CN116536319A - 免疫检查点cd47核酸适体的筛选及应用 - Google Patents
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Abstract
本发明公开了一种免疫检查点CD47核酸适体的筛选及应用,本发明筛选的CD47核酸适体,可高亲和力和高选择性的识别CD47分子。所述核酸适体是通过基于琼脂糖微珠分离‑流式细胞术分析的无糖基化蛋白SELEX方法筛选制备,并且利用该核酸适体实现了CD47阳性肿瘤细胞系特异性识别,验证了其精准检测肿瘤细胞CD47的可行性,并有望作为CD47的分子检测工具,应用于CD47检查点抑制剂疗效精准预测。
Description
技术领域
本发明涉及肿瘤免疫疗效精准预测领域中的分子工具技术领域,具体涉及免疫检查点CD47核酸适体的筛选及应用。
背景技术
近年来,基于免疫检查点抑制剂(Immune Checkpoint Inhibitors,简称ICIs)的肿瘤免疫疗法为癌症治疗提供了新的治疗途径,为癌症患者带来新的希望。免疫检查点是人机体为调节自身免疫功能的一系列分子,避免机体免疫系统过度反应而损伤自身。而肿瘤细胞会高度表达免疫检查点分子,逃过机体免疫系统杀伤。免疫检查点疗法就是针对免疫检查点施以特异性抑制阻断剂,恢复免疫系统杀伤能力从而治疗肿瘤。比如,CTLA-4、PD-1及PD-L1免疫检查点抗体类抑制剂,通过阻止免疫检查点之间的相互作用,从而恢复免疫细胞杀伤肿瘤细胞的特性,诱导机体产生有效的肿瘤免疫应答。目前,PD-1/PD-L1抗体抑制剂已用于多种类型肿瘤治疗,但只有部分患者从治疗中获益。
而对于没有从治疗中获益的患者,不仅没有减缓病痛,还要承受昂贵的治疗负担,所以辨识哪些患者对免疫药物有反应是很重要的。但免疫抑制是一个复杂的网络,抗PD-1/PD-L1治疗低响应表明肿瘤免疫应答中存在其他免疫抑制途径。因此,为降低治疗风险,使患者最大程度获益,亟待发展其他免疫检查点标志物用于协同预测免疫检查点抑制剂疗效。
经研究发现,肿瘤细胞为了保护自己,模仿红细胞在表面表达“别吃我”信号蛋白CD47,以躲避巨噬细胞的吞噬作用进而免疫逃逸。CD47是继PD-L1之后肿瘤治疗领域中上升热度最快的免疫检查点,在多种实体瘤细胞及恶性血液瘤细胞表面高表达,可与巨噬细胞上的信号调节蛋白α(SIRPα)结合并发出“别吃我”信号以逃避免疫监视。
但由于CD47免疫检查点为高度糖基化蛋白,而现有的CD47抗体尺寸及分子量大,易受糖基化修饰的位阻影响而导致检测灵敏度低;此外,具有不稳定、易失活及成本高的缺点。因此,发展更稳定、更有效、更低成本的“新型抗体”,将有望为检测CD47表达水平提供更高效的分子识别工具。
核酸适体(aptamer)是经指数富集配体系统进化技术(SELEX)体外筛选得到的小段寡核苷酸序列(DNA或RNA),可通过折叠形成特定的三维结构,特异且高效地与相应配体(如蛋白质、细胞等)结合,是一类特殊的“化学抗体”。与抗体相比,核酸适体具有诸多优点:1)易于筛选,可体外化学合成且合成时间短、成本低;2)分子量小、尺寸小,糖基化修饰位阻干扰小、识别效率高,小而灵活的结构使它们能够与一些抗体无法结合的隐藏结构域结合;3)生物相容性好、无生物毒性、无免疫原性。因此,近年来,核酸适体已成为一种具有巨大潜力的革命性分子探针工具,在肿瘤免疫预后及疗效精准预测领域显示出巨大的应用前景。
所以,如果获得性能优异的CD47核酸适体,就可以避免CD47抗体的不足之处,将其用于检测CD47检查点的表达水平,精准预测CD47检查点抑制剂疗效,有望为快速判断和指导是否需要免疫治疗带来新的希望和解决途径。
发明内容
