cancer

Abstract

The present invention relates to cancer, and more particularly to novel pharmaceutical compositions, therapies and methods for treating, preventing or alleviating cancer, especially metastatic disease. The present invention also relates to methods for diagnosing and prognosing cancer and metastatic disease, and biomarkers for these conditions.

Description

Technical Field

[0001] The present invention relates to cancer, and more particularly to novel pharmaceutical compositions, therapies and methods for treating, preventing or alleviating cancer, especially metastatic disease. The present invention also relates to methods for diagnosing and prognosing cancer and metastatic disease, and biomarkers for these conditions. Background Art

[0002] Cancer and malignancies are diseases involving abnormal cell growth that have the potential to invade or spread to other parts of the body (i.e., metastasize). In 2012, there were approximately 14 million new cases of cancer worldwide. Therefore, there is a need to provide improved diagnostics and therapies for the treatment of cancer and metastasis.

[0003] Acetylcholinesterase (AChE) is expressed in different forms at different stages of development, all of which have the same enzymatic activity, but have very different molecular compositions. The 'tailed' (T-AChE - SEQ ID No: 1) is expressed at the synapse, and the inventors have previously identified two peptides that can be cleaved from the C-terminus, one called "T14" (SEQ ID No: 3), which is located within the other called "T30" (SEQ ID No: 2), both of which have strong sequence homology to similar regions of β-amyloid protein. The C-terminal peptide "T14" of AChE has been identified as a protruding part of the AChE molecule that is responsible for its series of non-hydrolytic reactions. A synthetic 14 amino acid peptide analog (i.e. "T14"), and subsequently the larger, more stable, and more potent amino acid sequence into which it was embedded (i.e. "T30" - SEQ ID No: 3) showed effects similar to those reported for 'non-cholinergic' AChE, whereas an inert residue within the T30 sequence (i.e. "T15" - SEQ ID No: 4) had no effect.

[0004] The T14 peptide binds to an allosteric site on the α7 nicotinic receptor, which has no effect on its own. However, in the presence of primary ligands like acetylcholine or dietary choline, T14 enhances the calcium influx caused by these primary agents. Excess calcium can be absorbed into the mitochondria, where it disrupts oxidative phosphorylation and causes electron leakage. As a result, free radicals are formed, which then destabilize the cell membrane and then the cell dies. Summary of the invention

[0005] The inventors used protein immunoblotting to study the levels of toxic T14 peptides, α7 nicotinic receptors and acetylcholinesterase proteins in cell lysates and cell culture media of 7 cancer cell lines (MEC-1, KG1a, H929, MCF-7, MDA-MB231, CLL, and JJN3). They found that only toxic T14 peptides were released from cancer cells, and the extracellular T14 concentration was higher than the T14 level in the cancer cells. The inventors then continued to study the relationship between the known metastatic markers CD44, AchE and α7 nicotinic receptors with toxic T14 peptides in the cell membrane and cytosol of the cancer cell lines. They were surprised to observe that CD44 was significantly positively correlated with the toxic molecule T14. In short, these data strongly indicate that toxic T14 peptides, including their biosynthesis and metabolic pathways, represent good targets for treating cancer and metastasis, and in addition, T14 itself serves as a robust cancer metastasis biomarker.

[0006] Therefore, in a first aspect of the present invention, there is provided an inhibitor of the synthesis and / or activity of the peptide of SEQ ID No: 3 for use in treating, alleviating or preventing cancer or metastatic disease.

[0007] In a second aspect, a method of treating, ameliorating or preventing cancer or metastatic disease in a subject is provided, 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 the peptide of SEQ ID No: 3.

[0008] As described in Example 1, the inventors used Western blotting to detect the levels of toxic T14 peptide (SEQ ID No: 3), nicotinic α-7 receptor and acetylcholinesterase (AChE) proteins in cell lysates and cell culture media of 7 cancer cell lines (MEC-1, KG1a, H929, MCF-7, MDA-MB231, CLL, and JJN3) and normal B lymphocytes serving as controls. MDA-MB-231, KG1a, and MEC-1 cells are highly migratory cancer cell lines, while H929, JJN3, CLL, and MCF-7 are less migratory cancer cell lines, and B lymphocytes are normal non-cancerous cells. T14, α7 receptor, and AChE were all detected in cancer cells. Surprisingly, all three proteins had similar mobility, and their levels were positively correlated with each other, indicating that they were complexed with each other. However, outside the cancer cells (i.e., in the cell culture medium), only the toxic T14 peptide was detected. Even more surprising is that in 6 of 7 cancer cell lines, the level of T14 outside the cells was higher than the level of T14 inside the cells, indicating that T14 is produced and released from the cells. The inventors were also surprised to see that the level of T14 outside the cancer cells was significantly negatively correlated with the level of α-7 receptor inside the cancer cells.

[0009] Example 2 describes the use of Western immunoblotting to show the relationship between the well-known metastatic marker CD44, AchE and α-7 receptor with toxic T14 peptide in the cell membrane and cytosol of 6 cancer cell lines (JJN3, MDA-MB231, MCF-7, KG1a, MEC-1, and H929) and another cancer-derived cell line (PC12). In all cancer cell lines, the metastatic marker (CD44) is significantly positively correlated with the toxic molecule T14, and this correlation is also applicable in the cancer cell membrane and in the cytosol of the cancer cell. These findings strongly indicate that T14 is a good predictor of the extent of cancer and metastasis (i.e., tumor cell migration).

[0010] The cancer treated according to the first aspect or the second aspect can be a leukemia. For example, the cancer can be a lymphocytic leukemia or a chronic lymphocytic leukemia (CLL). The cancer can be a myeloid leukemia or an acute myeloid leukemia. The cancer can be a multiple myeloma. The cancer can be a breast cancer. The cancer can be a plasmacytoma.

[0011] Most preferably, inhibitors of the synthesis and / or activity of the T14 peptide (SEQ ID No: 3) are used to treat, alleviate or prevent metastatic disease or metastasis.

[0012] Acetylcholinesterase is a serine protease that hydrolyzes acetylcholine and is well known to those skilled in the art. The major form of acetylcholinesterase is found in the brain and is called tailed acetylcholinesterase (T-AChE). The protein sequence of an example of human tailed acetylcholinesterase (Gen Bank: AAA68151.1) has a length of 614 amino acids and is provided herein as SEQ ID No: 1 as follows:

[0013]

[0014] It will be appreciated that the first 31 amino acid residues of SEQ ID No: 1 are removed when the protein is released, leaving a 583 amino acid sequence.

[0015] 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:-

[0016] KAEFHRWSSYMVHWKNQFDHYSKQDRCSDL

[0017] [SEQ ID No: 2]

[0018] The amino acid sequence of the toxic T14 peptide (which corresponds to 14 amino acid residues near the end of SEQ ID No: 1 and lacks the last 15 amino acids present in T30) is provided herein as SEQ ID No: 3 as follows:-

[0019] AEFHRWSSYMVHWK

[0020] [SEQ ID No: 3]

[0021] 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:-

[0022] NQFDHYSKQDRCSDL

[0023] [SEQ ID No: 4]

[0024] The inventors have discovered that inhibition of the toxic T14 peptide is required to effectively treat cancer or metastasis. The inhibitors of the present invention can be used to prevent the development of cancer or the development and spread of metastasis. For example, the inhibitors can be administered to subjects at risk for the development of cancer or metastasis (e.g., with a genetic predisposition or exposure to a harmful environment). The inhibitors can also be used after surgery, radiotherapy, or chemotherapy to prevent the reappearance of cancer in a subject.

[0025] The inventors have shown that the toxic T14 peptide binds to an allosteric site on the α7 nicotinic receptor, which itself has no effect on the cell. However, in the presence of primary ligands like acetylcholine or dietary choline, T14 enhances the calcium influx into the cell caused by these primary agents. Excess calcium is taken up into the mitochondria, where it disrupts oxidative phosphorylation and causes electron leakage. Free radicals are then formed, which then destabilize the cell membrane, and the cell dies.

[0026] Based on the above understanding, the inventors have designed inhibitors that can reduce the synthesis or biological activity of toxic T14 peptides, which can achieve their effects through many biosynthetic and / or metabolic pathways. 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 manages to reach the allosteric site maintains interaction with the receptor, and this interaction can cause more receptor upregulation within 24 hours, that is, T14 can increase its own target. Therefore, preventing the interaction of T14 with the allosteric site is most preferred.

[0027] For example, such inhibitors can:

[0028] (a) reducing the interaction between the T14 peptide and the allosteric site on the α-7 receptor;

[0029] (b) compete with endogenous T14 peptide for the allosteric site on the α-7 receptor;

[0030] (c) binding to the T14 peptide to reduce its biological activity;

[0031] (d) reducing post-translational cleavage of the acetylcholinesterase polypeptide to produce the T14 peptide; or

[0032] (e) Inhibits the translocation of T14 to the allosteric site on the α-7 receptor.

[0033] In a preferred first embodiment of the invention, the inhibitor can directly interact with the T14 peptide (e.g., (a) to (c) above) to reduce the biochemical and / or metabolic activity of T14 in the subject's cells, and thus reduce the toxicity of T14. Preferably, the inhibitor reduces the interaction of T14 with the allosteric site on the alpha-7 receptor.

[0034] Preferred inhibitors include small molecule inhibitors. Such inhibitors can be determined as part of a small molecule library high throughput screening. For example, the screening method according to the fifth aspect of the invention (see below) represents a suitable means for determining such inhibitors that can be used to treat cancer or metastasis.

[0035] In a preferred embodiment, the inhibitor comprises a compound of formula (I):

[0036]

[0037] in:

[0038] R1 is -NR9R 10 or -OH;

[0039] R2 is

[0040] R3 is -H or C 1-5 Straight or branched chain alkyl or alkenyl;

[0041] R4 is

[0042] R5 is -H or C 1-5 Straight or branched chain alkyl or alkenyl;

[0043] R6 is

[0044] R7 is -H or C 1-5 Straight or branched chain alkyl or alkenyl;

[0045] R8 is -H,; C 1-5 Straight or branched chain alkyl or alkenyl or

[0046] X1-NR9R 10 , -OH or

[0047] The or each R9 and R 10 independently -H or C 1-5 Straight or branched chain alkyl or alkenyl;

[0048] R 11 is -NH2, -OH or aryl;

[0049] The or each m is independently between 0 and 5; and

[0050] Each n is independently between 0 and 10;

[0051] or a pharmaceutically acceptable salt, solvate, tautomeric form, or polymorphic form thereof.

[0052] It will be appreciated that the term "salt" refers to any salt of a compound provided herein that retains its biological properties, is not toxic for pharmaceutical use, and is not otherwise undesirable. Such salts may be derived from various organic and inorganic counterions 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 acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, sulfamic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, propionic acid, hexanoic acid, cyclopentylpropionic acid, glycolic acid, glutaric acid, pyruvic acid, lactic acid, malonic acid, succinic acid, sorbic acid, ascorbic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxyphenyl)-1-ol benzoic acid, picric acid, cinnamic acid, mandelic acid, phthalic acid, lauric acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphoric acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, lauryl sulfuric acid, glucoheptonic acid, or (2) base addition salts formed when the acid proton present in the parent compound is (a) replaced by a metal ion (e.g., an alkali metal ion, an alkaline earth ion or an aluminum ion) or an alkali metal or alkaline earth metal hydroxide (e.g., hydroxides of sodium, potassium, calcium, magnesium, aluminum, lithium, zinc, and barium), ammonia, or (b) coordinated with an organic base (e.g., an aliphatic, alicyclic, or aromatic organic amine, such as ammonia, methylamine, dimethylamine, diethylamine, picolin, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, lysine, arginine, ornithine, choline, N,N′-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, N-methylglucaminepiperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, and the like).

[0053] Salts may further include, by way of example only, but without limitation, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like, and when the compound contains a basic functional group, include salts of non-toxic organic or inorganic acids, such as hydrohalides (e.g., hydrochlorides and hydrobromides), sulfates, phosphates, sulfamates, nitrates, acetates, trifluoroacetates, trichloroacetates, propionates, hexanoates, cyclopentylpropionates, glycolates, glutarates, pyruvates, lactates, malonates, succinates, sorbates, ascorbates, malates, maleates, fumarates, tartrates, citrates, benzoates, 3-(4-hydroxybenzoyl)benzoate, picrates, cinnamates, mandelates, o-hydroxybenzoate, benzoic acid ... Phthalate, laurate, methanesulfonate (mesylate), ethylsulfonate, 1,2-ethane-disulfonate, 2-hydroxyethylsulfonate, 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, cyclohexylaminosulfonate, quinate, muconate, and the like.

[0054] It is understood that the term "solvate" refers to a compound or salt thereof provided herein that further includes a stoichiometric or non-stoichiometric amount of a solvent bound by non-covalent intermolecular forces. In the case where the solvent is water, the solvate is a hydrate.

[0055] It is understood that aryl refers to a substituent derived from an aromatic ring. Aryl can be C6-C12 aryl. Aryl is preferably phenyl, biphenyl or naphthyl.

[0056] Most preferably, the compound has formula (Ia):

[0057]

[0058] R1 may be -OH. However, preferably, R1 is -NR9R 10 , more preferably R1 is -NR9H, and most preferably R1 is -NH2.

[0059] Preferably, R2 is Preferably n is between 1 and 5. Thus, n may be 1, 2, 3, 4 or 5, most preferably n is 1.

[0060] It should be understood that when R2 is In the embodiment of , then m can be 0, 1, 2, 3, 4 or 5. Preferably m is 1. Preferably, X1 is located in the para position.

[0061] In a preferred embodiment, R2 is

[0062] Preferably, when R2 is In the embodiment, then R9 or R 10 At least one of them is -H, most preferably, R9 or R 10 All are -H.

[0063] In a preferred embodiment, R2 is

[0064] In a most preferred embodiment, R2 is

[0065] It should be understood that R3 can be methyl, ethyl, propyl, butyl or pentyl. Preferably, R3 is methyl. However, in a more preferred embodiment, R3 is -H.

[0066] In one embodiment, R4 is n is preferably between 1 and 5. Thus, n may be 1, 2, 3, 4 or 5, preferably n is 1.

[0067] In R4 In the embodiment of , then n is preferably between 1 and 7, more preferably between 2 and 6. Therefore, n can be 2, 3, 4, 5 or 6, preferably n is 3 or 4.

[0068] In one embodiment, R4 is preferably

[0069] More preferably, R4 is

[0070] It should be understood that when R4 is In the embodiment of , then m can be 0, 1, 2, 3, 4 or 5. Preferably m is 1. Preferably X1 is in the para position.

[0071] Preferably, R4 is

[0072] More preferably, R4 is

[0073] Preferably, when R4 is In the embodiment, then R9 or R 10 At least one of them is -H, most preferably, R9 or R 10 All are -H.

[0074] Therefore, R4 is preferably

[0075] More preferably, R4 is

[0076] Most preferably, R4 is

[0077] It should be understood that R5 can be methyl, ethyl, propyl, butyl or pentyl. Preferably, R5 is methyl. However, in a more preferred embodiment, R5 is -H.

