WO2003082920A1 - Chemokines and uses thereof - Google Patents

Abstract

The invention concerns a nuclear targeting signal found in the C-terminal sequence of constitutive chemokines. The chemokines have a nuclear targeting domain in their C-terminal sequence which provides the ability of the polypeptide to translocate to the cell nucleus. The invention provides nuclear targeting polypeptides isolated from chemokines and complexes comprising either the intact chemokine or just the nuclear targeting domain and substances to be transported to the cell nucleus.

Description

CHEMOKINES AND USES THEREOF

The present invention relates to chemokines and their use. Particularly, but not exclusively, the invention relates to a nuclear targeting peptide located predominantly in the C-terminal of constitutive chemokines. The invention provides constructs for targeting cells using the chemokine or its nuclear targeting domain as vehicles to carry agents such as cytotoxins to the cell nucleus.

Chemokines are members of a large family of peptides that are typically characterised as pro-inflammatory mediators (1,2) . The family is defined on the basis of sequence homology and on the presence of variations on a conserved cysteine motif which allows the family to be divided into four subfamilies. The most populous subfamilies each have four conserved cysteine residues and differ in the presence or absence of a single amino acid inserted between the first two cysteines. Thus in the CXC or α- chemokine family the first two cysteines are separated by a single variable amino acid whilst in the CC or β- chemokine family, these first two cysteines are juxtaposed.

Two other subfamilies are characterised by single members with lymphotactin being a member of the C family of chemokines (3) and Fractalkine being a member of the CX3C family (4,5). All chemokines mediate their actions through members of the seven transmembrane family of G protein-coupled receptors with CXC, or α-chemokine, receptors being referred to as CXCRs (currently 6) and CC, or β-chemokine, receptors being referred to as CCRs (currently 11) . There exist single C and CX3C chemokine receptors, a number of viral chemokine receptors as well as more promiscuous receptors such as D6 and DARC (6) . Whilst chemokines are classically regarded as pro- inflammatory mediators, they also exhibit a number of other well characterised functions. Thus chemokines have roles in development (7), angiogenesis (8) and control of haemopoietic stem and progenitor cell proliferation (9,10) and it is likely that other noninflammatory roles will be revealed in the future. The inventors have recently identified and characterised a novel member of the CC chemokine family which they cloned from an Embryonic Stem (ES) cell subtracted library (11) . The inventors have called this chemokine ESkine to denote its ES cell origin (12). ESkine is identical to proteins previously described as CTACK (13), ALP (14) and ILC (15) and is now systematically referred to as CCL27 in keeping with the recently adopted chemokine nomenclature system (16).

The ESkine/CCL27 gene resides on mouse chromosome 4 and human chromosome 9. In both species this gene overlaps at its 3' end with an Interleukin 11 receptor alpha chain gene. Indeed, one can infer much of the coding sequence of human ESkine/CCL27 by reverse translating the extreme 3' non-coding sequences of the human Interleukin 11 receptor alpha chain (12). In the mouse, this overlap with the interleukin 11 receptor is also evident (12,17), however the inventors' analyses indicate that this overlap in the genome of the 129sv mouse involves a novel member of the IL11 receptor alpha family that is currently under investigation (McLean et al , Manuscript in preparation) . Clear biological functions for ESkine/CCL27 have been difficult to demonstrate (14,15). However, it has been shown to be a chemoattractant for CLA positive T cells (13) and for a subset of CD4+ T cells (12). The inventors, have been involved in trying to identify the receptor for ESkine/CCL27 and whilst it does not bind to CCRsl-9, it does bind to GPR2 a previously identified orphan receptor (18) which has now been renamed CCR10 to indicate its chemokine receptor binding function (19, 20). ESkine shares binding to CCR10 with another recently described CC-chemokine CCL28 (21).

The inventor has surprisingly determined the existence of a differentially spliced variant that lacks the signal peptide and which is targeted to the nucleus following translation. This is one of the most intriguing features of ESkine/CCL27 , and one that makes it unique amongst currently identified chemokines. The inventors have named this nuclear targeted chemokine PESKY (12), and whilst

ESkine/CCL27 has very restricted expression patterns in vivo, PESKY is widely expressed suggesting a relatively general function for this unusual chemokine variant. This nuclear targeted chemokine therefore potentially represents a novel paradigm for chemokine function.

The inventor has further determined that the nuclear targeting motif in PESKY lies predominantly within the C terminal tail of the mature ESkine protein. Furthermore, they have demonstrated that mature ESkine is translocated to the nucleus following receptor mediated internalisation suggesting that at least aspects of both PESKY and ESkine function are likely to be shared and mediated within the nucleus. In addition, the inventor has examined the impact of PESKY over-expression in NIH- 3T3 cells and demonstrate that PESKY induces marked cytoskeletal changes in NIH-3T3 cells with associated reorganisation of the cellular actin cytoskeleton. These PESKY expressing cells display a much enhanced migratory competence suggesting that a potential role for PESKY is to relax the cytoskeleton and thus facilitate cellular migration and potentially diapedesis.

At its most general, the present invention provides a nuclear targeting peptide comprising a nuclear targeting signal derived from a chemokine, and materials and methods for specifically targeting substances to a cell nucleus using said nuclear targeting peptide.

The inventor has determined that ESkine and PESKY both possess a nuclear targeting signal in the C-terminus of the protein. ESkine enters the cell via the CCR10 receptor and is then transported to the cell nucleus. The inventors have surprisingly determined that a peptide comprising the nuclear targeting signal found in the C- terminus region of ESkine or PESKY can enter cells in a receptor independent fashion and target the cell nucleus.

Further, the inventor has identified the key amino acids present in the C-terminus region of ESkine/PESKY and has found that the presence of these amino acids in other chemokines, particularly constitutive chemokines, indicates the presence of nuclear targeting signal and the ability of these chemokines to target the cell nucleus .

In a first aspect of the present invention, there is provided a nuclear targeting peptide comprising or consisting essentially of an amino acid sequence derived from the C-terminus of a chemokine, preferably ESkine or PESKY. Thus, there is provided a peptide comprising a nuclear targeting domain isolated from a nuclear translocating chemokine.

Preferably, the nuclear targeting peptide will be coupled to a substance (coupling partner) . This substance may be a peptidyl or non-peptidyl substance. If the substance is a further polypeptide, it is preferably a heterologous polypeptide, i.e. a non-chemokine polypeptide. Examples of coupling partners are provided below.

In a preferred embodiment, the nuclear targeting peptide has an amino acid sequence having at least 70% sequence homology with the nuclear targeting signal (domain) of PESKY or ESkine as shown in Fig 7a. Even more preferably, the sequence has at least 75%, 80%, 90%, or 95% sequence homology with the nuclear targeting signal (domain) of PESKY or ESkine as shown in Fig 7a.

In a most preferred embodiment, the nuclear targeting peptide has the nuclear targeting signal (domain) of PESKY or ESkine as shown in Fig 7a. As mentioned above, ESkine and its splice variant PESKY share the same C- terminal sequence. It is this sequence that the inventors have found contains the unexpected nuclear targeting signal. Thus, the nuclear targeting peptide of the first aspect of the invention may comprise amino acid sequence corresponding to the minimum sequence required for nuclear targeting or it may comprise anything up to and including the whole ESkine or PESKY sequence, or a variant, allele, derivative thereof. Preferably, the C- terminal sequence of ESkine and PESKY is considered to be VHPQNRSLARWLERQGKRLQGTVPSLNLVLQKKMYSHPQQQN.

Specifically, the inventor has determined that two basic amino acids, e.g. lysine (K) and Arginine (R) , are essential for the nuclear targeting signal. The two amino acids may be found as K, R, KK, KR, RR or RK. In the case of ESkine and PESKY, the two basic amino acid residues are present in the last 12 amino acids of the C- terminal sequence. Therefore, the nuclear targeting signal preferably comprises amino acid sequence derived from at least the last 12 amino acids of the C-terminus, more preferably at least the last 30 amino acids of the C-terminus which contain a further Lysine and Arginine also considered to be important for nuclear targeting (see Fig. 7a) .

