CN114129718A - 一种用于体内自组装car-t的纳米递送系统及其制备方法和应用 - Google Patents
一种用于体内自组装car-t的纳米递送系统及其制备方法和应用 Download PDFInfo
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Abstract
本发明提供了一种用于体内自组装CAR‑T的纳米递送系统及其制备方法和应用。该系统包含脂质膜和由所述脂质膜包裹的带有CAR的质粒;所述脂质膜包含DlinMC3、DOPE、Chol、PEG‑DMG、DOTAP和DSPE‑MTAS‑NLS;所述用于体内自组装CAR‑T的纳米递送系统的外表面修饰有DSPE‑PEG‑CD3。本发明利用此纳米递送系统在体内直接产生CAR‑T细胞,为CAR‑T疗法提供了一种新颖、便捷、通用的实施方案。
Description
技术领域
本发明属于生物医药领域,具体涉及一种用于体内自组装CAR-T的纳米递送系统及其制备方法和应用。
背景技术
目前嵌合抗原受体T细胞(CAR-T)疗法是肿瘤免疫细胞治疗领域备受关注的热点。CAR-T主要是通过将一种人体自身的免疫细胞-T淋巴细胞,进行基因工程改造,使其表面表达特定的嵌合抗原受体(CAR)而形成的。CAR可识别肿瘤细胞表面的抗原,从而激活并介导CAR-T细胞对肿瘤细胞发挥特异性杀伤作用。与天然T细胞相比,CAR的存在还使CAR-T细胞不受人类主要组织相容性复合体(MHC)限制性的影响,从而能识别更广泛的目标,大大提高了T细胞攻击肿瘤细胞的效率和强度。目前使用最广泛的仍是第二代CAR,其结构包括一个肿瘤相关抗原(tumor-associated antigen,TAA)识别区(通常来源于单克隆抗体抗原结合区域的段),一个胞外铰链区,一个跨膜区和一个胞内信号区(一般为CD3ζ信号域加一个新的共刺激信号CD28或41BB)。目前临床研究最多的是能够识别B-ALL肿瘤细胞表面CD19抗原的CAR-T细胞(CD19 CAR-T),其治疗B-ALL的临床完全缓解(CR)率普遍高达70%-94%。
尽管如此,CAR-T技术的临床应用目前仍受到很多因素制约,其中最重要的是两个问题:(1)CAR-T需要复杂的体外制备过程和严格的质量管控;不同于传统药物,CAR-T是一种基于整合了CAR基因的自体T细胞疗法,这意味着CAR-T必须在尽可能短的时间内完成血样采集、分离、激活、转染、扩增、检测、制剂、运输和给药这一系列复杂流程,且CAR-T细胞的体外制备过程需要严格的GMP洁净度要求,需要高价投入,整个过程需要3周左右,花费时间较长;且每位患者的CAR-T治疗需要个人定制,无法量产,因此目前只能在世界各地的少数几个研究中心或特定实验室进行。这一问题也导致了CAR-T疗法的高昂价格,严重制约着CAR-T的临床应用和进一步推广,因此如何发展CAR-T细胞制备新技术,避免其复杂的体外制备流程和严格的质量管控,降低CAR-T疗法的技术壁垒,是细胞免疫治疗研究者需要解决的一大难题。(2)细胞因子释放综合征(CRS)问题。有研究表明CAR-T治疗中约有60-80%的患者会出现细胞因子释放综合症(Cytokine Release Syndrome,CRS)。其主要发生机制是CAR-T细胞在患者体内与肿瘤细胞抗原识别后被激活,不断扩增并释放大量细胞因子,从而引起全身性的炎症反应,其临床常表现为:持续高热、低血压、寒颤、以及一系列与细胞因子水平升高有关的神经系统症状等。严重的CRS反应会给患者带来死亡的危险。
发明内容
本发明要解决的技术问题之一是针对传统CAR-T疗法中体外制备存在的生产工艺复杂、周期长、质量控制要求严苛、成本高等一系列问题,本发明通过制备一种用于体内自组装CAR-T的纳米递送系统使得CAR-T的最终合成在体内完成,从而避免了上述一系列问题。
本发明要解决的技术问题之二是针对目前CAR-T治疗过程中普遍存在的严重的CRS问题,对传统CAR-T制备过程中所用的抗CD19-CAR等质粒DNA进行优化设计,添加沉默IL-6表达的shRNA片段,在保证CAR-T细胞对肿瘤细胞强大的杀伤效果前提下,沉默IL-6基因表达,减少CAR-T细胞IL-6分泌,在CAR-T细胞杀伤肿瘤细胞时避免引发严重的CRS副作用,提高这一实施方案的安全性。
一方面,为了解决上述问题中的至少一个,本发明具体提供了一种用于体内自组装CAR-T的纳米递送系统,包含脂质膜和由所述脂质膜包裹的带有CAR的质粒;所述脂质膜包含DlinMC3、DOPE、Chol、PEG-DMG、DOTAP和DSPE-MTAS-NLS;所述用于体内自组装CAR-T的纳米递送系统的外表面修饰有DSPE-PEG-CD3。
在一些实施方案中,所述带有CAR的质粒包含IL-6shRNA。
在一些实施方案中,所述IL-6shRNA的核酸序列如SEQ ID NO.1所示。
进一步地,所述带有CAR的质粒为PUT523。
在一些实施方案中,所述脂质膜中DlinMC3、DOPE、Chol、PEG-DMG、DOTAP和DSPE-MTAS-NLS的摩尔比为(20-35):(10-15):(20-38):(1-2):(10-20):3。在一个实施方案中,所述脂质膜中DlinMC3、DOPE、Chol、PEG-DMG、DOTAP和DSPE-MTAS-NLS的摩尔比为(20-30):10:(30-38):(1-2):(15-20):3。在另一个实施方案中,所述脂质膜中DlinMC3、DOPE、Chol、PEG-DMG、DOTAP和DSPE-MTAS-NLS的摩尔比为(30-35):(10-15):(20-38):(1.5-2):(10-15):3。
在一些实施方案中,所述带有CAR的质粒包含CD8 leader、抗靶蛋白的抗体相关区域、CD8 Hinge&TM、共刺激分子、CD3ζ;所述抗靶蛋白的抗体相关区域选自全抗体、抗体的F(ab)2段、scFv中的一种或多种;所述共刺激分子选自CD28、4-1BB、OX40中的一种或多种。
进一步地,CD8 leader的氨基酸序列如SEQ ID NO.4所示,CD8 leader的核酸序列如SEQ ID NO.5所示。CD8 Hinge&TM的氨基酸序列如SEQ ID NO.8所示,CD8Hinge&TM的核酸序列如SEQ ID NO.9所示。CD3ζ的氨基酸序列如SEQ ID NO.12所示,CD3ζ的核酸序列如SEQID NO.13所示。
进一步地,4-1BB的氨基酸序列如SEQ ID NO.10所示,4-1BB的核酸序列如SEQ IDNO.11所示。
在一些实施方案中,所述带有CAR的质粒的CAR的靶蛋白选自CD19、BCMA、CD20、CD22、CD138中的一种或者多种。
在一些实施方案中,所述靶蛋白为CD19;所述带有CAR的质粒的核酸序列如SEQ IDNO.2所示。进一步地,抗靶蛋白的抗体相关区域为CD19 scfv。CD19scfv的氨基酸序列如SEQID NO.6所示,CD19 scfv的核酸序列如SEQ ID NO.7所示。
在一些实施方案中,还包含乙酸钠溶液;所述带有CAR的质粒为溶质溶于所述乙酸钠溶液。
另一方面,本发明还提供了一种如上所述的用于体内自组装CAR-T的纳米递送系统的制备方法,包括以下步骤:
S1、所述带有CAR的质粒的构建;
S2、DSPE-MTAS-NLS的合成;
S3、制备脂质体悬液:称取DlinMC3、DOPE、Chol、PEG-DMG、DOTAP、DSPE-MTAS-NLS作为溶质溶于三氯甲烷作为油相溶液,用蒸发仪除去三氯甲烷,得到脂质膜;以乙酸钠溶液作为水相溶剂,溶解所述步骤S1得到的带有CAR的质粒作为水相溶液,使得所述带有CAR的质粒与所述油相溶液的溶质的质量比为1:(5-20);在所述脂质膜中加入所述水相溶液进行水化得到脂质体,使用微型挤出器将所述脂质体依次挤压通过核孔膜,得到脂质体悬液;
S4、DSPE-PEG-CD3的合成,具体为:将CD3抗体溶于NaHCO3溶液中,取DSPE-PEG-NHS溶解于DMSO,将DSPE-PEG-NHS滴加至CD3抗体溶液中,搅拌反应,透析,冷冻干燥,得到所述DSPE-PEG-CD3;
S5、将所述步骤S3得到的脂质体悬液装入超滤离心管中,加入PBS溶液,离心,将所述脂质体悬液的外液替换为PBS;然后加入所述步骤S4得到的DSPE-PEG-CD3,使得所述DSPE-PEG-CD3与所述带有CAR的质粒的摩尔比为1:(10-20),4℃±2℃过夜,收集悬液得到修饰有CD3的脂质体悬液。
