Extended Data Fig. 9: CAGE-prox-enabled protein activation as a pro-drug therapy in living mice.
From: Time-resolved protein activation by proximal decaging in living systems
a, The traditional pro-drug strategy on small-molecule drugs can be extended to proteins by adopting the CAGE-prox-enabled in situ activation of therapeutic proteins. b, Schematic of the CAGE-prox-activated LF as a potential pro-drug therapy. MEK1-dependent A375 human melanoma cells were planted into mice as a xenograft model; activated LF will cause the death of MAPK-dependent tumour cells by rapid cleavage on MEK kinases. c, Experimental validation of the CAGE-prox-activatable LF variants, with the activity evaluated by cleavage efficiency on MEK3 in HEK293T cells. Incorporation of ONBY at Y659 or Y728 blocked LF’s activity, which can be efficiently rescued by photo-decaging. n = 2. d, e, Delivery and activation of LF(Y659)-ONBY was demonstrated in HeLa cells and HEK293T cells. LF(Y659)-ONBY was expressed and purified in Esherichia coli and delivered into the target cell lines by PA. n = 2. f, The growth curves of HEK293T or HeLa cells treated with LF(Y659)-ONBY/PA. Activated LF had negligible influence on the proliferation of MEK-independent HEK293T and HeLa cells. The red arrows represent the time point at which the medium was exchanged and LF–PA treatments were performed. Mean ± s.d.; n = 3. g, Photo-activation of the CAGE-prox variant of FLuc in BALB/c nude mice and optimization of the UV irradiation protocol. Increased FLuc activity were observed after UV irradiation (bottom image). n = 2. h, The caged LF-ONBY was safer than the wild-type LF (LF-WT) as determined by a dose escalation protocol. Intraperitoneal injection of wild-type LF into healthy mice every 2 days for a 2-week period caused about 50% of animal death. By contrast, injection of LF(Y659)-ONBY with a fourfold-higher dosage during the same period had negligible adverse effects on mice (n = 4). i, Photo-activation of the LF-ONBY variant has negligible influence on mouse body weight in a two-week period treatment. Mean ± s.d.; n = 8. j, Photo-activation of the LF-ONBY variant markedly reduced the tumour weight in the xenograft model. Mean ± s.d.; n = 8; two-tailed t-test. k, Evaluation of UV light penetration in vitro. UV light was irradiated through the skin and re-activation of FLuc was used to evaluate the penetration ability. Mean ± s.d.; n = 3. l, Evaluation of UV light penetration in vivo. A caged fluorescent dye was intra-tumourally injected into mice followed by UV irradiation for 5 min. The tumour was then excised and the depth of light penetration was examined. n = 2. m, n, Bio-distribution of the injected caged LF in mice. The Cy5-labelled LF protein was injected into mice, followed by imaging the whole mouse body. Mean ± s.d.; n = 3. o, The immunogenicity of LF–PA can be reduced by fusing cell-surface-targeting elements. EGF, epidermal growth factor that can target its cell-surface receptor EGFR; ZHer, an affibody that can target its receptor HER2. Mean ± s.d.; n = 3. p, The immunogenicity of LF–PA can be reduced by the addition of immunosuppressors (pentostatin combined with cyclophosphamide). Mean ± s.d.; n = 3. All above-mentioned samples are biological replicates. P values are shown in the figure.
