The precise molecular interactions that occur between antigen and antibody in the active site determine whether a particular B cell will be selected to participate in an immune response. The nature of these interactions is the focus of this study using as a model system, monoclonal antibodies with a characteristic fine specificity that distinguishes between nitrophenyl phosphocholine (NPPC) and phosphocholine (PC); only NPPC blocks the binding of such antibodies to PC-protein. We call such antibodies Group II to distinguish them from T15-related anti-PC antibodies that bind both NPPC and PC (called Group I). Many """"""""natural' antibodies in nonimmunized animals, including man, are poly- and autoreactive. The polyreactivity of these molecules suggests that they may not obey the same molecular rules of antigen recognition as conventional antigen-induced antibodies which are not polyreactive; however, this is conjectural since few structural data are available regarding such antibodies. We propose to compare the binding sites of monoclonal poly/autoreactive antibodies that have the Group II fine specificity to antigen-induced Group II antibodies that are not polyreactive. Preliminary evidence derived from pairs of polyreactive and monoreactive Group II antibodies that use the same VH-VL gene combinations with a high degree of amino acid homology (ave 92%) suggests that VH CDR3 is an important distinguishing feature. We propose to test this hypothesis. We propose to test related hypotheses regarding the contributions of various regions of VH and VL to binding in conventional Group II antibodies and to examine the possible impact of these regions on polyreactive Group II antibodies. We propose to use site directed mutagenesis and chain recombination in transfectants to assess the contribution of H and L chains, and in particular the VH CDR3 of polyreactive antibodies. Through collaboration with an NMR spectroscopist we propose to compare the interactions of various ligands with the antibody active sites of conventional and polyreactive Group II antibodies allowing characterization in the solution state. We shall use computer assisted interactive molecular modeling to compare active site interactions in both types of antibodies and to guide molecular replacements to be achieved through directed mutation. Lastly we shall test the relative abilities of VH and VL in this system to undergo somatic mutation.
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