CD38 敲除原代 NK 细胞以防止“自相残杀”并提高 Daratumumab 活性,Blood

当前位置： X-MOL 学术 › Blood › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

CD38 敲除原代 NK 细胞以防止“自相残杀”并提高 Daratumumab 活性
Blood ( IF 21.0 ) Pub Date : 2019-11-13 , DOI: 10.1182/blood-2019-129456
Yuya Nagai ₁ , Meisam Naeimi Kararoudi ₂ , Ezgi Elmas ₂ , Marcelo Pereira ₂ , Syed Abbas Ali ₁ , Philip Hollingsworth Imus ₃ , Dean Anthony Lee ₄ , Gabriel Ghiaur ₁

Affiliation

多发性骨髓瘤 (MM) 是一种浆细胞肿瘤，通常以高且均匀的 CD38 表达为特征。Daratumumab (DARA) 是一种靶向 CD38 的人源化单克隆抗体，显着改善了难治性 MM 患者的预后，但在大多数情况下复发是不可避免的。DARA 通过多种机制消除 MM 细胞，包括抗体依赖性细胞毒性 (ADCC)、补体依赖性细胞毒性和抗体依赖性细胞吞噬作用。对 DARA 的抗性机制包括下调靶 MM 细胞上的 CD38 表达、通过上调补体抑制蛋白（CD55、CD59）导致补体定向细胞毒性受损，以及通过 DARA 诱导的 NK 细胞“自相残杀”导致 ADCC 受损。因为它与“自相残杀”有关，已经提出通过抗 DARA 的 NK 细胞恢复 ADCC 以增强 DARA 的治疗效果。为了实现这一目标，iPSC 衍生的 CD38 敲除 (CD38KO) NK 细胞已显示出这种方法的概念验证，但对临床转化提出了重大挑战。在这里，我们使用 Cas9-RNP 生成 CD38KO 原代 NK 细胞并表征它们对 DARA 诱导的“自相残杀”和 ADCC 恢复的抗性，同时还评估了 CD38 缺失对 NK 细胞信号传导和代谢的影响。首先，从健康供体的外周血中富集原代 NK 细胞，并在表达 mbIL-21 和 4-1BBL 的饲养细胞上扩增（PLoS One. 2012;7(1):e30264）。将靶向 CD38 的 Cas9/gRNA 复合物电穿孔到 NK 细胞中 (J Vis Exp. 2018 Jun 14;(136))。流式细胞仪分析显示 CD38KO 效率为 81.9±6.9%（平均值±标准差，n=5)。使用磁性细胞分离系统将 CD38KO 细胞进一步纯化至 ≥ 95% 纯度。全基因组测序未发现脱靶突变。单独的 CD38KO-NK 细胞在 DARA（图 1A）和没有 DARA 诱导的自相残杀（图 1B）存在下没有显示结合，与野生型 NK（WT-NK）细胞形成鲜明对比。与这些结果一致，用 DARA 处理 NSG 小鼠对 CD38KO-NK 细胞的持久性没有影响，而 WT-NK 细胞被完全消除（接种后 7 天外周血中的 NK 细胞频率；WT-NK NO DARA 12.11±5.15 %, KO-NK NO Dara 17.05±0.98%, WT-NK + Dara 0.21±0.08%, KO-NK + Dara 16.85±3.61%, 平均值±SD, n=4, p<0.01, ANOVA) (图 1C) . 为了检查 CD38KO-NK 细胞的功能状态，我们使用了 6 个具有不同 CD38 表达水平的 MM 细胞系（LP-1、RPMI8226、H929、MM.1s、OPM-2、和 KMS-11) 以及从新诊断或复发患者的骨髓中分离的三个 CD38+CD138+ 原发性 MM 样本。与 WT-NK 细胞相比，CD38KO-NK 细胞在 4 小时和 24 小时细胞毒性试验中显示出增强的 ADCC 活性（图 1D）。这甚至在表达低水平 CD38 的 MM 细胞中观察到，表明它们在 DARA 后复发性疾病患者中的潜在疗效（ADCC 在 E/T 5:1；MM.1s 7.8±1.8%（WT）对 21.5±0.4% (KO)，KMS-11 -3.5±0.6%(WT) 对比 11.1±0.4%(KO)，平均值±SD)。CD38KO-NK 细胞保留了与 WT-NK 对应部分相似的扩增效力。接下来，为了揭示操纵 CD38 表达对 NK 细胞的细胞毒性和代谢的任何影响，我们进行了 RNA 测序分析，这表明 CD38-KO NK 细胞中的几种细胞毒性基因如 IFNG 和 GzmB 的表达较高，葡萄糖转运蛋白基因和 GAPDH 的表达较高，表明这些细胞具有良好的代谢特征。与这些数据一致，我们观察到 CD38KO-NK 细胞通过 2-NBDG 葡萄糖摄取测定法测量的葡萄糖摄取能力提高（p=0.02，学生 t 检验）。总之，这些发现提供了概念验证，即由原代 NK 细胞产生的 CD38KO-NK 细胞增强了 DARA 的治疗效果，并有望用于针对 MM 的过继性免疫细胞治疗。YN 和 MNK 对这项研究做出了同等贡献。DAL 和 GG 对这项研究做出了同等贡献。图 1 披露 Ali: Celgene: Research Funding; 波塞达：研究经费。Lee:Kiadis Pharma：咨询、股权所有权、实体董事会或咨询委员会成员资格、专利和特许权使用费、研究资金。

