WO2002048191A2 - Inhibitory agents derived from specific growth factors - Google Patents

Abstract

Inhibitory agents for blocking proliferative, apoptic, mitogenic and morphogenetic processes which are derived from specific growth factors, and in particular to such agents which are derived from specific growth gactors, and in particular to such agents which are derived from NDF (neuregulin). The inhibitory agents of the present invention are optionally and preferably peptides, or homologues, analogues or other related biologically active agents thereof, which are derived from at least a portion of neuregulin. More preferably, such an agent is derived from a particular fragment of neuregulin, known as the ED fragment, which includes the EGF-like domain of neuregulin. Alternatively, the agent is derived from any portion of neuregulin having the ability to bind to at least one of the ErbB-3 and ErbB-2 receptors, but lacking the ability to induce morphogenetic events. Alternatively or additionally, the agent is derived from two or more separate portions of neuregulin having these characteristics. Thus, the inhibitory agent of the present invention only causes a partial phenotypic response, but blocks the ability of full-length neuregulin to induce those aspects of the full response which are related to morphogenetic events, such as cell motility and morphogenetic behaviors

Description

INHIBITORY AGENTS DERIVED FROM SPECIFIC GROWTH FACTORS

FIELD OF THE INVENTION

The present invention is related to inhibitory agents for blocking proliferative, apoptotic, mitogenic, migratory and/or morphogenetic processes which are derived from specific growth factors, and in particular to such agents which are derived from NDF (neu differentiation factor or neuregulin).

BACKGROUND OF THE INVENTION Neuregulins are actually a family of EGF-like ligands, also known as "NRGs", which interact with receptors of the ErbB -family. NRG ligands play an important role in the regulation of major biological processes, affecting cell motility, proliferation, differentiation and morphogenesis in vitro and in vivo [Burden, 1997; Riese, 1998]. The NRG subfamily is represented by at least four distinct genes. NRG 1 (also known as NDF, ARIA and heregulin), NRG 2, and NRG 3 encode several isoforms by alternative splicing [Peles, 1992; Carraway KL, 1997; Chang 1997; Zhang 1997; and Riese 1998]. The encoded peptides interact with high affinity with two members of the ErbB receptor family, ErbB-3 and ErbB-4 [Burden 1997; Riese 1998]. NRG 4 encodes a ligand interacting exclusively with ErbB-4 [Harari 1999,]. Other members of the ErbB family, ErbB-1 (EGF receptor) and especially the "orphan" receptor ErbB- 2, also play an important role in the transduction of signal, generated by neuregulins. They form heterodimers with either ErbB-3 or ErbB-4 and serve as essential co-receptors [Burden, 1997; Tzahar, 1998]. These co-receptors are indispensable for the signals transduced via ErbB-3 since this receptor has impaired tyrosine kinase activity [Sierke 1997; Guy, 1994].

Signaling through tyrosine kinase receptors of the ErbB family generally plays an important role in the regulation of cell proliferation, migration and tissue morphogenesis. These receptors are activated by ligands such as neuregulin. Overexpression of these receptors or other forms of excessive activation of these signals are often associated with neoplastic development. In particular, the ErbB-2 receptor, which does not interact with any ligands alone but which is an important co-receptor, has been shown to be involved in several forms of neoplasia, when overexpressed or mutated. The effect of ErbB signaling is usually pleiotropic and involves alterations in different characteristics of cell behavior including cell proliferation, adhesion and motility. Current research has attempted to find inhibitory agents for such signaling, although precise targets for such inhibition are not clear. For example, stimulation of proliferation alone by ErbB may cause relatively benign cell growth, while stimulation of adhesion may cause such cells to metastasize into dangerous cancerous tumors. Thus, clearly a more precise understanding of the processes and effects of ErbB signaling is required in order to derive more specific inhibitory agents of this pathway. Neuregulins are certainly an important component of the ErbB signaling pathway. In vivo studies showed that a major function of neuregulins is the induction of various morphogenetic processes [Burden, 1997; Lemke, 1996]. Thus, experiments with neuregulin- knock-out mice indicate that the lack of neuregulins leads to severe disorders in heart development, and deficiency in migratory activity of sympathogenic neural crest cells [Meyer 1995; Kramer, 1996; Britsch, 1998]. Experiments with organ cultures demonstrated that neuregulins are required for formation of alveolar structures in the course of mammary gland development [Yang, 1995; Niemann, 1998].

Morphogenetic effects of NRG- 1 (NDF-4) can be reproduced also in standard in vitro conditions [Chausovsky, 1998; Chausovsky, 2000]. In particular, neuregulin was shown to increase spreading and migratory ability of cells in monolayer culture [Chausovsky, 1998; Chausovsky, 2000]. Moreover, in several cell lines, NRG-1 induces complex morphogenetic effects, namely formation of multicellular ring-shaped complexes from epithelial colonies [Chausovsky, 1998]. Receptor combination, which is most efficient in induction of NRG- induced morphogenetic events, was shown to be an ErbB-3/ErbB-2 heterodimer. NRG- dependent formation of ring-shaped structures was reconstituted in CHO cells expressing ErbB- 3, ErbB-2 and a receptor maintaining cell-cell adhesion, N-cadherin [Chausovsky, 1998; Chausovsky, 2000].

Clearly, neuregulin is important for the formation of complex structures through cell migration and arrangement. However, this type of effect may also be involved in cellular processes which are actually detrimental to the organism, such as the uncontrolled cellular migration which is involved in metastatic processes. Cancerous cells are most dangerous after metastases are formed, which enable the tumor cells to migrate throughout the body. Clearly, being able to block such metastatic processes would be highly beneficial.

Another important component of these cellular processes is HSPG (heparan sulfate proteoglycans), which are actually a family of macromolecules, associated with the cell surface and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues (for a review, see for example PCT Application No. WO 00/03036). The basic HSPG structure features a protein core to which several linear heparan sulfate chains are covalently attached. The polysaccharide chains are typically composed of repeating hexuronic and D-glucosamine disaccharide units that are substituted to a varying extent with N- and O-linked sulfate moieties and N-linked acetyl groups. Studies on the involvement of ECM molecules in cell attachment, growth and differentiation revealed a central role of HSPG molecules in embryonic morphogenesis, angiogenesis, metastasis, neurite outgrowth and tissue repair

In particular, the ability of HSPGs to interact with ECM macromolecules such as collagen, laminin and fibronectin, and with different attachment sites on plasma membranes suggests a key role for this proteoglycan in the self-assembly and insolubility of ECM components, as well as in cell adhesion and locomotion. Therefore, interaction with HSPGs may play an important role in extravasation of normal and malignant blood-borne cells, and hence of metastatic processes.

