WO2002070679A2

WO2002070679A2 - Peptide fragments, derived from human telomerase reverse transcriptase

Info

Publication number: WO2002070679A2
Application number: PCT/NO2002/000069
Authority: WO
Inventors: Jon Amund Eriksen; Gustav Gaudernack; Mona Møller
Original assignee: Gemvax AS
Current assignee: Gemvax AS
Priority date: 2001-03-02
Filing date: 2002-02-19
Publication date: 2002-09-12
Anticipated expiration: 2003-09-02
Also published as: GB0105238D0; WO2002070679A3; AU2002233839A1

Abstract

The polypeptide R E E I L A K F L H W L S V Y V V E L and variants/fragments thereof are particularly useful in producing anticancer vaccine for populations of individuals having varying HLA profiles. This polypeptide comprises a surprisingly large number of HLA class I and HLA class II epitopes in overlapping or nested form.

Description

Vaccines

The present invention relates, inter alia, to vaccines, especially to anti-cancer vaccines.

Cancer develops through a multistep 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 that 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. The prior art suggests that such antibodies can be used 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, the effort to produce a cancer treatment based on the immune system of the body has been less successful than anticipated.

A fundamental feature of the immune system is that it does not normally react against self molecules and can distinguish self from non-self molecules and that it does not normally react against self molecules. 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 ahd secreted by B lymphocytes, and typically recognise free antigen in native conformation. They can therefore potentially recognise almost any site exposed on an 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. This antigen processing usually comprises proteolytic fragmentation of the protein, resulting in polypeptides that fit into a groove of the MHC molecules. This enables T cells to recognise polypeptides derived from intracellular protein fragments/antigens.

T cells can recognise aberrant polypeptides derived from any part of 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: 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. Every individual normally has 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 is reduced due to the occurrence of the same HLA allele twice. Roitt, I.M. et al. (1998, Immunology, 5^th Edition, Mosby International Ltd), provides a good general review of immunology, including the HLA systems.

HLA gene products are highly polymorphic. Different individuals express distinct HLA molecules that differ from those found in other individuals. This explains the difficulty of finding HLA matched organ donors for transplantation. 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 CD 8+ 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/NO92/00032 (published as WO92/14756), synthetic polypeptides and fragments of oncogene protein products derivable from point mutations or translocations in the corresponding proto-oncogene or tumour suppressor gene are described. These polypeptides correspond to, completely cover, or are fragments of the processed oncogene protein fragment or tumour suppressor gene fragment as presented by cancer cells or other antigen presenting cells, and are presented as a HLA-polypeptide complex by at least one allele in every individual. The polypeptides were shown to induce specific T cell responses to the actual oncogene protein fragment produced by the cell by processing and presented in the HLA molecule. In particular, it is described in WO92/14756 that polypeptides derived from the -pll-ras protein which had point mutations at particular amino acid positions, namely 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 through the administration of such polypeptides in vaccination or cancer therapy schemes. It has subsequently been shown that these polypeptides also elicit CD 8+ T cell immunity against cancer cells harbouring the mutated p21 ras oncogene protein through the administration mentioned above (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. These polypeptides correspond to, completely cover, or are fragments of the processed frameshift mutant protein fragment as presented by cancer cells or other antigen presenting cells, and are presented as a HLA-polypeptide complex by at least one allele in every individual. In particular polypeptides resulting from frameshift mutations in the BAX and hTGFbeta-RII genes are disclosed. These polypeptides were shown to be effective in stimulating CD4+ and CD 8+ T cells in a specific manner.

However, the polypeptides described above will be useful only in certain numbers of cancers involving oncogenes with point mutations, frameshift mutations or translocation in a protooncogene or tumour suppressor gene. There is a strong need for an anticancer treatment or vaccine that will be effective against a generic range of cancers.

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. Additional 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.

The human telomerase catalytic subunit is referred to hereafter as "telomerase" or "hTERT". It was identified as hTRT by Nakamura et al (1997, Science 277, 955-959) and hEST2 by

Meyerson et al (1997, Cell, Vol 90, 785-795), the cDNA sequences of which are deposited as GenBank accession numbers AF015950 and AF018167 respectively.

Due to the presence of telomerase in most types of cancer cells, the enzyme has been disclosed as a general cancer vaccine candidate (international patent application PCT/NO99/00220, published as O00/02581). 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.

According to one aspect of the present invention there is provided a polypeptide that:

a) consists of the sequence: R E E I L A K F L H W L M S V Y V N E L; b) is a fragment of the sequence set out in a) above that comprises at least two HLA epitopes and is at least 10 amino acids long;

c) comprises the sequence set out in a) above, as well as an additional C-terminal and/or N- terminal sequence, with the proviso that the polypeptide is not full length hTERT and does not include a contiguous sequence of hTERT that is more than 100 amino acids long; or

d) is a variant of a polypeptide as described in any of a) to c) above having one or more amino acid changes thereto, with the proviso that the variant retains the number and specificity of epitopes present in said polypeptide.

The sequence REEILAKFLHWLMSVYVNEL corresponds to the sequence from position 537 to 556 of telomerase. It includes a surprisingly large number of HLA epitopes, some of which are detailed in Example 1. This is useful in the preparation of vaccines suitable for large populations of individuals having different HLA profiles.

Polypeptides within the scope of each of a), b), c) and d) will now be discussed in further detail:

Polypeptides within the scope of a)

The most preferred polypeptide of the present invention consists of the polypeptide having the sequence RE E ILAKFLHWLMSVYVVEL.

For a relatively small polypeptide a very high number of HLA epitopes is present. This is possible because many of the epitopes are in overlapping or nested form, as is clear from Example 1.

Polypeptides within the scope ofb)

The present invention includes fragments of the polypeptide described in a) above.

These fragments comprise a plurality of T cell epitopes and are also particularly useful in vaccines for populations of individuals. Preferred polypeptide fragments include overlapping or nested epitopes. Examples of these are given in Example 1. Most preferred epitopes are two or more of those listed in Example 1.

A fragment of the present invention will generally be a polypeptide that is at least 10 amino acids long. Preferably it is at least 15, or at least 20 amino acids long. Desirably, however, it is no more than 30, 40 or 50 amino acids long (e.g. no more than 25 amino acids long). Most desirably, it is 10 to 30 amino acids long (e.g.12 to 25 amino acids long).

Advantageously, it will include at least one HLA class I epitope and at least one HLA class II epitope.

For example, it may include an HLA-A, HLA-B, and/or HLA-C epitope as well as an HLA-DR , HLA-DP, and/or HLA-DQ epitope. It may include a plurality of such epitopes in any desired combination.

It is desired to maximise the number of different class I and/or class II epitopes present so that the polypeptide can be administered to large populations. Thus the polypeptide will usually include a plurality of overlapping or nested epitopes within its structure.

Advantageously, at least 5, 10, 15 or 20 different HLA class I and/or class II epitopes are present. A plurality of epitopes within one or more given subtypes may advantageously be present.

Preferably the polypeptide does not include a substantial number of non-epitopic regions. Most preferably, the peptide consists solely of epitopic regions (e.g. of overlapping or of nested epitopes).

