Evaluation of a Maleimido Derivative of CHX-A′′ DTPA for Site-Specific Labeling of Affibody Molecules

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Abstract
Affibody molecules are a new class of small targeting proteins based on a common three-helix bundle structure. Affibody molecules binding a desired target may be selected using phage-display technology. An Affibody molecule ZHER2:342 binding with subnanomolar affinity to the tumor antigen HER2 has recently been developed for radionuclide imaging in vivo. Introduction of a single cysteine into the cysteine-free Affibody scaffold provides a unique thiol group for site-specific labeling of recombinant Affibody molecules. The recently developed maleimido-CHX-A′′ DTPA was site-specifically conjugated at the C-terminal cysteine of ZHER2:2395-C, a variant of ZHER2:342, providing a homogeneous conjugate with a dissociation constant of 56 pM. The yield of labeling with 111In was >99% after 10 min at room temperature. In vitro cell tests demonstrated specific binding of 111In-CHX-A′′ DTPA-Z2395-C to HER2-expressing cell-line SKOV-3 and good cellular retention of radioactivity. In normal mice, the conjugate demonstrated rapid clearance from all nonspecific organs except kidney. In mice bearing SKOV-3 xenografts, the tumor uptake of 111In-CHX-A′′ DTPA-Z2395-C was 17.3 ± 4.8% IA/g and the tumor-to-blood ratio 86 ± 46 (4 h postinjection). HER2-expressing xenografts were clearly visualized 1 h postinjection. In conclusion, coupling of maleimido-CHX-A′′ DTPA to cysteine-containing Affibody molecules provides a well-defined uniform conjugate, which can be rapidly labeled at room temperature and provides high-contrast imaging of molecular targets in vivo.

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