CN114807044A - 一种具有高nkt细胞比例的car-cik细胞制备方法及应用 - Google Patents
一种具有高nkt细胞比例的car-cik细胞制备方法及应用 Download PDFInfo
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Abstract
本发明属于肿瘤免疫治疗研究领域,具体涉及一种获得高比例的NKT细胞的CAR‑CIK细胞优化培养方法。所述方法为在CAR‑CIK培养过程中加入IL‑15和乙酰半胱氨酸(NAC)共培养,替代传统的IL‑2。本发明提供的新培养方案能明显提高NKT的比例,增强CAR‑CIK细胞的抗肿瘤活性,并且对靶点阴性的肿瘤细胞仍然有杀伤活性,抗肿瘤效果显著。
Description
本申请要求2021年4月30日提交的中国发明专利申请【CN2021104890924】、名称为“一种具有高NK-T细胞比例以及广谱抗肿瘤活性的CAR-CIK细胞制备方法”的优先权,该优先权发明专利申请以引用方式全文并入。
技术领域
本发明属于肿瘤免疫治疗研究领域,具体涉及一种获得高比例的NKT细胞的CAR-CIK细胞优化培养方法。
背景技术
以CAR-T为代表的免疫细胞治疗已成为抗肿瘤领域最具潜力的新兴疗法。然而,虽然CAR-T细胞治疗血液肿瘤已取得了突破性进展,但应用于实体瘤时仍面临着诸多挑战。实体瘤与血液***肿瘤不同,其固有特点是不均匀的表达肿瘤抗原,而CAR-T对肿瘤细胞的杀伤作用有着严格的靶点依赖性,CAR触发的识别和杀伤需要肿瘤细胞表达特异性的抗原作为靶点,这是CAR-T细胞对实体瘤疗效不佳的重要原因之一。如果肿瘤细胞通过下调靶抗原的表达从而使CAR-T细胞无法识别,即便与CAR-T细胞接触也能逃避免疫杀伤。有研究报道,CAR-T无法清除仅含有10%靶抗原阴性肿瘤细胞(Ag-)的混杂肿瘤细胞团,因为即便CAR-T细胞将其中90%靶抗原阳性的肿瘤细胞消灭殆尽,在该肿瘤环境中10%的阴性细胞则成为了优势生长细胞,而CAR-T细胞无法对其有效地杀伤,Ag-肿瘤细胞则会迅速克隆增殖以导致耐药或者病情复发。事实上,并不存在全阳性的肿瘤。因此,亟需找到一种可以对靶抗原阳性和靶抗原阴性肿瘤细胞同时有效的免疫治疗方案。
CIK细胞是在体外通过抗体激活和多种细胞因子培养后所获得的具有裂解靶细胞活性的混杂淋巴细胞群,上世纪90年代Schmidt-Wolf等首次以抗CD3-mAb、IFN-γ、IL-2等多种细胞因子培养PBMC得到。CIK细胞对肿瘤细胞的杀伤活性不高,单用CIK细胞在体内抗肿瘤功能有限。近年来,随着CAR-T细胞技术的兴起,用CAR修饰CIK细胞得到CAR-CIK细胞,对靶抗原阳性的肿瘤细胞具有显著增强的杀伤活性,但对靶抗原阴性的肿瘤细胞仍无法有效杀伤。
CIK细胞中包括NKT细胞、T细胞以及NK细胞等,其中NKT细胞是介导CIK抗肿瘤功能的最主要的细胞。NKT细胞同时表达NK细胞和T细胞的分子标记,对肿瘤细胞的杀伤具有广谱性,并且没有HLA限制性。然而目前常规技术培养的CIK细胞中NKT的比例有限,通过优化培养方案提高NKT在CIK细胞中的比例,那么以此细胞制备成的CAR-CIK细胞不仅对靶抗原阳性的肿瘤细胞具有很好的杀伤活性,而且对靶抗原阴性的肿瘤细胞仍能有效杀伤,就可以解决实体肿瘤内部分或大部门肿瘤细胞上靶抗原表达阴性的问题。
