CROSS-REFERENCE TO RELATED APPLICATION

This application is a filing under 35 U.S.C. § 371 of International Patent Application No. PCT/GB2017/050227, filed Jan. 30, 2017, which claims priority to United Kingdom Patent Application No. 1601585.1, filed Jan. 28, 2016. Each of the foregoing applications is incorporated herein by reference in its entirety.

The invention relates to cancer, and in particular to novel pharmaceutical compositions, therapies and methods for treating, preventing or ameliorating cancer, and especially metastatic disease. The invention also relates to diagnostic and prognostic methods for cancer and metastatic disease, and biomarkers for these conditions.

Cancer and malignant tumours form a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body, i.e. metastasis. In 2012, approximately 14 million new cases of cancer occurred globally. There is therefore a need to provide an improved diagnosis and therapies for the treatment of cancer and metastasis.

Acetylcholinesterase (AChE) is expressed at different stages of development in various forms, all of which have identical enzymatic activity, but which have very different molecular composition. The ‘tailed’ (T-AChE—SEQ ID No: 1) is expressed at synapses and the inventors have previously identified two peptides that could be cleaved from the C-terminus, one referred to as “T14” (SEQ ID No: 3), within the other which is known as “T30” (SEQ ID No: 2), and which both have strong sequence homology to the comparable region of β-amyloid. The AChE C-terminal peptide “T14” has been identified as being the salient part of the AChE molecule responsible for its range of non-hydrolytic actions. The synthetic 14 amino acids peptide analogue (i.e. “T14”), and subsequently the larger, more stable, and more potent amino acid sequence in which it is embedded (i.e. “T30”—SEQ ID No: 3) display actions comparable to those reported for ‘non-cholinergic’ AChE, whereas the inert residue within the T30 sequence (i.e. “T15”—SEQ ID No: 4) is without effect.

The T14 peptide binds to an allosteric site on the α7 nicotinic-receptor, where, on its own, it has no effect. However, in the presence of a primary ligand, such as acetylcholine or dietary choline, T14 enhances the calcium influx induced by these primary agents. Excessive calcium can be taken up into the mitochondria where it compromises oxidative phosphorylation, and causes a leakage of electrons. Free radicals are consequently formed that then destabilize the membrane, and the cell then dies.

The inventors investigated the levels of the toxic T14 peptide, α7 nicotinic-receptor and acetylcholinesterase proteins using Western Blotting in cell lysate and cell culture media of seven cancer cell lines (MEC-1, KG1a, H929, MCF-7, MDA-MB 231, CLL, and JJN3). They found that only the toxic T14 peptide was released from the cancer cells, and that T14 concentrations were higher outside of the cells than the T14 levels inside cancer cells. The inventors then went on to investigate the relationship between the known metastatic marker CD44 with the toxic T14 peptide, AChE, and the α7 nicotinic-receptor in membrane and cytosol fractions of cancer cell lines. They were surprised to observe that CD44 is significantly and positively correlated with the toxic molecule T14. Collectively, these data strongly suggest that the toxic T14 peptide, including its biosynthesis and metabolic pathways, represents a good target for treating cancer and metastasis, and furthermore, that T14 itself acts as a robust biomarker for cancer metastasis.

Thus, in a first aspect of the invention, there is provided an inhibitor of the synthesis and/or activity of a peptide of SEQ ID No: 3, for use in treating, ameliorating or preventing cancer or metastatic disease.

In a second aspect, there is provided a method of treating, ameliorating or preventing cancer or metastatic disease in a subject, the method comprising, administering to a subject in need of such treatment, a therapeutically effective amount of an inhibitor of the synthesis and/or activity of a peptide of SEQ ID No: 3.

As described in Example 1, the inventors detected the levels of the toxic T14 peptide (SEQ ID No: 3), nicotinic alpha-7 receptor and acetylcholinesterase (AChE) proteins using Western Blotting in cell lysate and cell culture media of seven cancer cell lines (MEC-1, KG1a, H929, MCF-7, MDA-MB 231, CLL, and JJN3), and normal B lymphocytes acting as control. MDA-MB-231, KG1a, and MEC-1 cells are highly migratory cancer cell lines, whereas H929, JJN3, CLL and MCF-7 are less migratory cancer cell lines, and B-lymphocytes are normal, non-cancerous cells. T14, alpha 7 receptors and AChE were all detected within the cancer cells. Surprisingly, all three proteins have similar mobility and their levels are positively correlated with each other, suggesting that they are complexed with each other. However, outside cancer cells (i.e. within the cell culture media), only the toxic T14 peptide was detected. Even more surprising was that the T14 levels were higher outside the cells than they were inside six out of the seven cancer cell lines, suggesting that T14 is produced to be released from the cell. The inventors were also surprised to see that the levels of T14 outside of the cancer cells were significantly negatively correlated with alpha-7 receptor levels within the cancer cells.

Example 2 describes the use of Western Blotting to show the relationship between the well-known metastatic marker CD44with the toxic T14 peptide, AChE, and the alpha-7 receptor in membrane and cytosol fractions of six cancer cell lines (JJN3, MDA-MB231, MCF-7, KG1a, MEC-1, and H929) and also one cancer-derived cell line (PC12). In all cancer cell lines, the metastatic marker (CD44) is significantly and positively correlated with the toxic molecule T14, and this correlation holds true for within the cancer cell membrane as well as within the cancer cell cytosol. These findings strongly suggest that T14 is a good predictor of the degree of cancer and metastasis, i.e. tumour cell migration.

The cancer, which is treated in accordance with the first or second aspect, may be leukaemia. For example, the cancer may be lymphocytic leukaemia or chronic lymphocytic leukaemia (CLL). The cancer may be myeloid leukaemia, or acute myeloid leukaemia. The cancer may be multiple myeloma. The cancer may be breast cancer. The cancer may be plasmacytoma.

Most preferably, the inhibitor of T14 peptide (SEQ ID No: 3) synthesis and/or activity, is for use in treating, ameliorating or preventing metastatic disease or metastasis.

