Combination therapy of anticancer treatment with toll-like receptor 7 or 8 agonist having active site temporarily inactivated for cancer treatment

Abstract

The present invention relates to a method for treating cancer, in which the anticancer effect is significantly enhanced by using a Toll-like receptor 7 or 8 agonist having the active site temporarily inactivated. The present invention provides a pharmaceutical composition for combination administration for preventing or treating cancer, comprising, as an active ingredient, a Toll-like receptor 7 or 8 agonist in which the active site is bound to cholesterol via a cleavable linker to be temporarily inactivated, wherein the pharmaceutical composition is administered after 1-20 days following anticancer treatment.

Description

Combination therapy of toll-like receptor 7 or 8 agonists with temporarily inactivated activation sites for the treatment of cancer with anticancer therapy

[0001] This invention claims priority to Republic of Korea Patent Application No. 10-2013-0164008, filed November 23, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a combination therapy of a toll-like receptor 7 or 8 agonist with a temporarily inactivated active site for treating cancer and an anticancer treatment, and more specifically, to a kit for preventing or treating cancer, comprising a) a first pharmaceutical composition comprising an anticancer treatment substance as an active ingredient, and b) a second pharmaceutical composition comprising a temporarily inactivated toll-like receptor 7 or 8 agonist with cholesterol bound to the active site by a cleavable linker as an active ingredient, and a method for treating cancer using the same.

[0003] Cancer is one of the leading causes of death in modern society. It is a disease caused by genetic mutations resulting from various factors, resulting in changes in normal cells. It refers to malignant tumors that do not follow the normal differentiation, proliferation, and growth patterns of cells. Cancer is characterized by "uncontrolled cell growth," and this abnormal cell growth leads to the formation of a tumor, which invades surrounding tissues and, in severe cases, can metastasize to other organs in the body. Despite treatments such as surgery, radiation, and drug therapy, cancer is often incurable, causing suffering and ultimately leading to death. It is an incurable chronic disease. Due to factors such as the growing elderly population and environmental degradation, the global cancer incidence rate has been increasing by more than 5% annually. According to a WHO report, the number of people diagnosed with cancer is projected to increase to 30 million within the next 25 years, with 20 million of them dying from cancer. Although various treatment methods are continuously being developed to effectively treat these cancers, the development of effective treatment methods that can treat various cancers is still insufficient.

[0004] Meanwhile, the immune response is a series of reactions that activated immune cells cause to foreign and endogenous substances, i.e. antigens. When microorganisms including bacteria, viruses, and foreign substances enter the body, immune cells recognize them and are activated, secreting factors such as cytokines to induce an inflammatory response. Recently, research on the mechanism at the stage of the innate immune response that acts non-specifically in the early stages of infection has been actively conducted. Among these, Toll-like receptors (TLRs) are genes that can recognize pathogens in the early stages of inflammation. They are known to recognize the plasma membrane components and nucleic acid components of pathogens and induce an immune response. Using these, research on various Toll-like receptor ligands to activate immune cells is actively being conducted (US Patent Publication No. 2012-0294885). Among these, toll-like receptor 7 or 8 agonist-based substances are used as immune adjuvants that induce cellular immune responses, and are known to include imiquimod, resiquimod, dactolisib, gardiquimod, sumanirole, motorimod, vesatolimod, loxoribine, SM360320, CL264, 3M-003, IMDQ, and Compound 54. These toll-like receptor 7 or 8 agonists are known to effectively induce not only humoral immunity but also cellular immunity as agonists of toll-like receptor 7 or 8 in the endosome. However, these toll-like receptor 7 or 8 agonists are difficult to disperse in aqueous solutions due to their molecular structure. In addition, it is soluble only in special organic solvents such as DMSO and methanol, and does not dissolve in commonly used organic solvents, so there is a limitation in producing immune-activating substances in various formulations.Therefore, cream-type formulations containing a mixture of various surfactants (e.g., Aldara) ® It is commercialized as a cream. In some studies, to overcome this problem, it was manufactured in the form of a salt so that it could be dissolved in an aqueous solution. However, the toll-like receptor 7 or 8 agonist manufactured in the form of a salt is absorbed into the blood vessels in the body, inducing a systemic immune response in the blood vessels, thereby causing many side effects (e.g., cytokine storm, various non-specific hypersensitivity immune responses, etc.), so it is not easy to use. In order to solve this problem of side effects, a temporarily inactivated toll-like receptor 7 or 8 agonist was developed by linking cholesterol to the active site of the toll-like receptor 7 or 8 agonist as a cleavable linker (Korean Patent No. 10-2323540).

[0005] Therefore, if a toll-like receptor 7 or 8 agonist that can be manufactured in various formulations and suppresses nonspecific immune responses without being absorbed into the blood vessels in the body is applied to the treatment of cancer, it is expected that an effective cancer treatment method that can be applied to various cancers through immune activation and reduces side effects can be developed.

[0006] The present invention has been made to solve the problems of the prior art as described above, and the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises as an active ingredient a temporarily inactivated toll-like receptor 7 or 8 agonist having cholesterol bound to the active site by a cleavable linker, administered 1 to 20 days after anticancer treatment; a kit for preventing or treating cancer, comprising: a) a first pharmaceutical composition comprising an anticancer therapeutic agent as an active ingredient; and b) a second pharmaceutical composition comprising as an active ingredient a temporarily inactivated toll-like receptor 7 or 8 agonist having cholesterol bound to the active site by a cleavable linker; uses thereof, methods for treating cancer using the same, etc.

[0007] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0008] The present invention provides a pharmaceutical composition for combination administration for the prevention or treatment of cancer, comprising as an active ingredient a temporarily inactivated Toll-like receptor 7 / 8 agonist in which cholesterol is bound to the active site by a cleavable linker, which is administered 1 to 20 days after anticancer treatment.

[0009] In addition, the present invention provides a kit for preventing or treating cancer, comprising: a) a first pharmaceutical composition comprising an anticancer therapeutic agent as an active ingredient; and b) a second pharmaceutical composition comprising a temporarily inactivated Toll-like receptor 7 / 8 agonist as an active ingredient, wherein cholesterol is bound to an active site by a cleavable linker, wherein the first pharmaceutical composition and the second pharmaceutical composition are administered sequentially. The kit is preferably administered such that the second pharmaceutical composition is administered 1 to 20 days after the first pharmaceutical composition, so that a tumor lysate generated by the first pharmaceutical composition can act as an antigen and significantly increase the immuno-cancer effect of the second pharmaceutical composition. The kit may be for single administration or multiple administration, and the number of administrations of the first pharmaceutical composition and the second pharmaceutical composition may be the same or different. The kit may preferably be for 1 to 15 administrations, 1 to 13 administrations, 1 to 11 administrations, 1 to 9 administrations, 1 to 7 administrations, 1 to 5 administrations, 1 to 3 administrations, or 1 to 2 administrations.In addition, the first pharmaceutical composition may preferably be for 1 to 15 administrations, 1 to 13 administrations, 1 to 11 administrations, 1 to 9 administrations, 1 to 7 administrations, 1 to 5 administrations, 1 to 3 administrations, or 1 to 2 administrations, and the second pharmaceutical composition may preferably be for the same number of administrations as the first pharmaceutical composition, and may be for 1 to 10 administrations, 1 to 9 administrations, 1 to 8 administrations, 1 to 7 administrations, 1 to 6 administrations, 1 to 5 administrations, 1 to 4 administrations, 1 to 3 administrations, or 1 to 2 administrations.

[0010] In one specific embodiment of the present invention, the anticancer treatment may preferably be anticancer drug treatment, radiation therapy, anticancer virus treatment, etc., and the anticancer treatment substance may be an anticancer drug, an anticancer virus, a radioisotope, etc., but is not limited thereto as long as it is an anticancer treatment method or anticancer treatment substance that can be used to treat cancer. Preferably, there is no limitation as long as it is an anticancer treatment method or anticancer treatment substance that can produce a tumor lysate by killing cancer cells and inducing destruction of tumor tissue.

[0011] In another specific embodiment of the present invention, the anticancer agent may preferably be doxorubicin, cisplatin, carboplatin, nedaplatin, goserelin, medroxyprogesterone, cyproterone, vinorelbine, cabazitaxel, denosumab, gemcitabine, capecitabine, oxaliplatin, vorinostat, entinostat, 5FU, taxol, topotecan, irinotecan, or a pharmaceutically acceptable salt thereof that induces cancer cell death, but there is no limitation as long as it is any registered drug for the purpose of cancer treatment (National Cancer Institute, https: / www.cancer.gov / about-cancer / treatment / drugs).

[0012] In another specific embodiment of the present invention, the anticancer virus may be at least one oncolytic virus selected from the group consisting of herpesvirus, adenovirus, vaccinia virus, poliovirus, measles virus, vesicular stomatitis virus, reovirus, and genetically modified viruses thereof, but is not limited thereto as long as it is a virus that dissolves a tumor and produces a tumor lysate.

