Method for stimulating type I interferon genes and composition containing cationic lipids

Abstract

The objective is to provide methods and compositions for modifying the type I IFN signaling pathway in a subject, including the administration of cationic lipids to the subject. [Solution] The cationic lipids include 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-1-(2,3-dioleoyloxy)-propyl-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), enantiomers, and combinations thereof. The composition may further contain one or more antigenic components, where such components are autologous or non-autologous.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and all interests of U.S. Provisional Patent Application No. 62 / 594,815, filed on 5 December 2017, which is expressly incorporated herein by reference in its entirety.

[0002] 1. Field of Invention The present invention generally relates to methods and compositions for modifying type I IFN signaling pathways, including the administration of cationic lipids. [Background technology]

[0003] 2. Explanation of related technologies Over the past decade, type I interferon (IFN-I) has been shown to be critically important in the mammalian immune response to disease. Type I interferon is directly induced by viral infection and is therefore called "viral" interferon. In contrast, "immune" IFN, or IFN-γ, is synthesized after T cells and natural killer (NK) cells bind to receptors during the immune response. IFN-I has also been well-established as an inducer of tumor cell apoptosis and anti-angiogenesis via interferon-alpha receptor (IFNAR) signaling. Due to IFN-I's crucial role in the therapeutic immune response, current focus is on identifying safe and effective ways to activate IFN-I to enhance both the prophylactic and therapeutic benefits of vaccines, immunotherapies, and other immune-based medicines. For example, preclinical studies in mice have shown that IFN-I directly activates other important cells in the immune system, such as antigen-presenting dendritic cells (DCs) and CD4 and CD8 T cells. IFN-I also increases antigen presentation by tumor cells recognized by T lymphocytes, thus showing a significant potential role in cancer immunotherapy.

[0004] In the field of vaccine development, methods and approaches to produce a large number of potent antigen-specific cytotoxic T cells in humans to treat pathogen-induced diseases or cancer remain an unmet medical need. Protein and peptide-based vaccines, used in combination with immunostimulants such as adjuvants, are one of the more promising approaches being evaluated for generating cytotoxic T cells. However, most adjuvants currently approved for human use are insufficient as type I interferons and cytotoxic T cell inducers, and have failed to establish long-term prophylactic or therapeutic effects.

[0005] Interferons (IFN-α / β / γ) are part of a more potent immunostimulatory cytokine group and play a crucial role in the development of CD8+ cytotoxic T cells. Interferons are also important for antiviral immunity. Effective T cell immunity requires activation of T cells presenting an antigen (signal 1) in the presence of appropriate co-stimulatory signals (signal 2) and cytokines (signal 3), driving a specific T cell immune response by promoting T cell proliferation, survival, differentiation, and effector function. IFN-I acts directly on T cells as signal 3, inducing T cell activation and driving optimal T cell proliferation, survival, and effector function. Initially identified as an antiviral cytokine, IFN-I has been shown to enhance the proliferation, survival, and effector function of CD8 T cells. Interferons also promote the maturation of antigen-presenting cells by modulating co-stimulatory molecules and enhancing antigen cross-presentation necessary for T cell activation. Furthermore, interferons have been demonstrated to possess both direct and indirect antitumor properties through IL-15 production. As a result of these important immunological characteristics, various IFN-I inducers and recombinant IFN-I therapies are currently being tested in human trials.

[0006] Type I interferons have been reported to inhibit cell transformation in vitro by maintaining the expression of the tumor suppressor gene p53 (Takaoka A, Hayakawa S, Yanai H, Stoiber D, Negishi H, Kikuchi H, et al. Integration of interferon-α / β signaling to p53 responses in tumor suppression and antiviral defense. Nature (2003) 424:516-23). ​​The specific role of IFN-I signaling in the negative regulation of tumor cell proliferation and induction of cell death in human cancer cell lines has also been demonstrated (Zitvogel L, Galluzzi L, Kepp O, Smyth MJ, Kroemer G. Type I interferons in anticancer immunity. Nat Rev Immunol (2015) 15:405-14). In vivo, deletion of IFNAR1 from intestinal epithelial cells increased tumorigenesis in mice (Tschurtschenthaler M, Wang J, Fricke C, Fritz TMJ, Niederreiter L, Adolph TE, et al. Type I interferon signaling in the intestinal epithelium affects Paneth cells, microbial ecology and epithelial regeneration. Gut (2014) 63:1921-31). Multiple studies provide strong evidence that IFN-I induces antitumor effects primarily through indirect stimulation of immune cells, rapidly eliminating malignant cells. Several studies strongly suggest that IFN-I promotes an anti-cancer immune response similar to the host's response to pathogens.

[0007] As a result of universal IFNAR expression, IFN-I has been shown to have important regulatory effects on immune cells in association with inflammatory and viral diseases (Decker T, Muller M, Stockinger S. The yin and yang of type I interferon activity in bacterial infection. Nat Rev Immunol (2005) 5:675-87; Stetson DB, Medzhitov R. Type I interferons in host defense. Immunity (2006) 25:373-81). Therefore, it is clear that cellular mediators of innate and adaptive immune responses can be very well regulated by IFN-I in defense against malignant diseases.

[0008] IFN-I has been shown to be important in tumor immunosurveillance (Dunn GP, ​​Bruce AT, Sheehan KCF, Shankaran V, Uppaluri R, Bui JD, et al. A critical function for type I interferons in cancer immunoediting. Nat Immunol (2005)6:722-9). In contrast to IFN-γ, IFN-I has been found to act on host hematopoietic cells rather than tumor cells themselves during the induction of a protective antitumor immune response in bone marrow transplantation experiments. Numerous studies have reported on the mechanisms by which type I interferons affect cells in the innate and adaptive immune systems in relation to tumor surveillance [Zitvogel L, Galluzzi L, Kepp O, Smyth MJ, Kroemer G. Type I interferons in anticancer immunity. Nat Rev Immunol (2015) 15:405-14; Parker BS, Rautela J, Hertzog PJ. Antitumour actions of interferons: implications for cancer therapy. Nat Rev Cancer (2016) 16:131-44].Several studies have identified the important role of type I interferon in the activation of host antigen-presenting cells (Fuertes MB, Kacha AK, Kline J, Woo SR, Kranz DM, Murphy KM, et al. Host type I IFN signals are required for antitumor CD8+ T cell responses through CD8α+ dendritic cells. J Exp Med (2011) 208:2005-16; Diamond MS, Kinder M, Matsushita H, Mashayekhi M, Dunn GP, ​​Archambault JM, et al. Type I interferon is selectively required by dendritic cells for immune rejection of tumors. J Exp Med (2011) 208:1989-2003). Early-produced type I IFN is tumor antigen-specific cytotoxic CD8. + CD8α is necessary for the activation of T lymphocytes (CTLs). + It has been determined to affect the concentration of dendritic cells (DCs). Type I IFN signaling is linked to CD8α +DCs specifically enhance the ability to cross-link antigens (Diamond MS, Kinder M, Matsushita H, Mashayekhi M, Dunn GP, ​​Archambault JM, et al. Type I interferon is selectively required by dendritic cells for immune rejection of tumors. J Exp Med (2011) 208:1989-2003). This effect is hypothesized to be a result of IFN-I enhancing DC survival and thus increasing antigen persistence on the cell surface during cross-presentation (Lorenzi S, Mattei F, Sistigu A, Bracci L, Spadaro F, Sanchez M, et al. Type I IFNs control antigen retention and survival of CD8α(+) dendritic cells after uptake of tumor apoptotic cells leading to cross-priming. J Immunol (2011) 186:5142-50; Schiavoni G, Mattei F, Gabriele L. Type I interferons as stimulators of DC-mediated cross-priming: impact on anti-tumor response. Front Immunol (2013) 4:483). Type I interferon promotes the maturation, differentiation, and migration of dendritic cells (Fuertes MB, Woo SR, Burnett B, Fu YX, Gajewski TF. Type I interferon response and innate immune sensing of cancer. Trends Immunol (2013) 34:67-73).

[0009] It is important to note that type I IFNs can induce interleukin-15 (IL-15) release by dendritic cells (Mattei F, Schiavoni G, Belardelli F, Tough DF. IL-15 is expressed by dendritic cells in response to type I IFN, double-stranded RNA, or lipopolysaccharide and promotes dendritic cell activation. J Immunol (2001) 167:1179-87). This is related to CD8 +It promotes the survival of memory cells and NK cells (Huntington ND. The unconventional expression of IL-15 and its role in NK cell homeostasis. Immunol Cell Biol (2014) 92:210-3). This effect is not limited to NK cells. CTLs have also been shown to acquire full effector function in response to type I IFN (Curtsinger JM, Mescher MF. Inflammatory cytokines as a third signal for T cell activation. Curr Opin Immunol (2010) 22:333-40; Fuertes MB, Kacha AK, Kline J, Woo SR, Kranz DM, Murphy KM, et al. Host type I IFN signals are required for antitumor CD8+ T cell responses through CD8α+ dendritic cells. J Exp Med (2011) 208:2005-16).In addition, neutrophils (Wu CF, Andzinski L, Kasnitz N, Kroger A, Klawonn F, Lienenklaus S, et al. The lack of type I interferon induces neutrophil-mediated pre-metastatic niche formation in the mouse lung. S. CXCR2-mediated tumor-associated neutrophil recruitment is regulated by IFN-β. Int J Cancer (2014) 134:1346-58), NKT, and γδ T cells (Woo SR, Corrales L, Gajewski TF. Innate immune recognition of cancer. Annu Rev Immunol (2015) By having the ability to influence other innate immune cell subsets (such as 33:445-74), type I IFNs exhibit tumor growth limiting properties.

