As Chimeric Antigen Receptor (CAR) T cell therapies advance from discovery through clinical development, analytical methods are increasingly expected to provide meaningful characterization of product identity and function. Flow cytometry is widely used to assess CAR expression on T cells, traditionally relying on surrogate detection reagents such as anti-linker antibodies, anti-tag antibodies, or reporter proteins (e.g., GFP) to monitor CAR-positive cells (1).

The U.S. Food and Drug Administration (FDA) has emphasized the importance of direct CAR detection for identity testing of CAR T cell products. In its guidance for industry, the FDA recommends direct detection of the CAR to determine the percentage of CAR-positive cells and advises that when surrogate markers or broad-specificity reagents (e.g., Protein L) are used, their correlation with CAR expression, sensitivity, and specificity should be evaluated and justified (2). Anti-idiotype antibodies have been used successfully for direct CAR detection; however, suitable anti-idiotype reagents are not always available for every CAR target (3). As a result, surrogate detection methods often remain the only practical option during CAR construct screening, selection, and optimization, particularly as CAR designs become increasingly complex, including dual-target CARs, cytokine-engager CARs, and other next-generation formats.

Recombinant bioactive target proteins offer an attractive alternative for direct, antigen-specific CAR T cell detection. For example, a recombinant CD19 dimer can be used to directly detect CAR expression on T cells by flow cytometry while simultaneously confirming target-binding specificity and potency. This approach aligns with the FDA’s emphasis on direct CAR detection by providing a single assay that measures both CAR expression and functional antigen recognition, resulting in a more biologically relevant characterization of CAR T cells throughout research, process development, and quality control testing.