Novel HPV16 non-HLA-restricted T-cell vaccine, its composition and method of use

Abstract

To provide novel human papillomavirus immunogenic compositions and use thereof.SOLUTION: The compositions comprise unique combinations of multi-epitope peptide sequences specially selected and designed to be effectively processed and cross-presented to T-cells. The peptides utilized in the compositions display high levels of binding to HLA-supertypes. The immunogenic compositions are broadly applicable to large proportions of target populations. The compositions comprise adjuvants such as cationic lipids.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] FIELD OF THE DISCLOSURE Embodiments of the present disclosure relate generally to novel HPV16 vaccines, particularly non-HLA-restricted T-cell vaccines, compositions and methods of use thereof. [Background technology]

[0002] Therapeutic vaccination with HPV E6 and E7 protein antigens has been demonstrated to offer strong potential for treating HPV-induced cancers, including cervical, anal, vulvar, vaginal, and head and neck cancers, as well as precancerous tumors. Due to the limited ability of antigen presentation to induce HPV-specific T cell responses, the earliest approaches to HPV therapeutic vaccination relied on the inclusion and presentation of short, single CD8+ peptide epitopes. HPV vaccines based on these peptides with restricted HLA-A2 epitopes have shown very limited applicability, even within the selected HLA-A2 populations evaluated. Recent approaches to overcome this significant drawback of cancer vaccines have focused on two key approaches or platforms: 1. delivery of full-length HPV protein-encoding DNA within live vectors, such as engineered viruses and bacteria, and 2. the use of multiple, overlapping, long, multi-epitope peptides encompassing the full-length HPV16 E6 and E7 protein sequences. Both approaches are directed at overcoming the genetic limitations faced by patients by using short, single-epitope HLA-A2 peptides to address a broad patient population, and both approaches show promising promise in human clinical trials.

[0003] In silico peptide binding analysis has been effectively used to understand the binding potential of immunogenic peptides. However, this technique has not been used to design more efficient cancer vaccines that are characterized by simplicity and yet have the potential to address the needs of a wide range of patient populations with diverse genetic backgrounds.

[0004] T cells mediate a range of immune responses, including those involved in the translocation of intracellular pathogens, virus-infected cells, and tumor cells, as well as those involved in transplant rejection and autoimmunity. The T cell immune system is adapted to recognize foreign cells as well as modified self-cells and eliminate them from the body. T cell recognition of peptide antigens occurs via the T cell receptor (TCR). This process requires that peptide antigens be presented to the TCR by major histocompatibility complex (MHC) molecules located on the surface of antigen-presenting cells (APCs), such as dendritic cells. Human MHC molecules are called human histocompatibility leukocyte antigens (HLA). Peptide antigens are bound to MHC molecules in a manner that allows the T cell receptor to recognize the unique structure formed by the combination of the MHC molecule and a specific peptide. The restrictive nature of T cell functionality, along with polymorphisms in MHC molecules and the wide spectrum of unique peptides that can associate with MHC, results in diverse recognition patterns such that a given MHC-peptide combination is recognized only by the function of a T cell clone.

[0005] There are two types of MHC molecules involved in antigen presentation: class I and class II. MHC class I molecules consist of an α chain with three domains and a transmembrane and cytoplasmic domain. MHC class I molecules are widely distributed and present on all nucleated cells. MHC class II molecules contain an α chain and a β chain that self-associate to form heterodimers. Each chain has two extracellular domains, as well as a transmembrane and cytoplasmic domain. MHC class II molecules are more restricted in distribution than class I molecules and are present, for example, on antigen-presenting cells (APCs).

[0006] Cytotoxic T lymphocytes ("CTLs") that are specifically activated against a particular antigen can kill cells that contain or express the antigen. The TCR of a CTL recognizes the antigen in the context of an MHC class I molecule. An important role for T helper lymphocytes ("Th cells") is the optimal induction of CTL responses; they may also play a role in maintaining CTL memory. The TCR of Th cells recognizes the antigen in the context of an MHC class II molecule.

[0007] Therapeutic vaccination to prime antigen-recognizing T cells has been demonstrated to be a viable option for active cancer immunotherapy, aiming to treat both early- and late-stage disease by activating the patient's immune system. Various mechanisms activated by therapeutic vaccination specifically attack antigen-expressing cancer cells and ignore normal cells. Therefore, therapeutic cancer vaccines could, in principle, be effective in suppressing tumor growth and treating recurrent tumors that are refractory to conventional therapies such as surgery, radiation therapy, and chemotherapy. A therapeutic cancer vaccine for treating prostate cancer has already been approved by the U.S. Food and Drug Administration. This major breakthrough paves the way for novel approaches to therapeutic vaccination that may offer improved safety and efficacy. Several such approaches are currently being evaluated preclinically and clinically. Unlike prophylactic antibody-inducing vaccines, which are generally administered to healthy individuals, therapeutic cancer vaccines are administered to cancer patients and are designed to eradicate cancer cells by enhancing the patient's own immune response, particularly T cell responses (Lollini PL, Cavallo F, Nanni P, Forni G. Vaccines for tumor prevention. Nature reviews. Cancer. 2006;6:204-216).

[0008] Tumor-associated antigens as therapeutic targets Recombinant vaccines based on proteins derived from defined tumor-associated antigens (TAA), or synthetic peptide vaccines derived from TAAs, typically administered in combination with adjuvants or immune modulators, offer significant advantages in cost and simplicity over autologous and DC vaccines. The availability of patient samples or specimens and the complex procedures for preparing personalized vaccines limit the widespread use of autologous cancer vaccines. MAGE-1 was the first gene reported to encode a human tumor antigen recognized by T cells (van der Bruggen P, Traversari C, Chomez P, Lurquin C, De Plaen E, Van den Eynde B, Knuth A, Boon T. A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science. 1991;254:1643-1647.), and has been well studied and used in clinical cancer vaccines. The identification of several TAAs has provided the ability to develop and design a variety of targeted therapeutic vaccines to address a wide range of cancers. Such TAAs have been classified into several major categories. Cancer-testis antigens such as NY-ESO-1, BAGE, MAGE, and SSX-2 are encoded by genes that are normally silenced in adult tissues but are transcriptionally reactivated in tumor cells (De Smet C, Lurquin C, van der Bruggen P, De Plaen E, Brasseur F, Boon T. Sequence and expression pattern of the human MAGE2 gene. Immunogenetics. 1994;39:121-129; Gnjatic S, Ritter E, Buchler MW, Giese NA, Brors B, Frei C, Murray A, Halama N, Zornig I, Chen YT, Andrews C, Ritter G, Old LJ, Odunsi K, Jager D.Seromic profiling of ovarian and pancreatic cancer. Proceedings of the National Academy of Sciences of the United States of America. 2010;107:5088-5093; Hofmann O, Caballero OL, Stevenson BJ, Chen YT, Cohen T, Chua R, Maher CA, Panji S, Schaefer U, Kruger A, Lehvaslaiho M, Carninci P, Hayashizaki Y, Jongeneel CV, Simpson AJ, Old LJ, Hide W. Genome-wide analysis of cancer / testis gene expression. Proceedings of the National Academy of Sciences of the United States of America. 2008;105:20422-20427; Karbach J, Neumann A, Atmaca A, Wahle C, Brand K, von Boehmer L, Knuth A, Bender A, Ritter G, Old LJ, Jager E. Efficient in vivo priming by vaccination with recombinant NY-ESO-1 protein and CpG in antigen naive prostate cancer patients. Clinical cancer research: an official journal of the American Association for Cancer Research.2011;17:861-870). Tissue differentiation antigens are antigens derived from normal cells and are shared by both normal cells and tumors, but melanoma (gp100, Melan-A / Mart-1, and tyrosinase) (Bakker AB, Schreurs MW, de Boer AJ, Kawakami Y, Rosenberg SA, Adema GJ, Figdor CG. Melanocyte lineage-specific antigen gp100 is recognized by melanoma-derived tumor-infiltrating lymphocytes. J Exp Med. 1994;179:1005-1009. Bakker AB, Schreurs MW, de Boer AJ, Kawakami Y, Rosenberg SA, Adema GJ, Figdor CG. Melanocyte lineage-specific antigen gp100 is recognized by melanoma-derived tumor-infiltrating lymphocytes. J Exp Med. 1994;179:1005-1009), prostate cancer (PSA, PAP) (Correale P, Walmsley K, Nieroda C, Zaremba S, Zhu M, Schlom J, Tsang KY. In vitro generation of human cytotoxic T lymphocytes specific for peptides derived from prostate-specific antigen. J Natl Cancer Inst. 1997;89:293-300; Kantoff PW, Higano CS, Shore ND, Berger ER, Small EJ, Penson DF, Redfern CH, Ferrari AC, Dreicer R, Sims RB, Xu Y, Frohlich MW, Schellhammer PF. Sipuleucel-T immunotherapy for castration-resistant prostate cancer.The New England journal of medicine. 2010a;363:411-422) and breast cancer (mammaglobin A) (Jaramillo A, Majumder K, Manna PP, Fleming TP, Doherty G, Dipersio JF, Mohanakumar T. Identification of HLA-A3-restricted CD8+ T cell epitopes derived from mammaglobin-A, a tumor-associated antigen of human breast cancer. International journal of cancer. Journal international du cancer. 2002;102:499-506). In addition to these differentiation-associated antigens, CEA (Tsang KY, Zaremba S, Nieroda CA, Zhu MZ, Hamilton JM, Schlom J. Generation of human cytotoxic T cells specific for human carcinoembryonic antigen epitopes from patients immunized with recombinant vaccinia-CEA vaccine. J Natl Cancer Inst. 1995;87:982-990), MUC-1 (Finn OJ, Gantt KR, Lepisto AJ, Pejawar-Gaddy S, Xue J, Beatty PL. Importance of MUC1 and spontaneous mouse tumor models for understanding the immunobiology of human adenocarcinomas. Immunologic research.2011;50:261-268), HER2 / Neu (Disis ML, Wallace DR, Gooley TA, Dang Y, Slota M, Lu H, Coveler AL, Childs JS, Higgins DM, Fintak PA, dela Rosa C, Tietje K, Link J, Waisman J, Salazar LG. Concurrent trastuzumab and HER2 / neu-specific vaccination in patients with metastatic breast tumor suppressor gene (p53) (Azuma K, Shichijo S, Maeda Y, Nakatsura T, Nonaka Y, Fujii T, Koike K, Itoh K. Mutated p53 gene encodes a nonmutated epitope recognized by HLA-B*4601-restricted and tumor cell-reactive CTLs at tumor site. Cancer Res. 2003;63:854-858), hTERT (Vonderheide RH, Hahn WC, Schultze JL, Nadler LM. The telomerase catalytic subunit is a widely expressed tumor-associated antigen recognized by cytotoxic T lymphocytes. Immunity. 1999;10:673-679), and certain anti-apoptotic proteins (e.g., survivin) (Vonderheide RH, Hahn WC, Schultze JL, Nadler LM.Several other tumor antigens, such as the telomerase catalytic subunit, a widely expressed tumor-associated antigen recognized by cytotoxic T lymphocytes. Immunity. 1999;10:673-679, are also highly elevated in tumor tissue compared with their normal counterparts. Unique tumor-specific antigens are often referred to as mutant oncogenes (ras, B-raf) (Brichard VG, Lejeune D. Cancer immunotherapy targeting tumor-specific antigens: toward a new therapy for minimal residual disease. Expert opinion on biological therapy. 2008;8:951-968). Targeting these tumor-specific antigens involved in promoting the tumorigenesis process has the advantage of being resistant to immune selection and may be more effective. While many such tumor-specific antigens have been identified and utilized, given the diverse genetic profiles among human populations, there is a continuing need to identify antigens with increased specificity for particular groups, such as ethnicity or geographic location, regardless of how such groups are identified. The need for identifying such additional antibodies continues to grow for a variety of reasons, including: There remains a need in the pharmaceutical industry to improve and simplify the vaccine manufacturing process, and therefore, it is a continuing goal to identify antigens, proteins, and peptides that are most precisely associated with eliciting an appropriate immunological response.

