AFFINITY MATURATION OF ANTIBODIES in vivo AND in vitro. Cara Marks¹, Kim Henric², Greg Winter³, Ray Stevens⁴, Thomas Simon⁵, and James D. Marks⁶. Struct. Biol. Div., Lawrence Berkeley Laboratory, Univ. of Calif., Berkeley¹, M.R.C.-Mill Hill, U.K², L.M.B., Cambridge, U.K³. Dept. of Chem., Univ. of Calif.,Berkeley⁴. E.L.I.A.S.,Freiburg,Germany⁵, Dept. of Pharm. Chem., Univ. of Calif., San Francisco⁶.

During the secondary and tertiary immune response, the humoral immune system produces high affinity antibodies by mutating the variable region genes (V) of lower affinity antibodies (Ab). Structural analysis of low and high affinity antibodies indicates these mutations predominantly yield changes in residues that do not contact antigen. Indeed, in vitro mutagenesis of contact residues to increase antibody affinity has been largely unsuccessful. To analyze how changes in non- contact residues could result in increased affinity, we determined the atomic structures of an antibody that binds a large protein antigen and a second antibody that recognizes a small hapten. In both Abs, the somatic mutations were located in residues that do not tend antigen but rather contribute indirectly to the rigidity of the antigen binding site. Examination of the 2.0Å structure of 48G7(Fab) -para-nitrophenyl phosphonate complex indicates that none of the nine somatic mutations that contribute to a 10⁴ increase in affinity contact this transition state analog. Similarly, the 2.0Å structure of FvRS1, a D1.3 variant which binds hen egg lysozyme with 8 fold higher affinity indicates that none of the somatic mutations or the mutations generated in vitro contact the protein antigen. Comparison of additional high affinity antibodies to models of their germline antibodies also reveal an antibody combining site composed of a well packed, easily perturbed network of residues that is stabilized by distal somatic mutations. Low frequency random mutagenesis of residues in this combining site is likely to perturb the synergy, and accounts for the largely unsuccesful in vitro results. In contrast, an increase in affinity could be obtained by drastically altering the binding site by simultaneous mutation of multiple residues coupled to a powerful selection scheme.