Development of a diphtheria toxin-based recombinant porcine IL-2 fusion toxin for depleting porcine CD25+ cells.
Regulatory T cells (Tregs) have been widely recognized as crucial players in controlling immune responses. Because their major role is to ensure that the immune system is not over reactive, Tregs have been the focus of multiple research studies including those investigating transplantation tolerance, autoimmunity and cancer treatment. On their surface Tregs constitutively express CD25, a high affinity receptor for the cytokine interleukin-2 (IL-2). The reagents constructed in this study were generated by genetically linking porcine IL-2 to the truncated diphtheria toxin (DT390). This reagent functions by first binding to the cell surface via the porcine IL-2/porcine CD25 interaction then the DT390 domain facilitates internalization followed by inhibition of protein synthesis resulting in cell death. Four versions of the porcine IL-2 fusion toxin were designed in an interest to find the most effective isoform: 1) monovalent glycosylated porcine IL-2 fusion toxin (Gly); 2) monovalent non-N-glycosylated porcine IL-2 fusion toxin (NonGly); 3) bivalent glycosylated porcine IL-2 fusion toxin (Bi-Gly); 4) bivalent non-N-glycosylated porcine IL-2 fusion toxin (Bi-NonGly). Using a porcine CD25(+) B cell lymphoma cell line (LCL13271) in vitro analysis of the fusion toxins' ability to inhibit protein synthesis demonstrated that the Bi-NonGly fusion toxin is the most efficient reagent. These in vitro results are consistent with binding affinity as the Bi-NonGly fusion toxin binds strongest to CD25 on the same LCL13271 cells. The Bi-Gly fusion toxin significantly prolonged the survival (p=0.028) of tumor-bearing NOD/SCID IL-2 receptor γ(-/-) (NSG) mice injected with LCL13271 cells compared with untreated controls. This recombinant protein has great potential to function as a useful tool for in vivo depletion of porcine CD25(+) cells for studying immune regulation.
Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA; DF/HCC-MGH Recombinant Protein Expression and Purification Core, Boston, MA, USA.
Jaclyn Stromp
JS
Peraino
Marian
M
Schenk
Guoying
G
Li
Huiping
H
Zhang
Evan A
EA
Farkash
David H
DH
Sachs
Christene A
CA
Huang
Raimon
R
Duran-Struuck
Zhirui
Z
Wang
D000818:Animals; D004167:Diphtheria Toxin / Q000235:genetics / Q000494:pharmacology*; D006031:Glycosylation; D064593:Heterografts; D007376:Interleukin-2 / Q000235:genetics / Q000494:pharmacology*; D053645:Interleukin-2 Receptor alpha Subunit / Q000819:agonists* / Q000276:immunology; D008212:Lymphocyte Depletion / Q000379:methods*; D016393:Lymphoma, B-Cell / Q000188:drug therapy / Q000276:immunology / Q000473:pathology; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D014176:Protein Biosynthesis / Q000187:drug effects* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000494:pharmacology*; D013552:Swine; D023041:Xenograft Model Antitumor Assays / Q000379:methods
D004167:Diphtheria Toxin; D007376:Interleukin-2; D053645:Interleukin-2 Receptor alpha Subunit; D011993:Recombinant Fusion Proteins
Diphtheria toxin; Fusion toxin; Pichia pastoris expression; Porcine IL-2; Regulatory T cell