Nasopharyngeal carcinoma (NPC) is endemic in China and Southeast Asia where it is tightly associated with infections by Epstein-Barr virus (EBV). The role of tumor-associated viral antigens in NPC renders it an appealing candidate for cellular immunotherapy. In earlier preclinical studies, a novel adenoviral vector-based vaccine termed AdE1-LMPpoly has been generated that encodes EBV nuclear antigen-1 (EBNA1) fused to multiple CD8 þ T-cell epitopes from the EBV latent membrane proteins, LMP1 and LMP2. Here, we report the findings of a formal clinical assessment of AdE1-LMPpoly as an immunotherapeutic tool for EBV-associated recurrent and metastatic NPC. From a total of 24 patients with NPC, EBV-specific T cells were successfully expanded from 16 patients with NPC (72.7%), whereas six patients with NPC (27.3%) showed minimal or no expansion of virus-specific T cells. Transient increase in the frequencies of LMP1&2-and EBNA1-specific T-cell responses was observed after adoptive transfer to be associated with grade I flu-like symptoms and malaise. The time to progression in these patients ranged from 38 to 420 days with a mean time to progression of 136 days. Compared with patients who did not receive T cells, the median overall survival increased from 220 to 523 days. Taken together, our findings show that adoptive immunotherapy with AdE1-LMPpoly vaccine is safe and well tolerated and may offer clinical benefit to patients with NPC. Cancer Res; 72(5); 1116-25. Ó2012 AACR.

Glioblastoma multiforme (GBM) is one of the most aggressive human brain malignancies. Even with optimal treatment, median survival is less than 6 months for patients with recurrent GBM. Immune-based therapies have the potential to improve patient outcome by supplementing standard treatment. Expression of human cytomegalovirus (CMV) antigens in GBM tissues provides the unique opportunity to target viral antigens for GBM therapy. Here, we report findings of a formal clinical assessment of safety and potential clinical efficacy of autologous CMV-specific T-cell therapy as a consolidative treatment for recurrent GBM. From a total of 19 patients with recurrent GBM, CMV-specific T cells were successfully expanded from 13 patients (68.4%), 11 of whom received up to four T-cell infusions. Combination therapy based on T-cell infusion and chemotherapy was well tolerated, and we detected only minor adverse events. The overall survival of these patients since first recurrence ranged from 133 to 2,428 days, with a median overall survival of 403 days. Most importantly, 4 of 10 patients that completed the treatment remained progression free during the study period. Furthermore, molecular profiling of CMV-specific T-cell therapy from these patients revealed distinct gene expression signatures, which correlated with their clinical response. Our study suggests that a combination therapy with autologous CMV-specific T cells and chemotherapy is a safe novel treatment option and may offer clinical benefit for patients with recurrent GBM. Cancer Res; 74(13); 3466–76. ©2014 AACR.

Role of the funding source:The funders of the study had no role in the study design, data collection, data analysis, data interpretation, or the writing of the manuscript. The corresponding authors had full access to all the data in the study and had final responsibility for the decision to submit for publication. Conflict of interest: MPP has received consulting fees and research funding from Atara Biotherapeutics and is a member of the Neurology Clinical Advisory Panel of Atara Biotherapeutics. CS has received consulting fees from Atara Biotherapeutics. SB is a member of the advisory boards of Roche, Sanofi Genzyme, Merck, and Teva. KAG has received personal fees from Roche and travel support from Sanofi Aventis. AS has received educational and travel support from Merck Serono Australia. KDH is a member of the advisory boards of Merck Serono Australia, Biogen Australia, and Roche Australia. BTA is an employee of, and owns equity shares in, Atara Biotherapeutics. SRB has received a license fee payment from Atara Biotherapeutics. AC has received a consulting fee from Sanofi Genzyme. RK is a consultant and member of the scientific advisory board of Atara Biotherapeutics and has received a license fee payment and research funding from Atara Biotherapeutics.

Purpose: The incidence of malignant melanoma continues to increase worldwide; however, treatment of metastatic melanoma remains unsatisfactory, and there is an urgent need for development of effective targeted therapeutics. A potential biological target on the surface of malignant melanoma cells is the up-regulated expression of intercellular adhesion molecule (ICAM)-1 and decay-accelerating factor (DAF), relative to surrounding benign tissue. Coxsackievirus A21 (a common cold virus) targets and destroys susceptible cells via specific viral capsid interactions with surfaceexpressed virus receptors comprising ICAM-1 and DAF.Experimental Design: The oncolytic capacity of a genetically unmodified wild-type common cold-producing human enterovirus (Coxsackievirus A21, CAV21) was assessed against in vitro cultures and in vivo xenografts of malignant human melanoma cells.Results: In vitro studies established that human melanoma cells endogenously express elevated levels of ICAM-1/DAF and were highly susceptible to rapid viral oncolysis by CAV21 infection, whereas ICAM-1/DAF-expressing peripheral blood lymphocytes were refractile to infection. In vivo studies revealed that the tumor burden of nonobese diabetic severe combined immunodeficient mice bearing multiple s.c. melanoma xenografts was rapidly reduced by oncolysis mediated by a single administration of CAV21.The antitumor activity of CAV21 was characterized by highly efficient systemic spread of progeny CAV21, with oncolysis of tumors also occurring at sites distant to the primary site of viral administration.Conclusions: Overall, the findings presented herein demonstrate an important proof of principle using administration of replication-competent CAV21 as a potential biological oncolytic agent in the control of human metastatic melanoma.

