WO2018109491A1 - Treatment of emt-associated disease - Google Patents

Abstract

The invention relates to treatment of EMT-associated disease, and more specifically to treatment of cancer exhibiting the EMT phenotype, using the polypeptide FKBP-L and peptide fragments thereof.

Description

Treatment of EMT-associated disease

Field of the invention

The invention relates to treatment of EMT-associated disease, and more specifically to treatment of cancer exhibiting the EMT phenotype, using the polypeptide FKBP-L and peptide fragments thereof.

Background of the invention

The transdifferentiation of cells with an epithelial phenotype into cells with a mesenchymal phenotype, referred to as epithelial-mesenchymal transition (EMT), is a critical process for embryonic development that also occurs in adult tissues. EMT involves changes that lead to loss of cell-cell adhesion and cell polarity, with acquisition of migratory and invasive properties. The reverse process, mesenchymal-epithelial transition (MET), involves the transition from motile, multipolar or spindle-shaped mesenchymal cells to stationary epithelial cells characterised by apical-basal polarity.

In adults, EMT occurs during tumour progression; tumour cells undergoing EMT are characterised by increased motility and invasiveness, which favour their dissemination to distal sites and the formation of metastases. In addition, they may become resistant to apoptosis and antitumour drugs and act as cancer stem-like cells. EMT is thus becoming a target of significant importance for anti-cancer therapy, and there is a clinical need to identify compounds that target the EMT pathway, i.e. compounds that inhibit EMT and/or promote MET (Marcucci et al., Nature Reviews Drug Discovery, 2016).

Summary of the invention

FKBP-L polypeptide and peptide fragments thereof have previously been described as anti-angiogenic agents with clinical potential in the treatment of cancer, specifically solid tumours (WO 2007/141533). It has now been observed that, in addition to possessing potent anti-angiogenic activity, peptide fragments of FKBP-L act as inhibitors of the epithelial-mesenchymal transition, particularly in cancer cells exhibiting the EMT phenotype. These experimental findings support the clinical utility of FKBP-L polypeptide, and biologically active peptide fragments thereof, in the treatment of EMT-associated disease. In particular, the observation of this new mode-of-action of FKBP-L supports a new therapeutic utility of the protein (and peptide fragments thereof) in the treatment of a molecular sub-group of cancers that exhibit the EMT-phenotype. Accordingly, in a first aspect of the invention there is provided an FKBP-L polypeptide or a biologically active peptide fragment thereof for use as an inhibitor of epithelial-mesenchymal transition (EMT).

The invention further provides an FKBP-L polypeptide or a biologically active peptide fragment thereof for use as a promoter of mesenchymal-epithelial transition (MET).

The invention further provides an FKBP-L polypeptide or a biologically active peptide fragment thereof for use in the treatment or prevention of EMT-associated disease.

The invention still further provides an FKBP-L polypeptide or a biologically active peptide fragment thereof for use in the treatment or prevention of cancer in a human subject, wherein said cancer (of said subject to be treated) exhibits an EMT phenotype.

In certain embodiments, the cancer is ovarian, breast, colon, lung, pancreatic, melanoma, renal, prostate or thyroid. In certain embodiments, the cancer may be platinum- resistant. In certain embodiments, the cancer may be cisplatin-resistant. In certain embodiments, the cancer may be carboplatin-resistant.

The invention also provides a method of inhibiting epithelial-mesenchymal transition (EMT) in a mammalian subject, comprising administering to a subject in need thereof a therapeutically effective amount of an FKBP-L polypeptide or a biologically active peptide fragment thereof.

The invention further provides a method of promoting mesenchymal-epithelial transition (MET) in a mammalian subject, comprising administering to a subject in need thereof a therapeutically effective amount of an FKBP-L polypeptide or a biologically active peptide fragment thereof.

The invention further provides a method of treating or preventing EMT-associated disease in a mammalian subject, comprising administering to a subject in need thereof a therapeutically effective amount of an FKBP-L polypeptide or a biologically active peptide fragment thereof.

The invention further provides a method of treating or preventing cancer in a human subject, wherein said cancer (or said subject to be treated) exhibits an EMT phenotype, comprising administering to a subject in need thereof a therapeutically effective amount of an FKBP-L polypeptide or a biologically active peptide fragment thereof.

In certain embodiments, the cancer is ovarian, breast, colon, lung, pancreatic, melanoma, renal, prostate or thyroid. In certain embodiments, the cancer may be platinum-resistant. In certain embodiments, the cancer may be cisplatin-resistant. In certain embodiments, the cancer may be carboplatin-resistant. In a particular embodiment, the cancer to be treated is platinum (cisplatin)-resistant human ovarian cancer.

The invention also provides a pharmaceutical composition for the treatment of EMT- associated disease comprising FKBP-L polypeptide or a biologically active peptide fragment thereof.

The invention further provides a pharmaceutical composition for the treatment of cancer in a human subject, wherein said cancer (or said subject to be treated) exhibits an EMT phenotype comprising FKBP-L polypeptide or a biologically active peptide fragment thereof.

In preferred embodiments of each of the above-described aspects of the invention, the subject to be treated, or one or more cells or tissues thereof, may exhibit an "EMT phenotype". Identification of the "EMT phenotype" provides a means to stratify a patient population (e.g. a group of cancer patients) in order to identify a patient sub-set who may benefit from treatment with the FKBP-L polypeptides described herein. In the case of cancer treatment, the "EMT phenotype" may be detectable in one or more tumour cells or tumour tissues of the subject to be treated.

In certain embodiments, the "EMT phenotype" is indicated by expression of one or more mesenchymal marker genes selected from N-cadherin, fibronectin, vimentin, SNAI1 , SNAI2 (SLUG), ZEB1 , Twist and TGF- 3.

In certain embodiments, the EMT phenotype is indicated by reduced expression of the epithelial marker gene E-cadherin.

In certain embodiments, the EMT phenotype is indicated by expression of one or more, any combination of, or all of the biomarker genes listed in Table A, which collectively make up the 45-gene EMT signature.

In certain embodiments, the EMT phenotype is indicated by expression of one or more, any combination of, or all of the biomarker genes listed in Table B, which collectively make up the 15-gene EMT signature.

In preferred embodiments of each of the above-described aspects of the invention, the biologically active peptide fragment of FKBP-L used in said treatment/prevention comprises or consists of the amino acid sequence IRQQPRDPPTETLELEVSPDPAS (SEQ ID NO:3), or a sequence at least 90% identical thereto.

In further embodiments, the FKBP-L polypeptide used in said treatment/prevention comprises or consists of the amino acid sequence shown as SEQ ID NO:1 or SEQ ID NO:2, or a sequence at least 90% identical thereto. In this context, a peptide which "consists of the defined amino acid sequence is to be interpreted as referring to an isolated peptide fragment of the defined amino acid sequence, thereby excluding the presence of any contiguous amino acid residues from the full length FKBP-L.

In further embodiments, the biologically active peptide fragment of FKBP-L used in said treatment/prevention comprises the amino acid sequence shown as any one of SEQ ID Nos 4 to 23, or a sequence at least 90% identical thereto.

Features of the invention will be described in further detail hereinafter. It is to be understood that the invention is not limited in its application to the details set forth in the following claims, description and figures. The invention is capable of other embodiments and of being practiced or carried out in various ways.

Brief description of the drawings The invention will be further understood with reference to the following drawings.

Figure 1 : Identification of molecular subgroups of HGSOC

A. Heat map showing unsupervised hierarchical clustering of gene expression data using the 1040 most variable genes in the Edinburgh 265 high grade serous ovarian carcinomas. Gene expression across all samples is represented horizontally.

Functional processes corresponding to each gene cluster are labelled along the right of the figure. Angio, Immune, and Angiojmmune subgroups are labelled for each of the sample clusters, and coded along the top as described in the legend box. A gene expression signature to detect each of the subgroups was generated. B. Kaplan-Meier Progression-Free Survival analysis of subgroups as defined by unsupervised clustering analysis of Edinburgh 265 HGSOC Samples and Kaplan- Meier overall survival analysis of subgroups as defined by unsupervised clustering analysis of Edinburgh 265 HGSOC Samples. C. Kaplan-Meier to show the prognostic utility of the 45 gene signature in HGSOC (PFS HR 1 .4 (1.092 to 1 .880) p=0.0256 and OS HR 1.4 (1 .05-1.87) p= 0.0224). D Kaplan-Meier to show the prognostic utility of the 15 gene signature in HGSOC Figure 2: The Angioimmune subgroup is associated with increased EMT signalling

A. Table showing increased expression of EMT genes in the Angioimmune subgroup compared to the other 2 subgroups. Expression of VIM, AXL, TWIST1 , SNAIL and SLUG is enhanced in the Angiojmmune subgroup (p<0.0001 ).

B. Semi-supervised clustering analysis was performed on the HGSOC dataset using the 3 public MEK gene lists. Gene clusters separating the ovarian samples from each individual clustering analysis were selected for further analysis. These were combined and a compilation gene list generated and semi-supervised analysis of the HGSOC dataset performed again. The table illustrates the overlap with the 'MEK ON' population with the 3 molecular subgroups. This demonstrated 77% overlap of the 'MEK ON' samples with the Angioimmune subgroup.

Reverse Phase Proteomic Array (RPPA) continuous Phospho-MEK (pMEK) scores (serine 217/221 ) and total MEK scores were downloaded from The Cancer Genome Atlas (TCGA) dataset (http://bioinformatics.mdanderson.org/main/TCPA). The Phospho-MEK scores were calculated as a ratio of total MEK. Gene signature scores were then correlated with the RPPA data and only the Angioimmune 45 gene signature was specifically found to correlate with pMEK serine 217/221 expression ROC analysis (p=0.048). This suggested that the Angioimmune subgroup was associated with activation of the MAPK pathway.

C. Box and whisker plot of 45-gene signature scores in MCF7 control and SNAIL

overexpressing cells (E-GEOD-58252). The 45 and 15-gene signature is enhanced by SNAIL overexpression.

Figure 3: The MEK subgroup is present in colon cancer and the EMT signature is prognostic

A. Heatmap representation of semi-supervised analysis of the MARISA dataset (GSE40967) using the Angiojmmune genes. Five individual clusters were identified, with Sample Cluster 3 (highlighted by the red box) defining the MEK driven subgroup. B. Kaplan-Meier to show the relapse-free survival of the five sample cluster groups. The MEK driven group represents poor prognosis in comparison to the other subgroups (p=0.037). C. Kaplan-Meier to show the relapse-free survival using the 45 and 15-gene signature scores from Marisa. The MEK ON group represents poor prognosis in comparison to the MEK OFF group D. Kaplan-Meier to show the disease-free survival using the 45 and 15-gene signature scores in the Jorissen dataset (GSE14333). The MEK ON group represents poor prognosis in comparison to the MEK OFF group.

Figure 4: The MEK subgroup is present in NSCLC cancer and the 45 and 15 EMT signature is prognostic

A. Heatmap representation of semi-supervised analysis of the Okayama dataset (GSE31210) using the Angiojmmune genes. Five individual clusters were identified, with Sample Cluster 4 (highlighted by the box) defining the MEK driven subgroup. B. Kaplan-Meier to show the relapse-free survival of the five sample cluster groups. The MEK driven group represents poor prognosis in comparison to the other subgroups (p=0.0004). C. Kaplan-Meier to show the progression-free survival using the 45 and 15-gene signature scores from Okayama. The SIG POS group represents poor prognosis in comparison to the SIG NEG group. D. Kaplan- Meier to show the overall survival using the 45 and 15-gene signature scores in the Okayama dataset. The SIG POS group represents poor prognosis in comparison to the SIG NEG group.

Figure 5: Activation of the EMT phenotype is enhanced in Cisplatin resistant ovarian cell lines

A. 10-day colony formation assay assessing sensitivity of OVCAR3-WT and

OVCAR3-CP cells to increasing concentrations of cisplatin (left panel). Western blot analysis showing increased MAPK signalling in the OVCAR4 cisplatin resistant cells compared to cisplatin sensitive counterparts (right panel).

B. Western blot analysis showing activation of MAPK and EMT signalling in

OVCAR3 CP and OVCAR4 CP (cisplatin resistant) with increased protein expression of Vimentin, N-cadherin and SLUG whilst decreasing protein expression of E-cadherin. B-actin was used as a loading control.

