A minimum of 20 million coincident events were recorded for each scan, which lasted between 10 and 45 min. 64Cu- and 89Zr-trastuzumab bioconjugates in HER2-positive BT-474 xenografts, with little background uptake in HER2-negative MDA-MB-468 xenografts or other tissues. This modular systemone in which the divergent point is a single covalently modified antibody stock that can be reacted selectively with various chelatorswill allow for both greater versatility and more facile cross-comparisons in the development of antibody-based radiopharmaceuticals. Introduction Over the past two decades, radiopharmaceuticals based on antibodies have assumed an increasingly prominent role in both diagnostic and therapeutic nuclear medicine. This trend is particularly evident in the field of positron emission tomography (PET), in which a wide variety of effective antibody-based radiotracers have been developed against an array of cancer biomarkers.1?3 Indeed, while some promising imaging agents have been labeled with long-lived nonmetallic radionuclides such as 124I, the majority of antibody-based PET bioconjugates have employed positron-emitting radiometals, including 64Cu, 86Y, and, most recently, 89Zr.4?8 In these systems, radiometals offer significant advantages over their nonmetallic cousins, most notably decay characteristics that result in high image quality, radioactive half-lives that complement the biological half-lives of the antibody vectors, and enhanced control and ease of radiolabeling through the use of chelating moieties. Despite their benefits, however, these chelating moieties are the source of a somewhat confounding Mouse monoclonal to NFKB1 issue in the study of radiometalated antibodies. Put simply, different radiometals require different chelators. For example, the small, hard 89Zr4+ cation shows very high affinity for the multiple oxygen donors of the chelator desferrioxamine (DFO), while the larger and softer 64Cu2+ cation exhibits higher thermodynamic and kinetic stability when bound to chelators bearing nitrogen donors in addition to oxygens, for example, 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA) and 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diyl)diacetic acid (CB-TE2A).6,9 Further, different chelators often require dramatically different synthetic strategies for antibody couplings.(10) In an isolated case of one antibody and one radiometal, these facts do not present a problem. However, they do create a significant obstacle to the versatility of radiometalated bioconjugates. To wit, given a particular monoclonal antibody, the development of a 64Cu-CB-TE2A-mAb conjugate for PET, a 89Zr-DFO-mAb conjugate for PET, and a 225Ac-DOTA-mAb conjugate for therapy would require three different routes for antibody modification. Not only would this require additional time to develop and optimize each pathway, but the disparate routes would also mandate differing reaction conditions for each antibody, opening the door for differences in immunoreactivity and chelator/antibody ratio and ultimately making meaningful comparisons among the various radiopharmaceuticals more difficult. Consequently, a modular systemone in which the divergent point is a single covalently modified antibody stock that can be reacted selectively with various chelatorswould resolve these issues and allow for more versatility and cross-comparisons in the development of antibody-based radiopharmaceuticals. The chemical requirements of such a modular systemselectivity, biocompatibility, bioorthogonalitymake it an almost perfect application for the use of click chemistry. Coined by K. Barry Sharpless, the UNC2881 term click chemistry broadly defines a group of chemical reactions by which two molecular components can be joined via a selective, rapid, clean, bioorthogonal, and biocompatible ligation.11?13 By far, the most popular example of click chemistry is the Cu(I)-catalyzed [3 + 2] Huisgen cycloaddition between an azide and alkyne.(14) This reaction has already been widely employed UNC2881 in the development of radiotracers, particularly 18F-based PET probes.15?18 The application of this technology to radiometal-based probes has lagged behind, however, most likely due to concerns over metal contamination by the catalyst itself, though clickable chelators based on both the Cu(I)-catalyzed reaction and other Cu(I)-free systems have become more common in the literature in recent years.19?22 Very recently, another promising click variant has come to light: the inverse electron demand DielsCAlder reaction between a tetrazine moiety and a UNC2881 strained alkene dienophile (Figure ?(Figure11).23?25 Like other click reactions, the ligation is selective, fast, biocompatible, and bioorthogonal, and unlike many DielsCAlder reactions, the coupling is irreversible, forming stable pyridazine products after the retro-DielsCAlder release of dinitrogen from the reaction intermediate. A.