[0078] R6 is n is between 1 and 5. Thus, n may be 1, 2, 3, 4 or 5, preferably n is 1.

[0079] Preferably, R6 is

[0080] It should be understood that when R6 is In the embodiment of , then m can be 0, 1, 2, 3, 4 or 5. Preferably, m is 0. More preferably, m is 1. Preferably, X1 is located in the para position.

[0081] In a preferred embodiment, R6 is

[0082] Preferably, R6 is In the embodiment, then R9 or R 10 At least one of them is -H, most preferably, R9 or R 10 All are -H.

[0083] Preferably, R6 is In the embodiment, then R 11 It is an aryl group, and most preferably a phenyl group.

[0084] In a preferred embodiment, R6 is

[0085] In a most preferred embodiment, R6 is

[0086] It should be understood that R7 can be methyl, ethyl, propyl, butyl or pentyl. Preferably, R7 is methyl. However, in a more preferred embodiment, R7 is -H.

[0087] In a preferred embodiment, R8 is -H. However, in a more preferred embodiment, R8 is

[0088] In a preferred embodiment, the inhibitor comprises a compound of formula (I), wherein:

[0089] R2 is

[0090] R3 is H or C 1-5 Straight or branched chain alkyl or alkenyl;

[0091] R4 is

[0092] R5 is H or C 1-5 Straight or branched chain alkyl or alkenyl;

[0093] R6 is and

[0094] R7 is H or C 1-5 Straight-chain or branched alkyl or alkenyl.

[0095] Preferably, R3 is H, R5 is H, and R7 is H.

[0096] In a more preferred embodiment, the inhibitor comprises a compound of formula (I), wherein:

[0097] R2 is

[0098] R3 is H or C 1-5 Straight or branched chain alkyl or alkenyl;

[0099] R4 is

[0100] R5 is H or C 1-5 Straight or branched chain alkyl or alkenyl; or

[0101] R6 is and

[0102] R7 is H or C 1-5 Straight-chain or branched alkyl or alkenyl.

[0103] Preferably, the inhibitor comprises a compound of formula (Ia). Preferably, R3 is H, R5 is H, and R7 is H.

[0104] Preferably, R1 is -OH, and R8 is H. More preferably, R1 is NH2, and R8 is

[0105] In an alternative more preferred embodiment, the inhibitor comprises a compound of formula (I), wherein:

[0106] R2 is

[0107] R3 is H or C 1-5 Straight or branched chain alkyl or alkenyl;

[0108] R4 is

[0109] R5 is H or C 1-5 Straight or branched chain alkyl or alkenyl; or

[0110] R6 is and

[0111] R7 is H or C 1-5 Straight-chain or branched alkyl or alkenyl.

[0112] Preferably, the inhibitor comprises a compound of formula (Ia). Preferably, R3 is H, R5 is H, and R7 is H.

[0113] Preferably, R1 is -OH, and R8 is H. More preferably, R1 is NH2, and R8 is

[0114] In an even more preferred embodiment, the inhibitor comprises a compound of formula (I), wherein:

[0115] R2 is

[0116] R3 is H or C 1-5 Straight or branched chain alkyl or alkenyl;

[0117] R4 is

[0118] R5 is H or C 1-5 Straight or branched chain alkyl or alkenyl;

[0119] R6 is and

[0120] R7 is H or C 1-5 Straight-chain or branched alkyl or alkenyl.

[0121] Preferably, R3 is H, R5 is H, and R7 is H.

[0122] In a more preferred embodiment, the inhibitor comprises a compound of formula (I), wherein:

[0123] R2 is

[0124] R3 is H or C 1-5 Straight or branched chain alkyl or alkenyl;

[0125] R4 is

[0126] R5 is H or C 1-5 Straight or branched chain alkyl or alkenyl; or

[0127] R6 is and

[0128] R7 is H or C 1-5 Straight or branched chain alkyl or alkenyl.

[0129] Preferably, the inhibitor comprises a compound of formula (Ia). Preferably, R3 is H, R5 is H, and R7 is H.

[0130] Preferably, R1 is -OH, and R8 is H. More preferably, R1 is NH2, and R8 is

[0131] In an alternative more preferred embodiment, the inhibitor comprises a compound of formula (I), wherein:

[0132] R2 is

[0133] R3 is H or C 1-5 Straight or branched chain alkyl or alkenyl;

[0134] R4 is

[0135] R5 is H or C 1-5 Straight or branched chain alkyl or alkenyl; or

[0136] R6 is and

[0137] R7 is H or C 1-5 Straight or branched chain alkyl or alkenyl.

[0138] Preferably, the inhibitor comprises a compound of formula (Ia). Preferably, R3 is H, R5 is H, and R7 is H.

[0139] Preferably, R1 is -OH, and R8 is H. More preferably, R1 is NH2, and R8 is

[0140] Preferably, the inhibitor comprises a compound of formula (101) or formula (103):

[0141]

[0142] More preferably, the inhibitor comprises a compound of formula (101a) or formula (103a):

[0143]

[0144]

[0145] It will be appreciated that compounds of formula (101a) and formula (103a) correspond to compounds Tri02 and Tri04, respectively, which are discussed in the examples. Thus, in a further aspect, an inhibitor as defined herein is provided for use in the treatment, alleviation or prevention of cancer or metastatic disease, preferably wherein the inhibitor is preferably formula (101a) or (103a).

[0146] In a preferred embodiment, the inhibitor comprises an antibody or an antigen-binding fragment thereof, i.e., an antibody that neutralizes T14. The antibody or antigen-binding fragment thereof preferably blocks T14 from interacting with an allosteric site on the alpha-7 receptor. Preferably, the antibody preferentially binds to T14, thereby forming a complex that is unable to bind to the receptor to cause toxicity.

[0147] The antibody or antigen-binding fragment thereof can be polyclonal or monoclonal.The antibody or antigen-binding fragment thereof can be produced in rabbit, mouse or rat.

[0148] 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 acids in the C-terminus of SEQ ID No: 3. Preferably, the antibody or antigen-binding fragment thereof specifically binds to one or more amino acids in SEQ ID No: 5 (i.e., SYMVHWK, which are the 7th to 14th C-terminal amino acids of SEQ ID No: 3). Preferably, the antibody or antigen-binding fragment thereof specifically binds to the C-terminal lysine (K) residue in the epitope.

[0149] The inventors surprisingly observed that the C-terminal amino acid sequence VHWK in SEQ ID No: 3 (described herein as SEQ ID No. 6 (i.e., the 10th to 14th C-terminal amino acids of SEQ ID No. 3)) acts as an epitope for an antibody or an antigen-binding fragment thereof. Therefore, more preferably, the antibody or its antigen-binding fragment specifically binds to one or more amino acids in SEQ ID No. 6. Most preferably, the antibody or its antigen-binding fragment specifically binds to SEQ ID No. 6. It is therefore understood that the epitope to which the antibody binds comprises or consists of SEQ ID No: 6.

[0150] Based on the discovery of the VHWK epitope (SEQ ID No: 6) in the T14 peptide, the inventors believe that these sequences can be used as antigens for the production of useful antibodies. As described in the Examples, the T14 peptide (SEQ ID No: 3) is cross-linked to keyhole limpet hemocyanin (KLH) via cysteine, and KLH acts as a carrier protein to stimulate 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 (C is added to connect KLH to the A at the N-terminus of the T14 peptide).

[0151] 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.

[0152] Polyclonal antibodies can be produced as polyclonal sera by injecting the antigen into an animal. Preferred polyclonal antibodies can be raised by inoculating an animal (e.g., a rabbit) with an antigen (e.g., T14 or a fragment thereof, including the C-terminus) using techniques known in the art. For example, the antibody or antigen-binding fragment thereof can be obtained by immunizing 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.

[0153] In another preferred embodiment, the inhibitor comprises a monoclonal antibody or an antigen-binding fragment thereof. Preferred monoclonal antibodies can be cultured using hybridoma cell technology using T14 or a fragment thereof as an antigen. Preferably, the antibody of the present invention is a human antibody. As used herein, the term "human antibody" may refer to an antibody such as a monoclonal antibody, which comprises heavy chain and light chain CDR amino acid sequences that are substantially identical to the heavy chain and light chain CDR amino acid sequences present in a specific human antibody exhibiting immunospecificity for SEQ ID No: 3. Amino acid sequences that are substantially identical to the heavy chain or light chain CDR exhibit a significant amount of sequence identity when compared to a reference sequence. It is clearly known or recognized that such identity represents the amino acid sequence of a specific human antibody. Substantially identical heavy chain and light chain CDR amino acid sequences may have, for example, minor modifications or conservative substitutions of amino acids. Such a human antibody will retain its function of selectively binding to SEQ ID No: 3.

[0154] Conventional hybridoma cell technology can be used to raise antibodies. The antigen used to generate monoclonal antibodies can be the whole T14 protein or a fragment thereof. Preferred fragments used to generate antibodies can also be the peptides discussed above, particularly SYMVHWK (SEQ ID No: 5) or VHWK (SEQ ID No: 6). The preferred antibody is preferably a gamma-immunoglobulin (IgG).

[0155] Another preferred inhibitor according to the present invention is an inactive peptide fragment of the T14 peptide, which competes with endogenous T14, thereby reducing its activity. For example, a truncation mutant of T14 that does not bind to the allosteric site on the α-7 receptor and inhibits the ability of T14 to interact with the allosteric site on the α-7 receptor can also be used as an inhibitor of the present invention.

[0156] In another embodiment, the inhibitor can prevent or reduce the post-translational cleavage of acetylcholinesterase to form T14 peptide, i.e. (d) above. For example, the inhibitor according to the present invention can be a gene silencing molecule that is configured to block or disrupt the enzymatic cleavage of acetylcholinesterase polypeptide into T14.

[0157] The term "gene silencing molecule" may refer to 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.

[0158] As discussed above, the gene encoding acetylcholinesterase is expressed and translated into a protein as shown in SEQ ID NO: 1. Post-translational modification of SEQ ID No: 1 may include cleavage by a protease to produce T14 (SEQ ID No: 3). Therefore, in a preferred embodiment, the inhibitor comprises a gene silencing molecule that reduces or prevents the expression of a protease responsible for cleaving T15 (SEQ ID No: 4) from T30 to produce T14.

[0159] In another embodiment, the inhibitor can inhibit the translocation of T14 to the alpha-7 receptor (ie, (e) above).

[0160] It should be understood that the inhibitors according to the present invention can be used in medicine, which can be used as a monotherapy (i.e., using only T14 inhibitors) for the treatment, alleviation, or prevention of cancer or metastasis. Alternatively, T14 inhibitors can be used as an adjuvant to known therapies (e.g., radiotherapy or chemotherapy) or any known anticancer drugs (e.g., lapatinib) for the treatment, alleviation, or prevention of cancer or metastasis, or can be used in combination with known therapies (e.g., radiotherapy or chemotherapy) or any known anticancer drugs (e.g., lapatinib) for the treatment, alleviation, or prevention of cancer or metastasis.

[0161] The various inhibitors according to the invention may be combined in compositions that have many different forms, depending in particular on the mode of use of the composition. 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, liposomal suspension, or any other suitable form that can be administered to a human or animal in need of treatment. It will be appreciated that the carrier for the medicament according to the invention should be a carrier that is well tolerated by the subject to which it is administered, preferably enabling delivery of the inhibitor to the target site, for example, across the blood-brain barrier in the treatment of brain tumors.

[0162] The inhibitor according to the invention may also be contained in a sustained release device or delayed release device. Such a device may, for example, be inserted above or below the skin and the drug may be released over a period of weeks or even months. The device may be located at least proximal to the treatment site. Such a device may be particularly advantageous when long-term treatment with the inhibitor according to the invention is desired and frequent dosing (e.g., at least daily injections) is typically required.

[0163] In a preferred embodiment, the medicine according to the present invention can be administered to a subject by injection into the bloodstream or directly into the site in need of treatment. For example, the medicine can be administered by injection into the bloodstream. The injection can be intravenous injection (bolus or infusion) or subcutaneous injection (bolus rapid injection or infusion) or intradermal injection (bolus or infusion).

[0164] It should be understood that the amount of the inhibitor required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physicochemical properties of the inhibitor, and whether it is used as a single treatment or for combined treatment. The frequency of administration can also be affected by the half-life of the inhibitor in the subject being treated. The optimal dose for administration can be determined by those skilled in the art, and can vary with the strength of the specific inhibitor used, the pharmaceutical composition, the mode of administration, and the progress of cancer or metastasis. Other factors depend on the specific subject being treated, and the need for adjusting the dosage can be generated, including the age, weight, sex, diet, and administration time of the subject.

[0165] Typically, a daily dose of between 0.001 μg / kg and 10 mg / kg body weight, or between 0.01 μg / kg and 1 mg / kg body weight of an inhibitor according to the invention can be used to treat, alleviate, or prevent cancer or metastasis, depending on which inhibitor is used.

[0166] The inhibitor can be used before, during or after the occurrence of cancer. The daily dose can be given as a single dose (e.g., a single daily injection or inhaled nasal spray). Alternatively, the inhibitor may need to be administered twice or more in one day. As an example, the inhibitor can be administered two (or more, depending on the severity of the cancer or metastasis being treated) daily doses, between 0.07 μg and 700 mg (i.e., assuming a body weight of 70 kg). The patient receiving treatment can take the first dose after waking up, and then take the second dose in the evening (if a two-dose regimen is adopted), or take the second dose at intervals of 3 hours or 4 hours thereafter. Alternatively, a sustained-release device can be used to provide the optimal dose of the inhibitor according to the present invention to the patient without the need for repeated doses.

[0167] Specific dosage forms and precise treatment regimens (such as daily doses and dosing frequency of the agents) of the inhibitors according to the invention can be developed using known procedures, such as those routinely employed by the pharmaceutical industry (e.g., in vivo testing, clinical trials, etc.). The inventors believe that, depending on the use of the inhibitors of the invention, it is recommended that anticancer therapeutic compositions first use them.

[0168] Therefore, in a third aspect of the present 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.

[0169] In a fourth aspect, the present invention also provides a method for manufacturing an anti-cancer or anti-metastatic pharmaceutical composition according to the third aspect, the method comprising combining a therapeutically effective amount of the inhibitor according to the first aspect with a pharmaceutically acceptable carrier.

[0170] The inhibitor of the first aspect inhibits the synthesis and / or activity of the peptide of SEQ ID No: 3, as described above.

[0171] "Subject" can be a vertebrate, mammal, or livestock. Therefore, the drug according to the present invention can be used to treat any mammal, such as livestock (e.g., horses), pets, or can be used for other veterinary applications. However, most preferably, the subject is a human being.

[0172] A "therapeutically effective amount" of an inhibitor is any amount which, when administered to a subject, is the amount of active agent required to treat cancer or metastasis or produce a desired effect. The inhibitor can be used as an adjuvant in the treatment of solid tumors or metastatic tumors (e.g., using chemotherapy or radiotherapy). This means that lower doses of chemotherapy and / or radiotherapy and lower exposure times are required.

