Cancer

Abstract

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

Claims

1. A method for diagnosing and treating an individual who suffers from metastatic disease, or a pre-disposition thereto, the method comprising: (a) obtaining a sample from the individual; (b) detecting, in the sample for the presence of an endogenous peptide of SEQ ID No: 3, or a variant thereof having at least 90% sequence identity to SEQ ID No: 3; (c) diagnosing the individual with metastatic disease when the presence of the endogenous peptide of SEQ ID No: 3, or a variant thereof having at least 90% sequence identity to SEQ ID No: 3, is detected in the sample; and (d) administering an effective amount of a treatment for metastatic disease to the diagnosed individual.

2. The method of claim 1, wherein the sample is blood, plasma, serum, spinal fluid, urine, sweat, saliva, tears, breast aspirate, prostate fluid, seminal fluid, vaginal fluid, stool, cervical scraping, cytes, amniotic fluid, intraocular fluid, mucous, moisture in breath, animal tissue, cell lysates, tumour tissue, hair, skin, buccal scrapings, lymph, interstitial fluid, nails, bone marrow, cartilage, prions, bone powder, ear wax, or combinations thereof.

3. The method of claim 1, wherein the sample comprises a blood sample.

4. The method of claim 1, wherein the sample comprises venous or arterial blood.

5. The method of claim 3, wherein the blood sample comprises blood serum or blood plasma.

6. The method of claim 1, wherein the method comprises assaying for a labelled compound having affinity with a ligand of the endogenous peptide of SEQ ID No: 3, or a variant thereof having at least 90% sequence identity to SEQ ID No: 3.

7. The method of claim 1, wherein the method comprises immunoassaying the sample to detect the presence of the endogenous peptide of SEQ ID No: 3, or a variant thereof having at least 90% sequence identity to SEQ ID No: 3.

8. The method of claim 1, wherein the endogenous peptide of SEQ ID No: 3, or a variant thereof having at least 90% sequence identity to SEQ ID No: 3 is detected by Western Blot analysis, enzyme-linked immunosorbent assay (ELISA), fluorometric assay, chemiluminescent assay, or radioimmunoassay analyses.

9. The method of claim 1, wherein the variant of SEQ ID No:3 comprises or consists of SEQ ID No:6.

10. The method of claim 1, wherein the method comprises detecting for the absence of SEQ ID No:2.

11. The method of claim 1, wherein the method comprises detecting for the absence of SEQ ID No:4.

12. The method of claim 1, wherein the method comprises detecting the presence of an endogenous peptide of SEQ ID No: 3, or a variant thereof having at least 90% sequence identity to SEQ ID No: 3 with an antibody or antigen-binding fragment thereof that specifically binds to SEQ ID No: 3.

13. The method of claim 1, wherein the treatment comprises administration of a therapeutic agent and/or treatment regime that prevents, reduces or delays the development of metastatic disease.

14. The method of claim 13, wherein the therapeutic agent comprises Herceptin, or wherein the treatment regime comprises radiotherapy or chemotherapy.

15. A method for diagnosing and treating an individual who suffers from metastatic disease, or a pre-disposition thereto, the method comprising: (a) obtaining a sample from the individual; (b) detecting the presence of an endogenous antigen consisting of SEQ ID No: 3 in the sample; (c) diagnosing the individual with metastatic disease when the presence of the endogenous antigen in the sample is detected; and (d) administering an effective amount of a treatment for metastatic disease to the diagnosed individual.

Description

(1) For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made, by way of example, to the accompanying Figures, in which:

(2) FIG. 1 are Western Blot data showing similar mobility between T14 (A), alpha 7 receptor (B) and AChE (C) in the cell lysate of seven cancer cell lines (CL), i.e. MEC-1, KG1a, H929, MCF-7, MDA-MB-231, CLL, and JJN3, and normal B-lymphocytes acting as control. MDA-MB-231, KG1a, and MEC-1 cells are highly migratory cancer cell lines. H929, JJN3, CLL and MCF-7 are less migratory cancer cell lines. B-lymphocytes are normal, non-cancerous cells. The peptide/protein levels were normalised to total protein (TP) and plotted together for each cell line. Peptide/protein levels were similar within each cell line with the exception of MCF-7 cells in which T14 levels were lower. Between cell lines, the peptide/protein levels were variable. The protein levels of the above were then subsequently quantified, as shown in FIG. 2 and FIG. 5;

(3) FIG. 2 are graphs of the Western Blot data showing variable levels of T14, alpha 7 receptor and AChE in cell lysate between the cancer cell lines, but similar levels within each cell line;

(4) FIG. 3 are graphs based on the Western Blot data shown in FIG. 1. The T14, alpha 7 receptor and AChE levels were normalised to total protein (TP) and the level of each peptide/protein was correlated with the other in x-y plots and linear regression analysis were conducted, displaying lines of best fit (solid line) along with their 95% confidence interval (dotted lines). A): T14 and alpha 7 receptor, R.sup.2=0.6262, P=0.0193. B): Alpha 7 receptor and AChE, R.sup.2=0.6705, P=0.0129.C): T14 and AChE. The circle in (C) highlights an anomaly data point, which, when excluded, gives a significant positive correlation (R.sub.u=0.7032, P=0.0184). The fact that all correlations were significantly linear suggests that the peptide/proteins exist as a complex with a ratio of 1:1:1 (D);

(5) FIG. 4 are Western Blot data showing that T14 was detected in cancer cell culture media of all seven cancer cell lines, but that AChE and alpha7 were not. T14 mobility in the cell media is slightly different from that in the cell lysate. The protein levels of the above were then subsequently quantified, as shown in FIG. 5;

(6) FIG. 5 are graphs of the Western Blot data showing that T14 concentrations within the cancer cell culture media are significantly greater than the concentrations of cellular T14 peptide/protein levels in MEC-1, KG1a, Normal B lymphocyte and MDA-MB-231 cell lines. No alpha 7 and AChE signals were detected in the cancer culture media. The peptide/protein levels were normalised to total protein (TP) and they were plotted together for each cell line;

(7) FIG. 6 are graphs based on the Western Blot data shown in FIG. 1 and FIG. 4. T14, alpha 7 receptor and AChE levels were normalised to total protein (TP) and the level of T14 within the cancer culture media (extracellular T14) was correlated with each intracellular peptide/protein in x-y plots and linear regression analysis were conducted, displaying lines of best fit (solid line) along with their 95% confidence interval (dotted lines). A): Extracellular T14 and intracellular alpha 7 receptor, R.sup.2=0.6518, P=0.0154. B): Extracellular T14 and intracellular T14. Cell lines were split into two groups (Dark grey and light grey circles). Dark grey on its own showed R.sup.2=0.9794, P=0.0105. C): Extracellular T14 and intracellular AChE;

(8) FIG. 7 is a graph showing that T14 levels vary significantly between cell lines and between cell media and cell lysate detected by ELISA;

(9) FIG. 8 are graphs demonstrating the significant inverse correlations between T14 levels in ELISA and Western Blot measurements for cell media and cell lysate in cancer cell lines;

(10) FIG. 9 shows Western Blot data showing similar mobility between CD44, T14 and AChE in the cell membrane and cytosol fractions of six cancer cell lines and one cancer-derived cell line. Alpha 7 receptor displayed different mobility from CD44, T14 and AChE. The order of 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, whereas H929, JJN3, MCF-7 and PC12 are less migratory cancer cell lines;

(11) FIG. 10 shows relative expressions of CD44, T14, AChE and Alpha 7 receptor to total protein (TP) level. Strongly metastatic cancers have more proteins at the membrane compared to the cytosol, whereas this relationship is variable for weakly metastatic cancers;

(12) FIG. 11 shows significant positive correlations between CD44, T14 and AChE, strongly suggesting that T14 and AChE are good predictors of the degree of cancer metastasis;

(13) FIG. 12 shows cell culture data (i.e. acetylcholinesterase activity) for peptidomimetic compound 1 (i.e. Tri02);

(14) FIG. 13 shows cell culture data (i.e. calcium ion influx) for peptidomimetic compound 1 (i.e. Tri02);

(15) FIG. 14 shows the results of voltage-sensitive dye imaging (VSDI) on brain slices for control cyclic peptide NBP-14;

