Title:  Immune responses against HPV antigens elicited by compositions comprising an HPV antigen and a stress protein or an expression vector capable of expression of these proteins

United States Patent:  6,524,825

Issued:  February 25, 2003

Inventors:  Mizzen; Lee A. (Victoria, CA); Chu; N. Randall (Victoria, CA); Wu; Huacheng Bill (Victoria, CA)

Assignee:  Stressgen Biotechnologies, Inc. (Vancouver, CA)

Appl. No.:  498918

Filed:  February 4, 2000

The present invention relates to compositions for inducing an immune response, preferably a cellular, in particular a cell-mediated, cytolytic immune response, to human papillomavirus (HPV) protein antigens displayed by HPV or exhibited by infected cells including cells from cervical and other tumors. In one embodiment, compositions comprise an HPV protein antigen joined to a stress protein (or heat shock protein (Hsp)). The HPV protein antigen may be joined to the stress protein by chemical conjugation or noncovalently using linking moieties, or the HPV protein antigen and the stress protein may be joined in a fusion protein containing both HPV protein antigen and stress protein sequences. In another embodiment, compositions comprise an expression vector including, in expressible form, sequences encoding the HPV protein antigen and sequences encoding the stress protein. The expression vector can be introduced into cells of a subject, or it can be used to transduce cells of the subject ex vivo, resulting in the expression of an HPV protein antigen-stress protein fusion protein that will stimulate the subject's immune response to the HPV protein antigen. The present invention also relates to compositions comprising a stress protein linked to an HPV antigen and another pharmacologically acceptable component, to stress protein-HPV protein antigen fusions and conjugates and to expression vectors encoding and capable of directing the expression in a subject's cells of a fusion protein comprising a stress protein and an HPV protein antigen sequence. The present invention also relates to uses of these compositions to induce immune responses against HPV and HPV protein antigen-exhibiting cells including HPV-associated tumors.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions that induce an immune response in a subject to human papillomavirus of cells of the subject exhibiting a protein antigen of an HPV. In one embodiment, the compositions comprise an HPV protein antigen and a stress protein. In another embodiment, the compositions comprise an expression vector capable of directing the expression of an HPV protein antigen-stress protein fusion protein.

The compositions of the present invention can be used prophylactically to raise immunity against an HPV protein antigen, preventing the establishment and proliferation of HPV or of cells of a subject expressing and exhibiting the HPV protein antigen or presenting portions thereof. The compositions can also be used therapeutically in a subject previously infected with an HPV to prevent further viral proliferation or to eliminate cells of the subject that proliferate as a consequence of HPV infection, including tumors expressing and exhibiting and HPV antigen or presenting a portion of the antigen. When reference is made herein to an HPV protein antigen as a target of an immune response induced by a composition of the present invention, the HPV protein antigen is understood to include an entire HPV protein or a polypeptide (molecular weight greater than 10 kDa) portion of the HPV protein exhibited on the surface of HPV or an infected cell of a subject as well as peptide displayed by an infected cell as a result of processing and presentation of the HPV protein, for example, through the typical MHC class I or II pathways.

The genomic sequences of many different types of HPV were cloned and were characterized by DNA sequence analysis. Bacterial vectors containing complete or partial HPV genomes are available from various sources including, for example, the American Tissue Culture Collection (ATCC). Additional types of HPV useful for the practice of the present invention can be isolated and typed by the methods previously established for this purpose, which methods are well known in the art.

HPV expresses six or seven non-structural and two structural proteins, and each of these proteins could, in principle, serve as a target for immunoprophylactic or immunotherapeutic approaches aimed at eliminating HPV and/or infected cells. Viral capsid proteins L1 and L2 are the structural proteins of HPV which are encoded by late genes. L1 is the major capsid protein that is highly conserved among different HPV species. The seven non-structural proteins are products of the early viral genes. Proteins E1, E2 and E4 play an important role in virus replication. Protein E4 functions additionally in the maturation of the virus. The role of E5 is less well known. Proteins E6 and E7 are oncoproteins critical for viral replication as well as for host cell immortalization and transformation. These proteins, that can be incorporated in the compositions of the present invention, are referred to as HPV protein antigens.

