4-1BB enhancement of CAR T function requires NF-κB and TRAFs
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
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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Research Article Immunology Oncology

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

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 7

TRAF2 overexpression modulates 4-1BB–based human CAR T function.

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TRAF2 overexpression modulates 4-1BB–based human CAR T function. (A) NF-...
(A) NF-κB signaling in human CD19 CAR–transduced (h19BBz-transduced) NF-κB/293/GFP-Luc reporter cells by increasing NF-κB. Cells were transduced with h19BBz CAR with or without TRAFs. NF-κB was measured by GFP fluorescence. Data are from 1 experiment in triplicate. (B) Viability and (C) cell expansion of h19BBz CAR T cells with TRAF overexpression upon antigen stimulation. CAR T cells were cocultured with 3T3-hCD19 at a 10:1 ratio and cell numbers and viability were measured daily for 3 days. (D) Cytotoxicity of h19BBz CAR T cells with TRAF overexpression. CAR T cells were cocultured with 3T3-hCD19 at a 5:1 ratio. Target cell killing was monitored by xCELLigence RTCA (real-time cell analysis) system. (E) Cytotoxicity of human CD33–targeted CAR (h33BBz) T cells with different scFvs. CAR T cells were cocultured with CHO-hCD33 at a 10:1 ratio. Target cell killing was monitored by RTCA. (F) Fold change of h33BBz T cell production with or without TRAF2 cotransduction. CAR T cells were produced and proliferation was evaluated by fold change from the initial cell number to final cell yield. (G) h33BBz CAR T cell expansion in vitro upon antigen stimulation. CAR T cells were stained with proliferation dye and cocultured with CHO-hCD33 at a 10:1 E/T ratio for 4 days. Proliferation of CAR T cells was evaluated by flow cytometry (MFI of proliferation dye in CAR T population). All experiments other than F were done in triplicate. For B, C, and D, data are 1 representative of 3 donors. For F and G, data are from 1 single experiment. For A and E, data are 1 representative of 2 independent experiments. Cytotoxicity data are shown as the mean only; others are shown as the mean ± SD. Untrans, untransduced; +TF1, CAR plus TRAF1; +TF2, CAR plus TRAF2; +TF3, CAR plus TRAF3. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by unpaired t test (A and G), 2-way ANOVA (B and C), or Kolmogorov-Smirnov test (D and E). ns, not significant.