WO2002094312A1

WO2002094312A1 - Polypeptides derived from human telomerase reverse transcriptase

Info

Publication number: WO2002094312A1
Application number: PCT/NO2002/000176
Authority: WO
Inventors: Jon Amund Eriksen; Gustav Gaudernack; Mona Møller; Stein Sæbøe-Larssen
Original assignee: Gemvax As
Priority date: 2001-05-21
Filing date: 2002-05-16
Publication date: 2002-11-28
Also published as: GB0112342D0

Abstract

The present invention relates to polypeptides and nucleic acids DNA encoding these polypeptides, capable of eliciting an immune reaction against cancer, methods for generating T lymphocytes capable of recognising and destroying tumour cells, and pharmaceutical compositions for the treatment, prophylaxis or diagnosis of cancer. The polypeptides of the invention are derived from human telomerase reverse transcriptase (hTERT) splice variants.

Description

Polypeptides derived from human telomerase reverse transcriptase

The present invention relates, inter alia, to polypeptides useful in treating cancer.

Cancer develops through a multi-step process involving several mutational events. These mutations result in altered expression/function of genes belonging to two categories: oncogenes and tumour suppressor genes. Oncogenes arise in nature from proto-oncogenes through point mutations or translocations, thereby resulting in a transformed state of the cell harbouring the mutation. Oncogenes code for and function through a protein. Proto-oncogenes are normal genes of the cell which have the potential of becoming oncogenes. In the majority of cases, proto-oncogenes have been shown to be components of signal transduction pathways. Oncogenes act in a dominant fashion. Tumour-suppressor genes on the other hand, act in a recessive fashion, i.e. through loss of function, and contribute to oncogenesis when both alleles encoding the functional protein have been altered to produce non-functional gene products.

In the field of human cancer immunology the last two decades have seen intensive efforts to characterise genuine cancer specific antigens. In particular, effort has been devoted to the analysis of antibodies to human tumour antigens. Such antibodies have been proposed for diagnostic and therapeutic purposes, for instance in connection with an anti-cancer agent.

However, antibodies can only bind to tumour antigens that are exposed on the surface of tumour cells. For this reason, efforts to produce a cancer treatment based on the immune system of the body have been less successful than anticipated.

A fundamental feature of the immune system is that it can distinguish self from non-self molecules. It does not normally target self molecules for attack. It has been shown that rejection of tissues or organs grafted from other individuals is an immune response to the foreign antigens on the surface of the grafted cells. The immune response comprises a humoral response, mediated by antibodies, and a cellular response. Antibodies are produced and secreted by B-lymphocytes, and typically recognise free antigen in native conformation. They can therefore potentially recognise almost any site exposed on the antigen surface. In contrast to antibodies, T cells, which mediate the cellular arm of the immune response, recognise antigens only in the context of major histocompatability complex (MHC) molecules, and only after appropriate antigen processing. Antigen processing usually consists of proteolytic fragmentation of the protein, resulting in polypeptides that fit into a groove of the MHC molecules. This enables T cells to also recognise polypeptides derived from intracellular protein fragments/antigens.

T cells can recognise aberrant polypeptides derived from anywhere in a tumour cell, in the context of MHC molecules on the surface of the tumour cell. The T cells can subsequently be activated to eliminate the tumour cell harbouring the aberrant polypeptide. In experimental models involving murine tumours it has been shown that point mutations in intracellular "self proteins may give rise to tumour rejection antigens, consisting of polypeptides differing in a single amino acid from the normal polypeptide. The T cells recognising these polypeptides in the context of MHC molecules on the surface of the tumour cells are capable of killing the tumour cells and thus rejecting the tumour from the host (Boon et al, 1989, Cell 58_ι 293-303).

MHC molecules in humans are normally referred to as HLA (human leukocyte antigen) molecules. There are two principal classes of HLA molecules: class I and class II. HLA class I molecules are encoded by HLA A, B and C subloci and primarily activate CD8+ cytotoxic T cells. HLA class II molecules, on the other hand, primarily activate CD4+ (cytotoxic or helper) T cells, and are encoded by the HLA DR, DP and DQ subloci. Individuals normally have six different HLA class I molecules, usually two alleles from each of the three subgroups A, B and C, although in some cases the number of different HLA class I molecules may be reduced due to the occurrence of the same HLA allele twice. (For a general review, see Roitt, I.M. et al. (1998) Immunology, 5^th Edition, Mosby, London.)

HLA gene products are highly polymorphic. Thus different individuals generally express different HLA molecules from those found in other individuals (with the main exception of identical twins). This explains the difficulty of finding HLA matched organ donors in transplantations. The significance of the genetic variation of the HLA molecules in immunobiology lies in their role as immune-response genes. Through their polypeptide binding capacity, the presence or absence of certain HLA molecules governs the capacity of an individual to respond to specific polypeptide epitopes. As a consequence, HLA molecules influence resistance or susceptibility to disease.

T cells may inhibit the development and growth of cancer by a variety of mechanisms. Cytotoxic T cells, both HLA class I restricted CD8+ and HLA class II restricted CD4+, may directly kill tumour cells presenting the appropriate tumour antigens. Normally, CD4+ helper T cells are needed for cytotoxic CD8+ T cell responses, but if the polypeptide antigen is presented by an appropriate APC, cytotoxic CD8+ T cells can be activated directly, which results in a quicker, stronger and more efficient response.

In international patent application PCT/N092/00032 (published as WO92/14756), synthetic polypeptides and fragments of oncogene protein products are described that arise from a point mutation or translocation compared to the relevant proto-oncogene or tumour suppressor gene. The polypeptides were shown to induce specific T cell responses to the actual oncogene protein fragment produced by the cell by processing and presented by the HLA molecule. In particular, WO92/14756 describes polypeptides derived from the p2\-ras protein, which had point mutations leading to changes at amino acid positions 12, 13 and 61. These polypeptides have been shown to be effective in regulating the growth of cancer cells in vitro. Furthermore, the polypeptides were shown to elicit CD4+ T cell immunity against cancer cells harbouring the mutated p21 -ras oncogene protein when administered in vaccination or cancer therapy schemes. It has subsequently been shown that these polypeptides also elicit CD8+ T cell immunity against cancer cells harbouring the mutated p21 ras oncogene protein with administered in such a manner (Gjertsen, M.K. et al, 1997, Int. J Cancer 72: 784-790).

International patent application PCT/NO99/00143 (published as WO99/58552) describes synthetic polypeptides and fragments of mutant protein products arising from frameshift mutations occurring in genes in cancer cells. In particular, polypeptides resulting from frameshift mutations in the BAX and hTGF -RII genes are described. These polypeptides were shown to be effective in stimulating CD4+ and CD 8+ T cells in a specific manner.

The concerted action of a combination of altered oncogenes and tumour-suppressor genes results in cellular transformation and development of a malignant phenotype. Such cells are however prone to senescence and have a limited life-span. In most cancers, immortalisation of the tumour cells requires the turning on of an enzyme complex called telomerase. In somatic cells, the catalytic subunit of the telomerase holoenzyme, hTERT (human telomerase reverse transcriptase), is not normally expressed. Events such as the action of proteins encoded by a tumour virus or demethylation of silenced (methylated) promoter sites, can result in expression of the genes encoding the components of the functional telomerase complex in tumour cells. Due to the presence of telomerase in most types of cancer cells, the enzyme has been disclosed as a general cancer vaccine candidate (see international patent application no. PCT NO99/00220, published as WO00/02581). WO00/02581 describes a method for preventing or treating cancer by generating a T cell response against telomerase-expressing cells in a mammal suffering (or likely to suffer from) cancer. It is demonstrated in WO00/02581 that both CD4+ and CD8+ T cells can be stimulated by administration of polypeptides having sequences derived from such a telomerase protein.

Alternative splice variants of telomerase pre-mRNA have been reported by Kilian, A. et al (Hum. Mol. Genet. 6: 2011-2019 [1997]). However, it is indicated that a full understanding of the significance of the variants awaits further characterisation. It is admitted that major questions remain in respect of the nature and role of the products encoded by the splice variants.

Analysis of the complete genomic sequence of the hTERT gene has verified that the different mRNA splice variants arise from the use of alternative splice sites in the hTERT pre-mRNA (Wick, M. et al, 1999, Gene 232: 97-106). However, although various hTERT splice variants were characterised it was indicated that further studies on the regulatory mechanisms controlling hTERT are needed before new cancer-specific therapies can be developed.

In summary, compared with full-length hTERT mRNA, at least five additional splice variants have now been detected although the significance of these variants remains unclear. A schematic drawing of these variants is provided here in Fig. 1. Fig. 2 shows an alignment of the proteins encoded. Two of the splice variants, designated -deletion (DELI) and -deletion (DEL2), represent deletions of specific coding sequences. The -del variant has deleted the first 36 nucleotides of exon 6 and encodes a protein, which lacks a stretch of 12 internal amino acids. In the -deletion variant 182 nucleotides representing the entire exons 7 and 8 are missing, leading to a shift in the open reading frame and a truncated protein with a 44-amino acid long carboxyl terminus not present in the full-length hTERT protein. The remaining splice variants result from the use of alternative splice sites located inside intron regions, providing an insertion of intron sequences within the open reading frame and premature termination of translation. The -insert (or INS1) variant results from an insertion of the first 38 nucleotides of intron 4. The -insert does not contain a stop codon, but instead, the open reading frame extends 22 nucleotides into the normal sequence using an alternative reading frame. The -insert (or INS3) variant is caused by insertion of the last 159 nucleotides from intron 14. Ins-4 contains the first 600 nucleotides from intron 14 while at the same time having deleted exon 15 and most of exon 16. The truncated proteins resulting from translation of these splice variants are shown in Fig. 2.

The present invention provides polypeptides useful in preparing vaccines with improved specificity in comparison with vaccines based on the functional variant of the telomerase (hTERT) protein.

According to one aspect of the present invention there is provided a polypeptide for use in medicine; wherein the polypeptide is capable of inducing a T cell response and:

a) comprises a sequence given in SEQ ID NO 1, 2 or 3 (see figure 3);

b) comprises at least 8 contiguous amino acids from SEQ ID NO 1 , 2 or 3, with the provisos that if said at least 8 contiguous amino acids are from SEQ ID NO 1 they include KD (Lys- Asp) and that if they are from SEQ ID NO 3 at least one (preferably at least two) of said amino acids is also from SEQ ID NO 2; or

c) comprises at least 8 contiguous amino acids that have only one, two or three amino acid changes relative to b) above, with the provisos that that either at least KD

(Lys-Asp) from SEQ ID NO 1 are present, or at least one (preferably at least 2) of the 8 contiguous amino acids from SEQ ID NO 2 is present .