The inventor has determined that the nuclear targeting domain/signal found in the C-terminal sequence of constitutive chemokines comprise one or more basic amino acids at two positions within the domain. The two positions are separated by 12, 13, 14 or 15 amino acids, preferably being non-basic amino acids, i.e. not K or R.

For example, the C-terminus of ESkine comprises two basic amino acids (KR) at a first position and two basic amino acids at a second position (KK) . The first and second positions are separated by 13 amino acids. This separation means that when the polypeptide is folded into an α helix, the two positions of basic amino acids will be in the same face of the helix. In other words, the separation of the positions equates to an even number of helical turns.

The inventor has found that the positioning of the one or more (standardly one, two or three) basic amino acids in the C-terminus of the chemokine indicates the presence of a nuclear targeting domain and consequently the ability of the chemokine and/or its isolated C-terminal sequence to translocated to the nucleus of a cell.

The nuclear targeting peptide preferably comprises between 5 and 50, 5 and 30 or 15 and 50 amino acids, more preferably between 15 and 34, 15 and 30 or between 8 and 15 amino acids derived from the C-terminus of constitutive chemokines, e.g. ESkine/PESKY. The C- terminus of ESkine is considered to be VHPQNRSLAR LERQGKRLQGTVPSLNLVLQKKMYSHPQQQN.

The invention resides primarily in the determination of a nuclear targeting signal and consequently the nuclear targeting domain, being present in ESkine/PESKY. By identifying the nuclear targeting motif, the inventors have determined that other chemokines also have nuclear localization capabilities (e.g. CCL28, TECK, SLC and ELC and MIP-3b see also Fig. 11 and Fig. 12) . This determination means that chemokines can provide a natural receptor-mediated internalization and nuclear localization vehicle which can be used to specifically target disease cells with cytotoxic payloads. The intact or substantially intact chemokine, as opposed to just the nuclear targeting signal, will enter cells in a chemokine receptor dependent fashion. In the case of ESkine, this will be via CCR10. In the case of MIP-3b, this will be via CCR7. However, other chemokines will be specific for other receptors. Thus, by using the intact ESkine coupled to a coupling partner, e.g. a cytotoxin, the construct will enter cells expressing CCR10 and the cytotoxin will be carried to the nucleus owing to the presence of the nuclear targeting signal in the C-terminal domain of ESkine.

Alternatively, it is possible to isolate the nuclear targeting domain from the chemokine and use this to carry coupling partners into cells and transport them to the nucleus. This may be achieved in a receptor independent fashion. It would also be possible to couple the nuclear targeting signal domain to a heterologous peptide or polypeptide e.g. a receptor ligand, that was capable of dictating cell-mediated entry via its specific receptor.

The use of the intact chemokine as the vehicle for carrying a coupling partner into cells has advantages. For example, the inventor has found that chemokine receptors are highly, constitutively and/or aberrantly expressed on tumour cells and other disease cells, and thus these cells can be specifically targeted. See, for example Fig. 10. As chemokine receptors are expressed in precise and restricted expression patterns in vivo there is a lower risk of non-specific cell internalization. Accordingly, the invention further provides a complex comprising a nuclear targeting polypeptide linked to a coupling partner.

The nuclear targeting polypeptide may be a nuclear targeting peptide isolated from a chemokine as described above, or it may be the whole or substantially intact chemokine .

The coupling partner may be any substance that it is desirable to transport to a cell nucleus. Examples of coupling partners include toxins, cell replication or differentiation factor, cell cycle inhibitors, labels e.g. dyes, radioactive labels, negative and positive transcription factors, nucleic acid sequences (anti-sense and sense oligonucleotides or RNA interference molecules (RNAi -double stranded RNA) ) double stranded nucleic acid (RNA or DNA or hybrid RNA/DNA) , polypeptides, small heterologous peptides or non-peptide molecules. Other examples will be apparent to the skilled person.

In a preferred embodiment, the coupling partner is a short range emitter, e.g. Auger emitter, that would be harmless to a host administered with the nuclear targeting peptide but that could kill a cell, e.g. a diseased cell once it is internalized to the nucleus.

In a second aspect of the present invention, there is provided a nucleic acid molecule comprising or consisting essentially of a first nucleic acid sequence encoding a nuclear targeting signal of a chemokine, and a second nucleic acid sequence encoding heterologous polypeptide, i.e. not being derived from said chemokine.

Thus, there is provided a nucleic acid molecule comprising a first nucleic acid sequence encoding a first polypeptide comprising a nuclear targeting domain of a chemokine; and a second nucleic acid sequence encoding a second polypeptide, wherein when expressed said first and second polypeptides form a fusion protein.

The nuclear targeting domain is preferably from chemokines ESkine or PESKY, although other chemokines having nuclear targeting capabilities include CCL28, TECK, SLC, ELC and MIP-3b (see Fig. 8 and Fig. 9 see also Fig. 11 and Fig. 12) .

The first sequence may encode the intact chemokine e.g. ESkine, or substantially intact chemokine, or a fragment of the chemokine, each comprising the nuclear targeting domain.

In accordance with the second aspect of the present invention, the encoded heterologous (second) polypeptide sequence may act as a toxin, label, or a cell replication or differentiation factor e.g. a transcription factor.

The nucleic acid molecule of the second aspect may conveniently be incorporated into a vector which can be taken up by the targeted cell. Suitable vectors will be known to the skilled person and include viral vectors. The first nucleic acid sequence encoding the nuclear targeting signal preferably has a sequence with at least 70% sequence homology with the nuclear targeting signal contained in the C-terminal of PESKY or ESkine as shown in Fig. 7a. It is more preferred that the sequence has at least 75%, 80%, 85%, 90% or 95% sequence homology with the nuclear targeting signal of PESKY or ESkine as shown in Fig. 7a.

In a most preferred embodiment of the second aspect, the first nucleic acid sequence comprises the nuclear targeting signal sequence of PESKY or ESkine as shown in Fig. 7a. As mentioned above, the sequence may comprise the minimum sequence required for the function of nuclear targeting or it may comprise up to and including the whole PESKY or ESkine nucleic acid sequence.

Thus, it is preferred that the first nucleic acid sequence comprises nucleic acid sequence encoding amino acid sequence contained within at least the last 12 amino acids of the C-terminus of ESkine or PESKY, more preferably at least the last 30 amino acids of the C- terminus, even more preferably at least the last 40 amino acids of the C-terminus. Most preferably, the amino acid sequence will be

VHPQNRSLARWLERQGKRLQGTVPSLNLVLQKKMYSHPQQQN.

The nucleic acid sequence encoding the nuclear targeting signal is preferably between 15 and 130 nucleotides in length, more preferably between 15 and 100, or between 15 and 60 nucleotides in length, or between 30 and 60 nucleotides in length. The present invention also provides a host cell comprising the nucleic acid molecule of the second aspect or the vector comprising it.

In a third aspect of the invention, there is provided a pharmaceutical composition comprising a nuclear targeting peptide in accordance with the first aspect of the invention or a nucleic acid molecule in accordance with the second aspect. Preferably, the peptide in accordance with the first aspect is linked to a coupling partner. The pharmaceutical compositions may comprise a chemokine having a nuclear-localization domain (targeting signal) linked to a coupling partner.

These compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. intravenous, intramuscular, intraperitoneal routes.

Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, e.g. the cells, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Whether it is a nuclear targeting construct comprising the whole chemokine or the nuclear targeting signal and the coupling partner, or nucleic acid molecule, according to the present invention that is to be given to an individual, administration is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed) , 1980.

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

As mentioned above, the inventor has found that the peptide comprising the nuclear targeting domain (signal) contained within the C-terminus of ESkine and PESKY can enter cells in a receptor independent fashion.