Acetylcholinesterase is a serine protease that hydrolyses acetylcholine, and will be well-known to the skilled person. The major form of acetylcholinesterase, which is found in the brain, is known as tailed acetylcholinesterase (T-AChE). The protein sequence of one embodiment of human tailed acetylcholinesterase (Gen Bank: AAA68151.1) is 614 amino acids in length, and is provided herein as SEQ ID No:1, as follows:

[SEQ ID No: 1]1mrppqcllht pslaspllll llwllgggvg aegredaell vtvrggrlrg irlktpggpv61saflgipfae ppmgprrflp pepkqpwsgv vdattfqsvc yqyvdtlypg fegtemwnpn121relsedclyl nvwtpyprpt sptpvlvwiy gggfysgass ldvydgrflv qaertvlvsm181nyrvgafgfl alpgsreapg nvglldqrla lqwvqenvaa fggdptsvtl fgesagaasv241gmhllsppsr glfhravlqs gapngpwatv gmgearrrat qlahlvgcpp ggtggndtel301vaclrtrpaq vlvnhewhvl pqesvfrfsf vpvvdgdfls dtpealinag dfhglqvlvg361vvkdegsyfl vygapgfskd neslisraef lagvrvgvpq vsdlaaeavv lhytdwlhpe421dparlreals dvvgdhnvvc pvaqlagrla aqgarvyayv fehrastlsw plwmgvphgy481eiefifgipl dpsrnytaee kifaqrlmry wanfartgdp neprdpkapq wppytagaqq541yvsldlrple vrrglraqac afwnrflpkl lsatdtldea erqwkaefhr wssymvhwkn601qfdhyskqdr csdl

It will be appreciated that the first 31 amino acid residues of SEQ ID No:1 are removed while the protein is released, thereby leaving a 583 amino acid sequence.

The amino acid sequence of T30 (which corresponds to the last 30 amino acid residues of SEQ ID No:1) is provided herein as SEQ ID No:2, as follows:

[SEQ ID No: 2]KAEFHRWSSYMVHWKNQFDHYSKQDRCSDL

The amino acid sequence of the toxic T14 peptide (which corresponds to the 14 amino acid residues located towards the end of SEQ ID No:1, and lacks the final 15 amino acids found in T30) is provided herein as SEQ ID No:3, as follows:

[SEQ ID No: 3]AEFHRWSSYMVHWK

The amino acid sequence of T15 (which corresponds to the last 15 amino acid residues of SEQ ID No:1) is provided herein as SEQ ID No:4, as follows:

[SEQ ID No: 4]NQFDHYSKQDRCSDL

The inventors have found that inhibition of the toxic T14 peptide is required for the effective treatment of cancer or metastasis. Inhibitors of the invention may be used to prevent the development of cancer or development and spread of metastasis. For instance, the inhibitors may be given to subjects who are at risk (e.g. a genetic predisposition or adverse environmental exposure) of developing cancer or metastasis. The inhibitors may also be used after surgery, radiotherapy or chemotherapy to prevent cancer re-establishing itself in a subject.

The inventors have shown that the toxic T14 peptide binds to an allosteric site on the α7 nicotinic-receptor, where, on its own, it has no effect on the cell. However, in the presence of a primary ligand, such as acetylcholine or dietary choline, T14 enhances the calcium influx into the cell induced by these primary agents. Excessive calcium is taken up into the mitochondria where it compromises oxidative phosphorylation, and causes a leakage of electrons. Free radicals are consequently formed that then destabilize the membrane, and the cell then dies.

Based on the above understanding, the inventors have designed inhibitors that are capable of decreasing the synthesis or biological activity of the toxic T14 peptide, and which may achieve their effect by a number of biosynthetic and/or metabolic means. Preferably, the inhibitor can bind to T14, and prevent it from binding to the α7 nicotinic-receptor. The inventors have found that any T14 peptide that has managed to reach the allosteric site stays interacting with the receptor, and that this interaction can cause upregulations of more receptors within 24 hours, i.e. T14 can increase its own target. Accordingly, prevention of T14 interaction with the allosteric site is most preferred.

For instance, such inhibitors may:(a) reduce the interaction between the T14 peptide and an allosteric site on the alpha-7 receptor;(b) compete with endogenous T14 peptide for the allosteric site on the alpha-7 receptor;(c) bind to the T14 peptide to reduce its biological activity;(d) decrease post-translational cleavage of acetylcholinesterase polypeptide to create the T14 peptide; or(e) inhibit T14 translocation to the allosteric site on the alpha-7 receptor.

In a preferred first embodiment of the invention, the inhibitor may directly interact with the T14 peptide (e.g. (a) to (c) above) to reduce T14's biochemical and/or metabolic activity, and thus toxicity, of T14, in the subject's cells. Preferably, the inhibitor results in a reduction in T14 interaction with the allosteric site on the alpha-7 receptor.

Preferred inhibitors comprise small molecule inhibitors. Such inhibitors may be identified as part of a high throughput screen of small molecule libraries. For example, the screening method according to the fifth aspect of the invention (see below) represents a suitable means of identifying such inhibitors which can be used to treat cancer or metastasis.

In a preferred embodiment, the inhibitor comprises a compound of Formula (I):


wherein:

R1is —NR9R10or —OH;

R2is



R3is —H or a C1-5straight or branched alkyl or alkenyl;

R4is



R5is —H or a C1-5straight or branched alkyl or alkenyl;

R6is



R7is —H or a C1-5straight or branched alkyl or alkenyl;

R8is —H; a C1-5straight or branched alkyl or alkenyl or



X1is —NR9R10, —OH or


the or each R9and R10are independently —H or a C1-5straight or branched alkyl or alkenyl;

R11is —NH2, —OH or an aryl group;

the or each m is independently between 0 and 5; and

each n is independently between 0 and 10;

or a pharmaceutically acceptable salt, solvate, tautomeric form, or polymorphic form thereof.

