Targeting BARF1 for the therapeutic control of EBV-associated malignancies (2010)

The Epstein-Barr virus is a y-herpesvirus that preferentially infects human Blymphocytes. It is estimated that 95% of worldwide population is infected, but usually the infection happens during childhood and is asymptomatic. Beside the association to a self-limiting lymphoproliferative disease, infectious mononucleosis, the virus is related to the development of some human cancerous forms, which are characterized by different patterns of viral latency, like Burkitt’s lymphoma (BL) and some forms of gastric carcinoma (GC), Hodgkin’s lymphoma (HL) and nasopharyngeal carcinoma (NPC), and, finally, PTLD, post-transplant lymphoproliferative diseases. In addition to the latency genes, it is known the expression of different non-polyadenilated RNA (EBER) and, especially in the cases of NPC and GC, the expression of a transmembrane protein belonging to the lytic cycle, BARF1. BARF1 is a 221-aa protein with a transmembrane domain at the C-terminal. Only recently, its transforming and immortalizing roles have been demonstrated in human cells. Moreover, its extracellular domain can be cleaved and can act in a paracrin way as a growth factor on bystander cells, and it owns mitogenic activities. Nevertheless, its mitogenic and mutagenic properties are not well understood yet, but the importance of this protein in the pathways of neoplastic progression and its expression only on infected cells (or at the surface of those that can bind its secreted forms) render BARF1 an optimal target for a therapeutic approach of EBV-related tumours. Different strategies for the treatment of EBV-related neoplasms are currently in use in clinic. Some strategies reside on the reduction of the immunosuppressive regimen, on the exploitation of gene therapy, on the use of chemotherapy or antiviral drugs, or on the approaches of immunotherapy. The use of autologous or HLA-matched cytotoxic T lymphocytes (CTL) proved to be efficient and usually devoided of side effects, especially for PTLD patients. A different aspect of immunotherapy is based on the use of monoclonal antibodies (mAb), as it was already demonstrated in different clinical settings by the use of rituximab. In this PhD work, the generation and the in vitro evaluation of different monoclonal antibodies specific for BARF1 are described. Moreover, once their activity was established on cell culture in vitro, this approach was translated to some pre-clinical models, using immunodeficient mice bearing EBV-positive tumours. Also in these experiments, the antibodies proved to be therapeutically efficient. On one side, the use of mAb for the diagnosis and for the cure of malignancies is acquiring an increasing importance in the clinic, thanks to the specificity of action of these molecules and to their relative easiness of use, if compared to the cellular adoptive therapy. On the other side, although BARF1 functions and interactions with other proteins or cells are not well studied yet, it can be regarded as a promising target for EBV-related malignancies, since it is expressed in NPC and GC and owns important transforming properties, while being a lytic cycle protein.