ASC SPECK in Cancer Immunotherapy

Abstract

The present invention relates to the use of ASC (a cell apoptosis-associated Speck-like protein containing CARD) in cancer immunotherapy. Furthermore, the present invention relates to a composition for treating cancer cells and tumors comprising an ASC speck carrier formed by an ASC protein and a tumor antigen.

Description

Technology Field

[0001] The present invention relates to the use of ASC speck in cancer treatment, and to a composition comprising an ASC speck carrier, in particular a composition comprising ASC speck and a tumor antigen. Background Technology

[0002] According to the World Health Organization (WHO), cancer is a leading cause of death worldwide, with an estimated 9.6 million deaths in 2018. There were 18 million new cases in the same year, and the most commonly diagnosed type of cancer is lung cancer, followed by breast cancer.

[0003] Today, cancer treatment options include surgery, radiation therapy, chemotherapy and targeted drug therapy, bone marrow transplantation, hormone therapy, and immunotherapy. As is well known in the field, most of these treatments have severe side effects and, as clearly demonstrated by mortality rates, still fail to effectively treat all types of cancer. Immunotherapy is relatively safe because it fights cancer by utilizing the body's own defense mechanisms.

[0004] Traditional cancer treatment methods are based on the principle of attacking cancer through chemotherapy, radiation, or surgical removal of tumors. Chemotherapy is a very common method used as a first- or second-line treatment. It attacks not only cancer cells and rapidly proliferating cells but also normal cells, such as bone marrow cells. Consequently, chemotherapy suppresses the patient's immune system as a primary side effect. On the other hand, immunotherapy assists the body's own immune system in attacking cancer cells, enabling the effective treatment of multiple cancer types. Unlike chemotherapy, immunotherapy improves the function of the entire immune system.

[0005] The immune system is a network composed of organs, tissues, cells, and proteins. Its primary function is to protect the body from invaders such as bacteria, viruses, fungi, or any other foreign substances. Due to research on cancer mechanisms, it has been recognized for many years that cancer cells are recognized by the immune system. However, since cancer cells originate from the body's own cells, it is not easy for defense mechanisms to identify them as foreign substances.

[0006] Another important aspect is that for the immune system to successfully protect the body from invaders or abnormal cells, it requires sufficient time to recognize these substances, establish necessary defense mechanisms, and destroy them. Given that cancer cells proliferate rapidly and are highly complex, the body's defense cells typically cease functioning against them before completing their role. Therefore, the immune system is considered to require assistance to protect the body from rapidly proliferating cancer cells.

[0007] Immunotherapy is a relatively new treatment method that is not widely known. First, the role of the immune system in cancer is not fully understood. Second, proteins involved in natural defense mechanisms are highly sensitive to environmental changes and easily lose their function. These factors present challenges in both developing new treatment options and applying these therapies to patients.

[0008] Recent research in immunology and tumor biology offers new insights into understanding the interactions between the immune system and tumor cells. The importance of T cells in mediating anti-tumor immunity has been established in numerous studies. The carcinogenic process induces changes in antigen expression in tumor cells recognizable by host T cells (Dermime, S., A. Armstrong, RE Hawkins and PL Stern, "Cancer vaccines and immunotherapy", British medical bulletin, Vol. 62, No. 1, pp. 149-162, 2002).

[0009] Recently, immune checkpoint inhibitors, engineered T cells, monoclonal antibodies, and cancer vaccines are commonly used as immunotherapies for many types of cancer (Kamta, J., M. Chaar, A. Ande, DA Altomare and S. Ait-Oudhia, "Advancing cancer therapy with present and emerging immuno-oncology approaches", Frontiers in oncology, Vol. 7, p. 64, 2017).

[0010] Cancer vaccines can be divided into two main groups: preventive vaccines or therapeutic vaccines. Theoretically, cancer preventive vaccines should prevent the development of tumors and provide long-lasting immunity. The first example of a preventive vaccine is for HBV and HPV infections (Chang, M.-H., "Hepatitis B Virus and Cancer Prevention", Recent results in cancer research. Fortschritte der Krebsforschung . Progres dans les recherches sur le cancer , Vol. 188, pp. 75-84, 2010).

[0011] Therapeutic vaccines can be divided into four major subgroups based on their composition: whole-cell tumor, dendritic cell, DNA, and subunit vaccines. Whole-cell tumor vaccines are mostly allogeneic, and achieving success is limited due to the significant level of tumor heterogeneity observed among patients (Srivatsan, S., JM Patel, EN Bozeman, IE Imasuen, S. He, D. Daniels and P. Selvaraj, "Allogeneic tumor cell vaccines", Human Vaccines & Immunotherapeutics , Vol. 10, No. 1, pp. 52-63, 1 2014). Dendritic cell vaccines are prepared by extracting cells from a patient and then stimulating them with a specific antigen and / or adjuvant (Kamta, J., M. Chaar, A. Ande, DA Altomare and S. Ait-Oudhia, "Advancing Cancer Therapy with Present and Emerging Immuno-Oncology Approaches.", Frontiers in oncology , Vol. 7, p. 64, 2017). There is Sipuleucel, an FDA-approved dendritic cell vaccine for prostate cancer (Guo, C., MH Manjili, JR Subjeck, D. Sarkar, PB Fisher and X.-Y. Wang, "Therapeutic Cancer Vaccines", Advances in cancer research, Vol. 119, pp. 421-475, 2013). DNA vaccines are newly emerging in cancer immunotherapy and basically require the sequence of a tumor-associated antigen.

[0012] Subunit vaccines for cancer immunotherapy contain antigens or epitopes of antigens, just like classical vaccines. Subunit vaccines are also utilized to treat melanoma, prostate cancer, testicular cancer, and breast cancer. Although the immune booster effect of subunit vaccines is weaker than that of whole-tumor vaccines, its efficacy can be enhanced with adjuvants (Temizoz, B., E. Kuroda and KJ Ishii, "Vaccine adjuvants as potential cancer immunotherapeutics", International Immunology , Vol. 28, no. 7, pp. 329-38, 2016).

[0013] Vaccines aim to induce adaptive immunity that can be enhanced by using adjuvants in some vaccine formulations. Mineral salts, cytokines, emulsions, microbial particles, and liposomes can be used as adjuvants (Guy, B., "The perfect mix: recent progress in adjuvant research.", Nature Reviews Microbiology, Vol. 5, No. 7, pp. 505-17, 6 2007). Although they have been used for over eight years, the mechanisms of action of adjuvants are only just beginning to be elucidated (Awate, S., LA Babiuk and G. Mutwiri, "Mechanisms of action of adjuvants.", Frontiers in immunology, Vol. 4, p. 114, 2013).

[0014] Aluminum salts (alum) are the most commonly used adjuvants in human vaccines. The inductive effect of aluminum hydroxide on diphtheria was first reported in guinea pigs in 1926, and various alum formulations have been used in human vaccines since 1931 (Marrack, P., AS McKee and MW Munks, "Towards an understanding of the adjuvant action of aluminum.", Nature reviews. Immunology, Vol. 9, No. 4, pp. 287-93, 2009).

[0015] However, the mechanism of action of alum reinforcing agents has not been fully understood to date. In recent publications, it has been reported that alum induces the immune system to caspase 1 and IL-1 cytokines, which are products of the inflammasome complex (Franchi, L., T. Eigenbrod, R. Munoz-Planillo and G. Nunez, "The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis.", Nature Immunology, Vol. 10, No. 3, pp. 241-7, 3 2009; .10, Eisenbarth, SC, OR Colegio, W. O'Connor, FS Sutterwala and RA Flavell, "Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminum adjuvants", Nature, Vol. 453, No. 7198, pp. 1122-1126, 6 2008; Grun, JL and PH Maurer, Different T helper cell subsets elicited in mice utilizing two different adjuvant vehicles: the role of endogenous interleukin 1 in proliferative responses.", Cellular immunology, Vol. 121, No. 1, pp. 134-45, 6 1989).

[0016] Following alum, a new adjuvant, AS04, was approved by the FDA for HPV in 1970. AS04 is formed from a combination of alum and monophosphoryl lipid A. Finally, the FDA authorized the use of AS03 for epidemic H5N1 influenza, but it is no longer commercially available. The formulation of MF59 used in influenza vaccines is very similar to AS03 but has not been approved by the FDA (Awate, S., LA Babiuk and G. Mutwiri, "Mechanisms of action of adjuvants", Frontiers in immunology, Vol. 4, p. 114, 2013).

[0017] Various mechanisms have been proposed for the booster effect of adjuvants on immune activation, which basically involve the enhancement of antigen uptake, the mobilization of immune cells to the injection site, and subsequent specific immune responses such as Th1 and Th2 cells following the activation of PRRs and inflammasomes (Reed, SG, MT Orr and CB Fox, "Key roles of adjuvants in modern vaccines", Nature Medicine, Vol. 19, No. 12, pp. 1597-1608, 12 2013).

[0018] Receptors of the innate immune system, such as TLRs, are targets of adjuvants. When an adjuvant activates TLRs, certain chemokines and cytokines are secreted into the extracellular matrix. Subsequently, these small molecules assist in the direction of cellular and humoral immunity. For example, the secretion of IL-4 from APCs and naive CD4 cells directs immunity to induce IgG production, as IL-12 and IFN-γ trigger a greater cellular immune response (Reed, SG, MT Orr and CB Fox, "Key roles of adjuvants in modern vaccines", Nature Medicine, Vol. 19, No. 12, pp. 1597-1608, 12 2013).

[0019] Since specific signature antigens were identified on human tumor cells, innovative approaches have been developed, leading to antigen-specific vaccination. The combination of specific tumor antigens and appropriate pathways for delivering these antigens to the immune system aims to optimize human-based vaccination (Dermime, S., A. Armstrong, RE Hawkins and PL Stern, "Cancer vaccines and immunotherapy", British medical bulletin, Vol. 62, No. 1, pp. 149-162, 2002).

[0020] Tumor cells can also be used as vaccines. Since these cells themselves do not induce an adequate immune response, researchers have focused on addressing this non-responsiveness by introducing co-stimulatory molecules or cytokine genes into the tumor cell genomes and then using them as vaccines to administer back to the same patients. However, the production of this type of vaccine is somewhat slow, labor-intensive, and logistically difficult.

[0021] Alternatively, recombinant viral vectors can be used to deliver tumor antigens into the body. The inherent ability of viral vectors to initiate an immune response and build an inflammatory response as a result of viral infection is an advantage of these vaccines. However, viral vectors must ideally be safe and should not promote an anti-vector immune response to boost an anti-tumor-specific immune response (Dermime, S., A. Armstrong, RE Hawkins and PL Stern, "Cancer vaccines and immunotherapy", British medical bulletin, Vol. 62, No. 1, pp. 149-162, 2002).

