肿瘤细胞治疗研究
英文原题：Leveraging CD16 fusion receptors to remodel the immune response for enhancing anti-tumor immunotherapy in iPSC-derived NK cells.
我们开发了一种新型 hnCD16FR 构建体，其细胞毒性强于已报道的 hnCD16，这是一种具有改善的 ADCC 特性的有前景的恶性肿瘤治疗方法。
背景：NK细胞的细胞毒作用主要依赖IgG Fc受体CD16a，该受体介导抗体依赖性细胞介导的细胞毒作用（ADCC）。高亲和力、不可剪切的CD16（hnCD16）已被开发，并显示出杀伤多种肿瘤的潜力。但hnCD16受体只激活单一CD16信号，对肿瘤的抑制有限。如何利用hnCD16特性并结合NK细胞特异性活化结构域，以进一步增强NK细胞抗肿瘤活性，是一个有前景的研究方向。方法：为拓展hnCD16介导的ADCC在癌症NK细胞免疫治疗中的应用，研究设计了hnCD16融合受体（FR）构型，将hnCD16胞外结构域与NK细胞特异性活化结构域融合于胞内。将FR构型转导至CD16阴性NK细胞系和人iPSC来源NK（iNK）细胞，并筛选有效构型。通过RNA测序和多重细胞因子释放检测，筛选并验证FR转导NK细胞中免疫活化及细胞因子释放相关通路的上调。通过与肿瘤细胞系共培养及荷有人B细胞淋巴瘤异种移植瘤的小鼠模型，分别评估体外和体内杀瘤效率。结果：研究筛选出杀伤B细胞淋巴瘤最有效的组合：hnCD16a胞外结构域与NK特异性共刺激分子2B4、DAP10及CD3胞内结构域融合。该构型在NK细胞系和iNK细胞中均表现出优异细胞毒性，并显著增加多种细胞因子释放。hnCD16和hnCD16FR转导NK细胞的转录组分析及验证实验显示，与hnCD16相比，hnCD16FR可重塑NK细胞免疫相关转录组，显著上调与细胞毒性、细胞因子释放、诱导肿瘤细胞凋亡及ADCC相关的基因。体内异种移植研究显示，低剂量单次给予工程化hnCD16FR iPSC来源NK细胞并联合抗CD20单克隆抗体，可产生强效抗肿瘤作用并显著延长生存期。结论：研究开发的新型hnCD16FR构型比已报道hnCD16具有更强细胞毒性，有望通过增强ADCC治疗恶性肿瘤；研究还为利用NK细胞活化结构域重塑免疫反应、增强NK细胞CD16信号提供了依据。
BACKGROUND: The cytotoxicity of NK cells is largely dependent on IgG Fc receptor CD16a, which mediates antibody-dependent cell-mediated cytotoxicity (ADCC). The high-affinity and non-cleavable CD16 (hnCD16) is developed and demonstrated a multi-tumor killing potential. However, the hnCD16 receptor activates a single CD16 signal and provides limited tumor suppression. How to exploit the properties of hnCD16 and incorporate NK cell-specific activation domains is a promising development direction to further improve the anti-tumor activity of NK cells. METHODS: To expand the applications of hnCD16-mediated ADCC for NK cell-based immunotherapy in cancer, we designed the hnCD16 Fusion Receptor (FR) constructs with the ectodomain of hnCD16 fused with NK cell-specific activating domains in the cytoplasm. FR constructs were transduced into CD16-negative NK cell line and human iPSC-derived NK (iNK) cells and effective FR constructs were screened. The up-regulation of immune activation- and cytokine-releasing-related pathways in FR-transduced NK cells was screened and validated by RNA sequencing and multiplex cytokines release assay, respectively. The tumor-killing efficiency was tested in vitro and in vivo via co-culture with tumor cell lines and xenograft mice-bearing human B-cell lymphoma, respectively. RESULTS: We screened the most effective combination to kill B cell lymphoma, which was fused with the ectodomain of hnCD16a, NK-specific co-stimulators (2B4 and DAP10) and CD3 in cytoplasmic domains. The screened construct showed excellent cytotoxicity effects and sharp multiple cytokines releasing both in the NK cell line and iNK cells. The transcriptomic analysis and validation assays of hnCD16- and hnCD16FR-transduced NK cells showed that hnCD16FR transduction remodeled immune-related transcriptome in NK cells, where significant upregulation of genes related to cytotoxicity, high cytokines releasing, induced tumor cell apoptosis, and ADCC in comparison with hnCD16 transduction were highlighted. In vivo xenograft studies demonstrated that a single low-dose regimen of engineered hnCD16FR iPSC-derived NK cells co-administered with anti-CD20 mAb treatment mediated potent activity and significantly improved survival. CONCLUSION: We developed a novel hnCD16FR construct that exhibits more potent cytotoxicity than reported hnCD16, which is a promising approach to treat malignancies with improved ADCC properties. We also offer a rationale for NK activation domains that remodel immune response to enhance CD16 signaling in NK cells.
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