CAR binding efficacy and off-target interactions for common CAR detection reagents

Area of Science:

• Immunotherapy
• Cellular Therapy
• Flow Cytometry

Background:

• Chimeric Antigen Receptor (CAR)-T cell therapy is an innovative immunotherapy.
• Expanding CAR-T therapies requires novel tumor antigens and CARs on other immune cells like macrophages and NK cells.
• Specific detection reagents are crucial for interrogating CAR surface expression as the field evolves.

Purpose of the Study:

• Compare commercially available CAR detection reagents for detecting CAR constructs with varying hinges, linkers, and scFv designs.
• Assess off-target interactions of these reagents within a peripheral blood mononuclear cell (PBMC) matrix.

Main Methods:

• Utilized flow cytometry to analyze CAR detection reagents.
• Tested reagents on stable CAR-Jurkat cell lines and primary human CAR-T cells.
• Evaluated reagent performance in a PBMC matrix to identify off-target binding.

Main Results:

• Antibody-based reagents showed specificity and signal in CAR-T cells and Jurkat lines.
• Protein L detected only anti-CD20 scFv-based CARs, with off-target staining in PBMCs.
• Recombinant antigens yielded clean results for CD19 and BCMA but not CD20.
• Antibody conjugates showed potential false positives in PBMCs due to Fc receptor binding.

Conclusions:

• CAR idiotype antibodies, recombinant antigens, and anti-linker antibodies provide specific CAR detection.
• Idiotype antibodies and recombinant antigens are limited to specific target antigens.
• Anti-linker antibodies enable universal CAR detection irrespective of the target antigen.