Of particular importance in the application of the present invention to the prophylactic and therapeutic treatment of HPV-associated cancers is the observation that HPV E6 and E7 proteins are consistently expressed in cervical cancers (Zur Hausen, Appl. Pathol. 5:19-24 (1987); Pater and Pater, Virology 145:313-8 (1985)). Kinoshita et al., Br. J. Cancer 71:344-9 (1995)) have demonstrated that the E6 and E7 genes are also expressed in lung carcinoma. Moreover, some natural immune (humoral) immune response to E7 was noted in cervical cancer patients (Jochmus-Kudielka et al., J. Nat'l Cancer Inst. 81:1698-704 (1989)). Finally, model studies demonstrate that immunization with a modified E7 protein protects mice against a challenge with lung cells transformed with an activated c-Ha-ras gene and HPV E6/E7 genes (Lin et al., Cancer Res. 56:21-26 (1996)). From the points of view that these proteins are typically expressed in cancers arising as a consequence of HPV infection, that the same proteins are also the oncogenes which most likely played a major role in the development and maintenance of the cancers, and that an immune response can be directed against these proteins, E6 and E7 are preferred targets for immunintervention or prophylaxis, and, hence, are preferred HPV protein antigens of compositions of the present invention to be used to prevent or treat HPV-associated cancer.

It has been shown in several animal models that cytotoxic T cell (CTL) peptides can induce protective immunity against certain viruses (Kast and Melief, Immunol. Lett. 30:229 (1991)). It has been observed that immunosuppressed individuals more often develop cervical carcinoma than immunocompetent individuals (Schneider et al., Acta Cytologica 27:220-4 (1983)). This strongly suggests that the cellular arm of the immune system, particularly the T cell system, are of major importance in the immunological dense against HPV-associated malignancy. Supporting evidence for the importance of a CTL response in producing protective immunity against E6- and E7-transformed cells came from an experiment in which mice vaccinated with a relevant CTL epitope of HPV 16 E7 were protected against transplantable HPV 16-induced tumors (Feltkamp et al., Eur. J. Immunol. 23:2242 (1993)). The present invention is based on our observation that linkage of a stress protein to an HPV protein antigen results in a composition that strongly stimulates cellular, in particular cell-mediated cytolytic, responses against the linked HPV protein antigen, which responses can kill cells exhibiting the HPV antigen.

As HPV protein antigen of the present invention can be any HPV-encoded polypeptide. In addition, it can be a portion of an HPV protein, provided that the portion, when joined with a stress protein, retains the ability to induce an immune response against the HPV protein antigen exhibited by infected cells or displayed by HPV. Compositions of the invention comprising various portions of an HPV antigen rather than a complete HPV protein antigen can be produced by routine methods such as those described hereinafter or in molecular biology and biochemistry textbooks. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press (1989); Deutscher, M., Guide to Protein Purification Methods Enzymology, vol. 182, Academic Press, Inc., San Diego, Calif. (1990). Each composition having a particular portion of an HPV protein antigen can be tested for the degree and quality of the immune response against the HPV protein antigen in experiments such as those described in the examples described hereinafter. Minimally, an HPV protein antigen in a composition of the present invention will contain at least one B or T cell epitope (e.g., a CTL or a T helper cell epitope) of an HPV protein. When reference is made to compositions of the present invention, the term "HPV protein antigen" includes portions of an HPV protein antigen, provided that such portions retain the ability to induce an immune response against the HPV protein antigen exhibited by infected cells or displayed by HPV.

