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Development of a Diphtheria Toxin Based Antiporcine CD3 Recombinant Immunotoxin

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Zhirui Wang*†‡, Raimon Duran-Struuck†, Rebecca Crepeau†, Abraham Matar†, Isabel Hanekamp†, Srimathi Srinivasan†, David M. Neville, Jr.§, David H. Sachs†‡, and Christene A. Huang†‡

Transplantation Biology Research Center, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, United States

DF/HCC-MGH Recombinant Protein Expression and Purification Core, Boston, Massachusetts, United States

Angimmune LLC, 9624 Parkwood Drive, Bethesda, Maryland 20814, United States

Bioconjugate Chem., 2011, 22 (10), pp 2014–2020

DOI: 10.1021/bc200230h

Publication Date (Web): August 25, 2011

Zhirui Wang, Ph.D., Transplantation Biology Research Center, Massachusetts General Hospital/Harvard Medical School MGH-East, Building 149-6113 13th Street, Boston, MA 02129, USA. Phone: +1-617-643-1957. Fax: +1-617-726-4067. E-mail: zhirui.wang@tbrc.mgh.harvard.edu.

Section:

Pharmacology

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 antiporcine CD3 recombinant immunotoxin was constructed by fusing the truncated diphtheria toxin DT390 with two identical tandem single chain variable fragments (scFv) derived from the antiporcine 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 antiporcine 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.

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