Vaccines incorporating protein-based immunologic adjuvants

Abstract

Compositions and methods for treating some HPV-associated solid tumors in a mammalian subject are disclosed. In one embodiment, a method includes intradermal delivery of a self-assembling vaccine to a subject. In at least one embodiment, the self-assembled vaccine comprises a fusion protein that is non-covalently linked to two or more biotinylated E6 / E7 peptides derived from a target virus or oncogenic protein epitope by biotin-avidin binding.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 359,560, filed on July 8, 2022, the disclosure of which is hereby incorporated by reference in its entirety. Technical Field

[0003] The present invention relates to the treatment of solid tumors (eg, head and neck cancer, cervical cancer, and carcinomas), and more particularly, to immunotherapeutic peptide-based vaccine methods and pharmaceutical compositions for treating tumors. Background Art

[0004] Adjuvants are chemical compounds approved for use in human vaccines in the United States that act as immunostimulants and enhance the production of antibodies when delivered with an antigen. Alum in particular has been widely used as an adjuvant over the past few decades. However, while alum induces a Th2-biased immune response, it fails to induce a Th1 cell-mediated immune response that is critical for treating some solid tumors. Inducing a Th1 cell-mediated immune response is desirable because they play a key role in inducing therapeutically important immune responses while allowing a high degree of safety to be maintained.

[0005] Numerous attempts have been made over the past 30 years to develop therapeutic vaccines as a treatment modality for a variety of solid tumors, but these approaches have lacked therapeutic effectiveness. Furthermore, current vaccine strategies are time-consuming and labor-intensive, can only be initiated when a threat emerges, and are impractical for generating personalized vaccines against diseases where the target antigens differ between individuals. Therefore, alternative approaches, such as platform vaccine technologies, are needed as they have the potential to generate therapeutically effective vaccines for a variety of conditions, including but not limited to treating some solid tumors, such as cervical cancer caused by human papillomavirus (HPV), or to contain rapidly evolving, fast-acting, and / or highly contagious threats. Summary of the invention

[0006] The following overview presents a simplified overview of various aspects of the present disclosure in order to provide a basic understanding of such aspects. This overview is not a comprehensive review of the present disclosure. It is neither intended to identify the key or important elements of the present disclosure, nor to delimit any scope of the specific embodiments of the present disclosure or any scope of the claims. Its only purpose is to present some concepts of the present disclosure in a simplified form as a preface to a more detailed description presented later.

[0007] In one aspect, the pharmaceutical composition comprises a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein non-covalently binds to two or more biotinylated peptides, and wherein the two or more biotinylated peptides each comprise a sequence derived at least in part from an epitope of a human viral antigen protein, with the proviso that each epitope belongs to an epitope of a different human viral antigen protein of the same human virus.

[0008] In another aspect, the pharmaceutical composition comprises a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein non-covalently binds to a biotinylated peptide, and wherein 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.

[0009] In at least one embodiment, the biotinylated peptide comprises two or more epitope sequences each separated by a linker sequence.

[0010] In at least one embodiment, the one or more HPV antigen proteins include E6 protein or E7 protein.

[0011] In another 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.

[0012] In another 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, wherein the biotinylated peptide is formed by joining 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.

[0013] In at least one embodiment, the heat shock protein is a mammalian heat shock protein or a bacterial heat shock protein.

[0014] In at least one embodiment, the heat shock protein is selected from the group consisting of Mycobacterium tuberculosis heat shock protein 70 (MTbHSP70) and human heat shock proteins.

[0015] In at least one embodiment, the heat shock protein is a member of the heat shock protein 70 (HSP70) family.

[0016] In at least one embodiment, the heat shock protein is or is derived from MTbHSP70.

[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.

[0018] 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.

[0019] In another aspect, the 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 a MTbHSP70-avidin fusion protein.

[0020] In at least one embodiment the biotin binding protein is selected from the group consisting of avidin, streptavidin and neutravidin.

[0021] 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.

[0022] In another aspect, a method of inducing an immune response in a subject comprises administering to the subject the pharmaceutical composition of any one of the preceding claims.

[0023] In at least one embodiment, the pharmaceutical composition comprises a pharmaceutically acceptable excipient, and wherein the therapeutic dose administered to a subject comprises about 150 μg to about 500 μg of the heat shock protein.

[0024] In another aspect, a method of treating a tumor in a subject wherein the HPV proteins E6 and / or E7 are expressed by cancer cells of an HPV-induced tumor comprises administering to the subject a pharmaceutical composition of any of the above pharmaceutical compositions. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The above and other features of the present disclosure, its nature and various advantages will become more apparent upon consideration of the following detailed description taken in conjunction with the accompanying drawings, in which:

[0026] Figure 1A The amino acid sequence of Mycobacterium tuberculosis heat shock protein 70 is shown;

[0027] Figure 1Bis a table summarizing exemplary E6 / E7 peptide sequences formed by click chemistry of two peptide fragments according to at least one embodiment;

[0028] Figure 1C Scheme illustrating a click chemistry reaction to form a triazole moiety according to at least one embodiment;

[0029] Figure 2 shows the amino acid sequence of a fusion protein according to at least one embodiment;

[0030] Figure 3 is a schematic diagram illustrating the spontaneous self-assembly of a fusion protein and a biotinylated peptide according to at least one embodiment;

[0031] Figure 4A including a graph showing IFNγ expression in splenocytes after stimulation according to at least one embodiment;

[0032] Figure 4B including a graph showing IL-2 expression in splenocytes after stimulation according to at least one embodiment;

