The Y54(L)W mutation of anti-leukotriene C4 single-chain antibody increases affinity to leukotriene E4

The X-ray crystal structure of an anti-leukotriene (LT) C₄ monoclonal antibody (mAbLTC) in complex with LTC₄ was determined, however, crystallographic studies alone are not enough to fully understand the structures of the antigen-binding site. To elucidate the individual contribution of Tyr-54 and Asn-58 in the light chain of mAbLTC, both of which formed a hydrogen bond with glutamic acid of LTC₄, we examined whether substitution of the residues affects the antigen binding affinity and specificity using an anti-LTC₄ single chain variable fragment (scFvLTC). Among the Tyr-54(L) mutants, Y54(L)W showed a dramatic increase in the affinity to LTE₄ which was comparable to that to LTD₄ Essentially the same results were obtained using the Y54(L)W mutant expressed in Escherichia coli and Pichia pastoris. The structural modeling suggested the formation of a novel hydrogen bond between the substituted tryptophan in the antibody and the cysteine residue in LTE₄ The affinity of Y54(L)R, Y54(L)E and Y54(L)L to LTC₄ was markedly reduced, whereas other tested Tyr-54(L) mutants as well as Asn-58(L) mutants did not show significant change in LT binding. The results may provide an insight into the molecular basis of specific LT recognition by the antibody.