Click Chemistry Enables [Zr]Zr-DOTA Radioimmunoconjugation for Theranostic Zr-immunoPET.

There have been predictions that the use of the macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) in zirconium-89 (Zr) immuno-positron emission tomography (Zr-immunoPET) could enhance the in vivo stability of Zr radioimmunoconjugates. However, conjugating [Zr]Zr-DOTA to a monoclonal antibody (mAb) remains a challenge as the heat treatment required for [Zr]Zr-DOTA chelation can lead to thermal denaturation of the mAb moieties. We developed a method for synthesizing [Zr]Zr-DOTA-mAb based on a tetrazine (Tz)-conjugated bifunctional DOTA derivative 2,2',2″-(10-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-3,21,26-trioxo-6,9,12,15,18-pentaoxa-29-carboxy-2,22,25-triazanonacosane-29-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTAGA-Tz) and the inverse electron-demand Diels-Alder (IEDDA) click chemistry reaction where -cyclooctene-modified mAbs are conjugated to [Zr]Zr-DOTAGA without being exposed to heat. The stability of IEDDA-derived [Zr]Zr-DOTAGA-trastuzumab was confirmed by in vitro, ex vivo, and in vivo testing and comparative analysis against the conventional deferoxamine (DFO) counterpart [Zr]Zr-DFO-trastuzumab. The in vivo immunoPET imaging using [Zr]Zr-DOTAGA-trastuzumab clearly visualized human epidermal growth factor receptor 2-positive malignancies in murine xenograft models. Greater tumor contrast was observed from [Zr]Zr-DOTAGA-trastuzumab at a 72-h delayed scan compared with [Zr]Zr-DFO-trastuzumab. These findings suggest that our IEDDA ligation approach can be an effective means of synthesizing [Zr]Zr-DOTA-mAb and can enhance the theranostic potential of Zr-immunoPET in DOTA-mediated radioimmunotherapy.