E6 and particularly E7 are transforming proteins. In compositions including as the HPV protein antigen a complete HPV E6 or E7 protein sequence or a portion sufficiently complete to retain transforming ability, the transforming nature of the antigen may or may not represent a substantial risk, depending on the method by which the HPV antigen is manufactured. For example, in cases in which an HPV protein antigen or a composition including such antigen is prepared by recombinant techniques that carry a risk of DNA contamination, it may be prudent to undertake steps to eliminate the transforming ability of the antigen. When using compositions including an expression vector directing the expression in a subject of a fusion protein including a complete HPV E6 or E7 protein sequence or a portion sufficiently complete to retain transforming ability, it may be necessary to eliminate sequences that render the protein product transforming. Nontransforming variants of E6 and E7 were obtained by fusing E6 and E7 sequences (PCT/AU95/00868). It is therefore possible that certain fusion proteins including E6 or E7 sequences and stress protein sequences may already be nontransforming. Alternatively, sequences may be selectively deleted from E6 or E7 using techniques that are well known in the art, and that have also been described in PCT/AU95/00868. The deletions can be made in expression vectors expressing E6 or E7 sequences alone or in vectors expressing E6- or E7 -stress protein fusion proteins. The results of such manipulations can be assessed by testing the transforming ability of deletion proteins in a transfection experiment. For example, NIH3T3 cells can be transfected with an expression vector including a gene for a deletion protein, and transforming ability can be estimated by colony-forming assay in soft agar. This particular test is also described in PCT/AU95/00868.

In one embodiment of the present invention, compositions are comprised of two moieties: a stress protein and a protein antigen of HPV against which a cellular immune response is desired. The two moieties are joined to form a single unit. The two moieties can be connected by conjugation, i.e., through a covalent bond between the stress protein and the HPV protein antigen. Hermanson, G. T., Bioconjugate Techniques, Academic Press, Inc., San Diego, Calif. (1996); Lussow, A. R. et al., Eur. J. Immun. 21:2297-2302 (1991); Barrios, C. et al., Eur. J. Immun. 22:1365-1372 (1992)). Alternatively, recombinant techniques can be used to connect and express the two moieties, which techniques result in a recombinant fusion protein which includes the stress protein and the HPV protein antigen in a single molecule. This makes it possible to produce and purify a single recombinant molecule in the production process. The two moieties can also be joined noncovalently. Any of several known high-affinity interactions can be adapted to, noncovalently connect the two moieties. For example, a biotin group can be added to an HPV protein antigen, and the stress protein to be joined can be expressed as an avidin--stress protein fusion protein. The avidin--stress protein fusion protein will strongly bind the biotinylated HPV protein antigen. Analogously, portions of HPV protein antigens can be joined to a complete stress protein or to portions of the stress protein, and portions of a stress protein can be joined to a complete HPV protein antigen or to portions of the HPV protein antigen, provided that the respective portions are sufficient to induce an immune response against the HPV protein antigen in a subject to whom it is administered. In another embodiment, compositions comprise an expression vector capable of directing the expression of an HPV protein antigen-stress protein fusion protein.

Any suitable stress protein (heat shock protein (hsp)) can be used in the compositions of the present invention. For example, as described in the examples, Hsp60 and/or Hsp70 can be used. Turning to stress proteins generally, cells respond to a stressor (typically heat shock treatment) by increasing the expression of a group of genes commonly referred to as stress, or heat shock, genes. Heat shock treatment involves exposure of cells or organisms to temperatures that are one to several degrees Celsius above the temperature to which the cells are adapted. In coordination with the induction of such genes, the levels of corresponding stress proteins increase in stressed cells. As used herein, a "stress protein," also known as a "heat shock protein" or "Hsp," is a protein that is encoded by a stress gene, and is therefore typically produced in significantly greater amounts upon the contact or exposure of the stressor to the organism. A "stress gene," also known as "heat shock gene" is used herein as a gene that is activated or otherwise detectably upregulated due to the contact or exposure of an organism (containing the gene) to a stressor, such as heat shock, hypoxia, glucose deprivation, heavy metal salts, inhibitor of energy metabolism and electron transport, and protein denaturants, or to certain benzoquinone ansamycins. Nover, L., Heat Shock Response, CRC Press, Inc., Boca Raton, Fla. (1991). "Stress gene" also includes homologous genes within known stress gene families, such as certain genes within the Hsp70 and Hsp90 stress gene families, even though such homologous genes are not themselves induced by a stressor. Each of the terms stress gene and stress protein as used in the present specification may be inclusive of the other, unless the context indicates otherwise.