[0033] Figure 4C including a graph showing TNFα expression in splenocytes after stimulation according to at least one embodiment;

[0034] Figure 4D including a graph showing CD62L expression in splenocytes after stimulation according to at least one embodiment;

[0035] Figure 5 including a graph showing the therapeutic effect of eSAV, 5% Alum-E6-E7 peptide, or saline on syngeneic tumor growth according to at least one embodiment;

[0036] Figure 6 including a graph showing the therapeutic effect of eSAV, 5% Alum-E6-E7 peptide, or saline on mouse survival according to at least one embodiment;

[0037] Fig. 7A Including showing that according to at least one embodiment, CD8 + Graphs showing the expression of IFNγ, TNFα, and IL-2 in splenocytes;

[0038] Figure 7B Including showing that according to at least one embodiment, CD8 + Graphs showing the expression of IFNγ, TNFα, and IL-2 in lymphocytes;

[0039] Fig. 8A Including showing that according to at least one embodiment, CD4+ Graphs showing the expression of IFNγ, TNFα, and IL-2 in splenocytes;

[0040] Figure 8B comprising demonstrating that CD4 + Graphs showing the expression of IFNγ, TNFα, and IL-2 in lymphocytes;

[0041] Fig.9A including graphs showing tumor growth kinetics for vaccine and anti-mPD1 treatment groups at different vaccine doses according to at least one embodiment;

[0042] Fig. 9B including graphs showing tumor growth kinetics for vaccine and anti-IgG2a treatment groups at different vaccine doses according to at least one embodiment;

[0043] Fig. 10A including a graph showing survival curves for vaccine and anti-mPD1 treatment groups at different vaccine doses according to at least one embodiment;

[0044] Fig. 10B Included are graphs showing survival curves for vaccine and anti-IgG2a treatment groups at different vaccine doses according to at least one embodiment.

[0045] definition

[0046] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates 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, etc.

[0047] In addition, as used herein, "about" when used in conjunction with a measured amount refers to the normal variation of that measured amount as would be expected by one of ordinary skill in the art when making the measurements and exercising a level of care commensurate with the purpose of the measurement and the precision of the measuring equipment. In certain embodiments, the term "about" includes ±10% of the recited value, such that "about 10" would include 9 to 11.

[0048] Furthermore, as used herein, "including" is used to mean "including but not limited to."

[0049] In addition, as used herein, "protein" has its ordinary and customary meaning in the art and includes and refers to polypeptides (i.e., a string of at least two amino acids interconnected by peptide bonds). Polypeptides can include natural amino acids, non-natural amino acids, synthetic amino acids, amino acid analogs, and combinations thereof. The term "peptide" is generally used to refer to polypeptides of less than about 50 amino acids in length. Proteins may include parts other than amino acids (e.g., glycoproteins) and may be processed or modified. Proteins may be complete polypeptide chains produced by cells or functional parts thereof. Proteins may include more than one polypeptide chain, which may be 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.

[0050] In addition, the term "biotin binding protein" as used herein refers to a protein that can non-covalently bind to biotin. Biotin binding proteins can be monomers, dimers or tetramers that can form monovalent, divalent or tetravalent pharmaceutical compositions, respectively, as described herein. Non-limiting examples include anti-biotin antibodies, avidin, streptavidin and neutravidin. Avidin can include mature avidin, or a sequence at least 80%, 85%, 90%, 95% or 99% identical to the sequence identified by NCBI accession number NP 990651. Streptavidin can include, for example, a sequence at least 80%, 85%, 90%, 95%, 99% identical to the sequence identified by NCBI accession number AAU48617. The term "biotin binding protein" is intended to cover wild-type and derivatives of avidin, streptavidin and neutravidin, which form monomers, dimers, tetramers.

[0051] In addition, as used herein, "vaccine" has its ordinary and customary meaning in the art and refers to any preparation used to stimulate the production of antibodies against one or more diseases. A vaccine can be a preparation that provides immunity against one or more diseases, or a preparation that stimulates an immune response against one or more diseases. Vaccines can include killed or attenuated disease pathogens, products or derivatives of the above pathogens, and synthetic substitutes. Synthetic substitutes may include preparations composed of synthetic peptides, carbohydrates, antigens, or RNA or DNA chains.

[0052] Furthermore, as used herein, "epitope" refers to a region of an antigen to which an antibody preferentially and specifically binds.

[0053] In addition, as used herein, "linker" refers to a molecule or group of molecules that connects two molecules, such as a molecule that forms a covalent bond between a heat shock protein and a biotin binding protein. A linker can consist of a single linker molecule, or can include a linker molecule and a spacer molecule, intended to separate the linker molecule and the moiety by a specific distance.

[0054] In addition, as used herein, "immunogenicity" refers to the ability of a substance to induce an immune response. An immune response refers to a subject's response to the presence of an antigen and may include at least one of the following: production of antibodies, immunity, hypersensitivity to the antigen, and tolerance.

[0055] Furthermore, as used herein, "endogenous" refers to proteins, nucleic acids or genes that occur in nature and within a living system (eg, an organism, tissue or cell), but the term is not intended to exclude proteins that do not occur naturally in a patient or host.

[0056] In addition, as used herein, "heat shock proteins" are encoded by "heat shock genes" or stress genes and refer to genes that are activated or otherwise detectably unregulated as a result of contact or exposure of an 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 proteins" also include homologous proteins encoded by genes within known stress gene families, even if these homologous genes themselves are not induced by stressors.

