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

Abstract

PurposeThe ∼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).MethodsNanobody 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.ResultsThe 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 (<2% radioactivity loss). In biodistribution studies the 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.ConclusionVia 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.

Siderophores for molecular imaging applications

“…Second, the labels were poorly retained within cells after Ab internalization and catabolism. More recently, a novel bifunctional chelate (BFC) p-isothiocyanatobenzyl-DFO (Df-Bz-NCS), originally developed for 89 Zr labelling, was used to prepare anti-EGF Nanobody conjugates of DFO for 68 Ga labelling for PET applications [74]. Fast radiolabelling, high tumour uptake and tumour to normal tissue ratios in nude mice bearing A431 xenografts were obtained with the fast kinetics of the 68 Ga-Nanobody conjugates, indicating a promising application of DFO conjugates with 68 Ga.…”

Synthesis, Functionalization, and Design of Magnetic Nanoparticles for Theranostic Applications

“…[517] Specifically, nanbobodies have been used as antagonistic drugs [518] and targeting agents of effector domains [519] in cancer immunotherapy. On the other hand, several molecular imaging modalities of SPECT [520] and PET, [521] optical imaging, [522] and ultrasound imaging [523] have been actively directed toward the target by means of nanobodies conjugated to them. Additionally, grafting nanobodies on nanoparticles have shown a considerable potential for increasing their affinity toward the target.…”

Nanobodies Targeting the Hepatocyte Growth Factor: Potential New Drugs for Molecular Cancer Therapy

“…Among others, linking aHGF-Nanobodies to our recently described aEGFRNanobody 7D12 might be promising (17). Like other researchers postulated; combination therapies are needed to overcome acquired resistance to inhibitors of signal transduction pathways.…”

“…Initial biodistribution studies with monospecific anti-EGFR (aEGFR)-Nanobodies in tumor-bearing mice showed, as expected, rapid blood clearance with a serum half-life time of less than an hour and low tumor uptake (15)(16)(17). Therefore, monospecific Nanobodies do not seem to be qualified for long-term blockage of growth factors and their receptors.…”