T cell-inducing anticancer vaccine against commensal viruses to treat mucosal cancers

Abstract

An immune-based approach to treat and reduce the risk of mucosal cancer by boosting T cell immunity against commensal HPV.

Description

[Technical field]

[0001] Claiming priority This application claims the benefit of U.S. Provisional Patent Application Publication No. 63 / 140,159, filed January 21, 2021. The entire contents of the foregoing are incorporated herein by reference.

[0002] Immune-based approaches to treat and prevent mucosal cancer by boosting T cell immunity against commensal HPV. [Background technology]

[0003] Mucosal cancer, including mucosal squamous cell carcinoma (mSCC), is a common type of cancer affecting the upper aerodigestive tract, anus, and cervix. 1 High-risk human papillomaviruses (HPV) are responsible for a proportion of head and neck cancers (HNC), anal cancer, and cervical cancer. Summary of the Invention

[0004] As shown herein, a vaccine based on commensal HPV is beneficial in protecting against mucosal cancers.

[0005] Thus, provided herein is a method of treating or reducing the risk of developing mucosal cancer in a subject, comprising administering to the subject an effective amount of a composition comprising (i) a plurality of antigenic peptides, each of which comprises a sequence of 9 to 30 amino acids derived from a protein derived from a commensal human papillomavirus, or (ii) a plurality of live or attenuated live commensal human papillomaviruses, and a T cell adjuvant that enhances a T cell response to the antigenic peptides. Further provided is a composition described herein for use in a method of treating or reducing the risk of developing mucosal cancer in a subject.

[0006] In some embodiments, the subject is at increased risk for developing mucosal cancer or is immunocompromised, for example, as a result of an acquired immune deficiency, a primary immune deficiency, or an organ transplant.

[0007] In some embodiments, the commensal human papillomavirus is a low-risk α-HPV, β-HPV, γ-HPV, and / or μ-HPV strain, for example, the commensal human papillomavirus is a β-HPV and / or γ-HPV strain listed in Table A.

[0008] In some embodiments, the multiple antigenic peptides include peptides derived from one or more of E1, E2, E4, E5, E6, or E7 proteins.

[0009] In some embodiments, the multiple antigenic peptides include peptides derived from proteins from multiple commensal human papillomaviruses.

[0010] In some embodiments, the composition comprises at least 200 peptides, each having a unique sequence, eg, including multiple peptides for each unique sequence.

[0011] In some embodiments, the T cell adjuvant comprises one or more of: nanoparticles that enhance T cell responses; poly-ICLC (carboxymethylcellulose, polyinosinic-polycytidylic acid, and poly-L-lysine double-stranded RNA), imiquimod, CpG oligodeoxynucleotides and formulations (IC31, QB10), AS04 (aluminum salts formulated with 3-O-desacyl-4'-monophosphoryl lipid A (MPL)), AS01 (MPL and saponin QS-21), MPLA, STING agonists, other TLR agonists, Candida albicans skin test antigen (candin), GM-CSF, Fms-like tyrosine kinase-3 ligand (Flt3L), and / or IFA (incomplete Freund's adjuvant). In some embodiments, the T cell adjuvant comprises topical imiquimod and / or topical 5-fluorouracil and / or topical calcipotriene (calcipotriol), for example in combination with 5-fluorouracil.

[0012] In some embodiments, the mucosal cancer is a cancer of the oral and paranasal mucosa, optionally, for example, mucosal squamous cell carcinoma (mSCC) of the tongue; head and neck cancer (HNC); cancer of the mucosa of the upper respiratory tract; or a genitourinary cancer, optionally anal cancer, cervical cancer, or cancer of the vulva, vestibule, vagina, perineum, or perianal tissues.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials are described herein for use in this invention, and other suitable methods and materials known in the art can also be used.Materials, methods, and examples are merely illustrative and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety.In case of conflict, the present specification, including definitions, will prevail.