[0013] In another specific embodiment of the present invention, the radiation therapy may be a treatment of cancer using high-energy radiation such as X-rays, gamma rays, electron rays, or proton rays, or may be a treatment of cancer using a radioisotope. The radioisotope is not limited to any radioisotope used in the treatment of cancer, but is preferably iridium-192 (Ir-192).

[0014] In another specific embodiment of the present invention, the administration may be by intrathecal injection, intravenous injection, intra-articular injection, subcutaneous injection, intradermal injection, intramuscular injection, intratumoral injection, intraocular injection, intraperitoneal injection, etc., and preferably, the first pharmaceutical composition may be administered in the form of intravenous administration, and the second pharmaceutical composition may be administered in the form of intravenous injection, intradermal injection, intratumoral administration, or subcutaneous administration. However, in general, the form of administering an anticancer agent or a toll-like receptor 7 or 8 agonist is not limited thereto.

[0015] In another specific example of the present invention, the cancer may be breast cancer, colon cancer, rectal cancer, lung cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, uterine cancer, stomach cancer, bone cancer, blood cancer, head and neck cancer, bone cancer, vaginal cancer, esophageal cancer, lymphoma, gallbladder cancer, endocrine cancer, adrenal cancer, melanoma, etc., but is not limited thereto as long as it is a type of cancer that can be treated using an anticancer agent and a toll-like receptor 7 or 8 agonist. More specifically, the composition or kit of the present invention can be applied to the treatment of various cancers regardless of the type of cancer because it generates tumor lysates and acts as antigens by using various anticancer treatments that induce the death of cancer cells, and also can be used to treat not only early-stage cancers but also advanced cancers because it can significantly increase immuno-anticancer activity by having a toll-like receptor 7 or 8 agonist act as an adjuvant, and further, can be used to effectively suppress the recurrence of cancer by continuously inducing immune action in the body like a vaccine.

[0016] In another specific embodiment of the present invention, the toll-like receptor 7 or 8 agonist may be preferably at least one selected from the group consisting of an imidazoquinoloine-based agonist, an 8-hydroxyadenine-based agonist, a pteridone-based agonist, a 2-aminopyrimidine-based agonist, a benzoazepine-based agonist, and a 7-thia-8-oxoguanosine-based agonist, and more preferably, imiquimod, resiquimod, dactolisib, gardiquimod, sumanirole, motolimod, Examples include, but are not limited to, vesatolimod, loxoribine, SM360320, CL264, 3M-003, IMDQ, Compound 54, and toll-like receptor 7 or 8 agonists that can temporarily exhibit an inactive form by chemically binding cholesterol to the active site, i.e., NH2.

[0017] In another specific embodiment of the present invention, the cleavable linker may preferably include at least one bond selected from the group consisting of carbamate, disulfide, ester, peptide, azide, or a combination thereof, but is not limited thereto, as long as the chemical bond with cholesterol is cleaved in response to the enzymes and pH of the tumor microenvironment, or the endosomes and lysosomes within the cell, thereby exposing the activation site of the toll-like receptor 7 or 8 agonist, thereby restoring the function of the toll-like receptor 7 or 8 agonist.

[0018] In another specific embodiment of the present invention, the pharmaceutical composition or kit may further comprise an immune checkpoint inhibitor, or a third pharmaceutical composition comprising an immune checkpoint inhibitor as an active ingredient. The immune checkpoint inhibitor may preferably be anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-KIR, anti-LAG3, anti-CD137, anti-OX40, anti-CD276, anti-CD27, anti-GITR, anti-TIM3, anti-41BB, anti-CD226, anti-CD40, anti-CD70, anti-ICOS, anti-CD40L, anti-BTLA, anti-TCR, anti-TIGIT, etc., but is not limited thereto as long as it is an immune checkpoint inhibitor used as an immune anticancer agent.

[0019] In another specific embodiment of the present invention, the pharmaceutical composition or kit is characterized by inhibiting cancer proliferation, metastasis, recurrence, etc., or inhibiting resistance to anticancer treatment, but is not limited thereto if it is part of a commonly used cancer treatment method.

[0020] In another specific embodiment of the present invention, the pharmaceutical compositions may be prepared in the form of nanoparticles to facilitate administration and formulation. However, this is merely for facilitating administration and formulation and is not related to therapeutic effects, so it is not limited thereto. The nanoparticles include, but are not limited to, nanoliposomes, nanomicelles, solid nanoparticles, nanoemulsions, and the like.

[0021] In addition, the present invention provides a method for preventing or treating cancer, comprising administering the pharmaceutical composition in a therapeutically effective amount to a subject in need thereof.

[0022] In addition, the present invention provides a method for preventing or treating cancer, comprising a step of administering a pharmaceutical composition in a therapeutically effective amount to an individual in need thereof using the kit.

[0023] Additionally, the present invention provides a use of the pharmaceutical composition or kit for preventing or treating cancer.

[0024] The present invention also provides a use for producing a drug comprising a transiently inactivated toll-like receptor 7 or 8 agonist having cholesterol linked to the active site by a cleavable linker, the drug being administered 1 to 20 days after an anticancer treatment to enhance the anticancer effect.

[0025] The pharmaceutical composition and / or kit according to the present invention is first administered by chemotherapy, radiation therapy, anticancer virus administration, etc. to induce the destruction of tumor tissue, and after the primary anticancer treatment, the resulting tumor lysate acts as an antigen, and at this time, by secondarily administering a toll-like receptor 7 or 8 agonist with the activation site temporarily inactivated, thereby significantly increasing the immune activation effect, thereby significantly increasing the therapeutic effect of cancer. In addition, it was confirmed that the anticancer effect can be further significantly increased by intratumorally administering a toll-like receptor 7 or 8 agonist with the activation site temporarily inactivated or by additionally administering an immune checkpoint inhibitor. Therefore, the pharmaceutical composition and / or kit according to the present invention is expected to be effectively applied to the therapeutic effect of various cancers.

[0026] Figure 1 is a drawing showing the results of confirming the effects of single administration and combination administration in a homologous transplant mouse tumor model according to one embodiment of the present invention, based on the size of the tumor.

[0027] Figure 2 is a drawing showing the results of confirming the effects of single administration and combination administration in a homologous transplant mouse tumor model according to one embodiment of the present invention by the number of MDSC cells in the spleen.

[0028] Figure 3 is a drawing showing the results of confirming the effects of single administration and combination administration in a homologous transplant mouse tumor model according to one embodiment of the present invention by the number of T cells in the spleen.

[0029] Figure 4 is a drawing showing the results of confirming the effects of single administration and combination administration in a homologous transplant mouse tumor model according to one embodiment of the present invention, as measured by the number of T cells and NK cells in tumor tissue.

[0030] FIG. 5 is a drawing showing the results of confirming the effect of combined administration of a toll-like receptor 7 or 8 agonist with temporarily inactivated activation site, an anticancer agent, and an immune checkpoint inhibitor on the size of the tumor in a homologous transplant mouse tumor model according to one embodiment of the present invention.

[0031] Figure 6 is a drawing showing the results of confirming the combined administration effect on various cancer types according to one embodiment of the present invention.

[0032] Figure 7 is a drawing showing the results of confirming the anticancer effect in primary retransplanted cancer according to one embodiment of the present invention.

[0033] Figure 8 is a drawing showing the results of confirming the anticancer effect in secondary retransplantation cancer according to one embodiment of the present invention.

[0034] FIG. 9 is a diagram showing the results of confirming the anticancer effect according to the dose of a temporarily inactivated toll-like receptor 7 or 8 agonist according to one embodiment of the present invention.

[0035] FIG. 10 is a drawing showing the results of confirming the anticancer effect according to the administration method of a temporarily inactivated toll-like receptor 7 or 8 agonist according to one embodiment of the present invention.

[0036] FIG. 11 is a diagram showing the results of confirming the anticancer effect according to the administration dose and administration schedule of a temporarily inactivated toll-like receptor 7 or 8 agonist according to one embodiment of the present invention.

[0037] FIG. 12 is a diagram showing the results of confirming the anticancer effect according to the administration dose and administration schedule of a temporarily inactivated toll-like receptor 7 or 8 agonist according to one embodiment of the present invention.

[0038] Figure 13 is a drawing showing the results of an experiment in which anticancer treatment was performed at one-week intervals according to one embodiment of the present invention.

[0039] Figure 14 is a diagram showing the results of confirming intratumoral immune cell infiltration after anticancer treatment according to one embodiment of the present invention. The white bar in the diagram represents 100 μm.

[0040] Figure 15 is a drawing briefly showing the therapeutic mechanism of the kit of the present invention.

[0041] Throughout this specification, whenever a part is said to "include" a component, this means that it may include other components, but not to the exclusion of other components, unless otherwise stated.

[0042] The terms "about," "substantially," and the like used in this specification are used to mean at or near the numerical value when manufacturing and material tolerances inherent to the meanings stated are presented. They are also used to prevent unscrupulous infringers from unfairly exploiting disclosures that contain precise or absolute values ​​to aid understanding of this specification. Furthermore, throughout this specification, the terms "step of" or "step of" do not imply "step for."

