GB2361701A

GB2361701A - DNA polymerase delta mutants

Info

Publication number: GB2361701A
Application number: GB0010327A
Authority: GB
Inventors: Jon Amund Eriksen; Gustav Gaudernack; Mona Moller; Jens Bjorheim
Original assignee: Norsk Hydro ASA
Current assignee: Norsk Hydro ASA
Priority date: 2000-04-28
Filing date: 2000-04-28
Publication date: 2001-10-31
Also published as: AU2001262806A1; WO2001083689A2; WO2001083689A3; GB0010327D0

Abstract

The invention relates to nucleic acid sequences coding for DNA polymerase . w. molecule having a frameshift mutation between C2953 and A2960 consisting of the insertion of a single G nucleotide, two 6 nucleotides, or the deletion of two G nucleotides. Polypeptides coded for by said nucleic acid sequences are also disclosed, in addition to a polypeptide coded for by a nucleic acid comprising a deletion of a single G nucleotide between C2953 and A2960. The nucleotides and polypeptides of the invention can be used in the treatment or diagnosis of diseases associated with defects in DNA polymerase . w. , such as colorectal cancer.

Description

2361701 PEPTIDES

SUMMARY OF THE INVENTION

This invention relates to peptides which are fragments of protein products arising from frameshift mutations in the DNA polymerase 8 gene. In particular it relates to peptides which elicit T cellular immunity, and to cancer vaccines and compositions for anticancer treatment comprising said peptides.

The invention also relates to DNA sequences encoding at least one trameshift mutant peptide, and vectors comprising at least one insertion site containing a DNA sequence encoding at least one frarneshift mutant peptide.

Further the invention relates to methods for the treatment or prophylaxis of cancers associated with frameshift mutations in the DNA polymerase 8 gene by administration of at least one frameshift mutant peptide or a recombinant virus vector comprising at least one insertion site containing a DNA sequence encoding at least one trameshift mutant peptide, or an isolated DNA sequence comprising a DNA sequence encoding at least one frameshift mutant peptide.

The present invention represents a further development of anticancer treatment or prophylaxis based on the use of peptides to generate activation and strengthening of the anticancer activity of the T cellular arm of the body's own immune system.

Technical Background

Tumour antigens, Status:

T cell defined antigens have now been characterised in a broad spectrum of cancer types. These antigens can be divided into several main groups, depending on their expression. The two main groups are constituted by developmental differentiation related antigens (tumour-testis antigens, oncofoetal antigens etc., such as MAGE antigens and CEA) and tissue specific differentiation antigens (Tyrosinase, gp 100 etc.). The group containing the truly tumour specific antigens contains proteins that are altered due to mutations in the genes encoding them. In the majority of these, the mutations are unique and have been detected in a single or in a small number of tumours. Several of these antigens seem to play a role in oncogenesis.

Cancer vaccines, Status:

The focus in cancer vaccine development has been on antigens expressed in a high degree within one form of cancer (such as melanoma) or in many kinds of cancers. The field is in rapid growth. A growing number of publications, exemplified by those detailed in the "Prior Art" section below, including WO 99/103 82 and GB 2253211, detail progress in the field and the technology available in the preparation of vaccines.

Inheritable cancer/cancer gene testing:

Inherited forms of cancer occur at a certain frequency in the population. For several of these cancer forms, the underlying genetic defects have been mapped. This is also the case in Lynch syndrome cancers which constitute an important group of inheritable cancer. In families afflicted with this syndrome, family members inherit defective genes encoding DNA Mismatch Repair (MMR) Enzymes. Carriers of such MMR defects frequently develop colorectal cancer (fINPCC) and other forms of cancer. Mutations in MMR enzymes can be detected using gene testing in the same way as other cancer related genes can be detected.

Gene testing of risk groups in this case represents an ethical dilemma, since no acceptable forms for prophylactic treatment exist. At present surgery to remove the organ in danger of developing cancer has been the only treatment option. In these patients, cancer vaccines will be a very useful form of prophylaxis.

The lack of efficient repair of mismatched DNA results in deletions and insertions in one strand of DNA, and this happens preferentially in stretches of DNA containing repeated units (repeat sequences). Until now, focus has been on repeat sequences in the form of non-coding microsattelite loci. Indeed, microsattelite instability is the hallmark of cancers resulting from NIMR defects. We have taken another approach, and have concentrated on frameshift mutations occurring in DNA sequences coding for proteins related to the oncogenic process. Such frameshift mutations result in completely new amino acid sequences in the C-terminal part of the proteins, prematurely terminating where a novel stop codon appears. This results in two important consequences:

I) The truncated protein resulting from the frameshift is generally nonfunctional, in most cases resulting in "knocking out" of an important cellular function. Aberrant proteins may also gain new functions such as the capacity to aggregate and form plaques. In both cases the frameshift results in disease.

2) The short new C-terrninal amino acid sequence resulting from the shift in the reading frame (the "frameshift sequence") is foreign to the body. It does not exist prior to the mutation, and it only exists in cells having the mutation, i.e. in tumour cells and their pre-malignant progenitors. Since they are completely novel and therefore foreign to the immune system of the carrier, they may be recognised by T-cells in the repertoire of the carrier. Such vaccines may also be used prophylactically in persons who inherit defective enzymes belonging to the NWR machinery.

It has been shown that single amino acid substitutions in intracellular "self '-proteins may give rise to tumour rejection antigens, consisting of peptides differing in their amino acid sequence from the normal peptide. The T cells which recognise these peptides in the context of the major histocomPatibility (NWC) molecules on the surface of the tumour cells, are capable of killing the tumour cells and thus rejecting the tumour from the host.

