Fig. 5: Characterization of constrained IL-2 antibody agonists.

a Heatmaps depicting IL-2R pathway agonism in Jurkat^bg-STAT5-Luc reporter cells. Jurkat^bg-STAT5-Luc cells were treated with 100 nM of bispecific WT IgG (left), contorsbodies (middle), and iAbs (right) overnight at 37 °C and IL-2R pathway agonism was quantified by luminescence and plotted as the fold increase in luminescence over untreated controls (n = 2 independent wells). The activity of wild-type human IL-2 at 100 nM is shown for comparison. X denotes no expression. b Concentration-dependent activity of lead constrained IL-2 pathway agonists and corresponding WT IgG controls in the Jurkat reporter assay (n = 2 independent wells). c Schematic (left, created with BioRender.com) and plot (right) of an IL-2Rg/IL-2Rb bridging ELISA (n = 3 independent wells). The Y-axis reflects fold-change of absorbance signal over control that lacks any antibody sample. d Activity of lead constrained IL-2 pathway agonists and corresponding WT IgG controls in primary NK cells (left), and primary CD8 T cells (right) (n = 1 biological replicate/donor per cell type, n = 3 independent wells). Each cell type was obtained from a different individual donor and isolated to >85% purity. For B-D, a legend showing symbols for each line is shown below and an antibody against an irrelevant viral antigen (gD) is shown as a control. For c, d, data is presented as mean values ± SEM. Source data are provided as a Source Data file. e Hierarchical clustering of various IL-2R pathway agonists and corresponding WT IgG bispecifics based on altered gene expression in primary CD8 + T cells determined by mRNA sequencing. Differential gene expression is depicted as the log₂ fold change (log₂FC) of the top 40 up- or down-regulated genes in each treatment group relative to an isotype control antibody (anti-gD) (n = 3 independent samples). Genes are arrayed by column and IL-2 ligand and mimetic antibodies by row.