FUSION PROTEINS WITH CYTOTOXIC ACTIVITY AGAINST CELLS OVEREXPRESSING erbB3 AND/OR erbB4

Abstract: The present invention provides a ligand toxin which has cytotoxic activity against cells overexpressing erbB3 and/or erbB4. The ligand toxin comprises a peptide having or including an amino acid sequence which binds to erbB3 and/or erbB4 fused to a toxin. In a preferred form the peptide has or includes an amino acid sequence substantially homologous to that of the epidermal growth factor-like domain of heregulin beta 2. It is also preferred that the toxin is Pseudomonas exotoxin or a derivative thereof.

Description: Fusion Proteins with Cytotoxic Activity Against Cells Overexpressing erbB3 and/ or erbB4 The present invention relates to a ligand toxin comprising the epidermal growth factor-like domain of heregulin β2 fused to a modified Pseudomonas exotoxin (PE40). The present invention further relates to pharmaceutical compositions including the ligand toxin and to methods of treating cancers, particularly breast cancers, in which cells often overexpress erbB2, erbB3 and erbB4 (erbB2-4), alone or in combination. Antibodies directed against erbB2 are known, e.g., 4D5 monoclonal antibody (Hudziak et al. Mol. Cell. Biol. 9, 1165-1172, 1989) and the use of these antibodies in anti-cancer therapy has been proposed. 4D5 specifically targets cells which overexpress erbB2 and inhibits their growth. The advantages of this approach are specificity towards erbB2 overexpressing cells and lack of toxic side effects. The disadvantages are that the approach is limited to cells which overexpress erbB2 and that the effect is cytostatic rather than cytotoxic. The use of immunotoxins directed against erbB2 has also been proposed, e.g., e23-LysPE40 (Batra et al. Proc. Natl. Acad. Sci. USA 89, 5867- 5871, 1992), TA-1-ricin (Rodriguez et al. Am. J. Obstet. Gynecol. 168, 228- 232, 1993). These are specific towards erbB2 overexpressing cells and cytotoxic; however there may be more toxic side effects than with e.g. 4D5 alone. The cloning and characterisation of a family of polypeptides proposed to represent natural erbB2 ligands (termed in this report the heregulins, HRGs) was first reported by Wen et al. (Cell 69, 559-572, 1992) and Holmes et al. (Science 256, 1205-1210, 1992). Members of this family have subsequently been cloned by two other groups; Marchionni et al. (Nature 362. 312-318, 1993) and Falls et al. (Cell 72. 801-815, 1993). However, recent work has shown that the HRGs do not bind directly to erbB2. Instead, erbB3 and erbB4 function as receptors for these ligands, the observed tyrosine phosphorylation of erbB2 following HRG binding occurring as a consequence of heterodimerisation with these receptors (Plowman et al. Nature 366 473-475, 1993; Carrawav et al. J. Biol. Chem. 269, 14303-14306, 1994; Slikowski et al. J. Biol. Chem. 269, 14661-14665, 1994). Two groups reported that the HRGs induce growth inhibition and phenotypic differentiation of human breast cancer cells overexpressing erbB2 (Wen et al., Cell 69, 559-572, 1992: Bacus et al.. Cell Growth Differ. 3, 401-411). This therefore represents a potential therapeutic strategy with minimal side effects. However, Holmes et al. only observed proliferative effects of the HRGs and the present inventors own unpublished data supports this finding. HRG-toxins represent an alternative approach for targeting human breast cancer cells and compared to anti-erbB2 immunotoxins should be easier to produce (due to the smaller size of the HRG component compared to an antibody and the suitability of high yield, prokaryotic expression systems) and less antigenic. The erbB2 receptor is overexpressed in approximately 20% of human breast cancers (Slamon et al. Science 244, 707-712, 1989) and also in other cancers including ovarian, gastric and colonic, (Beschuk et al. Cancer Res. 50, 4087-4091, 1990; Kameda et al. Cancer Res. 50, 8002-8009, 1990; D'Emilla et al. Oncogene 4, 1233-1239, 1989) making it an attractive target for therapy. The preliminary characterisation