另外,目前普遍采用IL-2来培养CAR-CIK细胞,但有研究表明IL-2可诱导免疫细胞的耗竭,使免疫细胞在体内存活时间短,影响抗肿瘤效果,因此需要进一步优化制备方法。
综上,本发明提出一种CAR-CIK细胞的优化培养方法,不仅能诱导产生高比例的NKT细胞,而且降低NKT上耗竭相关分子的表达,增加NKT细胞的寿命,从而对靶抗原阳性和阴性的肿瘤细胞都有良好杀伤活性,表现出更好的抗肿瘤活性。由此,建立了一种提高CAR-CIK细胞抗肿瘤效力的新技术方案。
发明内容
本发明的目的之一在于提供一种获得高比例的NKT细胞的CAR-CIK细胞优化培养方法。具体技术方案如下:
一种提高CAR-CIK细胞中NKT细胞比例的制备方法,在CAR-CIK细胞培养过程中加入IL-15和NAC共培养。
研究表明,IL-15能加速诱导记忆性T细胞的产生,增加T细胞的持续性从而提高抗肿瘤活性,并且IL-15的的作用在NK细胞、NKT细胞亚型中均得到了验证。因此本发明希望通过添加IL-15提高CAR-CIK细胞对肿瘤细胞的杀伤活性,并减少CAR-CIK细胞的的凋亡。
T细胞激活后会使细胞内活性氧(ROS)水平升高,ROS的积累会损害代谢重编程和T细胞的增殖,NAC能促进细胞内还原型谷胱甘肽(GSH)的合成,是ROS的清除剂。本发明通过添加NAC,降低CAR-CIK内ROS水平,增强CAR-CIK的持续性杀伤能力。
进一步,所述方法包括如下步骤:
1)获取CIK细胞,所述CIK细胞来源于外周血或脐带血;
2)用慢病毒感染制备工程化的CAR-CIK细胞;
3)使用50ng/mL的IL-15和大于等于2mM的NAC培养制备得到的CAR-CIK细胞,得到高NKT比例的CAR-CIK细胞。
进一步,步骤2)中制得的CAR-CIK细胞还含有包括CD3和/或4-1BB的共刺激胞内信号域。
本发明另一方面还提供一种联用方案,具体如下:
NAC与IL-15和NAC共培养的CAR-CIK细胞联用在制备抗实体肿瘤药中的应用。
进一步,所述实体肿瘤包括靶抗原阳性、靶抗原阴性和/或混杂靶抗原阳性和阴性的肿瘤细胞群的实体肿瘤。
进一步,所述实体瘤包括靶向Mesothelin(MSLN)和HER2的实体瘤。
进一步,所述IL-15和NAC可刺激所述CAR-CIK分化出更多的NKT细胞,用于增强所述药物的广谱抗癌能力。
进一步,所述IL-15和NAC可减少所述CAR-CIK的凋亡和耗竭,用于提高所述药物对肿瘤的杀伤力。
进一步,所述药物采用注射给药的方式,先注射用IL-15和NAC共培养的CAR-CIK细胞,再加强注射NAC。
进一步,在注射所述CAR-CIK细胞后,分别于第2天、第4-9天加强注射1g/kg NAC。
有益效果
本发明一方面提供了一种新的CAR-CIK细胞培养方法,采用IL-15和NAC替代IL-2培养CAR-CIK细胞,使NKT细胞的比例明显增加,从原来的28%增长到40%,从而使本发明制得的CAR-CIK细胞具有更好的广谱抗癌能力。实验证实,采用本发明的培养方法得到的CAR-CIK细胞对靶抗原阳性、靶抗原阴性和/或混杂靶抗原阳性和阴性的肿瘤细胞群的实体肿瘤均有杀伤效果,因此对于控制免疫细胞治疗后因靶抗原阴性细胞的优势增殖而导致的病情复发具有重要的临床意义。此外,本发明培养得到的CAR-CIK体外增殖能力变强,连续杀伤后的凋亡和耗竭减少,因此从整体上提高了CAR-CIK细胞杀伤肿瘤的能力和效率。
本发明制备得到的CAR-CIK细胞作为CAR-T治疗的潜在替代方案,对克服免疫细胞治疗在实体瘤中所面临的限制以及控制CAR-T治疗后的耐药或复发具有重大的意义。