[0010] Type I intercellular neurons (IFNs) are released very early in infection (Biron CA. Initial and innate responses to viral infections - pattern setting in immunity or disease. Curr Opin Microbiol (1999) 2:374-81) and are important regulators of innate immune cell subsets such as DCs and NK cells in anti-cancer host responses. Regarding NK cells, type I IFNs have been shown to be important in their ability to generate an initial response to viral infection, and are thought to enhance NK cell cytotoxicity and cytokine production (Lee CK, Rao DT, Gertner R, Gimeno R, Frey AB, Levy DE. Distinct requirements for IFNs and STAT1 in NK cell function. J Immunol (2000) 165:3571-7; Nguyen KB, Salazar-Mather TP, Dalod MY, Van Deusen JB, Wei X, Liew FY, et al. Coordinated and distinct roles for IFN-alpha beta, IL-12, and IL-15 regulation of NK cell responses to viral infection. J Immunol (2002) 169:4279-87).

[0011] In humans, type I interferon production is typically induced via the binding of pathogen-associated pattern recognition receptors, such as Toll-like receptors, NOD-like receptors, and retinoic acid-inducible gene I-like (RIG-I) receptors. Furthermore, various cytoplasmic secondary messengers (c-GMP) and cytoplasmic nucleic acid sensors can induce type I interferon production through activation of the interferon gene stimulation (STING) pathway.

[0012] IFN-I is thought to mediate antitumor activity in various ways. For example, interferons can directly act on tumor cells and alter their viability and proliferative capacity [Parker, BS, J. Rautela, and PJ Hertzog, Antitumour actions of interferons: implications for cancer therapy. Nat Rev Cancer, 2016. 16(3): p. 131-443].

[0013] Many methods have been studied in humans to utilize the immunological properties mediated by type I hepatitis C. The administration of recombinant IFN-I monotherapy, along with recombinant interferon-alpha, has been clinically proven in the treatment of chronic hepatitis C (Adrian M. Di Bisceglie, MD, Paul Martin, MD, Chris Kassianides, MD, Mauricio Lisker-Melman, MD, Linda Murray, RN, Jeanne Waggoner, BA, Zachary Goodman, MD, Steven M. Banks, Ph.D., and Jay H. Hoofnagle, MD, Recombinant Interferon Alfa Therapy for Chronic Hepatitis C, November 30, 1989 N Engl J Med 1989; 321:1506-1510). Another approach involves administering Toll-like receptor (TLR) agonists such as double-stranded RNA (Poly I:C), CpG oligonucleotides, Imiquimod, or single standard RNA to induce endogenous production of type I interferons (Munir Akkaya, Billur Akkaya, Patrick Sheehan, Pietro Miozzo, Mukul Rawat, Mirna Pena, Ann S Kim, Olena Kamenyeva, Juraj Kabat, Chen-Feng Qi, Silvia Bolland, Akanksha Chaturvedi and Susan K Pierce, The Toll-like receptor ligand CpG-A induces type 1 interferons in B cells contrasting the proinflammatory inducing activity of CpG-B, J Immunol May 1, 2017, 198 (1 Supplement) 152.4).More recently, STING pathway agonists have gained increasing importance as focused type I interferon activators for antitumor immunity [Corrales, L., et al., The host STING pathway at the interface of cancer and immunity. J Clin Invest, 2016. 126(7): p. 2404-11].

[0014] However, there remains a clear need for effective methods to promote potent activation of the type I interferon pathway. Given the numerous therapeutic benefits associated with activation of the type I interferon pathway, improved methods for this purpose are clearly desirable.

[0015] Therefore, what is needed are improved methods and compositions for vaccines that enable potent activation of IFN1 (IFN-α / β / γ). Furthermore, what is needed are improved methods and compositions such as vaccines that induce a robust cytotoxic T-cell immune response, with or without antigens such as peptide or protein antigens. Preferably, such compositions and vaccines incorporate the use of immunotherapeutic agents that are safe, easy to administer, and have minimal side effects or toxicity. [Overview of the Initiative]

[0016] This specification provides novel methods and compositions for vaccines comprising cationic lipids as potent activators of IFN1 (IFN-α / β / γ). As described herein, the compositions, when administered to a host, enable the induction of an effective and robust cytotoxic T-cell immune response. The compositions comprise cationic lipids, optionally combined with peptides or protein antigens; in certain embodiments, the cationic lipids are in the form of liposomes. As described in detail below, the compositions of the present invention result in IFN-I activated cytotoxic cells having the ability to degenerate tumors established in mammals such as mice. The present invention further provides the use of cationic liposomes for use in specifically activating IFN-I to develop more effective prevention against infectious pathogens and therapies for cancer and other diseases.

[0017] Other features and advantages of the present invention will become readily apparent as the invention is better understood after referring to the accompanying drawings and reading the following description. [Brief explanation of the drawing]