Thus, a complex comprising a nuclear targeting peptide in accordance to the first aspect of the present invention and a coupling partner, e.g. a tag/label, may be capable of entering cells in a receptor independent manner.

However, if the coupling partner is linked to the full chemokine comprising the nuclear targeting signal, e.g. ESkine, then it will enter cells expressing receptors for that chemokine. For ESkine this will be CCR10.

The present inventor has determined that ESkine is internalized following interaction with at least the CCR10 receptor. Other chemokines are internalized following interaction with other receptors. Examples of other chemokine receptors include CXCR3, CXCR4, CCR7, CXCR5, CCR6 and CCR9.

CCR10 is expressed in a number of tissues including skin, small intestine, colon, brain, lung, liver and testes. Further, CCR10 is over-expressed is Burkitt's lymphoma cells (at least a 10-fold increase in comparison to levels in control B-cells) and Nasopharyngeal carcinoma cells. Malignant melanomas also show high expression of CCR10 in addition to the other chemokine receptors, CXCR4 and CCR7. Of note, CXCR4 and CCR7 are also highly expressed in human breast cancer cells, malignant breast tumours and metastases. Thus, nuclear targeting signals containing chemokines specific for these receptors could be utilized in the treatment of these diseases. The inventors have also shown that receptor CCR10 is highly expressed in EBV positive cells (Fig. 10) and therefore ESkine/PESKY complexes may be used to treat EBV related pathologies .

Thus, the potential use of a pharmaceutical composition according to the present invention is surprisingly high.

In a fourth aspect of the present invention, there is provided a method of producing a nuclear targeting peptide of the invention coupled to a coupling partner, said method comprising the steps of expressing a polypeptide comprising the nuclear targeting signal derived from a chemokine, e.g. PESKY or ESkine and linking said polypeptide to a coupling partner.

The nuclear targeting peptide may comprise the intact or substantially intact chemokine. By substantially intact, it is meant the minimum chemokine polypeptide that comprises the nuclear targeting signal and the specific receptor binding ligand allowing cell internalization in a receptor dependent fashion. Alternatively, the nuclear targeting peptide may comprise a fragment of the chemokine, the fragment comprising the nuclear targeting signal/domain but not the chemokine sequence which determines receptor binding. By using a fragment of the chemokine, it is possible for the nuclear targeting peptide to enter the cell in a receptor independent fashion. Alternatively, a heterologous receptor ligand may be coupled with the nuclear targeting peptide. The receptor ligand will then determine internalization into a cell.

Where the coupling partner is a heterologous polypeptide, this can be expressed with the nuclear targeting signal as a fusion protein. Where the coupling partner is an effector molecule, a label e.g. radioactive label, a drug, a toxin and/or a carrier or transport molecule, these may be linked by a chemical bond such as a co- valent bond. Techniques for coupling the peptide or nucleic acid molecules of the invention to both peptidyl and non-peptidyl coupling partners are well known in the art.

In a fifth aspect of the invention, there is provided a method of treating a cell with a nuclear targeting peptide and coupling partner or a nucleic acid molecule according to the invention, said method comprising the steps of contacting the nuclear targeting peptide or nucleic acid with the cell. The contact may take place in vi tro, in vivo or ex vivo .

The cell may be a diseased cell such as a tumour cell. In this case the coupling partner may be a toxin capable of killing the cell. As mentioned above, the inventor has determined that ESkine is internalized by the cell through contact with the CCRIO. Other chemokines interact with other receptors. Therefore, the cell being contacted by the nuclear targeting peptide and coupling partner complex, preferably expresses a chemokine receptor for the chemokine in question.

Further, it is preferable that the cell being treated is an abnormal cell such as a tumour cell that expresses a chemokine receptor. Such a cell may be a Melanoma cell, a breast cancer cell, a B-cell lymphoma cell etc. The particular chemokine receptor being expressed by the tumour cell can be determined using standard techniques.

Where the cells are to be contacted in vivo, the peptide/coupling partner complex or nucleic acid molecule may be administered in the form of a pharmaceutical. The pharmaceutical may contact the cells by being injected into the tissue of cells, e.g. a tumour.

In accordance with the above, the present invention further provides the use of a nuclear targeting peptide complex or nucleic acid molecule as defined above in the preparation of a medicament for treating cancer. In particular, the cancer is Burkitt's lymphoma, B-cell lymphoma, melanoma, Hodgkin ' s/Non-Hodgkin ' s lymphomas, breast cancer, nasopharyngeal carcinomas etc.

The inventor has determined that the present invention is particularly suited to treating abnormal cells which, as a result of the abnormality, constitutively and/or aberrantly express chemokine receptors such as CCRIO, CCR7 and/or CXCR5.

Alternatively, the cells to be treated may be modified to express chemokine receptors, thus making them susceptible to internalization of a nuclear targeting complex in accordance with the invention. The complex may comprise a cell cycle inhibitor, i.e. a chemotherapeutic which will kill the tumour cells targeted.

Aspects and embodiments of the invention will now be illustrated, by way of example, with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

In the Figures: Figure 1 shows sequences in the body of ESkine/CCL27 mediate nuclear translocation: a) Diagrammatic representation of PESKY indicating the distribution of basic amino acid residues (marked +) and the subdivision of the molecule into regions for determination of nuclear translocation ability; b) Confocal imaging of the subcellular fate of EGFP/PESKY protein fusions in transfected CHO cells. Green coloration is EGFP and red is Propidium Iodide. The individual regions listed correspond to those delineated in Figure la; c) Confocal imaging of the subcellular fate of MlP-lα body/EGFP fusion proteins intransfected CHO cells . Figure 2 shows sequences in the C terminus of ESkine mediate nuclear translocation. The division of ESkine into N and C termini is as outlined in Figure la. Again Green is GFP and red, propidium Iodide. Figure 3 shows ESkine is translocated to the nucleus following receptor mediated internalisation The first column (green) records the presence of Biotinylated ESkine protein. The second column records the propidium iodide staining and the third represents the merging of the green and red signals. Conditions used are indicated to the left of each row.

Figure 4 shows PESKY mediates marked cytoskeletal rearrangements in transfected 3T3 cells a) Morphological examination of vector control and PESKY transfected 3T3 cells b) Visualisation of Actin Stress Fibres in vector control and PESKY transfected cells.

Figure 5 shows Anti IGF-1 receptor antibodies partially reverse the cytoskeletal effects of PESKY. Visualisation of the actin stress fibre content of vector control cells or PESKY transfected cells treated with PBS or anti IGF-1 receptor antibodies for 24 hours. Cells were viewed at a magnification of 40x.

Figure 6 shows PESKY induced cytoskeletal rearrangements correlate with enhanced migratory properties. Vector control and PESKY transfected cells were tested for migratory capacity in a monolayer 'wounding' assay. The white arrows represent distance of migration. Note that the magnification used for the 24hr pictures is less that that used for the others as the PESKY transfected cells had substantially closed the monolayer 'wound' by this stage. Figure 7 shows the sequences of ESkine and PESKY, a) the full length cDNA and protein sequence for ESkine. The signal peptide is underlined, and the putative signal pepidase cleavage site is marked with an arrow. Conserved cysteine residues typical of a β-chemokine are in boldface type and underlined, and the putative N-linked glycosylation site is highlighted in boldface italic type. The key amino acids determined to be associated with the nuclear targeting property of the chemokine are marked by a box, b) the alternative amino-terminal stretch seen in PESKY. From the alanine prior to the LPLP motif, PESKY is identical to ESkine. c) a line up of the murine, rat and human ESkine sequences. Only the mature protein sequences are shown here, and only the stretch of the human homologue inferred from the published human ILllRoc sequence is shown. Identical amino acids are indicated by a vertical line, and conserved alterations are indicated by a colon.

Figure 8 shows the nuclear translocation ability of constitutive chemokines as opposed to inducible chemokines .

