Refining chimeric antigen receptors via barcoded protein domain combination pooled screening

Area of Science:

• Immunology
• Molecular Biology
• Biotechnology

Background:

• Chimeric antigen receptor (CAR)-T cell therapy is a promising cancer immunotherapy approach.
• Current methods for developing novel CAR constructs are inefficient and time-consuming.
• There is a need for accelerated methods to evaluate and compare CAR designs.

Purpose of the Study:

• To develop a novel cloning strategy for efficient generation and evaluation of CAR constructs.
• To identify optimal CAR architectures for enhanced T cell proliferation and expansion.
• To discover novel CAR designs with superior in vivo antitumor activity.

Main Methods:

• A novel cloning strategy using sequential assembly of CAR domains via blunt ligation.
• Each CAR domain was assigned a unique DNA barcode for quantification.
• Generation of 360 CAR constructs targeting CD19 and GD2 tumor antigens.
• Next-generation sequencing was used to quantify barcode frequencies and assess CAR functionality.

Main Results:

• The screening identified a critical role for the hinge domain in CAR functionality.
• CD8a and IgG4 hinges demonstrated opposing effects on CAR performance depending on the target antigen (CD19 vs. GD2).
• Two novel CD19-CAR architectures with an IgG4 hinge showed superior in vivo antitumor activity compared to an FDA-approved therapy.

Conclusions:

• The developed cloning strategy significantly accelerates the evaluation and comparison of CAR designs.
• This approach enables rapid identification of high-performing CAR constructs for cancer immunotherapy.
• The findings pave the way for faster development of next-generation CAR-T cell therapies.