Immune responses in certain inbred mouse strains are dominated by antibodies which share common variable (V) region structures (idiotypes) detected serologically by anti-idiotypic reagents. Such idiotypic determinants characterizing certain antibody specificities are useful structural and genetic markers in studies of antibody diversity and regulation. The predominant cross reactive idiotype (Id-CR) in A/J mice immunized with rho-azophenylarsonate (Ars)-protein conjugates is heritable and encoded by a single combination of germline gene segments (""""""""canonical""""""""). Although Jerne proposed that interactions between Id and anti-Id regulate immune responses through recognition of Id determinants, evidence in the Ars system has accumulated that Id dominance may be due to antigen-driven selection of favorable somatic mutants with higher affinity derived from preferred (more """"""""adaptable"""""""") V region germline gene combinations. The Ars system is a model for examining regulation by defining the antibody structural changes involved in enhanced antigen affinity occurring temporally during the immune response (""""""""affinity maturation""""""""). Based upon the high resolution X-ray crystal structure of a somatically mutated Ars-binding Id-CR bearing antibody (36-71) and the germline tertiary structure deduced therefrom, we can now examine the detailed structural correlates of Ars binding and idiotypy, as related to somatic mutation and gene junctional variation. We will capitalize on methodologic advances in protein engineering using site-specific mutagenesis and expression, in concert with predictive computer modelling and the crystal structures to: 1) relate somatic mutation and gene junctional diversity to fine structure of the hapten binding site geometry using comparisons of the tertiary structures of germline and mutated antibodies. 2) Engineer novel mutations designed to increase affinity. 3) Examine sites of hypermutation found in vivo to assess whether or not they relate to affinity enhancement or altered idiotopes. 4) Engineer new binding specificities to Ars homologues as a measure of the differentiative capacity of a canonical V region structure. 5) Map idiotopes by mutagenesis and measurement of reactivity with monoclonal anti-Id reagents, and by determination of the crystal structures of Id-anti-Id. The use of antibody engineering to further understand and thus modify antigen-antibody complementarity is necessary to the design of antibodies for therapeutic use in targeting to drugs, toxins, hormones, and cellular receptors.
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