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

Autor: Markus G. Manz, Steve Pascolo, Judith A. Shizuru, Chiara F. Magnani, Dario Neri, Renier Myburgh, Norman F. Russkamp
Rok vydání: 2021
Předmět:
Zdroj: Blood. 138:2808-2808
ISSN: 1528-0020
0006-4971
Popis: 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. Disclosures Myburgh: University of Zurich: Patents & Royalties: CD117xCD3 TEA. Shizuru: Forty seven Inc: Other: Inventor on a patent licenses by Forty Seven. Forty seven was acquired by Gilead in 2020; Jasper Therapeutics, Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Chair of scientific advisory board. Neri: Philogen S.p.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Multiple patents on vascular targeting; ETH Zurich: Patents & Royalties: CD117xCD3 TEA. Manz: CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company; University of Zurich: Patents & Royalties: CD117xCD3 TEA.
Databáze: OpenAIRE
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