[0009] Protein / peptide-based vaccines have a clear cost advantage over autologous or personalized vaccines. However, the fact that they target only one or a few epitopes of a TAA may be considered a disadvantage. For a cancer vaccine to be optimally effective, antigen-specific CTLs and antigen-specific CD4 +It is generally believed that induction of both helper T cells is necessary, and some polypeptide vaccines (e.g., Stimuvax®) potentially contain both CD4 and CD8 epitopes. Another approach to enhance the immunogenicity of self-antigens is to modify the peptide sequence of TAA to introduce enhancer agonist epitopes, which increase peptide binding to MHC molecules or T cell receptors, resulting in higher levels of T cell responses and / or higher T cell avidity (Dzutsev AH, Belyakov IM, Isakov DV, Margulies DH, Berzofsky JA. Avidity of CD8 T cells sharpens immunodominance. International immunology. 2007;19:497-507; Jordan KR, McMahan RH, Kemmler CB, Kappler JW, Slansky JE. Peptide vaccines prevent tumor growth by activating T cells that respond to native tumor antigens. Proceedings of the National Academy of Sciences of the United States of America. 2010;107:4652-4657; Rosenberg SA, Yang JC, Schwartzentruber DJ, Hwu P, Marincola FM, Topalian SL, Restifo NP, Dudley ME, Schwarz SL, Spiess PJ, Wunderlich JR, Parkhurst MR, Kawakami Y, Seipp CA, Einhorn JH, White DE. Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat Med. 1998;4:321-327).

[0010] A common approach to cancer therapeutic vaccination is vaccination with precise MHC-human leukocyte antigen (HLA)-binding peptides derived from the sequence of a TAA. T cells recognize their target antigens as 8-10 amino acid peptides presented by MHC class I molecules on the cell surface. A major drawback of such an approach is the fact that humans are genetically diverse, with a wide range of HLA alleles that recognize and bind different peptide antigens. As a result, cancer vaccines based on short peptides have shown very limited applicability, and recent approaches have required the inclusion of multiple peptides, in some cases more than 10 peptides, in order to provide reasonable coverage of the population.

[0011] HPV vaccine HPV E6 and E7 proteins are constitutively co-expressed in all HPV-infected precancerous cells and are the most abundant viral transcripts found in biopsies from HPV-associated cervical cancer cells (K. Seedorf, T. Oltersdorf, G. Krammer, W. Rowekamp, ​​Identification of early proteins of the human papilloma viruses type 16 (HPV 16) and type 18 (HPV 18) in cervical carcinoma cells. EMBO J. 6, 139-144 (1987)). Due to their interaction with p53 and the retinoblastoma protein (D. Pim, A. Storey, M. Thomas, P. Massimi, L. Banks, Mutational analysis of HPV-18 E6 identifies domains required for p53 degradation in vitro, abolition of p53 transactivation in vivo, and immortalization of primary BMK cells. Oncogene 9, 1869-1876 (1994)), E6 and E7 are involved in cellular transformation and are required for the maintenance of HPV-associated malignancies (K. Munger, P. M. Howley, Human papillomavirus immortalization and transformation functions. Virus Res. 89, 213-228 (2002)).In particular, E6- and E7-specific cellular immune responses are associated with antibodies in HPV16-associated lesions (S. Peng, C. Trimble, L. Wu, D. Pardoll, R. Roden, CF Hung, TC Wu, HLA-DQB1*02-restricted HPV-16 E7 peptide-specific CD4+ T-cell immune responses correlate with regression of HPV-16-associated high-grade squamous intraepithelial lesions. Clin. Cancer Res. 13, 2479-2487 (2007)). Farhat et al. reported that the rate of positive enzyme-linked immunospot (ELISpot) responses to HPV16 E6 and E7 was significantly increased in women with recently elucidated HPV infections compared with women with persistent cervical HPV16 infection (S. Farhat, M. Nakagawa, AB Moscicki, Cell-mediated immune responses to HPV-16 E6 and E7 antigens as measured by interferon gamma enzyme-linked immunospot in women with cleared or persistent human papillomavirus infection. Int. J. Gynecol. Cancer 19, 508-512 (2009)). Therefore, HPV E6 and E7 antigens are considered promising immunotherapy targets.To date, several HPV therapeutic vaccines have been developed, including protein / peptide-based vaccines (L. Muderspach, S. Wilczynski, L. Roman, L. Bade, J. Felix, L.A. Small, W.M. Kast, G. Fascio, V. Marty, J. Weber, A phase I trial of a human papillomavirus (HPV) peptide vaccine for women with high-grade cervical and vulvar intraepithelial neoplasia who are HPV 16 positive. Clin. Cancer Res. 6, 3406-3416 (2000); W.J. van Driel, M.E. Ressing, G.G. Kenter, R.M.P. Brandt, E.J.T. Krul, A.B. van Rossum, E. Schuuring, R.O.V.ringa, T. Bauknecht, A. Tamm-Hermelink, P.A. van Dam, G.J. Fleuren, W.M. Kast, C.J.M. Melief and J.B. Trimbos, Vaccination with HPV 16). Peptides of Patients with Advanced Cervical Carcinoma: Clinical Evaluation of a Phase I-II Trial, Eur J Cancer, Vol. 35, No. 6, pp. 946-952, 1999) have been developed with a focus on stimulating the production and activation of HPV E6- and E7-specific T cells. However, HPV vaccines based on these peptides with restricted HLA-A2 epitopes have shown very limited applicability even within the evaluated and selected HLA-A2 population. In 2009, it was reported that an HPV peptide vaccine containing 13 overlapping peptides derived from the HPV16 E6 protein and 4 overlapping peptides derived from the HPV16 E7 peptide showed strong anti-HPV responses when tested in a non-restricted patient population with VIN3.This study provided the first demonstration of broadly active HPV peptide vaccines in a non-HLA-restricted population (Gemma G. Kenter, Marij JP Welters, A. Rob PM Valentijn, Margriet JG Lowik, Dorien MA Berends-van der Meer, Annelies PG Vloon, Farah Essahsah, Lorraine M. Fathers, Rienk Offringa, Jan Wouter Drijfhout, Amon R. Wafelman, Jaap Oostendorp, Gert Jan Fleuren, Sjoerd H. van der Burg, and Cornelis JM Melief; Vaccination against HPV-16 Oncoproteins for Vulvar Intraepithelial Neoplasia, N Engl J Med 2009;361:1838-47).

[0012] These references, particularly the recent one by Kenter et al., highlight the major drawbacks associated with current approaches to the development of peptide vaccines that provide broad HLA coverage. Current approaches focus on developing long peptides that cover the entire sequence of an antigenic protein due to the inability to determine immunogenic sequences that can provide broad coverage. As a result, there are four major drawbacks: 1. The vaccines are complex and involve the use of a large number of peptides, e.g., 13 peptides in Kenter et al.; 2. The vaccines are unnecessarily expensive; 3. Patients are subjected to receiving unnecessary peptides that may provide no therapeutic or immunogenic benefit; and 4. Active peptides may be inactivated by competitive binding with other peptides (this particular drawback is reported by Kenter et al., supra).

[0013] The present invention reports a simpler, more cost-effective, and efficient approach to the development of highly effective, broad-spectrum HLA-encapsulated peptide vaccines.

