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

• Published in: Journal for immunotherapy of cancer 2022-09, Vol.10 (9), p.e004446
• Main Authors: Mueller, Katherine P, Piscopo, Nicole J, Forsberg, Matthew H, Saraspe, Louise A, Das, Amritava, Russell, Brittany, Smerchansky, Madeline, Cappabianca, Dan, Shi, Lei, Shankar, Keerthana, Sarko, Lauren, Khajanchi, Namita, La Vonne Denne, Nina, Ramamurthy, Apoorva, Ali, Adeela, Lazzarotto, Cicera R, Tsai, Shengdar Q, Capitini, Christian M, Saha, Krishanu
• Format: Article
• Language: English
• Subjects: Antigens; Antigens, CD19; Cancer; Cell Engineering; Cloning; CRISPR; Gangliosides - metabolism; Gene Expression Profiling; Genome editing; Genomes; Genotype & phenotype; Hematology; Humans; Immune Cell Therapies and Immune Cell Engineering; Immunotherapy; Immunotherapy, Adoptive; Lymphocytes; Neuroblastoma; Neuroblastoma - pathology; Plasmids; Receptors, Antigen, T-Cell; Receptors, Chimeric Antigen; T cell receptors; T-Lymphocytes; Tumors; Vectors (Biology); Xenograft Model Antitumor Assays
• ISSN: 2051-1426; 2051-1426
• DOI: 10.1136/jitc-2021-004446

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.