Click-Chemistry Strategy for Labeling Antibodies with Copper-64 via a Cross-Bridged Tetraazamacrocyclic Chelator Scaffold

We report a click-chemistry based modular strategy for antibody labeling with Cu-64 (t(1/2) = 12.7 h; beta+ 0.656 MeV, 17.4%; beta 0.573 MeV, 39%; EC 43%) under ambient condition utilizing a cross-bridged tetraazamacrocyclic (CB-TE2A) analogue, which otherwise requires harsh conditions that make the CB-TE2A analogues under-utilized for protein labeling despite the fact that they form kinetically inert copper complexes with high in vivo stability. Our strategy involves prelabeling a CB-TE2A based scaffold (CB-TE2A-1C) with Cu-64 and its subsequent reaction with an antibody via the tetrazine-norbornene mediated click chemistry. The effectiveness of this strategy was demonstrated by labeling two monoclonal antibodies, an anti-PSMA antibody (YPSMA-1) and a chimeric anti-phosphatidylserine antibody (Bavituximab). The immunoreactivity of the antibodies remained unchanged after the tetrazine modification and click-chemistry Cu-64 labeling. To further demonstrate the practicality of the modular Cu-64 labeling strategy, we tested positron emission tomography (PET) imaging of tumor with the Cu-64-labeled bavituximab in a mouse xenograft model. The tumor visualization and uptake of the labeled antibody exhibited the versatility of the click-chemistry strategy.