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  • C07K14/475—Growth factors; Growth regulators
  • C07K14/4756—Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
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  • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
  • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

• the present invention relates to a ligand toxin comprising the epidermal growth factor-like domain of heregulin ⁇ 2 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.
• 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.
• 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.
• HRG-toxins as a therapeutic strategy.
• the present inventors have constructed a ligand toxin consisting of the epidermal growth factor-like domain of heregulin ⁇ 2 (HRG ⁇ 2, amino acids 177-237) fused to a modified Pseudomonas toxin (PE40).
• the HRG ⁇ 2- PE40 ligand toxin was engineered and expressed using the pFlag prokaryotic expression system.
• HRG ⁇ 2-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.
• HRG ⁇ 2-PE40 produced a marked cytotoxic effect relative to the addition of HRG ⁇ 2 or PE40 alone.
• 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.
• the peptide has or includes an amino acid sequence substantially homologous to that of the epidermal growth factor-like domain of heregulin ⁇ 2.
• 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.
• the ligand toxin has an amino acid sequence substantially as shown in Figure 1.
• the ligand toxin is produced recombinantly.
• 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.
• 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.
• 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.
• the subject is suffering from breast cancer.
• Fig. 1 Complete amino acid sequence of the mature HRG ⁇ 2-PE40 ligand toxin encoded by the pFLAG/HRG ⁇ 2-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 HRG ⁇ 2 protein sequence is in bold type. A PE40 leader sequence including the Hindi ⁇ 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 ( ⁇ , ⁇ l, ⁇ 2 or ⁇ 3) at the doses indicated. After a further 20 min at 37°C, the cells were lysed and aliquots of the cell extracts subjected to
• Fig. 4 Effects of HRG ⁇ 2 or HRG ⁇ 2/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.
• HRG ⁇ 2, HRG ⁇ 2/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 (HRG ⁇ 2 InM; HRG ⁇ 2/PE40 5nM; PE40 5nM; control vehicle alone). Each experiment was performed three or four times with essentially identical results.
• Fig. 5 Dose response for HRG ⁇ 2-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. HRG ⁇ 2, PE40 or HRG ⁇ 2/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; HRG ⁇ 2 - D; HRG ⁇ 2/PE40 - •; PE40 - ⁇ )
• 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
• 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 DH5 ⁇ , transformants harbouring plasmids encoding particular HRG isoforms were identified by sequencing of small scale plasmid preparations (Sequenase Version 2.0, USB).
• a 1184 bp Hind ⁇ l/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 P ⁇ 40 toxin which was used as a control for ligand toxin experiments.
• the HindEU cDNA fragment encoding HRG ⁇ or HRG ⁇ 2 was then inserted into this PE40 construct (creating HRG ⁇ - and HRG ⁇ 2-PE40, respectively).
• IPTG-induced cultures were grown at 37°C with shaking until the culture reached an OD eoo of 0.8. IPTG was then added to 500 ⁇ M 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
• extraction buffer A 50 mM Tris-HCl pH 8.0, 5 raM EDTA, 0.25 mg/ml lysozyme, 50 ⁇ g/ml NaN 3
• extraction buffer B 1.5 M NaCl, 0.1 M CaCl 2 , 0.1 M MgCl 2 , 0.02 ⁇ g/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.
• 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
• 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.
• 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
• 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 ⁇ 2 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 ⁇ l of TBS/0.05% BSA (20 pM to 10 nM final concentration). Control wells received vehicle alone.
• 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 ⁇ g/ml leupeptin, 10 ⁇ g/ml aprotinin.
• 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 ⁇ g/ml leupeptin, 10 ⁇ g/ml aprotinin.
• samples of the resulting cell lysates were separated by SDS-PAGE, transferred to nitrocellulose, and
• the immunocomplexes were then collected by incubation with goat anti-mouse IgG Sepharose or protein A-Sepharose beads (40 ⁇ l) 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 HRG ⁇ 2 (lOnM) administration.
• 184B5 immortalized human breast epithelial cells in MEGM
• human breast cancer cells in RPMI 1640 containing 5% FCS
• recombinant HRG ⁇ 2, HRG ⁇ 2-PE40 or PE40 were dispensed into individual wells of 96-well plates at an initial concentration of 103 cells/well in 50 ⁇ l of growth media, 3 days before (Day -3) the addition of recombinant HRG ⁇ 2, HRG ⁇ 2-PE40 or PE40.
• recombinant proteins were added in 50 ⁇ l of growth media to final concentrations ranging from 0.5 pM to 5nM. Controls received either an equivalent volume of 0 vehicle (0.5 ⁇ l of 0.1% BSA) in 50 ⁇ l of growth media or growth media alone.
• T-47D, MCF-7 and ZR-75-1 human breast cancer cells responded to HRG ⁇ 2 administration with increased proliferation rates compared to untreated controls (Figs 4 and 5).
• ZR-75-1 cells significant increases in cell number relative to control cells were observed at concentrations of 50 pM or greater (Fig. 5).
• 184B5 cells did not exhibit a proliferative response to HRG ⁇ 2 (Figs. 4 and 5).
• concentrations tested 0.5 pM to 5 nM
• 3 initial plating densities ranging from 200-1000 cells/well
• HRG ⁇ 2-PE40 is capable of targeting cells overexpressing erbB3.
• ZR-75-1 cells were most sensitive to HRG ⁇ 2-PE40. exhibiting an IC 5 ⁇ value of 2-4 pM (Figs 4 and 5 and Table 1).
• 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).
• HRG ⁇ 2-PE40 Effect of HRG ⁇ 2-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 HRG ⁇ 2-PE40. Three such cell lines, Ln-CaP, PC-3 and DU145 were subjected to increasing concentrations of HRG ⁇ 2-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.
• HRG ⁇ 2- 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), HRG ⁇ 2-PE40 may also be used to target these types of cancer cell.
• HRG ⁇ 2-_ ⁇ 40 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.
• HRG ⁇ 2-PE40 caused marked cytotoxic effects on the human breast cancer cells and not on the 184B5 control.

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• 1994
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