DEVELOPMENT OF AN IMMUNOASSAY FOR BMS-191352, A SINGLE-CHAIN IMMUNOTOXIN, AND ITS APPLICATION TO TOXICOKINETIC STUDIES

BMS-191352 is a single-chain fusion protein composed of the variable r egions of chimeric BR96 monoclonal antibody and the binding defective form of Pseudomonas Exotoxin A (PE40). The immunotoxin exhibits potent cytotoxicity against tumor cells expressing the Lewis(Y) antigen. A s ensitive and specific double antibody sandwich ELISA has been develope d and validated for the determination of BMS-191352 in rat and dog EDT A plasma. A monoclonal anti-PE40 antibody (EXA2-1H8) was used to captu re BMS-191352 in plasma samples. The captured BMS-191352 was then dete cted using a biotinylated monoclonal BR96 antiidiotypic antibody (757- 4-1) followed by the addition of streptavidin-horseradish peroxidase c onjugate and chromogen 3, 3', 5, 5'-tetramethylbenzidine. The optical density was measured at 450 nm. The standard curve range in rat and do g plasma was 2-32 ng/mL. The RSD for the inter-and intra-assay precisi on was within 9.2% and the accuracy was greater than 89.0%. The ELISA method was applied to the analysis of BMS-191352 in plasma samples fro m toxicokinetic studies conducted in rats and dogs. These studies reve aled that the systemic exposure of BMS-191352 was dose proportional an d the kinetics of BMS-191352 were linear between the dose range of 1.8 -7.2 mg/m(2) in the rat and 2.5-15 mg/m(2) in the dog.