[0043] Throughout this specification, the term “combination thereof” included in the expressions in the Makushi format means one or more mixtures or combinations selected from the group consisting of the components described in the expressions in the Makushi format, and means including one or more selected from the group consisting of said components.

[0044]

[0045] In the present specification, a “toll-like receptor 7 or 8 agonist with a temporarily inactivated activation site” is characterized in that cholesterol is bound to the activation site by a cleavable linker, so that the immune activation function is temporarily inhibited. The inhibition may mean that the function of the activation site of the toll-like receptor 7 or 8 agonist is delayed. The cleavable linker is in a form that can be naturally cleaved by the tumor microenvironment and / or the physiological environment (low pH, enzymes, glutathione, etc.) within the cell, particularly endosomes and lysosomes, so that the function of the toll-like receptor 7 or 8 agonist can be restored, and there is no limitation on the form thereof.

[0046] In this specification, “cholesterol” is a general term for a type of lipid and an organic substance of the steroid series with hydrophobic properties. The cholesterol may include various analogs based on the cholesterol structure and compounds that can be obtained by chemically changing a part of cholesterol. Preferably, it may include bile acid (cholic acid, deoxycholic acid, lithocholic acid, chenodeoxycholic acid), Vitamin D, steroid hormones (testosteron, estradiol, cortisol, aldosteron, prednisolone, prednisone), etc., but is not limited thereto. In addition, the cholesterol is a substance that helps to position toll-like receptor 7 or 8 agonists on the surface and inside of various types of nanoparticles, and may be replaced with a lipid substance having a similar function, for example, a natural lipid such as phospholipids, a synthetic lipid, etc.

[0047] In the present specification, "Toll-like receptor 7 or 8 agonist-based materials" refers to agonists of Toll-like receptor 7 or 8, which may be selected from the group consisting of imidazoquinoloine series, 8-hydroxyadenine series, pteridone series, 2-aminopyrimidine series, benzoazepine series, and 7-thia-8-oxoguanosine series, and the imidazoquinoline series compounds are compounds of the type mentioned in WO 2018 196823, WO 2011 049677, WO 2011 027022, WO 2017 102652, WO 2019 040491, etc. Including, but not limited to, pharmaceutically acceptable salts. In addition, the above hydroxyadene series compounds are disclosed in WO 2012 080730, WO 2013 068438, WO 2019 036023, WO 2019 035969, WO 2019 035970, WO 2019 035971, WO 2019 035968, CN 108948016, US 2014 8846697, WO 2016 023511, WO 2017 133683, WO 2017 133686, WO 2017 133684, WO 2017 133687, WO 2017 076346, WO 2018 210298, WO 2018 095426, WO 2018 068593, WO 2018 078149, WO 2018 041763, etc., including but not limited to compounds or pharmaceutically acceptable salts thereof.The above pteridone series compounds include, but are not limited to, compounds of the type mentioned in US 2010 0143301, WO 2016 007765, WO 2016 044182, WO 2017 035230, WO 2017 219931, WO 2011 057148, CN 1087 94486, etc. or pharmaceutically acceptable salts thereof. The above aminopyrimidine series compounds are disclosed in WO 2010 133885, WO 2012 066335, WO 2012 066336, WO 2012 067268, WO 2013 172479, WO 2012 136834, WO 2014 053516, WO 2014 053595, US 2018 0215720, WO 2012 156498, WO 2014 076221, WO 2016 141092, WO 2018 045144, WO 2015 014815, WO 2018 233648, WO 2014 207082, WO 2014 056593, WO 2018 002319, WO 2013 117615, etc., including but not limited to compounds or pharmaceutically acceptable salts thereof. The above benzoazepine series compounds include, but are not limited to, compounds of the type mentioned in WO 2007 024612, WO 2010 014913, WO 2010 054215, WO 2011 022508, WO 2011 022509, WO 2012 097177, WO 2012 097173, WO 2016 096778, WO 2016 142250, WO 2017 202704, WO 2017 202703, WO 2017 216054, WO 2017 046112, WO 2017 197624, etc. or pharmaceutically acceptable salts thereof. The above thioxoguanosine series compounds include, but are not limited to, compounds of the type mentioned in WO 2016 180691, WO 2016 055553, WO 2016 180743, WO 2016 091698, etc. or pharmaceutically acceptable salts thereof.In addition, it may include, but is not limited to, toll-like receptor 7 or 8 compounds or pharmaceutically acceptable salts mentioned in PCT / US2009 / 035563, PCT / US2015 / 028264, PCT / US2016 / 020499, WO 2015 023598, PCT / US2015 / 039776, etc., and includes all cases of toll-like receptor 7 or 8 agonists that can be easily guessed and used by a person working in the art.

[0048] In this specification, “co-administration” means administering a complex of a toll-like receptor 7 or 8 agonist and cholesterol together with various substances such as antigens, immune checkpoint inhibitors, immune adjuvants, immune activators, and chemotherapeutic agents for the treatment of cancer, and there are no limitations on the type and form thereof. The term “combination” as used herein can be achieved by administering (treating) individual components of the treatment regimen simultaneously, sequentially, or individually. It is defined as obtaining a combination treatment effect by performing two or more anticancer therapies simultaneously, sequentially, or alternately at regular or indefinite intervals, and the combination therapy is not limited thereto, but can be defined as providing a synergistic effect while being therapeutically superior to the efficacy obtained by administering one or the remaining components of the combination therapy at a regular dose, as measured by, for example, the degree of response, the rate of response, the time until disease progression, or the duration of survival.

[0049] For the purposes of this article, the compounds used in the treatment of cancer are not limited to those known to the industry, such as Paclitaxel, Docetaxel, 5-Flurouracil, Alendronate, Doxorubicin, Simvastatin, Hydrazinocurcumin, Amphotericin B, Ciprofloxacin, Rifabutin, Rifampicin, Efavirenz, Cisplatin, Theophylline, Pseudomonas exotoxin A, Zoledronic acid, Trabectedin, Siltuximab, Dasatinib, Sunitinib, Apatinib, 5,6-Dimethylxanthenone-4-acetic acid, Silibinin, PF-04136309, Trabectedin, Carlumab, BLZ945, PLX3397, Emactuzumab, AMG-820, IMC-CS4, GW3580, PLX6134, N-acetyl-l-cystein, Vitamin C, bortezomib, aspirin, salicylates, Indolecarboxamide derivatives, quinazoline analogues, Thalidomide, prostaglandin metabolites, 2ME2, 17-AAG, Camptothecin, Topotecan, Pleurotin, 1-methylpropyl, 2-imidazolyl disulphide, Tadalafil, Sildenafil, L-AME, Nitroaspirin, Celecoxib, NOHA, Bardoxolone methyl, D,L-1-methyl-tryptophan, Gemcitabine, Axitinib, Sorafenib, Cucurbitacin B, JSI-124, Anti IL-17 antibodies, Anti-glycan antibodies, Anti-VEGF antibodies, Bevacizumab,Antracycline, Tasquinimod, Imatinib, cyclophosphamide, etc., but are not limited to these.

[0050] As used herein, "antigen" refers to a substance capable of eliciting an immune response in a host. An antigen can be recognized and bound to antibodies. The antigen may originate within the body or from the external environment, and is preferably a tumor lysate generated by anticancer treatment.

[0051] In this specification, “prevention” means any act of suppressing or delaying the onset of cancer by administering a composition according to the present invention.

[0052] In this specification, “treatment” means any action in which the symptoms of cancer are improved or beneficially changed by administration of the composition according to the present invention.

[0053] As used herein, "individual" or "subject" refers to a subject to which the composition of the present invention can be administered, preferably a mammal including a human, but there are no limitations on the subject. "Subject in need thereof" may refer to, but is not limited to, a subject having cancer or at high risk of developing cancer and requiring treatment.

[0054] In this specification, the term "active ingredient" is used interchangeably with active drug, active ingredient, active preparation, drug and therapeutic preparation, and refers to any substance used to prevent, alleviate, improve or treat a target disease.

[0055] As used herein, “cancer” refers to various blood cancers, malignant solid tumors, etc. that can spread locally through invasion and systematically through metastasis. Specific examples of cancer include, but are not limited to, colon cancer, adrenal cancer, bone cancer, brain cancer, breast cancer, bronchial cancer, colon cancer and / or rectal cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, laryngeal cancer, liver cancer, lung cancer, nerve tissue cancer, pancreatic cancer, prostate cancer, parathyroid cancer, skin cancer, stomach cancer, thyroid cancer, etc. Other examples of cancer include adenocarcinoma, adenoma, basal cell carcinoma, cervical dysplasia and carcinoma in situ, Ewing's sarcoma, squamous cell carcinoma, acinar cell carcinoma, malignant brain tumor, hairy cell carcinoma, intestinal ganglioneuroma, hyperplastic corneal neurocarcinoma, islet cell carcinoma, Kaposi's cancer, leiomyoma, leukemia, lymphoma, malignant carcinoid tumor, malignant melanoma, malignant hypercalcemia, Marpanoid habitus cancer, medullary carcinoma, metastatic skin cancer, mucosal neuroma, myelodysplastic syndrome, myeloma, filiform sarcoma, neuroblastoma, osteosarcoma, osteogenic and other sarcomas, ovarian cancer, pheochromocytoma, polycythemia vera, primary brain tumor, small cell lung cancer, ulcerative and papillary squamous cell carcinoma, seminal vesicle carcinoma, soft tissue sarcoma, retinoblastoma, rhabdomyoblastoma, renal cell tumor or carcinoma, reticulocyte These include sarcomas and Wilms' tumors. They also include astrocytomas, gastrointestinal stromal tumors (GISTs), gliomas or glioblastomas, renal cell carcinomas (RCCs), hepatocellular carcinomas (HCCs), and pancreatic neuroendocrine cancers.

