WO2010094085A1 - Inhibition de voies multiples d'activation cellulaire - Google Patents

Inhibition de voies multiples d'activation cellulaire Download PDF

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WO2010094085A1
WO2010094085A1 PCT/AU2010/000203 AU2010000203W WO2010094085A1 WO 2010094085 A1 WO2010094085 A1 WO 2010094085A1 AU 2010000203 W AU2010000203 W AU 2010000203W WO 2010094085 A1 WO2010094085 A1 WO 2010094085A1
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polypeptide
binding domain
kinase
cell
cancer
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PCT/AU2010/000203
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English (en)
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Michael Valentine Agrez
Douglas Dorahy
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Inter-K Pty Limited
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Priority claimed from AU2009900762A external-priority patent/AU2009900762A0/en
Application filed by Inter-K Pty Limited filed Critical Inter-K Pty Limited
Priority to US13/202,843 priority Critical patent/US20120277161A1/en
Priority to AU2010215086A priority patent/AU2010215086A1/en
Priority to EP10743341.9A priority patent/EP2398484A4/fr
Priority to JP2011550386A priority patent/JP2012518602A/ja
Publication of WO2010094085A1 publication Critical patent/WO2010094085A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/10—Peptides having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the invention relates to inhibition of the growth and/or proliferation of cancer cells.
  • tumour cell migration 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.
  • 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 cancer cells are involved in complex cell signalling which may regulate cell proliferation, migration, invasion and metastasis and several families of adhesion molecules that contribute to these events have now been identified including integrins, cadherins, the immunoglobulin superfamily, hyaluronate receptors, and mucins. 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.
  • 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 ( ⁇ ) and beta ( ⁇ ) chain in close association that provide a structural and functional bridge between extracellular matrix molecules and cytoskeletal components with the cell.
  • the integrin family comprises 17 different ⁇ and 8 ⁇ subunits, and the ⁇ combinations are subsumed under 3 subfamilies.
  • the remaining integrins are arranged into two major subgroups, designated ⁇ 1 and ⁇ v based on sharing common chains.
  • the ⁇ 1 chain combines with any one of nine ⁇ chain members ( ⁇ l-9), and the ⁇ chain which associates with ⁇ 1 determines the matrix- binding specificity of that receptor.
  • ⁇ 2 ⁇ l binds collagen and laminin
  • ⁇ 3 ⁇ 1 binds collagen
  • ⁇ 5 ⁇ 1 binds fibronectin.
  • the abundant and promiscuous ⁇ v chain combines with any one of five ⁇ chains, and a distinguishing feature of ⁇ v integrins is that they all recognise and bind with high affinity to arginine-glycine-aspartate (RGD) (SEQ ID. No.
  • cysteines are arranged in four repeating patterns which are thought to be linked internally by disulphide bonds.
  • the ⁇ -subunits have a molecular mass ranging from 150 - 200 kDa. They exhibit a lower degree of similarity than the ⁇ chains, although all contain seven repeating amino acid sequences interspaced with non-repeating domains.
  • the ⁇ subunit cytoplasmic domain is required for linking integrins to the cytoskeleton. 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 ⁇ - actinin, talin, and focal adhesion kinase (FAK). At least three different regions that are required for focal contact localisation of ⁇ 1 integrins have been delineated (Reszka et al, 1992). These regions contain conserved sequences that are also found in the cytoplasmic domains of the ⁇ 2, ⁇ 3, ⁇ 5, ⁇ 6 and ⁇ 7 integrin subunits. The functional differences between these cytoplasmic domains with regard to their signalling capacity have not yet been established.
  • the integrin ⁇ 6 subunit was first identified in cultured epithelial cells as part of the ⁇ v ⁇ 6 heterodimer, and the ⁇ v ⁇ 6 complex was shown to bind fibronectin in an arginine-glycine-aspartate (RGD)-dependent manner in human pancreatic carcinoma cells (Sheppard et al, 1990).
  • the ⁇ 6 subunit is composed of 788 amino acids and shares 34 - 51% sequence homology with other integrin subunits ⁇ l - ⁇ 5.
  • the ⁇ 6 subunit also contains 9 potential glycosylation sites on the extracellular domain (Sheppard et al, 1990).
  • the cytoplasmic domain differs from other subunits in that it is composed of a 41 amino acid region that is highly conserved among integrin 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 ⁇ 6 to focal contacts. In fact, its removal appears to increase receptor localisation. However, removal of any of the three conserved regions identified as important for the localisation of ⁇ l integrins to focal contacts (Reszka et al, 1992) has been shown to eliminate recruitment of ⁇ 6 to focal contacts (Cone et al, 1994).
  • the integrin ⁇ v ⁇ 6 has previously been shown to enhance growth of colon cancer cells in vitro and in vivo (Agrez et al, 1994), and this growth -enhancing effect is due, at least in part, to ⁇ v ⁇ 6 mediated gelatinase B secretion (Agrez et al, 1999).
  • This epithelial-restricted integrin of particular interest in cancer is that it is either not expressed or expressed at very low levels on normal epithelial cells, but is highly expressed during wound healing and tumourigenesis, particularly at the invading edge of tumour cell islands (Breuss et al, 1995; Agrez et al, 1996). 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 cytoplasmic domains (inside-out signalling), while the binding of the extracellular matrix (ECM) elicits signals that are transmitted into the cell (outside- in signalling).
  • 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.
  • 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 (P 1 P 2 ) synthesis).
  • the second category of integrin signalling is 'collaborative signalling', in which integrin-mediated cell adhesion modulates signalling events initiated through other types of receptors, particularly receptor tyrosine kinases that are activated by polypeptide growth factors.
  • 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).
  • MEKs MAP kinase kinases
  • ERKs extracellular signal-regulated kinases
  • ERK 1/2 44kD and 42kD MAPks, respectively
  • serine/threonine kinases phosphorylate and modulate the function of many proteins with regulatory functions including other protein kinases (such as p90 rsk ) cytoskeletal proteins (such as microtubule-associated phospholipase A 2 ), upstream regulators (such as the epidermal growth factor receptor and Ras exchange factor) and transcription factors (such as c- myc and EIk-I).
  • ERKs play a major role in growth -promoting events, especially when the concentration of growth factors available to a cell is limited (Giancotti and
  • PD kinases can be activated by interaction with the Ras pro to- oncogene, it can be activated independently of Ras involvement, and PDK activity alone is sufficient to promote cellular survival in the absence of trophic support and to block apoptosis induced by toxic stimuli.
  • PDK activity provides a parallel cell survival/activation pathway emanating from receptor tyrosine kinases.
  • Survival stimuli generally mediate intracellular signalling through ligation of transmembrane receptors which either possess intrinsic tyrosine kinase activity, are indirectly coupled to tyrosine kinases, or are coupled to seven transmembrane g-protein coupled receptors. Activation of these receptors results in the recruitment of PDK isoforms to the inner surface of the plasma membrane as a result of ligand-regulated protein- protein interactions (Datta et al, 1999).
  • the family of PDKs is divided into several subgroups of which the Class I enzymes consists of the p85 adaptor subunit complexed with one of four pi 10 catalytic subunits (alpha, beta, delta, or gamma) and is capable of associating with receptor tyrosine kinases and oncoproteins (Zhao & Roberts, 2006). While PDK gamma and delta are mainly expressed in haematopoietic tissues, alpha and beta are ubiquitously expressed.
  • Akt was first implicated in signal transduction by the demonstration that the kinase activity of Akt is induced by growth factors such as basic fibroblast growth factor and PDGF. It is now known that a diverse array of physiological stimuli can induce Akt activity primarily in a PD Kinase-dependent manner. In turn, Akt regulates survival through the phosphorylation of multiple substrates involved in the regulation of apoptosis, for example, through phosphorylation of the Bcl-2 homolog Bad and caspase-9 (Datta et al, 1999).
  • colon cancers respond less well to the new anti-Akt compound, GK690693 in animal models than, for example, breast cancer which has a much higher frequency of PD Kinase/Akt mutations.
  • Colon cancers have mutations of these kinase in at least 20% of tumours and a much higher incidence of mutation rates for Ras/Raf/BRaf.
  • mutations of pi 10 alpha isoform of the PD Kinase subfamily is very common in cervical, breast and colon cancer (P. Workman, presentation at the HRMI Cancer Conference, Newcastle, NSW, September, 2008).
  • PDK beta has also been shown to be required for de novo DNA synthesis in colon cancer cells (Benistant et al, 2000). Importantly, pi 10 alpha also functions in insulin signalling, whereas inhibition of pi 10 beta appears not to affect insulin signalling (Zhao & Roberts, 2006) making PDK beta an attractive target.
  • Src kinases are cytoplasmic, membrane associated, non-receptor intracellular tyrosine kinases that mediate a variety of intracellular signalling pathways. They are cellular homologs of the products of the Rous sarcoma virus gene (v-Src), which is the mutated and activated version of a normal cellular gene (c-Src).
  • Src, Fyn, and Yes are ubiquitously expressed
  • Lck Hck, Fgr, Lyn and BIk have more tissue-restricted expression mainly in hematopoietic cells (Abram CL & Courtneidge SA, 2000).
  • the remaining Src family member is Frk which is in its own subfamily.
  • Src tyrosine kinases are known to be over expressed in a variety of tumour types, such as human colon adenocarcinoma (Windam TC et al, 2002; Haier J et al, 2002), breast cancer (Myoui A et al, 2003; Lu Y et al, 2003), pancreatic carcinoma (Lutz MP et al, 1998), and ovarian cancer (Budd RJ et al, 1994; Weiner JR et al, 2003).
  • Src family members are involved in numerous signalling pathways involved in proliferation, migration, tumour adhesion, and angiogenesis (Sato M et al, 2002) and mediate signalling from many types of receptors including receptor tyrosine kinases (RTKs), integrins, and G-protein-coupled receptors (Haier J et al, 2002).
  • RTKs receptor tyrosine kinases
  • integrins integrins
  • G-protein-coupled receptors Heaier J et al, 2002.
  • RTKs that signal through Src kinases include platelet-derived growth factor receptors (PDGFRs), epidermal growth factor receptors (EGFRs), and fibroblast growth factor receptors (Browaeys-Poly E et al, 2000).
  • the Src family also appears to be required for growth factor- simulated DNA synthesis, particularly for growth factors with RTKs such as platelet-derived growth factor receptor and EGFR (Nanjundan M et al, 2003; Erpel T, 1996).
  • c-Src tyrosine kinase is the prototypical member of the Src family, and is involved in a variety of cell signalling events, regulating both cell proliferation and differentiation. Inhibition of c-Src is associated with decreased activation of cell growth and survival pathways.
  • Src family kinases are required for the endomembrane activation of the Ras-MAPK pathway, where they phosphorylate and activate PLC- ⁇ l . PLC- ⁇ l then activates RasGRPl, Ras guanine nucleotide exchange factor, thereby promoting Ras activation.
  • c-Src Increased specific activity of c-Src is observed in >80% of colon adenocarcinomas relative to normal colonic mucosa (Bolen JB et al, 1987). Further increases in c-Src are seen in metastases relative to primary tumours. Thus, in the majority of colon tumour cells, c-Src is constitutively active. Recently, a subset of human colon tumours has been found to contain an activating mutation in the c-Src gene (Irby RB et al, 1999), although such mutations were not observed in other patient populations.
  • c-Src activity increases at progressive stages of the disease (Talamonti MS et al, 1993; Termulen PM et al, 1993) and is predictive of poor clinical prognosis (Allgayer H et al, 2002) suggesting that c-Src activation confers growth and/or survival advantages to metastatic colon tumour cells. Regardless of the mechanism of activation, there is substantial evidence suggesting that c-Src activation contributes to increased tumourigenicity of human colon cancer cell lines.
  • c-Src activation may contribute to colon tumour progression and metastasis in part by activating Akt-mediated survival pathways that decrease sensitivity of detached cells to anoikis (Windham TC et al, 2002).