The data presented here demonstrate for the first time the clinical safety of CMV-specific adoptive T-cell therapy and its potential therapeutic benefit for SOT patients with recurrent and/or drug-resistant CMV infection or disease.

The frequent detection of human cytomegalovirus (CMV) antigens in glioblastoma multiforme (GBM) has raised the possibility of exploiting CMV-specific T-cell immunotherapy to control this disease in CMV-seropositive patients. Here, we have conducted a comprehensive ex vivo profiling of CMV-specific CD8 þ T-cell responses in a cohort of GBM patients. Of the patients analyzed, approximately half exhibited serological evidence of past infection with CMV. Although no CMV-specific CD8 þ T-cell responses could be detected in the serologically negative GBM patients, virus-specific CD8 þ T-cell responses were detected in all seropositive GBM patients. Using major histocompatibility complex-peptide multimers, the frequency of CMV-specific T-cells in the patients detected ranged from 0.1 to 22% of CD8 þ T-cells and a high proportion of these cells were positive for the human natural killer-1 glycoprotein CD57. Furthermore, ex vivo polychromatic functional analysis of the CMV-specific T-cells from GBM patients revealed that large proportions of these cells were unable to produce multiple cytokines (macrophage inflammatory protein (MIP)-1b, tumor necrosis factor (TNF)a and interferon (IFN)c) and displayed limited cytolytic function (CD107a mobilization) following stimulation with CMV peptide epitopes. However, in vitro stimulation with CMV peptide epitopes in the presence of cC cytokine dramatically reversed the polyfunctional profile of these antigen-specific T-cells with high levels of MIP-1b, TNFa, IFNc and CD107a mobilization. Most importantly, adoptive transfer of these in vitro-expanded T-cells in combination with temozolomide (TMZ) therapy into a patient with recurrent GBM was coincident with a long-term disease-free survival. These studies provide an important platform for a formal assessment of combination therapies based on CMV-specific T-cells and TMZ for recurrent GBM.

Defective control of Epstein–Barr virus (EBV) infection by cytotoxic CD8+ T cells might predispose to multiple sclerosis (MS) by allowing EBV-infected autoreactive B cells to accumulate in the central nervous system. We have treated a patient with secondary progressive MS with in vitro-expanded autologous EBV-specific CD8+ T cells directed against viral latent proteins. This adoptive immunotherapy had no adverse effects and the patient showed clinical improvement with reduced disease activity on magnetic resonance imaging and decreased intrathecal immunoglobulin production. This is the first report of the use of EBV-specific adoptive immunotherapy to treat MS or any other autoimmune disease.

Latent membrane antigen 1 and -2 (LMP-1/2)-specific CD8؉ T cells from newly diagnosed and relapsed Hodgkin's lymphoma (HL) patients display a selective functional impairment. In contrast, CD8؉ T cells specific for Epstein-Barr virus (EBV) nuclear proteins and lytic antigens retain normal T-cell function. Reversion to a dysfunctional phenotype of LMP-1/2-specific T cells is coincident with the regression of HL. To delineate the potential basis for this differential susceptibility for the loss of function, we have carried out a comprehensive functional analysis of EBV-specific T cells using ex vivo multiparametric flow cytometry in combination with assessment of antigen-driven proliferative potential. This analysis revealed that LMP-1/2-specific T cells from healthy virus carriers display a deficient polyfunctional profile compared to that of T cells specific for epitopes derived from EBV nuclear proteins and lytic antigens. Furthermore, LMP-specific T-cells are highly susceptible to galectin-1-mediated immunosuppression and are less likely to degranulate following exposure to cognate peptide epitopes and poorly recognized endogenously processed epitopes from virusinfected B cells. More importantly, ex vivo stimulation of these T cells with an adenoviral vector encoding multiple minimal CD8 ؉ T-cell epitopes as a polyepitope, in combination with a ␥C cytokine, interleukin-2, restored polyfunctionality and shielded these cells from the inhibitory effects of galectin-1.