[0056] In this specification, the terms “active ingredient,” “active drug,” “effective ingredient,” “active agent,” “drug,” and “therapeutic agent” are used interchangeably herein and refer to any substance used to prevent, alleviate, or treat a target disease.

[0057] In this specification, the term “pharmaceutical composition” may be characterized as being in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition may be characterized as being intended for humans. The pharmaceutical composition is not limited to these, but may be formulated and used in the form of oral dosage forms such as powder, granule, capsule, tablet, aqueous suspension, etc., external preparation, suppository, and sterile injectable solution, respectively, according to a conventional method. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, and fragrances for oral administration, and buffers, preservatives, analgesics, solubilizers, isotonic agents, and stabilizers may be mixed and used for injections, and bases, excipients, lubricants, and preservatives may be used for topical administration. The formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing it with the pharmaceutically acceptable carriers described above. For example, the pharmaceutical composition of the present invention may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. for oral administration, and the injection may be prepared in the form of unit dose ampoules or multiple doses. In addition, the pharmaceutical composition may be formulated in the form of solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

[0058] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. In addition, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may be additionally included.

[0059] Routes of administration of the pharmaceutical composition according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Oral or parenteral administration is preferred. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0060] The pharmaceutical composition of the present invention may vary depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, dosage, administration time, administration route, excretion rate, drug combination, and severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route, and period, but may be appropriately selected by a person skilled in the art, and may be administered at 0.0001 to 500 mg / kg or 0.001 to 500 mg / kg per day. Preferably, as shown in Fig. 9, a therapeutically effective amount is administered, and the therapeutically effective amount refers to an amount that shows a greater response than that of a negative control group, and means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level cannot be limited to a specific amount because it can be determined according to factors including the type and severity of the patient's disease, the activity and sensitivity of the drug, the administration time, the administration route and the excretion rate, the treatment period, concurrently used drugs, and other factors well known in the medical field. Preferably, it means an amount sufficient to treat or prevent the target disease. Administration may be administered once a day or divided into several times. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, a dragee, a capsule, a solution, a gel, a syrup, a slurry, or a suspension.

[0061] In this specification, the term “kit” refers to a device that can individually administer an anticancer agent and a toll-like receptor 7 or 8 agonist. The kit may include each pharmaceutical composition in the form of a syringe individually packaged, or may include two pharmaceutical compositions packaged together. Alternatively, the kit may additionally include other immune checkpoint inhibitors, local anesthetics, etc. However, there are no limitations as long as the pharmaceutical composition including the anticancer agent and the pharmaceutical composition including the toll-like receptor 7 or 8 agonist as an active ingredient can be individually administered by controlling the administration time, administration dose, administration method, etc.

[0062]

[0063] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0064]

[0065] [Example]

[0066] Example 1: Synthesis of a conjugate of toll-like receptor 7 or 8 agonist and cholesterol.

[0067] Various toll-like receptor 7 or 8 agonists (imidazoquinoloine, 8-hydroxyadenine, pteridone, 2-aminopyrimidine, benzoazepine, 7-thia-8-oxoguanosine series, etc.) conjugated with cholesterol are prepared by chemical reactions such as reaction scheme 1 or 2, and can be prepared by reacting an amine group (NH2) site, which is an active site of a toll-like receptor 7 or 8 agonist or a derivative thereof, with cholesterol or a cholesterol analogue capable of forming a bond with a carbamate, disulfide, ester, peptide, azide, etc. The above analogues collectively refer to similar compounds obtained by chemically modifying a portion of a Toll-like receptor 7 or 8 agonist or cholesterol. More specifically, a Toll-like receptor 7 or 8 agonist conjugate with a temporarily inactivated active site was prepared in the same manner as disclosed in Korean Patent No. 10-2323540.

[0068] [Reaction Formula 1]

[0069]

[0070] The above R is a side chain containing an aliphatic or aromatic group, and may include -NH-、-CO-、-CONH-、-CSNH-、-COO-、-CSO-、-SO2NH-、-SO2-, -SO-, -O-, etc.

[0071] [Reaction Formula 2]

[0072]

[0073] The above R is a side chain containing an aliphatic or aromatic group, and may include -NH-、-CO-、-CONH-、-CSNH-、-COO-、-CSO-、-SO2NH-、-SO2-, -SO-, -O-, etc.

[0074]

[0075] 1.1. Synthesis of resquimod-cholesterol conjugates with carbamate bonds

[0076] In order to synthesize a cholesterol-conjugated Toll-like receptor 7 or 8 agonist, a conjugate of cholesterol and Toll-like receptor 7 or 8 agonist was synthesized using Resquimod (R848), one of the Toll-like receptor 7 or 8 agonists, by the method of Scheme 3 below. More specifically, 31.4 mg of Resquimod was dissolved in 3 mL of dichloromethane by adding 100 μL of pyridine, and 90.0 mg of cholesteryl chloroformate was dissolved in 1 mL of dichloromethane, and the resulting solution was slowly added dropwise. Then, the mixture was stirred at 4°C for 16 hours to prepare a mixture. And after adjusting the temperature of the above mixture to room temperature, distilled water was added to separate the water and dichloromethane layers, and sodium sulfate was added to the separated dichloromethane layer and reacted for 16 hours to remove the remaining water. And the remaining solution was purified using a silica gel column, and a white powder of cholesterol-bound resquimod was obtained. The structure of the resquimod used in the synthesis and the obtained cholesterol-bound resquimod are 1 H-NMR and 15 It was verified using N-HSQC (Heteronuclear single quantum coherence spectroscopy).

[0077] [Reaction Formula 3]

[0078]

[0079]

[0080] 1.2. Synthesis of imiquimod-cholesterol conjugates with carbamate bonds

[0081] In order to synthesize a cholesterol-conjugated toll-like receptor 7 or 8 agonist, a conjugate of cholesterol and toll-like receptor 7 or 8 agonist was synthesized using imiquimod (R837), one of the toll-like receptor 7 or 8 agonists, by the method of the following reaction scheme 4. More specifically, 31.4 mg of cholesterol was dissolved in 100 μL of pyridine in 3 mL of dichloromethane, and a solution of 90.0 mg of cholesteryl chloroformate dissolved in 1 mL of dichloromethane was slowly added dropwise. Then, the mixture was stirred at 4 °C for 16 hours to prepare a mixture. After adjusting the mixture to room temperature, distilled water was added to separate the water and dichloromethane layers, and sodium sulfate was added to the separated dichloromethane layer and reacted for 16 hours to remove the remaining water. The remaining solution was purified using a silica gel column, and imiquimod bound to cholesterol was obtained as a white powder. The structure of the imiquimod used in the synthesis and the obtained imiquimod bound to cholesterol are as follows. 1 H-NMR and 15 It was verified using N-HSQC.

[0082] [Reaction Formula 4]

[0083]

[0084] The above R1 or R2 is a side chain containing an aliphatic or aromatic group, and may include -NH-、-CO-、-CONH-、-CSNH-、-COO-、-CSO-、-SO2NH-、-SO2-, -SO-, -O-, etc.

[0085]

[0086] 1.3. Synthesis of resquimod and cholesterol conjugate cross-linked with disulfide

[0087] To synthesize cholesterol-disulfide crosslinked toll-like receptor 7 or 8 agonists, the method of Scheme 5 below was used. More specifically, 1.54 g of 2-hydroxyethyl disulfide was dissolved in 30 mL of tetrahydrofuran, and then slowly added dropwise to a phosgene solution (15 mL, 15 wt% in toluene) to prepare a mixture. The mixture was stirred at 25°C for 10 hours, and the solvent was evaporated under vacuum. 2.3 g of N-hydrosuccinimide was dissolved in tetrahydrofuran, mixed with the mixture, and 1.57 mL of triethylamine was added. After reacting at 40°C for 16 hours, the precipitate was removed, and the solvent was evaporated under vacuum. And after purification using silica gel column chromatography, recrystallization with cold hexane and drying under vacuum were obtained as a purified disulfide cross-linking linker as a white solid. And 387 mg of cholesterol was added and dissolved in 10 mL of dichloromethane, and 523 mg of disulfide cross-linking linking group was added and stirred at room temperature for 16 hours, and cholesterol-disulfide as a white powder in which cholesterol and disulfide were combined was purified using a silica gel column. And 31.4 mg of resquimod and 80 mg of cholesterol-disulfide were added to 5 mL of dichloromethane and stirred at room temperature for 16 hours. And distilled water was added to the stirred solution to separate the water and dichloromethane layers, and sodium sulfate was added to the separated dichloromethane layer and reacted for 16 hours to remove the remaining water. The remaining solution was purified using a silica gel column, and a white powder of cholesterol-disulfide cross-linked resquimod was obtained.The structure of the resquimod combined with the obtained cholesterol used in the synthesis. 1 H-NMR and 15 It was verified using N-HSQC.

