GMP Scale up for a Clinical Gene Therapy Trial - High Efficiency Human T Cell Expansion and Transduction in a Closed Culture System Utilizing Serumfree Medium and Low IL-2 Concentrations.
Authors: Bauer Gerhard , Walker Jon E. , Ritchey Julie , Nolta Jan A. , DiPersio John F. 
Published: April 2020

Abstract
Translational research is an ever increasing focus in the field of cellular and gene therapy. Here we demonstrate the scale-up process for a gene therapy clinical trial aimed at abrogation of Graft versus Host Disease (GvHD) using a suicide gene fused to a truncated CD34 receptor as a selectable marker in a Donor Lymphocyte Infusion setting to treat relapsed hematologic malignancies after allogeneic bone marrow transplantation. Transduced T cells can be purified to over 95% in a magnetic cell sorter (CliniMacs, Miltenyi, Auburn, CA) using the CD34 receptor, normally not present on T cells. After infusion, these T cells produce a Graft versus Leukemia effect, but can be eliminated by administration of Ganciclovir if GvHD develops. Donor T Cells were cultured in a closed system using 2 types of serumfree media: X-VIVO 15 (Cambrex, Walkersville, MD) and the newly developed GMP grade Stemline T Cell Expansion Medium (Sigma, St. Louis, MO). Stimulation was done using clinical grade magnetic beads coated with antibodies to CD3 and CD28 (Xcyte, Seattle, WA). High concentrations of IL-2 (500 U/ml) impair the in vivo functionality of cultured T cells, as shown in our NOD SCID/B2 M deficient mouse model of T cell expansion. To maintain in vivo functionality of transduced T cells, it was imperative to lower the IL-2 concentration to 50 U/ml during culture. At least a 2 fold T cell expansion with maintenance of a physiological CD4 and CD8 compartment is necessary, since transduction using a Moloney leukemia virus based retroviral vector depends on cell division. It is also important to transduce both CD4 and CD8 cells in the same ratio for normal function of the infused T cell population, and expression of the selectable surface marker for subsequent enrichment of the transduced cells in both fractions. 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. We demonstrated that a transduction frequency of 20–30% obtained at an MOI of 1–2 is required to generate this copy number (Rettig et al., 2003). After 48 hours of pre-stimulation, the cells were transduced 2 times, medium was replaced by spinning the culture bags, removing 2/3 of the supernatant and replacing it with fresh vector containing medium. Cells were harvested on day 5 by disrupting bead/cell clumps and bead removal in a magnetic field. With X-VIVO 15, strong variability in expansion and viability at the end of culture could be seen using low concentrations of IL-2. However, the consistency of cell expansion increased dramatically when the concentration of IL-2 was increased to 1000U/ml. In contrast, the newly formulated Stemline serumfree medium consistently worked equally well in high and low concentrations of IL-2. At 50 U/ml of IL-2, a 3 fold expansion of T cells with a 30% transduction efficiency, equally well distributed in the CD4 and CD8 compartment, was observed. CD4/CD8 ratio was maintained at input ratio, with greater than 95% cell viability. Removal of magnetic beads after culture was consistently more than 2 logs. The transduced cells could be purified by magnetic cell sorting using the selectable marker, which was highly expressed. In summary, a new serumfree media formulation allows for reproducible, high efficiency expansion and transduction of T cells in low IL-2 concentrations to optimize in vivo T cell functionality for human gene therapy trials.