New Bifunctional Chelators Incorporating Dibromomaleimide Groups for Radiolabeling of Antibodies with Positron Emission Tomography Imaging Radioisotopes

[Image: see text] Positron Emission Tomography (PET) imaging with antibody-based contrast agents frequently uses the radioisotopes [(64)Cu]Cu(2+) and [(89)Zr]Zr(4+). The macrobicyclic chelator commonly known as sarcophagine (sar) is ideal for labeling receptor-targeted biomolecules with [(64)Cu]Cu(2+). The siderophore chelator, desferrioxamine-B (dfo), has been widely used to incorporate [(89)Zr]Zr(4+) into antibodies. Here, we describe new bifunctional chelators of sar and dfo: these chelators have been functionalized with dibromomaleimides (dbm), that enable site-specific and highly stable attachment of molecular cargoes to reduced, solvent-accessible, interstrand native disulfide groups. The new sar–dbm and dfo–dbm derivatives can be easily conjugated with the IgG antibody trastuzumab via reaction with reduced interstrand disulfide groups to give site-specifically modified dithiomaleamic acid (dtm) conjugates, sar–dtm–trastuzumab and dfo–dtm–trastuzumab, in which interstrand disulfides are rebridged covalently with a small molecule linker. Both sar– and dfo–dtm–trastuzumab conjugates have been radiolabeled with [(64)Cu]Cu(2+) and [(89)Zr]Zr(4+), respectively, in near quantitative radiochemical yield (>99%). Serum stability studies, in vivo PET imaging, and biodistribution analyses using these radiolabeled immunoconjugates demonstrate that both [(64)Cu]Cu-sar–dtm–trastuzumab and [(89)Zr]Zr-dfo–dtm–trastuzumab possess high stability in biological milieu. Dibromomaleimide technology can be easily applied to enable stable, site-specific attachment of radiolabeled chelators, such as sar and dfo, to native interstrand disulfide regions of antibodies, enabling tracking of antibodies with PET imaging.