Vaccines incorporating protein-based immune adjuvants

Abstract

Compositions and methods for treating certain HPV-associated solid tumors in mammalian subjects are disclosed. In one embodiment, the method comprises intradermal delivery of a self-assembling vaccine to a subject. In at least one embodiment, the self-assembling vaccine comprises a fusion protein non-covalently linked to two or more biotinylated E6 / E7 peptides derived from target viral or oncoprotein epitopes using biotin-avidin engagement.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 359,560, filed July 8, 2022, the disclosure of which is incorporated by reference herein in its entirety.

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

[0003] Adjuvants are compounds approved for use in human vaccines in the United States that, when administered with an antigen, function as immune stimulants and enhance antibody production. Alum, in particular, has been widely used as an adjuvant over the past several decades. However, while alum induces a Th2-biased immune response, it is ineffective at inducing a Th1 cell-mediated immune response, which is essential for the treatment of some solid tumors. Inducing a Th1 cell-mediated immune response is desirable because it plays a key role in inducing a therapeutically relevant immune response while maintaining a high level of safety.

[0004] Over the past 30 years, numerous attempts have been made to develop therapeutic vaccines as a treatment modality for many solid tumors, but these approaches have lacked therapeutic efficacy. Furthermore, current vaccine strategies are time-consuming and labor-intensive, can only be initiated when a threat arises, and it is not feasible to generate personalized vaccines to combat diseases whose target antigens vary from person to person. Therefore, to generate therapeutic vaccines for a number of scenarios, including but not limited to the treatment of some solid tumors (e.g., cervical cancer caused by human papillomavirus (HPV)), or due to the potential for rapidly evolving, fast-acting, and / or highly contagious threats, alternative approaches (e.g., platform vaccine technology) are needed. Summary of the Invention

[0005] The following summary presents a simplified overview of various aspects of the disclosure to provide a basic understanding of such aspects. This summary is not an extensive overview of the disclosure. It is not intended to identify key or critical elements of the disclosure, nor to delineate the scope of any particular embodiments of the disclosure or the 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, the 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, each of the two or more biotinylated peptides comprising a sequence derived at least in part from an epitope of a human viral antigenic protein, wherein each epitope is of a different human viral antigenic protein of the same human virus.

[0007] In a further aspect, the 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 the biotinylated peptide comprises a sequence derived at least in part from one or more epitopes of one or more human papillomavirus (HPV) antigenic proteins.

[0008] In at least one embodiment, 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 antigenic proteins include an E6 protein or an E7 protein.

[0010] In a further aspect, the 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 wherein 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, the 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, the biotinylated peptide being formed by linking two peptide fragments, each of which has 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.

[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 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 at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1, which contains a point mutation from Val381 to Phe381.

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

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

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

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

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

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

[0024] The above and other features of the present disclosure, its nature and various advantages will become more apparent from the following detailed description considered in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0025] [Figure 1A] The amino acid sequence of Mycobacterium tuberculosis heat shock protein 70 is shown. [Figure 1B] 1 is a table summarizing exemplary E6 / E7 peptide sequences formed through click chemistry of two peptide fragments according to at least one embodiment. [Figure 1C] 1 illustrates a click chemistry reaction to form a triazole moiety according to at least one embodiment. [Figure 2] 1 shows the amino acid sequence of a fusion protein according to at least one embodiment. [Figure 3] FIG. 1 is a schematic diagram showing spontaneous self-assembly of a fusion protein and a biotinylated peptide according to at least one embodiment. [Figure 4A] 1 includes a plot showing expression of IFNγ in splenocytes after stimulation according to at least one embodiment. [Figure 4B] 1 includes a plot showing expression of IL-2 in splenocytes after stimulation according to at least one embodiment. [Figure 4C] 1 includes a plot showing expression of TNFα in splenocytes after stimulation according to at least one embodiment. [Figure 4D] 1 includes a plot showing expression of CD62L on splenocytes after stimulation according to at least one embodiment. [Figure 5] According to at least one embodiment, a plot showing the therapeutic effect of eSAV, 5% alum E6-E7 peptide, or saline on the growth of syngeneic tumors is included. [Figure 6] 1 includes a plot showing the effect of treatment with eSAV, 5% alum-E6-E7 peptide, or saline on mouse survival, according to at least one embodiment. [Figure 7A] 1 includes plots showing expression of IFNγ, TNFα, and IL-2 in CD8+ splenocytes after stimulation with different doses of eSAV, according to at least one embodiment. [Figure 7B] 1 includes plots showing expression of IFNγ, TNFα, and IL-2 in CD8+ lymphocytes after stimulation with eSAV at different doses, according to at least one embodiment. [Figure 8A] 1 includes plots showing expression of IFNγ, TNFα, and IL-2 in CD4+ splenocytes after stimulation with eSAV at different doses, according to at least one embodiment. [Figure 8B] 1 includes plots showing expression of IFNγ, TNFα, and IL-2 in CD4+ lymphocytes after stimulation with eSAV at different doses, according to at least one embodiment. [Figure 9A] According to at least one embodiment, a plot showing tumor growth kinetics for vaccine and anti-mPD1 treatment groups at different vaccine doses is included. [Figure 9B] According to at least one embodiment, a plot showing tumor growth kinetics for vaccine and anti-IgG2a treatment groups at different vaccine doses is included. [Figure 10A] According to at least one embodiment, a plot showing survival curves for vaccine and anti-mPD1 treatment groups at different vaccine doses is included. [Figure 10B] According to at least one embodiment, a plot showing survival curves for vaccine and anti-IgG2a treatment groups at different vaccine doses is included.

