Bi-functional IL2/CD25/IL10 fusion protein promotes Tregs, reduces inflammation, and is highly effective in limiting diabetes in NOD mice

Abstract

Abstract Clinical trials indicate that low-dose IL-2 is a promising approach for patients with autoimmunity. To improve upon IL-2, the mouse (m) IL-2/CD25 fusion protein was developed, which has a long half-life, is selective to the high-affinity IL-2R and expands Tregs to limit diabetes in NOD mice. However, after an extended time off-therapy, many NOD mice exhibited T1D. To improve efficacy, we developed a bifunctional fusion protein, consisting of IL-2/CD25 covalently linked to IL-10 to increase Tregs and limit inflammation. With respect to IL-2 activity, IL2/CD25/IL10 supported IL-2-dependent proliferation of CTLL and STAT5 activation by Tregs. With respect to IL-10 activity, it supported IL-10-dependent STAT3 activation by RAW264.7 cells and limited IFNγ and IL-6 production by activated T and B cells, respectively. In vivo, IL2/CD25/IL10 supported expansion of Tregs as well as activation of pSTAT3 in macrophages and reduced LPS-dependent production of IL-6 and TNFα, indicative of bifunctional activity. When 12-week-old female NOD mice received IL2/CD25/IL10 for 5-weeks, IL2/CD25/IL10 (mean survival >50 week) was more effective than IL-2/CD25 (mean survival 26 weeks) in preventing progressive diabetes. Collectively, these data support the notion that a combination approach, where Tregs are increased and autoreactive T cells are limited, in this case through IL-10, will enhance the efficacy of a Treg-targeted immunotherapy of autoimmunity.