This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator. I. HYPOTHESIS We have previously used adoptively transferred EBV-specific CTL to prevent and treat EBV-lymphoproliferative disease after stem cell transplant. Recently we have investigated whether the same approach could be of value to treatment for EBV+ Nasopharyngeal cancer(NPC). We have been successful in generating autologous EBV-specific CTL lines for NPC patients using a standardized method. Adoptive transfer of EBV-specific CTLs has proven safe in all 10 NPC patients treated. In six patients with refractory or relapsed disease, CTL infusion resulted in 1 complete response, 2 partial responses and stabilization of disease for >6 months in 1 case with 2 patients too early to evaluate. Four patients who were treated in remission have remained disease free for >5->13 months.The above results are promising and indicate the potential anti-tumor activity of EBV-specific CTLs in this patient group. However, not all patients with bulky disease responded completely. We believe this failure was due in part to the failure of infused T lymphocytes to undergo adequate expansion in vivo. The size of the T cell compartment is maintained at a steady state by a number of potent homeostatic mechanisms. If a host is made lymphopenic, mature T cells will proliferate to restore the steady state5. Hence, in the recipients of T cell depleted stem cell transplants we treated previously, we have found that infused EBV specific T cells expand by 4 logs or more. In contrast, when EBV specific T cells are given to NPC patients, in whom the T cell compartment is already replete, expansion of adoptively transferred T lymphocytes is lower, by 2 orders of magnitude or more. Published data suggest that in vivo depletion of T cells with cytotoxic drugs, as in stem cell transplantation, can permit massive expansion of subsequently infused T cells with anti-cancer activity, since under those circumstances homeostatic mechanisms work to restore lymphoid compartments. We now propose to take advantage of these homeostatic mechanisms in patients with advanced or refractory NPC. We will deplete their lymphoid compartment using short-lived CD45 monoclonal antibodies (MAbs), which we have shown (in mouse and in man) can profoundly deplete lymphocytes in peripheral blood and lymphoid organs, whilst sparing hematopoietic progenitor cells. Subsequent adoptive transfer of EBV-specific CTL post MAb induced T cell depletion should result in expansion of the infused cells to restore the T cell compartment. Moreover, the expanded cells should enter and function unimpeded in tumor sites depleted of regulatory T cells. II.
SPECIFIC AIMS Aim 1) To perform a phase I study of EBV-specific CTL infusion before and after administration of CD45 monoclonal antibody in patients with NPC. Primary clinical endpoints of this study will be safety and hematopoietic and immune reconstitution. In our previous studies in animal and humans, lytic CD45 MAbs safely depleted lymphocytes of all lineages, but spared hematopoietic progenitor cells, which have a low level of target antigen expression. The antibodies have a half-life of circa 8 hours and therefore cause only transient neutropenia with no measurable stem cell toxicity. The degree of T cell depletion will be determined by total and differential T cell counts and phenotyping. We plan to monitor hematopoietic and immune reconstitution by measuring responses to recall antigens such as cytomegalovirus (CMV) and tetanus toxoid (TT) using tetramers and ELISPOT. The clinical implications of transient lymphopenia in these patients will be monitored with emphasis on the occurrence of potential side effects such as infection.
Aim 2) To analyze the effect of CD45-mediated host T cell depletion on the expansion, persistence and anti-tumor effector function of subsequently infused EBV-specific CTL.
