Evolution Of Binding Agents

In the past decade, several different protein frameworks have been harnessed to derive novel binding agents for use in biotechnology and drug discovery (Figure 1.4). Antibodies represent by far the most abundant framework under development. Within antibodies, six hypervariable loops or complementarity determining regions (CDRs) define the combined site responsible for antigen recognition (Knappik et al. 2000;

FIGURE 1.4 Structures of scaffolds used for developing binding agents. Positions in the binding sites are shown as spheres, and the frameworks are shown as ribbons. (A) An antigen-binding fragment (Fab) of an antibody (PDB entry 1tzi) (Fellouse et al. 2004). (B) A designed ankyrin repeat protein (PDB 1svx) (Binz et al. 2004; Steiner et al. 2006). (C) A lipocalin scaffold based on a retinol binding protein (PDB 1lnm) (Schlehuber et al. 2000). (D) A fibronectin type III domain (PDB 2obg) (Koide et al. 2007). (E) An affibody based on the Z domain of protein A (PDB 2b87) (Lendel et al. 2006).

FIGURE 1.4 Structures of scaffolds used for developing binding agents. Positions in the binding sites are shown as spheres, and the frameworks are shown as ribbons. (A) An antigen-binding fragment (Fab) of an antibody (PDB entry 1tzi) (Fellouse et al. 2004). (B) A designed ankyrin repeat protein (PDB 1svx) (Binz et al. 2004; Steiner et al. 2006). (C) A lipocalin scaffold based on a retinol binding protein (PDB 1lnm) (Schlehuber et al. 2000). (D) A fibronectin type III domain (PDB 2obg) (Koide et al. 2007). (E) An affibody based on the Z domain of protein A (PDB 2b87) (Lendel et al. 2006).

Krebs et al. 2001; Hoet et al. 2005). These CDR loops, which are supported by a more conserved structural framework, provide the diversity responsible for the vast repertoire of binding molecules in the human immune system. Although bacterial expression is possible, antibodies are difficult to express in E. coli in their full-length form (Simmons et al. 2002). Instead, phage display relies on the expression of antibody fragments, such as single-chain variable fragments (scFv) or antigen-binding fragments (Fab) (Figure 1.4A) (Sheets et al. 1998; de Haard et al. 1999). Tight affinities in the low- to sub-nanomolar range have been selected from phage-displayed antibody fragment libraries without further affinity maturation (Rothlisberger et al. 2004; Sidhu and Fellouse 2006; Fellouse et al. 2007).

Despite the considerable success of phage display, the most common method for deriving antibodies is still animal immunization, which leads to the production of polyclonal antibodies directly from sera or monoclonal antibodies from cell lines derived by hybridoma methods (Winter and Milstein 1991). However, these popular technologies involve lengthy and expensive processes and also impose several limitations. For example, animal immunization does not allow for the selection of antibodies against molecules that are toxic to the host, and the vertebrate immune system might not produce high affinity antibodies against some antigens due to immuno-logical tolerance. In addition, the DNA encoding the antibodies is not obtained in a recombinant form, and thus it is not possible to identify the sequence of the antibody or to improve affinity, specificity, or stability. In contrast, phage display and other in vitro display methods (described in Chapters 2 and 3) do not suffer from these limitations, and thus they possess considerable advantages over animal immunization and hybridoma technologies.

Furthermore, in vitro display methods are not restricted to antibody repertoires, and many studies have investigated alternative scaffolds as substitutes for antibodies (described in detail in Chapter 5). In general, alternative scaffolds should have a stable protein core, which allows for the efficient formation of an antigen-binding site presented on the surface of the protein. As is the case for antibodies, the binding site must be tolerant to mutations and should not affect the overall folding of the protein. Additionally, these proteins should be well suited for high-level production in E coli. To date, several protein folds have been used as alternative scaffolds, and the most extensively studied include designed ankyrin repeat proteins (Figure 1.4B) (Binz et al. 2004), lipocalins (4C) (Beste et al. 1999), fibronectins (4D) (Koide and Koide 2007), and "affibodies" based on the Z domain of protein A (4E) (Lendel et al. 2006). In addition, other domains have also been used for more specialized applications, and these include kunitz domains (Ley et al. 1996), bovine pancreatic trypsin inhibitor (BPTI) (Roberts et al. 1992), and insect defensin (Dennis and Lazarus 1994). This list is far from complete, and we refer the interested reader to several recent reviews (Skerra 2000; Binz et al. 2005; Sidhu and Koide 2007; Skerra 2007).

Some alternative scaffolds (e.g., kunitz domains, BPTI, and insect defensin) are too small to form a substantial hydrophobic core and instead are stabilized mainly by disulfide bridges. These observations suggested that it might be possible to select small, structured binding peptides stabilized by disulfide bonds. In confirmation of this supposition, many studies have used random peptide libraries to select disul-fide-constrained binding peptides (Sidhu et al. 2000; Szardenings 2003; Mori 2004).

These peptides have been used to target a remarkably diverse array of targets, including cell-surface receptors, hormones, and enzymes, and structural analyses have revealed that the peptides usually form discrete, compact structures (Figure 1.5) (Dennis and Lazarus 1994; Livnah et al. 1996; Wiesmann et al. 1998; Eigenbrot et al. 2001; Deshayes et al. 2002; Schaffer et al. 2003). Although the affinities of these binding peptides are typically lower than those of antibodies, peptide ligands can be readily synthesized and may be useful for specialized applications (Lowman 1997; Schaffer et al. 2003; Uchiyama et al. 2005).

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