Antibody conjugation, characterization, and ²¹¹At labeling of a bis-maleimido-closo-decaborate(2-) derivative

Objectives We are investigating a bis-maleimido-closo-decaborate(2-) derivative as a monoclonal antibody (mAb) conjugate for labeling with ²¹¹At. A mono-maleimido-closo-decaborate(2-) derivative for ²¹¹At labeling of mAbs previously yielded promising therapeutic results, however unacceptable renal toxicity was observed in a canine model. In this study a bis-maleimide reagent was investigated for mAb conjugation and radiolabeling to potentially mediate the previously observed renal toxicity.

Methods Conjugation conditions and ²¹¹At labeling efficiency were evaluated using two mAb’s; the anti- EGFR mAb Erbitux and BC8, an anti-CD45 mAb. Both antibodies were conjugated with the bis-maleimide reagent via reactions with sulfhydryl groups produced by disulfide reduction. The mAbs were reacted with 5, 10, & 20 equivalents of the bis-maleimide reagent to evaluate reagent loading and determine the optimal reaction conditions. The mAb conjugates were characterized by SE-HPLC and electrophoresis (SDS-PAGE and IEF). The mAb conjugates were subsequently labeled with ²¹¹At using chloramine-T and Na[²¹¹At]At (400 μCi).

Results SE-HPLC analyses showed that the reduction and subsequent cross-linking of the disulfide bonds did not significantly affect the purity of either mAb conjugated via reduced disulfides. Further, both reducing and non-reducing SDS-PAGE analyses indicated very low quantities of non-specific protein fragments in the conjugates as compared to controls and mono-maleimide conjugates of the same mAbs. Ten molar equivalents of the bis-maleimide reagent was chosen as an optimal amount to achieve a stable conjugation with the mAbs; and those mAb conjugates were successfully labeled with ²¹¹At in 90.4% (Erbitux) and 83.7% (BC8) radiochemical yields.

Conclusions The stable cross-linking and efficient ²¹¹At labeling of mAbs conjugated with the bis-maleimido-closo-decaborate(2-) derivative indicates that further studies are warranted.

Research Support The authors wish to acknowledge the following source of funding for this work: NIH (CA113431)