Increased Endosomal Sorting of Ligand to Recycling Enhances Potency of an Interleukin-2 Analog

Abstract

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 ␣ relative to IL-2 receptor ␤ 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.

Computational Model for Effects of Ligand/Receptor Binding Properties on Interleukin-2 Trafficking Dynamics and T Cell Proliferation Response

“…Thus, internalization of IL-2 occurs solely via receptor-mediated endocytosis (66,67). Although this process is saturable at high ligand concentrations, the low density of high affinity IL-2 receptors on T cells makes saturation an unlikely phenomenon (68,69), consistent with experimental studies (37). As noted above, experimental evidence indicates that the components of the IL-2/IL-2R complex undergo differential sorting wherein IL-2RR is recycled to the cell surface while IL-2, IL-2R , and IL-2Rγ all remain associated en route to lysosomes (34).…”

“…This work demonstrated that a double mutant of IL-2, Leu18Met/Leu19Ser (termed 2D1), displayed reduced endocytic degradation when compared to wild-type IL-2 (WT) and that this effect was due to enhanced ligand recycling. The continuous flow of ligands through intracellular sorting pathways thus led to greater sustenance of 2D1 concentrations in cell culture and increased T-lymphocyte proliferation (37). The objective of our modeling work is not to mimic these experimental results in specific detail but rather to elucidate insights concerning key molecular properties that should be considered in a ligand-based protein engineering approach to altering cell function.…”

“…This result stemmed from a decreased internalization rate and increased recycling rate for both ligand and receptor, opposing the signal-attenuating effects of ligand depletion and receptor downregulation. An IL-2 variant with increased affinity for the R chain but reduced affinity for the γ-complex demonstrated greater potency based on similar effects in a different system (37). This result is also counterintuitive because the mutation increased the ligand's affinity for the subunit not directly involved in signal transduction while decreasing the affinity for the chains that generate a mitogenic signal.…”

Membrane localization is not required for Mpl function in normal hematopoietic cells

“…Other attributes of cell surface location of these receptors that may facilitate signal transduction include organization into specific microdomains on the cell surface, [2][3][4][5] preassembled signaling complexes, 6,7 cross-talk among heterologous cytokine receptors, [8][9][10] and commencement of normal intracellular trafficking of the receptor-ligand complex. [11][12][13][14] A stringent test of growth factor receptor function is the ability to support the proliferation and differentiation of normal hematopoietic cells. We hypothesized that the location of a hematopoietic growth factor receptor in the plasma membrane was important not only for communication with the extracellular milieu but also for expression of the full repertoire of receptor functions.…”

Improving Protein Pharmacokinetics by Genetic Fusion to Simple Amino Acid Sequences

“…In addition, post-translational modifications such as site-specific carbohydrate attachment (3,4), sialylation degree (5), and multimerization (6) may modulate this phenomenon. The presence of endogenous receptors for certain molecules also regulates their mean residence time in blood (7)(8)(9)(10). Although scarce evidence is at hand, the existence of linear amino acid motifs involved in the bloodstream half-life of proteins might be postulated (11,12).…”