CN116218786A - 一种多重基因编辑的通用型巨噬细胞及在制备抗肿瘤药物中的应用 - Google Patents
一种多重基因编辑的通用型巨噬细胞及在制备抗肿瘤药物中的应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域,具体涉及一种多重基因编辑的通用型巨噬细胞及在制备抗肿瘤药物中的应用。本发明提取脐带血中的CD34+细胞作为受体细胞,利用非整合型核电转方式重编程iPSCs,通过CRISPR/Cas9技术完成SIRPα表达抑制,并通过慢病毒感染的方式整合αPD‑1基因序列及特异性CAR基因序列,最终凭借iPSCs的多向分化潜能,分化成具备同时阻断免疫检查点SIRPα、PD‑1免疫抑制作用的CAR‑M细胞,为肿瘤患者的细胞和基因治疗提供了新策略。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种多重基因编辑的通用型巨噬细胞、所述巨噬细胞的构建方法、包含所述巨噬细胞的药物组合物及在制备抗肿瘤药物领域的应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
恶性肿瘤是严重威胁人类健康和生命的主要疾病之一,目前临床上的治疗手段主要为外科手术治疗、化疗及放疗。然而手术治疗存在切除残留,化疗、放疗方式缺乏肿瘤特异性、副作用较大以及易产生药物耐药等问题的存在,疾病的复发率和死亡率仍居高不下,对肿瘤的预防和治疗提出了严峻的挑战。随着肿瘤学、免疫学及分子生物学等学科的发展与融合,肿瘤免疫细胞治疗已成为一种新的治疗方案。嵌合抗原受体T细胞(CAR-T)在多种血液系统肿瘤(如白血病、淋巴瘤、多发性骨髓瘤)的治疗中表现不俗,取得了富有前景的疗效。但仍存在肿瘤抗原靶点选择、肿瘤抑制微环境及免疫逃逸、治疗维持深度不足、副反应明显、治疗后复发及实体瘤治疗效果差等情况,促使人们探索寻找新的替代方案。
巨噬细胞(Macrophage)是先天免疫系统的关键组成部分,主要通过吞噬细菌、死亡细胞及细胞残片等作用参与非特异性免疫调节,随后将吞噬的物质消化并将其特征递呈淋巴细胞及其他免疫细胞参与特异性免疫调节。嵌合抗原受体巨噬细胞(CAR-M)疗法是指将经过编辑的特定CAR基因植入巨噬细胞,以装备巨噬细胞,使之能够通过特异性抗原与肿瘤细胞表面结合并随后激活巨噬细胞的活性,进而直接吞噬、清除肿瘤细胞,并将肿瘤特异性抗原递呈给T细胞,激活T细胞对肿瘤的免疫反应,增强对肿瘤的杀伤力。与CAR-T细胞疗法相比,CAR-M能够进入实体瘤微环境、激活特异性免疫应答,且能在实体瘤环境中生存,具有广阔的应用发展前景。目前,可用于实验的巨噬细胞主要有两个来源:1.肿瘤衍生细胞系,如U937、THP-1细胞等,虽具有无限增殖的能力,但核型异常、表型不成熟,不能进行特定基因型的研究,且不适用于临床;2.原代细胞,如外周血单核细胞、单核细胞源性巨噬细胞等,是一种应用广泛的巨噬细胞功能实验模型,但该来源细胞是终末分化状态,不能进行自我更新,缺乏增殖能力,且较难对其进行基因编辑。因此,如何获取稳定增殖、数量充足,且可进行基因编辑的巨噬细胞是亟需解决的一个难题。
诱导多能干细胞(induced pluripotent stem cells,iPSCs)是通过转录因子或者小分子化合物诱导分化的体细胞逆转恢复到全能性状态,形成形态和功能类似于胚胎干细胞(embryonic stem cells,ESCs)的一类细胞,具有自我更新能力,能够“无限”增殖,同时也具有多向分化的潜能,在特定的诱导条件下可分化为不同表型的成熟细胞。近些年,iPSCs重编程技术已成为基础及临床研究的热点,备受研究者们关注。利用iPSCs重编程技术并诱导分化所需巨噬细胞,能够解决细胞稳定获取问题,为研究巨噬细胞特定功能、免疫应答机制、活化和极化分子及巨噬细胞相关疾病或肿瘤治疗提供良好的工具。此外,重编程的iPSCs易于通过基因组编辑方法(如CRISPR/Cas9、慢病毒转染等)进行基因修饰,能够衍生出足量的、稳定的CAR-M,利用肿瘤相关抗原靶向性,将其吞噬活性直接作用于肿瘤细胞,在肿瘤免疫细胞治疗中展现出独有的优势。
机体免疫系统与恶性肿瘤之间存在较为复杂的调控网络,肿瘤细胞可通过多种机制逃避免疫监视。免疫检查点是一类免疫抑制性分子,能够调节免疫的强度和力度,而免疫检查点失调是肿瘤产生免疫逃逸的重要原因。信号调节蛋白(Signal regulatoryproteins,SIRP)家族包括SIRPα、SIRPβ1、SIRPγ、SIRPβ2和SIRPδ五个成员,其中SIRPα主要表达于髓系细胞表面,如单核细胞、巨噬细胞、粒细胞以及髓系树突状细胞等。CD47是SIRPα最主要的配体,二者结合后可以使SIRPα胞内段免疫受体酪氨酸抑制基序发生磷酸化,随后募集并激活酪氨酸磷酸酶SHP-1/2,从而通过阻止Myosin-IIA在吞噬突触中的聚集,抑制巨噬细胞的吞噬效应。这种功能在正常的人体环境里可以用于标记“自我”和“非我”,避免引起“误伤”。然而,由于多数肿瘤,尤其是实体瘤肿瘤细胞,表面往往高表达CD47分子,CD47-SIRPα途径即可作为肿瘤细胞逃脱巨噬细胞吞噬效应的重要机制。已有研究证明,通过阻断CD47-SIRPα通路,能够解除巨噬细胞的免疫抑制,激发巨噬细胞的肿瘤吞噬作用。目前,靶向该通路的在研方案主要为三大类药物,包括CD47抗体、SIRPα融合蛋白以及SIRPα抗体。已开展的临床试验结果表明,系统的通过单药对肿瘤患者的CD47-SIRPα通路进行阻断效果甚微,且CD47的阻断会带来贫血症等副作用。因此,如何阻断免疫检查点免疫抑制作用,并同时保证高抗肿瘤活性及高肿瘤亲和性成为研究的关键点及难点所在。
程序性死亡受体1(programmed death 1,PD-1)是一种重要的免疫抑制分子,主要表达于活化的T淋巴细胞及前体B细胞。有研究表明,某些肿瘤细胞通过表面PD-1配体(PD-L1)的高表达,与PD-1结合后启动T细胞程序性死亡,使其失活并耗竭,从而获得免疫逃逸,继续恶性增殖。此外,PD-1在巨噬细胞非特异性免疫应答中也发挥重要作用。与活化状态的T淋巴细胞相比,PD-1在巨噬细胞中的表达水平较低,但在给予一定刺激后,如LPS、IFN-α等,巨噬细胞表面会上调PD-1表达,进而影响其功能成熟及共刺激分子的表达,抑制其吞噬肿瘤细胞的能力。因此,阻断肿瘤细胞表面PD-L1与免疫细胞表面PD-1结合,能够有效增强免疫细胞的抗肿瘤活性。常用的阻断方法是给予肿瘤患者PD-1抗体或PD-L1抗体治疗,阻断两者的联接,解除免疫功能抑制,增强T细胞毒性、巨噬细胞吞噬效应以及免疫细胞的肿瘤富集等。然而PD-1/PD-L1通路的广泛抑制会加速和加强自身免疫,导致很多免疫介导的副作用。
发明内容
随着基因编辑技术的快速发展,iPSCs重编程技术和基因编辑技术之间的协同作用为肿瘤发病机制和治疗方法的探索提供了更加便利、有效的条件。本发明设计提供了一种免疫检查点抑制与嵌合抗原受体治疗相结合的多重基因编辑的通用型巨噬细胞,凭借iPSCs的多向分化潜能,分化成具备同时阻断免疫检查点SIRPα、PD-1免疫抑制作用的CAR-M细胞,应用于肿瘤患者的细胞和基因治疗,具有良好的应用前景。
具体的,本发明的技术方案如下:
本发明第一方面,提供一种多重基因编辑的通用型巨噬细胞,所述巨噬细胞由iPSCs细胞诱导分化而来,细胞表面分子SIRPα表达抑制,细胞表面具有嵌合抗原受体(CAR)修饰,同时细胞可分泌表达抗PD-1抗体的单链可变片段(single-chain variablefragment(scFv)of the PD-1antibody,αPD-1)。
本发明提供的上述多重基因编辑的通用型巨噬细胞通过iPSCs细胞诱导分化而来,相比现有肿瘤衍生细胞系或原代细胞来源的巨噬细胞,iPSCs具有无限繁殖以及诱导分化的特性,能够获得足量治疗所需的免疫细胞,以保证治疗的持续进行。
针对所述多重基因编辑的通用型巨噬细胞,本发明还增加了免疫检查点联合阻断以解除肿瘤的免疫抑制效应,整合嵌合抗原受体以增强抗肿瘤靶向性。
本发明针对重编程获得的iPSCs进行了细胞表面分子SIRPα的表达抑制,能够阻断SIRPα激活导致的免疫抑制。本发明还将αPD-1基因序列及特异性CAR基因序列整合到重编程后的iPSCs中,持续性分泌的αPD-1能够阻断肿瘤PD-1/PD-L1通路激活导致的免疫抑制效应;诱导iPSCs分化为CAR-M,通过表达的肿瘤特异性抗原受体,提高靶向抗肿瘤作用。
优选的,所述CAR基因序列及αPD-1基因序列通过酰胺键直接连接,或通过连接子(linker)连接,所述连接子可基于本领域常规的考虑进行选择,如P2A,T2A,E2A或F2A,本发明提供的一种实施方式中,所述CAR基因序列与αPD-1基因序列通过P2A进行连接。
其中,所述CAR可以为靶向任意一个或多个肿瘤靶点的嵌合抗原受体,所述肿瘤可以为血液瘤和实体瘤,因此,所述肿瘤靶点包括但不限于CD19、BCMA、CD20、CD22、CD123、CD30、TAA、HER2、EGFR、GPC3、MUC1、PSMA。其中,CD19是B细胞表面的跨膜蛋白。它与B细胞活化、信号传导及生长调节密切相关,是B淋巴细胞表面的一种功能受体分子,在B细胞抗原受体识别抗原时构成B细胞双重抗原结合模型,参与B细胞内Ca2+的转运,调节B细胞的活化与增殖。CD19作为重要标记物被广泛应用于血液瘤(如白血病、淋巴瘤)及免疫系统疾病的诊断和预后判断。因此在本发明的一个具体实施方式中,所述肿瘤靶点选自CD19,因此所述CAR为靶向CD19的嵌合抗原受体(CD19 CAR)。
优选的,所述巨噬细胞还具有其他化学修饰或遗传修饰,所述修饰物包括但不限于以下类型:核酸、抗生素、抗炎剂、抗体或其抗体片段、生长因子、细胞因子、酶、蛋白质、肽、融合蛋白、合成分子、有机分子、碳水化合物或类似物、脂质、激素、微粒体、其衍生物或变体、以及其任何组合。
本发明第二方明,提供第一方面所述多重基因编辑的通用型巨噬细胞构建方法,所述构建方法包括以下步骤:获取脐带血CD34+细胞重编程为iPSCs,对所述iPSCs进行细胞表面分子SIRPα的表达抑制,得到iPS-SIRPαko(缩写为iPS-Sko)细胞,同时将αPD-1基因序列及特异性CAR基因序列整合到iPS-Sko细胞中得到iPS-SIRPαko-CAR-αPD-1(缩写为iPS-SkoCA)细胞,再对iPS-SkoCA细胞进行诱导分化得到所述多重基因编辑的通用型巨噬细胞。
上述构建方法中,iPSCs以脐带血中的CD34+细胞作为受体细胞来源,其优势主要包括以下两方面:1、脐带血中的免疫细胞较为幼稚,免疫功能尚不成熟,免疫源性较其他来源更低,可作为通用型免疫细胞的制备源,为异体移植治疗提供良好的渠道;2、脐带血CD34+细胞相对其他成人体细胞更为纯净,基本排除病毒以及其他病原微生物的感染及传播,制备出的iPSCs基因突变较少,应用于肿瘤的免疫治疗具有更好的安全性。
