantly reduced compared to the saline control group (P<0.001). [Table 4]

[0149] Figures 1-22 show the changes in subcutaneous tumor weight during the administration period. Compared to the saline control group, the T0009-1 Qtolimod 1.5 mg / kg group showed a significant reduction in subcutaneous tumor weight (***P<0.001), and the tumor inhibition rate was 90.43%.

[0150] The tumor suppression rate was calculated using the weight of the subcutaneous tumors with the following formula: Tumor suppression rate = (Tumor weight of negative control group - Tumor weight of treatment group) / Tumor weight of negative control group × 100%. The formula for calculating relative tumor volume (RTV) is V t / V0, where V0 is the tumor volume at the time of group division, V t θ was the tumor volume at each measurement. The tumor-suppressing effect of the compound was evaluated as T / C(%). The relative tumor growth rate was T / C(%) = T RTV / C RTV It was ×100% (T RTV This is the RTV of the treatment group, and C RTV (This was the RTV of the negative control group). In principle, the evaluation criteria are that if T / C(%) > 40%, it is invalid, and if T / C(%) ≤ 40%, statistically processed, P < 0.05, it is valid.

[0151] The effects of T0009-1 Qtolimod on tumor metastasis in various animal tissues were as follows: After allogeneic transplantation of mouse B16-Luc cells via the tail vein, visual observation and counting revealed that tumor metastasis mainly occurred in the lung tissue, but also affected the spleen, kidney, spine, femur or tibia, ovaries, abdominal cavity or abdominal fat, medial or lateral abdominal wall, and intestinal tract. The number of metastatic tumor nodules in the lungs was high. After the above subcutaneous intratumoral injection administration, the number of metastatic tumor nodules was examined, and compared to the saline control group, the number of tumor nodules in the lung tissue of the animals was significantly reduced in the T0009-1 Qtolimod 1.5 mg / kg group (**P<0.01), and the total number of metastatic tumor nodules in all animal tissues was significantly reduced in the T0009-1 Qtolimod 1.5 mg / kg group (**P<0.01). The results are shown in Figures 23 to 25.

[0152] Under the conditions of this experiment, when administered subcutaneously via intratumoral injection once every three days for four doses, no animals died in the T0009-1 Qtolimod 1.5 mg / kg group, and the animals showed a decrease in body weight. Analysis revealed that this weight loss was likely due to the reduction of subcutaneous tumors. T0009-1 Qtolimod 1.5 mg / kg significantly reduced the tumor volume and relative tumor volume of subcutaneous tumors in mouse melanoma B16-Luc allogeneically transplanted animals, and significantly reduced the tumor weight of the subcutaneous tumors, demonstrating a good inhibitory effect on subcutaneous tumors in mouse melanoma B16-Luc allogeneically transplanted animals. Intratumoral injection of T0009-1 Qtolimod 1.5 mg / kg significantly reduced the number of metastatic tumor nodules in the lung tissue and the total number of metastatic tumor nodules in all tissues of mouse animals that received tail vein allogeneic transplantation of melanoma B16-Luc, demonstrating a good inhibitory effect on systemic metastatic tumors in mouse melanoma B16-Luc allogeneically transplanted animals. In short, under the conditions of this experiment, when T0009-1 Qtolimod was administered subcutaneously to tumors at a dose of 1.5 mg / kg, once every three days for a total of four doses (days 1, 4, 7, and 10), it showed good inhibitory effects on both subcutaneous tumors and systemic metastatic tumors in mouse melanoma B16-Luc allografts, and also extended the survival time of the animals. Based on the common technical knowledge in this field, the above mouse administration plan can be adapted to administration plans for other animals, such as human administration plans.

[0153] Example 17: Therapeutic efficacy of the investigational drug in a mouse head and neck cancer SCC-7 subcutaneous tumor model. In this experiment, the methods described in Examples 2 to 5 were adopted, and T0009-1 was encapsulated using lipid nanoparticles. Intratumoral administration was employed, and its effect on tumor growth was investigated.

[0154] The T0009-1 Qtolimod preparation was pre-dispensed, stored at -20°C, and reconstituted on the day of use. SCC-7 cells (deposited at Moslet (Hangzhou) Biotechnology Co., Ltd.) were revived, cultured in vitro, and 5 × 10⁶ cells were prepared. 6We obtained several cells. Twenty-four 7-week-old female C3H mice were reared for one week to allow them to adapt, and then their body weight was measured. Mouse head and neck cancer SCC-7 cell lines were revived and subcultured. When the cells were in good condition, SCC-7 cells (inoculation dose: 2e5 cells) were transplanted subcutaneously into the right shoulder of the mice, and changes in the mice's body weight and tumor size were recorded (all tumor-bearing mouse models were constructed by subcutaneous inoculation of the SCC-7 cell line into the back). The tumor was approximately 80 mm. 3 At this point, the mice were divided into groups. Information regarding group division and administration is shown in Table 5 below. [Table 5]

