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 ⁶⁴Cu (t_1/2 = 12.7 h; β⁺ 0.656 MeV, 17.4%; β^- 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 ⁶⁴Cu 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 ⁶⁴Cu labeling. To further demonstrate the practicality of the modular ⁶⁴Cu labeling strategy, we tested positron emission tomography (PET) imaging of tumor with the ⁶⁴Cu-labeled bavituximab in a mouse xenograft model. The tumor visualization and uptake of the labeled antibody exhibited the versatility of the click-chemistry strategy.