TY - JOUR T1 - A Human Transferrin-Vascular Endothelial Growth Factor (hnTf-VEGF) Fusion Protein Containing an Integrated Binding Site for <sup>111</sup>In for Imaging Tumor Angiogenesis JF - Journal of Nuclear Medicine JO - J Nucl Med SP - 1745 LP - 1752 VL - 46 IS - 10 AU - Conrad Chan AU - Jasbir Sandhu AU - Abhijit Guha AU - Deborah A. Scollard AU - Judy Wang AU - Paul Chen AU - Karen Bai AU - Lydia Lee AU - Raymond M. Reilly Y1 - 2005/10/01 UR - http://jnm.snmjournals.org/content/46/10/1745.abstract N2 - Our objective was to synthesize a recombinant protein (hnTf-VEGF [VEGF is vascular endothelial growth factor]) composed of VEGF165 fused through a flexible polypeptide linker (GGGGS)3 to the n-lobe of human transferrin (hnTf) for imaging angiogenesis. The hnTf domain allowed labeling with 111In at a site remote from the VEGF receptor-binding domain. Methods: DNA encoding hnTf, peptide linker (GGGGS)3, and VEGF165 genes were cloned into the Pichia pastoris vector pPICZαB to generate the pPICZαB-hnTF-VEGF plasmid. The expression vector was transformed into P. pastoris KM71H strain. The protein was purified using Co2+ metal affinity resin. The growth-stimulatory effects of hnTf-VEGF on human umbilical vascular endothelial cells (HUVECs) and its binding to porcine aortic endothelial cells (PAECs) transfected with VEGF receptors were evaluated. hnTf-VEGF protein was labeled with 111InCl3 in 10 mmol/L HEPES/15 mmol/L NaHCO3 buffer, pH 7.4 (HEPES is N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid). The loss of 111In in vitro from 111In-hnTf-VEGF to transferrin in human plasma and to diethylenetriaminepentaacetic acid (DTPA) in buffer was determined. Tumor and normal tissue distributions of 111In-hnTf-VEGF were evaluated in athymic mice implanted subcutaneously with U87MG human glioblastoma xenografts. Tumor imaging was performed. Results: Sodium dodecylsulfate–polyacrylamine gel electrophoresis under reducing and nonreducing conditions showed bands for hnTf-VEGF monomer (Mr of 65 kDa) and dimer (Mr of 130 kDa). hnTf-VEGF stimulated the growth of HUVECs 3-fold and demonstrated binding to PAECs displaced by a 50-fold excess of VEGF165 but not by apotransferrin. There was 21.3% ± 3.4% loss of 111In per day from 111In-hnTf-VEGF to transferrin in plasma, but &lt;5% loss to DTPA over 4 h. 111In-hnTf-VEGF accumulated in U87MG tumors (6.7% injected dose per gram at 72 h after injection) and its tumor uptake decreased 15-fold by coadministration of a 100-fold excess of VEGF but not by apotransferrin. The tumor-to-blood ratio was 4.9:1 at 72 h after injection and tumors were imaged at 24–72 h after injection. Conclusion: 111In-hnTf-VEGF is a promising radiopharmaceutical for imaging tumor angiogenesis and represents a prototypic protein harboring the metal-binding site of transferrin for labeling with 111In without introducing DTPA metal chelators. ER -