AU2005204275B2 - A method of modulating integrin mediated cellular activity and agents useful for same - Google Patents

Description

AUSTRALIA

Patents Act 1990 (Cth) Complete Specification Applicant Inter-K Pty Limited Invention Title A Method Of Modulating Integrin Mediated Cellular Activity And Agents Useful For Same The invention is described in the following statement including the best method known to us: la A METHOD OF MODULATING INTEGRIN MEDIATED

CELLULAR

ACTIVITY AND AGENTS USEFUL FOR SAME Field Of The Invention The present invention relates to a method of modulating cell activity mediated by a cell adhesion molecule and more particularly, finds application in down regulation of the growth and/or the proliferation of a cell such as a neoplastic cell. In particular, the invention finds use inter alia in the prophylaxis or treatment of conditions such as cancers requiring modulation of cell activity. The present invention also provides agents for use in the above.

Background Of The Invention Colorectal cancer is the commonest internal malignancy affecting men and women in Australia. About 4% of individuals develop this disease during the course of their lifetime and it was responsible for 14% of cancer deaths in that country in 1990. In 1995 there were 10,615 cases of colorectal cancer and 4508 deaths in Australia.

Worldwide, an estimated 875,000 cases of colorectal cancer occurred in 1996, accounting for 8.5% of all new cases of cancer. Incidence rates vary approximately fold around the world, with the highest rates seen in the developed world and lowest in India. Australian incidence rates are towards the higher end of the scale internationally alongside those for North America and New Zealand. Five-year survival following diagnosis of colon cancer is around 55% in the developed world and has altered little during the past few decades despite advances in chemo-, immuno- and radiotherapy.

Colorectal cancer is a malignant tumour that starts in the bowel wall and is confined locally for a relatively long period before spreading through the bowel wall and metastasising to lymph nodes and other parts of the body. Survival rates are significantly improved where the disease is detected and treated early.

The aetiology of colorectal cancer is complex and appears to involve interactions between inherited susceptibility and environmental factors. Recognition of the genetic component of colorectal cancer is growing. Mutations are present as inherited germline defects or arise in somatic cells secondary to environmental insult. There are two main inherited predisposition syndromes: Familial Adenomatous Polyposis (FAP) and Hereditary Non-Polyposis Colorectal Cancer (HNPCC); the remaining cases are attributed to so-called sporadic colorectal cancer. FAP and HNPCC contribute to approximately 1% and respectively, of all colorectal cancers and a strong family history of bowel cancer in first-degree relatives is obtained in another 10 15% of patients.

However, in the vast majority of patients the aetiology of large bowel cancer remains unknown. Most colon cancer arises within pre-existing benign precursor lesions or adenomas. Adenomas are classified by histological architecture as tubular, tubulovillous or villous. Villous change is associated with a higher malignant potential, as are large and high-grade epithelial dysplasia. Environmental risk factors for development of colorectal cancer include diets low in fibre and vegetables and high in fat, red meat and alcohol and cigarette smoking which may induce mutations in somatic cells.

Studies have shown that persistent genetic instability and accumulation of mutations in several genes that are mainly concerned with cell growth or DNA repair, may be critical for the development of all colorectal cancers. For example, a normal mucosal cell with inactivation of tumour suppressor genes such as the Adenomatous Polyposis Coli (APC) gene or Mutated in Colon Cancer (MCC) gene can proliferate and become a small adenomatous polyp. Mutations in oncogenes such as k ras and in tumour suppressor genes such as p53 and the Deleted in Colon Cancer gene (DCC) may then occur and lead to the transformation of the polyp into a large adenoma, from which a carcinoma can eventually arise. The uncontrolled cell growth that leads to the development of neoplasia is believed to result, therefore, from a series of inherited and acquired accumulated genetic changes. This multistep process confers on cells the capacity to survive and proliferate in a manner freed from the constraints imposed on normal cell growth.

Spread of cancer cells involves tumour cell migration through the extracellular matrix scaffold, invasion of basement membranes, arrest of circulating tumour cells, and tumour cell extravasation and proliferation at metastatic sites. Detachment of cells from the primary tumour mass and modification of the peri-cellular environment aid penetration of tumour cells into blood and lymphatic vessels. It is the invasive and metastatic potential of tumour cells that ultimately dictates the fate of most patients suffering from malignant diseases. Hence, tumourigenesis can be viewed as a tissue remodelling process that reflects the ability of cancer cells to proliferate and digest surrounding matrix barriers. These events are thdiught to be regulated, at least in part, by cell adhesion molecules and matrix-degrading enzymes.

Cell adhesion receptors on the surface of colon cancer cells are involved in complex cell signalling which may regulate cell proliferation, migration, invasion and metastasis and several families of adhesion molecules have now been identified including integrins, cadherins, the immunoglobulin superfamily, hyaluronate receptors, and mucins (Agrez, 1996.) In general, these cell surface molecules mediate both cellcell and cell-matrix binding, the latter involving attachment of tumour cells to extracellular scaffolding molecules such as collagen, fibronectin and laminin. It is now clear that multiple and varied cell adhesion receptors exist on colon cancer cells at any one time and an understanding of the role of individual receptors in promoting growth and spread of colon cancer is only just beginning to be elucidated.

Of all the families of cell adhesion molecules, the best-characterised at the present time is the family known as integrins. Integrins are involved in several fundamental processes including leucocyte recruitment, immune activation, thrombosis, wound healing, embryogenesis, virus intemalisation and tumourigenesis. Integrins are transmembrane glycoproteins consisting of an alpha and beta chain in close association that provide a structural and functional bridge between extracellular matrix molecules and cytoskeletal components with the cell. The integrin family comprises 17 different a and 8 P subunits and the ac combinations are subsumed under 3 subfamilies.

Excluding the leucocyte integrin subfamily that is designated by the P2 nomenclature, the remaining integrins are arranged into two major subgroups, designated p1 and av based on sharing common chains.

In the pl subfamily, the 31 chain combines with any one of nine a chain members (al and the a chain which associates with pi determines the matrix-binding specificity of that receptor. For example, a2pl binds collagen and laminin, a3pl1 binds collagen, laminin and fibronectin, and a5pl binds fibronectin. In the av subfamily of receptors, the abundant and promiscuous av chain combines with any one of five P chains, and a distinguishing feature of av integrins is that they all recognise and bind with high affinity to arginine-glycine-aspartate (RGD) sequences present in the matrix molecules to which they adhere (Hynes, 1992).

The current picture of integrins is that the N-terminal domains of a and p subunits combine to form a ligand-binding head on each integrin. This head, containing the cation binding domains, is connected by two stalks representing both subunits, to the membrane-spanning segments and thus to the two cytoplasmic domains. The P subunits all show considerable similarity at the amino acid level (Loftus et al, 1994). All have a molecular mass between 90 and 110 kDa, with the exception of p4 which is larger at 210 kDa.. Similarly, they all contain 56 conserved cysteine residues, except for p4 which has 48. These cysteines are arranged in four repeating patterns which are thought to be linked internally by.disulphide bonds. The a-subunits have a molecular mass ranging from 150 200 kDa. They exhibit a lower degree of similarity than the p chains, although all contain seven repeating amino acid sequences interspaced with nonrepeating domains.

The 3 subunit cytoplasmic domain is required for linking integrins to the cytoskeleton (Hynes, 1992). In many cases, this linkage is reflected in localisation to focal contacts, which is believed to lead to the assembly of signalling complexes that include a-actinin, talin, and focal adhesion kinase (FAK) (Otey et al, 1990; Guan and Shalloway, 1992; Kornberg et al, 1992). At least three different regions that are required for focal contact localisation of p integrins have been delineated (Reszka et al, 1992). These regions contain conserved sequences that are also found in the cytoplasmic domains of the P2, 33, 05, P6 and p7 integrin subunits. The functional differences between these cytoplasmic domains with regard to their signalling capacity have not yet been established.

Ligation of integrins by their extracellular matrix protein ligands induces a cascade of intracellular signals that include tyrosine phosphorylation of focal adhesion kinase, S increases in intracellular Ca 2 levels, inositol lipid synthesis, synthesis of cyclins and expression of immediate early genes. In contrast, prevention of integrin-ligand interactions suppresses cellular growth or induces apoptotic cell death (Meredith et al, 1993; Montgomery et al, 1994; Brooks et al, 1994; Varner et al, 1995; Boudreau et al, 1995). Thus, integrins play roles in a number of cellular processes that impact on the development of tumours, including the regulation of proliferation and apoptosis.

The integrin 36 subunit was first identified in cultured epithelial cells as part of the avp6 heterodimer, and the avp6 complex was shown to bind fibronectin in an arginineglycine-aspartate (RGD)-dependent manner in human pancreatic carcinoma cells (Sheppard et al, 1990; Busk et al, 1992). The P6 subunit is composed of 788 amino acids and shares 34 51% sequence homology with other P integrin subunits 131 05. The 36 subunit also contains 9 potential glycosylation sites on the extracellular domain (Sheppard et al, 1990). The cytoplasmic domain differs from other p subunits in that it is composed of a 41- amino acid region that is highly conserved among integrin 1 subunits, and a unique 11 amino'acid carboxy-terminal extension. The 11 amino acid extension has been shown not to be necessary for localisation of p6 to focal contacts; in fact, its removal appears to increase receptor localisation. However, removal of any of the three conserved regions previously identified as important for the localisation of p1 integrins to focal contacts (Reszka et al, 1992) has been shown to eliminate recruitment of P6 to focal contacts (Cone et al, 1994).

The integrin av6 has previously been shown to enhance growth of colon cancer cells in vitro and in vivo (Agrez et al, 1994). What makes this epithelial-restricted integrin of particular interest in colon cancer is that it is not expressed in normal cells but is highly expressed during tumourigenesis (Breuss et al, 1995; Agrez et al, 1996).

Invasion of the extracellular matrix and metastatic spread of colon cancer is also likely to reflect the ability of tumour cells to digest their surrounding matrix scaffold through secretion of matrix-degrading enzymes such as matrix metalloproteinases (MMPs). The mechanisms whereby human colon cancer cells escape the constraints on growth imposed on normal cells by cell crowding and dense pericellular matrices is unclear. However, even colon cancer cells are subject to relative growth inhibition in vitro in a dense extracellular matrix environment (Agrez, 1989).

Integrins can signal through the cell membrane in either direction. The extracellular binding activity of integrins can be regulated from the cell interior as, for example, by phosphorylation of integrin cytolasmic domains (inside-out signalling), while the binding of the extracellular matrix (ECM) elicits signals that are transmitted into the cell (outside-in signalling) (Giancotti and Ruoslahti, 1999). Outside-in signalling can be roughly divided into two descriptive categories. The first is 'direct signalling' in which ligation and clustering of integrins is the only extracellular stimulus.

Thus, adhesion to ECM proteins can activate cytoplasmic tyrosine kinases focal adhesion kinase FAK) and serine/threonine kinases (such as those in the mitogenactivated protein kinase (MAPK) cascade) and stimulate lipid metabolism (eg phosphatidylinositol-4, 5-biphosphate (PP 2 synthesis. The second category of integrin signalling is 'collaborative signalling', in which integrin-mediated cell adhesion modulates signalling events initiated through other types of receptors, particularly receptor tyrosine kinases that are activated by polypeptide growth factors (Howe et al, 1998). In all cases, however, integrin-mediated adhesion seems to be required for efficient transduction of signals into the cytosol or nucleus.

MAP kinases behave as a convergence point for diverse receptor-initiated signalling events at the plasma membrane. The core unit of MAP kinase pathways is a three-member protein kinase cascade in which MAP kinases are phosphorylated by MAP kinase kinases (MEKs) which are in turn phosphorylated by MAP kinase kinase kinases Raf-1) (Garrington and Johnson, 1999). Amongst the 12 member proteins of the MAP kinase family are the extracellular signal-regulated kinases (ERKs) (Boulton et al, 1991) activated by phosphorylation of tyrosine and threonine residues (Payne et al, 1991) which is the type of activation common to all known MAP kinase isoforms. ERK 1/2 (44kD and 42kD MAPks, respectively) share 90% amino acid identity and are ubiquitous components of signal transduction pathways (Boulton et al, 1991). These serine/threonine kinases phosphorylate and modulate the function of many proteins with regulatory functions including other protein kinases (such as p 9 0 r s k) cytoskeletal proteins (such as microtubule-associated phospholipase A 2 upstream regulators (such as the epidermal growth factor receptor and Ras exchange factor) and transcription factors (such as C-Myc and Elk-1).

MAP kinases can be activated through non-receptor tyrosine kinases such as focal adhesion kinase (FAK), cytoplasmic tyrosine kinase (pp60 c-srk) (Schlaepher and Hunter, 1998), and growth factors acting through membrane-associated receptor tyrosine kinases. The FAK pathway is activated by most integrins. In addition to activating FAK, some pi and av integrins also activate the tyrosine kinase Fyn and through it, the adaptor protein She (Wang et al, 1996). It is likely that both FAK and She contribute to the activation of Ras and thence to the downstream kinase cascade of Raf-1, MEK, and MAP kinases (Schlaepfer et al, 1994; 1997). It is now generally accepted that the activation of ERK in response to integrin ligation requires Ras signalling (Wary et al, 1996; Schlaepfer and Hunter, 1997). The laminin receptor ta64, the laminin/collagen receptor o 1 1, the fibronectin receptor a5pl and the ROD binding receptor avp3 are linked to the Ras-Raf-MEK-ERK signalling pathway and control of immediate early gene expression (Wary et al, 1996; Maniero et al, 1995; 1997). The ability of integrins to activate ERK may be especially important when the concentration-of growth factors available to the cell is limited. In this setting, proliferation is likely to require costimulation of ERK through integrins and growth factor receptors (Giancotti and Ruoslahti, 1999). Moreover, activation of ERK in response to integrin ligation may play a role in regulating cell migration (Klemke et al, 1997) possibly by initiating matrixdegrading enzyme secretion.

While there is a good deal of evidence in support of a key role for FAK and the phosphotyrosine-domain-containing adaptor protein She (Howe et al, 1998; Giancotti Ruoslahti, 1999) in the Ras-Raf MEK-MAP kinase activation pathway there are also data implicating alternate pathways independent of MEKs. For example, MEKindependent regulation of MAP kinase activation in NIH3T3 fibroblasts has been shown to be mediated by phosphatidylinositol-3-kinases and the conventional protein kinase C (PKC) isoforms and is thought to be due to inactivation of a MAP kinase inhibitor (Grammer and Blenis, 1997).

Although the mechanism by which PKC regulates integrin function is not known, PKC has been shown to regulate integrin-induced activation of the MAP kinase pathway upstream of She. For example, PKC inhibition has been shown to inhibit ERK2 activation by fibronectin receptors without any effect on integrin-induced FAK or paxillin tyrosine phosphorylation (Miranti et al, 1999). Hence, MAP kinase activation is more complicated than a simple linear pathway, and the mechanistic basis for the commonly observed integrin-mediated activation of MAP kinases remains controversial.

Various intracellular proteins may be linked directly or spatially to integrin cytoplasmic domains. Direct interactions have been identified between cytoskeletal proteins such as a-actinin and talin and pi and p3 integrin tails (Horwitz et al, 1986; Otey et al, 1990; Knezevic et al, 1996; Pfaffet al, 1998). A direct association between FAK and the p1 integrin tail has been suggested based on in vitro 1p peptide studies, but this remains to be confirmed (Schaller et al, 1995). More recently, the cytoplasmic domain of the a4 subunit has been found to be physically associated with the signalling adaptor protein paxillin in Jurkat T cells, and this binding event regulates the kinetics of FAK tyrosine phosphorylation (Liu et al, 1999).

Direct integrin links with the intracellular calcium-binding protein, calreticulin, and integrin-linked kinase (ILK) (Hannigan et al, 1996) have been shown to regulate "inside-out" integrin signalling. For example, calreticulin has been shown to bind to a chain cytoplasmic domains (Rojiani et al, 1991) and modify a2pl integrin activation by phorbol esters and anti-integrin antibodies (Coppolino et al, 1995). Newly identified 11 integrin-binding molecules include the serine/threonine integrin-linked kinase, ILK, which can associate with the 3p, p2 and 33 subunits. When over-expressed, ILK has been shown to reduce anchorage-independent growth and tumourigenicity in nude mice (Hannigan et al, 1996). Co-immunoprecipitation strategies have also demonstrated interactions between integrins and the integral plasma membrane protein IAP, and members of the four transmembrane domain protein family (tetraspans). The extracellular Ig region of the IAP molecule mediates association with cavp3 and is required for cell binding to vitronectin-coated particles (Lindberg et al, 1996). An emerging model for tetraspans is that they recruit signalling enzymes such as phosphatidylinosito.l-4-kinase and PKC into complexes with integrins (Hemler, 1998).

Integrins have also been shown to be physically linked with matrix-degrading enzymes and growth factors. For example, the integrin avp6 has been shown to bind and activate latent TGFlI in keratinocytes (Munger et al, 1999) which is thought to be important in modulating the inflammatory process following epithelial injury. In melanoma cells, avp3 binds activated gelatinase A (Brooks et al, 1996), and both insulin and platelet-derived growth factor (PDGF) co-immunoprecipitate with this integrin in NIH3T3 mouse fibroblasts (Schneller et al, 1997). Synergism between integrinmediated signalling processes and growth factor responses is now well-recognised and Schneller et al (1997) showed that a small subset of each of the insulin receptor and PDGF P-receptor is tyrosine phosphorylated upon growth factor stimulation.

Interestingly, this subset can associate with the cav3 integrin, and PDGF activity is enhanced in association with increased MAP kinase activity in cells plated on the cavp3 ligand, vitronectin.

FROM ADAMS PLUCK (TUE)SEP 30 2008 10:53/ST.10:51/No,7500000852 P 00 12 0 0 Summary Of The Invention Broadly stated, the present invention relates to modulation of integrin expression in

C)

Sneoplastic cells to inhibit the growth of the cells, and the surprising finding that members of the mitogen activated protein (MAP) kinase family associate with the cytoplasmic domain of an integrin molecule. It is believed that no member of the MAP kinase family Shas previously been found to directly associate with any integrin or for that matter, with t any transmembrane protein. The identification of this functional relationship permits the Ci rational design of agents for therapeutically or prophylactically modulating cellular activity mediated by the MAP kinase and integrin interaction.

to In one aspect of the present invention there is provided an isolated polypeptide comprising a binding domain of a cytoplasmic region ofa B integrin subunit for an ERK2 MAP kinase or a modified amino acid sequence compared to the binding domain, wherein the binding domain incorporates an amino acid linker sequence that links opposite end regions of the binding domain together and which is non-essential to the binding of the MAP kinase, and the B integrin subunit is selected from 35 and 66, and the modified amino acid sequence is other than a fragment of 05 and 86 or other 13 integrin subunit and has sufficient amino acid sequence homology with both the end regions of the binding domain to bind to the MAP kinase.

