A unique epitope of cancer cell-derived immunoglobulins recognized by RP215 monoclonal antibody

4452 The molecular nature of cancer-associated antigen, CA215 which reacts with RP215 monoclonal antibody and its unique epitope(s) were characterized. RP 215 was initially selected and produced from one of 3,000 hybridomas which were generated from mice immunized with OC-3-VGH ovarian cancer cells. This cancer-associated antigen from various sources including cancer cell extract, shed cell culture medium and affinity-purified forms were analyzed by MALDI-TOF MS, Western Blot, carbohydrate profiling, enzyme immunoassays (EIA), as well as immunohistochemical studies of the microarray of breast cancer tissues and tissue sections of several other human cancers. The results of this study clearly showed that CA215 is homologous to the heavy chains of human immunoglobulins (Ig9s) with molecular sizes on SDS-PAGE ranging from 50 to 70 KDa, when probed with RP215 or anti-human Ig9s (G, A or M). Treatments of cancer cells with NaIO4 drastically reduce RP215 binding to the carbohydrate-associated epitope(s) of CA 215 located on the Fab or variable domain of the heavy chains of human Ig9s. Further studies indicated that CA 215 is expressed by cancer cells in both secretory and membrane-bound forms, and its carbohydrate-associated epitope(s) appears to be present only in cancer cell-derived Ig9s, but not found in normal human Ig9s. Compared to normal Ig9s, CA215 contains a significantly higher percentage of N-acetyl and N-glycol neuraminic acid (21% vs. 8%) in the O-linked carbohydrate moiety, but a lower content of N-acetyl glucosamine (27% vs. 45%) in the N-linked ones. Tissue microarray study revealed that greater than 60% (22/36) of breast cancer tissues were positively stained, comparable to those from other human cancer tissue sections including endometrium, cervix, ovary, lung, stomach, intestine and esophagus (20 to 100%, n=51). These observations were consistent with the elevated serum levels of CA215 among different cancer patients when evaluated by EIA. It was concluded from this study that RP215 monoclonal antibody reacts specifically with carbohydrate-associated epitope(s) of the heavy chains of Ig9s expressed by a variety of human cancer cells. Besides broad diagnostic applications as pan cancer marker, the membrane-bound CA215 on the cancer cell surface may also be the ideal target for therapeutic treatments of various human cancers with humanized RP215 in the future.