In Vitro Evaluation of Avidin Antibody Pretargeting Using 211At-Labeled and Biotinylated Poly-L-Lysine as Effector Molecule

BACKGROUND: Pretargeting is an approach for enhancing the therapeutic index of radioimmunotherapy by separating the administrations of tumor-targeting substance and radiolabel. In this study, a pretargeting model system of avidin-conjugated monoclonal antibody trastuzumab and biotinylated, At-211-labeled poly-L-lysine was constructed and analyzed in vitro. METHODS: Avidin activated by 4-(N-maleimidomethyl)cyclohexane-l-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC) and thiolated trastuzumab were incubated overnight at 4 C. The monomeric fraction was extracted using size exclusion fast protein liquid chromatography (FPLC) and further purified on an iminobiotin affinity column. Poly-L-lysine was biotinylated with succinimidyl-6-(biotinamido)hexanoate (NHS-LC-biotin), followed by direct At-211-labeling with N-succinimidyl-3-(trimethylstannyl)benzoate (m-MeATE), and succinylation with succinic anhydride. The avid in-trastuzumab conjugate was characterized by sodium dodecyl sulfate- polyacrylamide gel electrophoresis and FPLC, together with cell-binding and biotin-binding analyses. The labeled poly-L-lysine conjugate was assessed in terms of radiochemical purity and avidin binding. Furthermore, the full pretargeting system was evaluated in a tumor cell binding assay. RESULTS: The estimated size of the pretargeting molecule was 220 kDa, which corresponds to that of the expected avidin-trastuzumab monomer. Neither cell-binding ability (64%) nor biotin-binding ability (85%-95%) indicated any severe adverse effects from the chemical modifications. The radiochemical purity of the effector molecule was 92%-97%, and the avidin binding capacity was 91%-93%. The complete pretargeting assay resulted in a binding of 75.3 +/- 6.2% of added effector molecules to cells. CONCLUSIONS: The high binding of effector molecules to cells demonstrates a proof of concept for the synthesized molecules and pretargeting system, which will be further evaluated in vivo in future studies. Cancer 2010;116(4 suppl):1101-10. (C) 2010 American Cancer Society.