BACKGROUND TO THE INVENTION The tumour suppressor p53 is at the centre of a network of interactions that protect organisms against cancer. p53 is a transcription factor that activates many genes and leads to the apoptosis of (cancer) cells. p53 is an extremely unstable protein, with a denaturation temperature that is only slightly above body temperature (Friedler et al., (2002) Proceedings of the National Academy of Sciences (USA) 99,937-942). It is possible that this instability is necessary because the level of p53 in the cell is tightly regulated by proteolysis competing with biosynthesis; p53 is directed to the proteosome, where it is degraded, by binding to Mdm2, a ubiquitin ligase that is induced by p53. More than 50% of human cancers have mutations in the gene for p53 that inactivate it. Virtually all of the mutations are located in the core (DNA-binding) domain of p53, and many mutations simply lower its melting temperature.

In vitro studies have suggested that reactivation of p53 by small molecules could be an important therapy for cancer (Friedler et al., above; Bykov et al., (2002) Nature Medicine, 8, 282-288 ; Foster et al., (1999) Science 286,2507-10 ; Rippin et al., (2002) Oncogene 21, 2119-2129).

Potential small molecule drugs may be found by screening for molecules that bind to and stabilise the core domain (Foster et al.) or by rational design (Friedler et al.). An early report of a finding a drug that stabilised the core domain and then was subsequently successful in vivo (Foster et al.) was shown to be artefactual (Rippin et al.) : the drug does not bind to core domain in vitro and kills cells by a non-p53 route in vivo.

In this regard, recently, another small molecule, Prima-1, has been found to rescue p53 function in living cells (Bykov et al.). Furthermore, the Pfizer compound CP-31398 which

had been claimed to have been found by binding to the core domain of p53 has been shown not to bind to p53 (Rippin et al. (2002), Oncogene 21,2119-2129). Further, co- workers of the Pfizer group have now admitted that CP-31398 functions not by direct binding but by preventing the ubiquitination of p53 (Wang et al. (2003), Mol. Cell. Biol.

23,2171-2181).

In vitro studies have shown that a peptide, derived from a structural analysis of the complex between p53 and the p53 binding protein 2 (53BP2), does bind to p53 core domain and stabilises it (Friedler et al.). The peptide, F1-CDB3, corresponds to residues 490-498 of p53BP2 and has the fluorescein group attached to its N-terminus (i. e. , Fl- REDEDEIEW-NH2). It binds very tightly to p53 core domain with a dissociation constant of 0. 5 uM.

However, at the time of filing there is no evidence that such a molecule is effective is in vivo and there remains a need in the art to identify small molecules that rescue p53 function in vivo, that is in cell-based systems.

SUMMARY The present inventors have shown that, surprisingly and contrary to the effects expected from in vitro studies, the CDB3 peptide (having the sequence REDEDEIEW) increases the expression of p53 as well as oncogenic mutants thereof. The peptide also induces the expression of p21 and Mdm-2 tumour suppressor proteins in vivo in cells treated therewith.

CDB3 was moreover surprisingly found by the present inventors to induce the onset and/or progression of apoptosis in vivo in cells containing wild type p53 or oncogenic mutants thereof.

Thus, in a first aspect, the present invention provides a method for increasing the expression of p53 in vivo in one or more cells comprising treating the one or more cells with the peptide CDB3.

In a further aspect, the present invention provides the use of a CDB3 peptide in the preparation of a medicament for increasing the expression of p53 in vivo in one or more cells.

In a further aspect, the present invention provides a method for increasing the expression of one or more oncogenic mutant/s of p53 in one or more cells comprising the step of treating the one or more cells with a CDB3 peptide.

In a further aspect still, the present invention provides the use of a CDB3 peptide in the preparation of a medicament for increasing the expression of one or more oncogenic mutants of p53 in vivo in one or more cells.

According to the above aspects of the invention, the term'treating', when applied to cells, means bringing the cells into contact with one or more CDB3 peptides such that these one or more peptides enter the interior of the one or more treated cells. Preferably, the peptides are internalised into the nucleus of the cells. The procedures according to the invention are carried out in living cells.

Cells may be'treated'as herein defined simply by bringing the cells into contact with the peptide CDB3. Alternatively, the uptake of the one or more peptides into the cells may be enhanced by physical or chemical means that will be familiar to those skilled in the art.

Suitable physical means include microinjection and electroporation. Those skilled in the art will appreciate that this list is not intended to be exhaustive. The peptides may also be introduced into cells by expression from DNA-or RNA-based vectors, including viral vectors capable of transducing the cells. For example, retroviral, lentiviral or poxviral vectors may be used to transduce cells with nucleic acid encoding the CDB3 peptide. As an alternative, direct injection of the nucleic acid can be employed.

Alternatively, or in addition, chemical reagents may be employed in order to facilitate the uptake of the peptide or nucleic acid encoding the peptide into cells. Suitable chemical reagents include calcium phosphate and DEAE-dextran for nucleic acids; and

lipofectamineTM, liposome-based delivery systems, fusions with peptides such as viral fusogenic peptides, nuclear transfer peptides such as VP22 and penetratin, and the like, for the delivery of peptides. Those skilled in the art will appreciate that this list is not intended to be exhaustive.

The present inventors have found that the peptide CDB3 is able to penetrate inside the cells by itself, although the efficiency of the delivery is enhanced by the use of chemical reagents such as LipofectamineTM'. Furthermore, the present inventors have found that there is a pronounced nuclear localisation of CDB3 in cells that express wild-type p53 or the severely compromised mutant R175H than there is in cells lacking p53. As p53 normally exerts its effects in the nucleus, then this suggests that CDB3 forms a complex with p53 and is subsequently transported into the nucleus.

The CBD3 peptide may be labelled with fluorescein at its N-terminus, such that the sequence of the peptide is Fl-REDEDEIEW-NH2.

Cells suitable for treatment are any cells that comprise p53 and/or a p53 mutant and/or the nucleic acid encoding such p53 polypeptides. Examples of such cells include but are not limited to any of those selected from the group consisting of the following: H1299 lung carcinoma, which have both p53 alleles deleted; H1299-His175, which is H1299 transfected with R175H mutant; Saos-2, osteosarcoma, both p53 alleles deleted; Saos-2- His273, transfected with R273H mutant; HCT116p53+/+, which has wild-type 53 (as well as a high level of Mdm2 and ARF deleted); and HCTp53-/-, in which both p53 alleles are deleted by homologous recombination. Cells that lack p53 may be modified, for example by transfection or transformation, to express exogenous p53; and/or may be supplemented with exogenous p53 polypeptide.

According to the present invention, the term"R175H"refers to a mutant of tumour suppressor in which arginine at residue 175 is mutated to histidine, and is also denoted by H175 or His 175; R273H refers to a mutant of tumour suppressor in which arginine at residue 273 is mutated to histidine, and is also denoted by H273 or His 273.