[0014] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. [Brief description of the drawings]

[0015] [Figure 1-1] Papillomavirus colonization protects against carcinogen-induced tongue cancer. (FIG. 1A) Time to tumor onset (long rank test) for MmuPV1-infected vs. mock-infected tongues of immunocompetent p53+ / - mice after treatment with the DMBA / 4NQO carcinogenesis protocol. Red arrows point to invasive mSCC in mock-infected tongues. n=15 for MmuPV1 and n=16 for mock group. [Figure 1-2] Papillomavirus colonization protects against carcinogen-induced tongue cancer. (Figure 1B) Survival of MmuPV1-infected immunocompetent p53+ / - mice vs. mock-infected immunocompetent p53+ / - mice after treatment with DMBA / 4NQO carcinogenesis protocol (long rank test). Note that the burden of oral lesions induced by carcinogenesis is responsible for weight loss and mortality in the animals. Red arrows point to invasive mSCC in mock-infected tongues. n=15 for MmuPV1 and n=16 for the mock group. [Figure 1-3] Papillomavirus colonization protects against carcinogen-induced tongue cancer. (FIG. 1C) Representative H&E staining images of MmuPV1-infected (top) and mock-infected (bottom) tongues treated with DMBA / 4NQO. Red arrows point to invasive mSCC in mock-infected tongues. n=15 for MmuPV1 and n=16 for mock group. [Figure 2-1] Papillomavirus colonization protects against carcinogen-induced proliferation of mutant Trp53 clones in tongue epithelium. (FIG. 2A) Size of mutant Trp53 clones (Mann-Whitney U test) in MmuPV1-infected versus mock-infected tongues of immunocompetent p53+ / - mice after treatment with the DMBA / 4NQO carcinogenesis protocol. [Figure 2-2]Figure 2B: Papillomavirus colonization protects against carcinogen-induced proliferation of mutant Trp53 clones in tongue epithelium. (Figure 2B) Representative p53 staining images of MmuPV1-infected (top) and mock-infected (bottom) tongues treated with DMBA / 4NQO. Red arrows point to mutant Trp53 clones in MmuPV1-infected and mock-infected tongues. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] High-risk HPV vaccines targeting humoral immunity reduce the risk of mSCC by blocking α-HPV infection. However, effective modalities to prevent the majority of mSCC in smoking-induced HNC and individuals already infected with high-risk HPV are lacking. Treatments for mSCC include surgery and radiation for localized disease and chemotherapy, targeted therapy, and immunotherapy for unresectable and metastatic cancers. In addition to their side effects, the treatment of mSCC represents a growing public health challenge. Thus, new technologies to prevent and treat mSCC are urgently needed.

[0017] High-risk α-HPV is known to induce oropharyngeal mSCC. 2 However, carcinogen-induced HNC accounts for more than 70% of mSCC affecting the upper aerodigestive tract. 3 Importantly, low-risk commensal HPVs are found ubiquitously in the upper aerodigestive tract, anal canal, and genital tract. 4、5 Low-risk commensal HPV does not contribute any carcinogenic role to the development of mSCC, but the influence of immunity to commensal HPV on the development of mSCC has not been investigated.

[0018] Based on the potential of the commensal virome for cancer prevention and therapy, a live commensal HPV vaccine strategy is described herein to boost antiviral T cell immunity in skin and mucosal sites colonized by commensal HPV, reducing the risk of cancer development and treating early SCCs containing active virus. As shown herein, by studying patient skin and cancer samples and a novel mouse papillomavirus (MmuPV1) model, the role of commensal HPV and their immune defense against cancer in skin has properties similar to low-risk commensal HPV. In stark contrast to prevailing dogma, MmuPV1 skin colonization of immune-competent mice does not have a carcinogenic effect, but instead increases the CD8 + It was found to protect the skin against chemical and UV skin carcinogenesis in a T cell-dependent manner. Furthermore, adoptive transfer of memory T cells from the skin-draining lymph nodes of immune mice (i.e., wart-free) immunized wart-bearing mice and provided them with protection against skin carcinogenesis. 6 As described herein, a commensal HPV vaccine can also be used for cancer prevention and therapy at mucosal sites, because oral papillomavirus colonization protects an immunocompetent host from mSCC.

[0019] T cell-based vaccine against β-HPV Described herein is a composition that can be used to induce T cell-based immune response against β-HPV, thereby reducing the risk of developing mucosal cancer in subjects.Instead of preventing or eliminating infection, the vaccine induces T cell immunity against the symbiotic virus that already infects tissues, with the aim of using the presence of virus in all cells to boost the detection and elimination of early cancer clones by T cells.Current high-risk HPV vaccines for preventing cervical cancer and head and neck cancer are primarily intended to prevent infection, and have minimal effectiveness in individuals who are already infected with the virus.

