CN111196858A - 一种治疗血液肿瘤合并hiv感染的双特异性嵌合抗原受体、基因、构建方法及其应用 - Google Patents
一种治疗血液肿瘤合并hiv感染的双特异性嵌合抗原受体、基因、构建方法及其应用 Download PDFInfo
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Abstract
本发明公开了一种治疗HIV感染合并血液肿瘤的双特异性嵌合抗原受体重组基因的构建方法及其应用,所述嵌合抗原受体由信号肽、HIV gp120抗原特异性单链抗体及抗CD19单链抗体依次连接,再和CD28跨膜区以及CD28胞内结构域(ICD)、4‑1BB共刺激结构域、CD3ζ胞内信号传导结构域依次串联组成,或者所述嵌合抗原受体由信号肽、HIV gp120抗原特异性单链抗体和CD28跨膜区以及CD28‑ICD、4‑1BB共刺激结构域、CD3ζ胞内信号传导结构域(第一CAR)和信号肽、抗CD19单链抗体和CD8跨膜区以及CD28‑ICD、4‑1BB共刺激结构域、CD3ζ胞内信号传导结构域(第二CAR),第一CAR和第二CAR依次并联组成。
Description
技术领域
本发明涉及医药生物领域,具体是指一种治疗血液肿瘤合并HIV感染的双特异性嵌合抗原受体、其编码基因、构建方法及其应用。
背景技术
人体感染艾滋病病毒后,CD4+ T淋巴细胞受到病毒攻击,导致机体免疫能力降低,易发生各种病毒感染或恶性肿瘤疾病,淋巴瘤就是其中一种。艾滋病相关淋巴瘤的发病风险是普通人群的165倍,恶性淋巴瘤是目前艾滋病患者主要死亡原因之一。
根据肿瘤专科医师意见,采用内科治疗方案给予HIV合并恶性肿瘤疾病患者治疗,治疗方案给予抗肿瘤联合ART治疗;入院后给予协助患者完成各项常规检查后,根据患者病情、CD4+ T淋巴细胞数、药敏试验结果等,给予患者免疫支持、对症、抗病毒、抗机会性感染、高效抗转录病毒治疗等,病症稳定后,给予患者化疗治疗,化疗药物包括:亚叶酸钙、氟尿嘧啶、顺铂、阿霉素、环磷酰胺、长春新碱、吉西他滨等,化疗2周为1疗程。在此基础上给予患者三维适形放射治疗,每周照射总剂量3000-4800cGy,1次/d,5次/周,共治疗4周为1疗程。现目前,临床中并无对HIV合并恶性肿瘤兼治的最佳方案与药物,一般根据患者实际病毒载量与CD4+ T淋巴细胞计数给予对症治疗。若患者CD4+ T淋巴细胞<200/μL,则需对患者先行对症治疗与高效抗逆转录病毒(HAART)治疗,不宜即刻接受抗癌化疗,待患者病情稳定后再给予化疗或放疗,避免抗癌药物加剧机体感染及对免疫系统的损伤。若患者CD4+ T淋巴细胞>200/μL则可给予患者高效抗逆转录病毒(HAART)治疗同时给予抗癌治疗。
高效抗逆转录病毒治疗(HAART)是HIV/AIDS治疗史上的第一次革命,其极大的降低了HIV/AIDS的发病率和病死率,显著延长了患者寿命,甚至降低了HIV的传播。但也面临着很多挑战:(1)患者必须终生服药,需付出昂贵的经济代价;(2)严重的毒副作用;(3)耐药毒株的出现;(4)更为重要的是cART不能彻底清除病毒,主要是因为药物仅对复制中的病毒有效,而对HIV在感染早期建立的潜伏性病毒“储藏库”(reservoir)是无效的。一旦抗逆转录病毒治疗中断,病毒储藏库中的整合前病毒再度激活,几乎所有患者体内病毒血症会迅速反弹。对淋巴瘤的治疗主要是化疗的方法,副作用大且容易复发,无可供使用的疗法。但HIV合并恶性肿瘤患者一般病情严重,患者免疫系统遭受严重损伤,病情进展较快,患者病死率较高,对此,如何选择有效的治疗方案已迫在眉睫。
为了有效治疗血液肿瘤合并HIV感染患者,本发明提供一种治疗血液肿瘤合并HIV感染的双特异性嵌合抗原受体及其编码基因,其能够通过CAR-T疗法对血液肿瘤合并HIV感染患者的病情起到很好的缓解、减轻以及治愈的疗效,具有广阔的临床推广前景。
发明内容
针对现有技术的不足,本发明第一目的是提供一种治疗血液肿瘤合并HIV感染的双特异性嵌合抗原受体,所述双特异性嵌合抗原受体中包含抗HIV gp120单链抗体和抗CD19单链抗体。
本发明通过优化设计HIV gp120、CD19特异的单链抗体,提供了一种HIV gp120、CD19双靶点修饰的CAR-T细胞,能够特异地与HIV gp120抗原及CD19结合。在HIV感染和CD19阳性B淋巴细胞系恶性肿瘤的细胞杀伤试验中,采用HIV gp120、CD19双靶点修饰的CAR-T细胞进行CAR-T疗法能实现一种CAR-T细胞杀伤两种不同类型恶性细胞,实现一种CAR-T同时治疗两种疾病的效果。
进一步地,所述双特异性嵌合抗原受体还包括CSF2RA信号肽和CD8信号肽、CD28跨膜区和CD8跨膜区、CD28-ICD、4-1BB共刺激结构域和CD3ζ胞内信号转导结构域。
进一步地,所述双特异性嵌合抗原受体从N端到C端顺次拼接信号肽SP1、抗HIVgp120单链抗体、Strep II、连接肽、抗CD19单链抗体、CD8铰链区、CD28跨膜区、CD28-ICD、4-1BB共刺激结构域以及CD3ζ胞内信号传导结构域。
进一步地,所述双特异性嵌合抗原受体从N端到C端由第一CAR:信号肽SP1、抗HIVgp120抗原特异性单链抗体、Strep II、连接肽和CD28跨膜区、CD28-ICD、4-1BB共刺激结构域以及CD3ζ胞内信号传导结构域和第二CAR:信号肽SP2、抗CD19单链抗体、CD8跨膜区、CD28-ICD以及CD3ζ胞内信号传导结构域,第一CAR和第二CAR通过P2A依次并联组成。
更进一步地,所述信号肽优选为CSF2RA和CD8信号肽;所述连接肽为3×G4S;所述自切割短肽优选为P2A类短肽。
本发明还提供双特异性嵌合抗原受体的编码核苷酸。
进一步地,所述双特异性嵌合抗原受体的编码核苷酸的核苷酸序列如SEQ ID NO:37或如SEQ ID NO:38所示。
本发明第三目的是提供一种重组慢病毒载体,所述慢病毒载体以PTK881-EF1α载体为骨架,含有前述的双特异性嵌合抗原受体的编码核苷酸。
本发明第四目的是提供一种免疫细胞,所述免疫细胞转染有前述的重组慢病毒载体。
本发明第五目的是提供一种双特异性嵌合抗原受体编码核苷酸的构建方法。
进一步地,所述构建方法包括以下步骤:
1)基因合成如SEQ ID NO:1所示的信号肽-抗HIV gp120单链抗体编码核苷酸SP1-N6,以及如SEQ ID NO:2所示的信号肽-抗CD19单链抗体编码核苷酸SP2-FMC83-28Z,将合成的上述编码核苷酸分别克隆至pUC57载体;
2)以人cDNA文库为模板,设计引物分别扩增片段CD8铰链区、CD28跨膜区、CD28胞内结构域(ICD)、4-1BB共刺激结构域、CD8跨膜区、CD3ζ胞内信号传导结构域、以引物互补方式得到Strep II、连接肽3×G4S、P2A;
3)采用Overlap PCR技术将SP1-N6、Strep II、连接肽3×G4S、SP2-FMC63-28Z与CD8铰链区、CD28跨膜区、CD28-ICD、4-1BB共刺激结构域、CD3ζ胞内信号传导结构域顺次扩增连接,获得嵌合抗原受体的编码基因C5-CAR,其结构示意图如图1所示;采用Overlap PCR技术分别将SP1-N6与Strep II、连接肽3×G4S、CD8铰链区、CD28跨膜区、CD28-ICD、4-1BB共刺激结构域、CD3ζ胞内信号传导结构域顺次扩增成片段SP1-CD3ζ,P2A与SP2-FMC63-28Z、CD8铰链区、CD8跨膜区、CD28-ICD、CD3ζ胞内信号传导结构域顺次扩增成片段P2A-SP2-CD3ζ,片段SP1-CD3ζ与P2A-SP2-CD3ζ最后连接成编码基因C6-CAR,其结构示意图如图2所示;
优选地,所述单链抗体N6的氨基酸序列如SEQ ID NO:35所示、所述单链抗体FMC63-28Z的氨基酸序列如SEQ ID NO:36所示,信号肽SP1、信号肽SP2、Strep II、连接肽3×G4S的核苷酸序列分别如SEQ ID NO:7、9、11、23所示;C5-CAR中CD8铰链区、CD28跨膜结构域、CD28-ICD、4-1BB共刺激结构域、CD3ζ胞内信号传导结构域的核苷酸序列分别如SEQ IDNO:13、15、17、19、21所示;C6-CAR中P2A的核苷酸序列如SEQ ID No:25所示,单链抗体FMC63-28Z连接的CD8铰链区、CD8跨膜结构域、CD28-ICD、CD3ζ胞内信号传导结构域的核苷酸序列如SEQ ID No:27、29、31、33所示。
本发明第六目的是提供双特异性嵌合抗原受体、及其编码核苷酸、重组慢病毒载体、免疫细胞在制备治疗血液肿瘤合并HIV感染的药物或制剂中的应用。
进一步地,所述血液肿瘤为CD19+阳性的B淋巴细胞系恶性肿瘤。
本发明的有益效果在于:
本发明的创新在于使用针对HIV囊膜蛋白的广谱中和抗体和针对血液肿瘤靶向标志物CD19的特异性抗体以串联的形式组合成scFv,或并联分别生成两个CAR分子并将其装到γδT细胞和CD8+ T细胞上,优点在于:
首先,不需要抽取HIV感染者的血液;其次,只转导一次看可以制备针对两种恶性细胞的CAR分子;再者,高负荷的肿瘤在体内的存在会刺激CAR-T细胞的增殖,加速HIV感染细胞的清除;最后,不需要化疗和HAART药物,无副作用。
附图说明
图1为C5-CAR结构示意图;
图2为C6-CAR结构示意图;
图3为PTK881-EF1α-C5质粒图谱;
图4为PTK881-EF1α-C6质粒图谱;
图5为细胞转导效率检测结果,即从上至下依次是C5-CAR-γδT、C6-CAR-γδT细胞转导效率检测结果;
图6为细胞转导效率检测结果,即从上至下依次是C5-CAR-CD8+ T、C6-CAR-CD8+T细胞转导效率检测结果;
图7为C5-CAR-γδT细胞体外杀瘤效率检测结果;
图8为C5-CAR-CD8+T细胞体外杀瘤效率检测结果;
图9为C6-CAR-γδT细胞体外杀瘤效率检测结果;
图10为C6-CAR-CD8+T细胞体外杀瘤效率检测结果;
图11为γδT细胞体外杀瘤效率检测结果;
图12为CD8+T细胞体外杀瘤效率检测结果。
图13为gp41-CD19 CAR-γδT与C5-CAR-γδT、C6-CAR-γδT体外杀伤效率比较
图14为gp41-CD19 CAR-CD8+T与C5-CAR-CD8+T、C6-CAR-CD8+T体外杀伤效率比较
具体实施方式
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解,下面的实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1:PTK881-EF1α-C5、PTK881-EF1α-C6质粒的构建
1、序列SP1-N6(核苷酸序列SEQ ID NO:1)、SP2-FMC63-28Z(核苷酸序列SEQ IDNO:2)由苏州金唯智生物科技公司进行基因合成,合成的序列克隆到pUC57载体上。
2、以人cDNA文库为模板,设计引物分别扩增片段CD8铰链区、CD28跨膜区、CD28-ICD、4-1BB共刺激结构域、CD8跨膜区、CD3ζ胞内信号传导结构域、以引物互补方式得到Strep II、连接肽3×G4S、P2A;
3、采用Overlap PCR技术将SP1-N6、Strep II、连接肽3×G4S、SP2-FMC63-28Z与CD8铰链区、CD28跨膜区、CD28-ICD、4-1BB共刺激结构域、CD3ζ胞内信号传导结构域顺次扩增连接,获得嵌合抗原受体的编码基因C5-CAR,其结构示意图如图1所示,其核苷酸序列为37;采用Overlap PCR技术分别将SP1-N6与Strep II、连接肽3×G4S、CD8铰链区、CD28跨膜区、CD28-ICD、4-1BB共刺激结构域、CD3ζ胞内信号传导结构域顺次扩增成片段SP1-CD3ζ,P2A与SP2-FMC63-28Z、CD8铰链区、CD8跨膜区、CD28共刺激结构域、CD3ζ胞内信号传导结构域顺次扩增成片段P2A-SP2-CD3ζ,片段SP1-CD3ζ与P2A-SP2-CD3ζ最后连接成编码基因C6-CAR,其结构示意图如图2所示,其核苷酸序列为38;
其中,C5-CAR、C6-CAR为能够识别被HIV病毒感染的血液肿瘤细胞表面的具有信号肽的单链抗体ScFv-N6、ScFv-FMC63-28Z。单链抗体ScFv-N6:重链可变区VH氨基酸序列如SEQ ID NO:3所示,轻链可变区VL的氨基酸序列如SEQ ID NO:4所示。单链抗体ScFv-FMC63-28Z:重链可变区VH的氨基酸序列如SEQ ID NO:5所示,轻链可变区VL的氨基酸序列如SEQID NO:6所示。单链抗体ScFv-N6、ScFv-FMC63-28Z的氨基酸序列如SEQ ID NO:35、SEQ IDNO:36所示。
信号肽SP1的核苷酸序列、氨基酸序列如SEQ ID NO:7、SEQ ID NO:8所示,信号肽SP2的核苷酸序列、氨基酸序列如SEQ ID NO:9、SEQ ID NO:10所示,StrepⅡ的核苷酸序列、氨基酸序列如SEQ ID NO:11、SEQ ID NO:12所示,连接肽3×G4S的核苷酸序列、氨基酸序列如SEQ ID NO:23、SEQ ID NO:24所示。C5-CAR中CD8铰链区的核苷酸序列、氨基酸序列如SEQID NO:13、SEQ ID NO:14所示,C5-CAR中CD28跨膜结构域的核苷酸序列、氨基酸序列如SEQID NO:15、SEQ ID NO:16所示,C5-CAR中CD28-ICD的核苷酸序列、氨基酸序列如SEQ ID NO:17、SEQ ID NO:18所示,C5-CAR中4-1BB共刺激结构域的核苷酸序列、氨基酸如序列如SEQID NO:19、SEQ ID NO:20所示,C5-CAR中CD3ζ的核苷酸序列、氨基酸如序列如SEQ ID NO:21、SEQ ID NO:22所示如。
C6-CAR中P2A的核苷酸序列、氨基酸序列如SEQ ID NO:25、SEQ ID NO:26所示,C6-CAR中单链抗体scFv-N6连接的StrepⅡ、连接肽3×G4S、CD8铰链区、CD28跨膜结构域、CD28-ICD、4-1BB共刺激结构域、CD3ζ胞内信号传导结构域所对应的核苷酸序列、氨基酸序列均与C5-CAR相同,C6-CAR中单链抗体scFv-FMC63-28Z连接的CD8铰链区的核苷酸序列、氨基酸序列如SEQ ID NO:27、SEQ ID NO:28所示,C6-CAR中单链抗体scFv-FMC63-28Z连接的CD8跨膜区的核苷酸序列、氨基酸序列如SEQ ID NO:29、SEQ ID NO:30所示,C6-CAR中单链抗体scFv-FMC63-28Z连接的CD28-ICD的核苷酸序列、氨基酸序列如SEQ ID NO:31、SEQ ID NO:32所示,C6-CAR中单链抗体scFv-FMC63-28Z连接的CD3ζ的核苷酸序列、氨基酸序列如SEQID NO:33、SEQ ID NO:34所示。
3、将质粒PTK881-Kan使用EcoR I和BamH I限制性内切酶进行双酶切,产物经过0.8%的琼脂糖凝胶电泳,并割胶回收置于Eppendorf管内,用Axygen公司的琼脂糖凝胶回收试剂盒回收相应的片段,并测定产物的纯度和浓度。
4、将上述载体回收片段分别与C5-CAR、C6-CAR以1:2摩尔比加入Eppendorf管加入ExnaseⅡ连接酶(Vazyme)与同源重组酶5×CEⅡbuffer,37℃反应0.5小时;将连接液取出10μL加入100μL DH5α感受态细胞冰浴30min后42℃热激90s,完成后加入500μL soc培养基37℃、220rpm培养2小时;2小时后将Eppendorf管4000g离心1min移除400μL多余液体。将剩余液体涂布在含卡那霉素的LB平板37℃培养12小时;分别在平板上挑取单菌落,接种到5mLLB液体培养基中37℃、220rpm培养12小时。
5、用Axygen小提试剂盒提取质粒,获得质粒PTK881-EF1α-C5-1、PTK881-EF1α-C6-1,送生工生物工程(上海)股份有限公司科技公司一代测序验证无误后,进行含质粒PTK881-EF1α-C5-1、PTK881-EF1α-C6-1的DH5α菌株保种。PTK881-EF1α-C5-1的完整图谱示意图如图3所示,PTK881-EF1α-C6-1的完整图谱示意图如图4所示。
实施例2:质粒的制备及测序
1、质粒的制备
将含质粒PTK881-EF1α-C5-1、PTK881-EF1α-C6-1的DH5α菌种分别接种至250mL含100μg/mL卡那霉素的LB培养液中,37℃、220rpm培养过夜。培养液在4℃于6000g离心20min,弃上清。
取出EndoFree plasmid mega kit(Qiagen)中的Buffers P1,向离心得到的大肠杆菌沉淀中加120mL提前预冷的Buffers P1,盖上离心瓶盖,剧烈振荡离心瓶使大肠杆菌沉淀在Buffers P1中完全分散。
向离心瓶中加120mLBuffers P2,盖上瓶盖放置在滚轴混匀仪上,慢慢提速至50rpm,彻底混匀后室温放置5min。
向离心瓶中加120mLBuffers P3,盖上瓶盖放置在滚轴混匀仪上,慢慢提速至滚轴混匀仪最大转速70rpm,彻底混匀直至呈白色不粘稠蓬松的混合液。在4℃于9000g离心15min。
向QIAfilter Cartridge倒入50mLBufferFW,将离心所得上清液倒入QIAfilterCartridge中,轻轻地搅拌混匀。将混合液抽滤入已标记好对应的玻璃瓶中。
