03.09 Sleeping Beauty and Minicircle Design of Mesothelin CAR Enhances Cytotoxicity in Pancreatic Cancer

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.