Titel: Monitoring the in-vivo persistence of CAR-T cells by Taqman Real Time PCR
ID: VS-5-6
Art: Abstractvortrag
Redezeit: 5 + 5 min
Sektion Stammzelltransplantation und Zelltherapie

Referent: Sowmya Gowdavally (Ulm)

Abstract - Text

Offenlegung Interessenkonflikt:

There are no conflicts of interest. 


We developed a Taqman assay specific for the genomic sequence of the Single-chain variable fragment (scFv), which encodes the antigen recognition site of the CAR construct of tisagenlecleucel. A semi-quantitative real-time PCR protocol was established, which compares the amount of CAR-T DNA with reference genomic DNA sequences (KELL, IGF1, ASMA). A full blood count from each patient sample was performed prior to DNA analysis. PCR efficiencies of each PCR reaction were determined and quantification based upon corrected deltaCT values was performed. The results are reported as CAR construct copies per 1000 nucleated cells and as CAR-T copies per 1000 cells.


A total of 6 patients with DLBCL were evaluated. First measurement was performed in the week 2 after CAR-T infusion, when maximum in-vivo expansion of CAR-T cells is expected. 4 of the 6 patients showed clinically manifest cytokine release. Maximum CAR-T copies per 1000 cells were 28 cells (8042/µg DNA), 15 cells (4476/µg DNA), 155 (56329\µg DNA) and 224 (64872/µgDNA) respectively. The measured number of CAR-T copies decreased gradually over time. 2 patients, however, showed no clinical signs of in-vivo CAR-T expansion with a maximum count of CAR-T copies of 3/1000 cells (781/µg DNA) and 3/1000 (799/µg DNA) after day 7. Follow up measurements in 1 among the 2 patients revealed observation of no CAR-T cells, 8 weeks after their infusion.


Anti-CD19 chimeric antigen receptor (CAR) T cell therapy is a novel treatment form for patients with relapsed or refractory ALL and DLBCL. The active components are autologous T-cells genetically engineered to express an antigen specific receptor which is able to induce targeted cytotoxicity. Upon engagement of the antigen, CAR-T cells are stimulated and proliferate. During the course of this therapy it would be beneficial to monitor the number of CAR-T cells in-vivo.


The real-time measurements seem to correlate well with the clinical response of the patients. Establishment of a standard assay to monitor the amount of CD19 specific CAR T cells may aid assessment of efficacy of the treatment and may provide useful information in case of partial responses of relapse, when decisions regarding salvage treatment options are required. We aim to perform a validation of the assay using FACS staining of the CAR-T cells using a FITC conjugated recombinant CD19 antigen.