[0057] In addition, "fusion protein" as used herein refers to a hybrid protein comprising the sequences of at least two different proteins. These sequences may be proteins from the same or different organisms. In various embodiments, the fusion protein may include one or more amino acid sequences connected to the first protein. When more than one amino acid sequence is fused to the first protein, the fusion sequence may be multiple copies of the same sequence, or may be different amino acid sequences. The first protein may be fused to the N-terminus, C-terminus, or N-terminus and C-terminus of the second protein.

[0058] In addition, 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 can be joined to a biotin binding protein via the C-terminus or N-terminus to produce a heat shock protein fusion protein. When administered in conjunction with a biotinylated component provided herein, the heat shock protein fusion protein can stimulate or enhance a humoral and / or cellular immune response to an antigen of interest, including a CD8 cytotoxic T cell (TCL) response.

[0059] In addition, 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 co-stimulatory molecules. The biotinylated component will be administered to a subject in conjunction with the heat shock protein fusion protein described herein.

[0060] Furthermore, as used herein, "concatemer" refers to a DNA fragment consisting of multiple copies of a sequence linked together in tandem.

[0061] In addition, as used herein, "pharmaceutically acceptable excipient or carrier" refers to any inert ingredient in the composition combined with the active agent in the preparation. Pharmaceutically acceptable excipients may include, but are not limited to, carbohydrates (e.g., glucose, sucrose, or dextran), antioxidants (e.g., 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, emulsifiers, dispersants, 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 edition (1985).

[0062] In addition, the term "patient" as used herein refers to a subject, particularly a human (but non-humans may also be included), who has shown clinical manifestations of one or more specific symptoms suggesting a need for treatment, has been treated prophylactically for a disorder, or has been diagnosed with a disorder to be treated.

[0063] Furthermore, "subject" as used herein encompasses the definition of the term "patient" and does not exclude otherwise healthy individuals.

[0064] Furthermore, "treatment of" or "treating" as used herein includes the administration of a drug to lessen the severity of a disorder (eg, cervical cancer) or to prevent the disorder.

[0065] Furthermore, "prevention of" or "preventing" as used herein includes avoiding the onset of a disorder (eg, cervical cancer).

[0066] Furthermore, "condition" or "conditions" as used herein refers to those medical conditions that can be treated, alleviated or prevented by administering an effective amount of a drug to a subject.

[0067] In addition, "effective amount" as used herein refers to the amount of drug sufficient to produce a beneficial or desired effect, and the effect level can be easily detected by the method commonly used to detect such effects. In some embodiments, such effects result in at least a 10% change compared to the basal level value when the drug is not administered. In other embodiments, relative to the basal level, the change is at least 20%, 50%, 80% or even higher percentage. As described below, the effective amount of the drug may vary from subject to subject, depending on the age of the subject, the general condition of the subject, the severity of the disease treated, the specific drug administered, etc. Those of ordinary skill in the art can determine the appropriate "effective" amount in any individual case by reference to relevant texts and literature and / or by using routine experiments.

[0068] Furthermore, as used herein, "active agent" refers to any substance intended to produce a therapeutic, prophylactic, or other desired effect, whether or not approved for that purpose by a governmental agency.

[0069] Unless otherwise specified herein, the enumeration of value ranges herein is intended only to be used as a shorthand method for individually representing each individual value belonging to the range, and each individual value is incorporated into the specification as if it were individually described herein. Unless otherwise specified herein or clearly contradictory to the context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to illustrate certain materials and methods and is not intended to limit the scope. The language in the specification should not be considered to indicate that any unclaimed element is necessary for implementing the disclosed materials and methods. DETAILED DESCRIPTION

[0070] Human papillomavirus (HPV) is a common virus that is responsible for approximately 640,000 new cases of cancer 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 transmitted through vaginal, anal, or oral sex. Sexually transmitted HPV types are divided into two categories: low risk (mostly do not cause disease) and high risk. On the other hand, HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 are classified as high risk because they have been causatively associated not only with cervical disease (cervical intraepithelial neoplasia and cervical cancer) but also with precancerous lesions or cancers of the penis, anus, vulva, vagina, oropharynx, and larynx.

[0071] Because HPV infects squamous cells lining the inner surfaces of certain organs, such as the cervix, oropharynx, anus, penis, vagina, and vulva, most HPV-associated cancers are a class of cancers called squamous cell carcinomas. HPV proteins, such as the E6 and E7 proteins, are considered oncogenic and are reasonable cancer antigen targets for immunotherapy because they are viral rather than human proteins. HPV-associated intraepithelial neoplasia or cancer is a candidate for cancer immunotherapy because HPV oncoproteins, such as the E6 and E7 proteins of high-risk HPVs, can be used as foreign antigens. Recently, checkpoint inhibitors (CPIs) have emerged as a promising new treatment for solid cancers; however, these therapies have shown only modest efficacy against HPV-associated cancers.

[0072] The present disclosure relates to a peptide-based self-assembling vaccine (SAV) platform that produces a highly effective immune response when administered to a subject and can be used in combination with a checkpoint inhibitor in certain embodiments to treat HPV-induced tumors, where the HPV proteins E6 and E7 are expressed by cancer cells. The E6 and E7 viral oncoproteins of high-risk HPVs exert their effects by interfering with the activity of cellular tumor suppressor proteins. The E6 protein increases the turnover of the tumor suppressor protein p53 by targeting to accelerate ubiquitin-mediated degradation. The E7 protein inhibits gene transcription necessary for cell cycle progression, enabling the virus to replicate in cells outside of the dividing population.

