722. Overcoming EBV Tumor Specific T-Cell Anergy in Rapidly-Generated EBVST-Cells for Adoptive Transfer Therapy
Authors: Sandhya Sharma, Serena Perna, Natalia Lapteva, Minhtran Ngo, Vicky Torrano, Rayne Rouce, Carlos Ramos, Catherine Bollard, Helen Heslop, Cliona Rooney
Journal: Molecular Therapy (2015)

Up to 30% of Hodgkin and non-Hodgkin lymphomas carry the Epstein-Barr virus (EBV) genome and express the viral latency proteins EBNA1, LMP1, LMP2 and BARF1; a type 2 latency pattern of EBV gene expression. We previously reported that EBV-specific T-cells (EBVSTs) directed to LMP1 and LMP2 expanded from the blood of lymphoma patients produced complete tumor responses in over 50% of patients. To shorten and simplify the EBVST production time, we removed viral-vector components from our manufacturing process, replacing these components with dendritic cells pulsed with peptide libraries (pepmixes) spanning type 2 latency antigens. Responder T-cells are then expanded by restimulation with pepmix-pulsed, autologous, activated T-cells and HLA-negative K562 costimulatory cells in the presence of IL-4 and IL-7. Despite enhanced antigen specificity from EBVSTs generated from healthy donors, we were unable to consistently generate patient-derived EBVSTs with significant activity against the Type 2 antigens. We hypothesized that patient T-cells were anergized by their immunosuppressive tumors. IL-15 has been shown to rescue tolerant or anergized CD8+ T-cells and we found that substitution of IL-15 (5ng/mL) for IL-4 (in combination with IL-7 (10ng/mL)) improved CD4+ and CD8+ T-cells' specificity for the EBV Type 2 latency antigens by up to 10-fold. By increasing the concentration of IL-15, we achieved significantly higher fold expansion (3 fold mean increase in absolute EBVST number) and further enhanced specificity for type 2 latency EBV antigens (high vs. low IL-15 concentration: EBNA1: 216±273 vs. 29±48, LMP1: 145±253 vs. 44±63, LMP2: 636±548 vs. 106±74 and BARF1: 80±100 vs. 29±22; SFC/105 cells; n=5). There was no increase in the absolute numbers of NK-cells despite high doses of IL-15. Enhanced antigen-specificity correlated with increased cytotoxic effect, with an increase in mean % specific lysis of EBV pepmix-pulsed autologous activated T-cell targets from 5 to 66% at a 20:1 E:T ratio in the low vs. high dose IL-15 conditions (n=5). We have infused EBVSTs manufactured using all three conditions into 17 patients with multiply-relapsed, EBV-positive lymphoma as adjuvant therapy after stem cell transplantation or chemotherapy in 8 patients and as treatment for disease in 9 patients. Of patients in remission at the time of infusion, two with IL-4/7-grown EBVSTs and 6 with IL-15/7 grown EBVSTs remain in remission. Of patients with disease at the time of infusion, one receiving IL-4/7-grown EBVSTs had stable disease and 3 had progressive disease, while of 5 patients with IL-15/7-grown EBVSTs, one had stable disease, one had a partial response, one had a complete response and two are too early to assess. We will continue to modify our manufacturing process in an attempt to further increase the specificity and function of EBVSTs from patients with relapsed or refractory lymphoma.