Increased endosomal sorting of ligand to recycling enhances potency of an interleukin-2 analog

An interleukin-2 (IL-2) variant containing adjacent point mutations (L18M/L 19S, termed 2D1) displaying binding affinity to the heterotrimeric IL-2 rec eptor similar to that of wild-type IL-2 (WT) had been previously found to s urprisingly exhibit increased bioactivity in a peripheral blood lymphocyte proliferation assay. In order to provide an explanatory mechanism for this unexpected potency enhancement, we hypothesize that altered endocytic traff icking of the 2D1 variant might be responsible by increasing the number of ligand-receptor complexes. We demonstrate here that the internalization kin etics of 2D1 via the high affinity IL-2 receptor are equivalent to those of WT but that a significantly increased fraction of internalized 2D1 is sort ed to recycling instead of to lysosomal degradation. We further find a redu ced pH sensitivity of binding to IL-2 receptor alpha relative to IL-2 recep tor beta compared with WT, which could be responsible for the altered sorti ng behavior of 2D1 in the acidic endosomal compartment. Accordingly, the 2D 1 variant displays a half-life 36 h longer than that of IL-2 in T-lymphocyt e culture at concentrations equal to the K-D of the IL-2 receptor. The exte nded half-life of intact 2D1 provides enhanced mitogenesis as compared with IL-2. In addition, 2D1 stimulates natural killer cells to a lesser degree than IL-2 at equal concentrations. We conclude that this IL-2 variant provi des increased mitogenic stimulation that could not be easily predicted from its cell surface receptor binding affinity while minimizing undesired stim ulation of natural killer cells. This concept of altering trafficking dynam ics may offer a generalizable approach to generating improvements in the ph armacological efficacy of therapeutic cytokines.