Sitedirected Mutagenesis

Site-directed mutagenesis facilitates changes at specific sites or domains within the antibody genes, such as in specific CDR loops or framework regions, allowing the design of new molecules in a 'rational' strategy. It involves the use of oligonucleotides to incorporate either specific or random sequence changes at defined sites. One example is the random (saturation) mutagenesis to the CDR loops followed by phage display and selection (Barbas et al., 1992, 1994). In cases where the three-dimensional structures of the protein molecules have been determined by crystallography, an improved interface between molecules could be designed, or domains grafted from one molecule to another.

One model system for the evaluation of the potential applications of this technology has been the antibodies with specificity for influenza neuraminidase. The binding surfaces of two native antibodies (NC10 and NC41; Malby et al., 1994) and the recombinant scFv NC10 (Malby et al., 1993; Kortt et al., 1994) have been resolved by X-ray crystallography, making this an ideal system. Mutants have been designed in order to fill the cavities at the planar interface of the antibody-antigen combining site, or to modify the salt-bridge interactions. The antibody fragment coding regions were then transferred to a high-level expression vector such as pPOW (Power et al., 1992) in order to produce sufficient amounts of the proteins for further structural analysis and immunodiagnostic and therapeutic applications.

Sharon (1990) used site-directed mutation of three amino acids in the heavy chain to increase the affinity of a low-affinity monoclonal antibody to the hapten p-azophenylarsonate. The changes were based on a high-affinity monoclonal antibody to the hapten. Of the 19 amino-acid residues that differed between the two antibodies, only three were necessary and sufficient to bring about a 200-fold increase to produce equivalent affinities. Two mutations were in CDR2 and the other in CDR3. This illustrates how antigen-driven affinity maturation, in this case under in vivo conditions, provides the information to allow the design of specific mutations to produce a higher affinity antibody.

Barbas et al. (1994) have shown that directed sequential site-directed mutagenesis in the CDR3 region of a heavy chain resulted in 3 amino acid changes, and increased the affinity 1000-fold. However, single-chain molecules with high initial affinities (such as when taken from hybridomas) can be intransigent to affinity maturation in the laboratory (Hawkins et al., 1992).

0 0

Post a comment