Bioorthogonally Applicable Fluorescence Deactivation Strategy for Receptor Kinetics Study and Theranostic Pretargeting Approaches

Steffen van der Wal; Clarize M de Korne; Laurens G L Sand; Danny M van Willigen; Pancras C W Hogendoorn; Karoly Szuhai; Fijs W B van Leeuwen; Tessa Buckle

doi:10.1002/cbic.201800229

Bioorthogonally Applicable Fluorescence Deactivation Strategy for Receptor Kinetics Study and Theranostic Pretargeting Approaches

Chembiochem. 2018 Jun 4;19(16):1758-1765. doi: 10.1002/cbic.201800229. Online ahead of print.

Authors

Steffen van der Wal¹, Clarize M de Korne¹, Laurens G L Sand^{2

3}, Danny M van Willigen¹, Pancras C W Hogendoorn², Karoly Szuhai⁴, Fijs W B van Leeuwen¹, Tessa Buckle^{1

5}

Affiliations

¹ Interventional Molecular Imaging Laboratory, Department of Radiology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands.
² Department of Pathology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands.
³ Bone Marrow Transplantation and Cell Therapy, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN, 38105, USA.
⁴ Department of Molecular Cell Biology, Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands.
⁵ Division of Molecular Pathology, Netherlands Cancer Institute-Antoni van Leeuwenhoek Hospital (NKI-AvL), Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands.

Abstract

The availability of a receptor for theranostic pretargeting approaches was assessed by use of a new click-chemistry-based deactivatable fluorescence-quenching concept. The efficacy was evaluated in a cell-based model system featuring both membranous (available) and internalized (unavailable) receptor fractions of the clinically relevant receptor chemokine receptor 4 (CXCR4). Proof of concept was achieved with a deactivatable tracer consisting of a CXCR4-specific peptide functionalized with a Cy5 dye bearing a chemoselective azide handle (N₃ -Cy5-AcTZ14011). Treatment with a Cy7 quencher dye (Cy7-DBCO) resulted in optically silent Cy7-[click]-Cy5-AcTZ14011. In situ, a >90 % FRET-based reduction of the signal intensity of N₃ -Cy5-AcTZ14011 [K_D =(222.4±25.2) nm] was seen within minutes after quencher addition. In cells, discrimination between the membranous and the internalized receptor fraction could be achieved through quantitative assessment of quenching/internalization kinetics. Similar evaluation of an activatable tracer variant based on the same targeting moiety (Cy5-S-S-Cy3-AcTZ14011) was unsuccessful in vitro. As such, using the described deactivatable approach to screen membrane receptors and their applicability in receptor-(pre-)targeted theranostics can become straightforward.

Keywords: click chemistry; deactivatable; fluorescence; pretargeting; theranostics.