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Manufacturing Chimeric Antigen Receptor CAR T Cells for Adoptive Immunotherapy
Published on: December 17, 2019
Pooled screening of CAR T cells identifies diverse immune signaling domains for next-generation immunotherapies
Daniel B Goodman1,2,3,4,5, Camillia S Azimi1,2,3, Kendall Kearns1,2
1Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA.
Abstract:
Chimeric antigen receptors (CARs) repurpose natural signaling components to retarget T cells to refractory cancers but have shown limited efficacy in persistent, recurrent malignancies. Here, we introduce "CAR Pooling," a multiplexed approach to rapidly identify CAR designs with clinical potential. Forty CARs with signaling domains derived from a range of immune cell lineages were evaluated in pooled assays for their ability to stimulate critical T cell effector functions during repetitive stimulation that mimics long-term tumor antigen exposure. Several domains were identified from the tumor necrosis factor (TNF) receptor family that have been primarily associated with B cells. CD40 enhanced proliferation, whereas B cell-activating factor receptor (BAFF-R) and transmembrane activator and CAML interactor (TACI) promoted cytotoxicity. These functions were enhanced relative to clinical benchmarks after prolonged antigen stimulation, and CAR T cell signaling through these domains fell into distinct states of memory, cytotoxicity, and metabolism. BAFF-R CAR T cells were enriched for a highly cytotoxic transcriptional signature previously associated with positive clinical outcomes. We also observed that replacing the 4-1BB intracellular signaling domain with the BAFF-R signaling domain in a clinically validated B cell maturation antigen (BCMA)-specific CAR resulted in enhanced activity in a xenotransplant model of multiple myeloma. Together, these results show that CAR Pooling is a general approach for rapid exploration of CAR architecture and activity to improve the efficacy of CAR T cell therapies.
Insights
CAR Pooling identifies novel chimeric antigen receptor (CAR) designs by testing numerous signaling domains. This approach enhances T cell functions, improving CAR T cell therapy efficacy against persistent cancers.
Area of Science:
- Immunology
- Oncology
- Biotechnology
Background:
- Chimeric antigen receptors (CARs) show promise in cancer therapy but struggle with persistent or recurrent malignancies.
- Existing CAR designs have limitations in sustained T cell effector functions during prolonged tumor antigen exposure.
Purpose of the Study:
- To introduce and validate "CAR Pooling," a multiplexed screening approach for identifying potent CAR designs.
- To explore novel signaling domains for enhanced T cell effector functions in CAR T cell therapy.
Main Methods:
- Evaluated 40 CARs with diverse immune cell-derived signaling domains using pooled assays.
- Assessed T cell effector functions, including proliferation and cytotoxicity, under repetitive stimulation mimicking long-term antigen exposure.
- Analyzed CAR T cell signaling states, transcriptional signatures, and in vivo efficacy in a multiple myeloma xenotransplant model.
Main Results:
- Identified tumor necrosis factor (TNF) receptor family domains, particularly from B cells (CD40, BAFF-R, TACI), that significantly enhanced T cell functions.
- BAFF-R and TACI domains promoted cytotoxicity, while CD40 enhanced proliferation, outperforming clinical benchmarks after prolonged stimulation.
- BAFF-R CAR T cells exhibited a cytotoxic transcriptional signature linked to favorable clinical outcomes.
- Replacing the 4-1BB domain with BAFF-R in a BCMA-specific CAR improved activity in a multiple myeloma model.
Conclusions:
- CAR Pooling is an effective strategy for rapid discovery and optimization of CAR architectures.
- Novel signaling domains, especially from the TNF receptor superfamily, can significantly enhance CAR T cell efficacy against refractory cancers.
- This approach holds potential for improving the clinical success of CAR T cell therapies for challenging malignancies.
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