SUMMARY OF THE INVENTION

The background art does not teach or suggest effective inhibitory agents for inhibiting the signaling processes for neuregulin. The background art also does not teach or suggest such agents for blocking interactions with HSPGs which are important for metastatic processes. The background art also does not teach or suggest selective inhibitory agents which block those aspects of the neuregulin and HSPG mediated processes which are involved in metastasis, without blocking those aspects of these processes which are beneficial or at least benign. The present invention overcomes these deficiencies of the background art by providing inhibitory agents for blocking proliferative, apoptotic, mitogenic and morphogenetic processes which are derived from specific growth factors, and in particular to such agents which are derived from NDF (neuregulin). The inhibitory agents of the present invention are optionally and preferably peptides, or homologues, analogues or other related biologically active agents thereof, which are derived from at least a portion of neuregulin. More preferably, such an agent is derived from a particular fragment of neuregulin, known as the ED fragment, which includes the EGF-like domain of neuregulin. Alternatively, the agent is derived from any portion of neuregulin having the ability to bind to at least one of the ErbB-3 and ErbB-2 receptors, but lacking the ability to induce morphogenetic events. Alternatively or additionally, the agent is derived from two or more separate portions of neuregulin having these characteristics. Thus, the inhibitory agent of the present invention only causes a partial phenotypic response, but blocks the ability of full-length neuregulin to induce those aspects of the full response which are related to morphogenetic events, such as cell motility and morphogenetic behaviors. The ED fragment selectively blocks morphogenetic events which are induced by neuregulin, apparently without blocking those effects of neuregulin which are related to other pathways regulated by this peptide, such as receptor phosphorylation and MAPK activation. Without wishing to be limited by a single hypothesis, the morphogenetic effects of the full- length neuregulin peptide are presumably induced through interaction with one or more HSPG molecules (heparan sulfate proteoglycans). Thus, fragments of neuregulin which are able to bind to at least one of the ErbB-3 and ErbB-2 receptors, but which are unable to induce morphogenetic events, are therefore expected to be inhibitors of such events, by blocking binding of full-length neuregulin molecules which could induce these morphogenetic events. Therefore, the inhibitory agent of the present invention, such as the ED fragment of neuregulin for example, is able to suppress neuregulin-induced morphogenetic signals, and therefore can be used to suppress invasive growth and metastases of tumors, as well as for the suppression of neovascularization in tumors. The latter effect is supported by the finding that full-length neuregulin induces the formation of alveolar structures in the course of mammary gland development, and has also been shown to induce the formation of multicellular ring-like structures in vitro. Therefore, inhibition of neuregulin effects would be expected to include the suppression of neovascularization, which also depends upon the formation of tube-like structures for the blood vessels.

According to the present invention, there is provided an inhibitory agent for a neuregulin- mediated process, comprising a peptide derived from at least a portion of a neuregulin molecule, or homologues, analogues or mimetics thereof. Preferably, the peptide is derived from the ED fragment.

According to another embodiment of the present invention, there is provided a therapeutic agent for treating a cancerous condition in a subject, comprising a peptide derived from at least a portion of a neuregulin molecule, or homologues, analogues or mimetics thereof. Preferably, the peptide is derived from the ED fragment. Also preferably, the peptide has at least one of an effect selected from the group consisting of suppression of invasive growth, suppression of metastases of tumors, and suppression of neovascularization in tumors. Optionally and preferably, the peptide is derived from any portion of neuregulin having the ability to bind to at least one of the ErbB-3 and ErbB-2 receptors, but lacking the ability to induce a morphogenetic event.

According to yet another embodiment of the present invention, there is provided a method for treating a cancerous condition in a subject, comprising the step of administering a peptide derived from at least a portion of a neuregulin molecule, or homologues, analogues or mimetics thereof.

Hereinafter, the term "biologically active" refers to molecules, or complexes thereof, which are capable of exerting an effect in a biological system. Hereinafter, the term "fragment" refers to a portion of a molecule or a complex thereof, in which the portion includes substantially less than the entirety of the molecule or the complex thereof.

Hereinafter, the term "amino acid" refers to both natural and synthetic molecules which are capable of forming a peptide bond with another such molecule. Hereinafter, the term "natural amino acid" refers to all naturally occurring amino acids, including both regular and non-regular natural amino acids. Hereinafter, the term "regular natural amino acid" refers to those alpha amino acids which are normally used as components of a protein. Hereinafter, the term "non-regular natural amino acid" refers to naturally occurring amino acids, produced by mammalian or non-mammalian eukaryotes, or by prokaryotes, which are not usually used as a component of a protein by eukaryotes or prokaryotes. Hereinafter, the term "synthetic amino acid" refers to all molecules which are artificially produced and which do not occur naturally in eukaryotes or prokaryotes, but which fulfill the required characteristics of an amino acid as defined above. Hereinafter, the term "peptide" includes both a chain of a sequence of amino acids, whether natural, synthetic or recombinant. Hereinafter, the term "peptidomimetic" includes both peptide analogues and mimetics having substantially similar or identical functionality thereof, including analogues having synthetic and natural amino acids, wherein the peptide bonds may be replaced by other covalent linkages.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, with reference to the accompanying drawings, wherein: FIG. 1A is a schematic block diagram of the interaction of the ED fragment (truncated ligand) with the ErbB signaling pathway according to the present invention;

FIG. IB shows heparin binding by neuregulin and ErbB-family receptors, in which the amount of total (Upper panel) and heparin-sepharose bound (lower panel) neuregulin (lane-1), ED (2), and soluble extracellular part of ErbB-1, 2,3,4 receptors (lane 3-6) is shown, with visualization of amount of protein by western blotting (lane 1, 3-6) and by autoradiography of iodinated ligand (lane 2). Note that full-length neuregulin, but not ED fragment binds to heparin sepharose; among ErbB-family receptors only ErbB-3 demonstrates heparin binding;

FIG. 1C shows a schematic diagram of the structure of the transmembrane precursor of neuregulin (proNRG), full length secreted ectodomain (NRG-FL) and recombinant EGF-like domain (NRG-ED);

FIG. 2 shows that the EGF-like domain of neuregulin (ED) did not induce morphogenetic response and prevented induction of such response by the full length neuregulin as follows: effect of neuregulin, ED or their combination on the spreading of 32D cells (2A-D), formation of ring-shaped multicellular aggregates (2E-H) and migration of individual cells as assessed by phagokinetic track method (2I-L). (2A, E, I)-non treated cells, (2B, F, J)-cells treated with neuregulin, (2C, G, K)-cells treated with ED, (2D, H, L)-cells treated with combination of neuregulin, and ED. (2M)-quantification of phagokinetic experiment: average areas of phagokinetic tracks after different treatments. (2N)-western blot of lysates of control (c) and treated cells demonstrating the effect of neuregulin and ED on the activation of MAPK as revealed by staining with antibody against activated form of MAPK. Equality of loading was controlled by anti-α tubulin (Tub);

