ASC Specification in Cancer Immunotherapy

Abstract

The present invention relates to the use of ASC (apoptosis-associated speck-like protein containing CARD) specks in cancer immunotherapy. The present invention also relates to ASC speck carriers formed by the ASC protein and compositions containing tumor antigens for treating cancer cells and tumors.

Description

[Technical Field]

[0001] The present invention relates to the use of ASC speck in cancer treatment, and to compositions comprising ASC speck carriers, in particular compositions of ASC speck and tumor antigens. [Background technology]

[0002] According to the World Health Organization (WHO), cancer is the leading cause of death worldwide, resulting in an estimated 9.6 million deaths in 2018. The number of new cases rose to 18 million in the same year, with lung cancer being the most frequently diagnosed type of cancer, followed by breast cancer.

[0003] Today, cancer treatment options include surgery, radiation therapy, chemotherapy, targeted drug therapy, bone marrow transplantation, hormone therapy, and immunotherapy. As is well known in the art, most of these treatment methods have serious side effects and still cannot effectively cure all cancer types, as evidenced by mortality rates. Immunotherapy is relatively safe because it uses the body's own defense mechanisms to fight cancer.

[0004] Traditional cancer treatment methods are based on the principle of attacking cancer with chemicals (chemotherapy), radiation, or surgical removal of tumors. Chemotherapy is a very common method used as either a first-line or second-line treatment. It attacks rapidly proliferating cells, including cancer cells and normal cells such as bone marrow cells. Therefore, a major side effect of chemotherapy is suppression of the patient's immune system. On the other hand, immunotherapy helps the body's own immune system attack cancer cells, making it possible to effectively treat multiple types of cancer. Unlike chemotherapy, immunotherapy improves overall immune system function.

[0005] The immune system is a network of organs, tissues, cells, and proteins. The main function of the immune system is to protect the body from invaders such as bacteria, viruses, fungi, or any other foreign substances. Research into the mechanism of cancer has long known that cancer cells are also recognized by the immune system. However, because they originate from the body's own cells, it is not easy for the defense mechanism to identify cancer cells as foreign.

[0006] Another important aspect is that for the immune system to successfully protect the body from invaders or abnormal cells, it needs enough time to recognize such body, produce necessary defense mechanisms, and destroy them. Considering that cancer cells grow rapidly and are very complex, the body's defense cells usually stop before completing their function against cancer cells. Therefore, it is recognized that the immune system needs assistance to be able to protect the body from rapidly growing cancer cells.

[0007] Immunotherapy is a relatively new treatment approach with many unknowns. First, the role of the immune system in cancer is not fully understood. Second, the proteins involved in this natural defense mechanism are highly sensitive to environmental changes and prone to losing their function. This presents challenges both in the development of new treatment options and in the use of these therapies in patients.

[0008] Recent research in immunology and tumor biology has provided new insights into the understanding of the interaction between the immune system and tumor cells. Many studies have established the importance of T cells for mediating antitumor immunity. The process of carcinogenesis results in altered expression of antigens on tumor cells that can be recognized 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] Currently, immune checkpoint inhibitors, engineered T cells, monoclonal antibodies, and cancer vaccines are commonly used as immunotherapies for many cancer types (Kamta, J., M. Chaar, A. Ande, D.A. 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 and therapeutic. Theoretically, a cancer preventive vaccine should prevent tumor development and provide long-lasting immunity. The first examples of preventive vaccines are those against 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 main subgroups according to their composition: whole-cell tumor, dendritic cell, DNA, and subunit vaccines. Whole-cell tumor vaccines are mostly allogeneic and are somewhat effective due to the large amount of tumor heterogeneity observed between patients (Srivatsan, S., J. M. P. Tel, E. N. Bozeman, I. E. Imasuen, S. He, D. Daniels, and P. Selvaraj, "Allogeneic tumor cell vaccines," Human Vaccines & Immunotherapeutics, Vol. 10, No. 1, pp. 52-63, 2014). Dendritic cell vaccines are produced by extracting cells from patients and priming them with specific antigens and / or adjuvants (Kamta, J., M. Chaar, A. Ande, D.A. Altomare, and S. Ait-Oudhia, “Advancing Cancer Therapy with Present and Emerging Immuno-Oncology Approaches,” Frontiers in Oncology, Vol. 7, p. 64, 2017). One FDA-approved dendritic cell vaccine, sipuleucel, is for prostate cancer (Guo, C., M.H. Manjili, J.R. Subjeck, D. Sarkar, P.B. Fisher, and X.-Y. Wang, “Therapeutic Cancer Vaccines,” Advances in Cancer Research, Vol. 119, pp. 421–475, 2013). DNA vaccines are emerging in cancer immunotherapy and essentially require sequences of tumor-associated antigens.

[0012] Subunit vaccines for cancer immunotherapy contain antigens or epitopes of antigens, as in traditional vaccines. Subunit vaccines are used to treat melanoma, prostate cancer, testicular cancer, and breast cancer. Although the immune boosting effect of subunit vaccines is milder than that of whole tumor vaccines, their effectiveness 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, which can be enhanced by the use of adjuvants in certain vaccine formulations. Inorganic 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, 2007). The mechanism of action of adjuvants is only beginning to be elucidated, despite their use for over 80 years (Awate, S., L.A. Babiuk and G. Mutwiri, "Mechanisms of action of adjuvants." Frontiers in immunology, Vol. 4, p. 114, 2013).

[0014] Aluminum salts (alum) are the most frequently used vaccine adjuvants in human vaccines. The inducing effect of aluminum hydroxide against diphtheria was first demonstrated in guinea pigs in 1926, and various alum formulations have been used in human vaccines since 1931 (Marrack, P., A.S.M. McKee and M.W. 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 adjuvants has not been fully understood until now. Several recent publications have shown that alum induces immune responses more effectively than IL-1 cytokines and caspase-1, a product of the inflammasome complex (Franchi, L., T. Eigenbrod, R. Muñoz-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, 2009; Eisenbarth, S.C., O.R. Colegio, W. O'Connor, F.S. Sutterwala and R.A. Flavell, “Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminum adjuvants,” Nature, Vol. 453, No. 7198, pp. 1122-1126, 2008; Grun, J.L. and P.H. 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] More than 70 years after alum, a new adjuvant, AS04, was approved by the FDA for HPV. AS04 is formed from a combination of alum and monophosphoryl lipid A. Finally, the FDA approved AS03 for use against pandemic H5N1 influenza, but it is no longer commercially available. The formulation of MF59 used in influenza vaccines is very similar to AS03 but is not approved by the FDA (Awate, S., L.A. Babiuk and G. Mutwiri, "Mechanisms of action of adjuvants," Frontiers in Immunology, Vol. 4, p. 114, 2013).

[0017] There are several proposed mechanisms for the booster effect of adjuvants in immune activation, primarily through enhanced antigen uptake, recruitment of immune cells to the injection site, and activation of PRRs and inflammasomes followed by specific immune responses such as Th1 and Th2 (Reed, SG, MT Orr and CBFox, "Key roles of adjuvants in modern vaccines", Nature Medicine, Vol. 19, No. 12, pp. 1597-1608, 2013).

[0018] Innate immune receptors, such as TLRs, are targets of adjuvants. When adjuvants activate TLRs, specific chemokines and cytokines are secreted into the extracellular matrix. These small molecules then help direct cellular and humoral immunity. For example, IL-12 and IFN-γ induce more cellular immune responses, while IL-4 secretion from APCs and naive CD4+ cells induces IgG production (Reed, S.G., M.T. Orr, and C.B. Fox, "Key roles of adjuvants in modern vaccines," Nature Medicine, Vol. 19, No. 12, pp. 1597-1608, 2013).

[0019] Following the identification of specific signature antigens of human tumor cells, innovative approaches have been developed, leading to the development of antigen-specific vaccinations. The combination of specific tumor antigens with appropriate routes for delivering these antigens to the immune system aims to optimize human-based vaccinations (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. Because these cells themselves are unable to elicit a sufficient immune response, researchers have focused on overcoming this unresponsiveness by introducing costimulatory molecule or cytokine genes into the genome of tumor cells, which are then used as a vaccine and infused back into the same patient. However, producing this type of vaccine is quite slow, labor-intensive, and logistically challenging.

[0021] Alternatively, recombinant viral vectors can be used to deliver tumor antigens to the body. The inherent ability of viral vectors to initiate immune responses and form inflammatory reactions as a result of viral infection is an advantage of these vaccines. However, viral vectors must be completely safe and not promote anti-vector immune responses to enable the boosting of anti-tumor-specific immune responses (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 known in the art, cancer incidence and, in parallel, mortality rates are increasing every year. Thus, there remains a great need in the art for new cancer treatment methods.