本发明另一方面还提供了一种IL-15、NAC与CAR-CIK联用的技术方案。在体内动物模型中观察到CAR-CIK/IL-15+NAC的方案对体内肿瘤具有良好的抑制效果,进一步延长了实验动物的生存期,具有深远的临床应用价值。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍。在所有附图中,类似的元件或部分一般由类似的附图标记标识。
图1为优化培养的CAR-CIK细胞体外增殖能力图和诱导NKT细胞分化图;
图2为优化培养的CAR-CIK细胞分型情况;
图3为不同浓度NAC培养的CAR-CIK细胞对靶抗原阳性的肿瘤细胞的杀伤活性;
图4为流式结果图,显示多轮连续杀伤后凋亡和耗竭的CAR-CIK细胞(优化培养)减少;
图5为靶向MSLN的CAR-CIK细胞(优化培养)对靶抗原阳性的肿瘤细胞(SK-OV-3-MSLN)和靶抗原阴性的肿瘤细胞(SK-OV-3)的杀伤效果图;
图6为分别将靶抗原阳性(Ag+)和靶抗原阴性(Ag-)的肿瘤细胞标记上绿色和红色的荧光蛋白,1:1混合后作为靶细胞与CAR-CIK细胞(优化培养)共孵育,通过荧光显微镜观察并计数存活的肿瘤细胞图;
图7为在腹腔瘤NSG小鼠模型中,优化培养的CAR-CIK细胞体内抗瘤活性和小鼠生存时间图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述。显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
需要说明的是,在本文中,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者装置不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者装置所固有的要素。在没有更多限制的情况下,由语句“包括一个……”限定的要素,并不排除在包括该要素的过程、方法、物品或者装置中还存在另外的相同要素。
如在本说明书中使用的,术语“大约”,典型地表示为所述值的+/-5%,更典型的是所述值的+/-4%,更典型的是所述值的+/-3%,更典型的是所述值的+/-2%,甚至更典型的是所述值的+/-1%,甚至更典型的是所述值的+/-0.5%。
在本说明书中,某些实施方式可能以一种处于某个范围的格式公开。应该理解,这种“处于某个范围”的描述仅仅是为了方便和简洁,且不应该被解释为对所公开范围的僵化限制。因此,范围的描述应该被认为是已经具体地公开了所有可能的子范围以及在此范围内的独立数字值。例如,范围的描述应该被看作已经具体地公开了子范围如从1到3,从1到4,从1到5,从2到4,从2到6,从3到6等,以及此范围内的单独数字,例如1,2,3,4,5和6。无论该范围的广度如何,均适用以上规则。
实施例一
1)靶向MSLN的CAR-CIK细胞(采用本发明提供的方案进行优化培养)对靶抗原阳性的肿瘤细胞(SK-OV-3-MSLN)具有较强的杀伤,也能有效杀伤靶抗原阴性的肿瘤细胞(SK-OV-3);分别将靶抗原阳性(Ag+)和靶抗原阴性(Ag-)的肿瘤细胞标记上绿色和红色的荧光蛋白,1:1混合后作为靶细胞与CAR-CIK细胞共孵育,通过荧光显微镜观察并计数存活的肿瘤细胞,发现CAR-CIK细胞对于混合肿瘤细胞中的Ag-细胞也具有较强的杀伤能力,提示CAR-CIK细胞能有效避免由于肿瘤细胞靶抗原表达丢失所导致的免疫逃逸(图1和图3)。