C. Increased 45 and 15 gene signature score in OVCAR3 cisplatin resistant cells compared to wild-type cells.

D. Increased proliferation, migration and invasion in cisplatin resistant cells compared to wildtype counterparts. Bar charts to show the fold change increase in migration of OVCAR3 and OVCAR4 cisplatin resistant cells compared to the wildtype ovarian cell lines. Figure 6: ALM201 reverses mesenchymal markers in the Kuramochi and OVCAR3 cisplatin-resistant cells

A. Western Blot analysis showing reversal of the EMT pathway and downregulation of the MAPK pathway by ALM201 in ovarian Kuramochi cells.

B. Western blot demonstrating activity of ALM201 in reversing EMT markers, downregulation of the MAPK pathway following addition of 1 nM and 10nM ALM201 at 24 hour treatment time point in the OVCAR3 cisplatin resistant cell line.

C. 10-day colony formation assay assessing sensitivity of OVCAR3-WT, OVCAR3-CP, OVCAR4-WT and OVCAR4-CP cells to increasing concentrations of ALM201 . Table shows IC50 values for OVCAR3-WT and OVCAR3-CP cells for Cisplatin and

Cisplatin in combination with ALM201.

D. xCELLigence migration and invasion assay illustrating that 0.1 nM, 1 nM and 10nM ALM201 inhibits migration (p=0.08544, p=0.015522 and p=0.036739, respectively) and invasion (p=0.021 1 , p=0.0026 and p=0.3373, respectively) in the OVCAR3 platinum resistant cell line.

Figure 7: Tumour volume data of a selection of Champions TumorGraft® patient- derived ovarian cancer xenografts with or without ALM201 treatment

In vivo efficacy of ALM201 (dosed 3mg/kg s.c. once daily) to female athymic nude mice bearing different patient-derived ovarian cancer xenografts (selected from the Champions Oncology Inc. TumorGraft® collection). ALM201 was observed to elicit a strong reduction in tumour growth compared to the PBS/no drug treatment. Tumour volumes are plotted on the graphs relative to the tumour volume at the start of treatment (i.e. Day 0), whereas %TGI was calculated from the absolute tumour volume (see Table 2). Figure 8: RNA-seq transcriptome analysis of prostate cancer xenograft tumours cisplatin-resistant cells

A. Significantly differentially expressed genes in DU-145 Xenograft model mapped to Hallmarks of Cancer EMT pathway definition (P Value < 0.05).

B. Differentially expressed genes mapped to Hallmarks of Cancer EMT pathway in context of wider differentially expressed Geneset. Detailed description of the invention Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Practitioners are particularly directed to Current Protocols in Molecular Biology (Ausubel) for definitions and terms of the art. Abbreviations for amino acid residues are the standard 3-letter and/or 1 -letter codes used in the art to refer to one of the 20 common L-amino acids.

Any reference referred to as being "incorporated herein" is to be understood as being incorporated in its entirety.

It is further noted that, as used in this specification, the singular forms "a," "an," and "the" include plural referents unless expressly and unequivocally limited to one referent. The term "or" is used interchangeably with the term "and/or" unless the context clearly indicates otherwise.

Also, the terms "portion" and "fragment" are used interchangeably to refer to parts of a polypeptide, nucleic acid, or other molecular construct.

As used herein, the term "biologically active FKBP-L peptide" (e.g., fragment and/or modified polypeptides) is used to refer to a peptide or polypeptide that displays the same or similar amount and type of activity as the full-length FKBP-L polypeptide. In this context "biological activity" of an FKBP-L polypeptide, fragment or derivative refers to the ability to inhibit and/or reverse the EMT pathway, or to promote MET, and/or the ability to down- regulate the MAPK pathway. MAPK is known to induce EMT via phosphorylation of the SNAIL/SLUG transcription factors, (Virtakoivu et al., 2015).

Biological activity of FKBP-L fragments or derivatives may be tested in comparison to full length FKBP-L using any of the in vitro or in vivo assays described in the

accompanying examples, including cell-based assays of the mesenchymal phenotype, such as for example the colony formation assay, migration assay or invasion assay. In other embodiments, "biological activity" of an FKBP-L polypeptide, fragment or derivative may be demonstrated by assaying expression of one or more biomarkers of the EMT pathway (e.g. mesenchymal markers), or one or more biomarkers of the MAPK pathway, as discussed below.

As used herein a "subject" to be treated may be an animal. For example, the subject may be a mammal. Also, the subject may be a human. In alternate embodiments, the subject may be either a male or a female. In certain embodiments, the subject may be a patient, where a patient is an individual who is under medical care and/or actively seeking medical care for a disorder or disease.

"Polypeptide" and "protein" are used interchangeably herein to describe protein molecules that may comprise either partial or full-length proteins. The term "peptide" is used to denote a less than full-length protein or a very short protein unless the context indicates otherwise.

As is known in the art, "proteins", "peptides," "polypeptides" and "oligopeptides" are chains of amino acids (typically L-amino acids) whose alpha carbons are linked through peptide bonds formed by a condensation reaction between the carboxyl group of the alpha carbon of one amino acid and the amino group of the alpha carbon of another amino acid. Typically, the amino acids making up a protein are numbered in order, starting at the amino terminal residue and increasing in the direction toward the carboxyl terminal residue of the protein.

The terms "identity" or "percent identical" refers to sequence identity between two amino acid sequences or between two nucleic acid sequences. Percent identity can be determined by aligning two sequences and refers to the number of identical residues (i.e., amino acid or nucleotide) at positions shared by the compared sequences. Sequence alignment and comparison may be conducted using the algorithms standard in the art (e.g. Smith and Waterman, 1981 , Adv. Appl. Math. 2:482; Needleman and Wunsch, 1970, J. Mol. Biol. 48:443; Pearson and Lipman, 1988, Proc. Natl. Acad. Sci., USA, 85:2444) or by computerized versions of these algorithms (Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, Wl) publicly available as BLAST and FASTA. Also, ENTREZ, available through the National Institutes of Health, Bethesda MD, may be used for sequence comparison. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTN; available at the Internet site for the National Center for Biotechnology

Information) may be used. In one embodiment, the percent identity of two sequences may be determined using GCG with a gap weight of 1 , such that each amino acid gap is weighted as if it were a single amino acid mismatch between the two sequences. Or, the ALIGN program (version 2.0), which is part of the GCG (Accelrys, San Diego, CA) sequence alignment software package may be used.

As used herein, the term "conserved residues" refers to amino acids that are the same among a plurality of proteins having the same structure and/or function. A region of conserved residues may be important for protein structure or function. Thus, contiguous conserved residues as identified in a three-dimensional protein may be important for protein structure or function. To find conserved residues, or conserved regions of 3-D structure, a comparison of sequences for the same or similar proteins from different species, or of individuals of the same species, may be made.

As used herein, the term "similar" or "homologue" when referring to amino acid or nucleotide sequences means a polypeptide having a degree of homology or identity with the wild-type amino acid sequence. Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percent homology between two or more sequences (e.g. Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA, 80:726-730). For example, homologous sequences may be taken to include an amino acid sequences which in alternate embodiments are at least 70% identical, 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, or 98% identical to each other.

As used herein, the term "at least 90% identical thereto" includes sequences that range from 90 to 99.99% identity to the indicated sequences and includes all ranges in between. Thus, the term at least 90% identical thereto includes sequences that are 91 , 91.5, 92, 92.5, 93, 93.5. 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5 percent identical to the indicated sequence. Similarly the term "at least 70% identical includes sequences that range from 70 to 99.99% identical, with all ranges in between. The determination of percent identity is determined using the algorithms described herein.

As used herein, the term "linked" identifies a covalent linkage between two different groups (e.g., nucleic acid sequences, polypeptides, polypeptide domains) that may have an intervening atom or atoms between the two groups that are being linked. As used herein, "directly linked" identifies a covalent linkage between two different groups (e.g., nucleic acid sequences, polypeptides, polypeptide domains) that does not have any intervening atoms between the two groups that are being linked.

The term "peptide mimetics" refers to structures that serve as substitutes for peptides in interactions between molecules (Morgan et al., 1989, Ann. Reports Med.

Chem., 24:243-252). Peptide mimetics may include synthetic structures that may or may not contain amino acids and/or peptide bonds but that retain the structural and functional features of a peptide, or agonist, or antagonist. Peptide mimetics also include peptoids, oligopeptoids (Simon et al., 1972, Proc. Natl. Acad, Sci., USA, 89:9367); and peptide libraries containing peptides of a designed length representing all possible sequences of amino acids corresponding to a peptide of the invention. As used herein, an "effective amount" means the amount of an agent that is effective for producing a desired effect in a subject. The term "therapeutically effective amount" denotes that amount of a drug or pharmaceutical agent that will elicit therapeutic response of an animal or human that is being sought. The actual dose which comprises the effective amount may depend upon the route of administration, the size and health of the subject, the disorder being treated, and the like.

The term "pharmaceutical composition" is used herein to denote a composition that may be administered to a mammalian host, e.g. topically or systemically, in unit dosage formulations containing conventional non-toxic carriers, diluents, adjuvants, vehicles and the like.

The term "pharmaceutically acceptable carrier" as used herein may refer to compounds and compositions that are suitable for use in human or animal subjects, as for example, for therapeutic compositions administered for the treatment of a disorder or disease of interest.

A "stable" formulation is one in which the polypeptide or protein therein essentially retains its physical and chemical stability and biological activity upon storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301 , Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991 ) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature for a selected time period. For rapid screening, the formulation of interest may be kept at 40° C for 1 week to 1 month, at which time stability is measured. The extent of aggregation following lyophilization and storage can be used as an indicator of peptide and/or protein stability. For example, a "stable" formulation is one wherein less than about 10% and preferably less than about 5% of the polypeptide or protein is present as an aggregate in the formulation. An increase in aggregate formation following lyophilization and storage of the lyophilized formulation can be determined. For example, a "stable" lyophilized formulation may be one wherein the increase in aggregate in the lyophilized formulation is less than about 5% or less than about 3%, when the lyophilized formulation is incubated at 40° C for at least one week. Stability of the fusion protein formulation may be measured using a biological activity assay such as a binding assay as described herein.

FKBP-L polypeptides for the treatment of EMT-associated disease

The present invention is based on the observation that FKBP-L, and specifically peptide fragments of FKBP-L, can reverse the mesenchymal phenotype and expression of mesenchymal markers in cells (e.g. cancer cells) that have undergone epithelial- mesenchymal transition (EMT). These findings support the utility of FKBP-L, and peptide fragments thereof, as inhibitors of EMT and/or as promoters of MET. In particular, these findings support the clinical utility of FKBP-L, and peptide fragments thereof, in the treatment of EMT-associated diseases, including but limited to treatment of a molecular sub-type of cancer having the "EMT phenotype".

As used herein, the terms "epithelial-mesenchymal transition", "EMT" or "EMT pathway" refer to the process of transdifferentiation of epithelial cells into motile

mesenchymal cells (Lamouille et al., Nature Mol. Cell. Bio., Volume 15, 178-196, 2014). EMT is a stepwise cascade that leads to the transition of epithelial cells acquiring mesenchymal properties. EMT is driven by SNAI1 (Snail), zinc-finger E-box-binding (ZEB) and basic helix-loop-helix (bHLH) transcription factors that repress epithelial marker genes and activate genes associated with the mesenchymal phenotype.

The term "EMT phenotype" refers to the characteristic mesenchymal phenotype of cells that have undergone EMT. Cells having the "EMT phenotype" may be recognised on the basis of their characteristic properties and/or on the basis of biomarker expression. In general, cells that have undergone EMT are characterised by one or more of: dissolution of epithelial cell-cell junctions, loss of apical-basal polarity, reorganisation of the cytoskeletal architecture, down-regulation of epithelial marker gene expression (e.g. E-cadherin), activation of genes defining the mesenchymal phenotype, increased cell protrusions and motility, resistance to senescence and apoptosis, invasive, migratory and apoptotic properties.

In certain embodiments, cells may be identified as having the EMT phenotype on the basis of expression of one or more mesenchymal markers selected from: N-cadherin, fibronectin, vimentin, SNAI1 (Snail), SNAI2 (SLUG), ZEB1 , ZEB2, Twist and TGF- 3.