[0026] definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a drug" includes a single drug and mixtures of two or more different drugs, reference to "an adjuvant" includes a single adjuvant and mixtures of two or more different adjuvants, etc.

[0027] Also, as used herein, "about," when used in connection with a measurand, refers to the normal variation in the measurand that would be expected by one of ordinary skill in the art making the measurement using a level of care commensurate with the purpose of the measurement and the precision of the measuring device. In certain embodiments, the term "about" includes the stated value plus or minus 10%, such as "about 10" including 9-11.

[0028] Also, as used herein, "including" is used to mean "including but not limited to."

[0029] Also, as used herein, "protein" has its normal and accustomed meaning in the art and includes or refers to a polypeptide (i.e., a chain of at least two amino acids linked together by peptide bonds). Polypeptides include natural amino acids, unnatural 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 contain non-amino acid moieties (e.g., glycoproteins) and may be processed or modified. A protein may be an entire polypeptide chain produced in a cell, or a functional portion thereof. A protein may include multiple polypeptide chains that are chemically linked (e.g., by disulfide bonds), non-chemically linked (e.g., by hydrogen bonds), 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.

[0030] Also, as used herein, the term "biotin-binding protein" refers to a protein capable of non-covalently binding to biotin. Biotin-binding proteins may be monomers, dimers, or tetramers, capable of forming monovalent, divalent, or tetravalent pharmaceutical compositions, respectively, as described herein. Non-limiting examples include anti-biotin antibodies, avidin, streptavidin, and neutravidin. Avidin may comprise mature avidin or a sequence at least 80%, 85%, 90%, 95%, or 99% identical to the sequence identified in NCBI Accession No. NP990651. Streptavidin may comprise a sequence at least 80%, 85%, 90%, 95%, or 99% identical to the sequence identified in NCBI Accession No. AAU48617, for example. The term "biotin-binding protein" is intended to encompass wild-type and derivatives of avidin, streptavidin, and neutravidin that form monomers, dimers, and tetramers.

[0031] 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 more diseases. A vaccine is a preparation that provides immunity to one or more diseases or stimulates an immune response to one or more diseases. Vaccines may include killed or attenuated causative agents of the diseases, products, or derivatives of the drugs and synthetic alternatives. Synthetic alternatives may include preparations consisting of synthetic peptides, carbohydrates, antigens, or strands of RNA or DNA.

[0032] Also, as used herein, "epitope" refers to the region of an antigen to which an antibody binds preferentially and specifically.

[0033] Also, as used herein, "linker" refers to a molecule or group of molecules that connect two molecules, such as a molecule that forms a covalent bond between a heat shock protein and a biotin-binding protein. A linker may consist of a single linking molecule or may include a linking molecule and a spacer molecule intended to separate the linking molecule and the moiety by a specific distance.

[0034] Also, as used herein, "immunogenicity" refers to the ability of a substance to elicit an immune response. An immune response refers to a subject's reaction to the presence of an antigen and may include at least one of the following: the production of antibodies, the development of immunity, the development of hypersensitivity to the antigen, and the development of immune tolerance.

[0035] Also, as used herein, "endogenous" refers to a protein, nucleic acid, or gene that is present in nature and within a biological system (e.g., an organism, tissue, or cell), but the term is not intended to exclude proteins that do not naturally occur within a patient or host.

[0036] Also, as used herein, "heat shock protein" refers to a gene encoded by a "heat shock gene" or stress gene and that is activated or otherwise detectably regulated by contact or exposure of the organism (containing the gene) to a stressor (e.g., heat shock, hypoxia, glucose deprivation, heavy metal salts, inhibitors of energy metabolism and electron transport, and protein denaturants, or certain other compounds (e.g., benzoquinone ansamycins)). Nover, L., Heat Shock Response, CRC Press, Inc., Boca Raton, Fla. (1991). "Heat shock protein" also includes, for example, homologous proteins encoded by genes within known stress gene families, even if such homologous genes are not themselves induced by the stressor.

[0037] Also, as used herein, "fusion protein" refers to a hybrid protein containing sequences from at least two different proteins. The sequences may be from proteins of the same organism or different organisms. In various embodiments, the fusion protein may include one or more amino acid sequences linked to a first protein. When multiple amino acid sequences are fused to the first protein, the fusion sequences may be multiple copies of the same sequence or different amino acid sequences. The first protein may be fused to the N-terminus, C-terminus, or N- and C-terminus of the second protein.

[0038] Also, as used herein, "heat shock protein fusion protein" refers to a heat shock protein conjugated to another peptide or protein (e.g., a biotin-binding protein). For example, a heat shock protein may be conjugated to the C-terminus or N-terminus of a biotin-binding protein to generate a heat shock protein fusion protein. When administered in combination with a biotinylated component provided herein, the heat shock protein fusion protein can stimulate or enhance humoral and / or cellular immune responses, including CD8 cytotoxic T cell (TCL) responses, against an antigen of interest.

[0039] 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 administered to a subject in combination with a heat shock protein fusion protein described herein.

