SELECTION OF EPITOPE DIRECTED RECOMBINANT ANTIBODIES TO INHIBIT MUTATED NUCLEOPHOSMIN ACTIVITIES

Nucleophosmin (NPM1; also known as B23, NO38, or numatrin) is an abundant nuclear-cytoplasmic shuttling protein that is involved in different cellular processes such as centrosome duplication, cell cycle progression and stress response. NPM1 physically interacts with several nuclear proteins, including nucleolin, p120, p53 and Mdm2. Recently, it has been demonstrated that NPM1 forms a stable complex with the tumor suppressor p19/Arf and it is absolutely required for its correct localization and stabilization in the nucleolus. The observation of chromosomal translocations of the NPM1 gene in human hematopoietic cancers has suggested that NPM1 contributes to tumor development by activating the oncogenic potential of the fused protein partners. Moreover, it has been found that about one-third of primary adult Acute Myeloid Leukemia (AML) patients bear mutations in the last exon of the NPM1 gene, leading to an aberrant cytoplasmic localization of the protein (NPMc+). Interestingly, the accumulation of NPMc+ in the cytoplasm appears responsible for the delocalization of proteins that, under normal conditions, interact with wild type NPM1 in the nucleus. In particular, NPMc+ binds, delocalizes and inactivates the p19/Arf tumor suppressor, as well as the Fbw7γ F-box protein that is involved in the proteasome-dependent degradation of the c-Myc oncoprotein. These data prompted me to explore the possibility of blocking the aberrant activity of NPMc+ using NPMmutant-specific antibodies. I report here the isolation, from the ETH2-Gold phage display library, of a recombinant antibody in scFv format specific for the NPMc+ protein. It univocally targets the mutated region at the C-terminal end of NPMc+. The specificity of this antibody was evaluated in vitro by performing western immuno-blot analyses, immunofluorescence and immunoprecipitation assays. I show that the scFv antibody performs as well as the mouse monoclonal antibody specific for the same NPMc+ mutation and used as control. It is also able to immunoprecipitate the overexpressed and the endogenous NPMc+ proteins. Furthermore, I have found that the scFv can be efficiently expressed in mammalian cells as an intrabody, upon transfection of specific vectors that carry its cDNA sequence, and that it accumulates in the nucleus when fused to a nuclear localization signal (NLS). This evidence opened the opportunity to try the in vivo re-localization of NPMc+ from cytoplasm to the nucleus by using the recombinant antibody as a specific carrier. However, scFv-NLS fusions were unable to re-locate NPMc+ into the nucleus. The addition of multiple NLSs to the scFv did not modify the sub-cellular localization of NPMc+. In conclusion, I have been able to isolate a recombinant antibody that specifically targets NPMc+ protein. This antibody, directed against the C-terminal epitope of the mutated protein, has been biochemically and functionally characterized both in vitro and in vivo. The scFv successfully expresses as an intrabody in mammalian cells and specifically interacts with the native NPMc+ in vivo, thus giving the premises for its further development as a potential innovative therapeutic tool.