[0155] 50 μL of the drug was administered intratumorally to each mouse in Group 1 and Group 2 (in some cases, injections could be administered at two locations), with a total dose of 1.5 mpk. A total of 5 injections were given, with the first injection on day 0. Group 3 received 100 μL of the positive control drug, PD-1 monoclonal antibody, subcutaneously, with a dose of 10 mpk. The growth and proliferation of subcutaneous colon tumors on the backs of tumor-bearing mice in the experimental and control groups (Groups 1 and 3) were observed and recorded daily. This included measuring mouse body weight and tumor volume, calculating the tumor growth inhibition rate (TGI), and creating tumor growth curves, as shown in Figures 26 and 27. Under the conditions of this experiment, on day 17 after subcutaneous intratumoral injection of mouse head and neck cancer SCC-7, the PD-1 monoclonal antibody in group 3, the positive control group, did not significantly suppress tumor growth, with a TGI% of 21.18%. However, T0009-1 Qtolimod in group 2 significantly suppressed tumor growth, with a TGI% of 57.28%. During the administration period, the animals experienced a slight decrease in body weight, and analysis suggested that this weight loss was likely due to the reduction of subcutaneous tumors.

[0156] Example 18: Pharmacodynamic study of the investigational drug in female Balb / c mouse EMT-6 and EMT-6-HPV18 subcutaneous implantation models. In this experiment, Qtolimod was produced by encapsulating T0009-1 using lipid nanoparticles, employing the methods described in Examples 2-5 above. The efficacy of the test drug was investigated in female Balb / c mouse EMT-6 and EMT-6-HPV18 subcutaneous transplantation models using intratumor administration.

[0157] The T0009-1 Qtolimod formulation was pre-dispensed, stored at -20°C, and reconstituted on the day of use.

[0158] EMT-6, a mouse mammary cancer cell, and EMT-6-HPV18, a mouse mammary cancer HPV-positive cell (deposited at WuXi AppTec(Nantong) Co., Ltd.), were cultured in vitro in a monolayer, with culture conditions based on the manufacturer's technical description or references. The cells were subculturised twice a week using trypsin-EDTA. When the cell saturation reached 80-90% and the required number was reached, the cells were collected, counted, and inoculated. The criteria for tumor inoculation and group division were as follows: EMT-6 cells (5 × 10^5 cells + Matrigel / 200 μL, PBS:Matrigel = 1:1) or EMT-6-HPV18 cells (5 × 10^5 cells + Matrigel / 200 μL, PBS:Matrigel = 1:1) were subcutaneously inoculated into the right posterior dorsal region of each mouse. The average tumor volume is approximately 100 mm². 3 When the patient reached the required level, they were divided into groups and treatment was initiated. Information regarding group division and treatment is summarized in Table 6. [Table 6]

[0159] The volume of the subcutaneous tumor is approximately 100 mm². 3When the tumor reached a certain stage, tumor-bearing mice with well-growing tumors that did not rupture were selected and randomly divided into groups based on the volume of the subcutaneous tumor (see Example 16). The day of group division was designated as Day 0, and administration was started according to the animal's body weight. The positive control drug PD-1 monoclonal antibody was administered twice a week for a total of 6 times, and the other drugs were administered once a week for a total of 4 times. All drugs were administered by subcutaneous intratumoral injection at a dose of 2.5 mL / kg body weight, and the negative control group received an equal amount of saline.

[0160] The experimental results are shown in Figures 28A to 28D. Under the conditions of this experiment, mice with EMT6 mammary tumors were administered subcutaneously via intratumoral injection once a week for a total of four times (the positive control drug PD-1 monoclonal antibody was administered twice a week for a total of six times). During the administration period, there were deaths in all groups: the saline group, the QTsome group, and the PD-1 monoclonal antibody group. Of these, two mice died in the saline group, and one mouse each died in the QTsome group and the PD-1 monoclonal antibody group. On the other hand, no animal deaths occurred in the Qtolimod 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, and 3 mg / kg groups. During the administration period, the animals' body weight decreased, and this weight loss is likely due to the reduction of the subcutaneous tumors. Qtolimod 0.1 mg / kg (mpk) significantly reduced the tumor volume and relative tumor volume of mouse EMT6 mammary cancer allograft subcutaneous tumors, demonstrating a good inhibitory effect on mouse EMT6 mammary cancer allograft subcutaneous tumors. On day 18 after administration, the tumor growth inhibition rates for mouse EMT6 mammary cancer at Qtolimod 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, and 3 mg / kg were 49.9%, 67.9%, 68.4%, and 89.8%, respectively, all of which were higher than the tumor suppressive effect of PD-1 monoclonal antibody. On day 38 after administration, all mice in the saline and QTsome groups died, while 2 mice survived in the positive control PD-1 monoclonal antibody group, 2 mice survived in the Qtolimod 0.1 mpk group, 5 mice survived in the 0.3 mpk group, 5 mice survived in the 1 mpk group, and all mice survived in the 3 mpk group. Therefore, Qtolimod has a significant inhibitory effect on mouse mammary cancer EMT6 allograft subcutaneous tumors and can also extend the survival time of the animals.

