CN112402597B - 一种fap修饰的类外泌体纳米囊泡肿瘤疫苗 - Google Patents

一种fap修饰的类外泌体纳米囊泡肿瘤疫苗 Download PDF

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CN112402597B
CN112402597B CN202011348086.9A CN202011348086A CN112402597B CN 112402597 B CN112402597 B CN 112402597B CN 202011348086 A CN202011348086 A CN 202011348086A CN 112402597 B CN112402597 B CN 112402597B
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程平
胡世川
魏于全
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Abstract

本发明公开了一种FAP修饰的类外泌体纳米囊泡肿瘤疫苗。疫苗包括纳米囊泡，以及佐剂；纳米囊泡能表达如SEQ ID NO.1或SEQ ID NO.2序列所示的FAP基因。本发明以FAP修饰的类外泌体肿瘤纳米囊泡来制备能够用于治疗肿瘤的疫苗，制备得到的疫苗能够通过同时靶向肿瘤细胞和TAFs的杀伤，从而解除免疫抑制屏障，重塑肿瘤微环境来增强抗肿瘤效果。

Description

一种FAP修饰的类外泌体纳米囊泡肿瘤疫苗
技术领域
本发明属于分子生物技术领域，具体涉及一种FAP修饰的类外泌体纳米囊泡肿瘤疫苗。
背景技术
肿瘤来源的外泌体作为一种无细胞疫苗已被报道具有抗肿瘤效应，但其产量低，制备方式复杂限制其规模化的生产。研究发现将肿瘤细胞直接挤压形成的纳米囊泡拥有与外泌体一样的生物特性，并成功克服了生产外泌体产量不足的问题。肿瘤间质环境对肿瘤的发生和发展至关重要，肿瘤相关的成纤维细胞(TAFs)是肿瘤间质的主要成分之一。研究报道，TAFs可以塑造防止免疫细胞浸润的物理屏障，分泌免疫抑制性因子，从而募集免疫抑制细胞，支持肿瘤的生长。成纤维细胞激动蛋白(FAP)是一种可以促进TAFs的募集，分化和增殖的丝氨酸蛋白酶，在90％以上人类肿瘤中的TAFs上高表达。免疫疗法针对FAP抗原靶向TAFs的杀伤，展现出良好的抗肿瘤效果。靶向FAP的全细胞疫苗和FAP的DNA疫苗已被报道。FAP修饰的类外泌体肿瘤纳米囊泡疫苗作为一种全新的无细胞疗法还未见报道。
发明内容
针对现有技术中的上述不足，本发明提供一种FAP修饰的类外泌体纳米囊泡肿瘤疫苗，该疫苗通过同时靶向肿瘤细胞和TAFs的杀伤，从而解除免疫抑制屏障，重塑肿瘤微环境来增强抗肿瘤效果。
为实现上述目的，本发明解决其技术问题所采用的技术方案是：
一种FAP修饰的类外泌体纳米囊泡肿瘤疫苗，疫苗包括纳米囊泡，以及佐剂；纳米囊泡能表达如SEQ ID NO.1或SEQ ID NO.2所示的FAP基因。
进一步地，纳米囊泡来自肿瘤细胞。
进一步地，FAP基因为失去水解活性的基因。
进一步地，SEQ ID NO.1所示的FAP基因为人源FAP基因；SEQ ID NO.2所示的FAP基因为鼠源FAP基因，两者同源性高达91％，尤其是酶活结构域高达92％。
进一步地，FAP全基因编码的第624位丝氨酸突变为了丙氨酸。
由于在进行克隆时只将FAP基因的跨膜区和胞外区克隆至慢病毒目的载体pLJM1中，本申请只需要FAP基因的跨膜区和胞外区相应的序列，在提供的核苷酸序列和氨基酸序列中，只有跨膜区和胞外区的核苷酸序列和氨基酸序列，缺少胞内区的12个碱基和4个氨基酸，因而，突变氨基酸位点在提供的氨基酸序列中的620位。
进一步地，SEQ ID NO.1所示的FAP基因编码的氨基酸序列如SEQ ID NO.3所示。
进一步地，SEQ ID NO.2所示的FAP基因编码的氨基酸序列如SEQ ID NO.4所示。
一种治疗癌症的药物，包括上述的纳米囊泡，以及其在药学上能够接受的佐剂。
一种制备上述纳米囊泡的方法，包括以下步骤：
(1)利用基因定点突变技术将上述基因中的624位丝氨酸(Ser624)突变为丙氨酸(Ala624)，使其失去水解活性，接着用分子克隆技术将该基因的跨膜区和胞外区(13–2283bp)克隆至慢病毒目的载体pLJM1；
(2)HEK 293T细胞在100mm培养皿中生长到60％～70％汇合度时，被转染第三代慢病毒包装载体(pMDLg/pRRE＝6μg、pRSV-REV＝2.5μg、pMD2G＝2.5μg)和目的载体(pLJM1-FAP＝10μg)，加入后8h更换新鲜培养液，之后收集24h和48h的培养上清，过0.45μm的除菌滤器，并用60,000×g超速离心2h，收集病毒沉淀，感染肿瘤细胞48h后，加入相应浓度的嘌呤霉素至对照的肿瘤细胞完全死亡，扩大培养生长正常的稳定表达FAP的肿瘤细胞株；
