WO2001000677A1

WO2001000677A1 - A method of modulating integrin mediated cellular activity and agents useful for same

Info

Publication number: WO2001000677A1
Application number: PCT/AU2000/000729
Authority: WO
Inventors: Michael Valentine Agrez; Nuzhat Ahmed
Original assignee: The University Of Newcastle Research Associates Limited
Priority date: 1999-06-28
Filing date: 2000-06-28
Publication date: 2001-01-04
Also published as: DE60037970D1; EP1196449A4; US8618062B2; EP1196449A1; ATE385503T1; EP1196449B1; WO2001000677A9; DE60037970T2; US8119594B1; US20120232249A1

Abstract

There is disclosed agents capable of inhibiting the binding of a MAP kinase to a binding domain of an integrin for the MAP kinase, and methods of modulating the activity of a cell utilising the agents. The methods are particularly suitable for inhibiting the growth of cancer cells.

Description

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 20-

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 (FINPCC); the remaining cases are

attributed to so-called sporadic colorectal cancer. FAP and HNPCC contribute to

approximately 1% and 4%, 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 thought 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 cell-

cell 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 internalisation and tumourigenesis. Integrins are

transmembrane glycoproteins consisting of an alpha (α) and beta (β) 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 α and 8 β subunits and the αβ combinations are subsumed under 3 subfamilies.

Excluding the leucocyte integrin subfamily that is designated by the β2

nomenclature, the remaining integrins are arranged into two major subgroups, designated

βl and αv based on sharing common chains.

In the βl subfamily, the βl chain combines with any one of nine α chain members

(αl-9), and the α chain which associates with βl determines the matrix-binding

specificity of that receptor. For example, α2βl binds collagen and laminin, α3βl binds

collagen, laminin and fibronectin, and α5βl binds fibronectin. In the αv subfamily of

receptors, the abundant and promiscuous αv chain combines with any one of five β chains, and a distinguishing feature of αv 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 α and β 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 β 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 β4 which is larger at 210

kDa.. Similarly, they all contain 56 conserved cysteine residues, except for β4 which

has 48. These cysteines are arranged in four repeating patterns which are thought to be

linked internally by disulphide bonds. The α-subunits have a molecular mass ranging

from 150 - 200 kDa. They exhibit a lower degree of similarity than the β chains,

although all contain seven repeating amino acid sequences interspaced with non-

repeating domains.

The β 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 α-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 βl integrins have been delineated (Reszka et al,

1992). These regions contain conserved sequences that are also found in the cytoplasmic

domains of the β2, β3, β5, β6 and β7 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,

increases in intracellular Ca 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 β6 subunit was first identified in cultured epithelial cells as part of the

αvβ6 heterodimer, and the αvβ6 complex was shown to bind fibronectin in an arginine-

glycine-aspartate (RGD)-dependent manner in human pancreatic carcinoma cells

(Sheppard et al, 1990; Busk et al, 1992). The β6 subunit is composed of 788 amino acids

and shares 34 - 51% sequence homology with other β integrin subunits βl - β5. The β6

subunit also contains 9 potential glycosylation sites on the extracellular domain

(Sheppard et al, 1990). The cytoplasmic domain differs from other β subunits in that it

is composed of a 41 amino acid region that is highly conserved among integrin β

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 β6 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 βl integrins to focal contacts (Reszka et al, 1992) has been shown to eliminate recruitment

of β6 to focal contacts (Cone et al, 1994).

The integrin αvβ6 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 (e.g. focal

adhesion kinase FAK) and serine/threonine kinases (such as those in the mitogen-

activated protein kinase (MAPK) cascade) and stimulate lipid metabolism (eg phosphatidylinositol-4, 5-biphosphate (PjP₂) 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

(e.g. 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 p90^rs ) cytoskeletal proteins

(such as microtubule-associated phospholipase A₂ ), 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 (ρρ60 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 βl and αv 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 α6β4, the laminin/collagen

receptor αlβl, the fibronectin receptor α5βl and the RGD binding receptor αvβ3 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 co-

stimulation 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 matrix-

degrading 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, MEK-

independent 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 α-actinin and talin and βl and β3 integrin tails (Horwitz et al, 1986;

Otey et al, 1990; Knezevic et al, 1996; Pfaff et al, 1998). A direct association between

FAK and the βl integrin tail has been suggested based on in vitro βl peptide studies, but

this remains to be confirmed (Schaller et al, 1995). More recently, the cytoplasmic

domain of the α4 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 α

chain cytoplasmic domains (Rojiani et al, 1991) and modify α2βl integrin activation by

phorbol esters and anti-integrin antibodies (Coppolino et al, 1995). Newly identified integrin-binding molecules include the serine/threonine integrin-linked kinase, ILK,

which can associate with the β 1 , β2 and β3 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 αvβ3 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

phosphatidylinositol-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 αvβ6 has been shown to bind and

activate latent TGFβl in keratinocytes (Munger et al, 1999) which is thought to be

important in modulating the inflammatory process following epithelial injury. In

melanoma cells, αvβ3 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 integrin-

mediated 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 β-receptor is tyrosine phosphorylated upon growth factor stimulation.

Interestingly, this subset can associate with the αvβ3 integrin, and PDGF activity is

enhanced in association with increased MAP kinase activity in cells plated on the αvβ3

ligand, vitronectin. Summary Of The Invention

Broadly stated, the present invention relates to modulation of integrin expression

in neoplastic 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 has previously been found to directly associate with any integrin or for that

matter, with any transmembrane protein. The identification of this functional

relationship permits the rational design of agents for therapeutically or prophylactically

modulating cellular activity mediated by the MAP kinase and integrin interaction.

In an aspect of the present invention there is provided an agent capable of

inhibiting binding of a MAP kinase with an integrin.

Typically, the agent will be capable of binding with a binding site on the MAP

kinase that binds to a binding domain on the integrin for the MAP kinase. Alternatively,

the agent may be capable of binding to the binding domain on the integrin for the MAP

kinase or other site on the integrin such that inhibition of binding of the MAP kinase to

the integrin is thereby caused.

The agent may be provided either isolated or for instance, coupled to another

molecule for facilitating transport of the agent into a cell.

In another aspect of the present invention there is provided an isolated polypeptide

capable of binding with a binding site on a MAP kinase which binding site binds with a

binding domain on an integrin for the MAP kinase, 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 β6(770t) or β6(777t) deletion mutant. Preferably, the polypeptide will comprise the binding domain of the integrin or

sufficient core amino acid sequence of the binding domain to enable 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 aspect of the present invention there is provided a

fragment of an integrin subunit wherein the fragment is capable of binding with a MAP

kinase, or a homolog, analog, variant or derivative of the fragment, with the proviso that

the integrin subunit is other than a β6(770t) or β6(777t) deletion mutant.

Preferably, the polypeptide or fragment will have a length of about 150 amino

acids or less, more preferably about 100 or 50 amino acids or less and more usually

about 40 amino acids or less. Typically, the length will be between about 5 to about 30

amino acids.

The fragment 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 aspect of the present invention 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 β6 Δ746-764 deletion mutant. In still another aspect of the present invention there is provided a fusion protein

incorporating a polypeptide of the invention or a homolog, analog or variant of the

polypeptide.

In a further aspect of the present invention there is provided a fusion protein

incorporating a fragment of the invention or a homolog, analog or variant of the

fragment.

Typically, the polypeptide or fragment will be coupled to a carrier polypeptide for

facilitating entry of the fusion protein into a cell. Preferably, the carrier polypeptide will

be penetatin.

In another aspect of the present invention there is provided an isolated nucleic acid

sequence encoding a polypeptide of the invention or a homolog, analog, or variant of the

polypeptide.

In yet another aspect of the invention there is provided an isolated nucleic acid

sequence encoding a fragment of the invention or a homolog, analog, or variant of the

fragment.

In another aspect of the invention 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 β6 Δ746-764 deletion

mutant.

In a still further aspect of the present invention there is provided an isolated

nucleic acid sequence encoding a fusion protein of the invention. There are also provided antisense nucleic acid sequences complimentary to the

sense nucleic sequences of the invention. 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 of the invention may be labelled with a suitable reporter molecule

for enabling detection of hybridisation of the primer or probe to a target nucleic acid

sequence.

In yet another aspect of the present invention there is provided a vector

incorporating a nucleic acid sequence of the invention. Typically, the vector will be an

expression vector and the nucleic acid sequence will be capable of being transcribed.

In a further aspect of the present invention there is provided a host cell transformed

with a vector of the invention.

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 aspect of the present invention there is provided a transgenic animal

with cells containing heterologous nucleic acid of the invention.

In yet another aspect of the present invention there is provided an antibody

generated with the use of a polypeptide or fragment of the invention.

In a still further aspect of the invention there is provided an antibody capable of

binding to a binding domain on 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 aspect of the present invention there is provided a method of

isolating a MAP kinase from a sample utilising a molecule immobilised on a solid

support and which is capable of binding to a binding site on the MAP kinase which

binding site binds with a binding domain of an integrin for the MAP kinase, comprising:

(a) contacting the molecule immobilised on the solid support with the sample

under conditions suitable for binding of the MAP kinase to the molecule;

(b) eluting the MAP kinase from the solid support;

(c) 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 is capable of binding, or for instance an

analog, homolog, variant or derivative of the polypeptide or fragment.