Several oncogenic cell signalling pathways, including the MAPK pathway, the Wnt^-catenin pathway and the Notch pathway, are capable of inducing EMT, via activation of EMT-promoting transcription factors. Accordingly, in other embodiments, cells may be identified as having the EMT phenotype on the basis of expression of one or more markers of the MAPK pathway, or the MEK signalling pathway.

As used herein, the term "EMT inhibitor" refers to the ability of a compound (e.g. FKBP-L or a biologically active peptide fragment thereof) to prevent EMT from occurring, or to reverse the mesenchymal phenotype in cells which have undergone EMT. In certain embodiments, treatment of cells having the EMT phenotype with an EMT inhibitor may result in loss of mesenchymal cell characteristics and/or re-acquisition of epithelial cell characteristics. FKBP-L polypeptides that exhibit the ability to reverse the mesenchymal phenotype in cells which have undergone EMT may also be referred to herein as

"promoters of MET". In other embodiments, the "EMT inhibitor" (or "MET promoter") may also downregulate the MAPK signalling pathway and/or the MEK signalling pathway.

Embodiments of the present invention encompass the use of the full-length FKBP-L polypeptide, and also peptide fragments thereof which exhibit biological activity, as well as modified forms and derivatives of the full-length protein or biologically active peptide fragments that function as EMT inhibitors/MET promoters, as therapeutic agents in the treatment of EMT-associated disease, including but not limited to treatment of a molecular sub-type of cancer having the "EMT phenotype".

The term "FKBP-L" refers to the protein FK506 binding protein-like, (McKeen et al. Endocrinology, 2008, Vol 149(1 1 ), 5724-34; Gene ID:63943). FKBP-L and peptide fragments thereof have previously been demonstrated to possess potent anti-angiogenic activity (WO 2007/141533). The anti-angiogenic activity of FKBP-L peptide fragments appears to be dependent on an amino acid sequence located between amino acids 34-57, in the N-terminal region of the full-length protein. This anti-angiogenic activity suggested a clinical utility of the peptide in the treatment of cancers, particularly solid tumours. The present application extends beyond the findings of WO 2007/141533 by demonstrating a specific mode-of-action of FKBP-L and peptide fragments thereof in reversing the mesenchymal phenotype in cancer cells that have undergone EMT.

The expression "FKBP-L polypeptide" is used in the specification according to its broadest meaning. It designates the naturally occurring full-length protein as shown in SEQ ID NO:1 , together with homologues due to polymorphisms, other variants, mutants and portions of said polypeptide which retain their biological activities. For example, in certain embodiments, the FKBP-L polypeptide comprises SEQ ID NO:1 (GENBank

Accession No. NP_071393; NM_0221 10; [gi:34304364]), or SEQ ID NO:2 with a Threonine at position 181 and a Glycine at position 186 of the wild-type sequence. Example constructs of other FKBP-L polypeptides (e.g., fragments and other modifications) and polynucleotide constructs encoding for FKBP-L polypeptides are described in WO

2007/141533, the contents of which are incorporated herein in their entirely by reference, expressly for this purpose.

In SEQ ID NO: 2, the FKBP-L insert (originally cloned into PUC18 by Cambridge Bioscience and now cloned into pcDNA3.1 ); had two inserted point mutations compared to the sequence that is deposited on the PUBMED database (SEQ ID NO: 1 ). There is a point mutation at 540 bp (from start codon): TCT to ACT which therefore converts a serine (S) to a Threonine (T) (amino acid: 181 ). There is also a point mutation at 555 bp (from start codon): AGG to GGG which therefore converts an Arginine (R) to a Glycine (G) (amino acid: 186). Both FKBP-L polypeptides (SEQ ID NO: 1 and SEQ ID NO: 2) display biological activity.

An FKBP-L polypeptide or peptide for use according to the present invention may include natural and/or chemically synthesized or artificial FKBP-L peptides, peptide mimetics, modified peptides (e.g., phosphopeptides, cyclic peptides, peptides containing D- and unnatural amino-acids, stapled peptides, peptides containing radiolabels), or peptides linked to antibodies, carbohydrates, monosaccharides, oligosaccharides, polysaccharides, glycolipids, heterocyclic compounds, nucleosides or nucleotides or parts thereof, and/or small organic or inorganic molecules (e.g., peptides modified with PEG or other stabilizing groups). Thus, the FKBP-L (poly)peptides of the invention also include chemically modified peptides or isomers and racemic forms.

Embodiments of the present invention comprise an isolated FKBP-L polypeptide or a biologically active fragment of a FKBP-L polypeptide, or a biologically active derivative of such a FKBP-L polypeptide or fragment for use as a medicament for treatment of the EMT- associated diseases described herein.

Preferred, but non-limiting, embodiments of the present invention comprise use of a FKBP-L peptide or nucleotide that encodes a FKBP-L peptide as described herein, wherein the FKBP-L polypeptide comprises the amino acid sequence shown in SEQ ID NO:3

(IRQQPRDPPTETLELEVSPDPAS), or an amino acid sequence at least 90% identical to the amino acid sequence shown in SEQ ID NO:3.

As described herein, the methods and pharmaceutical compositions for use according to the present invention may utilize a full-length FKBP-L polypeptide, or biologically active fragments of the polypeptide. Thus, certain embodiments of the present invention comprise a FKBP-L derivative which comprises or consists of a biologically active portion of the N-terminal amino acid sequence of naturally occurring FKBP-L. This sequence may comprise, consist essentially of, or consist of an active N-terminal portion of the FKBP-L polypeptide. In alternate embodiments, the polypeptide may comprise, consist essentially of, or consist of amino acids 1 to 57 of SEQ ID NO: 2 (i.e., SEQ ID NO: 8), or amino acids 34-57 of SEQ ID NO:2 (i.e., SEQ ID NO: 4), or amino acids 35-57 of SEQ ID NO:2 (i.e. SEQ ID NO:3). Or, the peptide may comprise, consist essentially of, or consist of a sequence that comprises at least 18 contiguous amino acids of SEQ ID NO: 4 (e.g., SEQ ID NOs: 10, 12, or 19). In alternate embodiment, the polypeptide used in the methods and compositions of the present invention may comprise, consist essential of, or consist of one of the amino acid sequences shown in any one of SEQ ID NOs: 1 -23. In certain embodiments, the present invention comprises a biologically active fragment of FKBP-L, wherein said polypeptide includes no more than 200 consecutive amino acids of the amino acid sequence shown in SEQ ID NO:1 , or SEQ ID NO:2, with the proviso that said polypeptide includes the amino acid sequence shown as SEQ ID NO:3 .

As described herein, the peptides may be modified (e.g., to contain PEG and/or His tags, albumin conjugates or other modifications). Or, the present invention may comprise isolated polypeptides having a sequence at least 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99% identical to the amino acid sequences as set forth in any one of SEQ ID NOS: 1 -23, including in particular sequences at least 70%, or 75%, or 80%, or 85%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99% identical to the amino acid sequence shown as SEQ ID NO:3. In this regard, deliberate amino acid substitutions may be made in the peptide on the basis of similarity in polarity, charge, solubility,

hydrophobicity, or hydrophilicity of the residues, as long as the specific biological activity (i.e. function) of the peptide is retained.

The FKBP-L peptide may be of variable length as long as it retains its biological activity and can be used according to the various aspects of the invention described above.

Fragments of FKBP-L

Embodiments of the present invention recognize that certain regions of the N- terminus of the FKBP-L protein may display biological activity, therefore the invention encompasses use of biologically active fragments of FKBP-L, in particular any fragment which exhibits biological activity substantially equivalent to that of the 23-mer peptide (SEQ ID NO:3). In certain embodiments, the biological activity of the FKBP-L 23mer peptide (SEQ ID NO:3; referred to herein also as ALM201 ) is exhibited as a reduction in expression of mesenchymal markers in Kuramochi cells or OVCAR3 cisplatin resistant cells (Figure 6A). In further embodiments, the biological activity of the FKBP-L 23mer peptide (SEQ ID NO:3; referred to herein also as ALM201 ) is exhibited as a reversal of the mesenchymal phenotype in OVCAR3 or OVCAR4 cisplatin resistant cells (Figure 6B).

A "fragment" of a FKBP-L polypeptide means an isolated peptide comprising a contiguous sequence of at least 6 amino acids, preferably at least 10 amino acids, or at least 15 amino acids, or at least 20 amino acids, or at least 23 amino acids of FKBP-L. The "fragment" preferably contains no more than 50, or no more than 45, or no more than 40, or no more than 35, or no more than 30, or no more than 25, or no more than 23 contiguous amino acids of FKBP-L. Preferred fragments for use according to the invention are those having the amino acid sequences shown in any one of SEQ ID Nos: 4-23, or minor sequence variants thereof (e.g. variants containing one or more conservative amino acid substitutions). Derivatives

An FKBP-L derivative for use in the invention includes polypeptides modified by varying the amino acid sequence of FKBP-L, e.g. SEQ ID NO:1 , SEQ ID NO: 2, or SEQ ID NO:29, or a fragment thereof, or a polypeptide at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto, or such peptides that have be modified by the addition of a functional group (e.g., PEG, other peptides or proteins, polymers,

nanoparticles, small molecule probes etc.). Generation of such peptides may be performed by manipulation of the nucleic acid encoding the polypeptide or by altering the protein itself. In certain embodiments, FKBP-L derivatives may be generated by genetic fusion or chemical conjugation of the FKBP-L peptide to a functional group, as defined above.

FKBP-L derivatives include analogues of the natural FKBP-L amino acid sequence and may involve insertion, addition, deletion and/or substitution of one or more amino acids, while providing a polypeptide capable of effecting similar biological effects. Also included in the FKBP-L derivatives of the present invention are polypeptides derived from SEQ ID Nos: 1 -23.

Thus, FKBP-L derivatives used in the methods and compositions of the present invention also include fragments, portions or mutants of the naturally occurring FKBP-L. In certain embodiments, such derivatives involve the insertion, addition, deletion and/or substitution of 5 or fewer amino acids, more preferably of 4 or fewer, even more preferably of 3 or fewer, most preferably of 1 or 2 amino acids only.

FKBP-L derivatives also include multimeric peptides comprising the FKBP-L polypeptides of SEQ ID NOs: 1 -23, and prodrugs including such sequences. For example, in certain embodiments FKBP-L or fragments of FKBP-L may form multimers by the formation of disulfide bonds between monomers.

Derivatives of the FKBP-L polypeptides may include the polypeptide linked to a coupling partner, e.g., an effector molecule, a label, a drug, a toxin and/or a carrier or transport molecule. Techniques for coupling the polypeptides of the invention to both peptidyl and non-peptidyl coupling partners are well known in the art.

FKBP-L derivatives also include fusion peptides. For example, derivatives may comprise FKBP-L polypeptides of SEQ ID NOs: 1 -23 linked, for example, to antibodies that target the peptides to diseased tissue. Other derivatives may comprise an FKBP-L polypeptide of SEQ ID NOs: 1 -23 linked or fused to serum albumin or proteins that bind serum albumin, in order to increase circulating half-life.

The FKBP-L polypeptide or their analogues may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1 , CH2, CH3, or any combination thereof), resulting in chimeric polypeptides. These fusion polypeptides or proteins can facilitate purification and may show an increased half-life in vivo. Such fusion proteins may be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995).

Fusion proteins of the invention also include FKBP-L polypeptides fused with albumin, for example recombinant human serum albumin or fragments or variants thereof (see, e.g., US Patent No. 5876969, EP Patent 0413622 and US Patent No. 5766883).

The use of polynucleotides encoding such fusion proteins described herein is also encompassed by the invention. The use of a polynucleotide fused to a cytotoxic agent is also encompassed by the invention. In this instance the FKBP-L polypeptide may bind to a receptor and the cytotoxic drug could be internalised.

