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.

A novel monoclonal antibody 107-1A4 with high prostate specificity: generation, characterization of antigen expression, and targeting of human prostate cancer xenografts

“…Biodistribution data were obtained by determining the presence of the co-injected radionuclides in a panel of tissues harvested at sacri®ce (24, 72, 120 and 168 h post-injection) as previously described in detail. 14 …”

Comparison of anti-CD20 and anti-CD45 antibodies for conventional and pretargeted radioimmunotherapy of B-cell lymphomas

“…This pretargeting approach has been demonstrated to improve the ratios of radiation delivered to tumors compared with the doses delivered to normal organs in preclinical and clinical models. [12][13][14][15][16][17][18][19][20][21][22][23][24] Recent work using human carcinoma xenografts demonstrated that a single treatment of pretargeted 90 Y-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-biotin resulted in rapid effective blood clearance of non-tumorbound antibody, improved tumor-to-normal organ ratios of absorbed radioactivity, and superior objective and durable remissions with minimal toxicity. 25 Synthetic clearing agents have been introduced as an additional refinement to pretargeting protocols to more efficiently deplete immunoconjugates lingering in the bloodstream before administration of the radioactive moiety.…”

Synthesis and Testing of Mechanism‐Based Protein‐Profiling Probes for Retaining Endo‐glycosidases

“…The biotinamido amine 7 was synthesized and characterized as described previously. [18] 3-[N-(D-Biotinoyl)-13-amino-4,7,10-trioxatridecanylaminocarbonyl]propanoic acid (8 a). A solution of the biotinamido amine 7 (2.80 g, 6.3 mmol) in dry DMF (20 mL) was reacted with succinic anhydride (688 mg, 6.88 mmol), and the solution was stirred at room temperature for 1 h under N 2 atmosphere.…”