向每个玻璃瓶中加入20mL Buffer ER,上下颠倒混匀6次,在-20℃孵育30min。
将标记好的mega柱放入对应的架子上,向每个mega柱内加入35mL Buffers QBT平衡,重力作用使之流尽。
将玻璃瓶中的液体分批全部倒入对应标记的mega柱中,待柱中液体流尽后,向每个mega柱分批加入200mLBuffer QC进行清洗。待柱中液体流尽后,将废液收集盘中的废液倒入50mL洁净离心管内。
再向每个mega柱内加入40mL Buffer QN,使用50mL洁净离心管收集流出液,上下颠倒6次混匀,分装20mL至另一洁净已标记的50mL离心管内。
向每个50mL离心管加入14mL异丙醇(常温),上下颠倒6次混匀。在4℃于15000g离心50min。
超净工作台内吸尽上清,每管加入3.5mL Endotoxin-free water漂洗,不要将底部沉淀冲散。在4℃于15000g离心30min。将EndoFree plasmid mega kit中的BufferTE放入烘箱内预热。
在超净工作台内吸尽离心后的上清,于超净工作台内吹干(挥发残留的无水乙醇,时间在10min左右)。
在烘箱内拿出BufferTE,在超净工作台内向每管加入1mL BufferTE,用枪吹打10次后放入65℃烘箱,期间不间断地敲击管壁促使沉淀完全溶解。在4℃于4000g离心1min将管壁上的液体甩到管底后吹打混匀。
在超净工作台内将液体全部转移至无内毒素无热源无核酸酶对应标记的EP管中。吸出2μL,用微量分光光度计测质粒浓度,并标记在对应的EP管上,获得质粒PTK881-EF1α-C5-1、PTK881-EF1α-C6。
2、目的基因测序
分别取20μL(500ng)质粒DNA,外送测序,根据原始种子序列,检查质粒生产所得产品的目的基因有无发生改变,稳定的工艺下,工作种子在进行发酵培养放大过程中,目的基因不会发生改变,可用于下一环节的生产和正确表达蛋白。
实施例3、Lenti3-C5、Lenti3-C6慢病毒载体的制备与活滴检测
1、慢病毒载体的制备
在2个多层细胞培养瓶(Hyperflask)分别接入130.0~140.0×106数目的293T细胞(Takara),共560mL DMEM完全培养基(50mL胎牛血清、5mL Antibiotic-Antimycotic(100×)),在37℃含5%CO2培养箱中培养24小时。分别将混有320μg质粒(PTK881-EF1α-C5:BZ1质粒:BZ2质粒:BZ3质粒=12:10:5:6和PTK881-EF1α-C6:BZ1质粒:BZ2质粒:BZ3质粒=12:10:5:6)的DMEM基础培养基加入两个960μg PEI管中,漩涡震荡,室温平衡10min。将上述两管35mLPEI与质粒的混合液分别与525mL DMEM完全培养基混匀,换入上述多层细胞培养瓶中。将多层细胞培养瓶置于37℃含5%CO2培养箱培养3天后,收集细胞培养上清液。
分别将上清液4000rpm(或3000g)离心30min后,向离心后上清中加入cryonase酶(Takara)置于4℃。6个小时后,使用0.22μm的滤膜对慢病毒上清液进行抽滤,4℃于30000g离心2.5h。去除上清,加入1mL T细胞培养基重悬沉淀。重悬后,留20μL做病毒活性滴度检测,剩余慢病毒浓缩液分装,标记为Lenti3-C5、Lenti3-C6并置于-80℃保存备用。
2、慢病毒载体活性滴度检测
原理:anti-Strep tagⅡ抗体上标记有荧光素,而anti-Strep tagⅡ抗体能与CAR中Strep tagⅡ特异性结合,通过流式细胞仪检测到的荧光信号间接反应了CAR在293T细胞中的表达情况。
方法:在6孔板中接入5.0×105个/孔293T细胞,慢病毒浓缩液每孔分别加入0.1μL、0.5μL、1μL,并设1个阴性对照。置于37℃含5%CO2培养箱内培养。三日后,用Versene溶液(Gibco)收集293T细胞送流式细胞学检测CAR阳性293T细胞比例,并换算得到Lenti3-C5、Lenti3-C6慢病毒浓缩液活性滴度。
目前的慢病毒浓缩液活性滴度在1×108~10×108(TU/mL)范围内,检测分析结果见表1。
表1 Lenti3-C5和Lenti3-C6慢病毒活性滴度检测分析结果
样品编号 | 活性滴度(TU/mL) |
Lenti3-C5 | 1.8×10<sup>8</sup> |
Lenti3-C6 | 2.1×10<sup>8</sup> |
实施例4、CAR-γδT、CAR-CD8+T细胞的制备
1、CAR-γδT细胞制剂制备:
采集健康供者外周血200mL,采用Ficoll淋巴细胞分离液分离单个核细胞。计数后,使用适量TCRγδ+ T Cell Isolation Kit,human(美天旎)分选TCRγδ+ T细胞,并以1.0~2.0×106个/mL密度在γδT细胞激活培养液(OpTmizerTM CTSTM T-Cell ExpansionBasal Medium,OpTmizerTM CTS T-Cell Expansion Supplement(Invitrogen),500~1000IU/mL的IL-2(双鹭药业),IL-75~20ng/mL,唑唻膦酸5μM)中培养,活化γδT细胞。
24小时后,按MOI为5分别加入Lenti3-C5、Lenti3-C6慢病毒载体进行转导,混匀后置于CO2培养箱孵育,4小时后补加适量的γδT细胞激活培养液进行培养。
慢病毒转导24小时后将转导后的C5-CAR-γδT、C6-CAR-γδT细胞换入γδT细胞激活培养液,并调整活细胞密度为1.0-2.0×106/mL,继续培养扩增3天,每天进行观察和计数,并根据计得的细胞数量进行补液扩大培养,始终保持细胞培养密度为1.0-2.0×106/mL。第4天开始,每天补加γδT细胞扩增培养液(OpTmizerTM CTSTM T-Cell ExpansionBasal Medium,OpTmizerTMCTS T-Cell Expansion Supplement(Invitrogen),500~1000IU/mL的IL-2(双鹭药业),IL-75~20ng/mL),并调整活细胞密度为1.0-2.0×106/mL,扩增培养至14天。
根据预计细胞用量收集C5-CAR-γδT、C6-CAR-γδT细胞,重悬于含2%人血白蛋白的100mL生理盐水中,转入细胞回输袋中,热封后制成C5-CAR-γδT、C6-CAR-γδT细胞制剂成品。
2、CAR-CD8+T细胞制剂制备
采集健康供者脐带血60mL,采用Ficoll淋巴细胞分离液分离单个核细胞。计数后,使用适量CD8+ T Cell Isolation Kit human(美天旎)分选CD8阳性细胞,并以1.0~2.0×106个/mL密度在T细胞完全培养液(OpTmizerTM CTSTM T-Cell ExpansionBasal Medium,OpTmizerTMCTS T-Cell Expansion Supplement(Invitrogen),500~1000IU/mL的IL-2(双鹭药业))中培养,同时按每106个细胞加入25μl Dynabeads Human T-Activator CD3/CD28(Invitrogen)活化T细胞。
24小时后,按MOI为3分别加入Lenti3-C5、Lenti3-C6慢病毒载体进行转导,混匀后置于CO2培养箱孵育,4小时后补加适量的T细胞完全培养基进行培养。
慢病毒转导24小时后将转导后C5-CAR-CD8+T、C6-CAR-CD8+T细胞换入新鲜T细胞完全培养液,并调整活细胞密度为1.0-2.0×106/mL,继续培养扩增10~20天,每天进行观察和计数,并根据计得的细胞数量进行补液扩大培养,始终保持细胞培养密度为1.0-2.0×106/mL。
根据预计细胞用量收集C5-CAR-CD8+T、C6-CAR-CD8+T细胞,重悬于含2%人血白蛋白的100mL生理盐水中,转入细胞回输袋中,热封后制成C5-CAR-CD8+T、C6-CAR-CD8+T细胞制剂成品。
3、CAR-γδT、CAR-CD8+T细胞转导效率检测
取1.0×106个CAR-γδT、CAR-CD8+T细胞,分别与FITC-Strep II室温孵育30分钟,生理盐水清洗两次后,通过流式细胞仪检测FITC荧光信号,测量FITC阳性细胞比率,反映了CAR阳性细胞在总细胞中的比率。C5-CAR-γδT、C5-CAR-CD8+T、C6-CAR-γδT、C6-CAR-CD8+T细胞转导效率检测结果分别如图5、图6所示。图5、图6表明成功制备了C5-CAR-γδT、C5-CAR-CD8+T、C6-CAR-γδT、C6-CAR-CD8+T细胞。
实施例5、CAR-γδT、CAR-CD8+T细胞的体外功能检测
体外杀瘤检测:
采用钙黄绿素检测法分别对CD8+T、γδT、CAR-γδT、CAR-CD8+T细胞进行体外杀瘤功能检测。选取构建稳转细胞293T-gp160+,Raji细胞作为阳性靶细胞,以293T细胞作为阴性靶细胞。
取适量的上述293T-gp160+、Raji、293T靶细胞,在1×106/mL的细胞悬液(PBS,5%胎牛血清)加入钙黄绿素-乙酰羟甲基酯(Calcein-AM)至终浓度25μM,培养箱中孵育30min。常温,洗两遍后将细胞重悬至0.5×105/mL,向96孔板中分别加阴性靶细胞和阳性靶细胞,阳性靶细胞分三组,第一组:每孔加入5000个293T-gp160+;第二组:每孔加入5000个Raji细胞;第三组每孔加入2500个293T-gp160+和2500个Raji细胞。按25:1,5:1,1:1的效靶比分别加入T、γδT、C5-CAR-γδT、C5-CAR-CD8+T、C6-CAR-γδT、C6-CAR-CD8+T细胞,37℃孵育2~3小时。孵育完成后取上清,测量其中钙黄绿素的荧光强度,并根据自发释放对照和最大释放对照,计算靶细胞裂解百分数。靶细胞裂解百分数结果如图7至图12所示,结果显示,C5-CAR-γδT、C6-CAR-γδT细胞能明显促进gp160、CD19阳性靶细胞(293T-gp160+、Raji)裂解,对阴性靶细胞(293T)也有一定的杀伤作用;γδT细胞对阳性靶细胞和阴性靶细胞都有促进裂解作用。C5-CAR-CD8+T、C6-CAR-CD8+T细胞能显著促进gp160、CD19阳性靶细胞(293T-gp160+、Raji)裂解,而对阴性靶细胞作用不明显。
另外,比较了以293T-gp160+(gp160为囊膜蛋白gp120和gp41依次相连全长)为靶细胞时,gp41-CD19 CAR-γδT与C5-CAR-γδT、C6-CAR-γδT体外杀伤效率。以293T-gp160+为靶细胞时,gp41-CD19 CAR-CD8+T与C5-CAR-CD8+T、C6-CAR-CD8+T体外杀伤效率,结果分别见图13、图14。结果显示,gp120-CD19组合的CAR-T杀伤效果比gp41-CD19组合杀瘤好。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
序列表
<110> 武汉科技大学
<120> 一种治疗血液肿瘤合并HIV感染的双特异性嵌合抗原受体、基因、构建方法及其应用
<141> 2020-02-05
<160> 38
<170> SIPOSequenceListing 1.0
<210> 1
<211> 786
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccccgc cttcctgctg 60
atcccccgag cgcacctggt acaatcaggg actgcgatga agaaaccggg ggcctcagta 120
agagtctcct gccagacctc tggatacacc tttaccgccc acatattatt ttggttccga 180
caggcccccg ggcgaggact tgagtgggtg gggtggatca agccacaata tggggccgtg 240
aattttggtg gtggttttcg ggacagggtc acattgactc gagacgtata tagagagatt 300
gcgtacatgg acatcagagg ccttaaacct gacgacacgg ccgtctatta ctgtgcgaga 360
gaccgttcct atggcgactc ctcttgggcc ttagatgcct ggggacaggg aacgacggtc 420
gtcgtctccg cgggcggagg gggttcaggt ggaggaggct ctggcggtgg cggaagctac 480
atccacgtga cccagtctcc gtcctccctg tctgtgtcta ttggagacag agtcaccatc 540
aattgccaga cgagtcaggg tgttggcagt gacctacatt ggtatcaaca caaaccgggg 600
agagccccta aactcttgat ccaccatacc tcttctgtgg aagacggtgt cccctcaaga 660
ttcagcggct ctggatttca cacatctttt aatctgacca tcagcgacct acaggctgac 720
gacattgcca catattactg tcaagtttta caatttttcg gccgagggag tcgactccat 780
attaaa 786
<210> 2
<211> 792
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccatc 60
cccgacatcc agatgaccca gaccacctcc agcctgagcg ccagcctggg cgaccgggtg 120
accatcagct gccgggccag ccaggacatc agcaagtacc tgaactggta tcagcagaag 180
cccgacggca ccgtcaagct gctgatctac cacaccagcc ggctgcacag cggcgtgccc 240
agccggttta gcggcagcgg ctccggcacc gactacagcc tgaccatctc caacctggaa 300
caggaagata tcgccaccta cttttgccag cagggcaaca cactgcccta cacctttggc 360
ggcggaacaa agctggaaat caccggcagc acctccggca gcggcaagcc tggcagcggc 420
gagggcagca ccaagggcga ggtgaagctg caggaaagcg gccctggcct ggtggccccc 480
agccagagcc tgagcgtgac ctgcaccgtg agcggcgtga gcctgcccga ctacggcgtg 540
agctggatcc ggcagccccc caggaagggc ctggaatggc tgggcgtgat ctggggcagc 600
gagaccacct actacaacag cgccctgaag agccggctga ccatcatcaa ggacaacagc 660
aagagccagg tgttcctgaa gatgaacagc ctgcagaccg acgacaccgc catctactac 720
tgcgccaagc actactacta cggcggcagc tacgccatgg actactgggg ccagggcacc 780
agcgtgaccg tg 792
<210> 3
<211> 122
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Arg Ala His Leu Val Gln Ser Gly Thr Ala Met Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Arg Val Ser Cys Gln Thr Ser Gly Tyr Thr Phe Thr Ala His
20 25 30
Ile Leu Phe Trp Phe Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val
35 40 45
Gly Trp Ile Lys Pro Gln Tyr Gly Ala Val Asn Phe Gly Gly Gly Phe
50 55 60
Arg Asp Arg Val Thr Leu Thr Arg Asp Val Tyr Arg Glu Ile Ala Tyr
65 70 75 80
Met Asp Ile Arg Gly Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Arg Ser Tyr Gly Asp Ser Ser Trp Ala Leu Asp Ala Trp
100 105 110
Gly Gln Gly Thr Thr Val Val Val Ser Ala
115 120
<210> 4
<211> 103
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Tyr Ile His Val Thr Gln Ser Pro Ser Ser Leu Ser Val Ser Ile Gly
1 5 10 15