It may be understood that the term “salt” refers to any salt of a compound provided herein which retains its biological properties and which is not toxic or otherwise undesirable for pharmaceutical use. Such salts may be derived from a variety of organic and inorganic counter-ions well known in the art. Such salts include, but are not limited to: (1) acid addition salts formed with organic or inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic, acetic, trifluoroacetic, trichloroacetic, propionic, hexanoic, cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic, succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric, benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic, phthalic, lauric, methanesulfonic, ethanesulfonic, 1,2-ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic, 4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-toluenesulfonic, camphoric, camphorsulfonic, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic, glucoheptonic, 3-phenylpropionic, trimethylacetic, tert-butylacetic, lauryl sulfuric, gluconic, benzoic, glutamic, hydroxynaphthoic, salicylic, stearic, cyclohexylsulfamic, quinic, muconic acid and the like acids; or (2) base addition salts formed when an acidic proton present in the parent compound either (a) is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion or an aluminum ion, or alkali metal or alkaline earth metal hydroxides, such as sodium, potassium, calcium, magnesium, aluminum, lithium, zinc, and barium hydroxide, ammonia or (b) coordinates with an organic base, such as aliphatic, alicyclic, or aromatic organic amines, such as ammonia, methylamine, dimethylamine, diethylamine, picoline, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N,N′-dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, N-methylglucamine piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, and the like.

Salts may further include, by way of example only and without limitation, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium and the like, and when the compound contains a basic functionality, salts of non-toxic organic or inorganic acids, such as hydrohalides, e.g. hydrochloride and hydrobromide, sulfate, phosphate, sulfamate, nitrate, acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate, cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate, malonate, succinate, sorbate, ascorbate, malate, maleate, fumarate, tartarate, citrate, benzoate, 3-(4-hydroxybenzoyl)benzoate, picrate, cinnamate, mandelate, phthalate, laurate, methanesulfonate (mesylate), ethanesulfonate, 1,2-ethane-disulfonate, 2-hydroxyethanesulfonate, benzenesulfonate (besylate), 4-chlorobenzenesulfonate, 2-naphthalenesulfonate, 4-toluenesulfonate, camphorate, camphorsulfonate, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylate, glucoheptonate, 3-phenylpropionate, trimethylacetate, tert-butylacetate, lauryl sulfate, gluconate, benzoate, glutamate, hydroxynaphthoate, salicylate, stearate, cyclohexylsulfamate, quinate, muconate and the like.

It may be understood that the term “solvate” refers to a compound provided herein or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. Where the solvent is water, the solvate is a hydrate.

It may be appreciated that an aryl group refers to a substituent derived from an aromatic ring. The aryl group may be a C6-C12 aryl group. Preferably, the aryl group is phenyl, biphenyl or naphthyl.

Most preferably, the compound has Formula (Ia):



R1may be —OH. However, preferably, R1is —NR9R10, more preferably R1is —NR9H, and most preferably R1is —NH2.

Preferably, R2is and n is


preferably between 1 and 5. Accordingly, n may be 1, 2, 3, 4 or 5, and most preferably n is 1.

It will be appreciated that in embodiments where R2is then m may


be 0, 1, 2, 3, 4 or 5. Preferably m is 1. Preferably, X1is in the para position.

In a preferred embodiment R2is



Preferably, in embodiments when R2is


then at least one of R9or R10is —H, and most preferably R9or R10are both —H.

In a preferred embodiment, R2is



In a most preferred embodiment, R2is



It will be appreciated that R3may be a methyl, ethyl, propyl, butyl or pentyl group. Preferably, R3is methyl. However, in a more preferred embodiment, R3is —H.

In one embodiment R4is


and n is preferably between 1 and 5. Accordingly, n may be 1, 2, 3, 4 or 5, and preferably n is 1.

In embodiments where R4is


then n is preferably between 1 and 7, and is more preferably between 2 and 6. Accordingly, n may be 2, 3, 4, 5 or 6, and is preferably n is 3 or 4.

In one embodiment, R4is preferably



More preferably, R4is



It will be appreciated that in embodiments where R4is


then m may be 0, 1, 2, 3, 4 or 5. Preferably m is 1. Preferably, X1is in the para position.

Preferably, R4is



More preferably, R4is



Preferably, in embodiments when R4is


then at least one of R9or R10is —H, and most preferably both R9or R10are —H.

Accordingly, R4is preferably



More preferably, R4is



Most preferably, R4is



It will be appreciated that R5may be a methyl, ethyl, propyl, butyl or pentyl group. Preferably, R5is methyl. However, in a more preferred embodiment, R5is —H.

In R6is


and n is between 1 and 5. Accordingly, n may be 1, 2, 3, 4 or 5, and preferably n is 1.

Preferably, R6is



It will be appreciated that in embodiments where R6is


then m may be 0, 1, 2, 3, 4 or 5. Preferably, m is 0. More preferably, m is 1. Preferably, X1is in the para position.

In a preferred embodiment R6is



Preferably, in embodiments when R6is


then at least one of R9or R10is —H, and most preferably both R9or R10are —H.

Preferably, in embodiments when R6is


then R11is aryl, and most preferably phenyl.

In a preferred embodiment, R6is



In a most preferred embodiment, R6is



It will be appreciated that R7may be a methyl, ethyl, propyl, butyl or pentyl group. Preferably, R7is methyl. However, in a more preferred embodiment, R7is —H.

In one preferred embodiment R8is —H. However, in a more preferred embodiment R8is



In a preferred embodiment, the inhibitor comprises a compound of Formula (I), wherein:

R2is



R3is H or a C1-5straight or branched alkyl or alkenyl;

R4is



R5is H or a C1-5straight or branched alkyl or alkenyl;

R6is



R7is H or a C1-5straight or branched alkyl or alkenyl.

Preferably, R3is H, R5is H and R7is H.