[0023] ASC (apoptosis-associated speck-like protein containing CARD) is a 22 kDa adaptor protein with an N-terminal PYD domain and a CARD domain at the C-terminus that functions in the formation of inflammasome complexes, particularly in the NLRP3, NLRC4, and AIM2 inflammasomes. The ASC protein plays a role in the activation of caspase-1 protein in the inflammatory and pyroptosis signaling pathways (Franchi et al., 2009). It is a cytoplasmic protein that forms speck-like structures upon inflammasome activation (Miao et al., 2011).

[0024] ASC proteins polymerize into filaments, forming large supramolecular scaffolds called ASC specks (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). These ASC specks can reside 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, pe 0134912, 2015).

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

[0026] International Patent Application WO2009 / 014863 discloses that ASC protein and pyrotosomes can be used for the treatment and diagnosis of autoimmune diseases. According to this application, apoptosomes and pyrotosomes containing ASC dimers and procaspase-1 are formed during the early inflammatory response in macrophage cells. Caspase-1 activation is dependent on pyrotosome formation. Inflammatory conditions are diagnosed by isolating pyrotosomes from macrophage cells.

[0027] The ASC protein was hypothesized to play a role in eliciting antigen-specific humoral responses after MF59-adjuvanted influenza vaccination (Ellebedy et al., 2011).

[0028] Furthermore, the invention disclosed in patent application WO2013 / 160807 demonstrates that antigens and / or biologically active molecules can be loaded onto ASC speck carriers for use in vaccination. The invention was developed by the inventors of the present invention. It was found that the stability of biologically active molecules can be enhanced for at least 30 days by using ASC speck as a carrier and can withstand at least eight freeze-thaw cycles without degradation. In other words, the cited application discloses a composition useful for vaccines in general. However, it does not disclose the therapeutic effects of ASC speck itself or a composition containing ASC speck for the treatment of cancer cells or tumors.

[0029] Thus, none of the prior art documents mentions the role of ASC specs in the treatment of cancer. Summary of the Invention

[0030] An object of the present invention is to provide a novel method for treating cancer.

[0031] The present invention relates to the use of ASC (apoptosis-associated speck-like protein 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 the therapeutic treatment of 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 between an ASC protein and a tumor antigen.

[0034] In another embodiment of the present invention, ASC specs are used in the treatment of cancer and have the ability to boost the immune system against tumor cells.

[0035] Furthermore, in another embodiment of the present invention, a composition comprising an ASC spec, preferably a composition comprising a combination of an ASC spec and a tumor antigen, is provided for use in the treatment of cancer, the method comprising inducing and / or stimulating an immune response against cancer disease in a mammal, particularly a human subject, the induction or stimulation resulting in the inhibition of cancer progression or the arrest of cancer cell proliferation. [Brief explanation of the drawings]

[0036] [Figure 1] Production and purification of tOVA-ASC specks from HEK293FT cell line. a) tOVA-ASC encoding p-C3-d(1-238)OVA-ASC plasmid. Coomassie blue staining (b), anti-hASC blot (c), and bright-field image of purified tOVA-ASC specks (d). e) Fluorescence image of purified mCherry-ASC specks. [Figure 2] Phagocytosis of mCherry-ASC specks by PMA-differentiated THP-1 macrophages. a) Brightfield and fluorescent images of THP-1 macrophages were taken 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 specks. [Figure 3]Degradation of phagocytosed mCherry-ASC specks by THP-1 macrophages. Fluorescent images of phagocytosed and degraded mCherry-ASC specks were taken 12 hours after mCherry-ASC treatment. [Figure 4] Complete degradation of mCherry-ASC specks resulted in the formation of inflammasome complexes in host cells. Fluorescence images (a-b) and merged image (c) of anti-hASC staining of mCherry-ASC specks phagocytosed by THP-1 macrophages. d) 3x magnification of the blue frame in the merged image. [Figure 5] Secretion of pro-inflammatory cytokines by mCherry-ASC treatment. a) mRNA expression levels of NLRP3, pro-caspase-1, and ASC, along with RT-qPCR, IL-1β (b) and TNF-α (c) secretion levels were measured after mCherry-ASC treatment of THP-1 macrophages. (Error bars indicate SD between two biological replicates.) [Figure 6] Phagocytosis of tOVA-ASC specks by PMA-differentiated THP-1 macrophages. a) Immunocytochemistry of THP-1 macrophages 12 hours after treatment with tOVA-ASC, PBS, OVA and anti-hASC antibody. b) Anti-OVA and c) anti-hASC blots of THP-1 macrophages after PBS, OVA and tOVA-ASC treatment. [Figure 7] Secretion of pro-inflammatory cytokines from PMA-differentiated THP-1 macrophages upon treatment with tOVA-ASC. IL-1β (a), TNF-α (b), and IL-6 (c) secretion levels were measured by ELISA in THP-1 macrophages treated with PBS, 10 μg of OVA, or 2 μg, 5 μg, and 10 μg of tOVA-ASC specks. [Figure 8] Cytokine secretion levels from splenocytes of animals injected with tOVA-ASC after OVA pulse. Splenocytes pulsed or not pulsed with OVA. Splenocyte cytokine levels were measured by ELISA from the supernatant of splenocytes (n=4 for the OVA group, n=6 for the tOVA-ASC group). [Figure 9]Chicken ovalbumin producing EG7-OVA cell line used in the tumor model. a) Anti-OVA blot of EG7-OVA cell line. b) Timeline of EG7-OVA tumor model experiment. (OVA: 43 kDa) [Figure 10] EG7-OVA tumor progression after tOVA-ASC injection. a) Average tumor volume across all injections after injection (each dot represents one tumor). b) Progression of each tumor in the different groups. c) Average weight of excised tumors. (n=3 for PBS, n=5 for OVA, n=8 for tOVA-ASC injection.) [Figure 11] Eradication of subcutaneously inoculated EG7-OVA tumors after two intraperitoneal injections of tOVA-ASC speck. Photographs were taken of tumors excised from animals injected with PBS, OVA, and tOVA-ASC. Six of eight tumors in animals injected with tOVA-ASC were completely eradicated. [Figure 12] OVA-specific IgG responses in C57BL / 6 mice bearing EG7-OVA tumors and injected with 100 μg of tOVA-ASC. Anti-OVA IgG responses were measured by ELISA from plasma collected immediately before the animals were sacrificed (n = 1 for PBS, n = 4 for OVA, n = 6 for tOVA-ASC injection). [Figure 13] Intraperitoneally injected tOVA-ASC specks were detected in subcutaneously inoculated EG7-OVA tumors. Anti-TMS1 blots of tumor samples from animals injected with tOVA-ASC, OVA, and PBS. [Figure 14] Infiltration of CD11C+ dendritic cells into EG7-OVA tumors from animals injected with tOVA-ASC. 50-um sections of tumors from animals injected with PBS, OVA, and tOVA-ASC were collected using a cryostat and stained with anti-CD11c antibody. (CD11C: dendritic cell marker. White arrows indicate CD11c+ cells, and red arrows indicate remnants of nonspecific antibody in the interstitial spaces of tumor tissue.) [Figure 15]T cell proliferation in the spleens of animals injected with tOVA-ASC. Splenocytes from the excised spleens of animals injected with PBS and tOVA-ASC were stained with anti-CD4, anti-CD8, and anti-B220 antibodies, and the percentages of cells were analyzed using a ow cytometer. (CD4: T helper cells, CD8: cytotoxic T cells, B220: B cell marker. Error bars indicate SD values ​​between animals in the tOVA-ASC group.) [Figure 16] Intraperitoneally injected mCherry-ASC was located in the mesenteric lymph nodes of one animal. Animals injected with PBS, mCherry, and mCherry-ASC were anesthetized and examined at 580 nm under an IVIS device on days 1 (a) and 2 (b). [Figure 17] Drainage of intraperitoneally injected mCherry-ASC specks to secondary lymphoid organs. Lymph nodes (A) and spleens (B) from animals injected with PBS, mCherry, and mCherry-ASC were lysed by sonication and blotted with anti-TMS-1 antibody. [Figure 18] Vaccination schedule for immunization of EG7-OVA tumor-bearing mice. [Figure 19] Tumor volume after immunization. After immunization, the tOVA-ASC group had the highest antitumor activity compared to the other groups. The mCh-ASC+OVA group gave controversial results, as one of them completely eradicated the tumor after immunization. Each dot represents one mouse (n=4). [Figure 20] OVA-specific IgG responses for experimental groups in the EG7-OVA model. OVA-specific IgG responses were highest in tOVA-ASC-immunized mice. Compared with the OVA group, the mCh-ASC+OVA group had higher OVA-specific IgG levels. There was a significant increase in IgG levels in the OVA, mCh-ASC+OVA, and tOVA-ASC groups compared with the PBS group (n=4 per group). DETAILED DESCRIPTION OF THE INVENTION

[0037] The main objective of the present invention is to develop a novel cancer immunotherapy, which is achieved by using ASC protein, which has an important function in the inflammatory signaling pathway.

