Abstract

PurposeConstruction of antibody-based, molecular-targeted optical imaging probes requires the labeling of an antibody with a fluorophore. The most common method for doing this involves non-specifically conjugating a fluorophore to an antibody, resulting in poorly defined, heterogeneous imaging probes that often have suboptimal in vivo behavior. We tested a new strategy to site-specific label antibody-based imaging probes using the SpyCatcher/SpyTag protein ligase system.ProceduresWe used the SpyCatcher/SpyTag protein ligase system to site specifically label nimotuzumab, an anti-EGFR antibody and an anti-HER3 diabody. To prevent the labeling from interfering with antigen binding, we introduced the SpyTag and SpyCatcher at the C-terminus of the antibody and diabody, respectively. Expression and binding properties of the C-terminal antibody-SpyTag and diabody-SpyCatcher fusions were similar to the antibody and diabody, indicating that the SpyTag and SpyCatcher fusions were well tolerated at this position. Site-specific labeling of the antibody and diabody was performed in two steps. First, we labeled the SpyCatcher with IRDye800CW-Maleimide and the SpyTag with IRDye800CW-NHS. Second, we conjugated the IRDye800CW-SpyCatcher and the IRDye800CW-SpyTag to the antibody or diabody, respectively. We confirmed the affinity and specificity of the IRDye800CW-labeled imaging probes using biolayer interferometry and flow cytometry. We analyzed the in vivo biodistribution and tumor accumulation of the IRDye800CW-labeled nimotuzumab and anti-HER3 diabody in nude mice bearing xenografts that express EGFR and HER3, respectively.ResultsExpression and binding properties of the C-terminal antibody-SpyTag and diabody-SpyCatcher fusions were similar to the antibody and diabody, indicating that the SpyTag and SpyCatcher fusions were well tolerated at this position. We confirmed the affinity and specificity of the IRDye800CW-labeled imaging probes using biolayer interferometry and flow cytometry. We analyzed the in vivo biodistribution and tumor accumulation of the IRDye800CW-labeled nimotuzumab and anti-HER3 diabody in nude mice bearing xenografts that express EGFR and HER3, respectively. Site-specifically IRDye800CW-labeled imaging probes bound to their immobilized targets, cells expressing these targets, and selectively accumulated in xenografts.ConclusionsThese results highlight the ease and utility of using the modular SpyTag/SpyCatcher protein ligase system for site-specific fluorescent labeling of protein-based imaging probes. Imaging probes labeled in this manner will be useful for optical imaging applications such as image-guided surgery and have broad application for other imaging modalities.

Highlights

  • Non-invasive optical imaging is an emerging approach that aids clinicians in many aspects of cancer diagnosis and treatment [1,2,3]

  • Antibody-based, molecular targeted imaging (MTI) probes are being developed for optical imaging to allow visualization of disease-specific markers

  • We demonstrated site-specific labeling of nimotuzumab and an anti-HER3 diabody using the SpyCatcher/SpyTag system for in vivo cancer imaging

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Summary

Introduction

Non-invasive optical imaging is an emerging approach that aids clinicians in many aspects of cancer diagnosis and treatment [1,2,3]. Fluorophores are most commonly conjugated to antibodies in a non-specific manner, which can result in decreased antigen-binding affinity and poor pharmacological properties [4,5,6,7]. To overcome this problem, methods have been developed to label antibodies at specific locations. Peptide tag-based labeling allows the fluorophore to be attached at a specific location on the imaging probe, with minimal off-site labeling [6, 7]. Site-specific labeling using peptide tags results in MTI probes that are more homogenous [9] compared to random approaches [7]

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