CN113577265B - Til细胞及其制备方法以及在癌症治疗中的用途 - Google Patents
Til细胞及其制备方法以及在癌症治疗中的用途 Download PDFInfo
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Abstract
本发明涉及TIL细胞及其制备方法以及在癌症治疗中的用途。本发明提供了一种有效分离并制备的肿瘤浸润淋巴细胞,将所述细胞转TNF‑α高表达后与制备的CTLA‑4单克隆抗体和培美曲塞一起使用能够显著的抑制肺癌肿瘤的增殖,具有较好的效果。
Description
技术领域
本发明涉及生物领域,更具体的涉及TIL细胞及其制备方法以及在癌症治疗中的用途。
背景技术
肿瘤浸润淋巴细胞(TIL)是从肿瘤组织中分离出来的肿瘤抗原特异性CD4+、CD8+细胞群,其疗效是淋巴因子激活的杀伤细胞(LAK)的50~100倍,其中发挥杀伤肿瘤作用的主要为CD8+T细胞,大量临床研究表明TILs联合高剂量白细胞介素-2(IL-2)在晚期多发性骨髓瘤(MM)患者中可达到约50%的阳性反应率,且持续缓解时间较长,使治愈MM成为可能。在肿瘤抗原的长期刺激下,肿瘤浸润淋巴细胞表面的一些抑制性受体(PD-1,CTLA-4,Tim-3,LAG-3等)表达水平上调,处于“衰竭”状态,大量研究表明,阻断这些抑制性受体通路可使TILs功能恢复,可明显提高TILs的杀伤肿瘤能力,TILs在肿瘤免疫治疗中具有极其重要的作用,这为肿瘤的靶向治疗提供了依据。Ln-145是一种最新开发的TIL治疗方法,2019年,FDA授予肿瘤浸润淋巴细胞(TIL)治疗方法LN-145为突破性的治疗指定,这是用于实体瘤的细胞免疫疗法首次获此殊荣,预计2021年上市,一旦FDA批准,这将是首款用于实体瘤的细胞免疫疗法,将给癌症患者带来巨大的生存获益。
关于TIL的抗肿瘤机制,目前已经得到证实的是:①直接杀伤作用:TIL杀伤肿瘤细胞主要依赖其中的CTL的有效激活和有效行使功能。T细胞转化为效应细胞,需要有两个刺激信号:第一激活信号由TCR与抗原肽:主要组织相容性复合体(MHC)分子复合物结合提供;第二激活信号由T细胞上的CD28与抗原递呈细胞(APC)上的B7分子结合提供。分化成熟的效应T细胞经过识别与结合、细胞器重排与颗粒外吐、CTL解离、靶细胞解体四个阶段发挥其杀伤功能,特异性裂解靶细胞;②Fas介导的细胞凋亡:淋巴细胞通过表面的Fas配体与靶细胞上的Fas结合,引起细胞DNA的片段化,导致细胞发生凋亡;③穿孔素(performingprotein,PFP)/颗粒酶B(granymeB,GranB)介导的细胞凋亡:CTL可以通过外吐胞质颗粒,释放颗粒内容物,杀伤靶细胞。穿孔素可以在靶细胞膜上聚合,形成穿膜孔道从而裂解靶细胞。颗粒酶B能够通过依赖/非依赖caspase途径诱导凋亡;④TIL分泌的一些炎性细胞因子,如TNFct,IL2等介导的细胞溶解与凋亡,该途径可通过释放颗粒或直接细胞接触杀伤靶细胞。已经发展的各项研究表明,TIL治疗将有待不断改进和发展,最终将成为人类抗癌的新武器。
肺癌常伴恶性胸腔积液,这与胸膜转移后渗出有关。临床上常应用胸腔穿刺抽液及胸腔内注入顺铂(DDP)、丝裂霉素C(MMC)、博莱霉素(BLM)等化疗药物治疗,但许多肿瘤化疗药物短期使用可以使癌细胞死亡,长期使用容易使癌细胞产生耐药性,导致对肿瘤特异杀伤性不强。TIL与化疗药物的联合应用或交替应用可以提高患者生存期。常用的提取治疗肺癌用的TIL有三种途径:(1)从恶性胸腔积液中提取,取肝素抗凝无菌胸腔积液离心后,制备细胞悬浮于Hank液,辅助以淋巴细胞分离液,收集界面上细胞。以10%人AB血清及rIL-2培养,根据扩增及代谢情况按需换液。(2)从手术切除的瘤体或活检标本中提取,将获得的组织消化解离,消化后的单个细胞悬液经非连续密度梯度离心,即可获得较纯的TIL。(3)转移淋巴结,手术或活检获取的转移淋巴结同样含有丰富的TIL,1g淋巴结可分离约108个TIL,足以培养获取治疗量。但是目前的研究还不多,针对肺癌的治疗也不多。
