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.ABSTRACT I. HYPOTHESIS We propose to develop and test an innovative approach to adoptive immunotherapy for EBV +ve Hodgkin disease (HD) and Non-Hodgkin's Lymphoma (NHL). We propose that failure of adoptively transferred CTL to expand effectively in these patients in part reflects poor specificity of the CTL for the tumor and a T helper 2 environment due to the T cell infiltrate at sites of disease. In this inhibitory environment, ex vivo-expanded CTL may be required to circumvent in vivo inhibition and may fail to expand or be diverted along the T helper 2 pathway despite an initial tumor-specific response. In addition to these intratumoral Th2 inhibitory cells, we propose that homeostatic mechanisms act on adoptively transferred tumor specific CTLs to prevent their expansion and long-term function in vivo in patients with a normal T cell level. We hypothesize that the expansion, function and anti-tumor activity of infused LMP2 -specific CTL with defined specificity against the LMP2a antigen expressed by these tumor cells, may be increased dramatically by depleting the lymphoid compartment of all T cells including negative regulatory T cells and Th2 cells by pre-treating patients. The lytic CD45 monoclonal antibody can accomplish this, and its short half-life will allow rapid infusion of therapeutic cells. II.
SPECIFIC AIMS1. To determine the safety of autologous LMP-2 specific cytotoxic T-lymphocytes (CTL) in combination with CD45 monoclonal antibody (Mab) in patients with EBV positive Hodgkin's's disease (HD) or non-Hodgkin's's lymphoma (NHL).2.To obtain information on the expansion, persistence and anti-tumor effects of of autologous LMP-2 specific cytotoxic T-lymphocytes (CTL) given after lymphodepletion with CD45 monoclonal antibody (Mab) in patients with EBV positive Hodgkin's's disease or non-Hodgkin's's lymphoma. III. BACKGROUND AND SIGNIFICANCE Hodgkin Disease (HD)HD, a malignant neoplasm of lymphoreticular cell origin, has an annual incidence of 3 cases per 100,000 patients in the USA.1 For a majority of patients, the long-term prognosis is favorable. With combined conventional chemotherapy and /or radiotherapy 80% of patients with early disease and 60% with advanced disease can expect to be cured.2 Of patients who relapse after initial therapy, only 50% will enter a second remission. For the remainder, who either fail salvage chemotherapy or relapse for a second time, the prognosis is poor. While overall survival rates are good, current treatment regimens are still far from ideal. Long term follow-up studies of Hodgkin survivors show increased risks for treatment related morbidity and mortality for up to twenty years beyond diagnosis. Further, non-fatal sequelae of therapy, such as altered somatic growth, infertility and restrictive lung disease can seriously affect the quality of life of Hodgkin survivors.3 It is therefore desirable to develop novel therapies that could improve disease-free survival in relapsed/refractory patients and might ultimately reduce the incidence of long-term treatment related complications in all patients. The etiology of HL remains unknown. It is an unusual disease in that the bulk of the tumor consists of normal cells among which H-RS cells may be found. In-situ hybridization studies have localized EBV RNA and antigens to the neoplastic H-RS cells in about 40% of cases. 4,5 Four viral proteins are expressed: the EBV nuclear antigen (EBNA)1, the latent membrane proteins (LMPs)1 and 2, and a product of the BamHI A open reading frame, BARFO. Regardless of their role in the pathology of the disease, these antigens provide potential targets for immunotherapeutic approaches with CTL.Non-Hodgkin's lymphomaNon-Hodgkin Lymphomas broadly can be divided into B and T/NK cell types and all types can have an association with EBV. The average annual incidence of NHL in patients <20 years is 1 per 100,000. Only EBV positive NHLs arising in the immunocompetent host will be discussed, as it is this group that we aim to treat on this protocol. B cell NHL includes Burkitt's lymphoma, which expresses a type 1 latency and is therefore not a good target for adoptive immunotherapeutic approaches. However, other EBV +ve B cell lymphomas occurring in the immunocompetent host include Large B cell lymphoma and CD30+ Ki-1-positive anaplastic large cell lymphoma (ALCL) of B cell type. Up to 38% of these tumors are EBV positive expressing a type 2 latency pattern. The 3-year event free survival rates for various protocols in the treatment of Large B cell lymphoma and anaplastic large cell lymphoma in children ranges from 75-95% depending on the stage of the disease. In adults the overall survival at 3 years is approximately 50% for Large B cell lymphoma and 70% for B-cell anaplastic large cell lymphoma. Primary resistance to chemo and