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

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Abstract
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 68Ga 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 89Zr immuno-positron emission tomography (PET). Methods Nanobody 7D12 was premodified with Df-Bz-NCS at pH 9. Radiolabelling with purified 68Ga 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 67Ga and 68Ga was used. Biodistribution and immuno-PET studies of 68Ga-Df-Bz-NCS-7D12 were performed in nude mice bearing A431 xenografts using 89Zr-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 68Ga 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. 68/67Ga-Df-Bz-NCS-7D12 was stable in storage buffer as well as in human serum during a 5-h incubation period (68Ga-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 89Zr-Df-Bz-NCS-7D12. Tumours were clearly visualized in a PET imaging study. Conclusion Via a rapid procedure under mild conditions a 68Ga-Nanobody was obtained that exhibited high tumour uptake and tumour to normal tissue ratios in nude mice bearing A431 xenografts. Fast kinetic 68Ga-Nanobody conjugates can be promising tools for tumour detection and imaging of target expression.

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