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J Immunol Methods. 2018 Jul;458:74-82. doi: 10.1016/j.jim.2018.04.007. Epub 2018 Apr 21.

Development of an RNA-based kit for easy generation of TCR-engineered lymphocytes to control T-cell assay performance.

Author information

1
Translational Oncology at the University Medical Center of the Johannes-Gutenberg University Mainz (TRON gGmbH), Freiligrathstraße 12, Mainz 55131, Germany; BioNTech AG, An der Goldgrube 12, 55131 Mainz, Germany.
2
BioNTech AG, An der Goldgrube 12, 55131 Mainz, Germany.
3
Department of Medical Oncology, Leiden University Medical Center, Albinusdreef 2, Leiden, ZA 2333, The Netherlands.
4
ECMC, Cancer Sciences Unit, Faculty of Medicine, University of Southampton, Tremona Road, Southampton SO16 6YD, United Kingdom.
5
Department of Immunology, University of Tuebingen, Auf der Morgenstelle 15, Tuebingen 72076, Germany.
6
Immatics biotechnologies GmbH, Paul-Ehrlich-Str. 15, Tuebingen 72076, Germany.
7
Laboratory of Hematology, University Hospital Herlev, Ringvej 75, Herlev DK-2730, Denmark.
8
Dept. of Tumor Immunology, Radboud university medical center, Radboud Institute for Molecular Life Sciences, P.O. Box 9101, Nijmegen, HB 6500, The Netherlands.
9
Translational Oncology at the University Medical Center of the Johannes-Gutenberg University Mainz (TRON gGmbH), Freiligrathstraße 12, Mainz 55131, Germany.
10
Translational Oncology at the University Medical Center of the Johannes-Gutenberg University Mainz (TRON gGmbH), Freiligrathstraße 12, Mainz 55131, Germany; University Medical Center of the Johannes Gutenberg-University Mainz, Langenbeckstrasse 1, Mainz D-55131, Germany; BioNTech AG, An der Goldgrube 12, 55131 Mainz, Germany.
11
Department of Medical Oncology, Leiden University Medical Center, Albinusdreef 2, Leiden, ZA 2333, The Netherlands. Electronic address: shvdburg@lumc.nl.

Abstract

Cell-based assays to monitor antigen-specific T-cell responses are characterized by their high complexity and should be conducted under controlled conditions to lower multiple possible sources of assay variation. However, the lack of standard reagents makes it difficult to directly compare results generated in one lab over time and across institutions. Therefore TCR-engineered reference samples (TERS) that contain a defined number of antigen-specific T cells and continuously deliver stable results are urgently needed. We successfully established a simple and robust TERS technology that constitutes a useful tool to overcome this issue for commonly used T-cell immuno-assays. To enable users to generate large-scale TERS, on-site using the most commonly used electroporation (EP) devices, an RNA-based kit approach, providing stable TCR mRNA and an optimized manufacturing protocol were established. In preparation for the release of this immuno-control kit, we established optimal EP conditions on six devices and initiated an extended RNA stability study. Furthermore, we coordinated on-site production of TERS with 4 participants. Finally, a proficiency panel was organized to test the unsupervised production of TERS at different laboratories using the kit approach. The results obtained show the feasibility and robustness of the kit approach for versatile in-house production of cellular control samples.

KEYWORDS:

Immunoguiding; Immunomonitoring; Performance; Standardization; T-cell assays; Validation

PMID:
29684430
DOI:
10.1016/j.jim.2018.04.007
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