WO2019243547A1 - Therapeutically active complexes - Google Patents

Abstract

A biologically active complex having anti-tumour activity, consisting of a peptide of at least amino acids, comprising an alpha-helical structure; and oleic acid or an oleate salt, in a ratio of at least 3 oleic acid or oleate salt molecules per peptide molecule, wherein, when present in the complex, the peptide has an increased conformational fluidity of three dimensional structure as compared to the peptide alone as indicated by an increased peak width on at least some 1H NMR peaks of the complex as compared to the corresponding width of the peaks of a 1H NMR of the peptide alone;provided the peptide is other than alpha-lactalbumin, SAR 1, HOPS/CORVET, SEA (Sehl-associated), Snf7 domain subunits, SEQ ID NO: 10-16, or fragments thereof having 20 or more amino acids. The complexes are biologically active and may be useful in the treatment of cancer.

Description

THERAPEUTICALLY ACTIVE COMPLEXES

TECHNICAL FIELD

The present invention relates to a class of peptides which have therapeutic activity, in particular as anti-cancer or anti-tumour agents. Methods for preparing these peptides, as well as pharmaceutical compositions containing them form a further aspect of the invention.

BACKGROUND

Novel cancer treatments should ideally combine efficacy with selectivity for the targeted tumor and new, targeted therapies act with greater precision. Tissue toxicity and side effects are still the norm, however, and the notion of new, tumor specific mechanisms of cell death is justly regarded with skepticism. Yet, recent investigations into the tumoricidal effects of certain protein-lipid complexes suggest that tumor cells may share conserved mechanisms of cell death that distinguish them from normal, differentiated cells. These protein-lipid complexes insert into lipid bilayers and trigger cell death by perturbing the membrane structure of tumor cells. The subsequent internalization and inhibition of critical cellular functions distinguishes tumor cells from healthy differentiated cells and as a result, the tumor cells are killed while normal, differentiated cells survive.

These properties identify protein-lipid complexes as interesting drug candidates, with broad tumoricidal activity and documented tumor specificity. The feasibility of this approach is illustrated by HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells), which was discovered serendipitously, in a fraction of human milk. HAMLET kills many different tumor cells with rapid kinetics and shows therapeutic efficacy in animal models of colon cancer, glioblastoma and bladder cancer. Investigator-driven clinical trials have demonstrated that HAMLET is active topically, against skin papillomas and induces shedding of dead tumor cells into the urine of patients with bladder cancer.

Alpha-lactalbumin is the most abundant protein in human milk, essential for the survival of lactating mammals, due to its role as a substrate specifier in the lactose synthase complex. HAMLET is formed by partial unfolding of globular alpha-lactalbumin and binding of deprotonated oleic acid, with a stoichiometry of 1/4-8.

A number of peptides derived from alpha-lactalbumin have also been found to have therapeutic effects in their own right (see for example WO2012/069836).

The applicants have previously identified specific alpha-lactalbumin peptide domains as the functional ligands for tumor cell recognition and death. Shared peptide reactivity among tumor cells from different tissues suggests that the alpha-helical peptide is recognized by tumor cell membranes in the context of oleic acid and that this interaction triggers a conserved death response in cancer cells and established cancers, in vivo. Thus, complexes comprising these peptides show broad tumoricidal activity, as exemplified by work done with the known complexes based upon alpha-helical domains of alpha- lactalbumin. However, the applicants have surprising found that these effects can be generalized to other alpha-domain peptides with membrane perturbing activity.

As described in co-pending International Patent Application No. PCT/IB2017/058140 a new, general mechanism by which alpha-helical peptides can target and kill tumor cells has been determined. The applicants found that membrane interactive peptide-domains form oleate complexes with broad tumoricidal activity. This concept is exemplified by the N-terminal alpha helices of alpha-lactalbumin, which invades tumor cells and accumulates in nuclear speckles, where it suppresses transcription through a direct effect on the speckle

constituents SC-35, PKC and Pol II. This "gain of function" was reproduced for Sari in the COPII family, where the alpha-helical, membrane-integrating peptide gained tumoricidal activity, when mixed with oleate. The beta sheet domains of these proteins, in contrast, were sorted to the lysosomes for degradation. Synthetic alphal peptide formed therapeutic oleate complexes that reduced tumor load in a murine bladder cancer model. These findings suggested that tumor cells recognize alpha-helical peptide motifs in the context of oleate and respond by activating a conserved mechanism of tumor cell death.

The peptides were derived from proteins with a membrane perturbing activity. In particular, the peptides were from 20-40 amino acids in length. They include an alpha peptide domain derived from the SARI protein of SEQ ID NO 1.

MAGWDIFGWF RDVLASLGLW NKH (SEQ ID NO 1)

As used herein the expression 'alpha-helical domain' refers to a motif in the secondary structure of the peptide in which a right-hand coiled or spiral conformation (helix) is formed, in which every backbone N-H group donates a hydrogen bond to the backbone C=0 group of the amino acid located three or four residues earlier along the peptide sequence.

The applicants have now found that highly active complexes may be derived from smaller peptide fragments.

SUMMARY OF THE INVENTION

According to the present invention, there is provided a biologically active complex having anti-tumour activity, consisting of a peptide of at least 10 amino acids, comprising an alpha- helical structure; and oleic acid or an oleate salt, in a ratio of at least 3 oleic acid or oleate salt molecules per peptide molecule, wherein, when present in the complex, the peptide has an increased conformational fluidity of three-dimensional structure as compared to the peptide alone as indicated by an increased peak width on at least some *H NMR peaks of the complex as compared to the corresponding width of the peaks of a *H NMR of the peptide alone; provided the peptide is other than alpha-lactalbumin, SAR 1, HOPS/CORVET, SEA (Sehl- associated), Snf7 domain subunits, SEQ ID NOs: 10-16, or fragments thereof having 20 or more amino acids.

The inventors have provided biologically active complexes that surprisingly have the ability to target and kill tumor cells. Specifically, the inventors have identified that certain peptides with diverse primary sequences can form complexes with oleate that, despite their diversity of sequence, actually share certain structural properties which result in antitumor properties. These include having an alpha-helical structure, the ability to bind oleic acid or oleate, and having an increased conformational fluidity of three-dimensional structure upon binding. A marker of increased fluidity of three-dimensional structure, as described in more detail below, is an increased peak width on at least some *H NMR peaks of the complex as compared to the corresponding width of the peaks of a *H NMR of the peptide alone. In one embodiment, the peptide has at least one tryptophan residue and the *H NMR peak of at least one tryptophan indole proton in the complex is increased as compared to the corresponding width of the peak of a *H NMR of the peptide alone. Another indicator of increased fluidity is a reduced chemical shift dispersion of *H NMR peaks in a *H NMR of the complex as compared to the corresponding peaks of a *H NMR of the peptide alone.

In one embodiment, the oleic acid or oleate molecules are in a ratio of at least 4, preferably 5, molecules per molecule of peptide.

The peptide is other than SEQ ID NOs: 10-16, which are set out below.

SEQ ID NO 10 MSVAGLKKQF HKATQKVSEK VGGAEGTKLD DDFKE SEQ ID NO 11 SSLGLWASGL ILVLGFLKLI HLLLRRQT SEQ ID NO 12 SEKKK TRRCNGFKMF LAALSFSYIA KALG SEQ ID NO 13 GTPEYVKFAR QLAGGLQCLMWVAAAICLIA SEQ ID NO 14 VQIPYEVTLW ILLASLAKIG FHLYHRLPG SEQ ID NO 15 SEKKKTRRANGFKMFL AALSF SYIAKALG SEQ ID NO 16 GTPEYVKFARQLAGGLQALMWVAAAIALIA

In one embodiment the peptide is other than Endophilin-Resl-35, Lung-CYP4B1, Liver- SLC01B3, Stomach -ATP4A, Stomach-SLC9A4. In one embodiment, the peptide is other than COPI complex proteins, COPII complex proteins, SEA complex proteins, clathrin adaptor proteins, endophilin proteins, and ESCRT complex proteins, or variants thereof or fragments thereof having 20 or more amino acids.

In one embodiment, the peptide is other than coat complexes and BAR domain proteins, or variants thereof or fragments thereof having 20 or more amino acids. In a particular embodiment, the fragments have 18 or more amino acids. In a further particular embodiment, the fragments have 16 or more amino acids. In a particular embodiment, the fragment is an N-terminal fragment, i.e. comprising the N-terminal amino acid of the parent protein. In one embodiment, the peptide is other than a variant of the recited peptides. In one embodiment, the peptide is other than a variant wherein cysteine residues have been replaced by other residues, such as by alanine residues. In one embodiment, the peptide is other than alpha-lactalbumin and SAR 1 wherein all cysteine residues have been replaced by another residue, such as alanine.

In an embodiment, the increased conformational fluidity of the three-dimensional structure as compared to the peptide alone is further indicated by at least one of: (a) an increased transverse relaxation rate (R₂), as obtained by NMR as described herein, as compared to the corresponding transverse relaxation rate of the peptide alone; (b) a hydrodynamic radius 1.5 to 2.5 times larger than the corresponding hydrodynamic radius of the peptide alone; and/or (c) mean residue ellipticity (MRE), [Q], in deg cm² dmol^-1 as obtained using circular dichroism (CD) as described herein, at 220 nm that is at least 1 deg cm² dmol^-1 lower than the peptide alone. In a particular embodiment, the hydrodynamic radius is 1.6 to 2.0 times larger, preferably about 1.8 times larger, than the corresponding hydrodynamic radius of the peptide alone. In one embodiment, the mean residue ellipticity at 220nm is at least 2,

3, 4, 5, 6, 7, 8, 9 or 10 deg cm² dmol^-1 lower than the peptide alone. The CD is as described herein. In particular, the CD should be carried out in aqueous buffer, preferably 50 mM sodium phosphate buffer, pH 7.4, with 10 % D₂0. Preferably, the final concentration of protein is 0.2 mg/ml.