  • Akt-mediated survival pathways that decrease sensitivity of detached cells to anoikis
  • PKC delta -mediated signaling by the relatively specific PKC delta inhibitor, rottlerin, has been shown to block most of the estrogen-induced ERK activation (Venkateshwar et al, 2002) highlighting the importance of signaling "cross-talk" in cancer cells.
  • the PKC family consists of a number of serine-threonine kinases that are divided into three groups based on their activating factors. PKCs have been linked to carcinogenesis since PKC activators can act as tumor promoters and activation of the pKC alpha and beta isoenzymes ( ⁇ l and ⁇ 2) have often been linked to the malignant phenotype.
  • PKC over-expression has been shown to stimulate Akt activity and suppress apoptosis induced by interleukin 3 withdrawal in myeloid cells (Weiqun L et al, 1999).
  • Akt activity has been shown to stimulate Akt activity and suppress apoptosis induced by interleukin 3 withdrawal in myeloid cells (Weiqun L et al, 1999).
  • PD Kinase inhibition suppressed PKC -mediated activation of Akt.
  • PKCs have, for instance, been reported to modulate the Inhibitor of Apoptosis Protein family (IAPs) that bind and potently inhibit the proteolytic activities of the pro-apoptotic caspases 3, 6 and 7 implicated in many different types of cancer including those with the highest mortality rates.
  • IAPs Apoptosis Protein family
  • PMA phorbol myristate acetate induced IAP expression appears to be a general feature of colon cancer cells and it has been shown (Wang Q et al) that PMA increases PKC delta activity, and blocking this enzyme prevents PMA from increasing IAP expression in colon cancer cells demonstrating a role for PKC-dependent signaling in prevention of apoptosis in human colon cancer cells.
  • the PKC beta isoforms (beta I and beta II) have also been reported to be an effective target for chemoprevention of colon cancer, and inhibition of PKC beta prevents invasion by rat intestinal epithelial cells mediated via activation of MEK signaling (Zhang J, 2004).
  • PKC betall has been shown to significantly reduce both tumor initiation in a colon cancer mouse model and tumor progression by inhibiting expression of pro-proliferative genes (Fields AP et al, 2009).
  • PKC betall has also been implicated in proliferation of the intestinal epithelium. For example, evidence has been provided for a direct role for PKC betall in colonic epithelial cell proliferation and colon carcinogenesis, possibly through activation of the APC/beta catenin signaling pathway (Murray NR et al, 1999).
  • EGF-over-expressing invasive cancer cells have the ability to compensate for the loss of MAPK-mediated signaling through activation of PKC delta signaling for cell migration, which plays a major role in invasion and metastases (Kruger JS & Reddy KB, 2003). Those investigators have suggested that inhibition of MAPK and PKC delta signaling pathways should abrogate cell migration and invasion in EGFR-over- expressing human breast cancer cells.
  • Akt/PKB pathway functions as a cardinal nodal point for transducing extracellular (growth factor and insulin) and intracellular (receptor tyrosine kinases, Ras and Src) oncogenic signals.
  • extracellular (growth factor and insulin) and intracellular (receptor tyrosine kinases, Ras and Src) oncogenic signals ectopic expression of Akt, especially constitutively activated akt, is sufficient to induce oncogenic transformation of cells and tumor formation in transgenic mice as well as chemoresistance (Cheng JQ et al, 2005).
  • Activated Akt is detectable and a poor prognostic factor for many types of cancer (reviewed in Targeting Akt in Cancer: Promise, Progress, and Potential Pitfalls: Dennis PA, AACR Education Book, 2008: 25-35).
  • Akt2 PKB beta
  • the RAF serine/threonine family is composed of A-RAF, B-RAF and C-RAF (RAF-I).
  • RAF-I C-RAF
  • c-Raf mutations are rare due to its low basal activity.
  • pan-specific RAF agents would be more efficacious against melanomas than B-Raf - specific drugs (Sebolt- Leopold JS, 2008).
  • MAPK pathway inhibitors e.g., anti-MEK
  • RAS signals through multiple effectors, not just RAF. Consequently, activation by RAS of the PI3 Kinase /Akt survival signaling pathway may erode the therapeutic gain derived from shutting off MAPK activation in at least some tumours (Sebolt-Leopold JS, 2008).
  • the invention relates to the finding that an anti -cancer polypeptide providing a binding domain of a ⁇ integrin subunit for an extracellular signal-regulated kinase (ERK) of the mitogen activated protein (MAP) kinase family can inhibit the activity of protein kinase enzymes other than in the MAP kinase family, that are involved in a number of different cell activation pathways.
  • This startling finding provides for the inhibition of multiple activation pathways in a cancer cell with a single therapeutic agent and thereby, the inhibition of cross-signalling or "cross-talk" between the pathways for the prophylaxis or treatment of cancer. More particularly, this finding provides for the inhibition of growth and proliferation of cancer cells that are mediated by aberrant or up- regulated activity of one or more cell activation pathways besides the
  • Ras/Raf/MEK/MAPK pathway The use of a polypeptide inhibitor of an ERK MAP kinase to inhibit the activity of a different class of kinase and particularly one involved in a cell activation pathway other than the Ras/Raf/MEK/MAPK pathway is entirely counter-intuitive, and represents a significant advance in the art.
  • the invention in one or more forms relates to a method for inhibiting a plurality of cell activation pathways in a cancer cell, comprising treating the cancer cell with an effective amount of a polypeptide providing a MAP kinase cytoplasmic binding domain of a ⁇ integrin subunit for binding of ERK2, or a variant or modified form of the binding domain, to which ERK2 binds.
  • a method for inhibiting growth and/or proliferation of a cancer cell comprising: selecting an inhibitor for inhibiting at least one protein kinase in at least one cell activation pathway of the cancer cell other than a MAP kinase, the inhibitor being a polypeptide providing a MAP kinase cytoplasmic binding domain of a ⁇ integrin subunit for binding of ERK2, or a variant or modified form of the binding domain, to which ERK2 binds; and treating the cancer cells with an effective amount of the polypeptide to inhibit the protein kinase.
  • the protein kinase(s) inhibited by the polypeptide can be selected from the group consisting of kinases in the Src, PDK, Protein kinase B (PKB/AKT), and Protein kinase C (PKC) families.
  • PKA Protein kinase B
  • PKC Protein kinase C
  • c-Raf and MEKl can also be inhibited by a polypeptide providing a MAP kinase cytoplasmic binding domain of a ⁇ integrin subunit for binding of ERK2.
  • a method for inhibiting activity of at least one protein kinase comprising: selecting an inhibitor for inhibiting the protein kinase, the inhibitor being a polypeptide providing a MAP kinase cytoplasmic binding domain of a ⁇ integrin subunit for binding of ERK2, or a variant or modified form of the binding domain, to which ERK2 binds; and contacting the target kinase with an effective amount of the polypeptide to inhibit the protein kinase, the protein kinase being selected from the group consisting of c-Raf, MEKl and kinases in the Src, PDK, Protein kinase B (PKB/AKT), and Protein kinase C (PKC) families.
  • the inhibitor being a polypeptide providing a MAP kinase cytoplasmic binding domain of a ⁇ integrin subunit for binding of ERK2, or a variant or modified form of the binding domain, to which ERK
  • a method for inhibiting the activity of at least one protein kinase comprising contacting the protein kinase with a polypeptide providing a MAP kinase cytoplasmic binding domain of a ⁇ integrin subunit for binding of ERK2, or a variant or modified form of the binding domain, to which ERK2 binds, the protein kinase being selected from the group consisting of c- RAF, MEKl, and kinases in the Src, PDK, PKB and PKC families.
  • a method for inhibiting a plurality of cell activation pathways in a cancer cell comprising treating the cancer cell with an effective amount of at least one polypeptide providing a MAP kinase cytoplasmic binding domain of a ⁇ integrin subunit for binding of ERK2, or a variant or modified form of the binding domain, to which ERK2 binds.
  • the cancer cell(s) can be treated with the polypeptide or a nucleic acid for expression of the polypeptide within the cells for effecting the treatment of the cells.
  • the polypeptide or nucleic acid can be presented by a dendrimer or coupled to another form of facilitator moiety for facilitating passage of the polypeptide or nucleic acid into the cytoplasm of the cancer cell, and all such embodiments are expressly encompassed by the invention.
  • a method for prophylaxis or treatment of cancer in a mammal comprising administering to the mammal an effective amount of at least one dendrimer for inhibiting a plurality of cell activation pathways in cancer cells of the cancer, the dendrimer presenting at least one polypeptide providing a MAP kinase cytoplasmic binding domain of a ⁇ integrin subunit for binding of ERK2, or a variant or modified form of the binding domain, to which ERK2 binds.
  • the dendrimer and/or polypeptide is administered to inhibit the activity of at least two different protein kinases for inhibition of at least two activation pathways in the cancer cell(s).
  • the dendrimer When a dendrimer is administered the dendrimer will typically present more than 8 monomer units of the polypeptide.
  • the binding domain of the ⁇ integrin subunit incorporates an intervening amino acid linker sequence that links opposite end regions of the binding domain together wherein the linker sequence is not essential for the binding of ERK2.
  • one or more amino acids of the amino acid linker sequence may be deleted and/or differ in the polypeptide compared to the binding domain of the ⁇ integrin subunit.
  • all of the amino acids in the intervening amino acid sequence are deleted in the polypeptide compared to the binding domain.
  • the opposite end regions of the binding domain are defined by respective amino acid sequences, and typically, the amino acid sequence identity of the opposite end regions of the binding domain are unchanged in the polypeptide compared to the binding domain.
  • the ⁇ integrin subunit is expressed by the cancer cells of the cancer.
  • the cancer cells essentially do not express the ⁇ integrin subunit.
  • the cancer cells are treated with the dendrimer or polypeptide to inhibit at least one kinase in a cell activation pathway in the cancer cells other than, or besides, the Ras/Raf/MEK/MAPK pathway.
  • the cells are treated with the dendrimer or polypeptide to inhibit the Ras/Raf/MEK/ERK activation pathway and at least one other cell activation pathway in the cells.
  • the cells are treated with the dendrimer or polypeptide to inhibit one or more cell activation pathways selected from the group consisting of the PD kinase/ Akt and PI3 kinase/ Akt/mTOR pathways, and cell activation pathways involving one or more kinases in the Src, PKB/AKT and/or PKC kinase families.
  • the Src kinase(s) inhibited by the dendrimer or polypeptide can be one or more kinases selected from the group c-Src, c-Lyn, c-Yes and c-Fyn.
  • PKB is also known as the AKT protein kinase family, and the PKB kinase(s) inhibited by the polypeptide can be one or more kinases selected from the group consisting of PKB alpha (AKTl), PKB beta (AKT2), and PKB gamma (AKT3).
  • the PKC kinase(s) inhibited by the polypeptide can be one or more kinases selected from the group consisting of PKC alpha, PKC beta I, PKC beta II, PKC gamma and PKC delta.
  • the PDK may be selected from the group of PD kinases consisting of the adaptor subunit (e.g., p85) complexed with a catalytic subunit (e.g., pi 10 alpha, beta, delta or gamma). In some embodiments, a mixture of these kinases may be inhibited by a method as described herein.
  • the adaptor subunit e.g., p85
  • a catalytic subunit e.g., pi 10 alpha, beta, delta or gamma
  • a mixture of these kinases may be inhibited by a method as described herein.
  • the polypeptide will comprise, or consist of, an amino acid sequence selected from the group consisiting of RSKAKWQTGTNPLYR (SEQ ID No: 2), RARAKWDTANNPLYK (SEQ ID No: 3), RSRARYEMASNPLYR (SEQ ID No: 4), KEKLKS QWNNDNPLFK (SEQ ID No: 5), RSKAKNPLYR (SEQ ID No: 6), RARAKNPLYK (SEQ ID No: 7), RSRARNPLYR (SEQ ID No: 8), and KEKLKNPLFK (SEQ ID NO: 9).