of the HRGs as erbB2 ligands and as factors which inhibit the proliferation and promote the differentiation of human breast cancer cells overexpressing erbB2 led the present inventors to attempt the recombinant production of these ligands and to investigate their therapeutic potential. The finding of the present inventors that the HRGs stimulated the proliferation of human breast cancer cells, including some cell lines which overexpress erbB2, led the present inventors to focus on HRG-toxins as a therapeutic strategy. The recent identification of the HRGs as ligands for the erbB3 and erbB4 receptors led the present inventors to predict that the HRG-toxins would target a wider range of human breast cancer cells than if the HRGs were ligands for erbB2 alone. This is because the erbB3 and erbB4 receptors are also often overexpressed by human breast cancer cells (Kraus et al. Proc. Natl.Acad Sci (USA) 86, 9193-9197. 1989; Plowman et al. Proc.Natl Acad Sci (USA) 90, 1746-1750, 1993), and these cells may not also overexpress erbB2. This hypothesis has now been confirmed. The present inventors have constructed a ligand toxin consisting of the epidermal growth factor-like domain of heregulin β2 (HRGβ2, amino acids 177-237) fused to a modified Pseudomonas toxin (PE40). The HRGβ2- PE40 ligand toxin was engineered and expressed using the pFlag prokaryotic expression system. HRGβ2-PE40 induced the tyrosine phosphorylation of erbB2 and erbB3 receptors on MCF-7 human breast cancer cells at concentrations as low as 20 pM. The ligand toxin did not significantly inhibit the growth of 184B5 immortalised normal breast epithelial cells, which express low or undetectable levels of erbB2, erbB3 and erbB4 receptors. However, in growth experiments using human breast cancer cells which overexpress erbB3 and/or erbB4 relative to normal breast epithelial cells, addition of HRGβ2-PE40 produced a marked cytotoxic effect relative to the addition of HRGβ2 or PE40 alone. For example, for ZR-75-1 human breast cancer cells, which overexpress erbB2-4 relative to normal breast epithelial cells, the IC 50 (the concentration of ligand-toxin required for a 50% reduction in cell numbers relative to control cultures) for HRGβ2-PE40 was 2-4pM compared to an IC 50 for PE40 alone of 2500pM. These results suggest that HRGβ2-PE40 represents a novel therapeutic strategy against human breast, or other, cancers which overexpress erbB2-4 alone or in combination. Accordingly, in a first aspect the present invention consists in a ligand toxin, the ligand toxin comprising a peptide having or including an amino acid sequence which binds to erbB3 and/or erbB4 fused to a toxin. In a preferred embodiment of the present invention the peptide has or includes an amino acid sequence substantially homologous to that of the epidermal growth factor-like domain of heregulin β2. The toxin may be any of a large number of toxins known in the art, however, it is presently preferred that the toxin is Pseudomonas exotoxin or a derivative thereof. It is particularly preferred that the toxin is PE40. In yet a further preferred embodiment of the present invention the ligand toxin has an amino acid sequence substantially as shown in Figure 1. In a further preferred embodiment the ligand toxin is produced recombinantly. In a second aspect the present invention consists in a vector, the vector including a DNA sequence encoding a ligand toxin having an amino acid sequence substantially as shown in Figure 1. In a third aspect the present invention consists in a composition for use in treating a cancer in which there is an overexpression of erbB3 and/or erbB4, the composition comprising the ligand toxin of the first aspect of the present invention and an acceptable carrier. In a fourth aspect the present invention consists in a method of treating a cancer in which there is an overexpression of erbB3 and/or erbB4 in a subject comprising administering to the subject an effective amount of the composition of the third aspect of the present invention. In a preferred