Background
Cellular immunotherapy using chimeric antigen receptor T cell (CAR T) therapy has revolutionized treatment of patients with hematologic malignancy. Despite success, as many as half of leukemia patients experience disease progression. CAR T infusion product potency is often compromised by the poor health of T cells recovered from leukemia patients and intrinsic cellular mechanisms, such as activation-induced cell death (AICD), that limit the duration of the anti-tumor response. To enhance the overall efficacy of CAR T therapy and predict potency, innovative methods are needed to deliver CAR constructs with minimal cellular damage and accurately predict potency and resistance to exhaustion while limiting in vivo toxicity.
Methods
Adaptive immune cells are intrinsically heterogeneous, and their phenotypes change after interaction with target cells. We previously reported efficient genomic editing of primary T cells using microfluidic vortex shedding (‘hydroporation’). In this study, we targeted the TRAC locus in primary human T cells with a CD19-specific CAR using homology-driven repair. We directly compared the transfection efficiency, yield, viability, and functions of CAR T prepared by hydroporation and nucleofection (Lonza). To assess viability, potency, and functional exhaustion at the single cell level, we performed TIMING™ (Time-Lapse Imaging Microscopy In Nanowell Grids) assays.
Results
Hydroporation resulted in 1.7-2X greater yield of CAR T cells by day 5. We observed similar cytotoxicity of CAR T prepared by hydroporation and nucleofection, as well as nearly identical CD4+/CD8+ T cell ratios, memory phenotype, and pro-inflammatory cytokine secretion upon target engagement. At the single cell level, we found that CAR T manufactured by hydroporation and nucleofection formed synapses with similar kinetics and had similar efficiency of tumor cell killing and serial killing. However, hydroporated CAR T retained higher motility while in synapse with target cells and were more resistant to activation-induced cell death (AICD) after target engagement.
Conclusions
These results show that hydroporation is an efficient method of manufacturing CAR T cells that yields cells of equivalent cytotoxicity as CAR T manufactured by nucleofection. Hydroporation not only yielded more cells after outgrowth, but also yielded cells more likely to persist longer in vivo. As prior work on patient infusion products has shown that single-cell motility correlates with patient response, this work is highly significant in expanding the options for production of efficacious CAR T for treatment of malignancies and autoimmune disease.
Acknowledgements
This project has been funded in part with Federal funds from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services, under Contract No. 75N91020C00030 and 7591022C00053. CellChorus received support from the National Center for Translational Sciences (R44TR005137) and the National Institute of General Medical Sciences (R44GM149106) of the National Institutes of Health.
Ethics Approval
This study uses only de-identified human cells from a commercial source. No ethics review is required.