In yet another aspect of the present invention there is provided a MAP kinase

isolated by a method of the invention.

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 capable of inhibiting binding of a MAP kinase to a binding domain of an

integrin for the MAP kinase, comprising: (a) testing a number of agents for ability to bind to either the MAP kinase or the integrin; and

(b) determining if any said agent is capable of inhibiting binding of the MAP

kinase to the binding domain of the integrin on the basis of the testing.

In another aspect of the invention there is provided a method of screening for an

agent capable of inhibiting binding of a MAP kinase to a binding domain on an integrin

for the MAP kinase, comprising:

(a) testing a number of agents for ability to bind to either the MAP kinase or the

integrin;

(b) 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

(c) utilising the selected said agent or agents in an assay for indicating whether

the or any of the selected said agents is capable of inhibiting the binding of the MAP

kinase to the binding domain of the integrin.

In another aspect of the invention there is provided a method of evaluating whether

an agent is capable of inhibiting binding of a MAP kinase to a binding domain of an

integrin for the MAP kinase, comprising:

(a) selecting the agent;

(b) utilising the agent in an assay for indicating whether the agent is capable of

inhibiting the binding of the MAP kinase to the binding domain of the integrin; and

(c) determining if the agent is capable of inhibiting 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 aspect of the present invention there is provided a method of

screening for an agent capable of binding to a binding domain of an integrin for a MAP

kinase, comprising:

(a) testing a number of agents for ability to bind to the binding domain of the

integrin for the MAP kinase; and

(b) determining if any said agent is capable of binding to the binding domain of

the integrin on the basis of the testing.

In yet another aspect of the invention there is provided a method of screening for

an agent capable of binding to a binding domain of an integrin for a MAP kinase,

comprising:

(a) testing a number of agents for ability to bind to the integrin;

(b) selecting an agent or agents identified as being able to bind to the integrin on

the basis of the testing; and (c) utilising the selected said agent or agents in an assay for indicating whether

the or any of the selected said agents is capable of binding to the binding domain on the

integrin for the MAP kinase.

In another aspect of the present invention there is provided a a method of

evaluating whether an agent is capable of binding to a binding domain of an integrin for

a MAP kinase, comprising:

(a) testing the agent for ability to bind to the binding domain of the integrin for the

MAP kinase; and

(b) determining if the agent is capable of binding to the binding domain on the

basis of the testing.

Preferably, a polypeptide or fragment of the invention 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 of the invention will usually be provided in the form of a pharmaceutical

composition. Accordingly, in another aspect of the present invention there is provided a

pharmaceutical composition comprising an agent of the invention capable of inhibiting

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 agent will be coupled to

a carrier molecule for facilitating entry of the agent into a cell.

In a further aspect of the invention there is provided a pharmaceutical composition

comprising a nucleic acid sequence of the invention 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 aspect of the present invention 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 aspect of the present invention 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 is capable of inhibiting 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 be capable of binding with the binding site on the

MAP kinase which binding site binds to the binding domain on the integrin. In still another aspect of the invention there is provided a method of modulating

activity of a cell, comprising causing the expression of an integrin to which a MAP

kinase is able to bind to be down-regulated.

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 aspect of the invention 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 aspect of the present invention there is provided a method of modulating

activity of a cell, comprising:

contacting the cell with an effective amount of an agent capable of inhibiting

binding of a MAP kinase to a binding domain on an integrin expressed by the cell.

In a further aspect of the invention there is provided a method of modulating

activity of a cell, comprising: contacting the cell with an effective amount of an agent capable of binding to

a MAP kinase to thereby inhibit binding of the MAP kinase to a binding domain on the

integrin for the MAP kinase.

Preferably, the agent will be capable of binding 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 aspect of the present invention 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 aspect of the invention 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

capable of causing the expression of an integrin to which a MAP kinase is able to bind to

be down regulated.

In still another aspect of the present invention there is provided a method 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 is capable of

inhibiting binding of a MAP kinase with a binding domain of an integrin for the MAP

kinase to thereby cause down-regulation of MAP kinase integrin binding, or a homolog,

analog or variant of the polypeptide.

In another aspect of 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 of the

invention capable of inhibiting binding of a MAP kinase to binding domain on an

integrin for the MAP kinase and the method comprises administering an effective

amount of the agent to the mammal.

In another aspect there is provided use of a nucleic acid of the invention 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 aspect of the invention there is provided the use of an agent

capable of inhibiting 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 within the scope of the invention. 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. Usually, the integrin will

be a member of the αv subfamily. Preferably, the integrin is or incorporates an integrin subunit selected from the group consisting of β3, β5 and β6. Most preferably, the

integrin comprises β6.

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.

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 50%

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

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

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

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

is the uncontrolled proliferation of a cell.

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 β5

and β6 in HT29 colon cancer cells stably transfected with either vector alone (mock

transfectants) or antisense β6 construct;

Figure 2: Amplification of β6 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 β6 (141 basepairs) and GAPDH (216 basepairs) bands are indicated (WT, wild-type; S, sense β6;

A/S, antisense β6; mock, vector alone);

Figure 3: Non-transfected HT29 cells (wild) and cells transfected with vector alone

(mock), sense β6 and antisense β6 analysed by FACScan for expression of the β6

subunit. White and black histograms represent cells stained in the absence and presence,

respectively, of mAb E7P6 (anti-β6);

Figure 4: WiDr cells transfected with vector alone (mock) or antisense β6 and

analysed by FACScan for expression of the β6 subunit. White and black histograms

represent cells stained in the absence and presence, respectively, of mAb E7P6 (anti-

β6).;

Figure 5 : Tumor cell proliferation in vitro assessed by ( H)-thymidine uptake for

WiDr wild-type cells and transfectants (mock and antisense β6) cultured on plastic for

the times indicated.

Figure 6: Tumor cell proliferation in vitro assessed by ( H)-thymidine uptake for

HT29 wild-type cells and transfectants (mock, sense β6 and antisense β6) cultured on

plastic for the times indicated.

Figure 7: Tumour growth after 6 weeks following subcutaneous inoculation of 10

viable cells of HT29 mock (vector alone) and antisense β6 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 β6 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 β6 transfectants;

Figure 10: β6-associated ERK identified by immunoprecipitafions of integrin

subunits from SW480 β6 transfectants. (Top) Cell lysates were immunoprecipitated

with mAbs QE2E5 (anti-βl), L230 (anti-αv), P1F6 (anti-β5), R6G9 (anti-β6), or

matched isotype control Abs (IgGl and IgG2A), and (bottom) blotted with anti-ERK 1/2

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 1(B): (A) Western blotting: equal protein loads of cell lysates

from one representative clone each from WiDr mock and antisense β6 blotted with anti-

ERK mAb (SC-1647) against total ERK. Purified non-phosphorylated ERK2 is shown in

the left hand lane. (B) β6 immunoprecipitates (mAb R6G9) from equal protein loads of

the cell lysates in (A) probed with anti-ERK mAb (ElO). Purified phosphorylated ERK2

is shown in the left hand lane;

Figure 12: β6-bound ERK shown for the high and low SW480 β6-expressing

clones by probing β6 immunoprecipitates with anti-ERK mAb (ElO) against

phosphorylated forms of ERK1/2. Purified, phosphorylated ERK2 is shown in the left

hand lane;

Figures 13(A) and 13(B): (A) Surface biotinylation of WiDr wild-type cells and β6

immunodepletion of the cell lysates by three successive rounds of β6 and β5

immunoprecipitations using mAb R6G9 and P1F6, respectively or control mAb

(IgG2A). The β6 and partner αv bands are arrowed. (B) β6-immunodepleted WiDr cell lysates after 3 successive rounds of β6-immunoprecipitations probed with anti-ERK

mAb SC-1647 recognising both phosphorylated and non-phosphorylated forms of

ERK 1/2 and compared with non-β6 immunodepleted lysates and control lysates sequentially immunoprecipitated 3 times with either isotype matched control mAb

(IgG2 A) or mAb P 1 F6 (anti-β5);

Figure 14: Non-transformed (HaCaT) and Ras-transformed (HaRas) human

keratinocytes: β6 immunoprecipitation and ERK western blots probed with monoclonal

antibody ElO (against Phosphorylated ERK 1/2) and monoclonal antibody SC1647

(against total ERK 1/2), respectively.

Figure 15(A) and 15(B): (A) Depletion of β6 with sequential rounds of ERK

immunodepletion of cell lysates using anti-ERK mAb (SC-1647). (B) MAP kinase

activity in cell lysates before and after 5 rounds of sequential β6 immunodepletion from

SW480 β6 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 β6 immunodepletion was highly signigicant

(P<0.005, students T test).