In the context of the present invention the term CDB3 peptide includes within its scope fragments, derivatives and homologues (as herein defined) of the peptide in so far as the peptide possesses the requisite activity of increasing the expression of p53 or of an oncogenic mutant of p53.

Cells suitable for treatment may be any cell type that comprises p53, a p53 oncogenic mutant or the nucleic acid encoding either or both of them. In a preferred embodiment of the invention, cells for treatment are those undergoing neoplastic growth. In an especially preferred embodiment of the invention, the cells for treatment are p53 mediated cancer cells.

According to the present invention preferably the treatment of cells occurs in vivo, although the in vitro treatment of cells is also contemplated herein.

Common characteristics of'cancer'as herein defined include the ability of a cell to undergo endless replication, loss of contact inhibition, invasiveness and the ability to metastasise. Mutations within the nucleic acid of one or more cells are often involved in the onset of cancer. Often, more than one nucleic acid mutation or other aberrant cellular event is required for the development of tumours (bundles of aberrantly dividing cells), that is tumour formation is often a multi-signal event.

According to the present invention, the term'increasing the expression'means that the functional activity of p53 and/or an oncogenic mutant of the protein is increased in one or more cells treated with CDB3, or a derivative, peptide, fragment or homologue of it as compared with one or more cells under the same or similar conditions which have not been treated as herein defined with the peptide, or a derivative, fragment or homologue of CDB3. Preferably, the term refers to an increase in the level of polypeptide present in the cell. Advantageously, it refers to an increase in the rate of transcription of a nucleic acid encoding p53 or a mutant thereof.

Interestingly, the present inventors have also surprisingly found that CDB3 peptide is capable of inducing the expression of the endogenous target genes p21 and Mdm-2 in a p53 dependent manner.

Thus in a further aspect, the present provides a method for inducing the expression of p21 and/or Mdm-2 proteins in one or more cells comprising the nucleic acid encoding the respective proteins comprising the step of treating the one or more cells with a CDB3 peptide.

In yet a further aspect, the present invention provides the use of a CDB3 peptide in the preparation of a medicament for inducing the expression of p21 and/or Mdm-2 proteins in one or more cells comprising the nucleic acid encoding the respective protein.

Furthermore, the inventors have found that the CDB3 peptides are capable of inducing the onset or progression of apoptosis in one or more cells comprising p53 and/or an oncogenic mutant of p53 and/or the nucleic acid encoding them.

Thus, in a further aspect still, the present invention provides a method for inducing the onset of progression of apoptosis in one or more cells comprising the steps of treating those one or more cells with one or more CDB3 peptides.

In a yet a further aspect, the present invention provides the use of one or more CDB3 peptides in the preparation of a medicament for modulating the onset or progression of apoptosis in one or more cells.

"Apoptosis"or cell death is a controlled intracellular process characterised by the condensation and subsequent fragmentation of the cell nucleus during which the plasma membrane remains intact. A cascade of enzymes including caspases that cleave at aspartic acid residues is activated in the process.

By"modulating apoptosis"is meant that for a given cell, under certain environmental conditions, its normal tendency to undergo apoptosis is changed compared to an untreated cell. A decreased tendency to apoptose may also be a measurable increase in cell survival and may be the result of an inhibition of apoptosis by inhibiting one or more components of the apoptotic pathway. An increase in the tendency to undergo apoptosis is measurable by increased cell death, for instance as described below.

Methods for inducing apoptosis are well known in the art and include, without limitation, exposure to chemotherapy or radiotherapy agents and withdrawal of obligate survival factors (e. g. GM-CSF, NGF) if applicable. Differences between treated and untreated cells indicates effects attributable to the test compound.

Methods for measuring apoptosis are familiar to those skilled in the art and are described herein.

As a consequence of the properties described above, the present invention provides the peptide CBD3 for use in the prophylaxis or treatment of cancer.

Thus, in a further aspect, the present invention provides a method for the prophylaxis or treatment of cancer, preferably of p53 mediated cancer, comprising the steps of treating one or more p53 mediated cancer cells with one or more CDB3 peptides.

In yet a further aspect, the present invention provides the use of one or more CDB3 peptides in the preparation of a medicament for the prophylaxis or treatment of cancer, preferably p53 mediated cancer.

Moreover, the present inventors have found that the CDB3 peptides described herein are effective as an adjuvant in cancer therapy.

Thus in a further aspect the present invention provides the use of one or more CDB3 peptides in the preparation of a medicament effective as an adjuvant in the prophylaxis or treatment of cancer.

As herein defined, the term"p53 mediated cancer"means any form of cancer as herein defined in which the protein p53 plays a role.

Fl-CDB3 is moreover provided, in the present invention, as a protein transduction domain. Thus, the invention relates to the use of a Fl-CDB3 peptide for facilitating the transport of a polypeptide across a cell membrane.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Distribution of FL-CDB3 in cells after treatment with peptide for 24 h. The nuclei are visible in blue (staining with Hoechst), the peptide is green. Top left : H1299 cells containing p53 R175H. F1-CDB3 was localised in nuclei and large deposits could be seen in a nucleolus. Top right : cytoplasmic distribution was also observed in some cases.

Middle : after combined delivery with Lipofectamine 2000 D, the peptide was located in the cytoplasm, although some nuclear fraction was present as well. Bottom left and right : distribution of the peptide in parental p53-null H1299 cells. It appears that in p53 null cells peptide is localised mostly in cytoplasm (H1299), although in some cells nucleolar localisation is also evident (H12991-1). The peptide remained visible for at least 48 h.

Figure 2. Detection of induced protein expression by Western blots after 24 h incubation with F1-CDB3. Frames A, C, and D: Treatment with FL-CDB3 restored the ability of p53 mutants His175 and His273 to activate the transcription of endogenous genes p21 and Mdm-2. Lung carcinoma cells H1299 transfected with His175 p53 mutant and parental nontransfected cells were treated with the amounts of peptide indicated below, incubated for 24 h and tested for p53, p21, and Mdm-2 protein expression. The levels of actin show the equal loading of protein. Notably, mutant p53 levels were remarkably induced. B: Treatment with FL-CDB3 induces wtp53 in colon carcinoma HCT116 cells and activates

expression of Mdm-2 and p21. No induction of p21 nor Mdm-2 was observed in the absence of p53 expression in HCTp53-/-cells. For A and B: Lane 1 was the control with no F1-CDB3 ; Lane 2 was 24 h post treatment with 10 llg/mL FL-CDB3. For C and D, Lane 1 was the control (no F1-CDB3) ; Lane 2,10 pg/mL FL-CDB3; and Lane 3, 1 ug/mL FL-CDB3. The treatment with peptide was performed either with or without Lipofectamine. All the data presented here were obtained after treatment without Lipofectamine, except frames C and D. The induction of p53 target genes in C and D is seen to be dependent on the concentration of Fl-CDB3.