本发明的目的在于提供性能优异的CD47核酸适体,并提供其在指导肿瘤免疫治疗药物用药中的应用。本发明中提供CD47核酸适体,可高亲和力和高选择性的结合CD47蛋白。所述核酸适体是通过基于琼脂糖微珠分离-流式细胞术分析的无糖基化蛋白SELEX方法筛选制备。所述核酸适体具有生物相容性好、非免疫原性等特点。并且利用该核酸适体实现了CD47阳性肿瘤细胞系特异性识别,验证了其精准检测肿瘤细胞CD47的可行性,并有望作为CD47的分子检测工具,以期预测CD47检查点抑制剂疗效,补充指导免疫药物用药。
实现本发明目的的技术方案是:
免疫检查点CD47核酸适体,包括SEQ ID No.1至SEQ ID No.9所示的核苷酸序列。
所述CD47核酸适体还包括SEQ ID No.1至SEQ ID No.9所示的核苷酸序列的突变体或截短序列。
所述CD47核酸适体突变体为SEQ ID No.1至SEQ ID No.9所示的核苷酸序列具有80%以上同源性的序列。
所述CD47核酸适体突变体为SEQ ID No.1至SEQ ID No.9所示的核苷酸序列具有90%以上同源性的序列。
一种免疫检查点CD47核酸适体的筛选方法,为使用琼脂糖微珠分离-流式细胞术分析的无糖基化蛋白SELEX方法筛选。
上述免疫检查点CD47核酸适体在肿瘤免疫疗效精准预测研究领域的应用。
上述免疫检查点CD47核酸适体的应用,所述应用包括以下至少一种:
(1)所述免疫检查点CD47的核酸适体在肿瘤细胞CD47及其糖基化的流式分析检测、成像中的应用;
(2)所述免疫检查点CD47的核酸适体在组织切片CD47及其糖基化检测、成像中的应用;
(3)所述免疫检查点CD47的核酸适体在肿瘤来源细胞外囊泡或外泌体CD47的流式分析检测、ELISA检测、成像中的应用;
(4)所述免疫检查点CD47的核酸适体在肿瘤来源细胞外囊泡或外泌体CD47糖基化的流式分析检测、成像中的应用;
(5)所述免疫检查点CD47的核酸适体在肿瘤免疫疗效及预后精准预测、指导肿瘤免疫治疗药物用药研究中的应用。
本技术方案与现有技术相比,它具有如下优点:
1)本技术方案筛选获得的核酸适体是通过基于琼脂糖微珠分离-流式细胞术分析的无糖基化蛋白SELEX方法筛选得到,相比于抗体,更为简便快速。
2)相比于抗体,本技术方案筛选获得的核酸适体具有非免疫原性,易合成和标记、合成成本低,稳定性好、不易失活等特点,应用前景更广阔。
3)本技术方案筛选获得的核酸适体实现了CD47蛋白、CD47阳性肿瘤细胞系的特异性识别,有望作为CD47的分子检测工具,应用于CD47抗体抑制剂疗效的精准预测。
附图说明
图1为流式细胞术监测核酸适体筛选富集进程以及筛选富集文库与CD47原核蛋白琼脂糖微球、Ni琼脂糖微球的结合能力的数据图;
图2为通过流式细胞术考察测序分析得到的7条核酸适体HX1、HX2、HX3、HX4、HX5、HX6与HX7针对CD47原核蛋白微球、Ni琼脂糖微球的结合能力的数据图;
图3为通过流式细胞术考察测序分析得到的7条核酸适体HX1、HX2、HX3、HX4、HX5、HX6与HX7针对CD47真核蛋白的结合能力的数据图;
图4为通过流式细胞术考察测序分析得到的7条核酸适体HX1、HX2、HX3、HX4、HX5、HX6与HX7针对表达CD47蛋白的人乳腺癌系MCF-7与MDA-MB-231、人非小细胞肺癌细胞NCI-H1299、人肺腺癌细胞NCI-H1975、人结肠癌细胞SW480、人膀胱癌细胞T24的结合能力的数据图;
图5为通过流式细胞术考察测序分析得到的7条核酸适体HX1、HX2、HX3、HX4、HX5、HX6与HX7针对不表达CD47蛋白的心肌细胞H9C2、小鼠成纤维细胞NIH-3T3、小鼠单核巨噬细胞raw264.7的结合能力的数据图;