Asp Arg Val Thr Ile Asn Cys Gln Thr Ser Gln Gly Val Gly Ser Asp
20 25 30
Leu His Trp Tyr Gln His Lys Pro Gly Arg Ala Pro Lys Leu Leu Ile
35 40 45
His His Thr Ser Ser Val Glu Asp Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Phe His Thr Ser Phe Asn Leu Thr Ile Ser Asp Leu Gln Ala
65 70 75 80
Asp Asp Ile Ala Thr Tyr Tyr Cys Gln Val Leu Gln Phe Phe Gly Arg
85 90 95
Gly Ser Arg Leu His Ile Lys
100
<210> 5
<211> 117
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser
1 5 10 15
Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly
20 25 30
Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly
35 40 45
Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser
50 55 60
Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys
65 70 75 80
Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys
85 90 95
His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Ser Val Thr Val
115
<210> 6
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr
100 105
<210> 7
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccccgc cttcctgctg 60
atcccc 66
<210> 8
<211> 22
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro
20
<210> 9
<211> 63
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccatc 60
ccc 63
<210> 10
<211> 21
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Ile Pro
20
<210> 11
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
aactggagcc acccccagtt cgagaag 27
<210> 12
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Asn Trp Ser His Pro Gln Phe Glu Lys
1 5
<210> 13
<211> 135
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 14
<211> 45
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 15
<211> 81
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60
gcctttatta ttttctgggt g 81
<210> 16
<211> 27
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 15
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
20 25
<210> 17
<211> 123
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60
gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120
tcc 123
<210> 18
<211> 41
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 19
<211> 126
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 20
<211> 42
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 21
<211> 336
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
<210> 22
<211> 112
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 23
<211> 45
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
ggcggtggcg gaagcggcgg agggggttca ggtggaggag gctct 45
<210> 24
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 25
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacct 57
<210> 26
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn
1 5 10 15
Pro Gly Pro
<210> 27
<211> 165
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
ttcgtgcccg tgttcctgcc cgccaaacct accaccaccc ctgcccctag acctcccacc 60
ccagccccaa caatcgccag ccagcctctg tctctgcggc ccgaagcctg tagacctgct 120
gccggcggag ccgtgcacac cagaggcctg gacttcgcct gcgac 165
<210> 28
<211> 55
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro
1 5 10 15
Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu
20 25 30
Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg
35 40 45
Gly Leu Asp Phe Ala Cys Asp
50 55
<210> 29
<211> 84
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
atctacatct gggcccctct ggccggcacc tgtggcgtgc tgctgctgag cctggtgatc 60
accctgtact gcaaccaccg gaac 84
<210> 30
<211> 28
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
1 5 10 15
Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn
20 25
<210> 31
<211> 123
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
agaagcaagc ggagccggct gctgcacagc gactacatga acatgacccc aagacggcct 60
ggccccaccc ggaagcacta ccagccttac gcccctccca gagacttcgc cgcctaccgg 120
tcc 123
<210> 32
<211> 41
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 33
