CN110251666A - 一种用于癌症靶向或免疫治疗的组合物及其应用 - Google Patents
一种用于癌症靶向或免疫治疗的组合物及其应用 Download PDFInfo
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Abstract
本发明提供一种宫颈癌治疗组合物及其应用,所述组合物包括一种或多种抗癌抗体,所述抗癌抗体选自MESO抗体、MUC 1抗体、CD 133抗体和GD2抗体。所述组合物可用于制备宫颈癌治疗药物,尤其是宫颈癌免疫或靶向治疗药物。
Description
技术领域
本发明涉及生物医学领域,具体涉及一种用于癌症靶向或免疫治疗的组合物及其应用。
背景技术
嵌合型抗原受体(CAR)是由具有抗原特异性的CAR与T细胞信号区融合而成,而抗原特异性是由CAR中的靶向区决定。目前CAR-T细胞对于循环淋巴细胞肿瘤治疗有显著效果,尤其是对B细胞恶性肿瘤的治疗,但是CAR-T细胞应用于实体肿瘤的报道远低于血液系统。分析原因,存在以下几种情况:①实体瘤除肿瘤组织本身外,周围还包绕间质成分、正常组织和坏死组织,形成坚固的屏障,免疫细胞需要越过这些屏障才能到达目标肿瘤组织,免疫细胞不易进入瘤体内部限制了杀伤作用的发挥。②对于实体肿瘤目前尚无完美肿瘤特异性抗原,有时正常细胞表面也表达目标抗原,任何会在正常细胞表达的抗原都有被误伤的风险导致脱靶效应,对正常细胞产生毒性反应。③在转染技术中不同载体转染效率的限制,获得高效的CAR-T细胞极为困难。这些都成为阻碍CAR-T免疫治疗在实体瘤中应用的重要原因。
多项研究发现肿瘤患者存在各种类型的抗细胞自身抗原抗体,其中与肿瘤发生有关的抗原被称为肿瘤相关抗原。目前CAR-T细胞在宫颈癌中的临床应用国内外尚未见报道,考虑可能与以下因素有关:①缺乏宫颈癌特异性肿瘤相关抗原;②在发达国家,如美国,由于宫颈癌有完善的筛查体系,患者大部分在早期发现,晚期宫颈癌患者较少,因此,在发达国家研究甚少。我国属于发展中国家,2018年国家癌症中心数据显示宫颈癌发病排名女性恶性肿瘤第6位,60%以上宫颈癌患者发现时为中晚期,而且有年轻化趋势,35岁之前女性占24%,85%以上为鳞状细胞癌。早期宫颈癌患者以手术治疗为主,预后好,中晚期宫颈癌患者主要以放疗同步化疗或手术联合放化疗为主,部分患者预后差,对于晚期、复发、转移患者为了延长生命,免疫治疗成为最后可选择的一种方法,作为宫颈癌高发的国家以CAR-T细胞为首的免疫治疗在中国有很大的潜在需求,因此值得我们去关注、探讨和研究。
发明内容
本发明的目的在于寻找宫颈鳞状细胞癌肿瘤特异性相关抗原,并进一步研究CAR-T细胞在宫颈癌治疗中的临床应用。
根据本发明,提供一种宫颈癌治疗组合物,所述组合物包括一种或多种抗癌抗体,所述抗癌抗体选自MESO抗体、MUC1抗体、CD133抗体和GD2抗体。
根据本发明,还提供一种宫颈癌治疗组合物,所述组合物包括MESO-CAR-T细胞,所述MESO-CAR-T细胞是表达MESO-CD8-4-1BB-CD3ζ融合蛋白的T淋巴细胞。其中,所述组合物还包括一种或多种免疫促进剂。
根据本发明,还提供一种宫颈癌疫苗,所述疫苗包括一种或多种癌症相关抗原,所述癌症相关抗原选自MESO蛋白、MUC1蛋白、CD133蛋白和GD2蛋白。
根据本发明,还提供上述任意组合物在制备宫颈癌治疗药物中的应用。在一些实施方式中,所述宫颈癌治疗药物为宫颈癌免疫治疗药物。在另一些实施方式中,所述宫颈癌治疗药物为宫颈癌靶向治疗药物。在另一些实施方式中,所述宫颈癌治疗药物为杀伤宫颈癌细胞的药物。
根据本发明,还提供MSLN基因在制备宫颈癌治疗药物中的应用,以MSLN基因作为抗原结合域基因与跨膜区和信号转导区基因进行基因重组,生成重组质粒,再将该质粒通过基因转导的方法转入T细胞中,形成MESO-CAR-T细胞,用于宫颈癌治疗药物的制备。其中,以MSLN基因作为抗原结合域基因与跨膜区和信号转导区基因进行基因重组得到的重组基因序列为SEQ ID NO:1。
附图说明
图1示出实施例2的细胞划痕实验中针对同一视野在SiHa细胞迁移0h、24h和48h获得的图片;
图2示出实施例3中MESO-过表达慢病毒构建的试验流程图;
图3示出实施例3中MESO-过表达慢病毒构建的目的基因及工具载体连接次序;
图4示出实施例3中MESO-过表达慢病毒构建的菌落PCR鉴定结果;
图5示出实施例3中MESO-过表达慢病毒构建的慢病毒包装与质量检测中的pHelper 1.0载体图谱和pHelper 2.0载体图谱;
图6示出MESO-CAR基因结构示意图;
图7示出实施例4的乳酸脱氢酶(LDH)试验检测CAR-T细胞的杀伤能力中实验各体系情况;
图8示出实施例4中在靶细胞存在/不存在条件下,MESO-CAR-T细胞及CON-CAR-T细胞体外增殖情况;
图9示出实施例5的不同时间点不同治疗组小鼠体内肿瘤大小。
具体实施方式
下面结合具体的实施例对本发明做进一步的详细说明。应当指出,这些代表性的实施例仅用于对本发明技术方案的解释与说明,本发明并非受限于这些具体的实施例。根据本发明的实质性内容,对所述实施例进行的任意组合、改变、修饰,均属于本发明要求保护的范围。
为了使本发明更加易于理解,本发明说明书中会阐述一些本发明所用术语的定义。
在本发明中,提及基因序列,本领域技术人员应当理解,实际包括互补双链的任意一条,或者两条。为了方便,在本说明书和权利要求书中,虽然多数情况下至给出了一条链,但实际上也公开了与之互补的另一条链。例如提及MESO基因的DNA序列,实际上包括该序列以及其互补序列。本领域技术人员还可以理解,利用一条链可以检测另一条链,反之亦然。
本申请中的基因序列包括DNA形式或RNA形式,公开其中一种,意味着另一种也被公开。例如提及MESO基因的DNA序列,实际也包括相应的RNA序列。
本发明提供一种宫颈癌治疗组合物,在一种实施方式中,所述组合物包括一种或多种抗癌抗体,所述抗癌抗体选自MESO抗体、MUC1抗体、CD133抗体和GD2抗体。在一种实施方式中,所述组合物包括抗癌抗体MESO抗体和MUC1抗体中的一种或两种。在另一种实施方式中,所述组合物仅包括一种抗癌抗体MESO抗体。在上述实施方式中,所述抗癌抗体是指抗体本身及其药学上可接受的盐、水合物、溶剂合物、立体异构体或上述任意组合。在上述实施方式中,所述抗癌抗体可以为抗体片段、包含抗体的复合物或包含抗体的结合物,抗体可以为人造抗体,例如单克隆抗体。
本发明还提供一种宫颈癌疫苗,所述疫苗包括一种或多种癌症相关抗原。本发明提到的术语“癌症相关抗原”包括癌细胞表面抗原、癌细胞内部抗原或癌细胞生长介质。具体来说,所述癌症相关抗原是指(i)肿瘤特异性抗原、(ii)肿瘤相关抗原、(iii)表达肿瘤特异性抗原的细胞、(iv)表达肿瘤相关抗原的细胞、(v)肿瘤上的胚胎抗原、(vi)自体性肿瘤细胞、(vii)肿瘤特异性膜抗原、(viii)肿瘤相关膜抗原、(ix)生长因子受体、(x)生长因子配位体和(xi)任何其它类型的抗原或提供抗原的细胞或与癌症相关的物质。在根据本发明的一些实施方式中,所述癌症相关抗原为宫颈癌相关抗原,其选自间皮素(mesothelin,MESO)、黏蛋白(Mucoprotein,MUC1)、CD133蛋白、神经苷酯2(Ganglioside 2,GD2)中的一种或多种。在一些实施方式中,所述宫颈癌疫苗仅含有MESO蛋白。在上述实施方式中,所述抗原是指抗原本身及其药学上可接受的盐、水合物、溶剂合物、立体异构体或上述任意组合。在上述实施方式中,所述癌症相关抗原可以为细胞、蛋白、肽、融合蛋白、编码肽或蛋白的DNA、编码肽或蛋白的RNA、糖蛋白、脂蛋白、磷蛋白、糖、脂多糖、脂质、其两种或两种以上的化学连接组合、其两种或两种以上的融合或其两种或两种以上的混合物。
本发明还提供一种宫颈癌治疗组合物,所述组合物包括所述组合物包括MESO-CAR-T细胞。在一种实施方式中,所述组合物仅含有MESO-CAR-T细胞。所述MESO-CAR-T细胞是指表达MESO-CD8-4-1BB-CD3ζ融合蛋白的T淋巴细胞。在一种实施方式中,MESO蛋白具有序列SEQ ID NO:2。
在一些实施方式中,上文所述组合物用作治疗剂,即给与所述组合物来治疗癌症或阻止癌症复发。在另一些实施方式中,所述组合物用作预防剂,即给与所述组合物以阻止或延缓癌症的发展。在所述组合物用作治疗剂的实施方式中,将所述组合物给与癌症患者以引发免疫反应,从而通过阻止或减缓肿瘤的生长、抑制肿瘤转移、减小肿瘤尺寸来抑制癌症的复发或者消除早期治疗未能杀死的癌细胞。在所述组合物用作预防剂的实施方式中,将所述组合物给与未患有癌症的个体以引发免疫反应,从而靶向潜在的癌细胞或靶向源自与癌症相关病毒的抗原。