[0173] For example, a therapeutically effective amount of the inhibitor used may be from about 0.001 mg to about 800 mg, preferably from about 0.01 mg to about 500 mg.

[0174] As referred to herein, a "pharmaceutically acceptable carrier" is any known compound or combination of known compounds, which is known to those skilled in the art to be useful in formulating a pharmaceutical composition.

[0175] In one embodiment, the pharmaceutically acceptable carrier can be a solid, and the composition can be in the form of a powder or tablet. However, the pharmaceutical carrier can be a liquid, and the pharmaceutical composition is in the form of a solution. The liquid pharmaceutical composition is a sterile solution or suspension, which can be used by (for example) intramuscular injection, intrathecal injection, epidural injection, intraperitoneal injection, intravenous injection, especially subcutaneous injection.

[0176] The inhibitors and compositions of the invention can be administered orally in the form of sterile solutions or suspensions containing other solutes or suspending agents (e.g., sufficient saline or glucose to render the solution isotonic), bile salts, gum arabic, gelatin, sorbitan monooleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide). The inhibitors used according to the invention can also be administered orally in the form of liquid or solid compositions. Compositions suitable for oral administration include solid forms such as pills, capsules, granules, tablets, and powders, as well as liquid forms such as solutions, syrups and elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0177] As demonstrated by the CD44 data in Example 2, the understanding of the surprising role that T14 plays in cancer, particularly metastatic disease, enabled the inventors to develop a screen to identify whether a test compound is a putative inhibitor for treating, preventing or ameliorating cancer or metastasis.

[0178] Therefore, according to the fifth aspect of the present invention, there is provided a method for screening candidate compounds to test whether the compound has the efficacy of treating, preventing or alleviating cancer or metastatic disease, the method comprising:

[0179] (i) exposing the biological system to a candidate compound;

[0180] (ii) detecting the concentration, expression or activity of the T14 peptide of SEQ ID No: 3 in a biological system; and

[0181] (iii) comparing the concentration, expression or activity of the T14 peptide in a biological system treated with the compound with the concentration, expression or activity in a control biological system not treated with the compound,

[0182] Among them, the compound having the efficacy of treating, preventing or ameliorating cancer or metastatic disease reduces the concentration, expression or activity of T14 peptide relative to the control.

[0183] It will be appreciated that the method according to the fifth aspect of the invention may be adapted so that it can be used to test whether a compound actually causes cancer or metastasis.

[0184] Therefore, according to a sixth aspect, there is provided a method for screening a compound to test whether the compound causes cancer, the method comprising:

[0185] (i) exposing the biological system to a test compound;

[0186] (ii) detecting the concentration, expression or activity of the T14 peptide of SEQ ID No: 3 in a biological system; and

[0187] (iii) comparing the concentration, expression or activity of the T14 peptide in a biological system treated with the compound with the concentration, expression or activity of the T14 peptide in a control biological system not treated with the compound,

[0188] The oncogenic compound or the compound that increases metastasis increases the concentration, expression or activity of the T14 peptide relative to a control.

[0189] The screening method or assay of the present invention is based on the inventor's recognition that the expression level and / or activity level of T14 is closely related to the development of cancer and metastasis. The screening method of the fifth aspect of the present invention is particularly applicable to screening compound libraries to determine compounds approved for use as anticancer drugs or anti-metastatic agents according to the first aspect of the present invention. The sixth aspect of the present invention can be used to determine carcinogenic compounds that should be avoided. Therefore, the screening according to the sixth aspect of the present invention can be used for environmental monitoring (e.g., testing emissions from factories) or for toxicity testing (e.g., testing the safety of hypothetical drugs, cosmetics, foods, etc.).

[0190] The term "biological system" may refer to any test system that a skilled person would understand to be capable of providing an understanding of the effect that a compound may have on the concentration, expression or activity of a T14 peptide in a physiological environment. The system may include: (a) an experimental test subject (when an in vivo test is to be employed); (b) a biological sample derived from a test subject (e.g., blood or blood fractions (e.g., serum or plasma), lymph or cell / biopsy samples); (c) a cell line model (e.g., a cell that naturally expresses a T14 peptide, or a cell that is engineered to express T14); or (d) an in vitro system that contains T14 or its gene and stimulates a physiological environment so that the concentration, expression or activity of a T14 peptide can be measured.

[0191] Screening preferably assays biological cells or lysates thereof. When the method is based on an in vivo test, they may be contained in a test animal (e.g., mouse or rat) in the case where the screening involves assaying cells. Alternatively, the cells may be in a tissue sample (for an in vitro test), or the cells may be grown in culture. It should be understood that such cells should express, or may be induced to express, functional (i.e., toxic) T14. Cells that are naturally not prone to producing T14 may also be used, 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 present invention. This is because animal cells or even prokaryotic cells may be transformed to express human T14 peptides, and therefore represent a good cell model for testing the efficacy of candidate drugs for human treatment.

[0192] Most preferably, the biological cells used in the screening method according to the present invention are derived from a subject, in particular a cancer xenograft model (eg, mouse xenograft).

[0193] With regard to the screening method "detecting the concentration, expression or activity of T14 peptide" according to the present invention, the term "activity" may refer to detecting the interaction of T14 with the allosteric site on the α-7 receptor or determining the end-point physiological effect. The term "expression" may refer to the detection of T14 protein in any compartment of the cell (e.g., cytosol, endoplasmic reticulum or Golgi apparatus).

[0194] The expression of T14 in biological systems can be detected by western blotting, immunoprecipitation (IP) or co-IP, or immunohistochemistry.

[0195] The screening method may also be based on the use of a cell extract comprising the T14 peptide. Such an extract is preferably derived from the cells described above.

[0196] The concentration, expression or activity of the T14 peptide can be measured using many conventional techniques. The test can be an immunoassay-based test. For example, a labeled antibody can be used in an immunoassay to assess the binding of a compound to T14 in a sample. The T14 peptide can be separated and the amount of label bound to it can be detected. A reduction in the bound label (relative to the control) will indicate that the test compound competes with the label for binding to T14 and is also a putative anti-cancer or anti-metastatic agent. Alternatively, a functional activity that measures the activity of the T14 peptide can be used.

[0197] In addition, molecular biological techniques can be used in screening to detect T14 peptides. For example, cDNA can be generated from mRNA extracted from the tested cells or subjects and primers designed to amplify the test sequence for quantitative polymerase chain reaction amplification from cDNA. When using a subject (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 predetermined length of time, and then a sample should be obtained from the subject to assay the concentration, expression or activity of the T14 peptide. The sample can be, for example, blood or a biopsy.

[0198] As discussed in the examples, the inventors have clearly shown that the toxic T14 peptide is easily detected outside the cancer cells (i.e., in the cell culture medium), while AChE and α-7 nicotinic receptors are not. In addition, the inventors have demonstrated a strong correlation between the metastatic marker (CD44) and the toxic molecule T14 peptide. This correlation is also applicable within the cancer cell membrane and in the cell fluid of the cancer cell. Therefore, the inventors believe that T14 represents a robust biomarker for cancer itself, especially metastasis.

[0199] Therefore, according to a seventh aspect of the present 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.

[0200] Most preferably, the present invention provides the use of a peptide of SEQ ID No: 3, or a fragment or variant thereof, as a diagnostic or prognostic biomarker for metastatic disease.

[0201] According to the eighth aspect, a method for diagnosing an individual suffering from cancer or metastatic disease, or susceptible to cancer or metastatic disease, or for providing a prognosis of an individual's condition is provided, the method comprising detecting the presence of a peptide of SEQ ID No: 3, or a fragment or variant thereof, in a sample obtained from the individual, wherein the presence of a peptide of SEQ ID No: 3, or a fragment or variant thereof means that the individual suffers from cancer or metastatic disease, or is susceptible to cancer or metastatic disease, or that the individual's condition has a poor prognosis.

[0202] According to the ninth aspect of the present invention, a kit for diagnosing a subject suffering from cancer or metastatic disease, or susceptible to cancer or metastatic disease, or for providing a prognosis of a subject's condition is provided, the kit comprising a detection tool 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 the presence of a peptide of SEQ ID No: 3, or a fragment or variant thereof, in the sample indicates that the subject suffers from cancer or metastatic disease, or is susceptible to cancer or metastatic disease.

[0203] Advantageously, the uses, methods and kits of the present invention enable diagnosis or prognosis of individuals suffering from cancer or metastatic disease, or susceptible to cancer or metastatic disease, most preferably metastasis. The inventors have found that the toxic T14 peptide is a robust biomarker for identifying individuals suffering from cancer or metastatic disease. Therefore, individuals diagnosed with cancer or metastatic disease, or susceptible to cancer or metastatic disease, or with a poor prognosis may benefit from early therapeutic treatment according to the uses, methods and kits of the present invention in order to prevent the occurrence of cancer or metastatic disease.

[0204] The subject may be a vertebrate, a mammal, or a domestic animal. However, most preferably, the subject is a human. The subject may be a child or an adult. Preferably, the method is performed in vitro.

[0205] Preferably, the sample comprises a biological sample. The sample can be any material that can be obtained from an individual (from which the protein can be obtained). In addition, the sample can be blood, plasma, serum, spinal fluid, urine, sweat, saliva, tears, breast puncture aspirate, prostatic fluid, semen, vaginal fluid, feces, cervical scraping, cells, amniotic fluid, intraocular fluid, mucus, moisture in breath, animal tissue, cell lysate, tumor tissue, hair, skin, oral scraping, lymph, interstitial fluid, nail, bone marrow, cartilage, prions, bone meal, earwax, or a combination thereof.

[0206] The sample may comprise blood, urine, tissue, etc. Most preferably, the sample comprises a blood sample. The blood may be venous or arterial blood.

[0207] The kit may include a sample collection container for receiving the extracted sample. The blood sample may be immediately assayed for T14 levels. Alternatively, the blood sample may be stored at low temperatures, such as in a freezer or even frozen, before the T14 assay is performed. The detection of T14 may be performed with whole blood. However, preferably, the blood sample comprises blood serum. Preferably, the blood sample comprises blood plasma.

[0208] The blood may be further processed before being assayed for T14. For example, an anticoagulant may be added, such as a citrate (e.g., sodium citrate), hirudin, heparin, PPACK, or sodium fluoride. Thus, the sample collection container may contain an anticoagulant to prevent clotting of the blood sample. Alternatively, the blood sample may be centrifuged or filtered to prepare a plasma or serum portion that can be used for analysis. Thus, T14 is preferably analyzed or assayed in a blood plasma or blood serum sample. The concentration of T14 is preferably measured in vitro from a blood serum sample or a plasma sample taken from an individual.

[0209] Preferably, the kit or method is used to determine the presence or absence of T14-positive cells (i.e., cells comprising SEQ ID No: 3) in a sample, or to determine their concentration in a sample. The detection means may comprise an assay suitable for detecting the presence and / or absence of T14-positive cells in a sample. The kit or method may comprise the use of a positive control and / or a negative control, to which the assay may be compared. For example, the kit preferably comprises a reference to the concentration of T14-positive cells in a sample from an individual who does suffer from cancer or metastatic disease (i.e., a positive control) and / or a reference to the concentration of T14-negative cells in a sample from an individual who does not suffer from cancer or metastatic disease (i.e., a negative control).

[0210] The T14 peptide (SEQ ID No: 3) can be assayed by many methods known to those skilled in the art. For example, preferably, an immunoassay can be used to measure the level of the T14 peptide. However, it should be understood that non-immunological assays can be used, for example, by labeling a compound that has an affinity for a ligand of the T14 peptide molecule and then assaying the label.

[0211] The T14 peptide can also be determined using Western blot analysis. Thus, the total protein level of the T14 peptide can be determined using immunoassays and Western blot analysis. The concentration of the T14 peptide can also be detected by enzyme-linked immunosorbent assay (ELISA), fluorescence assay, chemiluminescence assay, or radioimmunoassay analysis.

[0212] The kit may further comprise a detectable label. The term "label" may refer to a group or a fragment thereof that can be attached to a detection tool. The group can be used for (for example) a therapeutic or diagnostic procedure. Therapeutic labels include (for example) a group that can be attached to an antibody or fragment thereof of the present invention and used to monitor the binding of the antibody to the T14 peptide (i.e., SEQ ID No: 3). As described herein, the antibody or its antigen-binding fragment specifically binds to SEQ ID No: 3, or a fragment or variant thereof, and can be used as a detection tool, or for a detection tool. Preferably, the antibody or its antigen-binding fragment does not bind to SEQ ID No: 2 (i.e., T30). Preferably, the antibody or its antigen-binding fragment does not bind to SEQ ID No: 4 (i.e., T15).

[0213] Diagnostic markers include, for example, groups that 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 markers that can be detected by analytical tools include enzymes, radioisotopes, fluorescent pigments, chemiluminescent markers and biotin.

[0214] Examples 3 and 4 describe ELISA and protein immunoblot data for T14 in a series of cancer patients. It will be appreciated that the concentration of T14 peptide in cancer patients is highly dependent on many factors, such as how far the cancer has progressed. It will also be appreciated that the concentration of T14 peptide in individuals who do not have cancer may fluctuate to some extent, but on average the concentration tends to be substantially constant over a given period of time. It will also be appreciated that the concentration of T14 peptide in a group of individuals who do not have cancer may differ from the concentration of T14 peptide in another group of individuals who do not have cancer. However, a skilled person will understand how to determine the average concentration of T14 peptide in individuals who do not have cancer, which is referred to as the 'normal' concentration of T14 peptide. The normal concentration corresponds to the reference value discussed above.

[0215] The concentration of serum or plasma of T14 peptide can be measured by double antibody sandwich ELISA, which will be known to the skilled person. The ELISA may comprise the use of a suitable antibody for coating an ELISA plate. For example, such a suitable antibody may comprise an affinity purified polyclonal rabbit anti-human T14 peptide antibody. In addition, the ELISA may comprise the use of a suitable antibody for detection. For example, such a suitable antibody may comprise a peroxidase labeled monoclonal mouse anti-human T14 peptide antibody. The human T14 peptide, which can be purified from plasma and then quantified by amino acid analysis, can be used to calibrate plasma standards by standard techniques known to the skilled person.

[0216] The label may be attached directly to the antibody, or to a second binding agent that specifically binds to T14. Such a second binding agent may be, for example, a second antibody. The second antibody may be polyclonal or monoclonal, and may be derived from human, murine, or chimeric sources.

[0217] Due to the current limitations of the assays (both protein immunoblot and ELISA), all values ​​in both cases can only be viewed as relatively different groups at present. However, although the ELISA data may not be completely conclusive, there are surprisingly clear and significant differences between the groups analyzed using protein immunoblot, where poor survival rates correspond to (P=0.01) lower T14 levels together with longer first treatment times. Since protein immunoblots measure aggregates of peptides, these data indicate that more free monomers will be present in the blood circulation of those who have not yet been treated and therefore have a worse prognosis, acting as signaling molecules. The fact that the ELISA data are currently inconclusive may be that the free monomers it measures have been absorbed by and / or combined with their corresponding targets, thereby mediating the migration of cells.

