Immune maturation is viewed as a Darwinian process that involves selection of the structures with best fit. The long term objective of this project is to study the molecular basis of antigen recognition and its contribution as a selective force in shaping the antibody response to phosphocholine-protein PC-KLH conjugates. The evolution of antibodies to PC-KLH will be analyzed in three families of clonally related hybridomas isolated during the memory antibody response. These families are representative of group II antibodies which have a strong requirement for the phenyl structure that links PC to the carrier protein. Each of these families uses a distinct V gene pair to form the active site. The interaction of members of these families with antigen will be studied at the molecular and atomic levels to determine the structural evolution of their antibody combining sites during maturation of the response. We shall also evaluate the concept of B cell """"""""wastage"""""""" during the antibody somatic diversification process by studying the ability of random mutations in the VH CDR2 to lead to antigen binding loss mutations in hybridomas producing anti-PC-KLH antibodies. We have observed that mutations in the VH CDR2 of certain antibodies leads to loss of antibody secretion. We shall study the molecular basis for this effect by studying the intracellular assembly and trafficking of immunoglobulin molecules bearing particular mutations. The proposed studies make use of biochemical and immunochemical methods to study the function of antibodies modified through the use of site directed and saturation mutagenesis. The mutant molecules will be examined by computer assisted molecular modeling and the interactions of antibody fragments with antigen will be examined using nuclear magnetic resonance (NMR). These studies will provide important information on the structure of antibody combining sites that will be important in redesigning antibodies to improve their function and may lead to new understanding of diseases where antibody secretion may be impaired.
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