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Kaeuferle, T.* ; Zwermann, M.* ; Stoll, N.* ; Ferrada-Ernst, P.* ; Jablonowski, L.* ; Zeidler, R. ; Willier, S.* ; Stenger, D.* ; Yassin, A.* ; Stripecke, R.* ; Feuchtinger, T.*

All-in-one CRISPR/Cas-engineered glucocorticoid-receptor knock-out EBV-gp350-CAR knock-in T cells are potent and resistant to dexamethasone.

Exp. Hematol. Oncol. 14:40 (2025)
Verlagsversion DOI PMC
Open Access Gold
Creative Commons Lizenzvertrag
BACKGROUND: Epstein-Barr virus (EBV) reactivation in immunocompromised patients and post-transplantation is associated with morbidity, mortality and with the onset of a variety of malignant diseases. Adoptive T-cell therapies have emerged as promising therapeutic options, but post-transplant immunosuppression jeopardizes the protective anti-EBV immune surveillance by adoptively transferred T cells. METHODS: Using an all-in-one CRISPR/Cas-mediated approach, we inserted an anti-EBV (gp350) CAR into the T-cell receptor (TRAC) locus and simultaneously knocked-out the glucocorticoid receptor (GR) on a good manufacturing practice (GMP)-compatible platform. RESULTS: CAR knock-in (CARKI) was confirmed in primary human T cells on genetic and on protein level with a mean efficiency of 41%. With 83%, additional GR knock-out was highly efficient in CARKI cells. On a functional level CARKIGRKO T cells showed target-specific potency in terms of cytokine secretion patterns, proliferative capacity and cytotoxic activity against gp350-expressing target cells. Further, CARKIGRKO T cells were insensitive to dexamethasone treatment and maintained T-cell functionality. In contrast, CARKIGRKO T cells were sensitive to the GR-independent immunosuppressant cyclosporine A (CsA), thereby providing a rescue treatment for patients in case of safety issues. CONCLUSIONS: The study lays the proof-of-concept for virus-free all-in-one GMP-manufacturing of glucocorticoid-resistant CAR T-cell products. Further, the glucocorticoid-resistant gp350-CAR T cells can provide a future therapeutic option for high-risk post-transplant patients with EBV-reactivations or patients with EBV-associated pathologies requiring steroid treatment.
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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Korrespondenzautor
Schlagwörter Adoptive T Cell Transfer ; Car T Cells ; Crispr/cas ; Dexamethasone ; Ebv ; Genetic Engineering ; Glucocorticoid Receptor ; Steroid Treatment; Lymphoproliferative Disease; Immunosuppression; Therapy; Load
ISSN (print) / ISBN 2162-3619
e-ISSN 2162-3619
Zeitschrift Experimental Hematology and Oncology
Quellenangaben Band: 14, Heft: 1, Seiten: , Artikelnummer: 40 Supplement: ,
Verlag BMC
Verlagsort Campus, 4 Crinan St, London N1 9xw, England
Nichtpatentliteratur Publikationen
Begutachtungsstatus Peer reviewed
Institut(e) Institute of Structural Biology (STB)
Förderungen Cancer Research Center Cologne Essen (CCCE)
German Cancer Aid (Deutsche Krebshilfe Grant)
Munich-Wurzburg, Germany
LETSimmun
German Centre for Infection Research (DZIF Grant)
Projekt DEAL