IL-2K35C-moFA, a Long-Acting Engineered Cytokine with Decreased Interleukin 2 Receptor α Binding, Improved the Cellular Selectivity Profile and Antitumor Efficacy in a Mouse Tumor Model

SIMPLE SUMMARY: The application of IL-2 for treating cancer is limited owing to its toxicity and short half-life. Its high binding ability to IL-2 receptor α expands immunosuppressive Treg cells, which represents an undesirable toxicity in cancer immunotherapy. Moreover, its small molecular size is the reason for its short half-life. We solved these problems by using a covalent modification strategy of IL-2 variant IL-2K35C with fatty acid by maleimide chemistry, namely, IL-2K35C-moFA. The experiments performed in vitro and in vivo proved that IL-2K35C-moFA is a novel immunotherapeutic agent with the potential to selectively stimulate CD8(+) T cells and NK cells. Compared to IL-2WT, IL-2K35C-moFA showed a specifically reduced potency for the stimulation of Treg cells. Our results also showed that fatty acid conjugation appears to be effective in half-life extension. The combination of selective lymphocyte expansion and its long half-life means IL-2K35C-moFA should be evaluated as a potential human immunotherapeutic in the future. ABSTRACT: Human interleukin 2 (IL-2) has shown impressive results as a therapeutic agent for cancer. However, IL-2-based cancer therapy is limited by strong Treg amplification owing to its high binding affinity to IL-2 receptor α (IL-2Rα) and its short half-life owing to its small molecular size. In this study, we solved these problems using a covalent modification strategy of the IL-2 variant, i.e., substituting cysteine (C) for lysine (K) at position 35, using octadecanedicarboxylic acid through maleimide chemistry, creating IL-2K35C-moFA. IL-2K35C-moFA was equipotent to human IL-2 wild type (IL-2WT) in activating tumor-killing CD8(+) memory effector T cells (CD8(+) T) and NK cells bearing the intermediate affinity IL-2 receptors, and less potent than IL-2WT on CTLL-2 cells bearing the high-affinity IL-2 receptors. Moreover, it was shown to support the preferential activation of IL-2 receptor β (IL-2Rβ) over IL-2Rα because of the mutation and fatty acid conjugation. In a B16F10 murine tumor model, IL-2K35C-moFA showed efficacy as a single dose and provided durable immunity for 1 week. Our results support the further evaluation of IL-2K35C-moFA as a novel cancer immunotherapy.