Figure 3. FACS analysis of effects of FL-CDB3 on cell cycle. We treated tumour cells with 10 ug/mL of peptide and analysed the cell cycle distribution and cell death (as subGl fraction) 24 h post treatment using FACS analysis. The left hand side of each pair of panels is the control without F1-CDB3. In one experiment, the percentage of dead cells was determined by trypan blue exclusion: the number of dead cells in H1299-His175 cells before treatment was 5%, after treatment, 37%; in control H1299 (p53'), before 3%, after treatment 11%; in Saos-2-His273 cells, before 3%, after 28%; in control Saos-2 (p53') ; before treatment 3%, after 13%.

Figure 4 shows the stabilisation of p53 core domain by FL-CDB3. (a) differential scanning calorimetry. The apparent Tm of wild-type and R249S core domain in the presence or absence of FL-CDB3 is determined as described in materials and methods. For the wild-type core domain Tm=40. 1 °C in the absence of the peptide and 41. 6 °C in its presence. For R249S Tm=34. 9 °C in the absence of the peptide and 35.9 °C in its presence.

Raw data are shown and are offset for clarity. (b-c) Urea dependence of p53-CDB3 binding. Wild-type p53 core domain is titrated into fluorescein-labelled CDB3 in presence of increasing urea concentrations, and changes in anisotropy are monitored. (b) anisotropy titration curves under various urea concentrations (c) log Kd for the p53 core domain- CDB3 interaction versus urea concentration (d) CDB3 induces refolding of p53 core domain. Wild-type p53 core domain is pre-incubated overnight with 3 M urea, then mixed with fluorescein-labelled CDB3 and the anisotropy change over time is monitored. As a control, the same protein is mixed with 3M urea and with fluorescein-labelled CDB3 without pre-incubation and anisotropy changes over time are monitored.

Figure 5 shows the uptake of Fl-labelled peptides by human tumor cells. A, Microscopy analysis of cellular uptake of peptides FL-CDB3, FL-poly-Glu, and control peptide in H1299-His175 cells. B, shows the in vivo interaction of biotinylated peptides with mutant p53.

Figure 6 shows the restoration of the wild type conformation to mutant p53 by p53- binding peptides. A, Rescue of His-175 mutant conformation. FL-CDB3, Fl-polu-Glu and control peptides were incubated with H1299-His175 cells for 24 hours. Conformation of p53 in cell lysates was evaluated by ELISA using monoclonal ant-p53 antibodies PAB1620, which recognises properly folded p53, and PAb240 for the detection of unfolded protein. B, shows the restoration of conformation to His-273 mutant. H1299- His175 cells were incubated with lOug/ml of biotinylated b-CDB3 for indicated time points. Intracellular peptide was captured from lysates using avidin-coated Dynobeads.

Bound proteins were analysed by Western blot.

Figure 7 Partial restoration of apoptosis-inducing activity of mutant p53 by FL-CDB3 A, FACS analysis demonstrated FL-CDB3-mediated induction of subGl fraction in mutant p53 carrying cells, but not in p53-null cells. Percentage of a sub-Gl population was determined by FACS analysis of ethanol fixed cells stained with propidium iodide (PI). Cells were treated with lOug/ml of FL-CDB3 for 24 h. B, p53-binding peptides sensitize HCT116 cells to ionising radiation.

DETAILED DESCRIPTION OF THE INVENTION CDB3 binds tightly to the p53 core domain (Friedler et al.). The experiments reported here demonstrate that p53 significantly enhances CDB3's ability to enter the nucleus, and that even the unstable mutant R175H is effective. Accordingly, it is likely that CDB3 is transported into the nucleus with p53 while the two are in a stable complex. As CDB3 is able to stabilise and hence activate R175H, CDB3 has been demonstrated to be a real candidate or lead for an anticancer drug in cells. Further, as CDB3 upregulates the levels of wild-type p53 as well as mutants in cells, CDB3 clearly stabilises directly or indirectly

the levels of p53 in the cell by interfering with the Mdm2 pathway, either as a consequence of stabilising p53 or by inhibiting its interaction with Mdm2.

General Techniques 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 (e. g. , in cell culture, molecular genetics, nucleic acid chemistry, hybridisation techniques and biochemistry).

Standard techniques are used for molecular, genetic and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc. which are incorporated herein by reference) and chemical methods. In addition Harlow & Lane. , A Laboratory Manual Cold Spring Harbor, N. Y, is referred to for standard Immunological Techniques.

CDB3 PEPTIDE CDB3 comprises a 9 amino-acid residue peptide, having the sequence REDEDEIEW- NH2. This peptide is referred to as CDB3. We also provide a fluorescein-labelled derivative of CDB3 (FL-CDB3) which can bind and stabilise p53 core domain.

CDB3 is derived from the p53 binding polypeptide 53BP2 and consists of residues 490- 498 of that protein. Resides 490-498 constitute one of the p53 binding loops in the protein.

Peptides according to the invention can be produced by expression in host cell systems, including mammalian, insect, bacterial and other cell types. Protein production is achieved by causing or allowing expression of a nucleic acid, for example by culturing host cells under conditions which lead to expression of the gene, so that the peptide encoded by the nucleic acid is produced. If the peptide is expressed coupled to an appropriate signal leader peptide it may be secreted from the cell into the culture medium.

Following production by expression, a polypeptide may be isolated and/or purified from the host cell and/or culture medium, as the case may be, and subsequently used as desired, e. g. in the formulation of a composition which may include one or more additional components, such as a pharmaceutical composition which includes one or more pharmaceutically acceptable excipients, vehicles or carriers (e. g. see below).

Introduction of nucleic acid may take place in vivo, as part of a gene therapy approach, as discussed below.

The peptide according to the invention can also be generated wholly or partly by chemical synthesis (see below). Such peptides can be readily prepared according to standard peptide synthesis protocols, general descriptions of which are available (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); and Applied Biosystems 430A Users Manual, ABI Inc. , Foster City, California), or they may be prepared in solution, by the liquid phase method or by any combination of solid-phase, liquid phase and solution chemistry. Apparata and protocols for peptide synthesis are widely available in the art, for example from Applied Biosystems.

CDB3 DERIVITIVES, HOMOLOGUES AND FRAGMENTS THEREOF As used in this document, the terms"peptide","polypeptide"and"protein"are synonymous with each other.

The term'peptide'in the context of this document includes two or more amino acids linked together by a peptide bond. Typically, they have more than 5,10 or 20 amino acids.