[0161] In this experiment, we also studied an EMT6-HPV18 mouse mammary cancer model containing HPV18 antigen fragments. The model was administered subcutaneously via intratumor injection once weekly for a total of four times, while the positive control drug mPD- was administered twice weekly for a total of six times. During the administration period, one mouse died in the QTsome group, but no animal deaths occurred in the saline, mPD-1, or Qtolimod 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, and 3 mg / kg groups. During the administration period, the animals lost body weight, which may be due to the reduction of subcutaneous tumors. Qtolimod 0.1 mg / kg significantly reduced both tumor volume and relative tumor volume in EMT6 allografted subcutaneous tumors of mouse mammary cancer, demonstrating a good inhibitory effect on EMT6 allografted subcutaneous tumors of mouse mammary cancer. On day 21 after administration, the tumor growth inhibition rates for mouse mammary cancer EMT6 in the Qtolimod 0.3 mg / kg, 1 mg / kg, and 3 mg / kg groups were 55.1%, 75.2%, and 85.3%, respectively, all of which were higher than the tumor suppression effect of the PD-1 monoclonal antibody. On day 38 after administration, all mice in the saline, QTsome, and Qtolimod 0.1 mpk groups had died. One mouse survived in the positive control PD-1 monoclonal antibody group, two mice survived in the Qtolimod 0.3 mpk group, four mice survived in the 1 mpk group, and all mice survived in the 3 mpk group.

[0162] A comparison of the efficacy of Qtolimod in two mouse allogeneic breast cancer subcutaneous tumor models, EMT6 and EMT6-HPV18, revealed that both tumor cells expressing HPV antigens and those not expressing them showed significant tumor suppression after treatment with Qtolimod.

[0163] Example 19: Efficacy of the investigational drug in a Balb / c mouse osteosarcoma K7M2 subcutaneous tumor model. In this experiment, Qtolimod was produced by encapsulating T0009-1 using lipid nanoparticles, employing the methods described in Examples 2-5 above. The efficacy of the test drug was investigated in a female Balb / c mouse K7M2 subcutaneous transplantation model using intratumor administration.

[0164] The formulation was pre-dispensed, stored at -20°C, and reconstituted on the day of use.

[0165] Mouse osteosarcoma cells K7M2 (deposited at WuXi AppTec(Nantong) Co., Ltd.) were cultured in a monolayer in vitro, with culture conditions referring to the manufacturer's technical description or references. The cells were subcultured twice weekly using trypsin-EDTA. When the cell saturation reached 80-90% and the required number was reached, the cells were collected, counted, and inoculated. The criteria for tumor inoculation and grouping were as follows: K7M2 cells (1 × 10^6 cells) were subcutaneously inoculated into the right posterior dorsal region of each mouse. The average tumor volume was approximately 110 mm². 3 When the patient reached a certain level, they were divided into groups and treatment was initiated. Information regarding group division and treatment is summarized in Table 7. [Table 7]

[0166] The volume of the subcutaneous tumor was approximately 110 mm². 3 When the tumor reached a certain stage, tumor-bearing mice with well-growing tumors that did not rupture were selected, randomly divided into groups based on the volume of the subcutaneous tumor, and administration was started according to the animal's body weight. The day of group division was recorded as day 0. The drug was administered once a week for a total of three times, and the positive control drug PD-1 monoclonal antibody was administered twice a week for a total of six times. For all administrations, a subcutaneous intratumoral injection of 2.5 mL / kg body weight was used, and the same volume of physiological saline was administered to the saline-negative control group.