Particularly preferred fragments include those consisting of any of the following three sequences: ILAKFLHWLMSVYVVEL, EILAKFLHWLMSVYVVEL, or EEILAKFLHWLMSVYVVEL.

It will be appreciated by the skilled person that, although WO00/02581 discloses various hTERT peptides comprising HLA epitopes, the vast majority of these fragments are not within the sequence REEILAKFLHWLMSVYVVEL. Furthermore, there is nothing in WO00/02581 to indicate that this sequence has any particular significance. There is also nothing in WO00/02581 to suggest providing a plurality of nested or overlapping epitopes.

Polypeptides within the scope ofc)

A polypeptide within the scope ofc) will have the sequence

R E E I L A K F L H W L M S V Y V V E L, together with an additional N-terminal and/or an additional C-terminal amino acid sequence.

As indicated supra, the full length hTERT sequence is known and is provided in Figure 1. This is not within the scope of the present invention. Furthermore, large fragments of that sequence (above 100 amino acids long) are also not within the scope of the present invention. Smaller fragments of hTERT that include the sequence R E E I L A K F L H W L M S V Y V V E L are however within the scope of the present invention. Prior to the present invention, there would have been be no particular reason to focus upon such fragments. Thus the additional N-terminal and/or C-terminal sequences need not be heterologous sequences. They may be hTERT sequences.

Additional N-terminal or C-terminal sequences may be provided for various reasons. Techniques for providing such additional sequences are well known in the art. These include using gene cloning techniques to ligate nucleic acid molecules encoding polypeptides or parts thereof and expressing a polypeptide encoded by the ligated sequences using an expression system.

Additional sequences may be provided in order to alter the characteristics of a particular polypeptide. This can be useful in improving expression or regulation of expression in particular expression systems. For example, an additional sequence may provide some 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 etal., 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. Different signal sequences can be used for different expression systems. Hydrophobic membrane spanning sequences may be provided to anchor a polypeptide in a membrane if they are not already present. This is useful if it is desired to provide the polypeptide on a surface (e.g. on the surface of a liposome). Alternatively, if membrane-spanning domains are present they can be deleted e.g. in order to allow secretion of the protein. Thus the present invention includes within its scope both soluble and membrane-bound polypeptides.

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 binding agents (e.g. immobilised antibodies or immobilised antibody fragments that bind to the epitope, desirably with a high degree of specificity). The resultant fusion protein can usually 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 IgG 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.

In some cases, it may be desired to provide a fusion protein comprising a polypeptide linked to binding agent (e.g. to an antibody or a part thereof) so that it can bind to a structure recognised by the binding agent (e.g. to a particular epitope). This can be useful not only in purification but also in targeting moieties to particular sites.

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 substance 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. Of course, if desired, non-essential sequences may be removed. This can be done by enzymatic techniques.

Alternatively, such sequences can be avoided, e.g. by using a nucleic acid encoding only essential sequences to express the polypeptide or by using chemical synthesis techniques to synthesise only essential sequences.

From the foregoing description, it will be appreciated that there are many different possible N- terminal and/or C-terminal sequences. However it is generally preferred that such sequences, if present, are relatively short (e.g. less than 25, less than 15, less than 10 or less than 5 amino acids long).

By providing a relatively short polypeptide, possible side effects or other disadvantages arising from the presence of additional sequences can be reduced. The total length of a polypeptide within the scope ofc) above is preferably no more than 50, 40, or 30 amino acids long.

Polypeptides within the scope ofd)

The present invention includes variants of polypeptides described in any of a) to c) above comparing one or more amino acid changes, with the proviso that the variants retain the number and specificity of epitopes present in said polypeptide.

In particular, it will be appreciated that amino acid changes (e.g. substitutions) to the polypeptide may be made to anchor residues of T cell epitopes which fit into HLA molecules for presentation to T cells, whilst still achieving binding of the polypeptide to HLA molecules (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). Example 2 describes a possible assay for HLA binding.

Whereas the polypeptides that are presented by HLA class II molecules are of varying length (12-25 amino acids), the polypeptides presented by HLA class I molecules will normally be nine amino acid residues long in order to fit into the class I HLA binding groove. A 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. (Longer polypeptides that can be processed in such a manner are, of course, within the scope of the present invention.) Only a limited number of deviations from the requirement of nine amino acids have been reported, and in those cases the length of the presented polypeptide has been either eight or ten amino acid residues long. Reviews on polypeptide binding to MHC molecules are provided by Rammensee et al. (1995, Immunogenetics, 41: 178-228), Barinaga (1992, Science 257: 880- 881) and by Male et al. (1996, Advanced Immunology, Mosby International Ltd.).

In addition to the foregoing considerations, a skilled person would be aware of general possibilities for amino acid substitutions, deletions and/or insertions. These are discussed below:

(i) Substitutions

Certain 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 the 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 (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since 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 a polypeptide is to be used in medicine, dosage levels can be reduced by deleting inessential regions. Reducing the length of the polypeptide may also reduce the risk of side effects and improve the chances of regulatory approval.

(Hi) Insertions

Amino acid insertions can also be made. This may be done to alter the nature of a polypeptide - e.g. to assist in identification, purification or expression.

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.

From the foregoing description it will be appreciated that a wide range of polypeptides are within the scope of the present invention, including not only the polypeptide R E E I L A K F L H W L M S V Y V V E L, but variants and fragments thereof. A polypeptide of the present invention may be produced by techniques known to those skilled in the art. For example, gene cloning techniques may be used for example to provide a nucleic acid sequence encoding a polypeptide of the present invention. The polypeptide may then be expressed and purified using standard expression and protein purification techniques. Techniques useful in gene-cloning, expression and protein purification are discussed in greater detail later on in the section entitled "Nucleic acid molecules".

Alternatively, chemical synthesis techniques may be used to produce polypeptides of the present invention. Such techniques generally utilise solid phase synthesis (see e.g. "Solid Phase Peptide ■ Synthesis" by Stewart et al, Chem. Press Co., 1984). Chemical synthesis techniques allowing polypeptides having particular sequences to be produced have now been automated. Apparatuses capable of chemically synthesising polypeptides are available from a number of companies, such as Applied Biosystems (e.g. the Applied Biosystems (ABI) 430A solid phase synthesiser). If desired, relatively short polypeptides can be synthesised initially and can then be ligated together to produce longer polypeptides.

Various uses of polypeptides of the present invention will now be described.

A) Medical uses

Polypeptides of the present invention may be used in medicine.

They are particularly useful in the preparation of vaccines.

Preferred vaccines include vaccines against cancer. The cancer may, for example, be a mammalian or human cancer.

Human cancers include breast cancer, prostate cancer, pancreatic cancer, colo-rectal cancer, lung cancer, malignant melanoma, leukaemias, lymphomas, ovarian cancer, cervical cancer and biliary tract carcinoma.

Desirably vaccines of the present invention are capable of substantially increasing the number and/or activity of T helper and/or T cytotoxic cells. Any increase can be advantageous, but increases of at least 5% (e.g. at least 10, 25, 50 or 100%) are preferred. Example 2 provides suitable assay procedures, although other procedures may be used.

Thus a polypeptide of the present invention may be used in the manufacture of a vaccine or other medicament. The vaccine may comprise an adjuvant. The adjuvant may be a cytokine (e.g. TNF alpha, IL-2, IL-12, and/or GM-CSF.)

The medicament will usually be supplied as part of a pharmaceutical composition. Pharmaceutical compositions are therefore within the scope of the present invention. They can be administered via any suitable route of administration.

Dosages of a polypeptide of the present invention can vary between wide limits, depending upon the nature of the treatment, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used.

However, without being bound by any particular dosages, a daily dosage of a polypeptide of the present invention of from lng to lOmg/kg body weight may be suitable. More preferably the dosage is from lOOng to lOOμg/kg body weight/day. The dosage may be repeated as often as appropriate. If side effects develop, the amount and/or frequency of the dosage can be reduced, in accordance with good clinical practice.

If desired, a plurality of different polypeptides of the present invention may be administered together.

One or more additional pharmaceutically active agents (e.g. anti-cancer agents) may optionally be present in a composition of the present invention.

For example, the pharmaceutical composition may include a polypeptide capable of inducing a T cell response directed against a polypeptide produced by an oncogene or against a mutant tumour suppressor protein. Examples of said oncogenes or mutant tumour suppressor proteins include p21-ras, Rb, p53, abl, gip, gsp, ret or trk. The oncogene target may be the p21-rαϊ polypeptides described in international application No. PCT/NO92/00032 (publication no. WO92/14756). Other examples of pharmaceutically active agents include polypeptides arising from frameshift mutations in the BAX and hTGF beta RII genes. Examples of these are provided in

WO99/58552. As discussed supra, these polypeptides have been shown to be effective in stimulating CD4+ and CD 8+ T cells in a specific manner.

Further examples of pharmaceutically active agents include telomerase itself as well as fragments thereof that are capable of eliciting T cell immunity. These are discussed in WO00/02581, for example.

Whilst it is possible to administer a plurality of pharmaceutically active agents in a single composition (e.g. a multi-component vaccine) this is not essential since one or more components may be administered separately. The present invention therefore includes the simultaneous, sequential or separate administration of active agents.

Polypeptides of the present invention may be provided in the form of a lipopolypeptide conjugate composition. This can induce a high-affinity cytotoxic T cell response (Deres, K. et al, 1989, Nature 342: 561-564).

B) Diagnostic uses

In addition to the medical uses discussed above, polypeptides of the present invention can be used in diagnosis. For example, they can be used to diagnose cancer by virtue of binding to antibodies and/or stimulating T cells.

C) Screening uses

Polypeptides of the present invention can be used in screening.

One method of screening includes providing a sample of fluid (e.g. blood), a cancer cell, an extract from such a cell, or a cancer cell model with a polypeptide of the present invention and assaying for properties useful in treating cancer (anti-cancer activity). Any appropriate assay can be used. For example, T cell activity can be assayed (see Example 2).

Another method of screening includes screening for substances that bind to a polypeptide of the present invention. Uses of such substances are set out in D) below.

Screening can also be done to identify agonists or antagonists that affect activity and/or expression of the polypeptide.

Screens of the present invention may be used in a drug development program. Such programs are within the scope of the present invention, as are drugs developed therefrom and medical uses of ' such drugs.

A drug development program may involve taking a moiety identified or identifiable by a screening method of the present invention, optionally modifying it (e.g. modifying its structure and/or modifying a composition which includes said moiety) and performing further studies (e.g. toxicity studies and/or studies on activity). Trials may be performed on non-human animals and/or humans. Such trials will generally include determining the effect(s) of different dosage regimes.

Drug development programs may use computers to analyse moieties identified by screening (e.g. to predict structure and/or function, to identify possible agonists/antagonists, to search for other moieties that may have similar structures and/or functions, etc.). The use of computers to perform such analyses is within the scope of the present invention, as are drugs developed based upon such analyses.

Polypeptides of the present invention may be provided together with other moieties in libraries for screening or other analyses. The present invention also includes within its scope a library comprising a polypeptide of the present invention. The polypeptide of the present invention is desirably present in the library in an environment in which it is not found in nature (e.g. it may be bound to a surface which it is not bound to in nature or it is present in a composition together with comprising one or more heterologous polypeptides with which it is not normally associated in nature). Preferred libraries comprise at least 100, at least 10,000 or at least 1,000,000 different polypeptides. Many different types of library can be provided. Moieties present in the library may provided in solution, on beads, on chips (see e.g. Fodor (1993) Nature 364:555-556), on bacteria (see e.g. US Patent 5223409), on spores (see e.g. US Patent 5223409), on 'phage (see e.g. Scott and Smith (1990) Science 249:386-90 and US Patent 5223409), etc. Libraries are particularly useful for screening (e.g. high throughput screening) of drug candidates. Screening may for example include binding studies, activity studies, toxicity studies, etc.

Polypeptides of the present invention may be arranged in an array or as part of an array, for example in order to facilitate screening. The present invention therefore includes an array comprising a polypeptide of the present invention. Preferably the array has a predetermined pattern. It may have a grid-like pattern. Moieties present in the array may be identified by spatial . coordinates, which may be stored in a computer. The moieties may be provided in immobilised form. However this is not essential. For example an array can be formed of wells or other means for containing polypeptides in a fluid environment. Preferred arrays comprise at least 100, at least 10,000 or at least 1 ,000,000 different moieties.

D) Uses in raising or selecting binding agents

A further use of the polypeptides of the present invention is in raising or selecting binding agents.

Binding agents that bind to polypeptides of the present invention are within the scope of the present invention. Preferred binding agents bind to polypeptides of the present invention with a sufficient degree of specificity to be useful in purifying such polypeptides. (For example, they may be immobilised and used to bind to polypeptides of the present invention. The polypeptides may then be eluted by washing with a suitable eluent under appropriate conditions.) Such binding agents can be considered to be "specific binding agents".

Binding agents are not limited to use in purification.

They can be used in therapy. For example, they may be used to bind to telomerase. They may also be used to target other agents (e.g. toxins or other active agents to a site where telomerase is present). This can be useful in treating cancer since telomerase may be expressed in cancer cells.

They can be used in diagnosis. For example, they may be used to identify telomerase and may therefore be useful in diagnosing conditions such as cancer.

They can be generally used in procedures for assaying and/or detecting polypeptides of the present invention. Various such 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).

E) Computer analysis of sequences

It will be appreciated by those skilled in the art that the present invention provides many molecules having novel and non-obvious nucleotide and amino acid sequences. These sequences are referred to herein as sequences of the present invention.

Sequences of the present invention can be provided on a data carrier, which is preferably machine-readable. A data carrier comprising a sequence of the present information is also within the scope of the present invention. The data carrier may, for example, be a floppy disk, a CD, a minidisk, a chip, a tape, a card, a hard disk, a "zip" disk, other means for storing data in compressed form, a server, a database, etc. All of the foregoing are within the scope of the present invention.

Sequences of the present invention can be used in various methods of technical analysis. These are also within the scope of the present invention. Examples are given below.

The degree of sequence identity and/or sequence homology between a sequence of the present invention and at least one other sequence can be determined. If desired, the method may allow for one or more gaps or insertions in one or more sequences.