[0020] Antigenic peptides In some embodiments, the composition comprises multiple antigenic peptides derived from a protein, e.g., E1, E2, E6, or E7 protein, from a commensal human papillomavirus, e.g., a low-risk cutaneotropic α-HPV, β-HPV, γ-HPV, and / or μ-HPV strain, e.g., those listed in Table A. In some embodiments, the composition does not include peptides derived from an HPV type associated with cancer, e.g., a high-risk HPV, such as HPV 16 or 18. See, for example, Ma et al., J Virol. 2014 May; 88(9): 4786-4797; Doorbar et al., Rev Med Virol. 2015 Mar; 25(Suppl Suppl 1): 2-23; Doorbar et al., The biology and life-cycle of human papillomaviruses. Vaccine 2012; 30(Suppl 5): F55-F70; de Villiers, Virology 2013; 445(1-2): 2-10; and U.S. Pat. No. 8,652,482, which are incorporated herein by reference. Other commensal HPV types that are not "high-risk" alpha-HPV can be used. Table A is an exemplary and not exhaustive list. Other HPVs in normal skin and mucosal sites are also known, see, e.g., Ma, Journal of virology 2014: 88; 4786-4797.

[0021] [Table 1-1]

[0022] [Table 1-2]

[0023] The peptides can be derived from any antigenic protein in the virus. In some embodiments, the peptides are derived from E1, E2, E4, E5, E6 or E7 proteins. Sequences for these proteins in multiple symbiotic strains are provided. In some embodiments, at least 50 or more, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more different peptides (i.e., peptides with different sequences) are included in the composition. In some embodiments, at least 50 or more, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more different peptides from each virus strain are included in the composition, and peptides from two or more virus strains are included.

[0024] In some embodiments, the peptides are of a length optimized for MHC1 / MHCII presentation, e.g., 9-30 amino acids, e.g., 12-25, 12-18, 12-16, 13-16, 14-16, or 15 amino acids. The sequences of the peptides can be, e.g., synthetic long overlapping peptides, e.g., 15 amino acid peptides with 10 amino acid overlaps, identified, e.g., bioinformatically, to predict antigenicity and / or generated using a moving window of overlapping peptides to cover the entire protein (similar to the "gene walk" method used to identify optimal antisense oligonucleotides). In some embodiments, synthetic long overlapping peptides (SLPs) are used (Zom et al., Cancer Immunol Res. 2014 Aug; 2(8): 756-64). The composition can include multiple peptides derived from one or more (e.g., multiple) different viral strains. The peptide is preferably a synthetic peptide, and methods of synthesizing peptides are known in the art, including solution-phase techniques and solid-phase peptide synthesis (SPPS).See, for example, Petrou and Sarigiannis, Ch. 1 - Peptide synthesis: Methods, trends, and challenges, In: Editor(s): Sotirios Koutsopoulos, Peptide Applications in Biomedicine, Biotechnology and Bioengineering, Woodhead Publishing, 2018, pages 1-21; and Chandrudu et al., Molecules 2013, 18, 4373-4388.

[0025] Antigen Protein In some embodiments, the compositions can include virus-like particles that contain multiple proteins, such as E1, E2, E6, or E7 proteins, from a commensal human papillomavirus, such as a low-risk alpha-HPV, beta-HPV, gamma-HPV, and / or mu-HPV strain, such as those listed in Table A (see, e.g., Yang et al., Virus Res 231, 148-165 (2017); Hancock et al., Therapeutic HPV vaccines. Best Pract Res Clin Obstet Gynaecol 47, 59-72 (Feb. 2018); Joh et al., Exp Mol Pathol.; 93(3): 416-21 (2012)).

[0026] Nucleic Acid-Based Vaccines In some embodiments, the compositions can include multiple DNA plasmids and / or RNA replicons containing nucleotide sequences to express a protein or antigenic peptide derived from (i.e., comprising) a protein, e.g., an E1, E2, E6, or E7 protein, from a commensal human papillomavirus, e.g., a low-risk alpha-HPV, beta-HPV, gamma-HPV, and / or mu-HPV strain, e.g., those listed in Table A (see, e.g., Yang et al., Virus Res 231, 148-165 (2017); Hancock et al., Therapeutic HPV vaccines. Best Pract Res Clin Obstet Gynaecol 47, 59-72 (2018)).