In contrast to antibodies produced by the B cells, which typically recognise a free antigen in its native conformation and further potentially recognise almost any site exposed on the antigen surface, T cells recognise an antigen only if the antigen is bound and presented by a M11C molecule. Usually this binding will take place only after appropriate antigen processing, which comprises a proteolytic fragmentation of the protein, so that the resulting peptide fragment fits into the groove of the MVIC molecule. Thereby T cells are enabled to also recognise peptides derived from intracellular proteins. T cells can thus recognise aberrant peptides derived from anywhere in the tumour cell, in the context of MBC molecules on the surface of the tumour cell, and can subsequently be activated to eliminate the tumour cell harbouring the aberrant peptide.

M. Barinaga (1992, Science, 257: 880-88 1) offers a short review of how MW binds peptides. A more comprehensive explanation of the Technical Background for this Invention may be found in D. Male et al., Advanced Immunology, 1987, J.B.fippincott Company, Philadelphia. Both references are included herein in their entirety by reference.

The MW molecules in humans are normally referred to as HLA (human leukocyte antigen) molecules. They are encoded by the HLA region on the human chromosome No 6.

The HLA molecules appear as two distinct classes depending on which region of the chromosome they are encoded by and which T cell subpopulations they interact with and thereby activate primarily. The class I molecules are encoded by the HLA A, B and C subloci and they primarily activate C138+ cytotoxic T cells. The HLA class 11 molecules are encoded by the DR, DP and DQ subloci and primarily activate CD4+ T cells, both helper cells and cytotoxic cells.

Normally every individual has six HLA Class 1 molecules, usually two from each of the three groups A, B and C. Correspondingly, all individuals have their own selection of HLA Class II molecules, again two from each of the three groups DP, DQ and DR. Each of the groups A, B, C and DP, DQ and DR are again divided into several subgroups. In some cases the number of different HLA Class 1 or II molecules is reduced due to the overlap of two HLA subgroups.

All the gene products are highly polymorphic. Different individuals thus express distinct HLA molecules that differ from those of other individuals. This is the basis for the difficulties in finding HLA matched organ donors in transplantations. The significance of the genetic variation of the HLA molecules in immunobiology is reflected by their role as immune-response genes. Through their peptide binding capacity, the presence or absence of certain HLA molecules governs the capacity of an individual to respond to peptide epitopes. As a consequence, HLA molecules determine resistance or susceptibility to disease.

T cells may control the development and growth of cancer by a variety of mechanisms. Cytotoxic T cells, both HLA class 1 restricted CD8+ and HLA Class 11 restricted CD4+, may directly kill tumour cells carrying the appropriate tumour antigens. CD4+ helper T cells are needed for cytotoxic C138+ T cell responses as well as for antibody responses, and for inducing macrophage and LAK cell killing.

A requirement for both HLA class 1 and 11 binding is that the peptides must contain a binding motif, which usually is different for different HLA groups and subgroups. A binding motif is characterised by the requirement for amino acids of a certain type, for instance the ones carrying large and hydrophobic or positively charged side groups, in definite positions of the peptide so that a narrow fit with the pockets of the HLA binding groove is achieved. The result of this, taken together with the peptide length restriction of 8- 10 amino acids within the binding groove, is that it is quite unlikely that a peptide binding to one type of HLA class I molecules will also bind to another type. Thus, for example, it may very well be that the peptide binding motif for the HLA-A I and HLAA2 subgroups, which both belong to the class I gender, are as different as the motifs for the HLA-A I and HLA-B I molecules.

For the same reasons it is not likely that exactly the same sequence of amino acids will be located in the binding groove of the different class 11 molecules. In the case of HLA class 11 molecules the binding sequences of peptides may be longer, and it has been found that they usually contain from 10 to 16 amino acids, some of which, at one or both terminals, are not a part of the binding motif for the HLA groove.

However, an overlap of the different peptide binding motifs of several HLA class I and class 11 molecules may occur. Peptides that have an overlap in the binding sequences for at least two different HLA molecules are said to contain "nested T cell epitopes". The various epitopes contained in a "nested epitope peptide" may be formed by processing of the peptide by antigen presenting cells and thereafter be presented to T cells bound to different HLA molecules. The individual variety of HLA molecules in humans makes peptides containing nested epitopes more useful as general vaccines than peptides that are only capable of binding to one type of HLA molecule.

Effective vaccination of an individual can only be achieved if at least one type of HLA class I and/or 11 molecule in the patient can bind a vaccine peptide either in it's full length or as processed and trimmed by the patient's own antigen presenting cells.

The usefulness of a peptide as a general vaccine for the majority of the population increases with the number of different HLA molecules it can bind to, either in its full length or after processing by antigen presenting cells.

In order to use peptides derived from a protein encoded by a mutated gene as vaccines or anticancer agents to generate anti turnour CD4+ and/or CD8+ T cells, it is necessary to investigate the mutant protein in question and identify peptides that are capable, eventually after processing to shorter peptides by the antigen presenting cells, to stimulate T cells.

PRIOR ART

There is a growing number of publications relating to cancer vaccines and frameshift mutations, and they include the following:

WO 95/3273 1, which discloses frameshift mutations in the APC (adenomatous polyposis coli) gene which had uses in the treatment of cancers, particularly in the manufacture of cancer vaccines, the frameshift mutations being found to induce the assembly of HLA A2 Class I molecules in vitro and thus possibly having use in the treatment of individuals in order to induce immune responses to mutant cellular proteins resulting from the frameshift mutations.

WO 96/18409 discloses antigens from p53 and Her-2/Neu useful in stimulating CTLs (cytotoxic T lymphocytes, also referred to as cytotoxic T cells) specific against cells producing same.