In the present invention, the CDB3 peptide is 9 amino acids long. However, the invention encompasses peptides which comprise the sequence of CDB3 but which contain amino acid deletions, additions or substitutions, whilst retaining the biological activity of CDB3 described herein. Preferably, peptides which comprise amino acid additions, deletions or

substitutions retain the ability to upregulate the expression of p53 in cells, preferably in vivo. A polypeptide or protein includes single-chain polypeptide molecules as well as multiple-polypeptide complexes where individual constituent polypeptides are linked by covalent or non-covalent means.

Amino acids may be naturally occurring or synthetic. Those skilled in the art will be aware of suitable sources of amino acids.

The polypeptide of the invention may be generated from naturally occurring or synthetic proteins, and/or polypeptides, and/or peptides. Degradation of the proteins, polypeptides or peptides may be performed by enzymatic and/or chemical digestion, using methods familiar to those skilled in the art. Those skilled will be aware of other suitable methods of degradation.

The term'peptide'in the context of this document, also includes within its scope, derivatives and variants thereof, as herein described.

Examples of derivatives include peptides that have undergone post-translational modifications such as the addition of phosphoryl groups. It may also include the addition of one or more of the ligands selected from the group consisting of : phosphate, amine, amide, sulphate, sulphide, biotin, a fluorophore, and a chromophore. One skilled in the art will appreciate that this list is not intended to be exhaustive. In a preferred embodiment of this aspect, a stabilising molecule that is a peptide is derivatised using a fluorophore. In an especially preferred embodiment, the fluorophore is fluorescein.

The terms"variant"or"derivative"in relation to the amino acid described here includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) amino acids from or to the sequence.

Variants of the peptides described here are likely to comprise conservative amino acid substitutions. Conservative substitutions may be defined, for example according to the Table below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other: ALIPHATIC Non-polar GAP ILV Polar-uncharged C S T M NQ Polar-charged D E KR AROMATIC H F W Y

The peptide according to the invention may moreover comprise non-natural amino acid substitutions or modified amino acids. For example, Cl-alanine, phenyl glycine and homoarginine may be included. Amino acids which are not gene-encoded may also be used in the present invention. All of the amino acids used in the present invention may be either the D-or L-optical isomer. The L-isomers are preferred. In addition, other peptidomimetics are also useful in the present invention (see Spatola, 1983, in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p. 267). Moreover, the peptide may be linear or cyclic in form.

CDB3 BINDING SITE ON P53 The CDB3 binding site, as mapped by NMR chemical shift analysis, is situated at the edge of the DNA-binding site and consists of three structural elements (loop 1, helix 2 and the edge of strand 8) which are remote sequentially but close spatially. The advantage of this site as a general target for p53-stabilising molecules is its location in proximity to the DNA binding site, enabling a local stabilising effect in that site. Indeed, chemical shift data shows the difference between the effects of DNA binding and CDB3 binding. CDB3 binding generates a strong localised effect on the DNA-binding site within p53 core

domain, while the chemical shift pattern upon DNA binding is significantly different, with shifts that are not as localised but are rather spread throughout the whole protein structure.

An intriguing observation is that CDB3 does not bind p53 core domain in the same location as the parent loop in the 53BP2 protein (Gorina, S. and Pavletich, N. P. (1996) Science, 274,1001-1005). The original 53BP2 loop binds the core domain between helix 2 and loop 3, with Trp498 of 53BP2 making contacts mainly with loop3 of p53, and the carboxylic acid side chains of 53BP2 making contacts with p53 Arg273 (a DNA-binding residue located in strand 10, close to helix 2). The CDB3 binding site might also be an additional binding site for 53BP2, and the two alternative binding sites might have a regulatory role. The observation that CDB3 and the original 53BP2 loop bind p53 at different sites might also be explained by the partly electrostatic nature of the interaction.

Owing to its high negative charge, CDB3 as a free peptide might act partly as a negatively charged"DNA-mimic", which binds the positively charged surface of the DNA-binding site.

LABELLED CDB3 PEPTIDES The CDB3 peptides may be labelled by a radio-isotope as known in the art, for example 32p or 31S or 99Tc, or a molecule such as a nucleic acid, polypeptide, or other molecule as explained below conjugated with such a radio-isotope. The CDB3 peptide may be opaque to radiation, such as X-ray radiation. The peptide may also comprise a targeting means by which it is directed to a particular cell, tissue, organ or other compartment within the body of an animal. For example, the CD3 peptide may comprise a radiolabelled antibody specific for defined molecules, tissues or cells in an organism.

PEPTIDE SYNTHESIS Peptides may be synthesised using methods known to those skilled in the art. A typical procedure is detailed below:

Peptides may be synthesised using a 432A Synergy peptide synthesiser (Applied Biosystems (ABI) ). Protected amino acid derivatives, reagents and solvents may be purchased from ABI, except for Fmoc-Ser (PO (OBzl) OH) -OH, which can be purchased from NOVAbiochem. Standard Fmoc chemistry can be employed, with coupling agents HBTU/HOBt. The peptides can be cleaved from the resin using a mixture of trifluoroacetic acid: Triisopropylsilane: water 90: 5: 5, precipitated in cold ethyl ether, washed 3 times with cold ethyl ether, dissolved in water or in a mixture of water: acetonitrile 1: 1 and lyophilised.

The peptides can be purified using reverse-phase HPLC (Waters 600 equipped with a 996 PDA detector). The column may be a preparative reverse phase C8 column (Vydac) and the gradient is 100% A to 100% B in 35 min (A = 0. 1 % TFA in water, B = 95% acetonitrile, 5% water, 0. 1% TFA). The purified peptides are characterised by MALDI-TOF MS and had the expected Mw.

For biotinylated peptides, the biotin may be coupled to the N-terminus through its carboxylic acid group during the solid-phase synthesis. The same conditions may be applied for the biotin coupling as for the coupling of the protected amino acids, except that it is repeated twice in some cases. Proteins and peptides may also be purchased commercially; for example, fluorescein-labelled CDB3 is purchased from Dr Graham Bloomberg (University of Bristol, UK).

Methods of protein and polypeptide synthesis are known in the art and are described in for example, Maniatis et al. For example, proteins such as human p53 core domain wild-type and mutants (residues 94-312) and human tetrameric p53 (residues 94-360) may be cloned, expressed and purified using methods familiar to those skilled in the art, in particular those described previously (Bullock, et al. (1997) Proc Natl Acad Sci U S A, 94, 14338-14342)."N-labelled human p53 core domain may be produced as described previously (Wong, K. B., et al., (1999) Proc Natl Acad Sci U S A, 96, 8438-8442).

BINDING OF CDB3 TO P53 AND ONCOGENIC MUTANTS THEREOF CDB3 is found to bind two p53 core domain hot-spot mutants: G245S, which is weakly destabilised (Bullock, et al., (2000) Oncogene, 19,1245-1256), and R249S, which is distorted in the DNA binding region (Bullock et al., 2000 ; Wong et al., 1999) in in vitro studies. FL-CDB3 affinity to the G245S mutant, which is folded almost as the wild-type, is the same as for the wild type. Binding to the R249S mutant, which is more destabilised, is weaker (but still in the low micromolar range). In addition CDB3 binds to a particular p53 core mutant (195T) which is highly destabilised. The mutation in this mutant is not in one of the typical oncogenic hot-spots.