2)流式结果显示,多轮连续杀伤后凋亡和耗竭的CAR-CIK细胞明显减少(图4)。
3)本发明提供的新培养方案的CAR-CIK细胞体外增殖能力更强,并能诱导更高比例的NKT细胞分化,从28%增长到40%(图5)。
4)在腹腔瘤NSG小鼠模型中,新培养方案的CAR-CIK细胞体内抗瘤活性增强,并能延长小鼠生存时间,提示优化培养的CAR-CIK细胞具有更好的应用潜力(图6)。
实施例二
1.HER2-CAR及MSLN-CAR质粒的获取
HER2-CAR分子序列中HER2 ScFv为鼠源单链可变区片段FRP5。本发明中涉及的MSLN-CAR分子序列中的MSLN ScFv为人源单链可变区片段m912。CAR分子包括:信号肽、ScFv、铰链区、CD8跨膜区、4-1BB共刺激信号区以及CD3ζ信号结构域。在获得原始质粒后加入50μL TB buffer稀释,标记好名称,置于-80℃进行长期保存。
2.慢病毒包装
使用磷酸钙沉淀法进行慢病毒包装。配制含20μg核心质粒,10μg包装质粒psPAX2,5μg包装质粒pMD2.G和50μL 2.5M CaCl2的500μL溶液,混匀后边振荡边逐滴加入500μL 2×BBS溶液,静置15min。将配好的混合液按每皿1mL的用量轻柔滴加至适宜密度(80%~90%)的HEK 293T细胞培养皿中。轻微摇晃培养皿,混匀培养基和混合液,置于37℃细胞孵箱中继续培养。8h后换液,48h后收集病毒原液,暂放于4℃保存,同时补充8-10mL新鲜培养基至皿中,24h后再次收集病毒原液。将两次收集到的病毒原液1500rpm离心5min,小心避开底部沉淀吸取上清,并经0.22μm滤头过滤至干净的容器中,使用超速离心机浓缩病毒。
3.CIK细胞的激活和培养
抽取健康志愿者的外周血于肝素钠抗凝管中,使用Ficoll分离液分离得到PBMC。取一干净的六孔板,加入2mL X-VIVO15完全培养基重悬后的PBMC,向其中加入1000IU/mL的IFN-γ激活,24h后向培养基中加入100ng/mL的CD3抗体以及500IU/mL的IL-2,此后继续用含有IL-2的培养基培养CIK细胞。
4.CAR-CIK细胞的制备和优化
4.1CAR-CIK细胞的制备
在非黏附的12孔板中加入1mL 25μg/mL RetroNectin铺板,置于4℃过夜。吸走孔板中的RetroNectin,用DPBS洗涤,吸走液体,重新加入1mL过滤后的2%的BSA溶液封闭30min。将浓缩的病毒加至12孔板中,32℃离心,1000g,2h,加速度、减速度均设为2。离心结束后向孔中再加入重悬好的2×106个CIK细胞,32℃离心,300g,10min,加速度、减速度均设为2。放于37℃的孵箱中培养2~3天后流式检测CAR表达。
4.2CAR-CIK细胞的优化培养
使用50ng/mL的IL-15和大于等于2mM乙酰半胱氨酸(NAC)进行体外培养,根据培养状态,每隔2-3天或3-4或4-5天重新补充新的细胞因子和NAC于培养基中。CAR-CIK细胞可以根据需要一直培养。
当分别使用50ng/mL的IL-15和2mM的NAC,50ng/mL的IL-15和5mM的NAC,50ng/mL的IL-15和10mM的NAC来培养CAR-CIK细胞,培养结果显示:CAR-CIK细胞的杀伤活性随NAC浓度的增加而增加(图3)。
实施例三
1.CAR-CIK细胞的体外杀伤
1.1多轮连续杀伤实验
