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4-1BB enhancement of CAR T function requires NF-κB and TRAFs
Gongbo Li, … , Daniel Abate-Daga, Marco L. Davila
Gongbo Li, … , Daniel Abate-Daga, Marco L. Davila
Published September 20, 2018
Citation Information: JCI Insight. 2018;3(18):e121322. https://doi.org/10.1172/jci.insight.121322.
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Research Article Immunology Oncology

4-1BB enhancement of CAR T function requires NF-κB and TRAFs

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Abstract

Chimeric antigen receptors (CARs) have an antigen-binding domain fused to transmembrane, costimulatory, and CD3ζ domains. Two CARs with regulatory approval include a CD28 or 4-1BB costimulatory domain. While both CARs achieve similar clinical outcomes, biologic differences have become apparent but not completely understood. Therefore, in this study we aimed to identify mechanistic differences between 4-1BB and CD28 costimulation that contribute to the biologic differences between the 2 CARs and could be exploited to enhance CAR T cell function. Using CD19-targeted CAR T cells with 4-1BB we determined that enhancement of T cell function is driven by NF-κB. Comparison to CAR T cells with CD28 also revealed that 4-1BB is associated with more antiapoptotic proteins and dependence on persistence for B cell killing. While TNF receptor–associated factor 2 (TRAF2) has been presupposed to be required for 4-1BB costimulation in CAR T cells, we determined that TRAF1 and TRAF3 are also critical. We observed that TRAFs impacted CAR T viability and proliferation, as well as cytotoxicity and/or cytokines, in part by regulating NF-κB. Our study demonstrates how 4-1BB costimulation in CAR T cells impacts antitumor eradication and clinical outcomes and has implications for enhanced CAR design.

Authors

Gongbo Li, Justin C. Boucher, Hiroshi Kotani, Kyungho Park, Yongliang Zhang, Bishwas Shrestha, Xuefeng Wang, Lawrence Guan, Nolan Beatty, Daniel Abate-Daga, Marco L. Davila

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Figure 1

Comparison of mouse T cells with mCD19-targeted CARs having different intracellular domains.

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Comparison of mouse T cells with mCD19-targeted CARs having different in...
(A) Cytotoxicity assay. CAR T cells were cocultured with EL4-mCD19 cells at indicated E/T ratios. Cytotoxicity was evaluated with a chromium-release assay. Data are representative of 3 independent experiments in triplicate. (B) Cytokine production. CAR T cells were cocultured with 3T3-mCD19 cells for 24 hours. Supernatants were collected for Luminex assay. Data are representative of 2 independent experiments in triplicate. (C) Survival and (D) in vivo B cell killing and T cell persistence 3 weeks after CAR T injection at 5 × 106 dose. Six days after injection with Eμ-ALL cells, mice were i.p. injected with cyclophosphamide (CTX) followed 1 day later with an i.v. injection of 5 × 106 T cells. Survival data are pooled from 2 independent experiments (n = 45 total). Negative control groups are CTX alone or with m19Δz CAR T cells (CTX ± m19Δz). (E) Survival and (F) in vivo B cell killing and T cell persistence 4 weeks after CAR T injection at 3 × 105 T cell dose. Seven days after injection with Eμ-ALL, mice were i.p. injected with CTX followed 1 day later with an i.v. injection of CAR T cells. Survival data are from 1 experiment (n = 39 total). B (B220+CD19+) and donor T (CD3+Thy1.1+) cells in the blood were quantified using CountBright counting beads. For D and F, each data point represents 1 mouse. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by log-rank test (C and E) or unpaired t test (B, D, and F). ns, not significant.

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