In particular embodiments, e.g., in cases involving chemical conjugates between a stress protein and an HPV protein antigen, the stress proteins used in the present invention are isolated stress proteins, which means that the stress proteins have been selected and separated from the host cell in which they were produced. Such isolation can be carried out as described herein and using routine methods of protein isolation known in the art. Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1982); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press (1989); Deutscher, M., Guide to Protein Purification Methods Enzymology, vol. 182, Academic Press, Inc., San Diego, Calif. (1990).

In bacteria, the predominant stress proteins are proteins with molecular sizes of about 70 and 60 kDa, respectively, that are commonly referred to as Hsp70 and Hsp60, respectively. These and other specific stress proteins and the genes encoding them are discussed further below. In bacteria, Hsp70 and Hsp60 typically represent about 1-3% of cell protein based on the staining pattern using sodium dodecyl sulfate polyacrylamide gel electrophoresis and the stain Coomassie blue, but accumulate to levels as high as 25% under stressful conditions. Stress proteins appear to participate in important cellular processes such as protein synthesis, intracellular trafficking, and assembly and disassembly of protein complexes. It appears that the increased amounts of stress proteins synthesized during stress serve primarily to minimize the consequences of induced protein unfolding. Indeed, the preexposure of cells to mildly stressful conditions that induce the synthesis of stress proteins affords protection to the cells from the deleterious effects of a subsequent more extreme stress.

The major stress proteins appear to be expressed in every organism and tissue type) examined so far. Also, it appears that stress proteins represent the most highly conserved group of proteins identified to date. For example, when stress proteins in widely diverse organisms are compared. Hsp90 and Hsp70 exhibit 50% or higher identity at the amino acid level and share many similarities at non-identical positions. It is noted that similar or higher levels of homology exist between different members of a particular stress protein family within species.

The genes encoding stress proteins may be present in a single copy or in multiple, non-identical copies in the genome of a cell or organism. For example, the human genome has been shown to contain at least one copy of an hsp100 gene, at least two different hsp90 genes, up to ten hsp70 genes of which at least several are non-identical copies, several T complex genes (Tcp genes) and at least one gene encoding the related mitochondrial protein Hsp60, as well as at least three copies of small hsp genes encoding Hsps in the 20-30 kDa range of molecular size. In most families of stress genes there is at least one gene whose expression level is relatively high and is either entirely constitutive or only mildly heat shock-inducible. Furthermore, several families of stress genes include members that are not up-regulated by heat but by other cues such as increased calcium levels, etc.

The stress proteins, particularly Hsp70, Hsp60, Hsp20-30 and Hsp10, are among the major determinants recognized by the host immune system in the immune response to infection by Mycobacterium tuberculosis and Mycobacterium leprae. Young, R. A. and Elliott, T. J., Stress Proteins, Infection, And Immune Surveillance, Cell 50:5-8 (1989). Further, some rat arthritogenic T cells recognize Hsp60 epitopes. Van Eden, W. et al., Nature 331:171-173 (1988). However, individuals, including healthy individuals, with no history of mycobacterial infection or autoimmune disease also carry T cells that recognize both bacterial and human Hsp60 epitopes; a considerable fraction of T cells in healthy individuals that are characterized by expression of the gamma-delta T cell receptor recognize both self and foreign stress proteins. O'Brien, R. et al., Cell 57:664-674 (1989). Thus, individuals, even healthy individuals possess T-cell populations that recognize both foreign and self stress protein epitopes.

This system recognizing stress protein epitopes presumably constitutes an "early defense system" against invading organisms. Murray, P. J. and Young, R. A., J. Bacteriol. 174: 4193-6 (1992). The system may be maintained by frequent stimulation by bacteria and viruses. As discussed before, healthy individuals have T cell populations recognizing self stress proteins. Thus, the presence of autoreactive T cells is compatible with normal health and does not cause autoimmune disease; this demonstrates the safety of stress proteins within an individual. The safety of stress proteins is additionally demonstrated by the success and relative safety of BCG (Bacille Calmette Guerin, a strain of Mycobacterium bovis) vaccinations, which induce an immune response against stress proteins that is also protective against Mycobacterium tuberculosis.

