Three-Step Tumor Imaging with Biotinylated Monoclonal Antibody, Streptavidin and 111In-DTPA–Biotin
Introduction
Scintigraphic tumor imaging using the avidin (streptavidin)–biotin system is a technique that makes possible clear and early imaging after injection of radiolabeled compound. However, the multistep targeting method is sometimes difficult to apply in a nude mouse tumor model, probably owing to the higher level of endogenous biotin in mice than in humans [20]. Although various approaches have been studied 9, 22, to date there are few reports demonstrating successful tumor targeting with radiolabeled biotin in mice. Recently tumor targeting was reported in a mouse system with two-step method using CC49 monoclonal antibody–streptavidin conjugate and radiolabeled biotin [1]. Compared with the two-step method, the three-step method has some advantages, such as an easy preparation of the administrative materials. Some experimental or clinical studies using three-step method have been reported 11, 18. To verify this approach in a mouse system, we describe in this report the three-step delivery of radiolabeled biotin to subcutaneous LS180 human colon cancer xenografts pretargeted with the monoclonal antibody MLS128 and with streptavidin and the successful imaging of the tumors as early as 2 h after administration of radiolabeled biotin.
Section snippets
Cells
LS180 human colon cancer cells were grown in RPMI 1640 medium (Nissui, Tokyo, Japan) supplemented with 10% fetal calf serum (GIBCO Laboratories, Grand Island, NY) and 0.03% l-glutamine at 37°C in 5% CO2. For in vitro and in vivo studies, subconfluent cells were harvested using calcium- and magnesium-free phosphate-buffered saline (PBS) containing 0.02% ethylenediamine triacetic acid (EDTA).
Monoclonal Antibodies
MLS128 is a murine IgG3 monoclonal antibody with a κ light chain, which was produced by immunizing mice
In Vitro Reactivity of Biotinylated MLS128 and Biotinylated OST6
More than 95% of the radioactivity of 125I-biotinylated MLS128 and of 111In-DTPA–biotin bound to immobilized avidin, and of 125I-labeled streptavidin also showed more than 90% binding to biotin-coated beads. The immunoreactive fraction of 125I-labeled biotinylated MLS128 was 47.2%, which was slightly lower than that of the unmodified MLS128 [22]. 125I-labeled biotinylated OST6 showed no significant binding with LS180 cells.
Biodistribution
The biodistribution of 125I-labeled streptavidin in the mice pretargeted
Discussion
The very high affinity of avidin or streptavidin for biotin (KA = 1015/M) makes the avidin–biotin system applicable to multistep targeting of tumors. In addition to its high-affinity binding to avidin or streptavidin, biotin has very low molecular weight. Radiolabeled biotin is cleared from normal tissues faster than the radiolabeled antibodies used in the conventional method. As a result, the pretargeting technique with radiolabeled biotin provides higher tumor-to-background ratios at an
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