The identity of the conjugate was confirmed by liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS). The conjugate was designated as DOTA-C-(HE) 3-ADAPT6. For site-specific labeling, a maleimido-derivative of the versatile DOTA-chelator was conjugated to the unique cysteine introduced at the N-terminus of the (HE) 3-ADAPT6. In addition, an ADAPT6 molecule with an N-terminal GSSHHHHHHDEAVDANS-sequence was also produced for blocking experiments, H 6-ADAPT. The variant was designated as C-(HE) 3-ADAPT6. A GCSSHEHEHEDEAVDANS-sequence was introduced at the N-terminus both as a tag for immobilized metal ion affinity chromatography (IMAC) purification and as a biodistribution modifier ( 30). The binding of the selected molecule, ADAPT ERBB2-FACS-6, to albumin was deleted by complementing the substitution 元7R ( 28) at the albumin-binding surface with two alanine substitutions (S18Y20). Thus, the use of engineered scaffold proteins is a promising approach for development of imaging agents. Several other targeting scaffold-based proteins, such as anticalins ( 17), cysteine-knot peptides ( 18), fibronectin domains ( 19), and designed ankyrin repeats ( 20) have successfully been used for imaging of therapeutic targets in preclinical studies. Affibody molecules have also demonstrated successful preclinical imaging of other molecular targets, including EGFR ( 13), HER3 ( 14), IGF-1R ( 15), and PDGFRβ ( 16). Clinical studies have confirmed the feasibility of imaging of HER2-expressing metastases using affibody molecules within hours after injection ( 12). In preclinical models, this scaffold protein has demonstrated tumor-to-organ ratios exceeding those of monoclonal antibodies by one-two orders of magnitude ( 11). The first scaffold protein for molecular imaging was an affibody molecule targeting HER2, a receptor tyrosine kinase that is overexpressed in cancer and serves as a target for several mAbs and tyrosine kinase inhibitors ( 10). 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.

Framework protein scaffold