(16) FIG. 15 shows the results of voltage-sensitive dye imaging (VSDI) on brain slices for peptidomimetic compound 1 (i.e. Tri02);

(17) FIG. 16 shows correlation analysis of changes induced by addition of peptides against respective baseline response amplitude using voltage-sensitive dye imaging (VSDI) on brain slices. Changes in response amplitude induced by T30 were found to be negatively correlated with the amplitude of their respective baselines (A). Therefore, subsequent correlation analyses were carried out 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. Units on y-axis=ΔF/Fo; x-axis=ΣδF/Fo;

(18) FIG. 17 shows quantification of effects mediated by the addition of Tri02 and T30;

(19) FIG. 18 shows a graph comparing the co-application of T30 and NBP14 against that of Tri02 and T30 at blocking the effects of T30 on activity within the basal forebrain. NBP14 co-application was able to totally block the T30-induced effects, whereas T30 w/Tri02 caused a similar but muted modulatory response;

(20) FIG. 19 shows PC12 cell culture data (i.e. calcium ion influx) for peptidomimetic compound 2 (i.e. Tri04);

(21) FIG. 20 shows (A) space-time maps of basal forebrain activity changes induced by addition of peptides (T30 and Tri04) against the baseline response amplitude using voltage-sensitive dye imaging (VSDI) on brain slices. In (B) is shown a graph comparing basal forebrain evoked activity for recordings with T30 and Tri04 (2 uM) with that of T30 alone in the basal forebrain;

(22) FIG. 21 shows the fluorescence fractional change (response time-series, n=29) for recordings made in the presence of T30 alone or after co-application of T30 and its blocker Tri04 in comparison to the baseline condition;

(23) FIG. 22 shows a bar graph of basal forebrain activity using the blocker Tri04 at 4 μM concentration. Tri04 co-application was able to totally block the T30-induced effects in the rat basal forebrain;

(24) FIG. 23 shows a bar graph of the range of levels of T14 as measured by ELISA relative to a known concentration of exogenous T14. Patients are rank ordered by relative T14 concentrations in the examined CLL patient cohort;

(25) FIG. 24 shows a table of values of T14 as measured by ELISA relative to a known concentration of exogenous T14 in the examined CLL patient cohort and a statistical grouping;

(26) FIG. 25 shows the Kaplan Meier estimate conducted on 100 CLL serum samples separated into the dark grey group (top curve) above the median and the light grey group (bottom curve) below the median. Hazard ratio, P value and the predicted median TTFT are indicated above;

(27) FIG. 26 shows the Kaplan Meier estimate conducted on 100 CLL serum samples separated into four groups non different groups. Hazard ratio, P value and the predicted median TTFT are indicated above;

(28) FIG. 27 shows a table of values of T14 from the examined CLL patient cohort and a statistical grouping;

(29) FIG. 28 shows Kaplan Meier estimate conducted on 100 CLL serum samples, separated into the dark grey group (top curve) above the median and the light grey group (bottom curve) below the median. Hazard ratio, P value and the predicted median TTFT are indicated above; and

(30) FIG. 29 shows the results of a cell proliferation assay. Both T14 and T30 exhibit equal toxic effects at high concentrations (10 μM), the trophic effect at low concentrations on astroglia is greater with T30 (120±4% at 1 pM and 135±9% at 10 pM) than with T14 (96±5% at 1 pM and 121±8% at 10 pM).

EXAMPLES

Materials and Methods

Synthesis of Polyclonal Antibody

(31) The antibody was synthesised on order by Genosphere Biotechnologies (Paris, France). Two New Zealand rabbits were used with four immunizations with KLH-peptide (“Pep4”:T14-hapten CAEFHRWSSYMVHWK—SEQ ID No. 7) as immunogen over 70 days. The C was included to link the T14 peptide to the KLH acting as immunogen. The animals were bled four times and the bleeds were pooled. The antiserum was then passed through a gravity column with covalently bound peptide-support, following washing, the antibodies were eluted in acidic buffer and the solution neutralized. Further dialysis against PBS buffer and lyophilisation completed the process.

Optimisation of the T14 Antibody Conditions

(32) The manufacturer's report on the antibody was used to perform the experiments of ELISA at the optimum conditions. In the report, the manufacturer specifies the optical density related to the concentration of antibody (see table below) and the ELISA protocol used for this procedure (see protocol below).

(33) Protocol from the Manufacturer:

(34) Antigens were coated on EIA strips at ioug per well. Wells were washed with 200 ul PBS buffer.

(35) TABLE-US-00005 Manufacturer ELISA Results: Absorbance 405 nm Antibody Rabbit No. 1 Rabbit No. 2 Dilution Pre-immune Purified Pre-immune Purified 1:1 000 0.120 1.64 0.143 1.66 1:10 000 0.045 0.77 0.036 0.74 1:100 000 0.016 0.19 0.017 0.19

(36) Antisera was diluted in series, added in separate wells, and incubated for 2 hours. Unbound antibodies were washed and anti-rabbit IgG-HRP conjugate was added. Plates were washed and colour development run for 15 minutes with TMB substrate. Absorbance was read at 405 nm (2.00 AUFS). Colour intensity was directly proportional to the amount of antibodies. Antibody was positive if absorbance was >2 folds over that of pre-immune serum. Background absorbance for pre-immune serum could reach 0.1 to 0.3.

Conclusion

(37) For all the experiments described herein, the 1:1000 was the chosen dilution of antibody.

Western Blotting

Cell Fractionation

(38) Whole cell pellets from the cancer cell lines (JJN3, MDA-MB231, MCF-7, KG1a, MEC-1, H929, PC12) were resuspended in the 300 μl of subcellular fractionation buffer (Hepes-NaOH 10 mM, MgCl.sub.2 1.5 mM, KCl 10 mM, EDTA 1 mM, DTT 1 mM, 1× Proteinase Inhibitor Cocktail, Nonidet P-40 0.05%) and left on ice for 10 min. Following that, the mixtures were homogenised using a polytron to ensure cell lysis. Then, the mixtures were centrifuged at 500 g for 5 min. The resulting supernatant contained the cytosol portion and was retained. The resulting pellet contained the membrane fractions, which was then resuspended in a further 300 μl of subcellular fractionation buffer and retained.

(39) Cell Lysate Preparation

(40) Whole cell pellets from seven cancer cell lines (MEC-1, CLL, KG1a, JJN3, H929, MCF-7 and MDA-MB) and normal B lymphocyte were kindly donated from Prof Chris Pepper (School of Bioscience, Cardiff University). Whole cell pellets were solubilised in 1×Lysis Buffer (20 mM Tris Base, 137 mM NaCl, 1% TWEEN-20 detergent, 2 mM EDTA) containing protease inhibitor cocktails (Phosphatase 1:1, PMSF 1:1, aprotinin 1:1) with a 17% v/v ratio. Subsequently, the mixture was triturated using a Polytron for 10 seconds and shaken at 4° C. for 2 hours. Then, the samples were centrifuged at 13,000 g for 30 minutes at 4° C. and the supernatants were taken and stored at −80° C.

Measuring Protein Sample Concentration

(41) Protein concentrations from the above samples were measured using the Pierce™ 660 nm Protein Assay (Thermo Scientific). In short, a serial dilution (0 to 2 mg/ml) was made from a 10 mg/ml stock of bovine serum albumin (BSA). Three replicates of each BSA concentration were prepared by transferring 10 μl of the protein into a clear 96 well plate (Greiner). Then, samples were diluted with three concentrations (1:1, 1:2, 1:10) and three replicates of each concentration were placed into the same 96 well plate with each replicate containing 10 μl of sample. Subsequently, 150 μl of Pearce Reagent was added to the standards and all samples and the mixture was left to incubate for 5 min with gentle shaking. Finally, the plate was read on a spectrophotometer (Molecular Devices) at 660 nm. The protein concentrations of the samples were determined using the slope and y-intercept from the BSA standard curve, both calculated via Microsoft Excel.

