The general aim of this project is to study the effects of immunotoxin induced transient T-cell depletion on the modulation of T cells from immunization responses towards tolerizing responses, and to use this knowledge for the experimental and clinical treatment of T cell driven autoimmune diseases, graft-versus-host disease, and the induction of tolerance to mismatched organ and cell transplants. We previously showed that a 2-3 day course of anti-rhesus CD3 immunotoxin constructed with CRM9, a binding site mutant of diphtheria toxin, depletes lymph node and blood T cells by 99% after a 48 hour lag period. This process was associated with a marked prolongation of survival of mismatched functioning rhesus kidney transplants, 50% over 9 months without further immunosuppressive therapy. Rejections, were preceded by the appearance of anti-graft antibodies (done with S. Knechtle and J. Thomas). To circumvent this adverse T cell mediated B cell response that is apparently initiated during the delay in immunotoxin induced T cell killing, we have added a short course of agents that are believed to block antigen presentation such as deoxyspergualin (DSG). Out of 5 bilaterally rephrectomized recipients long term tolerance was achieved in 3 without further immunosuppresion as judged by a graft survival of 4 years or longer. Congeneic pancreatic islet cell transplants in monkeys suffering from spontaneous insulin dependent diabetes have been shown to reverse diabetes as judged by returning non-fasting blood glucose and glycosylated hemoglobin values to normal in the absence of exogenous insulin. A short course of immunotoxin, methyl prednisolone and cyclosporine provide induction of stable operational tolerance to islets that has lasted over one year in one case. This is the first reported reversal of diabetes using a tolerizing protocol that does not depend on chronic immunosuppression, a process previously shown to compromise transplanted islet function (done with J. Thomas). Streptozotocin induced diabetes in monkeys has been successfully reversed by using anti-T cell immunotoxin in combination with 14 days of DSG and allografted pancreatic islets (done with J. Thomas). Three out of seven are long term tolerant survivors (6-7 years). A recombinant divalent anti-human anti-T cell immunotoxin has been developed that has 20-fold the potency of the previous chemical conjugates. This is expressed in Pichia pastoris. This material has now been produced for a phase I clinical trial in T cell leukemia and an INDA has been filed. In order to aid the production of immunotoxin and other bioactive proteins in Pichia pastoris, new strains have been constructed that exhibit toxin resistance and increased heterologous protein production in the presence of an active unfolded-protein response. ? Recently, a recombinant anti-CD3 immunotoxin active against monkey T cells has been developed. This was done in order to facilitate pre-clinical studies in organ transplantation and autoimmune diseases. A side benefit of this study was that it required explorations of different recombinant antibody geometries for optimal activity. A fold-back diabody structure was found to be most optimal toward monkey CD3. Model building studies predicted that this structure should have very high binding activity towards targets that contain homodimeric epitopes rotated by 180 degrees. This prediction was found to be true in the case of a human solid tumor epitope and clinical studies at another institution are planned.

National Institute of Health (NIH)
National Institute of Mental Health (NIMH)
Intramural Research (Z01)
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Wang, Zhirui; Kim, Geun-Bae; Woo, Jung-Hee et al. (2007) Improvement of a recombinant anti-monkey anti-CD3 diphtheria toxin based immunotoxin by yeast display affinity maturation of the scFv. Bioconjug Chem 18:947-55
Kim, Geun-Bae; Wang, Zhirui; Liu, Yuan Yi et al. (2007) A fold-back single-chain diabody format enhances the bioactivity of an anti-monkey CD3 recombinant diphtheria toxin-based immunotoxin. Protein Eng Des Sel 20:425-32
Liu, Yuan Yi; Wang, Zhirui; Thomas, Judith et al. (2007) Polymorphisms of CD3epsilon in cynomolgus and rhesus monkeys and their relevance to anti-CD3 antibodies and immunotoxins. Immunol Cell Biol 85:357-62
Woo, Jung Hee; Liu, Yuan Yi; Neville Jr, David M (2006) Minimization of aggregation of secreted bivalent anti-human T cell immunotoxin in Pichia pastoris bioreactor culture by optimizing culture conditions for protein secretion. J Biotechnol 121:75-85
Wang, Z; Mathias, A; Stavrou, S et al. (2005) A new yeast display vector permitting free scFv amino termini can augment ligand binding affinities. Protein Eng Des Sel 18:337-43
Hettiaratchy, Shehan; Melendy, Elizabeth; Randolph, Mark A et al. (2004) Tolerance to composite tissue allografts across a major histocompatibility barrier in miniature swine. Transplantation 77:514-21
Wang, Zhirui; Neville Jr, David M (2004) Expression and characterization of recombinant soluble monkey CD3 molecules: mapping the FN18 polymorphic epitope. Mol Immunol 40:1179-88
Woo, Jung Hee; Liu, Yuan Yi; Stavrou, Scott et al. (2004) Increasing secretion of a bivalent anti-T-cell immunotoxin by Pichia pastoris. Appl Environ Microbiol 70:3370-6
Cho, Patricia S; Mueller, Nicolas J; Cameron, Andrew M et al. (2004) Risk factors for the development of post-transplant lymphoproliferative disorder in a large animal model. Am J Transplant 4:1274-82
Contreras, Juan L; Jenkins, Stacie; Eckhoff, Devin E et al. (2003) Stable alpha- and beta-islet cell function after tolerance induction to pancreatic islet allografts in diabetic primates. Am J Transplant 3:128-38

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