[0027] Live Vector-Based Vaccines In some embodiments, the compositions can include multiple viral vectors that have been engineered to express a protein, such as a protein or antigenic peptide derived from (i.e., comprising) an E1, E2, E6, or E7 protein, from a commensal human papillomavirus, such as a low-risk alpha-HPV, beta-HPV, gamma-HPV, and / or mu-HPV strain, such as those listed in Table A (see, e.g., Yang et al., Virus Res 231, 148-165 (2017); Hancock et al., Therapeutic HPV vaccines. Best Pract Res Clin Obstet Gynaecol 47, 59-72 (2018)).

[0028] Viral vectors for use in the present methods and compositions include recombinant retroviruses, adeno-Lewis, adeno-associated viruses, alphaviruses, and lentiviruses.

[0029] A preferred viral vector system useful for delivery of nucleic acid in the present method is adeno-associated virus (AAV). AAV is a small non-enveloped virus with a 25 nm capsid. No disease is known or understood to be associated with this wild type virus. AAV has a single-stranded DNA (ssDNA) genome. AAV has been shown to exhibit long-term episomal transgene expression, and AAV has demonstrated excellent transgene expression in many tissues, including the brain, especially in neurons. Vectors containing as few as 300 base pairs of AAV can be packaged and integrated. Space for exogenous DNA is limited to approximately 4.7 kb. DNA can be introduced into cells using AAV vectors such as those described in Tratschin et al., Mol. Cell. Biol. 5: 3251-3260 (1985). A variety of nucleic acids have been introduced into various cell types using AAV vectors (see, e.g., Hermonat et al., Proc. Natl. Acad. Sci. USA 81: 6466-6470 (1984); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al., Mol. Endocrinol. 2:32-39 (1988); Tratschin et al., J. Virol. 51:611-619 (1984); and Flotte et al., J. Biol. Chem. 268:3781-3790 (1993)). Numerous alternative AAV variants exist (more than 100 have been cloned), and AAV variants have been identified based on desirable properties. For example, AAV9 has been found to efficiently cross the blood-brain barrier. Additionally, AAV capsids can be engineered to increase transduction efficiency and selectivity (e.g., biotinylated AAV vectors, directed molecular evolution, self-complementary AAV genomes, etc.). In some embodiments, AAV1 is used.

[0030] Alternatively, retroviral vectors and adeno-associated viral vectors can be used as recombinant gene delivery systems for the transfer of exogenous genes in vivo, especially to humans. These vectors provide efficient delivery of genes to cells, and the transferred nucleic acid is stably integrated into the host's chromosomal DNA. The development of specialized cell lines (called "packaging cells") that produce only replication-defective retroviruses has increased the usefulness of retroviruses for gene therapy, and defective retroviruses are characterized for use in gene transfer for gene therapy purposes (for review, see Miller, Blood 76: 271 (1990)). Replication-defective retroviruses can be packaged into virions, which can be used to infect target cells with helper viruses by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Ausubel, et al., eds., Current Protocols in Molecular Biology, Greene Publishing Associates, (1989), Sections 9.10-9.14, and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM, which are known to those skilled in the art. Examples of packaging virus systems suitable for preparing both ecotropic and amphotropic retrovirus systems include ΨCrip, ΨCre, Ψ2 and ΨAm.Retroviruses have been used to transfer a variety of genes into a wide variety of cell types in vitro and / or in vivo, including epithelial cells (see, e.g., Eglitis, et al. (1985) Science 230: 1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85: 6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci. USA 85: 3014-3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87: 6141-6145; Huber et al. (1991) Proc. Natl. Acad. Sci. USA 88: 8039-8043; Ferry et al. (1991) Proc. Natl. Acad. Sci. USA 88: 8377-8381;Chowdhury et al. (1991) Science 254: 1802-1805;van Beusechem et al. (1992) Proc. Natl. Acad. Sci. USA 89: 7640-7644;Kay et al. (1992) Human Gene Therapy 3: 641-647;Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89: 10892-10895;Hwu et al. (1993) J. Immunol. 150: (See, e.g., U.S. Pat. Nos. 4,868,116, 4,980,286; PCT Application Publication No. WO 89 / 07136; PCT Application Publication No. WO 89 / 02468; PCT Application Publication No. WO 89 / 05345; and PCT Application Publication No. WO 92 / 07573).