This aim will be achieved by comparing expansion, persistence and function of infused T cells given to the same patient before and after CD45 MAb treatment. We will use tetramer staining and functional ELISPOT assays to monitor the expansion and persistence of adoptive transferred EBV-specific CTL. Anti-tumor effects will be evaluated by measuring cell-free EBV-DNA in plasma, tumor size on imaging and clinical outcome III. BACKGROUND AND SIGNIFICANCE 1. Nasopharyngeal carcinoma1.1 NPC treatment and outcome. The incidence of nasopharyngeal carcinoma varies widely with geographical location, with an incidence as high as 50 per 100,000 men in Southern China and less than 1 per 100,000 adults in low incidence areas including the USA10. NPC is a radiosensitive tumor, and by modern imaging and radiation techniques, local control rates of greater than 80% are obtained11. Despite this, distant failures remain the major problem in patients with loco-regional bulky disease, which is the most common form at presentation due to the lack of early symptoms. At present, radiotherapy combined with induction or concurrent chemotherapy results in a 5-year survival of 55-70% in patients with advanced stage disease. However, 40-50% of patients relapse12 and the treatment-related morbidity and mortality of the current regimens are of major concern2. 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.1.2 Nasopharyngeal carcinoma and Epstein-Barr virus. Multiple factors including EBV exposure, environmental triggers and genetic susceptibility are thought to play a role in the pathogenesis of NPC13. EBV has been detected in virtually all cases of undifferentiated non-keratinizing NPC14 and in a proportion of squamous cell NPC17. The latter represents a more heterogeneous group of tumors, in which other co-factors such as smoking and human papilloma virus (HPV) contribute to the pathogenic process. The strong association with EBV makes NPC an attractive candidate for immunotherapeutic strategies. Although EBV positive NPC cells lack the expression of the immunodominant EBNA3 antigens, LMP 1 is expressed in 65% of NPC tumors and transcriptional analysis has shown expression of LMP2 mRNA in the majority of tumors16. Therefore, these membrane proteins provide target antigens for immunotherapeutic strategies. Importantly, T lymphocytes specific for LMP2 and to a lesser extent for LMP1 are present in the peripheral blood of NPC patients and could therefore potentially be activated and expanded for immunotherapeutic approaches.2. Immunotherapy with antigen-specific T cells.2.1 Successes. Immunotherapy with T cells has been most successful in stem cell transplant recipients, for whom the normal marrow donors have been used as the source of T cells. Adoptive immunotherapy with donor lymphocyte infusions (DLI) after allogeneic hematopoietic stem cell transplant (HSCT) has provided an effective means of augmenting the graft versus leukemia response to eliminate residual disease and for the treatment of EBV-associated lymphoproliferative diseases occurring after HSCT. However, DLI are associated with a high risk of GvHD. Selectively expanded CMV and EBV-specific CTL have successfully restored immune responses and prevented diseases associated with these viruses without causing GvHD. Infusion of CMV-specific CD4+ and CD8+ clones demonstrated that the survival of the CD8+ CTL was dependent on the presence of CD4+ CMV-specific 'helper' T cells. Similarly, polyclonal EBV-specific CTL, containing both CD4+ and CD8+ T cells survived for up to eighty-six months after infusion and were able to reduce the high virus load observed in about 20% of patients. EBV-CTL also appeared to prevent progression to EBV-lymphoma, since no patient who received prophylactic CTL developed this malignancy, compared to 11.5% of controls2. Further, five of six patients who received CTL as treatment for overt lymphoma achieved complete remissions. In the patient who failed to respond, the tumor was transformed with a virus that had deleted the two CTL epitopes for which the donor CTL line was specific. 2.2 Rationale for immunotherapy of Nasopharyngeal carcinoma with EBV-specific CTLs. A major advantage offered by CTL therapy is its lack of toxicity. If CTL are effective in the treatment of relapsed disease, they could subsequently be used as part of front line therapy for nasopharyngeal carcinoma and allow a reduction in radiotherapy and chemotherapy dosage and hence in long-term sequelae. Since adoptively transferred CTL have proved long-lived and effective in the prevention and treatment of EBV-associated PTLD in HSCT recipients, they have the potential to be beneficial in patients with other EBV-associated malignancies that express viral latency proteins that are potential CTL targets. Importantly, NPC tumor cells express MHC class I molecules as well as the peptide transporters TAP1 and TAP2, and are therefore capable of processing and presenting endogenously synthesized protein in the context of HLA class I MHC molecules, for immune recognition by CTL.3. Disruption of lymphoid homeostasis to facilitate expansion and engraftment of infused CTLs.3.1 Rationale for T cell depletion prior to CTL infusion. Lymphocyte numbers and subset composition are maintained at stable levels in healthy adults. Perturbations of cell numbers due to infection, loss of cells, or other influences are restored to steady-state levels by poorly understood homeostatic mechanisms. Each lymphoid compartment maintains homeostasis more or less independently, so that in T cell deficient mice, B cell