EBV is associated with Hodgkin’s disease as well as NHL in immunocompetent patients,28,29
with the majority of EBV-associated lymphomas in these patients being type II latency lymphomas ().
Latency of Epstein-Barr virus (EBV)-positive lymphomas developing in immunocompetent hosts.
Hodgkin’s lymphoma is a unique malignancy as the bulk of the tumour is composed of normal cells within which the malignant Hodgkin-Reed-Sternberg cells are found. The cellular origin of the neoplastic cells has been controversial, but most studies support an origin from germinal centre B cells.30
EBV is the only infectious agent that has been associated consistently with Hodgkin’s disease, and EBV-encoded RNA is detected in the Hodgkin-Reed-Sternberg cells in up to 40% of cases.31
Other EBV-positive B-cell lymphomas express type II latency including large B-cell lymphoma, CD30+ Ki-1+ anaplastic large cell lymphoma of B-cell type, T-cell-rich B-cell NHL and lymphatoid granulomatosis. The association of these lymphomas with EBV varies between 10% and 95% ().
EBV-associated NK/T-cell lymphomas include extranodal NK/T-cell lymphoma (nasal type), angioimmunoblastic lymphoma and large granular lymphocyte leukaemia/lymphoma (NK- or T-cell type). The neoplastic cells of EBV-positive T- and NK-cell lymphomas have a cytotoxic phenotype and are often associated with haemophagocytosis.
While chemotherapy and radiation remain the mainstays in the initial treatment of Hodgkin’s disease and NHL, patients with relapsed disease or those who fail to enter remission are rarely cured using these conventional methods.32,33
Therefore, approaches using adoptive immunotherapies offer very attractive alternative options for this subgroup of patients. Another strategy would be to use vaccination to enhance the proliferation of endogenous or adoptively transferred EBV-specific T cells. Approaches using peptides or DNA as the source of EBV antigen, and LCLs or dendritic cells (DCs) as antigen-presenting cells are being explored in preclinical settings.
Type II latency is associated with expression of multiple EBV-associated proteins which serve as potential targets for cellular immunotherapy Adoptive T-cell immunotherapy approaches to treating type II latency EBV-positive lymphomas have been evaluated in both the allogeneic and autologous settings.
EBV-HD and NHL develop in the immunocompetent host where viral gene expression is limited to immunosubdominant proteins including LMP1 and LMP2, which are weak targets for CTL activity, thereby allowing malignant cells to evade the immune system. In the initial studies, the authors’ group adapted the approach that had proved successful in type III latency EBV tumours. CTLs were generated from patients with relapsed EBV-HD using EBV-transformed B cells (LCLs) as the antigen-presenting cell. However, the frequency of T-cell clones recognizing the LMP2 antigens was relatively low in the majority of polyclonal EBV-CTL lines generated using this approach.34,35
In a phase I dose-escalation study, the authors’ group evaluated the use of autologous EBV-specific CTLs in 14 patients with relapsed EBV-HD. Seven of the patients received EBV-specific CTLs which were gene marked using a retrovirus expressing the neomycin resistance gene to allow tracking of persistence. Clinically, administration of EBV-specific CTLs was well tolerated and resulted in anti-tumour activity as evidenced by five complete remissions (two of whom had detectable disease at the time of CTL infusion), one partial response and five with stable disease. Tetramer and functional analyses revealed that T cells reactive against LMP2 expanded in peripheral blood following infusion, and could track to sites of disease.36
In addition, the ability to track the gene-marked CTLs proved that infused effector cells could expand by several logarithms in vivo, with persistence up to 12 months.34
Another group administered EBV-specific CTLs to three patients with extranodal NK/T-cell lymphoma and saw disease stabilization that persisted for over 3 years in two patients.37
As it is difficult to expand autologous CTLs in sufficient numbers for heavily pretreated patients, partially HLA-matched allogeneic EBV-specific CTLs were used in a phase I study in patients with relapsed EBV-HD. Five of six patients had a reduction in measurable disease, with a maximum duration of response of 22 months. However, these patients also received fludarabine conditioning prior to CTL infusion, making it difficult to interpret the role of CTLs in the clinical response. Additionally, this approach may be limited by the short-term persistence of the allogeneic cells, as donor EBV-specific CTLs could not be detected in vivo.38
Since the authors’ previous experience using EBV-specific CTLs for relapsed EBV-HD generated CTL lines with low frequencies of cells specific for the tumour-associated EBV antigen LMP2, it has been hypothesized that expanding CTLs specifically targeting these tumour-associated EBV antigens may result in greater tumour-specific activity.39–42
It has been shown that LMP2-specific CTLs can be generated from normal donors using DCs as potent antigen-presenting cells that were genetically modified to express LMP2A after transduction with a recombinant adenovirus encoding LMP2A (Ad5LMP2A).39
However, this approach required the generation of large numbers of DCs to expand LMP2-specific CTLs to numbers required for a clinical trial. This strategy was therefore not practical in these heavily pretreated patients, so a subsequent modification to the manufacturing protocol was made. The resultant procedure was the use of Ad5f35LMP2-transduced DCs for the initial stimulation, followed by stimulation with LCLs that had been genetically modified to overexpress LMP2a by transduction with the same vector ().43
Generation of LMP2-specific cytotoxic T lymphocytes (CTLs)
The authors have infused LMP2-specific CTLs manufactured using this methodology in a dose-escalation study for 16 patients with high-risk EBV-HD and NHL.44
Using overlapping peptide pools for LMP2, a significantly increased number of LMP2-specific CTLs was detected in the LMP2-CTL lines compared with EBV-CTL lines generated with genetically unmodified LCLs from the same patients.
Patients received doses from 4×107
. Ten patients received CTLs as adjuvant therapy, with nine sustaining a complete remission for 1 to >4 years.44
In addition, five of six patients with active, relapsed disease at the time of LMP2-specific CTL administration showed evidence of tumour response, with complete regression of measurable disease in four patients that was sustained for more than 9 months. No short-or long-term toxicities were observed after CTL infusion.44
To expand on this concept, the authors are now using autologous T cells enriched for both LMP1 and LMP2 for the treatment of EBV-positive lymphoma, with a clinical trial currently actively enrolling.