Families of stress genes and proteins for use in the present invention are those well known in the art and include, for example, Hsp100-200, Hsp100, Hsp90, Lon, Hsp70, Hsp60, TF55, Hsp40, FKBPs, cyclophilins, Hsp20-30, ClpP, GrpE, Hsp10, ubiquitin, calnexin, and protein disulfide isomerases. Macario, A. J. L., Cold Spring Harbor Laboratory Res. 25:59-70, 1995; Parsell, D. A. & Lindquist, S. Ann. Rev. Genet. 27:437-496 (1993); U.S. Pat. No. 5,232,833 (Sanders et al.). A particular group of stress proteins includes Hsp90, Hsp70, Hsp60, Hsp20-30, further preferably Hsp70 and Hsp60.

Hsp100-200 examples include Grp170 (for glucose-regulated protein). Grp170 resides in the lumen of the ER, in the pre-golgi compartment, and may play a role in immunoglobulin folding and assembly.

Hsp100 examples include mammalian Hsp110, yeast Hsp104, ClpA, ClpB, ClpC, ClpX and ClpY. Yeast Hsp104 and E. coli ClpA, form hexameric and E. coli ClpB, tetrameric particles whose assembly appears to require adenine nucleotide binding. Clp protease provides a 750 kDa heterooligomer composed of ClpP (a proteolytic subunit) and of ClpA. ClpB-Y are structurally related to ClpA, although unlike ClpA they do not appear to complex with ClpP.

Hsp90 examples include HtpG in E. coli, Hsp83 and Hsc83 yeast, and Hsp90alpha, Hsp90beta and Grp94 in humans. Hsp90 binds groups of proteins, which proteins are typically cellular regulatory molecules such as steroid hormone receptors (e.g., glucocorticoid, estrogen, progesterone, and testosterone receptors), transcription factors and protein kinases that play a role in signal transduction mechanisms. Hsp90 proteins also participate in the formation of large, abundant protein complexes that include other stress proteins.

Lon is a tetrameric protein functioning as an ATP-dependent protease degrading non-native proteins in E. coli.

Hsp70 examples include Hsp72 and Hsc73 from mammalian cells, DnaK from bacteria, particularly mycobacteria such as Mycobacterium leprae, Mycobacterium tuberculosis, and Mycobacterium bovis (such as Bacille-Calmette Guerin; referred to herein as Hsp71), DnaK from Escherichia coli, yeast, and other prokaryotes, and BiP and Grp78. Hsp70 is capable of specifically binding ATP as well as unfolded polypeptides and peptides, thereby participating in protein folding and unfolding as well as in the assembly and disassembly of protein complexes.

Hsp60 examples include Hsp65 from mycobacteria. Bacterial Hsp60 is also commonly known as GroEL, such as the GroEL from E. coli. Hsp60 forms large homooligomeric complexes, and appears to play a key role in protein folding. Hsp60 homologues are present in eukaryotic mitochondria and chloroplasts.

TF55 examples include Tcpl, TRiC and thermosome. The proteins typically occur in the cytoplasm of eukaryotes and some archaebacteria, and form multi-membered rings, promoting protein folding. They are also weakly homologous to Hsp60.

Hsp40 examples include DnaJ from prokaryotes such as E. coli and mycobacteria and HSJ1, HDJ1 and Hsp40. Hsp40 plays a role as a molecular chaperone in protein folding, thermotolerance and DNA replication, among other cellular activities.

FKBPs examples include FKBP12, FKBP13, FKBP25, and FKBP59, Fprl and Nepl. The proteins typically have peptidyl-prolyl isomerase activity and interact with immunosuppressants such as FK506 and rapamycin. The proteins are typically found in the cytoplasm and the endoplasmic reticululum.

Cyclophilin examples include cyclophilins A, B and C. The proteins have peptidyl-prolyl isomerase activity and interact with the immunosuppressant cyclosporin A. The protein cyclosporin A binds calcineurin (a protein phosphatase).

Hsp20-30 is also referred to as small Hsp. Hsp20-30 is typically found in large homooligomeric complexes or, possibly, also heterooligomeric complexes where an organism or cell type expresses several different types of small Hsps. Hsp20-30 interacts with cytoskeletal structures, and may play a regulatory role in the polymerization/depolymerization of actin. Hsp20-30 is rapidly phosphorylated upon stress or exposure or resting cells to growth factors. Hsp20-30 homologues include alpha-crystallin.

