CN110540960A - 结合有免疫检查点阻断剂的t淋巴细胞及其在制备抗肿瘤药物中的应用 - Google Patents
结合有免疫检查点阻断剂的t淋巴细胞及其在制备抗肿瘤药物中的应用 Download PDFInfo
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Abstract
本发明公开了一种结合有免疫检查点阻断剂的T淋巴细胞,其通过将T淋巴细胞分离并体外扩增,并在体外与免疫检查点阻断剂和激动型抗体结合而制得。通过将免疫检查点阻断剂与T淋巴细胞结合,确保了免疫检查点阻断剂随着T淋巴细胞快速进入宿主的免疫系统,人为将免疫检查点阻断剂结合于T淋巴细胞的表面,可快速而有效的弥补T淋巴细胞在自然状态下分泌的免疫检查点阻断剂不足的缺陷。在将本发明的结合有免疫检查点阻断剂的T淋巴细胞回输至宿主后有效抑制了肿瘤细胞的生长,降低癌症复发及转移。利用本发明的T淋巴细胞进行抗肿瘤时,无需了解肿瘤相关抗原,与传统过继转移细胞疗法相比,过程简便易行。
Description
技术领域
本发明属于生物技术领域,具体涉及一种结合有免疫检查点阻断剂的T淋巴细胞及其在制备抗肿瘤药物中的应用。
背景技术
虽然目前人类的医学技术水平已取得较大进步和发展,但是恶性肿瘤仍是威胁人类健康的重大疾病之一。近年来,肿瘤免疫治疗已逐渐成为研究热点,并取得了较大的成果。
过继细胞转移(adoptive cell transfer,ACT)疗法是肿瘤免疫治疗的热点研究领域之一。过继细胞转移疗法是指通过采集宿主自身的免疫细胞,经体外培养扩增或是为了增强其靶向性而进一步修饰处理后,将其重新输回宿主体内以提高肿瘤细胞的免疫原性和对效应细胞杀伤的敏感性。
肿瘤浸润淋巴细胞(tumor-infiltrating lymphocytes,TILs)是过继细胞转移疗法中的一种重要的治疗细胞,使用患者自体肿瘤浸润淋巴细胞的癌症免疫疗法已显示在难治性转移性黑素瘤中介导持久的完全反应。TILs是存在于肿瘤微环境的T淋巴细胞,能够识别肿瘤相关抗原,包括新生肿瘤抗原(neoantigen),因此具有高度特异的抗肿瘤反应和低毒性。
然而,使用TILs进行抗肿瘤治疗中的主要限制因素之一是复杂的TILs制造过程。该过程需要在高剂量白介素-2(IL-2)存在下,从消化的肿瘤碎片或单细胞悬浮液的多孔培养物;经过初始培养3-5周后,通过将TILs样品与自体肿瘤细胞共培养来测试不同孔中TILs的肿瘤反应性;然后才能选择具有肿瘤反应性的样品用于大规模二级多克隆扩增,过程非常繁琐。不仅如此,TILs疗法需要确保TILs具有显著的自体肿瘤反应性。因此,在扩增阶段之前尽可能富集肿瘤特异性T细胞亚群可以增强最终细胞产物的肿瘤反应性并简化TILs生产过程。
所以一个重要的问题是如何在不知道特异性肿瘤抗原的情况下将分选出肿瘤反应性T细胞。许多研究发现肿瘤反应性T细胞表达几种活化相关分子,例如CD137,可以作为肿瘤细胞特异性识别的产物。类似地,在T细胞活化时诱导耗竭型检查点分子,其中PD-1最有代表性。PD-1是一类表达于T细胞表面的球状蛋白,对于免疫抑制起着重要的作用。PD-1通过与肿瘤细胞分泌的配体PD-L1结合,并去磷酸化TCR信号通路上的多个关键分子,从而发挥对肿瘤抑制过程的免疫应答的负性调节作用。最近有研究发现CD8+PD-1+T细胞可以从肿瘤浸润物中特异性分离,并且在这些分离的组织中大多数都具有抗肿瘤反应性。由于从黑色素瘤和其他实体瘤中分离出的绝大多数CD8+T细胞表达PD-1,因此PD-1可以作为一个反映TILs对肿瘤保持特异性的标志物。