According to a further aspect, there is provided a biologically active complex comprising a peptide which comprises a truncated form of SEQ ID NO 1 or SEQ ID NO 7 of up to 19 amino acids in length,

MAGWDIFGWF RDVLASLGLW NKH (SEQ ID NO 1)

KQFTKAELSQ LLKDIDGYGG IALPELIATM FHTSGYDTQ (SEQ ID NO 7) and oleic acid or a salt thereof. In one embodiment, this aspect is a preferred embodiment of the first aspect of the invention. In one embodiment, the peptide comprises a truncated form of SEQ ID NO 1. The truncated sequence of SEQ ID NO 1 or SEQ ID NO 7 is up to 19 amino acids in length, for example up to 18 amino acids, or up to 15 amino acids in length. Typically, the peptide will be from 10-19 amino acids in length. Suitably, the peptide is an N-terminal fragment of SEQ ID NO 1.

A particular example of such a peptide is a peptide of SEQ ID NO 2 or SEQ ID NO 3. MAGWDIFGWF RDVLA (SEQ ID NO 2)

KQFTKAELSQ LLKDI (SEQ ID NO 3)

In one embodiment, the peptide comprises a peptide of SEQ ID NO 2.

The applicants have found that complexes may be formed with peptides comprising truncated forms of SEQ ID NO 1 and 7, whereas complexes made using truncated forms of other peptides known to produce active complexes, such as other truncated forms of alpha peptides derived from alphalactalbumin, as described for example in EP-B-2643010 and also elsewhere herein, have reduced activity. The fact that peptides such as SEQ ID NO 2 and SEQ ID NO 3 produce complexes having such activity is therefore unexpected.

The use of shorter peptides may be desirable, both from the point of view of cost and complexity of production, and also from the point of view of reliability and consistency of product.

If desired however, the peptide may comprise small numbers of additional amino acid residues not obtained from SEQ ID NO 1 or SEQ ID NO 7, for example up to 6 amino acids, such as up to 3 amino acids, for example 1 or 2 additional amino acids, may be attached at N and/or C terminal of the peptide, if convenient, for example for expression or purification purposes, as would be understood in the art.

The complexes of the invention further comprise oleic acid or a salt thereof. In particular, the complex further comprises a water soluble oleate salt. Particular examples of suitable salts may include alkali or alkaline earth metal salts. In a particular embodiment, the salt is an alkali metal salt such as a sodium- or potassium salt.

According to a further aspect of the present invention there is provided a method for preparing a biologically active complex as described above. Said method may comprise combining together peptide as defined above; with oleic acid or a salt thereof, under conditions in which they form a biologically active complex.

Typically, the preparation may be carried out simply by mixing together a suitable peptide and oleic acid or a salt thereof, for example in a solution such as an aqueous solution. The ratio of oleate: peptide added to the mixture is suitably in the range of from 20: 1 to 3 : 1, but preferably an excess of oleate is present, for instance in a ratio of oleate: peptide of about 5: 1. The mixing can be carried out at a temperature of from 0-50°C, conveniently at ambient temperature and pressure. This simple preparation method provides a particular advantage for the use of such peptides in the complexes. The methods can be carried out in situ, when required for treatment. Thus kits may be provided comprising peptides and salts for mixing immediately prior to administration.

Such kits, and reagents for use in the kits form a further aspect of the invention. Peptides are suitably synthetic peptides although they may be prepared by recombinant DNA technology.

Peptides useful in forming the complexes of the invention are novel and these form yet a further aspect of the invention.

The complex of the invention can be used in the treatment of cancer. For this purpose, the complex is suitably formulated as a pharmaceutical composition.

Thus, complexes as described above and/or oleate salts also as described above, may be formulated into useful pharmaceutical compositions by combining them with

pharmaceutically acceptable carriers in the conventional manner. Such compositions form a further aspect of the invention.

The compositions in accordance with this aspect of invention are suitably pharmaceutical compositions in a form suitable for topical use, for example as creams, ointments, gels, or aqueous or oily solutions or suspensions. These may include the commonly known carriers, fillers and/or expedients, which are pharmaceutically acceptable.

Topical solutions or creams suitably contain an emulsifying agent for the protein complex together with a diluent or cream base.

The daily dose of the complex varies and is dependent on the patient, the nature of the condition being treated etc. in accordance with normal clinical practice. As a general rule from 2 to 200 mg/dose of the biologically active complex is used for each administration.

In a further aspect of the invention, there is provided a method for treating cancer which comprises administering to a patient in need thereof, a biologically active complex as described above.

In particular, the complex may be used to treat cancers such as human skin papillomas, human bladder cancer, colon cancer, kidney cancer, lung cancer and glioblastomas. In the latter case, administration may be by infusion as is known in the art. In particular, the cancer can be lung cancer, kidney cancer or bladder cancer. The invention further provides the biologically active complex as defined above for use in therapy, in particular in the treatment of cancer.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", mean "including but not limited to", and do not exclude other components, integers or steps. Moreover the singular encompasses the plural unless the context otherwise requires: in particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Within the scope of this application it is expressly intended that the various aspects, embodiments, examples and alternatives set out in the preceding paragraphs, in the claims and/or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and/or features of any embodiment can be combined in any way and/or combination, unless such features are incompatible.

BRIEF DESCRIPTION OF THE DRAWINGS

One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

Figure 1 is a schematic diagram mapping the truncated peptides tested with the longer active peptides from which they are derived;

Figure 2 shows the results obtained in a cell death assay using complexes obtained using the peptides of Figure 1, where (A) shows the results of an ATPlite assay and (B) shows the results of a Prestoblue assay:

Figure 3 is a graph showing the average of 3 repeats of the cell death assay carried out on the complexes of the invention and complexes comprising the alpha-1 peptide of alphalactalbumin;

Figure 4 shows the results complexes of the invention on membrane perturbation using a giant unilamellar vesicle model.

Figure 5 shows the structure of peptide-oleate complexes and therapeutic efficacy in a murine bladder cancer model : (a) Ribbon representation of the crystallographically determined three-dimensional structure of human a-lactalbumin (PDB id : 1B90), indicating the alphal (blue), beta (green) and alpha2 (gray) domains. The calcium ion is not shown (b) Far-UV circular dichroism spectra of synthetic alphal peptide, beta peptide, and their respective oleate complexes. (c,f) Rapid death of human lung (A549), kidney (A498) and murine bladder carcinoma cells (MB49) treated with the alphal-oleate or sarlalpha-oleate complexes (3 h, 35 pM), quantified as a reduction in ATP levels or PrestoBlue fluorescence (% of control). Means ± S.E.Ms. (n=3, **P<0.01). (d) Structural models of sarlalpha and sarlbeta peptides generated by I-TASSER. (e) Far-UV circular dichroism spectra of synthetic sarlalpha, sarlbeta peptide, and their respective oleate complexes. (g,h) Alphal- oleate and sarlalpha-oleate triggers rapid membrane blebbing in lung carcinoma cells (h) and giant unilamellar vesicles (GUVs) (h). (i) Colocalization of AlexaFluor488-labeled alphal-oleate or sarlalpha-oleate (green) and the GUV membrane (red), (j-k) K⁺ efflux and (j) tumoricidal effects (k) in lung carcinoma cells are inhibited by Amiloride or BaCL· (10 pM).

Figure 6 shows NMR spectra and free-energy surface analyses of naked alphal- and sarlalpha peptides and their oleate complexes: (a,b) One-dimensional *H NMR spectra. The naked alphal- (a, black) and sarlalpha- (b, black) peptides assume an ensemble of structures that interconvert rapidly and are therefore seen as sharp peaks. The alphal- oleate (a, red) and sarlalpha-oleate complexes (b, red) show broader peaks. Arrows indicate the indole *H signals arising from the three Trp side chains present in the sarlalpha peptide, (c-d) Two-dimensional *H NOESY spectra of alphal-oleate and sarlalpha-oleate complexes, showing atomic-level proximities of the fatty acid to the respective peptide. The spectra highlight NOEs between the 9,10 olefinic protons (5.23 ppm) of oleic acid with the Ha protons and aromatic protons of the (C) alphal-oleate complex and the (d) sarlalpha- oleate complex, (e-h) Free-energy surfaces as a function of the first two principal components for (e) alphal-oleate, (f) naked alphal, (g) sarlalpha-oleate and (h) naked sarlalpha. PCI and PC2 represent the axes of the two greatest variances after

mathematical transformation for dimension reduction. The representative structures of peptides or peptide-oleate complexes, along with their respective local minima annotations, are colored from the N termini (blue) to the C termini (red). The free-energy surface of the alphal-oleate complex contains 2 minima basins, A1 and Bl, with A1 representing the major conformational ensemble. The free-energy surface of the sarlalpha-oleate complex contains 3 minima basins, A3, B3, and C3 (with the A3 basin harboring the major structural ensemble), and is characterized by a prominent alpha-helical secondary structural element, as shown from simulation calculated alpha-helical propensities. By contrast, the free-energy surface of the naked sarlalpha shows large structural heterogeneity. Here, minima basins A4 and D4 are represented by helical structures, B4 by beta structure, and C4 and E4 by random coil structures.

