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.

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qualitative indicators of dynamics and the quantitative methods of filtered fluorescence correlation spectroscopy (fFCS) and dynamic photon distribution analysis (PDA), can be extended and applied to three-color FRET experiments. Specifically, in three-color pulsed interleaved excitation experiments (PIE) with multiparameter fluorescence detection (MFD), the multidimensional information of color, lifetime and anisotropy offers superior contrast to separate different conformational states. This increases the robustness of the analysis and the sensitivity to minor conformational changes that are otherwise not detectable in two-color FRET. The glycosylation of antibodies is known to influence their ability to instigate an immune response. The biantennary sugars present in the crystallizable fragment (Fc) region of antibodies are thought to stabilize the conformation of the Fc region so it can successfully bind to its receptors, thereby initiating an immune response. Prior studies have suggested that sugar removal leads to an increase in the flexibility of the Fc region. Other results show a collapse of the Fc region upon sugar removal. Either of these conformational alterations would impact receptor binding, thereby explaining the decreased immune response observed upon sugar removal. To examine the structure of the Fc region of murine immunoglobulin G (IgG) antibodies, click chemistry was used to attach dye molecules to azide-modified sugars in the Fc region. In addition, the enzyme EndoS was used to cleave the majority of the sugars from the Fc region. This enzyme leaves behind a single N-acetylglucosamine moiety, which was modified with an azide group and then reacted via click chemistry with dye molecules. These dye-labeled glycosylated and deglycosylated fully intact IgG antibodies were then examined using single molecule FRET to observe the structural impact of sugar removal.