Abstract
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Objectives The detection of cell surface markers with high specificity and affinity is essential in tumor imaging. In this study, we developed a unique reporter system using a hapten imaging probe and an anti-hapten antibody (scFv) for HER2-expressed tumor imaging by two-step targeting.
Methods A Hapten-specific scFv was produced by panning phage displayed Human Single Fold scFv Libraries (Tomlinson I + J) against a hapten-peptide (histamine-succinyl-GSYK, Him). The selected scFv was recombinantly fused to a HER2 affibody (ZHER2:477) via a peptide linker, (G4S)3. The binding specificity of the fusion protein (scFv-L-A) was characterized by SPR. HER2 targeting of the scFv-L-A and hapten (Him) was evaluated by in vitro confocal microscopy and flow cytometry using cypate- and sulfo-Cy5-conjugated haptens, Cy-Him and Cy5-Him, respectively. NIR tomographic imaging and ex vivo tumor tissue labeling were performed with mice bearing SK-BR3 xenograft tumors and Cy-Him.
Results Hapten (Him) and its fluorescent conjugates were characterized by HPLC, LC/MS, and UV/Vis spectrometry. The highest affinity scFv (KD~2.3 nM) to the hapten was obtained and used for the fusion protein construction, which showed dual target specificity to both HER2 and the hapten with KD’s of ~17.3 nM and ~2.52 nM, respectively by SPR. The inhibition SPR study confirmed the bispecificity of the fusion protein. In vitro cell labeling and flow cytometry demonstrated the stepwise targeting of the scFv-L-A and hapten probes. The tomographic FMT data showed the tumor localized NIR signals from the fusion pre-treated mice followed by Cy-Him. Immunofluorescent staining of tumor tissue validated the HER2 co-localization of scFv-L-A and Cy-Him in NIR confocal microscopy.
Conclusions Our results suggest that this hapten-peptide and anti-hapten scFv can be a universal reporter system in a wide range of imaging and therapeutic applications.