WO2016026919A1

WO2016026919A1 - Vaccines and manufacturing methods for treatment and prevention of disease

Info

Publication number: WO2016026919A1
Application number: PCT/EP2015/069106
Authority: WO
Inventors: Magnus Von Knebel-Doeberitz; Miriam Reuschenbach; Lutz Gissmann; Martin Müller; Jürgen KOPITZ; Franziska FAULSTICH
Original assignee: Deutsches Krebsforschungszentrum DKFZ; Universitaet Heidelberg
Current assignee: Deutsches Krebsforschungszentrum DKFZ; Universitaet Heidelberg
Priority date: 2014-08-20
Filing date: 2015-08-20
Publication date: 2016-02-25
Anticipated expiration: 2017-02-20

Abstract

The present invention relates to a vaccine comprising a viral coat protein and an antigen, wherein the viral coat protein and the antigen are covalently or non-covalently linked to each other, wherein the antigen is a cellular tumor antigen. It further relates to a nucleic acid encoding said vaccine, an expression vector comprising said nucleic acid and a recombinant host cell comprising said expression vector. Further, the present invention relates to a method of manufacturing a vaccine of the present invention, a pharmaceutical composition comprising said vaccine and the use of said vaccine for treatment and/or prevention of a disease associated with said antigen.

Description

Vaccines and manufacturing methods for treatment and prevention of disease

Background of the invention

Cancer is a multifaced disease with numerous possibilities of origin. One of these are infectious agents like bacteria, e.g. the bacterium Helicobacter pylori which causes 5.5% of all cancers. Viruses are even more prominent as some play an important role as oncoviruses. Examples are the human papilloma viruses (HPV; cancers of anogenital tract and head and neck region), the hepatitis B and C viruses (HBV, HCV; hepatocellular carcinoma), Epstein-Barr virus (EBV; Burkitt's lymphoma, nasopharynx carcinoma), or the human herpes virus 8 (HHV8; Kaposi's sarcoma). Infections with oncogenic human papillomaviruses (HR-HPVs) are highly prevalent in the general population all over the world, affecting over 70% of all women and men. While most of the infections resolve spontaneously, they may cause intraepithelial neoplasia and eventually invasive cancers in a fraction of affected individuals. The prevalence of high grade cervical intraepithelial neoplasia (CIN) as one of the leading diseases caused by HR-HPV reaches 1 to 2% and requires invasive treatment which results in substantial tissue damage with a high risk for premature delivery. A fraction of affected women, particularly immunosuppressed patients, suffer from multiple HPV-induced genital intraepithelial lesions, including vulvar, anal and cervical cells. They are hardly to manage successfully by current treatment options. Two prophylactic vaccines, based on HPV LI virus like particles (VLP), are currently available for the prevention of HPV infection. These vaccines induce HPV type-specific prophylactic immunity but have no therapeutic effect on established HPV infections, as LI is only expressed during early infection stages and rarely in transformed cells of pre-neoplasia or cancer. The cellular protein pl6^INK4a is expressed in HPV infected cells if the cells achieve a transformed and possibly malignant phenotype due to the disruption of a negative feedback loop by the HPV oncogene product E7. Thus, pl6^INK4a is strongly expressed in virtually all HPV-induced carcinomas and high grade pre-neoplasias, including cervical, vulvar, vaginal, penile, anal and head and neck tumors. Under physiological conditions pi 6™*^ is only expressed in senescent cells and, thus, is barely found in normal tissues. Besides the HPV-oncogene driven pl6-expression in HPV-associated tumors, pi 6™*^ is also found overexpressed in various tumors not associated with an HPV infection or in tumors where HPV has been found but a viral carcinogenesis is not proven, including a fraction of melanoma and non-melanoma skin cancers, lung cancers, esophageal, gastric and colorectal cancers and kidney, bladder, ovarian, endometrial and breast cancer. It is known that mutations of the retinoblastoma tumor suppressor gene result in upregulation of pi()iNK4a expression. However, the underlying mechanisms for the strong pi 6™*^ expression in these instances are most likely more heterogenic and not finally understood. Despite substantial progress in prophylactic vaccination, there are several challenges left: The prophylactic vaccines only induce type- specific immunity and have therefore a negligible prophylactic effect on other virus (HPV) types. Furthermore they are costly due to the production in mammalians, respectively insect cells. A cold chain for transportation is necessary as these vaccines are not stable at room temperature which further increases the cost factor. Apart from that, it would be necessary to apply prophylactic vaccination widely to achieve an effect for the society and this is hard to accomplish in developing countries, where health systems are problematic, vaccine coverage is poor but need is significant: The present inventors have observed that a vaccine in which a viral coat protein and a tumor or bacterial antigen are covalently or non-covalently connected elicit a strong humoral and/or cellular immune response against both the viral coat protein and the tumor or bacterial antigen. This vaccine can be used inter alia for prophylactic and for combined prophylactic and therapeutic vaccination. In particular if the antigen is a cellular tumor antigen, it provides the advantage that the therapeutic effect of the vaccine is independent from the type of the virus that induced the respective tumor.

Summary of the Invention

In a first aspect, the present invention provides a vaccine comprising a viral coat protein and an antigen, wherein the viral coat protein and the antigen are covalently or non-covalently linked to each other, wherein the antigen is a cellular tumor antigen.

In a second aspect, the present invention provides a nucleic acid encoding the vaccine of the first aspect of the present invention.

In a third aspect the present invention relates to an expression vector comprising the nucleic acid of the second aspect of the present invention.

In a fourth aspect the present invention provides a recombinant host cell comprising the expression vector of the third aspect of the present invention.

In a fifth aspect, the present invention provides a method for manufacturing a vaccine of the first aspect of the present invention, comprising the steps of a) providing a recombinant host cell and

b) expressing said vaccine.

In a sixth aspect the present invention provides a pharmaceutical composition comprising the vaccine, the nucleic acid or the expression vector of the present invention, a pharmaceutically acceptable carrier and/or suitable excipient(s).

In a seventh aspect the present invention relates to a vaccine of the first aspect, a nucleic acid of the second aspect or an expression vector of the third aspect for use in eliciting a T cell response against said antigen for treating and/or preventing a disease associated with said antigen.

Detailed Description of the Invention

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank Accession Number sequence submissions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Definitions:

In the following, some definitions of terms frequently used in this specification are provided. These terms will, in each instance of its use, in the remainder of the specification have the respectively defined meaning and preferred meanings. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are to be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step.

The term "vaccine" as referred to in this specification is a biological preparation that improves immunity to an antigen associated with a particular disease. A vaccine stimulates the body's immune system to recognize the antigen as foreign, destroy the antigen or cells displaying this antigen, and keep a record of it, so that the immune system can more easily recognize and destroy any of these antigens, cells displaying these antigens or microorganisms bearing these antigens on its surface that it later encounters. Vaccines can be prophylactic (e.g. to prevent or ameliorate (i) the effects of a future infection by a pathogen or (ii) the occurrence of cells displaying said antigen, e.g. tumor cells) or therapeutic (e.g. vaccines against an established cancer). A cancer vaccine is a vaccine that treats existing cancer and/or prevents the development of cancer. Vaccines that treat existing cancer are known as therapeutic cancer vaccines. Some types of cancer, such as cervical cancer and some liver cancers, are caused by viruses (known as oncoviruses), and traditional vaccines against those viruses will prevent those types of cancer.

The term "human papillomavirus" as used in this specification is a DNA virus from the papillomavirus family that is capable of infecting humans. Like all papillomaviruses, HPVs establish productive infections only in keratinocytes of the skin or mucous membranes. In some people subclinical infections will become clinical and may cause benign papillomas (such as warts or squamous cell papilloma) or cancers of the cervix, vulva, vagina, penis, oropharynx and anus. Persistent infection with-high-risk-HPV types which are different from the ones that cause skin warts may progress to precancerous lesions and invasive cancer. HPV infection is a cause of nearly all cases of cervical cancer. HPVs are commonly transmitted by sexual contact and infect exclusively epithelial cells. Most of the cervical HPV infections are cleared as a result of humoral and cell-mediated immune response. However, some infections persist over long periods of time and can progress to cervical intraepithelial neoplasia (CIN) or cancer. The largest is the alpha group that contains most of the genital / mucosal HPV types. Benign and neoplastic diseases of the anogenital tract include cervical, vaginal, penile and anal cancers. Besides their oncogenic properties in the genital tract, HPVs also infect squamous epithelial tissues in the head and neck region. Furthermore, it was found that viruses from the B supergroup are involved in the development of non-melanoma skin cancer (NMSC) whereby epidermodysplasia verruciformis (EV) HPV types are most plausibly linked to the development of NMSC. EV is an inherited disease and patients show an increased susceptible to HPVs of the skin.

The term Papoviridae " as referred to in this specification is mainly associated with various neoplasms in mammals. The family of Papovaviridae is split into the families of Papillomaviridae and Polyomaviridae.