  • RSKAKWQTGTNPLYR SEQ ID No: 2
  • RARAKWDTANNPLYK SEQ ID No: 3
  • RSRARYEMASNPLYR SEQ ID No: 4
  • KEKLKS QWNNDNPLFK SEQ ID No: 5
  • RSKAKNPLYR SEQ ID No: 6
  • RARAKNPLYK SEQ ID No: 7
  • the ⁇ integrin subunit will normally be selected from the group consisting of ⁇ 2, ⁇ 3, ⁇ 5, and ⁇ 6, and most usually, will be ⁇ 6.
  • binding domain of the ⁇ integrin subunit (or a variant or modified form of the binding domain) can be incorporated in a fusion protein, and the invention expressly extends to the use of such fusion proteins in a method embodied by the invention, whether presented in a dendrimer or not.
  • the dendrimer can be any type suitable for use in a method embodied by the invention.
  • the dendrimer may, for example, have branched organic framework to which the binding domain (or modified or variant form thereof) is coupled, such as framework formed by poly (amidoamine) (PAMAM), tris(ethylene amine) ammonia or poly (propylene imine) (AstramolTM).
  • PAMAM poly (amidoamine)
  • AstramolTM poly (propylene imine)
  • the dendrimer can have framework incorporating polyamino acids forming branching units to which the peptide is coupled.
  • the dendrimer has a framework of branching units formed by polyamino acids.
  • the dendrimer will have a plurality of layers/generations of polyamino acid branching units to which the peptide is coupled.
  • the polyamino acid branching units are normally formed by lysine residues.
  • the respective units of the peptide presented by the dendrimer can provide the same or different binding domains (or variant forms thereof) of ⁇ integrin subunits to which ERK2 binds.
  • the dendrimer will present monomers of the peptide(s).
  • the dendrimer can also have a core from which the branching framework of the dendrimer extends.
  • cancer any type of malignant, unregulated cell proliferation.
  • the cancer can be selected from the group consisting of, but is not limited to, epithelial cell cancers, sarcomas, lymphomas and blood cell cancers, including leukemias such as myeloid leukemias, eosinophilic leukemias and granulocytic leukemias.
  • leukemias such as myeloid leukemias, eosinophilic leukemias and granulocytic leukemias.
  • the ⁇ subunit of the integrin may be ⁇ 2 the expression of which is restricted to white blood cells (Hynes et al, 1992).
  • a polypeptide providing a MAP kinase cytoplasmic binding domain of a ⁇ integrin subunit for binding of ERK2 to inhibit at least one target protein kinase in at least one cell activation pathway other than a MAP kinase, and thereby inhibit growth and/or proliferation of a cancer cell, or a variant or modified form of the binding domain, to which ERK2 binds, or a nucleic acid for expression of the polypeptide or the modified or variant form thereof in the cancer cell.
  • a polypeptide providing a MAP kinase cytoplasmic MAP kinase binding domain of a ⁇ integrin subunit for binding of ERK2 in the manufacture of a medicament for inhibiting at least one target protein kinase in at least one cell activation pathway other than a MAP kinase to inhibit growth and/or proliferation of a cancer cell, or a variant or modified form of the binding domain, to which ERK2 binds, or a nucleic acid for expression of the polypeptide or the modified or variant form thereof in the cancer cell.
  • the mammal can be any mammal treatable with a method of the invention.
  • 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, or a primate or human being.
  • the mammal will be a human being.
  • Figure 1 is a diagram illustrating cell activation pathways.
  • Figure 2 is a schematic illustration of peptide dendrimer.
  • Figure 3 (a) Shows a schematic illustration of a multiple antigen peptide dendrimer (MAP), incorporating eight peptide monomers, (b) An increase in the number of Lys branching units increases the number of surface amine groups.
  • MAP multiple antigen peptide dendrimer
  • Lys branching units increases the number of surface amine groups.
  • FIG 4 is a schematic illustration of a peptide dendrimer presenting 10 peptide monomers of the peptide RSKAKNPLYR (SEQ ID NO: 6) (referred to herein as dendrimer Dend 10- 10(4)) .
  • Figure 5 is a graph showing dose response inhibition of c-Src tyrosine kinase activity by peptide RSKAKNPLYR (SEQ ID No: 4) in a cell-free assay.
  • Figure 6 is a graph showing the efficacy of cisplatin and peptide AAVALLPAVLLALLARSKAKNPLYR (SEQ ID NO: 10) (IK2) alone and in combination against chemotherapeutic drug-resistant ADDP human ovarian carcinoma cells compared to A2780 ovarian cancer cells treated with cisplatin alone.
  • Figures 7 (A) and (B) are graphs showing effect of oxaliplatin in combination with peptide AAVALLPAVLLALLARSKAKNPLYR (SEQ ID No: 10) (IK2) against ADDP human ovarian cancer cells.
  • Figure 8 is a graph showing synergy between cisplatin and the peptide
  • AAVALLPAVLLALLARSKAKNPLYR SEQ ID No: 10
  • IK2 ADDP human ovarian cancer cells.
  • Figure 9 is a graph showing synergy between cisplatin and the peptide AAVALLPAVLLALLARSKAKNPLYR (SEQ ID No: 10) (IK2) against HT29 human colon cancer cells.
  • Figure 10 is a graph showing inhibition of ERK activity in HT29 colon cancer cells in a dose dependent manner by the peptide dendrimer Dend 8-10(4) presenting 8 monomer units of the peptide RSKAKNPLYR (SEQ ID No. 6).
  • Figure 11 is a graph showing induction of apoptosis in human colon cancer cells by a peptide dendrimer presenting 10 monomer units of the peptide
  • RSKAKNPLYR (SEQ ID No. 6) in which the peptide is comprised entirely of D amino acids and is pegylated (dendrimer Dend 10-10(4)DP).
  • Figure 12 is a graph showing inhibition of proliferation of HT29 colon cancer cells by the dendrimer Dend 10-10(4) presenting 10 monomer units of the peptide RSKAKNPLYR (SEQ ID NO. 6).
  • Figure 13 is a graph showing the effect of dendrimers Dend 9-10(4) and Dend 12-10(4) (presenting 9 and 12 monomers of the peptide RSKAKNPLYR (SEQ ID No. 6), respectively) on proliferation of HT29 human colon cancer cells cultured for 48 hours.
  • Figure 14 is a graph showing the efficacy of the dendrimer
  • Dend 10-10(4)DP (identified as Mod. IK248) in inhibiting proliferation of HT29 colon cancer cells compared to cisplatin, irinotecan (CPT-11) and 5-fluorouracil (5FU).
  • Figure 15 is a graph showing treatment of HT29 colon cancer cells with peptide dendrimer presenting 8 monomer units of the peptide RARAKNPLYK (SEQ ID No. 7) (Dend8- ⁇ 3) (solid squares) or 8 monomers of peptide RSRARNPLYR (SEQ ID No. 8) (Dend8- ⁇ 5) (solid diamonds).
  • Figure 16 is a graph showing inhibition of HT29 colon cancer tumour growth in a BALB/c mouse model by the dendrimer Dend 10-10(4) (identified as IK248) (solid squares) compared to a vehicle only control (solid diamonds) when injected intra- tumorally.
  • a peptide providing a MAP kinase cytoplasmic binding domain of a ⁇ integrin subunit for binding of ERK2 besides being an inhibitor of the ERK MAP kinase is also an inhibitor of protein kinases in the Src and PI3K families as well as other protein kinases, including but not limited to c-RAF, MEKl, and kinases in the PKB (e.g., PKB alpha, PKB beta and PKB gamma) and protein kinase C (PKC) (e.g., PKC alpha, PKC beta I, PKC beta II, and PKC delta) families.
  • PKB e.g., PKB alpha, PKB beta and PKB gamma
  • PKC protein kinase C
  • a polypeptide used in a method as described herein can provide the MAP kinase binding domain of the ⁇ -integrin subunit for binding of ERK2, or vary from the binding domain by one or more amino acids.
  • the polypeptide may also, or alternatively, differ by one or more amino acids from one or both regions of the ⁇ -integrin subunit that flank the binding domain .
  • binding domain is meant the minimum length of contiguous amino acid sequence of the ⁇ -integrin subunit required for binding of the MAP kinase substantially without compromising the optimum level of binding with the MAP kinase (e.g., ERK1/2). Moreover, the term “binding domain” includes those binding domains encoded by naturally occurring mutant and polymorphic alleles.
  • variable form of the binding domain is meant an amino acid sequence that differs from the binding domain by one or more amino acids essentially without adversely effecting binding by the MAP kinase, and includes isolated or purified naturally occuring such sequences.
  • modified form is meant an amino acid sequence in which the binding domain has been modified by one or more amino acid changes essentially without adversely affecting the binding by the MAP kinase.
  • MAP kinase as used herein is meant a member of the mitogen activated protein kinase family (e.g., ERKl, ERK2, JNK and p38 isoforms) and excludes MAP kinase kinases and MAP kinase kinase kinase enzymes.
  • Variant and modified forms of the binding domain include derivatives and peptidomimetics of the binding domain.
  • a variant or modified form of the binding domain will generally include 2 or or more charged amino acid residuess (each independently positively or negatively charged) and typically, a minimum of 3 positively charged amino acids (e.g., His, Lys, and/or Arg).
  • the polypeptide is a direct inhibitor of the protein kinase(s). That is, the polypeptide can inhibit the activity of the kinase(s) via the direct interaction of the polypeptide with the kinase(s).
  • the binding domain will have opposite end regions that are linked together by a number of contiguous intervening amino acids (i.e., an amino acid linker sequence) which are not essential for binding of ERK2 and can be deleted.
  • an amino acid linker sequence i.e., an amino acid linker sequence
  • a polypeptide useful in a dendrimer or method embodied by the invention as described herein can be achieved by the addition, deletion and/or the substitution of one or more amino acids of the binding domain 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.
  • one or more amino acids of the non-essential intervening amino acid linker sequence of the binding domain can be deleted or substituted for another amino acid or amino acids, (e.g., conservative amino acid substitution(s)).
  • modified polypeptides can be prepared by introducing nucleotide changes in a nucleic 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 amino acid sequence incorporating the desired amino acid changes, which possibilities are well within the capability of the skilled addressee.
  • a modified binding domain or polypeptide as described herein can incorporate an amino acid or amino acids not encoded by the genetic code, or amino acid analog(s).
  • D- amino acids rather than L-amino acids can be utilised.
  • a peptide useful in an embodiment of the invention may consist partly or entirely of D amino acids.
  • D-peptides can be produced by chemical synthesis using techniques that are well-known in the art. Accordingly, in some embodiments, the peptide(s) may include L-amino acids, D-amino acids or a mixture of L- and D-amino acids.
  • peptides including D-amino acids can inhibit peptidase activity (e.g., endopeptidase) and thereby enhance stability and increase the half -life of the peptide in vivo compared to the corresponding L-peptide.
  • peptidase activity e.g., endopeptidase
  • the N -terminal or C-terminal ends of the polypeptides/peptides can be modified to protect against or inhibit in vivo degradation (e.g., by peptidases).
  • the C-terminus of the polypeptides can be amidated to protect against peptidase degradation.
  • the N- or C-terminal end of a polypeptide as described herein can also be pegylated with a plurality of ethylene glycol monomer units to render it less resistant to degradation by proteases in vivo or to inhibit their clearance from the circulation via the kidneys. Methods for pegylation of polypeptides/peptides are well known in the art and all such methods are expressly encompassed.
  • a pegylated polypeptide used in a method embodied by the invention will be coupled to 2 or more monomer units of polyethylene glycol (PEG) and generally, from about 2 to about 11 monomers of PEG (i.e., (PEG)n where n equals from 2 to 11). Most usually, n will be 2.
  • Substitution of an amino acid may involve a conservative or non-conservative amino acid substitution.
  • conservative amino acid substitution is meant replacing an amino acid residue with another amino acid having similar stereochemical properties (e.g., structure, charge, acidity or basicity characteristics) and which does not substantially adversely effect the binding activity of the binding domain.
