AFFINITY ENGINEERING OF ANTIPOLYSACCHARIDE MONOCLONAL ANTIBODY

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Many pathogenic bacteria are surrounded by an outer polysaccharide layer referred to as the capsule. The capsule plays a role in both virulence of the bacteria, helping them evade the non-specific immune response, and in host defense, as anti-capsular antibody responses are involved with clearance and protection. Using bacterial levan as a model polysaccharide, we are studying how directed changes in antibody protein sequence can be used to affect avidity increases in anti-polysaccaride mAb. These avidity engineered antibodies are being produced by site directed mutagenesis of antibody expression constructs and subsequent expression and antibody production in an appropriate cell substrate. Mu and Kappa chain cDNAs of an anti-levan monoclonal have been produced and cloned into a CMV promotor based expression vector. These expression vectors have been mutated in the VH region to affect a N->D and a N->H change at position 53. These expression vectors are currently being transfected into a Kappa chain only producing hybdridoma cell substrate for production of the mutated mAb. The engineered mAb will be used to study how individual amino acid changes affect avidity and specificity. These predicted changes are also being modeled using the "Look" software provided by the Center for Molecular Modeling, DCRT, NIH on a high powered Silicon Graphics workstation. The effects of single mutations on the conformation of the antibody combining site will be determined.