Polyacrylamide Gel Electrophoresis of Protein Samples

(42) Polyacrylamide gels (mini-PROTEAN® TGX stain free™ gels, BIO-RAD) were placed into the electrophoresis tank (BIO-RAD, mini-PROTEAN tetra system) and Running buffer (25 mM TRIS-base, pH 8.6, 192 mM glycine, 0.1% SDS) was added the gel and tank reservoirs (BioRad). Protein samples were prepared by mixing with distilled water and 4× Laemmli sample buffer (final concentrations: 69.5 mM TRIS-Ha pH 6.8, 1.1% LDS, 11.1% (w/v) glycerol, 0.005% bromophenol blue, BIO-RAD) and 2.5% mercaptoethanol (BIO-RAD). The mixtures were heated at 95° C. for 5 min before cooling on ice. Samples and the positive control were loaded into the gels and were electrophoresed alongside with a molecular weight marker (Precision Plus Protein™ Dual Xtra Standards, BIO-RAD) at 35 mV for 90 min. Ice block was placed inside the running tank to prevent any overheating.

Transfer of Protein Samples onto PVDF Membrane

(43) Stacking gels were trimmed off and the separating gels were transferred onto PVDF Transfer Membrane (Thermo Scientific) in a Mini Transblot Cell (BIO-RAD). Briefly, the PVDF Transfer Membrane was activated by soaking with methanol for 1 min followed by soaking with distilled water for 2 min. All layers were subsequently saturated with transfer buffer (20 mM TRIS-base pH 8.6, 154 mM glycine, 0.8% w/v SDS and 20% methanol). The transfer sandwich, in the order of bottom to top, consists of transfer sponge, blotting paper, the gel, PVDF Transfer Membrane, blotting paper, transfer sponge were placed into a transfer cassette, which was inserted into the Mini Transblot Cell filled with Transfer Buffer. Finally, electrophoretic transfer took place for 90 min at 200 mA. Ice block was placed inside the transfer tank to prevent any overheating.

Staining of PVDF Membrane

(44) BLOT-Faststain™ (G-Biosciences, USA) was used to stain for total protein, acting as the loading control. Immediately after electrophoretic transfer, the PVDF transfer membrane was stained with the diluted BLOT-Faststain™ fixer solution (10 fold) for 2 min with gentle shaking. The membrane was then incubated with the diluted BLOT-Faststain™ developer solution (4 fold) for 1 min with gentle shaking. Subsequently, the membrane was stored at 4° C. in the dark in the developer solution for 30 min to allow protein bands to reach maximum intensity. Finally, the membrane was washed with cold water to eliminate background staining and imaged using the G box (Syngene). The membrane can then be destained using warm deionised water (40-45° C.) and ready is for the blocking stage.

(45) Detection of Protein Bands

(46) The PVDF transfer membrane was blocked in TBS (TRIS-buffered saline, 20 mM TRIS-base pH 7.5, 0.5 mM NaCl) containing 5% skimmed milk powder for 1 h, then washed twice for 7 min each in TTBS (TBS supplemented with 0.05% v/v TWEEN-20 detergent). The membrane was incubated overnight at 4° C. with a primary antibody diluted in TTBS containing 1% skimmed milk powder. On the following day, the primary antibody was removed. The membrane was washed three times for 5 min each in TTBS, then incubated for 1 h at room temperature with the secondary antibody. The secondary antibody of choice depends on the type of primary antibody used. It can either be goat anti mouse secondary antibody conjugated to HRP (a9309, Sigma, diluted in TTBS containing 1% skimmed milk powder (working concentration: 1:1000) or goat anti rabbit secondary antibody conjugated to HRP (ab6721, abcam) diluted in TTBS containing 1% skimmed milk powder (working concentration: 1:5000). After secondary antibody incubation, membranes were washed three times for 5 min in TTBS before a final 10 min wash in TBS. Protein bands were detected using the G box (Syngene).

(47) TABLE-US-00006 TABLE 1 Primary antibodies used for Western blotting detection. Species Catalogue Working Antibody Raised in Company Number conc. Anti-T14 Rabbit Neuro-Bio N/A 1:1000 Anti-Nicotinic Rabbit Abcam Ab10096 1:1000 Acetylcholine Receptor alpha 7 Anti-AChE Rabbit Abnova PAB 5222 1:1000 Anti-CD44 Rabbit Sigma HPA005785 1:1000

Protein Band Imaging and Data Analysis

(48) The PVDF membrane was placed in the G box (Syngene). The focus and zoom settings were adjusted to ensure that the membrane was at its largest at the centre of the screen. Luminol and Peroxide solutions from Clarity™ Western ECL substrate (BIO-Rad) were mixed the equal parts and applied to the membrane. Images were taken in the dark at 1 min time intervals for 5 min to obtain the optimal signal for the protein bands. Following that, the membrane was exposed in white light using an automatic setting in order to obtain an image for the molecular ladder. The blot images were then analysed using image J. Box of equal sizes were placed around protein bands in each lane, allowing measurement of protein band intensities. The background was then subtracted from the band intensities and the results were analysed in Microsoft Excel and the Graphpad software.

(49) Antibody Stripping for Reprobing

(50) The protein signal from the PVDF transfer membrane can be stripped and reprobed for a different protein. Briefly, the membrane was washed with mild stripping buffer (200 mM Glycine, 3.5 mM SDS, 1% v/v TWEEN-20 detergent, pH 2.2) twice for 10 min each. Subsequently, the membrane was washed with PBS twice for 10 min each and then washed with TTBS twice for 5 min each. Clarity™ Western ECL substrate (BIO-Rad) was added to the membrane and imaged using the G Box (Syngene) in order to check for residual protein signals. If residual signal was too strong, then the whole stripping process was repeated. Then, the membrane was ready for subsequent blocking stage and primary antibody probing (see above).

ELISA for T14 Peptide Antibody

(51) The standard curves and the samples were run in triplicate. The Cell Culture Media and Cell Lysate samples were diluted 1:10 in PBS Buffer. The standard curve for determination of T14 peptide in brain tissue samples were diluted in PBS buffer. The standard curve ranged from 8 to 100 nM of T14, and the blank was PBS buffer alone. 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 following day the sample was removed by flicking the plate over a sink with running water, and 200 μl of the blocking solution containing 2% bovine serum albumin (BSA) in Tris-buffered saline and Tween 20 (TBS-T) was added and incubated for 4 h at room temperature. Blocking solution was then removed and 100 μl of antibody, diluted in blocking solution to 1 μg/ml, was added and incubated overnight at 4° C. The primary antibody was removed the next day and wells were washed 3 times with 200 μl of TBS-T. After 100 μl of secondary enzyme-conjugated antibody diluted in blocking solution to 0.1 μg/ml was added and incubated for 2 h at room temperature; the plate was covered with parafilm during all incubations. After 2 h, the plate was washed 4 times with TBS-T. The addition of 3,3,5,5-tetramethylbenzidine started the colour reaction. The reaction was stopped 20 min later with stopping solution containing 2 M H.sub.2SO.sub.4, and the absorbance was measured at 450 nm in a Vmax plate reader (Molecular Devices, Wokingham, UK).

Example 1

(52) The inventors set out to detect the levels of the toxic T14 peptide (AEFHRWSSYMVHWK—SEQ ID No:3), nicotinic alpha-7 receptor and acetylcholinesterase (AChE) proteins using Western Blotting in cell lysate and cell culture media of seven cancer cell lines (MEC-1, KG1a, H929, MCF-7, MDA-MB 231, CLL, JJN3), and normal B lymphocytes acting as control. In addition, they detected the levels of the T14 peptide using ELISA.

Results from Western Blotting

(53) Western Blotting (WB) was carried out to detect the levels of T14, AChE and alpha 7 nicotinic acetylcholine receptor in cell lysate (CL) of seven different cancer cell lines, and normal B lymphocytes as control. Qualitative data showed that T14, AChE and alpha 7 receptor were all detected in the CL of all cell lines. Surprisingly, all three proteins have virtually identical mobility (see FIGS. 1A, B and C). The inventors do not consider this to be antibody cross-reactivity because the three proteins showed different mobility in the human brain homogenate (data not shown). Subsequently, T14, AChE and alpha 7 receptor levels were quantified and normalised against total protein levels (Colins et al 2015).