SEQ ID NOs 1 and 2 result from the hTERT and β deletion splice variants respectively. SEQ ID NO 3 generally corresponds to SEQ ID NO 2 but includes a nine amino acid extension at the N terminus, corresponding to the amino acids found at the corresponding position in the hTERT β deletion expression product.

A polypeptide for use in the present invention (referred to herein as "a polypeptide of the present invention") can induce a T cell response by virtue of fitting into an MHC or HLA class I or class II binding groove, optionally after intracellular processing.

Polypeptides that are presented by HLA class II molecules are of varying lengths

(12-25 amino acids), but polypeptides presented by HLA class I molecules will normally be about nine amino acid residues long in order to fit into the class I HLA binding groove. A significantly longer polypeptide will not bind if it cannot be processed internally by an APC or target cell, such as a cancer cell, before presenting in the class I restricted HLA groove. Only a limited number of deviations from the number of nine amino acids have been reported. In those cases, the length of the presented polypeptide has been either eight or ten amino acid residues long. (For reviews on polypeptide binding to MHC molecules see Rammensee, H.-G. et al. (1995) Immunogenetics 41: 178-228 and Barinaga (1992), Science 257: 880-881. Male, D.K. et al. (1996, Advanced Immunology, Mosby, London) provides background information.)

The T cell response induced by a polypeptide of the present invention may be any therapeutically beneficial T cell response. It may, for example, be an increase in the number or activity of T helper and/or T cytotoxic cells.

Those skilled in the art can assay T cell responses using known procedures. Various non-limiting assay procedures are provided in the examples.

In the light of the foregoing comments, it will be appreciated that polypeptides of the present invention will be at least 8 amino acids long, but may be longer. For example, they may be at least 10, at least 12, at least 15, at least 20, or at least 25 amino acids long.

In some cases the polypeptides may comprise the amino acid sequences shown for SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3. The term "comprise" indicates that, in addition to a polypeptide consisting of the specified amino acid sequence, polypeptides having an additional N-terminal and/or an additional C-terminal amino acid sequence are included.

Additional N-terminal and/or C-terminal sequences may be provided for various reasons. Techniques for providing such sequences are well known in the art. These include using gene cloning techniques to ligate together nucleic acid molecules encoding polypeptides or parts thereof and expressing a polypeptide encoded by the ligated sequences using an expression system. Alternatively, chemical techniques can be used to link together polypeptide sequences or individual amino acids by providing peptide bonds, if necessary using immobilisation techniques and protective groups to prevent undesired side reactions. Additional sequences may be provided in order to alter the characteristics of a particular polypeptide. For example, an additional sequence may provide protection against proteolytic cleavage. This has been done for the hormone somatostatin by fusing it at its N-terminus to part of the β galactosidase enzyme (Itakwa et al, Science 198: 105-63: (1977)). Additional sequences can also be useful in altering the properties of a polypeptide to aid in identification or purification. For example, a signal sequence may be present to direct the transport of the polypeptide to a particular location within, a cell or to export the polypeptide from the cell.

Another example of the provision of an additional sequence is where a polypeptide is linked to a moiety capable of being isolated by affinity chromatography. The moiety may be an epitope and the affinity column may comprise a binding agent (e.g. an immobilised antibody, or antibody fragment that binds to the epitope, desirably with a high degree of specificity). The resultant fusion protein may then be eluted from the column by addition of an appropriate buffer. Staphylococcus protein A can also be used to provide a fusion protein that can be isolated by affinity chromatography. Here the fusion protein can be purified using immobilised Ig G or an immobilised part thereof (e.g. a part comprising the Fc fragment). Further examples of fusion proteins include fusions to a poly-arginine sequence. (The strongly basic arginine residues increase the affinity of a polypeptide for anionic resins so that purification can be effected by cation exchange chromatography. Carboxypeptidase B treatment can then be used to remove C-terminal Arg residues.) Glutathione-S-transferase can also be used in fusion proteins and is sometimes preferred where high stability and/or solubility is desired (see e.g. US-A-5654176). The fusion proteins can be purified under non-denaturing conditions via affinity chromatography using immobilised glutathione.

Additional N-terminal and/or C-terminal sequences need not, however, provide any particular advantageous characteristic and may be present simply as a result of a particular technique used to obtain a polypeptide of the present invention. For example, some cloning techniques may result in the presence of an N-terminal Met residue that is not present in the corresponding mature natural polypeptide.

Polypeptides for use in the present invention include variants having one or more amino acid changes from other polypeptides, as indicated in (c) above. For example, amino acid changes can be made to anchor residues that fit into HLA or MHC molecules for presentation to T cells. Enhanced binding and immunogenic properties of the polypeptide to HLA or MHC molecules may thus be achieved (see Bristol, J.A. et al, 1998, J. Immunol. 160(5 2433-2441; Clay, T.M. et al, 1999, J. Immunol. 162 (3): 1749-1755).

More generally, it is known in tlie art that various changes can often be made to the amino acid sequence of a polypeptide that has a desired property to produce variants that still have said property. They include variants that may occur in nature (although non-naturally occurring variants are also included). Thus variants within a given species are included (intra-species variants), as well as variants occurring between different species (inter-species variants). For example variants may occur within or between species of: bacterium, insect, worm, fish, amphibian, bird, reptile, rodent (e.g. mouse, rat, rabbit or guinea pig), pig, sheep, cow, horse, chimp, monkey, and ape (e.g. chimpanzee, orang-utan or human). Variants occurring within or between mammalian species (especially ape species) are particularly preferred. Polypeptides for use in the present invention include both wild-type polypeptides and non wild-type variants thereof. The variants may be allelic variants or non-allelic variants.

Polypeptide variants are discussed in greater detail in sections (i) to (iii) below:

(i) Substitutions

An example of a variant is a polypeptide as defined in a) above, apart from the substitution of one or more amino acids with one or more other amino acids. A skilled person is aware that various amino acids have similar characteristics. One or more such amino acids can often be substituted by one or more other such amino acids without eliminating a desired property of a polypeptide. Substitutions of this nature are often referred to as "conservative" or "semi-conservative" substitutions. For example, the amino acids glycine, alanine, valine, leucine and/or isoleucine can often be substituted for one another (amino acids having aliphatic side chains). Of these possible substitutions it is preferred that glycine and alanine are used to substitute for one another (because they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (because they have larger aliphatic side chains which are hydrophobic). Other amino acids that can often be substituted for one another include phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and/or cysteine and methionine (amino acids having sulphur-containing side chains). (ii) Deletions

Amino acid deletions can be advantageous since the overall length and the molecular weight of a polypeptide can be reduced, whilst still retaining a desired property. This can enable the amount of polypeptide required for a particular purpose to be reduced. For example, if the polypeptide is to be used in medicine, dosage levels can be reduced.

Thus, if desired, non-essential or non-important sequences may be removed from a given polypeptide. This can be done by enzymatic techniques followed by ligation. More preferably, such sequences can be excluded initially, e.g. by using a nucleic acid encoding only essential or important sequences in order to express the polypeptide or by using chemical synthesis techniques in order to synthesise only desired or essential sequences.

(iii) Insertions

Amino acid insertions relative to a polypeptide as defined in a) above can also be made. This may be done to alter the nature of the polypeptide (e.g. to assist in identification, purification or expression, as explained above in relation to fusion proteins).

Polypeptides incorporating amino acid changes (whether substitutions, deletions and/or insertions) relative to the sequence of a polypeptide as defined in a) above can be provided using any suitable techniques. For example, a nucleic acid sequence incorporating a desired sequence change can be provided by site-directed mutagenesis. This can then be used to allow the expression of a polypeptide having a corresponding change in its amino acid sequence.

Various polypeptides of the present invention may have substantial sequence identity with SEQ ID NO: 1, 2, or 3. Such polypeptides may, for example, have at least 40%, 50%, 60%, 70% or 80% sequence identity therewith. In some cases sequence identities may be at least 90% or at least 95%. Where high degrees of sequence identity are present there may be relatively few sequence differences. Thus, for example, there may be less than 15, less than 10, or less than 5 differences. As indicated supra, polypeptides of the present invention are useful in medicine, especially for cancer treatments, including anti-cancer vaccines.

In particular, the polypeptides may be used in a vaccine capable of eliciting either CD4+ or CD 8+ T cell immunity. If desired, the polypeptide may be produced by chemical synthesis techniques or by recombinant DNA technology. Transforming cancer genes or other moieties that might produce deleterious effects can therefore be avoided.

The cancer may be a mammalian cancer, e.g. human cancer. It may, for example, be breast cancer, prostate cancer, pancreatic cancer, colo-rectal cancer, lung cancer, malignant melanoma, leukaemia, lymphoma, ovarian cancer, cervical cancer or a biliary tract carcinoma.

A polypeptide of the present invention (or other active agent) may be used in the manufacture of a medicament. The medicament will usually be supplied as part of a pharmaceutical composition. Pharmaceutical compositions are therefore also within the scope of the present invention. Preferred pharmaceutical compositions are vaccines.

The pharmaceutical composition may comprise a pharmaceutically acceptable carrier, diluent, additive, stabiliser, and or adjuvant.

Compounds such as cytokines and/or growth factors, (e.g. interleukin-2 (IL-2), interleukin-12 (IL-12), and granulocyte macrophage colony stimulating factor (GM-CSF)) are particularly preferred for use in vaccines in order to strengthen an immune response.

Polypeptides of the present invention may be supplied in the form of a lipopeptide conjugate, if desired. This is known to induce a l igh-affinity, cytotoxic T cell response (Deres, K. et al, 1989, Nature 342: 561-564).

Pharmaceutical compositions of the present invention may include additional polypeptides. Such polypeptides include other polypeptides arising from a frameshift mutation, e.g. a frameshift mutation in the BAX or hTGFβ-RII gene. The stimulatory effect on CD4+ and CD8+ T cells in a specific manner by polypeptides resulting from frameshift mutations in the BAX and hTGF -RII genes is disclosed in WO99/58552 (see above). Pharmaceutical compositions of the present invention may further comprise a polypeptide capable of inducing a T cell response directed against a polypeptide produced by an oncogene or against a mutant tumour suppressor protein, or a nucleic acid encoding such a polypeptide, or a binding agent that binds such a polypeptide, or a T cell that is capable of killing a cell expressing such a polypeptide or of helping in the killing of such a cell. Example of such oncogenes or mutant tumour suppressor proteins include p21-rαs', Rb, p53, abl, gip, gsp, ret or trk. The oncogene target may be the p21-ras polypeptides described in international patent application no. PCT/NO92/00032 (publication no. WO92/14756).

