Complete Tumor Responses in Lymphoma Patients Who Receive Autologous Cytotoxic T Lymphocytes Targeting EBV Latent Membrane Proteins
Authors: Bollard Catherine M. , Stanojevic Maja , Leen Ann M. , Lopez Teresita , Sheehan Andrea N. , Carrum George , Liu Hao , Chang Chung-Che , Gee Adrian P. , Brenner Malcolm K. , Rooney Cliona M. , Heslop Helen E. , Gottschalk Stephen 
Published: April 2020

Abstract
EBV-associated Hodgkin’s Lymphoma (HL) and some non-Hodgkins lymphoma (NHL) have type II viral latency expressing the subdominant EBV antigens EBNA1, LMP1 and LMP2. These antigens may serve as targets for immunotherapy approaches and in previous studies, we used polyclonal EBV-specific CTL in patients with relapsed EBV +ve HL obtaining 2 complete and 1 partial response in 11 patients. 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 enriched for effector cells specifically targeting LMP antigens would have greater efficacy in these patients. LMP-CTL were generated using dendritic cells for initial stimulations then EBV-transformed lymphoblastoid cell lines (LCL) both of which had been genetically modified to overexpress either LMP2 alone or inactive LMP1 (ΔLMP1) and LMP2 by transduction with an Ad5f35LMP2 (n=16) or Ad5f35ΔLMP1-I-LMP2 (n=14) vector respectively. All LMP-CTL lines were polyclonal comprising CD4+ (mean 17±18%; range 1–92%) and CD8+ (mean 74 ± 25%; range 1–99%) T-cells. Flow cytometric analysis of memory markers revealed mixed populations of CD45RA- CD62L- T-cells (45±15%; range 31–63%) and CD45RA- CD62L+ T-cells (34±5%; range 28–41%). The CTL lines had specificity for CD4+ and CD8+ restricted LMP2 epitopes alone (n=19; mean 1; range 0–7) or both LMP1 and LMP2 epitopes (n=13; mean 2; range 0–6) per CTL line, as determined using overlapping LMP1 and LMP2 peptide pools in ELISPOT assays. Twenty-four patients with EBV+ Hodgkin’s Lymphoma and non-Hodgkin Lymphoma have been treated on dose escalation studies. 16 with LMP2 CTLs and 8 with LMP1/2 CTLs. No immediate toxicity was observed. After CTL infusion, increased numbers of LMP-specific T cells were detected in the blood of 15/22 evaluable patients, (range 2 to 70 fold) persisting for up to 3 months. Additionally, two patients had lymph node biopsies 3–6 months post CTL, which showed selective accumulation of LMP2-multimer positive cells in lymph nodes. 12/13 high-risk and/or multiply relapsed patients who received LMP-CTL as adjuvant treatment after chemotherapy remain in remission for a median of 2years (range &gt;3months to &gt;5years) after CTL. 11 patients had detectable disease at the time of CTL, 2 of these had progressive disease by 8 weeks and 9 had clinical responses. The median duration of the clinical responses is 1 year with one stable disease (&gt;12months), one partial response (36 months) and 7 complete responses (range 9 months to &gt;4.5 years). One of the complete responders was biopsied 7 weeks after receiving CTL, which were predominantly CD4+ (92%). Increased CD4+ T cells were seen compared to pre-CTL biopsy specimens and imaging studies confirmed remission. In conclusion, immunotherapy with CTL targeting LMP antigens is well tolerated in patients with EBV+ lymphoma and infused LMP-CTL can accumulate at tumor sites and induce complete and sustained clinical responses.