[0218] Therefore, preferably, Western blotting is used to detect T14 and / or quantify T14 levels.

[0219] The invention further extends to methods of treatment.

[0220] Thus, according to a tenth aspect of the present invention, there is provided a method of treating an individual susceptible to the development of cancer or metastatic disease, the method comprising:

[0221] (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 the presence of a peptide of SEQ ID No: 3, or a fragment or variant thereof, in the sample indicates that the subject has cancer or metastatic disease, or is susceptible to cancer or metastatic disease; and

[0222] (ii) administering to a subject a therapeutic agent or treatment regimen that prevents, reduces or delays the development of cancer or metastatic disease.

[0223] Examples of suitable therapeutic agents that may be administered include, but are not limited to, Herceptin. Examples of suitable treatment regimens include radiation therapy or chemotherapy.

[0224] It should be understood that the present invention extends to any nucleic acid or peptide or variant, derivative or analog thereof, which comprises the amino acid or nucleic acid sequence of any sequence mentioned herein, including its functional variant or functional fragment. The terms "substantially amino acid / nucleotide / peptide sequence", "functional variant" and "functional fragment" can be a sequence having at least 40% sequence identity with the amino acid / nucleotide / peptide sequence of any sequence mentioned herein (e.g., 40% identity with a sequence identified as SEQ ID No: 1 to 7, etc.).

[0225] Amino acid / polynucleotide / polypeptide sequences with a sequence identity of greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and even more preferably greater than 80% to any of the sequences mentioned are also contemplated. Preferably, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity to any of the sequences mentioned, 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 to any of the sequences mentioned herein.

[0226] The skilled person will understand how to calculate the percent identity between two amino acid / polynucleotide / polypeptide sequences. In order to calculate the percent identity between two amino acid / polynucleotide / polypeptide sequences, first an alignment of the two sequences must be prepared, followed by calculation of the sequence identity value. The percent identity of two sequences may take different values, depending on: (i) the method used to align the sequences, e.g. ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment by 3D alignment; and (ii) the parameters used for the alignment method (e.g. local alignment versus global alignment), the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet, etc.), and the gap penalty, e.g. functional form and constant.

[0227] After alignment, there are many different ways to calculate the percent identity between two sequences. For example, the number of identities can be divided by: (i) the length of the shortest sequence; (ii) the length of the alignment; (iii) the average sequence length; (iv) the number of non-gap positions; or (iv) the number of equivalent positions excluding overhangs. It should also be understood that the percent identity also depends strongly on length. Therefore, the shorter the sequence pair, the higher the probability that sequence identity will occur can be expected.

[0228] It should be understood that accurate alignment of protein or DNA sequences is a complex process. The commonly used 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 method for generating multiple alignments of proteins or DNA according to the present invention. Suitable ClustalW parameters can be as follows: for DNA alignment: gap opening penalty = 15.0, gap extension penalty = 6.66, and matrix = identity. For protein alignment: gap opening penalty = 10.0, gap extension penalty = 0.2, and matrix = Gonnet. For DNA and protein alignment: ENDGAP = -1, GAPDIST = 4. Those skilled in the art will appreciate that these parameters and other parameters may need to be changed to perform optimal sequence alignment.

[0229] Preferably, the calculation of the percent identity between two amino acid / polynucleotide / polypeptide sequences can then be calculated based on this alignment as (N / T)*100, where N is the total number of positions at which the sequences have identical residues, and T is the total number of positions compared (including gaps but excluding overhangs). Thus, the most preferred method for calculating the percent identity between two sequences comprises (i) preparing the sequence alignment using the ClustalW program using a suitable set of parameters (e.g., those listed above); and (ii) inserting the N and T values ​​into the following formula: - Sequence identity = (N / T)*100.

[0230] 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 that hybridizes to a DNA sequence or its complement under stringent conditions. By stringent conditions, we mean that the nucleotides hybridize to filter-bound DNA or RNA in 3x sodium chloride / sodium citrate (SSC) at about 45°C, followed by at least 1 wash in 0.2xSSC / 0.1% SDS at about 20 to 65°C. Alternatively, a substantially similar polypeptide may differ from the sequence shown in SEQ ID No: 1 to 4 by at least 1, but less than 5, 10, 20, 50 or 100 amino acids.

[0231] Due to the degeneracy of the gene code, it is clear that any nucleotide sequence described herein can be changed or altered without significantly affecting the protein sequence encoded by it to provide its functional variant. Suitable nucleotide variants are those having sequences that are altered to produce silent changes, and the alteration is carried out by replacing different codons of the same amino acid in the coding sequence. Other suitable variants are those having homologous nucleotide sequences but containing all or part of the sequences that are altered to produce conservative changes, and the alteration is carried out by replacing different codons that encode amino acids with biophysical properties similar to the amino acids it replaces to its side chain. 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. Polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine. Positively charged (basic) amino acids include lysine, arginine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be understood which amino acids may be substituted with amino acids having similar biophysical properties, and the skilled artisan will be aware of the nucleotide sequences encoding these amino acids.

[0232] All features described herein (including any accompanying claims, abstract and drawings) and / or all steps of any method or process so disclosed may be combined in any combination with any one or more aspects, except combinations where at least some of such features and / or steps are mutually exclusive. BRIEF DESCRIPTION OF THE DRAWINGS

[0233] For a better understanding of the invention, and in order to show how embodiments of the invention may function, reference will now be made, by way of example, to the accompanying drawings, in which:-

[0234] Figure 1 It is protein immunoblot data, which shows similar mobility between T14 (A), α7 receptor (B) and AChE (C) in cell lysates of 7 cancer cell lines (CL) (i.e. MEC-1, KG1a, H929, MCF-7, MDA-MB-231, CLL and JJN3) and normal B lymphocytes acting as controls. MDA-MB-231, KG1a and MEC-1 cells are highly mobile cancer cell lines. H929, JJN3, CLL and MCF-7 are cancer cell lines with lower mobility. B lymphocytes are normal non-cancerous cells. Peptide / protein levels are normalized to total protein (TP) and mapped together for each cell line. Peptide / protein levels are similar in each cell line, except for MCF-7 cells with lower T14 levels. Between each cell line, peptide / protein levels are variable. Then the above protein levels are followed as Figure 2 and Figure 5 The shown are quantified;

[0235] Figure 2 is a graph of Western blot data showing that the levels of T14, α7 receptor, and AChE in cell lysates vary between cancer cell lines, but are similar within each cell line;

[0236] Figure 3 is based on Figure 1 Figure 2 shows a graph of the western blot data. The levels of T14, α7 receptor, and AChE were normalized to total protein (TP), and the levels of each peptide / protein were correlated to the other in the xy plot and linear regression analysis was performed. The best fit line (solid line) and its 95% confidence interval (dashed line) are shown. A): T14 and α7 receptor, R 2 =0.6262, P=0.0193. B): α7 receptor and AChE, R 2 =0.6705, P=0.0129. C): T14 and AChE. The circles in (C) highlight outlier data points that, when excluded, give a significant positive correlation (R 2=0.7032, P=0.0184). The fact that all correlations were significantly linear suggests that the peptide / protein exists as a complex in a 1:1:1 ratio (D);

[0237] Figure 4 The following is the Western blot data, which shows that T14 was detected in the cancer cell culture medium of all 7 cancer cell lines, but AChE and α7 were not detected. The mobility of T14 in the cell culture medium was slightly different from that in the cell lysate. The above protein levels were then Figure 5 The shown are quantified;

[0238] Figure 5 is a graph of western blot data showing that T14 concentrations in cancer cell culture media were significantly higher than T14 peptide / protein concentration levels of cells in MEC-1, KG1a, normal B lymphocytes, and MDA-MB-231 cell lines. α7 and AChE signals were not detected in cancer culture media. Peptide / protein levels were normalized to total protein (TP) and plotted together for each cell line;

[0239] Figure 6 is based on Figure 1 and Figure 4 Figure 2 shows a graph of the western blot data. The levels of T14, α7 receptor, and AChE were normalized to total protein (TP), and the levels of T14 in the cancer culture medium (extracellular T14) were correlated to each intracellular peptide / protein in the xy graph and linear regression analysis was performed, showing the best fit line (solid line) and its 95% confidence interval (dashed line). A): Extracellular T14 and intracellular α7 receptor, R 2 =0.6518, P=0.0154. B): Extracellular T14 and intracellular T14. The cell lines are divided into two groups (dark gray circles and light gray circles). Dark gray itself shows R 2 =0.9794, P = 0.0105. C): Extracellular T14 and intracellular AChE;

[0240] Figure 7 is a graph showing that T14 levels detected by ELISA vary significantly between cell lines and between cell culture medium and cell lysate;

[0241] Figure 8 is a graph demonstrating a significant inverse correlation between T14 levels in ELISA and T14 levels in Western blot measurements for cell culture media and cell lysates in cancer cell lines;

[0242] Fig. 9Western blot data are shown, which show similar mobility between CD44, T14, and AChE in the cell membrane and cytosolic fractions of 6 cancer cell lines and 1 cancer-derived cell line. The α7 receptor shows different mobility from CD44, T14, and AChE. The order of the lanes from left to right is: JJN3, MDA-MB231, MCF-7, KG1a, MEC-1, H929, and PC12. MDA-MB-231, KG1a, and MEC-1 cells are highly migratory cancer cell lines, while H929, JJN3, MCF-7, and PC12 are less migratory cancer cell lines;

[0243] Fig.10 The expression of CD44, T14, AChE and α7 receptors relative to total protein (TP) levels is shown. Strongly metastatic carcinomas have more protein in the cell membrane than in the cytosol, while this relationship is altered for weakly metastatic carcinomas.

[0244] Fig.11 The results showed a significant positive correlation between CD44, T14 and AchE, which strongly suggested that T14 and AchE were good predictors of the extent of cancer metastasis.

[0245] Fig.12 Cell culture data (i.e., acetylcholinesterase activity) are shown for peptide mimetic compound 1 (i.e., Tri02);

[0246] Fig.13 Cell culture data (i.e., calcium influx) are shown for peptide mimetic compound 1 (i.e., Tri02);

[0247] Fig.14 The results of voltage-sensitive dye imaging (VSDI) for the control cyclic peptide NBP-14 on brain slices are shown;

[0248] Fig.15 Shows the voltage sensitive dye imaging (VSDI) results for peptide mimetic compound 1 (i.e. Tri02) on brain slices;

[0249] Fig.16 Shown is a correlation analysis of changes induced by added peptides relative to the corresponding baseline response amplitudes in brain slices using voltage-sensitive dye imaging (VSDI). The change in response amplitude induced by T30 was found to be negatively correlated with its corresponding baseline amplitude (A). Therefore, subsequent correlation analyses were performed for each experiment in which exogenous peptides were perfused: B) T15, C) NBP14, D) Tri02, E) T30 in the presence of NBP14, and F) T30 in the presence of Tri02. y-axis units = ΔF / F0; x-axis = ΣδF / F0;

[0250] Fig.17Quantification of the effects mediated by the addition of Tri02 and T30 is shown;

[0251] Fig.18 A graph showing the comparison of co-administration of T30 and NBP14 versus co-administration of Tri02 and T30 in blocking the effects of T30 on activity within the basal forebrain. Co-administration of NBP14 was able to completely block the effects elicited by T30, whereas T30 w / Tri02 elicited a similar but attenuated regulatory response;

[0252] Fig.19 PC12 cell culture data (i.e., calcium ion influx) are shown for peptide mimetic compound 2 (i.e., Tri04);

[0253] Figure 20 shows (A) spatiotemporal graphs of changes in basal forebrain activity caused by addition of peptides (T30 and Tri04) relative to baseline response amplitude obtained on brain slices using voltage sensitive dye imaging (VSDI). (B) shows a graph comparing basal forebrain activity recordings induced by T30 and Tri04 (2uM) with T30 alone in the basal forebrain;

[0254] Fig.21 Shown are small fluorescence changes in recordings obtained in the presence of T30 alone or after co-application of T30 and its blocker Tri04 compared with recordings under baseline conditions (response time series, n=29);

[0255] Fig. 22 Bar graph showing basal forebrain activity using the blocker Tri04 at a concentration of 4 μM. Co-administration of Tri04 was able to completely block the effects induced by T30 in the rat basal forebrain;

[0256] Fig.23 Bar graph showing the range of levels of T14 measured by ELISA relative to known concentrations of exogenous T14. Patients are ranked by relative T14 concentrations in the CLL patient cohort examined;

[0257] Fig.24 Table showing T14 values ​​measured by ELISA relative to known concentrations of exogenous T14 in the CLL patient cohorts and statistical groupings examined;

[0258] Fig.25 Kaplan Meier estimates performed on 100 CLL serum samples divided into a dark grey group above the median (top curve) and a light grey group below the median (bottom curve) are shown. Hazard ratios, P values, and estimated median TTFTs are given above;

[0259] Fig.26Kaplan Meier estimates performed on 100 CLL serum samples divided into 4 different groups are shown. Hazard ratios, P values, and estimated median TTFTs are given above;

[0260] Fig. 27 Table showing T14 values ​​for the CLL patient cohorts examined and statistical groupings;

[0261] Fig.28 Kaplan Meier estimates performed on 100 CLL serum samples divided into a dark grey group above the median (top curve) and a light grey group below the median (bottom curve) are shown. Hazard ratios, P values, and estimated median TTFTs are given above;

[0262] Fig.29 Results of cell proliferation assays are shown. T14 and T30 both exhibited equal toxic effects at high concentrations (10 μM), and T30 had a greater trophic effect on astrocytes at low concentrations (120 ± 4% at 1 pM and 135 ± 9% at 10 pM) than T14 (96 ± 5% at 1 pM and 121 ± 8% at 10 pM). DETAILED DESCRIPTION

[0263] Examples

[0264] Materials and methods

[0265] Synthesis of polyclonal antibodies

[0266] Antibodies were synthesized on order by Genosphere Biotech (Paris, France). Two New Zealand rabbits were used, which were immunized four times over 70 days with a KLH peptide ("Pep4": T14-hapten CAEFHRWSSYMVHWK - SEQ ID No. 7) as an immunogen. C was included to link the T14 peptide to KLH acting as an immunogen. The animals were bled four times and the blood was pooled. The antiserum was then passed through a gravity column with a covalently bound peptide carrier, and after washing, the antibodies were eluted into an acidic buffer and the solution was neutralized. This process was completed by further dialysis against PBS buffer and freeze drying.

[0267] Optimization of T14 antibody conditions

[0268] The manufacturer's instructions for the antibodies were used to perform the ELISA experiments under optimal conditions. In the instructions, the manufacturer specifies the optical density associated with the antibody concentration (see table below) and the ELISA protocol (see protocol below) used for the procedure.

[0269] From the manufacturer:

[0270] The antigen was coated onto the EIA strip at 10 ug per well and each well was washed with 200 ul PBS buffer.