ClpP is an E. coli protease involved in degradation of abnormal proteins. Homologues of ClpP are found in chloroplasts. ClpP forms a heterooligomeric complex with ClpA.

GrpE is an E. coli protein of about 20 kDa that is involved in both the rescue of stress-damaged proteins as well as the degradation of damaged proteins GrpE plays a role in the regulation of stress gene expression in E. coli.

Hsp10 examples include GroES and Cpn10. Hsp10 is typically found in E. coli and in mitochondria and chloroplasts of eukaryotic cells. Hsp10 forms a seven-membered ring that associates with Hsp60 oligomers. Hsp10 is also involved in protein folding.

Ubiquitin has been found to bind proteins in coordination with the proteolytic removal of the proteins by ATP-dependent cytosolic proteases.

In particular embodiments, the stress proteins of the present invention are obtained from enterobacteria, mycobacteria (particularly M. leprae, M. tuberculosis, M. vaccae, M. smegmatis and M. bovis), E. coli, yeast, Drosophila, vertebrates, avians, chickens, mammals, rats, mice, primates, or humans.

The stress proteins may be in the form of acidic or basic salts, or in neutral form. In addition, individual amino acid residues may be modified by oxidation or reduction. Furthermore, various substitutions, deletions, or additions may be made to the amino acid or nucleic acid sequences, the net effect of which is to retain or further enhance the increased biological activity of the stress protein. Due to code degeneracy, for example, there may be considerable variation in nucleotide sequences encoding the same amino acid sequence. The present invention is also suitable for use with portions of stress proteins or peptides obtained from stress proteins, provided such portions or peptides include the epitopes involved with enhancing the immune response to the chosen HPV protein antigen. Portions of stress proteins may be obtained by fragmentation using proteinases, or by recombinant methods, such as the expression of only part of a stress protein-encoding nucleotide sequence (either alone or fused with another protein-encoding nucleic acid sequence). Peptides may also be produced by such methods, or by chemical synthesis. The stress proteins may include mutations introduced at particular loci by a variety of known techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press (1989); Drinkwater and Klinedinst Proc. Natl. Acad. Sci. USA 83:3402-3406 (1986); Liao and Wise, Gene 88:107-111 (1990); Horwitz et al., Genome 3:112-117 (1989). The term stress protein as used herein is intended to include such portions and peptides of a stress protein.

Methods of identifying a gene or a protein under consideration as a stress gene or protein are well known in the art. For example, the conservation of the genes and proteins of a particular stress protein family permits comparison of the nucleotide or amino acid sequence of the gene/protein under consideration with well known stress genes such as DnaK, GroEL or DnaJ, e.g., by nucleic acid hybridization or nucleic acid or amino acid sequencing followed by computer comparison analysis. Voellmy, R., et al., Proc. Nat'l Acad. Sci. USA 82:4949-4953 (1985). Alternatively, an assay may be used to identify and/or discriminate between essential structural features and/or functional properties of a selected stress protein. For example, an expression library may be screened using anti-Hsp antibodies. Hsp90 is well known to bind the benzoquinone ansamycin geldanamycin with high affinity. An expression library could therefore be screened with geldanamycin to discover putative homologs of Hsp90 as proteins binding the benzoquinone ansamycin. The nature of the protein encoded by the isolated nucleic acid could be further confirmed by other assays including antibody-based assays. Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press (1988). In addition, the biological activity of a given stress protein group may be exploited. Guidon, P. T., and Hightower, L. E., Biochem. 25:3231-3239 (1986). For example, Hsp70 is capable of specifically binding ATP as well as unfolded polypeptides and peptides in the assembly of protein complexes. Thus, mixing a protein under consideration with a sample comprising appropriate polypeptides, peptides, or ATP, followed by determination of the presence or absence of production of protein-protein or protein-nucleotide complexes indicates the apparent presence or absence of an Hsp70 protein or gene, which presence or absence can be confirmed utilizing other assays such as antibody-based assays.