The term "Polyomaviridae" as referred to in this specification is a taxonomic family of viruses and comprises polyomavirus species which lead to a variety of tumors.

The term "Papillomaviridae" is a taxonomic family of non-enveloped DNA viruses, collectively known as papillomaviruses. Several hundred species of papillomaviruses have been identified infecting all carefully inspected mammals but also other amniotes such as birds, snakes and turtles. The virus family is divided into subfamilies selected from the group consisting of Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus, Deltapapillomavirus, Epsilonpapillomavirus, Etapapillomavirus, Iotapapillomavirus, Kappapapillomavirus, Lambdapapillomavirus, Mupapillomavirus, Nupapillomavirus, Omikronpapillomavirus, Pipapillomavirus, Thetapapillomavirus, Xipapillomavirus and Zetapapillomavirus. The genus alpha papillomavirus is further subdivided into three groups: high risk, low risk and cutaneous. Low-risk HPV types, e.g. HPV 6 and 11, are rarely associated with cervical cancer but are nevertheless medically important as they can cause genital warts. High risk HPVs belonging to the alpha 5, 6, 7, 9 and 11 species specifically target genital squamous epithelia and are present in almost all cervical cancers. HPV16 is the most prevalent type of the high-risk category in the general population and is responsible for approximately half of all cervical cancers, followed by HPV 18 as the second most common HPV type. Strains of HPV types responsible for skin and mucous infections are selected from the group consisting of type 1, 2, 3, 4, 6, 7, 8, 10, 11, 16, 18, 22, 31, 32, 33, 35, 39, 45, 51, 52, 56, 58, 59, 60, 63, 66, 68, 73, and 82.

The "viral coat protein" as referred to in this specification is a structural virus capsid protein of the family of Papoviridea. The virus capsid is composed of two late proteins, LI and L2 which play important roles in mediating infectivity. The major capsid protein LI, naturally occurring as a monomer, is the primary structural element and has the ability to form pentamers also referred to as "capsomeres" which then self-assemble into virus like particles (VLP). A VLP is the empty capsid of a virus without genetic material inside. VLPs can be produced by genetic engineering and are highly immunogenic: they resemble the native, infectious virions in size and shape and are thereby able to induce high titers of virus-neutralizing antibodies, which provide protection from further virus challenge. LI consists of 503 amino acids and has a molecular weight of 56 kDa (HPV 16). The minor capsid protein L2 was found to be located in the central internal cavity of the LI pentamer. It has a size of 473 amino acids (HPV16) and a molecular weight of 51 kDa. The natural virus capsid consists of 72 LI pentamers (360 LI molecules) and approximately 30 L2 molecules. LI side chains lining the axial cavity are highly conserved between different HPV types whereby the rim of the LI pocket is more variable. LI capsids assembled in vitro are the basis of prophylactic vaccines against several HPV types. Compared to other papillomavirus genes, the amino acid sequences of most portions of LI are well-conserved between types. However, the surface loops of LI can differ substantially, even for different members of a particular papillomavirus species

A "variant" as used herein, can alternatively or additionally be characterized by a certain degree of sequence identity to the parent polypeptide from which it is derived. More precisely, a variant in the context of the present invention exhibits at least 80% sequence identity, more preferably at least 85% sequence identity, even more preferably at least 90% sequence identity, and most preferably at least 95% sequence identity to the reference polypeptide. Preferably, the variants of the present invention exhibit the indicated sequence identity, and preferably the sequence identity is over a continuous stretch of 100, 150, 200, 250, 300 or more amino acids. Most preferably the indicated identity is determined over the entire length of the alignment between the two amino acids, i.e. the reference amino acid and the amino acid that is assessed for its identity. Such amino acid sequence alignments can be carried out with several art-known algorithms, preferably with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) or with the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994) Nucleic Acids Res. 22, 4673-80) available e.g. on http://www.ebi.ac.uk/Tools/clustalw/ or on http://www.ebi.ac.uk/Tools/clustalw2/index.html or on http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html. Preferred parameters used are the default parameters as they are set on http://www.ebi.ac.uk/Tools/clustalw/ or http://www.ebi.ac.uk/Tools/clustalw2/index.html. The grade of sequence identity (sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX). Preferably, sequence matching analysis may be supplemented by established homology mapping techniques like Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov random fields. When percentages of sequence identity are referred to in the present application, these percentages are calculated in relation to the full length of the longer sequence, if not specifically indicated otherwise.

An "antigen" as referred to in the context of the present specification is any substance which provokes an adaptive immune response. An antigen may be foreign or toxic to the body or may be a cellular protein that is associated with a particular disease. Antigens are recognized by highly variable antigen receptors (B-cell receptor or T-cell receptor) of the adaptive immune system and may elicit a humoral or cellular immune response. A fraction of the proteins inside cells, irrespective of whether they are foreign or cellular, are processed into smaller peptides and presented to by the major histocompatibility complex (MHC). To a cellular immune response is elicited, if the small peptide fragment is bound by a T-cell receptor. Foreign antigens can enter the body from the outside, e.g. by inhalation, ingestion, or injection. Usually an antigen comprises one or more epitopes.

The term "epitope" as used herein refers to an antigenic determinant, which is part of an antigen and is that part of the antigen that is recognized by molecules and/or cells of the adaptive immune system. Epitopes that elicit a humoral immune response are referred to as "B cell epitopes". Epitopes that elicit a cellular immune response are referred to as "T cell epitopes". B cell epitopes typically consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. The B-cell epitopes of an antigen may be a conformational epitope or a non-conformational, i.e. a linear, epitope. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. A conformational B-cell epitope is composed of discontinuous sections of the antigens' amino acid sequence. These B-cell epitopes interact with the ligand based on the 3- D surface features and shape or tertiary structure of the antigen. Most B cell epitopes are conformational. By contrast, linear epitopes interact with the ligand based on their primary structure. A linear B-cell epitope is formed by a continuous sequence of amino acids from the antigen. Conformational epitopes preferably comprise between 8 and 20 discontinuous amino acids, preferably between 8 and 15 amino acids. Linear epitopes have a length of between 6 to 20 amino acids, more preferably between 8 and 15 amino acids. T cell epitopes are presented on the surface of an antigen-presenting cell, where they are bound to MHC molecules. T cell epitopes presented by MHC class I molecules are typically peptides between 8 and 11 amino acids in length, whereas MHC class II molecules present longer peptides, 13-17 amino acids in length.

A "cellular tumor antigen" is an antigenic substance produced in tumor cells, i.e. it triggers an immune response in the host and which is encoded by the genome of the cell, i.e. is not of viral origin like, e.g. HPV E6 or E7. Initially tumor antigens were broadly classified into two categories based on their pattern of expression: Tumor- specific antigens (TSA), which are present only on tumor cells and not on any other cell and tumor- associated antigens (TAA), which are present on some tumor cells and also some normal cells. However, many antigens thought to be tumor- specific turned out to be expressed on some normal cells as well. The modern classification of tumor antigens is based on their molecular structure and source, e.g. products of mutated oncogenes and tumor suppressor genes, products of other mutated genes, overexpressed or aberrantly expressed cellular proteins, oncofetal antigens, altered cell surface glycolipids and glycoproteins, cell type- specific differentiation antigens, lineage-restricted antigens, mutated antigens, posttranslational altered antigen, idiothypic antigen, overexpressed antigen, cancer-testis antigens or frame-shift antigens. Overexpressed antigens comprise cancer-rescue antigens, which are expressed in tumor cells in order to protect the non-tumor cell, but due to an inactivated downstream mechanism circumventing cell growth, fail to protect the non-tumor cells. Cancer rescue antigens comprise but are not limited to pl5™^K4b, pl6™^K4a, pl8™^K4c, pl9™^K4d, p21^CIP, p27^KIP, and p57^KIP2. Mutated antigens comprise but are not limited to pRb, Bcr-Abl, mt IDH1.

