Abstract 4225: IL-2Rβ/IL-2R γsynthetic cytokines induce activation of human T and NK cells
Authors: Malefyt Rene de Waal , Aspuria PJ , Vivona Sandro , Balasubrahmanyam Priyanka , Buffone Cindy , Ramadass Mahalakshmi , Riener Romina , Oft Martin , Lupardus Patrick , Kastelein Robert 
Published: June 2022

Abstract
Introduction: Heterodimerization of the intermediate affinity Interleukin-2 Receptors (IL-2R), IL-2Rβ and IL-2Rγ, initiates a signaling cascade in T and NK cells that ultimately results in proliferation and production of Interferon-gamma (IFN-γ). Binding of IL-2 in a dimeric complex with intermediate affinity or in the high affinity trimeric complex, which also includes IL-2Rα and is predominantly expressed on antigen activated T cells and Tregs leads to trans-phosphorylation of signaling motifs on the IL-2Rβ and IL-2Rγ intracellular domains by the associated JAK kinases. The function of the wild type IL-2 is to bring the receptor chains in close proximity to produce an optimal level of phosphorylation and activation of associated STAT transcription factors. Here we established a way to bring IL-2Rβ and IL-2Rγ chains together in a cytokine-independent manner that results in functional activation.
Experimental Procedures: Human IL-2Rβ and IL-2Rγ specific heavy chain single domain antibodies (VHH) were generated by camel immunization and screening of VHH libraries prepared from peripheral blood cells for binding. Ten IL-2Rβ VHH segments and six IL-2Rγ VHH segments, all with low nanomolar affinity, were identified and coupled as IL-2Rβ/IL-2Rγ VHH dimers in all possible combinations in both amino-carboxy and carboxy-amino orientations yielding 120 different proteins that were tested in human T and NK cell functional assays.
Results: IL-2Rβ/IL-2Rγ VHH dimers were biologically active and induced pSTAT-5 phosphorylation in the IL-2 dependent NK cell line NKL at various levels. The biological activity of these IL-2Rβ/IL-2Rγ VHH dimers was further confirmed on primary cells. IL-2Rβ/IL-2Rγ VHH dimers induced pSTAT5 phosphorylation on NK cells isolated from human peripheral blood and culture with IL-2Rβ/IL-2Rγ VHH dimers resulted in proliferation and production of high but varied levels of IFN-γ. The activity of IL-2Rβ/IL-2Rγ VHH dimers on primary T cells was also examined. Similar to NK cells, IL-2Rβ/IL-2Rγ VHH dimers induced pSTAT5 phosphorylation, proliferation and IFN-γ production by CD4 positive and CD8 positive T cell blasts generated after CD3/CD28 activation of human PBMC.
Conclusions: In conclusion, we have generated a series of functional IL-2Rβ/IL-2Rγ VHH dimers representing surrogate cytokine agonists each with unique signaling strengths that will now be further analyzed for potential therapeutic application in tumor immunology and auto-immunity
Citation Format: Rene de Waal Malefyt, PJ Aspuria, Sandro Vivona, Priyanka Balasubrahmanyam, Cindy Buffone, Mahalakshmi Ramadass, Romina Riener, Martin Oft, Patrick Lupardus, Robert Kastelein. IL-2Rβ/IL-2R γsynthetic cytokines induce activation of human T and NK cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4225.