Reagents for Astatination of Biomolecules. 6. An Intact Antibody Conjugated with a Maleimido-c/oso-Decaborate(2-) Reagent via Sulfhydryl Groups Had Considerably Higher Kidney Concentrations than the Same Antibody Conjugated with an Isothiocyanato-c/oso-Decaborate(2-) Reagent via Lysine Amines

摘要

We are investigating the use of an ~(211)At-labeled anti-CD45 monoclonal antibody (mAb) as a replacement of total body irradiation in conditioning regimens designed to decrease the toxicity of hematopoietic cell transplantation (HCT). As part of that investigation, dose-escalation studies were conducted in dogs using ~(211)At-labeled anticanme CD4S mAb, CA12.10C12, conjugated with a maleimido-closo-decaborate(2-) derivative, 4. Unacceptable renal toxicity was noted in the dogs receiving doses in the 0.27-0.62 mCi/kg range. This result was not anticipated, as no toxicity had been noted m prior biodistribution and toxicity studies conducted in mice. Studies were conducted to understand the cause of the renal toxicity and to find a way to circumvent it. A dog biodistribution study was conducted with ~(123)I-labeled CA12.10C12 that had been conjugated with 4. The biodistribution data showed that 10-fold higher kidney concentrations were obtained with the maleimido-conjugate than had been obtained in a previous biodistribution study with ~(123)I-labeled CA12.10C12 conjugated with an amine-reactive phenylisofhiocyanato-CHX-A" derivative. The difference in kidney concentrations observed in dogs for the two conjugation approaches led to an investigation of the reagents. SE-HPLC analyses showed that the purity of the CA12.10C12 conjugated via reduced disulfides was lower than that obtained with amine-reactive conjugation reagents, and nonreducing SDS-PAGE analyses indicated protein fragments were present in the disulfide reduced conjugate. Although we had previously prepared closo-decaborate(2-) derivatives with amine-reactive functional groups (e.g., 6 and 8), a new, easily synthesized, amine-reactive (phenylisofhiocyanate) derivative, 10, was prepared for use in the current studies. A biodistribution was conducted with coadministered ~(125)I- and ~(211)At-labeled CA12.10C10 conjugated with 10. In that study, lower kidney concentrations were obtained for both radionuclides than had been obtained in the earlier study of the same antibody conjugated with 4 after reduction of disulfide bonds.