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

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

[0040] In unstimulated cells, ASC is soluble in the cytosol. Upon stimulation, ASC proteins aggregate to form spherical structures called ASC specks. ASC specks can be secreted to the outside of the cell to promote immune cell recruitment.

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

[0042] Because one of the molecular mechanisms of action of current adjuvants is the enhancement of antigen uptake by APCs, we used the THP-1 monocytic cell line for cell culture experiments. To understand whether ASC specks were phagocytosed by APCs, we separately exposed PMA-differentiated THP-1 macrophages to tOVA-ASC specks and mCherry-ASC specks. This experiment demonstrated that both tOVA-ASC specks and mCherry-ASC specks were phagocytosed by macrophages in a concentration-dependent manner (Figure 2). Furthermore, phagocytosed mCherry-ASC specks were degraded by THP-1 macrophages and were observed in different locations within the cells using fluorescence and confocal microscopy (Figure 3).

[0043] The inflammasome is one of the targets of the commonly used adjuvant alum, as well as the currently manufactured adjuvants AS03 and MF59. To understand whether the proposed adjuvant ASC-Speck, according to the present invention, can induce an inflammatory response in THP-1 macrophages, we measured changes in the mRNA expression levels of the pro-inflammatory proteins NLRP3, ASC, and procaspase-1 by RT-qPCR. In this regard, we have shown that the expression levels of pro-inflammatory proteins increase by at least 1.5-fold after mCherry-ASC-Speck treatment compared to mCherry protein treatment alone (Figure 5).

[0044] To confirm the RT-qPCR data, the secretion levels of IL-1β, TNF-α, and IL-6 cytokines were measured by ELISA after treatment with either mCherry-ASC specks or tOVA-ASC specks. IL-1β secretion increased in a time- and concentration-dependent manner. The increase in TNF-α secretion was concentration-dependent in the early phase (12 h after treatment), but no difference in TNF-α secretion was observed in the later phase (24 h after treatment) (Figure 6). tOVA-ASC treatment increased IL-1β secretion in a concentration-dependent manner. Treatment with tOVA-ASC specks at each concentration produced more IL-1β than treatment with 10 μg OVA. TNF-α and IL-6 levels increased concentration-dependently after tOVA-ASC treatment, and cytokine secretion levels were higher than those of 10 μg OVA treatment. Consequently, ASC specks formed by fusion of antigen with ASC induce the secretion of inflammatory cytokines from THP-1 macrophages (Figure 6). Using these RT-qPCR and ELISA data, it can be concluded that ASC speck treatment causes an increased inflammatory response in macrophages.

[0045] Antibody titers are not the only criterion for a successful vaccine; cytokine production by CD4+ cells may be a stronger correlate of successful immunization (Plotkin, 2010). To understand the cytokine production of T helper cells induced by tOVA-ASC specks in immune cells, we performed an in vitro recall assay and measured the levels of TNF-α, IFN-gamma, and IL-4 in both the presence and absence of OVA pulses. IFN-gamma is a cytokine produced primarily by T lymphocytes and natural killer cells and plays an important role in inhibiting tumor progression and metastasis (Farrar, MA and RD Schreiber, "The Molecular Cell Biology of Interferon and its Receptor," Annual Review of Immunology, Vol. 11, No. 1, pp. 571-611, 4, 1993). It has also been used as a direct antiviral agent.It is produced specifically by type 1 T helper cells after vaccination, triggering delayed-type hypersensitivity responses and resulting in 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 J.J. 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 November 1996). Using an in vitro recall assay, the present inventors demonstrated that both OVA-pulsed and non-pulsed splenocytes from animals injected with tOVA-ASC specks secreted IFN-gamma, and the secretion level decreased after OVA pulsing, whereas the other groups secreted IFN-gamma only after OVA pulsing. In this regard, it is believed that tOVA-ASC specks may induce the formation of a cellular immune response without antigen specificity.

[0046] TNF-α is a proinflammatory cytokine that may be required for both humoral and cell-mediated immune responses. However, TNF-α also induces a loss of function in NK cells, resulting in decreased IFN-γ secretion and cytotoxic activity against certain cancers (Romero-Reyes, M., C. Head, N.A. Cacalano, and A. Jewett, "Potent induction of TNF-α during interaction of immune effectors with oral tumors as a potential mechanism for loss of NK cell viability and function," Apoptosis, Vol. 12, No. 11, pp. 2063-2075, September 2007).

[0047] In the present experiment, splenocytes from each animal secreted TNF-α after OVA pulsing, but the secretion from splenocytes from both animals injected with tOVA-ASC and animals injected with alum + OVA was less than that from animals injected with OVA.

[0048] The IL-4 cytokine, produced primarily by type 2 T helper cells and known as a reciprocal antagonist of IFN-gamma, regulates B cell proliferation and eosinophil recruitment in vivo (Smiley, ST and MJ Grusby, "Interleukin 4," Encyclopedia of Immunology, pp. 1451-1453, Elsevier, 1998). IL-4 secretion from splenocytes of each animal decreased after OVA pulsing, but splenocytes from animals injected with tOVA-ASC secreted the highest amounts of the cytokine, both with and without OVA pulsing.

[0049] As a result, the inventors of the present invention showed that the secretion levels of TNF-α and IL-4 from splenocytes of animals injected with tOVA-ASC were very similar to those of animals injected with alum + OVA, a commonly used adjuvant (Figure 8). The difference between the proposed adjuvants, ASC specs, and alum adjuvant is the secretion of IFN-gamma before OVA pulse. Because IFN-gamma induces a more cell-mediated immune response, ASC specs can be used to enhance cellular immunity.

[0050] As mentioned above, immunotherapy is one of the most widely studied areas of cancer research. Vaccine adjuvants have been developed to enhance patients' cellular immune responses against cancer, and IFN-gamma is one of the major cytokines that primarily directs the immune system toward cellular immunity. Because tOVA-ASC-specks induce IFN-gamma secretion from splenocytes, we confirmed the in vivo effect of tOVA-ASC-specks on tumor formation by conducting an EG7-OVA tumor model in C57BL / 6 mice. To this end, 2.5 × 10 EG7-OVA cells were subcutaneously inoculated into the dorsal flank of mice. Once tumors reached a palpable size, intraperitoneal tOVA-ASC-specks injections were initiated. Because tumor size showed heterogeneity and the number of animals was limited, mice were divided into experimental and control groups so that each group had at least one small, one medium, and one large tumor. PBS and OVA injections were used as negative controls.

[0051] Four tumors in animals injected with tOVA-ASC were completely eradicated 7 days after the initial injection, while another tumor formed during the same period in animals injected with OVA. A booster injection was then administered on day 7. Two days after the booster injection, two more tumors were completely eradicated in the tOVA-ASC group. One animal died in the PBS group. Tumor progression in tOVA-ASC-injected animals indicated that tOVA-ASC specks had somehow exerted a therapeutic effect on the EG7-OVA tumors, while only two tumors remained in the tOVA-ASC group. Therefore, the experiment was terminated to understand the cellular and molecular mechanisms underlying this therapeutic effect of ASC specks. During necropsy, only connective tissue was found at the tumor inoculation site in animals whose tumors had been completely eradicated before necropsy, without any residual tumor tissue. The average tumor weight in the PBS and OVA groups was approximately 2 g, but 0.03 g in the tOVA-ASC group (Figure 9).

[0052] Further experiments using animal serum showed that the OVA-specific IgG response in animals injected with tOVA-ASC was successfully increased to approximately 18 μg / ml, compared with 5 μg / ml and 8 μg / ml in the PBS and OVA groups, respectively (Figure 12). The increase in IgG response in the tOVA-ASC group may be primarily due to two reasons. First, because EG7-OVA tumors continuously produce OVA protein, if tOVA-ASC specks induce a general immune burst in mice, it may induce antibody production against OVA. Second, this may be caused by doubling the amount of tOVA-ASC specks injected intraperitoneally.

[0053] Furthermore, the present inventors observed that intraperitoneally injected tOVA-ASC specks migrated to the subcutaneously inoculated EG7-OVA tumor site in one of the animals and remained intact, at least in the form of tOVA-ASC protein (Figure 13). This may be due to enhanced angiogenesis around the tumor microenvironment and leakage of these blood vessels, resulting in leakage of tOVA-ASC specks from the intraperitoneal site into the circulation and into the tumor. Another possible explanation is that tOVA-ASC specks are phagocytosed by APCs, which then accumulate in the tumor for some reason. Because mature DCs infiltrating tumors lead to increased immune activation and recruitment of immune effector cells, we confirmed whether DCs infiltrated tumors by IHC staining. We used a CD11C+ antibody, which stains conventional / classical DC subtypes.