For example, in alternate embodiments, derivatives may include: site-specific PEGylation (or the like) of peptide to increase half-life; or incorporation unnatural amino acids and back bone modifications to stabilize against proteolysis; or cyclic derivatives (to provide proteolytic resistance); or to block the N- and C-termini to prevent or reduce exopeptidase and/or proteinase activity; or to join together multiple copies of peptides either in a contiguous chain via linkers chain or in a dendrimer type of approach to increase 'avidity'. For example, trimeric covalently linked derivatives of 24mer may be used as derivatives of FKBP-L. Or, the FKBP-L 24mer may be attached to a domain which homotrimerises to form non-covalent trimers. Or, biotin derivatives of peptides which will form tetrameric complexes with streptavidin may be used as derivatives of FKBP-L. Or, FKBP-L or fragments of FKBP-L may form multimers by the formation of disulphide bonds between monomers. In addition, FKBP-L may form oligomers through non-covalent associations, possibly through the predicted tetratricopeptide repeat domains within the protein sequence.

Reverse Peptide Analogues

Analogues for use in the present invention further include reverse-or retro- analogues of natural FKBP-L proteins, portion thereof or their synthetic derivatives. See, for example, EP 0497 366, U.S. 5,519,1 15, and Merrifield et al., 1995, PNAS, 92:3449-53, the disclosures of which are herein incorporated by reference. As described in EP 0497 366, reverse peptides are produced by reversing the amino acid sequence of a naturally occurring or synthetic peptide. Such reverse-peptides may retain the same general three- dimensional structure (e. g., alpha-helix) as the parent peptide except for the conformation around internal protease-sensitive sites and the characteristics of the N-and C-termini.

Reverse peptides are purported not only to retain the biological activity of the non-reversed "normal" peptide but may possess enhanced properties, including increased biological activity. (See Iwahori et al., 1997, Biol. Pharm. Bull. 20: 267-70). Derivatives for use in the present invention may therefore comprise reverse peptides of natural and synthetic FKBP- L proteins.

Peptides (including reverse peptides and fragments of either) for use in the invention may be generated wholly or partly by chemical synthesis or by expression from nucleic acid. The peptides for use in the present invention can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods known in the art (see, for example, J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984). Multimeric Peptides

As described above, the peptides may be in the form of multimers. Thus multimers of 2, 3 or more individual FKBP-L polypeptide monomeric units, or two or more fragments of FKBP-L, are within the scope of the invention.

In one embodiment, such multimers may be used to prepare a monomeric peptide by preparing a multimeric peptide that includes the monomeric unit, and a cleavable site (i.e., an enzymatically cleavable site), and then cleaving the multimer to yield a desired monomer.

In one embodiment, the use of multimers can increase the binding affinity for a receptor.

The multimers can be homomers or heteromers. As used herein, the term homomer, refers to a multimer containing only polypeptides corresponding to a specific amino acid sequence (e.g., SEQ ID NO: 1 , SEQ ID NO: 2, or SEQ ID NO: 3), or variants, splice variants, fusion proteins, or other FKBP-L analogues or derivatives described herein. These homomers may contain FKBP-L peptides having identical or different amino acid sequences. For example, the multimers can include only FKBP-L peptides having an identical amino acid sequence, or can include different amino acid sequences. The multimer can be a homodimer (e.g., containing only FKBP-L peptides, these in turn may have identical or different amino acid sequences), homotrimer or homotetramer.

As used herein, the term heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., non-FKBP-L peptide or polypeptides) in addition to the FKBP-L (poly)peptides described herein.

The multimers may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation. Thus, in one embodiment, multimers are formed when the FKBP-L peptides described herein contact one another in solution. In another embodiment, heteromultimers are formed when FKBP-L and non-FKBP-L (poly)peptides contact antibodies to the (poly)peptides described herein (including antibodies to the heterologous (poly)peptide sequence in a fusion protein described herein) in solution. In other embodiments, multimers described herein may be formed by covalent associations with and/or between the FKBP-L peptides (and optionally non-FKBP-L peptides) described herein.

Such covalent associations can involve one or more amino acid residues contained in the FKBP-L sequence (e.g., that recited in SEQ ID NOs: 1 -23). In one embodiment, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations can involve one or more amino acid residues contained in the heterologous polypeptide sequence in a FKBP-L fusion protein. In one example, covalent associations are between the heterologous sequence contained in a fusion protein described herein (see, e.g., US Patent No. 5478925). In another specific example, covalent associations of fusion proteins described herein are using heterologous polypeptides sequence from another protein that is capable of forming covalently associated multimers, for example, oesteoprotegerin (see, e.g., International Publication NO: WO 98/49305). In another embodiment, two or more polypeptides described herein are joined through peptide linkers. Examples include those peptide linkers described in US Patent No. 5073627. Proteins comprising multiple FKBP-L peptides separated by peptide linkers can be produced using conventional recombinant DNA technology.

Multimers may also be prepared by fusing the FKBP-L (poly)peptides to a leucine zipper or isoleucine zipper polypeptide sequence. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric proteins described herein are those described in PCT application WO 94/10308. Recombinant fusion proteins comprising a polypeptide described herein fused to a polypeptide sequence that dimerizes or trimerizes in solution can be expressed in suitable host cells, and the resulting soluble multimeric fusion protein can be recovered from the culture supernatant using techniques known in the art.

The multimers may also be generated using chemical techniques known in the art. For example, polypeptides to be contained in the multimers described herein may be chemically cross-linked using linker molecules and linker molecule length optimisation techniques known in the art (see, e.g., US Patent No. 5478925). Additionally, the multimers can be generated using techniques known in the art to form one or more inter- molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., US Patent No. 5478925). Further, polypeptides described herein may be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., US Patent No. 5478925). Additionally, techniques known in the art can be used to prepare liposomes containing two or more C-12-C peptides desired to be contained in the multimer (see, e.g., US Patent No. 5478925).

Alternatively, those multimers including only naturally-occurring amino acids can be formed using genetic engineering techniques known in the art. Alternatively, those that include post-translational or other modifications can be prepared by a combination of recombinant techniques and chemical modifications. In one embodiment, the FKBP-L peptides are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent No. 5478925, which is herein incorporated by reference in its entirety). For example, polynucleotides coding for a homodimer described herein can be generated by ligating a polynucleotide sequence encoding a FKBP-L peptide described herein to sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., US Patent No. 5478925). The recombinant techniques described herein or otherwise known in the art can be applied to generate recombinant FKBP-L (poly)peptides that contain a transmembrane domain (or hydrophobic or signal peptide) and that can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., US Patent No. 5478925). Pro-Drugs

The polypeptides described herein are intended, at least in some embodiments, to be administered to a human or other mammal to treat or prevent an EMT-associated disease, including but limited to treatment of a molecular sub-type of cancer having the ΈΜΤ phenotype". As discussed below, for the purposes of reversing the EMT phenotype FKBP-L peptides are typically administered via systemic administration, e.g. oral administration or a parenteral route, such as intraperitoneal administration.

Peptides or polypeptides can be conjugated to various moieties, such as polymeric moieties, to modify the physiochemical properties of the peptide drugs, for example, to increase resistance to acidic and enzymatic degradation and to enhance penetration of such drugs across mucosal membranes. For example, Abuchowski and Davis have described various methods for derivatizating enzymes to provide water-soluble, non- immunogenic, in vivo stabilized products ("Soluble polymers-Enzyme adducts," Enzymes as Drugs, Eds. Holcenberg and Roberts, J. Wiley and Sons, New York, N.Y. (1981 )).

Thus, in certain embodiments, the FKBP-L peptides may be conjugated to polymers, such as dextrans, polyvinyl pyrrolidones, glycopeptides, polyethylene glycol and polyamino acids. The resulting conjugated polypeptides retain their biological activities and solubility in water for parenteral applications. In an embodiment, the FKBP-L peptides may be coupled to polyethylene glycol or polypropropylene glycol having a molecular weight of 500 to 20,000 Daltons to provide a physiologically active non-immunogenic water soluble polypeptide composition (see e.g., U.S. Patent No. 4,179,337 and Garman, A.J., and Kalindjian, S.B., FEBS Lett., 1987, 223, 361 -365). The polyethylene glycol or polypropylene glycol may protect the polypeptide from loss of activity and the composition can be injected into the mammalian circulatory system with substantially no immunogenic response. In other embodiments, the FKBP-L is coupled to an oligomer that includes lipophilic and hydrophilic moieties (see e.g., U.S. Patent Nos. 568181 1 , 5438040 and 5359030).

Prodrugs can be prepared for example, by first preparing a maleic anhydride reagent from polydispersed MPEG5000 and then conjugating this reagent to the polypeptides disclosed herein. The reaction of amino acids with maleic anhydrides is well known. The hydrolysis of the maleyl-amide bond to reform the amine-containing drug is aided by the presence of the neighbouring free carboxyl group and the geometry of attack set up by the double bond. The peptides can be released (by hydrolysis of the prodrugs) under physiological conditions. The polypeptides can also be coupled to polymers, such as polydispersed PEG, via a degradable linkage, for example, the degradable linkage shown (with respect to pegylated interferon) in Roberts, M.J., et al., Adv. Drug Delivery Rev., 2002, 54, 459-476.

The polypeptides can also be linked to polymers such as PEG using 1 ,6 or 1 ,4 benzyl elimination (BE) strategies (see, for example, Lee, S., et al., Bioconjugate Chem., (2001 ), 12, 163-169; Greenwald, R.B., et al., U.S. Patent No. 6,180,095, 2001 ; Greenwald, R.B., et al., J. Med. Chem., 1999, 42, 3657-3667.); the use of trimethyl lock lactonization (TML) (Greenwald, R.B., et al., J. Med. Chem., 2000, 43, 475-487); the coupling of PEG carboxylic acid to a hydroxy-terminated carboxylic acid linker (Roberts, M.J., J. Pharm. Sci., 1998, 87(1 1 ), 1440-1445), and PEG prodrugs involving families of MPEG phenyl ethers and MPEG benzamides linked to an amine-containing drug via an aryl carbamate (Roberts, M.J., et al., Adv. Drug Delivery Rev., 2002, 54, 459-476), including a prodrug structure involving a meta relationship between the carbamate and the PEG amide or ether (US Patent No. 6413507 to Bently, et al.); and prodrugs involving a reduction mechanism as opposed to a hydrolysis mechanism (Zalipsky, S., et al., Bioconjugate Chem., 1999, 10(5), 703-707).

The FKBP-L polypeptides of the present invention have free amino, amido, hydroxy and/or carboxylic groups, and these functional groups can be used to convert the peptides into prodrugs. Prodrugs include compounds wherein an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues which are covalently joined through peptide bonds to free amino, hydroxy or carboxylic acid groups of various polymers, for example, polyalkylene glycols such as polyethylene glycol. Prodrugs comprising the polypeptides of the invention or pro-drugs from which peptides of the invention (including analogues and fragments) are released or are releaseable are considered to be analogues of the invention.

Prodrugs also include compounds wherein PEG, carbonates, carbamates, amides and alkyl esters which are covalently bonded to the above peptides through the C-terminal carboxylic acids. Thus, embodiments of the present invention comprise site-specific PEG addition.

Treatment of EMT-associated disease

Embodiments of the present invention relate to the treatment of "EMT-associated disease", which term encompasses any disease condition associated with/mediated by the EMT phenotype. Particular embodiments relate to the treatment or prevention of certain cancers, specifically a sub-set of cancers that exhibit the EMT phenotype. It is well known that certain tumour cells can be induced to undergo EMT by a variety of stimuli (reviewed by Marcucci et al., Nature Reviews Drug Discovery, 2016). Tumour cells that have undergone EMT may acquire the capacity to disarm the body's antitumour defences, resist apoptosis and anticancer drugs, disseminate throughout the organism and act as a reservoir that replenishes and expands the tumour cell population. Inhibition of EMT/promotion of MET is thus a target of prime interest for anti-cancer therapy. The present invention provides a means to treat a sub-population of cancers/cancer patients that are characterised by the presence of cells exhibiting the EMT phenotype, by use of FKBP-L, or biologically active peptide fragments or derivatives thereof that are capable of reversing EMT.

In certain embodiments, the cancer exhibiting an EMT phenotype may be an ovarian cancer, breast cancer, colon cancer, pancreatic cancer, lung cancer (e.g. non- small cell lung cancer), melanoma, renal cancer, prostate cancer or thyroid cancer.

