Production and characterization of virus-free, CRISPR-CAR T cells capable of inducing solid tumor regression

Abstract

BackgroundChimeric antigen receptor (CAR) T cells have demonstrated high clinical response rates against hematological malignancies (e.g., CD19+ cancers) but have shown limited activity in patients with solid tumors. Recent work showed that precise insertion of a CAR at a defined locus improves treatment outcomes in the context of a CD19 CAR; however, it is unclear if such a strategy could also affect outcomes in solid tumors. Furthermore, CAR manufacturing generally relies on viral vectors for gene delivery, which comprise a complex and resource-intensive part of the manufacturing supply chain.MethodsAnti-GD2 CAR T cells were generated using CRISPR/Cas9 within 9 days using recombinant Cas9 protein and nucleic acids, without any viral vectors. The CAR was specifically targeted to the T cell receptor alpha constant gene (TRAC). T cell products were characterized at the level of the genome, transcriptome, proteome, and secretome using CHANGE-seq, targeted next-generation sequencing, scRNA-seq, spectral cytometry, and ELISA assays, respectively. Functionality was evaluated in vivo in an NSG™ xenograft neuroblastoma model.ResultsIn comparison to retroviral CAR T cells, virus-free CRISPR CAR (VFC-CAR) T cells exhibit TRAC-targeted genomic integration of the CAR transgene, elevation of transcriptional and protein characteristics associated with a memory-like phenotype, and low tonic signaling prior to infusion arising in part from the knockout of the T cell receptor. On exposure to the GD2 target antigen, anti-GD2 VFC-CAR T cells exhibit specific cytotoxicity against GD2+ cells in vitro and induce solid tumor regression in vivo. VFC-CAR T cells demonstrate robust homing and persistence and decreased exhaustion relative to retroviral CAR T cells against a human neuroblastoma xenograft model.ConclusionsThis study leverages virus-free genome editing technology to generate CAR T cells featuring a TRAC-targeted CAR, which could inform manufacturing of CAR T cells to treat cancers, including solid tumors.

Deciphering and advancing CAR T-cell therapy with single-cell sequencing technologies

“…scRNA-seq performed on IKZF2-KO (knockout) and TLE4-KO CAR T-cells revealed that these cells displayed enhanced cytotoxicity and immune stimulation transcriptional signatures while prohibiting exhaustion, indicating their superior effector function against tumor cells [ 130 ]. In addition, two pilot studies used CRISPR-Cas9 gene editing technology for CAR gene transduction instead of traditional viral transduction [ 131 , 132 ]. scRNA-seq found that CAR T-cells integrating the CAR gene at the T-cell receptor α constant (TRAC) locus and the PD1 gene locus both expressed a memory-like phenotype and less exhaustion-associated transcriptional signatures, which was related to higher potency [ 131 , 132 ].…”

“…In addition, two pilot studies used CRISPR-Cas9 gene editing technology for CAR gene transduction instead of traditional viral transduction [ 131 , 132 ]. scRNA-seq found that CAR T-cells integrating the CAR gene at the T-cell receptor α constant (TRAC) locus and the PD1 gene locus both expressed a memory-like phenotype and less exhaustion-associated transcriptional signatures, which was related to higher potency [ 131 , 132 ]. Obviously, the site-directed integration of CAR genes by CRISPR may be superior to random insertion by viral vector transduction.…”

“… The feasibility of preparing TRAC-targeted CAR T-cells by CRISPR/Cas9 technology and virus-free method was proved and TRAC-integrated CAR T-cells showed higher proportion of memory phenotype, less depletion phenotype and lower degree of differentiation. [ 132 ] scRNA-seq, CRISPR/Cas9 genome editing system B-NHL CD19 IPs, engineered CAR T-cells, PBMCs, 3 patients Cell 63,789 scRNA-seq was performed on CAR T-cells from 3 NHL patients before CAR T-cell infusion and on day 7/12/28/29 after infusion. The feasibility of preparing PD1-targeted CAR T-cells by CRISPR/Cas9 technology and virus-free method was proved and non-viral, PD1-integrated CAR-T cells exhibited enhanced antitumor ability.…”

Applications of single‐cell omics for chimeric antigen receptor T cell therapy

“…This results in uniform CAR expressions in the product [71]. Multi‐omics approaches have confirmed how CRISPR‐mediated CAR integration in the TRAC locus results in the production of CAR T cells with an enhanced antitumor response and lower exhaustion signatures compared to CAR T cells produced using retroviral vectors [72]. Unstimulated virus‐free CRISPR CAR T cells created by Mueller et al exhibited lower basal TCR and CAR signalling and cytokine production than retroviral CAR T cells, but their cytokine production rose to a similar or higher level than retroviral CAR T cells after antigen stimulation.…”

“…Based on the scRNA‐seq data, unstimulated CRISPR CAR T cells had almost double the number of memory‐like cell clusters and half of the effector‐like clusters compared to retroviral CAR T cells. Upon stimulation, memory‐like transcriptional signatures of CRISPR CAR T cells remained stable, unlike retroviral CAR T cells that shifted more towards effector signatures [72]. These observations, which are due to the decrease in tonic signalling within CAR T cells after TRAC disruption, emphasize the importance of CRISPR/Cas9 in quality CAR T cell production.…”