The term " l6^INK4a " as referred to in the context of the present specification is a tumor suppressor protein which shows increased expression with worsening grades of cervical intraepithelial neoplasia (CIN) and is strongly overexpressed in HPV induced cervical cancer. Therefore, pl6^INK4a is considered as a marker for early diagnosis and is routinely used as a highly specific marker for CIN and transforming HPV infections. pl6^INK4a overexpression increases with the severity of cytological/histological abnormality. pl6^INK4a is a tumor suppressor protein with a molecular weight of 16 kDa (156 amino acids). The protein structure shows four contiguous ankyrin repeats, a protein motif consisting of two alpha helices separated by loops. pl6^INK4a is encoded by chromosome 9p21 within the INK4a/ARF locus. The INK4 family of CDK inhibitors includes four members with similar biochemical properties: pi 5^M4b, pi 6™^, pi 8™^° and piS)iNK4d. Mutations and deletions of ρ15™^Κ4Β and pl6^INK4a are frequently observed in different malignancies and pl6^mK4:i is one of the most direct links between cell cycle control and cancer. Cyclin-dependent kinases (CDKs) form complexes with cyclins and are thereby activated. pl6™^K4 (also known as cyclin-dependent kinase inhibitor 2A) acts as a negative regulator of the proliferation of normal cells by interacting strongly with CDK4 and CDK6. This inhibits their ability to interact with cyclins D and to phosphorylate the retinoblastoma protein (RB). HPV oncogene expression and evidence of its deregulation can be monitored through detection of the cellular protein pl6^INK4a as E6 and E7 disrupt cell cycle checkpoints and affect almost all CDK inhibitors which are linked to the Gl and G2 checkpoints. As pl6^INK4a is released from its negative feedback control by pRB inactivation and KDM6B overexpression, elevated levels of this protein do not lead to cell cycle arrest in tumor cells. Under physiological conditions pl6^INK4a is only expressed in senescent cells and, thus, is barely found in normal tissues. Besides the HPV- oncogene driven pl6-expression in HPV-associated tumors, ρΐό™^ is also found overexpressed in various tumors not associated with an HPV infection or in tumors where HPV has been found, including a fraction of melanoma and non-melanoma skin cancers, lung cancers, esophageal, gastric and colorectal cancers and kidney, bladder, ovarian, endometrial and breast cancers. As used in this specification the term "cyclin dependent kinase inhibitor protein" is a protein which inhibits cyclin-dependent kinase. Several function as tumor suppressor genes. Cell cycle progression is negatively controlled by cyclin-dependent kinases inhibitors (called CDIs, CKIs or CDKIs). CDIs are involved in cell cycle arrest at the Gl phase. In many human cancers, CDKs are overactive or CDK-inhibiting proteins are not functional. Therefore, it is rational to target CDK function to prevent unregulated proliferation of cancer cells.

As used in this specification the term "nucleic acid" comprises polymeric or oligomeric macromolecules, or large biological molecules, essential for all known forms of life. Nucleic acids, which include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid), are made from monomers known as nucleotides. Most naturally occurring DNA molecules consist of two complementary biopolymer strands coiled around each other to form a double helix. The DNA strand is also known as polynucleotides consisting of nucleotides. Each nucleotide is composed of a nitrogen-containing nucleobase as well as a monosaccharide sugar called deoxyribose or ribose and a phosphate group. Naturally occurring nucleobases comprise guanine (G), adenine (A), thymine (T), uracil (U) or cytosine (C). The nucleotides are joined to one another in a chain by covalent bonds between the sugar of one nucleotide and the phosphate of the next, resulting in an alternating sugar-phosphate backbone. If the sugar is desoxyribose, the polymer is DNA. If the sugar is ribose, the polymer is RNA. Typically, a polynucleotide is formed through phosphodiester bonds between the individual nucleotide monomers. In the context of the present invention the term "nucleic acid" includes but is not limited to ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixtures thereof such as e.g. RNA-DNA hybrids (within one strand), as well as cDNA, genomic DNA, recombinant DNA, cRNA and mRNA. A nucleic acid may consist of an entire gene, or a portion thereof, the nucleic acid may also be a miRNA, siRNA, or a piRNA. MiRNAs are short ribonucleic acid (RNA) molecules, which are on average 22 nucleotides long but may be longer and which are found in all eukaryotic cells, i.e. in plants, animals, and some viruses, which functions in transcriptional and post-transcriptional regulation of gene expression. MiRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger RNA transcripts (mRNAs), usually resulting in translational repression and gene silencing. Small interfering RNAs (siRNAs), sometimes known as short interfering RNA or silencing RNA, are short ribonucleic acid (RNA molecules), between 20-25 nucleotides in length. They are involved in the RNA interference (RNAi) pathway, where they interfere with the expression of specific genes. PiRNAs are also short RNAs which usually comprise 26-31 nucleotides and derive their name from so-called piwi proteins they are binding to. The nucleic acid can also be an artificial nucleic acid. Artificial nucleic acids include polyamide or peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Each of these is distinguished from naturally- occurring DNA or RNA by changes to the backbone of the molecule.

As used in this specification the term "expression vector", also referred to as an expression construct, is usually a plasmid or virus designed for protein expression in cells. The vector is used to introduce a specific gene into a target cell, and can use the cell's mechanism for protein synthesis to produce the protein encoded by the gene. The expression vector is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. The goal of a well-designed expression vector is the production of significant amount of stable messenger RNA, and therefore proteins. A commonly used expression vector is pGEX-4T2.

The term "recombinant host-cell" as referred to in the context of the present invention is selected from the group consisting of a bacterial, yeast, baculovirus, plant, mammalian host cell. Bacterial host cells comprise but are not limited to E. coli, preferably BL21 -competent E. coli, or Bacillus subtilis,

The term "pharmaceutical composition" as used within this specification refers to a composition formulated to be suitable for administration to a patient in order to prevent and/ or treat precancerous neoplasia and/or persisting tumors. The pharmaceutical composition can be used as a tumor vaccination. Further, pharmaceutical compositions can be formulated for oral, parenteral, topical, inhalative, rectal, sublingual, transdermal, subcutaneous or vaginal application routes according to their chemical and physical properties. Pharmaceutical compositions comprise solid, semisolid, liquid, transdermal therapeutic systems (TTS). Solid compositions are selected from the group consisting of tablets, coated tablets, powder, granulate, pellets, capsules, effervescent tablets or transdermal therapeutic systems. Also comprised are liquid compositions, selected from the group consisting of solutions, syrups, infusions, extracts, solutions for intravenous application, solutions for infusion or solutions of the carrier systems of the present invention. Semisolid compositions that can be used in the context of the invention comprise emulsion, suspension, creams, lotions, gels, globules, buccal tablets and suppositories.

The term„disease associated with the antigen" as referred to in the present specification is a proliferative disorder. A proliferative disorder is one in which too many of some type of cell are produced. For example, leukemia is a proliferative disorder characterized by an abnormal proliferation (production) i.e. overproduction of white blood cells. The proliferative disorders can be "pre-cancerous" or "pre -neoplastic" or malign. Examples of such conditions include proliferative disorders or cellular proliferative disorders with a high degree of dysplasia. Neoplastic diseases of the anogenital tract include cervical, vaginal, penile and anal cancers. Besides their oncogenic properties in the genital tract, HPVs also infect squamous epithelial tissues in the head and neck region. Furthermore, it was found that viruses from the B super group are involved in the development of non-melanoma skin cancer (NMSC) whereby epidermodysplasia verruciformis (EV) HPV types are most plausibly linked to the development of NMSC. EV is an inherited disease and patients show an increased susceptible to HPVs of the skin. The tumor disease comprises malignant tumors. Malignant tumors are also defined as cancer. Cancer comprises a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Cancers are selected from the group consisting of carcinomas, sarcomas, neuroendocrine tumors, leukemia and lymphomas, and teratomas.

Embodiments

In the following passages different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Based on these results the present invention provides in a first aspect a vaccine comprising a viral coat protein and an antigen, wherein the viral coat protein and the antigen are covalently or non-covalently linked to each other, wherein the antigen is a cellular tumor antigen. If the antigen is covalently linked and the antigen is a polypeptide, it is preferred that the covalent link is a peptide bond. The antigen may be linked to the C-terminus or N-terminus of the viral coat protein. It is also envisioned that the antigen is inserted into the viral coat protein. In the latter case the antigen is preferably inserted in such that the viral coat protein does not lose its ability to assemble into capsomers. This ability can be determined as outlined below. It is particularly preferred that the antigen is inserted into the viral coat protein. This is particular preferred if the viral coat protein is a LI protein of a human Papilloma virus (HPV).

Preferred regions for inserting antigens are selected from the group consisting of either at the N-terminus of the protein, the C-terminus of the protein or a region within the C-terminal sequence of the viral coat protein.

Preferably, an antigen is inserted at the N-terminus of the viral coat protein. As outlined in more detail below the viral coat protein may also be a fragment of a viral coat protein, preferably a LI protein, more preferably a LI protein of a HPV, in which N-terminal amino acids are deleted. Preferred N-terminal deletions are outlined below in more detail. Preferably the deletion comprises 10 to 100 amino acids, i.e. 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 95, 100. Most preferably the deletion comprises 10 N-terminal amino acids (SEQ ID NO: 4) using LI protein of HPV 16. Thus, in this embodiment the antigen is covalently or non-covalently coupled to the N-terminus of the fragment of the viral coat protein. As outlined in more detail below the viral coat protein may also be a fragment of a viral coat protein, preferably a LI protein, more preferably a LI protein of a HPV, in which C-terminal amino acids are deleted. Preferred C-terminal deletions are outlined in more detail below. More preferably, an antigen is inserted at the C-terminal end of the LI protein wherein the C-terminus is modified by deletion, even more preferably the deletion comprises 10 to 50 deletions, i.e. 10, 15, 20, 25, 30, 35, 40, 45, 50, most preferably the deletion comprises 29 C-terminal amino acids (SEQ ID NO: 6) using LI protein of HPV 16. Thus, in this embodiment the antigen is covalently or non-covalently coupled to the C-terminus of the fragment of the viral coat protein.