[0040] Also, as used herein, a "concatamer" refers to a DNA segment made up of multiple copies of a sequence linked in tandem.

[0041] Also, as used herein, "pharmaceutically acceptable excipient or carrier" refers to an inactive ingredient in a composition that is combined with an active drug in a formulation. Pharmaceutically acceptable excipients may include, but are not limited to, carbohydrates (such as glucose, sucrose, or dextran), 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 pharmaceutically acceptable carriers 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 are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th Ed. (1985).

[0042] Also, as used herein, "patient" refers to a subject, particularly a human (but will also include non-humans), who is exhibiting clinical signs of a particular condition or symptoms indicating the need for treatment, is being treated prophylactically for a condition, or has been diagnosed with a condition to be treated.

[0043] Also, as used herein, "subject" encompasses the definition of the term "patient" and does not exclude otherwise healthy individuals.

[0044] Also, as used herein, "treatment" and "treating" include the administration of a drug intended to reduce the severity of or prevent a condition (e.g., cervical cancer).

[0045] As used herein, "prevention" and "preventing" also include, for example, the avoidance of the onset of a condition (eg, cervical cancer).

[0046] Also, as used herein, "condition" or "conditions" refers to medical conditions that can be treated, alleviated, or prevented by administering to a subject an effective amount of a drug.

[0047] Also, as used herein, "effective amount" refers to an amount of drug sufficient to produce a beneficial or desired effect at a level readily detectable by methods commonly used to detect such effects. In some embodiments, such an effect results in at least a 10% change from basal levels when the drug is not administered. In other embodiments, the change is at least 20%, 50%, 80%, or even higher from basal levels. As explained below, the effective amount of a drug may vary from subject to subject, depending on the subject's age, general condition, the severity of the disease being treated, the particular drug being 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 relevant texts or literature and / or by routine experimentation.

[0048] Also, as used herein, "active drug" refers to any substance intended to produce a therapeutic, prophylactic, or other intended effect, regardless of whether it has been approved by a government agency for that purpose.

[0049] The recitation of ranges of values herein is intended merely 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 herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted otherwise by context. The use of any and all examples, or exemplary language (e.g., "such as") presented herein is intended merely to facilitate understanding of particular materials and methods and does not impose a limitation on the scope of the claims. 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 OF THE INVENTION

[0050] Human papillomavirus (HPV) is a common virus responsible for approximately 640,000 new cancer cases worldwide each year and is characterized by abnormal tissue growths (e.g., warts) and other cellular changes. 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 are divided into two groups: low-risk, which rarely cause disease, and high-risk. HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 are classified as high-risk due to their causative association with cervical disease (cervical intraepithelial neoplasia and cervical cancer) as well as precancerous conditions or cancers of the penis, anus, vulva, vagina, oropharynx, and larynx.

[0051] Because HPV infects squamous epithelial cells that line certain organs (e.g., the cervix, oropharynx, anus, penis, vagina, and vulva), most HPV-associated cancers are a type of cancer called squamous cell carcinoma. HPV proteins (e.g., E6 and E7 proteins) are thought to contribute to carcinogenesis and are rational cancer antigen targets for immunotherapy, based on the fact that they are viral rather than human proteins. HPV-associated intraepithelial neoplasia (HIPNs) or cancers are candidates for cancer immunotherapy because they can utilize HPV oncoproteins (e.g., E6 and E7 proteins of high-risk HPV) as foreign antigens. Recently, checkpoint inhibitors (CPIs) have been integrated as promising new treatments for solid tumors, but these therapies have shown limited efficacy against HPV-associated cancers.

[0052] The present disclosure relates to a peptide-based self-assembling vaccine (SAV) platform that generates a highly potent immune response when administered to a subject. In certain embodiments, HPV proteins E6 and E7 are expressed in cancer cells and can be used in combination with checkpoint inhibitors to treat HPV-induced tumors. The E6 and E7 viral oncoproteins of high-risk HPV function by inhibiting the activity of cellular tumor suppressor proteins. The E6 protein increases the turnover of the tumor suppressor protein p53 by targeting it for ubiquitin-mediated degradation. The E7 protein represses gene transcription required for cell cycle progression, allowing viral replication in cells outside the dividing population.

[0053] HPV-associated tumors clearly express tumor-specific antigens (HPV proteins E6 and E7), which contain multiple immunogenic peptide sequences that can be targeted by both the human and mouse immune systems. + and CD8 + T cells contribute to viral control during viral infection by producing effector cytokines (e.g., IFNγ and TNF) and exerting cytotoxic activity against virus-infected cells. HPV viral oncoproteins E6 and E7 are constitutively expressed in transformed cells, making them desirable targets for immunotherapy against HPV-induced malignancies. The intended mechanism of resistance is the expression of PD-1 by tumor-infiltrating E6-specific activated T cells and PD-L1 by tumor-infiltrating immune cells. This has led, in certain embodiments, to the approach of the present disclosure in combination with immune checkpoint inhibitors.

