Low-Background Cancer Imaging With a Bioorthogonal Fluorescence Probe and Engineered Reporter Enzyme Bearing a Targeting Moiety

Combinatorial use of an antibody-reporter enzyme conjugate and a fluorescence probe activated by the enzyme is a powerful strategy for fluorescence-guided cancer surgery. However, conventional probes for typical reporter enzymes are insufficiently bioorthogonal, resulting in high background signals in non-target tissues. We screened a library of HMRef (rhodol derivative)-based fluorescence probes bearing various sugar moieties, and discovered that HMRef-β-D-fucose is bioorthogonal in mammalian systems, but is activated by a metagenomic glycosidase, Td2F2. Directed evolution generated a mutant with a k _cat / K _m value for HMRef-β-D-fucose of 3.3 x 10 ⁵ /M/sec, 7.3 times higher than that of wild-type Td2F2 and comparable to that of β-galactosidase (LacZ) with a corresponding probe. Theoretical calculation suggested that E296G mutation in Td2F2 causes structural changes that facilitate the probe’s access to the enzyme’s active site. In a proof-of-concept study, cancer cells were visualized with a minimal background in the mesentery of a mouse model of peritoneally disseminated human-ovarian-cancer-derived SKOV-3 cells, which endogenously express HER2, by using HMRef-β-D-fucose together with engineered Td2F2 conjugated/fused to a HER2-binding antibody/nanobody.