AU2005280819A2 - Novel peptides and methods for the treatment of inflammatory disease - Google Patents

Description

WO 2006/028393 PCT/NZ2005/000234 -1- Novel Peptides and Methods for the Treatment of Inflammatory Disease

FIELD

The present invention relates to novel peptides, nucleic acids encoding same, pharmaceutical compositions comprising said peptides or nucleic acids, methods for modulating 37 integrin function, including methods for the treatment of inflammatory disorders, antibodies directed to said peptides, and methods for the identification of integrin p7 functional interactors.

BACKGROUND

The precise control of leukocyte adhesivity is critical in maintaining effective homeostasis of the immune response, for lymphocyte motility, homing, and recirculation, the localization of leukocytes at sites of inflammation, and antigen presentation. A small subset of integrins, namely a4, (32, and 37 integrins, largely controls leukocyte adhesion, and related functions The integrins are a superfamily of transmembrane receptors which mediate cellextracellular matrix and cell-cell interactions. Each integrin consists of noncovalently paired alpha and beta subunits. There are presently 8 beta and 18 alpha subunits known. The 37 subunit partners with two a chains to form two heterodimeric molecules, namely a437 and aEp7.

Integrin adhesivity is regulated by a complex array of intracellular signalling pathways that impinge on integrin subunit cytoplasmic domains, and trigger changes in integrin conformation, clustering affinity for ligands and cell spreading all of which contribute to increased cell adhesion In the case of P7 integrins, it has been demonstrated that small GTP-binding proteins induce integrin xa47-mediated T cell adhesion to the mucosal addressin MAdCAM-1 in a hierarchical fashion, by a mechanism predominantly involving changes in receptor avidity due to ligand-induced clustering The control of 37-integrin adhesion is critical as the two P7 integrins, ca4P7 and aEp7, play key roles in forming and maintaining gut immunity (10, 11), and a437 contributes to leukocyte infiltration into WO 2006/028393 PCT/NZ2005/000234 -2the islets of Langerhans in Type I diabetes and the central nervous system in demyelinating diseases such as multiple sclerosis ca47 mediates the adherence of lymphocytes to high endothelial venules (HEV) at such chronically inflamed sites via its preferred ligand MAdCAM-1 (14, 15), whereas aE37 mediates the adhesion of intraepithelial lymphocytes to the intestinal epithelium by an interaction with Ecadherin (17,18).

Additionally, "inside-out" transmembrane signalling pathways reveal that the short (-47-66 amino acid residues) integrin P subunit cytoplasmic domains serve as substrates for cellular kinases, and are phosphorylated upon cell activation (19-22).

Certain p subunit cytoplasmic domains directly interact with, and share, cytoskeletal elements including talin, a-actinin, paxillin, and filamin. Some share intracellular signalling molecules such as integrin-linked kinase (ILK), and Rackl, yet potentially associate in an exclusive fashion with an array of other intracellular signalling molecules that may ultimately dictate the nature of integrin-specific "inside-out" and "outside-in" signalling pathways. The human WD repeat protein WAIT-1 specifically interacts with the cytoplasmic tails of p7-integrin a and P subunits (p7, a4, and aE), but not those of integrin 31, 32, and cL subunits (23).

Notwithstanding the above knowledge, the nature and identity of the molecules which associate with the integrins and regulate their activity are largely unidentified.

Similarly, the regulatory sites or motifs present within the integrin subunits have not been fully characterised. Accordingly, there is still much to be understood of the precise mechanisms which allow for regulation of integrin activity and concomitantly cell-cell or cell-extracellular matrix interactions.

In light of the role integrins play, particularly 17 integrins, in regulating leukocyte activity and targeting, and their implication in the development of certain inflammatory disorders, elucidating the precise mechanisms by which their function may be regulated may allow for control thereof, with concomitant amelioration of relevant inflammatory disorders.

WO 2006/028393 PCT/NZ2005/000234 -3- Bibliographic details of the publications referred to herein are collected at the end of the description.

Abbreviations used herein: p7cyt, integrin 37 subunit cytoplasmic domain; CARD, cell adhesion regulatory domain; GST-glutathione S transferase; ICAM-1, intercellular adhesion molecule-1; MAdCAM-1, mucosal addressin cell adhesion molecule-1; PMA, 4p-phorbol 12 myristate 13-acetate; VCAM-1, vascular cell adhesion molecule- 1.

OBJECT

It is an object of the present invention to provide novel peptides, nucleic acids encoding same, derivatives of said peptides, pharmaceutical compositions comprising said peptides, derivatives thereof, or nucleic acids, methods for modulating p7 integrin function, including methods for the treatment of inflammatory disorders, antibodies directed to said peptides, and/or methods for the identification of integrin 37 functional interactors and mimetics of said peptides and derivatives thereof.

It is a further, or alternative object to at least provide the public with a useful choice of any one or the above.

STATEMENT OF INVENTION In accordance with the present invention the inventors have identified a functional motif in the P7 cytoplasmic domain that controls clustering and adhesion of 37 integrins, particularly the integrin a437. It has been surprisingly discovered that this functional motif, which encompasses residues 735-740 of the transmembraneproximal region of the cytoplasmic tail of the 17 subunit, provides a peptide YDRREY (37-8) which inhibits the adhesion of p7 integrins to their ligands, as is exemplified hereinafter in relation to a4p7-mediated adhesion of mouse TK-1, and human H9 T cells, to MAdCAM-1, VCAM-1, and RGD-polymer. Peptides carrying the YDRREY motif, or nucleic acids encoding same, may provide novel anti-inflammatory reagents WO 2006/028393 PCT/NZ2005/000234 -4for the treatment of inflammatory disorders.

Accordingly, in one aspect of the present invention there is provided an isolated peptide comprising at least the amino acid sequence YDRREY, or a derivative of said peptide.

In another aspect, the present invention provides a peptide consisting of the amino acid sequence RLSVEI YDRREY, or a derivative of said peptide.

In another aspect, the invention provides a peptide comprising at least the amino acid sequence YDRLEY, or a derivative of said peptide.

In a related aspect, the invention provides a peptide as herein before described or a derivative thereof, including a cell membrane translocating motif. Preferably, said cell membrane translocating motif is peptide-based. More preferably, said cell membrane translocating motif is penetratin or a polymer of arginine.

In another aspect, the present invention provides isolated nucleic acids which encode a peptide or derivative thereof in accordance with the invention.

In a related aspect, the invention provides constructs or vectors comprising nucleic acids which encode a peptide or derivative thereof in accordance with the invention.

In a further aspect, the present invention provides a pharmaceutical composition comprising at least a peptide in accordance with the invention, or derivative thereof, together with one or more pharmaceutically acceptable diluents, carriers and/or excipients.

In a related aspect, the present invention provides a pharmaceutical composition comprising at least a nucleic acid or construct in accordance with the invention together with one or more pharmaceutically acceptable diluents, carriers and/or excipients.

WO 2006/028393 PCT/NZ2005/000234 In a further aspect of the present invention there is provided a method for modulating the function of integrin 37 in a subject comprising at least the step of administering to said subject an effective amount of a peptide, or derivative thereof, or composition comprising same as herein before described.

Alternatively, the method of modulating the function of integrin (7 in a subject comprises at least the step of administering to said subject an effective amount of a nucleic acid, construct, or composition comprising same as herein before described.

In a further aspect, the present invention provides a method of modulating the function of integrin P7 in an in vitro system the method comprising at least the step of administering to said system a peptide or derivative thereof, nucleic acid, construct, or composition in accordance with the invention.

In a further aspect of the invention there is provided a method for the treatment of integrin P7-mediated inflammatory disorders, the method comprising at least the step of administering to a subject in need thereof a therapeutically effective amount of a peptide, or derivative thereof, or composition comprising same as herein before described.

In a further aspect of the invention there is provided a method for the treatment of integrin P37-mediated inflammatory disorders, the method comprising at least the step of administering to a subject in need thereof a therapeutically effective amount of a nucleic acid, construct, or composition comprising said nucleic acid or construct as herein before described.

In another aspect, the present invention provides the use of a peptide or derivative thereof, or a nucleic acid or construct as herein before described in the manufacture of a medicament for the treatment of integrin 37-mediated inflammatory disorders.

WO 2006/028393 PCT/NZ2005/000234 -6- In yet a further broad aspect, the present invention provides a method for the identification of potential 37 integrin functional interactors, or mimetics of the peptides of the invention, the method comprising at least the step of bringing a potential functional interactor or mimetic in contact with a peptide of the invention, or derivative thereof, and observing whether or not binding occurs.

In a related aspect of the invention the method further comprises the step of determining whether or not the functional interactor molecule or mimetic influences the level of adhesion of leukocytes to p7 integrin ligands. Preferably the method comprises the step of determining whether or not the functional interactor molecule or mimetic lowers the level of, or disrupts or prevents, adhesion of leukocytes to 37 integrin ligands.

In another aspect, the invention provides the use of a peptide or derivative thereof in accordance with the invention in identifying or screening for potential 37 integrin functional interactor molecules or mimetics of said peptides or derivatives thereof.

In a related aspect, the invention provides the use of a peptide or derivative thereof in accordance with the invention in designing mimetics of said peptide or derivative.

In another aspect, the invention provides an antibody directed against a peptide or derivative of the invention.

In another aspect, the invention provides a kit for modulating the function of integrin P7 or for the treatment of integrin p7-mediated inflammatory disorders, the kit comprising at least a peptide or derivative thereof in accordance with the invention.

In a related aspect, the invention provides a kit for modulating the function of integrin p7 or for the treatment of integrin p7-mediated inflammatory disorders, the kit comprising a nucleic acid or construct in accordance with the invention.

WO 2006/028393 PCT/NZ2005/000234 -7- The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

FIGURES

These and other aspects of the present invention, which should be considered in all its novel aspects, will become apparent from the following description, which is given by way of example only, with reference to the accompanying figures, in which: Figure 1: Illustrates cell-permeable p7cyt peptide sequences, and their uptake by TK-1 T cells. Peptides representing different regions of p7cyt used in the study.

Peptides were either fused to penetratin (Pen) or a D-isomeric form of a nine amino acid arginine polymer Upper and lowercase denote L- and D-enantiomers, respectively. The P3-CARD peptide containing the CARD from the integrin 33 subunit was used as a control. Intracellular peptide was detected by staining fixed, permeabilized cytocentrifuged cell smears with FITC-streptavidin, followed by confocal laser scanning microscopy. The transverse sections of single cells presented are representative of 70-80% of the cell population. The panels illustrate cells that have taken up peptides P7-2 P7-3 P7-4 P7-5 P7-7 p7-8 P7- 9 and the P3-CARD Panel A has cells only, and no peptide. The peptides sequences of p7-3, p7-4, P7-5, P7-7, p7-9, p7-10, p711 and P3-CARD (excluding the polymer of arginine and penetratin sequences) have been allocated the sequence identifiers SEQ ID NO 21 to SEQ ID NO 28, in accordance with the sequence listing accompanying this specification.

