Figure 5.

Exploring the structure–activity relationship of Nectin-4/CD137 Bicycle TICAs by introducing point mutations to CD137-binding Bicycle. (A) Structure of hinge 5. (B) Box and whisker plot of the geometric mean of EC50 and E_max of each Bicycle TICA (excluding substitution Asp9 → Asn and Asp9 → Glu that substantially impacted binding affinity) synthesized in the 1:1 format using linker 3 and 1:2 format using hinge 5 in the determined in HT1376/CD137 reporter coculture assay. The test for significance was performed using the Mann–Whitney U test. (C) EC50 and E_max of Bicycle TICAs from the HT1376/CD137 reporter coculture assay shown as individual data points with geometric mean represented as crossbars. The x-axis represents point mutations to the CD137 Bicycle of the Bicycle TICA as compared to BCY12379 (linker 3, 1:1) and BCY12485 (hinge 5, 1:2).