本发明所述任意组合物或疫苗均可包含一种或多种免疫促进剂。所述免疫促进剂可以为toll样受体(TLR)激动剂(例如,TLR4、TLR7、TLR9)、N-乙酰基胞壁酰基-L-丙氨酸-D-异谷氨酰胺(MDP)、脂多糖(LPS)、遗传修饰和/或降解的LPS、明矾、葡聚糖、集落刺激因子(例如,EPO、GM-CSF、G-CSF、M-CSF、聚乙二醇化G-CSF、SCF、IL-3、IL6、PIXY321)、干扰素(例如,γ-干扰素、α-干扰素)、白细胞介素(例如,IL-2、IL-7、IL-12、IL-15、IL-18)、MHCII型结合肽、皂苷(例如,QS21)、未甲基化的CpG序列、1-甲基色氨酸、精氨酸酶抑制剂、环磷酰胺或阻断免疫抑制功能的抗体(例如,抗CTLA4抗体)以及上述任意两种或两种以上的组合。
适用于本发明所述组合物或疫苗的给药方式包括非经肠给药,例如皮下注射、经皮、静脉内、肿瘤内、肿瘤周围、鼻内、眼部、肌肉内、皮内、腹膜内、肺部;和经肠投药,例如经粘膜、透皮、吸入、阴道内、直肠或口服投药。在一些实施方式中,采用注射给药。
本发明所述组合物或疫苗用于刺激、引发或增强受试者个体的免疫反应。适用于本发明的受试者包括哺乳动物或鸟类,优选为人类。本发明所述受试者可能存在患有癌症的风险、经诊断患有癌症、处于癌症治疗期间或处于癌症治疗后恢复期间。
本发明所述“免疫反应”包括细胞和体液免疫反应,包括刺激细胞因子的产生、刺激免疫细胞的增殖、刺激免疫细胞的活化或刺激免疫细胞的溶解活性。通过本发明的方法刺激的免疫反应的实例是细胞因子的分泌,NK细胞的活化,B细胞、T细胞、巨噬细胞、单核细胞和其它免疫细胞的增殖,以及其它免疫反应。举例来说,为了检测细胞免疫反应,可使用标准分析来检测抵抗表达抗原的细胞的T细胞效应物活性,例如靶细胞杀死、巨噬细胞活化、B细胞活化或淋巴因子产生。可以通过使用诸如ELISA的常规方法检测例如抗原特异性抗体的出现或其效价的增加来测量体液反应。可以通过测量级别转变(诸如从早期IgM反应转变成后期IgG反应)来确定抗体反应的进展。
本发明所述“刺激、引发或增强受试者个体的免疫反应”包括刺激、引发、增加、增强、保持和/或改善新免疫反应或先前存在的免疫反应。因此,在癌症免疫治疗或靶向治疗中,本发明所述“刺激、引发或增强受试者个体的免疫反应”指的是增强治疗功效、增加存活时间、减缓癌性肿瘤的发展或缩小癌性肿瘤的尺寸、阻止肿瘤扩散或转移扩散、阻止或减缓所治疗癌症的复发、消除早期治疗未杀死的癌细胞、靶向潜在癌细胞或靶向源自与癌症相关的病毒的抗原。
本发明上文所述的组合物和疫苗可以作为宫颈癌治疗药物或主要有效成分来制备宫颈癌免疫治疗药物或者宫颈癌靶向治疗药物。
在一些实施方式中,所述组合物或疫苗可与宫颈癌常规疗法或药物配方组合使用。这些常规疗法包括但不限于外科手术、放射疗法和/或切除疗法(例如,激光疗法、红外线疗法和类似疗法)。
下面通过具体实施例来对本发明进行阐述。
实施例1 宫颈癌特异肿瘤相关抗原筛选
根据国内外文献及已有临床试验报告,选取MESO、MUC1、CD133和GD2四种膜蛋白为待选宫颈癌肿瘤相关特异抗原(TAA)。搜集“宫颈鳞癌”患者癌组织、癌旁及“子宫肌瘤”患者正常宫颈组织各8例,利用免疫印迹(Western Blot,WB)技术检测四种膜蛋白在宫颈鳞癌、癌前病变及正常组织中表达情况,选择在癌组织中最特异高表达的蛋白作为宫颈癌TAA。
免疫印迹实验发现,GD2蛋白质在宫颈鳞状细胞癌、癌旁及宫颈炎症组织中均无表达,MESO及MUC1在宫颈鳞癌及癌旁组中高表达。MUC1在宫颈鳞癌及癌旁组织中阳性表达率与在宫颈炎症组阳性表达率近似,MESO在宫颈鳞癌及癌旁组中阳性表达率高于在宫颈炎症组,而CD133在宫颈炎症组高表达,见表1。
表1.宫颈组织中不同宫肿瘤相关抗原表达情况
免疫印迹(WB)定量分析结果显示:GD2在宫颈鳞癌、癌旁及宫颈炎症组织中均无表达。MESO和CD133蛋白质在宫颈鳞癌、癌旁及宫颈炎症组织中比较差异无统计学意义(P>0.05),MUC1蛋白质在宫颈鳞癌、癌旁及宫颈炎症组中比较差异有统计学意义(F=7.415,P=0.025),MUC1组组间比较,宫颈炎症和宫颈鳞癌之间MUC1表达差异有统计学意义(P=0.003),宫颈炎症和癌旁之间MUC1表达差异无统计学意义(P=0.234),宫颈鳞癌和癌旁之间MUC1表达差异无统计学意义(P=0.279),见表2。
表2.宫颈组织中不同肿瘤相关抗原表达情况的免疫印迹定量分析[M(P25,P75)]
*P<0.05,差异有统计学意义
上述实验结果表明,MESO可作为宫颈癌相关特异抗原,作为识别靶点,构建特异性的MESO-CAR-T细胞。
比较MESO蛋白在SiHa、Caski和Hela细胞中的表达,其中在SiHa细胞中表达最高,故选取SiHa宫颈鳞癌细胞系作为研究对象,进行后续试验。
实施例2 MESO蛋白基因(MSLN)功能检测
构建MESO低表达慢病毒,利用FACS流式细胞学进行细胞凋亡检测、MTT方法进行细胞增殖检测和细胞划痕实验了解细胞转移情况。
慢病毒干扰技术敲低SiHa细胞中MESO的表达,RT-PCR提示敲低效率为78.3%。MESO低表达组(Knock-down,KD)与SiHa细胞组(MOCK组)和空病毒感染SiHa细胞(Normalcontrol,NC组)比较,结果如下。
1.FACS流式细胞学细胞凋亡检测
ShRNA慢病毒感染SiHa细胞,培养3天后,进行凋亡率比较,实验进行三次重复。相比NC组凋亡率为4.77%,KD组细胞凋亡率明显增加为5.81%,差异有统计学意义p<0.05。MOCK和NC组比较,前者凋亡率为2.8%,后者凋亡率为4.77%,NC组细胞凋亡率高,差异有统计学意义,p=0.000;NC和KD组比较,后者细胞凋亡率高于前者,差异有统计学意义,p=0.014;MSLN低表达促进细胞凋亡,见表3。
表3.三组细胞凋亡情况
2.MTT检测细胞增殖情况
SiHa细胞各实验组在酶标仪对波长490nm的光的吸收率随时间变化的对比。OD490在这里反映了具有细胞活力的细胞的数量,实验重复三次取平均值。相比NC组,KD组于第5日的细胞增殖倍数经T检验分析,p<0.05。第四天MTT结果提示MOCK和NC组比较p=0.0017,NC组和KD组比较p=0.0084,MOCK和KD组比较p=0.000;第五天MTT结果提示MOCK和NC组比较p=0.000,NC和KD比较p=0.0015,MOCK和KD比较p=0.000。上述结果均提示KD组细胞增殖低于NC和MOCK组。虽然差异有统计学意义,但是KD和NC组及MOCK组比较,测定差异均<20%,故细胞增殖不判读为有明显差异,见表4和表5。
表4.OD490MTT结果
表5.OD490/FOLD MTT结果
3.细胞划痕实验
图1为针对同一视野在SiHa细胞迁移0小时(h)、24h和48h获得的图片,对划痕区域的宽度进行Photoshop分析,实验重复五次取平均值。通过迁移率计算,划痕24小时迁移率,MOCK组为29%,NC组为25%,KD组为20%;划痕48小时迁移率,MOCK组为44%,NC组为43%,KD组为38%。MOCK组和NC组比较,p24h=0.032,p48h=0.599;NC组和KD组比较,p24h=0.139,p48h=0.1437;MOCK和KD组比较,p24h=0.018,p48h=0.0722。MOCK和NC组,及MOCK和KD组在24小时比较,p<0.05差异有统计学意义,但是两组测定差异值比较<20%,因此不建议判读为阳性结果,见图1和表6。
表6.细胞迁移率计算
上述实验结果表明,MESO基因(MSLN)参与宫颈癌细胞凋亡,MESO基因低表达促进宫颈癌SiHa细胞凋亡,但不参与SiHa宫颈鳞癌细胞增殖和细胞迁移。
MESO-CAR-T细胞杀伤性研究(实施例3-5)
实施例3 MESO-CAR-T细胞构建
实验方法:
3.1 MESO-过表达慢病毒构建(试验流程见图2)
3.1.1克隆构建
(1)目的基因及工具载体信息(见图3)
载体名称:GV401
克隆位点:BamHI/BamHI
元件顺序:EF1a-ScFv-二代CarT-2A-EGFP
(2)载体酶切
