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4-1BB-encoding CAR causes cell death via sequestration of the ubiquitin-modifying enzyme A20
Zhangqi Dou1,2, Thomas Raphael Bonacci3, Peishun Shou1
1Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA.
Abstract:
CD28 and 4-1BB costimulatory endodomains included in chimeric antigen receptor (CAR) molecules play a critical role in promoting sustained antitumor activity of CAR-T cells. However, the molecular events associated with the ectopic and constitutive display of either CD28 or 4-1BB in CAR-T cells have been only partially explored. In the current study, we demonstrated that 4-1BB incorporated within the CAR leads to cell cluster formation and cell death in the forms of both apoptosis and necroptosis in the absence of CAR tonic signaling. Mechanistic studies illustrate that 4-1BB sequesters A20 to the cell membrane in a TRAF-dependent manner causing A20 functional deficiency that in turn leads to NF-κB hyperactivity, cell aggregation via ICAM-1 overexpression, and cell death including necroptosis via RIPK1/RIPK3/MLKL pathway. Genetic modulations obtained by either overexpressing A20 or releasing A20 from 4-1BB by deleting the TRAF-binding motifs of 4-1BB rescue cell cluster formation and cell death and enhance the antitumor ability of 4-1BB-costimulated CAR-T cells.
Insights
The 4-1BB costimulatory domain in CAR-T cells causes cell death and aggregation by sequestering A20. Modulating A20 levels or 4-1BB binding rescues these effects and improves anti-tumor activity.
Area of Science:
- Immunology
- Cell Biology
- Molecular Biology
Background:
- Chimeric antigen receptor (CAR)-T cells utilize costimulatory endodomains like CD28 and 4-1BB for sustained antitumor activity.
- The molecular mechanisms underlying the effects of ectopic CD28 or 4-1BB in CAR-T cells are not fully understood.
Purpose of the Study:
- To investigate the molecular events triggered by the 4-1BB costimulatory domain within CAR-T cells.
- To elucidate the impact of 4-1BB on cell aggregation, cell death pathways, and antitumor function.
Main Methods:
- Studied CAR-T cells engineered with the 4-1BB endodomain.
- Investigated cell cluster formation, apoptosis, and necroptosis.
- Performed mechanistic studies involving A20, TRAF, NF-κB, ICAM-1, and the RIPK1/RIPK3/MLKL pathway.
- Utilized genetic modifications including A20 overexpression and deletion of TRAF-binding motifs in 4-1BB.
Main Results:
- 4-1BB incorporation in CAR-T cells induced cell cluster formation and cell death (apoptosis and necroptosis) independently of tonic CAR signaling.
- 4-1BB sequestered A20 to the cell membrane via TRAF, leading to A20 deficiency, NF-κB hyperactivity, ICAM-1 overexpression, cell aggregation, and necroptosis.
- Genetic interventions, such as overexpressing A20 or modifying 4-1BB's TRAF-binding sites, prevented cell aggregation and death.
Conclusions:
- The 4-1BB endodomain triggers specific molecular events leading to cell aggregation and death through A20 sequestration and NF-κB/necroptosis pathway activation.
- Genetic modulation of the 4-1BB-A20 interaction can mitigate adverse cellular effects and enhance the therapeutic potential of 4-1BB-costimulated CAR-T cells.
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