WO2024010962A2 - Vaccine incorporating protein-based immune adjuvant - Google Patents

Abstract

Disclosed are a composition and method of treating some HPV-related solid tumors in mammalian subjects. In one embodiment, a method comprises delivering a self-assembling vaccine intradermally to a subject. In at least one embodiment, the self-assembling vaccine comprises a fusion protein non-covalently attached to two or more biotinylated E6/E7 peptides, derived from targeted viral or oncogenic protein epitopes, using a biotin-avidin engagement.

Description

VACCINE INCORPORATING PROTEIN-BASED IMMUNE ADJUVANT

CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] The present application claims the benefit of priority of U.S. Provisional Patent Application No. 63/359,560, filed on July 8, 2022, the disclosure of which is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to the treatment of solid tumors (e.g., head and neck cancer, cervical cancer, and carcinoma), and, more specifically to immunotherapeutic peptide- based vaccine methods and pharmaceutical compositions for the treatment of tumors.

BACKGROUND OF THE INVENTION

[0003] Adjuvents are chemical compounds approved for use in human vaccines in the United States to serve as immunostimulants and enhance antibody production when delivered with antigens. Alum in particular has been widely used as adjuvants for the past several decades. However, although alum induces a Th2 -biased immune response, it is ineffective in inducing a Thl cell-mediated immune response vital for the treatment of some solid tumors. Inducing Thl cell-mediated immune responses is desirable as they play a crucial role in inducing therapeutically- significant immune responses while allowing for a high degree of safety to be maintained.

[0004] In the last 30 years, numerous attempts have been made to develop therapeutic vaccines as a modality for the treatment of a number of solid tumors, but these approaches lacked therapeutic effectiveness. In addition, current vaccine strategies are time intensive, labor intensive, can only commence once a threat emerges, and are also impractical for generating personalized vaccines to combat disease for which target antigens vary among individuals. Thus, there is a need for alternative approaches, such as platform vaccine technologies, due to their potential to generate therapeutically effective vaccines for a number of scenarios including, but not limited to, the treatment of some solid tumors, such as cervical cancer caused by human papillomavirus (HPV), or to contain quickly evolving, fast acting, and/or highly contagious threats.

SUMMARY OF THE DISCLOSURE

[0005] The following summary presents a simplified summary of various aspects of the present disclosure in order to provide a basic understanding of such aspects. This summary is not an extensive overview of the disclosure. It is intended to neither identify key or critical elements of the disclosure, nor delineate any scope of the particular embodiments of the disclosure or any scope of the claims. Its sole purpose is to present some concepts of the disclosure in a simplified form as a prelude to the more detailed description that is presented later.

[0006] In one aspect, a pharmaceutical composition comprises a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to two or more biotinylated peptides, and wherein the two or more biotinylated peptides each comprise a sequence at least partially derived from an epitope of a human virus antigen protein with the proviso that each epitope is of a different human virus antigen protein of the same human virus.

[0007] In a further aspect, a pharmaceutical composition comprises a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to a biotinylated peptide, and wherein the biotinylated peptide comprises a sequence at least partially derived from one or more epitopes of one or more human papillomavirus (HPV) antigen proteins. [0008] In at least one embodiment, wherein the biotinylated peptide comprises two or more epitope sequences each separated by a linker sequence.

[0009] In at least one embodiment, the one or more HPV antigen proteins comprise E6 protein or E7 protein.

[0010] In a further aspect, a pharmaceutical composition comprises a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to at least one biotinylated peptide, and where the at least one biotinylated peptide comprises at least one amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0011] In a further aspect, a pharmaceutical composition comprises a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to a biotinylated peptide, wherein the biotinylated peptide is formed by joining two peptide fragments, the peptide fragments each having amino acid sequences selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0012] In at least one embodiment, the heat shock protein is a mammalian heat shock protein or a bacterial heat shock protein.

[0013] In at least one embodiment, the heat shock protein is selected from the group consisting of Mycobacterium tuberculosis heat shock protein 70 (MTbHSP70) and a human heat shock protein.

[0014] In at least one embodiment, the heat shock protein is a member of the heat shock protein 70 (HSP70) family.

[0015] In at least one embodiment, the heat shock protein is or is derived from MTbHSP70. [0016] In at least one embodiment, the heat shock protein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1.

[0017] In at least one embodiment, the heat shock protein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1 containing a Val381 to Phe381 point mutation.

[0018] In a further aspect, pharmaceutical composition comprises: a fusion protein derived from a heat shock protein; a first peptide non-covalently bound to the fusion protein, the first biotinylated peptide comprising a human papillomavirus (HPV) E6 protein epitope; and a second peptide non-covalently bound to the fusion protein, the second biotinylated peptide comprising an HPV E7 protein epitope. In at least one embodiment, the fusion protein comprises MTbHSP70- avidin fusion protein.

[0019] In at least one embodiment, the biotin-binding protein is selected from a group consisting of avidin, streptavidin, and neutravidin.

[0020] In at least one embodiment, any of the aforementioned pharmaceutical compositions comprises a pharmaceutically acceptable excipient. In at least one embodiment, the pharmaceutical composition is a vaccine composition.

[0021] In a further aspect, a method for inducing an immune response in a subject comprises administering to the subject the pharmaceutical composition of any one of the preceding claims.

[0022] In at least one embodiment, the pharmaceutical composition comprises a pharmaceutically-accepted excipient, and wherein a therapeutic dose administered to the subject comprises about 150 pg to about 500 pg of the heat shock protein.