embodiment of this aspect of the present invention the subject is suffering from breast cancer. In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following examples and Figures: Fig. 1. Complete amino acid sequence of the mature HRGβ2-PE40 ligand toxin encoded by the pFLAG/HRGβ2-PE40 expression vector. After cleavage of the ompA peptide the mature ligand toxin protein is expressed in the periplasmic space of the transformed bacteria. Purification of this recombinant protein is then performed by anti FLAG-affinity chromatography. The 8 amino acid FLAG peptide is double underlined. The HRGβ2 protein sequence is in bold type. A PE40 leader sequence including the Hindiπ cloning site is underlined and the 361 amino acid PE40 protein sequence is displayed in normal type. The translation termination codon is indicated by an asterisk. Fig. 2. HRG-induced tyrosine phosphorylation of erbB2-4. Monolayer cultures of MCF-7 human breast cancer cells were serum starved for 18h in RPMI 1640/0.5% FCS before addition of each recombinant HRG isoform (α, βl, β2 or β3) at the doses indicated. After a further 20 min at 37°C, the cells were lysed and aliquots of the cell extracts subjected to Western blot analysis using an anti-phosphotyrosine antibody. The intensity of the major 180-185kDa band corresponding to erbB2-4 was estimated by densitometry and is represented graphically, (α - D; βl - •; β2 - Δ; β3 - ▼) Fig. 3. Comparison of HRGβ2- and HRGβ2-PE40-induced tyrosine phosphorylation of erbB2-4. Monolayer cultures of MCF-7 human breast cancer cells were serum starved for 18h in RPMI 1640/0.5% FCS before addition of HRGβ2 or HRGβ2-PE40 at the doses indicated. After a further 20 min at 37°C the cells were lysed and aliquots of the cell extracts subjected to Western blot analysis using an anti-phosphotyrosine antibody. The intensity of the major 180-185kDa band corresponding to erbB2-4 was estimated by densitometry and is represented graphically. (HRG - D; HRG-PE40 - •) Fig. 4. Effects of HRG β 2 or HRGβ2/PE40 on the rate of proliferation of 184B5 immortalized human breast epithelial cells and human breast cancer cells. Cells were dispensed into individual wells of 96-well culture plates. HRGβ2, HRGβ2/PE40 or PE40 (0.5 pM - 5nM) were added at Day 0 and cell numbers were evaluated at Days 3, 5 and 7 using an indirect MTT assay. The graph shows typical results for doses giving the maximum response (HRGβ2 InM; HRGβ2/PE40 5nM; PE40 5nM; control vehicle alone). Each experiment was performed three or four times with essentially identical results. (A - 184B5; B - T-47D; C - MCF-7; D - ZR-75-1; Control - D; HRGβ2 - «; HRGβ2/PE40 - Δ; PE40 - ♦;) Fig. 5. Dose response for HRGβ2-PE40-induced cytotoxicity on 184B5 immortalized human breast epithelial cells and ZR-75-1 human breast cancer cells. Cells were dispensed into individual wells of 96-well culture plates. HRGβ2, PE40 or HRGβ2/PE40 (0.5pM - 5nM) were added at Day 0 and cell numbers determined on Day 7 using an indirect MTT assay. Cell numbers are presented as a percentage of control values. (A - 184B5; B - ZR-75-1; HRGβ2 - D; HRGβ2/PE40 - •; PE40 - Δ) Isolation of cDNA fragments encoding the EGF-like domains of HRG isoforms The guanidinium isothiocyanate-caesium chloride procedure was used to isolate total RNA from the human breast cancer cell line MDA-MB- 231, which expresses high levels of the HRGs. Poly (A) + RNA was then prepared by oligo dT cellulose chromatography on Dynabeads (Dynal). First strand cDNA was synthesized using an oligo dT primer and M-MuLV reverse transcriptase. Double stranded cDNA was then prepared using RNase H and DNA polymerase I and used as a template for the synthesis of HRG-derived cDNAs by PCR. cDNA synthesis reagents were obtained from Clontech; PCR reagents were obtained from Perkin-Elmer Cetus. Amplification of DNA fragments utilised a forward primer common to each isoform ,5'-AGACTAAGCTTAGCCATCTTGTAAAATGTGCG-3') containing a five nucleotide leader sequence and a Hindlll site (underlined) to facilitate sub- cloning, and reverse