Toll-like receptor agonists and use thereof

Abstract

Toll-like receptor agonists. The Toll-like receptor agonists are C-type CpG-ODNs, and can effectively activate TLR9, and the nucleotide sequences thereof comprise any one of modified or unmodified nucleotide sequences shown as SEQ ID NO: 1-4. The Toll-like receptor agonists have a remarkable effect of inhibiting tumor growth. The provided lipid-encapsulated Toll-like receptor agonists alone or in combination with other therapeutic agents can inhibit tumor growth, prevent and treat tumor recurrence and improve immune activation efficiency, and have improved properties than free Toll-like receptor agonists.

Description

Toll-like receptor agonists and their applications

[0001] Cross-references to related patents

[0002] This application claims priority to application CN202311008396.X filed on August 10, 2023, application CN202410324833.7 filed on March 20, 2024, and application CN202410567565.1 filed on May 9, 2024. The prior applications are deemed part of the disclosure of this application and are incorporated in their entirety into this application. Technical Field

[0003] The present disclosure relates to the field of biomedicine, and in particular to Toll-like receptor agonists. Background Art

[0004] Toll-like receptors (TLRs) are highly conserved pattern recognition receptors found in a variety of cell types and play a key role in the surveillance of microbial pathogens by innate immune cells. Ten TLRs have been identified in humans and are expressed on T cells, B cells, APCs, and a variety of non-immune cells, including epithelial and endothelial cells. TLRs are located in two different regions of the cell: the cell membrane and the intracellular body. TLR1, 2, 5, and 6 are present in the plasma membrane, TLR3, 7, 8, and 9 are expressed in the intracellular body, and TLR4 is present in both regions. TLRs are expressed on immune cells and tumor cells in the tumor microenvironment, and their activation can inhibit Treg cell function and enhance CD8 + T cell survival, proliferation, and cytokine release.

[0005] To date, numerous studies have investigated the use of TLR agonists in the treatment of tumors. Because TLR9 activation can activate dendritic cells, T cells, and B cells, and promote T cell migration into the tumor microenvironment by inducing interferon-α secretion, TLR9 agonists have shown great potential in the field of tumor treatment. TLR9 agonists primarily consist of bacterial unmethylated CpG DNA, and synthetic CpG oligonucleotides (CpG-ODN) have also been shown to effectively activate TLR9. CpG-ODNs are generally classified into three categories: A, B, and C. Category A CpG-ODNs have a core consisting of a palindromic sequence containing CpG dinucleotides, flanked by poly G tails at both ends, and a partially thiolated phosphodiester backbone. This palindromic sequence and poly G form a higher-order structure that can activate plasmacytoid dendritic cells to induce the production of large amounts of type I interferon, but exhibits weak activity against B cells. Category B CpG-ODNs are fully thiolated, linear CpG-ODNs that exhibit strong immunostimulatory activity against B cells but fail to activate plasmacytoid dendritic cells. Category C CpG-ODNs are fully thiolated, forming dimers through the palindromic sequence. They possess the activity of both type A and type B CpG-ODNs, activating both plasmacytoid dendritic cells and B cells. CpG-ODNs are used as adjuvants in vaccines to enhance immune responses, and some products, such as the hepatitis B vaccine HEPLISAV-B, have been approved for marketing. In addition to vaccines, TLR9 agonists can also be used to treat inflammatory-related diseases and infectious diseases, and are currently in the clinical stage.

[0006] Although TLR9 agonists have great potential in tumor treatment, there are the following difficulties in developing them into anti-tumor drugs: 1) Systemic administration of TLR9 agonists can cause systemic inflammation, resulting in significant side effects, and only a small amount of the drug enters the tumor microenvironment, resulting in poor efficacy; 2) Some researchers use intratumoral injection to administer the drug, which reduces the dosage to a certain extent, but the drug diffuses rapidly, and only a small amount of the drug can be retained in the tumor; 3) TLR9 is an intracellular receptor, and only a small amount of naked CpG-ODN can enter the cell and exert its effect.

[0007] Currently, TLR9 agonists are mainly optimized from two aspects: preventing the rapid degradation of oligonucleotides and enhancing endocytosis. Mologen Ag's product Lefitolimod extends the in vivo half-life of oligonucleotides by transforming linear oligonucleotides into dumbbell-shaped oligonucleotides. However, the results of phase 2 and 3 clinical trials of monotherapy for advanced colorectal cancer after first-line treatment and small cell lung cancer after first-line treatment were poor, and the efficacy was not obvious, so there are currently no ongoing tumor clinical trials. Checkmate's intratumoral injection product CMP-001 is a CpG-A type TLR9 agonist that prevents oligonucleotides from being degraded by nucleases by encapsulating them with virus-like particles, greatly extending their half-life. However, the use of virus-like particles to load and deliver oligonucleotides has a cumbersome acquisition process and high production costs.

[0008] Summary of the Invention

[0009] The present disclosure provides a Toll-like receptor agonist with a tumor inhibitory effect, and further provides a lipid-encapsulated Toll-like receptor agonist or a combination of a Toll-like receptor agonist and other therapeutic agents with multiple active ingredients. The Toll-like receptor agonist or a combination of a Toll-like receptor agonist and other therapeutic agents encapsulated by a lipid component provided by the present disclosure has a significant broad-spectrum tumor inhibitory effect, can prevent tumor recurrence, and prevent tumor metastasis. The Toll-like receptor agonist or a combination of a Toll-like receptor agonist and other therapeutic agents encapsulated by a lipid component provided by the present disclosure has a prolonged intratumoral retention compared to free Toll-like receptor agonists, has an immune activation effect, can upregulate cytokine expression, has a broad-spectrum tumor inhibitory effect after systemic administration, prevents tumor recurrence and prevents tumor metastasis, and effectively prolongs the survival of test animals, and has good safety and tolerability. The Toll-like receptor agonist or a combination of a Toll-like receptor agonist and other therapeutic agents encapsulated by a lipid component provided by the present disclosure has fewer side effects than free Toll-like receptor agonists, and is expected to have positive efficacy in combating human tumors.

[0010] In the first aspect of the present disclosure, a Toll-like receptor agonist is provided, characterized in that it comprises any one of the following (a)-(c): (a) any one of the modified or unmodified nucleotides as shown in SEQ ID NOs: 1-4; (b) a functional variant of any one of the modified or unmodified nucleotides as shown in SEQ ID NOs: 1-4; (c) a pharmaceutically acceptable salt of (a) or (b) the nucleotide; and the nucleotides are all or partly ribonucleotides or all or partly deoxyribonucleotides.

[0011] In a second aspect of the present disclosure, an active ingredient encapsulated by a lipid component is provided, wherein the active ingredient comprises the Toll-like receptor agonist provided in the first aspect of the present disclosure. In some embodiments, the lipid component comprises DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG.

[0012] In a third aspect of the present disclosure, a method for preparing an active ingredient encapsulated by a lipid component is provided, characterized in that it comprises the following steps (a) to (f):

[0013] (a) preparing the Toll-like receptor agonist provided by the first aspect of the present disclosure into an acidic solution;

[0014] (b) preparing the components of the lipid composition into a lipid ethanol solution;

[0015] (c) mixing the solutions prepared in steps (a) and (b) to form a preliminary solution of nanolipid particles;

[0016] (d) optionally repeating the above steps (a)-(c) to obtain more initial solution of nanolipid particles;

[0017] (e) removing ethanol from the initial nanolipid particle solution and adjusting the active ingredient concentration and pH to obtain a nanolipid particle suspension;

[0018] (f) selectively mixing other therapeutic agents with the nanolipid particle suspension to prepare multi-active ingredient liposomes.

[0019] In a fourth aspect of the present disclosure, a composition is provided, characterized in that it comprises the active ingredient encapsulated by the lipid component provided in the second aspect of the present disclosure and other therapeutic agents.

[0020] In a fifth aspect of the present disclosure, a delivery system is provided, characterized in that it comprises an active ingredient and a lipid component, wherein the active ingredient comprises the Toll-like receptor agonist provided by the first aspect of the present disclosure.

[0021] In the sixth aspect of the present disclosure, a pharmaceutical composition is provided, characterized in that it comprises the Toll-like receptor agonist provided by the first aspect of the present disclosure, the active ingredient encapsulated by the lipid component provided by the second aspect, the composition provided by the fourth aspect or the delivery system provided by the fifth aspect, and a pharmaceutically acceptable carrier or excipient.

[0022] In the seventh aspect of the present disclosure, a vaccine adjuvant or vaccine is provided, characterized in that it comprises the Toll-like receptor agonist provided by the first aspect of the present disclosure, the active ingredient encapsulated by the lipid component provided by the second aspect, the composition provided by the fourth aspect, the delivery system provided by the fifth aspect, or the pharmaceutical composition provided by the sixth aspect.

[0023] In an eighth aspect of the present disclosure, there is provided a use of the Toll-like receptor agonist of the first aspect of the present disclosure, the active ingredient encapsulated by the lipid component of the second aspect, the composition of the fourth aspect, the delivery system of the fifth aspect, or the drug combination of the sixth aspect in any one of the following (a) to (j):

[0024] (a) Use in the preparation of a drug for preventing tumor occurrence;

[0025] (b) use in the preparation of drugs for treating tumors;

[0026] (c) use in the preparation of a drug for inhibiting tumor growth;

[0027] (d) Use in the preparation of a medicament for preventing tumor cell metastasis;

[0028] (e) Use in the preparation of a medicament for preventing tumor recurrence;

[0029] (f) use in the preparation of vaccines;

[0030] (g) use in the preparation of a medicament for treating or preventing inflammation-related diseases or infectious diseases;

[0031] (h) Use in the preparation of a drug for immune activation;

[0032] (i) Use in the preparation of a medicament for upregulating cytokine expression;

[0033] (j) For non-therapeutic purposes to upregulate cytokine expression.

[0034] In a ninth aspect of the present disclosure, there is provided a method of any one of the following (a) to (h):

[0035] (a) Methods for preventing tumorigenesis;

[0036] (b) methods for treating tumors;

[0037] (c) methods for inhibiting tumor growth;

[0038] (d) Methods for preventing tumor cell metastasis;

[0039] (e) methods for preventing tumor recurrence;

[0040] (f) methods for treating or preventing inflammation-related diseases or infectious diseases;

[0041] (g) methods of immune activation;

[0042] (h) methods for upregulating cytokine expression;

[0043] It is characterized in that it comprises administering to a subject in need thereof a therapeutically effective amount of the Toll-like receptor agonist of the first aspect of the present disclosure, the active ingredient encapsulated by the lipid component of the second aspect, the composition of the fourth aspect, the delivery system of the fifth aspect, or the drug combination of the sixth aspect.

[0044] In a tenth aspect of the present disclosure, a method for preparing a delivery system is provided, characterized in that it comprises the following steps (a) to (d):

[0045] (a) taking the Toll-like receptor agonist according to the first aspect of the present disclosure and preparing it with a buffer to prepare a Toll-like receptor agonist buffer;

[0046] (b) preparing a lipid ethanol solution by taking the lipid component and using anhydrous ethanol;

[0047] (c) mixing the lipid ethanol solution and the Toll-like receptor agonist buffer solution at a mass ratio of lipid component to Toll-like receptor agonist of (8-12):1;

[0048] (d) Remove ethanol, dilute and adjust pH to 6.8-7.2. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIG1 is a curve showing the change of tumor volume over time during the intratumoral administration of MC38 tumor-bearing mice when physiological saline or different CpG-ODNs, namely T0009-1, T0009-2, T0009-3, and T0009-4, were administered in Example 1.

[0050] Figure 2 shows the change in tumor volume over time during intratumoral administration of Qtolimod (T0009-1 Qtolimod, T0009-2 Qtolimod, T0009-3 Qtolimod, and T0009-4 Qtolimod) in MC38 tumor-bearing mice when administered with saline or different CpG-ODN-based Qtolimods in Example 8. Where it represents intratumoral injection, and q3d*5 represents injection every three days for a total of five injections.

[0051] Figure 3 shows the body weight change (%) over time during intratumoral administration of Qtolimod in MC38 tumor-bearing mice when administered with saline or different CpG-ODN-based Qtolimod in Example 8. Where it represents intratumoral administration, and q3d*5 represents injection every three days for a total of five injections.

[0052] FIG4 is a curve showing the change of tumor volume over time in B16F10 tumor-bearing mice during administration of normal saline or Qtolimod prepared with different CpG-ODNs in Example 9.

[0053] Figure 5 is a curve showing the change in body weight (%) over time in B16F10 tumor-bearing mice during administration of normal saline or Qtolimod prepared with different CpG-ODNs in Example 9.

[0054] Figure 6 is a curve showing the change in survival rate over time during intratumoral administration of B16F10 tumor-bearing mice when Qtolimod prepared with physiological saline or different CpG-ODNs was administered in Example 9.

[0055] Figure 7 is a curve showing the change of tumor volume over time in MC38 tumor-bearing mice during administration of normal saline, T0009-1 Qtolimod, Tritolimod or Doxotolimod in Example 10 (the first tumor measurement time is the day before the first administration, Day-1).

[0056] FIG8 is a curve showing the change in body weight (%) over time in MC38 tumor-bearing mice during administration of physiological saline, T0009-1 Qtolimod, Tritolimod or Doxotolimod in Example 10.

[0057] Figure 9 is a curve showing the change of tumor volume over time in B16F10 tumor-bearing mice during tail vein administration of Tritolimod or normal saline in Example 11.

[0058] FIG10 is a curve showing the change in survival rate over time during tail vein administration of B16F10 tumor-bearing mice when Tritolimod or normal saline was administered in Example 11.

[0059] Figure 11 is a curve showing the body weight change rate (%) over time during intratumoral administration of MC38 tumor-bearing mice when administered with physiological saline, lipid solution (QTsome group), 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg or 2 mg / kg of T0009-1 Qtolimod in Example 12 (the first tumor measurement time was the day before the first administration, Day-1).

[0060] Figure 12 is a curve showing the change in tumor volume over time during intratumoral administration of MC38 tumor-bearing mice when administered with normal saline, lipid solution (QTsome group), 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg or 2 mg / kg of T0009-1 Qtolimod in Example 12 (the first tumor measurement was the day before the first administration, Day-1).

[0061] Figure 13A is a curve showing the change of tumor volume over time in tumor-bearing mice after MC38 cells were subcutaneously transplanted into the left shoulder of mice in the control group and each Qtolimod group after the control group was re-established on Day 36 in Example 12 (no further medication was given to each group).

