Department of Clinical Sciences and Translational Medicine, University of Rome "Tor Vergata", Rome, Italy.
Saint Camillus International University of Health and Medical Sciences, Rome, Italy.
Front Immunol. 2022 Jul 28;13:886319. doi: 10.3389/fimmu.2022.886319. eCollection 2022.
Adoptive transfer of engineered NK cells, one of clinical approaches to fight cancer, is gaining great interest in the last decade. However, the development of new strategies is needed to improve clinical efficacy and safety of NK cell-based immunotherapy. NK cell-mediated recognition and lysis of tumor cells are strictly dependent on the expression of ligands for NK cell-activating receptors NKG2D and DNAM-1 on tumor cells. Of note, the PVR/CD155 and Nectin-2/CD112 ligands for DNAM-1 are expressed primarily on solid tumor cells and poorly expressed in normal tissue cells. Here, we generated human NK cells expressing either the full length DNAM-1 receptor or three different DNAM-1-based chimeric receptor that provide the expression of DNAM-1 fused to a costimulatory molecule such as 2B4 and CD3ζ chain. Upon transfection into primary human NK cells isolated from healthy donors, we evaluated the surface expression of DNAM-1 and, as a functional readout, we assessed the extent of degranulation, cytotoxicity and the production of IFNγ and TNFα in response to human leukemic K562 cell line. In addition, we explored the effect of Nutlin-3a, a MDM2-targeting drug able of restoring p53 functions and known to have an immunomodulatory effect, on the degranulation of DNAM-1-engineered NK cells in response to human neuroblastoma (NB) LA-N-5 and SMS-KCNR cell lines. By comparing NK cells transfected with four different plasmid vectors and through blocking experiments, DNAM-1-CD3ζ-engineered NK cells showed the strongest response. Furthermore, both LA-N-5 and SMS-KCNR cells pretreated with Nutlin-3a were significantly more susceptible to DNAM-1-engineered NK cells than NK cells transfected with the empty vector. Our results provide a proof-of-concept suggesting that the combined use of DNAM-1-chimeric receptor-engineered NK cells and Nutlin-3a may represent a novel therapeutic approach for the treatment of solid tumors, such as NB, carrying dysfunctional p53.
过继转输工程化 NK 细胞是一种癌症治疗的临床方法，在过去十年中受到了广泛关注。然而，为了提高 NK 细胞免疫疗法的临床疗效和安全性，需要开发新的策略。NK 细胞介导的肿瘤细胞识别和裂解严格依赖于肿瘤细胞上 NK 细胞激活受体 NKG2D 和 DNAM-1 的配体表达。值得注意的是，DNAM-1 的 PVR/CD155 和 Nectin-2/CD112 配体主要表达在实体瘤细胞上，而在正常组织细胞中表达水平较低。在这里，我们构建了表达全长 DNAM-1 受体或三种不同基于 DNAM-1 的嵌合受体的人 NK 细胞，这些受体提供了将 DNAM-1 与共刺激分子（如 2B4 和 CD3ζ 链）融合表达的能力。将其转染到从健康供体中分离的原代人 NK 细胞中后，我们评估了 DNAM-1 的表面表达，并作为功能读数，评估了脱颗粒、细胞毒性以及对人白血病 K562 细胞系的 IFNγ 和 TNFα 产生的程度。此外，我们还研究了 Nutlin-3a 的作用，Nutlin-3a 是一种靶向 MDM2 的药物，能够恢复 p53 功能，并且已知具有免疫调节作用，它对 DNAM-1 工程化 NK 细胞在响应人神经母细胞瘤（NB）LA-N-5 和 SMS-KCNR 细胞系时的脱颗粒作用的影响。通过比较转染了四种不同质粒载体的 NK 细胞并进行阻断实验，发现 DNAM-1-CD3ζ 工程化 NK 细胞表现出最强的反应。此外，与转染空载体的 NK 细胞相比，用 Nutlin-3a 预处理的 LA-N-5 和 SMS-KCNR 细胞对 DNAM-1 工程化 NK 细胞更敏感。我们的结果提供了一个概念验证，表明 DNAM-1 嵌合受体工程化 NK 细胞与 Nutlin-3a 的联合使用可能代表一种治疗携带功能失调 p53 的实体瘤（如 NB）的新治疗方法。