[0167] The results are shown in Figures 29A to 29C. Under the conditions of this experiment, mice with osteosarcoma K7M2 were administered subcutaneously via intratumoral injection once a week for a total of three times, while the positive control drug PD-1 monoclonal antibody was administered twice a week for a total of six times. During the administration period, no animal deaths occurred in any of the groups receiving Qtolimod (0.3 mg / kg, 1 mg / kg, or 3 mg / kg). During the administration period, the animals' body weight decreased slightly, which may be due to the reduction in subcutaneous tumor size. Qtolimod 0.1 mg / kg significantly reduced the tumor volume and relative tumor volume of allografted subcutaneous tumors of mouse osteosarcoma K7M2, demonstrating a good inhibitory effect on allografted subcutaneous tumors of mouse osteosarcoma K7M2. On day 19 after administration, the tumor growth inhibition rates for mouse osteosarcoma K7M2 in the Qtolimod 0.3 mg / kg, 1 mg / kg, and 3 mg / kg groups were 55.1%, 78.6%, and 92.2%, respectively, all of which were higher than the tumor suppression effect of the PD-1 monoclonal antibody (TGI% = 28.7%). On day 35 after group division, all mice in the saline group, QTsome group, positive control drug mPD-1 group, and Qtolimod 0.3 mpk group had died, two mice survived in the 1 mpk group, and all mice survived in the 3 mpk group, with an average tumor size of 539 mm. 3 Therefore, Qtolimod has a significant subcutaneous inhibitory effect on mouse osteosarcoma K7M2 allograft and can also extend the survival time of the animals.

[0168] Example 20: Study of the mechanism of action of the drug at different time points in the treatment of a C57BL / 6J mouse intestinal cancer MC38 subcutaneous tumor model using the test drug. In this experiment, Qtolimod was produced by encapsulating T0009-1 using lipid nanoparticles, employing the methods described in Examples 2-5 above. Intratumor administration was used, and the distribution at the administration site and in splenic immune cells was investigated at different time points after administration.

[0169] The formulation was pre-dispensed, stored at -20°C, and reconstituted on the day of use. MC38 cells were revived, cultured in vitro, and 5 × 10⁶ cells were collected. 6We obtained individual cells. 216 seven-week-old female C57BL / 6 mice were reared for one week to allow them to adapt, and then weighed. Mouse colon cancer MC38 cell line was revived and subcultured, and when the cell condition was good, MC38 cells (inoculation amount: 1 × 10⁶) were used. 6 (100 mm) were transplanted subcutaneously into the right shoulder of mice, and changes in the mice's body weight and tumor size were recorded (all tumor-bearing mouse models were constructed by subcutaneous inoculation of MC38 cell line into the back). The tumor volume was approximately 100 mm. 3 When the patient reached the required level, they were divided into groups and treatment was initiated. Information regarding group division and treatment is summarized in Table 8. [Table 8]

[0170] Each mouse was administered 50 μL intratumorally (in some cases, injections could be administered at two locations), with a total dose of 1.5 mpk. This was done once every three days for a total of five injections, with the first injection on day 0. After each administration, three mice with large, moderate, or small tumor sizes were selected from the same group and euthanized within 6, 24, and 48 hours, respectively. After the mice died, tumor tissue and spleen samples were collected and performed RT-qPCR.

[0171] 1. RNA extraction. 100 mg of liver tissue was mixed with beads and 500 μL of Trizol lysis buffer, then placed in a bead mill and ground at 65 Hz for 5 minutes. 100 μL of the ground solution was removed, 900 μL of Trizol lysis buffer was added, and it was dissolved for 10 minutes. 200 μL of chloroform was added to each tube, and after thorough mixing, it was left on ice for 10 minutes to completely dissociate the nucleoprotein complex. Centrifugation was performed at 4°C and 13000 rpm for 15 minutes. Meanwhile, a new EP tube was prepared, 500 μL of isopropanol was added, and it was pre-cooled on ice. After centrifugation, the upper aqueous phase (approximately 500 μL) was transferred to this new EP tube. It was left on ice and precipitated with an equal volume of isopropanol for 10 minutes. Centrifugation was performed at 13000 rpm for 10 minutes. The supernatant was removed, the RNA precipitate was washed once with 1 mL of 75% ethanol, and the mixture was centrifuged at 12,000 rpm for 5 minutes. The supernatant was discarded, and the RNA precipitate was air-dried or vacuum-dried for 5–10 minutes. The RNA was dissolved in 30–50 μL of deionized water treated with DEPC, and additional water may be needed depending on the volume of RNA precipitate. The concentration and purity of the sample were then determined using a spectrophotometer.

[0172] 2. Reverse transcription is performed. RNA was reverse transcribed into cDNA using the following materials and steps. Genomic DNA is removed. That is, the following mixture was prepared in an RNase-free centrifuge tube (all reagents are provided from kit R312-01 from Nanjing Vazyme Biotech Co., Ltd.). RNase-free ddH2O was added to 2 μL of 5× gDNA wiper mix to make 10 μL, and the total RNA was 1 μg. Using a pipette, the mixture was gently pipetted to ensure uniformity, and centrifugation was performed at 42°C for 2 minutes. The first-strand cDNA synthesis reaction mixture is prepared. Specifically, a mixture was prepared in an RNase-free centrifuge tube consisting of 10 μL of the mixture prepared in the previous step, 2 μL of 10×RT Mix, 2 μL of HiScript III Enzyme Mix, 1 μL of Oligo(dT)20 VN, 1 μL of Random hexamers, and 4 μL of RNase-free ddH2O (all reagents were provided from kit R312-01 by Nanjing Vazyme Biotech Co., Ltd.). The mixture was gently pipetted using a pipette to ensure uniform mixing. First-strand cDNA was obtained by centrifugation at 37°C for 15 minutes and then at 85°C for 5 seconds. The reagents 2×qPCR Mix (abm, G891) and PCR primers (Hangzhou Leqi Biomedical Technology Co., Ltd., MQP026401, MQP103938, MQP076780, MQP101232, MQP093050, MQP091959, MQP031788, MQP093658, MQP072830, MQP025979, MQP091399, MQP029630, MQP028834, MQP025254, MQP077323, MQP076358, MQP092655, MQP027158) were thawed and gently mixed uniformly to prepare the qPCR reaction system on ice as shown in Table 9 below. [Table 9]

