A cell-based immunogenicity assay to detect antibodies against chimeric antigen receptor expressed by tisagenlecleucel.

The use of T-cells expressing Chimeric Antigen Receptors (CARs) offers new opportunities for cancer treatment, as well as new challenges for the bioanalysis of this new class of drugs. The analysis of humoral immunogenicity (anti-drug antibodies) against CARs could be performed with a bridging ELISA, using labeled CAR fragments. However, outside of its native cell membrane environment and without potential interaction partners on the cell surface, a labeled or coated recombinant CAR fragment may structurally differ from the membrane-bound CAR expressed on CAR-T cells. Consequently, immunogenicity to CARs may be missed due to the artificial nature of a ligand binding assay setup. T-cell lines expressing the CAR offer the opportunity to measure anti-drug antibodies to the CAR in its natural cell environment, as an alternative to ligand-binding assays. Here we describe a novel, flow cytometry-based humoral immunogenicity assay for tisagenlecleucel (CTL019, Kymriah®) using a human T-cell line that expresses murine CAR19. The assay described here was fully validated according to health authority guidelines for the development and validation of immunogenicity assays and has a sensitivity of 100 ng/mL. A good correlation of screening assay signal strengths to titer assay results was observed while exploring options to increase titration assay throughput. Pre-existing antibodies against the cell line used in the assay as well as against the CAR itself complicate the assay and data interpretation.