title: MUC1/X protein immunization enhances cDNA immunization in generating anti-MUC1 alpha/beta junction antibodies that target malignant cells.
abstract: 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 alpha chain tandem repeat array. A major shortcoming of such antibodies is that the MUC1 alpha 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 alpha subunit binding the membrane-tethered beta 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 alpha/beta 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 alpha/beta 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 alpha/beta 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.
authors.affiliation: Food and Drug Administration, Silver Spring, Maryland, USA.
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Rubinstein
authors.forename: Maya
authors.initials: M
authors.lastname: Karmely
authors.forename: Ravit
authors.initials: R
authors.lastname: Ziv
authors.forename: Itai
authors.initials: I
authors.lastname: Benhar
authors.forename: Orit
authors.initials: O
authors.lastname: Leitner
authors.forename: Shoshana
authors.initials: S
authors.lastname: Baron
authors.forename: Ben-Zion
authors.initials: BZ
authors.lastname: Katz
authors.forename: Daniel H
authors.initials: DH
authors.lastname: Wreschner
mesh_terms: D000818:Animals; D000906:Antibodies / Q000097:blood* / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D000918:Antibody Specificity / Q000276:immunology; D001854:Bone Marrow Cells / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics; D006801:Humans; D007114:Immunization / Q000379:methods; D051379:Mice; D018396:Mucin-1 / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D009101:Multiple Myeloma / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D009154:Mutation / Q000235:genetics; D009369:Neoplasms / Q000097:blood / Q000276:immunology / Q000473:pathology; D011485:Protein Binding
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D000939:Epitopes; D018396:Mucin-1