[0054] This subtype has a defined, characteristic morphology with MHC class II expression (Collin, M., N. McGovern and M. Haniffa, "Human dendritic cell subsets," Immunology, Vol. 140, No. 1, pp. 22-30, September 2013). We demonstrated that CD11C+ DCs were localized in the tumor cryostat of one of the animals injected with tOVA-ASC (Figure 14). Interestingly, tOVA-ASC was found in CD11C+ DC-infiltrated tumors. The IHC and Western blot data raise the possibility that tOVA-ASC specks were transported to the tumor microenvironment by DCs.

[0055] To understand the molecular and cellular mechanisms of the therapeutic effect of tOVA-ASC specks, we repeated the EG7-OVA tumor model. EG7-OVA cells were inoculated into each dorsal flank of 24 mice, but only four tumors formed. To understand the effect of tOVA-ASC specks on immune cell proliferation and tumor infiltration, these four tumor-bearing animals were injected with PBS and tOVA-ASC. Seven days after the first injection, the tumor in one of the tOVA-ASC-injected animals began to shrink and was no longer palpable by day 10. The experiment was then terminated, and the percentages of helper T cells, cytotoxic T cells, and B cells in the spleen were measured using flow cytometry analysis. The percentages of CD4+ and CD8+ cells in the spleens of tOVA-ASC-injected animals were increased compared with those of PBS-injected animals (Figure 15). Based on both cytokine secretion from splenocytes and flow cytometry analysis, we can conclude that tOVA-ASC injection induces the formation of helper T cell activation in C57BL / 6 mice.

[0056] Vaccines are generally injected subcutaneously, intramuscularly, or intraperitoneally to stimulate immunity. The success of vaccines is enhanced by the influx of antigens into lymph nodes, where APCs interact with lymphocytes (Johansen, P. and T. M. Kundig, “Intralymphatic immunotherapy and vaccination in mice,” Journal of visualized experiments: JoVE, No. 84, p. e51031, February 2014; Tozuka, M., T. Oka, N. Jounai, G. Egawa, K. J. 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, April 2016). Furthermore, delivery of antigens from the injection site to lymphoid organs by migratory DCs enhances the efficiency of CTL priming (Allan, RS, J. Waithman, S. Bedoui, C.M. Jones, J.A. Villadangos, Y. Zhan, A.L. Rew, K. Shortman, W.R. Heath, and F.R. 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, July 2006.). To understand whether ASC specks induce antigen influx into secondary lymph nodes, we intraperitoneally injected mCherry-ASC specks into C57BL / 6 mice and visualized them using IVIS technology.Interestingly, red fluorescent signals from the spleen and mesenteric lymph nodes of animals injected with mCherry-ASC were recognized 1 day after immunization, but when excited at 580 nm, fluorescent signals were only present in animals injected with mCherry, but not with PBS (Figure 16). Furthermore, we validated the IVIS results by performing anti-hASC blots from mesenteric lymph node and spleen tissues after sacrificing the animals 2 days after injection (Figure 17).

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

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

[0059] The truncated forms of OVA contain both T cell epitopes of OVA, and the OVA peptides of 257-268 amino acids and 323-339 amino acids are MHC class I and MHC class II epitopes, respectively (Lipford, G.B., 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, 2019; McFarland, B.J., A.J. Sand, T.P. 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 December 1999). The tOVA-ASC protein is used to produce tOVA-ASC specks.

[0060] Furthermore, tOVA+ASC specks are produced by loading tOVA protein into ASC specks, which are then used to investigate the adjuvant effect of ASC specks in cancer immunotherapy.

[0061] It is important to note herein that there are various ways to load tumor antigens into ASC specs. For some methods of loading bioactive molecules in general, and tumor antigens specifically according to the present invention, into ASC specs, see published patent application WO2013 / 160807 (Methods for antigen delivery).

[0062] For example, tumor antigens can be loaded onto the ASC speck carrier via hydrophobic interactions or by forming a fusion protein of at least one antigen with the ASC protein that forms the ASC speck carrier.

[0063] Hydrophilic antigens cannot be supported on ASC speck carriers solely through hydrophobic interactions. Therefore, in one embodiment, hydrophilic antigens can be loaded onto ASC speck carriers via hydrophobic interactions by creating a fusion protein between the antigen and a peptide containing at least 13 amino acids in length and a hydrophobic sequence.

[0064] Second, produce and purify mCherry-ASC specks according to the method described in WO 2013 / 160807. mCherry is a red fluorescent protein used as a tracker for imaging systems.

[0065] Experiments performed using the above-mentioned ASC specks demonstrated that purified ASC specks were phagocytosed and degraded by macrophages. Furthermore, it has been demonstrated that stimulation of THP-1 macrophages with purified ASC specks induces the secretion of the proinflammatory cytokines IL-1β, IL-6, and TNF-α. Furthermore, injection of tOVA-ASC specks has been shown to induce the secretion of type I and type II cytokines from splenocytes of immunized wild-type C57BL / 6 mice. Finally, we demonstrated that injection of tOVA-ASC specks resulted in the complete eradication or reduction in size of EG7-OVA tumors in C57BL / 6 mice.

[0066] As mentioned above, chicken ovalbumin (OVA) is used as a model antigen for the purposes of this invention. To produce tOVA-ASC (a fusion protein of a truncated form of chicken ovalbumin (OVA) containing amino acid sequences 239-386 and human ASC protein), the p-C3-d(1-238)OVA-ASC plasmid was cloned.

[0067] To test the immunostimulatory effects of ASC specks in vivo and on THP-1 cell lines, we produced mCherry-ASC specks and tOVA-ASC specks in the HEK293FT cell line. To measure the purity and concentration of tOVA-ASC specks, purified specks and a given concentration of BSA were loaded onto SDS-PAGE and stained with Coomassie blue, as shown in Figure 1. To understand whether the purified protein was tOVA-ASC, we performed anti-hASC blots of the isolated protein.

[0068] To examine the effect of ASC specks on antigen uptake, PMA-differentiated THP-1 macrophages were treated with 10 μg / ml, 25 μg / ml, 50 μg / ml, and 100 μg / ml of mCherry-ASC specks. PBS and 50 μg / ml of commercial mCherry protein were used as negative controls. After 12 hours of treatment, no mCherry signal was observed in wells treated with PBS and mCherry protein, whereas cells treated with mCherry-ASC specs phagocytosed the mCherry-ASC specks, as seen in Figure 2. hASC blots of treated cells also show phagocytosed and degraded mCherry-ASC protein.

[0069] To confirm and quantify the fluorescence data, we repeated the same experiment and counted mCherry-positive cells by flow cytometry 12 hours after treatment. The number of mCherry-positive cells increased with increasing concentrations of mCherry-ASC specks in the treatment. These experiments demonstrate that ASC specks promote the phagocytosis of mCherry protein by THP-1 macrophages.

[0070] Degradation of phagocytosed mCherry-ASC specks by THP-1 macrophages was detected under a fluorescent microscope (Figure 3). Four hours after mCherry-ASC treatment, using a 40x objective lens on a fluorescent microscope, the red fluorescent signal originated from the cell cytoplasm and spread throughout the cell cytoplasm, rather than forming speck-like structures.

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

[0072] To understand whether ASC specks induce a pro-inflammatory response after phagocytosis, we measured secreted cytokines from the culture medium of PMA-differentiated THP-1 macrophages 12 and 24 hours after mCherry-ASC treatment. PBS, mock-purified samples, and commercially available mCherry protein were used as negative controls. Treatment with mCherry-ASC specks increased the secretion of IL-1β and TNF-α levels in a time- and concentration-dependent manner, whereas cells treated with mCherry and mock-purified samples secreted basal levels of cytokines, as seen in Figure 5.

[0073] As a result, it is speculated that even if the peptide used is not antigenic to the ASC speck of THP-1 macrophages, the phagocytosis of the peptide may be enhanced, leading to the formation of an inflammatory response.

[0074] To further investigate whether tOVA-ASC specks are also phagocytosed by APCs, PMA-differentiated THP-1 macrophages were treated with 10 μg / ml tOVA-ASC specks and subjected to immunocytochemistry using an anti-hASC antibody 12 hours after treatment. 10 μg / ml OVA and PBS were used as negative controls. Anti-mouse IgG conjugated to Alexa-488 was used as the secondary antibody. The weak green fluorescent signal in Figure 6 indicates endogenous ASC, which is normally found in cells distributed throughout the cytoplasm but forms speck-like structures after inflammatory activation. The patchy, intense green fluorescent signal in Figure 6 indicates the phagocytosis of tOVA-ASC specks by THP-1 macrophages.