[0018] [Figure 1] Figure 1 is a graph showing that Versamune® (R-DOTAP) induced rapid and sustained recruitment of immune cells in afferent lymph nodes. The data in Figure 1 corresponds to an experiment in which 100 μl of 12 mM R-DOTAP liposome nanoparticles were subcutaneously injected into the posterior neck of C57BL / 6J mice (n=4). Mice injected with PBS and LPS (50 μg / mouse) (at 24 hours) were used as negative and positive controls, respectively. Axillary and brachial lymph nodes from each mouse were pooled 4 or 24 hours after injection and treated by collagenase digestion. CD11c-positive cells (~20,000 cells) from these lymph node cell suspensions were sorted and purified, lysed in RLT buffer, and processed for relative gene expression analysis using the nCounter® mouse inflammation kit and nanostring technology. The data shows the upregulation of type I interferon genes by R-DOTAP, with an average of 4 mice per group. [Figure 2] Figure 2 is a graph showing R-DOTAP-induced IFN gene expression in draining lymph nodes. The data in Figure 2 correspond to an experiment in which C57BL / 6J mice (n = 2) were subcutaneously injected with 50 μl of 6 mM R-DOTAP or S-DOTAP nanoparticles into the footpad. Mice injected with PBS and LPS (50 μg / mouse) (at the 24-hour time point) were used as negative and positive controls, respectively. At 3 or 24 hours after injection, popliteal lymph nodes from each mouse were pooled, lysed in RLT buffer, and processed for relative gene expression analysis using Taqman gene expression assay and RT-PCR. The data show upregulation of type I interferon genes by both S-DOTAP and R-DOTAP. [Figure 3A] Figures 3A, 3B, and 3C provide graphs showing R-DOTAP-induced cell mobilization and CD69 expression in draining lymph nodes. The data in Figure 3 correspond to an experiment in which A) C57BL / 6J mice (n = 3) were subcutaneously injected with 50 μl of 6 mM R-DOTAP or 280 mM sucrose into the footpad. At the indicated times, popliteal lymph node draining lymph nodes from each mouse were isolated, and a single suspension of the lymph nodes was counted using a hemocytometer to obtain the total cell number. B) C57BL / 6J (n = 3) or IFNAR− / − (n = 3) mice were injected with 50 μl of 6 mM R-DOTAP or 280 mM sucrose into the footpad, draining popliteal lymph nodes were harvested from each mouse, and lymph nodes enzymatically digested for total cell number at 24 hours (B) were evaluated using a hemocytometer, and the expression of CD69 on CD3+ T cells (B) was evaluated using flow cytometry. A-B) The data show the total cell number of each draining lymph node. C) The percentage (%) of CD69+ cells in each group 4 hours after vaccination 2 is shown (n = 3 mice / group). [Figure 3B]Figures 3A, 3B, and 3C provide graphs showing R-DOTAP-induced cell recruitment and CD69 expression in afferent lymph nodes. The data in Figure 3 correspond to experiments in which 50 μl of 6 mM R-DOTAP or 280 mM sucrose was subcutaneously injected into the foot pads of C57BL / 6J mice (n=3). At the instructed time, popliteal lymph node drainage lymph nodes were isolated from each mouse, and the single suspension of lymph nodes was counted using a hemocytometer to obtain the total cell count. B) C57BL / 6J (n=3) or IFNAR- / - (n=3) mice were injected with 50 μl of 6 mM R-DOTAP or 280 mM sucrose in a foot pad. Drainage popliteal lymph nodes were collected from each mouse, and the total cell count of the enzymatically digested lymph nodes was evaluated using a hemocytometer 24 hours later (B). CD69 expression on CD3+ T cells (B) was evaluated using flow cytometry. AB) The data show the total cell count of each regional lymph node. C) The percentage of CD69+ cells in each group 4 hours after vaccination 2 is shown (n=3 mice / group). [Figure 3C] Figures 3A, 3B, and 3C provide graphs showing R-DOTAP-induced cell recruitment and CD69 expression in afferent lymph nodes. The data in Figure 3 correspond to experiments in which 50 μl of 6 mM R-DOTAP or 280 mM sucrose was subcutaneously injected into the foot pads of C57BL / 6J mice (n=3). At the instructed time, popliteal lymph node drainage lymph nodes were isolated from each mouse, and the single suspension of lymph nodes was counted using a hemocytometer to obtain the total cell count. B) C57BL / 6J (n=3) or IFNAR- / - (n=3) mice were injected with 50 μl of 6 mM R-DOTAP or 280 mM sucrose in a foot pad. Drainage popliteal lymph nodes were collected from each mouse, and the total cell count of the enzymatically digested lymph nodes was evaluated using a hemocytometer 24 hours later (B). CD69 expression on CD3+ T cells (B) was evaluated using flow cytometry. AB) The data show the total cell count of each regional lymph node. C) The percentage of CD69+ cells in each group 4 hours after vaccination 2 is shown (n=3 mice / group). [Figure 4A]Figure 4 shows a graph illustrating the induction of type I interferon production by dendritic cells using cationic lipids. The data in Figure 4 corresponds to experiments in which A) bone marrow-derived dendritic cells (BMDCs) from IFNAR-Ko mice were stimulated for 24 hours with the indicated concentrations of cationic lipids (6-400 μM) or LPS-positive control (1-500 ng / ml). B) FLT3-induced BMDCs (pDCs) and GM-CSF / IL-4-induced BMDCs (cBMDCs) were stimulated for 24 hours with the specified concentrations of cationic lipids (6-400 μM) or LPS-positive control (1-500 ng / ml). To measure type I interferon production, cell supernatant (100 μl) was added to reporter cells (B16.Blue-IFNα / β cells from In Vivogen, USA) and incubated for 18 hours to stimulate type I interferon-inducible secretory alkaline phosphatase (SEAP) production by the reporter cells. SEAP activity in reporter cell supernatant was quantified using a colorimetric SEAP assay kit according to the manufacturing protocol. BMDC secreted type I interferon was quantified using a standard curve based on recombinant mouse IFN-β stimulated SEAP activity in reporter cell lines. [Figure 4B]Figure 4 shows a graph demonstrating the induction of type I interferon production by dendritic cells with cationic lipids. The data in Figure 4 correspond to experiments in which A) bone marrow-derived dendritic cells (BMDC) from IFNAR-Ko mice were stimulated for 24 hours with the indicated (6 - 400 μM) concentrations of cationic lipids or an LPS positive control (1 - 500 ng / ml). B) FLT3-induced BMDC (pDC) and GM-CSF / IL-4-induced BMDC (cBMDC) were stimulated for 24 hours with the indicated concentrations (6 - 400 μM) of cationic lipids or an LPS positive control (1 - 500 ng / ml). To measure type I interferon production, cell supernatants (100 μl) were added to reporter cells (B16.Blue-IFNα / β cells from InvivoGen, USA) and incubated for 18 hours to stimulate the production of type I interferon-inducible secreted alkaline phosphatase (SEAP) by the reporter cells. The SEAP activity in the reporter cell supernatants was quantified using a colorimetric SEAP assay kit according to the manufacturing protocol. BMDC-secreted type I interferon was quantified using a standard curve with recombinant mouse IFN-β-stimulated SEAP activity in the reporter cell line. [Figure 5A]Figure 5 provides a graph showing the efficacy of an R-DOTAP-based vaccine in type I IFN signaling-deficient mice. The data in Figure 5 correspond to experiments in which Jackson-derived C57BL / 6J mice (n=6) or IFNAR- / - mice were subcutaneously injected with 100 μl of a vaccine formulation (Versamune® or complete Freund's adjuvant) containing SIINFEKL peptide (A) (SEQ ID NO: 1) or RF9 peptide (B) (SEQ ID NO: 2) on days 0 and 7. Seven days after the second vaccination, spleens from vaccinated mice and control mice were processed to measure antigen-specific CD8 T cell responses induced by the vaccine formulation in wild-type and IFNAR- / - mice. Antigen-specific CD8 T cell responses were evaluated by measuring SIINFEKL-specific (A) (SEQ ID NO: 1) and RF9 peptide-specific (B) (SEQ ID NO: 2) CD8 T cell production in the ELISPOT assay. The data are representative of multiple experiments with similar results. The statistical significance of the data was estimated using one-way ANOVA, and multiple comparisons were analyzed using Tukey's test. **The WT Versamune® vaccination group was statistically significant (p<0.05).** [Figure 5B]Figure 5 provides a graph showing the efficacy of an R-DOTAP-based vaccine in type I IFN signaling-deficient mice. The data in Figure 5 correspond to experiments in which Jackson-derived C57BL / 6J mice (n=6) or IFNAR- / - mice were subcutaneously injected with 100 μl of a vaccine formulation (Versamune® or complete Freund's adjuvant) containing SIINFEKL peptide (A) (SEQ ID NO: 1) or RF9 peptide (B) (SEQ ID NO: 2) on days 0 and 7. Seven days after the second vaccination, spleens from vaccinated mice and control mice were processed to measure antigen-specific CD8 T cell responses induced by the vaccine formulation in wild-type and IFNAR- / - mice. Antigen-specific CD8 T cell responses were evaluated by measuring SIINFEKL-specific (A) (SEQ ID NO: 1) and RF9 peptide-specific (B) (SEQ ID NO: 2) CD8 T cell production in the ELISPOT assay. The data are representative of multiple experiments with similar results. The statistical significance of the data was estimated using one-way ANOVA, and multiple comparisons were analyzed using Tukey's test. **The WT Versamune® vaccination group was statistically significant (p<0.05).** [Modes for carrying out the invention]

[0019] The present invention can be more readily understood by referring to the following detailed description of specific embodiments included herein. Although the present invention has been described with reference to specific details of its particular embodiments, such details should not be considered as limitations on the scope of the invention.

[0020] The full texts of the references cited herein are incorporated herein by reference and include U.S. Provisional Patent Application No. 62 / 594,815, U.S. Patents No. 7,303,881, No. 8,877,206, No. 9,789,129, and U.S. Patent Applications No. 14 / 344,327, No. 14 / 407,419, No. 14 / 429,123, and No. 15 / 775,680.

[0021] This specification provides novel methods and compositions comprising the use of cationic lipids for modification of type I interferon genes. In some embodiments, type I interferon genes are upregulated to induce or activate type I interferon signaling. In some embodiments, type I interferon genes are downregulated to suppress or inactivate type I interferon signaling. Also provided are novel methods and compositions comprising the use of cationic lipids to promote and enhance the potency of disease-specific CD8+ T cell populations through the effects of type I interferon. In some embodiments, the methods and compositions claimed herein may include the use of disease-specific antigens intended to direct cytolytic activity against specific infected or affected cells.

[0022] Cationic liposomes have been widely used in vivo for the delivery of low molecular weight drugs, plasmid DNA, oligonucleotides, proteins, and peptides, and as vaccine adjuvants. In this invention, as a result of research conducted to better understand how cationic lipids interact with the immune system, and due to the mechanistic reasons for the ability of certain cationic lipids to facilitate cross-presentation, the unique ability of cationic lipids to upregulate type I interferons was discovered. Initially produced type I IFNs trigger tumor antigen-specific cytotoxic CD8 + CD8α is necessary for the activation of T lymphocytes (CTLs). + Acting on the concentration of dendritic cells (DCs), type I IFN signaling is controlled by CD8α + It has been reported that cationic lipids specifically enhance the antigen cross-expression ability of DCs. This ability of cationic lipids to promote antigen cross-presentation that elicits an antigen-specific CD8+ T cell response has now been demonstrated in both preclinical and human clinical studies. The ability to use cationic lipids to safely activate type I interferons to promote antigen cross-presentation and CD8+ T cell induction in humans offers the potential to successfully address a significant unmet medical need in the field of therapeutic immunology.

[0023] In embodiments intended herein, cationic lipids are administered to activate the type I interferon pathway in a subject.

[0024] In another embodiment, cationic lipids are combined with disease-specific antigens to activate type I interferons, promote antigen cross-presentation, and prime disease-specific CD8+ T cells to treat the disease in the target.

[0025] In another embodiment, a method for treating cancer is provided, which includes the step of treating a target with a type I interferon-activated cationic lipid combined with a protein antigen.

[0026] In another embodiment, a method for treating cancer is provided, which includes the step of treating a target with a cationic lipid combined with a T cell activating vaccine.

[0027] In another embodiment, a method for treating cancer is provided, which includes the step of treating a subject with a cationic lipid combined with a protein or peptide tumor antigen, and in combination with an adjuvant.

[0028] In another embodiment, a method for treating cancer is provided, which includes the step of treating a target with a cationic lipid combined with any tumor antigen, including a DNA or RNA-based antigen.

[0029] In another embodiment, a method for treating cancer is provided, which includes the step of treating a target with a cationic lipid to activate a type I interferon pathway combined with a tumor antigen, as needed, in combination with an adjuvant and / or any agent that combats tumor immunosuppression by reducing MDSCs, Tregs, or blocking immune checkpoints.

[0030] In another embodiment, a method for treating cancer is provided, which includes the step of treating a target with a cationic lipid-based vaccine combined with a DNA or RNA-based tumor antigen, in combination with an adjuvant and / or any agent that combats tumor immunosuppression.

[0031] In yet another embodiment, a method for enhancing the antitumor immune response in mammals is provided. This method includes treating a mammal with a growth factor together with a type I interferon-activated cationic lipid, or one or more cationic lipids, in some cases such as GM-CSF and cytokines.

[0032] In various embodiments, the composition comprises one or more lipids having at least one type I interferon-activated cationic lipid and at least one antigen. In one embodiment, it comprises two or more antigens.

