Human pancreatic RNase1-human epidermal growth factor fusion: an entirely human 'immunotoxin analog' with cytotoxic properties against squamous cell carcinomas

The gene encoding human pancreatic ribonuclease 1 (hpRNase1) was fused with a gene encoding human epidermal growth factor (hEGF), The hybrid human pro tein was isolated from Escherichia coli inclusion bodies, refolded and puri fied to homogeneity, The fusion protein competed with I-125-hEGF for bindin g to hEGF receptors (EGFR) and had ribonucleolytic activities approaching t hose of hpRNase1, Several conformations having different enzymatic activiti es could be detected after reversed-phase highperformance liquid chromatogr aphic analysis, the less hydrophobic molecules being the most active. The h ybrid protein was specifically cytotoxic to A431, an EGFR overexpressing sq uamous carcinoma cell line, with an IC50 of similar to 10(-7) M. In contras t, recombinant hpRNase1 had an IC50 higher than 10(-4) M. A mixture of free hEGF and free hpRNase1 was not more cytotoxic than hpRNase1 alone and no c ytotoxicity was detected in EGFR-deficient control cells. Taken together, t hese data suggest that this construct might be useful for targeted therapy of esophageal, lung and other squamous cell carcinomas and also breast canc ers overexpressing EGFR, which correlate with a poor prognosis and cannot b e cured by surgery alone. Engineering hybrid molecules with endogenous huma n proteins for targeted therapy may alleviate the dose-limiting immunogenic ity and toxicity of conventional immunotoxins.