Site-Specific Fluorescent Labeling of Antibodies and Diabodies Using SpyTag/SpyCatcher System for In Vivo Optical Imaging

• DOI: 10.1007/s11307-018-1222-y
• Publication Date: 2018-06-12T11:57:51Z
• Authors: Md. Kausar Alam; Ayman El-Sayed; Kris Barreto; Wendy Bernhard; Humphrey Fonge; C. Ronald Geyer
• Keywords: 0301 basic medicine; 0303 health sciences; Cell Transplantation; Acute Lung Injury; Graft Survival; Green Fluorescent Proteins; Optical Imaging; Antibodies, Monoclonal; Fluorescent Antibody Technique; 3. Good health; Ligases; 03 medical and health sciences; Hepatocyte Nuclear Factor 4; Cell Tracking; Organ Specificity; Antibodies, Bispecific; Luminescent Measurements; Animals; Heterografts; Humans; Female; Luciferases; Cells, Cultured; Fluorescent Dyes

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