primers common to isoforms α, βl and β2 f5'-AGACTAAGCTTTCACTGGTACAGCTCCTCCGC-3' or β3 f5'-AGACTAAGCTTCTATTCAGGCAGAGACAGAAAGGG-31 containing a leader sequence, a HindlΗ site (underlined) and an in-frame termination codon (double underlined). In order to clone cDNAs suitable for the production of HRG-PE40 ligand toxins, the following reverse primer was used in which the stop codon was deleted; 5'-AGACTAAGCTTCTGGTACAGCTCCTCCGC-3'. These cDNA fragments encode the EGF-like domain of the HRGs which has previously been shown to be responsible for biological activity (Holmes et al., Science 256, 1205- 1210, 1992). Assembly of HRG and HRG-toxin expression vectors PCR products encoding each HRG isoform were digested with Hindlll and ligated into the corresponding site in the pFLAG-1 expression vector. After transformation into E. coli DH5α, transformants harbouring plasmids encoding particular HRG isoforms were identified by sequencing of small scale plasmid preparations (Sequenase Version 2.0, USB). For assembly of the ligand toxin expression vector, a 1184 bp Hindϋl/EcoRI cDNA fragment encoding Pseudo onas exotoxin PE40 was ligated into the corresponding sites in pFLAG-1. This initial construct, when expressed in E. coli DH5, produced recombinant PΕ40 toxin which was used as a control for ligand toxin experiments. The HindEU cDNA fragment encoding HRGα or HRGβ2 was then inserted into this PE40 construct (creating HRGα- and HRGβ2-PE40, respectively). Expression of HRG isoforms and HRG-PE40 in E. coli Recombinant bacteria were grown at 37°C with shaking until the culture reached an OD eoo of 0.8. IPTG was then added to 500 μM and the culture incubated for a further 2h. In an initial experiment to determine the level of expression in each E. coli cell fraction, IPTG-induced cultures (100ml) were divided into three and the cells collected by centrifugation. The whole cell fraction was isolated by resuspension of one cell pellet in SDS-PAGE sample buffer. The second pellet was used for fractionation of the whole cell pellet into soluble or insoluble fractions by adding 5 ml of extraction buffer A (50 mM Tris-HCl pH 8.0, 5 raM EDTA, 0.25 mg/ml lysozyme, 50 μg/ml NaN 3 ) followed by 0.5ml of extraction buffer B (1.5 M NaCl, 0.1 M CaCl 2 , 0.1 M MgCl 2 , 0.02 μg/ml DNAse I, 0.2 mM NaVO 3 , 0.2 mM PMSF, 0.2 mM leupeptin, 0.2 mM aprotinin). The resulting suspension was centrifuged at 18000 x g for lh giving the soluble (supernatant) and insoluble (pellet) cell fractions. The insoluble cell fraction was resuspended in SDS-PAGE sample buffer while an aliquot of the soluble fraction was mixed with an equal volume of 2x sample buffer. Finally, the third cell pellet was prepared for osmotic shock by resuspension in 40ml of OS buffer (0.5 M sucrose, 0.03 M Tris-HCl pH 8.0, 1 mM EDTA) and centrifugation at 3500 x g for 10 min at 10°C. The cells were then resuspended rapidly in 25ml of ice-cold water to release periplasmic proteins. Following centrifugation at 3500 x g for 10 min at 4°C, the supernatant was collected and a 50 μl aliquot mixed with an equal volume of SDS-PAGE sample buffer. After SDS-PAGE and transfer of proteins to nitrocellulose membranes, FLAG epitope-tagged proteins were detected by anti-FLAG M2 monoclonal antibody (10 μg/ml) followed by Enhanced Chemiluminescence (ECL) (Amersham). The M2 antibody detects FLAG proteins consisting of both ompA cleaved and non-cleaved forms. Recombinant protein was detected in all fractions for both the different HRG isoforms and the HRG-derived ligand toxins. In the case of the HRGs, proteins of the expected mobility (approximately 7kDa) were produced. The HRG-PE40 ligand toxins migrated at a relative mobility of 52kDa due to the aberrant migration of PE40, which migrates at an apparent molecular weight of approximately 47kDa when expressed alone. Since the protein found in the periplasmic fraction is the simplest to extract and is most likely to be correctly folded and biologically active, affinity purification was performed on periplasmic extracts. Purification