[0014] Immunostimulatory adjuvants for protein / peptide-based vaccines Given the inherently poor immunogenicity of TAAs, immunostimulatory adjuvants may be necessary in some cases for the generation of effective immune responses. Aluminum salts (alum) have been used as adjuvants with great success for nearly a century and have been effective in promoting protective humoral immunity. However, alum is least effective against diseases in which cell-mediated immunity is required for protection. The recognition over the past 20 years that activation of innate immunity is necessary to promote adaptive immune responses has fundamentally changed the theory of how adjuvants promote adaptive immunity. In particular, Charles Janeway's pioneering work demonstrated that adaptive immune responses precede and depend on innate immune receptors triggered by microbial components (Janeway CA., Jr. The immune system evolved to discriminate infectious nonself from noninfectious self. Immunol Today. 1992;13:11-16). Recognition of conserved moieties associated with pathogens or pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors, such as Toll-like receptors (TLRs), is involved in coordinated innate and adaptive immunity against microbial pathogens or infectious cells (Kawai T, Akira S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity. 2011;34:637-650). TLR-mediated activation of antigen-presenting cells (e.g., DCs) is a critical step in this process.Indeed, many established experimental vaccines incorporate PAMPs not only to protect against infectious diseases but also as part of therapeutic vaccinations against cancer (Wille-Reece U, Flynn BJ, Lore K, Koup RA, Miles AP, Saul A, Kedl RM, Mattapallil JJ, Weiss WR, Roederer M, Seder RA. Toll-like receptor agonists influence the magnitude and quality of memory T cell responses after prime-boost immunization in nonhuman primates. J. Exp. Med. 2006; 203:1249-1258). The use of these molecularly and functionally defined molecules as adjuvants greatly facilitates the rational design of vaccines.

[0015] In support of this view, Bacillus Calmette-Guerin (BCG), which has been used for many years to treat bladder cancer, has been shown to be relatively effective and to activate TLR2 and TLR4 (Heldwein KA, Liang MD, Andresen TK, Thomas KE, Marty AM, Cuesta N, Vogel SN, Fenton MJ. TLR2 and TLR4 serve distinct roles in the host immune response against Mycobacterium bovis BCG. Journal of Leukocyte Biology. 2003;74:277-286). LPS, the natural ligand for TLR4, was reported to have anticancer properties as early as the 1960s (Mizuno D, Yoshioka O, Akamatu M, Kataoka T. Antitumor effect of intracutaneous injection of bacterial lipopolysaccharide. Cancer Research. 1968;28:1531-1537). Monophosphoryl lipid A (MPL) is a chemically modified derivative of S. mnemonicum endotoxin that exhibits significantly reduced toxicity while retaining most of the immunostimulatory properties of LPS (Mata-Haro V, Cekic C, Martin M, Chilton PM, Casella CR, Mitchell TC. The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4. Science. 2007;316:1628-1632).Many researchers have shown that MPL potently enhances patient immune responses to viral and tumor-associated antigens (Schwarz TF. Clinical update of the AS04-adjuvanted human papillomavirus-16 / 18 cervical cancer vaccine, Cervarix. Advances in therapy. 2009;26:983-998). The FDA has approved the Cervarix vaccine, formulated with MPL and aluminum salts, as a preventive vaccine against human papillomavirus (Schiffman M, Wacholder S. Success of HPV vaccination is now a matter of coverage. The Lancet Oncology. 2012;13:10-12). Imiquimod (a TLR agonist) was approved by the FDA in 2004 for human use in the treatment of actinic keratosis and superficial basal cell carcinoma (Hoffman ES, Smith RE, Renaud RC., Jr. From the analyst's couch: TLR-targeted therapeutics. Nat Rev Drug Discov. 2005;4:879-880). These TLR agonists have strong potential in promoting the immunogenicity of weakly immunogenic TAAs. In fact, several peptide / protein-based cancer vaccines combined with TLR agonists are being tested in clinical trials. These include Ampligen (NCT01355393), which targets TLR3; Histonol (NCT00773097, NCT01585350, NCT01437605), which targets TLR3; MELITAC 12.1 (NCT01585350), which targets TLR4; and Resiquimod (NCT00960752), which targets TLR9. The family of PRRs has expanded greatly in recent years, and therefore, significant efforts have been expended in investigating the role of innate immune pathways in defining the mechanisms of adjuvant action as well as the role of other PRRs (e.g., NLRs, RLRs) in the adjuvant activity of cancer therapeutic vaccines.

[0016] In addition to sensing pathogen-associated signals, PRPs also recognize endogenous "alarmins," such as stress / heat shock proteins (HSPs) and HMGB-1 (Lotze MT, Zeh HJ, Rubartelli A, Sparvero LJ, Amoscato AA, Washburn NR, Devera ME, Liang X, Tor M, Billiar T. The grateful dead: damage-associated molecular pattern molecules and reduction / oxidation regulate immunity. Immunol Rev. 2007;220:60-81; Todryk SM, Melcher AA, Dalgleish AG, Vile RG. Heat shock proteins refine the danger theory. Immunology. 2000;99:334-337). As intrinsic and highly conserved protein components of cells, these damage-associated molecular patterns (DAMPs) also communicate the nature and magnitude of cellular damage to the host immune system.HSPs are known to act as molecular chaperones involved in the quality control of intracellular proteins (Calderwood SK, Murshid A, Prince T. The shock of aging: molecular chaperones and the heat shock response in longevity and aging--a mini-review. Gerontology. 2009;55:550-558; Mayer MP, Bukau B. Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol Life Sci. 2005;62:670-684). Research over the past 20 years has established the concept that certain HSPs can integrate both innate and adaptive immune responses and can be used as immunostimulatory agents for immunotherapy (Mayer MP, Bukau B. Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol Life Sci. 2005;62:670-684; Wang XY, Facciponte JG, Subjeck JR. Molecular chaperones and cancer immunotherapy. Handb Exp Pharmacol. 2006b;172:305-329).

[0017] Although there have been significant advances in the rational design of vaccines, there is a continuing need for the development of vaccines that are optimized for both prophylactic and therapeutic use. There is a need for the development of vaccines that have broad applicability to large patient populations, and for such vaccines to be specific and effective. Summary of the Invention

[0018] Disclosed herein are novel compositions comprising HPV peptide sequences, optionally combined with one or more adjuvants, wherein the HPV peptide sequences correspond to HPV E6 peptide and / or HPV16 E7 peptide, and the peptide sequences have a binding affinity with five HLA supertypes with an IC50 of approximately 5000 nM, and some of the peptides are multi-epitope. In certain embodiments, the compositions include an adjuvant comprising a cationic lipid, and in certain embodiments, the cationic lipid comprises DDA, R-DOTAP, DOTAP, DOTMA, or DOEPC, or variations or analogs thereof. The novel compositions disclosed herein are superior to currently available vaccines in that they are effective in over 80-90% of the general population.

[0019] Disclosed herein is a method for inducing an immune response to HPV infection in a subject, comprising administering to the subject a composition comprising HPV peptide sequences, optionally in combination with an adjuvant, corresponding to HPV16 E6 peptide and / or HPV16 E7 peptide, and having a binding affinity with five HLA supertypes with an IC50 of about 5000 nM, and some of the peptides being multi-epitope. The method can be prophylactic or therapeutic. [Brief explanation of the drawings]

[0020] [Figure 1]Figure 1 provides a graph showing the interferon-γ assay by subject and visit anti-HPV16 E6 and E7 responses by 1 mg and 3 mg R-DOTAP cohorts. Abbreviations: Bkg = background (striped bars); PBMC = peripheral blood mononuclear cells; R-DOTAP = R-enantiomer of 1,2-dioleoyl-3-trimethylammonium-propane chloride; SFC = SFU = spot-forming units; Stim = stimulated (clear bars). Data represent the average SFU per 4 x 10 PBMC in triplicate wells. The striped bars represent background SFU in wells stimulated with medium alone. The clear bars represent SFU in wells stimulated with the peptide pool. Visit 2 = Day 1, before vaccination; Visit 3 = Day 15, 14 days after vaccination 1; Visit 5 = Day 36, 14 days after vaccination 2; Visit 7 = Day 57, 14 days after vaccination 3; Visit 9 = Day 133, 90 days after vaccination 3. [Figure 2] Figure 2 provides graphs showing interferon-γ assay by subject and visit anti-HPV16 E6 and E7 responses with the 10 mg R-DOTAP cohort. Data represent the mean SFU per 4 x 10 PBMC in triplicate wells. Striped bars represent background SFU in wells stimulated with medium alone. Clear bars represent SFU in wells stimulated with peptide pools. Visit 2 = Day 1, pre-vaccination; Visit 3 = Day 15, 14 days after vaccination 1; Visit 5 = Day 36, 14 days after vaccination 2; Visit 7 = Day 57, 14 days after vaccination 3; Visit 9 = Day 133, 90 days after vaccination 3. [Figure 3] Figure 3 provides sample tumor regression plots showing the results of peptide effects on clinically measured median tumor size after tumor implantation for peptides corresponding to SEQ ID NO: 1 (open squares), SEQ ID NOs: 7, 8, 31, 32, and 31 (filled circles), SEQ ID NO: 7 (open circles), and SEQ ID NO: 8 (filled triangles). In Figure 3, compositions containing only R-DOTAP are represented by plot lines marked with striped squares. DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed Description of the Invention The following detailed description is exemplary and explanatory and is intended to provide further explanation of the disclosure set forth herein. It is expected that other advantages and novel features will be readily apparent to those skilled in the art from the following detailed description of the disclosure. References mentioned herein, including U.S. Provisional Patent Application No. 62 / 404,458, are incorporated by reference in their entirety.

[0022] Disclosed herein are methods for the design and use of unique peptide sequences, including unique multi-epitope peptides derived from HPV16 E6 and E7, designed to be efficiently processed and cross-presented to T cells, as screened and demonstrated in HLA-A2 humanized transgenic mice and confirmed in various human subjects. In certain embodiments, novel compositions containing novel peptide sequences consist of 2-8 peptide sequences, 2-6 peptide sequences, or 4 peptide sequences. The peptides can be incorporated into immunogenic compositions, such as vaccines. As demonstrated below, in silico peptide binding analysis against major HLA supertypes confirms that the resulting compositions and vaccines address greater than 80-90% of the population. In non-HLA-restricted human clinical trials involving the use of the peptide compositions described herein, robust T cell induction was observed in all subjects. Utilizing the novel approach of in silico binding analysis combined with testing in humanized transgenic mice, we developed the first simple, effective, and broadly applicable peptide-based HPV16 cancer therapeutic vaccine.

