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Optimization of anti-TIM3 chimeric antigen receptor with CD8α spacer and TNFR-based costimulation for enhanced
Kristine Cate S Pe1, Sirirut Jewmoung2, Sm Ali Hosseini Rad3
1Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand.
Abstract:
CAR T cell therapy for AML remains limited due to the lack of a proper target without on-target off-tumor toxicity. TIM3 is a promising target due to its high expression on AML cells and absence in most normal hematopoietic cells. Previous reports have shown that each CAR component impacts CAR functionality. Here, we optimized TIM-3 targeting CAR T cells for AML therapy. We generated CARs targeting TIM3 with two different non-signaling domains: an IgG2-CH3 spacer with CD28 transmembrane domain (CH3/CD28) and a CD8α spacer with CD8α transmembrane domain (CD8/CD8), and evaluated their characteristics and function. Incorporating the non-signaling CH3/CD28 domain resulted in unstable CAR expression in anti-TIM3 CAR T cells, leading to lower surface CAR expression over time and reduced cytotoxic function compared to anti-TIM3 CARs with the CD8/CD8 domain. Both types of anti-TIM3 CAR T cells transiently exhibited fratricide, which subsided overtime, and both CAR T cells achieved substantial T cell expansion. To further optimize the design, we explored the effects of different costimulatory domains. Compared with CD28 costimulation, 4-1BB and CD27 combined with a CD8/CD8 non-signaling domain showed higher cytokine secretion, superior antitumor activity, and enhanced T-cell persistence after repeated antigen exposure. These findings emphasize the impact of the optimal design of CAR constructs that provide efficient function. In the context of anti-TIM3 CAR T cells, using a CD8α spacer and transmembrane domain with TNFR-based costimulation is a promising CAR design to improve anti-TIM3 CAR T cell function for AML therapy.
Insights
Optimizing Chimeric Antigen Receptor (CAR) T cells targeting TIM3 for Acute Myeloid Leukemia (AML) therapy is crucial. A CD8α spacer and transmembrane domain with TNFR-based costimulation demonstrated superior anti-leukemia activity and T cell persistence.
Area of Science:
- Immunotherapy
- Cancer Biology
- Cellular Engineering
Background:
- CAR T cell therapy for Acute Myeloid Leukemia (AML) is hindered by the lack of specific targets, leading to on-target off-tumor toxicity.
- TIM3 is a highly expressed target on AML cells with minimal expression on normal hematopoietic cells, making it a promising target for AML therapy.
Purpose of the Study:
- To optimize the design of TIM3-targeting CAR T cells for improved efficacy and safety in AML treatment.
- To evaluate the impact of different non-signaling and costimulatory domains on CAR T cell functionality.
Main Methods:
- Generated CAR T cells targeting TIM3 with distinct non-signaling domains (IgG2-CH3/CD28 vs. CD8α/CD8α).
- Assessed CAR expression stability, cytotoxic function, T cell expansion, and cytokine secretion.
- Investigated the effects of different costimulatory domains (CD28, 4-1BB, CD27) in combination with the optimized non-signaling domain.
Main Results:
- The CD8α/CD8α non-signaling domain maintained stable CAR expression and superior cytotoxic function compared to the CH3/CD28 domain.
- Both CAR designs exhibited transient fratricide, which resolved over time, and achieved significant T cell expansion.
- Combining the CD8α/CD8α domain with 4-1BB and CD27 costimulation resulted in enhanced cytokine secretion, potent antitumor activity, and improved T cell persistence.
Conclusions:
- The design of CAR constructs significantly impacts CAR T cell functionality for AML therapy.
- A CAR T cell design incorporating a CD8α spacer and transmembrane domain with TNFR-based costimulation shows promise for enhancing anti-TIM3 CAR T cell efficacy in AML.