Figure 9 shows the entry of exogenously added ESkine (a) or MIP-3b (b) through their receptors expressed on E3e cells. The ESkine receptor is called CCR10 and the MIP-3b receptor is called CCR7. Entry into the nucleus by ESkine or MIP-3b takes about 5 to 10 minutes. Thus, this figure shows the kinetics of nuclear entry and also demonstrates that MIP-3b is another nuclear translocating chemokine . Figure 10 shows a PCR reaction using RNA from

Epstein Barr Virus (EBV) positive or negative lymphomas. As can be seen, there is strong expression of CCR10 in the EBV positive cells compared to the EBV negative cells. Actin is a control. This provides evidence that any EBV related pathologies such as Burkitts lymphoma or nasopharyngeal carcinoma are likely to express high levels of CCRIO and thus be amenable to attack using nuclear targeted ESkine.

Figure 11 and Figure 12 show alignment of amino acid sequences for a number of chemokines. The first and second positions of basic amino acids are shown by underline.

METHODS

Cell culture and transfection : NIH3T3 cells were cultured in DMEM (Life Technologies) supplemented with 10% foetal bovine serum (FBS) and antibiotics and the L1.2/hCCR10 cells were cultured in RPMI-1640 (Life Technologies) supplemented with 10% FBS as previously described (19). CHO cells were cultured in SLM medium (Life Technologies) supplemented with 10% FBS. All transfections were performed using the SuperFect reagent (Qiagen, Crawley,

West Sussex) . For expression of EGFP fusion constructs in CHO cells, 2μg EGFPchemokine constructs were used to transiently transfect semi confluent CHO cells grown in chambered slides (Nunc) . Stable transfectants of NIH3T3 cells were derived by transfecting semi-confluent NIH3T3 cells, grown in 100mm dishes with lOμg of a PESKY- pcDNA3.1 construct or control vector. Stable transfected clones were obtained after selection in 1.5mg/ml G418 and verified by RT-PCR and northern blotting. Morphological changes in NIH3T3-Pesky cells were analysed by staining with Giemsa and examined on a Zeiss Axiovert 25 microscope with a Fuji digital SLR camera. Generation of EGFP-Fusion constructs: Full length PESKY (12) and all other fragments were cloned into pEGFP-Cl or pEGFP-N2 (Clontech) as appropriate and sequenced fully. PCR primers used to facilitate cloning were as follows:

1) PESKY Full length (for C terminal fusion with EGFP) 5 ' -CCGGGATTCATGTCTCCAACA-3 ' and 5'-CTCGTTTTGATTCCTAGGT-3' .

2) ESkine Body (for N terminal fusion with EGFP) 5 ' -GGATCCAGCTGCTGTACT-3 ' and 5'-CTCGTTTTGATTCCTAGGT-3' .

3) PESKY specific N terminus (for C terminal fusion with EGFP)

5 ' -CCGGGATTCATGTCTCCAACA-3 ' and 5' -GGGAGGTCGTGACGCCTAGGACCGGTC-3' .

4) ESkine N Terminus (for N terminal fusion with EGFP) 5 ' -GGATCCAGCTGCTGTACT-3 ' and 5'-CCGAGCCGCGTCATTCCTAGG-3' .

5) ESkine C Terminus (for N terminal fusion with EGFP) : 5'-CCGGGATCCGTCTGTGTTCAT-3' and 5'-CTCGTTTTGATTCCTAGGT-3' .

Analysis of subcellular localisation of GFP fusion constructs: 24hrs after transfection (as outlined above) CHO cells were fixed with 3% paraformaldehyde

(PFA; Sigma) in Phosphate Buffered Saline (PBS), treated with lOOμg/ml RNAse-A for 15min and mounted with mounting medium containing propidium iodide (Vector Laboratories) . Subcellular localisation of GFP fusion constructs was analysed using a BioRad MRC600 confocal microscope (BioRad, UK) with detection of GFP at an excitation of 488nm and an emission of 520/35nm. Propidium Iodide staining was detected at an excitation of 568nm and an emission of 585nm.

Nuclear import assays: L1.2/hCCR10 cells from cultures in linear growth were washed once in fresh medium, twice in ice cold binding medium (RPMI-1640 without bicarbonate but containing lOmM HEPES, pH 7.0; 0.2%BSA) and resuspended at 5xl0δ cells/ml in Binding Medium. lxlOε cell aliquots in 200μl binding medium were incubated with 2μg/ml biotinylated human ESkine (CTACK-Biotin; Gryphon Sciences) by adding 20μl of a stock solution of 200μg/ml Biotinylated ESkine in PBS. Cells were incubated for 1 hour at 37oC with or without sodium azide (0.2%) or for 3 hours at 4oC following which they were centrifuged, to remove unbound chemokine, resuspended in 500μl BM and cytospun with a Cyto-Tek centrifuge at 500rpm for 5min. Slides were fixed in 3% PFA in PBS for lOmin, washed twice in PBS and incubated in quench solution (QS) (50mM NHC1 in PBS) for 20min. After quenching, slides were incubated for 30min in PBS-gelatin containing 0.2% fish- skin gelatine and 0.05% saponin. Permeabilized cells were incubated a monoclonal antibiotin antibody (Sigma) (1:100) for lhr. After staining, slides were washed 4 times with PBS-gelatine and mounted with mounting medium containing propidium iodide.

Actin staining: Stably PESKY transfected NIH3T3 cells were cultured in 8 well chambered slides (Nunc) . Cells in log phase were fixed for lOmin in 3% PFA in PBS and washed in PBS. Fixed cells were quenched in QS for 30min followed by a 30min incubation in PBS-gelatine . Permeabilized cells were incubated for 30 min with lμg/ml phalloidin-FITC (Sigma) in PBS. After incubation slides were washed 3x5min in PBS-gelatine and mounted with mounting medium containing propidium iodide. Actin staining in NIH3T3 cells and LI.2 cells was analysed using a Leica SP2 confocal microscope (Leica, UK) with green fluorescence being detected at an excitation of 488nm and an emission of 505-570nm, and Propidium Iodide being detected at an excitation of 543nm and an emission of 600-715nm.

Anti IGF receptor antibody treatment: IGF-I R antibody treatment: Vector control or PESKY transfected NIH-3T3 cells were seeded at 3 x 104 cells/well, in 2- well chambered slides (Nunc) . Cells were stimulated with PBS alone, or with either 1 μg/ml monoclonal or 5μg/ml polyclonal anti-human IGF-I R antibodies (R&D Systems Europe, Abingdon, UK) for 24 hours. Cells were washed in PBS and then fixed for 10 min in 3 % PFA. Fixed cells were then stained for actin using phalloidin- FITC, as previously described above.

Monolayer Wounding Assays: Vector control or PESKY expressing 3T3 cells were plated in 24 well dishes and grown to confluence. The monolayers were scored with a disposable pipette tip (blue) and the migration of the cells into the resulting 'wound' followed by photographing over 24 hours.

RESULTS The PESKY nuclear targeting signal lies within the mature ESkine sequence

Having identified PESKY as a nuclear targeted variant of ESkine/CCL27 the inventor has sought to determine the structural basis for this nuclear translocation. Nuclear translocation signals (NTS) typically comprise clusters of basic amino acid residues (22) and, as shown in Figure la, PESKY contains a relatively high density of basic amino acids. There are two possible explanations for the nuclear translocation of PESKY. The first is that the NTS lies within the specific PESKY sequence and that basic amino acids in this region are responsible for the carriage of PESKY to the nucleus. In this model therefore, the presence of the PESKY specific N terminus is central to the nuclear translocation process. The second possibility is that the NTS resides within the mature ESkine sequence and that the major function of the PESKY specific N terminal sequence is to replace the signal peptide and thus subvert the secretory process allowing PESKY to be translocated to the nucleus.

