CN102936281A - 一种rTRAIL突变体及其海兔毒素偶联物 - Google Patents
一种rTRAIL突变体及其海兔毒素偶联物 Download PDFInfo
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Abstract
一种rTRAIL突变体及其海兔毒素偶联物。本发明公开了一种rTRAIL突变体，其氨基酸序列如SEQ ID No.1所示。本发明还公开了所述rTRAIL突变体的编码基因及含有该编码基因的表达单元、重组载体或表达***。本发明还公开了一种rTRAIL突变体-vcMMAE偶联物及其制备方法和应用。本发明偶联物具有rTRAIL突变体和MMAE两者的生物学功能，通过rTRAIL突变体与肿瘤细胞表面的死亡受体特异结合，将MMAE定向转运到肿瘤细胞，并在肿瘤细胞内释放发挥作用，既可杀伤对TRAIL敏感性低甚至抵抗的肿瘤细胞，也减少MMAE单独给药产生的毒副作用。
Description
技术领域
本发明属于生物技术和药物领域，尤其涉及一种rTRAIL突变体及其海兔毒素偶联物。
背景技术
肿瘤坏死因子（TNF）相关的凋亡诱导配体（TNF relatedapoptosis-inducing ligand，TRAIL，或Apo2L，TNFSF10）是继TNF、FasL之后发现的第三个TNF家族的凋亡因子，为一种抗癌应用前景良好的生物药物。TRAIL由Wiley等从心肌cDNA文库中克隆出来的，因其氨基酸序列具有TNF超家族的结构特征并能诱导Jurkat细胞和EB病毒转化的人类淋巴细胞凋亡而得名。TRAIL属Ⅱ型跨膜蛋白，由281个氨基酸组成，其N端（1~17位氨基酸）位于胞内，C端（第39~281位氨基酸）延伸到胞外，其中第114~281位是发挥主要功能的部位。
许多临床前期研究表明TRAIL能有效诱导多种癌细胞系凋亡，但对正常细胞无毒副作用。目前国外已进行关于TRAIL及其受体激动型抗体的Ⅰ、Ⅱ期临床试验，并取得初步疗效。另外，TRAIL对NF-κB仅有很微弱的激活作用，即使是全身用药，也不会像TNF-α和Fas-L那样产生严重的炎症反应，这些特性使得TRAIL有成为新一代抗肿瘤药物的潜质。
TRAIL在免疫***细胞中表达，包括NK细胞、T细胞、巨噬细胞和树突细胞，并且位于细胞膜中，可经半胱氨酸蛋白酶加工成可溶形式。TRAIL是通过与与其细胞膜受体结合发挥诱导凋亡作用的。目前已发现5种TRAIL受体，包括：TRAIL-R1（DR4，TNFSF10a）和TRAIL-R2（DR5，TNFRSF10b）；TRAIL-R3（DcR1，TNFRSF10c）和TRAIL-R4（DcR2，TNFRSF10d）；循环骨保护素（OPG，TNFRSF11b）。DR4和DR5具有一段死亡结构域（DD），是TRAIL结合受体后诱导凋亡所必需的。其余三个受体由于缺乏功能性死亡结构域，虽然能与TRAIL结合，但无法诱导凋亡。
TRAIL与DR4或DR5结合后可激活线粒体依赖和非线粒体依赖两条细胞凋亡信号途径，介导死亡信号传导入细胞内，启动效应物半胱天冬酶-3（caspase-3），最终引起肿瘤细胞凋亡。
近年来，人们发现重组可溶TRAIL诱导了广谱的人肿瘤细胞系凋亡，但同时也存在一些对TRAIL敏感性较低或耐受的肿瘤细胞，如所有的人黑色素瘤细胞系、大部分乳腺癌细胞系、***癌细胞系LNcaP等，这些细胞表面虽然或多或少也表达DR4或DR5，却由于细胞内凋亡途径中关键酶的缺失、突变和其他抑制凋亡的蛋白高表达等原因，导致TRAIL结合DR4或DR5之后并不能引起细胞的最终凋亡。
对于这类细胞所形成的实体瘤，亟须其他治疗手段或改造TRAIL使之具备杀伤敏感性低以及耐受肿瘤细胞的能力。研究发现，可溶型重组TRAIL（rTRAIL）与放化疗联合应用能提高肿瘤细胞对rTRAIL的敏感性，二者具有协同作用。目前国际通用的rTRAIL是95~281位氨基酸片段，rTRAIL单体倾向于形成生物活性较好的三聚体而存在，在三聚体的顶部有一个锌离子结合位点，对稳定三聚体构象起重要作用。