[0073] HPV-associated tumors have a pronounced expression of tumor-specific antigens (HPV proteins - E6 and E7) that contain a variety of immunogenic peptide sequences that can be targeted by the human and murine immune systems. + and CD8 +T cells contribute to the control of viruses during viral infection by producing effector cytokines (e.g., IFNγ and TNF) and exerting cytotoxic activity on virally infected cells. HPV's viral oncoproteins E6 and E7 are constitutively expressed in transformed cells and are therefore ideal targets for immunotherapy of HPV-induced malignancies. A so-called resistance mechanism is that tumor-infiltrating E6-specific activated T cells express PD-1, and tumor-infiltrating immune cells express PD-L1. This results in the method of the present disclosure being combined with immune checkpoint inhibitors in certain embodiments.

[0074] 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 documented to be immunogenic in C57B1 / 6J mice chemically conjugated to biotin. The biotinylated peptide can be self-assembled with a protein construct 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:

[0075]

[0076] It corresponds to SEQ ID NO: 1 and is also depicted in Figure 1A middle.

[0077] In another embodiment, two peptides consisting of two MHC class I epitopes concatenated with one MHC class II epitope are chemically synthesized as biotin-PEG4 conjugates to form biotinylated antigenic peptides. The biotinylated antigenic peptides are then joined to a protein construct of a modified MTbHSP70 genetically fused to avidin (MAV) to form a SAV unit. Considering that the avidin pair has four biotin high affinity binding sites, this may produce a variety of different arrangements of E6-only and E7-only conjugates or E6 and E7 peptides in different ratios. In certain embodiments, the ratios of the different arrangements may be 3: 1, 2: 2, 2: 1, 1: 1, or any range defined therebetween. As used herein, "eSAV" refers to a self-assembling vaccine containing peptides derived from HPV E6 and / or E7 proteins.

[0078] In another embodiment, two desired arrangements of SAV units are combined in a 1: 1 ratio to produce a mixed vaccine composition. In other embodiments, the 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 defined therebetween.

[0079] In certain embodiments, the vaccine compositions of the invention incorporate two immunogenic peptides that are synthetic chimeras, each of which combines two documented MHC class I epitopes and one documented MHC class II epitope of HPV16 E6 / E7 into a single biotinylated concatemer. In certain embodiments, the synthesis of these peptides involves dividing each of the two "long" peptides into smaller parts or fragments for solid phase synthesis, followed by assembly by click chemistry and purification by high performance liquid chromatography (HPLC).

[0080] In certain embodiments, the MTbHSP70 portion of the MAV comprises a Val381 to Phe381 point mutation, which is expected to reduce endogenous peptide binding, thereby increasing the specificity of the platform for incorporated biotinylated peptides. One or more other or additional point mutations may be present to further increase specificity, as will be appreciated by those of ordinary skill in the art.

[0081] The heat shock protein fusion protein and biotinylated component produced as described above can be purified to a suitable purity for use as a pharmaceutical composition. In general, the purified composition will have a species that accounts for more than about 85% of all species present in the composition, accounting for about 85%, 90%, 95%, 99% or more of all species present. The target species can be purified to substantially homogeneity (the contaminant species in the composition cannot be detected by conventional detection methods), wherein the composition consists essentially of a single species. Those skilled in the art can use standard purification techniques (e.g., immunoaffinity chromatography, size exclusion chromatography, etc.) to purify the heat shock protein fusion protein and biotinylated component, or its non-covalent complex. The purity of the protein can be determined by a variety of methods known to those skilled in the art, including, for example, amino terminal amino acid sequence analysis, gel electrophoresis, and mass spectrometry.

[0082] Although many embodiments of modified MTbHSP70 are described herein, it should be understood that other point mutations can be considered. The one or more proteins used can also be functional variants of the proteins mentioned herein, and can show significant amino acid sequence identity compared to the original protein of SEQ ID NO: 1. For example, the amino acid identity with SEQ ID NO: 1 can 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%. In this article, the term "functional variant" refers to a variant of a protein that can partially or completely realize the function of the corresponding protein that occurs in nature. The functional variant of a protein can include, for example, a protein that has one or more amino acid substitutions, deletions or additions that differ from its naturally occurring counterpart.

[0083] Amino acid substitutions can be conservative or non-conservative. Preferably, the substitution is a conservative substitution, i.e., replacing an amino acid residue with an amino acid having similar polarity, which serves 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 to be replaced. For example, a hydrophobic residue can be replaced by another hydrophobic residue, or a polar residue can be replaced by another polar residue with the same charge. Functional homologous amino acids that can be used for conservative substitutions include, for example, non-polar 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.

[0084] Proteins that differ from their naturally occurring counterparts by one or more (e.g., 2, 3, 4, 5, 10, or 15) additional amino acids are also considered variants. These additional amino acids may be present within the amino acid sequence of the original protein (i.e., as insertions), or they may be added to one or both ends of the protein. Basically, insertions can be made at any position if the addition of amino acids does not impair the ability of the polypeptide to function as the naturally occurring protein in the subject being treated. In addition, protein variants also include proteins that lack one or more amino acids compared to the original polypeptide. Such deletions may affect any amino acid position, provided that the amino acid position does not impair the ability of the protein to function normally.