To discriminate between these two possibilities the inventor has subdivided PESKY into the PESKY specific sequences and the region of PESKY corresponding to the mature ESkine/CCL27 protein which will henceforth be referred to as the ESkine body (Figure la) . These peptide regions have been incorporated into GFP fusion constructs and their nuclear targeting capability studied following transfection into, and expression in, CHO cells. Full length PESKY is translocated to the nucleus following translation with the majority of GFP staining being seen in the nucleus and very little in the cytoplasm (Figure lb and ref . 12) . Analysis of the two fragments of PESKY reveal that this translocation is not mediated by basic residues within the PESKY specific sequence which does not translocate to the nucleus as shown by the diffuse GFP staining throughout the cells. In contrast, the ESkine body mediates extensive nuclear targeting which is indistinguishable from that seen with the full length PESKY protein. Thus, nuclear targeting of PESKY is mediated by residues within the ESkine body and not by those in the PESKY specific sequence.

To investigate whether this ability of mature ESkine to translocate to the nucleus is shared by other chemokines, the inventor has also generated GFP fusions with MlP-lα incorporating the complete coding sequence for the mature secreted MlP-lα protein. Following expression in transfected CHO cells, and in contrast to the ESkine body, the MlP-lα body does not translocate to the nucleus and is observed to form aggregates within the cytoplasm (Figure lc) . Thus nuclear translocation of PESKY is driven by sequences within the mature ESkine sequence and such nuclear translocation is not a generic property of the chemokine family.

Nuclear translocation of PESKY is predominantly driven by sequences within the C terminus of ESkine.

To more precisely map the NTS within the ESkine body, the inventor has further subdivided it into the C terminus which is particularly rich in basic amino acids and which demonstrates a high degree of evolutionary conservation of basic residues, and the remainder of the N terminus of ESkine (Figure la) . Again, these fragments were used to generate GFP fusion constructs which were then transfected into CHO cells to examine nuclear targeting competence. The analysis of these cells (Figure 2) demonstrates that the N terminus of ESkine is incapable of mediating nuclear translocation whilst the C terminus did support nuclear translocation. Thus, these results demonstrate that nuclear translocation of PESKY is mediated by sequences that reside predominantly within the C terminal tail of mature ESkine.

Mature ESkine is translocated to the nucleus following receptor mediated internalisation

Given that the NTS for PESKY resides within the mature ESkine sequence it is possible that mature secreted ESkine may be able to interact with receptor bearing target cells and be translocated to the nucleus following receptor mediated internalisation. Indeed, whilst this would be an unprecedented finding within the chemokine family, there are precedents from a number of other growth factor families such as the FGFs (23,24) which are translocated to the nucleus following receptor mediated internalisation. To examine possible receptor mediated internalisation and subsequent nuclear translocation of mature ESkine/CCL27 , the inventor has studied the fate of this protein following interaction with its receptor (CCR10) expressed on LI .2 cells (19). This approach has capitalised on the availability of biotinylated forms of ESkine/CCL27 that are fully functional in that they induce a calcium flux following CCR10 binding and display a dose response that is indistinguishable from that seen with the unmodified protein (DJ unpublished observations). This biotinylated protein was therefore applied to CCR10 bearing LI.2 cells as described below

(see methods section) and the subcellular fate of the ESkine/CCL27 examined using an anti-biotin monoclonal antibody. To control for non specific staining or staining associated artefacts the inventor has performed these experiments at both 37oC at which temperature internalisation should take place and also at 4oC or at

37oC in the presence of sodium azide, both of which should block energy dependent internalisation resulting in exclusively membrane associated staining. The results of these experiments are shown in Figure 3 and demonstrate that, as expected, at 37oC in the presence of sodium azide, or at 4oC, biotinylated ESkine remains predominantly associated with the Ll.2 cell membrane. In contrast, results from CCRIO bearing Ll.2 cells treated with biotinylated ESkine at 37oC show it to be effectively internalised. Additionally, whilst Ll.2 cells display a high nuclear to cytoplasmic ratio, it is still clear from the Biotin/PI merged picture that this protein is localised predominantly within the nucleus. As a further control the inventor has examined the fate of Biotinylated ESkine following interaction at 37oC with Ll.2 cells carrying the CXCR3 receptor which does not bind ESkine/CCL27. These cells displayed neither surface nor intracellular staining for ESkine/CCL27 confirming the requirement for CCR10 for internalisation and nuclear trafficking (data not shown) . Thus mature secreted ESkine can interact with cells in a paracrine manner and can enter the nucleus of target cells following receptor mediated internalisation. This indicates that at least aspects of the functions of ESkine and PESKY are likely to be common and mediated within the nucleus. PESKY induces morphological changes and actin cytoskeletal re-organisation in 3T3 cells

Given the demonstrable expression of PESKY in the majority of murine tissues (12) the inventor has reasoned that it is likely to have a role that is evident in many diverse cell types. Indeed, his preliminary data on cell specific expression indicated expression in cells as diverse as micro-glial cells in the brain and spermatogonia in the testes (AG and GJG unpublished) . Given the likely widespread effects of this protein they regarded it as appropriate to study its biological function in IH-3T3 fibroblasts for which numerous cellular and biochemical parameters can be measured. Thus, to examine the potential biological roles for PESKY, the inventor has generated NIH-3T3 cell clones stably expressing this protein. During repeated attempts it has proven difficult to obtain large numbers of stable transfected clones and, typically, those clones that are obtained express only low levels of PESKY transcripts. Simple morphological examination of all isolated clones of PESKY expressing NIH-3T3 in comparison to vector control NIH-3T3 cells revealed marked cytoskeletal abnormalities in the transfectants (Figure 4a) with the extent of cytoskeletal abnormalities correlating directly with the expression levels of PESKY in the individual clones. The PESKY expressing cells typically display shrunken nuclei and sparse cytoplasm with numerous filopodia extending out from the cells.

Morphologically, these PESKY expressing cells resemble

Ras transformed fibroblasts (25). However, these cells do not appear to be transformed as they do not display any capacity for anchorage independent growth and show no alterations in sensitivity to serum withdrawal (data not shown) . In fact although these cells are morphologically quite different from the vector control cells, they display an identical proliferative rate, doubling approximately every 24 hours during log phase growth. There is no evidence of excessive cell death amongst these transfectants and they can be passaged to high numbers without obvious loss of viability.

Typically, marked cytoskeletal alterations such as those seen in the PESKY expressing fibroblasts result from a re-arrangement of components of the cellular actin cytoskeleton (26) . This actin cytoskeleton which is represented in the form of stress fibres of aggregated actin in fibroblasts can be visualised using Phalloidin (27) .

Thus, to attempt to determine the role of actin re- arrangements in the cytoskeletal abormalities in PESKY transfectants, the inventor has examined the actin cytoskeleton in these cells. As can be seen from Figure 4b, Phalloidin staining of the vector control NIH-3T3 cells reveals a typically dense arrangement of actin stress fibres. In contrast, examination of the PESKY transfected NIH-3T3 cells reveals a striking reorganisation of the actin cytoskelton with a marked absence of actin stress fibres in comparison to the vector control cells. This absence of stress fibre associated actin staining is co-incident with the emergence of numerous densely stained filopodia emanating from the PESKY transfected cells and additionally with an increase in membrane associated filamentous actin. As with the gross morphological observations, the extent of the reorganisation of the actin cytoskleton correlates well with the levels of PESKY expression in the different transfected clones examined.

Thus, this data shows that nuclear targeted PESKY induces profound cytoskeletal changes in NIH-3T3 cells resulting predominantly from a radical re-alteration of the cellular actin cytoskeleton.

