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Thioether-Bonded Constructs of Fab' and Fc Modules Utilizing Differential Reduction of Interchain Disulfide Bonds¹

Kwok S. Kan^*, Valerie A. Anderson, Weng S. Leong, Andrew M. Smith, Andrew T. Worth and George T. Stevenson^2,

^* Biological Sciences, University of North London, London, United Kingdom; Tenovus Research Laboratory, Southampton University Hospitals, Southampton, United Kingdom; and The Edward Jenner Institute for Vaccine, Compton, United Kingdom

We describe a two-stage preparation of chemically engineered Ab constructs, employing as modules Fab' from mAb or rAb, and Fc from human normal IgG1. A multivalent, optionally multispecific F(ab')_n core is formed in stage one, and one or more Fc modules added in stage two. Examples include bispecific Fab₂Fc₂ (for simplicity, primes and Greek letters are omitted from names of final constructs) and trivalent Fab₃Fc₂, which are designed to kill neoplastic cells. An essential element in the construction is the availability of the Fab' in two reduced forms, Fab'(-sulfhydryl (SH))₅ and Fab'-SH. The first is obtained by full reduction of the interchain disulfide bonds (SS) in the F(ab')₂ fragment of IgG. Fab'-SH is obtained by disulfide-interchange reactions on Fab'(-SH)₅, whereby the -light SS is reconstituted, an unusual intrachain SS forms in the -chain hinge, and one hinge SH remains. F(ab')₂ and F(ab')₃ cores are built using partially reduced modules, being given intermodular thioether links that resist reduction. These cores are then fully reduced, making available SH groups for addition of the Fc modules. In the final constructs, all intermodular links embody tandem thioether bonds arising at hinge-region cysteines. Cytotoxic activities of representative constructs, and some enhancements deriving from multiple modules, are assessed. In guinea pigs, catabolism of Fab₂Fc₂ yielded a t_1/2 similar to that of human IgG1, although the serum Fab₂Fc₂ revealed some proteolytic breakdown not shown by the IgG1. Immunotherapy of a guinea-pig leukemia confirmed the ability of these constructs to kill target cells in vivo.

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