1. ¹Oncology, Washington University, St. Louis, MO
2. ²Xcyte Therapies, Inc., Seattle, WA

Abstract

Here we demonstrate the GMP scale-up process for a human gene therapy clinical trial utilizing retroviral vector transduced allogeneic T cells eliciting a Graft vs. Leukemia effect in a Donor Lymphocyte Infusion (DLI) setting. After elimination of leukemic cells by DLI, a fusion CD34-TK suicide gene conferring sensitivity to ganciclovir will allow killing of transduced T cells and abrogation of Graft vs. Host Disease (GvHD), a serious side effect of DLI. The truncated CD34 receptor, normally not present on T cells, allows for purification of transduced cells to >95% in a magnetic cell sorter (CliniMacs, Miltenyi, Auburn, CA). PBMCs from 8 donors were stimulated in a closed bag system using the new GMP grade Stemline serumfree T cell medium (Sigma, St. Louis, MO). This medium allowed for elimination of donor variability, which we experienced in T cell expansions using other serumfree media. T cell stimulation was done using clinical grade magnetic beads coated with CD3 and CD28 antibodies (Xcyte Therapies, Inc., Seattle, WA). At least a 2 fold T cell expansion is needed, since transduction and expression of the transgene using a Moloney leukemia virus based retroviral vector depends on cell division and activation. Maintenance and transduction of CD4 and CD8 cells in a physiological ratio is imperative for normal function of infused T cells. Requirements stipulated by regulatory agencies demanded not to introduce more than 1-2 vector copies per cell to keep the risk for insertional mutagenesis to a minimum. A transduction frequency of 20-30% obtained at an MOI of 1-2 was required to generate this copy number (Rettig et al., 2003). In the past we showed that high concentrations of IL-2 (500 U/ml, as previously used in clinical T cell expansions) impair the in vivo functionality of T cells, using our NOD SCID/B2 M deficient mouse model of T cell expansion. We therefore lowered the IL-2 concetration to 50 U/ml. After 48 hours of pre-stimulation, T cells were transduced twice, medium was replaced by bag spinning, removing 2/3 of the supernatant and replacing it with fresh vector containing medium. Cells were harvested on day 4 by disrupting bead/cell clumps and removing the magnetic beads by application of a strong magnetic field. Although a low IL-2 concentration was used, a 3 fold expansion of T cells with 30% transduction efficiency, equally well distributed in the CD4 and CD8 compartment, was observed. The CD4 and CD8 ratios were maintained at input ratio, cell viability was greater than 95%. The most remarkable result was the outstanding activation and in vivo functionality of the expanded T cells. All NOD SCID/B2 M deficient mice (n=12) injected at a cell dose of 10⁷ developed lethal GvHD at day 15 post injection. Activated cells clearly outperformed the GvHD potential of na|[iuml]|ve T cells (Nervi et al., 2005). For the first time, these results demonstrate consistent GvHD elicited in a mouse xenotransplant model by 10⁷ expanded and activated human T cells produced in a closed system, serumfree GMP manufacturing process.