In some cases it may be decided to administer one or more active ingredients separately - e.g. at different times and/or at different sites. The present invention therefore encompasses therapies using active agents that need not be administered together in a single composition. Sequential, separate or combined use of a plurality of active agents is therefore contemplated.

Dosages of polypeptides of the present invention may vary between wide limits, depending upon the nature of a treatment, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used. Without being bound by any particular dosages, a daily dosage of a polypeptide of the present invention of from one nanogram to 1 gram (1 g) / kg body weight may be administered to an adult patient. It is however preferred to use a dose in the range of 1 microgram (1 g) to 1 milligram (lmg) /kg body weight. The dosage may be repeated as often as is appropriate. If side effects develop, the amount and/or frequency of the dosage can be reduced, in accordance with good clinical practice.

Polypeptides of the present invention are also useful in diagnosis. They can be used to bind to antibodies present in a sample taken from a patient (e.g. a blood sample). They may therefore be used in diagnosing cancer. They may be provided in a diagnostic kit. The kit may comprise means for generating a detectable signal (e.g. a fluorescent label, a radioactive label) or a detectable change (e.g. an enzyme-catalysed change). The kit may include instructions for use in diagnosing cancer.

In addition to utilising polypeptides, the present invention can utilise nucleic acids. The present invention includes a nucleic acid molecule for use in medicine; wherein the nucleic acid molecule:

a) has a strand that encodes a polypeptide as described above;

b) has a strand that is complementary with a strand as described in a) above; or

c) has a strand that hybridises with a molecule as described in a) or b) above (e.g. under stringent conditions).

Various nucleic acid molecules of the present invention and uses thereof are discussed below:

a) Coding nucleic acid molecules

Polypeptides of the present invention can be coded for by a large variety of nucleic acid molecules, taking into account the well-known degeneracy of the genetic code. All of these coding nucleic acid molecules are within the scope of the present invention.

They may be administered to an individual and used to express said polypeptides. They may be used for the same treatments as the polypeptides of the present invention. Thus the foregoing discussions in respect of treatments involving polypeptides apply mutatis mutandis to treatments involving nucleic acids (or involving cells or vectors comprising such nucleic acids).

Preferred treatments use nucleic acid vaccines (e.g. DNA vaccines). The nucleic acid may therefore be delivered together with cytokines, such as IL-2, and/or other co-stimulatory molecules. The cytokines and/or co-stimulatory molecules may themselves be delivered in the form of plasmid or oligonucleotide DNA. Response to a DNA vaccine has been shown to be increased by the presence of immunostimulatory DNA sequences (ISS). These can take the form of hexameric motifs containing methylated CpG, according to the formula : 5'-purine-purine-CG-pyrimidine-pyrimidine-3'. DNA vaccines according to the present invention may therefore incorporate these or other ISS, in the DNA encoding the hTERT

-deletion protein and/or the hTERT -deletion protein, in the DNA encoding the cytokine or other co-stimulatory molecules, or in both. A review of the advantages of DNA vaccination is provided by Tighe et al. (1998, Immunology Today, 19(2^: 89-97). Nucleic acid molecules of the present invention may be administered to a patient by any appropriate method. Such methods include topical application of a nucleic acid using any appropriate vehicle (e.g. via a pharmaceutically acceptable excipient, such as phosphate buffered saline) They also include particle bombardment (which is sometimes known as "gene gun" technology and is described in US Patent No. 5371 15). Here inert particles, such as gold beads coated with a nucleic acid, can be accelerated at speeds sufficient to enable them to penetrate cells. They can be used for example to penetrate the skin of a patient and may be administered by means of discharge under high pressure from a projecting device. Other physical methods of administering the nucleic acid directly to a recipient include ultrasound, electrical stimulation (including iontophoresis) and microseeding (see e.g. US Patent No. 5697901). Alternatively, nucleic acid molecules may simply be injected at appropriate site (e.g. muscle). They may be incorporatedin or on a carrier (which may be a lipid-based carrier, such as a liposome).

Nucleic acid molecules may be introduced into host cells (optionally in the form of vectors) to enable the expression of polypeptides of the present invention. Alternatively, cell-free expression systems may be used. By using an appropriate expression system the polypeptides can be produced in a desired form. For example, the polypeptides can be produced by micro-organisms such as bacteria or yeast, by cultured insect cells (which may be baculovirus-infected), by mammalian cells (such as CHO cells) or by transgenic animals that, for instance, secrete the proteins in milk (see e.g. international patent application WO 88/00239). Where glycosylation is desired, eukaryotic (e.g. mammalian or insect) expression systems are preferred. Whatever means are used to obtain expression, transcriptional and translational control sequences will normally be present and will be operatively linked to a sequence encoding a polypeptide to be expressed. These control sequences may be heterologous to the sequence encoding the polypeptide or may be found associated with it in vivo. Promoter, operator and /or enhancer sequences may, for example, be provided, as may polyadenylation sites, splice sites, stop and start codons, upstream and downstream regulatory regions, etc. If desired, a constitutive promoter may be provided. Alternatively, a regulatable promoter may be provided to enable transcription to be controlled by administration of a regulator. The promoter (if present) may be tissue-specific or non tissue-specific. Polypeptides comprising N-terminal methionine may be produced using certain expression systems, whilst in others the mature polypeptide may lack this residue. Polypeptides may initially be expressed so as to include signal sequences. Different signal sequences may be provided for different expression systems. Alternatively, signal sequences may be absent, if not needed. Once expressed, polypeptides may be purified by a wide variety of techniques. Purification techniques may be used under reducing conditions (in order prevent disulphide bond formation) or non-reducing conditions. Available purification techniques include, for example, electrophoretic techniques, such as SDS PAGE (see e.g. Hunkapiller et al, Methods Enzymol. 91:227 (1983), which discloses "Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis"); affinity techniques (e.g. immunoaffinity chromatography); HPLC; gel filtration; ion-exchange chromatography; isoelectric focussing; etc. If desired, combinations of different purification steps may be used and or individual purification steps may be repeated. In summary, techniques for cloning, expressing and purifying polypeptides are well known to the skilled person. Various such techniques are disclosed in standard text-books, such as in Sambrook et al [Molecular Cloning 2nd Edition, Cold Spring Harbor Laboratory Press (1989)]; in Old & Primrose [Principles of Gene Manipulation 5th Edition, Blackwell Scientific Publications (1994)]; and in Stryer [Biochemistry 4th Edition, W H Freeman and Company (1995)].

b) Complementary nucleic acid molecules

In addition to nucleic acid strands coding for polypeptides of the present invention, the present invention also includes nucleic acid strands complementary thereto, whether or not the coding and complementary strands are associated in the form of a duplex. Thus, for example, mRNA and cDNA molecules are included.

c) Hybridising nucleic acid molecules

Nucleic acid molecules that can hybridise to one or more of the nucleic acid molecules discussed above are also covered by the present invention. Such nucleic acid molecules are referred to herein as "hybridising" nucleic acid molecules. Desirably hybridising molecules of the present invention are at least 10 nucleotides in length and preferably are at least 20, at least 50, at least 100, or at least 200 nucleotides in length.

A hybridising nucleic acid molecule of the present invention may have a high degree of sequence identity along its length with a nucleic acid molecule within the scope of b) or a) above (e.g. at least 50%, at least 75% or at least 90% sequence identity), although this is not essential. The greater the degree of sequence identity that a given single stranded nucleic acid molecule has with a strand of a nucleic acid molecule, the greater the likelihood that it will hybridise to the complement of said strand.

Hybridising nucleic acid molecules can be useful as probes or primers, for example.

Probes can be used to purify and/or to identify nucleic acids. They may be used in diagnosis and may be present in diagnostic kits, optionally in labelled form. For example, probes may be used to determine whether or not an individual has a gene encoding a polypeptide of the present invention, or whether or not one or more deletions, insertions and/or replacements of bases relative thereto are present. They may therefore be used to identify individuals that do or that do not express polypeptides of the present invention. Thus probes can be used in the diagnosis of cancer.

Primers are useful in synthesising nucleic acids or parts thereof based upon a template to which a probe hybridises. They can be used in techniques such as PCR to provide large numbers of nucleic acid molecules.

Hybridising molecules also include antisense strands. These hybridise with "sense" strands so as to inhibit transcription and /or translation. An antisense strand can be synthesised based upon knowledge of a sense strand and base pairing rules. It may be exactly complementary with a sense strand, although it should be noted that exact complementarity is not always essential. It may also be produced by genetic engineering, whereby a part of a DNA molecule is provided in an antisense orientation relative to a promoter and is then used to transcribe RNA molecules. Large numbers of antisense molecules can be provided (e.g. by cloning, by transcription, by PCR, by reverse PCR, etc.

Hybridising molecules include ribozymes. Ribozymes can also be used to regulate expression by binding to and cleaving RNA molecules that include particular target sequences recognised by the ribozymes. Ribozymes can be regarded as special types of antisense molecule. They are discussed, for example, by Haselhoff and Gerlach (Nature (1988) 334:585 - 91).

Antisense molecules may be DNA or RNA molecules. They may be used in antisense therapy to prevent or reduce undesired expression or activity e.g. to reduce expression of the polypeptides encoded by the α and β deletion variants discussed supra. Antisense molecules may be administered directly to a patient (e.g. by injection). Alternatively, they may be synthesised in situ via a vector or cell that has been administered to a patient.

In addition to tlie uses described above, nucleic acid molecules of the present invention (of whatever nature) may be used in screening. Screening can be done to identify moieties that bind to said nucleic acid molecules (e.g. to identify hybridising molecules). It can also be done to identify moieties that affect transcription or translation from said nucleic acid molecules.

It can be used to analyse expression, including analysing expression levels or expression patterns (e.g. by analysing mRNA or cDNA), etc. It can be used to identify particular nucleic acid molecules in a sample. This is useful for in identifying biological material from a given source (e.g. from a human or non-human animal) and can be used in forensic science, for security applications (e.g. for confirming the identity of a human or non-human animal), for checking for contamination (e.g. of food or drink), for geneological studies, etc. For example.; a reference nucleic acid molecule (or part of it) can be digested with restriction enzymes and the resultant nucleic acid fragments can be run on a gel. This can provide a restriction fragment pattern or "fingerprint" that can be compared with a sample. If the comparison provides a match that is unlikely to have occurred by chance, a conclusion can be reached that the sample and the reference molecule are likely to have originated from a common source. By performing statistical analysis a specific degree of confidence that such a conclusion is correct can be provided.