[0173] The qPCR reaction system was gently and uniformly mixed and centrifuged briefly. As shown in Table 10 below, set a 3-step qPCR program or use a Roche L96 qPCR instrument to set the program. 2 -ΔΔCt Process the data by the method.

Table 10

[0174] Mice were administered intratumorally at 1.5 mpk, Q3D×5. Samples were collected within 6 hours, 24 hours, and 48 hours after each administration until the 4th administration. After the 5th administration, samples were collected within 6 hours, 24 hours, 48 hours, and 96 hours after administration. After euthanizing the mice, tumor tissues and spleen tissues were collected and RT-qPCR was performed to measure various immune cell markers, and the activation status of mouse immune cells after administration was analyzed. The results are shown in Figures 30 and 31. Monocyte-derived macrophages in tumor tissues were significantly upregulated (about 60-fold) after administration, and the upregulation became more obvious as the number of administrations increased. Macrophages are widely involved in immune responses, immune effects, and immune regulation and play an important role in tumor treatment. Activated macrophages can effectively exert a tumor-killing effect, phagocytize tumor cells, and directly or indirectly exert a tumor-killing effect by secreting harmful cytokines, and can play a role in directly killing or suppressing the growth of tumor cells by generating specific enzymes and reactive oxygen species. Also, as a result of examining the spleen, monocyte-derived macrophages were not significantly upregulated after administration, indicating that the activation of monocyte-derived macrophages is concentrated at the administration site. Qtolimod can activate plasmacytoid dendritic cells (pDC), and the activated pDC can exert a tumor-killing effect by generating a large amount of IFN-α and other cytokines and mobilizing a large number of T cells to enter the tumor microenvironment. From the experimental results, intratumoral administration induces significant upregulation of plasmacytoid dendritic cells (pDC), as well as macrophages, CD3 + T cells, CD8 +It has been found that immune cells such as T cells and NK cells are significantly activated, granzyme B (GZMB) is released, and the killing of tumor cells is promoted. In addition, macrophages phagocytize tumor cell fragments and promote the presentation of tumor antigens and the immune activation of anti-tumor cells. In contrast, various immune cells in the spleen are always in an unactivated or mildly activated state after administration, suggesting that Qtolimod concentrates locally in tumors to induce anti-tumor immune responses and has a low risk of causing systemic side effects.

[0175] Example 21: Study on Immune Activation of Mouse and Human PBMC Cells after in Vitro Stimulation with Test Drugs In this experiment, the methods of Examples 2 to 5 were adopted, and Qtolimod was produced by encapsulating T0009-1 using lipid nanoparticles. PBMC cells were stimulated in vitro with the obtained Qtolimod, and after 24 hours, cell samples were collected and proteome analysis was performed. The Qtolimod formulation was pre-dispensed and stored at -20 °C, and reconstituted on the day of use.

[0176] Human PBMC cells were purchased from Zhejiang Free Trade Zone Maishun Biotechnology Co., Ltd. The steps for separating mouse PBMC cells were as follows. 4 mL of separation buffer was added to a 10 mL sterile silicon centrifuge tube, and then 0.5 - 2.0 mL of blood sample was slowly added. The blood sample was carefully added above the liquid surface of the separation buffer. Centrifuged at 450 g for 30 minutes, and then the ring-shaped milky white mononuclear cell layer was carefully aspirated from the centrifuge tube and transferred to a new centrifuge tube. 10 mL of washed and uniformly mixed cells were added to the centrifuge tube containing mononuclear cells. Centrifuged at 250 g for 10 minutes, and the supernatant was discarded. The cells obtained after resuspension were washed with 5 mL of PBS. Centrifuged at 250 g for 10 minutes, and the supernatant was discarded. The washing was repeated twice, the supernatant was discarded, and the obtained cells were resuspended in 0.5 mL of RPMI-1640 medium. Mouse and human PBMC cells were seeded at 1×10 per well 6Cells were seeded in 6-well plates, 5 μg of Qtolimod was added to stimulate the cells, and after 24 hours, the cells were collected and subjected to 4D-Smart DIA proteome analysis by Hangzhou Jingjie Biotechnology Co., Ltd. The results are shown in Figure 32. The test drug stimulated human and mouse PBMC cells in vitro, and after 24 hours, the cells were collected and subjected to proteome analysis. The results showed that, compared to the saline (Vehicle) group, Qtolimod could activate immune cells, including B cell pathway genes, T cell pathway genes, bone marrow-derived lymphocyte pathway genes, and lymphoid-derived lymphocyte pathway genes, to some extent, and there was no significant difference in the degree of activation of immune cells between humans and mice.

