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

Abstract

Clinical trials of CD19-specific chimeric antigen receptor (CAR19) T cells have demonstrated remarkable efficacy against relapsed and refractory B cell malignancies. The piggyBac transposon system offers a less complex and more economical means for generating CAR19 T cells compared to viral vectors. We have previously optimized a protocol for the generation of CAR19 T cells using the piggyBac system, but we found that CAR19 T cells had poor in vivo efficacy and persistence, probably due to deleterious FcγR interactions with the CAR's IgG1 Fc-containing spacer domain. We therefore designed three CD19-specifc CARs that lacked the IgG1 Fc region, and we incorporated combinations of CD28 or 4-1BB transmembrane and co-stimulatory domains. PiggyBac-generated CAR19 T cells expressing these re-designed constructs all demonstrated reactivity in vitro specifically against CD19 cell lines. However, those combining CD28 transmembrane and co-stimulatory domains showed CD4 predominance and inferior cytotoxicity. At high doses, CAR19 T cells were effective against B-ALL in a xenograft mouse model, regardless of co-stimulatory domain. At diminishing doses, 4-1BB co-stimulation led to greater potency and persistence of CAR19 T cells, and it provided protection against B-ALL re-challenge. Production of potent CAR T cells using piggyBac is simple and cost-effective, and it may enable wider access to CAR T cell therapy.

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

“…This increased the separation of these elements from <100 bp to approximately 230 bp. Because our previous work identified CAR19h28TM41BBz as our most effective construct, 22 this CAR was used for evaluation of the new dbDNA configuration. PBMCs were coelectroporated with piggyBac transposase (plasmid or dbDNA) and CAR19h28TM41BBz-transposon (plasmid or larger dbDNA), such that all plasmid and dbDNA pairs were evaluated.…”

“…When expressed, the transposase excises the transposon from the second plasmid and integrates it into the cellular genome. We have previously described a simple and inexpensive method for generating CAR T cells using the piggyBac system 21 and demonstrated that CAR19 T cells produced in this manner are capable of eradicating B cell acute lymphoblastic leukemia (B-ALL) xenografts in vivo 22 and CD19 + malignancies in humans. 23 Plasmids can be produced inexpensively at large scale, but their reliance on bacteria for amplification has several disadvantages for clinical translation.…”

T-Cell Receptor–Based Immunotherapy for Hematologic Malignancies

“…Transduction with a viral vector encoding a polycistronic construct of both α and β chains of the transgenic TCR separated by a ribosomal skip motif, such [71][72][73] and nanoparticles, 74 are also in development.…”

Consider Changing the Horse for Your CAR-T?

“…However, clinical grade retroviral vectors are costly to produce and can be generated at only a few facilities. The work in this issue of Molecular Theapy by Bishop et al 1 reveals that non-viral piggyBac transposon-generated CD19-specific chimeric antigen receptor (CAR) T cells have excellent in vivo efficacy in the setting of patient derived CD19+ B-acute lymphoblastic leukemia (ALL) in an animal model. Although piggyBac transposon-generated CAR T cells had previously demonstrated in vitro efficacy, 2-4 sustained activity in vivo had not been reported until now.…”

“…Therefore, a human immune system and malignancy is not fully recapitulated. Nonetheless, the study by Bishop et al 1 is an important step towards clinical testing and application.…”