[0062] Figure 13B shows the tumor volume on the left shoulder of the tumor-bearing mice at the end of the experiment after MC38 cells were subcutaneously transplanted into the left shoulder of the control group and each Qtolimod group mice in Example 12 on Day 36 (no further medication was given to each group).

[0063] Figure 14A shows in vivo drug imaging at different time points during intratumoral administration of T0009-1 Qtolimod and free T0009-1 in MC38 tumor-bearing mice as described in Example 13. The left mouse received free T0009-1, while the right mouse received T0009-1 Qtolimod. Colored areas on the mouse correspond to the primary distribution of the drug, with colors approaching yellow indicating high fluorescence intensity, red indicating medium fluorescence intensity, and black indicating low fluorescence intensity.

[0064] FIG14B is a histogram of drug imaging fluorescence intensity at different time points during intratumoral administration of T0009-1 Qtolimod and free T0009-1 in MC38 tumor-bearing mice in Example 13.

[0065] Figure 15A shows CD11b immunohistochemistry images (left) and CD11b-positive area ratios (right) of tumor sections of mice after administration of saline or T0009-1 Qtolimod in Example 14. Brown in the immunohistochemistry images represents CD11b positivity.

[0066] Figure 15B is the CD8 immunohistochemistry image (left) and the percentage of CD8 positive T cells in the total tumor cells (right) of the mouse tumor sections after administration of normal saline or T0009-1 Qtolimod in Example 14.

[0067] Figure 15C shows the F4 / 80 immunohistochemistry images (left) and F4 / 80 positive area ratios (right) of tumor sections from tumor-bearing mice after administration of saline or T0009-1 Qtolimod in Example 14. Brown in the immunohistochemistry images represents F4 / 80 positivity.

[0068] FIG16A shows the concentrations of IL-12, IL-10, and IFN-γ in serum and tumor samples of tumor-bearing mice after administration of physiological saline, lipid solution (QTsome group), or T0009-1 Qtolimod in Example 14.

[0069] FIG16B shows the concentrations of IL-1β, IL-6, and TNF-α in serum and tumor samples of tumor-bearing mice after administration of physiological saline, lipid solution (QTsome group), or T0009-1 Qtolimod in Example 14.

[0070] Figure 17 is a graph showing changes in body weight over time in female and male mice when subcutaneously injected with physiological saline (vehicle control group), lipid solution (QTsome group), free T0009-1 (API group) or T0009-1 Qtolimod (Qtolimod group) in Example 15.

[0071] FIG18 is a survival curve of animals in each group during the administration period in Example 16.

[0072] FIG19 shows the body weight change trends of animals in each group during the administration period in Example 16.

[0073] FIG20 shows the subcutaneous tumor volume change trend of each group of animals during the administration period in Example 16.

[0074] FIG21 shows the changes in relative tumor proliferation rate T / C values ​​of each group of animals during the administration period in Example 16.

[0075] Figure 22 shows the subcutaneous tumor weights of animals in each group in Example 16.

[0076] FIG23 shows the distribution of metastatic tumor nodules in various tissues of animals in Example 16.

[0077] FIG24 shows the number of metastatic tumor nodules in the lung tissue of each group of animals in Example 16.

[0078] FIG. 25 shows the total number of metastatic tumor nodules in each group of animals in Example 16.

[0079] FIG26 shows the subcutaneous tumor volume change trend of each group of animals during the administration period in Example 17.

[0080] FIG27 shows the body weight change trends of animals in each group during the administration period in Example 17.

[0081] FIG28A shows the trend of subcutaneous tumor volume changes in each group of EMT6 animals during the drug administration period in Example 18.

[0082] FIG28B shows the body weight change trends of animals in each EMT6 group during the drug administration period in Example 18.

[0083] FIG28C shows the subcutaneous tumor volume change trend of each EMT6-HPV18 group of animals during the drug administration period in Example 18.

[0084] FIG28D shows the body weight change trends of animals in each EMT6-HPV18 group during the drug administration period in Example 18.

[0085] FIG29A shows the trend of subcutaneous tumor volume changes in each group of K7M2 animals during the drug administration period in Example 19.

[0086] FIG29B shows the body weight change trends of animals in each group of K7M2 during the drug administration period in Example 19.

[0087] FIG29C shows the survival status of animals in each group during the observation period after administration in Example 19.

[0088] Figure 30 shows the detection of immune cells in tumor tissues of tumor-bearing mice at different time points after different administrations in Example 20. There are 3 mouse samples at each time point, and each dot in the figure represents one sample.

[0089] Figure 31 shows the detection of immune cells in spleen tissue of tumor-bearing mice at different time points after different administrations in Example 20. There are 3 mouse samples at each time point, and each dot in the figure represents one sample.

[0090] Figure 32 is the proteomic sequencing data analysis after in vitro stimulation of mouse and human PBMC cells in Example 21.

[0091] Figure 33 is an anatomical pathological image of orthotopic bladder cancer in mice after drug administration in Example 22. DETAILED DESCRIPTION

[0092] Unless otherwise stated, the terms used herein have the usual understanding meaning to those skilled in the art. For those skilled in the art, it can vary according to the desired properties and effects sought to be obtained by the application, and each numerical parameter should be interpreted according to the number of significant digits and conventional rounding methods or the manner understood by those skilled in the art. In general, the nomenclature used herein and the experimental procedures of organic chemistry, medicinal chemistry, biology described herein are well known in the art and are generally adopted in the art. Unless otherwise defined, all technical and scientific terms used herein generally have the same meanings that are commonly understood by those of ordinary skill in the art of the application. Where there are multiple definitions for the terms used herein, unless otherwise stated, the definitions in this section shall prevail.

[0093] Unless otherwise indicated, all numbers used in this specification and claims indicating content, concentration, ratio, weight, particle size, percentage, technical effect, etc. should be understood as being modified by the term "about" or "approximately" in any case. Therefore, unless otherwise indicated, the numerical parameters listed in the following specification and the appended claims are approximate values. "About" or "approximately" can be understood as a range of plus or minus 10%, 20%, 30%, 40% or 50% of the value shown.

[0094] 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 can be deduced analogously.

[0095] As used herein, the terms "comprises," "includes," and "comprising" mean that in addition to the listed elements, other elements are not excluded.

[0096] definition

[0097] As used herein, the term "pharmaceutically acceptable salts" includes acid addition salts and base addition salts. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include, but are not limited to, acetate, adipate, aspartate, benzoate, benzenesulfonate, bicarbonate / carbonate, bisulfate / sulfate, borate, camphorsulfonate, citrate, cyclamates, edisylate, formate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, 2-(4-hydroxybenzyl)benzoate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, 2-hydroxyethanesulfonate, lactate, malate, maleate, malonate, methanesulfonate, methylsulfate, naphthoate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, hexadecanoate, phosphate / hydrogenphosphate / dihydrogenphosphate, pyroglutamate, glucarate, stearate, salicylate, tannate, tartrate, toluenesulfonate, and trifluoroacetate. Suitable base addition salts are formed with bases which form non-toxic salts. Examples include, but are not limited to, aluminum, arginine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine, potassium, sodium, tromethamine, and zinc salts. Hemi-salts of acids and bases, such as hemisulfate and hemicalcium salts, can also be formed. For a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection and Use by Stahl and Wermuth (Wiley-VCH, 2002).

[0098] As used herein, the term "functional variant" refers to a Toll-like receptor agonist having substantial or significant sequence identity or similarity to 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.

[0099] Toll-like receptor agonists

[0100] The present disclosure provides a Toll-like receptor agonist, characterized in that it comprises any one of the following (a)-(c): (a) any one of the modified or unmodified nucleotides as shown in SEQ ID NOs: 1-4; (b) a functional variant of any one of the modified or unmodified nucleotides as shown in SEQ ID NOs: 1-4; (c) a pharmaceutically acceptable salt of (a) or (b) the nucleotide; the nucleotide is all or partly ribonucleotides or all or partly deoxyribonucleotides.

[0101] In some embodiments, the modification includes one or more of the following: replacing a P=O bond between all or part of two adjacent nucleotides in the sequence with a P=S bond; replacing all or part of the deoxyribonucleosides in the sequence with ribonucleosides, or replacing all or part of the ribonucleosides in the sequence with deoxyribonucleosides; replacing the 2'-OH group of a ribonucleoside or deoxyribonucleoside with a methoxy group, a methoxyethyl group, or a halogen; bridging the second and fourth carbon atoms of a nucleotide to form a locked nucleic acid or (S)-cEt-BNA. In some embodiments, the halogen is selected from fluorine, chlorine, bromine, and iodine.

[0102] Lipid-encapsulated active ingredients

[0103] The present disclosure provides an active ingredient encapsulated by a lipid component, characterized in that the active ingredient comprises the Toll-like receptor agonist provided by the present disclosure.

[0104] In some embodiments, the mass ratio of the lipid component to the Toll-like receptor agonist provided by the present disclosure is (8-12):1, preferably 10:1.

[0105] In some embodiments, the lipid component comprises one or more of the following: a cationic polymer, a nanolipid particle (LNP), a cationic liposome, a QTsome, a lipopolyplex, a microparticle, a microsphere, or a nanoemulsion. QTsome is a lipid nanoparticle in which tetravalent (Quaternary) and trivalent (Tertiary) cationic phospholipids coexist. In some embodiments, the present disclosure uses QTsome lipid nanotechnology to encapsulate CpG-ODNs, by introducing a combination of cationic lipids that can be ionized under specific conditions and utilizing the sensitivity of liposomes to the external environment pH to maximize oligonucleotide loading and promote intracellular delivery of CpG-ODNs.

[0106] In some embodiments, the lipid component comprises one or more of a cationic lipid, an ionizable lipid, a neutral lipid, or a PEGylated lipid.

[0107] Cationic lipids include but are not limited to DOTAP (CAS: 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, CLIP 1, DORIE, DOSPA, CLIP6, CLIP9, and those disclosed in US Patent 5,049,386, International Publication Nos. WO91 / 16024, WO97 / 019675, WO2005 / 121348, WO2009 / 086558, and WO2011 / 13636.

[0108] Ionizable lipids include, but are not limited to, tertiary amines and their derivatives, pyrrolidines and their derivatives, piperazines and their derivatives, piperidines and their derivatives, such as DODMA (1,2-dioleyloxy-3-dimethylaminopropane, 1,2-dioleyl-3-dimethylamino-propane, CAS: 104162-47-2), ALC-0159, A066 (Z016) (1-(2,3-bis(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)propyl)pyrrolidine), L-319 (CAS: 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), DLin-MC3-DMA((6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate), DLin-K-DMA, DLin-KC2-DMA, DLin-KC3-DMA, DLin-KC4-DMA, DLinDMA, other DLinDMAs, other DLin-K-DMAs, lipid 5A2-SC8.

[0109] In some embodiments, neutral lipids include but are not limited to 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC), dioleoyl L-α-phosphatidylethanolamine (DOPE, CAS: 4004-05-1), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPC, CAS: 4235-95-4), 1-hexadecanoyl 2-(cis-9-octadecenoyl)-sn-glycero-3-phosphocholine (POPC), egg yolk phosphatidylcholine (EPC), distearoylphosphatidylcholine (DSPC, CAS: 816-94-4), and cholesterol. In some embodiments, the neutral lipids include neutral phospholipids, including but not limited to phosphatidylcholine and / or phosphatidylethanolamine, such as distearoylphosphatidylcholine (DSPC), egg yolk phosphatidylcholine (EPC), soybean lecithin, hydrogenated soybean lecithin (HSPC), dioleoylphosphatidylethanolamine (DOPE), dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), diole ... Phosphatidylcholine (DOPC), diarachidylphosphatidylcholine (DAPC), dimyristoylphosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), 1-palmitoyl-2-oleoylethanolamine (POPE), 1-hexadecane-2-(cis-9-octadecenoyl)-sn-glycero-3-phosphocholine (POPC).

[0110] In some embodiments, polyethylene glycol lipids include but are not limited to PEG-behenyl acetamide, PEG-myristoyl diglyceride, PEG-diacylglycerol, PEG-dialkyloxypropyl, PEG-phospholipids, PEG-ceramide, DMG-PEG, PEG-DSPE, DSG-PEG. In some embodiments, polyethylene glycol lipids can be selected from at least one of DSG-PEG and DMG-PEG.

[0111] 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.

[0112] In some embodiments, the active ingredient encapsulated by the lipid component comprises a Toll-like receptor agonist provided herein and another therapeutic agent. In some preferred embodiments, the molar ratio of the other therapeutic agent to the Toll-like receptor agonist provided herein is (0.1-5):1, preferably 1:2.

[0113] In some embodiments, the other therapeutic agent is selected from one or more of an antibody, a chemotherapeutic agent, and a small molecule drug. In some embodiments, the other therapeutic agent is selected from one or more of polyinosinic-polycytidylic acid (Poly(I:C)), a STING agonist, imiquimod, resiquimod, gasimod, sorafenib, sunitinib, erlotinib, doxorubicin, paclitaxel, gemcitabine, and radiation therapy. In some preferred embodiments, the other therapeutic agent is one or more of resiquimod and doxorubicin.

[0114] In some embodiments, antibodies include but are not limited to antibodies with anti-tumor effects. Antibodies with anti-tumor effects include but are not limited to: antibodies targeting PD-1, such as nivolumab, pembrolizumab, cemiplizumab, toripalimab, sintilimab, carrelizumab, etc.; antibodies targeting PD-L1, such as atezolizumab, avelumab and durvalumab, etc.; antibodies targeting CTLA4, such as ipilimumab and tesimumab, etc. In some embodiments, the Toll-like receptor agonists, CpG-ODNs provided by the present disclosure, combined with antibody drugs with anti-tumor effects, such as antibody drugs targeting PD-1 or PD-L1, can effectively enhance the responsiveness of immune checkpoint inhibitors or overcome the resistance of immune checkpoint inhibitors, which is conducive to further expanding the patient population of PD-1 or PD-L1 inhibitors, improving drug efficacy, and broadening indications.

[0115] In some embodiments, the antibody includes but is not limited to an anti-tumor associated antigen (TAA) antibody. TAA includes but is 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, etc.

[0116] In some embodiments, chemotherapy drugs include but are not limited to doxorubicin, paclitaxel, gemcitabine, and radiation therapy.

