451. Robust Manufacture of CAR-T Cells

Abstract

Although adoptive transfer of chimeric antigen receptor (CAR)-modified T cells has produced promising clinical responses, the broader application of this therapy has been hindered by prolonged and complicated cell production methods. In the current work, we have overcome this limitation through the incorporation of a gas permeable culture device (G-Rex) to support T cell expansion. This culture system consists of a suite of devices, all of which contain a gas permeable silicone membrane, which allows gas exchange to occur at the base. This configuration allows for the culture of T cells with an unconventionally large volume of media per unit of surface area (10ml of media/cm2), thereby supporting uninterrupted cell growth without media exchange. Importantly, this system is simple to use and can be placed in a regular incubator as the G-Rex does not require active agitation or perfusion. To evaluate the utility of this system for the expansion of CAR T cells, we transduced healthy donor-derived primary T cells with a CAR targeting the prostate cancer antigen - PSCA (previously generated and characterized by our group). Three days after retroviral transduction, transgenic CAR T cells (transduction efficiency of 83.6±6%) from 3 donors were transferred to G-Rex100M devices (surface area of 100cm2) in 1000ml of complete T cell media (10ml/cm2) at low (total of 25E+06 CAR T cells), intermediate (50E+06 CAR T cells) and high (100E+06 CAR T cells) cell densities (250E+03 cells/cm2, 500E+03 cells/cm2 and 1000E+03 cells/cm2, respectively). Subsequently, the cell cultures were monitored by glucose and lactic acid assessment. After 10 days of culture, the average fold-expansion was similar for all conditions (24.3±10.4, 35.5±7.8, 29.8±2.1 for low, intermediate, and high cell densities, respectively). Interestingly though, the donor-to-donor variability was decreased significantly at the higher cell density (SD of 10.4, 7.8, and 2.1 for cell densities of 250E+03 cells/cm2, 500E+03 cells/cm2 and 1000E+03 cells/cm2, respectively), highlighting the importance of identifying the optimal seeding density to support robust manufacture. Notably, no media replenishment was required and the only culture manipulation performed was the addition of IL2 (50U/ml) 3 times/week. Importantly, T cells manufactured using this optimized method expressed higher levels of central memory and activation markers (CD62L and CD25) and demonstrated superior anti-tumor activity when compared to cells maintained in conventional tissue-culture plates. To further simplify the manufacturing process, we have now developed a semi-automated, closed system (GatheRex) for cell collection, which can be paired with G-Rex and allows collection of cells in a small volume (100ml) in under five minutes.