发明内容
本发明克服现有技术的缺陷。提供了一种用于治疗肺癌的TIL细胞及相应的单克隆抗体。
一方面,本发明提供一种TIL细胞的制备方法,所述方法包括如下步骤:将新鲜肺癌肿瘤组织置于含抗生素的冷Hanks’液中浸泡30min,去除坏死、出血及脂肪组织,称重,剪成lmm×lmm×lmm小块,加入以RPMI1640配制的有胶原酶Ⅱ型的消化酶液中震荡消化12h,再加入透明质酸酶和DNA酶,搅拌4h消化处理。完全消化后的组织过120目铜网,即得到含TIL和肿瘤细胞的单细胞悬液。将混合细胞悬液3ml轻轻加于lml小牛血清界面上,500r/min离心15s后,底层为瘤细胞,将上层细胞悬液再行连续梯度分离,获取TIL。将分离的TIL在含10%人AB血清的RPMI1640完全培养基中以1×106/ml初始浓度于24孔板中培养,并加入IL-2 10μg/ml,同时加OKT3 10μg/ml和VC 1μg/ml,37℃、5%CO2孵箱培养,3~5天传代一次,共培养20d,即得到目的TIL细胞。
另外一方面,本发明提供一种转基因的TIL细胞,所述TIL细胞为TNF-a转基因高表达;具体的,通过TNF-a基因重组逆转录病毒转染前述分离制备的TIL细胞后获得;所述TNF-a基因重组逆转录病毒是将TNF-a基因连接到逆转录病毒载体上获得,本领域常用的构建方式都可以实现。
更进一步的,本发明提供一种用于治疗肺癌的药物组合,其含有转基因的TIL细胞以及CTLA-4单克隆抗体和培美曲塞,所述TIL细胞TNF-a转基因高表达;所述CTLA-4单克隆抗体的重链可变区序列如SEQ ID NO:1所示,轻链可变区序列如SEQ ID NO:2所示。
另外一方面,含有转基因的TIL细胞以及CTLA-4单克隆抗体和培美曲塞的组合在制备用于治疗肺癌的药物组合物中的用途;其中,TIL细胞为TNF-a转基因高表达;所述CTLA-4单克隆抗体的重链可变区序列如SEQ ID NO:1所示,轻链可变区序列如SEQ ID NO:2所示。
在一些实施方案中,足以治疗有效剂量的TIL的数量为约1×107至约1×1010。可以给予任何合适剂量的TIL。优选约1×107至约13×1010施用PIL,平均约7-8×106个/g。
在一些实施方案中,该方法还包括使用冷冻保存方法冷冻保存包含收获的TIL群体的输液袋的步骤。
在一些实施方案中,使用收获的TIL群体与冷冻保存培养基的1:1比率来进行冷冻保存过程。
药物组合物是TIL在无菌缓冲液中的悬浮液。
使用本发明的方法扩大的TIL可以通过本领域已知的任何合适的途径给药。优选地,TIL作为单次动脉内或静脉内输注给药,其优选持续约30-60分钟。其它合适的给药途径包括腹膜内,鞘内和膜内给药。
本发明药物组合物中提供的TIL在宽剂量范围内是有效的。确切的剂量将取决于给药途径,化合物的给药形式,待治疗对象的性别和年龄,待治疗对象的体重以及主治医师的偏好和经验。如果合适,也可以使用临床确定剂量的TIL。使用本文方法施用的药物组合物的量,例如PBL的剂量将取决于所治疗的人或哺乳动物,病症或病症的严重程度,给药速率,活性药物成分的处置和处方医师的判断。
在一些实施方案中,TIL可以单剂量给药。这样的给药可以通过注射,例如静脉注射。在一些实施方案中,TIL可以以多个剂量给药。剂量可以是每年一次,两次,三次,四次,五次,六次或六次以上。剂量可以是每月一次,每两周一次,每周一次或每隔一天一次。TIL的给药可以根据需要继续进行,有效量的TIL可以通过具有类似效用的药剂的任何可接受的给药方式以单剂量或多剂量给药,包括鼻内和经皮途径,通过动脉内注射,静脉内,腹膜内,胃肠外,肌内,皮下,局部,移植或直接注射到肿瘤中,或通过吸入。
在一些实施方案中,本发明提供了上述任一段落中所述的药物组合物,所述药物组合物可应用于上述改进,使得所述药物组合物包含5×107至20×109TIL.