radiotherapies cause most treatment failures. Therefore, other therapeutic strategies are required for the successful management of these refractory patients. Further, the doxorubicin containing chemotherapeutic regimens are highly toxic especially in the adult population with 20% of patients developing a cardiac event within 1 year of treatment. EBVEpstein-Barr virus (EBV) is a latent herpes virus that infects over 90% of the population. Primary infection usually results in a mild, self-limiting illness that is followed by life-long virus latency in oral epithelial cells and in B cells. In primary infection, the main route of entry for EBV is via the oropharyngeal epithelium. Viral replication in these cells subsequently allows infection of B-lymphocytes thus resulting in a polyclonal expansion of transformed B-cells. These B- cells express a number of EBV-gene products including the nuclear antigens EBNA1, 2, 3A, 3B, 3C, the leader protein, the membrane proteins LMP-1, 2A and BARFO. In individuals with a normal immune response, malignant outgrowth of infected cells is prevented by the mounting of a complex immune response comprised HLA- restricted EBV-specific cytotoxic T-cells and MHC-unrestricted effectors. The genes expressed by EBV depend on the type of cell infected, and the pattern of gene expression determines the growth of the infected cell and its susceptibility to host immune responses.6 Three patterns of EBV gene expression have been observed in human cells: Type 1 latency, the least immunogenic pattern was defined in Burkitt lymphoma cells and involves the expression of only one EBV latent-cycle protein, EBNA1.6 This protein is sufficient to maintain the virus episome in dividing cells.6 It does not induce the expression of cell adhesion molecules that are important for the generation of an efficient immune response and cannot be processed into target peptides for CTL.7 Cells expressing type 1 latency are currently not good candidates for CTL therapy. In Type 2 latency, seen in nasopharyngeal cancer, some cases of EBV +ve NHL and in the Reed Sternberg cells of HD, EBNA1, LMP1, LMP2a and LMP2b are expressed.8,9 LMP2a and LMP2b proteins are identical, apart from a short leader sequence of 119 amino acids present only in LMP2a.10 Although experimentally, the LMP epitopes provide effective targets for CTL, little is known about the immunogenicity of tumors expressing type 2 latency, since they are not amenable to growth in tissue culture. Type 3 latency, probably the most immunogenic pattern of latent cycle gene expression, is seen in B cells transformed into lymphoblastoid cell lines (LCL) by EBV in vitro.6 LCL express 11 latent-cycle genes, 9 of which encode proteins. Several of these proteins induce B cell activation markers and cell adhesion molecules. Both the latent nuclear antigens (EBNAs 2, 3A, 3B, 3C and LP) and membrane antigens (LMP1, 2a and 2b) provide targets for CTL.11,12 Virus-infected B cells expressing type 3 latency are highly immunogenic and have not been observed in immunocompetent individuals in vivo, but these are characteristic of the cells which proliferate in the immunosuppressed host.In most virus systems studied, the immune response generated on a range of HLA backgrounds, is directed against a subset of viral proteins. In EBV, there is an apparent hierarchy of immunodominance among the 8 viral proteins expressed in type 3 latency. The most dominant of these (the EBNA3s) are downregulated in many virus-associated malignancies including EBV associated HD and NHL. Immunotherapy approaches must therefore access the less abundant CTL precursors that are directed against subdominant antigens. In this protocol we aim to do this by selectively reactivating subdominant CTL precursors using extremely potent antigen presenting cells that overexpress the subdominant protein LMP2 in the absence of the dominant proteins.LMP2A is consistently expressed on Reed-Sternberg cells and also detected in NHL tumor cells, which have a type II latency pattern of expression. This therefore represents a potential source of target epitopes. Most donors have a low but measurable frequency of circulating LMP2A-specific CTL that can be activated and expanded in vitro. Hence LMP2A may be the protein of choice to be targeted by CTL in patients with EBV-positive Lymphoma, which arises in the immunocompetent host.In this study we will generate autologous LMP2A-specific cytotoxic T-cells and adoptively transfer them to patients with relapsed EBV-positive Hodgkin's or non-Hodgkin's Lymphoma.LMP2A-peptide pulsed DC have been successfully used to stimulate LMP2A-specific CTL. However, this approach is limited to known MHC restricted peptides. A more promising strategy is the initiation of CTL by genetically modified DC that direct the CTL response to virally transduced genes.13,14 This approach allows expression