Facile labelling of an anti-epidermal growth factor receptor Nanobody with 68Ga via a novel bifunctional desferal chelate for immuno-PET

Eur J Nucl Med Mol Imaging. 2011 Apr;38(4):753-63. doi: 10.1007/s00259-010-1700-1. Epub 2011 Jan 6.


Purpose: 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.

Conclusion: 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies / blood
  • Antibodies / chemistry*
  • Antibodies / immunology
  • Buffers
  • Cell Line, Tumor
  • Chelating Agents / chemistry
  • Cross-Linking Reagents / chemistry
  • Deferoxamine / analogs & derivatives*
  • Deferoxamine / chemistry
  • Drug Stability
  • ErbB Receptors / immunology*
  • Gallium Radioisotopes
  • Humans
  • Isothiocyanates / chemistry*
  • Isotope Labeling / methods*
  • Mice
  • Positron-Emission Tomography / methods*
  • Radioimmunodetection / methods*
  • Temperature


  • 4-isothiocyanatobenzyl-desferrioxamine
  • Antibodies
  • Buffers
  • Chelating Agents
  • Cross-Linking Reagents
  • Gallium Radioisotopes
  • Isothiocyanates
  • ErbB Receptors
  • Deferoxamine