![]() The use of scaffold proteins has enabled the generation of binders with small size (4–20 kDa), well-defined specificity and low nanomolar or subnanomolar affinity. Scaffold proteins contain a robust structurally defined framework, which provides rigidity, and a variable surface area, where amino acids are randomized to create libraries for selection of binders. ©2015 AACR.Ī possible way to generate high-affinity targeting proteins is to combine molecular display techniques (e.g., phage, ribosomal, yeast, or bacterial display) with the use of engineered scaffold proteins ( 9). Our results offer a preclinical proof of concept for the use of ADAPT probes for noninvasive in vivo imaging. ![]() PET experiments demonstrated feasibility for discrimination of xenografts with high or low HER2 expression. HER2-expressing xenografts were visualized by gamma-camera or PET at 1 hour after infusion. Unbound tracer underwent rapid renal clearance followed by high renal reabsorption. ![]() Pharmacologic studies in mice demonstrated that the fully engineered molecule 111In/ 68Ga-DOTA-(HE) 3-ADAPT6 was specifically bound and taken up by HER2-expressing tumors, with a high tumor-to-normal tissue ratio in xenograft models of human cancer. Incorporation of a unique cysteine allowed site-specific conjugation to a maleimido derivative of a DOTA chelator, enabling radionuclide labeling, 111In for SPECT imaging and 68Ga for PET imaging. Furthermore, ABD was engineered to enable rapid purification, to eradicate its binding to albumin, and to enable rapid blood clearance. Surface-exposed amino acids of ABD were randomized to create a combinatorial library enabling selection of high-affinity binders to various proteins. In this study, we report the development of a clinical lead probe termed ADAPT6 that binds HER2, an oncoprotein overexpressed in many breast cancers that serves as a theranostic biomarker for several approved targeting therapies. One class of these imaging probes, termed ABD-Derived Affinity Proteins (ADAPT), has been created using the albumin-binding domain (ABD) of streptococcal protein G as a stable protein scaffold. Small engineered scaffold proteins have attracted attention as probes for radionuclide-based molecular imaging.
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