Retinoblastoma-independent antiproliferative activity of novel intracellular antibodies against the E7 oncoprotein in XXXX -positive cells.

@entity1 @entity66 @entity5 @entity996 @entity189 @entity1799 @entity1082 @entity10390
BACKGROUND: "High risk" @entity1082 strains are the causative agents of the vast majority of @entity189 of the uterine cervix. In these @entity5 , the physical integration of the HPV genome is a frequent, though not invariable occurrence, but the constitutive expression of the E6 and E7 viral genes is always observed, suggesting key roles for the E6 and E7 oncoproteins in the process of malignant transformation. The "intracellular antibody" technology using recombinant antibodies in single-chain format offers the possibility of targeting a protein in its intracellular environment even at the level of definite domains thus representing a valuable strategy to "knock out" the function of specific proteins. METHODS: In this study, we investigate the in vitro activity of two single-chain antibody fragments directed against the "high-risk" @entity1799 E7 oncoprotein, @entity10390 43M2 and @entity10390 51. These scFvs were expressed by retroviral system in different cell compartments of the @entity1799 -positive @entity1 cells, and cell proliferation was analyzed by Colony Formation Assay and EZ4U assay. The binding of these scFvs to E7, and their possible interference with the interaction between E7 and its main target, the @entity5 suppressor @entity996 protein, were then investigated by immunoassays, PepSet    technology and Surface Plasmon Resonance. RESULTS: The expression of the two scFvs in the nucleus and the endoplasmic reticulum of @entity1 cells resulted in the selective growth inhibition of these cells. Analysis of binding showed that both scFvs bind E7 via distinct but overlapping epitopes not corresponding to the @entity996 binding site. Nevertheless, the binding of @entity10390 43M2 to E7 was inhibited by @entity996 in a non-competitive manner. CONCLUSIONS: Based on the overall results, the observed inhibition of HPV-positive @entity1 cells proliferation could be ascribed to an interaction between @entity10390 and E7, involving non- @entity996 targets. The study paves the way for the employment of specific scFvs in immunotherapeutic approaches against the @entity66 .