[0023] In a further aspect, a method of treating an HPV-induced tumor of a subject for which HPV proteins E6 and/or E7 are expressed by cancerous cells of the tumor comprises administering to the subject the pharmaceutical composition of any of the aforementioned pharmaceutical compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The above and other features of the present disclosure, their nature, and various advantages will become more apparent upon consideration of the following detailed description, taken in conjunction with the accompanying drawings, in which:

[0025] FIG. 1A shows the amino acid sequence for Mycobacterium tuberculosis heat shock protein 70;

[0026] FIG. IB is a table summarizing exemplary E6ZE7 peptide sequences formed through click chemistry of the two peptide fragments in accordance with at least one embodiment; [0027] FIG. 1C illustrates a click chemistry reaction forming a triazole moiety in accordance with at least one embodiment;

[0028] FIG. 2 shows the amino acid sequence of a fusion protein in accordance with at least one embodiment;

[0029] FIG. 3 is a schematic illustrating spontaneous self-assembly of a fusion protein and biotinylated peptides in accordance with at least one embodiment;

[0030] FIG. 4A includes plots demonstrating the expression of IFNy in splenocytes following stimulation in accordance with at least one embodiment;

[0031] FIG. 4B includes plots demonstrating the expression of IL-2 in splenocytes following stimulation in accordance with at least one embodiment;

[0032] FIG. 4C includes plots demonstrating the expression of TNFa in splenocytes following stimulation in accordance with at least one embodiment;

[0033] FIG. 4D includes plots demonstrating the expression of CD62L in splenocytes following stimulation in accordance with at least one embodiment;

[0034] FIG. 5 includes plots demonstrating the therapeutic effects of eSAV, 5% alum-E6-E7 peptide, or saline on syngeneic tumor growth in accordance with at least one embodiment;

[0035] FIG. 6 includes a plot demonstrating the therapeutic effects of eSAV, 5% alum-E6-E7 peptide, or saline on mouse survival in accordance with at least one embodiment;

[0036] FIG. 7A includes plots demonstrating the expression of IFNy, TNFa and IL-2 in CD8⁺ splenocytes following stimulation of eSAV at different doses in accordance with at least one embodiment;

[0037] FIG. 7B includes plots demonstrating the expression of IFNy, TNFa and IL-2 in CD8⁺ lymphocytes following stimulation of eSAV at different doses in accordance with at least one embodiment;

[0038] FIG. 8A includes plots demonstrating the expression of IFNy, TNFa and IL-2 in CD4⁺ splenocytes following stimulation of eSAV at different doses in accordance with at least one embodiment;

[0039] FIG. 8B includes plots demonstrating the expression of IFNy, TNFa and IL-2 in CD4⁺ lymphocytes following stimulation of eSAV at different doses in accordance with at least one embodiment;

[0040] FIG. 9A includes plots demonstrating the tumor growth kinetics of the vaccine and anti-mPDl treated groups at different vaccine doses in accordance with at least one embodiment; [0041] FIG. 9B includes plots demonstrating the tumor growth kinetics of the vaccine and anti-IgG2a treated groups at different vaccine doses in accordance with at least one embodiment; [0042] FIG. 10A includes a plot demonstrating the survival curve of the vaccine and anti- mPDl treated groups at different vaccine doses in accordance with at least one embodiment;

[0043] FIG. 10B includes a plot demonstrating the survival curve of the vaccine and anti- IgG2a treated groups at different vaccine doses in accordance with at least one embodiment.

DEFINITIONS

[0044] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a drug” includes a single drug as well as a mixture of two or more different drugs; and reference to “an adjuvant” includes a single adjuvant as well as a mixture of two or more different adjuvants, and the like.

[0045] Also as used herein, “about,” when used in connection with a measured quantity, refers to the normal variations in that measured quantity, as expected by one of ordinary skill in the art in making the measurement and exercising a level of care commensurate with the objective of measurement and the precision of the measuring equipment. In certain embodiments, the term “about” includes the recited number ±10%, such that “about 10” would include from 9 to 11.

[0046] Also as used herein, “including” is used to mean “including but not limited to.”

[0047] Also as used herein, “protein” has its ordinary and customary meaning in the art and includes, and refers to a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds). Polypeptides may include natural amino acids, non-natural amino acids, synthetic amino acids, amino acid analogs, and combinations thereof. The term “peptide” is typically used to refer to a polypeptide having a length of less than about 50 amino acids. Proteins may include moieties other than amino acids (e.g., glycoproteins) and may be processed or modified. A protein can be a complete polypeptide chain as produced by a cell, or can be a functional portion thereof. A protein can include more than one polypeptide chain which may be chemically linked (e.g., by a disulfide bond), non-chemically linked (e.g., by hydrogen bonding), or both. Polypeptides may contain L-amino acids, D-amino acids, or both, and may contain any of a variety of amino acid modifications or analogs known in the art.

[0048] Also as used herein, the term “biotin-binding protein” refers to a protein that can non- covalently bind to biotin. A biotin-binding protein may be a monomer, dimer, or tetramer capable of forming monovalent, divalent, or tetravalent pharmaceutical compositions, respectively, as described herein. Non-limiting examples include anti-biotin antibodies, avidin, streptavidin, and neutravidin. The avidin may comprise mature avidin, or a sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the sequence identifies by NCBI Accession No. NP 990651. The streptavidin may comprise, for example, a sequence that is at least 80%, 85%, 90%, 95%, 99% identical to the sequence identified by of NCBI Accession No. AAU48617. The term “biotin- binding protein” is intended to encompass wild-type and derivatives of avidin, streptavidin, and neutravidin, which form monomers, dimers, tetramers.

[0049] Also as used herein, “vaccine” has its ordinary and customary meaning in the art and refers to any preparation used to stimulate antibody production against one or several diseases. A vaccine can be a preparation which provides immunity to one or several diseases, or a preparation that stimulates an immune response against one or several diseases. Vaccines can include killed or attenuated causative agents of a disease, products, or derivatives of said agents, and synthetic substitutes. Synthetic substitutes may include preparations consisting of synthetic peptides, carbohydrates, antigens, or strands of RNA or DNA.

[0050] Also as used herein, “epitope” refers to the region of an antigen to which an antibody binds preferentially and specifically.

[0051] Also as used herein, a “linker” refers to a molecule or group of molecules connecting two molecules, such as a molecule forming a covalent linkage between a heat shock protein and a biotin-binding protein. The linker may be comprised of a single linking molecule or may comprise a linking molecule and a spacer molecule, intended to separate the linking molecule and a moiety by a specific distance.

[0052] Also as used herein, “immunogenic” refers to the ability of a substance to elicit an immune response. Immune response refers to the reaction of a subj ect to the presence of an antigen and may include at least one of the following: making antibodies, developing immunity, developing hypersensitivity to the antigen, and developing tolerance.

[0053] Also as used herein, “endogenous” refers to proteins, nucleic acids, or genes that occur in nature and within a living system such as an organism, tissue or cell, but the term is not intended to exclude proteins that do not occur naturally in the patient or host.

