Physiological (TCR-like) regulated lentiviral vectors for the generation of improved CAR-T cells

Abstract

BackgroundChimeric antigen receptor (CAR) T cells directed against CD19 have achieved impressive outcomes for the treatment of relapsed/refractory B lineage lymphoid neoplasms. However, CAR-T therapy still has important limitations due to severe side effects and the lack of efficiency in 40-50% of the patients. Most CARs-T products are generated using retroviral vectors with strong promoters. However, high CAR expression levels can lead to tonic signalling, premature exhaustion and over-stimulation of CAR-T cells, reducing efficacy and increasing side effects. TCR-like expression of the CAR through genome editing resulted in enhanced anti-tumour potency, reducing tonic signalling and improving CAR-T phenotype. In this manuscript, we searched for LVs that mimic the TCR expression pattern as a closer-to-clinic alternative for the generation of improved CAR-T cells.MethodsDifferent LVs containing viral and human promoters were analysed to select those that closely mimic a TCR-like kinetic profile upon T-cell activation. WAS gene proximal promoter-driven LVs (AW-LVs) were selected to express a second generation 4-1BB aCD19 CAR (ARI-0001) into T cells to generate AWARI CAR-T cells. TCR-like AWARI and EF1α-driven ARI CAR T cells were analysed for in vitro and in vivo killing efficiency using leukaemia and lymphoma cellular models. Tonic signalling, exhaustion markers and phenotype were determined by flow cytometry. Large-scale automated manufacturing of AWARI CAR-T cells was performed in a CliniMACs Prodigy bioreactor.ResultsOur data showed that LVs expressing the transgene through the WAS gene proximal promoter mimic very closely the TCR (CD3) expression pattern kinetic upon TCR stimulation or antigen encounter. Compared to EF1α-driven ARI CAR-T cells, AWARI CAR-T cells exhibited a higher proportion of naïve/stem cell memory T cells with less exhausted phenotype after efficient killing of CD19+ cells both in vitro and in vivo. AWARI CAR-T cells also showed lower tonic signalling and reduced secretion of pro-inflammatory cytokines and were efficiently manufactured in large-scale GMP-like conditions.ConclusionsWAS-gene-promoter driven LVs can be used to generate physiological 4-1BB-CAR-T cell products with lower tonic signalling, improved phenotype and a safer profile. We propose the use of TCR-like LVs as an alternative to strong-promoter driven LVs for the generation of CAR-T products.

CAR Density Influences Antitumoral Efficacy of BCMA CAR T cells and Correlates with Clinical Outcome

“…These promoters induce high levels of CAR expression that can lead to tonic signaling and premature exhaustion. The possibility of reducing gene expression via weaker promoters as well as through more physiological promoters has been associated with decreased tonic signaling (22,23,52). In fact, the use of the MND promoter has been shown to reduce CAR surface density, while EF1a promoter increased its density, leading to a higher cytotoxic activity, cytokine production and expression of exhaustion markers (23).…”

“…In fact, the use of the MND promoter has been shown to reduce CAR surface density, while EF1a promoter increased its density, leading to a higher cytotoxic activity, cytokine production and expression of exhaustion markers (23). Thus, the use of physiological promoters would be a strategy to prevent an increase in CAR High T cells, thus limiting exhaustion of CAR-T and favoring long term persistence and antitumor efficacy (22).…”

“…For instance, recent studies have demonstrated that a more physiological expression of the CAR, by the integration of the transgene in the TRAC locus or the use of different promoters, can be associated with improved efficacy and reduced toxicity (21)(22)(23). These results also suggest that the density of CAR molecule in the membrane of CAR T cells might influence CAR signaling, affecting their antitumoral efficacy (23,24).…”

Secretory co-factors in next-generation cellular therapies for cancer

“…Since retrovirus vectors are generally used for T-cell transduction, the expression of secretory co-factors and CAR is often driven by the retroviral long terminal repeat (LTR) promoter or the CMV (cytomegalovirus), EF1α (elongation factor 1α), PGK (phosphoglycerate kinase), and SFFV (spleen focus-forming virus) promoters ( 97 – 100 ). In many cases, the EF1α and SFFV promoters are stronger, but do not always lead to a better transcriptional activity.…”

“…In many cases, the EF1α and SFFV promoters are stronger, but do not always lead to a better transcriptional activity. In addition, CAR overexpression can lead to tonic signals and premature exhaustion ( 100 , 101 ). Various gene drivers can be constructed by combining promoters, introns, and enhancers and can be optimized for the application of interest.…”