In a more preferred embodiment, the inhibitor comprises a compound of Formula (I), wherein:

R2is



R3is H or a C1-5straight or branched alkyl or alkenyl;

R4is



R5is H or a C1-5straight or branched alkyl or alkenyl; or

R6is


and

R7is H or a C1-5straight or branched alkyl or alkenyl.

Preferably, the inhibitor comprises a compound of Formula (Ia). Preferably, R3is H, R5is H and R7is H.

Preferably, R1is —OH and R8is H. More preferably, R1is NH2and R8is



In an alternative more preferred embodiment, the inhibitor comprises a compound of Formula (I), wherein:

R2is



R3is H or a C1-5straight or branched alkyl or alkenyl;

R4is



R5is H or a C1-5straight or branched alkyl or alkenyl; or

R6is


and

R7is H or a C1-5straight or branched alkyl or alkenyl.

Preferably, the inhibitor comprises a compound of Formula (Ia). Preferably, R3is H, R5is H and R7is H.

Preferably, R1is —OH and R8is H. More preferably, R1is NH2and R8is



In an even more preferred embodiment, the inhibitor comprises a compound of Formula (I), wherein:

R2


is

R3is H or a C1-5straight or branched alkyl or alkenyl;

R4is



R5is H or a C1-5straight or branched alkyl or alkenyl;

R6is


and

R7is H or a C1-5straight or branched alkyl or alkenyl.

Preferably, R3is H, R5is H and R7is H.

In a more preferred embodiment, the inhibitor comprises a compound of Formula (I), wherein:

R2is



R3is H or a C1-5straight or branched alkyl or alkenyl;

R4is



R5is H or a C1-5straight or branched alkyl or alkenyl; or

R6is


and

R7is H or a C1-5straight or branched alkyl or alkenyl.

Preferably, the inhibitor comprises a compound of Formula (Ia). Preferably, R3is H, R5is H and R7is H.

Preferably, R1is —OH and R8is H. More preferably, R1is NH2and R8is



In an alternative more preferred embodiment, the inhibitor comprises a compound of Formula (I), wherein:

R2is



R3is H or a C1-5straight or branched alkyl or alkenyl;

R4is



R5is H or a C1-5straight or branched alkyl or alkenyl; or

R6is


and

R7is H or a C1-5straight or branched alkyl or alkenyl.

Preferably, the inhibitor comprises a compound of Formula (Ia). Preferably, R3is H, R5is H and R7is H.

Preferably, R1is —OH and R8is H. More preferably, R1is NH2and R8is



Preferably, the inhibitor comprises a compound of Formula (101) or (103):



More preferably, the inhibitor comprises a compound of Formula (101a) or (103a):



It will be appreciated that compounds of Formula (101a) and (103a) correspond to compounds Tri02 and Tri04, respectively, which are discussed in the Examples. Hence, in a further aspect, there is provided an inhibitor as defined herein, for use in treating, ameliorating or preventing cancer or metastatic disease, preferably wherein the inhibitor is Formula (101a) or (103a).

In one preferred embodiment, the inhibitor comprises an antibody or antigen-binding fragment thereof, i.e. a T14-neutralising antibody. The antibody or antigen-binding fragment thereof preferably blocks T14 interaction with the allosteric site on the alpha-7 receptor. Preferably, the antibody binds to T14, thereby forming a complex, which is unable to bind to the receptor to cause toxicity.

The antibody or antigen-binding fragment thereof may be polyclonal or monoclonal. The antibody or antigen-binding fragment thereof may be generated in a rabbit, mouse or rat.

Preferably, the antibody or antigen-binding fragment thereof specifically binds to SEQ ID No:3. Preferably, the antibody or antigen-binding fragment thereof specifically binds to one or more amino acid in the C-terminus of SEQ ID No:3. Preferably, the antibody or antigen-binding fragment thereof specifically binds to one or more amino acid in SEQ ID No: 5 (i.e. SYMVHWK, which are the C-terminal amino acids numbers 7-14 of SEQ ID No: 3). Preferably, the antibody or antigen-binding fragment thereof specifically binds to a C-terminal lysine (K) residue in the epitope.

The inventors have surprisingly observed that the C-terminal amino acid sequence VHWK in SEQ ID No:3, which is described herein as SEQ ID No. 6 (i.e. the C-terminal amino acids numbers 10-14 of SEQ ID No.3), acts as an epitope for the antibody or antigen-binding fragment thereof. Accordingly, more preferably the antibody or antigen-binding fragment thereof specifically binds to one or more amino acid in SEQ ID No.6. Most preferably, the antibody or antigen-binding fragment thereof specifically binds to SEQ ID No.6. Hence, it will be appreciated that the epitope to which the antibody binds comprises or consists of SEQ ID No: 6.

Based on the discovery of the VHWK epitope (SEQ ID No: 6) within the T14 peptide, the inventors believe that these sequences can be used as an antigen for the production of useful antibodies. As described in the Examples, the T14 peptide (SEQ ID No:3) was cysteine-cross-linked to Keyhole Limpet Hemocyanin (KLH) acting as a carrier protein which stimulates an immune response in the host. The KLH protein cross-linked to T14 is referred to herein as SEQ ID No:7, i.e. CAEFHRWSSYMVHWK (the C was added to link KLH to the A at the N-terminus of the T14 peptide).

Preferably, the antibody or antigen-binding fragment thereof does not bind to SEQ ID No:2. Preferably, the antibody or antigen-binding fragment thereof does not bind to SEQ ID No:4.

Polyclonal antibodies may be produced as polyclonal sera by injecting antigen into animals. Preferred polyclonal antibodies may be raised by inoculating an animal (e.g. a rabbit) with antigen (e.g. T14 or fragments thereof, including the C-terminus) using techniques known to the art. For example, the antibody or antigen-binding fragment thereof may be obtained by immunising a host animal with SEQ ID No:3, and then collecting the antibody or antigen-binding fragment thereof. The host animal is most preferably a rabbit.