The stress protein, stress protein portion, stress protein homologue and the protein antigen of HPV to which the stress protein is conjugated or joined noncovalently present in the composition can be produced or obtained using known techniques. For example, the stress protein and/or the antigen can be obtained (isolated) from a source in which it is known to occur, can be produced and harvested from cell cultures or, in the case of the antigen, can be obtained from infected cells, can be produced by cloning, if necessary, and expressing a gene encoding the desired stress protein or the antigen, or can be synthesized chemically. Furthermore, a nucleic acid sequence encoding the desired stress protein or the antigen can be synthesized chemically. A fusion protein including a stress protein and an HPV protein antigen can be produced by recombinant means. For example, a nucleic acid encoding the stress protein can be joined to either end of a nucleic acid sequence encoding the HPV protein antigen such that the two protein-coding sequences are sharing a common translational reading frame and can be expressed as a fusion protein including the HPV protein-antigen and the stress protein. The combined sequence is inserted into a suitable vector chosen based on the expression features desired and the nature of the host cell. In the examples provided hereinafter, the nucleic acid sequences are assembled in a vector suitable for protein expression in the bacterium E. coli. Following expression in the chosen host cell, fusion protein can be purified by routine biochemical separation techniques or by immunoaffinity methods using an antibody to one or the other part of the fusion protein. Alternatively, the selected vector can add a tag to the fusion protein sequence, e.g., an oligohistidine tag as described in the examples presented hereinafter, permitting expression of a tagged fusion protein that can be purified by affinity methods using an antibody or other material having an appropriately high affinity for the tag. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press (1989); Deutscher, M., Guide to Protein Purification Methods Enzymology, vol. 182, Academic Press, Inc., San Diego, Calif. (1990). If a vector suitable for expression in mammalian cells is used, e.g., one of the vectors discussed below, the fusion protein can be expressed and purified from mammalian cells. Alternatively, the mammalian expression vector (including fusion protein-coding sequences) can be administered to a subject to direct expression of the fusion protein in the subject's cells. A nucleic acid encoding a fusion protein including a stress protein and an HPV protein antigen can also be produced chemically and then inserted into a suitable vector for fusion protein production and purification or administration to a subject. Finally, a fusion protein can also be prepared chemically.

The compositions comprising a stress protein and an HPV antigen described herein can be used to enhance an immune response, particularly a cell-mediated cytolytic response, against an HPV, or HPV-infected or transformed cell expressing an HPV antigen. Preferably, compositions will contain HPV antigen sequences from the particular HPV type against whose proteins an immune response is to be elicited.

The compositions comprising a stress protein and an HPV antigen described herein can be administered to a subject in a variety of ways. The routes of administration include intradermal, transdermal (e.g., slow release polymers), intramuscular, intraperitoneal, intravenous, subcutaneous, oral, epidural and intranasal routes. Any other convenient route of administration can be used, for example, infusion or bolus injection, or absorption through epithelial or mucocutaneous linings. In addition, the compositions described herein can contain and be administered together with other pharmacologically acceptable components such as biologically active agents (e.g., adjuvants such as alum), surfactants (e.g., glycerides), excipients (e.g., lactose), carriers, diluents and vehicles. Furthermore, the compositions can be used ex vivo as a means of stimulating white blood cells obtained from a subject to elicit, expand and propagate HPV protein antigen-specific immune cells in vitro that are subsequently reintroduced into the subject.

