Anti-CD117 CAR T Cells Incorporating a Safety Switch Eradicate Acute Myeloid Leukemia and Deplete Human Hematopoietic Stem Cells

Introduction

Acute Myeloid Leukemia (AML) arises from the accumulation of mutations within the hematopoietic stem and progenitor cells (HSPC), leading to the emergence of a population of malignant leukemia-initiating cells (LIC). AML-LICs maintain high phenotypic similarity with their cells-of-origin and can cause post-treatment relapse. Immunotherapy with chimeric antigen receptor (CAR) T cells is an innovative approach to tackle cancer via surface-expressed cancer-associated antigens. We recently proposed the use of CAR T cells specific for the CD117 antigen to deplete LIC and replace HSPC by allogeneic hematopoietic stem cell transplantation (HSCT) (Myburgh R et al. Leukemia 2020). This concept implies early termination of CAR T-cell activity to prevent subsequent graft rejection. Here, we exploit a non-viral technology for the generation of anti-CD117 CAR T cells incorporating a safety switch.

Methods

We designed a Sleeping Beauty (SB) transposon vector that includes the inducible Caspase 9 (iC9) switch and the anti-CD117CAR, separated by a 2A peptide. SB allows the generation of CAR T cells with potent anti-leukemic activity (Magnani CF et al. J Clin Invest. 2020). The vector has an optimized donor vector architecture and allows for the stoichiometric expression of the two transgenes. iC9 allows for rapid termination of CAR T cells by activation of the apoptotic pathway in case of treatment with a small molecule that acts as a chemical inducer of dimerization (CID). The hyperactive SB100X transposase, supplied as plasmid DNA or mRNA, catalyzes transgene integration. As an alternative approach, we used mRNA encoding an anti-CD117 CAR in human T cells.

Results

With the purpose of transduction optimization, we compared total PBMC and selected T cells as starting material in the presence of different concentrations of plasmids or mRNA. The procedure of generating CAR T cells with SB did not affect T cell memory differentiation but increased the CD8/CD4 proportion compared to non-transduced (NT) cells (75.63% vs. 41.63%, p= 0.0124). Based on higher transduction efficiency and favored in vitro expansion, we defined the lead protocol (selected T cells, PT4:SB100X plasmid 3:1 ratio, or PT4:SB100X mRNA 1:2 ratio). CAR T cells had a high level of viability, retained a high proportion of naïve-like (mean 36.38%, SEM 8.80) and T stem cell memory populations (mean 39.21%, SEM 8.43), and showed low levels of the exhaustion markers PD-1 (mean 2.21%, SEM 1.04), LAG3 (mean 61.20%, SEM 9.61), and TIM3 (mean 35.72%, SEM 10.79). Anti-CD117 CAR T cells exhibited potent cytotoxicity against the AML cell line MOLM-14, transduced and sorted to express human CD117, luciferase, and GFP. The addition of 200nM of the CID to cultures of anti-CD117 CAR T cells induced apoptosis of transduced CAR T cells within 24h but had no effect on the viability of NT cells. Anti-CD117 CAR T cells mediated depletion of CD117+ MOLM-14 cells in vivo, leading to a significant survival advantage compared to mice treated with NT cells (median overall survival for NT= 22.5 days vs. SB= not reached, p= 0.0122, Mantel-Cox). Notably, SB-transduced CAR T cells were as efficient as CAR T cells transduced with lentiviral vectors. In NSG mice reconstituted with human CD34+ cord blood cells, anti-CD117 CAR T cells were able to achieve complete CD117+ HSPC depletion. Treatment with a combination of CID and anti-thymocyte globulin (ATG) eliminated anti-CD117 CAR T cells and T cells of the previous transplant donor. Finally, transient expression of anti-CD117 CAR by mRNA conferred T cells the ability to kill CD117+ targets throughout 72 hours post mRNA electroporation. The cytotoxic activity decreased over time as mRNA-electroporated CAR T cells proliferate and lose CAR expression upon 3-5 divisions. Treatment of humanized NSG mice with two subsequent doses of anti-CD117 CAR mRNA T cells resulted in HSPC depletion.

Conclusions

Anti-CD117 CAR T cells engineered with the SB vector showed anti-leukemic activity and completely depleted healthy HSPC in vivo. iC9 transgene induced CAR T cell apoptosis and allowed rapid CAR T cell depletion that alternatively also could be achieved with mRNA electroporation of the anti-CD117 CAR. The ability to control CAR T cell pharmacokinetic properties is attractive to enable subsequent HSCT and to terminate unexpected toxicities. Anti-CD117 CAR T cells could be used prior to HSCT in refractory or minimal residual disease AML.