Sequence identity/homology studies are also useful, for example, in identifying SNPs (single nucleotide polymorphisms) or in identifying single amino acid differences. These can arise within populations of individuals due to point mutations.

A sequence can be used to predict the structure of all or a part of the molecule. For example it may be used to predict secondary, tertiary or quartenary structures of polypeptides. Thus it may be used to predict structures such as B-sheet structures, helix structures, disulphide bonds, antigenic sites, glycosylation sites, binding sites of ligands, recessed regions (including grooves and pockets), projecting regions, etc. In the case of nucleic acids, sequence data may, for example be used to predict which part of a sequence is likely to hybridise with another part and various structures can then be predicted (e.g. stem-loop structures, single stranded regions, etc). A sequence can be used to predict various chemical or physical properties. These include molecular weight, hydrophobicity and/or hydrophilicity profiles, isoelectric points, hybridisation properties, etc.

Sequences of the present invention can be analysed for the presence of restriction sites. Such sites may be used to generate restriction fragments by partial or complete digestion with appropriate restriction enzymes. Restriction fragment length polymorphisms (RFLPs) may be identified by comparing sequences of the present invention with other sequences. RFLPs are useful in nucleic acid fingerprinting and in other techniques. The present invention therefore also includes methods of identifying RFLPs by utilising using the sequences of the present invention.

A sequence may be analysed to predict functional regions. For example in the case of nucleic acids a sequence may be analysed used to predict operator regions, promoters, enhancers, splice sites, introns, exons, regulatory regions, open reading frames, coding regions, ribosome binding sites, start codons, stop codons, etc. In the case of polypeptides a sequence may be, for example, be used to predict regions that bind to another molecule (e.g. an antibody, a chemokine, a receptor, an inhibitor or an activator), that are involved in transmitting a signal, that stimulate a biological response, that act enzymatically, that are cleaved by enzymes, that activate or inhibiting the activity or expression of other molecules, etc.

From the foregoing description it will be appreciated that sequences of the present invention can be analysed using a wide variety of methods. Methods, computers, computer programs, databases and internet sites that utilise these methods in respect of sequences of the present invention are within the scope of the present invention.

F) Other uses

Polypeptides of the present invention are also useful in selecting or inducing T cells. These are within the scope of the present invention and may be used in anti-cancer therapy. A pharmaceutical composition comprising such T cells is therefore also within the scope of the present invention. A mixture of T cells with specificities to different HLA epitopes may be provided. Alternatively, identical T cells may be provided from a clonal cell line. This may be grown in vitro, if desired. Example 2 provided herein describes induction of T cells and T cell proliferation assays.

Nucleic acids

The present invention provides a nucleic molecule that:

a) has a strand that encodes a polypeptide of the present invention,

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).

Unless the context indicates, 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.

3) They may be provided in recombinant form i.e. covalently linked to a heterologous 5' and/or 3' flanking sequence to provide a chimaeric 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 which 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.

From the foregoing discussion it will be appreciated that a large number of nucleic acids 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).

Nucleic acid molecules of the present invention and uses thereof are discussed in further detail below:

a) Coding nucleic acid molecules

The 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 polypeptides of the present invention. Thus, they may be used for the same treatments as the polypeptides of the present invention. They may therefore be provided in pharmaceutical compositions (e.g. as nucleic acid vaccines). Nucleic acids encoding a plurality of pharmaceutically active polypeptides may be provided, if desired. Nucleic acid molecules of the present invention may be provided in the form of vectors, although this is not essential. Preferred vectors for use in treatment include replication-deficient adenoviruses, retroviruses and adeno-associated viruses.

Nucleic acid molecules of the present invention may be administered to a patient by physical methods. These methods include topical application of the nucleic acid in an appropriate vehicle - for example in solution in a pharmaceutically acceptable excipient, such as phosphate buffered saline (PBS). They also include particle bombardment (which is sometimes known as 'gene gun' technology and is described in US patent no. 5371015). Here inert particles, such as gold beads coated with a nucleic acid, are 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, the nucleic acid molecules may simply be injected at appropriate site (e.g. muscle). They may be incorporated in 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 WO88/00239). Where glycosylation is desired, eukaryotic (e.g. mammalian or insect) expression systems are preferred.

Whatever means is 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, 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 will lack this residue. Polypeptides may initially be expressed to include signal sequences. Different signal sequences may be provided for different expression systems. Alternatively, signal sequences may be absent.

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 [1983] Methods Enzymol 91:227, 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 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, or at least 100 nucleotides in length.

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. For example, probes may be used to determine whether or not an individual has a wild-type gene encoding a polypeptide of the present invention, or whether or not one or more deletions, insertions and/or replacements of bases relative to a wild-type sequence are present. It may therefore be used to identify individuals that do not express polypeptides of the present invention or that express polypeptides having reduced activity (including inactive polypeptides).

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 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. 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 the uses described above, nucleic acid molecules of the present invention (of whatever nature) are useful in screening. (The discussion provided herein in respect of screening using polypeptides of the present invention applies mutatis mutandis to screening using nucleic acid molecules of the present invention.)

Screening can, for example, be done to identify moieties that bind (e.g. hybridise) to said nucleic acid molecules. It can also be done to identify moieties that affect transcription or translation from said molecules. Screening can also be done to analyse expression, including analysing 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. a person) and can be used in forensic science, for security applications (e.g. for confirming the identity of a person), 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.

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. A further aspect of nucleic acids of the present invention lies in the utilisation of their sequences.

This is discussed supra under the section headed "Polypeptides". For example, sequence information can be used in predicting structure and/or function, in sequence homology or identity studies, etc.

Vectors

As indicated above, nucleic acid molecules of the present invention may be provided in the form of vectors. Vectors comprising such nucleic acid molecules are within the scope of the present invention and include plasmids, phasmids, cosmids, viruses (including bacteriophages), YACs, PACs, etc. They will usually include an origin of replication and may 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. Here a further marker may be provided that is different from the marker that is inactivated (e.g. it encodes a different type of drug resistance).

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.

Cells The present invention includes within its scope cells comprising nucleic acid molecules or vectors of the present invention. These may for example be used for expression, as described herein.

A cell capable of expressing a polypeptide of the present invention can be cultured and used to provide the polypeptide, which can then be purified.

Alternatively, the cell may be used in therapy for the same purposes as the polypeptide. 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 cells can then produce the polypeptide in vivo. Preferably the cells comprise a regulatable promoter enabling transcription to be controlled via administration of one or more regulator molecules. If desired, the promoter may be tissue specific.

Expression is not however essential since cells of the present invention may be provided simply for maintaining a given nucleic acid sequence, for replicating the sequence, for manipulating it, etc.

Other cells within the scope of the present invention are T cells, as described supra. T-cytotoxic and/or T helper cells are included, as are combinations of T cells. Preferred combinations are specific for all of the epitopes of a polypeptide of the present invention.