Even more preferably, the antigen is inserted within a C-terminal region of the protein sequence of the viral coat protein, preferably a LI, more preferably a LI protein of a HPV. A particular preferred C-terminal region is a region C-terminal to helix 4 of LI proteins. Helix 4 of LI of HPV 16 has the amino acid sequence according to SEQ ID NO: 7. To identify the region C- terminal of helix 4 the skilled person can align the sequences of various LI proteins and determine the sequence of a given LI protein that is similar to the sequence of helix 4. Such an exemplary alignment between the LI amino acid sequences of four different HPV types is shown in Fig. 12.

It is noted that the linkage of the antigen to the viral coat protein is carried out in a way not to diminish the adjuvant activity of the viral coat protein, e.g. the increase in T cell response, and/or to inhibit the assembly into capsomeres. Thus, each vaccine can be tested using the methods outlined below for one or both of these functional criteria.

The antigen may also be non-covalently linked to the viral coat protein. Suitable non- covalent links can be formed between various non-covalent binding pairs known in the art, which include, e.g. streptavidin and biotin. In this embodiment it is preferred that either biotin or streptavidin is covalently linked to the antigen the respective other binding partner is covalently linked to the viral coat protein. This allows the separate production of the viral coat proteins as well as capsomeres or VLPs comprising the VLPs and the subsequent loading of antigens onto the viral coat proteins, capsomeres or VLPs.

The viral coat protein is in a specific embodiment a protein of the virus family of Papoviridae, preferably of the subfamily of the Papillomaviridae. The coat proteins of these viruses typically comprise late protein 1 (LI) and late protein 2 (L2). Preferably the viral coat protein used in the context of the vaccine of the present invention is the LI protein. More preferably the LI protein is from a Papillomaviridae selected from the group consisting of Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus, Deltapapillomavirus, Epsilonpapillomavirus, Etapapillomavirus, Iotapapillomavirus, Kappapapillomavirus, Lambdapapillomavirus, Mupapillomavirus, Nupapillomavirus, Omikronpapillomavirus, Pipapilloma virus, Thetapapillomavirus, Xipapillomavirus and Zetapapillomavirus, with Alphapapillomavirus being preferred. In the most preferred embodiment the viral coat protein is the LI from HPV

Particular preferred LI proteins are derived from HPV types responsible for skin and mucous infections. Examples of such HPV types comprise 1, 2, 3, 4, 6, 7, 8, 10, 11, 16, 18, 22, 31, 32, 33, 35, 39, 45, 51, 52, 56, 58, 59, 60, 63, 66, 68, 73, and 82. Particular preferred types are 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 66 that are known to cause cancer. The most preferred types to derive LI protein from are either HPV type 6, 10, 11, 16 or 18 which account for 70% of all cervical cancers.

Thus, specific LI proteins that can be used as viral coat proteins are selected from the group consisting of SEQ ID NO: 2 (HPV Type 16), SEQ ID NO: 16 (HPV Type 6), SEQ ID NO: 17 (HPV Type 10), SEQ ID NO: 18 (HPV Type 18, and SEQ ID NO: 19 (HPV Type 11) or a variant within at least 80% sequence identity over aligned sequence between SEQ ID NO: 2, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or , SEQ ID NO: 19 and which enhances the T-cell response to a test antigen fused to the LI protein at least to the same extent as the LI protein SEQ ID NO: 2, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or , SEQ ID NO: 19, respectively. Specifically a variant in the context of the present invention exhibits preferably at least at least 80% sequence identity, more preferably at least 85% sequence identity, more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity, and most preferably at least 98% sequence identity to its parent protein over the aligned sequence.

Preferably, at least two, e.g. 2, 3, 4, 5 or 6 viral coat protein monomers form capsomeres, more preferably five viral coat protein monomers form a capsomere (pentamer). The term "capsomere" thus refers to a structural subunit of a viral capsid. Accordingly, one or more capsomers can form capsids, which are usually stabilized among each other by disulfide bonds between neighboring viral coat proteins, e.g. LI molecules. After expression in eukaryotic and prokaryotic systems, some viral coat proteins like HPV LI, are able to self-assemble into empty VLPs with similar immunogenicity compared to infectious virions. HPV virions and VLPs usually consist of 72 LI pentamers, also called capsomeres, which are arranged in a T = 71 icosahedral architecture with a diameter of 55 nm. VLPs are endocytosed, processed, and presented by APCs to naive T cells. The vaccines of the present invention are preferably in the form of capsomeres or VLPs. Capsomeres represent a potentially lower cost alternative to VLP-based vaccines as they can be produced in large amounts from prokaryotic expression systems and appear to be more stable at room temperature then VLPS.

The present inventors were able to show that the capsid-neutralizing antigenic domains are fully conserved in capsomeres and that antisera generated with capsomeres elicit neutralization titers comparable to that of entire VLPs. Certain LI constructs form highly immunogenic capsomeres. The LI protein can be modified in different ways and immunogenicity of the constructs was compared. A HPV construct with an N-terminal deletion of 10 amino acids, named LIANIO appeared to be the most immunogenic. LIANIO of HVP 16 induced antibody titers equivalent to those generated in response to VLPs. Several other studies present further evidence for the potential of capsomeres as antigen carriers.

Accordingly, the term "variant" in relation to the viral coat proteins of the present invention also comprises fragments of the viral coat proteins. Fragments can be N-terminally deleted, C- terminally deleted, internally deleted, N- and C-terminally deleted, N-terminally and internally deleted, C-terminally and internally deleted, and N- and C-terminally and internally deleted. Functionally, the deletion(s) are selected in such that they do not alter the ability of the fragment to form capsomeres. This functional property can easily be tested by expressing the respective fragment in a suitable host, e.g. E. coli, and analyzing the cellular extract or (partially) purified protein on a non-denaturing polyacrylamide gel. Multimers of the viral coat protein can then be identified and quantified by Western blot with viral coat protein specific antibodies or by protein staining techniques like, e.g. Coomassie or silver staining. The latter is used in particular if purified or partially purified viral coat proteins are used. Preferably, the fragment of the viral coat protein, more preferably the fragment of the LI protein of a HPV, most preferably the fragment of the LI protein with an amino acid according to SEQ ID NO: 2 carries a deletion of 5 to 20, e.g. 5, 8, 9, 10, 11, 12, 15 or 20, especially 10 N-terminal amino acids (SEQ ID NO: 4). Alternatively or additionally the viral coat protein, more preferably the fragment of the LI protein of a HPV, most preferably the fragment of the LI protein with an amino acid according to SEQ ID NO: 2 carries a deletion of 5 to 40, e.g. 5, 40, 5, 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 40, especially 29 C-terminal amino acids (SEQ ID NO: 6). Preferred combinations are 9 N-terminal deleted amino acids with a deletion of 5 to 40, e.g. 5, 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40 especially 29 C-terminal amino acids. Preferred combinations are 10 N-terminal deleted amino acids with a deletion of 5 to 40, e.g. 5, 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 40 especially 29 C-terminal amino acids. Preferred combinations are 11 N-terminal deleted amino acids with a deletion of 5 to 40, e.g. 5, 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 40 especially 29 C-terminal amino acids. As indicated above, the fragments may also comprise internal deletions. A preferred deletion is an internal deletion of helix 4. In a particular preferred embodiment the fragment of the LI protein with an amino acid according to SEQ ID NO: 2 carries a deletion of 5 to 40, e.g. 5, 40, 5, 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 40, especially 29 C-terminal amino acids and an internal deletion of helix 4 (SEQ ID NO: 7). Preferred combinations are 9 N-terminal deleted amino acids with a deletion of 5 to 40, e.g. 5, 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40 especially 29 C-terminal amino acids and an internal deletion of helix 4. Preferred combinations are 10 N-terminal deleted amino acids with a deletion of 5 to 40, e.g. 5, 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 40 especially 29 C- terminal amino acids and an internal deletion of helix 4. Preferred combinations are 11 N-terminal deleted amino acids with a deletion of 5 to 40, e.g. 5, 10, 15, 20, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 40 especially 29 C-terminal amino acids and an internal deletion of helix 4. The fragments of the present invention are preferably based on either LI protein of HPV 16 or HPV 18. The preferred deletions are indicated above with reference to the sequence of the LI protein of HPV Type 16. As is apparent from the alignment shown in Figure 12, the deletion of 10 amino acids from the N-terminus of LI of HPV Type 16 maintains a protein motive conserved among HPVs, namely T/L V Y V P P. Thus, it is preferred that the N-terminal deletion of the LI proteins of other HPV Types deletes a region(s) that align with the N-terminal region(s) deleted in LI of HPV Type 16. For example the skilled person can easily determine that the deletion of 70 amino acids of the N-terminus of LI of HPV Type 18 according to SEQ ID NO: 18, leads to the deletion of an N-terminal region that aligns with the 10 N-terminal amino acids of LI of HPV Type 16 according to SEQ ID NO: 2. Accordingly, preferred N-terminal deletion fragments of LI of HPV Type 18 lack 55 to 70 amino acids from the N-terminus of the amino acid sequence according to SEQ ID NO: 18. As is apparent from the alignment shown in Figure 12, the deletion of 29 amino acids from the C-terminus of LI of HPV Type 16 maintains a protein motive conserved among HPVs, namely K F L L/V Q X G X R/K X. Thus, it is preferred that the C-terminal deletion of the LI proteins of other HPV Types deletes a region that aligns with the C-terminal region(s) deleted in LI of HPV Type 16.