[0117] 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, taxane 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, TNFR2 inhibitors, etc.

[0118] In some embodiments, anthracycline compounds include but are not limited to doxorubicin, epirubicin, pirarubicin, daunorubicin, aclarubicin, idarubicin, amrubicin, and the like.

[0119] In some embodiments, camptothecin compounds include but are not limited to: topotecan, irinotecan, belotecan, isatecan (DX-8951), lurtotecan, sinotecan, rubitecan (9-NC), 9-aminocamptothecin (9-AC), gimatecan, karenitecin, DB-67, etc.

[0120] In some embodiments, the tyrosine kinase inhibitor includes but is not limited to sorafenib, sunitinib, gefitinib, erlotinib, lapatinib, afatinib, dacomitinib, vandetanib, neratinib, pelitinib (EKB-569), canertinib (CI-1033), osimertinib, rociletinib (CO-1686, Clovis Oncology), olmutinib (HM61713, Hanmi Pharmaceutical), naquotinib (ASP8273, Astellas), tesevatinib (XL647 / KD019, Kadmon Corporation), nazartinib (EGF816, Novartis) and PF-06747775 (Pfizer), etc.

[0121] In some embodiments, immunogenic cell death inducing agents include, but are not limited to, DNA damaging agents, bleomycin, and the like.

[0122] In some embodiments, taxane compounds include but are not limited to paclitaxel, docetaxel, and the like.

[0123] Pattern recognition receptor agonists other than the Toll-like receptor agonists provided herein include, but are not limited to, Toll-like receptors, RIG-I-like receptors, Nod-like receptors, AIM2-like receptors, and C-type lectin receptors, as well as cGas and other intracellular DNA sensor agonists.

[0124] In some embodiments, resiquimod (a TLR7 / 8 agonist, also known as R848) can be co-encapsulated with the CpG-ODNs provided by the present disclosure into a dual-active ingredient liposome (Tritolimod), thereby further enhancing the immune response of the tumor microenvironment, thereby effectively improving the therapeutic effect of the tumor.

[0125] In some embodiments, the molar ratio of Resiquimod to CpG-ODN is (0.1-5):1, preferably 1:2.

[0126] In some embodiments, the chemotherapy drug doxorubicin hydrochloride (DOX) can be co-encapsulated with the CpG-ODNs provided by the present disclosure into a dual-active ingredient liposome (Doxotolimod), thereby promoting the release of tumor antigens by doxorubicin and enhancing the activation of CD8 + T cells recognize and kill tumor cells, thereby effectively improving the treatment effect of tumors.

[0127] In some embodiments, the molar ratio of doxorubicin to CpG-ODNs is (0.1-5):1, preferably 1:2.

[0128] Delivery system

[0129] The present disclosure provides a delivery system, characterized in that it comprises an active ingredient and a lipid component, wherein the active ingredient comprises the Toll-like receptor agonist provided by the present disclosure.

[0130] In some embodiments, the mass ratio of the lipid component to the Toll-like receptor agonist provided by the present disclosure is (8-12):1, preferably 10:1.

[0131] In some embodiments, the lipid component comprises one or more of the following: a cationic polymer, a nanolipid particle (LNP), a cationic liposome, a QTsome, a lipopolyplex, a microparticle, a microsphere, or a nanoemulsion.

[0132] In some embodiments, the lipid component comprises one or more of a cationic lipid, an ionizable lipid, a neutral lipid, and a polyethylene glycol lipid. In some embodiments, the polyethylene glycol lipid can be selected from at least one of DSG-PEG and DMG-PEG.

[0133] 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.

[0134] In some embodiments, the active ingredient in the delivery system comprises a Toll-like receptor agonist provided herein and another therapeutic agent. In some preferred embodiments, the molar ratio of the other therapeutic agent to the Toll-like receptor agonist provided herein is (0.1-5):1, preferably 1:2.

[0135] In some embodiments, the other therapeutic agent is selected from one or more of an antibody, a chemotherapeutic agent, and a small molecule drug. In some embodiments, the other therapeutic agent is selected from one or more of polyinosinic-polycytidylic acid (Poly(I:C)), a STING agonist, imiquimod, resiquimod, gasimod, sorafenib, sunitinib, erlotinib, doxorubicin, paclitaxel, gemcitabine, and radiation therapy. In some preferred embodiments, the other therapeutic agent is one or more of resiquimod and doxorubicin.

[0136] In some embodiments, resiquimod (a TLR7 / 8 agonist, also known as R848) can be co-encapsulated with the CpG-ODNs provided by the present disclosure into a dual-active ingredient liposome (Tritolimod), thereby further enhancing the immune response of the tumor microenvironment, thereby effectively improving the therapeutic effect of the tumor.

[0137] In some embodiments, the molar ratio of Resiquimod to CpG-ODN is (0.1-5):1, preferably 1:2.

[0138] In some embodiments, the chemotherapy drug doxorubicin hydrochloride (DOX) can be co-encapsulated with the CpG-ODNs provided by the present disclosure into a dual-active ingredient liposome (Doxotolimod), thereby promoting the release of tumor antigens by doxorubicin and enhancing the activation of CD8 + T cells recognize and kill tumor cells, thereby effectively improving the treatment effect of tumors.

[0139] In some embodiments, the molar ratio of doxorubicin to CpG-ODNs is (0.1-5):1, preferably 1:2.

[0140] The delivery system provided herein, comprising a specific lipid composition, can provide a stable coating for CpG-ODNs, protecting them from degradation by enzymes in the body, improving their bioavailability, and extending their in vivo half-life. Furthermore, it can also increase drug particle size and cellular adsorption, thereby enhancing drug retention within tumors. The delivery system provided herein, combined with intratumoral administration, can effectively improve drug retention within tumors and efficiently deliver drugs to tumor-infiltrating immune cells, enhancing the immune response in tumor tissue and improving tumor treatment efficacy.

[0141] The delivery system provided by the present disclosure can effectively increase the infiltration of immune cells such as dendritic cells, macrophages, CD3+T cells, CD8+T cells, NK cells, etc. in the tumor site, and at the same time promote the release of factors such as interleukins, interferons, chemokines, granzymes, etc., to achieve the purpose of tumor immunotherapy. Traditional adjuvants such as alum, squalene, and QS21 are microemulsions, which have local retention and sustained release properties. Although they can induce antigen-specific immunity and have good safety, they cannot induce a strong type I immune response. Traditional CpG adjuvants can induce correct CD8+T cell responses, but they will quickly spread throughout the body, causing adverse side effects and thus hindering treatment. Furthermore, the adjuvant of traditional CpG adjuvants is separated from the antigen, inducing non-specific autoimmunity. The delivery system provided by the present disclosure induces correct type I response immunity without inducing systemic cytokines, combining the advantages of previous adjuvants to induce antigen-specific immunity.

[0142] The CpG-ODNs-containing delivery system provided by the present disclosure can effectively inhibit tumor growth and can be prepared into a medicine with one or more pharmaceutically acceptable excipients for use in the treatment of tumors.

[0143] The CpG-ODNs-containing delivery system provided by the present disclosure can effectively prevent tumor cells from metastasizing to other tissues and organs or prevent tumor recurrence.

[0144] Preparation method

[0145] The present disclosure provides a method for preparing an active ingredient encapsulated by a lipid component, characterized in that it comprises the following steps (a) to (f):

[0146] (a) preparing the Toll-like receptor agonist provided by the first aspect of the present disclosure, CpG-ODNs, into an acidic solution;

[0147] (b) preparing the components of the lipid composition into a lipid ethanol solution;

[0148] (c) mixing the solutions prepared in steps (a) and (b) to form a preliminary solution of nanolipid particles;

[0149] (d) optionally repeating the above steps (a)-(c) to obtain more initial solution of nanolipid particles;

[0150] (e) removing ethanol from the initial nanolipid particle solution and adjusting the active ingredient concentration and pH to obtain a nanolipid particle suspension;

[0151] (f) selectively mixing other therapeutic agents with the nanolipid particle suspension to prepare multi-active ingredient liposomes.

[0152] In some embodiments, step (f) is an optional step when the above method is used to prepare a single active ingredient encapsulated by a lipid component.

[0153] In some embodiments, step (f) is performed when preparing multiple active ingredients encapsulated by lipid components using the above method. In some embodiments, the other therapeutic agent used in step (f) is preferably resiquimod or doxorubicin.

[0154] The present disclosure also provides a method for preparing a delivery system, comprising the following steps: taking the Toll-like receptor agonist disclosed herein and preparing it with a buffer to prepare a Toll-like receptor agonist buffer; taking a lipid component and preparing it with anhydrous ethanol to prepare a lipid ethanol solution; taking the lipid ethanol solution and the Toll-like receptor agonist buffer at a mass ratio of the lipid component to the CpG-ODN of (8-12):1, mixing, removing the ethanol, diluting and adjusting the pH to 6.8-7.2, to obtain.

[0155] Composition, pharmaceutical composition

[0156] The present disclosure provides a composition, characterized in that it comprises an active ingredient encapsulated by a lipid component provided by the present disclosure and other therapeutic agents. In some embodiments, the other therapeutic agents are selected from one or more of antibodies, chemotherapeutic drugs and small molecule drugs. In some embodiments, the other therapeutic agents are selected from one or more of polyinosinic-acid (poly IC), STING agonists, imiquimod, resiquimod, gasimod, sorafenib, sunitinib, erlotinib, doxorubicin, paclitaxel, gemcitabine, and radiotherapy. In some preferred embodiments, the other therapeutic agents are one or more of resiquimod and doxorubicin.

[0157] The present disclosure provides a pharmaceutical composition, characterized by comprising the Toll-like receptor agonist provided by the present disclosure, an active ingredient encapsulated by a lipid component, a composition or a delivery system, and a pharmaceutically acceptable carrier or excipient.

[0158] As used herein, the term "pharmaceutical composition" refers to a mixture of a Toll-like receptor agonist, an active ingredient encapsulated by a lipid component, a composition or a delivery system provided by the present disclosure and other chemical components such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and / or excipients. The pharmaceutical composition facilitates administration of the Toll-like receptor agonist, the active ingredient encapsulated by a lipid component, a composition or a delivery system to an organism. There are various ways of administering pharmaceutical compositions in the art, including, but not limited to, intratumoral injection, intravenous injection, subcutaneous injection, intramuscular injection, arterial injection, intraperitoneal injection, injection into the central nervous system, such as intrathecal injection, intravesical instillation, interventional therapy, oral administration, transdermal administration, transpulmonary administration, ocular administration and topical administration.

[0159] As described herein, the pharmaceutical composition can be configured into a dosage form suitable for administration to a subject via a desired route of administration, including but not limited to tablets, capsules, caplets, pills, lozenges, powders, syrups, brews, suspensions, solutions, emulsions, transdermal patches, suppositories, inhalants, creams, ointments, lotions, pastes, sprays, freeze-dried solutions, injections, and gels.

[0160] The term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable materials, compositions or vehicles, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials, which are involved in carrying or transporting the Toll-like receptor agonist of the present invention, the active ingredient encapsulated by the lipid component, the composition, the delivery system within the subject or carrying or transporting the Toll-like receptor agonist of the present invention, the active ingredient encapsulated by the lipid component, the composition, the delivery system to the subject so that it can perform its intended function. Each salt or carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the subject. Some 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; powdered tragacanth; 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; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate; Esters and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; diluents; granulating agents; lubricants; binders; disintegrants; wetting agents; emulsifiers; colorants; release agents; coating agents; sweeteners; flavorings; perfuming agents; preservatives; antioxidants; plasticizers; gelling agents; thickeners; hardening agents; setting agents; suspending agents; surfactants; humectants; carriers; stabilizers; and other nontoxic, compatible substances used in pharmaceutical formulations, or any combination thereof.

[0161] Vaccines and vaccine adjuvants

[0162] The CpG-ODNs provided herein can also be used in the field of vaccines and vaccine adjuvants to enhance immune responses and increase antibody titers. In some embodiments, CpG-ODNs can be used as a main ingredient in the preparation of vaccine adjuvants, wherein CpG-ODNs can be present in the vaccine formulation in a lipid-conjugated form, a directly encapsulated form, or directly in a free form.

[0163] The present disclosure provides a vaccine adjuvant or vaccine, characterized by comprising the Toll-like receptor agonist provided by the present disclosure, an active ingredient encapsulated by a lipid component, a composition, a delivery system or a pharmaceutical composition.

[0164] In some embodiments, the above-mentioned vaccine or vaccine adjuvant is a vaccine or vaccine adjuvant against KRAS, RSV, TERT, TP53, hepatitis virus, personalized new antigens, etc.

[0165] Uses and methods

[0166] The present disclosure provides use of a Toll-like receptor agonist, an active ingredient encapsulated by a lipid component, a composition, a delivery system, or a pharmaceutical combination of the present disclosure in any one of the following (a) to (j):

[0167] (a) Use in the preparation of a drug for preventing tumor occurrence;

[0168] (b) use in the preparation of drugs for treating tumors;

[0169] (c) use in the preparation of a drug for inhibiting tumor growth;

[0170] (d) Use in the preparation of a medicament for preventing tumor cell metastasis;

[0171] (e) Use in the preparation of a medicament for preventing tumor recurrence;

[0172] (f) use in the preparation of vaccines;

[0173] (g) use in the preparation of a medicament for treating or preventing inflammation-related diseases or infectious diseases;

[0174] (h) Use in the preparation of a drug for immune activation;

[0175] (i) Use in the preparation of a medicament for upregulating cytokine expression;

[0176] (j) For non-therapeutic purposes to upregulate cytokine expression.

[0177] In some embodiments, immune activation includes but is not limited to B cell pathway activation, T cell pathway activation, myeloid lymphocyte pathway activation, and lymphoid lymphocyte pathway activation. In some embodiments, immune activation includes but is not limited to activation of immune cells such as macrophages, plasmacytoid dendritic cells (pDCs), B cells, T cells, CD3+ T cells, CD8+ T cells, NK cells, and granulocytes.

[0178] In some embodiments, the drug can be administered by various means, including but not limited to, intratumoral injection, intravenous injection, subcutaneous injection, intramuscular injection, arterial injection, intraperitoneal injection, injection into the central nervous system, such as intrathecal injection, intravesical instillation, interventional therapy, oral, transdermal, pulmonary, ocular and topical administration.

