Transposon-Based CAR T Cells in Acute Leukemias: Where are We Going?

Abstract

Chimeric Antigen Receptor (CAR) T-cell therapy has become a new therapeutic reality for refractory and relapsed leukemia patients and is also emerging as a potential therapeutic option in solid tumors. Viral vector-based CAR T-cells initially drove these successful efforts; however, high costs and cumbersome manufacturing processes have limited the widespread clinical implementation of CAR T-cell therapy. Here we will discuss the state of the art of the transposon-based gene transfer and its application in CAR T immunotherapy, specifically focusing on the Sleeping Beauty (SB) transposon system, as a valid cost-effective and safe option as compared to the viral vector-based systems. A general overview of SB transposon system applications will be provided, with an update of major developments, current clinical trials achievements and future perspectives exploiting SB for CAR T-cell engineering. After the first clinical successes achieved in the context of B-cell neoplasms, we are now facing a new era and it is paramount to advance gene transfer technology to fully exploit the potential of CAR T-cells towards next-generation immunotherapy.

Keywords: CAR T-cells; acute leukemia; gene transfer; immunotherapy; sleeping beauty; transposon.

Figure 1

Sleeping Beauty transposon system for CAR T-cell engineering. Two-component DNA transposon-based gene delivery systems: Transposon plasmid carrying the gene of interest flanked by the ITRs and transposase expression plasmid; mechanism of ’cut and paste’ transposition of Sleeping Beauty for a stable genomic integration of the CAR; different transposase sources currently available: plasmid DNA, synthetic mRNA and synthetic recombinant protein; recombination of parental plasmids into minicircles and miniplasmids. ITR, inverted terminal repeat. Created with Biorender.com

Figure 2

Flow diagram for viral vector and plasmid manufacture. The process of viral production relies on the vector production by stable producer cell lines in the case of retroviral vectors or by transient cell line transfection for lentiviruses. The viral supernatant undergoes concentration, purification and formulation steps. *Benzonase treatment applies only to lentiviral vectors. For plasmid production, initially the E Coli master cell bank of the intended plasmid is produced, from which plasmid replication is promoted by fermentation. After cell lysis, the pre-purification step can be performed by means of filtration, precipitation or solid–liquid separation, followed by the purification and formulation phases. Created with Biorender.com

Figure 3

Overview of donor-derived SB-engineered CARCIK-CD19 therapy; 50–60mL of donor PB are collected and PBMCs are isolated and nucleofected at day 0 with SB vectors. Gamma-irradiated PBMCs are added as feeder together with IFN-γ; at day 1 cells are stimulated with IL-2 and OKT3 and CARCIK-CD19 cells are expanded in vitro and frozen on days 18–21; during the pre-treatment phase (days 2 to 14) the patient undergoes lymphodepletion and then, on day 1 of the treatment phase, CARCIK-CD19 are thawed and subsequently infused into the patient; after the infusion, the patient is subjected to a long-term follow-up until the fifth year and to 12 months immune monitoring. Created with Biorender.com