Abstract 5544: IL10/IL2 surrogate cytokine agonists

Abstract

Abstract Introduction: IL10 is a pleiotropic cytokine that is secreted as a homodimer and initiates signaling on various cell types by engaging two copies of a heterodimeric receptor complex consisting of an IL-10Rα subunit and a IL-10Rβ subunit. IL10 signaling through the natural ligand has both immunosuppressive and immunostimulatory effects and this pleiotropy makes it difficult to develop wild type IL-10 for therapeutic use. In clinical trials that have studied IL10 in cancer, anemia, thrombocytopenia and hemophagocytosis associated with macrophage activation led to dose interruption and dose reduction. To drive more selective cytokine signaling only on cells of interest, we have generated surrogate cytokine agonists that bind to IL10Rα and IL2Rγ, resulting in the selective activation of T cells over macrophages. We establish a novel means of bridging the IL10Rα and IL2Rγ receptors using a IL10Rα/IL2Rγ surrogate cytokine agonist that consists of a domain that binds to IL10Rα and a second domain that binds to IL2Rγ. Upon contact with a cell that allows bridging of IL10Rα and IL2Rγ, the surrogate cytokine agonist causes the functional association of IL10Rα and IL2Rγ, resulting in downstream signaling in select cell types. Experimental Procedures: Human IL-10Rα and IL-2Ry specific single domain VHHs were generated by camel immunization and screening of VHH libraries prepared from peripheral blood cells for binding. Seven IL-10Rα VHHs and six IL-2Ry VHHs were identified and coupled as IL10Rα/IL2Ry dual VHHs in an all by all matrix and in both amino-carboxy and carboxy-amino orientations, yielding 84 unique surrogate cytokine agonists. These surrogate cytokine agonists were screened on a (p)STAT3 assay on primary human cells and further tested in T cell and monocyte functional assays. Results: Several IL10Rα/IL2Rγ surrogate cytokine agonists were biologically active on primary human lymphoid cells, inducing (p)STAT3 signal in B cells, NK cells, CD4 and CD8 T cells with little to no (p)STAT3 signal in monocytes. These IL10Rα/IL2Rγ surrogate cytokine agonists were also functionally active in promoting cell survival and at inducing IFN-γ and Granzyme production by CD8 T cell blasts generated upon CD3/CD28 activation. Consistent with the lack of STAT3 signaling in monocytes, the IL10Rα/IL2Rγ surrogate cytokine agonists did not inhibit LPS induced secretion of IL1β and TNFα by monocytes, suggesting selectivity and a lack of immunosuppressive activities. Conclusions: Designing surrogate cytokine agonists that pair non-natural cytokine receptors provides the possibility of generating molecules that can decouple the pleiotropy of cytokines like IL10 by stimulating only the desired cell population. Here, we have generated IL10Rα/IL2Rγ surrogate cytokine agonists that are biologically active and signal with varying strengths in the lymphoid cells with little to no activity on monocytes, thus providing an opportunity to decouple the pleiotropy of IL10 for use in cancer therapy. Citation Format: Mahalakshmi Ramadass, Sandro Vivona, Deepti Rokkam, Heiko Greb, Anu Gangopadhyay, Romina Riener, Rene de Waal Malefyt, Patrick Lupardus, Martin Oft, Robert Kastelein. IL10/IL2 surrogate cytokine agonists [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 5544.