In certain embodiments, the cancer exhibiting an EMT phenotype may be platinum- resistant, for example carboplatin-resistant or cisplatin-resistant. In a particular

embodiment, the cancer may be cisplatin-resistant ovarian cancer.

Cancers (or cancer patients) exhibiting an "EMT phenotype" may be identified on the basis of biomarker expression. Various biomarkers of the EMT phenotype have been described in the art. In certain embodiments, cells having an "EMT phenotype" may identified on the basis of reduced expression of the epithelial marker E-cadherin, and/or increased expression of one or more mesenchymal markers selected from: N-cadherin, fibronectin, vimentin, SNAI1 (Snail), SNAI2 (SLUG), ZEB1 , ZEB2, Twist and ΤΘΡ-β3. In this context, "expression" of a biomarker may be measured at the protein level and/or the mRNA level.

In other embodiments, cancers (or cancer patients) exhibiting an "EMT phenotype" may be identified on the basis of a characteristic gene expression (biomarker) signature.

By "biomarker signature" is meant an identifier comprised of one or more biomarkers (such as a DNA or RNA sequence, a protein or other biological molecule, a cell etc.). The expression level of the one or more biomarkers is measured and the measured expression levels allow the sample to be defined as signature positive or signature negative. Thus, at its simplest, an increased level of expression of one or more biomarkers defines a sample as positive for the biomarker signature. For certain biomarkers, a decreased level of expression of one or more biomarkers defines a sample as positive for the biomarker signature. However, where the expression level of a plurality of biomarkers is measured, the combination of expression levels is typically aggregated in order to determine whether the sample is positive for the biomarker signature. Thus, some biomarkers may display increased expression and some biomarkers may display decreased expression. This can be achieved in various ways, as discussed in detail herein.

In a general sense, a biomarker signature may be considered as indicative of a particular biological state (such as the presence of a disease condition or developmental state or belonging to a particular biological subgroup). "Positive" for a biomarker signature thus may be interpreted to mean that the sample reflects the relevant biological state that the biomarker signature identifies. Similarly, "negative" for a biomarker signature means that the sample is not in (or reflective of) the relevant biological state. In the present invention, the biological state indicated by the biomarker signature is a molecular subgroup of cancer characterised by misregulation of the epithelial-mesenchymal transition (EMT) pathway. Thus, the cancer identified by the signature may display higher expression of EMT associated genes. This may include increased expression of VIMENTIN, AXL,

TWIST1 , SNAIL and/or SLUG. The increased signalling or expression is as compared to other cancers of the same type. So, for example, the cancer may be an ovarian cancer and the subgroup displays increased signalling or expression as compared to other ovarian cancers.

By way of non-limiting example, cancers (or cancer patients) having the "EMT phenotype" can be identified based on the 45-gene EMT biomarker signature or the 15- gene EMT biomarker signature described in the accompanying examples. Therefore, in particular non-limiting embodiments, the invention provides methods of using FKBP-L and peptide fragments thereof (in particular the fragment identified herein as ALM201 ) to treat cancer in a patient, wherein said cancer (or said patient) has been identified as having the "EMT phenotype" on the basis of the 45-gene EMT signature (Table A) or on the basis of the 15-gene EMT signature (Table B) described herein. The "EMT phenotype" may be identified based on expression of one or more or any combination, or all of the biomarker genes listed in Table A, or based on expression of one or more or any combination, or all of the biomarker genes listed in Table B (optionally together with one or more additional biomarkers).

As demonstrated in the accompanying examples, both the 45-gene signature and the 15-gene signature identify a molecular sub-group of cancer that exhibits enhanced EMT signalling. Therefore, either signature could be used to identify cancer patients having the ΈΜΤ phenotype" who would benefit from treatment with FKBP-L, or peptide fragments thereof, especially the FKBP-L peptide identified herein as ALM201 . Preferred embodiments of the methods and uses of FKBP-L fragments described herein relate to treatment of patient sub-groups identified as having the ΈΜΤ phenotype" based on screening for the 15-gene EMT signature or the 45-gene EMT signature.

In certain embodiments, signatures other than the 45-gene signature and the 15-gene signature may be used to identify cancer patients having the "EMT phenotype" who would benefit from treatment with FKBP-L or peptide fragments thereof, especially the FKBP-L peptide identified herein as ALM201 .

Formulations/routes of administration

As used herein, "treatment" or "therapy" includes any regime that can benefit a human or non-human animal. The treatment may be in respect of an existing condition or may be prophylactic (preventative treatment). Treatment may include any intervention aimed at improving, stabilising or minimise deterioration of a medical condition, including curative, alleviation or prophylactic effects.

The dose of the FKBP-L polypeptide administered may vary depending upon the precise nature of the disorder being treated. In alternate embodiments, a dosage to be achieved in vivo would be equivalent to an in vitro level of greater than 10^"12 M, or 10^"11 M, or 10^"10 M, or 10^"9 M, or 10^"8 M, or 10^"7 M, or 10^"6 M, or 10^"5 M. Thus, a dosage to be achieved in vivo may be equivalent to an in vitro level of 10^"12 M to 10^"5 M, or 10^"11 M to 10^"6 M, or 10^"10 M to 10^"7 M, or 10^"9 M to 10^"7 M or ranges therein. In alternate embodiments, the dosage used may be equivalent to an in vitro level of about 1 -10000 ng/ml, or about 10- 5000 ng/ml, or about 100-1000 ng/ml. Or, in certain embodiments, the dosage may comprise from about 0.00001 to 500 mg/kg/day, or from about 0.0001 to 300 mg/kg/day, or from about 0.003 to 100 mg/kg/day, or from about 0.03 to 30 mg/kg/day, or from about 0.1 mg/kg/day to 10 mg/kg/day, or from about 0.3 mg/kg/day to 3 mg/kg/day.

The route of administration of a therapeutic agent may also vary depending upon the precise nature of the disorder being treated. Suitable routes of administration may include, but are not limited to, oral administration, parenteral administration, transdermal administration, etc.

For administration to a human subject, the FKBP-L polypeptide or peptide fragment thereof may be formulated into a pharmaceutical composition/dosage form suitable for administration via the chosen delivery route. Suitable dosage forms and dosage regimes are known for therapeutic agents and can be determined by a practising physician. Therapeutic agents are approved and marketed for administration in a given dosage form, together with detailed prescribing instructions. Thus, the invention is not limited in relation to how, or in what form, the therapeutic agent is administered since the skilled person would be in a position to determine this based on the therapeutic agent of interest and the nature of the disease to be treated.

It is envisaged that the FKBP-L polypeptide or peptide fragment thereof may be used as a stand-alone treatment (monotherapy), or as a component of a combination treatment. Combination therapies may comprise administration of the FKBP-L polypeptide or peptide fragment, in conjunction with at least one further therapeutic active agent, such as for example a further anti-cancer agent or chemotherapeutic. In specific embodiments, the further anti-cancer agent may be cisplatin, carboplatin or anthracycline. In further embodiments, the FKBP-L polypeptide or peptide fragment may be administered as an adjunct to an anti-cancer treatment such as radiotherapy or surgery. The invention will be further understood with reference to the following non-limiting experimental examples:

EXAMPLES Example 1

EMT molecular subgroup of cancer

A cancer with a given histopathological diagnosis may represent multiple diseases at a molecular level. The present example describes a molecular subgroup of cancer characterised by misregulation of the MAPK signalling pathway and the epithelial- mesenchymal transition (EMT) pathway. Biomarker signatures shown in Table A and Table B below can be used to identify cancers having the EMT phenotype.

Table A - Weighting and bias for the 45 gene signature

45 Gene Signature

Rank Gene Name Weight Bias

1 TMEM200A 0.059481295 3.681329367

2 GJB2 0.055985433 4.479833955

3 MMP13 0.038284076 3.724107067

4 GFPT2 0.037990641 4.860237265

5 POSTN -0.035480409 4.359882019

6 BICC1 0.030426737 3.698203663 7 CDH1 1 0.028340142 4.996780524

8 MRVI1 0.025598535 5.076083782

9 PMP22 0.024034610 5.564463361

10 COL1 1A1 -0.023672753 3.500170591

1 1 IGFL2 0.022225316 3.310383445

12 LUM -0.022014619 8.336273473

13 NTM -0.021750365 4.230245127

14 BGN 0.021089508 10.15236225

15 COL3A1 -0.021023256 8.323635399

16 COL10A1 0.019650845 6.353832828

17 RAB31 0.018014921 5.3171 19481

18 ANGPTL2 0.016630934 5.639562288

19 PLAU 0.016596202 5.848820224

20 COL8A1 0.016373799 6.419330171

21 MIR1245 0.015290888 5.455187262

22 POLD2 0.014555548 9.38782491

23 NKD2 0.014468847 7.371707677

24 FZD1 0.014334768 4.151874676

25 COPZ2 0.013866713 5.103944696

26 ITGA5 0.013561913 8.36627973

27 VGLL3 0.012488674 4.501866677

28 INHBA -0.01 1763261 4.684272993

29 MMP14 0.01 1010832 10.08406264

30 VCAN 0.009977966 5.551759846

31 THBS2 -0.008700202 8.130920944

32 RUNX2 0.008333275 4.73450528

33 TIMP3 0.008141253 6.498316457

34 SFRP2 -0.007890741 5.601725816

35 COL1A2 0.007788938 6.01000198

36 COL5A2 -0.007217722 3.567060064

37 SERPINF1 0.006801251 10.8333948

38 KIF26B -0.005249312 4.97815094

39 TNFAIP6 0.004963450 5.361760185

40 MMP2 0.003988003 5.362247865

41 FN1 0.003130435 4.984016427

42 ALPK2 0.002394440 3.513604572

43 CTSK 0.001542586 5.732155915

44 LOXL1 -0.001415170 9.593869933

45 FAP -0.000007237 5.23E+00 Table B - Weighting and bias for the 15 gene signature

Materials & Methods

High Grade Serous Ovarian Cancer (HGSOC) Tumour Material

This study performed gene expression analysis of a cohort of 265 macrodissected ovarian cancer FFPE tissue samples sourced from the Edinburgh Ovarian Cancer Database. Ethical approval for Edinburgh dataset analysis was obtained from Lothian Local Research Ethics Committee 2 (Ref: 07/S1 102/33).

This cohort of samples can be further described using the following inclusion criteria:

• Primary ovarian, peritoneal or fallopian tube cancer

• High grade serous histology

• Treatment-na^'ive FFPE tissue samples

• Matched pre chemotherapy and post-chemotherapy samples

Gene Expression Profiling of HGSOC samples

Total RNA was extracted from the macrodissected FFPE tumour samples using the Roche High Pure RNA Paraffin Kit (Roche Diagnostics GmbH, Mannheim, Germany) as described previously (Kennedy et al, 201 1 ). Total RNA was amplified using the NuGEN WT-Ovation™ FFPE System (NuGEN Technologies Inc., San Carlos, CA, USA). It was then hybridised to the Almac Ovarian Cancer DSA™ as described previously (Kennedy et al, 201 1 ) (Tanney et al, 2008). Arrays were scanned using the Affymentrix Genechip® Scanner 7G

(Affymetrix Inc., Santa Clara, CA).

Data Preparation & Hierarchical Clustering

Quality Control (QC) of profiled samples was carried out using MAS5 pre-processing algorithm to assess technical aspects of the samples i.e. average noise and background homogeneity, percentage of present call (array quality), signal quality, RNA quality and hybridization quality. Distributions and Median Absolute Deviation of corresponding parameters were analyzed and used to identify possible outliers. Sample pre-processing was carried out using RMA (Irizarry et al, 2003). The pre-processed data matrix was sorted by decreasing variance, decreasing intensity and increasing correlation to cDNA yield. Following filtering of probe sets (PS) correlated with cDNA yield (to remove any technical bias in the expression data), hierarchical clustering analysis was performed (Pearson correlation distance and Ward's linkage methods (Ward et al, 1963). Subsets of the data matrix were tested for cluster stability using the GAP statistic (Tibshirani et al, 2001 ), which gives an indication of the within-cluster tightness and between-cluster separateness. The GAP statistic was applied to calculate the optimal number of sample clusters in each sub- matrix, while the stability of cluster composition was assessed using a partition comparison tool (Carrigo et al, 2006; Pinto et al, 2008). The smallest number of PS generating the optimal sample cluster number was selected as the list of most variable PS to take forward for hierarchical cluster analysis.