在一些实施方案中,本发明提供了上述任一段落中所述的可应用的药物组合物,所述药物组合物经修饰使得所述药物组合物还包含冷冻保存剂。
在一些实施方案中,本发明提供了上述任一段落中所述的可应用的药物组合物,所述药物组合物经修饰使得所述药物组合物还包含至少约5%(v/v)二甲基亚砜(DMSO)。
有益效果
本发明提供了一种有效分离并制备的肿瘤浸润淋巴细胞,将所述细胞转TNF-α高表达后与制备的CTLA-4单克隆抗体和培美曲塞一起使用能够显著的抑制肺癌肿瘤的增殖,具有较好的效果。
附图说明
图1 TIL细胞与CTLA-4抗体联合治疗效果图
图2 TIL细胞与CTLA-4抗体和培美曲塞联合治疗效果图
具体实施方式
以下将给出实施例并进一步说明本发明,但本实施例仅是本发明实施例的示例,并不限定本发明的范围。
实施例1肺癌肿瘤浸润淋巴细胞的分离与增殖
将新鲜肺癌肿瘤组织置于含抗生素的冷Hanks’液中浸泡30min,去除坏死、出血及脂肪组织,称重,剪成lmmxlmmxlmm小块,加入以RPMI1640配制的有胶原酶Ⅱ型的消化酶液中震荡消化12h,再加入透明质酸酶和DNA酶,搅拌4h消化处理。完全消化后的组织过120目铜网,即得到含TIL和肿瘤细胞的单细胞悬液。
将混合细胞悬液3ml轻轻加于lml小牛血清界面上,500r/min离心15s后,底层为瘤细胞,将上层细胞悬液再行连续梯度分离,获取TIL。所得TIL用台盼蓝拒染法测定活率,并涂片行MGG染色,光镜下确定淋巴细胞百分率。结果显示,TIL的获得率达到89.3%,纯度为82.3%。
将分离的TIL在含10%人AB血清的RPMI1640完全培养基中以1×106/ml初始浓度于24孔板中培养,并加入IL-2 10μg/ml,同时加OKT3 10μg/ml和VC 1μg/ml,37℃、5%CO2孵箱培养,3~5天传代一次,共培养20d。结果显示,VC能够显著的协助缩短TIL的抑制期,20d扩增倍数达到了53倍,TIL体外扩增达到了治疗量109所需的浓度。
淋巴细胞表型测定采用间接免疫荧光染色法,荧光显微镜计数细胞。抗CD3、CD4、CD8、CDl6、CDl9、CD25单抗及羊抗鼠FITC-Ig,按说明书操作。结果显示,经三种因素诱导后,CD3+细胞比例显著增加,最高达99.0%,CD8+细胞明显增加达到71.3%;CD4+细胞增加不明显;HLA-DR细胞显著增多到99.4%;CD25+细胞增加到64.3%。说明所述的TIL细胞增殖较好。
实施例2转基因TIL细胞的制备
按照“转TNF-α基因肿瘤浸润淋巴细胞的构建及其生物学特性的研究,罗意革等,广医科大学学报,2002,19(2),p151-154”中的构建方法来制备所述的转基因TIL细胞。具体的,先制备含人TNF-α基因重组逆转录病毒的细胞株FIyRUFN14。将所述细胞株进行培养,取培养液上清,加人Polybrane至终浓度8mg/L,将上清液加入实施例1制备的对数生长期TIL细胞转染3d后加入400ug/ml G418筛选。2周后用有限稀释法克隆,继续用含1000u/ml IL-2的培养液培养。另取末转基因的TIL细胞,加人同样浓度的G418作对照培养。
TNF-α分泌量的测定:分别取TIL及转基因TIL配制浓度为细胞数5×105/m1,加入细胞培养板中培养于72h分别进行TNF-α检测。按试剂盒提供方法进行检测。结果显示,TIL细胞在72h检测时结果为阴性,而转基因的TIL细胞TNF-α分泌量达到了523.7pg,具有显著的高表达效果。将所述转基因阳性的TIL细胞扩增备用。