[0179] In some embodiments, the tumor is selected from a solid tumor or a tumor into which a drug can be injected, such as at least one of head and neck cancer, esophageal cancer, oral cancer, nasopharyngeal cancer, thyroid cancer, lung cancer, gastric 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, chondroma, liposarcoma, neuroblastoma, synovial sarcoma, astrocytoma, glioblastoma multiforme, anaplastic astrocytoma, peritoneal cavity cancer, soft tissue sarcoma, sarcoma, rhabdomyosarcoma, advanced myxoid disease, melanoma, skin cancer, such as non-melanoma skin cancer.

[0180] Inflammation-related diseases include, but are not limited to, delayed hypersensitivity reactions, allergic diseases, lupus, graft-versus-host disease, vasculitis caused by hepatitis C, type I diabetes, type II diabetes, multiple sclerosis, rheumatoid arthritis, alopecia areata, atherosclerosis, psoriasis, organ transplant rejection, Sjögren's syndrome, Behçet's disease, spontaneous abortion, atopic diseases, asthma, and inflammatory bowel disease.

[0181] Infectious diseases include, but are not limited to, fungal infections, bacterial infections, mycoplasma or chlamydia infections, and viral infections, such as viral hepatitis.

[0182] The present disclosure also provides a method of any one of the following (a)-(h):

[0183] (a) Methods for preventing tumorigenesis;

[0184] (b) methods for treating tumors;

[0185] (c) methods for inhibiting tumor growth;

[0186] (d) Methods for preventing tumor cell metastasis;

[0187] (e) methods for preventing tumor recurrence;

[0188] (f) methods for treating or preventing inflammation-related diseases or infectious diseases;

[0189] (g) methods of immune activation;

[0190] (h) methods for upregulating cytokine expression;

[0191] The method is characterized in that it comprises administering a therapeutically effective amount of the Toll-like receptor agonist, the active ingredient encapsulated by the lipid component, the composition, the delivery system or the pharmaceutical combination of the present disclosure to a subject in need thereof.

[0192] In some preferred embodiments, when preventing tumor occurrence, treating tumors, inhibiting tumor growth, preventing tumor cell metastasis, and preventing tumor recurrence, the Toll-like receptor agonist of the present invention or the active ingredient encapsulated by the lipid component is administered to a subject in need thereof at the following dose: at least 0.1 mg / kg (mpk) 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, 8 mg / kg body weight. In some preferred embodiments, when preventing tumor occurrence, treating tumors, inhibiting tumor growth, preventing tumor cell metastasis, and preventing tumor recurrence, the Toll-like receptor agonist of the present invention or the active ingredient encapsulated by the lipid component is administered to a subject in need at the following frequency: at least once a week, for a total of at least 3 times, for example, once every 3 days, for a total of 4 times, once every 3 days, for a total of 5 times, once a week, for a total of 3 times, once a week, for a total of 4 times, once a week, for a total of 5 times. In some preferred embodiments, the subject in need is a mouse. Based on the above-mentioned dosage and frequency, those skilled in the art can convert the dosage and frequency for one animal, such as a mouse, into the dosage frequency and dosage for another animal, such as a human, in combination with conventional technical means in the art.

[0193] In some embodiments, the tumor is selected from a solid tumor or a tumor into which a drug can be injected, such as at least one of head and neck cancer, esophageal cancer, oral cancer, nasopharyngeal cancer, thyroid cancer, lung cancer, gastric 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, chondroma, liposarcoma, neuroblastoma, synovial sarcoma, astrocytoma, glioblastoma multiforme, anaplastic astrocytoma, peritoneal cavity cancer, soft tissue sarcoma, sarcoma, rhabdomyosarcoma, advanced myxoid disease, melanoma, skin cancer, such as non-melanoma skin cancer.

[0194] Inflammation-related diseases include, but are not limited to, delayed hypersensitivity reactions, allergic diseases, lupus, graft-versus-host disease, vasculitis caused by hepatitis C, type I diabetes, type II diabetes, multiple sclerosis, rheumatoid arthritis, alopecia areata, atherosclerosis, psoriasis, organ transplant rejection, Sjögren's syndrome, Behçet's disease, spontaneous abortion, atopic diseases, asthma, and inflammatory bowel disease.

[0195] Infectious diseases include, but are not limited to, fungal infections, bacterial infections, mycoplasma or chlamydia infections, and viral infections, such as viral hepatitis.

[0196] As used herein, the term "subject" includes animals, such as vertebrates, preferably mammals, such as dogs, cats, pigs, cows, sheep, horses, rabbits, rodents (e.g., mice, rats, or guinea pigs), or primates (e.g., monkeys, gorillas, chimpanzees, and humans).

[0197] As used herein, the term "treat," ...

[0198] As used herein, the term "therapeutically effective dose" refers to an amount that results in a benefit or treatment of a disease compared to a corresponding subject not receiving that amount, but is sufficiently low within the scope of sound medical judgment to avoid serious side effects. The therapeutically effective dose of the Toll-like receptor agonist, active ingredient encapsulated by a lipid component, composition, delivery system or drug combination described herein will vary with the selected Toll-like receptor agonist, active ingredient encapsulated by a lipid component, composition, delivery system or drug combination; route of administration; severity of the disease being treated; age, size, weight and physical condition of the patient being treated; medical history of the patient being treated; duration of treatment; nature of concurrent treatment; desired therapeutic effect and the like, but can still be determined in a routine manner by those skilled in the art.

[0199] As used herein, the term "preventing" includes providing protection against the occurrence or recurrence of a disease in an individual who may be susceptible to the disease but has not yet been diagnosed with the disease.

[0200] The present disclosure further provides a Toll-like receptor agonist, an active ingredient encapsulated by a lipid component, a composition, a delivery system or a pharmaceutical combination of the present disclosure, for use in any one of the following (a) to (h):

[0201] (a) Prevent tumorigenesis;

[0202] (b) treating tumors;

[0203] (c) inhibiting tumor growth;

[0204] (d) prevent tumor cell metastasis;

[0205] (e) prevent tumor recurrence;

[0206] (f) treating or preventing inflammation-related diseases or infectious diseases;

[0207] (g) immune activation;

[0208] (h) Upregulation of cytokine expression.

[0209] The various embodiments described above for the Toll-like receptor agonist, active ingredient encapsulated by lipid components, and delivery system of the present disclosure are also applicable to the pharmaceutical compositions, compositions, uses, and methods of the present disclosure (as long as they are not inherently inconsistent with each other), and the various embodiments formed by such combination are considered part of the present disclosure.

[0210] Example

[0211] The following describes exemplary embodiments of the present application in conjunction with the accompanying drawings, including various details of the embodiments of the present application to facilitate understanding. It should be understood that they are considered merely exemplary and are in no way intended to limit the scope of protection of the present application. The scope of protection of the present application is defined solely by the claims. Therefore, it should be appreciated by those skilled in the art that various changes and modifications may be made to the embodiments described herein without departing from the scope of the present application. Similarly, for the sake of clarity and conciseness, descriptions of well-known functions and structures are omitted in the following description.

[0212] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0213] Example 1. Effect of CpG-ODN on the Growth of Colon Cancer Tumors in Mice

[0214] The experimental drugs were the four CpG-ODNs (T0009-1 to T0009-4) described in Table 1 , and in the sequences shown in Table 1 , all phosphodiester bonds were thio-modified.

[0215] Table 1 CpG-ODN sequence information

[0216] The above-mentioned experimental drugs were diluted to 1 μg / μl using physiological saline (0.9% sodium chloride aqueous solution).

[0217] 1. Experimental plan

[0218] (1) Fifty 7-week-old C57BL / 6 mice (Vitamin B, half male and half female) were taken and weighed after one week of adaptive feeding.

[0219] (2) Resuscitate and passage the mouse colon cancer MC38 cell line (Nanjing Kebai, catalog number CBP60825). When the cells are in good condition, inoculate MC38 cells (inoculation volume 10 6 cells) were transplanted subcutaneously on the back of mice, and the changes in mouse body weight and tumor size were recorded;

[0220] (3) When the tumor grows to about 100 mm 3 When the tumor size was different, the mice were divided into groups. Mice with tumors that were too large or too small were eliminated, and mice with the same number of mice were retained and divided into 5 groups using a completely randomized method, namely: T0009-1 group, T0009-2 group, T0009-3 group, T0009-4 group and normal saline group, with 7 mice in each group;

[0221] (4) (Day 0) Each mouse was intratumorally administered with 25 μL of (T0009-1, T0009-2, T0009-3, T0009-4) or normal saline (0.9% sodium chloride aqueous solution) at a dose of 1 mg drug / kg body weight (1 mg / kg), once every 3 days for a total of 5 injections. The day of the first injection was designated as day 0.

[0222] (5) Observe and record the growth and proliferation of subcutaneous colon tumors on the backs of tumor-bearing mice in the drug-treated group and the normal saline control group every day, including measuring the weight of the mice and the tumor volume, and drawing the tumor volume change curve and the weight change curve;

[0223] (6) On Day 18, the mice were killed, the tumor masses were removed, the tumor mass volume was measured, and the net weight of the tumor mass was weighed. The tumor growth inhibition rate (TGI) was calculated based on the relative tumor volume (RTV) of each group of mice on Day 18 relative to the grouping. The calculation formula of TGI (%) is: TGI (%) = [1-RTV (drug group) / RTV (control group)] * 100%

[0224] 2. Experimental results

[0225] As shown in Figure 1, all treatment groups demonstrated significant tumor suppression, with T0009-1 showing the highest efficacy. The TGI% for T0009-1, T0009-1, T0009-4, and T0009-1 were 73.72%, 56.12%, 58.41%, and 65.37%, respectively, demonstrating the superior tumor suppression efficacy of T0009-1. No mice in any treatment group died from the start of treatment until day 18.

[0226] Example 2-5. Preparation of lipid nanoparticles (Qtolimod) encapsulating CpG-ODNs

[0227] The CpG-ODN used in Examples 2-5 were T0009-1, T0009-2, T0009-3 and T0009-4, respectively, and the CpG-ODN Qtolimod prepared in Examples 2-5 were T0009-1 Qtolimod, T0009-2 Qtolimod, T0009-3 Qtolimod and T0009-4 Qtolimod, respectively.

[0228] The preparation method of Qtolimod encapsulating CpG-ODN comprises the following steps:

[0229] (1) CpG-ODN was prepared using sodium acetate buffer (pH = 5.0) to prepare a CpG-ODN sodium acetate solution with a pH of 5.0, a CpG-ODN concentration of 0.25 mg / mL, and a sodium acetate concentration of 25 mM;

[0230] (2) DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG were prepared with anhydrous ethanol to prepare a lipid ethanol solution with a total lipid concentration of 7.5 mg / mL; wherein the molar ratio of DOTAP, DODMA, DSPC, cholesterol, and DMG-PEG was 15:25:20:38.5:1.5;

[0231] (3) Take 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), and preheat them in a water bath at 25°C; then inject the lipid ethanol solution into the CpG-ODN sodium acetate solution, incubate for 1 min under magnetic stirring, and then dilute 4 times with a sodium acetate buffer solution with a pH of 5.0 and a concentration of 25 mM to obtain a Qtolimod initial solution of CpG-ODN encapsulated by QTsome; the above steps (1)-(3) can be repeated to obtain more initial solutions of nanolipid particles (LNPs) loaded with CpG-ODN;

[0232] (4) Using an ultrafiltration tube with a molecular weight cutoff of 100 kD and a centrifugal force of 2000 g, the ethanol in the LNP initial solution was removed by ultrafiltration. The solution was 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 LNP suspensions loaded with different CpG-ODNs, namely, CpG-ODN Qtolimod suspensions. The lipid solution used in the QTsome group, which served as a control in the in vitro and in vivo experiments, was a lipid solution. The lipid solution in the QTsome group and the lipid solution used in Example 14 did not undergo the mixing step with CpG-ODN described in (3), but was directly obtained by ultrafiltration to remove ethanol and adjust the pH value in this step from the lipid ethanol solution described in (2).

[0233] Example 6. Preparation of dual-active ingredient liposomes (Tritolimod) encapsulating Resiquimod and CpG-ODN

[0234] The T0009-1-loaded LNP suspension (T0009-1 Qtolimod suspension) prepared in Example 2 was mixed with resiquimod at a molar ratio of 2:1. Resiquimod, with its high logP value, actively disperses into the LNPs.

[0235] Example 7. Preparation of dual-active ingredient liposomes (Doxotolimod) encapsulating doxorubicin (DOX) and CpG-ODN

[0236] The T0009-1-loaded LNP suspension (T0009-1 Qtolimod suspension) prepared in Example 2 was mixed with DOX at a molar ratio of 2:1. This formed dual-active ingredient liposomes containing Doxotolimod. DOX can be remotely incorporated into the LNPs by binding to CpG and / or a captured conjugate (e.g., acetate).

[0237] Example 8. Effects of Qtolimod Prepared with Different CpG-ODNs on Colon Cancer Tumor Growth in Mice

[0238] The experimental drug was Qtolimod prepared in Example 2-5. The preparation was packaged in advance, stored at -20°C, and reconstituted on the day of use.

[0239] 1. Experimental plan

[0240] (1) Fifty 7-week-old C57BL / 6 mice (Vitamin B, half male and half female) were taken and weighed after one week of adaptive feeding.

[0241] (2) Resuscitate and passage the mouse colon cancer MC38 cell line (Nanjing Kebai, catalog number CBP60825). When the cells are in good condition, inoculate MC38 cells (inoculation volume 10 6 cells) were transplanted subcutaneously on the back of mice, and the changes in mouse body weight and tumor size were recorded;

[0242] (3) When the tumor grows to about 100 mm 3 When the tumor size was too large or too small, the mice were grouped. Mice with tumors of equal size were eliminated, and the remaining mice were kept and randomly divided into 5 groups using the complete randomization method, namely: T0009-1 Qtolimod group, T0009-2 Qtolimod group, T0009-3 Qtolimod group, T0009-4 Qtolimod group and normal saline group, with 7 mice in each group;

[0243] (4) (Day 0) Each mouse was intratumorally administered with 100 μL of (T0009-1 Qtolimod, T0009-2 Qtolimod, T0009-3 Qtolimod, T0009-4 Qtolimod) or normal saline (0.9% sodium chloride aqueous solution) (injected at two points depending on the situation) at a dose of 1 mg / kg, once every 3 days for a total of 5 injections. The day of the first injection was designated as Day 0.

