Design of an IL-2 based immunocytokine for targeted expansion of regulatory T cells.

Introduction: Interleukin-2 (IL-2) is a pleiotropic cytokine that orchestrates the differentiation, proliferation, survival, and activity of immune cells by signaling through a high-affinity heterotrimeric receptor (IL-2 receptor-a [IL-2Rα], IL-2Rβ, and γ_c chains) or an intermediate-affinity heterodimeric receptor (IL-2Rβ, and γ_c chains) (Fig. 1A). Since IL-2Rα is highly expressed on T_Reg cells, but virtually absent from naïve immune effector cells, low-dose IL-2 administration preferentially stimulates polyclonal expansion of T_Reg cells over immune effector cells to elicit an immunosuppressive response, and this effect has been leveraged as a therapeutic strategy to treat autoimmune disorders.^1,2 However, despite the cytokine’s proclivity towards T_Reg cells, IL-2 can still activate immune effector cells. Studies have shown that co-administration of IL-2 with the anti-cytokine antibody F5111 selectively biases IL-2 activity towards T_Reg cells, presenting an enticing opportunity for targeted cytokine therapy.³ Our lab engineered a single-agent human IL-2/F5111 fusion protein (F5111 immunocytokine (IC)) (Fig. 1B), which selectively expands T_Reg cells and protects against autoimmune diseases in mice.⁴ However, the large size and high affinity of F5111 IC can result in inefficient delivery to target tissues. To overcome these translational challenges, we report the development a miniaturized version of F5111 IC (miniF5111 IC), comprising IL-2 linked to the antibody single chain variable fragment (scFv) of F5111. This molecule will allow for tissue-targeted expansion of T_Reg cells, forming the basis for a new category of immunotherapeutics to treat a vast range of autoimmune diseases.

Materials and Methods: The miniF5111 ICs were designed and cloned based on F5111 IC by PCR.⁴ The miniF5111 ICs were transfected into human embryonic kidney 293-F cells and purified via nickel-affinity chromatography followed by size-exclusion chromatography. IL-2Rα+ YT-1 and IL-2Rα- YT-1 cells were then stimulated with the miniF5111 ICs and IL-2 signaling was measured based on abundance of the phosphorylated form of the transcription factor signal transducer and activator of transcription 5 (STAT5), as quantified by flow cytometry. MiniF5111 IC activity was further evaluated in primary human peripheral blood mononuclear cells. All results were analyzed using GraphPad Prism.

Results, Conclusions, and Discussions: The miniF5111 ICs were designed in 4 orientations, which differ in the orientation of the IL-2/F5111 scFv fragment fusion (Fig. 1C). MiniF5111 IC HL2 exhibited poor expression (not shown) and therefore was not included in subsequent IL-2-responsive cell signaling assays. MiniF5111 IC 2HL and LH2 demonstrated relatively low activity against IL-2Rα+ YT-1 human natural killer-like cells and showed a 1-2 order magnitude difference in reduced activity against IL-2Rα- YT-1 cells. However, miniF5111 IC 2LH showed comparable signaling to F5111 IC in IL-2Rα+ cells and a 3-4 order magnitude difference in reduced activity against IL-2Rα- cells (Fig. 1D), consistent with the activity of F5111 IC.



Conclusion. We have engineered a miniaturized version of the F5111 IC (miniF5111 IC 2LH) that recapitulates the biased immune cell activation profile of the parent molecule. Ongoing cellular and animal studies are characterizing the behavior of miniF5111 IC 2LH and developing disease-specific molecules for treatment of autoimmune disorders.


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References (Optional): : 1 Boyman O, Sprent J. The role of interleukin-2 during homeostasis and activation of the immune system. Nat Rev Immunol 2012;12:180–90. https://doi.org/10.1038/nri3156.

2 Liao W, Lin J-X, Leonard WJ. Interleukin-2 at the crossroads of effector responses, tolerance, and immunotherapy. Immunity 2013;38:13–25. https://doi.org/10.1016/j.immuni.2013.01.004.

3 Spangler JB, Tomala J, Luca VC, Jude KM, Dong S, Ring AM, et al. Antibodies to Interleukin-2 Elicit Selective T Cell Subset Potentiation through Distinct Conformational Mechanisms. Immunity 2015;42:815–25. https://doi.org/10.1016/j.immuni.2015.04.015.

4 VanDyke D, Iglesias M, Tomala J, Young A, Smith J, Perry JA, et al. Engineered human cytokine/antibody fusion proteins expand regulatory T cells and confer autoimmune disease protection. Cell Reports 2022;41:111478. https://doi.org/10.1016/j.celrep.2022.111478.