Fig. 3: EGFR CYpHER based on VHH nanobody.
From: CYpHER: catalytic extracellular targeted protein degradation with high potency and durable effect
a CT-1212-1 design. b CT-1212-1 SDS-PAGE Coomassie stain. NR: non-reduced. R: DTT-reduced. c SE-HPLC of CT-1212-1; right is zoomed. d EGFR-GFP trafficking by CYpHER in 293T-EGFR-GFP cells. e 293T-EGFR-GFP cells treated 24 h with PBS or 10 nM CT-1212-1 before either GFP microscopy (left) or flow cytometry after staining for surface EGFR (right). Black contour: unstained cells. f 293T-EGFR-GFP cells treated with PBS or 10 nM CT-1212-1 for 24 h and flow sorted for viable (DAPI-) cells prior to Western blotting. Full blot in Supplementary Fig. 13. g Same 293T-EGFR-GFP cells and treatment as e, stratified by surface EGFR quintile and showing normalized surface EGFR stain per cell. Two-tailed Kolmogorov–Smirnov test, PBS vs CT-1212-1 pairwise were all P < 0.0001, Quintile 1 vs Quintile 3 was P = 0.0062, all other CT-1212-1 quintile pairwise comparisons were P < 0.0001. h, i 293T-EGFR-GFP cells dosed with PBS or 10 nM CT-1212-1 for 30 min, 4 h, 24 h, or 24 h followed by 24 h without drug (“Withdrawal”) were flow analyzed for surface EGFR (h) or total EGFR-GFP (i) as in e, with EGFR stain (h) or total EGFR-GFP (i) fluorescence per cell shown. N cells per sample in h and i: PBS 30 min [m], 14806; 1212-1 30 m, 12117; PBS 4 hour [h], 15289; 1212-1 4 h, 13400; PBS 24 h, 14449; 1212-1 24 h, 12588; PBS Withdrawal [WD], 14487; 1212-1 WD, 13972. For surface EGFR (h), PBS vs CT-1212-1 pairwise were all P < 0.0001 via two-tailed Kolmogorov–Smirnov [KS] test. CT-1212-1 samples pairwise by Kruskal–Wallis test with Dunn’s correction [KWD]: 30 min vs Withdrawal was not significant (P > 0.9999), all others P < 0.0001. For total EGFR-GFP (i), PBS vs CT-1212-1 pairwise were all P < 0.0001 via two-tailed KS test. All four CT-1212-1 samples by KWD: 24 h vs Withdrawal was P = 0.0008, all others P < 0.0001. j 293T-EGFR-GFP Cells (24 well dish, 500 μL media per well) were treated with 50 μL CellLight Lysosomes-RFP (delivering gene for LAMP1-RFP) for 24 h, after which they were untreated or treated with 10 nM DyLight 755-labeled CT-1212-1 for 1 or 4 h and then imaged on the GFP, RFP, and near IR channels. Arrows indicate location of LAMP1-RFP foci (i.e., lysosomes). Each experiment in e–j was performed once (e, g–j) or twice (f) producing similar results. All box plots (h and i) feature a median (black line), 25th and 75th percentiles (box boundaries), and 5th and 95th percentiles (whiskers). See the Supplementary Data for full statistical breakdown. Source data are provided as a Source Data file.