在一些实施方案中,所述步骤S2具体为:将核定位肽Cys-MTAS-NLS溶于缓冲液中得到溶液一;取Mal-PEG6-DSPE溶于二甲基甲酰胺得到溶液二;将所述溶液一与所述溶液二加到磁力搅拌器中反应得到反应液;取出所述反应液,将过量的多肽和二甲基甲酰胺通过透析除去,再冷冻干燥得到所述DSPE-MTAS-NLS;所述透析的截止分子量为3.5kDa-8kDa。
在一些实施方案中,所述步骤S1得到的带有CAR的质粒包含IL-6shRNA。
第三方面,本发明还提供了如上所述的用于体内自组装CAR-T的纳米递送系统在制备治疗肿瘤的药物中的应用。
在一个实施方案中,所述肿瘤选自乳腺癌、结肠直肠癌、肺癌、胰腺癌、食管癌、子宫内膜癌、卵巢癌、胃癌、前列腺癌、肾癌、宫颈癌、骨髓瘤、淋巴瘤、白血病、甲状腺癌、子宫癌、膀胱癌、神经内分泌癌、头颈癌、肝癌、鼻咽癌、睾丸癌、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、皮肤纤维肉瘤突出症、梅克尔细胞癌、胶质母细胞瘤、神经胶质瘤、肉瘤、间皮瘤或骨髓发育不良综合征等血液肿瘤和实体瘤中的一种或多种。
第四方面,本发明还提供一种试剂盒,其包含如上所述的用于体内自组装CAR-T的纳米递送系统。
如本文所用,术语“重组蛋白”是指人工设计/构建的蛋白,而不是天然存在的蛋白质。本发明的“重组蛋白”中的“重组”不代表其生产方式,其仅用于表示“重组蛋白”并不天然存在。本发明的重组蛋白可以是表达的蛋白,可以是组装的蛋白。
如本文中所用,术语“抗体”通常是指包含一个或多个基本上由免疫球蛋白基因或免疫球蛋白基因片段编码的多肽的蛋白质。免疫球蛋白基因可以包括κ、λ、α、γ、δ、ε和μ恒定区基因,以及无数的免疫球蛋白可变区基因。如本文所用,轻链可被分类为κ或λ。重链可被分类为γ、μ、α、δ或ε,其依次分别定义免疫球蛋白类别:IgG、IgM、IgA、IgD和IgE。本申请中使用的抗体可具有包含四聚体的结构单元。每个四聚体可由两对相同的多肽链组成,每对具有一条“轻”链(约25kD)和一条“重链”(约50-70kD)。每个成员的N末端可以界定约100至110个或更多个氨基酸的可变区,其主要负责抗原识别。如本文中所用,术语轻链可变区(VL)和重链可变区(VH)通常分别指轻链和重链的这些区域。抗体可作为完整免疫球蛋白存在或作为通过用各种肽酶消化或从头表达产生的许多充分表征的片段存在。因此,例如,胃蛋白酶可以消化铰链区中二硫键以下的抗体以产生F(ab)2段(Fab的二聚体,所述Fab本身是通过二硫键连接至VH-CH1的轻链)。F(ab)2段可在温和条件下被还原以断裂铰链区中的二硫键,从而将(Fab)2段二聚体转化为Fab单体。Fab单体基本上是具有铰链区的部分的Fab(关于其它抗体片段的更详细描述,参见Fundamental Immunology,W.E.Paul编辑,RavenPress,N.Y.(1993))。虽然根据完整抗体的消化来定义各种抗体片段,但是本领域普通技术人员将理解,可以化学地或通过利用重组DNA方法从头合成这样的Fab片段。因此,如本文中所用,术语抗体还可包括通过修饰整个抗体或使用重组DNA方法从头合成产生的抗体片段,包括但不限于F(ab)2段、IgG、IgM、IgA、IgE、scFv、dAb、纳米抗体、单抗体和双链抗体。在一些实施方案中,抗体包括但不限于F(ab)2段、IgG、IgM、IgA、IgE和单链抗体,例如单链Fv(scFv)抗体,其中重链可变区和轻链可变区(直接地或通过肽接头)连接在一起以形成连续的多肽。
如本文所用,术语“Fc区”(fragment crystallizable,Fc)由IgG恒定区CH2和CH3结构域及铰链区组成。
如本文所用,术语“抗原结合片段”或“fab段”或“fab”由轻链的可变区(VL)、轻链的恒定区(CL)、重链的可变区(VH)、重链的恒定区1(CH1)结构域组成,可与抗原结合。
术语“抗原特异性”是指被抗原结合分子选择性地识别的特定抗原或其表位。
如本文所用,术语“取代”在用于氨基酸残基时是指在肽、多肽或蛋白中将天然存在的或引入的一个或多个氨基酸用另一个取代,形成新的肽、多肽或蛋白(如本文术语“突变体”或“突变型”)。多肽或蛋白中的取代可导致多肽或蛋白功能的减弱或不变。取代还可以是“保守取代”,其针对氨基酸序列时是指将一个氨基酸残基用另一个具有相似理化性质的侧链的不同氨基酸残基取代,或者对那些对多肽的活性并非至关重要的氨基酸的取代。例如,可以在非极性侧链氨基酸残基间(例如Met、Ala、Val、Leu和Ile、Pro、Phe、Trp)、不带电的极性侧链残基间(例如Cys、Ser、Thr、Asn、Gly和Gln)、酸性侧链残基间(例如Asp、Glu)、碱性侧链氨基酸间(例如His、Lys和Arg)、β分支侧链氨基酸间(例如Thr、Val和Ile)、含硫侧链氨基酸间(例如Cys和Met)或芳香侧链残基间(例如Trp、Tyr、His和Phe)进行保守取代。在某些实施方式中,取代、删除或添加也可以认为是“保守取代”。插入或删除的氨基酸的数量的范围可以为约1至5。保守取代通常不引起蛋白构象结构上的显著变化,并且因此能保持蛋白的生物学活性。
如本文所使用的术语“宿主细胞”通常包括可以是或已经是受试者质粒或载体的接受者的单个细胞、细胞系或细胞培养物,其包含本申请公开的多核苷酸,或表达本申请的蛋白质异二聚体(例如,异二聚体蛋白)。宿主细胞可以包括单个宿主细胞的后代。由于天然、偶然或有意的突变,后代可以不一定与原始亲本细胞完全相同(在形态上或在基因组总DNA互补体上)。宿主细胞可包括用本申请公开的载体在体外转染的细胞。宿主细胞可以是细菌细胞(例如大肠杆菌(E.coli))、酵母细胞或其它真核细胞,例如HEK293细胞、COS细胞、中国仓鼠卵巢(CHO)细胞、HeLa细胞或骨髓瘤细胞。在一些实施方案中,宿主细胞是哺乳动物细胞。在一些实施方案中,所述哺乳动物细胞是CHO细胞。
如在本文中所用,术语“载体”通常是指能够在合适的宿主中自我复制的核酸分子,其将插入的核酸分子转移至宿主细胞中和/或在宿主细胞之间转移。该术语可包括主要用于将DNA或RNA插入细胞的载体,主要用于DNA或RNA的复制的载体,以及用于DNA或RNA的转录和/或翻译的表达载体。还包括提供不止一种上述功能的载体。“表达载体”是当被引入合适的宿主细胞时可被转录并翻译成多肽的多核苷酸。“表达系统”通常意味着合适的宿主细胞,其包含能够产生期望的表达产量的表达载体。
术语“有效量”或“治疗有效量”是指足以实现预期应用(包括但不限于疾病治疗)的组合物(例如,本文所述的双特异性重组蛋白)的量。治疗有效量可根据预期的应用(例如,体外或体内)或所治疗的受试者和疾病状况,例如受试者的体重和年龄、疾病状况的严重性、施用方式等而变化,其可以容易地由本领域普通技术人员来确定。该术语还可应用于在靶细胞中诱导特定应答(例如,靶基因诱导、增殖和/或凋亡)的剂量。具体剂量将根据所选择的具体化合物、所遵循的给药方案、其是否与其它化合物组合施用、施用的时间安排、其所施用至的组织以及其所处的物理递送系统而变化。
术语“治疗”或“医治”或“缓解”或“改善”在本文中可互换使用,并且是指获得有益或所需的结果(包括但不限于治疗益处和/或预防益处)的方法。如本文中所用,治疗益处通常是指根除或减轻所治疗的潜在病症的严重性。此外,通过根除、减轻严重性或减少与潜在病症相关的一种或多种生理症状的发生率,以使得在受试者中观察到改善(尽管受试者仍然可能受到潜在病症折磨)来实现治疗益处。对于预防益处,可向处于发展特定疾病的风险中的受试者,或报告疾病的一种或多种生理症状的受试者施用组合物,即使可能尚未进行该疾病的诊断。
如本文中所用,术语“治疗作用”通常包括如上所述的治疗益处和/或预防益处。预防作用包括延迟或消除疾病或病况的出现,延迟或消除疾病或病况的症状的发作,减缓、停止或逆转疾病或病况的进展,或其任何组合。
如本文中所用,术语“共施用”、“与……组合施用”及其语法上等同物通常包括指向动物施用两种或更多种试剂,以使得试剂和/或其代谢物同时存在于受试者中。共施用包括在单独的组合物中同时施用,在单独的组合物中在不同时间施用,或在其中两种试剂均存在的组合物中施用。
如本文中所用,术语“细胞增殖”通常是指细胞数目由于分裂而改变的现象。例如,细胞增殖可导致细胞数目的增加。该术语还包括细胞形态通过其已发生改变(例如,尺寸增加)的细胞生长,其与增殖信号一致。
