Disulfide bond bridged divalent antibody-toxin, (Fab-PE38fl)₂, with the toxin PE38 fused to the light chain

B3 antibody specifically binds the Lewis^Y-related carbohydrate antigen of many carcinomas, and it is used as a model antibody in this study. In a previous study, the Fab fragment of the antibody was fused to a 38 kDa truncated form of Pseudomonas exotoxin A, PE38, to make Fab-PE38, where PE38 is fused to the Fd fragment of the Fab domain. This parent monomer molecule, Fab-PE38, had no cysteine in the hinge region, and it could not make a disulfide bond to form a disulfide bond bridged homodimer [7]. In this study, we constructed three different kinds of divalent Fab-toxin fusion homodimers where the toxin is fused to the light chain of Fab, (Fab-PE38fl)₂. In addition to the PE38 toxin fused to the light chain, these three molecules have different hinge sequences h1, h2, and h3 making Fabh1-, Fabh2-, and Fabh3-PE38fl monomers, respectively. These hinges contain only one cysteine on different positions of the hinge sequence. The disulfide bond between the hinge region of two monomers forms homodimers (Fabh1-PE38fl)₂, (Fabh2-PE38fl)₂, and (Fabh3-PE38fl)₂. The refolding yields of these dimers were 5 - 16-fold higher than a previously constructed dimer where the PE38 was fused to the Fd fragment (Fabh1-PE38)₂ [8]. Our data suggest that the steric repulsion between the two PE38s in (Fabh1-PE38)₂ during disulfide bridge formation is relieved by fusing it at the end of the light chain. The best cytotoxicity value of these dimers showed about 2.5-fold higher on an MCF7 cell line than that of the monovalent reference molecule in ng/ml scale, which is 15-fold higher in pM scale.