Figure 2: Illustrates that a cell-permeable peptide from the membrane-proximal region of P7cyt inhibits the adhesion of TK-1 cells to a407 ligands MAdCAM-1, VCAM-1, and RGD polymer. TK-1 cells were preincubated in the presence of WO 2006/028393 PCT/NZ2005/000234 8increasing concentrations of peptides P7-2 to P7-5, activated with Mn" D, F), PMA E, and A1F4" and added to glass slides coated with MAdCAM-1-Fc VCAM-1-Fc and fibronectin-like polymer (RGD) Cells bound were counted, and represent the mean +SD from four fields. Experiments were performed in triplicate.

Figure 3: Illustrates integrin c437-mediated cell adhesion is unaffected by the 33 subunit CARD. A cell-permeable peptide containing the CARD from the p3 subunit failed to inhibit the adhesion of Mn, PMA, and A1F4-activated TK-1 cells to MAdCAM-1 VCAM-1 and fibronectin-like polymer (RGD) Cells bound were counted, and represent the mean +SD from four fields. Experiments were performed in triplicate.

Figure 4: Illustrates the P7-2 peptide blocks cell adhesion across species, and is integrin-specific. Human H9 T cells were preincubated in the presence of increasing concentrations of peptides 37-2 to 3-5, activated with Mn4 PMA and added to glass slides coated with MAdCAM-1-Fc and VCAM-1-Fc D).

Cells bound were counted, and represent the mean +SD from four fields. Experiments were performed in triplicate. Inhibition of cell adhesion is integrin-specific. H9 T cells were preincubated in the presence of increasing concentrations of peptides P7-2 to 3-5, activated with Mn and added to glass slides coated with ICAM-1-Fc. Cells bound were counted, and represent the mean +SD from four fields. Experiments were performed in triplicate.

Figure 5: Illustrates peptide 37-2 blocks ligand-induced clustering of xa4P7 at the cell surface, but cannot disrupt established focal adhesions. Peptide P7-2 blocks ligand-induced clustering of ca4P7. TK-1 cells were preincubated with either the P7-2 or p7-3 peptides, or no peptide, and activated with A1F4'. Ligand-induced clustering of ca437 was induced by addition of MAdCAM-1-Fc, and cluster formation after 30 min was determined by staining cells with FITC-conjugated M293 mAb (anti-P7 subunit), followed by confocal microscopy. Each panel shows transverse (left quandrants) and f WO 2006/028393 PCT/NZ2005/000234 -9surface (right quadrants) views of either a single cell (upper quadrants) at high magnification, or a population of cells (lower quadrants) at lower magnification. B-E) Peptide P7-2 cannot disrupt focal adhesions to detach cells. TK-1 D) and H9 E) cells activated with Mn and PMA were adhered to glass slides coated with MAdCAM-1-Fc and VCAM-1-Fc and then incubated for 1 h with peptide p7-2. Cells bound were counted, and represent the mean +SD from four fields.

Experiments were performed in triplicate.

Figure 6: Illustrates a six amino acid CARD motif exists within peptide p7-2.

The p7-2 peptide was divided into two to give peptides P7-7 and P7-8. TK-1 cells were preincubated in the presence of increasing concentrations of peptides P7-7 and P7-8, activated with Mn D, PMA E, and A1F4" and added to glass slides coated with MAdCAM-1-Fc VCAM-1-Fc and fibronectin-like polymer Cells bound were counted, and represent the mean +SD from four fields. Experiments were performed in triplicate.

Figure 7: Illustrates tandem tyrosines are critical for the activity of the 07 CARD.

TK-1 cells were preincubated in the presence of increasing concentrations of peptide p7-8, and a mutant peptide, P7-9, in which tyrosines 735 and 740 had been substituted with phenylalanines. Cells were activated with Mn PMA and AlF4" and added to glass slides coated with MAdCAM-1-Fc and VCAM-1-Fc Cells bound were counted, and represent the mean +SD from four fields.

Experiments were performed in triplicate. Similar results were obtained for H9 cells.

Figure 8: Illustrates tyrosines 735 and 740 are both essential for p7 CARD activity. Confocal images showing intemalisation by TK-1 cells of variant forms of the p7 CARD (YDRREY) fused to a biotinylated R9 peptide. Intracellular peptide was detected (stained green) after a 30 min incubation as described in Fig. 1B. Panels illustrate cells that have internalized an L-amino acid form of the p7 CARD (peptide P7-12) and a D-amino acid form of the p7 CARD (peptide P7-13) Panel C illustrates cells without peptide. Bar; panels A-C, 10 pm, panels D and E, 5 pm. (B) WO 2006/028393 PCT/NZ2005/000234 TK-1 cells were preincubated in the presence of increasing concentrations of peptide 37-12, and mutant peptides 37-10, and P7-11, in which tyrosines 735 and 740 had been individually deleted, respectively. Cells were activated with Mn*, PMA, and A1F4", as indicated, and added to glass slides coated with MAdCAM-1-Fc. Cells bound were counted, and represent the mean +SD from four fields. Experiments were performed in triplicate. A D-amino acid analogue of the p7 CARD retains activity.

Both L (P7-12) and D (37-13) amino acid analogs of YDRREY fused to a biotinylated R9 peptide inhibit the adhesion of CMFDA-labeled murine TK-1 cells to human VCAM-1 Fc. Data represent the means SD from two independent experiments performed in duplicate.

Figure 9: Illustrates tyrosines 735 and 740 in the 37 CARD are both phosphorylatable. The p7 cytoplasmic domain is strongly tyrosine phosphorylatable. (Upper panel) A GST fusion protein containing the cytoplasmic domain of the human 37 subunit was immobilized on Sepharose and subjected to an in vitro kinase assay with trace amounts of an H9 cell lysate 100 dilution). The GSTfusion protein (arrowed) was resolved on a 10% polyacrylamide SDS gel that was subsequently treated with 1M KOH, and exposed to X-ray film. The sizes in kDa of molecular weight markers are given in the left-hand margin. (Lower panel) The GSTfusion protein was stained with Coomassie blue. The phosphorylated GST-p7cyt fusion protein in was removed from Sepharose beads by boiling the beads in SDS.

The sample was diluted, and subjected to immunoprecipitation with either normal rabbit serum (lane 1) or a polyclonal rabbit anti-p7cyt antibody (lane 2).

Immunoprecipitates were resolved by SDS-PAGE, as above. The p7 cytoplasmic domain is specifically phosphorylated on tyrosine. (Upper panel) A GST-p7cyt fusion protein immobilized on Sepharose was subjected to an in vitro kinase assay with trace amounts of an H9 cell lysate 100 dilution) in the absence (lane 1) or presence of (lane 10 (lane and 20 (lane 4) IM genistein. The GST-fusion protein was resolved on a 10% polyacrylamide SDS gel, and exposed to X-ray film. The sizes in kDa of molecular weight markers are given in the left-hand margin. (Lower panel) The GST-fusion protein was stained with Coomassie blue. The two tyrosine residues WO 2006/028393 PCT/NZ2005/000234 11in the YDRREY motif are phosphorylatable. Biotinylated peptides P7-3, P7-4, P7-7, P7-8, 07-10, and p7-11 were immobilized on streptavidin-Sepharose, and subjected to an in vitro kinase assay with lysates of TK-1 cells that had been either activated with A1F4- (filled bars) or left unactivated (empty bars). Each experiment was done in triplicate, and the results represent the mean cpm SD.

Figure 10: Illustrates the sequence of human and mouse p7cyt, and comparison with the cytoplasmic domains of other integrin P subunits. The positions of the 03 and p7 CARDs, and residues in the CARD that are shared with other P integrin tails are highlighted. Tyrosine phosphorylation sites in p7cyt are underlined. Residues that are common to several P integrin tails are emboldened. M, mouse; H, human. The peptide sequences p7 M, p7 H, p6, P5, p3, 32, and pi have been allocated the sequence identifiers SEQ ID NO 29 to SEQ ID NO 35, in accordance with the sequence listing accompanying this specification.

PREFERRED EMBODIMENT(S) The following is a description of the present invention, including preferred embodiments thereof, given in general terms. The invention is further elucidated from the disclosure given under the section entitled "Examples" herein after, which provides experimental support for, and specific examples of, the invention.

The inventors of the present invention have identified a regulatory motif within the P7 integrin subunit. This motif, YDRREY (SEQ ID NO encompasses residues 735- 740 of the transmembrane-proximal region of the cytoplasmic tail of the P7. While not wishing to be bound by any particular theory, the inventors propose that this phosphorylatable motif constitutes a major cell adhesion regulatory domain (CARD) that modulates the interaction of p7-expressing leukocytes with their extracellular matrix, endothelial and epithelial cells, dendritic cells and other cells expressing appropriate ligands.

WO 2006/028393 PCT/NZ2005/000234 -12- The inventors have surprisingly found that peptides comprising at least the motif YDRREY are able to disrupt the interaction of 07 integrins with their ligands, for example MAdCAM-1 and VCAM-1. This action has been demonstrated across species, namely mice and humans. The implication is that peptides of the invention compete for intracellular proteins or factors that are critical in controlling the function of 17 integrins thereby modulating their cellular adhesion function.

On the basis of the above findings, a peptide of the invention, or a pharmaceutical composition comprising same, may be used to modulate the cellular adhesion function of p7 integrins, particularly the adhesion of leukocytes to each other, to the extracellular matrix and to epithelial and endothelial cells, both in in vitro systems and in vivo. Such modulation has application in controlling p7 integrin-mediated inflammatory events, and particularly in the treatment of p7 integrin-mediated inflammatory disorders.

Similarly, the inventors contemplate the use of nucleic acids encoding peptides of the invention, and constructs or vectors comprising such nucleic acids, in methods for modulating the cellular adhesion function of p7 integrins, likewise including treatment of 17 integrin-mediated inflammatory disorders.

Additionally, a peptide of the invention may be used in assays for the identification of p7 integrin functional interactor molecules which may bind to and/or regulate the function of p7 integrins. As used herein the term "p7 integrin functional interactors" should be taken in its broadest context. It is intended to include molecules such as intracellular signalling molecules and other cellular components which may modulate the cellular adhesion function of the p7 integrins, and also potential therapeutic agents which may have application in treatment of disorders mediated by this function.

Furthermore, peptides of the invention may be used in assays to specifically identify interference molecules directed against the cytoplasmic domain of the integrin 37 subunit. As used herein, "interference molecules" are those molecules which are WO 2006/028393 PCT/NZ2005/000234 -13adapted to bind to a region of the cytoplasmic domain of the integrin subunit including a peptide motif of the invention. Preferably such "interference molecules" block the interaction of at least a region of the cytoplasmic domain with other molecules, and more preferably block the function of cytoplasmic domain of the integrin subunit.

"Interference molecules" include, but are not limited to, antibodies and nucleic acid aptamers (for example, RNA and DNA aptamers).

"Interference molecules" may find use in modulating or inhibiting the activity and function of p7 integrins, including disrupting or preventing the interaction of p7 integrins with their ligands, for example MAdCAM-1 and VCAM-1, thus modulating the cellular adhesion function of the P7 integrins, and having application in controlling p7 integrin-mediated inflammatory events, and particularly in the treatment of P7 integrin-mediated inflammatory disorders.