[0244] (5) Observe and record the growth and proliferation of subcutaneous colon tumors on the backs of tumor-bearing mice in the drug-treated group and the normal saline control group every day, including measuring the weight of the mice and the tumor volume, and drawing the tumor volume change curve and the weight change curve;

[0245] (6) On Day 30, the mice were killed, the tumor masses were removed, the tumor volume was measured, the net weight of the tumor mass was weighed, and the tumor growth inhibition rate was calculated.

[0246] 2. Experimental results

[0247] As shown in Figures 2-3, each drug-treated group began to show significant drug efficacy on Day 8 of the experiment. By Day 30, the tumor volume of mice in the normal saline control group reached the humane endpoint (2500mm 3 ), all groups receiving Qtolimod demonstrated significant tumor suppression. Calculated TGI% for Qtolimod in T0009-1, T0009-4, and T0009-1 were 84.42%, 85.522%, 89.152%, and 85.802%, respectively. No mice in any of the treatment groups died from the start of dosing until sacrifice on day 30.

[0248] During the entire experimental period, there were no obvious abnormalities in the body weight and clinical status of mice in the normal saline control group and the drug-treated group, indicating that under the conditions of this tumor model, the animals showed good tolerance to the test drug dose.

[0249] In summary, T0009-1 Qtolimod, T0009-2 Qtolimod, T0009-3 Qtolimod, and T0009-4 Qtolimod significantly inhibited the growth of xenograft tumors in mice, and the animals exhibited good drug tolerance. There were no significant differences among the treatment groups in improving the survival of tumor-bearing animals, tumor suppression, or clinical symptoms.

[0250] Example 9. Effects of Qtolimod Prepared with Different CpG-ODNs on the Growth of Mouse Skin Melanoma Tumors

[0251] The experimental drug was Qtolimod prepared in Example 2-5. The preparation was packaged in advance, stored at -20°C, and reconstituted on the day of use.

[0252] 1. Experimental plan

[0253] (1) Fifty 7-week-old C57BL / 6 mice were weighed after one week of adaptive feeding.

[0254] (2) Inoculation conditions are shown in Table 2 below;

[0255] Table 2 Cell seeding information

[0256] (3) After inoculation, tumor volume and body weight were measured once a week. When the average tumor volume reached about 40-60 mm 3 Mice were grouped. Mice with tumors that were too large or too small were removed, and the remaining mice, equal to the number of mice enrolled, were randomly divided into five groups using a completely randomized method: the T0009-1 Qtolimod group, the T0009-2 Qtolimod group, the T0009-3 Qtolimod group, the T0009-4 Qtolimod group, and the normal saline group, with 8 mice in each group. Immediately after grouping, drug administration or normal saline (0.9% sodium chloride aqueous solution) was started. Each mouse received an intratumoral injection (it) of 50 μL (injection could be divided into two injections depending on the situation) at a dose of 1 mg / kg, once every 3 days, for a total of 5 injections, with the first injection being the day of day 0.

[0257] (4) After the start of drug administration, the body weight and tumor volume of mice were measured three times a week. When the tumor volume of a single animal reached 3000 mm 3At , the animals were euthanized and regarded as dead. The median survival time (MST) of each group of animals was calculated, and the survival curves were drawn and statistically compared.

[0258] 2. Experimental results

[0259] As shown in Figures 4-6, all drug-treated groups showed significant drug efficacy on Day 7 of the experiment, among which the T0009-1 Qtolimod group showed better tumor inhibition effect.

[0260] Analysis of the mean survival of mice in each group showed that the saline control group had an average survival of 9 days, while the average survival of animals in the T0009-1, T0009-4, and T0009-4 Qtolimod groups was 16 days, 12 days, 11 days, and 12 days, respectively. The TGI% in the T0009-1 Qtolimod group was 75%, a highly significant difference compared to the saline control group (all P values ​​less than 0.01). The T0009-1 Qtolimod group had the longest survival of all the treatment groups.

[0261] During the entire experimental period, there were no obvious abnormalities in the body weight and clinical status of mice in the normal saline control group and the drug-treated group, indicating that under the conditions of this tumor model, the animals showed good tolerance to the test drug dose.

[0262] In summary, T0009-1 Qtolimod, T0009-2 Qtolimod, T0009-3 Qtolimod, and T0009-4 Qtolimod significantly inhibited the growth of xenograft tumors in mice, and the animals exhibited good drug tolerance. Among them, T0009-1 Qtolimod was the most effective in improving the survival, tumor suppression, and clinical symptoms of tumor-bearing animals.

[0263] Example 10. Effect of dual-active ingredient liposomes on the growth of colon cancer tumors in mice

[0264] 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 preparations were packaged in advance, stored at -20°C, and reconstituted on the day of use.

[0265] 1. Experimental plan

[0266] The experimental protocol is the same as that of Example 8.

[0267] 2. Experimental results

[0268] As shown in Figures 7-8, each dosing group began to show significant drug efficacy on Day 6 of the experiment. By Day 12, the Qtolimod group given T0009-1, the Tritolimod group given Tritolimod, and the Doxotolimod group given Doxotolimod all showed very significant (P<0.001) tumor inhibition effects. The calculated TGI% for the Qtolimod group, Tritolimod group, and Doxotolimod group were 90.97%, 94.09%, and 91.03%, respectively. Throughout the experimental period, there were no significant abnormalities in the body weight or clinical status of the mice in the saline control group and the dosing group, indicating that the animals showed good tolerance to the test drug dose under the conditions of this tumor model.

[0269] Example 11. Effect of systemic administration of dual-active ingredient liposome Tritolimod on the growth of skin melanoma in mice

[0270] The experimental drug was the dual-active ingredient liposome Tritolimod prepared in Example 6. The preparation was packaged in advance, stored at -20°C, and reconstituted on the day of use.

[0271] 1. Experimental plan

[0272] Similar to Example 9, the difference is that this example adopts the tail vein injection administration method.

[0273] 2. Experimental results

[0274] As shown in Figures 9-10, systemic administration of Tritolimod effectively inhibited 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). The survival of mice in the treatment group was also significantly prolonged.

[0275] Example 12. Study on the dose dependence of antitumor efficacy and anti-recurrence effect

[0276] This example uses intratumoral administration to study the effect of T0009-1 Qtolimod on tumor growth. The experimental drug is the lipid nanoparticle T0009-1 Qtolimod prepared in Example 2. The preparation is pre-packaged, stored at -20°C, and reconstituted on the day of use.

[0277] 1. Experimental plan

[0278] (1) Resuscitate MC38 cells and culture in vitro to obtain 5×10 6 cell;

[0279] (2) Thirty-six 7-week-old female C57BL / 6 mice were weighed after one week of adaptive feeding;

[0280] (3) Resuscitate and passage the mouse colon cancer MC38 cell line. When the cells are in good condition, inoculate MC38 cells (inoculation volume 10 6 The cells were transplanted subcutaneously into the right shoulder of mice, and the changes in the weight and tumor size of the mice were recorded;

[0281] (4) When the tumor grows to about 100 mm 3 When the size of the tumor was determined, the mice were grouped, and the mice with tumors that were too large or too small were eliminated. The remaining mice equal to the number of mice enrolled were retained and randomly divided into 6 groups using a completely random grouping method, namely: 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 normal saline control group, with 6 female mice in each group, wherein the mice in the QTsome group were given the lipid solution described in step (4) of Example 2, and the mice in the control group were given 0.9% sodium chloride aqueous solution;

[0282] (5) (Day 0) Each mouse was intratumorally administered with 0.5 mg / kg, 1 mg / kg, 1.5 mg / kg, or 2 mg / kg of T0009-1 Qtolimod, lipid solution, or saline (0.9% sodium chloride aqueous solution) in 50 μL (injected at two points depending on the situation), once every 3 days for a total of 5 injections. The day of the first injection was designated as Day 0.

[0283] (6) Observe and record the growth and proliferation of subcutaneous colon tumors on the right shoulder of tumor-bearing mice in the treatment group and the control group every day, including measuring the weight of the mice and measuring the tumor volume, calculating the tumor growth inhibition rate and drawing the tumor volume change curve (the results are shown in Figures 11-12);

[0284] (7) To understand whether this drug has an inhibitory effect on tumor recurrence, MC38 cells (inoculation size 10) were subcutaneously transplanted into the left shoulder of mice in the T0009-1Qtolimod (0.5 mg / kg, i.e., 0.5 mpk) group, the T0009-1 Qtolimod (1 mpk) group, the T0009-1Qtolimod (1.5 mpk) group, and the T0009-1 Qtolimod (2 mpk) group on Day 36. 6Due to animal deaths in the saline control and QTsome groups, a new control group was established for this study. This new control group consisted of six female C57BL / 6 mice of the same age as the mice in the T0009-1 Qtolimod groups described above. The same number of MC38 cells were inoculated in the same area on the left side of the mice in the new control group, but no drug was administered to these six female mice. After tumor inoculation, tumor growth was monitored in all mice, and tumor growth curves were plotted (the results are shown in Figures 13A-B).

[0285] 2. Experimental results

[0286] In an in vivo efficacy study in female C57BL / 6 mice inoculated with MC38 colorectal cancer cells, Qtolimod treatment began to demonstrate significant efficacy on Day 6. Calculated TGI% for each Qtolimod-treated group was greater than 63%. On Day 26, tumor volumes in the saline control group reached the humane endpoint. The remaining Qtolimod-treated groups (0.5 mpk, 1 mpk, 1.5 mpk, and 2 mpk) all demonstrated highly significant tumor suppression, with no significant differences. Calculated TGI% for the Qtolimod-treated groups (0.5 mpk, 1 mpk, 1.5 mpk, and 2 mpk) were 96.20%, 92.45%, 97.73%, and 98.82%, respectively. Monitoring of individual mouse tumor progression revealed that on Day 56, half of the mice in the 0.5 mpk group died due to relapse of their right shoulder tumors, while one-third of the mice showed continued right tumor reduction. In the 1.0mpk-dosing group, 1 / 3 of the mice died due to the re-exacerbation of the right shoulder tumor, while the right tumors of the remaining 2 / 3 mice continued to shrink. In the 1.5mpk and 2.0mpk-dosing groups, no mice died due to tumor deterioration. The right shoulder tumors of 5 mice in the 1.5mpk group were completely eliminated, and the right shoulder tumors of all 6 mice in the 2mpk group were completely eliminated. The above data show that the animal survival rate and tumor elimination rate are significantly dose-dependent with the drug. In the study on simulating tumor recurrence, except for some mice in the 0.5mpk and 1mpk groups who died due to the growth of the right tumor, the left tumors of almost all the mice in the dosing groups gradually disappeared after slow growth, suggesting that mice treated for right tumors produced tumor-specific memory immune cells (especially CD8+T cells) that can recognize and eliminate the same type of tumor cells in other parts of the body.

[0287] Example 13. Comparison of the content of Qtolimod and free CpG ODN in tumors after intratumoral injection

[0288] The test drugs in this experiment were T0009-1 Qtolimod and free T0009-1, prepared according to the method described in Example 2. This experiment used intratumoral administration to investigate drug retention within tumor tissue. Both T0009-1 Qtolimod and free T0009-1 were labeled with Cy5 (CpG ODN). The formulations were aliquoted in advance, stored at -20°C, and reconstituted on the day of use.

[0289] 1. Experimental plan

[0290] (1) Resuscitate MC38 cells and culture in vitro to obtain 5×10 6 cell;

[0291] (2) Two 7-week-old female C57BL / 6 mice were weighed after one week of adaptive feeding;

[0292] (3) Resuscitation and passage of mouse colon cancer MC38 cell line. When the cells are in good condition, MC38 cells (inoculation volume 10 6 The cells were transplanted subcutaneously into the right shoulder of mice, and the changes in the weight and tumor size of the mice were recorded;

[0293] (4) When the tumor reaches about 150 mm 3 At , mice were divided into T0009-1 Qtolimod group and free T0009-1 group, and intratumorally administered (1 mg / kg);

[0294] (5) In vivo imaging was performed using Tanon (ABL-X) at 0.5 h, 3 h, 1 day, and 3 days after administration (excitation light 630 nm, emission light 699 nm). The imaging results are shown in FIG14A .

[0295] 2. Experimental results

[0296] After labeling with cy5, the retention of free T0009-1 and T0009-1 Qtolimod inside the tumor was compared. According to Figure 14A-B, the content of free T0009-1 in the tumor tissue decreased rapidly after intratumoral administration. It decreased to 50% at 3 hours and only 8% at 72 hours. However, after intratumoral administration, the content of T0009-1 Qtolimod in the tumor tissue did not decrease at 3 hours, then decreased by 20% at 24 hours, and about 50% of the drug remained in the tumor tissue at 72 hours. The results show that QTsome encapsulated CpG ODN can significantly increase the retention of drugs in tumor tissues and significantly reduce the proportion entering the systemic circulation. It not only reduces systemic toxicity and improves drug safety, but also ensures the exposure of drugs in tumor tissues, achieves the effect of sustained drug release, and significantly improves the efficacy of tumor treatment.

[0297] Example 14. Study on the immune mechanism activated by the test drug

[0298] The test drug in this example was T0009-1 Qtolimod in Example 2, and the control group used normal saline or the lipid solution described in step (4) of Example 2. The animal experiment protocol was the same as that in Example 8.

[0299] I. Immunohistochemistry

[0300] After all the administrations were completed, the mouse tumors were isolated and immunohistochemistry experiments were performed as follows:

[0301] 1. Sample processing

[0302] The received tumor-bearing samples were further sampled and trimmed. The needle insertion site was used as the starting point and half of the tissue was cut along the largest surface for experiment. The mouse tissue was dehydrated using a Leica automatic dehydrator (Leica, HistoCore PEGASUS).

[0303] 2. Immunohistochemical staining of tumor-bearing tissues for F4 / 80, CD8 alpha, and CD11b

[0304] (1) The dehydrated tissue was paraffin-embedded and paraffin sections were prepared (4 μm sections, 5 sections per tissue) for immunohistochemistry.

[0305] (2) Immunohistochemical staining of mouse tumor-bearing tissue paraffin sections was performed using F4 / 80 (CST, 70076T), CD8 alpha (abcam, ab209775), and CD11b (abcam, ab133357) as primary antibodies. The treated tissue sample sections were added with primary antibodies. After incubation at room temperature for 1 hour, the sections were placed in 0.1% Triton TBS and washed on a shaker for 5 minutes, three times. Goat anti-rabbit secondary antibody (Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (Jacksonimmuno, 111-035-003), diluted 1:1000 in PBS) was used for incubation at room temperature for 30 minutes, and the sections were placed in 0.1% Triton TBS and washed on a shaker for 5 minutes, three times.