[0054] In one embodiment, the peptide is a single concatemer of three E6 / E7 epitopes (two MHC class I and one MHC class II) that have been demonstrated to be immunogenic in C57B1 / 6J mice chemically conjugated to biotin. The biotinylated peptide can self-assemble with the protein structure of recombinant Mycobacterium tuberculosis heat shock protein 70 (MTbHSP70) fused to avidin (MAV) to form a self-assembling vaccine (SAV). The amino acid sequence of MTbHSP70 is: MARAVGIDLGTTNSVVSVLEGGDPVVVANSEGSRTTPSIVAFARNGEVLV GQPAKNQAVTNVDRTVRSVKRHMGSDWSIEIDGKKYTAPEISARILMKLK RDAEAYLGEDITDAVITTPAYFNDAQRQATKDAGQIAGLNVLRIVNEPTA AALAYGLDKGEKEQRILVFDLGGGTFDVSLLEIGEGVVEVRATSGDNHLG GDDWDQRVVDWLVDKFKGTSGIDLTKDKMAMQRLREAAEKAKIELSSSSQS TSINLPYITVDADKNPLFLDEQLTRAEFQRITQDLLDRTRKPFQSVIADT GISVSEIDHVVLVGGSTRMPAVTDLVKELTGGKEPNKGVNPDEVVAVGAA LQAGVLKGEVKDVLLLDVTPLSLGIETKGGVMTRLIERNTTIPTKRSETF TTADDNQPSVQIQVYQGEREIAAHNKLLGSFELTGIPPAPRGIPQIEVTF DIDANGIVHVTAKDKGTGKENTIRIQEGSGLSKEDIDRMIKDAEAHAEED RKRREEADVRNQAETLVYQTEKFVKEQREAEGGSKVPEDTLNKVDAAVAE AKAALGGSDISAIKSAMEKLGQESQALGQAIYEAAQAASQATGAAHPGGE PGGAHPGSADDVVDAEVVDDGREAK This corresponds to SEQ ID NO: 1 and is also shown in Figure 1A.

[0055] In another embodiment, two peptides consisting of two MHC class I epitopes linked to an MHC class II epitope are chemically synthesized into a biotin-PEG4 conjugate to form a biotinylated antigenic peptide. The biotinylated antigenic peptide is then bound to a protein construct of modified MTbHSP70 genetically fused with avidin (MAV) to form an SAV unit. Avidin is believed to have four high-affinity binding sites for biotin, which can result in multiple different configurations of E6-only and E7-only conjugates, or different ratios of E6 and E7 peptides. In specific embodiments, the ratios of the different configurations can be 3:1, 2:2, 2:1, 1:1, or any range therebetween. As used herein, "eSAV" refers to a self-assembling vaccine containing peptides derived from HPV E6 and / or E7 proteins.

[0056] In another embodiment, two SAV units in the desired configuration are combined in a 1:1 ratio to obtain a combination vaccine composition. In other embodiments, this ratio can 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 any range therebetween.

[0057] In certain embodiments, vaccine compositions of the invention incorporate two immunogenic peptides that are synthetic chimeras, each combining two validated MHC class I epitopes and one validated MHC class II epitope of HPV16 E6 / E7 into a single biotinylated concatemer. In certain embodiments, synthesis of these peptides involves splitting each of the two "long" peptides into smaller portions or fragments for solid-phase synthesis, followed by assembly by click chemistry and purification by high-performance liquid chromatography (HPLC).

[0058] In certain embodiments, the MTbHSP70 portion of the MAV contains a point mutation from Val381 to Phe381, which is expected to reduce binding of endogenous peptides and thereby improve the specificity of the platform for the incorporated biotinylated peptide. As will be appreciated by those skilled in the art, one or more other or additional point mutations may be present to further improve specificity.

[0059] The heat shock protein fusion protein and biotinylated component produced as described above can be purified to a purity suitable for use as a pharmaceutical composition. Generally, a purified composition will have one species that comprises at least about 85 percent of all species present in the composition, at least about 85 percent, at least 90 percent, at least 95 percent, or at least 99 percent of all species present. The target species can be purified to essential homogeneity (contaminating species in the composition cannot be detected by conventional detection methods), and the composition will essentially consist of a single species. A skilled artisan can purify the heat shock protein fusion protein and biotinylated component, or their noncovalently bound complexes, using standard purification techniques (e.g., immunoaffinity chromatography, size exclusion chromatography, etc.). Protein purity can be determined by a number of methods known to those skilled in the art, including, for example, amino-terminal amino acid sequence analysis, gel electrophoresis, and mass spectrometry.

[0060] While many embodiments herein are described in terms of modified MTbHSP70, it is understood that other point mutations are also contemplated. The proteins used may also be functional variants of the proteins referred to herein and may exhibit significant amino acid sequence identity compared to the original protein of SEQ ID NO: 1. For example, the amino acid identity may reach 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% with SEQ ID NO: 1. In this context, the term "functional variant" means that a variant of a protein is capable of partially or completely achieving the function of the corresponding naturally occurring protein. A functional variant of a protein may include, for example, one or more amino acid substitutions, deletions, or additions that differ from the naturally occurring protein.