[0075] To ensure that all specks were not endogenously formed, Western blot analysis was performed using anti-TMS1 (a rabbit-produced hASC antibody). For Western blot analysis, THP-1 macrophages were treated with 2.5 μg / ml, 5 μg / ml, and 10 μg / ml of tOVA-ASC. 10 μg / ml of OVA and PBS were again used as negative controls. After 12 hours of treatment, wells were washed three times with PBS to remove any tOVA-ASC specks that had not yet been phagocytosed, and Western blot analysis was performed using anti-TMS1 and anti-OVA.

[0076] As can be seen in Figure 6, the band size is approximately 39 kDa, which indicates a patchy green fluorescent signal emanating from the tOVA-ASC speck. OVA can also be phagocytosed by macrophages without the need for adjuvants or vaccine carriers (lane 3 in Figure 6).

[0077] The cell culture medium was also collected, and the secreted IL-1β, IL-6, and TNF-α levels were measured by ELISA. tOVA-ASC speck treatment significantly increased IL-1β and IL-6 secretion (Figure 7). IL-1β secretion from THP-1 macrophages was higher with each concentration of tOVA-ASC treatment than with 10 μg of OVA treatment, and increased in a concentration-dependent manner. IL-6 secretion levels after tOVA-ASC treatment increased dose-dependently and were higher with 10 μg / ml tOVA-ASC treatment than with 10 μg / ml OVA treatment alone. TNF-α levels also increased with increasing concentrations of tOVA-ASC. However, there was no significant difference in TNF-α secretion between 10 μg / ml OVA treatment and 10 μg / ml tOVA-ASC treatment.

[0078] EG7-OVA, a C57BL / 6 mouse thymoma cell line that synthesizes chicken ovalbumin, is one of the tumor models primarily used for antigen-specific tumor vaccine research. Treatment with G418 induces OVA expression. Anti-OVA blot analysis demonstrated OVA expression in the EG7-OVA cell line prior to inoculation into mice, as shown in Figure 9.

[0079] To induce tumor formation in 8- to 10-week-old C57BL / 6 mice, 2.5 million EG7-OVA cells were inoculated into each dorsal flank. Twelve days after inoculation, 18 tumors reached a palpable size, and intraperitoneal injections of 100 μg of tOVA-ASC in 300 μl of PBS were initiated, as shown in Figure 9. 50 μg of OVA and PBS were used as a negative control. After the initial immunization, tumor sizes were measured every two days using two digital calipers.

[0080] One tumor in the tOVA-ASC-injected animal was completely eradicated four days after the first injection. Three additional tumors in the tOVA-ASC-injected animals were completely eradicated seven days after the first injection, and one additional tumor formed in one of the OVA-injected animals. One week after the first immunization, a booster injection was administered in the same manner as the first. One day after the second immunization, one of the PBS-injected animals died, likely due to the size of its tumor. As can be seen in Figure 10, two additional tumors in the tOVA-ASC-injected animals were completely eradicated two days after the second injection.

[0081] Four days after the second immunization, only two tumors remained after the second injection, so the experiment was terminated for further analysis using the tumors from the animals injected with tOVA-ASC. The progression of each tumor in each group was shown. All animals were sacrificed. No tumor tissue was observed in the animals injected with tOVA-ASC, whose tumors had already been eradicated. One of the animals injected with OVA had two separate tumor tissues on the same dorsal flank. As shown in Figure 11, the average volumes of the excised tumors for injected PBS, OVA, and tOVA-ASC were 1935 mm, respectively. 3 , 1690mm 3 and 62mm 3 As shown in Figure 11, the average tumor weights of tumors injected with PBS, OVA, and tOVA-ASC were 2.5 g, 2.11 g, and 0.07 g, respectively.

[0082] As a result, as shown in Figure 10, while tumors from PBS- and OVA-injected animals continued to grow in the dorsal flanks of C57BL / 6 mice, two shots of tOVA-ASCspec resulted in a reduction in the size of each tumor and complete eradication of 6 out of 8 EG7-OVA tumors.

[0083] Because the antitumor effect of tOVA-ASC specks may be caused by an antibody response to OVA, the OVA-specific IgG levels of the animals were measured by ELISA. One animal in the PBS group died before sacrifice, so blood could not be collected from the animal. Further experiments were performed using only one animal from the PBS group. As a result, as can be seen in Figure 12, animals injected with tOVA-ASC had a greater OVA IgG response than animals injected with PBS or OVA.

[0084] Further studies were performed to investigate the adjuvant effect of ASC specks.

[0085] There were four experimental groups, including PBS, OVA, tOVA-ASC, and mCh-ASC + OVA. Each group of mice was intraperitoneally injected with PBS alone, 100 μg of OVA alone, 100 μg of tOVA-ASC alone, and 100 μg of mCh-ASC and 100 μg of OVA. Because both OVA and ASC specks were suspended in PBS, PBS served as the negative control. To examine the adjuvant effect of ASC specks, OVA injection was performed to determine whether immunization with OVA alone was sufficient to reduce tumor size. The mCh-ASC and OVA group also served as a model to confirm the adjuvant properties of ASC specks compared with the OVA group. In this experiment, the tOVA-ASC group served as a model group to confirm the carrier capacity of ASC specks as an antigen-specific antitumor vaccine. The vaccination schedule for immunization of EG7-OVA tumor-bearing mice can be seen in Figure 18.

[0086] Fourteen days after inoculation with EG7-OVA cells, tumor formation reached a palpable size and immunization was initiated. As shown in Figure 19, 7 days after the first immunization, tumor size increased in the PBS, OVA, and mCh-ASC+OVA groups, while mice immunized with tOVA-ASC speck showed little progression in tumor size. A second dose was then injected into each group.

[0087] Seven days after the second immunization, one mCh-ASC+OVA-immunized mouse and three tOVA-ASC-immunized mice completely eradicated tumors. One tumor from a mouse immunized with OVA alone remained the same size after two injections. One mCh-ASC+OVA-immunized mouse maintained a stable tumor size after two injections. All animals were sacrificed after the final immunization. Finally, one tOVA-ASC-immunized mouse showed a tumor growth pattern after injection.

[0088] Antibodies are currently undergoing clinical trials to target tumor cells. These antibodies bind to tumor cell antigens and induce direct cell lysis or antibody-facilitated phagocytosis, leading to antigen processing and presentation via MHC class I or II molecules on APCs. This situation induces host antitumor immunity through the production of either antibodies and / or cytotoxic T cells against the tumor. Because the antitumor effect of tOVA-ASC speck was observed, this antitumor activity may be caused by antibody-facilitated antitumor immunity. To demonstrate antigen-specific antibody production in the host, ELISA was performed to examine OVA-specific IgG responses against EG7-OVA tumors.

[0089] As shown in Figure 20, PBS was the negative control group, and increased OVA-specific IgG levels were obtained in the OVA, mCh-ASC+OVA, and tOVA-ASC groups compared with the other groups containing PBS. When comparing the OVA-containing groups with each other, the mCh-ASC+OVA group had a significantly higher IgG response than the OVA group. The tOVA-ASC group also had a significantly higher IgG response than the OVA group. This indicated that ASC specks can have antitumor activity by enhancing the immune system as an adjuvant and delivering antigens to induce antitumor immunity in the host.

[0090] Finally, intraperitoneally injected mCherry-ASC specks have been shown to home to secondary lymph nodes, which are important for eliciting the adaptive immune system.

[0091] Because secondary lymph nodes are the primary site of T cell responses, we investigated whether intraperitoneally injected mCherry-ASC specks could be transported to the mesenteric lymph nodes and spleen using IVIS and Western blot analysis. To this end, eight 16-18 week-old C57BL / 6 mice were intraperitoneally injected with 200 μl of PBS containing 10 μg of mCherry-ASC specks. As negative controls, four and eight animals were injected with PBS and commercially available mCherry protein, respectively. The animals were monitored with an IVIS system 1, 2, and 10 days after injection. On day 2, half of the animals were sacrificed, and their spleens, mesenteric lymph nodes, livers, and brains were collected for Western blot analysis. The other half was sacrificed on day 10, and the same tissues were collected.

[0092] As seen in Figure 16, yellow indicates a high intensity red fluorescent signal in IVIS, while the abdominal and genital regions of C57BL / 6 animals produce background fluorescence when the animals are excited at wavelengths between 56 and 580 nm. However, one day after injection, strong fluorescence was observed in one of the mesenteric lymph node regions of an animal injected with mCherry-ASC. At day 10, the fluorescence intensity was nearly identical in each animal.

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

[0094] Thus, in one embodiment of the present invention, ASC specks (formed by ASC proteins) are used to treat cancer, as experiments have demonstrated their ability to enhance the immune system against tumor cells.

