TCR TRANSDUCTION FOR EBV-SPECIFIC IMMUNOTHERAPY. Adoptive immunotherapy with polyclonal cytotoxic T cell lines (CTL) has met with clinical success in the treatment of post-transplant lymphoma, an Epstein-Barr virus (EBV)-associated malignancy that expresses the most immunodominant EBV latency antigens. This strategy is not applicable to two other EBV-associated malignancies, Hodgkin's disease (appx. 50 percent of cases are EBV-associated) and nasopharyngeal carcinoma (100 percent EBV-associated). These malignancies only express the EBV latency antigens LMP-1, LMP-2, and EBNA-1; none of which induce a strong immune response. These represent sub-dominant tumor-associated antigens. The goal of this project is to provide an immunotherapeutic option to patients suffering from these diseases by cloning individual T cell receptor molecules (TCR) that recognize LMP-1 and LMP-2 in an HLA-restricted manner, and introducing these recombinant TCR into HLA-A2 lymphocytes. Toward that goal, CTL clones specific for LMP-2 will be generated, the TCR alpha and beta chains molecularly cloned, and then transferred to retroviral expression vectors. These vectors will then be used to transduce CTL clones of known specificity as well as activated primary lymphocytes in bulk culture.
The specific aims of this project seek to determine which TCRs are the best candidates for genetic transduction by comparing the CTL activity of the original cell to the lytic activity newly conferred upon the transduced cell. It remains to be determined whether it is the primary sequence of the TCR or the physiology of the transduced cell that determines the cytolytic activity conferred by the new receptor. We will also determine the structure of the TCR-CD3 complex in transduced cells, and in examining the bulk transduced lymphocyte population determine which cells are capable of expressing the transduced receptor. Should the retroviral vector used in these studies not give long-term expression of the transduced TCR, we also propose a newer generation of retroviral vectors that would be used instead. Once transduced, the newly expressed TCR-alpha and beta chains will have to compete with the endogenous TCR elements for association with the CD3 receptor complex and subsequent transit to the cell surface. Data obtained from this project will allow correlation between levels of retroviral gene transduction, mRNA expression, intracellular protein expression (the assembly of ICR subunits in the endoplasmic reticulum), cell surface expression of transduced TCR, and lytic function to be made. With a better understanding of these first principles of functional ICR assembly in primary lymphocytes, other malignancies with known tumor-associated antigens could be targeted by this approach as well.
| Jurgens, Lisa A; Khanna, Rajiv; Weber, James et al. (2006) Transduction of primary lymphocytes with Epstein-Barr virus (EBV) latent membrane protein-specific T-cell receptor induces lysis of virus-infected cells: A novel strategy for the treatment of Hodgkin's disease and nasopharyngeal carcinoma. J Clin Immunol 26:22-32 |
| Orentas, R J; Bircher, L A; Roskopf, S (2003) Retroviral transfer of T-cell receptor genes produces cells with a broad range of lytic activity. Scand J Immunol 58:33-42 |