[0061] Amino acid substitutions can be conservative or non-conservative. Preferably, the substitutions are conservative, i.e., substitution of an amino acid residue with an amino acid of similar polarity that acts as a functional equivalent. Preferably, the amino acid residue used as a substitute is selected from the same amino acid group as the amino acid residue being substituted. For example, a hydrophobic residue may be replaced with another hydrophobic residue, or a polar residue may be replaced with another polar residue having the same charge. Functionally homologous amino acids that can be used for conservative substitutions include nonpolar amino acids such as glycine, valine, alanine, isoleucine, leucine, methionine, proline, phenylalanine, and tryptophan. Examples of uncharged polar amino acids include serine, threonine, glutamine, asparagine, tyrosine, and cysteine. Examples of charged polar (basic) amino acids include histidine, arginine, and lysine. Examples of charged polar (acidic) amino acids include aspartic acid and glutamic acid.

[0062] Also considered 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 may be added to one or both ends of the protein. Essentially, insertions can be made at any position, provided that the addition of the amino acids does not inhibit the function of the polypeptide that performs the function of the naturally occurring protein in the treated subject. Furthermore, protein variants also include proteins that lack one or more amino acids compared to the original polypeptide. Such deletions can affect any amino acid position, provided that they do not impair the protein's ability to achieve its normal function.

[0063] Variants of proteins (e.g., heat shock proteins, HPVE6 / E7, etc.) also refer to protein and peptide sequences that differ from naturally occurring proteins only by structural modifications (e.g., modified amino acids). Modified amino acids are amino acids that have been modified either by natural processes (e.g., processing or post-translational modification) or by chemical modification processes known in the art. Exemplary amino acid modifications include phosphorylation, glycosylation, acetylation, O-linked N-acetylglucosaminylation, glutathionylation, acylation, branching, ADP-ribosylation, cross-linking, disulfide bridge formation, formylation, hydroxylation, carboxylation, methylation, demethylation, amidation, cyclization, and / or covalent or non-covalent attachment to phosphotidylinositol, flavin derivatives, lipoteichoic acid, fatty acids, or lipids.

[0064] Therapeutic vaccines contain one or more SAV units containing biotinylated antigenic peptides derived from target viral or oncoprotein epitopes noncovalently bound to MTbHSP70 via a stable biotin-avidin bond. Thus, MTbHSP70 can be expressed as a fusion protein with avidin (MAV), allowing spontaneous self-assembly with the biotinylated peptide. It is believed that MTbHSP70 can be expressed alone or as a fusion protein using techniques well known to those skilled in the art.

[0065] Heat shock proteins (HSPs) are ubiquitously expressed proteins that function as chaperones in biological systems. HSPs present a wide repertoire of antigens to dendritic cells (DCs) and have the ability to activate both innate and adaptive immune responses. HSP70, particularly MTbHSP70, is thought to have important immunological functions that contribute to protective adaptive immune responses.

[0066] Examples of HSP70s include HSP72 and Hsc73 from mammalian cells and DnaK from bacteria, particularly Mycobacteria (e.g., Mycobacterium leprae and Mycobacterium tuberculosis (MTb)). Without wishing to be bound by theory, HSP70 also binds ATP and unfolds proteins and is thought to be involved in protein folding and unfolding and the assembly and disassembly of protein complexes. In at least one embodiment, the heat shock protein comprises (or is derived from) MTbHSP70. Heat shock protein fusion proteins to be used in conjunction with the methods described herein may comprise a sequence at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO:1.

[0067] The heat shock protein fusion proteins and biotinylated components described herein can be administered to a subject to induce or enhance the subject's immune response (particularly a cell-mediated cytolytic response) against cells expressing the antigen to which the biotinylated component is directed. The fusion protein may simply enhance the immune response (thereby functioning as an immunogenic composition) or may confer protective immunity (thereby functioning as a vaccine).

[0068] In the vaccines of the present invention, the adjuvant may, in certain embodiments, induce a Th1-type immune response. The fusion protein SAV approach, which utilizes the adjuvant capacity of MTbHSP70, provides an adjuvant system (CD4 binding to a protein target while avoiding undesired reactogenicity). + T cell responses and CD8 + This is advantageous over previous approaches that lacked the ability to safely induce both IFNγ, TNFα, and IL-2-expressing CD4 T cells. Compared to alum, the use of the SAV unit described herein significantly increased the number of CD4 T cells expressing IFNγ, TNFα, and IL-2 when both groups were compared to a saline control group. + T cells and CD8 + Demonstrates improved immune response in T cells.

[0069] Pharmaceutical compositions containing the vaccines of the present invention can be prepared as either liquid solutions or suspensions. Pharmaceutical compositions of the present invention can contain commonly used pharmaceutically acceptable excipients (e.g., diluents and carriers). In particular, the compositions can contain pharmaceutically acceptable carriers (e.g., PBS buffer). In addition to carriers, pharmaceutical compositions can also contain emulsifiers, pH buffers, stabilizers, dyes, etc.

[0070] In certain embodiments, the pharmaceutical composition comprises a therapeutically effective dose of HSP70 per vaccine, which is a dose that, when administered to a subject, is capable of generating a very strong immune response and / or treating some HPV-associated cancers, for example, in combination with a checkpoint inhibitor, without causing toxicity to the subject. Treatment of solid tumors in some HPV-associated cancers can be evaluated as a change in phenotypic characteristics (e.g., tumor volume) or a change in immune response or expression associated with HPV-associated solid tumors. Thus, a therapeutically effective HSP70 per vaccine dose is typically an amount sufficient to increase the immunogenic response and reduce tumor growth volume in a treated subject when administered in a physiologically acceptable composition.