[0085] Protein variants (e.g., heat shock proteins, HPV E6 / E7, etc.) also refer to proteins and peptide sequences that differ from naturally occurring proteins by structural modifications (e.g., modified amino acids). Modified amino acids are amino acids modified by natural processes (e.g., processing or post-translational modifications) or by chemical modification processes known in the art. Typical amino acid modifications include phosphorylation, glycosylation, acetylation, O-linked N-acetylglucosaminylation, glutathionylation, acylation, branching, ADP ribosylation, cross-linking, disulfide bond formation, formylation, hydroxylation, carboxylation, methylation, demethylation, amidation, cyclization, and / or covalent or non-covalent binding to phosphatidylinositol, flavin derivatives, lipoteichoic acid, fatty acids, or lipids.

[0086] The therapeutic vaccine contains one or more SAV units, which contain biotinylated antigenic peptides derived from target viral or oncoprotein epitopes, which are non-covalently bound to MTbHSP70 through stable biotin-avidin binding. Therefore, MTbHSP70 can be expressed as a fusion protein with avidin (MAV), thereby being able to spontaneously self-assemble with the biotinylated peptide. It is contemplated that MTbHSP70 can be expressed alone or as a fusion protein using techniques familiar to those of ordinary skill in the art.

[0087] Heat shock proteins (HSPs) are ubiquitously expressed proteins that act as molecular chaperones in living systems. HSPs are able to present a range of antigens to dendritic cells (DCs) and activate innate and adaptive immune responses. HSP70, especially MTbHSP70, is believed to have significant immunological potency and contribute to protective and adaptive immune responses.

[0088] Examples of HSP70 include HSP72 and Hsc73 from mammalian cells, DnaK from bacteria, particularly mycobacteria such as Mycobacterium leprae and Mycobacterium tuberculosis (MTb). Without being bound by theory, it is believed that HSP70 binds ATP and unfolded proteins and participates in protein folding and unfolding, as well as the assembly and disassembly of protein complexes. In at least one embodiment, the heat shock protein includes (or is derived from) MTbHSP70. The heat shock protein fusion protein 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.

[0089] The heat shock protein fusion proteins and biotinylated components described herein can be administered to a subject to induce or enhance an immune response, particularly a cell-mediated cytolytic response, against cells expressing an antigen to which the biotinylated component is directed. The fusion protein may simply enhance an immune response (thus acting as an immunogenic composition), or confer protective immunity (thus acting as a vaccine).

[0090] In the vaccines of the present invention, the adjuvant may induce a Th1 type immune response in certain embodiments. The fusion protein SAV approach utilizing the adjuvant capacity of MTbHSP70 is superior to previous approaches because previous approaches lack a CD4+ / -1-antibody that can safely induce CD4+ / -antibodies against a protein target. + and CD8 + Compared with alum, CD4 + and CD8 + In T cells, the use of SAV units described herein resulted in an improved immune response of the T cells when both groups were compared to a saline control group.

[0091] The pharmaceutical composition containing the vaccine of the present invention can be prepared as a liquid solution or suspension. The pharmaceutical composition of the present invention can include conventional pharmaceutically acceptable excipients, such as diluents and carriers. Specifically, the composition can include a pharmaceutically acceptable carrier, such as PBS buffer. In addition to the carrier, the pharmaceutical composition can also contain an emulsifier, a pH buffer, a stabilizer, a dye, etc.

[0092] In certain embodiments, each vaccine of the pharmaceutical composition will contain a therapeutically effective dose of HSP70 that can produce an efficient immune response when administered to a subject, and / or can treat some HPV-related cancers (e.g., when used in combination with a checkpoint inhibitor) without causing toxicity to the subject. The treatment of solid tumors in some HPV-related cancers can be assessed as changes in phenotypic characteristics (e.g., tumor volume) or changes in immune response or expression associated with HPV-related solid tumors. Therefore, the therapeutically effective HSP70 in each vaccine dose is typically sufficient to increase the immunogenic response and reduce the volume of tumor growth in the subject being treated when administered in a physiologically tolerable composition.

[0093] A suitable dose of HSP70 for therapeutic efficacy is likely to be in the range of about 150-500 μg. Preliminary experiments have shown that HSP70 has a positive adjuvant effect and that as a recall response to E6 and E7 peptides, CD4+ cells expressing IFNγ in the spleen and lymph nodes + and CD8 +The percentage of T cells increased significantly. A favorable immunogenic response was observed at approximately 215 μg HSP70 per dose of vaccine.

[0094] Heat shock protein fusion proteins and biotinylated components or non-covalent complexes thereof as described herein can be administered to a subject in a variety of ways. Routes of administration include systemic, peripheral, parenteral, enteral, topical, and transdermal (e.g., sustained-release polymers). Any other convenient route of administration can be used, such as infusion or bolus injection, or absorption through epithelial or mucocutaneous linings.

[0095] In at least one embodiment, a composition comprising SAV can be transduced intradermally into a subject at 14-day intervals according to a Prime-Boost-Boost schedule. The intradermal (ID) route of administration is particularly advantageous for allowing MTbHSP70 to directly stimulate epidermal Langerhans cells / dendritic cells. Compared with the intramuscular (IM) route, administering ID vaccination has significant advantages. ID vaccination generally enhances vaccine responses, providing a more direct route to the extensive skin immune system through epidermal dendritic cells and draining skin lymph. Extracellular heat shock proteins (such as MTbHSP70) are potent inducers of innate and adaptive immunity. The dermal dendritic cell network is particularly rich in immune activation receptors for HSPs, including CD40, CD36, LOX-1, SR-A, TLR-2, and TLR-4. Therefore, using SAV for ID vaccination advantageously takes full advantage of the rich dermal antigen processing system and heat shock protein interactions.

