优化 CAR-NK 细胞的转导和扩增:利用细胞因子调节提高性能

Tiziano Ingegnere, Benjamin Segain, Adeline Cozzani, Mattias Carlsten, Suman Mitra, Silvia Gaggero
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引用次数: 0

摘要

细胞免疫疗法已成为治疗癌症患者最有效的方法之一。嵌合抗原受体(CAR)T 细胞的适应性转移以及单倍体自然杀伤(NK)细胞的使用可诱导淋巴瘤和白血病患者病情缓解。虽然 CAR T 细胞的应用已经确立,但由于存在严重不良事件的风险,包括细胞因子释放综合征和免疫效应细胞相关神经毒性综合征,目前这种方法的广泛应用受到限制。此外,如果不采用先进的 CRISPR 工程,异体 T 细胞输注引发移植物与宿主反应的风险也限制了自体 CAR T 细胞的使用。相比之下,基于 NK 细胞的癌症免疫疗法已成为一种安全的方法,即使在异体治疗中也是如此。然而,用T细胞修饰常用的水泡性口炎病毒G糖蛋白(VSV-G)伪型慢病毒高效转导原代血液NK细胞仍具有挑战性。本文介绍了一种详细的方法,通过在细胞因子补充培养基中进行短期培养,大大提高了 NK 细胞的转导效率。它还包括制备高滴度和高质量的慢病毒颗粒,以实现最佳的 NK 细胞转导。总之,本方案详细介绍了在细胞因子补充培养基中逐步培养 NK 细胞、用 VSV-G 慢病毒载体转导 NK 细胞以及随后扩增 NK 细胞进行功能测试的过程。© 2024 Wiley Periodicals LLC.基本方案 1:从人外周血单核细胞(PBMCs)中分离 NK 细胞基本方案 2:扩增 NK 细胞并用慢病毒转导生成 CAR-NK 细胞辅助方案 1:质粒扩增辅助方案 2:慢病毒制备辅助方案 3:慢病毒滴定
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Optimizing CAR-NK Cell Transduction and Expansion: Leveraging Cytokine Modulation for Enhanced Performance

Cellular immunotherapy has emerged as one of the most potent approaches to treating cancer patients. Adoptive transfer of chimeric antigen receptor (CAR) T cells as well as the use of haploidentical natural killer (NK) cells can induce remission in patients with lymphoma and leukemia. Although the use of CAR T cells has been established, this approach is currently limited for wider use by the risk of severe adverse events, including cytokine release syndrome and immune effector cell-associated neurotoxicity syndrome. Moreover, the risk of triggering graft vs host reactions in settings of allogeneic T cell infusion limits the use to autologous CAR T cells if advanced CRISPR engineering is not applied. In contrast, NK cell-based cancer immunotherapy has emerged as a safe approach even in allogeneic settings. However, efficient transduction of primary blood NK cells with vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped lentivirus commonly used for T cell modification remains challenging. This article presents a detailed method that significantly enhances the transduction efficiency of NK cells by utilizing a short-term culture in cytokine-supplemented medium. It also encompasses the preparation of high-titer and high-quality lentiviral particles for optimal NK cell transduction. Overall, this protocol details the step-by-step culture of NK cells in cytokine-supplemented medium, their transduction with VSV-G lentiviral vectors, and subsequent expansion for functional assays. © 2024 Wiley Periodicals LLC.

Basic Protocol 1: Isolation of NK cells from human peripheral blood mononuclear cells (PBMCs)

Basic Protocol 2: NK cell expansion and transduction with lentivirus for generating CAR-NK cells

Support Protocol 1: Plasmid amplification

Support Protocol 2: Lentivirus preparation

Support Protocol 3: Lentivirus titration

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