Since their general mechanism of action is simply binding the native state and shifting the equilibrium, CDB3-like compounds could be used for the rescue of weakly destabilised (e. g. G245S) and globally unfolded (e. g. V143A) mutants that are unable to bind DNA (see below). The application of CDB3 for the rescue of locally distorted mutants, such as R249S, depends on the specific binding mode of the peptide as well on the specific distortion caused by the mutation. In general, locally distorted mutants require more specific molecules, which alter the conformation near the distorted site. The present inventors have demonstrated that it is possible for R249S: FL-CDB3 stabilises it since it binds in proximity to the distortion site (near loops 2 and 3 in the DNA binding site and see Wong et al., 1999). FL-CDB3, which binds the DNA binding site at its edge, might contribute to a local conformational change at this site of distortion.

The mode of action of CDB3 is different from that of the previously reported p53 C- terminal peptides. CDB3 stabilises p53 by binding its native but not its denatured state, while the C-terminal peptides specifically regulate the activity and the DNA binding of p53 core domain (Abarzua, et al., (1996) Oncogene, 13, 2477-2482 ; Hupp, et al., (1995) Cell, 83,237-245 ; Selivanova, et al., (1997) Nat Med, 3, 632-638 ; Selivanova, et al., (1999). Mol Cell Biol, 19,3395-3402). CDB3, and especially its labelled derivative FL- CDB3, are lead compounds, and they can be used as a basis for the future design of peptides and small molecules that have a larger stabilising effect on p53 core domain.

DETECTION OF BINDING OF CDB3 PEPTIDES TO P53 OR RELATED ONCOGENIC MUTANTS THEREOF.

The binding of the CDB3 peptide to p53 or the oncogenic mutants thereof may detected using any suitable means known in the art. Preferred means include physical methods such as NMR spectroscopy. In a preferred embodiment the NMR involves the use of heteronuclear NMR spectroscopy. The binding may also be detected using surface plasmon resonance. Alternatively, the binding of the CDB3 peptide to the native form of the polypeptide is detected using Differential Scanning Calorimetry (DSC) and or fluorescence anisotropy. All of these methods will be familiar to those skilled in the art Alternatively, the binding of CDB3 peptide to each state of the polypeptide, i. e. , native or denatured, may be detected by examining the fraction of the polypeptide sample which expresses an epitope for one or more monoclonal antibodies, which epitopes are only present in one form of the polypeptide. Other suitable methods for detecting conformational changes in proteins include, but are not limited to electrophoresis and thin- layer chromatography. Those skilled in the art will be aware of other suitable methods.

CDB3 PEPTIDE INCREASES THE EXPRESSION OF P53 OR ONCOGENIC MUTANTS THEREOF Methods for measuring increased expression of a protein such as p53 or the oncogenic mutants thereof are familiar to those skilled in the art.

Where it is desired to monitor the levels of expression of a known gene product, conventional assay techniques may be employed, including nucleic acid hybridisation studies and activity-based protein assays. Kits for the quantitation of nucleic acids and polypeptides are available commercially.

Where the gene product to be monitored is unknown, however, methods are employed which facilitate the identification of the gene product whose expression is to be measured.

For example, where the gene product is a nucleic acid, arrays of oligonucleotide probes may be used as a basis for screening populations of mRNA derived from cells.

CDB3 PEPTIDE INDUCES APOPTOSIS IN CELLS COMPRISING P53 OR ONCOGENIC MUTANTS THEREOF A number of methods are known in the art for monitoring the onset of apoptosis. These include morphological analysis, DNA ladder formation, cell cycle analysis, externalisation of membrane phospholipid phosphatidyl serine and caspase activation analysis. Cell survival may be monitored by a number of techniques including cell cycle analysis and measuring cell viability. Measurements of cell proliferation may be made using a number of techniques including a plaque assay in which adherent cells are plated out in tissue culture plates and left to grow prior to fixing and staining. The number of colonies formed reflects the amount of cell proliferation.

FURTHER USES OF CDB3 PEPTIDES CDB3 peptides and compositions thereof may be employed for in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assay and reagent applications, and the like.

Therapeutic and prophylactic uses of CDB3 peptides and compositions thereof involve the administration of the above to a recipient mammal, such as a human.

The term"prevention"involves administration of the protective composition prior to the induction of the disease. "Suppression"refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease."Treatment"involves administration of the protective composition after disease symptoms become manifest.

Animal model systems that can be used to screen the effectiveness of the selected stab peptides or compositions in protecting against or treating the disease are available and will be familiar to those in the art.

Generally, the peptides or compositions will be utilised in purified form together with pharmacologically appropriate carriers. Typically, these carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, any including saline and/or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable adjuvants, if necessary to keep a polypeptide complex in suspension, may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).

The selected CDB3 peptides described here may be used as separately administered compositions or in conjunction with other agents. These can include various immunotherapeutic drugs, such as cylcosporine, methotrexate, adriamycin or cisplatinum, and immunotoxins or in conjunction with radiotherapy or radio-iostopes or ionising or other radiation. Pharmaceutical compositions can include"cocktails"of various agents.

The route of administration of pharmaceutical compositions may be any of those commonly known to those of ordinary skill in the art. For therapy, including without limitation immunotherapy, the selected stabilising molecules or compositions can be administered to any patient in accordance with standard techniques. The administration can be by any appropriate mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or also, appropriately, by direct infusion with a catheter. The dosage and frequency of administration will depend on the

age, sex and condition of the patient, concurrent administration of other drugs, counterindications and other parameters to be taken into account by the clinician.

The selected peptides or compositions can be lyophilised for storage and reconstituted in a suitable carrier prior to use. Known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of functional activity loss and that use levels may have to be adjusted upward to compensate.

The compositions containing the peptides or a cocktail thereof can be administered for prophylactic and/or therapeutic treatments. In certain therapeutic applications, an adequate amount to accomplish at least partial inhibition, suppression, modulation, killing, or some other measurable parameter, of a population of selected cells is defined as a "therapeutically-effective dose". Amounts needed to achieve this dosage will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from 0.005 to 5.0 mg of selected peptide or other stabilising molecule per kilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly used. For prophylactic applications, compositions containing the present selected stabilising molecules or cocktails thereof may also be administered in similar or slightly lower dosages.

Peptides and/or compositions can be used in the treatment of any disease where errors in the conformation, folding and aggregation of p53 contribute to the disease. Examples include cancer, and other proliferative disorders. In a particularly preferred embodiment, the disease is cancer. One skilled in the art will appreciate that thus list is not intended to be exhaustive.

