Biotin Reagents for Antibody Pretargeting. 3. Synthesis, Radioiodination, and Evaluation of Biotinylated Starburst Dendrimers D. Scott Wilbur,* ,† Pradip M. Pathare, † Donald K. Hamlin, † Kent R. Buhler, ‡ and Robert L. Vessella ‡ Departments of Radiation Oncology, 2121 North 35th Street, and Urology, University of Washington, Seattle, Washington 98195 . Received May 26, 1998; Revised Manuscript Received September 10, 1998 We are investigating the hypothesis that biotin multimers can be used with streptavidin and monoclonal antibody conjugates in cancer pretargeting protocols to provide a method of increasing the amount of radioactivity bound on cancer cells in patients. As part of that investigation, a series of biotinylated Starburst dendrimers (BSBDs) have been prepared and evaluated in vitro and in vivo. In this study, a new biotinidase-stabilized, water-solubilizing biotinylation reagent was prepared and reacted with Starburst (PAMAM) dendrimers, generations 0, 1, 2, 3, and 4. The reaction conditions employed resulted in perbiotinylation of generation 0 (four biotin moieties conjugated), generation 1 (eight biotin moieties conjugated), generation 2 (16 biotin moieties conjugated), and generation 3 (32 biotin moieties conjugated). With generation 4, incomplete biotinylation was achieved resulting in the largest portion of that BSBD having 51 biotin moieties (of 64 possible) conjugated. The ability of each BSBD to cross-link streptavidin (SAv) was examined in an in vitro assay. In that assay, an assessment was made of the quantity of [ 125 I]SAv bound with polystyrene-bound SAv after treatment with the synthesized BSBDs. All BSBDs cross-linked the polystyrene-bound SAv with [ 125 I]SAv; however, the amount of [ 125 I]SAv bound varied with the different BSBDs. Roughly 1 equiv of [ 125 I]- SAv was bound when Starburst dendrimers containing three or four biotin moieties (generation 0) were used. Two equivalents were bound with BSBD generation 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 using the NHS ester of p-[ 125 I]- iodobenzoate ([ 125 I]PIB). It was found that the radioiodinated BSBDs had low blood concentrations (i.e., 0.13-0.20% ID/g) at the 4 h time point. In fact, most tissues examined had low concentrations of biotinylated dendrimers, except kidney and liver. Kidney had the highest concentration of [ 125 I]- labeled BSBDs, and its concentration increased with increasing size and charge of dendrimer (e.g., 8-48% ID/g). On the basis of the increased radioactivity observed in the in vitro assay and the rapid clearance from blood in mice, additional in vivo studies with perbiotinylated Starburst dendrimer, generation 2, are planned. INTRODUCTION Reagents containing biotin, avidin, or streptavidin and tumor-specific monoclonal antibodies (mAbs), 1 in combi- nation with a variety of radionuclides, are under inves- tigation in an approach to cancer therapy that is termed “pretargeting” (1-3). The pretargeting approach is being investigated as a means of circumventing some of the inherent problems associated with directly radiolabeled mAbs in therapy protocols (4-6). Importantly, in the pretargeting approach, the tumor selective targeting of mAbs is retained, but the delivery of the radionuclide is relegated to another molecule, which in many investiga- tions is either a radiolabeled biotin derivative or a radiolabeled avidin or streptavidin molecule (7-17). The selection of these reagents for the pretargeting approach has been made because of the very high binding avidity of biotin with avidin (18, 19) and streptavidin (20). Tumor localization of the radiolabeled biotin or (strept)- avidin is achieved by binding with an antibody conjugate that has been previously localized on tumor cells. Be- cause avidin and streptavidin are composed of four identical subunits, four biotin molecules can bind each protein molecule. This tetrameric binding nature of avidin and streptavidin permits their use in various combinations of mAbs/biotin/(strept)avidin, which are administered in “2-step” or “3-step” pretargeting protocols (6, 21). The introduction of multiple “steps” in tumor targeting provides additional variables for optimizing the pharmacokinetics and distributions of the therapeutic radionuclide not previously obtainable with directly labeled mAbs (5). In addition to improving the pharmacokinetics and distribution of the therapeutic radionuclide, the use of * Author to whom correspondence should be addressed. Phone: 206-685-3085. Fax: 206-685-9630. E-mail: dswilbur@ u.washington.edu. † Department of Radiation Oncology. ‡ Department of Urology. 1 Abreviations: BSA, bovine serum albumin; BSBD(s), bioti- nylated Starburst dendrimer(s); ChT, chloramine-T; cpm, counts per minute; EDC, 1-(3-dimethylaminopropyl)-3-ethylcarbodiim- ide hydrochloride; G ) 0, 1, 2, 3, or 4, Starburst dendrimer generation 0, 1, 2, 3, or 4; IBz-Trimer, iodobenzoyl-biotin trimer; MALDI-TOF MS, matrix-assisted laser desorption/ionization- time-of-flight mass spectrometry; NCS, N-chlorosuccinimide; PBS, phosphate-buffered saline; r-SAv, recombinant streptavi- din; rt, room temperature; (strept)avidin, denotes avidin or streptavidin; SAv, streptavidin; SBD(s), Starburst dendrimer- (s); TFP, tetrafluorophenyl; TFP-OH, tetrafluorophenol; TsCl, p-toluenesulfonyl chloride. 813 Bioconjugate Chem. 1998, 9, 813-825 10.1021/bc980055e CCC: $15.00 © 1998 American Chemical Society Published on Web 10/22/1998