Figure 7 shows inhibition of gene expression : (a) Whole genome transcriptomic analysis of lung carcinoma cell treated with alphal-oleate (35 mM, 1 hour). FC > 1.41 compared to untreated cells. Red = upregulated, Blue=down-regulated genes, (b) Inhibition of gene expression relative to beta-oleate; heat map of differentially regulated genes, (c-d) Inhibition of histone H3-centric- (c) and proteasome-centric (d) gene networks by alphal- oleate. (e) Strong co-localization in nuclear speckles (yellow) of alphal-oleate (red) with histone H3 (green), (f) Strong co-localization (yellow) of alphal-oleate (red) with 20S proteasomes (green) in nuclear speckles. Beta-oleate served as negative control. Scale Bar = 10 pm. Nuclear counterstaining with DRAQ-5. Scale Bar = 10 pm.

Figure 8 shows peptide-specific interactions with different subcellular

compartments: (a,c) Internalization of alphal-oleate or sarlalpha-oleate and accumulation in a ring-like structure in the nuclear periphery (red) and (b,d) co-localization with the nuclear speckle marker, SC35 (green), (a-d) Internalization of beta-oleate and sarlbeta- oleate into cytoplasm Scale Bars = 10 pm. (e) Model depicting the interactions of SC-35 with nuclear speckle constituents Histone H3, PKC and RNA Pol II³⁵. (f) Alpha-lactalbumin- oleate bind to PKC isoforms in a proteomic screen and inhibit PKC phosphorylation (5/6 PKC isoforms, 1 hour, 21 pM). (g) Loss of nuclear- and cytoplasmic p-PKCP staining after alphal-oleate treatment, (h) Inhibition of p-PKCP activity by alpha-oleate and alpha- lactalbumin-oleate. (i) Inhibition of SC-35 phosphorylation by alphal-oleate and sarlalpha- oleate. Western blot analysis of whole cell extracts, (j) Inhibition of SC-35- and RNA Pol II phosphorylation (n=50 cells), (k) Alphal-oleate or sarlalpha-oleate disrupt the co- localization between phosphorylated SC-35 and RNA Pol II. Scale bar = 5 pm. (I) Model predicting disruption of nuclear speckles by alphal-oleate and sarl-oleate-interactions with SC-35, Histone H3, PKC and RNA Pol II.

Figure 9 shows therapeutic efficacies of alphal-oleate and sarlalpha-oleate in bladder cancer mice model : (a) Bladder cancer was established by intravesical instillation of MB49 cells and tumor development was quantified at sacrifice, on day 13. Mice were treated with alphal-oleate or sarl-oleate and sham-treated mice received PBS. All evaluations were investigator-blinded for treated or sham-treated mice across biological replicates, (b)

Macroscopic appearance of bladders, (c) Tumor area in whole bladder mounts; stained with H&E. Demarcation to healthy tissue is indicated (— ). Scale bar = 200 pm. (d) Therapeutic efficacy of alphal- or sarlalpha-oleate complexes, compared to PBS. Means ± S.E.Ms, two experiments. *P<0.05 and **P<0.01. (e) Reduction in proliferation markers Cyclin Dl,

Ki67, VEGF and COX-2 in tumor-bearing mice treated with alphal-oleate or sarlalpha- oleate complexes. DRAQ5 is the nuclear marker (Scale Bar, 100 pm), (f) Retention of VivoTag 680 labeled alphal-oleate in bladders of tumor bearing mice (n=5), compared to tumor-free control mice (n=3). Two mice per group are shown, (g) Tumor-specific uptake (upper panel) of alphal-oleate (arrowhead), 24 hours after intravesical instillation. Tissues were stained with specific anti-alphal-antibodies. Healthy tissues (lower panel) show negligible uptake. Scale bar = 100 pm.

Figure 10 shows *H and NOESY NMR data relating to sarlalphal, sarlalpha2, alphal and oleate complexes thereof. DETAILED DESCRIPTION

The inventors have made the unexpected observation that two, conformationally fluid alpha-helical peptides without sequence homology form oleic acid complexes with shared functional and structural characteristics. A strong agreement between NMR spectroscopy,

CD spectrometry and computational simulations predicted a strikingly malleable ensemble of structures. The ensemble of structures, which cannot be found in the folded native proteins, suggest that an "adapt-and-adopt" fluidity may be one of the key reasons why these complexes effectively kill a variety of tumor cell lines and cancer tissues. This offers a molecular context to the hypothesis that a certain polypeptide chains can perform vastly different biological functions, depending on its folding states and the availability of suitable tissue cofactors.

Molten globules retain secondary structural elements but lack tight packing of the interior, resulting in a loss of overall tertiary structure. The polypeptide backbone chain and the side chains of such proteins are in conformational exchange, resulting in broad peaks and poor chemical shift dispersion, as observed by biomolecular NMR spectroscopy. The high- resolution NMR and molecular modeling studies reveal similarities of the alphal-oleate complexes with the HAMLET (Human alpha-lactalbumin made lethal to tumor cells) family of complexes, suggesting that HAMLET acquires tumoricidal activity by exposure of the N- terminal, alpha-helical domains. The conclusions are supported by extensive structural studies of alpha-lactalbumin, where stable folding intermediates expose alpha-helical domains. Thus, in addition to supporting the conformational fluidity of the alpha-helical domains and defining the three-dimensional surface, the lipid cofactor endows a novel function and defines a distinct category of molecules.

Cell death is initiated at the plasma membrane where the peptide oleate complexes show direct, membrane perturbing effects and trigger rapid ion fluxes. Here we show that the complexes engage nuclear speckles and inhibit the transcription of key survival genes.

Spliceasomes are important sub-nuclear compartments, which work in concert to coordinate gene expression, including transcription, pre-mRNA processing and mRNA. Transcriptionally active genes localize to the speckles, where a continuous and rapid molecular exchange takes place with the surrounding nucleoplasm. We propose that alphal-oleate and sarlalpha-oleate disturb this stochastic self-organization, by establishing high affinity complexes with histone H3, damaging the architecture of the transcriptional machinery and preventing the dissociation of bound components. These effects were further supported by a direct effect on PKC-dependent phosphorylation of SC-35, which in turn, inhibits Pol II activation. The resulting inhibition of gene expression, which involved Ras, histone H3 and proteasome-centric gene networks, may mark a ''point of no return" for the dying tumor cell, as de novo synthesis of critical cellular constituents is severely impaired. Transitional cell carcinomas are common urological malignancies and the most costly cancers, due to a high recurrence rate and few curative therapies. Tumors confined to the mucosa are often treated by transuretheral resection, followed by intravesical instillation of Bacille-Calmette-Guerin (BCG) bacteria or cytostatic drugs. While these treatments may result in prolonged tumor free periods, there is an urgent need for less toxic and more specific therapies. The murine MB49 bladder cancer cells were used here to establish rapidly growing, highly reproducible tumors for therapeutic studies and tumor growth was effectively inhibited by intra-vesical instillation of the respective complex. The peptide- oleate complexes also showed selectivity for tumor tissue, shown by increased retention in the bladders in tumor bearing mice and selective uptake of the complexes in the tumor area, with no evidence of side effects. These therapeutically relevant and selective complexes are therefore immediately encouraging for patients with urological malignancies.

Example 1

Peptide synthesis

The peptides to individual domain of alpha lactalbumin were commercially synthesized using the mild Fmoc chemistry method (Mimotopes, Melbourne, Australia). For biotinylated peptides, an aminohexanoic acid (Ahx) spacer was added to ensure adequate separation between the biotin and the peptide moiety. The sequences for the peptides are set out in the following table 1

Table 1

Of these, peptides of SEQ ID No 3-5 represent fragments of the known active alpha peptide of alphalactalbumin, which is comprised in SEQ ID NO 7. Similarly, peptides of SEQ ID no 2 and 6 were fragments of the known active peptide of SEQ ID NO 1. This is illustrated schematically in Figure 1. Complex Preparation

5mg of sodium oleate was dissolved in 1 ml of PBS to give a 16 mM clear solution, which was mixed with peptides in a ratio of oleate: peptide of 5: 1.

Cellular assays

Human lung carcinoma cells (A549, ATCC) were cultured in RPMI-1640 with non-essential amino acids (1 : 100), 1 mM sodium pyruvate, 50 pg/ml Gentamicin and 5-10% fetal calf serum (FCS) at 37 °C, 5 % C0₂. For cell death experiment, cells were grown on 96-well plate (2xl0⁴/well, Tecan Group Ltd) overnight. Cells were incubated with peptide-oleate complexes in serum-free RPMI-1640 at 37 °C. FCS was added after 1 hour. After 3, 7 and 12 hours treatment cell death was quantified by two biochemical methods: Cell viability was quantified by Presto Blue fluorescence (Invitrogen, A13262) and cellular ATP levels using luminescence based ATPlite™ kit (Perkin Elmer). Fluorescence and luminescence was measured using a microplate reader (Infinite F200, Tecan).

The results are illustrated in Figure 2. It is clear from this result, that the peptide of SEQ ID NO 2 produced efficiacy similar to that obtainable using the favoured alpha-peptide of alpha-lactalbumin, in spite of the fact that it is considerably shorter. This result was confirmed when the average of 3 trials was compared as shown in Figure 3.

The complex of the invention was shown to produce membrane perturbation using a giant unilamellar vesicle model.