[0271]

[0272] Antiserum was serially diluted, added to different wells, and incubated for 2 hours. Unbound antibodies were washed, and anti-rabbit IgG-HRP conjugate was added. Each plate was washed and developed with TMB substrate for 15 minutes. Absorbance was read at 405nm (2.00AUFS). The color intensity is proportional to the amount of antibody. If the absorbance is greater than 2 times of pre-immune serum, the antibody is positive. The background absorbance of pre-immune serum can reach 0.1 to 0.3.

[0273] in conclusion:

[0274] For all experiments described in this paper, the antibody dilution chosen was 1:1000.

[0275] Western blotting

[0276] Cell fractionation

[0277] All cell masses from each cancer cell line (JJN3, MDA-MB231, MCF-7, KG1a, MEC-1, H929, PC12) were resuspended in 300 μl subcellular fractionation buffer (Hepes-NaOH 10 mM, MgCl2 1.5 mM, KCl 10 mM, EDTA 1 mM, DTT 1 mM, 1x proteinase inhibitor cocktail (Proteinase Inhibitor Cocktail), Nonidet P-40 0.05%) and placed on ice for 10 minutes. Afterwards, the mixture was homogenized using a polytron to ensure cell lysis. Then, the mixture was centrifuged at 500 g for 5 minutes. The resulting supernatant contained the cell fluid portion and was retained. The resulting mass contained the cell membrane portion, which was then resuspended in another 300 μl subcellular fractionation buffer and retained.

[0278] Preparation of cell lysates

[0279] All cell pellets from seven cancer cell lines (MEC-1, CLL, KG1a, JJN3, H929, MCF-7 and MDA-MB) and normal B lymphocytes were kindly donated by Professor Chris Pepper (School of Biological Sciences, Cardiff University). All cell pellets were dissolved in 1x lysis buffer (20mM TrisBase, 137mM NaCl, 1% Tween-20, 2mM EDTA) containing a 17% v / v ratio of protease inhibitor cocktail (phosphatase 1:1, PMSF 1:1, aprotinin 1:1). Subsequently, the mixture was ground for 10 seconds using a Polytron and shaken at 4°C for 2 hours. The samples were then centrifuged at 13,000g and 4°C for 30 minutes, and the supernatant was taken and stored at -80°C.

[0280] Measuring protein sample concentration

[0281] The protein concentration of the above samples was measured using Pierce TM 660nm Protein Assay (Thermo Scientific) was used for measurement. In short, serial dilutions (0 to 2mg / ml) were performed from a 10mg / ml bovine serum albumin (BSA) stock solution. Three replicates of each BSA concentration were prepared by transferring 10μl of protein to a transparent 96-well plate (Greiner). The sample was then diluted with 3 concentrations (1:1, 1:2, 1:10), and 3 replicates of each concentration were placed in the same 96-well plate, each containing 10μl of sample. Subsequently, 150μl of Pearce Reagent was added to the standard and all samples, and the mixture was incubated for 5 minutes under gentle shaking. Finally, the plate was read at 660nm on a spectrophotometer (Molecular Devices). The protein concentration of each sample was determined using the slope and y-intercept of the BSA standard curve, and the slope and y-intercept were calculated by Microsoft Excel.

[0282] Polyacrylamide gel electrophoresis of protein samples

[0283] Polyacrylamide gel ( TGX Stain-Free TMThe gel was placed in an electrophoresis tank (BIO-RAD, mini-PROTEAN quad system) and running buffer (25 mM TRIS-base, pH 8.6, 192 mM glycine, 0.1% SDS) was added to the gel and tank (BioRad). Protein samples were prepared by mixing with distilled water and 4x Laemmli sample buffer (final concentration: 69.5 mM TRIS-HCl pH 6.8, 1.1% LDS, 11.1% (w / v) glycerol, 0.005% bromophenol blue, BIO-RAD) and 2.5% mercaptoethanol (BIO-RAD). The mixture was heated at 95°C for 5 minutes and then cooled on ice. The samples and positive controls were loaded onto the gel and compared with molecular weight markers (Precision Plus Protein ELISA, BioRad) at 35 mV. TM Dual Xtra Standards, BIO-RAD) for 90 minutes. Ice was placed in the running tank to prevent any overheating.

[0284] Transfer protein samples to PVDF membrane

[0285] The stacked gel is cut off, and the separated gel is transferred to the PVDF transfer membrane (Thermo Fisher Scientific) in Mini Transblot Cell (BIO-RAD). In short, by soaking 1 minute with methanol and then soaking 2 minutes with distilled water, the PVDF transfer membrane is activated. All layers are saturated with transfer buffer (20mM TRIS-base pH 8.6, 154mM glycine, 0.8%w / vSDS and 20% methanol) subsequently. The multilayer structure of transfer is composed of transfer sponge, blotting paper, gel, PVDF transfer membrane in the order from bottom to top, and blotting paper, transfer sponge are placed in transfer box, and the transfer box is inserted in the Mini Transblot Cell that is filled with transfer buffer. Finally, electrophoretic transfer was carried out 90 minutes under 200mA. Ice cube is placed in transfer tank to prevent any overheating.

[0286] Staining of PVDF membrane

[0287] Using BLOT-Faststain TM (G-Biosciences, USA) was used to stain total protein and served as a loading control. Immediately after electrophoretic transfer, the PVDF transfer membrane was covered with diluted BLOT-Faststain TM Fixative solution (10x) was used for staining for 2 minutes. The membrane was then incubated with diluted BLOT-Faststain under gentle shaking. TMThe membrane was then incubated with developer solution (4x) for 1 minute. Subsequently, the membrane was stored in the developer solution in the dark for 30 minutes at 4°C to allow the protein bands to reach maximum intensity. Finally, the membrane was washed with cold water to eliminate background staining and imaged using a G box (Syngene). The membrane can then be destained using deionized warm water (40-45°C) and is ready for the blocking stage.

[0288] Detection of protein bands

[0289] The PVDF transfer membrane was blocked in TBS (TRIS buffered saline, 20 mM TRIS base pH 7.5, 0.5 mM NaCl) containing 5% skim milk powder for 1 hour, and then washed twice in TTBS (TBS supplemented with 0.05% v / v Tween-20) for 7 minutes each time. The membrane was incubated overnight at 4°C with the primary antibody diluted in TTBS containing 1% skim milk powder. The primary antibody was removed the next day. The membrane was washed 3 times in TTBS for 5 minutes each time, and then incubated with the secondary antibody for 1 hour at room temperature. The selected secondary antibody depends on the type of primary antibody used. It can be a goat anti-mouse secondary antibody conjugated to HRP (a9309, Sigma) diluted in TTBS containing 1% skim milk powder (working concentration: 1:1000) or a goat anti-rabbit secondary antibody conjugated to HRP (ab6721, abcam) diluted in TTBS containing 1% skim milk powder (working concentration: 1:5000). After the secondary antibody incubation, the membrane was washed 3 times in TTBS for 5 minutes each, followed by a final 10-minute wash in TBS. Protein bands were detected using a G box (Syngene).

[0290] Table 1 - Primary antibodies used for western blot detection.

[0291]

[0292] Protein band imaging and data analysis

[0293] The PVDF membrane was placed in a G-box (Syngene). The focus and zoom settings were adjusted to ensure that the membrane was maximized in the center of the screen. TMThe luminol of Western ECL substrate (BIO-Rad) and peroxide solution are mixed in equal parts, and applied to the film. With 1 minute time interval, in the dark, image is taken, and carried out for 5 minutes, to obtain the best signal of protein band. Afterwards, use automatic setting to expose film with white light, so as to obtain the image of molecular ladder. Then use image J to analyze the blot image. Place equal-sized frames around the protein band in each road, so as to measure the protein band intensity. Then background is removed from band intensity, and analysis of results in Microsoft Excel and Graphpad software.

[0294] Removal of antibodies for reprobing

[0295] The protein signal of the PVDF transfer membrane can be removed and re-probed with different proteins. Briefly, the membrane was washed twice for 10 minutes with a mild stripping buffer (200 mM glycine, 3.5 mM SDS, 1% v / v Tween-20, pH 2.2). Subsequently, the membrane was washed twice for 10 minutes with PBS and then twice for 5 minutes with TTBS. TM Western ECL substrate (BIO-Rad) was added to the membrane and imaged using a G-box (Syngene) to check for residual protein signal. If the residual signal was too strong, the entire removal process was repeated. The membrane was then ready for the subsequent blocking phase and primary antibody detection (see above).

[0296] ELISA for T14 peptide antibody

[0297] Standard curves and samples were run in triplicate. Cell culture media and cell lysate samples were diluted to 1:10 in PBS buffer. The standard curve for the determination of T14 peptide in brain tissue samples was diluted in PBS buffer. The standard curve ranged from 8 to 100 nm of T14, and the blank was PBS buffer alone. Briefly, a 96-well immunoplate (NUNC) was coated with 100 μl / well of sample or standard T14, covered with parafilm, and incubated overnight at 4°C. The next day, the samples were removed by flicking the plate with running water over a sink, 200 μl of blocking solution containing 2% bovine serum albumin (BSA) in Tris-buffered saline and Tween20 (TBS-T) was added, and incubated for 4 hours at room temperature. The blocking solution was then removed, 100 μl of antibody diluted to 1 μg / ml in blocking solution was added, and incubated overnight at 4°C. The primary antibody was removed the next day, and each well was washed 3 times with 200 μl of TBS-T. After adding 100 μl of enzyme-conjugated secondary antibody diluted to 0.1 μg / ml in blocking solution, incubate at room temperature for 2 hours; the plate was covered with parafilm during all incubations. After 2 hours, the plate was washed 4 times with TBS-T. The color reaction was initiated by adding 3,3,5,5-tetramethylbenzidine. After 20 minutes, the reaction was stopped with a stop solution containing 2M H2SO4 and the absorbance was measured at 450nm in a Vmax plate reader (Molecular Devices, Wokingham, UK).

[0298] Example 1

[0299] The inventors began to use Western blotting to detect the levels of toxic T14 peptide (AEFHRWSSYMVHWK-SEQ ID No: 3), nicotinic α-7 receptor and acetylcholinesterase (AChE) protein in cell lysates and cell culture medium of 7 cancer cell lines (MEC-1, KG1a, H929, MCF-7, MDA-MB231, CLL, JJN3) and normal B lymphocytes serving as controls. In addition, they also used ELISA to detect the level of T14 peptide.

[0300] Western blot results

[0301] Western blot (WB) was performed to detect the levels of T14, AChE and α7 nicotinic acetylcholine receptors in cell lysates (CL) of seven different cancer cell lines and normal B lymphocytes as a control. Qualitative data showed that T14, AChE and α7 receptors were all detected in the CL of all cell lines. Surprisingly, all three proteins had virtually identical mobility (see Figure 1A, 1B and 1C). The inventors do not believe this is antibody cross-reactivity, as these three proteins show different mobility in human brain homogenates (data not shown). Subsequently, the levels of T14, AChE and α7 receptors were quantified and normalized to total protein levels (Colins et al. 2015).

[0302] Quantitative data showed that the levels of T14, α7 receptor, and AChE were similar in each cell line, except for the MCF-7 line which had low cellular T14 levels (see Figure 2 However, the levels of T14, α7 receptors, and AChE varied between cell lines (see Figure 2 ).

[0303] Interestingly, within cell lysates, there was a significant difference between T14 levels and α7 receptor levels (see Figure 3 A), between α7 receptor level and AChE level (see Figure 3 B) between T14 level and AChE level (see Figure 3 C) There is a significant positive correlation. These correlations and the identical mobility of the protein bands indicate that T14, AChE, and α7 receptor exist as a protein complex in a 1:1:1 ratio within cancer cells.

[0304] Subsequently, Western blotting was performed to detect the levels of T14 and possibly α-7 receptor and AChE released into the cancer cell culture medium (CCM) of the above cell lines. Qualitative data showed that only T14 was released into the cell culture medium (see Figure 4 ), but not AChE (see Figure 4 A small amount of α7 receptors is released into the cell culture medium (see Figure 4 ), suggesting that they may have come from floating dead cancer cells.

[0305] Quantitative data showed that T14 released into the cell culture medium was greater than cellular T14, α7 receptor, and AChE in MEC-1, KG1a, normal B lymphocytes, and MDA-MB-231 cell lines (see Figure 5 ). However, T14 released into the cell culture medium was at levels similar to T14, α7 receptor, and AChE in primary CLL, JJN3, H929, and MCF-7 cell lines (see Figure 5 ).

[0306] Subsequently, the levels of T14 in the cell culture medium were correlated with the levels of T14, AChE, and α7 receptors in the cell lysates. There was a significant inverse correlation between the levels of T14 released in the cell culture medium and the levels of α7 receptors in the cell lysates (see Figure 6a). The inventors believe that this may be a general pathological mechanism, since this effect also occurs in the cerebral cortex and locus coeruleus of Alzheimer's patients (data not shown). There was no significant correlation between T14 in cell culture medium and T14 levels in cell lysates, although there was a significant inverse correlation when only four cell lines were included in the analysis (see Figure 6 B, dark grey panel). In addition, there was no significant correlation between T14 in cell culture medium and AChE levels in cell lysates, even when each cell group was analyzed separately (see Figure 6 C).

[0307] ELISA results

[0308] T14 levels in cell lysates and cell culture media

[0309] The aim was to determine the endogenous levels of T14 in cancer cells (kindly provided by Professor Chris Pepper of Cardiff University) with different metastatic propensities using an ELISA method, including the levels of T14 in intracellular material recovered from lysed cells and the levels of T14 released into the cell growth medium (CCM). The method used combined the results of the total protein (pierce assay) to provide a measure of μg T14 per mg protein in the sample. The results showed a high degree of variation in T14 levels between cell lines and between cell lysates and cell culture medium within a cell line, such as Figure 7 shown.

[0310] T14 is the highest in the cell lysate in the MDA-MB cell line, but comparatively speaking, the same cell line shows lower T14 levels in the cell culture medium. It is shown that the T14 level is the highest in the MCF-7 cell line, but the cell line also has one of the lowest T14 concentrations in the cell lysate, indicating that although T14 is highly expressed in the cell line, it is about 5 times more than in the cell in the extracellular than in the cell. When comparing the intracellular and extracellular levels, only JJN3 and KG1a have significantly different T14 levels. In JJN3, the same curve is seen in the MDA-MB cell line, and the extracellular level of T14 is significantly higher than the intracellular level. The KG1a cell line is the only cell line that shows significantly higher T14 levels in the cell lysate when compared with the culture medium. The T14 in the culture medium of the CLL cell line is lower than the detection limit of the assay.

[0311] Comparison of ELISAT14 data of cancer cell lines with WB data of cancer cell lines

[0312] Using the results obtained from ELISA and the results collected from Western blot, the inventors compared T14 / mg protein to μg T14 / μg total protein in cell lysates and cell culture media. Figure 8As can be seen, a significant inverse correlation was seen between WB T14 levels and ELISA T14 levels measured in cell culture medium and in cell lysates.

[0313] After linear regression analysis, both cell culture media and cell lysates showed an inverse correlation between T14 levels measured by WB and ELISA (P < 0.0001, R 2 =0.4956, and P =0.0315, R 2 = 0.1451), showing the best fit line (solid line) and 95% confidence interval (dashed line).

