Generation of antibodies that target and kill MUC1 expressing cancer cells via a novel cell-tethered MUC1 α/β junction epitope

3034 Background: MUC1 protein has generated considerable interest as a target for tumor killing. However, a serious shortcoming of anti-MUC1 antibodies generated to date is that they recognize epitopes within the strongly immunogenic tandem-repeat-array of the MUC1 a- chain which is released from the cell into the circulation. Soluble shed MUC1 a-chain sequesters anti-tandem-repeat-array antibodies, severely limiting their ability to reach MUC1-expressing malignant cells. Rather than target freely circulating MUC1 a-chain, we identified the junction of the MUC1 a-subunit to cell-membrane-bound MUC1 β-subunit as a preferable cell-tethered MUC1 epitope; since the site is not shed, antibodies recognizing it are more effective in targeting MUC1 expressing cells. Methods: To circumvent the immunogenicity of the a-chain tandem repeat array we formulated a novel antibody generating protocol utilizing immunization with both MUC1 cDNA and with the alternatively-spliced MUC1/X protein isoform from which the tandem repeat array is deleted. After immunization and hybridoma formation, anti- MUC1 a/β junction antibodies were selected. Results: DMC209 monoclonal antibodies uniquely specific for the MUC1 a/β junction were generated. The antibodies specifically bind the MUC1 a/β junction on full-length MUC1 expressed by breast and ovarian cancer cells, and on MUC1-positive malignant plasma cells of multiple myeloma. To demonstrate that anti-MUC1 a/β junction antibodies kill malignant cells, immunotoxin conjugates were formed with anti-MUC1 a/β junction polyclonal antibodies generated in our cDNA/protein immunization protocol and PE38, a powerful pseudomonas exotoxin. The antibody-exotoxin conjugates were potently cytocidal to MUC1-expressing malignant cells. Significantly, cell killing was abrogated by addition of soluble MUC1/X protein, highlighting that the cell-killing immunotoxin gains cell entry via the MUC1 a/β junction. Conclusions: The MUC1 a/β junction has been identified as an important cell-tethered MUC1 epitope against which highly specific antibodies can be generated capable of killing MUC1-expressing cells. These studies point to effective anti-MUC1-based immuno-therapeutic strategies. No significant financial relationships to disclose.