Animals

The present invention also includes within its scope non-human transgenic animals. Such animals may be useful for producing polypeptides of the present invention (e.g. via secretion in milk, as described herein). Alternatively, they may be useful as test animals for analysing the effect(s) of polypeptides of the- present invention. Techniques for producing transgenic animals are well known and are described e.g. in US patents 4870009 and 4873191. For example, a nucleic acid encoding a polypeptide of the present invention may be microinjected into a pronucleus of a fertilised oocyte. The oocyte may then be allowed to develop in a pseudopregnant female foster animal. The animal resulting from development of the oocyte can be tested (e.g. with antibodies) to determine whether or not it expresses a polypeptide of the present invention. Alternatively, it can be tested with a probe to determine if it has a transgene (even if there is no expression).

A transgenic animal can be used as a founder animal that can be bred from to produce further transgenic animals. Two transgenic animals may be crossed. In some cases transgenic animals being crossed may be haploid for a given transgene and it may be desired to provide a diploid animal as a result of crossing.

A transgenic animal may be cloned - e.g. by using the procedures set out in WO97/07668 and WO97/07699 (see also Nature 385:810-813 (1997)). Thus a quiescent cell can be provided and combined with an oocyte from which the nucleus has been removed combined. This can be achieved using electrical discharges. The resultant cell can be allowed to develop in culture and can then be transferred to a pseudopregnant female.

Figures

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

Figure 1 shows a sequence of a fragment of hTERT that is 100 amino acids long and comprises the sequence REEILAKFLHWLMSVYVVEL at its N-terminus and Figure 2 shows a sequence of a fragment of hTERT that is 100 amino acids long and comprises the sequence REEILAKFLHWLMSVYVVEL at its C-terminus

The present invention includes fragments of hTERT including the sequence REEILAKFLHWLMSVYVVEL, when the fragments are up to 100 amino acids long. Thus Figures 1 and 2 are useful in illustrating possible N-terminal and

C-terminal extensions of the sequence REEILAKFLHWLMSVYVVEL. Of course, it is possible to have both N-terminal and C-terminal extensions and the length of a hTERT fragment may be less than 100 amino acids. Examples

Example 1: Epitopes

The polypeptide REEILAKFLHWLMSVYVVEL is believed to encode several potentially strong HLA class I and II binders.

Peptides containing nested epitopes can be processed to obtain the various epitopes and can thus function as a vaccine for the population in general.

Various possible epitopes that can be incorporated in a polypeptide of the present invention are set out below:

Nested HLA class I epitopes.

HLA-A* 0201 nonamers:

REEILAKFLHWLMSVYVVEL ILAKFLHWL

LMSVYVVEL KFLHWLMSV

HWLMSVYVV LHWLMSVYV

HLA-A*0201 decamers

REEILAKFLHWLMSVYVVEL WLMSVYVVEL FLHWLMSVYV

EILAKFLHWL

AKFLHWLMSV

LHWLMSVYVV ILAKFLHWLM -

HLA-A 1 nonamers

REEILAKFLHWLMSVYVVEL FLHWLMSVY

HLA-A 1 decamers

REEILAKFLHWLMSVYVVEL KFLHWLMSVY

HLA-A26 nonamers

REEILAKFLHWLMSVYVVEL FLHWLMSVY

ILAKFLHWL EILAKFLHW

HLA-A26 decamers

REEILAKFLHWLMSVYVVEL EILAKFLHWL WLMSVYVVEL

KFLHWLMSVY

ILAKFLHWLM

HLA-B* 0702 nonamers

REEILAKFLHWLMSVYVVEL

LMSVYVVEL

HLA-B*0702 decamers

REEILAKFLHWLMSVYVVEL WLMSVYVVEL

HLA-B* 1510 nonamers

REEILAKFLHWLMSVYVVEL

LMSVYVVEL ILAKFLHWL

HLA-B*2705 nonamers

REEILAKFLHWLMSVYVVEL REEILAKFL ILAKFLHWL

HLA-B*2709 nonamers REEILAKFLHWLMSVYVVEL REEILAKFL

HLA-B*5101 octamers

REEILAKFLHWLMSVYVVEL LAKFLHWL

HLA-B 8 octamers

REEILAKFLHWLMSVYVVEL LAKFLHWL EILAKFLH

HLA-B 8 nonamers

REEILAKFLHWLMSVYVVEL ILAKFLHWL EILAKFLHW

LMSVYVVEL

Nested HLA class II epitopes.

HLA-DRB1*010115 - mers:

REEILAKFLHWLMSVYVVEL AKFLHWLMSVYVVEL

LAKFLHWLMSVYNVE

EEILAKFLHWLMSNY

REEILAKFLHWLMSN

HLA-DRB1*0301 (DR17 15 -mers:

REEILAKFLHWLMSVYVVEL REEILAKFLHWLMSV LAKFLHWLMSVYVVE

HLA-DRB1*0401 (DR4Dw4 15 -mers:

REEILAKFLHWLMSVYVVEL AKFLHWLMSVYVVEL

EEILAKFLHWLMSVY

Example 2: Testing Procedures

Polypeptide testing and cancer therapy:

To test a particular polypeptide according to the present invention one or more of the tests described below may be used. Testing of a polypeptide is not limited to the methods given in the examples but may also include other tests or assays known to a person skilled in the art. The T cells and T cell clones used in the examples below may be obtained from in vitro stimulation of T cells in blood samples from humans. Blood samples from both humans that have not been pre-treated with in vivo administration of the peptide(s) and humans that have been given the peptide(s) in vivo prior to blood sampling may be used.

Since all peptide specific T cell responses are dependent on binding of peptide epitopes to HLA molecules (in humans), it can be determined if the particular polypeptide binds to various HLA molecules in vitro. A number of established methods are known in the art for this purpose. For HLA class II binding, this can be done in established test systems using purified HLA class II molecules and radiolabelled peptides, as described by Johansen BH, Gedde-Dahl T, Sollid LM, Vartdal F, Thorsby E, Gaudernack G. (Binding of ras oncogene peptides to purified HLA- DQ(alpha l *0102,beta 1 *0602) and -DR(alpha,beta 1 *0101) molecules. Scand J Immunol. 1994 Jun;39(6):607-12). To directly verify the presence of peptide fragments corresponding to HLA class I epitopes, these shorter fragments may be synthesised and tested for binding in standard class I binding assays using a readily available cell line assay based on flow cytometry such as described by Stuber G, Leder GH, Storkus WT, Lotze MT, Modrow S, Szekely L, Wolf H, Klein E, Karre K, Klein G.(Identification of wild-type and mutant p53 peptides binding to HLA-A2 assessed by a peptide loading-deficient cell line assay and a novel major histocompatibility complex class I peptide binding assay. Eur J Immunol. 1994 Mar;24(3):765- 8). Alternatively, high throughput methods such as described by Stryhn A, Pedersen LO, Romme T, Holm CB, Holm A, Buus S. (Peptide binding specificity of major histocompatibility complex class ϊ resolved into an array of apparently independent subspecificities: quantitation by peptide libraiies and improved prediction of binding. Eur J Immunol. 1996 Aug;26(8):1911- 8) may be used. These in vitro assays function to verify the presence of potential HLA class I and II epitopes within a candidate vaccine. A more direct assay for the presence of functional epitopes involves eliciting 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 protein or antigen presenting cells that have processed naturally occurring hTERT protein. The specificity of T cells induced in vivo by hTERT polypeptide vaccination may also be determined.

