Systematic Exploration of the Antigen Binding Activity of Synthetic Peptides Isolated from the Variable Regions of Immunoglobulins

Abstract

Sets of short (12 residues) cellulose-bound synthetic overlapping peptides derived from the sequences of the variable regions of the heavy and light chains of three different antibodies (an anti-thyroglobulin antibody, the HyHEL-5 anti-lysozyme antibody, and an anti-angiotensin II antibody) were used to systematically assess the antigen binding capacity of peptides from the antibody paratope outside their natural molecular context. Peptides enclosing one or several of the complementarity determining region (CDR) residues had antigen binding activity, although the most active peptides were not necessarily those bearing the greatest number of CDR residues. Several residues from the framework region, preceding or following the CDR, were found to play a role in binding. Affinity constants from 4.1 ؋ 10 ؊7 to 6.7 ؋ 10 ؊8 M ؊1 for the soluble form of 9 lysozymebinding dodecapeptides were measured by BIAcore analysis. Alanine scanning of lysozyme-binding hexapeptides from the HyHEL-5 sequence identified 38 residues important for binding, of which 22 corresponded to residues that had been shown by x-ray crystallography to be at the interface between HyHEL-5 and lysozyme. Our results could be of interest for the rational identification of biologically active peptides derived from antibody sequences and in providing an experimental basis for mutagenesis of the antibody paratope.Antibody molecules bind antigens with high affinity and specificity by synergistically using multiple noncovalent forces. The combining site (paratope), whose shape is complementary to the epitope on the antigen, is made up of the hypervariable regions, also called complementarity determining regions (CDRs) 1 (1). It is commonly accepted that there are three CDRs in the light chain (L1, L2, and L3) and in the heavy chain (H1, H2, and H3). These CDRs fold into turn structures that are stabilized by the ␤-sheet framework of the variable domains. The interface between antibodies and antigens has been precisely described by x-ray crystallographic studies, and several complexes between Fab fragments of monoclonal antibodies and peptide or protein antigens have been recently described (for reviews see Refs. 2-4). The structures of antibody-antigen complexes indicate that at least four of the CDRs, and in some cases all six CDRs, contribute to antigen binding (5). Residues in the framework have rarely been reported to participate in this interaction (6, 7).Antibody-peptide or antibody-protein complexes are excellent model systems to study the physicochemical requirements for molecular recognition. Unfortunately, it is a difficult task to obtain crystals suitable for the structural elucidation of antibody fragments in complex with proteins or peptides. Therefore, other approaches to obtain information about the key residues involved in the interaction would be very useful, in particular for paratope mutagenesis. Some workers have demonstrated that synthetic peptides derived from the amino acid sequences of CDRs bind antigens with specificities similar to t...

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