Expression of the cancer-testis antigen FMR1NB on the surface of malignant cells.

Abstract

e13045 Background: Cancer-testis (CT) antigens represent ideal candidates for tumor-specific therapy due to their inherent immunogenicity and exceptional tissue restriction. Despite these advantageous properties the development of targeted therapeutics has been hampered by their predominantly intracellular localization. CT antigen FMR1NB might represent a promising exception due to its predicted transmembrane topology. Methods: We screened acute (AML) and chronic (CML) myeloid leukemia samples as well as healthy tissues for the expression of FMR1NB. Mammalian cell lines were transfected with FLAG- or GFP-tagged FMR1NB fusion constructs. We evaluated the subcellular localization of FMR1NB by fractionation and confocal microscopy. Epitopes of commercial anti-FMR1NB antibodies were characterized by ELISA using overlapping peptides. Results: FMR1NB mRNA was expressed in AML cell lines and patient samples and confirmed by western blot on the protein level. Specificity was confirmed by the lack of FMR1NB expression in healthy tissues. Confocal fluorescence microscopy of GFP chimeras in mammalian cell lines, immunofluorescence staining of AML cell lines, and subcellular fractionation strongly support a membraneous localization of FMR1NB including the plasmamembrane. In contrast to results of structural prediction software our characterization of epitopes recognized by polyclonal anti-FMR1NB antibodies and the subsequent immunostaining of intact transfected and native AML cells suggest an extracellular amino-terminus of FMR1NB. Flow cytometric analysis of AML and CML cell lines as well as AML patient samples and healthy donors further confirmed a strong and specific expression of FMR1NB on leukemia samples. Conclusions: FMR1NB mRNA is specifically expressed in AML cells and the resulting protein localizes to the membrane compartment including the plasma membrane. A putative extracellular domain of over 100aa length of the protein can be detected using FMR1NB-specific antibodies. The membrane localization of FMR1NB is a rare exception within the CT antigen family and our data suggest that it represents an ideal target for the diagnosis and therapy of myeloid leukemias. No significant financial relationships to disclose.