"点击查看英文标题和摘要"

CD38 Knockout Primary NK Cells to Prevent "Fratricide" and Boost Daratumumab Activity

Multiple myeloma (MM) is a plasma cell neoplasm typically characterized by high and uniform CD38 expression. Daratumumab (DARA), a humanized monoclonal antibody targeting CD38 has dramatically improved the outcome of patients with refractory MM, but relapse is inevitable in most cases. DARA eliminates MM cells through several mechanisms including antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity, and antibody-dependent cellular phagocytosis. Mechanisms of resistance to DARA include downregulated CD38 expression on target MM cells, impaired complement directed cytotoxicity via upregulation of complement inhibitory proteins (CD55, CD59), and impaired ADCC via DARA-induced NK cell "fratricide". As it relates to "fratricide", restoring ADCC by DARA-resistant NK cells has been proposed to bolster the therapeutic effect of DARA. Towards this goal, iPSC-derived CD38 knockout (CD38KO) NK cells have shown proof-of-concept for this approach but present major challenges towards clinical translation. Here, we used Cas9-RNP to generate CD38KO primary NK cells and characterized their resistance to DARA-induced "fratricide" and restoration of ADCC, but also assessed the impact of CD38 deletion on NK cell signaling and metabolism. First, primary NK cells were enriched from peripheral blood of healthy donors and expanded on feeder cells expressing mbIL-21 and 4-1BBL (PLoS One. 2012;7(1):e30264). Cas9/gRNA complexes targeting CD38 were electroporated into NK cells (J Vis Exp. 2018 Jun 14;(136)). Flow cytometric analysis revealed CD38KO efficiency was 81.9±6.9% (mean±SD, n=5). CD38KO cells were further purified to ≥ 95% purity using magnetic cell separation system. Whole genome sequencing identified no off-target mutations. CD38KO-NK cells alone showed no conjugation in the presence of DARA (Figure 1A) and no DARA-induced fratricide (Figure 1B), in stark contrast with wild type NK (WT-NK) cells. Consistent with these results, treatment of NSG mice with DARA had no impact on persistence of CD38KO-NK cells, whereas WT-NK cells were completely eliminated (NK cell frequency in peripheral blood 7 days after inoculation; WT-NK NO DARA 12.11±5.15%, KO-NK NO Dara 17.05±0.98%, WT-NK + Dara 0.21±0.08%, KO-NK + Dara 16.85±3.61%, mean±SD, n=4, p<0.01, ANOVA) (Figure 1C). To examine the functional status of CD38KO-NK cells, we used 6 MM cell lines with various CD38 expression levels (LP-1, RPMI8226, H929, MM.1s, OPM-2, and KMS-11) as well as three CD38+CD138+ primary MM samples isolated from bone marrow of newly diagnosed or relapsed patients. CD38KO-NK cells exhibited enhanced ADCC activity compared to WT-NK cells across all cell lines and primary samples tested in 4h and 24h cytotoxicity assay (Figure 1D). This was even observed in MM cells that express low levels of CD38, suggesting their potential efficacy in patients with relapsed disease after DARA (ADCC at E/T 5:1; MM.1s 7.8±1.8%(WT) vs 21.5±0.4%(KO), KMS-11 -3.5±0.6%(WT) vs 11.1±0.4%(KO), mean±SD). The CD38KO-NK cells preserved similar expansion potency as WT-NK counter parts. Next, to reveal any effect of manipulating CD38 expression on NK cells cytotoxicity and metabolism, we performed RNA sequencing analysis, which showed higher expression of several cytotoxic genes such as IFNG and GzmB in CD38-KO NK cells, and higher expression of glucose transporter genes and GAPDH, suggesting a favorable metabolic profile of these cells. Consistent with these data, we observed that CD38KO-NK cells have elevated capacity to take up glucose as measured by 2-NBDG glucose uptake assay (p=0.02, student t test). Taken together, these findings provide the proof-of-concept that CD38KO-NK cells generated from primary NK cells augment therapeutic effect of DARA and could be promising for adoptive immune cellular therapy targeting MM. Y.N. and M.N.K. contributed equally to this study. D.A.L. and G.G. contributed equally to this study. Figure 1 Disclosures Ali: Celgene: Research Funding; Poseida: Research Funding. Lee:Kiadis Pharma: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

更新日期：2019-11-13

点击分享查看原文

点击收藏

公开下载

阅读更多本刊新发论文本刊介绍/投稿指南