Peptides of the invention may also be used to design mimetics, including small molecule mimetics, or to screen libraries for such suitable mimetics of the peptides, which may be of use therapeutically.

The phrases "modulate adhesion of leukocytes to each other and to epithelial and endothelial cells", "modulating the cellular adhesion function of p7 integrins" or "regulate the function of p7 integrins", and the like, are generally used herein to refer to down-regulation of function. However, the inventors contemplate situations where up-regulation of function of the p7 integrins may occur through use of peptides, nucleic acids, or constructs of the invention; for example, where the peptides competitively bind to functional interactors which may have a negative effect on 07 integrin function. Accordingly, up-regulation of function of the p7 integrins is also encompassed by the present invention. To this end, while pharmaceutical compositions and methods are described herein after in relation to the treatment of inflammatory disorders, which implies down-regulation of p7 integrin function, it should be understood that they may equally be applicable to treatments where upregulation of p7 integrin function is desirable.

WO 2006/028393 PCT/NZ2005/000234 -14- The term "inflammatory disorder(s)" should be taken to mean any undesired physiological condition which involves inflammation, aberrant or otherwise.

"Inflammation" should be broadly taken to mean a characteristic reaction of tissues to injury or disease, or foreign particles and noxious stimuli, resulting in one or more of redness, swelling, heat, and pain. In accordance with the present invention, such inflammatory disorders will be mediated by the action of P7 integrins, and include, but are not limited to, demyelinating diseases such as multiple sclerosis, Type I diabetes mellitus, inflammatory bowel disease, asthma, arthritis, gastritis, mucositis, graftversus-host disease, hepatitis, psoriasis, Graves disease, septic shock, hemorrhagic shock, ischemica-reperfusion injury, arterial/vascular injury, transplant rejection, and inflammation that impedes tissue/skin healing.

As used herein, the term "treatment" is to be considered in its broadest context. The term does not necessarily imply that subject is'treated until total recovery.

Accordingly, "treatment" broadly includes the modulation or control of inflammation, or other P7 integrin-mediated event, aberrant or otherwise, amelioration of the symptoms or severity of a particular disorder, or preventing or otherwise reducing the risk of developing a particular disorder.

It will be appreciated by those of general skill in the art to which the invention relates, having regard to the nature of the invention and the results reported herein, that the present invention is applicable to a variety of different animals. Accordingly, a "subject" includes any animal of interest. In particular the invention is applicable to mammals, more particularly humans.

It should be understood that a peptide or protein in accordance with the invention, is an "isolated" or "purified" peptide or protein. An "isolated" or "purified" peptide or protein is one which has been identified and separated from the environment in which it naturally resides. It should be appreciated that 'isolated' does not reflect the extent to which the peptide has been purified or separated from the environment in which it naturally resides. Peptides of use in the invention may be purified from natural WO 2006/028393 PCT/NZ2005/000234 sources, or derived by chemical synthesis or recombinant techniques.

It should be understood that a nucleic acid in accordance with the invention, is an "isolated" or "purified" nucleic acid. An "isolated" or "purified" nucleic is one which has been identified and separated from the environment in which it naturally resides.

It should be appreciated that 'isolated' does not reflect the extent to which the nucleic has been purified or separated from the environment in which it naturally resides.

Nucleic acids of.use in accordance with the invention may be purified from natural sources, or derived by chemical synthesis or recombinant techniques.

Peptides and derivatives thereof In a particularly preferred embodiment a peptide of the invention comprises the amino acid sequence YDRREY. While the peptide may consist solely of the motif YDRREY, it should be appreciated that larger peptides in which the motif YDRREY is incorporated are also encompassed by the present invention. By way of example, the core motif YDRREY may be extended at either or both of its N-terminus or Cterminus by additional amino acids, for example 1 to 6 amino acids, taken from the native 37 subunit amino acid sequence at the relevant position. Exemplary 37 sequence information is found in GenBank, see accession numbers NM_000889 (human), NM_013566 (mouse), and XM_343336 (rat). Consistent with this, the peptide p7-2 comprising amino acids RLSVEIYDRREY (SEQ ID NO as described further herein after, forms part of the present invention. Alternatively, the core motif may be extended by, or fused to, heterologous amino acid motifs or sequences where desired. In this regard, a peptide of the invention should be taken to include fusion peptides or proteins.

It should be appreciated that a "peptide" according to the invention extends to any peptide that has been fused with, conjugated to, or otherwise incorporates, a motif which renders it cell-permeable. The motif may allow for active or passive movement of the peptide across or through the cell membrane. The motif may be referred to herein as a cell membrane translocating motif. Such a motif is preferably a peptidebased membrane translocating motif. However, those of skill in the art to which the WO 2006/028393 PCT/NZ2005/000234 -16invention relates will readily recognise motifs of an alternative nature which may effectively provide cell-permeability; for example, motifs that are bound by and internalized by cell-surface receptors, or lipid moieties. The Chariot transfection reagent is designed to transmit biologically active proteins and peptides into living cells, for example.

A peptide-based membrane translocating motif in accordance with the invention will effectively render a peptide cell-permeable, whilst retaining at least a degree of the desired function of said peptide. Those of skill in the art to which the present invention relates will readily appreciate appropriate peptide-based membrane translocating motifs of use in the invention. However, the inventors have found penetratin and a polymer of arginine (as detailed herein after under the heading "Examples") to be of particular use. Further suitable peptide-based membrane translocating motifs are described in the review by Joliot and Prochiantz-Transduction peptides: from technology to physiology. Nat Cell Biol. 2004; 6(3):189-96 (eg Tat RKKRRQRRR (SEQ ID NO, Buforin II TRSSRAGLQFPVGRVHRLLRK (SEQ ID NO 4), Transportan GWTLNSAGYLLGKINKALAALAKKIL (SEQ ID NO MAP (model amphipathic peptide) KLALKLALKALKAALKLA (SEQ ID NO K-FGF AAVALLPAVLLALLAP (SEQ ID NO Ku70 VPMLK (SEQ ID NO 8) or PMLKE (SEQ ID NO Prion MANLGYWLLALFVTMWTDVGLCKKRPKP (SEQ ID NO 10), pVEC LLIILRRRIRKQAHAHSK (SEQ ID NO 11), Pep-1 KETWWETWWTEWSQPKKKRKV (SEQ ID NO 12), SynB.1 RGGRLSYSRRRFSTSTGR (SEQ ID NO 13), Pep-7 SDLWEMMMVSLACQY (SEQ ID NO 14), HN-1 TSPLNIHNGQKL (SEQ ID NO A peptide of the invention may be composed of L-amino acids, D-amino acids or a mixture thereof.

The core YDRREY amino acid sequence may be modified by substitution of one or more of the amino acids of the core DRRE sequence with alternative amino acids, provided the modified peptide retains at least a degree of the desired function of the peptide YDRREY. In one embodiment of the invention the amino acid substitution is WO 2006/028393 PCT/NZ2005/000234 -17conservative. Persons skilled in the art will appreciate appropriate conservative amino acid substitutions based on the relative similarity between different amino acids, including the similarity of the amino-acid side chain substituents (for example, their size, charge, hydrophilicity, hydrophobicity and the like). However by way of example, D may be replaced with E, R may be replaced with K, and E may be replaced with D. In another embodiment the amino acid substitution is nonconservative. Persons of skill in the art will appreciate such non-conservative substitutions. However, by way of example, R could be replaced with L.

In one particular embodiment of the invention one of the R residues, particularly the second R residue, within the peptide is substituted by another amino acid. The inventors note that the rat amino acid sequence corresponding to YDRREY, is YDRLEY (SEQ ID NO 20). Accordingly the inventors contemplate that at least the second R residue is not essential to function.

The inventors also contemplate shortening of a peptide of the invention, by deletion of amino acids within the DRRE core sequence, provided such shortened peptides retain at least a degree of the desired function of the peptide YDRREY.

"Peptides" of the invention may be chemically modified where desirable. For example peptides may be modified by acetylation, glycosylation, cross-linking, disulfide bond formation, cyclization, branching, phosphorylation, conjugation or attachment to a desirable molecule (for example conjugation to bispecific antibodies), acylation, ADP-ribosylation, amidation, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, GPI anchor formation, hydroxylation, methylation, myristoylation, oxidation, pegylation, proteolytic processing, prenylation, racemization, conversion from L-isomer to D-isomer, sulfation, or otherwise to mimic natural post-translational modifications, for example. The peptides may also be modified to include one or more non-naturally occurring amino acids, as will be known in the art. Amino acids of a peptide may also be modified by substitution of R WO 2006/028393 PCT/NZ2005/000234 -18groups for other chemical groups as may be known in the art. In addition, amino acids may be substituted with chemical groups which mimic them; for example, benzimidazole is a known mimic of R and 1,4-benzodiazepine a mimic of G-D (see Curr Protein Pept Sci 2005 Apr; 6(2):151-169. Peptides of the invention may also be modified by arrangement of amino acid groupings from the peptide on a non-peptide scaffold. Considerations for designing such modified peptides are discussed in Curr Protein Pept Sci. 2005 Apr; 6(2):151-169 (Sillerud and Larson).

The invention should be taken to include pharmaceutically acceptable salts of peptides as well as stereoisomers of peptides. Persons of skill in the art will appreciate such salts and stereoisomers.

Peptides of the invention which have been modified as described herein before (for example, by chemical modification, addition of side groups, addition/inclusion of a cell membrane translocating motif, addition of further amino acids (including heterologous amino acids), inclusion of non-naturally occurring amino acids, substitution of amino acids, substitution of amino acid R groups, salts, isomers, reduction to peptidomimetics, and the like), or by other means known in the art, may be referred to herein as "derivatives" of the peptides.

Use herein of the words "peptide" or "peptides" should be taken to include reference to "derivatives" of such peptides, unless the context requires otherwise. In addition, "peptides" and "derivatives" thereof should be taken to include "prodrugs", that is peptides or derivatives which are in an inactive form and which are converted to an active form by biological conversion following administration to a subject.

"Derivatives" of the peptides of the invention will retain at least a degree of the desired function of said peptides; that is the ability to modulate the function of 37 integrins (as described herein) and preferably down-regulate, lower or inhibit function.

Accordingly, an alternative term for "derivatives" may be "functional derivatives".

The function of a derivative can be assessed, for example, using in vitro cell adhesion WO 2006/028393 PCT/NZ2005/000234 -19assays as described in the "Examples" section herein after. Skilled persons may readily appreciate alternative assays, including in vivo assays in animals.

A peptide of the invention may be purified from natural sources, or preferably derived by chemical synthesis (for example, ffmoc solid phase peptide synthesis as described in Fields GB, Lauer-Fields JL, Liu RQ and Barany G (2002) Principles and Practice of Solid-Phase peptide Synthesis; Grant G (2002) Evaluation of the Synthetic Product.

