MUC1/X Protein Immunization Enhances cDNA Immunization in Generating Anti-MUC1 A/B Junction Antibodies that Target Malignant Cells Daniel B. Rubinstein, 1 Maya Karmely, 2 Ravit Ziv, 2 Itai Benhar, 3 Orit Leitner, 5 Shoshana Baron, 6 Ben-Zion Katz, 6 and Daniel H. Wreschner 2,4 1 Food and Drug Administration, Silver Spring, Maryland; Departments of 2 Cell Research and Immunology and 3 Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat Aviv, Israel; 4 Biomodifying, LLC, San Diego, California; 5 Antibody Unit, Weizmann Institute of Science, Rechovot, Israel; and 6 Department of Hematology, Sourasky Medical Center, Tel Aviv, Israel Abstract MUC1 has generated considerable interest as a tumor marker and potential target for tumor killing. To date, most anti- bodies against MUC1 recognize epitopes within the highly immunogenic A chain tandem repeat array. A major short- coming of such antibodies is that the MUC1 A chain is shed into the peripheral circulation, sequesters circulating anti- tandem repeat array antibodies, and limits their ability to even reach targeted MUC1-expressing cells. Antibodies recog- nizing MUC1 epitopes tethered to the cell surface would likely be more effective. MUC1 A subunit binding the membrane- tethered B subunit provides such an epitope. By use of a novel protocol entailing immunization with cDNA encoding full- length 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 ex- pressed 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 immu- nization 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) Introduction MUC1 is a glycoprotein highly expressed in several human epithelial malignancies, including breast, prostate, ovarian, and pancreatic carcinomas, as well as on the malignant plasma cells of multiple myeloma (1–6). Although alternative splicing can generate a variety of MUC1 isoforms (7–13), the most intensively studied MUC1 protein is a type I transmembrane protein (MUC1/TM) composed of a heavily glycosylated extracellular domain containing a tandem repeat array, a transmembrane domain, and a cytoplasmic domain (9, 14, 15). MUC1/TM is proteolytically cleaved soon after its synthesis, generating two subunits, a and h, which specifically recognize and bind each other in a strong noncovalent interaction (Fig. 1, MUC1/TM; refs. 9, 16–19). Cleavage of MUC1 into the two subunits occurs in the SEA module (9, 18, 19), a highly conserved domain found in several cell-tethered mucin-like proteins (20). Shedding of a subunit from the cell membrane results in soluble tandem repeat–containing MUC1 in the peripheral circulation, and it is this molecule that is used to determine serum MUC1 levels in patients (21, 22). The presence of the soluble a subunit MUC1 protein in the circulation presents a singular difficulty in delivering adequate amounts of anti-MUC1 antibodies to directly target MUC1- expressing malignant cells. Because the most immunogenic part of MUC1 is the tandem repeat array, all anti-MUC1 antibodies generated to date almost exclusively recognize epitopes in that immunogenic region. Sequestration of antitandem repeat anti- bodies by the soluble, circulating MUC1 a subunit severely limits the amount of antibody that can successfully bind MUC1 on the cell surface. Furthermore, deposition of immune complexes of antitandem repeat antibodies and its soluble, circulating MUC1 target can lead to significant end-organ damage. In recent years, numerous efforts have been made to generate effective anti-MUC1 antibodies using the full-length MUC1/TM molecule as immunogen (23–25). The major obstacle hampering those attempts is that immunization with the whole MUC1/TM molecule invariably results in an antibody response composed almost in its entirety of antibodies recognizing epitopes on the highly immunogenic tandem repeat array. For ultimate application in in vivo targeting of MUC1-expressing tumor cells, such antibodies pose all the shortcomings inherent in antirepeat antibodies as detailed above. Antibodies recognizing MUC1 epitopes tethered to the cell surface potentially obviate these difficulties. Although conceptually simple, generation of monoclonal antibodies (mAb) to tethered MUC1 first requires characterization of cell-bound, nonshedding epitopes. The junction formed by the MUC1 a subunit binding the membrane-tethered h subunit provides such an epitope. We recently investigated the mechanism whereby the cleaved junction composed of the MUC1 a and h subunits is formed (9). In the course of those studies, we analyzed the ‘cleavageability’ of the MUC1/TM, MUC1/Y, and MUC1/X proteins (Fig. 1; ref. 9). The MUC1/Y and MUC1/X isoforms are generated from mRNAs spliced at two distinct sites that use donor and acceptor sites located Requests for reprints: Daniel H. Wreschner, Department of Cell Research and Immunology, Tel Aviv University, Tel Aviv 69978, Israel. Phone: 972-3-6407425; Fax: 972- 3-6422046; E-mail: danielhw@post.tau.ac.il or c/o A. Greenboim, Biomodifying LLC, San Diego, CA 92122. Phone: 858-678-0731; Fax: 858-678-0791. I2006 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-1486 www.aacrjournals.org 11247 Cancer Res 2006; 66: (23). December 1, 2006 Research Article