Immunotoxin Targeting Cell-Bound MUC1 on Myelomonocytic and Monocytic Leukemic Cells

Cell surface molecules aberrantly or overexpressed by myeloid leukemic cells represent potential disease-specific therapeutic targets for antibodies. MUC1, a polymorphic type I high molecular weight glycoprotein represents such a target. MUC1 is cell surface molecule with extracellular, transmembrane, and cytoplasmic domains; the cytoplasmic tail participates in intracellular signaling. Cleavage of MUC1 yields two unequal chains: a large extracellular alpha subunit bound in strong non-covalent interaction to a smaller beta subunit containing the transmembrane and cytoplasmic domains. Essentially all anti-MUC1 antibodies reported to date target the highly immunogenic MUC1 alpha chain. Because the MUC1 alpha chain binds the cell only intermittently in an 'on-and-off' manner, agents directed against the alpha chain will not effectively target MUC1+ cells. In contrast, the MUC1 SEA domain represents a stable structure fixed to the cell surface at all times. We therefore generated mAbs that specifically recognize the cell-bound MUC1 SEA domain. One of them, a partially humanized murine mAb termed DMB-5F3 was used to examine the expression of MUC1 on leukemic cells by flow cytometry. DMB-5F3 was found to strongly bind not all AML, but rather a specific set of AML subtypes: Chronic Myelomonocytic Leukemia (CMML); Acute Myelomonocytic and Monocytic Leukemia (AML-M4 and AML-M5), and Juvenile Myelomonocytic Leukemia (JMML). Other AML subtypes did not express MUC1. To test the cytotoxic ability of anti-MUC1 antibodies against MUC-expressing AML, we generated a humanized, dimeric single-chain DMB-5F3 scFv fragments fused to recombinant gelonin, a type I ribosome-inactivating toxin which itself is incapable of cell binding or internalization into mammalian cells to which a 6xHis-tag was added. The anti-MUC1 scFv-toxin cytometrically identified using an anti-6xHis-tag was found to bind MUC1 positive cell line. We examined the cytotoxic effects of anti MUC1 scFV-toxin using the MUC-1 positive AML cell lines MV411 and MOLM14 (monocytic leukemic subtype). MUC1 scFV-toxin was found to be highly cytotoxic on MUC1+ AML cell lines, resulting in direct cell death. Our data strongly support the use of an anti-MUC 1 antibody-drug conjugate to selectively target and inhibit the growth of MUC1-expressing AML.