如本文中所用,术语“增殖抑制”或“抑制细胞增殖”通常是指癌细胞的生长速率和/或增殖速率的降低。例如,这可包括癌细胞的死亡(例如通过凋亡)。在一些实施方案中,该术语还可指抑制实体瘤的生长和/或增殖和/或诱导肿瘤的尺寸减小或消除。
本申请使用的术语“受试者”或“个体”或“动物”或“患者”是指需要诊断、预后、缓解、预防和/或治疗疾病或病症的人或非人动物,包含哺乳动物或灵长类。哺乳类受试者包含人、畜养动物、农场动物,以及动物园、体育或宠物动物,如狗、猫、豚鼠、兔、大鼠、小鼠、马、猪、牛、熊等等。
如本文中所用,术语“体内”通常是指在受试者体内发生的事件。
如本文中所用,术语“体外”通常是指发生在受试者体外的事件。例如,体外测定包括在受试者外进行的任何测定。体外测定包括其中使用死细胞或活细胞的基于细胞的测定。体外测定还包括其中不使用完整细胞的无细胞测定。
如本文所用,术语“施用”是指将治疗有效量的包含本发明的重组蛋白或融合蛋白的药物组合物递送至受试者。施用可以全身施用也可以局部施用。施用可以通过施用装置进行,例如注射器。施用方式包括但不限于包埋、鼻吸、喷雾、注射等。施用途径包括吸入、鼻内、口服、静脉内、皮下或肌内施用等。
本发明首先设计并构建IL-6shRNA和CD19-CAR组合基因质粒;接着合成靶向脂质分子并表征,制备了纳米递送系统(比如CD3-NP/IL-6)并进行结构表征;然后进行体外细胞水平的功能验证,以探究其T细胞靶向性、转导成为IL-6敲减的CAR-T细胞的能力,并评价CAR-T细胞对肿瘤细胞的杀伤效果。最后构建小鼠血液肿瘤模型,评估纳米递送系统(以CD3-NP/IL-6为例)的体内效果,主要评价指标有小鼠外周血CAR-T定量以及IL-6表达、肿瘤生物荧光强度、小鼠生存期等。
与现有技术相比,本发明具有如下有益效果:
(1)利用此纳米递送系统在体内直接产生CAR-T细胞。目前只能通过在体外利用慢病毒通过基因工程将T细胞改造成CAR-T细胞,该方法存在生产工艺复杂、成本高、质量控制要求严格、不能量产等一系列问题。为此,本发明提出制备一种CD3抗体修饰的包载IL-6shRNA和抗靶蛋白的CAR(以CD19-CAR为例)组合基因质粒的纳米递送系统,其静脉注射后可在CD3抗体介导作用下主动寻靶T细胞,在体内直接将T细胞改造成CAR-T细胞,为CAR-T疗法提供了一种新颖、便捷、通用的实施方案。
(2)敲减CAR-T细胞的IL-6分泌功能从而降低CRS反应。当前CAR-T疗法存在的问题之二是因IL-6分泌而导致严重的CRS反应。针对该问题,本发明通过在CAR质粒中添加IL-6基因沉默元件,即构建IL-6shRNA和抗靶蛋白的CAR(以CD19-CAR为例)组合基因质粒,将其包裹在CD3抗体修饰的纳米递送系统中,使其在体内产生IL-6分泌功能缺失的CAR-T细胞,从而降低IL-6引起严重CRS的风险,提高CAR-T疗法的安全性。
(3)动物试验的结果显示经过本发明所涉及的用于体内自组装CAR-T的纳米递送系统治疗的肿瘤模型小鼠的主要脏器未发生病变,表明本发明所涉及的用于体内自组装CAR-T的纳米递送系统本身具有良好的生物安全性。为其开展临床研究提供了有利支持。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明,以充分地了解本发明的目的、特征和效果。
附图说明
图1是IL-6shRNA和CD19-CAR组合基因质粒结构示意图。
图2是IL-6shRNA和CD19-CAR组合基因质粒的大小及内切酶鉴定结果。
图3是DSPE-PEG6-Mal和DSPE-MTAS-NLS的H1-NMR图谱。
图4是DSPE-PEG6-Mal和DSPE-MTAS-NLS的红外图谱。
图5是DSPE-PEG2000-CD3和CD3的分子量的MOLDI-TOF-MS图。
图6是实施例4中CD3-NP/IL-6的粒径和Zeta电位图。
图7是实施例4中CD3-NP/IL-6转染HCT-116细胞的电子显微镜图片。左为转染后明场(白光)下的细胞形态,右为转染后同一视野下的细胞内GFP蛋白表达的荧光图(质粒上带有GFP绿色荧光)。
图8是实施例5中CD3-NP/IL-6的粒径和Zeta电位图。
图9是实施例5中CD3-NP/IL-6转染HCT-116细胞的电子显微镜图片。左为转染后明场(白光)下的细胞形态,右为转染后同一视野下的细胞内GFP蛋白表达的荧光图(质粒上带有GFP绿色荧光)。
图10是实施例6中CD3-NP/IL-6的粒径和Zeta电位图。
图11是实施例6中CD3-NP/IL-6转染HCT-116细胞的电子显微镜图片。左为转染后明场(白光)下的细胞形态,右为转染后同一视野下的细胞内GFP蛋白表达的荧光图(质粒上带有GFP绿色荧光)。
图12是实施例7中CD3-NP/IL-6的粒径和Zeta电位图。
图13是实施例8中CD3-NP/IL-6转染HCT-116细胞的电子显微镜图片。左为转染后明场(白光)下的细胞形态,右为转染后同一视野下的细胞内GFP蛋白表达的荧光图(质粒上带有GFP绿色荧光)。
图14是对实施例9得到的CD3-NP/IL-6的表征。其中,A:NP/IL-6和CD3-NP/IL-6的电镜图;B:粒径分布图;C:统计粒径和电位。
图15是CD3-NP/IL-6纳米粒被T淋巴细胞摄取图。
图16是CD3-NP/IL-6纳米粒转导T细胞的效率。其中,A:NP/IL-6的流式细胞散点图;B:CD3-NP/IL-6的流式细胞散点图;C:转导效率柱状图。
图17是CD3-NP/IL-6纳米粒转导T细胞后对CD19高表达的K562靶细胞、Raji靶细胞在不同效靶比下的杀伤能力。
图18是纳米粒在体外转导T细胞后对IL-6基因的沉默效果,靶细胞为表达CD19的K562细胞和Raji细胞。
图19是NP/IL-6和CD3-NP/IL-6的体内组织分布,左为NP/IL-6,右为CD3-NP/IL-6。
图20是CD3-NP/IL-6在荷瘤小鼠体内产生CAR-T细胞的数目。
图21是CD3-NP/IL-6产生CAR-T的体内肿瘤杀伤能力荧光图。
图22是CD3-NP/IL-6产生CAR-T的体内肿瘤杀伤能力。
图23是CD3-NP/IL-6在体内产生CAR-T的小鼠生存期。
图24是CD3-NP/IL-6在体内产生CAR-T的体内IL-6蛋白释放情况。
图25是给药后外周血中IL-4、IL-2、IFN-γ、IL-10、IL-17α、TNF-α的含量。
图26是CD3-NP/IL-6与NP/IL-6的主要脏器的HE染色切片图。
具体实施方式
为了使发明实现的技术手段、创造特征、达成目的和功效易于明白了解,下结合具体图示,进一步阐述本发明。但本发明不仅限于以下实施的案例。
实施例1 IL-6shRNA和CD19-CAR组合基因质粒的构建
通过Genebank(NCBI)查找IL-6基因的CDS区基因序列,将基因序列导入siRNASelection Program网站搜索有效靶点序列,利用电脑软件设计shRNA序列。通过ApaLI、PvuII、XhoI内切酶将IL-6shRNA序列(核酸序列如SEQ ID NO.1所示)插入基本载体质粒PUT523中,得到IL-6shRNA和CD19-CAR组合基因质粒(核酸序列如SEQ ID NO.2所示),如图1所示。IL-6shRNA和CD19-CAR组合基因质粒通过ApaLI、PvuII、XhoI内切酶酶切,分别得到2个片段(大小依次为3860和2454、608和5706、1981和4333,说明质粒构建成功),如图2所示。
实施例2 DSPE-MTAS-NLS的合成与表征
将10mg核定位肽Cys-MTAS-NLS(氨基酸序列如SEQ ID NO.3所示)溶于0.5ml的1XPBS溶液中(pH7.0),取15.8mg的Mal-PEG6-DSPE溶于1ml的二甲基甲酰胺(DMF),两者混合后加到磁力搅拌器中反应,反应完全后取出反应液,将过量的多肽和DMF通过超纯水透析2天(截止分子量3.5kDa)除去。透析3天之后进行冷冻干燥,得到目标化合物DSPE-MTAS-NLS。
采用H1-核磁共振(H1-NMR)对DSPE-PEG-MTAS-NLS进行表征,结果显示(图3)DSPE-PEG6-Mal中的Mal在6.7ppm有峰(见图3箭头处),与核定位肽MTAS-NLS结合之后,6.7ppm处的峰消失,显示合成成功。