[0177] Representative genes of the B cell pathway (mouse: Bcap29, Cd72, Blnk, Bcl11a, Bcl7b, Bcap31, Cd22) (human: BCL3, CD72, BCAP31, BCAP29, CD22, CD79B, BCL9L, BANK1, BCL10, PBXIP1), representative genes of the T cell pathway (mouse: Tgtp2, Cd2, Itfg1, Trac, Nfatc1, Art2b, Cd247) (human: LAT2, CD8B2, SART3, CD247, LAT, CD7, VSIR, CD3E, NFATC2, NFATC3, CD5, CD3D, CD4, CD6, CD3G, CD2), myeloid-derived lymphocytes Representative genes for the cell pathway (mouse: Mndal, Mlf2, Myd88) (human: MYADM, MNDA, CD33, MLF2, MYD88, MYDGF), and lymphoid pathway genes (mouse: Ly6d, Lsp1, Ly9, Cd19) (human: LCP2, CD19, BTLA, LSP1, LY75).

[0178] Example 22: Pharmacodynamic study of the investigational drug using a bladder carcinoma in situ (MB49) model. In this experiment, Qtolimod was produced by encapsulating T0009-1 using lipid nanoparticles, employing the methods described in Examples 2-5 above. A mouse model of bladder carcinoma in situ was established by transplanting mouse bladder cancer MB49 cells into the bladder mucosa. The therapeutic effect of Qtolimod in the bladder cancer MB49 orthotopic allogeneic transplant tumor model was evaluated by intravesical administration.

[0179] Qtolimod preparations were pre-dispensed, stored at -20°C, and reconstituted on the day of use. Mouse MB49-Luc cells were cultured in vitro under high-glucose DMEM medium at 37°C and 5% CO2. Subculturing was performed once or twice a week with normal medium changes and digestion. When the required cell count was reached, cells in the logarithmic growth phase were collected, counted, and inoculated. After digesting logarithmic growth phase mouse bladder cancer MB49-Luc cells with 0.5% trypsin, the cells were divided into 4 × 10⁶ cells. 7 A mouse model of orthotopic bladder tumor was constructed by resuspending the D-luciferin potassium substrate in sterile PBS buffer to a concentration of 1 / mL and injecting 50 μL / mouse into the bladder mucosa of C57BL / 6J mice. On day 5 after modeling, all mice were injected with D-luciferin potassium, followed by in vivo imaging. Well-growing tumor-bearing mice were selected and randomly divided into groups based on fluorescence intensity, ensuring that the difference in tumor fluorescence intensity between groups did not exceed 10-fold (see Table 9 for details). Based on palpable conditions, animals with abnormally large orthotopic tumors were removed. Administration began the following day (day 6 after modeling), with each administration day designated as Day 1 (D1). Administration started at 50 μL / mouse and continued every 7 days for a total of 3 doses (days 1, 8, and 15), all administered by intravesical instillation. The saline control group received an equal volume of saline.

[0180] For tumor monitoring, all animals were monitored before grouping, and then again after grouping. Tumor status was monitored at the time of grouping (i.e., before the first dose), every 7 days thereafter, and before euthanasia (days 1, 7, 14, and 21). The fluorescence intensity of bladder intraepithelial neoplasia was monitored using a live imaging system, and differences in orthotopic tumor growth and changes between groups were compared. In addition, the size grade of orthotopic tumors was determined by palpation twice a week after administration and before euthanasia (days 1, 5, 8, 12, 15, 19, 22, and 35). Tumors were divided into four grades based on their size. Grade 0: Almost no palpable lump. Grade 1: Very small lump palpable (estimated tumor diameter less than 2 mm). Grade 2: Moderately sized lump palpable (estimated tumor diameter 2-5 mm). Grade 3 is characterized by a large, palpable lump (estimated tumor diameter of 5-10 mm). Grade 4 is characterized by a very large, palpable lump (estimated tumor diameter exceeding 10 mm). The difference in orthotopic tumor size between the groups was compared based on the grade of the palpable tumor mass size.