WO 97/12992 discloses frameshift mutations in genes including PApp, Tau and Ubiquitin, together with methods for treatment of diseases associated with same.

WO 99/10382 discloses various synthetic peptides of oncogene protein products, particularly the p2l ras proto-oncogene, which elicit CTL immunity, for use in cancer vaccines and compositions for anti-cancer treatment.

WO 99/58552, the contents of which are incorporated herein in their entirety by reference, discloses peptides from cancer related protein products of frameshift mutated genes, which elicit T cell immunity are disclosed for use in cancer vaccines, as are compositions for anticancer treatment. A range of examples of genes where frameshift mutations may result in gene products that result in development of turnours is given at pages 40-42. These include a number of DNA synthesis and repair enzymes (paragraph bridging pages 41/42) but do not include or suggest DNA polymerase 8.

None of these publications identifies frameshift mutations in the DNA polymerase 5 gene, and thus cannot provide any teaching or suggestion of treatment or diagnosis of cancers or a predisposition to same caused by frameshift mutations in the DNA polymerase 6 gene.

Flohr, T. et al. (1999, Int. J. Cancer, 80: 919-929), the contents of which are incorporated herein in their entirety by reference, discloses mutations in the DNA polymerase 8 gene of human sporadic colorectal cancers and colon cancer cell lines, the mutations including frameshift mutations (Table IV). In particular, a frameshift mutation is identified resulting from the deletion of G2959 of the wild-type DNA polymerase 8 coding sequence. No other possible frameshift mutations in the 3'region of the DNA polymerase 6 coding sequence are suggested. No possible therapeutic uses are suggested for the identified mutations.

References herein to nucleotide positions within the DNA polymerase 6 coding sequence are made with the first nucleotide of the ATG start codon designated +1 (reference cDNA sequence as reported by Chung, D.W. et al., 1991, PNAS, 88: 11197- 11201; GenBank accession number M80397).

DEFINITION OF PROBLEM SOLVED BY THE INVENTION.

There is a continuing need for new anticancer agents based on antigenic peptides giving rise to specific T cellular responses and toxicity against tumours and cancer cells carrying genes with mutations related to cancer. The present invention supplies new peptides useful in the combat and prevention of cancer particularly for use as ingredients in anticancer vaccines, particularly multiple target anti-cancer vaccines.

Another problem solved by the present invention is that a protection or treatment can be offered to individuals identified as being at risk of developing (or suspected of having developed) cancers, for example individuals belonging to families or groups at high risk of hereditary cancers. The present invention also makes it possible to diagnose cancers, or the risk of (predisposition to) developing cancers.

BRIEF SUNEVIARY OF THE INVENTION A main objective of the present invention is to provide peptides corresponding to peptide fragments of mutant DNA polymerase 8 proteins, in particular produced by cancer cells, and which may be used to stimulate T cells.

Another main object of the invention is to develop a cancer therapy for cancers based on the T cell immunity which may be induced in patients by stimulating their T cells either in vivo or in vitro with the peptides according to the invention.

A third main object of the invention is to develop a vaccine to prevent the establishment of or to eradicate cancers based solely or partly on peptides corresponding to peptides of the present invention which can be used to generate and activate T cells which produce cytotoxic T cell immunity against cells harbouring the mutated genes.

A fourth main object of the invention is to provide an anticancer treatment or prophylaxis specifically adapted to a human individual in need of such treatment or prophylaxis, which comprises administering at least one peptide according to this invention to said individual.

A fifth main object of the invention is to provide an anticancer treatment or prophylaxis specifically adapted to a human individual in need of such treatment or prophylaxis, which comprises administering at least one nucleic acid, such as a pure DNA vaccine or a vector or an mRNA vaccine according to this invention to said individual.

DESCRIPTION OF THE INVENTION

According to the present invention there is provided an isolated nucleotide sequence encoding DNA polymerase 8 and having a trameshift mutation selected from one of the group consisting (i) the insertion between C2953 and A2960 of two G nucleotides; (ii) the insertion between C2953 and A2960 of a single G nucleotide; and (iii) the deletion of two G nucleotides between C2953 and A2960. Also provided is an isolated protein encoded by same.

The frameshift mutations are located in the 3'region of the DNA polymerase 8 coding sequence and cause the synthesis of a protein having an altered C-terminal amino acid sequence, affecting the DNA interaction domain I (Flohr et al., 1999, supra) and producing a premature stop codon resulting in C-terminal truncation of the synthesised protein. Part of the wild-type nucleotide sequence is shown in SEQ ID NO: 9. Mutated nucleotide sequences are given in SEQ ID NOs: 10- 13. In SEQ ID NOs: 9-13 nucleotide I corresponds to nucleotide 2929 of the cDNA encoding the wild- type DNA polymerase 8 (Chung et al., 199 1, supra) These mutations have been identified in individuals suffering from colorectal cancer, and thus the detection of such mutations may be used as the basis of diagnostic tests and diagnostic test methods for cancer or a predisposition of an individual to developing cancer. Thus nucleotide sequences encoding the mutations of the present invention may be for use in a method of treatment or diagnosis of the human body.

Probes specific to the mutations of the present invention may be readily prepared by persons skilled in the art using generally available techniques as described in for example Sambrook, J. et al., "Molecular Cloning. A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor Press, New York, 1989.

A diagnostic test for the presence in a patient sample of a frameshift mutation (i.e. mutated nucleotide sequence) according to the present invention may for example comprise the steps of.

i) contacting said sample with a probe specific to said frameshift mutation; ii) detecting any probe-sample hybridisation; and iii) correlating the results of detection step (ii) with the presence of said mutation in said sample.

Any diagnostic tests may of course be performed under suitable conditions, which may include the use of high stringency hybridisation conditions to minimise any possibility of non-specific hybridisation and thus of incorrect results.

