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Humoral immunotherapy using the monoclonal anti-CD20 antibody rituximab induces remissions in ∼60% of patients with relapsed follicular lymphoma; however, most patients eventually relapse despite continued expression of CD20 on lymphoma cells. We have hypothesized that cellular immunotherapy targeting CD20+ cells might provide a more effective mechanism for eliminating lymphoma cells than anti-CD20 antibodies and are therefore investigating the utility of cytotoxic T lymphocytes (CTL) genetically modified to target the CD20 antigen. Peripheral blood mononuclear cells were activated with anti-CD3 antibody (OKT3) and recombinant human interleukin-2 and electroporated with a plasmid containing a CD20-specific scFvFc:ζ chimeric T cell receptor gene and a neomycin phosphotransferase gene (neoR). Transfected cells were selected using the antibiotic G418 and cloned by limiting dilution. Using this approach, we have generated CD8+ CTL clones with CD20-specific cytotoxicity, which specifically lysed CD20+ target cells, including actual tumor cells from patients with follicular lymphoma, small lymphocytic lymphoma, splenic marginal zone lymphoma, diffuse large B cell lymphoma, and chronic lymphocytic leukemia. The CTL clones have been expanded to numbers sufficient for therapy (∼109 cells). Our data indicate the feasibility of generating and expanding CD20-specific CTL and, for the first time, demonstrate that such CTL exhibit specific cytotoxicity against actual tumor cells isolated from patients with a variety of B lymphoid malignancies. In view of these promising findings, a Phase I clinical trial for relapsed follicular lymphoma is being initiated. [Copyright &y& Elsevier]

Follicular Lymphoma (FL) is the second most common type of Non-Hodgkin's Lymphoma (NHL) in the United States, with over 120,000 Americans living with the disease. FL is considered incurable with conventional therapies and innovative new approaches are needed. We have initiated a pilot Phase I clinical trial to test the feasibility, safety, toxicity, and efficacy of treating patients with relapsed indolent B cell lymphomas with autologous CD8+ cytotoxic T lymphocytes (CTL) that have been genetically modified to express a chimeric T cell receptor (cTCR) recognizing the CD20 antigen present on B cell lymphomas. Five patients have been registered to the protocol to date. Peripheral blood mononuclear cells were obtained from patients by apheresis, activated with anti-CD3 monoclonal antibody and interleukin 2 and transfected by electroporation with a plasmid encoding a CD20-specific scFvFc:zeta chimeric immunoreceptor as well as a neomycin-resistance selection gene. Transfectants were selected using G418, and cloned by limiting dilution. CD8+ clones expressing the cTCR and manifesting CD20-specific cytotoxicity were identified by flow cytometry and chromium-51 cytotoxicity assays. Selected clones were expanded and infused into two of the patients at escalating doses (108, 109, and 3.3 × 109 cells/m2) on three occasions over a period of 10 days. Two additional patients are scheduled for T cell infusions in the near future. One patient is now off study because the clones failed to expand sufficiently to numbers required for therapy. No significant toxicities attributable to the T cell infusions were observed. There have been no cellular or humoral immune responses to the genetically modified T cells in the two treated patients. T cells expressing the cTCR were detectable in the peripheral blood of patients by PCR for 14 days after infusions. One patient was maintained in complete remission for over a year after T cell infusions, and a second patient was maintained in a partial remission for four months. Future planned interventions include infusion of polyclonal populations of transfected CD4 helper T cells with cytotoxic CD8 T cells rather than CD8 clones, subcutaneous administration of low dose interleukin 2, and introduction of a modified plasmid containing CD28 and CD137 co-stimulatory domains in addition to the CD3 zeta chain.Molecular Therapy (2006) 13, S22–S23; doi: 10.1016/j.ymthe.2006.08.068 [ABSTRACT FROM AUTHOR]