[0033] In one embodiment, a method and composition for modifying a type I IFN signaling pathway in a subject such as a mammal are provided, the method comprising administering a composition containing a cationic lipid to the subject. In one embodiment, the cationic lipid includes 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), N-1-(2,3-dioleoyloxy)-propyl-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), and combinations thereof. The composition may contain specific enantiomers of the cationic lipid. In one embodiment, the cationic lipid is 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), and in one embodiment, the cationic lipid is the R enantiomer of DOTAP, (R)-1,2-dioleoyl-3-trimethylammoniumpropane (R-DOTAP). In one embodiment, the composition further comprises an antigen such as a protein or peptide antigen.

[0034] In some embodiments, modification of the type I IFN signaling pathway in a subject includes downregulation or activation of the pathway. In some embodiments, modification of the type I IFN signaling pathway in a subject includes downregulation or inactivation of the pathway. In some embodiments, the T cell response is increased, the antigen-specific CD8+ T cell response is increased, and / or the immunogenicity of the composition is enhanced. In one embodiment, a method is provided for treating cancer in a subject, the method comprising administration of a composition comprising a cationic lipid, the administration of the cationic lipid resulting in stimulation of the type I IFN signaling pathway.

[0035] antigen In one embodiment, the novel method claimed herein involves the administration of a cationic lipid with one or more autoantigens, such as antigens derived from the tumor of the subject itself. In another embodiment, the method involves, but is not limited to, the administration of a composition comprising a cationic lipid combined with one or more non-autoantigens, such as synthetic peptides, recombinant proteins, RNA or DNA or active fragments thereof. In each case, the objective is to activate type I interferon to promote the induction of a potent antigen-specific CD8+ T cell response. The antigen may be any disease-related antigen known to those skilled in the art.

[0036] As used herein, “tumor-associated antigens” are molecules or compounds associated with tumors or cancer cells (e.g., proteins, peptides, polypeptides, lipoproteins, lipopeptides, glycoproteins, glycopeptides, lipids, glycolipids, carbohydrates, RNA, and / or DNA, or active fragments thereof) that, when expressed on the surface of antigen-presenting cells in association with MHC molecules, can evoke an immune response (humoral and / or cellular). Tumor-associated antigens include autoantigens, as well as other antigens that are not specifically associated with cancer but, nevertheless, when administered to animals, may enhance the immune response against tumors or cancer cells and / or reduce the proliferation of tumors or cancer cells. Further specific embodiments are provided herein.

[0037] As used herein, “microbial antigen” refers to an antigen of a microbial organism and includes, but is not limited to, infectious viruses, infectious bacteria, infectious parasites, and infectious fungi. A microbial antigen may be an intact microorganism, a synthetic compound identical or similar to a naturally occurring microbial antigen, and preferably a natural isolate, fragment, or derivative thereof that induces a natural immune response specific to the corresponding microorganism (from which the naturally occurring microbial antigen originates). In preferred embodiments, a compound is similar to a naturally occurring microbial antigen if it induces an immune response (humoral and / or cellular) similar to that of the naturally occurring microbial antigen. Compounds or antigens similar to natural microbial antigens are well known to those skilled in the art, such as proteins, peptides, polypeptides, lipoproteins, lipopeptides, glycoproteins, glycopeptides, lipids, glycolipids, carbohydrates, RNA, and / or DNA. Another non-limiting example of a compound similar to a natural microbial antigen is a peptide mimetic of a polysaccharide antigen. More specific embodiments are provided herein.

[0038] The term “antigen” is intended to further encompass peptide or protein analogs of known or wild-type antigens, such as those described herein. Analogues may be more soluble or more stable than wild-type antigens, and may contain mutations or modifications that make the antigen more immunologically active. Antigens can be modified in any way, including by the addition of lipid or sugar moieties, mutations in peptide or protein amino acid sequences, mutations in DNA or RNA sequences, or any other modifications known to those skilled in the art. Antigens can be modified using standard methods known to those skilled in the art.

[0039] Furthermore, useful in the compositions and methods of the present invention are peptides or proteins having an amino acid sequence homologous to the amino acid sequence of a desired antigen, and homologous antigens induce an immune response against their respective tumors, microorganisms, or infected cells.

[0040] In one embodiment, the method described herein comprises a composition in which a cationic lipid is administered without an antigen to activate type I interferon, thereby activating natural killer cells to attack and kill diseased or infected cells, for example, thereby promoting a desired immune response to treat a disease in a subject. In another embodiment, the method of the present invention comprises a composition in which a cationic lipid is administered together with an antigen, the antigen may be associated with tumors or cancer, i.e., tumor-associated antigens for developing immunotherapies to prevent or treat tumors. Thus, in one embodiment, the tumor or immunotherapy of the present invention further comprises at least one epitope of at least one tumor-associated antigen. In another embodiment, the immunotherapy method of the present invention further comprises multiple epitopes derived from one or more tumor-associated antigens. The cationic lipids and tumor-associated antigens used in the methods of the present invention may be intrinsically immunogenic, non-immunogenic, or slightly immunogenic. As shown herein, the composition can activate type I interferon (IFN), which is known to mediate antitumor effects against several tumor types, and therefore tumor-associated autoantigens can also be advantageously used in the immunotherapy for therapeutic effects. Examples of antigens include synthetic, recombinant, exogenous, or homologous antigens, and antigenic substances include, but are not limited to, proteins, peptides, polypeptides, lipoproteins, lipopeptides, lipids, glycolipids, carbohydrates, RNA, and DNA. Examples of such therapies include, but are not limited to, the treatment or prevention of breast cancer, head and neck cancer, melanoma, cervical cancer, lung cancer, prostate cancer, colon cancer, or any other cancer known in the art that is sensitive to immunotherapy. In such therapies, it is also possible to conjugate the antigen with a cationic lipid without encapsulation.

[0041] Tumor-associated antigens suitable for use in the present invention include both naturally occurring and modified molecules that can represent a single tumor type, are shared among several types of tumors, and / or can be expressed or overexpressed only in tumor cells compared to normal cells. In addition to proteins, glycoproteins, lipoproteins, peptides, and lipopeptides, tumor-specific expression patterns of carbohydrates, gangliosides, glycolipids, and mucins have also been reported. Exemplary tumor-associated antigens for use in cancer vaccines include protein products of oncogenes, tumor suppressor genes, and other genes with tumor-cell-specific mutations or rearrangements, reactivated embryonic gene products, oncoemetic antigens, tissue-specific (but not tumor-specific) differentiation antigens, growth factor receptors, cell surface carbohydrate residues, exogenous viral proteins, and numerous other autoproteins.

[0042] Specific embodiments of tumor-associated antigens include, for example, mutated or modified antigens, such as the Ras p21 proto-oncogene, tumor suppressor p53 and BCR-2 / neu oncogenes, and BCR-abl oncogene, as well as the protein products of CDK4, MUM1, caspase 8, and beta-catenin; overexpressed antigens, such as galectin 4, galectin 9, carbonic anhydrase A, aldolase A, PRAME, Her2 / neu, ErbB-2, and KSA; tumor embryo antigens, such as alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG); autoantigens, such as carcinoembryonic antigen (CEA) and melanocyte differentiation antigens, such as Mart 1 / Melan A, gp100, gp75, tyrosinase, TRP1, and TRP2; Mart 1 / Melan Examples include melanocyte differentiation antigens such as A, gp100, gp75, tyrosinase, TRP1 and TRP2; prostate-related antigens, e.g., PSA, PAP, PSMA, PSM-P1 and PSM-P2; reactivated embryonic gene products, e.g., MAGE 1, MAGE 3, MAGE 4, GAGE ​​1, GAGE ​​2, BAGE, RAGE; other cancer testicular antigens, e.g., NY-ESO1, SSX2 and SCP1; mucins, e.g., Muc-1 and Muc-2; gangliosides, e.g., GM2, GD2 and GD3; neutral glycolipids; and glycoproteins, e.g., Lewis(y); as well as glycoproteins, e.g., Tn, Thompson-Fridenreich antigen (TF) and sTn. Furthermore, tumor-associated antigens as used herein include whole cells and tumor cell lysates, as well as their immunogenic portions, as well as immunoglobulin idiotypes expressed in monoclonal proliferation of B lymphocytes for use against B-cell lymphoma.

[0043] Tumor-associated antigens and their respective tumor cell targets include, for example, cytokeratins, particularly cytokeratins 8, 18, and 19, as cancer antigens. Epithelial membrane antigen (EMA), human embryonic antigen (HEA-125), human milk fat globules, MBr1, MBr8, Ber-EP4, 17-1A, C26, and T16 are also known cancer antigens. Desmin and muscle-specific actin are antigens of myogenic sarcoma. Placental alkaline phosphatase, β-human chorionic gonadotropin, and α-fetoprotein are antigens of trophoblast and germ cell tumors. Prostate-specific antigen is the antigen of carcinoembryonic antigen in prostate cancer and colon adenocarcinoma. HMB-45 is the antigen of melanoma. In cervical cancer, useful antigens may be encoded by human papillomavirus. Chromagranin A and synaptophycin are antigens of neuroendocrine tumors and neuroectodermal tumors. Of particular interest are invasive tumors that form solid tumor masses with necrotic areas. The lysis of such necrotic cells provides an abundant source of antigens for antigen-presenting cells, and therefore this treatment can find advantageous use in conjunction with conventional chemotherapy and / or radiotherapy.

