Development of a Diphtheria Toxin Based Antiporcine CD3 Recombinant Immunotoxin

Abstract

Anti-CD3 immunotoxins, which induce profound but transient T cell depletion in vivo by inhibiting eukaryotic protein synthesis in CD3+ cells, are effective reagents in large animal models of transplantation tolerance and autoimmune disease therapy. A diphtheria toxin based anti-porcine CD3 recombinant immunotoxin was constructed by fusing the truncated diphtheria toxin DT390 with two identical tandem single chain variable fragments (scFv) derived from the anti-porcine CD3 monoclonal antibody 898H2-6-15. The recombinant immunotoxin was expressed in a diphtheria-toxin resistant yeast Pichia pastoris strain under the control of the alcohol oxidase promoter. The secreted recombinant immunotoxin was purified sequentially with hydrophobic interaction chromatography (Butyl 650 M) followed by strong anion exchange (Poros 50 HQ). The purified anti-porcine CD3 immunotoxin was tested in vivo in four animals; peripheral blood CD3+ T cell numbers were reduced by 80% and lymph node T cells decreased from 74% CD3+ cells pretreatment to 24% CD3+ cells remaining in the lymph node following 4 days of immunotoxin treatment. No clinical toxicity was observed in any of the experimental swine. We anticipate that this conjugate will provide an important tool for in vivo depletion of T cells in swine transplantation models.

Enhanced De Novo Alloantibody and Antibody-Mediated Injury in Rhesus Macaques

“…First, it reproduces biological phenomena associated with depleting agents in humans, namely sustained peripheral T-cell depletion, homeostatic repopulation of memory T cells, and a high incidence of donor-specific antibody production. Secondly, the FN18-CRM9 immunotoxin previously used in our lab exhibited a high incidence of donor-specific antibodies (73%), but the drug was replaced with a new recombinant IT to overcome the production/yield difficulty, linkage heterogeneity, and potential immunogenicity of its chemically-conjugated predecessor (58–60), making it unavailable for testing of B cell therapeutics. Lastly, alemtuzumab would be clinically most relevant, but hemolytic complications prohibited its use in our rhesus macaques, as most old world monkeys ubiquitously expressed alemtuzumab’s target molecule CD52, including on erythrocytes (61).…”

Lymphodepletional Strategies in Transplantation

“…We ventured to investigate the activation of the humoral response and eventual chronic rejection in the FN19-CRM9 immunotoxin-treated animals, but the drug was replaced with a new recombinant immunotoxin (A-dmDT390-scfbDb[C207]) because of production yield difficulties and potential immunogenicity of the former chemically conjugated construct (Ma et al 1997;Kim et al 2007;Wang et al 2011). Although the use of the new immunotoxin provided profound peripheral T-cell depletion, the rapid proliferation of memory T cells prevented sustained graft survival.…”

Recipient-matching of Passenger Leukocytes Prolongs Survival of Donor Lung Allografts in Miniature Swine

“…Mobilized hematopoietic stem cells were collected from peripheral blood by apheresis over 3 days, starting after the fifth dose of pIL-3/pSCF. Stem cell recipients underwent a reduced intensity conditioning regimen, consisting of 100 cGy total body irradiation on day −2, partial T cell depletion with recombinant CD3-immunotoxin (pCD3-DT390, 50 μg/kg IV) twice daily from day −4 to −1 (8), and a 45-day course of cyclosporine A (target trough 400–800 ng/mL day 0–30 then taper to discontinuation). Unmodified cytokine-mobilized peripheral blood mononuclear cells were transplanted over 2 or 3 days following conditioning (days 0–2) as required to achieve the target dose of 15 × 10 9 cells/kg.…”