A universally applicable toolbox for single-molecule quantification of chimeric antigen receptors using...

Area of Science:

• Immunotherapy
• Cellular Biology
• Microscopy

Background:

• Chimeric antigen receptor (CAR)-T cell therapies show promise for B-cell malignancies.
• Variations in CAR design and surface expression impact therapeutic outcomes.
• Current detection methods like flow cytometry lack quantitative precision for CAR density.

Purpose of the Study:

• To develop a quantitative method for assessing CAR surface expression across diverse CD19 CAR-T cell products.
• To enable antigen-independent detection of CARs by targeting conserved linker regions.
• To compare the sensitivity and accuracy of the new method against traditional flow cytometry.

Main Methods:

• Established a linker-targeted direct stochastic optical reconstruction microscopy (dSTORM) approach.
• Utilized commercially available antibodies to target conserved scFv linker regions (e.g., (G4S)3, Whitlow).
• Generated primary human T cells expressing constructs similar to approved CAR-T products for comparison with flow cytometry.

Main Results:

• dSTORM demonstrated superior sensitivity to flow cytometry, visualizing CAR expression levels below flow cytometry detection thresholds.
• Linker-targeted dSTORM revealed construct-specific differences in CAR surface density, with a (G4S)3 linker showing higher expression than Whitlow linkers.
• dSTORM provided more reliable resolution of differences at low CAR expression levels compared to flow cytometry.

Conclusions:

• Linker-targeted dSTORM is a highly sensitive and versatile platform for quantifying CAR surface expression.
• This method offers new insights into how CAR design influences receptor density and spatial organization.
• The technique can improve the understanding and assessment of CAR-T cell products for enhanced therapeutic efficacy.