[0022] Despite cancer treatment methods disclosed in the relevant technical field, the incidence of cancer and the associated mortality rate are increasing every year. Therefore, there is still a significant demand for new cancer treatment methods in this technical field.

[0023] ASC (a CARD-containing apoptosis-associated speck-like protein) is a 22 kDa adapter protein possessing a CARD domain at the C-terminus and a PYD domain at the N-terminus, which function to form inflammasome complexes, specifically NLRP3, NLRC4, and AIM2 inflammasomes. The ASC protein plays a role in activating the caspase-1 protein in inflammatory and pyroptosis signaling pathways (Franchi et al., 2009). It is a cytoplasmic protein that forms a speck-like structure upon the activation of the inflammasome (Miao et al., 2011).

[0024] ASC proteins polymerize into filaments to form a large supramolecular scaffold referred to as an ASC speck (Sahillioglu, AC, F. Sumbul, N. Ozoren and T. Haliloglu, "Structural and dynamics aspects of ASC speck assembly", Structure, Vol. 22, No. 12, pp. 1722-1734, 2014). ASC specks can exist inside the cell or be released externally (Sahillioglu, AC and N. Ozoren, "Artificial loading of ASC specks with cytosolic antigens", PloS one, Vol. 10, No. 8, p. e0134912, 2015).

[0025] The NLRP3 inflammasome, which also contains ASC protein, can be triggered by stimuli such as extracellular ATP, membrane damage toxins (nigericin), lysosomal damage, monosodium urea (MSU), and UV rays. NLRP3 inflammasome activation is accompanied by the synthesis of ASC speck. It has been found that ASC speck can be synthesized in vitro by incubating purified recombinant ASC protein in a hypotonic solution (Fernandes-Alnemri et al., 2007).

[0026] International patent application WO 2009 / 014863 discloses that ASC proteins and pyroptosomes can be used to treat and diagnose autoimmune diseases. According to this application, pyroptosome and pyroptosome structures containing ASC dimers and procaspase-1 occur in macrophages during the initial inflammatory response. Caspase-1 activation depends on the formation of pyroptosomes. The inflammatory state is diagnosed by isolating pyroptosomes from macrophages.

[0027] It was hypothesized that after influenza vaccination with an MF59-adjuvant, the ASC protein plays a role in triggering an antigen-specific humoral response (Ellebedy et al., 2011).

[0028] In addition, the invention disclosed in patent application WO 2013 / 160807 revealed that antigens and / or viable molecules can be loaded onto an ASC speck carrier for use in vaccination. This invention was developed by the inventor of the present invention. In this document, it was confirmed that the stability of viable molecules can be enhanced for more than 30 days by using ASC speck as a carrier, and that they can withstand at least 8 freeze-thaw cycles without degradation. That is, the aforementioned application discloses a composition generally useful for vaccines. However, the therapeutic effect of ASC speck itself or a composition containing ASC speck on cancer cells or tumors is not mentioned.

[0029] In conclusion, none of the prior art literature mentions the role of ASC speck in cancer treatment.

[0030] The objective of the present invention is to provide a new method for treating cancer.

[0031] The present invention relates to the use of ASC (a Speck-like protein related to apoptosis containing CARD) speck in cancer immunotherapy.

[0032] In one embodiment of the present invention, a composition comprising an ASC speck carrier and a tumor antigen is provided for therapeutically treating cancer cells or tumors.

[0033] In another embodiment of the present invention, a composition comprising an ASC speck carrier is provided, wherein the ASC speck is formed by a fusion protein of an ASC protein and a tumor antigen.

[0034] In another embodiment of the present invention, ASC speck is used in cancer treatment for its ability to enhance the immune system against tumor cells.

[0035] In addition, in another embodiment of the present invention, a composition comprising an ASC speck, preferably a combination of an ASC speck and a tumor antigen, is provided for use in cancer treatment comprising inducing and / or stimulating an immune response against cancer in mammals, particularly human individuals, wherein the induction or stimulation causes a reduction in cancer progression or a cessation of cancer cell proliferation. Brief explanation of the drawing

[0036] Fig. 1. HEK293FT Production and purification of tOVA-ASC speck from cell lines. a) p-C3-d (1-238) OVA-ASC plasmid encoding tOVA-ASC. Comacy blue staining (b), anti-hASC blot (c), and brightfield image (d) of the purified tOVA-ASC speck. e) Fluorescence image of the purified mCherry-ASC speck. Fig. 2. Engulfment of mCherry-ASC speck by PMA-differentiated THP-1 macrophages. a) Brightfield and fluorescence images of THP-1 macrophages acquired 12 hours after treatment. b) Anti-hASC blots of THP-1 macrophages treated with PBS, mCherry, and mCherry-ASC. c) Flow cytometry analysis of phagocytosed mCherry-ASC speck. Fig. 3. Degradation of mCherry-ASC speck phagocytosed by THP-1 macrophages. Fluorescence image of phagocytosed and degraded mCherry-ASC speck obtained 12 hours after mCherry-ASC treatment. Fig. 4.Complete degradation of mCherry-ASC speck leading to inflammasome formation in host cells. Fluorescence image (ab) and merged image (c), d) 3X magnified image of the blue frame in the merged image for anti-hASC staining of mCherry-ASC speck phagocytosed by THP-1 macrophages. Fig. 5. Secretion of pro-inflammatory cytokines induced by mCherry-ASC treatment. a) mRNA expression levels of NLRP3, pro-caspase-1, and ASC using RT-qPCR, and secretion levels of IL-1β (b) and TNF-α (c) measured after mCherry-ASC treatment of THP-1 macrophages. (Error bars represent the standard deviation of two biological replicas). Fig. 6. Phagocytosis of tOVA-ASCs by PMA-differentiated THP-1 macrophages. a) Immunochemical method of THP-1 macrophages using anti-hASC antibodies 12 hours after treatment with tOVA-ASCs, PBS, and OVA. b) Anti-OVA and c) anti-hASC blots of THP-1 macrophages after treatment with PBS, OVA, and tOVA-ASCs. Fig. 7. Secretion of pro-inflammatory cytokines in PMA-differentiated THP-1 macrophages upon tOVA-ASC treatment. Secretion levels of IL-1β (a), TNF-α (b), and IL-6 (c) in THP-1 macrophages treated with PBS, 10 µg OVA, 2 µg, 5 µg, and 10 µg tOVA-ASC speck, measured by ELISA. Fig. 8. Cytokine secretion levels in splenocytes of tOVA-ASC-injected animals after OVA pulsing. Splenocytes were pulsed with or without OVA. Cytokine levels in splenocytes were measured in the cell supernatant by ELISA (n=4 for the OVA group, n=6 for the tOVA-ASC group). Fig. 9.Chicken ovalbumin-producing EG7-OVA cell line used in a tumor model. a) Anti-OVA blot against the EG7-OVA cell line. b) Timeline for the EG7-OVA tumor model experiment (OVA: 43kDa). Fig. 10. EG7-OVA tumor progression after tOVA-ASC injection. a) Average tumor volume of all injection groups after injection (each dot represents a single tumor). b) Progression of each tumor across multiple groups. c) Average weight of excised tumors (n=3 for PBS, n=5 for OVA, n=8 for the tOVA-ASC injection group). Fig. 11. Eradication of subcutaneously inoculated EG7-OVA tumors following two intraperitoneal injections of tOVA-ASC speck. Photographs of tumors excised from PBS, OVA, and tOVA-ASC injected animals were taken. Six out of eight tumors in the tOVA-ASC injected animals were completely eliminated. Fig. 12. OVA-specific IgG response upon injection of 100 µg tOVA-ASC in C57BL / 6 mice with EG7-OVA tumors. Anti-OVA IgG response was measured by ELISA in plasma taken immediately before sacrifice of animals (n=1 for PBS, n=1 for OVA, and n=6 for the tOVA-ASC injection group). Fig. 13 . Intraperitoneally injected tOVA-ASC speck was detected in subcutaneously inoculated EG7-OVA tumors. Anti-TMS1 blots on tumor samples from tOVA-ASC, OVA, and PBS-injected animals. Fig. 14. Infiltration of CD11C+ dendritic cells into EG7-OVA tumors in tOVA-ASC-injected animals. 50 µm fragments of tumors from PBS, OVA, and tOVA-ASC-injected animals were taken using a cryostat and then stained with anti-CD11c antibody (CD11c: dendritic cell marker. White arrows indicate CD11c+ cells, and red arrows indicate non-specific antibody residues in gaps of tumor tissue). Fig. 15. Amplification of T cells in the spleen of tOVA-ASC-injected animals. Splenocytes derived from spleens extracted from PBS and tOVA-ASC-injected animals were stained with anti-CD4, anti-CD8, and anti-B220 antibodies, and the percentage of these cells was analyzed by flow cytometry (CD4: T helper cells, CD8: cytotoxic T cells, B220: B cell marker; error bars indicate the SD values ​​between animals in the tOVA-ASC group). Fig. 16 mCherry-ASC injected intraperitoneally was placed in the mesenteric lymph nodes of one animal. Animals injected with PBS, mCherry, and mCherry-ASC were anesthetized, and results were obtained at 580 nm using an IVIS device on day 1 (a) and day 2 (b). Fig. 17 . Drainage of mCherry-ASC spect injected intraperitoneally into secondary lymphatic organs. Lymph nodes (A) and spleens (B) of animals injected with PBS, mCherry, and mCherry-ASC were lysed by sonication and then blotted with anti-TMS-1 antibody. Fig. 18. Timeline for immunization of mice with EG7-OVA tumors. Fig. 19. Tumor volume after immunization. After immunization, the tOVA-ASC group exhibited the best anti-tumor activity compared to other groups. In the mCh-ASC+OVA group, one animal showed complete tumor eradication after immunization, yielding a debatable result. Each dot represents a mouse (n=4). Fig. 20. OVA-specific IgG responses in the experimental groups of the EG7-OVA model. OVA-specific IgG responses were best in tOVA-ASC immunized mice. The mCh-ASC+OVA group exhibited superior OVA-specific IgG levels compared to the OVA group. IgG levels in the OVA, mCh-ASC+OVA, and tOVA-ASC groups were significantly increased compared to the PBS group (n=4 in each group). Specific details for implementing the invention

[0037] The main objective of the present invention is to develop a new cancer immunotherapy. This is achieved by utilizing an ASC protein that plays a critical role in inflammatory signaling pathways.

[0038] The ASC protein, also known as PYCARD, is an adapter protein encoded by the PYCARD gene. It has an N-terminal PYRIN domain, a C-terminal caspase recruitment domain (CARD), and a linker between them. The ASC protein forms a bridge between pro-caspase-1 via CARD-CARD interactions and nucleotide-binding oligomerized domain-like receptor (NLR) proteins via PYRIN-PYRIN interactions.