As demonstrated in the present invention, F1-CBD3 peptides are taken up into cells very efficiently. Thus, the invention also provides a F1-CDB3 peptide for use in membrane penetration and/or facilitating the transport of a fusion protein across a cell membrane.

This finding by the present inventors is surprising since it is rare for peptides to be taken up by cells. Usually there have to be positively charged residues at the N-terminal region for import. Nevertheless, F1-CDB3 is taken up efficiently by cancer cell lines. The efficient import appears analogous to the selective uptake by cancer cells of polyglutamate (polyGlu), which resembles the sequence Glu-Asp-Glu-Asp-Glu-Ile-Glu in F1-CDB3. Cell Therapeutics, Inc. (Seattle, WA, USA) is marketing XYOTAXTM (PG-TXL), which links paclitaxel, the active ingredient in Taxol (g), to a biodegradable polyglutamate polymer. PG-TXL is currently in numerous clinical trials for a variety of indications including ovarian, lung, and colorectal cancers as a single agent or as combination therapy. Unlike Taxol, PG-TXL has been administered over a ten-minute infusion without premedication. Preclinical data suggest that the polyglutamate (PG) polymer delivers substantially more paclitaxel to the tumor site than is carried when paclitaxel, the active ingredient in Taxol, is administered alone.

The inventors envisage that FL-CDB3 could be similarly used and targeted. It could be infused prior to radiotherapy or chemotherapy, be selectively taken up by cancer cells, and activate them for more effective radiotherapy or chemotherapy. Preliminary results indicate that FL-CDB3 is indeed active in combination with radiation (Figure 5). As assessed by FACS analysis, FL-CDB3 did not induce apoptosis in wild-type p53 carrying HCT116 cells. However, combination of FL-CDB3 with gamma irradiation sensitised HCT116 cells, but not their p53-null counterparts HCT116 p53-/-cells to apoptosis induction by irradiation.

In an alternative embodiment FL-CDB3 peptide is fused to a heterologous polypeptide, in order to promote transport of the heterologous polypeptide across a cell membrane. The fusion protein may comprise a cleavable site, such as a protease cleavable linker, between the Fl-CDB3 peptide and the heterologous polypeptide. Such a site will allow the Fl- CDB3 peptide to be cleaved from the heterologous polypeptide once the fusion polypeptide has entered the cell.

Thus, the invention moreover provides a method for delivering a polypeptide across a cell membrane, comprising fusing the polypeptide to Fl-CDB3 and allowing Fl-CDB3 to promote transport across the membrane.

EXAMPLES Example 1 General Methods The general methods used in the examples are described in detail in Bykov et al., Nature Med 8, 282-288 (2002) which is herein incorporated by reference.

Peptide synthesis. The peptides were synthesised using a Pioneer peptide synthesizer (Perseptive) using standard Fmoc chemistry. Fluorescein was coupled to the N-terminus of the peptides on the pioneer peptide synthesizer using 4-fold excess of Fluorescein-Osu (molecular probes) and 4-fold excess of HoBt. The peptides were purified and characterised.

Protein expression and purification. Human p53 core wild type and mutants (residues 94-312) were cloned, expressed and purified. 15N labelled human p53 core was produced.

NMR Spectroscopy. All HSQC spectra were acquired at 20 °C on a Bruker DRX 600 MHz spectrometer. Samples for NMR experiments contained 15N labelled wild type or mutant p53 core at a concentration of 150 uM and FL-CDB3 or one of its derived peptides in a concentration of 600 uM or 1.2 mM. The buffer was 150 mM KCl, 5 mM dithiothreitol (DTT), 2% D20 in 25 mM sodium phosphate pH 7.2. Chemical shift analysis was performed.

Fluorescence anisotropy measurements. Fluorescence anisotropy measurements were performed in 50 mM Hepes pH 7.2 with fluorescein-labelled CDB3 derivatives (Table 1) at 10 °C using a Perkin-Elmer LS-50b luminescence spectrofluorimeter equipped with a

Hamilton microlab M dispenser controlled by laboratory software. The peptide (1 ml, 0.1- 0. 5, uM) was placed in the cuvette and the appropriate p53 core protein (240 p1, 20-200 uM) was placed in the dispenser. Additions of 6 tL of protein were titrated into the peptide solution every 1 min, the solution was stirred for 20 s and the anisotropy measured. Dissociation constants for the peptide-p53 core complex were calculated by fitting the anisotropy titration curves to a simple 1: 1 equilibrium model. To study the ionic strength dependence of peptide binding to p53 core, the titrations were performed under various ionic strength conditions in 10 mM Hepes pH 7.2 with increasing concentrations of NaCl.

Cells and plasmids The human Saos-2-His-273 and H1299-His-175 cell lines carry the indicated tetracycline-regulated mutant p53 constructs. The human HCT-116 cell line carries wild type p53 and in the HCT116p53-/-both p53 alleles were deleted by means of homologous recombination. SW480 and A431 colon carcinoma cell line carry His-273 mutant p53 and A431 carries a p53-responsive lacZ reporter. The p53-EGFP plasmid contains 13 synthetic p53 consensus DNA binding sites in front of the EGFP coding sequence. Transient transfections experiments were performed with Lipofectamine 2000 according to the manufacturer's recommendations (InvitrogenTM Life Technologies, Groningen, The Netherlands).

In vitro assays For FACS analysis, cells were stained with propidium iodide and analysed on a Becton Dickinson FACScan (Mountain View, California) according to standard procedures.

TUNEL staining, immunostaining, lacZ staining, preparation of cell extracts, ELISA and Western blotting were performed according to standard procedures. For PAbl620 staining, cells were fixed with 4% formaldehyde. The anti-p53 monoclonal antibodies PAbl620, PAb240, DO1 and PAbl801 were obtained from Calbiochem (Darmstadt, Germany). The anti-p53 rabbit polyclonal antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, California), the anti-MDM-2 monoclonal antibody was from Neo Markers (Fremont,

California) and the anti-p21 monoclonal antibody was from Transduction Laboratories (Lexington, Kentukki). Secondary antibodies (FITC-conjugated horse anti-mouse Ig, Texas Red-conjugated goat anti-rabbit Ig) were from Vector (Burlingame, California). All other reagents were from Sigma-Aldrich Sweden AB (Stockholm, Sweden).

Cell lines.

The following cell lines were used in the experiments described in the following examples: H1299 lung carcinoma, which had both p53 alleles deleted; H1299-His175, which was H1299 transfected with R175H mutant; Saos-2, osteosarcoma, both p53 alleles deleted; Saos-2-His273, transfected with R273H mutant; HCT116p53+/+, which has wild-type 53 (as well as a high level of Mdm2 and ARF deleted); and HCTp53-/-, in which both p53 alleles were deleted by homologous recombination.