[0044] Tumor-associated antigens can be prepared by methods well known in the art. For example, these antigens can be prepared from cancer cells by preparing a crude extract of cancer cells (e.g., as described in Cohen et al., Cancer Res., 54:1055 (1994)), by partially purifying the antigen, by recombinant technology, or by de novo synthesis of known antigens. Antigens may also be in the form of nucleic acids encoding antigenic peptides in a form suitable for expression in a target and presentation to the immune system of an immunized target. Furthermore, the antigen may be a complete antigen, or it may be a fragment of a complete antigen containing at least one epitope.

[0045] Antigens derived from pathogens known to predispose individuals to certain types of cancer may also be advantageously included in the cancer vaccines of the present invention. It is estimated that nearly 16% of global cancer incidence is attributable to infectious pathogens, and many common malignancies are characterized by the expression of specific viral gene products. Therefore, the inclusion of one or more antigens from pathogens involved in causing cancer may help expand the host immune response and enhance the prophylactic or therapeutic effects of the cancer vaccine. Among the pathogens of particular interest for use in the cancer vaccines provided herein are: This includes hepatitis B virus (hepatocellular carcinoma), hepatitis C virus (heptoma), Epstein-Barr virus (EBV) (Burkitt lymphoma, nasopharyngeal carcinoma, PTLD in immunosuppressed individuals), HTLVL (adult T-cell leukemia), oncogenic human papillomavirus types 16, 18, 33, and 45 (adult cervical cancer), and Helicobacter pylori (B-cell gastric lymphoma). Other medically relevant microorganisms that serve as antigens in mammals and, more specifically, in humans, are extensively described in the literature, e.g., CG A Thomas, Medical Microbiology, Bailliere Tindall, Great Britain 1983, the full contents of which are incorporated herein by reference.

[0046] In another embodiment, the antigen includes antigens derived from or associated with a pathogen, i.e., a microbial antigen. Thus, in one embodiment, the pathogen vaccine of the present invention further comprises at least one epitope of at least one microbial antigen. Pathogens that can be targeted by this immunotherapy include, but are not limited to, viruses, bacteria, parasites, and fungi. In another embodiment, the pathogen vaccine of the present invention further comprises multiple epitopes derived from one or more microbial antigens.

[0047] Microbial antigens used in cationic lipid immunotherapy and its methods may be intrinsically immunogenic, non-immunogenic, or slightly immunogenic. Exemplary antigens include synthetic, recombinant, exogenous, or homologous antigens, and antigenic substances include, but are not limited to, proteins, peptides, polypeptides, lipoproteins, lipopeptides, lipids, glycolipids, carbohydrates, RNA, and DNA.

[0048] Exemplary viral pathogens include, but are not limited to, viruses that infect mammals, and more specifically, humans. Examples of viruses include, but are not limited to, Retroviridae (e.g., human immunodeficiency virus, e.g., HIV-1 (also called HTLV-III, LAV, or HTLV-III / LAV, or HIV-III)); and isolates, e.g., HIV-LP; Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviridae (e.g., dengue fever virus, encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronavirus); Rhabdoviradae (e.g., vesicular stomatitis virus, rabies virus); Coronaviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviri dae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bungaviridae (e.g., Hantan virus, Bungavirus, phlebovirus and nairovirus); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reovirus, orbius and rotavirus, etc.); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvovirida (parvovirus); Papovaviridae (papillomavirus, polyomavirus); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2), varicella-zoster virus, cytomegalovirus (CMV), herpesvirus; Poxyiridae (smallpox virus, vaccinia virus, poxvirus); and Iridoviridae (e.g., African swine fever virus);This includes viruses that cannot be classified (e.g., the pathogen of spongiform encephalopathy, the pathogen of hepatitis delta (considered to be a defective satellite of hepatitis B virus), and pathogens of non-A and non-B hepatitis (Class 1 = internal infection; Class 2 = parenteral infection (i.e., hepatitis C); Norwalk and related viruses, and astroviruses)).

[0049] Furthermore, Gram-negative and Gram-positive bacteria can be targeted by this composition and method in vertebrates. Such Gram-positive bacteria include, but are not limited to, Pasteurella, Staphylococci, and Streptococcus species. Gram-negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas, and Salmonella species.The strains of the fermentation products include Helicobacter pyloris, Borella burgdorferi, and Legionella pneumophiliaii、Mycobacteriaincluding(such as M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae) Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Grade A), Streptococcus agalactiae (B, Streptococcus, Streptococcus, Streptococcus, Streptococcus, and Streptococcus faecalis diphtheriae、corynebacterium sp.、Erysipelothrix rhusiopathiae、Clostridium perfringers、Clostridium tetani、Enterobacter aerogenes、Klebsiella pneumoniae、Pasturella multocida、Bacteroides sp.、Fusobacterium nucleatumii, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Rickettsia, and Actinomyces israelli.

[0050] Polypeptides of bacterial pathogens that can be used as microbial antigen sources in the composition of the subject include, but are not limited to, iron-regulated outer membrane protein ("IROMP"), outer membrane protein ("OMP"), protein A of Aeromonis salmonicida causing furunculosis, p57 protein ("BKD") of Renibacterium salmoninarum causing bacterial kidney disease, major surface-associated antigen ("msa"), surface-expressed cytotoxin ("mpr"), surface-expressed hemolysin ("ish"), and flagellar antigen of Yersiniasis; extracellular protein ("ECP"), iron-regulated outer membrane protein ("IROMP"), and structural protein of Pasteurellosis; OMP and flagellar proteins of Vibrosis anguillarum and V. ordalii; flagellar proteins, OMP proteins, aroA, purA of Edwardsiellosis ictaluri and E. tarda; and surface antigen of Ichthyophthirius; and structural and regulatory proteins of Cytophaga columnari; This also includes the structure and regulatory proteins of rickettsiae. Such antigens can be isolated or prepared recombinantly or by any other means known in the art.

[0051] Examples of pathogens include, but are not limited to, fungi that infect mammals, more specifically humans. Examples of fungi include, but are not limited to, Cryptococcus neoformansi, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans. Examples of infectious parasites include Plasmodium, such as Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax. Other infectious microorganisms (i.e., protists) include Toxoplasma gondii. Polypeptides of parasitic pathogens include, but are not limited to, the surface antigen of Ichthyophthirius.

[0052] Other medically relevant microorganisms that act as antigens in mammals and, more specifically, in humans, are widely described in the literature; see, for example, CG A Thomas, Medical Microbiology, Bailliere Tindall, Great Britain 1983. In addition to the treatment of infectious human diseases and human pathogens, the compositions and methods of the present invention are useful for treating infections in non-human mammals. Many vaccines for the treatment of non-human mammals are disclosed in Bennett, K. Compendium of Veterinary Products, 3rd ed, North American Compendiums, Inc., 1995; see also WO02 / 069369. These disclosures are expressly incorporated herein by reference.

[0053] Exemplary non-human pathogens include, but are not limited to, mouse mammary tumor virus ("MMTV"), Rous sarcoma virus ("RSV"), avian leukemia virus ("ALV"), avian myeloblastosis virus ("AMV"), mouse leukemia virus ("MLV"), feline leukemia virus ("FeLV"), mouse sarcoma virus ("MSV"), gibbon leukemia virus ("GALV"), splenic necrosis virus ("SNV"), reticuloendotheliopathy virus ("RSV"), monkey sarcoma virus ("SSV"), and Mason Pfizer monkey virus ("MPMV"). This includes HIV-1, HIV-1, monkey retrovirus type 1, lentiviruses (such as HIV-1, HIV-2, SIV, and Visna virus), feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV), T-cell leukemia viruses such as HTLV-1, HTLV-II, and monkey T-cell leukemia virus (STLV), as well as bovine leukemia virus (BLV), and foam viruses such as human foam virus (HFV), monkey foam virus (SFV), and bovine foam virus (BFV).

[0054] In some embodiments, as used herein in relation to infectious pathogens, “treatment,” “to treat,” and “to treat” mean preventive measures that increase the subject’s resistance to infection by the pathogen or reduce the likelihood of the subject being infected with the pathogen; and / or measures taken after the subject has been infected to help the subject fight the infection, for example, to reduce or eliminate the infection or to prevent the infection from worsening.

[0055] Microbial antigens can be prepared by methods well known in the art. For example, these antigens can be prepared directly from viral and bacterial cells by preparing crude extracts, by partially purifying the antigens, by recombinant technology, or by de novo synthesis of known antigens. Antigens may also be in the form of nucleic acids encoding antigenic peptides suitable for expression in a target and presentation to the immune system of an immunized target. Furthermore, an antigen may be a complete antigen, or it may be a fragment of a complete antigen containing at least one epitope.

[0056] Antigens can be modified to increase their hydrophobicity or negative charge in order to improve their uptake into cationic lipid vesicles and their delivery to immune system cells. The hydrophobicity of an antigen can be increased, for example, by conjugating it to lipid chains or hydrophobic amino acids to improve the solubility of the antigen in the hydrophobic acyl chains of cationic lipids while maintaining the antigenic properties of the molecule. Modified antigens may be lipoproteins, lipopeptides, proteins or peptides modified with amino acid sequences having increased hydrophobicity, or combinations thereof. Modified antigens may have linkers conjugated between the lipid and the antigen; for example, an N-terminal α or ε-palmitoyllysine can be linked to the antigen via a dipeptide serine-serine linker. Furthermore, antigens can be manipulated to increase their negative charge by altering the formulation buffer in which the antigen is encapsulated within the cationic lipid complex, or by covalently bonding an anionic moiety, such as an anionic amino acid, to the antigen.

