A click-chemistry based strategy for labeling an anti-phosphatidylserine (PS) antibody with copper-64 via a cross-bridged tetraazamacrocyclic chelator scaffold

Objectives Cross-bridged tetraazamacrocyclic chelator (CBTE2A) scaffold forms a kinetically inert copper complex with high in vivo stability, and is regarded as one of the “gold standard’’. Unfortunately, due harsh labeling conditions it is not under utilized for antibody labeling. In this work we demonstrate a unique method for antibody labeling under ambient conditions utilizing CBTE2A scaffold. The method involves prelabeling CBTE2A based scaffold (CBTE1A) with ⁶⁴Cu and its subsequent reaction with antibody via tertrazine-norbornene mediated click reaction. We demonstrate the effectiveness of this technique by labeling and imaging bavituximab, a chimeric monoclonal antibody designed for the treatment of cancers, with ⁶⁴Cu.

Methods Norbornene moiety was attached to the free acid group of the tert butyl protected CBTE1A scaffold via carbodiimide chemistry. The compound was then deprotected and labeled with ⁶⁴Cu in ammonium acetate buffer. Tetrazine moiety was attached to bavituximab via carbodiimide chemistry and the mixture purified by FPLC. Copper-64 labeled CBTE1A was subsequently mixed with tetrazine modified bavituximab in phosphate buffer and the mixture analyzed with radio FPLC. The resultant mixture was purified and in vivo imaging was performed on LNCaP tumor bearing mice.

Results The ⁶⁴Cu-labeled CBTE1A was obtained in 90% radio labeling yield and greater than 99% purity. The radio FPLC analysis of the reaction mixture showed a strong radio peak for bavituximab. In vivo imaging showed uptake and retention of labeled bavituximab at 48 h post injection.

Conclusions We have successfully demonstrated a method to radiolabel antibodies under ambient condition using CBTE2A based scaffold. The in vivo results suggest the antibody remains intact throughout the chemical modification.

Research Support This work was partially supported by the Dr. Jack Krohmer Professorship Funds.