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

Mueller KP, Piscopo NJ, Forsberg MH, Saraspe LA, Das A, Russell B, Smerchansky M, Cappabianca D, Shi L, Shankar K, Sarko L, Khajanchi N, La Vonne Denne N, Ramamurthy A, Ali A, Lazzarotto CR, Tsai SQ, Capitini CM, Saha K
Source: J Immunother
Publication Date: (2022)
Issue: 1: 1
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Gene Expression
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
4D-Nucleofector® X-Unit

T cell nucleofection RNPs and HDR templates were electroporated 2 days after T cell isolation and stimulation. During crRNA and tracrRNA incubation, T cells were centrifuged for 3 min at 200 g and counted using a Countess II FL Automated Cell Counter with 0.4% Trypan Blue viability stain (Thermo Fisher). One million cells per replicate were aliquoted into 1.5 mL tubes. During the RNP complexation step (see RNP production), T cell aliquots were centrifuged for 10 min at 90 g. During the spin step, 2 µL of HDR template (total 4 µg) per condition were aliquoted to PCR tubes, followed by RNPs (2.8 µL per well; pipette should be set to a higher volume to ensure complete expulsion of viscous solution). Templates and RNPs were incubated at room temperature for at least 30 s. After cell centrifugation, supernatants were removed by pipette, and cells were resuspended in 20 µL P3 buffer (Lonza), then transferred to PCR tubes containing RNPs and HDR templates, bringing the total volume per sample to 24 µL. Each sample was transferred directly to a 16 well electroporation cuvette. Typically, no more than eight reactions were completed at a time to minimize the amount of time T cells spent in P3 buffer. T cells were electroporated with a Lonza 4D Nucleofector with X Unit using pulse code EH-115. Immediately after electroporation, 80 µL of prewarmed recovery medium with 500 U/mL IL-2 and 25 µL/mL ImmunoCult CD3/CD28/CD2 activator was added to each well of the cuvette. Cuvettes were rested at 37°C in the cell culture incubator for 15 min. After 15 min, cells were moved to 200 µL total volume of media with IL-2 and activator (see above) in a round bottom 96 well plate.


Background Chimeric 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.

Methods Anti-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.
Results In 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

Conclusions This 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.