IL-2 is a highly potent, pleiomorphic cytokine approved for the treatment of melanoma and renal cell carcinoma. The therapeutic potential of this cytokine has been restricted by dose-limiting toxicities and off-tumor effects associated with systemic IL-2 administration. Scientists have long-pursued the development of targeted IL-2 variants to control biodistribution and improve the therapeutic index, e.g. IL-2-antibody fusions or immunocytokines. However, the potential benefit of targeting IL-2 is limited by the high affinity of the cytokine for its cellular receptor, which dominates biodistribution and results in activation of cells outside the tumor microenvironment. To address this challenge, we took advantage of the remarkable stability of Neoleukin-2/15 (Neo-2/15), a fully de novo cytokine mimetic that potently activates the beta and gamma subunits shared by the IL-2 and IL-15 receptors. We demonstrate that Neo-2/15 can be divided into two pieces that cannot signal individually, but that can recover their binding and cell-signaling potential when co-localized. Fusing the split molecules to independent targeting domains creates a modular and highly specific implementation of conditional activation. We demonstrate that if one split fragment is targeted to one tumor-associated antigen, and the complementary fragment is targeted to a second tumor-associated antigen, the ability to bind the IL-2 receptor and trigger signaling is only restored when these split fragments bind to the surface of cells expressing both targets. The split Neo-2/15 molecules have anti-tumor activity in syngeneic cancer models and have the potential to enhance the therapeutic index of cytokine mimetics in immunotherapy. To our knowledge, these are the first designed examples of strictly conditionally-active, targeted, cytokine-like molecules for immunotherapy.

*Current affiliation for D-A.S.: Neoleukin Therapeutics Inc, Seattle, WA, USA.

Citation Format: Alfredo Quijano-Rubio, Lestat R. Ali, Md A. Bhuiyan, Marc J. Lajoie, Michael Dougan, David Baker, Daniel-Adriano Silva. Conditionally active de novo IL-2 cytokine mimetics for targeted immunotherapy: de novo split technology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1075.