[0095] In another embodiment, a composition comprising an ASC speck for use in a method of cancer treatment is provided, the method comprising inducing and / or stimulating an immune response against cancer disease in a mammal, the induction or stimulation resulting in the inhibition of cancer progression or the arrest of cancer cell proliferation.

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

[0097] In another embodiment, a composition of ASC specs for use in cancer treatment is provided, the composition comprising a tumor antigen carried by the ASC specs by forming hydrophobic interactions with the ASC proteins that form the ASC specs.

[0098] According to one embodiment of the present invention, a composition comprises an ASC speck formed by a fusion protein between a tumor antigen and at least one ASC protein, said composition being used in the treatment of cancer, in particular in the treatment of tumors.

[0099] The fusion protein can be formed by the tumor antigen fused to the ASC protein at the N-terminus or C-terminus.

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

[0101] The present invention relates to the treatment of any kind of cancer, preferably oncogenic cancer, more preferably thymic carcinoma.

[0102] According to one embodiment, the compositions of the present invention are used in cancer immunotherapy, wherein administration of the composition to a mammal in need thereof results in inhibition of tumor progression or a reduction in tumor size or complete eradication of the tumor.

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

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

[0105] Examples of tumor antigens that can be used in the present invention are HER2+ (membrane-bound protein) for breast cancer, PSA (glycoprotein with a size of 34 kDa) for prostate cancer, MAGE (melanoma-associated antigen) for melanoma cancer, MUC1 (transmembrane glycoprotein) for ductal glycoprotein or neoantigen. These are given as examples only, and any other tumor-associated antigen can be used for the purposes of the present invention.

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

[0107] experiment material: Details of the materials used in the experiments are given below. [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9]

[0108] method: 1. Purification of tOVA-ASC and mCherry-ASC Species 1.1. Amplification and isolation of plasmid DNA The p-C3-d(1-238)OVA-ASC and p-C3-mCherry-ASC plasmids were cloned and used in this study. Intermediate and large-scale preparations were performed using a MACHEREY-NEGEL (MN) kit as suggested by the manufacturer. Bacterial cultures were grown in 200 ml and 400 ml LB supplemented with kanamycin for intermediate and large-scale preparations, respectively.

[0109] 1.2.Maintenance of HEK293FT cell line The HEK293FT cell line was 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 washed with PBS and trypsinized for 2 min at 37°C for subculture.

[0110] 1.3.Calcium phosphate transfection The HEK293FT cell line was easily transfected using the calcium phosphate transfection method, which resulted in the formation of an insoluble precipitate of plasmid DNA and calcium phosphate. These precipitates were phagocytosed by HEK293FT cells. Fourteen million cells were seeded onto a 150 mm cell culture plate. Plasmid concentrations were adjusted according to the experiment by diluting in ddH2O, and 2M CaCl2 was added dropwise. After 5 minutes, 2x HBS was added and bubbles were formed. After 5 minutes, the plasmid and calcium phosphate mixture was added dropwise to the subcultured HEK293FT cells. [Table 10]

[0111] 1.4. Generation and isolation of ASC specks Fourteen million HEK293FT cells were counted and seeded into each of six 150 mm cell culture plates. (pC3-(d1-235)OVA-ASC) plasmid DNA was transfected using the calcium phosphate transfection method. 48 hours after transfection, the cell medium was aspirated and 3 ml of PBS was added. Cells were detached from the surface by scraping and placed in a 50 ml Falcon. They were lysed five times by 30% sonication for 5 seconds at 50% power. The lysed cells were centrifuged at 2400 g at 4°C using a Beckman tabletop centrifuge. After centrifugation, the pellet was lysed until filaments were easily detectable by vortexing. Large cell fragments were sedimented by gravity settling on ice for 5 minutes. 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 again dissolved by vortexing until filaments were visible, and the filaments were again allowed to settle by gravity for 5 minutes.

[0112] The supernatant was transferred to a new Falcon and centrifuged at 300 g. The final vortexing, gravity settling, and centrifugation were repeated until no filaments remained after vortexing. The final sample was filtered through a 1.2 or 5 μm syringe filter. 3–4 ml of sample was passed through each filter until backpressure was detected. 2–3 ml of PBS was filtered in the reverse direction with a 200 μl micropipette, and the ASC specks were transferred to 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 specks in PBS were kept at +4°C. The purity of the ASC specks was confirmed by SDS-PAGE and Coomassie blue staining. All steps except sonication were performed in a cell culture hood.

[0113] 1.5. Determining the Purity and Quantity of Isolated ASC Specks Using SDS-PAGE and Coomassie Blue Staining 40 μl of isolated ASC specks and BSA at different concentrations were mixed separately with 10 μl of 5x Leamli buffer in a 1.5 ml tube. They were boiled at 95°C for 10 minutes and loaded onto a 12% SDS-PAGE gel along with a prestained protein ladder. Samples were run as described in the protein gel electrophoresis section. The gel was stained with Coomassie blue in a plastic container for 1 hour and destained with Coomassie destaining solution. Photographs of the gel were taken, and the concentration of ASC specks was calculated using ImageJ software according to a standard curve derived from the BSA concentration.

[0114] 2. Effect of ASC speck on THP-1 macrophages 2.1.Maintenance of THP-1 monocytic cell line THP-1 is a monocytic cell line derived from a leukemia patient. The THP-1 eGFP-ASC stable cell line, which provides THP-1 cells and constitutively synthesizes eGFP-ASC, was derived from THP-1 monocytes by lentiviral transduction with pLenti-Ef1a-EGFP-ASC. The cell line was maintained in RPMI 1640 medium supplemented with 10% FBS, MEM-NEA, 100 U / ml penicillin, and 100 μg / ml streptomycin.

[0115] 2.2. Differentiation of THP-1 monocytes into THP-1 macrophages 1.5 × 10 THP-1 monocyte cells were differentiated into THP-1 macrophages in each well of a 6-well plate containing 100 nm / ml PMA in RPMI 1640 for 9 hours, and then the cell medium was replaced with RPMI 1640 without PMA.

[0116] Treatment of THP-1 macrophages with ASC specks After 48 hours of differentiation, to examine the effect of ASC specks on macrophages, mCherry-ASC or tOVA-ASC specks were added to 1 ml of cell culture medium at different concentrations. The cell culture medium was collected and stored at -20°C for further cytokine ELISA assays at different time points. Twelve hours after ASC speck treatment, THP-1 macrophages were prepared for flow cytometry analysis by incubating the cells with PBS-EDTA on ice for 30 minutes. Cells were then detached from the cell culture plate by gentle scraping and placed in a 15 ml centrifuge tube. The cells were centrifuged at 300 g for 5 minutes and washed twice with FACS buffer containing human serum. Their cell surface proteins were stained and analyzed as described in the Cell Surface Staining and Flow Cytometry Analysis section.

[0117] Immunocytochemistry of THP-1 macrophages 1.6×10 5THP-1 monocytes were differentiated into THP-1 macrophages in 48-well plates and treated with ASC speckle. 12 hours after ASC treatment, the cell medium was aspirated, and the wells were washed three times with PBS. The cells were then fixed with 4% PFA for 15 minutes at room temperature. The cells were then washed three times with PBS and permeabilized with 0.25% Triton-X100 surfactant in PBS. The cells were then blocked with blocking buffer (PBS-T containing 5% BSA (wt / vol)) for 30 minutes at room temperature. The cells were then incubated with a 1 / 50 dilution of anti-hASC antibody in blocking buffer for 2 hours at room temperature. The cells were washed again with PBS and incubated with a 1 / 500 dilution of Alexa-488 fluorophore-conjugated anti-mouse IgG for 1 hour at room temperature. After secondary antibody incubation, the cells were washed and visualized under a fluorescent microscope.

[0118] Cytokine ELISA Cytokine levels in cell culture media were measured by sandwich ELISA. Briefly, ELISA plates were coated with PBS containing the capture antibody at the manufacturer's suggested concentration and incubated overnight at room temperature. Three wells of a 96-well plate were coated for each supernatant sample, and three wells were coated with PBS alone as a negative control. After overnight incubation, the plate was washed three times with 400 μl of PBS-T and blocked for 1 hour at room temperature with 200 μl of reagent diluent (PBS containing 1% BSA) to prevent nonspecific binding. During plate blocking, the cell supernatant was diluted in reagent diluent so that the final OD value was less than 2.00. After blocking, the plate was washed three times with PBS-T, and 100 μl of diluted samples and cytokine standards were added to the wells and incubated for 2 hours at room temperature. The plate was then washed three times again with PBS-T and incubated with 100 μl of reagent diluent containing the detection antibody at the manufacturer's suggested concentration for 2 hours at room temperature.