[0054] Also as used herein, a “heat shock protein” is encoded by a “heat shock gene” or a stress gene, and refers to a gene that is activated or otherwise detectably unregulated due to the contact or exposure of an organism (containing the gene) to a stressor, such as heat shock, hypoxia, glucose deprivation, heavy metal salts, inhibitors of energy metabolism and electron transport, and protein denaturants, or to certain other compounds (such as benzoquinone ansamycins). Nover, L., Heat Shock Response, CRC Press, Inc., Boca Raton, Fla. (1991). “Heat shock protein” also includes homologous proteins encoded by genes within known stress gene families, even though such homologous genes are not themselves induced by a stressor.

[0055] Also as used herein, a “fusion protein” refers to a hybrid protein which comprises sequences from at least two different proteins. The sequences may be from proteins of the same or of different organisms. In various embodiments, the fusion protein may comprise one or more amino acid sequences linked to a first protein. In the case where more than one amino acid sequence is fused to a first protein, the fusion sequences may be multiple copies of the same sequence, or, alternatively, may be different amino acid sequences. A first protein may be fused to the N-terminus, the C-terminus, or the N-terminus and C- terminus of a second protein.

[0056] Also as used herein, “heat shock protein fusion protein” refers to a heat shock protein linked to another peptide or protein (e.g., a biotin-binding protein). For example, a heat shock protein may be C- or N- terminally joined to a biotin-binding protein to generate a heat shock protein fusion protein. When administered in conjunction with a biotinylated component provided herein, a heat shock protein fusion protein is capable of stimulating or enhancing humoral and/or cellular immune responses, including CD8 cytotoxic T cell (TCL) responses, to an antigen of interest.

[0057] Also as used herein, the term “biotinylated component” refers to a biotinylated protein, cell, or virus. Non-limiting examples of biotinylated proteins include biotinylated antigens, antibodies, and costimulatory molecules. The biotinylated component is to be administered to a subject in conjunction with a heat shock protein fusion protein as described herein.

[0058] Also as used herein, “concatemer” refers to a segment of DNA made up of multiple copies of the sequence linked together in tandem.

[0059] Also as used herein, a “pharmaceutically acceptable excipient or carrier” refers to any inert ingredient in a composition that is combined with an active agent in a formulation. A pharmaceutically acceptable excipient can include, but is not limited to, carbohydrates (such as glucose, sucrose, or dextrans), antioxidants (such as ascorbic acid or glutathione), chelating agents, low-molecular weight proteins, high-molecular weight polymers, gel-forming agents, or other stabilizers and additives. Other examples of a pharmaceutically acceptable carrier include wetting agents, emulsifying agents, dispersing agents, or preservatives, which are particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid. Examples of carriers, stabilizers or adjuvants can be found in Remington’s Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985).

[0060] Also as used herein, a “patient” refers to a subject, particularly a human (but could also encompass a non-human), who has presented a clinical manifestation of a particular symptom or symptoms suggesting the need for treatment, who is treated prophylactically for a condition, or who has been diagnosed with a condition to be treated.

[0061] Also as used herein, a “subject” encompasses the definition of the term “patient” and does not exclude individuals who are otherwise healthy.

[0062] Also as used herein, “treatment of’ and “treating” include the administration of a drug with the intent to lessen the severity of or prevent a condition, e.g., cervical cancer. [0063] Also as used herein, “prevention of’ and “preventing” include the avoidance of the onset of a condition, e.g., cervical cancer.

[0064] Also as used herein, a “condition” or “conditions” refers to those medical conditions that can be treated, mitigated, or prevented by administration to a subject of an effective amount of a drug.

[0065] Also as used herein, an “effective amount” refers to the amount of a drug that is sufficient to produce a beneficial or desired effect at a level that is readily detectable by a method commonly used for detection of such an effect. In some embodiments, such an effect results in a change of at least 10% from the value of a basal level where the drug is not administered. In other embodiments, the change is at least 20%, 50%, 80%, or an even higher percentage from the basal level. As will be described below, the effective amount of a drug may vary from subject to subject, depending on age, general condition of the subject, the severity of the condition being treated, the particular drug administered, and the like. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation.

[0066] Also as used herein, an “active agent” refers to any material that is intended to produce a therapeutic, prophylactic, or other intended effect, whether or not approved by a government agency for that purpose.

[0067] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to illuminate certain materials and methods and does not pose a limitation on scope. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosed materials and methods.

DETAILED DESCRIPTION

[0068] Human papillomavirus (HPV) is a common virus that accounts for approximately 640,000 new cancer cases per year globally and is characterized by abnormal tissue growth (e.g., warts) and other changes to cells. HPV is a group of more than 200 related viruses, some of which are spread through vaginal, anal, or oral sex. Sexually transmitted HPV types fall into two groups: low risk, which mostly cause no disease, and high risk. On the other hand, HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 are categorized as high risk due to their causative association with not only cervical disease (cervical intraepithelial neoplasia and cervical cancer), but also penile, anal, vulvar, vaginal, oropharyngeal, and laryngeal precancers or cancers.

[0069] Because HPV infects the squamous cells that line the inner surfaces of certain organs (e.g., cervix, oropharynx, anus, penis, vagina, and vulva), most HPV-related cancers are a type of cancer called squamous cell carcinoma. HPV proteins, such as the E6 and E7 proteins, are believed to contribute to carcinogenicity and are also rational cancer antigen targets for immunotherapy due to the fact that they are viral rather than human proteins. HPV-associated intraepithelial neoplasia or cancers are candidates for cancer immunotherapy since HPV oncoproteins, such as E6 and E7 proteins of high-risk HPVs, could be utilized as foreign antigens. Recently, checkpoint inhibitors (CPIs) have merged as a promising new treatment for solid cancers; however, these therapies have demonstrated only modest efficacy against HPV-associated cancers.

[0070] The present disclosure relates to a peptide-based self-assembling vaccine (SAV) platform to generate a highly potent immune response when administered to a subject and may be used in combination with checkpoint inhibitors in certain embodiments to treat HPV-induced tumors where the HPV proteins E6 and E7 are expressed by cancerous cells. The E6 and E7 viral oncoproteins of high risk HPV function by interfering with cellular tumor suppressor protein activity. The E6 proteins increase the turnover of the tumor suppressor protein p53 by targeting it for accelerated ubiquitin-mediated degradation. The E7 proteins repress gene transcription necessary for cell cycle progression enabling viral replication in cells outside of the dividing population.