Using similar considerations and alignment the skilled person can determine preferred N- terminal, C-terminal and internal deletions in SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19 that align with the preferred N-terminal, C-terminal and internal deletions of HPV Type 16.

As set out above in a particular embodiment 72 LI pentamers assemble to a VLP with similar immunogenicity compared to infectious virions. Accordingly, the fragments in a specific embodiment can form capsomeres that can form VLPs.

It is preferred that the cellular tumor antigen is selected from the group consisting of tumor- specific antigens, preferably lineage-restricted, mutated, posttranslational altered, idiothypic antigens; or tumor-associated antigens, preferably oncofetal, overexpressed, cancer-testis or frame-shift antigens. Preferred example of the lineage-restricted antigen is HD39, the mutated antigen is selected from the group consisting of pRb, Bcr-Abl, IDH1; the oncofetal antigen is selected from the group consisting of onco-throphoblast glycoprotein, alpha-fetoprotein, the overexpressed antigen is selected from the group consisting of Her-2, Muc-1, p53 (mutated), Ras, WT-1, preferably the overexpressed antigen is a cancer-rescue antigen. Preferably, also antigenic fragments thereof comprising at least one T and/or B cell epitope are comprised. It is preferred that the cancer-rescue antigen is selected from the group consisting of a cyclin dependent kinase inhibitor protein, preferably pl5^mK4h, pl6^mK4a, plS^mK4c, pl9™^K4d, p21^CIP, CIP2, p27^KIP, p57^KIP2, more preferably pl6^INK4a. Even more preferably the antigen is selected from the group consisting of SEQ ID NO: 10 or fragments thereof comprising at least one T and/or B cell epitope, preferably at least on T cell epitope.

In a second aspect, the present invention provides a nucleic acid encoding the vaccine of the first aspect of the present invention

In a third aspect, the present invention provides an expression vector comprising the nucleic acid of the second aspect. Preferably, the expression vector is selected from the group consisting of a bacterial, yeast, baculovirus, plant and mammalian expression vector, more preferably the expression vector is a bacterial expression vector or a cell-free expression vector. Even more preferably the expression vector is pGEX-4T2. More preferably, the expression vector construct is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, most preferably the vector construct is encoded by SEQ ID NO: 15.

In a fourth aspect, the present invention provides, a recombinant host cell comprising the expression vector and the nucleic acid. Preferably, the host-cell is selected from the group consisting of a bacterial, yeast, baculovirus, plant, mammalian host cell. More preferably, the host cell is a bacterial host-cell, even more preferably a host-cell from E. coli.

In a fifth aspect, the present invention provides a method for manufacturing a vaccine of the first aspect of the invention comprising the steps of providing a host cell and expressing the vaccine. In a preferred embodiment of that aspect the vaccine is expressed in a bacterial host cell, preferably in E. coli. It is preferred that the vaccine is expressed in inclusion bodies, preferably in E. coli and the purification of the vaccine includes lysis of the cell and separation of the protein containing inclusion bodies. It was surprisingly shown by the inventors that that particularly stable capsomeres could be isolated from inclusion bodies and thus, the vaccine, if purified from inclusion bodies, shows increased stability against heat which decreases the urgent need of a cooling chain.

In a sixth aspect, the present invention provides a pharmaceutical composition comprising the vaccine of the first aspect of the present invention, the nucleic acid of the second aspect, the expression vector of the third aspect and pharmaceutical acceptable carriers and/or suitable excipients. The pharmaceutical composition is selected from the group consisting of solid, liquid, semi- solid or transdermal therapeutic systems. It is envisioned that the pharmaceutical compositions of the invention comprise two or more different vaccines, wherein the viral coat protein of one vaccine is of one virus type and the viral coat protein of another vaccine is of another virus types, preferably viral coat proteins of two or more HPV types. Preferred combinations are viral coat proteins are viral coat proteins of HPV 16 and 18, HPV 6, 10, 11, 16 and 18.

In a seventh aspect, the invention provides a vaccine comprising a viral coat protein and an antigen, wherein the viral coat protein and the antigen are covalently or non-covalently linked to each other for use in eliciting a T cell response against said antigen, a nucleic acid encoding said vaccine, or an expression vector comprising said nucleic acid for treating and/or preventing a disease associated with said antigen. A particular advantage provided by the inventors is the produced heat-stable vaccine if purified by the method of the invention, i.e. from bacterial inclusion bodies.

In a preferred embodiment the disease is a hyperproliferative disorder. Preferred hyperproliferative disorders are a benign, pre-cancerous or malign disorder, preferably of the anal region, bladder, blood, bone, brain, breast, cervix, colon, endometrium, esophagus, kidney, larynx, liver, lung, lymphoma, mouth, ovary, pancreas, penis prostate, rectum, skin, stomach, testis, thyroid, vagina, or vulva, preferably tonsil or pharynx, more preferably of the cervix. Benign hyperproliferative disorders can be selected from the group consisting of cervical hyperplasia including warts e.g. Condylomata acuminata and Verruca vulgaris. Pre-cancerous or malign hyperproliferative disorders can be anal cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, mouth (oral) cancer, myeloma, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, vaginal cancer, and vulvar cancer, preferably cervical cancer. In particular, if the disease is a hyperproliferative disease it is preferred that the vaccine, the nucleic acid encoding said vaccine, the expression system comprising said nucleic acid or the pharmaceutical composition comprising any of those both treats and prevents the disease. This is in particular the case, if the disease is caused by HPV and the viral coat protein is a LI protein of a HPV and the antigen is a cellular tumor antigen.

List of figures

Figure 1: Capsomere constructs

HPV16 LI - pl6INK4a capsomere constructs depicted in cartoon format. The wild-type LI protein is displayed at the top (SEQ ID NO: 1). Modified LI and pl6INK4a alone were used as controls during immunization studies and were therefore also cloned and expressed in E. coli. Three constructs containing LI and pl6INK4a were generated. Therefore, the complete pl6INK4a encoding cDNA sequence was cloned a) upstream and b) downstream of a modified HPV16 LI sequence into a pGex-4T-2 expression vector. For the third construct c) the helix 4 region of LI was replaced by pl6INK4a

pGex-LlAN10Ah4AC29: modified HPV16 LI, forms capsomeres (SEQ ID NO: 11),

pGex-pl6INK4a: full length human pl6INK4a gene (SEQ ID NO: 12),

pGex-L 1 ΔΝ 10Ah4AC29-p 16INK4a (construct 1 ) : contains the full-length human p 16INK4a gene fused to the C terminus of the modified LI (SEQ ID NO: 13),

pGex-pl6INK4a-LlAN10Ah4AC29 (construct 2): contains the full-length human pi 6INK4a gene fused to the N terminus of the modified LI (SEQ ID NO: 14),

pGex-LlAN10Ah4-pl6INK4a-LlAC29 (construct 3): contains the full-length human pl6INK4a gene inserted at the helix 4 position of the modified LI (SEQ ID NO: 15).

Figure 2: Cloning into pGEX-4T-2 and expression in E. coli

Three chimeric HPV16 Ll-pl6^INK4a constructs were generated, expressed and purified for immunological applications. The complete human pl6^mK4:i encoding sequence was cloned (1) downstream of the HPV16 LI sequence, (2) upstream of the HPV16 LI sequence and within position of helix 4 of HPV16 LI. The major HPV16 capsid protein LI used had different deletions for which successful capsomere formation has been shown previously, leading to three construct referred to as (1) LlAN10Ah4AC29-pl6^INK4a (Figure 1), (2) pl6^INK4a-LlAN10Ah4AC29 and (3) LlAN10Ah4-pl6^INK4a-LlAC29. The constructs were cloned into pGEX-4T-2 (Stratagene).