The present invention also includes within its scope a library having a nucleic acid molecule of the present invention. The present invention further includes an array comprising a nucleic acid molecule of the present invention (which may be a library). Preferably the array is a regular array. The array may have a predetermined pattern. It may have a grid-like pattern. The discussion provided herein in respect of libraries and arrays comprising a polypeptide of the present invention applies mutatis mutandis to libraries and arrays comprising a nucleic acid molecule of the present invention.

One or more nucleic acid molecules of the present invention may be immobilised upon a surface (e.g. the surface of a bead or a chip). The surface may, for example, be silicon surface, glass, quartz, a membrane, etc. Techniques for immobilising nucleic acid molecules upon a surface are known and are disclosed, for example, in EP-A-0487104, WO96/04404, WO90/02205, WO96/12014, WO98/44151. In some cases they may include a step of nucleic acid amplification, which may involve PCR. Immobilisation is not however essential. For example nucleic acids may be provided in wells or other containment means (e.g. in a fluid environment).

In the light of the foregoing comments it will be appreciated that a large number of nucleic acids and uses thereof are within the scope of the present invention. These molecules include not only molecules with classical DNA or RNA structures, but also variants with modified (non-phosphodiester) backbones - e.g. morpholino derivatives and peptide nucleic acids (PNAs), which contain an N-(2-aminoethyl)glycine-based pseudopeptide backbone. (See Nielsen, P.E., Annual Review of Biophysics & Biomolecular Structure, 24:167-83 (1995)). Nucleic acid variants with modified backbones can have increased stability relative to unmodified nucleic acids and are particularly useful where hybridisation is desired over a relatively long period (e.g. in antisense therapy).

Unless the context indicates otherwise, nucleic acid molecules of the present invention may have one or more of the following characteristics:

1) They may be DNA or RNA (including variants of naturally occurring DNA or RNA structures, which have non-naturally occurring bases and/or non-naturally occurring backbones).

2) They may be single-stranded or double-stranded (or in some cases higher stranded, e.g. triple- stranded).

3) They may be provided in recombinant form i.e. covalently linked to a heterologous 5' and/or 3' flanking sequence to provide a chimeric molecule (e.g. a vector) that does not occur in nature.

4) They may be provided with or without 5' and/or 3' flanking sequences that normally occur in nature.

5) They may be provided in substantially pure form, e.g. by using probes to isolate cloned molecules having a desired target sequence or by using chemical synthesis techniques. Thus they may be provided in a form that is substantially free from contaminating proteins and/or from other nucleic acids.

6) They may be provided with introns (e.g. as a full-length gene) or without introns (e.g. as DNA).

7) They may be provided in linear or non-linear (e.g. circular) form.

Vectors are also within the scope of the present invention. They comprise nucleic acid molecules of the present invention and may be in the form of plasmids, phasmids, cosmids, viruses (including bacteriophages), YACs, PACs, etc. They will usually include an origin of replication and may also include one or more selectable markers e.g. drug resistance markers and/or markers enabling growth on a particular medium. A vector may include a marker that is inactivated when a nucleic acid molecule according to the present invention is inserted into the vector. A second marker may be provided that is different from the first marker in order to aid in selection/identification (e.g. a marker that encodes a different type of drug resistance from the first marker).

Vectors may include one or more regions necessary for transcription of RNA encoding a polypeptide of the present invention. Such vectors are often referred to as expression vectors. They will usually contain a promoter and may contain additional regulatory regions - e.g. operator sequences, enhancer sequences, etc. Translation can be provided by a host cell or by a cell free expression system.

Vectors need not be used for expression. They may be provided for maintaining a given nucleic acid sequence, for replicating that sequence, for manipulating, it or for transferring it between different locations (e.g. between different organisms).

Large nucleic acid molecules may be incorporated into high capacity vectors (e.g. cosmids, phasmids, YACs or PACs). Smaller nucleic acid molecules may be incorporated into a wide variety of vectors.

Vectors can generally be used for the same purposes as polypeptides or nucleic acids of the present invention. For example they may be used in anticancer vaccines. Nucleic acid molecules of the present invention may be provided in the form of vectors, although this is not essential. Examples of vectors for use in treatment include replication-deficient adenoviruses, retroviruses, adeno-associated viruses, vaccinia viruses, modified forms of the aforesaid, etc.

The present invention further includes cells comprising nucleic acid molecules or vectors of the present invention.

A cell capable of expressing polypeptide as described herein can be cultured and used to provide the polypeptide, which can then be purified. (Expression of polypeptides is not however essential since cells may be provided simply for maintaining a given nucleic acid sequence, for replicating the sequence, for manipulating it, etc.)

Alternatively, the cell may itself be used in therapy for the same purposes as the polypeptide. Thus the cell may be used to provide the polypeptide in vivo. For example, cells may be provided from a patient (e.g. via a biopsy), transfected with a nucleic acid molecule or vector of the present invention and, if desired, cultured in vitro, prior to being returned to the patient (e.g. by injection). The cell may comprise a regulatable promoter enabling transcription to be controlled via administration of one or more regulator molecules. One type of preferred cell for use in therapy is an antigen presenting cell, e.g. a dendritic cell.

Cells may be provided in any appropriate form. For example, they may be provided in isolated form, in culture, in stored form, etc. Storage may, for example, involve cryopreservation, buffering, sterile conditions, etc. Cells of the present invention may be provided by manipulation involving gene cloning techniques, transfection techniques, stem cell technology, and/or by other means.

It is important to note that cells for use in the present invention are not limited to cells comprising nucleic acid molecules or cells comprising polypeptides of the present invention. In one embodiment of the present invention there is provided a T lymphocyte that is capable of killing, or of helping to kill a cell expressing a polypeptide as described above. The T lymphocyte may be a T cytotoxic cell or a T helper cell. Preferably a plurality of T lymphocytes are provided.

T lymphocytes capable of recognising and destroying tumour cells in a mammal can be provided by a method involving taking a sample of T lymphocytes from a mammal (e.g. a human) and culturing the T lymphocyte sample in the presence of at least one polypeptide described above in an amount sufficient to generate hTERT -deletion protein specific T lymphocytes and/or hTERT -deletion protein specific T lymphocytes. Such a method is within the scope of the present invention.

Suitable T lymphocytes may be provided in the form of a clonal cell line. In some cases, however, it may be desired to provide a mixture of different T cells. Mixtures of T cytotoxic, T helper and/or of both T helper and T cytotoxic cells may be provided. All of the aforesaid can be used in anti-cancer therapy.

B lymphocytes producing antibodies that bind to polypeptides of the present invention can also be used. These can be used, for example, to produce hybridomas useful in generating antibodies.

Antibodies are only one type of binding agent that is useful in the present invention. Many different kinds of binding agents can be used. They may be naturally or non-naturally occurring, They include antibodies, HLA molecules, receptors, lectins, moieties predicted by a computer to fit a particular binding site, parts or variants of any of the aforesaid that retain binding activity, etc. As will be appreciated by a skilled person, once a binding site has been identified a plethora of different binding agents can be provided or synthesised. If desired they may be provided in recombinant form.

Binding agents of the present invention may be used in diagnosis. They may be used to detect a polypeptide of the present invention when present in a sample obtained from a patient. They may therefore be useful in diagnosing cancer. They may be provided in a kit, optionally including instructions for use and may be labelled, if desired.

They can also be used generally in procedures for assaying and/or detecting polypeptides of the present invention. Various assay and detection procedures are disclosed, for example, by Nakamura et al ("Immunochemical Assays and Biosensor Technology for the 1990s" (1992), published by the American Society for Microbiology).

A particularly preferred binding agent of the present invention is an HLA molecule (including an HLA tetramer, another HLA oligomer, or a part thereof) that binds to a polypeptide of the present invention with a sufficient degree of specificity to be useful in binding to and stimulating T cells specific for the polypeptide. For example HLA molecules may be immobilised via streptavidin biotin binding and used to bind to polypeptides of the present invention.

Preferred HLA molecules are in the form of tetramers. Tetramers are discussed, for example, in the following documents:

Ogg GS, McMichael AJ. HLA-peptide tetrameric complexes. Curr Opin Immunol 1998 Aug;10(4):393-6 (Review)

Altaian JD, Moss PA, Goulder PJ, Barouch DH, McHeyzer- Williams MG, Bell JI, McMichael AJ, Davis MM. Phenotypic analysis of antigen-specific T lymphocytes. Science 1996 Oct 4;274(5284):94-6

Pittet MJ, Speiser DE, Lienard D, Valmori D, Guillaume P, Dutoit V, Rimoldi D, Lejeune F, Cerottini JC, Romero P. Expansion and functional maturation of human tumor antigen-specific CD8+ T cells after vaccination with antigenic peptide. Clin Cancer Res. 2001 Mar;7(3 Suppl):796s-803s

Youde SJ, Dunbar PR, Evans EM, Fiander AN, Borysiewicz LK, Cerundolo V, Man S. Use of fluorogenic histocompatibility leukocyte antigen- A* 0201 /HPV 16 E7 peptide complexes to isolate rare human cytotoxic T-lymphocyte-recognizing endogenous human papillomavirus antigens. Cancer Res 2000 Jan 15;60(2):365-71

Kim SK, Devine L, Angevine M, DeMars R, Kavathas PB. Direct detection and magnetic isolation of Chlamydia trachomatis major outer membrane protein-specific CD8+ CTLs with HLA class I tetramers. J Immunol 2000 Dec 15;165(12):7285-92

Molldrem JJ, Lee PP, Wang C, Champlin RE, Davis MM. A PR1 -human leukocyte antigen- A2 tetramer can be used to isolate low-frequency cytotoxic T lymphocytes from healthy donors that selectively lyse chronic myelogenous leukemia. Cancer Res 1999 Jun 1;59(11):2675-81

Valmori D, Pittet MJ, Rimoldi D, Lienard D, Dunbar R, Cerundolo V, Lejeune F,

Cerottini JC, Romero P. An antigen-targeted approach to adoptive transfer therapy of cancer.