优选的,脐带血CD34+细胞重编程为iPSCs采用电转诱导方式,具体的操作方式如下:获取脐带血中的CD34+细胞进行扩增培养,将扩增后的CD34+细胞加入电转杯中处理,电转处理后的细胞铺入基质胶中进行扩增培养一段时间,筛选其中的阳性克隆得到所述iPSCs。
进一步的,上述iPSCs构建完成后还包括对其干细胞活性的鉴定,所述鉴定方式包括对多潜能标记物进行检测、细胞核型鉴定等,所述多潜能标记物为包括但不限于SOX2、OCT4、NANOG、SSEA4、TRA-1-60中的一种或几种的组合。
优选的,所述细胞表面分子SIRPα表达抑制可通过诸如RNAi(包括shRNA、siRNA)、TALEN、CRISPR(包括CRISPR/Cas9、CRISPR/Cas13)的方法,在本发明提供的一种实施方式中,通过CRISPR/Cas9系统进行编辑,具体实例中,所述SIRPα的CRISPR gRNA序列为:GGTAGAGGGCCGCCATCAGT(SEQ ID NO.1)。
优选的,所述αPD-1基因序列及特异性CAR基因序列可通过慢病毒感染、质粒转染等方式转入iPS-Sko细胞中。本发明提供的一种具体的实施方式中,上述基因序列采用慢病毒转染整合到iPS-Sko细胞中;
更具体的,将CD19 CAR(hFcGamma)过表达序列通过P2A序列与αPD-1过表达序列连接后,构建CD19CAR及αPD-1过表达载体,命名为pCDH-EF1-CD19 hFcGamma CAR-P2A-αPD-1(其核苷酸序列如SEQ ID NO.2所示),并进行慢病毒包装,进而转染iPS-Sko细胞。
优选的,所述分化诱导的具体步骤如下:将iPS-SkoCA细胞加入拟胚体培养基中获得拟胚体(EB),将拟胚体加入分化培养基A进行培养获得巨噬细胞前体,收集所述巨噬细胞前体加入分化培养基B中获得巨噬细胞。
进一步的,所述分化培养基A采用无血清培养基,所述培养基中包括80~120ng/mLM-CSF,20~30ng/mL IL-3,1~3mM GlutaMAX,0.8~1.2%青链霉素混合液,0.055mM 2-mercaptoethanol。
进一步的,所述分化培养B采用1640培养基,所述培养基中包括8~12%FBS、80~120ng/mL M-CSF。
本发明第三方面,提供一种药物组合物,所述药物组合物中包括活性剂量的第一方面所述多重基因编辑的通用型巨噬细胞。
上述药物组合物中,所述多重基因编辑的通用型巨噬细胞应当是有效剂量的,本发明已经证实上述多重基因编辑的通用型巨噬细胞对肿瘤细胞具有良好的吞噬效率,其施用剂量是基于药物施用目的、施用方式、受试对象等因素可常规获知的技术内容。
优选的,所述药物组合物中,还包括药学上所必须的载体,所述药学上可接受的载体剂量应当对受试者是无害的,具体为包括但不限于缓冲剂(如乙酸盐、Tris、磷酸盐、柠檬酸盐和其它有机酸)、抗氧化剂(如抗坏血酸和甲硫氨酸)、防腐剂(如氯化十八烷基二甲基苄基铵、酚、丁醇或苄醇、对羟基苯甲酸甲酯或对羟基苯甲酸丙酯、邻苯二酚、间苯二酚、环己醇、3-戊醇和间甲酚)、杀菌剂(如氯己双铵、苯扎氯铵、苄索氯铵)、蛋白质(如血清蛋白、明胶或免疫球蛋白)、亲水性聚合物(如聚乙烯吡咯烷酮)、氨基酸(如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸)单糖、二糖和其它碳水化合物(包括葡萄糖、甘露糖或糊精)、螯合剂(如EDTA)、张力调节剂(如海藻糖和氯化钠)、糖类(如蔗糖、甘露醇、海藻糖或山梨醇)、表面活性剂(如聚山梨醇酯)、成盐抗衡离子(如钠)、金属复合物(如Zn-蛋白质复合物)和/或非离子表面活性剂(如或聚乙二醇)。
本发明第四方面,提供第一方面所述多重基因编辑的通用型巨噬细胞、第三方面所述药物组合物在制备抗肿瘤药物中的应用。
所述肿瘤为包括但不限于肺癌、黑色素瘤、胶质瘤、胃癌、卵巢癌、结肠癌、肝癌、肾癌、膀胱癌、乳腺癌、经典霍奇金淋巴瘤、血液肿瘤、淋巴癌;进一步的,为淋巴癌或血液肿瘤。
本发明第五方面,提供一种抗肿瘤药物,所述药物中包括活性剂量的第一方面所述多重基因编辑的通用型巨噬细胞或第三方面所述药物组合物。
上述抗肿瘤药物的剂型为固体制剂或液体制剂,优选为液体制剂,进一步的,为注射剂。
上述一个或多个技术方案的有益技术效果:
上述技术方案设计提供了一种免疫检查点抑制与嵌合抗原受体治疗相结合的多重基因编辑的通用型巨噬细胞,凭借iPSCs的多向分化潜能,分化成具备同时阻断免疫检查点SIRPα、PD-1免疫抑制作用的CAR-M细胞,应用于肿瘤患者的细胞和基因治疗,具有良好的实际应用之价值。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为本发明实施例中(A)中白色箭头所指的是脐带血CD34+细胞电转19天后,形成的推定的iPSCs克隆。(B)显示的是脐带血CD34+细胞来源的iPSCs。
图2为本发明实施例中显示SIRPαgRNA靶点在SIRPα基因组第六外显子上。
图3为本发明实施例中显示在电转入PX459-SIRPα质粒后,iPSCs基因组测序在gRNA靶点附近出现了错配峰,说明iPSCs被CRISPR/Cas9技术编辑。
图4为本发明实施例中证实iPS-SKO细胞中的SIRPα在蛋白水平敲除成功。
图5为本发明实施例中(A)为pCDH-EF1-CD19 hFcGamma CAR-P2A-αPD-1载体图谱。(B)为pCDH-EF1-CD19 hFcGamma CAR-P2A-αPD-1载体中CD19 hFcGamma CAR-P2A-αPD-1部分主要结构示意图。
