Abstract
BackgroundLewis Y (Ley) is a blood group-related carbohydrate that is expressed at high surface densities on the majority of epithelial carcinomas and is a promising target for antibody-based immunotherapy. A humanized Ley-specific antibody (hu3S193) has shown encouraging safety, pharmacokinetic and tumor-targeting properties in recently completed Phase I clinical trials.Methodology/Principal FindingsWe report the three-dimensional structures for both the free (unliganded) and bound (Ley tetrasaccharide) hu3S193 Fab from the same crystal grown in the presence of divalent zinc ions. There is no evidence of significant conformational changes occurring in either the Ley carbohydrate antigen or the hu3S193 binding site, which suggests a rigid fit binding mechanism. In the crystal, the hu3S193 Fab molecules are coordinated at their protein-protein interface by two zinc ions and in solution aggregation of Fab can be initiated by zinc, but not magnesium ions. Dynamic light scattering revealed that zinc ions could initiate a sharp transition from hu3S193 Fab monomers to large multimeric aggregates in solution.Conclusions/SignificanceZinc ions can mediate interactions between hu3S193 Fab in crystals and in solution. Whether metallic ion mediated aggregation of antibody occurs in vivo is not known, but the present results suggest that similar clustering mechanisms could occur when hu3S193 binds to Ley on cells, particularly given the high surface densities of antigen on the target tumor cells.
Highlights
Recent studies of the normal biological functions of the Lewis Y (Ley or CD174) carbohydrate antigen have revealed new insights into its role in cellular function [1,2,3]
Whether metallic ion mediated aggregation of antibody occurs in vivo is not known, but the present results suggest that similar clustering mechanisms could occur when hu3S193 binds to Ley on cells, given the high surface densities of antigen on the target tumor cells
Defective or malformed sperm were shown to display Ley on the plasma membrane. Given that both Lex and Ley have been shown to interact with human dendritic cells via DC-SIGN to induce T-cell tolerance [16], these oligosaccharides may play a role in the immune privilege of the male reproductive tract [3]
Summary
Recent studies of the normal biological functions of the Lewis Y (Ley or CD174) carbohydrate antigen have revealed new insights into its role in cellular function [1,2,3]. This type 2 histo-blood group related carbohydrate antigen is expressed at high surface densities on 60% to 90% of carcinomas of the breast, ovary, colon, lung and prostate [4,5,6]. Lewis Y (Ley) is a blood group-related carbohydrate that is expressed at high surface densities on the majority of epithelial carcinomas and is a promising target for antibody-based immunotherapy. A humanized Ley-specific antibody (hu3S193) has shown encouraging safety, pharmacokinetic and tumor-targeting properties in recently completed Phase I clinical trials
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