In another preferred embodiment, the inhibitor comprises a monoclonal antibody or an antigen-binding fragment thereof. Preferred monoclonal antibodies may be raised using hybridoma technology using T14 or fragments thereof as antigen. Preferably, the antibody of the invention is a human antibody. As used herein, the term “human antibody” can mean an antibody, such as a monoclonal antibody, which comprises substantially the same heavy and light chain CDR amino acid sequences as found in a particular human antibody exhibiting immunospecificity for SEQ ID No:3. An amino acid sequence, which is substantially the same as a heavy or light chain CDR, exhibits a considerable amount of sequence identity when compared to a reference sequence. Such identity is definitively known or recognizable as representing the amino acid sequence of the particular human antibody. Substantially the same heavy and light chain CDR amino acid sequence can have, for example, minor modifications or conservative substitutions of amino acids. Such a human antibody maintains its function of selectively binding to SEQ ID No:3.

Conventional hybridoma techniques may be used to raise the antibodies. The antigen used to generate monoclonal antibodies may be the whole T14 protein or a fragment thereof. Preferred fragments for generating the antibodies may also be the peptides discussed above, and particularly SYMVHWK (SEQ ID No: 5) or VHWK (SEQ ID No: 6). It is preferred that the antibody is a γ-immunoglobulin (IgG).

Another preferred inhibitor according to the invention is an inactive peptide fragment of T14 peptide which will compete with endogenous T14 and thereby reduce its activity. For instance, truncation mutants of T14 that do not bind to the allosteric site on the alpha-7 receptor and which inhibit the ability of T14 to interact with the allosteric site on the alpha-7 receptor may also be used as inhibitors of the invention.

In another embodiment, the inhibitor may prevent or reduce post-translational cleavage of acetyl cholinesterase enzyme to form the T14 peptide, i.e. (d) above. For example, the inhibitor according to the invention may be a gene-silencing molecule configured to block or disrupt enzymatic cleavage of acetyl cholinesterase polypeptide into T14.

The term “gene-silencing molecule” can mean any molecule that interferes with the expression of the T14 peptide. Such molecules include, but are not limited to, RNAi molecules, including siNA, siRNA, miRNA, ribozymes and antisense molecules.

As discussed above, the gene encoding acetylcholinesterase is expressed and translated into the protein shown as SEQ ID NO: 1. Post-translational modifications of SEQ ID No:1 can include cleavage by a protease to create T14 (SEQ ID No:3). Accordingly, in a preferred embodiment, the inhibitor comprises a gene-silencing molecule which reduces or prevents expression of the protease responsible for cleaving off T15 (SEQ ID No:4) from T30 to create T14.

In another embodiment, the inhibitor may inhibit of T14 translocation to the alpha-7 receptor (i.e. (e) above).

It will be appreciated that inhibitors according to the invention may be used in a medicament, which may be used as a monotherapy (i.e. use of the T14 inhibitor alone), for treating, ameliorating, or preventing cancer or metastasis. Alternatively, the T14 inhibitor may be used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing cancer or metastasis, such as radiotherapy or chemotherapy, or any of the known anticancer drugs, such as Lapatinib.

The inhibitors according to the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment. It will be appreciated that the vehicle of medicaments according to the invention should be one which is well-tolerated by the subject to whom it is given, and preferably enables delivery of the inhibitor to the target site, for example across the blood-brain barrier when treating brain tumours.

Inhibitors according to the invention may also be incorporated within a slow- or delayed-release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months. The device may be located at least adjacent the treatment site. Such devices may be particularly advantageous when long-term treatment with inhibitors used according to the invention is required and which would normally require frequent administration (e.g. at least daily injection).

In a preferred embodiment, medicaments according to the invention may be administered to a subject by injection into the blood stream or directly into a site requiring treatment. For example, the medicament may be administered by injection into the blood stream. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion).

It will be appreciated that the amount of the inhibitor that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the inhibitor and whether it is being used as a monotherapy or in a combined therapy. The frequency of administration will also be influenced by the half-life of the inhibitor within the subject being treated. Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular inhibitor in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the cancer or metastasis. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.

Generally, a daily dose of between 0.001 μg/kg of body weight and 10 mg/kg of body weight, or between 0.01 μg/kg of body weight and 1 mg/kg of body weight, of the inhibitor according to the invention may be used for treating, ameliorating, or preventing cancer or metastasis, depending upon which inhibitor is used.

The inhibitor may be administered before, during or after onset of cancer. Daily doses may be given as a single administration (e.g. a single daily injection or inhalation of a nasal spray). Alternatively, the inhibitor may require administration twice or more times during a day. As an example, inhibitors may be administered as two (or more depending upon the severity of the cancer or metastasis being treated) daily doses of between 0.07 μg and 700 mg (i.e. assuming a body weight of 70 kg). A patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter. Alternatively, a slow release device may be used to provide optimal doses of inhibitor according to the invention to a patient without the need to administer repeated doses.

Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific formulations of the inhibitor according to the invention and precise therapeutic regimes (such as daily doses of the agents and the frequency of administration). The inventors believe that they are the first to suggest an anti-cancer treatment composition, based on the use of inhibitors of the invention.

Hence, in a third aspect of the invention, there is provided an anti-cancer or anti-metastatic pharmaceutical composition comprising a therapeutically effective amount of the inhibitor according to the first aspect, and optionally a pharmaceutically acceptable vehicle.

The invention also provides in a fourth aspect, a process for making the anti-cancer or anti-metastatic pharmaceutical composition according to the third aspect, the process comprising combining a therapeutically effective amount of inhibitor according to the first aspect, with a pharmaceutically acceptable vehicle.

The inhibitor of the first aspect inhibits the synthesis and/or activity of a peptide of SEQ ID No: 3, as described above.

A “subject” may be a vertebrate, mammal, or domestic animal. Hence, medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or may be used in other veterinary applications. Most preferably, however, the subject is a human being.