图6为本发明实施例中显示流式细胞术证实iPS-SKOCA细胞上CD19 CAR过表达成功。
图7为本发明实施例中显示Western Blot检测证实iPS-SKOCA细胞胞内αPD-1过表达成功。
图8为本发明实施例中(A)显示ELISA实验证实iPS-SKOCA细胞培养上清液中检测到αPD-1蛋白。(B)显示iPS-SKOCA细胞中过表达的αPD-1蛋白可以分泌到胞外。
图9为本发明实施例中显示iPS、iPS-SKO及iPS-SKOCA细胞均表达多潜能标记物SOX2、OCT4、NANOG、SSEA4及TRA-1-60。
图10为本发明实施例中显示UCB CD34+、iPS、iPS-SKO及iPS-SKOCA细胞的染色体核型均为46,XX。UCB:umbilical cord blood,脐带血。
图11为本发明实施例中iPSCs诱导分化为巨噬细胞的流程图。(a)图为iPSCs克隆。(b)图为iPSCs形成的EB球。(c)图为EB球在分化培养基A中逐渐贴壁。(d)图为EB球开始释放悬浮生长于上清中的细胞。(e)图为收集上清中的悬浮细胞加入分化培养基B中进行终分化。(f)图为终分化培养9-11天后,细胞呈现典型的巨噬细胞形态特征。
图14为本发明实施例中QPCR检测K562、K562-CD19及Raji细胞CD19表达水平。
图15为本发明实施例中(A)为及细胞对K562、K562-CD19及Raji细胞的吞噬作用图片,红色为巨噬细胞,绿色为淋巴瘤细胞。(B)为及细胞对K562、K562-CD19及Raji细胞的吞噬效率的柱状统计图。巨噬细胞吞噬效率=(发生吞噬的巨噬细胞/(发生吞噬的巨噬细胞+未发生吞噬的巨噬细胞))×100%
图16为本发明实施例中(A)为流式细胞术检测及细胞对K562、K562-CD19及Raji细胞的吞噬作用。(B)为及细胞对K562、K562-CD19及Raji细胞的吞噬效率的柱状统计图。巨噬细胞吞噬效率=(发生吞噬的巨噬细胞/(发生吞噬的巨噬细胞+未发生吞噬的巨噬细胞+未被吞噬的淋巴瘤细胞))×100%。
图17为本发明多重基因编辑的通用型巨噬细胞制备总体流程图。
具体实施方式
应该指出,以下详细说明都是例示性的,旨在对本发明提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本发明的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案,以下将结合具体的实施例详细说明本发明的技术方案。
实施例1
一、脐带血CD34+细胞来源iPSCs的制备
1.脐带血CD34+细胞提取及培养:
用肝素血袋取脐带血适量,室温放置24h,用EasySepTMHuman Cord Blood CD34Positive Selection Kit II(Stemcell Technologies)提取脐带血中的CD34+细胞。
提取的CD34+细胞按每孔1×105个接种于6孔板中,每孔加入2ml CD34+细胞扩增培养基(StemSpanTMSFEM II,StemCell Technologies:StemSpanTMCD34+ExpansionSupplement(10X),StemCell Technologies=9:1),放入温度37℃,含5%CO2培养箱中培养,此时记为Day 0。Day 2,换液,将孔板中的细胞收集到15ml离心管中,300g离心5min,用CD34+扩增培养基重悬后,2ml一孔铺到六孔板中,放入温度37℃,含5%CO2培养箱中继续培养两天。
2.脐带血CD34+细胞重编程为iPSCs:
Day 4,将6孔板的细胞收集到15ml离心管中,300g离心5min,用CD34+扩增培养基重悬后,计数,取1×106个细胞,300g离心5min,用细胞转染试剂盒(LONZA)中的细胞特异性转染液100μl重悬,加入pmaxGFP阳性对照质粒2μg或Epi5TMEpisomal iPSC重编程试剂盒(Invitrogen)两种游离性质粒各1μl,转入电转杯中,按照Lonza Nucleofector 2b细胞核转染仪操作说明书进行电转,电转结束后,向电转杯中加入500μl预热CD34+细胞扩增培养基,用一次性吸管将电转杯中的液体转移至含5.5ml预热CD34+细胞扩增培养基的15ml离心管中,得到电转后细胞6ml。
取电转后的细胞2ml(约3.3×105个细胞)铺到用Matrigel基质胶(Corning)预包被的六孔板中的一个孔,共铺三个孔,此时记为Day 0,放入温度37℃,含5%CO2培养箱中培养。Day 2,每个孔补加1ml CD34+细胞扩增培养基。Day 3,每个孔补加1ml ReproTeSR培养基(StemCell Technologies)。Day 5,每个孔补加1ml ReproTeSR培养基。Day 7,吸去孔内原培养基,每个孔加入2ml ReproTeSR培养基。Day 8-Day 18,每天吸去孔内原培养基,每个孔加入2ml ReproTeSR培养基。