[0031] Another viral gene delivery system useful in this method utilizes vectors derived from adenovirus. The genome of adenovirus can be engineered so that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in normal lytic viral life cycle. See, for example, Berkner et al., BioTechniques 6: 616 (1988); Rosenfeld et al., Science 252: 431-434 (1991); and Rosenfeld et al., Cell 68: 143-155 (1992). Suitable adenovirus vectors derived from adenovirus strain Ad type 5dl324 or other strains of adenovirus (such as Ad2, Ad3, or Ad7) are known to those skilled in the art. Recombinant adenoviruses may be suitable in certain circumstances in that they are unable to infect non-dividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al., supra, (1992)). Moreover, viral particles are relatively stable, suitable for purification and concentration, and can be modified to affect infectivity spectrum, as described above. Moreover, the introduced adenoviral DNA (and the foreign DNA contained therein) is not integrated into the genome of the host cell, but remains episomal, thereby avoiding potential problems that may occur as a result of in situ insertional mutagenesis when the introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham, J. Virol. 57: 267 (1986)).

[0032] Alphaviruses can also be used. Alphaviruses are enveloped single-stranded RNA viruses with a wide host range, and when used in gene therapy protocols, they can provide high levels of transient gene expression. Exemplary alphaviruses include Semliki Forest virus (SFV), Sindbis virus (SIN) and Venezuelan equine encephalitis (VEE) virus, all of which have been genetically modified to provide efficient replication-deficient and replication-competent expression vectors. Alphaviruses exhibit significant neurotropism and are therefore useful for CNS-related diseases. See, e.g., Lundstrom, Viruses. 2009 Jun; 1(1): 13-25; Lundstrom, Viruses. 2014 Jun; 6(6): 2392-2415; Lundstrom, Curr Gene Ther. 2001 May; 1(1): 19-29; Rayner et al., Rev Med Virol. 2002 Sep-Oct; 12(5): 279-96.

[0033] Commensal HPV vaccine strategy A live commensal HPV vaccine strategy can be used to optimally boost antiviral T cell immunity in the mucosa and prevent cancer development with active virus to treat early mucosal cancers or precancerous lesions, including adenocarcinoma in situ, vaginal, penile and anal intraepithelial lesions, squamous intraepithelial lesions (SIL), cervical intraepithelial neoplasia (CIN), and dysplasia and carcinoma in situ. Platforms for generating and propagating low-risk live HPV in culture to generate live and attenuated live HPV vaccines for use in patients are described in WO2020 / 112720. For example, the live and attenuated live HPV vaccines described in WO2020 / 112720 can be used.

[0034] T cell adjuvants The composition can also include an adjuvant to enhance T cell response. For example, it can include nanoparticles that enhance T cell response, as described in, for example, Stano et al., Vaccine (2012) 30: 7541-6 and Swaminathan et al., Vaccine (2016) 34: 110-9. See also Panagioti et al., Front. Immunol., 16 February 2018; doi.org / 10.3389 / fimmu.2018.00276. Alternatively, or in addition, adjuvants including poly ICLC (carboxymethylcellulose, polyinosinic-polycytidylic acid, and poly-L-lysine double stranded RNA), imiquimod, resiquimod (R-848), CpG oligodeoxynucleotides and formulations (IC31, QB10), AS04 (aluminum salts formulated with 3-O-desacyl-4'-monophosphoryl lipid A (MPL)), AS01 (MPL and saponin QS-21), MPLA, STING agonists, other TLR agonists, GM-CSF, Fms-like tyrosine kinase-3 ligand (Flt3L), and / or IFA (incomplete Freund's adjuvant) can also be used. In some embodiments, topical imiquimod and / or topical 5-fluorouracil and / or topical calcipotriene (calcipotriol) in combination with 5-fluorouracil (e.g., as described in Cunningham et al., J Clin Invest. 2017; 127(1): 106-116) may function as an adjuvant for the vaccine (which is particularly applicable in subjects with pre-malignant lesions that are commonly treated with these topical agents).See, e.g., Khong and Willem, Journal for ImmunoTherapy of Cancer 4:56 (2016); Coffman et al., Immunity. 2010 Oct 29; 33(4): 492-503; Martins et al., EBioMedicine 3: 67-78, 2016; and Del Giudice, Seminars in Immunology, 2018, doi.org / 10.1016 / j.smim.2018.05.001.

[0035] composition Pharmaceutical compositions typically include a pharma- ceutically acceptable carrier. As used herein, the language "pharma- ceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

[0036] A pharmaceutical composition is typically formulated to be compatible with its intended route of administration, examples of which include parenteral, e.g., intravenous, intradermal, subcutaneous, intratumoral, intramuscular or subcutaneous administration.