Coverslips were cleaned with 1M NaOH and plasma etched for 1 min using BD-20 laboratory corona treater (Electro Technic Products Inc., USA) to render the surface clean and hydrophilic. A thin film of 1% solution of molten agarose was made on the coverslip to provide a safe reaction bed for the giant unilamellar vesicles (GUVs) to form. 10 mM solution of Phosphatidyl Choline and O. lmM l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-(cap biotinyl) (DPPE-biotin) in chloroform was labelled using 1 to 25 concentrations of lmg/ml of Rhodamine. GUVs from this solution were formed onto the coverslips using a syringe through an air-blow dispersion method. Syke-Moore chambers were sonicated in 1M Sodium Hydroxide and rinsed with Milli-Q water. The coverslips were locked in the chambers and the GUVs were mobilized and rehydrated using 200 mM of Sucrose solution for 25 minutes. Further, the GUVs were collected and allowed to settle in 200 mM of glucose solution for 60 minutes. Alexa-633 labelled and unlabeled peptides- oleate complex in the ratio of 1 :4 was added to the solution in the observation chambers and monitored chronologically for at most 90 minutes. The GUVs were washed with PBS and monitored again to visualize co-localization. The results are shown in Figure 4. Example 2 - Peptide-specific tumor cell death

Fig. 5a is a graphical representation of human alpha-lactalbumin, of which were synthesized peptides corresponding to the alphal domain (residues 1-39, alphal), or the beta sheet (40-80, beta). The peptides formed complexes with oleate, with an approximate

stoichiometry of about five oleate residues per peptide. Alpha-helical secondary structure was confirmed by circular dichroism spectroscopy of alphal peptide and was enhanced by binding to oleate (alphal-oleate), (Fig. 5b). The beta-peptide structure remained largely unchanged when mixed with oleate (beta-oleate), as shown by the near overlapping spectra.

By forming oleate complexes, the alphal peptide gained tumoricidal activity for human lung- and kidney- carcinoma cells (A549, A498) and murine bladder cancer cells (MB49). ATP concentrations and Prestoblue fluorescence was reduced by about 80%, after 3 hours at a concentration of 35 mM (Fig. 5c). The beta-oleate complex, in contrast, did not significantly affect tumor cell viability despite equimolar oleate concentrations. The results identify the N-terminal, alpha-helical peptide domain of human alpha-lactalbumin as a tumoricidal entity, which achieves its effect by forming complexes with oleic acid.

Example 3 - The N-terminal sari alpha-helical peptide-oleate reproduces the effects of alphal-oleate

We obtained synthetic sari peptides comprising the N-terminal amphipathic alpha helix (residue 1-23, sarlalpha) or a beta-sheet (a. a. 46-78, sarlbeta) (Fig. 5d). The sarlalpha peptide gained tumoricidal activity when mixed with oleate at a 1 : 5 molar ratio (sarlalpha- oleate). Structurally, the alpha-helical content of the newly formed sarlalpha-oleate complexes was higher than that of the naked peptide. The sarlbeta-oleate complex was largely inactive and structure remained unchanged (Fig. 5, e,f and table 1).

Example 4 - Membrane perturbations determine the fate of tumor cells

Effects of the complexes on lipid bilayers were documented by real-time imaging, using protein-free, giant unilamellar vesicles (GUVs). The alphal-oleate complex showed extensive co-localization with GUV lipid membrane constituents and induced membrane bleb formation, suggesting direct, protein -receptor independent membrane interactions (Fig. 5, g to i). The membrane interactions of sarlalpha-oleate resembled those of alphal-oleate, with extensive bleb formation in tumor cells and GUV membranes. Major structural alterations were also observed in cancer cell membranes, including the formation of membrane blebs, with rapid kinetics and extensive bleb formation (Fig. 5g).

The membrane effects of alphal-oleate and sarlalpha-oleate were accompanied by rapid ion fluxes in tumor cells (Fig. 5j). Furthermore, inhibition of ion fluxes rescued the tumor cells from death, as shown using the Na+/H+ exchange inhibitor amiloride and the K+ efflux inhibitor BaCI2 (Fig. 5, j and k). These findings emphasize the intimate connection between membrane perturbations, ion fluxes and cell death (Fig. 5, g to k). The results show that N-terminal, alpha-helical peptide domains of alpha-lactalbumin and Sari form tumoricidal complexes, which act by perturbing lipid bilayers and triggering ion fluxes.

Example 5 - Biomolecular NMR analysis of the peptide-oleate complexes

To understand the structural basis behind the shared activity we performed biomolecular NMR analyses of the alphal- and sarlalpha- peptides and peptide-oleate complexes. 1H NMR spectra detected a shift from sharp signals for the naked peptides to broad signals and poor chemical shift dispersion for the oleate complexes (Fig 6, a and b), suggesting conformational exchange changing from a random-coil fast-exchange time regime to an intermediate millisecond time scale. An example was observable in the indole 1H region of naked sarlalpha (one sharp signal) compared to sarlalpha-oleate (two broad signals) for the three Trp side chains. Such broadening in the amide, side-chain methyl and aromatic regions suggests that interactions between fatty acids and peptides occur throughout the molecules. 2D NOESY spectra identified non-covalent, relatively short through-space interactions between the respective peptides and fatty acids. Important NOEs were detected between the olefinic protons (5.23 ppm) of oleic acid and the Ha and aromatic protons of alphal and also between the sarlalpha aromatic region and the oleic acid olefinic protons (Fig. 6, c and d). The downfield chemical shift of amide protons observed between 7.6 and 8.8 ppm suggests the presence of secondary structure in alphal and the alphal-oleate. Well-resolved signals obtained from the one-dimensional 1H NMR spectra provided a stoichiometry of 3.7 oleate molecules per alphal peptide. Chemical shift mapping revealed a cluster of residues with aliphatic side chains that change upon the binding of oleate, providing further evidence of interactions between peptides and fatty acids.

Hydrodynamic volume measurements carried out with SE-HPLC and diffusion-ordered NMR spectroscopy (DOSY) showed that while the hydrodynamic radius (RH) of the alphal peptide agreed well with the estimate of a small peptide of 39 residues (16.1 A from SE- HPLC and 14.4 A from DOSY), the alphal-oleate complex RH was considerably larger (27.4 A and 29.3 A, respectively). For comparison, human serum albumin (which tightly binds five or six oleic acid molecules; RH = 40 A and 40.9 A respectively) and oleate in aqueous solution (well above its critical micelle concentration of 6 mM; RH = 104.3 A from DOSY) displayed larger RH values. The fact that in the DOSY spectrum of the complex, the oleate molecules behaved in unison with the alphal peptide signals provided evidence that in terms of translational diffusion, the fatty acids were bound and were not a separate component in solution. The conformational exchanging properties of the alphal-oleate complex were further probed by determining the transverse relaxation rate R2 for the well-defined signals (the broadness of the spectrum precluded chemical shift assignments). Whereas the T2 relaxation curves of a wide sampling of the aliphatic and aromatic signals in the alphal peptide spectrum displayed typical rates found for a random-coil peptide, the signals of the alpha-oleate complex (aliphatic, aromatic, and bound oleate) exhibited a precipitous exponential decay despite the complex being smaller in hydrodynamic size than native, well-folded HSA. These large R2 values distinct from those of alphal peptide and HSA suggested that along with lower frequency (nanosecond) motions, contributions from slower millisecond to

microsecond exchange processes were present and likely responsible for the line broadening in the NMR spectrum (12). Importantly, the R2 values for the complex were also different from those found in oleate in aqueous solution, again suggesting that the dynamics of the complex was clearly different from micelle/vesicle-like particles formed from oleic acid/oleate with no peptide/ protein component.

Example 6 - Free energy surface analyses of the peptide- and peptide-oleate system

Computational simulations also pointed toward structural heterogeneity, showing that the naked peptides and peptide-oleate complexes belonged to wide conformational spaces with relatively deep basins (Fig. 6, e to h). Representative structures mapped to different free- energy surface minima revealed prominent alpha-helical secondary structural elements and a hydrophobic oleate core for the peptide-oleate complexes. It can be seen that the peptide-oleates fold upon this core differently than the naked peptides, which exhibit multiple local minima (Minima A2, B2, C2, and D2 for alphal; A4, B4, C4, D4 and E4 for sarlalpha) (Fig. 6, e to h). Naked alphal ensembles were characterized by various partially folded helix-turn conformations, whereas naked sarlalpha ensembles exhibited a mixture of random coil, helical and beta structures. A contact probability analysis revealed that the interactions between alphal or sarlalpha and oleate were mainly hydrophobic, with a >0.9 contact probability with olefinic protons. Strikingly, the peptide-oleate complexes displayed relatively wide and deep free-energy minima basins, suggesting that a multitude of conformations would be equally possible to visit (Fig. 6, e and g). When combined with the ?2 relaxation rates, the possibility of multiple sampling of various conformations within a short period of time provides an argument that rather than targeting specific partners, these alpha-helical complexes may potentially be interacting with multiple putative binding partners available on the cancer cell surfaces.

Based on these extensive investigations and the strong agreement of the experimental aspects with the simulated predicted ensembles, it was clear that completely non- homologous peptides form complexes with shared structural characteristics when bound to oleic acid. Example 7 - Sequence requirement for active peptide-oleate complex formation

To further characterize the alphal- and sarl-oleate complexes, we therefore synthesized truncated forms of these peptides (alphalpl : K1-I15, alphalp2: L11-E25 and alphalplp2: K1-E25, sarlalphal : M1-A15 and sarlalpha2: W9-H23) (Fig 1). The peptides were mixed with oleate at a 1 : 5 molar ratio and tested for effects on A549 lung carcinoma cells, using the ATP and Prestoblue assays. Sarlalphal-oleate retained about 80 % of the tumoricidal activity of Sarlalpha-oleate with similar dose-response characteristics (Figs 2 and 3). The alphalpl-oleate complex retained 45 % of the activity of alphal-oleate in the Prestoblue assay but the effect on ATP was less pronounced. The alphalp2 and alphalplp2 peptides were inactive, as was the sarlalpha2 peptide.