电转后19天,六孔板内已经形成若干推定的iPSCs克隆(见图1(A))。在立体显微镜下用5ml注射器针头将iPSCs克隆划成小碎片,再用带滤芯的200μL枪尖将碎片刮下来并吸走,转移至含有mTeSR Plus培养基(为促进碎片的初始贴壁,加入Y-27632(StemCellTechnologies),使其终浓度为10μM,24h后换为普通mTeSR Plus培养基)的用Matrigel基质胶预包被的六孔板中,放入温度37℃,含5%CO2培养箱中培养。分离后的iPSCs克隆,前三次传代继续使用上述方法进行操作,后续可以使用ReLeSR(StemCell Technologies),按照说明书进行传代,用mTeSR Plus培养基,按照说明书进行细胞培养,细胞形态如图1(B)所示。
二、对脐带血CD34+细胞来源iPSCs进行基因编辑
1.运用CRISPR/Cas9技术编辑iPSCs:
为了阻断CD47-SIRPα通路,本发明运用CRISPR/Cas9技术编辑了iPSCs的SIRPα基因。使用的SIRPα的CRISPR guideRNA(gRNA)序列为GGTAGAGGGCCGCCATCAGT(SEQ ID NO.1,见图2),将此序列构建到CRISPR/Cas9系统载体pSpCas9(BB)-2A-Puro(PX459)上,记为PX459-SIRPα。六孔板中培养的iPSCs,每孔用1ml DPBS洗一遍,再加入1ml Accutase,放入37℃培养箱中消化5min,然后加入mTeSR Plus培养基(含10μM Y-27632)终止消化,用1ml枪尖吹成单细胞后计数,取两组各1×106个细胞,200g离心5min,各用细胞转染试剂盒(LONZA)中的细胞特异性转染液100μl重悬后分别加入PX459空载阴性对照质粒(去内毒素)5μg或PX459-SIRPα质粒(去内毒素)5μg,混匀后加入电转杯中,按照Lonza Nucleofector2b细胞核转染仪操作说明书进行电转。电转结束后,迅速向电转杯中加入500μL预热的mTeSR Plus培养基(含10μM Y-27632),用一次性吸管吸出电转杯中的液体转移至含1.5mlmTeSR Plus培养基(含10μM Y-27632)的用Matrigel基质胶预包被的六孔板一个孔中,放入温度37℃,含5%CO2培养箱中培养,此时记为Day 0。Day 1,将六孔板中液体吸出,每孔加入2ml新鲜的mTeSR Plus培养基(含10μM Y-27632),放入温度37℃,含5%CO2培养箱中培养。Day 2,六孔板中的细胞汇合度达到70%-90%,用ReLeSR消化后取少量细胞200g离心5min后,加入50μl QuickExtractTMDNA Extraction Solution 1.0(Lucigen)按照说明书进行基因组DNA提取,PX459-SIRPα组iPSCs剩余的细胞按1:2的比例传代于六孔板中。提取的基因组DNA用PrimeSTAR Max DNA Polymerase(Takara)按照说明书进行PCR扩增,所用引物序列如下F:AGCAGTGGTGGGTTTGGTTT,R:ACGATGTCCTCCCTGAGACA。扩增后的产物纯化后进行Sanger测序,测序结果显示PX459-SIRPα组iPSCs的SIRPα基因组序列在gRNA序列附近出现错配峰(见图3),证明一部分细胞已经被编辑,可以继续创建克隆细胞系。Day 4,PX459-SIRPα组iPSCs传代的细胞,吸出培养基后,每孔加入2ml新鲜的mTeSR Plus培养基(含0.5μg/ml puromycin)进行药物筛选。Day 5,药筛后的细胞,用Accutase消化成单细胞后,用mTeSR Plus培养基(含10μM Y-27632)重悬至25-50个细胞/ml,取8ml铺于用Matrigel基质胶预包被的100mm细胞培养皿中,放入温度37℃,含5%CO2培养箱中培养,隔天换一次液。铺于100mm细胞培养皿中的单细胞培养10-12天后,长成2-4mm直径的克隆,选分离较好、圆形的iPSCs克隆,在立体显微镜下用5ml注射器针头将iPSCs克隆划成小碎片,再用带滤芯的200μL枪尖将碎片刮下来并吸走,一小部分转移至含有20μl QuickExtractTMDNAExtraction Solution 1.0的1.5ml EP管中,按照说明书进行基因组DNA提取,剩余的全部转移至用Matrigel基质胶预包被的24孔板的一个孔中,孔内预先加入0.5ml mTeSR Plus培养基(含10μM Y-27632)。重复克隆挑选,获得尽可能多的克隆。提取的基因组DNA用前述方法及引物进行PCR扩增,扩增后的产物纯化后进行Sanger测序,对测序结果阳性的克隆株进行扩增。对扩增的阳性株进行蛋白提取,同时提取人单核细胞白血病(THP-1)细胞蛋白作为阳性对照,并用SIRPα抗体(Cell Signaling Technology)进行Western Blot检测并验证了阳性株SIRPα蛋白表达水平,结果证实阳性株SIRPα蛋白水平降低(见图4),iPSCs的SIRPα基因编辑成功,阳性株命名为iPS-SIRPαKO,以下简写为iPS-SKO。
2.在iPS-SKO细胞上过表达CD19 CAR及αPD-1:
1)对iPS-SKO细胞进行慢病毒感染:
将CD19 CAR(hFcGamma)过表达序列通过P2A序列与αPD-1过表达序列连接后,构建CD19 CAR及αPD-1过表达载体,命名为pCDH-EF1-CD19 hFcGamma CAR-P2A-αPD-1(载体结构图见图5,载体序列如SEQ ID NO.2所示),并进行慢病毒包装。