(54) Quantitative data showed that the levels of T14, alpha 7 receptor and AChE within each cell line was similar, with the exception of the MCF-7 line having low cellular T14 levels (see FIG. 2). However, the levels of T14, alpha 7 receptor and AChE were variable between cell lines (see FIG. 2).

(55) Interestingly, within the cell lysate, significant positive correlations were found between levels of T14 and the alpha 7 receptor (see FIG. 3A), alpha 7 receptor and AChE (see FIG. 3B), T14 and AChE levels (see FIG. 3C). These correlations and the identical mobility of protein bands suggest that T14, AChE and alpha 7 receptor exist as a protein complex within the cancer cells with a ratio of 1:1:1.

(56) Subsequently, Western Blotting was carried out to detect the levels of T14 and possibly the alpha-7 receptor and AChE released into the cancer cell culture media (CCM) of the above cell lines. Qualitative data showed that there was only T14 released into the cell culture media (see FIG. 4) but AChE was not (see FIG. 4). There was a minute amount of alpha 7 receptor released into the cell culture media (see FIG. 4), suggesting that they could be from dead floating cancer cells.

(57) Quantitative data showed that T14 released into the cell culture media was greater than cellular T14, alpha 7 receptor and AChE in MEC-1, KG1a, Normal B lymphocyte and MDA-MB-231 cell lines (see FIG. 5). However, T14 released into the cell culture media was similar to T14, alpha 7 receptor and AChE levels in primary CLL, JJN3, H929 and MCF-7 cell lines (see FIG. 5).

(58) Subsequently, levels of T14 within the cell culture media was correlated with T14, AChE and alpha 7 receptor levels within the cell lysate. A significant inverse correlation was found between the released T14 levels in the cell culture media and the alpha 7 receptor levels in the cell lysate (see FIG. 6a). The inventors believe that this could be a general pathological mechanism since this effect also occurs in the Cerebral Cortex and Locus Coeruleus of Alzheimer's patients (data not shown). No apparent correlation was found between T14 within the cell culture media and T14 levels within the cell lysate, although there was a significant inverse correlation when only four cell lines were included in the analysis (see FIG. 6B, dark grey group). Moreover, no apparent correlation was found between T14 within the cell culture media and AChE levels within the cell lysate even when cell groups were analysed separately (see FIG. 6C).

ELISA Results

T14 Levels in Cell Lysate and Cell Culture Media

(59) The aim was to determine endogenous levels of T14 in cancer cells of varying metastatic propensity, kindly provided by Prof Chris Pepper of Cardiff University, in both the intracellular material recovered from lysed cells and levels of T14 released into the growth medium of the cells (CCM) using the ELISA Method. The method used combined results from a total protein (pierce assay) to provide a measure of μtg T14 for every mg of protein in the sample. The results show a high degree of variance in T14 levels between cell lines and between cell lysate and cell media within cell lines, as shown in FIG. 7.

(60) T14 was highest in the cell lysate in the MDA-MB cell line but the same cell line showed lower levels of T14 in the cell media comparatively. Levels of T14 were shown to be highest in the MCF-7 cell line, however this cell line also had one of the lowest concentrations of T14 in the cell lysate, suggesting that although T14 is highly expressed in this cell line, there is approximately 5× more outside of the cells than within the cells. Only JJN3 and KG1a had significantly different levels of T14 when comparing intracellular and extracellular levels. In JJN3, the same profile is seen as in the MDA-MB cell line with extracellular levels of T14 significantly higher than intracellular levels. The KG1a cell line is the only cell line to demonstrate a significantly higher level of T14 in the cell lysate when compared with the media. T14 in the media of the CLL cell line was below the limit of detection for this assay.

Comparison of Cancer Cell Line ELISA T14 Data with WB Cancer Cell Line Data

(61) Using the results taken from the ELISA and those collected from the Western Blots, the inventors compared μg of T14/mg protein and T14/total protein in both the cell lysate and in the cell media. A significant inverse correlation was seen between WB T14 and ELISA T14 levels measured in the cell media as well as in the cell lysate as can be seen in FIG. 8.

(62) The cell media and cell lysate both demonstrate inverse correlations between levels of T14 as measured by WB and ELISA (P<0.0001, R.sup.2=0.4956 and P=0.0315, R.sup.2=0.1451 respectively) after linear regression analysis were conducted, displaying lines of best fit (solid lines) along with the 95% confidence interval (dotted lines).

Summary

(63) 1) Within cancer cells, T14, alpha 7 receptors and AChE were all detected albeit with varying amounts. Surprisingly, all three proteins have similar mobility and their levels are positively correlated with each other, suggesting that they are complexed with each other. 2) Outside cancer cells (i.e. within the cell culture media), only the toxic T14 peptide was detected, but AChE and alpha-7 were not released into the external media. Moreover, T14 levels were higher outside the cells than inside for six out of the seven cancer cell lines, suggesting that T14 is produced to be released from the cell. Furthermore, the mobility of T14 outside the cell was different compared to that inside the cells. 3) Interestingly, the levels of T14 outside cancer cells were significantly negatively correlated with alpha-7 receptor levels within cancer cells. The inventors postulate that this could underlie a general disease mechanism as it is also the case within cerebral cortex and locus coeruleus homogenate of Alzheimer's disease patients (but not control patients), where a positive correlation exists instead between T14 and alpha-7. The inventors believe that this may be due to high concentrations of the T14 peptide down-regulating its target receptor in diseased individuals. 4) T14 levels inside and outside cancer cells were also measured by ELISA. The T14 levels in cancer cell media and cell lysate measured by ELISA were significantly inversely correlated with that from the Western Blot. This would suggest that ELISA may be measuring T14 monomers, whereas Western Blot is measuring T14 aggregate levels, and so the levels are complimentary.

Example 2

(64) The objective of this example was to investigate, using Western Blotting, the relationship between the metastatic marker CD44 with the toxic T14 peptide, AChE, and the alpha-7 receptor in membrane and cytosol fractions of six cancer cell lines (JJN3, MDA-MB231, MCF-7, KG1a, MEC-1, H929) and one cancer-derived cell line (PC12).

Western Blotting Results

(65) Qualitative data showed that, in the membrane fraction of all cell lines, CD44, AChE and T14 all have similar mobility (as shown in FIG. 9A, C, E), but that alpha-7 showed different protein mobility (see FIG. 9G). The same relationship between CD44, AChE and T14 was found in the cytosol portion of all cell lines (see FIG. 9B, D, F), with alpha-7 again being the exception (see FIG. 9H).

(66) Subsequently, all four peptide/protein levels (CD44, AChE, alpha-7 and T14) were quantified and normalised against total protein levels (Colins et al 2015). Quantitative data showed that strongly metastatic cancer cell lines (KG1a, MDA-MB231, MEC-1) have more peptide/proteins in their membrane compared to the cytosol, whereas weakly metastatic cancer cell lines/cancer derived cell line (JJN3, H929, MCF-7 and PC12) showed variable relationships between membrane and cytosolic peptide/proteins (see FIG. 10).

(67) Most importantly, the levels of metastatic marker CD44 is significantly and positively correlated with AChE in both the membrane (see FIG. 11A) and cytosol fractions (see FIG. 11B) as well as with T14 in both the membrane (FIG. 11C) and cytosol fractions (FIG. 11D). Moreover, AChE and T14 levels are significantly and positively correlated with each other in both the membrane (FIG. 11E) and cytosol fractions (FIG. 10). This finding strongly suggests that AChE and T14 are both good predictors of the degree of cancer metastasis, and perhaps the pivotal molecular intermediaries. Therefore, blocking the signalling actions of AChE and T14 can be utilized as anti-cancer therapeutics.

Summary

(68) 1) In six cancer cell lines and PC12 cells, the metastatic marker (CD44) is significantly and positively correlated with the toxic molecule T14 as well as with AChE. 2) This correlation is true for both within the cancer cell membrane and within the cancer cell cytosol. 3) This finding strongly suggests that T14 and AChE are good predictors of the degree of cancer metastasis, and perhaps the pivotal molecular intermediaries.

Example 3—T14 ELISA—CLL Cohort

(69) Given T14's clear role in cancer metastasis, the inventors explored, using ELISA, the potential of T14 as a cancer biomarker and its link to patient survival rate.

