肿瘤细胞治疗研究
英文原题：High-affinity CD16 integration into a CRISPR/Cas9-edited CD38 locus augments CD38-directed antitumor activity of primary human natural killer cells.
使用体外扩增的CD38 KO/CD16 KI NK细胞进行过继性免疫治疗，有潜力提高DARA的临床疗效。通过将互补的基因工程策略整合到CD38 KO生产平台中，我们生成了具有显著增强的CD38导向抗肿瘤活性的NK细胞，为在临床中探索这一免疫治疗策略建立了强有力的依据。
过继转移具有增强抗体依赖性细胞介导的细胞毒性（ADCC）能力且对CD38靶向具有抵抗力的自然杀伤（NK）细胞，有可能增强daratumumab（DARA）的临床抗骨髓瘤活性。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）），并同时为CD38 KO NK细胞装备高亲和力CD16（CD16-158V）受体。
使用Cas9核糖核蛋白复合物生成CD38 KO人类NK细胞。通过将CD38 KO NK细胞的信使RNA（mRNA）转染与CD38位点的靶向基因插入相结合以介导基因敲入（KI），该平台得到了扩展。在体外和MM.1S异种移植小鼠模型中，测试了这些基因编辑NK细胞在DARA存在下持续存在并介导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，且不影响其增殖或功能能力。CD38 KO赋予NK细胞对DARA诱导的NK细胞自相残杀的抵抗力，使其在体外和MM.1S异种移植小鼠模型中，在DARA存在下能够持续存在并增强对骨髓瘤细胞系的ADCC。CD38 KO 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CD38位点的同源定向修复模板促进了高效的2-in-1 CD38 KO，同时实现了截短型CD34报告基因和CD16-158V受体的KI，CD38 KO/CD16 KI NK细胞在体外和体内均表现出DARA介导的ADCC进一步增强。
BACKGROUND: 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 KO NK cells with a high-affinity CD16 (CD16-158V) receptor. METHODS: CD38 KO human NK cells were generated using Cas9 ribonucleoprotein complexes. The platform was expanded by incorporating messenger RNA (mRNA) transfection of CD38 KO NK cells and targeted gene insertion at the CD38 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. RESULTS: 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 KO 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 CD38 locus facilitated an efficient 2-in-1 CD38 KO coupled with KI of a truncated CD34 reporter and CD16-158V receptor, with CD38 KO /CD16 KI NK cells demonstrating a further enhancement of DARA-mediated ADCC both in vitro and in vivo. CONCLUSIONS: Adoptive immunotherapy using ex vivo expanded CD38 KO /CD16 KI 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.
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