  • a polar amino acid may be substituted with another polar amino acid, conservative amino acids changes being well known to the skilled addressee.
  • sequence identity between amino acid sequences as described herein can be determined by comparing amino acids at each position in the sequences when the sequences are optimally aligned for the purpose of comparison.
  • the sequences are considered the same at a position if the amino acids at that position are the same amino acid residue.
  • Alignment of sequences can be performed using any suitable program or algorithm such as for instance, by the Needleman and Wunsch algorithm (Needleman and Wunsch, 1970).
  • Computer assisted sequence alignment can be conveniently performed using standard software programs such as GAP which is part of the
  • a polypeptide useful in a dendrimer as described herein will have an overall amino acid sequence identity with the ⁇ integrin subunit of at least about 40% and more usually, at least about 50%, 60%, or 70% or greater and most preferably, at least about 80%, 90% or 95% sequence identity or greater.
  • the sequence identity with the binding domain of the ⁇ integrin subunit may be greater than the overall amino acid sequence identity between the two sequences, and will usually be at least about 60%, 70% or 80% or greater, and more usually will be at least about 90%, or 95% or greater.
  • the overall sequence identity of the polypeptide, or the sequence identity of the polypeptide with the binding domain can be any specific value or range within the particular values specified above.
  • the amino acid sequence identity of the polypeptide may be at least 66% or 75% or greater, and all such sequence identities and ranges are expressly encompassed by the invention.
  • a derivative of a polypeptide useful in a method embodied by the invention may be provided by cleavage cyclisation and/or coupling of the parent molecule 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.
  • polypeptide is used interchangeably herein with peptide.
  • peptide agents such as RSKAKNPL YR (SEQ ID No: 6) and KEKLKNPLFK (SEQ ID No: 9) fall within the scope of the term polypeptide.
  • Peptide dendrimers are particularly suitable for use in methods of the invention.
  • Peptide dendrimers in at least some embodiments of the invention present units of the polypeptide inibitor coupled to a branched framework of polyamino acids (typically lysine branching units).
  • the dendrimer will typically have at least 3 layers/generations of amino acid branching units, the units of the polypeptide inhibitor being coupled to the outermost layer/generation of the amino acid branching units such that the dendrimer presents more than 8 units of the polypeptide.
  • momoner units of the polypeptide are preferred, in other embodiments, dendrimers incorporating multiple units of the polypeptide (multimers) (e.g., (RSKAKNPLYR)n ((SEQ ID No.
  • n is the number of repeats of the polypeptide (typically 1 -3)) coupled to polyamino acid branching units of the dendrimer
  • the units of the polypeptide presented by the dendrimer can be monomer units, multimer units and/or mixtures of monomer and multimer units of the polypeptide.
  • the anti-cancer polypeptide can be bonded to the outermost layer/generation of polyamino acid branching units forming the framework of the dendrimer, or be synthetically assembled on the polyamino acid branching units of the dendrimer. More particularly, the synthesis of dendrimers useful in one or more methods embodied by the invention can be achieved by divergent or convergent synthesis strategies.
  • the divergent strategy is a direct approach by which the dendrimer is built stepwise in a continuous operation on a solid support through solid-phase synthesis.
  • Stepwise synthesis involves synthesis of the branching core of the dendrimer followed by synthesis of the polypeptide inhibitor in a continous manner.
  • the divergent strategy is particularly suitable for the synthesis of dendrimers with a framework of a trifunctional acid (e.g., polyamino acid).
  • Such solid phase synthesis schemes are the method of choice for the synthesis of lysine branching units where di-protected lysine is used to produce a branching framework of multiple levels of lysines.
  • the diamino nature of lysine results in each additional level of lysine effectively doubling the number of sites upon which the polypeptide inhibitor may be synthesized directly.
  • the convergent strategy is an indirect, modular approach by which the polypeptide and branching core unit are prepared separately and then coupled together.
  • Core units with branching framework used in the convergent synthesis of dendrimers are commercially available, and are typically formed from organic amino compounds such as poly (amidoamine) (PAMAM), tris(ethylene amine) ammonia or poly (propylene imine) (AstramolTM) to which separately prepared inhibitor is normally covalently linked.
  • PAMAM poly (amidoamine)
  • AstramolTM poly (propylene imine)
  • Suitable peptide dendrimer framework to which a polypeptide as described herein can be coupled and methods for the provision of peptide dendrimers, are for example described in Lee et al, 2005; Sadler and Tarn, 2002; and Cloninger, 2002, the entire contents of which are incorporated herein in their entirety by reference.
  • Examples of peptide dendrimers of the type suitable for use in embodiments of the present invention are schematically illustrated in Fig. 2. and Fig. 3 (Sadler, K., and Tarn, J. P., 2002), and in Fig. 4. Suitable dendrimers are also described in co-pending International
  • a dendrimer used in a method embodied by the invention typically present more than 8 units of the polypeptide (e.g., 9, 10 or 12 units). While the polypeptide units will normally all be the same, mixtures of polypeptides as described herein can also be used. For example, half of the units of the polypeptide can provide the binding domain of the ⁇ 6 integrin subunit for the ERK MAP kinase while the remaining units of the polypeptide present the binding domain of the ⁇ 5 integrin subunit (or variant or modified forms of these binding domains). However, it will be understood that the ratio of the different anti-cancer polypeptide agents can be varied.
  • the peptide(s) presented by a dendrimer used in a method embodied by the invention will also typically be N- or C- terminal protected against proteolytic degradation (e.g., by amidation, pegylation (i.e., the addition of PEG units) or the like). Methods such as pegylation of polypeptides are within the scope of the skilled addressee, and all such methods are expressly encompassed.
  • the polypeptide presented in the dendrimer in accordance with embodiments of the invention will have a length of about 60 amino acids or less.
  • the polypeptide will have a length of more than 5 amino acids and will normally, be up to about 50 amino acids, 40, 35, 30, 25, 20 or 15 amino acids in length.
  • the polypeptide may have a length in a range of from 6, 7, 8, 9 or 10 amino acids up to about 14, 15, 16, 17, 18, 19, 20, or 25 amino acids.
  • polypeptides of all specific lengths and length ranges within those identified above that are suitable for use in a dendrimer as described herein are expressly encompassed (e.g., 13 or 14 amino acids or from 10 to 15, 10 to 20 or 10 to 22 amino acids etc.).
  • the binding domain of the ⁇ integrin subunits ⁇ 2, ⁇ 3, ⁇ 5 and ⁇ 6 for the MAP kinase ERK2 are described in International Patent Application WO 2001/000677, and International Patent Application WO 2002/051993.
  • the binding domain of the ⁇ 2 integrin subunit for ERK2 is described in International Patent Application WO 2005/037308. The disclosures of all of these international patent applications are expressly incorporated herein by reference in their entirety.
  • polypeptide agents for inhibiting the binding of a ⁇ integrin subunit to a MAP kinase and which are suitable for being incorporated into a dendrimer as described herein are also described in those applications, as well as methodology for the localisation and characterization of the binding domains.
  • the binding domain may be localised by assessing the capacity of respective overlapping peptide fragments of the cytoplasmic binding domain of a ⁇ integrin subunit for the ERK MAP kinase.
  • the specific amino acid sequence which constitutes the binding domain may then be determined utilising progressively smaller peptide fragments.
  • test peptides are readily synthesised to a desired length involving deletion of an amino acid or amino acids from one or both of the N-terminal and C-terminal ends of the larger peptide fragment(s), and tested for their ability to bind with the ERK MAP kinase. This process is repeated until the minimum length peptide capable of binding with the ERK MAP kinase substantially without compromising the optimum observed level of binding is identified.
  • amino acids that play an essential role in the ERK MAP kinase - ⁇ integrin interaction may be 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 binding ability of the peptide.
  • substitution mutagenesis will involve substitution of selected ones of the amino acid sequence with alanine or other neutrally charged amino acid.
  • Nucleotide and amino acid sequence data for the ⁇ 6 integrin subunit for example is found in Sheppard et al, 1990.
  • ERKl and ERK2 have high overall amino acid sequence identity, with ERKl having about 96% sequence identity to a 26 mer amino acid sequence of ERK2 providing the binding site for ⁇ 6 (see International Patent Application No. WO 2002/051993.
  • the nucleotide and amino acid sequence for ERK2 is for instance found in Boulton et al, 1991. Reference to such published data allows the ready design of polypeptides useful in the dendrimers described herein and the provision of the corresponding nucleic acid sequences encoding the polypeptides.
  • polypeptide or other agent 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 incorporating cysteine residues which form a disulfide bridge.
  • a polypeptide or other agent may also be cyclised to provide enhanced rigidity and thereby stability in vivo, and various such methods are known in the art.
  • a polypeptide useful in a method embodied by the invention can comprise, or consist of, the binding domain of the ⁇ integrin subunit, or a variant or modified form thereof in which one or more amino acids of the intervening amino acid sequence of the binding domain that are not essential for binding of the MAP kinase are deleted.
  • the binding domain of ⁇ 6 comprises the amino acid sequence RSKAKWQTGTNPLYR (SEQ ID No: 2).
  • the intervening amino acid sequence WQTGT (SEQ ID No: 11) is not essential for binding of the MAP kinase ERK2.
  • a peptide with the amino acid sequence RSKAKNPLYR (SEQ ID No: 6) is still bound by ERK2.
  • the binding domains of ⁇ 2, ⁇ 3 and ⁇ 5 for ERK2 are provided by KEKLKSQWNNDNPLFK (SEQ ID NO. 5), RARAKWDTANNPLYK (SEQ ID No: 3) and RSRARYEMASNPLYR (SEQ ID No: 4), respectively. Deletion of the intervening sequences SQWNND (SEQ ID No.
  • the peptide RSKAKNPLYR (SEQ ID No. 6) has 80% sequence identity with peptide RSRARNPLYR (SEQ ID No: 8) and 70% sequence identity with the RARAKNPLYK (SEQ ID No. 7).
  • the peptide RSRARNPLYR (SEQ ID No. 8) has 70% sequence identity with peptide RARAKNPLYK (SEQ ID No. 7).
  • the amino acid designated by "-" can be a serine residue or may be another amino acid such as threonine, tyrosine, asparagine or glutamine.
  • the polypeptide has an amino acid sequence represented by R/K x R/K * R/K - xx*x* NPL Y/F R/K wherein each * is independently a hydrophobic amino acid or an amino acid selected from the group consisting of serine, tyrosine and threonine.
  • Hydrophobic amino acids are non-polar amino acids and examples include alanine, valine, leucine, isoleucine, and phenylalanine.
  • the entire intervening amino acid sequence indicated by -xxxxx may also be deleted such that the polypeptide comprises, or consists of, the sequence R/K x R/K x R/K NPL Y/F R/K.
  • acilitator moiety for facilitating passage or translocation of the polypeptide across the outer cell/plasma membrane into the cytoplasm of cells, such as a carrier peptide which has the capacity to deliver cargo molecules across cell membranes in an energy-independent manner.
  • Carrier peptides that are known in the art include penetratin and variants or fragments thereof, human immunodeficiency virus Tat derived peptide, transportan derived peptide, and signal peptides. Particularly suitable signal peptides are described in United States Patent
  • Signal peptide for Kaposi fibroblast growth factor consisting of, or incorporating, the amino acid sequence AA VALLP A VLLALLA (SEQ ID No: 15) or AAVALLPAVLLALLAP (SEQ ID NO: 16) is preferred. It is not necessary that a signal peptide used in a method of the invention be a complete signal peptide, and fragments or modified or variant forms thereof and the like which retain the ability to pass across the outer cellular membrane to effect delivery of the attached peptide or other agent into the cytoplasm of the cell may be utilised.
  • Cationic peptides have also been used successfully to transfer macromolecules such as DNA into living cells and a 15 mer arginine peptide has been reported to be the preferred number of amino acid residues to mediate expression of DNA encoding green fluorescent protein and the ⁇ -galactosidase gene in cancer cell lines (Choi HS et al, 2006; Kim HH et al, 2003).