[0023] An important consideration in the design of peptide-based vaccines designed to elicit CD8+ T cell generation and responses in humans is the polymorphism of HLA class I molecules in the population. Because different HLA alleles bind different peptides, it is important that peptide vaccines contain enough different peptides to be immunogenic in a high proportion of the population. Here, the inventors designed a novel multipeptide vaccine to cover the immunogenic regions of the HPV16 E6 and E7 proteins to provide correct processing and presentation of CD8+ T cell epitopes in humans. In one embodiment, the vaccine contains four HPV-associated peptides selected based on their binding and immunogenic activities. As detailed in the Examples, the inventors used ELISpot and tumor regression studies, as well as in silico analysis, to predict potential HLA alleles that could bind various HPV peptides. Next, the ability of the human immune system to present and recognize peptide epitopes was examined in humanized transgenic mice, confirming presentation, processing, and subsequent induction of antigen-specific CD8+ T cell responses. Finally, the ability to induce human T cell responses was investigated in a non-HLA-restricted human clinical trial, and the ability of the formulation to induce strong T cell responses in subjects with various HLA subtypes was confirmed.

[0024] In one embodiment, the novel compositions described herein comprise an HPV peptide sequence in combination with an adjuvant, wherein the HPV peptide sequence corresponds to the HPV16 E6 peptide and / or the HPV16 E7 peptide, and the peptide sequence has a binding affinity with five HLA supertypes with an IC50 of about 5000 nM. In a specific embodiment, the peptide is multi-epitope. The peptide may include SEQ ID NOs: 5, 9, 10, and 11, or lipidated versions thereof including SEQ ID NOs: 23, 24, 25, and 26. In one embodiment, the peptides may be present in the composition as individual peptides, or may be conjugated to each other (in any order) with or without spacers to form a single long peptide encompassing the claimed sequence by methods known to those skilled in the art. In certain embodiments, the HLA supertypes include HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-B*07:02, and HLA-B*58:01. In some embodiments, the compositions may further comprise enhancer agonist epitopes, such as HBV core helper peptides, and / or single epitope peptides, including, but not limited to, peptides encoded by SEQ ID NO: 14, or analogs thereof, such as SEQ ID NO: 15 and SEQ ID NOs: 27 and 28. The compositions described herein further include modified peptides, peptide analogs, and active fragments thereof. In certain embodiments, peptides may be modified by oxidation, cross-linking via disulfide bonds, pegylation, glycosylation, phosphorylation, palmitoylation, methylation, biotinylation, or other methods known to those skilled in the art to improve efficacy and immunogenicity. In one embodiment, the adjuvant of the composition consists of a cationic lipid, wherein the cationic lipid may be selected from the group consisting of DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, and variations or analogs thereof.In one embodiment, the novel compositions of the present disclosure comprise peptides corresponding to SEQ ID NOs: 5, 9, 10, and 11, and the adjuvant comprises a cationic lipid, wherein the cationic lipid comprises R-DOTAP. In one embodiment, the novel compositions of the present disclosure comprise peptides corresponding to SEQ ID NOs: 23, 24, 25, or 26, and the adjuvant comprises a cationic lipid, wherein the cationic lipid comprises R-DOTAP. In one embodiment, the novel compositions of the present disclosure comprise peptides corresponding to SEQ ID NOs: 5, 9, 10, 11, 23, 24, 25, or 26, and the adjuvant comprises a cationic lipid, wherein the cationic lipid comprises R-DOTAP. The above embodiments may optionally be encapsulated in liposomes. The above embodiments may optionally be combined with pharmaceutically acceptable carriers and excipients, various buffers such as acetate, phosphate, and tonicity agents such as sucrose, trehalose, or surfactants such as Tween, and others known to those of skill in the art.

[0025] In one embodiment, the disclosure herein provides a recombinant vector comprising a nucleic acid molecule encoding a polynucleotide comprising one or more of SEQ ID NOs: 5, 9, 10, 11, or one or more of SEQ ID NOs: 23, 24, 25, or 26, operably linked to a promoter. Also provided is a recombinant vector comprising a nucleic acid molecule encoding a peptide comprising one or more of SEQ ID NOs: 5, 9, 10, 11, or one or more of SEQ ID NOs: 23, 24, 25, or 26, operably linked to a promoter further comprising a nucleic acid molecule encoding an enhancer for the epitope and / or single epitope peptide. In one embodiment, the vector may comprise a recombinant adenovirus. As known to those skilled in the art, nucleic acid sequences can be cloned using conventional molecular biology techniques or synthesized de novo by DNA synthesis, which may be performed using conventional procedures by commercial companies in the fields of DNA synthesis and / or molecular cloning.

[0026] In one embodiment, the present disclosure provides a method for inducing an immune response against HPV infection in a subject, comprising administering to the subject a novel composition comprising an HPV peptide sequence in combination with one or more adjuvants, wherein the HPV peptide sequence corresponds to HPV16 E6 peptide and / or HPV16 E7 peptide, and the peptide sequence has a binding affinity with five HLA supertypes with an IC50 of approximately 5000 nM, and some of the peptides are multi-epitope. The methods for inducing an immune response disclosed herein can include inducing an immune response for prophylactic or therapeutic purposes. In certain embodiments, the peptides used in the novel compositions can include one or more peptides selected from the group consisting of SEQ ID NOs: 5, 9, 10, 11, 23, 24, 25, 26, 31, 32, or others listed herein, e.g., in Tables 1-5. The peptides may be present as individual peptides, or specific selected peptides may be conjugated to each other (in any order) with or without spacers to form a single long peptide encompassing the claimed sequences. The method may include the use of a composition in which the adjuvant comprises a cationic lipid, for example, the cationic lipid comprises DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, S-DOTMA, variations or analogs thereof. In one embodiment, a method for inducing an immune response may include administering to a subject a composition comprising a peptide consisting of SEQ ID NO:5, 9, 10, 11 and R-DOTAP, or a composition comprising a peptide consisting of SEQ ID NO:23, 24, 25, or 26 and R-DOTAP.

[0027] The methods described herein can include the use of novel HPV peptide compositions to treat a subject having an HPV infection, including conditions including, but not limited to, common warts, plantar warts, flat warts, genital warts, anogenital warts, anal dysplasia, genital cancer (vulva, vagina, cervix, penis, anus), head and neck cancer, epidermodysplasia verruciformis, focal epithelial hyperplasia, oral papilloma, oropharyngeal cancer, bullous cyst, and laryngeal papillomatosis.

[0028] For example, it will be understood by those skilled in the art that, using standard molecular biology techniques, modifications can be made to a peptide, e.g., by amino acid substitution, deletion, addition, etc. In general, conservative amino acid substitutions can be made without loss of function or immunogenicity of the polypeptide, which can be confirmed according to standard procedures well known to those skilled in the art.

[0029] The scope of the peptides described herein also includes modifications of the peptides and peptide fragments. Such modifications include, but are not limited to, the substitution of natural amino acids at specific positions with other molecules, including natural and unnatural amino acids. Such substitutions may alter the biological activity of the peptide and generate biological or pharmacological agonists or antagonists. Such substitutions may include conservative substitutions known to those of skill in the art, such as valine for alanine. Acceptable substitutions may also include amino acid modifications, such as norleucine for leucine. It will be understood that substitutions of D amino acids for L amino acids are encompassed within the scope of the present invention. Some substitutions are described in Dictionary of Biochemistry and Molecular Biology, 2nd ed., J. Stenesh, John Wiley & Sons, 1989, which is incorporated herein by reference in its entirety. Further modifications include the addition of amino acids such as tyrosine or other amino acids at specific positions in the peptide or fragment thereof to enhance labeling ability with radioactive and non-radioactive labels, the addition of molecules such as lysine, and the addition of radioactive and / or non-radioactive labels.

[0030] Furthermore, one skilled in the art will recognize that individual substitutions, deletions, or additions in the amino acid sequences of the disclosed peptides or in the nucleotide sequences encoding the amino acids in the peptides, altering, adding, or deleting a single amino acid or a small number of amino acids in the encoded sequence (generally less than 5%, more usually less than 1%) are conservatively modified variations, where the modification results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are known in the art. The following six groups each contain amino acids that are conservative substitutions for one another: 1) alanine (A), serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M); Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W)