Functional Analysis of 3 Molecular Gene Clusters

To establish the functional significance of the gene clusters an enrichment analysis, based on the hypergeometric function (False Discovery Rate applied (Benjamini and Hochberg, 1995, J. R. Stat. Soc. 57:289:300)), was performed. Over-representation of biological processes and pathways were analysed for each gene group generated by the hierarchical clustering using Gene Ontology biological processes. Hypergeometric p-values were assessed for each enriched functional entity class. Functional entity classes with the highest p-values were selected as representative of the group and a general functional category representing these functional entities was assigned to the gene clusters based on significance of representation (i.e. p-value). Gene Selection for Signature Generation

Genes that are variable and highly expressed across multiple disease indications were determined prior to model development. The disease indications that were included in this evaluation were: ovarian cancer; colon cancer; lung cancer and melanoma. Two data sets per disease indication were assessed with the exception of prostate cancer where only one dataset was evaluated. Data sets were pre-processed using RMA and summarised to Entrez Gene ID level using the median of probe sets for each Entrez Gene ID on the Ovarian Cancer DSA™. Within each data set, Entrez Gene IDs were ranked based on the average rank by variance and mean intensity across samples (high rank = high variance, high mean intensity). A single combined rank value per gene was calculated based on the average variance-intensity rank within each disease indication. Genes with no expression level were removed from further analysis. Scatterplots were generated to show the combined variance-intensity rank of the 19920 Entrez gene IDs in the disease indications evaluated with two datasets where the x and y axis represent the rank for the two data sets evaluated within each indication. A final classification of expressed genes as high/low rank was defined within each disease indication. Finally the overlap in high ranking genes across disease indications was determined and the top 75% ranked genes were identified. This list was then used as the starting list for signature generation.

Signature Generation of the 45-gene and 15-gene signatures

The genes that had common high expression and variance in ovarian, colon, lung, melanoma and prostate were used as a starting set for model development. The Edinburgh ovarian cancer sample cohort was used to train the signature under 5 fold cross validation (CV) with 10 repeats. Partial least squares (PLS) (de Jong, 1993) was paired with Forward Feature Selection (FFS) to generate signatures for the top 75% ranked list. Tables A and B indicate the weightings and bias for each probeset incorporated within the 45-gene signature (A) and the 15-gene signature (B). Model Selection and Signature Validation for the 15-gene signature

The C-index performance was calculated using the progression free survival (PFS) time endpoint and signature scores generated within cross validation for each evaluated signature length. This data was then used to determine the signature length at which optimal performance is reached with respect to association between signature scores and PFS. The highest C-index values were compared for signatures of length less than 100 and greater than 10 features. The signature with the shortest length and highest C-index within this subset was selected as the final model for identifying the subgroup.

The C-index performance metric was the primary metric analysed for model selection. The C-index was significant across the majority of feature lengths in the training set and the C- index performance was highest at a feature length of 15 (56.62 [57.86-55.55]).

A threshold was generated for classification of signature scores by using the value where the sum of sensitivity and specificity with respect to predicting the subtype in the training data is highest. This threshold was set at 0.5899 using the curve of sensitivity and specificity. Samples with scores above the selected threshold would be classified as MEK ON whereas samples with scores below or equal to the threshold would be labelled as MEK OFF. Functional Analysis

Functional enrichment analysis of the selected model was performed using the Gene Ontology biological processes classification to gain an understanding of the underlying biology behind the selected signature. The top 20 biological processes include:

• Angiogenesis (p = 2.09e-05)

· Blood vessel development (p = 5.55e-06)

• Cell-cell junction organization (p = 2.55e-05)

Cisplatin was acquired from Belfast City Hospital Pharmacy department and diluted in PBS to produce a 10 μΜ stock solution. Cisplatin was stored at room temperature and protected from light.

Generation of OVCAR3 and OVCAR4 Cisplatin Resistant Cell Lines

OVCAR3 and OVCAR4 cells were trypsinised and relevant cell numbers were seeded into P90 plates. Cells were allowed to adhere overnight. The following day media was removed and replaced with media containing 25 nM cisplatin. The concentration of cisplatin was increased every 2 weeks, doubling the concentration at every increment. Batches of cells were frozen every two weeks upon increasing the concentration of cisplatin. Once cells were stably growing at 200 nM cisplatin, sensitivity to cisplatin was tested by clonogenic assay. Cells were continuously grown in 200 nM cisplatin. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Reverse transcription was performed using the First Strand cDNA synthesis kit (Roche). 500 ng of RNA was reverse transcribed according to manufacturer's instructions. Exon- spanning qPCR primers were designed using Roche Universal Probe Library Assay Design Centre and were used at a concentration of 0.5 μΜ. The following primer sequences were used:

N-cadherin

Forward CTC-CAT-GTG-CCG-GAT-AGC (SEQ ID NO. 24)

Reverse CGA-TTT-CAC-CAG-AAG-CCT-CTA-C (SEQ ID NO. 25)

SLUG

Forward TGT-TGC-AGT-GAG-GGC-AAG-AA (SEQ ID NO. 26)

Reverse GAC-CCT-GGT-TGC-TTC-AAG-GA (SEQ ID NO. 27)

SNAIL

Forward ACC-ACT-ATG-CCG-CGC-TCT-T (SEQ ID NO. 28)

Reverse GGT-CGT-AGG-GCT-GCT-GGA-A (SEQ ID NO. 29)

Vimentin

Forward TGG-TCT-AAC-GGT-TTC-CCC-TA (SEQ ID NO. 30)

Reverse GAC-CTC-GGA-GCG-AGA-GTG (SEQ ID NO. 31 )

TWIST

Forward AGC-TAC-GCC-TTC-TCG-GTC-T (SEQ ID NO. 32)

Reverse CCT-TCT-CTG-GAA-ACA-ATG-ACA-TC (SEQ ID NO. 33)

TGF-B3

Forward AAG-TGG-GTC-CAT-GAA-CCT-AA (SEQ ID NO. 34)

Reverse AAA-TTC-ACT-CTG-CCC-AGG-ACG (SEQ ID NO. 35)

PUM1 (Housekeeping gene)

Forward 5' CCA GAA AGC TCT TGA GTT TAT TCC 3' (SEQ ID NO. 36)

Reverse 5' CAT CTA GTT CCC GAA CCA TCT C 3' (SEQ ID NO. 37) To perform absolute quantification from qPCR, we used a standard curve method. The efficiency of each primer set was derived from the standard curve using the following equation:

E= 10^Λ(-1 /slope)

The product of Reverse Transcription was diluted 1 :10 in Nuclease Free Water (NFW). Each 10 μΙ PCR reaction, consisted of 0.5 μΙ of 10 μΜ Forward primer, 0.5μΙ of 10 μΜ Reverse primer, 5 μΙ of 2X LightCycler® 480 SYBR Green I Master mix (Roche), 1 .5 μΙ NFW and 2.5 μΙ diluted Reverse Transcription product. These 10 μΙ reactions were pipetted into wells of a LightCycler® 480 multiwell 96 plate (Roche), the plate was then sealed using clear adhesive film (Roche). The plate was placed into the LightCycler® 480 (Roche) and run with the following protocol. (95°C for 10 mins, 45 cycles of; 95°C for 15 sees, 55°C for 30 sees and 72°C for 30 sees, finishing with a melt curve for confirmation of primer specificity. All qPCR data was analysed using the LightCycler® 480 software provided by Roche. For analysis, the Cp value from a technical duplicate was calculated and the relative amount of a gene was calculated Cp value to an in-run standard curve. Each mean value was then normalised to the mean concentration of the housekeeping gene PUM1 within the corresponding sample, by dividing the concentration of the target gene by the concentration of the house keeping gene. Relative expression refers to the gene expression levels which have been normalised to the housekeeping gene and made relative to the associated control samples. From these normalized values, the fold- changes for each gene were calculated and the average of three individual fold-changes were derived from three independent experimental triplicates. The unpaired, two-tailed students T-test available on GraphPad Prism 5.0 software was used to detect statistical significance. Colony Formation Assays

Cells were seeded at predetermined densities, 24 hours later treated with drug, which was replenished every 3-4 days. After 10 days, cells were washed with PBS, fixed in methanol, stained with crystal violet and colonies counted. The surviving fraction (SF) for a given dose was calculated and dose-response curves plotted and IC50 generated using GraphPad Prism™ 5. Receiver operator curves (ROC) were plotted by dicotomising the IC50 values based on the median of the IC50 and defining the higher IC50 values as resistant and the lower IC50 values as sensitive. The gene signatures associated with the cell lines were plotted based on sensitive and resistant cells. Additionally ROCs were plotted by dicotomising the signature scores based on the median of the scores and defining the higher signature score as signature positive and the lower signature scores as signature negative. The IC50s associated with the cell lines were plotted based on signature positive and signature negative cells. Migration assay

The migration assay was performed using the xCELLigence RTCA DP system and carried out with CIM-plate 16 (ACEA bioscience). Normal cell media growth conditions (RPMI 1640, 1 % L-Glut and 20% FCS) was the chemoattractant condition used in the bottom chamber. 160μΙ of the chemoattractant was added to each bottom chamber of a CIM-plate 16. The CIM-Plate 16 is assembled by placing the top chamber onto the bottom chamber and snapping the two together. 30μΙ pf optimem (serum-free) is placed in the top chamber to hydrate and pre-incubate the membrane for 2 hours in the C02 incubator at 37 °C before obtaining a background measurement.

The protocol is optimized for the two paired cancer cell lines: OVCAR3, OVCAR4 parental and OVCAR3, OVCAR4 platinum resistant cell lines. Platinum resistant cell lines were grown for 24 hours in media containing 0.1 nM, 1 nM and 10nM ALM201. On the experimental day, cells are washed x1 with PBS. Cells are lightly trypsinized, pelleted and re-suspended at 100μΙ, containing 50,000 cells, in optimem (serum-free) medium in the presence of 0.1 nM, 1 nM and 10nM ALM201 . Once the CIM-Plate 16 has been equilibrated, it is placed in the RTCA DP station and the background cell-index values are measured. The CIM-Plate 16 is then removed from the RTCA DP station and the cells are added to the top chamber. The CIM-Plate 16 is placed in the RTCA DP station and migration is monitored every 5 minutes for several hours.

Each experimental condition was performed in triplicate. For quantification of the migration rate, the slope of the curve was used to determine the rate if change in cell index. The average and standard deviation slope values were then quantified relative to that at the control condition.

Invasion Assay

The invasion assay was performed using the xCELLigence RTCA DP system and carried out with CIM-plate 16 (ACEA bioscience).

Normal cell media growth conditions (RPMI 1640, 1 % L-Glut and 20% FCS) was the chemoattractant condition used in the bottom chamber. 160μΙ of the chemoattractant was added to each bottom chamber of a CIM-plate 16. The CIM-Plate 16 is assembled by placing the top chamber onto the bottom chamber and snapping the two together. 20μΙ Matrigel growth factor reduced (GFR) (phenol-red free) basement membrane matrix (Cornig, ref: 356231 ) was diluted in 400μΙ optimem (serum free) giving a final working concentration of GFR Matrigel of 5%. 20μΙ of the Matrigel-optimem master mix is placed in the top chamber to hydrate and pre-incubate the membrane for 2 hours in the C02 incubator at 37 °C before obtaining a background measurement.

The protocol is optimized for the two paired cancer cell lines: OVCAR3, OVCAR4 parental and OVCAR3, OVCAR4 platinum resistant cell lines. Platinum resistant cell lines were grown for 24 hours in media containing 0.1 nM, 1 nM and 10nM ALM201. On the experimental day, cells are washed x1 with PBS. Cells are lightly trypsinized, pelleted and re-suspended at 10ΟμΙ, containing 50,000 cells, in optimem (serum-free) medium in the presence of 0.1 nM, 1 nM and 10nM ALM201 . Once the CIM-Plate 16 has been equilibrated, it is placed in the RTCA DP station and the background cell-index values are measured. The CIM-Plate 16 is then removed from the RTCA DP station and the cells are added to the top chamber. The CIM-Plate 16 is placed in the RTCA DP station and migration is monitored every 5 minutes for several hours.