  • the invention extends to the use of such cationic peptides as facilitator moieties for facilitating the passage into the target cancer cells of the polypeptide providing the binding domain of the ⁇ integrin subunit for the binding of ERK2 in accordance with the invention, or DNA encoding the polypeptide for expression of the polypeptide within the cells.
  • the invention in at least some embodiments also extends to coupling cationic peptide(s) such as a 15 mer arginine peptide (e.g., via a lysine bond) to a dendrimer presenting multiple units of the polypeptide (which may pegylated or unpegylated) providing the binding domain, or DNA encoding the polypeptide, for delivery/further assisting passage of the polypeptide or DNA into the target cancer cells, as a "double hit" strategy.
  • cationic peptide(s) such as a 15 mer arginine peptide (e.g., via a lysine bond)
  • a dendrimer presenting multiple units of the polypeptide (which may pegylated or unpegylated) providing the binding domain, or DNA encoding the polypeptide, for delivery/further assisting passage of the polypeptide or DNA into the target cancer cells, as a "double hit" strategy.
  • the facilitator moiety can be a lipid moiety or other non-peptide moiety which enhances cell membrane solubility of the selected anti-cancer peptide, such that passage of the peptide across the cell membrane is facilitated.
  • the lipid moiety can for instance be selected from triglycerides, including mixed triglycerides. Fatty acids and particularly, C 16 - C 2 o fatty acids can also be used. Typically, the fatty acid will be a saturated fatty acid and most usually, stearic acid.
  • the invention is not limited to the use of any such non-peptide facilitator molecule, and any molecule that provides the desired cell membrane solubility and which is physiologically acceptable can be used.
  • a polypeptide presenting the binding domain of a ⁇ integrin subunit for an ERK MAP kinase (or a variant or modified form of the binding domain) as described herein can be linked to the facilitator moiety in any conventionally known manner.
  • the polypeptide can be linked directly to a carrier peptide through an amino acid linker sequence by a peptide bond or non-peptide covalent bond using a cross- linking reagent.
  • chemical ligation methods may be used to create a covalent bond between the carboxy terminal amino acid of the carrier peptide or linker sequence and a peptide comprising, or consisting of, the binding domain of the ⁇ integrin subunit for the ERK MAP kinase.
  • Targeting or delivery of polypeptides, nucleic acids or dendrimers to cancer cells as described herein may be achieved by coupling a targeting moiety such as a ligand (e.g., that binds to a receptor expressed by the cancer calls), or a binding peptide, an antibody or binding fragment thereof (such as Fab and F(ab) 2 fragments), to the facilitator moiety or directly to the dendrimer, polypeptide or the like.
  • a targeting moiety such as a ligand (e.g., that binds to a receptor expressed by the cancer calls), or a binding peptide, an antibody or binding fragment thereof (such as Fab and F(ab) 2 fragments)
  • a targeting moiety such as a ligand (e.g., that binds to a receptor expressed by the cancer calls), or a binding peptide, an antibody or binding fragment thereof (such as Fab and F(ab) 2 fragments)
  • One approach employs coupling the facilitator moiety-peptid
  • peptides with the sequence DLXXL can be used to target the extracellular domain of the ⁇ 6 integrin subunit.
  • ⁇ 6 expression enhances effective proteolysis at the cell surface by matrix metalloproteinase-9 (MMP-9) (Agrez MV et al, 1999)
  • MMP-9 matrix metalloproteinase-9
  • targeting approaches include engineering an MMP-9 cleavage site between the targeting moiety and the carrier to facilitate internalisation of the carrier-agent complex.
  • the ligand recognition motif for ⁇ V ⁇ 6 integrin, RTDLDSLRTYTL may be used in conjunction with or without an engineered MMP-9 cleavage site to deliver the facilitator moiety-peptide complex to the surface of the target cell.
  • Targeting of cancer cells in a method of the invention may also be achieved by coupling an antibody or binding peptide specific for the EGF receptor as are known in the art to the polypeptide or dendrimer (e.g., such as to the lysine (Lys) residue at the apex of the dendrimer illustrated in Fig. 4).
  • Uptake into a cell can occur via a number of mechanisms, including via lysosomes which are rich in cathepsin, and targeting moieties employed can include a cathepsin cleavage site for release of the polypeptide or dendrimer to effect treatment of the cell. All such methods, dendrimers and polypeptides are expressly encompassed by the invention. Further, the polypeptide providing the binding domain of the ⁇ integrin subunit (and/ or a variant or modified form of the binding domain) can also be pegylated as described above (whether the polypeptide is included in a dendrimer or not).
  • liposomes, ghost bacterial cells, caveospheres, synthetic polymer agents, ultracentifuged nanoparticles and other anucleate nanoparticles may loaded with dendrimers or polypeptides as described herein and used for targeted delivery of the cargo to cancer cells (e.g., via labeling of the minicells, caveospheres or nanoparticles with bispecific antibodies, targeting peptides or the like as described above) (e.g., see also MacDiarmid, JA, 2007).
  • minicells and the like may be formulated for injection, or oral consumption for passage through the acid environment of the stomach for release and uptake of the dendrimer via the small intestine.
  • Still another approach is to load minicells as described above with nucleic acid encoding a polypeptide presenting the binding domain of the ⁇ integrin subunit for the ERK MAP kinase (or a variant or modified form of the binding domain) as described herein for delivery of the nucleic acid into cancer cells for expression of the polypeptide within the cells.
  • minicells caveospheres, bacteriophages, bacterial envelopes, recombinant vectors, and other conventional nanotechnology delivery methods can be used for delivery of the nucleic acid insert into the target cells.
  • any suitable vector incorporating the nucleic acid for expression of the polypeptide in the cancer cells may be utilized, including plasmids.
  • the expression vector may be designed for heterologous or homologous recombination events for integration of the nucleic acid into genomic DNA, and will typically include transcriptional regulatory control sequences to which the inserted nucleic acid sequence is operably linked.
  • 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.
  • transcriptional regulatory control sequences include promoters for facilitating binding of RNA polymerase to initiate transcription, expression control elements for enabling binding of ribosomes to transcribed mRNA, and enhancers for modulating promoter activity.
  • fusion proteins incorporating a polypeptide which binds to the binding domain of an ERK MAP kinase for a ⁇ integrin subunit as described herein is also expressly provided for by the invention.
  • Polypeptides and fusion proteins or the like can be chemically synthesised or produced using conventional recombinant techniques.
  • Nucleic acid encoding a fusion protein may for instance be provided by joining separate DNA fragments encoding peptides or polypeptides having the desired amino acid sequence(s) by employing blunt-ended termini and oligonucleotide linkers, digestion to provide staggered termini as appropriate, and ligation of cohesive ends.
  • 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 Ausubel et al.
  • Polypeptides and fusion proteins can be expressed in vitro and purified from cell culture for administration to a subject, or cells may be transfected with nucleic acid encoding a polypeptide or fusion protein for in vitro or in vivo expression thereof.
  • the nucleic acid will typically first be introduced into a cloning vector and amplified in host cells, prior to the nucleic acid being excised and incorporated into a suitable expression vector for transfection of cells.
  • Methods for the cloning, expression and purification of polypeptides useful in dendrimers as described herein are also well within the scope of the skilled addressee.
  • the toxicity profile of a polypeptide or dendrimer for use in a method embodied by the invention may be tested on cells by evaluation of cell morphology, trypan-blue exclusion, assessment of apoptosis and cell proliferation studies (e.g., cell counts, H-thymidine uptake and MTT assay).
  • Polypeptides as described herein e.g., including in dendrimer form
  • 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 whereby the polypeptide(s) and drugs exert their effect over overlapping therapeutic windows.
  • chemotherapeutic drugs which may used in accordance with one or more embodiments of the invention can be selected from the group consisting of metal and non-metal based drugs.
  • the metal complexes can be organic, inorganic, or mixed ligand co-ordination compounds or chelates. Transition metal complexes include for example complexes of platinum, palladium, copper, zinc, rhodium and ruthenium.
  • platinum based chemotherapeutic drugs examples include cisplatin (cis- diamminedichloroplatinum (H)), oxaliplatin, ([Pt(l)xalto (IR), (2R)- diaminocyclohexane] complex), carboplatin (cis-diammine(l,l- cyclobutanedicarboxylato)platinum (II), and bleomycin.
  • cisplatin cis- diamminedichloroplatinum (H)
  • oxaliplatin oxaliplatin
  • IR [Pt(l)xalto (IR), (2R)- diaminocyclohexane] complex
  • carboplatin cis-diammine(l,l- cyclobutanedicarboxylato)platinum (II)
  • bleomycin bleomycin.
  • non-metal chemotherapeutic drugs include Paclitaxel, Gleevac, Docetaxel, Taxol, 5-fluorouracil, Doxorubicin, cyclophosphamide, Vincristine (Oncovin), Vinblastine, Vindesin, Camplothecin, Gemcitabine, Adriamycin, and topoisomerase inhibitors such as Irinotecan (CPT-11).
  • a peptide as described herein can be co-administered with one or more of such conventional anti-cancer drugs or other drugs.
  • the dendrimer or polypeptide may be co-administered to the mammal in combination or in conjunction with the chemotherapeutic drug to which cells of the cancer are otherwise resistant.
  • the chemotherapeutic drug for example, inhibition of Src tyrosine kinase has been shown to enhance cytotoxicity of chemotherapeutic agents such as cisplatin in drug- sensitive ovarian cancer cells and to restore sensitivity in drug-resistant cells In normal cells c-Src is maintained in an inactive configuration by multiple intramolecular interactions.
  • the proto-oncogene c-Src is rarely mutated in human cancers although mutated c-Src that exhibits constitutive catalytic activity has been reported in small subsets of colon and endometrial cancers (reviewed by Ishizawar, R and Parsons, SJ, 2004). More commonly, this non-receptor tyrosine kinase exhibits elevated protein levels and increased activity of wild-type c-Src is seen in numerous types of human cancers.
  • c-Src This arises from interactions between c-Src and many membrane bound receptors and cellular factors (e.g., growth factor receptors, integrins, steroid hormone receptors, G-protein receptors, focal adhesion kinase (FAK) and other adaptor proteins) (reviewed by Ishizawar and Parsons, 2004).
  • c-Src becomes transiently activated and phosphorylates downstream targets.
  • the Src family of cytoplasmic, membrane-associated non-receptor tyrosine kinases are upstream of MAP kinases and, therefore, ERK activation.
  • c-Src of targets occurs in a unidirectional manner and is initiated by interactions between c-Src and the many membrane bound receptors and cellular factors near the plasma membrane as described above.
  • c-Src and Src family members are critical mediators of multiple signaling pathways that regulate all stages of cancer progression (from initiation to metastasis) in multiple cell types.
  • the Src oncoprotein is extremely potent causing rapid transformation in cell culture and activated Src protein is over expressed in many human epithelial malignancies, particularly breast and colon cancers.
  • Activated Src induces cellular invasion through a number of effectors, i.e., GTPase Rho and atypical protein kinase C.
  • GTPase Rho atypical protein kinase C.
  • Myc appears to be important in mediating Src's ability to cause cells to undergo unregulated cell proliferation.
  • ECM extracellular matrix
  • Integrin engagement leads to a number of intracellular signaling events, including the activation of Src family kinases (SFKs) and ERK1/2, responses that are dependent on the tyrosine phosphorylation and activation of focal adhesion kinase (FAK)(Miranti CK & Brugge JS, 2002). Further, aberrant integrin function and/or over-expression of focal adhesion kinase result in Src activation in focal adhesion complexes contributing to cell survival, and activating pathways that contribute to proliferation of some cell types (primarily through the Ras pathway) (Summy & Gallick, 2006). A functional interaction between integrins and c-Src has been recognised (Huveneers S et al, 2007).