*1-1 NMR spectra detected a shift from sharp signals for the naked sarlalphal to broad signals and poor chemical shift dispersion for the sarlalphal-oleate, consistent with the change in conformational exchange time-scale, which suggests peptide-oleate complex formation (Fig. 10A-C). As with the fully active full-length peptide-oleate, such broadening in different regions (amide, side-chain methyl and aromatic) suggests that interactions with oleate occur throughout sarlalphal. 2D NOESY spectra of sarlalphal-oleate suggested close connectivity between the aromatic side chains and the aliphatic fatty acyl chain (Fig. 10D). In contrast, such characteristic conformational exchange behavior was not detected for alphalpl-oleate, where the *1-1 NMR spectrum was sharp and 2D NOESY spectra relatively well resolved (not shown).

These results suggest that the biological effect is determined by the three-dimensional fold of the peptide-oleate complex and that the absence of such structural characteristics undermines oleate binding and the functional conversion process. Active sarlalphal-oleate shared structural characteristics with the full-length sarlalpha-oleate and alphal-oleate. Truncating the N-terminal of sarlalpha (=sarlalpha2) abolished the bioactivity, even though oleate binding is detected, consistent with structural analysis of the sarlalpha- oleate. Similarly, alphalpl that showed reduced bioactivity did not to bind oleate efficiently or exhibit conformational fluidity.

The experiments reveal why and how non-homologous peptide sequences can allow cognate oleate binding and bioactivity. We reason that cognate oleate binding and presentation as well as the bioactivity require a contiguous stretch of amino acid sequence, rather than specific placements of individual amino acid residues between "inactive" flanking sequences.

This conclusion is further supported by previous studies, where mutation and chemical modification strategies were used to probe how specific amino acid residues influence alpha- lactalbumin-oleic acid complex formation and tumoricidal activity (Proteins 2013; 81 : 1-17, J Mol. Biol. 2009; 394:994-1010). Activity was abolished after chemical modification of positively charged lysine residues with negatively charged citraconyl- or neutral acetyl groups (Proteins 2013; 81 : 1-17). In contrast, a cys-ala alpha-lactalbumin mutant created by single-residue mutagenesis retained full activity, despite the change in tertiary structure definition that results from a loss of disulphide bond formation (J Mol Biol 2009; 394:994- 1010). Finally, a trial with covalent conjugations of three oleic acids to lysine residues within the alphal peptide yielded inactive complexes, supporting the notion that oleate binding is dependent on the overall three-dimensional structure rather than specific binding (data not shown).

Example 8 - Effects of alpha-helical peptide-oleate complexes on gene expression

The programmed cellular response that leads to tumor cell death was further defined by genome-wide transcriptomic profiling. After exposure of lung carcinoma cells to the peptide- oleate complexes, we detected a marked reduction in gene expression, resulting in a pronounced inhibitory effect on chromatin remodeling and histone-related functions, as well as Pol II-dependent gene expression (Fig. 7, a to d). In addition, a proteasome-centric network was inhibited and suppression of proteasome- and ubiquitin-mediated proteolysis was supported by gene-set enrichment analysis.

Example 9 - Targeting of spliceasomes in tumor cell nuclei

The notion of a common mechanism of action for the two unrelated alpha-helical peptide- oleate complexes was further supported, by defining nuclear targets of the alphal-oleate and sarl-oleate complexes. We therefore exposed human lung carcinoma cells (A549) to Alexa-labeled peptides or peptide-oleate complexes and monitored nuclear translocation by live cell imaging. Alphal-oleate and sarlalpha-oleate complexes were rapidly detected in the nuclei (Fig. 8a) but the naked alphal or sarlalpha peptides were not, despite binding to the plasma membrane and formation of local membrane aggregates. The naked beta or sarlbeta peptide were internalized by tumor cells and sorted to the lysosomal compartment but did not reach tumor cell nuclei (Fig. 8a).

The alphal-oleate complex accumulated in a ring-like structure in the nuclear periphery, which was identified as nuclear speckles by staining for SC35 (Fig. 8, b and d). Nuclear speckles reside in the inter-chromatin space of eukaryotic nuclei and serve as important nodes in the splicing of pre-mRNA and transport of spliced RNA. Based on imaging data and in vitro kinase assays, it has been proposed that protein kinase C (PKC) phosphorylates SC35, which relocates to sites of active transcription, where it interacts with RNA

polymerase II (RNA Pol II). We therefore examined if the alpha-oleate complexes might affect the nuclear speckle environment by interacting directly with SC35, as well as the speckle constituents PKC, and RNA Pol II (Fig. 8e). To identify additional interaction partners, we performed a proteomic screen of 8000 human proteins. Several PKC isoforms were identified as alpha-lactalbumin targets and PKC phosphorylation was reduced, as shown using an antibody array specific for phosphorylated kinases and substrates (Fig. 8f). The inhibition of PKC was confirmed by Western blot analysis of phosphorylated PKCP for both alpha-lactalbumin- and alphal-oleate complexes (Fig. 8, g and h). SC35 phosphorylation was inhibited by both alphal-oleate and

sarlalpha-oleate, as shown by Western blot analysis (Fig. 8, i to j). Furthermore, alphal- oleate and sarlalpha-oleate reduced Pol II Serine 2 phosphorylation (Fig. 8, j to k).

Histones are exposed in transcriptionally active chromatin and 20S proteasome recruitment is important for spliceosome function. We observed by confocal imaging that alphal-oleate triggers a redistribution of histone H3 and 20S proteasomes to the nuclear speckles, where strong co-localization with the peptides was detected (Fig. 7, e to f). These observations are consistent with previous studies, showing that HAMLET forms high-affinity complexes with histone H3 and catalytic subunits of 20S proteasomes.

The results suggest that alphal complexes interfere with spliceosome assembly and inhibit transcription by directly affecting SC35, RNA Pol II and upstream regulators such as PKC (Fig. 8i). Genes involved in Ras and kinase signaling, RNA transport and cellular stress, were also inhibited, linking the effect on transcription to ion fluxes and to the membrane effect of the complex. The resulting inhibition of gene expression, which affected H3 and proteasome-centric gene networks, may mark a "point of no return" for the dying tumor cell, as de novo synthesis of critical cellular constituents is severely impaired.

Example 10 - Therapeutic efficacy in a bladder cancer model

In the screen of cancer cells from different tissues (Fig. 5), we observed that the murine MB49 bladder cancer cell line was highly sensitive to the alphal-oleate and sarlalpha-oleate complexes (8). Bladder cancer was therefore established in C57BL/6 mice, by instillation of MB49 cells on day 0, after preconditioning of the bladders with poly-L-lysine for 20 minutes. In the treatment group, mice received five intra-vesical instillations of alphal-oleate or PBS on days 3, 5, 7, 9 and 11 (Fig. 9a). Bladders were harvested on day 13 and macroscopic evaluation and tissue imaging were investigator-blinded for treated or sham-treated mice across biological replicates.

The sham-treated mice developed palpable tumors that altered the macroscopic appearance of the bladders, compared to controls not receiving tumor cells (Fig. 9b). The tumors were growing invasively, from the mucosa and the tumor mass gradually filled the bladder lumen, replacing functional bladder tissue. The tumors showed increased nuclear density and a loss of tissue structure definition, including mucosal folds (H&E staining of whole bladder mounts, Fig. 9c).

Tumor growth was markedly attenuated after treatment with alpha 1-oleate or sarlalpha- oleate and bladder tissue organization remained more intact (**P<0.01, compared to sham- treated mice in Fig. 9d). Alphal-oleate and sarlalpha-oleate also reduced the expression of the tumor proliferation markers Cyclin Dl, Ki67, VEGF and COX-2 (Fig. 9e). Beta-oleate, in contrast, did not significantly affect tumor progression, as shown by tumor size and tissue structure definition (H&E staining of whole tissue mounts).

Alphal-oleate was shown to accumulate in tumor tissue, after intra-vesical instillation of fluorescently labeled alphal-oleate in mice with palpable tumors. Fluorescence was monitored by IVIS technology and compared to controls without tumor (day 8, Fig. 9f). The complex was retained for at least 24 hours in the tumor-bearing mice, as shown by a strong fluorescence signal. Healthy mice, in contrast, rapidly excreted the fluorescently labeled complex, with no evidence of tissue accumulation. This tumor-specific accumulation of alphal-oleate was confirmed by immuno-histochemistry, using alpha-l-specific, polyclonal antibodies to stain frozen tissue sections, obtained 24 hours after instillation of alphal- oleate (Fig. 9g). A similar affinity for tumor tissue was documented for the HAMLET complex. The results identify alphal-oleate and sarlalpha-oleate as potent tumoricidal complexes, with therapeutic efficacy.