[0071] A suitable therapeutically effective dose of HSP70 may be in the range of approximately 150-500 μg. Preliminary studies have shown that HSP70 has a positive adjuvant effect and inhibits CD4+ cells expressing IFNγ as a recall response to E6 and E7 peptides. + T cells and CD8 + Both spleen and lymph node proportions of T cells were significantly increased. A favorable immunogenic response was observed at approximately 215 μg of HSP70 per vaccine dose.

[0072] The heat shock protein fusion protein and biotinylated component, or their non-covalent complexes, can be administered to a subject in a variety of ways, as described herein. Routes of administration include systemic, peripheral, parenteral, enteral, topical, and transdermal (e.g., slow-release polymers). Any other convenient route of administration (e.g., infusion or bolus injection, or absorption through epithelial or mucocutaneous linings) can also be used.

[0073] In at least one embodiment, a composition containing SAV can be intradermally administered to a subject using a prime-boost-boost schedule at 14-day intervals. The intradermal (ID) administration route is particularly advantageous because it allows MTbHSP70 to directly stimulate epidermal Langerhans cells / dendritic cells. Using ID vaccination over the intramuscular (IM) route offers significant advantages. ID vaccination generally enhances vaccine responses and provides a more direct route to the broader cutaneous immune system via epidermal dendritic cells and draining dermal lymphatics. Extracellular heat shock proteins (e.g., MTbHSP70) are potent inducers of innate and adaptive immunity. The dermal dendritic cell network is particularly rich in HSP immune activating receptors (including CD40, CD36, LOX-1, SR-A, TLR-2, and TLR-4). Therefore, ID vaccination with SAV advantageously exploits the interaction of heat shock proteins with the abundant skin antigen processing system.

[0074] In at least one embodiment, the pharmaceutical compositions can be administered as is or in admixture with other agents. Thus, combined (combined) therapy includes sequential, simultaneous and separate, or simultaneous administration such that the therapeutic effect of the first administered agent is not completely abolished when the second is administered.

[0075] In at least one embodiment, a composition comprising SAV can be transduced into ID in combination with an approved anti-PD-1 drug, an immune checkpoint inhibitor. Immune checkpoint inhibitors maintain antitumor activity by disrupting T cell co-inhibitory signaling pathways. One of the key mechanisms by which cancer cells evade immune surveillance is 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) function primarily in a context of mutual inhibition by engaging with PD-1 / PD-L1 within tumor masses. When mutual inhibition is relieved by either anti-PD-1 or anti-PD-L1 specific antibodies, CD8 +CTLs are released by cleaving the binding chains, thereby restoring cytotoxicity. + The CTLs will attack and eliminate the tumor cells.

[0076] HPV-associated cancers that may be treated with the methods disclosed herein may include, but are not limited to, head and neck cancer, cervical cancer, anal cancer, other HPV-induced cancers that express E6 and E7 oncoproteins, and combinations thereof. [Example]

[0077] The following examples are provided to aid in the understanding of the present disclosure and, of course, should not be construed as specifically limiting the embodiments described and claimed herein. Such variations of the embodiments now known or later developed (within the knowledge of those skilled in the art), as well as minor variations in formulation or experimental design, should be considered to be included within the scope of the embodiments incorporated herein.

[0078] Example 1: Sequences of peptides used and associated epitopes In this example, the two immunogenic peptides are synthetic chimeras, each combining two validated MHC class I mouse epitopes and one validated MHC class II mouse epitope of HPV16 E6 / E7 into a single biotinylated concatemer. Table 1 shows peptide sequences with three epitope identities, each separated by a linker sequence. The first column shows the epitope and linker sequence for the E6 peptide, and the second column shows the epitope and linker sequence for the E7 peptide. [Table 1]

[0079] The peptide sequence used in the following studies incorporated linked MHC class I and II epitopes to HPV proteins E6 and E7. This peptide sequence is shown in Table 2, which shows the structure of the spacer and epitope, and has the following sequence: LEQLERVKREVYDFAFRDLAAYRVKRQAEPDRAHYNIVTFCCKCDGPGPGRVKRYMLDLQPET (SEQ ID NO: 2). [Table 2]

[0080] Example 2: Design of human immunogenic HPV E6 / E7 peptides Consider the target E6 / E7 peptide sequence selected for presentation on human MHC class I and II. The final two "long" biotinylated peptides (i.e., one for E6 and one for E7) can be formed using two small peptide fragments through a click chemistry mechanism. Figure 1B shows both the small peptide fragments, including the DBCO- and azide (NNN)-containing peptides, and the final sequence, which is a conjugate of peptides 1 and 2 via a triazole moiety. The schematic in Figure 1C illustrates the mechanism of the click chemistry reaction. The fully formed peptide was then added to MAV via a non-covalent biotin-avidin bond. The peptide fragments in Figure 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).

[0081] Example 3: MAV sequences and properties In this example, a MAV protein containing a histidine tag, MTbHSP70, a polyglycine linker, and avidin was synthesized as a vaccine adjuvant component using techniques readily available and understood by those skilled in the art. Figure 2 shows the point mutation of Val381 to Phe381 in the MTbHSP70 moiety, which is predicted to inactivate endogenous peptide binding and thereby improve the platform's specificity for the incorporated biotinylated peptide. The 771 amino acid sequence in Figure 2 corresponds to SEQ ID NO:7.

