STREPTAVIDIN IN ANTIBODY PRETARGETING - 2 - EVALUATION OF METHODS FORDECREASING LOCALIZATION OF STREPTAVIDIN TO KIDNEY WHILE RETAINING ITSTUMOR BINDING-CAPACITY

An investigation has been conducted to determine if the kidney localiz ation of recombinant streptavidin can be decreased to improve its char acteristics in pretargeting protocols. Three different methods of acco mplishing this were evaluated. The first method, blocking kidney uptak e with a preadministration of recombinant streptavidin in which biotin occupied all of the binding sites, was unsuccessful. In a second meth od, L-lysine administration was used to block kidney localization. Thi s method worked well, decreasing the concentration to 29% of the unmod ified amount at 8 h postinjection. However, this method suffered from a requirement for constant infusion of lysine during the period of obs ervation. A third method, use of succinylated recombinant streptavidin , was found to be the best approach. Succinylation of streptavidin was readily accomplished with very good protein recovery. With the succin ylated streptavidin, the kidney concentration was only 14% of that of nonmodified streptavidin at 4 h postinjection. While these results dem onstrated that the concentration of streptavidin could be decreased in the kidney, it was important to assess whether the tumor colocalizati on of streptavidin with biotinylated antibody was affected under those conditions. As part of our continuing investigation of pretargeting, a new water-solubilized biotinidase-stabilized biotinylation reagent w as prepared. Using that reagent in a pretargeting experiment, an equiv alent quantity of succinylated recombinant streptavidin as biotinylate d antibody Fab' was localized in a tumor xenograft model. In that expe riment, the kidney concentration was decreased to less than 10% of tha t obtained with unmodified recombinant streptavidin at 24 h postinject ion. The results of our investigation have demonstrated that succinyla tion of streptavidin improves its distribution characteristics for pre targeting applications. The fact that succinylated streptavidin has no specific tissue localization should allow its use as a carrier of rad ioactivity in ''two-step'' pretargeting protocols.