Fig. 2: PD-L1 CYpHER design and target depletion in cell pools overexpressing PD-L1-GFP.
From: CYpHER: catalytic extracellular targeted protein degradation with high potency and durable effect
a Two designs of PD-L1 CYpHERs, named CT-4212-1 and CT-4212-3, using a high-affinity TfR-binding CDP and a pH-dependent PD-L1-binding CDP. b Illustration of PD-L1-GFP trafficking induced by CYpHER. Pools of 293T cells (c), H1650 cells (d), and MDA-MB-231 cells (e) transduced with lentivirus driving PD-L1-GFP were untreated or incubated with 10 nM CYpHER for 24 h before GFP-channel microscopy (above) and flow cytometry (below) after staining for surface PD-L1. Black contour in flow profiles: cells stained without PD-L1 antibody. Flow cytometry quantitation of normalized surface PD-L1 (f, h, and j) or total PD-L1-GFP (g, i, and k) signal in 293T-PDL1-GFP cells (f and g), H1650-PDL1-GFP cells (h and i), and MDA-MB-231-PDL1-GFP cells (j and k) with or without CYpHER treatment. N cells per sample as follows. c, f, g Untreated, 842; CT-4212-1, 808; CT-4212-3, 598. d, h, i Untreated, 1867; CT-4212-1, 2318; CT-4212-3, 2734. e, j, k Untreated, 823; CT-4212-1, 536; CT-4212-3, 893. For f–k, within each line and assay, significance by Kruskal–Wallis test with Dunn’s correction were all P < 0.0001 except: 293T-PDL1-GFP surface PD-L1, CT-4212-1 vs CT-4212-3 (P = 0.1647); 293T-PDL1-GFP total GFP, CT-4212-1 vs CT-4212-3 (P = 0.0027); H1650-PDL1-GFP surface PD-L1, CT-4212-1 vs CT-4212-3 (P = 0.0908); H1650-PDL1-GFP total GFP, Untreated vs CT-4212-3 (P = 0.0002); MDA-MB-231-PDL1-GFP total GFP, Untreated vs CT-4212-1 (P = 0.3139). Each experiment in c–k was performed once. All box plots (f–k) 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.