[0082] Table 3 shows the properties of the MAV units, including molecular weight and concentration, which were evaluated and confirmed using gel electrophoresis and light absorption at 280 nm, respectively. [Table 3]

[0083] Example 4: Assembly of eSAV To prepare eSAV, a 5-fold molar excess of biotinylated peptide 3 from Table 2 was added to MAV in PBS with end-over-end mixing for 12 hours at room temperature. The precipitated peptide was removed by centrifugation. Assembly reactions were prepared for 80 μg of MAV per 50 μL volume of eSAV. The resulting eSAV was stored at -80°C until use.

[0084] The mechanism by which self-assembly forms eSAV units through stable biotin-avidin engagement is shown in Figure 3. To verify this assembly, the absorbance of the eSAV units after addition of HABA dye was compared to that of unassembled MAV, and this absorbance difference was used to calculate the moles of biotinylated peptide attached per mole of MAV. Self-assembling constructs conjugated with different peptides can be mixed together to target vaccines to multiple epitopes.

[0085] Example 5: Immune response to eSAV This example demonstrates the immune response of mice to various T cell types after eSAV vaccination. For each vaccination, C57BL / 6 mice were injected intradermally with the drug (50 μL volume) suspended in sterile PBS.

[0086] Figure 4A shows the expression of IFNγ in splenocytes after stimulation. The eSAV-vaccinated group showed significantly higher CD4 + Cells and CD8 + There was a statistically significant increase in IFNγ expression in CD4 cells. + 9.5% of cells (p ≤ 0.01) and CD8 + 6.5% (p≦0.05) of cells were positive for IFNγ expression after recall exposure to peptide. The other groups did not reach statistical significance. These results indicate that eSAV elicited a significant and balanced immune response compared with saline. There was a significant difference in CD4 + IFNγ + (p=0.68) and CD8 + IFNγ + There was no statistically significant difference (p=0.83).

[0087] Figure 4B shows the results of peptide stimulation on IL-2 expression in splenic T cells. For this cytokine, only eSAV achieved a statistically significant increase in IL-2 expression compared to the saline group, which was related to CD4 + There was no statistically significant difference in IL-2 secretion between the eSAV-treated group and the 5% alum + peptide-treated group.

[0088] Figure 4C shows the results of TNFα expression in T cells. Only T cells from the eSAV-treated group showed a statistically significant difference in expression compared to saline. CD4 + 10% of cells (p ≤ 0.01) and CD8 +5.5% (p≦0.05) of cells expressed TNFα in response to peptide stimulation. There was no statistically significant difference in TNFα secretion between the eSAV-treated group and the 5% alum + peptide-treated group.

[0089] Figure 4D shows the results of CD62L expression in splenic T cells. No group showed a significant increase in expression of this ligand. There was no statistically significant difference in CD62L expression between the eSAV-treated group and the 5% alum + peptide-treated group.

[0090] In these examples shown in Figures 4A-4D, each data point represents cells from an individual mouse, and horizontal bars indicate mean expression within groups and classes. Significant p-values compared to saline are shown after adjustment for multiple hypothesis testing. In all graphs, a single * represents a p-value of 0.05 or less, and a ** represents a p-value of 0.01 or less.

[0091] Example 6: Tumor response to eSAV This example demonstrates the effect of either eSAV or alum (5%) + peptide vaccination on the growth of TC-1 tumors in female C587BL / 6 mice. In this tumor treatment study, cultured cancer cells (TC-1) were suspended in sterile PBS at a concentration of 10,000 cells per 100 μL and injected with an insulin syringe into the loose skin on the flank of 8-10 week-old female C57BL / 6 mice. Tumor growth was measured twice weekly and calculated using the formula (volume = (a × b 2 Tumor volume was calculated using the formula: a / b / 2 (where a and b are the maximum and minimum diameters of the tumor, respectively).

[0092] The top plot in Figure 5 shows tumor growth in each mouse individually, while the bottom plot shows the average tumor size for each treatment group. The data show that eSAV vaccination was significantly more effective at reducing tumor growth volume over the study period than peptide + alum (5%) vaccination.

[0093] Example 7: Survival Probability In this example, the survival probability of mice in a tumor treatment study is shown and compared between eSAV and peptide + alum (5%) vaccination. In Figure 6, the mean survival rate of the eSAV-treated group was statistically significantly higher than that of the group receiving saline alone and approached statistical significance compared to the alum (5%) + peptide group.

[0094] Example 8: Dosage-based CD8 + immune response In this example, CD8 in spleen cells and lymphocytes after stimulation with E6 / E7 peptides at different vaccine doses was measured. + T cell immune responses are shown. In Figure 7A, the immune response to splenocytes is shown. For IFNγ, only the 130, 215, and 350 μg groups showed a statistically significant increase in expression compared to the saline control group. The two highest dose groups showed the most pronounced responses (p≦0.0001). No group showed a significant TNFα response. For IL-2, only the 215 μg group had a statistically significant recall response.

[0095] In Figure 7B, CD8 in lymphocytes + T cell responses are shown. A similar pattern was observed with significant IFNγ only in the 130, 215, and 350 μg groups. In this case, the response at 215 μg was the best. There was no significant TNFα expression seen in any group. IL-2 was only significant at 215 μg.