Materials and Methods

Chemicals and antibodies

Sodium oleate (Sigma, Cat# 07501), AlexaFluor568 protein labeling kit (Thermo Scientific, Cat# A10238), AlexaFluor488 protein labeling kit (Thermo Scientific, Cat# A10235), Lysotracker (Thermo Scientific, Cat# L7526), ECL Plus detection reagent (GE healthcare, Cat# RPN2132), Richard-Allan Scientific Signature Series Hematoxylin and Eosin-Y (Thermo Scientific, Cat# 7211 and 7111), DAPI (Sigma, Cat# D9542), VivoTag 680XL (Perkin Elmer, Cat# 13625), ATPlite (Perkin Elmer, Cat# 6016947), Presto Blue Cell Viability Assay

(Invitrogen, Cat# A13262), Anti-peptide antibodies (GeneCust), Anti-GAPDH antibody (Santa Cruz, Cat# sc-25778), Anti-histone H3 antibody (Abeam, Cat# Abl791), HRP- conjugated anti-mouse antibody (Cell Signaling), Polyclonal rabbit anti-mouse IgG-HRP (Dako, Cat# P0260), Polyclonal goat anti-rabbit IgG-HRP (Cell Signaling, Cat# 7074),

Mouse monoclonal phospho anti-SC-35 (Abeam, Abll826), Rabbit monoclonal anti-SC-35 (Abeam, Cat# Ab204916), Rabbit polyclonal anti-20S proteasome (Enzo Life Sciences, Cat# PW8155), Rabbit polyclonal anti-VEGF (Abeam, Cat# Ab46154), Mouse monoclonal anti-Ki- 67 (BD Biosciences, Cat# 556003), Rabbit monoclonal anti-cyclin Dl (Thermo Fisher, Cat# sc8396), Mouse monoclonal anti-PKC (Santa Cruz Biotechnology, Cat# sc-17769), Mouse monoclonal anti-PKC (Santa Cruz Biotechnology Cat# sc-365463), Goat anti-rabbit IgG Alexa488 (Thermo Fisher, Cat# a-11034), Goat anti-rabbit IgG Alexa568 (Thermo Fisher, Cat# a-11077), DRAQ5 (Abeam, Cat# abl08410).

Peptide synthesis and complex generation

All peptides were synthesized using Fmoc solid phase chemistry (Mimotopes, Melbourne, Australia). For biotinylated peptides, an aminohexanoic acid (Ahx) spacer was added to ensure adequate separation between the biotin and the peptide moieties. A five-fold stoichiometric concentration of sodium oleate in phosphate-buffered saline was prepared, followed by the addition of each respective peptide. The more-hydrophobic peptides were initially dissolved in DMSO, then transferred to the oleate buffer. The sequences for the peptides are as follows:

Alphal : Ac-KQFTKAELSQLLKDIDGYGGIALPELIATMFHTSGYDTQ-OH

Beta: Ac-IVENNESTEYGLFQISNKLWAKSSQVPQSRNIADISADKFLDDD-OH

Sarlalpha: Ac-MAGWDIFGWFRDVLASLGLWNKH-OH

Sarlbeta: Ac-DRLATLQPTWHPTSEELAIGNIKFTTFDLGGHI-OH

Alphalpl : Ac-KQFTKAELSQLLKDI-OH

Alphalp2: Ac-LLKDIDGYGGIALPE-OH

Alphalplp2: Ac-KQFTKAELSQLLKDIDGYGGIALPE-OH

Sarlalphal : Ac-MAGWDIFGWFRDVLA-OH

Sarlalpha2: Ac-WFRDVLASLGLWNKH-OH

Circular dichroism spectroscopy

Far-ultraviolet (UV) circular dichroism (CD) spectra were collected on alphal-, beta-, sarlalpha- and sarlbeta- peptides with and without oleate at 25 °C using a Jasco 815 CD Spectropolarimeter. The peptides were dissolved in 50 mM sodium phosphate buffer, pH 7.4, with 10 % D₂0, at a final concentration of 0.2 mg/ml. Far-UV CD was performed from 185 to 260 nm for the samples without oleate and from 200 to 260 nm for the samples with oleate. The buffer was subtracted from the values obtained and the mean residue ellipticity (MRE), [Q], in deg cm² dmol^-1, was calculated as described previously. The MRE values were then submitted to K2D3, an online server enabling the prediction of secondary structure from far-UV CD data (42).

Cell lines and cell culture

Human lung carcinoma cells (A549, ATCC), human kidney carcinoma cells (A498, ATCC) and mice bladder carcinoma cells (MB49) were cultured in RPMI-1640 with non-essential amino acids (1 : 100), 1 mM sodium pyruvate, 50 pg/ml Gentamicin and 5-10% fetal calf serum (FCS) at 37 °C, 5 % C0₂. Cellular viability assays

For cell death experiments, cells were grown overnight on 96-well plates (2xl0⁴/vvell, Tecan Group Ltd). Cells were incubated with peptide-oleate complexes in serum-free RPMI-1640 at 37 °C. FCS was added after 1 hour. After 3 hours of treatment, cell death was quantified by two biochemical methods: cell viability was quantified via Presto Blue fluorescence

(Invitrogen, A13262) and cellular ATP levels via a luminescence-based ATPlite™ kit (Perkin Elmer). Fluorescence and luminescence was measured using a microplate reader (Infinite F200, Tecan).

Preparation of giant unilamellar vesicles (GUVs)

Coverslips were cleaned with 1M NaOH and plasma etched for 1 min using BD-20 laboratory corona treater (Electro Technic Products Inc., USA) to render the surface clean and hydrophilic. A thin film of 1% solution of molten agarose was made on the coverslip to provide a safe reaction bed for the giant unilamellar vesicles (GUVs) to form. 10 mM solution of Phosphatidyl Choline and O. lmM l,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-(cap biotinyl) (DPPE-biotin) in chloroform was labelled using 1 to 25 concentration of lmg/ml of Rhodamine. GUVs from this solution were formed onto the coverslips using a syringe through an air-blow dispersion method (43). Syke-Moore chambers were sonicated in 1M Sodium Hydroxide and rinsed with Milli-Q water. The coverslips were locked in the chambers and the GUVs were mobilized and rehydrated using 200 mM of Sucrose solution for 25 minutes. Further, the GUVs were collected and allowed to settle in 200 mM of glucose solution for 60 minutes. Alexa-488 labelled and unlabeled peptides-oleate complex in the ratio of 1 :4 was added to the solution in the observation chambers and monitored chronologically for at most 90 minutes. The GUVs were washed with PBS and monitored again to visualize co-localization.

Membrane blebbing experiment

A549 cells were seeded on to 8-chamber plates at the concentration of 2.5xl0⁴ cells per well and allowed to grow overnight. The cells were detached, washed with PBS and serum free RPMI medium was added to them. They were treated with the peptide-oleate complexes and monitored chronologically during the process using a confocal microscope.

Confocal imaging

Cells were grown overnight on an 8-well chamber slide (3xl0⁴/well, Lab-Tek). Lung carcinoma cells were treated with peptides (35 mM, 10% Alexa Fluor® -488 or -568 labeled). Labeling was done via amine coupling according to the manufacturer's instructions (Life Technologies). After treatment, cells were fixed with 2 % paraformaldehyde, permeabilized with Triton X-100 (0.25% in PBS) for 10 minutes, washed with PBS and blocked with 10% FCS in PBS for 10 minutes at room temperature. Cells were then incubated with anti-Histone H3 (abl791, Abeam), anti-SC-35 (abl l826, Abeam) or anti-20S proteasome (PW8155, Enzo Life Sciences) antibodies (1 : 50 in 10% FCS/PBS) for 2 hours at room temperature, washed three times with PBS and incubated with appropriate secondary antibodies conjugated to Alexa Fluor-488 (1 : 100 in 10% FCS/PBS, Molecular Probes) for 1 hour at room temperature. Nuclei were stained with DRAQ-5 (abl08410, Abeam). Cells were washed with PBS three times and mounted using Fluoromount. Slides were examined using an LSM 510 META laser scanning confocal microscope (Carl Zeiss).

Cellular uptake of peptide-oleate complexes

For uptake experiments, lung carcinoma cells were treated with Alexa Fluor® -488 or -568 labeled peptide-oleate complexes, washed and visualized via real-time confocal microscopy imaging using an LSM 510 META laser scanning confocal microscope (Carl Zeiss). The localization of peptides in lysosomes was detected by pre-labeling the cells with LysoTracker Green DND-26 (Thermo Fisher Scientific). The accumulation of peptide or peptide-oleate complexes in lysosomes was investigated by treating lung carcinoma cells with Alexa Fluor® 568 labeled peptide (20% labeled and 80% unlabeled) or peptide-oleate complexes for 1 hour. Cells were counterstained with Lysotracker. Slides were examined using an LSM 510 META or LSM 800 laser scanning confocal microscope (Carl Zeiss).

Western blotting

Cells were grown overnight on 6-well plates (3xl0⁵/vvell, TPP). Cells treated with peptides- oleate complexes were lysed with mammalian NP-40 lysis buffer supplemented with protease and phosphatase inhibitors (both from Roche Diagnostics). Blots were probe for p- SC35 (1 :4000 in NN8 buffer, Abeam), p-PKC8 (1 : 1000, 5% BSA, Santa Cruz) or PKC (1 : 1000, 5% BSA, Santa Cruz). After the primary antibodies incubation, the blots were washed and incubated with either HRP-conjugated secondary anti-rabbit or anti-mouse antibodies (1 :4000, 5 % NFDM, Cell Signaling) were also incubated. Visualization using ECL Plus detection reagent (GE Healthcare) then followed. Densitometry was performed using the ImageJ software 1.46r (NIH).

