Design and application of diabodies, triabodies and tetrabodies for cancer targeting.

Multivalent recombinant antibody fragments provide high binding avidity and unique specificity to a wide range of target antigens and haptens. This review describes the design and expression of diabodies, triabodies and tetrabodies using examples of scFv molecules that target viruses (influenza neuraminidase) and cancer (Ep-CAM; epithelial cell adhesion molecule). We discuss the preferred choice of linker length between V-domains to direct the formation of either diabodies (60 kDa), triabodies (90 kDa) or tetrabodies (120 kDa), each with size, flexibility and valency suited to different applications for in vivo imaging and therapy. The increased binding valency of these scFv multimers results in high avidity (low off-rates). A particular advantage for tumour targeting is that molecules of 60-100 kDa have increased tumour penetration and fast clearance rates compared to the parent Ig (150 kDa). We highlight a number of cancer-targeting scFv multimers that have recently successfully undergone pre-clinical trials for in vivo stability and efficacy. We also review the design of multi-specific Fv modules suited to cross-link two or more different target antigens. These bi- and tri-specific multimers can be formed by association of different scFv molecules and, in the first examples, have been designed as cross-linking reagents for T-cell recruitment into tumours (immunotherapy), viral retargeting (gene therapy) and as red blood cell agglutination reagents (immunodiagnostics).