[0181] The results are shown in Figure 33. Under the conditions of this experiment, intravesical instillation was performed once a week for a total of three times. Because the orthotopic transplanted tumors grew rapidly, the fluorescent substrate could not penetrate the tumor in the later stages, and palpation was used for observation. When the tumors were dissected and observed on day 35 of the administration period, the situation was consistent with palpation. In the saline control group, the diameter of the animal tumors reached 1.7 cm, they were oval-shaped, and the bladder distended due to tumor growth, necrosis and congestion occurred inside the tumors, and metastatic tumors developed in the kidneys and large intestines, respectively. In the Qtolimod 4mpk administration group, the diameters of the tumors were 0.5 cm and 0.7 cm, respectively, they were nearly circular in shape, and no metastasis was observed. This indicates that the test drug significantly suppressed tumor growth and mitigated the effect of bladder tumor growth on the behavior of mice.

[0182] The above-described embodiments are merely some of the embodiments of the present disclosure. Those skilled in the art can make several modifications and improvements without departing from the concept of the present disclosure, all of which fall within the scope of the present disclosure.

Claims

1. (a) Any one of the nucleotides represented by SEQ ID NOs: 1 to 4, whether modified or unmodified. (b) A functional variant of any one of the nucleotides represented by SEQ ID NOs: 1-4, whether modified or unmodified, and (c) A pharmaceutically acceptable salt of the nucleotide described in (a) or (b) It includes any one of the following: The nucleotides are, in whole or in part, ribonucleotides, or in whole or in part, deoxyribonucleotides. A Toll-like receptor agonist characterized by the following features.

2. The Toll-like receptor agonist according to claim 1, characterized in that the modifications include one or more of the following: replacement of all or some P=O bonds between two adjacent nucleotides in the sequence with P=S bonds; replacement of all or some deoxyribonucleosides in the sequence with ribonucleosides and replacement of all or some ribonucleosides in the sequence with deoxyribonucleosides; replacement of the 2'-OH group of a ribonucleoside or deoxyribonucleoside with a methoxy group, a methoxyethyl group or a halogen; and formation of a locked nucleic acid or (S)-cEt-BNA by crosslinking the second and fourth carbon atoms of a nucleotide.

3. An active ingredient encapsulated by a lipid component, characterized in that it is an active ingredient containing the Toll-like receptor agonist described in claim 1 or 2.

4. The active ingredient encapsulated by the lipid component according to claim 3, wherein the mass ratio of the lipid component to the Toll-like receptor agonist according to claim 1 or 2 is (8-12):

1.

5. The active ingredient encapsulated by the lipid component according to claim 3, characterized in that the lipid component comprises one or more of the following: cationic polymer, nanolipid particles (LNP), cationic liposome, QTsome, lipopolyplex, microparticle, microsphere, and nanoemulsion.

6. The active ingredient encapsulated by the lipid component according to claim 3, wherein the lipid component comprises one or more of cationic lipids, ionizable lipids, neutral lipids, and PEGylated lipids.

7. The lipid component comprises DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG, and the molar ratio of DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG is (1-30):(5-50):(5-40):(15-60):(0-10), and preferably, the lipid component comprises DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG, and the molar ratio of DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG is 15:25:20:38.5:1.5, an active ingredient encapsulated by the lipid component according to feature 6.

8. The active ingredient further comprises another therapeutic agent, and preferably the molar ratio of the other therapeutic agent to the Toll-like receptor agonist according to claim 1 or 2 is (0.1 to 5):1, preferably 1:2, characterized in that the active ingredient is encapsulated with a lipid component according to claim 3 or 7.

9. The other therapeutic agent is selected from one or more antibodies, chemotherapeutic agents and small molecule drugs, preferably at least one selected from pattern recognition receptor agonists other than the Toll-like receptor agonists described in claim 1 or 2, antibodies, anthracycline compounds, camptothecin compounds, immunogenic cell death inducers, tyrosine kinase inhibitors and paclitaxel compounds, and more preferably the other therapeutic agent is one or more of reximod and doxorubicin, characterized in that the active ingredient is encapsulated with a lipid component as described in claim 8.

10. (a) a step of preparing the Toll-like receptor agonist according to claim 1 or 2 as an acidic solution, (b) a step of preparing each component of the lipid as a lipid ethanol solution, Step (c) involves mixing and reacting the solutions prepared in steps (a) and (b) above to obtain a primary solution of nanolipid particles, Selectively repeat the above steps (a) to (c) to obtain a larger number of nanolipid particle primary solutions (d), Step (e) removes ethanol from the above nanolipid particle primary solution, adjusts the concentration and pH of the active ingredient, and then obtains a nanolipid particle suspension. A method for producing an active ingredient encapsulated by a lipid component according to claim 3, characterized by comprising the step (f) selectively mixing another therapeutic agent and the above-mentioned nanolipid particle suspension to obtain a multi-active ingredient liposome.