[0039] Cytoplasmic NLR family proteins detect pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs). Some NLRs, such as NLRP3 and AIM2, induce the formation of inflammasome complexes. Caspase-1 is activated within the inflammasome multiprotein complex through interaction with ASC proteins. Activated caspase-1 cleaves pro-IL-1β and pro-IL-18 into their mature forms, and these cytokines are released into the extracellular space to induce inflammation.

[0040] In unstimulated cells, ASC is cytoplasmically soluble. Upon stimulation, ASC proteins aggregate to form spherical structures referred to as ASC specks. ASC specks are secreted extracellularly and can accelerate the recruitment of immune cells.

[0041] For the purposes of the present invention, chicken ovalbumin protein (OVA) is used as a model antigen.

[0042] Since one of the molecular mechanisms of action of current adjuvants is the enhancement of antigen uptake by APCs, THP-1 mononuclear cell lines were used for cell culture experiments. To determine whether ASC speck is phagocytosed by APCs, PMA-differentiated THP-1 macrophages were exposed to tOVA-ASC speck and mCherry-ASC speck, respectively. Through these experiments, it was found that both tOVA-ASC speck and mCherry-ASC speck were phagocytosed by macrophages in a concentration-dependent manner (Fig. 2). Furthermore, it was found that the phagocytosed mCherry-ASC speck was degraded by THP-1 macrophages and was detected at various intracellular locations by fluorescence and confocal microscopy (Fig. 3).

[0043] The inflammasome is one of the targets of commonly used adjuvants, alum, and currently produced adjuvants AS03 and MF59. To determine whether the adjuvant ASC speck proposed according to the present invention can induce an inflammatory response in THP-1 macrophages, changes in the mRNA expression levels of pro-inflammatory proteins—NLRP3, ASC, and pro-caspase-1—were confirmed by RT-qPCR. In this regard, treatment with mCherry-ASC speck was found to increase the levels of pro-inflammatory proteins by at least 1.5-fold compared to treatment with mCherry protein alone (Fig. 5).

[0044] To support the RT-qPCR data, IL-1β, TNF-α, and IL-6 cytokine secretion levels were determined by ELISA after treatment with either mCherry-ASC speck or tOVA-ASC speck. IL-1β secretion increased in a time- and concentration-dependent manner. The increase in TNF-α secretion levels was concentration-dependent at the initial time point (12 hours after treatment), but no difference in TNF-α release levels was observed at the later time point (24 hours after treatment) (Fig. 5). tOVA-ASC treatment enhanced IL-1β secretion in a concentration-dependent manner. At each concentration, tOVA-ASC speck treatment induced higher levels of IL-1β compared to 10 µg OVA treatment. TNF-α and IL-6 levels increased in a concentration-dependent manner after tOVA-ASC treatment, and cytokine secretion levels were higher compared to after 10 µg OVA treatment. In conclusion, the ASC speck generated by the fusion of the antigen and ASC induces the secretion of inflammatory cytokines from THP-1 macrophages (Fig. 7). Based on these RT-qPCR and ELISA data, it was concluded that ASC speck treatment leads to an increase in the inflammatory response in macrophages.

[0045] Antibody titers are not the only measure of a superior vaccine, and cytokine production by CD4+ cells may be more closely related to good immunization (Plotkin, 2010). To investigate cytokine production by T helper cells induced by tOVA-ASC speck in immune cells, an in vitro recall assay was performed, and levels of TNF-α, IFN-γ, and IL-4 were measured during OVA pulsing and non-pulsing. IFN-γ is a cytokine primarily produced by T lymphocytes and natural killer cells, playing a critical role in inhibiting tumor progression and metastasis (Farrar, MA and RD Schreiber, "The Molecular Cell Biology of Interferongamma and its Receptor", Annual Review of Immunology , Vol. 11, No. 1, pp. 571-611, 4 1993). In addition, it has been used as a direct antiviral agent. It is specifically generated by type 1 T helper cells after vaccination, induces a delayed hypersensitivity response, and consequently induces cell-mediated immunity (Romagnani, S., "Type 1 T helper and type 2 T helper cells: functions, regulation and role in protection and disease", International journal of clinical & laboratory research , Vol. 21, No. 2, pp. 152-8, 1991; Godfroid, J., G. Czaplicki, P. Kerkhofs, V. Weynants, G. Wellemans, E. Thiry and JJ Letesson, "Assessment of the cell-mediated immunity in cattle infection after bovine herpesvirus 4 infection, using an in vitro antigen-specific interferon-gamma assay", Veterinary microbiology , Vol. 53, No. 1-2, pp. 133-41, 11 1996). Through in vitro recall analysis, the inventors of the present invention confirmed that both OVA pulsed and non-pulse treated splenocytes of tOVA-ASC speck-injected animals secrete IFN-γ, and that secretion levels decreased after the OVA pulse, whereas the other group secreted IFN-γ only after OVA treatment. In this regard, tOVA-ASC speck appears to be capable of inducing a cellular immune response without antigen specificity.

[0046] TNF-α is a pro-inflammatory cytokine that may be necessary for implementing humoral and cellular immune responses. However, it also leads to loss of function in NK cells, consequently causing reduced IFN-γ secretion and cytotoxic activity against certain types of cancer (Romero-Reyes, M., C. Head, NA. Cacalano and A. Jewett, "Potent induction of TNF-α during interaction of immune effectors with oral tumors as a potential mechanism for the loss of NK cell viability and function", Apoptosis , Vol. 12, No. 11, pp. 2063-2075, 9 2007).

[0047] In the experiment of the present invention, the splenocytes of each animal secreted TNF-α after OVA pulsing, but the levels in the splenocytes of tOVA-ASC and Alum+OVA injected animals were weaker than those in OVA injected animals.

[0048] The cytokine IL-4 is primarily produced by type 2 T helper cells and is known to be a mutual antagonist of IFN-γ. It regulates B cell proliferation and eosinophil mobilization in vivo (Smiley, ST and MJ Grusby, "Interleukin 4", Encyclopedia of Immunology , pp.1451-1453, Elsevier, 1998). Although the secretion of IL-4 from splenocytes of each animal decreases after OVA pulsing, splenocytes of tOVA-ASC injected animals secrete cytokines at the highest levels during OVA pulsing and non-pulsing.

[0049] In conclusion, the inventors of the present invention confirmed that the secretion of TNF-α and IL-4 from splenocytes of tOVA-ASC-injected animals is very similar to that of the commonly used adjuvant Alum+OVA (Fig. 8). The difference between the proposed adjuvant ASC speck and the alum adjuvant is the secretion of IFN-γ prior to OVA pulsing. Since IFN-γ leads to a greater cell-mediated immune response, cell immunity can be boosted using ASC speck.

[0050] As mentioned above, immunotherapy is one of the most extensively studied fields regarding cancer. Vaccine adjuvants have been developed to boost patients' cellular immune responses against cancer, and IFN-γ is one of the major cytokines that directs the immune system largely toward the cellular immune component. Since tOVA-ASC speck induces the secretion of IFN-γ in splenocytes, the in vivo effects of tOVA-ASC speck on tumorigenesis were investigated using the C57BL / 6 EG7-OVA tumor model. For this purpose, 2.5 x 10 6EG7-OVA cells were subcutaneously inoculated onto the dorsal side of mice, and intraperitoneal injection of tOVA-ASC speck was initiated when the tumor reached a palpable size. Due to the heterogeneity of tumor sizes and the limited number of animals, mice were divided into an experimental group and a control group, with at least one small, medium, and large tumor included in each group. PBS and OVA injections were used as negative controls.

[0051] In animals injected with tOVA-ASC, four tumors were completely eradicated on day 7 following the first injection, whereas one additional tumor formed in animals injected with OVA at the same time. A booster injection was subsequently administered on day 7. On day 2 following the booster injection, two or more tumors were completely eradicated in the tOVA-ASC group. In the PBS group, one animal died. Based on the progression of tumors in tOVA-ASC injected animals, it was confirmed that the tOVA-ASC speck somehow possessed a therapeutic effect on EG7-OVA tumors, and with only two tumors remaining in the tOVA-ASC group, the experiment was terminated to determine the cellular and molecular mechanisms of this therapeutic effect of the ASC speck. In the autopsy of the animals, in which tumors were completely eradicated prior to autopsy, only connective tissue was present at the tumor inoculation site, and no tumor tissue remained. For the PBS and OVA groups, the average tumor weight was about 2 g, whereas for the tOVA-ASC group it was 0.03 g (Fig. 10).

[0052] In additional experiments on animal serum, the OVA-specific IgG response was successfully increased to approximately 18 µg / ml in animals injected with tOVA-ASC, whereas it was 5 µg / ml and 8 µg / ml in the PBS and OVA groups, respectively (Fig. 12). The increase in the IgG response in the tOVA-ASC group may be due to two main reasons. First, since the EG7-OVA tumor continues to produce OVA protein, if the tOVA-ASC speck induces a general immune burst in mice, it may trigger antibody production against OVA. Second, it may be because the amount of tOVA-ASC speck injected intraperitoneally was doubled.

[0053] In addition, the inventors of the present invention confirmed that in one of the animals, the tOVA-ASC speck injected intraperitoneally migrated to the EG7-OVA tumor site inoculated subcutaneously, while the remainder remained intact in at least the form of tOVA-ASC protein (Fig. 13). Basically, this may occur due to the influx of tOVA-ASC speck from the intraperitoneal site into the circulatory system, and leakage into the tumor resulting from intensive vascularization surrounding the tumor microenvironment and the exposure of these vessels. Another reason may be that the tOVA-ASC speck is phagocytosed by APCs and somehow accumulated within the tumor by these APCs. Since tumor-infiltrating mature DCs confer immune activation and increased mobilization of immune effector cells, IHC staining was used to confirm whether the DCs had infiltrated the tumor. A CD11C+ antibody that stains conventional / classical DC subtypes was used.

[0054] This subtype has a characteristic morphology defined by MHC class II expression (Collin, M., N. McGovern and M. Haniffa, "Human dendritic cell subsets.", ImmunologyVol. 140, No. 1, pp. 22-30, 9 2013). The inventors of the present invention confirmed that CD11C+ DCs were located in the tumor frozen fragments of one of the animals injected with tOVA-ASC (Fig. 14). Interestingly, tOVA-ASCs were found in CD11C+ DC-invasive tumors. IHC and Western blot data increase the likelihood that tOVA-ASC specks are transported into the tumor microenvironment by DCs.

[0055] The molecular and cellular mechanisms of the therapeutic effects of tOVA-ASC speck were investigated using the EG7-OVA tumor model again. EG7-OVA cells were inoculated onto the dorsal sides of 24 mice, resulting in the formation of only four tumors. To understand the effects of tOVA-ASC speck on immune cell amplification and tumor invasion, PBS and tOVA-ASC were injected into these four animals. On day 7 following the first injection, one of the tumors in the tOVA-ASC-injected animals began to shrink, and by day 10, it was no longer propellable. Subsequently, the experiment was terminated, and the proportions of helper T cells, cytotoxic T cells, and B cells in the spleen were confirmed by flow cytometry analysis. In the tOVA-ASC-injected animals, the percentage of CD4+ and CD8+ cells in the spleen increased compared to the PBS-injected animals (Fig. 15). Based on cytokine secretion and flow cytometry analysis in splenocytes, it can be concluded that tOVA-ASC injection induces the formation of helper T cells in C57BL / 6 mice.