Synthetic Peptides, A User's Guide, Grant GA, Second Edition, 93-219; 220-291, Oxford University Press, New York) or genetic expression techniques (as are outlined broadly herein after), methods for which are readily known in the art to which the invention relates. The inventor's contemplate production of a peptide of the invention by an appropriate transgenic animal, microbe, or plant.

To the extent that a peptide of the present invention may be produced by recombinant techniques the invention provides nucleic acids encoding peptides of the invention and constructs or vectors which may aid in the cloning and expression of such nucleic acids. Certain such constructs may also be of use to a therapeutic end as herein after detailed.

Those of general skill in the art to which the invention relates will readily be able to identify nucleic acids which encode peptides of the invention, including desired fusion peptides or proteins, on the basis of the amino acid sequences of the desired peptides, sequence information contained herein, the genetic code, and the understood degeneracy therein. However, by way of example: CGG CTC TCG GTG GAA ATC TAT GAC CGC CGG GAA TAC (for a peptide having the amino acid sequence RLSVEI YDRREY) and TAT GAC CGC CGG GAA TAC (for a peptide having the amino acid sequence YDRREY), are appropriate nucleic acids. These two nucleic acids may be referred to by the sequence identifiers SEQ ID NO 16 and SEQ ID NO 17, respectively.

Nucleic acid constructs in accordance with this embodiment of the invention will generally contain heterologous nucleic acid sequences; that is nucleic acid sequences WO 2006/028393 PCT/NZ2005/000234 that are not naturally found adjacent to the nucleic acid sequences of the invention.

The constructs or vectors may be either RNA or DNA, either prokaryotic or eukaryotic, and typically are viruses or a plasmid. Suitable constructs are preferably adapted to deliver a nucleic acid of the invention into a host cell and are either capable or not capable of replicating in such cell. Recombinant constructs comprising nucleic acids of the invention may be used, for example, in the cloning, sequencing, and expression of nucleic acid sequences of the invention. Additionally, as is herein after detailed, recombinant constructs or vectors of the invention may be used to a therapeutic end.

Those of skill in the art to which the invention relates will recognise many constructs suitable for use in the present invention. However, the inventors contemplate the use of cloning vectors such as pUC and pBluescript and expression vectors such as pCDM8, adeno-associated virus (AAV) or lentiviruses to be particularly useful; exemplification in regard to cloning and expression vectors is also provided herein under the heading "Examples".

The constructs may contain regulatory sequences such as promoters, operators, repressors, enhancers, termination sequences, origins of replication, and other appropriate regulatory sequences as are known in the art. Further, they may contain secretory sequences to enable an expressed protein to be secreted from its host cell. In addition, expression constructs may contain fusion sequences (such as those that encode a heterologous amino acid motif, for example penetratin, mentioned herein before) which lead to the expression of inserted nucleic acid sequences of the invention as fusion proteins or peptides.

In accordance with the invention, transformation of a construct into a host cell can be accomplished by any method by which a nucleic acid sequence can be inserted into a cell. For example, transformation techniques include transfection, electroporation, microinjection, lipofection, adsorption, and biolistic bombardment.

WO 2006/028393 PCT/NZ2005/000234 -21- As will be appreciated, transformed nucleic acid sequences of the invention may remain extrachromosomal or can integrate into one or more sites within a chromosome of a host cell in such a manner that their ability to be expressed is retained.

Any number of host cells known in the art may be utilised in cloning and expressing nucleic acid sequences of the invention. For example, these include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors; yeast transformed with recombinant yeast expression vectors; insect cell systems infected with recombinant virus expression vectors baculovirus); animal cell systems such as CHO (Chinese hamster ovary) cells using the pEE14 plasmid system; plant cell systems infected with recombinant virus expression vectors cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors Ti plasmid). Those host cells detailed herein after under "Examples" are found to be particularly useful.

A recombinant peptide in accordance with the invention may be recovered from a transformed host cell, or culture media, following expression thereof using a variety of techniques standard in the art. For example, detergent extraction, sonication, lysis, osmotic shock treatment and inclusion body purification. The protein may be further purified using techniques such as affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, and chromatofocusing.

As mentioned herein before, a peptide of the invention may be in the form of a fusion peptide or protein; for example, a peptide of the invention attached to a peptide-based membrane translocating motif, or alternatively, or in addition, a motif which may aid in subsequent isolation and purification of the peptide (for example, ubiquitin, his-tag, or biotin). Means for generating such fusion peptides are readily known in the art to which the invention relates, and include chemical synthesis and techniques in which fusion peptides are expressed in recombinant host cells, as mentioned herein before.

The inventors contemplate Strep-tag (Sigma-Genosys), Impact T M system (New WO 2006/028393 PCT/NZ2005/000234 -22- England Biolabs), his-tag, and the eg pMAL -p2 expression system (New England BioLabs), to be particularly useful in the present instance. Further exemplification is provided herein after under "Examples". In addition, fusion tags of use in recombinant protein expression and purification have been described by R.C. Stevens. "Design of high-throughput methods of protein production for structural biology" Structure, 8, R177-R185 (2000).

Membrane translocating motifs may also be fused, conjugated or otherwise incorporated in or attached to a peptide by alternative means known in the art to which the invention relates. For example, where cell-permeabilising moieties comprise an entire protein, fatty acids and/or bile acids, such molecules may be linked to the active peptide by an amino acid bridge, or by a non-peptidyl linkage.

A peptide of the invention, for example YDRREY, may be simultaneously joined to two tags, where one tag allows for cell secretion (eg signal peptide), and another tag renders the peptide cell-permeable. In this scenario the peptide could be produced and secreted by a non-leukocyte to be subsequently taken up by a leukocyte. This could be advantageous for instance where one may wish parenchymal or endothelial cells within an inflamed tissue to secrete the peptide to inhibit the adhesion of infiltrating leukocytes.

Compositions and Methods of Treatment Inasmuch as the present invention relates to the modulation of integrin p7 function, including the treatment of inflammatory disorders, it also provides a pharmaceutical composition comprising a peptide of the invention, or a derivative thereof, in association with one or more pharmaceutically acceptable diluents, carriers and/or excipients.

The invention also provides a pharmaceutical composition comprising a nucleic acid encoding a peptide of the invention, or construct comprising same, in association with one or more pharmaceutically acceptable diluents, carriers and/or excipients.

WO 2006/028393 PCT/NZ2005/000234 -23- As-used herein, the phrase "pharmaceutically acceptable diluents, carriers and/or excipients" is intended to include substances that are useful in preparing a pharmaceutical composition, may be co-administered with an appropriate agent for example a peptide, nucleic acid encoding said peptide, or construct comprising same, of the invention while allowing the agent to perform its intended function, and are generally safe, non-toxic and neither biologically nor otherwise undesirable.

Pharmaceutically acceptable diluents, carriers and/or excipients include those suitable for veterinary, use as well as human pharmaceutical use. Examples of pharmaceutically acceptable diluents, carriers and/or excipients include solutions, solvents, dispersion media, delay agents, emulsions and the like.

In addition to standard diluents, carriers and/or excipients, a pharmaceutical composition in accordance with the invention may be formulated with additional constituents, or in such a manner, so as to enhance the activity of a peptide, or nucleic acid or construct of the invention, or help protect the integrity of such agents. For example, the composition may further comprise constituents which provide protection against proteolytic degradation, enhance bioavailability, decrease antigenicity, or enable slow release upon administration to a subject. For example, slow release vehicles include macromers, poly(ethylene glycol), hyaluronic acid, poly(vinylpyrrolidone), or a hydrogel.

Furthermore, cell permeability of a peptide, derivative thereof, or nucleic acid or construct of the invention may be achieved, or facilitated, through formulation of the composition.

Additionally, it is contemplated that a pharmaceutical composition in accordance with the invention may be formulated with additional active ingredients which may be of benefit to a subject in particular instances. Persons of ordinary skill in the art to which the invention relates will readily appreciate suitable additional active ingredients having regard to the description of the invention herein and the nature of a particular disorder to be treated, for example. As a general example, antibodies, small molecule inhibitors, immunosuppressors, pharmaceutical drugs (eg steroids), may be used.

WO 2006/028393 PCT/NZ2005/000234 -24- In one embodiment, the present invention also pertains to methods for the treatment of inflammatory disorders comprising at least the step of administering to a subject in need thereof a therapeutically effective amount of a peptide of the invention, or a pharmaceutical composition comprising same. In a related embodiment the method involves the administration to a subject in need thereof of a therapeutically effective amount of a nucleic acid encoding a peptide of the invention, or a construct comprising same.

It should be appreciated that peptides (and derivative thereof) of the invention may be administered and formulated as pro-drugs, which are converted to active agents following administration.

As used herein, a "therapeutically effective amount", or an "effective amount" is an amount necessary to at least partly attain a desired response.

The inventors contemplate administration of a peptide, derivative thereof, nucleic acid encoding a peptide of the invention, or a construct comprising same, or pharmaceutical compositions of any one or more of these agents by any means capable of delivering such agents to leukocytes at a target site within the body of a subject; a "target site" is a site at which an inflammatory event has, or is predicted to, occur, or a site which may otherwise benefit from the delivery of said peptide. By way of example, compounds of this invention may be administered as pharmaceutical compositions by one of the following routes: oral, topical, systemic (eg. transdermal, intranasal, or by suppository), parenteral (eg. intramuscular, subcutaneous, or intravenous injection), by administration to the CNS (eg. by intraspinal or intracistemal injection); by implantation, and by infusion through such devices as osmotic pumps, transdermal patches, and the like. Further examples may be provided herein after. Skilled persons may identify other appropriate administration routes.

In accordance with such modes of administration, and the suitable pharmaceutical excipients, diluents and/or carriers mentioned herein before, compositions of the WO 2006/028393 PCT/NZ2005/000234 invention may be converted to customary dosage forms such as solutions, orally administrable liquids, injectable liquids, tablets, coated tablets, capsules, pills, granules, suppositories, trans-dermal patches, suspensions, emulsions, sustained release formulations, gels, aerosols, powders and immunoliposomes. Additionally, sustained release formulations may be utilised. The dosage form chosen will reflect the mode of administration desired to be used. Particularly preferred dosage forms include orally administrable tablets, gels, pills, capsules, semisolids, powders, sustained release formulation, suspensions, elixirs, aerosols, ointments or solutions for topical administration, and injectable liquids. Further specific examples will be provided herein after.

As will be appreciated, the dose of an agent or composition administered, the period of administration, and the general administration regime may differ between subjects depending on such variables as the severity of symptoms of a subject, the type of disorder to be treated, the mode of administration chosen, and the age, sex and/or general health of a subject.

Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in, cell cultures or animal models to achieve a cellular concentration range that includes the the concentration of the test compound that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1, p.1).

WO 2006/028393 PCT/NZ2005/000234 -26- Specific examples of compositions and modes of administration relevant to 1) peptides, and 2) nucleic acids are now provided. These are given by way of example only.

Peptide Compositions and Modes of Administration Those skilled in the art of peptide-based treatments will readily appreciate a variety of pharmaceutically acceptable diluents, carriers and/or excipients which may be employed in compositions of the invention comprising one or more peptides. By way of example, suitable liquid carriers, especially for injectable solutions, include water, aqueous saline solution, aqueous dextrose solution, and the like, with isotonic solutions being preferred for intravenous, intraspinal, and intracisternal administration.

