Discovery of a Conditionally Activated IL-2 that Promotes Antitumor Immunity and Induces Tumor Regression.

IL-2 is a cytokine clinically approved for the treatment of melanoma and renal cell carcinoma. Unfortunately, its clinical utility is hindered by serious side effects driven by the systemic activity of the cytokine. Here, we describe the design and characterization of a conditionally activated IL-2 prodrug, WTX-124, that takes advantage of the dysregulated protease milieu of tumors. WTX-124 was engineered as a single molecule containing an inactivation domain and a half-life extension domain that are tethered to a fully active IL-2 by protease-cleavable linkers. We show that the inactivation domain prevented IL-2 from binding to its receptors in nontumor tissues, thereby minimizing the toxicity associated with systemic exposure to IL-2. The half-life extension element improves the pharmacokinetic profile of WTX-124 over free IL-2, allowing for greater exposure. WTX-124 was preferentially activated in tumor tissue by tumor-associated proteases, releasing active IL-2 in the tumor microenvironment. In vitro assays confirmed that the activity of WTX-124 was dependent on proteolytic activation, and in vivo WTX-124 treatment resulted in complete rejection of established tumors in a cleavage-dependent manner. Mechanistically, WTX-124 treatment triggered the activation of T cells and natural killer (NK) cells, and markedly shifted the immune activation profile of the tumor microenvironment, resulting in significant inhibition of tumor growth in syngeneic tumor models. Collectively, these data demonstrate that WTX-124 minimizes the toxicity of IL-2 treatment in the periphery while retaining the full pharmacology of IL-2 in the tumor microenvironment, supporting its further development as a cancer immunotherapy treatment. See related Spotlight by Silva, p. 544.