[0037] Methods for formulating suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY).For example, the solution or suspension used for parenteral, intradermal, intramuscular or subcutaneous application can contain the following components: sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic solvent; antibacterial agent, such as benzyl alcohol or methyl paraben; antioxidant, such as ascorbic acid or sodium bisulfite; chelating agent, such as ethylenediaminetetraacetic acid; buffer, such as acetate, citrate or phosphate, and agent for adjusting tonicity, such as sodium chloride or dextrose.pH can be adjusted with acid or base, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0038] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. The composition should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic agents in the composition, such as sugars, polyalcohols, such as mannitol, sorbitol, sodium chloride. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption in the composition, such as aluminum monostearate and gelatin.

[0039] Sterile injectable solution can be prepared by incorporating active compound in the required amount in suitable solvent with one or combination of above-listed components as required, followed by sterile filtration.Generally, dispersion is prepared by incorporating active compound into sterile vehicle that contains basic dispersion medium and other required components from above-listed.For the preparation of sterile injectable solution, the preferred preparation method is vacuum drying and freeze-drying, which obtains powder of active ingredient and any additional desired ingredient from their previously sterile filtered solution.

[0040] In one embodiment, the therapeutic compound is prepared with a carrier that protects the therapeutic compound against rapid elimination from the body, such as sustained release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using standard techniques or are commercially available, for example, from Alza and Nova Pharmaceuticals. Liposomal suspensions, including liposomes that target selected cells using monoclonal antibodies against cellular antigens, can also be used as pharma- ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0041] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0042] subject The vaccine compositions described herein can be used to boost immunity against mucosal cancer in immunocompetent subjects and immunosuppressed or immunocompromised patients who have reduced T cell immunity against β-HPV and are prone to develop multiple mucosal warts and cancers (viral load) with poor prognosis. In some embodiments, the subject does not have cancer (e.g., does not have mucosal cancer). In some embodiments, the subject is at high risk (i.e., has a risk above that of the general population) of developing mucosal cancer. For example, the subject may have a family history of cancer, a personal history of smoking, alcohol use, ionizing radiation, occupational exposure (e.g., acid mist), dietary exposure (e.g., salted fish), precancerous lesions, related sexual history, or a family or personal history of mucosal cancer. In some embodiments, the subject has or has a history of recurrent respiratory papillomatosis.

[0043] In some embodiments, the subject may be immunosuppressed due to, for example, an organ transplant, an acquired immune deficiency, such as HIV / AIDS, or a primary human immune deficiency.

[0044] Subjects that can be treated using the present method include mammals, eg, human and non-human veterinary subjects.

[0045] Methods for inducing anti-cancer immunity The composition can be used to induce anti-cancer immunity and reduce the risk of developing mucosal cancer, i.e., cancer of mucosal tissue.Mucosal cancer includes cancer of oral and paranasal mucosa, including mucosal squamous cell carcinoma (mSCC); head and neck cancer (HNC); cancer of upper respiratory tract mucosa; and cancer of urogenital tract, including anal cancer, cervical cancer, and cancer of vulva, vestibule, vagina, perineum and perianal area.The method includes administering one or more doses of the vaccine composition described herein to a subject, for example a subject in need thereof.

[0046] The composition is administered in an effective amount. An "effective amount" is an amount sufficient to produce a beneficial or desired result. For example, an effective amount is one that achieves a desired therapeutic effect, e.g., the amount necessary to treat a disease or reduce the risk of developing a disease or disease symptoms (also referred to as a therapeutically effective amount or a prophylactically effective amount, respectively). An effective amount may be administered in one or more administrations, applications, or dosages. The therapeutically effective amount (i.e., effective dosage) of a therapeutic compound will depend on the therapeutic compound selected. The composition may be administered one or more times per day to one or more times per week, including once every other day. Those skilled in the art will appreciate that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, general health and / or age of the subject, and other diseases present, may affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of a therapeutic compound described herein may include a single treatment or a series of treatments. For example, the methods can include administering a first dose followed by a second dose (e.g., a "booster" dose), e.g., 1 week, 2 weeks, 4 weeks, 6 weeks, 8 weeks, 12 weeks, 18 weeks, 24 weeks, or 52 weeks later.

[0047] The dosage, toxicity and therapeutic effectiveness of therapeutic compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example to determine LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compositions that exhibit high therapeutic indices are preferred. Compositions that exhibit toxic side effects may be used, but care should be taken to minimize and reduce side effects.

