Abstract
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Objectives The ability to rapidly image tumors with intact antibodies is often hindered by their sluggish pharmacokinetics. Antibody fragments circumvent this dilemma by maintaining affinity and specificity while exhibiting customized targeting and blood clearance profiles suitable for same-day PET imaging. Our overall goal is to engineer an anti-αvβ6 diabody and a covalently bound anti-αvβ6 cys-diabody and evaluate the utility of each fragment to rapidly image αvβ6-positive tumors in vivo with PET.
Methods The anti-αvβ6 diabodies were expressed from 293F stable cells and purified by Ni-NTA chromatography. Binding affinities were assessed using competitive ELISA and specificities were determined with flow cytometry on Dx3Puroβ6 (αvβ6+) and Dx3Puro (αvβ6-) cell lines. The diabodies were radiolabeled with N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB, 10 mins, 37○C, pH 8.7) and incubated with Dx3Puroβ6 and Dx3Puro cell lines to determine in vitro immunoreactivity.
Results Potent antigen binding was observed for the diabody (IC50=0.8nM) and cys-diabody (IC50=0.6nM) towards immobilized αvβ6. [18F]SFB radiolabeling was rapidly accomplished in 10 mins with yields of 22.6±3.6% (diabody) and 8.0±1.7% (cys-diabody) and radiochemical purities >98%. Specificity and immunoreactivity were well preserved following radiolabeling with [18F]SFB (67.6±0.5% diabody, 87.1±1.6% cys-diabody), further demonstrating their utility as potential PET imaging agents.
Conclusions We have engineered two novel anti-αvβ6 diabodies and demonstrated the potent and highly specific binding of these molecular imaging agents towards the αvβ6 integrin. Comprehensive small animal PET imaging, biodistribution, and immunohistochemical analyses are currently under investigation for each diabody.
Research Support Office of Science, United States Department of Energy, DE-SC0008385.