实施例3 CTLA-4单克隆抗体的制备
抗原为CTLA-4重组蛋白,货号JN1568,百奥莱博。BALB/c小鼠颈背部注射免疫抗原10mg,每隔2周加强免疫1次,3免后7~10d断尾取血,间接ELISA方法检测血清抗体效价,具体步骤如下:检测原质量浓度为5μg/mL,96孔酶标板每孔100μL,4℃包被过夜;以含体积分数0.1%吐温-20的0.01mol/L磷酸盐缓冲液(PBS)作为洗液,洗涤3次,每次用量300mL,拍干(下同);每孔加入含体积分数10%胎牛血清的洗液封闭,37℃孵育2h;洗涤、拍干;将待测血清稀释1000倍,每孔加入100μL,并且设置空白对照孔(PBS)和阴性对照孔(阴性血清),37℃孵育45min;洗涤、拍干;每孔加入100μL稀释10000倍的HRP标记山羊抗小鼠IgG,37℃孵育30min;洗涤、拍干;每孔加入底物显色液100μL,37℃避光反应15min;每孔加入50μL终止液,终止反应;450nm波长处测定吸光度(A),阴性对照孔吸光度为A1,阳性对照孔为A2,以A2/A1≥2.1为阳性结果。选取免疫效果最佳的小鼠进行细胞融合。采用聚乙二醇法进行融合,融合8d后,出现肉眼可见细胞集落时换用HT培养基;上清液用间接ELISA方法检测,筛选阳性杂交瘤细胞;检测结果为阳性的孔,挑选吸光度较高的单个克隆,通过有限稀释法进行亚克隆,直至得到稳定分泌抗体的杂交瘤细胞株3A7;将3A7杂交瘤细胞注射到小鼠体内诱生腹水,采用辛酸-硫酸铵沉淀的方法纯化,获得了纯化的3A7单克隆抗体。用超微量核酸蛋白测定仪测定其蛋白质量浓度达到了27.4mg/mL,间接ELISA测定效价为106以上,具有较好的效果。采用间接ELISA方法测定单克隆抗体的亚型为IgG1。采用特异性验证,发现所述单抗也只与CTLA-4蛋白进行特异性结合,与其他蛋白均不结合。此外,采用本领域常规的单克隆抗体亲和力测定方法,检测得到所述抗体与抗原的解离常数为3.7nM,具有较好的结合特性。
采用本领域常规的轻重链序列鉴定方法,获得了抗体的轻链可变区序列和重链可变区序列分别如下所示,
SEQ ID NO:1:重链可变区序列
EVKLVESGGGLVKPGGSLKLYCAASGFTFSHYHTIWVRQIPEKRLEWVAIFMTWYAVMCLRREYSRFTISRDNARNICYLQMNSLRSDATAMYYCARPDWSRAREGIPWGQGTTLTVSS
SEQ ID NO:2:轻链可变区序列
DIVLTQSPASLAVQLGQRATISCTVDTTICWIARIGDGWYQQKPGQPPKLLIYLDPNSLEGVPARFSGSGSGTDFSRNIHPVEEDDIAMYFCAPMNAWYMKFGAGTKLELK。
实施例4肺癌模型制备及治疗实验
小鼠Lewis肺癌移植瘤制备:小鼠Lewis肺癌细胞用8%胎牛血清DMEM培养基进行培养,进行传代2-3次,取其中对数生长期的细胞,用浓度0.25%的胰蛋白酶进行消化后1000r/min进行离心,时间10min,取下液加0.9%生理盐水进行细胞悬浮并稀释浓度至1.0×106/ml,取0.1ml对实验对象小鼠进行接种。
对照组1:10只对照组小鼠进行腹腔注射生理盐水;
