New bifunctional chelators incorporating dibromomaleimide groups for radiolabeling of antibodies with PET imaging radioisotopes

Positron Emission Tomography (PET) imaging with antibody-based contrast agents frequently uses the radioisotopes [Cu]Cu and [Zr]Zr. The macrobicyclic chelator commonly known as sarcophagine (sar) is ideal for labelling receptor-targeted biomolecules with [Cu]Cu; the siderophore chelator, desferrioxamine-B (dfo), has been widely used to incorporate [Zr]Zr 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, solventaccessible, 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-dtmtrastuzumab, in which interstrand disulfides are re-bridged covalently with a small molecule linker. Both sarand dfo-dtm-trastuzumab conjugates have been radiolabeled with [Cu]Cu and [Zr]Zr respectively, in near quantitative radiochemical yield (>99%). Serum stability studies, in vivo PET imaging and biodistribution analyses using these radiolabeled immunoconjugates demonstrate that both [Cu]Cu-sar-dtm-trastuzumab and [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.