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Mutational Analysis of Avidity and Fine Specificity of Anti-Levan Antibodies

Division of Monoclonal Antibodies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892

Using the polyfructose, bacterial levan, as a model polysaccharide, we analyzed how V regions affect binding in anti-polysaccharide mAbs. Previously, panels of mAb were constructed from bacterial levan-immunized BALB/c and CBA/Ca mice. The BALB/c mAb were mostly germline V_HJ606:V11, and a subset contained presumed somatic mutations in the complementarity-determining regions (CDRs) that correlated with increases in avidity for the ß(21) inulin linkage of levan. The CBA/Ca mAb were more heterogeneous in V gene usage, but a subset of inulin-nonreactive mAb were V_HJ606:V and had V_H sequence differences in the CDRs from the V_HJ606 regions of the BALB/c mAb. In this report, V_HJ606 Abs containing various combinations of specifically mutated H and L chains were produced by engineered transfectants and tested for inulin avidity and levan binding. Two presumed somatic mutations seen in CDRs of the BALB/c hybridomas were shown to directly cause marked increases in avidity for inulin (V_H N53H, 9-fold; V_L N53I, 20-fold; together, 46-fold) but not for ß(26) levan. Exchange of either positions 50 or 53 in V_H or the H3 loop between the BALB/c and CBA/Ca mAb resulted in either fine specificity shift or total loss of bacterial levan binding. Three-dimensional models of the V regions suggested that residues that affect binding to inulin alone are near the edge of the CDR surface, while residues involved with binding both forms of levan and affecting fine specificity are in the V_H:V_L junctional area.

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