Figure 3: Protein purification

Insoluble LI -containing proteins were purified with a special protocol that included several washing and re-suspension steps. The purified inclusion bodies were denatured with 5 % N- Lauroylsarcosine and refolded by dialysis. Thereby, E. coli host cell proteins could be removed and high recovery of the target proteins was ensured. The GST-tagged proteins present in the bacterial lysate after French press is shown in Coomassie stained protein gels before and after inclusion body purification. The bacterial lysates show strong overexpression of the target proteins; however the supernatants did not contain suitable amounts as shown before. After several washing and re-suspension steps, the purified inclusion body pellets still contain the majority of the GST-fusion protein. Several smaller bands were observed to co-purify and were identified as truncated target proteins. Figure 4: Analysis of protein preparations

After purification of the inclusion bodies, the aggregated and probably misfolded proteins had to be transformed into a soluble, at its best, native form. Therefore the proteins were first denatured and thereby solubilized with the anionic detergent N-Lauroylsarcosine. Subsequent refolding was achieved utilizing dialysis with decreasing concentrations of the detergent and salts. Analysis of protein preparations obtained from inclusion body purification after Lauroylsarcosine- denaturation and refolding of the target proteins. The whole protein content analyzed by coomassie staining, the anti-Ll (MD2H11) and the anti-pl6 (D7D7) western blots are shown. In each case, the major proportion - respectively the dominant band in the Coomassie gel - is made up of the target protein as analyzed by western blotting. Smaller fragments could also be identified to be predominantly of LI or pl6 origin speaking for truncated, co-purified proteins. Figure 4 shows the results of the protein preparations obtained from inclusion bodies. For each of the LI containing constructs, Coomassie staining, anti-Ll and anti-pl6 western blot are shown in comparison. The intense bands at -92 kDa, respectively 75 kDa for GST-LI, indicate the GST-tagged fusion proteins. Below this upper dominant band, several smaller fragments could be identified in all the preparations. Western blot analysis revealed that these fragments must be of LI or pl6 origin, and thereby presenting truncated or degraded target proteins.

Figure 5: pl6^INK4a GSTrap purification

As GST-pl6^INK4a was expressed in soluble form, it had to be purified in a different way than the insoluble LI capsomeres. All protein lysates were treated equally as far as possible, e.g. they were lysed in the same buffer. Coomassie and western blot analysis of the bacterial lysate after French press, the supernatant containing the soluble protein and the purified GST-pl6^INK4a after GSTrap affinity chromatography are shown. The intense band at 42 kDa in the Coomassie gel was confirmed to be ρ16^ίΝΚΆ with western blot analysis. After disruption of the bacterial cells, the supernatant was collected and loaded onto a 5 ml GSTrap FF column (GE Healthcare). The GST- pl6^INK4a protein was eluted with 20 mM glutathione to sustain the GST-tag. It was also possible to cleave pl6^INK4a from the GST tag with thrombin. However, to ensure best possible comparability for subsequent immunological in vivo experiments, pl6^INK4a was purified along with its GST-tag, at least for the application as control group protein in the mouse trials.

Figure 6: Structural characterization of chimeric capsomeres: Sedimentation analysis

The purified capsomeres were analyzed for their structural integrity, sedimentation characteristics and the presence of conformation-specific epitopes by means of transmission electron microscopy, sedimentation analysis and conformation specific ELISA. To verify the capsomere structures, purified protein extracts were subjected to sucrose gradient centrifugation. The collected fractions (20 in total, 600 μΐ each, fraction 1 from the top of the tube) were analyzed by western blotting. All expressed and purified fusion proteins contain the modified LI sequence with a deletion of the helix 4 region that prevents assembly into larger particles. Sedimentation analysis of the refolded proteins revealed the presence of capsomeres as demonstrated by western blot analysis with the Ll-specific antibody MD2H11. BSA was loaded as calibration marker and assembled VLPs were loaded as control. VLPs were found at the bottom of the centrifuge tube whereas the capsomeres accumulated at the top of the sucrose gradient (fractions 1 to 4) due to their low sedimentation coefficient. These sedimentation characteristics indicate the presence of small particles and are in line with previous studies.

Figure 7: Structural characterization of chimeric capsomeres: Transmission electron microscopy

The purified proteins were analyzed by transmission electron microscopy (TEM) and two representative replicas of each sample are shown in Figure 44. All preparations contained large amounts of 10 - 12 nm capsomeric structures which is in line with previous reports. The characteristic donut-like appearance was also observed in some of the capsomeres but this was not a consistent finding. The black dot in the center of the capsomeres originates from the staining of their cylindrical cavity. As we used the C-terminally truncated LI construct this dot was only sporadically visible as described before. The capsomeres were found to be of variable size with a certain tendency to the formation of aggregates. Especially pl6Ll and Llpl6Ll showed higher grades of agglomeration compared to LI and Llpl6. In general, many non-assembled LI monomers were observed as "background noise". This often resulted in EM images with lower resolution as the uranyl acetate staining was challenging.

Figure 8: Structural characterization of chimeric capsomeres: Conformation specific

ELISA

The different chimeric constructs were obtained from inclusion bodies including denaturation and refolding steps. Therefore it was not clear whether conformation- specific epitopes are present after the refolding procedure or not. An ELISA using the LI -directed monoclonal antibody was applied to elucidate this. VLPs from insect cells were used as a positive control to determine functionality of the test. The results of the conformation specific or 'antigen capture ELISA' are summarized in Figure 8. As the absorption values are quite low even for the undiluted proteins, it can be supposed that conformation-specific epitopes are only present to low extend, if at all. These data indicate that LI without the pi fusion presents, at least partly, the desired epitopes and that the pl6Ll capsomeres are slightly more immunoreactive to the conformation specific antibody. The low overall reactivity of the capsomeres in this ELISA indicates the presence of rather missfolded LI protein. At least the antibody- specific epitope seems to be masked although this does not exclude the presence of LI in the form of higher order structures, such as capsomeres. However, for these proteins the value of that assay is limited. Figure 9: Comparison of the different HPV16 LI - pl6^INK4a capsomere constructs: cellular immune response determined by IFNy ELISpot I

Another experiment was performed to elucidate immunological differences between the three different capsomere constructs. Figure 9 depicts that all three fusion protein constructs induce high Ll-specific T cell numbers in immunized mice whereby construct #3 (Llpl6Ll) shows the highest T cell response with a mean of 172 spots. Also the other constructs #1 (Llpl6) and #2 (pl6Ll) with mean spot numbers of 82 and 67 gave higher LI responses than the LI control group with a mean of 27 spots. Fishers LSD test revealed significance for the comparison of the LI control group to the test groups pl6Ll (p<0.0001) and Llpl6Ll (p=0.0002). The LI protein was administered in lower dose for the control group, as it has to be considered that the 50 μg fusion protein dose does not contain 50 μg pure LI but GST, ρΐβ™^ and LI and thereby presents a comparable LI proportion altogether. The presence of pl6^mK4:i seems to boost the LI specific T cell response what could be due to the presentation of T helper epitopes in this formulation. Concerning the pl6^INK4a T cell response (shown in Figure 52) the same analysis problem occurred with this ELISpot as it was found previously. However, significantly higher T cell numbers could be detected for construct #2 (pl6Ll, p<0.0001) and #3 (Llpl6Ll, p=0.0002) compared to the control group which only received LI protein. It should be noted that the number of counted spots is very low and was therefore not normalized for RPMI control. As a number of up to 13 spots was found to be regular assay background, these spot numbers must be considered to be background response that would also have appeared without peptide stimulation. This shows that with using the pool of overlapping pl6 peptides for the ELISpot test, only low numbers of pi 6- specific T cells can be detected.

Figure 10: Comparison of the different HPV16 LI - pl6^INK4a capsomere constructs: cellular immune response determined by IFNy ELISpot II

Figure 10 shows the HPV16 Ll-ELISpot results split for each group, whereby LI 25 μg and pi()iNK4_a 50 μg represent the control groups. Grey bars indicate assay negative control spot numbers that were counted from unstimulated (RPMI) cells, black bars indicate Ll -specific T lymphocytes. Splenocytes from mice vaccinated with LI or one of the LI containing chimeric constructs produced significantly more IFNy spots when stimulated with the AGVDNRECI peptide compared to unstimulated cells. This is true for all test groups. The control group of 10 mice that only received pi 6™^K4a protein, did not develop Ll-reactive T cells, which demonstrates specificity of the ELISpot assay. The graphic also displays a boxplot diagram after Tukey to visualize the distribution of RPMI spot numbers in a large cohort of animals (n=50), respectively the assay negative control, obtained in this experiment. From this it is concluded, that spot numbers up to 13 can appear although no peptide specific stimulation preceded. Mice number 14 and 25 were thereby identified as outliers.

