Preclinical evaluation of anti-α_vβ₆ diabodies and anti-α_vβ₆ cys-diabodies for imaging α_vβ₆-positive tumors with positron emission tomography

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-α_vβ₆ diabody and a covalently bound anti-α_vβ₆ cys-diabody and evaluate the utility of each fragment to rapidly image α_vβ₆-positive tumors in vivo with PET.

Methods The anti-α_vβ₆ 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 (α_vβ₆+) and Dx3Puro (α_vβ₆-) 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 α_vβ₆. [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-α_vβ₆ diabodies and demonstrated the potent and highly specific binding of these molecular imaging agents towards the α_vβ₆ 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.