Fig. 3: Identification and in vitro characterization of THOR-707, a pegylated IL-2 with “not alpha” pharmacology.

a Cell-based screening of pegylated IL-2 variants identified compounds with a reduced contribution of IL-2Rα engagement to signaling potency. Pegylated variants were analyzed in parallel in the Discoverx PathHunter® IL-2 receptor agonist assays. Compounds were tested in duplicate in a dose–response format. Shown are the potency statistics (EC50) for each compound in the IL-2 receptor βγ (orange) and αβγ (blue) assays. Initially, pegylated variants were tested with a 10 kDa mPEG modification (compounds not marked with asterisk). During the screen, it was determined that larger mPEG modifications enable improvements in pharmacokinetics and target cell expansion (not shown), and larger mPEGs (30 kDa) were therefore employed for the remaining compounds (compounds marked with an asterisk). The calculated βγ: αβγ EC50 ratios for each test compound are listed below each plot. (n = 2 independent experiments). b Biochemical characterization of THOR-707 interactions with IL-2 Rα and β receptor subunits using SPR demonstrate “not alpha” signature of THOR-707. Human IL-2 Rα (top row) and β (bottom row) extracellular domains were immobilized on the surface of a SPR sensor and probed with a dilution series starting at 2 µM of either rhIL-2 (left column) or THOR-707 (right column). Test samples were injected for 60 s to allow measurement of association, followed by buffer only (wash, initiation time at 60 s) to measure dissociation kinetics. Response units (RU, y axis) are plotted versus time (s, x axis). Colors correspond to test concentration as shown (inset). c The cell-specific signaling of THOR-707 in human primary lymphocytes shows THOR-707 reprogramming to reduce IL-2Rα-mediated bias for Treg potency. Human PBMC samples from 6 healthy donors were stimulated in triplicate with concentration series of rhIL-2 (left panel) or THOR-707 (right panel). Treated cell populations were analyzed using multi-color flow cytometry to detect and quantify pSTAT5 activation in different T and NK cell subsets (cyan, blue, orange represent the data from Tregs, NK, and CD8⁺ T cells, respectively). The plots shown represent the average pSTAT5 signal, fit to a baseline restrained 4-parameter logistic regression and normalized to maximum signal to facilitate comparison of different cell subsets, with error bars representing SEM. (n = 6 independent donor samples). Source data are provided as a Source Data file.