Bouwmeester, Sarina (2022) Development of a ddPCR assay to precisely quantify CAR T-cells in vivo. thesis, Medicine.
Full text available on request.Abstract
CAR T-cells are a type of immunotherapy, in which a T-cell is transduced with a chimeric antigen receptor (CAR) to specifically target tumor cells. The efficacy of CAR T-cell therapy depends on the expansion and persistence of the cells in the blood. The expansion levels of CAR T-cells in the first 28 days after treatment correlate to the efficacy of the therapy. A low expansion and poor persistence could lead to poor response. It is therefore important to monitor CAR T-cell levels in vivo. Digital droplet PCR (ddPCR) is a promising technique for monitoring this. Accurately monitoring of the CAR T-cell levels is a secondary endpoint in an upcoming clinical trial, where ARI-0001 (a novel academic anti-CD19 CAR T-cell) and Yescarta (a commercial CAR T-cell) will be compared. To that end, the aim of this project was to develop a ddPCR assay to precisely quantify CAR T-cells in vivo. Primers were first designed and validated in qPCR. A primer on 4-1BB/CD3ζ (intracellular domains) was designed to amplify ARI-0001, of which a sensitivity of 0.001% was found in qPCR, whereas the limit of detection by flow cytometry was 0.1%. For Yescarta, a specific primer on FMC63 (extracellular domain) was developed. The sensitivity has yet to be investigated. In conclusion, a specific primer was developed and optimized to detect ARI-0001 with very good sensitivity. For Yescarta, a specific primer was developed, of which the sensitivity has yet to be determined. In addition, both primers need to be validated with ddPCR.
| Item Type: | Thesis (UNSPECIFIED) |
|---|---|
| Supervisor name: | van Meerten, dr. T and Bremer, prof. dr. E. |
| Faculty: | Medical Sciences |
| Date Deposited: | 23 May 2023 11:31 |
| Last Modified: | 23 May 2023 11:31 |
| URI: | https://umcg.studenttheses.ub.rug.nl/id/eprint/3534 |
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