Diluents, carriers and/or excipients may be chosen to enhance peptide stability. For example, one or more of the following may be used: buffer(s), blocking agent(s), solvent(s), salt(s), chelator(s), detergent(s), and preservative(s). Stabilizing diluents for polypeptides and antigens are described for example in United States Patent: 6,579,688.

As mentioned herein before, peptides of the invention may be formulated to allow for slow release. Pharmaceutical compositions for prolonged peptide release and preparation method are described for example in United States Patents: 6,503,534 and 6,482,435, and 6,187,330, and 6,011,011. In addition, to prolong the in vivo half-life of proteins and to reduce their antigenicity proteins may be conjugated to soluble synthetic polymers, in particular poly(ethylene glycol), poly(vinyl pyrrolidone), poly(vinyl alcohol), poly(amino acids), divinylether maleic anhydride, ethylene-maleic anhydride, N-(2-hydroxypropyl)methacrylamide and dextran. Methods for synthesis of polymer bio-active conjugates are described for example in United States Patent: 6,172,202. Peptides may also be delivered via implants as described in United States Patent: 6,077,523.

Furthermore, while a peptide of the invention may be rendered cell-permeable by fusion or conjugation to an appropriate membrane translocating motif, cell permeability may alternatively be achieved, or further be facilitated, through WO 2006/028393 PCT/NZ2005/000234 -27formulation of the composition. Pharmaceutical formulation of a therapeutic polypeptide together with a permeation-enhancing mixture to enhance bioavailability is described for example in United States Patent: 6,008,187.

Methods of formulating a peptide composition of the invention will be readily appreciated by persons of ordinary skill in the art to which the invention relates.

Nonetheless, guidance may be found in Gennaro AR: Remington: The Science and Practice of Pharmacy, 20th ed., Lippincott, Williams Wilkins, 2000.

As will be appreciated, the dose of a peptide (or composition comprising same) administered, the period of administration, and the general administration regime may differ between subjects depending on such variables as mentioned herein before.

However, by way of general example, the inventors contemplate administration of from approximately 30 [tg to 300mg per kilogram (mg/Kg) mass of the animal, for example, 0.3 to 30 mg/Kg, with lower doses such as 0.003 to 0.3 mg/Kg, e.g. about 0.03 mg/Kg, being appropriate for administration through the cerebrospinal fluid (for example, which may be appropriate in treatment of encephalitis including multiple sclerosis) such as by intracerebroventricular administration, and higher doses such as 3 to 300 mg/Kg, e.g. about 30 mg/Kg, being appropriate for administration by methods such as oral, systemic (eg. transdermal), or parenteral intravenous) administration.

Gene Therapy Compositions and Modes of Administration As mentioned herein before, methods of the invention may involve the administration of nucleic acids encoding peptides of the invention and/or constructs comprising same.

The use of such nucleic acid techniques may be referred to herein as "gene therapy".

Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below. For general reviews of the methods of gene therapy, see Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.

Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and WO 2006/028393 PCT/NZ2005/000234 -28- Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH 11(5):155- 215).

Methods commonly known in the art of recombinant DNA technology which can be used in generating appropriate constructs or vectors are described generally herein before and more specifically for example in Ausubel et al. 1993, Current Protocols in Molecular Biology, John Wiley Sons, NY; and Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY.

In one aspect, a composition comprising at least nucleic acid sequences encoding a peptide of the invention in expression vectors are administered to suitable hosts. The expression of nucleic acid sequences encoding a peptide of the invention may be optimized by enlarging the sequence either by including repeats of the peptide sequence or including flanking heterologous sequences to enable the sequence to be expressed, and processed by the translational machinery. The sequence may be fused with a signal peptide and cell-permeable peptide to allow for secretion, and cell uptake. The expression of nucleic acid sequences encoding a peptide of the invention may be regulated by any inducible, constitutive, or tissue-specific promoter known to those of skill in the art. In a specific embodiment, the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription. In a particular embodiment, nucleic acid molecules encoding a peptide of the invention are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of said coding regions (Koller and Sniithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid molecules or constructs containing them, or indirect, in which case, cells are first transformed with the nucleic acid molecules in vitro to express secretable cell-permeable forms of the peptide, and then transplanted WO 2006/028393 PCT/NZ2005/000234 -29into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid molecules are directly administered in vivo, where they are expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art; for example, they may be constructed as part of an appropriate nucleic acid expression vector and administered so that they become intracellular, by infection using defective or attenuated retroviral or other viral vectors (see U.S. Patent No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, Wu and Wu, 1987, J. Biol. Chem.

262:4429-4432) (which can be used to target cell types specifically expressing the receptors), and the like.

In another embodiment, nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid molecules to avoid lysosomal degradation.

In yet another embodiment, the nucleic acid molecules can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (as described for example in WO 92/06180 dated April 16, 1992 (Wu et WO 92/22635 dated December 23, 1992 (Wilson et W092/20316 dated November 26, 1992 (Findeis et W093/14188 dated July 22, 1993 (Clarke et and, WO 93/20221 dated October 14, 1993 (Young)). Alternatively, the nucleic acid molecules can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438).

WO 2006/028393 PCT/NZ2005/000234 In a specific embodiment, viral vectors are used to express nucleic acid sequences.

Persons of skill in the art to which the invention relates may appreciate a variety of suitable viral vectors having regard to the nature of. the invention described herein.

However, by way of example, a retroviral vector can be used (see Miller et al., 1993, Meth. Enzymol. 217:581-599). Such retroviral vectors have deleted retroviral sequences that are not necessary for packaging of the viral genome and integration into host cell DNA. More detail about retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6:291-302, for example. Other references illustrating the use of retroviral vectors in gene therapy include, for example: Clowes et al., 1994, J. Clin.

Invest. 93:644-651; Kiem et al., 1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr. Opin.

In Genetics and Devel. 3:110-114.

Another example of a suitable viral vector of use in gene therapy techniques applicable to the invention includes adenoviruses. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3:499-503 present a review of adenovirusbased gene therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., 1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155; Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT Publication W094/12649; and Wang, et al., 1995, Gene Therapy 2:775-783.

Adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300; U.S. Patent No. 5,436,146).

AAV present the most preferable viral vectors for use in the present invention. AAV vectors have been reported to lead to persistent 6 months) expression of a transgene in both gut epithelial cells and hepatocytes, resulting in long-term phenotypic recovery in a diabetic animal model (Xu, RA et al., 2001, Peroral transduction of diffuse cells and hepatocyte insulin leading to euglycemia in diabetic rats, Mol Ther 3:S180; During, MJ et al., 1998, Peroral gene therapy of lactose intolerance using an adeno- WO 2006/028393 PCT/NZ2005/000234 -31associated virus vector, Nature Med. 4:1131- 1135; During MJ et al., 2000, An oral vaccine against NMDAR1 with efficacy in experimental stroke and epilepsy, Science 287:1453-1460). AAV is a nonpathogenic, helper-dependent member of the parvovirus family with several major advantages, such as stable integration, low immunogenicity, long-term expression, and the ability to infect both dividing and nondividing cells. It is capable of directing long-term transgene expression in largely terminally differentiated tissues in vivo without causing toxicity to the host and without eliciting a cellular immune response to the transduced cells (Ponnazhagan S et al., 2001, Adeno-associated Virus for Cancer Gene Therapy, Cancer Res 61:6313- 6321; Lai CC et al., 2001, Suppression of choroidal neovascularization by adenoassociated virus vector expressing angiostatin, Invest Ophthalmol Vis Sci 42(10):2401-7; Nguyen JT et al., 1998, Adeno-associated virus-mediated delivery of antiangiogenic factors as an antitumor strategy, Cancer Research 58:5673-7).

In a preferred embodiment of the invention, the cells into which a nucleic acid can be introduced for purposes of gene therapy are leukocytes. However, any desired, available cell type, could be used, especially where the nucleic acid is adapted to express a peptide to be secreted from the cell and subsequently taken up by a leukocyte. For example, the nucleic acid may be introduced into epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, and macrophages.

As mentioned herein before, nucleic acids and nucleic acid constructs of use in this aspect of the invention may be formulated into appropriate compositions in association with one or more pharmaceutically acceptable diluents, carriers and/or excipients.

Skilled persons will readily appreciate such suitable diluents, carriers and/or excipients. However, by way of specific example, suitable liquid carriers, especially for ihjectable solutions, include water, aqueous saline solution, aqueous dextrose solution, and the like, with isotonic solutions being preferred.

The nucleic acids, constructs and viruses may be formulated to help assist in delivery, or protect the integrity of the nucleic acid in vivo. For example, they may be WO 2006/028393 PCT/NZ2005/000234 -32formulated into liposomes, microparticles, microcapsules, or recombinant cells, or as a part of appropriate viral vectors. They may also be formulated to make use of delivery by receptor-mediated endocytosis (see, Wu and Wu, J. Biol. Chem. 262:4429- 4432 (1987)). Lipid Polycation DNA (LPD) may be employed in which DNA is condensed prior to encapsulation in the lipid (as used by Targeted Genetics Corporation, Seattle, WA, USA).

Specific examples of methods of administering a gene-therapy-based composition of the invention include, but are not limited to, parenteral administration intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal intranasal and oral routes). In a specific embodiment, prophylactic or therapeutic compositions of the invention are administered intramuscularly, intravenously, or subcutaneously. The composition may be administered by any convenient route, for example by infusion or injection, by absorption through epithelial or mucocutaneous linings (e.g.,_oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents.

Administration can be systemic or local. Systemic gene-therapy via intravenous administration provides a preferable mode of administration.

Methods of Identification of Functional Interactor Molecules and Mimetics Methods of identifying 37 integrin functional interactor molecules, including interference molecules (such as aptamers), and mimetics of the peptides of the invention, will generally comprise at least the step of bringing a potential functional interactor or interference molecule in contact with a peptide of the invention and observing whether or not binding occurs. For example, such molecules can be identified by "pull-down" assays whereby a peptide of the invention is immobilised on a matrix eg Sepharose beads and used to affinity isolate interactors from a cell lysate.

The interactors can be electrophoresed on an SDS-gel and identified by Western blotting with mAbs against candidate interactors, or the interactors are identified directly by mass spectroscopy. The peptides of the invention may be immobilised on a column, and used to affinity purify interactors. BiaCORE technology based on surface plasmon resonance can be used to establish or characterise molecular interactions.

WO 2006/028393 PCT/NZ2005/000234 -33- It should be appreciated that the peptides may also be used to screen libraries of molecules for potential interactors; for example aptamer libraries (such as those of Archemix, Cambridge, Massachusetts), libraries of chemical mimetics (as may be known in the art) and libraries of synthetic antibodies (for example, HuCAL® antibody libraries (Morphosys AG, Martinsried/Planegg, Germany)).

