Abstract
Treatment with axicabtagene ciloleucel (Axi-cel) CD19-CAR-T (chimeric antigen receptor T) cells has been approved for refractory/relapsed diffuse large B cell lymphoma (DLBCL) and primary mediastinal large B cell lymphoma (PMBCL). Because treatment success as well as side effects might depend on CAR-T cell expansion in vivo, we aimed at developing digital PCR (dPCR) assays for detection and quantification of CAR-T cells. To this end, we cloned and sequenced the complete cDNA of the CAR construct. We designed different combinations of primers and dual-labeled hydrolysis probes located in various CAR regions. Three combinations were successfully tested on CAR-positive and -negative cells in duplex reactions with a reference gene (REF) to concomitantly assess cell numbers. All assays demonstrated excellent specificity and reproducibility with neglectable inter-assay variations. For all three assays, almost perfect correlation between the two dPCRs (Axi-cel versus REF) was observed, and the limit of detection was one single CAR-transduced cell corresponding to a sensitivity of 0.01% for 100 ng genomic DNA. After cross-validation, we used one assay to monitor Axi-cel CAR-T numbers in patients. CAR-T expansion and contraction followed the expected kinetics with median peak value of 11.2 Axi-cel CAR-T cells/μL at 11.3 days (median). Clinically, we observed only two partial responses (PRs) in the five patients with CAR-T cell peak numbers below median, whereas four of the five patients with comparatively good expansion showed clinical responses (two complete responses [CRs] and two PRs) on day 30. In conclusion, we established a novel dPCR assay for the sensitive detection of transgenic CAR-T cells, which should be very useful in the context of Axi-cel treatment.
Highlights
Chimeric antigen receptor T (CAR-T) cells have become a new treatment modality in hematology.[1]
CAR-T cell peak concentrations and area under the curve in the first 28 days after axicabtagene ciloleucel (Axi-cel) infusion have been associated with long-term efficacy.[3,4]
Mixed DNA from 20 buffy coats as well as pre-infusion samples from patients were negative with the exclusion of one slightly positive PCR (0.07%) with amplicon A in a pre-infusion sample of patient #001; the sample was negative with both amplicons B and C
Summary
Chimeric antigen receptor T (CAR-T) cells have become a new treatment modality in hematology.[1] Recently, two CD19-CAR-T cell products (tisagenlecleucel/Tisa-cel/Kymriah and axicabtagene ciloleucel/ Axi-cel/Yescarta) have been licensed for the treatment of different B cell malignancies.[1,2] CAR-T cell engraftment and expansion in vivo were shown to represent crucial parameters for efficacy of Axi-cel treatment.[3,4] In particular, CAR-T cell peak concentrations and area under the curve in the first 28 days after Axi-cel infusion have been associated with long-term efficacy.[3,4] low initial CAR-T expansion seems to be associated with poorer outcome. Additional data are necessary in order to adapt treatment protocols based on CAR-T engraftment. Diagnostic assays to quantitatively assess CAR-T cells in vivo are currently missing and are urgently needed
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