CLINICAL TRIAL: ADMINISTRATION OF LMP1- AND LMP2-SPECIFIC CYTOTOXIC T-LYMPHOCYTE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This Phase I dose-escalation trial is designed to evaluate the safety of escalating doses of autologous LMP1- and LMP2-specific CTL. We have been using a modified version of the continual reassessment method (mCRM) in the design of new trials on T-cell based therapies. Our rationale for the use of this model-based, adaptive design stems from our considerable experience with these cytostatic therapies, which unlike cytotoxic agents, have shallow dose-toxicity profiles over the range of doses proposed. As such, designs with more accelerated dose escalations should not compromise the safety of our patients. Simulations based on previous T-cell immunotherapy trials, indicate that this design provides higher probabilities of declaring the appropriate dose as the MTD and allows smaller numbers of patients to be accrued at lower and possibly suboptimal dose levels. More importantly, our simulations indicate that use of the mCRM strategy will not lead to increased toxicities, compared with standard 3+3 designs. There is no randomization or control groups. We and others have demonstrated the feasibility of CTL therapy for EBV-positive NPC in immunocompetent patients, providing preliminary evidence of anti-tumor activity of EBV-CTL in this patient population.34,35 Not all patients responded, however, suggesting the need for further improvement. We propose that CTL failure can be overcome by increasing the specificity of the infused CTL product. That is, infusion of CTL specific for LMP1 and LMP2 will produce greater clinical benefit than EBV-specific CTL. The rationale for this approach is straight forward: EBV-specific CTL lines generated by standard methods are dominated by T-cell clones not reactive to the subdominant EBV proteins LMP1 and LMP2 expressed in NPC. SPECIFIC AIMS a. To determine the safety of autologous LMP1- and LMP2- specific cytotoxic T-lymphocytes (CTL) in patients with EBV-positive nasopharyngeal carcinoma (NPC). b. To obtain information on the expansion, persistence and anti-tumor effects of autologous LMP1- and LMP2 specific CTL in patients with EBV-positive NPC. BACKGROUND AND SIGNIFICANCE EBV-associated malignancies EBV is a latent gamma herpesvirus that infects more than 90% of the world's population.1-4 During primary infection in the oropharynx, EBV establishes lifelong latency in B cells that are preferentially infected through the CD21 receptor and subsequently programmed to the B-cell memory compartment. The virus persists as an episome in infected B cells, establishing a latent infection characterized by the expression of only a limited array of subdominant EBV antigens. Healthy individuals mount a vigorous humoral and cellular immune response to primary EBV infection. Although antibodies to the viral membrane proteins neutralize virus infectivity, the cellular immune response, consisting of CD4+ and CD8+ T cells, is essential for controlling both primary infection and latent EBV-infected cells, and studies with tetramer technology have shown that high frequencies of EBV-specific CD8+ T cells persist long-term. Latent EBV infection is associated with a heterogeneous group of malignancies. In type I latency, found in EBV-positive Burkitt lymphoma, only EBNA1, EBERs and the BamHI-A rightward transcripts (BARTs) are expressed. Type II latency, characterized by EBNA1, LMP1, LMP2, EBERs and BARTs expression, is found in EBV-positive Hodgkin disease, NPC, and peripheral T/NK-cell lymphomas. While malignancies associated with type I and II latency occur in individuals with minimal or only no immune dysfunction, type III latency is associated with malignancies in severely immunocompromised patients. It is characterized by the expression of the entire array of EBV latency genes, including EBNAs 1, 2, 3A, 3B, 3C, leader protein (LP), LMP1, LMP2, EBERs and BARTs. This pattern of gene expression is found in EBV-associated lymphoproliferative disease (EBV-LPD) developing after hematopoietic stem cell transplantation (HSCT) or solid organ transplant and in EBV-positive lymphomas occurring in patients with congenital immunodeficiency or human immunodeficiency virus (HIV) infection. In addition type III latency can also be found in lymphoblastoid cell lines (LCLs), which can be readily prepared by infecting B cells in vitro with EBV and were instrumental in generating of EBV-specific CTL for the prophylaxis and therapy of EBV-LPD. Nasopharyngeal Cancer: Background and Association with EBV Nasopharyngeal carcinoma is a malignant disease with a variable range of incidence depending on age, geographical place, race and EBV exposure.5;6 It has an annual incidence of nearly 1 case per 100,000 children 5 years are obtained when treated with combined modality therapy for stage IV disease.6 Of major concern are the treatment-related morbidity and mortality of the current treatment regimens. Late medical complications after treatment for NPC include growth hormone deficiency, hypothyroidism and pulmonary fibrosis.7-11 A large retrospective study in Taiwan of a cohort of 1,549 patients was performed to assess the risk of secondary malignancies in NPC patients post radiotherapy +/- chemotherapy. The patients ranged in age from 10-80 years (median 46.3 years) with a maximum follow-up of 16 years. Fatal neoplastic complications included secondary leukemia related to alkylating agent chemotherapy and head and neck cancers (most likely radiation induced), and gastric cancer. It is therefore desirable to develop novel therapies that could improve disease free survival in relapsed/refractory patients and which might ultimately reduce the incidence of long-term treatment related complications in all patients. The etiological factors of endemic NPC include EBV, environmental risk factors and genetic susceptibility. The etiological link between NPC and EBV was first based on serological evidence and more recently on EBV-DNA measurements in plasma.12-22 The association between EBV and NPC was subsequently confirmed by showing that EBV-DNA was present in the NPC tumor cells and that EBV-DNA in NPC biopsy samples is clonal, arising from a single EBV infected cell. EBV has been detected in virtually all cases of undifferentiated non-keratinizing NPC. On the other hand, squamous cell NPC shows a geographical variability with regard to their EBV association with more EBV-positive tumors seen in high incidence areas such as Asia. In addition, in squamous cell NPC, additional factors contributing to the pathogenic process such as smoking and human papilloma virus (HPV) have been identified. Nevertheless, there is a strong association of NPC with EBV. Thus NPC may represent a final common response to several pathological processes that include viral infections, occupational and environmental stimuli with, perhaps, a genetic contribution. The Cellular Immune Response is Critical for Controlling Persistent EBV Infection Both humoral and cell mediated immunity have important roles to play in the control of EBV.1-4 EBV-specific CD8+ CTL are thought to be the most important defense mechanism against outgrowth of EBV-infected B cells. These cells recognize peptide fragments, derived from viral antigens, expressed on the surface of antigen presenting cells in association with MHC molecules. Studies using EBV latent proteins expressed in vaccinia constructs have shown that a number of antigens can serve as targets and that the pattern of antigens recognized is dependent on the individual's HLA type.