In another aspect of the present invention there is provided an isolated polypeptide comprising a modified binding domain of a cytoplasmic region of a B integrin subunit for a MAP kinase, the polypeptide having a modified amino acid sequence compared to the binding domain, wherein the binding domain of the B integrin subunit incorporates an amino acid linker sequence that links opposite end regions of the binding domain COMS ID No: ARCS-207933 Received by IP Australia: Time 11:01 Date 2008-09-30 FROM ADAMS PLUCK (TUE)SEP 30 2008 10:53/ST.10:51/No. 7500000852 P 8 00 13 0 0 together and which is non-essential to the binding of the MAP kinase, and wherein one ci or more amino acids in the amino acid linker sequence of the binding domain are deleted oor differ in the polypeptide compared to the binding domain and the polypeptide has sufficient amino acid sequence homology with both the end regions of the binding domain to bind to the MAP kinase.

In a further aspect of the present invention there is provided a method for inhibiting growth of a cancer cell expressing a B integrin subunit, comprising treating the ttn ocancer cell with an effective amount of a polypeptide moiety, the polypeptide moiety being a fragment of a cytoplasmic region of the B integrin subunit wherein the fragment comprises a binding domain of the B integrin subunit for a MAP kinase, or a modified amino acid sequence compared to the binding domain that has sufficient amino acid sequence homology with the binding domain to bind to the MAP kinase.

In yet another aspect of the present invention there is provided a method for prophylaxis or treatment of cancer in a mammal, the method x)mprising: selecting an agent for inhibiting growth of cancer cells of the cancer, the cancer cells expressing a B integrin subunit; and administering an effective amount of the agent to the mammal, the agent comprising a polypcptide moiety being a fragment of a cytoplasmic region of thc B integrin subunit wherein the fragment comprises a binding domain of the 3 integrin subunit for a MAP kinase, or a modified amino acid sequence compared to the binding domain and having sufficient amino acid sequence homology with the binding domain to bind to the MAP kinase.

In still another aspect of the present invention there is provided a method for prophylaxis or treatment of cancer in a mammal, comprising administering to the COMS ID No: ARCS-207933 Received by IP Australia: Time 11:01 Date 2008-09-30 mammal an effective amount of a polypeptide moiety being a fragment of a cytoplasmic region of a 1 integrin subunit expressed by cancer cells of the cancer wherein the fragment comprises a binding domain of the p integrin subunit for a MAP kinase, or a modified amino acid sequence compared to the binding domain, and wherein the 3 integrin subunit is selected from the group consisting of 35 and P6 and the MAP kinase is ERK2, and the modified amino acid sequence has sufficient amino acid sequence homology with the binding domain to bind to the MAP kinase.

In a related embodiment there is provided an agent capable of inhibiting binding of a MAP kinase with an integrin.

Typically, the agent will bind to a binding site on the MAP kinase that binds to a binding domain on the integrin for the MAP kinase. Alternatively, the agent may bind to the binding domain on the integrin for the MAP kinase or other site on the integrin such that binding of the MAP kinase to the integrin is thereby inhibited.

In another related embodiment there is provided an isolated polypeptide that binds to a binding site of a MAP kinase for an integrin or a homolog, analog, variant or derivative of the polypeptide, with the proviso that the polypeptide is other than a full length integrin subunit or a 06(770t) or 06(777t) deletion mutant.

Preferably, the polypeptide will comprise the binding domain of the integrin or sufficient core amino acid sequence of the binding domain for binding of the polypeptide with the MAP kinase.

Preferably, the polypeptide will comprise amino acid sequence RSKAKWQTGTNPLYR, more preferably RSKAKNPLYR, or one or both amino acid sequences RSKAK and NPLYR. Most preferably, the polypeptide will be a fragment of an integrin subunit.

Accordingly, in a further related embodiment there is provided a fragment of an integrin subunit wherein the fragment binds to a MAP kinase, or a homolog, analog, variant or derivative of the fragment, with the proviso that the integrin subunit is other than a p6(770t) or p6(777t) deletion mutant.

Preferably, a polypeptide or fragment as described herein will have a length of about 150 amino acids or less, more preferably about 100 or 50 amino acids or less, more usually about 40 amino acids or less, and generally, between about 5 to about amino acids. Preferably, the length will be up to about 20 amino acids, more preferably greater than 5, and up to 15, amino acids and most preferably, from 10 to 15 amino acids in length.

Some fragments may comprise an amino acid sequence incorporating extracellular and cytoplasmic regions of the integrin subunit. Preferably, the fragment will be a fragment of the cytoplasmic domain of the integrin subunit.

In another related embodiment there is provided an integrin subunit with a mutagenised binding domain for a MAP kinase or in which the binding domain is deleted,wherein capability of the integrin subunit to bind with the MAP kinase is thereby reduced, or a homolog, analog, variant or derivative of the integrin subunit, with the proviso that the integrin subunit is other than a 06 A746-764 deletion mutant.

In still another related embodiment there is provided a fusion protein incorporating a polypeptide as described herein or a homolog, analog or variant of the polypeptide.

In a further related embodiment there is provided a fusion protein incorporating a fragment as described herein or a homolog, analog or variant of the fragment.

Typically, a polypeptide or fragment as described herein will be coupled to a molecule such as penetratin or other facilitator molecule for facilitating entry into the cytoplasm of the cell.

In another related embodiment there is provided an isolated nucleic acid sequence encoding a polypeptide as described herein or a homolog, analog, or variant of the polypeptide.

In yet another related embodiment there is provided an isolated nucleic acid sequence encoding a fragment as described herein or a homolog, analog, or variant of the fragment.

In still yet another related embodiment there is provided a nucleic acid sequence encoding an integrin subunit with a mutagenised binding domain for a MAP kinase or in which the binding domain is deleted,wherein capability of the integrin subunit to bind with the MAP kinase is thereby reduced, or a homolog, analog, or variant of the integrin subunit, with the proviso that the integrin subunit is other than a P6 A746-764 deletion mutant.

In a still further related embodiment there is provided an isolated nucleic acid sequence encoding a fusion protein as described herein.

There are also provided antisense nucleic acid sequences complimentary to the sense nucleic sequences described herein. Such antisense sequences find application in antisense therapy of cells in which down regulation of cellular activity is desired, and include oligonucleotides. Sense oligonucleotides coding for the binding domain of an integrin subunit or that of a homolog, analog or variant thereof, and complimentary antisense oligonucleotides find particular application as primers or probes. A nucleic acid primer or probe may be labelled with a suitable reporter molecule for enabling detection ofhybridisation of the primer or probe to a target nucleic acid sequence.

In yet another related embodiment there is provided a vector incorporating a nucleic acid sequence described herein. Typically, the vector will be an expression vector and the nucleic acid sequence will be capable of being transcribed.

In a further related embodiment there is provided a host cell transformed with a vector described herein.

Preferably, the host cell will be selected from the group consisting of a mammalian cell, an epithelial cell, a neoplastic cell, and a cancer cell. Preferably, the host cell will be a mammalian cell and most preferably, a colon cancer cell.

In a further related embodiment there is provided a transgenic animal with cells containing heterologous nucleic acid described herein.

In yet another related embodiment there is provided an antibody generated with the use of a polypeptide or fragment described herein.

In a still further related embodiment there is provided an antibody that binds to a binding domain of an integrin for a MAP kinase.

The antibody may be a polyclonal or monoclonal antibody. Preferably, the antibody is a monoclonal antibody.

In still another related embodiment there is provided a method of isolating a MAP kinase from a sample utilising a molecule immobilised on a solid support and which 18 binds to a binding site on the MAP kinase which binding site binds with a binding domain of an integrin for the MAP kinase, comprising: contacting the molecule immobilised on the solid support with the sample under conditions suitable for binding of the MAP kinase to the molecule; eluting the MAP kinase from the solid support; and collecting the eluted MAP kinase.

The molecule may be the integrin, or a fusion protein, a polypeptide, or a fragment of the invention to which the MAP kinase binds, or for instance an analog, homolog, variant or derivative of the polypeptide or fragment.

In yet another related embodiment there is provided a MAP kinase isolated by a method described herein.

Rather than isolating the MAP kinase from a sample, the MAP kinase may be immobilised on a solid support and used to isolate the molecule from a sample, and all such methods are also expressly encompassed as is the molecule when isolated in this way.

In another aspect of the present invention there is provided a method of screening for an agent that binds to a binding domain of a P integrin subunit for a MAP kinase, comprising: utilising the agent in an assay for determining whether the agent binds to the binding domain of the P integrin subunit; and determining if the agent binds to the binding domain on the basis of the assay.

In another related embodiment there is provided a method of screening for an agent that inhibits binding of a MAP kinase to a binding domain of an integrin for the MAP kinase, comprising: testing a number of agents for ability to bind to either the MAP kinase or the integrin; and determining if any said agent inhibits binding of the MAP kinase to the binding domain of the integrin on the basis of the testing.

In another related embodiment there is provided a method of screening for an agent that inhibits binding of a MAP kinase to a binding domain on an integrin for the MAP kinase, comprising: testing a number of agents for ability to bind to either the MAP kinase or the integrin; selecting an agent or agents identified as being able to bind to the MAP kinase or the integrin on the basis of the testing; and utilising the selected said agent or agents in an assay for indicating whether the or any of the selected said agents inhibits the binding of the MAP kinase to the binding domain of the integrin.

In another related embodiment there is provided a method of evaluating whether an agent inhibits binding of a MAP kinase to a binding domain of an integrin for the MAP kinase, comprising: selecting the agent; utilising the agent in an assay for indicating whether the agent inhibits binding of the MAP kinase to the binding domain of the integrin; and determining if the agent inhibits the binding of the MAP kinase to the binding domain of the integrin on the basis of the assay.

Testing or assaying of an agent for ability to bind to the MAP kinase or the integrin and thereby inhibit binding of the MAP kinase to the integrin, may comprise exposing the integrin to the agent(s) to enable binding of the agent(s) to the integrin to occur either in the presence of the MAP kinase or prior to the addition of the MAP kinase. Rather than using the integrin, a polypeptide or fragment of the invention or other molecule capable of binding with the binding site on the MAP kinase that binds to the integrin may be used. Alternatively, the testing or assaying may comprise exposing the MAP kinase to the agent(s) to enable binding of the agent(s) to the MAP kinase to occur either in the presence of the integrin or other molecule capable of binding with the binding site on the MAP kinase, or prior to the addition of the integrin or molecule.

In still another related embodiment there is provided a method of screening for an agent that binds to a binding domain of an integrin for a MAP kinase, comprising: testing a number of agents for ability to bind to the binding domain of the integrin for the MAP kinase; and determining if any said agent binds to the binding domain of the integrin on the basis of the testing.

In yet another related embodiment there is provided a method of screening for an agent that binds to a binding domain of an integrin for a MAP kinase, comprising: testing a number of agents for ability to bind to the integrin; selecting an agent or agents identified as being able to bind to the integrin on the basis of the testing; and 21 utilising the selected said agent or agents in an assay for indicating whether the or any of the selected said agents binds to the binding domain on the integrin for the MAP kinase.

In another related embodiment there is provided a a method of evaluating whether an agent binds to a binding domain of an integrin for a MAP kinase, comprising: testing the agent for ability to bind to the binding domain of the integrin for the MAP kinase; and determining if the agent binds to the binding domain on the basis of the testing.

Preferably, a polypeptide or fragment described herein consisting of the binding domain of the integrin or core amino acid sequence of the binding domain or a homolog, analog or variant of the polypeptide or fragment is used in the testing or assaying for whether an agent is capable of binding to the binding domain of the integrin. Most preferably, the polypeptide or fragment will consist of the amino acid sequence RSKAKWQTGTNPLYR or RSKAKNPLYR.

An agent as described herein will usually be provided in the form of a pharmaceutical composition. Accordingly, in another related embodiment there is provided a pharmaceutical composition comprising an agent that inhibits binding of a MAP kinase to a binding domain on an integrin for the MAP kinase, and a pharmaceutically acceptable carrier or diluent.

The agent may or not be proteinaceous in nature. Preferably, the agent will comprise a fusion protein, or polypeptide. Most preferably, the polypeptide will be coupled to a molecule for facilitating entry of the agent into a cell.

22 In a further related embodiment there is provided a pharmaceutical composition comprising a nucleic acid sequence described herein and a pharmaceutically acceptable carrier or diluent. Preferably, the nucleic acid sequence is incorporated into a vector as described herein. Alternatively, the nucleic acid sequence may be joined to a carrier molecule for facilitating entry of the nucleic acid sequence into a target cell.

In a further related embodiment there is provided a method of modulating activity of a cell, comprising: transfecting the cell with a nucleic acid sequence encoding an integrin subunit for being expressed by the cell, wherein the integrin subunit has a mutagenised binding domain for a MAP kinase or in which the binding domain is deleted, or a homolog, analog or variant of the integrin subunit.

In yet another related embodiment there is provided a method of modulating activity of a cell, comprising: transfecting the cell with a nucleic acid encoding a polypeptide for being expressed by the cell wherein the polypeptide inhibits binding of a MAP kinase with a binding domain on an integrin for the MAP kinase, or a homolog, analog or variant of the polypeptide.

Preferably, the polypeptide will bind to the binding site on the MAP kinase which binding site binds to the binding domain on the integrin.

In still another related embodiment there is provided a method of modulating activity of a cell, comprising down-regulating the expression of an integrin to which a MAP kinase binds.

Preferably, down-regulation of the expression of the integrin is achieved using an antisense nucleic acid sequence that inhibits expression of the gene encoding the integrin. The antisense nucleic acid sequence may be administered to the cell or generated in vivo within the cell. Preferably, the cell will be transformed with a vector of the invention for generation of the antisense nucleic acid sequence in vivo.

Preferably, the antisense nucleic acid sequence will specifically hybridise with the sense nucleic acid sequence coding for the binding domain of the integrin for the MAP kinase and/or intron sequence between such coding sequence.

In a still further related embodiment there is provided a method of modulating activity of a cell, comprising contacting the cell with an effective amount of an agent for down regulating functional activity of an integrin expressed by the cell and thereby the activity of the cell.

In another related embodiment there is provided a method of modulating activity of a cell, comprising: contacting the cell with an effective amount of an agent that binds to a MAP kinase or a cytoplasmic region of an integrin expressed by the cell such that binding of the MAP kinase to a binding domain of the integrin for the MAP kinase is inhibited.

In a further related embodiment there is provided a method of modulating activity of a cell, comprising: contacting the cell with an effective amount of an agent that binds to a MAP kinase inhibiting binding of the MAP kinase to a binding domain on the integrin for the MAP kinase.

24 Preferably, the agent will bind to the binding site on the MAP kinase which binding site binds to the binding domain on the integrin for the MAP kinase.

In a further related embodiment there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cell activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid sequence encoding an integrin subunit for being expressed, wherein the integrin subunit has a mutagenised binding domain for a MAP kinase or in which the binding domain is deleted, or a homolog, analog or variant of the integrin subunit.

In another related embodiment there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cell activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid of the invention for down-regulating expression of an integrin to which a MAP kinase binds.

In still another related embodiment there is provided a method of prophylaxis or treatment of a disease or condition in a mammal wherein modulation of cell activity is desirable, comprising: administering to the mammal an effective amount of a nucleic acid sequence encoding a polypeptide for being expressed, wherein the polypeptide inhibits binding of a MAP kinase to a binding domain of an integrin for the MAP kinase down-regulating MAP kinase integrin binding, or a homolog, analog or variant of the polypeptide.

In another related embodiment there is provided a method of treatment or prophylaxis of a disease or condition in a mammal, wherein said condition is responsive to an agent as described herein, the agent inhibiting binding of a MAP kinase to a binding domain of an integrin for the MAP kinase, and the method comprising administering an effective amount of the agent to the mammal.

In another related embodiment there is provided use of a nucleic acid described herein in the manufacture of a medicament for administration to a mammal in the prophylaxis or treatment of a disease or condition in which down regulation of cellular activity is desirable.

In a still further related embodiment there is provided the use of an agent that inhibits binding of a MAP kinase to a binding domain of an integrin in the manufacture of a medicament for administration to a mammal for the prophylaxis or treatment of a disease or condition where down regulation of cellular activity is desirable.

The cellular activity desired to be down-regulated will typically but not exclusively, be cell growth. Indeed, any activity influenced by signalling mediated by MAP kinase activation is expressly included.

The cell may be any cell type in which functional activity of a MAP kinase arising from binding with an integrin may occur. Preferably, the cell will be an epithelial cell and most preferably, a neoplastic cell. By modulating the activity a neoplastic cell, methods of the invention find particular application in the prophylaxis or treatment of cancer. In particular, methods of the invention find particular application in the treatment of colon cancer.

Usually, the MAP kinase will be selected from the group consisting of an extracellular signal-regulated kinase (ERK) and a JNK MAP kinase. Preferably, the MAP kinase is ERK2 or JNK-1. Most preferably, the MAP kinase is ERK2.

The mammal may be any mammal treatable with a method of the invention. For instance, the mammal may be a member of the bovine, porcine, ovine or equine families, a laboratory test animal such as a mouse, rabbit, guinea pig, a cat or dog, a primate or a human being. Preferably, the mammal will be a human being.

The term "modulating" is to be taken as reference to down-regulating the activity of the cell or the functional activity of the integrin. Reference to "down-regulating" should be understood to include preventing, reducing or otherwise inhibiting one or more aspects of the activity of the cell or the functional activity of the integrin molecule or the MAP kinase In the broadest sense, the term "integrin" unless otherwise specified, is to be taken to encompass an integrin family member or a homolog, derivative, variant or analog of an integrin subunit, or an integrin family member incorporating at least one such homolog, derivative, variant or analog of an integrin subunit. Preferably, the integrin is or incorporates an integrin subunit selected from the group consisting of 33, 35 and 136.

Most preferably, the integrin comprises 136.

By "binding domain" is meant the minimum length of contiguous amino acid sequence required for binding by the MAP kinase. By "core amino acid sequence" is meant regions or amino acids of the binding domain that directly participate in the binding as distinct from any amino acids that do not directly participate in the binding interaction with the MAP kinase. Typically, the core amino acid sequence of the binding domain will comprise regions of the binding domain linked together by a number of intervening amino acids which do not directly participate in the binding.