[0048] Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans.The dosage of such compounds is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.Dosage can vary within this range depending on the dosage form used and the route of administration utilized.For any composition used in the methods described herein, therapeutically effective dose can be estimated first from cell culture assays.Dose can be formulated in animal models.Using such information, useful doses in humans can be more accurately determined.

[0049] The method may also include administering one or more other treatments known in the art for mucosal cancer, e.g., in a subject with mucosal cancer, or a treatment for reducing the risk of developing mucosal cancer. For example, a combination of the compositions described herein and treatments for benign or precancerous lesions (to reduce the risk of developing mucosal cancer) (e.g., surgical procedures (e.g., resection (e.g., loop electrocautery excision procedure (LEEP) or large loop electrocautery excision of the transition zone (LLETZ) for cervical lesions, or cold steel surgery, laser ablation and laser deposition for vaginal lesions) or destruction (e.g., using carbon dioxide vaporization, cryotherapy, electrocautery, or low temperature (thermal) coagulation)); photodynamic therapy; local or systemic medications (e.g., podophyllin resin, podophyllotoxin, organic acids such as salicylic acid, trichloroacetic acid and dichloroacetic acid, 5-fluorouracil, topical imiquimod, calcipotriene plus 5-fluorouracil, bleomycin, IFN-α, or cidofovir)) can be used in combination with the methods. In some embodiments, these agents boost antigen presentation (innate signals), while the compositions boost antigen recognition by T cells. In subjects with mucosal cancer, surgical treatment (e.g., resection (e.g., loop electrocautery excision (LEEP) or large loop electrocautery excision of the transition zone (LLETZ) for cervical lesions, or cold steel surgery, laser ablation and laser deposition for vaginal lesions) or destruction (e.g., using carbon dioxide vaporization, cryotherapy, electrocautery or low temperature (thermal) coagulation)); radiation therapy; photodynamic therapy; local or systemic medications (e.g., podophyllin resin, podophyllotoxin, organic acids such as salicylic acid, trichloroacetic acid and dichloroacetic acid, 5-fluorouracil, topical imiquimod, calcipotriene plus 5-fluorouracil, bleomycin, IFN-α, or cidofovir); or chemotherapy (e.g., using platinum-containing compounds such as cisplatin) can be used in combination with the method.See, e.g., IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Human Papillomaviruses. Lyon (FR): International Agency for Research on Cancer; 2007. (IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, No. 90.) 1, Human Papillomavirus (HPV) Infection., available at ncbi.nlm.nih.gov / books / NBK321770 / . EXAMPLES

[0050] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0051] [Example 1] To expand the application of commensal HPV vaccines for cancer prevention and therapy from the skin (e.g., as described in WO 2020 / 112720) to mucosal sites, we investigated whether papillomavirus colonization of the oral cavity protects immunocompetent hosts from mSCC.

[0052] Methods: We used MmuPV1, which does not cause any oral lesions, to generate immunocompetent p53 + / - The tongues of mice were colonized. Briefly, mice were anesthetized with isoflurane and then an 18-gauge sterile needle was used to inject a 0.2 cm area of ​​sputum into the base of the tongue. 2 ~0.3cm 2 Microablation was performed on B6.Cg-Foxn1 nu / nuPurified virus inoculum from MmuPV1-induced muzzle warts in mice was applied to the sacrified tongue and spread homogenously. The same virus inoculum was used for all infected mice. Mock infections were performed with MmuPV1 VLPs (105 μg in 10 μL PBS per mouse) applied to the scraped tongue. Two weeks after MmuPV1 and mock oral infections, we performed p53 + / - Mice were subjected to a DMBA / 4NQO carcinogenesis protocol optimized to induce carcinogenesis on the mouse tongue (Figure 1A-C). 7 Animals' tongues were treated with a single dose of 50 μg of 7,12-dimethylbenz(benza)anthracene (DMBA) in 50 μL of sesame oil applied to the tongue, followed by 0.5% 4-nitroquinoline 1-oxide (4NQO) treatment in drinking water for 20 weeks. Mice were monitored for signs of oral cancer and harvested after 26 weeks of DMBA or any time when mice developed severe oral lesions that interfered with their ability to eat and resulted in greater than 10% weight loss.

