The structural basis of affinity maturation in the anti-phenloxazolone antibodies system

Affinity maturation of antibodies occurs in the immune system of Vertebrates upon antigenic stimulation and leads to the production of more efficient antibodies. Detailed studies of the antibody response to a model antigen (the hapten 2-phenyl-l,3-oxazol-S-one, phOx) were carried out several years ago in C. Milstein's laboratory and allowed the definition of three different classes of anti-phOx antibodies with different affinity and kinetics profiles. However, only the crystal structure of one antibody, specifically the class III NQ10/12.S Fab fragment, has been determined so far. In this thesis, the cloning, expression and determination of the crystal structure of the scFvs of nine representative anti-ph Ox antibodies was attempted in order to clarify the structural basis of antibody affinity maturation. Out of these nine antibodies, the structure of the scFvs of the low and high affinity class III antibodies NQlO/1.12 and NQ16/113.8, complexed with the hapten, was determined by molecular replacement. They differ for nine amino acid substitutions, all located in the VH domain. The comparison of these two structures show how affinity maturation in this class is mainly based on an increase in surface complementarity, as a result of a finely tuned and highly concerted process chaperoned by the somatic mutations. Moreover, the comparison between these two class III scFv structures on one side, and the class II antibody Fab structure of NQlO/12.S on the other, shows that these two antibody classes respond in a completely different way to the architectural problem of binding phOx. The two novel structures presented in this thesis contribute to a better understanding of the antibody affinity maturation in the phOx system and suggest the importance of further research towards the structural solution of other anti-phOx antibodies.EThOS - Electronic Theses Online ServiceGBUnited Kingdo