IMAGING IMMUNITY – from Nanoscale to Macroscale | Insights from Biophysics
To search for a specific ID please enter the hash sign followed by the ID number (e.g. #123).

Talks from Abstract Submissions

Session chair: Gemma Dias (Oxford, UK)
Date: Tuesday, 14 January, 2020, 5:00 PM - 5:45 PM


Click on an contribution to preview the abstract content.

5:00 PM -01

Human PD-L1 Nanobody For Immuno-PET Imaging: Strategies for Site-specific Radiolabeling (#17)

Jessica Bridoux1, Katrijn Broos2, Maxine Crauwels1, 3, Quentin Lecocq2, Charlotte Martin6, Frederik Cleeren7, Steven Ballet6, Guy Bormans7, Geert Raes5, Karine Breckpot2, Serge Muyldermans3, Nick Devoogdt2, Vicky Caveliers4, Marleen Keyaerts4, Catarina Xavier1

1 Vrije Universiteit Brussel (VUB), In Vivo Cellular and Molecular Imaging (ICMI), Jette, Belgium
2 VUB, Laboratory of Molecular and Cellular Therapy (LMCT), Jette, Belgium
3 VUB, Cellular and Molecular Immunology (CMIM), Ixelles, Belgium
4 UZ Brussel, Nuclear Medicine Department,, Jette, Belgium
5 VIB Inflammation Research Center, Myeloid Cell Immunology Lab, Ghent, Belgium
6 VUB, Research group of Organic Chemistry (ORGC), Ixelles, Belgium
7 University of Leuven, Radiopharmaceutical Research, Department of Pharmacy and Pharmacology, Leuven, Belgium


Immune checkpoints such as Programmed death-ligand 1 (PD-L1) limit the T-cell function, and tumor cells have developed this receptor to escape the anti-tumor 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 probe to assess human PD-L1 (hPD-L1) expression using PET imaging. The Nb has been site-specifically modified for Gallium-68 (68Ga) or Fluorine-18 (18F) radiolabeling.


The hPD-L1 Nb with a sortag-motif at its C-terminus was site-specifically coupled to a bifunctional chelator or a tetrazine (Tz) via the Sortase A enzyme coupling reaction. 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 labeled with 68Ga, RESCA-(hPD-L1) Nb with [18F]AlF and Tz-(hPD-L1) Nb with [18F]F-PEG3-BCN. Radiochemical purity (RCP) was assayed by SEC and iTLC. In vivo tumor targeting of [68Ga]Ga-NOTA-(hPD-L1) Nb was assessed in xenografted-athymic nude mice bearing PD-L1 positive cells, or PD-L1 negative cells as a control. PET/CT imaging was performed with 68Ga-labeled NOTA-(hPD-L1) Nb 1h post-injection.


Site-specifically functionalized hPD-L1 Nbs with NOTA or RESCA were obtained with high purity (≥99%) in 52% and 59% yield (Figure 1). (hPD-L1)-Tz was obtained in 63% yield. Functionalization did not affect affinity.
Labelling of NOTA-(hPD-L1) with 68Ga was performed at room temperature (RT) for 10 min in a 80% decay corrected radiochemical yield (DC-RCY), ≥99% RCP and apparent molar specific activity of 85 GBq/μmol. Over 4 hours, the radiolabeled probe and metal complex were stable (≥99% RCP). In vivo tumor targeting studies revealed high tumor uptake of (3.66 ± 0.76) %IA/g organ, and no unspecific organ targeting, except in the kidneys and excretion to the bladder (route of excretion).
Labeling of RESCA-(hPD-L1) with [18F]AlF was performed at RT for 12 min at in a 29% DC-RCY and with a RCP ≥99%. Biodistribution in healthy animals showed higher bone uptake than for the 68Ga-labeled Nb.
Preliminary results with [18F]F-PEG3-BCN-Nb showed about 30% labeling and more tests will be performed


A site-specific functionalized (hPD-L1) Nb was obtained using Sortase enzyme approach, which could be radiolabeled with 68Ga or 18F. [68Ga]Ga-NOTA-(hPD-L1) Nb proved to specifically target the hPD-L1 receptor in vivo. Further studies will be performed with the 18F-labeled Nb after optimization of the labelling conditions. The best probe in terms of ease of production and in vivobehavior will be selected for clinical translation.


K. Broos et. al., Evaluating a Single Domain Antibody Targeting Human PD-L1 as a Nuclear Imaging and Therapeutic Agent, Cancers 2019, 11(6), 872.


The authors would like to thank Cindy Peleman for animal handling and PET imaging.Research project funded by the EU H2020 MSCA-ITN-2015 program 675417 PET3D. This work was funded by a grant from the Scientific Fund W. GeptsUZ Brussel and FWO G066615N.Q. Lecocqis funded by FWO-SB 1S24218N. Marleen Keyaerts is a senior clinical investigator of the Research Foundation – Flanders. Research at ICMI-BEFY is supported by the Strategic Research Program (SRP) of the VUB Research Council.