Cancer Res 1999 May 1;59(9):2167-73 Yee C, Savage PA, Lee PP, Davis MM, Greenberg PD. Isolation of high avidity melanoma- reactive CTL from heterogeneous populations using peptide-MHC tetramers. J Immunol 1999 Feb 15;162(4):2227-34

Reichstetter S, Ettinger RA, Liu AW, Gebe JA, Nepom GT, Kwok WW. Distinct T cell interactions with HLA class II tetramers characterise a spectrum of TCR in the human antigen-specific T cell response. J Immunol. 2000 Dec 15;165(12):6994-8.

Preferred uses of HLA molecules of the present invention (whether or not they are in the form of tetramers) include:

a) isolation of peptide specific cells (e.g. by flow cytometry, magnetic bead, panning and other techniques) from patients or normal donors

b) ex vivo expansion of peptide specific cells for therapy

c) monitoring of T cell responses during therapy (e.g. vaccine or adoptive therapy by ex vivo expanded cells) and

d) diagnosis (i.e. detection of peptide specific T cells that may be associated with a disease, e.g. at an early stage)

From the foregoing description it will be appreciated that many different moieties can be used in the present invention, including polypeptides, nucleic acids, vectors, cells, binding agents, pharmaceutical compositions, etc. Where the moieties are novel the present invention includes the moieties per se and is therefore not limited to particular uses thereof. If desired, the moieties may be provided in substantially pure form or in isolated form. They may be provided as part of a pharmaceutical composition.

The present invention will now be described by way of example only, with reference to the accompanying drawings; wherein:

Fig. 1 is a schematic drawing of the full-length hTERT mRNA and splice variants found in cancer cell lines. The position of introns present in the hTERT pre-mRNA is indicated by the letter "i" followed by an appropriate number. Insertion and deletion variants are shown as square boxes; shaded fill represents sequences that encode protein sequence not present in the full-length hTERT protein. Position and orientation of oligonucleotide primers used to analyse the different splice variants is indicated by arrows.

Fig. 2 shows a protein alignment between a portion of the hTERT protein and proteins resulting from translation of splice variants. Amino acid numbering is shown above the sequence. Amino acids are represented by their standard one letter abbreviation known in the art.

Fig. 3 shows the region where the carboxyl terminus and the amino terminus on each side of the< hTERT -deletion are joined together (SEQ ID NO 1), and the region of the carboxyl terminus of the hTERT -deletion splice variant protein (SEQ ID NOs 2 and 3). In Fig. 3, SEQ ID NO 1 reflects the region where the "protein ends", resulting from the hTERT -deletion are joined together, and contains 24 amino acids from the sequence of each side of the -deletion. SEQ ID NO 2 reflects the truncated tail of the hTERT -deletion protein. SEQ ID NO 3 reflects SEQ ID NO 2 with an extension at the amino terminus with the nine amino acids normally found in these positions in the naturally occurring hTERT -deletion expression product (underlined).

Fig. 4 shows the nucleotide strands coding for SEQ ID NO 1 and SEQ ID NO 3 from figure 3

Fig. 5 shows results from RT-PCR analysis of the region comprising the and deletion splice variants of hTERT.

Examples

The examples outlined herein describe the characterisation of hTERT splice variants in various cancer cell lines compared with normal cells. Synthesis of polypeptides according to the present invention, and experiments for testing the efficacy of the polypeptides for use in cancer therapy are detailed.

RT-PCR Analysis of the -deletion and -deletion splice variants of hTERT

RNA Analysis:

Poly(A) mRNA from completely lysed cells was isolated directly from crude lysates using magnetic oligo(dT) beads (Dynal AS; Jakobsen, K.S. et al, 1990, Nucleic Acids Res. l_8ι 3669). Cytosolic mRNA fractions were prepared by incubating cells in 1% IGEPAL (Sigma) at 0°C for one minute, followed by centrifugation [lOOOOg; 1 min.; 4°C] to remove nuclei.

Poly(A)⁺ mRNA was then isolated from the supernatant using oligo(dT) beads as described above.

cDNA synthesis and PCR:

First strand cDNA synthesis was carried out by standard procedures using M-MLV RNaseH÷ reverse transcriptase (Promega Corp.), and the PCR reactions were performed by using HotStar Taq DNA polymerase (Qiagen) and run for 35 cycles on a PTC -200 thermal cycler (MJ Research). To obtain detectable products from PBM and CD34+ cells, 10% of the reaction was used as template in a second PCR reaction and amplified by 15 additional cycles.

For analysis of the and deletion splice variants the plus-strand primer hTERT p2184 (5-CCG CCT GAG CTG TAC TTT GTC) and minus-strand primer hTERT m2681 (5-GCT CTA GAT CCA CCA AAC GCA GGA GCA) were used. Applied on the full-length hTERT cDNA and the and ώletion variants, these primers produce fragments of 505, 469 and 323 nucleotides, respectively. To verify that the PCR products represent genuine splice variants, all fragments were isolated from the gel and analysed by direct sequencing using an ABI prism 310 automated sequencer (PE Corp.). Results:

Telomerase activity is subject to complex regulation at the post-transcriptional level, and methods used to detect the presence or absence of telomerase proteins should involve direct measurements of the protein itself, or alternatively, mRNA variants. Furthermore, the abundance of the different hTERT splice variants found in cells is not necessarily correlated with the levels found in the cytosolic fraction of the same cells (see Fig. 5). Such deviations may be explained by differences in the efficiency with which mRNA variants are transported from the nucleus to the cytosolic compartment, and/or by differential stability of the specific splice, variants in the cytosol. It is well known in the art that such mechanisms are part of the concept of gene regulation. Nevertheless, the studies conducted to explain hTERT regulation, including those cited above, have used total RNA or mRNA isolated from completely lysed cells for their analysis. Kits and reagents required to perform this kind of RNA isolation are widely available in the commercial market. To obtain a correct picture of gene expression, studies on mRNA abundance should include analysis of mRNA specific to the cytosolic compartment.

Fig. 5 shows results from RT-PCR analysis of the region comprising the and deletion variants of hTERT. HL60, K562, and Jurkat denote the cancer cell lines analysed. HL60 is a promyelocytic leukemia cell line (Sokoloski, J.A. et al, 1993, Blood 82: 625-632), K562 an erythroid leukemia cell line (Lozzio, CB. et al, 1975, Blood 45: 321-324), while Jurkat is derived from acute T-lymphocyte leukemia cells (Gillis, S. et al, 1980, J. Exp. Med. 152: 1709-1719). The HL60, K562, and Jurkat cancer cell lines are commercially available (for example, from ATCC, Oslo). PBM1, PBM2, PBM3 and PBM4 represent peripheral blood mononuclear (PBM) cell populations isolated from four different healthy donors. CD34 denotes CD34-positive stem cells isolated from a healthy donor, and CC1/CC2 denotes colon cancer biopsies obtained from two cancer patients at the Norwegian Radium Hospital, Oslo. RT-PCR reactions performed with mRNA isolated by complete lysis of cells and with mRNA isolated from cytosolic fractions are marked with the letters "T" and "C", respectively. "M" indicates lane with molecular weight marker. Position of PCR fragments representing the and deletion splice variants and the respective full-length hTERT products (+) is indicated on the right side of the panels. " " denotes the splice variant having both the - and -deletions, and "+/ " is a heterodimer produced by hybridisation between the full-length product and the -deletion variant during the last annealing step in the PCR reaction. The RT-PCR analysis showed that both and ώletion splice variants were readily detectable in all cancer cell lines and tumour samples analysed, either in the form of single splice variants, or as the double deletion variant.

Polypeptide synthesis and analysis for applications relating to cancer

Polypeptide synthesis:

The polypeptides were synthesised by using continuous flow solid phase peptide synthesis. N-a-Fmoc-amino acids with appropriate side chain protection were used. The Fmoc-amino acids, were activated for coupling as pentafluorophenyl esters or by using either TBTU or diisopropyl carbodiimide activation prior to coupling. 20% piperidine in DMF was used for selective removal of Fmoc after each coupling. Cleavage from the resin and final removal of side chain protection was performed by 95% TFA containing appropriate scavengers. The polypeptides were purified and analysed by reversed phase HPLC. The identity of the polypeptides was confirmed by using electro-spray mass spectroscopy.

Polypeptide testing and cancer therapy:

In order for a cancer vaccine according to the present invention, and methods for specific cancer therapy based on T cell immunity to be effective, two conditions must be met:

(a) the polypeptide is at least 8 amino acids long and is a fragment of the hTERT -deletion protein or the hTERT -deletion protein and

(b) the polypeptide is capable of inducing, either in its full length or after processing by antigen presenting cell, a T cell response.

The following experimental methods may be used to determine if these two conditions are met for a particular polypeptide. It can be determined if the particular polypeptide gives rise to T cell immune responses in vitro. It can also be established if the synthetic polypeptides correspond to, or are capable after processing to yield, polypeptide fragments corresponding to polypeptide fragments occurring in cancer cells harbouring the hTERT -deletion protein and/or the hTERT -deletion protein or antigen presenting cells that have processed naturally occurring hTERT

-deletion protein and/or hTERT -deletion protein. The specificity of T cells induced in vivo by hTERT -deletion and/or hTERT -deletion polypeptide vaccination may also be determined.

In vitro T cell response analysis:

It can be determined if hTERT -deletion and/or hTERT -deletion expressing tumour cell lines can be killed by T cell lines or clones obtained from peripheral blood of a normal donor after repeated in vitro stimulation with hTERT -deletion and/or hTERT -deletion polypeptide or with T cell lines or clones from carcinoma cancer patients after hTERT -deletion and/or hTERT -deletion polypeptide vaccination. T cell lines are obtained after repeated in vitro stimulation with hTERT -deletion and or hTERT -deletion polypeptide from normal donors or cancer patients after hTERT -deletion and/or hTERT -deletion polypeptide vaccination. T cell clones are obtained after cloning of T-cells blasts present in T cell lines. The polypeptide vaccination protocol includes several in vivo injections of polypeptides intracutaneously with GM-CSF or another commonly used adjuvant. Cloning of T cells is performed by plating responding T cell blasts at 5 blasts per well onto Terasaki plates. Each well contains 2 x 10⁴ autologous, irradiated (30 Gy) PBMC as feeder cells. The cells are propagated with the candidate hTERT -deletion and/or hTERT -deletion polypeptide at 25 mM and 5 U/ml recombinant interleukin-2 (rIL-2) (Amersham, Aylesbury, UK) in a total volume of 20 ml. After 9 days T cell clones are transferred onto flat-bottomed 96-well plates (Costar, Cambridge, MA) with 1 mg/ml phytohemagglutinin (PHA, Wellcome, Dartford, UK), 5 U/ml rIL-2 and allogenic irradiated (30 Gy) PBMC (2 x 10⁵) per well as feeder cells. Growing clones are further expanded in 24-well plates with PHA / rIL-2 and 1 x 10⁶ allogenic, irradiated PBMC as feeder cells and screened for polypeptide specificity after 4 to 7 days.

