CAR+ extracellular vesicles predict ICANS in patients with B cell lymphomas treated with CD19-directed CAR T cells
Gianluca Storci, … , Massimiliano Bonafè, Francesca Bonifazi
Gianluca Storci, … , Massimiliano Bonafè, Francesca Bonifazi
Published June 4, 2024
Citation Information: J Clin Invest. 2024;134(14):e173096. https://doi.org/10.1172/JCI173096.
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Clinical Research and Public Health Hematology Immunology

CAR+ extracellular vesicles predict ICANS in patients with B cell lymphomas treated with CD19-directed CAR T cells

Abstract

BACKGROUND Predicting immune effector cell–associated neurotoxicity syndrome (ICANS) in patients infused with CAR T cells is still a conundrum. This complication, thought to be consequent to CAR T cell activation, arises a few days after infusion, when circulating CAR T cells are scarce and specific CAR T cell–derived biomarkers are lacking.METHODS CAR+ extracellular vesicle (CAR+EV) release was assessed in human CD19.CAR T cells cocultured with CD19+ target cells. A prospective cohort of 100 patients with B cell lymphoma infused with approved CD19.CAR T cell products was assessed for plasma CAR+EVs as biomarkers of in vivo CD19.CAR T cell activation. Human induced pluripotent stem cell–derived (iPSC-derived) neural cells were used as a model for CAR+EV-induced neurotoxicity.RESULTS In vitro release of CAR+EVs occurs within 1 hour after target engagement. Plasma CAR+EVs are detectable 1 hour after infusion. A concentration greater than 132.8 CAR+EVs/μL at hour +1 or greater than 224.5 CAR+EVs/μL at day +1 predicted ICANS in advance of 4 days, with a sensitivity and a specificity outperforming other ICANS predictors. ENO2+ nanoparticles were released by iPSC-derived neural cells upon CAR+EV exposure and were increased in plasma of patients with ICANS.CONCLUSION Plasma CAR+EVs are an immediate signal of CD19.CAR T cell activation, are suitable predictors of neurotoxicity, and may be involved in ICANS pathogenesis.TRIAL REGISTRATION NCT04892433, NCT05807789.FUNDING Life Science Hub–Advanced Therapies (financed by Health Ministry as part of the National Plan for Complementary Investments to the National Recovery and Resilience Plan [NRRP]: E.3 Innovative health ecosystem for APC fees and immunomonitoring).

Authors

Gianluca Storci, Francesco De Felice, Francesca Ricci, Spartaco Santi, Daria Messelodi, Salvatore Nicola Bertuccio, Noemi Laprovitera, Michele Dicataldo, Lucrezia Rossini, Serena De Matteis, Beatrice Casadei, Francesca Vaglio, Margherita Ursi, Francesco Barbato, Marcello Roberto, Maria Guarino, Gian Maria Asioli, Mario Arpinati, Pietro Cortelli, Enrico Maffini, Enrica Tomassini, Marta Tassoni, Carola Cavallo, Francesco Iannotta, Maria Naddeo, Pier Luigi Tazzari, Elisa Dan, Cinzia Pellegrini, Serafina Guadagnuolo, Matteo Carella, Barbara Sinigaglia, Chiara Pirazzini, Caterina Severi, Paolo Garagnani, Katarzyna Malgorzata Kwiatkowska, Manuela Ferracin, Pier Luigi Zinzani, Massimiliano Bonafè, Francesca Bonifazi

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Figure 4

Combined confocal/STORM analysis of 19BBζ.CAR T and 19BBζ.eGFP.CAR T cells and CAR+EVs.

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Combined confocal/STORM analysis of 19BBζ.CAR T and 19BBζ.eGFP.CAR T cel...
(A) Analysis of 19BBζ.eGFP.CAR T cell coculture supernatants (24 hours): 19BBζ.eGFP.CAR protein (eGFP.CAR; green, wide-field signal) and CD63/CD9/CD81 pool (red, STORM signal). Scale bars: 500 nm (n = 6). (B) 19BBζ.CAR T cell coculture supernatants (24 hours): 2D-STORM analysis of anti-CD19.CAR Ab–mediated pull-down CAR+EVs (CAR; red; scale bar: 500 nm); 3D-STORM analysis of a single CAR+EV (colors represent Z-depth; scale bar: 50 nm). White arrowheads highlight CD19.CAR antigen clusters on the EV (n = 17). (C) 3D-STORM analysis (colors represent Z-depth; scale bar: 50 nm) of CAR T cell supernatants purified by size-exclusion chromatography. White arrowheads highlight CD19.CAR antigen clusters on the EV (n = 3). (D) Schematic representation of the combined confocal/STORM analysis workflow. (E) Combined confocal/STORM analysis with 3D rendering of a 19BBζ.CAR T cell (CAR, red; DAPI, blue); detail of a CAR+ intracellular vesicle observed in 2D- and 3D-STORM (blue box, magnification ×13) (n = 3). Scale bars: 5 μm and 50 nm. (F) Confocal microscopy imaging of a 19BBζ.CAR T cell (CAR, red; CD63, green; DAPI, blue). Colocalization details (blue box, ×3 magnification; purple box, ×22 magnification) measured by Manders’ overlap coefficients (CD63 over CAR, and CAR over CD63, n = 13, MW test) (n = 7). Scale bars: 5 μm, 3 μm, and 0.3 μm. (G) Combined microscopy of 19BBζ.eGFP.CAR T cells: confocal imaging of eGFP.CAR (green) and DAPI (blue), and correlative wide-field (eGFP.CAR, green)/STORM analysis (CD63, white, and CAR, red). Colocalization details (blue boxes, magnification ×2) measured by Manders’ overlap coefficients (CD63 over CAR, and CAR over CD63, n = 5, MW test) (n = 2). Scale bars: 5 μm. Data are presented as boxes and whiskers; boxes show median and IQR, and whiskers represent minimum and maximum values.