Leone Beagley

Effective Treatment of Metastatic Forms of Epstein-Barr Virus–Associated Nasopharyngeal Carcinoma with a Novel Adenovirus-Based Adoptive Immunotherapy

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(+) 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.

Autologous T cell Therapy for Cytomegalovirus as a Consolidative Treatment for Recurrent Glioblastoma

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.

Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement.

The manufacture of clinical grade cellular products for adoptive immunotherapy requires ex vivo culture and expansion of human T cells. One of the key components in manufacturing of T cell therapies is human serum (HS) or fetal bovine serum (FBS), which can potentially expose immunotherapy recipient to adventitious infectious pathogens and are thus considered as non‐cGMP compliant for adoptive therapy. Here we describe a novel xeno‐free serum replacement (SR) with defined components that can be reproducibly used for the production of clinical grade T‐cell therapies in combination with several different cell culture media. Dynabeads CD3/CD28 Cell Therapy System (CTS)‐activated or antigen‐specific T cells expanded using the xeno‐free SR, CTS Immune Cell SR, showed comparable growth kinetics observed with cell culture media supplemented with HS or FBS. Importantly the xeno‐free SR supplemented medium supported the optimal expansion of T cells specific for subdominant tumour‐associated antigens and promoted expansion of T cells with central memory T‐cell phenotype, which is favourable for in vivo survival and persistence following adoptive transfer. Furthermore, T cells expanded using xeno‐free SR medium were highly amenable to lentivirus‐mediated gene transduction for potential application for gene‐modified T cells. Taken together, the CTS Immune Cell SR provides a novel platform strategy for the manufacture of clinical grade adoptive cellular therapies.

Ex vivo functional analysis, expansion and adoptive transfer of cytomegalovirus-specific T-cells in patients with glioblastoma multiforme

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+ 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+ T‐cell responses could be detected in the serologically negative GBM patients, virus‐specific CD8+ 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+ 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)‐1β, tumor necrosis factor (TNF)α and interferon (IFN)γ) 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 γC cytokine dramatically reversed the polyfunctional profile of these antigen‐specific T‐cells with high levels of MIP‐1β, TNFα, IFNγ 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.

Acquisition of Polyfunctionality by Epstein-Barr Virus-Specific CD8+ T Cells Correlates with Increased Resistance to Galectin-1-Mediated Suppression

ABSTRACT Latent membrane antigen 1 and -2 (LMP-1/2)-specific CD8+ T cells from newly diagnosed and relapsed Hodgkins 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 virus-infected 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 γC cytokine, interleukin-2, restored polyfunctionality and shielded these cells from the inhibitory effects of galectin-1.

Epstein–Barr virus-specific adoptive immunotherapy for progressive multiple sclerosis

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.

Discerning regulation of cis- and trans -presentation of CD8 + T-cell epitopes by EBV-encoded oncogene LMP-1 through self-aggregation

Activation of the nuclear factor-kappaB pathway by Epstein-Barr virus-encoded latent membrane protein-1 (LMP-1) leads to an up-regulation of the major histocompatibility complex class I antigen-processing pathway. Paradoxically, LMP-1 itself induces a subdominant CD8+ T-cell response and appears to have evolved to avoid immune recognition. Here we show that, although expression of LMP-1 in human cells dramatically enhanced the trans-presentation of CD8+ T-cell epitopes, cis-presentation of LMP-1-derived epitopes was severely impaired. Testing of a series of LMP-1 mutants revealed that deletion of the first transmembrane domain of LMP-1, which prevented self-aggregation, significantly enhanced cis-presentation of T-cell epitopes from this protein, whereas it lost its ability to up-regulate trans-presentation. Interestingly, we also found that cis-presentation of LMP-1 epitopes was rescued by blocking the proteasome function. Taken together, these results delineate a novel mechanism of immune evasion, which renders a virally encoded oncogene inaccessible to the conventional major histocompatibility complex class I pathway limiting its cis-presentation to effector cells.

Adoptive T-cell immunotherapy for ganciclovir-resistant CMV disease after lung transplantation

Infections with cytomegalovirus (CMV) can induce severe complications after solid organ transplantation (SOT). The prognosis for ganciclovir‐resistant CMV infection and disease is particularly poor. Whereas adoptive transfer of CMV‐specific T cells has emerged as a powerful tool in hematopoietic stem cell transplant patients, its translation into the SOT setting remains a significant challenge as underlying immunosuppression inhibits the virus‐specific T‐cell response in vivo. Here, we demonstrate successful expansion and adoptive transfer of autologous CMV‐specific T cells from a seronegative recipient of a seropositive lung allograft with ganciclovir‐resistant CMV disease, resulting in the long‐term reconstitution of protective anti‐viral immunity, CMV infection, disease‐free survival and no allograft rejection.

Pre-emptive and therapeutic adoptive immunotherapy for nasopharyngeal carcinoma: Phenotype and effector function of T cells impact on clinical response

ABSTRACT Adoptive T cell therapy has emerged as a powerful strategy to treat human cancers especially haematological malignancies. Extension of these therapies to solid cancers remains a significant challenge especially in the context of defining immunological correlates of clinical responses. Here we describe results from a clinical study investigating autologous Epstein-Barr virus (EBV)-specific T cells generated using a novel AdE1-LMPpoly vector to treat patients with nasopharyngeal carcinoma (NPC) either pre-emptively in at-risk patients with no or minimal residual disease (N/MRD) or therapeutically in patients with active recurrent/metastatic disease (ARMD). Tolerability, safety and efficacy, including progression-free survival (PFS) and overall survival (OS), were evaluated following adoptive T-cell immunotherapy. Twenty-nine patients, including 20 with ARMD and nine with N/MRD, successfully completed T-cell therapy. After a median follow-up of 18.5 months, the median PFS was 5.5 months (95% CI 2.1 to 9.0 months) and the median OS was 38.1 months (95% CI 17.2 months to not reached). Post-immunotherapy analyses revealed that disease stabilization in ARMD patients was significantly associated with the functional and phenotypic composition of in vitro-expanded T cell immunotherapy. These included a higher proportion of effector CD8+ T-cells and an increased number of EBV-specific T-cells with broader antigen specificity. These observations indicate that adoptive immunotherapy with AdE1-LMPpoly-expanded T cells stabilizes relapsed, refractory NPC without significant toxicity. Promising clinical outcomes in N/MRD patients further suggest a potential role for this approach as a consolidation treatment following first-line chemotherapy.

Novel autologous T-cell therapy for drug-resistant cytomegalovirus disease after lung transplantation.

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