of recombinant HRG and HRG-PE40 ligand toxin For each HRG isoform, HRG-PE40 or PE40 toxin alone, protein expressed in the periplasmic space was isolated and purified by affinity chromatography using the anti-FLAG Ml monoclonal antibody which binds only ompA-cleaved FLAG fusion proteins. Since this antibody binds to the FLAG peptide only in the presence of calcium, the periplasmic cell fractions were made up to 2 mM CaCl 2 in 50 mM Tris-HCl (pH 7.5) prior to column application. Bound proteins were eluted in 50mM Tris-HCl (pH 7.5) containing 2 mM EDTA and analysed for size and purity by Western blot and Coomassie staining of SDS-PAGE gels. For each protein, a readily soluble protein was purified to an essentially single band by SDS-PAGE analysis. In general, yields of purified protein were in the range of 0.2-2.0 mg/1 of culture. However, for the HRG isoforms, the highest yield of soluble protein was consistently obtained with the β2 isoform. with yields of up to 3.0 mg/1 of culture. HRG and HRG-PE40 stimulation of erbB receptor tyrosine phosphorylation The MCF-7 human breast cancer cell line was maintained as previously described (Buckley et al., Oncogene 8, 2127-2133, 1993) and grown to near confluence in 6-well tissue culture plates. The cells were then starved for 18h in medium containing 0.5% FCS. Recombinant HRG or HRG-PE40 was then added in 100 μl of TBS/0.05% BSA (20 pM to 10 nM final concentration). Control wells received vehicle alone. Following a 20 min incubation at 37°C, the medium was removed by aspiration and cells were lysed in 200 μl of lysis buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.2 mM Na 3 VO 4 , 1 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml aprotinin). Following incubation on ice for 5 min, cellular debris was removed by centrifugation (10,000 x g, 5 min, 4°C). In order to determine the tyrosine phosphorylation levels of erbB 2-4 following HRG or HRG-PE40 administration, samples of the resulting cell lysates were separated by SDS-PAGE, transferred to nitrocellulose, and Western blotted with a monoclonal antibody against phosphotyrosine (PY20, Transduction Laboratories). Detection of the bound antibody was by ECL (Amersham). Since erbB2-4 migrate upon SDS-PAGE analysis with a similar mobility (180-185kDa), the combined tyrosine phosphorylation levels of these receptors can be determined by analysis of this region of the gel. The ability of the HRG isoforms to activate erbB 2-4 receptors was evident from the induction of a tyrosine phosphorylated band at 180-185kDa in MCF-7 cells at HRG doses greater than 20 pM (Fig. 2). HRGs α, β2 and β3 produced an approximately 10 fold increase in erbB2-4 tyrosine phosphorylation at the highest concentration tested (10 nM). The ability of HRGβ2-PE40 to stimulate tyrosine phosphorylation of erbB2-4 was also tested using MCF-7 cells (Fig. 3). The ligand toxin also induced the tyrosine phosphorylation of proteins of 180-185 kDa in these cells, and importantly, a similar dose response of tyrosine phosphorylation to that induced by HRGβ2 was observed. These results indicate that despite the presence of PE40 at the carboxy- terminus of the HRG protein, the HRG domain retains similar activity in terms of activation of erbB2-4 to that of the recombinant HRG expressed alone. In order to determine which erbB receptors were activated, immunoprecipitation experiments were performed. ZR-75-1 human breast cancer cells were used since they express readily detectable levels of erbB2- 4. Cells were grown to near confluence in T150 tissue culture flasks. After serum starvation, HRGβ2 (lOnM) was added for 15 minutes. The cells were then lysed in 1ml of lysis buffer as described above. Antibodies (l-2μg) were incubated with lysates (100 μl of lysate for all reactions except for the detection of erbB4 in PY20 immunoprecipitates where 250μl was used) for at least 2 hours at 4°C. The immunocomplexes were then collected by incubation with goat anti-mouse IgG Sepharose or protein A-Sepharose beads (40μl) for at least lh at 