Materials and Methods

(70) T14 antibody: The antibody was synthesised by Genosphere Biotechnologies (Paris, France). Two New Zealand rabbits were used with four immunisations of keyhole limpet hemocyanin (KLH)-peptide (T14-hapten: CAEFHRWSSYMVHWK (SEQ ID No: 7); C was included to link to KLH as the immunogen) over 70 days. The animals were bled four times and the bleeds pooled. The antiserum was then passed through a gravity column with covalently bound peptide-support and, following washing, the antibodies were eluted in acidic buffer and the solution neutralised. Further dialysis against phosphate buffered saline (PBS) buffer and lyophilisation completed the process.

(71) ELISA for T14 peptide antibody: The standard curves and the samples were run in triplicate. The human serum samples were diluted 1:10,000, and the standard curve for determination of relative T14 content in the samples was diluted in PBS buffer. The standard curve ranged from 3.3 to 40 nM of 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 following day the sample was removed by flicking the plate over a sink with running water, and 200 ml of the blocking solution containing 2% bovine serum albumin (BSA) in Tris-buffered saline and TWEEN-20 detergent (TBS-T) was added and incubated for 4 h at room temperature. Blocking solution was then removed and 100 μl of antibody, diluted in blocking solution to 1 μg/ml, was added and incubated overnight at 4° C. The primary antibody was removed the next day and wells were washed 3 times with 200 μl of TBS-T. After 100 ml of secondary enzyme-conjugated antibody diluted in blocking solution to 0.1 mg/ml was added and incubated for 2 h at room temperature; the plate was covered with parafilm during all incubations. After 2 h, the plate was washed 4 times with TBS-T. The addition of 3,3,5,5-tetramethylbenzidine started the colour reaction. The reaction was stopped 30 min later with stopping solution containing 2MH2SO4, and the absorbance was measured at 450 nm in a Versamax plate reader (Molecular Devices, Wokingham, UK).

(72) Analysis: The results of the assay are recorded in an arbitrary unit of optical density (OD). The OD of the blank was subtracted from the signal of each sample prior to analysis. The OD of each sample in the assay was compared to a baseline of the lowest known concentration of T14 in the standard curve. The optical density could then be represented in relation to a known signal, and recorded as a proportion of this signal. All samples were compared for their relative level of T14 as a direct reading from the standard curve is not yet possible with the current method in human blood samples. The grouping of the samples into the groups “High”, “Med-High”, “Med-Low”, and “Low” are defined by the distance of the relative value from the mean value of the entire cohort. As the values are normally distributed, all values within 1 standard deviation of the mean are in the medium range, and are either “Med-High” if they are above the mean, and “Med-Low” if they are below the mean. “Low” T14 values are those that are below, and beyond the standard deviation of, the mean. Conversely “High” are those that are above, and also beyond the standard deviation of, the mean.

Results and Discussion

(73) Referring to FIG. 23, there is shown a bar graph of the range of levels of T14 as measured by ELISA relative to a known concentration of exogenous T14. Patients are rank ordered by relative T14 concentrations in the examined CLL patient cohort.

(74) Referring to FIG. 24, there is shown a table of values of T14 as measured by ELISA relative to a known concentration of exogenous T14 in the examined CLL patient cohort and a statistical grouping.

(75) Referring to FIG. 25, there is shown ELISA values in 2 different groups divided by relative T14 concentration analysed statistically by using the Kaplan Meier estimate conducted on 100 CLL serum samples.

(76) Referring to FIG. 26, there is shown ELISA values in 4 different groups divided by relative T14 concentration analysed statistically by using the Kaplan Meier estimate conducted on 100 CLL serum samples.

Example 4—Western Blot Data on the Cancer Samples (CLL Cohort)

(77) Given T14's role in cancer metastasis, the inventors explored, using Western blotting, the potential of T14 as a cancer biomarker and its link to patient survival rate.

Materials and Methods

(78) Samples: A total of 100 patients with CLL had their serum taken before their treatment and subsequently treated and monitored for 16 years.

(79) Western blotting: Protein concentrations were determined in the serum samples above using the Pierce™ 660 nm Protein Assay (Thermo Scientific, 22660). Subsequently, Western blot analysis was conducted on the samples using the previous established method [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 profiling of a novel modulator of the α7 nicotinic receptor: Blockade of a toxic acetylcholinesterase-derived peptide increased in Alzheimer brains’, Neuropharmacology. doi: 10.1016/j.neuropharm.2016.02.006.] The primary antibodies used were 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 derived from the cell lysates were quantified using Image J, measuring total optical intensity, and were subsequently normalized to total protein levels using Blot FastStain to control for loading error.

(80) Data analysis: T14 50 KDa/TP values were ranked from low to high and the median was calculated. Subsequently, the values were divided into two groups with the light grey group containing values below the median and the dark grey group containing values above the median (FIG. 28). Finally, Kaplan Meier estimate was performed on the above two groups. The analysis can assess the effect of a treatment on the number of subjects survived or saved after that treatment over a period of time (TTFT: time to first treatment). The survival curve is calculated by computing of probabilities of occurrence of event at a certain point of time and multiplying these successive probabilities by any earlier computed probabilities to get the final estimate.

Results

(81) Referring to FIG. 27, there is shown a table with raw values of T14 50 KDa normalised to total protein from 100 serum samples from leukaemia patients detected by WB, separated groups below and above the median.

(82) Referring to FIG. 28, Kaplan Meier estimate conducted on the two groups showed high prognostic significance in this cohort. Patients with serum T14 50 KDa/TP values less than the median (light grey group) were 2.37 times more likely to require treatment in unit time than those with serum T14/TP values above the median (dark grey group) as illustrated by the hazard ratio (HR=2.37, P=0.01, FIG. 42). Moreover, this survival analysis showed that T14 50 KDa/TP values are predicative TTFT. The estimated median survival times were not reached for the low risk group and 8.05 years for the high risk group.

Discussion

(83) The inventors have shown previously that T14 is involved in the cancer mechanism and is a good cancer biomarker. Therefore, T14 together with conventional prognostic tools, can predict patient survival and time to first treatment.

Conclusions

(84) In conclusion, western blot is a surprisingly reliable method for detecting status in leukaemia. Although, currently, the ELISA data may not be reliably used currently as a clinical indication, it could nonetheless show the underlying mechanism mediating cell migration.

Example 5—Design and Production of Peptidomimetic T14 Inhibitors

(85) The inventors have designed and synthesised peptidomimetic compounds which inhibit T14 activity, and thereby outcompete T30 for the allosteric active site of the nicotinic receptor.

(86) Compound 1—Tri02 (Score: −10.2)

(87) ##STR00058##

(88) 4-((S)-2((S)-2-acetamido-3-(naphthalene-2-yepropanamido)-3-(((S)-1-amino-3-(1H-indol-3-yl)-1-oxopropan-2-yl)amino)-3-oxopropyebenzenaminium

(89) Compound 2—Trio4 (Score: −9.4)

(90) ##STR00059##

(91) 4-((S)-2-((S)-2-((S)-2-acetamido-3-(4-benzoylphenyl)propanamido)-6-((amino(iminio)methyeamino)hexanamido)-3-amino-3-oxopropyl)benzenaminium

Example 6—Synthesis of Identified Compounds

(92) Materials and Methods

(93) The T14 inhibitor compounds 1 and 2 (Tri 02 and Tri 04) from Example 5 were synthesised by Genosphere Biotechnologies and analysed for purity using RP-HPLC (>99% pure), and mass by mass spectroscopy (average MS 604.79 for Tri02 and 628.83 for Tri04).

Brief Stepwise Description of Synthesis of TRI02—Sequence: [acetyl]-[2Nal][4nh2-F]-Trp-[amide]

(94) 1) Boc-Trp-OH+ClooEt+NH.sub.3.H.sub.2O —Boc-Trp-NH.sub.2, reaction in THF, extracted by acetic ether.

(95) 2) Boc-Trp-NH.sub.2,4NHcl, removed Boc-, obtained H-Trp-NH.sub.2.Hcl, precipitation reaction by diethyl ether.

(96) 3) (2-Naphtyl)-Ala+Acetic Anhydride—Ac-(2-Naphtyl)-Ala-OH, reaction THF/H.sub.2O, extracted byacetic ether.