A “therapeutically effective amount” of the inhibitor is any amount which, when administered to a subject, is the amount of active agent that is needed to treat the cancer or metastasis, or produce the desired effect. The inhibitor may be used as an adjuvant for the treatment of solid or metastatic tumours, for example with chemotherapy or radiotherapy. This means that lower doses and exposure times of chemotherapy and/or radiotherapy are required.

For example, the therapeutically effective amount of inhibitor used may be from about 0.001 mg to about 800 mg, and preferably from about 0.01 mg to about 500 mg.

A “pharmaceutically acceptable vehicle” as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.

In one embodiment, the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet. However, the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution. Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection.

The inhibitor and compositions of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like. The inhibitor used according to the invention can also be administered orally either in liquid or solid composition form. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

Knowledge of the surprising role that T14 plays in cancer, and especially metastatic disease, as evidenced by the CD44 data in Example 2, has enabled the inventors to develop a screen for identifying whether or not test compounds are putative inhibitors for treating, preventing or ameliorating cancer or metastasis.

Thus, according to a fifth aspect of the present invention, there is provided a method of screening a candidate compound to test whether or not the compound has efficacy for treating, preventing or ameliorating cancer or metastatic disease, the method comprising:(i) exposing a biological system to the candidate compound;(ii) detecting the concentration, expression or activity of the T14 peptide of SEQ ID No:3 in the biological system; and(iii) comparing the concentration, expression or activity of T14 peptide in the biological system treated with the compound relative to the concentration, expression or activity found in a control biological system that was not treated with the compound,wherein a compound with efficacy for treating, preventing or ameliorating cancer or metastatic disease decreases the concentration, expression or activity of the T14 peptide relative to the control.

It will be appreciated that the method according to the fifth aspect of the invention may be adapted such that it can be used to test whether or not a compound actually causes cancer or metastasis.

Therefore, according to a sixth aspect, there is provided a method of screening a compound to test whether or not the compound causes cancer, the method comprising:(i) exposing a biological system to the test compound;(ii) detecting the concentration, expression or activity of the T14 peptide of SEQ ID No:3 in the biological system; and(iii) comparing the concentration, expression or activity of the T14 peptide in the biological system treated with the compound relative to the concentration, expression or activity of the T14 peptide found in a control biological system that was not treated with the compound,wherein a compound that is carcinogenic or which increases metastasis increases the concentration, expression or activity of the T14 peptide relative to the control.

The screening methods or assays of the invention are based upon the inventors' realisation that the extent of T14 expression and/or activity is closely related to the development of cancer and metastasis. The screening method of the fifth aspect of the invention is particularly useful for screening libraries of compounds to identify compounds that may be used as anti-cancer or anti-metastasis agents used according to the first aspect of the invention. The sixth aspect of the invention may be used to identify compounds that are carcinogenic, and which should be avoided. Accordingly, the screen according to the sixth aspect of the invention may be used for environmental monitoring (e.g. to test effluents from factories) or in toxicity testing (e.g. to test the safety of putative pharmaceuticals, cosmetics, foodstuffs and the like).

The term “biological system” can mean any experimental system that would be understood by a skilled person to provide insight as to the effects a compound may have on the concentration, expression or activity of the T14 peptide in the physiological environment. The system may comprise: (a) an experimental test subject when an in vivo test is to be employed; (b) a biological sample derived from a test subject (for instance: blood or a blood fraction (e.g. serum or plasma), lymph or a cell/biopsy sample); (c) a cell line model (e.g. a cell naturally expressing the T14 peptide or a cell engineered to express T14); or (d) an in vitro system that contains T14 or its gene and simulates the physiological environment such that the concentration, expression or activity of the T14 peptide can be measured.

The screen preferably assays biological cells or lysates thereof. When the screen involves the assay of cells, they may be contained within an experimental animal (e.g. a mouse or rat) when the method is an in vivo based test. Alternatively, the cells may be in a tissue sample (for ex vivo based tests) or the cells may be grown in culture. It will be appreciated that such cells should express, or may be induced to express, functional (i.e. toxic) T14. It is also possible to use cells that are not naturally predisposed to produce T14 provided that such cells are transformed with an expression vector. Such cells represent preferred test cells for use according to the fifth or sixth aspects of the invention. This is because animal cells or even prokaryotic cells may be transformed to express human T14 peptide and therefore represent a good cell model for testing the efficacy of candidate drugs for use in human therapy.

It is most preferred that biological cells used according to the screening methods of the present invention are derived from a subject and in particular xenograft models of cancer (e.g. mouse xenografts).

With regards to “detecting the concentration, expression or activity of the T14 peptide” according to the screening methods of the present invention, the term “activity” can mean the detection of T14 interaction with the allosteric site on the alpha-7 receptor or determination of an end-point physiological effect. The term “expression” can mean detection of the T14 protein in any compartment of the cell (e.g. in the cytosol, the Endoplasmic Reticulum or the Golgi Apparatus).

Expression of T14 in the biological system may be detected by Western Blot, immuo-precipitation (IP) or co-IP, or immunohistochemistry.

The screening methods may also be based upon the use of cell extracts comprising T14 peptide. Such extracts are preferably derived from the cells described above.

The concentration, expression or activity of the T14 peptide may be measured using a number of conventional techniques. The test may be an immunoassay-based test. For instance, labelled antibodies may be used in an immunoassay to evaluate binding of a compound to T14 in the sample. T14 peptide may be isolated and the amount of label bound to it detected. A reduction in bound label (relative to controls) would suggest that the test compound competes with the label for binding to T14 and that it was also a putative anti-cancer or anti-metastasis agent. Alternatively, a functional activity measuring T14 peptide activity may be employed.

Furthermore, molecular biology techniques may be used to detect T14 peptide in the screen. For instance, cDNA may be generated from mRNA extracted from tested cells or subjects and primers designed to amplify test sequences used in a quantitative Polymerase Chain Reaction to amplify from cDNA. When a subject is used (e.g. an animal model or even an animal model engineered to express human T14), the test compound should be administered to the subject for a pre-determined length of time and then a sample taken from the subject for assaying concentration, expression or activity of the T14 peptide. The sample may for instance be blood or biopsy tissue.