图6为通过流式细胞术测定所得核酸适体HX2对表达CD47蛋白的人乳腺癌系MCF-7与MDA-MB-231、人非小细胞肺癌细胞NCI-H1299、人结肠癌细胞SW480、人膀胱癌细胞T24的解离常数(Kd)的数据图;
图7为通过流式细胞术测定所得核酸适体HX7对表达CD47蛋白的人乳腺癌系MCF-7、人膀胱癌细胞T24的解离常数的数据图;
图8为通过流式细胞术考察截短优化得到的核酸适体HX2C和HX7C针对CD47原核蛋白、Ni琼脂糖微球的结合能力的数据图;
图9为通过流式细胞术考察经截短优化得到的核酸适体HX2C和HX7C与表达CD47蛋白的人乳腺癌系MCF-7与MDA-MB-231、人非小细胞肺癌细胞NCI-H1299、人结肠癌细胞SW480的结合能力的数据图;
图10为通过流式细胞术考察经截短优化得到的核酸适体HX2C和HX7C与不表达CD47蛋白的小鼠成纤维细胞NIH-3T3的结合能力的数据图;
图11为通过流式细胞仪测定经截短优化得到的核酸适体HX2C对表达CD47蛋白的人乳腺癌系MCF-7与MDA-MB-231、人非小细胞肺癌细胞NCI-H1299、人结肠癌细胞SW480的解离常数的数据图;
图12为通过流式细胞术考察截短优化得到的核酸适体HX2C针对CD47真核蛋白的结合能力及解离常数的数据图;
图13为通过流式细胞术考察截短优化得到的核酸适体HX2C对CD47原核蛋白的解离常数的数据图;
图14为通过流式细胞术考察截短优化得到的核酸适体HX2C针对EpCAM蛋白及PD-L1蛋白的结合能力的数据图。
具体实施方式
下面结合附图和实施例对本发明内容做进一步阐述,但不是对本发明的限定。
实施例1:CD47核酸适体的筛选
1)化学合成DNA初始文库,如SEQ ID No.10所示,其中每一条DNA链由85个碱基组成,包含两端各20个碱基的固定序列和中间45个碱基的随机序列,具体为:5'-AAGGAGCAGCGTGGAGGATA-45nt-TTAGGGTGTGTCGTCGTGGT-3',库容量为1015以上,取3nmol DNA初始文库溶于结合缓冲液(PBS,pH 7.4,含2.5mmol/L MgCl2)中,进行变性处理:95℃处理10min,后置于冰上10min,最后于室温下放置10min;
2)通过His与Ni的亲和相互作用,将原核融合蛋白His-CD47固定在Ni琼脂糖微球(Ni-beads)上,得到CD47蛋白琼脂糖微球(CD47-beads);将步骤1)得到的预处理初始文库液体与CD47-beads于37℃孵育40min;
3)在步骤2)中,进行到第四轮筛选时候,需要加入反筛的过程,具体操作为:将处理好的上一轮DNA文库与Ni-beads于37℃孵育15min,进行反筛,然后收集未与Ni-beads结合的液体,再将得到的液体与CD47-beads于37℃下孵育,进行正筛;
4)去掉未与CD47-beads结合的DNA序列,使用结合缓冲液洗涤孵育后的CD47-beads,再将结合了序列的CD47-beads做PCR扩增反应(扩增程序为94℃预变性3min,94℃30s,60℃30s,72℃30s,扩增8个循环,最后72℃延伸5min,正向引物如SEQ ID No.11所示:
5'-FAM-AAGGAGCAGCGTGGAGGATA-3';反向引物如SEQ ID No.12所示:5'-Biotin-ACCACGACGACACACCCTAA-3';)
5)PCR扩增反应结束后,产物为3’端带有生物素标记,5’端带有FAM标记的双链DNA,加入链酶亲和素琼脂糖微球,室温反应30min,然后用0.1mol/L NaOH进行碱变性单链化,经3K超滤管脱盐过滤,纯化,即得到用于下一轮筛选的单链DNA文库,即次一级单链DNA文库;