T cell clones are selected for further characterisation. The cell-surface phenotype of the T cell clone is determined to ascertain if the T cell clone is CD4+ or CD8+. T cell clone is incubated with autologous tumour cell targets at different effector to target ratios to determine if lysis of tumour cells occurs. Lysis indicates that the T cell has reactivity directed against a tumour derived antigen, for example, hTERT -deletion and/or hTERT -deletion proteins. Correlation between polypeptides and in vivo hTERT deletion fragments:

In order to verify that the antigen recognised is associated with hTERT -deletion protein or hTERT -deletion protein, and to identify the HLA class I or class II molecule presenting the putative hTERT -deletion or hTERT -deletion polypeptide to the T cell clone, different hTERT -deletion and/or hTERT -deletion expressing tumour cell lines carrying one or more HLA class I or II molecules in common with those of the patient, are used as target cells in cytotoxicity assays. Target cells are labelled with ⁵¹Cr or ³H-thymidine (9.25 x 10⁴ Bq/mL) overnight, washed once and plated at 5000 cells per well in 96 well plates. T cells are added at different effector to target ratios and the plates are incubated for 4 hours at 37°C and then harvested before counting in a liquid scintillation counter (Packard Topcount). For example, the bladder carcinoma cell line T24 (12Val⁺, HLA-A1⁺, B35⁺), the melanoma cell line FMEX (12Val⁺, HLA-A2⁺, B35⁺) and the colon carcinoma cell line SW 480 (12Val⁺, HLA-A2⁺, B8⁺) or any other telomerase positive tumour cell line may be used as target cells. A suitable cell line which does not express hTERT -deletion and/or hTERT -deletion proteins may be used as a control, and should not be lysed. Lysis of a particular cell line indicates that the T cell clone being tested recognises an endogenously-processed hTERT -deletion and/or hTERT - deletion epitope in the context of the HLA class I or class II subtype expressed by that cell line.

Characterisation of T cell clones:

The HLA class I or class II restriction of a T cell clone may be determined by blocking experiments. Monoclonal antibodies against HLA class I antigens, for example the panreactive HLA class I monoclonal antibody W6/32, or against class II antigens, for example, monoclonals directed against HLA class II DR, DQ and DP antigens (B8/11, SPV-L3 and B7/21), may be used. The T cell clone activity against the autologous tumour cell line is evaluated using monoclonal antibodies directed against HLA class I and class II molecules at a final concentration of 10 mg/ml. Assays are set up as described above in triplicate in 96 well plates and the target cells are preincubated for 30 minutes at 37°C before addition of T cells.

The fine specificity of a T cell clone may be determined using polypeptide pulsing experiments. To identify the hTERT -deletion and/or hTERT -deletion polypeptide actually being recognised by a T cell clone, a panel of nonamer polypeptides is tested. ⁵¹Cr or ³H-thymidine labelled, mild acid eluted autologous fibroblasts are plated at 2500 cells per well in 96 well plates and pulsed with the polypeptides at a concentration of 1 mM together with b2-microglobulin (2.5 mg/mL) in a 5% CO incubator at 37°C before addition of the T cells. Assays are set up in triplicate in 96 well plates and incubated for 4 hours with an effector to target ratio of 5 to 1. Controls can include T cell clone cultured alone, with APC in the absence of polypeptides or with an irrelevant melanoma associated polypeptide MART-1/Melan-A polypeptide.

An alternative protocol to determine the fine specificity of a T cell clone may also be used. In this alternative protocol, the TAP deficient T2 cell line is used as antigen presenting target cells. This cell line expresses only small amounts of HLA-A2 antigen, but increased levels of HLA class I antigens at the cell surface can be induced by addition of b2-microglobulin. ³H-labelled target cells are incubated with the different test polypeptides and control polypeptides at a concentration of 1 mM together with b2-microglobulin (2.5 mg/mL) for one hour at 37°C. After polypeptide pulsing, the target cells are washed extensively, counted and plated at 2500 cells per well in 96 well plates before addition of the T cells. The plates are incubated for 4 hours at 37°C in 5% CO before harvesting. Controls include T cell clone cultured alone or with target cells in the absence of polypeptides. Assays were set up in triplicate in 96 well plates with an effector to target ratio of 20 to 1.

The sensitivity of a T cell clone to a particular polypeptide identified above may also be determined using a dose-response experiment. Polypeptide sensitised fibroblasts can be used as target cells. The target cells are pulsed with the particular peptide as described above for fine specificity determination, with the exception that the peptides are added at different concentrations before the addition of T cells. Controls include target cells alone and target cells pulsed with the irrelevant melanoma associated peptide Melan- A/Mart- 1. Terminology

For the avoidance of doubt, certain terminology used herein is discussed below. Similar terminology should be construed accordingly, unless the context indicates otherwise.

"Polypeptide "

This is used in a broad sense to indicate that a particular molecule comprises a plurality of amino acids joined together by peptide bonds and/or disulphide bridges. It therefore includes within its scope molecules that may sometimes be referred to as peptides, polypeptides or proteins.

Polypeptides may be provided in any appropriate form. They may be linear or non-linear. For example, the ends of a linear structure may be may be joined together to provide a structure that is sometimes referred to as a "cyclic" or "endless" structure. Such a structure may sometimes have increased stability and/or increased immunogenicity relative to a linear structure. Various methods for producing cyclic polypeptides are disclosed in WO98/54577, for example. Sequences provided herein in respect of the present invention should be construed as including both linear and non-linear forms.

"Substantially Pure Form " and "Isolated Form "

The term "substantially pure form" is used to indicate that a given component is present at a high' level. The component is desirably the predominant component present in a composition. Preferably it is present at a level of more than 30%, of more than 50%, of more than 75%, of more than 90%, or even of more than 95%, said level being determined on a dry weight / dry weight basis with respect to the total composition under consideration. At very high levels (e.g. at levels of more than 90 %, of more than 95% or of more than 99%) the component may be regarded as being in "isolated form". Biologically active substances of the present invention (including polypeptides, nucleic acid molecules, binding agents, moieties identified/identifiable via screening, etc.) may be provided in a form that is substantially free of one or more contaminants with which the substance might otherwise be associated. Thus for example they may be substantially free of one or more potentially contaminating polypeptides and/or nucleic acid molecules. They may be provided in a form that is substantially free of other cell components (e.g. of cell membranes, of cytoplasm, etc.). When a composition is substantially free of a given contaminant, the contaminant will be at a low level (e.g. at a level of less than 10%, less than 5%, or less than 1% on the dry weight/dry weight basis set out above)

"Sequence Identity"

Sequence identity is the preferred manner of analysing sequence comparisons for the present invention. The percentage sequence identity between two amino acid or nucleotide sequences can be determined in a number of ways. For example:

1) It can be determined in a simple manner by aligning a given sequence with a reference sequence without allowing for gaps in a manner so that the maximum number of amino acids* or nucleotides match up with each other. The percentage sequence identity (S) can then be calculated by using the equation: S = 100 X (M/N); where M is the number of nucleotides or amino acids in the given sequence that are identical with nucleotides or amino acids at the corresponding positions in the reference sequence and N is the total number of amino acids or nucleotides in the reference sequence.

If gaps are allowed then gap penalties may be incurred. For example, gaps may be penalised in a simple manner simply by considering the gaps to represent amino acid or nucleotide mismatches over the full length of any gaps present. Two gaps of 5 and 10 amino acids would therefore be considered to represent mismatches totalling 15 amino acids and the proportion of matches would be reduced (relative to a system in which gaps were not penalised). More sophisticated systems for penalising gaps are also known and may involve separate penalties for gap lengths and for the numbers of gaps present.

2) It can be determined by using various algorithms, which may be incorporated in computer programs. (If desired, the default parameters of these programs can be used.)

For example, the algorithm of Karlin and Altschul (1990 Proc. Natl. Acad. Sci. USA 87: 2264-68), or a modified version thereof can be used (see e.g. Karlin and Altschul 1993 Proc. Natl. Acad. Sci. USA 90:5873-77). This algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990 J. Mol. Biol. 215:403-10.) Nucleotide searches can be performed with the NBLAST program (e.g. score = 100, wordlength = 11). BLAST protein searches can be performed with the XBLAST program (e.g. score = 50, word length = 3). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilised as described in Altschul et al., 1997 Nucleic Acids Research 25(17):3389-3402. When utilising BLAST and gapped BLAST programs it is preferred that the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used (see e.g. http://www.ncbi.nlm.nih.gov/BLAST)

Alternatively, the FASTA program can also be used. This is based upon a modified version of the Wilbur and Lipman algorithm (see e.g. http://www2.ebi.ac.uk/fasta 3).

Another algorithim that can be used is that of Myers and Miller, (CABIOS, 4:11-17 (1989)). This algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package (see e.g. http://www2.igh.cnrs.fr/bin/align-guess.cgi). When using the ALIGN program for comparing sequences, a PAM 120 weight residue table, a ^• gap length penalty of 12, and a gap penalty of 4 can be used, for example.

For the purposes of the present invention, it is preferred that sequence identity is determined by using Gapped BLAST (version 2.0), using the default parameters provided.

Where high degrees of sequence identity are present there may be relatively few sequence differences. Thus, for example, there may be less than 20, less than 10, or less than 5 differences.

"Hybridising Molecule "

This refers to a nucleic acid strand that is capable of at least partially base-pairing with another strand to form a structure that is at least partially double stranded. Preferred hybridising molecules hybridise under conditions of moderate or high stringency. Hybridisation conditions are discussed in detail at pp 1.101 -1.110 and 11.45 - 11.61 of Sambrook et al [Molecular Cloning, 2nd Edition, Cold Spring Harbor Laboratory Press (1989)].