4°C. Sepharose beads were collected by centrifugation and washed 3 times in cold lysis buffer, resuspended in SDS- PAGE sample buffer and subjected to SDS-PAGE. After transfer to nitrocellulose, the samples were Western blotted with the desired antibody. Western blotting of erbB2 and erbB3 immunoprecipitates from ZR- 75-1 cells with an antiphosphotyrosine monoclonal antibody revealed a marked increase in tyrosine phosphorylation of these receptors upon HRGβ2 (lOnM) administration. Since the anti-erbB4 antibody used in these studies is only suitable for Western blotting, the reciprocal experiment was performed in which anti-phosphotyrosine immunoprecipitates from control and HRG-stimulated cells were blotted with the anti-erbB4 antibody. HRG addition clearly resulted in increased recruitment of erbB4 into the anti- phosphotyrosine immunoprecipitate. Therefore these results demonstrate that erbB2-4 become tyrosine phosphorylated in ZR-75-1 human breast cancer cells upon HRG treatment. The same experiments were performed using cell lysates of MCF-7 breast cancer cells. Similar results were obtained except that insufficient expression levels of erbB4 precluded detection of activation by HRGβ2. The most obvious interpretation of these results is that recombinant HRGβ2 binds to erbB3 and erbB4 receptors on human breast cancer cells and induces their tyrosine phosphorylation. The observed tyrosine phosphorylation of erbB2 is probably due to heterodimerization with these receptors. These findings are in accordance with previous reports. Effects of HRGβ2 and HRGβ2-PE40 on the proliferation of immortalized human breast epithelial and human breast cancer cells 184B5 immortalized human breast epithelial cells (Stampfer and Yaswen, in Transformation of Human Epithelial Cells: Molecular and Oncogenetic Mechanisms, pp 117-140, Milo, Casto and Shuler Eds, CRC Press, 1992) and several human breast cancer cell lines were used for these studies. The erbB receptor status of these cell lines, derived from Northern and Western blot analysis (Janes et al., Oncogene 9, 3601-3608, 1994; Fiddes, Janes and deFazio, unpublished results) are shown in Table 1. The object of these experiments was therefore to determine the selectivity of HRGβ2-PE40 in targeting breast cancer cells overexpressing different combinations of erbB receptors. 184B5 immortalized human breast epithelial cells (in MEGM) or human breast cancer cells (in RPMI 1640 containing 5% FCS) were dispensed into individual wells of 96-well plates at an initial concentration of 103 cells/well in 50 μl of growth media, 3 days before (Day -3) the addition of recombinant HRGβ2, HRGβ2-PE40 or PE40. At Day 0, recombinant proteins were added in 50 μl of growth media to final concentrations ranging from 0.5 pM to 5nM. Controls received either an equivalent volume of 0 vehicle (0.5 μl of 0.1% BSA) in 50 μl of growth media or growth media alone. Table 1. Cytotoxicity of HRGβ2/PE40 ligand toxin and toxin component PE40 alone. Cell numbers are expressed relative to vehicle treated control cultures, 7 days after HRGβ2-PE40 addition, and IC 50 values represent the dose required for 50% reduction in cell numbers. Also indicated are the 5 relative expression levels of the EGFR and erbB2-4 receptors; - not detected; + very low levels; + + low expression; + + + moderate expression; + + + + high level of expression, sd; standard deviation of the mean. Receptor levels for the prostate cancer cell lines have not been determined. 