Example 2-Distribution of FL-CD 83 in cell after treatment with FLCD 83 peptide for 24hrs.

The results can be seen in figure 1. Details of the methods used are described in Bykov et al., Nature Med 8,282-288 (2002).

Figure 1 shows the distribution of FL-CDB3 in cells after treatment with peptide for 24 h.

The nuclei are visible in blue (staining with Hoechst), the peptide is green. Top left : H1299 cells containing p53 R175H. Fl-CDB3 was localised in nuclei and large deposits could be seen in a nucleolus. Top right : cytoplasmic distribution was also observed in some cases.

Middle : after combined delivery with Lipofectamine 20000, the peptide was located in the cytoplasm, although some nuclear fraction was present as well. Bottom left and right : distribution of the peptide in parental p53-null H1299 cells. It appears that in p53 null cells peptide is localised mostly in cytoplasm (H1299), although in some cells nucleolar localisation is also evident (H12991-1). The peptide remained visible for at least 48 h.

In conclusion, the peptide FL-CDB3 is able to penetrate inside the cells by itself, although the efficiency of delivery could be enhanced by LipofectamineTM (Fig 1). Importantly, there is a much more pronounced nuclear localisation of Fi-CDB3 in cells that express wild-type p53 or the severely compromised mutant R175H than there is in cells lacking p53. p53 normally exerts its activity in the nucleus.

Example 3 Detection of induced protein expression by Western blots after 24 h incubation with FI-CDB3.

The results can be seen in figure 2.

Frames A, C, and D: Treatment with FL-CDB3 restored the ability of p53 mutants His175 and His273 to activate the transcription of endogenous genes p21 and Mdm-2. Lung carcinoma cells H1299 transfected with His175 p53 mutant and parental nontransfected cells were treated with the amounts of peptide indicated below, incubated for 24 h and tested for p53, p21, and Mdm-2 protein expression. The levels of actin show the equal loading of protein. Notably, mutant p53 levels were remarkably induced. B: Treatment with FL-CDB3 induces wtp53 in colon carcinoma HCT116 cells and activates expression of Mdm-2 and p21. No induction of p21 nor Mdm-2 was observed in the absence of p53 expression in HCTp53-/-cells. For A and B: Lane 1 was the control with no Fl-CDB3, Lane 2 was 24 h post treatment with 10 Fg/mL FL-CDB3. For C and D, Lane 1 was the control (no F1-CDB3) ; Lane 2, 10 ug/mL FL-CDB3, and Lane 3,1 llg/mL FL-CDB3.

The treatment with peptide was performed either with or without Lipofectamine. All the data presented here were obtained after treatment without Lipofectamine, except frames C and D. The induction of p53 target genes in C and D is seen to be dependent on the concentration of F1-CDB3.

Overall, the peptide induces endogenous p53 target genes p21 and Mdm-2 in a p53- dependent manner (Fig 2). Two mutants were tested, H273 and H175. Surprisingly, the transcription activity of both of them was reactivated. All experiments were repeated at

least three times. Interestingly, transcriptional function of wild-type p53 is also activated.

The levels of wild-type and mutant p53 were considerably raised.

Example 4. FACS analysis of effects of FL-CDB3 on cell cycle.

Tumour cells were treated with 10 llg/mL of peptide and analysed the cell cycle distribution and cell death (as subGl fraction) 24 h post treatment using FACS analysis.

The left hand side of each pair of panels is the control without F1-CDB3. In one experiment, the percentage of dead cells was determined by trypan blue exclusion: the number of dead cells in H1299-His175 cells before treatment was 5%, after treatment, 37%; in control H1299 (p53-), before 3%, after treatment 11%; in Saos-2-His273 cells, before 3%, after 28%; in control Saos-2 (p53~) ; before treatment 3%, after 13%.

From the results of this experiment, it is clear that CDB3 peptide induces apoptosis in tumour cells in p53-dependent manner (Fig 3). There is a difference between p53-positive and p53-negative cells. Surprisingly, no growth arrest was detected.

Example 5 Peptide binding in vitro We synthesized a range of variants of F1-CDB3 to analyse and optimise its binding to p53 to select the best derivative for the experiments in vivo (Table 1). The derivatives also provide important controls for linking activities in vivo and in vitro. Fl-CDB3 is highly negatively charged and binds to a positively charged region of p53. We made mutants that differ in their net charges as well as in key amino acid residues. These include: FL- polyGlu, which is a model of a relatively non-specific peptide that binds by electrostatic interactions with positively charged sites; F1-E491/3/5/7A, which lacks negatively charged side chains; and a control peptide Fl-control (Fl-RKSKKKITW) which is positively charged and should not bind to the positively charged binding site. Binding was assayed by fluorescence anisotropy at various ionic strengths, L At low ionic strength, the electrostatic interactions from the glutamates or aspartates dominated: FL-polyGlu bound

with Kd = 12 nM, 50 times more tightly than did FL-CDB3 at I = 23 mM and the positively charged control, Fl-control, had no detectable binding. Binding affinity to p53 core decreased with removal of negatively charged residues. At physiological ionic strength (I = 0.15 M), the parent Fl-CDB3 bound the tightest. Biotinylated CDB3 (Biotin- CDB3) bound far more weakly. F1-CDB3 bound tightly to p53 R273H with dissociation constants of 3.4 nm at I= 23 mM FI-CDB3 is taken into cancer cells We treated the human tumor cell lines with F1-CDB3, Fl-polyGlu, and the control peptide and other derivatives (Table 1). All the peptides were able to enter the cells (Fig. 5). The distribution of peptides was similar, predominantly cytoplasmic, with some nuclear and/or nucleolar localization in all cell lines tested. The ability of peptides to enter the cells and their intracellular distribution did not depend on p53 status, as it did not differ in H1299 p53 null cells and H1299-His-175.

Biotin-CDB3 binds p53 in cells We next tested whether peptides can bind p53 in the context of cellular proteins. H1299- His175 cells were treated with Biotin-CDB3. Mutant p53 from H1299-His-175 cells co- precipitated with Biotin-CDB3, despite its poorer affinity (Fig. 6B). The binding was specific, since Mdm2 protein was not bound by Biotin-CDB3. Thus, the p53-binding peptides selected in vitro, can bind to the p53 protein in living cells.

