LMP2-Cytotoxic T Lymphocyte Therapy for Relapsed EBV Positive Lymphoma.

Abstract

EBV-associated Hodgkin's Disease (HD) and non-Hodgkins Lymphoma (NHL) show type II latency. They express the subdominant EBV antigens EBNA1, LMP1 and LMP2, which may serve as targets for immunotherapy approaches. In our previous studies, we used polyclonal EBV-specific CTL (EBV-CTL) in patients with relapsed EBV +ve HD and saw 2 complete and 1 partial response in 11 patients. Tetramer and functional analyses of EBV-CTL lines showed that small populations of T cells reactive against the tumor-associated antigen LMP2 were present in the majority of the infused lines, with some expansion in the peripheral blood following infusion. We therefore hypothesized that CTL specifically targeting type II viral latency antigens might have greater efficacy in these patients. LMP2-CTL could be generated from normal donors using dendritic cells (DC) genetically modified with a recombinant adenovirus encoding LMP2a (Ad5LMP2a) to direct the CTL response to LMP2. However, this approach required the generation of large numbers of DC to expand LMP2-CTL and was not practical in these heavily pretreated patients. We therefore modified the LMP2-CTL generation protocol to use DC for the initial stimulation, followed by stimulation with LCL that had been genetically modified to overexpress LMP2a by transduction with an Ad5f35LMP2a vector. This approach allowed us to specifically expand LMP2-CTL from patients to the numbers required for clinical use. We have generated LMP2 specific CTL lines in 10 patients with EBV+ve HD or NHL. LMP2 peptide tetramers were used for analysis of the polyclonal LMP2-CTL lines where HLA-restricted tetramers were available, and the lines recognized 2–6 (median 4) LMP2 epitopes, as determined by ELISPOT assay. A mean of 22.8% (5–42.1%) of CD8+ T cells bound these LMP2 tetramers, compared to a mean of 0.11% (0.01–0.24%) of LMP2-tetramer positive CD8+ T cells found in CTL generated with genetically unmodified LCL from the same patients. So far, 6 patients have been treated, initially receiving 2 doses of 2x107 CTL/m2 two weeks apart. All patients were off other therapies for at least 1 month prior to receiving CTL. No immediate toxicity was observed. In patients with identified LMP2-epitopes, measurement of IFNγ secretion by CD8+ T cells after stimulation with appropriate LMP2-peptides in ELISPOT assays showed a 4–25-fold increase in spot forming cells after infusions. In contrast, the frequency of CMV and superantigen-specific T cells did not increase. Four patients without radiological evidence of disease who received CTL as adjuvant therapy post stem cell transplant or chemotherapy remain well up to 9 months post CTL. Two patients with measurable disease at the time of CTL infusion had stable disease 8 weeks post CTL. They received 2 further doses of LMP2-CTL at 2x107/m2/dose. Re-evaluation was performed 8 weeks after the additional CTL infusions and one patient continues with stable disease. In the second patient, radiological review now revealed no evidence of disease, and a supraclavicular lymph node resection showed selective accumulation of LMP2-tetramer positive cells (0.3% compared to 0.01% in the peripheral blood) with scanty residual tumor cells. Immunotherapy with autologous LMP2-CTL is therefore well tolerated in patients with relapsed EBV+ve HD/NHL and infused LMP2-CTL cells can localize to the tumor and induce a clinical response.