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The high binding specificity and affinity of antibody molecules is a biological wonder. Because of these properties they have been used as prophylactic, diagnostic, and analytical reagents for almost a century now. Their use as "magic bullets" for targeted therapy was conceived by Ehrlich in the late 19th century. With the advent first of the hybridoma technology (1) and then of recombinant DNA technology dawned the era of antibody-based therapies. Not long after the beginning of this new field of medicine, the hurdles associated with the pharmakokinetic properties of antibodies became clear. It was realized that the large size of the antibody molecules precluded their proper penetration into masses of biological tissues such as solid tumors (2). It was also realized that additional effector functions can be hooked on to antibody molecules. There was the urge to widen the spectrum of effector functions of antibodies by substituting the constant domains with enzymes (to make prodrug), toxins (immunotoxins) and radionucleides (3). These requirements led to the development of the recombinant Fab and the Fv technology. The Fv technology greatly aided in solving not only the problems associated with the large size of the antibody but also in reducing the immunogenecity of mouse monoclonal antibodies (MAbs) in humans. However, it had its own drawbacks, one of which was the loss of stability of many Fvs as compared to the corresponding IgGs (4). This is a serious drawback in view of the fact that for a molecule to be therapeutically successful it has to be stable at 37°C. It is therefore conceivable that some Fvs that have the potential for therapeutic use because of their antigen binding specificity can face the severe limitations arising out of their inherent instability. It is generally believed that the hydrophobic interaction between the VH and VL strongly contribute to the stability of a Fv. But in many Fvs this interaction is not strong enough. Because of this Fvs are modified into scFvs where a flexible peptide linker, typically a (Gly4Ser)3 repeat is used to hold the two V domains together (5). With the application of the scFv technology to many different Fvs, it was realized that although the linker was effective in covalently holding the two V-domains together, it was not always effective in keeping a scFv active. In other words the chains could still unfold and lead to aggregation. This had led to the hypothesis that increasing the interaction between the two V-domains may be important to enhancing the stability of a biologically active Fv (being capable of remaining as a monomer and bind antigen). Several approaches trying to increase the stability of Fvs have been described. One of the earlier approaches involved engineering disulphide bonds into the framework regions of Fvs such that they are covalently held together (6). Other technologies involve either decreasing hydrophobic patches on scFvs to minimize aggregation (7,8) and to increase the strength of non-covalent interaction between the V domains (8). This involves identifying the potentially "problematic" residues that either reduce the interdomain interaction and/or causes aggregation. This chapter is meant to help the reader in identifying the potentially problematic residues and in making a judgement call on substitution(s) that is (are) likely to stabilize the Fv. The discussion that follows and the strategy that is presented is largely based on information that was gathered from the Kabat database (Kabat database maintained by Johnson and Wu, http://immuno.bme.nwu.edu).… [ABSTRACT FROM AUTHOR]

The potential of antibodies as magic bullets for targeting therapeutic drugs or imaging agents has been well-documented and is now an emerging area of molecular medicine. Because antibodies act first by binding to their specific antigens it is easy to envisage that the specific activity of antibody-based drugs depends to a large extent on the antigen-binding affinity of antibodies. For a variety of reasons under many circumstances instead of whole antibodies, smaller antigen-binding units of antibodies such as Fabs and Fvs are better suited to perform the job of the magic bullet. The conversion of whole antibodies into Fabs and Fvs (scFvs or dsFvs) is often associated with a drastic reduction in the antigen-binding affinity. It is therefore pertinent that for efficient use of Fvs as targeting agents their binding affinities need to be improved. Until the recent past there had been two different approaches to improve binding affinity of antibodies (whole IgGs, Fabs, and Fvs). One approach relied on the high-resolution crystal structure of antibody-antigen complex followed by engineering of key contact residues in the Fv portion to enhance the interaction. Although this approach is very logical and has greater chances of success, it is not readily feasible. The other approach that has been used to improve affinities of antibodies involved mutating the CDRs randomly or semirandomly and create large expression libraries (for example, scFv or Fab phagedisplay libraries), which served as a potential source of high-affinity variants. This approach has been used successfully several times and in fact has become the method of choice to improve antibody affinity. However, it requires the construction, maintenance, and handling of large and/or multiple phage-display libraries. These tasks require technical expertise and at times they can be time-consuming and expensive. [ABSTRACT FROM AUTHOR]

For over a century now, antibodies have proven to be extremely useful reagents in biomedical research. They are also being tried as therapeutic agents for a number of intractable diseases. Their uses include identification and cloning of new genes from expression libraries, purification and structure-function analysis of proteins, histochemical localization of proteins, as diagnostic markers and/or probes, as targeting agents to deliver drugs and as magic bullets to bind and kill cells specifically among a long list of other uses to which they have been put. [ABSTRACT FROM AUTHOR]

The potential of Fvs as magic bullets for targeting therapeutic drugs or imaging agents is well-documented. However, the conversion of whole antibodies (Abs) into Fvs (scFvs or dsFvs) is often associated with a drastic reduction in antigen (Ag)-binding affinity. For efficient use of Fvs as targeting agents, this property must be improved. [ABSTRACT FROM AUTHOR]