of the whole protein leading to presentation of multiple, undefined antigen epitopes. We have constructed an E1/E3 deleted recombinant adenovirus vector encoding LMP2A and shown that this vector when used for transduction of DC is capable of inducing CTL that lyse LMP2A expressing target cells. These LMP2A-specific CTL will have the ability to recognize and kill LMP2A expressing cells including the tumor cells of Hodgkin and non-Hodgkin's lymphoma.Additional Mechanisms Whereby Malignant Hodgkin's Cells Escape Immune RecognitionAlthough H-RS cells display the hallmarks of good APC, they generate a Th2 environment with a T cell infiltrate consisting exclusively of CD4+ T cells with a Th2 profile.15 In this inhibitory environment, ex vivo-expanded CTL may fail to expand in vivo and/or be diverted along the Th2 pathway despite an initial oncolytic response.In addition to intratumoral Th2 inhibitory cells, CD4+25+ negative regulatory T cells and other poorly understood homeostatic mechanisms may act on adoptively-transferred tumor-specific CTLs to prevent their expansion and long-term function in vivo. Limited Expansion of Infused EBV Specific CTL in Lymphoma PatientsOne of the reasons why infusion of small numbers of EBV specific CTL after stem cell transplantation effectively prevents and treats EBV malignancy is likely attributable to the massive in vivo expansion of these cells. This in turn may be associated with the proliferative environment created by the combination of cytoablation followed by infusion of T cell depleted marrow. Hence, EBV-specific CTL expand by four logs after infusion into patients post transplant and persist for up to 86 months. In other words, the deficit in the lymphoid compartment promotes homeostatic lymphoproliferation of the infused EBV-specific CTLs to restore the steady state of T lymphocyte numbers. In contrast in most patients with cancer, including those to be treated here, the immune system is closer to a steady state, and infused EBV-specific CTL have not expanded to the same degree, and only a 4 to 50 fold expansion is observed. This may limit the capacity of the infused CTL to destroy the tumor cells. Recently, Rosenberg's group described how T cell homeostasis could be disrupted and a proliferative environment artificially induced by administration of Fludarabine and cyclophosphamide.16 Patients with advanced melanoma received lymphoreductive doses of these cytotoxic drugs and were then infused with autologous tumor infiltrating lymphocytes. In 6 patients there was marked expansion of the infused cells associated with tumor responses: in two patients the tumor responses were complete, and the infused TIL came to dominate the lymphoid compartment, suggesting a relationship between cell expansion and anti-tumor activity. In this protocol we will investigate whether the same benefits can be seen in patients with advanced Hodgkin's disease or non-Hodgkin's Lymphoma receiving LMP2 specific CTL with and without T cell depletion using a CD45 MAb.Depletion of T Cells Using CD45 AntibodyBecause the patients we plan to treat will have received large dose of chemo-radiotherapy, it would be preferable if cytoreduction of resident T lymphocytes could be achieved by more specific means than those used by Dudley et al16. One possibility is to use monoclonal anti-T antibodies. However, while numerous such antibodies are available for clinical use, all have the disadvantage of a long half-life. Thus, at low dose they will deplete inadequate numbers of host T cell, while at saturating doses their in vivo persistence would impair the survival of infused CTL. We propose to use an alternative monoclonal antibody, targeted to the CD45 antigen, which in pre-clinical and clinical studies has been shown to be highly lytic of T cells, to pare hemopoieisis and to have a half life of just 6 to 9hrs in vivo. The CD45 antigen is present on all cells of the hematopoietic lineage. Using a murine model, we found that anti-CD45 MAb markedly diminished all leukocyte subsets in peripheral blood, but that only the effect on the lymphoid compartment and lymphoid organs was sustained (for up to 30 days)17. Circulating T lymphocyte counts were reduced by circa 99%. By contrast, marrow progenitor cells were spared from destruction, and hemopoiesis rapidly recovered17. Given the transient effects of the monoclonal antibody on myelopoiesis and the more persistent effects on lymphopoiesis, we asked whether this agent could contribute to donor hemopoietic engraftment following subablative transplantation. The combination of anti-CD45 and an otherwise inactive dose of total-body irradiation allowed engraftment of H2 fully allogeneic donor stem cells17. We attribute this result to the recipient immunodepletion produced by depletion of CD45-positive lymphocytes.
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