Reagents for Astatination of Biomolecules.2.Conjugation of Anionic Boron Cage Pendant Groups to a Protein Provides a Method for Direct Labeling that is Stable to in Vivo Deastatinationz

摘要

Cancer-targeting biomolecules labeled with 211At must be stable to in vivo deastatination,as control of the 211At distribution is critical due to the highly toxic nature of alpha-particle emission.Unfortunately,no astatinated aryl conjugates have shown in vivo stability toward deastatination when(relatively)rapidly metabolized proteins,such as monoclonal antibody Fab'fragments,are labeled.As a means of increasing the in vivo stability of 211At-labeled proteins,we have been investigating antibody conjugates of boron cage moieties.In this investigation,protein-reactive derivatives containing a nido-carborane(2),a bis-nido-carborane derivative(Venus Flytrap Complex,3),and four 2-nonahydro-closo-decaborate(2-)derivatives(4-7)were prepared and conjugated with an antibody Fab'fragment such that subsequent astatination and in vivo tissue distributions could be obtained.To aid in determination of stability toward in vivo deastatination,the Fab'-borane conjugates were also labeled with 125I,and that material was coinjected with the 211At-labeled Fab'.For comparison,direct labeling of the Fab'with 125I and 211At was conducted.Direct labeling with Na[125I]I and Chloramine-T gave an 89% radiochemical yield.However,direct labeling of the Fab'with Na[211At]At and Chloramine-T resulted in a yield of<1% after quenching with NaS2O5.As another comparison,the same Fab'was conjugated with p-[211At]astatobenzoate NHS ester,[211At]1c-Fab',and(separately)with p-[125I]iodobenzoate NHS ester,[125I]1b-Fab'.An evaluation in athymic mice demonstrated that[211At]1c-Fab'underwent deastatination.In contrast,the high in vivo stability of[125I]-1b-Fab'allowed it to be used as a tracer control for the natural distribution of Fab'.Although found to be much more stable in vivo than[211At]1c-Fab',the biodistributions of nido-carborane conjugated Fab'([125I]2-Fab7[211At]2-Fab')and the bis-nido-carborane(VFC)([125I]3-Fab'/[211At]3-Fab')had very different in vivo distributions than the control[125I]1b-Fab'.Biodistributions of closo-decaborate(2-)conjugates([125I]4-Fab'/[211At]4-Fab',[125I]-6-Fab'/[211At]6-Fab',and[125I]7-Fab'/[211At]7-Fab')demonstrated that they were stable to in vivo deastatination and had distributions similar to that of the control[125I]1b-Fab'.In contrast,a benzyl-modified closo-decaborate-(2-)derivative evaluated in vivo([125I]5-Fab'/[211At]5-Fab')had a very different tissue distribution from the control.This study has shown that astatinated protein conjugates of closo-decaborate(2-)are quite stable to in vivo deastatination and that some derivatives have little effect on the distribution of Fab'.Additionally,direct 211At labeling of Fab'conjugated with closo-decaborate(2-)derivatives provide very high(e.g.,58-75%)radiochemical yields.However,in vivo data also indicate that the closo-decaborate(2-)may cause some retention of radioactivity in the liver.Studies to optimize the closo-decaborate(2-)conjugates for protein labeling are underway.