[0071] HPV-related tumors have clear expression of tumor specific antigens (HPV proteins - E6 and E7) which contain multiple immunogenic peptide sequences that can be targeted by both the human and murine immune systems. CD4⁺ and CD8⁺ T cells contribute to controlling a virus during viral infection by producing effector cytokines (e.g., IFNy and TNF) and by exerting cytotoxic activity against virus-infected cells. Viral oncoproteins E6 and E7 of HPV are constitutively expressed in transformed cells, and are thus desirable targets for immunotherapy against HPV-induced malignancies. A purported mechanism of resistance is expression of PD-1 by tumor-infiltrating E6-specific activated T cells, as well as expression of PD-L1 by tumorinfiltrating immune cells. This has led to the approach of the current disclosure in combination with an immune checkpoint inhibitor in certain embodiments.

[0072] In one embodiment, the peptide is a single concatemer of three E6ZE7 epitopes (two MHC class I and one MHC class II) documented as being immunogenic in C57B1/6J mice chemically conjugated to biotin. The biotinylated peptide can self-assemble with a protein construct of a recombinant Mycobacterium tuberculosis heat shock protein 70 (MTbHSP70) fused with avidin (MAV) to form a self-assembled vaccine (SAV). The amino acid sequence for

MTbHSP70 is:

MARAVGIDLGTTNSVVSVLEGGDPVVVANSEGSRTTPSIVAFARNGEVLV GQPAKNQAVTNVDRTVRSVKRHMGSDWSIEIDGKKYTAPEISARILMKLK RDAEAYLGEDITDAVITTPAYFNDAQRQATKDAGQIAGLNVLRIVNEPTA AALAYGLDKGEKEQRILVFDLGGGTFDVSLLEIGEGVVEVRATSGDNHLG GDDWDQRVVDWLVDKFKGTSGIDLTKDKMAMQRLREAAEKAKIELSSSQS TSINLPYITVDADKNPLFLDEQLTRAEFQRITQDLLDRTRKPFQSVIADT GISVSEIDHVVLVGGSTRMPAVTDLVKELTGGKEPNKGVNPDEVVAVGAA LQAGVLKGEVKDVLLLDVTPLSLGIETKGGVMTRLIERNTTIPTKRSETF TTADDNQPSVQIQVYQGEREIAAHNKLLGSFELTGIPPAPRGIPQIEVTF DIDANGIVHVTAKDKGTGKENTIRIQEGSGLSKEDIDRMIKDAEAHAEED RKRREEADVRNQAETLVYQTEKFVKEQREAEGGSKVPEDTLNKVDAAVAE AKAALGGSDISAIKSAMEKLGQESQALGQAIYEAAQAASQATGAAHPGGE PGGAHPGSADDVVDAEVVDDGREAK which corresponds to SEQ ID NO: 1, and is also depicted in FIG. 1 A.

[0073] In another embodiment, two peptides composed of two MHC class I epitopes concatemerized with an MHC class II epitope are chemically synthesized to a biotin-PEG4 conjugate to form biotinylated antigenic peptides. The biotinylated antigenic peptides are then joined to a protein construct of a modified MTbHSP70 genetically fused with avidin (MAV) to form a SAV unit. It is contemplated that avidin has 4 high affinity binding sites for biotin which may result in E6 only and E7 only conjugates or multiple different arrangements of E6 and E7 peptides in different ratios. In certain embodiments, the ratio of different arrangements may be 3:1, 2:2, 2: 1, 1 : 1, or in any range defined therebetween. As used herein, “eSAV” refers to a selfassembled vaccine with peptides derived from HPV E6 and/or E7 proteins.

[0074] In another embodiment, two SAV units of a desired arrangement are combined in a 1 : 1 ratio to yield a mixed vaccine composition. In other embodiments the ratio may be in the range of about 1 :10 to about 10: 1, about 1 : 10, 2: 10, 3: 10, 4: 10, 5: 10, 6: 10, 7: 10, 8: 10, 9: 10, 10:9, 10:8, 10:7, 10:6, 10:5, 10:4, 10:3, 10:2, or 10: 1, or in any range defined therebetween.

[0075] In certain embodiments, the vaccine compositions of the present invention incorporate two immunogenic peptides that are synthetic chimeras, each combining two documented MHC class I and one documented MHC class II epitopes for HP VI 6 E6ZE7 into a single biotinylated concatemer. In certain embodiments, synthesis of these peptides involves dividing each of the two “long” peptides into smaller portions or fragments for solid phase synthesis, followed by assembly through click chemistry and purification by high-performance liquid chromatography (HPLC).

[0076] In certain embodiments, the MTbHSP70 portion of MAV contains a Val381 to Phe381 point mutation that is expected to reduce endogenous peptide binding and thereby improve the specificity of the platform for the incorporated biotinylated peptides. One or more other or additional point mutations may be present to further improve specificity, as would be appreciated by those of ordinary skill in the art.

[0077] The heat shock protein fusion protein and biotinylated components produced as described above may be purified to a suitable purity for use as a pharmaceutical composition. Generally, purified compositions will have one species that comprises more than about 85 percent of all species present in the composition, more than about 85%, 90%, 95%, 99%, or more of all species present. The object species may be purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single species. A skilled artisan may purify a heat shock protein fusion protein and biotinylated components, or a non-covalent complex of the same, using standard techniques for purification, for example, immunoaffinity chromatography, size exclusion chromatography, etc. Purity of a protein may be determined by a number of methods known to those of skill in the art, including, for example, amino-terminal amino acid sequence analysis, gel electrophoresis, and mass-spectrometry analysis.

[0078] Although, numerous embodiments herein are described with respect to the modified MTbHSP70, it is understood that other point mutations may be contemplated. The protein or proteins used may also be functional variants of the proteins mentioned herein and may exhibit a significant amino acid sequence identity compared to the original protein of SEQ ID NO: 1. For instance, the amino acid identity may amount to at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% to SEQ ID NO: 1. In this context, the term “functional variant” means that the variant of the protein is capable of, partially or completely, fulfilling the function of the naturally occurring corresponding protein. Functional variants of a protein may include, for example, proteins that differ from their naturally occurring counterparts by one or more amino acid substitutions, deletions, or additions.