Figure 16: Shows the amino acid sequence of the cytoplasmic domain of the β6

subunit as well as the amino acid sequences for the βl to β3 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 β6 subunit; Figure 18: Graph showing binding of ERK2 (GST.ERK2) to the β6 cytoplasmic

domain and the peptide fragments indicated in Fig. 16 over a range of concentrations of

ERK2;

Figure 19: Graph showing binding of ERK2 (GST.ERK2) to the β6 cytoplasmic

domain and fragments thereof;

Figure 20: Graph showing binding of ERK2 (GST.ERK2) to a 15 mer fragment of

the β6 cytoplasmic domain and which has the amino acid sequence

RSKAKWQTGTNPLYR; and

Figure 21 : Shows regions of the cytoplasmic domain of the β6 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 β6 subunit.

Figure 24: Graph showing binding of JNK-1 to the β6 cytoplasmic domain.

Figure 25: location of β6 Δ746-764, β6(770t) and β6(777t) deletions in the

cytoplasmic domain of the β6 subunit.

Figure 26: SW480 cells transfected with wild-type full length coding sequence for

β6 or β6 Δ746-764 deletion mutant analysed by FACScan for expression of wild-type or

mutant β6. White and black histograms represent cells stained in the absence and

presence of the integrin subunit, respectively. Figures 27(A) and 27(B): (A) Western blotting: equal protein loads of cell lyates

from SW480 cells expressing wild-type β6 or β6 Δ746-764 deletion mutant. (B) β6

immunoprecipitates (mAb R6G9) from equal protein loads of cell lysates (A) probed with anti-ERK mAb (ElO).

Figure 28: Proliferation of HT29 colon cancer cells cultured for 48 hours and

treated with penetratin, the fragment of β6 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 β6 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 (A) to 29 (C): (A) SW480 cells cultured with control additive for 24

hours; (B) SW480 cells cultured with penetratin for 24 hours; (C) SW480 cells cultured

with RSKAKWQTGTNPLYR bound to penetratin for 24 hours.

Figures 31 (A) and 31 (B): (A) S W480 mock (-β6) and S W480 β6 transfectants

(+β6) cultured in presence of the RSKAKWQTGTNPLYR peptide coupled to

penetratin. (B) Photomicrographs of cells shown in (A) cultured in the presence/absence

of the peptide penetratin complex.

Figures 32(A) to 32(C): (A) SW480 cells cultured in a 3-dimensional collagen type

I matrix photographed in the gel at the end of 10 days. (B) The SW480 cells following

dissolution of the collagen with collagenase. (C) Graph showing numbers of colonies of

the SW480 cells having a diameter exceeding 200μ. Figures 33(A) and 33(B): Graphs showing inhibition of proliferation of SW480

cells expressing full length wild-type β6 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 β3 and β5.

Detailed Description Of Preferred Embodiments Of The Present Invention

The distribution of β6 integrin subunit within various tissues has been assessed by

both in situ hybridisation and immunostaining and reported in the art. For instance, β6

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

β6 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

β6 expression is 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, β6 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 β6 has been observed in several types of carcinoma.

For example, β6 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 β6 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 β6 is also up-regulated in migrating keratinocytes at the wound edge

during experimental epidermal wound healing. αvβ6 is not expressed in normal

epithelium (Jones et al, 1997). However, following experimental wounding, αv appears

to switch its heterodimeric association from β5 to β6 subunit during re-epithelialisation.

At day 3 after wounding, β6 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 re-

epithelialisation is complete, all suprabasalar cells overlying the wound express β6 but

not β5. In human mucosal wounds, maximal expression of β6 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 αvβ6 expression. Interestingly, freshly isolated keratinocytes have not

been found to express β6 but begin to express this after subculturing (Haapasalmi et al,

1996). Moreover, TGF-βl has been shown to induce the de novo expression of αvβ6 at

the cell surface on keratinocytes (Zambruno et al, 1995). This is particularly relevant in

view of the recent observation that αvβ6 binds and activates latent TGF-βl which may

be a means of locally regulating TGF-βl function in vivo during tissue response to injury

(Munger et al, 1999).

αvβ6 expression is also upregulated in type II alveolar epithelial cells during lung

injury caused by injection of live bacteria and αvβ6 mRNA is induced within 5 hours of

acute injury (Breuss et al, 1995). Interestingly, αvβό has been shown to be expressed on

proximal airway epithelial cells in 50% of smokers undergoing lung resection (Weinacker et al, 1995). Just as in human keratinocytes, in primary cultures of human

airway epithelial cells, TGF-βl has been shown to dramatically increase expression of

αvβό without affecting surface expression of any other integrin. Moreover, epidermal

growth factor (EGF) also induces αvβό in these cells, and the effect of both growth

factors on β6 expression has been shown to be additive (Wang et al, 1996). αvβό

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 αvβό affects cell spreading, migration and growth during re-

organisation of epithelia in development, tissue repair and neoplasia.

Recent studies have shown that αvβό is a major fibronectin-binding receptor in

colorectal cancer (Agrez et al, 1996). Moreover, normal colonic epithelium from cancer

patients does not express αvβό in immunostaining studies, and as with squamous cell

carcinomas from the oral mucosa (Thomas et al, 1997) maximal β6 expression in colon

cancer has been observed at the invading edges of tumour cell islands (Agrez et al,

1996). Importantly, heterologous expression of the βό 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 1 1 amino acid C-terminal cytoplasmic

extension unique to the β6 subunit (Agrez et al, 1994). These findings suggest that one

mechanism of the enhanced growth effect involves induced secretion of matrix-

degrading enzymes as has been previously suggested for invasive melanoma cells.

Support for this arises from findings that the proliferative capacity of colon cancer cells within 3-dimensional collagen matrices is inversely related to the density of the

extracellular matrix (Agrez, 1989), and the observation that induced expression of αVβό

in colon cancer cells markedly enhances gelatinase B secretion (Agrez et al, 1999).

The βό subunit is widely observed in cancers of various origins (Breuss et al,

1995). As described above, β6 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 β6 is observed in the normal

lining cells of the mouth but in both primary and metastatic tumours from the

oropharyngeal mucosa, strong β6 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, β6 expression is similarly maximal at the advancing edges of

tumour cell islands (Agrez et al, 1996).

Hence, modulation of MAP kinase interaction with the β6 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 pre- disposition 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 BRCA1 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 βό 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 βό integrin and/or inhibiting the β6 MAP

kinase interaction are of particular interest. In particular, the fact that βό 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 intra-

tumoural injection of recombinant virus (Spitz et al, 1996). 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

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 βό and hence an integrin heterodimer such as αvβό, is achieved using antisense

nucleic acid sequences (e.g. 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 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 βό subunit in malignant cells

such as in colon cancer by means of adenoviral-mediated antisense therapy is preferred

because down-regulating β6 by means of a non-adenoviral approach may increase cell

surface expression of the β5 subunit. The relevance to such therapy is that the

vitronectin-binding integrin αvβ5 promotes adenovirus internalisation (Thomas et al,

1993). Given that abundant αvβ5 is always present on the surface of colon cancer cells

for example, a secondary benefit of inhibiting βό expression during the course of therapy may be the concomitant rise of β5 in cells already transduced with antisense βό nucleic

acid. This is likely to facilitate further virus uptake into such cells since the amount of

integrin αvβ5 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

been spliced out by normal cellular processing events.

It is not 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 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. 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 micro injection 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 N-

terminal and C-terminal amino acid residues of the identified amino acid sequence. Nucleotide and amino acid sequence data for the β6 integrin subunit for instance is

found in Sheppard et al, 1990. The amino acid sequence for βό 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 tliree 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 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 β-pleated sheet or helical structure such as an α-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 N-

terminal 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 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 [ H]-thymidine uptake or other measurement of cellular activity. As will be understood, a difference in

observed functional activity in the presence of the test agent is indicative of the

modulating effect provided by the test agent.

It will also be understood that the integrin in the context of such assays may be an

integrin subunit or polypeptide or fragment incorporating the binding domain of the

integrin for the MAP kinase, or a homolog, analog, variant or derivative of such a

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.

32

Probes may be labelled with for instance commonly used isotopes such as P , 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 NaCl/0.0015M sodium

citrate) /0.1 % SDS (sodium dodecylsulfate) wash buffer at 42°C. High stringency wash

conditions employ for instance, 0.1 x SSC wash buffer at 68°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 molecule capable of facilitating the transport of a nucleic acid sequence inserted therein

into a cell and includes expression vectors and cloning vectors.

Suitable expression vectors include plasmids and cosmids capable of expression of

a DNA (eg. genomic DNA or cDNA) insert. An expression vector will typically include

transcriptional regulatory control sequences to which the inserted nucleic acid sequence

is operably linked. By "operably linked" is meant the nucleic acid insert is linked to the

transcriptional regulatory control sequences for permitting transcription of the inserted

sequence without a shift in the reading frame of the insert. Such transcriptional

regulatory control sequences include promotors for facilitating binding of RNA

polymerase to initiate transcription, expression control elements for enabling binding of

ribosomes to transcribed mRNA, and enhancers for modulating promotor activity. A

promotor may be a tissue specific promotor which facilitates transcription of the nucleic

acid insert only in specific cell lineages and not in other cell types or only to a relatively

low level in such other cell types. The design of an expression vector will depend on the

host cell to be transfected, the mode of transfection and the desired level of transcription

of the nucleic acid insert.