然而分离表达PD-1+的T细胞用于过继治疗的一个问题是这些细胞处于耗竭状态或功能受损,这是由于肿瘤微环境会诱导TILs高表达PD-1分子。
虽然T细胞能够特异性的杀伤肿瘤细胞,然而肿瘤细胞也会逐渐抑制T淋巴细胞的活性,以实现自我保护,比如通过启动免疫检查点(immune checkpoint)等诸多途径使免疫细胞失活,达到逃避免疫识别和免疫清除的目的。近年来,人们已发现了多个免疫检查点信号通路。至今已有多个阻断免疫检查点的抗体治疗被批准作为临床上进行肿瘤治疗的药物。免疫检查点阻断剂通过阻断肿瘤微环境中的免疫检查点信号通路,改变肿瘤微环境,恢复并增强T淋巴细胞杀伤肿瘤细胞的功能。免疫检查点阻断剂中的PD-1抗体能通过对PD-1/PD-L1通路的抑制而提高抗肿瘤效果,但是PD-1抗体在临床应用中的抗癌效果并不理想,且其带来的治疗副作用较大。
发明内容
基于上述背景的情况下,本发明的目的之一,在于提供一种结合有免疫检查点阻断剂的T淋巴细胞,所述免疫检查点阻断剂结合于T淋巴细胞的表面。通过将免疫检查点阻断剂与T淋巴细胞结合,确保了免疫检查点阻断剂随着T淋巴细胞快速进入宿主免疫系统,通过人为将免疫检查点阻断剂结合于T淋巴细胞的表面,可快速而有效的弥补T淋巴细胞在自然状态下分泌的免疫检查点阻断剂不足的缺陷,因而提高了T淋巴细胞的抗肿瘤性能。
进一步的,所述T淋巴细胞为肿瘤浸润淋巴细胞。肿瘤浸润淋巴细胞是存在于肿瘤微环境的T淋巴细胞,能够识别肿瘤相关抗原,具有高度特异的肿瘤反应性和低毒性,此外,表达PD-1的肿瘤浸润淋巴细胞虽然表现出功能耗竭,但可以作为一个反映TILs对肿瘤保持特异性的标志物,因此无需了解肿瘤新生抗原即可确保本发明的T淋巴细胞具有较高的肿瘤反应性。
更进一步的,所述免疫检查点阻断剂为PD-1抗体、LAG-3抗体、TIM-3抗体、TIGIT抗体、CTLA-4抗体和VISTA抗体中的至少一种。将肿瘤浸润T淋巴细胞分离并体外扩增,在体外与免疫检查点阻断剂反应结合后,增强了TILs的抗肿瘤功能,而且,免疫检查点阻断剂可与肿瘤浸润T淋巴细胞进行靶向结合,使得免疫检查点阻断剂充分结合于T淋巴细胞的表面,因而确保进入宿主免疫系统中的免疫检查点阻断剂有效的发挥抗肿瘤作用并最大可能的降低了进入生物体内的免疫检查点阻断剂的使用量,因而降低了其副作用。
更进一步的,所述T淋巴细胞上还结合有激动型抗体,所述激动型抗体选自OX40抗体、4-1BB抗体、ICOS抗体和GITR抗体中的至少一种。
更进一步的,所述肿瘤浸润淋巴细胞按照如下方法制备:
(1)切下肿瘤组织块,去除脂肪等其他组织,置于含双抗的PBS缓冲液中浸泡5分钟;
(2)将步骤(1)得到的肿瘤组织块通过全自动组织处理器处理后,收集细胞悬液并过滤后收集;
(3)将步骤(2)得到的细胞悬液中加入红细胞裂解液,孵育后加入PBS终止裂解,离心处理,弃上清,以PBS洗涤细胞后,用PBS重悬;