The Cytoskeletal effects of PESKY are mediated in part by Insulin Like Growth Factor-1

To examine the molecular basis for the cytoskeletal effects of PESKY expression in NIH-3T3 cells, the inventor compared the transcriptomes of vector control and PESKY expressing cells using gene array technology. Surprisingly few differences in gene expression were detected between these two cellular populations (data not shown) . Indeed only 17 genes were identified as being present at significantly higher levels (>2.5 fold higher) in the PESKY expressing cells compared to the vector control cells. In addition, only 21 genes were identified that were present at significantly lower levels (>2.5 fold lower) in the PESKY cells compared to the control cells. It has been difficult to identify a role for the majority of these genes in the induction and maintenance of the PESKY related phenotype. However one of the most highly over-expressed genes in the PESKY expressing cells is Insulin Like Growth Factor-1 (IGF-1; 5.4 fold higher expression in the PESKY cells compared to control cells) and IGF-1 is interesting in this context as treatment of NIH-3T3 cells or other cells with this growth factor induces cytoskeletal alterations similar to those seen in the PESKY transfected cells (28). Further, PCR analysis has confirmed elevated expression of IGF-1 throughout a panel of PESKY transfectants (data not shown) .

To attempt to examine a role for IGF-1 as an intermediate in the mechanism of action of PESKY, the inventor has used antibodies to the IGF-1 receptor and have studied the impact of these antibodies on the cellular cytoskeleton. As shown in Figure 5, anti-IGF-1 receptor antibodies have no discernable effects on the actin cytoskeleton in vector control NIH-3T3 cells. However, as also shown in Figure 5, treatment of the PESKY expressing NIH-3T3 cells with this antibody induces significant reformation of actin stress fibres indicating at least a partial role for IGF-1 as an intermediate in the cytoskeletal effects of PESKY.

PESKY transfected NIH-3T3 cells display higher motility than control NIH-3T3 cells

As reorganisation of the cytoskeleton is typically associated with cellular migration (29) with stress fibres being regarded as anti migratory structures, the inventor has sought to examine the migratory potential of the PESKY transfected NIH-3T3 cells. To do this he performed monolayer 'wounding' assays as described below (see methods section) . This assay involves growing the adherent NIH-3T3 cells to confluence and then 'wounding' the monolayer by scoring it with a disposable pipette tip. Wild type NIH-3T3 cells will migrate into the space created over 48 hours (30) and the effect of expressed genes on this motility can therefore be measured by photographing the cells throughout the migratory process. As shown in Figure 6, whilst it took 8 hours to see the initial signs of migration in the control NIH-3T3 cells, the PESKY transfected cells displayed initial migration at 4 hours which was marked by 8 hours and which had significantly closed the gap generated by the wounding by 24 hours. Even by 24 hours the migration seen with the control NIH- 3T3 cells was, at best, equivalent to that seen with the PESKY transfected cells at 4 hours. Thus this data demonstrates that PESKY mediates an increase in motility of transfected NIH-3T3 cells and this apparently correlates with a rearrangement of the cellular actin cytoskeleton.

DISCUSSION

The inventor has recently described the chemokine ESkine/CCL27 as exhibiting radical differential splicing of a form that is currently unprecedented within the wider chemokine family (12). This splicing generates two variants one of which bears a signal peptide and is secreted from producer cells and which is identical to the previously described CTACK (13), ALP (14) and ILC

(15) proteins. The second splice variant, in contrast, does not have a signal peptide and has replaced this with a non signal peptide competent stretch of amino acids. This non secreted isoform is called PESKY and displays strong nuclear localisation tendencies following expression in a range of cell types. This differential splicing is mediated by the use of alternative first exons (12). As detailed above, the inventor has analysed the nuclear targeting ability of PESKY in more detail and demonstrate that the NTS resides within the main body of mature ESkine, predominantly at the C terminus. The presence of the NTS at the C terminus of ESkine is consistent with the high density of basic amino acids in this region and the strong evolutionary conservation of these charges (as seen in mouse, rat and human proteins) argues for the importance of this region in ESkine function.

Given the fact that the NTS resides in the mature ESkine sequence, the inventor has reasoned that it is possible, as is seen with other growth factors (23,24), that mature ESkine may be able to enter target cells by interacting with its receptor (CCR10) following which it may be able to migrate to the nucleus where it mediates a number of its biological effects. Indeed, using CCR10 transfectants the inventor demonstrates here that exogenous ESkine can enter CCR10 bearing cells and translocate to the nucleus following receptor mediated internalisation. Thus, at least for aspects of ESkine function, the receptor may serve more as a gateway into the cell, facilitating nuclear translocation, rather than as the primary mediator of biological function. It is important to note however that ESkine/CCL27 does trigger a downstream signal following receptor binding (19,20) and thus it may be that there are dissociable activities associated with this protein that separately require receptor signalling or receptor mediated internalisation. Whilst the inventor has demonstrated the above for ESkine/CCL27 and have shown that MlP-lα does not translocate to the nucleus, it has also been shown that post receptor nuclear translocation of chemokines is a more widespread phenomenon. Indeed, there have been occasional reports of the detection of other chemokines (CTAPIII and GRO alpha) in the nucleus of leukocytes (31,32). Further more, a number of beta chemokines such as SLC, MIP-3β and CCL28 (which also binds to CCRIO) have high densities of basic amino acids at their C-termini and may also be candidate nuclear localising chemokines. Indeed the inventors' preliminary results indicate that a number of other beta- chemokines will translocate to the nucleus as GFP fusion proteins indicating that nuclear localisation of chemokines is indeed be a more widespread phenomenon than previously suspected. The inventor has also determined basic amino acid motifs within the C-terminal sequence of the chemokines which indicates the presence of the nuclear localising signal.

With a view to examining the possible roles for nuclear targeted ESkine/PESKY, the inventor has generated stable NIH-3T3 PESKY transfectants. The first observation that they made was that it is very difficult to derive stable clones of PESKY transfectants and those that were obtained displayed only low levels of expression. Nevertheless, these cells revealed marked morphological alterations compared to wild type or vector control NIH- 3T3 cells. The morphology of these PESKY transfectants resembles that of NIH-3T3 cells transformed with oncogenes such as Ras (25) . However these PESKY transfectants do not appear to be transformed as they do not display anchorage independent growth and have a sensitivity to serum withdrawal that is indistinguishable from that seen with control cells. The inventors have also demonstrated that a radical re-organisation of the cellular actin cytoskeleton underlies the morphological alterations and that this apparent relaxation of the fibroblast cytoskeleton is associated with an enhanced migratory potential as revealed in the monolayer wounding assays. It appears from the antibody studies reported in Figure 5 that IGF-1 is involved in the effects of PESKY. It is important to emphasise that the antibody treated PESKY cells do not display a complete reversal of phenotype. However, the increase in stress fibre formation seen in the antibody treated cells is marked indicating that IGF1 has a significant role to play in mediating the effects of PESKY. Intriguingly, a recent study has reported further interactions between IGF1 and the chemokine family with IGF1 being capable of inducing expression of chemokines (33). One other over-expressed gene in the PESKY expressing 3T3 cells is the chemokine CIO and thus it is possible that IGF1 may be an intermediate step in a more complex network of cytoskeletal regulators induced by PESKY.

Interestingly, the cytoskeletal and actin rearrangements associated with PESKY expression are very similar to those seen in cells treated with other growth factors such as Growth Hormone (34), Transforming Growth Factor-α (35) and, as mentioned above, Insulin Like Growth Factor- 1 (Goh et al , 2000) . In addition, cdc42 dependent transient rearrangement of the actin cytoskeleton been shown to be involved in the migratory response to chemokines (Weber et al , 1998; Burger et al , 1999; Yayoshi-Yamamoto et al , 2000). So, what do the inventor's observations tell us about the role of PESKY in cell function/migration? It appears that PESKY relaxes the cellular cytoskeleton and previous reports indicating the negative influence of stress fibers on cell motility (Stossel, 1993; Coutant et al , 1997) are in keeping with our observations of enhanced motility in the PESKY expressing cells. By identifying the precise cell types expressing PESKY in different tissues one can determine the role of PESKY in regulating the cells motility requirements. In the wider chemokine context, as nuclear translocation of chemokines is a more widespread phenomenon, then it is possible that the nuclear mediated disruption of the actin cytoskeleton may be a common mechanism for facilitating cell movement and transendothelial migration.