消化肿瘤细胞,计数后用适量X-VIVO15双无培养基重悬,调整浓度至4×104个/200μL,混合均匀后根据实验设计均匀铺于48孔板,置于37℃孵箱培养;4h后镜下确认肿瘤细胞有无贴壁,贴壁后计数优化培养后的CAR-CIK细胞,用适量X-VIVO15双无培养基重悬,根据效靶比调整浓度,混合均匀后向已铺有肿瘤细胞的孔中加入效应细胞共孵育,每孔总体积为400μL。共孵育24h后在新的孔板中重复步骤1,铺板Round 2所需的靶细胞。用枪尖轻轻吹打混匀Round1孔内的细胞,分别吸至1.5mL离心管中,2000rpm离心4min,加入200μL新的X-VIVO15双无培养基重悬,加至Round 2肿瘤细胞孔中共孵育。重复以上步骤,继续构建Round 3、Round 4的杀伤体系。
1.2对靶抗原阳性和阴性混合肿瘤细胞团的杀伤
分别计数靶抗原阳性或阴性的肿瘤细胞,于48孔板中提前铺好肿瘤细胞。计数CAR-CIK细胞并用适量X-VIVO15双无培养基重悬,调整浓度为适宜效靶比,混匀后加入已贴壁的肿瘤细胞孔板中共孵育,杀伤24小时后于荧光显微镜下观察并拍照,利用Image J软件计数视野下存活的荧光细胞。
2.CAR-CIK细胞的分群
取约1×106个CAR-CIK细胞至流式管中,加1mL DPBS洗涤,1500rpm离心4min,离心后吸走上清,加100μL DPBS,向五个流式管中同时加入1μL以下抗体:FITC anti-humanCD3、BV510anti-human CD8、APC anti-human CD56、PE anti-human CD62L、PE-cy5 anti-human CD45RO抗体,拍打混匀。4℃避光孵育30min后,加1mL DPBS洗涤,1500rpm离心4min。吸走上清,加400μL PBS混匀后上机检测。
通过CD45RO和CD62L的表达不同将CAR-CIK细胞分为四种不同的表型。优化培养的CAR-CIK细胞有更高比例的CD45RO+记忆细胞表型,具有记忆表型的Tcm被认为具有更强的存活能力,更高比例的Tcm往往预示着更佳的抗肿瘤反应(图2)。
3.CAR-CIK细胞的凋亡和耗竭
取约1×106个效应细胞或杀伤后的CAR-CIK细胞,转移至流式管中,加入DPBS洗涤,按Binding buffer:DPBS为1:9的比例配制100μL洗涤液,混匀后加入流式管中,重悬细胞。向流式管中加入1μL的Annexin和1μL的7-AAD,室温下孵育15min,避光。加入1mL的DPBS洗涤,离心后加入400μL的Binding buffer重悬,上机检测凋亡分子的表达。
取约1×106个效应细胞至流式管中,加1mL DPBS洗涤。离心后吸走上清,加100μLDPBS,向流式管中分别加入1μL以下抗体:PE anti-human PD-1、PE anti-human TIM-3、APCanti-human LAG-3抗体,拍打混匀。4℃避光孵育30min后,DPBS洗涤后加400μL DPBS重悬,混匀后上机。
4.动物实验
实验动物均为4-6周龄的体重18-20g的NSG小鼠,需要提前在动物房SPF层流室适应环境一周。实验动物符合喂养标准,每周更换两次垫料、饲料、饮用水及笼具,保证生活环境洁净度,并密切注意观察小鼠生理状态。
消化SK-OV-3-luc肿瘤细胞后制成单细胞悬液,计数细胞浓度。往小鼠腹腔注射2×105个/200μL肿瘤细胞。接种肿瘤2天后对小鼠进行活体成像,观察成瘤情况。并将小鼠按荧光值大小均匀分组。day3向对应组的小鼠腹腔注射1×106个/200μL效应细胞(即优化培养的CAR-CIK细胞),PBS组腹腔注射等量PBS溶液。在接种后day 2,day 4-9分别向小鼠腹腔加强注射NAC溶液,NAC的注射剂量为1g/kg,一日两次。分别于接种后第3,11,18,25,33,40天对各组小鼠进行生物素荧光成像,拍照观察小鼠的肿瘤大小,调整标尺至合适大小。用活体成像方法对小鼠的肿瘤生长进行观察,时间间隔为一周。观察小鼠的生存状况,按时记录体重变化和生存时间。
总结
本发明分别构建了靶向人表皮生长因子受体(HER2)和间皮素(MSLN)的含共刺激分子4-1BB的二代CAR重组质粒,并利用HEK293T细胞包装CAR慢病毒。将外周血单核细胞(PBMC)来源的CIK细胞经慢病毒感染后制备为CAR-CIK细胞。通过体外杀伤实验检测杀伤效率以及杀伤过程中细胞因子释放情况,以此验证制备的CAR-CIK细胞的抗肿瘤活性。流式检测CAR-CIK的CD4/CD8细胞分群和CD3/CD56表达情况。利用慢病毒***构建过表达MSLN的SK-OV-3-MSLN肿瘤细胞,选用HER2和MSLN阳性的人卵巢癌SK-OV-3和SK-OV-3-MSLN作为靶细胞,分别和HER2-CAR-CIK和MSLN-CAR-CIK细胞共孵育,并设置不同的效靶比,并使用乳酸脱氢酶(LDH)试剂盒检测杀伤效率。
为探究CAR-CIK细胞的抗肿瘤特点,用CD3和CD28抗体激活PBMC中的T细胞,再经慢病毒感染后制备为CAR-T细胞,以比较CAR-T和CAR-CIK细胞的扩增能力,并用流式检测细胞分群以及效应细胞的功能表型变化。本发明使用靶抗原阳性或阴性的肿瘤细胞作为靶细胞,分别与CAR-T或CAR-CIK细胞共孵育进行体外杀伤实验。利用慢病毒***构建稳定表达绿色荧光蛋白的SK-OV-3-MSLN-GFP和稳定表达红色荧光的SK-OV-3-mcherry,并将以上肿瘤细胞按1:1的数量混合作为靶细胞,分别与MSLN-CAR-T和MSLN-CAR-CIK共孵育进行体外杀伤实验。在荧光显微镜下观察杀伤情况和拍摄图片,并利用Image J分别计数视野下的荧光细胞,比较各组的杀伤效率。本发明还通过多轮连续杀伤实验模拟体内杀伤环境,并通过流式细胞术检测CAR-CIK细胞经重复抗原刺激后效应细胞的凋亡情况以及表面的耗竭分子PD-1、TIM-3、LAG-3的表达水平。
本发明比较了新培养方案(IL-15和NAC)和传统IL-2培养的CAR-CIK的增殖能力以及NKT细胞分化的变化。通过多轮杀伤体系探究新培养方案对CAR-CIK细胞杀伤过程的作用。使用带Luciferase基因的肿瘤细胞在NSG小鼠中建立卵巢癌腹腔瘤模型,利用活体成像***监测PBS组、CIK组、CAR-CIK组和CAR-CIK/IL-15+NAC组肿瘤生长过程,以研究新培养方案对CAR-CIK细胞体内抗肿瘤作用的影响。
结果及结论:
本发明中构建的CAR-CIK细胞具有较强的体外增殖能力,接触靶抗原后能被激活并发挥抗肿瘤作用。由于CAR-CIK细胞中含有CD3+CD56+NKT细胞,对靶抗原阴性的肿瘤细胞杀伤明显强于CAR-T细胞,而且对于混合了靶抗原阳性(Ag+)和阴性(Ag-)肿瘤细胞的混杂细胞群,CAR-CIK细胞分别对其中Ag+、Ag-肿瘤细胞以及混杂肿瘤细胞群整体的杀伤作用均明显强于CAR-T细胞。