[0314] Summarize

[0315] 1) In cancer cells, T14, α7 receptor, and AChE were all detected, albeit in different amounts. Surprisingly, all three proteins had similar mobility, and their levels were positively correlated with each other, suggesting that they were complexed with each other.

[0316] 2) Outside the cancer cells (i.e., inside the cell culture medium), only the toxic T14 peptide was detected, while AChE and α-7 were not released into the external culture medium. In addition, for 6 of the 7 cancer cell lines, T14 levels were higher outside the cells than inside the cells, indicating that T14 was produced to be released from the cells. In addition, the mobility of T14 outside the cells was different from that inside the cells.

[0317] 3) Interestingly, T14 levels outside of cancer cells were significantly negatively correlated with alpha-7 receptor levels inside cancer cells. The inventors propose that this may underlie a general disease mechanism, as this was also true in homogenates of the cerebral cortex and locus coeruleus of Alzheimer's disease patients (but not controls), where the difference was a positive correlation between T14 and alpha-7. The inventors believe that this may be due to high concentrations of the T14 peptide downregulating its target receptor in diseased individuals.

[0318] 4) T14 levels inside and outside cancer cells were also measured by ELISA. The levels of T14 in cancer cell culture medium and cell lysates measured by ELISA were significantly inversely correlated with the levels of T14 in cancer cell culture medium and cell lysates measured by Western blotting. This would suggest that ELISA may measure the levels of T14 monomers, while Western blotting measures the levels of T14 aggregates, and therefore the levels are complementary.

[0319] Example 2

[0320] The purpose of this example is to use western blotting to investigate the relationship between the metastatic marker CD44 and the toxic T14 peptide, AChE, and alpha-7 receptor in the cell membrane and cytosolic fractions of six cancer cell lines (JJN3, MDA-MB231, MCF-7, KG1a, MEC-1, H929) and one cancer-derived cell line (PC12).

[0321] Western blot results

[0322] Qualitative data showed that CD44, AChE, and T14 had similar mobility in the cell membrane fraction of all cell lines (e.g. Fig. 9 A, C, and E), while α-7 showed different protein mobility (see Fig. 9 G). The same relationship between CD44, AChE and T14 was found in the cytosolic fraction of all cell lines (see Fig. 9 B, D, F), again, except for α-7 (see Fig. 9 H).

[0323] Subsequently, the levels of all four peptides / proteins (CD44, AchE, α-7, and T14) were quantified and normalized to total protein levels (Colins et al. 2015). The quantitative data showed that strongly metastatic cancer cell lines (KG1a, MDA-MB231, MEC-1) had more peptides / proteins in their cell membranes compared to the cytosol, while weakly metastatic cancer cell lines / cancer-derived cell lines (JJN3, H929, MCF-7, and PC12) showed different relationships between peptides / proteins in the cell membrane and the cytosol (see Fig.10 ).

[0324] Most importantly, the levels of the metastatic marker CD44 were significantly correlated with the cell membrane fraction (see Fig.11 A) and cytosolic fraction (see Fig.11 B) were significantly positively correlated with AChE and with the cell membrane part ( Fig.11 C) and cytosolic fraction ( Fig.11 In addition, the levels of AChE and T14 in the cell membrane ( Fig.11 E) and cytosolic fraction ( Fig.11 F) are significantly positively correlated with each other. This discovery strongly indicates that AChE and T14 are good predictors of cancer metastasis and may be key molecular intermediates. Therefore, blocking the signal transduction effect of AChE and T14 can be used as an anticancer therapy.

[0325] Summarize

[0326] 1) In 6 cancer cell lines and PC12 cells, the metastatic marker (CD44) was significantly positively correlated with the toxic molecule T14 and also significantly positively correlated with AChE.

[0327] 2) This correlation exists both within the cancer cell membrane and within the cytosol of cancer cells.

[0328] 3) This finding strongly suggests that T14 and AchE are good predictors of the extent of cancer metastasis and may be key molecular intermediates.

[0329] Example 3-ELISA of T14-CLL cohort

[0330] Given the clear role of T14 in cancer metastasis, the inventors investigated the potential of T14 as a cancer biomarker using ELISA, and its association with patient survival.

[0331] Materials and methods

[0332] T14 Antibody: The antibody was synthesized by Genosphere Biotech (Paris, France). Two New Zealand rabbits were used and immunized 4 times over 70 days with keyhole limpet hemocyanin (KLH)-peptide (T14-hapten: CAEFHRWSSYMVHWK (SEQ ID No: 7); C included for linkage to KLH as immunogen). The animals were bled 4 times and the blood was pooled. The antiserum was then passed through a gravity column with a covalently bound peptide carrier and, after washing, the antibodies were eluted into an acidic buffer and the solution was neutralized. This process was completed by further dialysis against phosphate buffered saline (PBS) buffer and freeze drying.

[0333] ELISA for T14 peptide antibody: Standard curve and samples were run in triplicate. Human serum samples were diluted to 1:10,000, and the standard curve used to determine the relative T14 content in the samples was diluted in PBS buffer. The standard curve ranged from 3.3 to 40 nM T14. Briefly, 96-well immunoplates (NUNC) were coated with 100 μl / well of sample or standard T14, covered with parafilm, and incubated overnight at 4°C. The next day, the samples were removed by flicking the plates over a sink with running water, 200 ml of blocking solution containing 2% bovine serum albumin (BSA) in Tris-buffered saline and Tween20 (TBS-T) was added, and incubated for 4 hours at room temperature. The blocking solution was then removed, 100 μl of antibody diluted to 1 μg / ml in blocking solution was added, and incubated overnight at 4°C. The primary antibody was removed the next day, and each well was washed 3 times with 200 μl of TBS-T. After adding 100 μl of the enzyme-conjugated secondary antibody diluted to 0.1 μg / ml in blocking solution and incubating for 2 hours at room temperature, the plate was covered with parafilm during all incubations. After 2 hours, the plate was washed 4 times with TBS-T. 3,3,5,5-tetramethylbenzidine was added to start the color reaction. After 30 minutes, the reaction was stopped with a stop solution containing 2M H2SO4 and the absorbance was measured at 450nm in a Versamax plate reader (Molecular Devices, Wokingham, UK).

[0334] Analysis: The results of the assay are recorded as optical density (OD) in arbitrary units. The OD of the blank is removed from the signal of each sample prior to analysis. The OD of each sample in the assay is compared to the baseline of the lowest known concentration of T14 in the standard curve. The optical density can then be described in relation to the known signal and recorded as the ratio of the signal. All samples are compared for their relative T14 levels, since direct reading from the standard curve is not possible using current methods in human blood samples. The samples are grouped as "high", "medium-high", "medium-low" and "low" as defined by the distance of the relative value from the mean of the entire group. Since the values ​​are normally distributed, all values ​​within 1 standard deviation of the mean are in the medium range, if they are above the mean they are "medium-high", and if they are below the mean they are "medium-low". "Low" T14 values ​​are those that are below the mean and exceed the standard deviation of the mean. Conversely, "high" T14 values ​​are those that are above the mean and exceed the standard deviation of the mean.

[0335] Results and discussion

[0336] refer to Fig.23, shows a bar graph of a range of T14 levels measured by ELISA relative to known concentrations of exogenous T14. Patients are ranked by relative T14 concentrations in the CLL patient cohort examined.

[0337] refer to Fig.24 , a table showing ELISA-measured T14 values ​​relative to known concentrations of exogenous T14 in the CLL patient cohort examined and in statistical groupings.

[0338] refer to Fig.25 , shows the ELISA values ​​in 2 different groups divided by relative T14 concentrations by statistical analysis using Kaplan Meier estimation performed on 100 CLL serum samples.

[0339] refer to Fig.26 , shows the ELISA values ​​in 4 different groups divided into relative T14 concentrations by statistical analysis using Kaplan Meier estimation performed on 100 CLL serum samples.

[0340] Example 4 - Western blot data on cancer samples (CLL cohort)

[0341] Given the role of T14 in cancer metastasis, the inventors investigated the potential of T14 as a cancer biomarker and its association with patient survival using Western blotting.

[0342] Materials and methods

[0343] Samples: Serum was drawn from a total of 100 CLL patients before they were treated and subsequently treated and monitored for 16 years.

[0344] Western blotting: The serum samples above were analyzed using Pierce TMProtein concentration was determined by 660 nm Protein Assay (Thermo Fisher Scientific, 22660). Subsequently, Western blot analysis was performed on the samples using previously established methods [Garcia Rates et al. 2016–Garcia-Ratés, S., Morrill, P., Tu, H., Pottiez, G., Badin, A., Tormo-Garcia, C., Heffner, C., Coen, C., and Greenfield, S. (2016) '(I) Pharmacological dissection of new α7 nicotinic receptor modulators: Blockade of toxic acetylcholinesterase-derived peptides is increased in Alzheimer's brain', Neuropharmacology. doi: 10.1016 / j.neuropharm.2016.02.006.]. The primary antibody used was anti-T14 antibody (1:1000) [Garcia Rates et al. 2016]. The secondary antibody used was goat anti-rabbit antibody conjugated to horseradish peroxidase (Abcam, ab6721, 1:5000). Protein bands obtained from cell lysates were quantified by measuring total light intensity using Image J and then normalized to total protein levels using Blot FastStain to control loading errors.

[0345] Data analysis: T14 50KDa / TP values ​​were graded from low to high and the median was calculated. Subsequently, each value was divided into 2 groups, the light grey group containing values ​​below the median and the dark grey group containing values ​​above the median ( Fig.28 ). Finally, Kaplan Meier estimates are performed for the above two groups. The analysis can evaluate the effect of a treatment on the number of subjects who survive or are saved after the treatment within a time period (TTFT: time to first treatment). The probability of an event occurring at a certain time point is calculated, and these continuous probability values ​​are multiplied by any previously calculated probability values ​​to obtain a final estimate, thereby calculating the survival curve.

[0346] result

[0347] refer to Fig. 27 , shows a table of raw values ​​of T14 50KDa of 100 leukemia patient serum samples detected by WB, which were normalized to total protein and divided into groups below and above the median value.

[0348] refer to Fig.28, the Kaplan Meier estimation performed on the two groups showed high prognostic significance in this group. As shown in the hazard ratio (HR = 2.37, P = 0.01, numerical value 42), the probability of patients with serum T14 50KDa / TP values ​​less than the median (light gray group) requiring treatment per unit time is 2.37 times higher than those with serum T14 / TP values ​​higher than the median (dark gray group). In addition, the survival analysis showed that the T14 50KDa / TP value is a predictive TTFT. The estimated median survival time was not reached for the low-risk group and was 8.05 years for the high-risk group.

[0349] discuss

[0350] The inventors have previously shown that T14 is involved in cancer mechanisms and is a good cancer biomarker. Therefore, T14 together with conventional prognostic tools can predict patient survival and time to first treatment.

[0351] in conclusion

[0352] In summary, Western blotting is a surprisingly reliable method to detect conditions in leukemia. Although ELISA data may not be reliable for clinical indication at present, it may indicate the potential mechanisms mediating cell migration.

[0353] Example 5 - Design and Generation of Peptide Mimetic T14 Inhibitors

[0354] The inventors have designed and synthesized peptidomimetic compounds that inhibit T14 activity, thereby competing with and outcompeting T30 for the allosteric active site of the nicotinic receptor.

[0355] Compound 1 – Tri02 (score: -10.2)

[0356]

[0357] 4-((S)-2((S)-2-acetylamino-3-(naphthalen-2-yl)propionylamino)-3-(((S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl)amino)-3-oxopropyl)anilinium

[0358] Compound 2 – Tri04 (score: -9.4)

[0359]

[0360] 4-((S)-2-((S)-2-((S)-2-acetylamino-3-(4-benzoylphenyl)propionylamino)-6-((amino(imino)methyl)amino)hexanoylamino)-3-amino-3-oxopropyl)anilinium

[0361] Example 6 - Synthesis of identified compounds

[0362] Materials and methods

[0363] The T14 inhibitor compounds 1 and 2 (Tri02 and Tri04) of Example 5 were synthesized by Genosphere Biotechnology, and analyzed for purity using RP-HPLC (>99% pure) and mass by mass spectrometry (average MS of 604.79 for Tri02 and 628.83 for Tri04).

[0364] TRI02-Sequence: A brief step-by-step description of the synthesis of [acetyl]-[2Nal][4NH2-F]-Trp-[amide]

[0365] 1) Boc-Trp-OH+ClooEt+NH3.H2O------Boc-Trp-NH2, react in THF and extract with ethyl acetate.

[0366] 2) Boc-Trp-NH2, 4NHcl, remove Boc- to obtain H-Trp-NH2.Hcl, and carry out precipitation reaction through ether.

[0367] 3) (2-naphthyl)-Ala + acetic anhydride----Ac-(2-naphthyl)-Ala-OH, react with THF / H2O, and extract with ethyl acetate.

[0368] 4) Boc-(4-NH2)-Phe-OH+H-Trp-NH2.Hcl----Boc-(4-NH2)-Phe-Trp-NH2, react in DMF and extract with ethyl acetate.

[0369] 5) Boc-(4-NH2)-Phe-Trp-NH2, 4NHcl, remove Boc- to obtain H-(4-NH2)-Phe-Trp-NH2.Hcl, and carry out precipitation reaction through ether.

[0370] 6) Ac-(2-naphthyl)-Ala-OH + H-(4-NH2)-Phe-Trp-NH2.Hcl--Ac-(2-naphthyl)-Ala-(4-NH2)-Phe-Trp-NH2 was reacted in DMF and extracted with ethyl acetate.

[0371] 7) Purification

[0372] TRI04-Sequence: A brief step-by-step description of the synthesis of [acetyl]-[bpa]R[4NH2-F]-[amide]

[0373] 1) Rink amide MBHA. The resin was soaked in DCM for 30 minutes, pumped dry, washed 3 times with DMF and pumped dry.

[0374] 2) Add Fmoc-(4-NH2)Phe-OH, DIEA, HBTU, DMF, and N2, react for 30 minutes, pump dry, wash with DMF 6 times, and pump dry.

[0375] 3) Add piperidine / DMF to remove Fmoc-, react for 20 minutes, pump dry, wash 3 times with DMF, and pump dry.

[0376] 4) Add Fmoc-Arg(Pbf)-OH, DIEA, HBTU, DMF, and N2, react for 30 minutes, pump dry, wash with DMF 6 times, and pump dry.

[0377] 5) Repeat step 3.

[0378] 6) Add Fmoc-Bpa-OH, DIEA, HBTU, DMF, and N2, react for 30 minutes, pump dry, wash with DMF 6 times, and pump dry.

[0379] 7) Repeat step 3.

[0380] 8) Add acetic anhydride / DMF, N2, react for 30 min, pump dry, wash 3 times with DMF, pump dry, wash 3 times with DCM, pump dry, wash 3 times with MeOH, pump dry.

[0381] 9) The peptide was cut from the resin, pumped dry, precipitated with ether to obtain the crude peptide, and centrifuged to dry.

