Immunization with MUC1/X protein enhances cDNA immunization in generating anti-MUC1 alpha/beta junction antibodies that target cancer cells

Abstract

10072 Background: MUC1, a glycoprotein highly expressed in epithelial malignancies including breast, prostate, and ovarian, and on the malignant cells of multiple myeloma has generated considerable interest as a tumor marker and target for tumor killing. The most intensively studied MUC1 protein is a type I transmembrane protein (MUC1/TM) which is proteolytically cleaved soon after synthesis into α and β subunits which bind in a strong non-covalent interaction. Almost all antibodies generated to date against MUC1 recognize epitopes within the highly immunogenic tandem-repeat-array. A major shortcoming in use of such antibodies is the fact that the tandem-repeat-array-containing part of MUC1 is shed from the cell surface into the circulation. Soluble, shed MUC1 sequesters circulating anti-tandem-repeat-array antibodies, limiting their ability to reach targeted MUC1-expressing cells. Antibodies to MUC1 epitopes tethered to the cell surface would likely be more effective therapeutic agents. Despite efforts in recent years, such antibodies have remained elusive; generation of anti-cell antibodies requires characterization of cell-bound epitopes. The junction of the MUC1 α-subunit binding the membrane-tethered β-subunit provides such an epitope. Methods: By use of a novel protocol, entailing immunization with MUC1/TM cDNA and boosting with MUC1/X protein, a MUC1 isoform lacking the tandem-repeat-array, we generated monoclonal antibodies designated DMC209 which recognize the MUC1 α/β junction. Results: DMC209 is exquisitely unique for the target site; all amino acid mutations which abrogate MUC1 cleavage also abrogate DMC209 binding. Additionally, DMC209 binds the MUC1 α/β junction on cell-tethered tandem-repeat-array-containing MUC1 (MUC1/TM) on breast and ovarian cancer, and on myeloma cells. Conclusion: DMC209 is likely to have clinical application by targeting MUC1-expressing cells directly, and as an immunotoxin conjugate. Moreover, the novel high-titer immunization procedure used in generating DMC209 can be used to generate anti-MUC1 α/β junction antibodies acting as ligand and, analogously to herceptin, may have direct cytotoxic activity. No significant financial relationships to disclose.

Targeting cell-bound MUC1 on myelomonocytic, monocytic leukemias and phenotypically defined leukemic stem cells with anti-SEA module antibodies

“…To that end, a limited number of putative tumor antigens have been described on the AML cell surface that might potentially serve as targets for antibody therapy, including CD33 and CD123 [1,2]. MUC1, a polymorphic type I high-molecular-weight glycoprotein, is a heterodimer composed of extracellular, transmembrane, and cytoplasmic domains that is proteolytically cleaved into two subunits, a and b subunits, which bind to each other noncovalently (Figure 1) [7]. Although the b-subunit remains bound to the cell surface, the a-subunit undergoes an "on-and-off" mechanism whereby it sometimes binds the cell surface b-subunit and at times freely disassociates from the cell, such that, in vivo, it is shed into the peripheral circulation.…”

“…MUC1 cell surface expression on AML cells was determined with two antibodies: HMFG-1, which recognizes the tandem repeat of the MUC1 a-chain [10,11], and DMB5F3, which targets the SEA domain formed by the interaction of the a-subunit with the extracellular portion of the b-subunit (Figure 1) [7]. A murine mammary tumor cell line, DA3-PAR, and its counterpart, DA3-TM, stably transfected with and expressing human MUC1 DNA served as negative and positive controls, respectively (Figure 2A).…”

“…In contrast, the MUC1 SEA domain (a highly conserved protein module so called from its initial identification in a sea urchin sperm protein, in enterokinase, and in agrin) represents a stable structure fixed to the cell surface at all times. With this in mind, we generated anti-MUC1 antibodies directed against the a/b junctions [7,8] and found that they bound avidly to cell surface MUC1. Furthermore, a partially humanized anti-MUC1 SEA domain antibody linked to ZZ-Pseudomonas exotoxin toxin was found to be cytotoxic to MUC1-expressing solid tumor cells [8].…”