根据下表7,配制50μl酶切体系。按表7顺序依次加入各种试剂,用移液器轻轻吹打混均,短暂离心,置于37℃反应3h或过夜,对载体酶切产物进行琼脂糖凝胶电泳,回收目的条带。
表7.酶切体系配置
(3)目的基因片段的获取
引物:
表8.引物序列
ID | seq |
gene(16335-21)-P1 | AGGTCGACTCTAGAGGATCCCGCCACCATGGCCTTACCAGTGACCGCC |
gene(16335-21)-P2 | GAAGTTGGTGGCCATGGATCCGTACGCGGGGGCGTCTGCGCTC |
引物说明:含交换配对碱基、酶切位点,并含有目的基因5’端部分序列用于PCR钓取目的基因。
PCR扩增目的基因片段:配制如下反应体系,见表9,轻轻吹打混均,短暂离心,置于PCR仪中进行反应
表9.反应体系配置
表10.反应条件
(4)PCR产物与载体进行交换
于冰水浴中配制如下反应体系,见表11。用移液器轻轻吹打混匀,短暂离心,避免产生气泡。于37℃反应30min,随后置于冰水浴中冷却5min后立即转化。
表11.反应体系配置
(5)转化
将10μl交换反应产物加入到100μL感受态细胞中,轻弹管壁数下混均,在冰上放置30min,42℃热激90s,冰水浴孵育2min,加入500μl LB培养基,置于37℃摇床振荡培养1h。取适当菌液均匀涂布在含有相应抗生素的平板上,在恒温培养箱中倒置培养12-16h。
(6)菌落PCR鉴定
引物:
表12.引物序列
ID | seq |
gene(16340-2)-P3 | CCTTCACCAGCTACTGGATG |
pEGFP-N-3 | CGTCGCCGTCCAGCTCGACCAG |
PCR鉴定:配比如表13反应体系,震荡混均,短暂离心,在超净工作台中,用无菌的墙头挑取单个菌落至20μl鉴定体系中,吹打混均,置于PCR仪中尽心光反应。
表13.反应体系配置
表14.PCR反应条件
鉴定结果见图4。
表15.图4中样品说明
*空白对照以ddH2O为模板,用于检测鉴定体系是否存在污染。
**阴性对照以未插入目的基因的空载体为模板,用于证明扩增过程中无假阳性现象。
(7)测序
将测定出的阳性克隆转化子接种于适量含相应抗生素的LB液体培养基中,37℃培养12-16h,取适量菌液进行测序。对测序结果与目的基因序列进行比对分析,比对结果测序通过。
3.1.2质粒抽提
将测序正确的菌液转接于10ml含相应抗生素的LB液体培养基中,37℃培养过夜,用天根无内毒素质粒小提中量试剂盒进行质粒抽提,抽提合格的质粒进入下游流程:
(1)收集过夜培养的菌液于标记好的5ml离心管,12000rpm,离心2min收菌;
(2)弃上清,加入250μl细胞重悬液,充分振荡,使菌块悬浮均匀;加入250μl细胞裂解液,再加入10μl蛋白酶K,上下颠倒5-6次,轻轻混匀;
(3)加入250μl细胞裂解液,再加入10μl蛋白酶K,上下颠倒5-6次,轻轻混匀;静置1-2min,致使菌体裂解澄清;
(4)加入350μl中和液,上下颠倒混匀,使蛋白完全析出,冰浴静置5min;
(5)10000rpm离心10min,弃蛋白,收集上清于另一干净无菌的1.5ml EP管;
(6)12000rpm离心5min,同时准备标记好的回收柱,将上清转移至回收柱中,12000rpm离心1min,弃下层废液;
(7)加入600μl预先配置好的漂洗液,12000rpm离心1min,弃下层废液,重复一次,12000rpm离心2min,进一步除去残留的漂洗液;
(8)在超净台中将回收柱转移至新的1.5ml EP管中,静置10-20min,自然晾干;
(9)往回收柱中加入95μl Nuclease-Free Water,静置2min,12000rpm离心2min,收集样品做好编号,电泳、测定浓度,进行质检。
3.1.3慢病毒包装与质量检测
(1)细胞株:293T,慢病毒的包装细胞,为贴壁依赖型成上皮样细胞,生长培养基为DMEM(含10%FBS)。贴壁细胞经培养生长增殖形成单层细胞。
菌株:大肠杆菌菌株DH5α,用于扩增慢病毒载体和辅助包装质粒。
载体:GV载体图谱:同前GV401,辅助质粒(Helper 1.0和2.0),见图5。
(2)质粒转染与慢病毒收获:
①转染前24h,用胰蛋白酶消化对数生长期的293T细胞,以含10%血清的培养基调整细胞密度约5×106个细胞/15ml,重新接种于10cm细胞培养皿,37℃、5%CO2培养箱内培养。24h待细胞密度达70-80%时即可用于转染。
②转染前2h更换为无血清培养基。
③向一灭菌离心管中加入所制备的各DNA溶液(GV载体质粒20μg、pHelper1.0载体质粒15μg、pHelper 2.0载体质粒10μg),与相应体积的吉凯转染试剂混合均匀,调整总体积为1ml,在室温下温育15min。
④混合液缓慢滴加至293T细胞培养液中,混匀,于37℃、5%CO2细胞培养箱中培养。
⑤培养6h后弃去含有转染混和物的培养基,加入10ml的PBS液清洗一次,轻柔晃动培养皿以洗涤残余的转染混和物后倒弃。
⑥缓慢加入含10%血清的细胞培养基20ml,于37℃、5%CO2培养箱内继续培养48-72h。
(3)慢病毒浓缩与纯化
①根据细胞状态,收集转染后48h(转染即可计为0h)的293T细胞上清液。
②于4℃、4000g离心10min,除去细胞碎片。
③以0.45μm滤器过滤上清液于40ml超速离心管中。
④分别配平样品,将带有病毒上清液的超速离心管逐一放入至Beckman超速离心机内,设置离心参数为25000rpm,离心时间为2h,离心温度控制在4℃。
⑤离心结束后,弃去上清,尽量去除残留在管壁上的液体,加入病毒保存液(可用PBS或细胞培养基替代),轻轻反复吹打重悬。
⑥经充分溶解后,高速离心10000rpm、5min后,取上清按要求分装。
⑦准备样品待检测。
(4)慢病毒质量检测
①物理指标检测
颜色判定:通过肉眼判定,吉凯公司研发的慢病毒保存液呈粉红色澄清液体状。
粘稠度判定:用20-200μl规格移液器缓慢吸取50μl慢病毒保存液体,无明显粘稠感或吸液滞后现象。
②无菌检测
将病毒加入293T细胞验证,正常培养24h后镜检,无任何细菌及真菌污染情况,同时参照空细胞组,细胞间隙无明显颗粒存在,培养基澄清透明。
③荧光法/药物筛法测定滴度
a测定前一天,使用293T贴壁细胞铺板,96孔板,每孔4×104个细胞,体积100μl。
b根据病毒的预期滴度,准备7-10个无菌的EP管,每管中加入90μl无血清培养基。
c取待测定的病毒原液10μl加入到第一个管中,混匀后,取10μl加入到第二个管中,继续相同的操作直到最后一管。
d选取所需的细胞孔,弃去90μl培养基,加入90μl稀释好的病毒溶液,培养箱培养。
e 24h后,加入完全培养基100μl,小心操作,不要吹起细胞。3-4天后可观察荧光表达情况,荧光表达数随稀释倍数的增加而减少。
f感染后72h加入抗性药物puromycin,维持药物浓度5μg/ml。继续培养1天,观察细胞生长状况。
④病毒样本稀释:第一个EP管中加入10μl病毒原液,记为10μl;第二个EP管中进行了第一次十倍稀释,所得病毒原液为第一个EP中的1/10,记为1μl;依次类推,第八个EP观众进行了第七次十倍稀释,记为1×10-6μl。
⑤滴度分析:
根据荧光图片中GFP表达情况,在1×10-6μl病毒原液感染孔中观察到2个荧光细胞,说明该孔至少有2个病毒颗粒感染了细胞,病毒滴度=荧光细胞数/病毒=2/1×10-6μl=2×106/L(TU/μl)=2×109TU/ml。
若为带有puromycin抗性的慢病毒,通过感染后的活细胞数量来计算病毒滴度。在加入1×10-5μl病毒原液的孔中观察到3个细胞存活,说明该孔至少有3个病毒感染了细胞,则:病毒滴度=活细胞数/病毒原液量=3/1×10-5=3×105(TU/ul)=3×108(TU/ml)。
3.2 PBMC分离
(1)将新鲜血液转移到50mL离心管中,用生理盐水按照1:1(体积比)进行稀释,轻轻上下吹打3-5次混匀。
(2)将6mL Ficoll加入到15mL离心管中,竖直放置到水平台面上静止备用。将稀释好的血液(总体积8mL)用移液管缓慢均匀加入到竖直放置在台面的含Ficoll溶液的离心管中。
(3)将加好血液与Ficoll的离心管轻轻转移至离心机中。在转移离心管过程尽量减少晃动,保持离心管竖直,以避免打破液面分层,室温400g离心30分钟。
(4)离心结束后,轻轻转移离心管至超净台中。用移液器吸取中间的白膜层(即淋巴细胞层)转移至新的50mL离心管中。尽量避免吸取到下层或上层的细胞,避免造成污染。