Transcriptomic Analysis

Lung carcinoma cells (300,000/well) were allowed to adhere overnight on a 6-well plate (TPP, Trasadingen, Switzerland). After treatment with peptide- (35 mM) oleate (175 pM) complexes for one hour, total RNA was extracted (RNeasy Mini kit, Qiagen). 100 ng of RNA was amplified using a GeneChip 3 ^' IVT Express Kit and then fragmented. Next, labeled aRNA was hybridized onto Human Genome (HG)-219 array strips for 16 hours at 45°C, washed, stained and scanned using the Geneatlas system (all Affymetrix). Transcriptomic data was normalized using Robust Multi Average implemented in the Partek Express Software (Partek). Fold change was calculated by comparing treated cells to PBS control cells. Genes with absolute fold change > 1.41 were considered differentially expressed.

Heat-maps were constructed by Gitools 2.1.1 software. Differentially expressed genes and regulated pathways were analyzed using Ingenuity Pathway Analysis software (IPA, Qiagen) and Gene Set Enrichment Analysis (GSEA, Broad Institute).

Bladder Cancer Model

C57BL/6 female mice were bred at the Department of Laboratory Medicine and used at ages from 7 to 12 weeks. For procedures, mice were anesthetized by intraperitoneal injection of a ketamine and xylazine cocktail. MB49 tumors were established as described¹². On day 0, the bladder was emptied and preconditioned by intravesical instillation of 100 pi poly-L- lysine solution (0.1 mg/ml) through a soft polyethylene catheter (Clay Adams, Parsippany, New Jersey) with an outer diameter of 0.61 mm for 30 minutes before MB49 tumor cells (2x l0⁵ in 100 mI PBS) were instilled. On days 3, 5, 7, 9 and 11, 100 mI of alphal-oleate (alphal : 1.7 mM, sodium oleate: 8.5 mM), sarlalpha-oleate (sarlalpha: 1.7 mM, sodium oleate: 8.5 mM) or PBS (sham-treated controls) were instilled. Mice remained under anesthesia on preheated blocks with the catheter in place to prolong tumor exposure to peptide-oleate complexes (approximately 1 hour). Groups of 5-7 mice for each treatment and control were sacrificed at each time point, and bladders were imaged and processed for histology. Two independent experiments were performed. Experiments were approved by the Malmo/Lund Animal Experimental Ethics Committee at the Lund District Court, Sweden (#M59-14). Procedures followed institutional, national, and European Union guidelines.

Histology and Immunohistochemistry

Bladders were embedded in O.C.T. compound (VWR), and 5-pm-thick fresh cryosections on positively-charged microscope slides (Superfrost/Plus; Thermo Fisher Scientific) were fixed with 4 % paraformaldehyde or acetone-methanol (1 : 1 v/v). For hemotoxylin-eosin (H&E) staining, Richard-Allan Scientific Signature Series Hematoxylin 7211 and Eosin-Y 7111 (Thermo Fisher Scientific) were used to counterstain the tissue sections. Imaging was done with AX10 (Carl Zeiss). Cryosections were permeabilized (0.25 % Triton X-100, 5 % fetal calf serum/PBS) and incubated with primary anti-VEGF antibody (1 : 100, ab46154, Abeam), anti-Ki-67 (1 : 100, BD Biosciences) or anti-Cyclin D1 (1 : 100, sc8396, Santa Cruz

Biotechnology). This was followed by staining with Alexa Fluor® 488-labeled secondary goat anti-mouse (1 :200, A-11001, Molecular Probes) or goat anti-rabbit antibodies (1 :200, A-11011, Molecular Probes). Tissues were counterstained with DAPI (4',6-diamidine-2'- phenylindole) (0.05 mM, Sigma-Aldrich) or DRAQ5 (1 : 1000, Abeam) and examined using an LSM 510 confocal microscope (Carl Zeiss). The quantification of images and fluorescence was performed in ImageJ or Photoshop CS5.

Real-time in vivo fluorescence imaging of peptides

Peptides were labeled using a VivoTag 680XL Protein Labeling Kit (Perkin Elmer). Mice were anesthetized using Isofluorane, and a 100 mI solution of labeled alphal-oleate (1.7 mM, 10

% labeled) was instilled in the bladders of tumor-bearing or healthy control mice. Hair was removed from the ventral sides of anesthetized mice. Mice were imaged at various time points using an IVIS Spectrum imaging system (Perkin Elmer). Fluorescence signals from alphal-oleate were acquired at a 680 nm excitation wavelength. For tissue specific uptake, Alexa Flour® 568 labeled alphal was instilled in the bladders of tumor-bearing or healthy control mice. Mice were sacrificed after 24 hours of treatment and bladder sections were imaged using a Zeiss AX10 fluorescence microscope.

Size-exclusion HPLC

Calibration standards and samples were injected onto a TSKgel Super SW3000 HPLC column (4.6 mm x 30 cm, Particle size 4 pm, Pore size 25 nm, Tosoh Bioscience) eluted with 0.05 M sodium phosphate buffer pH 7.0 containing 0.1 M Na₂S0₄ at a flow rate of 0.25 mL/min and detection at 280 nm. The chromatography was performed on a Dionex Ultimate HPLC 3000 Standard System running Chromeleon 6 software (Dionex, Thermo Scientific). The standard calibration curve was generated with the proteins given (Supplementary Fig. 5) including human serum albumin (HSA), which has a hydrodynamic radius (RH) of 40 A. The RH VS. elution volume linearity of the standard calibration curve is known to vanish after approximately 3.8 mL of elution volume (44). As a result, the RH of oleate in methanol eluent (a solvent which ensures that the fatty acid is monomeric) will be less than what is estimated from the standard curve (12.3 A). The retention times of small R_H-analytes are closely reproduced regardless of eluent, be it aqueous buffer or methanol.

Biomolecular NMR spectroscopy

The alphal- and sari- naked peptide samples were dissolved in 50 mM sodium phosphate buffer (pH 7.4, 90 % H₂0, 10 % D₂0), and the peptide-oleate complexes were reconstituted from a lyophilisate of phosphate-buffered saline. All experiments were carried out in the phase-sensitive mode (45). One-dimensional *H, two-dimensional NOESY (Nuclear

Overhauser Effect Spectroscopy) and ^-^C HSQC (Heteronuclear Single Quantum

Correlation) spectra were acquired on an Agilent Technologies 18.8 T (800 MHz) DD2 Premium Compact spectrometer with a triple-resonance, 5 mm enhanced cold probe. The ^-^C HSQC spectra were collected at 20°C with 16 scans, an initial delay of 3.0 s, a 90° pulse width of 7.5 and 9.8 ps and an acquisition time of 0.4 s— with broadband decoupling for alphal and sari peptide samples. For the alphal-OA and sarl-OA complexes, we used a 90° pulse width of 12.80 and 13.30 ps and an acquisition time of 0.4 s. All acquisition parameters were kept constant for all samples. Two-dimensional DPFGSE-NOESY (Double Pulse Field Gradient Spin Echo-NOESY) pulse sequences were used to acquire data at 20 °C with 16 scans, with an optimized mixing time of 300 ms for the alphal and alphal-OA complexes and a delay period of 1.5 s. For the sarl-OA complex, water-gate NOESY was used with 12 scans, with a mixing time of 150 ms. A trace amount of TSP was added to serve as a chemical shift reference. Each 2D HSQC spectrum consisted of 4K complex points in the acquisition dimension and 512 complex points in the indirect dimension. For the NOESY spectra, 4K complex points were used in the acquisition dimension and IK complex points in the indirect dimension. The two-dimensional data were processed with Gaussian apodization in both dimensions. The stoichiometry of the peptide with the oleic acid was determined by comparing the peak areas (using the ID 1H spectra) or peak volumes (using the 2D HSQC spectra) of well-resolved, isolated regions found in the spectra.

Diffusion-ordered spectroscopy (DOSY) measurements were performed at 293 K. Samples were prepared in 50 mM phosphate buffer at pH 7.4. The DgscteSL_dpfgsc DOSY pulse program was used, which consists of gradient compensated stimulated echo with spin lock using the excitation sculpting solvent suppression method (46). A spectral window of 13020 Hz was used, with an acquisition time of 2.46 s with a relaxation delay of 3 s. The FIDs were collected with 32000 complex data points with 64 scans. Logarithmically the gradient pulse strength was increased from 3 % to 86 % of the maximum strength of 32767 G/cm in 60 steps. A diffusion time (D) of 100 ms and bipolar half-sine-shaped gradient pulses (d) of 5 ms were applied. 1,4-Dioxane, which is known to behave independently of protein concentration and the folded state of the protein, was used as an internal chemical shift reference and hydrodynamic radius calibration reference (3.75 ppm; R_H = 2.12 A) (47, 48). DOSY processing was performed using a two-component fit with discrete approach, which further processed using non-uniform gradients approach. Three replicate acquisitions were given for each sample, and the resulting diffusion coefficient (D) values calculated. For alphal peptide the average D value for was 2.162 m²/s and 14.10 m²/s for 1,4-dioxane. In case of alphal-oleate complex the average D value was 0.986 m²/s for complex and 13.61 m²/s for 1.4-dioxane. The calculated RH are as follows: alphal peptide RH = 13.82 ± 0.447 A, alphal-oleate complex RH = 29.3 ± 0.606 A, human serum albumin R_H = 40.9 ± 1.44 A, oleate in aqueous solution R_H = 104.3 ± 7.22 A, oleate in methanol RH = 5.58 ± 0.0649 A. Note that the D (diffusion coefficient) values for 1,4-dioxane are slightly variable dependent upon the co-solute (lower panel where D is between 14.4 and 12.6), which rightly reflects the different solution micro-environment conditions that both solutes are mutually experiencing for each sample.