11. A composition comprising an active ingredient encapsulated with a lipid component according to any one of claims 3 to 7, and another therapeutic agent, wherein the other therapeutic agent is preferably one or more of reximod and doxorubicin.

12. A delivery system comprising an active ingredient and a lipid component, wherein the active ingredient comprises a Toll-like receptor agonist as described in claim 1 or 2.

13. The delivery system according to claim 12, characterized in that the mass ratio of the lipid component to the Toll-like receptor agonist according to claim 1 or 2 is (8-12):1, preferably 10:

1.

14. The delivery system according to claim 12, characterized in that the lipid component comprises one or more of cationic lipids, ionizable lipids, neutral lipids, and PEGylated lipids, preferably comprising DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG, with a molar ratio of DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG being (1-30):(5-50):(5-40):(15-60):(0-10), and more preferably comprising DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG, with a molar ratio of DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG being 15:25:20:38.5:1.

5.

15. The delivery system according to any one of claims 12 to 14, wherein the active ingredient further comprises other therapeutic agents, preferably one or more of reximod and doxorubicin.

16. (a) a step of preparing a Toll-like receptor agonist buffer using a buffer, (b) a step in which the lipid component is prepared as a lipid ethanol solution with anhydrous ethanol, Step (c) involves mixing the lipid ethanol solution and the Toll-like receptor agonist buffer so that the mass ratio of the lipid component to the Toll-like receptor agonist is (8-12):

1. A method for producing a delivery system according to claim 12, characterized by comprising the step (d) of removing ethanol, diluting, and adjusting the pH to 6.8 to 7.

2.

17. A pharmaceutical composition comprising a Toll-like receptor agonist according to claim 1 or 2, an active ingredient encapsulated with a lipid component according to any one of claims 3 to 9, the composition according to claim 11, or a delivery system according to any one of claims 12 to 15, and a pharmaceutically acceptable carrier or excipient.

18. A vaccine adjuvant or vaccine comprising a Toll-like receptor agonist according to claim 1 or 2, an active ingredient encapsulated with a lipid component according to any one of claims 3 to 9, the composition according to claim 11, a delivery system according to any one of claims 12 to 15, or the pharmaceutical composition according to claim 17.

19. (a) Use in the manufacture of drugs to prevent the development of tumors (b) Use in the manufacture of drugs for the treatment of tumors (c) Use in the manufacture of drugs to inhibit tumor growth (d) Use in the manufacture of drugs to prevent metastasis of tumor cells (e) Use in the manufacture of drugs to prevent tumor recurrence (f) Use in vaccine manufacturing (g) Use in the manufacture of drugs for the treatment or prevention of inflammatory diseases or infections (h) Use in the manufacture of immunoactivating drugs (i) Use in the manufacture of drugs for upregulating cytokine expression (j) Use to upregulate cytokine expression for purposes other than therapy Use of a Toll-like receptor agonist according to claim 1 or 2 in any one of (a) to (j) above, an active ingredient encapsulated with a lipid component according to any one of claims 3 to 9, the composition according to claim 11, the delivery system according to any one of claims 12 to 15, or the pharmaceutical composition according to claim 17.

20. The aforementioned immune activation includes activation of the B cell pathway, T cell pathway, bone marrow-derived lymphocyte pathway, lymphoid-derived lymphocyte pathway, macrophages, plasmacytoid dendritic cells (pDCs), B cells, T cells, and CD3 cells. ＋ T cells, CD8 ＋ The use according to claim 19, characterized by comprising activation of T cells, NK cells, and granulocytes.

21. The use according to claim 19, characterized in that the tumor is selected from solid tumors or tumors to which the drug can be injected, and is, for example, at least one of the following: head and neck cancer, esophageal cancer, oral cancer, nasopharyngeal cancer, thyroid cancer, lung cancer, stomach cancer, liver cancer, pancreatic cancer, spleen cancer, non-small cell lung cancer, colorectal cancer, colon cancer, intestinal cancer, kidney cancer, brain tumor, glioma, bladder cancer, prostate cancer, breast cancer, ovarian cancer, fallopian tube cancer, cervical cancer, uterine cancer, bone cancer, osteosarcoma, osteochondroma, liposarcoma, neuroblastoma, synovial sarcoma, astrocytoma, glioblastoma pleomorphic, anaplastic astrocytoma, peritoneal cancer, soft tissue sarcoma, sarcoma, rhabdomyosarcoma, end-stage myxoid disease, melanoma, and skin cancer.

22. The use according to claim 19, characterized in that the drug is administered by one or more routes of administration from among intratumor injection, intravenous injection, subcutaneous injection, intramuscular injection, arterial injection, intraperitoneal injection, central nervous system injection, intravesical instillation, interventional therapy, oral administration, transdermal administration, transpulmonary administration, intraocular administration, and local administration.

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