[0306] (3) Substrate color development: About 200 μL of DAB color developer (Fuzhou Maixin, DAB-1031, DAB kit (20x)) was added dropwise, color was developed at room temperature for 5 min, and then immediately immersed in tap water to terminate the reaction;

[0307] (4) Nuclear counterstaining and dehydration and mounting: After counterstaining the cell nucleus with hematoxylin for 1 min, dehydrate with gradient ethanol and xylene and manually mount the slides with neutral gum;

[0308] (5) All sections were digitally scanned (Olympus VS200), and the digital quantitative analysis software QuPath was used to analyze the tissue necrosis rate of any immunohistochemical (IHC) stained tissue scan (semi-automatically marking necrotic tissue in the anuclear area); QuPath was used to perform statistical analysis on the marked F4 / 80, CD8 alpha, and CD11b positive signals (brown).

[0309] 3. Data Analysis

[0310] GraphPad Prism software was used to calculate mean ± Sem, and differences were analyzed using a T-test. A P < 0.05 was considered significant between the two groups. The results are shown in Figures 15A-C.

[0311] II. After administration, the expression of cytokines IL-12, IL-10, IFN-γ, TNF-α, IL-6, and IL-1β in mouse blood and tumor tissue samples was detected by ELISA.

[0312] 1. Sample processing

[0313] Blood was collected from the hearts of mice after administration of T0009-1 Qtolimod, saline, or lipid solution. The blood was allowed to stand at room temperature for 1 hour, and then the blood was heated at 5500 rpm for 20 minutes to collect serum samples. The mouse tumors were removed and minced into about 1 mm with surgical scissors. 3 After sizing, 2 mL of PBS was added and vortexed at 2000 rpm for 2 min, and the supernatant was collected as the tumor sample.

[0314] 2. ELISA test

[0315] The ELISA test kit was provided by QuanLab.CUSABIO. The experimental method is as follows:

[0316] (1) Move all reagents to room temperature (18-25°C) and equilibrate for at least 30 minutes. Prepare the reagents according to the kit instructions for use. Sample addition: set up blank wells (S0), standard wells (two replicates), and test sample wells. Add 100 μL of the standard or test sample (serum sample, tumor sample) in the kit to each well. Add an equal amount of sample diluent to the blank well (S0), gently shake to mix, cover with a plate sticker, and incubate at 37°C for 2 hours.

[0317] (2) Discard the liquid, spin dry, and do not wash;

[0318] (3) Add 100 μL of biotinylated antibody working solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour;

[0319] (4) Discard the liquid in the wells, spin dry, and wash the plate three times. Soak for 2 minutes each time, 200 μL / well, and spin dry.

[0320] (5) Add 100 μL of horseradish peroxidase-labeled avidin working solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour;

[0321] (6) Discard the liquid in the wells, spin dry, and wash the plate 5 times. Soak for 2 minutes each time, 200 μL per well, and spin dry.

[0322] (7) Add 90 μL of substrate solution to each well and incubate at 37°C in the dark for 15-30 minutes.

[0323] (8) Add 50 μL of stop solution to each well to terminate the reaction;

[0324] (9) Within 5 minutes after the reaction, the optical density (OD value) of each well was measured in sequence using a microplate reader at a wavelength of 450 nm.

[0325] 3. Data processing

[0326] Subtract the S0 value from the standard and sample values ​​and plot the curves. Average the replicates and calculate the concentration. After plotting the standard curve using relevant software, substituting the measured OD values ​​into the calibration curve formula, calculate the sample concentration. The results are shown in Figures 16A-B.

[0327] III. Experimental Results

[0328] Immunohistochemistry results showed that compared with the saline control group, the infiltration of macrophages and CD8+ T cells in the tumor site of the drug-treated group increased significantly, playing a role in directly killing the tumor, clearing tumor tissue fragments, and stimulating adaptive immunity through antigen presentation. The infiltration of CD11b-positive cells (monocytes, granulocytes, activated lymphocytes, NK cells, dendritic cells) also increased, playing an important role in inducing efficient and specific cellular immune responses to tumor-associated antigens and recruiting immune cells. Cytokine detection results showed that compared with the saline and LNP control groups, IL-12, IL-10, IFN-γ, and TNF-α in the Qtolimod-treated group were significantly upregulated in tumor tissue, while IL-1β and IL-6 did not change significantly, and their expression levels in the blood were all below the detection limit. IL-12 promotes the proliferation and cytotoxic activity of NK cells and CD8+T cells; induces Th1 differentiation and IFN-γ secretion, promoting antigen presentation; IL-10 is produced by antigen-presenting cells, promotes CD8+T cell activation and inhibits the expression of inflammatory cytokines such as TNF-α, IL-6 and IL-1 by activating macrophages; 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, and indirectly by making tumor cells susceptible to immune responses that induce apoptosis.

[0329] Example 15. Study on the impact of delivery system on safety

[0330] In order to study the effect of the QTsome delivery system on the safety of the CpG ODN, this experiment set up a solvent control group, a QTsome group, an active pharmaceutical ingredient (API) group, and a Qtolimod group. The solvent control group used normal 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 weight changes of the mice after administration were observed.

[0331] 1. Experimental plan

[0332] (1) Each group consisted of 6 CD1 (ICR) mice (Wei Tonglihua, half male and half female) aged 6-8 weeks, weighing 20-30 g;

[0333] (2) Saline, lipid solution, free T0009-1, or T0009-1 Qtolimod were administered subcutaneously (SC) on days 1, 8, 15, 22, and 29, once weekly (QW) for a 29-day cycle, for a total of five doses. The dose was 20 mg / kg. The lipid content in the QTsome group was the same as that in the Qtolimod group. The day of the first SC injection was designated Day 1.

[0334] 2. Experimental results

[0335] As shown in Figure 17, except for the mice in the API group, all other mice tolerated the drug well during the experiment, with no significant weight loss. However, mice given free T0009-1 experienced weight loss after dosing, and one male and two female mice died on day 12, accounting for 50% of the total. Autopsy and weighing of the remaining surviving mice in the API group revealed significantly paler kidneys and slightly lighter weights, and significant liver and spleen enlargement. Male liver weights were 2.5 times, and spleen weights were 5.4 times, compared to those in the vehicle control group; female liver weights were 3 times, and spleen weights were 6.8 times. At the endpoint (after five doses), autopsy of Qtolimod-treated mice revealed that male liver weights were 1.7 times, and spleen weights were 2.9 times, compared to those in the vehicle control group; and female liver weights were 1.7 times, and spleen weights were 3.4 times, compared to those in the vehicle control group. Based on these experimental results, the QTsome delivery system can effectively improve the safety of this product.

[0336] Example 16. Efficacy of the test drug in treating the mouse melanoma B16-Luc metastasis model

[0337] In this experiment, the test drug, T0009-1 Qtolimod, was prepared by encapsulating T0009-1 with lipid nanoparticles using the methods described in Examples 2-5. Subcutaneous and systemic metastatic mouse models were established by subcutaneously transplanting murine melanoma B16-Luc cells into the tail vein of each mouse. The therapeutic effects of the test drug in both subcutaneous and systemic melanoma B16-Luc homologous transplant models were evaluated.

[0338] 1. Construction of Subcutaneous Tumor and Metastasis Models

[0339] Mouse melanoma B16-Luc cells were distributed in several tubes with a bottom area of ​​75 cm 2 The cells were placed in a culture flask and cultured according to conventional methods, using DMEM containing 10% fetal bovine serum in a constant temperature and humidity incubator at 37°C and 5% CO2. When the cells were in the logarithmic growth phase, they were digested with 0.25% trypsin. Digestion was terminated after the cells detached 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. The suspension was adjusted to the desired cell concentration for animal transplantation. The cells were resuspended in sterile PBS buffer and Matrigel at a ratio of 1:1 to 1×10 7 / ml, 0.1ml / mouse was subcutaneously implanted into the left armpit of C57BL / 6J mice to establish a subcutaneous tumor transplant mouse model, and the cells were resuspended in sterile PBS buffer to 5×10 6 / mL, and 0.2 ml / mouse was injected into C57BL / 6J mice via tail vein to establish a systemic metastatic tumor mouse model.

[0340] 2. Subcutaneous Tumor and Systemic Metastasis Experiments

[0341] T0009-1 Qtolimod preparations were pre-packaged and stored at -20 degrees Celsius. They were reconstituted on the day of use. 3Around 6:00 p.m., tumor-bearing mice with healthy, well-growing, non-ulcerated tumors were selected. Mice with tumors that were either too large or too small were removed, leaving the remaining mice equal to the enrollment number. The mice were randomly assigned to two groups using a complete randomization method: the T0009-1 Qtolimod 1.5 mg / kg group and the saline control group, with 16 mice in each group. The T0009-1 Qtolimod 1.5 mg / kg group received 1.5 mg / kg of T0009-1 Qtolimod, while the control group received 0.9% sodium chloride solution. The day of grouping was designated Day 1 (D1). Drug was administered at a dose of 2.5 ml / kg of body weight every three days, on Days 1, 4, 7, and 10, for a total of four doses. Drug administration was via subcutaneous intratumoral injection. Mice in the saline control group received an equal volume of saline.

[0342] The experimental results showed that one animal died in the saline control group on days 8, 9, 10, and 11 after dosing, while no animals in the T0009-1 Qtolimod 1.5mg / kg group died. The survival status of the animals in each group is shown in Figure 18. The health, mortality, mental state, behavioral activity, fecal characteristics, and other abnormalities of all animals in the T0009-1 Qtolimod 1.5mg / kg group were observed daily before, during, and until euthanasia. No significant abnormalities were observed.

[0343] Changes in animal body weight during the dosing period are shown in Table 3 and Figure 19. Before the first dose (at the time of grouping), there were no significant differences in animal body weight among the groups (P>0.05). On days 3, 5, and 9 after dosing, and at the end of the experiment (D11), the body weight of the animals in the T0009-1 Qtolimod 1.5 mg / kg group decreased significantly compared to the saline control group (D3: P<0.05, D5: P<0.05, D9: P<0.001, and end of the experiment: P<0.001).

[0344] Table 3 Effect of T0009-1 Qtolimod on the body weight of animals in the mouse melanoma B16-Luc homograft model

[0345] * indicates P < 0.05, *** indicates P < 0.001

[0346] Changes in subcutaneous tumor volume during the dosing period are shown in Table 4 and Figure 20. Before the first dose (at the time of grouping), there were no significant differences in subcutaneous tumor volume between the groups (P>0.05). On days 5, 7, and 9 after dosing, and at the end of the experiment, the subcutaneous tumor volume in the T0009-1 Qtolimod 1.5 mg / kg group was significantly reduced compared to the saline control group (P<0.001).

[0347] Table 4 Effects of T0009-1 Qtolimod on subcutaneous tumor volume in the murine melanoma B16-Luc homograft model

[0348] ***P < 0.001

[0349] The changes in subcutaneous tumor weight during the administration period are shown in Figures 1 to 22. Compared with the saline control group, the subcutaneous tumor weight of animals in the T0009-1 Qtolimod 1.5 mg / kg group was significantly reduced (***P<0.001), and the tumor inhibition rate was 90.43%.

[0350] The tumor inhibition rate was calculated based on the weight of subcutaneous tumors according to the following formula: Tumor inhibition rate = (tumor weight of negative control group - tumor weight of drug-treated group) / tumor weight of negative control group × 100%. The relative tumor volume (RTV) was calculated as: V t / V0, where V0 is the tumor volume at the time of grouping, V t The tumor volume at each measurement. The anti-tumor efficacy of the compound was evaluated by T / C (%). Relative tumor growth rate T / C (%) = T RTV / C RTV *100%. (T RTV :RTV in treatment group; C RTV : Negative control group RTV). In principle, the evaluation criteria are: T / C (%) > 40% is invalid; T / C (%) ≤ 40%, and P < 0.05 after statistical analysis is valid.

[0351] The effects of T0009-1 Qtolimod on tumor metastasis in various animal tissues are as follows. Following tail vein homologous transplantation of mouse B16-Luc cells, visual inspection and counts revealed that the primary tissue for metastasis was the lungs, with the spleen, kidneys, spine, femur or tibia, ovaries, abdominal cavity or intraperitoneal fat, medial or lateral abdominal wall, and intestines also involved. The number of metastatic nodules in the lungs was high. Following subcutaneous intratumoral injection, the number of metastatic nodules was significantly reduced in the T0009-1 Qtolimod 1.5 mg / kg group compared to the saline control group (**P < 0.01). Furthermore, the total number of metastatic nodules in all tissues in the T0009-1 Qtolimod 1.5 mg / kg group was significantly reduced (**P < 0.01). The results are shown in Figures 23-25.

[0352] Under the conditions of this experiment, four subcutaneous and intratumoral injections of T0009-1 Qtolimod (1.5 mg / kg) were administered once every three days. No animals died, but weight decreased, which analysis suggests was likely due to subcutaneous tumor shrinkage. T0009-1 Qtolimod (1.5 mg / kg) significantly reduced tumor volume, relative tumor volume, and weight in subcutaneous B16-Luc mouse melanoma homografts, demonstrating a strong inhibitory effect on subcutaneous tumor growth in B16-Luc mouse melanoma homografts. Intratumoral administration of T0009-1 Qtolimod (1.5 mg / kg) significantly reduced the number of lung metastatic nodules and the total number of metastatic nodules in all tissues in B16-Luc mouse melanoma homografts via the tail vein, demonstrating a strong inhibitory effect on systemic metastasis in B16-Luc mouse melanoma homografts. In summary, under the experimental conditions described above, T0009-1Qtolimod, administered subcutaneously and intratumorally at a dose of 1.5 mg / kg every three days for a total of four doses (D1, D4, D7, and D10), demonstrated significant inhibition of subcutaneous tumor growth and systemic metastases in murine melanoma B16-Luc homografts, while also prolonging animal survival. The above dosing regimen for mice can be translated to other animal models, such as humans, based on prior knowledge in the art.

[0353] Example 17. Efficacy of the Test Drug in Treating the Mouse Head and Neck Cancer SCC-7 Subcutaneous Tumor Model

[0354] In this experiment, the method of Examples 2-5 above was used to encapsulate T0009-1 using lipid nanoparticles, and intratumoral administration was used to study its effect on tumor growth.