Once binding to a peptide of the invention has been established, the function of a candidate molecule, can be assessed, for example, using in vitro cell adhesion assays as described in the "Examples" section herein after. Interactors can also be overexpressed or inhibited (eg with antisense, RNAi etc) to determine whether they regulate the function of p7 integrins. Skilled persons may readily appreciate alternative assays, including in vivo assays in animals.

"Interference molecules" will exhibit at least some ability to disrupt or inhibit the activity and function of a P7 integrin. Preferably they will disrupt or prevent the interaction of p7 integrins with their ligands.

Antibodies Peptides of the invention may be used as antigens for the production of antibodies.

Such antibodies may have specific application in experimental studies of the functions of p7 integrins, or as prophylactic or therapeutic reagents when rendered cellpermeable. Anti-idiotypic antibodies raised against antibodies that recognise peptides of the invention may be used to identify potential interactors, or for therapy.

The term "antibody" should be understood in the broadest possible sense and is intended to encompass, for example, intact monoclonal antibodies, polyclonal antibodies, and derivatives of such antibodies; for example, hybrid and recombinant antibodies (for example, humanised antibodies, diabodies, triabodies, and single chain antibodies) and antibody fragments so long as they exhibit the desired biological activity. An antibody may also be modified so as to render it cell-permeable (a "Transbody"). This may be achieved using the membrane translocation motif WO 2006/028393 PCT/NZ2005/000234 -34technology described herein before. In addition, the methodology described by Heng and Cao (Med Hypotheses. 2005;64(6):1105-8) may be used.

Antibody "fragments" is intended to encompass a portion of an intact antibody, generally the antigen binding or variable region of the antibody. Examples of antibody. fragments include Fab, Fab' F(ab') 2 and Fv fragments. Those of ordinary skill in the art to which the invention relates will recognise methods to generate such antibody fragments. However, by way of general example proteolytic digestions of intact antibodies may be used, or the fragments may be directly produced via recombinant nucleic acid technology.

"Humanised" antibodies are essentially hybrid or chimeric antibodies containing domains derived from human sources and domains derived from the animal in which an antibody may have been generated. In the present case, they are either fully-human or mouse/human-hybrid antibodies. Humanised antibodies in accordance with the invention will generally comprise the mouse CDR (complementarity determining region or antigen binding site) of an antibody against of peptide of the invention fused to appropriate human antibody domains or regions necessary to form a functional antibody, for example. Humanization of murine antibodies can be achieved using techniques known in the art, for example by epitope-guided selection (Wang et al, 2000). The methods of Jones et al (1986), or Maynard and Georgiou (2000) provide further examples.

Humanisation of antibodies may help reduce the immunogenicity of the antibodies of the invention in humans for example. Reduced immunogenicity can be obtained by transplanting murine CDR regions to a homologous human P sheet framework (termed CDR grafting; refer to Riechmann et al 1988 and Jones et al 1986).

Those of skill in the art to which the invention relates will appreciate the terms "diabodies" and "triabodies". These are molecules which comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a short peptide linker that is too short to allow pairing between the two domains on the same WO 2006/028393 PCT/NZ2005/000234 chain. This promotes pairing with the complementary domains of one or more other chain encouraging the formation of dimeric or trimeric molecules with two or more functional antigen binding sites. The resulting antibody molecules may be monospecific or multispecific (eg bispecific in the case of diabodies). Such antibody molecules may be created from two or more of the antibodies of the present invention using methodology standard in the art to which the invention relates; for example, as described by Holliger et al (1993), and Tomlinson and Holliger (2000).

The production of antibodies in accordance with the invention may be carried out according to standard methodology in the art. For example, in the case of the production of polyclonal antibodies the method of Diamond et al (1981) may be used.

Monoclonal antibodies may be prepared, for example, as described in Current Protocols in Immunology (1994, published by John Wiley Sons and edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober), by Winter and Milstein (1991), or in "Monoclonal Antibody Production Techniques and Applications", Marcel Dekker Inc.

Production of an antibody or derivative thereof may also be achieved using standard recombinant techniques known in the art, and discussed previously herein. It will be appreciated that nucleic acids encoding an antibody, and thus suitable for recombinant production of the antibody, may be identified by isolating and sequencing nucleic acids from an appropriate hybridoma, or by having regard to the amino acid sequence of the antibody and knowledge of the genetic code and degeneracy therein. The amino acid sequence of an antibody of the invention may be determined using standard methodology; for example, the technique of Edman degradation and HPLC or mass spectroscopy analysis (Hunkapiller et al, 1983), may be used.

The inventors consider recombinant techniques to be a preferable means of producing antibodies on a commercial scale for therapeutic applications.

Antibodies or derivatives thereof may be formulated into pharmaceutical compositions in a similar manner as described herein before, particularly in relation to formulation WO 2006/028393 PCT/NZ2005/000234 -36of the peptides of the invention (see in particular the sections entitled "compositions and methods of treatment" and "peptide compositions and modes of administration").

Antibodies may also be administered in accordance with the principles described in those sections. Improved delivery methods for antibodies include controlled-release and local delivery strategies as described, for example, by Grainger (in "Controlledrelease and local delivery of therapeutic antibodies", Expert Opin Biol Ther. 2004 Jul;4(7):1029-44).

Antibodies may also be delivered to a subject in the form of "intrabodies", or nucleic acid constructs which are adapted to express the antibodies in desired cells following plasmid or viral delivery, for example. Inasmuch as this is the case, appropriate nucleic acids can be formulated into acceptable pharmaceutical compositions and administered as herein before described in the sections entitled "compositions and methods of treatment" and "gene-therapy compositions and modes of administration.

Stocks (in Intrabodies: production and promise. Drug Discov Today. 2004 Nov 15;9(22):960-6.) provides further guidance on the production of"intrabodies".

Kits The peptides and nucleic acids of the invention may be used in kits suitable for modulating the function of integrin 17 or for the treatment of integrin 37-mediated inflammatory disorders. Such kits will comprise at least a peptide or nucleic acid of the invention in a suitable container. The nucleic acid or peptide may be formulated in a pharmaceutical composition ready for direct administration to a subject.

Alternatively, the kit may comprise the peptide or nucleic acid in one container and a pharmaceutical carrier composition in another; the contents of each container being mixed together prior to administration. The kit may also comprise additional agents and compositions in further separate containers as may be necessary for a particular application. Further, kits of the invention can also comprise instructions for the use and administration of the components of the kit.

Any container suitable for storing and/or administering a pharmaceutical composition may be used in a kit of the invention. Suitable containers will be appreciated by WO 2006/028393 PCT/NZ2005/000234 -37persons skilled in the art. By way of example, such containers include vials and syringes. The containers may be suitably sterilised and hermetically sealed.

EXAMPLES

MATERIALS AND METHODS Cell Lines and synthetic peptides. The mouse spontaneous AKR/Cum Y CD8 LPAM-I+/VLA4- T lymphoma cell line TK-1, and the human T lymphoma cell line H9, were purchased from the American Type Culture Collection, Rockville, MD.

They were cultured at 37°C in RPMI 1640 medium supplemented with 50 U/ml penicillin, 50 pg/ml streptomycin, 200 gg/ml L-glutamine, 10% FCS and 0.05 mM P-mercaptoethanol. All synthetic peptides were custom made by Mimotopes Pty Ltd., Victoria, Australia. The p7 cytoplasmic domain peptides were N-terminally fused during synthesis to biotinylated penetratin (RQIKIWFQNRRMKWKKFDRREF (SEQ ID NO 18)) or to a biotinylated R9 polymer (SEQ ID NO 19) to render them cell-permeable.

GST-fusion protein encoding the integrin 17 subunit cytoplasmic domain. A pGEX-2T vector encoding the complete p7 cytoplasmic domain was kindly provided by Dr Andrew Lazarovits (Imperial Cancer Research Fund, London).

Recombinant MAdCAM-1-Fc, VCAM-1-Fc, and ICAM-1-Fe chimeras. The production of soluble recombinant mouse MAdCAM-1-Fc from insect cells using a baculovirus expression system has been described previously The soluble human VCAM-1-Fc and ICAM-1-Fc chimeras were produced using the glutamine synthetase gene amplification system. The extracellular portions of human VCAM-1 and ICAM- 1 fused to the Fc domain of human IgG were expressed from the pEE14 vector (kindly provided by Dr. Chris Bebbington, Celltech Ltd., UK) in CHO K1 cells as described previously MAdCAM-1-Fc-coated microspheres were prepared by mixing 1 p~g of recombinant MAdCAM-1-Fc with 1 [d of a 1 suspension of Power- WO 2006/028393 PCT/NZ2005/000234 -38bind protein A microparticles (Seradyn, 0.979 mM diameter) in 100 tl for 30 min at 4 0

C.

Peptide internalization and visualization. Biotinylated peptides at 20 pM were added to the cells in serum-free RPMI 1640 medium for 30 min at 37 0 C, or 60 min at room temperature. After washing with PBS, cells were resuspended into PBS/1% FCS and cytocentrifuged onto glass slides. Cytospin smears were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, washed twice with PBS and permeabilized in PBS containing 0.2% Triton X-100 for 10 min. Biotinylated peptide was detected by incubating the slides with streptavidin-FITC (Sigma, MO), and visualized using a Leica TCS 4D confocal laser microscope. Images were processed using Leica ScanwareTM 4.2 A software and Adobe Photoshop Cell adhesion assays. Lab-Tek 16-well glass slides (Nunc) or flat-bottomed 96-well maxisorb plates (Nunc) were coated with purified mouse MAdCAM-1-Fc, human VCAM-1-Fc, and human ICAM-1-Fc (70 to 100 al of 5 to 10 pg/ml), and incubated at 4°C overnight. Slides were washed once with PBS, and blocked with FCS for 2 h at room temperature. Slides were washed with Hanks balanced salt solution (HBSS) containing 10 mM Hepes. Cells were either left unlabeled or labeled with the fluorescent dye chloromethyl fluorescein diacetate (CMFDA; Molecular Probes, Oregon), and activated by resuspension in HBSS containing 10 mM Hepes supplemented with 2 mM Ca" and 2 mM Mn Cells were alternatively activated with 50 ng/ml PMA, or by incubating with A1F4- (10 mM NaF and 40 OM AlC13).

They were preincubated with peptides at different concentrations for 10 min at room temperature, checked for viability by trypan blue exclusion, and then 106 cells added to each coated well and left to adhere for 30 min at room temperature. Non-adherent unlabeled cells in 16-well glass slides were removed by twice dipping slides gently into PBS, and adherent cells fixed in PBS containing 2% glutaraldehyde for at least 3h. The number of adherent cells was determined by counting the number of adherent cells in four independent fields at 100x magnification under an inverted microscope. Alternatively, non-adherent CMFDA-labeled cells in 96-well plates were removed by inverse centrifugation at 70 x g for 5 min into 50 mM Tris pH 7.5, 150 WO 2006/028393 PCT/NZ2005/000234 -39mM NaCI, 2 mM MnC1 2 followed by gentle washing with a pipette. The fluorescence of CMFDA-labeled adherent cells was measured using a VICTOR 1420 multilabel counter (Wallac). Unless otherwise stated, experiments were repeated in triplicate, using duplicate wells.

