c Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC). We assess the safety and tolerability of adoptive transfer of autologous cytotoxic T lymphocytes (CTLs) specific for the EBV latent membrane protein (LMP) in a patient with recurrent NPC. After infusion, the majority of pulmonary lesions were no longer evident, although the primary tumor did not regress. CASE REPORT In 2002, a middle-aged man (42 years old) was diagnosed with stage IV (T4 N2c M1) Epstein-Barr virus (EBV)-positive undifferentiated carcinoma with all of the pathological characteristics of nasopharyngeal carcinoma (NPC). This NPC patient (HLA A11, B60, B62) presented with multiple pulmonary metastases. He underwent a course of chemotherapy (3 cycles of induction cisplatin and fluorouracil [5FU]) and high-dose palliative radiotherapy to the nasopharynx and upper (60 Gy in 30 fractions over 6 weeks) and lower (50 Gy) cervical nodes, followed by 3 more cycles of cisplatin and 5FU. This treatment resulted in a complete radiological regression of pulmonary metastases and partial regression of NPC at the primary site. Three years later, the pulmonary metastases became radiologically apparent again and were treated with six cycles of carboplatin and 5FU. Following this, six cycles of gemcitabine were administered. In each case, while a complete regression of the lung metastases was seen, the lesion subsequently recurred, prompting a further nine cycles of gemcitabine. However, despite this, his disease progressed, suggesting resistance to gemcitabine. Carboplatin was added for another nine cycles in late 2006; radiological evidence demonstrated disease progression and resistance to chemotherapy.In September 2007, the patient commenced adoptive immunotherapy, for which he received six infusions (on a fortnightly basis, during a 3-month period) of autologous EBV-specific cytotoxic T lymphocytes (CTLs) directed toward the latent membrane protein (LMP) of this virus (1). This treatment resulted in regression of the majority of pulmonary metastases. To generate these LMP-specific cells, autologous peripheral blood mononuclear cells (PBMCs) were screened for reactivity to two different HLAcompatible LMP 1-and 2-specific CTL epitopes. A response against the LMP 2 peptide epitope SSCSSCPLSK (SSC) (1) was detected in this patient. Subsequently, autologous PBMCs were stimulated with SSC-coated autologous lymphoblastoid cell lines (LCLs) and expanded to yield large numbers of LMP-specific CTL cultures (see the legend to Fig. 1 for details). These cultures were screened for specificity by 51 Cr release assays and for phenotype by flow cytometry after 21 days. In the functional 51 Cr release studies, a single LMP 2 response to the HLA A11-restricted peptide (SSC) was detected (2), as measured by specific 51 Cr release of autologous phytohemagglutinin (PHA) target cells coated with SSC or another LMP-specific peptide epitope, IEDPPFNSL (IED) (Fig. 1) (1). Flow cytometry revealed that these patient-derived CTLs contained a high percentage o...

Epstein-Barr virus (EBV) is associated with several malignant diseases including nasopharyngeal carcinoma (NPC), a common neoplasm throughout southeast Asia. Radiotherapy and chemotherapy can achieve remission, but a reemergence of disease is not uncommon. Therefore, there is a need for specific therapies that target the tumor through the recognition of EBV antigens. In NPC, latent membrane protein 1 (LMP1) and LMP2 offer the best opportunity for specific targeting since they are typically expressed and T-cell determinants in each of these proteins have been defined. We have attempted to maximize the opportunity of incorporating every possible CD4 and CD8 determinant in a single formulation. We have achieved this by generating a scrambled protein incorporating random overlapping peptide sets from EBNA1, LMP1, and LMP2, which was then inserted into a replication-deficient strain of adenovirus (adenovirus scrambled antigen vaccine [Ad-SAVINE]). This report describes the construction of this Ad-SAVINE construct, its utility in generating LMP1 and LMP2 responses in healthy individuals as well as NPC patients, and its capacity to define new epitopes. This formulation could have a role in NPC immunotherapy for all ethnic groups since it has the potential to activate all possible CD4 and CD8 responses within EBNA1 and LMPs.

Nasopharyngeal carcinoma (NPC) is Epstein-Barr virus (EBV) positive in all undifferentiated cases, expressing the latency II phenotype of latent membrane proteins (LMPs) 1 and 2, in addition to EBV nuclear antigen (EBNA) 1. Several studies have attempted to treat NPC with EBV-specific cytotoxic T lymphocyte (CTL) with a partial response. To improve this therapy, there is a need to expand CTL targeted to the latency II antigens of EBV, rather than the immunodominant EBV nuclear antigens 3-6 peptides typically expanded by lymphoblastoid cells. In order to maximize the expansion of LMP-specific CTL in vitro for use in adoptive immunotherapy of nasopharyngeal carcinoma patients, we used lymphoblastoid cell lines coated with synthetic peptides corresponding to CTL determinants from the LMP proteins. We investigated several issues pertaining to the expansion of an immunologically weak CTL response, including peptide and interleukin-2 concentration, and screening assays for selecting the optimal peptide for use in expansion of LMP-specific CTL. Although screening of ex vivo peripheral blood mononuclear cells did not prove to be useful in the selection of an LMP peptide for use in CTL cultures, the peptide and interleukin-2 concentrations were critical for the maximum expansion of CTL. Therefore, it is imperative that stimulation protocols are optimized for the expansion of LMP-specific CTL.