(3)FAP稳定表达的肿瘤细胞以5×106细胞/mL的浓度悬浮在PBS中，使用微型挤出机(Avanti Polar Lipids)依次通过10μm，5μm和1μm聚碳酸酯膜滤膜(Whatman)连续挤压细胞悬液5次，以生产人FAP类外泌体的纳米级细胞外囊泡。
一种FAP修饰的类外泌体纳米囊泡肿瘤疫苗的制备方法，在超速离心管中由下至上铺设50％，10％碘克沙醇溶液和挤压得到的纳米级细胞外囊泡，4℃，100,000×g密度离心2h。从10％至50％的碘克沙醇层中获得FAP纳米囊泡疫苗(NVs-FAP)，并进一步在4℃下于100,000×g超速离心2小时，重悬，-80℃保存。
本发明的有益效果为：
本发明以FAP修饰的类外泌体肿瘤纳米囊泡来制备能够用于治疗肿瘤的疫苗，制备得到的疫苗能够通过同时靶向肿瘤细胞和TAFs的杀伤，从而解除免疫抑制屏障，重塑肿瘤微环境来增强抗肿瘤效果。
附图说明
图1为稳定表达的肿瘤细胞株检测结果；
图2为NVs-FAP疫苗预防性抗肿瘤的接种流程；
图3为NVs-FAP疫苗预防性抗肿瘤检测结果；其中，A为接种疫苗24天后的小鼠，PBS为空白对照组；NVs为对照组；NVs-FAP为实验组；B为肿瘤体积的变化，曲线由上至下依次为PBS组、NVs组和NVs-FAP组；C为小鼠存活率，曲线由左至右依次为PBS组、NVs组和NVs-FAP组；*表示差异显著。
图4为NVs-FAP疫苗治疗性性抗肿瘤的接种流程；
图5为NVs-FAP疫苗治疗性抗肿瘤检测结果；其中，A为接种疫苗27天后的小鼠，PBS为空白对照组；NVs为对照组；NVs-FAP为实验组；B为肿瘤体积的变化；C为小鼠存活率；*表示差异显著；
图6为接种NVs-FAP疫苗对T细胞中细胞因子IFNγ释放量的影响；其中，PBS为空白对照组；NVs为对照组；NVs-FAP为实验组；*表示差异显著。
具体实施方式
下面对本发明的具体实施方式进行描述，以便于本技术领域的技术人员理解本发明，但应该清楚，本发明不限于具体实施方式的范围，对本技术领域的普通技术人员来讲，只要各种变化在所附的权利要求限定和确定的本发明的精神和范围内，这些变化是显而易见的，一切利用本发明构思的发明创造均在保护之列。
实施例1 疫苗制备
1、小鼠FAP基因包含胞内区(1-12bp)，跨膜区(13-75bp)，胞外区(76-2286bp)，共2286个碱基。利用基因定点突变技术将该基因的624位丝氨酸(Ser624)突变为丙氨酸(Ala624)，使其失去水解活性，接着用分子克隆技术将该基因的跨膜区和胞外区(13-2286bp)片段克隆至慢病毒目的载体pLJM1。
2、HEK 293T细胞在100mm培养皿中生长到60％～70％汇合度时，被转染第三代慢病毒包装载体(pMDLg/pRRE＝6μg、pRSV-REV＝2.5μg、pMD2G＝2.5μg)和目的载体(pLJM1-FAP＝10μg)，加入后8h更换新鲜培养液，之后收集24h和48h的培养上清，过0.45μm的除菌滤器，并用60,000×g超速离心2h，收集病毒沉淀，感染肿瘤细胞48h后，加入相应浓度的嘌呤霉素至对照的肿瘤细胞完全死亡，扩大培养生长正常的稳定表达FAP的肿瘤细胞株，其筛选结果见图1。
3、FAP稳定表达的肿瘤细胞以5×106细胞/mL的浓度悬浮在PBS中，使用微型挤出机(Avanti Polar Lipids)依次通过10μm，5μm和1μm聚碳酸酯膜滤膜(Whatman)连续挤压细胞悬液5次，以生产FAP类外泌体的纳米级细胞外囊泡。然后在超速离心管中由下至上铺设50％，10％碘克沙醇溶液和挤压样本，4℃，100,000×g密度离心2h。从10％至50％的碘克沙醇层中获得FAP纳米囊泡疫苗(NVs-FAP)，并进一步在4℃下于100,000×g超速离心2小时，重悬，-80℃保存。
实施例2 NVs-FAP疫苗用于预防性抗肿瘤
Babl/c小鼠接种前第28天，14天和7天免疫实施例1制备得到的NVs-FAP疫苗(50μg/只)，在最后一次免疫后第7天皮下接种1×106个CT26细胞。其免疫过程见图2，免疫疗效见图3。
根据图3的检测结果可知，NVs-FAP疫苗在进行预防性抗肿瘤时，能够有效的抑制肿瘤细胞的生长，并显著延长了小鼠的存活时间。
实施例3 NVs-FAP疫苗用于治疗性抗肿瘤
Babl/c小鼠皮下接种1×106个CT26细胞，第8天，11天，14天和17天瘤内注射NVs-FAP疫苗(50μg/只)，见图4和图5。
根据图5的检测结果可知，NVs-FAP疫苗在进行治疗性抗肿瘤时，能够有效的抑制肿瘤细胞的生长，并显著延长了小鼠的存活时间。
实施例4 NVs-FAP疫苗刺激T细胞释放IFNγ
分离用NVs-FAP疫苗(50μg/只)免疫3次后的小鼠脾脏，在体外用NVs-FAP(50μg/只)刺激小鼠的淋巴细胞24h，对其分泌的细胞因子IFNγ检测，见图6。
根据图6的检测结果可知，NVs-FAP疫苗能够刺激小鼠T细胞产生更多的细胞因子IFNγ。说明本申请制备得到的NVs-FAP疫苗具有优异的抗肿瘤效果。
序列表
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Claims (6)