Confocal microscopy analysis of receptor clustering. TK-1 cells were incubated in the presence or absence of peptides (50 uM) for 1 h at room temperature, and cells were activated for 30 min at room temperature in HBSS containing 2 mM Ca++ and A1F4". Cells were incubated with MAdCAM-l-coated microspheres for 1 h on ice.

They were washed with PBS containing 0.02% sodium azide, and fixed in 4 (w/v) paraformaldehyde in PBS for an additional 1 h. They were then stained with FITCconjugated anti-p7 subunit M293 mAb, washed and fixed as above. Cells were mixed 1:1 with Citifluor glycerol/PBS solution, mounted on slides, and analyzed using a Leica TCS4D confocal laser scanning microscope equipped with an external argonkrypton laser (488 nm). Images were digitally recorded and printed on an Epson colour printer using Microsoft Power Point software.

Phosphorylation of recombinant GST fusion protein encoding p7cyt. H9 cells (5 x 107) were lysed at 4 0 C in 1 ml of lysis buffer (10 mM Tris pH 7.4, 50 mM NaCI, mM NaF, 1 mM sodium orthovanadate, 300 jtg/ml PMSF, 20 tg/ml aprotinin, 4ig/ml leupeptin, and 1% NP-40), centrifuged, and the supernatant precleared with GST-Sepharose. A GST fusion protein (50 utg) encoding the P7 subunit cytoplasmic domain bound to glutathione-Sepharose was incubated with trace amounts (1:100 dilution) of precleared lysate for 2 h at 4 0 C. The beads were washed thrice with lysis buffer and thrice with kinase buffer (10 mM Tris pH 7.4, 50 mM NaC1, 5 mM MgC1 2 mM MnC12, 0.1 mM Na 3

VO

4 and incubated in 30 ±tl kinase buffer containing I.Ci 32 P] TATP for 20 min at 30 0 C. The sample was subjected to SDS-PAGE on a acrylamide gel. The gel was stained with Coomassie Blue to visualize the GST fusion protein, and treated with 1 M KOH at 55 0 C for 2 h to remove alkali-labile phosphate moieties on serine and threonine. The gel was dried, and subjected to autoradiography.

WO 2006/028393 PCT/NZ2005/000234 To confirm that the p7cyt was phosphorylated, GST-p7cyt beads subjected to an in vitro kinase assay were washed, and boiled in 10 pl of 10% SDS for 5 min. The supernatant was diluted to 1 ml, and precleared with 10% Streptococcal protein-G and rabbit Ig-Sepharose. The sample was divided, and one half immunoprecipitated with pl of rabbit polyclonal antisera against P7cyt, and the other half with 50 pl of normal rabbit sera. The immunoprecipitates were resolved by SDS-PAGE, and autoradiographed.

Assay to test whether P7 cytoplasmic domain peptides bind a kinase(s). TK-1 cells (3 x 106) were lysed in 1 ml of lysis buffer, and centrifuged at 12,000 x g for min at 4 0 C. Supematants were precleared with 10 Cl of Sepharose and then 10 ptl of streptavidin-Sepharose. Cytoplasmic domain peptides were screened for their ability to associate with a kinase in lysates of TK-1 cells that were either left unactivated or had been activated with A1F4". Biotinylated peptides immobilized on 5 pl of streptavidin- Sepharose were incubated with 1 ml of lysis buffer containing 1 Cl of cell lysate for 2 h at 4 0 C with gentle shaking. The beads were washed thrice with ice-cold lysis buffer, once with kinase buffer, and were incubated with 30 p. kinase buffer and 0.5 to 1 pCi -ATP for 20 min at 30 0 C. Beads were washed five times, and radioactivity measured in a scintillation counter.

RESULTS

A cell-permeable peptide containing a membrane-proximal region of the 37 cytoplasmic domain inhibits a4P7-mediated T cell adhesion. Seven overlapping peptides (p7-2 to P7-9) encompassing the entire 52 amino acid residue cytoplasmic domain of the 37 subunit (p7cyt) (Fig. 1A) were fused at their N-termini to penetratin, a 16 amino acid residue fragment of the third helix of the homeodomain of Antennapedia (26, 27). The peptides (50 pM) were incubated with TK-1 T cells (a4 p37 p31) to determine whether they had been rendered cell-permeable by penetratin.

All seven peptides were imported into the cytoplasm of cells after 1 h, but were excluded from the nucleus (Fig. 1B). When peptides 37-2 to P7-5, which together WO 2006/028393 PCT/NZ2005/000234 -41spanned the entire cytoplasmic domain, were tested for their abilities to block "insideout" signalling (Fig. 2A-G), only the P7-2 peptide encompassing residues 729-740 abrogated TK-1 cell adhesion to the immobilised ca47 ligands MAdCAM-1 (Fig. 2A- VCAM-1 (Fig. 2D, and an engineered polymer of RGD (Fig. 2F, Cellbinding to MAdCAM-1 and VCAM-1 was almost completely blocked at 100 ntm of peptide, whereas there was only a 50% reduction in binding to the RGD polymer (Fig.

2F, Similar levels of inhibition were achieved irrespective of whether the cell activator was the pan-activator Mn (Fig. 2A, D, the protein kinase C (PKC) activator PMA (Fig. 2B, E, or the G-protein activator A1F4- in the case of MAdCAM-1 (Fig. 2C).

A cell adhesion regulatory domain (CARD) from the extreme C-terminus (residues 747 to 762) of the integrin 33 subunit has been reported to inhibit the adhesion of erythroleukaemia and endothelial cells to immobilized fibrinogen As expected, a cell-permeable control peptide (p3-CARD) containing the 33 CARD fused Nterminally to penetratin (Fig. 1A, B) had no effect on 37-mediated adhesion of Mn A1F4-, or PMA-activated TK-1 cells to MAdCAM-1, and VCAM-1 (Figs 3A, The peptide did not block 03 integrin-mediated cell-binding to the RGD polymer (Fig. 3C), as TK-1 cells do not express p3 integrins (data not shown). Rather binding of TK-1 cells to RGD polymers is mediated by 37 integrins, as reported previously (29).

Similar experiments to those described above were carried out using the human T lymphoma cell line H9 (a4 37 p31) to demonstrate that peptide p7-2-mediated blockade of lymphocyte adhesion was not species-specific. Peptides were imported into this cell line at a rate similar to that observed with the TK-1 cell line (data not shown). Peptide p7-2 blocked Mn (Fig. 4A, and PMA-activated (Fig. 4B, D) adhesion of H9 cells to MAdCAM-1 (Fig. 4A, and VCAM-1 (Fig. 4C, D), respectively, whereas peptides p7-3, and p7-5 were completely ineffective. p7-2 peptide-mediated inhibition of H9 cell adhesion to VCAM-1 was not quite as effective as blockade of TK-1 cell adhesion, as evidenced by the higher concentrations of peptide required to block cell adhesion, presumably because H9 cells express a4pl WO 2006/028393 PCT/NZ2005/000234 -42which preferentially mediates cell binding to VCAM-1. In contrast, the 33 CARD peptide failed to block the adhesion of Mn" and PMA-activated H9 cells to MAdCAM-1 and VCAM-1 (data not shown).

Inhibition of cell adhesion by peptide P7-2 is 37 integrin-specific.

H9 cells express the p2 integrin aLp2, and bind to the aLp2 ligand ICAM-1 (Fig. 4E).

None of the P7 cytoplasmic domain peptides (P7-2 to p7-5) inhibited Mn+-activated H9 cells from binding to ICAM-1, demonstrating that the inhibitory activity of the P7- 2 peptide is restricted to p7 integrins (Fig. 4E).

Cell-permeable peptide p7-2 blocks ligand-induced clustering of a407, but is unable to disrupt established focal adhesions. Stimulation of TK-1 cells in the presence of MAdCAM-1 leads to ligand-induced clustering of a4p7 at the cellsurface, a process which appears largely responsible for increased ca47-mediated cell adhesion Peptide p7-2 completely blocked the MAdCAM-1-induced redistribution of a4p7 on the surface of A1F4"-activated TK-1 cells, suggesting that the peptide prevents the formation of focal adhesion complexes (Fig. 5A). In contrast, the nonactive peptide 37-3 was without effect. The p7-2 peptide was not able to detach Mn" and PMA-activated TK-1 (Fig. 5B, and H9 (Fig. 5C, E) cells already adhered to MAdCAM-1 (Fig. 5B, C) and VCAM-1 (Fig. 5D, suggesting it could not disrupt established focal adhesions.

A six amino acid residue motif is essential for cell adhesion. The p7-2 peptide was divided into two to give the cell-permeable peptides P7-7, and p7-8 (Fig. 1A, in order to sublocalize the region responsible for peptide blockade of cell adhesion.

Peptide p7-8 encompassing residues 735-740 retained the ability to block Mn PMA, and A1F4" -activated TK-1 cell adhesion to MAdCAM-1, VCAM-1, and RGD polymer (Fig. with a similar dose-dependency to that exhibited by peptide p7-2 (Fig. In contrast, peptide P7-7 (residues 729-735) was completely ineffective. The p7-8 peptide also blocked the adhesion of H9 cells to the latter ligands (data not shown).

WO 2006/028393 PCT/NZ2005/000234 -43- Tandem tyrosine residues in the p7 CARD are critical for the inhibitory activity of peptide p7-8. A mutant peptide (37-9) in which both tyrosine residues 735 and 740 of YDRREY had been substituted for phenylalanines was not able to inhibit the adhesion of Mn* PMA, and AlF4"-activated TK-1 (Fig. 7) and H9 cells (data not shown) to either MAdCAM-1 (Fig. 7A-C), or VCAM-1 (Fig. 7D,E).

Recently, it was discovered that polymers of L-arginine of 6 amino acids in length or greater are highly cell-permeable Peptide 37-12 containing the YDRREY peptide fused to a D-isomeric form of a nine arginine polymer was rapidly internalized by cells (Fig. 8A), and inhibited the adhesion of Mn PMA, and A1F4" -activated TK- 1 cells to MAdCAM-1 (Fig. 8B), indicating that the cell adhesion blocking effect of the p7 CARD was independent of the type of carrier peptide. Truncation of peptide P7-12 to individually remove the flanking tyrosine residues 735 (peptide 37-10) and 740 (peptide P7-11) destroyed the activity of the peptide, as peptides 37-10 and p7-11 were not able to inhibit the adhesion of Mn PMA, and A1F4- -activated TK-1 cells to MAdCAM-1 (Fig. 8B). Thus, both flanking tyrosine residues are critical for activity, and YDRREY defines the minimal motif.

A D-amino acid form of the 17 CARD retains activity. A D-amino acid version of the p7 CARD fused to a D-isomeric form of R9 was internalized by TK-1 cells (Fig 8A), and was as equally potent as the L-enantiomer at inhibiting the adhesion of TK-1 T cells to immobilized VCAM-1-Fc (Fig. 8C). The D-amino acid form was cytotoxic at concentrations exceeding 50 pM, causing 50% cell death at 100 4M (data not shown).

