RT Journal Article SR Electronic T1 A Human Transferrin-Vascular Endothelial Growth Factor (hnTf-VEGF) Fusion Protein Containing an Integrated Binding Site for 111In for Imaging Tumor Angiogenesis JF Journal of Nuclear Medicine JO J Nucl Med FD Society of Nuclear Medicine SP 1745 OP 1752 VO 46 IS 10 A1 Chan, Conrad A1 Sandhu, Jasbir A1 Guha, Abhijit A1 Scollard, Deborah A. A1 Wang, Judy A1 Chen, Paul A1 Bai, Karen A1 Lee, Lydia A1 Reilly, Raymond M. YR 2005 UL http://jnm.snmjournals.org/content/46/10/1745.abstract AB 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 <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.