27 The term "homolog" is to be taken to mean a molecule that has amino acid sequence similarity. The homology between amino acid sequences can be determined by comparing amino acids at each position in the sequences when optimally aligned for the purpose of comparison. The sequences are considered homologous at a position if the amino acids at that position are the same. Typically, a homolog will have an overall amino acid sequence homology of at least about 30% more preferably at least about or 70% and most preferably, greater than about 80%, 90% or 98% sequence homology.

Homology with the binding domain of an integrin may be greater than the overall amino acid sequence homology of the homolog, and will usually be greater than about 60% or 80%, and more usually greater than about 90%, 95% or 98%.

The term "analog" is to be taken to mean a molecule that has one or more aspects of biological function characteristic of the molecule on which at least part of the analog is based or which was otherwise utilised in the design or preparation of the analog. An analog may have substantial overall structural similarity with the molecule or only structural similarity with one or more regions or domains thereof responsible for the desired characteristic biological function. By "structural" similarity is meant similarity in shape, conformation and/or other structural features responsible for the provision of the biological function or which otherwise have involvement in the provision of the biological function. Alternatively, it will be understood that with knowledge of the region(s) or domain(s) of a molecule that provide(s) the characteristic biological function, analogs may be designed that while differing in structure nevertheless possess such biological function. Indeed, it is not necessary that an analog have amino acid 28 sequence homology, and an analog may not be proteinaceous at all. An analog may for instance be a mimetic of a molecule.

By the term "variant" is meant an isoform of an integrin subunit, an integrin subunit encoded by an allelic variant of a gene for an integrin subunit, a naturally occurring mutant form of a gene for an integrin subunit, or an integrin subunit or polypeptide having an amino acid sequence that differs in one or more amino acids but which retains one or more aspects of desired characteristic biological function. This may be achieved by the addition of one or more amino acids to an amino acid sequence, deletion of one or more amino acids from an amino acid sequence and/or the substitution of one or more amino acids with another amino acid or amino acids. Inversion of amino acids and any other mutational change that results in alteration of an amino acid sequence are also encompassed. A variant may be prepared by introducing nucleotide changes in a nucleic acid sequence that encodes for an integrin subunit or amino acid sequence such that the desired amino acid changes are achieved upon expression of the mutagenised nucleic acid sequence, or for instance by synthesising an integrin subunit or amino acid sequence incorporating the desired amino acid changes, both of which possibilities are well within the capability of the skilled addressee.

Substitution of an amino acid may involve a conservative or non-conservative amino acid substitution. By conservative amino acid substitution is meant replacing an 28a.

amino acid residue with another amino acid having similar stereochemical properties (eg. structure, charge, acidity or basicity characteristics) and which does not substantially effect conformation or the desired aspect or aspects of characteristic biological function.

Preferred variants include ones having amino acid sequences in which one or more amino acids have been substituted with alanine or other neutrally charged amino acid residue(s), or to which one or more such residues have been added. A variant may also incorporate an amino acid or amino acids that are not encoded by the genetic code.

By the term "derivative" is meant a molecule that is derived or obtained from another molecule and which retains one or more aspects of characteristic biological function of that molecule. A derivative may for instance arise as a result of the cleavage of the parent molecule, cyclisation and/or coupling with one or more additional moieties that improve solubility, lipophilic characteristics to enhance uptake by cells, stability or biological half-life, decreased cellular toxicity, or for instance to act as a label for subsequent detection or the like. A derivative may also result from post-translational or post-synthesis modification such as the attachment of carbohydrate moieties or chemical reaction(s) resulting in structural modification(s) such as the alkylation or acetylation of amino acid residues or other changes involving the formation of chemical bonds.

The term "polypeptide" is used interchangeably herein with "peptide" and encompasses amino acid sequences incorporating only a few amino acid residues or many amino acid residues coupled by peptide bonds.

The term "neoplastic cell" is to be taken to mean a cell exhibiting abnormal growth and may or may not be a malignant cell. "Growth" is to be taken in its broadest sense 28b.

and includes proliferation of the cell. In this regard, an example of abnormal cell growth is the uncontrolled proliferation of a cell.

All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in this specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of this application.

Unless the context clearly requires otherwise, throughout the description and the claims, the words 'comprise', 'comprising', and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".

The features and advantages of the present invention will become further apparent from the following detailed description of preferred embodiments and the accompanying drawings.

Brief Description Of The Accompanying Drawings Figure 1: Surface biotinylation and immunoprecipitation of integrin subunits and 06 in HT29 colon cancer cells stably transfected with either vector alone (mock transfectants) or antisense 06 construct; Figure 2: Amplification of 16 and glyceraldehyde dehydrogenase (GAPDH) mRNA: Ethidium-stained agarose gels with amplification products following RT-PCR from total RNA extracted from transfected HT29 and WiDr cell lines. Equal amounts of PCR product obtained from RT-PCR reactions were loaded on each lane and the 16 (141 29 basepairs) and GAPDH (216 basepairs) bands are indicated (WT, wild-type; S, sense P6; A/S, antisense p6; mock, vector alone); Figure 3: Non-transfected HT29 cells (wild) and cells transfected with vector alone (mock), sense p6 and antisense p6 analysed by FACScan for expression of the 36 subunit. White and black histograms represent cells stained in the absence and presence, respectively, of mAb E7P6 (anti-36); Figure 4: WiDr cells transfected with vector alone (mock) or antisense p6 and analysed by FACScan for expression of the p6 subunit. White and black histograms represent cells stained in the absence and presence, respectively, of mAb E7P6 (anti- P6).; Figure 5: Tumor cell proliferation in vitro assessed by 3 H)-thymidine uptake for WiDr wild-type cells and transfectants (mock and antisense p6) cultured on plastic for the times indicated.

Figure 6: Tumor cell proliferation in vitro assessed by 3 H)-thymidine uptake for HT29 wild-type cells and transfectants (mock, sense 16 and antisense p6) cultured on plastic for the times indicated.

Figure 7: Tumour growth after 6 weeks following subcutaneous inoculation of 106 viable cells of HT29 mock (vector alone) and antisense p6 transfectants; Figure 8: Graph showing average tumour size at weekly intervals for each subgroup of 10 animals following subcutaneous inoculation of clone 1 from WiDr mock (vector alone) and antisense 16 transfectants; Figure 9: Graph showing average tumour size at weekly intervals for each subgroup of 10 animals following subcutaneous inoculation of HT29 mock (vector alone) and antisense 36 transfectants; Figure 10: 06-associated ERK identified by immunoprecipitations of integrin subunits from SW480 06 transfectants. (Top) Cell lysates were immunoprecipitated with mAbs QE2E5 (anti-pl), L230 (anti-av), P1F6 (anti-35), R6G9 (anti-P6), or matched isotype control Abs (IgGI and IgG2A), and (bottom) blotted with anti-ERKl/2 mAb, SC-1647, which recognises total ERK (phosphorylated and non-phosphorylated).

Purified, non-phosphorylated ERK2 is shown in the left hand lane; Figures 11(A) and 1 Western blotting: equal protein loads of cell lysates from one representative clone each from WiDr mock and antisense p6 blotted with anti- ERK mAb (SC-1647) against total ERK. Purified non-phosphorylated ERK2 is shown in the left hand lane. p6 immunoprecipitates (mAb R6G9) from equal protein loads of the cell lysates in probed with anti-ERK mAb (E 10). Purified phosphorylated ERK2 is shown in the left hand lane; Figure 12: p6-bound ERK shown for the high and low SW480 p6-expressing clones by probing p6 immunoprecipitates with anti-ERK mAb (E10) against phosphorylated forms of ERKl/2. Purified, phosphorylated ERK2 is shown in the left hand lane; Figures 13(A) and 13(B): Surface biotinylation of WiDr wild-type cells and 36 immunodepletion of the cell lysates by three successive rounds of 16 and immunoprecipitations using mAb R6G9 and P1F6, respectively or control mAb (IgG2A). The 06 and partner av bands are arrowed. 6-immunodepleted WiDr cell 31 lysates after 3 successive rounds of 06-immunoprecipitations probed with anti-ERK mAb SC-1647 recognising both phosphorylated and non-phosphorylated forms of ERK1/2 and compared with non-p6 immunodepleted lysates and control lysates sequentially immunoprecipitated 3 times with either isotype matched control mAb (IgG2A) or mAb P1F6 Figure 14: Non-transformed (HaCaT) and Ras-transformed (HaRas) human keratinocytes: 06 immunoprecipitation and ERK western blots probed with monoclonal antibody E10 (against Phosphorylated ERK 1/2) and monoclonal antibody SC1647 (against total ERK respectively.

Figure 15(A) and 15(B): Depletion of 16 with sequential rounds of ERK immunodepletion of cell lysates using anti-ERK mAb (SC-1647). MAP kinase activity in cell lysates before and after 5 rounds of sequential p6 immunodepletion from SW480 06 and SW480 mock transfectants (full grey and hatched bars, respectively).

MAP kinase activity is shown as the mean of three independent experiments. The reduction in MAP kinase activity following p6 immunodepletion was highly signigicant (P-0.005, students T test).

Figure 16: Shows the amino acid sequence of the cytoplasmic domain of the P6 subunit as well as the amino acid sequences for the p1 to 13 subunits, respectively; Figure 17: Graph showing binding of non-phosphorylated ERK2 (GST.ERK2) to overlapping fragments corresponding to different regions of the cytoplasmic domain of the 16 subunit; 32 Figure 18: Graph showing binding of ERK2 (GST.ERK2) to the p6 cytoplasmic domain and the peptide fragments indicated in Fig. 16 over a range of concentrations of ERK2; Figure 19: Graph showing binding ofERK2 (GST.ERK2) to the p6 cytoplasmic domain and fragments thereof; Figure 20: Graph showing binding of ERK2 (GST.ERK2) to a 15 mer fragment of the p6 cytoplasmic domain and which has the amino acid sequence RSKAKWQTGTNPLYR; and Figure 21: Shows regions of the cytoplasmic domain of the 36 subunit corresponding to synthesised fragments thereof evaluated for capacity to be bound by ERK2.

Figure 22: Graph showing assay results for ERK2 (GST.ERK) binding to synthesised 10 mer fragments identified in fig. 21.

Figure 23: Graph showing binding of ERK2 (thrombin cleaved) to synthesised peptide having the amino acid sequence RSKAKNPLYR compared to the 15 mer RSKAKWQTGTNPLYR fragment of the cytoplasmic domain of the 06 subunit.

Figure 24: Graph showing binding of JNK-1 to the 36 cytoplasmic domain.

Figure 25: location of P6 A746-764, p6(770t) and p6(777t) deletions in the cytoplasmic domain of the p6 subunit.

Figure 26: SW480 cells transfected with wild-type full length coding sequence for p6 or p6 A746-764 deletion mutant analysed by FACScan for expression of wild-type or mutant p6. White and black histograms represent cells stained in the absence and presence of the integrin subunit, respectively.

Figures 27(A) and 27(B): Western blotting: equal protein loads of cell lyates from SW480 cells expressing wild-type p6 or p6 A746-764 deletion mutant. P6 immunoprecipitates (mAb R6G9) from equal protein loads of cell lysates probed with anti-ERK mAb Figure 28: Proliferation of HT29 colon cancer cells cultured for 48 hours and treated with penetratin, the fragment of p6 cytoplasmic domain having amino acid sequence RSKAKWQTGTNPLYR alone or the fragment coupled to penetratin for the final 24 hours of the incubation period.

Figure 29: Proliferation of SW480 cells expressing wild-type p6 cultured on plastic for 48 hours and treated with penetratin, the RSKAKWQTGTNPLYR peptide alone or the peptide coupled to penetratin for the final 24 hours of the incubation period.

Figures 30 to 29 SW480 cells cultured with control additive for 24 hours; SW480 cells cultured with penetratin for 24 hours; SW480 cells cultured with RSKAKWQTGTNPLYR bound to penetratin for 24 hours.

Figures 31 and 31 SW480 mock and SW480 p6 transfectants cultured in presence of the RSKAKWQTGTNPLYR peptide coupled to penetratin. Photomicrographs of cells shown in cultured in the presence/absence of the peptide penetratin complex.

Figures 32(A) to 32(C): SW480 cells cultured in a 3-dimensional collagen type I matrix photographed in the gel at the end of 10 days. The SW480 cells following dissolution of the collagen with collagenase. Graph showing numbers of colonies of the SW480 cells having a diameter exceeding 200up.

34 Figures 33(A) and 33(B): Graphs showing inhibition of proliferation of SW480 cells expressing full length wild-type p6 in the presence of RSKAKWQTGTNPLYR peptide bound to penetratin.

Figure 34: Graph showing binding of ERK2 to RSKAKWQTGTNPLYR peptide and peptides corresponding to regions of the cytoplasmic domain of 03 and Detailed Description Of Preferred Embodiments Of The Present Invention The distribution of p6 integrin subunit within various tissues has been assessed by both in situ hybridisation and immunostaining and reported in the art. For instance, 36 mRNA in adult primate tissues was detected only in epithelial cells and at very low or undetectable levels in most normal tissues (Breuss et al, 1993). High-level expression of P6 has been observed in secretory endometrial glands while low-level expression was detected in the ductal epithelia of salivary gland, mammary gland and epididymis, in gall and urinary bladder, and in the digestive tract. Immunostaining data has also shown that 16 expressionis restricted to epithelia and is up-regulated in parallel with morphogenetic events, tumourigenesis and epithelial repair (Breuss et al, 1993; 1995). During development of the kidney, lung and skin, p6 is expressed by specific types of epithelial cells, whereas it is mostly undetectable in normal adult kidney, lung and skin. In contrast, high level expression of 06 has been observed in several types of carcinoma.

For example, p6 is almost invariably neo-expressed in squamous cell carcinomas derived from the oral mucosa, and often focally localised at the infiltrating edges of tumour cell islands (Breuss et al, 1995; Thomas et al, 1997). Moreover, expression of the p6 subunit has been observed in renal cell carcinoma and testicular tumour cell lines (Takiuchi et al, 1994) and 50% of lung cancers have been shown to express this subunit (Smythe et al, 1995).

Expression of 16 is also up-regulated in migrating keratinocytes at the wound edge during experimental epidermal wound healing. avp6 is not expressed in normal epithelium (Jones et al, 1997). However, following experimental wounding, av appears to switch its heterodimeric association from 15 to p6 subunit during re-epithelialisation.

At day 3 after wounding, p6 is absent but then appears around the perimeter of the basal cells of the migrating epidermis (Clark et al, 1996). By day 14 after wounding, when reepithelialisation is complete, all suprabasalar cells overlying the wound express 06 but not p5. In human mucosal wounds, maximal expression of p6 has been observed relatively late when epithelial sheets are fused and granulation tissue is present (Haapasalmi et al, 1996). Furthermore, those investigators observed maximal expression of tenascin with cav6 expression. Interestingly, freshly isolated keratinocytes have not been found to express 06 but begin to express this after subculturing (Haapasalmi et al, 1996). Moreover, TGF-13 has been shown to induce the de novo expression of avp6 at the cell surface on keratinocytes (Zambruno et al, 1995). This is particularly relevant in view of the recent observation that otv16 binds and activates latent TGF-pl which may be a means of locally regulating TGF-11 function in vivo during tissue response to injury (Munger et al, 1999).

av6 expression is also upregulated in type II alveolar epithelial cells during lung injury caused by injection of live bacteria and avp6 mRNA is induced within 5 hours of acute injury (Breuss et al, 1995). Interestingly, avp6 has been shown to be expressed on proximal airway epithelial cells in 50% of smokers undergoing lung resection 36 (Weinacker et al, 1995). Just as in human keratinocytes, in primary cultures of human airway epithelial cells, TGF-P31 has been shown to dramatically increase expression of ctvp6 without affecting surface expression of any other integrin. Moreover, epidermal growth factor (EGF) also induces cav6 in these cells, and the effect of both growth factors on P6 expression has been shown to be additive (Wang et al, 1996). tvp6 expression has also been observed in adult lungs and kidneys at focal sites of sub-clinical inflammation as well as in a variety of clinical specimens from patients with chronic or acute inflammation of the lungs or kidneys (Breuss et al, 1995). Taken together these data indicate that avp6 affects cell spreading, migration and growth during reorganisation of epithelia in development, tissue repair and neoplasia.

Recent studies have shown that av6 is a major fibronectin-binding receptor in colorectal cancer (Agrez et al, 1996). Moreover, normal colonic epithelium from cancer patients does not express ctvp6 in immunostaining studies, and as with squamous cell carcinomas from the oral mucosa (Thomas et al, 1997) maximal p6 expression in colon cancer has been observed at the invading edges of tumour cell islands (Agrez et al, 1996). Importantly, heterologous expression of the 36 subunit in colon cancer cells lacking constitutive expression of this receptor has been shown to stimulate tumour cell proliferation in vitro and tumour growth in athymic immune-deficient mice, and this growth-promoting effect is mediated through the 11 amino acid C-terminal cytoplasmic extension unique to the 16 subunit (Agrez et al, 1994). These findings suggest that one mechanism of the enhanced growth effect involves induced secretion of matrixdegrading enzymes as has been previously suggested for invasive melanoma cells.

Support for this arises from findings that the proliferative capacity of colon cancer cells 37 within 3-dimensional collagen matrices is inversely related to the density of the extracellular matrix (Agrez, 1989), and the observation that induced expression of aV36 in colon cancer cells markedly enhances gelatinase B secretion (Agrez et al, 1999).

The (36 subunit is widely observed in cancers of various origins (Breuss et al, 1995). As described above, 36 is detected in at least 50% of bowel cancer tumours.

Others have reported its presence in oropharyngeal cancers where it is also present and strongly expressed in the invading margins of the cancer cell islands as is commonly found in bowel cancer. In the oropharyngeal mucosa, no 36 is observed in the normal lining cells of the mouth but in both primary and metastatic tumours from the 0o oropharyngeal mucosa, strong P6 expression is seen which does not correlate with degree of differentiation and in particular, is restricted to the basal layer of epithelial cells. In colon cancer, p6 expression is similarly maximal at the advancing edges of tumour cell islands (Agrez et al, 1996).

Hence, modulation of MAP kinase interaction with the p6 subunit in epithelial cells may be used in the prophylaxis or treatment of cancer of the lip, tongue, salivary glands, gums, floor and other areas of the mouth, oropharynx, nasopharynx, hypopharynx and other oral cavities, oesophagus, stomach, small intestine, duodenum, colon, rectum, gallbladder, pancreas, larynx, trachea, bronchus, lung, female and male breast, uterus, cervix, ovary, vagina, vulva, prostate, testes, penis, bladder, kidney, thyroid and skin.

