Laboratory of Transplantation Immunotherapy, Cellular and Molecular Therapeutics Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
Biologics Process Research and Development, Merck & Co Inc, Kenilworth, New Jersey, USA.
J Immunother Cancer. 2022 Feb;10(2). doi: 10.1136/jitc-2021-003804.
Adoptive transfer of natural killer (NK) cells with augmented antibody-dependent cellular cytotoxicity (ADCC) capabilities and resistance to CD38 targeting has the potential to enhance the clinical anti-myeloma activity of daratumumab (DARA). Therefore, we sought to develop an efficient CRISPR/Cas9-based gene editing platform to disrupt CD38 expression (CD38 knockout (KO)) in ex vivo expanded NK cells and simultaneously arm CD38 NK cells with a high-affinity CD16 (CD16-158V) receptor.
CD38 human NK cells were generated using Cas9 ribonucleoprotein complexes. The platform was expanded by incorporating messenger RNA (mRNA) transfection of CD38 NK cells and targeted gene insertion at the locus to mediate gene knockin (KI). The capacity of these gene-edited NK cells to persist and mediate ADCC in the presence of DARA was tested in vitro and in a MM.1S xenograft mouse model.
Highly efficient CD38 gene disruption was achieved in ex vivo expanded NK cells without affecting their proliferative or functional capacity. CD38 KO conferred resistance to DARA-induced NK cell fratricide, enabling persistence and augmented ADCC against myeloma cell lines in the presence of DARA in vitro and in a MM.1S xenograft mouse model. CD38 NK cells could be further modified by transfection with mRNA encoding a CD16-158V receptor, resulting in augmented DARA-mediated ADCC. Finally, we observed that a homology-directed repair template targeted to the locus facilitated an efficient 2-in-1 CD38 KO coupled with KI of a truncated CD34 reporter and CD16-158V receptor, with CD38/CD16 NK cells demonstrating a further enhancement of DARA-mediated ADCC both in vitro and in vivo.
Adoptive immunotherapy using ex vivo expanded CD38/CD16 NK cells has the potential to boost the clinical efficacy of DARA. By incorporating complementary genetic engineering strategies into a CD38 KO manufacturing platform, we generated NK cells with substantially augmented CD38-directed antitumor activity, establishing a strong rationale for exploring this immunotherapy strategy in the clinic.
过继输注具有增强的抗体依赖性细胞细胞毒性（ADCC）能力和抗 CD38 靶向性的自然杀伤（NK）细胞有可能增强达雷妥尤单抗（DARA）治疗多发性骨髓瘤（MM）的临床疗效。因此，我们试图开发一种高效的基于 CRISPR/Cas9 的基因编辑平台，以破坏体外扩增 NK 细胞中的 CD38 表达（CD38 敲除（KO）），同时为 CD38 NK 细胞赋予高亲和力 CD16（CD16-158V）受体。
使用 Cas9 核糖核蛋白复合物生成 CD38 人 NK 细胞。该平台通过将 CD38NK 细胞的信使 RNA（mRNA）转染和靶向基因插入 基因座以介导基因敲入（KI）进行扩展。在体外和 MM.1S 异种移植小鼠模型中测试这些基因编辑 NK 细胞在存在 DARA 的情况下持续存在并介导 ADCC 的能力。
在体外扩增的 NK 细胞中实现了高效的 CD38 基因敲除，而不影响其增殖或功能能力。CD38 KO 赋予了对 DARA 诱导的 NK 细胞自噬的抗性，使 NK 细胞在存在 DARA 的情况下在体外和 MM.1S 异种移植小鼠模型中持续存在并增强对骨髓瘤细胞系的 ADCC。CD38 NK 细胞可进一步通过转染编码 CD16-158V 受体的 mRNA 进行修饰，从而增强 DARA 介导的 ADCC。最后，我们观察到靶向 基因座的同源定向修复模板促进了高效的 2-in-1 CD38 KO 与截断的 CD34 报告基因和 CD16-158V 受体的 KI 的结合，CD38/CD16 NK 细胞在体外和体内均进一步增强了 DARA 介导的 ADCC。
使用体外扩增的 CD38/CD16 NK 细胞过继免疫疗法有可能提高 DARA 的临床疗效。通过将互补的基因工程策略纳入 CD38 KO 制造平台，我们生成了具有显著增强的 CD38 定向抗肿瘤活性的 NK 细胞，为探索这种免疫疗法策略在临床上的应用提供了强有力的依据。