(5)加入30mL生理盐水至上述转移出来的淋巴细胞中,轻轻吹打混匀,60-100g室温离心10分钟。重复洗涤淋巴细胞,去除上清,加入一定量的完全培养基重悬细胞。
(6)计数调整密度为5×105个细胞/ml,预先处理过的12孔板中每孔加入1ml。将培养板放置于37℃,5%CO2培养箱培养48小时。
3.3慢病毒感染
(1)收集上述PBMC,室温300g离心4min,调整密度为5×105个细胞/ml,新的12孔板中每孔加入1ml细胞,实验组加入7.5μl MESO二代CAR的慢病毒(病毒滴度为2×108/mL,MOI=3);对照组加入7.5μl对照病毒(病毒滴度为2×108/mL,MOI=3),每孔加入一定的TRANSB感染试剂。
(2)将细胞培养板置于37℃,5%CO2培养箱培养。
(3)第三天每孔加入1ml新鲜的培养基,继续培养。
(4)第七天计数计算扩增倍数。
3.4 CAR-T表型和分型检测
(1)计数取出1.0×106个细胞,1000rpm,离心3min,加入100μl FACS buffer重悬细胞,加入15μl APC anti-human CD8、15μl PE anti-human CD4、15μl PerCY5.5anti-human CD3。
(2)4℃避光孵育30min,1000rpm,离心3min,去掉上清加入FACS buffer再洗两遍。
(3)加入150μl FACS buffer重悬细胞,FACS检测荧光。
结果:
3.5 MESO-CAR基因结构
MESO-CAR基因包含针对间皮素SS1P段的人全长scFv,CD3ζ激活信号(胞内信号域)和4-1BB一个胞内共刺激信号区以及CD8铰链区,见图6。
3.6 MESO-CAR慢病毒构建及包装
(1)实验组MESO-CAR序列如SEQ ID NO:1所示,对照组CON-CAR序列如SEQ ID NO:3所示,其中,MESO-CAR序列依次包括CD8-pre(SEQ ID NO:4)、MESO-scFv(SEQ ID NO:5)、CD8铰链+跨膜(SEQ ID NO:6)、4-1BB(CD137)-CD3ζ(SEQ ID NO:7),CON-CAR序列依次包括CD8-pre(SEQ ID NO:4),MESO-scFv(SEQ ID NO:5),CD8-ΔCD3ζ(SEQ ID NO:8)。实验组MESO-CAR和对照组CON-CAR阳性克隆序列均通过测序。
(2)病毒滴度
两种包装好病毒质量检测均合格,见表16。对照组CON-CAR慢病毒滴度为2×108TU/mL,MESO-CAR慢病毒滴度为1×109TU/mL,可以继续应用慢病毒感染T细胞。
表16.慢病毒质量控制检测结果
3.7 MESO-CAR-T细胞及CON-CAR-T细胞的制备
本次制备初始PBMC细胞量均为1×106,实验组MESO-CAR-T细胞第三天检测慢病毒感染效率为46.0%,第七天细胞增殖至1.68×107,细胞扩增倍数约为16倍,见表17。CON-CAR-T第三天慢病毒感染效率为31.5%,第七天细胞增殖至1.73×107,细胞扩增倍数约为17倍,见表17。
表17.MESO-CAR-T细胞及CON-CAR-T细胞增殖
3.8 CAR-T的细胞分群检测
MESO-CAR-T细胞及CON-CAR-T细胞分群检测结果,MESO-CAR-T CD3+T细胞占97.0%,CON-CAR-T CD3+T细胞占97.8%,且CD3+CD4+和CD3+CD8+细胞在两组比例相似。MESO-CAR-T组CD4+/CD8比例为0.5,CON-CAR-T CD4+/CD8比例为0.55,如表18和表19所示。
表18.MESO-CAR-T细胞亚群分布比例
表19.CON-CAR-T细胞亚群分布比例
讨论:
嵌合抗原受体(CARs)是由胞外抗原识别域和一个胞内信号域组成。通过将识别肿瘤相关抗原(Tumor associated antigen,TAA)的抗体单链可变区(scFv)和胞内信号域“免疫受体酪氨酸活化基序(Immunoreceptor tyrosine-based activation motifs,ITAM)”在体外进行基因重组,生成重组质粒。再将这种质粒通过基因转导的方法转入T细胞中,这样经过基因改造的T细胞称之为CAR-T细胞。CAR-T细胞在体外经大规模扩增后,回输到患者体内,能够以非MHC限制性的模式表现强效的抗癌作用。CARs技术至今已发展至第四代,用于临床试验的主要是第二代和第三代CAR。第二代CARs是在第一代的基础上添加了一个胞内信号区,使其本身就可以介导T细胞的激活,而不需要共刺激。第三代CARs则在胞内添加了两个串联的共刺激信号区,同时提高了T细胞自身的增殖能力和杀伤毒性。最近又出现了第四代CARs,整合表达一个白介素12因子(IL-12)的基因,分泌IL-12因子,增加CAR-T杀伤性和T细胞增殖分化。
CARs的胞外部分用来识别特异性的肿瘤抗原,随后胞内信号域会刺激T细胞增殖,并且通过细胞溶解和细胞因子释放来消除肿瘤细胞。二代CARs共刺激信号包括CD28、CD27、CD134(OX40)或CD137(4-1BB)。不同共刺激分子功能不同,影响激活阈值。反应类型、生存时间以及CAR-T细胞亚型。CD28、CD134对提高初始T细胞获得持久的体外增殖和较强的细胞因子分泌非常重要。CD28是T细胞表面的一个蛋白,能够为T细胞提供刺激信号,对T细胞的活化和存活具有重要作用。T细胞刺激信号通过CD28,能够促进多种白介素的分泌。4-1BB(CD137)是TNF受体家族成员,其表达与活化后的T细胞,也表达于树突细胞、滤泡树突细胞、NK细胞、粒细胞及炎症位置的血管壁细胞等。4-1BB可救援和放大CD3ζ的激活信号,双倍的信号通过增强T细胞增殖和细胞因子(IFN-γ和IL-2)的产生来防止T细胞失能并增加其持久性和发挥其功能,其在体内增殖性和持久性作用超过共刺激因子CD28,还通过招募PI3K,TRAF和其它通路的信号分子来降低活化引起的细胞死亡。目前临床试验中试验CAR-T细胞大部分为二代CARs,在治疗B淋巴细胞白血病的临床试验中含有CD28或4-1BB共刺激信号的CAR-T细胞均表现出优异疗效,含有4-1BB共刺激信号的二代CAR-T细胞应用范围及杀伤效果略优于含CD28的CAR-T细胞,因此我们选择4-1BB作为本研究二代MESO-CAR的共刺激信号。
本发明构建了二代MESO-CAR的慢病毒,在成功构建CAR慢病毒实验过程中,首先构建了PNBS328-meso CAR质粒,经酶切鉴定正确,后经测序比对序列完全正确,并无碱基突变存在,近而用来进行后续实验。MESO-CAR基因包含针对间皮素SS1P段的人全长scFv,以及CD3ζ胞内激活信号区和4-1BB一个胞内共刺激信号区(Costimulatory molecule,CM)的第二代CAR。
研究组MESO-CAR-T细胞与对照组CON-CAR-T比较,慢病毒感染效率高,均成功感染,细胞增殖满意。细胞分群检测,前者MESO-CAR-T CD3+T细胞占97.0%,后者CON-CAR-TCD3+T细胞占97.8%,且二者在CD3+CD4+和CD3+CD8+细胞亚群的分布上没有明显区别,CD3+CD4+/CD3+CD8+比值约0.5。实验组MESO-CAR-T和对照组CON-CAR-T细胞成功构建,为后续试验提供基础。
结论
二代MESO-CAR慢病毒成功感染PBMC细胞,成功构建实验组MESO-CAR-T细胞和对照组CON-CAR-T细胞。
实施例4 MESO-CAR-T细胞体外杀伤功能检测
实验方法:
4.1靶细胞验证
(1)正常培养目的细胞,确保细胞状态良好。
(2)细胞计数,每组每管取出5×105个细胞,1000rpm,离心3min,用FACS buffer洗一次细胞,再相同条件离心一次,并用200μl FACS buffer重悬细胞,再加入2μl抗体。
(3)室温避光孵育45min后,1000rpm,离心3min,去掉上清加入FACS buffer再洗两遍。
(4)加入400μl FACS buffer重悬细胞,FACS检测荧光。
4.2 T细胞体外功能验证
4.2.1 LDH释放
(1)收集靶细胞(SiHa),用稀释缓冲液将细胞洗涤一次,1000rpm 3min离心,弃上清,以含2%FBS的1640重悬细胞、计数,最终将细胞密度调整为1×105个细胞/ml。