numbers remain the same as in normal mice. Similarly, regulation of the na ve T cell subset, which derives from thymic proliferation and maintains T cell receptor diversity, is independent of memory T cell regulation, which is maintained by antigen-independent as well as antigen-dependent mechanisms. After T cells are depleted, lymphoid reconstitution with a fully diversified T cell receptor repertoire is dependent on thymopoiesis, but this continues at a steady rate, regardless of the size of the lymphoid compartment. Conversely, expansion of memory T cells is the major source of peripheral repopulation. Regulation of lymphocyte numbers may be controlled by cytokines or ligands, by counting mechanisms, or by the availability of 'space'. While these mechanisms are bypassed during immune responses, with increases in the size of spleen, lymph nodes and blood, there is a rapid return to a steady state when antigen stimulation subsides. Expansion of infused T cells is likely to be limited in a steady state situation. In the context of a T cell deficit however, infused cells have been shown to proliferate rapidly to restore steady situation5. Animal experiments provide us with important rules regulating the restoration of homeostasis when confronted with a lymphopenic situation. First, T cell proliferation to restore a deficit is not dependent on an antigen stimulus, although activated T cells have a proliferative advantage which is increased in the presence of antigen24. Second, once the peripheral pool is replenished, further expansion is curtailed, which prevents unlimited expansion of infused lymphocytes. Third, rapid repopulation of the peripheral pool can be achieved with a single infusion of mature T lymphocytes, even using a small number of cells. Conversely, in case of high dose lymphocyte infusion, steady state is achieved by apoptotic rather than proliferative mechanisms. Since entry into the memory pool can only be established by proliferation, administration of an appropriate cell number may be crucial to allow for in vivo expansion and thus long-term anti-tumor protection. Finally, additional poorly understood mechanisms maintain peripheral diversity and prevent an antigen-driven progression to oligoclonality. In sublethally irradiated lymphopenic mice, polyclonal but not clonal autologous T cells prevented tumor growth when the animals were challenged with melanoma or colon carcinoma cells. Thus homeostasis by proliferation may explain why infusions of small numbers of polyclonal EBV-specific CTLs expanded by 4 logs after T cell-depleted BMT, and persisted for up to eighty-six months post infusion: the T cell compartment was depleted; the infused T cells were activated; and EBV antigens were present. By contrast, homeostasis by apoptosis may explain why large numbers of clonal T cells infused into T cell depleted patients rapidly disappear after infusion and never enter the memory pool. The above described mechanisms of lymphocyte homeostasis imply that the administration of a small number of EBV-specific T cells post lymphocyte depletion may allow for significant in vivo expansion and thereby improve their anti-tumor function. Furthermore, depletion of tumor-infiltrating T cells with an inhibitory effect on the function of adoptively transferred CTL may be an additional advantageous effect of lymphocyte depletion prior to CTL infusion. This secondary gain is further discussed in the rationale of Aim 2. Lymphoid depletion as a strategy to create space for the expansion of adoptively transferred cells and to facilitate their entry into the tumor site has already shown evidence of success. When melanoma patients received cyclophosphamide and fludarabine prior to the adoptive transfer of large numbers of highly-activated melanoma-specific tumor infiltrating T cells (TIL), repopulation and proliferation of the transferred cells was observed, as well as regression of metastatic melanoma. However, some patients remained profoundly immunocompromised and failed to regenerate an effective immune system from these infused TILs, their expansion notwithstanding. This poor immune reconstitution resulted in part from the extensive and non-specific destruction of the resident immune system by the cytotoxic drugs used. A method of T cell depletion with less systemic toxicity, particularly to the myeloid system, may allow in vivo expansion of infused CTL and recovery of the residual lymphoid compartment. CD45 MAb, which targets a common leucocyte antigen, profoundly depletes the lymphocyte compartment for approximately 30 days while the myeloid system regenerates within 72 hours. In combination with its short half-life (about 8 hours), which allows for CTL infusion shortly after MAb administration, these characteristics make CD45 Mab a potentially safe and effective method of lymphodepletion. 4. Summary and implicationsWe hypothesize that the anti-tumor activity of EBV-specific CTL infused into patients with EBV+ NPC can be increased dramatically by prior depletion of the T cell compartment to allow for expansion of infused tumor-specific T cells. We believe that CD45 MAbs will safely and substantially deplete normal T cells, and their short half-life will allow infusion of EBV-specific CTL shortly after CD45 MAb administration, enabling their expansion prior to the recovery of endogenous T cells. This study will provide important information on the ability of MAb mediated T cell depletion to enhance the proliferation of adoptively transferred, tumor specific T cells.
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