Aberrant epidermal growth factor receptor (EGFR) signaling is a common feature of multiple tumor types, including glioblastoma (GBM). As such, EGFR has emerged as an attractive target for antitumor therapy. In the present study, we sought to develop an immunotoxin capable of specifically targeting EGFR-expressing cells and mediating inhibition of cell growth and cell killing. The Luffin P1 (LP1) ribosome inactivating protein was chosen to generate a fusion protein, antiEGFR/LP1, based upon its potent protein synthesis inhibition and small size (5 kDa). LP1 was fused to the C-terminus of an anti-EGFR single-chain antibody (scFv). The recombinant antiEGFR/LP1 protein was expressed in Escherichia coli, and refolded and purified on an immobilized Ni2+-affinity chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis revealed that antiEGFR/LP1 was sufficiently expressed. Confocal microscopy and flow cytometry demonstrated that antiEGFR/LP1 bound specifically to EGFR-positive cells (U251), as almost no binding to EGFR-negative (Jurkat cells) was observed under identical time and dosage conditions. Finally, the MTT cell viability assay showed that antiEGFR/LP1 elicited obvious cytotoxicity toward EGFR-positive tumor cells. Collectively, these results suggest that antiEGFR/LP1 is biologically active and specific toward EGFR-positive tumor cells and may represent an effective EGFR-targeted cancer therapy. (C) 2012 Elsevier Inc. All rights reserved.