PRODUCTION OF “UNIVERSAL” CHIMERIC ANTIGEN RECEPTOR (CAR) T CELLS THROUGH A HIGHLY EFFICIENT CRISPR/CAS9-INDUCED KNOCKOUT OF THE ENDOGENOUS TRAC GENE

Allogeneic Chimeric Antigen Receptor (CAR) T cell therapy represents a promising approach to overcome the accessibility limitations of conventional autologous treatment. Some advantages of such allogeneic therapy include the availability of an off-the-shelf product, cost reduction associated with production scalability, and the selection potential of fitter third-party T cells as long as immune incompatibility restrictions are addressed. Genome editing technologies are applied to overcome HLA-incompatibility barriers, giving rise to a universal product, mainly by depleting the T Cell Receptor (TCR) to prevent graft-versus-host disease development. Here, we describe a refined platform for universal anti-CD19 CAR T cell production by disrupting the TCR alpha-chain constant region (TRAC gene) through a high-fidelity CRISPR/Cas9 system. Activated human T cells from three healthy donors were modified in a two-step engineering process: transduction with a lentiviral vector coding the anti-CD19 CAR, followed by Ribonucleoprotein (RNP) electroporation. RNPs were produced prior to electroporation through the complexation of the high-fidelity Cas9 protein with the single guide RNA (sgRNA) and poly-L-glutamic acid polymer association to prevent protein aggregates. We followed the viability and growth kinetics over twelve days of manufacturing and assayed the produced cells on days nine and twelve for CAR/TCR expressions and antitumor efficiency, respectively. Cell viability was consistent between non-transduced (UTD), transduced (CAR), and transduced+electroporated (uCAR) T cells, with the frequency of live cells reaching 85% after two days of transduction and 63% three days post-electroporation. Despite that, CAR T cells exhibited slightly higher expansion compared to UTD and uCAR T cells (11.13 ± 7.18 vs. 5.93 ± 3.72 and 8.23 ± 4.21-fold expansion on day twelve, respectively), which was also affected by donor-to-donor variability. We observed similar transduction efficiency between CAR (48.7±8.6%) and uCAR T cells (43.4±9.4%). Within the latter group, we got 39.2% (±4.9%) of CAR+/TCR− cells and an overall 86.1% (± 3.4%) TCR− cells, highlighting the high efficiency of TCR disruption in our genome editing platform. We then analyzed the in vitro antitumor potential of CAR and uCAR T cells by coculturing them with luciferase-expressing tumor cell lines (1:1 effector-to-target ratio) for 48h. CAR and uCAR T cells exhibited potent cytotoxic activity against CD19+ Nalm6 cells, with uCAR T cells displaying superior performance (72.36 ± 1.41% of cell lysis vs. 54.21 ± 2.45% for CAR T cells; p≤0.05). Enhanced antitumor activity of uCAR T cells was associated with an increased release of IFN-y in coculture supernatant (217.29 ± 14.58 pg/mL) compared to unedited CAR T cells (125.67 ± 1.2 pg/mL; p ≤ 0.01). Both CAR and uCAR T cells spared CD19− K562 cells, demonstrating the robust cytotoxic function and target specificity of the CAR T cell products. Collectively, these findings validate our manufacturing platform for generating universal anti-CD19 CAR T cells with high genome editing efficiency. These results lay the groundwork to consolidate an off-the-shelf allogeneic CAR-T cell therapy for B-cell malignancies and potentially for any other tumor type.