Kaeuferle Theresa, Zwermann Maximilian, Stoll Nadine, Ferrada-Ernst Paulina, Jablonowski Lena, Zeidler Reinhard, Willier Semjon, Stenger Dana, Yassin Abdallah, Stripecke Renata, Feuchtinger Tobias
Department of Pediatric Hematology, Oncology, Hemostaseology and Stem Cell Transplantation, Dr. von Hauner Children's Hospital, University Hospital LMU Munich, Munich, Germany.
Center for Cell and Gene Therapy Freiburg, University Medical Center Freiburg, Albert-Ludwigs-University Freiburg, Freiburg, Germany.
Exp Hematol Oncol. 2025 Mar 19;14(1):40. doi: 10.1186/s40164-025-00631-w.
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.
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.
CAR knock-in (CAR) 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 CAR cells. On a functional level CARGR T cells showed target-specific potency in terms of cytokine secretion patterns, proliferative capacity and cytotoxic activity against gp350-expressing target cells. Further, CARGR T cells were insensitive to dexamethasone treatment and maintained T-cell functionality. In contrast, CARGR T cells were sensitive to the GR-independent immunosuppressant cyclosporine A (CsA), thereby providing a rescue treatment for patients in case of safety issues.
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.
免疫功能低下患者及移植后患者体内的爱泼斯坦-巴尔病毒（EBV）再激活与发病率、死亡率以及多种恶性疾病的发生有关。过继性T细胞疗法已成为有前景的治疗选择，但移植后的免疫抑制会损害过继转移T细胞对EBV的保护性免疫监视。
我们采用一体化CRISPR/Cas介导的方法，在符合药品生产质量管理规范（GMP）的平台上，将抗EBV（gp350）嵌合抗原受体（CAR）插入T细胞受体（TRAC）基因座，同时敲除糖皮质激素受体（GR）。
在原代人T细胞中，通过基因和蛋白质水平确认了CAR敲入（CAR），平均效率为41%。在CAR细胞中，GR额外敲除的效率高达83%。在功能水平上，CARGR T细胞在细胞因子分泌模式、增殖能力以及对表达gp350的靶细胞的细胞毒性活性方面表现出靶标特异性效力。此外，CARGR T细胞对地塞米松治疗不敏感，并维持T细胞功能。相比之下，CARGR T细胞对不依赖GR的免疫抑制剂环孢素A（CsA）敏感，从而在出现安全问题时为患者提供挽救治疗。
该研究为糖皮质激素抗性CAR T细胞产品的无病毒一体化GMP生产提供了概念验证。此外，糖皮质激素抗性gp350-CAR T细胞可为移植后有EBV再激活风险的高危患者或需要类固醇治疗的EBV相关病症患者提供未来的治疗选择。
