BIOTIN REAGENTS FOR ANTIBODY PRETARGETING - 3 - SYNTHESIS, RADIOIODINATION, AND EVALUATION OF BIOTINYLATED STARBURST DENDRIMERS

We are investigating the hypothesis that biotin multimers can be used with streptavidin and monoclonal antibody conjugates in cancer pretarg eting protocols to provide a method of increasing the amount of radioa ctivity bound on cancer cells in patients. As part of that investigati on, a series of biotinylated Starburst dendrimers (BSBDs) have been pr epared and evaluated in vitro and in vivo. In this study, a new biotin idase-stabilized, water-solubilizing biotinylation reagent was prepare d and reacted with Starburst (PAMAM) dendrimers, generations 0, 1, 2, 3, and 4. The reaction conditions employed resulted in perbiotinylatio n of generation 0 (four biotin moieties conjugated), generation 1 (eig ht biotin moieties conjugated), generation 2 (16 biotin moieties conju gated), and generation 3 (32 biotin moieties conjugated). With generat ion 4, incomplete biotinylation was achieved resulting in the largest portion of that BSBD having 51 biotin moieties (of 64 possible) conjug ated. The ability of each BSBD to cross-link streptavidin (SAv) was ex amined in an in vitro assay. In that assay, an assessment was made of the quantity of [I-125]SAv bound with polystyrene-bound SAv after trea tment with the synthesized BSBDs. All BSBDs cross-linked the polystyre ne-bound SAV with [I-125]SAv; however, the amount of [I-125]SAv bound varied with the different BSBDs. Roughly 1 equiv of [I-125]- SAv was b ound when Starburst dendrimers containing three or four biotin moietie s (generation 0) were used. Two equivalents were bound with BSBD gener ation 1, and 4 equiv were bound with BSBDs generations 2, 3, and 4. To assess the distribution of BSBDs generations 0, 1, and 2 in mice (at 4 h postinjection), a method was developed for radioiodinating them us ing the NHS ester of p-[I-125]-iodobenzoate ([I-125]PIB). It was found that the radioiodinated BSBDs had low blood concentrations (i.e., 0.1 3-0.20% ID/g) at the 4 h time point. In fact, most tissues examined ha d low concentrations of biotinylated dendrimers, except kidney and liv er. Kidney had the highest concentration of [I-125]- labeled BSBDs, an d its concentration increased with increasing size and charge of dendr imer (e.g., 8-48% ID/g). On the basis of the increased radioactivity o bserved in the in vitro assay and the rapid clearance from blood in mi ce, additional in vivo studies with perbiotinylated Starburst dendrime r, generation 2, are planned.