Numerous expression vectors suitable for transfection of prokaryotic (eg. bacterial)

or eukaryotic (eg yeast, insect or mammalian cells) are known in the art. Expression

vectors suitable for transfection of eukaryotic cells include pSV2neo, pEF.PGK.puro,

pTk2, pRc/CNV, pcDNAI/neo, non-replicating adenoviral shuttle vectors incorporating

the polyadenylation site and elongation factor 1-α promotor and pAdEasy based

expression vectors most preferably incorporating a cytomegalovirus (CMV) promotor

(eg. see He et al, 1998). For expression in insect cells, baculovirus expression vectors may be utilised examples of which include pVL based vectors such as pVL1392, and

pVL941, and pAcUW based vectors such as pAcUWl. 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. Amp^r for ampicillin

resistance), a gene encoding an enzyme such as chloramphenicol acetyltransferase

(CAT), β-lactamase, adenosine deaminase (ADA), aminoglycoside phosphotransferase

(APH), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH),

thymidine kinase (TK), or for instance β-galactosidase encoded by the E. coli lacZ gene

(LacZ'). Yeast reporter genes include imidazole glycerolphosphate dehydratase (HIS3),

N-(5'-phosphoribosyl)-anthranilate isomerase (TRP1) and β-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. 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 internalisation 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 β6-expressing cancer cells may also be achieved by coupling

humanised anti-β6 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 β6 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 pentratin-

agent complex. Another approach may employ coupling the penetratin-agent complex to

β6 integrin receptor-targetted peptides, targetted for binding to the extracellular βό

domain by virtue of their DLXXL sequence. For example, a ligand recognition motif for

αVβό 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. 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, 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 proposed mode of administration. Preferred oral compositions according to the

invention will contain between about 0.1 μg 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 absoφtion of the composition may be brought

about by the use in the compositions of agents for delaying absoφtion 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 incoφorated into any suitable

sustained-release preparation or formulation. 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 pharmaceutically 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.

Supplementary active ingredients can also be incoφorated into the compositions if

desired.

It is particularly preferred to formulate parenteral compositions in dosage unit form

for ease of administration 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 μg to about 2000mg/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 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 32 P, 125 I or 131 I, an enzyme, a

fluorescent label, chemiluminescent molecule or for instance an affinity label such as

biotin, avidin, streptavidin and the like. 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, β-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 (eg., 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 (eg., 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 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 adsoφtion.

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 incoφorating 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 (eg., 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 (eg., with a higher salt concentration

and at an altered pH, typically pH 2.5 or pH 11) that facilitates the release of the binding 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 of immunoassay.

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

examples.

Example 1

Anti-integrin antibodies and antibodies against the β 1 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 α2βl 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 α5βl receptor to regulation of

growth appears to be ligand (fibronectin)-dependent. For example, induced expression

of α5βl 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 α5βl expression was associated with a

marked reduction of tumourigenicity in immune-deficient mice. Failure to ligate all of

the tumour cell α5βl 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 αvβό expression on colon cancer growth was

examined.

1.1 Methods

1.1.1 Generation of sense and antisense βό constructs in pEF.PGK.puro vector.

For βό antisense constructs, βό cDNA was excised from the vector pcDNAlneo βό

(Weinacker et al, 1994) using the restriction enzymes SnaBl and Xbal. This produced a

5' overhang (Xbal) and a 3' blunt end (SnaBl). 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 βό 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 β6. Digestion with BstEl 1

produced the expected two fragments, one of 6.0 basepairs and the other 3.5 basepairs.

To provide β6 sense constructs, βό cDNA was also excised from the pcDNAlneo

b6 vector using the restriction enzymes Snabl 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 produced

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 αvβό. Stable transfectants of WiDr and HT29 cells expressing βό

in sense and antisense direction were generated using lipofectamine and puromycin as

the selection antibiotic. One stable antisense βό 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 curves performed using a range of concentrations of puromycin established that WiDr

and HT29 cells could be stably transfected with puromycin concentrations of 1.Oμg and

2.5μg/ml, respectively.

1.1.3 Assessment of β6 expression in the transfected cell lines

βό 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°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 0.5ml

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 (lOmM sodium borate,

150mM sodium chloride, pH 8.8) for 30 mins at 4°C with continuous slow mixing. Cell

pellets were then centrifuged at 4°C, washed twice with cold PBS and exposed to lysis

buffer (containing lOOmM Tris, 150mM NaCl, lmM CaCl₂ , 1% Triton, 0.1% SDS and

0.1% NP-40 at pH 7.4 and containing lmM phenylmethylsulfonyl fluoride (PMSF)) for 30 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 βό expression were evaluated by reverse transcriptase-

polymerase 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). 0.4-2μg of RNA was used to prepared cDNA by reverse transcription.

Briefly, a reaction mixture in a final volume of 40μl containing 8μl of 5 x RT reaction

buffer (250mM Tris, 15mM MgCl₂ , 375mM KCL, pH 8.3), 8μl of 2.5mM of each

dNTP, 4μl of lOOmM DTT, 40U of an Rnase inhibitor, Rnasin (Promega, Madison,

Wisconsin, USA), 0.5μg 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°C for 5 min

and cDNA stored at 4°C until PCR. 2-5μl of this cDNA was combined with 5μl of 10 x

PCR buffer (lOOmM Tris, 500mM KC1, 15mM MgCl₂ , pH8.3) 8μl of 1.25mM dNTP

each and 1.25μl of 20μM of both forward and reverse primers. The forward primer

sequence was 5ΑGGATAGTTCTGTTTCCTGC3' and the reverse primer sequence

5ΑTCATAGGAATATTTGGAGG3'. The reaction was initiated by 2.5U of Taq

polymerase in a final volume of 50μl. After an initial 5 min incubation at 94°C, 30 cycles of amplification were performed under the following conditions: 94°C 1 min,

54°C 1 min and 72°C for 1 min. The reaction was stopped by incubating at 72°C for 10

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 35

cycles.

1.2 Results

1.2.1 αvβό expression in HT29 and WiDr transfected cell lines

Transfection of the colon cancer cell lines HT29 and WiDr with the βό gene

construct in an antisense orientation resulted in a marked reduction of βό expression at

the transcript level and on the cell surface as shown in Figs 1 to 4. Transfection of cells

with β6 in the sense orientation did not enhance βό surface expression. However, a

consequence of down-regulation of the β6 subunit in antisense transfectants was a

marked increase in surface expression of the β5 subunit. The changes in surface

expression of β6 and β5 subunits noted on FACScan analyses of antisense β6

transfectants was confirmed by surface-labelling cells with biotin and

immunoprecipitating integrin subunits with either anti-βό mAb (R6G9) or anti-β5 mAb

(P1F6).

1.2.2 Effect of Suppression of αvβό expression on cell binding to fibronectin

The major substrate for αvβό is fibronectin and to investigate the effect that

reduction in βό surface expression in antisense βό transfectants might have on cell-

matrix adhesion, WiDr and HT29 antisense βό transfectants were seeded on fibronectin in adhesion assays. Since both cell lines can adhere to fibronectin through members of

the βl integrin subfamily (either α3βl or α5βl) αvβό-mediated adhesion to fibronectin

was assessed in the absence/presence of blocking anti-βl antibody.

The cell-adhesion assays were performed in wells of non-tissue culture-treated

polystyrene 96-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°C.

Harvested cells were seeded at a density of 10 cells/well for HT29 and 1.5 x 10⁵

cells/well for WiDr cells in 200μl of standard DMEM which lacked FBS but contained

0.5% bovine serum albumin. To block adhesion, cells were incubated with anti-βl

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°C in humidified 5% carbon

dioxide. Non-adherent cells were removed by centrifugation top side down at 48 x g for

5 min. The attached cells were fixed and stained with 0.5% crystal violet (in 20%

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 βό, βl -independent adhesion of cells

to fibronectin was reduced compared with mock transfectants (containing vector alone)

which express surface βό at similar levels to wild-type cells. The further addition of

blocking anti-αv antibody completely prevented binding to fibronectin. 1.2.3 Effect of suppression of αvβό expression on tumour cell proliferation and tumour growth in vivo

To investigate the effect of diminished αvβό surface expression on cell

proliferation in vitro, WiDr and HT29 antisense β6 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

lμCi ( 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 β6 transfectants, and HT29 wild-type, mock, sense βό and antisense β6

transfectants were harvested second daily during a six-day culture period.

A marked increase in thymidine incoφoration was observed for WiDR and HT29

cells expressing normal levels of αvβό compared with antisense βό transfectants (see

Figs. 5 and 6).

1.2.4 Effect of Suppression of αvβό expression on Tumour Formation

The ability of HT29 antisense βό transfactants to form tumours in immune-

deficient mice was assessed.

BALB/C female athymic mice (8 weeks of age purchased from the Animal

Resource Centre, Perth, Western 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 βό transfected

clones (clones 1 - 3) and HT29 mock, sense and antisense βό 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

β6 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 βό WiDr clones 2 and 3 each inoculated into 10 mice are not shown.

Of a total of 40 mice injected with cells expressing antisense βό (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 in size during the six week

period compared with tumours at least 15mm in size from cells expressing normal

levels of β6. To confirm the presence of tumour xenografts histologically at one week

following subcutaneous inoculation of mock and antisense β6 transfectants, tumour

nodules were excised, fixed in formalin and stained with haematoxylin and eosin.