(4)将步骤(3)获得的肿瘤浸润淋巴细胞计数,用荧光标记CD3和PD-1的流式抗体染色,用PBS洗涤,离心处理弃上清,重悬于PBS中,进行CD3+PD-1+子集的分选;
(5)向步骤(4)获得的肿瘤浸润淋巴细胞中加入经辐照的鼠PBMC、人白介素-2和可溶性CD3抗体,刺激淋巴细胞增殖。
本发明的目的之二,在于提供本发明的目的之一的结合有免疫检查点阻断剂的T淋巴细胞的制备方法,其包括如下步骤:将所述免疫检查点阻断剂与所述肿瘤浸润淋巴细胞进行混匀、孵育和充分洗涤。利用本发明的制备方法可将免疫检查点阻断剂与T淋巴细胞进行充分结合,并将免疫检查点阻断剂充分附着于T淋巴细胞的表面,因而免疫检查点阻断剂能随着T淋巴细胞被一起转运。
本发明的目的之三,在于提供一种将结合有免疫检查点阻断剂的T淋巴细胞在制备抗肿瘤药物中的应用。利用本发明的结合有免疫检查点阻断剂的T淋巴细胞进行抗肿瘤治疗时无需了解肿瘤相关抗原,与传统过继转移细胞疗法相比,过程简便易行。
进一步的,所述T淋巴细胞为肿瘤浸润淋巴细胞,所述肿瘤浸润淋巴细胞按照如下方法制备:
(1)切下肿瘤组织块,去除脂肪等其他组织,置于含双抗的PBS缓冲液中浸泡5分钟;
(2)将步骤(1)得到的肿瘤组织块通过全自动组织处理器处理后,收集细胞悬液并过滤后收集;
(3)将步骤(2)得到的细胞悬液中加入红细胞裂解液,孵育后加入PBS终止裂解,离心处理,弃上清,以PBS洗涤细胞后,用PBS重悬;
(4)将步骤(3)获得的肿瘤浸润淋巴细胞计数,用荧光标记CD3和PD-1的流式抗体染色,用PBS洗涤,离心处理弃上清,重悬于PBS中,进行CD3+PD-1+子集的分选;
(5)向步骤(4)获得的肿瘤浸润淋巴细胞中加入经辐照的鼠PBMC、人白介素-2和可溶性CD3抗体,刺激淋巴细胞增殖。利用肿瘤浸润淋巴细胞与免疫检查点阻断剂结合后得到的治疗细胞使具有高度特异的肿瘤反应性和低毒性,因此无需了解肿瘤新生抗原即可确保本发明的结合有免疫检查点阻断剂的T淋巴细胞具有较高的肿瘤反应性。
更进一步的,所述T淋巴细胞上结合的免疫检查点阻断剂为PD-1抗体、LAG-3抗体、TIM-3抗体、TIGIT抗体、CTLA-4抗体和VISTA抗体中的至少一种,在所述T淋巴细胞上结合还结合有激动型抗体,所述激动型抗体选自OX40抗体、4-1BB抗体、ICOS抗体和GITR抗体中的至少一种。
本发明的目的之四在于提供一种药物组合物,其含有有效量的本发明的目的之一所述的结合有免疫检查点阻断剂的T淋巴细胞及药学上可接受的载体。利用本发明的目的之一的结合有免疫检查点阻断剂的T淋巴细胞回输至同一生物体后,能够有效提高肿瘤浸润淋巴细胞的抗肿瘤的疗效,而且能有效宿主体内抑制癌细胞的生长,降低癌症复发及转移,对于肿瘤切除手术后模拟的细胞转移模型取得了显著的抗癌效果。
术语“PD-1抗体”指抗程序性细胞死亡蛋白-1(programmed cell death protein1,PD-1)抗体;
术语“TIM-3抗体”指T细胞免疫球蛋白及黏蛋白分子-3(T cell immunoglobulindomain and mucin domain-3)抗体;
术语“LAG-3抗体”指淋巴细胞活化基因-3(Lymphocyte activation gene-3)抗体;
