Introduction: Immune checkpoints such as Programmed death-ligand 1 (PD-L1) limit the T-cell function, and tumour cells have developed this receptor to escape the anti-tumour immune response. Monoclonal antibody-based treatments have shown long-lasting responses, but only in a subset of patients. Therefore, there is a need to predict the response to treatments. This study aims to develop a Nanobody (Nb)-based radiopharmaceutical to assess human PD-L1 (hPD-L1) expression using PET imaging. The Nb has been site-specifically coupled to the NOTA-chelator for gallium-68 labelling, or with the RESCA-chelator for [18F]AlF labelling.

Methods: The hPD-L1 Nb with a sortag-motif at its C-terminus was site-specifically coupled to a bifunctional chelator (BFC) via the Sortase A enzyme coupling reaction. BFCs were synthesised by attaching p-SCN-Bn-NOTA or RESCA-(tBu)-COOH to the side chain of the lysine in a GGGYK peptide. Modified Nbs were purified by immobilized metal affinity chromatography (IMAC) and size-exclusion chromatography (SEC), characterized by Mass Spectrometry (ESI-Q-TOF), SDS-PAGE and Western Blot. NOTA-(hPD-L1) Nb was labelled with gallium-68 and RESCA-(hPD-L1) Nb with [18F]AlF. Radiochemical purity (RCP) was assayed by SEC and iTLC. Probe stability was evaluated in vitro, and in vivo stability was performed with [67Ga]Ga-NOTA-(hPD-L1) by SEC analysis of blood and urine samples from different time points up to 2h. An in vivo biodistribution study in C57BL/6 mice was performed with [68Ga]Ga-NOTA-(hPD-L1) and [18F]AlF-RESCA-(hPD-L1). In vivo tumour targeting was assessed in xenografted-athymic nude mice bearing PD-L1 positive cells, or PD-L1 negative cells as a control. PET/CT and SPECT/CT imaging was performed with gallium-68 and gallium-67 labelled NOTA-(hPD-L1) Nb respectively.

Results: Site-specifically functionalized hPD-L1 Nbs with NOTA and RESCA were obtained with high purity (≥99%) in 52% and 59% yield respectively (Figure 1). Functionalization did not affect affinity nor specificity.
Labelling of NOTA-(hPD-L1) with gallium-68 was performed at room temperature for 10 min at pH 4.4-4.7 in a 80% decay corrected radiochemical yield (DC-RCY), ≥99% RCP and apparent molar specific activity of 85 GBq/μmol. Over 4 hours, the radiolabelled probe and metal complex were stable in injection buffer and in presence of DTPA excess (≥99% RCP). RCP after 1 hour at 37°C in human serum was ≥94%, and the probe remained intact in vivo in blood (≥95%) and urine (≥90%). In vivo tumour targeting and biodistribution studies revealed high tumour uptake of (3.66 ± 0.76) %IA/g organ, and no unspecific organ targeting, except in the kidneys and excretion to the bladder (expected route of excretion).
Labelling of RESCA-(hPD-L1) with [18F]AlF was performed at room temperature for 12 min at pH 4.4-4.7 in a 29% DC-RCY and with a RCP ≥99%. The radiolabelled probe was stable over 2.5 hours in injection buffer (RCP ≥98%). Biodistribution in healthy animals was similar as for [68Ga]Ga-NOTA-(hPD-L1), except for slightly higher bone uptake.

Conclusion: The Sortase enzyme approach allowed to obtain a site-specifically functionalized (hPD-L1) Nb, which could be easily radiolabelled with gallium-68 or [18F]AlF. [68Ga]Ga-NOTA-(hPD-L1) Nb proved to specifically target the hPD-L1 receptor in vivo and the targeting experiment will be repeated with [18F]AlF-RESCA-(hPD-L1). The next step before clinical translation would be to test the probe in a humanized mouse model.

Acknowledgments: The authors would like to thank Cindy Peleman for animal handling and PET imaging.
Original languageEnglish
Publication statusPublished - 26 May 2019
EventInternational Symposium on Radiopharmaceutical Sciences, ISRS - Bejing, China
Duration: 26 May 201931 May 2019


ConferenceInternational Symposium on Radiopharmaceutical Sciences, ISRS
Internet address

    Research areas

  • PET, Nanobody, PD-L1, Site-specific, Fluorine-18, Gallium-68

ID: 49490976