- Background:
Epstein-Barr virus (EBV) is associated with several human malignancies, amongst which 40-90% of Hodgkin's disease (HD). EBV is transcriptionally active in the Hodgkin and Reed-Sternberg cells (H-RS cells), leading to the abundant expression of a selected set of EBV encoded proteins, especially the nuclear antigen 1 (EBNA1), and the latent membrane proteins 1 and 2 (LMP1,2). Due to the expression of virus encoded non-self proteins, EBV+ malignancies are obvious candidates for immunotherapy. Because EBNA1 escapes T-cell recognition, LMP1 and LMP2 are the prime targets. However, the frequency of LMP1,2 specific T-cells is very low. Similarly, antibodies against LMP1,2 are barely detectable and only present in about 30-40% of HD patients at low titers. Using T-cell stimulation studies with LMP1,2 peptide libraries and our unique intact purified LMP1 we recently defined new epitopes of these proteins, specifically recognized by CD4 and CD8 T-cells of healthy EBV+ donors. Our most recent data indicate that highly purified recombinant LMP1 is taken up and digested by dendritic cells (DC) in vitro, thus allowing the generation of LMP1-specific T-cell responses using DC as antigen presenting cells. In vivo EBV+ (H-RS) tumor cells apparently resist immune responses. Detailed studies by us and others on HD strongly suggest that in vivo local events mediate the escape of the EBV+ H-RS cells from cytotoxic immune responses. Recently, we demonstrated that LMP1 itself has immunomodulating activities; indirectly by inducing the expression human IL-10 in H-RS cells and directly by inhibiting T-cell activation via a domain in the first transmembrane region (TM1). This might explain the local escape of LMP1,2 from immune recognition. However, for immunotherapeutic purposes, induction of an immune response towards LMP is expected to be achieved by presenting LMP1, 2 to the immune system by DC in a locally immunostimulating environment. The enhanced T-cell reactivity, thus generated, will provide a strong afferent limb of the immune response effective against H-RS cells, despite local circumstances.
- Purpose:
Since LMP1 and LMP2 expressed in EBV+ H-RS cells are considered to be relevant targets for immunotherapy and can evoke cytotoxic T-cell (CTL) responses in healthy individuals, we wish to stimulate the immune reactivity to LMP1,2 in patients with HD and to increase immunogenicity of LMP1 by using a deletion mutant protein, devoid of the immunosuppressive domain.
- Plan of investigation:
Immune reactivity to LMP1,2 will be investigated along the following lines:
1. Expression and purification of recombinant (fragments of) LMP1 and LMP2.
Recently we succeeded to express full length LMP1 and LMP2 in insect cells. Purification of LMP1 was optimized using a unique monoclonal antibody-based immunoaffinity purification protocol. A mutant form of LMP1 (DTM1), lacking the (immunosuppressive) first transmembrane region, has been constructed and is expressed in insect cells. Its purification and that of full-length LMP2 will be evaluated. LMP1,2 peptide libraries and defined MHC-I/-II restricted peptides are available.
2. Optimal presentation of LMP1 and LMP2 to T cells by exploiting DC as antigen presenting cell.
Optimization of antigen presentation by DC will be investigated using DC of various sources, stimulated with various mixtures of cytokines. The effect of (the mutant form of) LMP1,2 on the maturation of DC, the production of cytokines and induction of T-cell responses will be investigated.
3. Optimal induction of LMP1 and LMP2 reactive CD4 and CD8 T-cells.
Functional assays will be performed on PBMCs of HD patients and healthy controls, focusing on anti-LMP1,2 recognition. These cells will be tested for interferon-g production, granzyme B expression and cytotoxic activity after DC-mediated stimulation with purified recombinant LMP1,2 or synthetic peptide epitopes of LMP1,2. CTLs specifically activated in this way will be analyzed on autologous target cells (LCLs or PHA-blasts) naturally expressing LMP1 and LMP2 or transfected with recombinant Vaccinia virus.
4. Induction of LMP 1 and LMP2 immune response in vivo.
In the last year of this project the induction of an anti-LMP1,2 immune responses will be assessed in vivo in patients with HD, nodular sclerosing subtype, unresponsive to chemotherapy, using a DC-based LMP1,2 vaccine.
- Relevance for cancer research and therapy:
The results of the study will provide important clues for the development of immunotherapeutic strategies in EBV positive malignancies related to EBV latency type II, such as HD. |
