ANTIBODY FRAGMENTS IN TUMOR PRETARGETING - EVALUATION OF BIOTINYLATEDFAB' COLOCALIZATION WITH RECOMBINANT STREPTAVIDIN AND AVIDIN

An evaluation of the use of a biotinylated monoclonal antibody Fab' fr agment in tumor pretargeting was conducted. As a model system, tumor c olocalization of avidin or recombinant streptavidin (r-streptavidin) a nd 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 reacti ve biotinylation reagent, 13-N-maleimdo-4,7,10-trioxatridecanyl)biotin amide, was synthesized and used for biotinylation of Fab'. A biodistri bution 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-strept avidin at 4 or 20 h, was conducted. Specific colocalization of r-strep tavidin to tumors containing the A6H Fab'-S-biotin was evident from th e data obtained. In a similar biodistribution study, specific colocali zation of avidin to tumors pretargeted with A6H Fab'S-biotin was also observed. The avidin used in the study was radioiodinated with the N-h ydroxysuccinimidyl ester of p-[I-125]iodobenzoate ([I-125]PIB-NHS). Ve ry low concentrations (e.g. 0.35% ID/g) of avidin colocalized at the t umor. To further show that specific colocalization within the tumor xe nografts had occurred with biotinylated A6H Fab', radioiodinated avidi n and r-streptavidin were co-injected into athymic mice bearing tumor xenografts to obtain their distributions without having biotinylated F ab' present. At 20 h postinjection, only small differences in the bloo d and tumor concentrations of either protein were observed, indicating that the specific tumor colocalization seen in the previous two biodi stributions must have been due to the presence of Fab'-S-biotin. Calcu lations 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 studi ed.