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Bulletin of the Korean Chemical Society (BKCS)

ISSN 0253-2964(Print)
ISSN 1229-5949(Online)
Volume 22, Number 12
BKCSDE 22(12)
December 20, 2001 

 
Title
A Divalent Immunotoxin Formed by the Disulfide Bond between Hinge Regions of Fab Domain
Author
SeongHyeok Choi, JiEun Kim, YongChan Lee, Young-Ju Jang, Ira Pastan, MuHyeon Choe
Keywords
Divalent immunotoxin, [B3(FabH1)-PE38]2, B3 antibody, Pseudomonas exotoxin A, Antibody refolding.
Abstract
Recombinant immunotoxins are hybrid cytotoxic proteins designed to selectively kill cancer cells. A divalent immunotoxins, [B3(FabH1)-PE38]2, was constructed by recombining Fab domain of B3 antibody as a cell-targeting domain and Pseudomonas exotoxin A (PE) as a cytotoxic domain. Monoclonal antibody, B3, is the murine antibody (IgG1k) directed against Lewis Y-related carbohydrate antigens, which are abundant on the surface of many carcinomas. Fab fragment of this antibody was used in this study with the modified hinge sequence where last two cysteines out of three were mutated to serine. PE is a 66 kDa bacterial toxin that kills eukaryotic cells by inhibiting protein synthesis with ADP ribosylation of ribosomal elongation factor 2 (EF2). Fc region of B3 antibody was substituted with the truncated form of PE (38 kDa, PE38) on DNA level. [B3(FabH1)-PE38]2 was formed by disulfide bond between cysteines in the modified hinge region of B3(FabH1)-PE38. Each polypeptide for recombinant immunotoxins was overexpressed in Escherichia coli and collected as inclusion bodies. Each inclusion body was solubilized and refolded, and cytotoxic effects were measured. Divalent immunotoxins, [B3(FabH1)-PE38]2, had ID50 values of about 10 ng/mL on A431 cell lines and about 4 ng/mL on CRL1739 cell lines. Control immunotoxins, B3(scFv)-PE40, had ID50 values of about 28 ng/mL on A431 cell lines and about 41 ng/mL on CRL1739 cell lines. Divalent immunotoxins, [B3(FabH1)-PE38]2, had higher cytotoxic effects than B3(scFv)-PE40 control immunotoxins.
Page
1361 - 1365
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