DIABODIES AND BISPECIFIC ANTIBODY CONSTRUCTS
A variety of engineered antibody constructs are presently being investigated. A diabody consists of two single-chain Fv (scFv) fragments assembled together. Shortening the linker from 15 amino acids to five drives dimerization of two scFv chains. This no longer allows intrachain assembly of the linked VH and VL regions. The dimer consists of two scFv fragments arranged in a crisscross manner (Fig. 6.13). The resulting diabody has two antigen binding sites pointing in opposite directions. If two different scFv fragments are used, the result is a bispecific diabody that will bind to two different target proteins simultaneously. Note that formation of such a bispecific diabody requires that VH-A be linked to VL-B and VH-B to VL-A. It is of course possible to engineer both sets of VH and VL regions onto a single polypeptide chain encoded by a single recombinant gene, as shown in Fig. 6.13. Bispecific diabodies have a variety of potential uses in therapy, because they may be used to bring together any two other molecules; for example, they might be used to target toxins to cancer cells.
Another way to construct an engineered bispecific antibody is to connect the two different scFv fragments to other proteins that bind together (Fig. 6.14). Two popular choices are streptavidin and leucine zippers. Streptavidin is a small biotin binding protein from the bacterium Streptococcus. It forms tetramers, so it allows up to four antibody fragments to be assembled together. Furthermore, binding to a biotin column can purify the final constructs. Leucine zipper regions are used by many transcription factors that form dimers. Often, such proteins form mixed dimers when their leucine zippers recognize each other and bind together. Leucine zipper regions from two different transcription factors that associate (e.g., the Fos and Jun proteins) may therefore be used to assemble two different scFv fragments.
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