Also provided is a method of diagnosis of a mutation according to the present invention comprising performing said diagnostic test.

The mutated nucleotides of the present invention, together with a frameshift mutation comprising the deletion of G2959 (Flohr et al., supra) also provide opportunities for therapy of any condition resulting from them. They may for example be used in the manufacture of medicaments such as DNA vaccines specific against them using techniques known in the art (see for example Leitner, W.W. et al., 1999, Vaccine, 18: 765-777; Montgomery, D.L. et al., 1997, Pharmacol. Ther., 74(2): 195-205; Donnelly, J.J. et al., 1997, Annu. Rev. Immunol., 15: 617-648; Manickan, E. et al., 1997, Crit. Rev. Immunol., 17(2): 139-154; Fries, P.C., 1999, New England Journal of Medicine, 341: 1623-1624). They may also be used in the manufacure of mRNA vaccines (Boczkowski, D. et al., 2000, Cancer Res., 60(4): 1028-1034; PMID 10706120) (although of course a complementary RNA strand, i.e. an mRNA, must be produced) which can be administered to patients to effect prophylactic or therapeutic treatment.

Also provided is a method of treatment of a condition resulting from the mutations of the present invention comprising administering a medicament according to the present invention to a patient in need of same, The exact dosages and administration regime of medicaments of the present invention may be readily determined using for example doseresponse assays.

According to the present invention there is also provided a peptide derived from a DNA polymerase 8 having a frameshift mutation selected from one of the group consisting (i) the insertion between C2953 and A2960 of two G nucleotides; (ii) the insertion between C2953 and A2960 of a single G nucleotide; (iii) the deletion of two G nucleotides between C2953 and A2960; and (iv) the deletion of one G nucleotide between C2953 and A2960, and which peptide induces, either in its full length or after processing by an antigen presenting cell, T cell responses, characterised in that:

i) it is at least 8 amino acids long; ii) it comprises at least one amino acid of the sequence of any one of SEQ ID NOs: 1-4; and iii) it comprises 0-10 amino acids from the carboxy terminus of the normal part of the protein sequence preceding the amino terminus of the corresponding one of SEQ ID NOs: 14; but not having the sequence of SEQ ID NO: 1.

Thus the peptide may have the sequence of any one of SEQ ID NOs: 5-8 or 14-22. These sequences correspond to SEQ ID NOs: 1-4 but additionally including 10 amino acids Nterminal to the point of the frameshift mutation. The peptides may not extend to the Cterminus of SEQ ID NOs: 1-4 but must of course include at least part of any one of the corresponding SEQ ID NOs: 1-4. Clearly, the peptide is not limited solely to the sequences of SEQ ID NOs: 1-4, 5-8 or 14-22. For example, they include fragments of SEQ ID NOs: 1-4 having 0- 10 amino acids from the carboxyl terminus of the normal part of the protein sequence preceding the amino terminus of the fragment.

As discussed above, the length restrictions of HLA grooves mean that the peptides may contain 8-10 or 10-16 amino acids. Alternatively they may have 8-25, 9-20, 9-16, 8-12 or 20-25 amino acids. They may for example consist of 9, 12, 13, 16 or 21 amino acids.

It is most preferred that the peptides of the present invention are at least 9 amino acids long, for instance 9-18 amino acids long, but due to the processing possibility of the antigen presenting cells, longer peptides are also very suitable for the present invention. Thus the whole mutant amino acid sequence may be used as a frameshift mutant peptide according to the present invention.

Also provided according to the present invention is an isolated nucleic acid sequence encoding a peptide according to the present invention. It may for example have the sequence of any one of SEQ ID NOs: 10- 13.

The peptides and nucleic acid sequences of the present invention may be for use in a method of treatment or diagnosis of the human body. In particular they may be for use in the treatment of cancer, for example colorectal cancer.

Also provided according to the present invention is the use of at least one peptide or nucleic acid sequence encoding same according to the present invention in the manufacture of a medicament for the treatment of a condition caused by a mutation according to the present invention, particularly cancer. Also provided is a method of manufacture of a medicament for the treatment caused by a mutation according to the present invention, characterised in the use of at least one peptide according to the present invention, or a nucleic acid sequence encoding same. Medicaments according to the present invention may contain a dosage of an active (therapeutic) ingredient suitable for the patient to whom the medicament is to be administered. Such dosages may be determined using for example dose-response assays. Medicaments may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient (see for example Remington's Pharmaceutical Sciences and US Pharmacopeia, 1984, Mack Publishing Company, Easton, PA, USA).

A medicament according to the present invention may comprise at least one peptide according to the present invention, optionally with a pharmaceutically acceptable carrier, diluent or excipient.

Peptides of the present invention used in medicaments may be present in amounts of I nanogram to I gram, for example in amounts of I microgram to I milligram.

Such medicaments may be vaccines, and may be administered to a patient either prophylactically or therapeutically.

The nucleic acid sequences of the present invention may be provided in the form of (i.e. comprise part of) a plasmid or virus vector. Vectors include, but are not limited to, E. coli plasmid, a Listeria vector and recombinant viral vectors. Recombinant viral vectors include, but are not limited to, orthopox virus, canary virus, capripox virus, suipox virus, vaccinia, baculovirus, human adenovirus, SV40, bovine papilloma virus and the like comprising the DNA sequence encoding a frameshift mutant peptide.

Medicaments of the present invention may be administered together, either simultaneously or separately, with compounds such as cytokines and/or growth factors, i.e. interleukin-2 (IL-2), interleukin-12 (IL-12), granulocyte macrophage colony stimulating factor (GM-CSF), Flt-3 ligand or the like in order to strengthen the immune response as known in the art.