Each experimental condition was performed in triplicate. For quantification of the migration rate, the slope of the curve was used to determine the rate if change in cell index. The average and standard deviation slope values were then quantified relative to that at the control condition.

Proliferation (3-day) assay

The proliferation assay was performed using 6-well plates. The experiment was set-up with two controls mechanisms to ensure accuracy of results. Each cell line was seeded in duplicates and the experiment was performed in triplicate. For quantification of proliferation, cell numbers were counted manually using a coulter counter on day 1 , day 2 and day 3 in three different concentrations of ALM201 (0.1 nM, 1 nM and 10nM). Media is changed on day 2 and day 3 with fresh media containing the 3 concentrations of ALM201.

On day 0, each cell line was lightly trypsinized, counted and seeded at a concentration of 5x10⁴ per well in the presence of ALM201 (0.1 nM, 1 nM and 10nM concentration). 2mls of cells was added to each well in three 6 well plates (representing day 1 , 2 and 3) and left to incubate for 24 hours in the C02 incubator at 37 °C prior to counting cells for day 1 , 48 hour incubation prior to count day 2 and 72 hour incubation prior to counting day 3. At each time point, media was aspirated from the wells and wells were washed with PBS x1 . 500μΙ 5% trypsin was added to each well and incubated 3-5 mins. 1 .5mls media was added to each well to neutralise the trypsin. Cells were counted using the coulter counter. To estimate significance, the unpaired, two-tailed student T-test was calculated using the T-test calculator available on GraphPad Prism 5.0 software.

Western Blotting

Cells were grown in I nM and 10nM of ALM201 for 24 hours prior to harvesting lysates. 50 μg of protein lysates were mixed with LDS loading dye (Invitrogen) and

Reducing Agent (Invitrogen) and denatured for 10 minutes at 70 °C. Samples were briefly centrifuged, and loaded onto a Bolt 4-12% Bis-Tris gel and electrophoresed at 165 V for 1 hour 30 minutes using MOPS running buffer. SeeBlue Pre Stained protein ladder

(Invitrogen) was used as a reference for protein size. After electrophoresis proteins were transferred onto immobilon-P PVDF membrane (Millipore) at 30 V for 2 hours using the XCell surelock mini-cell transfer system (Invitrogen). To ensure proper transferring of proteins onto membrane, the membrane was stained with Ponceau S solution (Sigma). Membranes were incubated in blocking solution (5% bovine serum albumin) for 1 hour at room temperature on a rocking platform in order to prevent non-specific binding of antibody to membrane. Membranes were then incubated in primary antibody overnight (see appendix 3 for dilutions) at 4 °C. The following day, the membranes were washed 3 times in TBS-T for 10 minutes and incubated in secondary antibody at a 1 :5000 dilution 1 hour at room temperature. Membranes were then washed 5 times for 5 minutes in TBS-T, and incubated for 5 minutes in Luminata Cresendo or Forte (Millipore) detection reagent.

Analysis was performed using Alpha Innotech Imager FluorChem Software.

Antibodies

ERK (Cell Signalling 2496) - monoclonal mouse antibody for total p44/42 MAP Kinase (ERK1/2), used at a 1 :1000 dilution in 5% milk. pERK (Cell Signalling 4370) - polyclonal rabbit antibody for p44 and p42 MAP Kinase (ERK1/2), used at a 1 :1000 dilution in 5% BSA.

MEK (Cell Signalling 4694) - mouse monoclonal antibody for total MEK1/2, used at a dilution of 1 :1000 in 5% milk. pMEK (Cell Signalling 9121 ) - rabbit polyclonal antibody for phosphO-MEK1/2 at Ser217/221 , used at a dilution of 1 :1000 in 5% BSA. N-cadherin (Cell Signalling) - rabbit monoclonal, used at a dilution of 1 :1000 in 5% milk.

E-cadherin (Cell Signalling 24E10) - rabbit monoclonal antibody used at a dilution of 1 :1000 in 5% milk. Vimentin (Cell Signalling R28) - rabbit monoclonal antibody to detect Vimentin, used at a dilution of 1 :1000 in 5% milk.

SLUG (Cell Signalling C10G7) - rabbit monoclonal antibody to detect SLUG EMT marker, used at a dilution of 1 :1000 in 5% milk.

VEGFa (Abeam) - rabbit polyclonal antibody to detect VEGFa, used at a dilution of 1 :1000 in 5% BSA.

B-actin (Sigma A2228) - mouse monoclonal antibody detecting the N-terminus of β-actin, used at a dilution o 1 :5000 in 5% milk.

Secondary antibodies - anti-rabbit and anti-mouse (Cell Signalling) were used at a dilution of 1 :5000 in 5% milk. Results

The 45 and 15 gene signatures identify a molecular subgroup of High Grade Serous Ovarian Cancer (HGSOC) with an EMT phenotype

We previously defined 3 molecular subgroups of High grade serous ovarian cancer (HGSOC), an Angiogenesis subgroup (HGS1 ), an Immune subgroup (HGS2) and an

Angiojmmune subgroup (HGS3) (Figure 1 a) using gene expression data from 265 FFPE HGSOC samples obtained from treatment naive patients but who were treated with carboplatin + paclitaxel or carboplatin only Standard of Care (SoC) chemotherapy (Gourley, McCabe et al., manuscript in preparation). Functional analysis of the gene clusters revealed one subgroup was characterised by up-regulation of immune response genes (Immune subgroup), a second by up-regulation of angiogenesis/vascular development genes (Angio subgroup) and a third by up-regulation of both immune response and angiogenesis/vascular development genes (Angiolmmune subgroup). Functional analysis of the gene clusters revealed that two of the 4 gene clusters had no significantly enriched processes (PS clusters 1 & 3), the third was characterised by Immune processes (PS cluster 2) and the fourth by Angiogenesis processes (PS cluster 4).

Patients within these 3 molecular subgroups respond differently to SoC surgery and chemotherapy. The Immune group has the best prognosis, represented by both increased progression-free survival (PFS) (HR=0.60 (0.44-0.82) compared to Angiojmmune subgroup and HR=0.64 (0.49-0.92) compared to Angio subgroup) and overall survival (OS) (HR=0.58 (0.41 -0.82) compared to Angiojmmune subgroup and (HR=0.55 (0.37-0.80) compared to Angio subgroup), while the Angio and Angiojmmune subgroups respond similarly (Figure 1 b). We have defined this as the Discovery dataset.

We hypothesised that the Angiojmmune group would be prognostic in the context of SoC treatment in ovarian cancer. We therefore investigated this in the treatment naive

Discovery dataset. We developed 2 signatures to detect the Angiolmmune subgroup. A 45 gene signature developed in our HGSOC dataset to detect the subgroup with high sensitivity and specificity. We also developed a 15 gene signature which was optimised to detect the subgroup across multiple diseases including HGSOC, prostate cancer, lung cancer, colon cancer and melanoma. This signature also detects the subgroup with high sensitivity and specificity in multiple diseases. The 45-gene signature was associated with worse prognosis (PFS HR 1 .6403 (1 .2252- 2.1960) p=0.0002 and OS HR 1 .6562 (1 .2169-2.2540) p= 0.0004) and therefore predicted response to cisplatin based therapy (Figure 1 c). The 15-gene signature was also associated with worse prognosis (PFS, HR = 1 .3564 [1.0156-1 .81 17]; p = 0.0279 and OS, HR = 1.3464 [0.9901 -1 .8308]; p = 0.0441 ) and could predict response to cisplatin based therapy (Figure 1 d).

The 45 and 15-gene EMT signatures detect s poor prognosis subgroup in Colon Cancer (CRC) and Non-Small Cell Lung Cancer (NSCLC)

We hypothesised that the 45 and 15-gene EMT phenotype signature may also define a subgroup of patients in alternative disease indications, namely colon cancer (CRC) and non-small cell lung cancer (NSCLC) which have high incidence of alterations in the MAPK pathway. We therefore investigated this in two publically available colon datasets both in the context of treatment (Marisa GSE40967 and Jorissen GSE14333) and one NSCLC dataset which incorporates an untreated population (Okayama GSE31210). The Marisa dataset consisting of 566 Stage l-IV colon cancers, had the MEK defined subgroup present in sample cluster 3 (C3) following hierarchical clustering (Figure 3a, box). The MEK subgroup (C3) was associated with worse prognosis (Figure 3b). Additionally, the 45 and 15-gene EMT signature described as 'MEK ON' was associated with poor prognosis, (Figure 3c). The Jorissen dataset consisting of 260 Stage l-IV colon cancers also showed a poor prognostic subgroup detected by the 45 and 15-gene EMT signature, (Figure 3d).

In relation to NSCLC, the Okayama dataset consisting of Stage I and II untreated NSCLC samples also had the MEK defined subgroup present in sample cluster 4 (C4) following hierarchical clustering (Figure 4a, box). The MEK subgroup (C4) was associated with worse prognosis (p=0.0004) (Figure 4b). Additionally, the 45 and 15 -gene EMT signature described as 'SIGN POS' was associated with poor prognosis, (PFS HR 3.045 (1 .631 - 5.686), p=0.0005) (Figure 4c). The Okayama dataset confirmed a poor prognostic subgroup detected by the 45-gene EMT signature (Figure 4d). In sum, the 45 and 15-gene EMT signature detects a poor prognosis subgroup of patients in both colon cancer and NSCLC.

Activation of the EMT phenotype is enhanced in Cisplatin resistant ovarian cell lines

We investigated the relationship between cisplatin resistance in-vitro and an associated EMT phenotype utilising both cisplatin-sensitive and -resistant cell lines generated in- house; HGSOC OVCAR3 and OVCAR4 cell lines (Figure 5a). It appears that cisplatin resistant ovarian cells (OVCAR3 CP and OVCAR4 CP) exhibit activation of EMT signalling as demonstrated by the upregulated protein expression of MAPK signalling and Vimentin, N-cadherin and SLUG in conjunction with decreased levels of E-cadherin, compared to their wildtype counterparts (Figure 5b). These observed changes in EMT markers are all indicative of EMT activation. MAPK is known to phosphorylate SLUG and other key players of the SNAIL/SLUG transcription factors, to induce epithelial-mesenchymal transition (EMT) which is known to be a contributing mechanism of progressive disease (Virtakoivu et al., 2015). Using the E-GEOD-58582 dataset, MCF7 breast cells overexpressing SNAIL show a positive association with the 45 and 15-gene signature (Figure 5c). In sum, the 45 and 15-gene signature is also associated with enhanced EMT signalling. As a result, the 45 and 15-gene signature may also be used to identify cancer cells having the "EMT phenotype". Moreover, cisplatin resistance in OVCAR3 and OVCAR4 shows greater cell migration and an enhanced migratory phenotype (Figure 5d), hence suggesting the activation of the EMT phenotype.

ALM201 reverses the EMT phenotype in OVCAR3 and 4 cisplatin resistant cells

We used the EMT on cell line Kuramochi and the OVCAR3 and OVCAR4 cisplatin resistant cells to examine the effects of ALM201 on EMT markers and associated phenotypes.

ALM201 treatment caused reduced MAPK signalling and EMT signalling in the Kuramochi cell line (Figure 6a). The same effect was seen in the OVCAR3 cisplatin resistant cell line (Figure 6b). Additionally in the OVCAR3 and OVCAR4 cells treatment with ALM201 inhibited the proliferation capacity (Figure 6c), and in the OVCAR3 cisplatin resistant cells, ALM201 inhibited the migration and invasion potential of the cells (Figure 6d). Example 2

ALM201 inhibits growth of tumour cells in vivo

Nonclinical studies utilising patient-derived ovarian cancer xenografts (PDX) engrafted in female athymic nude mice were conducted. Each of the xenografts was assessed for EMT status prospectively using the 15-gene EMT biomarker, and retrospectively by performing an open Gene Set Enrichment Analysis (GSEA). Of the xenografts for which treatment history was available, each was derived from a patient who has previously been treated with carboplatin; each of the PDX models shown was derived from a metastatic site. The clinical history of the patient-derived xenografts used and response to carboplatin is provided in Table 1 below:

Table 1

NA - Not available; PD - Poorly differentiated; PSAC - Papillary serous adenocarcinoma;

Tumours were implanted in the mice and, once tumours reached a mean volume of 199 mm³ (range of 151 to 264 mm³), treatment was initiated. ALM201 or vehicle (PBS) was subcutaneously administered once daily. ALM201 was dosed at 3 mg/kg. Mice

administered vehicle only served as the no treatment control. Carboplatin (50 mg/kg) was administered once weekly by intraperitoneal injection. Tumour volumes were collected throughout the study and the percentage of tumour growth inhibition (%TGI) against the vehicle control was assessed at the termination of each model. The study was terminated when the vehicle tumour size reached the humane size endpoint. One animal was utilised for each arm of each ovarian patient-derived xenograft model.

