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

  • Marta Kijanka
  • Frank-Jan Warnders
  • Mohamed El Khattabi
  • Marjolijn Lub-de Hooge
  • Gooitzen M. van Dam
  • Vasilis Ntziachristos
  • Liesbeth de Vries
  • Sabrina Oliveira
  • Paul M. P. van Bergen en HenegouwenEmail author
Original Article



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.


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.


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.


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.


Nanobody VHH HER2 Breast cancer Optical imaging 



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).

Supplementary material

259_2013_2471_MOESM1_ESM.pdf (664 kb)
ESM 1 (PDF 663 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Marta Kijanka
    • 1
  • Frank-Jan Warnders
    • 2
  • Mohamed El Khattabi
    • 3
  • Marjolijn Lub-de Hooge
    • 2
  • Gooitzen M. van Dam
    • 4
  • Vasilis Ntziachristos
    • 5
  • Liesbeth de Vries
    • 6
  • Sabrina Oliveira
    • 1
    • 7
  • Paul M. P. van Bergen en Henegouwen
    • 1
    Email author
  1. 1.Cell Biology, Department of Biology, Faculty of ScienceUtrecht UniversityUtrechtThe Netherlands
  2. 2.Hospital and Clinical PharmacyUniversity Medical Center of GroningenGroningenThe Netherlands
  3. 3.QVQ BVUtrechtThe Netherlands
  4. 4.Department of Surgery, Division of Surgical Oncology, BioOptical Imaging CenterUniversity of GroningenGroningenThe Netherlands
  5. 5.Biological Imaging & Institute for Medical and Biological ImagingTechnische Universität München and Helmholtz Zentrum MünchenMunichGermany
  6. 6.Department of Medical OncologyUniversity Medical Center of GroningenGroningenThe Netherlands
  7. 7.Department of Pathology, Division Laboratories and PharmacyUniversity Medical Center UtrechtUtrechtThe Netherlands

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