Temporary inaccessibility for avidin to a biotinylated antibody in mice

1402 Objectives A clearance step is often included in a pretargeting procedure. Avidin as an clearing agent clears the biotinylated pretargeting antibody rapidly but the clearance efficiency is lower than the desired level (for example, > 90%). To reduce the non-specific background, we have investigated the in vivo interaction of the avidin with a biotinylated antibody. Methods A biotin and a synthetic DNA analogue termed morpholino phosphorodiamidate oligomer (MORF) were conjugated to a pretargeting IgG antibody. Avidin was used as a clearing agent for the antibody in the circulation. The first study tested the prolongation of the pretargeting interval to improve clearance. In the second study, a radiolabeled biotin-Ab-MORF was directly combined with excess avidin prior to injection into mice. In the third study, multiple avidin injections were applied to the mice that received the biotin-Ab-MORF. Results The prolongation of the pretargeting interval demonstrated limited improvement and the clearance efficiency remained low. However, if the biotinylated antibody was bound to avidin, it was cleared rapidly (in ~ 0.5 h); the avidin-treated antibody remaining in the blood was only 12% of the avidin-untreated control antibody. Thus, the lower clearance efficiency in the first study could not be explained by the 88% clearable molecules measured in the second. Multiple avidin injections confirmed most of the antibody that survived the first clearance was still clearable. Three injections of avidin achieved a collective efficiency of 80%, i.e. 91% (80/88) of the avidin-clearable MORF modified antibody. Conclusions Although clearable, the biotinylated antibody in some compartments was not accessible temporarily to the IV-injected avidin, but a continuous infusion of avidin would be predicted to achieve the clearance of all the clearable molecules. Research Support The research support was mainly from NIH DK94994 and CA94199.