1.2.5 Effect of suppression of αvβό expression on gelatinase B secretion

Serum-free tumour-conditioned medium was collected from each of the 3 WiDr

mock and antisense βό 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 αvβό expression resulted in a marked reduction in

gelatinase B secretion. 1.3 Discussion of Results

Induced expression of βό in Chinese hamster ovary (CHO) cells has been shown to

result in decreased surface expression of the β5 integrin subunit which also partners αv

(Weinacker et al, 1994). The concept of integrin switching depends on the availability

of the promiscuous αv partner subunit. In the present study, the reverse was observed.

As a consequence of down-regulation of βό in colon cancer cells which constitutively

express αvβό, β5 surface expression increased, most likely secondary to increased

availability of the αv subunit partner.

Heterologous expression of αvβό in colon cancer cells has previously been

reported to enhance tumour growth in immune-deficient mice (Agrez et al, 1994).

Suppression of αvβό 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 αvβό expression had not been perturbed. Similar

findings have been described with loss of the classical vitronectin receptor αvβ3 in

melanoma. For example, in experimental animal models, the loss of αvβ3 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 αvβό-mediated tumour growth remain to be

elucidated, the present in vitro data show that loss of βό 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 βό, the findings reported in the present study suggest that intracellular signalling pathways

activated via the integrin αvβό play a major role in promoting progression of this tumour

type.

Example 2

The association of αvβό expression and MAP kinase activity were evaluated using

WiDr, HT29 and SW480 cell lines.

2.1 Methods

2.1.1 S W480 colon cancer βό transfectants

Stable transfectants of S W480 colon cancer cells (ATCC) expressing gene

constructs of either wild-type or mutant forms of the βό 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 β6 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 β6 transfectants were established

by seeding 1 x 10 cells/5ml of culture medium in 25cm tissue culture flasks. Cells

were incubated at 37°C in humidified CO₂ 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

(lOmM Tris-HCl, 150mM NaCl, 2mM EDTA, 2mM DTT, lmM orthovanadate, lmM PMSF, 4μg/ml aprotonin, 2μg/ml leupeptin and lμg/ml pepstatin, pH 7.4) and sonicated

at a setting of 7, using a Soniprep 150 watt ultrasonic disintegrator for a total of 90

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

32

[γ PJ-ATP to a synthetic peptide that contains specifically a p42/p44 MAP kinase

phosphorylation site was measured as described in the manufacturer's instructions.

[ P]-labelled peptides were spotted onto PEl -cellulose paper, unbound radioactivity was

washed with 75mM phosphoric acid and bound [ 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 40μM, were added one hour before

the addition of serum to the medium.

2.1.2 Western blotting

To detect the MAP kinases ERK 1/2, cells were lysed in lysis buffer containing

lOOmM Tris, 150mM NaCl, lmM CaC12, 1% Triton, 0.1% SDS and 0.5% NP-40 at

pH7.4, supplemented with enzyme inhibitors (lmM PMSF, lmM sodium orthovanadate,

1 μg/ml pepstatin A, 2.5 μg/ml aprotonin, lmM benzamidine, lμg/ml leupeptin). Lysates

were clarified by ultracentrifugation and equal protein loads electrophoresed in 8% or

10% SDS-PAGE under non-reducing conditions. Electrophoresed proteins were

transferred to nitrocellulose membranes (Biotrace NT, Gelman Sciences, Ann Arbor, 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 (ElO 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 β-sύbunit 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 β5 and β6. 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-βό monoclonal antibody,

R6G9 (using isotype matched antibody, IgG2A, and anti-β5 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 ElO and SC-1647. In parallel experiments, the sequentially

immunoprecipitated βό 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 ElO 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 UO126 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 β6 expression on MAP kinase activity following serum

stimulation.

In these experiments, WiDr transfectants (3 mock and 3 antisense βό clones),

HT29 transfectants (mock and antisense β6) and SW480 β6 transfectants (mock and

sense βό) were serum-starved for 16 hrs followed by 30 mins exposure to serum.

Increased expression of αvβό was associated with a marked increase in MAP kinase activity upon serum stimulation compared with cells lacking αvβό altogether (SW480

mock). Cells in which βό expression had been down-regulated (antisense βό

transfectants) exhibited suppressed MAP kinase activity. Induced expression of β6 in

the non-β6-expressing colon cancer cell line SW480 resulted in a three fold increase in

serum-dependent MAP kinase activity.

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

A highly surprising finding arising from phage display screening which undeφins

the present work is that the integrin βό cytoplasmic domain binds a MAP kinase. Use of

phage display to screen a γgt cDNA colon cancer cell library with the βό 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 of ERK1/2. The specificity of this

interaction was examined in integrin immunoprecipitations against βl, β5, βό and αv

integrin subunits. As shown in Fig. 10, the anti- ERK mAb SC-1647 identified a β6-

specific band migrating at the position of purified ERK2 protein. The integrin-

associated ERK band was identified only in immunoprecipitations of the βό subunit and

its partner αv and not in immunoprecipitations of the βl/β5 subunits or in

immunoprecipitations using isotype-matched control antibodies. 2.3.4 Effect of altered βό expression on total cellular ERK and βό-bound ERK.

Equal protein loads from one representative clone each from WiDr mock and

antisense β6 transfectants were electrophoresed, transferred to nitrocellulose and blotted

with anti-ERK mAb (SC-1647) as shown in Fig.11(A). β6 immuno-precipitates from

equal protein loads of the WiDr mock and antisense βό clones were electrophoresed,

transferred and probed with anti-ERK mAb (ElO) as shown in Fig. 11(B). As indicated,

suppression of βό expression resulted in a reduction of both total cellular and

phosphorylated integrin-associated ERK compared with WiDr mock transfectants.

Similarly, βό immunoprecipitations from SW480 βό transfected clones expressing high

and low levels of βό (confirmed by FACScan and βό immunoprecipitations) showed

parallel changes in βό-bound phosphorylated ERK (Fig. 12).

2.3.5 Effect of βό immunodepletion on total cellular ERK and β6-bound ERK

WiDr wild-type cells were surface biotinylated and the cell lysates

immunodepleted of β6 in three rounds of sequential immunoprecipitation using anti-βό

mAb (R6G9) resulting in a marked loss of β6 from the lysates as shown in Fig. 13(A).

βό-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 βό immunodepletion, levels

of ERK1/2 in βό-depleted lysates compared with non-immunodepleted lysates, were

markedly reduced suggesting a significant contribution of β6-bound ERK to total

cellular ERK. In contrast, immunodepletion of β5 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 β6-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. ERK-

immunodepleted cell lysates probed with either ElO or SC-1647 mAbs contained

markedly less ERKs. To examine the effect of ERK-immunodepletion on β6-bound

ERK, the ERK-immunodepleted cell lysates were immunoprecipitated with anti-βό mAb

(R6G9) and the βό-immunoprecipitates probed with anti-ERK mAb (ElO recognising

phosphorylated ERK1/2). ERK immunodepletion effectively reduced levels of βό-

bound ERK.

2.3.7 Effect of preventing cell attachment on αvβό-bound ERK

SW480 mock- and β6 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 /batch). The first group

was plated on a 75 cm 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°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 30

mins. Cells were collected washed with PBS and lysed in cell lysis buffer (100 mM

Tris-HCl pH-7.5, 150 mM NaCl, lmM Cacl2, 1% Triton, 0.1% SDS, 0.1% Np-40, 1

mM vanadate, 1 μg/ml pepstatin, 1 mM PMSF, 5 μg/ml aprotonin and lμg/ml of leupeptin). 10 μl (10 μg of protein) was used for the analyses of cell lysates after adding

equal volumes of non-reducing Laemmli buffer. For SW480-β6-transfected cells the

rest of the cell lysate was used for immunoprecipitation of αvβό integrin. Cell lysates

and βό immunoprecipitates were subjected to western blotting and probed with ElO

monoclonal antibody (recognising phosphorylated ERK 1/2).

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 βό

colon cancer cells do not require serum factors to maintain the activation

(phosphorylation) state of βό-bound ERK.

2.3.8 Effect of PP2A phosphatase on αvβό-bound ERK

In experiments to examine the effect of protein phosphatase 2 A (PP2A) on βό-

bound ERK, SW480 β6-transfected cells were sonicated in buffer comprising 50mM

Tric-HCl (pH 8.0), lOmM MgCl₂ and O.OlmM 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, PP2A-

treated cell lysates were immunoprecipitated with mAb R6G9 (anti-β6) followed by

Western blot with anti-ERK mAb El O. MAP kinase activity assays were performed

32 according to the manufacturer's instructions (Amersham Pharmacia Biotec) using γ 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 βό-bound ERK was not observed in βό immunoprecipitates prepared from serum-supplemented or serum-starved cells βό

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 βό transfectants were cultured under serum-free conditions for 48 hours in

the absence/presence of the MEK inhibitor PD98059 (40μM) 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 βό-ERK2 association in HaCaT and HaRas Cell Lines

β6 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-βό) and the immunoprecipitates probed

with mab ElO (against phosphorylated ERK 1/2). Fig. 14 shows that ERK2 associates

with βό 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 βό 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 serum-starved β6-expressing cells. In contrast, only a one-fold increase in serum-

dependent MAP kinase activity was observed for cells in which βό had been down-

regulated consequent upon transfection with antisense βό. Moreover, induced

expression of β6 in the non-β6-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 αvβό 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 βό 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 βό subunit. Slight differences in mobility of the βό-associated ERK band compared

with the pure ERK protein could arise if β6-bound ERK is hyper-phosphorylated and/or

exists in an altered conformation consequent upon its association with βό. ERK bound

to βό 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 β6 expression in colon cancer cell lines

was associated with increased/decreased β6-bound ERK, respectively. In addition,

immunodepletion experiments suggest that βό-bound ERK makes a substantial

contribution to total phosphorylated ERK within the cell and overall MAP kinase

activity (see Figs. 15(A) and 15(B)). The observation that β6-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 βό-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 βό-expressing cells, it

seems that activation of MAP kinase signalling plays a role, at least in part, in β6-

mediated induction of gelatinase B secretion.