Figure 11: Comparison of three different capsomere constructs: T-cell response

Direct comparison of the 3 different capsomere constructs. A: pl6^INK4a-ELISpot using 3 different predicted pl6 peptides; B: HPV16 Ll-ELISpot using the AGVDNRECI peptide. 5 C57BL/6 mice per group were immunized three times s.c. at biweekly intervals with 50 μg of either Llpl6, pl6Ll or Llpl6Ll protein. Dots represent individual mice with group specific mean and standard deviation indicated. Dotted line indicates cut off (= mean + 3*SD) calculated from the assay negative controls (medium). Figure 12: Alignment of LI proteins of common Human papilloma virus types (HPV 6, 10,

16, 18)

An exemplary alignment of protein sequences of LI proteins of different HPV Types using CLUSTAL Omega and standard alignment parameters is shown. The N-terminal region of the HPV LI proteins that is deleted in preferred embodiments of the invention is highlighted in grey. C-terminal of the highlighted region is the conserved sequence motive that is preferably kept intact in the N-terminal deletion fragments of HPV LI proteins. This motive is indicated by underline. Also highlighted is helix 4 of the aligned HPV LI proteins. Helix 4 is deleted in preferred embodiments of the invention. The C-terminal region of the HPV LI proteins that is deleted in preferred embodiments of the invention is highlighted in grey. N-terminal of the highlighted region is the conserved sequence motive that is preferably kept intact in the N-terminal deletion fragments of HPV LI proteins. This motive is indicated by underline. Corresponding N-terminal, C-terminal and/or internal regions of LI proteins of other HPV can be determined by the skilled person using standard alignment parameters and programs as set out above.

Examples

Example 1: Purification of HPV16 LI capsomeres and chimeric fusion proteins combining

HPV16 LI and pl6^INK4a from E. coli: Protein expression

For the expression of GST-HPV16 LI and the chimeric proteins containing GST, HPV 16 LI and ρΐβ™^, E. coli Rosetta or BL21 bacteria were transformed with the pGex-4T-2 vector containing the respective genes. TB medium was inoculated in a ratio of 1: 100 with an overnight culture (LB) of the plasmid containing bacteria and incubated while shaking at 200 rpm at 37°C. As soon as the cultures reached an optical density (OD6oonm) of ~ 2.0 to 3.0 (respectively two following Οϋδοο values indicated stationary phase entry), bacteria were cooled for 5 min on ice before adding IPTG to a final concentration of 0.1 mM to induce protein expression. After 16 to 18 h incubation at 200 rpm at 22 °C, bacteria were harvested by centrifugation for 20 min at 4,000 rpm at 4°C (874 rotor). The HPV16 LI expressed in E. coli was predominantly found to build intracellular inclusion bodies what also holds true for the chimeric capsomeres containing pi 6^INK4a and LI. Although a small percentage of the protein was expressed in soluble form, sufficient amounts could not be purified with the available techniques. Therefore, we decided to establish an inclusion body purification protocol to overcome quantity issues. Shortly, the inclusion bodies were washed, cells were disrupted with a French press and the protein was extracted from the resulting pellet. Denatured protein was then refolded by dialysis and could be used for further studies.

Example 2: Purification of HPV16 LI capsomeres and chimeric fusion proteins combining

HPV16 LI and pl6^INK4a from E. coli: Inclusion body preparation

HPV16 LI capsomeres and chimeric capsomeres were purified using an adapted protocol described in the iFOLD® Protein Refolding System 1 User Protocol by Novagene. Fresh or frozen cell pellets were re-suspended in IB Resuspension Buffer (5.4.2; 10 ml / 1 g cell paste) supplemented with protease inhibitor cocktail (1 tablet per 50 ml). 20 μΐ Lysonase were added per gram cell pellet and stirred for 15 minutes. Bacteria were lysed using a high-pressure homogenizer (French press). Subsequently ATP and MgC12 were added to the lysate to final concentrations of 2 and 5 mM, respectively. After 1 h incubation at room temperature, Triton X-100 was added to a final concentration of 1 % and incubated for 15 minutes on a roller mixer. Cell lysates were centrifuged at 8.000 x g (-10.000 rpm - SS-34 rotor, Sorvall) for 15 minutes and the supernatant was discarded. The resulting pellets were washed with IB Wash Buffer (10 ml buffer / 1 g cell pellet) whereby complete resuspension was achieved with the help of a Potter Elvehjem Homogenizer. The suspension was centrifuged again and the Triton X-100 washing was repeated one more time. After the second washing and centrifugation, remaining Triton X-100 was removed by washing the pellet twice in Resuspension Buffer, as described above. The purified inclusion body pellets were stored at -80°C until further use.

Example 3: Denaturing of inclusion bodies and refolding of the protein

Per 0.5 g washed inclusion body pellet 10 ml Denaturing Buffer was added and complete homogenization was achieved with a Potter Homogenizer. The solution was supplemented with 1.75 ml 30 % N-Lauroylsarcosine (5 % final cone.) and agitated on a roller mixer until it cleared after 30 min to 2 h. The solubilized, denatured protein was cleared by centrifugation at 25.000 x g (-18.000 rpm - SS-34 rotor, Sorvall) for 15 minutes at 4 °C. The supernatant was collected and dialyzed (MWCO 3.500 Da) against Dialysis Buffer whereby a volume of 0.5 L was used per 10 ml denatured protein solution. After 4 hours of dialysis, the buffer was exchanged by freshly prepared buffer and dialyzed over night at 4°C. The refolded protein was then removed from the dialysis tube and further analyzed by electron microscopy. Protein concentration was determined with the 2-D Quant Kit (GE Healthcare). Fresh or frozen cell pellets were re-suspended in IB Resuspension Buffer (5.4.2; 10 ml / 1 g cell paste) supplemented with protease inhibitor cocktail (1 tablet per 50 ml). 20 μΐ Lysonase were added per gram cell pellet and stirred for 15 minutes. Bacteria were lysed using a high-pressure homogeniser (French press). Subsequently ATP and MgC12 were added to the lysate to final concentrations of 2 and 5 mM, respectively. After 1 h incubation at room temperature, cell lysates were centrifuged at 8.000 x g (-10.000 rpm - SS-34 rotor, Sorvall) for 15 minutes and the pl6^INK4a containing supernatant was used for further purification solution. Afterwards columns were washed with 20-30 CVs of PBS and stored in 20% ethanol.

Example 4: Explorative protein purification methods

Denaturing of inclusion bodies

Inclusion bodies were denatured with 5 % N-Lauroylsarcosine, urea (4, 6 and 8 M) and 6 M guanidine hydrochloride. The protein lysate was incubated with the respective denaturing agent at RT on a roller mixer until the solution became clear.

Dialysis / Refolding screen

Dialysis conditions for denatured proteins were tested in small scale to determine optimal protein concentration and buffer receipts for refolding. Thereto, a 3,500 Da dialysis membrane was cut into 2 x 2 cm squares and equilibrated in aqua bidest. 1.5 ml Protein LoBind Eppendorf reaction tubes were cut at the 1.5 mark and the lid was filled with 100 μΐ of the denatured protein; diluted in denaturing buffer if required. Then, the dialysis membrane was clamped between the lid and the remaining reaction tube and put into the dialysis buffer of choice over night at 4°C. The next day, absorption at 340 nm was measured to quantify the amount of precipitated protein. The lower the value, the more soluble protein was present in the solution. This method allows testing of different buffer conditions and protein dilutions in an easy and economy way.

Example 5: Transmission electron microscopy

For evaluation of capsomere preparations by EM, suspensions of interest were prepared by 'negative staining' with uranyl acetate. Purified proteins were diluted in their appropriate buffer to about 0.1 mg/ml protein and 3 μΐ drops were applied to 400-mesh carbon-coated copper-grids, which were rendered hydrophilic by glow-dicharge (0.8 mbar/ 8 μΑ/ 10 sec). After passage through two drops of bi-distilled water, grids were wetted with 1% aqueous uranyl acetate, drained with filter paper (Whatman) and air-dried for direct observation in an electron microscope (Zeiss EM 912, operated at 120kV; CCD-Camera by Proscan, Germany). Example 6: Immunization of mice

Immunization by subcutaneous injection

Mice were immunized subcutaneously at the neck using a blue G23 needle. 5 to 50 μg of recombinant antigen was diluted in PBS and mixed; if necessary, with adjuvant. Mice were usually immunized 3 times at biweekly intervals with an injection volume of 100 μΐ unless mentioned otherwise. Negative control mice were treated with PBS with purified irrelevant antigens.

Immunization by topical application

Mice were immunized by topical application with 50 μg recombinant protein formulated in 50% DMSO. Thereto, it was necessary to shave the back of the mice with an electric clipper. Mice were held at their tail and carefully shaved across the grain. Between the first shaving and the first antigen application, three days were paused to heal potential wounding of the skin. Mice were creamed daily for about 1 minute till the mixture was absorbed through the skin. This procedure was carried out 2 x 5 days with 2 days rest in-between. Blood and spleens were sampled 4 days after the last topical application and analyzed for humoral and cellular immune response.