6)使用200pmol的次一级单链DNA文库,重复进行步骤2)~5)的筛选,并在筛选过程中逐步降低文库以及CD47-beads的投入量,并且逐步增加Ni-beads的投入量以及洗涤次数以增强筛选强度。本实施例中,共进行10轮筛选,而后通过流式细胞术监测核酸适体筛选富集进程,结果如图1所示,显示从第6轮开始,富集文库与CD47蛋白有结合,而与Ni-beads没有结合,最后对第8轮筛选得到的单链DNA富集文库进行测序表征,得到与CD47特异结合的核酸适体序列,即如SEQ ID No.1至SEQ ID No.7所示的HX1、HX2、HX3、HX4、HX5、HX6与HX7。
实施例2:通过流式细胞术考察单链DNA富集文库与CD47原核蛋白的结合能力
首先分别用PCR扩增带荧光标记的第0,6,8,10轮DNA富集文库,使用正向引物(SEQID No.11):5'-FAM-AAGGAGCAGCGTGGAGGATA-3'与反向引物(SEQ ID No.12):5'-Biotin-ACCACGACGACACACCCTAA-3',PCR产物为5’端带有FAM并且3’端带有生物素的双链DNA,加入链酶亲和素琼脂糖微球,室温反应20min,然后用0.1mol/L NaOH进行碱变性单链化,经3K超滤管脱盐过滤,纯化,即得到第0,6,8,10轮单链DNA富集文库;
用200μL结合缓冲液配置浓度为200nmol/L的第0,6,8,10轮单链DNA富集文库溶液,分别加入约105个CD47-beads(原核蛋白),37℃下孵育40min,去掉未与CD47-beads结合的单链DNA,使用结合缓冲液洗涤微球2次,而后把CD47-beads结合重悬在200μL结合缓冲液中,使用流式细胞仪对CD47-beads进行荧光测定。
结果如图1所示,其中图1A是第0,6,8,10轮DNA富集文库与CD47-beads的结合情况,图1B是第0,6,8,10轮DNA富集文库与Ni-beads的结合情况(第0轮文库为DNA初始文库)。结果表明第6,8,10轮DNA富集文库与CD47-beads有结合,而Ni-beads没有结合,说明经过10轮富集,成功地富集了CD47的核酸适体。
实施例3:通过流式细胞术考察测序核酸适体与CD47原核蛋白的结合能力
首先将合成好的带荧光标记的测序核酸适体,即如SEQ ID No.1至SEQ ID No.7所示的HX1、HX2、HX3、HX4、HX5、HX6与HX7,用200μL结合缓冲液配置浓度为200nmol/L的核酸适体溶液,分别加入约105个CD47-beads(原核蛋白),37℃下孵育40min,去掉未与CD47-beads结合的单链DNA,使用结合缓冲液洗涤微球2次,而后把CD47-beads结合重悬在200μL结合缓冲液中。使用流式细胞仪对CD47-beads进行荧光测定。
结果如图2所示,7条核酸适体HX1、HX2、HX3、HX4、HX5、HX6与HX7针对CD47原核蛋白均能结合(图2A),而与Ni琼脂糖微球均不结合(图2B),说明7条核酸适体选择性好。至此结果表明经过8轮富集,获得7条与CD47结合力及特异性良好的核酸适体,将对其进行下一步的表征分析。
实施例4:通过流式细胞术考察测序核酸适体与CD47真核蛋白的结合能力
首先将合成好的带荧光标记的测序核酸适体,即如SEQ ID No.1至SEQ ID No.7所示的HX1、HX2、HX3、HX4、HX5、HX6与HX7,用200μL结合缓冲液配置浓度为200nmol/L的核酸适体溶液,分别加入约105个CD47-beads(真核蛋白),37℃下孵育40min,去掉未与CD47-beads结合的单链DNA,使用结合缓冲液洗涤微球2次,而后把CD47-beads结合重悬在200μL结合缓冲液中。使用流式细胞仪对CD47-beads进行荧光测定。结果如图3所示,7条核酸适体HX1、HX2、HX3、HX4、HX5、HX6与HX7针对CD47真核蛋白均能结合(图3)。