0 HRGP2 PE40 PE40 Relative Cell IC-o (pM) Number (5 nM 1C 50 (pM) HRGβ2/PE40. % EGF erbB2 erbB3 erbB4 of control ± sd) (range) R Immortalised 184B5 + + + 100 ± 0.5 >5000 >5000 Normal Breast Epithelial ZR-75-1 + + ++ + + + + + + + 0+0 2-4 -2500 MDA-MB- . ++ + + + + + + 5+3 7-20 >5000 453 Human Breast T-47D + + + + + . + 7+2 50-100 -2500 Cancer SK-BR-3 + + + + + + + + + β+2 100-200 >5000 + MCF-7 + + + + + 18+.5 300-400 -5000 nCaP 3 200 >5000 Human Prostate Dul45 100 >5000 >5000 Cancer PC - 100 >5000 >5000 11 Three plates of identical treatments were prepared and one plate analysed for cell growth on each of Days 3, 5 and 7 after drug addition. Cell numbers (six wells for each treatment) were measured indirectly using a non-radioactive cell proliferation assay (Promega). The absorbance at 570 nm was recorded for each well using a Dynatech MR7000 Spectrophotometer. T-47D, MCF-7 and ZR-75-1 human breast cancer cells responded to HRGβ2 administration with increased proliferation rates compared to untreated controls (Figs 4 and 5). In the case of ZR-75-1 cells significant increases in cell number relative to control cells were observed at concentrations of 50 pM or greater (Fig. 5). In contrast 184B5 cells did not exhibit a proliferative response to HRGβ2 (Figs. 4 and 5). At all concentrations tested (0.5 pM to 5 nM), and at 3 initial plating densities (ranging from 200-1000 cells/well), no significant difference in cell number between the HRGβ2-treated and control cells was observed during the 7 day incubation period. Similar studies were performed in parallel with HRGβ2-PE40 and PE40 alone. The cytotoxicity of HRGβ2-PE40 towards human breast cancer cell lines displaying erbB3 and/or erbB4 overexpression relative to normal breast epithelial cells was demonstrated by a marked decrease in cell number relative to control cultures (Fig. 4 and 5, Table 1). Inhibition of cell proliferation by PE40 only occurred at very high concentrations (Table 1), indicating that the HRGβ2 component of the ligand toxin targets the toxin to the breast cancer cells. Two cell lines exhibiting marked erbB3 overexpression relative to normal breast epithelial cells, T-47D and MCF-7, demonstrated IC 50 values of 50-100 and 300-400 pM respectively. Thus HRGβ2-PE40 is capable of targeting cells overexpressing erbB3. However. ZR-75-1 cells were most sensitive to HRGβ2-PE40. exhibiting an IC 5ϋ value of 2-4 pM (Figs 4 and 5 and Table 1). Although these cells overexpress erbB2-4, the very high expression level of erbB4 is likely to be the major explanation for the increased sensitivity of these cells relative to T-47D or MCF-7 cells, since erbB4 constitutes a higher affinity receptor for the HRGs than erbB3 (Ttzahar et al. J. Biol. Chem. 269, 25226-25233. 1994). Importantly, when 184B5 cells were exposed to HRGβ2-PE40, no cytotoxic effects were seen at any dose (Figs. 4 and 5 and Table 1). The most obvious interpretation of these results, in light of the binding activity of HRGs towards erbB receptors, is that HRGβ2-PE40 selectively targets human breast cancer cells which overexpress erbB3 and/or erbB4, and that erbB2 overexpression may also be observed in these cell lines. Effect of HRGβ2-PE40 on the proliferation of human prostate cancer cells. Increased expression of erbB3 represents an early event in the development of prostatic adenocarcinomas (Myers et al. J. Nat.Cancer Inst. 86, 1140-1145). The present inventors were therefore interested in examining the sensitivity of human prostate cancer cell lines to HRGβ2-PE40. Three such cell lines, Ln-CaP, PC-3 and DU145 were subjected to increasing concentrations of HRGβ2-PE40, as described above for the breast cancer cell lines. Marked cytotoxicity of the ligand toxin was observed against the Ln-CaP cell line, with an IC 50 value of 200pM. Although the erbB receptor status of this cell line has not been ascertained, this IC 50 value is comparable to that observed for breast cancer cells which overexpress erbB3 e.g. T-47D, MCF-7. HRGβ2- PE40 is therefore capable of targeting a subset of prostate cancer cell lines, and, as with breast cancer cells, it is likely that the sensitivity of such cells is largely determined by the expression levels of erbB3 and erbB4. Since overexpression of erbB3 has also been observed in gastric (Sanidas et al. Int. J. Cancer 54, 953-940, 1993) and pancreatic cancers (Lemoine et al. J. Pathology 168. 