Toward high-throughput engineering techniques for improving CAR intracellular signaling domains

Area of Science:

• Immunology
• Molecular Biology
• Biotechnology

Background:

• Chimeric antigen receptors (CARs) are crucial for T-cell-based immunotherapies.
• CAR T-cell activity is determined by intracellular signaling domains.
• Optimizing CAR signaling often involves combinatorial domain approaches, but sequence engineering within domains is less explored.

Purpose of the Study:

• To develop a novel in situ screening method for directed evolution of CAR intracellular signaling domains.
• To identify CAR variants with enhanced and selective T-cell activation.
• To enable high-throughput discovery of improved CAR signaling components.

Main Methods:

• Utilized a human NF-κB and NFAT reporter T-cell line for screening.
• Constructed a model library of CAR signaling domain variants.
• Employed a mammalian display approach for directed evolution and screening.

Main Results:

• Demonstrated the ability to discern between CARs with different co-stimulatory domains.
• Successfully identified rare, higher-signaling variants (frequency as low as 1 in 1000).
• Validated the screening approach for high-throughput identification of CAR signaling mutations.

Conclusions:

• The developed screening method shows promise for CAR signaling domain discovery.
• Mammalian display offers a powerful tool for engineering CAR variants.
• This approach has implications for advancing CAR T-cell therapy development.