[0056] Vaccines are generally injected subcutaneously, intramuscularly, or intraperitoneally to stimulate immunity. Vaccine success is enhanced by the drainage of antigens into lymph nodes where APCs interact with lymphocytes (Johansen, P. and TM Kundig, "Intralymphatic immunotherapy and vaccination in mice.", Journal of visualized experiments: JoVE,No. 84, p. e51031, 2 2014; Tozuka, M., T. Oka, N. Jounai, G. Egawa, KJ Ishii, K. Kabashima and F. Takeshita, "Efficient antigen delivery to the draining lymph nodes is a key component in the immunogenic pathway of the intradermal vaccine", Journal of Dermatological Science, Vol. 82, No. 1, pp. 38-45, 4 2016). In addition, antigen transfer by migratory DCs from the injection site to lymphatic organs also enhances the efficiency of CTL sensitization (Allan, RS, J. Waithman, S. Bedoui, CM Jones, JA Villadangos, Y. Zhan, AM Lew, K. Shortman, WR Heath and FR Carbone, "Migratory Dendritic Cells Transfer Antigen to a Lymph Node-Resident Dendritic Cell Population for Efficient CTL Priming", Immunity , Vol. 25, No. 1, pp. 153-162, 7 2006). To determine whether ASC speck triggers antigen drainage to secondary lymph nodes, mCherry-ASC speck was injected intraperitoneally into C57BL / 6 mice and visualized using IVIS. Interestingly, red fluorescence signals were detected from the spleen and mesenteric lymph nodes in mCherry-ASC injected animals the day after immunization, whereas no fluorescence signals were observed upon excitation at 580 nm in PBS and mCherry alone injected animals (Fig. 16). Additionally, the inventors verified the IVIS results by sacrificing the animals on day 2 after injection and performing anti-hASC blots on the mesenteric lymph nodes and spleen (Fig. 17).

[0057] A detailed description of the tests is provided in the experimental section of this specification. Brief information regarding these experiments and their results is described below.

[0058] First, the tOVA-ASC protein (a fusion protein of the human ASC protein and the truncated form of OVA containing the amino acid sequence of 239-386) is produced by the p-C3-d (1-238) OVA-ASC plasmid.

[0059] The truncated form of OVA contains all of the T cell epitopes of OVA; the OVA peptides of amino acids 257–268 and 323–339 are MHC class I and MHC class II epitopes, respectively (Lipford, GB, M. Hoffman, H. Wagner and K. Heeg, "Primary in vivo responses to ovalbumin. Probing the predictive value of the Kb binding motif.", Journal of Immunology (Baltimore, Md . : 1950) , Vol. 150, no. 4, pp. 1212-22, 2 1993; McFarland, BJ, AJ Sant, TP Lybrand and C. Beeson, "Ovalbumin (323-339) peptide binds to the major histocompatibility complex class II IA(d) protein using two functionally distinct registers.", Biochemistry , Vol. 38, No. 50, pp. 16663-70, 12 1999). tOVA-ASC speck is produced using tOVA-ASC protein.

[0060] In addition, tOVA+ASC speck is produced by carrying the tOVA protein onto ASC speck, and then the adjuvant effect of ASC speck in cancer immunotherapy is investigated using this.

[0061] It is important to note that there are various methods for loading tumor antigens onto an ASC speck. In general, some of the methods for loading live molecules, particularly tumor antigens, onto an ASC speck according to the present invention refer to published patent application WO 2013 / 160807 (method for delivering antigens).

[0062] For example, a tumor antigen can be loaded onto an ASC speck carrier by forming a fusion protein between an ASC protein that forms an ASC speck carrier and one or more antigens, or through hydrophobic interactions.

[0063] Antigens with hydrophilic characteristics cannot be transported using an ASC speck carrier by hydrophobic interactions alone. Accordingly, in one embodiment, an antigen with hydrophilic characteristics can be loaded onto an ASC speck carrier through hydrophobic interactions by constructing a fusion protein between the antigen and a peptide containing 13 or more amino acids and a hydrophobic sequence.

[0064] Second, mCherry-ASC speck is produced and purified according to the method described in WO 2013 / 160807. mCherry is a red fluorescent protein used as a tracer in imaging systems.

[0065] In experiments using the aforementioned ASC speck, it was confirmed that purified ASC speck is phagocytosed and degraded by macrophages. Furthermore, it was demonstrated that stimulation of THP-1 macrophages by purified ASC speck induces the secretion of inflammatory cytokines: IL-1β, IL-6, and TNF-α. In addition, it was confirmed that tOVA-ASC speck injection induces the secretion of type 1 and type 2 cytokines in splenocytes of immunized wild-type C57BL / 6 mice. Finally, the inventors of the present invention demonstrated that tOVA-ASC injection induces complete eradication or reduction in size of EG7-OVA tumors in C57BL / 6 mice.

[0066] As described above, chicken ovalbumin protein (OVA) is used as a model antigen for the purposes of the present invention. To produce tOVA-ASC (a fusion protein of a human ASC protein and a terminally truncated form of egg albumin protein, i.e., OVA, containing a sequence of 239-386 amino acids), the p-C3-d (1-238)OVA-ASC plasmid is cloned.

[0067] To examine the immunostimulatory effects of ASC speck in vivo and in THP-1 cell lines, mCherry-ASC speck and tOVA-ASC speck were produced in HEK293 FT cell lines. To determine the purity and concentration of tOVA-ASC speck, the purified speck and a predetermined concentration of BSA were loaded onto SDS-PAGE and stained with Comarsh blue as shown in Figure 1. An anti-hASC blot was performed on the isolated protein to confirm whether the purified protein was tOVA-ASC.

[0068] To investigate the effect of ASC speck on antigen uptake, PMA-differentiated THP-1 macrophages were treated with mCherry-ASC speck at concentrations of 10 µg / ml, 25 µg / ml, 50 µg / ml, and 100 µg / ml. PBS and commercial mCherry protein at 50 µg / ml were used as negative controls. After 12 hours, no mCherry signal was observed in the cases treated with PBS and mCherry protein, while cells treated with mCherry-ASC speck phagocytized mCherry-ASC speck as shown in Figure 2. hASC blots of the treated cells also showed phagocytization and degradation of mCherry-ASC protein.

[0069] To validate and quantify the fluorescence data, the same experiment was repeated, and mCherry-positive cells were counted by flow cytometry 12 hours after treatment. The number of mCherry-positive cells increased with increasing concentration of mCherry-ASC speck during treatment. In these experiments, ASC speck was demonstrated to enhance the phagocytosis of mCherry protein by THP-1 macrophages.

[0070] The degradation of mCherry-ASC speck phagocytosed by THP-1 macrophages was detected under a fluorescence microscope (Fig. 3). After 4 hours of treatment with mCherry-ASC, a red fluorescence signal was observed in the cytoplasm of the cell when using a fluorescence microscope at 40X magnification, and it was spread throughout the cytoplasm of the cell rather than to speck-like structures.

[0071] The expression levels of NLRP3 ASC and pro-caspase-1 (pro-inflammatory protein) were determined by RT-qPCR after treatment with 10 µg mCherry-ASC speck, 10 µg mCherry, and 10 µg PBS. HPRT, a housekeeping gene, was used as a control for qPCR, and the expression of all genes was standardized according to HPRT expression. As shown in Figure 5, the expression levels of NLRP3, pro-caspase-1, and ASC significantly increased after treatment with mCherry-ASC compared to treatment with mCherry.

[0072] To determine whether ASC speck induces a pro-inflammatory response after phagocytosis, secreted cytokines were measured in the medium of PMA-differentiated THP-1 macrophages at 12 and 24 hours after treatment with mCherry-ASC. PBS, mock purification samples, and commercial mCherry protein were used as negative controls. Treatment with mCherry-ASC speck increased the secretion levels of IL-1β and TNF-α in a time- and concentration-dependent manner, whereas cells treated with mCherry and mock purification samples secreted cytokines at basal levels, as shown in Figure 5.

[0073] In conclusion, it is presumed that although the peptide used is not antigenic to ASC speck in THP-1 macrophages, phagocytosis of the peptide is enhanced and an inflammatory response may be induced.

[0074] In addition, to confirm whether tOVA-ASC speck is also phagocytosed by APCs, PMA-differentiated THP-1 macrophages were treated with 10 µg / ml tOVA-ASC speck, and immunohistochemistry analysis was performed using an anti-hASC antibody 12 hours after treatment. 10 µg / ml OVA and PBS were used as negative controls. Alexa-488-conjugated anti-mouse IgG was used as the secondary antibody. In Figure 6, the weak green fluorescence signal is observed to be distributed throughout the cytoplasm in cells under normal conditions, indicating endogenous ASC, and forms speck-like structures after inflammatory activation. In Figure 6, the strong green fluorescence signal in the dot pattern indicates the phagocytosis of tOVA-ASC speck by THP-1 macrophages.

[0075] To confirm that not all specks were endogenously formed, a Western blot was performed using anti-TMS1 (an hASC antibody produced in rabbits). For Western blot analysis, THP-1 macrophages were treated with 2.5 µg / ml, 5 µg / ml, and 10 µg / ml tOVA-ASCs. 10 µg / ml OVA and PBS were also used as negative controls. After 12 hours, the wells were washed three times with PBS to remove unphagocytosed tOVA-ASC specks, and then Western blot analysis was performed with anti-TMS1 and anti-OVA.

[0076] As can be seen in Fig. 6, the band size is approximately 39 kDa, which means that the punctate green fluorescent signal originates from the tOVA-ASC speck. Additionally, OVA can also be phagocytosed by macrophages without an adjuvant or vaccine carrier (3rd lane in Fig. 6).

[0077] Cell medium was also collected to measure secreted IL-1β, IL-6, and TNF-α levels by ELISA. Treatment with tOVA-ASC speck significantly increased IL-1β and IL-6 secretion (Fig. 7). IL-1β secretion from THP-1 macrophages was higher at each tOVA-ASC treatment concentration than at 10 µg OVA treatment and increased in a concentration-dependent manner. IL-6 secretion levels increased in a dose-dependent manner after tOVA-ASC treatment and were higher at 10 µg / ml tOVA-ASC treatment than at 10 µg / ml OVA treatment alone. TNF-α levels also increased with increasing tOVA-ASC dosage. However, there was no significant difference in TNF-α secretion between 10 µg / ml OVA and 10 µg / ml tOVA-ASC treatments.

