IL-2 is the master-regulator cytokine for T cell dependent responses and is crucial for proliferation and survival of T cells. However, IL-2-based treatments remained marginal, in part due to short half-life. Thus, we aimed to extend IL-2 half-life by flanking the IL-2 core with sequences derived from the extensively glycosylated hinge region of the NCR2 receptor. We termed this modified IL-2: “S2A”. Importantly, S2A blood half-life was extended 14-fold compared to the clinical grade IL-2, Proleukin. Low doses inoculation of S2A significantly enhanced induction of Tregs (CD4+ Regulatory T cells) in vivo, as compared to Proleukin, while both S2A and Proleukin induced low levels of CD8+ T cells. In a B16 metastatic melanoma model, S2A treatment was unable to reduce the metastatic capacity of B16 melanoma, while enhancing induction and recruitment of Tregs, compared to Proleukin. Conversely, in two autoimmune models, rheumatoid arthritis and DSS-induced colitis, S2A treatment significantly reduced the progression of disease compared to Proleukin.
Fig: IL-2 variant design and in vitro functional analysis. (A) A scheme structure of the recombinant IL-2 S2A. The human IL-2 encoding sequence is flanked by two highly glycosylated fragments. S2A sequence also includes the IL-2 signal peptide at the N-terminal and His-tag sequence at the C-terminal. (B) S2A encoding plasmids were transfected into HEK293F cells, and protein was purified from media using the Ni-ion exchange column. Following purification, purity and size were determined by running eluted fraction on SDS-PAGE. (C) A representative ELISA binding curve (fitted to 4-parameter logistic) using IL-2 ELISA kit (Biolegend) (D) A representative CTLL-2 proliferation test, done using MTT assay (fitted to 4-parameter logistic. (E) Aggregated results of 5 independent CTLL-2 assays for different preparations of commercial IL-2 (Proleukin) and S2A. Mann–Whitney U test was used to determine significance (solid line—mean, crushed line—median; *, P < 0.05).
Our results suggest new avenues for generating long-acting IL-2 for long-standing treatment and a new technique for manipulating short-life proteins for clinical and research uses.
Ottolenghi, A., Bolel, P., Sarkar, R. et al. Life-extended glycosylated IL-2 promotes Treg induction and suppression of autoimmunity. Sci Rep11, 7676 (2021). https://doi.org/10.1038/s41598-021-87102-4