FIG. 3 is a schematic block diagram for showing that ErbB-3 binds heparin due to specific heparin-binding motif and for demonstrating the scheme of production of mutated ErbB- 3 lacking the heparin-binding motif (Adar and Yayon, 2001). Putative heparin-binding motif in the extracellular portion of ErbB-3, 466KHNRPRR472, was substituted by corresponding region from ErbB-1 sequence (alignment was performed according to (reference number 45));

FIG. 4 shows that a mutation in heparin binding domain of ErbB-3 prevents neuregulin- induced morphogenetic response, in which (A-D) Effect of neuregulin on the colonies of FL4 cells transfected with WT of ErbB-3 (A,B) and mutated ErbB-3 (C,D). Non-treated (A,C) and neuregulin-treated (B,D) colonies stained with TRITC phalloidin and DAPI are shown. Note that WT ErbB-3 mediates neuregulin signaling leading to the formation of multicellular rings, while the mutated ErbB-3 transfected cells do not form rings. (E)-percentage of ring-shaped colonies in the cultures of different clones transfected with WT or mutated variants of ErbB-3. Each symbol represents the result obtained in the experiment with individual clone. (F)-mutated variant of ErbB-3 does not bind heparin. Elution of soluble alkaline-phosphatase-conjugated WT and mutated ErbB-3 bound to heparin carrier by different concentration of NaCl. WT ErbB- 3 binds heparin and can be eluted only by more than 0.4 M NaCl. Mutated ErbB-3 does not bind heparin. (G) Western blot of lysates of control (-) and ED treated cells (+) transfected with WT or mutated (Mut) ErbB-3 demonstrating the MAPK activation as revealed by staining with antibody against activated form of MAPK. Equality of loading was controlled by anti-α tubulin (Tub);

FIG. 5 shows the effect of anti-ErbB-3 antibody on the binding of ¹²⁵I-ED or ¹²⁵I-β4 NDF to T47D breast carcinoma cells, in which the results are given as per cent of ligand binding in the absence of antibody taken as 100%. Error bars denote SEM. Note that ErbB-3 -directed antibody which prevents binding of ED fragment to cells, inhibited the binding of the full-length neuregulin to a much lower extent;

FIGS. 6 A and 6B show the effect of heparin and or sodium chlorate pretreatment of the formation of circular multicellular arrays: Figure 6 A shows the effect of heparin and sodium chlorate pretreatment with administration of the full length NDF (NDF-FL); Figure 6B shows the effect of sodium chlorate pretreatment with the administration of the full length NRG 1 (β4 isoform of NDF; NRG-FL); and

FIG. 7 shows the inhibitory effect of ED fragment on tumor size in an animal model, in which six to 8-week old nude mice (CD-I) were implanted in the upper part of the front leg with FL4-ErbB-3 cells (2«10⁷ cells/mice) or with human gastric carcinoma (10⁷ cells/mice). Three days after implantation six animals in each group were injected with 10, 100 or 300 ng/mice of ED in 300μl of serum-free DMEM, or 300 μl of serum-free DMEM was injected as control of treatment. All injections were done in the upper part of the front leg. The treatment was done three times at 3, 7 and 12^th day after cell implantation. Tumor volume (XxYxZ) was measured weekly starting at the day of the last injection and ending around two months after cell implantation. Each point is the result of measurement of 6 animals (±SEM).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention discloses inhibitory agents for blocking proliferative, apoptotic, mitogenic and morphogenetic processes which are derived from specific growth factors, and in particular to such agents which are derived from NDF (neuregulin). The inhibitory agents of the present invention are optionally and preferably peptides, or homologues, analogues or other related biologically active agents thereof, which are derived from at least a portion of neuregulin. More preferably, such an agent is derived from a particular fragment of neuregulin, known as the ED fragment, which includes the EGF-like domain of neuregulin. Alternatively, the agent is derived from any portion of neuregulin having the ability to bind to at least one of the ErbB-3 and ErbB-2 receptors, but lacking the ability to induce morphogenetic events. Alternatively or additionally, the agent is derived from two or more separate portions of neuregulin having these characteristics. Thus, the inhibitory agent of the present invention only causes a partial phenotypic response, but blocks the ability of full-length neuregulin to induce those aspects of the full response which are related to morphogenetic events, such as cell motility and morphogenetic behaviors.

As described in greater detail below, the ED fragment selectively blocks morphogenetic events which are induced by neuregulin, apparently without blocking those effects of neuregulin which are related to other pathways regulated by this peptide, such as receptor phosphorylation and MAPK activation. Furthermore, the ED fragment was also shown, as described in greater detail below, to have an inhibitory effect on tumor size in mice. Without wishing to be limited by a single hypothesis, the morphogenetic effects of the full-length neuregulin peptide are presumably induced through interaction with one or more HSPG molecules (heparan sulfate proteoglycans). Thus, fragments of neuregulin which are able to bind to at least one of the ErbB- 3 and ErbB-2 receptors, but which are unable to induce morphogenetic events, are therefore expected to be inhibitors of such events, by blocking binding of full-length neuregulin molecules which could induce these morphogenetic events.

This process may be more fully described with regard to Figure 1 A, which is a schematic block diagram of the presumed interaction of the ED fragment with the ErbB signaling pathway according to the present invention. Without wishing to be limited by a single hypothesis, the full-length ligand of neuregulin binds to both of the ErbB-3 and ErbB-2 receptors, and also binds to an HSPG molecule. The combined binding to these three components induces the full array of morphogenetic and other signaling effects by neuregulin. On the other hand, the truncated ligand, such as the ED fragment or other portion of neuregulin, only results in partial signaling, and therefore cannot induce the full array of effects. Furthermore, since binding to all components of this system by neuregulin appears to be required, binding of the truncated ligand to only a portion of the components of the system, such as one or both of the ErbB-3 and ErbB-2 receptors, effectively inhibits the induction of the full signaling pathway by the full-length neuregulin ligand.