[0088] [Reaction Formula 5]

[0089]

[0090]

[0091] 1.4. Synthesis of disulfide-cross-linked imiquimod and cholesterol conjugates

[0092] To synthesize cholesterol-disulfide cross-linked toll-like receptor 7 or 8 agonists, the method of the following reaction scheme 6 was used. More specifically, 1.54 g of 2-hydroxyethyl disulfide was dissolved in 30 mL of tetrahydrofuran, and then slowly added dropwise to a phosgene solution (15 mL, 15 wt% in toluene) to prepare a mixture. The mixture was stirred at 25 °C for 10 hours, and the solvent was evaporated under vacuum. 2.3 g of N-hydrosuccinimide was dissolved in tetrahydrofuran, mixed with the mixture, and 1.57 mL of triethylamine was added. After reacting at 40 °C for 16 hours, the precipitate was removed, and the solvent was evaporated under vacuum. And after purification using silica gel column chromatography, recrystallization with cold hexane and drying under vacuum were obtained as a purified disulfide cross-linking linker as a white solid. And 387 mg of cholesterol was added and dissolved in 10 mL of dichloromethane, and 523 mg of disulfide cross-linking linking linker was added and stirred at room temperature for 16 hours, and cholesterol-disulfide as a white powder in which cholesterol and disulfide were combined was purified using a silica gel column. And 31.4 mg of imiquimod and 80 mg of cholesterol-disulfide were added to 5 mL of dichloromethane and stirred at room temperature for 16 hours. And distilled water was added to the stirred solution to separate the water and dichloromethane layers, and sodium sulfate was added to the separated dichloromethane layer and reacted for 16 hours to remove the remaining water. And the remaining solution was purified using a silica gel column, and imiquimod with cholesterol-disulfide cross-linking was obtained as a white powder. The structure of the imiquimod used in the synthesis and the obtained imiquimod with cholesterol bound are 1 H-NMR and15 It was verified using N-HSQC.

[0093] [Reaction Formula 6]

[0094]

[0095] The above R1 or R2 is a side chain containing an aliphatic or aromatic group, and may include -NH-、-CO-、-CONH-、-CSNH-、-COO-、-CSO-、-SO2NH-、-SO2-, -SO-, -O-, etc.

[0096]

[0097] Example 2: Preparation of nanoparticles comprising a conjugate of cholesterol-toll-like receptor 7 or 8 agonists.

[0098] The cholesterol-toll-like receptor 7 or 8 conjugate was prepared in the form of nanoliposomes to maximize interaction with immune cells. As an example, although nanoliposomes were prepared, the cholesterol-toll-like receptor 7 or 8 conjugate contains cholesterol, so it can be easily loaded, inserted, or encapsulated into various drug delivery vehicles such as lipid-containing nanoliposomes, nanoemulsions, nanomicelles, hydrogels, and polymer nanoparticles, thereby enabling the preparation of various drug forms. More specifically, DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) were first dissolved in ED solution (10 vol% DMSO and 90 vol% ethanol) to a concentration of 50 mg / mL each. And the resquimod-cholesterol conjugate linked by a disulfide bond synthesized in the same manner as Example 1.3 was dissolved in an ED solution at a concentration of 10 mg / mL to prepare a solution of a toll-like receptor 7 or 8 agonist with a temporarily inactivated active site. Then, 160 μL of the prepared DOPC solution, 160 μL of the prepared DOTAP solution, 400 μL of the resquimod-cholesterol conjugate solution, and 280 μL of the ED solution were mixed to prepare a lipid stock. Then, the lipid stock and the phosphate buffer of pH 6.0 were mounted in a ratio of 1:3 on the Ignite device of Precision Nanosystem, and operated under the conditions of a syringe volume of 3 mL, a flow rate ratio of 3:1, a total volume of 3.5 mL, and a total flow rate of 12 mL / min to fabricate nanoliposomes. The fabricated liposomes had a mass of 0.After filtering using a 2 μm syringe filter, the solution was dialyzed using 10K Dialysis Cassettes at 160 rpm for 2 to 4 hours, and the buffer was replaced again and dialyzed for 16 hours to remove the organic solvent used in the production. The manufactured nanoliposomes (ProLNG-001) were stored refrigerated in a light-shielded state until use.

[0099]

[0100] Example 3: Efficacy of combination therapy with anticancer agents and toll-like receptor 7 or 8 agonists with temporarily inactivated activation sites.

[0101] To confirm the effective combination therapy of temporarily inactivated cholesterol-toll-like receptor 7 or 8 agonists and anticancer drugs, ProLNG-001, manufactured in the same manner as Example 2, and anticancer drugs were co-administered under various conditions. All animal studies were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines. First, 4T1 cells, a triple-negative breast cancer (TNBC) cell line, were cultured at 5X10 5 Syngenic mouse models were created by subcutaneous (SC) injection into the dorsal region of 7-8 week-old BALB / c mice with the same number of cells. Animal breeding and experiments were all performed in a specific pathogen-free (SPF) facility, and each experimental group used eight animals. The experimental group to confirm the effect of combined administration was transplanted with 4T1 cell lines and Doxil, a widely used anticancer agent, was administered on the 7th day. ⓡ80 μg was administered intravenously (IV), and on the 8th day, 140 μg of ProLNG-001 was administered intratumorally (IT) or subcutaneously (SC). On the 11th day, 140 μg of ProLNG-001 was administered intratumorally or subcutaneously again, completing the first dose. On the 14th day, Doxil was administered for the second dose. ⓡ 80 μg was injected intravenously, 140 μg of ProLNG-001 was injected intratumorally or subcutaneously on the 15th day, and 140 μg of ProLNG-001 was injected intratumorally or subcutaneously on the 18th day. The experimental group to confirm the effect of single administration was transplanted with 4T1 cell line, and then Doxil was administered on the same schedule as the combination administration experimental group. ⓡ Or ProLNG-001 was injected, Doxil ⓡ The single-administration group was injected with the same amount of phosphate buffer solution at the time of ProLNG-001 injection, and the ProLNG-001 single-administration group was injected with Doxil ⓡ The same volume of phosphate buffer solution was injected at the time of injection. The control group received only the same volume of phosphate buffer solution at each administration time point. Tumor size was measured at 2-3 day intervals until day 21. Tumor size was calculated by measuring the length and width of the tumor using a caliper and substituting it into the following equation. All results were expressed as the mean ± standard deviation. Statistical significance was confirmed using Student's t-test, and statistical significance was determined if P < 0.05. The results are shown in Figure 1.

[0102]

[0103] After completing the second administration, all mice were euthanized on the 21st day, and lymph nodes (LNs), tumor tissues, and spleens were isolated. The isolated tumor tissues were first crushed using scissors, and the crushed tissues were treated with 1 mg / mL collagenase type I and incubated at 37°C for 1 hour to separate them into single cells. The separated cells were then strained using a 70 μm strainer and washed using a phosphate buffer. The obtained spleen or lymph nodes were first crushed using scissors, and the crushed tissues were treated with a red blood cell lysis buffer and incubated at 37°C for 10 minutes to lyse red blood cells. The spleen or lymph nodes were then strained using a 70 μm strainer and washed using a phosphate buffer. The washed tumor cells, spleen cells, and lymph node cells were each labeled using antibodies. Myeloid-derived suppressor cells (MDSCs) in the spleen were labeled using anti-CD11b and anti-GR-1 antibodies. T cells in the spleen were labeled using anti-CD3 antibodies, or anti-CD4 antibodies and anti-TNF-α antibodies, or anti-CD8 antibodies and anti-INF-γ antibodies. T cells in tumor tissues were labeled using anti-CD3 antibodies or anti-CD49b antibodies, and natural killer cells (NK cells) were labeled using anti-CD69 antibodies for CD3-positive T cells, or anti-CD69 antibodies for CD49b-positive cells. They were also labeled using anti-PD-L1 antibodies. The results were analyzed using a fluorescence flow cytometer. The results are shown in Figures 2 to 4.

[0104] As shown in Figure 1, Doxil ⓡThe monotherapy group (Doxil) and the subcutaneous injection group of ProLNG-001 (ProLNG-SC) showed suppressed tumor growth compared to the control group, but tumor growth was observed to be slight. However, the intratumoral injection group of ProLNG-001 (ProLNG-IT) and Doxil ⓡ Subcutaneous injection combination therapy group of ProLNG-001 (DP-SC), Doxil ⓡ The intratumoral combination therapy group (DP-IT) of ProLNG-001 and ProLNG-001 showed a continuous decrease in tumor size. In particular, in the case of DP-IT, it was confirmed that the tumor grew little.