As discussed in the Examples, the inventors have clearly shown that toxic T14 peptide is readily detectable outside cancer cells (i.e. within the cell culture media), whereas AChE and alpha-7 nicotinic receptor were not. Moreover, the inventors have demonstrated a strong correlation between the metastatic marker (CD44) and the toxic molecule T14 peptide. This correlation holds true for both within the cancer cell membrane and within the cancer cell cytosol. Accordingly, the inventors believe that T14 represents a robust biomarker for cancer per se, and in particular, for metastasis.

Thus, according to a seventh aspect of the invention, there is provided the use of a peptide of SEQ ID No: 3, or a fragment or variant thereof, as a diagnostic or prognostic biomarker for cancer or metastatic disease.

Most preferably, the invention provided use of a peptide of SEQ ID No: 3, or a fragment or variant thereof, as a diagnostic or prognostic biomarker for metastatic disease.

According to an eighth aspect, there is provided a method for diagnosing an individual who suffers from cancer or metastatic disease, or has a pre-disposition thereto, or for providing a prognosis of an individual's condition, the method comprising detecting, in a sample obtained from the individual, for the presence of a peptide of SEQ ID No: 3, or a fragment or variant thereof, wherein the presence of the peptide of SEQ ID No: 3, or a fragment or variant thereof, means that the individual suffers cancer or metastatic disease, or has a pre-disposition thereto, or the individual's condition has a negative prognosis.

According to a ninth aspect of the invention, there is provided a kit for diagnosing a subject suffering from cancer or metastatic disease, or a pre-disposition thereto, or for providing a prognosis of the subject's condition, the kit comprising detection means for determining the concentration of a peptide of SEQ ID No: 3, or a fragment or variant thereof, present in a sample from a test subject, wherein presence of the peptide of SEQ ID No: 3, or a fragment or variant thereof, in the sample suggests that the subject suffers from cancer or metastatic disease, or a pre-disposition thereto.

Advantageously, the use, method and kit of the invention enable the diagnosis or prognosis of an individual who suffers from cancer or metastatic disease, or has a pre-disposition thereto, most preferably metastasis. The inventors have found that the toxic T14 peptide is a robust biomarker for identifying individuals who suffer from cancer or metastatic disease. Thus, individuals diagnosed with cancer or metastatic disease, or who have a predisposition thereto, or have a negative prognosis, according to the use, methods and kit of the invention, can benefit from the early therapeutic treatment in order to prevent the onset of cancer or metastatic disease.

The individual may be a vertebrate, mammal, or domestic animal. Most preferably, however, the individual is a human being. The individual may be a child or adult. Preferably, the method is carried out in vitro.

Preferably, the sample comprises a biological sample. The sample may be any material that is obtainable from an individual from which protein is obtainable. Furthermore, the sample may be blood, plasma, serum, spinal fluid, urine, sweat, saliva, tears, breast aspirate, prostate fluid, seminal fluid, vaginal fluid, stool, cervical scraping, cytes, amniotic fluid, intraocular fluid, mucous, moisture in breath, animal tissue, cell lysates, tumour tissue, hair, skin, buccal scrapings, lymph, interstitial fluid, nails, bone marrow, cartilage, prions, bone powder, ear wax, or combinations thereof.

The sample may comprise blood, urine, tissue etc. Most preferably, the sample comprises a blood sample. The blood may be venous or arterial blood.

The kit may comprise a sample collection container for receiving the extracted sample. Blood samples may be assayed for T14 levels immediately. Alternatively, the blood sample may be stored at low temperatures, for example in a fridge or even frozen before the T14 assay is conducted. Detection of T14 may be carried out on whole blood. Preferably, however, the blood sample comprises blood serum. Preferably, the blood sample comprises blood plasma.

The blood may be further processed before the T14 assay is performed. For instance, an anticoagulant, such as citrate (such as sodium citrate), hirudin, heparin, PPACK, or sodium fluoride may be added. Thus, the sample collection container may contain an anticoagulant in order to prevent the blood sample from clotting. Alternatively, the blood sample may be centrifuged or filtered to prepare a plasma or serum fraction, which may be used for analysis. Hence, it is preferred that the T14 is analysed or assayed in a blood plasma or a blood serum sample. It is preferred that T14 concentration is measured in vitro from a blood serum sample or a plasma sample taken from the individual.

Preferably, the kit or method is used to identify the presence or absence of T14-positive cells (i.e. cells comprising SEQ ID No:3) in the sample, or determine the concentration thereof in the sample. The detection means may comprise an assay adapted to detect the presence and/or absence of T14-positive cells in the sample. The kit or method may comprise the use of a positive control and/or a negative control against which the assay may be compared. For example, the kit preferably comprises a reference for the concentration of T14-positive cells in a sample from an individual who does (i.e. positive control) suffer from cancer or metastatic disease and/or a reference for the concentration of T14-negative cells in a sample from someone who does not (i.e. a negative control) suffer from cancer or metastatic disease.

T14 peptide (SEQ ID No: 3) may be assayed by a number of ways known to one skilled in the art. For example, preferably, immunoassays may be employed to measure T14 peptide levels. However, it will be appreciated that non-immuno based assays may be employed, for example, labelling a compound having affinity with a ligand of the T14 peptide molecule, and then assaying for the label.

T14 peptide may also be determined with Western Blot analysis. Hence, immunoassays and Western blot analyses may be used to determine the total protein level of T14 peptide. T14 peptide concentration may also be detected by enzyme-linked immunosorbent assay (ELISA), fluorometric assay, chemiluminescent assay, or radioimmunoassay analyses.