(97) 4) Boc-(4-NH.sub.2)-Phe-OH+H-Trp-NH.sub.2.Hcl—Boc-(4-NH.sub.2)-Phe-Trp-NH.sub.2, reaction in DMF, extracted by acetic ether.

(98) 5) Boc-(4-NH.sub.2)-Phe-Trp-NH.sub.2,4NHcl, removed Boc-, obtained H-(4-NH.sub.2)-Phe-Trp-NH.sub.2.Hcl, precipitation reaction by diethyl ether.

(99) 6) Ac-(2-Naphtyl)-Ala-OH+H-(4-NH.sub.2)-Phe-Trp-NH.sub.2.Hcl—Ac-(2-Naphtyl)-Ala-(4-NH.sub.2)-Phe-Trp-NH.sub.2 reaction in DMF, extracted by acetic ether.

(100) 7) Purification

Brief Stepwise Description of Synthesis of TRI04—Sequence: [acetyl]-[bpa]R[4NH.SUB.2.-F]-[amide]

(101) 1) Rink Amide MBHA.Resin Soak in DCM for 30 mins, pumped dry, washed by DMF for 3 times, pumped dry.

(102) 2) Add Fmoc-(4-NH.sub.2)Phe-OH,DIEA,HBTU,DMF,N.sub.2, reaction for 30 mins, pumped dry, washed by DMF for 6 times, pumped dry.

(103) 3) Add piperidine/DMF to remove Fmoc-, reaction for 20 mins, pumped dry, washed by DMF for 3 times, pumped dry.

(104) 4) Add Fmoc-Arg(Pbf)-OH,DIEA,HBTU,DMF,N.sub.2, reaction for 30 mins, pumped dry, washed by DMF for 6 times, pumped dry.

(105) 5) Repeat step 3.

(106) 6) Add Fmoc-Bpa-OH,DIEA,HBTU,DMF,N.sub.2, reaction for 30 mins, pumped dry, washed by DMF for 6 times, pumped dry.

(107) 7) Repeat step 3.

(108) 8) Add Acetic Anhydride/DMF,N.sub.2, reaction for 30 mins, pumped dry, washed by DMF for 3 times, pumped dry, washed by DCM for 3 times, pumped dry, washed by MeOH for 3 times, pumped dry.

(109) 9) Peptide cleaved from resin, pumped dry, precipitation reaction by diethyl ether, obtain the crude peptide, centrifugal drying.

(110) 10) Purification

Example 7—T30 vs T14

(111) With reference to FIG. 43, the inventors' initial work was performed with the 14mer T14, proving to be trophic-toxic in a range of preparations. Whilst the active sequence of AChE-peptide can be attributed to a specific 14 amino acid sequence originating from the C-terminus tail of AChE (T14) (Greenfield, 2013—Greenfield, S. A. (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’, Chemico-biological interactions. 203(3), pp. 543-6. doi: 10.1016/j.cbi.2013.03.015), exogenous AChE-peptide treatment in investigations has more recently involved a 30 amino acid peptide (T30) which includes the active T14 motif: the larger T30 is less likely to form fibrils when in solution, thereby possessing a higher stability and greater efficacy than T14 (Bond et al., 2009Bond, C., Zimmermann, M. and Greenfield, S. (2009) ‘Upregulation of alpha7 Nicotinic receptors by Acetylcholinesterase c-terminal peptides’, PloS one., 4(3). doi: 0.1371/journal.pone.0004846).

(112) Hence, the T30 peptide was used throughout this study (Badin, A. S., Morrill, P., Devonshire, I., Greenfield, S. A., 2016 Jan 7. (II) Physiological profiling of an endogenous acetylcholinesterase-derived peptide in the basal forebrain: age-related bioactivity and blockade with a novel modulator. Neuropharmacology. 105, 47e60). The 15 amino acid residues at the C terminal of T30, ‘T15’, has been used as a control as it proved inert on its own and therefore not contributing to the bioactivity of T14. Moreover, the efficacy of T14 itself has been shown in the need for the antibody to bind to a terminal lysine on the C terminus that would not be exposed within the longer T30 sequence. Hence, T30 is useful in exploring the effects of the exogenous peptide or its endogenous counterpart.

(113) In summary, T30 is a convenient experimental tool for exploring trophic-toxic effects, providing an inert control, and allowing for the antibody to detect endogenous T14 without cross-contamination with the exogenous (T30) peptide probe. Accordingly, any data showing that T30 activity is inhibited is a demonstration that T14 activity is also inhibited.

Example 8—Evaluation of Compound 1(Tri02) and Compound 3 (Tri04) in Cell Cultures

(114) The inventors tested T30, NBP-14, and Tri02 in cell culture studies to determine their effects on acetylcholinesterase activity and calcium influx, and the effects of Tri04 on calcium influx.

(115) Materials and Methods

(116) 1. AChE Activity Assay

(117) AChE activity was measured using the Ellman reagent that measures the presence of thiol groups as a result of AChE activity. In the case of the G4 experiment, AChE (G4) activity was tested alone and also together with either NBP14 or Tri peptides. PC12 cells were plated the day before the experiment as for the cell viability assay. Cells were treated with T30 (1 μM) alone or combined with NBP14 or Tri peptide (0.5 μM). After treatment, supernatant (perfusate) of each treatment was collected and 25 μL from is each condition were added to a new flat bottomed 96 well plate followed by the addition of 175 μl of Ellman reagent (Solution A: KH.sub.2PO.sub.4 139 mM and K.sub.2HPO.sub.4 79.66 mM, pH 7.0; solution B (substrate): Acetylthiocholine Iodide 11.5 mM; Solution C (Reagent): 5,5′-dithiobis (2-nitrobenzoic acid) 8 mM and NaHCO.sub.15 mM). The Ellman reagent was prepared as a mixture of the 3 solutions in a ratio 33(A):3(B):4(C). Absorbance measurements were taken for an interval of 60 minutes across experiments at 405 nm in a Vmax plate reader (Molecular devices, Wokingham, UK).

(118) 2. Calcium Fluorometry

(119) PC12 cells were plated in 200 μl of Dulbecco's Modified Eagle's medium (DMEM) plus 2 mM of L-glutamine medium the day before the experiment in 96 well plates. On the day of the experiment, the Fluo-8 solution (Abcam) was prepared as described by the provider by adding 20 μl of Fluo-8 in the assay buffer that contains 9 ml of Hank's Balanced Salt Solution (HBSS) and 1 ml of pluronic F127 Plus. Subsequently, 100 μl of growth medium was removed and 100 μl of Fluo-8 solution were added. Treatments with T30 in conjunction with NBP14 or Tri peptides were added and incubated for 30 minutes in the incubator and 30 minutes room temperature. After 1 h, the plate was placed in the fluorescence plate reader (Fluostar, Optima, BMG Labtech, Ortenberg, Germany). Before reading the fluorescence, PNU282987 1 μM, an alpha7 specific agonist of the nicotinic receptors, was prepared and placed in the Fluostar injector. For each well, the reading was formed by a basal fluorescence reading followed by PNU282987 injection that induced an increase of calcium via nicotinic receptors.

(120) 3. Data Analysis

(121) In each of the different cell techniques, the statistics analysis was performed with the average of the percentage values of 3 or more experiments. Comparisons between multiple treatment groups and the same control were performed by one-way analysis of variance (ANOVA) and Tukey's post-hoc tests using GraphPAD Instat (GraphPAD software, San Diego, Calif.). Statistical significance was taken at a p value<0.05.

Results

(122) The results for Trio2 are shown in FIGS. 12 and 13, in which n values shown on the subsequent graphs refer to number of repeated experiments. As can be seen, 1 μM T30 increases calcium influx and AChE activity, and, as shown in previous work (see WO 2005/004430), 1 μM NBP14 protects against these toxic effects.

(123) In addition, as can be seen in the Figures, Tri02 also clearly protects against the toxic effects of T30 by reducing both calcium influx and AChE activity. As such, the inventors are convinced that Tri02 acts as a T14 activity inhibitor and can be used to treat cancer or matastasis.