对照组2:在肿瘤接种后第3天,将未转基因的TIL细胞细胞密度为5×107个/mL,从每只荷瘤小鼠尾静脉注射0.1mL/次,每天1次,连续10天。
实验组1:在肿瘤接种后第3天与第10天,分别将100μg CTLA-4抗体(3A7)注入小鼠腹腔内。
实验组2:在肿瘤接种后第3天,将实施例2制备的转基因阳性的TIL细胞细胞密度为5×107个/mL,从每只荷瘤小鼠尾静脉注射0.1mL/次,每天1次,连续10天。
实验组3:在肿瘤接种后第3天与第10天,分别将100μg CTLA-4抗体(3A7)注入小鼠腹腔内;同时在肿瘤接种后第3天,将实施例2制备的转基因阳性的TIL细胞细胞密度为5×107个/mL,从每只荷瘤小鼠尾静脉注射0.1mL/次,每天1次,连续10天。
移植瘤指标的测定。治疗开始日起每3d建立模型测量一次肿瘤的最大直径(a)与横径(b),并计算体积。共治疗21d,第22d行颈椎脱臼法处死后取出小鼠脾,测量肿瘤体积,计算出肿瘤生长抑制率。生长抑制率=(对照组肿瘤重量-实验组肿瘤重量)/对照组瘤重×100%。结果如图1所示。
从图1可以看出,对照组对肿瘤生长抑制率为0,而未转基因的TIL细胞对肿瘤的抑制率在5.4%左右,而转基因的TIL细胞对肿瘤的抑制率达到了56.7%,单独使用单抗对肿瘤的抑制率达到了45.2%,而将单抗和转基因的TIL细胞一起使用对肿瘤的抑制率达到了89.6%,具有较好的协同治疗效果。
实施例5组合治疗实验
小鼠Lewis肺癌移植瘤制备:小鼠Lewis肺癌细胞用8%胎牛血清DMEM培养基进行培养,进行传代2-3次,取其中对数生长期的细胞,用浓度0.25%的胰蛋白酶进行消化后1000r/min进行离心,时间10min,取下液加0.9%生理盐水进行细胞悬浮并稀释浓度至1.0×106/ml,取0.1ml对实验对象小鼠进行接种。
对照组3:10只对照组小鼠进行腹腔注射生理盐水;
实验组4:在肿瘤接种后第3天与第10天,分别将100μg CTLA-4抗体(3A7)注入小鼠腹腔内;同时在肿瘤接种后第3天,将实施例2制备的转基因阳性的TIL细胞细胞密度为5×107个/mL,从每只荷瘤小鼠尾静脉注射0.1mL/次,每天1次,连续10天。
实验组5:在肿瘤接种后第3天与第10天,分别将100μg CTLA-4抗体(3A7)和培美曲塞1mg注入小鼠腹腔内;同时在肿瘤接种后第3天,将实施例2制备的转基因阳性的TIL细胞细胞密度为5×107个/mL,从每只荷瘤小鼠尾静脉注射0.1mL/次,每天1次,连续10天。
移植瘤指标的测定。治疗开始日起每3d建立模型测量一次肿瘤的最大直径(a)与横径(b),并计算体积。共治疗21d,第22d行颈椎脱臼法处死后取出小鼠脾,测量肿瘤体积,计算出肿瘤生长抑制率。生长抑制率=(对照组肿瘤重量-实验组肿瘤重量)/对照组瘤重×100%。结果如图2所示。
从图2可以看出,对照组3对肿瘤生长抑制率为0,将单抗和转基因的TIL细胞一起使用对肿瘤的抑制率达到了89.7%,而将单抗和转基因的TIL细胞以及培美曲塞一起使用对肿瘤的抑制率达到了95.6%,这说明培美曲塞具有增强单抗和TIL细胞作用的效果。
以上结合具体实施方式和范例性实例对本发明进行了详细说明,不过这些说明并不能理解为对本发明的限制。本领域技术人员理解,在不偏离本发明精神和范围的情况下,可以对本发明技术方案及其实施方式进行多种等价替换、修饰或改进,这些均落入本发明的范围内。本发明的保护范围以所附权利要求为准。