[0079] The amino acid substitutions can be conservative or non-conservative. It is preferred that the substitutions are conservative substitutions, i.e., a substitution of an amino acid residue by an amino acid of similar polarity, which acts as a functional equivalent. Preferably, the amino acid residue used as a substitute is selected from the same group of amino acids as the amino acid residue to be substituted. For example, a hydrophobic residue can be substituted with another hydrophobic residue, or a polar residue can be substituted with another polar residue having the same charge. Functionally homologous amino acids, which may be used for a conservative substitution comprise, for example, non-polar amino acids such as glycine, valine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, and tryptophan. Examples of uncharged polar amino acids comprise serine, threonine, glutamine, asparagine, tyrosine and cysteine. Examples of charged polar (basic) amino acids comprise histidine, arginine, and lysine. Examples of charged polar (acidic) amino acids comprise aspartic acid and glutamic acid.

[0080] Also considered as variants are proteins that differ from their naturally occurring counterparts by one or more (e.g., 2, 3, 4, 5, 10, or 15) additional amino acids. These additional amino acids may be present within the amino acid sequence of the original protein (i.e., as an insertion), or they may be added to one or both termini of the protein. Basically, insertions can take place at any position if the addition of amino acids does not impair the capability of the polypeptide to fulfill the function of the naturally occurring protein in the treated subject. Moreover, variants of proteins also comprise proteins in which, compared to the original polypeptide, one or more amino acids are lacking. Such deletions may affect any amino acid position provided that it does not impair the ability to fulfill the normal function of the protein.

[0081] Variants of proteins (e.g, heat shock proteins, HPV E6/E7, etc.) also refer to proteins and peptide sequences that differ from the naturally occurring protein by structural modifications, such as modified amino acids. Modified amino acids are amino acids which have been modified either by natural processes, such as processing or post-translational modifications, or by chemical modification processes known in the art. Typical amino acid modifications comprise phosphorylation, glycosylation, acetylation, O-linked N-acetylglucosamination, glutathionylation, acylation, branching, ADP ribosylation, crosslinking, disulfide bridge formation, formylation, hydroxylation, carboxylation, methylation, demethylation, amidation, cyclization, and/or covalent or non-covalent bonding to phosphotidylinositol, flavine derivatives, lipoteichonic acids, fatty acids, or lipids.

[0082] The therapeutic vaccine contains one or more SAV units which comprise biotinylated antigenic peptides derived from targeted viral or oncogenic protein epitopes that are non- covalently bound to MTbHSP70 using a stable biotin-avidin engagement. Thus, MTbHSP70 can be expressed as a fusion protein with avidin (MAV) enabling spontaneous self-assembly with biotinylated peptides. It is contemplated that the MTbHSP70 could be expressed by itself separately or as a fusion protein using techniques familiar to those of ordinary skill in the art. [0083] Heat shock proteins (HSPs) are ubiquitously expressed proteins that act as chaperones in living systems. HSPs have the ability to present a broad repertoire of antigens to dendritic cells (DCs) and activate both innate and adaptive immune responses. HSP70, and in particular MTbHSP70, is believed to have significant immunopotency that contributes to a protective, adaptive immune response.

[0084] HSP70 examples include HSP72 and Hsc73 from mammalian cells, DnaK from bacteria, particularly mycobacteria such as Mycobacterium leprae and Mycobacterium tuberculosis (MTb). Without wishing to be bound by theory, it is believed that HSP70 binds ATP as well as unfolded proteins and participates in protein folding and unfolding, as well as in the assembly and disassembly of protein complexes. In at least one embodiment, the heat shock protein comprises (or is derived from) MTbHSP70. A heat shock protein fusion protein to be used in conjunction with the methods described herein may comprise a sequence that is at least 80%, 85%, 90%, 95% or 99% identical to SEQ ID NO: 1.

[0085] The heat shock protein fusion protein and biotinylated components described herein can be administered to a subject to induce or enhance that subject’s immune response, particularly a cell-mediated cytolytic response, against a cell expressing an antigen against which the biotinylated components are directed. The fusion protein may simply enhance the immune response (thus serving as an immunogenic composition), or confer protective immunity (thus serving as a vaccine).

[0086] In the vaccines of the present invention, the adjuvant may induce a Thltype immune response in certain embodiments. The fusion protein SAV approach utilizing MTbHSP70’s adjuvant ability is advantageous to previous approaches which lacked an adjuvant system that could safely induce both CD4⁺ and CD8⁺ T cell responses against the protein targets while avoiding unwanted reactogenicity. In comparison to alum, use of SAV units as described herein shows improved immune responses in CD4⁺ and CD8⁺ T cells expressing IFNy, TNFa, and IL-2 when both groups are compared to saline control group.

[0087] Pharmaceutical compositions that contain the vaccines of the present invention may be prepared as either liquid solutions or suspensions. The pharmaceutical composition of the invention can include commonly used pharmaceutically acceptable excipients, such as diluents and carriers. In particular, the composition can comprise a pharmaceutically acceptable carrier, such as PBS buffer. In addition to the carrier, the pharmaceutical composition may also contain emulsifying agents, pH buffering agents, stabilizers, dyes, and the like.

[0088] In certain embodiments, a pharmaceutical composition will comprise a therapeutically effective HSP70 dose per vaccine, which is a dose that is capable of generating a highly potent immune response when administered to a subject, and/or capable of treating some HPV-related cancers, for example, in combination with checkpoint inhibitors, without being toxic to the subject. Treatment of solid tumors in some HPV-related cancers may be assessed as a change in a phenotypic characteristic associated with HPV-related solid tumors (e.g., volume of the tumor) or a change in immune response or expression. Thus, a therapeutically effective HSP70 per vaccine dose is typically one that, when administered in a physiologically tolerable composition, is sufficient to increase immunogenic responses and decrease tumor growth volume in the treated subject.

[0089] A suitable dose of the therapeutically effective HSP70 may be in range of about 150- 500 pg. Preliminary experiments indicate that the HSP70 had a positive adjuvanting effect and that there was a significant increase in the percentage of splenic and lymph node in both CD4⁺ and CD8⁺ T cells expressing JFNy as a recall response to E6 and E7 peptides. An advantageous immunogenic response was observed at about 215 pg HSP70 per vaccine dose.