Figure 1C shows the domain structure of the transmembrane precursor of neuregulin itself, full length secreted neuregulin (NRG-FL), and recombinant truncated neuregulin (NRG- ED). The full length secreted ligand is effective for induction of the full signaling pathway, while the truncated ligand, such as NRG-ED, cannot induce the full signaling cascade, yet is still able to partially bind to the receptor. Therefore, the inhibitory agent of the present invention, such as the ED fragment of neuregulin for example, is able to suppress neuregulin-induced morphogenetic signals, and therefore can be used to suppress invasive growth and metastases of tumors, as well as for the suppression of neovascularization in tumors. The latter effect is supported by the finding that full-length neuregulin induces the formation of alveolar structures in the course of mammary gland development, and has also been shown to induce the formation of multicellular ring-like structures in vitro. Therefore, without wishing to be limited to a single hypothesis, inhibition of neuregulin effects would be expected to include the suppression of neovascularization, which also depends upon the formation of tube-like structures for the blood vessels. According to other embodiments of the present invention, the inhibitory agent may optionally include any portion, or combination of portions, of neuregulin which inhibit one or more of cell migration, cell proliferation, invasive cell growth and neovascularization. The inhibitory agent may be any type of peptide as previously described, including homologues, analogues, derivatives or mimetics thereof, or any biologically active substance having a substantially similar effect as previously defined. According to particularly preferred embodiments of the present invention, the inhibitory agent is the ED fragment of neuregulin, and/or homologues, analogues, derivatives or mimetics thereof.

EXAMPLE 1 IN VITRO EXPERIMENTAL DATA

This section describes in vitro experimental data, supporting the concept that the ED fragment can only partially induce signaling of the neuregulin-controlled pathway, and specifically is inhibitory for the morphogenetic effects of neuregulin.

Materials and Methods

Cell culture, transfections, and ligand treatment

Sublines of the 32D murine hematopoietic progenitor cell line 63 that express various ErbB-proteins were established through a two-step transfection protocol with ErbB-expression vectors, as described 50. 32D ErbB-2/ErbB-3 cells were routinely cultivated in RPMI medium supplemented with antibiotics, 10% heat-inactivated fetal bovine serum and 0.1% medium that was conditioned by IL-3 -producing cells. 32D cells do not attach or spread on tissue culture dishes or on glass coverslips. The experiments were performed with binuclear 32D cells, produced by cytochalasin D pretreatment as it was originally described 41. The various ligands were added to the cultures for 1 hour in serum-free medium and the cells were fixed.

FL4-ErbB-3 cells and human breast carcinoma (T47D, 6 ) _were described previously 41. To produce cells expressing both N-cadherin and either wild type or heparin binding site mutated ErbB-3 (mut ErbB-3), the FL4 subline was used (Levenberg et al., 1998).

Site-directed mutagenesis and construction of a heparin-binding mutant of ErbB-3

The putative heparin-binding domain of ErbB-3 located at amino acids 466-472 (KHNRPRR), was replaced with the homologous region from ErbB-1 (EGFR) [amino acids 467- 473 (ISNRGEN)] which totally lacks any heparin-binding ability. Site directed mutagenesis was performed according to Higuchi et al., 1988. Briefly, first PCR was performed using pairs of primers containing the mismatched oligonucleotide and the corresponding 5' or 3' primer. The primer at the 5' was GAGATCACAGGTTACCTGAAC located at position 1372-1392 of the ErbB-3 cDNA and the primer at the 3' was CCCTCAGGGATCCACACTCC located at position 2374-2390. ErbB-3 cDNA served as a template. After completion of the primary PCRs, aliquots were size fractionated and isolated on 1% low melting agarose gel, and then served as target DNA for the secondary PCR with the 5' and 3' above primers. The final mutated PCR product (~1 kb) was cloned into a pGEM-T vector (Promega, Medison), sequenced, excised from the vector using BstE II - BamH I sites and cloned into the ErbB-3 cDNA, which was digested with the same restriction enzymes.

For construction of the soluble ErbB-3 mutant the above 5' primer and a reverse primer GAAGATCTGGTTTTGCCGATCAGCACC at position 2100-2118 containing an additional Bgl II restriction site, were used. The ErbB-3 heparin-binding mutant (mut ErbB-3) cDNA served as a template. The resulting PCR product was digested with BstE II - Bgl II and subcloned in place of the homologous region in the wild type ErbB-3 alkaline phosphatase expression vector (Tzahar E. et al., 1994).

Binding of soluble ErbB-3 and its heparin binding site mutant to heparin Conditioned medium, containing the extracellular part of both receptors expressed as alkaline phosphatase fusion protein in 293T cells, was incubated with heparin-Sepharose beads

0 for 18h at 4 C. After excess washing with PBS, the beads were washed three times with increasing concentrations of NaCl. All the samples were then washed twice with PBS and the bound receptor level was estimated according to the associated alkaline phosphatase activity determined by incubation with the phosphatase substrate p-Nitrophenyl phosphate (Sigma 104) and reading the absorbance at 410 nm.

Microscopic observations

4 For microscopic observations of FL4 cell derivatives, 5x10 cells were plated on 18 mm coverslips, maintained for 24 hours in DMEM with 10% bovine calf serum, washed once with serum-free DMEM and incubated for an additional 24 hours. At that time, compact cell colonies were formed. Neuregulin (20 ng/ml) or EGF-like domain of neuregulin (20ng/ml) was added in serum-free DMEM for an additional 1-3 hours. Recombinant rat neuregulin (NDF β4) expressed in CHO cells and recombinant EGF-like domain of βl NDF (NDF-βl 177-246) (Amgen Co,

USA ) were used. Monoclonal antibodies against ErbB-3 were described previously 4 ,

Biochemical and immunochemical procedures

Precipitation and Western blotting were performed as previously described 40,41 Polyclonal anti-NDF (Amgen, Co, USA), anti-α-tubulin antibody (clone DM-1A, Sigma, Israel), and anti-activated MAP kinase (Gabay et al., 1997), were used as primary antibodies. The secondary antibodies used were horseradish peroxidase (HRP)-labeled anti-mouse IgG (Amersham, UK), anti-human HRP (Jackson, USA). Radiolabeling of ligands, covalent crosslinking and ligand binding assays were done according to Tzahar et al. (1996). In all experiments, the protein loading was controlled by the staining of the same blots with the anti-α- tubulin antibody (clone DM-1 A, Sigma, Israel).

Microscopy and immunofiuorescence

Immunofluorescence staining was performed as described 40,41 Actin was stained with TRITC-labeled phalloidin (Sigma, Israel) and nuclei were stained with DAPI. The specimens were examined by fluorescence microscopy using a Zeiss Axiophot microscope (Zeiss, Germany) equipped with a lOOx/1.3 Planapochromat objective, a CCD camera (Photometries, USA), and the Priism software package (Applied Precision, Issaquah, WA) on a Silicon Graphics computer. Cell motility measurements

To assess the rate of cell motility, the phagokinetic track method was used 65. Cells were

3 seeded on eighteen-millimeter coverslips coated with colloidal gold at a density of 2x10 per coverslip and incubated for 4 h. The medium was then changed to serum-free medium, with or without β4 NDF (20 ng/ml), ED (20 ng/ml) or mixture of them (20 +200 ng/ml) and the cells were incubated overnight (20 h). Tracks were examined by dark field microscopy using a lOx objective. The areas of 40-50 randomly selected tracks were measured for each determination.

