Fine mapping of the epitopes of humanized anti-L-selectin monoclonal antibodies HuDREG-55 and HuDREG-200.

Blocking the function of L-selectin with a monoclonal antibody (mAb) is a promising way to prevent neutrophils from causing tissue damage during inflammation. HuDREG-55 and HuDREG-200 are humanized mAb which bind to human L-selectin and block its function as an adhesion molecule. To understand the mechanism of the action of HuDREG-55 and HuDREG-200, we determined their epitopes on L-selectin at the amino acid level. The analysis of human E- and L-selectin chimeric proteins demonstrated that the lectin domain of L-selectin is necessary for the binding of HuDREG-55 and HuDREG-200. Mutational analysis of Escherichia coli-expressed L-selectin showed that HuDREG-55 binding is sensitive to amino acid changes at positions 11, 56, 87, 89, 105, 107 and 111 (counting from the amino-terminus of mature L-selectin) while HuDREG-200 binding is sensitive to amino acid changes at 45, 46 and 47. Both epitopes are located close to the predicted carbohydrate binding site, indicating that HuDREG-55 and HuDREG-200 block the function of L-selectin by directly inhibiting the binding to carbohydrate ligands.