In summary therefore the inventor has demonstrated that ESkine and PESKY are targeted to the cell nucleus by sequences within the C-terminus of the mature ESkine protein and that this nuclear targeting can also take place following receptor mediated internalisation. Furthermore the nuclear translocation of PESKY is associated with marked cytoskeletal rearrangements involving alterations to the cellular actin cytoskeleton. These rearrangements are associated with enhanced motility of the PESKY expressing cells. These actions, that are likely to be mediated within the nucleus, reveal ESkine/CCL27 to display an unprecedented mode of action that therefore enhances our understanding of the wider capabilities of members of the chemokine family of proteins.

Following his work on ESkine and PESKY, the inventor has also determined key sequences within the nuclear targeting domain which enable us to predict the ability of other chemokines to translocate to the nucleus .

This information allows the use of nuclear targeting peptides isolated from chemokines other than ESkine/PESKY, or indeed the intact chemokines themselves to act as transporters to carry substances to a cell nucleus.

The inventor has also determined that the nuclear targeting peptides may possess the ability to enter a cell in a receptor independent fashion. Further, they can be manipulated (e.g. through coupling with a receptor specific ligand) to enter cells in a receptor dependent fashion. This knowledge allows the development of complexes between the nuclear targeting peptide and a substance, or for example, a chemotherapeutic which can be used in therapy.

References

1) Rollins, B.J. (1997). BLOOD, 90, 909-928.

2) Baggiolini, M. (1998) NATURE., 392, 565-568.

3) Kelner, G. S., Kennedy, J. , Bacon, K.B., Kleyensteuber, S., Largaespada, D.A.,

Jenkins, N.A., Copeland, N.G., Bazan, J.F., Moore, K.W., Schall, T.J. and Zlotnik, A. (1994). SCIENCE, 266,1395-1399.

4) Bazan, J. F., Bacon, K.B., Hardiman, G., Wang, W., Soo, K., Rossi, D., Greaves,

D.R., Zlotnik, A. and Schall, T.J. (1997). NATURE, 385, 640-644.

5) Pan Y., Lloyd, C, Zhou, H., Dolich, S., Deeds, J. , Gonzalo, J.A. , Vath, J. , Gosselin, M., Ma, J.Y., Dussault, B., Woolf, E., Alperin, G., Culpepper, J. ,

Guitierrez-Ramos, J.C. and Gearing, D. (1997) . NATURE, 387, 611-615.

6) Murphy, P.M., Baggiolini, M. , Charo, I.F., Herbert, C.A., Horuk, R., Matsushima,

K., Miller, L.H., Oppenheim, J.J. and Power, C.A. (2000).

PHARMACOLOGICAL

REVIEWS, 52, 145-176.

7) Nagasawa, T., Hirota, S., Tachibana, K. , Takakura, N., Nishikawa, S., Kitamura,

Y., Yoshida, N., Kikutani, H. and Kishimoto, T. (1996). NATURE, 382, 635-638.

8) Strieter, R.M., DiGiovine, B., Polverini, P.J., Kunkel, S.L., Shanafelt, A., Hesselgesser, J. , Horuk, R. and Arenberg D.A.. (1999). In: Rollins, B.J. (ed) CHEMOKINES AND CANCER chapter 9, pp. 143-167. Humana Press: New Jersey.

9) Graham, G.J. (1997). BALLIERE'S CLINICAL HAEMATOLOGY, 10, 539-559. 10) Broxmeyer, H.E. and Kim, CH. (1999) . In: B.J. Rollins Ed. CHEMOKINES AND CANCER, Humana Press, NJ. pp. 263-291.

11) Baird, J.W., Ryan, K.R., Hayes, I., Hampson, L., Heyworth, CM., Clark, A., Wootton, M., Ansell, J.D., Menzel, U., Hole, N. and Graham, G.J. (2001). J. BIOL. CHEM., 276, 9189-9198.

12) Baird, J.W., Nibbs, R.J.B., Komai-Koma, M., Connolly, J.A., Ottersbach, K. , Clark-Lewis, I., Liew, F.Y. and Graham, G.J. (1999). J. BIOL. CHEM. 274, 33496- 33503.

13) Morales, J. , Homey, B., Vicari, A. P., Hudak, S., Oldham, E., Hedrick, J. , Orozco, J., Copeland, N.G., Jenkins, N.A., McEvoy, L. and Zlotnik, A. (1999). PROC . NATL. ACAD. SCI. (USA) 96, 14470-14475.

14) Hromas, R. , Broxmeyer, H.E., Kim, C, Christopherson, K. and Hou, Y-H. (1999). Biochem. Biophys . Res. Comm. 258, 737-740.

15) Ishikawa-Mochizuki, I., Kitaura, M. , Baba, M., Nakayama, T., Izawa, D., Imai,

T., Yamada, H., Hieshima, K., Suzuki, R. , Nomiyama H. and Yoshie, 0. (1999) .

FEBS LETT. 460, 544-548.

16) Zlotnik, A. and Yoshie, 0. (2000). IMMUNITY, 12, 121-

127. 17) Nakano, H. and Gunn, M.D. (2001). J. IMMUNOL. 166, 361-369.

18) Marchese, A., Docherty, J.M., Nguyen, T., Heiber, M., Cheng, R. , Heng, H.H.Q., Tsui, L-C, Shi, X., George, S.R. and 0' Dowd, B.F. (1994). GENOMICS, 23, 609- 615.

19) Jarmin, D.I., Rits, M., Bota, D. , Gerard, N.P., Graham, G.J., Clark-Lewis, I. and Gerard, CG. (2000). J. IMMUNOL. 164, 3460-3464.

20) Homey, B., Wang, W., Soto, H., Buchanan, M.E., Wiesenborn, A. , Catron, D. ,

Muller, A., McClanahan, T.K., Dieu-Nosjean, M-C, Orozco, R., Ruzicka, T., Lehmann, P., Oldham, E. and Zlotnik, A. (2000). J. IMMUNOL. 164, 3465-3470.

21) Wang, W., Soto, H., Oldham, E.R., Buchanan, M.E., Homey, B., Catron, D. ,

Jenkins, N., Copeland, N.G., Gilbert, D.J., Nguyen, N., Abrams, J. , Kershenovich, D.,

Smith, K., McClanahan, T., Vicari, A. P. and Zlotnik, A. (2000) . J. BIOL. CHEM. 275, 22313-22323.

22) Jans, D.A. and Hassan, G. (1998). BIOESSAYS, 20, 400- 411.

23) Kiefer, P., Acland, P., Pappin, D., Peters, G. and Dickson, C (1994) . EMBO J.

13, 4126-4136. 24) Antoine, M., Reimers, K. , Dickson, C and Kiefer, P.

(1997) . J. BIOL. CHEM. ,

272, 29475-29481.

25) Johnston, S.R.D. (2001). LANCET ONCOL., 2, 18-26. 26) Hall, A. (1998). SCIENCE, 279, 509-514.

27) Wulf, E., Deboben, A., Bautz, F.A. , Faulstich, H. and Wieland, T. (1979) .

PROC NATL. ACAD. SCI. USA, 76, 4498-4504. 28) Goh, E.L.K., Zhu, T., Yakar, S., LeRoith, D. and Lobie, P.E. (2000). J. BIOL. CHEM. 275, 17683-17692.

29) Nabi, I.R. (1999). J. CELL SCI. 112, 1803-1811.

30) Malliri, A., Symons, M., Hennigan, R.F., Hurlstone, A.F.L., Lamb, R.F.,

Wheeler, T. and Ozanne, B.W. (1998) . J. CELL. BIOL. 143, 1087-1099.

31) Walz, A., Schmutz, P., Mueller, C and SchnyderCandrian, S. (1997). J. LEUK. BIOL. 62, 604-611.

32) Nilsson, C.L., Murphy, CM. and Eckman, R. (1997). RAPID

COMMUNICATIONS IN MASS SPECTROMETRY . 11, 610-612.