RESULTS

The morphogenetic effects of neuregulin require its heparin-binding domain. It is well established that the EGF-like domain of NRG is necessary and sufficient for the binding of the ligand to its receptors 16,17 _while Ig-like domain at the N-terminus is responsible for heparin binding 23,42,43 As determined herein, although the full length NRG 1 (β4 isoform of NDF that is termed hereafter "NRG-FL") is retained by heparin-agarose beads (Fig. IB, lane 1), the recombinant EGF-like domain (NRG-ED) consisting of amino acid residues 177-246 of human

NDF βl (NDF-βl i77_246) ^ does not bind heparin (Fig. IB, lane 2). As described in further detail below, the morphogenetic responses of cells to NRG-ED and NRG-FL were compared.

In previous studies 40,41 by t_ne inventors, a number of morphogenetic effects induced by NRG-FL (NDF β4) in cells of different types were described. The simplest of these effects is an increase of lamellipodial activity and spreading, induced by NRG-FL in hematopoietic 32D cells ectopically expressing ErbB-3 and ErbB-2 receptors (Fig 2 A,B). Treatment of these cells with NRG-ED did not induce increase of cell spreading. While about 90% of round cells became spread and displayed characteristic actin recruitment into peripheral lamellipodia and ruffles upon 40 min incubation with NRG-FL (Fig. 2 B), only some activation of petaloid ruffles formation (not shown), but not cell spreading can be registered upon treatment with NRG-ED in the same conditions (Fig. 2 C). Moreover, treatment of cells with NRG-FL in the presence of excess of NRG-ED completely abolished NRG-FL effect (Fig. 2 D).

A more complex effect induced by NDF-FL is an activation of cell migration ability as visualized by phagokinetic track method. It has been shown that a variant of CHO cells stably transfected with ErbB-3 receptor and expressing ErbB-2 receptor endogenously (FL4-ErbB-3) became significantly more motile and produced larger phagokinetic tracks when treated with NRG-FL (Fig. 21,J,M). The same cells incubated with NRG-ED did not demonstrate any increase of migratory ability (Fig. 2 1,K,M). An excess of NRG-ED prevented the stimulatory effect of NRG-FL on cell migration (Fig. 2 1,L,M). Enhancement of cell motility induced by NRG-FL leads to the scattering of epithelioid cell colonies formed 24 hours after cell plating 40, χ_nls effect was observed in different cell types including FL4-ErbB-3, and also in a number of cultured human carcinomas, endogenously expressing ErbB-2 and ErbB-3. Treatment with NRG-ED, on the contrary, did not induce colony scattering and prevented scattering, induced by NRG-FL (not shown). As shown in previous studies by the inventors, treatment of cell colonies with NRG-FL can induce also other types of effects, namely the transformation of epithelioid colonies into the multicellular rings. These types of responses were observed when colonies were preincubated for longer time interval (more than 24 h) prior to the NRG-FL treatment and cell-cell adherens junctions and desmosoms became stabilized 40. Treatment of such colonies with NRG-FL led to an increase in lamellipodial activity of peripheral cells and spreading of the whole colony that culminated in formation of a central lumen-like gap (Fig. 2 E,F). The bulk of cell-cell adherens junctions are preserved in these ring-shaped complexes.

Treatment of the colonies with NRG-ED, however, did not induce formation of ring- shaped structures as demonstrated in experiments with FL4-ErbB-3 cells (Fig. 2 E,G) and several lines of cultured carcinoma cells (not shown). Moreover, when given together NRG-ED prevented NRG-FL-induced formation of ring-shaped complexes (Fig. 2 E,H). Thus, unlike NRG-FL, NRG-ED did not induce cell spreading, cell migration and colony reorganization and prevented these effects when added together with NRG-FL.

In spite of strict differences between morphogenetic effects of NRG-FL and NRG-ED, NRG-ED preserved the full ability to activate some other signal transduction pathways. Both NRG-FL and NRG-ED induced tyrosine phosphorylation of ErbB-3 receptor (not shown). Moreover, both NRG-FL and NRG-ED treatment led to transient activation of Erkl/Erk2 MAP kinase (Fig. 2 N). Combined treatment of cells with the mixture of NRG-FL and NRG-ED also led to the same response (not shown). ErbB-3 binds heparin and this binding is required for neuregulin-induced morphogenetic effects ErbB-3 receptor has a unique ability among the ErbB-family receptors to directly bind heparin as revealed by binding of soluble extracellular domains of ErbB 1 -ErbB-4 receptors to heparin-Sepharose (Fig. IB). Also, Figure 3 shows that ErbB-3 binds to heparin because of a specific heparin-binding motif. Figure 3 is a schematic diagram for showing a scheme of production of mutated ErbB-3 lacking the heparin-binding motif (Adar and Yayon, 2001). Putative heparin-binding motif in the extracellular portion of ErbB-3, 466KHNRPRR472, was substituted by corresponding region from ErbB-1 sequence (alignment was performed according to (reference number 45). Primary sequence analysis based on consensus heparin binding motifs predicted a putative linear heparin-binding site located at the carboxy-terminal end of the third extracellular subdomain 44,45₅ comprising a putative linker between the third flanking domain and the forth, cysteine rich domain. Primary sequence analysis based on consensus heparin binding motifs predicted a putative linear heparin-binding site located at the carboxy-terminal end of the third extracellular subdomain 44,45_^ comprising a putative linker between the third flanking domain and the forth, cysteine rich domain (Fig. 3).

To determine the capacity of the mutant ErbB-3 versus the wild type receptor to bind heparin, the extracellular part of both receptors, expressed as an alkaline phosphatase fusion protein in 293T cells was loaded onto heparin-Sepharose beads. Wild type ErbB-3 was retained on the column and could be eluted by using only high salt solution (0.4 M NaCl), implying the specific binding (Fig. 4 F). By contrast, the mutant receptor did not bind to the heparin beads (Fig. 4 G), demonstrating that this mutation in ErbB-3 completely abolished its heparin-binding capability.