Protocols for generation of T ceil responses in vitro:

A large number of different protocols may be used to elicit peptide specific T cell responses in vitro. The majority of current protocols employ pulsing of dendritic cells to generate potent T cell responses. A standard protocol for generating both CTL (CD8+, HLA-class I restricted) and T-helper (CD4+, HLA-class II restricted) responses is given below. This protocol reliably generates specific T cell cultures against reference melanoma peptide antigens in 6-8 out of 10 blood donors, and the responding T cells may easily be cloned for detailed functional studies. Similar or better results may be obtained using blood harvested form cancer patients that have had their cancer removed by surgery. Another source of lymphocytes may be a patient that has undergone hTERT peptide vaccination in an experimental clinical protocol. Alternatively T cells may be obtained from a tumour biopsy from a cancer patient having a hTERT positive tumour. However any other known protocol giving comparable results may also be used for the same purpose.

Day -7

Stimulation cell (DC) culture:

# Thaw 250 x 10° PBMC (or use fresh PBMC), wash with standard medium.

# Plate PBMC in 6-well plates at 20 x 10⁶ in 3ml standard medium per well.

# Incubate for 2 hours at 37°C, gently swirl plates and collect medium containing non-adherent cells. # Gently wash cells in wells with 2 ml standard medium to remove remaining non-adherent cells.

# Freeze non-adherent cells for future use as responder cells. # Add 2.5 ml standard medium containing 800 U/ml of GM-CSF and 500 U/ml of

IL 4 to wells with adherent cells.

Day -5 # Add 2.5 ml of fresh standard medium containing 1600 U/ml GM-CSF and

1000 U/ml IL 4.

Day -3

# Remove 2.5 ml of culture medium from each well and replace by 2.5 ml of fresh standard medium containing 1600 U/ml GM-CSF and 1000 U/ml IL 4. (For Class

II protocol: Add candidate hTERT peptide at 50μg/ml.)

Day -2

# Remove 1 ml of culture medium from each well and replace by 1 ml of fresh medium containing 10 ng/ml of TNF-alpha.

Day -1

# Add poly-IC overnight (last 16 hours) at a final concentration of 12.5 μg/ml.

Day O

# Harvest the cultured DC and wash twice with standard medium. Note: DC adhere easily to the plastic tube; therefore "pre-coat" the tubes with standard medium. Record the percentage of DC present using MHC class II-antibody L243 and at least antibodies for CD19. CD83 and CD86. # (For Class I protocol: Resuspend cells in 1 ml standard medium containing

50 μg/ml candidate hTERT peptide and 3 μg/ml β₂m).

# Incubate at 37°C for 4 hours; gently resuspend every hour.

# Irradiate at 25 Gy and wash twice with standard medium, resuspend cells (stimulator cells) at 0.3 x 10⁶/ml in standard medium. # Thaw non-adherent fraction (those frozen on day -7). Initiation of T cell-stimulation cultures

1) For the class I protocol, CD 8+ cells will be enriched from PBMC using any technique described in the art.

2) For the class II protocol, PBMC will be used without separation

Class I protocol:

# Use at least 15 x 10⁶ responders (and therefore 1.5 x 10⁶ stimulator cells to give R:S ratio of 10: 1) per induction culture (ie. per peptide).

# Resuspend responder cells at 3 x 10⁶/ml in standard medium containing 20 ng/ml of IL 7 and 100 pg/ml of IL 12 and plate in 24-well plates (1 ml/well). # Add 10% stimulator cells (ie. 0.3 x 10⁶ in 1 ml per well).

Day +7

# Remove 1 ml of medium and replace with 1 ml of standard medium containing 20 ng/ml of IL 7.

Day 12

# Harvest responder cells, separate over Ficoll/Hypaque, wash once and count viable cells.

# Resuspend at 1.5 x 10⁶/ml in standard medium and keep tube at 37°C. # Thaw 4 x 10⁶ autologous PBMC per 1.5 x 10⁶ responder cells. Use serum-free RPMI for thawing.

# Wash once in serum-free RPMI and irradiate at 60 Gy.

# Wash again in serum-free RPMI and resuspend at 4 x 10⁶/ml in standard medium. # Plate in 24-well plates ( 1 ml/well) and incubate for 2 hours at 37°C.

# Swirl plates gently and collect medium containing non-adherent cells. Gently wash cells with 2 ml standard medium to remove remaining non-adherent cells. # Add 0.5 ml of standard medium containing 20 μg/ml of candidate hTERT peptide and 3 μg/ml of β₂m to the adherent cells and incubate for 2 hours at 37°C.

# Remove medium, gently wash once and add 1 ml of responder cell suspension (ie. 1.5 x lθ7mi).

Day 14

# Add 1 ml of standard medium containing 20 IU/ml of IL 2 to each well.

Day 19 # Restimulate as on day 12. Whether isolation of live cells over Ficoll/Hypaque is necessary will depend on the quality of the cultures and total number of cells.

Day 21

# Add fresh medium + IL 2 as on day 14.

Day 26

# Restimulate as on day 12.

Note: check the CD4/CD8 ratio of responder cells at this time. In the class I protocol these must be predominantly CD 8. ^'

Day 28

# Add fresh medium + IL 2 as on day 14.

Day 33 # Harvest responder cells. Whether isolation of live cells over Ficoll/Hypaque is necessary will depend on the quality of the cultures and total number of cells.

Class II protocol:

# Use at least 15 x 10 responders (and therefore 1.5 x 10⁶ stimulator cells to give

R:S ratio of 10: 1 ) per induction culture (ie. per peptide).

# Resuspend responder cells at 3 x 10⁶/ml in standard medium and plate in 24-well plates (1 ml/well).

# Add 10% stimulator cells (ie. 0.3 x 10⁶ in 1 ml per well). Day +7

# Remove 1 ml of medium and replace with 1 ml of standard medium containing 20 IU/ml of IL 2.

Day 12

# Resuspend at 1.5 x 107ml in standard medium and keep tube at 37°C. # Thaw 4 x 10 autologous PBMC per 1.5 x 10 responder cells. Use serum-free RPMI for thawing.

# Wash once in serum-free RPMI and irradiate at 60 Gy.

# Wash again in serum-free RPMI and resuspend at 4 x 10⁶/ml in standard medium. # Plate in 24-well plates (1 ml/well) and incubate for 2 hours at 37°C.

# Swirl plates gently and collect medium containing non-adherent cells. Gently wash cells with 2 ml standard medium to remove remaining non-adherent cells.

# Add 0.5 ml of standard medium containing 20 μg/ml of candidate hTERT peptide and add 1 ml of responder cell suspension (ie. 1.5 x 107ml).

Day 14

# Add 1 ml of standard medium containing 20 IU/ml of IL 2 to each well.

Day 19

# Restimulate as on day 12. Whether isolation of live cells over Ficoll/Hypaque is necessary will depend on the quality of the cultures and total number of cells.

Day 21 # Add fresh medium + IL 2 as on day 14.