%TGI was calculated as follows: %TGI = 1 -(Tf-Ti)/(Cf-Ci)^*100

T = Treated tumour volume; C = Control tumour volume;

f = Final measurement; i = Initial tumour measurement

Calculation was performed using absolute tumour volume and not relative tumour volume.

As shown in Figure 7 and Table 2, administration of ALM201 inhibited tumour growth by over 60%. Of particular importance are PDX models CTG-0791 and CTG-0992, which were scored as high EMT and were shown to be insensitive to carboplatin treatment. This further demonstrates that ALM201 can inhibit tumour growth in platinum resistant, high EMT phenotype tumours, as well as in platinum sensitive tumours,

Table 2

Example 3

ALM201 treatment down regulates EMT phenotype-associated gene expression

The DU-145 cancer cell line was classified as having a mesenchymal profile and was used in a xenograft study in which tumour-bearing animals were treated with ALM201

(100mg/ml_) or with PBS vehicle (control); ALM201 or PBS was delivered by constant infusion using sub-cutaneous mini-pumps (Alzet). At the end of the study, RNA- Sequencing (RNA-seq) was performed on three tumour samples harvested from each of the control and treated groups. Differential mRNA expression analysis was performed between the control and treated arms of the study followed by a comprehensive gene set enrichment analysis. As shown in Table 3 and Figure 8, treatment with ALM201 resulted in downregulation of genes indicative of an EMT phenotype, such as SNAI2 (SLUG). ALM201 treatment also resulted in downregulation of genes from both the 45 gene signature of Table A (for example, LOXL1 , VCAN, COL1 1AA, FAP, THBS2) and from the 15 gene signature of Table B (e.g. VCAN, FAP, THBS2, COL5A2).

These results show that ALM201 treatment is downregulating genes indicative of an EMT phenotype, suggesting that ALM201 can inhibit epithelial-mesenchymal transition and/or promote mesenchymal-epithelial transition. Table 3

Gene Direction of Change

CDH6 Down

DCN Down

FBN2 Down

VCAN Down

COL6A3 Down

FMOD Down

COL1 1A1 Down

FAP Down

GJA1 Down

SPARC Down

THBS2 Down

SNAI2 Down

LOXL1 Down

VEGFC Down

COL8A2 Down

MATN2 Down

COL12A1 Down

ITGB5 Down

MYL9 Down

CAP2 Down

FERMT2 Down

WIPF1 Down

COL1A1 Down

FBLN1 Down

COL5A2 Down

ITGB1 Down

TPM1 Down

COL6A2 Up

COL7A1 Up

TNFAIP3 Up

GADD45A Up

CXCL1 Up

INHRA Up Table 4: FKBP-L peptides

SEQUENCE SEQ ID NO:

METPPVNTIGEKDTSQPQQEWEKNLRENLDSVIQIRQQPRDPPTETLELEVSPDPASQILEHTQGAEKLV 1

AELEGDSHKSHGSTSQMPEALQASDLWYCPDGSFVKKIVIRGHGLDKPKLGSCCRVLALGFPFGSGPPEG

WTELTMGVGPWREETWGELIEKCLESMCQGEEAELQLPGHSGPPVRLTLASFTQGRDSWELETSEKEALA

REERARGTELFRAGNPEGAARCYGRALRLLLTLPPPGPPERTVLHANLAACQLLLGQPQI-AAQSCDRVLE

REPGHLKALYRRGVAQAALGNLEKATADLKKVLAIDPKNRAAQEELGKWIQGKNQDAGLAQGLRKMFG

METPPVNTIGEKDTSQPQQEWEKNLRENLDSVIQIRQQPRDPPTETLELEVSPDPASQILEHTQGAEKLV 2

AELEGDSHKSHGSTSQMPEALQASDLWYCPDGSFVKKIVIRGHGLDKPKLGSCCRVLALGFPFGSGPPEG

WTELTMGVGPWREETWGELIEKCLESMCQGEEAELQLPGHTGPPVGLTLASFTQGRDSWELETSEKEALA

REERARGTELFRAGNPEGAARCYGRALRLLLTLPPPGPPERTVLHANI-AACQLLLGQPQLAAQSCDRVLE

REPGHLKALYRRGVAQAALGNLEKATADLKKVLAIDPKNRAAQEELGKWIQGKNQDAGLAQGLRKMFG

IRQQPRDPPTETLELEVSPDPAS (referred to herein as ALM201 ) 3

QIRQQPRDPPTETLELEVSPDPAS 4

METPPVNTIGEKDTSQPQQEWEKNLRENLDSVIQIRQQPRDPPTETLELEVSPDPASQILEHTQGAEKLV 5

AELEGDSHKSHGSTSQMPEALQASDLWYCPDGSFVKKIVIRGHGLDKPKLGSCCRVLALGFPFGSGPPEG

WTELTMGVGPWREETWGELIEKCLESMCQGEEAELQLPGHTGPPVGLTLASFTQGRDSW

METPPVNTIGEKDTSQPQQEWEKNLRENLDSVIQIRQQPRDPPTETLELEVSPDPASQILEHTQGAEKLV 6

AELEGDSHKSHGSTSQMPEALQASDLWYCPDGSFVKKIVIRGHGLDKPKLGSCCRVLALGFPFGSGPPEG

WTELTMGVGP

METPPVNTIGEKDTSQPQQEWEKNLRENLDSVIQIRQQPRDPPTETLELEVSPDPASQILEHTQGAEKLV 7 AELEGDSHKSHGSTS

METPPVNTIGEKDTSQPQQEWEKNLRENLDSVIQIRQQPRDPPTETLELEVSPDPAS 8

METPPVNTIGEKDTSQPQQEWEKNLRENLDSVIQIRQQPRDPPTETL 9

QQPRDPPTETLELEVSPD 10

QIRQQPRDPPTETLELEVSPD 1 1

QIRQQPRDPPTETLELEV 12

QIRQQPRDPPTETLE 13

QIRQQPRDPPTE 14

QQPRDPPTETLELEVSPDPAS 15

RDPPTETLELEVSPDPAS 16

PTETLELEVSPDPAS 17

TLELEVSPDPAS 18

RQQPRDPPTETLELEVSPD 19

RQQPRDPPTETLELEVSP 20 RQQPRDPPTETLELEVS 21

PRDPPTETLELEVSPD 22

RDPPTETLELEVSPD 23

Claims

CLAIMS:

1 . FKBP-L polypeptide or a biologically active peptide fragment thereof for use as an inhibitor of epithelial-mesenchymal transition (EMT).

2. FKBP-L polypeptide or a biologically active peptide fragment thereof for use as a promoter of mesenchymal-epithelial transition (MET).

3. FKBP-L polypeptide or a biologically active peptide fragment thereof for use in the treatment or prevention of EMT-associated disease.

4. FKBP-L polypeptide or a biologically active peptide fragment thereof for use in the treatment or prevention of cancer in a human subject, wherein said cancer exhibits an EMT phenotype.

5. FKBP-L polypeptide or a biologically active peptide fragment thereof for use according to claim 4 wherein said EMT phenotype is indicated by expression of one or more mesenchymal marker genes selected from N-cadherin, fibronectin, vimentin, SNAI1 , SNAI2 (SLUG), ZEB1 , Twist and TGF- 3.

6. FKBP-L polypeptide or a biologically active peptide fragment thereof for use according to claim 4 wherein said EMT phenotype is indicated by reduced expression of the epithelial marker gene E-cadherin.

7. FKBP-L polypeptide or a biologically active peptide fragment thereof for use according to claim 4 wherein said EMT phenotype is indicated by expression of one or more, any combination of, or all of the biomarker genes listed in Table A.

8. FKBP-L polypeptide or a biologically active peptide fragment thereof for use according to claim 4 wherein said EMT phenotype is indicated by expression of one or more, any combination of, or all of the biomarker genes listed in Table B.

9. FKBP-L polypeptide or a biologically active peptide fragment thereof for use according to any one of claims 4 to 8, wherein said cancer is ovarian, breast, colon, lung, pancreatic, melanoma, renal, prostate or thyroid.

10. FKBP-L polypeptide or a biologically active peptide fragment thereof for use according to any one of claims 4 to 9, wherein said cancer is platinum-resistant, optionally

carboplatin-resistant or cisplatin-resistant.

1 1. FKBP-L polypeptide or a biologically active peptide fragment thereof for use according to any one of claims 1 to 10, wherein said biologically active peptide fragment comprises the amino acid sequence IRQQPRDPPTETLELEVSPDPAS (SEQ ID NO:3), or a sequence at least 90% identical thereto.

12. FKBP-L polypeptide or a biologically active peptide fragment thereof for use according to any one of claims 1 to 10 wherein said FKBP-L polypeptide comprises the amino acid sequence shown as SEQ ID NO:1 or SEQ ID NO:2, or a sequence at least 90% identical thereto.

13. FKBP-L polypeptide or a biologically active peptide fragment thereof for use according to any one of claims 1 to 10 wherein said biologically active peptide fragment comprises the amino acid sequence shown as any one of SEQ ID NOs 4 to 23, or a sequence at least 90% identical thereto.

14. A method of inhibiting epithelial-mesenchymal transition (EMT) in a mammalian subject, comprising administering to a subject in need thereof a therapeutically effective amount of an FKBP-L polypeptide or a biologically active peptide fragment thereof.

15. A method of promoting mesenchymal-epithelial transition (MET) in a mammalian subject, comprising administering to a subject in need thereof a therapeutically effective amount of an FKBP-L polypeptide or a biologically active peptide fragment thereof.

16. A method of treating or preventing EMT-associated disease in a mammalian subject, comprising administering to a subject in need thereof a therapeutically effective amount of an FKBP-L polypeptide or a biologically active peptide fragment thereof.

17. A method of treating or preventing cancer in a human subject, wherein said cancer exhibits an EMT phenotype, comprising administering to a subject in need thereof a therapeutically effective amount of an FKBP-L polypeptide or a biologically active peptide fragment thereof.

18. A method according to claim 17, wherein said EMT phenotype is indicated by expression of one or more mesenchymal marker genes selected from N-cadherin, fibronectin, vimentin, SNAI1 , SNAI2 (SLUG), ZEB1 , Twist and ΤΘΡ-β3.

19. A method according to claim 17, wherein said EMT phenotype is indicated by reduced expression of the epithelial marker gene E-cadherin.

20. A method according to claim 17, wherein said EMT phenotype is indicated by expression of one or more, any combination of, or all of the biomarker genes listed in Table A.

21. A method according to claim 17, wherein said EMT phenotype is indicated by expression of one or more, any combination of, or all of the biomarker genes listed in Table B.

22. A method according to any one of claims 17 to 21 , wherein said cancer is ovarian, breast, colon, lung, pancreatic, melanoma, renal, prostate or thyroid.

23. A method according to any one of claims 17 to 22, wherein said cancer is platinum- resistant, optionally carboplatin-resistant or cisplatin-resistant.

24. The method of any one of claims 14 to 23 wherein said biologically active peptide fragment comprises the amino acid sequence IRQQPRDPPTETLELEVSPDPAS (SEQ ID NO:3), or a sequence at least 90% identical thereto.

25. The method of any one of claims 14 to 23 wherein said FKBP-L polypeptide comprises the amino acid sequence shown as SEQ ID NO:1 or SEQ ID NO:2, or a sequence at least 90% identical thereto.

26. The method of any one of claims 14 to 23 wherein said biologically active peptide fragment comprises the amino acid sequence shown as any one of SEQ ID NOs 4 to 23, or a sequence at least 90% identical thereto.