Rituximab, a chimeric monoclonal antibody directed against CD20, has shown significant therapeutic activity in patients with follicular lymphoma (FL), yet it's exact mechanism of action has not been completely defined. Although killing of the CD20+ FL cells, either through complement dependent cytolysis, antibody dependent cellular cytotoxicity or direct induction of apoptosis may contribute to its effectiveness, these mechanisms are unlikely to be the only mechanisms of action as; (a) the clinical and molecular responses to Rituximab may continue for months after the last dose of Rituximab and; (b) the median duration of the second response to Rituximab is longer than that of the first response, clinical findings that cannot be explained solely by the mechanisms described above. Rituximab induced FL cell death is expected to result in the release of tumor antigens, for example antigens derived from the variable regions of the heavy (VH) and light chains (VL) of the FL associated immunoglobulin (Ig), and also as yet undefined tumor antigens, in a pro-inflammatory environment. We hypothesize that the Rituximab induced release of lymphoma-associated antigens will provoke a cell-mediated lymphoma specific immune response. We will test this hypothesis by monitoring newly diagnosed FL patients for the presence of both peripheral blood and tumor infitrating lymphoma specific effector and/or memory CD4+ and CD8+ T-cells, before and after Rituximab treatment using Elispot, Fluorispot, Lysispot and intracellular cytokine flow cytometric assays. We will also test two corollaries of this hypothesis, being that a Rituximab elicited active lymphoma specific cellular response would; (a) result in an increase in the effector functions of both the lymphoma-specific CD4+ and CD8+ T cells; and (b) provide the necessary T- cell help required to generate lymphoma-specific humoral responses. The demonstration that Rituximab generates a lymphoma specific immune response, as well as the knowledge of the target antigens of the response would have profound implications as to how we optimally treat patients with FL. For example, it would suggest that combining Rituximab with agents which would be anticipated to augment the elicitation of a lymphoma specific T-cell response, such as GM-CSF, CpG oligonucleotides, and anti-CTLA-4 monoclonal antibodies, would be of potential clinical benefit. In addition, the results of this study should be applicable to antibody therapy of other malignancies, and as such, we anticipate that these studies will lead to novel treatment strategies designed to enhance the effectiveness of monoclonal antibody therapy for cancer in general. Follicular lymphoma (FL) is an indolent, but essentially incurable disease, however new treatments, including Rituximab therapy, are offering new hope for patients suffering with this disease. Rituximab is an antibody, which is a protein that targets another protein on the lymphoma cell, resulting in lymphoma cell death, however the exact mechanism(s) by which Rituximab destroys lymphoma cells is not yet clearly defined. Our group is determining whether Rituximab induces lymphoma cell death by stimulating the patient's own immune system to eradicate the disease, as a better understanding of how Rituximab works in patients with lymphoma will allow us to develop new approaches to treatment that we anticipate will lead to increased benefit for patients with this disease. ? ? PROJECT SUMMARY/ABSTRACT Rituximab, a chimeric monoclonal antibody directed against CD20, has shown significant therapeutic activity in patients with follicular lymphoma (FL), yet it's exact mechanism of action has not been completely defined. Although killing of the CD20+ FL cells, either through complement dependent cytolysis, antibody dependent cellular cytotoxicity or direct induction of apoptosis may contribute to its effectiveness, these mechanisms are unlikely to be the only mechanisms of action as; (a) the clinical and molecular responses to Rituximab may continue for months after the last dose of Rituximab and; (b) the median duration of the second response to Rituximab is longer than that of the first response, clinical findings that cannot be explained solely by the mechanisms described above. Rituximab induced FL cell death is expected to result in the release of tumor antigens, for example antigens derived from the variable regions of the heavy (VH) and light chains (VL) of the FL associated immunoglobulin (Ig), and also as yet undefined tumor antigens, in a pro-inflammatory environment. We hypothesize that the Rituximab induced release of lymphoma-associated antigens will provoke a cell-mediated lymphoma specific immune response. We will test this hypothesis by monitoring newly diagnosed FL patients for the presence of both peripheral blood and tumor infitrating lymphoma specific effector and/or memory CD4+ and CD8+ T-cells, before and after Rituximab treatment using Elispot, Fluorispot, Lysispot and intracellular cytokine flow cytometric assays. We will also test two corollaries of this hypothesis, being that a Rituximab elicited active lymphoma specific cellular response would; (a) result in an increase in the effector functions of both the lymphoma-specific CD4+ and CD8+ T cells; and (b) provide the necessary T- cell help required to generate lymphoma-specific humoral responses. The demonstration that Rituximab generates a lymphoma specific immune response, as well as the knowledge of the target antigens of the response would have profound implications as to how we optimally treat patients with FL. For example, it would suggest that combining Rituximab with agents which would be anticipated to augment the elicitation of a lymphoma specific T-cell response, such as GM-CSF, CpG oligonucleotides, and anti-CTLA-4 monoclonal antibodies, would be of potential clinical benefit. In addition, the results of this study should be applicable to antibody therapy of other malignancies, and as such, we anticipate that these studies will lead to novel treatment strategies designed to enhance the effectiveness of monoclonal antibody therapy for cancer in general.PROJECT NARRATIVE Follicular lymphoma (FL) is an indolent, but essentially incurable disease, however new treatments, including Rituximab therapy, are offering new hope for patients suffering with this disease. Rituximab is an antibody, which is a protein that targets another protein on the lymphoma cell, resulting in lymphoma cell death, however the exact mechanism(s) by which Rituximab destroys lymphoma cells is not yet clearly defined. Our group is determining whether Rituximab induces lymphoma cell death by stimulating the patient's own immune system to eradicate the disease, as a better understanding of how Rituximab works in patients with lymphoma will allow us to develop new approaches to treatment that we anticipate will lead to increased benefit for patients with this disease. ? ? ?