One example of hybridisation conditions that can be used for the present invention involves using a pre-washing solution of 5 X SSC, 0.5% SDS, l.OmM EDTA (pH 8.0) and attempting hybridisation overnight at 55°C using 5 X SSC. However, there are many other possibilities. Some of these are listed in Table 1 of WO98/45435, for example. (See especially the conditions set out under A-F of that table and, less preferably, those listed under G to L or M to R.) Another approach is to determine the Tm for a given perfect duplex (i.e. with no mismatches) of a certain length under given conditions and then to perform attempted hybridisation with a single strand of the duplex under those conditions, but at a temperature sufficiently below the Tm to allow for formation of a range of stable hybrids at an acceptable rate, whilst still requiring a reasonable degree of hybridisation specificity. The Tm for such a duplex can be determined empirically by providing the duplex and gradually increasing the temperature until the Tm is achieved. The Tm can also be estimated e.g. by using:

Tm = 81.5 + 16.6 (log₁₀ [Na+] ) + 0.41(fraction G + C) - (600/N), where N is the chain length.

(This formula is reasonably accurate for Na+ concentrations of IM or less and for polynucleotide lengths of 14 to 70, but is less accurate when these parameters are not satisfied.)

For nucleic acid molecules of greater than 200 nucleotides in length, hybridisation may, for example, be carried out at 15 to 25°C below the Tm of a perfect hybrid (i.e. with no mismatches) under given conditions. However, as the length is decreased the Tm is lowered, so that it is sometimes inconvenient to carry out hybridisation at 25 °C below the Tm . Hybridisation with shorter nucleic acid molecules is therefore often carried out at only 5 to 10°C below the Tm.

Moderate or high stringency conditions will usually only allow a small proportion of mismatches. As a rule of thumb, for every 1% of mismatches there is a reduction of Tm by 1- 1.5°C. Preferably hybridisation conditions are chosen to allow less than 25% mismatches, more preferably to allow less than 10% or less than 5% mismatches.

Hybridisation can be followed by washes of increasing stringency. Thus initial washes may be under conditions of low stringency, but these can be followed with higher stringency washes, up to the stringency of the conditions under which hybridisation was performed.

The foregoing discussions of hybridisation conditions are provided for general guidance but are not intended to be limiting. This is because a skilled person will be able to vary parameters as appropriate in order to provide suitable hybridisation conditions, and can take into account variables such as polynucleotide length, base composition, nature of duplex (i.e. DNA/DNA, RNA/RNA or DNA/RNA), type of ion present, etc.

"Treatment"

This includes any therapeutic applications that can benefit a human or non-human animal. The treatment of mammals is particularly preferred. Thus both human and veterinary treatments are within the scope of the present invention. Non-human animals include farm animals, pets, wild animals, animals bred for racing or hunting, zoo and aquarium animals, etc. Particular non-human animals include rodents, sheep, goats, horses, cows, pigs, cats, dogs, reptiles, fish, amphibians, birds, monkeys and non-human apes.

Treatment may be in respect of an existing condition or it may be prophylactic. It may be of an adult, a juvenile, an infant, a foetus, a cell, tissue, or organ, or a part of any of the aforesaid (e.g. a nucleic acid molecule).

Where treatments are discussed herein, it will be appreciated that pharmaceutical compositions comprising the active agent can be provided and are within the scope of the present invention. The active agent or composition may be administered via any appropriate route of administration and at any appropriate dosage.

Disorders to be treated may be genetic in origin. Thus they may arise due to one or more mutations that result in a deleterious effect, e.g. mutations in genes or in other regions. Mutations may result in excessive, insufficient, or otherwise aberrant expression or activity of a gene product. Disorders to be treated may also/alternatively arise due to environmental factors.

Treatments may be by via any appropriate techniques. For example, gene therapy techniques (including antisense therapy) may be used. Gene therapy techniques include introducing nucleic acid into a patient by any appropriate means. The nucleic acid may be included in a cell or vector (e.g. a retroviral or non-retroviral vector), although this is not essential. It may be used to combine with nucleic acid in a host (e.g. via homologous or non-homologous recombination) or may remain separate from the host nucleic acid (e.g. as an episome). Gene therapy techniques include decreasing the expression or activity of deleterious gene products (e.g. by "knocking out" the relevant genes / transcription products). They also include increasing the activity or expression of beneficial gene products (e.g. by modifying existing genes or by inserting additional genes). Gene therapy techniques are disclosed, for example, in US Patent 5399346, in WO93/09222, in US Patent 5371015, etc.

Non gene therapy techniques may also be used and may sometimes be preferable to gene therapy techniques. They include administering pharmaceutical compositions via various routes, as will be described later.

"Pharmaceutical Composition "

This is a composition that comprises or consists of a pharmaceutically active agent. It preferably includes a pharmaceutically acceptable carrier. This pharmaceutical composition will desirably be provided in a sterile form. It may be provided in unit dosage form, will generally be provided in a sealed container. A plurality of unit dosage forms may be provided.

Pharmaceutical compositions within the scope of the present invention may include one or more of the following: preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts (polypeptides of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents, antioxidants, adjuvants, excipients and diluents. They may also contain other therapeutically active agents in addition to polypeptides of the present invention. Where two or more therapeutic agents are used they may be administered separately (e.g. at different times and/or via different routes) and therefore do not always need to be present in a single composition. Thus combination therapy is within the scope of the present invention.

Therapeutically active agents may be provided in any suitable form - i.e. they may be used as such or may be used in the form of a pharmaceutically effective derivative. For example they may be used in the form of a pharmaceutically acceptable salt or hydrate. Pharmaceutically acceptable salts include alkali metal salts (e.g. sodium or potassium salts), alkaline earth metal salts (e.g. calcium or magnesium salts) aluminium salts, zinc salts, ammonium salts (e.g. tetra-alkyl ammonium salts), etc. Inorganic acid addition salts (e.g. hydrochlorides, sulphates, or phosphates) or organic acid addition salts (e.g. citrates, maleates, fumarates, succinates, lactates, propionates or tartrates) may be used. Pharmaceutical compositions of the present invention may be provided in controlled release form. This can be achieved by providing a pharmaceutically active agent in association with a substance that degrades under physiological conditions in a predetermined manner. Degradation may be enzymatic or may be pH-dependent.

Pharmaceutical compositions may be deigned to pass across the blood brain barrier (BBB). For example, a carrier such as a fatty acid, inositol or cholesterol may be selected that is able to penetrate the BBB. The carrier may be a substance that enters the brain through a specific transport system in brain endothelial cells, such as insulin-like growth factor I or II. The carrier may be coupled to the active agent or may contain/be in admixture with the active agent. Liposomes can be used to cross the BBB. WO91/04014 describes a liposome delivery system in which an active agent can be encapsulated/embedded and in which molecules that are normally transported across the BBB (e.g. insulin or insulin-like growth factor I or II) are present on the liposome outer surface. Liposome delivery systems are also discussed in US Patent No. 4704355.

"Route of Administration "

A pharmaceutical composition within the scope of the present invention may be adapted for administration by any appropriate route. Such a composition may be prepared by any method known in the art of pharmacy, for example by admixing one or more active ingredients with a suitable carrier.

Different drug delivery systems can be used to administer pharmaceutical compositions of the present invention, depending upon the desired route of administration. Drug delivery systems are described, for example, by Langer (Science 249:1527 - 1533 (1991)) and by Ilium and Davis (Current Opinions in Biotechnology 2: 254 - 259 (1991)). Different routes of administration for drug delivery will now be considered in greater detail:

(i) Transdermal Administration

Pharmaceutical compositions adapted for transdermal administration may be provided as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis. (Iontophoresis is described in Pharmaceutical Research, 3(6):318 (1986).)

(ii) Topical Administration

Pharmaceutical compositions adapted for topical administration may be provided as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For topical administration to the skin, mouth, eye or other external tissues a topical ointment or cream is preferably used. When formulated in an ointment, the active ingredient may_. be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops. Here the active ingredient can be dissolved or. suspended in a suitable carrier, e.g. in an aqueous solvent. Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouthwashes.

(iii) Rectal Administration

Pharmaceutical compositions adapted for rectal administration may be provided as suppositories or enemas.

(iv) Nasal Administration

Pharmaceutical compositions adapted for nasal administration may use solid carriers , e.g. powders (preferably having a particle size in the range of 20 to 500 microns). Powders can be administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nose from a container of powder held close to the nose. Compositions adopted for nasal administration may alternatively use liquid carriers, e.g. nasal sprays or nasal drops. These may comprise aqueous or oil solutions of the active ingredient.

Compositions for administration by inhalation may be supplied in specially adapted devices - e.g. in pressurised aerosols, nebulizers or insufflators. These devices can be constructed so as to provide predetermined dosages of the active ingredient. (v) Vaginal Administration

Pharmaceutical compositions adapted for vaginal administration may be provided as pessaries, tampons, creams, gels, pastes, foams or spray formulations.

(vi) Par enter al Administration

Pharmaceutical compositions adapted for parenteral administration include aqueous and non- aqueous sterile injectable solutions or suspensions. These may contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially isotonic with the blood of an intended recipient. Other components that may be present in such compositions include water, alcohols, polyols, glycerine and vegetable oils, for example. Compositions adapted for parenteral administration may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of a sterile liquid carrier, e.g. sterile water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

"Binding agents "

Various binding agents can be used in the present invention.

One type of binding agent is an antibody. Antibodies within the scope of the present invention may be monoclonal or polyclonal.

Polyclonal antibodies can be raised by stimulating their production in a suitable animal host (e.g. a mouse, rat, guinea pig, rabbit, sheep, goat or monkey) when a polypeptide of the present invention is injected into the animal. If desired, an adjuvant may be administered together with a polypeptide of the present invention. Well-known adjuvants include Freund's adjuvant (complete or incomplete) and aluminium hydroxide. The antibodies can then be purified by virtue of their binding to a polypeptide of the present invention.

Monoclonal antibodies can be produced from hybridomas. These can be formed by fusing together myeloma cells and spleen cells that produce the desired antibody in order to form an immortal cell line. Thus the well-known Kohler & Milstein technique (Nature 256 (1975)) or subsequent variations upon this technique can be used.

Techniques for producing monoclonal and polyclonal antibodies that bind to a particular polypeptide are now well developed in the art. They are discussed in standard immunology textbooks - e.g. in Roitt, I.M. et al. (1998, Immunology, 5 Edition, Mosby International Ltd). Antibodies can be purified by adsorption to staphlylococcal protein A .The staphlylococcal protein will usually be coupled to a solid support, such as Sepharose beads. This can be done via cyanogen bromide coupling. Antibodies bind to protein A chiefly by hydrophobic interactions, which can be disrupted when desired so as to elute the antibodies (e.g. via transient exposure to low pH).