  • a cancer treated in accordance with the invention will typically exhibit up- regulated activity of one or more of the kinase(s) inhibited by a dendrimer or polypeptide embodied by the invention.
  • the cancer can be a "Src” or a "PI3K” cancer.
  • the cancer can be a drug resistant cancer (i.e., a cancer resistant to one or more other anti-cancer drugs., e.g., a multi-drug resistant cancer).
  • the inhibition of c-Src and/or one or more other Src family kinases by a dendrimer or polypeptide in accordance with an embodiment of the invention may render a drug resistant cancer more susceptible, or otherwise sensitise the cancer to, conventional chemotherapeutic drug(s) to which the cancer is otherwise resistant.
  • Src cancer is meant a cancer arising from, or associated with, aberrant and/ or elevated levels of expression or activation of c-Src and/or one or more other Src family kinases.
  • Src cancer cell is meant a cancer cell arising from, or associated with, aberrant and/or elevated levels of expression or activation of c-Src and/or one or more other Src family kinases.
  • Aberrant or elevated expression of activated the Src kinase may arise from the expression of mutant forms of c-Src (the mutation(s) causing constitutive activation of the protein) or for instance, as a result of interaction of c-Src with adjacent membrane bound receptor and/or cellular factors.
  • the membrane bound receptor or cellular factor may be constitutively activated (e.g., as a result of a mutation in the receptor or cellular factor).
  • the term "PD kinase or PKC cancer” is meant a cancer arising from, or associated with, aberrant or elevated levels of expression and/or activation of a PD kinase or kinase in the PKC family.
  • PD kinase cancer cell and "PKC cancer cell” is meant a cancer cell arising from, or associated with, aberrant and/or elevated levels of expression and/or activation of a PD kinase or kinase in the PKC family.
  • Aberrant or elevated expression and/or activation of a PD kinase or kinase in the PKC family may arise from the expression of mutant forms of the PDK or kinase in the PKC family(the mutation(s) causing constitutive activation of the kinase) or for example, as a result of interaction of the PD kinase or PKC family member with cellular receptor(s) and/or factor(s).
  • the cancer treated by a method of the invention may for instance be selected from the group consisting of carcinomas, sarcomas, lymphomas, solid tumors, head and neck cancers, blood cell cancers, leukaemias, myeloid leukaemias, eosinophilic leukaemias, granulocytic leukaemias, and cancer of the liver, tongue, salivary glands, gums, floor and other areas of the mouth, oropharynx, nasopharynx, hypopharynx and other oral cavities, oesophagus, gastrointestinal tract, stomach, small intestine, duodenum, colon, colonrectum, rectum, gallbladder, pancreas, larynx, trachea, bronchus, lung (including non-small cell lung carcinoma), breast, uterus, cervix, ovary, vagina, vulva, prostate, testes, penis, bladder, kidney, thyroid, bone marrow, and skin (including
  • the cancer will be an epithelium cancer and most usually, a non-dermal cancer. Most usually, the cancer will be selected from the group consisting of lung cancers, colon cancers, pancreatic cancers, breast cancers, colon adenocarcinomas and ovarian cancers.
  • the polypeptide used will typically be formulated into a pharmaceutical composition comprising a pharmaceutically acceptable carrier and/or excipient for administration to the intended subject.
  • the peptide can be administered orally, intravenously, parenterally, rectally, subcutaneously, by infusion, topically such as in the treatment of skin cancers, intramuscularly, intraperitonealy, intranasally and by any other route deemed appropriate.
  • the pharmaceutical composition can for example be in the form of a liquid, suspension, emulsion, syrup, cream, ingestable tablet, capsule, pill, suppository, powder, troche, elixir, or other form that is appropriate for the selected route of administration.
  • compositions embodied by the invention include aqueous solutions. Injectable compositions will be fluid to the extent that syringability exists and typically, will normally stable for a predetermined period to provide for storage after manufacture.
  • pharmaceutically acceptable carriers include any suitable conventionally known solvents, dispersion media, physiological saline and isotonic preparations or solutions, and surfactants. Suitable dispersion media can for example contain one or more of ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol and the like), vegetable oils and mixtures thereof.
  • any orally acceptable carrier can be used.
  • the polypeptide can be formulated with an inert diluent, an assimilable edible carrier or it may be enclosed in a hard or soft shell gelatin capsule.
  • Topically acceptable carriers conventionally used for forming creams, lotions or ointments for internal or external application can be employed. Such compositions can be applied directly to a site to be treated or via by dressings and the like impregnated with the composition.
  • a pharmaceutical composition as described herein can also incorporate one or more preservatives suitable for in vivo and/or topical administration such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.
  • preservatives suitable for in vivo and/or topical administration such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.
  • prolonged absorption of the composition may be brought about by the use in the compositions of agents for delaying absorption such as aluminium monosterate and gelatin.
  • Tablets, troches, pills, capsules and the like containing the polypeptide can also contain one or more of the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; a disintegrating agent such as corn starch, potato starch or alginic acid; a lubricant such as magnesium sterate; a sweetening agent such as sucrose, lactose or saccharin; and a flavouring agent.
  • a binder such as gum tragacanth, acacia, corn starch or gelatin
  • a disintegrating agent such as corn starch, potato starch or alginic acid
  • a lubricant such as magnesium sterate
  • a sweetening agent such as sucrose, lactose or saccharin
  • a flavouring agent such as sucrose, lactose or saccharin
  • ingredients and media as described above in pharmaceutical compositions is well known. Except insofar as any conventional media or ingredient is incompatible with the dendrimer, use thereof in therapeutic and prophylactic compositions as described herein is included.
  • 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 used.
  • dosage unit form is for example, a capsule, tablet or pill
  • various ingredients may be used as coatings (e.g., shellac, sugars or both) to otherwise modify the physical form of the dosage unit or to facilitate administration to the individual.
  • a pharmaceutical composition will generally contain at least about 1% by weight of the polypeptide. The percentage may of course be varied and can conveniently be between about 5% to about 80% w/w of the composition or preparation. As will be understood, the amount of the peptide in the composition will be such that a suitable effective dosage will be delivered to the subject taking into account the proposed route of administration. Preferred oral compositions embodied by the invention will contain between about 0.1 ⁇ g and 15 g of the polypeptide.
  • the dosage of the polypeptide will depend on a number of factors including whether the polypeptide is to be administered for prophylactic or therapeutic use, the condition for which the polpeptide 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 individual as may be determined by the physician or attendant in accordance with accepted principles. For instance, a low dosage may initially be given which is subsequently increased at each administration following evaluation of the individual's response. Similarly, the frequency of administration may be determined in the same way that is, by continuously monitoring the individual's response between each dosage and if necessary, increasing the frequency of administration or alternatively, reducing the frequency of administration.
  • the polypeptide will be administered in accordance with a method of the invention to provide a dosage of the polypeptide of up to about 100 mg/kg body weight of the individual, more usually in a range up to about 50 mg/kg body weight, and most usually in a range of about 5 mg/kg to 40 mg/kg body weight.
  • the polypeptide will be administered to provide a dosage of the polypeptide in a range of from about 5 to 25 mg/kg body weight, usually in a range of from about 5 mg/kg to about 20mg/kg and more usually, in a range of from 10 mg/kg to about 20 mg/kg.
  • up to about 20g of the dendrimer may be administered per day, (e.g., 4 oral doses per day, each dose comprising 5g of the dendrimer).
  • suitable routes are via injection into blood vessels which supply a tumour or a cancer in to be treated in particular organs.
  • the polypeptide, dendrimer, fusion protein or the like can be delivered into isolated organs, limbs and tissue by any suitable infusion or perfusion techniques.
  • the polypeptide may also be delivered into cavities such for example the pleural or peritoneal cavity, or be injected directly into tumour tissue.
  • Suitable pharmaceutically acceptable carriers and formulations useful in compositions of the present invention can for instance, be found in handbooks and texts well known to the skilled addressee, such as "Remington: The Science and Practice of Pharmacy (Mack Publishing Co., 1995)", the contents of which is incorporated herein in its entirety by reference.
  • EXAMPLE 1 Inhibition of c-Src by RSKAKNPLYR (SEQ ID No.4)
  • the RSKAKNPLYR peptide (50 ⁇ M) (SEQ ID No: 4) (designated 10(4)) was assayed for inhibitory activity against the MAP kinase ERK2, cellular Src tyrosine kinase (c-Src) and the tyrosine kinases Lyn and Yes (at equivalent activity concentrations).
  • the assay conditions for each kinase were as follows (Upstate Kinase Profiling Services, Dundee, Scotland).
  • c-SRC (h) (5-10 mU) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 250 ⁇ M KVEKIGEGTYGWYK (SEQ ID No. 19) (Cdc2 peptide), 10 mM MgAcetate and [ ⁇ -33P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required).
  • the reaction is initiated by the addition of the MgATP mix. After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 5 ⁇ L of a 3% phosphoric acid solution. 10 ⁇ L of the reaction is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.
  • human Lyn is incubated with the RSKAKNPLYR peptide (SEQ ID No: 6) in 50 mM Tris buffer pH 7.5, 0.1 mM EGTA, 0.1 mM Na3VO4, 1% mercaptoethanol, 0.1 mg/ml poly(Glu,Tyr) 4:1, 10 mM Mg acetate and [ ⁇ -33P-ATP] (specific activity approx. 500 cpm/pmol).
  • the reaction was initiated by the addition of the MgATP mix, incubated for 40 minutes at room temperature prior to being stopped and kinase activity assessed by scintillation counting as per the protocol described for c-Src described in Example 1.1.
  • human Yes is incubated with the RSKAKNPLYR peptide (SEQ ID No: 6) in 8 mM MOPS buffer pH 7.0, 0.2 mM EDTA, 0.1 mg/ml poly(Glu,Tyr) 4:1, 10 mM Mg acetate and [ ⁇ -33P-ATP] (specific activity approx. 500 cpm/pmol).
  • the reaction is initiated by the addition of the MgATP mix, incubated for 40 minutes at room temperature prior to being stopped and kinase activity assessed by scintillation counting again as per the protocol described for c-Src in Example 1.1.
  • a peptide dendrimer of the type shown in Fig. 4 and presenting 10 monomer units of the peptide RSKAKNPLYR (SEQ ID No. 6) was assayed for inhibitory activity against ERK2 and the PDKs PDK beta and PDK gamma.
  • the dendrimer is referred to herein as dendrimer IK248B (or Dend 10 10(4)).
  • the treatment protocols were as as described below (Upstate Kinase Profiling Services, Dundee, Scotland).
  • Results were expressed as a percentage activity of control (PDK or ERK2 in the absence of dendrimer).
  • dendrimer Dend 10-10(4) inhibited ERK2 to 47% activity relative to control cells whereas PIK3 beta and PIK3 gamma were inhibited to 83% and 11% activity relative to control by the dendrimer.
  • RSKAKNPLYR peptides SEQ ID No.
  • dendrimer Dend 10 D- 10(4)DP D-amino acids
  • c-RAF (h) (5-10 mU) is incubated with 25 mM Tris pH 7.5, 0.02 mM EGTA,0.66 mg/mL myelin basic protein, 10 mM
  • MgAcetate and [ ⁇ -33P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required).
  • the reaction is initiated by the addition of the MgATP mix. After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 5 ⁇ L of a 3% phosphoric acid solution. 10 ⁇ L of the reaction is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.
  • MEKl (h) (1-5 mU) is incubated with 50 mM Tris pH 7.5, 0.2 mM EGTA, 0.1% mercaptoethanol, 0.01% Brij-35, 1 ⁇ M inactive ERK2 (m), 10 mM MgAcetate and cold ATP (concentration as required).
  • the reaction is initiated by the addition of the MgATP.
  • 5 ⁇ L of this incubation mix is used to initiate an ERK2 (m) assay.
  • PKB(h) (5-10 mU) is incubated with 8 niM MOPS pH 7.0, 0.2 niM EDTA, 30 ⁇ M GRPRTSSFAEGKK (SEQ ID No. 26), 10 rnM MgAcetate and [ ⁇ - 33 P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required).