经新培养方案优化的CAR-CIK细胞与传统IL-2培养的CAR-CIK细胞相比,具有更强的体外扩增能力,能分化出更多NKT细胞,并提高CAR-CIK体外杀伤靶细胞的效率。新培养方案能减少CAR-CIK细胞经多轮连续杀伤后的凋亡和耗竭分子的表达水平。在NSG小鼠的腹腔瘤模型中,新培养方案能提高CAR-CIK细胞的体内杀伤效应,进一步延长小鼠生存期,具有良好的体内抗肿瘤效应。
本发明中制备的CAR-CIK细胞可对靶细胞产生有效杀伤。对于仅有靶抗原阳性或仅有靶抗原阴性的肿瘤细胞以及Ag+/Ag-混合的肿瘤细胞,CAR-CIK细胞的杀伤能力均高于CAR-T细胞。而新培养方案优化的CAR-CIK细胞增殖能力进一步提高,并且能更早地诱导出更高比例的NKT细胞,从而增强对靶抗原阳性、阴性和Ag+/Ag-混杂肿瘤细胞的杀伤效应,而且减少了连续杀伤后CAR-CIK细胞的凋亡和耗竭。此外,在体内动物模型中也观察到CAR-CIK/IL-15+NAC细胞良好的体内抗瘤活性。综上所述,CAR-CIK细胞有助于克服CAR-T治疗在异质性较大的实体瘤中所面临的“抗原困境”,其对于控制免疫细胞治疗后因靶抗原阴性细胞的优势增殖而导致的病情复发具有重要的临床意义。而新培养方案能诱导更高比例的NKT细胞,增强CAR-CIK细胞的作用,从而提高CAR-CIK细胞治疗在临床应用中的可行性。
上面结合附图对本发明的实施例进行了描述,但是本发明并不局限于上述的具体实施方式,上述的具体实施方式仅仅是示意性的,而不是限制性的,本领域的普通技术人员在本发明的启示下,在不脱离本发明宗旨和权利要求所保护的范围情况下,还可做出很多形式,这些均属于本发明的保护之内。
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Claims (10)
1.一种提高CAR-CIK细胞中NKT细胞比例的制备方法,其特征在于,在CAR-CIK细胞培养过程中加入IL-15和NAC共培养。
2.如权利要求1所述的制备方法,其特征在于,包括如下步骤:
1)获取CIK细胞,所述CIK细胞来源于外周血或脐带血;
2)用慢病毒感染制备工程化的CAR-CIK细胞;
3)使用50ng/mL的IL-15和大于等于2mM的NAC培养制备得到的CAR-CIK细胞,得到高NKT比例的CAR-CIK细胞。
3.如权利要求2所述的制备方法,其特征在于,步骤2)中制得的CAR-CIK细胞还含有包括CD3和/或4-1BB的共刺激胞内信号域。
4.NAC与IL-15和NAC共培养的CAR-CIK细胞联用在制备抗实体肿瘤药中的应用。
5.如权利要求4所述的应用,其特征在于,所述实体肿瘤包括靶抗原阳性、靶抗原阴性和/或混杂靶抗原阳性和阴性的肿瘤细胞群的实体肿瘤。
6.如权利要求4所述的应用,其特征在于,所述实体瘤包括靶向Mesothelin和HER2的实体瘤。
7.如权利要求4所述的应用,其特征在于,所述IL-15和NAC可刺激所述CAR-CIK分化出更多的NKT细胞,用于增强所述药物的广谱抗癌能力。
8.如权利要求4所述的应用,其特征在于,所述IL-15和NAC可减少所述CAR-CIK的凋亡和耗竭,用于提高所述药物对肿瘤的杀伤力。
9.如权利要求4所述的应用,其特征在于,所述药物采用注射给药的方式,先注射用IL-15和NAC共培养的CAR-CIK细胞,再加强注射NAC。
10.如权利要求9所述的应用,其特征在于,在注射所述CAR-CIK细胞后,分别于第2天、第4-9天加强注射1g/kg NAC。
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