In terms of prophylactic use, a method of the invention may find application in protecting against ultraviolet-induced skin cancer, lung cancer in smokers, cancer of the gut where polyps are present as in polyposis coli or other inheritable disease where a predisposition to the development of polyps exists, breast cancer in high risk patients with a familial history of breast cancer or otherwise identified as carrying known mutations of the breast cancer susceptibility gene BRCAI or BRCA2 associated with breast cancer, transitional cell cancers arising from bladder papillomas, and cancer of the cervix in individuals deemed to be at high risk.

The epithelial restricted integrin subunit p6 is shown herein to interact with at least MAP kinases ERK2 and JNK-1, and its down-regulation in the present study dramatically suppresses growth of colon cancer. Hence, therapeutic strategies to inhibit growth and proliferation of colon cancer and growth of other malignant cancer cells growth by switching off either the permissive p6 integrin and/or inhibiting the p6 MAP kinase interaction are of particular interest. In particular, the fact that p6 expression may be significantly upregulated on tumour cells compared to normal cells offers the potential for tumour cell specificity substantially without impairment of normal cellular function.

Down-regulation of the functional activity of an integrin can be achieved in a number of ways and in particular, by preventing or down-regulating the expression of the integrin molecule or by inhibiting the signalling function of the integrin.

Gene therapy is one strategy for treating cancers of different types. The use of recombinant adenoviruses has for instance been utilised for restoring expression of polypeptide encoded by a wild-type p53 tumour suppressor gene (Bookstein et al, 1996), and adenoviral vectors for expression of wild-type p53 have been shown to suppress growth of human colon cancers by as much as 60% in animal models following intratumoural injection of recombinant virus (Spitz et al, 1996).

39 Rather than replacing a defective gene with one encoding a polypeptide the expression of which restores normal function of the cell as in the above examples, the possibility exists to achieve down regulation of cancer cells such as colon cells by introducing a gene that encodes an integrin subunit in which the binding domain for interacting with a MAP kinase has been rendered defective by mutagenesis, or in which the binding domain has been wholly or partially deleted, to thereby achieve down regulation through the inhibition of the MAP kinase integrin interaction. The defective integrin subunit will nevertheless usually be able to associate with its normal partner integrin subunit and be expressed on the cell membrane. Preferably, the defective integrin subunit will be expressed at a higher level than the corresponding wild-type integrin subunit such that down regulation is achieved by a dominant negative effect.

Alternatively, such therapy may involve the introduction and expression of a nucleic acid sequence that encodes a fragment or truncated form of an integrin subunit that excludes the binding domain for the MAP kinase or in which the binding domain has s1 been partially or wholly deleted or otherwise mutagenised so as to be defective.

Another option is to introduce a nucleic acid sequence encoding a polypeptide capable of binding to the binding domain on the integrin for the MAP kinase or to the binding site on the MAP kinase upon being expressed within the cell to thereby inhibit binding of the MAP kinase to the integrin and thereby achieve down regulation of cellular activity.

A gene or nucleic acid sequence encoding an integrin subunit may also be modified such that although the encoded binding domain for the MAP kinase remains unaltered, the amino acid sequence of a region of the integrin subunit distant from the binding domain, or the amino acid sequence of either one or both regions flanking the binding domain, is altered to achieve a change in the three dimensional conformation of the integrin subunit such that the binding of the MAP kinase with the binding domain is inhibited.

The gene or nucleic acid sequence may be altered to achieve the desired outcome by the deletion, insertion or substitution of one or more nucleotides such that the corresponding amino acid sequence is modified to the extent that the binding of the MAP kinase to the integrin is inhibited. Inhibition in this context may be partial or total inhibition.

The gene or nucleic acid sequence can be introduced into a cell in an appropriate expression vector for expression of the gene or nucleic acid sequence extrachromosomally or more preferably, for facilitating integration of the gene or nucleic acid sequence into genomic DNA by heterologous or homologous recombination events.

In another strategy, down-regulation of the expression of an integrin subunit such as p6 and hence an integrin heterodimer such as acvp6, is achieved using antisense nucleic acid sequences oligonucleotides) for inhibiting expression of the subunit.

Typically, this will involve expression of a nucleic acid construct incorporating all or part of a coding region of the gene for the integrin subunit inserted in the reverse orientation resulting in the synthesis of antisense RNA which inhibits translation of mRNA encoding the integrin subunit by hybridisation of the antisense RNA to the mRNA.

More broadly, a method of down regulating an activity of a cell may comprise contacting the cell with a first nucleic acid molecule that is capable of interacting with a 41 target nucleic acid sequence, or which first nucleic acid molecule is capable of being transcribed to a nucleic acid molecule capable of interacting with the target sequence whereby the interaction of the first nucleic acid molecule with the target sequence inhibits expression of the integrin subunit.

Reference to the first nucleic acid molecule is to be understood as a reference to any nucleic acid molecule which directly or indirectly facilitates reduction, inhibition or other form of down regulation of the expression of the integrin molecule. Nucleic acid molecules which fall within the scope of this definition include antisense sequences administered to a cell and antisense sequences generated in situ which have sufficient complementarity with target sequence such as mRNA encoding the integrin subunit or for instance, a transcription regulatory sequence controlling transcription of the gene encoding the integrin subunit, to thereby be capable of hybridising with the target sequence and inhibit the expression of the integrin subunit. The first nucleic acid molecule may also be a ribozyme capable for instance, specifically binding to that region of nucleic acid encoding the binding domain of an integrin subunit and cleaving the nucleic acid.

On a priori grounds, targeting the expression of the P6 subunit in malignant cells such as in colon cancer by means of adenoviral-mediated antisense therapy is preferred because down-regulating p6 by means of a non-adenoviral approach may increase cell surface expression of the P5 subunit. The relevance to such therapy is that the vitronectin-binding integrin avp5 promotes adenovirus internalisation (Thomas et al, 1993). Given that abundant avp5 is always present on the surface of colon cancer cells for example, a secondary benefit of inhibiting p6 expression during the course of therapy 42 may be the concomitant rise of p5 in cells already transduced with antisense 16 nucleic acid. This is likely to facilitate further virus uptake into such cells since the amount of integrin av35 present has been shown to be closely related to levels of gene expression following adenovirus-mediated gene transfer (Takayama et al, 1998).

Typically, an antisense nucleic acid sequence will hybridise with all or part of that region of the target sense nucleic acid encoding the binding domain. An antisense nucleic sequence may for instance be capable of hybridising to exon and/or intron sequences of pre-mRNA. Preferably, an antisense nucleic acid sequence will be designed for specifically hybridising to mature mRNA in which intron sequences have So been spliced out by normal cellular processing events.

It is riot necessary that an antisense nucleic acid sequence have total complementarity with its target sequence only that substantial complementarity exists for specificity and to allow hybridisation under cellular conditions. Preferably, an antisense nucleic sequence will have a complementarity of about 70% or greater, more preferably about 80% or greater and most preferably, about 90% or 95% or greater.

Preferred antisense sequences are oligonucleotides wherein the complementary sense nucleotides encode for about 50 amino acids or less, preferably about 35 or 30 amino acids or less, more preferably less than about 25 or 20 amino acids, most preferably about 15 amino acids or less and usually between about 5 to about 15 amino acids.

Antisense nucleic acid sequences may be generated in vivo by transcription of a suitable expression vector within a cell transformed with the vector, or ex vivo and then be introduced into a target cell to effect down regulation of the MAP kinase integrin interaction. Antisense sequences will desirably be designed to be resistant to 43 endogenous exonucleases and/or endonucleases to provide in vivo stability in target cells. Modification to the phosphate backbone, sugar moieties or nucleic acid bases may also be made to enhance uptake by cells or for instance solubility, and all such modifications are expressly encompassed. Such modifications include modification of the phosphodiester linkages between sugar moieties, the utilisation of synthetic nucleotides and substituted sugar moieties, linkage to liphophilic moieties and the such like as described in US Patent No. 5,877,309. Methods for the construction of oligonucleotides for use in antisense therapy have previously been described (see Van der Krol et al, 1998 Biotechniques 6:958-976; and Stein et al, 1998 Cancer Res 48:2659- 2668; Bachman et al, 1998, J. Mol. Med. 76:126-132).

Any means able to achieve the introduction of a gene or a nucleic acid into a target cell may be used. Gene transfer methods known in the art include viral and non-viral transfer methods. Suitable virus into which appropriate viral expression vectors may be packaged for delivery to target cells include adenovirus (Berkner, 1992; Gorziglia and Kapikian, 1992); vaccinia virus (Moss, 1992); retroviruses of avian (Petropoulos et al, 1992); murine (Miller, 1992) and human origin (Shimada et al, 1991); herpes viruses including Herpes Simplex Virus (HSV) and EBV (Margolskee, 1992; Johnson et al, 1992; Fink et al, 1992; Breakfield and Geller, 1997; Freese et al, 1990); papovaviruses such as SV40 (Madzak et al, 1992), adeno-associated virus (Muzyczka, 1992); BCG and poliovirus. Particularly preferred virus are replication deficient recombinant adenovirus (eg. He et al, 1998).. Engineered virus may be administered locally or systemically to achieve delivery of the gene or nucleic acid sequence of interest into a target cell.

44 Gene transfer methods as described above may be used in the provision of transgenic animals for studying in vivo the effect of for instance, modifying the binding site of an integrin for a MAP kinase interaction. A transgenic animal is one with cells that contain heterologous nucleic acid as a result of the deliberate introduction of the nucleic acid. The nucleic acid may be introduced indirectly by viral transfer as indicated above or directly by microinjection into a pronucleus of a fertilised egg prior to transfer of the egg to a surrogate mother for development to term. Alternatively, a gene or nucleic acid sequence of interest may be introduced into an embryonal stem (ES) cell in culture and the transformed cell injected into a recipient blastocyst which is then transferred to a surrogate mother for development to term. Techniques for generation of transgenic animals are for instance described in US Patent No. 4,873,191; Van der Putten, 1985; Thompson et al, 1989 and Lo, 1983.

A transgenic animal homozygous for a transferred gene for instance may be obtained by the mating of animals heterozygous for the gene as will be appreciated.

Both animal cells expressing a heterologous gene or nucleic acid sequence and transgenic animals in which a particular gene has been mutagenised by homologous recombination may be provided. A transgenic animal may for instance be a mouse, rat, hamster or pig. Typically, the transgenic animal will be a mouse.

Agents for modulating the functional activity of a cell arising from the interaction of a MAP kinase like ERK2 or JNK-1 with a integrin include antagonists and inhibitors capable of associating with the integrin to thereby inhibit the MAP kinase and integrin interaction. Antagonists and inhibitors include those agents that act by binding the integrin adjacent to the binding domain and stearically hindering the interaction of the MAP kinase with the integrin, as well as allostearic inhibitors that distort the binding domain upon associating with the integrin.

The binding domain of an integrin may be identified and characterised using protocols and techniques described herein. Specifically, a binding domain may be localised by assessing the capacity of respective overlapping peptide fragments corresponding to different regions of the cytoplasmic domain of an integrin subunit to associate with a MAP kinase. The specific amino acid sequence which constitute the binding domain for the MAP kinase may then be determined utilising progressively smaller peptide fragments of the region of the cytoplasmic domain of the integrin subunit observed to interact with the MAP kinase. In particular, test peptides are readily synthesised to a desired length involving deletion of an amino acid or amino acids from either or both ends of the amino acid sequence corresponding to that region each time, and tested for their ability to associate with the MAP kinase. This process is repeated until the minimum length peptide capable of associating with the MAP kinase substantially without compromising the optimum observed level of association is identified. The specific amino acids that play an active role in the interaction with the MAP kinase is achieved with the use of further synthesised test peptides in which one or more amino acids of the sequence are deleted or substituted with a different amino acid or amino acids to determine the effect on the ability of the peptide of associate with the MAP kinase. Typically, substitution mutagenesis will involve substitution of selected ones of the amino acid sequence with alanine or other relatively neutrally charged amino acid. By deletion is meant deletion of one or more of the amino acids between the Nterminal and C-terminal amino acid residues of the identified amino acid sequence.

46 Nucleotide and amino acid sequence data for the P6 integrin subunit for instance is found in Sheppard et al, 1990. The amino acid sequence for p6 is also set out in SEQ ID NO: 1. Reference to such published data allows the ready design of peptide fragments of an integrin subunit cytopasmic domain for use in the identification and localisation of the binding domain for a MAP kinase, and the indentification of the corresponding nucleic acid sequence encoding such peptide fragments as well as the amino acid sequence of the binding domain.

Localisation and characterisation of the binding domain for a MAP kinase enables the design of agents for use in down regulating the functional activity of the integrin and more particularly, the binding interaction of the MAP kinase with the integrin. This will typically involve determining the physical properties of the binding domain such as size and charge distribution, and the tertiary structure of the binding domain.

Specifically, at least the region of the integrin containing the binding domain is modelled taking into account the stereochemistry and physical properties of the binding domain such as size and charge distribution as well as its three dimensional structure as determined using x-ray chrstallography, nuclear magnetic resonance and/or commercially available computer modelling software. In a variation of this approach, the modelling will take into account the interaction of the binding domain with the MAP kinase such that any change in conformation arising from the interaction may be taken in to account in the design of an agent. Modelling flanking regions adjacent the binding domain also allows.the design of agents for associating with such flanking regions but which are nevertheless capable of inhibiting the binding domain MAP kinase interaction 47 either by stearic hinderence or by distorting the conformation of the binding domain (eg.

allostearic inhibitors).

The design of a mimetic of the binding domain will usually involve selecting or deriving a template molecule onto which chemical groups are grafted to provide required physical and chemical characteristics. The selection of template molecule and chemical groups is based on ease of synthesis, likely pharmacological acceptability, risk of or potential for degradation in vivo, stability and maintenance of biological activity upon administration. Pharmacological acceptability and the like are also taken into consideration in the design of other agent types.

In order to constrain a polypeptide or other agent in a three dimensional conformation required for binding, it may be synthesised with side chain structures or otherwise be incorporated into a molecule with a known stable structure in vivo. In particular, a polypeptide or the like may be incorporated into an amino acid sequence at least part of which folds into a P-pleated sheet or helical structure such as an a-helix (eg.

see Dedhar et al., 1997).

A polypeptide or other agent may also be cyclised to provide enhanced rigidity and thereby stability in vivo. Various methods for cyclising peptides, fusion proteins or the like are known (eg. Schiller et al., 1985). For example, a synthetic peptide incorporating two cysteine residues distanced from each other along the peptide may be cyclised by the oxidation of the thiol groups of the residues to form a disulfide bridge between them.

Cyclisation may also be achieved by the formation of a peptide bond between the Nterminal and C-terminal amino acids of a synthetic peptide or for instance through the formation of a bond between the positively charged amino group on the side chain of a 48 lysine residue and the negatively charged carboxyl group on the side chain of a glutamine acid residue. As will be understood, the position of the various amino acid residues between which such bonds are formed will determine the size of the cycle.

Variation of cycle size for optimisation of binding affinity may be achieved by synthesising peptides in which the position of amino acids for achieving cyclisation has been altered. The formation of direct chemical bonds between amino acids or the use of any suitable linker to achieve cyclisation is also well within the scope of the skilled addressee.

Further strategies for identifying possible agents include large scale screening techniques as are known in the art. For example, peptide library technology provides an efficient way of testing a vast number of potential agents. Such libraries and their use are well known. Prospective agents identified may be then further evaluated in suitable activity, competitive and other immunoassays. A method of screening for an agent or evaluating whether an agent is capable of binding to a MAP kinase or an integrin and thereby inhibiting binding of the MAP kinase to the binding domain of the integrin may for instance involve utilising the agent in an assay whereby the agent has the opportunity of binding to the MAP kinase or the integrin in the presence of the integrin or the MAP kinase as the case may be or prior to the addition of the integrin or the MAP kinase, and determining whether inhibition of binding of the MAP kinase to the integrin results. An alternate screening method may for instance involve selecting a test agent capable of binding with the integrin or MAP kinase, measuring cellular activity in the presence of the test agent, and comparing that activity with cellular activity in the absence of the test agent. Cellular activity may be assessed by cell growth as indicated by 3 H]-thymidine tf 49 0 uptake or other measurement of cellular activity. As will be understood, a difference in Sobserved functional activity in the presence of the test agent is indicative of the Smodulating effect provided by the test agent.

It will also be understood that the integrin in the context of such assays may be an 5 integrin subunit or polypeptide or fragment incorporating the binding domain of the 0 integrin for the MAP kinase, or a homolog, analog, variant or derivative of suci a 0 molecule to which the MAP kinase is capable of binding. In addition, determination of whether an agent is capable of binding to the binding domain of an integrin may be readily achieved by using a polypeptide or fragment as described herein consisting of the binding domain of the integrin or core amino acid sequence of the binding domain that directly participates in the binding interaction with the MAP kinase or analogs or the like of such molecules.

It is not necessary that an agent be proteinaceous in character and indeed, mimetics may be prepared which may not be a polypeptide at all but which nevertheless possess the capability of binding with the integrin.

Polypeptides including fusion proteins and fragments of an integrin subunit comprising the binding domain for a MAP kinase or incorporating sufficient core amino acid sequence of the binding domain for binding by the MAP kinase are encompassed by the present invention. Typically, a polypeptide of the invention will have a length of about 150 amino acids or less, more preferably about 100 or 50 amino acids or less and generally, less than about 40 amino acids. Preferably, the length will be from between about 5 to about 30 amino acids, and more preferably from between about 5 amino acids and about 25 amino acids. Preferably, a polypeptide will comprise or incorporate the amino acid sequence RSKAKWQTGTNPLYR, more usually the amino acid sequence RSKAKNPLYR, or one or both of sequences RSKAK and NPLYR.

Polypeptides and fusion proteins or the like may be synthesised or produced using conventional recombinant techniques. Nucleic acid encoding a fusion protein may for instance be provided via the joining together of separate DNA fragments encoding peptides or polypeptides having desired three dimensional conformations and/or other characteristics by employing blunt-ended termini and oligonucleotide linkers, digestion to provide staggered termini as appropriate, and ligation of cohesive ends prior to insertion of the resultant chimeric sequence into a suitable expression vector.

Alternatively, PCR amplification of DNA fragments can be utilised employing primers which give rise to amplicons with complementary termini which can be subsequently ligated together (eg. see Current Protocols in Molecular Biology. John Wiley Sons, 1992).

Nucleic acid sequences encoding for the polypeptides, mutagenised integrin subunits and the like as described herein are also encompassed by the present invention as are the respective complementary antisense nucleic acid sequences.