Regarding DNA vaccines, the above cytokines and/or co-stimulatory molecules may themselves be delivered in the form of plasmid or oligonucleotide DNA. The response to a DNA vaccine has been shown to be increased by the presence of immunostimulatory DNA sequences (ISS). These can take the form of hexameric motifs containing methylated CpG, according to the formula: 5'-purine-purine-CG-pyrimidine-pyrimidine3'. Our DNA vaccines may therefore incorporate these or other ISS, in the DNA encoding the peptides, in the DNA encoding the cytokine or other co- stimulatory molecules, or in both. A review of the advantages of DNA vaccination is provided by Tighe et al. (1998, Immunology Today, 19(2): 89-97).

The peptides according to the present invention can be used in a vaccine or a therapeutical composition either alone or in combination with other materials, such as for instance standard adjuvants or in the form of a lipopeptide conjugate which as known in the art can induce high-affmity cytotoxic T lymphocytes, (K. Deres, Nov 1989, Nature, 342).

The peptides of the present invention may usefully be included in either a peptide or recombinant fragment based vaccine.

Thus the present invention also provides a method of treatment of a condition caused in a patient by a frameshift mutation in a DNA polymerase 8 gene, said frameshift mutation being selected from one of the group consisting (i) the insertion between C2953 and A2960 of two G nucleotides; (ii) the insertion between C2953 and A2960 of a single G nucleotide; (iii) the deletion of two G nucleotides between C2953 and A2960; and (iv) the deletion of one G nucleotide between C2953 and A2960, the method comprising administering to said patient a medicament according to the present invention.

Methods of vaccination or therapy of a condition caused by a mutation according to the present invention may consist of administering a medicament according to the present invention one or more times (for example over the course of one or two months) in an amount sufficient to induce T-cell immunity to the mutation.

In order to determine whether the peptides thus identified are useable inthe compositions and methods according to the present invention for the treatment or prophylaxis of cancer, the following steps are performed with the peptides of the present invention, including SEQ ID NOs: 1-4, 5- 8 and 14-22:

i) it is determined whether the peptides are able to stimulate T cells; and (optionally) ii) peptides containing nested epitopes for different major HLA class I and/or HLA class 11 molecules are determined.

Cancers other than colorectal cancer which may be treated according to the present invention include, but are not limited to, breast cancer, small-cell lung cancer, non smallcell lung cancer, liver cancer (primary and secondary), renal cancer, melanoma, ovarian cancer, cancer of the brain, head and neck cancer, Pancreatic cancer, gastric cancer, oesophageal cancer, prostate cancer and leukemias and lymphomas.

The compositions of the present invention may be employed in conjunction with other compositions in order to enhance their efficacy. In particular, they may be employed together with compositions of WO 99/58552. They may also be used with anti-cancer drugs, vectors and vaccines such as those of WO 99/10382 and GB 2253211. Thus they may be employed in combination with existing anti-cancer treatments (such as for instance cytostatica, radiation therapy, surgical therapy and other therapies known in the art) in order to enhance their efficacy.

Thus the present invention achieves the following advantages:

- Patients suffering from conditions, particularly cancers, arising from frame-shift mutations in their genes may be treated both therapeutically and prophylactically. This provides good treatment alternatives for cancers most of which at present do not have good treatment alternatives.

- Humans carrying genetic dispositions or belonging to other high risk groups may be prophylactically vaccinated.

- Combination treatments for specific conditions, particularly for specific cancers, such as for instance colorectal cancers, may be prepared.

- The medicaments of the present invention can be used in combination with established vaccines and future vaccines to obtain a multiple targeting treatment.

- Similarly, patients suffering from conditions, particularly cancers, associated with multiple frameshift mutations in genes can be treated more efficiently through a combination treatment.

The invention will be further apparent from the following description, which show, by way of example only, uses of the mutations of the present invention.

DEFINITIONS:

"Amino acid residue" as used herein means an amino acid, e.g., one formed upon chemical digestion (hydrolysis) of a polypeptide at its peptide linkages. The amino acid residues described herein are preferably in the "L" isomeric form. However, residues in the "D" isomeric form can be substituted for any L-amino acid residue, as long as the desired functional Property is retained by the polypeptide. N112 refers to the free amino group present at the ainino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. Nomenclature is as defined in WIPO Standard ST. 25 unless stated otherwise.

The phrase "amino acid residue" is broadly defined to include modified and unusual amino acids as defined in WIPO Standard ST.25, and incorporated herein by reference. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a ftirther sequence of one or more amino acid residues or to an amino-terminal group such as N112or to a carboxy-terminal group such as COOK The term "fragment" when referring to a peptide or protein means a shorter part of a longer peptide or protein.

"Operatively linked" means, with reference to a regulatory element and DNA coding sequence, that following such a link the regulatory element can direct the expression of the linked DNA coding sequence.

A "regulatory element" from a gene is the DNA sequence which is necessary for the expression of that gene.

As used herein, the term "vector" refers to a DNA molecule capable of autonomous replication and to which a DNA segment, e.g. gene or polynucleotide, can be operatively linked so as to bring about replication of the attached segment.

Vectors capable of directing the expression of DNA segments (genes) encoding one or more proteins are referred to herein as "expression vectors". Also included are vectors which allow the cloning of cDNA (complementary DNA) from mRNAs produced using reverse transcriptase.