  • the reaction is initiated by the addition of the MgATP mix. After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 5 ⁇ L of a 3% phosphoric acid solution. 10 ⁇ L of the reaction mixture is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.
  • PKC kinase (h) (5-10 mU) is incubated with 20 mM HEPES pH 7.4, 0.03% Triton X-100, 0.1 mM, 0.1 mg/mL phosphatidylserine, lO ⁇ g/mL diacylglycerol, 0.1 mg/mL histone Hl or 50 ⁇ M of the peptide ERMRPRKRQGSVRRRV (SEQ ID No. 20), 10 mM MgAcetate and [ ⁇ - 33 P- ATP] (specific activity approx. 500 cpm/pmol, concentration as required). The reaction is initiated by the addition of the MgATP mix.
  • the reaction is stopped by the addition of 5 ⁇ L of a 3% phosphoric acid solution. 10 ⁇ L of the reaction is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.
  • dendrimer Dend 10-10(4)DP at a concentration of 20 ⁇ M was without any effect on the activities of mTOR, JNK or FAK (data not shown).
  • EXAMPLE 5 Inhibition of c-Src by integrin ⁇ 2, ⁇ 3, ⁇ 5 and ⁇ 6 based peptides
  • EXAMPLE 6 Treatment of ADDP drug resistant ovarian cancer cells with cisplatin or oxaliplatin in combination with c-Src tyrosine kinase inhibitor peptide
  • 6.1 Cell proliferation (MTT) assay Single cell suspensions of viable trypsinised cells were seeded into 96-well tissue culture plates at a density of 2 x 10 3 cells per well in a volume of 100 ⁇ l of a suitable culture media supplemented with heat inactivated foetal calf serum (FCS) (e.g., Dulbecco's Modified Eagles Medium (DMEM), with 10% v/v FCS, 1% L-glutamine, 2% (v/v) Hepes, and antibiotics). A set of triplicate wells was prepared for each concentration of the peptide dendrimer being tested. Additional sample wells containing untreated cells or media alone were set up in each treatment plate and processed in parallel as reference controls.
  • FCS heat inactivated foetal calf serum
  • FCS heat inactivated foetal calf serum
  • DMEM Dulbecco's Modified Eagles Medium
  • a zero-time plate of untreated cells and media-alone wells was simultaneously prepared and an MTT assay carried out on this plate at the time of addition of the kinase inhibitor (i.e., polypeptide or dendrimer) to treatment plates. All plates were cultured for 24 hours before addition of the test dendrimer.
  • the kinase inhibitor i.e., polypeptide or dendrimer
  • kinase inhibitor concentrations of the kinase inhibitor were prepared by dilution of freshly prepared sterile ImM stock solutions into cell culture media to give a final well volume of 200 ⁇ l containing 10% v/v FCS.
  • the zero-plate was processed by addition of MTT at this time.
  • Cell culture was continued for a further 24 or 48 hours before addition of 20 ⁇ l of MTT in PBS (5mg/ml, 0.2um filter sterilised).
  • the MTT cell proliferation assay measures cell proliferation rate and, in instances where cell viability is compromised, the assay indicates a comparative reduction in cell viability.
  • the efficacy of cisplatin and the peptide AAVALLPAVLLALLARSKAKNPLYR (SEQ ID NO: 10) (IK2) alone and in combination against the ADDP human ovarian carcinoma cell line was evaluated using the MTT assay described above.
  • the ADDP cell line has induced resistance to cisplatin (Lu Y et al, 1988) and is derived from the A2780 ovarian cancer cell line which is sensitive to cisplatin. The results are shown in Fig. 6. As can be seen, the IK2 peptide in combination with cisplatin resulted in high level inhibition of the growth of the ADDP cancer cells compared to either cisplatin or IK2 peptide alone.
  • the inhibition of the growth of the cells increased with increasing concentration of the IK2 peptide showing that the peptide sensitises the ADDP cells to treatment with cisplatin.
  • the effect of cisplatin on the cisplatin sensitive ovarian cancer cell line A2780 is also shown in this figure.
  • the A2780 cell line was highly susceptible to cisplatin with substantially less inhibition of growth of the ADDP cell line by cisplatin being observed.
  • Greater inhibition of growth of ADDP cells by cisplatin in combination with IK2 compared to cisplatin alone was observed at all concentrations of the IK2 peptide utilised.
  • ADDP ovarian cancer cells were treated with oxaliplatin and the IK2 peptide alone or in combination, and inhibition of growth of the cancer cells again evaluated utilising the MTT assay described above.
  • the IK2 peptide in combination with oxaliplatin resulted in high level inhibition of the growth of the ADDP cancer cells compared to either oxaliplatin or IK2 peptide alone.
  • the inhibition of the growth of the ADDP cells again also increased with increasing concentration of the IK2 peptide.
  • Figs. 7A and 7B show a synergistic effect between cisplatin and 30 ⁇ M IK2 peptide against ADDP cisplatin-resistant ovarian cancer cells compared to the calculated additive effect of cisplatin and IK2.
  • EXAMPLE 7 Phospho-ERK 1/2 levels in HT29 human colon adenocaricnoma cells treated with peptide dendrimers presenting peptide RSKAKNPLYR (SEQ ID No. 6)
  • Peptide dendrimers of the type illustrated in Fig. 4 comprising lysine branching units presenting either 8 (identified herein as Dend 8-10(4)) or 10 (Dend 10-10(4)) monomer units of the peptide RSKAKNPLYR (SEQ ID No. 6) were utilised.
  • HT29 cells were harvested using 0.5% trypsin EDTA (Invitrogen) and 5000 cells in 200 ⁇ L media were plated into each well of a clear NUNC tissue culture treated 96 well plate (NUNC).
  • Cells were seeded in DMEM media (In vitro gen) supplemented with 10% (v/v) heat inactivated foetal calf serum (FCS, Invitrogen), 1% (v/v) L-glutamine (Invitrogen) and 2% (v/v) IM Hepes buffer solution (Invitrogen). Cells were then incubated overnight at 37 0 C.
  • the growth medium was replaced with lOO ⁇ L of DMEM supplemented with 1% (v/v) L-glutamine and 2% (v/v) IM HEPES buffer solution (Invitrogen) (serum free medium, SFM) and plates incubated for a further 24 hours at 37 0 C.
  • the dendrimers were re-constituted in SFM and the desired concentration was added to the plate in lOO ⁇ L SFM to make the total volume of each well 200 ⁇ L.
  • the control wells (minus peptide) had only SFM added.
  • the assay plate was then incubated at 37 0 C for 4 hours.
  • 22 ⁇ L FCS (10% v/v final concentration) was added to wells for the final 10 minutes at 37°C.
  • SFM only (22 ⁇ L) was added to control wells.
  • Phospho-ERK levels were evaluated by ELISA utilising an Active Motif
  • FACE ERK1/2 ELISA kit (Australian Biosearch, WA, Australia) as per the manufacturers instructions. Briefly, media was replaced and cells fixed with 4% formaldehyde in PBS, and after a one hour incubation with antibody blocking buffer (supplied), the primary phospho-ERK antibody was added and the plate incubated overnight at 4 0 C. Antibody dilution buffer only was added to control test wells containing no primary antibody. The following day, HRP-conjugated secondary antibody was added to all wells for one hour before plates were developed and the absorbance measured at 450nm using a Labsystems Multiskan EX microplate reader (Labsystems, Thermo Labsystems, UK).
  • FCS heat inactivated fetal calf serum
  • phospho-ERKl/2 levels upon FCS stimulation of HT29 cells were significantly reduced in the presence of Dend 8-10(4) compared to much less inhibition of ERK 1/2 activity upon FCS stimulation for skin fibroblast cells, thereby indicating substantial selectivity of the dendrimer for cancer cells compared with normal cells (data not shown).
  • a dendrimer of the same type but presenting 8 monomeric units of a scrambled form of the RSKAKNPLYR (SEQ ID No.6) peptide was relatively ineffective at inhibiting ERK1/2 activation upon FCS stimulation in HT29 colon cancer cells compared with the Dend 8-10(4) dendrimer presenting the unscrambled monomeric peptide (data not shown).
  • EXAMPLE 8 Effect of peptide dendrimers on activity ERK2 and Src family kinases in a cell -free system
  • c-Src human c-Src, c-Yes and c-Lyn was assesed as described in Example 1.
  • the activity of c-Fyn was assessed as follows. In a final reaction volume of 25 ⁇ L, Fyn (h) (5-10 mU) is incubated with 50 mM Tris pH 7.5, 0.1 mM EGTA, 0.1 mM Na3VO4, 250 ⁇ M KVEKIGEGTYGVVYK (SEQ ID No. 19) (Cdc2 peptide), 10 mM MgAcetate and [ ⁇ -33P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required). The reaction is initiated by the addition of the MgATP mix.
  • the reaction is stopped by the addition of 5 ⁇ L of a 3% phosphoric acid solution. 10 ⁇ L of the reaction is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanolprior to drying and scintillation counting.
  • the dendrimer Dend 10-10(4) (presenting 10 monomer units the peptide RSKAKNPLYR (SEQ ID No. 6)) was found to inhibit c-Src activity to a level of 57% and ERK activity to a level of 53%. However, surprisingly c-Src activity was stimulated to 305% of the control (three times the control value) by the dendrimer Dend8-10(4) (presenting 8 monomer units of RSKAKNPLYR (SEQ ID No. 6)). This stimulation of c-Src activity occurred concomitant with significant inhibition of ERK2 activity by the Dend 8-10(4) dendrimer (Table 4).
  • RSRARNPLYR SEQ ID No. 8 monomer alone (4%) relative to control (at a concentration of 50 ⁇ M).
  • the c-Src oncoprotein is extremely potent at causing rapid transformation in cell culture and is over-expressed and activated in many human epithelial malignancies, particularly breast, pancreatic and colon cancers.
  • Activated c-Src induces cellular invasion through a number of effectors, i.e., GTPase Rho and atypical protein kinase C.
  • GTPase Rho i.e., GTPase Rho and atypical protein kinase C.
  • GTPase Rho atypical protein kinase C
  • one of the major alterations found in cells transformed by c-Src is that they can proliferate in the absence of external growth factors.
  • One of the consequences of elevated Src activity in colon cancer cells is disruption of E-cadherin- associated cell-cell contacts.
  • EXAMPLE 10 ERK/PI3K cell adhesion assay
  • EXAMPLE 11 Induction of apoptosis in HT-29 human adenocarcinoma cells
  • RPMI 1640 cell culture medium foetal calf serum (FCS), PBS and HBSS (Invitrogen Australia, Mt Waverley, VIC, Australia). Penicillin- streptomycin and Trypan Blue (Sigma- Aldrich, Castle Hill, NSW, Australia). FACScalibur flow cytometer (Becton-Dickinson, North Ryde, NSW, Australia). FITC Annexin V Apoptosis Detection Kit II (BD Pharmingen, North Ryde, NSW, Australia).
  • HT-29 cells were cultured in RPMI 1640 cell culture medium, supplemented with 10% v/v heat inactivated (FCS) and 50 IU/mL penicillin- streptomycin. All cells were grown at 37 0 C in a humidified cell culture incubator supplied with 95% air / 5% CO 2 . The cells used in this study were used after passage 3.
  • FCS v/v heat inactivated
  • IxIO 6 HT-29 cells were incubated in the presence of either Dend 10-10(4)DP or Staurosporine (positive control) for 4 hours at 37 0 C in a humidified cell culture incubator supplied with 95% air / 5% CO 2 . Cells were then harvested by trypsinisation, washed twice in cold phosphate buffered saline (PBS) and then resuspended in IX binding buffer. 100 ⁇ L of the cell suspensions containing 1x10 cells were transferred to a 5 mL plastic tube. A 5 ⁇ L volume of FITC annexin V and 5 ⁇ L of propidium iodine (PI) were added to each tube and incubated for 15 minutes at room temperature in the dark.