33) Mira, E., Lacalle, R.A. , Gonzalez, M.A., Gomez- Mouton, C, Abad, J.L., Bernad,

A., Martinez-A, C. and Manes, S. (2001). EMBO REPORTS, 2, 151-156.

34) Goh, E.L.K., Pircher, T.J., Wood, T.J.J., Norstedt, G., Graichen, R. and Lobie, P.E. (1997). ENCODRINOLOGY, 138, 3207-3215.

35) Warren, R.H. (1997). EXP. CELL RES. 236, 294-303.

36) Weber, K.S.C, Klickstein, L.B., Weber, P.C and Weber, C (1998) . EUR. J.

IMMUNOL. 28, 2245-2251. 37) Burger, J.A., Burger, M. and Kipps, T.J. (1999). BLOOD, 94, 3658-3667. 38) Yayoshi-Yamamoto, S., Taniuchi, I. and Watanabe, T (2000) . MOL. CELL.

BIOL. 20, 6872-6881.

39) Stossel, T.P. (1993). SCIENCE, 260, 1086-1094.

40) Coutant, K.D., Corvaia, N. and Ryder, N.S. (1997). BIOCHEM. BIOPHYS.

RES. COMM. 237, 257-161.

Claims

Claims

1. A nuclear targeting peptide comprising a nuclear targeting domain isolated from a chemokine.

2. A nuclear targeting peptide according to claim 1 wherein the nuclear targeting domain is isolated from the C-terminal sequence of the chemokine.

3. A nuclear targeting peptide according to claim 1 or claim 2 wherein the chemokine is a constitutive chemokine.

4. A nuclear targeting peptide according to any one of the preceding claims wherein the chemokine is ESkine/CCL27, PESKY, CCL19, CCL28, TECK, SLC, ELC or MIP-3b.

5. A nuclear targeting peptide according to any one of the preceding claim wherein the nuclear targeting domain is isolated from the C-terminal sequence of the chemokine and comprises one or more basic amino acids at a first position and one or more basic amino acids at a second position within the sequence, wherein the first and second positions are separated by between 12 to 15 amino acids.

6. A nuclear targeting peptide according to claim 5 wherein the first and second positions are separated by 13 or 14 amino acids.

7. A nuclear targeting peptide according to claim 5 or claim 6 wherein the one or more basic amino acids are selected from K, R, KK, KR, RR or KR.

8. A nuclear targeting peptide according to any one of claims 5 to 7 wherein the nuclear targeting domain comprises between 15 and 50 amino acids.

9. A nuclear targeting peptide according to any one of claims 1 to 4 wherein the nuclear targeting domain has an amino acid sequence having at least 70% homology with the C-terminal sequence of ESkine as shown in Fig. 7a.

10. A nuclear targeting peptide according to claim 9 wherein the C-terminal sequence consists of the sequence VHPQNRSLARWLERQGKRLQGTVPSLNLVLQKKMYSHPQQQN.

11. A nuclear targeting peptide according to any one of the preceding claims further comprising a heterologous polypeptide sequence.

12. A nuclear targeting peptide according to any one of claims 1 to 10 coupled to a nucleotide sequence.

13. A nuclear targeting peptide according to claim 12 wherein the nucleotide sequence is selected from the group consisting of anti-sense and sense oligonucleotides, RNA interference molecules and double stranded nucleic acid.

14. A complex comprising a nuclear targeting peptide according to any one of the preceding claims linked to a coupling partner.

15. A complex according to claim 14, wherein the coupling partner is a heterologous polypeptide sequence.

16. A complex according to claim 15 wherein said heterologous polypeptide sequence is selected from the group consisting of a cell toxin, cell replication factor, cell cycle inhibitor, cell differentiation factor, label, negative transcription factor, positive transcription factor and a receptor binding ligand.

17. A complex according to claim 14 wherein the coupling partner is a nucleic acid sequence.

18. A complex according to claim 17 wherein the nucleic acid sequence is antisense oligonucleotide, sense oligonucleotide, RNA interference molecules or double- stranded RNA.

19. A complex according to claim 14 wherein the coupling partner is a non-peptidyl substance.

20. A complex according to claim 20 wherein the coupling partner is a short-range radioactive emitter.

21. A complex for transporting a substance to the nucleus of a cell, said complex comprising the substance coupled to a first polypeptide having a nuclear localisation domain of a constitutive chemokine.

22. A complex according to claim 21 wherein said substance is a second polypeptide linked to said first polypeptide as a fusion protein.

23. A complex according to claim 21 or claim 22 wherein said second polypeptide is a cell toxin, cell replication factor, cell differentiation factor, label, negative transcription factor or a positive transcription factor.

24. A complex according to claim 23 wherein said first polypeptide comprises a fragment of a consitutive chemokine which comprises the nuclear localisation domain.

25. A complex according to claim 24 wherein said fragment comprises at least 12 amino acids from the C-terminus of the chemokine.

26. A complex according to claim 23 or claim 24 wherein said fragment has a sequence having at least 70% homology with the sequence VHPQNRSLARWLERQGKRLQGTVPSLNLVLQKKMYSHPQQQN.

27. A complex according to any one of claims 24 to 26 wherein the fragment consists of the sequence VHPQNRSLARWLERQGKRLQGTVPSLNLVLQKKMYSHPQQQN.

28. A complex according to any one of claims 21 to 23 wherein the first polypeptide comprises a constitutive chemokine having a nuclear localisation domain.

29. A complex according to any one of claim 21 to 28 wherein the chemokine is selected from ESkine, PESKY,

CCL19, CCL28, CCL27, TECK, SLC, ELC or MIP-3b.

30. A nucleic acid sequence encoding a nuclear targeting peptide according to any one of claims 1 to 10.

31. A nucleic acid sequence comprising a first sequence encoding a nuclear targeting peptide according to any one of claims 1 to 10 and a second sequence encoding a heterologous polypeptide.

32. A nucleic acid sequence according to claim 31 wherein the heterologous polypeptide is a cellular toxin, label, cell replication factor, cell differentiation factor, cell transcription factor or a receptor binding ligand.

33. An expression vector comprising a nucleic acid sequence according to any one of claims 30 to 32.

3 . A host cell comprising an expression vector according to claims 33 or a nucleic acid sequence according to any one of claims 30 to 32.

35. A pharmaceutical composition comprising a nuclear targeting peptide according to any one of claims 1 to 13.

36. A pharmaceutical composition comprising a complex according to any one of claims 14 to 29.

37. A pharmaceutical composition comprising a nucleic acid sequence according to any one of claims 30 to 32.

38. A pharmaceutical composition comprising an expression vector according to claim 33.

39. A pharmaceutical composition comprising a host cell according to claims 34.

40. A method of producing a nuclear targeting peptide according to any one of claims 1 to 10 comprising the steps of expressing a nucleic acid sequence encoding said nuclear targeting peptide in a cell and isolating said peptide from the cell.

41. A method according to claim 40 further comprising linking said peptide to a coupling partner.

42. A method of transporting a substance to a cell nucleus comprising the steps of contacting said cell with a complex according to any one of claims 14 to 29.

43. A method according to claim 42 wherein said cell is a tumour cell.

44. A method of transporting a substance to a cell nucleus comprising the steps of contacting said cell with a complex comprising the substance coupled to a chemokine or fragment thereof having a nuclear targeting domain, wherein the cell displays receptors for said chemokine.

45. A method according to claim 44 wherein said chemokine is ESkine or PESKY and the chemokine receptor is CCR10.

46. A method according to claim 44 wherein said chemokine is MIP-3b and the chemokine receptor is CCR7.

47. A method according to any one of claims 44 to 46 wherein the cell is a tumour cell.

48. Use of a nuclear targeting peptide according to any one of claims 1 to 13 in the preparation of a medicament for treating cancer.

49. Use of a complex according to any one of claims 14 to 29 in the preparation of a medicament for treating cancer.

50. Use according to claim 48 or claim 49 wherein the cancer is Burkitts lymphoma or nasopharyngeal carcinoma.