1.一种FAP修饰的类外泌体纳米囊泡肿瘤疫苗，其特征在于，所述疫苗包括纳米囊泡，以及佐剂；所述纳米囊泡能表达如SEQ ID NO.1或SEQ ID NO.2所示的FAP基因编码的FAP；
所述纳 米囊泡来自肿瘤细胞。
2.根据权利要求1所述的FAP修饰的类外泌体纳米囊泡肿瘤疫苗，其特征在于，所述FAP基因为失去水解活性的基因。
3.根据权利要求1所述的FAP修饰的类外泌体纳米囊泡肿瘤疫苗，其特征在于，所述SEQID NO.1所示的FAP基因编码的氨基酸序列如SEQ ID NO.3所示。
4.根据权利要求1所述的FAP修饰的类外泌体纳米囊泡肿瘤疫苗，其特征在于，所述SEQID NO.2所示的FAP基因编码的氨基酸序列如SEQ ID NO.4所示。
5.一种治疗癌症的药物，其特征在于，包括权利要求1中 的纳米囊泡，以及佐剂。
6.一种制备权利要求1所述纳米囊泡肿瘤疫苗的方法，其特征在于，包括以下步骤：
（1）将权利要求1中如SEQ ID NO.1或SEQ ID NO.2所示的FAP基因克隆至慢病毒载体上；
（2）使慢病毒载体经转染后感染肿瘤细胞，并筛选能稳定表达FAP基因的肿瘤细胞株，最后挤压肿瘤细胞株形成的细胞悬液，得到该纳米囊泡。
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