Theranostics 2018; 8(17):4856-4869. doi:10.7150/thno.24252

Research Paper

Evaluation of antibody fragment properties for near-infrared fluorescence imaging of HER3-positive cancer xenografts

Ayman El-Sayed1, Wendy Bernhard1, Kris Barreto1, Carolina Gonzalez1, Wayne Hill1, Landon Pastushok1, Humphrey Fonge2,3✉, C. Ronald Geyer1✉

1. Department of Pathology, University of Saskatchewan, Saskatoon, Canada
2. Department of Medical Imaging, University of Saskatchewan, Saskatoon, Canada
3. Department of Medical Imaging, Royal University Hospital Saskatoon, Canada

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El-Sayed A, Bernhard W, Barreto K, Gonzalez C, Hill W, Pastushok L, Fonge H, Geyer CR. Evaluation of antibody fragment properties for near-infrared fluorescence imaging of HER3-positive cancer xenografts. Theranostics 2018; 8(17):4856-4869. doi:10.7150/thno.24252. Available from

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In vivo imaging is influenced by the half-life, tissue penetration, biodistribution, and affinity of the imaging probe. Immunoglobulin G (IgG) is composed of discrete domains with known functions, providing a template for engineering antibody fragments with desired imaging properties. Here, we engineered antibody-based imaging probes, consisting of different combinations of antibody domains, labeled them with the near-infrared fluorescent dye IRDye800CW, and evaluated their in vivo imaging properties. Antibody-based imaging probes were based on an anti-HER3 antigen binding fragment (Fab) isolated using phage display.

Methods: We constructed six anti-HER3 antibody-based imaging probes: a single chain variable fragment (scFv), Fab, diabody, scFv-CH3, scFv-Fc, and IgG. IRDye800CW-labeled, antibody-based probes were injected into nude mice bearing FaDu xenografts and their distribution to the xenograft, liver, and kidneys was evaluated.

Results: These imaging probes bound to recombinant HER3 and to the HER3-positive cell line, FaDu. Small antibody fragments with molecular weight <60 kDa (scFv, diabody, and Fab) accumulated rapidly in the xenograft (maximum accumulation between 2-4 h post injection (hpi)) and cleared primarily through the kidneys. scFv-CH3 (80 kDa) had fast clearance and peaked in the xenograft between 2-3 hpi and cleared from xenograft in a rate comparable to Fab and diabody. IgG and scFv-Fc persisted in the xenografts for up to 72 hpi and distributed mainly to the xenograft and liver. The highest xenograft fluorescence signals were observed with IgG and scFv-Fc imaging probes and persisted for 2-3 days.

Conclusion: These results highlight the utility of using antibody fragments to optimize clearance, tumor labeling, and biodistribution properties for developing anti-HER3 probes for image-guided surgery or PET imaging.

Keywords: antibody fragments, near-infrared fluorescence imaging, HER3, ErbB3, IRDye800CW