Structure and application of single-chain Fvs as diagnostic and therapeutic agents

The genetic engineering revolution promises to deliver a new range of antibody molecules, each specifically designed for a select purpose (reviewed by Winter and Milstein, 1991). For medical applications, the hope lies in the production of novel diagnostic and therapeutic reagents with properties surpassing the current range of specificity and affinity provided by monoclonal antibodies against a variety of antigens and haptens. Fab and Fv fragments can be used to replace monoclonal antibodies in situations where monovalent binding specificity is required. The advantages of producing small antibody fragments such as Fab and Fv molecules by genetic engineering technology are summarized in Table 1 . The unique features required for diagnostic reagents relate more to the binding affinity and target specificity, whereas for therapeutic applications the requirement is to mimic natural human antibodies to avoid the unwanted anti-idiotype immune reactions. Expression systems have been established recently for genetically engineered antibodies, utilizing the now trusted vectors in bacteria, yeast and mammalian cells. The expression rules hold as true for antibodies as for many secreted proteins which comprise compact domain structures; bacteria are the most cost-effective and efficient system for small non-glycosylated proteins (such as Fab or Fv fragments) and eukaryotic systems for larger proteins such as whole antibodies (as discussed in Chapter 8). Fab and Fv fragments offer a number of advantages over whole antibodies for diagnostics and therapeutics, especially in cases where it is important to minimize immune cross-reactions. Fvs are the smallest molecular unit which comprises the variable heavy (V_H) and variable light (V_L) domains and thereby retain the binding affinity of the parent antibody. The small Fv fragments carry the minimum amount of additional protein and minimize immune cross-reactions. This is especially important in whole blood clinical diagnosis where cross-reactions to mouse antibodies require the addition of blocking antibody to mask the anti-mouse reaction. Small fragments also offer advantages in tissue penetration and fast clearance rates, which are ideal features for imaging reagents.