<211> 336
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
agagtgaagt tcagcagatc cgccgacgcc cctgcctacc agcagggaca gaaccagctg 60
tacaacgagc tgaacctggg cagacgggaa gagtacgacg tgctggacaa gcggagaggc 120
cgggaccccg agatgggcgg aaagcccaga cggaagaacc cccaggaagg cctgtataac 180
gaactgcaga aagacaagat ggccgaggcc tacagcgaga tcggcatgaa gggcgagcgg 240
aggcgcggca agggccacga tggcctgtac cagggcctga gcaccgccac caaggacacc 300
tacgacgccc tgcacatgca ggccctgccc cccaga 336
<210> 34
<211> 112
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 35
<211> 240
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 35
Arg Ala His Leu Val Gln Ser Gly Thr Ala Met Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Arg Val Ser Cys Gln Thr Ser Gly Tyr Thr Phe Thr Ala His
20 25 30
Ile Leu Phe Trp Phe Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val
35 40 45
Gly Trp Ile Lys Pro Gln Tyr Gly Ala Val Asn Phe Gly Gly Gly Phe
50 55 60
Arg Asp Arg Val Thr Leu Thr Arg Asp Val Tyr Arg Glu Ile Ala Tyr
65 70 75 80
Met Asp Ile Arg Gly Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Arg Ser Tyr Gly Asp Ser Ser Trp Ala Leu Asp Ala Trp
100 105 110
Gly Gln Gly Thr Thr Val Val Val Ser Ala Gly Gly Gly Gly Ser Gly
115 120 125
Gly Gly Gly Ser Gly Gly Gly Gly Ser Tyr Ile His Val Thr Gln Ser
130 135 140
Pro Ser Ser Leu Ser Val Ser Ile Gly Asp Arg Val Thr Ile Asn Cys
145 150 155 160
Gln Thr Ser Gln Gly Val Gly Ser Asp Leu His Trp Tyr Gln His Lys
165 170 175
Pro Gly Arg Ala Pro Lys Leu Leu Ile His His Thr Ser Ser Val Glu
180 185 190
Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Phe His Thr Ser Phe
195 200 205
Asn Leu Thr Ile Ser Asp Leu Gln Ala Asp Asp Ile Ala Thr Tyr Tyr
210 215 220
Cys Gln Val Leu Gln Phe Phe Gly Arg Gly Ser Arg Leu His Ile Lys
225 230 235 240
<210> 36
<211> 243
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 36
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly
100 105 110
Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys
115 120 125
Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser
130 135 140
Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser
145 150 155 160
Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile
165 170 175
Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu
180 185 190
Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn
195 200 205
Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr
210 215 220
Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser
225 230 235 240
Val Thr Val
<210> 37
<211> 2388
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccccgc cttcctgctg 60
atcccccgag cgcacctggt acaatcaggg actgcgatga agaaaccggg ggcctcagta 120
agagtctcct gccagacctc tggatacacc tttaccgccc acatattatt ttggttccga 180
caggcccccg ggcgaggact tgagtgggtg gggtggatca agccacaata tggggccgtg 240
aattttggtg gtggttttcg ggacagggtc acattgactc gagacgtata tagagagatt 300
gcgtacatgg acatcagagg ccttaaacct gacgacacgg ccgtctatta ctgtgcgaga 360
gaccgttcct atggcgactc ctcttgggcc ttagatgcct ggggacaggg aacgacggtc 420
gtcgtctccg cgggcggagg gggttcaggt ggaggaggct ctggcggtgg cggaagctac 480
atccacgtga cccagtctcc gtcctccctg tctgtgtcta ttggagacag agtcaccatc 540
aattgccaga cgagtcaggg tgttggcagt gacctacatt ggtatcaaca caaaccgggg 600
agagccccta aactcttgat ccaccatacc tcttctgtgg aagacggtgt cccctcaaga 660
ttcagcggct ctggatttca cacatctttt aatctgacca tcagcgacct acaggctgac 720
gacattgcca catattactg tcaagtttta caatttttcg gccgagggag tcgactccat 780
attaaaaact ggagccaccc ccagttcgag aagggcggtg gcggaagcgg cggagggggt 840
tcaggtggag gaggctctga catccagatg acccagacca cctccagcct gagcgccagc 900
ctgggcgacc gggtgaccat cagctgccgg gccagccagg acatcagcaa gtacctgaac 960
tggtatcagc agaagcccga cggcaccgtc aagctgctga tctaccacac cagccggctg 1020
cacagcggcg tgcccagccg gtttagcggc agcggctccg gcaccgacta cagcctgacc 1080