(2)收集CAR-T(效应细胞),用稀释缓冲液将细胞洗涤一次,1000rpm 3min离心,弃上清,以含2%FBS的1640重悬细胞、计数,将细胞密度分别调整为2×106个细胞/ml、0.5×106个细胞/ml、0.2×106个细胞/ml、0.1×106个细胞/ml。
(3)将上述细胞分别加入U型底96孔板中(200μl体系),分组如表20,E(MESO-CAR-T细胞):T(靶细胞SiHa)分别为20:1、5:1、2:1、1:1,见表20。
表20.CAR-T细胞与靶细胞的配比分组
(4)细胞放入含5%CO2的37℃培养箱中进行培养4-6小时。
(5)实验终点前45min,在靶细胞最大释放组中加入20μl 10×lysis。
(6)培养终点1500rpm 5min离心培养板,每孔取50μl上清液转移到新的96孔板中,每孔加入50μl混合后的底物,室温避光孵育30min,加入50μl终止液,490nm检测吸收值。
4.2.2靶细胞存在下的CAR-T增殖
(1)收集靶细胞,用稀释缓冲液将细胞洗涤一次,1000rpm离心3min,弃上清,以含2%FBS的1640重悬细胞计数,最终将细胞密度调整为1×105个细胞/ml。
(2)收集带有GFP的CAR-T(效应细胞),用稀释缓冲液将细胞洗涤一次,1000rpm离心3min,弃上清,以含2%FBS的1640重悬细胞计数,将细胞密度分别调整为2×105个细胞/ml。
(3)上述效应细胞和靶细胞各1ml加入12孔板中,37℃、5%CO2培养箱中培养72小时。
(4)FACS检测GFP阳性细胞。
4.2.3细胞因子分泌
(1)收集SiHa(靶细胞)及CAR-T细胞(效应细胞),用稀释缓冲液将细胞洗涤一次,1000rpm离心3min,弃上清,以含2%FBS的1640重悬细胞,计数,最终将细胞稀释到1×106个细胞/ml浓度。
(2)将100ul靶细胞和100μl CAR-T细胞1:1混合加入96孔板中,37℃、5%CO2共同培养20~24小时。
(3)1000rpm离心5min,收集上清检测细胞因子。
4.2.4细胞因子检测
准备捕获珠:
(1)确定好需要检测的细胞因子及样品个数。
(2)取出待检测的细胞因子捕获珠子瓶,用力混匀。每种珠子取出(待检测样品数+阴性对照数)×10μl的捕获珠子量。并将每种珠子进行涡轮震荡混匀。
样品准备及检测。
(3)用标准品稀释液对样品进行稀释(稀释倍数如1:10)。
(4)取N个1.5ml EP管(N=样品数+对照数)。
(5)向每管(样品、阴性对照)中加入50μl混合珠子;并向各管中加入相应待测试剂(样品、对照)50μl以及PE Detection Reagent 50μl,充分混合后,避光室温孵育3小时。
(6)每管中加入1ml wash buffer,轻柔吹匀后200g离心5分钟。
(7)小心吸去上清,并向每管中加入200μl wash buffer,并重悬沉淀。
(8)将样品进行流式细胞仪检测。
结果:
4.3靶细胞验证
我们选择宫颈鳞癌SiHa和Caski细胞系,宫颈腺癌HeLa细胞系进行MESO表达检测,其表达率最高的细胞系作为本研究靶细胞。从FACS(流式细胞学检测)结果来看,12.8%的SiHa细胞膜上天然能检测到MESO的表达,但在Caski细胞上MESO的表达阳性率极低,仅为0.1%。相对阴性对照细胞MIA PaCa-2及阳性对照SiHa细胞,Hela细胞中MESO表达阳性率低于SiHa细胞表达。因此,后续研究选择MESO表达阳性率较高的SiHa细胞作为靶细胞,而Caski细胞因MESO表达阳性率极低作为非靶细胞(non-target)。
4.4乳酸脱氢酶(LDH)试验检测CAR-T细胞的杀伤能力
MESO-CAR-T细胞:靶细胞分别按1:1,2:1,5:1和20:1,大概比例5:1时,共培养4h后,LDH乳酸脱氢酶实验提示MESO-CAR-T组对靶细胞细胞裂解率高于对照组,当配比为20:1时,靶细胞裂解率达到最大为22%,见图7、表21-23,无论效靶比多少,CON-CAR-T细胞在靶细胞存在情况下杀伤率维持在10%,MESO-CAR-T细胞在非靶细胞存在情况下杀伤率维持在14%,CON-CAR-T细胞在非靶细胞存在情况下杀伤率维持在5%-10%。实验重复三次取平均值,实验各体系情况见图7和表21。
表21.分组情况
组别 | 体系组成 |
BackGround | 培养基200μl |
Target Auto Release | 培养基100μl+(非)靶细胞100μl |
Y | 培养基100μl+(非)靶细胞100μl(实验结束前加入lysis) |
Effector | 培养基100μl+不同倍数的CAR-T-Test(-CON)100μl |
X | (非)靶细胞100μl+不同倍数的CAR-T-Test(-CON)100μl |
计算公式=(X-Effector-Target Auto Release+BackGround)/(Y-BackGround)
表22.三次LDH计算转化后结果平均值
表23.三次LDH计算转化后方差分析
4.5存在靶细胞时MESO-CAR-T细胞的增殖
CAR-T细胞与SiHa(靶细胞)比值为2:1时,MESO-CAR-T细胞和CON-CAR-T细胞增殖结果,在靶细胞存在条件下,MESO-CAR-T细胞及CON-CAR-T细胞增殖不明显,见图8。
4.6细胞因子分泌实验
本次实验分别检测了IL-2、IL-4、IL-5、IL-10、TNF-α、IFN-γ的分泌,实验结果表明MESO-CAR-T细胞在存在靶细胞时,IL-2、IL-4、IL-5、IL-10、TNFα、IFN-γ的分泌高于对照组CON-CAR-T细胞,结果显示MESO-CAR-T细胞具有一定的杀伤活性,见表24-25,实验重复三次取平均值。
表24.三次不同组细胞因子分泌检测平均值
表25.三次不同组细胞因子分泌检测方差
讨论:
本研究选择宫颈癌SiHa、HeLa及Caski三种细胞系进行表面抗原MESO表达阳性率的检测,在SiHa细胞系中MESO蛋白表达阳性率最高,因此选择SiHa细胞作为靶细胞进行下一步研究。
间皮素(MESO)的过表达最初是在间皮瘤和卵巢癌中发现的,接着又在肺癌、食管癌、胰腺癌、胃癌、胆管癌、子宫内膜癌、胸腺癌、结肠癌和乳腺癌中发现到。仅在美国,间皮素过表达的发病率从每年34万增长至两百万。间皮素在90%的上皮样恶性胸膜间皮瘤中都表达,在肺腺癌中的表达率为69%,乳腺癌中60%,食管癌中46%。因此,MESO是一个较特异的肿瘤标志物,可作为肿瘤治疗的潜在靶点。本发明研究显示宫颈鳞癌MESO的表达阳性率为12.8%,但至今CAR-T细胞在宫颈癌研究少有研究。因此本文旨在探究MESO-CAR-T细胞在宫颈癌中的应用价值。
在靶细胞(SiHa细胞)存在条件下,MESO-CAR-T细胞及CON-CAR-T细胞增殖不明显。流式细胞学(FACS)检测在SiHa细胞的MESO抗原刺激下,MESO-CAR-T细胞中IL-2、IL-4、IL-5、IL-10、TNF-α、IFN-γ六种细胞因子的分泌情况。结果显示,与对照组CON-CAR-T细胞组相比,MESO-CAR-T细胞组中上述细胞因子分泌都显著升高。IFN-γ、TNF-α和IL-2的升高可在一定程度上反映出免疫功能的增强,证明MESO-CAR-T细胞可高效杀伤MESO高表达的SiHa细胞,而对几乎无MESO靶抗原的Caski细胞杀伤作用小。
通过LDH细胞毒性检测试剂盒检测MESO-CAR-T细胞对靶细胞SiHa细胞的毒性作用,4h共培养条件下,1:1、2:1、5:1和20:1四种效靶比时,MESO-CAR-T细胞对SiHa靶细胞的杀伤率分别达到了9%、10%、18%和22%,MESO-CAR-T细胞:SiHa细胞比值为5:1时,MESO-CAR-T细胞对SiHa细胞杀伤性高于对照组,MESO-CAR-T细胞:SiHa比例为20:1时,杀伤性最强,靶细胞(SiHa细胞)裂解率为22%。相比之下,无论效靶多少,CON-CAR-T细胞在SiHa靶细胞存在情况下杀伤率维持在10%,MESO-CAR-T细胞在非靶细胞存在情况下杀伤率维持在14%,CON-CAR-T细胞在非靶细胞存在情况下杀伤性维持5%-10%。这些结果都表明我们构建的MESO-CAR-T细胞具有一定的特异性,且效果显著。
结论:
体外杀伤性研究显示,MESO-CAR-T细胞组在SiHa靶细胞存在情况下,细胞因子分泌明显升高,但增殖不显著;MESO-CAR-T细胞与SiHa细胞(靶细胞)比例为20:1时,细胞杀伤性最强。MESO-CAR-T细胞在体外有特异性杀伤力,且效果显著