The kit may further comprise a label which may be detected. The term “label” can mean a moiety that can be attached to the detection means, or fragment thereof. Moieties can be used, for example, for therapeutic or diagnostic procedures. Therapeutic labels include, for example, moieties that can be attached to an antibody or fragment thereof of the invention and used to monitor the binding of the antibody to T14 peptide (i.e. SEQ ID No:3). As described herein the antibody or antigen-binding fragment thereof binds specifically to SEQ ID No:3, or a fragment or variant thereof, and can be used as the detection means, or in the detection means. Preferably, the antibody or antigen-binding fragment thereof does not bind to SEQ ID No:2 (i.e. T30). Preferably, the antibody or antigen-binding fragment thereof does not bind to SEQ ID No:4 (i.e. T15).

Diagnostic labels include, for example, moieties which can be detected by analytical methods. Analytical methods include, for example, qualitative and quantitative procedures. Qualitative analytical methods include, for example, immunohistochemistry and indirect immunofluorescence. Quantitative analytical methods include, for example, immunoaffinity procedures such as radioimmunoassay, ELISA or FACS analysis. Analytical methods also include both in vitro and in vivo imaging procedures. Specific examples of diagnostic labels that can be detected by analytical means include enzymes, radioisotopes, fluorochromes, chemiluminescent markers, and biotin.

Examples 3 and 4 describe ELISA and Western Blot data for T14 in a range of cancer patients. It will be appreciated that the concentration of T14 peptide in cancer patients is highly dependent on a number of factors, for example, how far the cancer has progressed. It will also be appreciated that the concentration of T14 peptide in individuals who do not suffer from cancer may fluctuate to some degree, but that on average over a given period of time, the concentration tends to be substantially constant. In addition, it should be appreciated that the concentration of T14 peptide in one group of individuals who do not suffer from cancer may be different to the concentration of T14 peptide in another group of individuals who do not suffer from cancer. However, the skilled technician will know how to determine the average concentration of T14 peptide in individuals who do not suffer from cancer, and this is referred to as the ‘normal’ concentration of T14 peptide. The normal concentration corresponds to the reference values discussed above.

T14 peptide serum or T14 peptide plasma concentrations may be measured by double-antibody sandwich ELISA, which will be known to the skilled technician. The ELISA may comprise using a suitable antibody for coating a microtiter plate. For example, such a suitable antibody may comprise an affinity-purified polyclonal rabbit anti-human T14 peptide antibody. Furthermore, the ELISA may comprise using a suitable antibody for detection. For example, such a suitable antibody may comprise peroxidase-labeled monoclonal mouse anti human T14 peptide antibody. Human T14 peptide, which may be purified from plasma, and which then may be quantified by amino acid analysis, may be used to calibrate a plasma standard using standard techniques known to the skilled technician.

A label can be attached directly to the antibody, or be attached to the secondary binding agent that specifically binds T14. Such a secondary binding agent can be, for example, a secondary antibody. A secondary antibody can be either polyclonal or monoclonal, and of human, rodent or chimeric origin.

Due to current limitations of the assays (both western blot and ELISA), all values in both cases can currently only be considered relative different groups. However, whilst ELISA data may not be totally conclusive, there was a surprisingly clear and significant difference between groups using western blot analysis where poor survival with longer time to first treatment corresponded (P=0.01) to lower levels of T14. Since western blots will measure aggregates of the peptide, these data suggest that there will be more free monomer to act as a signalling molecule in the circulation of those yet to be treated and therefore with poorer prognosis. The fact that data with ELISA is, at present, inconclusive could be that the free monomer that it would be measuring has been taken up and/or is bound to its respective targets, mediating cell migration.

Preferably, therefore, western blot is used to detect T14 and/or quantify T14 levels.

The invention further extends to methods of treatment.

Hence, according to a tenth aspect of the invention, there is provided a method of treating an individual having a susceptibility to developing cancer or metastatic disease, the method comprising:

(i) determining the concentration of a peptide of SEQ ID No: 3, or a fragment or variant thereof, present in a sample from a test subject, wherein presence of the peptide of SEQ ID No: 3, or a fragment or variant thereof, in the sample suggests that the subject suffers from cancer or metastatic disease, or a pre-disposition thereto; and

(ii) administering, to the individual, a therapeutic agent or treatment regime that prevents, reduces or delays the development of cancer or metastatic disease.

Examples of suitable therapeutic agents which may be administered include but are not limited to Herceptin. Examples of suitable treatment regimes include radiotherapy or chemotherapy.

It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof. The terms “substantially the amino acid/nucleotide/peptide sequence”, “functional variant” and “functional fragment”, can be a sequence that has at least 40% sequence identity with the amino acid/nucleotide/peptide sequences of any one of the sequences referred to herein, for example 40% identity with the sequence identified as SEQ ID No:1-7, and so on.

Amino acid/polynucleotide/polypeptide sequences with a sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged. Preferably, the amino acid/polynucleotide/polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein.

The skilled technician will appreciate how to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences. In order to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences, an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on: (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants.

Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.

Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the invention. Suitable parameters for ClustalW may be as follows: For DNA alignments: Gap Open Penalty=15.0, Gap Extension Penalty=6.66, and Matrix=Identity. For protein alignments: Gap Open Penalty=10.0, Gap Extension Penalty=0.2, and Matrix=Gonnet. For DNA and Protein alignments: ENDGAP=−1, and GAPDIST=4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment.

Preferably, calculation of percentage identities between two amino acid/polynucleotide/polypeptide sequences may then be calculated from such an alignment as (N/T)*100, where N is the number of positions at which the sequences share an identical residue, and T is the total number of positions compared including gaps but excluding overhangs. Hence, a most preferred method for calculating percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and T into the following formula:—Sequence Identity=(N/T)*100.

Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleotide sequence will be encoded by a sequence, which hybridizes to DNA sequences or their complements under stringent conditions. By stringent conditions, we mean the nucleotide hybridises to filter-bound DNA or RNA in 3× sodium chloride/sodium citrate (SSC) at approximately 45° C. followed by at least one wash in 0.2×SSC/0.1% SDS at approximately 20-65° C. Alternatively, a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in SEQ ID No: 1-4.

Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof. Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change. Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change. For example small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.

All of the features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

