Xue et al. used a single-cell microfluidics device to simultaneously measure 16 cytokines released by CD19 CAR T cells in response to target-specific stimulation, and developed two novel bioinformatics tools to visualize the data. CD19 CAR T cells exhibited a high degree of polyfunctionality (production of multiple cytokines, a predictor of therapeutic efficacy) dominated by antitumor effector and stimulatory functions, and significant heterogeneity, both within cells from the same donor and between donors.

BACKGROUND: It remains challenging to characterize the functional attributes of chimeric antigen receptor (CAR)-engineered T cell product targeting CD19 related to potency and immunotoxicity ex vivo, despite promising in vivo efficacy in patients with B cell malignancies. METHODS: We employed a single-cell, 16-plex cytokine microfluidics device and new analysis techniques to evaluate the functional profile of CD19 CAR-T cells upon antigen-specific stimulation. CAR-T cells were manufactured from human PBMCs transfected with the lentivirus encoding the CD19-BB-z transgene and expanded with anti-CD3/anti-CD28 coated beads. The enriched CAR-T cells were stimulated with anti-CAR or control IgG beads, stained with anti-CD4 RPE and anti-CD8 Alexa Fluor 647 antibodies, and incubated for 16 h in a single-cell barcode chip (SCBC). Each SCBC contains ~12,000 microchambers, covered with a glass slide that was pre-patterned with a complete copy of a 16-plex antibody array. Protein secretions from single CAR-T cells were captured and subsequently analyzed using proprietary software and new visualization methods. RESULTS: We demonstrate a new method for single-cell profiling of CD19 CAR-T pre-infusion products prepared from 4 healthy donors. CAR-T single cells exhibited a marked heterogeneity of cytokine secretions and polyfunctional (2+ cytokine) subsets specific to anti-CAR bead stimulation. The breadth of responses includes anti-tumor effector (Granzyme B, IFN-gamma, MIP-1alpha, TNF-alpha), stimulatory (GM-CSF, IL-2, IL-8), regulatory (IL-4, IL-13, IL-22), and inflammatory (IL-6, IL-17A) functions. Furthermore, we developed two new bioinformatics tools for more effective polyfunctional subset visualization and comparison between donors. CONCLUSIONS: Single-cell, multiplexed, proteomic profiling of CD19 CAR-T product reveals a diverse landscape of immune effector response of CD19 CAR-T cells to antigen-specific challenge, providing a new platform for capturing CAR-T product data for correlative analysis. Additionally, such high dimensional data requires new visualization methods to further define precise polyfunctional response differences in these products. The presented biomarker capture and analysis system provides a more sensitive and comprehensive functional assessment of CAR-T pre-infusion products and may provide insights into the safety and efficacy of CAR-T cell therapy.

Author Info: (1) Novartis Pharmaceuticals, 64 Sidney Street, Cambridge, MA, 02139, USA. chelsea.xue@novartis.com. Present Address: Novartis Institute of BioMedical Research, 300 Technology Square, Cambridge, MA, 02139, USA

Author Info: (1) Novartis Pharmaceuticals, 64 Sidney Street, Cambridge, MA, 02139, USA. chelsea.xue@novartis.com. Present Address: Novartis Institute of BioMedical Research, 300 Technology Square, Cambridge, MA, 02139, USA. chelsea.xue@novartis.com. (2) IsoPlexis Corporation, 35 NE Industrial Rd, Branford, CT, 06405, USA. (3) IsoPlexis Corporation, 35 NE Industrial Rd, Branford, CT, 06405, USA. (4) IsoPlexis Corporation, 35 NE Industrial Rd, Branford, CT, 06405, USA. (5) IsoPlexis Corporation, 35 NE Industrial Rd, Branford, CT, 06405, USA. (6) IsoPlexis Corporation, 35 NE Industrial Rd, Branford, CT, 06405, USA. (7) Novartis Pharmaceuticals, 64 Sidney Street, Cambridge, MA, 02139, USA. Present Address: Novartis Institute of BioMedical Research, 64 Sidney street, Cambridge, MA, 02139, USA. (8) Novartis Pharmaceuticals, 64 Sidney Street, Cambridge, MA, 02139, USA. Present Address: Novartis Pharmaceuticals, 45 Sidney Street, Cambridge, MA, 02139, USA. (9) NanoSystems Biology Cancer Center, Division of Chemistry, California Institute of Technology, Pasadena, CA, 91125, USA. (10) Department of Biomedical Engineering, Yale University, New Haven, CT, 06520, USA. (11) IsoPlexis Corporation, 35 NE Industrial Rd, Branford, CT, 06405, USA. (12) Novartis Pharmaceuticals, 64 Sidney Street, Cambridge, MA, 02139, USA. Present Address: Adaptimmune, 351 Rouse Blvd, Philadelphia, PA, 19112, USA. (13) Novartis Pharmaceuticals, 64 Sidney Street, Cambridge, MA, 02139, USA. Present Address: Mustang Bio, 95 Sawyer Road, Waltham, MA, 02453, USA. (14) IsoPlexis Corporation, 35 NE Industrial Rd, Branford, CT, 06405, USA. jing@isoplexis.com.

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