[0090] The heat shock protein fusion protein and biotinylated components, or a non-covalent complex of the same, as described herein can be administered to a subject in a variety of ways. The routes of administration include systemic, peripheral, parenteral, enteral, topical, and transdermal (e.g., slow release polymers). Any other convenient route of administration can be used, for example, infusion or bolus injection, or absorption through epithelial or mucocutaneous linings.

[0091] In at least one embodiment, a composition comprising SAV may be transduced into a subject intradermally in a Prime-Boost-Boost schedule with 14-day intervals. The intradermal (ID) route of administration is particularly advantageous for allowing MTbHSP70 to directly stimulate epidermal Langerhans/dendritic cells. There are significant advantages to using ID vaccination over the intramuscular (IM) route. ID vaccination generally enhances vaccine responses, providing a more direct route to the extensive cutaneous immune system through epidermal dendritic cells and draining cutaneous lymphatics. Extracellular heat shock proteins, such as MTbHSP70, are potent inducers of innate and adaptive immunity. The dermal dendritic cell network is especially rich in immune-activating receptors for HSPs, including CD40, CD36, LOX-1, SR- A, TLR-2, and TLR-4. Thus, ID vaccination with SAV advantageously leverages the rich dermal antigen processing system and heat shock protein interaction.

[0092] In at least one embodiment, the pharmaceutical compositions may be administered as such or in admixtures in conjunction with other agents. Conjunctive (combination) therapy thus includes sequential, simultaneous and separate, or co-administration in a way that the therapeutic effects of the first administered one has not entirely disappeared when the subsequent is administered. [0093] In at least one embodiment, compositions comprising SAV may be transduced ID in combination with an approved anti-PD-1 agent, an immune checkpoint inhibitor. Immune checkpoint inhibitors sustain antitumor activities by interfering with T-cell coinhibitory signaling pathways. One of the important mechanisms by which cancer cells evade immune surveillance is the activation of immune checkpoint pathways, which suppress antitumor responses by causing T- cell exhaustion or anergy. Tumor cells and tumor- specific CD8⁺ cytotoxic T cells (CTLs) primarily function in a situation of mutual suppression through tethering engagement with PD-1/PD-L1 within the tumor mass. When the mutual suppression is abrogated by either anti- PD-1 or anti-PD-Ll specific antibodies, CD8⁺ CTLs are released by disconnecting the tethering chains, thus regaining their cytotoxicity. These activated CD8⁺ CTLs will attack and eliminate tumor cells.

[0094] HPV-related cancers that may be treated by the methods disclosed herein may include, without limitations, head and neck cancer, cervical cancer, anal carcinoma, other HPV-induced cancers expressing the E6 and E7 oncoproteins, and combinations thereof.

ILLUSTRATIVE EXAMPLES

[0095] The following example is set forth to assist in understanding the disclosure and should not, of course, be construed as specifically limiting the embodiments described and claimed herein. Such variations of the embodiments now known or later developed, which would be within the purview of those skilled in the art, and changed in formulation or minor changes in experimental design, are to be considered to fall within the scope of the embodiments incorporated herein.

Example 1: Sequence of peptides used and related epitopes

[0096] In this example, the two immunogenic peptides are synthetic chimeras, each combining two documented MHC class I and one documented MHC class II murine epitopes for HPV 16 E6ZE7 into a single biotinylated concatemer. Table 1 illustrates the peptide sequences, along with the three epitope identities each separated by linker sequences. The first column indicates the epitope and linker sequences for an E6 peptide and the second column indicates the epitope and linker sequences for an E7 peptide.

Table 1 : Exemplary peptide sequences containing three epitope identities each separated by linker sequences

[0097] The peptide sequence used in the studies below incorporated concatemerized MHC class I and II epitopes against HPV proteins E6 and E7. This peptide sequence is shown in Table 2, which illustrates the spacer and epitope structure, and has the following sequence:

LEQLERVKREVYDFAFRDLAAYRVKRQAEPDRAHYNIVTFCCKCDGPGPGRVKRYML DLQPET

(SEQ ID NO: 2).

Table 2: Peptide sequence used in immunogenicity studies

Example 2: Design of Human Immunogenic HPV E6/E7 Peptides

[0098] Targeted E6ZE7 peptide sequences selected for presentation on human MHC class I and II are contemplated. The final, two “long” biotinylated peptides (i.e., one E6 and one E7), can be formed using two smaller peptide fragments through a click chemistry mechanism. FIG. IB illustrates both the smaller peptide fragments, involving the DBCO-containing peptide and the azide (NNN)-containing peptide, and the final sequence which is a conjugate of peptides one and two via a triazole moiety. The schematic in FIG. 1C depicts the mechanism of the click chemistry reaction. The fully formed peptide was then added to the MAV through a non-covalent biotinavidin bond. The peptide fragments of FIG. 1C correspond to the following sequences: LEQLERVKREVYDFAFRDLCIVYRDGNPYAVRVKRGG (SEQ ID NO: 3);

PYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLL (SEQ ID NO: 4); LEQLERVKRPTLHEYMLDLQPETTDLYCYGG (SEQ ID NO: 5); and LEQLERVKRAGQAEPDRAHYNIVTFRVKRLRLCVQSTHVDIRTL (SEQ ID NO: 6).

Example 3: Sequence and Properties ofMAV

[0099] In this example, a MAV protein as the adjuvanting component of the vaccine was synthesized to include a histidine tag, a MTbHSP70, a, polyglycine linker, and an avidin using techniques readily available to and understood by those of ordinary skill in the art. FIG. 2 depicts a Val381 to Phe381 point mutation in the MTbHSP70 portion which is expected to inactivate endogenous peptide binding and thereby improve the specificity of the platform for the incorporated biotinylated peptides. The 771 amino acid sequence of FIG. 2 corresponds to SEQ ID NO: 7.

[0100] Table 3 shows the properties of the MAV unit including the molecular weight and concentration, which were assessed and confirmed using gel electrophoresis and optical extinction at 280nm respectively.