Sense oligonucleotides encoding for the binding site of an integrin subunit or a partial amino acid sequence of a binding domain, and the complementary antisense oligonucleotides are particularly suitable for use as primers in polymerase chain reaction (PCR) amplification methods or as probes for detection of the presence the respective target nucleic acid sequences with which they hybridise such as in Southern blotting protocols, or in affinity chromatography purification of the target nucleic acid sequence.

Probes may be labelled with for instance commonly used isotopes such as P2, 51 fluorophores, chemiluminescent agents and enzymes (see eg. Essential Molecular Biology. A Practical Approach Vol. II, Oxford University Press, 1993; Current Protocols in Molecular Biology, Ausubel FM., John Wiley Sons Inc., 1998). The choice of a label will vary depending on the degree of sensitivity required, ease of conjugation with the probe, safety and other factors.

Oligonucleotides for use as probes or primers will usually have a length of less than about 60 nucleotides, usually less than about 50 or 40 nucleotides preferably, between about 14 and about 30 nucleotides, and more preferably, between about 14 and about 25 nucleotides. While it is desirable that a primer or probe has 100% complementarity with its target sequence, oligonucleotides may be designed with less complementarity but which nevertheless hybridise with the target sequence. Typically, a primer or probe will have a complementarity of about 70% or greater, more preferably about 80% or greater and most preferably about 90% or 95%, or greater. A probe will generally be designed for being capable of hybridising with its target nucleic acid sequence under moderate or high stringency wash conditions. Moderate stringency wash conditions are for example those that employ 0.2 x SSC (0.015M NaC1/0.0015M sodium citrate) SDS (sodium dodecylsulfate) wash buffer at 42*C. High stringency wash conditions employ for instance, 0.1 x SSC wash buffer at 68 0 C. Generally, the content of purine relative to the content of pyrimidine nucleotides in the region of target nucleic acid of interest will be taken into account in the design of such primers and probes as will be their length in accordance with well accepted principles known in the art.

In addition, the present invention provides vectors incorporating nucleic acid sequences of the invention. The term "vector" is to be taken to mean a nucleic acid FROM DAMS PLUCK (TUE)SEP 30 2008 10:53/ST.10:51/No.7500000852 P 7 00 52, 0 0 Typical cloning vectors incorporate an origin of replication (ori) for permitting efficient replication of the vector, a reporter or marker gene for enabling selection of host

C)

Scells transformed with the vector, and restriction enzyme cleavage sites for facilitating the insertion and subsequent excision of the nucleic acid sequence of interest.

Preferably, the cloning vector has a polylinker sequence incorporating an array of restriction sites. The marker gene may be drug-resistance gene (eg. Amp' for ampicillin 0 Sresistance), a gene encoding an enzyme such as chloramphenicol acetyltransferase O (CAT), P-lactamase, adenosine deaminase (ADA), aminoglycoside phosphotransferase (APH), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase

(HPH),

thymidine kinase or for instance P-galactosidase encoded by the E. coli lacZ gene (LacZ'). Yeast reporter genes include imidazole glycerolphosphate dehydratase (HIS3), isomerase (TRP and P-isopropylmalate dehydrogenase (LEU2). As will be appreciated, expression vectors of the invention may also incorporate such marker genes.

Cloning vectors include cloning vectors for mammalian, yeast and insect cells.

Particular vectors that may find application include pBR322 based vectors and pUC vectors such as pUC118 and pUC119. Suitable expression and cloning vectors are for instance described in Molecular Cloning. A Laboratory Manual,, Sambrook et al., 2nd Ed. Cold Spring Harbour Laboratory., 1989.

Host cells suitable for being transformed by vectors of the invention include bacteria such as E. coli, Bacillus such as B. subtilis, Streptomyces and Pseudomonas bacterial strains, yeast such as Sacchromyces and Pichia, insect cells, avian cells and mammalian cells such as Chinese Hamster Ovary cells (CHO), COS, HeLa, HaRas, W138, SW480, and NIH3T3 cells. Host cells may be cultured in a suitable culture COMS ID No: ARCS-207933 Received by IP Australia: Time 11:01 Date 2008-09-30 53 may be utilised examples of which include pVL based vectors such as pVL 1392, and pVL941, and pAcUW based vectors such as pAcUW1. Viral expression vectors are particularly preferred.

Typical cloning vectors incorporate an origin of replication (ori) for permitting efficient replication of the vector, a reporter or marker gene for enabling selection of host cells transformed with the vector, and restriction enzyme cleavage sites for facilitating the insertion and subsequent excision of the nucleic acid sequence of interest.

Preferably, the cloning vector has a polylinker sequence incorporating an array of restriction sites. The marker gene may be drug-resistance gene (eg. Ampr for ampicillin resistance), a gene encoding an enzyme such as chloramphenicol acetyltransferase (CAT), P-lactamase, adenosine deaminase (ADA), aminoglycoside phosphotransferase (APH), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase

(HPH),

thymidine kinase or for instance P-galactosidase encoded by the E. coli lacZ gene (LacZ'). Yeast reporter genes include imidazole glycerolphosphate dehydratase (HIS3), N-(5'-phosphoribosyl)-anthranilate isomerase (TRP 1) and P-isopropylmalate dehydrogenase (LEU2). As will be appreciated, expression vectors of the invention may also incorporate such marker genes.

Cloning vectors include cloning vectors for mammalian, yeast and insect cells.

Particular vectors that may find application include pBR322 based vectors and pUC vectors such as pUC 118 and pUC 119. Suitable expression and cloning vectors are for instance described in Molecular Cloning. A Laboratory Manual., Sambrook et al., 2nd Ed. Cold Spring Harbour Laboratory., 1989.

54 Host cells suitable for being transformed by vectors of the invention include bacteria such as E. coli,.Bacillus such as B. subtilis, Streptomyces and Pseudomonas bacterial strains, yeast such as Sacchromyces and Pichia, insect cells, avian cells and mammalian cells such as Chinese Hamster Ovary cells (CHO), COS, HeLa, HaRas, WI38, SW480, and NIH3T3 cells. Host cells may be cultured in a suitable culture medium and under conditions for facilitating expression of nucleic acid sequences of the invention or replication of cloning vectors, prior to purification from the host cells, and/or supernatants as the case may be using standard purification techniques.

Rather than utilising viral mediated transfection of cells, nucleic acid sequences and other molecules of the invention may also be delivered to a cell in vitro or in vivo by liposome mediated transfection. The liposomes may carry targeting molecules for maximising delivery of the agent or agents contained therein to specific cell types of interest. Such targeting molecules may be for instance antibodies, ligands or cell surface receptors for facilitating fusion of liposomes to the specific cells of interest. Agents may also be intracellularly delivered in vitro using conventional cold or heat shock techniques or for instance, calcium phosphate coprecipitation or electroporation protocols. Yet another strategy is to design the agent to have the inherent ability to pass across the lipid bilayer of a cell.

A particularly preferred way of achieving intracellular delivery of an agent is to use "carrier peptides" which have the ability to deliver macro-molecules across cell membranes in an energy-independent manner (Prociantz, 1996). Indeed, such peptides provide the possibility of both testing potential agents in cell culture without drastically altering cell membrane integrity and of delivering agents in vivo. Carrier peptides that are known in the art include penetratins and variants thereof (Derossi et al, 1994, 1996), human immunodeficiency virus Tat derived peptide (Prociantz, 1996), and transportan derived peptide (Pooga et al. 1998). Indeed, carrier peptides have been successfully used to facilitate intemalisation of mimetics of Src homology 2 binding sites, and peptides which inhibit protein kinase C mediated axon development and CD44 (hyaluronate receptor) dependent migration (Theodore et al, 1995; Williams et al, 1997; Peck Isacke, 1998; Derossi et al, 1998).

Specific targetting to P6-expressing cancer cells may also be achieved by coupling humanised anti-P6 antibody to carrier molecules such as penetratin coupled to an agent capable of inhibiting binding of a MAP. kinase with an integrin expressed by the cell or down regulation of the expression of the integrin. Coupling may for instance be by a peptide bond or disulfide bridge. Given that 36 expression enhances effective proteolysis at the cell surface by matrix metalloproteinase-9 (Agrez et al, 1999), such targetting approaches may include engineering an MMP-9 cleavage site between the antibody and the carrier peptide penetratin to facilitate internalisation of the pentratinagent complex. Another approach may employ coupling the penetratin-agent complex to p6 integrin receptor-targetted peptides, targetted for binding to the extracellular 36 domain by virtue of their DLXXL sequence. For example, a ligand recognition motif for aV36 integrin, RTDLDSLRTYTL (Kraft et al, 1999) may be used in conjunction with or without an engineered MMP-9 cleavage site to release the penetratin-agent complex at the cell surface. Further protocol for targetting nucleic acids to cells by targetting integrins is described in Bachmann et al, 1998.

56 The toxicity profile of an agent of the invention may be tested on normal and malignant cells by evaluation of cell morphology, trypan-blue exclusion, assessment of apoptosis and cell proliferation studies (eg cell counts, 3 H-thymidine uptake and MTT assay).

Agents of the invention may be co-administered with one or more other compounds or drugs. For example, an agent or agents may be administered in combination with antisense therapy or chemotherapeutic drugs. Alternatively, an agent may be administered in conjunction with antisense therapy and/or chemotherapeutic drugs. By "co-administered" is meant simultaneous administration in the same formulation or in two different formulations by the same or different routes, or sequential administration by the same or different routes. By "sequential" administration is meant a time difference of between the administration of the agents, drugs and other therapies which can be administered in any order. The time difference may range from very short times up to hours or for instance days or weeks.

The agent or agents will typically be formulated into pharmaceutical composition incorporating pharmaceutically acceptable carriers, diluents and/or excipients for administration to the intended subject.

Pharmaceutical forms include sterile aqueous solutions suitable for injection, (where the agent or agents is water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. Such injectable compositions will be fluid to the extent that the syringability exists and typically, will be stable to allow for storage after manufacture. The carrier may be a solvent or dispersion medium containing one or more of ethanol, polyol (eg glycerol, propylene glycol, liquid polyethylene glycol and the like), vegetable oils, and suitable mixtures thereof Fluidity may be maintained by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants.

Sterile injectable solutions will typically be prepared by incorporating the active agents in the desired amount in the appropriate solvent with various other components enumerated above, prior to sterilising the solution by filtration. Generally, dispersions will be prepared by incorporating the sterile active agents into a sterile vehicle which contains the dispersion medium and other components. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying techniques which yield a powder of the active agent plus any additional desired ingredient from previously sterile filtered solutions thereof.

For oral administration, the active agents may be formulated into any orally acceptable carrier deemed suitable. In particular, the active ingredient may be formulated with an inert diluent, an assimilable edible carrier or it may be enclosed in a hard or soft shell gelatin capsule. Alternatively, it may be incorporated directly into food. Moreover, an active agent may be incorporated with excipients and used in the form of ingestable tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and the like.

Such compositions will generally contain at least about 1% by weight of the active agent or agents. The percentage may of course be varied and may conveniently be between about 5 to about 80% w/w of the composition or preparation. As will be appreciated, the amount of active agent or agents in such compositions will be such that a suitable effective dosage will be delivered to the subject taking into account the 58 proposed mode of administration. Preferred oral compositions according to the invention will contain between about 0.1 ig and 2000mg of each active agent, respectively.

Active agents may also be formulated into topically acceptable carriers conventionally used for forming creams, lotions, ointments and the like for internal or external application.

Typically, a composition of the invention will incorporate one or more preservatives such as parabens, chlorobutanol, phenol, sorbic acid, and thimersal. In many cases, a composition may furthermore include isotonic agents such as sugars or sodium chloride. In addition, prolonged absorption of the composition may be brought about by the use in the compositions of agents for delaying absorption such as aluminium monosterate and gelatin.

Tablets, troches, pills, capsules and the like may also contain one or more of the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the'like; a lubricant such as magnesium sterate; a sweetening agent such as sucrose, lactose or saccharin or a flavouring agent such as peppermint, oil of wintergreen, orange or cherry flavouring. When the dosage unit form is a capsule, it may contain in addition to one or more of the above ingredients a liquid carrier. Various other ingredients may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills or capsules may be coated with shellac, sugars or both. In addition, an active agent may be incorporated into any suitable sustained-release preparation or formulation.

59 Pharmaceutically acceptable carriers, diluents and/or excipients include any Suitable conventionally known solvents, dispersion media and isotonic preparations or solutions. Use of such ingredients and media for pharmnac eutically active substances is well known. Except insofar as any conventional media or agent is incompatible with the active agent, use thereof in therapeutic and Prophylactic compositions is contemplated.

Supplementr active ingredients can also be incorporated into the compositions if desired.

It is Particularly preferred to formulate parenteral compositions in dosage unit form for ease of admnistration and uniformity of dosage. Dosage unit form as used herein is to be taken to mean physically discrete units suited as unitary dosages for the subject to be treated, each unit containing a predetermined quantity of active agent calculated to produce the desired therapeutic or prophylactic effect in association with the relevant carrier, diluent and/or excipient.

A unit dosage formed will generally contain each active agent in amounts ranging from about 0.5 p~g to about 20 0 0 mg/ml of carrier respectively.

A Pharmaceutical composition may also comprise vectors capable of transfecting target cells where the vector carries a nucleic acid molecule for modulating functional activity or expression of an integrin or MAP kinase. The vector may for instance, be packaged into a suitable virus for delivery of the vector into target cells as described above.

The dosage of an active agent will depend on a number of factors including whether the agent is to be administered for prophylactic or therapeutic use, the condition for which the agent is intended to be administered, the severity of the condition, the age of the subject, and related factors including weight and general health of the subject as may be determined by the physician or attendant in accordance with accepted principles.

Indeed, a low dosage may initially be given which is subsequently increased at each administration following evaluation of the subjects response. Similarly, frequency of administration may be determined in the same way that is, by continuously monitoring the subjects response between each dosage and if necessary, increasing the frequency of administration or alternatively, reducing the frequency of administration.

The route of administration of a pharmaceutical composition will again depend on the nature of the condition for which the composition is to be administered. Suitable routes of administration include but are not limited to respiritoraly, intratracheally, nasopharyngeally, intraveneously, intraperitonealy, subcutaneously, intracranialy, intradermially, intramuscularly, intraoccularly, intrathecally, intranasally, by infusion, orally, rectally, via IV group patch, topically and by implant. With respect to intravenous routes, particularly suitable routes are via injection into blood vessels which supply a tumour or particular organs to be treated. Agents may also be delivered into cavities such for example the pleural or peritoneal cavity, or be injected directly into tumour tissue.

The production of antibodies and monoclonal antibodies is well established in the art (eg. see Antibodies, A Laboratory Manual. Harlow Lane Eds. Cold Spring Harbour Press, 1988). For polyclonal antibodies, a mammal such as a sheep or rat for instance is immunised with a polypeptide of the invention and antisera subsequently isolated prior to purification of the antibodies therefrom by standard affinity chromatography techniques such as Sepharose-Protein A chromatography. Desirably, the mammal is 61 periodically challenged with the relevant antigen to establish and/or maintain high antibody titre. To produce monoclonal antibodies, B lymphocytes can be isolated from the immunised mammal and fused with immortalising cells (eg. myeloma cells) by standard somatic cell fusion techniques (eg. utilising polyethylene glycol) to produce hybridoma cells (Kohler and Milstein, 1975; see also Handbook of Experimental Immunology, Weir et al Eds. Blackwell Scientific Publications. 4th Ed. 1986). The resulting hybridoma cells may then be screened for production of antibodies specific for the peptide by an enzyme linked immunosorbant assay (ELISA) or other immunoassay.

Conventionally used methods for preparing monoclonal antibodies include those involving the use of Epstein-Barr virus (Cole et al. Monoclonal Antibodies and Cancer Therapy, Allen R. Liss Inc. pp. 77-96, 1985). The term "antibody" or "antibodies" as used herein is to be taken to include within its scope entire intact antibodies as well as binding fragments thereof such as Fab and (Fab') 2 fragments which may be obtained by papain or pepsin proteolytic cleavage, respectively.

An antibody of the invention may be labelled for enabling detection of antibody binding in immunoassays including competitive inhibition assays. A "label" may be any molecule which by its nature is capable of providing or causing the production of an analytically identifiable signal which allows the detection of an antibody and antigen complex. Such detection may be qualitative or quantitative. An antibody can for instance be labelled with radioisotopes including 32p, I 2 I or an enzyme, a fluorescent label, chemiluminescent molecule or for instance an affinity label such as biotin, avidin, streptavidin and the like.

62 An enzyme can be conjugated with an antibody by means of coupling agents such as gluteraldehyde, carbodiimides, or for instance periodate although a wide variety of conjugation techniques exist. Commonly used enzymes include horseradish peroxidase, glucose oxidase, P-galactosidase and alkaline phosphatase amongst others. Detection utilising enzymes is achieved with use of a suitable substrate for the selected enzyme.

The substrate is generally chosen for the production upon hydrolysis of a detectable colour change. However, fluorogenic substrates may also be used which yield a fluorescence product rather than a chromogen. Suitable fluorescent labels are those capable of being conjugated to an antibody substantially without altering the binding capacity of the antibody and include fluorescein, phycoerythrin (PE) and rhodamine which emit light at a characteristic wavelength in the colour range following illumination with light at a different wavelength. Methods for labelling of antibodies can be found in Current Protocols in Molecular Biology. Ausubel FM., John Wiley Sons Inc.

Immunoassays in which antibodies of the invention may be utilised include radioimmunoassays (RIA) and ELISA see Handbook of Experimental Immunology, Weir et al., Vol. 1-4, Blackwell Scientific Publications 4th Edition, 1986). Such assays include those in which a target antigen is detected by direct binding with a labelled antibody, and those in which the target antigen is bound by a first antibody, typically immobilised on a solid substrate a microtitre tissue culture plate formed from a suitable plastics material such as polystyrene or the like) and a labelled second antibody specific for the first antibody used to form an antigen-first antibody-second antibody complex that is detected by a signal emitted by the label. Sandwich techniques in which the antigen is immobilised by an antibody for presentation to a labelled second antibody 63 specific for the antigen are also well known. An antibody can be bound to a solid substrate covalently utilising commonly used amide or ester linkers, or by adsorption.

Optimal concentrations of antibodies, temperatures, incubation times and other assay conditions can be determined by the skilled addressee with reference to conventional assay methodology and the application of routine experimentation.

Antibodies and other molecules of the invention including polypeptides and oligonucleotides as described herein when bound to a solid support can be used in affinity chromatography for the purification of a binding partner for which they are specific. In particular, a polypeptide either alone or as a fusion protein comprising the binding domain for a MAP kinase for instance can be utilised in the purification, isolation or concentration of the MAP kinase. It may also be used for assaying levels of MMP kinase in cell extracts. Similarly, an antibody that specifically binds to the binding domain of an integrin has use in the purification of the integrin or fragments thereof incorporating the binding domain from a relatively crude preparation or mixture.