六孔板中培养的iPS-SKO细胞,每孔用1ml DPBS洗一遍,再加入1ml Accutase,放入37℃培养箱中消化5min,然后加入1ml mTeSR Plus培养基(含10μM Y-27632)终止消化,用1ml枪尖吹成单细胞后,200g离心5min,再用mTeSR Plus培养基(含10μM Y-27632)重悬,将5×104-1×105个细胞接种于含2ml mTeSR Plus培养基(含10μM Y-27632)并用Matrigel基质胶预包被的六孔板的一个孔中,放入温度37℃,含5%CO2培养箱中培养24h。24h后,冰上化开慢病毒pCDH-EF1-CD19 hFcGamma CAR-P2A-αPD-1,按照MOI值30计算加入慢病毒的体积,吸去六孔板中的旧培养基,分别加入所需体积的慢病毒,晃匀后加入1ml mTeSR Plus培养基,放入温度37℃,含5%CO2培养箱中培养4-6h,再向六孔板中每孔补加1ml mTeSR Plus培养基,放入温度37℃,含5%CO2培养箱中培养过夜。第二天,吸去六孔板中的旧培养基,每个孔用1-2ml knockout DMEM/F-12洗两遍,接下来按照日常操作对细胞进行换液直到细胞可以进行传代,传代按照iPSCs日常培养操作进行。成功感染慢病毒pCDH-EF1-CD19 hFcGammaCAR-P2A-αPD-1的iPS-SKO细胞命名为iPS-SIRPαKO-CAR-αPD-1,以下简写为iPS-SKOCA。
2)检测iPS-SKOCA细胞CD19 CAR表达情况:
利用Protein L可识别抗体的Kappa轻链,可与含有Kappa可变区的scFv结合而达到检测CAR表达目的的原理,对iPS-SKOCA细胞进行CD19 CAR阳性率检测。将iPS及iPS-SKOCA细胞用Accutase消化后,分别取1×106个细胞重悬于200μl预冷的PBS(含2%BSA及0.02μgBiotin-Protein L(Genscript)),孵育30min。用1ml PBS清洗细胞3次,300g离心5min。200μl PBS重悬后,加入1μl PE Streptavidin(BioLegend),室温孵育30min。用1ml PBS清洗细胞2次,300g离心5min。仔细吸去或倒掉上清。200μl PBS重悬细胞后用流式细胞仪(AgilentNOVOcyte D3000)检测并分析数据,结果显示iPS-SKOCA细胞上CD19 CAR过表达成功(见图6)。
3)检测iPS-SKOCA细胞αPD-1表达情况:
对iPS-SKOCA细胞进行蛋白提取,同时提取iPSCs蛋白作为阴性对照,用His-tag抗体(Cell Signaling Technology)进行Western Blot检测,结果显示iPS-SKOCA细胞胞内有αPD-1蛋白表达(见图7)。为证实iPS-SKOCA细胞过表达的αPD-1蛋白可以分泌到胞外,收集iPS-SKOCA细胞培养上清液,用Microsep Advance离心浓缩管(PALL)浓缩约10倍后,用αPD-1ELISA检测试剂盒(Acro Biosystems)按照说明书对iPS-SKOCA细胞培养上清液中的αPD-1蛋白进行检测。首先用人PD-1蛋白对96孔板进行包被,然后向孔内加入上清浓缩液及生物素标记的αPD-1抗体孵育,清洗后加入链霉亲和素标记的HRP并用TMB进行显色,用酶标仪检测每孔OD值,并根据OD值计算上清浓缩液中αPD-1浓度,结果显示iPS-SKOCA细胞培养上清液中检测到αPD-1蛋白,证实iPS-SKOCA细胞中过表达的αPD-1蛋白可以分泌到胞外(见图8)。
三、对iPSCs进行干性鉴定
用免疫细胞化学方法,检测iPS、iPS-SKO及iPS-SKOCA细胞多潜能标记物SOX2、OCT4、NANOG、SSEA4和TRA-1-60的表达情况,结果显示三种iPSCs均表达上述多潜能标记物(见图9),具体实施步骤如下:
将iPSCs接种于用Matrigel基质胶预包被的八孔板(ibdi)中,放入温度37℃,含5%CO2培养箱中培养。待细胞生长密度合适时,用PBS浸洗3次,每次3min。用4%的多聚甲醛固定细胞15min,PBS浸洗3次,每次3min。0.5%Triton X-100(PBS配制)室温通透20min(细胞膜上表达的抗原省略此步骤)。PBS浸洗3次,每次3min,吸净PBS,在玻片上滴加正常山羊血清,室温封闭30min。吸净封闭液,每孔分别滴加足够量的稀释好的一抗SOX2(Abcam)、OCT4(Abcam)、NANOG(Thermofisher)、SSEA4(Abcam)和TRA-1-60(Abcam),并放入湿盒,4℃孵育过夜。第二天,PBST浸洗八孔板3次,每次3min,吸净孔内多余液体后滴加稀释好的荧光二抗(山羊抗小鼠IgG H&L(Alexa488,Abcam);山羊抗兔IgG H&L(Alexa488,Abcam)),湿盒中20-37℃孵育1h,PBST浸洗3次,每次3min。向孔内滴加适量含抗荧光淬灭剂的DAPI(Beyotime)后,在激光共聚焦显微镜(Leica LCSSP8)下观察采集图像。
四、对iPSCs进行核型鉴定
脐带血CD34+细胞的核型鉴定过程简述如下:秋水仙碱作用150min后离心收集细胞,加入低渗液(5.5g/L KCl)后37℃水浴低渗10min,用固定液(甲醇:冰乙酸=3:1)预固定3min,第一次固定30min,第二次固定20min,用冰片滴片后放入75℃烤箱进行制片,制片后用胰蛋白酶-吉姆萨染液进行显带,显带后通过Leica GSL120自动扫描分析系统进行分析。iPSCs的核型鉴定由北京赛贝生物技术有限公司完成。结果显示iPS、iPS-SKO及iPS-SKOCA细胞仍然保持着与脐带血CD34+细胞相同的46,XX的染色体核型(见图10),核型鉴定每隔一段时间鉴定一次,确保三种iPSCs核型未发生变化。
