Structural Basis of Affinity Maturation of Antibodies in the 2- Phenyl-5-Oxazolone System

Background: Affinity maturation of antibodies is the process whereby more efficient antibodies are produced through somatic hypermutation and antigen-guided selection. No extensive study is available at the moment concerning the relationship between somatic mutations and their structural counterpart. The antibody response to 2-phenyl-5-oxazolone has been thoroughly investigated from the genetic point of view. It consists of three antibody classes, with each member of each class derived from a unique pair of VH and VL germline genes by somatic hypermutation. In this project, we are investigating the structure of the VH and VL domains of 10 representative antibodies. Methods: The VH and VL domains of each antibody are being expressed as recombinant scFvs, crystallised, and their structure determined by X-ray crystallography. Results: The structures and models available allow an initial definition of the strategies adopted. In class I, maturation is bound to improvement of surface complementarity, especially at the top of the binding pocket, and in surface charge changes. In class II the maturation strategy seems to be based on the increase of the interacting surface, and on the introduction of a specific bond with the oxazolone ring. In class III, where the low and high affinity antibodies differ by 8 mutations, the increase in affinity is mainly determined by the improvement in the surface complementarity by removal of a bulky phenylalanine, which allows a better tightening of the two sides of the binding site. Conclusions: These results are relevant to determine the principles underlying affinity maturation of antibodies.