[0053] Results: Colonization of immunocompetent p53+ / - mice with MmuPV1 protected the tongue from DMBA / 4NQO-induced cancer compared to mock-infected controls. Mice colonized with MmuPV1 showed a significant delay in tongue tumor development and survived much longer compared to mock-infected animals (see Figure 1A-C). Furthermore, Figure 2A-B show the effect of papillomavirus tongue colonization on reducing the size of malignant clones (labeled Trp53 mutant clones) in mouse tongues, indicating a protective impact of papillomavirus colonization against early malignant transformation in mucosal epithelium. These findings demonstrate the cancer protective effect of commensal papillomavirus colonization in mucosal epithelium, which can be boosted by commensal HPV vaccine.

[0054] References:

[0055] [Table 2]

[0056] Other embodiments Although the present invention has been described in conjunction with its detailed description, it will be understood that the foregoing description is intended to be illustrative of the invention and not to limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. For use in a method of treating mucosal cancer or reducing the risk of developing mucosal cancer in a subject. a plurality of (i) antigenic peptides each comprising a sequence of 9 to 30 amino acids derived from a protein derived from a commensal human papillomavirus, or (ii) a live or attenuated live commensal human papillomavirus; a T cell adjuvant that enhances T cell responses to the antigenic peptide; A composition comprising:

2. The composition for use according to claim 1 , wherein the subject is at increased risk of developing mucosal cancer or is immunocompromised.

3. The composition for use according to claim 2, wherein the subject is immunocompromised as a result of acquired immune deficiency or organ transplantation.

4. The composition for use according to any one of claims 1 to 3, wherein the commensal human papillomavirus is a low-risk α-HPV, β-HPV, γ-HPV and / or μ-HPV strain.

5. The composition for use according to any one of claims 1 to 3, wherein the commensal human papillomavirus is a β-HPV and / or γ-HPV strain listed in Table A.

6. The composition for use according to any one of claims 1 to 5, wherein the plurality of antigenic peptides comprises peptides derived from one or more of E1, E2, E4, E5, E6 or E7 proteins.

7. The composition for use according to any one of claims 1 to 6, wherein the plurality of antigenic peptides comprises peptides derived from proteins from multiple commensal human papillomaviruses.

8. 8. The composition for use according to claim 7, comprising at least 200 peptides, each having a unique sequence.

9. 9. The composition for use according to claim 8, comprising multiple peptides for each unique sequence.

10. 10. The composition for use according to any one of claims 1 to 9, wherein the T cell adjuvant comprises one or more of: nanoparticles that enhance T cell responses; poly-ICLC (carboxymethylcellulose, polyinosinic-polycytidylic acid, and poly-L-lysine double stranded RNA), imiquimod, CpG oligodeoxynucleotides and formulations (IC31, QB10), AS04 (aluminum salt formulated with 3-O-desacyl-4'-monophosphoryl lipid A (MPL)), AS01 (MPL and saponin QS-21), MPLA, STING agonists, other TLR agonists, Candida albicans skin test antigen (candin), GM-CSF, Fms-like tyrosine kinase-3 ligand (Flt3L), and / or IFA (incomplete Freund's adjuvant).

11. The composition for use according to any one of claims 1 to 10, wherein the T cell adjuvant comprises topical imiquimod or topical 5-fluorouracil.

12. 12. The composition for use according to any one of claims 1 to 11, wherein the mucosal cancer is a cancer of the oral and paranasal mucosa, optionally mucosal squamous cell carcinoma (mSCC); a head and neck cancer (HNC); a cancer of the mucosa of the upper respiratory tract; or a genitourinary cancer, optionally anal cancer, cervical cancer, or a cancer of the vulva, vestibule, vagina, perineum, or perianal tissues.

13. a plurality of (i) antigenic peptides each comprising a sequence of 9 to 30 amino acids derived from a protein derived from a commensal human papillomavirus, or (ii) a live or attenuated live commensal human papillomavirus; a T cell adjuvant that enhances T cell responses to the antigenic peptide; A composition comprising: The composition, wherein the commensal human papillomavirus is a low-risk α-HPV, β-HPV, γ-HPV, and / or μ-HPV strain, and optionally the commensal human papillomavirus is a β-HPV and / or γ-HPV strain listed in Table A.

14. 14. The composition of claim 13, wherein the plurality of antigenic peptides comprises peptides derived from one or more of E1, E2, E4, E5, E6 or E7 proteins, optionally comprising at least 200 peptides each having a unique sequence, and comprising multiple peptides for each unique sequence.

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