CN117143815B - 工程化记忆样nk细胞的制备方法及应用 - Google Patents
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Abstract
本发明提供了工程化记忆样NK细胞的制备方法及应用,采用IL‑2、IL‑12、IL‑15、IL‑18、IL‑21、IL‑33等白介素家族因子进行顺序免疫刺激,并使用抗CD16抗体和抗NKG2A抗体强化免疫记忆,可提高记忆样NK细胞的增殖活性,强化肿瘤抑制作用,在体内外可对多种肿瘤细胞产生抑制作用,为相关药物开发和工程化应用提供基础。
Description
技术领域
本发明属于生物技术研发领域,具体提供了一种工程化记忆样NK细胞的制备方法及应用。
背景技术
自然杀伤细胞(Natural killer cells,NK 细胞)于 20 世纪 70 年代被发现,当时得出的结论是 NK 细胞能够自然裂解某些肿瘤靶细胞,无需事先致敏即可介导肿瘤细胞裂解,故命名为“自然杀伤”细胞(参见Herberman RB, Ortaldo JR. Natural killercells: their roles in defenses against disease. Science (1981) 214:24–30)。NK细胞在识别患病细胞后使用两个主要功能来保护宿主,一是产生细胞因子来沟通和协调免疫反应,二是直接杀伤靶细胞。NK 细胞分泌促炎细胞因子(例如干扰素-γ(Interferon-γ,IFN- γ)和肿瘤坏死因子(tumor necrosis factor))和趋化因子(例如 CCL3-5),刺激巨噬细胞进行吞噬和裂解,上调主要组织相容性复合体 (MHC) I 类表达作用于抗原呈递细胞,招募额外的免疫效应子,并促进细胞毒性。NK 细胞还通过含有穿孔素和颗粒酶的预先形成的细胞毒性颗粒的胞吐作用以及死亡受体配体介导对靶细胞的接触依赖性杀伤。
NK 细胞传统上被认为是先天免疫系统的一部分,不具有与适应性 T 和 B 淋巴细胞相关的特异性或增强的记忆反应。然而,大量的工作已经改变了先天免疫和适应性免疫之间长期存在的分歧,NK 细胞记忆和类似记忆的反应在半抗原暴露、病毒感染和联合细胞因子激活后明确建立。
NK 细胞获得抗原特异性记忆的能力最初是在对化学半抗原的接触超敏反应(contact hypersensitivity,CHS) 中被观察到的,上皮细胞暴露于小分子刺激物,这些刺激物结合并修饰蛋白质,触发半抗原特异性 T 细胞记忆的形成。O'Leary 及其同事发现,缺少 T 细胞和 B 细胞(但不包括 NK 细胞)的小鼠能够表现出半抗原诱导的 CHS 反应,并且这些回忆反应不仅能够区分不同的半抗原(DNFB [2,4-二硝基-1-氟苯]和 OXA [恶唑酮]);来自 DNFB 致敏供体的肝 NK 细胞的过继转移导致受体在重新暴露于相同半抗原后产生 CHS 反应 (参见O’Leary JG, Goodarzi M, Drayton DL, von Andrian UH. Tcell– and B cell– independent adaptive immunity mediated by natural killercells. Nat Immunol 2006;7:507–16)。这些观察首次得出这样的结论:NK 细胞能够介导长期的、抗原特异性的适应性记忆反应。随后的实验表明,NK 细胞对半抗原的记忆反应取决于 CXCR6 的表达,CXCR6 是一种趋化因子受体,对于肝 NK 细胞的持久性和稳态至关重要。
病毒刺激也能使NK细胞产生记忆。Sun和Lanier发现,小鼠NK细胞在小鼠巨细胞病毒(MCMV)感染后表现出特异性的适应性反应(参见 Sun JC, Beilke JN, Lanier LL.Adaptive immune features of natural killer cells. Nature 2009;457:557–61)。MCMV 感染后,带有激活受体 Ly49H(与 MCMV 编码的 MHC I 类糖蛋白 m157 结合)的 NK细胞在感染后约一周内表现出扩增。虽然这种扩增的 Ly49H + NK 群体在初始扩展后下降,但 Ly49H 仍然持续表达。Sun 和同事使用这个模型在感染后 4 周识别了 NK 细胞的“记忆”库,这些记忆 NK 细胞的分离和离体再刺激显示出对 Ly49H 刺激的增强的功能反应,这是通过增加的 IFN-γ来测量的。
细胞因子是激活NK细胞,使其产生免疫记忆的重要手段。Cooper 和 Yokoyama 与MCMV 诱导的记忆 NK 细胞同时报道,发现用有效的细胞因子组合激活的小鼠 NK 细胞表现出类似记忆的特性(参见Cooper MA, Elliott JM, Keyel PA, Yang L, Carrero JA,Yokoyama WM. Cytokine-induced memory-like natural killer cells. Proc NatlAcad Sci. U S A 2009;106:1915–19)。用 IL-12、IL-15 和 IL-18 激活的小鼠 NK 细胞或仅用 IL-15 支持的 NK 细胞(维持存活所需)被过继转移到同系小鼠中,并使用 CFSE和同源标记进行追踪,发现细胞因子诱导的记忆样(ML 和 CIML)NK 细胞在体内增殖,但在过继转移后 1 周恢复到静息状态。与过继转移的对照 NK 细胞或内源性宿主 NK 细胞相比,这些 ML NK 细胞在再刺激时表现出增强的 IFN- γ反应。
研究人员发现NK细胞的“记忆功能”后,开始专注于如何获得大量的可用于临床试验或治疗的工程化的记忆样NK细胞。最先采用的是细胞因子的组合,细胞因子 IL-2 和IL-15 可驱动 NK 细胞分化、增殖和激活,而活化的树突状细胞反式提呈的 IL-15 对于NK 细胞的存活至关重要(参见 Rautela J, Huntington ND. IL-15 signaling in NKcell cancer immunotherapy. Curr Opin Immunol. (2017) 44:1–6)。树突状细胞在病毒感染期间分泌的 IL-12 和 IL-18 诱导有效的 NK 细胞 IFN-γ 产生和细胞毒性,特别是组合使用,并协同增强 IL-2 和 IL-15 诱导的 NK 细胞活化(参见Terren I, Mikelez I,Odriozola I, Gredilla A, Gonzalez J, Orrantia A, et al., Implication ofinterleukin-12/15/18 and ruxolitinib in the phenotype, proliferation, andpolyfunctionality of human cytokine-preactivated natural killer cells. FrontImmunol. (2018) 9:737)。此外,IL-2、IL-33也被认为在培养记忆样NK细胞中具有积极意义(参见Trinchieri G, Matsumoto-Kobayashi M, Clark SC, Seehra J, London L,Perussia B. Response of resting human peripheral blood natural killer cellsto interleukin 2. J Exp Med. (1984) 160:1147–69;Martinez-Gonzalez I, Matha L,Steer CA, Ghaedi M, Poon GF, Takei F. Allergen-experienced group 2 innatelymphoid cells acquire memory-like properties and enhance allergic lunginflammation. Immunity (2016) 45:198–208)。此外,特异性抗体也被用于刺激NK细胞的活化并产生免疫基因,研究人员将NK细胞暴露于治疗性抗体(如四价双特异性抗体AFM13(CD30/CD16A))后发现,CD16A的参与增强了随后IL2-和IL15驱动的NK细胞增殖和扩增。这种作用涉及NK细胞上CD25(IL2Rα)和CD132(γc)的上调,导致对低剂量IL-2或IL-15的敏感性增加,NK细胞也对不同的肿瘤靶点产生了增加的细胞毒性(参见Pahl JHW, Koch J,Gotz JJ, Arnold A, Reusch U, Gantke T, et al., CD16A activation of NK cellspromotes NK cell proliferation and memory-like cytotoxicity against cancercells. Cancer Immunol Res. (2018) 6:517–27)。
尽管研究人员对于记忆样NK的制备技术做出了诸多改进和尝试,但是仍难以满足大规模临床试验的需求,且目前的制备方法多依赖于白介素家族细胞因子,虽然能够刺激NK细胞增殖,但诱导产生的免疫记忆特异性和靶向性不强,难以发挥对肿瘤细胞的强烈杀伤作用,NK细胞的增殖能力也有待于进一步强化。为此,本发明提供了一种工程化记忆样NK细胞的制备方法,可生产增殖能力、抗肿瘤活性高的记忆样NK细胞,满足临床应用的需要。
发明内容
本发明中的第一方面提供了一种工程化记忆样NK细胞的制备方法,其特征在于,所述方法包括以下步骤:
(1)从外周血中提取NK细胞;
(2)将NK细胞接种至培养瓶中,加入含IL-2和IL-12的培养基,所述IL-2的终浓度为50-100ng/mL,所述IL-12的终浓度为10-50ng/mL,37℃培养箱中孵育1周;
(3)更换新鲜细胞培养基,加入含IL-15、IL-18和抗CD16抗体的培养基,所述IL-15的终浓度为10-50ng/mL,所述IL-18的终浓度为10-50ng/mL,所述抗CD16抗体的终浓度为10-50μg/ml,37℃培养箱中孵育1周;
(4)更换新鲜细胞培养基,加入含IL-21、IL-33、抗CD16抗体和抗NKG2A抗体的培养基,所述IL-21的终浓度为10-50ng/mL,所述IL-33的终浓度为10-100ng/mL,所述抗CD16抗体的终浓度为10-50μg/ml,所述抗NKG2A抗体的终浓度为10-50μg/ml,37℃培养箱中孵育3-5天,收获得到记忆样NK-细胞;
所述抗CD16抗体的重链可变区氨基酸序列如SEQ ID NO.1所示,轻链可变区氨基酸序列如SEQ ID NO.2所示;所述抗NKG2A抗体的重链可变区氨基酸序列如SEQ ID NO.3所示,轻链可变区氨基酸序列如SEQ ID NO.4所示。
进一步的,所述步骤(1)包括:收集外周血,使用Ficoll 分离液分离获得单个核细胞层;加入红细胞裂解液,离心收获细胞;利用免疫磁珠,筛选CD3-CD56+细胞。
进一步的,所述步骤(2)中,所述IL-2的终浓度为80-100ng/mL,所述IL-12的终浓度为10-30ng/mL。
进一步的,所述步骤(2)中,所述IL-2的终浓度为100ng/mL,所述IL-12的终浓度为30ng/mL。
进一步的,所述步骤(3)中,所述IL-15的终浓度为10-30ng/mL,所述IL-18的终浓度为10-30ng/mL,所述抗CD16抗体的终浓度为10-30μg/ml。
进一步的,所述步骤(3)中,所述IL-15的终浓度为20ng/mL,所述IL-18的终浓度为20ng/mL,所述抗CD16抗体的终浓度为20μg/ml。
进一步的,所述步骤(4)中,所述IL-21的终浓度为10-30ng/mL,所述IL-33的终浓度为10-50ng/mL,所述抗CD16抗体的终浓度为10-30μg/ml,所述抗NKG2A抗体的终浓度为10-30μg/ml。
进一步的,所述步骤(4)中,所述IL-21的终浓度为30ng/mL,所述IL-33的终浓度为50ng/mL,所述抗CD16抗体的终浓度为20μg/ml,所述抗NKG2A抗体的终浓度为20μg/ml。
本发明中的第二方面提供了一种所述方法制备的记忆样NK细胞在制备抗肿瘤药物中的应用,所述肿瘤包括血液瘤和实体瘤。
进一步的,所述肿瘤选自白血病、淋巴瘤、多发性骨髓瘤、胃癌、肝癌、乳腺癌、前列腺癌中的至少一种。
有益效果
本发明提供了一种工程化记忆样NK细胞的制备方法,具有以下优势:
(1)使用多种白介素家族因子的组合,替代传统的IL-12、IL-15和IL-18孵育,提高记忆样NK细胞的增殖活性;
(2)顺序施加细胞因子进行免疫诱导,防止一次性高剂量诱导而导致的副作用;
(3)使用抗CD16抗体和抗NKG2A抗体进行免疫刺激,强化抗肿瘤免疫记忆,提高抗肿瘤活性。
附图说明
图1:不同方法获得的NK细胞增殖能力图;
图2:免疫因子分泌水平图;
图3:NK细胞对于血液肿瘤细胞的抑制作用图;
图4:NK细胞对于实体肿瘤细胞的抑制作用图;
图5:记忆样NK细胞抑制体内肿瘤生长图。
实施方式
以下非限制性实施例可以使本领域的普通技术人员更全面地理解本发明,但不以任何形式限制本发明。凡基于本发明上述内容所实现的技术均应当属于本申请要求保护的范围之中。
以下实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂生物材料、检测试剂盒,如无特殊说明,均可从商业途径获得。
实施例1记忆样 NK细胞的培养
1.1 传统培养方法
为便于研究比较,本发明中首先采用传统方法提取和培养记忆样 NK细胞,该方法依赖于IL-12、IL-15和IL-18等细胞因子,相关培养步骤可参见文献Uppendahl LD,Felices M, Bendzick L, et al. Cytokine-induced memory-like natural killercells have enhanced function, proliferation, and in vivo expansion againstovarian cancer cells. Gynecol Oncol (2019)153:149–57。具体的步骤包括:
(1)抽取健康志愿者外周血置于含肝素抗凝剂的采血管中;另用 50ml 的离心管,将 Ficoll 分离液(购自Solarbio公司)倒进离心管中,然后沿管壁缓慢加入经PBS溶液2倍稀释后的外周血;4℃下 3000rpm,离心10min;离心结束后,吸取中间白膜层获得单个核细胞,放入新的15ml 的离心管中,加入 5ml 的 PBS 重悬单个核细胞层,以 3000rpm离心5min,弃上清,获得细胞沉淀;向沉淀中加入5mL红细胞裂解液(购自Solarbio公司),重悬后3000rpm离心 5min,去除上清后用 PBS 清洗一次,获得细胞;用PBS溶液重悬细胞,分别加入CD3磁珠和CD56磁珠(购自MiltenyiBiotec公司),利用流式细胞仪筛选CD3-CD56+细胞,所述细胞用PBS洗涤1次,即为NK细胞。
(2)将所述NK细胞接种于含10%FBS的RPMI-1640培养基中,同时加入终浓度为10ng/mL的IL-12、10 ng/mL的IL-15、50 ng/mL的IL-18,37℃、5%CO2培养箱中培养,观察细胞状态和密度,每 2-3 天进行补液和补加细胞因子,于14天后收获细胞,记为NK-1。
1.2 顺序施加复合细胞因子促进记忆样 NK细胞增殖
相比于传统方法采用IL-12、IL-15和IL-18诱导培养的方式,本节中尝试采用多种细胞因子的组合和顺序使用,以便提高记忆样NK细胞的增殖能力,具体步骤如下:
(1)NK细胞的提取和分离,方法同1.1节中第(1)步;
(2)将所述NK细胞接种于含10%FBS的RPMI-1640培养基中,同时加入终浓度为100ng/mL的IL-2、30ng/mL的IL-12,37℃、5%CO2培养箱中孵育3天;
(3)更换新鲜的含10% FBS的RPMI-1640培养基,同时加入终浓度为20ng/mL的IL-15、20ng/mL的IL-18,37℃、5%CO2培养箱中孵育7天;
(4)更换新鲜的含10% FBS的RPMI-1640培养基,同时加入终浓度为30ng/mL的IL-21、50ng/mL的IL-33,37℃、5%CO2培养箱中孵育4天,收获NK细胞,记为NK-2。
1.3 使用抗CD16抗体和抗NKG2A抗体活化记忆样 NK细胞
有报道称使用特异性抗体(如靶向CD16抗体)有利于记忆样 NK细胞的活化和增殖,本节中尝试使用抗CD16和NKG2A抗体改善记忆样 NK细胞的性能,所述抗体为申请人在前期实验中筛选获取,其中抗CD16抗体重链可变区氨基酸序列如SEQ ID NO.1所示,轻链可变区氨基酸序列如SEQ ID NO.2所示;抗NKG2A抗体重链可变区氨基酸序列如SEQ ID NO.3所示,轻链可变区氨基酸序列如SEQ ID NO.4所示。经检测,所述抗 CD16 抗体与CD16蛋白的亲和力为 5.31nM,所述抗 NKG2A 抗体与NKG2A蛋白的亲和力为 8.09nM。
具体步骤如下:
(1)NK细胞的提取和分离,方法同1.1节中第(1)步;
(2)将所述NK细胞接种于含10%FBS的RPMI-1640培养基中,同时加入终浓度为100ng/mL的IL-2、30ng/mL的IL-12,37℃、5%CO2培养箱中孵育3天;
(3)更换新鲜的含10% FBS的RPMI-1640培养基,同时加入终浓度为20ng/mL的IL-15、20ng/mL的IL-18和20μg/ml抗CD16抗体,37℃、5%CO2培养箱中孵育7天;
(4)更换新鲜的含10% FBS的RPMI-1640培养基,同时加入终浓度为30ng/mL的IL-21、50ng/mL的IL-33、20μg/ml抗CD16抗体和20μg/ml抗NKG2A抗体,37℃、5%CO2培养箱中孵育4天,收获NK细胞,记为NK-3。
实施例2记忆样 NK细胞的表征
2.1 增殖能力检测
细胞增殖能力是考察免疫是否能够在体内体外发挥免疫作用的重要因素,本实施例中采用MTT法检测记忆样NK细胞的增殖能力,分别取105个NK-1、NK-2和NK-3细胞,接种于96孔板中,各组设置3个复孔,置于37℃、5%CO2恒温培养箱中培养12h后,弃去原培养液,每孔加20μL 5g/L MTT溶液,置于37℃、5%CO2恒温培养箱中4h后,每孔加150μLDMSO,振荡10min,用酶标仪测量波长490 nm处吸光度值(OD),并按公式计算细胞相对增殖率,细胞相对增殖率=实验组OD值/对照组OD值×100%,以NK-1细胞为对照组。
结果如图1所示,相对于传统培养方法,使用本发明中所提供的细胞因子组合以及顺序处理策略,可显著提高记忆样NK细胞的增殖活性,然而施加抗CD16抗体和抗NKG2A抗体似乎对于细胞增殖活性影响不大,NK-2和NK-3组相比无明显差异。
2.2 免疫因子分泌能力
免疫因子是NK细胞发挥抗肿瘤作用的重要途径和载体,本实施例中检测不同培养方式对于免疫因子分泌的影响,以便考察其免疫调节能力。将5×105个NK-1、NK-2和NK-3细胞,接种于6孔板中,各组设置3个复孔,置于37℃、5%CO2恒温培养箱中培养24h后,收集细胞培养液,3000rpm离心取上清,使用ELISA试剂盒(购自美国Abcam公司)检测血清中的TNF-α和IFN-γ含量,具体操作步骤按照试剂盒说明书进行。
结果如图2所示,NK-1组和NK-2组中的免疫因子分泌水平类似,但NK-3组中TNF-α和IFN-γ水平均大幅度提高,说明使用抗CD16抗体和抗NKG2A抗体可刺激NK细胞分泌免疫调节因子,有利于形成肿瘤相关免疫记忆,进而提高抗肿瘤活性。
实施例3 记忆样 NK细胞的抗肿瘤作用
3.1 记忆样 NK细胞抑制血液肿瘤细胞生长
为验证本发明中所提供的记忆样NK细胞对血液肿瘤的抑制能力,选用人白血病细胞系THP-1、淋巴瘤细胞系Raji、多发性骨髓瘤细胞U266验证其抗肿瘤效果。本实施例中采用乳酸脱氢酶释放法检测 NK 细胞的肿瘤杀伤能力,按照10:1的比例将靶细胞(肿瘤)与效应细胞(NK细胞)接种于96孔板中,同时设置效应细胞自然释放组、靶细胞自然释放组及靶细胞最大释放组。96 孔板置于 37 ℃、5%CO2的培养箱培养24h,离心5 min,每孔吸取100 μL 细胞上清 于96孔板 ,加入100 μL LDH 溶液反应5 min,加入1 mol/L HCL 30 μl酶标仪检测 490 nm 处光密度值(OD值)。NK 细胞的杀伤活性=(实验孔 OD 值−效应细胞自然释放组OD 值)/(靶细胞最大释放组 OD 值−靶细胞自然释放组 OD值)。
结果如图3所述,记忆样NK对于血液肿瘤细胞具有普遍的抑制作用,其中对THP-1和Raji细胞的抑制作用更强,而且与传统方法获取的NK-1细胞相比,经过本申请中所述的细胞因子组合处理和抗CD16抗体、抗NKG2A抗体活化后,NK-2和NK-3组表现出了更强的抗肿瘤能力。
3.2 记忆样 NK细胞抑制实体肿瘤细胞生长
为验证本发明中所提供的记忆样NK细胞对实体肿瘤的抑制能力,选用胃癌细胞系BGC-823、肝癌细胞系HepG2、乳腺癌细胞系BT549、前列腺癌细胞系PC-3作为实验对象,研究记忆样NK细胞的肿瘤杀伤能力。具体实验方法参见3.1节,结果如图4所示,记忆样NK对于实体肿瘤细胞具有普遍的抑制作用,这方面与血液肿瘤类似,但是其抑制程度相对血液肿瘤较低,但对于PC-3和HepG2的杀伤能力相对较强,经过抗CD16抗体、抗NKG2A抗体活化后的NK-3也展现出了更强的抗肿瘤活性。
实施例4 记忆样NK细胞体内抑制肿瘤