[0057] In some embodiments described herein, cationic lipids may be in the form of nanoparticle aggregates. As used herein, the term “nanoparticle” refers to particles having a size measured on a nanometer scale. As used herein, “nanoparticle” refers to particles having a structure having a size less than about 10,000 nanometers. In some embodiments, the nanoparticles are liposomes.

[0058] The cationic lipid composition of the present invention can form liposomes that can be mixed with an antigen as needed, and may contain chiral cationic lipids alone or in combination with neutral lipids. Suitable chiral cationic lipid species include, but are not limited to, R and S enantiomers.

[0059] As used herein, the term "cationic lipid" refers to any of a number of lipid species that have a net positive charge at physiological pH or have protonable groups and have a positive charge at a pH lower than the pKa. Suitable cationic lipids according to the present disclosure may include, but are not limited to: 3-β 4 N-( 1 N, 8 N,N-diguanyidinospemidine)-carbamoyl]cholesterol (BGSC); 3-β[N,N-diguanidinoethyl-aminoethane)-carbamoyl]cholesterol; (BGTC); N,N 1 N 2 N 3Tetramethyltetrapalmitylspermine (Celfectin); Nt-butyl-N'-tetradecyl-3-tetradecyl-aminopropion-amidine (CLONfectin); Dimethyldioctadecylammonium bromide (DDAB); 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE); 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-p-ropanaminonium truffle Luoroacetate) (DOSPA); 1,3-Dioleoyloxy-2-(6-carboxyspermyl)-propylamide (DOSPER); 4-(2,3-bis-palmitoyloxypropyl)-1-methyl-1H-imidazole (DPIM) N,N,N',N'-Tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3-Dioleoyloxy-1,4-butane-iodide diammonium) (Tfx-50); N-1-(2,3-Dioleoyloxy)propyl-N,N,N- Trimethylammonium chloride (DOTMA) or other N-(N,N-1-dialkoxy)-alkyl-N,N,N-trisubstituted ammonium surfactants; 1,2-dioleoyl-3-(4'-trimethylammonio)butanol-sn-glycerol (DOBT) or cholesteryl(4'trimethylammonia)butanoate (ChOTB) (where the trimethylammonium group is a double chain via a butanol spacer arm (in the case of DOTB) or a cholesteryl group (in the case of ChOTB) (In the case of) linked to); DORI (DL-1,2-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) or DORIE (DL-1,2-O-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium-m) (DORIE) or its analogues disclosed in WO93 / 03709; 1,2-dioleoyl-3-succinyl-sn-glycerolcholine ester (DOSC); cholesteryl hemysuccinate ester (ChOSC);Lipopolyamines, for example, dioctadecylamide glycylspermine (DOGS) and dipalmitoyl phosphatidylethanolamylspermine (DPPES), cholesteryl-3β-carboxylamide ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesterylcarboxylate iodide, cholesteryl-3-O-carboxyamide ethyleneamine, cholesteryl-3-β-oxysuccinate-ethylenetrimethylammonium Iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3-β-oxysuccinate iodide, 2-(2-trimethylammonio)-ethylmethylaminoethyl-cholesteryl-3-β-oxysuccinate iodide, 3-β-N-(N',N'-dimethylaminoethane)carbamoylcholesterol (DC-chol), and 3-β-N-(polyethyleneimine)-carbamoylcholesterol; O,O'-dimyristyl-N-lysylaspartate (DMKE); O,O '-Dimiristyl-N-lysyl-glutamic acid (DMKD); 1,2-Dimiristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE); 1,2-Dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC); 1,2-Dimiristoyl-sn-glycero-3-ethylphosphocholine (DMEPC); 1,2-Dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC); 1,2-Dipalmitoyl-sn-glycero-3-ethylphosphocholine (DPEPC) ); 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (DSEPC); 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); dioleoyldimethylaminopropane (DODAP); 1,2-palmitoyl-3-trimethylammoniumpropane (DPTAP); 1,2-distearoyl-3-trimethylammoniumpropane (DSTAP), 1,2-myristoyl-3-trimethylammoniumpropane (DMTAP); and sodium dodecyl sulfate (SDS). Furthermore, structural variations and derivatives of any of the listed cationic lipids are also intended.

[0060] In some embodiments, the cationic lipid is selected from the group consisting of DOTAP, DOTMA, DOEPC, and combinations thereof. In other embodiments, the cationic lipid is DOTAP. In yet another embodiment, the cationic lipid is DOTMA. In yet another embodiment, the cationic lipid is DOEPC. In some embodiments, the cationic lipid is purified.

[0061] In some embodiments, cationic lipids are enantiomers of cationic lipids. The term "enantiomer" refers to a stereoisomer of a cationic lipid that is an incompatible mirror image of its corresponding stereoisomer, such as the R and S enantiomers. In various examples, the enantiomer is R-DOTAP or S-DOTAP. In one example, the enantiomer is R-DOTAP. In another example, the enantiomer is S-DOTAP. In some embodiments, the enantiomer is purified. In various examples, the enantiomer is R-DOTMA or S-DOTMA. In one example, the enantiomer is R-DOTMA. In another example, the enantiomer is S-DOTMA. In some embodiments, the enantiomer is purified. In various examples, the enantiomer is R-DOEPC or S-DOEPC. In one example, the enantiomer is R-DOEPC. In another example, the enantiomer is S-DOEPC. In some embodiments, the enantiomer is purified.

[0062] It should be noted that, for illustrative purposes, several examples will be conducted using model HPV antigens that have been well-studied and serve as suitable antigens to illustrate the effect of type I interferon upregulation on antigen cross-presentation to CD8+ T cells.

[0063] Generally, when referring to treatment, the compositions described herein may be administered orally, parenterally (e.g., intravenously or subcutaneously), intramuscularly, intraperitoneally, percutaneously, extracorporeally, intracavitarially, percutaneously, topically, or by inhalation. Topical administration may be ophthalmologically, vaginally, rectally, or nasally. In this specification, “topical intranasal administration” means delivery of the composition to the nose and nasal cavity through one or both nostrils, and may include delivery by spray or droplet mechanism, or delivery by aerosolization of nucleic acids or vectors. Administration of the composition by inhalation may be done through the nose or mouth via spray or droplet mechanism. Delivery may also be directly delivered to any area of ​​the respiratory system (e.g., the lungs) via intubation.

[0064] As used herein, “parenteral administration” of a composition is generally characterized by injection. Injectable preparations can be prepared in conventional forms, such as liquid solutions or suspensions, solid forms suitable for dissolving suspensions in liquids before injection, or emulsions. Parenteral administration includes the use of sustained-release, time-release, or sustained-release systems to maintain a constant dose.

[0065] The term "therapeutably effective" means that the amount of composition used is sufficient to improve one or more causes or symptoms of a disease or disorder, such as abnormal cell proliferation, tumorigenesis, and cancer. Such improvement does not necessarily require elimination, but only reduction or modification. Such improvement may include the induction of an immune response. Effective doses and schedules for administering the disclosed composition can be determined empirically, and making such determinations is within the scope of the art. The dose range for administering the composition is large enough to produce the desired effect, affecting the symptoms of the disorder. The dose should not be so large as to cause undesirable side effects such as cross-reactions or anaphylactic reactions. In general, the dose can be determined by the art, depending on the patient's age, condition, sex and severity of the disease, route of administration, or whether other drugs are included in the regimen. In the case of any counterindication, the dose may be adjusted by the individual physician. Dosages vary and may be administered once or multiple times daily for one day or several days. Guidance is provided in the literature on appropriate dosages for a particular class of medicinal products.

[0066] The specific effective dose of a composition administered to any particular subject or patient depends on various factors, including the disease or disorder being treated and its severity; the identity and activity of the particular composition used; the patient's age, weight, general health, sex, and diet; the time of administration; the route of administration; the rate of excretion of the particular composition used; the duration of treatment; any drugs used in combination with or concurrently with the particular composition used; and similar factors well known in the medical field.

[0067] For example, it is within the scope of the art to start with a dose of a composition at a level lower than the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. Furthermore, certain aspects of medical history, signs, symptoms, and objective clinical examinations can be evaluated, which are known to be useful in assessing the condition of a subject requiring attention for the treatment of ischemia-reperfusion injury, trauma, drug / toxicity-induced injury, neurodegenerative diseases, cancer, or other diseases and / or conditions. These signs, symptoms, and objective clinical examinations vary depending on the specific disease or condition being treated or prevented, as is known to clinicians treating such patients or researchers conducting experiments in this field. For example, based on comparisons with appropriate control groups and / or knowledge of the normal progression of the disease in the general population or a particular subject or patient, a particular treatment regimen is considered effective if (1) the subject's physical condition is shown to have improved (e.g., a tumor has partially or completely regressed), (2) the progression of the disease or condition has stabilized, slowed, or reversed, or (3) the need for other drugs to treat the disease or condition has decreased or been eliminated.