Day 26

# Restimulate as on day 12.

Note: check the CD4/CD8 ratio of responder cells at this time. In the class II protocol these must be predominantly CD4+. Day 28

# Add fresh medium + IL 2 as on day 14.

Day 33

# Harvest responder cells. Whether isolation of live cells over Ficoll/Hypaque is necessary will depend on the quality of the cultures and total number of cells.

# Responder cells in the different protocols are tested in cytotoxicity assay, proliferation assay and cytokine secretions assays. For class I epitopes also fluorescence labelled HLA-class I hTERT peptide tetramers may be used. These assays are all standard assays which are well known in the art.

In vitro T cell response analysis:

To determine if hTERT expressing tumour cell lines can be killed by T cell clones obtained from peripheral blood from cancer patients or healthy individuals can be done by using the following method. T cell clones are obtained after cloning of T-cell blasts present in peripheral blood mononuclear cells (PBMC) after hTERT polypeptide stimulation or vaccination. The polypeptide vaccination protocol includes several in vivo injections of polypeptides intracutaneously and/or subcutanously with GM-CSF or another commonly used adjuvant.

Cloning of T cells is performed by plating responding T cell blasts at 0,5 and 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 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+. Both types of T cell clones may kill target cells provided they express the relevant HLA molecules. Killing is verified at two levels. First T cell clones are incubated with HLA matched or autologous cell targets pulsed with hTERT peptides at different effector to target ratios to determine if lysis of tumour cells occurs. Lysis in this case demonstrates that the

T cell are functionally active and specific for the antigen used for in vitro stimulation. Peptide specific CD4+ T cell clones that do not kill are tested in proliferative assays and cytokine secretion assays, such as the ELISPOT assay (Geginat G, Schenk S, Skoberne M, Goebel W, Hof H. (A Novel Approach of Direct Ex Vivo Epitope Mapping Identifies Dominant'and

Subdominant CD4 and CD8 T Cell Epitopes from Listeria monocytogenes. Immunol. 2001 Feb 1;166(3):1877-1884). Functionally active T cell clones are used for further characterization.

Correlation between polypeptides and in vivo hTERT fragments:

In order to verify that the antigen recognised is associated with hTERT protein, and to identify the HLA class I or class II molecule presenting the putative hTERT polypeptide to the T cell clone, different hTERT 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 10 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 hTERT positive tumour cell line may be used as target cells. A suitable cell line not expressing hTERT may be used as a control, and should not be lysed. hTERT positive cell lines not expressing the relevant HLA molecules are also used as negative controls. Lysis of a particular cell line indicates that the T cell clone being tested recognises an endogenously-processed hTERT epitope in the context of the HLA class I or class II subtype expressed by that cell line. For non cytotoxic, CD4+ T cell clones proof for processing of the hTERT epitope is obtained in several ways: The T cell clone is mixed with irradiated (50Gy) target cells (50x10 T cells and

50x10^" target cells) and incubated for 3 days before adding

³H-thymidine (9.25 x 10⁴ Bq/mL) overnight and harvested and counted in a scintillation counter. The target cells can be autologous or HLA matched cell lines made to express HLA class II molecules by incubation with recombinant human IFNγ for 1 -3 days as described by

Sollid LM, Gaudernack G, Markussen G, Kvale D, Brandtzaeg P,

Thorsby E. (Induction of various HLA class II molecules in a human colonic adenocarcinoma cell line. Scand J Immunol. 1987 Feb;25(2): 175-80). Alternatively, autologous or HLA matched Dendritic Cells can be pulsed with recombinant hTERT expressing the same peptide sequence or made to express hTERT by transfection with DNA or mRNA encoding hTERT and be used as APC for the T cell clones.

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 can be 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 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% CO₂ 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 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 l.

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.

The procedures discussed above can be applied mutatis mutandis for development of vaccines for indications other than cancer. Terminology

For the avoidance of doubt, certain terminology used in the present application 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 may be joined 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. 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.

"Comprising" or "Having"

These terms covers not only anything consisting of a specified feature or characteristic, but also anything including that feature or characteristic, but also possessing one or more additional features or characteristics. Thus in the case of a nucleotide or amino acid sequence comprising or having a given sequence, the sequence itself is covered, as are longer sequences.

"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. 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 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)

"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 -l.HO and 11.45 - 11.61 ofSambrook et al [Molecular Cloning, 2nd Edition, Cold Spring Harbor Laboratory Press (1989)]. One example of hybridisation conditions that can be used 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 1M 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 Tm - 25°C. 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 to 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 discussion of hybridisation conditions is provided for general guidance but is 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, RNARNA 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.

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, or a part of any of the aforesaid (e.g. a nucleic acid molecule). Where treatments are discussed, 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/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 aberrant expression of a gene product.

Disorders to be treated may also, or 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.

(The foregoing comments in respect of treatments apply mutatis mutandis to medical uses.)

"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, colorants, odourants, salts (polypeptides of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents, adjuvants, excipients, diluents or antioxidants. They may also contain other therapeutically active agents in addition to polypeptides of the present invention.

Therapeutically active agents may themselves 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. For example, it may be administered by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradennal) routes. 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) Oral Adminish'ation

Pharmaceutical compositions adapted for oral administration may be provided as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids); as edible foams or whips; or as emulsions. Tablets or hard gelatine capsules may comprise lactose, maize starch or derivatives thereof, stearic acid or salts thereof. Soft gelatine capsules may comprise vegetable oils, waxes, fats, semi-solid, or liquid polyols etc. Solutions and syrups may comprise water, polyols and sugars. For the preparation of suspensions oils (e.g. vegetable oils) may be used to provide oil-in-water or water-in-oil suspensions.

An active agent intended for oral administration may be coated with or admixed with a material that delays disintegration and/or absorption of the active agent in the gastrointestinal tract (e.g. glyceryl monostearate or glyceryl distearate may be used). Thus the sustained release of an active agent may be achieved over many hours and, if necessary, the active agent can be protected from being degraded within the stomach. Pharmaceutical compositions for oral administration may be formulated to facilitate release of an active agent at a particular gastrointestinal location due to specific pH or enzymatic conditions. (ii) 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).)

(Hi) 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 paraffimc 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.

(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. include 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) P ar 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^th 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. Cbiswell et al in Tibtech 10, 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 Vj regions, which contributes to the stability of the molecule. Other 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 chimaeric 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 chimaeric 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).

General remarks

a) Substances described herein (including nucleic acid molecules, vectors, polypeptides, moieties identified/identifiable by screening, binding agents, etc.) may be provided in substantially pure form or in isolated form. However, they are not limited to being provided in such forms. A substance described herein may be provided as part of a kit. Such a kit is within the scope of the present invention. A kit will normally (although not necessarily) include instructions for use (e.g. instructions for use in treatment, diagnosis, screening, etc). It may comprise one or more sealed containers containing components of the kit (e.g. under sterile conditions).

b) Where features are described in connection with particular aspects of the present invention, they shall be deemed to apply mutatis mutandis to other aspects of the invention, unless the context indicates otherwise.

c) In some cases lists of categories are discussed herein. Categories are not intended to be mutually exclusive, unless the context indicates otherwise. Thus, for example, the categories of screening uses and research uses overlap, as do the categories of complementary and hybridising nucleic acids.

d) The description of the invention provided herein is merely illustrative thereof and it should therefore be appreciated that various variations and modifications can be made without departing from the spirit or scope of the invention as set forth in the accompanying claims.

e) All documents cited herein are hereby expressly incorporated by reference, to the extent allowable under the relevant patent law and practice.