[0096] In at least one embodiment, the pharmaceutical composition can be used in the form of itself or in the form of a mixture combined with other agents. Therefore, combined (combination) therapy includes sequential, simultaneous and separate or co-administration, such that the therapeutic effect of the drug first administered has not completely disappeared during subsequent administration.

[0097] In at least one embodiment, the composition comprising SAV can be combined with an approved anti-PD-1 agent (immune checkpoint inhibitor) for ID transduction. Immune checkpoint inhibitors maintain anti-tumor activity by interfering with T cell co-inhibitory signaling pathways. One of the important mechanisms by which cancer cells evade immune surveillance is to activate immune checkpoint pathways, which inhibit anti-tumor responses by causing T cell exhaustion or anergy. Tumor cells and tumor-specific CD8 + Cytotoxic T cells (CTLs) mainly work by binding to the PD-1 / PD-L1 tether within the tumor mass under conditions of mutual inhibition. When anti-PD-1 or anti-PD-L1 specific antibodies eliminate the mutual inhibition, CD8 + CTLs, thereby restoring their cytotoxicity. +CTLs attack and eliminate tumor cells.

[0098] HPV-related cancers that can be treated by the methods disclosed herein can include, but are not limited to, head and neck cancer, cervical cancer, anal cancer, other HPV-induced cancers expressing E6 and E7 oncoproteins, and combinations thereof.

[0099] Illustrative Embodiments

[0100] The following examples are set forth to aid in understanding the present disclosure and, of course, should not be construed as particularly limiting the embodiments described and claimed herein. Such variations of the embodiments now known or later developed that would be within the knowledge of those skilled in the art, as well as changes in formulation or minor changes in experimental design should be considered to fall within the scope of the embodiments incorporated herein.

[0101] Example 1: Sequences of used peptides and relevant epitopes

[0102] In this example, two immunogenic peptides are synthetic chimeras, each combining two documented MHC class I and one documented MHC class II murine epitopes of HPV 16 E6 / E7 into a single biotinylated concatemer. Table 1 illustrates the peptide sequences, and the identities of the three epitopes, each separated by a linker sequence. The first column represents the epitope and linker sequence of the E6 peptide, and the second column represents the epitope and linker sequence of the E7 peptide.

[0103] Table 1: Exemplary peptide sequences containing three epitope identities each separated by a linker sequence

[0104] Peptide 1 Peptide 2 N-terminus Biotin-PEG4 Biotin-PEG4 Connectors LEQLERVKR LEQLERVKR Epitope EVYDFAFRDL RAHYNIVTF Connectors AAYRVKR AAYRVKR Epitope GPAGQAEPDRAHYNIVTFCCK IVYRDGNPYAVCDK Connectors GPGPGRVKR GPGPGRVKR Epitope GTLGIVCPI QYNKPLCDLLI C-terminus AA-Amide AA-Amide

[0105] The peptide sequence used in the following study incorporates concatenated MHC class I and class II epitopes for HPV proteins E6 and E7. The peptide sequence is shown in Table 2, which illustrates the spacer and epitope structure, and has the following sequence:

[0106] LEQLERVKREVYDFAFRDLAAYRVKRQAEPDRAHYNIVTFCC KCDGPGPGRVKRYMLDLQPET

[0107] (SEQ ID NO:2).

[0108] Table 2: Peptide sequences used in immunogenicity studies

[0109] Peptide 3 N-terminus Biotin-PEG Connectors LEQLERVKR Epitope EVYDFAFRDL Connectors AAYRVKR Epitope QAEPDRAHYNIVTFCCKCD Connectors GPGPGRVKR Epitope YMLDLQPET C-terminus AA-Amide

[0110] Example 2: Design of human immunogenic HPV E6 / E7 peptides

[0111] Consider targeting E6 / E7 peptide sequences selected for presentation on human MHC class I and class II. Finally, two "long" biotinylated peptides (ie, one E6 and one E7) can be formed by a click chemistry mechanism using two smaller peptide fragments. Figure 1B Schematic diagram of smaller peptide fragments, including a DBCO-containing peptide and an azide (NNN)-containing peptide, and the final sequence of conjugation of peptide one and peptide two via a triazole moiety. Figure 1C The schematic diagram in depicts the mechanism of the click chemistry reaction. The fully formed peptide is then added to the MAV via a non-covalent biotin-avidin bond. Figure 1C The peptide fragment corresponds to the following sequence:

[0112] LEQLERVKREVYDFAFRDLCIVYRDGNPYAVRVKRGG(SEQ ID NO:3);

[0113] PYAVCDKCLKFYSKISEYRHYCYSLYGTTLEQQYNKPLCDLL(SEQ ID NO:4);

[0114] LEQLERVKRPTLHEYMLDLQPETTDLYCYGG (SEQ ID NO: 5); and

[0115] LEQLERVKRAGQAEPDRAHYNIVTFRVKRLRLCVQSTHVDIR TL (SEQ ID NO: 6).

[0116] Example 3: Sequence and properties of MAV

[0117] In this example, MAV protein as a vaccine adjuvant component was synthesized using techniques readily available and understood by those of ordinary skill in the art, including a histidine tag, MTbHSP70, a polyglycine linker, and avidin. Figure 2 Depicted is a Val381 to Phe381 point mutation in the MTbHSP70 portion that is predicted to inactivate endogenous peptide binding, thereby increasing the specificity of the platform for incorporated biotinylated peptides. Figure 2 The 771 amino acid sequence corresponds to SEQ ID NO:7.