Suitable solid supports include agarose, sepharose and other commercially available supports such as beads formed from latex, polystyrene, polypropylene, dextran, glass or synthetic resins, typically packed in an affinity column through which the relevant sample containing the binding partner is passed at a pH and conditions low salt concentration) under which, the binding partner becomes bound by the antibody, polypeptide or other such molecule. The column is then washed utilising a suitable buffer whereby the binding partner is retained bound on the column, prior to being eluted therefrom utilising a suitable elution buffer with a higher salt concentration and at an altered pH, typically pH 2.5 or pH 11) that facilitates the release of the binding 64 partner from the affinity column, and collected. Protocols for affinity chromatography are described in Current Protocols in Molecular Biology-Ausubel FM., John Wiley Sons Inc. Buffers and conditions utilised for the purification, isolation or concentration of a binding partner will vary depending on the affinity the antibody, polypeptide or the like for the binding partner.

A kit for use in assays as described herein may include one or more of an antibody, polypeptide, vector; nucleic acid or other molecule of the invention. The kit may also comprise one or more other reagents such as washing solutions, dilution buffers and the like together with instructions for use. The antibody or other molecule of the invention may or may not be labelled, bound to a solid support or be coupled with another molecule. Particularly preferred kits are those provided for use in affinity chromotography or RIA, ELISA or other type ofimmunoassay.

The present invention will be described herein after with reference to a number of examples.

Example 1 SAnti-integrin antibodies and antibodies against the pi subunit have been shown to inhibit proliferation.of retinal pigment epithelial cells (Hergott et al, 1993). In endothelial cells, inhibition of cell-matrix interactions by anti-integrin antibodies specifically against a(x2 1 converts the cells from a proliferative to a differentiated phenotype (Gamble et al, 1993). In another study, synthetic peptides containing the integrin recognition sequence arginine-glycine-aspartate (RGD) have been shown to inhibit tumour cell invasion in vitro and tumour metastases from melanoma in an animal model (Humphries et ;al, 1986; Gehlsen et al, 1988).

In colon cancer, the contribution made by the a5pl receptor to regulation of growth appears to be ligand (fibronectin)-dependent. For example, induced expression of c5pl in human colon cancer cells constitutively lacking this integrin has been shown to result in decreased tumour cell proliferation in vitro (Varner et al, 1995).

Interestingly, when the appropriate ligand was present, cell proliferation was restored, indicating that the unoccupied receptor mediated a negative growth signal in these cells (Varner et al, 1995). Moreover, induction of oa5pl expression was associated with a marked reduction oftumourigenicity in immune-deficient mice. Failure to ligate all of the tumour cell a5pl molecules with sufficient murine fibronectin most likely accounts for the in vivo tumour suppression in these studies (Varner et al, 1995). In the present study the effect of down-regulating avp6 expression on colon cancer growth was examined.

1.1 Methods 1.1.1 Generation of sense and antisense p6 constructs in pEF.PGK.puro vector.

For p6 antisense constructs, p6 cDNA was excised from the vector pcDNAlneo 136 (Weinacker et al, 1994) using the restriction enzymes SnaB1 and Xbal. This produced a overhang (Xbal) and a 3' blunt end (SnaB1). The 5' overhang was blunted with Klenow (Promega) prior to ligation. pEF.PGK.puro vector (a gift from D. Huang, the Walter Eliza Hall Institute, Melbourne Australia) was cut with EcoRV which produced blunt ends. The pEF.puro vector was de-phosphorylated using calf intestinal alkaline phosphatase (CIAP, Promega) and the p6 cDNA ligated overnight into pEF.puro using T4 DNA ligase (Promega) at a ratio of vector:insert of 1:5. After ligation all the reaction mix was used for transformation into competent JM109 cells and the cells plated onto LB plates containing ampicillin. After overnight incubation at 37°C, colonies were selected, the plasmid DNA extracted by microprepping, and the DNA cut with BstEl 1 to confirm the antisense orientation of P6. Digestion with BstEl 1 produced the expected two fragments, one of 6.0 basepairs and the other 3.5 basepairs.

To provide p6 sense constructs, p6 cDNA was also excised from the pcDNAlneo b6 vector using the restriction enzymes Snabi and Xbal as described above. The pEF.puro vector was then cut with Xbal and EcoRV and the insert was ligated in sense direction into the pEF.PGK.puro vector without having to blunt the insert. The ligation reaction was prepared at a ratio of vector:insert of 1:1. Digestion with BstEl 1 pioduced two expected fragments, one of 7.5 basepairs and the other 2.0 basepairs.

1.1.2 Transfection of WiDr and HT29 colon cancer cells The human colon cancer cell lines, WiDr and HT29 were obtained from the American Type Culture Collection (ATCC), Rockville, Maryland, USA and maintained as monolayers in standard medium comprising Dulbecco's Modified Eagle's medium (DMEM; 4.5gm/litre of glucose) with 10% heat-inactivated foetal bovine serum (FBS) supplemented with HEPES and penicillin/streptomycin. WiDr and HT29 cell lines constitutively express avp6. Stable transfectants of WiDr and HT29 cells expressing 36 in sense and antisense direction were generated using lipofectamine and puromycin as the selection antibiotic. One stable antisense p6 transfectant from HT29 cells and three stable clones from WiDr cells were successfully established for use in all experiments.

Mock transfectants using vector alone were also generated as controls. Initial killing 67 curves performed using a range of concentrations of puromycin established that WiDr and HT29 cells could be stably transfected with puromycin concentrations of 1.Op.g and 2.51g/ml, respectively.

1.1.3 Assessment of 36 expression in the transfected cell lines 36 expression was assessed by means of FACScan analyses of parent cell lines and clones generated therefrom by means of limiting-dilution experiments. Stability of the transfectants was confirmed regularly by repeated FACScan analyses, and surface biotinylation and immunoprecipitation as described below.

1.1.4 FACScan analyses Monolayer cultures of cell lines were harvested with trypsin/EDTA and then blocked with goat serum at 4 0 C for 10 min. Cells were washed once with PBS, incubated with primary antibody against integrin subunits for 20 min at 4°C and then washed twice with PBS. Cells were then stained with secondary antibody conjugated with phycoerythrin for 20 min at 4°C, washed twice with PBS and resuspended in PBS prior to FACScan analysis (Becton Dickinson, Rutherford, New Jersey, USA).

1.1.5 Integrin immunoprecipitation Cells were harvested with trypsin/EDTA, the trypsin neutralised with standard culture medium, and the cell pellets washed once with cold PBS. Cell pellets were then exposed to biotin-CNHS-ester (Sigma) in biotinylation buffer (10mM sodium borate, 150mM sodium chloride, pH 8.8) for 30 mins at 4 0 C with continuous slow mixing. Cell pellets were then centrifuged at 4"C, washed twice with cold PBS and exposed to lysis buffer (containing 100mM Tris, 150mM NaC1, ImM CaCl 2 1% Triton, 0.1% SDS and 0.1% NP-40 at pH 7.4 and containing ImM phenylmethylsulfonyl fluoride (PMSF)) for 68 min at 4°C. Lysates were then clarified by ultracentrifugation (10,000g) for 30 min and the protein content measured using the BCA protein assay reagent kit. Lysates containing equal protein amounts were pre-cleared with rabbit anti-mouse (RAM) immunoglobulin coupled to Sepharose-4B beads for 12 hrs. Immunoprecipitations were carried out indirectly using RAM-Sepharose 4B and analysed by 7.5% SDS-PAGE under non-reducing conditions.

1.1.6 Reverse transcriptase-PCR (RT-PCR) mRNA levels for p6 expression were evaluated by reverse transcriptasepolymerase chain reaction (RT-PCR). Total RNA was extracted from cell cultures using the commercial TriPure isolation reagent based on the method of Chomozynski and Sacchi (1987). 0.4-2pg of RNA was used to prepared cDNA by reverse transcription.

Briefly, a reaction mixture in a final volume of 40p1 containing 8pl of 5 x RT reaction buffer (250mM Tris, 15mM MgCl2, 375mM KCL, pH 8 1 p of 2.5mM of each dNTP, 41l of 100mM DTT, 40U of an Rnase inhibitor, Rnasin (Promega, Madison, Wisconsin, USA), 0.5.g of random hexamers (Promega) and 200U of Moloney murine leukaemia virus (M-MLV) reverse transcriptase (Promega) were mixed and incubated at 38*C for a minimum of 90min. The reaction was stopped by heating at 95 0 C for 5 min and cDNA stored at 4°C until PCR. 2-51l. of this cDNA was combined with 51l of 10 x PCR buffer (100mM Tris, 500mM KCI, 15mM MgCI 2 pH8.3) 8 pl of 1.25mM dNTP each and 1.25ptl of 20pM of both forward and reverse primers. The forward primer sequence was 5'AGGATAGTTCTGTTTCCTGC3' and the reverse primer sequence 5'ATCATAGGAATATTTGGAGG3'. The reaction was initiated by 2.5U of Taq polymerase in a final volume of 50u1l. After an initial 5 min incubation at 94°C, cycles of amplification were performed under the following conditions: 94C 1 min, 54 0 C 1 min and 72 0 C for 1 min. The reaction was stopped by incubating at 72 0 C for min. To verify that equal amounts of RT product from cells were subjected to PCR amplification, the same amounts of cDNA were amplified for the "house-keeping" gene GAPDH using specific primers. The same reaction conditions were used except that the annealing temperature was changed to 48*C and PCR amplification performed for cycles.

1.2 Results 1.2.1 avp6 expression in HT29 and WiDr transfected cell lines Transfection of the colon cancer cell lines HT29 and WiDr with the 36 gene construct in an antisense orientation resulted in a marked reduction of P6 expression at the transcript level and on the cell surface as shown in Figs 1 to 4. Transfection of cells with p6 in the sense orientation did not enhance p6 surface expression. However, a consequence of down-regulation of the p6 subunit in antisense transfectants was a marked increase in surface expression of the 35 subunit. The changes in surface expression of 16 and 15 subunits noted on FACScan analyses of antisense 36 transfectants was confirmed by surface-labelling cells with biotin and immunoprecipitating integrin subunits with either anti-p6 mAb (R6G9) or anti-P5 mAb (P1F6).

1.2.2 Effect of Suppression of av16 expression on cell binding to fibronectin The major substrate for avp6 is fibronectin and to investigate the effect that reduction in p6 surface expression in antisense 16 transfectants might have on cellmatrix adhesion, WiDr and HT29 antisense p6 transfectants were seeded on fibronectin in adhesion assays. Since both cell lines can adhere to fibronectin through members of the 31 integrin subfamily (either a3pl or a5pl) av6-mediated adhesion to fibronectin was assessed in the absence/presence of blocking anti-pl antibody.

The cell-adhesion assays were performed in wells of non-tissue culture-treated polystyrene 9 6 -well flat bottom microtitre plates (Nunc, Roskilde, Denmark). Culture wells were coated with fibronectin, washed with phosphate-buffered saline (PBS) and then blocked with 0.5% bovine serum albumin (Sigma) in PBS for lhr at 37 0

C.

Harvested cells were seeded at a density of 10 5 cells/well for HT29 and 1.5 x 10 s cells/well for WiDr cells in 2 0 0pl of standard DMEM which lacked FBS but contained 0.5% bovine serum albumin. To block adhesion, cells were incubated with anti-pl blocking antibodies for 15 min at 4°C before plating. The plates were centrifuged (top side up) at 10 x g for 5 min, then incubated for 1 hr at 37 0 C in humidified 5% carbon dioxide. Non-adherent cells were removed by centrifugation top side down at 48 x g for min. The attached cells were fixed and stained with 0.5% crystal violet (in methanol and 1% formaldehyde) and the wells washed with phosphate-buffered saline.

The relative number of cells in each well was evaluated by measuring the absorbance at 595nm in a Microplate Reader (Bio-Rad).

With reduction in cell surface expression of P6, 1 3 -independent adhesion of cells to fibronectin was reduced compared with mock transfectants (containing vector alone) which express surface P6 at similar levels to wild-type cells. The further addition of blocking anti-av antibody completely prevented binding to fibronectin.

1.2.3 Effect of suppression of oxav6 expression on tumour cell proliferation and tumour growth in vivo To investigate the effect of diminished av16 surface expression on cell proliferation in vitro, WiDr and HT29 antisense p6 transfectants were seeded as monolayers in 96-well microtitre culture plates (5,000 viable cells per well) in standard culture medium containing the puromycin selection antibiotic. Cells were pulsed with I Ci 3 H)-thymidine (Amersham) per well for the last 24 hours of each experiment before automated harvesting and measurement of radioactivity. WiDr wild-type, mock and antisense p6 transfectants, and HT29 wild-type, mock, sense p6 and antisense P6 transfectants were harvested second daily during a six-day culture period.

A marked increase in thymidine incorporation was observed for WiDR and HT29 cells expressing normal levels of avp6 compared with antisense p6 transfectants (see Figs. 5 and 6).

1.2.4 Effect of Suppression of avp6 expression on Tumour Formation The ability of HT29 antisense p6 transfactants to form tumours in immunedeficient mice was assessed.

BALB/C female athymic mice (8 weeks of age purchased from the Animal Resource Centre, Perth,'Wester Australia) were maintained under pathogen-free conditions and fed standard mouse chow and water ad lib. The mice were divided into groups often each and all mice within each group inoculated with a single cell line.

Cells used were WiDr mock (transfected with vector alone) and antisense p6 transfected clones (clones 1 3) and HT29 mock, sense and antisense 16 cell lines. Mice received subcutaneous flank injections of 106 viable tumour cells suspended in 0.2ml of standard DMEM culture medium. Animal weights and tumour sizes (breadth and length as measured with calipers) were recorded weekly. Six weeks following the last injection, visible subcutaneous tumours were excised, weighed and fixed in 4% formalin. At the time of euthanasia, all internal organs were routinely inspected for presence of metastases.

Tumour growth after six weeks following inoculation of HT29 mock and antisense P6 transfectants is shown in Fig. 7. Measurements of tumour growth for WiDr and HT29 cells are shown in Figs 8 and 9, respectively. Similar tumour growth profiles for mock and antisense 06 WiDr clones 2 and 3 each inoculated into 10 mice are not shown.

Of a total of 40 mice injected with cells expressing antisense p6 (HT29 10 mice and WiDr cells lines, 3 clones 30 mice) tumours completely disappeared in 93% of animals.

In the remaining 3 animals tumour sizes diminished to 1 mm 2 in size during the six week period compared with tumours at least 15mm 2 in size from cells expressing normal levels of p6. To confirm the presence of tumour xenografts histologically at one week following subcutaneous inoculation of mock and antisense p6 transfectants, tumour nodules were excised, fixed in formalin and stained with haematoxylin and eosin.

1.2.5 Effect of suppression of avp6 expression on gelatinase B secretion Serum-free tumour-conditioned medium was collected from each of the 3 WiDr mock and antisense p6 clones and concentrated x 44 for measurement of gelatinase B using the Biotrak MMP-9 activity assay system (Amersham Pharmacia Biotech, Uppsala Sweden). Down-regulation of aov6 expression resulted in a marked reduction in gelatinase B secretion.

73 1.3 Discussion of Results Induced expression of 36 in Chinese hamster ovary (CHO) cells has been shown to result in decreased surface expression of the 35 integrin subunit which also partners av (Weinacker et al, 1994). The concept of integrin switching depends on the availability of the promiscuous cav partner subunit. In the present study, the reverse was observed.

As a consequence of down-regulation of P6 in colon cancer cells which constitutively express av6, 15 surface expression increased, most likely secondary to increased availability of the av subunit partner.

Heterologous expression of av6 in colon cancer cells has previously been reported to enhance tumour growth in immune-deficient mice (Agrez et al, 1994).

Suppression of avp6 expression in the present study was shown to result in nearly complete disappearance of tumours in 93% of animals following subcutaneous inoculation of tumour cells. Moreover, in the remaining 7% of animals, a 95% reduction in tumour size was observed over a six week period compared with large tumours seen in all animals injected with cells in which avp6 expression had not been perturbed. Similar findings have been described with loss of the classical vitronectin receptor avp3 in melanoma. For example, in experimental animal models, the loss of avp3 expression in melanoma cells has been shown to lead to reduced in vivo proliferation which is restored upon re-expression of the receptor (Felding-Habermann et al, 1992).

Although the mechanisms involved in avp6-mediated tumour growth remain to be elucidated, the present in vitro data show that loss of 16 expression is associated with decreased proliferative capacity of the cells. Taken together with the marked reduction in gelatinase B secretion seen for colon cancer cells transfected with antisense 16, the tt 74 O findings reported in the present study suggest that intracellular signalling pathways activated via the integrin avp6 play a major role in promoting progression of this tumour type.

Example 2 The association of cavp6 expression and MAP kinase activity were evaluated using SWiDr, HT29 and SW480 cell lines.

2.1 Methods 2.1.1 SW480 colon cancer p6 transfectants Stable transfectants of SW480 colon cancer cells (ATCC) expressing gene constructs of either wild-type or mutant forms of the p6 integrin subunit or the expression plasmid only (pcDNAlneo) have been previously described (Agrez et al, 1994). The transfected SW480 cell lines were maintained in standard medium supplemented with the neomycin analogue G418. Stable transfectants of WiDr and HT29 cells expressing wild-type p6 in antisense orientation were generated as described in Example 1.1.2 and maintained in standard medium supplemented with puromycin.

2.1.1 MAP kinase assay.

Cultures of WiDr and HT29 mock and antisense p6 transfectants were established by seeding 1 x 106 cells/5ml of culture medium in 25cm 2 tissue culture flasks. Cells were incubated at 37C in humidified CO 2 for 24 hours before serum starvation in serum-free medium for the next 16 hours. Foetal calf serum was then added to a final concentration of 10% for 30 mins before MAP kinase assays were performed. Before each experiment the cells were washed twice with PBS, resuspended in extraction buffer Tris-HC1, 150mM NaCI, 2mM EDTA, 2mM DTT, ImM orthovanadate, ImM PMSF, 4tg/ml aprotonin, 2pg/ml leupeptin and lig/ml pepstatin, pH 7.4) and sonicated at a setting of 7, using a Soniprep 150 watt ultrasonic disintegrator for a total of seconds in three 30 second pulses with an interval of 30 seconds between each pulse.

