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Chan C, Sandhu J, Guha A, Scollard DA, Wang J, Chen P, Bai K, Lee L, Reilly RM: A human transferrin-vascular endothelial growth factor (hnTf-VEGF) fusion protein containing an integrated binding site for (111) In for imaging tumor angiogenesis. J Nucl Med. 2005 Oct;46(10):1745-52.
Our objective was to synthesize a recombinant protein (hnTf-VEGF [VEGF is vascular endothelial growth factor]) composed of VEGF (165) 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 (111) In at a site remote from the VEGF receptor-binding domain. METHODS: DNA encoding hnTf, peptide linker (GGGGS)(3), and VEGF (165) genes were cloned into the Pichia pastoris vector pPICZalphaB to generate the pPICZalphaB-hnTF-VEGF plasmid. The expression vector was transformed into P. pastoris KM71H strain. The protein was purified using Co (2+) 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 (111) InCl (3) in 10 mmol/L HEPES/15 mmol/L NaHCO (3) buffer, pH 7.4 (HEPES is N-(2-hydroxyethyl) piperazine-N'-(2-ethanesulfonic acid). The loss of (111) In in vitro from (111) In-hnTf-VEGF to transferrin in human plasma and to diethylenetriaminepentaacetic acid (DTPA) in buffer was determined. Tumor and normal tissue distributions of (111) In-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 (M (r) of 65 kDa) and dimer (M (r) of 130 kDa). hnTf-VEGF stimulated the growth of HUVECs 3-fold and demonstrated binding to PAECs displaced by a 50-fold excess of VEGF (165) but not by apotransferrin. There was 21.3% +/- 3.4% loss of (111) In per day from (111) In-hnTf-VEGF to transferrin in plasma, but <5% loss to DTPA over 4 h. (111) In-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: (111) In-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 (111) In without introducing DTPA metal chelators. |
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