实施例5 MESO-CAR-T细胞小鼠模型体内杀伤性
实验方法:
(1)准备足量带有荧光素酶报告基因的SiHa细胞,将处于对数生长期的各实验组成瘤细胞胰酶消化后,完全培养基重悬成细胞悬液。
(2)用血球计数板对细胞进行计数,并最终用一定体积的D-Hanks或PBS重悬,使细胞悬液的浓度为2×107个细胞/ml。
(3)准备好肿瘤细胞后(2×107个细胞/mL),使用一次性无菌注射器,吸取细胞,注射至动物皮下,每只200μl。
(4)根据细胞成瘤能力,5-20天后,观察成瘤情况,直径大于5mm的,可以开始给药并测量肿瘤大小,动物体重。测量肿瘤大小和动物体重频率根据瘤体增殖情况而定,至少保证5次测量数据。
(5)在处死动物前对动物进行活体成像:按10μl/g的量腹腔注射0.7%戊巴比妥钠麻醉动物,数分钟后待动物麻醉,将其放置于活体成像仪下进行成像,观察荧光,保存数据。Luciferase发光检测,需先按10μl/g剂量腹腔注射D-Luciferin(15mg/Ml),等待15-20分钟左右后再进行麻醉检测。
(6)使用成像仪自带软件进行成像:设置活体成像各项参数,调整焦距,并根据设定好的检测程序对动物进行活体成像检测,观察动物体内或皮下的荧光表达。
(7)使用软件进行定量分析并收集数据。
(8)将成像完成的动物依次放回饲养笼具中,2小时后进行动物健康状态观察。
(9)皮下注射肿瘤细胞到达观察天数后,实验动物进行注射过量2%戊巴比妥钠安乐死,并进行颈椎脱落确认死亡。在白板上排列好动物,并用2把标尺放置于左边及上方,以便观察具体刻度,用数码照相机拍摄动物照片。并将肿瘤放在上方便于观察。
(10)用医用剪刀和医用镊子取出肿瘤,并在白板上排列好后拍照留存。同样需要标尺作为参照,读取具体刻度。
(11)将取出的肿瘤称重,并放于多聚甲醛固定常温保存或液氮冷冻后-80℃保存。
结果:
5.1小鼠预实验(注射一次MESO-CAR-T细胞)
选择四只严重联合免疫缺陷小鼠(Severe Combined Immunodeficient,SCID),分别标号1-4号。1-2号注射SiHa细胞成瘤数量为5×106个细胞/只,3-4号注射SiHa细胞数量为1×107个细胞/只,2号和4号为MESO-CAR-T细胞,1号和3号为CON-CAR-T对照组细胞。于SCID裸鼠右侧皮腋下种植SiHa鳞癌细胞,培养18天后,于第19天瘤体内注射MESO-CAR-T细胞1×107个细胞,每周进行一次活体成像到第69天,两组均在D22-D29天左右,MESO-CAR-T细胞组肿瘤大小<对照组,此后肿瘤增长迅速,MESO-CAR-T细胞组肿瘤增大明显>对照组。提示MESO-CAR-T细胞1周内起效,1周左右达到最佳疗效。于D69天处死裸鼠,皮下注射SiHa细胞数量为5×106个细胞/只中,对照组肿瘤重量(CON-CAR-T)3.379g,研究组肿瘤重量(MESO-CAR-T)3.611g;皮下注射SiHa细胞数量为1×107个细胞/只中,对照组肿瘤重量(CON-CAR-T)2.795g,研究组肿瘤重量(MESO-CAR-T)3.975g。接种106-107SiHa细胞可成瘤,研究组和对照组裸鼠及瘤体照相并进行测量肿瘤大小差异不显著,研究组(MESO-CAR-T)甚至有增大趋势,上述小鼠未见一例提前死亡。
5.2小鼠体内MESO-CAR-T细胞杀伤性研究(注射二次MESO-CAR-T细胞)
另选择四只SCID裸鼠,分别标号1-4号。1-2号裸鼠右侧皮腋下注射SiHa细胞成瘤数量为2×106个细胞/只,3-4号注射细胞数量为5×106个细胞/只,1号和3号为MESO-CAR-T细胞,2号和4号为CON-CAR-T对照组。瘤细胞成瘤培养14天后,于第15天和第22天瘤体内注射实验组MESO-CAR-T细胞(1×107个细胞)或CON-CAR-T细胞为对照细胞各二次,每周进行二次活体成像到第36天。每次活体成像同时测量肿瘤体积,见图9和表26,在D26天皮下注射SiHa细胞2×106个细胞/只组,在D22天皮下注射SiHa细胞5×106个细胞/只组,研究组(MESO-CAR-T细胞)肿瘤小于对照组(CON-CAR-T细胞),说明MESO-CAR-T细胞在注射1周内起效,1周左右疗效最佳。上述小鼠未见一例提前死亡。
表26.肿瘤体积测量
讨论:
在SCID小鼠体内进行MESO-CAR-T细胞杀伤性,选择5×106个细胞/只和2×106个细胞/只SiHa细胞进行裸鼠皮下成瘤,后者肿瘤大小在同一时间点小于前者,分别在第15天和第22天注射两次CAR-T细胞(1×107)。注射两次CAR-T细胞后,2×106个细胞/只组在D26天和5×106个细胞/只组在D22天,MESO-CAR-T细胞肿瘤组织大小小于CON-CAR-T细胞组,且在2×106个细胞/只组注射MESO-CAR-T细胞后肿瘤组织缩小明显。由于在体内条件影响因素很多,构建困难,在体内观察MESO-CART细胞对宫颈癌适度抑制。根据目前结果,考虑在SiHa细胞为2×106个细胞/只组中MESO-CAR-T细胞效果最佳,MESO-CAR-T细胞在注射后1周内有效果,减缓肿瘤增长。在小鼠试验过程中未见一例小鼠提前死亡,考虑MESO-CAR-T细胞无致死性副作用。
目前在小鼠体内宫颈肿瘤(鳞癌)抑制效果不显著可能与以下几个因素有关:①宫颈鳞癌中MESO阳性表达率不高(12.8%),远低于MESO在以下肿瘤中表达,包括:上皮样恶性胸膜间皮瘤85-90%,胰腺癌80-85%,肺腺癌60-65%,卵巢癌60-65%,乳腺癌25-60%,食管癌中35-40%,子宫内膜癌20-25%,上述肿瘤都有相应临床试验进行中。②开始注射MESO-CAR-T细胞时,瘤灶较大;注射CAR-T细胞次数少及剂量小有关,相关参数仍需调整。③更换二代CARs 4-1BB为共刺激因子CD27、CD28、CD134或者增加共刺激因子应用三代CAR-T细胞可能会改善效果。④另外本研究制备MESO-CAR-T细胞与CON-CAR-T细胞扩增后CD4+/CD8+T细胞比例为0.5,文献报道CD4+/CD8+T细胞比例接近1:1时可增加疗效,因此调整CD4+/CD8+T细胞可否改善效果需要进一步研究。
结论:
体外研究表明,MESO-CAR-T细胞在小鼠接种SiHa细胞为2×106个细胞/只时体内杀伤性最强,连续两次瘤内注射MESO-CAR-T细胞效果较一次注射明显,有效时间为注射MESO-CAR-T细胞1周内,1周左右达到最佳疗效,因体内因素众多、复杂,小鼠体内MESO-CAR-T细胞效果并不理想,需进一步摸索条件,待进一步研究。在小鼠试验过程中未见一例小鼠提前死亡,考虑MESO-CAR-T细胞无致死性副作用。
以上结合优选实施方式和范例性实例对本发明进行了详细说明。不过需要声明的是,这些具体实施方式仅是对本发明的阐述性解释,并不对本发明的保护范围构成任何限制。在不超出本发明精神和保护范围的情况下,可以对本发明技术内容及其实施方式进行各种改进、等价替换或修饰,这些均落入本发明的保护范围内。本发明的保护范围以所附权利要求为准。
序列表
<110> 首都医科大学附属北京妇产医院
<120> 一种用于癌症靶向或免疫治疗的组合物及其应用
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gccatcatca tcacccccgt ggtgcccaag ttcgactact ggggccaggg caccaccctg 840
accgtgagca gcaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 900
tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 960
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aggagcgcag acgcccccgc gtacaagcag ggccagaacc agctctataa cgagctcaat 1260
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Asp Gly Val Leu Ala Asn Pro Pro Asn Ile Ser Ser Leu Ser Pro Arg