Cellular debris was removed by centrifugation at 900g for 10 min at 4°C. The assay was performed on equal cell numbers using a MAP kinase assay system (Amersham Pharmacia Biotech, Uppsala Sweden). The ability of cells to transfer phosphate from [y 32 P]-ATP to a synthetic peptide that contains specifically a p42/p44 MAP kinase phosphorylation site was measured as described in the manufacturer's instructions.

2 P]-labelled peptides were spotted onto PEI -cellulose paper, unbound radioactivity was washed with 75mM phosphoric acid and bound [32 P]-labelled peptides were measured by liquid scintillation counting. Protein estimation was performed on each cell lysate used and enzyme activity calculated as described in the manufacturer's instructions.

Where MEK inhibitors, PD98059 and U0126 were used, cells were cultured as described above and the inhibitors, at a final concentration of 40uM, were added one hour before the addition of serum to the medium.

2.1.2 Western blotting To detect the MAP kinases ERKI/2, cells were lysed in lysis buffer containing 100mM Tris, 150mM NaCI, ImM CaC12, 1% Triton, 0.1% SDS and 0.5% NP-40 at pH7.4, supplemented with enzyme inhibitors (ImM PMSF, ImM sodium orthovanadate, l.g/ml pepstatin A, 2.5 g/ml aprotonin, ImM benzamidine, 1 g/ml leupeptin). Lysates were clarified by ultracentrifugation and equal protein loads electrophoresed in 8% or SDS-PAGE under non-reducing conditions. Electrophoresed proteins were transferred to nitrocellulose membranes (Biotrace NT, Gelman Sciences, Ann Arbor, 76 MI) in transfer buffer (25mM Tris, 192mM glycine, 20% methanol, 0.1% SDS) for 2 hours at a constant voltage of 40 volts in a Transfer Blot Cell (Bio-Rad). Membranes were blocked with 5% casein for 1 hr at room temperature and probed with monoclonal anti-ERK antibodies (E10 which recognises only phosphorylated ERK1/2 (New England BioLabs) and SC-1647 which recognises total ERKs, non-phosphorylated and phosphorylated (Santa Cruz Biotechnology). .In some experiments, membranes were probed with polyclonal anti-ERK antibody (New England BioLabs) which also recognises total ERKs. Membranes were then washed three times in Tris buffered saline, containing 0.1% Tween and incubated with HRP-conjugated goat anti-mouse or goat anti-rabbit antibody. Blots were visualised by the enhanced chemi-luminescence detection system according to the manufacturer's instructions (Du Pont).

2.1.3 Integrin P-subunit immunoprecipitations Tumour cells were harvested and divided into two equal aliquots based on cell counts. One aliquot was surface biotinylated and the cells lysed for integrin immunoprecipitations as described in Example 1.1.5, with the exception that three sequential rounds of immuno-precipitation were performed to deplete the lysates of integrin subunits P5 and P6. Lysis buffer was the same as that used for Western blotting.

The other aliquot, intended for subsequent ERK2 immunoblotting to examine the effect of integrin immunodepletion on ERKs was not biotinylated but lysed immediately and divided into three samples each at a protein concentration of lmg/ml. Three sequential rounds of immunoprecipitation were performed against anti-P6 monoclonal antibody, R6G9 (using isotype matched antibody, IgG2A, and anti-p5 monoclonal antibody, P1F6, in control immunoprecipitations).

The integrin-depleted lysates were electrophoresed in 8% or 10% SDS-PAGE under non-reducing conditions and transferred to nitrocellulose membranes. Membranes were blocked with casein as for Western blotting and probed with anti-ERK monoclonal antibodies E10 and SC-1647. In parallel experiments, the sequentially immunoprecipitated p6 subunit bound to rabbit anti-mouse (RAM) coupled Sepharose B4 beads was also electrophoresed in 10% SDS-PAGE under non-reducing conditions, transferred to nitrocellulose membranes, and the membranes probed with anti-ERK monoclonal antibody E10 which recognises only phosphorylated forms of ERK1/2.

2.3 Results 2.3.1 Effect of serum on MAP kinase activity The effect of MEK (MAP kinase kinase) inhibitors U0126 and PD98059 on MAP kinase activity in WiDr and HT29 wild-type cells was tested in the absence/presence of serum. Adherent cell monolayers on plastic were grown for 24 hrs in standard culture medium, then washed three times in PBS followed by 16 hrs in culture under serum-free conditions. Serum was then added for 30 min and MAP kinase activity assessed before and after addition of serum. The addition of serum markedly stimulated MAP kinase activity for both cell lines and was inhibitable by both MEK inhibitors.

2.3.2 Effect of altered 06 expression on MAP kinase activity following serum stimulation.

In these experiments, WiDr transfectants (3 mock and 3 antisense p6 clones), HT29 transfectants (mock and antisense p6) and SW480 06 transfectants (mock and sense p6) were serum-starved for 16 hrs followed by 30 mins exposure to serum.

Increased expression of av36 was associated with a marked increase in MAP kinase 78 activity upon serum stimulation compared with cells lacking av6 altogether (SW480 mock). Cells in which p6 expression had been down-regulated (antisense 36 transfectants) exhibited suppressed MAP kinase activity. Induced expression of p6 in the non-p6-expressing colon cancer cell line SW480 resulted in a three fold increase in serum-dependent MAP kinase activity.

2.3.3 azvp6 binds an extracellular signal-related kinase (ERK).

A highly surprising finding arising from phage display screening which underpins the present work is that the integrin p6 cytoplasmic domain binds a MAP kinase. Use of phage display to screen a ygt" cDNA colon cancer cell library with the p6 cytoplasmic domain as bait yielded a clone which coded for ERK2 at the 3' end with 100% nucleotide sequence identity (across 393 bases) to the published sequence of ERK2 (Boulton et al, 1991). This putative association was investigated further.

Integrin immunoprecipitations were performed on equal protein loads of tumour cell lysates and the transferred proteins blotted with antibodies recognising phosphorylated and non-phosphorylated forms ofERK1/2. The specificity of this interaction was examined in integrin immunoprecipitations against 1, 35,. 6 and av integrin subunits. As shown in Fig. 10, the anti- ERK mAb SC-1647 identified a p6specific band migrating at the position of purified ERK2 protein. The integrinassociated ERK band was identified only in immunoprecipitations of the p6 subunit and its partner av and not in immunoprecipitations of the p 1/p5 subunits or in immunoprecipitations using isotype-matched control antibodies.

79 2.3.4 Effect of altered 06 expression on total cellular ERK and p6-bound ERK.

Equal protein loads from one representative clone each from WiDr mock and antisense p6 transfectants were electrophoresed, transferred to nitrocellulose and blotted with anti-ERK mAb (SC-1647) as shown in Fig.l p6 immuno-precipitates from equal protein loads of the WiDr mock and antisense p6 clones were electrophoresed, transferred and probed with anti-ERK mAb (E10) as shown in Fig. 11(B). As indicated, suppression of p6 expression resulted in a reduction of both total cellular and phosphorylated integrin-associated ERK compared with WiDr mock transfectants.

Similarly, p6 immunoprecipitations from SW480 p6 transfected clones expressing high and low levels of p6 (confirmed by FACScan and 06 immunoprecipitations) showed parallel changes in p6-bound phosphorylated ERK (Fig. 12).

2.3.5 Effect of p6 immunodepletion on total cellular ERK and 06-bound ERK WiDr wild-type cells were surface biotinylated and the cell lysates immunodepleted of 06 in three rounds of sequential immunoprecipitation using anti-36 mAb (R6G9) resulting in a marked loss of 06 from the lysates as shown in Fig. 13(A).

p6-immunodepleted lysates were then transferred and blotted with anti-ERKl/2 antibody (SC-1647), which recognises both phosphorylated and non-phosphorylated forms of ERK1/2. As shown in Fig. 13(B), following three rounds of 16 immunodepletion, levels of ERK1/2 in p6-depleted lysates compared with non-immunodepleted lysates, were markedly reduced suggesting a significant contribution of 16-bound ERK to total cellular ERK. In contrast, immunodepletion of p5 by three successive rounds of immunoprecipitation with mAb P1F6 or isotype-matched control antibody IgG2A did not result in any reduction of cellular ERK levels compared with non-immunodepleted cell lysates.

2.3.6 Effect of ERK immunodepletion on p6-bound ERK Cell lysates from WiDr wild-type cells were immunodepleted of ERK1/2 by means of sequential immunoprecipitations using the anti-ERK mAb, SC-1647. ERKimmunodepleted cell lysates probed with either E10 or SC-1647 mAbs contained markedly less ERKs. To examine the effect of ERK-immunodepletion on p6-bound ERK, the ERK-immunodepleted cell lysates were immunoprecipitated with anti-36 mAb (R6G9) and the I6-immunoprecipitates probed with anti-ERK mAb (E10 recognising phosphorylated ERK1/2). ERK immunodepletion effectively reduced levels of p6bound ERK.

2.3.7 Effect of preventing cell attachment on avp6-bound ERK SW480 mock-and P6 expressing transfected cells were grown to 80-90% confluency. Cells were washed twice with PBS and harvested after trypsinization. Cells were divided into three equal portions (approximately 4 x 10 6 /batch). The first group was plated on a 75 cm 2 plastic flask in normal 10% serum containing medium (attached cells) while the other two groups were plated on 0.3% agarose underlay in serum free medium (non-attached cells). Cells were allowed to grow for 24 hours at 37 0 C, after which in one group of 0.3% agarose underlay, 15 ml of serum-free medium was added while in the other an equal volume of 10% serum-containing medium was added for mins. Cells were collected washed with PBS and lysed in cell lysis buffer (100 mM Tris-HCI pH-7.5, 150 mM NaCI, ImM Cacl2, 1% Triton, 0.1% SDS, 0.1% Np-40, 1 mM vanadate, 1 pg/ml pepstatin, 1 mM PMSF, 5 g/ml aprotonin and 1 g/ml of 81 leupeptin). 10 pl (10 pg of protein) was used for the analyses of cell lysates after adding equal volumes of non-reducing Laemmli buffer. For SW480-p6-transfected cells the rest of the cell lysate was used for immunoprecipitation of av36 integrin. Cell lysates and p6 immunoprecipitates were subjected to western blotting and probed with monoclonal antibody (recognising phosphorylated ERKI Cell lysates from the non-attached cells were found to require serum factors to maintain phosphorylation of total cellular ERK. In contrast, non-attached SW480 36 colon cancer cells do not require serum factors to maintain the activation (phosphorylation) state of p6-bound ERK.

2.3.8 Effect of PP2A phosphatase on acv6-bound ERK In experiments to examine the effect of protein phosphatase 2A (PP2A) on p6bound ERK, SW480 36-transfected cells were sonicated in buffer comprising Tric-HCI (pH 10mM MgCl 2 and 0.01mM EGTA together with enzyme inhibitors, and cell lysates from both serum-starved and serum-induced cells treated with 0.5 units of PP2A (Promega). at 30*C for 10 minutes. The reaction mixture was stopped by addition of equal volumes of non-reducing Laemmli sample buffer. In parallel, PP2Atreated cell lysates were immunoprecipitated with mAb R6G9 (anti-p6) followed by Western blot with anti-ERK mAb E10. MAP kinase activity assays were performed according to the manufacturer's instructions (Amersham Pharmacia Biotec) using y 32

P-

ATP.

Exposure of cell lysates prepared from serum-supplemented and serum-starved cells to the PP2A catalytic subunit resulted in dephosphorylation of total ERK. In contrast, dephosphorylation of p6-bound ERK was not observed in P6 82 immunoprecipitates prepared from serum-supplemented or serum-starved cells p6 Bound ERK may therefore serve to maintain adjacent growth factor receptors in an activated state and thereby alter their sensitivity to exogenous co-factors.

2.3.9 Inhibition of MAP kinase activity inhibits secretion of gelatinase B.

SW480 p6 transfectants were cultured under serum-free conditions for 48 hours in the absence/presence of the MEK inhibitor PD98059 (40pM) or DMSO (vehicle control). Tumour-conditioned medium was assayed for gelatinase B by analysis of equal protein loads in a gelatin zymogram.

Inhibition of MAP kinase activity by the MEK inhibitor reduced gelatinase B secretion compared with controls.

2.3.10 P6-ERK2 association in HaCaT and HaRas Cell Lines p6 immunoprecipitates were prepared from human kerotinocyte cell lines (HaCaT and HaRas were obtained from Prof. N. Fusenig, The German Cancer Research Institute, Heidelberg, Germany) using mAb R6G9 (anti-p6) and the immunoprecipitates probed with mab E10 (against phosphorylated ERK Fig. 14 shows that ERK2 associates with p6 in both of HaCat and HaRas cells.

2.4 Discussion of Results The MAP kinase pathway has been shown to be important in experimental tumour metastases (Mansour et al, 1994) and recent data implicate MAP kinases in tumour growth and invasiveness of colon cancer cells (Sebolt-Leopold et al, 1999). In the present study, up-/down-regulation of 06 expression in various colon cancer cell lines was shown to enhance/suppress respectively, MAP kinase activity. The presence of serum induced a three-fold increase in MAP kinase activity above that observed for 83 serum-starved p6-expressing cells. In contrast, only a one-fold increase in serumdependent MAP kinase activity was observed for cells in which p6 had been downregulated consequent upon transfection with antisense p6. Moreover, induced expression of 16 in the non-P6-expressing colon cancer cell line SW480, was associated with a three-fold increase in serum-dependent MAP kinase activity. Serum contains a mixture of growth factors raising the possibility that one role for av06 in colon cancer cells is to lower the threshold for activation of MAP kinase signalling pathways at times when the supply of serum-containing growth factors is limited.

The ERK band co-immunoprecipitated with p6 migrated either at or 1 2 kD higher than the mobility of purified phosphorylated ERK2 depending on the acrylamide concentration used in SDS-PAGE showing that the kinase does indeed, associate with the 06 subunit. Slight differences in mobility of the p6-associated ERK band compared with the pure ERK protein could arise if p6-bound ERK is hyper-phosphorylated and/or exists in an altered conformation consequent upon its association with p6. ERK bound to p6 may also be an alternatively spliced variant of ERK2 causing it to migrate differently. Finally, the purified ERK2 protein is derived from mouse which differs slightly from human ERK2 by being two amino acid residues shorter and also containing a single amino acid substitution.

In the present study, increased/decreased 36 expression in colon cancer cell lines was associated with increased/decreased p6-bound ERK, respectively. In addition, immunodepletion experiments suggest that p6-bound ERK makes a substantial contribution to total phosphorylated ERK within the cell and overall MAP kinase activity (see Figs. 15(A) and 84 The observation that p6-mediated colon cancer growth in vitro is inhibitable by a matrix metalloproteinase inhibitor (Agrez et al, 1999) suggests that the increased gelatinase B secretion by 36-expressing colon cancer cells contributes to tumour progression. Taken together with the finding that inhibition of MAP kinase activity by the MEK inhibitor PD98059 diminished gelatinase B secretion in 16-expressing cells, it seems that activation of MAP kinase signalling plays a role, at least in part, in p6mediated induction of gelatinase B secretion.

Example 3 3.1 Identification of the binding domain on the P6 subunit cytoplasmic tail domain for ERK2 Peptide fragments corresponding to regions of the cytoplasmic tail domain of the 36 subunit were screened in an enzyme-linked immunosorbent assay (ELISA) for binding with ERK2. The 52 amino acid long p6 cytoplasmic tail is shown in Fig. 16 as are the amino acid sequences for the cytoplasmic domains of the p31 to p3 subunits. In particular, four synthetic peptides designated fragment 1 to fragment 4 were prepared and biotinylated at the N-terminal end of each, respectively (Auspep Pty Ltd, Melbourne Australia).

The region of the p6 tail to which each corresponds is indicated in Fig. 16 and set out below.

Fragment 1: HDRKEVAKFEAERSKAKWQTGT Fragment 2: RSKAKWQTGTNPLYRGSTST Fragment 3: NPLYRGSTSTFKNVTYKHRE Fragment 4: FKNVTYKHREKQKVDLSTDS The fragments overlap by 10 amino acids and are each 20 amino acids long with the exception of the fragment 1 with a length of 22 amino acids. Fragment 4 was synthesised with a terminal serine rather than a cysteine as found in wild-type 16 to avoid formation of a disulfide bridge between peptides.

Overlapping biotinylated fragments 1 to 4 were coated onto streptavidin coated polystyrene plates (Pierce, Rockford IL USA, Cat No. 15125) and the ELISA performed substantially according to manufacturers instructions. Briefly, wells are washed with 3 x 2 0 0pl of wash buffer (TBS, 0.1% BSA, 0.05% Tween 20 or SuperBlock T blocking buffer in TBS, Pierce, Prod. No. 37535) prior to addition of biotinylated peptides (100pl) and incubation for 1 hr at room temperature to allow for capture of the peptides on the plate. Following another washing step, peptides were overlayed with GST-ERK, ERK (or JNK-1) alone at a volume of 1001.l per well, and the plates incubated for a further 1 hr at room temperature before removal of any unbound ERK by further washing.

Binding of ERK to the peptides is detected using 100pl anti-ERK1/2 mAb SC1647 (Santa Cruz) as the primary antibody at a dilution of 1.:700 (isotype matched antibody IgG2b is used as a control). This is followed by another washing step and addition of 100l rabbit anti-mouse antibody (Biorad) conjugated to alkaline phosphatase at a concentration of 1:1.000 for 30 minutes again at room temperature. A 100 l aliquot of detection reagent (alkaline phosphatase detection kit-Biorad) is then introduced into each well after a final washing step and allowed to react for 15-30 minutes at room temperature in the dark before absorbance is measured at 405nm. All dilutions of 86 peptides, MAP kinase and antibodies were performed using the wash buffer. GST-ERK is a fusion protein consisting of ERK coupled to glutathione-S-transferase and purified from host cells transfected with pGEX vector.

As shown in Fig. 17, significant binding of non-phosphorylated GST.ERK2 (0.25pg/100il) to peptide fragment 2 was observed (1 p.g/100.1) while only negligible or low level binding for the other fragments was found.

Significant binding of non-phosphorylated ERK2 to both fragment 2 and 36 cytoplasmic tail peptide compared to fragments 1, 3 and 4 over a range of concentrations of ERK2 was also observed (see Fig.18). Similar results were observed using a range of concentrations of the peptide fragments as shown in Fig. 19.

To further localise the binding domain on the cytoplasmic tail of the p6 subunit, progressively shorter peptides from the region of the 36 cytoplasmic tail corresponding to peptide fragment 2 were synthesised, biotinylated and the capacity to associate or otherwise bind to ERK2 assessed as described above. The binding of GST.ERK2 to a mer test peptide (seq. 4) having the amino acid sequence RSKAKWQTGTNPLYR and a mer test peptide having the sequence RSKAKWQTGT is shown in Fig. 20 compared to fragment 2 over a range of concentrations of the peptides. As can be seen, no reduction in binding to the seq. 4 peptide compared to fragment 2 was found. Binding of ERK2 to the seq. 3 peptide was substantially less than that observed for seq. 4.

