Jack M. Webster

A 2-Helix Small Protein Labeled with 68Ga for PET Imaging of HER2 Expression

Affibody molecules are a class of scaffold proteins being developed into a generalizable approach to targeting tumors. Many 3-helix–based Affibody proteins have shown excellent in vivo properties for tumor imaging and therapy. By truncating one α-helix that is not responsible for receptor recognition in the Affibody and maturating the protein affinity through synthetic strategies, we have successfully identified in our previous research several small 2-helix proteins with excellent binding affinities to human epidermal growth factor receptor type 2 (HER2). With preferential properties such as faster blood clearance and tumor accumulation, lower immunogenic potential, and facile and economically viable synthetic schemes, we hypothesized that these 2-helix protein binders could become excellent molecular imaging probes for monitoring HER2 expression and modulation. Methods: In this study, a 2-helix small protein, MUT-DS, was chemically modified with a metal chelator, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). DOTA-MUT-DS was then site-specifically radiolabeled with an important PET radionuclide, 68Ga. The resulting radiolabeled anti-HER2 2-helix molecule was further evaluated as a potential molecular probe for small-animal PET HER2 imaging in a SKOV3 tumor mouse model. Results: The 2-helix DOTA-MUT-DS showed high HER2-binding affinity (dissociation constant, 4.76 nM). The radiolabeled probe displayed high stability in mouse serum and specificity toward HER2 in cell cultures. Biodistribution and small-animal PET studies further showed that 68Ga-DOTA-MUT-DS had rapid and high SKOV3 tumor accumulation and quick clearance from normal organs. The specificity of 68Ga-DOTA-MUT-DS for SKOV3 tumors was confirmed by monitoring modulation of HER2 protein on treatment of tumor mice with heat shock protein 90 inhibitor 17-N,N-dimethyl ethylene diamine-geldanamycin in vivo. Conclusion: This proof-of-concept research clearly demonstrated that synthetic 2-helix 68Ga-DOTA-MUT-DS is a promising PET probe for imaging HER2 expression in vivo. The Affibody-derived small 2-helix protein scaffold has great potential for developing targeting agents for a variety of tumor-associated biomarkers.

Engineered Two-Helix Small Proteins for Molecular Recognition

Less is more: By starting with a high‐affinity HER2‐binding 3‐helix affibody molecule, we successfully developed 2‐helix small protein binders with 5 nM affinities by using a combination of several different strategies. Our efforts clearly suggest that 2‐helix small proteins against important tumor targets can be obtained by rational protein design and engineering.

Functional Imaging of Oxidative Stress with a Novel PET Imaging Agent, 18F-5-Fluoro-l-Aminosuberic Acid

Glutathione is the predominant endogenous cellular antioxidant, playing a critical role in the cellular defensive response to oxidative stress by neutralizing free radicals and reactive oxygen species. With cysteine as the rate-limiting substrate in glutathione biosynthesis, the cystine/glutamate transporter (system xc-) represents a potentially attractive PET biomarker to enable in vivo quantification of xc- activity in response to oxidative stress associated with disease. We have developed a system xc- substrate that incorporates characteristics of both natural substrates, l-cystine and l-glutamate (l-Glu). l-aminosuberic acid (l-ASu) has been identified as a more efficient system xc- substrate than l-Glu, leading to an assessment of a series of anionic amino acids as prospective PET tracers. Herein, we report the synthesis and in vitro and in vivo validation of a lead candidate, 18F-5-fluoro-aminosuberic acid (18F-FASu), as a PET tracer for functional imaging of a cellular response to oxidative stress with remarkable tumor uptake and retention. Methods: 18F-FASu was identified as a potential PET tracer based on an in vitro screening of compounds similar to l-cystine and l-Glu. Affinity toward system xc- was determined via in vitro uptake and inhibition studies using oxidative stress–induced EL4 and SKOV-3 cells. In vivo biodistribution and PET imaging studies were performed in mice bearing xenograft tumors (EL4 and SKOV-3). Results: In vitro assay results determined that l-ASu inhibited system xc- as well as or better than l-Glu. The direct comparison of uptake of tritiated compounds demonstrated more efficient system xc- uptake of l-ASu than l-Glu. Radiosynthesis of 18F-FASu allowed the validation of uptake for the fluorine-bearing derivative in vitro. Evaluation in vivo demonstrated primarily renal clearance and uptake of approximately 8 percentage injected dose per gram in SKOV-3 tumors, with tumor-to-blood and tumor-to-muscle ratios of approximately 12 and approximately 28, respectively. 18F-FASu uptake was approximately 5 times greater than 18F-FDG uptake in SKOV-3 tumors. Dynamic PET imaging demonstrated uptake in EL4 tumor xenografts of approximately 6 percentage injected dose per gram and good tumor retention for at least 2 h after injection. Conclusion: 18F-FASu is a potentially useful metabolic tracer for PET imaging of a functional cellular response to oxidative stress. 18F-FASu may provide more sensitive detection than 18F-FDG in certain tumors.

