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 4

m19-humBBz CAR T cells have higher antiapoptotic protein expression than m1928z CAR T cells.

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m19-humBBz CAR T cells have higher antiapoptotic protein expression than...
Viability (A) and proliferation (B) of mCD19-targeted CAR T cells. CAR T cells were produced and proliferation was evaluated by fold change from the initial cell number to final cell yield at day 4. Cell viability was measured by trypan blue staining on an automated cell counter (Bio-Rad). Data were pooled from 17 (viability) and 19 (proliferation) independent productions. (C) BCL2 and BCL-XL expression in CAR T cells by flow cytometry. Day 4 CAR T cells were intracellularly stained with anti-BCL2 and anti–BCL-XL antibodies and analyzed by flow cytometry. Cells were pregated on live CAR T cells. Data are representative of 2 independent experiments. (D) BCL2 and BCL-XL protein expression after antigen stimulation. One million day 4 CAR T cells were stimulated on 1 × 105 3T3-mCD19 cells for 4 hours. Cell lysates from CAR T cells were prepared, protein quantified by BCA, normalized to total protein, and analyzed by Western blot. Western blots are representative of 2 independent experiments. Semiquantitation of Western blots was done using ImageJ software. BCL2 and BCL-XL expression in different CAR T cells was compared by normalizing to β-actin. Data were analyzed by unpaired t test. ns, not significant.