Diphtheria toxin-based bivalent human IL-2 fusion toxin with improved efficacy for targeting human CD25(+) cells.
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.
Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA; MGH-DF/HCC Recombinant Protein Expression and Purification Core, Boston, MA, USA.
Jaclyn Stromp
JS
Peraino
Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA; MGH-DF/HCC Recombinant Protein Expression and Purification Core, Boston, MA, USA.
Huiping
H
Zhang
Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA; MGH-DF/HCC Recombinant Protein Expression and Purification Core, Boston, MA, USA.
Priyani V
PV
Rajasekera
Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA; MGH-DF/HCC Recombinant Protein Expression and Purification Core, Boston, MA, USA.
Min
M
Wei
Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.
Joren C
JC
Madsen
Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA; MGH-DF/HCC Recombinant Protein Expression and Purification Core, Boston, MA, USA.
David H
DH
Sachs
Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA; MGH-DF/HCC Recombinant Protein Expression and Purification Core, Boston, MA, USA.
Christene A
CA
Huang
Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA; MGH-DF/HCC Recombinant Protein Expression and Purification Core, Boston, MA, USA. Electronic address: zhirui.wang@tbrc.mgh.harvard.edu.
Zhirui
Z
Wang
D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D015153:Blotting, Western; D049109:Cell Proliferation; D004167:Diphtheria Toxin / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D007376:Interleukin-2 / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D053645:Interleukin-2 Receptor alpha Subunit / Q000276:immunology* / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011485:Protein Binding / Q000276:immunology; D055503:Protein Multimerization / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D050378:T-Lymphocytes, Regulatory / Q000276:immunology* / Q000378:metabolism
D004167:Diphtheria Toxin; C508017:IL2RA protein, human; D007376:Interleukin-2; D053645:Interleukin-2 Receptor alpha Subunit; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C078456:denileukin diftitox
Diphtheria toxin; Fusion toxin; Human IL-2; Pichia pastoris expression; Regulatory T cell