Adoptive Immunotherapy for Epstein-Barr Virus-Associated Malignancy by T-Cell Receptor Transduction
Section of Hematology-Oncology, Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, United States
AIM: Adoptive immunotherapy has proven efficacy against Epstein Barr virus (EBV)-associated malignancy. Polyclonal T cell lines specific for the immunodominant latency antigens of EBV can both prevent the onset of and treat the occurrence of post-transplant lymphoma. However EBV-associated Hodgkin’s disease (approximately half of all cases) and nasopharyngeal carcinoma (universally EBV-associated) express a more limited repertoire of EBV latency antigens and are not readily recognized by the cellular immune system. In order to develop an immunotherapeutic strategy for these patients, we have turned to gene-based therapy whereby the alpha and beta chains of the T cell receptor for antigen (TCR) are molecularly cloned into a retroviral expression vector and this vector used to transduce activated lymphocytes from peripheral blood (PBMC), thus bypassing the need to produce HLA-restricted antigen specific T cell from each patient. METHODS: LMP-2 cytotoxic T cell clones (CTL) were produced by incubation of PBMC with HLA-A2 restricted peptide followed by limiting dilution cloning, or by co-culture with EBV-transformed B lymphoblastoid cell lines (B-LCL) and agarose cloning, Orentas, R.J., et al., 2001, and Khanna, R., et al., 1996, respectively. Full-length TCR alpha and beta chains were molecularly cloned and transferred to the A7 retroviral vector, and then transfected into the Phoenix-ampho packaging cell line to produce infectious vector supernatant. PBMC were activated with OKT3 and IL-2 and transduced by centrifugation in the presence of vector and polybrene for three consecutive days followed by culture in G418 for 5-6 additional days. The lytic activity of transduced PBMC was evaluated in chromium release assays. RESULTS: TCR transferred by retroviral transduction into activated human PBMC recapitulated the lytic specificity of the donor CTL clone. Importantly, TCRs cloned from two CTL lines able to lyse B-LCL transferred the ability to mediate tumor cell, i.e. B-LCL, lysis. Kinetic studies of CTL lytic activity demonstrated that lytic activity was primarily a function of transduced gene expression, as demonstrated by quantitative real-time PCR analysis. The overall lytic activity of transduced lymphocyte clones was decreased (both in Km and Vmax), suggesting that competition for assembly of both TCR chains into a functional TCR-CD3 complexes may occur between the endogenous and the transduced TCR chains. CONCLUSIONS: Retroviral transduction of the TCR is a rapid means to transfer the clonal specificity of a T cell line to activated PBMC from any donor sharing the same HLA allele. This process can be generalized to the production of antigen specific T cells for any known peptide-MHC combination for which TCRs are available. We propose that the TCR vectors described here are suitable for use in adoptive immunotherapy trials for patients with EBV-associated Hodgkin’s disease and nasopharyngeal carcinoma.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 995 (Immunotherapy).