采用红外光谱分析(图4),结果DSPE-PEG6-Mal红外图谱上显示出较弱的N-H(3200-3600cm-1)和C=O(1666.8cm-1)伸缩振动峰,而在DSPE-MTAS-NLS图谱上,这两个峰的信号强度均有显著增强,而在核定位肽MTAS-NLS的结构中含有大量N-H和C=O结构,表明核定位肽已与DSPE-PEG6-Mal发生成功连接。
实施例3 DSPE-PEG-CD3靶向脂质分子的合成与表征
将CD3抗体溶于1ml的NaHCO3溶液(pH=8)中,取4mol的DSPE-PEG2000-NHS溶解于DMSO,将DSPE-PEG2000-NHS缓慢滴加至CD3抗体溶液中,搅拌4小时,反应完全后通过超纯水透析1天(截止分子量3.5kDa),进行冷冻干燥,得到目标化合物。
采用电离飞行时间质谱(MALDI-TOF-MS)技术检测DSPE-PEG2000-CD3和CD3的分子量,结果显示(图5)CD3抗体的平均分子量为152258.DSPE-PEG2000-CD3的分子量为154482。
实施例4 CD3-NP/IL-6的制备之一
用分析天平精密称取DlinMC3、DOPE、Chol、PEG-DMG、DOTAP、DSPE-MTAS-NLS(摩尔比为20:10:30:1:20:3)以上材料溶于2ml的三氯甲烷作为脂类油相溶液,用旋转蒸发仪逐渐除去三氯甲烷,得到均匀的膜。配制pH4,浓度为25mM的乙酸钠溶液作为水相溶剂,溶解标记GFP的质粒作为水相溶液,质粒与磷脂材料的质量比为1:10。在除去三氯甲烷的脂质薄膜中加入配好的水相溶液进行水化1小时,使用微型挤出器将脂质体依次挤压通过400、200的核孔膜,得到NP/IL-6的悬液。将NP/IL-6的悬液装入超滤离心管中(Mw=10000),加入4倍量体积的PBS溶液,并离心1h,将NP/IL-6外液替换为PBS。随后将DSPE-PEG2000-CD3(与质粒的摩尔比为1:10)加入以上溶液中,4℃过夜,收集悬液得到CD3-NP/IL-6,置于4℃冰箱保存。
对本实施例制备得到的CD3-NP/IL-6进行表征,结果(见图6和7):CD3-NP/IL-6的粒径为212nm,PDI为0.227,Zeta电位为-0.073mV;CD3-NP/IL-6转染HCT-116细胞的效率为40%,表明CD3-NP/IL-6能够成功递送至细胞。
实施例5 CD3-NP/IL-6的制备之二
用分析天平精密称取DlinMC3、DOPE、Chol、PEG-DMG、DOTAP、DSPE-MTAS-NLS(摩尔比为35:15:20:1.5:10:3)以上材料溶于2ml的三氯甲烷作为脂类油相溶液,用旋转蒸发仪逐渐除去三氯甲烷,得到均匀的膜。配制pH4,浓度为25mM的乙酸钠溶液作为水相溶剂,溶解标记GFP的质粒作为水相溶液,质粒与磷脂材料的质量比为1:10。在除去三氯甲烷的脂质薄膜中加入配好的水相溶液进行水化1小时,使用微型挤出器将脂质体依次挤压通过400、200的核孔膜,得到NP/IL-6的悬液。将NP/IL-6的悬液装入超滤离心管中(Mw=10000),加入4倍量体积的PBS溶液,并离心1h,将NP/IL-6外液替换为PBS。随后将DSPE-PEG2000-CD3(与质粒的摩尔比为1:10)加入以上溶液中,4℃过夜,收集悬液得到CD3-NP/IL-6,置于4℃冰箱保存。
对本实施例制备得到的CD3-NP/IL-6进行表征,结果(见图8和9):CD3-NP/IL-6的粒径为275nm,PDI为0.206,Zeta电位为-0.711mV;CD3-NP/IL-6转染HCT-116细胞的效率为21%,表明CD3-NP/IL-6可在细胞中表达目标基因。
实施例6 CD3-NP/IL-6的制备之三
用分析天平精密称取DlinMC3、DOPE、Chol、PEG-DMG、DOTAP、DSPE-MTAS-NLS(摩尔比为30:10:38:2:15:3)以上材料溶于2ml的三氯甲烷作为脂类油相溶液,用旋转蒸发仪逐渐除去三氯甲烷,得到均匀的膜。配制pH4,浓度为25mM的乙酸钠溶液作为水相溶剂,溶解标记GFP的质粒作为水相溶液,质粒与磷脂材料的质量比为1:10。在除去三氯甲烷的脂质薄膜中加入配好的水相溶液进行水化1小时,使用微型挤出器将脂质体依次挤压通过400、200的核孔膜,得到NP/IL-6的悬液。将NP/IL-6的悬液装入超滤离心管中(Mw=10000),加入4倍量体积的PBS溶液,并离心1h,将NP/IL-6外液替换为PBS。随后将DSPE-PEG2000-CD3(与质粒的摩尔比为1:10)加入以上溶液中,4℃过夜,收集悬液得到CD3-NP/IL-6,置于4℃冰箱保存。
对本实施例制备得到的CD3-NP/IL-6进行表征,结果(见图10和11):CD3-NP/IL-6的粒径为295nm,多分散系数(PDI)为0.183,Zeta电位为-2.32mV;CD3-NP/IL-6转染HCT-116细胞的效率为36%,表明CD3-NP/IL-6可在细胞中表达目标基因。
实施例7 CD3-NP/IL-6的制备之四
用分析天平精密称取DlinMC3、DOPE、Chol、PEG-DMG、DOTAP、DSPE-MTAS-NLS(摩尔比为30:10:35:1.5:15:3)以上材料溶于2ml的三氯甲烷作为脂类油相溶液,用旋转蒸发仪逐渐除去三氯甲烷,得到均匀的膜。配制pH4,浓度为25mM的乙酸钠溶液作为水相溶剂,溶解质粒作为水相溶液,质粒与磷脂材料的质量比分别为1:5。在除去三氯甲烷的脂质薄膜中加入配好的水相溶液进行水化1小时,使用微型挤出器将脂质体依次挤压通过400、200的核孔膜,得到NP/IL-6的悬液。将NP/IL-6的悬液装入超滤离心管中(Mw=10000),加入4倍量体积的PBS溶液,并离心1h,将NP/IL-6外液替换为PBS。随后将DSPE-PEG2000-CD3(与质粒的摩尔比为1:10)加入以上溶液中,4℃过夜,收集悬液得到CD3-NP/IL-6,置于4℃冰箱保存。
对本实施例制备得到的CD3-NP/IL-6进行表征,结果(见表1和图12):CD3-NP/IL-6的粒径为174nm,PDI为0.166,Zeta电位为3.39mV;CD3-NP/IL-6转染Jurkat细胞的效率为32%,表明CD3-NP/IL-6可在jurkat细胞中表达目标基因,实现基因的成功递送。
实施例8 CD3-NP/IL-6的制备之五
用分析天平精密称取DlinMC3、DOPE、Chol、PEG-DMG、DOTAP、DSPE-MTAS-NLS(摩尔比为30:10:35:1.5:15:3)以上材料溶于2ml的三氯甲烷作为脂类油相溶液,用旋转蒸发仪逐渐除去三氯甲烷,得到均匀的膜。配制pH4,浓度为25mM的乙酸钠溶液作为水相溶剂,溶解质粒作为水相溶液,质粒与磷脂材料的质量比分别为1:20。在除去三氯甲烷的脂质薄膜中加入配好的水相溶液进行水化1小时,使用微型挤出器将脂质体依次挤压通过400、200的核孔膜,得到NP/IL-6的悬液。将NP/IL-6的悬液装入超滤离心管中(Mw=10000),加入4倍量体积的PBS溶液,并离心1h,将NP/IL-6外液替换为PBS。随后将DSPE-PEG2000-CD3(与质粒的摩尔比为1:10)加入以上溶液中,4℃过夜,收集悬液得到CD3-NP/IL-6,置于4℃冰箱保存。
对本实施例制备得到的CD3-NP/IL-6进行表征,结果(见表2和图13):CD3-NP/IL-6的粒径为212nm,PDI为0.202,Zeta电位为-1.41mV;CD3-NP/IL-6转染Jurkat细胞的效率为18%,表明CD3-NP/IL-6可在jurkat细胞中表达目标基因,实现基因的成功递送。
实施例9 CD3-NP/IL-6的制备之六
用分析天平精密称取DlinMC3、DOPE、Chol、PEG-DMG、DOTAP、DSPE-MTAS-NLS(摩尔比为35:10:25:1.5:15:3)以上材料溶于2ml的三氯甲烷作为脂类油相溶液,用旋转蒸发仪逐渐除去三氯甲烷,得到均匀的膜。配制pH4,浓度为25mM的乙酸钠溶液作为水相溶剂,溶解质粒作为水相溶液,质粒与磷脂材料的质量比为1:10。在除去三氯甲烷的脂质薄膜中加入配好的水相溶液进行水化1小时,使用微型挤出器将脂质体依次挤压通过400、200的核孔膜,得到NP/IL-6的悬液。将NP/IL-6的悬液装入超滤离心管中(Mw=10000),加入4倍量体积的PBS溶液,并离心1h,将NP/IL-6外液替换为PBS。随后将DSPE-PEG2000-CD3(与质粒的摩尔比为1:10)加入以上溶液中,4℃过夜,收集悬液得到CD3-NP/IL-6,置于4℃冰箱保存。