术语“TIGIT抗体”指T细胞免疫球蛋白ITIM结构域(T cell immunoreceptor withIg and ITIM domains)、
术语“VISTA抗体”指V区Ig抑制子(V-Domain Immunoglobulin-ContainingSuppressor of T Cell Activation)抗体;
术语“CTLA-4抗体”指抗细胞毒性T淋巴细胞抗原-4抗体(cytotoxic T-lymphocyte-associated protein 4)抗体;
术语“激动型抗体”是与受体结合并激活受体的抗体;
术语“OX40抗体”指肿瘤坏死因子受体超家族,成员4(Tumor necrosis factorreceptor superfamily,member 4)抗体;
术语“4-1BB抗体”指肿瘤坏死因子受体超家族,成员9(tumor necrosis factorreceptor superfamily member 9)抗体;
术语“ICOS抗体”指诱导协同刺激分子(Inducible T-cell costimulator)抗体;
术语“ICOS抗体”指糖皮质激素诱导的肿瘤坏死因子受体(glucocorticoid—induced tumor necrosis factor receptor)抗体;
术语“药学上可接受的载体”指一种或多种有机或无机成分,它可以是天然的或合成的,与抗体组合后可促进其应用。可接受的载体包括无菌的生理盐水或是其它药学上可获得的且为本领域所熟知的水或非水的等渗溶液或灭菌混悬剂。
附图说明
图1(a)为肿瘤浸润淋巴细胞的共聚焦显微镜检图;
图1(b)为结合有PD-1抗体的肿瘤浸润淋巴细胞的共聚焦显微镜检图;
图2为TILs与aPD-1抗体结合回输后对小鼠体内的肿瘤组织肿瘤组织荧光成像分析结果;
图3为TILs与aPD-1结合回输后对小鼠体内的肿瘤组织体积大小的影响;
图4为TILs与aPD-1结合回输后对小鼠体重的影响;
图5为TILs与aPD-1结合回输后对小鼠存活率的影响。
在图2-5中,UnTx表示未经任何处理,TILs表示仅利用肿瘤浸润淋巴细胞进行处理、aPD-1表示仅利用PD-1抗体处理、TILs-aPD-1表示利用TILs和PD-1抗体结合后在进行处理。
具体实施方式
本发明各实施例中的材料来源为:
小鼠黑色素瘤细胞B16OVA购自美国模式菌种保藏中心(简称ATCC)。B16OVA于添加有10%胎牛血清、100U/ml青霉素和100U/ml的链霉素的DMEM培养基中保藏。使用第三代或第四代传代培养的细胞用于本发明的各实施例。
6-10周龄的C57BL/6小鼠购自常州卡文斯实验动物有限公司。小鼠按照中国科学院生化与细胞所实验动物管理委员会(IACUC)的指导操作方法进行处理。
PD-1抗体(aPD-1)购自Biox cell公司。
人白介素-2、CD3抗体(OKT-3)购自Biolegend公司。
实施例1:肿瘤浸润淋巴细胞(TILs)的提取