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Gln Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu Ser Thr Glu
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Arg Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys Asn Val Lys Leu
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Ser Thr Glu Gln Leu Arg Cys Leu Ala His Arg Leu Ser Glu Pro Pro
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Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu Leu Leu Phe Leu Asn Pro
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Asp Ala Phe Ser Gly Pro Gln Ala Cys Thr Arg Phe Phe Ser Arg Ile
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Thr Lys Ala Asn Val Asp Leu Leu Pro Arg Gly Ala Pro Glu Arg Gln
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Arg Leu Leu Pro Ala Ala Leu Ala Cys Trp Gly Val Arg Gly Ser Leu
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Leu Ser Glu Ala Asp Val Arg Ala Leu Gly Gly Leu Ala Cys Asp Leu
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Pro Gly Arg Phe Val Ala Glu Ser Ala Glu Val Leu Leu Pro Arg Leu
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Val Ser Cys Pro Gly Pro Leu Asp Gln Asp Gln Gln Glu Ala Ala Arg
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Leu Arg Pro Arg Phe Arg Arg Glu Val Glu Lys Thr Ala Cys Pro Ser
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Gly Lys Lys Ala Arg Glu Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys
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Trp Glu Leu Glu Ala Cys Val Asp Ala Ala Leu Leu Ala Thr Gln Met
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Asp Arg Val Asn Ala Ile Pro Phe Thr Tyr Glu Gln Leu Asp Val Leu
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Lys His Lys Leu Asp Glu Leu Tyr Pro Gln Gly Tyr Pro Glu Ser Val
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Ile Gln His Leu Gly Tyr Leu Phe Leu Lys Met Ser Pro Glu Asp Ile
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Arg Lys Trp Asn Val Thr Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu
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Val Asn Lys Gly His Glu Met Ser Pro Gln Ala Pro Arg Arg Pro Leu
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Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val Lys Gly Arg Gly Gln
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Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala Phe Tyr Pro Gly Tyr
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Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser Val Pro Pro Ser Ser
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Ile Trp Ala Val Arg Pro Gln Asp Leu Asp Thr Cys Asp Pro Arg Gln
465 470 475 480
Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala Phe Gln Asn Met Asn
485 490 495
Gly Ser Glu Tyr Phe Val Lys Ile Gln Ser Phe Leu Gly Gly Ala Pro
500 505 510
Thr Glu Asp Leu Lys Ala Leu Ser Gln Gln Asn Val Ser Met Asp Leu
515 520 525
Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val Leu Pro Leu Thr Val
530 535 540
Ala Glu Val Gln Lys Leu Leu Gly Pro His Val Glu Gly Leu Lys Ala
545 550 555 560
Glu Glu Arg His Arg Pro Val Arg Asp Trp Ile Leu Arg Gln Arg Gln
565 570 575
Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gln Gly Gly Ile Pro Asn
580 585 590
Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu Ala Leu Ser Gly Thr
595 600 605
Pro Cys Leu Leu Gly Pro Gly Pro Val Leu Thr Val Leu Ala Leu Leu
610 615 620
Leu Ala Ser Thr Leu Ala
625 630
<210> 3
<211> 1101
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaactcg 60
gccatggcct taccagtgac cgccttgctc ctgccgctgg ccttgctgct ccacgccgcc 120
aggccggaca tcgtgatgac ccagagccac cagttcatga gcaccagcgt gggcgaccgc 180
