Targeted therapy to the IL‐2R using diphtheria toxin and caspase‐3 fusion proteins modulates Treg and ameliorates inflammatory colitis

Abstract

Pathogenic lymphocytes in the enteric wall of inflammatory bowel disease patients display various abnormalities, including reduced sensitivity to apoptosis. We evaluated a therapeutic approach to elimination of cytotoxic cells, using two IL-2 fusion proteins, a diphtheria toxin (IL2-DT) and a caspase-3 (IL2-cas) conjugate. In models of acute (dextran sodium sulfate and trinitrobenzene sulfonic acid) and chronic (dextran sodium sulfate) toxic colitis, therapeutic doses of the fusion proteins improved survival and prevented colon shortening. While both chimeric proteins eradicated CD41 CD251 Foxp3 1 T cells in mesenteric LN, IL2-DT caused severe lymphopenia. In contrast, IL2-cas was equally protective and increased fractional expression of Foxp3. Similar effects of the fusion proteins were observed in healthy mice: IL2-DT caused lymphopenia and IL2-cas increased fractional expression of FoxP3. The fusion proteins induced apoptosis in CD25 1T cells in vitro, with lower toxicity of IL2-cas to Foxp3 1 T cells. These data infer that targeted depletion of cells expressing the IL-2 receptor has therapeutic potential in models of inflammatory colitis, despite depletion of CD25 1 Treg. The IL2-cas fusion protein is particularly relevant to inflammatory bowel disease, as direct internalization of toxic moieties overcomes multiple pathways of resistance to apoptosis of colitogenic T cells.

Targeting of IL-2 receptor with a caspase fusion protein disrupts autoimmunity in prediabetic and diabetic NOD mice

“…The glucose tolerance test was performed by intraperitoneal administration of 2 g glucose and measurement of blood glucose at 30, 60 and 120 min. IL2-cas is composed of full-frame IL-2 conjugated head to tail to fullframe caspase-3 (without the leading procaspase segment) [23,25]. The sequence was inserted into a PET-28 plasmid under control of the T7 promoter and protein retrieved from the inclusion bodies of Escherichia coli was purified under denatured conditions and refolded to a biologically active molecule.…”

“…On the one hand, an agent that mediates targeted killing of CD25 + T cells is expected to precipitate early onset of diabetes similar to cyclophosphamide [4][5][6] and anti-CD25 antibodies [27,28]. On the other hand, decreased activity of reactive cells suggests that the fusion protein might ameliorate the course of insulitis as observed with diphtheria toxin in NOD mice [20] and IL2-cas in inflammatory colitis [22,23,25]. Our female NOD colony was monitored over an extended period of time to determine onset of diabetes at age 14 weeks, with an overall incidence of 83% diabetic mice at the age of 30 weeks (n=312).…”

“…Notably, increased fractional expression of FOXP3 in the lymph modes and pancreas early after IL2-cas administration was associated with reduced the incidence of hyperglycaemia. Elevated fractional expression of FOXP3 in the lymph nodes following administration of IL2-cas (0.9 μg/g, intravenous) has also been observed in a model of toxic colitis in BALB/c mice [23]. To determine whether the acute effects caused by the fusion protein and Cy are specific to NOD mice, wild-type C57BL/6 mice were infused (intraperitoneally) with 4 μg/g IL2-cas and 200 μg/g Cy.…”

“…A fusion protein composed of IL-2 and diphtheria toxin showed protective effects against development of diabetes in NOD mice and humans [20,21], as well as in models of inflammatory colitis [22]. However, we recently showed that this fusion protein causes severe lymphopenia at the therapeutic doses, which might have detrimental consequences in the context of autoimmunity [23]. Homeostatic expansion of effector cells outpaces the recovery of Treg under conditions of lymphopenia [24], creating a transient period of instability with predominant autoimmune effector activity [9,10].…”

“…Homeostatic expansion of effector cells outpaces the recovery of Treg under conditions of lymphopenia [24], creating a transient period of instability with predominant autoimmune effector activity [9,10]. In variance, a fusion protein composed of IL-2 and activated caspase-3 (IL2-cas) provided the same defence against inflammatory colitis without causing lymphopenia [23,25]. In this study we evaluated the effect of IL2-cas in NOD mice, where targeted deletion of cells expressing the IL-2 receptor has two possible outcomes.…”