Evaluation of maleimide derivative of DOTA for site-specific labeling of recombinant affibody molecules

Affibody molecules are a new class of small (7 kDa) scaffold affinity proteins, which demonstrate promising properties as agents for in vivo radionuclide targeting. The Affibody scaffold is cysteine-free and therefore independent of disulfide bonds. Thus, a single thiol group can be engineered into the protein by introduction of one cysteine. Coupling of thiol-reactive bifunctional chelators can enable site-specific labeling of recombinantly produced Affibody molecules. In this study, the use of 1,4,7,10-tetraazacyclododecane-1,4,7-tris-acetic acid-10-maleimidoethylacetamide (MMA-DOTA) for In-111-labeling of anti-HER2 Affibody molecules HiS(6)-Z(HER2:342)-Cys and Z(HER2:2395)-CYS has been evaluated. The introduction of a cysteine residue did not affect the affinity of the proteins, which was 29 pM for HiS(6)-Z(HER2:342)-Cys and 27 pM for Z(HER2:2395)-CYS, comparable with 22 pM for the parental Z(HER2:342). MMA-DOTA was conjugated to DTT-reduced Affibody molecules with a coupling efficiency of 93% using a 1:1 molar ratio of chelator to protein. The conjugates were labeled with. In-111 to a specific radioactivity of up to 7 GBq/mmol, with preserved binding for the target HER2. In vivo, the non His-tagged variant In-111-[MMA-DOTA-CyS61]-Z(HER2:2395)-Cys demonstrated appreciably lower liver uptake than its His-tagcontaining counterpart. In mice bearing HER2-expressing LS174T xenografts, In-111-[MMA-DOTA-Cys(61)]- Z(HER2:2395)-CYS showed specific and rapid tumor localization, and rapid clearance from blood and nonspecific compartments, leading to a tumor-to-blood-ratio of 18 +/- 8 already 1 h p.i. Four hours p.i., the tumor-to-blood ratio was 138 +/- 8. Xenografts were clearly visualized already 1 h p.i.