title: Facile labelling of an anti-epidermal growth factor receptor Nanobody with 68Ga via a novel bifunctional desferal chelate for immuno-PET.
abstract: OBJECTIVE
  The ∼15 kDa variable domains of camelid heavy-chain-only antibodies (called Nanobodies®) have the flexibility to be formatted as monovalent, monospecific, multivalent or multispecific single chain proteins with either fast or slow pharmacokinetics. We report the evaluation of the fast kinetic anti-epidermal growth factor receptor (EGFR) Nanobody 7D12, labelled with (68)Ga via the novel bifunctional chelate (BFC) p-isothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS). Df-Bz-NCS has recently been introduced as the chelate of choice for (89)Zr immuno-positron emission tomography (PET).
  
  
  METHODS
  Nanobody 7D12 was premodified with Df-Bz-NCS at pH 9. Radiolabelling with purified (68)Ga was performed at pH 5.0-6.5 for 5 min at room temperature. For in vitro stability measurements in storage buffer (0.25 M NaOAc with 5 mg ml(-1) gentisic acid, pH 5.5) at 4°C or in human serum at 37°C, a mixture of (67)Ga and (68)Ga was used. Biodistribution and immuno-PET studies of (68)Ga-Df-Bz-NCS-7D12 were performed in nude mice bearing A431 xenografts using (89)Zr-Df-Bz-NCS-7D12 as the reference conjugate.
  
  
  RESULTS
  The Df-Bz-NCS chelate was conjugated to Nanobody 7D12 with a chelate to Nanobody molar substitution ratio of 0.2:1. The overall (68)Ga radiochemical yield was 55-70% (not corrected for decay); specific activity was 100-500 MBq/mg. Radiochemical purity of the conjugate was >96%, while the integrity and immunoreactivity were preserved. (68/67)Ga-Df-Bz-NCS-7D12 was stable in storage buffer as well as in human serum during a 5-h incubation period (<2% radioactivity loss). In biodistribution studies the (68)Ga-labelled Nanobody 7D12 showed high uptake in A431 tumours (ranging from 6.1 ± 1.3 to 7.2 ± 1.5%ID/g at 1-3 h after injection) and high tumour to blood ratios, which increased from 8.2 to 14.4 and 25.7 at 1, 2 and 3 h after injection, respectively. High uptake was also observed in the kidneys. Biodistribution was similar to that of the reference conjugate (89)Zr-Df-Bz-NCS-7D12. Tumours were clearly visualized in a PET imaging study.
  
  
  CONCLUSIONS
  Via a rapid procedure under mild conditions a (68)Ga-Nanobody was obtained that exhibited high tumour uptake and tumour to normal tissue ratios in nude mice bearing A431 xenografts. Fast kinetic (68)Ga-Nanobody conjugates can be promising tools for tumour detection and imaging of target expression.
authors.affiliation: Department of Otolaryngology/Head and Neck Surgery, VU University Medical Center, De Boelelaan 1117, P.O. Box 7057, 1007 MB, Amsterdam, The Netherlands. gams.vandongen@vumc.nl
authors.forename: Maria J W D
authors.initials: MJ
authors.lastname: Vosjan
authors.forename: Lars R
authors.initials: LR
authors.lastname: Perk
authors.forename: Rob C
authors.initials: RC
authors.lastname: Roovers
authors.forename: Gerard W M
authors.initials: GW
authors.lastname: Visser
authors.forename: Marijke
authors.initials: M
authors.lastname: Stigter-van Walsum
authors.forename: Paul M P
authors.initials: PM
authors.lastname: van Bergen En Henegouwen
authors.forename: Guus A M S
authors.initials: GA
authors.lastname: van Dongen
mesh_terms: D000818:Animals; D000906:Antibodies / Q000097:blood / Q000737:chemistry* / Q000276:immunology; D002021:Buffers; D045744:Cell Line, Tumor; D002614:Chelating Agents / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D003676:Deferoxamine / Q000031:analogs & derivatives* / Q000737:chemistry; D004355:Drug Stability; D066246:ErbB Receptors / Q000276:immunology*; D005710:Gallium Radioisotopes; D006801:Humans; D017879:Isothiocyanates / Q000737:chemistry*; D007553:Isotope Labeling / Q000379:methods*; D051379:Mice; D049268:Positron-Emission Tomography / Q000379:methods*; D016719:Radioimmunodetection / Q000379:methods*; D013696:Temperature
chemical_list: C549125:4-isothiocyanatobenzyl-desferrioxamine; D000906:Antibodies; D002021:Buffers; D002614:Chelating Agents; D003432:Cross-Linking Reagents; D005710:Gallium Radioisotopes; D017879:Isothiocyanates; D066246:ErbB Receptors; D003676:Deferoxamine