Diphtheria toxin-based bivalent human IL-2 fusion toxin with improved efficacy for targeting human CD25+ cells

Abstract

Regulatory T cells (Treg) constitute a major inhibitory cell population which suppresses immune responses. Thus, Treg have proven to be key players in the induction of transplantation tolerance, protection from autoimmune disease and prevention of the development of effective anti-tumor immune reactions. Treg express high levels of the high affinity interleukin-2 receptor (IL-2R) consisting of IL-2Rα (CD25) together with IL-2Rβ (CD122) and the common γ-chain (CD132). An effective reagent capable of depleting Treg in vivo would facilitate better cancer treatment and allow mechanistic studies of the role of Treg in transplantation tolerance and the development of autoimmune disease. In this study, we have developed a novel bivalent human IL-2 fusion toxin along with an Ontak®-like monovalent human IL-2 fusion toxin and compared the functional ability of these reagents in vitro. Here we show that genetically linking two human IL-2 domains in tandem, thereby generating a bivalent fusion toxin, results in significantly improved capacity in targeting human CD25+ cells in vitro. Binding analysis by flow cytometry showed that the bivalent human IL-2 fusion toxin has notably increased affinity for human CD25+ cells. In vitro functional analysis demonstrated that the bivalent isoform has an increased potency of approximately 2 logs in inhibiting cellular proliferation and protein synthesis in human CD25+ cells compared to the monovalent human IL-2 fusion toxin. Additionally, we performed two inhibition assays in order to verify that the fusion toxins target the cells specifically through binding of the human IL-2 domain of the fusion toxin to the human IL-2 receptor on the cell surface. These results demonstrated that 1) both monovalent and bivalent human IL-2 fusion toxins are capable of blocking the binding of biotinylated human IL-2 to human CD25 by flow cytometry; and 2) human IL-2 blocked the fusion toxins from inhibiting protein synthesis and cellular proliferation in vitro, thus confirming that the human IL-2 fusion toxins target the cells specifically through binding to the human IL-2 receptor. We believe that the bivalent human IL-2 fusion toxin will be a more potent, and therefore, more optimal agent than the current clinically-used monovalent fusion toxin (denileukin diftitox, Ontak®) for in vivo depletion of Treg.

Ontak-like human IL-2 fusion toxin

“…With this expression system, the expressed human IL-2 fusion toxins were secreted into the culture medium which facilitated the down-stream purification. The production level and quality were significantly improved comparing with the Ontak ® -production system E.coli (Peraino et al, 2014). In vitro efficacy analysis demonstrated that both monovalent and bivalent human IL-2 fusion toxins were potent and the bivalent isoform is approximately two logs more potent than the monovalent isoform using human CD25 + HUT102/6TG cells (Peraino et al, 2014).…”

“…1, Peraino et al, 2014). With this expression system, the expressed human IL-2 fusion toxins were secreted into the culture medium which facilitated the down-stream purification.…”

“…We assumed that the bivalent isoform would be more potent than the monovalent isoform in vivo as we demonstrated in vitro (Peraino et al, 2014). However, the in vivo data demonstrated that the in vivo efficacies for both isoforms are comparable.…”

“…Recently we have developed monovalent and bivalent human IL-2 fusion toxins using an advanced unique diphtheria toxin resistant yeast Pichia Pastoris expression system (Fig. 1) and characterized their efficacy in vitro (Peraino et al, 2014). In this study, we further assessed the in vivo efficacy of the yeast-produced human IL-2 fusion toxins in human CD25 + CTCL tumor-bearing NSG mouse model.…”

DAB389IL-2 recombinant fusion toxin effect on lymphocyte- and macrophage-producing cytokine subpopulation cells in experimentally induced demyelinating disease in mice

“…Denileukin diftitox, a diphtheria toxin fragment IL-2 fusion protein, targets and kills CD25 ( + ) cells 19 Recently, a newly constructed diphtheria toxin DT 390 porcine IL-2 truncated fusion toxin (Bi-NonGly) has also shown great potential for in vivo depletion of porcine CD25 + cells. 20 Bivalent human IL-2 fusion toxin has proved to be more potent than the current, clinically-used monovalent fusion toxin (denileukin diftitox, Ontak®) for in vivo depletion of Treg cells 21–24 .…”