atctccaacc tggaacagga agatatcgcc acctactttt gccagcaggg caacacactg 1140
ccctacacct ttggcggcgg aacaaagctg gaaatcaccg gcagcacctc cggcagcggc 1200
aagcctggca gcggcgaggg cagcaccaag ggcgaggtga agctgcagga aagcggccct 1260
ggcctggtgg cccccagcca gagcctgagc gtgacctgca ccgtgagcgg cgtgagcctg 1320
cccgactacg gcgtgagctg gatccggcag ccccccagga agggcctgga atggctgggc 1380
gtgatctggg gcagcgagac cacctactac aacagcgccc tgaagagccg gctgaccatc 1440
atcaaggaca acagcaagag ccaggtgttc ctgaagatga acagcctgca gaccgacgac 1500
accgccatct actactgcgc caagcactac tactacggcg gcagctacgc catggactac 1560
tggggccagg gcaccagcgt gaccgtgacc acgacgccag cgccgcgacc accaacaccg 1620
gcgcccacca tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg 1680
gggggcgcag tgcacacgag ggggctggac ttcgcctgtg atttttgggt gctggtggtg 1740
gttggtggag tcctggcttg ctatagcttg ctagtaacag tggcctttat tattttctgg 1800
gtgaggagta agaggagcag gctcctgcac agtgactaca tgaacatgac tccccgccgc 1860
cccgggccca cccgcaagca ttaccagccc tatgccccac cacgcgactt cgcagcctat 1920
cgctccaaac ggggcagaaa gaaactcctg tatatattca aacaaccatt tatgagacca 1980
gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga 2040
ggatgtgaac tgagagtgaa gttcagcagg agcgcagacg cccccgcgta ccagcagggc 2100
cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 2160
aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 2220
ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 2280
aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 2340
accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgc 2388
<210> 38
<211> 3216
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccccgc cttcctgctg 60
atcccccgag cgcacctggt acaatcaggg actgcgatga agaaaccggg ggcctcagta 120
agagtctcct gccagacctc tggatacacc tttaccgccc acatattatt ttggttccga 180
caggcccccg ggcgaggact tgagtgggtg gggtggatca agccacaata tggggccgtg 240
aattttggtg gtggttttcg ggacagggtc acattgactc gagacgtata tagagagatt 300
gcgtacatgg acatcagagg ccttaaacct gacgacacgg ccgtctatta ctgtgcgaga 360
gaccgttcct atggcgactc ctcttgggcc ttagatgcct ggggacaggg aacgacggtc 420
gtcgtctccg cgggcggagg gggttcaggt ggaggaggct ctggcggtgg cggaagctac 480
atccacgtga cccagtctcc gtcctccctg tctgtgtcta ttggagacag agtcaccatc 540
aattgccaga cgagtcaggg tgttggcagt gacctacatt ggtatcaaca caaaccgggg 600
agagccccta aactcttgat ccaccatacc tcttctgtgg aagacggtgt cccctcaaga 660
ttcagcggct ctggatttca cacatctttt aatctgacca tcagcgacct acaggctgac 720
gacattgcca catattactg tcaagtttta caatttttcg gccgagggag tcgactccat 780
attaaaaact ggagccaccc ccagttcgag aagggcggtg gcggaagcgg cggagggggt 840
tcaggtggag gaggctctac cacgacgcca gcgccgcgac caccaacacc ggcgcccacc 900
atcgcgtcgc agcccctgtc cctgcgccca gaggcgtgcc ggccagcggc ggggggcgca 960
gtgcacacga gggggctgga cttcgcctgt gatttttggg tgctggtggt ggttggtgga 1020
gtcctggctt gctatagctt gctagtaaca gtggccttta ttattttctg ggtgaggagt 1080
aagaggagca ggctcctgca cagtgactac atgaacatga ctccccgccg ccccgggccc 1140
acccgcaagc attaccagcc ctatgcccca ccacgcgact tcgcagccta tcgctccaaa 1200
cggggcagaa agaaactcct gtatatattc aaacaaccat ttatgagacc agtacaaact 1260
actcaagagg aagatggctg tagctgccga tttccagaag aagaagaagg aggatgtgaa 1320
ctgagagtga agttcagcag gagcgcagac gcccccgcgt accagcaggg ccagaaccag 1380
ctctataacg agctcaatct aggacgaaga gaggagtacg atgttttgga caagagacgt 1440
ggccgggacc ctgagatggg gggaaagccg agaaggaaga accctcagga aggcctgtac 1500
aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 1560
cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 1620
acctacgacg cccttcacat gcaggccctg ccccctcgcg ctactaactt cagcctgctg 1680
aagcaggctg gagacgtgga ggagaaccct ggacctatgg ccttaccagt gaccgccttg 1740
ctcctgccgc tggccttgct gctccacgcc gccatccccg acatccagat gacccagacc 1800
acctccagcc tgagcgccag cctgggcgac cgggtgacca tcagctgccg ggccagccag 1860
gacatcagca agtacctgaa ctggtatcag cagaagcccg acggcaccgt caagctgctg 1920