269-273, 1992), HRGβ2-PE40 may also be used to target these types of cancer cell. HRGβ2-_Ε40 can be used to target cancer cells which overexpress erbB3 or erbB4, alone or in combination. It therefore represents a novel potential therapy for human breast cancer, since breast cancer cells often overexpress these receptors. It may also be effective against other cancer types which also overexpress erbB receptors, e.g., prostate, gastric and pancreatic cancers. These predictions are supported by laboratory-based data on the efficacy of HRGβ2-PE40. Thus in experiments in which the proliferation of either immortalised normal breast epithelial cells (184B5) or human breast cancer cells overexpressing erbB3 and/or erbB4 (e.g. MCF-7. T-47D and ZR-75-1 cells) were measured, HRGβ2-PE40 caused marked cytotoxic effects on the human breast cancer cells and not on the 184B5 control. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims: CLAIMS: 1. A ligand toxin, the ligand toxin comprising a peptide having or including an amino acid sequence which binds to erbB3 and/or erbB4 fused to a toxin. - 2. A ligand toxin as claimed in claim 1 in which the peptide has or includes an amino acid sequence substantially homologous to that of the epidermal growth factor-like domain of heregulin β2. 3. A ligand toxin as claimed in claim 1 in which the toxin is Pseudomonas exotoxin or a derivative thereof. 4. A ligand toxin as claimed in any one of claims 1 to 3 in which the toxin is PE40. 5. A ligand toxin as claimed in any one of claims 1 to 4 in which the ligand toxin has an amino acid sequence substantially as shown in Figure 1. 6. A ligand toxin as claimed in any one of claims 1 to 5 in which the ligand toxin is produced recombinantly. 7. A vector, the vector including a DNA sequence encoding a ligand toxin having an amino acid sequence substantially as shown in Figure 1. 8. A composition for use in treating a cancer in which there is an overexpression of erbB3 and/or erbB4, the composition comprising the ligand toxin as claimed in any one of claims 1 to 6 and an acceptable carrier. 9. A method of treating a cancer in which there is an overexpression of erbB3 and/or erbB4 in a subject comprising administering to the subject an effective amount of the composition as claimed in claim 8. 10. A method as claimed in claim 9 in which the subject is suffering from breast cancer. AMENDED CLAIMS [received by the International Bureau on 1 Apri l 1996 (01 .04.96) ; original claims 1 - 10 replaced by amended claims 1 - 9 ( 1 page) ] 1. A ligand toxin, the ligand toxin comprising a peptide having or including an amino acid sequence substantially homologous to that of the epidermal growth factor-like domain of heregulin β2 which binds to erb B3 and/or erb B4 fused to a toxin. 2. A ligand toxin as claimed in claim 1 in which the toxin is Pseudomonas exotoxin or a derivative thereof. 3. A ligand toxin as claimed in claim 1 or claim 2 in which the toxin is PE40. 4. A ligand toxin as claimed in any one of claims 1 to 3 in which the ligand toxin has an amino acid sequence substantially as shown in Figure 1. 5. A ligand toxin as claimed in any one of claims 1 to 4 in which the ligand toxin is produced recombinantly. 6. A vector, the vector including a DNA sequence encoding a ligand toxin having an amino acid sequence substantially as shown in Figure 1. 7. A composition for use in treating a cancer in which there is an overexpression of erbB3 and/or erbB4, the composition comprising the ligand toxin as claimed in any one of claims 1 to 5 and an acceptable carrier. 8. A method of treating a cancer in which there is an overexpression of erbB3 and/or erbB4 in a subject comprising administering to the subject an effective amount of the composition as claimed in claim 7. 9. A method as claimed in claim 8 in which the subject is suffering from breast cancer.

Inventors: DALY ROGER JOHN, FIDDES RODNEY JOHN, DALY, ROGER, JOHN, FIDDES, RODNEY, JOHN
Applicants: CRC FOR BIOPHARMACEUTICAL RESE, DALY ROGER JOHN, FIDDES RODNEY JOHN, CRC FOR BIOPHARMACEUTICAL RESEARCH PTY. LTD., DALY, ROGER, JOHN, FIDDES, RODNEY, JOHN
Application year: 1995
Main classification: C12N15
IPC codes: A61K38, A61P35, A61P43, C07K14, C07K19, C12N15, C12P21, C12R1