Evaluation of Methods to Decrease Formation of a Higher Molecular Weight Species When²¹¹At-Labeling of Antibody-B10 Conjugates Using Chloramine-T as Oxidant

Background: Our research group has conducted studies that show a boron cage labeling moiety, the isothiocyanato-phenethyl-ureido-closo-decaborate(2-) (abbreviated as B10-NCS) can be conjugated with MAbs, and ²¹¹At-labeled using chloramine-T (ChT), to provide ²¹¹At-MAb-B10 conjugates with high in vivo stability of the ²¹¹At label. However, in the studies it was observed that a radiolabeled high molecular weight (HMW) species was formed based on SE-HPLC chromatograms. Of note is the fact that the percent of the ²¹¹At-labeled HMW species in the radiochromatogram was considerably higher than the percentage of HMW species on the UV (280 nm) chromatogram. This fact suggested that the HMW species formation was unique to ²¹¹At. Indeed, the reaction of the same MAb-B10 with radioiodine under the essentially identical reaction conditions (using ChT) did not show a difference between the HMW percentage in the radiochromatogram and the UV chromatogram. We felt that the quantity of ChT oxidant used may be problematic, so we evaluated different quantities of ChT in the labeling reaction. Additionally, a new conjugation moiety that contains two closo-decaborate(2-) moieties was synthesized, conjugated to a MAb and radiolabeled with ²¹¹At to determine if it could be used to alleviate the ²¹¹At-labeled HMW species.

Methods: The B10-NCS reagent was prepared as previously published. The new bis(B10)-NCS reagent was prepared in a multi-step reaction sequence. Two anti-CD45 MAbs were used in the studies, CA12.10C12 and BC8. MAb conjugations of the two different B10 reagents were conducted in PBS, pH 8.5 at room temperature overnight. The MAb conjugates were characterized by SE-HPLC, and IEF. The MAb-B10 and MAb-bis(B10) conjugates were labeled with ²¹¹At, adding 20, 10, 5 or 0 μg of ChT to the reaction solution. SE-HPLC with UV and radioactive detection was used to evaluate products obtained in ²¹¹At-labeling reactions. ²¹¹At-labeling of MAb-bis(B10) was conducted with 20 μg of ChT.

Results: ²¹¹At-labeling of a MAb-B10 conjugate, CA12.10C12-B10, using decreasing quantities of the oxidant ChT (20, 10, 5, or 0 μg) resulted in high overall labeling radiochemical yields (90+%), with varying quantities (27%, 17%, 13% & 9%, respt.) of a HMW species being formed. The high ²¹¹At-labeling yield when using no oxidant was confirmed in several MAb-B10 labeling reactions. Importantly, radioiodination of the same reagent using 20 μg of ChT had essentially no HMW species formed. Under the same reaction conditions, a MAb-bis(B10) conjugate had no HMW species formed when 20 μg of ChT was used in the ²¹¹At-labeling reaction.

Conclusions: Our studies demonstrated that no oxidant is required to label MAb-B10 conjugates with ²¹¹At, and high radiochemical yields are obtained in the reactions. The studies clearly showed that a lack of oxidant (ChT) in the ²¹¹At-labeling of MAb-B10 results in minimal or no formation of ²¹¹At-labeled HMW species. That result is further confirmed by the fact that the HMW species is not obtained when the MAb was conjugated with a reagent that contained two B10 moieties, MAb-bis(B10). The study results suggest that ²¹¹At-labeled HMW species seen by SE-HPLC may be a MAb-B10-At-B10-MAb dimer, where ²¹¹At is presumably forming an astatonium ion. Acknowledgement: This research is supported by the U.S. Department of Energy Isotope Program, managed by the Office of Science for Nuclear Physics (DE-SC0018013) and NIH (CA113431). We thank Dr. Brenda Sandmaier (Fred Hutch, Seattle, WA) for providing the MAbs used.