将B16OVA细胞(5×105)皮下注射到C57BL/6小鼠的背部,待肿瘤长至5mmX5mm大小后切下,去除表皮、脂肪等其他组织,置于含双抗的PBS缓冲液中浸泡5分钟。接着将肿瘤组织块通过全自动组织处理器处理,收集细胞悬液并过200目尼龙网,收集细胞于15ml离心管中。加入1×红细胞裂解液2ml,孵育5分钟后加入6mlPBS终止裂解,然后在400g离心机上离心5分钟,弃上清,PBS洗涤细胞2次后,用含1%胎牛血清的PBS重悬。将获得的肿瘤浸润淋巴细胞计数,用荧光标记CD3和PD-1的流式抗体染色30分钟。然后PBS洗涤,离心处理,弃上清,重悬于PBS中。最后在FACSAria流式细胞分选器中进行CD3+和PD-1+子集的分选。将获得的肿瘤浸润淋巴细胞置于培养皿中,加入经辐照的鼠PBMC,人白介素-2(3000IU/ml)和可溶性CD3抗体(30ng/ml),刺激细胞增殖,增殖细胞以维持0.5-2×106/ml的浓度。
实施例2:aPD-1(PD-1抗体)与肿瘤浸润淋巴细胞的体外结合
收集扩增9-10天后的肿瘤浸润淋巴细胞,并在没有IL-2的培养基中过夜。将扩增后的细胞300g离心5分钟,弃上清,置于EP管中;加入PD-1抗体,混匀,37℃震荡孵育30分钟;用PBS洗涤1次,获得与aPD-1结合后的T淋巴细胞。
实施例3:结合有PD-1抗体的肿瘤浸润淋巴细胞的定性分析
(1)aPD-1和TILs结合后的荧光成像分析
首先将TILs用Hoechst33342染色,利用共聚焦显微镜观察,结果如图1(b)所示,在将aPD-1用FITC(异硫氰酸荧光素)染色标记后按照实施例2的方法将aPD-1与肿瘤浸润淋巴细胞结合后,利用共聚焦显微镜观察,结果如图1(b)所示,由图中可看出,用FITC标记的aPD-1均匀分布在肿瘤浸润淋巴细胞的外表面,说明aPD-1与肿瘤浸润淋巴细胞在体外与TILs充分结合,形成了TILs-aPD-1细胞药物。
实施例4:结合有PD-1抗体的肿瘤浸润淋巴细胞的抗癌效果分析
对小鼠皮下注射B16-luc(1*106),对小鼠随机分组,培养7天后回输实施例3得到的细胞药物。设置对照组(分别为未经任何处理、仅注入TILs、仅注入aPD-1、TILs与aPD-1未结合同时处理),监测肿瘤体积大小和小鼠存活率进行监测。小鼠体内的肿瘤组织荧光成像分析方法为:肿瘤组织利用其荧光信号在IVIS荧光成像分析系统下进行分析。将15mg/ml的D-荧光素按照10μg/g鼠体重的剂量注射于小鼠腹部10分钟后,利用IVIS荧光成像分析系统进行分析,曝光时间5分钟。肿瘤体积大小计算公式为:(长径*短径2)/2。由图3和图4可看出,在回输了本发明的结合有PD-1抗体的肿瘤浸润淋巴细胞后的小鼠的肿瘤体积明显低于仅仅使用肿瘤浸润淋巴细胞或仅仅使用PD-1抗体处理的小鼠的肿瘤体积,由图5可以看出经回输本发明的结合有PD-1抗体的肿瘤浸润淋巴细胞处理的小鼠的存活率也明显更高,说明本发明的细胞药物具有良好的抗癌效果。
作为本发明的一种优选的实施方式,还可在肿瘤浸润淋巴细胞上结合激动型抗体,如OX40抗体、4-1BB抗体、ICOS抗体或GITR抗体中的至少一种而增加T淋巴细胞的抗癌活性。
以上实施方式只为说明本发明的技术构思及特点,其目的在于让熟悉此项技术的人了解本发明的内容并加以实施,并不能以此限制本发明的保护范围,凡根据本发明精神实质所做的等效变化或修饰,都应涵盖在本发明的保护范围内。
Claims (10)
1.一种结合有免疫检查点阻断剂的T淋巴细胞,其特征在于,所述免疫检查点阻断剂结合于T淋巴细胞的表面。