Claims

Patent Claims:

1. A polypeptide that:

a) consists of the sequence: R E E I L A K F L H W L M S V Y V V E L;

b) is a fragment of the sequence set out in a) above that comprises at least two HLA epitopes and is at least 10 amino acids long;

c) comprises the sequence set out in a) above as well as an additional

C-terminal and/or N-terminal sequence, with the proviso that the polypeptide is not full length hTERT and does not include a contiguous sequence of hTERT that is more than 100 amino acids long; or

2. A polypeptide according to any preceding claim that comprises at least one HLA class I epitope and at least one HLA class II epitope.

3. A polypeptide according to claim 1 or claim 2, wherein said at least two HLA epitopes are HLA epitopes described in Example 1.

4. A polypeptide according to claim 1 or claim 2, wherein the polypeptide is at least 12, or at least 15 amino acids long.

5. A polypeptide according to any preceding claim, when the polypeptide is no more than 25 amino acids long.

6. A polypeptide according to any preceding claim that comprises an HLA-A epitope.

7. A polypeptide according to claim 6 that comprises a plurality of HLA-A epitopes.

8. A polypeptide according to any preceding claim that comprises an HLA-B epitope.

9. A polypeptide according to claim 8 that comprises a plurality of HLA-B epitopes.

10. A polypeptide that comprises an HLA-DR.

11. A polypeptide according to claim 11 that comprises a plurality of HLA-DR epitopes.

12. A polypeptide according to preceding claim that comprises an HLA-A epitope, an HLA-B epitope and an HLA-DR epitope.

13. A polypeptide according to claim 1 that consists of the amino acid sequence REEILAKFLHWLMSVYVVEL.

14. A polypeptide according to claim 1 that consists of the amino acid sequence: ILAKFLHWLMSVYVVEL, EILAKFLHWLMSVYVVEL, or

EEILAKFLHWLMSVYVVEL.

15. A nucleic acid molecule that:

a) has a strand that encodes a polypeptide as described in any of claims 1 to 14,

c) has a strand that hybridises with a molecule as described in a) or b) above.

16. A vector or a cell comprising a nucleic acid molecule according to claim 15.

17. A binding agent that binds to a polypeptide as described in any of claims 1 to 14.

18. A plurality of T cells that are specific for T cell epitopes of a polypeptide according to any of claims 1 to 14.

19. A polypeptide according to any of claims 1 to 14, a nucleic acid according to claim 15, a vector or cell according to claim 16, a binding agent according to claim 17, or a plurality of T cells according to claim 18; for use in medicine.

20. The use of a polypeptide according to any of claims 1 to 14, of a nucleic acid according to claim 15, of a vector or cell according to claim 16, of a binding agent according to claim 17, or of a plurality of T cells according to claim 18, 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 claim 24, wherein the vaccine is a vaccine for a population of individuals having different HLA profiles.

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

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

28. The use according to claim 27, 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).

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

30. A pharmaceutical composition comprising a polypeptide according to any of claims 1 to 14, a nucleic acid according to claim 15, a vector or cell according to claim 16, a binding agent according to claim 17, or a plurality of T cells according to claim 18.

31. A pharmaceutical composition according to claim 30, further comprising a polypeptide capable of inducing a T cell response against a polypeptide produced by an oncogene or against a mutant tumour suppressor protein; a nucleic acid encoding said polypeptide capable of inducing a T cell response; or a T cell that is capable of killing a cell expressing said polypeptide produced by an oncogene.

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

33. A pharmaceutical composition according to any of claims 30 to 32 further comprising telomerase, or a fragment thereof that is not a polypeptide of the present invention, or a nucleic acid encoding telomerase or a fragment thereof, or a T cell capable of inducing a T cell response against telomerase or a fragment thereof.

34. A combined preparation of:

a) a polypeptide according to any of claims 1 to 14, a nucleic acid according to claim 15, a vector or cell according to claim 16, a binding agent according to claim 17, or a plurality of T cells according to claim 18, and

b) one or more components from any of claims 31 to 33;

for simultaneous, separate or sequential use in anticancer therapy.

35. A pharmaceutical composition according to any of claims 30 to 33 or a combined preparation according to claim 34, including a pharmaceutically acceptable carrier, diluent, additive, stabiliser, and/or adjuvant.

36. A pharmaceutical composition according to any of claims 30 to 33 or 35, or a combined preparation according to claim 34 or 35, which is a vaccine.

37. A vaccine according to claim 36, which comprises a cytokine or growth factor (e.g. TNF alpha, GM-CSF, IL-2 or IL-12).

38. A pharmaceutical composition according to any of claims 30 to 32 or 35, which comprises an antisense molecule, or is capable of producing an antisense molecule in vivo.

39. A diagnostic composition comprising a polypeptide according to any of claims 1 to 14, a nucleic acid according to claim 15, a vector or cell according to claim 16, or a binding agent according to claim 17.

40. A kit for diagnosing cancer, comprising a polypeptide according to any of claims 1 to 14, a nucleic acid according to in cla n 15, a vector or cell according to claim 16, or a binding agent according to claim 17, said kit optionally including instructions for use.

41. A kit according to claim 40, 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).

42. A machine-readable data carrier (e.g. a diskette) comprising the sequence of a polypeptide according to any of claims 1 to 14, or of a nucleic acid according to claim 15.

43. A method comprising using a sequence as described in claim 42 to perform sequence identity studies, sequence homology studies, and/or hybridisation studies.

44. A method comprising using a sequence as described in claim 42 to predict structure and/or function (e.g. to predict anti-cancer activity).

45. A method of screening comprising providing a cancer cell, an extract from such a cell, or a cancer cell model, with a polypeptide a polypeptide according to any of claims 1 to 14, a nucleic acid according to claim 15, a vector or cell according to claim 16, or a binding agent according to claim 17, or a plurality of T cells according to claim 18, and assaying for anticancer activity.

46. The use of a method according to any of claims 43 to 45 in a drug development or drug screening procedure.

47. A drug or drug candidate identified or selected by a procedure including a method as described in any of claims 43 to 45.

48. A library suitable for screening (e.g. high throughput screening) comprising a polypeptide according to any of claims 1 to 14, a nucleic acid according to claim 15, a vector or cell according to claim 16, or a binding agent according to claim 17.

49. An organised array comprising a polypeptide according to any of claims 1 to 14, a nucleic acid according to claim 15, a vector or cell according to claim 16, or a binding agent according to claim 17.

50. A polypeptide according to any of claims 1 to 14, a nucleic acid according to claim 15, a vector or cell according to claim 16, or a binding agent according to claim 17; when in immobilised form.

51. A polypeptide according to any of claims 1 to 14, a nucleic acid according to claim 15, a vector or cell according to claim 16, or a binding agent according to claim 17; when in purified or isolated form.

52. A computer or database that displays or stores a sequence as described in claim 42, or that is set up to perform a method according to claim 43 or 44.

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