Fusion of the CH1 domain of IgG1 to epidermal growth factor (EGF) prolongs its retention in the blood but does not increase tumor uptake.
An expression vector (pJW4) for a human epidermal growth factor (hEGF)-CH1 fusion protein was constructed by fusing the gene for hEGF with the gene for CH1 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-CH1 expressed by induction of the lac Iq promotor with 50 microM isopropyl beta-D-thiogalactopyranoside (IPTG). hEGF- CH1 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-CH1 exhibited preserved binding to A431 (2-3 x 10(6) EGFR/cell) and MDA-MB-468 breast cancer cells (1-2 x 10(6) EGFR/cell). hEGF-CH1 without the linker exhibited poor receptor binding. hEGF-linker-CH1 also exhibited strong binding to soluble EGFR equivalent to that of hEGF. The tumor and normal tissue distribution of hEGF-linker-CH1 labeled with 123I was compared with 123 I-hEGF at 24 h after i.v. injection to mice implanted with s.c. MDA-MB-468 xenografts. Fusion of hEGF with CH1 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-CH1. We conclude that hEGF is more attractive than hEGF-linker-CH1 for imaging EGFR-positive tumors.
Wang
Judy
J
Division of Nuclear Medicine, University Health Network, Toronto, ON, Canada.
Reilly
Raymond M
RM
Chen
Paul
P
Yang
Shaoxian
S
Bray
Mark R
MR
Gariépy
Jean
J
Chan
Conrad
C
Sandhu
Jasbir
J
D000818:Animals; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D005260:Female; D006801:Humans; D007127:Immunoglobulin Constant Regions / Q000378:metabolism*; D007074:Immunoglobulin G / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism*; D007457:Iodine Radioisotopes; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism*; D051379:Mice; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains; D007457:Iodine Radioisotopes; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors