Natural killer cell-derived extracellular vesicles (NK-EVs) have shown promising potential as biotherapeutics for cancer due to their unique attributes as cytotoxic nanovesicles against cancer cells and immune-modulatory activity towards immune cells. However, a biomanufacturing workflow is needed to produce clinical-grade NK-EVs for pre-clinical and clinical applications. This study established a novel biomanufacturing workflow using a closed-loop hollow-fibre bioreactor to continuously produce NK-EVs from the clinically relevant NK92-MI cell line under serum-free, Xeno-free and feeder-free conditions following GMP-compliant conditions. The NK92 cells grown in the bioreactor for three continuous production lots resulted in large quantities of both NK cell and NK-EV biotherapeutics at the end of each production lot (over 10⁹ viable cells and 10¹³ EVs), while retaining their cytotoxic payload (granzyme B and perforin), pro-inflammatory cytokine (interferon-gamma) content and cytotoxicity against the human leukemic cell line K562 with limited off-target toxicity against healthy human fibroblast cells. This scalable biomanufacturing workflow has the potential to facilitate the clinical translation of adoptive NK cell-based and NK-EV-based immunotherapies for cancer with GMP considerations.

中文翻译：

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临床相关的大规模生物制造工作流程，用于生产用于癌症免疫治疗的自然杀伤细胞和自然杀伤细胞衍生的细胞外囊泡

自然杀伤细胞来源的细胞外囊泡（NK-EV）因其独特的属性（针对癌细胞的细胞毒性纳米囊泡和针对免疫细胞的免疫调节活性）而显示出作为癌症生物治疗药物的巨大潜力。然而，需要生物制造工作流程来生产用于临床前和临床应用的临床级 NK-EV。本研究建立了一种新型生物制造工作流程，使用闭环中空纤维生物反应器，在符合 GMP 的条件下，在无血清、无异源和无饲养层的条件下，从临床相关的 NK92-MI 细胞系中连续生产 NK-EV。NK92 细胞在生物反应器中连续生长三个生产批次，在每个生产批次结束时产生大量 NK 细胞和 NK-EV 生物治疗药物（超过 10 9 个^活细胞和 10 ¹³ EV），同时保留其细胞毒性有效负载（颗粒酶 B 和穿孔素）、促炎细胞因子（干扰素-γ）含量和对人白血病细胞系 K562 的细胞毒性，对健康人成纤维细胞的脱靶毒性有限。这种可扩展的生物制造工作流程有可能促进基于 NK 细胞和 NK-EV 的过继性癌症免疫疗法的临床转化，并考虑到 GMP。