[0096] In these examples, as shown in Figures 7A-7B, T cell responses are expressed as the percentage of viable T cells. Each data point represents cells from an individual mouse, and horizontal bars indicate the mean expression within the group and class. Significant p-values compared to the saline group after adjustment for multiple hypothesis testing are shown. In these examples, significance markers are as follows: * = p-value less than or equal to 0.05, ** = p-value less than or equal to 0.01, *** = p-value less than or equal to 0.001, and **** = p-value less than or equal to 0.0001.

[0097] Example 9: Dosage-based CD4 + immune response In this example, CD4 in spleen cells and lymphocytes after stimulation with E6 / E7 peptides at different vaccine doses was measured. + T cell immune responses are shown. Figure 8A shows the immune response in splenocytes. For IFNγ, only the 215 μg and 350 μg groups showed a statistically significant increase in expression compared to the saline control group. Both groups showed highly significant responses (p≦0.0001). No group showed a significant TNFα response. For IL-2, only the 215 μg group had a statistically significant recall response.

[0098] In Figure 8B, CD4 in lymphocytes + T cell responses showed a slightly broader range of immune responses. For IFNγ, significant responses were seen in the 130, 215, and 350 μg groups, with 215 μg clearly being the best. For TNFα, a significant response was seen in the 215 μg group, and for IL-2, significant responses were seen in the 130 μg and 250 μg groups.

[0099] In these examples, as shown in Figures 8A-8B, T cell responses are expressed as the percentage of viable T cells. Each data point represents cells from an individual mouse, and horizontal bars indicate the mean expression within the group and class. Significant p-values compared to the saline group after adjustment for multiple hypothesis testing are shown. In these examples, significance markers are as follows: * = p-value less than or equal to 0.05, ** = p-value less than or equal to 0.01, *** = p-value less than or equal to 0.001, and **** = p-value less than or equal to 0.0001.

[0100] Example 10: Reactogenicity of SAV in combination with PD-1 checkpoint inhibitor therapy In this example, reactogenicity of the vaccine / antibody treatment was evaluated. No reactogenicity was observed with any of the treatments in any group during the study period.

[0101] Example 11: Tumor growth rate This example shows tumor growth kinetics for groups treated with vaccine and anti-mPD1, and vaccine and anti-IgG2a. Figures 9A-B show that eSAV significantly reduced TC-1 tumor volume, with tumor volume reductions in mice receiving 80, 130, and 215 μg of eSAV. There were no statistically significant differences between mice receiving anti-mPD1-treated eSAV compared to the matched anti-IgG2a-treated group.

[0102] Example 12: Survival curves In this example, survival curves for vaccine and anti-mPD1, and vaccine and anti-IgG2a treatment groups are shown. In Figures 11A-B, SAV has a highly significant and positive survival effect in mice injected with TC-1 tumors at 80, 130, and 215 μg per vaccine dose (p<0.0001). There was also a dose response to SAV, with TC-1 tumor-bearing mice receiving 215 μg of SAV receiving the best overall survival benefit. There was a statistically significant survival benefit for anti-mPD1in peptide-treated mice compared to the anti-IgG2a treatment group.

[0103] In the foregoing description, numerous specific details (e.g., specific materials, dimensions, process parameters, etc.) are set forth to provide a thorough understanding of the present invention. Particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments. The word “example” or “exemplary” is 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 phrase “example” or “exemplary” is merely intended to illustrate a concept. As used in this application, the term “or” is intended to mean an inclusive “or” rather than an exclusive “or.” That is, unless expressly stated otherwise or apparent from context, it is intended to mean any of the natural inclusive permutations of “X includes A or B.” That is, if X includes A, X includes B, or X includes both A and B, then "X includes A or B" is satisfied in any of the above cases. Throughout this specification, the term "embodiment," "particular embodiment," or "one embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment. Thus, the appearances of this phrase "embodiment," "particular embodiment," or "one embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment.

[0104] The present invention has been described with reference to specific exemplary embodiments thereof. Accordingly, the specification and drawings are 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 be included 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, each of the two or more biotinylated peptides comprising a sequence at least partially derived from an epitope of a human viral antigen protein, with the proviso that each epitope is of a different human viral antigen protein of the same human virus.

2. A pharmaceutical composition comprising a heat shock protein fused to a biotin-binding protein, the biotin-binding protein being non-covalently bound to a biotinylated peptide, the biotinylated peptide comprising a sequence derived at least in part from one or more epitopes of one or more human papillomavirus (HPV) antigenic 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 antigenic proteins include an E6 protein or an 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 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, the biotinylated peptide being formed by linking two peptide fragments, each of the peptide fragments having 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.

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

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

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

11. 7. The pharmaceutical composition of any one of claims 1 to 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. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein the heat shock protein has an amino acid sequence at least 80%, 85%, 90%, 95%, or 99% identical to SEQ ID NO: 1 containing a point mutation from Val381 to Phe381.

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

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

15. 10. The pharmaceutical composition of any one of the preceding claims, wherein the biotin-binding protein is selected from the group consisting of avidin, streptavidin, and neutravidin.

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

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

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

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

20. 18. A method of treating an HPV-induced tumor in a subject, wherein HPV proteins E6 and / or E7 are expressed in cancer cells of said tumor, said method comprising administering to said subject a pharmaceutical composition according to any of claims 1 to 17.

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