A number of 10 mer biotinylated peptides corresponding to regions of fragment 2 or fragment 3 were then tested. The amino acid sequence for each peptide is as follows and their location in the 36 cytoplasmic domain is indicated in Fig. 21.

10(1): NPLYRGSTST 10(2): WQTGTNPLYR 10(3): KFEAERSKAK The results are set out in Fig. 22 and show that GST.ERK binding to the 10 mer peptides is substantially reduced compared to binding to the seq. 4 peptide suggesting that opposite end regions ofseq.' 4 participate in the binding of ERK2.

Comparable binding of ERK2 to seq. 4 was found using a further 10 mer peptide identified as 10(4) in which amino acid sequence WQTGT of seq. 4 is ormtted indicating that WQTGT is a linker sequence that does not participate directly in the binding of ERK to seq. 4. Negligible binding of ERK2 to the 5 mer peptide RSKAK was observed as shown in Fig. 23. ERK2 cleaved from GST-ERK2 by thrombin was used in this assay. Results (not shown) indicate that greater than a 3 fold increase in assay sensitivity can be achieved using thrombin cleaved ERK2 rather than GST-ERK2.

Example 4 4.1 MAP kinase JNK-1 binds to the cytoplasmic tail domain of P6 In view of the observation that ERK2 associates with the cytoplasmic tail of the P6 subunit, the MAP kinase JNK-1 was tested to evaluate whether it also could associate with the cytoplasmic tail of p6.

Briefly, 0.05-1.5 m/100pl of JNK-1 (Santa Cruz) was aliquoted into wells of a 96 well culture place containing increasing concentrations of the p6 cytoplasmic domain tail peptide used in Example 3. For comparison purposes, non-phosphorylated

GST-

ERK2 (0.05-1.5pm/100pl) was aliquoted into wells containing p6 cytoplasmic tail peptide or peptide fragments 1 or 2 resectively. Binding of JNK-I was detected using 88 mouse anti-JNK-1 mAb SC 474-G (Santa Cruz) and HRP-conjugated goat anti-mouse antibody. Absorbance was read at 405nm and the results are shown in Fig. 24.

Significant binding of JNK-1 to the p6 cytoplasmic tail peptide was found.

Example 5.1 Evaluation of ability of ERK2 to bind to p6 A746-764 deletion mutant.

To examine the role of the amino acid sequence RSKAKWQTGTNPLYR in the p6 cytoplasmic domain in situ, a p6 deletion construct lacking the coding sequence for AERSKAKWQTGTNPLYRG was transfected into colon cancer cell line SW480 which does not constitutively express the aVp6 integrin using the calcium phosphate method previously described for transfections into this cell line (Agrez et al, 1994). The location of the p6 A746-764 deletion is indicated in Fig. 25. Construction of the p6 A746-764 deletion mutant in the vector pcDNAlneo and failure of the expressed receptor to localise to focal adhesions in Chinese hamster ovary cells has been reported (Cone et al, 1994). Facscan analysis revealed comparable levels of surface expression of mutant 36 to that seen for the full length wild-type receptor (see Fig. 26).

Equal protein loads of cell lysates prepared from SW480 cells were immunoprecipitated with either anti-p6 monoclonal antibody (mAb R6G9) or matched isotype control antibody. Surface biotinylation prior to immunoprecipitation confirmed equal surface expression of mutant and wild-type p6 (see Fig. 27 Aliquots of the immunoprecipitates were electrophoresed and transferred to nitrocellulose for Western blotting using monoclonal antibody E10 which recognises ERKI/2. As seen in Fig.

27(B), loss of the RSKAKWQTGTNPLYR sequence in the p6 cytoplasmic domain 89 reduced levels of P6-bound ERK by greater than approximately 75% of that observed for the wild type receptor.

Example 6 6.1 Growth inhibition study.

HT29 and SW480 P6-expressing colon cancer cell lines were seeded into wells of 96-well microtitre plates (Nunclon) in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% foetal bovine serum, glutamine, Hepes, and antibiotics. Seeding cell densities were 3 x 103 cells per triplicate well for each condition tested and after 24 hours incubation of cell cultures in 5% CO 2 100% humidity at 37 0 C, the culture medium was exchanged for serum-free DMEM medium supplemented with insulin, transferrin, selenous acid, hydrocortisone, non-essential amino acids, glutamine, Hepes and antibiotics containing either peptide RSKAKWQTGTNPLYR alone or penetratinpeptide complex at.a concentration of 10m for HT29 cells or 30tm for SW480 P6expressing cells. Cell cultures were incubated for a further 24 hours following which cultures were photographed (Kodak Techpan Film at 100 ASA setting) and the experiments terminated by addition of the cell proliferation reagent WST-1 (Boehringer Mannheim) to monitor effects of the peptide on cell growth. The cell proliferation reagent WST-1 is designed to be used for the non-radioactive, spectrophotometric quantification of cell growth and viability in proliferation and chemosensitivity assays.

The colourmetric assay is based on the cleavage of the tetrazolium salt WST-1 by mitochondrial dehydrogenase in viable cells.

In particular, at the termination of experiments, 30il of WST-1 was added to 270l culture medium volume in each microtitre well and the colour change quantitated in an ELISA plate reader by measuring absorbance of the formazan product at 450mn (using a reference wavelength of more than 600nm). The mean absorbance readings from triplicate wells standard error of the means) after subtraction of background control wells (culture medium without cells)was determined. Only the carrier penetratin-peptide complex was effective in inhibiting cell proliferation in contrast to either peptide or penetratin alone as shown in Figs. 28 and 29 indicating that the penetratin-peptide complex was internalised by both the HT29 and SW480 cells resulting in the observed suppression of colon cancer growth. Photographs of the SW480 cells treated with penetratin alone or the penetratin-peptide complex are shown in Fig. 30 to Example 7 SW480 mock and SW480 36 transfectants were cultured in DMEM medium supplemented with 1% foetal bovine serum in the presence of 20 pM seq. 4 coupled to penetratin. Percentage inhibition was assessed by the WST-1 colorimetric dehydrogenase assay described in Example 6. The percentage inhibition of growth observed for the -P6 and +P6 expressing cells was 17% and 50%, respectively as indicated in Fig 31A. The -36 and +0 cultured cells are shown in Fig 3 B.

Example 8 The proliferation of SW480 cells expressing a 16 A746-764 deletion mutant was compared with non-p6 expressing SW480 cells and SW480 cells expressing full length wild-type P6. Cells were cultured for 10 days within a 3-dimensional collagen type I matrix. Collagen gels were prepared as bilayers (upper layer cell-containing and lower layer minus cells) in 24 well culture plates as previously described (Agrez, 1989; Agrez, 1994) except for the use of 5% foetal bovine serum as the supplement for DMEM.

Colonies were photographed in the gel Fig.32(A) at 10 days (bar represents 200k) and following dissolution of the collagen with collagenase Fig.32(B) prior to visual colony counting of all colonies exceeding 200p. in diameter within each well Fig.32(C). As can be seen, significant proliferation of the SW480 cells expressing the full length wild-type p6 was observed compared to the non-p6 expressing cells and cells expressing the p6 A746-764 deletion mutant.

Example 9 Growth inhibition of SW480 cells expressing full length wild-type P6 exposed to seq.4 coupled to penetratin or RSKAKWQTGTNPLYR peptide coupled to penetratin 20, 30 pM in DMEM minus foetal bovine serum) but which peptide contained alanine substitutions at the four positions indicated was assessed. As shown in Fig.33(A) and Fig 33(B), progressive inhibition of proliferation in a dose-response manner was observed for the seq.4 penetratin complex compared with the alanine substituted peptide-penetratin complex which was without effect at all doses tested.

Example Binding of ERK2 to the seq.4 peptide (RSKAKWQTGTNPLYR) was compared with peptides corresponding to regions of the cytoplasmic domain of integrin subunits P1, p2, 03 and p5. The amino acid sequences for those peptides is shown below: pl KFEKEKMNAKWDTGENPIYK p2 KEKLKSQWNNDNPLFK P3 RARAKWDTANNPLYK

RSRARYEMASNPLYR

As shown in Fig.34, significant binding of ERK2 to the seq. 4 peptide was observed.

Binding of ERK2 to the p5 and p3 peptides was also found. The results have been corrected for non-specific binding and indicate a hierarchy of binding of ERK2 to integrin subunits.

Although the present invention has been described hereinbefore with reference to a number of preferred embodiments, the skilled addressee will understand that numerous variations and modifications are possible without departing from the scope of the invention.

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Claims (17)

1. 2. A polypeptide according to claim 1 comprising the modified amino acid sequence and wherein all of the amino acids in the amino acid linker sequence of the binding domain of the 3 integrin subunit are deleted in the polypeptide compared to the binding domain.
2. 3. A polypeptide according to claim I or 2 wherein the polypeptide is from 10 to amino acids in length.
3. 4. A polypeptide according to claim 3 wherein the polypeptide is 10 amino acids or amino acids in length, A polypeptide according to any one of claims 1 to 3 wherein the modified amino acid sequence has greater than 50% overall amino acid sequence homology with the binding domain. COMS ID No: ARCS-207933 Received by IP Australia: Time 11:01 Date 2008-09-30 FROM RDAMS PLUCK (TUE)SEP 30 2008 10:53/ST.10:51/No,7500000852 P 8 00 S106.
4. 6. A polypeptide according to claim 5 wherein the modified amino acid sequence C) C has at least 80% overall amino acid sequence homology with the binding domain. 0
5. 7. A polypeptide according to any one of claims I to 4, wherein the B integrin subunit is B6. 1- O 5 8, An isolated polypeptide comprising a modified binding domain of a cytoplasmic Sregion of a 8 integrin subunit for a MAP kinase, the polypeptide having a modified Ci amino acid sequence compared to the binding domain of the B integrin subunit, wherein the binding domain incorporates an amino acid linker sequence that links opposite end regions of the binding domain together and which is non-essential to the binding of the MAP kinase, and wherein one or more amino acids in the amino acid linker sequence of the binding domain are deleted or differ in the polypeptide compared to the binding domain and the polypeptide is up to 20 amino acids in length and has sufficient amino acid sequence homology with both the end regions of the binding domain to bind to the MAP kinase. 9, A polypeptide according to claim 8 wherein all of the amino acids in the amino acid linker sequence of the binding domain are deleted in the modified amino acid sequence compared to the binding domain. A polypeptide according to claim 8 or 9 wherein the end regions of the binding domain are unchanged in the modified amino acid sequence compared to the binding domain. COMS ID No: ARCS-207933 Received by IP Australia: Time 11:01 Date 2008-09-30 FROM ADAMS PLUCK (TUE)SEP 30 2008 10:53/ST.10:51/No.750000952 P 00 S107.
6. 11. A polypeptide according to any one of claims 8 to 10 wherein the polypeptide is C) Sfrom 10 to 15 amino acids in length. 0
7. 12. A polypeptide according to any one of claims 8 to 11 wherein the modified amino acid sequence has greater than 50% overall amino acid sequence homology with the binding domain. S13. A polypeptide according to claim 12 wherein the modified amino acid sequence 0 Ci has at least 80% overall amino acid sequence homology with the binding domain.
8. 14. A polypeptide according to any one of claims 8 to 13 wherein the MAP kinase is ERK2 and wherein the 1 integrin subunit is selected from the group consisting of 83, and 86. A polypeptide according to claim 14 wherein the B integrin subunit is 86.
9. 16. A polypeptide according to claim 8 comprising the amino acid sequence RSKAKNPLYR.
10. 17. An isolated polypeptide consisting of an amino acid sequence selected from the group consisting of RSKAKWQTGTNPLYR, RARAKWDTANNPLYK and RSRARYEMASNPLYR.
11. 18. An isolated non-naturally occurring agent for inhibiting growth of a cancer cell, comprising: a polypeptide moiety that binds to a MAP kinase; and COMS ID No: ARCS-207933 Received by IP Australia: Time 11:01 Date 2008-09-30 FROM ADAMS PLUCK (TUE)SEP 30 2008 10:53/ST.10:51/No .7500000852 P 11 00 0 108, ~a facilitator moiety coupled to the polypeptide for facilitating passage of the C) C polypeptide moiety across the outer cell membrane of the cancer cell into the cytoplasm 0 Sof the cancer cell; wherein the polypeptide moiety is a polypeptide as defined in any one of claims 1 to 17. 0
12. 19. A pharmaceutical composition comprising a polypeptide as defined in any one of 0 Sclaims 1 to 17 together with a pharmaceutically acceptable carrier or diluent. C-q A method of screening for an agent that binds to a binding domain of a 13 integrin subunit for a MAP kinase, comprising: utilising the agent in an assay for determining whether the agent binds to the binding domain of the 3 integrin subunit; and determining if the agent binds to the binding domain on the basis of the assay.
13. 21. A method according to claim 20 wherein the B integrin subunit is selected from the group consisting of 03, B5 and 06 and wherein the MAP kinase is an ERK family member or a JNK family member.
14. 22. A method for inhibiting growth of a cancer cell expressing a B integrin subunit, comprising treating the cancer cell with an effective amount of a polypeptide moiety, the polypeptide moiety being a fragment of a cytoplasmic region of the B integrin subunit wherein the fragment comprises a binding domain of the B integrin subunit for a MAP kinase, or a modified amino acid sequence compared to the binding domain that has sufficient amino acid sequence homology with the binding domain to bind to the MAP kinase. COMS ID No: ARCS-207933 Received by IP Australia: Time 11:01 Date 2008-09-30 FROM ADAMS PLUCK FROM DAMS LUCK(TUE)SEP 30 2008 10:54/ST. 10:51/Ho, 7500000352 P 12 00 0
15. 109. 23. A method accord ing to claim 22 wherein the polypeptide moiety comprises the V) modified amino acid sequence. 0 rn 24. A method according to claim 23 wherein the binding domain incorporates an amino acid linker sequence that links opposite end regions of the binding domain together and which is non-essential to the binding of the MAP kinase, and the modified CA amino acid sequence has sufficient amino acid sequence homology with both the end o regions of the binding domain to effect the binding to the MAP kinase. A method according to claim 24 wherein one or more amino acids in the amino acid linker sequence have been deleted in the, modified amino acid sequence compared to the binding domain, 26. A method according to claim 25 wherein all of the amino acids in the amino acid linker sequence have been deleted. 27. A method according to claim 25 or 26 wherein the end regions of the binding domain are defined byrespective amino acid sequences that remain unchanged in the modified amino acid sequence compared to the binding domain. 28. A method according to any one of claims 22 to 27 wherein the modified amino acid sequence has greater than 50% overall amino acid sequence homology with the binding domain. 29. A met'hod according to claim 28 wherein the modified amino acid sequence has at least 80% overall amino acid sequence homology with the binding domain. COMS ID No: ARCS-207933 Received by IP Australia: Time 11:01 Date 2008-09-30 FROM ADAMS PLUCK (TUE)SEP 30 2008 10:54/ST.10:51/No, 7500000852 P 13 00
16. 110. A method according to claim 22 wherein the polypeptide moiety comprises the C) fragment of the B integrin subunit. O 31. A method according to any one of claims 22 to 30 wherein the polypeptide moiety has a length of greater than 5 amino acids and up to 20 amino acids. o 5 32. A method according to claim 31 wherein the polypeptide moiety has a length of o. from 10 to 15 amino acids. 0 33. A method according to any one of claims 22 to 32 wherein the polypeptide moiety is coupled to a facilitator molecule that facilitates passage of the polypeptide moiety across the cell membrane of the cancer cell into the cytoplasm of the cell. 34. A method according to any one of claims 22 to 33 wherein the MAP kinase is selected from the group consisting of members of the ERK MAP kinase family. A method according to claim 34 wherein the MAP kinase is ERK2. 36. A method according to any one of claims 22 to 35 wherein the B integrin subunit is selected from the group consisting of 33, 85 and J36. 37. A method according to claim 36 wherein the B integrin subunit is selected from and 86. 38. A method according to claim 22 or 36 wherein the polypeptide moiety comprises an amino acid sequence selected from the group consisting of RSKAKWQTGTNPLYR, RARAKWDTANNPLYK, RSRARYEMASNPLYR, and RSKAKNPLYR. COMS ID No: ARCS-207933 Received by IP Australia: Time 11:01 Date 2008-09-30 FROM ADAMS PLUCK (TUE)SEP 30 2008 10:54/ST. 10:51/No,7500000852 P 14 00 C 39. A method for prophylaxis or treatment of a cancer in a mammal, the method C) comprising: 0 r selecting an agent for inhibiting growth of cancer cells of the cancer, the cancer cells expressing a B integrin subunit; and Cl 5 administering an effective amount of the agent to the mammal, the agent Scomprising a polypeptide moiety being a fragment of a cytoplasmic region of the B 0 integrin subunit wherein the fragment comprises a binding domain of the B integrin l subunit for a MAP kinase, or a modified amino acid sequence compared to the binding domain and having sufficient amino acid sequence homology with the binding domain to bind to the MAP kinase. A method for prophylaxis or treatment of cancer in a mammal, comprising administering to the mammal an effective amount of a polypeptide moiety being a fragment of a cytoplasmic region ofa B integrin subunit expressed by cancer cells of the cancer wherein the fragment comprises a binding domain of the 3 integrin subunit for a MAP kinase, or a modified amino acid sequence compared to the binding domain, and wherein the B integrin subunit is selected from the group consisting of B5 and 86 and the MAP kinase is ERK2, and the modified amino acid sequence has sufficient amino acid sequence homology with the binding domain to bind to the MAP kinase. 41. Use of an agent comprising a polypeptide moiety in the manufacture of a medicament for inhibiting growth of cancer cells of a cancer in a mammal, the cancer cells expressing a 13 integrin subunit and the polypeptide moiety being a fragment of a cytoplasmic region of the B integrin subunit wherein the fragment comprises a binding domain for the MAP kinase, or a modified amino acid sequence compared to the COMS ID No: ARCS-207933 Received by IP Australia: Time 11:01 Date 2008-09-30 FROM ADAMS PLUCK (TUE)SEP 30 2008 10:54/ST.10:51/No.7500000852 P 00
17. 112. binding domain which has sufficient amino acid sequence homology with the binding C/3) domain to bind to the MAP kinase. 0 O1- COMS ID No: ARCS-207933 Received by IP Australia: Time 11:01 Date 2008-09-30