[0078] The C57BL / 6 mouse thymoma cell line EG7-OVA, which synthesizes chicken ovalbumin, is one of the most widely used tumor models in antigen-specific tumor vaccine research. It expresses OVA upon G418 treatment. Before inoculating mice with the EG7-OVA cell line, OVA expression in this cell line was demonstrated by an anti-OVA blot as shown in Figure 9.

[0079] To induce tumor formation in 8-10 week old C57BL / 6 mice, 2,500,000 EG7-OVA cells were inoculated onto each dorsal side of the mice. Twelve days after inoculation, 18 tumors reached a palpable size, and intraperitoneal injection of 100 µg of tOVA-ASC in 300 µl of PBS was initiated as shown in Figure 9. 50 µg of OVA and PBS were used as negative controls. Two lengths were measured from the tumors using a two-point caliper every two days after the first immunization.

[0080] One tumor in the tOVA-ASC injected animal was completely eliminated on day 4 after the first injection. On day 7 after the first injection, three more tumors were completely eliminated in the tOVA-ASC injected animal, while one additional tumor developed in one of the OVA injected animals. One week after the first immunization, the booster injection was repeated as in the first. On day 1 after the second immunization, one PBS injected animal died, presumably due to the size of the tumor. On day 2 after the second injection, two or more tumors were completely eliminated in the tOVA-ASC injected animal, as shown in Figure 10.

[0081] On day 4 following secondary immunization, only two tumors remained after the second injection, so the experiment was terminated and the tumors in the tOVA-ASC-injected animals were further analyzed. The progression of each tumor in each group was confirmed. All animals were sacrificed. No tumor tissue was detected in the tOVA-ASC-injected animals from which the tumors had already been removed. One OVA-injected animal had two individual tumor tissues on the same dorsal side. The average volume of excised tumors in the PBS, OVA, and tOVA-ASC-injected animals was 1935 mm³, respectively, as shown in Fig. 11. 3 , 1690 mm 3 and 62 mm 3 The average tumor weights of the tumors injected with PBS, OVA, and tOVA-ASC were 2.5 g, 2.11 g, and 0.07 g, respectively, as shown in Figure 11.

[0082] In conclusion, as shown in Figure 10, tOVA-ASC speck 2 shots caused a reduction in the size of each tumor, resulting in the complete elimination of 6 out of 8 EG7-OVA tumors, whereas the tumors of animals injected with PBS and OVA continued to proliferate on the dorsal side of C57BL / 6 mice.

[0083] Since the anti-tumor effect of tOVA-ASC speck can be induced by an antibody response to OVA, the OVA-specific IgG levels in animals were measured by ELISA. In the case of the PBS group, one animal had already died before being sacrificed, so blood could not be collected; therefore, additional experiments were performed on only one animal in the PBS group. In conclusion, as shown in Figure 12, the tOVA-ASC injected animals exhibited a greater OVA IgG response compared to the PBS and OVA injected animals.

[0084] Additional tests were performed to investigate the reinforcing effect of ASC speck.

[0085] Four groups were used, including the PBS, OVA, tOVA-ASC, and mCh-ASC+OVA experimental groups. PBS alone, 100 µg OVA alone, 100 µg tOVA-ASC alone, and 100 µg mCh-ASC + 100 µg OVA were injected intraperitoneally into each mouse group. Since OVA and ASC speck are suspended in PBS, PBS was used as a negative control. To investigate the adjuvant effect of ASC speck, OVA injections were performed to determine whether OVA immunization alone was sufficient to reduce tumor size. Additionally, the mCh-ASC + OVA group was used as a model to compare the adjuvant characteristics of ASC speck with those of the OVA group. In the experiment, the tOVA-ASC group served as a model to verify the carrier capacity of ASC speck as an antigen-specific anti-tumor vaccine. The vaccination timeline for the immunization of mice with EG7-OVA tumors can be seen in Figure 18.

[0086] On day 14 after inoculation with EG7-OVA cells, the tumors reached a proliferable size to initiate immunization. As shown in Figure 19, on day 7 after primary immunization, although tumor size increased in the PBS, OVA, and mCh-ASC+OVA groups, tOVA-ASC speck-immunized mice showed almost no progress in tumor size. Subsequently, a second dose was injected into each group.

[0087] Seven days after the second immunization, tumors were completely eradicated in one mCh-ASC+OVA-immunized mouse and three tOVA-ASC-immunized mice. In the case of mice immunized with OVA alone, one tumor remained of the same size after two injections. One mCh-ASC+OVA-immunized mouse maintained a stable tumor size after two injections. After the final immunization, all animals were sacrificed. Finally, one of the tOVA-ASC-immunized mice exhibited a tumor proliferation pattern after injection.

[0088] Antibodies have been attempted to target tumor cells in clinical trials. These antibodies bind to antigens on tumor cells, inducing direct cytolysis or antibody-catalyzed phagocytosis, thereby leading to antigen processing and presentation onto APCs via MHC class I or II molecules. This situation induces the production of tumor-specific antibodies and / or host anti-tumor immunity through cytotoxic T cells. Anti-tumor effects of tOVA-ASC speck are observed, and this anti-tumor activity may be induced by antibody-catalyzed anti-tumor immunity. To demonstrate antigen-specific antibody production in the host, an ELISA was performed to confirm OVA-specific IgG responses against EG7-OVA tumors.

[0089] As shown in Figure 20, PBS is a negative control, and an increase in OVA-specific IgG levels was achieved in the OVA, mCh-ASC+OVA, and tOVA-ASC groups compared to other groups using PBS. When comparing the OVA-containing groups with one another, the IgG response was significantly higher in the mCh-ASC+OVA group compared to the OVA group. Additionally, the IgG response was significantly higher in the tOVA-ASC group compared to the OVA group. This demonstrates that ASC speck can possess anti-tumor activity by boosting the immune system as an adjuvant and by carrying antigens to induce anti-tumor immunity in the host.

[0090] Finally, mCherry-ASC speck injected intraperitoneally was proven to migrate to secondary lymph nodes important for triggering the adaptive immune system.

[0091] Since secondary lymph nodes are the primary sites for T cell responses, the inventors of the present invention confirmed through IVIS and Western blot analysis whether the mCherry-ASC speck injected intraperitoneally could migrate to mesenteric lymph nodes and the spleen. To this end, 10 µg of mCherry-ASC speck in 200 µl of PBS was injected intraperitoneally into 16-18 week old C57BL / 6 mice. As negative controls, 4 and 8 animals were injected with PBS and commercial mCherry protein, respectively. The animals were monitored using an IVIS device on days 1, 2, and 10 after injection. On day 2, half of the animals were sacrificed, and the spleen, mesenteric lymph nodes, liver, and brain were taken for Western blot analysis. The remaining half were sacrificed on day 10, and the same tissues were taken.

[0092] As shown in Figure 16, yellow indicates areas with strong red fluorescence signals in IVIS, and the abdominal and genital regions of C57BL / 6 animals showed background fluorescence signals upon excitation at wavelengths of 56–580 nm. However, on day 1 after injection, a strong fluorescence signal was observed in one mesenteric lymph node region of an mCherry-ASC-injected animal. On day 10, the fluorescence intensity was nearly identical in each animal.

[0093] To support the IVIS data, all collected tissues were blotted with anti-hASC produced in rabbits to detect the injected mCherry-ASC speck. As shown in Figure 17, mCherry-ASC protein was detected in the mesenteric lymph nodes of one animal and in the spleen of another animal. Additionally, degradation of mCherry-ASC protein can be seen in the lower band of the anti-hASC blotting of the spleen.

[0094] Accordingly, in one embodiment of the present invention, since the ability to boost the immune system against tumor cells has been demonstrated in experiments, the ASC speck (formed by the ASC protein) is used in cancer treatment.

[0095] In another embodiment, a composition comprising an ASC speck for use in a cancer treatment method comprises inducing and / or stimulating an immune response against cancer in mammals, such induction or stimulation causes a reduction in cancer progression or a halt in the proliferation of cancer cells.

[0096] In another embodiment, a composition comprising a tumor antigen and an ASC speck molecule formed by an ASC protein is provided for use in cancer treatment.

[0097] In another embodiment, an ASC speck composition for use in cancer therapy is provided, comprising a tumor antigen carried to an ASC speck through hydrophobic interactions with an ASX protein that forms an ASC speck.

[0098] In one embodiment of the present invention, the composition comprises an ASC speck formed through the fusion of a tumor antigen and one or more ASC proteins. This composition is used to treat cancer, particularly tumors.

[0099] The aforementioned fusion protein can be prepared by fusing a tumor antigen with an ASC protein at the N-terminus or C-terminus.

[0100] In another embodiment of the present invention, the composition comprises a tumor antigen carried by forming hydrophobic interactions with an ASC protein that forms an ASC speck, preferably within the ASC speck.

[0101] The present invention relates to the treatment of any type of cancer, preferably a tumor forming cancer, more preferably a tumor forming thymic cancer.

[0102] In one embodiment, the composition of the present invention is used in cancer immunotherapy by inducing a reduction in tumor progression, a reduction in tumor size, or the complete eradication of the tumor when administered to a required mammal.

[0103] In another embodiment of the present invention, a method for treating cancer is provided, comprising the step of administering a composition containing an ASC speck to a required mammal in a therapeutically effective amount. The composition preferably further comprises a tumor antigen; preferably, the tumor antigen is carried by the ASC speck by forming a fusion protein with an ASC protein that forms the ASC speck.

[0104] In another embodiment, a composition comprising a tumor antigen bound to an ASC speck carrier is provided, characterized in that the ASC speck carrier is formed by the oligomerization of a fusion protein consisting of an ASC protein and a tumor antigen.

[0105] Examples of tumor antigens available in the present invention include HER2+ (a membrane-bound protein) for breast cancer, PSA (a 34 kDa glycoprotein) for prostate cancer, MAGE (melanoma-associated antigen) for melanoma cancer, and MUC1 (a transmembrane glycoprotein) for ductal glycoproteins or neoantigens. These are provided as simple examples, and any other tumor-associated antigen may also be used for the purposes of the present invention.

[0106] In another embodiment of the present invention, a composition comprising a tumor antigen bound to an ASC speck carrier is used for the treatment of thymic tumors, wherein the ASC speck is formed by a fusion protein consisting of an ASC and a tumor antigen.

[0107] experiment

[0108] ingredient:

[0109] Detailed information regarding the materials used in the experiment is described below.

