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bioRxiv · 10.1101/2025.03.04.641560

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

Wang, Z.·Kojima, R.·Kiji, R.·Fujita, K.·Tachibana, R.·Uchiyama, T.·Minagawa, Y.·Mizuno, T.·Igarashi, K.·Noji, H.·Kamiya, M.·Urano, Y.
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Abstract

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-{beta}-D-fucose is bioorthogonal in mammalian systems, but is activated by a metagenomic glycosidase, Td2F2. Directed evolution generated a mutant with a kcat/Km value for HMRef-{beta}-D-fucose of 3.3 x 105/M/sec, 7.3 times higher than that of wild-type Td2F2 and comparable to that of {beta}-galactosidase (LacZ) with a corresponding probe. Theoretical calculation suggested that E296G mutation in Td2F2 causes structural changes that facilitate the probes access to the enzymes 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-{beta}-D-fucose together with engineered Td2F2 conjugated/fused to a HER2-binding antibody/nanobody.

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Wang, Z., Kojima, R., Kiji, R., Fujita, K., Tachibana, R., Uchiyama, T., Minagawa, Y., Mizuno, T., Igarashi, K., Noji, H., Kamiya, M., Urano, Y.. 2025-03-12. Low-Background Cancer Imaging With a Bioorthogonal Fluorescence Probe and Engineered Reporter Enzyme Bearing a Targeting Moiety. https://doi.org/10.1101/2025.03.04.641560

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