Fig. 1. Basic principles of CYpHER and component binders.
a CYpHER design including a pH-independent TfR-binding domain and a pH-dependent target-binding domain separated by a linker. b CYpHER mechanism. CYpHER induces ternary complex formation with target and TfR. Upon TfR-mediated uptake and endosomal acidification, target is released for endolysosomal system trafficking. TfR and CYpHER recycle to the surface for engagement with another target molecule. c 293F cells displaying a high-affinity TfR-binding CDP were stained with TfR and rinsed at pH 7.4 or pH 5.5 for 10 min, showing similar binding via flow cytometry in both conditions. Mean fluorescence intensity [MFI] ±95% confidence interval [CI], pH 7.4, 80.1 ± 1.9; pH 5.5, 90.6 ± 2.0; precise N per sample unavailable. d 293F cells displaying medium or high affinity TfR-binding CDPs were stained with human TfR (hTfR) or mouse TfR (mTfR). Kruskal–Wallis test with Dunn’s correction: Med affn hTfR vs High affn hTfR was not significant (P > 0.9999), all others P < 0.0001. N cells per sample: GFP-, 479; Med affn hTfR, 317; Med affn mTfR, 295; High affn hTfR, 266; High affn mTfR, 236. e pH-dependent PD-L1 binding flow profile of 293F cells displaying a pool of histidine-doped variants of a PD-L1-binding CDP after four rounds of flow sorting; two for high binding after pH 7.4 rinse, two for low binding after pH 5.5 rinse. pH 7.4, N = 38,979 cells; pH 5.5, N = 37,450 cells. f Three His substitutions were tested as singletons and combinations for PD-L1-binding on 293F cells displaying a given binder after 10 min pH 7.4 or pH 5.5 rinse followed by flow cytometry quantitation. N cells per sample: Untransfected [UTF] 7.4, 19727; UTF 5.5, 17843; Parental 7.4, 41; Parental 5.5, 46; His sub 1 [HS1], 7.4, 27; HS1 5.5, 17; HS2 7.4, 29; HS2 5.5, 25; HS3 7.4, 58; HS3 5.5, 62; HS1 + 2 7.4, 62; HS1 + 2 5.5, 58; HS1 + 3 7.4, 25; HS1 + 3 5.5, 29; HS2 + 3 7.4, 45; HS2 + 3 5.5, 40; HS1 + 2 + 3 7.4, 87; HS1 + 2 + 3 5.5, 92. Non-Parental samples within a given pH (7.4 or 5.5) vs Parental sample by Kruskal–Wallis test with Dunn’s correction: at pH 7.4, His sub 1 (P > 0.9999), His sub 3 (P = 0.1161), and His sub 1 + 3 (P > 0.9999) were not significant, all others P < 0.0001. At pH 5.5, His sub 1 (P = 0.1666) and His sub 3 (P > 0.9999) were not significant, all others P < 0.0001. Variant with His substitutions 1 and 3 was chosen for further work. Each experiment in c–f was performed once. All box plots (d and f) 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.