More recently, techniques such as 'phage display have been used to express antibodies. These techniques are becoming increasingly popular and are described for example by M J Geisow in Tibtech 10, 75 - 76 and by D. Chiswell et al in Tibtech JO, 8 - 84, (1992). They can be used to express antibodies recognising desired epitopes.

The above discussion focuses on whole antibodies. However the present invention includes other moieties that are capable of binding to polypeptides of the present invention. Thus the present invention includes antibody fragments and synthetic constructs. Examples of antibody fragments and synthetic constructs are given by Dougall et al in Tibtech 12,372-379 (September 1994).

Antibody fragments include, for example, Fab, F(ab') and Fv fragments. (These are discussed in Roitt et al, supra?) Fv fragments can be modified to produce a synthetic construct known as a single chain Fv (scFv) molecule. This includes a peptide linker covalently joining V_h and Vi regions, which contributes to the stability of the molecule. Otlier synthetic constructs that can be used include CDR peptides. These are synthetic peptides comprising antigen-binding determinants. Peptide mimetics may also be used. These molecules are usually conformationally restricted organic rings that mimic the structure of a CDR loop and that include antigen-interactive side chains.

Synthetic constructs include chimeric molecules. Thus, for example, humanised (or primatised) antibodies are within the scope of the present invention. An example of a humanised antibody is an antibody having human framework regions, but rodent hypervariable regions. Ways of producing chimeric antibodies are discussed for example by Morrison et al in PNAS, 81_? 6851-6855 (1984), by Takeda et al in Nature. 314, 452-454 (1985) and by Cunningham et al in Tibtech 10, 112-113 (1992).

Synthetic constructs also include molecules comprising an additional moiety that provides the molecule with some desirable property in addition to antigen binding. For example the moiety may be a label (e.g. a fluorescent or radioactive label). Alternatively, it may be a pharmaceutically active agent.

A further type of binding agent that can be used in the present invention is a lectin. Lectins are carbohydrate binding proteins of non-immune (e.g. plant) origin (see e.g. the discussion of lectins by Deutscher in Methods in Enzymology, Guide to Protein Purification, 182 (1990).) Different lectins can be used to select particular glycoproteins based upon the presence of particular carbohydrate moieties (e.g. sialic acid, galactose, mannose, fucose, N-acetyl glucosamine, N-acetyl galactosamine, etc) . In some cases a plurality of different lectins may be used - e.g. if a glycoprotein is known to include three different sugars, then three different lectins may be used to purify it. They may be used sequentially (e.g. in sequential affinity columns).

A particularly preferred binding agent is an HLA molecule (including an HLA tetramer, other HLA oligomer, or a part thereof).

From the above discussion it will be appreciated that many different types of binding agent can be used in the present invention. Preferred binding agents are specific for polypeptides of the present invention.

Claims

1. A polypeptide a polypeptide for use in medicine; wherein the polypeptide is capable of inducing a T cell response and:

a) comprises a sequence given in SEQ ID NO: 1, 2 or 3;

b) comprises at least 8 contiguous amino acids from SEQ ID NO: 1, 2 or 3, with the provisos that if said at least 8 contiguous amino acids are from SEQ ID NO: 1 they include KD (Lys- Asp) and that if they are from SEQ ID NO 3 at least one (preferably at least two) of said amino acids is also from SEQ ID NO: 2; or

c) comprises at least 8 contiguous amino acids that have only one, two or three amino acid changes relative to b) above, with the provisos that that either at least KD (Lys-Asp) from SEQ ID NO 1 are present, or at least one (preferably at least 2) of the 8 contiguous amino acids from SEQ ID NO: 2 is present .

2. A polypeptide for use in medicine according to claim 1 ; wherein the polypeptide has at least 40%) sequence identity with a molecule as shown in SEQ ID NO 1,2 or 3.

3. A polypeptide for use in medicine according to claim 1 or claim 2; wherein the polypeptide comprises a sequence as given in SEQ ID NO: 1 , 2, 3, 4, 5 or 6 or is a fragment of a sequence as shown in SEQ ID NO: 1, 2 or 3.

4. A polypeptide for use in medicine according to any preceding claim; wherein the T cell response is generated after intracellular cleavage of the polypeptide to provide a fragment that fits into an MHC or HLA binding groove.

5. A polypeptide for use in medicine according to any of claims 1 to 4; wherein the polypeptide does not need intracellular cleavage to fit into an MHC or HLA class I binding groove.

6. A polypeptide for use in medicine according to claim 5; wherein the polypeptide is from 8 to 10 amino acids long.

7. A polypeptide for use in medicine according to any of claims 1 to 4; wherein the polypeptide does not need intracellular cleavage to fit into an MHC or HLA class II binding groove.

8. A polypeptide for use in medicine according to claim 7; wherein the polypeptide is from 12 to 25 amino acids long.

9. A polypeptide for use in medicine according to any preceding claim; wherein the T cell response increases the number and/or activity of T helper and/or T cytotoxic cells.

10. A polypeptide for use in medicine according to any preceding claim; wherein the polypeptide does not stimulate a substantial cytotoxic T cell response in a patient against one or more of the following: bone marrow stem cells, epithelial cells in colonic crypts or lymphocytes.

11. A nucleic acid molecule for use in medicine; wherein the nucleic acid molecule:

a) has a strand that encodes a polypeptide as described in any of claims 1 to 10;

12. A vector or cell for use in medicine comprising a nucleic acid molecule according to claim 11.

13. A binding agent for use in medicine; wherein the binding agent binds to a polypeptide as described in any of claims 1 to 10.

14. A binding agent for use in medicine according to claim 13; wherein the binding agent is specific for a polypeptide as described in any of claims 1 to 10.

15. A binding agent for use in medicine according to claim 13 or claim 14 that comprises an HLA molecule or a part thereof, said molecule or part being optionally in the form of a multimer (e.g. a tetramer)

16. A T lymphocyte for use in medicine; wherein the T lymphocyte is capable of killing a cell expressing a polypeptide according to any of claims 1 to 10 or of helping in the killing of such a cell.

17. T lymphocyte for use in medicine according to claim 16; wherein the T lymphocyte is a T cytotoxic cell or a T helper cell.

18. A clonal cell line for use in medicine comprising a plurality of T lymphocytes according to claim 16 or claim 17.

19. A mixture of T lymphocytes for use in medicine comprising:

a) a T helper cell as described in claim 17 or a clonal cell line of T helper cells as described in claim 18; and

b) a T cytotoxic cell as described in claim 17 or a clonal cell line of T cytotoxic cells as described in claim 18.

20. The use of a polypeptide as described in any of claims 1 to 10, a nucleic acid as described in claim 11 , a vector or cell as described in claim 12, a binding agent as described in any of claims 13 to 15, a T lymphocyte as described in claim 16 or 17, a cell line as described in claim 18, or a mixture of T lymphocytes as described in claim 19, in the preparation of a medicament for treating cancer, or in the preparation of a diagnostic for diagnosing cancer.

21. The use according to claim 20; wherein the cancer is mammalian cancer.

22. The use according to claim 21; wherein the cancer is human cancer.

23. The use according to any of claims 20 to 22; wherein the cancer is breast cancer, prostate cancer, pancreatic cancer, colo-rectal cancer, lung cancer, malignant melanoma, leukaemia, lymphoma, ovarian cancer, cervical cancer or a biliary tract carcinoma.

24. The use according to any of claims 20 to 23; wherein the medicament is a vaccine.

25. The use according to any of claims 20 to 23; wherein the medicament is an antisense molecule or is capable of generating an antisense molecule in vivo.

26. The use according to any of claims 20 to 23 ; wherein the diagnostic is provided in a kit.

27. The use according to claim 26; wherein the kit comprises means for generating a detectable signal (e.g. a fluorescent label, a radioactive label) or a detectable change (e.g. an enzyme-catalysed change).

28. The use according to claim 26 or claim 27; wherein the kit includes instructions for use in diagnosing cancer.

29. A pharmaceutical composition comprising a polypeptide as described in any of claims 1 to 10, a nucleic acid as described in claim 11, a vector or cell as described in claim 12, a binding agent as described in any of claims 13 to 15, a T lymphocyte as described in claim 16 or 17, a cell line as described in claim 18, or a mixture of T lymphocytes as described in claim 19.

30. A pharmaceutical composition according to claim 29 further comprising a polypeptide capable of inducing a T cell response directed against a polypeptide produced by an oncogene or against a mutant tumour suppressor protein, or a nucleic acid encoding such a polypeptide, or a binding agent that binds such a polypeptide, or a T cell that is capable of killing a cell expressing such a polypeptide or of helping in the killing of such a cell.

31. A pharmaceutical composition according to claim 30; wherein said oncogene or mutant tumour suppressor protein is p21-r s', Rb, p53, abl, gip, gsp, ret or trk.

32. A combined preparation comprising a component from claim 29 and a component from claim 30 or from claim 31, for simultaneous, separate or sequential use in anticancer therapy.

33. A pharmaceutical composition according to any of claims 29 to 31, or a combined preparation according to claim 32, comprising a pharmaceutically acceptable carrier, diluent, additive, stabiliser, and/or adjuvant; said composition or combined preparation optionally further including one or more of: a cytokine or growth factor (e.g. IL-2, IL-12, and/or GM-CSF) and another polypeptide arising from a frameshift mutation (e.g. a frameshift mutation in the BAX or lϊTGFβ-RII gene.)

34. A pharmaceutical composition according to any of claims 29 to 31 or 33, or a combined preparation according to claim 32 or 33, which is a vaccine.

35. A pharmaceutical composition according to any of claims 29 to 31 or 33, or a combined preparation according to claim 32 or 33, which comprises or is capable of producing antisense molecules.

36. A diagnostic composition comprising a polypeptide as described in any of claims 1 to 10, a nucleic acid as described in claim 11, a vector or cell as described in claim 12, a binding agent as described in any of claims 13 to 15, a T lymphocyte as described in claim 16 or 17, a cell line as described in claim 18, or a mixture of T lymphocytes as described in claim 19.

37. A diagnostic kit as described in any of claims 26 to 28.

38. A polypeptide as described in any of claims 1 to 10, optionally in isolated form:

39. A binding agent as described in any of claims 13 to 15, optionally in isolated form.

40. A T lymphocyte as described in claim 16 or 17, optionally in isolated form.

41. A clonal cell line as described in claim 18, optionally in isolated form.

42. A mixture of T lymphocytes as described in claim 19.

43. The invention as substantially hereinbefore described with reference to the accompanying figures and examples.