(124) The results for Tri04 in PC12 cell culture are shown in FIG. 19. The cells were derived from a PC12 cell line, which come from a tumour of the adrenal gland and act as neurons (Bornstein et al., Mol. Psychiatry (2012), 17, 354-358). As can be seen, Tri04 also protects the toxic effects of T30 by reducing calcium influx in these cells indicating that it acts as a T14 inhibitor, and can therefore be used to treat cancer or metastasis.

Example 9—Evaluation of Compound 1 in Brain Slices

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

(126) Materials and Methods

(127) 1. Brain Slice Preparation

(128) Male Wistar rats (14 days old) were anaesthetised using isoflurane (−15 ml, 100% w/w). Isoflurane was applied to the cotton bed at the bottom of an anaesthetic chamber (glass box 20×15×15 cm) where rats were then placed for approximately 45 s until complete anaesthesia was reached. The hind paw of each anaesthetised rat was pinched to check for the appropriate depth of anaesthesia. Upon confirmation of anaesthesia, rats were quickly decapitated, with the brain being quickly removed and immersed in ice cold oxygenated ‘slicing’ artificial cerebrospinal fluid (aCSF in mmol: 120 NaCl, 5 KCL, 20 NaHCO.sub.3, 2.4 CaCl.sub.2 2 MgSO.sub.4, 1.2 KH.sub.2PO.sub.4, 10 glucose, 6.7 HEPES salt and 3.3 HEPES acid; pH=7.1). Coronal slices (400 μm thick) were then taken from a block of brain containing the basal forebrain, namely the MS-dBB complex (between +9.20 and +9.48 mm Interaural and +0.48 and +0.2 mm Bregma, FIG. 4A) and the somatosensory barrel field cortex (SiBF, between +8.08 and +7.20 mm Interaural and −0.92 mm and −1.80 mm Bregma) (Paxinos and Watson, 1998) using a Vibratome (Leica VT1000S). Slices were then transferred to a bubbler pot containing oxygenated aCSF at room temperature (recording aCSF in mmol: 124 NaCl, 5 KCL, 20 NaHCO.sub.3, 2.4 CaCl.sub.2 2 MgSO.sub.4, 1.3 KH.sub.2PO.sub.4, 10 glucose; pH=7.4) which was identical to that used in VSDI (voltage sensitive dye imaging) recording. Slices were then left for approximately 1-1.5 hours before preparing them for VSD staining.

(129) 2. VSD Setup

(130) Slices were placed in a dark, high humidity chamber filled with aCSF bubbled with 95% O.sub.2, 5% CO.sub.2. Once there, the dye solution (4% 0.2 mM styryl dye pyridinium 4-[2-[6-(dibutylamino)-2-napthalenyl]-ethenyl]-1-(3-sulfopropyl)hydroxide(Di-4-NEPPS), Invitrogen, Paisley, UK in 48% aCSF, 48% foetal bovine serum, 3.5% DMSO and 0.4% cremophore EL) (Tominaga et al., 2000) was applied to the slices for 20-25 minutes before being transferred back to a bubbler pot containing oxygenated aCSF kept at room temperature for 30 minutes.

(131) When starting the VSDI recordings, slices were placed in the recording bath on a small piece of filter paper to allow the flow of oxygenated aCSF on the underside of the slice and in order to keep it alive. The slice was then weighed down by a home-made plastic grid that was placed on top of the slice. The perfusing bath solution was heated to 30±1° C. by a stage heater. A concentric bipolar stimulating electrode (FHC, Maine, USA) was placed in the ventral diagonal band of the basal forebrain with stimulation being set to 30V. For the acquisition of VSD data, 2 dimensional images, equivalent to 88×60 pixels, were recorded using the MiCamo2 High Resolution camera (Brain Vision, Japan) with BV_Analyze imaging software. Acquisition of images was coupled to Spike2 V4.23 software (CED Ltd, Cambridge, UK) in order to align the image capture with the stimulation protocol (every 28 s with 30 repeats) via the Micro 1401 MkII. (CED Ltd, Cambridge, UK). Light was generated using an Osram halogen xenophot 64634 HLX EFR Display/Optic lamp and was filtered to emit green (530±10 nm) light using a MHF-G150LR (Moritex Corporation) coupled to the MiCamo2 High resolution imaging system and filtered the emitted fluorescence through a >590 nm high pass filter.

(132) 3. Drug Preparation and Application

(133) Acetylcholinesterase (AChE) C-terminus 30 amino acid peptide (T30; sequence: ‘N’—KAEFHRWSSYMVHWKNQFDHYSKQDRCSDL—SEQ ID No: 2), the cyclic version of the active 14 amino acid region 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 (Tis; sequence: ‘N’—NQFDHYSKQDRCSDL—SEQ ID No: 4) were custom synthesised and purchased from Genosphere Biotechnologies (Paris, France) at >99% purity. The linear peptidomimetic, Tri02 was designed in silico by Iproteos (Barcelona, Spain) and synthesised and purchased from Genosphere Biotechnologies at >99% purity. All drug and peptide stocks were prepared in frozen aliquots prior to experiments. For the production of perfusion solutions, stock solutions were thawed and added to recording aCSF as appropriate and bath applied at a constant rate of 1.5 ml/min perfusion using the Minipulse 3 peristaltic pump (Gilson Scientific Ltd., Bedfordshire, UK). Each experimental trial lasted 52 minutes, with 20 minutes to establish a baseline recording (perfusion with recording aCSF only), 12 minutes to allow the drug solution to perfuse into the bath as well as to let the dye molecules reseat themselves in the cell membranes and finally, a 20 minute recording period measuring the response in the presence of the drug solution.

(134) 4. Data Analysis and Statistics

(135) From the 2 dimensional images generated with each drug condition, the critical data such as the time-course of activation, intensity and spread of the overall fluorescent signal were extracted. These data were processed using a custom script to convert them into usable MatLab (Mathworks Inc. Massachusetts, US) files and then analysed using a Matlab toolbox specifically made for VSDI data analysis (Bourgeois et al., 2014). This toolbox allows for the selection of a fixed region of interest (ROI) geometry that can be applied to every slice, in order to extract and collate the data from an identical ROI across all slices used in each experiment. For the basal forebrain slices, the ROI that will be used is the MSdBB complex, chosen as it encompasses the MS (medial septal nuclei), VDB (ventral diagonal band) and HDB (horizontal diagonal band). More crucially, this ROI was chosen in order to include the entirety of the evoked response. This response can be plotted as a single averaged time series or over space and time in a ‘space-time map’ so as to provide a qualitative description of the data. However, in order to produce quantifiable values, the area underneath the time series was calculated (summed fluorescence fractional change) between the moment of stimulation (t=0) and 156 ms after that. Due to the variability of responses seen between each individual slice, the raw data generated from each experiment was normalised with respect its own baseline to give normalised fluorescence values. This method of quantification was chosen in order to account for the multiple components of the signal generated by VSDI (Chemla and Chavane, 2010) namely the immediate peak and the long latency response (Badin et al., 2016). Statistics were carried out in Prism Graphpad 6.

(136) 5. Analysis of Modulatory Peptides

(137) Throughout the experiments in which T30 was used, an increase or a decrease in signal was observed. Thus upon averaging these results together, no change was detected. However, given the past 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 induced by application of T30 n this type of preparation are moderately negatively correlated (r=−0.4286, p=0.0257, Spearman's rank correlation, n=27, FIG. 13A) with baseline response amplitude, it was decided that these results should be dichotomised by whether an increase or a decrease was seen.

(138) Subsequently, a similar correlation analysis was performed for each experiment in which an exogenous compound was added (FIG. 16). Upon determination of a significant correlation, data was then categorised based on whether and increase or a decrease was seen.

Results

(139) Referring to FIGS. 14 and 15, addition of 4 μM Triol recapitulates results seen with application of 4 μM NBP14.

(140) Referring to FIG. 14, addition of NBP14 (4 uM) to the perfusate induced small, non-significant alterations to the magnitude (summed fluorescence) of evoked responses. Though insignificant, these small induced changes were found to be inversely correlated with magnitude of baseline response; as a result, data were split into trials which caused slight decreases (left histograms) and those which caused increase (right histograms), both in real (top) and normalised (bottom) data format. If considered together, the dataset would show no change from baseline (as increases and decreases would cancel each other out), yet it was crucial to check that no significant effects were induced by NB14 even when the fluorescence changes were considered separately.

