A Novel Approach to Manufacture CAR-T Cells for Clinical Applications

Chimeric antigen receptors (CARs) are artificial molecules that can be used to redirect T cell immune response against antigens expressed on the surface of tumor cells. Although promising, most current protocols expand engineered T cells non-specifically using IL-2 and OKT3, which   decreases the frequency of transgenic populations over time. Additionally, cell expansion using conventional cultureware is complicated and labor intensive, which limits the broader application of this therapy. With the grant support from Production Assistance for Cell Therapy (PACT), we assessed whether CAR T cell manufacture could be optimized and streamlined by: (i) supplementing non-specific stimuli (IL-2) with an artificial antigen presenting cell (a-APC) engineered to express cognate antigen and co-stimulatory molecules, and (ii) efficiently and rapidly expanding cells in a simple and scalable gas permeable culture device (G-Rex). As a proof of principle, we sought to expand T cells engineered with a CAR targeting the prostate cancer antigen, PSCA. We first generated an a-APC cell line by modifying K562 cells, which expressed a range of co-stimulatory molecules including CD80, CD86, and 41BBL, with a retroviral vector encoding the PSCA antigen. After the co-culture of CAR-PSCA T cells with the irradiated a-APC, we found that a-APCs co-expressing PSCA antigen, CD80, and 41BBL were the most effective in inducing T cell expansion, with a 1.9 fold increase in total cell numbers when compared with CAR T cells expanded in the presence of IL2 alone. We also saw an increase in the frequency of transgenic CAR T cells which increased from 36.5% to 88.1% after 10 days of culture. In contrast, the percentage of transgenic T cells was sustained when cultured in the presence of IL2 (36.5% on day 0 and 37.2% on day 10). Thus, culture of CAR-T cells with antigen-expressing a-APCs not only improves total cell output, but also enriches for transgene-expressing. Next, to assess whether we could scale up cell production we transferred the engineered a-APCs and CAR-PSCA T cells (at a 2:1 ratio) into a static GMP-compliant G-Rex with a surface area of 100cm². In these G-Rex devices, O₂ and CO₂ are exchanged across a silicone membrane at the base, which allows for the addition of an increased depth of medium above the cells. These culture conditions have been shown to increase cell output without increasing the number of cell doublings. From an initial seeding density of 25E+06 CAR T cells, we obtained a total of 2200-2500E+06 cells within 10 days of culture. Thus, without any intervention we obtained a 93 fold increase in cell numbers using only 1 liter of T cell culture media. We also observed an enrichment of transgenic T cells (from 33.2% to 81.7%, a 2.4±1.2 fold increase, after 10 days of culture). Taken together the total T cell fold expansion (93) and the enrichment for the transgene (2.4±1.2), we calculate a 223.5±111.6 fold expansion of CAR T cells.