The immune system creates binding sites of high specificity and affinity in the variable domains of antibodies by generating sequence and consequently structural diversity in the complementarity-determining region (CDR) loops, which are located at the N-terminal ends of these domains. Sequence variations in the CDR loops of an antibody generally do not have a significant influence on the overall structure of the variable domain that carries them. This feature of variable domains is actually observed in a more general sense in immunoglobulin-like domains, which are known to have a similar general shape in the core beta-barrel and high structural variability in the loops. Furthermore, overall sequence similarity of domains with an immunoglobulin fold is mainly below 25%, while their structural similarity is high, with a root mean square (rms) deviation of Cα atoms always below 3.9 Å (Halaby et al., 1999).

We therefore set out to explore whether this inherent stability and conservation of the immunoglobulin fold allows loops of immunoglobulin domains other than the CDR loops to accommodate sequence variation without negatively impacting the overall structure and stability of the protein. As shown in Figure 17.1, the candidate loops of an IgG1 for this kind of engineering are manifold, including the N- and C-terminal loops of the constant domains as well as C-terminal loops of the variable domains.

In the examples described in this chapter, we engineered the AB and the EF loops of the third constant domain (CH3) of human IgG1 by randomizing a number of residues and also by inserting random sequences in the loops, thereby generating new binding sites.