Dextran-conjugated vascular endothelial growth factor receptor antibody for in vivo melanoma xenografted mouse imaging

Abstract

Intact immunoglobulin G antibody has a relatively large molecule size of approximately 150 kDa that remains in the bloodstream for many weeks, which is a considerable disadvantage when it is used to carry radioactive materials for imaging. To lower background activity and increase the contrast of images, we investigated antivascular endothelial growth factor (VEGF) receptor 2 antibody (DC101) conjugated dextran for VEGF receptor 2 imaging in tumor xenografted mice. DTPA-conjugated aminodextran was synthesized, reacted with sulfo-LC-SPDP, and then reacted with DC101. The binding affinity of DTPA-dextran-DC101 to Flk-1 was measured. The gamma imaging and biodistributions of (99m)Tc-DTPA-dextran-DC101, (99m)Tc-DTPA-DC101, and (125)I-DC101 were studied in B16F10 melanoma xenografted mice. The dissociation values for DC101, DTPA-DC101, and DTPA-dextran-DC101 were 22.48, 3.05, and 14.74 pM, respectively. In gamma images, (99m)Tc-DTPA-dextran-DC101 showed weak liver uptake and rapid kidney elimination. In biodistribution results, the liver uptake of (99m)Tc-DTPA-dextran-DC101 was similar with that of (99m)Tc-DTPA-DC101 at each time point. However, the blood activity of (99m)Tc-DTPA-dextran-DC101 has shown significant differences, compared with (99m)Tc-DTPA-DC101 at all time points. The tumor accumulation of dextran-conjugated antibody was increased with time, whereas that of dextran nonconjugated antibody decreased. In particular, the pattern of tumor uptake of (99m)Tc-DTPA-dextran-DC101 was similar to that of (125)I-DC101, so this was thought to reflect the kinetics of DC101, unlike the nonconjugated form. The results of this study suggested that introduction of dextran moiety to make (99m)Tc-radiolabeled DC101 imaging agent could provide better images with the impaired background and the steady increasing binding to the receptor. However, further studies are necessary to improve clinical pharmacokinetics, such as enhancement of tumor uptake and impaired renal uptake.

FIG. 1.

¹H-NMR spectra of dextran-COOH (a) and aminodextran (b). Each proton is marked with asterisks.

FIG. 2.

Synthetic schematic of aminodextran (a) and DTPA-dextran-DC101 (b).

FIG. 2.

Synthetic schematic of aminodextran (a) and DTPA-dextran-DC101 (b).

FIG. 3.

Binding to Flk-1 by DC101, DTPA-DC101, and DTPA-dextran-DC101. Dose-dependant binding of DC101 and DTPA-dextran-DC101 to immobilized Flk-1. All experiments were done in duplicate. The K_d values of DC101, DTPA-DC101, and DTPA-dextran-DC101 were 22.48, 3.05, and 14.74 pM, respectively.

FIG. 4.

Planar images of ^99mTc-DTPA-DC101– and ^99mTc-DTPA-dextran-DC101–treated plates (a). Cellular uptakes of these two conjugates were inhibited by cold DC101. Blocking experiments with 2 nmol (300 μg) of DC101 revealed significant reduction (b). *p=0.004, **p=0.001.

FIG. 5.

In vivo imaging of athymic nude mice bearing B16F10 melanoma xenografts at 1 hour after i.v. injection of ^99mTc-DTPA-dextran-DC101 (a), ^99mTc-DTPA-DC101 (b), and ¹³¹I-DC101 (c), respectively. Black arrows indicate several organ locations. T, tumor; L, liver; K, kidney; B, bladder.