title: Rapid optical imaging of human breast tumour xenografts using anti-HER2 VHHs site-directly conjugated to IRDye 800CW for image-guided surgery.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 GE, Utrecht, The Netherlands.
authors.forename: Marta
authors.initials: M
authors.lastname: Kijanka
authors.forename: Frank-Jan
authors.initials: FJ
authors.lastname: Warnders
authors.forename: Mohamed
authors.initials: M
authors.lastname: El Khattabi
authors.forename: Marjolijn
authors.initials: M
authors.lastname: Lub-de Hooge
authors.forename: Gooitzen M
authors.initials: GM
authors.lastname: van Dam
authors.forename: Vasilis
authors.initials: V
authors.lastname: Ntziachristos
authors.forename: Liesbeth
authors.initials: L
authors.lastname: de Vries
authors.forename: Sabrina
authors.initials: S
authors.lastname: Oliveira
authors.forename: Paul M P
authors.initials: PM
authors.lastname: van Bergen En Henegouwen
mesh_terms: D000818:Animals; D000915:Antibody Affinity; D001557:Benzenesulfonates / Q000493:pharmacokinetics* / Q000627:therapeutic use; D001943:Breast Neoplasms / Q000473:pathology / Q000601:surgery; D002161:Camelids, New World; D005456:Fluorescent Dyes / Q000493:pharmacokinetics* / Q000627:therapeutic use; D006801:Humans; D007211:Indoles / Q000493:pharmacokinetics* / Q000627:therapeutic use; D061986:MCF-7 Cells; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D061848:Optical Imaging / Q000379:methods*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D061905:Single-Domain Antibodies / Q000276:immunology* / Q000627:therapeutic use; D025321:Surgery, Computer-Assisted / Q000379:methods*; D023041:Xenograft Model Antitumor Assays
chemical_list: D001557:Benzenesulfonates; D005456:Fluorescent Dyes; D007211:Indoles; D018719:Erb-b2 Receptor Tyrosine Kinases; D061905:Single-Domain Antibodies; C562366:IRDye 800CW; C508053:ERBB2 protein, human