(141) As shown in FIG. 15, addition of Tri02 (4 uM) to the perfusate induced small alterations to the magnitude (summed fluorescence) of evoked responses, with induced decreases (n=8 of 11 total) showing a significant deviation from normalised baseline level (bottom left histogram, p<0.05). These changes were also found to be inversely correlated with magnitude of baseline response; as a result, data were split into trials which caused decreases (left histograms) and those which caused increases (right histograms), both in real (top) and normalised (bottom) data format. If considered together, the dataset would show no change from baseline (as increases and decreases would cancel each other out), yet it was crucial to check that no significant effects were induced by NB14 even when the fluorescence changes were considered separately.

(142) Analysis of Modulatory Pep Tidomimetics

(143) Referring to FIG. 16, there is shown correlation analysis for Tri02 (4 uM) and T30 (2 uM) data (n=15) showing that their co-perfusion induces some changes to the magnitude of evoked responses, with some slices featuring slight increases in activity (n=6) whilst most showed slight decreases (n=9). This correlation was found to be significant (p=0.0405; r.sup.2=0.534), providing justification to split the data into those that showed increases and decreases in evoked activity as a result of Tri02 and T30 application, just as was done for the addition of NBP14 and Tri02 (FIGS. 14 & 15, respectively).

(144) Referring to FIG. 17, there is shown quantification of effects mediated by the addition of Tri02 and T30: Both in the case of induced increases and decreases, Tri02 was not found to protect against T30-induced deviations from baseline, with significant decreases (left panel, p<0.01, n=9) and increases (right panel, p<0.05, n=6) reported in overall effects.

(145) As shown in FIG. 18, overall line graph of normalised effects respective to baseline for experiments testing the effects of normal aCSF (black line), 2 uM T30 (red lines), T30 (2 uM) and 4 uM NBP14 (blue lines), T30 (2 uM) and 4 uM Tri02, control NBP14 (4 uM) experiments (FIG. 11, orange lines), control Tri02 (4 uM) experiments (FIG. 15, purple lines). This graph shows the normalised decreases relative to baseline and each other, with T30 alone inducing the greatest deviation, and Tri02 showing some efficacy in blocking those T30-induced deviation, yet with significant changes still taking place in their co-perfusion (green lines).

Example 10—Evaluation of Compound 2 in Brain Slices

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

(147) Materials and Methods

(148) 1. Brain Slice Preparation

(149) Brain slices were prepared as in Example 8.

(150) 2. VSD Setup

(151) Slices were placed in a dark, high humidity chamber filled with aCSF bubbling with 95% O.sub.2e5% CO.sub.2. Once there, the dye solution (4% 0.2 mM styryl dye pyridinium 4-[2-[6-(dibutylamino)-2-aphthalenyl]-ethenyl]-1-(3-sulfopropyl)phydroxide (Di-4-ANEPPS, Invitrogen, Paisley, UK) (Tominaga et al., 2000) in aCSF 48%, fetal bovine serum 48%, DMSO 3.5% and cremophore EL 0.4%) was applied to the slices for 20-25 min before being transferred to an aCSF bubbler pot (room temperature, 22 C+/−1.5 C) for 1 h to wash off excess dye and recover.

(152) When starting VSD recordings, slices were placed in the recording bath on a small piece of filter paper to keep slice alive and was weighed down appropriately using a home-made plastic grid placed atop the slice. The perfusing bath solution was heated to 30+/−1 C by a stage heater. A concentric bipolar stimulating electrode (FHC, Maine, US) was placed in the ventral diagonal band of the basal forebrain with stimulation being set at 30V. For acquiring of VSD data, 16-bit images were recorded with ims resolution with a digital camera (Brain Vision MiCAM Ultima R3-V20 Master) with Ultima 2004/08 imaging software (Brain Vision) coupled to Spike 2 V6.0 (CED Ltd, Cambridge, UK) which was used to trigger stimulations with respect to appropriate ISI. Light was generated using an Osram halogen xenophot 64634 HLX EFR Display/Optic lamp and was filtered to emit green (530+/−10 nm) light using a MHF-G150LR (Moritex Corporation) coupled to MiCAM Ultima ultra-fast imaging system and filtered the emitted fluorescence through a >590 nm high-pass filter.

(153) 3. Drug Preparation and Application

(154) The linear peptidomimetic, Tri04, was designed in silico by Iproteos (Barcelona, Spain) and synthesised and purchased from Genosphere Biotechnologies at >99% purity. All drug and peptide stocks were prepared in frozen aliquots prior to experiments. For the production of perfusion solutions, stock solutions were thawed and added to recording aCSF as appropriate and bath applied at a constant rate of 1.5 ml/min perfusion using the Minipulse 3 peristaltic pump (Gilson Scientific Ltd., Bedfordshire, UK). Each experimental trial lasted 52 minutes, with 20 minutes to establish a baseline recording (perfusion with recording aCSF only), 12 minutes to allow the drug solution to perfuse into the bath as well as to let the dye molecules reseat themselves in the cell membranes and finally, a 15 minute recording period measuring the response in the presence of the drug solution.

(155) 5. Analysis of Modulatory Peptides

(156) Throughout the majority of experiments in which T30 was used, a decrease in signal was observed. T30 induced a net inhibition (n=21) in recorded VSDI signal in the basal forebrain of p14 rats, this value actually includes a minority of instances where negligible or marginally positive effects were seen during T30 perfusion (Badin et al., 2016).

Results and Discussion

(157) Referring to FIGS. 20, 21 and 22, addition of 4 μM Tri04 recapitulates results previously seen with the application of 4 μM NBP14, while 2 μM Tri04 in the perfusion solution determines a significant effect on basal forebrain population activity.

(158) Analysis of Modulatory Peptidomimetics

(159) Referring to FIG. 20A, there is shown that space-time maps exhibit a recovery in basal forebrain activity due to the presence of 2 uM Tri04 in the perfusate containing 2 μM of T30 (n=29). More specifically, 2 μM Tri04 determines a reversal of the inhibitory effect of T30 over activity measured by direct stimulation of the rat basal forebrain.

(160) Referring to FIG. 20B, bar graphs relative to the 3 recording epochs show changes in the evoked response after Tri04 application, confirming that 2 μM Tri04 co-perfusion induces an increase in network activity (n=29, p=0.06, two-tailed paired t-test) caused by a inhibition of T30-induces effects.

(161) Referring to FIG. 21, there is shown that response time-series across the three recording conditions (baseline, T30 application to the artificial cerebro-spinal fluid (aCSF) and co-application of T30 and Tri04 to the aCSF show a similar activation profile for T30 recordings and T30+Tri04 for the first 100 msec, while a higher activity in recordings made in presence of Tri04 is detectable afterwards, confirming a protective role of Tri04 over T30.

(162) Referring to FIG. 22, there is shown bar graphs relative to three recording conditions. The co-perfusion of 4 μM Tri04 in the artificial cerebro-spinal fluid (aCSF) containing 2 uM T30 determines a significant effect reversing T30 activity. In particular, Tri04 has been found to be protective against T30-induced deviations from the baseline with a significant increase (n=20, p<0.05, two-tailed paired t-test) in basal forebrain activity in comparison to recordings in the presence of T30 alone. Therefore, Tri04 shows some efficacy blocking T30 toxic effects on meso-scale network activity.

Conclusions

(163) Treatment of Cancer

(164) The inventors have demonstrated herein that the antibody, and the two peptidomimetics, Tri02 and Tri04, act as inhibitors of the activity of the T14 peptide, due to the clear inhibitory effects on T30 in the data shown herein. Accordingly, these compounds can be used to treat, ameliorate or prevent cancer, and especially metastatic disease.

(165) Diagnosis/Prognosis of Cancer

(166) The inventors have also shown that T14, or variants thereof, such as its truncations, can be used as a diagnostic or prognostic marker of cancer, and especially metastatic disease. Low T14 levels as measured by western blots in the blood are indicative of poorer survival rates and corresponding longer time to first treatment (i.e. clinical status).