实施例5:通过流式细胞术考察测序核酸适体与肿瘤细胞系的结合能力及选择性
首先将合成好的带荧光标记的测序核酸适体,即如SEQ ID No.1至SEQ ID No.7所示的HX1、HX2、HX3、HX4、HX5、HX6与HX7,用200μL结合缓冲液配置浓度为200nmol/L的核酸适体溶液,加入约105个肿瘤细胞,37℃下孵育40min,去掉未与肿瘤细胞结合的单链DNA,使用结合缓冲液洗涤肿瘤细胞2次,而后把肿瘤细胞重悬在200μL结合缓冲液中。使用流式细胞仪对肿瘤细胞进行荧光测定。结果如图4、5所示,其中HX2、HX3、HX4、HX5、HX6、HX7等6条核酸适体与六种CD47阳性细胞(包括人乳腺癌系MCF-7与MDA-MB-231、人非小细胞肺癌细胞NCI-H1299、人肺腺癌细胞NCI-H1975、人结肠癌细胞SW480、人膀胱癌细胞T24)均有结合,且HX2与HX7结合最好;
且HX1、HX2、HX3、HX4、HX5、HX6等6条核酸适体与三种CD47阴性细胞(包括心肌细胞H9C2、小鼠成纤维细胞NIH-3T3、小鼠单核巨噬细胞raw264.7)均不有结合,唯独HX7适体与心肌细胞H9C2及小鼠成纤维细胞NIH-3T3有微量非特异吸附。
实施例6:通过流式细胞术测定HX2及HX7核酸适体与CD47阳性肿瘤细胞系的解离常数
首先将合成好的带荧光标记的核酸适体,即如SEQ ID No.2所示的HX2及SEQ IDNo.7所示的HX7,用200μL结合缓冲液配置浓度为0,3,5,7.5,10,20,25,30,40,50,75,100,150,200,250,300nmol/L等浓度梯度的核酸适体溶液,分别加入约105个CD47阳性肿瘤细胞,37℃下孵育40min,去掉未与肿瘤细胞结合的单链DNA,使用结合缓冲液洗涤肿瘤细胞2次,后重悬在200μL结合缓冲液中。使用流式细胞仪对肿瘤细胞进行荧光测定,并计算出HX2及HX7核酸适体的解离常数。
结果如图6、7所示,为如SEQ ID No.2所示的HX2对人乳腺癌系MCF-7与MDA-MB-231、人非小细胞肺癌细胞NCI-H1299、人结肠癌细胞SW480、人膀胱癌细胞T24的解离常数分别为2.4±1.0nM(图6A)、23.2±5.4nM(图6B)、8.9±4.4nM(图6C),11.0±3.0nM(图6D)、16.0±5.4nM(图6E);而HX7对人乳腺癌系MCF-7与人膀胱癌细胞T24的解离常数分别为5.3±1.9nM(图7A)、9.0±3.9nM(图7B)。结果表明HX2及HX7核酸适体对肿瘤细胞系表达的CD47蛋白具有高亲和力,因此HX2及HX7核酸适体可以用于后续截短优化分析。
实施例7:HX2及HX7核酸适体的截短优化及结合力、亲和力表征
综上所述,最终选择HX2及HX7核酸适体进行后续截短优化以及实验。具体操作是:先利用NUPACK对HX2及HX7核酸适体进行二级结构的模拟,对其碱基组成和空间结构进行分析,在保证HX2及HX7核酸适体原有的亲和力和选择性基本不变的前提下,去除不必要的碱基,主要去除序列两端的引物,得到最优核酸适体,如SEQ ID No.8所示的HX2C及SEQ IDNo.9所示的HX7C。接下来就是考察最优核酸适体HX2C及HX7C与CD47蛋白及CD47阳性肿瘤细胞系的结合能力、特异性以及亲和力。具体操作与实施例3-6类似。