gtgagcgtga cctgcaaggc cagccacgac gtgggcacca gcgtggcctg gtaccagcag 240
aagcccggcc agagccccaa gctgctgatc tactgggcca gcacccgcca caccggcgtg 300
cccgaccgct tcaccggcag cggcagcggc accgacttca ccctgaccat cagcaacgtg 360
cagagcgagg acctggccga ctacttctgc cagcagtaca gcagctaccc cctgaccttc 420
ggcgccggca ccaagctgga gctgaagggt ggaggcggtt caggcggcgg tggctctagc 480
ggtggtggat cgcaggtgca gctgcagcag cccggcgccg agctggtgaa gcccggcgcc 540
agcatgaagc tgagctgcaa ggccagcggc tacaccttca ccagctactg gatgcactgg 600
gtgaagcagc gccccggcca gggcctggag tggatcggca tgatccaccc caacagcgac 660
aacaccatct actacgagaa gttcaagagc aaggccaccc tgaccgtgga caagagcagc 720
agcaccgcct acatgcagct gagcagcctg accagcgagg acagcgccgt gtactactgc 780
gccatcatca tcacccccgt ggtgcccaag ttcgactact ggggccaggg caccaccctg 840
accgtgagca gcaccacgac gccagcgccg cgaccaccaa caccggcgcc caccatcgcg 900
tcgcagcccc tgtccctgcg cccagaggcg tgccggccag cggcgggggg cgcagtgcac 960
acgagggggc tggacttcgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 1020
ggggtccttc tcctgtcact ggttatcacc ctttactgca gagtgaagtt cagcaggagc 1080
gcagacgccc ccgcgtacta a 1101
<210> 4
<211> 63
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaactcg 60
gcc 63
<210> 5
<211> 789
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggacatcg tgatgaccca gagccaccag ttcatgagca ccagcgtggg cgaccgcgtg 120
agcgtgacct gcaaggccag ccacgacgtg ggcaccagcg tggcctggta ccagcagaag 180
cccggccaga gccccaagct gctgatctac tgggccagca cccgccacac cggcgtgccc 240
gaccgcttca ccggcagcgg cagcggcacc gacttcaccc tgaccatcag caacgtgcag 300
agcgaggacc tggccgacta cttctgccag cagtacagca gctaccccct gaccttcggc 360
gccggcacca agctggagct gaagggtgga ggcggttcag gcggcggtgg ctctagcggt 420
ggtggatcgc aggtgcagct gcagcagccc ggcgccgagc tggtgaagcc cggcgccagc 480
atgaagctga gctgcaaggc cagcggctac accttcacca gctactggat gcactgggtg 540
aagcagcgcc ccggccaggg cctggagtgg atcggcatga tccaccccaa cagcgacaac 600
accatctact acgagaagtt caagagcaag gccaccctga ccgtggacaa gagcagcagc 660
accgcctaca tgcagctgag cagcctgacc agcgaggaca gcgccgtgta ctactgcgcc 720
atcatcatca cccccgtggt gcccaagttc gactactggg gccagggcac caccctgacc 780
gtgagcagc 789
<210> 6
<211> 207
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
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> 7
<211> 462
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactgagag tgaagttcag caggagcgca gacgcccccg cgtacaagca gggccagaac 180
cagctctata acgagctcaa tctaggacga agagaggagt acgatgtttt ggacaagaga 240
cgtggccggg accctgagat ggggggaaag ccgagaagga agaaccctca ggaaggcctg 300
tacaatgaac tgcagaaaga taagatggcg gaggcctaca gtgagattgg gatgaaaggc 360
gagcgccgga ggggcaaggg gcacgatggc ctttaccagg gtctcagtac agccaccaag 420
gacacctacg acgcccttca catgcaggcc ctgccccctc gc 462
<210> 8
<211> 249
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgatatcta catctgggcg cccttggccg ggacttgtgg ggtccttctc 180
ctgtcactgg ttatcaccct ttactgcaga gtgaagttca gcaggagcgc agacgccccc 240
gcgtactaa 249
Claims (10)
1.一种宫颈癌治疗组合物,所述组合物包括一种或多种抗癌抗体,所述抗癌抗体选自MESO抗体、MUC1抗体、CD133抗体和GD2抗体。
2.一种宫颈癌治疗组合物,所述组合物包括MESO-CAR-T细胞,所述MESO-CAR-T细胞是表达MESO-CD8-4-1BB-CD3ζ融合蛋白的T淋巴细胞。
3.根据权利要求1或2所述的组合物,其特征在于,所述组合物还包括一种或多种免疫促进剂。
4.一种宫颈癌疫苗,所述疫苗包括一种或多种癌症相关抗原,所述癌症相关抗原选自MESO蛋白、MUC1蛋白、CD133蛋白和GD2蛋白。
5.权利要求1、2或4中任一项所述的组合物在制备宫颈癌治疗药物中的应用。
6.根据权利要求5所述的应用,其特征在于,所述宫颈癌治疗药物为宫颈癌免疫治疗药物。
7.根据权利要求5所述的应用,其特征在于,所述宫颈癌治疗药物为宫颈癌靶向治疗药物。
8.根据权利要求5所述的应用,其特征在于,所述宫颈癌治疗药物为杀伤宫颈癌细胞的药物。
9.MSLN基因在制备宫颈癌治疗药物中的应用,以MSLN基因作为抗原结合域基因与跨膜区和信号转导区基因进行基因重组,生成重组质粒,再将该质粒通过基因转导的方法转入T细胞中,形成MESO-CAR-T细胞,用于宫颈癌治疗药物的制备。
10.根据权利要求9所述的应用,其特征在于,以MSLN基因作为抗原结合域基因与跨膜区和信号转导区基因进行基因重组得到的重组基因序列为SEQ ID NO:1。
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