[0105] And as shown in Figure 2, the number of MDSC cells with immunosuppressive function in the spleen was not significantly reduced in Control, Doxil, and ProLNG(sc), but in the intratumoral injection experimental group of ProLNG-001 (ProLNG(it)), Doxil ⓡ Subcutaneous injection combination therapy group of ProLNG-001 (Doxil / PriLNG(sc)) and Doxil ⓡ In the experimental group (Doxil / PriLNG(it)) administered intratumoral combination of ProLNG-001, a significant decrease was confirmed.

[0106] As shown in Fig. 3, the number of CD3+ T cells, CD4+TNF-α+ T cells, and CD8+INF-γ+ T cells in the spleen also showed an increase in ProLNG(it), Doxil / ProiLNG(sc), and Doxil / ProiLNG(it), and it was confirmed that Doxil / ProiLNG(it) showed a significantly higher number of T cells.

[0107] In addition, as shown in Fig. 4, the number of CD3+ T cells and NK cells exhibiting anticancer effects within tumor tissues was confirmed to be significantly increased in the case of ProLNG(it).

[0108] Through the above results, it was confirmed that in the case of a toll-like receptor 7 or 8 agonist with a temporarily inactivated activation site, tumor administration can effectively increase the immune activation effect, and that administering an inactivated toll-like receptor 7 or 8 agonist that exhibits an immune activation effect after administering an anticancer drug can increase the effect of combined administration.

[0109]

[0110] Example 4: Efficacy of combination therapy with a toll-like receptor 7 or 8 agonist with a temporarily inactivated activation site, an anticancer agent, and an immune checkpoint inhibitor.

[0111] To confirm the effect of combination therapy with immune checkpoint inhibitors, the experiment was conducted using the same method as Example 3. Briefly, Doxil was administered on the 7th day after transplantation of 4T1 cell lines. ⓡ 80 μg was injected intravenously, 200 μg of Anti-PD-L1 (Atezolizumab; Atz), which is widely used as an immune checkpoint inhibitor, was injected intraperitoneally (IP), and on the 8th day, 140 μg of ProLNG-001 was injected intratumorally. On the 9th day, 200 μg of Atz was injected intraperitoneally, and on the 11th day, 140 μg of ProLNG-001 was injected intratumorally again and 200 μg of Atz was injected intraperitoneally, completing the first administration. And on the 14th day, for the second administration, Doxil ⓡ 80 μg was injected intravenously, 200 μg of Atz was injected intraperitoneally, and 140 μg of ProLNG-001 was injected intratumorally on the 15th day. Then, 200 μg of Atz was injected intraperitoneally on the 16th day, and 140 μg of ProLNG-001 and 200 μg of Atz were injected intratumorally on the 18th day. The remaining conditions were the same as in Example 3. Then, the tumor size was measured at 2-3 day intervals until the 25th day. The results are shown in Fig. 5.

[0112] As shown in Fig. 5, the ATZ monotherapy group showed tumor growth similar to the control group, whereas the ProLNG-001 monotherapy group (ProLNG-001(P)) and the ProLNG-001 and ATZ combination therapy group (P+ATZ) showed tumor growth inhibition. And Doxil ⓡ Co-administration experimental group (D+P) of ProLNG-001 and Doxil ⓡ In the experimental group (D+P+ATZ) administered in combination with ProLNG-001, ProLNG-001, and ATZ, tumor growth was minimal. These results demonstrate that the anticancer effect can be significantly enhanced by co-administering an immune checkpoint inhibitor with an anticancer drug and a toll-like receptor 7 or 8 agonist with a temporarily inactivated activation site.

[0113]

[0114] Example 5: Confirmation of anticancer effects in various cancer types.

[0115] In order to confirm whether the same anticancer effect was shown in various cancer types, the anticancer effect was confirmed using the bladder cancer cell line MB49 and the melanoma cell line B16F10 using the same method as Example 3. More specifically, MB49 and B16F10 cells were each cultured at 5X10 5 The number of cells was injected subcutaneously into the back of 7-8 week old C58BL / 6 mice, and 4T1 cells were injected at 5X10 5 A mouse tumor model was created by subcutaneously injecting the number of cells into the dorsal region of 7-8 week old BALB / c mice. The control group was injected with the same amount of phosphate buffer solution, and the co-administration experimental group was injected with Doxil. ⓡ 80 μg was administered intravenously, and 140 μg of ProLNG-001 was administered intratumorally, and the combination was administered on the same schedule as Example 3. All three types of tumors were 100 mm in diameter on the 7th day after transplantation. 3After confirming that the size had been reached, an experiment was conducted. The results are shown in Fig. 6.

[0116] As shown in Fig. 6, tumor growth was virtually suppressed in the melanoma model, and in breast cancer and bladder cancer models, tumor growth did not occur at all even for a long period of approximately 40 days. These results confirm that the combination therapy of the present invention can effectively suppress tumor growth in various cancer types.

[0117]

[0118] Example 6: Confirmation of anticancer effect in relapsed cancer

[0119] In order to confirm whether it shows an anticancer effect in relapsed cancer, the bladder cancer cell line MB49 was used and the experiment was conducted in the same manner as in Example 5. After the first tumor cell line was transplanted to the experimental group that achieved complete response, 5X10 were transplanted again about 50 days later. 5 Re-challenge was performed by subcutaneously injecting the cells into the back of six mice. In addition, Doxil was administered to three mice. ⓡ 80 μg was administered intravenously, and 140 μg of ProLNG-001 was administered intratumorally, and co-administered according to the same schedule as in Example 3. The remaining three mice were injected with the same amount of phosphate buffer solution. The results are shown in Fig. 7.

[0120] The left drawing of Fig. 7 shows the results of the control group (4 mice) that were transplanted with the bladder cancer cell line MB49 and injected with a phosphate buffer solution into new mice; the middle drawing shows the results of the experimental group (3 mice) that achieved CR and were then transplanted with the MB49 cell line again and injected with a phosphate buffer solution; and the right drawing shows the results of the experimental group (3 mice) that achieved CR and were then transplanted with the MB49 cell line again and co-administered on the same schedule as Example 3. Since all the original mice in the control group died, the experiment was conducted using new mice without re-transplantation. As shown in the right drawing of Fig. 7, in the experimental group that was re-transplanted with the tumor cell line and co-administered, the tumor seemed to grow slightly at first, but the growth of the tumor was suppressed again, and it was confirmed that there was no growth at all even 70 days after the re-transplantation of the tumor cell line. Moreover, in two of the three animals that received the same amount of phosphate buffered solution injection without any post-transplantation treatment, tumor growth was suppressed again, and CR occurred up to 70 days after transplantation.

[0121] On day 72 after retransplantation, 5 mice with confirmed CR were re-injected with 5X10 MB49 cells. 5 Secondary reimplantation was performed by subcutaneously injecting cells into the dorsal region of mice. Tumor growth was observed without any treatment. The results are shown in Figure 8.

[0122] As shown in Fig. 8, in all three mice that received chemotherapy after the first re-transplantation (Re-treat) and two mice that did not receive treatment (Re-challenge), the cancer appeared to grow for about a week, but it was confirmed that the cancer cells did not grow at all from about two weeks onwards. Through the above results, it was confirmed that the combination administration of the present invention is not a simple chemotherapy, but that the tumor lysates generated through the chemotherapy that primarily kills cancer cells act as antigens, and the secondarily injected inactivated toll-like receptor 7 or 8 agonist acts as an immune adjuvant, thereby effectively increasing the immune response in the body, thereby effectively suppressing not only the first chemotherapy but also the recurrence of cancer without additional treatment.

[0123]

[0124] Example 7: Confirmation of anticancer effects according to administered dose

[0125] To determine the anticancer effect according to the administered dose, the experiment was conducted using the same method as Example 3. However, to determine the anticancer effect according to the dose of the temporarily inactivated toll-like receptor 7 or 8 agonist, ProLNG-001 was injected intratumorally at doses of 7, 14, 28, 35, 70, or 140 μg, respectively. The results are shown in Figure 9.

[0126] As shown in Figure 9, the anticancer effect increased with increasing dose of ProLNG-001, reaching a plateau at concentrations above 35 μg. These results confirm that a certain concentration of dose is required to exhibit anticancer effects in treatments using temporarily inactivated toll-like receptor 7 or 8 agonists.