序列表
<110> 诺赛联合(北京)生物医学科技有限公司
<120> TIL细胞及其制备方法以及在癌症治疗中的用途
<160> 2
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<211> 119
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<213> 人工序列(Artificial Sequence)
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Claims (3)
1.一种产品,其为用于治疗肺癌的药物组合,由转基因的TIL细胞以及CTLA-4单克隆抗体和培美曲塞组成,所述转基因的TIL细胞为转TNF-a基因高表达的TIL细胞;所述CTLA-4单克隆抗体的重链可变区序列如SEQ ID NO:1所示,轻链可变区序列如SEQ ID NO:2所示;所述TIL细胞采用如下方法制备获得:将新鲜肺癌肿瘤组织置于含抗生素的冷Hanks’液中浸泡30min,去除坏死、出血及脂肪组织,称重,剪成lmm×lmm×lmm小块,加入以RPMI1640配制的有胶原酶Ⅱ型的消化酶液中震荡消化12h,再加入透明质酸酶和DNA酶,搅拌4h消化处理;完全消化后的组织过120目铜网,即得到含TIL和肿瘤细胞的单细胞悬液;将混合细胞悬液轻轻加于小牛血清界面上,500r/min离心15s后,底层为瘤细胞,将上层细胞悬液再行连续梯度分离,获取TIL;将分离的TIL在含10%人AB血清的RPMI1640完全培养基中以1×106/ml初始浓度于24孔板中培养,并加入IL-2 10μg/ml,同时加OKT3 10μg/ml和VC 1μg/ml,37℃、5%CO2孵箱培养,3~5天传代一次,共培养20d,即得到目的TIL细胞。
2.转基因的TIL细胞以及CTLA-4单克隆抗体和培美曲塞的组合在制备用于治疗肺癌的药物组合物中的用途;所述转基因的TIL细胞为转TNF-a基因高表达的TIL细胞;所述CTLA-4单克隆抗体的重链可变区序列如SEQ ID NO:1所示,轻链可变区序列如SEQ ID NO:2所示;所述TIL细胞采用如下方法制备获得:将新鲜肺癌肿瘤组织置于含抗生素的冷Hanks’液中浸泡30min,去除坏死、出血及脂肪组织,称重,剪成lmm×lmm×lmm小块,加入以RPMI1640配制的有胶原酶Ⅱ型的消化酶液中震荡消化12h,再加入透明质酸酶和DNA酶,搅拌4h消化处理;完全消化后的组织过120目铜网,即得到含TIL和肿瘤细胞的单细胞悬液;将混合细胞悬液轻轻加于小牛血清界面上,500r/min离心15s后,底层为瘤细胞,将上层细胞悬液再行连续梯度分离,获取TIL;将分离的TIL在含10%人AB血清的RPMI1640完全培养基中以1×106/ml初始浓度于24孔板中培养,并加入IL-2 10μg/ml,同时加OKT3 10μg/ml和VC 1μg/ml,37℃、5%CO2孵箱培养,3~5天传代一次,共培养20d,即得到目的TIL细胞。
3.如权利要求2所述的用途,其特征在于:TIL细胞的量为足以治疗有效剂量的TIL的数量,所述数量为1×107至1×1010。
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