MUC1 has generated considerable interest as a tumor marker and potential target for tumor killing. To date, most antibodies against MUC1 recognize epitopes within the highly immunogenic A chain tandem repeat array. A major shortcoming of such antibodies is that the MUC1 A chain is shed into the peripheral circulation, sequesters circulating antitandem repeat array antibodies, and limits their ability to even reach targeted MUC1-expressing cells. Antibodies recognizing MUC1 epitopes tethered to the cell surface would likely be more effective. MUC1 A subunit binding the membranetethered B subunit provides such an epitope. By use of a novel protocol entailing immunization with cDNA encoding fulllength MUC1 (MUC1/TM) followed by boosting with the alternatively spliced MUC1/X isoform from which the tandem repeat array has been deleted, we generated monoclonal antibodies, designated DMC209, which specifically bind the MUC1 A/B junction. DMC209 is exquisitely unique for this site; amino acid mutations, which abrogate MUC1 cleavage, also abrogate DMC209 binding. Additionally, DMC209 specifically binds the MUC1 A/B junction on full-length MUC1/TM expressed by breast and ovarian cancer cell lines and on freshly obtained, unmanipulated MUC1-positive malignant plasma cells of multiple myeloma. DMC209 is likely to have clinical application by targeting MUC1-expressing cells directly and as an immunotoxin conjugate. Moreover, the novel immunization procedure used in generating DMC209 can be used to generate additional anti-MUC1 A/B junction antibodies, which may, analogously to Herceptin, have cytotoxic activity. Lastly, sequential immunization with MUC1/TM cDNA acting as a nonspecific adjuvant followed by protein of interest may prove to be a generalizable method to yield high-titer specific antibodies. (Cancer Res 2006; 66(23): 11247-53)
MUC1, a heavily glycosylated mucin, has generated considerable interest as a target for tumor killing because of its overexpression in malignancies. Full-length MUC1 (MUC1/TM) is proteolytically cleaved after synthesis generating a and b subunits, which specifically bind in a noncovalent interaction. Although the b chain remains on the cell surface, the a chain binds in an on-and-off interaction. Most anti-MUC1 antibodies (Abs) described to date recognize epitopes within the highly immunogenic a-chain tandem repeat. Because the a-chain is shed, such Abs are sequestered and fail to reach MUC1-expressing cells. Immunizing with cDNA encoding MUC1/TM and the spliced MUC1/X isoform from which the tandem repeat has been deleted yielded antibodies to the MUC1 a/b junction. Pseudomonas toxin PE38 linked to polyclonal anti-MUC1 a/b junction Abs both bound and killed MUC1-positive malignant cells. Monoclonal DMC209 binds the MUC1 a/b junction in both MUC1/X and MUC1/TM. When injected into SCID mice xenotransplanted with human breast cancer MDA-MB-231, monoclonal DMC209 showed significant in vivo tumorsuppressive activity. The MUC1/X a/b junction presents a biologically-significant target in MUC1-expressing malignancies because (i) antibodies directed against cell-bound a/b junction epitopes reach the intended cellular target, (ii) antibodies to junction epitope are internalized into cells, (iii) anti a/b junction antibodies can effectively kill high MUC1-expressing cancer cells as antibody-toxin conjugates and (iv) antibodies targeting the MUC1 cell-bound a/b junction results in tumor suppression in vivo. Our results indicate that cell-bound MUC1 a/b junction, unlike shed alpha chain, represents a highly effective moiety for targeting and killing MUC1-expressing malignancies. ' 2008 Wiley-Liss, Inc.Key words: MUC1; a/b; junction; oncogene; cancer cell killing; target-specific therapy Mucins are heavily glycosylated proteins found on both normal and malignantly transformed epithelial cells and preferentially expressed by a variety of adenocarcinomas, including breast, prostate, ovarian and pancreatic carcinomas, as well as on the malignant plasma cells of multiple myeloma. 1-5 Because of their presence on the cell surface, mucins play an important role in cell adhesion and matrix formation. 6 In addition to cell-to-cell functions, the transmembrane mucin molecule undergoes phosphorylation on its intracellular cytoplasmic tail, 7 thereby initiating intracellular transduction cascades that influence cellular proliferation and survival. 8-14 MUC1 mucin, because of its high expression on a number of different human tumor types, is widely recognized as a potentially important target for targeting human epithelial cancer cells. [15][16][17][18][19][20] The most intensively studied MUC1 protein is a type I transmembrane protein (MUC1/TM) composed of extracellular, transmembrane and cytoplasmic domains. 5,21 MUC1/TM is proteolytically cleaved soon after its synthesis, 22-26 generating two subunits, a and b, which specifically reco...
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.
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