[0119] After washing the plate, 100 μl of 1:50 diluted HRP in reagent diluent was added to the wells and incubated at room temperature for 1 hour. The plate was then washed again, and 100 μl of a 1:1 mixture of ELISA substrate A and ELISA substrate B was added to the wells, causing the color to begin to change to blue. Twenty minutes after the addition of the substrate, the reaction was terminated with 1N sulfuric acid, causing the color to change 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 was extracted between 450 and 540 nm. A standard curve was constructed using SoftMaxPro software according to the absorbance values ​​and concentrations of the standards, and cytokine levels in the supernatants were measured from the standard curve.

[0120] 2.6. Determining the expression patterns of pro-inflammatory proteins in THP-1 macrophages after ASC-speck treatment by RT-qPCR Protein expression patterns at the mRNA level were measured by real-time quantitative PCR in PMA-differentiated THP-1 cells treated with ASC specks. THP-1 macrophages were treated with 10, 25, 50, or 100 μg of mCherry-ASC specks for 12 hours. PBS and 50 μg of mCherry protein alone were used as controls.

[0121] 2.6.1. RNA extraction. Cells were washed three times with PBS, harvested by scraping, and RNA was extracted using the MN RNA extraction kit as suggested by the manufacturer. Briefly, cells were lysed in lysis buffer and mixed with 70% ethanol, after which the lysate was bound to the column. After several washing steps, the column was treated with DNase incubation buffer containing DNase I for 15 minutes at room temperature. The column was again washed with wash buffer and dried. Total RNA was then isolated with 60 μl of RNase-free water. RNA purity and concentration were measured using a Nanodrop spectrophotometer.

[0122] 2.6.2.cDNA Synthesis. cDNA was synthesized from total RNA using the BIOLINE SensiFAST cDNA Synthesis Kit as suggested by the manufacturer. Briefly, 500 ng of total RNA was mixed with 4 μl of 5x TransAmp buffer and 1 μl of reverse transcriptase in DEPC-treated H20 (final volume: 20 μl). The sample was incubated at 25°C for 10 min, 42°C for 15 min, and 85°C for 5 min for inactivation.

[0123] 2.6.3. Quantitative PCR The relative expression levels of the focal genes after ASC-spec treatment were examined using primers specific for each gene and HPR-T as a control. cDNA samples were amplified with BIOLINE SensiFAST SYBR MasterMix using an Exicycler 96 qPCR instrument. The expression levels of genes in PBS-treated cells were adjusted to 1 and 2. -ddct The relative expression of the genes of interest was measured by using the method (Livak and Schmittgen 2001).

[0124] 3. EG7-OVA Tumor Model and Immunization with tOVA-ASC 3.1. Maintenance of EG7-OVA cell line The EG7-OVA cell line was derived from the C57BL / 6 mouse lymphoma cell line EL-4. The EL4 cell line was stably transfected with the pAc-neo-OVA plasmid, which contains the intact form of chicken ovalbumin (OVA) and a neomycin resistance gene, to form the EG7-OVA cell line, which constitutively synthesizes OVA protein as a model tumor antigen when treated with G418. The cell line was maintained in RPMI 1640 medium 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 two days.

[0125] 3.2. EG7-OVA Tumor Inoculation and Immunization with OVA, PBS, and tOVA-ASC Twelve C57BL / 6 mice were inoculated with 2.5 million EG7-OVA cells in 100 μl of PBS into each dorsal flank. Eighteen tumors reached a palpable size (<50 mm) within 12 days. Intraperitoneal injections were performed as follows: 100 μg of tOVA-ASC (n = 10), 50 μg of commercial OVA (n = 5), and PBS alone (n = 3) in 300 μl of PBS. Two tumor sizes were measured using two digital calipers. The volume was calculated based on the formula: 1 / 2 ((shorter length^2) longer length). The second injection was repeated as the first. One of the PBS-injected animals likely died on day 20. Because only two tumors remained after the second injection, the experiment was terminated for further analysis using the tumors from the tOVA-ASC-injected animals. Blood was collected from all animals via the orbital sinus. All animals were sacrificed, and their spleens were collected. The tumors were excised, and their volumes and weights were calculated. No tumor tissue was observed in tOVA-ASC-injected animals whose tumors had already been eradicated. Plasma from each animal was stored at -20°C. Splenocytes from the animals were harvested and cultured in 96-well plates for further in vitro recall assays. The tumors from the animals were embedded in OCT for further immunohistochemistry assays. A small portion from the tumors of each group was divided for Western blot analysis and stored at -80°C.

[0126] 3.3. EG7-OVA Tumor Inoculation and Immunization with PBS, OVA, tOVA-ASC, and mCherry-ASC Similar to the above experiment, another test was performed for additional mCherry-ASC immunizations. 6 Each mouse was inoculated with 100 μl of 1×PBS containing EG7-OVA cells. Before inoculation, OVA expression in these cells was confirmed by Western blot analysis using an anti-OVA antibody. The cells were prepared under sterile conditions. Each mouse was inoculated with these cells into the right dorsal flank. After 12–14 days, tumors were palpable (≥50 mm). 3), intraperitoneal injections were performed as follows: 100 μg OVA, 100 μg mCherry ASC, 100 μg OVA, and 100 μg tOVA ASC in 300 μl of 1×PBS. Tumor size was measured with a caliper. The formula 1 / 2[(short length 2 The volume of each tumor was calculated according to the following formula: (1) longest length. The second injection was repeated in the same manner as the first injection.

[0127] Because most tumors in mice immunized with tOVA ASC were eradicated, the experiment was terminated one week after the second injection. Animal tumors were calculated using calipers. All animals were then sacrificed. Blood was collected from their hearts. Their spleens and livers were also collected for further analysis. Animal tumors were excised and embedded in OCT. All tissues were kept on ice until the last mouse was treated. The collected blood samples were incubated at 37°C for 1 hour and then centrifuged at 11,000 rpm for 1 minute. Blood serum was collected into new tubes and kept at -80°C for long-term storage.

[0128] 3.4. Tumor and spleen immunohistochemistry 3.4.1 OCT Embedding: Tumor samples were added to OCT compound and kept at -20°C overnight, then maintained at -80°C for further analysis.

[0129] 3.4.2. Frozen Sectioning. The OCT-embedded tumor specimen was placed right-side up in a frozen sectioning machine, and the tissue-free frozen OCT was removed with a blade. Then, using a cryostat, 10-micron tumor sections were harvested onto positively charged slides. The slides were dried at 55°C and kept at -80°C for further analysis.

[0130] 3.4.3 Fixation and Antigen Retrieval. The slides were dried at 55°C for 2 hours, and the tissue sections were surrounded by a hydrophobic barrier. The tissues were then fixed with 4% PFA at room temperature for 15 minutes. The PFA was removed, and the slides were washed three times with PBS-T in a jar on a shaker. The slides were transferred to another jar filled with citrate buffer (pH: 6.0) and kept at 70°C for 30 minutes.

[0131] 3.4.4. Antibody staining of cryostat sections. After washing the slides three times with PBS-T, they were blocked with blocking buffer (PBS-T containing 3% BSA) for 30 minutes at room temperature on a shaker. The slides were then incubated overnight at 4°C with blocking buffer containing a 1 / 400 diluted fluorescently labeled primary antibody. After overnight incubation, the slides were washed three times for 5 minutes with PBS-T and visualized under a fluorescent microscope.

[0132] 3.5. Preparation of Single Cells from EG7-OVA Tumors Tumors were excised and minced using scissors until all tissue sections were processed into 1-2 mm pieces. The minced samples were disaggregated against a cell strainer using the plunger from a disposable 10 ml syringe. The tumor tissue on the cell strainer was washed with 3-5 ml of cold RPMI 1640. The washing process was repeated several times until only small fragments of connective tissue were observed on the underside of the filter. The cells were washed three times with RPMI 1640 and centrifuged at 300 x g for 5 minutes at room temperature.

[0133] 3.6. ELISA for OVA-specific IgG responses OVA-specific IgG responses were measured by ELISA. Briefly, 96-well ELISA plates were coated with 100 ng / well of OVA protein diluted in 100 μl of PBS, with three wells containing PBS alone used as a negative control. The plates were sealed and incubated overnight at 4°C. Each well was aspirated and washed three times with 300 μl of wash buffer (PBS-T). After the final wash, residual wash buffer and air bubbles were removed by blotting the plate with clean paper towels. 200 μl of reagent diluent (PBS containing 1% BSA) was added to each well, and the plate was incubated at room temperature for a minimum of 1 hour and a maximum of 2 hours. The washing process was repeated. After the final wash, residual wash buffer and air bubbles were removed by blotting the plate with clean paper towels. 100 μl of standard (25 pg / ml to 5000 pg / ml) or sample (1:500 dilution) was added, the plate was covered with stretch film, and incubated at room temperature for 1 hour. The washing process was repeated, and the same washing 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 room temperature for 1 hour. The washing process was repeated, and the same washing protocol was performed after the final wash. Protecting the plate from direct light, 100 μl of substrate mix (a 1:1 mixture of color reagents A and B) was added to each well, and the plate was incubated at room temperature for 20 minutes. Then, 20 μl of stop solution (2N H2SO4) was added to each well. The plate was gently tapped to ensure complete mixing. The absorbance of each well was measured at 450 nm and 540 nm using a plate reader.