[0127]

[0128] Example 8: Confirmation of anticancer effects according to administration location

[0129] To determine the anticancer effect of temporarily inactivated toll-like receptor 7 or 8 agonists depending on the site of administration, 4T1 cells were cultured at 5X10 5 An allograft mouse tumor model was created by subcutaneously implanting the cells into the dorsal region of 7-8 week old BALB / c mice. In the previous examples, the tumor size was 100 mm. 3 When the tumor size reached 150 mm, that is, on the 7th day, anticancer treatment was performed, but in this example, in order to mimic the situation after the cancer progressed further, the tumor size was 150 mm. 3 On the 12th day, chemotherapy was started. More specifically, on the 12th day, Doxil ⓡ 80 μg was administered intravenously, and on the 13th day, 140 μg of ProLNG-001 was administered intratumorally (i.t.), intravenously (iv), or subcutaneously (sc). On the 16th day, 140 μg of ProLNG-001 was administered again using the same method, completing the first dose. On the 19th day, Doxil was administered for the second dose. ⓡ 80 μg was administered intravenously, and 140 μg of ProLNG-001 was administered on the 20th and 23rd days using the same method as before to complete the second administration. The experimental group to confirm the effect of single administration was administered Doxil on the same schedule as the combination administration experimental group after transplanting the 4T1 cell line. ⓡ Or ProLNG-001 was injected, Doxil ⓡ The single-administration group was injected with the same amount of phosphate buffer solution at the time of ProLNG-001 injection, and the ProLNG-001 single-administration group was injected with Doxil ⓡ An equal volume of phosphate buffer solution was injected at each injection point. The control group received only an equal volume of phosphate buffer solution at each administration point. The results are shown in Figure 10.

[0130] As shown in Fig. 10, compared to the single-administration groups, it was confirmed that tumor growth was suppressed in the combination administration group, and in particular, when a temporarily inactivated toll-like receptor 7 or 8 agonist was injected intratumorally, it was confirmed that cancer growth was suppressed even after 50 days. Through the above results, it was confirmed that the combination administration method of the present invention can be used to treat cancer not only in the early stage but also in the advanced stage, and that when a temporarily inactivated toll-like receptor 7 or 8 agonist was administered intratumorally, the anticancer effect could be most effectively increased.

[0131]

[0132] Example 9: Confirmation of anticancer effects according to administration dose and administration schedule

[0133] To determine the anticancer effect according to the dose and administration schedule of temporarily inactivated toll-like receptor 7 or 8 agonists, 4TI cells were subcutaneously injected into the back of mice using the same method as Example 3, and tumors were grown to a size of 100 mm. 3 When the tumor size was reached, chemotherapy was initiated. Chemotherapy was administered intratumorally at doses of 35, 140, or 240 μg of ProLNG-001, and the chemotherapy schedule was as shown in Figure 11. The results are shown in Figure 11.

[0134] In Fig. 11, 1 is a control group administered with a phosphate buffer solution, 2 is an experimental group administered with an anticancer drug and then administered ProLNG-001 at a concentration of 35 μg twice on day 1 and day 4, repeating the schedule twice (D / P 35 x 2 cycle 2), 3 is an experimental group administered with an anticancer drug and then administered ProLNG-001 at a concentration of 35 μg four times on day 1, day 2, day 3, and day 4, repeating the schedule twice (D / P 35 x 4 cycle 2), 4 is an experimental group administered with an anticancer drug and then administered ProLNG-001 at a concentration of 35 μg twice on day 1 and day 4, repeating the schedule four times (D / P 35 x 2 cycle 4), 5 is an experimental group administered with an anticancer drug and then administered ProLNG-001 at a concentration of 140 μg one day later. The experimental group that repeated the schedule of administering 1 μg of ProLNG-001 twice (D / P 140 x 1 cycle 2) once, 6 is the experimental group that repeated the schedule of administering 140 μg of ProLNG-001 twice 1 day and 4 days after administering the anticancer drug twice (D / P 140 x 2 cycle 2), 7 is the experimental group that repeated the schedule of administering 280 μg of ProLNG-001 once 1 day after administering the anticancer drug twice (D / P 280 x 1 cycle 2), 8 is the experimental group that repeated the schedule of administering 140 μg of ProLNG-001 and 140 μg of ProLNG-001 again 4 days after administering the anticancer drug twice (D / P 140 x 2 cycle 2(s)). The anticancer drug was administered repeatedly at weekly intervals. As shown in Figure 11, it was confirmed that long-term, repeated low-dose treatment significantly increased the anticancer effect, rather than short-term, high-dose treatment. This confirmed that repeated administration had an effect similar to a booster dose of a vaccine, significantly enhancing the therapeutic effect of cancer by boosting the immune response.

[0135] To clarify the effects of different dosing schedules, experiments were conducted using various dosing schedules. Anticancer treatment was administered to tumors with a size of 100 mm. 3 It started when I reached Doxil. More specifically, ⓡ G2, Doxil administered 80 μg iv 7 times at 1-week intervals ⓡ G3, Doxil administered 80 μg iv three times at two-week intervals ⓡ G4, Doxil administered 80 μg iv twice at three-week intervals ⓡ G5, Doxil administered 80 μg iv, followed by 35 μg ProLNG-001 it the next day, administered 7 times at one-week intervals ⓡ G6, Doxil administered 80 μg iv, followed by 140 μg ProLNG-001 it the next day, 7 times at 1-week intervals ⓡ G7, Doxil administered 80 μg iv, followed by 140 μg ProLNG-001 it the next day, administered 4 times at two-week intervals ⓡ G8, Doxil administered 80 μg iv, followed by 140 μg ProLNG-001 it the next day, administered three times at three-week intervals ⓡ G9, which was administered 80 μg iv, then 140 μg ProLNG-001 it the next day, and then 140 μg ProLNG-001 it again 3 days later, for 7 doses at 1-week intervals, and the control group (G1, control) which was administered the same phosphate buffer solution on each administration day, were used to determine the size of the tumor and the survival rate. The results are shown in Fig. 12.

[0136] As shown in Fig. 12, the anticancer effect was increased in the experimental group administered at one-week intervals, and the therapeutic effect could be increased when doxorubicin, an anticancer drug, was administered in combination with a temporarily inactivated toll-like receptor 7 or 8 agonist rather than when it was used alone. It was confirmed that the experimental group that administered the anticancer drug and then the temporarily inactivated toll-like receptor 7 or 8 agonist twice showed the most remarkable therapeutic effect.

[0137] To illustrate this more clearly, the tumor sizes of the experimental groups that received anticancer treatment at one-week intervals before the first death occurred are shown in Figure 13.

[0138] As shown in Figure 13, when co-administration of an anticancer drug and a temporarily inactivated toll-like receptor 7 or 8 agonist is performed at one-week intervals, the anticancer treatment effect can be enhanced, and it was confirmed that the experimental group that was administered the temporarily inactivated toll-like receptor 7 or 8 agonist twice showed the most remarkable treatment effect.

[0139]

[0140] Example 10: Confirmation of immune response to anticancer treatment

[0141] To confirm the intratumoral immune response following administration of a temporarily inactivated toll-like receptor 7 or 8 agonist, 4TI cells were subcutaneously injected into the flank area of ​​mice using the same method as in Example 3, and tumors were grown to a size of 100 mm. 3 When I reached that point, I started chemotherapy. Chemotherapy was Doxil ⓡ Group (D) administered 80 μg and Doxil ⓡThe group (DP) was administered 80 μg, and the next day, 140 μg of ProLNG-001 was administered it, and 3 days later, 140 μg of ProLNG-001 was administered it again. The group was then divided into a control group (C) that was administered phosphate buffer solution, and the second dose of ProLNG-001 was administered and observed using a fluorescence microscope. The fluorescence observation was commissioned to IVIM Technology Inc. 4T1 cells were observed with a green fluorescence wavelength, CD3-positive T cells were observed with a red fluorescence wavelength, CD49-positive cells, which are NK cells, were observed with a red fluorescence wavelength, and CD31-positive cells, which are vascular epithelial cells, were observed with a blue fluorescence wavelength. The results are shown in Fig. 14.

[0142] As shown in Fig. 14, in the control group, there was almost no change in the morphology of cancer cells, and CD49b was observed in large quantities around blood vessels. In the Doxil group, there was also almost no change in the morphology of cancer cells, and excessive new blood vessels were formed around the cells, and CD49b was observed around the blood vessels. On the other hand, in the DP group, dead cancer cells, not normal cells, were observed, and it was confirmed that CD49b was co-localized within the blood vessels or with cancer cells. Through the above results, it was confirmed that the combined administration of the present invention activates the immune response in the body and induces infiltration of immune cells such as T cells and NK cells into the tumor.

[0143]

[0144] Through the above results, as shown in Fig. 15, when the toll-like receptor 7 or 8 agonist with the activation site temporarily inactivated of the present invention is co-administered with an anticancer agent, cancer cells are first killed and tumor tissues are destroyed through chemotherapy, radiation therapy, anticancer virus administration, etc., and the resulting tumor lysate acts as an antigen, thereby enhancing the immunological therapeutic effect of the tumor treatment. Secondarily, by administering the toll-like receptor 7 or 8 agonist with the activation site temporarily inactivated, the toll-like receptor 7 or 8 agonist acts as an adjuvant, inducing a more potent immuno-cancer effect through its immune activation action, thereby significantly enhancing the anticancer treatment effect. In other words, it was confirmed that the therapeutic effect of cancer can be significantly enhanced by utilizing the time difference between the immuno-cancer activity of the primary anticancer treatment and the secondary toll-like receptor 7 or 8 agonist. In addition, it was confirmed that the anticancer effect can be significantly enhanced by intratumoral injection of a toll-like receptor 7 or 8 agonist with the activation site temporarily inactivated or by additional administration of an immune checkpoint inhibitor. Therefore, the present invention provides a method for significantly enhancing the anticancer treatment effect by utilizing a toll-like receptor 7 or 8 agonist with the activation site temporarily inactivated, thereby reducing the side effects of the toll-like receptor 7 or 8 agonist while maintaining immune activation for a longer period of time, thereby enabling it to be effectively used in the treatment of various cancers.

