A human transferrin-vascular endothelial growth factor (hnTf-VEGF) fusion protein containing an integrated binding site for (111)In for imaging tumor angiogenesis.

Abstract

UNLABELLED:

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.

• Authors: Conrad Chan, Jasbir Sandhu, Abhijit Guha, Deborah A Scollard, Judy Wang, Paul Chen, Karen Bai, Lydia Lee, Raymond M Reilly
• Journal: Journal of nuclear medicine : official publication, Society of Nuclear Medicine (J Nucl Med) Vol. 46 Issue 10 Pg. 1745-52 (Oct 2005) ISSN: 0161-5505 [Print] United States
• PMID: 16204726 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S.)

Chemical References

• Indium Radioisotopes
• Radiopharmaceuticals
• Recombinant Fusion Proteins
• Transferrin
• VEGFA protein, human
• Vascular Endothelial Growth Factor A

Topics

• Animals
• Binding Sites
• Cell Line, Tumor
• Female
• Glioblastoma (blood supply, diagnostic imaging, metabolism)
• Humans
• Indium Radioisotopes (chemistry, pharmacokinetics)
• Metabolic Clearance Rate
• Mice
• Mice, Nude
• Neovascularization, Pathologic (diagnostic imaging, metabolism)
• Organ Specificity
• Protein Binding
• Radionuclide Imaging
• Radiopharmaceuticals (pharmacokinetics)
• Recombinant Fusion Proteins (chemistry, pharmacokinetics)
• Tissue Distribution
• Transferrin (chemistry, genetics, pharmacokinetics)
• Vascular Endothelial Growth Factor A (chemistry, genetics, pharmacokinetics)