Expanding the conformational diversity by random insertions to CDRH2 results in improved anti-estradiol antibodies

The length of the heavy chain complementarity-determining region 2 (CDRH2) was extended beyond what is found in germline genes to improve the binding properties of an anti-estradiol antibody. The previous immunochemical chara cterization and the molecular modeling of the high affinity (K-a = 3.9 x 10 (8)) murine anti-estradiol antibody 57-2 suggested that a part of the antig en was loosely recognized by the antibody. The CDRH2, because of its close location but scarce contacts with the hapten, was considered as a conceivab le target for mutagenesis. Libraries with either two, three or four random amino acid insertions in the tip of the CDRH2 loop were constructed and dis played on the M13 filamentous phage as Fab fragments. Mutations were introd uced also into the rest of the V-H domain by error-prone polymerase chain r eaction to allow the surrounding structures to adapt to the extended CDRH2. After the panning of the libraries with an antigen off-rate-based selectio n, a number of active clones, most of which showed significantly improved a ffinity and specificity, were isolated, characterized and sequenced. The re sults indicate that the structure of the antibody can tolerate a number of different insertions in the CDRH2 region. They also suggest that the repert oire of antibody libraries can be expanded by extending the length of the C DR loops beyond that naturally provided by the given set of germline genes. This kind of mutagenesis can be generally useful for the engineering of ha pten-binding antibodies. (C) 1999 Academic Press.