五、iPSCs诱导分化成巨噬细胞及鉴定
1.iPSCs诱导分化成巨噬细胞:
1)准备试剂:
分化培养基A:X-VIVO 15无血清细胞培养基(Lonza)含100ng/ml M-CSF(PeproTech),25ng/ml IL-3(PeproTech),2mM GlutaMAX(Gibco),1%青链霉素混合液(Solarbio),0.055mM 2-mercaptoethanol(Gibco)。
分化培养基B:RPMI 1640培养基(Gibco),含10%FBS(Gibco,56℃灭活30min),100ng/ml M-CSF。2)拟胚体(embryoidbody,EB)形成:
用Accutase将iPS、iPS-SKO及iPS-SKOCA细胞分别消化成单细胞并计数,用AggreWell plate(STEMCELL Technologies)进行EB形成,此时记为Day 0。Day1,进行75%换液。Day2,进行75%换液。
3)释放巨噬细胞前体:
第三天,小心收集AggreWell plate中的EB球,将EB球转移至40μm细胞网筛上,并用PBS清洗几次,最后转移10-15个EB球到用Matrigel基质胶预包被的六孔板中的一个孔(预先加入2.5ml分化培养基A),铺匀后放入温度37℃,含5%CO2培养箱中培养,4-5天换一次液,培养2-3周,3-4周后EB球开始释放悬浮生长于上清中的巨噬细胞前体。
4)终分化成巨噬细胞:
收集EB板中悬浮生长于上清中的巨噬细胞前体,然后向板中加入新的分化培养基A。收集的细胞200g离心3min用分化培养基B重悬,计数,并调整浓度为2×105个细胞/ml,将细胞以每孔3ml铺于未包被的六孔板中,每隔5天换一次液,培养9-11天后可以进行功能验证。三种iPSCs诱导分化的巨噬细胞分别命名为及EB板中的悬浮细胞每隔3-4天收集一次做终分化,可以持续收集三个月以上。整个分化过程如图11所示。
2.iPSCs诱导分化成巨噬细胞的CD19 CAR表达情况及表面标志物鉴定:
1)流式细胞术检测巨噬细胞CD19 CAR表达情况:
细胞用胰酶消化后,取1×106个细胞重悬于200μl预冷的PBS(含2%BSA及0.02μgBiotin-Protein L(Genscript,M00097)),孵育30min。用1ml PBS清洗细胞3次,300g离心5min。200μl PBS重悬后,加入1μl PE Streptavidin(BioLegend),室温孵育30min。用1ml PBS清洗细胞2次,300g离心5min。仔细吸去或倒掉上清。200μl PBS重悬细胞后用流式细胞仪(BD FACS Arial III)检测并分析数据,结果显示iPS-SKOCA细胞在诱导分化成巨噬细胞后,仍然保持着较高水平的CD19 CAR表达阳性率(见图12)。
2)流式细胞术检测巨噬细胞标志物:
分别取1×106 及细胞,重悬于1ml预冷的BD PharmingenStain Buffer(以下简称Stain Buffer),4℃,300g离心5min,重复2次。加入100μl预冷的Stain Buffer调整细胞终浓度为1×107cells/ml。每管加入5μl Human TruStain FcX(BioLegend),室温孵育5-10min。每管加入适量特异性表面抗体CD11b-FITC(Abcam)、CD14-APC(Invitrogen)、CD68-PE(BioLegend)、CD163-PE(BioLegend)或相应的同型对照抗体,冰上避光孵育20min。用1ml Stain Buffer清洗细胞2次,300g离心5min。仔细吸去或倒掉上清。200μl Stain Buffer重悬细胞后上流式细胞仪(Agilent NOVOcyte D3000)检测并分析数据,结果显示三种巨噬细胞均表达上述几种巨噬细胞标志物(见图13)。
为了验证细胞对携带特异性抗原的肿瘤细胞是否具有高效的吞噬能力,本发明利用三种CD19表达水平不同的淋巴瘤细胞与iPSCs诱导分化成的巨噬细胞共培养后进行体外吞噬实验验证iPSCs诱导分化成的巨噬细胞的吞噬功能。人Burkitt's淋巴瘤细胞Raji细胞本身高表达CD19,人慢性髓原白血病细胞K562细胞本身并不表达CD19,感染CD19过表达慢病毒后,经过药筛,构建出K562细胞的CD19过表达稳转株,命名为K562-CD19,QPCR检测三种淋巴瘤细胞的CD19过表达水平,结果显示K562细胞上成功过表达CD19,Raji细胞CD19表达水平高于K562-CD19细胞(见图14)。将细胞膜红色荧光探针Dil(Beyotime)标记的及细胞铺到八孔板(ibidi)中,2×104细胞/孔。24小时后,将细胞增殖示踪绿色荧光探针CFDA SE(Beyotime)标记的K562、K562-CD19及Raji细胞与标记好的及按照1:5的比例铺到上述八孔板中。两者共孵育24小时后,PBS洗去未被吞噬的淋巴瘤细胞,激光共聚焦显微镜(Leica LCSSP8)观察吞噬淋巴瘤的巨噬细胞的比例。结果显示对于K562细胞,及的吞噬效率均明显高于而和的吞噬效率无明显差异;对于K562-CD19细胞,及的吞噬效率均显著高于且的吞噬效率也高于对于Raji细胞,及的吞噬效率均显著高于且的吞噬效率也显著高于(见图15),说明针对表达CD19抗原的肿瘤细胞,细胞具有特异性高效杀伤能力。同时用流式细胞术进行体外吞噬实验,将CellTracker Deep Red Dye(Thermo Fisher)标记的及细胞铺到六孔板中,3×105细胞/孔。24小时后,将CellTrackerCMFDA Dye(Thermo Fisher)标记的K562、K562-CD19及Raji细胞与标记好的及细胞按照1:5的比例铺到上述六孔板中。两者共孵育24小时后,收集贴壁和悬浮细胞,离心后PBS重悬,流式细胞仪分析发生吞噬的巨噬细胞的比例。结果同样显示对表达CD19抗原的肿瘤细胞,细胞具有特异性高效杀伤能力(见图16)。