Table 3: Properties of synthesized MAV unit

Example 4: Assembly of eSAV

[0101] To prepare eSAV, five times molar excess of biotinylated peptide 3 from Table 2 was added to MAV in PBS at room temperature for 12h with end-over-end mixing. Any precipitated peptide was removed by centrifugation. The assembly reaction was prepared for 80 pg MAV per 50 pL dose of eSAV. The resultant eSAV was stored at -80 °C until use.

[0102] The mechanism of self-assembly through a stable biotin-avidin engagement to form an eSAV unit is illustrated in FIG. 3. To validate this assembly, the absorbance of the eSAV unit after adding HABA dye was compared to that of the unassembled MAV and this difference in absorbance was used to calculate the moles of biotinylated peptides attached per mole of MAV. Self-assembled constructs joined with different peptides can be mixed together to target a vaccine against a large number of epitopes. Example 5: Immune Responses to eSAV

[0103] In this example, the immune responses for different T cell types in mice are shown following vaccination with eSAV. For vaccination, C57BL/6 mice were injected intradermally with agent suspended in sterile PBS to a volume of 50 pL on each occasion.

[0104] In FIG. 4A, the expression of fFNy in splenocytes following stimulation is demonstrated. The eSAV-vaccinated group demonstrated a statistically significant increase in IFNy expression in CD4⁺ and CD8⁺ cells compared to the saline control group. In the eSAV group, 9.5% of the CD4⁺ cells (p < 0.01) and 6.5% of the CD8⁺ cells (p < 0.05) were positive for IFNy expression after recall exposure to peptides. No other group reached statistical significance. These results show that a significant, balanced immune response was elicited by the eSAV compared to the saline. There was no statistically significant difference in CD4⁺ IFNy⁺ (p=0.68) and CD8⁺ IFN'y⁺ (p=0.83) between the eSAV and the 5% Alum + peptide treated group.

[0105] In FIG. 4B, the results of peptide stimulation on IL-2 expression in splenic T cells are demonstrated. For this cytokine, only the eSAV achieved a statistically significant increase in expression of IL-2 compared to the saline group and this occurred only in CD4⁺ cells. There was no statistically significant difference in the IL-2 secretion between the eSAV and the 5% Alum + peptide treated group.

[0106] In FIG. 4C, the results of expression in T cells of TNFa are demonstrated. Only T cells from the eSAV treated group showed statistically significant expression difference from saline. 10% of the CD4⁺ cells (p < 0.01) and 5.5% of the CD8⁺ cells (p < 0.05) expressed TNFa in response to peptide stimulation. There was no statistically significant difference in the TNFa secretion between the eSAV and the 5% Alum + peptide treated group.

[0107] In FIG. 4D, the results of expression in splenic T cells of CD62L are demonstrated. For this ligand, none of the groups showed any significant increase in expression. There was no statistically significant difference in the CD62L expression between the eSAV and the 5% Alum + peptide treated group.

[0108] In these examples, illustrated by FIGS. 4A-4D, each data point represents cells from an individual mouse and the horizontal bars indicate mean expression within group and class. Significant p values, relative to saline are shown after adjustment for multiple hypothesis testing. In all graphs, a single * represents a p value of < 0.05 and ** represents a p value of < 0.01.

Example 6: Tumor Response to eSAV

[0109] In this example, the effect of either eSAV or alum (5%) + peptide vaccination on the growth of the TC-1 tumors in female C587BL/6 mice are shown. For this tumor treatment study, cultured cancer cells (TC-1) were suspended in sterile PBS at a concentration of 10 thousand per 100 pL and injected into loose skin over the flank of 8 to 10 week old female C57BL/6 mice with an insulin syringe. The tumor growth was measure twice a week and tumor volume was calculated using the formula, volume = (a x b²)/2, in which a and b are the largest and the smallest tumor diameters, respectively.

[0110] In the top plot of FIG. 5, the growth of the tumor in each mouse is shown separately, while in the bottom plot the mean tumor size for each treatment group is shown. This data shows that eSAV vaccination had a greater effect on reducing the volume of tumor growth over the duration of the study in comparison with peptide + alum (5%) vaccination.

Example 7: Probability of Survival

[oni] In this example, the probability of survival of mice in the tumor treatment study is shown and compared between eSAV and peptide + alum (5%) vaccination. In FIG. 6, the mean survival of the eSAV treated group was statistically significant over those who received only saline and approached statistical significance compared to the alum (5%) + peptide group.

Example 8: Dosage- Based CD8⁺ Immune Response

[0112] In this example, the CD8⁺ T cell immune responses in splenocytes and lymphocytes are shown following stimulation with E6/E7 peptides at different vaccine doses. In FIG. 7A, the immune responses for splenocytes are demonstrated. For IFNy, only the 130, 215 and 350 pg groups showed a statistically-significant increase in expression compared to the saline control group. The two highest dose groups showed the most significant responses (p < 0.0001). None of the groups showed a significant TNFa response. For IL-2, only the 215 pg group had a statistically significant recall response.

[0113] In FIG. 7B, the CD8⁺ T cell responses in lymphocytes are shown. A similar pattern was observed, with significant IFNy only in the 130, 215 and 350 pg groups. In this case, responses at 215 pg were best. There was no significant TNFa expression found in any group. IL-2 was only significant at 215 pg.

[0114] In these examples, as illustrated in FIGS. 7A-7B, T cell responses are shown as a percentage of live T cells. Each data point represents cells from an individual mouse and the horizontal bars indicate mean expression within group and class. Significant p values, relative to saline group are shown after adjustment for multiple hypothesis testing. In these examples, significance markers are as follows: * = p value of < 0.05, ** = p value of < 0.01, *** = p value of < 0.001, **** = p value of < 0.0001. Example 9: Dosage- Based CD4⁺ Immune Response

[0115] In this example, the CD4⁺ T cell immune responses in splenocytes and lymphocytes are shown following stimulation with E6ZE7 peptides at different vaccine doses. In FIG. 8A, the immune responses in splenocytes are demonstrated. For IFNy, only the 215 and 350 pg groups showed a statistically-significant increase in expression compared to the saline control group. Both groups showed a very significant response (p < 0.0001). None of the groups showed a significant TNFa response. For IL-2, only the 215 pg group had a statistically significant recall response.

