R. B. Batchu1,2, O. Gruzdyn1,2, D. W. Weaver1, S. A. Gruber1,2 1Wayne State University,Surgery,Detroit, MI, USA 2John D. Dingell VA Medical Center,Research,Detroit, MI, USA
Introduction: Chimeric Antigen Receptor (CAR) T cell-based immunotherapy has achieved clinical success due to its ability to overcome negative effects of the tumor microenvironment along with its HLA-independent recognition of tumor cells. Human T cells can be engrafted with CARs using efficient viral vectors, but these pose safety concerns. Alternatively, safer, non-viral, plasmid DNA vectors are being used, but are less efficient. Sleeping Beauty (SB) transposon/transposase engineering of CARs retains the advantage of viral vector efficiency, but with safe, non-viral technology. Minicircle DNA vectors (MC) lacking bacterial backbone and antibiotic-resistant gene sequences have been shown to increase the safety and efficiency of CAR T cells. We have previously developed CAR T cell therapy using a conventional plasmid DNA vector by targeting mesothelin (MSN), a differentiating antigen that is overexpressed in pancreatic cancer (PC). Here, we re-engineer our MSN CAR vector by adding both SB and MC design enhancements to achieve superior expression and cytotoxicity against PC cells.
Methods: We generated MC encoding MSN CAR with SB from the pSB11 vector for use in all experiments. Human T cells, harvested from the non-adherent fraction of PBMC, were cultured in the presence of CD3/CD28 and IL-2 and electroporated with MSN CAR vector. Protein L binding to single-chain antibody fragments was employed for immunofluorescence analysis. Cytotoxicity was assessed using effector (E) T cells against the target (T) BxPC-3 human PC cell line.
Results: After initial construction of the MSN CAR SB conventional plasmid vector of ~8.1 kb (Fig. 1A), we removed bacterial elements to generate MSN CAR SB MC (Fig. 1B), resulting in a size reduction of ~2.5 kb. When compared with mock-electroporated T cells (Control; Fig. 1Ci), we observed ~25% CAR engraftment of T cells with MSN CAR SB conventional plasmid electroporation (Fig. 1Cii), which was increased to ~80% engraftment with MSN CAR SB MC (Fig. 1Ciii). Compared with control (Fig. 1Di), MSN CAR SB conventional T cells displayed a specific lysis of ~40% at an E:T ratio of 5:1 and ~60% at an E:T ratio of 10:1 against BxPC-3 cells (Fig. 1Dii). We observed a significant increase in MSN CAR SB MC-induced cytotoxicity to ~65% and ~85% at E:T ratios of 5:1 and 10:1, respectively (Fig. 1Diii).
Conclusion: A significant increase in MSN CAR T cell engraftment along with enhanced cytotoxicity can be achieved by engineering CAR T cell vectors incorporating both SB and MC technology. This has the potential to the improve the clinical safety profile of CAR T cell therapy by eliminating both viral and bacterial concerns while simultaneously increasing engraftment and cell killing.
