Protein Engineering, Vol 11, 1285-1292, Copyright © 1998 by Oxford University Press
K Psarras, M Ueda, T Yamamura, S Ozawa, M Kitajima, S Aiso, S Komatsu and M Seno
The gene encoding human pancreatic ribonuclease 1 (hpRNasel) was fused with
a gene encoding human epidermal growth factor (hEGF). The hybrid human
protein was isolated from Escherichia coli inclusion bodies, refolded and
purified to homogeneity. The fusion protein competed with 125I-hEGF for
binding to hEGF receptors (EGFR) and had ribonucleolytic activities
approaching those of hpRNase1. Several conformations having different
enzymatic activities could be detected after reversed-phase
high-performance liquid chromatographic analysis, the less hydrophobic
molecules being the most active. The hybrid protein was specifically
cytotoxic to A431, an EGFR overexpressing squamous carcinoma cell line,
with an IC50 of approximately 10(-7) M. In contrast, recombinant hpRNase1
had an IC50 higher than 10(-4) M. A mixture of free hEGF and free hpRNasel
was not more cytotoxic than hpRNasel alone and no cytotoxicity was detected
in EGFR-deficient control cells. Taken together, these data suggest that
this construct might be useful for targeted therapy of esophageal, lung and
other squamous cell carcinomas and also breast cancers overexpressing EGFR,
which correlate with a poor prognosis and cannot be cured by surgery alone.
Engineering hybrid molecules with endogenous human proteins for targeted
therapy may alleviate the dose-limiting immunogenicity and toxicity of
conventional immunotoxins.
ARTICLES
Human pancreatic RNase1-human epidermal growth factor fusion: an entirely human 'immunotoxin analog' with cytotoxic properties against squamous cell carcinomas
Department of Surgery, School of Medicine, Keio University, Tokyo, Japan.
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