[0110] Table 1. Cell lines

[0111] cell line Major suppliers Catalog number HEK293FT Invitrogen, USA R700-07 THP-1 ATCC, USA TIB-202 THP-1eGFP-ASC THP-1 cell line - EG7-OVA ATCC, USA CRL-2113

[0112] Table 2. Cell culture medium

[0113] badge Catalog number supplier DMEM, high glucose content, pyruvate 41966029 Gibco, ThermoFischer Scientific, USA RPMI MEDIUM 1640 21875034 Gibco, ThermoFischer Scientific, USA FBS (Fetal Bovine Serum) 10270106 Gibco, ThermoFischer Scientific, USA Penicillin-Streptomycin 15140122 Gibco, ThermoFischer Scientific, USA MEM NEAA- 100X 11140050 Gibco, ThermoFischer Scientific, USA 2-Mercaptoethanol 21985-023 Gibco, Lifetechnologies, USA G418 Geneticin 1150MG250 Neofroxx, Germany

[0114] Table 3. Cell culture solution

[0115] 10X PBS 1.37 M NaCl27mM KCl15mM KH2PO481mM Na2HPO4pH:7,4 ACK Cell Lysis Buffer 154mM NaH4Cl10mM KHCO30.1mM Na2EDTA freezing medium 10% DMSO (A3672, AppliChem, Germany) 90% Growth Medium 0.05% trypsin 0.53 mM EDTA 0.5 g / L Trypsin 8 g / L NaCl 0.4 g / L KCl 0.06 g / L KH2PO4 1 g / L D-Glucose 0.048 g / L Na4HPO4 0.35 g / L NaHCO3 pH: 7.5-8 2M CaCl2 110.98 g / 500 ml ddH2O 2X HBS 25 ml Hepes7.3 g NaCl0.1 g Na2HPO4 / 500 ml, pH:7,05

[0116] Table 4. Western blot solution

[0117] NP40 Cell Lysis Buffer 150mM NaCl, 0.2% NP-40, 50mM Tris, pH:8 30:0,8 Acrylamide:Bisacrylamide 30 g acrylamide, 0.8 g bisacrylamide / 100 mL ddH2O 4% Stacking Gel 66 mL 30:0.8 Acrylamide:Bisacrylamide, 126 mL 0.5 M Tris-HCl, pH 6.8, 5 mL 10% SDS / 500 mL ddH2O 15% Resolving Gel 500 mL 30:0.8 Acrylamide:Bisacrylamide, 200 mL 1.875 M Tris, pH 8.8, 10 mL 10% SDS / 1 L ddH2O 5X Laemmli buffer 1.5 M Tris-HCl, pH 6.8, 10 ml glycerol, 5 ml β-mercaptoethanol, 2 g SDS, 1 ml 1% bromophenol blue / 20 ml ddH2O APS Applichem, Germany TEMED Sigma, Germany Running Cushion (10 X Tris-Glycine) 30 g Tris base, 144 g glycine, / 1L dH2O Semi-dry transfer buffer 2.93 g glycine (39 mM), 5.81 g Tris base (47 mM), 6.25 ml 10% SDS / 1 L dH2O pH 9.2 titrated methanol Merck, USA Bovine Serum Albumin (BSA) Factor V Roche, Germany 10% BSA 10% BSA / TBS-T 10X TBS buffer 1.5 M NaCl 0.5 M Tris, pH: 7.5 Tween-20 TBS-T 100 ml 10X TBS Buffer 0.1% Tween-20 Sodium azide Roche, Germany Pre-stained protein ladder PageRuler, Thermo Fischer Scientific, USA Enhanced Chemiluminescent Substrate (ECL) Advansta Inc., USA Komash blue dye solution 0.1% Komash Blue, 10% Acetic Acid, 50% Methanol / dH2O Komash Blue Decolorizing Solution 10% acetic acid, 40% methanol / dH2O Elution buffer for SDS-PAGE Titrated with 50 mM Tris-HCl, 150 mM NaCl, and 0.1 mM EDTA pH 7.5

[0118] Table 5. ELISA solution

[0119] solution ingredient cleaning solution 0.05% Tween 20 / PBS, pH 7.2-7.4 Reagent diluent Filtered 1% BSA / PBS, pH 7.2-7.4, Substrate solution A 1:1 mixture of color reagent A (H2O2) and color reagent B (tetramethylbenzidine). Stop solution 2N H2SO4

[0120] Table 6. Antibodies

[0121] antibodies composite Catalog number Catalog number Anti-rabbit IgG HRP 7074S Cell Signaling Technology, Netherlands Anti-mouse IgG HRP 7076S Cell Signaling Technology, Netherlands Anti-hASC - - Kindly provided byProf. Mosumoto, Japan anti-β-actin - 4970S Cell Signaling Technology, Netherlands Anti-TMS1 - ab155970 Abcam, UK Anti-OVA - ab17293 Abcam, UK Hang-mCherry - ab125096 Abcam, UK Anti-CD8-α APC 100711 Biolgend, USA Anti-CD16 / CD32 - 553141 BD Biosciences, USA Anti-CD8-α Alexa647 - Provided out of goodwill by Professor Batu Herman, Türkiye Hang-B220 PE - Provided out of goodwill by Professor Batu Herman, Türkiye Anti-CD4 PE - Provided out of goodwill by Professor Batu Herman, Türkiye ant-H57 FITC - Provided out of goodwill by Professor Batu Herman, Türkiye Leu4 FITC - Provided out of goodwill by Professor Batu Herman, Türkiye Leu4 PE - Provided out of goodwill by Professor Batu Herman, Türkiye

[0122] Table 7. Plasmids

[0123] p-C3-d(1-238)OVA-ASC AKIL, Türkiye p-C3-mCherry-ASC AKIL, Türkiye

[0124] Table 8. Injection Solution Components

[0125] aluminum potassium sulfate dodecahydrate 237086 Sigma, Germany Full Freund reinforcing agent (CFA) 344289 Calbiochem, Germany Incomplete Pround Reinforcement (ICFA) SC37726 Santa Cruz, USA Chicken ovalbumin A5503 Sigma, Germany mCherry 4993-100 Bio Vision, USA

[0126] Table 9. Kit

[0127] Mouse IL-4 DuoSet ELISA DY404-05 R&D Systems, USA Mouse IFN-gamma DuoSet ELISA DY485-05 R&D Systems, USA Mouse TNF-alpha DuoSet ELISA DY410-05 R&D Systems, USA Human IL-1 beta / IL-1F2 DuoSet ELISA DY201 R&D Systems, USA Human IL-6 DuoSet ELISA DY206 R&D Systems, USA Human TNF-alpha DuoSet ELISA DY210 R&D Systems, USA NucleoBond Xtra Midi 740410.50 Macherey-Nagel, Germany NucloeSpin RNA 740955.50 Macherey-Nagel, Germany

[0128] method:

[0129] 1. tOVA - ASC and mCherry - ASC Speck tablets

[0130] 1.1. Plasmid DNA Amplification and Isolation

[0131] The p-C3-d (1-238) OVA-ASC and p-C3-mCherry-ASC plasmids were cloned and used in this experiment. Midi- and maxi preparations were performed using the MACHEREY-NEGEL (MN) kit according to the manufacturer's recommendations. Bacterial cultures were cultured in 200 ml and 400 ml of LB, respectively, supplemented with kanamycin for midi and maxi preparations.

[0132] 1.2. HEK293FT Maintenance of cell lines

[0133] HEK293FT cell lines were maintained in DMEM supplemented with 10% FBS, MEM-NEA, 100 U / ml penicillin, and 100 µg / ml streptomycin. When the cells reached 90% confluence, the plates were rinsed with PBS and then subcultured by trypsin treatment at 37°C for 2 minutes.

[0134] 1.3. Calcium- Phosphate Transfection

[0135] The HEK293FT cell line is readily transfected by the calcium phosphate transfection method, which induces the formation of insoluble precipitates of plasmid DNA and calcium phosphate. The precipitates are absorbed by the HEK293FT cells. 14 million cells were seeded into a 150 mm cell culture plate. The plasmid concentration was adjusted by diluting it in ddH2O according to the experiment, and 2M CaCl2 was added dropwise. After 5 minutes, 2X HBS was added, and bubbles were produced. After 5 minutes, the mixture of plasmid and calcium phosphate was dropwise added to the HEK293FT cells during subculture.

[0136] Table 10. Transfection mixture.

[0137] Plate type DNA ddH2O CaCl2 2X HBS 150 mm plate 30-40 ㎍ 1317 ㎕ 183 ㎕ 1500 ㎕

[0138] 1.4. ASC Speck manufacturing and isolation

[0139] 14 million HEK293FT cells were counted and inoculated into six 150 mm cell culture plates. The cells were transfected with the (pC3-(d1-235) OVA-ASC) plasmid DNA via the calcium phosphate transfection method. After 48 hours, the cell medium was aspirated, and 3 ml of PBS was added. The cells were scraped off the surface and taken into a 50 ml Falcon. The cells were lysed five times using 30% sonication at 50% power for 5 seconds. The lysed cells were centrifuged at 2400 g at 4°C using a Beckman table centrifuge. After centrifugation, the pellet was lysed until the filaments were easily detectable by vortexing. The pellet was placed on ice for 5 minutes to allow a large amount of cells to settle by gravity sedimentation. The supernatant was carefully transferred to a new 50 ml Falcon and centrifuged at 300 g for 5 minutes. The supernatant was aspirated, and the pellet was dissolved in 10 ml of PBS. The pellet was vortexed until filaments were observed and dissolved again. The filaments were then allowed to settle by gravity for 5 minutes.

[0140] The supernatant was transferred to a new Falcon and centrifuged at 300 g. Vortexing, gravity sedimentation, and centrifugation were repeated until no filaments were observed after vortexing. The final sample was filtered through a 1.2 or 5 µm syringe filter. 3–4 ml of the sample was passed through each filter until back pressure was detected. 2–3 ml of PBS was applied to the filter in reverse, and the ASC speck was recovered in a 15 ml Falcon. The reverse filtrate was centrifuged at 3900 g for 1 hour. The pellet was dissolved in 500 µl of PBS. The ASC speck in PBS was stored at +4°C. The purity of the ASC speck was confirmed by SDS-PAGE and Coomassie Blue staining. All steps, except for sonication, were performed in a cell culture hood.

[0141] 1.5. SDS-PAGE and Komashi Blue using dye Danrihan ASC Determination of speck purity and quantity

[0142] 40 µl of isolated ASC speck and various concentrations of BSA were mixed with 10 µl of 5X Leamli buffer in separate 1.5 ml test tubes. The mixture was boiled at 95°C for 10 minutes and then loaded onto a 12% SDS-PAGE gel along with a pre-stained protein ladder. The samples were developed as described in the protein gel electrophoresis section. The gel was stained with Coomassie blue in a plastic container for 1 hour and then destained with Coomassie destaining solution. A gel photograph was taken, and the ASC speck concentration was calculated using ImageJ software based on a standard curve prepared by converting the ASC speck concentration to the BSA concentration.