  • PBS cold phosphate buffered saline
  • HT29 colon cancer cells suspended in RPMI cell culture medium supplemented with 10% FCS, 100 IU/ml penicillin- streptomycin, 2mM L-Glutamine and 1 mM sodium pyruvate were seeded into 35 mm Fluorodishes and incubated at 37° C in 95% air/5% CO 2 .
  • 10 ⁇ L of either FITC conjugated Dend 10-10(4)DP dendrimer see Example 2.3 (Dend 10-10(4)DP-FITC (resuspended in phosphate buffered saline) or 10 ⁇ L of FITC alone (resuspended in DMSO) were added to separate dishes to give a final compound concentration of 1 ⁇ M per dish.
  • the treated dishes were incubated at 37°C for a further hour and the Hoechst nuclear stain 33258 (In vitro gen) added to the cells 20 minutes prior to imaging at a final concentration of 2.5 ⁇ g/ml.
  • the confocal microscopy showed the FITC-conjugated dendrimer was located at the cell membrane, within the cytoplasm and within nuclei after 1 hour exposure to the compound, in contrast to lack of cellular fluorescence for cells exposed to FITC alone.
  • EXAMPLE 13 Peptide dendrimer comprising 4 or 8 monomer units of the polypeptide RSKAKNPL YR (SEQ ID No. 6) inhibits the proliferation of HT29 colon cancer cells
  • Peptide dendrimers of the type shown in Fig. 4 comprising 4 (Dend 4-10(4)) or 8 (referred to herein as dendrimer Dend 8-10(4), see Example 7.1) monomer units of the polypeptide RSKAKNPLYR (SEQ ID No. 6) were found to inhibit proliferation of HT29 colon cancer cells as assessed by the MTT assay described in Example 6.1.
  • Dend 8-10(4) was found to be substantially more effective at inhibiting cell growth/proliferation than the peptide dendrimer comprising 4 monomers of the polypeptide (24 hour incubation period) .
  • the Dend 10-10(4)DP dendrimer also inhibited growth/proliferation of MKN45 gastric carcinoma cells (Cancer Research Laboratory, University of New south Wales, Sydney, Australia), MCF-7 breast cancer cells (breast adenocarcinoma cells obtained from the American Type Culture Collection, ATCC, Manassas VA, United States), and DU145 prostate cancer cells.
  • the cells were incubated in the presence of the dendrimer for 48 hours. Cell lines were maintained at 37 0 C in a humid atmosphere containing 5% CO 2 . Cells were passaged at pre-confluent densities using a solution containing 0.05% trypsin and 0.5 mM EDTA (Invitrogen).
  • EXAMPLE 14 Phospho-ERKl/2 levels in HT29 human colon cancer cells treated with various agents
  • the facilitator moieties utilised were the signal peptide fragment AA V ALLP A VLLALLA (SEQ ID No. 15), the TAT-G peptide GRKKRRQRRRPPQG (SEQ ID No. 23), a modified pentratin sequence Tr-Pen RRQKWKKG (SEQ ID No. 24), and the penetratin peptide RQIKIWFQNRRMKWKKCs-sC (SEQ ID No. 25) wherein S-S indicates a disulphide bridge between the adjacent cysteine residues.
  • the percentage inhibition of activated phospho-ERK 1/2 by dendrimer Dend 8-10(4) and the polypeptide- facilitator moieties in the HT29 cells at the 1 hour time point is shown in As can be seen, the Dend 8-10(4) dendrimer exhibited at least 30% greater inhibition than the test agent which displayed the closest level of inhibition, namely the TAT-G RSKAKNPLYR (SEQ ID No. 6) polypeptide. At 4 hours, 5 ⁇ M Dend 8-10(4)exhibited approx. 95% inhibition of activated phospho- ERK 1/2 compared to relatively low level inhibition by the polypeptide-facilitator moieties (data not shown).
  • EXAMPLE 15 Inhibition of proliferation in HT29 human adenocarcinoma cells
  • HT29 cells were cultured for 48 hours in the presence of selected dendrimers and proliferation of the cells was assessed by MTT assay essentially as described in Example 6.1. The results were calculated as percentage proliferation of control cells (not treated with dendrimer).
  • HT29 cells were treated with peptide dendrimer Dend 10-10(4) and the results are shown in Fig. 12. As can be seen, proliferation of the cells was inhibited by the dendrimer.
  • Dendrimers of the type Shown in Fig. 4 presenting 9 (Dend 9-10(4) or 12 (Dend 12-10(4)) monomer units of the peptide RSKAKNPLYR (SEQ ID No. 6) were assessed for capacity to inhibit proliferation of the HT29 cells. As shown in Fig. 13, Dend 12- 10(4) was more effective than Dend 9-10(4) in inhibiting proliferation of the cells. When compared to dendrimer Dend 10-10(4) (presenting 10 monomer units of peptide RSKAKNPLYR (SEQ ID No.
  • Dend 12-10(4) showed a small improvement in IC 50 value (1 ⁇ M versus 1.8 ⁇ M) but no increase in the dendrimer concentration required for total kill (namely 10 ⁇ M for both Dend 12-10(4) and Dend 10-10(4)) was obtained.
  • Dendrimer Dend 10-10(4) was in turn more effective than dendrimer Dend 8-10(4) (presenting 8 monomer units of the RSKAKNPLYR peptide (SEQ ID No. 6)) (IC 50 's of 1.8 ⁇ M and 5 ⁇ M, respectively).
  • EXAMPLE 16 Treatment of HT29 colon cancer cells with peptide dendrimers presenting RARAKNPLYK (SEQ ID No. 7) or RSRARNPLYR (SEQ ID No. 8)
  • HT29 colon cancer cells were treated with peptide dendrimers of the type illustrated in Fig. 4 presenting 8 monomer units of the 10 mer ⁇ 3 based peptide RARAKNPLYK (SEQ ID No. 7)) (Dend 8- ⁇ 3) or the ⁇ 5 based peptide RSRARNPLYR (SEQ ID No. 8) (identified as Dend 8- ⁇ 5).
  • Test cells were exposed to the dendrimers for 48 hours and proliferation of the cells was evaluated using the MTT assay essentially as described above in Example 6.1. Absorbance was read at 550 nm using a microtitre plate reader, and the percentage inhibition of proliferation of the test cells was calculated relative to untreated control cells. The results are shown in Fig. 15.
  • EXAMPLE 17 Inhibition of tumour growth in a mouse model
  • Reagents for the culture of HT-29 human colorectal adenocarcinoma cells were obtained from the following suppliers: RPMI 1640 cell culture medium, FBS and HBSS from Invitrogen Australia (Mt Waverley, VIC, Australia); penicillin- streptomycin, phosphate buffered saline (PBS) and trypan blue from Sigma-Aldrich (Castle Hill, NSW, Australia) .
  • HT29 human colorectal adenocarcinoma cells were cultured in RPMI 1640 cell culture medium supplemented with 10% v/v heat inactivated FCS and 50 IU/mL penicillin- streptomycin. The cells were harvested by trypsinisation, washed twice in HBSS and counted. The cells were then resuspended in HBSS to a final concentration of 2 x 10 7 cells/mL. 17.3 Test system
  • Body weight range 20.61-25.27g at onset of treatment (mean 22.27g)
  • Age range 10- 12 weeks at onset of treatment.
  • mice Prior to inoculation the skin on the injection site (dorsal right flank) was swabbed with alcohol. The needle was introduced through the skin into the subcutaneous space just below the animal' s right shoulder, and lOO ⁇ L of cells (2 x 10 6 cells) were discharged. The treatment of mice began nine days after HT29 cell inoculation, the average tumour volume was 68 mm 3 (average variability of 6.1%).
  • Body weight and tumour dimensions were measured for all animals on the first day of treatment (day 0) and then three times per week, including the termination day of the study (Day 24).
  • mice were randomized, based on body weight, into two groups of ten mice on
  • PBS phosphate buffered saline
  • Dend 10-10(4) dendrimer (20mg/kg) were each administered by intra-tumoural injection once daily for five consecutive days, beginning on Day 0.
  • the vehicle control and Dend 10-10(4) dendrimer were administered at a dosing volume of 4.762 mL/kg (lOO ⁇ L) based on a 21g mouse. Each animal's body weight was measured immediately prior to dosing. The volume of dosing solution administered to each mouse was calculated and adjusted based on individual body weight.
  • Tumours were excised from all mice post mortem and weighed. Tumour volume was calculated using the equation:
  • tumour growth was markedly inhibited by the dendrimer Dend 10-10(4) (solid squares, and identified as IK248 in Fig. 16) compared to the vehicle only control (solid diamonds).
  • the growth of the tumours slowed noticeably between Day 5 to Day 15 in the treatment groups relative to the control group.
  • the average tumour weight in the control group at the end of the study was 0.962g +0.124 SEM compared to 0.437g + 0.072 SEM for the Dend 10-10(4) treatment group, a highly significant outcome (P ⁇ O.003).
  • Nanjundan M Sun J, Zhao J, et al (2003), J Biol Chem 278: 37413-37418.

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Abstract

La présente invention a pour objet une méthode permettant d'inhiber la croissance et/ou la prolifération d'une cellule cancéreuse. La méthode comprend le traitement d'une cellule cancéreuse par une quantité efficace d'un polypeptide présentant un domaine de liaison cytoplasmique d'une sous-unité β d'intégrine pour la liaison de l'ERK2 afin d'inhiber au moins une protéine kinase, autre qu'une MAP kinase, dans une voie d'activation cellulaire de la cellule cancéreuse. Les protéines kinases inhibées par le polypeptide peuvent être choisies dans le groupe constitué par c-Raf, MEK 1 et des kinases dans les familles Src, PI3K, PKB/AKT et PKC. L'invention a également pour objet des méthodes destinées à la prévention et au traitement du cancer.
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CN104271590A (zh) * 2012-01-24 2015-01-07 Inter-K私人有限公司 用于癌治疗的肽剂
WO2019218016A1 (fr) * 2018-05-15 2019-11-21 Interk Peptide Therapeutics Limited Agent d'activation de peptide
WO2020107079A1 (fr) * 2018-11-30 2020-06-04 Interk Peptide Therapeutics Limited Polypeptides et méthodes pour soulager les affections cutanées
WO2023019323A1 (fr) * 2021-08-20 2023-02-23 Interk Peptide Therapeutics Limited Compositions et procédés de traitement de l'auto-immunité, comprenant l'auto-immunité associée au cancer et à la thérapie du cancer

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KR20210021311A (ko) * 2018-05-15 2021-02-25 인터크 펩타이드 테라퓨틱스 리미티드 활성화 제제

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JP2015510393A (ja) * 2012-01-24 2015-04-09 インター・ケー・ピーティーワイ・リミテッド 癌治療用ペプチド剤
EP2807180A4 (fr) * 2012-01-24 2016-01-06 Inter K Pty Ltd Agents peptidiques utilisés en thérapie anticancéreuse
US9403877B2 (en) 2012-01-24 2016-08-02 Inter-K Pty Limited Peptide agents for cancer therapy
CN104271590B (zh) * 2012-01-24 2018-06-05 Inter-K私人有限公司 用于癌治疗的肽剂
WO2019218016A1 (fr) * 2018-05-15 2019-11-21 Interk Peptide Therapeutics Limited Agent d'activation de peptide
CN112399970A (zh) * 2018-05-15 2021-02-23 英特肽治疗有限公司 肽活化剂
US11912789B2 (en) 2018-05-15 2024-02-27 Interk Peptide Therapeutics Limited Peptide activating agent
WO2020107079A1 (fr) * 2018-11-30 2020-06-04 Interk Peptide Therapeutics Limited Polypeptides et méthodes pour soulager les affections cutanées
WO2023019323A1 (fr) * 2021-08-20 2023-02-23 Interk Peptide Therapeutics Limited Compositions et procédés de traitement de l'auto-immunité, comprenant l'auto-immunité associée au cancer et à la thérapie du cancer

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