Fig. 4: Designed 5HCS binder to PD-L1.

a Biophysical Characterization of 5HCS_PDL1_1. Left: Model of 5HCS_PDL1_1 (cartoon) binding to PD-L1 (PDB ID: 3BIK), with 5HCS_PDL1_1 colored by Shannon entropy from site saturation mutagenesis results. Middle: Biolayer interferometry characterization of 5HCS_PDL1_1. Biotinylated PD-L1 was loaded to Streptavidin (SA) tips, and these were incubated with 8 nM, 2.7 nM, and 0.9 nM of 5HCS_PDL1_1 to measure the binding affinity. Right: Circular dichroism spectra from 25 °C to 95 °C for 5HCS_PDL1_1. b Heat map representing the log enrichments for the 5HCS_PDL1_1 SSM library selected with 6 nM PD-L1 at representative positions. The annotated amino acid in each column indicates the residue from the parent sequence. c Unbound crystal structure of 5HCS_PDL1_1 and designed interactions between 5HCS_PDL1_1 (green) and PD-L1 (white). Color schemes are the same as Fig. 2. d WT A431 (green) and PD-L1 KO A431 (gold) cell lines were stained with fluorophore-labeled 5HCS_PDL1_1 and anti-PD-L1 antibody respectively and then analyzed through FACS. The comparison between anti-PD-L1 vs. A431 WT and Unstain A431 WT resulted in a significance level with p = 2.874 × 10⁻⁴. The 5HCS_PDL1_1 vs. A431 and Unstain A431 WT comparison yielded a significant p-value of p = 4.269 × 10⁻². A431 WT samples were tested in two wells, and A431 KO samples in four wells, with 5000 cells per well, ensuring reproducibility and statistical reliability. ns, no significance, *P  <  0.05, and ***P  <  0.001. (t test independent samples with Bonferroni correction). Bars, mean ± SEM. e The increase of TCR activation induced signal (via NFAT pathway) from engineered PD-1 effector cell lines by 5HCS_PDL1_1 (green), control antibody (gold) is shown. The mean values were calculated from triplicates for the cell signaling inhibition assays measured in parallel, and error bars represent standard deviations. Color schemes and experimental details are as in Fig. 3. Source data (a, d, e) are provided in the Source Data file.