[0031] Lipid adjuvants Cationic lipids have been reported to have strong immunostimulatory adjuvant effects. The cationic lipids of the present invention can form liposomes that are optionally mixed with antigens and can contain cationic lipids alone or in combination. Suitable cationic lipid species include: 3-β[ 4 N-( 1 N, 8 -diguanidinospermidine)-carbamoyl] cholesterol (BGSC); 3-β[N,N-diguanidinoethyl-aminoethane)-carbamoyl] cholesterol (BGTC); N,N 1 N 2 N 3Tetra-methyltetrapalmitylspermine (Cellfectin); Nt-butyl-N'-tetradecyl-3-tetradecyl-aminopropane-amidine (CLONfectin); Dimethyldioctadecylammonium bromide (DDAB); 1,2-Dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE); 2,3-Dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate) (DOSPA); 1,3-Dioleoyloxy-2-(6-carboxyspermyl)-propylamide (DOSPER); 4-(2,3-bis-palmitoyloxy-propyl)-1-methyl-1H-imidazole (DPIM); N,N,N',N'-tetramethyl-N,N'-bis(2-hydroxyethyl)-2,3 Dioleoyloxy-1,4-butanediammonium iodide) (Tfx-50); N-1-(2,3-dioleyloxy)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'-trimethylammonium) butanoate (ChOTB) where the trimethylammonium group is attached to either the duplex (in the case of DOTB) or the cholesteryl group (in the case of ChOTB) via a butanol spacer arm; DORI (DL-1,2-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) or DORIE (DL-1,2-O-dioleoyl-3-dimethylaminopropyl-β-hydroxyethylammonium) (DORIE) or analogs thereof described in WO 93 / 03709; 1,2-dioleoyl-3-succinyl-sn-glycerol choline ester (DOSC); cholesteryl hemisuccinate ester (ChOSC);Lipopolyamines, such as dioctadecylamidoglycylspermine (DOGS) and dipalmitoylphosphatidylethanolamylspermine (DPPES) or cationic lipids described in U.S. Pat. No. 5,283,185, cholesteryl-3β-carboxyl-amido-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesterylcarboxylate iodide, cholesteryl-3-O-carboxyamidoethyleneamine, cholesteryl-3-β-oxysuccinamido-ethylenetrimethylammonium iodide, 1-dimethylamino-3-trimethylammonio-DL-2-propyl-cholesteryl-3-β-soxysuccinate iodide, 2-(2-trimethylammonio)-ethylmethylaminoethyl-cholesteryl-3-β-oxysuccinate iodide, 3-β-N-(N',N'-dimethylaminoethane) Carbamoyl cholesterol (DC-chol), and 3-β-N-(polyethyleneimine)-carbamoyl cholesterol; O,O'-Dimyristyl-N-lysyl aspartate (DMKE); O,O'-Dimyristyl-N-lysyl glutamate (DMKD); 1,2-Dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE); 1,2-Dilauroyl-sn-glycero-3-ethylsulfocholine (DLEPC); 1,2-Dimyristoyl-sn-glycero-3-ethylsulfocholine (DMEPC); 1,2-Dioleoyl-sn-glycero-3-ethylsulfocholine (DOEPC); 1,2-Dipalmitoyl-sn-glycero-3-ethylsulfocholine (DPEPC); 1,2-Distearoyl-sn-glycero-3-ethylsulfocholine (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). The present invention contemplates the use of structural variants and derivatives of the cationic lipids disclosed herein.

[0032] A particular embodiment of the present invention is a compound of the formula: [ka] (In the formula, R 1 is a quaternary ammonium group, and Y 1 is selected from a hydrocarbon chain, an ester, a ketone, and a peptide; R 2 and R 3 are independently selected from saturated fatty acids, unsaturated fatty acids, ester-linked hydrocarbons, phosphate diesters, and combinations thereof DOTAP, DMTAP, DSTAP, DPTAP, DPEPC, DSEPC, DMEPC, DLEPC, DOEPC, DMKE, DMKD, DOSPA, and DOTMA are examples of lipids with this general structure.

[0033] In one embodiment, the chiral cationic lipid of the present invention is a lipid in which the bond between the hydrophilic group and the amino group is stable in aqueous solution. Therefore, a characteristic of the complexes of the present invention is their stability during storage (i.e., their ability to maintain a small diameter and maintain physiological activity over time after their formation). Such bonds used in cationic lipids include amide bonds, ester bonds, ether bonds, and carbamoyl bonds. Those skilled in the art can easily understand that liposomes containing two or more cationic lipid species can be used to prepare the complexes of the present invention. For example, liposomes containing two cationic lipid species, lysyl-phosphatidylethanolamine and β-alanyl cholesterol ester, have been disclosed for specific drug delivery (Brunette, E. et al., Nucl. Acids Res., 20:1151 (1992)).

[0034] In considering chiral cationic liposomes suitable for use in the present invention, and optionally for mixing with an antigen, it can be further understood that the methods of the present invention are not limited to the use of only the above-mentioned cationic lipids, but that any lipid composition can be used so long as the cationic liposomes produced have a resulting cationic charge density sufficient to activate and induce an immune response.

[0035] Thus, the lipids of the present invention can include other lipids in addition to cationic lipids, including, but not limited to, lysolipids, such as lysophosphatidylcholine (1-oleoyllysophosphatidylcholine), cholesterol, or neutral phospholipids including dioleoylphosphatidylethanolamine (DOPE) or dioleoylphosphatidylcholine (DOPC), and various lipophilic surfactants containing a polyethylene glycol moiety, such as Tween-80 and PEG-PE.

[0036] The cationic lipid of the present invention can also include negatively charged lipids and cationic lipids, as long as the net charge of the complex formed is positive and / or the surface of the complex is positively charged.The negatively charged lipid of the present invention comprises at least one lipid species or a combination thereof that has a net negative charge at or near physiological pH.Suitable negatively charged lipid species include, but are not limited to, CHEMS (cholesteryl hemisuccinate), NGPE (N-glutaryl phosphatidylethanolamine), phosphatidylglycerol and phosphatidic acid or similar phospholipid analogs.

[0037] Methods for producing liposomes for use in preparing the lipid-containing drug delivery complexes of the present invention are well known to those skilled in the art. Overviews of liposome preparation methodologies can be found in "Liposome Technology" (CFC Press, New York, 1984); "Liposomes by Ostro" (Marcel Dekker, 1987); "Methods Biochem Anal. 33:337-462 (1988)" and U.S. Patent No. 5,283,185. Such methods include freeze-thaw extrusion and sonication. Both unilamellar liposomes (average diameter less than about 200 nm) and multilamellar liposomes (average diameter greater than about 300 nm) can be used as starting components for preparing the complexes of the present invention.

[0038] In cationic liposomes utilized to produce the cationic lipid vaccines of the present invention, the cationic lipid is present in the liposome at about 10 mol % to about 100 mol %, or about 20 mol % to about 80 mol % of the total liposomal lipids. Neutral lipids, when present in the liposome, may be present at a concentration of about 0 mol % to about 90 mol %, or about 20 mol % to about 80 mol %, or 40 mol % to 80 mol % of the total liposomal lipids. Negatively charged lipids, when present in the liposome, may be present at a concentration ranging from about 0 mol % to about 49 mol %, or about 0 mol % to about 40 mol % of the total liposomal lipids. In one embodiment, the liposome contains cationic and neutral lipids in a ratio of about 2:8 to about 6:4. It is further understood that the complexes of the present invention may contain modified lipids, proteins, polycations, or receptor ligands that function as targeting factors to direct the complex to specific tissues or cell types. Examples of targeting factors include, but are not limited to, monoclonal and polyclonal antibodies against acyloglycoproteins, insulin, low-density lipoprotein (LDL), folic acid, and cell surfaces. Additionally, to modify the circulatory half-life of the conjugate, positive surface charges can be sterically blocked by incorporating lipophilic surfactants containing polyethylene glycol moieties.

[0039] Cationic lipid vaccines can be stored in an isotonic sucrose or dextrose solution upon recovery from the sucrose gradient, or they can be lyophilized and then reconstituted in an isotonic solution before use. In one embodiment, the cationic lipid complexes are stored in solution. The stability of the cationic lipid complexes of the present invention is measured by specific assays to determine the physical stability and biological activity of the cationic lipid vaccine over time during storage. The physical stability of the cationic lipid vaccine can be measured by methods well known to those skilled in the art, such as electron microscopy, gel filtration chromatography, or by measuring the diameter and charge of the cationic lipid complexes by quasi-elastic light scattering, such as with a Coulter N4SD particle size analyzer, as described in the Examples. The physical stability of the cationic lipid complexes, as determined at the time the cationic lipid vaccine is purified, is considered "substantially unchanged" upon storage if the diameter of the stored cationic lipid vaccine does not increase by more than 100%, or by no more than 50%, or by no more than 30% over the diameter of the cationic lipid complex.

[0040] While cationic lipids can be administered in pure or substantially pure form, it is preferable to present them as pharmaceutical compositions, formulations, or preparations. Pharmaceutical compositions using the chiral cationic lipid complexes of the present invention can include the cationic lipid vaccine in a physiologically compatible sterile buffer, such as phosphate-buffered saline, isotonic saline, or a low ionic strength buffer such as acetate or Hepes (exemplary pH ranges from about 5.0 to about 8.0). The chiral cationic lipid vaccine can be administered as an aerosol or as a solution for intratumoral, intraarterial, intravenous, intratracheal, intraperitoneal, subcutaneous, and intramuscular administration.

[0041] The formulations of the present invention may incorporate any stabilizer known in the art. Exemplary stabilizers are cholesterol and other sterols, which can stiffen the liposome bilayer and help prevent bilayer collapse or destabilization. Agents such as polyethylene glycol, poly-, and mono-saccharides can also be incorporated into liposomes to modify the liposome surface and prevent it from being destabilized due to interactions with blood components. Other exemplary stabilizers are proteins, saccharides, inorganic acids, or organic acids, which can be used either alone or in mixtures.

[0042] Many pharmaceutical methods can be used to control, modify, or extend the duration of immune stimulation. Controlled-release preparations can be achieved by using polymer complexes, such as polyesters, polyamino acids, methylcellulose, polyvinyl, poly(lactic acid), and hydrogels, to encapsulate or entrap cationic lipids and slowly release them. Similar polymers can also be used to adsorb liposomes. To modify the release profile of the stimulating agent, liposomes can be incorporated into emulsion formulations. Alternatively, the duration of the stimulating agent's presence in the blood circulation can be enhanced by coating the surface of the liposomes with compounds such as polyethylene glycol or other polymers, as well as other substances, such as saccharides, that can enhance the circulation time or half-life of liposomes and emulsions.

[0043] If an oral formulation is required, the chiral cationic lipids can be combined with common pharmaceutical carriers known in the art, such as, for example, sucrose, lactose, methylcellulose, carboxymethylcellulose, or gum arabic. The cationic lipids can also be encapsulated in capsules or tablets for systemic delivery.