[0355] T0009-1 Qtolimod preparation was pre-packaged and stored at -20 degrees. It was reconstituted on the day of use. SCC-7 cells (stored by Hangzhou Moslet Biotechnology Co., Ltd.) were revived and cultured in vitro to obtain 5×10 6 Cells. After one week of adaptive feeding, 24 7-week-old female C3H mice were weighed. The mouse head and neck cancer SCC-7 cell line was revived and passaged. When the cells were in good condition, SCC-7 cells (inoculation volume 2e5) were transplanted subcutaneously on the right shoulder of the mice. Changes in mouse body weight and tumor size were recorded (tumor-bearing mouse models were constructed by subcutaneously implanting SCC-7 cells on the back). When the tumor grew to approximately 80 mm 3 The mice were divided into groups according to their size. The group and dosing information is shown in Table 5.

[0356] Table 5: Grouping and dosing of mice

[0357] Each mouse in G1-G2 received 50 μL of intratumoral administration (injected at two different sites, depending on the situation) at a dose of 1.5 mpk for a total of five injections, with the first injection occurring on day 0. In G3, a 100 μL dose of 10 mpk of PD-1 monoclonal antibody was administered subcutaneously. The growth and proliferation of subcutaneous colon tumors on the backs of tumor-bearing mice in the experimental and control groups (G1 and G3) were observed and recorded daily, including measurement of mouse body weight and tumor volume. Tumor growth inhibition rates (TGI) were calculated, and tumor growth curves were plotted in Figures 26 and 27. Under these experimental conditions, after subcutaneous intratumoral administration of PD-1 monoclonal antibody to mice bearing head and neck cancer cells (SCC-7), on day 17, the PD-1 monoclonal antibody in the G3 group had no significant inhibitory effect on tumor growth (TGI% = 21.18%). However, T0009-1 Qtolimod significantly inhibited tumor growth in the G2 group (TGI% = 57.28%). Furthermore, the animals experienced a slight decrease in body weight during the administration period, which was likely due to tumor shrinkage.

[0358] Example 18. Pharmacodynamic study of the test drug in the EMT-6 and EMT-6-HPV18 subcutaneous transplantation models of Balb / c female mice

[0359] This study used the methods described in Examples 2-5 above to prepare Qtolimod using lipid nanoparticles to encapsulate T0009-1. The drug was administered intratumorally to investigate its efficacy in the EMT-6 and EMT-6-HPV18 subcutaneous transplant models of Balb / c female mice.

[0360] T0009-1 Qtolimod preparation should be aliquoted in advance, stored at -20 degrees, and reconstituted on the day of use.

[0361] Mouse breast cancer cells EMT-6 and mouse breast cancer HPV-positive cells EMT-6-HPV18 (both preserved by Nantong WuXi AppTec Pharmaceutical Technology Co., Ltd.) were cultured in monolayer in vitro. The culture conditions were based on the supplier's technical instructions or references. Routine digestion and passage were performed with trypsin-EDTA twice a week. When the cell saturation was 80%-90% and the number reached the requirement, the cells were counted and inoculated. Tumor inoculation and grouping criteria: EMT-6 cells (5×10^5 cells+Matrigel / 200μL, PBS:Matrigel=1:1) or EMT-6-HPV18 cells (5×10^5 cells+Matrigel / 200μL, PBS:Matrigel=1:1) were subcutaneously inoculated into the right back of each mouse. The average tumor volume reached approximately 100mm 3 The group dosing information is summarized in Table 6.

[0362] Table 6 Grouping and dosing of mice

[0363] When the subcutaneous tumor volume reaches about 100mm 3 Around 14 days, tumor-bearing mice with good tumor growth, no ulceration, and healthy growth were randomly divided according to subcutaneous tumor volume (see Example 16). The day of grouping was designated as Day 0, and dosing began according to animal body weight. The positive drug PD-1 monoclonal antibody was administered twice a week for a total of 6 doses, and the other drugs were administered once a week for a total of 4 doses, all by subcutaneous intratumoral injection at a rate of 2.5 ml / kg body weight. The negative saline control group was given an equal amount of saline.

[0364] The experimental results are shown in Figures 28A-D. Under the conditions of this experiment, mice with breast cancer EMT6 were injected subcutaneously into the tumor 4 times, once a week (positive drug PD-1 monoclonal antibody was administered twice a week for a total of 6 times). During the administration period, mice in the saline group, QTsome group, and PD-1 monoclonal antibody group all died, of which 2 mice died in the saline group, and one mouse died in the QTsome group and PD-1 monoclonal antibody group. No animals died in the Qtolimod 0.1mg / kg, 0.3mg / kg, 1mg / kg, and 3mg / kg groups, and the weight of the animals decreased during the administration period, which was likely due to the reduction of the subcutaneous tumor. Qtolimod 0.1mg / kg (mpk) significantly reduced the tumor volume and relative tumor volume of subcutaneous tumors of mouse breast cancer EMT6 homografts, and had a good inhibitory effect on mouse breast cancer EMT6 homografts. On day 18 of administration, Qtolimod 0.1mg / kg, 0.3mg / kg, 1mg / kg, and 3mg / kg inhibited EMT6 mouse breast cancer tumor growth by 49.9%, 67.9%, 68.4%, and 89.8%, respectively, all exceeding the tumor inhibition effect of PD-1 monoclonal antibodies. On day 38 of administration, all mice in the saline and QTsome groups had died, while 2 mice in the positive drug PD-1 monoclonal antibody group survived, 2 mice in the Qtolimod 0.1mpk group survived, 5 mice in the 0.3mpk group survived, 5 mice in the 1mpk group survived, and all mice in the 3mpk group survived. Therefore, Qtolimod has a significant inhibitory effect on subcutaneous EMT6 mouse breast cancer homografts and can also prolong the survival of animals.

[0365] This study also investigated the EMT6-HPV18 mouse breast cancer model, which harbors HPV18 antigen fragments. The drug was administered via subcutaneous intratumoral injection four times weekly, while the active agent mPD-1 was administered twice weekly for a total of six doses. During the dosing period, one mouse died in the QTsome group, while no animals died in the saline, mPD-1, or Qtolimod 0.1mg / kg, 0.3mg / kg, 1mg / kg, or 3mg / kg groups. The animals also experienced weight loss during the dosing period, likely due to shrinkage of the subcutaneous tumors. Qtolimod 0.1mg / kg significantly reduced the tumor volume and relative tumor volume of subcutaneous tumors transplanted from mouse breast cancer EMT6 homologous transplants, and had a good inhibitory effect on subcutaneous tumors transplanted from mouse breast cancer EMT6 homologous transplants. On day 21 of administration, Qtolimod 0.3mg / kg, 1mg / kg, and 3mg / kg inhibited EMT6 mouse breast cancer tumor growth by 55.1%, 75.2%, and 85.3%, respectively, all exceeding the tumor inhibition effect of PD-1 monoclonal antibodies. On day 38 of administration, all mice in the saline group, QTsome group, and Qtolimod 0.1mpk group had died. One mouse survived in the positive drug PD-1 monoclonal antibody group, two survived in the Qtolimod 0.3mpk group, four survived in the 1mpk group, and all survived in the 3mpk group.

[0366] By comparing the efficacy results of two mouse breast cancer subcutaneous tumor models, EMT6 and EMT6-HPV18, it was found that tumor cells expressing or not expressing HPV antigens showed significant tumor inhibition effects after treatment with Qtolimod.

[0367] Example 19. Efficacy of the Test Drug in Treating the Balb / c Mouse Osteosarcoma K7M2 Subcutaneous Tumor Model

[0368] This experiment used the methods described in Examples 2-5 above to prepare Qtolimod using lipid nanoparticles to encapsulate T0009-1. The drug was administered intratumorally to explore its efficacy in a subcutaneous transplant model of Balb / c female K7M2 mice.

[0369] The preparation should be divided into smaller packages in advance, stored at -20 degrees, and reconstituted on the day of use.

[0370] Mouse osteosarcoma K7M2 cells (stored by Nantong WuXi AppTec Pharmaceutical Technology Co., Ltd.) were cultured in a monolayer in vitro. Culture conditions were based on the supplier's technical specifications or references. Twice a week, routine digestion and passage were performed using trypsin-EDTA. When the cell saturation reached 80%-90% and the number reached the required level, the cells were harvested, counted, and inoculated. Tumor inoculation and grouping criteria: 1×10^6 K7M2 cells were subcutaneously inoculated into the right back of each mouse. The average tumor volume reached approximately 110 mm 3The group dosing information is summarized in Table 7.

[0371] Table 7 Grouping and Dosing of Mice

[0372] The subcutaneous tumor volume reached about 110 mm 3 Around 6:00 p.m., tumor-bearing mice with healthy, well-growing, non-ulcerated tumors were randomly divided according to subcutaneous tumor volume. Dosing began according to animal body weight, with grouping date designated as Day 0. Dosing was once weekly for a total of three doses, while the active PD-1 monoclonal antibody was administered twice weekly for a total of six doses. Dosing was administered via subcutaneous intratumoral injection at a rate of 2.5 mL / kg body weight. A negative saline control group received an equal volume of saline.

[0373] The results are shown in Figures 29A-C. Under the experimental conditions, mice with osteosarcoma K7M2 were injected subcutaneously into the tumor three times, once a week, while the positive drug PD-1 monoclonal antibody was administered twice a week for a total of 6 times. During the administration period, no animals died in the Qtolimod 0.3mg / kg, 1mg / kg, and 3mg / kg groups, and the weight of the animals decreased slightly during the administration period, which may be due to the reduction of subcutaneous tumors. Qtolimod 0.1mg / kg can significantly reduce the tumor volume and relative tumor volume of subcutaneous tumors of mouse osteosarcoma K7M2 homologous transplants, and has a good inhibitory effect on subcutaneous tumors of mouse osteosarcoma K7M2 homologous transplants. Among them, on the 19th day of administration, the tumor growth inhibition rates of Qtolimod 0.3mg / kg, 1mg / kg, and 3mg / kg on mouse osteosarcoma K7M2 were 55.1%, 78.6%, and 92.2%, respectively, which were all higher than the tumor inhibition effect of PD-1 monoclonal antibody (TGI% = 28.7%). On the 35th day after grouping, all mice in the normal saline group, QTsome group, positive drug mPD-1 group, and Qtolimod 0.3mpk group had died, 2 mice in the 1mpk group survived, and all mice in the 3mpk group survived with an average tumor size of 539mm. 3 Therefore, Qtolimod has a significant inhibitory effect on subcutaneous mouse osteosarcoma K7M2 homografts and can also prolong the survival of animals.

[0374] Example 20. Study on the mechanism of action of the test drug at different time points in the treatment of the C57BL / 6J mouse MC38 subcutaneous tumor model

[0375] This experiment used the methods described in Examples 2-5 above to prepare Qtolimod using lipid nanoparticles to encapsulate T0009-1. Intratumoral administration was used to investigate the distribution of immune cells at the administration site and in the spleen at different time points after administration.

[0376] The preparation was aliquoted in advance and stored at -20 degrees. It was reconstituted on the day of use. MC 38 cells were revived and cultured in vitro to obtain 5×10 6 Cells. 216 7-week-old female C57BL / 6 mice were weighed after one week of adaptive feeding. Mouse colon cancer MC38 cell line was revived and passaged. When the cells were in good condition, MC38 cells (inoculation size 1×10 6 ) were transplanted subcutaneously on the right shoulder of mice, and the changes in mouse body weight and tumor size were recorded (the tumor-bearing mouse models were constructed by subcutaneously implanting MC38 cells on the back). The tumor volume reached about 100 mm 3 The group dosing information is summarized in Table 8.

[0377] Table 8 Grouping and Dosing of Mice

[0378] Each mouse was intratumorally administered 50 μL of the drug (injected at two different sites depending on the situation) at a dose of 1.5 mpk, once every three days for a total of five injections, with the first injection being on Day 0. Within 6, 24, and 48 hours after each administration, three mice with large, medium, or small tumors were euthanized, respectively. Tumor tissue and spleen samples were collected for RT-qPCR analysis.

[0379] 1. RNA Extraction: 100 mg of liver tissue was added to grinding beads and 500 μL of Trizol lysis buffer. Grind in a grinder at 65 Hz / 5 min. Add 900 μL of Trizol lysis buffer to 100 μL of grinding buffer and lyse for 10 min. Add 200 μL of chloroform to each tube, mix thoroughly, and incubate on ice for 10 minutes to completely dissociate the nucleoprotein complex. Centrifuge at 13,000 rpm for 15 minutes at 4°C. During this time, add 500 μL of isopropanol to a new EP tube and pre-chill on ice. After centrifugation, transfer the upper aqueous phase (approximately 500 μL) to the new EP tube. Incubate on ice and precipitate with an equal volume of isopropanol for 10 minutes. Centrifuge at 13,000 rpm for 10 minutes. Remove the supernatant and wash the RNA pellet once with 1 mL of 75% ethanol and centrifuge at 12,000 rpm for 5 minutes. Discard the supernatant and air-dry or vacuum-dry the RNA pellet for 5-10 minutes. Dissolve the RNA in 30-50 μL of DEPC-treated deionized water, adding more water depending on the amount of RNA precipitate. Next, perform spectrophotometric analysis to determine sample concentration and purity.

[0380] 2. Reverse Transcription: Reverse transcribe RNA into cDNA using the following materials and procedures. To remove genomic DNA, prepare the following mixture in an RNase-free centrifuge tube (all reagents are from the Novozymes kit, R312-01): 2 μL of 5×g DNA wiper mix, RNase-free ddH₂O to 10 μL, and 1 μg of total RNA. Gently pipette to mix thoroughly and incubate at 42°C for 2 min. Prepare the first-strand cDNA synthesis reaction mixture by preparing the following mixture in an RNase-free centrifuge tube (all reagents are from the Novozymes kit, R312-01): 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)20VN, 1 μL of random hexamers, and 4 μL of RNase-free ddH₂O. Gently pipette to mix thoroughly. Incubate at 37°C for 15 minutes and 85°C for 5 seconds to obtain first-strand cDNA. Thaw and gently mix the following reagents: 2× qPCR Mix (abm, G891) and PCR Primers (Hangzhou Leqi, MQP026401, MQP103938, MQP076780, MQP101232, MQP093050, MQP091959, MQP031788, MQP093658, MQP072830, MQP025979, MQP091399, MQP029630, MQP028834, MQP025254, MQP077323, MQP076358, MQP092655, MQP027158). Prepare the qPCR reaction system on ice according to Table 9.

