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Rapid optical imaging of human breast tumour xenografts using anti-HER2 VHHs site-directly conjugated to IRDye 800CW for image-guided surgery

Abstract

Purpose

Molecular optical imaging using monoclonal antibodies is slow with low tumour to background ratio. We used anti-HER2 VHHs conjugated to IRDye 800CW to investigate their potential as probes for rapid optical molecular imaging of HER2-positive tumours by the determination of tumour accumulation and tumour to background levels.

Methods

Three anti-HER2 VHHs (11A4, 18C3, 22G12) were selected with phage display and produced in Escherichia coli. Binding affinities of these probes to SKBR3 cells were determined before and after site-specific conjugation to IRDye 800CW. To determine the potential of VHH-IR as imaging probes, serial optical imaging studies were carried out using human SKBR3 and human MDA-MB-231 xenograft breast cancer models. Performance of the anti-HER2 VHH-IR was compared to that of trastuzumab-IR and a non-HER2-specific VHH-IR. Image-guided surgery was performed during which SKBR3 tumour was removed under the guidance of the VHH-IR signal.

Results

Site-specific conjugation of IRDye 800CW to three anti-HER2 VHHs preserved high affinity binding with the following dissociation constants (KD): 11A4 1.9 ± 0.03, 18C3 14.3 ± 1.8 and 22G12 3.2 ± 0.5 nM. Based upon different criteria such as binding, production yield and tumour accumulation, 11A4 was selected for further studies. Comparison of 11A4-IR with trastuzumab-IR showed ∼20 times faster tumour accumulation of the anti-HER2 VHH, with a much higher contrast between tumour and background tissue (11A4-IR 2.5 ± 0.3, trastuzumab-IR 1.4 ± 0.4, 4 h post-injection). 11A4-IR was demonstrated to be a useful tool in image-guided surgery.

Conclusion

VHH-IR led to a much faster tumour accumulation with high tumour to background ratios as compared to trastuzumab-IR allowing same-day imaging for clinical investigation as well as image-guided surgery.

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Acknowledgments

We would like to thank Mies Steenbergen, Anton Terwisscha van Scheltinga and Titia Lamberts for technical support. We thank Prof. Dr. Paul van Diest and Prof. Dr. Willem Mali for interesting discussions. We thank QVQ BV for providing pQVQ72 vector. This research was supported by the Center for Translational Molecular Medicine (MAMMOTH project).

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Correspondence to Paul M. P. van Bergen en Henegouwen.

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Kijanka, M., Warnders, F., El Khattabi, M. et al. Rapid optical imaging of human breast tumour xenografts using anti-HER2 VHHs site-directly conjugated to IRDye 800CW for image-guided surgery. Eur J Nucl Med Mol Imaging 40, 1718–1729 (2013) doi:10.1007/s00259-013-2471-2

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Keywords

  • Nanobody
  • VHH
  • HER2
  • Breast cancer
  • Optical imaging