Comparative immunological studies of tumor-associated Lewis X, Lewis Y, and KH-1 antigens

Tumor-associated carbohydrate antigens Lewis X (Le^x), Lewis Y (Le^y), and KH-1 are useful targets for cancer immunotherapy. In this regard, an insight into the structure-immunogenicity relationships of these antigens is important but this has not been systematically investigated yet. In the current study, Le^x, Le^y, and KH-1 antigens with a lactose unit at the reducing end as a spacer were synthesized and coupled with keyhole limpet hemocyanin (KLH) protein. Immunological evaluations of the resultant conjugates revealed that they all could elicit robust immune responses whilst the Le^y conjugate could provoke the highest titers of total and IgG antibodies. The binding assays of their antisera to each antigen and to cancer cells showed that each antiserum had extensive cross-reaction with all three antigens as protein conjugates and strong but somewhat antigen-selective binding towards MCF-7 cancer cell. Moreover, none of these antisera had obvious binding to SKMEL-28 cancer cell that does not express Le^x, Le^y and KH-1. The results of assays of these antisera to mediate complement-dependent cytotoxicity (CDC) to MCF-7 and SKMEL-28 cancer cells were very similar to the results of binding assays. Thus, it was concluded that all three antigens could form effective conjugate vaccines whereas the Le^y conjugate induced the most robust immune responses and the antiserum of Le^x had the highest binding and cytotoxicity to target cancer cells. In addition, as the antibodies induced by each antigen had extensive cross-reaction with other two antigens, either Le^x or Le^y or the two combined can be utilized to formulate effective conjugate vaccines for cancer immunotherapy. Another paradigm-shifting discovery of this study is that the presentation of Le^x, Le^y, and KH-1 antigens on cancer cell can be different from that in synthetic conjugates, which should be taken into consideration during the design and optimization of related cancer vaccines or immunotherapies.