A Binding Domain on Mesothelin for CA125/MUC16

Ovarian cancer and malignant mesothelioma frequently express both mesothelin and CA125 (also known as MUC16) at high levels on the cell surface. The interaction between mesothelin and CA125 may facilitate the implantation and peritoneal spread of tumors by cell adhesion, whereas the detailed nature of this interaction is still unknown. Here, we used truncated mutagenesis and alanine replacement techniques to identify a binding site on mesothelin for CA125. We examined the molecular interaction by Western blot overlay assays and further quantitatively analyzed by enzyme-linked immunosorbent assay. We also evaluated the binding on cancer cells by flow cytometry. We identified the region (296–359) consisting of 64 amino acids at the N-terminal of cell surface mesothelin as the minimum fragment for complete binding activity to CA125. We found that substitution of tyrosine 318 with an alanine abolished CA125 binding. Replacement of tryptophan 321 and glutamic acid 324 with alanine could partially decrease binding to CA125, whereas mutation of histidine 354 had no effect. These results indicate that a conformation-sensitive structure of the region (296–359) is required and sufficient for the binding of mesothelin to CA125. In addition, we have shown that a single chain monoclonal antibody (SS1) recognizes this CA125-binding domain and blocks the mesothelin-CA125 interaction on cancer cells. The identified CA125-binding domain significantly inhibits cancer cell adhesion and merits evaluation as a new therapeutic agent for preventing or treating peritoneal malignant tumors.Ovarian cancer largely is confined to the peritoneal cavity for much of its natural history (1). Peritoneal mesothelioma is a highly invasive tumor originating from the mesothelial linings of the peritoneum (2). The development of effective drug regimens against ovarian cancer and mesothelioma has proven extremely difficult.Mesothelin was first identified in 1992 by the monoclonal antibody (mAb)² K1 that was generated by the immunization of mice with human ovarian carcinoma (OVCAR-3) cells (3). The mesothelin gene encodes a 71-kDa precursor protein that is processed to a 40-kDa protein termed mesothelin, which is a glycosylphosphatidylinositol (GPI)-anchored glycoprotein present on the cell surface (4). Mesothelin is a differentiation antigen that is present on a restricted set of normal adult tissues such as the mesothelium. In contrast, it is overexpressed in a variety of cancers including mesothelioma, ovarian cancer, and pancreatic cancer (5). In addition, mesothelin is also expressed on the surface of non-small cell lung cancer cells (6, 7), especially most lung adenocarcinomas (8).We and others have shown that mesothelin is shed from tumor cells (9, 10), and antibodies specific for mesothelin are elevated in the sera of patients with mesothelioma and ovarian cancer (11). Shed serum mesothelin has been approved by the United States Food and Drug Administration (FDA) as a new diagnostic biomarker in mesothelioma. In a Phase I clinical study of an intrapleural interferon-β gene transfer using an adenoviral vector in patients with mesotheliomas, we found that antitumor immune responses targeting mesothelin were elicited in several patients (12). A recent study indicated that anti-mesothelin antibodies and circulating mesothelin relate to the clinical state in ovarian cancer patients (13). Pastan and colleagues (14) developed an immunotoxin (SS1P) with a Fv for mesothelin. Two Phase I clinical trials were completed at the National Cancer Institute (National Institutes of Health, Bethesda, MD) and there was sufficient antitumor activity of SS1P to justify a Phase II trial. A chimeric antibody containing the mouse SS1 Fv for mesothelin was also developed and is currently examined in a Phase I clinical trial for ovarian cancer, mesothelioma, pancreatic cancer, and non-small cell lung cancer (15).Mucins are heavily glycosylated proteins found in the mucus layer or at the cell surface of many epitheliums (16). There are two structurally distinct families of mucins, secreted and membrane-bound forms. CA125 (also known as MUC16) was first identified in 1981 by OC125, a mAb that had been developed from mice immunized with human ovarian cancer cells (17). The first cDNA clones were reported in 2001 (18, 19). CA125 is a very large membrane-bound cell surface mucin, with an average molecular mass between 2.5 and 5 million daltons. It is also heavily glycosylated with both O-linked and N-linked oligosaccharides (20). The peptide backbone of CA125 is composed of the N-terminal region, extensive Ser/Thr/Pro-rich tandem repeats (TR) with 156 amino acids each with both N- and O-glycosylations, a SEA domain with high levels of O-glycosylation and a C-terminal region with a short cytoplasmic tail (19). The SEA domain was first identified as a module commonly found in sea urchin sperm protein, enterokinase and agrin (21, 22). The significance of the SEA domain in CA125 is not clear.CA125 was originally used as a biomarker in ovarian cancer due to its high expression in ovarian carcinomas and that it is shed into the serum (23). A majority (88%) of mesotheliomas are also CA125 positive on the cell membrane (24). It was shown that 25% of peritoneal mesotheliomas have high CA125 expression (25). The intensity of CA125 membranous expression is indistinguishable between ovarian carcinomas and peritoneal mesotheliomas. Gene expression analysis using the SAGE tag data base has shown that mesothelioma has the second highest co-expression of CA125 and mesothelin after ovarian cancer (26). Rump and colleagues (26) have shown that mesothelin binds to CA125 and that this interaction may mediate cell adhesion. Scholler et al. (27) recently showed that CA125/mesothelin-dependent cell attachment could be blocked with anti-CA125 antibodies. Because mesothelin is present on peritoneal mesothelium, there may be an important role for the mesothelin-CA125 interaction in the tumorigenesis of ovarian cancer and mesothelioma in the peritoneal cavity. The mesothelin binding site on CA125 may lie within the 156-amino acid TR units, indicating multimeric binding of mesothelin to CA125. It has been found that the extraordinarily abundant N-glycans on CA125, presumably in the TR region, are required for binding to both glycosylated and non-glycosylated mesothelin (28).Here, we identified the binding site of CA125 on mesothelin by use of truncated mutagenesis and alanine replacement approaches. We measured binding qualitatively by Western blot overlay assays and quantitatively by enzyme-linked immunosorbent assay (ELISA). We also evaluated the interaction of CA125 and mesothelin on cancer cells by flow cytometry. Furthermore, we have shown that a single chain mAb (SS1) recognized the CA125-binding domain and blocked the mesothelin-CA125 interaction on cancer cells. The identified CA125-binding domain-Fc fusion protein also significantly inhibited cancer cell adhesion. Our results suggest that conformation-sensitive structures of the region (296–359) are required and sufficient for specific binding of mesothelin to CA125. The domain proteins or the antibodies that block the mesothelin-CA125 interaction merit evaluation as new therapeutic agents in treating peritoneal malignant tumors.