atctaccaca ccagccggct gcacagcggc gtgcccagcc ggtttagcgg cagcggctcc 1980
ggcaccgact acagcctgac catctccaac ctggaacagg aagatatcgc cacctacttt 2040
tgccagcagg gcaacacact gccctacacc tttggcggcg gaacaaagct ggaaatcacc 2100
ggcagcacct ccggcagcgg caagcctggc agcggcgagg gcagcaccaa gggcgaggtg 2160
aagctgcagg aaagcggccc tggcctggtg gcccccagcc agagcctgag cgtgacctgc 2220
accgtgagcg gcgtgagcct gcccgactac ggcgtgagct ggatccggca gccccccagg 2280
aagggcctgg aatggctggg cgtgatctgg ggcagcgaga ccacctacta caacagcgcc 2340
ctgaagagcc ggctgaccat catcaaggac aacagcaaga gccaggtgtt cctgaagatg 2400
aacagcctgc agaccgacga caccgccatc tactactgcg ccaagcacta ctactacggc 2460
ggcagctacg ccatggacta ctggggccag ggcaccagcg tgaccgtgtt cgtgcccgtg 2520
ttcctgcccg ccaaacctac caccacccct gcccctagac ctcccacccc agccccaaca 2580
atcgccagcc agcctctgtc tctgcggccc gaagcctgta gacctgctgc cggcggagcc 2640
gtgcacacca gaggcctgga cttcgcctgc gacatctaca tctgggcccc tctggccggc 2700
acctgtggcg tgctgctgct gagcctggtg atcaccctgt actgcaacca ccggaacaga 2760
agcaagcgga gccggctgct gcacagcgac tacatgaaca tgaccccaag acggcctggc 2820
cccacccgga agcactacca gccttacgcc cctcccagag acttcgccgc ctaccggtcc 2880
agagtgaagt tcagcagatc cgccgacgcc cctgcctacc agcagggaca gaaccagctg 2940
tacaacgagc tgaacctggg cagacgggaa gagtacgacg tgctggacaa gcggagaggc 3000
cgggaccccg agatgggcgg aaagcccaga cggaagaacc cccaggaagg cctgtataac 3060
gaactgcaga aagacaagat ggccgaggcc tacagcgaga tcggcatgaa gggcgagcgg 3120
aggcgcggca agggccacga tggcctgtac cagggcctga gcaccgccac caaggacacc 3180
tacgacgccc tgcacatgca ggccctgccc cccaga 3216
Claims (9)
1.一种用于治疗HIV感染合并血液肿瘤的双特异性嵌合抗原受体,其特征在于:所述双特异性嵌合抗原受体中包含抗HIV gp120单链抗体和抗CD19单链抗体,进一步地,所述双特异性嵌合抗原受体还包括信号肽、CD8铰链区、CD28跨膜区、CD28-ICD、4-1BB共刺激结构域和CD3ζ胞内信号传导结构域。
2.根据权利要求1所述的双特异性嵌合抗原受体,其特征在于:所述双特异性嵌合抗原受体从N端到C端顺次拼接信号肽SP1、抗HIV gp120单链抗体、Strep II、连接肽、抗CD19单链抗体、CD8铰链区、CD28跨膜区、CD28-ICD、4-1BB共刺激结构域以及CD3ζ胞内信号传导结构域。
3.根据权利要求1所述的双特异性嵌合抗原受体,其特征在于:所述双特异性嵌合抗原受体由第一CAR:信号肽SP1、抗HIV gp120抗原特异性单链抗体、Strep II、连接肽、CD8铰链区、CD28跨膜区、CD28-ICD、4-1BB共刺激结构域以及CD3ζ胞内信号传导结构域和第二CAR:信号肽SP2、抗CD19单链抗体、CD8铰链区、CD8跨膜区、CD28-ICD以及CD3ζ胞内信号传导结构域,第一CAR和第二CAR由自切割短肽依次并联组成。
4.根据权利要求2和3所述的双特异性嵌合抗原受体,其特征在于:所述信号肽优选为CD8信号肽和CSF2RA信号肽;所述连接肽为3×G4S;所述自切割短肽优选为P2A类短肽。
5.权利要求1-4任一项所述双特异性嵌合抗原受体的编码核苷酸,其特征在于:权利要求2所述的双特异性嵌合抗原受体的编码核苷酸序列如SEQ ID NO:37所示;权利要求3所述的双特异性嵌合抗原受体的编码核苷酸序列如SEQ ID NO:38所示。
6.一种重组慢病毒载体,其特征在于:以PTK881-EF1α载体为骨架,含有权利要求5所述的编码核苷酸。
7.一种免疫细胞,其特征在于:所述免疫细胞转染有权利要求6所述的重组载体,优选地,所述免疫细胞为T细胞,更优选地,所述T细胞为健康供者γδT细胞和脐血来源的CD8+T细胞。
8.权利要求5所述编码核苷酸的构建方法,其特征在于:包括以下步骤:
1)基因合成如SEQ ID NO:1所示的信号肽-抗HIV gp120单链抗体编码核苷酸SP1-N6,以及如SEQ ID NO:2所示的信号肽-抗CD19单链抗体编码核苷酸SP2-FMC83-28Z,将合成的上述编码核苷酸分别克隆至pUC57载体;
2)以人cDNA文库为模板,设计引物分别扩增片段CD8铰链区、CD28跨膜区、CD28胞内结构域(ICD)、4-1BB共刺激结构域、CD8跨膜区、CD3ζ胞内信号传导结构域、以引物互补方式得到Strep II、连接肽3×G4S、P2A;
3)采用Overlap PCR技术将SP1-N6、Strep II、连接肽3×G4S、SP2-FMC63-28Z与CD8铰链区、CD28跨膜区、CD28-ICD、4-1BB共刺激结构域、CD3ζ胞内信号传导结构域顺次扩增连接,获得嵌合抗原受体的编码基因C5-CAR,其结构示意图如图1所示;采用Overlap PCR技术分别将SP1-N6与Strep II、连接肽3×G4S、CD8铰链区、CD28跨膜区、CD28-ICD、4-1BB共刺激结构域、CD3ζ胞内信号传导结构域顺次扩增成片段SP1-CD3ζ,P2A与SP2-FMC63-28Z、CD8铰链区、CD8跨膜区、CD28-ICD、CD3ζ胞内信号传导结构域顺次扩增成片段P2A-SP2-CD3ζ,片段SP1-CD3ζ与P2A-SP2-CD3ζ最后连接成编码基因C6-CAR,其结构示意图如图2所示;
优选地,所述单链抗体N6的氨基酸序列如SEQ ID NO:35所示、所述单链抗体FMC63-28Z的氨基酸序列如SEQ ID NO:36所示,信号肽SP1、信号肽SP2、Strep II、连接肽3×G4S的核苷酸序列分别如SEQ ID NO:7、9、11、23所示;C5-CAR中CD8铰链区、CD28跨膜结构域、CD28-ICD、4-1BB共刺激结构域、CD3ζ胞内信号传导结构域的核苷酸序列分别如SEQ ID NO:13、15、17、19、21所示;C6-CAR中P2A的核苷酸序列如SEQ ID No:25所示,单链抗体FMC63-28Z连接的CD8铰链区、CD8跨膜结构域、CD28-ICD、CD3ζ胞内信号传导结构域的核苷酸序列如SEQID No:27、29、31、33所示。
9.权利要求1-4任一项所述的双特异性嵌合抗原受体、权利要求5所述的编码核苷酸、权利要求6所述的重组慢病毒载体、权利要求7所述的免疫细胞在用于制备治疗血液肿瘤合并HIV感染的药物或制剂中的应用,优选地,所述血液肿瘤为CD19阳性B淋巴细胞系恶性肿瘤。
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WO2021155670A1 (zh) * | 2020-02-05 | 2021-08-12 | 武汉科技大学 | 治疗血液肿瘤合并hiv感染的双特异性嵌合抗原受体 |
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