To study the requirement of heparin for neuregulin-induced morphogenetic effects, a full- length wild type ErbB-3 and its heparin binding site mutant were stably expressed in FL4 cells. The selection of positive clones was performed by binding of iodinated NRG-ED, therefore selected stable transfectants containing mutated ErbB-3 receptor preserved the ability to bind NRG-ED in the same degree as transfectants expressing wild-type ErbB-3. Clones expressing the wild-type (8 clones) or the mutant (12 clones) receptors were tested for their ability to form multicellular ring-shaped complexes (Fig. 4 A-E). Clones expressing the wild-type receptor formed these ring-shaped complexes upon the addition of NRG-FL (Fig. 4 A, B, E). The mutant receptor on the other hand was much less efficient in induction of ring formation after NRG-FL addition (Fig. 4 C, D, E), although in some clones increase of spreading was apparent.

In spite of an impaired ability to induce morphogenetic response, mutated ErbB-3 receptor preserved the capacity to mediate signal, leading to activation of MAPK (Fig. 4 G). Moreover in this experiment we used truncated variant of the neuregulin (NRG-ED), lacking the heparin-binding site. Thus, heparin binding by either ErbB-3 receptor or its ligand NRG is dispensable for MAPK activation, but critically important for the induction of morphogenetic effects.

Heparin bearing molecules of cell surface as possible co-receptors for neuregulin

Heparin-binding molecules involved in transduction of NRG-mediated morphogenetic signals are most probably cell surface components. This can be inferred from the observation that in addition to ErbB-3, NRG-FL interacts with some other receptors of cell surface, while NRG-ED interacts exclusively with ErbB-3. In fact, binding of ¹²⁵I-NRG-FL (¹²⁵I-NDF β4) to FL4-ErbB-3 cells expressing ErbB-3 and ErbB-2 (but not ErbB-4) was not inhibited by addition of excess of specific ErbB-3 monoclonal antibodies 47 (pig, 5). ι_n the same time, binding of iodinated EGF-like domain of neuregulin (NRG-ED) can be efficiently suppressed by the excess of these antibodies (Fig. 5). Thus, there are sites on the cell surface, other than ErbB-3 receptors, which are involved in NRG-FL association with cells, while NRG-ED lacks an ability to bind to these sites.

Excess of exogenous heparin that competes with interaction of both ligand (NRG-FL) and its receptor (ErbB-3) with heparin-bearing molecules of the cell surface interfered with the morphogenetic effects of NRG-FL in a dose dependent manner. Thus, in the assay with formation of the ring-shaped multicellular complexes by FL4-ErbB-3 cells treated with NRG-FL, addition of 20 ng/ml of heparin significantly inhibited the effect, while 5 ng/ml was only partially active (Fig. 6). Addition of exogenous heparin (100 ng/ml) blocked ErbB-3 phosphorylation induced by NRG-FL completely, but did not interfere with ErbB-3 phosphorylation induced by NRG-ED (not shown). Finally, to elucidate a nature of the surface heparin-binding molecules involved in NRG-

FL morphogenetic signaling sodium chlorate, known to be an inhibitor of ATP-sulfurylase, was used as a tool to suppress sulfation of heparan sulfate 48,49, ι_n these experiments FL4-ErbB-3 cells were pretreated with different concentration of sodium chlorate for 24 h in serum free medium and then NRG-FL was added for additional 2 hours. In the absence of sodium chlorate, NRG-FL induced ring formation in more then 60% of cell colonies. Pretreatment with sodium chlorate and heparin reduced the percentage of ring-positive colonies in a dose- dependent manner (Figure 6A). Figure 6B shows the effect of sodium chlorate alone as a pretreatment of the formation of circular multicellular arrays. Colonies of ErbB-3 transfected FL4 cells were left untreated or pretreated with different concentrations of sodium chlorate (an inhibitor of HSPG formation), were then incubated with NRG-FL and percentages of the colonies converted into ring-shaped arrays were scored. However, NRG-FL treatment induced formation of rings in about 60% of control colonies, while pretreatment with sodium chlorate significantly inhibited this effect. Error bars denote SEM.

These results indicate to the involvement of HSPGs in the transduction of morphogenetic signal generated by NDF-FL.

Discussion

Signaling through NRG mediated by ErbB-2/ErbB-3 receptors induces two types of response in target cells. First, it induces a proliferative signal, which, without wishing to be limited by a single hypothesis, most probably occurs via activation of MAP kinase pathway 50. Second, NRG signaling via ErbB-2/ErbB-3 activates a variety of morphogenetic processes such as nerve cell migration 3 _ancl formation of alveolar structures in mammary gland development 35,37, A simple model of morphogenetic responses to NRG in culture has been described: formation of multicellular ring-shaped complexes 40,

ErbB-3 has impaired kinase activity, but contrary to ErbB-2 can efficiently bind NRG. According to the commonly accepted paradigm, binding of NRG to ErbB-3 promotes formation of ErbB-3/ErbB-2 dimers that transmit signals due to strong kinase activity of ErbB-2 10. However the function of ErbB-3 in the transduction of the morphogenetic signal is more complex than just transactivation of ErbB-2 since the activation of ErbB-2 alone, bypassing

ErbB-3, was not sufficient to produce the morphogenetic effect 41.

In the present work, it has been demonstrated that the morphogenetic effect of NRG depends on involvement of some heparin/heparan sulfate bearing molecules, and the major evidence for this conclusion was based on the unique ability of ErbB-3 receptor to bind heparin. The crucial experiment demonstrating the involvement of heparin binding in morphogenetic effects produced by NRG/ErbB-3 pathway was a substitution of heparin-binding site of ErbB-3 for analogous domain of ErbB-1, lacking the heparin-binding ability. It has been shown that this mutated ErbB-3 receptor lost its ability to transmit the NRG-dependent morphogenetic signal, but preserved the ability to activate MAPK.

Furthermore, it has been shown that not only ErbB-3, but NRG itself should interact with heparin/heparan sulfate in order to induce the complete morphogenetic effect. This conclusion is based on the comparison of the effects of full length and truncated NRG. NRGs share with other EGF family peptides a domain of homology that consists of approximately 50 amino acids. It is accepted that this domain is both necessary and sufficient for the ligand binding to the receptors and their activation. Moreover in the majority of experiments in vitro addressing the NRG- mediated signal transduction, EGF-like domain of NRG (NRG-ED) was usually used as a substitute of the full-length ligands. Functions of other domains of NRG molecules are less clear, but it was shown that N-terminal part of molecule containing Ig-like domain is responsible for the heparin binding 23,42 In the present work, it has been shown that morphogenetic effects of NRG, in contrast to its ability to activate MAPK, can be induced only by full-length ligand NRG-FL, but not by its EGF-like domain NRG-ED. Moreover, experiments with combined action of truncated and full- length NRG demonstrated that NRG-ED fragment efficiently prevents locomotory and morphogenetic responses induced by the full-length NRG. This inability of NRG-ED fragment of NRG to induce the morphogenetic effects correlates with the fact that NRG-ED, lacking N- tenninal part, does not interact with heparin.

