Journal Article

Improved binding of a bivalent single-chain immunotoxin results in increased efficacy for in vivo T-cell depletion

Jerry Thompson, Scott Stavrou, Marla Weetall, J.Mark Hexham, Mary Ellen Digan, Zhuri Wang, Jung Hee Woo, Yongjun Yu, Askale Mathias, Yuan Yi Liu, Shenglin Ma, Irina Gordienko, Philip Lake and David M. Neville

in Protein Engineering, Design and Selection

Volume 14, issue 12, pages 1035-1041
Published in print December 2001 | ISSN: 1741-0126
Published online December 2001 | e-ISSN: 1741-0134 | DOI: https://dx.doi.org/10.1093/protein/14.12.1035
Improved binding of a bivalent single-chain immunotoxin results in increased efficacy for in vivo T-cell depletion

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Anti-CD3 immunotoxins exhibit considerable promise for the induction of transplantation tolerance in pre-clinical large animal models. Recently an anti-human anti-CD3ε single-chain immunotoxin based on truncated diphtheria toxin has been described that can be expressed in CHO cells that have been mutated to diphtheria toxin resistance. After the two toxin glycosylation sites were removed, the bioactivity of the expressed immunotoxin was nearly equal to that of the chemically conjugated immunotoxin. This immunotoxin, A-dmDT390-sFv, contains diphtheria toxin to residue 390 at the N-terminus followed by VL and VH domains of antibody UCHT1 linked by a (G4S)3 spacer (sFv). Surprisingly, we now report that this immunotoxin is severely compromised in its binding affinity toward CD3+ cells as compared with the intact parental UCHT1 antibody, the UCHT1 Fab fragment or the engineered UCHT1 sFv domain alone. Binding was increased 7-fold by adding an additional identical sFv domain to the immunotoxin generating a divalent construct, A-dmDT390-bisFv (G4S). In vitro potency increased 10-fold over the chemically conjugated immunotoxin, UCHT1–CRM9 and the monovalent A-dmDT390-sFv. The in vivo potency of the genetically engineered immunotoxins was assayed in the transgenic heterozygote mouse, tgε 600, in which the T-cells express human CD3ε as well as murine CD3ε. T-cell depletion in the spleen and lymph node observed with the divalent construct was increased 9- and 34-fold, respectively, compared with the monovalent construct. The additional sFv domain appears partially to compensate for steric hindrance of immunotoxin binding due to the large N-terminal toxin domain.

Keywords: CD3; depletion; divalent; immunotoxin; T cell; transplantation

Journal Article.  5696 words.  Illustrated.

Subjects: Proteins

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