One pretargeting approach to
cancer radioimmunotherapy utilizes an antibody-
streptavidin conjugate that is first localized to the
tumor. A "clearing agent" is then administered to remove the excess bioconjugate from blood, followed by injection of the radiolabeled
biotin therapeutic. In this study, the role of
streptavidin-
biotin affinity in this pretargeting system was investigated for the first time in vivo, with a reduced affinity, site-directed
streptavidin mutant and with radiolabeled bis-
biotin reagents. The S45A
streptavidin mutant (SA-S45A), which displays a faster off-rate for
biotin, was utilized with a bivalent
biotin carrier that retains high avidity for the
streptavidin mutant. Mice were fed either a normal or
biotin-deficient diet, yielding serum endogenous
biotin concentrations of 31 nM and 5 nM, respectively.
Lymphoma-bearing nude mice pretargeted with 1F5 Antibody-SA-Wild Type (WT) bioconjugates produced (125)I-bis-biotin
tumor concentrations of 2.2%ID/g and 7.0%ID/g in mice fed normal diets vs
biotin-deficient diets. (125)I-bis-biotin
tumor concentrations of mice pretargeted with 1F5-SA-S45A were 12%ID/g and 10%ID/g for mice fed normal and
biotin-deficient diets, respectively. However, poor clearance of the 1F5-SA-S45A with the biotinylated clearing agent led to high normal organ concentrations of (125)I-bis-biotin. A galactosylated
human serum albumin (HSA) modified with bis-
biotin was then tested, and normal organ (125)I-bis-biotin concentrations were significantly reduced.
Tumor-to-organ ratios achieved for 1F5-SA-S45A with the HSA-bis-
biotin clearing agent in mice with high serum
biotin were similar to those achieved with 1F5-SA-WT in mice with low serum
biotin. These results demonstrate that exchange of bound endogenous
biotin with lower affinity
streptavidin mutants is possible, and that corresponding use of bis-
biotin carriers can nearly eliminate the differences in therapeutic radioactivity at the
tumor site in animals on normal vs
biotin-deficient diets. The results also interestingly demonstrate, however, that improved clearance agents capable of removing the lower affinity
streptavidin-antibody conjugate are needed to achieve comparable specificity in
tumor to blood or normal organ ratios.