[0118] Table 3 shows the properties of the MAV units, including molecular weight and concentration, which were assessed and confirmed using gel electrophoresis and extinction at 280 nm, respectively.

[0119] Table 3: Properties of the synthesized MAV unit

[0120] nature Molecular type His tag PI 5.23 EC 0.63 endotoxin <1EU / mg concentration 4.854mg / mL Buffer PBS, pH 7.4

[0121] Example 4: Assembly of eSAV

[0122] To prepare eSAV, a five-fold molar excess of biotinylated peptide 3 from Table 2 was added to MAV in PBS for 12 h at room temperature with end-over-end mixing. Any precipitated peptide was removed by centrifugation. The assembly reaction was prepared for 80 μg MAV / 50 μL dose of eSAV. The resulting eSAV was stored at -80°C until use.

[0123] Figure 3 Schematic illustration of the self-assembly mechanism of eSAV units formed by stable biotin-avidin binding. To verify this assembly, the absorbance of the eSAV units after addition of HABA dye was compared to that of unassembled MAV, and the absorbance difference was used to calculate the number of moles of biotinylated peptide attached per mole of MAV. Self-assembling constructs conjugated with different peptides can be mixed together to target vaccines against a large number of epitopes.

[0124] Example 5: Immune response to eSAV

[0125] In this example, the immune response of different T cell types after mice were vaccinated with eSAV was shown. For vaccination, C57BL / 6 mice were injected intradermally with 50 μL of the vaccine suspended in sterile PBS.

[0126] exist Figure 4A Figure 3 shows the expression of IFNγ in spleen cells after stimulation. Compared with the saline control group, the eSAV-vaccinated group showed CD4 + and CD8 + There was a statistically significant increase in IFNγ expression in the cells. In the eSAV group, 9.5% of CD4 + cells (p≤0.01) and 6.5% of CD8 + The cells (p ≤ 0.05) were positive for IFNγ expression. None of the other groups reached statistical significance. These results suggest that eSAV elicits a significant and balanced immune response compared to saline. There was no significant difference in CD4 + IFNγ + (p=0.68) and CD8 + IFNγ + There was no statistically significant difference in the P values ​​(p=0.83).

[0127] exist Figure 4B In Figure 2, the results of peptide stimulation on IL-2 expression in splenic T cells are presented. For this cytokine, only eSAV achieved a statistically significant increase in IL-2 expression compared to the saline group, and this occurred only in CD4 +There was no statistically significant difference in IL-2 secretion between the eSAV and 5% Alum + Peptide treatment groups.

[0128] Figure 4C Results of TNFα expression in T cells are shown in Figure 2. Only T cells from the eSAV-treated group showed statistically significant differences in expression compared to saline. + cells (p≤0.01) and 5.5% of CD8 + Cells (p≤0.05) expressed TNFα in response to peptide stimulation. There was no statistically significant difference in TNFα secretion between the eSAV and 5% Alum+Peptide treatment groups.

[0129] Figure 4D Results for CD62L expression in splenic T cells are presented in . None of the groups showed any significant increase in expression for this ligand. There was no statistically significant difference in CD62L expression between the eSAV and 5% Alum + Peptide treated groups.

[0130] In these embodiments, Figures 4A-4D Graphical legend, each data point represents cells from a single mouse, horizontal bars represent mean expression within groups and classes. 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 a ** represents a p-value of ≤0.01.

[0131] Example 6: Tumor Response to eSAV

[0132] In this example, the effect of eSAV or alum (5%) + peptide vaccination on TC-1 tumor growth in female C587BL / 6 mice is shown. For 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 into the loose skin of the flank of 8- to 10-week-old female C57BL / 6 mice using an insulin syringe. Tumor growth was measured twice a week and calculated using the formula volume = (axb 2 ) / 2, where a and b are the maximum and minimum tumor diameters, respectively.

[0133] exist Figure 5 The top graph shows tumor growth for each mouse, while the bottom graph shows the average tumor size for each treatment group. The data show that eSAV vaccination had a greater effect on reducing tumor growth volume during the study period compared to peptide + alum (5%) vaccination.

[0134] Example 7: Survival Probability

[0135] In this example, the survival probability of mice in a tumor treatment study is shown and compared between eSAV and peptide + alum (5%) vaccination. Figure 6 The mean survival rate of the eSAV treated group was statistically significant compared to the group receiving saline only and approached statistical significance compared to the Alum (5%) + peptide group.

[0136] Example 8: Dose-based CD8 + Immune response

[0137] In this example, CD8 + T cell immune response. Fig. 7A The immune response of splenocytes is shown in Figure 2. 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 significant response (p≤0.0001). No group showed a significant TNFα response. For IL-2, only the 215 μg group had a statistically significant recall response.

[0138] exist Figure 7B Figure 2 shows CD8 in lymphocytes. + T cell responses. A similar pattern was observed, with significant IFNγ only in the 130, 215, and 350 μg groups. In this case, the response was best at 215 μg. No significant TNFα expression was found in any group. IL-2 was significant only at 215 μg.

[0139] In these embodiments, Figure 7A-7B As illustrated, T cell responses are shown as a percentage of live T cells. Each data point represents cells from a single mouse, and the horizontal bars represent the average expression within groups and classes. After adjustment for multiple hypothesis testing, significant p values ​​relative to the saline group are shown. In these examples, significance is marked as follows: * = p value is ≤ 0.05, ** = p value is ≤ 0.01, *** = p value is ≤ 0.001, **** = p value is ≤ 0.0001.