18F-5-fluoro-aminosuberic acid (FASu) as a potential tracer to gauge oxidative stress in breast cancer models.

The cystine transporter (system xC−) is an antiporter of cystine and glutamate. It has relatively low basal expression in most tissues and becomes upregulated in cells under oxidative stress (OS) as one of the genes expressed in response to the antioxidant response element promoter. We have developed 18F-5-fluoroaminosuberic acid (FASu), a PET tracer that targets system xC−. The goal of this study was to evaluate 18F-FASu as a specific gauge for system xC− activity in vivo and its potential for breast cancer imaging. Methods: 18F-FASu specificity toward system xC− was studied by cell inhibition assay, cellular uptake after OS induction with diethyl maleate, with and without anti-xCT small interfering RNA knockdown, in vitro uptake studies, and in vivo uptake in a system xC−–transduced xenograft model. In addition, radiotracer uptake was evaluated in 3 breast cancer models: MDA-MB-231, MCF-7, and ZR-75-1. Results: Reactive oxygen species–inducing diethyl maleate increased glutathione levels and 18F-FASu uptake, whereas gene knockdown with anti-xCT small interfering RNA led to decreased tracer uptake. 18F-FASu uptake was robustly inhibited by system xC− inhibitors or substrates, whereas uptake was significantly higher in transduced cells and tumors expressing xCT than in wild-type HEK293T cells and tumors (P < 0.0001 for cells, P = 0.0086 for tumors). 18F-FASu demonstrated tumor uptake in all 3 breast cancer cell lines studied. Among them, triple-negative breast cancer MDA-MB-231, which has the highest xCT messenger RNA level, had the highest tracer uptake (P = 0.0058 when compared with MCF-7; P < 0.0001 when compared with ZR-75-1). Conclusion: 18F-FASu as a system xC− substrate is a specific PET tracer for functional monitoring of system xC− and OS imaging. By enabling noninvasive analysis of xC− responses in vivo, this biomarker may serve as a valuable target for the diagnosis and treatment monitoring of certain breast cancers.

A simple route to [11C]N-Me labeling of aminosuberic acid for proof of feasibility imaging of the xC− transporter

Oxidative stress has been implicated in a variety of conditions, including cancer, heart failure, diabetes, neurodegeneration and other diseases. A potential biomarker for oxidative stress is the cystine/glutamate transporter, system x(C)(-). L-Aminosuberic acid (L-ASu) has been identified as a system x(C)(-) substrate. Here we report a facile method for [(11)C]N-Me labeling of L-ASu, automation of the radiochemical process, and preliminary PET imaging with EL4 tumor bearing mice. The results demonstrate uptake in the tumor above background, warranting further studies on the use of radiolabeled analogs of L-ASu as a PET imaging agent for system x(C)(-).