Fusion of the C H 1 Domain of IgG 1 to Epidermal Growth Factor (EGF) Prolongs its Retention in the Blood but Does Not Increase Tumor Uptake

Abstract

An expression vector (pJW4) for a human epidermal growth factor (hEGF)- C_H1 fusion protein was constructed by fusing the gene for hEGF with the gene for C_H1 of murine IgG₁ with/without a peptide linker sequence [(GGGGS)₃] and inserting the recombinant gene into vector pGEX2T. Expression vector pGEX2T was transfected into E. coli (BL-21) and hEGF-C_H1 expressed by induction of the lac I^q promotor with 50 μM isopropyl β-D-thiogalactopyranoside (IPTG). hEGF- C_H1 fused to glutathione S-transferase (GST) was isolated and purified by affinity chromatography. GST was cleaved using thrombin. SDS-PAGE demonstrated a protein with the expected M_r (18 kDa) positive for hEGF by Western blot. hEGF-linker-C_H1 exhibited preserved binding to A431 (2-3 × 10⁶ EGFR/cell) and MDA-MB-468 breast cancer cells (1-2 × 10⁶ EGFR/cell). hEGF-C_H1 without the linker exhibited poor receptor binding. hEGF-linker-C_H1 also exhibited strong binding to soluble EGFR equivalent to that of hEGF. The tumor and normal tissue distribution of hEGF-linker-C_H1 labeled with ¹²³I was compared with ¹²³I-hEGF at 24 h after i.v. injection to mice implanted with s.c. MDA- MB-468 xenografts. Fusion of hEGF with C_H1 increased its retention in the blood 14-fold but did not significantly increase tumor uptake. Tumor/blood ratios were higher for hEGF than for hEGF-linker-C_H1. We conclude that hEGF is more attractive than hEGF-linker-C_H1 for imaging EGFR-positive tumors.