Example 7: Analysis of humoral and cellular immune responses: VLP-capture ELISA to determine Ll-specific antibodies

96-well microtiter plates (Nunc) were coated with 50 μΐ/well of the purified polyclonal HPV16 Ll-specific serum in a 1:500 dilution in PBS at 4°C overnight. All incubation steps were performed 1 h at 37°C unless mentioned otherwise and plates were washed after each incubation step 4 times with PBS-T. After washing off unbound coating antibody, plates were blocked 1 h with 5 % milk in PBS-T. HPV16 VLPs were diluted 1:500 (0.3 μ^&\\ in blocking solution) and 50 μΐ per well were added and incubated. Plates were washed and sera of immunized mice were applied in duplicates in a 1: 10 dilution (100 μΐ/well). The positive control (HPV16 LI monoclonal antibody) was also diluted 1: 10. Incubation was followed by another washing step and a horseradish peroxidase conjugated anti-mouse IgG antibody (50 μΐ/ well; 1:5000) was applied. After 1 h, plates were washed and 50 μΐ TMB substrate were added to the wells and incubated at room temperature. The reaction was stopped after 5 to 10 minutes with 50 μΐ per well of 1 M H2S04 and absorbance at 450 nm (reference wavelength: 620 nm) was measured in an ELISA reader. Sera were considered to be LI antibody positive if the absorption value was above cut-off. The cut-off value is based on the distribution of absorbance values of control sera and was calculated as the mean value of negative controls plus three times standard deviation. Example 8: Analysis of humoral and cellular immune responses: pl6^INK4a-peptide pool ELISA to determine pl6^INK4a -specific antibodies

pi 6™^K4a specific antibodies were detected with overlapping peptides covering the complete sequence. To this end, 5 peptide pools consisting of 2 or 3 peptides were used by default. 96well microtiter plates (Nunc) were coated with a dilution of 40 μg per petide per ml in PBS. 50 μΐ per well were incubated at 4°C overnight. Plates were washed 4 times with PBS-T after each step and incubation was performed for 1 h at 37°C. After peptide coating, remaining binding sites were blocked with 0.5 % casein in PBS-T (coating for 1 h at room temperature. After washing, sera of immunized mice were applied in duplicates in a 1: 10 dilution (50 μΐ/well) and incubated. Plates were washed again and a horseradish peroxidase conjugated anti-mouse IgG antibody (50 μΐ/ well; 1:5000) was applied and incubated. TMB substrate was used to detect HRP activity and the reaction was stopped after 30 minutes with 50 μΐ 1 M H2S04. The yellow reaction product was read at 450 nm in an ELISA reader (reference wavelength: 620nm). Individual sera were scored as antibody-positive or negative using a cut-off value that was calculated as the mean value of negative controls plus three times standard deviation. Sera were considered to be positive if at least one of the tested peptide pools showed a value above cut-off.

Example 9: Detection of antigen-specific cytotoxic T-lymphocytes by IFNy-ELISpot

An ex vivo IFNy-ELISpot assay was performed 7 days after the last immunization and all steps on day one and two were carried out under sterile conditions in a flow hood. On day 1, 96well MultiScreen-HA plates (Millipore) were incubated for 5 minutes with 70 μΐ of 70% Ethanol, washed 4 times with 200 μΐ PBS /well and coated with 100 μΐ/well anti-mouse IFNy capture antibody (0.6 μg per well) diluted in PBS. The plates were wrapped in aluminium foil and incubated at 4°C over night or at 37°C for 1 - 2 hours. On day 2, plates were washed with PBS 4 times and then blocked with 200 μΐ RPMI culture medium supplemented with 10% FCS and Pen/Strep for 1-2 hours at 37°C. Splenocytes of the sacrificed mice and peptide working solutions were further processed during this incubation time. The blocking medium was removed stepwise and 1.3 - 1.5 x 106 cells/well were seeded in a volume of 100 μΐ. Per mouse three wells were used for the assay positive control which were stimulated unspecifically with 2 μg ConA per well in 100 μΐ; three wells were toped up with 100 μΐ culture medium for the background negative control. Also the test peptides or the pl6 peptide pool were each tested in triplicates with 10 ng/well antigenic peptide in 100 μΐ medium. A typical loading scheme is depicted in Figure 11, yet it was adapted corresponding to the number of mice and peptides tested. After incubation for 18 to 20 hours at 37°C cells were removed and plates were washed 4 times with PBS-0.01% Tween20 and once with PBS. 0.1 μg of a biotinylated rat anti-mouse IFNy antibody diluted in PBS (100 μΐ) were added per well and incubated 1 - 2 h at room temperature. After removing the secondary antibody, plates were washed 6 times with PBS-T and once with PBS. Then, 100 μΐ per well streptavidin- alkaline phosphatase diluted 1: 1,000 in PBS were added and incubated for 30 min at room temperature in the dark. Plates were subsequently washed 3 times with PBS-T and 3 times with PBS and 100 μΐ/well BCTP/NBT substrate (Sigma) were added and incubated in the dark. After 5 to 10 minutes, the staining reaction was stopped by rinsing the plates extensively with distilled water and plates were left to dry overnight. Spots were then quantified with an ELISpot reader.

Claims

A vaccine comprising a viral coat protein and an antigen, wherein the viral coat protein and the antigen are covalently or non-covalently linked to each other, wherein the antigen is a cellular tumor antigen.

The vaccine according to claim 1, wherein two or more of the viral coat proteins form a capsomere.

The vaccine according to claims 1 to 2, wherein the viral coat protein is late protein 1 (LI) of a virus of the family of Papovaviridae, preferably a Papillomaviridae of the subfamily selected from Alphapapillomavirus, Betapapillomavirus, Gammapapillomavirus, Deltapapillomavirus, Epsilonpapillomavirus, Etapapillomavirus, Iotapapillomavirus, Kappapapillomavirus, Lambdapapillomavirus, Mupapillomavirus, Nupapillomavirus, Omikronpapillomavirus, Pipapillomavirus, Thetapapillomavirus, Xipapillomavirus and Zetapapillomavirus.

The vaccine according to claim 3, wherein the Papillomaviridae is a strain of papillomavirus capable of infecting human skin and mucous membranes.

The vaccine according to claim 4, wherein the strain is HPV of type 1, 2, 3, 4,

6,

7, 8, 9 10, 11, 16, 18, 22, 31, 32, 33, 35, 39, 45, 51, 52, 56, 58, 59, 60, 63, 66, 68, 73, and 82.

The vaccine according to any of claims 1 to 5, wherein the cellular tumor antigen is selected from the group consisting of a:

(i) tumor- specific antigens, preferably lineage-restricted, mutated, posttranslationally altered, idiothypic; or

(ii) tumor-associated antigen, preferably oncofetal, overexpressed, cancer-testis, frame-shift antigens.

The vaccine according to claim 6, wherein:

(i) the lineage-restricted antigen is HD39

(ii) the mutated antigen is selected from the group consisting of pRb, Bcr-Abl, mt IDH1, (iii) oncofetal antigen is selected from the group consisting of onco-throphoblast glycoprotein, alpha-fetoprotein;

(iv) overexpressed antigen is selected from the group consisting of Her-2, Muc-1, p53 (mutated), Ras, WT-1, preferably the overexpressed antigen is a cancer-rescue antigen;

or an antigenic fragment thereof comprising at least one T and/or B cell epitope.

8. The vaccine according to claim 7, wherein the cancer-rescue antigen is selected from the group consisting of a cyclin dependent kinase inhibitor protein, preferably plS™^, pl6^mK4\ plS^mK4c and ρ19™, p21^CIP, CIP2, p27^KIP and P57^KIP2.

9. A nucleic acid encoding the vaccine of any of claims 1 to 8.

10. An expression vector comprising the nucleic acid of claim 9 and which is selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.

11. A recombinant host cell comprising the expression vector of claim 10.

12. A method for manufacturing a vaccine according to any of claims 1 to 8, comprising the steps of

a) providing a recombinant host cell of claim 1 1 and

b) expressing said vaccine.

13. A pharmaceutical composition comprising the vaccine according to any of claims 1 to 18, the nucleic acid of claim 09 or the expression vector of claim 10, a pharmaceutically acceptable carrier and/or suitable excipient(s).

14. A vaccine according to any of claims 1 to 8, the nucleic acid of claim 9 or the expression vector of claim 10 for use in eliciting a T cell response against said antigen, a nucleic acid encoding said vaccine, or an expression vector comprising said nucleic acid for treating and/or preventing a disease associated with said antigen.

15. The vaccine according to claim 14, wherein the disease is a hyperproliferative disorder, preferably the hyperproliferative disorder is a benign, pre-cancerous or malign disorder, preferably of the anal region, bladder, blood, bone, brain, breast, cervix, colon, endometrium, esophagus, kidney, larynx, liver, lung, lymphoma, mouth, ovary, pancreas, penis prostate, rectum, skin, stomach, testis, and thyroid vagina, and vulvar, preferably cervix.