Miniaturized Fab’ imaging probe derived from a clinical antibody: Characterization and imaging in CRISPRi-attenuated mammary tumor models

Clinical imaging-assisted oncosurgical navigation requires cancer-specific miniaturized optical imaging probes. We report a near-infrared (NIR) Fab’-based epidermal growth factor receptor (EGFR)-specific probe carrying 3 NIR fluorophores (Fab’-800CW), which retained high-affinity binding to EGFR ectodomain (equilibrium KDE = 1 nM). Fab’-800CW showed a robust 4-times gain of fluorescence intensity (FI) and a 20% lifetime (FLT) increase under the conditions mimicking intracellular degradation. The probe was tested by using triple-negative breast cancer (TNBC) cell lines obtained by applying CRISPR interference (CRISPRi) effect of EGFR-targeting sgRNA and dCas9-KRAB chimera coexpression in MDA-MB-231 cells (WT cells). FI imaging in cell culture proved a 50% EGFR expression attenuation by CRISPRi. FI imaging in animals harboring attenuated or WT TNBC tumors with ex vivo corroboration identified differences between WT and CRISPRi tumors FI at 30 min post injection. Our results suggest the feasibility of EGFR expression imaging using a Fab’-based probe relevant for imaging-guided cancer surgery. [Display omitted] •Both fluorescence intensity and lifetime increase after fragmentation of the Fab’ NIR probe•Fluorescence lifetime contrast increases upon internalization of Fab’ NIR probe•CRISPRi yielded two times lower EGFR expression in triple-negative breast cancer cells•Fab’ probe showed the feasibility of CRISPRi effect imaging in vivo at an early time point Surgery; Medical imaging; Cancer.