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/L19S, termed 2D1) displaying binding affinity to the heterotrimeric IL-2 receptor similar to that of wild-type IL-2 (WT) had been previously found to surprisingly 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 trafficking of the 2D1 variant might be responsible by increasing the number of ligand-receptor complexes. We demonstrate here that the internalization kinetics 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 sorted to recycling instead of to lysosomal degradation. We further find a reduced pH sensitivity of binding to IL-2 receptor alpha relative to IL-2 receptor beta compared with WT, which could be responsible for the altered sorting behavior of 2D1 in the acidic endosomal compartment. Accordingly, the 2D1 variant displays a half-life 36 h longer than that of IL-2 in T-lymphocyte culture at concentrations equal to the K(D) of the IL-2 receptor. The extended 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 provides increased mitogenic stimulation that could not be easily predicted from its cell surface receptor binding affinity while minimizing undesired stimulation of natural killer cells. This concept of altering trafficking dynamics may offer a generalizable approach to generating improvements in the pharmacological efficacy of therapeutic cytokines.