Latent membrane protein 1 (LMP1) oncogene belongs to a group of antigens expressed on the surface of cells infected with the Epstein-Barr virus (EBV) during the latency period and is considered to be one of the most important EBV-transforming proteins [Meij et al., J. Infectious Diseases, 179:1108-15, 1999]. LMP1 belongs to a group of EBV antigens. Currently known members of this group of antigens include two EBV encoded noncoding RNAs (EBER1, 2), six Epstein-Barr nuclear antigens (EBNA1, 2, 3a, 3b, LP), and three latent membrane proteins (LMP1, 2A, 2B) [Id.].
LMP1 protein has a short cytoplasmic amino terminus which is involved in transcriptional activation. The carboxy terminus contains a C-terminal activation region 1 (CTAR1) region adjacent to the plasma membrane, including a PXQTX (SEQ ID NO. 1) core TRAF protein-binding motif and an outermost CTAR2 region for tumor necrosis factor associated death domain protein (TRADD) and receptor interacting protein (RIP) binding. In addition to these functional domains, LMP1 also contains three plasma membrane spanning domains, which expose short loops to the extracellular space [Knecht et al. Oncology 60:289-302, 2001]. While these short loops are present on the surface of the infected cell, the LMP1 oncogene is known to have a very low immunogenicity [Meij et al. J. Infectious Diseases, 179:1108-15, 1999]. This has hampered development of antibodies against it. Moreover, the extracellular loops of the LMP1 are part of a transmembrane domain. Membrane association of LMP1 makes production of antibodies against a recombinant antigen difficult because such a recombinant antigens do not present themselves in a native membrane-bound conformation.
Epstein Barr Virus (EBV) belongs to γ-herpesviruses and it is associated with various malignant and benign lymphoproliferative disorders [Liebowitz, N. Eng. J. Med. 338:1413-21 (1998)]. It is the etiologic agent of infectious mononucleosis. It is also strongly associated with malignancies like Burkitt's Lymphoma (BL), nasopharyngeal carcinoma, and immunoblastic B cell lymphomas (non-Hodgkin's lymphoma, NHL) in immunocompromised individuals. EBV has also been detected in substantial percentage of Hodgkin's disease (HD), in certain types of T and NK (natural killer) cell NHL (T-NHL and B-NHL), and gastric carcinoma patients. In addition to its association of human malignancies, EBV is also associated with a spectrum of diseases, collectively called chronic EBV syndrome, and with oral hairy cell leukoplakia predominantly seen in patients with AIDS.
Based upon viral latent gene expression and the expression of surface antigens, three main types of EBV latency have been characterized. In type I latency, the EBV infected cells express EBNA-1 and EBER1 and 2, and the disease is usually a phenotypically representative BL. Type II latency is seen in nasopharyngeal carcinoma, Hodgkin's disease, T-cell non-Hodgkin's lymphoma, and B-cell non-Hodgkin's lymphoma, and in immunocompetent patients and is characterized by expression of EBNA1, EBER1 and EBER2, and LMP1, LMP2A, and LMP2B. Type II latency, in which all gene products are expressed, is seen in B-NHL in immunocompromised patients.
In many cases, EBV infection results in a lymphoproliferative disease that may be only temporarily debilitating. However, in immunosuppressed individuals, the result can be full-blown malignancy. This occurs in individuals who are immunosuppressed intentionally, particularly children receiving organ transplants who are treated with cyclosporine A, or opportunistically, as in the case with individuals infected with EBV, or genetically, as in the case of affected males carrying the XLP (X-linked lymphoproliferative syndrome) gene. In these cases the resulting malignancies derive from the polyclonal proliferation of EBV-infected B cells. In addition, in such patients uncontrolled epithelial replication of the virus is detectable in lesions of oral hairy leukoplakia Thus, the immune response plays a central role in the control of EBV infection.
Vaccination against EBV might be useful for several groups of people who are seronegative for EBV. These include patients undergoing bone marrow or organ transplantation, persons with X-linked lymphoproliferative disease, people in areas of the world with high incidence of Burkitt's lymphoma (equatorial Africa) or nasopharyngeal carcinoma (southern China), and adolescents and adults at risk for infectious mononucleosis.
Current treatment of BL and other EBV associated malignancies include chemotherapy, for example cyclophosphamide, and/or radiation therapy. Radiation therapy is frequently used, especially in patients with AIDS, because chemotherapy alone is rarely successful. However, these treatments are focused on unspecific general destruction of rapidly dividing cells and are associated with a number of undesirable side effects.
Some anti-B-cell antibodies, such as monoclonal antibodies to CD21 (EBV receptor), CD24 (pan-B-cell antibody), and CD20 (Rituxan®, rituximab, from Genetech oncobiology, Inc.) have recently been introduced [Cohen J. I., New England J. Med. 343:481-492, 2000; Benkerrou et al., Blood 92:3137-47 (1998)]. However, while being B-cell specific, they do not discriminate between EBV infected and uninfected B-cells and their use results in destruction of all B-cells with such surface antigens. Therefore it would be desirable to develop antibodies that are specific for EBV infected cells and that elicit sufficient immune responses to specifically target infected with EBV infected cells. It would also be desirable to have new means for determining cells infected by EBV and means which can be used to differentiate between different disease states.