STRUCTURAL REQUIREMENTS OF ANTIBODY COMBINING SITES

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The long term objective of this study is to attain an understanding of how the amino acid sequence of antibody variable regions leads to antibody specificity and affinity for antigen, and to idiotypic expression. Oligonucleotide-directed mutagenesis of antibody genes will be used to introduce mutations in the complementarity determining regions (CDRS) of a mouse antibody specific for p-azophenylarsonate (Ars). Mutant antibodies will be expressed following transfection of the mutagenized genes into myeloma or hybridoma cell lines, and transfectomas will be recovered either in liquid microcultures or in soft agarose. Secreted antibodies, in supernatants or in situ, will be screened by standard immunological assays. The hypothesis that antibody affinity is particularly sensitive to changes at certain positions will be tested by introducing all possible substitutions at each of four positions that have been found to affect affinity of the anti-Ars antibody. Minimal requirements for improved antibody affinity and for changed specificity will be determined by introducing one or a few random mutations at a time in the CDRs of the anti-Ars antibody, and screening for mutants with increased affinity for Ars or with new antigen specificities. To probe the roles of each of the six CDRs of the anti-Ars antibody in antigen binding and idiotypic expression, the CDRs will be individually "blanked out" by replacement with glycine residues. The reconstruction of an anti-Ars combining site will then be attempted by introducing into such glycine-replaced CDRS, the amino acid residues that have been implicated in Ars contact. The glycine-replaced CDRs will also be randomly mutagenized at low frequency, to obtain antibodies specific for Ars or for other antigens. Insights gained from the proposed studies should further our understanding of protein folding in general and of antibody complementarity in particular, and should bring us a step closer to the long term objective of tailoring antibodies for research and medical purposes.