[0044] Administration of the chiral cationic lipid compositions of the present disclosure can be for either prophylactic or therapeutic purposes. When provided prophylactically, the cationic lipid is provided before any signs or symptoms of disease. When provided therapeutically, the cationic lipid is provided at or after the onset of disease. Therapeutic administration of an immunostimulant serves to alleviate or treat disease. For both purposes, the cationic lipid may be administered together with an additional therapeutic agent or antigen. For both purposes, the cationic lipid may be administered together with an additional therapeutic agent or antigen. When the cationic lipid is administered together with an additional therapeutic agent or antigen, a prophylactic or therapeutic effect may occur against certain diseases, including, for example, diseases or disorders caused by HPV.

[0045] The formulations of the present invention comprise only pure chiral cationic lipids as described above, as a mixture of R and S enantiomers, together with one or more therapeutic moieties, such as antigens or drug molecules, for both veterinary and human use. The formulations may conveniently be provided in unit dosage form and may be prepared by any method known in the pharmaceutical arts.

[0046] term The terms "a" or "an" refer to one or more. As such, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein.

[0047] The words "comprise", "comprises" and "comprising" are to be interpreted inclusively rather than exclusively. The words "consist", "consisting" and variations thereof are to be interpreted exclusive rather than inclusive.

[0048] As used herein, the term "about" means a 10% variability from the given reference, unless otherwise specified.

[0049] As used herein, the terms "subject" and "patient" are used interchangeably and include, for example, mammals such as humans, mice, rats, guinea pigs, dogs, cats, horses, cows, pigs, or non-human primates such as monkeys, chimpanzees, baboons, or gorillas.

[0050] As used herein, the terms "disease," "disorder," and "condition" are used interchangeably to refer to an abnormal state in a subject.

[0051] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art and by reference to publications which provide those skilled in the art with general guidance to many of the terms used in this application.

[0052] The compositions of the present disclosure comprise an amount of HPV peptides effective to produce an immunogenic response in a subject. Specifically, the dosage of the composition to achieve a therapeutic effect depends on factors such as the formulation, the pharmacological potency of the composition, the age, weight, and sex of the patient, the condition being treated, the severity of the patient's symptoms, the route of delivery, and the patient's response pattern. Treatment and dosage amounts of the composition may be administered in unit dosage forms, and those skilled in the art may adjust the unit dosage form accordingly to reflect the relative activity level. The determination of the specific dosage to be used (and the number of times administered daily) is within the discretion of those skilled in the art and may vary depending on the dosage titration for a particular situation to produce a therapeutic effect. Furthermore, those skilled in the art can calculate any changes in the effective amount of the composition due to changes in the composition components or dilution. In one embodiment, the composition may be diluted 2-fold. In another embodiment, the composition may be diluted 4-fold. In yet another embodiment, the composition may be diluted 8-fold.

[0053] Thus, an effective amount of the compositions disclosed herein can be about 1 mg to about 1000 mg per dose based on a 70 kg mammal, e.g., a human subject. In other embodiments, the therapeutically effective amount is about 2 mg to about 250 mg per dose. In further embodiments, the therapeutically effective amount is about 5 mg to about 100 mg per dose. In still other embodiments, the therapeutically effective amount is about 25 mg to 50 mg, about 20 mg, about 15 mg, about 10 mg, about 5 mg, about 1 mg, about 0.1 mg, about 0.01 mg, or about 0.001 mg.

[0054] An effective amount (when administered therapeutically) may be provided on a regular schedule, i.e., daily, weekly, monthly, or yearly, or on an irregular schedule that varies the administration days, weeks, months, etc. Alternatively, the therapeutically effective amount to be administered may vary. In one embodiment, the therapeutically effective amount for the first dose is higher than the therapeutically effective amount for one or more of the subsequent doses. In another embodiment, the therapeutically effective amount for the first dose is lower than the therapeutically effective amount for one or more of the subsequent doses. Equivalent dosages may be administered over various time periods, including, but not limited to, about every 2 hours, about every 6 hours, about every 8 hours, about every 12 hours, about every 24 hours, about every 36 hours, about every 48 hours, about every 72 hours, about every week, about every 2 weeks, about every 3 weeks, about every 2 months, about every 3 months, and about every 6 months. The number and frequency of doses corresponding to a completed course of treatment may be determined according to the judgment of a healthcare professional.

[0055] The composition may be administered by any route, taking into account the particular condition for which it is selected. The composition may be delivered orally by injection, inhalation (including orally, intranasally, intratracheally), ocularly, transdermally (via simple passive diffusion formulations or by enhanced delivery using, for example, iontophoresis, microporation with microneedles, radiofrequency ablation, or the like), intravascularly, cutaneously, subcutaneously, intramuscularly, sublingually, intracranially, epidurally, intrarectally, intravesically, and intravaginally, among others.

[0056] The compositions may be formulated neat or with one or more pharmaceutical carriers and / or excipients for administration. The amount of pharmaceutical carrier will depend on the solubility and chemical properties of the peptide, the selected route of administration, and standard pharmacological practice. The pharmaceutical carrier may be liquid or may incorporate both solid and liquid carriers / matrices. A variety of suitable liquid carriers are known and can be readily selected by those skilled in the art. Such carriers may include, for example, dimethyl sulfoxide (DMSO), saline, buffered saline, cyclodextrins, hydroxypropylcyclodextrin (HPβCD), n-dodecyl-β-D-maltoside (DDM), and mixtures thereof. Similarly, a variety of solid (hard or soft) carriers and excipients are known to those skilled in the art.

[0057] The composition can be administered alone or in the presence of one or more physiologically compatible pharmaceutical carriers. The carriers can be in dry or liquid form and must be pharmaceutically acceptable. Liquid pharmaceutical compositions can be sterile solutions or suspensions. When liquid carriers are used, they must be sterile liquids. Liquid carriers can be used to prepare solutions, suspensions, emulsions, syrups, and elixirs. In one embodiment, the composition can be dissolved in a liquid carrier. In another embodiment, the composition can be suspended in a liquid carrier. Those skilled in the formulation arts can select an appropriate liquid carrier depending on the route of administration. Alternatively, the composition can be formulated in a solid carrier. In one embodiment, the composition can be compressed into a unit dosage form, i.e., a tablet or caplet. In another embodiment, the composition can be incorporated into a unit dosage form, i.e., a capsule. In a further embodiment, the composition can be formulated for administration as a powder. The solid carrier can serve multiple functions, i.e., the function of two or more of the excipients described below. For example, solid carrier can also act as flavoring agent, lubricant, solubilizer, suspending agent, filler, glidant, compression aid, binder, disintegrating agent or encapsulating material.In one embodiment, solid carrier acts as lubricant, solubilizer, suspending agent, disintegrating agent or encapsulating material.Composition can also be subdivided to contain appropriate amount of composition.For example, unit dose can be packaged composition, for example, packaged powder, vial, ampoule, pre-filled syringe or sachet containing liquid.

[0058] In one embodiment, the composition can be administered by a modified-release delivery device. As used herein, "modified-release" refers to controlled delivery of the disclosed compositions, for example, over at least about 8 hours (e.g., extended delivery) to at least 12 hours (e.g., sustained delivery). Such devices can also provide immediate release (e.g., therapeutic levels achieved in less than about 1 hour, or less than about 2 hours). Suitable modified-release delivery devices are well known to those skilled in the art.

[0059] Kits containing the compositions disclosed herein are also provided. The kits may further include packaging or containers containing the compositions formulated for the delivery route. Suitably, the kits include instructions for administration and inserts regarding the compositions.

[0060] Numerous packages or kits are known in the art for dispensing pharmaceutical compositions for periodic use. In one embodiment, the package has markings for each period. In other embodiments, the package is a foil or blister package, a labeled ampoule, vial, or bottle.

[0061] The packaging means of the kit may be adapted for administration itself, such as an inhaler, syringe, pipette, eyedropper, catheter, cytoscope, trocar, cannula, pressure release device, or other such device by which the formulation may be applied to an affected area of ​​the body such as the lungs, infused into a subject, delivered to bladder tissue, or applied to and mixed with other components of the kit.

[0062] One or more components of these kits may also be provided in dried or lyophilized form. When an agent or component is provided as a dried form, reconstitution is generally by adding a suitable solvent. It is contemplated that the solvent may also be provided in a separate package. The kit may also include a means for receiving the vial or other suitable packaging means for commercial sale in tightly confined confinement, such as an injection- or blow-molded plastic container into which the vial is retained. Regardless of the number or type of packaging, and as noted above, the kit may also include or be packaged with a separate device for assisting in the injection / administration or placement of the composition into an animal's body. Such a device may be an inhaler, syringe, pipette, forceps, measuring spoon, eyedropper, catheter, cytoscope, trocar, cannula, pressure delivery device, or any such medically approved delivery means.

[0063] The terms "treat," "treating," or any variation thereof, are meant to include a therapy utilized to improve a health problem or condition in a patient or subject. In one embodiment, the health problem or condition may be resolved, either permanently or in the short term. In other embodiments, the severity of the health problem or condition, or one or more symptoms characteristic of the health problem or condition, may be reduced, either permanently or in the short term. The effectiveness of pain treatment may be determined using any standard pain index, such as those described herein, or may be determined based on the patient's subjective pain. A patient is considered "treated" if they report a decrease in pain or a decreased response to stimuli that would otherwise cause pain.

[0064] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the present invention in any way. On the contrary, it is clearly understood that the present invention can have various other embodiments, modifications, and equivalents thereof, which may suggest themselves to those skilled in the art after reading the description herein without departing from the essence of the present invention. [Example]

[0065] To facilitate a more complete understanding of the present invention, examples are provided below. To date, to facilitate the development of peptide vaccines for a wide range of patients, vaccines have been developed to contain overlapping peptide sequences of 15 to 30 amino acids covering the entire protein sequence of HPV. Although in silico peptide binding analysis is available, which can determine peptide binding to various HLA molecules, this approach has not been widely utilized in the design of HLA-independent vaccines because it is well established that not all peptides capable of binding to MHC molecules are naturally processed in cells, and the actual HLA coverage may be significantly lower than expected.

