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


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

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How to cite this article:
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