Example 1

In order to determine the ability of the peptides of the present invention to stimulate a T-cell response, a series of peptides were made using SEQ ID NOs: 1-4 as templates. Each of the synthesised peptides comprised: (i) at least 8 amino acids; (ii) at least one amino acid of the sequence of any one of SEQ ID NOs: 14; and (iii) 0- 10 amino acids from the carboxyl terminus of the normal part of the DNA polymerase 6 sequence preceding the amino terminus of the corresponding one of SEQ ID NOs: 1-4, but did not have the sequence of SEQ ID NO: 1. The synthesised peptides included, but were not limited to, peptides having the sequences of SEQ ID NOs: 5-8, 14-22 and other fragments of SEQ ID NOs: 1-4.

Peripheral blood mononuclear cells (PBMCs) from each of a series of donors were stimulated with a mixture of peptides (above). The concentration of each individual peptide in the mixture was 20 ptM. After two weeks, and weekly thereafter, the bulk cultures were restimulated with autologous P13MCs pulsed with 10-25 [LM of the peptide mixture. After up to 8 restimulations the bulk cultures were tested in a standard proliferation assay with P13MCs alone or as a control or PBMCs pulsed with 25 [IM of the peptides as antigen presenting cells (APCs).

Example 2

In order to generate T cell clones against separate peptides of the mixture used in the bulk stimulation experiments (above), the bulk culture from a donor is seeded with 5 cells per well in U-bottomed, 96- well microtitre plates and using as feeder cells autologous PBMCs pulsed with 25 gM of a peptide having for example the sequence of any one of SEQ ID NOs: 5-8 and 14-22. Autologous B-lymphoblastoid cells were used as APCs in the proliferation assay.

Synthesis Peptides were synthesised by using continuous flow solid phase peptide synthesis. N-aFnioc-amino acids with appropriate side chain protection were used. The Frnoc-arnino acids were activated for coupling as pentafluorophenyl esters or by using either TBTU or diisopropyl carbodiiinide activation prior to coupling. 20% piperidine in DMF was used for selective removal of Fmoc after each coupling. Cleavage from the resin and final removal of side chain protection was performed by 95% TFA containing appropriate scavengers. The peptides were purified and analysed by reversed phase (C 18) HPLC. The identity of the peptides was confirmed by using electro-spray mass spectroscopy (Finnigan mat SSQ710).

Peptides used for in vitro studies of T cell stimulation were synthesised by this method.

Several other well known methods can be applied by a person skilled in the art to synthesise the peptides.

21- SEQUENCE LISTING <110> NORSK HYDRO ASA <120> PEPTIDES <130> NOO/0140/GB <140> <141> <160> 22 <170> PatentIn Ver. 2.1 <210> 1 <211> 57 <212> PRT <213> Homo sapiens <400> 1 Thr Thr Arg Ala Ala Arg Arg Cys Ser Arg Ala Arg Trp Ala Ala Ser 1 5 10 15 Trp Pro Ser Pro Asn Ala Ala Thr Ala Ala Leu Ala Ala Ala Gln Cys 25 30 Ser Ala Thr Arg Glu Pro Cys Val Ser Ser Ala Ser Pro Gly Ser Leu 40 45 Ser Cys Ile Arg Arg Arg Tyr Pro Ile 55 <210> 2 <211> 58 <212> PRT <213> Homo sapiens <400> 2 Gly Thr Thr Arg Ala Ala Arg Arg Cys Ser Arg Ala Arg Trp Ala Ala 1 5 10 15 Ser Trp Pro Ser Pro Asn Ala Ala Thr Ala Ala Leu Ala Ala Ala Gln 25 30 Cys Ser Ala Thr Arg Glu Pro Cys Val Ser Ser Ala Ser Pro Gly Ser 40 45 Leu Ser Cys Ile Arg Arg Arg Tyr Pro Ile 55 <210> 3 <211> 40 <212> PRT <213> Homo sapiens 2.2- <400> 3 Gly Pro His Ala Leu Gln Asp Gly Ala His Gly Gln Gly Gly Arg Pro 1 5 10 15 Pro Gly Leu Arg Gln Thr Pro Gln Leu Leu His Trp Leu Pro His Ser 25 30 Ala Gln Pro Pro Gly Ser Arg Val 40 <210> 4 <211> 39 <212> PRT <213> Homo sapiens <400> 4 Pro His Ala Leu Gln Asp Gly Ala His Gly Gln Gly Gly Arg Pro Pro 1 5 10 15 Gly Leu Arg Gln Thr Pro Gln Leu Leu His Trp Leu Pro His Ser Ala 25 30 Gln Pro Pro Gly Ser Arg Val 35 <210> 5 <211> 67 <212> PRT <213> Homo sapiens <400> 5 Gly Arg Ala Glu Ala Val Leu Leu Arg Gly Thr Thr Arg Ala Ala Arg 1 5 10 15 Arg Cys Ser Arg Ala Arg Trp Ala Ala Ser Trp Pro Ser Pro Asn Ala 25 30 Ala Thr Ala Ala Leu Ala Ala Ala Gln Cys Ser Ala Thr Arg Glu Pro 40 45 Cys Val Ser Ser Ala Ser Pro Gly Ser Leu Ser Cys Ile Arg Arg Arg 55 60 Tyr Pro Ile 65 <210> 6 <211> 68 <212> PRT <213> Homo sapiens <400> 6 Gly Arg Ala Glu Ala Val Leu Leu Arg Gly Gly Thr Thr Arg Ala Ala 1 5 10 15 23- Arg Arg Cys Ser Arg Ala Arg Trp Ala Ala Ser Trp Pro Ser Pro Asn 25 30 Ala Ala Thr Ala Ala Leu Ala Ala Ala Gln Cys Ser Ala Thr Arg Glu 40 45 Pro Cys Val Ser Ser Ala Ser Pro Gly Ser Leu Ser Cys Ile Arg Arg 55 60 Arg Tyr Pro Ile <210> 7 <211> 50 <212> PRT <213> Homo sapiens <400> 7 Gly Arg Ala Glu Ala Val Leu Leu Arg Gly Gly Pro His Ala Leu Gln 1 5 10 15 Asp Gly Ala His Gly Gln Gly Gly Arg Pro Pro Gly Leu Arg Gln Thr 25 30 Pro Gln Leu Leu His Trp Leu Pro His Ser Ala Gln Pro Pro Gly Ser 40 45 Arg Val <210> 8 <211> 49 <212> PRT <213> Homo sapiens <400> 8 Gly Arg Ala Glu Ala Val Leu Leu Arg Gly Pro His Ala Leu Gln Asp 1 5 10 15 Gly Ala His Gly Gln Gly Gly Arg Pro Pro Gly Leu Arg Gln Thr Pro 25 30 Gln Leu Leu His Trp Leu Pro His Ser Ala Gln Pro Pro Gly Ser Arg 40 45 Val <210> 9 <211> 207 <212> DNA <213> Homo sapiens <300> <303> Proc. Natl. Acad. Sci. U.S.A.

