Background Chimeric Antigen Receptor (CAR)-T cell therapy is a powerful new modality for cancer immunotherapy. To date, much of the success has been for the treatment of hematological malignancies. Effective strategies targeting solid tumors, however, will require a deeper understanding of CAR-T cell biology. Many commercially available CAR cell purification reagents either lack specificity or are not versatile in their ability to purify CAR cells of differing antigen specificity. There is a need for specific, yet flexible CAR cell purification reagents that can be used to research and engineer cells with a better therapeutic index. Recombinant rabbit monoclonal antibodies specific to two linker sequences that are commonly integrated into single-chain variable fragment (scFv)-based CARs, either repeats of a Gly4Ser peptide or a Whitlow/218 peptide, were generated and characterized in multiple applications including immunoaffinity purification. Here, we describe a magnetic bead-based immunoaffinity method for the enrichment of CAR expressing cells. This specific yet broadly applicable strategy will enable researchers to expand and characterize viable, bead-free CAR expressing cells containing common scFv-based CAR linker sequences.

Methods The monoclonal antibodies, E7O2V and E3U7Q targeting either Gly4Ser or Whitlow/218 linkers, respectively, were biotinylated and used in concert with cleavable streptavidin coated magnetic beads to immunoaffinity purify CAR expressing cells. Validation of specificity and versatility was performed using non-transduced versus CAR-transduced cell lines and primary human T cells.

Results Flow cytometric analysis of bead-free, purified cells revealed that biotinylated E7O2V and E3U7Q could enrich CAR cells containing the appropriate linker sequence, independently of scFv specificity. No carryover of non-transduced cells were observed.

Conclusions A versatile immunoaffinity method to positively select viable CAR cells containing either a Gly4Ser linker or a Whitlow/218 linker was developed. The specificity of monoclonal antibodies, E7O2V and E3U7Q, combined with gentle separation from magnetic beads enabled the purification of live cells expressing scFv-based CARs containing either a Gly4Ser linker or a Whitlow linker regardless of CAR antigen specificity. The CAR-T cell selection approach demonstrated here can potentially be used in an automated fashion, in high-throughout and without the need of expensive equipment such as a flow cell sorter. Lastly, the purified cells obtained from this method are potentially suitable for downstream applications such as single-cell RNAseq and flow cytometry.