The p7 CARD binds a kinase(s), and tyrosine residues in the p7 CARD are phosphorylatable. Peptide p7-8 possesses two of the three potential 37cyt tyrosine phosphorylation sites (residues 735, 740, and 760) in P7cyt. An in vitro kiriase assay revealed that p7cyt can be phosphorylated on tyrosine by a kinase(s) present in lymphocyte lysates (Fig. 9A). The prominent phosphorylated band formed with the WO 2006/028393 PCT/NZ2005/000234 -44- GST-P7cyt fusion protein in Fig. 9A (lane 37) was specifically immunoprecipitated by a rabbit anti-p7cyt antibody (Fig. 9B). Inclusion of genistein at 20 pM completely blocked the phosphorylation of p7cyt, indicating that the phosphorylation was tyrosine-specific (Fig. 9C). The P7-4, 37-5, p7-7, 37-8, P7-10, and p7-11 peptides were each tested for their ability to associate with a kinase(s) and/or be phosphorylated. Peptides immobilized on Sepharose beads were incubated with lysates from unactivated and A1F4"-activated cells, the beads washed, and subjected to an in vitro kinase assay to detect the presence of an associated kinase(s). Peptides p7-8, P7and p7-11 formed a phosphorylated complex following treatment with a TK-1 cell lysate, as did peptide P7-4 containing tyrosine 760 within the NPLY motif (Fig. 9D).

Thus, kinases interact with both the YDRREY and QLNWKQDSNPLYKSAITTT sequences within the N- and C-terminal regions of the cytoplasmic domain of the P7 subunit. Phosphorylated complexes were formed irrespective of whether lysates of unactivated or A1F4- -activated cells were used, suggesting either that activation was not required, or the kinase(s) were activated after disruption of the cells. In contrast, peptide p7-7 and the extreme C-terminal control peptide P7-5 lacking potential tyrosine phosphorylation sites did not form a phosphorylated complex.

DISCUSSION

The results herein reveal that the 6-amino acid peptide motif 735-YDRREY-740 in the membrane proximal region of the cytoplasmic tail of P7 plays a critical role in mediating the clustering and adhesive function of P7 integrins. This motif is unique to the P7 cytoplasmic domain (Fig. 10), with the pi and P5 subunits sharing the DRRE core, but lacking the two flanking tyrosine residues. The YDRREY motif has a certain symmetry, in that a core of two basic arginines, flanked by acidic residues, are contained by the tandem tyrosine residues. The P7 CARD is completely conserved between human and mouse P7cyt (Fig. 10). The p2 subunit is the only typical integrin p subunit that lacks the dibasic core, having a DLRE motif instead. The DLRE motif has been proposed to bind the GFFKR motif common to all integrin a subunits, and to constrain LFA-1 into a low affinity state Interestingly, substitution of a single amino acid to create the DRRE motif within the P2 subunit induced clustering of LFA- WO 2006/028393 PCT/NZ2005/000234 1 at the cell-surface, and restored the PMA responsiveness of LFA-1 in K562 cells suggesting that this motif controls receptor avidity via clustering as implied from the results of the present study.

Tyrosine residues 735 and 740 were both phosphorylatable and acted in tandem to mediate the adhesive function of a4p7.

Data looked at by the inventors indicates the CARD in 37 is unique to p7 integrins, suggesting that the adhesive function of 17 integrins may be regulated by a different intracellular signalling pathway compared to the likes of 11, 33 and 36 integrins. In accord, neither the cell permeable YDDREY peptide nor the 33 CARD were able to block 32 integrin-mediated adhesion of H9 cells to ICAM-1 in the present study, indicating that the adhesivity of each of the 32, 33 and 17 integrins is differentially regulated.

Whilst not wishing to be bound by any particular theory the inventors believe that cellpermeable soluble forms of p7cyt compete for intracellular proteins that are critical for the cell-surface clustering of a4p7, and thereby abrogate c4p7-mediated cell adhesion. Whilst a large array of at least 21 proteins are known to bind one or more integrin P tails so far only the actin-binding protein filamin and the WD repeat protein WAIT-1 (25) have been reported in the published literature as binding to the p7 subunit. WAIT-1 specifically interacts with the cytoplasmic tail of the 37 subunit, but not the 1p, and P2 subunits The WAIT-1 binding site 729- RLSVEIYDR-740 in 17cyt only partially overlaps the CARD in p7cyt. The filamin binding site in p7cyt has yet to be defined. Filamin-binding regions in the pi and 32 integrin subunit cytoplasmic domains appears to differ. Thus, the filamin binding site in the p2 subunit was localized to the membrane proximal region (residues 724 to 747) whereas all but three C-terminal residues were required in the case of the p1 subunit Filamin is recruited to focal adhesions, provides a mechanical link between integrins and the cytoskeleton, and acts as an adapter protein for signaling molecules that regulate cytoskeletal dynamics If the CARD in p7cyt binds WO 2006/028393 PCT/NZ2005/000234 -46filamin, this might explain, in part, the ability of the cell-permeable YDRREY peptide to inhibit a4p7 cell-surface clustering.

Whilst the inventors have demonstrated that an. extreme C-terminal p7cyt peptide failed to exhibit a dominant-negative affect on a437-mediated adhesion, it is clear that this region also plays a role in regulating P7 integrin function. Thus, deletion of a 34amino acid residue segment from the C-terminus of P7cyt abrogated adhesion of 38C13 B lymphoma cells to p7 integrin ligands It is possible that different regions of p7cyt assume cell-type specific functions.

In summary, a CARD in the cytoplasmic tail of the integrin p7 subunit has been identified that contributes to cell-surface clustering of a4p7, and thereby plays a critical role in mediating the adhesion of activated T cells. The CARD YDRREY motif represents a molecule of use to modulate the adhesive functions of p7 integrins "from within" to treat several major inflammatory diseases to which p7 integrins contribute, and also provides a template to design further pharmaceutical drugs.

The invention has been described herein, with reference to certain preferred embodiments, in order to enable the reader to practice the invention without undue experimentation. However, a person having ordinary skill in the art to which the invention relates will readily recognise that many of the components and parameters may be varied or modified to a certain extent without departing from the scope of the invention. Furthermore, titles, headings, or the like are provided to enhance the reader's comprehension of this document, and should not be read as limiting the scope of the present invention.

The entire disclosures of all applications, patents and publications, cited above and below, if any, are hereby incorporated by reference.

WO 2006/028393 PCT/NZ2005/000234 -47- The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in the field of endeavour to which the invention relates.

Throughout this specification, and any claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integeror step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

WO 2006/028393 PCT/NZ2005/000234 -48-

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Claims (23)

1. An isolated peptide comprising at least the amino acid sequence YDRREY, or a derivative of said peptide.

2. An isolated peptide consisting of the amino acid sequence RLSVEI YDRREY, or derivative of said peptide.

3. A peptide or derivative thereof as claimed in claim 1 or 2 wherein the peptide or derivative is fused to a cell membrane translocating motif.

4. A peptide or derivative as claimed in claim 3 wherein the cell membrane translocating motif is peptide-based.

5. A peptide or derivative as claimed in claim 4 wherein the cell membrane translocating motif is penetratin or a polymer of arginine.

6. An isolated nucleic acid which encodes a peptide or derivative thereof as claimed any one of claims 1 to

7. A construct comprising a nucleic acid which encodes a peptide or derivative thereof as claimed in any one of claims 1 to

8. A construct as claimed in claim 7, wherein the construct is an expression construct.

9. A pharmaceutical composition comprising a peptide or derivative thereof as claimed in any one of claims 1 to 5, together with one or more pharmaceutically acceptable diluents, carriers and/or excipients. A pharmaceutical composition comprising a nucleic acid or construct as claimed in any one of claims 6 to 8, together with one or more pharmaceutically acceptable diluents, carriers and/or excipients.

11. A method of modulating the function of integrin P7 in a subject comprising the step of administering to said subject an effective amount of a peptide or derivative thereof as claimed in any one of claims 1 to 5, or a composition as claimed in claim 9.

12. A method of modulating the function of integrin 37 in a subject comprising administering to said subject an effective amount of a nucleic acid or construct as claimed in any one of claims 6 to 8, or composition as claimed in claim

13. A method of modulating the function of integrin 37 in an in vitro system comprising the step of administering to said system a peptide or derivative WO 2006/028393 PCT/NZ2005/000234 thereof as claimed in any one of claims 1 to 5, or a composition as claimed in claim 9.

14. A method of modulating the function of integrin p7 in an in vitro system comprising the step of administering to said system a nucleic acid or construct as claimed in any one of claims 6 to 8, or composition as claimed in claim A method for the treatment of integrin p7-mediated inflammatory disorders comprising the step of 'administering to a subject in need thereof a therapeutically effective amount of a peptide or derivative thereof as claimed in any one of claims 1 to 5, or a composition as claimed in claim 9.

16. A method for the treatment of integrin p7-mediated inflammatory disorders comprising the step of administering to a subject in need thereof a therapeutically effective amount of a nucleic acid or construct as claimed in any one of claims 6 to 8, or composition as claimed in claim

17. The use of a peptide or derivative thereof as claimed in any one of claims 1 to 5 in the manufacture of a medicament for the treatment of integrin p7-mediated inflammatory disorders.

18. The use of a nucleic acid or construct as claimed in any one of claims 6 to 8 in the manufacture of a medicament for the treatment of integrin p7-mediated inflammatory disorders.

19. A method for the identification of potential p7 integrin functional interactor molecules, or mimetics of peptides or derivatives as claimed in any one of claims 1 to 5, the method comprising at least the step of bringing a potential functional interactor or mimetic in contact with a peptide or derivative thereof as claimed in any one of claims 1 to 5, and observing whether or not binding occurs. A method as claimed in claim 19 further comprising the step of determining whether or not the functional interactor molecule or mimetic influences the level of adhesion of leukocytes to 37 integrin ligands.

21. A method as claimed in claim 19, wherein the method comprises determining whether or not the functional interactor molecule or mimetic lowers or inhibits the adhesion of leukocytes to 17 integrin ligands. WO 2006/028393 PCT/NZ2005/000234 -56-

22. The use of a peptide or derivative thereof as claimed in any one of claims 1 to in identifying or screening for potential P7 integrin functional interactor molecules or mimetics of said peptides or derivatives thereof.

23. The use of a peptide or derivative thereof as claimed in any one of claims 1 to 5 in designing a mimetic thereof.

24. An antibody directed against a peptide or derivative thereof as claimed in any one of claims 1 to A kit for modulating the function of integrin 37 or for the treatment of integrin p7-mediated inflammatory disorders, the kit comprising at least a peptide or derivative thereof as claimed in any one of claims 1 to 5 or a composition as claimed in claim 9.

26. A kit for modulating the function of integrin 37 or for the treatment of integrin p7-mediated inflammatory disorders, the kit comprising a nucleic acid or construct as claimed in any one of claims 6 to 8 or a composition as claimed in claim

27. An isolated peptide comprising at least the amino acid sequence YDRLEY, or a derivative of said peptide.