Imagenología del Cáncer de Bajo Fondo con una Sonda de Fluorescencia Bioortogonal y una Enzima Reportera Modificada...

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 lack sufficient bioorthogonality, leading to high background signals in nontarget tissues. We screened a library of HMRef (rhodol derivative)-based fluorescence probes with various sugar moieties and found 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 of 3.3 × 10⁵/M/sec, 7.3 times higher than wild-type Td2F2 and comparable to β-galactosidase (LacZ) with its corresponding probe. Theoretical calculation suggested the E296G mutation facilitates probe access to the enzyme's active site. In a proof-of-concept study, SKOV-3 cells, which endogenously express HER2, were visualized with minimal background in the mesentery of a mouse model using HMRef-β-d-Fucose and engineered Td2F2 conjugated or fused to a HER2-binding antibody or nanobody.