Figure 1: Site-specific functionalization and radiolabeling of the hPD-L1 Nb
Sortase A mediated site-specific functionalization of the hPD-L1 Nb with the bifunctional chelators (GGGYK-NOTA or GGGYK-RESCA) or with a tetrazine, and radiolabeling with Gallium-68, [18F]AlF or [18F]F-PEG3-BCN respectively.
Figure 2: Biodistribution of [68Ga]Ga-NOTA-(hPD-L1) Nb in tumor bearing mice.
Biodistribution of [68Ga]Ga-NOTA-(hPD-L1) Nb in athymic nude mice bearing MEL624 hPD-L1 positive (hPD-L1POS) tumors or negative (hPD-L1POS) tumors as a control, showing high specific tumor uptake of (3.66 ± 0.76) %IA/g organ, and no unspecific organ targeting, except in the kidneys and excretion to the bladder (route of excretion). (N = 6 / group). 
Keywords: PET-CT, nanobody, PD-L1, gallium-68, fluorine-18
5:20 PM -02

Functionalized perfluorocarbon nanoemulsions for sensitive fluorine-19 MRI immune cell detection in vivo (#21)

Eric Ahrens1, Chao Wang1, Dina Hingorani1, Fanny Chaplin2, Benjamin Leach1, Stephen Adams3

1 Uc San Diego, Radiology, La Jolla, California, United States of America
2 UC San Diego, Bioegineering, La Jolla, California, United States of America
3 UC San Diego, Pharmacology, La Jolla, California, United States of America


Advances in cell immunotherapy against cancer has stimulated the need for imaging tools to determine cell biodistribution and survival post-transfer. 19F MRI enables background-free, quantitative hot-spot imaging of cell therapies.1 We describe next-generation perfluorocarbon nanoemulsion (NE) probes to detect cells with an order of magnitude sensitivity improvement. We use a two-pronged approach for boosting detection via (i) the incorporation of paramagnetic metal chelate into the NE fluorous phase and (ii) formulation of NE displaying cell-penetrating peptides to enhance cell uptake.


We synthesized fluorinated, metal-binding β-diketones conjugated to linear perfluoropolyether (PFPE) yielding “FETRIS” construct2 and blended this with unconjugated PFPE oil. As a co-surfactant, we synthesized a modified peptide from the transactivator of transcription (TAT) component of the HIV virus type-1; TAT residues 49-58 to facilitate endocytosis, and a fluorous anchor was covalently attached. Fluorous TAT and poloxamer surfactant was used to form NE with the blended PFPE oil. Metalation of NE occurred via the addition of Fe(III) into the aqueous buffer. Intracellular cell uptake of the probe ex vivo was studied in human chimeric antigen receptor (CAR) T cells. In vivo 19F MRI mouse studies using inoculated labeled CAR T cells were performed in a xenograft model of glioma at 11.7 T.


Additional of Fe(III) into NE decreases the 19F T1 to >10-fold via the intermolecular paramagnetic relaxation enhancement mechanism, with modest T2 broadening. Shortening T1 increases the 19F image sensitivity per time with repetitive signal averaging. By incorporating TAT a labeling efficiency ~1012 fluorine atoms per CAR T cell was achieved which is a >8-fold increase compared to NE without TAT. In vitro assays show that T cells are unaltered after NE labeling. The 19F MRI signal detected from TAT-labeled CAR T cells in mouse was >8 times higher than cells labeled with control NE (Figure 1).


Lymphocytes are challenging to label and detect with MRI due to their weak phagocytic properties and small size. Using multipronged improvements to NE formulation via incorporation of Fe-chelate and TAT peptide, one can significantly enhance cell labeling and imaging sensitivity. These same agents should be useful for tagging other weakly phagocytic cells such as stem and progenitor cells.


1. Chapelin F, Capitini CM, Ahrens ET. Fluorine-19 MRI for detection and quantification of immune cell therapy for cancer. J Immunother Cancer. 2018;6:105.

2. Kislukhin AA, Xu H, Adams SR, et al. Paramagnetic fluorinated nanoemulsions for sensitive cellular fluorine-19 magnetic resonance imaging. Nat Mater. 2016;15(6):662-668.


We thank Hongyan Xu and Deanne Lister for technical assistance. ETA was funded through National Institutes of Health grants R01-EB024015 and the California Institute for Regenerative Medicine grant LA1-C12-06919. ETA is founder, consultant, member of the advisory board, and shareholder of Celsense, Inc.

Figure 1.

Panel (a) displays 19F (hot-iron) and 1H (grayscale) from a slice in mouse with bilateral flank gliomas, where the left and right tumor (LT, RT) each received 107 CAR T cells labeled with PFC (control) or TAT-PFC nanoemulsions, respectively. A reference (REF) is also shown. MRI data were acquired using RARE sequences. A histogram of the 19F signal-to-noise ratio for voxels in the tumors is displayed (b). Comparison of apparent 19F atoms per tumor in vivo (N = 4) is displayed (c) showing ~8-fold sensitivity enhancement (* indicates p<0.001) for TAT-PFC nanoemulsions compared to control.

Keywords: MRI, fluorine-19, lymphocytes, peptide, chelate