title: Antibody fragments in tumor pretargeting. Evaluation of biotinylated Fab' colocalization with recombinant streptavidin and avidin.
abstract: An evaluation of the use of a biotinylated monoclonal antibody Fab' fragment in tumor pretargeting was conducted. As a model system, tumor colocalization of avidin or recombinant streptavidin (r-streptavidin) and the biotinylated Fab' fragment (Fab'-S-biotin) of A6H, an antirenal cell carcinoma antibody, was evaluated in athymic mice bearing human renal cell carcinoma xenografts. A new water soluble sulfhydryl reactive biotinylation reagent, N-(13-N-maleimdo-4, 7,10-trioxatridecanyl)-biotinamide, was synthesized and used for biotinylation of Fab'. A biodistribution of ChT-labeled A6H Fab'-S-biotin was conducted. Data from that distribution indicated that the Fab'-S-biotin localized well (i.e. 28% ID/g at 24 h) to human tumor xenografts in athymic mice. Subsequently, a biodistribution study involving pretargeting radioiodinated A6H Fab'-S-biotin to tumor xenografts, followed by administration of r-streptavidin at 4 or 20 h, was conducted. Specific colocalization of r-streptavidin to tumors containing the A6H Fab'-S-biotin was evident from the data obtained. In a similar biodistribution study, specific colocalization of avidin to tumors pretargeted with A6H Fab'-S-biotin was also observed. The avidin used in the study was radioiodinated with the N-hydroxysuccinimidyl ester of p-[125I]iodobenzoate ([125I]PIB-NHS). Very low concentrations (e.g. 0.35% ID/g) of avidin colocalized at the tumor. To further show that specific colocalization within the tumor xenografts had occurred with biotinylated A6H Fab', radioiodinated avidin and r-streptavidin were co-injected into athymic mice bearing tumor xenografts to obtain their distributions without having biotinylated Fab' present. At 20 h postinjection, only small differences in the blood and tumor concentrations of either protein were observed, indicating that the specific tumor colocalization seen in the previous two biodistributions must have been due to the presence of Fab'-S-biotin. Calculations were conducted to estimate how much r-streptavidin (as a molar ratio) was colocalized. From the data obtained it was estimated that 36-61% of the tumor-localized Fab'-S-biotin molecules were bound with r-streptavidin and 4-23% bound with avidin, under the conditions studied.
authors.affiliation: Department of Radiation Oncology, University of Washington, Seattle 98195, USA. dswilbur@u.washington.edu
authors.forename: D S
authors.initials: DS
authors.lastname: Wilbur
authors.forename: D K
authors.initials: DK
authors.lastname: Hamlin
authors.forename: R L
authors.initials: RL
authors.lastname: Vessella
authors.forename: J E
authors.initials: JE
authors.lastname: Stray
authors.forename: K R
authors.initials: KR
authors.lastname: Buhler
authors.forename: P S
authors.initials: PS
authors.lastname: Stayton
authors.forename: L A
authors.initials: LA
authors.lastname: Klumb
authors.forename: P M
authors.initials: PM
authors.lastname: Pathare
authors.forename: S A
authors.initials: SA
authors.lastname: Weerawarna
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000493:pharmacokinetics*; D001360:Avidin / Q000008:administration & dosage / Q000493:pharmacokinetics*; D001426:Bacterial Proteins / Q000008:administration & dosage / Q000493:pharmacokinetics*; D001710:Biotin; D016503:Drug Delivery Systems; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000008:administration & dosage / Q000378:metabolism*; D008301:Maleimides; D051379:Mice; D019809:Streptavidin; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D007140:Immunoglobulin Fab Fragments; D008301:Maleimides; D001360:Avidin; D001710:Biotin; D019809:Streptavidin