Scholarlink

Background <p>Epstein-Barr virus (EBV) reactivation in immunocompromised patients and <i>post</i>-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 <i>post</i>-transplant immunosuppression jeopardizes the protective anti-EBV immune surveillance by adoptively transferred T cells.</p> Methods <p>Using an all-in-one CRISPR/Cas-mediated approach, we inserted an anti-EBV (gp350) CAR into the T-cell receptor (<i>TRAC</i>) locus and simultaneously knocked-out the glucocorticoid receptor (GR) on a good manufacturing practice (GMP)-compatible platform.</p> Results <p>CAR knock-in (CAR<sup>KI</sup>) 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<sup>KI</sup> cells. On a functional level CAR<sup>KI</sup>GR<sup>KO</sup> T cells showed target-specific potency in terms of cytokine secretion patterns, proliferative capacity and cytotoxic activity against gp350-expressing target cells. Further, CAR<sup>KI</sup>GR<sup>KO</sup> T cells were insensitive to dexamethasone treatment and maintained T-cell functionality. In contrast, CAR<sup>KI</sup>GR<sup>KO</sup> T cells were sensitive to the GR-independent immunosuppressant cyclosporine A (CsA), thereby providing a rescue treatment for patients in case of safety issues.</p> Conclusions <p>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 <i>post</i>-transplant patients with EBV-reactivations or patients with EBV-associated pathologies requiring steroid treatment.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

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

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2025年3月19日

查看全文
  • Theresa Kaeuferle,
  • Maximilian Zwermann,
  • Nadine Stoll,
  • Paulina Ferrada-Ernst,
  • Lena Jablonowski,
  • Reinhard Zeidler,
  • Semjon Willier,
  • Dana Stenger,
  • Abdallah Yassin,
  • Renata Stripecke,
  • Tobias Feuchtinger

摘要

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.