<304> 88 <306> 11197-11201 <307> 1991 <308> GenBank M80397 <400> 9 ggccgtgccg aggctgtgct actgcggggg gaccacacgc gctgcaagac ggtgctcacg 60 ggcaaggtgg gcggcctcct ggccttcgcc aaacgccgca actgctgcat tggctgccgc 120 acagtgctca gccaccaggg agccgtgtgt gagttctgcc agccccggga gtctgagctg 180 tatcagaagg aggtatccca tctgaat 207 <210> 10 <211> 204 <212> DNA <213> Homo sapiens <400> 10 ggccgtgccg aggctgtgct actgcggggg accacacgcg ctgcaagacg gtgctcacgg 60 gcaaggtggg cggcctcctg gccttcgcca aacgccgcaa ctgctgcatt ggctgccgca 120 cagtgctcag ccaccaggga gccgtgtgtg agttctgcca gccccgggag tctgagctgt 180 atcagaagga ggtatcccat ctga 204 <210> 11 <211> 207 <212> DNA <213> Homo sapiens <400> 11 ggccgtgccg aggctgtgct actgcggggg gggaccacac gcgctgcaag acggtgctca 60 cgggcaaggt gggcggcctc ctggccttcg ccaaacgccg caactgctgc attggctgcc 120 gcacagtgct cagccaccag ggagccgtgt gtgagttctg ccagccccgg gagtctgagc 180 tgtatcagaa ggaggtatcc catctga 207 <210> 12 <211> 153 <212> DNA <213> Homo sapiens <400> 12 ggccgtgccg aggctgtgct actgcggggg ggaccacacg cgctgcaaga cggtgctcac 60 gggcaaggtg ggcggcctcc tggccttcgc caaacgccgc aactgctgca ttggctgccg 120 cacagtgctc agccaccagg gagccgtgtg tga 153 <210> 13 <211> 150 <212> DNA <213> Homo sapiens <400> 13 ggccgtgccg aggctgtgct actgcgggga ccacacgcgc tgcaagacgg tgctcacggg 60 caaggtgggc ggcctcctgg ccttcgccaa acgccgcaac tgctgcattg gctgccgcac 120 agtgctcagc caccagggag ccgtgtgtga 150 25- <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Polymerase delta frameshift sequences <400> 14 Glu Ala Val Leu Leu Arg Gly Gly Thr Thr Arg Ala Ala Arg Arg Cys 1 5 10 15 Ser <210> 15 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Polymerase delta frameshift sequences <400> 15 Glu Ala Val Leu Leu Arg Gly Thr Thr Arg Ala Ala Arg Arg Cys Ser 1 5 10 15 Arg Ala Arg Trp Ala 20 <210> 16 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Polymerase delta frameshift sequences <400> 16 Arg Cys Ser Arg Ala Arg Trp Ala Ala Ser Trp Pro Ser Pro Asn Ala 1 5 10 15 Ala Thr Ala Ala Leu Ala Ala Ala Gln 25 <210> 17 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Polymerase

Z6- delta frameshift sequences <400> 17 Thr Ala Ala Leu Ala Ala Ala Gin Cys, Ser Ala Thr Arg Glu Pro Cys 1 5 10 is Val Ser Ser Ala Ser Pro Gly 20 <210> 18 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Polymerase delta frameshift sequences <400> 18 Pro Cys Val Ser Ser Ala Ser Pro Gly Ser Leu Ser Cys Ile Arg Arg 1 5 10 15 Arg Tyr Pro Ile 20 <210> 19 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Polymerase delta frameshift sequences <400> 19 Glu Ala Val Leu Leu Arg Gly Gly Pro His Ala Leu Gin Asp Gly Ala 1 5 10 15 <210> 20 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Polymerase delta frameshift sequences <400> 20 Glu Ala Val Leu Leu Arg Gly Pro His Ala Leu Gln Asp Gly Ala His 1 5 10 15 Gly Gin Gly Gly Arg 20 27- <210> 21 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Polymerase delta frameshift sequences <400> 21 Gly Ala His Gly Gln Gly Gly Arg Pro Pro Gly Leu Arg Gln Thr Pro 1 5 10 15 Gln Leu Leu His 20 <210> 22 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Polymerase delta frameshift sequences <400> 22 Leu Arg Gln Thr Pro Gln Leu Leu His Trp Leu Pro His Ser Ala Gln 1 5 10 15 Pro Pro Gly Ser Arg Val 20 2-9

Claims

A nucleotide sequence encoding DNA polymerase 8 and having a frameshift mutation selected from one of the group consisting (i) the insertion between C2953 and A2960 of two G nucleotides; (ii) the insertion between C2953 and A2960 of a single G nucleotide; and (iii) the deletion of two G nucleotides between C2953 and A2960.
2. A nucleotide sequence according to claim 1, for use in a method of treatment or diagnosis of the human or animal body.
3. The use of a nucleic acid sequence according to claim 1 in a method of manufacture of a medicament for the treatment of a condition caused by said frameshift mutation.
4. A diagnostic test for the presence in a patient sample of a nucleic acid sequence having a frameshift mutation according to claim 1, comprising the steps of.

i) contacting said sample with a probe specific to said frameshift mutation; ii) detecting any probe-sample hybridisation; and iii) correlating the results of detection step (ii) with the presence of said mutation in said sample.
5. A diagnostic test according to claim 4, being a diagnostic test for colorectal cancer or a susceptibility to same.
6. A protein encoded by a nucleic acid sequence according to claim 1.