For T₂ relaxation measurements, the standard CPMGT2 pulse sequence was used to run the experiments with 15 relaxations delays, which were chosen logarithmically for different maximum T₂ time intervals: 8 s (alphal peptide), 1.2 s (alphal-oleate complex), 3.0 s (HSA), 7.0 s (oleate in aqueous solution), and 10 s (oleate in methanol) respectively. The data were acquired with 32000 complex points with baseline correction of 4. The T₂ analyses were performed on VNMRJ version 4.0 (Agilent Technologies) software by exponential fitting of these values with their corresponding intensity. All other NMR parameters were kept constant for all samples throughout the experiments. The

experiments were acquired at sample temperature of 293 K. The data are presented in Table S5.

Computational Simulations: Model Building of Peptide and Peptide-OA Complexes The initial structure of the alphal peptide was obtained from the corresponding domain in the crystal structure of human alpha-lactalbumin (PDB ID: 1B90). All cysteines were mutated to alanines, consistent with findings that a reduced human alpha-lactalbumin mutant in which all cysteines mutated to alanines could form a cytotoxic complex in the presence of the lipid cofactor (49). The initial structure of the sarlalpha peptide was obtained from an I-TASSER-built homology model (50). The alphal and the sarlalpha peptide were centered in a cubic box with box edges 1.2 nm from the peptide. For the oleate-containing systems of alphal and sarlalpha, 4 molecules of oleate are placed randomly in the box surrounding the alphal peptide to obtain a peptide-oleic acid ratio of 1 :4. The Amber 99SB-ildn force field and the TIP3P water model were used. For the coordinates, the starting structure was built using Discovery Studio 4.1 (Accelrys).

Geometry optimization for the ligand was performed using Gaussian09 at the level of HF-6- 31G*, and the partial charges were determined by the RESP method implemented in the antechamber tool of AmberToolsl6 (AMBER 2016). Topologies for the oleate were built using the General Amber Force Field (51). All systems were neutralized and Na⁺ and Cl ions were added to a concentration of 0.15 M. Energy minimization was performed using the steepest descent algorithm for 1000 steps to remove any initial bad contacts. Long-range electrostatics were treated with the particle mesh ewald algorithm, with a real-space cutoff of 1.2 nm, and Van Der Waal's interactions were truncated at 1.2 nm. All systems with oleate-containing peptides or naked peptides were initially heated at 500 K for 40 ns to eliminate starting structure bias and provide a partially unfolded state for the peptides. Temperature coupling of the system was performed using a velocity rescaling thermostat.

Hamiltonian replica exchange molecular dynamics simulations

The Gromacs 5.1.2 molecular dynamics package (51) with the Plumed 2.3 plugin for Hamiltonian Replica Exchange Molecular Dynamics was used to perform the simulations. All atoms of the alphal- and sarlalpha peptide residues, along with the oleate residues of the two oleate-containing systems, were selected for Hamiltonian scaling. Twenty replicas were used for each system, and scaling factors were generated for an effective temperature range of 300 K to 800 K. Temperatures for scaling were selected based on a geometric progression. The temperature factors were 300, 315.893, 332.629, 350.251, 368.807, 388.346, 408.919, 430.583, 453.395, 477.415, 502.707, 529.34, 557.384, 586.913, 618.006, 650.747, 685.223, 721.525, 759.75 and 800 K. Each replica was simulated for 400 ns, resulting in an effective simulation of 8 ps. Exchanges were attempted every 2 ps, and the result was an average acceptance probability of approximately 30%.

Simulation analysis

Analysis of simulation data was performed using the built-in Gromacs tools of the Gromacs package (51). The ensemble for each system with the canonical unsealed potential energy was used for the analysis, and data analysis was performed on the last 300 ns for each system. Dihedral Principal Component Analysis was performed using the gmx angles, gmx covar and gmx anaeig tools to prepare and diagonalize the covariance matrix and analyze eigenvectors and eigenvalues. The free-energy surface was constructed through projection onto the first and second principal components with the formula F_t = -RT \n( PJP₀), where R is the gas constant, T is temperature (300 K), P_t is the population in each bin and P₀ is the population of the most populated bin. The gmx cluster tool of Gromacs 5.1.2 was used to identify the representative structure of each minima for geometric clustering for the Gromos algorithm. We used the Define Secondary Structure of Proteins (DSSP) algorithm to calculate secondary structure propensities. For our analysis, we classified the 3io helix, a helix and n helix structures as helices; the b-sheet and residue in isolated b-bridge structures as sheets and the remaining structures as others. The contact probability was calculated using the gmx mindist tool in the Gromacs package. The minimum distance between protons of side chains for each residue and oleic acid was calculated for each frame. To calculate the contact probability, a contact was defined if the measured distance was less than 0.55 nm. The contact probabilities between Aromatic ring protons and Olefinic protons of alphal- and sarlalpha- oleate-containing systems were also calculated similarly. Proton distances were calculated to facilitate comparison of simulation data to Nuclear Overhauser Spectroscopy data.

Statistical analysis

Results are presented as a Mean ± SEM. Statistical analysis was performed using Student's t-test or the Mann-Whitney test at different statistical levels of significance: *P<0.05 and **P<0.01.

Claims

1. A biologically active complex having anti-tumour activity, consisting of a peptide of at least 10 amino acids, comprising an alpha-helical structure; and oleic acid or an oleate salt, in a ratio of at least 3 oleic acid or oleate salt molecules per peptide molecule, wherein, when present in the complex, the peptide has an increased conformational fluidity of three- dimensional structure as compared to the peptide alone as indicated by an increased peak width on at least some ^ NMR peaks of the complex as compared to the corresponding width of the peaks of a ^ NMR of the peptide alone; provided the peptide is other than alpha-lactalbumin, SAR 1, HOPS/CORVET, SEA (Sehl- associated), Snf7 domain subunits, SEQ ID NOs: 10-16, or fragments thereof having 20 or more amino acids.

2. A biologically active complex according to claim 1, wherein the increased conformational fluidity of the three-dimensional structure as compared to the peptide alone is further indicated by at least one of: a. an increased transverse relaxation rate (R₂), as obtained by NMR as described herein, as compared to the corresponding transverse relaxation rate of the peptide alone; b. a hydrodynamic radius 1.5 to 2.5 times larger than the corresponding hydrodynamic radius of the peptide alone; or c. mean residue ellipticity (MRE), [Q], in deg cm² dmol^_1 as obtained using

circular dichroism (CD) as described herein, at 220 nm that is at least 1 deg cm² dmol^-1 lower than the peptide alone.

3. A biologically active complex comprising a peptide which comprises a truncated form of SEQ ID NO 1 or SEQ ID NO 7 of up to 19 amino acids in length,

MAGWDIFGWF RDVLASLGLW NKH (SEQ ID NO 1)

KQFTKAELSQ LLKDIDGYGG IALPELIATM FHTSGYDTQ (SEQ ID NO 7)

and oleic acid or a salt thereof.

4. A biologically active complex according to claim 3, wherein the peptide is of SEQ ID NO 2 or SEQ ID NO 3

MAGWDIFGWF RDVLA (SEQ ID NO 2) KQFTKAELSQ LLKDI (SEQ ID NO 3) .

5. A biologically active complex according to any one of the preceding claims which comprises a water soluble oleate salt.

6. A biologically active complex according to claim 5 wherein the oleate salt is an alkali metal salt such as a sodium- or potassium oleate.

7. A method for preparing a biologically active complex according to any one of the preceding claims which comprises combining together a peptide as defined in any of claims 1 to 4, with oleic acid or an oleate salt, under conditions in which they form a biologically active complex.

8. A kit comprising a peptide as defined in any of claims claim 1 to 4 and oleic acid or a salt thereof.

9. A peptide of at least 10 amino acids, comprising an alpha-helical structure, and which is capable of forming a complex with an oleic acid or an oleate salt, wherein, when present in the complex, the peptide has an increased conformational fluidity of three-dimensional structure as compared to the peptide alone as indicated by an increased peak width on at least some ^ NMR peaks of the complex as compared to the corresponding width of the peaks of a ^ NMR of the peptide alone; provided the peptide is other than alpha-lactalbumin, SAR 1, HOPS/CORVET, SEA (Sehl- associated), Snf7 domain subunits, SEQ ID NOs: 10-16, or fragments thereof having 20 or more amino acids

10. A peptide which comprises a N-terminal fragment of SEQ ID NO 1 or SEQ ID NO 7 of up to 19 amino acids in length.

11. A peptide according to claim 10 which is of SEQ ID NO 2 or SEQ ID NO 3.

12. A pharmaceutical composition comprising a biologically acceptable complex according to any one of claims 1 to 6 in combination with a pharmaceutically acceptable carrier or excipient.

13. A method for treating cancer which comprises administering to a patient in need thereof, an effective amount of a biologically active complex according to any one of claims 1 to 6, or a pharmaceutical composition according to claim 12.

14. A method according to claim 13 wherein the cancer is a human skin papilloma, human bladder cancer, kidney cancer, lung cancer and glioblastomas.

15. A method according to claim 13 or 14, wherein the cancer is lung cancer, kidney cancer or bladder cancer.

16. A biologically active complex as defined in any one of claims 1 to 6 for use in therapy, in particular in the treatment of cancer.

17. A biologically active complex for use according to claim 16, wherein the cancer is lung cancer, kidney cancer or bladder cancer.