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 deastatination

Cancer-targeting biomolecules labeled with At-211 must be stable to in vivo deastatination, as control of the At-211 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 At-211-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 I-125, and that material was coinjected with the At-211-labeled Fab'. For comparison, direct labeling of the Fab' with I-125 and At-211 was conducted. Direct labeling with Na[I-125]I and Chloramine-T gave an 89% radiochemical yield. However, direct labeling of the Fab' with Na[At-211]At and Chloramine-T resulted in a yield of < 1% after quenching with NaS2O5. As another comparison, the same Fab' was conjugated with p-[At-211]astatobenzoate NHS ester, [At-211]1c-Fab', and (separately) with p-[I-125]iodobenzoate NHS ester, [I-125]1b-Fab'. An evaluation in athymic mice demonstrated that [At-211]1c-Fab' underwent deastatination. In contrast, the high in vivo stability of [I-125]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 [At-211]1c-Fab', the biodistributions of nido-carborane conjugated Fab' ([I-125]2-Fab'/ [At-211]2-Fab') and the bis-nido-carborane (VFC) ([I-125]3-Fab'/[At-211]3-Fab') had very different in vivo distributions than the control [I-125]1b-Fab'. Biodistributions of closo-decaborate(2-) conjugates ([I-125]4-Fab'/[At-211]4-Fab', [I-125]6-Fab'/[At-211]6-Fab', and [I-125]7-Fab'/[At-211]7-Fab') demonstrated that they were stable to in vivo deastatination and had distributions similar to that of the control [I-125]1b-Fab'. In contrast, a benzyl-modified closo-decaborate(2-) derivative evaluated in vivo ([I-125]5-Fab'/[At-211]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 At-211 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.