Example 3

3.1 Identification of the binding domain on the βό subunit cytoplasmic tail domain

for ERK2

Peptide fragments corresponding to regions of the cytoplasmic tail domain of the

βό subunit were screened in an enzyme-linked immunosorbent assay (ELISA) for

binding with ERK2. The 52 amino acid long β6 cytoplasmic tail is shown in Fig. 16 as

are the amino acid sequences for the cytoplasmic domains of the βl to β3 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 β6 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 βό 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

200μl of wash buffer (TBS, 0.1% BSA, 0.05% Tween 20 or SuperBlock™ blocking

buffer in TBS, Pierce, Prod. No. 37535) prior to addition of biotinylated peptides

(lOOμl) 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 lOOμl 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 lOOμl anti-ERKl/2 mAb SCI 647

(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

lOOμl rabbit anti-mouse antibody (Biorad) conjugated to alkaline phosphatase at a

concentration of 1 : 1000 for 30 minutes again at room temperature. A lOOμl 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 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

(0.25μg/100μl) to peptide fragment 2 was observed (lμg/lOOμl) while only negligible or

low level binding for the other fragments was found.

Significant binding of non-phosphorylated ERK2 to both fragment 2 and βό

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 βό subunit,

progressively shorter peptides from the region of the βό 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 15

mer test peptide (seq. 4) having the amino acid sequence RSKAKWQTGTNPLYR and a

10 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 βό 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 of seq. 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 omitted 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 β6

In view of the observation that ERK2 associates with the cytoplasmic tail of the βό

subunit, the MAP kinase JNK-1 was tested to evaluate whether it also could associate

with the cytoplasmic tail of βό.

Briefly, 0.05-1.5μm/100μl of JNK-1 (Santa Cruz) was aliquoted into wells of a 96

well culture place containing increasing concentrations of the β6 cytoplasmic domain

tail peptide used in Example 3. For comparison purposes, non-phosphorylated GST-

ERK2 (0.05-1.5μm/100μl) was aliquoted into wells containing βό cytoplasmic tail

peptide or peptide fragments 1 or 2, respectively. Binding of JNK-1 was detected using 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 βό cytoplasmic tail peptide was found.

Example 5

5.1 Evaluation of ability of ERK2 to bind to βό Δ746-764 deletion mutant.

To examine the role of the amino acid sequence RSKAKWQTGTNPLYR in the

β6 cytoplasmic domain in situ, a β6 deletion construct lacking the coding sequence for

AERSKAKWOTGTNPLYRG was transfected into colon cancer cell line SW480 which

does not constitutively express the αVβό integrin using the calcium phosphate method

previously described for transfections into this cell line (Agrez et al, 1994). The location

of the βό Δ746-764 deletion is indicated in Fig. 25. Construction of the β6 Δ746-764

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 βό

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-β6 monoclonal antibody (mAb R6G9) or matched

isotype control antibody. Surface biotinylation prior to immunoprecipitation confirmed

equal surface expression of mutant and wild-type β6 (see Fig. 27 (A). Aliquots of the

immunoprecipitates were electrophoresed and transferred to nitrocellulose for Western

blotting using monoclonal antibody ElO which recognises ERK1/2. As seen in Fig.

27(B), loss of the RSKAKWQTGTNPLYR sequence in the βό cytoplasmic domain reduced levels of βό-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 βό-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 10 cells per triplicate well for each condition tested and after 24

hours incubation of cell cultures in 5% CO₂, 100% humidity at 37°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 penetratin-

peptide complex at a concentration of lOμm for HT29 cells or 30μm for SW480 βό-

expressing 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, 30μl of WST-1 was added to

270μl 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 (A) to (C).

Example 7

SW480 mock and S W480 βό transfectants were cultured in DMEM medium

supplemented with 1% foetal bovine serum in the presence of 20 μM 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 -βό and +βό expressing cells was 17% and 50%, respectively as

indicated in Fig 31 A. The -β6 and +β cultured cells are shown in Fig 3 IB. Example 8

The proliferation of SW480 cells expressing a β6 Δ746-764 deletion mutant was

compared with non-β6 expressing SW480 cells and SW480 cells expressing full length

wild-type βό. 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 200μ) and

following dissolution of the collagen with collagenase Fig.32(B) prior to visual colony

counting of all colonies exceeding 200μ in diameter within each well Fig.32(C). As can

be seen, significant proliferation of the SW480 cells expressing the full length wild-type

β6 was observed compared to the non-βό expressing cells and cells expressing the β6

Δ746-764 deletion mutant.

Example 9

Growth inhibition of S W480 cells expressing full length wild-type βό exposed to

seq.4 coupled to penetratin or RSKAKWQTGTNPLYR peptide coupled to penetratin (5,

10, 20, 30 μM 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 10

Binding of ERK2 to the seq.4 peptide (RSKAKWQTGTNPLYR) was compared

with peptides corresponding to regions of the cytoplasmic domain of integrin subunits

βl, β2, β3 and β5. The amino acid sequences for those peptides is shown below:

βl KFEKEKMNAKWDTGENPIYK

β2 KEKLKSQWNNDNPLFK

β3 RARAKWDTANNPLYK

β5 RSRARYEMASNPLYR

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

Binding of ERK2 to the β5 and β3 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

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS :-

1. An isolated polypeptide capable of binding with a binding site on a MAP kinase

which binding site binds with a binding domain of an integrin for the MAP kinase, 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 βό(770t) or βό(777t) deletion

mutant.

2. A polypeptide according to claim 1 wherein the polypeptide comprises a binding

domain of an integrin subunit for the MAP kinase.

3. A polypeptide according to claim 1 wherein the polypeptide is other than a

fragment of an integrin subunit.

4. A polypeptide according to claim 3 consisting of or incoφorating the amino acid

sequence RSKAKNPLYR.

5. A polypeptide according to claim 1 or 2 wherein the polypeptide comprises one or

both of amino acid sequences RSKAK and NPL YR.

6. A polypeptide according to any one of claims 1 to 5 wherein the polypeptide has a

length of about 150 amino acids or less.

7. A polypeptide according to claim 6 wherein the polypeptide has a length of about

100 amino acids or less.

8. A polypeptide according to claim 7 wherein the polypeptide has a length of about

50 amino acids or less.

9. A polypeptide according to claim 8 wherein the polypeptide has a length of about

40 amino acids or less.

10. A polypeptide according to claim 9 wherein the polypeptide has a length of about

30 amino acids or less.

11. A polypeptide according to claim 10 wherein the polypeptide has a length of about

20 amino acids or less.

12. A polypeptide according to claim 11 wherein the polypeptide has a length of about

15 amino acids or less.

13. A polypeptide according to claim 12 wherein the polypeptide has a length of about

10 amino acids or less.

14. A polypeptide according to any one of claims 1 to 13 or an analog or derivative of

the polypeptide.

15. A polypeptide according to any one of claims 1 to 14 wherein the MAP kinase is

an ERK family member or a JNK family member.

16. A polypeptide according to claim 15 wherein the MAP kinase is ERK2.

17. A fragment of an integrin subunit wherein the fragment is capable of binding with

a MAP kinase, or a homolog, analog variant or derivative of the fragment, with the

proviso that the integrin subunit is other than a βό(770t) or βό(777t) deletion mutant.

18. A fragment according to claim 17 wherein the fragment comprises a binding

domain of the integrin subunit for the MAP kinase.

19. A fragment according to claim 18 wherein the fragment has a length of about 150

amino acids or less.

20. A fragment according to claim 19 wherein the fragment has a length of about 100

amino acids or less.

21. A fragment according to claim 20 wherein the fragment has a length of about 50 amino acids or less.

22. A fragment according to claim 21 wherein the fragment has a length of about 40

amino acids or less.

23. A fragment according to claim 22 wherein the fragment has a length of about 30

amino acids or less.

24. A fragment according to claim 23 wherein the fragment has a length of about 25

amino acids or less.

25. A fragment according to claim 24 wherein the fragment has a length of about 20

amino acids or less.

26. A fragment according to any one of claims 17 to 25 comprising the amino acid

sequence RSKAKWQTGTNPLYRGSTST or a contiguous partial amino acid sequence

thereof.

