A central core structure in an antibody variable domain determines antigenspecificity

Antibody binding sites provide an adaptable surface capable of interacting with essentially any molecular target. Using CDR shuffling, residues import ant for the assembly of mucin-1 specific paratopes were defined by random r ecombination of the complementarity determining regions derived from a set of mucin-1 specific clones, previously selected from an antibody fragment l ibrary. It was found that positions 33 and 50 in the heavy chain and 32, 34 , 90, 91 and 96 in the light chain were conserved in many of the clones. Th ese particular residues seem to be located centrally in the binding site as indicated by a structure model analysis. The importance of several of thes e conserved residues was supported by their presence in a mouse monoclonal antibody with a known structure and the same epitope specificity. Several o f these corresponding residues in the mouse monoclonal antibody are known t o interact with the antigen. In conclusion, critical residues important for maintaining a human antigen-specific binding site during the process of in vitro antibody evolution were defined. Furthermore, an explanation for the observed restricted germline gene usage in certain antibody responses agai nst protein epitopes is provided.