[0140] Example 9: Dose-based CD4 + Immune response

[0141] In this example, CD4+ Tgp3+ expression in splenocytes and lymphocytes after stimulation with E6 / E7 peptides at different vaccine doses is shown. + T cell immune response. Fig. 8AIn the Figure 2, the immune response of splenocytes is shown. For IFNγ, the 215 and 350 μg 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 TNFα response. For IL-2, only the 215 μg group had a statistically significant recall response.

[0142] exist Figure 8B CD4 in lymphocytes + T cell responses showed a slightly broader range of immune responses. For IFNγ, significant responses were observed in the 130, 215, and 350 μg groups, with 215 μg appearing to be the best. For TNFα, the 215 μg group had a significant response, and for IL-2, the 130 and 250 μg groups showed significant responses.

[0143] In these embodiments, Figures 8A-8B In the figure, T cell responses are shown as the percentage of live T cells. Each data point represents cells from a single mouse, and the horizontal bar represents the average expression within the group and class. After adjustment for multiple hypothesis testing, significant p values ​​relative to the saline group are shown. In these examples, significance is marked as follows: * = p value is ≤ 0.05, ** = p value is ≤ 0.01, *** = p value is ≤ 0.001, **** = p value is ≤ 0.0001.

[0144] Example 10: Reactogenicity of SAV when used in combination with PD-1 checkpoint inhibitor therapy

[0145] In this example, the reactogenicity of vaccine / antibody treatments was evaluated. During the study, no reactogenicity was observed for any treatment in all groups.

[0146] Example 11: Tumor Growth Kinetics

[0147] In this example, tumor growth kinetics of vaccine and anti-mPD1 and vaccine and anti-IgG2a treatment groups are shown. Fig.9A In Figure 2B, eSAV significantly reduced TC-1 tumor volume, with reductions in tumor volume seen in mice receiving 80, 130, and 215 μg of eSAV. There were no statistically significant differences between anti-mPD1-treated mice receiving eSAV compared to the matched anti-IgG2a-treated group.

[0148] Example 12: Survival rate curve

[0149] In this example, survival curves for vaccine and anti-mPD1 and vaccine and anti-IgG2a treatment groups are shown. In Figures 11A-B, SAV had a highly significant positive survival effect on mice injected with TC-1 tumors at 80, 130 and 215 μg / vaccine doses (p<0.0001). There was also a dose response for SAV, with 215 μg SAV providing the best overall survival benefit for mice bearing TC-1 tumors. Anti-mPD1 had a statistically significant survival benefit in peptide-treated mice compared to the anti-IgG2a treatment group.

[0150] In the foregoing description, many specific details, such as specific materials, dimensions, process parameters, etc., are set forth to provide a thorough understanding of the present invention. Specific characteristics, structures, materials or features can be combined in any suitable manner in one or more embodiments. The words "example" or "illustrative" are used herein to indicate use as examples, examples or illustrations. Any aspect or design described herein as "example" or "illustrative" need not be interpreted as being more preferred or more advantageous than other aspects or designs. Instead, the use of the words "example" or "illustrative" is intended only to present concepts in a specific manner. The term "or" as used in this application is intended to represent an inclusive "or", not an exclusive "or". That is, unless otherwise specified or clear from the context, "X includes A or B" is intended to represent any natural inclusive arrangement. 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 foregoing cases. "An embodiment", "some embodiments" or "one embodiment" mentioned throughout this specification means that a specific characteristic, structure or feature described in conjunction with the embodiment is included in at least one embodiment. Thus, appearances of the phrases "an embodiment," "certain embodiments," or "one embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment.

[0151] The present invention has been described with reference to specific exemplary embodiments of the present invention. Therefore, the description and the accompanying drawings are to be regarded as illustrative rather than restrictive. Various modifications of the present 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 attached embodiments.

Claims

1. A pharmaceutical composition comprising a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein non-covalently binds to two or more biotinylated peptides, and wherein the two or more biotinylated peptides each comprise a sequence that is at least partially derived from an epitope of a human viral antigen protein, provided that each epitope belongs to 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, wherein the biotin-binding protein non-covalently binds to a biotinylated peptide, and wherein 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.

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 include 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 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.

6. A pharmaceutical composition comprising a heat shock protein fused to a biotin-binding protein, wherein the biotin-binding protein non-covalently binds to a biotinylated peptide, wherein the biotinylated peptide is formed by joining 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.

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 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 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 according to any one of claims 1 to 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 point mutation from Val381 to Phe381.

13. A pharmaceutical composition comprising: Fusion proteins derived from heat shock proteins; A first peptide non-covalently bound to the fusion protein, the first biotinylated peptide comprising a human papillomavirus (HPV) E6 protein epitope; and The second peptide is non-covalently bound to the fusion protein, and the second biotinylated peptide comprises 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 biotin binding protein is selected from the group consisting of avidin, streptavidin and neutravidin.

16. A pharmaceutical composition as claimed in any one of the preceding claims, further comprising a pharmaceutically acceptable excipient.

17. A pharmaceutical composition as claimed in any one of the preceding claims, wherein the pharmaceutical composition is a vaccine composition.

18. A method of 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 acceptable excipient, and wherein the therapeutic dose administered to the subject comprises about 150 μg to about 500 μg of the heat shock protein.

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

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