Modular Strategy for the Construction of Radiometalated Antibodies for Positron Emission Tomography Based on Inverse Electron Demand Diels-Alder Click Chemistry

A modular system for the construction of radio-metalated antibodies was developed based on the bioorthogonal cycloaddition reaction between 3-(4-benzylamino)-1,2,4, 5-tetrazine and the strained dienophile norbomene. The well-characterized, HER2-specific antibody trastuzumab and the positron emitting radioisotopes Cu-64 and Zr-89 were employed as a model system. The antibody was first covalently coupled to norbomene, and this stock of norbornene-modified antibody was then reacted with tetrazines bearing the chelators 1,4,7,10-tetraazacyclo-dodecane:1,4,7,10-tetiaacetic acid (DOTA) or desferrioxamine (DFO) and subsequently radiometalated with Cu-64 and Zr-89, respectively. The modification strategy is simple and robust, and the resultant radiometalated constructs were obtained in high specific (2.7-75.3 mCi/mg). For a given initial stoichiometric ratio of norbornene to antibody, the Cu-64-DOTA- and Zr-89-DFO-based probes Were shown to be nearly identical in terms of stability, the number of chelates per antibody, and immunoreactivity (9356 hi all cases). In vivo PET imaging and acute biodistribution experiments revealed significant, specific uptake of the Cu-64- and Zr-89-trastuzumab bioconjugates in HER2-positive BT-474 xenografts, with little background uptake in HER2-negative MDA-M-408 xenografts or other tissues. This modular system-one in which the divergent point is a single covalently modified antibody stock that can be reacted selectively with various chelators-will allow for both greater versatility and more facile cross-comparisons in the development of antibody-based radiopharmaceuticals.