[0382] 10) Purification

[0383] Example 7 - T30 vs. T14:

[0384] Referring to Figure 43, the inventors conducted preliminary studies using 14-mer T14, which was shown to be trophotoxic in a range of preparations. Although the active sequence of the AChE-peptide can be attributed to a specific 14 amino acid sequence derived from the C-terminal tail of AChE (T14) (Greenfield, 2013–Greenfield, SA (2013) 'Discovering and targeting the basic mechanism of neurodegeneration: The role of peptides from the c-terminus of acetylcholinesterase: Non-hydrolytic effects of ache: The actions of peptides derived from the c-terminal and their relevance to neurodegeneration', the active sequence of the AChE-peptide can be attributed to a specific 14 amino acid sequence derived from the C-terminal tail of AChE (T14) (Greenfield, 2013–Greenfield, SA (2013) 'Discovering and targeting the basic mechanism of neurodegeneration: The role of peptides from the c-terminus of acetylcholinesterase: Non-hydrolytic effects of ache: The actions of peptides derived from the c-terminal and their relevance to 15), but studies on exogenous AChE-peptide therapy have recently involved a 30 amino acid peptide (T30) that includes the active T14 motif: the larger T30 is less likely to form fibrils in solution and thus has a higher stability and greater potency than T14 (Bond et al., 2009Bond, C., Zimmermann, M. and Greenfield, S. (2009) 'Upregulation of alpha7 Nicotinic receptors by Acetylcholinesterase c-terminal peptides', PloSone, 4(3). doi: 0.1371 / journal.pone.0004846).

[0385] Therefore, the T30 peptide was used throughout this study (Badin, AS, Morrill, P., Devonshire, I., Greenfield, SA, January 7, 2016. (II) Physiological profiling of anendogenous acetylcholinesterase-derived peptide in the basal forebrain: age-related bioactivity and blockade with a novel modulator. Neuropharmacology. 105, 47e60). The 15 amino acid residues 'T15' at the C-terminus of T30 were used as a control because it was shown to be inert by itself and therefore did not contribute to the biological activity of T14. In addition, the efficacy of T14 itself has been shown to require antibody binding to the terminal lysine on the C-terminus, which is not exposed within the longer T30 sequence. Therefore, T30 can be used to study the effects of exogenous peptides or their endogenous counterparts.

[0386] In summary, T30 is a convenient experimental tool to study nutrient toxicity effects, providing an inert control and allowing the antibody to detect endogenous T14 without cross-contamination with exogenous (T30) peptide probes. Therefore, any data showing inhibition of T30 activity demonstrates that T14 activity is also inhibited.

[0387] Example 8 - Evaluation of Compound 1 (Tri02) and Compound 3 (Tri04) in Cell Culture

[0388] The inventors tested T30, NBP-14 and Tri02 in cell culture studies to determine their effects on acetylcholinesterase activity and calcium influx, and the effect of Tri04 on calcium influx.

[0389] Materials and methods

[0390] 1. AChE activity assay

[0391] AChE activity was measured using Ellman's reagent, which measures the presence of thiol groups, which are responsible for the activity of AChE. In the case of the G4 experiment, AChE (G4) activity was tested alone and also with NBP14 or Tri peptide. PC12 cells were plated the day before the experiment as in the cell viability assay. Cells were treated with T30 (1 μM) alone or with NBP14 or Tri peptide (0.5 μM). After treatment, the supernatant (perfusate) of each treatment was collected and 25 μL of each condition was added to a new flat-bottom 96-well plate, followed by the addition of 175 μl of Ellman's reagent (solution A: KH2PO4 139 mM and K2HPO4 79.66 mM, pH 7.0; solution B (substrate): iodinated acetylthiocholine 11.5 mM; solution C (reagent): 5,5'-dithiobis(2-nitrobenzoic acid) 8 mM and NaHCO3 15 mM). Ellman's reagent was prepared as a mixture of 3 solutions in the ratio 33(A):3(B):4(C).Absorbance measurements were performed at 405 nm in a Vmax plate reader (Molecular Devices, Wokingham, UK) at 60 min intervals throughout the experiment.

[0392] 2. Calcium Fluorescence Assay

[0393] PC12 cells were plated in 200 μl of Dulbecco's modified Eagle medium (DMEM) plus 2mM L-glutamine medium in 96-well plates the day before the experiment. On the same day of the experiment, Fluo-8 solution (Abcam) was prepared by adding 20 μl of Fluo-8 in the assay buffer containing 9ml of Hank's balanced salt solution (HBSS) and 1ml of pluronic F127Plus as described by the supplier. Subsequently, 100 μl of growth medium was removed, and 100 μl of Fluo-8 solution was added. The treated material treated with T30 and NBP14 or Tri peptide was added, and incubated in an incubator for 30 minutes, then incubated at room temperature for 30 minutes. After 1 hour, the plate was placed in a fluorescent plate reader (Fluostar, Optima, BMGLabtech, Ottenberg, Germany). Prior to reading the fluorescence, PNU282987 1 μM (α7 specific agonist of nicotinic receptors) was prepared and placed in a Fluostar syringe. For each well, a readout was made by reading the basal fluorescence followed by an injection of PNU282987 to induce an increase in calcium via the nicotinic receptors.

[0394] 3. Data Analysis

[0395] In each of the different cell techniques, statistical analysis was performed to obtain the mean value of the percentage values ​​of 3 or more experiments. The comparison between multiple treatment groups and the same control was performed by one-way analysis of variance (ANOVA) and Tukey's post-hoc test using GraphPAD Instat (GraphPAD Software, San Diego, California). Statistical significance was obtained at p value < 0.05.

[0396] result

[0397] The results of Tri02 are Fig.12 and 13 , where n values ​​shown on subsequent figures refer to the number of replicate experiments. It can be seen that 1 μM T30 increases calcium influx and AchE activity, and as shown in previous studies (see WO2005 / 004430), 1 μM NBP14 prevents these toxic effects.

[0398] In addition, as can be seen from the figure, Tri02 also clearly prevents the toxic effects of T30 by reducing both calcium influx and AChE activity. As such, the inventors confirmed that Tri02 acts as a T14 activity inhibitor and can be used to treat cancer or metastasis.

[0399] The results of Tri04 in PC12 cell culture were Fig.19 The cells are derived from a PC12 cell line that is derived from a tumor from the adrenal gland and acts as a neuron (Bornstein et al., Mol. Psychiatry (2012), 17, 354-358). It can be seen that Tri04 also prevents the toxic effects of T30 in these cells by reducing calcium influx, indicating that it acts as a T14 inhibitor and can therefore be used to treat cancer or metastasis.

[0400] Example 9 - Evaluation of Compound 1 in Brain Slices

[0401] The inventors tested NBP-14 and Tri02 in brain slice studies using voltage-sensitive dye imaging (VSDI).

[0402] Materials and methods

[0403] 1. Preparation of Brain Slices

[0404] Male Wistar rats (14 days old) were anesthetized using isoflurane (~15 ml, 100% w / w). Isoflurane was applied to the cotton bed at the bottom of the anesthesia chamber (glass box, 20x15x15 cm), and the rats were then placed therein for approximately 45 seconds until complete anesthesia was achieved. The hind paws of each anesthetized rat were pinched to check for appropriate depth of anesthesia. After confirming anesthesia, the rats were quickly decapitated, the brains were quickly removed, and immersed in ice-cold oxygenated 'slicing' artificial cerebrospinal fluid (aCSF, in mmol: 120 NaCl, 5 KCL, 20 NaHCO3, 2.4 CaCl2 2MgSO4, 1.2 KH2PO4, 10 glucose, 6.7 HEPES salts, and 3.3 HEPES acid; pH = 7.1). Then a piece of brain containing the basal forebrain (i.e., the MS-dBB complex (Interaural between +9.20 and +9.48 mm and Bregma between +0.48 and +0.2 mm, Figure 4 Coronal sections (400 μm thick) were obtained from the posterior cingulate cortex (A) and the somatosensory barrel field cortex (S1BF, Interaural between +8.08 and +7.20 mm and Bregma between -0.92 mm and -1.80 mm) (Paxinos and Watson, 1998). The sections were then transferred to a bubbler containing oxygenated aCSF (recording aCSF, in mmol: 124 NaCl, 5 KCL, 20 NaHCO3, 2.4 CaCl22 MgSO4, 1.3 KH2PO4, 10 glucose; pH = 7.4) at room temperature, which was the same as that used for VSDI (voltage sensitive dye imaging) recordings. The sections were then left for approximately 1 to 1.5 hours before they were prepared for VSD staining.

[0405] 2. VSD settings

[0406] The sections were placed in a dark, high humidity chamber filled with aCSF, bubbled with 95% O2, 5% CO2. Once this was done, a dye solution (4% 0.2 mM styryl dye 4-[2-[6-(dibutylamino)-2-naphthyl]-vinyl]-1-(3-sulfopropyl)pyridinium hydroxide (Di-4-NEPPS), Invitrogen, Paisley, UK, in 48% aCSF, 48% fetal bovine serum, 3.5% DMSO and 0.4% Cremophore EL) (Tominaga et al., 2000) was applied to the sections for 20 to 25 minutes and then transferred back to the bubbler containing oxygenated aCSF at room temperature for 30 minutes.

[0407] When starting VSDI recording, slices are placed on a small piece of filter paper in the recording tank to allow oxygenated aCSF to flow under the slice and keep it alive. Then the slices are weighted by a homemade plastic grid placed on the top of the slice. The perfusion tank solution is heated to 30 ± 1 ° C by a graded heater. Coaxial bipolar stimulating electrodes (FHC, Maine, USA) are placed in the ventral diagonal band of the basal forebrain, and the stimulation is set to 30V. In order to obtain VSD data, MiCam02 high-resolution camera (Brain Vision, Japan) and BV_Analyze imaging software are used to record two-dimensional images equivalent to 88x60 pixels. Image acquisition is combined with Spike2V4.23 software (CED Co., Ltd., Cambridge, UK) to make image capture consistent with the stimulation scheme (repeated 30 times every 28 seconds) by Micro 1401MkII. (CED Co., Ltd., Cambridge, UK). Light was generated using an Osram halogen xenophot 64634HLX EFR Display / Optic lamp and filtered using a MHF-G150LR (Moritex) connected to a MiCam02 high-resolution imaging system to emit green (530±10 nm) light and to filter out emitted fluorescence by a high-pass filter >590 nm.

[0408] 3. Preparation and application of drugs

[0409] The acetylcholinesterase (AChE) C-terminal 30 amino acid peptide (T30; sequence: 'N' - KAEFHRWSSYMVHWKNQFDHYSKQDRCSDL - SEQ ID No: 2), the active 14 amino acid region of T30 in the cyclic form of T30 (NBP14; sequence: c[AEFHRWSSYMVHWK] - SEQ ID No: 3; c[] = cyclic, N-terminal to C-terminal), and the inert 15 amino acid peptide contained within the T30 sequence (T15; sequence: 'N' - NQFDHYSKQDRCSDL - SEQ ID No: 4) were custom synthesized and purchased from Genosphere Biotechnologies (Paris, France) with a purity of >99%. The linear peptide mimetic Tri02 was designed in silico by Iproteos (Barcelona, ​​Spain), synthesized, and purchased from Genosphere Biotechnologies with a purity of >99%. Stocks of all drugs and peptides were prepared and frozen in aliquots prior to experiments. To generate the perfusion solution, the stock solution was thawed, added to the recording aCSF as needed, and bath applied at a constant rate of 1.5 ml / min perfusion using a Minipulse3 peristaltic pump (Gilson Scientific Ltd., Bedfordshire, UK). Each pilot experiment lasted 52 minutes, with 20 minutes to establish baseline recording (using only the recording aCSF perfusion), 12 minutes to allow the drug solution to perfuse into the bath and for the dye molecules to reset themselves in the cell membrane, and a final 20-minute recording period to measure responses in the presence of the drug solution.

[0410] 4. Data Analysis and Statistics

[0411] Key data such as activation time course, intensity and spread of the overall fluorescent signal were extracted from the two-dimensional images generated for each drug condition. These data were processed using custom scripts to convert them into usable MatLab (Mathworks, MA, USA) files and then analyzed using a Matlab toolbox specifically made for VSDI data analysis (Bourgeois et al., 2014). The toolbox allows the selection of fixed region of interest (ROI) geometries that can be applied to each slice so that data can be extracted and collated from the same ROI in all slices used in each experiment. For basal forebrain slices, the ROI to be used is the MSdBB complex, which was chosen because it includes the MS (septal nucleus), VDB (ventral diagonal band) and HDB (horizontal diagonal band). More importantly, the ROI was chosen so as to include the entirety of the evoked response. The response can be plotted as a single average time series, or plotted over space and time in a 'space-time map' to provide a qualitative description of the data. However, in order to generate quantifiable values, the area under the time series (total fluorescence microchange) between the stimulus moment (t = 0) and the subsequent 156 milliseconds was calculated. Due to the variability of responses between each individual slice, the raw data generated from each experiment were normalized to their baseline to give normalized fluorescence values. This quantification method was chosen to account for the multiple components of the signal generated by VSDI (Chemla and Chavane, 2010), namely the immediate peak and long-latency responses (Badin et al., 2016). Statistics were performed in Prism Graphpad 6.

[0412] 5. Analysis of regulatory peptides

[0413] Increases or decreases in signal were observed throughout the experiments using T30. Therefore, after averaging these results together, no changes were detected. However, given the previously observed modulatory effects of this peptide in various preparations (Bon and Greenfield, 2003, Day and Greenfield, 2004, Greenfield et al., 2004, Badin et al., 2013) and the fact that the changes caused by the application of T30 in such preparations were moderately negatively correlated with the baseline response amplitude (r = -0.4286, p = 0.0257, Spearman rank correlation, n = 27, Fig.13 A), decide whether these outcomes should be dichotomized by seeing an increase or decrease.

[0414] Subsequently, similar correlation analyses were performed for each experiment in which exogenous compounds were added, ( Fig.16 ). After determining that there is a significant correlation, the data are categorized based on whether an increase or decrease is observed.

[0415] result

[0416] refer to Fig.14 and 15 , addition of 4 μM Tri02 reproduced the results seen with 4 μM NBP14.

[0417] refer to Fig.14 , the addition of NBP14 (4uM) to the perfusate caused a small, non-significant change in the amplitude of the evoked response (total fluorescence). Although not significant, these small changes were found to be inversely correlated with the baseline response amplitude; therefore, the data are divided into experiments that caused a slight decrease (left histogram) and experiments that caused an increase (right histogram), both of which are given in actual (top) and normalized data formats (bottom). When considered together, the data sets do not show changes from the baseline (because increases and decreases will cancel each other out), but it is important to check that NBP14 did not cause a significant effect even when considering fluorescence changes separately.