对本实施例制备得到的CD3-NP/IL-6进行表征,结果(见图14):CD3-NP/IL-6的粒径为250.5±1.5nm,Zeta电位为0.5±0.1mV;CD3-NP/IL-6和NP/IL-6的电镜图表明该纳米粒呈类球形,大小为200nm左右,形态均一。
试验例1 CD3-NP/IL-6被T细胞摄取效果评价
制备包裹Dio荧光染料的CD3-NP/IL-6纳米粒以及包裹Dio荧光染料的NP/IL-6纳米粒,利用激光共聚焦显微镜判断有无CD3修饰纳米粒被T细胞摄取情况。从图15可以看出,CD3靶向的纳米粒被T细胞的摄取显著增加。
试验例2 CD3-NP/IL-6体外转染T细胞效果评价
将带有绿色荧光蛋白(GFP)基因的CAR质粒用CD3-NP/IL-6和NP/IL-6包裹后与人源的T淋巴细胞共同孵育48小时,用流式细胞仪观察其表达GFP的细胞数量,证明体外转导效率。从图16可以看出相比于NP/IL-6,CD3-NP/IL-6在T细胞上表达CAR的效率更高,表明加入了CD3修饰的纳米递送系统更能靶向T细胞。
试验例3 CD3-NP/IL-6体外转导T细胞后产生的杀伤效果
分别取经过CD3-NP/IL-6和NP/IL-6、以及未敲减的CD3-NP纳米粒转导48小时的T细胞分别与CD19过表达的K562靶细胞、Raji靶细胞以10:1、5:1的比例共孵育48小时,通过LDH法测定对靶细胞的杀伤效率。从图17可以看出,CD3-NP/IL-6纳米粒在10:1的效靶比下能够有效杀伤两种肿瘤细胞。
试验例4 CD3-NP/IL-6产生的CAR-T体外IL-6沉默效果评价
分别取经过CD3-NP/IL-6和NP/IL-6、以及未敲减IL-6的CD3-NP纳米粒(CD3-NP)转导48小时的T细胞分别与CD19过表达的K562靶细胞、Raji靶细胞以10:1的比例共孵育,用定量聚合酶链式反应(qPCR)检测IL-6的mRNA表达情况。从图18可以看出,CD3-NP/IL-6能够在体外有效敲减IL-6水平。
试验例5 CD3-NP/IL-6的体内组织分布评价
将NSG小鼠(NOD-SCID IL-2receptor gamma null mice)随机分为三组,分别为实验组CD3-NP/IL-6、对照组NP/IL-6和空白生理盐水组,利用近红外染料DiR标记,然后尾静脉注射,利用活体成像仪检测CD3-NP/IL-6的体内组织分布(图19)。其结果表明在CD3的介导下,纳米粒能够有效分布于体内的淋巴结中。
试验例6 CD3-NP/IL-6在荷瘤小鼠体内产生CAR-T细胞的数目
将NSG小鼠通过尾静脉注射Raji-Luc细胞,在第7天分别尾静脉注射人源T细胞+CD3-NP/IL-6、T细胞+NP/IL-6、敲减IL-6的CAR-T细胞(CAR-T/IL-6)、T细胞+未敲减IL-6的纳米粒CD3-NP,每组5只。分别于第7天、第14天、第21天、第35天、第90天取小鼠外周血0.1mL,采集的外周血使用单核细胞分离液进行分离,通过流式细胞仪检测CAR-T细胞数目(图20),计算外周血中CAR-T细胞的总数量。结果:CD3-NP/IL-6实现了在荷瘤小鼠体内稳定转染T细胞并改造成为CAR-T细胞,并在21天达到高峰期。
试验例7 CD3-NP/IL-6产生CAR-T的体内肿瘤杀伤(活体成像)和生存期评价
体外培养带有荧光素酶(Luciferase)的Raji-Luc细胞,尾静脉接种NSG小鼠构建血液肿瘤模型,使用小动物成像仪检测是否种瘤成功。将建立好的肿瘤模型小鼠分为四组,每组5只。分别注射PBS、人源T细胞+CD3-NP/IL-6、T细胞+NP/IL-6、敲减IL-6的CAR-T细胞(CAR-T/IL-6)。分别在给药后第6天(D6)、第9天(D9)、第12天(D12)、第15天(D15)、第27天(D27)、第41天(D41)进行活体成像(图21),统计肿瘤荧光强度(图22),观察并记录小鼠生存期(图23)。通过这些指标来评估各组肿瘤抑制效果。
试验例8 CD3-NP/IL-6产生CAR-T的体内IL-6释放功能评价
将建立好的Raji白血病模型小鼠随机分为7组,每组5只。分别注射PBS、人源T细胞+CD3-NP/IL-6、T细胞+NP/IL-6、敲减IL-6的CAR-T细胞(CAR-T/IL-6)、T细胞+未敲减IL-6的纳米粒CD3-NP以及单独注射纳米粒CD3-NP/IL-6、NP/IL-6。各组给药后于第7天、第10天、第14天分别取小鼠外周血,按照1:9加入抗凝剂(EDTA-2K),将采集血液用离心机4℃,2500rpm离心10min,取上清用CBA试剂盒检测IL-6表达水平。结果见图24,可以看出,CD3-NP/IL-6在体内产生CAR-T细胞,并能够有效减少IL-6蛋白的释放。
试验例9 CD3-NP/IL-6在体内产生CAR-T的安全性
分别在不同的时间测定给药后外周血中IL-4、IL-2、IFN-γ、IL-10、IL-17α、TNF-α的含量,比较各组中产生的细胞因子,观察小鼠是否产生了细胞因子风暴。结果如图25所示,可以看出,CD3-NP/IL-6在体内产生CAR-T之后并未在体内产生大量炎症因子,在发挥治疗效果的同时,未导致细胞因子风暴。
此外,在给药治疗的30天后,对治疗的小鼠进行解剖,并取小鼠的主要器官心、肝、脾、肺和肾进行HE染色,观察其是否有病变。结果如图26所示,CD3-NP/IL-6未导致主要脏器发生病变,证明了本发明所涉及的用于体内自组装CAR-T的纳米递送系统具有良好的生物安全性。
英文缩写:
DlinMC3:4-(N,N-二甲基氨基)丁酸(二亚油基)甲酯
DSPE-PEG:二硬脂酰磷脂酰乙醇胺-聚乙二醇
PEG-DMG:二肉豆蔻酰甘油-聚乙二醇2000
DOTAP:(2,3-二油酰基-丙基)-三甲基铵-氯盐;(2,3-二油酰基-丙基)-三甲基氯化铵
DOTMA:1,2-双十八烯氧基-3-甲基铵丙烷(氯盐)
DODMA:1,2-二油醇-3-二甲基氨基-丙烷
DSPE-MTAS-NLS:二硬脂酰磷脂酰乙醇胺-微管相关-核定位肽
DSPE-PEG-CD3:二硬脂酰磷脂酰乙醇胺-聚乙二醇耦连CD3抗体
DSPE-PEG-CD4:二硬脂酰磷脂酰乙醇胺-聚乙二醇耦连CD4抗体
DSPE-PEG-CD8:二硬脂酰磷脂酰乙醇胺-聚乙二醇耦连CD8抗体
本文提供的任何和所有实施例或示例性语言(例如,“诸如”)的使用仅旨在更好地说明本发明,而不对本发明的范围构成限制,除非另有要求。说明书中的语言不应被解释为指示任何未要求保护的元件对于实施本发明是必要的。
本说明书中引用的所有出版物和专利申请通过引用并入本文,如同每个单独的出版物或专利申请被具体地和单独地指明通过引用并入。此外,本文所述的任何理论、机制、证明或发现旨在进一步增强对本发明的理解,并且不意图以任何方式将本发明限制到这样的理论、机制、证明或发现。尽管已经在附图和前面的描述中详细地示出和描述了本发明,但是本发明应当被认为是说明性的而不是限制性的。
序列表
<110> 华东师范大学
<120> 一种用于体内自组装CAR-T的纳米递送系统及其制备方法和应用
<130> WHYY-NP-21-101080-2
<160> 13
<170> PatentIn version 3.5
<210> 1
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ccgggcacag aacttatgtt gttctctcga gaacaacata agttctgtgc ccttttttg 59
<210> 2
<211> 6314
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 60
gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 120
atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 180
tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg 240
tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta 300
cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg 360
agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc 420
ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt 480
tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc 540
aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg 600
cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag 660
cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg 720
gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag 780
ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga 840
cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt 900
cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt 960
agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg 1020
agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat 1080
tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag 1140
tggttcaaag tttttttctt ccatttcagg tgtcgtgagg atccgccacc atggccttac 1200
cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg ccggacatcc 1260
agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc accatcagtt 1320
gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa ccagatggaa 1380
ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca tcaaggttca 1440
gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag caagaagata 1500
ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga ggggggacca 1560
agctggagat cacaggtggc ggtggctcgg gcggtggtgg gtcgggtggc ggcggatctg 1620
aggtgaaact gcaggagtca ggacctggcc tggtggcgcc ctcacagagc ctgtccgtca 1680
catgcactgt ctcaggggtc tcattacccg actatggtgt aagctggatt cgccagcctc 1740
cacgaaaggg tctggagtgg ctgggagtaa tatggggtag tgaaaccaca tactataatt 1800
cagctctcaa atccagactg accatcatca aggacaactc caagagccaa gttttcttaa 1860
aaatgaacag tctgcaaact gatgacacag ccatttacta ctgtgccaaa cattattact 1920
acggtggtag ctatgctatg gactactggg gccaaggaac ctcagtcacc gtctcctcaa 1980
ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg cagcccctgt 2040
ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg agggggctgg 2100
acttcgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg gtccttctcc 2160
tgtcactggt tatcaccctt tactgcaaac ggggcagaaa gaaactcctg tatatattca 2220
aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt agctgccgat 2280
ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg agcgcagacg 2340
cccccgcgta caagcagggc cagaaccagc tctataacga gctcaatcta ggacgaagag 2400
aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg ggaaagccga 2460
gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag atggcggagg 2520
cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac gatggccttt 2580
accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg caggccctgc 2640
cccctcgcta agtcgacgct agcccccttc accgagggcc tatttcccat gattccttca 2700
tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac 2760
acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca 2820
gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc 2880
gatttcttgg ctttatatat cttgtggaaa ggacgaaact ccgggcacag aacttatgtt 2940
gttctctcga gaacaacata agttctgtgc ccttttttga gcggccgcga ctctagatca 3000
taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc 3060
ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt 3120
ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac 3180
tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttaaggcgta aattgtaagc 3240
gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa 3300
taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt 3360
gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg 3420
cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt 3480
ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga 3540
gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg 3600
ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg 3660
cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc 3720
cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc 3780
tgataaatgc ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt 3840
ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc 3900
aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag 3960
gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc 4020
cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa 4080
ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt 4140
gaggaggctt ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga 4200
tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag 4260
aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc 4320
cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg 4380
aatgaactgc aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc 4440
gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg 4500
ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct 4560
gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg 4620
aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat 4680
ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc 4740
atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg 4800
gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc 4860
tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct 4920
gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat 4980
cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga 5040
cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct 5100
tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg 5160
agttcttcgc ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag 5220
gaacccgcgc tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg 5280
ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc 5340
attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg 5400
aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact 5460
catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 5520
tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 5580
cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 5640
gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 5700
taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc 5760
ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 5820
tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 5880
ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 5940
cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 6000
agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 6060
gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 6120
atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 6180
gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 6240
gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 6300
ttaccgccat gcat 6314
<210> 3
<211> 45
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Cys Gly Arg Tyr Leu Thr Gln Glu Thr Asn Lys Val Glu Thr Tyr Lys
1 5 10 15
Glu Gln Pro Leu Lys Thr Pro Gly Lys Lys Lys Lys Gly Lys Pro Gly
20 25 30
Lys Arg Lys Glu Gln Glu Lys Lys Lys Arg Arg Thr Arg
35 40 45
<210> 4
<211> 21
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro
20
<210> 5
<211> 63
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccg 63
<210> 6
<211> 242
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu
115 120 125
Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys
130 135 140
Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg
145 150 155 160
Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser
165 170 175
Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile
180 185 190
Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln
195 200 205
Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly
210 215 220
Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
225 230 235 240
Ser Ser
<210> 7
<211> 726
<212> DNA
<213> 人工序列(Artificial Sequence )
<400> 7
gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60
atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120
gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180
aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240
gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300
gggaccaagc tggagatcac aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360
ggatctgagg tgaaactgca ggagtcagga cctggcctgg tggcgccctc acagagcctg 420
tccgtcacat gcactgtctc aggggtctca ttacccgact atggtgtaag ctggattcgc 480
cagcctccac gaaagggtct ggagtggctg ggagtaatat ggggtagtga aaccacatac 540
tataattcag ctctcaaatc cagactgacc atcatcaagg acaactccaa gagccaagtt 600
ttcttaaaaa tgaacagtct gcaaactgat gacacagcca tttactactg tgccaaacat 660
tattactacg gtggtagcta tgctatggac tactggggcc aaggaacctc agtcaccgtc 720
tcctca 726
<210> 8
<211> 69
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
35 40 45
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
50 55 60
Ile Thr Leu Tyr Cys
65
<210> 9
<211> 207
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180
ctgtcactgg ttatcaccct ttactgc 207
<210> 10
<211> 42
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 11
<211> 126
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 12
<211> 112
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 13
<211> 336
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
Claims (13)
1.一种用于体内自组装CAR-T的纳米递送系统,其特征在于,包含脂质膜和由所述脂质膜包裹的带有CAR的质粒;所述脂质膜包含DlinMC3、DOPE、Chol、PEG-DMG、DOTAP和DSPE-MTAS-NLS;所述用于体内自组装CAR-T的纳米递送系统的外表面修饰有DSPE-PEG-CD3。
2.如权利要求1所述的用于体内自组装CAR-T的纳米递送系统,其特征在于,所述带有CAR的质粒包含IL-6 shRNA。
3.如权利要求2所述的用于体内自组装CAR-T的纳米递送系统,其特征在于,所述IL-6shRNA的核酸序列如SEQ ID NO.1所示。
4.如权利要求1所述的用于体内自组装CAR-T的纳米递送系统,其特征在于,所述脂质膜中DlinMC3、DOPE、Chol、PEG-DMG、DOTAP和DSPE-MTAS-NLS的摩尔比为(20-35):(10-15):(20-38):(1-2):(10-20):3。
5.如权利要求1所述的用于体内自组装CAR-T的纳米递送系统,其特征在于,所述带有CAR的质粒包含CD8 leader、抗靶蛋白的抗体相关区域、CD8 Hinge&TM、共刺激分子、CD3ζ;所述抗靶蛋白的抗体相关区域选自全抗体、抗体的F(ab)2段、scFv中的一种或多种;所述共刺激分子选自CD28、4-1BB、OX40中的一种或多种。
6.如权利要求1所述的用于体内自组装CAR-T的纳米递送系统,其特征在于,所述带有CAR的质粒的CAR的靶蛋白选自CD19、BCMA、CD20、CD22、CD138中的一种或者多种。
7.如权利要求1所述的用于体内自组装CAR-T的纳米递送系统,其特征在于,所述靶蛋白为CD19;所述带有CAR的质粒的核酸序列如SEQ ID NO.2所示。
8.如权利要求1所述的用于体内自组装CAR-T的纳米递送系统,其特征在于,还包含乙酸钠溶液;所述带有CAR的质粒为溶质溶于所述乙酸钠溶液。
9.一种如权利要求1所述的用于体内自组装CAR-T的纳米递送系统的制备方法,其特征在于,包括以下步骤:
S1、所述带有CAR的质粒的构建;
S2、DSPE-MTAS-NLS的合成;
S3、制备脂质体悬液:称取DlinMC3、DOPE、Chol、PEG-DMG、DOTAP、DSPE-MTAS-NLS作为溶质溶于三氯甲烷作为油相溶液,用蒸发仪除去三氯甲烷,得到脂质膜;以乙酸钠溶液作为水相溶剂,溶解所述步骤S1得到的带有CAR的质粒作为水相溶液,使得所述带有CAR的质粒与所述油相溶液的溶质的质量比为1:(5-20);在所述脂质膜中加入所述水相溶液进行水化得到脂质体,使用微型挤出器将所述脂质体依次挤压通过核孔膜,得到脂质体悬液;
S4、DSPE-PEG-CD3的合成,具体为:将CD3抗体溶于NaHCO3溶液中,取DSPE-PEG-NHS溶解于DMSO,将DSPE-PEG-NHS滴加至CD3抗体溶液中,搅拌反应,透析,冷冻干燥,得到所述DSPE-PEG-CD3;
S5、将所述步骤S3得到的脂质体悬液装入超滤离心管中,加入PBS溶液,离心,将所述脂质体悬液的外液替换为PBS;然后加入所述步骤S4得到的DSPE-PEG-CD3,使得所述DSPE-PEG-CD3与所述带有CAR的质粒的摩尔比为1:(10-20),4℃±2℃过夜,收集悬液得到修饰有CD3的脂质体悬液。
10.如权利要求9所述的制备方法,其特征在于,所述步骤S2具体为:将核定位肽Cys-MTAS-NLS溶于缓冲液中得到溶液一;取Mal-PEG6-DSPE溶于二甲基甲酰胺得到溶液二;将所述溶液一与所述溶液二加到磁力搅拌器中反应得到反应液;取出所述反应液,将过量的多肽和二甲基甲酰胺通过透析除去,再冷冻干燥得到所述DSPE-MTAS-NLS;所述透析的截止分子量为3.5kDa-8kDa。
11.如权利要求9所述的制备方法,其特征在于,所述步骤S1得到的带有CAR的质粒包含IL-6 shRNA。
12.如权利要求1-8任一项所述的用于体内自组装CAR-T的纳米递送系统在制备治疗肿瘤的药物中的应用。
13.一种试剂盒,其特征在于,包含如权利要求1-8任一项所述的用于体内自组装CAR-T的纳米递送系统。
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