27. A fragment according to any one of claims 17 to 25 wherein the fragment has a

length of about 15 amino acids or less.

28. A fragment according to claim 27 wherein the fragment comprises amino acid

sequence RSKAKWQTGTNPLYR.

29. A fragment according to any one of claims 17 to 28 wherein the MAP kinase is an

ERK family member or a JNK family member.

30. A fragment according to any one of claims 17 to 29 wherein the MAP kinase is

ERK2.

31. A fragment according to any one of claims 17 to 30 wherein the integrin subunit is

βό.

32. An agent capable of inhibiting binding of a MAP kinase with an integrin.

33. An agent according to claim 32 wherein the agent is capable of binding to a

binding domain on the integrin for the MAP kinase.

34. An agent according to claim 33 wherein the agent is capable of binding with a

binding site on the MAP kinase which binding site binds to a binding domain on the

integrin for the MAP kinase.

35. An agent according to any one of claims 32 to 34 wherein the agent is adapted to

be capable of passing across a cell membrane.

36. An agent according to claim 35 comprising a carrier moiety for facilitating passage

of the agent across the cell membrane and an inhibitor moiety for inhibiting the binding

of the MAP kinase with the integrin.

37. An agent according to claim 36 wherein the agent is adapted for release of the

inhibitor moiety from the carrier moiety when the agent has passed across the cell

membrane.

38. An agent according to claim 32 comprising:

a targeting moiety for targeting cells expressing the integrin;

an inhibitor moiety for inhibiting binding of the MAP kinase with the integrin; and

a carrier moiety for facilitating passage of the inhibitor moiety across the cell

membrane of a said cell;

wherein the inhibitor and carrier moieties are capable of being released from the

targeting moiety at the cell.

39. An agent according to claim 38 wherein the agent comprises an enzyme cleavage

site for being cleaved to thereby release the carrier moiety and the inhibitor moiety at the

cell.

40. An agent according to claim 39 wherein the enzyme cleavage site is a cleavage site

for matrix metalloproteinase-9 (MMP-9).

41. An agent according to any one of claims 38 to 40 wherein the targeting moiety is

an antibody or a binding fragment of an antibody.

42. An agent according to claim 41 wherein the antibody or the binding fragment is

specific for an extracellular region of the integrin.

43. An agent according to any one of claims 38 to 40 wherein the targeting moiety is

an integrin receptor targeted peptide for binding to the integrin.

44. An agent according to any one of claims 38 to 43 wherein the inhibitor moiety is

capable of binding with a binding site on the MAP kinase which binding site binds to a

binding domain on the integrin for the MAP kinase.

45. An agent according to any one of claims 38 to 43 wherein the inhibitor moiety is

capable of binding to a binding domain on the integrin for the MAP kinase.

46. An agent according to any one of claims 36 to 45 wherein the inhibitor moiety

comprises a polypeptide.

47. An agent according to claim 46 wherein the inhibitor moiety is a polypeptide as

defined in any one of claims 1 to 16, or a homolog. analog, variant or derivative of the

polypeptide.

48. An agent according to any one of claims 36 to 45 wherein the inhibitor moiety is a

fragment as defined in any one of claims 17 to 31 or a homolog, analog, variant or

derivative of the fragment.

49. An agent according to any one of claims 36 to 48 wherein the carrier moiety is a

polypeptide.

50. An agent according to claim 49 wherein the carrier moiety is penetratin.

51. An agent according to any one of claims 32 to 48 wherein the agent is a fusion

protein.

52. An integrin subunit with a mutagenised binding domain for a MAP kinase or in

which the binding domain is deleted, or a homolog, analog, variant or derivative of the

integrin subunit, wherein capability to bind with the MAP kinase is thereby reduced with

the proviso that the integrin subunit is other than a βό Δ746-764 deletion mutant.

53. An integrin subunit according to claim 52 wherein the MAP kinase is an ERK

family membrane or a JNK family member.

54. An integrin subunit according to claim 53 wherein the MAP kinase is ERK2.

55. An integrin subunit according to any one of claims 52 to 55, wherein the integrin

subunit is βό.

56. An integrin subunit according to any one of claims 52 to55, wherein the integrin

subunit is a truncated integrin subunit.

57. An isolated nucleic acid sequence encoding a polypeptide as defined in any one of

claims 1 to 16, or a homolog, analog, variant or derivative of the polypeptide.

58. An isolated nucleic acid sequence encoding a fragment of an integrin subunit as

defined in any one of claims 17 to 31, or a homolog, analog, variant or derivative of the fragment.

59. An isolated nucleic acid sequence encoding an integrin subunit as defined in any

one of claims 52 to 56.

60. An isolated nucleic acid sequence encoding a fusion protein incoφorating a

polypeptide as defined in any one of claims 1 to 16, or a homolog, analog, variant or

derivative of the polypeptide.

61. An isolated nucleic acid sequence encoding a fusion protein incoφorating a

fragment as defined in any one of claims 17 to 31 . or a homolog, analog or variant of the

fragment.

62. An isolated nucleic acid sequence according to claim 60 or claim 61 wherein the

fusion protein further comprises a carrier polypeptide for facilitating passage of the

fusion protein across a cell membrane.

63. An isolated antisense nucleic acid sequence complementary to a nucleic acid

sequence as defined in claim 57 or 58.

64. An isolated antisense nucleic acid sequence according to claim 63 wherein the

antisense nucleic acid sequence is labelled.

65. A vector incoφorating a nucleic acid sequence as defined in any one of claims 57

to 59.

66. A vector incoφorating a nucleic acid sequence as defined in any one of claims 60

to 62.

67. A vector according to claim 65 or claim 66 wherein the vector is an expression

vector.

68. A host cell transformed with a vector as defined in any one of claims 65 to 67.

69. A host cell transformed with a vector as defined in claim 65 wherein the host cell

is selected from the group consisting of a mammalian cell, an epithelial cell, a neoplastic

cell and a cancer cell.

70. A host cell according to claim 69 wherein the host cell is a colon cancer cell.

71. A pharmaceutical composition comprising a polypeptide as defined in any one of

claims 1 to 16 or a homolog, analog, variant or derivative thereof, together with a

pharmaceutically acceptable carrier or diluent.

72. A pharmaceutical composition comprising a fragment as defined in any one of

claims 17 to 31 or a homolog, analog, variant or derivative thereof, together with a

pharmaceutically acceptable carrier or diluent.

73. A pharmaceutical composition comprising an agent as defined in any one of claims

32 to 51 together with a pharmaceutically acceptable carrier or diluent.

74. A pharmaceutical composition comprising a vector as defined in claim 65 together

with a pharmaceutically acceptable carrier or dil ent.

75. A pharmaceutical composition comprising an antisense nucleic acid sequence as

defined in claim 63 together with a pharmaceutically acceptable carrier or diluent.

76. An antibody capable of binding to a binding domain of an integrin for a MAP

kinase, or a binding fragment of the antibody.

77. An antibody capable of binding to a polypeptide as defined in any one of claims 1

to 16 or a homolog, analog, variant or derivative of the polypeptide.

78. An antibody capable of binding to a fragment as defined in any one of claims 17 to

31 or a homolog, analog, variant or derivative of the polypeptide.

79. An antibody according to any one of claims 76 to 78 wherein the antibody is a

monoclonal antibody.

80 A method of screening for an agent capable of inhibiting binding of a MAP kinase

to a binding domain of an integrin for the MAP kinase, comprising:

integrin; and

(b) determining if any said agent is capable of inhibiting binding of the MAP

kinase to the binding domain of the integrin on the basis of the testing.

81. A method of screening for an agent capable of inhibiting binding of a MAP kinase

to a binding domain of an integrin for the MAP kinase, comprising:

integrin;

(b) 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

(c) utilising the selected said agent or agents in an assay for indicating whether the

or any of the selected said agents is capable of inhibiting the binding of the MAP kinase

to the binding domain of the integrin.

82. A method of evaluating whether an agenl i s capable of inhibiting binding of a

MAP kinase to a binding domain of an integrin for the MAP kinase, comprising:

(a) selecting the agent; (b) utilising the agent in an assay for indicating whether the agent is capable of

(c) determining if the agent is capable oϊ inhibiting the binding of the MAP kinase

to the binding domain of the integrin on the basis of the assay.

83. A method of screening for an agent capable of binding to a binding domain of an

integrin for a MAP kinase, comprising:

(a) testing a number of agents for ability to bind to the binding domain of the

integrin for the MAP kinase; and

(b) determining if any said agent is capable of binding to the binding domain of the

integrin on the basis of the testing.

84. A method of screening for an agent capable of binding to a binding domain of an

integrin for a MAP kinase, comprising:

(a) testing a number of agents for ability to bi d to the integrin;

the basis of the testing; and

or any of the selected said agents is capable of binding to the binding domain of the

integrin for the MAP kinase.

85. A method of evaluating whether an agent is capable of binding to a binding

domain of an integrin for a MAP kinase, comprising:

MAP kinase; and (b) determining if the agent is capable of binding to the binding domain on the basis of the testing.

86. A method according to any one of claims 83 to 85 wherein the testing comprises

utilising a polypeptide consisting of the binding domain of the integrin, or a homolog,

analog, variant or derivative of the polypeptide.