[0145]

[0146] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0147] The pharmaceutical composition and / or kit according to the present invention effectively enhances intratumoral immune responses, thereby significantly enhancing the therapeutic efficacy of various types of cancer when administered in combination with existing anticancer treatments, and can also effectively suppress recurrence. Therefore, the pharmaceutical composition and / or kit according to the present invention is expected to be effectively applied to the treatment of various cancers.

Claims

1. A pharmaceutical composition for combination administration for the prevention or treatment of cancer, comprising as an active ingredient a temporarily inactivated Toll-like receptor 7 or 8 agonist in which cholesterol is bound to the active site by a cleavable linker, A pharmaceutical composition, wherein the pharmaceutical composition is administered 1 to 20 days after anticancer treatment.

2. In paragraph 1, A pharmaceutical composition, characterized in that the above anticancer treatment is an anticancer drug treatment, radiation therapy, or anticancer virus treatment capable of inducing destruction of tumor tissue.

3. In paragraph 2, A pharmaceutical composition, characterized in that the anticancer agent is at least one selected from the group consisting of doxorubicin, cisplatin, carboplatin, nedaplatin, goserelin, medroxyprogesterone, cyproterone, vinorelbine, cabazitaxel, denosumab, gemcitabine, capecitabine, oxaliplatin, vorinostat, entinostat, 5FU, taxol, topotecan, irinotecan, and pharmaceutically acceptable salts thereof.

4. In paragraph 2, A pharmaceutical composition, characterized in that the oncolytic virus is any one oncolytic virus selected from the group consisting of herpesvirus, adenovirus, vaccinia virus, poliovirus, measles virus, vesicular stomatitis virus, reovirus, and genetically modified viruses thereof.

5. In paragraph 1, A pharmaceutical composition, characterized in that the pharmaceutical composition is in the form of intravenous injection, intradermal injection, intratumoral injection or subcutaneous injection.

6. In paragraph 1, A pharmaceutical composition, characterized in that the cancer is any one selected from the group consisting of breast cancer, colon cancer, rectal cancer, lung cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, uterine cancer, stomach cancer, bone cancer, blood cancer, head and neck cancer, bone cancer, vaginal cancer, esophagus cancer, lymphoma, gallbladder cancer, endocrine cancer, adrenal cancer, and melanoma.

7. In paragraph 1, A pharmaceutical composition, characterized in that the toll-like receptor 7 or 8 agonist is at least one selected from the group consisting of an imidazoquinoloine-based agonist, an 8-hydroxyadenine-based agonist, a pteridone-based agonist, a 2-aminopyrimidine-based agonist, a benzoazepine-based agonist, and a 7-thia-8-oxoguanosine-based agonist.

8. In paragraph 1, A pharmaceutical composition, characterized in that the cleavable linker comprises at least one bond selected from the group consisting of carbamate, disulfide, ester, peptide, azide, and combinations thereof.

9. In paragraph 1, A pharmaceutical composition characterized in that the above temporary inactivation is caused by cleavage of the chemical bond with cholesterol in response to enzymes and pH of the tumor microenvironment, or the endosomes and lysosomes within the cell, thereby exposing the activation site of the toll-like receptor 7 or 8 agonist and restoring its function.

10. In paragraph 1, A pharmaceutical composition, characterized in that the pharmaceutical composition further comprises an immune checkpoint inhibitor.

11. In Article 10, A pharmaceutical composition, characterized in that the above immune checkpoint inhibitor is at least one selected from the group consisting of anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-KIR, anti-LAG3, anti-CD137, anti-OX40, anti-CD276, anti-CD27, anti-GITR, anti-TIM3, anti-41BB, anti-CD226, anti-CD40, anti-CD70, anti-ICOS, anti-CD40L, anti-BTLA, anti-TCR, and anti-TIGIT.

12. In paragraph 1, A pharmaceutical composition characterized in that the pharmaceutical composition inhibits cancer proliferation, metastasis, recurrence or resistance to anticancer treatment. 13.a) A first pharmaceutical composition comprising an anticancer therapeutic substance as an active ingredient; and b) a second pharmaceutical composition comprising, as an active ingredient, a temporarily inactivated Toll-like receptor 7 / 8 agonist in which cholesterol is bound to an active site by a cleavable linker; A kit for preventing or treating cancer, characterized in that the first pharmaceutical composition and the second pharmaceutical composition are administered sequentially.

14. In paragraph 13, A kit characterized in that the sequential administration is performed 1 to 20 days after the first pharmaceutical composition is administered.

15. In paragraph 13, A kit, characterized in that the second pharmaceutical composition is administered 1 to 10 times.

16. In paragraph 13, A kit, characterized in that the second pharmaceutical composition is in the form of intravenous injection, intradermal injection, intratumoral injection or subcutaneous injection.

17. In paragraph 13, A kit, characterized in that the anticancer therapeutic substance is an anticancer agent, an anticancer virus, or a radioisotope.

18. In paragraph 17, A kit characterized in that the anticancer agent is at least one selected from the group consisting of doxorubicin, cisplatin, carboplatin, nedaplatin, goserelin, medroxyprogesterone, cyproterone, vinorelbine, cabazitaxel, denosumab, gemcitabine, capecitabine, oxaliplatin, vorinostat, entinostat, 5FU, taxol, topotecan, irinotecan, and pharmaceutically acceptable salts thereof.

19. In paragraph 17, A kit characterized in that the oncolytic virus is any one oncolytic virus selected from the group consisting of herpesvirus, adenovirus, vaccinia virus, poliovirus, measles virus, vesicular stomatitis virus, reovirus, and genetically modified viruses thereof.

20. In paragraph 17, A kit, characterized in that the radioactive isotope is iridium-192 (Ir-192).

21. In paragraph 13, A kit characterized in that the cancer is any one selected from the group consisting of breast cancer, colon cancer, rectal cancer, lung cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, uterine cancer, stomach cancer, bone cancer, blood cancer, head and neck cancer, bone cancer, vaginal cancer, esophagus cancer, lymphoma, gallbladder cancer, endocrine cancer, adrenal cancer, and melanoma.

22. In paragraph 13, A kit characterized in that the toll-like receptor 7 or 8 agonist is at least one selected from the group consisting of an imidazoquinoloine-based agonist, an 8-hydroxyadenine-based agonist, a pteridone-based agonist, a 2-aminopyrimidine-based agonist, a benzoazepine-based agonist, and a 7-thia-8-oxoguanosine-based agonist.

23. In paragraph 13, A kit, wherein the cleavable linker comprises at least one bond selected from the group consisting of carbamate, disulfide, ester, peptide, azide, and combinations thereof.

24. In paragraph 13, The above temporary inactivation is characterized in that, in response to the tumor microenvironment, or enzymes and pH of the endosomes and lysosomes within the cell, the chemical bond with cholesterol is cleaved, exposing the activation site of the toll-like receptor 7 or 8 effector, and the function is restored.

25. In paragraph 13, A kit characterized in that the kit further comprises c) a third pharmaceutical composition comprising an immune checkpoint inhibitor as an active ingredient.

26. In paragraph 25, A kit characterized in that the above immune checkpoint inhibitor is at least one selected from the group consisting of anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-KIR, anti-LAG3, anti-CD137, anti-OX40, anti-CD276, anti-CD27, anti-GITR, anti-TIM3, anti-41BB, anti-CD226, anti-CD40, anti-CD70, anti-ICOS, anti-CD40L, anti-BTLA, anti-TCR, and anti-TIGIT.

27. In paragraph 13, The kit is characterized in that the kit inhibits cancer proliferation, metastasis, recurrence or resistance to anticancer treatment. 28.a) a step of administering a first pharmaceutical composition containing an anticancer therapeutic substance as an active ingredient to a subject in need thereof in a therapeutically effective amount; and b) A method for preventing or treating cancer, comprising administering to the subject a therapeutically effective amount of a second pharmaceutical composition comprising, as an active ingredient, a temporarily inactivated Toll-like receptor 7 / 8 agonist in which cholesterol is linked to an active site by a cleavable linker, 1 to 20 days after administration of the first pharmaceutical composition.

29. Use of a temporarily inactivated toll-like receptor 7 or 8 agonist, wherein cholesterol is linked to the active site by a cleavable linker, to increase the therapeutic effect of anticancer treatment by administering the agent 1 to 20 days after anticancer treatment.

30. Use for producing a drug product comprising a temporarily inactivated toll-like receptor 7 or 8 agonist having cholesterol linked to the active site by a cleavable linker, said drug product being administered 1 to 20 days after the anticancer treatment to increase the therapeutic effect of the anticancer treatment.

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