[0143] 2. THP -1 For macrophages ASC The effect of speck

[0144] 2.1. THP -1 Maintenance of monocyte cell lines

[0145] THP-1 is a monocyte cell line derived from leukemia patients. THP-1 cells were provided, and a THP-1 eGFP-ASC stable cell line was obtained that constitutively synthesizes eGFP-ASC from THP-1 monocytes through lentiviral transduction of pLenti-Ef1a-EGFP-ASC. The cell line was maintained in RPMI 1640 supplemented with 10% FBS, MEM-NEA, 100 U / ml penicillin, and 100 µg / ml streptomycin.

[0146] 2.2. THP -1 In monocytes THP -1 Differentiation into macrophages

[0147] 1.5 x 10⁶ in each well of a 6-well plate for 9 hours using RPMI 1640 containing 100 nm / ml PMA 6 THP-1 monocyte cells were differentiated into THP-1 macrophages. Then, the cell medium was replaced with PMA-free RPMI 1640.

[0148] 2.3. THP -1 to macrophages ASC Speck processing

[0149] Forty-eight hours after differentiation, mCherry-ASC or tOVA-ASC speck was applied to macrophages at various concentrations in 1 ml of cell medium to confirm the effects of ASC speck on macrophages. The cell medium was collected and stored at -20°C for future cytokine ELISA analysis at various time points. Twelve hours after ASC speck treatment, THP-1 macrophages were prepared for flow cytometry analysis and incubated on ice with PBS-EDTA for 30 minutes. Subsequently, the cells were detached by gently scraping the cell culture plate and collected in a 15 ml centrifuge tube. The cells were centrifuged at 300 g for 5 minutes and rinsed twice with FACS buffer containing human serum. Their cell surface proteins were stained and analyzed as described in the section on cell surface staining and flow cytometry analysis.

[0150] 2.4. THP -1 Immunohistochemistry against macrophages

[0151] THP-1 monocytes 1.6 x 10 5Dogs were differentiated into THP-1 macrophages in a 48-well plate and then treated with ASC speck. After 12 hours following treatment with ASC speck, the cell medium was aspirated, and the wells were rinsed three times with PBS. Subsequently, the cells were fixed with 4% PFA for 15 minutes at room temperature. After rinsing the cells three times with PBS, they were permeable with PBS containing 0.25% Triton-X100 detergent. The cells were then blocked with blocking buffer (5% BSA (w / v) / PBS-T) for 30 minutes at room temperature. The cells were incubated with an anti-hASC antibody diluted 1 / 50 in the blocking buffer for 2 hours at room temperature. The cells were rinsed again with PBS and then incubated with an Alexa-488 fluorescence-conjugated anti-mouse IgG 1 / 500 dilution for 1 hour at room temperature. After incubating with the secondary antibody, the cells were rinsed and visualized under a fluorescence microscope.

[0152] 2.5. Cytokine ELISA

[0153] Cytokine levels in the cell culture medium were measured using a sandwich ELISA. Briefly, ELISA plates were coated with capture antibodies in PBS at the manufacturer's recommended concentrations and incubated overnight at RT. Three wells of a 96-well plate were coated for each supernatant sample, while the other three wells were coated with PBS alone as negative controls. After overnight incubation, the plates were rinsed with 400 µl of PBS-T and blocked with 200 µl of reagent diluent (1% BSA / PBS) at RT for 1 hour to prevent non-specific binding. During the plate blocking process, the cell supernatant was diluted with the reagent diluent until the final OD value was less than 2.00. After blocking, the plates were rinsed three times with PBS-T, and 100 µl of the diluted sample and cytokine standard were added to the wells and incubated at room temperature for 2 hours. Afterwards, the plate was rinsed three times with PBS-T and incubated at RT for 2 hours in 100 µl of detection antibody and reagent diluent at the concentration recommended by the manufacturer.

[0154] After rinsing the plate, 100 µl of HRP diluted 1:50 in reagent diluent was added to the wells and incubated at RT for 1 hour. Subsequently, the plate was rinsed again, and 100 µl of a 1:1 mixture of ELISA substrate A and ELISA substrate B was added to the wells, resulting in a blue color change. After 20 minutes following substrate addition, 1N sulfuric acid was added to stop the reaction, and the color changed from blue to yellow. The OD values ​​of the wells were calculated at 450 nm and 540 nm using a microplate reader, and the absorbance at 540 nm was subtracted from the absorbance at 450 nm. A standard curve was constructed based on the absorbance values, and the concentrations of standard substances and cytokines in the supernatant were determined from the standard curve using SoftMaxPro software.

[0155] 2.6. ASC RT- after speck processing qPCR used THP -1 Confirmation of expression patterns of pro-inflammatory proteins in macrophages

[0156] The expression patterns of proteins at the mRNA level were confirmed by real-time quantitative PCR in PMA-differentiated THP-1 cells after ASC speck treatment. THP-1 macrophages were treated with mCherry-ASC speck at concentrations of 10, 25, 50, and 100 µg for 12 hours. PBS alone and 50 µg of mCherry protein were used as negative controls.

[0157] 2.6.1. RNA Extraction

[0158] Cells were rinsed three times with PBS, scraped, and collected. RNA was extracted using the MN RNA extraction kit according to the manufacturer's recommendations. Briefly, cells were lysed in cell lysis buffer, the cell lysate was mixed with 70% ethanol, and bound to a column. After washing the column several times, DNase I in DNase incubation buffer was treated at RT for 15 minutes. The column was rinsed again with wash buffer and dried. Subsequently, total RNA was isolated using 60 µl of RNase-free water. The purity and concentration of RNA were measured using a Nanodrop spectrophotometer.

[0159] 2.6.2. cDNA Synthesis

[0160] cDNA was synthesized from whole RNA using the BIOLINE SensiFAST cDNA synthesis kit according to the manufacturer's recommendations. Briefly, 500 ng of whole RNA was mixed with 4 µl of 5X TransAmp buffer and 1 µl of reverse transcriptase in DEPC-treated H2O (final volume: 20 µl). The samples were incubated for inactivation at 25°C for 10 minutes, 42°C for 15 minutes, and 85°C for 5 minutes.

[0161] 2.6.3. Quantitative PCR

[0162] The relative expression levels of target genes after ASC speck treatment were investigated using primers specific to each gene and HPR-T as a control. cDNA samples were amplified using BIOLINE SensiFAST SYBR MasterMix in an Exicycler 96 qPCR system. The gene expression levels in PBS-treated cells were 1 and 2 - ddct The relative expression of the target gene was determined by adjusting as in the method (Livak and Schmittgen 2001).

[0163] 3. EG7-OVA Tumor Model and tOVA - ASC Immunization

[0164] 3.1. Maintenance of EG7-OVA Cell Line

[0165] The EG7-OVA cell line was provided. EG7-OVA is derived from the C57BL / 6 mouse lymphoma cell line EL-4. The EL4 cell line was stably transfected with a pAc-neo-OVA plasmid containing chicken ovalbumin (OVA) and neomycin resistance genes in their full length to construct an EG7-OVA cell line that constitutively expresses OVA protein upon treatment with G418 as a model tumor antigen. The cell line was maintained in RPMI 1640 supplemented with 10% FBS, MEM-NEA, 100 U / ml penicillin, 100 µg / ml streptomycin, 2-mercaptoethanol, and 0.4 mg / ml G418. The cell medium was changed every 2 days.

[0166] 3.2. EG7-OVA Tumor Inoculation and OVA, PBS and tOVA - ASC Immunization using

[0167] 2.5 million EG7-OVA cells in 100 µl of PBS were inoculated onto each dorsal side of 12 C57BL / 6 mice. On day 12, 18 tumors reached a palpable size (<50 mm 3). Intraperitoneal injections were performed as follows: 100 µg of tOVA-ASC in 300 µl of PBS (n=10), 50 µg of commercial OVA (n=5), and PBS alone (n=3). Two lengths were measured from the tumor using a 2-digit caliper. Volume was calculated using the formula: 1 / 2((shorter length 2 ) long length). The second injection was repeated as with the first injection. Potentially, one PBS-injected animal died on day 20. As only two tumors remained after the second injection, the experiment was terminated, and the tumors in the tOVA-ASC-injected animals were further analyzed. Blood was collected from the orbital sinus of all animals. All animals were sacrificed, and their spleens were collected. The tumors were excised, and their volume and weight were calculated. In tOVA-ASC-injected animals where the tumors had already been eradicated, no tumor tissue was detected. The plasma of each animal was stored at -20°C. Splenocytes were recovered and cultured in 96-well plates for further in vitro recall analysis. The animal tumors were embedded in OCT for further immunohistochemical analysis. A portion of the tumors from each group was set aside for Western blot analysis and stored at -80°C.

[0168] 3.3. EG7-OVA Tumor Inoculation, and PBS, OVA, tOVA - ASC and mCherry - ASC Immunization using

[0169] As with the aforementioned experiment, another test was performed for additional mCherry-ASC immunization. 2.5 x 10⁶ EG7-OVA cells in 100 µl of 1X PBS 6Dogs were inoculated into each mouse. Prior to inoculation, OVA expression in these cells was confirmed by Western blot analysis using anti-OVA antibodies. Cells were prepared under sterile conditions. Inoculation of these cells was performed on the right dorsal side of each mouse. After 12–14 days, tumors of a palpable size (≥ 50 mm) 3 When ) was reached, intraperitoneal injections were performed as follows: in 300 µl of 1X PBS, 1X PBS alone, 100 µg of OVA, 100 µg of OVA + 100 µg of mCherry-ASC speck, and 100 µg of tOVA ASC. The tumor size was measured with a caliper. The volume of each tumor was calculated using the formula: 1 / 2[(short length 2 It was calculated as ).long length]. The second injection was repeated, just like the first injection.

[0170] In mice immunized with tOVA ASC after the second injection, tumors were eradicated, and the experiment was terminated one week later. The tumors in the animals were measured using a caliper. Subsequently, all animals were sacrificed. Blood was collected from the hearts of the animals. Additionally, the livers and spleens were collected for further analysis. The tumors were excised and embedded in an OCT. All tissues were kept on ice until the experiment was terminated in the last mouse. The collected blood samples were incubated at 37°C for one hour and then centrifuged at 11,000 rpm for one minute. Blood serum was collected in new test tubes and stored at -80°C for an extended period.

[0171] 3.4. Immunohistochemistry of Tumors and Spleen

[0172] 3.4.1. OCT Pomae . Tumor samples were placed in the OCT compound and left at -20°C overnight, then stored at -80°C for subsequent analysis.

[0173] 3.4.2. freeze cuttingThe OCT-embedded tumor sample was placed in the cryosection device in the correct orientation, and the tissue-free cryosection was removed with a blade. Then, 10 µm tumor sections were taken onto a positively charged slide using a cryostat appropriately. The slides were dried at 55°C and stored at -80°C for subsequent analysis.

[0174] 3.4.3. Fixation and Antigen Retrieval. The slides were dried at 55°C for 2 hours, and the tissue fragments were surrounded by a hydrophobic barrier. Then, the tissues were fixed with 4% PFA at RT for 15 minutes. After removing the PFA, the slides were rinsed three times with PBS-T in a jar over a stirrer. The slides were transferred to another jar filled with citrate buffer (pH: 6.0) and incubated at 70°C for 30 minutes.

