This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. ABSTRACT Improvements in radiochemotherapy have correspondingly improved the prognosis of patients with Hodgkin's Disease (HD) and non-Hodgkin's lymphoma (NHL). However, patients resistant to the standard therapeutic approaches have an extremely poor outcome. Moreover, life expectancy and quality of life of patients cured of lymphoma are both significantly reduced by treatment related mortality and morbidity. These limitations of current treatment protocols illustrate the need for more effective therpeutic approaches for patients who relapse. In HD and NHL, up to 49% of specimens have been shown to carry EBV-DNA and express EBV-genes. Our group has successfully generated EBV-specific CTL in patients with EBV-positive Hodgkin's lymphoma. After infusion these CTL home to the tumor sites persist in the circulation for up to 12 months, and produce transient clinical benefits. In that protocol, lymphoblastoid cell lines (LCL) were used as EBV-antigen presenting cells (APC). LCL activate polyclonal CTL populations that are preferentially directed against the immunodominant EBNA3A, 3B and 3C EBV-proteins. These immunogenic proteins are not expressed in HD/NHL tumor cells. Instead, the EBV-antigens on HD/NHL tumor cells are restricted to the expression of a subset of latent proteins such as LMP2. LCL have limited efficacy in stimulating CTL directed against these subdominant proteins. LMP2 epitopes were shown to be conserved among Lymphoma biopsy samples displaying little heterogeneity between viral strains. Hence we thought that LMP2 could be targeted by CTL inpatients with Lymphoma and in an ongoing study, our group is successfully generating CTL targeting LMP2 from the peripheral blood of patients with EBV +ev HD and NHL. In this current protocol, LMP2A -specific CTL were able to elimiante some EBV-positive lymphoma's and the LMP2-specific CTL accumulated at tumor sites, increased in the peripheral blood and produced transient clinical benefits. The CTL were however least effective in patients with bulky disease. It is therefore possible that LMP2A-specific CTL have good cytotoxicity in vitro but that when they are subjected to the highly immunosuppressive activity of Transforming Growth Factor-beta (TGFB) secreted by the tumor environment, this precludes effective tumor elimination. We therefore want to render the CTL resistant to TGFB, to overcome the problem of effective tumor kill. In preclinical studies we have cloned a truncated dominant negative TGFB receptor (DNR) into the retrovirus vector, SFG and transduced CTL. Under normal circumstances TGFB has profound effects on the proliferation, and cytolytic activity of established CTL, and the continued presence of TGFB in vitro leads to the death of cultures within two to three weeks. In contrast, when CTL transduced with DNR are cultured in the presence of TGFB, they retain apparently normal intracellular signaling, grow normally, continued to secrete GM-CSF and yIFN, and retained their cytolytic function. Thus, the transdominant-negative TGFB receptor mutant is capable of protecting CTL from the inhibitory effects of TGFB in vitro. Importantly, for the feasibility of using this approach clinically, culture of CTL in the absence of antigenic stimulation and growth factors, led to cell death as rapidly as in non-transduced T cells. We also developed and completed an in vivo safety study in a murine model to confirm the suitaility of the approach for clinical use. In this study we will generate LMP2A-specific cytotoxic T-cells using the same methodology as on our current study and genetically modify them using a retroviral vector expressing a dominant negative TGFB receptor II (DNRII) to render them resistant to the immunosuppressive effects of TGFB. We will then adoptively transfer these TGFB resistant LMP2-CTL to patients with relapsed EBV-positive Hodgkin's or non-Hodgkins Lymphoma.
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