制备基于Raji细胞的肿瘤动物模型,取5×106 个肿瘤细胞接种于裸鼠右前肢腋窝处皮下,每天观察成瘤情况并记录,肿瘤体积生长至100mm3以上时,表示造模成功。取40只造模成功小鼠,随机分为4组,分别为:NK-1组,尾静脉注射1×106个NK-1细胞,每周给药一次;NK-2组,尾静脉注射1×106个NK-2细胞,每周给药一次;NK-3组,尾静脉注射1×106个NK-3细胞,每周给药一次;对照组,每周给药一次注射等体积生理盐水。各种共治疗4周。
每周检测动物肿瘤组织体积,如图5所示,NK细胞治疗可延缓肿瘤生长过程,其中NK-3细胞作用最为明显,NK-2和NK-1的体内抗肿瘤作用类似,虽能够抑制肿瘤生长,但作用幅度有限,但从第4周的实验数据来看,似乎NK-3的抗肿瘤治疗更具优势。
以上所述技术方案,仅是本发明的较佳实施例而已,并非对本发明作任何形式上的限制,虽然本发明已以较佳实施例揭露如上,然而并非用以限定本发明,任何熟悉本专业的技术人员,在不脱离本发明技术方案范围内,当可利用上述揭示的技术内容作出些许更动或修饰为等同变化的等效实施例,仍属于本发明技术方案的范围内。
Claims (9)
1.一种工程化记忆样NK细胞的制备方法,其特征在于,所述方法包括以下步骤:
(1)从外周血中提取NK细胞;
(2)将NK细胞接种至培养瓶中,加入含IL-2和IL-12的培养基,所述IL-2的终浓度为50-100ng/mL,所述IL-12的终浓度为10-50ng/mL,37℃培养箱中孵育1周;
(3)更换新鲜细胞培养基,加入含IL-15、IL-18和抗CD16抗体的培养基,所述IL-15的终浓度为10-50ng/mL,所述IL-18的终浓度为10-50ng/mL,所述抗CD16抗体的终浓度为10-50μg/ml,37℃培养箱中孵育1周;
(4)更换新鲜细胞培养基,加入含IL-21、IL-33、抗CD16抗体和抗NKG2A抗体的培养基,所述IL-21的终浓度为10-50ng/mL,所述IL-33的终浓度为10-100ng/mL,所述抗CD16抗体的终浓度为10-50μg/ml,所述抗NKG2A抗体的终浓度为10-50μg/ml,37℃培养箱中孵育3-5天,收获得到记忆样NK细胞;
所述抗CD16抗体的重链可变区氨基酸序列如SEQ ID NO.1所示,轻链可变区氨基酸序列如SEQ ID NO.2所示;所述抗NKG2A抗体的重链可变区氨基酸序列如SEQ ID NO.3所示,轻链可变区氨基酸序列如SEQ ID NO.4所示。
2.根据权利要求1所述的方法,其特征在于,所述步骤(1)包括:收集外周血,使用Ficoll 分离液分离获得单个核细胞层;加入红细胞裂解液,离心收获细胞;利用免疫磁珠,筛选CD3-CD56+细胞。
3.根据权利要求1所述的方法,其特征在于,所述步骤(2)中,所述IL-2的终浓度为80-100ng/mL,所述IL-12的终浓度为10-30ng/mL。
4.根据权利要求3所述的方法,其特征在于,所述步骤(2)中,所述IL-2的终浓度为100ng/mL,所述IL-12的终浓度为30ng/mL。
5.根据权利要求1所述的方法,其特征在于,所述步骤(3)中,所述IL-15的终浓度为10-30ng/mL,所述IL-18的终浓度为10-30ng/mL,所述抗CD16抗体的终浓度为10-30μg/ml。
6.根据权利要求5所述的方法,其特征在于,所述步骤(3)中,所述IL-15的终浓度为20ng/mL,所述IL-18的终浓度为20ng/mL,所述抗CD16抗体的终浓度为20μg/ml。
7.根据权利要求1所述的方法,其特征在于,所述步骤(4)中,所述IL-21的终浓度为10-30ng/mL,所述IL-33的终浓度为10-50ng/mL,所述抗CD16抗体的终浓度为10-30μg/ml,所述抗NKG2A抗体的终浓度为10-30μg/ml。
8.根据权利要求7所述的方法,其特征在于,所述步骤(4)中,所述IL-21的终浓度为30ng/mL,所述IL-33的终浓度为50ng/mL,所述抗CD16抗体的终浓度为20μg/ml,所述抗NKG2A抗体的终浓度为20μg/ml。
9.根据权利要求1-8任一项所述方法制备的记忆样NK细胞在制备抗肿瘤药物中的应用,所述肿瘤选自白血病、淋巴瘤、多发性骨髓瘤、胃癌、肝癌、乳腺癌、前列腺癌中的至少一种。
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