Further, a stress protein-HPV protein antigen fusion protein can be administered by in vivo expression of a nucleic acid encoding such protein sequences into a human subject. Expression of such a nucleic acid can also be achieved ex vivo as a means of stimulating white blood cells obtained from a subject to elicit, expand and propagate HPV antigen-specific immune cells in vitro that are subsequently reintroduced into the subject. Expression vectors suitable for directing the expression of HPV protein antigen-stress protein fusions can be selected from the large variety of vectors currently used in the field. Preferred will be vectors that are capable of producing high levels of expression as well as are effective in transducing a gene of interest. For example, recombinant adenovirus vector pJM17 (All et al., Gene Therapy 1:367-84 (1994); Berkner K. L., Biotechniques 6:616-24 1988), second generation adenovirus vectors DE1/DE4 (Wang and Finer, Nature Medicine 2:714-6 (1996)), or adeno-associated viral vector AAV/Neo (Muro-Cacho et al., J. Immunotherapy 11:231-7 (1992)) can be used. Furthermore, recombinant retroviral vectors MFG (Jaffee et al., Cancer Res. 53:2221-6 (1993)) or LN, LNSX, LNCX, LXSN (Miller and Rosman, Biotechniques 7:980-9 (1989)) can be employed. Herpes simplex virus-based vectors such as pHSV1 (Geller et al., Proc. Nat'l Acad. Sci 87:8950-4 (1990) or vaccinia viral vectors such as MVA (Sutter and Moss, Proc. Nat'l Acad. Sci. 89:10847-51 (1992)) can serve as alternatives.

Frequently used specific expression units including promoter and 3' sequences are those found in plasmid CDNA3 (Invitrogen), plasmid AH5, pRC/CMV (Invitrogen), pCMU II (Paabo et al., EMBO J. 5:1921-1927 (1986)), pZip-Neo SV (Cepko et al., Cell 37:1053-1062 (1984)) and pSRa (DNAX, Palo Alto, Calif.). The introduction of genes into expression units and/or vectors can be accomplished using genetic engineering techniques, as described in manuals like Molecular Cloning and Current Protocols in Molecular Biology (Sambrook, J., et al., Molecular Cloning, Cold Spring Harbor Press (1989); Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley-Interscience (1989)). A resulting expressible nucleic acid can be introduced into cells of a human subject by any method capable of placing the nucleic acid into cells in an expressible form, for example as part of a viral vector such as described above, as naked plasmid or other DNA, or encapsidated in targeted liposomes or in erythrocyte ghosts (Friedman, T., Science: 244:1275-1281 (1989); Rabinovich, N. R. et al., Science, 265:1401-1404 (1994)). Methods of transduction include direct injection into tissues and tumors, liposomal transfection (Fraley et al., Nature 370:111-117 (1980)), receptor-mediated endocytosis (Zatloukal et al., Ann. N.Y. Acad. Sci. 660:136-153 (1992)), and particle bombardment-mediated gene transfer (Eisenbraun et al., DNA & Cell. Biol. 12:791-797 (1993)).

The amount of stress protein and HPV protein antigen (fused, conjugated or noncovalently joined as discussed before) in the compositions of the present invention is an amount which produces an effective immunostimulatory response in a subject. An effective amount is an amount such that when administered, it results in an induction of an immune response. In addition, the amount of stress protein and HPV protein antigen administered to the subject will vary depending on a variety of factors, including the HPV protein antigen and stress protein employed, the size, age, body weight, general health, sex, and diet of the subject as well as on its general immunological responsiveness. Adjustment and manipulation of established dose ranges are well within the ability of those skilled in the art. For example, the amount of stress protein and antigen can be from about 1 microgram to about 1 gram, preferably from about 100 microgram to about 1 gram, and from about 1 milligram to about 1 gram. An effective amount of a composition comprising an expression vector is an amount such that when administered, it induces an immune response against the HPV protein antigen which it encodes. Furthermore, the amount of expression vector administered to the subject will vary depending on a variety of factors, including the HPV protein antigen and stress protein expressed, the size, age, body weight, general health, sex, and diet of the subject as well as on its general immunological responsiveness. Additional factors that need to be considered are the route of application and the type of vector used. For example, when prophylactic or therapeutic treatment is carried out with a viral vector containing a nucleic acid encoding an HPV protein antigen-stress protein fusion protein, the effective amount will be in the range of 10⁴ to 10¹² helper-free, replication-defective virus per kg body weight, preferably in the range of 10⁵ to 10¹¹ virus per kg body weight and most preferably in the range of 10⁶ to 10¹⁰ virus per kg body weight.

Claim 1 of 100 Claims

What is claimed is:

1. A fusion protein comprising a human papillomavirus (HPV) antigen, or an antigenic portion thereof, and a stress protein thereof, wherein the fusion protein induces an immune response to the HPV antigen in a mammal to whom the fusion protein is administered.
 

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