Enhanced Expression of Anti-CD19 Chimeric Antigen Receptor in piggyBac Transposon-Engineered T Cells

Abstract

Adoptive T cell therapy using chimeric antigen receptor (CAR)-modified T cells is a promising cancer immunotherapy. We previously developed a non-viral method of gene transfer into T cells using a piggyBac transposon system to improve the cost-effectiveness of CAR-T cell therapy. Here, we have further improved our technology by a novel culture strategy to increase the transfection efficiency and to reduce the time of T cell manufacturing. Using a CH2CH3-free CD19-specific CAR transposon vector and combining irradiated activated T cells (ATCs) as feeder cells and virus-specific T cell receptor (TCR) stimulation, we achieved 51.4% ± 14% CAR+ T cells and 2.8-fold expansion after 14 culture days. Expanded CD19.CAR-T cells maintained a significant fraction of CD45RA+CCR7+ T cells and demonstrated potent antitumor activity against CD19+ leukemic cells both in vitro and in vivo. Therefore, piggyBac-based gene transfer may provide an alternative to viral gene transfer for CAR-T cell therapy.

Integration Mapping of piggyBac-Mediated CD19 Chimeric Antigen Receptor T Cells Analyzed by Novel Tagmentation-Assisted PCR

“…Briefly, we used program EO-115 to electroporate 1 × 10 7 PBMC with 5 μg each of pIRII-CAR.CD19 transposon plasmid and pCMV- piggyBac transposase plasmid. Further, the transfected cells were co-cultured with irradiated autologous activated T cells (ATCs, stimulated with OKT3 and anti-CD28 antibody and cultured with IL-15 for 3 days) as feeder cells that were pulsed with four viral peptide pools (MACS GMP PepTivator; AdV5 Hexon, CMV pp65, EBV EBNA-1, and EBV BZLF1) (Miltenyi Biotec, Auburn, CA, USA), as previously reported [ 20 ]. Following stimulation, the cells were cultured in TexMACS Medium (Miltenyi Biotec) supplemented with human interleukin (IL)-7 (10 ng/mL, Miltenyi Biotec) and IL-15 (5 ng/mL, Miltenyi Biotec) in 24-well plates at 37 °C in a humidified 5% CO 2 incubator.…”

PiggyBac-Engineered T Cells Expressing CD19-Specific CARs that Lack IgG1 Fc Spacers Have Potent Activity against B-ALL Xenografts

“…Although studies of piggyBac-generated CAR T cells have consistently shown in vitro efficacy against their relevant targets,15,20,21,[32][33][34][35] this has not previously translated into sustained complete remissions of malignancy in vivo 21,34. A recent report by Morita et al described a novel method for manufacture of CAR19 T cells incorporating CD28 spacer and co-stimulatory domains 36. These CAR19 T cells displayed antitumor function in vivo, inhibiting expansion of the Daudi cell line without complete remission in murine studies.…”

CAR T Cell Generation by piggyBac Transposition from Linear Doggybone DNA Vectors Requires Transposon DNA-Flanking Regions

“…23 CAR19 T cell activity occurred, despite production utilizing plasmid-based piggyBac components rather than traditional viral vectors and together with other studies, demonstrated that if sufficient CAR is expressed on the T cell surface, then vector choice does not appear to dictate anti-tumor activity. 7,22,23,39,47 In the current study, CAR19 T cells generated using dbDNA-only piggyBac components had similar surface expression of CAR19h28TM41BBz to those generated with plasmid-only components, and we expect that CAR19h28TM41BBz T cells produced with dbDNA-only piggyBac components should retain potent activity against CD19 + targets.…”

“…Both the piggyBac and Sleeping Beauty transposon systems have been used to generate CAR19 T cells that have potent activity against B cell malignancies. 7,21,22,[37][38][39] Nevertheless, plasmids have several undesirable qualities that include bacterial genetic elements, antibiotic resistance genes, and the requirement for expansion in bacteria with subsequent endotoxin removal.…”