[0068] The effective dose of a prescribed therapeutic agent may be administered daily, every other day, weekly, monthly, every other month, annually, or at any other interval determined by the physician or provider to be effective. For example, an effective daily dose may be divided into multiple doses for the purpose of administration. In conclusion, a single-dose therapeutic agent may comprise such a quantity or submultiples thereof and constitute a daily dose. The disclosed therapeutic agent may also be administered as part of a combination of antitumor or anticancer therapies. In one embodiment, the disclosed composition may be administered to a subject or patient before treatment with an antitumor or anticancer therapy. In one embodiment, the disclosed composition may be administered concurrently with an antitumor or anticancer therapy. In one embodiment, the disclosed composition may be administered after an antitumor or anticancer therapy. In one embodiment, the patient or subject receives both therapies on an alternating or rotating schedule. In one embodiment, the subject or patient receives a specific therapy with the disclosed composition. In one embodiment, the subject or patient receives at least one therapy with the disclosed composition. In one embodiment, the subject or patient receives at least one therapy with the disclosed composition and at least one other antitumor or anticancer therapy.

[0069] Dosage may be adjusted according to the individual physician or patient in the case of any counterindication. Dosages vary and can be administered once or multiple times daily for one day or several days. Guidance is provided in the literature on appropriate dosages for a particular class of medicine.

[0070] The terms used herein are intended to describe only specific aspects and are not intended to be limiting.

[0071] As used in the specification and the attached claims, unless the context explicitly indicates otherwise, the singular forms "a," "an," and "the" can include multiple criteria. For example, a reference to "compound" includes a mixture of compounds, and a reference to "pharmaceutical carrier" includes a mixture of two or more such carriers, and so on.

[0072] A range can be expressed herein as being "approximately" from one specific value "about" and / or "about" another specific value. The term "approximately" is used herein to mean roughly, within, approximately, or around that area. When the term "approximately" is used with a numerical range, it modifies that range by extending the boundary above and below the numerical value being described. Generally, the term "approximately" is used herein with a 20% variance to modify the numerical values ​​above and below. When such a range is expressed, the aspect includes one specific value and / or other specific values. Similarly, when a value is expressed as an approximation, it is understood that the specific value forms the aspect by using the preceding "approximately". Furthermore, it is understood that the endpoints of each range are significant in relation to other endpoints and independently of other endpoints.

[0073] As used herein, the term "or" means any one member of a particular list, and also includes any combination of members of that list.

[0074] "Inhibit," "to inhibit," and "inhibition" mean reducing or decreasing activity, reaction, state, disease, or other biological parameters. This may include, but is not limited to, complete ablation of activity, reaction, state, or disease. This may also include, for example, a 10% inhibition or reduction in activity, reaction, state, or disease compared to natural or control levels. Thus, in one embodiment, inhibition or reduction may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%, or any amount of reduction compared to natural or control levels. In one embodiment, inhibition or reduction may be 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100% compared to natural or control levels. In one embodiment, inhibition or reduction is 0-25, 25-50, 50-75, or 75-100% compared to natural or control levels.

[0075] "Modify," "to modify," and "modify" as used herein mean a change in activity, function, or number. The change may be an increase or decrease, enhancement, or inhibition of activity, function, or number.

[0076] "Promote," "enhance," and "to promote" refer to an increase in activity, response, state, disease, or other biological parameter. This includes, but is not limited to, the onset of activity, response, state, or disease. It may also include, for example, a 10% increase in activity, response, state, or disease compared to natural or control levels. Thus, in some embodiments, an increase or promotion may be a promotion of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% or more, or any amount compared to natural or control levels. In some embodiments, an increase or promotion may be 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100% compared to natural or control levels. In some embodiments, the increase or enhancement is 0-25, 25-50, 50-75, or 75-100% or more, for example, 200, 300, 500, or 1000% or more compared to native or control levels. In some embodiments, the increase or enhancement can exceed 100% compared to natural or control levels, such as 100, 150, 200, 250, 300, 350, 400, 450, 500%, or more compared to natural or control levels.

[0077] As used herein, the term “determine” may mean measuring or confirming a change in quantity, amount, or activity. For example, determining the amount of a disclosed polypeptide in a sample as used herein may mean the steps taken by a person skilled in the art to measure or confirm a certain quantifiable value of the polypeptide in a sample. A person skilled in the art is familiar with methods for measuring the amounts of disclosed polypeptides and nucleotides disclosed in a sample.

[0078] The term "sample" can refer to tissue or organs derived from the subject; cells (within the subject, directly collected from the subject, or maintained in culture or from a cultured cell line); cell lysates (or lysate fractions) or cell extracts; or a solution containing one or more molecules derived from cells or cellular materials (e.g., polypeptides or nucleic acids). A sample may also be any bodily fluid or excretion containing cells or cellular components (e.g., blood, urine, feces, saliva, tears, bile).

[0079] The present invention will be further described with reference to the following embodiments, but it should be understood that the present invention is not limited to such embodiments. Rather, many changes and modifications are presented to those skilled in the art in light of this disclosure which describes the current best mode for carrying out the present invention, without departing from the scope and spirit of the invention. All changes, modifications and variations that fall within the meaning and scope of equivalence of the claims should be considered to be within that scope.

[0080] The present invention has been described in an illustrative manner. It should be understood that the terms used are intended to be descriptive rather than restrictive.

[0081] Many modifications and variations of the present invention are possible in light of the above teachings. Therefore, within the scope of the appended claims, the present invention can be implemented in ways other than those specifically described. [Examples]

[0082] The following materials, methods, and protocols were used in the examples described below.

[0083] Materials and methods Animals: 6-12 week old C57BL6 / J mice (B6 mice), B6. Cg-Tg(HLA-A / H2-D)2Enge / J transgenic breeder mice expressing the human HLA-A2 gene (AAD mouse), and IFNAR- / -(B6.129S2-Ifnar1tm1Agt / Mmjax) gene knockout breeder mice obtained from Jackson Laboratory (Bar Port, ME) were reared under specific pathogen-free conditions. Animal rearing, breeding, and experimental procedures were carried out according to the DLAR-approved IACUC protocol.

[0084] Peptides and Reagents: cGMP-grade R-DOTAP and S-DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) were supplied by Merck & Cie in Shaffhausen, Switzerland. cGMP-grade R-DOTAP liposome nanoparticles were produced using a standard liposome manufacturing process. Peptide antigens (KF18:KSSGPDAERAHYNIVTF (SEQ ID NO: 3), SF9:SIINFEKL (SEQ ID NO: 1), RF9:RAHYNIVTF (SEQ ID NO: 2)) were synthesized and purified to >95% purity using GenScript in Piscataway, New Jersey, USA. All other cationic lipids were purchased from Avanti Polar Lipids, Birmingham, AL.

[0085] Cell lines and bone marrow-derived dendritic cell cultures: Primary bone marrow-derived dendritic cells were obtained by culturing hematopoietic bone marrow cells for 8 days in complete RPMI medium (RPMI medium containing 10% FBS, 1 mM L-glutamine, 1 mM sodium pyruvate, 1 mM MEM non-essential amino acids, 50 μM β-mercaptoethanol, 100 u / ml penicillin, and 100 u / ml streptomycin) supplemented with recombinant mouse GM-CSF and IL-4 (conventional BMDC (cDC)) or recombinant flt3 ligand (plasmacytoid BMDC (pDC)). B16-Blue™ IFN-α / β cells, used for the detection of cationic lipid-induced type I IFNs, were purchased from InvivoGen in San Diego, California, USA, and cell cultures were maintained according to the manufacturer's protocol.

[0086] IFN Reporter Cell Assay and Cell Phenotypic Analysis: To detect IFN, supernatant from type I IFN-producing cells was added to B16-Blue® IFN-α / β cells in a 96-well plate. After 24 hours of incubation, the supernatant was assayed for SEAP activity using QUANTI-Blue® reagent (InvivoGen, USA) according to the manufacturer's instructions. Briefly, 50 μl of cell supernatant containing SEAP was mixed with 150 μl of QUANTI-Blue® reagent, incubated at 37°C for 3-4 hours, and absorbance was measured at 650 nm using a spectrometer. The concentration of type I IFN in the test samples was quantified using a standard curve generated from recombinant type I IFNβ-treated B16-Blue® IFN-α / β cells in the same assay. To measure CD69 expression, single-cell suspensions from lymph nodes were stained with fluorescent dye-conjugated CD3, and CD69 and percentage CD69+CD3+ T cells in each afferent lymph node were measured using flow cytometry.

[0087] RNA isolation, nanostring analysis, and gene expression analysis: For gene expression analysis, outflow popliteal lymph nodes (n=4) were enzymatically digested at 37°C for 60 minutes using a cell dissociation cocktail, and CD11c+ cells were sorted using a Sony SY3200 cell sorter with a single-cell suspension. The sorted and purified CD11c+ cells (25K cells) were lysed in Qiagen's RLT cell lysis buffer (Qiagen, USA), and total RNA was isolated using the Qiagen Total RNA Isolation Kit (Qiagen, USA). The obtained total RNA was sent to the University of Kentucky Genomics Core Laboratory (Lexington, KY), where it was mixed with the ncounter™ mouse inflammation panel (Nano String Technologies, Seattle, WA), which can measure more than 547 genes involved in the immune response. mRNA binding was detected using the Nanso String nCounter analysis system, live count data were normalized, and statistical analysis was performed using the SAS program by the Genomics Core Laboratory, UK. For confirmation testing using RT-PCR, lymph nodes were directly lysed in RLT cell lysis buffer, and the entire isolated RNA was reverse transcribed into cDNA using the QuantiTect reverse transcription kit (Qiagen, USA). Subsequently, the cDNA was amplified using the TaqMan gene expression system (Applied Biosystems, USA), and mouse IFNα and IFNβ transcripts in relation to GAPDH expression were detected and quantified using quantitative PCR.