2.根据权利要求1所述的结合有免疫检查点阻断剂的T淋巴细胞,其特征在于,所述T淋巴细胞为肿瘤浸润淋巴细胞。
3.根据权利要求2所述的结合有免疫检查点阻断剂的T淋巴细胞,其特征在于,所述免疫检查点阻断剂为PD-1抗体、LAG-3抗体、TIM-3抗体、TIGIT抗体、CTLA-4抗体和VISTA抗体中的至少一种。
4.根据权利要求3所述的结合有免疫检查点阻断剂的T淋巴细胞,其特征在于,在所述T淋巴细胞上还结合有激动型抗体,所述激动型抗体选自OX40抗体、4-1BB抗体、ICOS抗体和GITR抗体中的至少一种。
5.根据权利要求2-4中任一项所述的结合有免疫检查点阻断剂的T淋巴细胞,其特征在于,所述肿瘤浸润淋巴细胞按照如下方法制备:
(1)切下肿瘤组织块,去除脂肪等其他组织,置于含双抗的PBS缓冲液中浸泡5分钟。
(2)将步骤(1)得到的肿瘤组织块通过全自动组织处理器处理后,收集细胞悬液并过滤后收集;
(3)将步骤(2)得到的细胞悬液中加入红细胞裂解液,孵育后加入PBS终止裂解,离心处理,弃上清,以PBS洗涤细胞后,用PBS重悬;
(4)将步骤(3)获得的肿瘤浸润淋巴细胞计数,用荧光标记CD3和PD-1的流式抗体染色,用PBS洗涤,离心处理弃上清,重悬于PBS中;进行CD3+PD-1+子集的分选;
(5)向步骤(4)获得的肿瘤浸润淋巴细胞中加入经辐照的鼠PBMC、人白介素-2和可溶性CD3抗体,刺激淋巴细胞增殖。
6.权利要求2或3所述的结合有免疫检查点阻断剂的T淋巴细胞的制备方法,其特征在于,包括如下步骤:将所述免疫检查点阻断剂与所述肿瘤浸润淋巴细胞进行混匀、孵育和充分洗涤。
7.权利要求1所述的结合有免疫检查点阻断剂的T淋巴细胞在制备抗肿瘤药物中的应用。
8.根据权利要求7所述的T淋巴细胞在制备抗肿瘤药物中的应用,其特征在于,所述T淋巴细胞为肿瘤浸润淋巴细胞,所述肿瘤浸润淋巴细胞按照如下方法制备:
(1)切下肿瘤组织块,去除脂肪等其他组织,置于含双抗的PBS缓冲液中浸泡5分钟;
(2)将步骤(1)得到的肿瘤组织块通过全自动组织处理器处理后,收集细胞悬液并过滤后收集;
(3)将步骤(2)得到的细胞悬液中加入红细胞裂解液,孵育后加入PBS终止裂解,离心处理,弃上清,以PBS洗涤细胞后,用PBS重悬;
(4)将步骤(3)获得的肿瘤浸润淋巴细胞计数,用荧光标记CD3和PD-1的流式抗体染色,用PBS洗涤,离心处理弃上清,重悬于PBS中,进行CD3+PD-1+子集的分选;
(5)向步骤(4)获得的肿瘤浸润淋巴细胞中加入经辐照的鼠PBMC、人白介素-2和可溶性CD3抗体,刺激淋巴细胞增殖。
9.根据权利要求8所述的T淋巴细胞在制备抗肿瘤药物中的应用,其特征在于,所述T淋巴细胞上结合的免疫检查点阻断剂为PD-1抗体、LAG-3抗体、TIM-3抗体、TIGIT抗体、CTLA-4抗体和VISTA抗体中的至少一种,在所述T淋巴细胞上结合还结合有激动型抗体,所述激动型抗体选自OX40抗体、4-1BB抗体、ICOS抗体和GITR抗体中的至少一种。
10.一种药物组合物,其特征在于,含有有效量的权利要求1-4中任一项所述的结合有免疫检查点阻断剂的T淋巴细胞及药学上可接受的载体。
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