综上,本发明实施例提供了一种多重基因编辑的通用型巨噬细胞的制备方法(见图17),并证明该多重基因编辑的通用型巨噬细胞具有肿瘤特异性高效杀伤能力,因而具有相关肿瘤免疫治疗特别是细胞和基因治疗的巨大潜力和广阔应用前景。
实施例中pCDH-EF1-CD19-hFcGamma CAR-P2A-αPD-1载体序列:
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种多重基因编辑的通用型巨噬细胞,其特征在于,所述巨噬细胞由iPSCs诱导分化而来,细胞表面分子SIRPα表达抑制,细胞表面具有嵌合抗原受体CAR修饰,细胞分泌表达抗PD-1抗体的单链可变片段αPD-1。
2.如权利要求1所述多重基因编辑的通用型巨噬细胞,其特征在于,CAR序列及αPD-1序列通过酰胺键直接连接,或通过连接子连接,所述连接子选自P2A,T2A,E2A或F2A。
3.如权利要求2所述多重基因编辑的通用型巨噬细胞,其特征在于,所述CAR与αPD-1通过P2A进行连接;
其中,所述CAR为靶向任意一个或多个肿瘤靶点的嵌合抗原受体,所述肿瘤靶点包括CD19、BCMA、CD20、CD22、CD123、CD30、TAA、HER2、EGFR、GPC3、MUC1、PSMA;进一步选自CD19。
4.权利要求1-3任一项所述多重基因编辑的通用型巨噬细胞构建方法,其特征在于,所述构建方法包括以下步骤:获取脐带血CD34+细胞重编程为iPSCs,对所述iPSCs表面分子SIRPα进行敲除得到iPS-Sko细胞,同时将αPD-1基因序列及特异性CAR基因序列整合到iPS-Sko细胞中得到iPS-SkoCA细胞,再对iPS-SkoCA细胞进行分化诱导得到所述多重基因编辑的通用型巨噬细胞。
5.如权利要求4所述多重基因编辑的通用型巨噬细胞构建方法,其特征在于,脐带血CD34+细胞重编程为iPSCs细胞采用电转诱导方式,具体的操作方式如下:获取脐带血中的CD34+细胞进行扩增培养,将扩增后的CD34+细胞加入电转杯中处理,电转处理后的细胞铺入基质胶中进行扩增培养一段时间,筛选其中的阳性克隆得到所述iPSCs。
6.如权利要求4所述多重基因编辑的通用型巨噬细胞构建方法,其特征在于,所述细胞表面分子SIRPα表达抑制通过CRISPR/Cas9系统进行编辑,所述SIRPα的CRISPR gRNA序列为:GGTAGAGGGCCGCCATCAGT(SEQ IDNO.1);
或,所述αPD-1基因序列及特异性CAR基因序列可通过慢病毒感染整合到iPS-Sko细胞中;
具体的,将CD19 CAR过表达序列通过P2A序列与αPD-1过表达序列连接后,构建CD19CAR及αPD-1的过表达载体,命名为pCDH-EF1-CD19 hFcGamma CAR-P2A-αPD-1,其核苷酸序列如SEQ ID NO.2所示,并进行慢病毒包装,进而转染iPS-Sko细胞;
或,所述分化诱导的具体步骤如下:将iPS-SkoCA细胞加入拟胚体培养基中获得拟胚体,将拟胚体加入分化培养基A进行培养获得巨噬细胞前体,收集所述巨噬细胞前体加入分化培养基B中获得巨噬细胞。
7.如权利要求6所述多重基因编辑的通用型巨噬细胞构建方法,其特征在于,所述分化培养基A采用无血清培养基,所述培养基中包括80~120ng/mL M-CSF,20~30ng/mL IL-3,1~3mM GlutaMAX,0.8~1.2%青链霉素混合液,0.055mM2-mercaptoethanol;
或,所述分化培养B采用1640培养基,所述培养基中包括8~12%FBS、80~120ng/mL M-CSF。
8.一种药物组合物,其特征在于,所述药物组合物中包括活性剂量的权利要求1-3任一项所述多重基因编辑的通用型巨噬细胞;
所述药物组合物中,还包括药学上所必须的载体,所述药学上可接受的载体剂量应当对受试者是无害的,具体为包括缓冲剂、抗氧化剂、防腐剂、杀菌剂、蛋白质、亲水性聚合物、氨基酸、单糖、二糖和其它碳水化合物、螯合剂、张力调节剂、糖类、表面活性剂、成盐抗衡离子、金属复合物和/或非离子表面活性剂。
9.权利要求1-3任一项所述多重基因编辑的通用型巨噬细胞、权利要求8所述药物组合物在制备抗肿瘤药物中的应用;
所述肿瘤为包括肺癌、黑色素瘤、胶质瘤、胃癌、卵巢癌、结肠癌、肝癌、肾癌、膀胱癌、乳腺癌、经典霍奇金淋巴瘤、血液肿瘤、淋巴癌中的一种。
10.一种抗肿瘤药物,其特征在于,所述药物中包括活性剂量的权利要求1-3任一项所述多重基因编辑的通用型巨噬细胞或权利要求8所述药物组合物;
所述抗肿瘤药物的剂型为液体制剂,或为注射剂。
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