[0088] Vaccination and Evaluation of Antigen-Specific T Cell Response: Mice were anesthetized with isoflurane, the injection sites were shaved and washed with 70% ethanol before vaccination. To evaluate the antigen-specific T cell response using ELISPOT, mice were vaccinated twice (100 μl / dose) at 7-day intervals with a vaccine formulation containing cationic lipids and antigenic peptides, or once with a CFA formulation prepared by emulsifying equal amounts of complete Freund's adjuvant and antigenic peptides delivered on day 0. Antigen-specific responses were evaluated 14 days after the first vaccination. To measure the antigen-specific response, spleen cells derived from euthanized mice were used to detect the antigen-specific T cell response using the IFN-γ ELISPOT assay (Mabtec, Cincinnati, Ohio, USA). 2.5 × 10⁶ cells were used for the IFN-γ ELISPOT assay. 5 Treated spleen cells were stimulated with a CD8 T cell epitope of interest at 37°C for 18–24 hours in 96-well plates pre-coated with mouse IFN-γ capture antibody. After stimulation, the wells were washed with PBS and incubated with biotin-conjugated anti-IFNγ antibody, followed by streptavidin-HRP antibody. To visualize antigen-specific IFN-γ producing cells, the wells were incubated with TMB substrate for 6 minutes, washed with water, and air-dried. Spots were scanned and counted using a CTL ImmunoSpot Analyzer and ImmunoSpot Ver.4 software (Cellular Technology Limited, Cleveland, Ohio, USA). Spot counts were summarized as the median from three samples. Each sample had unstimulated and PMA / ionomycin control wells to detect background and positive controls. A well was considered positive if the number of spots exceeded 5 and the antigen-specific reaction was positive, or if the number of spots exceeded 3 times that of the control.

[0089] Example 1 R-DOTAP induces type I interferon gene expression in lymph nodes in the lymphatic region. To evaluate cationic nanoparticle-induced gene expression, 100 μl of 12 mM R-DOTAP nanoparticles were injected subcutaneously into the posterior neck of C57BL / 6J mice, and inflammatory gene expression in the afferent lymph nodes was analyzed 4 or 24 hours after vaccination. Mice injected with PBS and LPS (50 μg / mouse) (at 24 hours) were used as negative and positive controls, respectively. Axillary and brachial lymph nodes from each mouse were pooled 4 or 24 hours after injection and treated by collagenase digestion. Activated dendritic cells (CD11c) in lymph node cell suspension were also analyzed. + Cells were sorted and purified, lysed in RLT buffer, and processed for relative gene expression analysis using the nCounter® mouse inflammation kit and nanostring technology. R-DOTAP injection was found to significantly alter the expression of several inflammatory genes at both time points. Several genes, including IFN-1α and IFN-1β, showed increases of more than 5-fold compared to LPS, which was used as a positive control. Expression of other genes, including Cxcl10, also increased significantly, to levels comparable to those induced by LPS (Figure 1).

[0090] Example 2 Both R-DOTAP and S-DOTAP induce interferon-α and interferon-β gene expression in lymph nodes in the lymphatic region.

[0091] To compare type I interferon gene expression induced by both R- and S-DOTAP, a similar study outlined in Example 1 was conducted. C57BL / 6J mice were subcutaneously injected with 50 μl of 6 mM RDOTAP nanoparticles into a foot pad. Mice injected with PBS and LPS (50 μg / mouse) (at 24 hours) were used as negative and positive controls, respectively. Popliteal lymph nodes from each mouse were pooled 3 or 24 hours post-injection, lysed in RLT buffer, and processed for relative gene expression analysis using Taqman® gene expression assay and RT-PCR. These studies confirmed that both R- and S-DOTAP are potent inducers of type I interferon (Figure 2).

[0092] Example 3 R-DOTAP and S-DOTAP upregulate type I interferon-associated CD69 expression in T cells. To further understand the effects and capabilities of cationic lipids in activating type I interferons, C57BL / 6J mice or IFNAR- / - mice were subcutaneously injected with 50 μl of 6 mM R-DOTAP nanoparticles, 6 mM S-DOTAP, or 280 mM sucrose via a foot pad. Twenty-four hours after injection, popliteal lymph node outflow lymph nodes were isolated from each mouse, and the single-cell suspension of the lymph nodes was stained with fluorescent dye-conjugated CD3 and CD69. R-DOTAP alone (without antigen) resulted in a visible increase in DLN size, which was attributed to a steady increase in total cell count over seven days (Figure 3A). Studies of T cell influx into the DLN using pertussis toxin suggest that lipids structurally related to R-DOTAP induce lymph node homing chemokines, likely a direct consequence of type I IFN signaling. This increase in total cell number was significantly reduced in IFNαR knockout mice, confirming its dependence on type I IFN signaling (Figure 3B). Type I IFN is known to inhibit lymphocyte efflux from lymphoid organs via CD69 upregulation, which in turn inhibits the sphingosine-1-phosphate receptor necessary for lymphocyte efflux. Therefore, we hypothesized that injection of both S-DOTAP and R-DOTAP would result in CD69 upregulation in the DLN. Indeed, subcutaneous injection of both cationic lipids into wild-type mice resulted in the strongest CD69 upregulation in T cells. In contrast, no CD69 upregulation was observed after R-DOTAP injection in IFNαR knockout mice, demonstrating that R-DOTAP-induced CD69 upregulation is type I IFN-dependent (Figure 3C). Data represent the percentage of CD69+CD3+ T cells in each regional lymph node.

[0093] Example 4 Various cationic lipids upregulate type I interferons. In vitro studies were conducted to evaluate the potential of various cationic lipids to induce type I interferon. This study investigated the cationic lipids DOTAP, DOEPC, and DOTMA. The neutral lipid DOPC was also evaluated to confirm the specificity of interferon upregulation for cationic lipids. In one study, bone marrow-derived dendritic cells (BMDCs) from IFNAR-Ko mice were stimulated for 24 hours with cationic lipids or LPS-positive controls (1-500 ng / ml) at indicated concentrations (6-400 μM) (Figure 4A). In a second study, FLT3-induced BMDCs (pDCs) and GM-CSF / IL-4-induced BMDCs (cBMDCs) were stimulated for 24 hours with R-DOTAP or LPS-positive controls (1-500 ng / ml) at specified concentrations (6-400 μM) (Figure 4B). To measure type I interferon production, cell supernatant (100 μl) was added to reporter cells (Invivogen's B16.Blue-IFNα / β cells, USA) and incubated for 18 hours to stimulate type I interferon-inducible secretory alkaline phosphatase (SEAP) production by the reporter cells. SEAP activity in the reporter cell supernatant was quantified using a colorimetric SEAP assay kit according to the manufacturing protocol. BMDC secretory type I interferon was quantified in the reporter cell line using a standard curve with recombinant mouse IFN-β stimulated SEAP activity. These studies confirm that the ability to upregulate type I interferon is not specific to DOTAP and can be activated by multiple cationic lipids. It has also been confirmed that neutral lipids cannot activate the type I interferon pathway.

[0094] Example 5 A decrease in the CD8+ T cell response mediated by cationic lipids is observed in type I IFN-deficient mice. The demonstration of cationic lipid-mediated upregulation of type I IFN genes suggests that cationic lipids target the type I IFN pathway and induce a robust CD8+ T cell response. To further confirm this hypothesis, an R-DOTAP formulation containing a well-characterized mouse CD8+ T cell epitope (SIINFEKL (SEQ ID NO: 1)) derived from chicken egg albumin or HPV16-E7 protein (RF9:RAHYNIVTF (SEQ ID NO: 2)) was used in wild-type C57BL / 6J mice and type I interferon signaling-deficient IFNARs. - / - Mice were administered the vaccine on days 0 and 7. Mice were also inoculated with complete Freund's adjuvant (CFA) + SIINFEKL. A peptide-only vaccine was used as a positive control. On day 14, antigen-specific (SIINFEKL) CD8+ T cell responses induced by R-DOTAP or CFA were evaluated using the IFN-γELISPOT assay. As shown in Figure 5A, vaccination of wild-type mice with the DOTAP formulation induced a stronger antigen-specific CD8+ T cell response, which was larger than that induced by the CFA adjuvant formulation. In contrast, the CD8+ T cell response induced by R-DOTAP was dramatically reduced in IFNAR- / - mice, but no such difference was observed with the CFA-adjuvant formulation. Similar results were observed for tumor-associated antigens expressed in HPV16-positive tumors (Figure 5B). CFA adjuvants have been well-established for targeting multiple TLR pathways and thus avoiding the need for type I IFN signaling to induce antigen-specific CD8+ T cell responses.

[0095] The loss of cationic lipid-inducing effects in mice lacking type I IFN signaling, coupled with its ability to potently activate type I IFN genes, strongly suggests that R-DOTAP effectively promotes antigen cross-presentation and simultaneously activates the type I IFN pathway to drive a robust antigen-specific CD8+ T cell immune response (Figure 5).

Claims

[Claim 1] A method for modifying the type I IFN signaling pathway in a subject, including administration of cationic lipids to a mammal.

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