DataSheet_1_DNAM-1-chimeric receptor-engineered NK cells, combined with Nutlin-3a, more effectively fight neuroblastoma cells in vitro: a proof-of-concept study.docx

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.

工程化自然杀伤（NK）细胞的过继输注是当前癌症临床治疗策略之一，近十年来受到学界广泛关注。然而，当前仍需开发全新策略以提升基于NK细胞的免疫治疗的临床疗效与安全性。NK细胞介导的肿瘤细胞识别与裂解，严格依赖于肿瘤细胞表面NK细胞活化受体NKG2D与DNAM-1的配体表达。值得注意的是，DNAM-1的配体PVR/CD155与Nectin-2/CD112主要表达于实体肿瘤细胞，在正常组织细胞中表达水平极低。本研究构建了可表达全长DNAM-1受体，或三种不同基于DNAM-1的嵌合受体的人源NK细胞；此类嵌合受体将DNAM-1与2B4、CD3ζ链等共刺激分子进行融合表达。将上述构建体转染至健康供者来源的原代人NK细胞后，我们首先检测了DNAM-1的表面表达水平；随后以功能读值为指标，评估了细胞对人白血病细胞系K562的脱颗粒程度、细胞毒性，以及干扰素γ（IFNγ）与肿瘤坏死因子α（TNFα）的分泌水平。此外，本研究还探讨了靶向MDM2的药物Nutlin-3a——该药物可恢复p53功能，且已被证实具有免疫调节活性——对DNAM-1工程化NK细胞在应对人神经母细胞瘤（NB）细胞系LA-N-5与SMS-KCNR时的脱颗粒反应的影响。通过对比转染四种不同质粒载体的NK细胞，并结合阻断实验，我们发现表达DNAM-1-CD3ζ的工程化NK细胞应答效应最强。此外，经Nutlin-3a预处理的LA-N-5与SMS-KCNR细胞，相较于转染空载体的NK细胞，对DNAM-1工程化NK细胞的易感性显著提升。本研究结果提供了概念验证依据，提示将DNAM-1嵌合受体工程化NK细胞与Nutlin-3a联合使用，可为携带p53功能异常的实体肿瘤（如神经母细胞瘤）提供全新的治疗策略。