Journal: Journal of Biological Engineering

Article Title: Directed evolution–driven reprogramming of PD-L1 for compact and tunable checkpoint modulation

doi: 10.1186/s13036-025-00577-x

Figure Lengend Snippet: Isolation of PD-L1 variants with improved PD-1 binding affinity. ( A ) Flow cytometry analysis comparing PD-1 binding to yeast-displayed wild-type PD-L1 fused to Aga2 at either the N-terminus or C-terminus. The C-terminal fusion format resulted in significantly superior receptor engagement. ( B ) Crystal structure of the PD-1/PD-L1 complex (PDB ID: 3BIK), highlighting the N- and C-termini of PD-L1 relevant to display orientation. ( C ) FACS density plots showing progressive enrichment of PD-L1 variants with enhanced PD-1 binding over successive rounds of library screening. Yeast cells were co-stained with anti-FLAG-Alexa Fluor 647 and PD-1-GST-Alexa Fluor 488 probes. ( D ) ELISA-based quantification of PD-1 binding by seven affinity-matured PD-L1 variants (Y2–Y9), in comparison with wild-type PD-L1 and a previously reported variant (L3B3)

Article Snippet: Interleukin-4 (IL-4), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and human cytokine DuoSet ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Isolation, Binding Assay, Flow Cytometry, Library Screening, Staining, Enzyme-linked Immunosorbent Assay, Comparison, Variant Assay

Journal: Journal of Biological Engineering

Article Title: Directed evolution–driven reprogramming of PD-L1 for compact and tunable checkpoint modulation

doi: 10.1186/s13036-025-00577-x

Figure Lengend Snippet: Identification and binding characterization of engineered PD-L1 variants with enhanced affinity for PD-1. ( A ) Amino acid sequence alignment of affinity-matured PD-L1 variants in comparison to wild-type PD-L1. Two conserved mutations, N45D and V50M, are found recurrently among the top-performing variants. ( B ) ELISA analysis of PD-1 binding by selected PD-L1 variants (Y2, Y3, Y6), a single mutant (D), and a double mutant (DM), compared with wild-type PD-L1 and L3B3. ( C ) Comparative ELISA of PD-1 binding for the wild-type, L3B3, L3C7c, and DM variants, highlighting the superior affinity of the DM variant. ( D ) Kinetic parameters derived from bio-layer interferometry (BLI), including association ( k a ), dissociation ( k d ), and equilibrium dissociation constants ( K D ), confirming the DM variant as the highest-affinity binder. The BLI assay data, including fitted kinetic constants and their associated error values, are presented in Supplementary Figure

Article Snippet: Interleukin-4 (IL-4), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and human cytokine DuoSet ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Binding Assay, Sequencing, Comparison, Enzyme-linked Immunosorbent Assay, Mutagenesis, Variant Assay, Derivative Assay

Journal: Journal of Biological Engineering

Article Title: Directed evolution–driven reprogramming of PD-L1 for compact and tunable checkpoint modulation

doi: 10.1186/s13036-025-00577-x

Figure Lengend Snippet: PD-1 binding inhibition and T cell activation induced by Fc-fused PD-L1 variants. ( A and B ) Competitive ELISA demonstrating the ability of Fc-fused PD-L1 variants to inhibit PD-1 binding to immobilized ligands. Increasing concentrations of Fc-fused wild-type and DM PD-L1 variants were tested for their ability to block PD-1 interaction with ( A ) plate-bound PD-L1-Fc or ( B ) PD-L2-Fc. Absorbance at 450 nm reflects residual PD-1 binding after competition, with error bars indicating variation between duplicate samples. ( C and D ) Mixed lymphocyte reaction (MLR) assay evaluating the immunostimulatory capacity of Fc-fused PD-L1 variants. CD4⁺ T cells were co-cultured with mature dendritic cells in the presence of the indicated Fc-fused ligands. Cytokine levels in the supernatants were quantified by ELISA. ( C ) Interferon-γ (IFN-γ) and (D) interleukin-2 (IL-2) production, expressed in pg/mL. Statistical significance for cytokine secretion was assessed using one-way ANOVA as follows: p > 0.05 (ns, not significant), p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***)

Article Snippet: Interleukin-4 (IL-4), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and human cytokine DuoSet ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Binding Assay, Inhibition, Activation Assay, Competitive ELISA, Blocking Assay, Mlr Assay, Cell Culture, Enzyme-linked Immunosorbent Assay