Thus, without wishing to be limited by a single hypothesis, it can be suggested that optimal transduction of morphogenetic signal induced by NRG requires the interaction of both ligand and receptor with some heparin/heparan-sulfate-bearing molecules. Association with heparan sulfate proteoglycans is shown to be critical for the signal transduction mediated by a number of tyrosine kinase receptors such as FGF 51,52_{^ or} Met 53. However, the signaling by NRG via ErbB-3 seems to be unique as it can be dissected into MAPK activating pathway, that does not depend on heparin binding and "morphogenetic" pathway that requires the ability of both ligand and receptor to bind heparin. This conclusion is consistent with our previous data showing that MAPK activation is necessary but not sufficient for the morphogenetic effect of NRG 41.

Mechanisms of involvement of heparin/heparan sulfate-bearing molecules in the NRG- induced morphogenetic signaling are not clear yet. Without wishing to be limited to a single hypothesis, it appears that inhibition of heparan-sulfate synthesis by sodium chlorate as well as addition of exogenous heparin led to the block of morphogenetic reactions, suggesting the involvement of HSPG cell-surface molecules in the transmission of morphogenetic signals. These data are in agreement with the results showing that HSPG plays an important role in the response of Schwann cells to NRG [Sudhalter, 1996 #184] . HSPG molecules, in particular syndecans, are shown to be important for the processes of cell adhesion and migration 54,55 jt is documented that cytoplasmic parts of syndecan 1 and syndecan 4 are associated with actin cytoskeleton 54, Syndecan 1 mediates morphological and cytoskeletal reorganization and involved in the process of spreading of Schwann cells 56 a d lymphoblastoid cells 57, Syndecan 4 in cooperation with integrins is involved in Rho-dependent assembly of focal adhesions and stress fibers 58, it was shown that syndecan 2 also can alter cell morphology by inducing maturation of dendritic spines in rat hypocampal neurons 59, Another candidate for the role of a co-receptors of ErbB-3 in NRG-induced morphogenetic signaling are heparan sulfate proteoglycan isoforms of the CD44 hyaluronan receptor. Activation of CD44 by hyaluronic acid was recently shown to induce formation of lamellipodia and cell migration 60,61 There are some indications that scatter factor HGF/SF can also co-operate with CD44 via its heparan sulfate chain 62,

In conclusion, without wishing to be limited to a single hypothesis, it may be possible that certain heparin-bearing cell surface molecules can work as co-receptors for NRG, being also associated with the ErbB-3 receptors. This allows formation of multimolecular complexes gathering ErbB-3, ErbB-2, and NRG on the same molecular scaffold. Alternatively there is a potential possibility that complex of ErbB-3 receptor and NRG can cluster some heparin-bearing molecules of the cell surface or even induce their phosphorylation due to the activity of associated ErbB-2 receptor kinase.

EXAMPLE 2

ΓN VΓVO EXPERIMENTAL DATA

This section describes further experimental data for in vivo studies, demonstrating that the ED fragment of neuregulin is also able to inhibit tumor size in mice as an animal model. Thus, this inhibitory agent according to the present invention is clearly able to at least reduce cancerous growths in a mammalian model, and therefore is an effective treatment for cancer, regardless of the mechanism of action thereof.

Figure 7 shows the clear inhibitory effect of ED fragment on tumor size in mice as an animal model. The experimental procedure was performed as follows.

Six to 8-week old nude mice (CD-I) were implanted in the upper part of the front leg with FL4-ErbB-3 cells (2*10⁷ cells/mice) or with human gastric carcinoma (10⁷ cells/mice). Three days after implantation six animals in each group were injected with 10, 100 or 300 ng/mice of ED in 300μl of serum-free DMEM, or 300 μl of serum-free DMEM was injected as control of treatment. All injections were done in the upper part of the front leg. The treatment was done three times at 3, 7 and 12^th day after cell implantation. Tumor volume (XxYxZ) was measured weekly starting at the day of the last injection and ending around two months after cell implantation. Each point is the result of measurement of 6 animals (±SEM). As shown, the ED fragment had a clear dose-dependent inhibitory effect against the growth of tumors in the mice, which is particularly striking when compared to tumor growth in the control mice.

EXAMPLE 3 FURTHER EXPERIMENTAL DATA This section describes further experimental data, again supporting the effect of the ED fragment for only partially inducing signaling of the neuregulin-controlled pathway as the non- limiting hypothesis for the mechanism of action of blocking the ability of neuregulin to induce that signaling pathway.

First, it has been found that the EGF-like domain of NDF βl (ED) does not induce colony scattering. In particular, with regard to the effect of β4 NDF and ED on the scattering of cell colonies, the ED fragment was shown to be unable to induce such scattering. Cells of N87, human gastric carcinoma, T47D, human breast carcinoma and FL4-ErbB-3, Chinese hamster ovary cells transfected with ErbB-3 and N-cadherin c-DNA's were incubated for 24 h in serum free medium, or in serum free medium with 20 ng/ml of β4 NDF or with 20 ng/ml of ED. After incubation cells were fixed and stained with DAPI to visualize the distribution of cell nuclei. ED did not induce scattering of cell colonies in concentration from 0.1 to 100 ng/ml (Table 1).

Table 1 ED NDF βl does not induce colony scattering.

Next, the neuregulin EGF-like domain (ED) was shown to prevent spreading induced by full-length neuregulin (β4 isomer type; NDF-FL). For the effect of neuregulin (NDF-FL) and its EGF-like domain (ED) on cell spreading of 32D hematopoetic cells ectopically expressing ErbB- 2 and ErbB-3 receptors, 32D ErbB-2/ErbB-3 cells were fixed after 150 min incubation in serum free medium or after 90 min pre-incubation in serum-free medium and subsequent 60 min in the medium containing 20 ng/ml NDF-FL, 20 ng/ml of ED, or a mixture of 20 ng/ml of NDF-FL and 200 ng/ml of ED. Fluorescent staining with rhodamin-phalloidin and DAPI, visualizing F-actin (red) and nuclei (blue) respectively and scanning electron microscopy demonstrated significant spreading and formation of actin-positive ruffles after NDF-FL treatment and absence of spreading after treatment with ED or with mixture of NDF-FL and ED. Furthermore, the ED fragment was shown to have an inhibitory effect on neuregulin- induced colony morphogenesis and cell migration.