87. A method according to any one of claims 83 to 85 wherein the testing comprises

utilising a polypeptide comprising core amino acid sequence of the binding domain

wherein binding of the agent to the polypeptide indicates the agent is capable of binding

to the binding domain of the integrin, or a homolog, analog variant or derivative of the

polypeptide.

88. A method according to any one of claims 80 to 87 wherein the integrin comprises

β6.

89. A method according to any one of claims 80 to 88 wherein the MAP kinase is an

ERK family member of a JNK family member.

90. A method according to claim 89 wherein the MAP kinase is ERK2.

91. An agent identified to be capable of binding to a binding domain of an integrin for

a MAP kinase by a method as defined in any one of claims 80 to 82.

92. An agent identified to be capable of inhibiting binding of a MAP kinase to a

binding domain of an integrin for the MAP kinase by a method as defined in any one of

claims 83 to 85.

93. A method of isolating a MAP kinase from a sample utilising a molecule

immobilised on a solid support and which is capable of binding to a binding site on the MAP kinase which binding site binds with a binding domain of an integrin for the MAP kinase, comprising:

(a) contacting the molecule immobilised on the solid support with the sample

under conditions suitable for binding of the MAP kinase to the molecule;

(b) eluting the MAP kinase from the solid support; and

(c) collecting the eluted MAP kinase.

94. A method according to claim 93 wherein ihc molecule is an integrin subunit of the

integrin which integrin subunit incoφorates the binding domain, or a homolog, analog,

variant or derivative of the integrin subunit.

95. A method according to claim 93 wherein the molecule is a fragment of an integrin

subunit of the integrin which integrin subunit incorporates the binding domain, or a

homolog, analog, variant or derivative of the fragment.

96. A method according to claim 93 wherein the molecule is a polypeptide.

97. A method according to claim 96 wherein the polypeptide is other than an integrin

subunit incoφorating the binding domain or a fragment of the integrin subunit.

98. A method according to claim 97 wherein the polypeptide comprises amino acid

sequence RSKAKNPLYR.

99. A method according to claim 93 wherein the molecule is an antibody capable of

binding to the binding domain of the integrin for the MAP kinase.

100. A method according to claim 93 wherein the integrin comprises β6.

101. A method according to any one of claims 93 to 100 wherein the MAP kinase is an

ERK family member or a JKN family member.

102. A method according to claim 101 wherein the MAP kinase is ERK2.

103. A MAP kinase isolated from a sample by a method as defined in any one of claims 93 to 103.

104. A kit for determining if a test agent is capable of binding to a binding domain of an

integrin for a MAP kinase, wherein the kit comprises:

a molecule for forming with a test agenl a complex for being detected to indicate

the test agent is capable of binding to the binding domain of the integrin; and

instructions for use.

105. A kit for determining if a test agent is capable of inhibiting binding of a MAP

kinase to a binding domain of an integrin for the MAP kinase, wherein the kit comprises:

a molecule for forming with a test agent a complex for being detected to indicate

the test agent is capable of inhibiting binding of the \ A kinase to the binding domain

of the integrin; and

instructions for use.

106. A kit according to claim 104 or 105 wherein the molecule is a polypeptide

consisting of the binding domain of the integrin or a homolog. analog or variant or

derivative of the polypeptide.

107. A kit according to claim 104 or 105 wherein the molecule is a polypeptide

comprising core amino acid sequence of the binding domain, or a homolog, analog

variant or derivative of the polypeptide.

108. A kit according to any one of claims 104 to 107 wherein the integrin comprises βό.

109. A kit according to any one of claims 104 to 108 wherein the MAP kinase is an

ERK family member or a JNK family member.

110. A kit according to claim 109 wherein the MAP kinase is ERK2.

111. A method of modulating activity of a cell, comprising causing the expression of an

integrin to be down-regulated, wherein the integrin has a binding domain for a MAP

kinase.

112. A method according to claim 111 wherein the down-regulation of the expression

of the integrin is provided by an antisense nucleic acid sequence that inhibits expression

of a gene encoding an integrin subunit of the integrin.

113. A method according to claim 1 12 wherein the integrin subunit is βό.

114. A method according to any one of claims 1 1 1 to 1 13 wherein the antisense nucleic

acid sequence is administered to the cell or is transcribed from an expression vector

introduced into the cell.

115. A method according to any one of claims 1 1 1 to 1 14 wherein the antisense nucleic

acid sequence specifically hybridises with sense nucleic acid encoding for at least part of

the binding domain for the integrin.

116. A method according to any one of claims 1 1 1 to 115 wherein the cell is a cancer

cell.

117. A method according to any one of claims 1 1 1 to 1 1 6 wherein the MAP kinase is an

ERK family member or a JNK family member.

118. A method according to claim 117 wherein the MAP kinase is ERK2.

119. A method of modulating activity of a cell expressing an integrin, comprising:

transfecting the cell with a nucleic acid sequence encoding an integrin subunit or a

homolog, analog or variant thereof for being expressed by the cell, and which has a

mutagenised binding domain for a MAP kinase or in which the binding domain has been deleted whereby binding of the MAP kinase is thereby reduced, with the proviso that the

integrin subunit is other than a βό Δ746-764 deletion mutant.

120. A method according to claim 119 wherein the transfecting comprises transfecting

the cell with an expression vector incoφorating the nucleic acid sequence.

121. A method according to claim 119 or 120 wherein the MAP kinase is an ERK

family member or a JNK family member.

122. A method according to claim 121 wherein the MAP kinase is ERK2.

123. A method according to any one of claims 1 19 to 1 12 wherein the integrin subunit

is βό.

124. A method according to any one of claims 1 19 to 123 wherein the integrin subunit

is truncated.

125. A method of modulating activity of a cell expressing an integrin, comprising:

transfecting the cell with a nucleic acid sequence encoding a polypeptide for being

kinase with a binding domain of the integrin f r the MAP kinase.

126. A method according to claim 125 wherein the polypeptide is capable of binding

with the binding site on the MAP kinase which binding site binds to the binding domain

of the integrin.

127. A method according to claim 125 or 126 wherein the polypeptide comprises the

binding domain of the integrin.

128. A method according to claim 126 or 127 wherein the polypeptide comprises the

amino acid sequence RSKAKWQTGTNPLYR., or a homolog, variant or analog of the

polypeptide.

129. A method according to claim 125 wherein the polypeptide comprises sufficient

core amino acid sequence of the binding domain of the integrin to enable the polypeptide

to bind to the binding site on the MAP kinase.

130. A method according to claim 129 wherein the polypeptide comprises the amino

acid sequence RSKAKNPLYR, or a homolog. analog, variant or derivative of the

polypeptide.

131. A method according to any one of claims 125 to 1 0 wherein the nucleic acid

sequence is incorporated in an expression vector.

132. A method according to any one of claims 125 to 13 1 wherein the integrin

comprises β6.

133. A method according to any one of claims 1 25 to 1 32 w herein the MAP kinase is an

ERK family member or a JNK family member.

134. A method according to claim 133 wherein the MAP kinase is ERK2.

135. A method according to any one of claims 125 to 134 wherein the cell is a cancer

cell.

136. A method of modulating activity of a cell comprising:

contacting the cell with an effective amount of an agent capable of inhibiting

binding of a MAP kinase to a binding domain of an integrin expressed by the cell.

137. A method according to claim 136 wherein the agent is an agent as defined in any

one of claims 32 to 51.

138. A method according to claim 136 wherein the agent is a polypeptide as defined in

any one of claims 1 to 16 or a homolog, analog, variant or derivative thereof.

139. A method according to claim 136 wherein ihc ageni is a fragment as defined in any

one of claims 17 to 31 or a homolog, analog, variant or derivative thereof.

140. A method according to any one of claims 136 to 139 wherein the MAP kinase is an

ERK family member or a JNK family member.

141. A method according to claim 140 wherein the MAP kinase is ERK2.

142. A method according to any one of claims 136 to 141 wherein the integrin

comprises β6.

143. A method according to any one of claims 136 to 142 wherein the activity of the

cell is growth of the cell.

144. A method according to any one of claims 136 to 143 wherein the cell is a cancer

cell.

145. A method according to claim 144 wherein the cancer cell is a colon cancer cell.

146. 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 ihc ceil

147. A method according to claim 146 wherein ihe agent inhibits binding of a MAP

kinase to a binding domain of the integrin for the MAP kinase.

148. A method according to claim 146 wherein the agent is a polypeptide as defined in

any one of claims 1 to 16 or a homolog, analog, variant or derivative thereof.

149. A method according to claim 136 wherein the agent is a fragment as defined in any

one of claims 17 to 31 or a homolog, analog, variant or derivative thereof.

150. A method according to claim 146 wherein the agent is an agent as defined in any

one of claims 32 to 51.

151. A method according to any one of claims 146 to 150 wherein the MAP kinase is an

ERK or a JNK family member.

152. A method according to claim 151 wherein the MAP kinase is ERK2.

153. A method according to any one of claims 146 to 152 wherein the integrin

comprises β6.

154. A method according to any one of claims 146 to 1 53 wherein the activity of the

cell is growth of the cell.