Site-specific radiolabelling strategies of the anti-human PD-L1 Nanobody for PET imaging

Nanobodies (Nbs) are ideal probes for positron emission tomography (PET) imaging and can be radiolabeled with short-lived radioisotopes such as gallium-68 (68Ga) or fluorine-18 (18F). In this study, we aim to develop a site-specifically radiolabeled human PD-L1 (hPD-L1) Nb for non-invasive characterization of whole body PD-L1 expression. A site-specific functionalization technique of Nbs involves the use of the Sortase A enzyme. The lead hPD-L1 Nb is engineered with the LPETG motif at its C-terminus, allowing recognition of the enzyme. Tris-glycine peptides, used as nucleophilic probes in the Sortase-mediated ligation, are synthesized to contain the NOTA or the RESCA chelator for direct labeling under mild conditions with 68Ga or [18F]Al-F respectively. Peptides containing unsaturated moieties such as azide (N3) or tetrazine (Tz) functions can also be introduced for indirect labeling via copper-free click chemistry with 18F-labeled DBCO or BCN prosthetic groups. This functionalization strategy allowed to obtain the hPD-L1 Nb coupled to NOTA or RESCA chelator, azide (N3) or tetrazine (Tz) function with high purity (≥99%) and in high yields (52% - 63%). Functionalization did not affect affinity nor specificity. 68Ga labeling of NOTA-(hPD-L1) was performed in 10 min in 80% decay-corrected radiochemical yield (DC-RCY), ≥99% radiochemical purity (RCP) and apparent molar specific activity of 85 GBq/μmol. In vivo targeting revealed high tumour uptake and no unspecific organ uptake, except in kidneys and bladder because of excretion. [18F]Al-F labelling of RESCA-(hPD-L1) was performed at R.T. in 12 min in a 29% DC-RCY and with a RCP > 99%. First attempts to label Tz-(hPD-L1) with [18F]F-PEG3-BCN showed about 30% conversion after 10 minutes, and labelling conditions still require optimization. The most promising strategy in terms of ease of synthesis and in vivo behaviour will be selected for future clinical translation of the hPD-L1 Nb. Acknowledgments: This project is funded by the EU-H2020-MSCA-ITN-PET3D.