Abstract
IL15 and IL2 are two similar cytokines that stimulate the proliferation of lymphocytes, and their therapeutic potential has been well established in animal models and human trials. Both cytokines exert their cell signaling function through binding to a trimeric complex consisting of two shared receptors, the common gamma chain (γc) and IL2Rβ, as well as an alpha chain receptor unique to each cytokine: IL2Rα or IL15Rα. Both cytokines share a similar biology, with the exception that IL2 has a greater preference for Tregs due to their high constitutive expression of IL2Rα. IL15 functions as a stabilized heterodimeric complex with membrane-bound IL15Rα on the surface of monocytes and DCs, and this IL15/IL15Rα complex is presented in trans to lymphocytes expressing IL2Rβ and γc. It has been shown that recombinant IL15/IL15Rα heterodimer is stable and highly active. As potential drugs, both IL2 and IL15 are extremely potent and suffer from low tolerability and very fast clearance that limits therapeutic window. Seeking to engineer a more druggable version of IL15, we created various IL15/IL15Rα heterodimeric Fc-fusions (IL15/IL15Rα-Het-Fc) with reduced potency to improve tolerability, slow receptor-mediated clearance, and prolong half-life.
We engineered IL15/IL15Rα-Het-Fc by fusing IL15 to one side of a heterodimeric Fc-region, and the sushi domain of IL15Rα to the other side, or by creating a single-chain IL15/IL15Rα that was attached to one side of a heterodimeric Fc-region. These Fc-fusions were tuned for optimal activity by engineering amino acid substitutions in IL15 - at the IL2Rβ or γc interface - that reduced overall in vitro potency. In vitro proliferation of lymphocytes in normal human PBMCs was monitored by counting Ki67+ cells after incubation with Fc-fusions for 4 days and by measuring signaling in a STAT5 phosphorylation assay for 15 minutes. In vivo activity was evaluated using a huPBMC-NSG mouse model by measuring the extent of human leukocyte engraftment by flow cytometry and IFNγ. Tolerability, immune stimulation, and pharmacokinetics were evaluated in non-human primates (NHP).
IL15/IL15Rα-Het-Fc were produced with good yield and purity. The Fc-fusions enhanced proliferation of CD8+ T and NK cells in vitro, and potency of variants with substitutions at the IL2Rβ and/or γc interface was reduced up to ~700-fold compared to wild-type IL15/IL15Rα-Het-Fc. Treatment of huPBMC-NSG mice with IL15/IL15Rα-Het-Fc promoted enhanced T cell engraftment and elevated IFNγ in a dose dependent manner. NHP studies indicated half-lives of several days for IL15/IL15Rα-Het-Fc, which are significantly longer than the <1 hr half-life of IL15. In both in vivo settings, a marked inverse correlation of pharmacodynamics and clearance was observed, with weaker potency variants allowing higher doses and enhanced in vivo lymphocyte proliferation as a result of more sustained exposure.
Citation Format: Matthew J. Bernett, Rajat Varma, Christine Bonzon, Rumana Rashid, Liz Bogaert, Ke Liu, Suzanne Schubbert, Kendra N. Avery, Irene W. Leung, Nicole Rodriguez, Seung Y. Chu, Umesh S. Muchhal, Gregory L. Moore, John R. Desjarlais. Potency-reduced IL15/IL15Rα heterodimeric Fc-fusions display enhanced in vivo activity through increased exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5565.
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