2 ck
7. A peptide derived from a DNA polymerase 5 having a karneshift mutation selected from one of the group consisting (i) the insertion between C2953 and A2960 of two G nucleotides; (ii) the insertion between C2953 and A2960 of a single G nucleotide; (iii) the deletion of two G nucleotides between C2953 and A2960; and (iv) the deletion of one G nucleotide between C2953 and A2960, and which peptide induces, either in its full length or after processing by an antigen presenting cell, T cell responses, characterised in that:

i) it is at least 8 amino acids long; ii) it comprises at least one amino acid of the sequence of any one of SEQ ID NOs: 14; and iii) it comprises 0-10 amino acids from the carboxy terminus of the normal part of the protein sequence preceding the amino terminus of the corresponding one of SEQ ID NOs: 14; but not having the sequence of SEQ ID NO: 1.
8. A peptide according to claim 7, having the amino acid sequence of any one of SEQ ID NOs: 5-8 or 14-22.
9. A peptide according to claim 7, comprising 8- 10 or 10- 16 amino acids.
10. A peptide according to claim 7, comprising 8-25, 9-20, 9-16, 8-12 or 20-25 amino acids.
11. A peptide according to claim 10, comprising 9, 12, 13, 16 or 21 amino acids.
12. A peptide according to claim 7, comprising at least 9 amino acids.
13. A peptide according to claim 12, comprising 9-18 amino acids.

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14. A nucleic acid sequence encoding a peptide according to any one of claims 7-13.
15. A nucleic acid sequence according to claim 14 and having the sequence of anyone of SEQ ID NOs: 10- 13.
16. A peptide or nucleic acid sequence according to any one of claims 715, for use in a method of treatment or diagnosis of the human or animal body.
17. A peptide or nucleic acid sequence according to claim 16, for use in the treatment of cancer.
18. A peptide or nucleic acid sequence according to claim 16, for use in the treatment of colorectal cancer.
19. The use of at least one peptide or nucleic acid sequence encoding same according to any one of claims 7-15 in the manufacture of a medicament for the treatment of a condition caused by said frameshifi mutation.
20. The use of at least one peptide or nucleic acid sequence encoding same according to claim 19 in the manufacture of a medicament for the treatment of cancer.
21. A method of manufacture of a medicament characterised in the use of at least one peptide, or nucleic acid sequence encoding same according to any one of claims 7-15, said medicament being for the treatment of a condition caused by said frameshift mutation.
22. A method of manufacture of a medicament according to claim 21, said medicament being for the treatment of cancer.

-1 k
23. The use or method according to any one of claims 19-22, said medicament additionally comprising a dosage of an active ingredient suitable for the patient to whom the medicament is to be administered.
24. The use or method according to any one of claims 19-23, said medicament comprising between 1 nanogram and 1 gram of said peptide.
25. The use or method according to any one of claims 19-23, said medicament comprising between 1 microgram and 1 milligram of said peptide.
26. The use or method according to any one of claims 19-25, said medicament additionally comprising a pharmaceutically acceptable carrier, diluent or excipient.
27. The use or method according to any one of claims 19-26, said medicament being a vaccine.
28. The use or method according to any one of claims 19-27, said nucleic acid sequence being provided in the form of a plasmid or virus vector.
29. The use or method of claim 28, said vector being selected from the group comprising an E. coli plasmid, a Listeria vector, orthopox virus, canary virus, capripox virus, suipox virus, vaccinia, baculovirus, human adenovirus, SV40, and bovine papilloma virus.
30. The use or method according to any one of claims 19-29, said medicament additionally comprising a cytokine and/or growth factor.

3 -Z
31. The use or method according to claim 30, said cytokine and/or growth factor being selected from the group consisting interleukin-2 (IL-2), interleukin- 12 (IL 12), granulocyte macrophage colony stimulating factor (GM-CSF), and Flt-3 ligand.
32. A method of treatment of a condition caused in a patient by a frameshift mutation in a DNA polymerase 6 gene, said frameshift mutation being selected from one of the group consisting (i) the insertion between C2953 and A2960 of two G nucleotides; (ii) the insertion between C2953 and A2960 of a single G micleotide; (iii) the deletion of two G nucleotides between C2953 and A2960; and (iv) the deletion of one G nucleotide between C2953 and A2960, the method comprising administering to said patient a medicament according to any one of claims 19-3 1.
33. A method of treatment according to claim 32, additionally comprising the step of separately administering a cytokine and/or growth factor to said patient.
34. A method of treatment according to claim 33, said cytokine and/or growth factor being selected from the group consisting interleukin-2 (IL2), interleukin- 12 (IL12), granulocyte macrophage colony stimulating factor (GM-CSF), and Flt-3 ligand.
35. A method of treatment according to any one of claims 32-34, comprising administering said medicament at least once in an amount sufficient to induce T-cell immunity to said frameshift mutation.