[0066] Therefore, an alternative approach was needed to successfully develop a simple HPV therapeutic vaccine that could provide broad patient coverage without utilizing a large number of peptides and without the need to completely represent the entire E6 and E7 protein sequence in the vaccine. The first step in the process developed herein to design a simpler formulation was the design of several libraries of peptides from the HPV16 E6 and E7 proteins. The next step was to conduct extensive in vivo studies to screen the peptides and confirm or understand the ability of the immune system to correctly process specific peptides and present the correct T epitopes via MHC class I and class II to CD8+ and CD4+ T cells, respectively. The only way to accurately obtain this information is through actual in vivo studies to evaluate T cell responses.

[0067] In this study, T cell responses were evaluated by tumor regression assays using the TC-1 tumor model in C57 / B6 mice and interferon-gamma ELISPOT assays in HLA-A2 humanized transgenic mice. The next step was to select appropriate or preferred peptides based on the resulting T cell immune responses and eliminate those that elicited no or very weak T cell responses. The selected active peptides were then analyzed by in silico binding analysis to determine their binding affinities for various HLA molecules. At the end of the combined in vivo and in silico analysis analysis, the goal was to select the minimum number of active peptide sequences from the in vivo studies that, when used in combination, could cover at least 90% of the human population, as predicted by the in silico analysis. Finally, the selected peptides were combined into a vaccine formulation. This formulation was then tested for T cell induction potency to confirm that the potency of the selected sequences had not been substantially reduced as a result of competition for binding sites and that T cell responses against all included peptides were still obtained. Finally, the predicted broad range of applications was confirmed in human clinical trials.

[0068] Herein, sequence selection was achieved using both tumor regression tests in C57 / B6 mice and interferon-gamma ELISPOT tests in HLA-A2 humanized transgenic mice. In peptide evaluation studies using tumor regression, a positive T cell response was a T cell response that resulted in tumor regression. In the HLA-A2 mouse model ELISPOT test, at least 20 spots per million splenocytes were considered a positive reaction.

[0069] Provided herein are summary results showing positive or negative T cell responses. The details provided will enable one skilled in the art to understand the importance of extensive screening of peptide sequences by in vivo means as a first step and to follow along with all steps of the invention.

[0070] Also provided herein are illustrative diagrams showing that peptide HLA binding predictions by in silico binding analysis confirm and correspond to at least 90% patient coverage. These diagrams allow anyone skilled in the art to correctly follow the process of first selecting potentially immunogenic E6 and E7 peptide sequences and then testing them in vivo to confirm processing and presentation. Third, selected sequences that are shown to be appropriately immunogenic in vivo are selected, and less immunogenic sequences are eliminated. Fourth, the peptides are subjected to in silico binding analysis to determine the binding affinity of each peptide to various HLA types. Finally, peptide combinations predicted to provide at least 90% coverage are selected. In the present case, human clinical trials were conducted to test and confirm the predictions.

[0071] The following examples demonstrate exemplary modes of making and practicing the invention. However, as alternative methods can be used to achieve similar results, the scope of the invention is not limited to the specific embodiments disclosed in these examples, which are illustrative only. Peptide sequences used to illustrate the invention are provided in Table 1. [Table 1]

[0072] Example 1 Screening of peptide sequences from TC-1 tumor regression in C57 / B6 mice This example illustrates the evaluation or screening of several HPV E7 peptides in the TC-1 mouse tumor regression model for their efficient processing and presentation of key epitopes that lead to HPV-specific T cell induction and subsequent tumor regression.

[0073] In tumor regression screening studies, mice were implanted with 100,000 TC-1 cells on day 0. On day 7, once tumors were well established, mice were vaccinated with specific peptide sequences formulated either alone or in combination with cationic lipids such as DOTMA, DOEPC, or R-DOTAP to promote uptake and presentation. In these studies, vaccines contained selected peptides at concentrations of 0.1–3.0 mg / mL and cationic liposomes at concentrations of 0.2–3.0 mg / mL. When the peptide sequence was lipidated by attaching a palmitoyl chain, the peptide was simply mixed with the cationic liposomes before vaccination. In the case of non-lipidated peptides, liposomes were prepared and the peptides were encapsulated by conventional thin-film liposome preparation methods (F Szoka, and D Papahadjopoulos, Comparative Properties and Methods of Preparation of Lipid Vesicles (Liposomes), Annual Review of Biophysics and Bioengineering, Vol. 9:467-508, 1980). Other methods known to those skilled in the art can also be used.

[0074] Sample tumor regression plots showing results for peptides corresponding to SEQ ID NO: 1 (open squares), SEQ ID NOs: 7, 8, 31, 32, and 31 (filled circles), SEQ ID NO: 7 (open circles), and SEQ ID NO: 8 (filled triangles) are shown in Figure 3. The results highlight the fact that some HPV E7 sequences resulted in effective tumor regression, while others did not, likely due to an ineffective ability to present the required CD8+ T cell epitopes. The results of the selection are summarized in Table 1.

[0075] Example 2 Peptide sequence screening by IFN-G ELISPOT assay in HLA-A2 mice This example highlights an approach for assessing T cell responses using ELISPOT assays in humanized HLA-A2 transgenic mice. In these assays, the vaccine contained selected peptides at concentrations of 0.1–3.0 mg / mL and cationic liposomes at concentrations of 0.2–3.0 mg / mL at the time of use. Humanized HLA-A2 transgenic mice, which possess components of the human immune system capable of recognizing human antigens, were inoculated with 100 μL of vaccine on days 0 and 7. Mice were sacrificed on day 14, and HPV-specific immune responses were assessed by interferon-gamma ELISPOT assay using standard techniques using splenocytes from mice (4–8 per group). Splenocytes were stimulated with specific CD8+ T cell epitopes or long multiepitope peptides identified in Table 2. For specific examples, the number of IFN-g spots per million cells is listed. A minimum of 20 spots was the cutoff for sufficient titer and selection for binding epitope testing. The ability of humanized transgenic mice to successfully generate T cell immune responses (>20 spots) when stimulated with both long multi-epitope peptides and short single-epitope peptides confirms that the selected peptides are effectively processed and presented to T cells. In these studies, unstimulated splenocytes and splenocytes stimulated with an irrelevant peptide were used as negative controls in both assays. ConA was used as a positive control in the assays.

[0076] [Table 2]

[0077] Example 3 Effect of chemical modifications such as oxidation and selected sequence multimerization on the immunogenicity of HPV16 vaccines This study was conducted to evaluate the effect of chemical modification of selected peptides on their immunogenicity and their ability to be processed, presented, and stimulate human CD8+ HPV-specific T cell responses. The peptides corresponding to SEQ ID NOs: 23 and 25 contain cysteines, which upon oxidation result in the formation of dimers and other multimers. Two vaccine formulations containing the peptides corresponding to SEQ ID NOs: 23 and 25 were prepared.

[0078] In Formulation A: Both peptides were monomers of high purity as confirmed by HPLC analysis. The vaccine contained 1.5-1.6 mg / mL of each peptide.

[0079] In Formulation B: Significant oxidation and multimerization of both peptides was confirmed by HPLC analysis.

[0080] Both formulations were mixed 1:1 with 5.8 mg / mL R-DOTAP before vaccination.

[0081] The immunogenicity of the peptides was compared in 10 HLA-A2 mice per formulation by assessing their ability to induce CD8+ T cells, as described in the previous examples. ELISPOT tests were performed as described above. Table 3 below shows the results of the ELISPOT tests and comparisons using a T-test. Statistical analysis results show that the difference between formulation A and formulation B did not meet a p-value of P<0.05 for significance. This test suggests that modifications such as oxidation do not adversely affect the immunogenicity of the peptide sequences.

[0082] [Table 3]

[0083] Example 4 Summary of in silico analysis of potential HLA alleles capable of binding to HPV peptides present in a multi-epitope peptide vaccine containing four selected peptide sequences Several HPV E16 and E17 peptide sequences were designed and evaluated in vivo for their ability to be efficiently processed in humanized HLA-A2 mice and for the specific epitopes presented to stimulate antigen-specific T cells, as described in the Examples above. As will be apparent to those skilled in the art, peptide-epitope interactions are complex, depending on numerous factors, including stereochemistry, the presence of cofactors, and the biochemical properties of the environment. Therefore, selecting appropriate peptides for optimal efficacy in a vaccine is not a routine or predictable exercise. Based on extensive in vivo testing to identify appropriate peptide sequences capable of efficient processing and presented T cell epitopes, selected peptides were further selected based on their ability to induce regression of established tumors or induce IFN-gamma-induced HPV-specific T cells, and then analyzed by in silico binding analysis to assess their HLA coverage. These predictions were then confirmed in human clinical trials.

[0084] The HLA supertype, HLA-A2, accounts for approximately 42% of the population (Sette, A. and J. Sidney, Nine major HLA class I supertypes account for the vast preponderance of HLA-A and -B polymorphism. Immunogenetics, 1999. 50(3-4): pp. 201-12). HPV16 E6 and E7 express experimentally confirmed epitopes that can be presented by HLA2 A2, as well as epitopes that can be presented by other HLA types.