[0116] In FIG. 8B, the CD4⁺ T cell responses in lymphocytes showed a slightly broader range of immune responses. For IFNy, significant response were seen in the 130, 215 and 350 pg groups with 215 pg appearing to be the best. For TNFa, there was a significant response in the 215 pg group and for IL-2, the 130 and 250 pg groups showed significant responses.

[0117] In these examples, as illustrated in FIGS. 8A-8B, T cell responses are shown as a percentage of live T cells. Each data point represents cells from an individual mouse and the horizontal bars indicate mean expression within group and class. Significant p values, relative to saline group are shown after adjustment for multiple hypothesis testing. In these examples, significance markers are as follows: * = p value of < 0.05, ** = p value of < 0.01, *** = p value of < 0.001, **** = p value of < 0.0001.

Example 10: Reactogenicity of SAV in combination with PD-1 Checkpoint Inhibitor Therapy [0118] In this example, the reactogenicity of the vaccine/antibody treatment was evaluated. No reactogenicity was observed for the any of the treatments in all groups for the duration of the study.

Example 11: Tumor Growth Kinetics

[0119] In this example, the tumor growth kinetics of the vaccine and anti-mPDl, and the vaccine and anti-IgG2a treated groups are shown. In FIGS. 9A-B, eSAV significantly reduced TC-1 tumor volume with reduced tumor volume in 80, 130 and 215 pg eSAV receiving mice. There was no statistically significant difference among anti-mPDl treated eSAV receiving mice compared to the matched anti-IgG2a treated group.

Example 12: Survival Curve

[0120] In this example, the survival curves of the vaccine and anti-mPDl, and the vaccine and anti-IgG2a treated groups are shown. In FIGS. 11 A-B, SAV has a highly significant and positive survival effect at 80, 130 and 215 pg per vaccine dose in the TC-1 tumor injected mice (p <0.0001). There was also a dose-response to SAV with the overall best survival benefit to the TC-1 tumor bearing mice 215 pg SAV. There was a statistically significant survival benefit of anti-mPDlin peptide treated mice compared to anti-IgG2a treated group.

[0121] In the foregoing description, numerous specific details are set forth, such as specific materials, dimensions, processes parameters, etc., to provide a thorough understanding of the present invention. The particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments. The words “example” or “exemplary” are used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “example” or “exemplary” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Rather, use of the words “example” or “exemplary” is simply intended to present concepts in a concrete fashion. As used in this application, the term “or” is intended to mean an inclusive “or” rather than an exclusive “or.” That is, unless specified otherwise, or clear from context, “X includes A or B” is intended to mean any of the natural inclusive permutations. That is, if X includes A; X includes B; or X includes both A and B, then “X includes A or B” is satisfied under any of the foregoing instances. Reference throughout this specification to “an embodiment,” “certain embodiments,” or “one embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrase “an embodiment,” “certain embodiments,” or “one embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment.

[0122] The present invention has been described with reference to specific exemplary embodiments thereof. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art and are intended to fall within the scope of the appended embodiments.

Claims

1. A pharmaceutical composition comprising a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to two or more biotinylated peptides, and wherein the two or more biotinylated peptides each comprise a sequence at least partially derived from an epitope of a human virus antigen protein with the proviso that each epitope is of a different human virus antigen protein of the same human virus.

2. A pharmaceutical composition comprising a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to a biotinylated peptide, and wherein the biotinylated peptide comprises a sequence at least partially derived from one or more epitopes of one or more human papillomavirus (HPV) antigen proteins.

3. The pharmaceutical composition of claim 2, wherein the biotinylated peptide comprises two or more epitope sequences each separated by a linker sequence.

4. The pharmaceutical composition of claim 2, wherein the one or more HPV antigen proteins comprise E6 protein or E7 protein.

5. A pharmaceutical composition comprising a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to at least one biotinylated peptide, and where the at least one biotinylated peptide comprises at least one amino acid sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

6. A pharmaceutical composition comprising a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein is non-covalently bound to a biotinylated peptide, wherein the biotinylated peptide is formed by joining two peptide fragments, the peptide fragments each having amino acid sequences selected from the group consisting of: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

7. The pharmaceutical composition of any one of the preceding claims, wherein the heat shock protein is a mammalian heat shock protein or a bacterial heat shock protein.

8. The pharmaceutical composition of any one of claims 1-6, wherein the heat shock protein is selected from the group consisting of Mycobacterium tuberculosis heat shock protein 70 (MTbHSP70) and a human heat shock protein.

9. The pharmaceutical composition of any one of claims 1-6, wherein the heat shock protein is a member of the heat shock protein 70 (HSP70) family.

10. The pharmaceutical composition of any one of claims 1-6, wherein the heat shock protein is or is derived from MTbHSP70.

11. The pharmaceutical composition of any one of claims 1-6, wherein the heat shock protein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1.

12. The pharmaceutical composition of any one of claims 1-6, wherein the heat shock protein has an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1 containing a Val381 to Phe381 point mutation.

13. A pharmaceutical composition comprising: a fusion protein derived from a heat shock protein; a first peptide non-covalently bound to the fusion protein, the first biotinylated peptide comprising a human papillomavirus (HPV) E6 protein epitope; and a second peptide non-covalently bound to the fusion protein, the second biotinylated peptide comprising an HPV E7 protein epitope.

14. The pharmaceutical composition of claim 13, wherein the fusion protein comprises MTbHSP70-avidin fusion protein.

15. The pharmaceutical composition of any one of the preceding claims, wherein the biotinbinding protein is selected from a group consisting of avidin, streptavidin, and neutravidin.

16. The pharmaceutical composition of any one of the preceding claims, further comprising a pharmaceutically acceptable excipient.

17. The pharmaceutical composition of any one of the preceding claims, wherein the pharmaceutical composition is a vaccine composition.

18. A method for inducing an immune response in a subject, the method comprising administering to the subject the pharmaceutical composition of any one of the preceding claims.

19. The method of claim 18, wherein the pharmaceutical composition comprises a pharmaceutically-accepted excipient, and wherein a therapeutic dose administered to the subject comprises about 150 pg to about 500 pg of the heat shock protein.

20. A method of treating an HPV-induced tumor of a subject for which HPV proteins E6 and/or E7 are expressed by cancerous cells of the tumor, the method comprising administering to the subject the pharmaceutical composition of any of claims 1-17.