[0381] Table 9 qPCR reaction system

[0382] Gently mix the qPCR reaction system and centrifuge briefly. Set up the three-step qPCR program according to Table 10 below or use the Roche L96 qPCR quantifier to set the program. Data processing: 2 -ΔΔCt .

[0383] Table 10 Three-step qPCR program

[0384] Mice were intratumorally administered 1.5 mpk of the drug every three days for five days. Samples were collected within 6, 24, and 48 hours of each administration for the first four doses, and within 6, 24, 48, and 96 hours after the fifth dose. Tumor and spleen tissues were euthanized and analyzed for various immune cell markers using RT-qPCR to analyze immune cell activation in mice following administration. The results are shown in Figures 30-31. Monocytes and macrophages in tumor tissue were significantly upregulated (approximately 60-fold) after administration, with this increase becoming more pronounced with increasing dosing frequency. Macrophages are widely involved in immune responses, immune effects, and immune regulation, and play a key role in cancer treatment. Activated macrophages can effectively kill tumor cells, phagocytose tumor cells, secrete cytotoxic cytokines that directly or indirectly kill tumor cells, and produce certain enzymes and reactive oxygen species that directly kill or inhibit tumor cell growth. Simultaneously, spleen analysis showed no significant increase in monocytes and macrophages after administration, indicating that monocyte and macrophage activation is concentrated at the site of administration. Qtolimod can activate plasmacytoid dendritic cells (pDC). The activated pDC recruits a large number of T cells into the tumor microenvironment by producing a large amount of IFN-α and other cytokines, thereby playing a role in killing tumors. Experimental results found that intratumoral administration induced a significant upregulation of plasmacytoid dendritic cells (pDC), while immune cells such as macrophages, CD3+T cells, CD8+T cells, and NK cells were significantly activated, and granzyme B (GZMB) was released to promote the killing process of tumor cells. At the same time, macrophages phagocytize tumor cell fragments, promote tumor antigen presentation and anti-tumor cell immune activation. In contrast, various immune cells in the spleen are always in an unactivated or slightly activated state after administration, indicating that Qtolimod induces anti-tumor immune responses locally in the tumor, and the risk of causing systemic side effects is relatively low.

[0385] Example 21. Study on immune activation after in vitro stimulation of mouse PBMC cells and human PBMC cells by test drugs

[0386] In this experiment, Qtolimod was prepared using the methods described in Examples 2-5 above, using lipid nanoparticles encapsulating T0009-1. PBMCs were stimulated with the prepared Qtolimod in vitro, and cell samples were collected 24 hours later for proteomic sequencing analysis. The Qtolimod formulation was aliquoted and stored at -20°C before reconstitution on the day of use.

[0387] Human PBMC cells were purchased from Maishun Biotechnology Co., Ltd. in Zhejiang Free Trade Zone. The steps for separating mouse PBMC cells are as follows: take a 10ml sterile siliconized centrifuge tube, add 4ml of separation solution, and then slowly add 0.5-2.0ml of blood sample. The blood sample is carefully added to the interface of the separation liquid. Centrifuge at 450g for 30min. After centrifugation, carefully aspirate the ring-shaped milky white mononuclear cell layer in the centrifuge tube and transfer it to a new centrifuge tube. Add 10ml to the centrifuge tube containing mononuclear cells to wash and mix the cells. Centrifuge at 250g for 10min. Discard the supernatant. Wash and resuspend the obtained cells with 5ml PBS. Centrifuge at 250g for 10min and discard the supernatant. Repeat the washing twice, discard the supernatant, and resuspend the obtained cells with 0.5mL RPMI-1640 culture medium. Mouse and human PBMC cells were cultured at 1×10 6 / well, plated in a 6-well plate, and 5 μg Qtolimod was added to stimulate the cells. The cells were collected after 24 hours and sent to Jingjie Bio for 4D-Smart DIA proteomics sequencing analysis. The results are shown in Figure 32. The test drug stimulated human and mouse PBMC cells in vitro, and the cells were collected after 24 hours for protein sequencing analysis. The results showed that compared with the normal saline (Vehicle) group, Qtolimod was able to activate immune cells to a certain extent, including B cell pathway genes, T cell pathway genes, myeloid lymphocyte pathway genes, and lymphocyte pathway genes, and there was no significant difference in the degree of immune cell activation between humans and mice.

[0388] 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 lymphocytes (Myeloid cell) pathway representative genes (mouse: Mndal, Mlf2, Myd88) (human: MYADM, MNDA, CD33, MLF2, MYD88, MYDGF); lymphocyte (Lymphocyte) pathway genes (mouse: Ly6d, Lsp1, Ly9, Cd19) (human: LCP2, CD19, BTLA, LSP1, LY75).

[0389] Example 22. Study on the efficacy of the test drug on the orthotopic bladder cancer (MB49) model

[0390] In this study, Qtolimod was prepared using lipid nanoparticles encapsulating T0009-1, following the methods described in Examples 2-5. An orthotopic bladder cancer mouse model was established by transplanting MB49 bladder cancer cells into the bladder mucosa. The therapeutic effect of Qtolimod in the MB49 orthotopic bladder cancer homograft model was evaluated by intravesical administration.

[0391] Qtolimod preparations were packaged in advance, stored at -20 degrees, and reconstituted on the day of use. Mouse MB49-Luc cells were cultured in vitro in high-glucose DMEM medium at 37°C and 5% CO2. Routine culture medium replacement and digestion and subculture were performed 1-2 times a week. When the cell number reached the requirement, cells in the logarithmic growth phase were collected, counted, and inoculated. Mouse bladder cancer MB49-Luc cells in the logarithmic growth phase were digested with 0.5% trypsin and resuspended to 4×10 7 / ml, 50μl / mouse was injected into the bladder mucosa of C57BL / 6J mice to establish a bladder orthotopic transplanted tumor mouse model. Five days after modeling, all mice were injected with the substrate potassium luciferin for in vivo imaging, and healthy tumor-bearing mice were selected for grouping. They were randomly divided into groups according to the fluorescence intensity value (see Table 9 for details), so that the intra-group difference in the fluorescence intensity value of the tumor in each group did not exceed 10 times. Based on the touch situation, animals with abnormally large orthotopic tumors were eliminated. The drug was administered the next day (the 6th day after modeling), and the day of administration was designated as Day 1 (D1). The drug was administered at 50μL / mouse, and was administered once every 7 days for a total of 3 times (D1, D8, D15), all by bladder instillation. The normal saline control group was given an equal amount of normal saline.

[0392] For tumor monitoring, all animals were monitored before grouping, and the enrolled animals were monitored after grouping. Tumor status was monitored at the time of grouping (i.e., before the first dose), every 7 days after grouping, and before euthanasia (D1, D7, D14, D21). The fluorescence intensity of the in situ bladder cancer was monitored by in vivo imaging, and the differences in in situ tumor growth changes between the groups were compared. At the same time, the grade and size of the in situ tumor were determined by palpation twice a week after administration and before euthanasia (D1, D5, D8, D12, D15, D19, D22, D35). Tumors are divided into 4 grades according to size: Grade 0, almost no lump can be felt; Grade 1, a very small lump can be felt (the tumor diameter is estimated to be within 2mm); Grade 2, a medium-sized lump can be felt (the tumor diameter is estimated to be 2-5mm); Grade 3, a larger lump can be felt (the tumor diameter is estimated to be 5-10mm); Grade 4, an extra-large lump can be felt (the tumor diameter is estimated to be more than 10mm). The size of the in situ tumors was compared among the groups by palpating the size grade of the tumors.

[0393] The results are shown in Figure 33. Under the conditions of this experiment, the drug was administered intravesically once a week for a total of three times. Since the in situ transplanted tumors grew rapidly and the fluorescent substrate could not enter the interior of the tumor in the later stage, touch was used for observation during the observation period. The anatomical observation on the 35th day of the administration period was consistent with the touch situation. The tumor diameter of the animals in the normal saline control group reached 1.7 cm and the shape was oval. The tumor growth caused the bladder to expand, and there was necrosis and congestion inside the tumor. At the same time, metastases appeared in the kidney and large intestine respectively. The tumor diameters of the Qtolimod 4mpk administration group were 0.5 cm and 0.7 cm respectively, and the shape was round, and no metastasis was found. This shows that the test drug significantly inhibited tumor growth and alleviated the behavioral effects of mice caused by bladder tumor growth.

[0394] The above are only some embodiments of the present disclosure. For those skilled in the art, several modifications and improvements can be made without departing from the inventive concept of the present disclosure, and these all fall within the scope of protection of the present disclosure.

Claims

1. A Toll-like receptor agonist, characterized in that Contains any of the following (a)-(c): (a) any one of the modified or unmodified nucleotides shown in SEQ ID NOs: 1-4; (b) a functional variant of any one of the modified or unmodified nucleotides shown in SEQ ID NOs: 1-4; (c) a pharmaceutically acceptable salt of the nucleotide described in (a) or (b); The nucleotides are all or partly ribonucleotides or all or partly deoxyribonucleotides.

2. The Toll-like receptor agonist according to claim 1, characterized in that The modification includes one or more of the following items: replacing the P=O bond between all or part of two adjacent nucleotides in the sequence with a P=S bond; replacing all or part of the deoxyribonucleosides in the sequence with ribonucleosides, and replacing all or part of the ribonucleosides in the sequence with deoxyribonucleosides; replacing the 2'-OH group of the ribonucleoside or deoxyribonucleoside with a methoxy group, a methoxyethyl group or a halogen; bridging the second and fourth carbon atoms of the nucleotide to form a locked nucleic acid or (S)-cEt-BNA.

3. An active ingredient encapsulated by a lipid component, characterized in that The active ingredient comprises the Toll-like receptor agonist according to claim 1 or 2.

4. The active ingredient encapsulated by the lipid component according to claim 3, 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.

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 particle (LNP), cationic liposome, QTsome, lipopolyplex, microparticle, microsphere, nanoemulsion.

6. The active ingredient encapsulated by the lipid component according to claim 3, characterized in that: The lipid component comprises one or more of cationic lipids, ionizable lipids, neutral lipids, and polyethylene glycol lipids (PEGylated lipids).

7. The active ingredient encapsulated by the lipid component according to claim 6, characterized in that: The lipid component includes 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); preferably, the lipid component includes 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.

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

2.

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

10. The method for preparing the active ingredient encapsulated by the lipid component according to claim 3, characterized in that: The method comprises the following steps (a) to (f): (a) preparing the Toll-like receptor agonist according to claim 1 or 2 into an acidic solution; (b) preparing the components of the lipid composition into a lipid ethanol solution; (c) mixing the solutions prepared in the above steps (a) and (b) to prepare a nanolipid particle initial solution; (d) selectively repeating the above steps (a)-(c) to obtain more nanolipid particle initial solution; (e) removing ethanol from the initial solution of nanolipid particles and adjusting the concentration of active ingredients and pH to obtain a nanolipid particle suspension; (f) selectively mixing other therapeutic agents with the above nano lipid particle suspension to prepare multi-active ingredient liposomes.

11. A composition, characterized in that The method comprises an active ingredient encapsulated by a lipid component according to any one of claims 3 to 7 and other therapeutic agents, preferably, the other therapeutic agents are one or more of resiquimod and doxorubicin.

12. A delivery system, characterized in that The invention comprises an active ingredient and a lipid component, wherein the active ingredient comprises the Toll-like receptor agonist according to 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 a cationic lipid, an ionizable lipid, a neutral lipid, and a PEGylated lipid; 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 (1-30):(5-50):(5-40):(15-60):(0-10); more 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.

15. The delivery system according to any one of claims 12 to 14, characterized in that The active ingredient further comprises other therapeutic agents, preferably, the other therapeutic agents are one or more of resiquimod and doxorubicin.

16. A method for preparing the delivery system according to claim 12, characterized in that The method comprises the following steps (a) to (d): (a) taking the Toll-like receptor agonist according to claim 1 or 2 and preparing it with a buffer to prepare a Toll-like receptor agonist buffer; (b) taking the lipid component and preparing a lipid ethanol solution with anhydrous ethanol; (c) mixing the lipid ethanol solution and the Toll-like receptor agonist buffer solution at a mass ratio of lipid component to Toll-like receptor agonist of (8-12):1; (d) Remove ethanol, dilute and adjust pH to 6.8-7.

2.

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

18. A vaccine adjuvant or a vaccine, characterized in that A method comprising: comprising the Toll-like receptor agonist according to claim 1 or 2, the active ingredient encapsulated by 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.

19. Use of the Toll-like receptor agonist according to claim 1 or 2, the active ingredient encapsulated by 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 in any one of the following (a) to (j): (a) Use in the preparation of a drug for preventing tumor occurrence; (b) Use in the preparation of drugs for treating tumors; (c) Use in the preparation of a drug for inhibiting tumor growth; (d) Use in the preparation of a drug for preventing tumor cell metastasis; (e) Use in the preparation of a drug for preventing tumor recurrence; (f) Use in the preparation of vaccines; (g) Use in the preparation of a medicament for treating or preventing inflammation-related diseases or infectious diseases; (h) Use in the preparation of a drug for immune activation; (i) Use in the preparation of a drug for upregulating cytokine expression; (j) Upregulation of cytokine expression for non-therapeutic purposes.

20. The use according to claim 19, characterized in that The immune activation includes B cell pathway activation, T cell pathway activation, myeloid lymphocyte pathway activation, lymphoid lymphocyte pathway activation, macrophages, plasmacytoid dendritic cells (pDC), B cells, T cells, CD3+T cells, CD8+T cells, NK cells and granulocyte activation.

21. The use according to claim 19, characterized in that The tumor is selected from solid tumors or tumors into which drugs can be injected, such as head and neck cancer, esophageal cancer, oral cancer, nasopharyngeal cancer, thyroid cancer, lung cancer, gastric 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, chondroma, liposarcoma, neuroblastoma, synovial sarcoma, astrocytoma, glioblastoma multiforme, anaplastic astrocytoma, peritoneal cavity cancer, soft tissue sarcoma, sarcoma, rhabdomyosarcoma, advanced myxoid disease, melanoma, skin cancer at least one.

22. The use according to claim 19, characterized in that The drug is administered by one or more of the following routes: 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, ocular administration, and topical administration.

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