结果如图8至图14所示,其中图8是HX2C及HX7C核酸适体针对CD47原核蛋白、Ni琼脂糖微球的结合情况;图9是HX2C及HX7C核酸适体与CD47阳性肿瘤细胞系(包括人乳腺癌系MCF-7与MDA-MB-231、人非小细胞肺癌细胞NCI-H1299、人结肠癌细胞SW480)的结合情况;图10是HX2C及HX7C核酸适体与CD47阴性肿瘤细胞-小鼠成纤维细胞NIH-3T3的结合情况;图11是HX2C核酸适体对表达CD47蛋白的人乳腺癌系MCF-7与MDA-MB-231、人非小细胞肺癌细胞NCI-H1299、人结肠癌细胞SW480的解离常数;图12是HX2C核酸适体针对CD47真核蛋白的结合情况及解离常数;图13是HX2C核酸适体针对CD47原核蛋白的解离常数;图14是HX2C核酸适体针对EpCAM及PD-L1蛋白的结合情况。
结果表明HX2与HX7的截短序列HX2C和HX7C保持良好的CD47原核、真核蛋白及CD47阳性肿瘤细胞系结合能力,均相比于全长具有更好的结合力。HX2C对CD47真核、原核蛋白的解离常数分别为19.7±6.3nM、23.9±7.7nM。此外,HX2C对人乳腺癌系MCF-7与MDA-MB-231、人非小细胞肺癌细胞NCI-H1299、人结肠癌细胞SW480的解离常数分别为56.5±19.8nM(图11A)、22.2±9.4nM(图11B)、27.5±9.7nM(图11C)、29.4±3.0nM(图11D)。结果表明HX2C核酸适体对原核、真核及肿瘤细胞系表达的CD47蛋白仍具高亲和力;相比于HX7C适体,HX2C适体具有更好的CD47选择性及特异性。所以,最终获得了一条高亲和、高特异性的CD47核酸适体HX2C。
以上所述,仅为本发明较佳实施例而已,故不能依此限定本发明实施的范围,即依本发明专利范围及说明书内容所作的等效变化与修饰,皆应仍属本发明涵盖的范围内。
Claims (6)
1. 免疫检查点CD47核酸适体,其特征在于,所述CD47核酸适体包括SEQ ID No.1至SEQID No.9所示的核苷酸序列。
2. 根据权利要求1所述的免疫检查点CD47核酸适体,其特征在于:所述CD47核酸适体还包括SEQ ID No.1至SEQ ID No.9所示的核苷酸序列的突变体或截短序列。
3. 根据权利要求2所述的免疫检查点CD47核酸适体,其特征在于:所述CD47核酸适体突变体为SEQ ID No.1至SEQ ID No.9所示的核苷酸序列具有80%以上同源性的序列。
4. 根据权利要求2所述的免疫检查点CD47核酸适体,其特征在于:所述CD47核酸适体突变体为SEQ ID No.1至SEQ ID No.9所示的核苷酸序列具有90%以上同源性的序列。
5.权利要求1至4中任一项所述的免疫检查点CD47核酸适体的应用,所述应用为在肿瘤免疫疗效精准预测研究领域的应用。
6.权利要求1至4中任一项所述的免疫检查点CD47核酸适体的应用,所述应用包括以下至少一种:
(1)所述免疫检查点CD47的核酸适体在肿瘤细胞CD47及其糖基化的流式分析检测、成像中的应用;
(2)所述免疫检查点CD47的核酸适体在组织切片CD47及其糖基化检测、成像中的应用;
(3)所述免疫检查点CD47的核酸适体在肿瘤来源细胞外囊泡或外泌体CD47的流式分析检测、ELISA检测、成像中的应用;
(4)所述免疫检查点CD47的核酸适体在肿瘤来源细胞外囊泡或外泌体CD47糖基化的流式分析检测、成像中的应用;
(5)所述免疫检查点CD47的核酸适体在肿瘤免疫疗效及预后精准预测、指导肿瘤免疫治疗药物用药研究中的应用。
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CN116478999A (zh) * | 2023-01-16 | 2023-07-25 | 广西师范大学 | 免疫抑制分子cd24核酸适体的筛选及应用 |
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