[0418] like Fig.15 As shown, the addition of Tri02 (4uM) to the perfusate caused a small change in the amplitude of the evoked response (total fluorescence), and the reduction caused (n = 8 of a total of 11) showed a significant deviation from the normalized baseline level (bottom left histogram, p < 0.05). It was found that these changes were also inversely correlated with the baseline response amplitude; therefore, the data were divided into experiments that caused a reduction (left histogram) and experiments that caused an increase (right histogram), both of which were given in actual (top) and normalized data formats (bottom). When considered together, the data set does not show changes from the baseline (because the increase and decrease will cancel each other out), but it is important to check that NBP14 did not cause a significant effect even when considering the fluorescence changes separately.

[0419] Analysis of regulatory peptide mimetics

[0420] refer to Fig.16 , shows a correlation analysis of Tri02 (4uM) and T30 (2uM) data (n=15), showing that their co-perfusion caused some changes in the amplitude of the evoked response, with some slices characterized by a slight increase in activity (n=6), while the majority showed a slight decrease (n=9). This correlation was found to be significant (p=0.0405; r 2 =-0.534), thus providing a clear reason to separate the data into those showing increases and decreases in the activity induced by the application of Tri02 and T30, just as for the addition of NBP14 and Tri02 (respectively Fig.14 and Fig.15 ) did.

[0421] refer to Fig.17, showing the quantification of the effects mediated by the addition of Tri02 and T30: in both cases where increases and decreases were induced, Tri02 was not found to prevent the deviations from the baseline induced by T30, with significant decreases (left panels, p<0.01, n=9) and increases (right panels, p<0.05, n=6) being recorded in the overall effect.

[0422] like Fig.18 As shown, the normal aCSF (black line), 2uM T30 (red line), T30 (2uM) and 4uM NBP14 (blue line), T30 (2uM) and 4uM Tri02, and control NBP14 (4uM) experiments ( Fig.11 , orange line), control Tri02 (4uM) experiment ( Fig.15 , purple line) relative to baseline. The figure shows the normalized decrease relative to baseline and relative to each other, with only T30 causing the largest deviations, Tri02 showing some efficacy in blocking those deviations caused by T30, but still causing significant changes in their co-perfusion (green line).

[0423] Example 10 - Evaluation of Compound 2 in Brain Slices

[0424] The inventors tested Tri04 in brain slice studies using voltage-sensitive dye imaging (VSDI).

[0425] Materials and methods

[0426] 1. Preparation of Brain Slices

[0427] Brain slices were prepared as in Example 8.

[0428] 2. VSD settings

[0429] The sections were placed in a dark, high humidity chamber filled with aCSF, bubbled with 95% O2e5% CO2. Once in place, a dye solution (4% 0.2 mM styryl dye 4-[2-[6-(dibutylamino)-2-naphthyl]-vinyl]-1-(3-sulfopropyl)pyridinium hydroxide (Di-4-ANEPPS, Invitrogen, Paisley, UK) (Tominaga et al., 2000) 48%, fetal bovine serum 48%, DMSO 3.5% and cremophore EL 0.4%) in aCSF was applied to the sections for 20 to 25 minutes and then transferred to an aCSF bubbler (room temperature, 22C + / - 1.5C) for 1 hour to allow for washing out of excess dye and recovery.

[0430] When VSD recording was started, the slice was placed on a small piece of filter paper in the recording tank to keep the slice alive and appropriately weighted using a homemade plastic grid placed on top of the slice. The perfusion tank solution was heated to 30 + / - 1 ° C by a step heater. A coaxial bipolar stimulating electrode (FHC, Maine, USA) was placed in the ventral diagonal band of the basal forebrain and the stimulation was set to 30 V. To acquire VSD data, 16-bit images were recorded with a resolution of 1 ms using a digital camera (Brain Vision MiCAM Ultima R3-V20Master) and Ultima 2004 / 08 imaging software (Brain Vision) connected to Spike 2V6.0 (CED Ltd., Cambridge, UK) used to trigger stimulation relative to the appropriate ISI. Light was generated using an Osram halogen xenophot 64634HLX EFR Display / Optic lamp, filtered to emit green (530 + / - 10 nm) light using a MHF-G150LR (Moritex) connected to a MiCAM Ultima ultrafast imaging system, and fluorescent light was filtered by a >590 nm high pass filter.

[0431] 3. Preparation and application of drugs

[0432] Linear peptide mimics Tri04 is computer designed by Iproteos (Barcelona, ​​Spain), synthesized, and purchased from Genosphere Biotech, purity>99%. All drugs and peptide reserves are prepared into frozen aliquots before the experiment. In order to produce perfusion solution, the stock solution is melted, added to the recording aCSF as appropriate, and applied with a constant rate bath of 1.5ml / min perfusion using Minipulse 3 peristaltic pumps (Gilson Science Co., Ltd., Bedfordshire, UK). Each experimental experiment lasts 52 minutes, 20 minutes to establish baseline recording (using only recording aCSF perfusion), 12 minutes to allow drug solution to be perfused in the bath and allow dye molecules to reset themselves in the cell membrane, and the last 15 minutes of recording period measure the response in the presence of drug solution.

[0433] 5. Analysis of regulatory peptides

[0434] Signal reduction was observed during the majority of experiments using T30. T30 caused a net inhibition in VSDI signals recorded in p14 rat basal forebrain (n=21), a value that actually included a few cases where negligible or slightly positive effects were seen during T30 perfusion (Badin et al., 2016).

[0435] Results and discussion

[0436] 20, 21 and 22, the addition of 4 μM Tri04 reproduced the results seen previously with 4 μM NBP14, while 2 μM Tri04 in the perfusion solution determined a significant effect on basal forebrain population activity.

[0437] Analysis of regulatory peptide mimetics

[0438] refer to Fig. 20A , showing a spatiotemporal graph demonstrating restoration of basal forebrain activity due to the presence of 2 μM Tri04 in a perfusate containing 2 μM T30 (n=29). More specifically, 2 μM Tri04 determined the reversal of the inhibitory effect of T30 on activity measured by direct stimulation of the rat basal forebrain.

[0439] refer to Fig. 20B , The bar graphs relative to the three recording sessions show the changes in evoked responses after application of Tri04, confirming that co-infusion of 2 μM Tri04 caused an increase in network activity due to the inhibition of the effects induced by T30 (n=29, p=0.06, two-tailed paired t-test).

[0440] refer to Fig.21 , showing that the response time series under three recording conditions (baseline, application of T30 to artificial cerebrospinal fluid (aCSF), and co-application of T30 and Tri04 to aCSF) showed similar activation curves for the first 100 milliseconds of T30 recordings and T30+Tri04, while higher activity was subsequently detected in recordings obtained in the presence of Tri04, confirming the protective effect of Tri04 on T30.

[0441] refer to Fig. 22 , histograms relative to 3 recording conditions are shown. Co-perfusion of 4 μM Tri04 in artificial cerebrospinal fluid (aCSF) containing 2 uM T30 determined the significant effect of reversing T30 activity. In particular, it has been found that Tri04 prevents the deviation from baseline caused by T30, and basal forebrain activity is significantly increased compared to the recording in the presence of T30 alone (n=20, p<0.05, two-tailed paired t test). Therefore, Tri04 shows some efficacy in blocking the toxic effects of T30 on mesoscale network activity.

[0442] in conclusion

[0443] Cancer Treatment

[0444] The inventors have demonstrated herein that antibodies and two peptide mimetics Tri02 and Tri04 act as inhibitors of the activity of the T14 peptide, due to the obvious inhibitory effect on T30 in the data presented herein. Therefore, these compounds can be used to treat, alleviate or prevent cancer, particularly metastatic disease.

[0445] Cancer Diagnosis / Prognosis

[0446] The inventors have also shown that T14 or its variants (such as its truncations) can be used as diagnostic or prognostic markers for cancer, particularly metastatic disease. Low T14 levels measured in the blood by Western blotting indicate poorer survival and a correspondingly longer first treatment time (i.e., clinical status). Sequence Listing <110> Neuro-Bio Co., Ltd. <120> cancer <130> AXH / 78632PCT1 <150> GB1601585.1 <151> 2016-01-28 <160> 7 <170> PatentIn Version 3.5 <210> 1 <211> 614 <212> PRT <213> Homo sapiens <400> 1 Met Arg Pro Pro Gln Cys Leu Leu His Thr Pro Ser Leu Ala Ser Pro 1 5 10 15 Leu Leu Leu Leu Leu Leu Trp Leu Leu Gly Gly Gly Val Gly Ala Glu 20 25 30 Gly Arg Glu Asp Ala Glu Leu Leu Val Thr Val Arg Gly Gly Arg Leu 35 40 45 Arg Gly Ile Arg Leu Lys Thr Pro Gly Gly Pro Val Ser Ala Phe Leu 50 55 60 Gly Ile Pro Phe Ala Glu Pro Pro Met Gly Pro Arg Arg Phe Leu Pro 65 70 75 80 Pro Glu Pro Lys Gln Pro Trp Ser Gly Val Val Asp Ala Thr Thr Phe 85 90 95 Gln Ser Val Cys Tyr Gln Tyr Val Asp Thr Leu Tyr Pro Gly Phe Glu 100 105 110 Gly Thr Glu Met Trp Asn Pro Asn Arg Glu Leu Ser Glu Asp Cys Leu 115 120 125 Tyr Leu Asn Val Trp Thr Pro Tyr Pro Arg Pro Thr Ser Pro Thr Pro 130 135 140 Val Leu Val Trp Ile Tyr Gly Gly Gly Phe Tyr Ser Gly Ala Ser Ser 145 150 155 160 Leu Asp Val Tyr Asp Gly Arg Phe Leu Val Gln Ala Glu Arg Thr Val 165 170 175 Leu Val Ser Met Asn Tyr Arg Val Gly Ala Phe Gly Phe Leu Ala Leu 180 185 190 Pro Gly Ser Arg Glu Ala Pro Gly Asn Val Gly Leu Leu Asp Gln Arg 195 200 205 Leu Ala Leu Gln Trp Val Gln Glu Asn Val Ala Ala Phe Gly Gly Asp 210 215 220 Pro Thr Ser Val Thr Leu Phe Gly Glu Ser Ala Gly Ala Ala Ser Val 225 230 235 240 Gly Met His Leu Leu Ser Pro Pro Ser Arg Gly Leu Phe His Arg Ala 245 250 255 Val Leu Gln Ser Gly Ala Pro Asn Gly Pro Trp Ala Thr Val Gly Met 260 265 270 Gly Glu Ala Arg Arg Arg Ala Thr Gln Leu Ala His Leu Val Gly Cys 275 280 285 Pro Pro Gly Gly Thr Gly Gly Asn Asp Thr Glu Leu Val Ala Cys Leu 290 295 300 Arg Thr Arg Pro Ala Gln Val Leu Val Asn His Glu Trp His Val Leu 305 310 315 320 Pro Gln Glu Ser Val Phe Arg Phe Ser Phe Val Pro Val Val Asp Gly 325 330 335 Asp Phe Leu Ser Asp Thr Pro Glu Ala Leu Ile Asn Ala Gly Asp Phe 340 345 350 His Gly Leu Gln Val Leu Val Gly Val Val Lys Asp Glu Gly Ser Tyr 355 360 365 Phe Leu Val Tyr Gly Ala Pro Gly Phe Ser Lys Asp Asn Glu Ser Leu 370 375 380 Ile Ser Arg Ala Glu Phe Leu Ala Gly Val Arg Val Gly Val Pro Gln 385 390 395 400 Val Ser Asp Leu Ala Ala Glu Ala Val Val Leu His Tyr Thr Asp Trp 405 410 415 Leu His Pro Glu Asp Pro Ala Arg Leu Arg Glu Ala Leu Ser Asp Val 420 425 430 Val Gly Asp His Asn Val Val Cys Pro Val Ala Gln Leu Ala Gly Arg 435 440 445 Leu Ala Ala Gln Gly Ala Arg Val Tyr Ala Tyr Val Phe Glu His Arg 450 455 460 Ala Ser Thr Leu Ser Trp Pro Leu Trp Met Gly Val Pro His Gly Tyr 465 470 475 480 Glu Ile Glu Phe Ile Phe Gly Ile Pro Leu Asp Pro Ser Arg Asn Tyr 485 490 495 Thr Ala Glu Glu Lys Ile Phe Ala Gln Arg Leu Met Arg Tyr Trp Ala 500 505 510 Asn Phe Ala Arg Thr Gly Asp Pro Asn Glu Pro Arg Asp Pro Lys Ala 515 520 525 Pro Gln Trp Pro Pro Tyr Thr Ala Gly Ala Gln Gln Tyr Val Ser Leu 530 535 540 Asp Leu Arg Pro Leu Glu Val Arg Arg Gly Leu Arg Ala Gln Ala Cys 545 550 555 560 Ala Phe Trp Asn Arg Phe Leu Pro Lys Leu Leu Ser Ala Thr Asp Thr 565 570 575 Leu Asp Glu Ala Glu Arg Gln Trp Lys Ala Glu Phe His Arg Trp Ser 580 585 590 Ser Tyr Met Val His Trp Lys Asn Gln Phe Asp His Tyr Ser Lys Gln 595 600 605 Asp Arg Cys Ser Asp Leu 610 <210> 2 <211> 30 <212> PRT <213> Homo sapiens <400> 2 Lys Ala Glu Phe His Arg Trp Ser Ser Tyr Met Val His Trp Lys Asn 1 5 10 15 Gln Phe Asp His Tyr Ser Lys Gln Asp Arg Cys Ser Asp Leu 20 25 30 <210> 3 <211> 14 <212> PRT <213> Homo sapiens <400> 3 Ala Glu Phe His Arg Trp Ser Ser Tyr Met Val His Trp Lys 1 5 10 <210> 4 <211> 15 <212> PRT <213> Homo sapiens <400> 4 Asn Gln Phe Asp His Tyr Ser Lys Gln Asp Arg Cys Ser Asp Leu 1 5 10 15 <210> 5 <211> 7 <212> PRT <213> Homo sapiens <400> 5 Ser Tyr Met Val His Trp Lys 1 5 <210> 6 <211> 4 <212> PRT <213> Homo sapiens <400> 6 Val His Trp Lys 1 <210> 7 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 7 Cys Ala Glu Phe His Arg Trp Ser Ser Tyr Met Val His Trp Lys 1 5 10 15

Claims

1. Use of the peptide of SEQ ID No: 3 in preparing a kit for diagnosing a subject suffering from cancer or for providing a prognosis of the condition of the subject; the kit comprises a detection tool for determining the concentration of the endogenous peptide of SEQ ID No: 3 in a sample of a test subject, wherein the presence of the endogenous peptide of SEQ ID No: 3 in the sample indicates that the subject suffers from cancer; wherein, The sample is blood or cells.

2. Use of the peptide of SEQ ID No: 3 according to claim 1 in preparing a kit, wherein: The detection means comprises an assay suitable for detecting the presence and / or absence of T14 positive cells in the sample.

3. Use of the peptide of SEQ ID No: 3 according to claim 2 in preparing a kit, wherein: The identification is an immunoassay test.

4. Use of the peptide of SEQ ID No: 3 according to claim 2 in preparing a kit, wherein: Said identification comprises the use of antibodies.

5. Use of the peptide of SEQ ID No: 3 according to claim 2 in preparing a kit, wherein: The identification involved the use of Western blotting.

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