[0134] 4. Tracking mCherry-ASC Spectroscopy In Vivo mCherry-ASC specks were used to track ASC specks after intraperitoneal injection. Eight C57BL / 6 mice were intraperitoneally injected with 300 μl of PBS containing 10 μg of mCherry-ASC. As controls, 300 μl of PBS containing 10 μg of mCherry and 300 μl of PBS alone were injected. Eight and four mice were used for mCherry injection and PBS injection, respectively.

[0135] 4.1. In vivo imaging Injected mCherry-ASC specks were visualized in vivo using an IVIS Spectrum in vivo imaging system, and animals were observed with the IVIS Spectrum instrument on days 1, 2, and 10.

[0136] 4.2. Tissue Sampling for Western Blot and Immunohistochemistry On day 2, half of the animals in each group were sacrificed, and on day 10, the remaining half were sacrificed. The spleens, mesenteric lymph nodes, livers, and brains of the animals were excised. The spleens were observed ex vivo under IVIS. A representative section from the spleen was taken for Western blot analysis, and the other sections were embedded in OCT compound and stored at -80°C. The mesenteric lymph nodes, livers, and brains of the animals were kept at -80°C for further Western blot analysis.

[0137] 5. Western Blot Analysis 5.1. Sample preparation from cell cultures Gene expression patterns and regulation at the protein level are commonly detected by Western blot analysis. To analyze protein expression in cells, each well of a 6-well plate was washed with 1 ml of PBS, or the cells were centrifuged and washed with 1 ml of PBS for adherent and suspension cell cultures, respectively. After aspirating the PBS, 160 μl of 0.2% NP40 lysis buffer supplemented with protease inhibitor complex was added to the cells. For adherent cell lines, cells were collected into 1.5 ml tubes using a scraper and a micropipette. The collected cells were kept on ice for 1 hour and vortexed for 15 minutes each to completely lyse the cells. Larger cell fragments were pelleted by centrifugation at 13,000 rpm for 30 minutes at 4°C. The supernatant was collected into another 1.5 ml tube. Then, 40 μl of 5x Laemmli buffer was added to the sample, and proteins were denatured in Laemmli buffer at 95°C for 10 minutes.

[0138] 5.2. Sample preparation from tissue In a 2 ml tube, 200 μl of lysis buffer was added to representative sections of minced and frozen large tissues (spleen, liver, brain, and tumor) and whole mesenteric lymph nodes. The tissues were lysed by sonication at 50% power for 1 minute at 70% output and kept on ice for 15 minutes. The tissue lysate was then centrifuged at 13,000 rpm for 30 minutes to remove larger fragments and connective tissue. The supernatant was transferred to a new 1.5 ml tube and diluted. 5x Laemmli buffer was then added appropriately, and the proteins were denatured at 95°C for 15 minutes.

[0139] 5.3. Preparation of SDS-PAGE gel Eight milliliters of 10%, 12%, or 15% resolving gel solution was mixed with 80 μl of 10% APS and 8 μl of TEMED. After mixing, the gel was loaded between a 1.5 mm spacer and a short plate. Isopropanol was then applied to the gel surface. After polymerization of the resolving gel, the isopropanol was removed, and 4 ml of a 4% stacking gel was prepared by mixing 40 μl of 10% APS and 4 μl of TEMED. The stacking gel was loaded onto the resolving gel, and a 10-well comb was inserted. After polymerization of the stacking gel was complete, the SDS-PAGE gel was used for protein gel electrophoresis.

[0140] 5.4. Protein Gel Electrophoresis Proteins were loaded onto an SDS-PAGE gel and separated according to their kDa content. The SDS-PAGE cast gel was placed in a vertical protein gel electrophoresis chamber and filled with running buffer. The comb was carefully removed, and 35 μl of denatured protein sample in Leamli buffer and protein ladder was loaded into the well. The sample was run at 70 V until it passed through the stacking gel, and then at 120 V until the dye reached the bottom of the resolving gel.

[0141] 5.5.Semi-dry transfer For antibody blotting, proteins from the SDS-PAGE gel were transferred to a nitrocellulose membrane. Briefly, the nitrocellulose membrane was activated with methanol, and the membrane and two pieces of filter paper were wetted with transfer buffer. One filter paper, the membrane, the SDS-PAGE gel, and the other filter paper were placed in a semi-dry transfer apparatus. The cover of the apparatus was closed, and the transfer of proteins from SDS-PAGE to the nitrocellulose membrane was completed at 10 V for 50 minutes.

[0142] 5.6.Membrane Blocking The protein transfer membrane was placed in a plastic container and blocked for 1 hour with TBS-T containing 5% (wt / vol) skim milk powder to prevent nonspecific binding of the detection antibody.

[0143] 5.7. Antibody Incubation The primary antibody was diluted in TBS-T containing 5% (wt / vol) BSA, and sodium azide was added to the antibody solution to inhibit microbial growth at 4°C. After blocking, the membrane was washed three times for 5 minutes with TBS-T. The membrane was then incubated overnight at 4°C with the appropriate dilution of primary antibody. The prepared antibody dilution was kept at 4°C and used for 5–10 incubations. After incubation with the primary antibody, the membrane was washed three times for 5 minutes with TBS-T. The secondary antibody solution was prepared by diluting HRP-conjugated anti-mouse or anti-rabbit antibody 1 / 2000 (v / v) in TBS-T containing 5% (wt / vol) skim milk powder. The membrane was then incubated with the secondary antibody dilution at room temperature for 1 hour.

[0144] 5.8. Protein visualization After secondary antibody incubation, the membrane was washed three times for 5 min with TBS-T, and then incubated with 2 ml of 1:1 mixed ECL Western blotting substrate. Protein bands on the membrane were then visualized using a Syngene Gbox Chemi chemiluminescence imaging device.

[0145] 6. Cell Surface Staining and Flow Cytometry Analysis One million cells were washed twice in a FACS tube with 3 ml of FACS buffer. The supernatant was discarded, and 5 μl of Fc blocker and 10 μl of fluorescent dye-conjugated antibody were added. The cells were incubated with the antibody for 30 minutes at +4°C. Again, the cells were washed three times with 3 ml of FACS buffer. The supernatant was discarded, and the cells were resuspended in 1 ml of FACS buffer. Upregulation of cell surface markers was analyzed using BD Acuri and FlowJo software.

Claims

1. A composition comprising an ASC speck formed by an ASC protein for use in a method of cancer treatment.

2. 2. The composition for use according to claim 1, wherein the method comprises inducing and / or stimulating an immune response against a cancer disease in a mammal, wherein the induction or stimulation results in the inhibition of cancer progression or the arrest of cancer cell proliferation.

3. The composition for use according to claim 1 or 2, wherein the cancer is thymic carcinoma.

4. The composition for use according to any one of claims 1 to 3, wherein the composition further comprises a tumor antigen.

5. 5. The composition for use according to claim 4, wherein the tumor antigen is carried by the ASC speck by forming hydrophobic interactions with the ASC proteins that form the ASC speck.

6. 5. The composition for use according to claim 4, wherein the tumor antigen is carried by the ASC speck as part of a fusion protein formed by the tumor antigen and at least one ASC protein forming the ASC speck.

7. The composition for use according to claim 6, wherein the tumor antigen is fused to the ASC protein at the N-terminus or C-terminus.

8. The composition for use according to any one of claims 4 to 7, wherein the tumor antigen is carried within the ASC speck.

9. 9. The composition for use according to any one of claims 1 to 8, wherein the cancer is a tumor-forming cancer and the method comprises inhibiting tumor progression or reducing tumor size.

10. A composition for generating an immune response against cancer in a mammal, comprising an ASC speck carrier and a tumor antigen.

11. 11. The composition of claim 10, wherein the tumor antigen is carried by the ASC speck carrier as part of a fusion protein formed by the tumor antigen and at least one ASC protein that forms an ASC speck.

12. A method of treating cancer comprising administering to a mammal in need thereof a therapeutically effective amount of a composition comprising an ASC speck.

13. 13. The method of claim 12, wherein the composition is administered once a week.

14. The method of claim 12 or 13, wherein the composition further comprises a tumor antigen.

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