A candidate antigen for antibody mediated immunotherapy (serotherapy) of myeloma has recently been identified in our laboratory, namely a non-glycosylated (core protein) variant of Muc-1 (episialin, DF3 antigen, polymorphic epithelial mucin, epithelial membrane antigen). Muc-1 is a member of a family of high molecular weight (>300 kDa) glycoproteins. Highly glycosylated Muc-1 is normally expressed on the apical surfaces of glandular tissues. Muc-1 is also found on the cell surface of many adenocarcinomas including those of breast, ovarian and pancreatic origin in an aberrant form which has been attributed to incomplete glycosylation leading to an "unmasking" of antigenic determinants not seen in normal glandular tissue Muc-1 domains.

Suprisingly, Muc-1 which is normally found on epithelial cells, is also present on most myeloma cell lines, fresh patient myeloma cells and plasmacytomas and in certain other hematological malignancies. The function of Muc-1 is not well understood, but a role as an anti-adhesive factor has been suggested since glandular cells only express this antigen on their luminal surface. In an effort to characterize the expression of Muc-1 on myeloma cells, and on normal hematological cells, we recently employed a battery of four antibodies which identify glycosylated (DF3, 3C6) and core protein Muc-1 (DF3-P, 4H5) determinants by FACS analysis. These studies demonstrated that myeloma cell lines, and freshly obtained patient myeloma cells strongly express both glycosylated and core protein antigenic determinants of Muc-1. In contrast to myeloma cells, a survey of normal CD34+ stem cells, unstimulated B-cells and T-cells, failed to show either Muc-1 determinants. Also, in comparison to myeloma cells, Muc-1 bearing breast and ovarian cancer cell lines weakly expressed glycosylated Muc-1 determinants by both DF3, and 3C6 Abs) and more interestingly did not display on their cell surface Muc-1 core protein determinants. In contrast to our FACS analysis studies, western blot analyses performed by us and others using DF3, and DF3-P antibodies on cell lysates taken from both breast cancer and myeloma cells showed presence of both Muc-1 forms. Taken together, the above studies imply that myeloma cells strongly express on their cell surface both glycosylated and core protein Muc-1 antigenic determinants, the latter being seen only on myeloma cell lines.

In addition to the above work, we also studied the regulation of Muc-1 in myeloma and breast cancer cell lines. Consensus sequences for steroid response elements for the estrogen, progesterone and glucocorticoid receptors had previously been identified on the promoter of the Muc-1 gene. No functional analyses for these steroid response elements has so far been reported. As such we treated myeloma and breast cancer cell lines with agonists and antagonists for the estrogen, progesterone, and glucocorticoid receptors. A dramatic upregulation of Muc-1 expression (both glycosylated and core protein forms) was seen by FACS analysis on all myeloma cells exposed to dexamethasone which was maximal after 24 hours at a dose of 0.1 uM. In contrast, no change in Muc-1 expression was observed on breast cancer cell lines.

In these contemplated studies we hope to expand on our earlier work by demonstrating the feasibility of using Muc-1 targeted serotherapy for the treatment of multiple myeloma. We propose to study in vivo the anti-myeloma effects of administered unconjugated and radioimmunoconjugated monoclonal antibodies (DF3-P and 4H5) which target the core protein of Muc-1. In anticipation of using these monoclonal antibodies in two SCID mouse myeloma models, we will first characterize and select the most optimal of these antibodies by studying in vitro their ability to mount complement dependent (DCD) and antibody dependent cell mediated (ADCC) cytotoxicity, as well as perform antibody retention and endocytosis studies which are important to determining their usefulness as part of a radioimmunotherapy strategy. We will then treat human myeloma xenografted SCID mice with unconjugated and radioimmunoconjugated antibodies to Muc-1 core protein and determine tumor response and survival lengths. In addition, we will examine the benefit of pretreating myeloma xenografted SCID mice with dexamethasone prior to antibody administration, this since dexamethasone has been shown by us to dramatically upregulate Muc-1 expression. Hence, dexamethasone pretreatment might augment antibody capture by myeloma cells by increased display of core protein determinants, thereby enhancing tumor kill. It is hoped that these experiments will help us determine the feasibility of using either unconjugated or radioimmunoconjugated Muc-1 directed antibody immunotherapy for MM patients with the goal being sponsorship of a related clinical trial.
Research Progress Report
1998 Brian D. Novis Grant
Study of Muc-1 and Multiple Myeloma
Steven P. Treon M.D., Ph.D.
We have sought to identify proteins on multiple myeloma cells which can serve as targets for immunotherapy. One such target which we have identified is the Muc-1 core protein whose selective expression we have shown on myeloma plasma cells and B-cells. In addition, we have shown that expression of Muc-1 could be increased on myeloma cells by dexamethasone, a drug commonly used to treat myeloma patients. As part of these studies we have developed a preclinical model in mice to demonstrate the feasibility of targeting Muc-1 core protein by antibody mediated immuno-therapy (serotherapy). In these studies, mice bearing human myeloma cell tumors (plasmacytomas) were treated with either saline or dexamethasone prior to having their plasmacytomas removed and examined for the presence of Muc-1 core protein. These studies, which were performed by flow cytometric analysis, showed that Muc-1 core protein expression on myeloma cells could be increased with dexamethasone. Since MM cells tend to be radiation sensitive, our rationale was to exploit the above findings by devicing a radioimmunotherapy strategy for treating myeloma. As part of this work, we linked an antibody which binds to Muc-1 core protein with iodine-131, a potent radioactive molecule which can kill tumor cells. Such a strategy has been successfully employed in treating patients with B-cell lymphomas, a group of diseases similar to myeloma. Mice bearing human plasmacytomas were injected through their tail vein with this radioactive antibody (131-I-VU-4H5) and examined by a Siemens gamma camera which records emissions of radioactivity. These studies showed that the radioactive anti-Muc-1 antibody was taken up by the plasmacytomas, and that pretreatment of mice with dexamethasone increased the uptake of this radioactive antibody by the myeloma tumors. In ongoing work, we are attempting to increase the levels of radioactivity being delivered to these tumors, so as to effect tumor killing. Our anticipation is that these studies will lead to a clinical trial using serotherapy aimed at Muc-1 core protein in myeloma patients.