[0175] 3.4.4. Cryostat Antibody staining of the section. After rinsing the slides three times with PBS-T, they were blocked with blocking buffer (3% BSA / PBS-T) on a stirrer at room temperature for 30 minutes. Then, the slides were incubated at 4°C with a fluorescently labeled primary antibody diluted 1 / 400 in the blocking buffer. After incubation overnight, the slides were rinsed three times with PBS-T for 5 minutes each and visualized under a fluorescence microscope.

[0176] 3.5. Single-cell preparation from EG7-OVA tumors

[0177] The tumor was excised and cut with scissors until all tissue fragments were processed into 1-2 mm fragments. The cut samples were crushed by pushing them toward a cell strainer using the plunger of a disposable 10 ml syringe. The tissue tumor on the cell strainer was rinsed with 3-5 ml of cold RPMI 1640. The washing step was repeated several times until only small fragments of connective tissue remained on the back of the filter. The cells were rinsed three times with RPMI 1640 and then centrifuged at 300 g for 5 minutes at RT.

[0178] 3.6. OVA Unique IgG ELISA for the reaction

[0179] OVA-specific IgG reactions were measured by ELISA. Briefly, 96-well ELISA plates were coated with OVA protein diluted in 100 µl of PBS at a concentration of 100 ng / well, and three wells were used as negative controls containing only PBS. The plates were sealed and incubated at 4°C for 10 minutes. Each well was aspirated and rinsed three times with 300 µl of wash buffer (PBS-T). After the final wash, the plates were patted with a clean towel to remove any remaining wash buffer and foam. 200 µl of reagent diluent (1% BSA / PBS) was added to each well, and the plates were incubated at RT for a minimum of 1 hour and a maximum of 2 hours. The washing steps were repeated. After the final wash, the plates were patted with a clean towel to remove any remaining wash buffer and foam. 100 µl of standard substance (25 pg / ml - 5000 pg / ml) or sample (1:500 dilution) was added, the plate was covered with stretch film, and incubated at RT for 1 hour. The washing steps were repeated, and the same cleaning protocol was performed after the final wash. 100 µl of 1:2000 anti-mouse IgG HRP conjugated secondary antibody was added to each well, the plate was covered with stretch film, and incubated at RT for 1 hour. The washing steps were repeated, and the same cleaning protocol was performed after the final wash. 100 µl of substrate mixture (a 1:1 mixture of color reagents A and B) was added to each well, the plate was protected from direct light, and incubated at RT for 20 minutes. Then, 20 µl of stop solution (2N H2SO4) was added to each well. The plate was gently tapped to ensure thorough mixing. The absorbance of each well was measured at 450 and 540 nm on a plate reader.

[0180] 4. mCherry - ASC Speck's In vivo tracking

[0181] ASC speck was tracked after intraperitoneal injection using mCherry-ASC speck. 10 µg of mCherry-ASC in 300 µl of PBS was injected intraperitoneally into 8 C57BL / 6 mice. As controls, 10 µg of mCherry in 300 µl of PBS and 300 µl of PBS alone were injected. 8 mice were used for the mCherry injection group and 4 mice for the PBS injection group.

[0182] 4.1. In vivo Imaging

[0183] Using an IVIS spectrum in vivo imaging system, the observed mCherry-ASC speck was visualized in vivo, and animals were observed on the IVIS spectrum device on days 1, 2, and 10.

[0184] 4.2. Western Blot Tissue sample collection for immunohistochemistry

[0185] On day 2, half of the animals in each group were sacrificed, and on day 10, the remaining half were sacrificed. The spleen, mesenteric lymph nodes, liver, and brain of the animals were excised. The spleen was observed in vitro using IVIS. Representative portions of the spleen were taken for Western blot analysis, while other portions were embedded in OCT compounds and stored at -80°C. The mesenteric lymph nodes, liver, and brain of the animals were stored at -80°C for further Western blot analysis.

[0186] 5. Western Blot analyze

[0187] 5.1. Preparation of Samples from Cell Cultures

[0188] Expression patterns and regulation at the protein level are generally detected by Western blot analysis. To analyze protein expression in cells, each well of a 6-well plate was rinsed with 1 ml of PBS, or the cells were centrifuged and rinsed with 1 ml of PBS for both adherent and suspension cell cultures, respectively. After aspirating the PBS, 160 µl of a protease inhibitor complex supplemented with 0.2% NP40 lysis buffer was added to the cells. For adherent cells, the cells were collected into 1.5 ml test tubes using a scraper and a micropipette. The collected cells were placed on ice for 1 hour and then vortexed for 15 minutes each to completely lyse the cells. Large cell fragments were pelleted by centrifuging at 13,000 rpm at 4°C for 30 minutes. The supernatant was transferred to another 1.5 ml test tube. Then, 40 µl of 5X Laemmli buffer was added to the sample, and the protein was denatured in Laemmli buffer at 95°C for 10 minutes.

[0189] 5.2. Preparation of samples from the organization

[0190] 200 µl of cell lysis buffer was added to representative excisions and frozen portions of large tissues (spleen, liver, brain, and tumors) and entire mesenteric lymph nodes in a 2 ml test tube. The tissues were lysed by sonication at 50% power for 1 minute at 70% power, followed by incubation on ice for 15 minutes. Subsequently, the tissue lysates were centrifuged at 13,000 rpm for 30 minutes to remove large fragments and connective tissue regions. The supernatant was transferred to a new 1.5 ml test tube and diluted. Then, 5X Laemmli buffer was added as an appropriate amount, and proteins were denatured at 95°C for 15 minutes.

[0191] 5.3. SDS-PAGE Gel Preparation

[0192] 8 ml of 10%, 12%, or 15% separation gel solution was mixed with 80 µl of 10% APS and 8 µl of TEMED. After mixing the solutions, the gel was loaded between a 1.5 mm spacer and a short plate. Then, isopropanol was applied to the surface of the gel. After the separation gel polymerized, the isopropanol was removed, and 40 µl of 10% APS and 4 µl of TEMED were mixed to prepare 4 ml of 4% stacking gel. The stacking gel was loaded onto the separation gel, and a 10-well comb was inserted. Once the polymerization of the stacking gel was complete, an SDS-PAGE gel was used for protein gel electrophoresis.

[0193] 5.4. Protein Gel Electrophoresis

[0194] Proteins were loaded onto an SDS-PAGE gel and separated according to their kDa. The cast SDS-PAGE gel was mounted in a vertical protein gel electrophoresis tank, and the tank was filled with running buffer. The comb was carefully removed, and 35 µl of the denatured protein sample in Leamli buffer and the protein ladder were loaded into the wells. The samples were developed at 70 V until they passed through the stacking gel, and at 120 V until the dye migrated to the bottom of the separation gel.

[0195] 5.5. Semi-dry transport

[0196] Proteins on an SDS-PAGE gel were transferred to a nitrocellulose membrane for antibody blotting. Briefly, the nitrocellulose membrane was activated in methanol, and the membrane and two filter papers were soaked in transfer buffer. One filter paper, the membrane, the SDS-PAGE gel, and another filter paper were placed on a semi-dry transfer apparatus, respectively. The apparatus was covered, and protein transfer from SDS-PAGE to the nitrocellulose membrane was performed at 10V for 50 minutes.

[0197] 5.6. Barrier Blocking Process

[0198] The protein-transferred membrane was taken out of a plastic container and then blocked with 5% (w / v) degreased powder / TBS-T for 1 hour to prevent non-specific binding of the detection antibody.

[0199] 5.7. Antibodies Incubation

[0200] The primary antibody was diluted in 5% (w / v) BSA / TBS-T, and sodium azide was added to the antibody solution at 4°C to prevent microbial growth. After blocking the membrane, it was rinsed three times with TBS-T for 5 minutes. The membrane was incubated overnight at 4°C with the appropriately diluted primary antibody. The prepared antibody dilution was stored at 4°C and used 5–10 times for incubation. The membrane incubated with the primary antibody was rinsed three times with TBS-T for 5 minutes. A secondary antibody solution was prepared by diluting HRP-conjugated anti-mouse or anti-rabbit antibody to 1 / 2000 (v / v) in 5% (w / v) defatted powder / TBS-T. The membrane was incubated with the secondary antibody dilution at RT for 1 hour.

[0201] 5.8. Protein Visualization

[0202] The membrane was incubated with a secondary antibody, rinsed three times with TBS-T for 5 minutes, and treated with 2 ml of a 1:1 mixture of ECL Western blotting substrates. Subsequently, protein bands on the membrane were visualized using a Syngene Gbox Chemi chemiluminescence imaging device.

[0203] 6. Cell surface staining and Parenchyma Measurement Analysis

[0204] One million cells were rinsed twice with 3 ml of FACS buffer in FACS tubes. The supernatant was discarded, and 5 µl of Fc blocker and 10 µl of chromophore-conjugated antibody were added. The cells were incubated with the antibodies at +4°C for 30 minutes. The cells were rinsed again three times with 3 ml of FACS buffer. The supernatant was discarded, and the cells were resuspended in 1 ml of FACS buffer. The upregulation of cell surface markers was analyzed using BD Acuri and FlowJo software.

Claims

Claim 1 A composition for use in a cancer treatment regimen comprising an ASC speck and a tumor antigen formed by an ASC (apoptosis-associated Speck-like protein containing CARD) protein. Claim 2 A composition according to claim 1, wherein the therapy induces and / or stimulates an immune response to cancer in mammals, and said induction or stimulation causes a reduction in cancer progression or a cessation of cancer cell proliferation. Claim 3 A composition according to claim 1, wherein the cancer is thymic cancer. Claim 4 A composition according to claim 1, wherein the tumor antigen is loaded onto an ASC speck by forming a hydrophobic interaction with an ASC protein that forms an ASC speck. Claim 5 A composition according to claim 1, wherein the tumor antigen is loaded onto an ASC speck comprising a fusion protein formed by the tumor antigen, which is fused with at least one ASC protein forming the ASC speck. Claim 6 A composition according to claim 5, wherein the tumor antigen is fused to an ASC protein at the N-terminus or C-terminus. Claim 7 A composition according to any one of claims 1 to 6, wherein the cancer is a tumor forming cancer, and the therapy includes slowing down tumor progression or reducing tumor size. Claim 8 A composition for inducing an immune response against cancer in mammals, comprising an ASC speck formed by an ASC protein and a tumor antigen. Claim 9 A composition according to claim 8, wherein the tumor antigen is loaded onto the ASC speck as part of a fusion protein formed by at least one ASC protein that forms the ASC speck and the tumor antigen. Claim 10 delete Claim 11 delete Claim 12 delete Claim 13 delete Claim 14 delete

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