Colonies of FL4-ErbB-3 cells produced after 48 h incubation in the culture were treated for additional 24 h with 20 ng/ml NDF-FL, 20 ng/ml ED, mixture of 20 ng/ml NDF-FL and 200 ng/ml ED, or left untreated. After fixation cultures were stained with rhodamin phalloidin and DAPI. Addition of 10-fold excess of ED over NDF-FL prevented formation of multicellular ring- shaped complexes. Phagokinetic tracks of individual FL4-ErbB-3 cells incubated in either serum free medium or in the serum free medium incubated for 24 hours with 20 ng/ml of NDF-FL, 20 ng/ml of ED and mixture of 20 ng/ml of NDF-FL with 200 ng/ml of ED are shown. Average areas (±SEM) are presented; 50 tracks were measured for each type of treatment.

EXAMPLE 4 METHODS AND COMPOSITIONS FOR ADMINISTRATION The peptides of the present invention, and their homologues or related compounds, hereinafter referred to as the "therapeutic agents of the present invention", can be administered to a subject by various ways, which are well known in the art. Hereinafter, the term "therapeutic agent" includes a peptide as previously defined, including homologues, analogues or mimetics thereof, or any biologically active substance having a substantially similar effect as previously defined.

Hereinafter, the term "treatment" includes both the prevention of the genesis of the condition to be treated, as well as the substantial reduction or elimination of effects and/or symptoms associated with the condition.

Hereinafter, the term "subject" refers to the human or lower animal to whom the therapeutic agent is administered. For example, administration may be done topically (including opthalmically, vaginally, rectally, intranasally and by inhalation), orally, or parenterally, for example by intravenous drip or intraperitoneal, subcutaneous, or intramuscular injection. Formulations for topical administration may include but are not limited to lotions, ointments, gels, creams, suppositories, drops, liquids, sprays and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, sachets, capsules or tablets. Thickeners, diluents, flavorings, dispersing aids, emulsifiers or binders may be desirable.

Formulations for parenteral administration may include but are not limited to sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

Dosing is dependent on the severity of the symptoms and on the responsiveness of the subject to the therapeutic agent. Persons of ordinary skill in the art can easily determine optimum dosages, dosing methodologies and repetition rates. EXAMPLE 5 METHOD OF TREATMENT OF CANCEROUS CONDITIONS As noted above, the therapeutic agents of the present invention are useful for the treatment of cancerous and pre-cancerous conditions. The following example is an illustration only of a method of treating a cancerous condition with the therapeutic agent of the present invention, and is not intended to be limiting.

The method includes the step of administering a therapeutic agent, in a pharmaceutically acceptable carrier as described in Example 4 above, to a subject to be treated. The therapeutic agent is administered according to an effective dosing methodology, preferably until a predefined endpoint is reached, such as the absence of a symptom of the cancerous condition in the subject, or the prevention of the appearance of such a symptom in the subject.

The therapeutic agent may also optionally (additionally or alternatively) be characterized as an inhibitory agent for blocking a growth-factor mediated process, wherein the process is characterized by requiring binding to a plurality of receptors, comprising an agent for binding to at least one receptor of the plurality of receptors, wherein the agent is not capable of binding to all of the plurality of receptors, such that the agent inhibits the process.

It will be appreciated that the above descriptions are intended only to serve as examples, and that many other embodiments are possible within the spirit and the scope of the present invention,

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Claims

WHAT IS CLAIMED IS:

1. An inhibitory agent for a neuregulin-mediated process, comprising a peptide derived from at least a portion of a neuregulin molecule, or homologues, analogues or mimetics thereof.

2. The inhibitory agent of claim 1 , wherein said peptide is derived from the ED fragment of said neuregulin molecule.

3. A therapeutic agent for treating a cancerous condition in a subject, comprising a peptide derived from at least a portion of a neuregulin molecule, or homologues, analogues or mimetics thereof.

4. The therapeutic agent of claim 3, wherein said peptide is derived from the ED fragment.

5. The therapeutic fragment of claim 3, wherein said peptide has at least one of an effect selected from the group consisting of suppression of invasive growth, suppression of metastases of tumors, and suppression of neovascularization in tumors.

6. The therapeutic fragment of claim 3, wherein said peptide is derived from any portion of neuregulin having the ability to bind to at least one of the ErbB-3 and ErbB-2 receptors, but lacking the ability to induce a morphogenetic event.

7. A method for treating a cancerous condition in a subject, comprising the step of administering a peptide derived from at least a portion of a neuregulin molecule, or homologues, analogues or mimetics thereof.

8. The method of claim 7, wherein said peptide is derived from the ED fragment.

9. The method of claim 7, wherein said peptide has at least one of an effect selected from the group consisting of suppression of invasive growth, suppression of metastases of tumors, and suppression of neovascularization in tumors.

10. The method of claim 7, wherein said peptide is derived from any portion of neuregulin having the ability to bind to at least one of the ErbB-3 and ErbB-2 receptors, but lacking the ability to induce a morphogenetic event.

11. An inhibitory agent for blocking a growth-factor mediated process, wherein the process is characterized by requiring binding to a plurality of receptors, comprising an agent for binding to at least one receptor of said plurality of receptors, wherein said agent is not capable of binding to all of said plurality of receptors, such that said agent inhibits the process.

12. The inhibitory agent of claim 11 , wherein said agent is derived from the growth factor mediating the process.

13. The inhibitory agent of claim 12, wherein said agent is a peptide derived from at least a portion of a neuregulin molecule.

14. The inhibitory agent of claim 13, wherein said peptide is the ED fragment of neuregulin.

15. The inhibitory agent of any of claims 11-14, wherein the process is a cell proliferative process.

16. The inhibitory agent of any of claims 11-14, wherein the process is an apoptotic process.

17. The inhibitory agent of any of claims 11-14, wherein the process is a mitogenic process.

18. The inhibitory agent of any of claims 11-14, wherein the process is a morphogenetic process.

19. The inhibitory agent of any of claims 11-14, wherein said peptide has at least one of an effect selected from the group consisting of suppression of invasive growth, suppression of metastases of tumors, and suppression of neovascularization in tumors.

20. A composition for treating a cancerous condition in a subject, wherein the cancerous condition is affected by a growth factor, the growth factor mediating a process, wherein the process is characterized by requiring binding to a plurality of receptors, the composition comprising: an agent for binding to at least one receptor of said plurality of receptors, wherein said agent is not capable of binding to all of said plurality of receptors, such that said agent inhibits the process.

21. The composition of claim 20, wherein said agent is a peptide derived from at least a portion of a neuregulin molecule, or homologues, analogues or mimetics thereof.

22. The composition of claims 20 or 21 , wherein said agent is a peptide derived from a plurality of portions of said neuregulin molecule, or homologues, analogues or mimetics thereof.

23. An inhibitory agent for inhibiting a neuregulin-mediated process, comprising a peptide derived from at least a portion of a neuregulin molecule, or homologues, analogues, derivatives or mimetics thereof.