Table 1: FL-CDB3 derived peptides studied for p53 core domain binding Peptide Sequence Kd (RM) FL-CDB3 R E D E D E I E W 0. 6 single mutants R490A A E D E D E I E W 0. 2 E491A2 R A D E D E I E W 3. 4 D492A R E A E D E I E W 2.4 E493A R E D A D E I E W 2. 2 D494A R E D E A E I E W 2. 9 E495A R E D E D A I E W 1. 8 I496A R E D E D E A E W 0. 6 E497A R E D E D E I A W 2. 0 W498A R E D E D E I E A 1. 2 double mutants E49/3A2 R A D A D E I E W 8. 7 E491/7A R A D E D E I A W 8. 6 E493/5A R E D A D A I E W 7. 5 E495/7A R E D E D A I A W 6. 1 490/6A A E D E D E A E W 0. 19 496/8A R E D E D E A E A 1. 6 triple mutants 490/6/8A A E D E D E A E A 0. 3 491/2/3A R A A A D E I E W 33 493/4/5AR E D A A A I E W 28 494/5/7A R E D E A A I A W 16 other mutants E491/3/5/7A R A D A D A I A W 136 FL-poly Glu E E E E E E E E E 0. 012

'Peptide nomenclature is according to the residue numbers in the original 53BP2 sequence (Gorina et al., Science 1996). Mutated residues are shown in bold. Dissociation constants, Kd, are at 50 mM Hepes pH = 7.2 (ionic strength = 21 mM) and were determined using fluorescence anisotropy as described (Friedler et al. , PNAS 2002).

2 Kd for these peptides at physiological ionic strength was 930-950 uM.

Table 2: FL-CDB3 derivatives tested for in vivo activity Peptide2 Charge Kd (pM) Kd (M) Induction of native Target gene I= 0. 15 Ml I= 0. 021 M Conformation upregulation4 in vivo3 FL-CDB3-5 100 0.6 ++ ++ F1-E491/3/5/7A-1 1800 136 + + FL-R490A-6 140 0.2 + + FL-polyGlu-9 1800 0.012 + FL-control +5 N. D. N. B.

1 Kd is reported for binding of p53 core to the peptide at physiological ionic strength (1) = 0.15 M as well I= 0.021 M.

2 For peptide sequences see Table 1.

3 As measured by the ability of p53 to recognize the antibody against the native state pat 1620 after peptide treatment. The scale ranges between ++ = high activity and-= no activity at all.

4 As measured by the ability of p53 to induce its target genes p21 and MDM2 after peptide treatment. The scale ranges between ++ = high activity and-= no activity at all.

Example 6 Upregulation of p53 Importantly and unexpectedly, treatment of cells with both F1-CDB3 and polyGlu resulted in a substantial increase in total p53 levels as detected by ELISA by the conformationally insensitive antibody DO1. Western blot analysis confirmed the induction of mutant p53

levels in H1299-His175 and Saos-2-His273 cells upon treatment with FL-CDB3 and polyGlu, but not by the control peptide (Fig6A). In addition, wtp53 in HCT116 cells was also induced by F1-CDB3 (Fig6B). The p53-reactivating compound PRIMA-1, on the other hand, did not upregulate p53. Since we failed to detect any binding of it to the core domain, PRIMA-1 clearly has a different mode of action from F1-CDB3, perhaps binding to another domain.

Example 7 Partial restoration of p53-dependent apoptosis To determine whether FL-CDB3 can rescue the apoptosis-inducing function of p53, we analysed cell survival after treatment with peptide using FACS. Fig. 7A shows the DNA content profile of Saos-2 and Saos-2-His-273 cells treated with 10 llg/ml Fl- CDB3 for 24 hours. Fl-CDB3 caused a mild increase (19%) in the fraction of cells with a sub-Gl DNA content in the presence of mutant p53, indicating DNA fragmentation and cell death. We analyzed the biological response to FL-CDB3 of human tumor cell lines with different p53 status (p53 null, wild type p53, mutant p53).

Comparisons between the isogenic lines that differ only in p53 status, i. e. , Saos-2 and Saos-2-His273 ; and H1299 and H1299-His175, showed that F1-CDB3 induced apoptosis in cell lines carrying mutant p53, but not in their p53-null counterparts (Fig. 7a). Thus, the biological effect of Fl-CDB3 was dependent on mutant p53. The control peptide and Fl-PolyGlu did not induce apoptosis, and Fl-E491/3/5/7A and Fl- R490A were only half as effective as Fl-CDB3.

Sensitization to gamma-radiation As assessed by FACS analysis, F1-CDB3 did not induce apoptosis in wild-type p53 carrying HCT116 cells. However, combination of FL-CDB3 with gamma irradiation sensitised HCT116 cells, but not their p53-null counterparts HCT116 p53-/-cells to apoptosis induction by irradiation (Fig. 7B).

Example 8 Validity of the cl : aperoning strategy One of the classic dilemmas of designing a drug from experiments in vitro is whether it binds to the same target in cells and is active in the desired manner. The peptide Fl-CDB3 and its designed derivatives are imported by cancer cell lines. They induce the upregulation of wild-type p53, a representative destabilized structural mutant, R175H and a representative contact mutant R273H. The upregulated proteins are all in the native conformation and only a small fraction in the denatured state, as evidenced from antibody binding studies. Wild-type and mutant proteins are all active, as shown by the induction of p53-transactivation products, such as p21 and HDM2. The absence of effect of p53- binding peptides on gene expression or cell growth in p53-negative cells provides a strong evidence that the effects observed in p53-positive cells are exerted through p53. Indeed, our results show that Biot-CDB3 binds p53 in vivo. Importantly, the activities in cells parallel the binding affinities in vitro (Table 1). Taken together, our data provide a solid support to the idea that Fl-CDB3 functions as a p53-specific molecular chaperone.

The upregulation of p53 and its mutants is a bonus as a massive increase in the concentration of, for example, R273H would compensate for its lower activity. We do not yet know the reason for the upregulation of p53 levels. We did not observe any changes in the fraction of p53 bound to HDM2 or p300 upon peptide treatment (data not shown). The fraction of poly-ubiquitinated p53 did not change either. One possibility is that the binding of FL-CDB3 to p53 allows mono-ubiquitination, but not poly-ubiquitination, which is required for p53 degradation. This possibility is currently being investigated. Notably, the p53-reactivating compound PRIMA-1 [], did not upregulate p53. Since we have failed to detect any binding of it to the core domain (unpublished data), PRIMA-1 clearly has a mode of action different from F1-CDB3. It is possible that PRIMA-1 binds to another domain of p53.

Interestingly, the EDEDEIE sequence of F1-CDB3 resembles the structure of polyGlu.

Cancer cells selectively take up polyGlu, and this is the principle behind the use of

XYOTAXTM which is paclitaxel, the active ingredient in Taxolt), linked to a biodegradable polyglutamate polymer. XYOTAXTM is currently in numerous clinical trials for more effective drug delivery in a variety of indications including ovarian, lung, and colorectal cancers as a single agent or as combination therapy. We envisage that F1-CDB3 or an optimised derivative could be infused prior to radiotherapy or chemotherapy, be selectively taken up by cancer cells, and activate them for more effective radiotherapy or chemotherapy.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in biochemistry and molecular biology or related fields are intended to be within the scope of the following claims.