[0085] To do this, peptide-binding affinities were assessed for nine different HLA molecules representing the nine major HLA supertypes (Sette, A. and J. Sidney, HLA supertypes and supermotifs: a functional perspective on HLA polymorphism. Curr Opin Immunol, 1998. 10(4): pp. 478-82), which account for more than 98% of the peptide-binding capacity of the human population (Sette, A. and J. Sidney, Nine major HLA class I supertypes account for the vast preponderance of HLA-A and -B polymorphism. Immunogenetics, 1999. 50(3-4): pp. 201-12). Extensive in vivo screening studies leading to the identification of sequences suitable for assessing HLA binding, as well as confirmatory studies to ensure that competition for binding sites does not reduce the immune system's ability to process and present specific T cell epitopes when combined in a vaccine formulation, are described in the experimental examples provided herein. This work demonstrates that although peptide selection and screening techniques are available, the process of identifying, selecting, and combining peptides to optimize the process for creating an effective vaccine with broad applicability is neither general nor straightforward.

[0086] [Table 4]

[0087] For purposes of this disclosure, Table 4 summarizes the results of peptide binding analysis of four preferred peptide sequences identified by T cell induction studies, as well as the potential epitopes covered by those sequences using the epitope prediction tool present in the Immune Epitope Database (www.iedb.org). This tool calculates the predicted binding affinity (IC50) of a peptide to a specific HLA class I allele. An IC50 of 5,000 nM or less is generally reported to be sufficient for biologically relevant binding and presentation, with binding affinities below 500 nM considered to represent high affinity binding. In the current analysis, 2,000 nM was selected as the cutoff for evaluating potential HLA-binding peptides. As shown in Table 4, at least five different HLA molecules representing five different HLA supertypes are found to have the potential for biologically significant binding and presentation of different peptides within SEQ ID NOS: 5, 9, 10, and 11 or 23-26. These five supertypes are known to represent over 90% of the human population, regardless of ethnicity (Sette, A. and J. Sidney, Nine major HLA class I supertypes account for the vast preponderance of HLA-A and -B polymorphism. Immunogenetics, 1999. 50(3-4): pp. 201-12).

[0088] Example 5 Compatibility of other cationic lipids beyond R-DOTAP with HPV-E6 and E7 peptide sequences and their ability to induce HPV-specific T cell responses To determine whether the identified long HPV peptides are compatible with cationic lipids other than DOTAP, the peptide corresponding to SEQ ID NO: 26 was used as a model peptide in combination with two other cationic lipids, DOTMA and DOEPC. To assess the ability of the peptides to be efficiently processed and presented in the presence of both cationic lipids, ELISPOT assays were performed using normal C57 / B6 mice. ELISPOT assays were performed as described above and used to determine effective CD8+ T cell induction.

[0089] In Formulation 1, 1.35 mg / mL of DOTMA was mixed 1:1 v / v with 0.5 mg / mL of the peptide corresponding to SEQ ID NO:26.

[0090] In formulation 2, 1.67 mg / mL of DOEPC was mixed 1:1 v / v with 0.5 mg / mL of the peptide corresponding to SEQ ID NO:26.

[0091] For each formulation, four mice were vaccinated on days 0 and 7. On day 14, mice were sacrificed and splenocytes were used for ELISPOT assays.

[0092] [Table 5]

[0093] This study demonstrates that various cationic lipids can be used in conjunction with the identified HPV E6 and E7 peptides to induce strong HPV-specific T cell responses.

[0094] Example 8 Use of lipidated peptides (SEQ ID NOS: 23-28) and R-DOTAP cationic lipids in an HPV therapeutic vaccine and evaluation of HPV-specific T cell responses in human clinical trials A six-peptide formulation containing four peptides (SEQ ID NOS: 23-26) predicted in Examples 1-4 that provide >90% population coverage based on in vivo T cell responses and peptide binding, and two additional single epitope peptides (SEQ ID NOS: 27 and 28) already contained in SEQ ID NOS: 24 and 25, was evaluated in a human clinical trial (ClinicalTrials.gov number NCT02065973). The two single epitope peptides included in the formulation do not provide additional coverage, as they are already contained in two of the longer peptide sequences included.

[0095] In this exploratory study, due to the diverse nature of biological systems and human responses, immune responses to the vaccine were evaluated by both IFN-γ and granzyme-b ELISPOT assays. Each subject received three vaccinations with the R-DOTAP / peptide formulation. All subjects received three vaccinations, one every three weeks. For immune monitoring by ELISPOT, blood samples were collected before vaccination (baseline), 14 to 19 days after each vaccination, and 90 days after the final vaccination. To confirm broad vaccine coverage, subjects were not restricted by HLA type. ELISPOT assays were performed using subject PBMCs stimulated with a six-peptide mixture to determine effective presentation and T cell recognition of HPV-peptides.

[0096] The results of the human clinical trials are shown in Table 6 below. A vaccine-induced response is generally defined as a two- or three-fold increase in immune response over baseline. In this study, an immune response was defined as a three-fold or greater increase in T cell response after vaccination compared to baseline samples by either IFN-γ or granzyme-β analysis. Two subjects, subjects 2 and 5, who showed very strong T cell responses of over 420 IFN-γ spots at baseline (the immune system likely responding to a recent HPV infection), were considered outliers (Table 6). All subjects were tested for HLA type. Half of the subjects were HLA-A2, as expected, since HLA-A2 is the most common HLA type. However, all subjects, including non-HLA-A2 subjects (HLA-A1, 30, 3, 74, 80, etc.), developed strong T cell responses to the vaccine. This study confirmed that the combination of four multi-epitope peptides, SEQ ID NOs: 23-26, provides a broadly applicable human HPV16 vaccine that can be recognized by patients of various genetic backgrounds.

[0097] [Table 6]

Claims

1. 1. A pharmaceutical composition for use in eliciting an immune response against HPV infection, comprising at least two HPV multi-epitope peptide antigens and an adjuvant, wherein the at least two HPV multi-epitope peptide antigens comprise two different HPV peptide antigens, wherein at least one HPV peptide antigen is selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 23, and at least one HPV peptide antigen is selected from the group consisting of SEQ ID NOs: 5, 9, 10, 11, 23, 24, 25, and 26, and the adjuvant is a cationic lipid.

2. 2. The pharmaceutical composition of claim 1, wherein the HPV multi-epitope peptide antigen comprises a peptide consisting of SEQ ID NO: 5, a peptide consisting of SEQ ID NO: 9, a peptide consisting of SEQ ID NO: 10, and a peptide consisting of SEQ ID NO:

11.

3. 3. The pharmaceutical composition of claim 1, wherein the HPV multi-epitope peptide antigens are present in the composition as individual peptides.

4. 3. The pharmaceutical composition of claim 1, wherein the HPV multi-epitope peptide antigens are conjugated to each other with or without a spacer to form a single long peptide encompassing the sequence.

5. 2. The pharmaceutical composition of claim 1, wherein the cationic lipid is selected from DOTAP, DDA, DOEPC, DOTMA, R-DOTAP, R-DDA, R-DOEPC, R-DOTMA, S-DOTAP, S-DDA, S-DOEPC, or S-DOTMA.

6. 2. The pharmaceutical composition of claim 1, wherein each of the HPV multi-epitope peptide antigens has binding affinity to at least one HLA supertype selected from HLA-A*02:01, HLA-A*03:01, HLA-A*24:02, HLA-B*07:02, and HLA-B*58:

01.

7. The pharmaceutical composition of claim 1, further comprising an enhancer agonist epitope.

8. 2. The pharmaceutical composition of claim 1, further comprising at least one single epitope peptide antigen selected from the peptide consisting of SEQ ID NO: 14 and the peptide consisting of SEQ ID NO:

15.

9. 2. The pharmaceutical composition of claim 1, wherein the HPV multi-epitope peptide antigen comprises a peptide consisting of SEQ ID NO: 5, a peptide consisting of SEQ ID NO: 9, a peptide consisting of SEQ ID NO: 10, and a peptide consisting of SEQ ID NO: 11, and the cationic lipid comprises R-DOTAP.

10. 2. The pharmaceutical composition of claim 1, wherein the cationic lipid comprises R-DOTAP.

11. 2. The pharmaceutical composition of claim 1, wherein the HPV infection is common warts, plantar warts, flat warts, genital warts, anogenital warts, anal dysplasia, vulvar cancer, vaginal cancer, cervical cancer, anal cancer, penile cancer, head and neck cancer, epidermodysplasia verruciformis, focal epithelial hyperplasia, oral papilloma, oropharyngeal cancer, bullous cyst, or laryngeal papillomatosis.

12. 2. The pharmaceutical composition of claim 1, wherein the peptide antigen comprises a peptide consisting of SEQ ID NO: 23, a peptide consisting of SEQ ID NO: 24, a peptide consisting of SEQ ID NO: 25, and a peptide consisting of SEQ ID NO:

26.

13. 2. The composition of claim 1, further comprising at least one single epitope peptide antigen selected from the peptide consisting of SEQ ID NO: 27 and the peptide consisting of SEQ ID NO:

28.

14. The composition of claim 1, wherein the HPV multi-epitope peptide antigen comprises a peptide consisting of SEQ ID NO: 23, a peptide consisting of SEQ ID NO: 24, a peptide consisting of SEQ ID NO: 25, and a peptide consisting of SEQ ID NO: 26, and the cationic lipid comprises R-DOTAP.

15. 2. The pharmaceutical composition of claim 1, comprising at least two HPV multi-epitope peptide antigens and an adjuvant, wherein one of the at least two HPV multi-epitope peptide antigens is a peptide consisting of SEQ ID NO: 11, and one of the at least two HPV multi-epitope peptide antigens is selected from a peptide consisting of SEQ ID NO: 5, a peptide consisting of SEQ ID NO: 9, and a peptide consisting of SEQ ID NO: 10, and the adjuvant comprises a cationic lipid.

16. 2. The pharmaceutical composition of claim 1, comprising at least two HPV multi-epitope peptide antigens and an adjuvant, wherein the at least two HPV multi-epitope peptide antigens are selected from a peptide consisting of SEQ ID NO: 23, a peptide consisting of SEQ ID NO: 24, a peptide consisting of SEQ ID NO: 25, and a peptide consisting of SEQ ID NO: 26, and the adjuvant comprises a cationic lipid.

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