但联合治疗方案仅是将rTRAIL和这些药物同时给药，并未在二者之间建立联系，无法将这些药物单一地导向肿瘤细胞，因此一般只选择低剂量低毒性的药物，以免对正常细胞产生毒害。相应地，也就无法得到更好的治疗效果。因此，若能将一些能够强力杀伤肿瘤细胞的药物加以有效利用，将更有利于肿瘤的治疗。例如化疗药物海兔毒素（Monomethyl auristatinE，MMAE）是一种人工合成抗肿瘤小分子，它通过抑制细胞内微管蛋白二聚化而诱导细胞凋亡。但由于它具有很强的无选择毒性，会对正常细胞造成伤害，所以其本身并不能成药。另外，针对TRAIL的突变体、TRAIL受体的抗体的研究都在进行中，但治疗效果仍不理想。
发明内容
本发明提供了一种rTRAIL突变体，将特定氨基酸突变为半胱氨酸，从而实现与海兔毒素的偶联，且不会大幅降低蛋白对肿瘤细胞的杀伤力。
一种rTRAIL突变体，氨基酸序列如SEQ ID NO.1所示。
该突变体取自全长TRAIL蛋白的第95~281个氨基酸，且第109位的天冬酰胺（Asn）突变为半胱氨酸（Cys）。
本发明还提供了一种编码所述rTRAIL突变体的基因，碱基序列如SEQ ID NO.2所示。该基因第109位编码天冬酰胺（Asn）的密码子AAT突变为编码半胱氨酸（Cys）的密码子TGT。
本发明还提供了一种含有所述基因的表达单元、重组载体或表达***。
所述表达单元的启动子为T7，在这些启动子的作用下，rTRAIL突变体可以直接在大肠杆菌宿主细胞中实现胞内可溶表达。
所述重组载体的原始载体为：pET-28a（+）。
所述表达***可选细菌、酵母、昆虫细胞或哺乳动物细胞表达***，优选为细菌表达***，最优选为大肠杆菌表达***。
本发明还提供了一种rTRAIL突变体-海兔毒素偶联物，由海兔毒素通过连接臂偶联rTRAIL突变体三聚体构成。所述海兔毒素的合成方法参考美国专利文献：Tumer inhibiting tetrapeptide bearing modified phenethylamides，（专利号：5,635,483）。
本发明所使用的连接臂为马来酰亚胺修饰的缬氨酸-瓜氨酸二肽，其合成方法参考文献：Gene M.D.,et al,Cathepsin B-Labile Dipeptide Linkersfor Lysosomal Release of Doxorubicin from Internalizing Immunoconjugates:Model Studies of Enzymatic Drug Release and Antigen-Specific In VitroAnticancer Activity,Bioconjugate Chem.,13(4)855-869(2002).。
本发明带连接臂的海兔毒素（vcMMAE）由江阴康诺泰生物技术有限公司代为合成，也可以参考文献（Svetlana O.D.,et al,Development of potentmonoclonal antibody auristatin conjugates for cancer therapy,NatureBiotechnology.,21(7)778-784(2003).），偶联时再通过缬氨酸上的马来酰亚胺与rTRAIL突变体的半胱氨酸巯基进行烷基化反应，最终形成本发明偶联物。
本发明还提供了所述rTRAIL突变体-海兔毒素偶联物的制备方法，包括：
（1）将rTRAIL突变体多聚体解聚，重新聚合得到rTRAIL突变体三聚体，具体包括：将rTRAIL突变体溶于含锌离子的缓冲液中，加入磷酸三（β-氯乙基)酯，30~40℃水浴反应1~3h；
由于rTRAIL突变体之间会通过二硫键形成多聚体，加入磷酸三（β-氯乙基）酯（TCEP）使不稳定的多聚体解聚，保持单体状态，反应体系中的锌离子进一步促使rTRAIL突变体单体形成稳定的三聚体。
（2）将rTRAIL突变体三聚体与带连接臂的海兔毒素混合，进行偶联反应，偶联后，每个三聚体可以结合1-3个海兔毒素分子；偶联反应的温度优选为0~4℃，时间为30~60min。
（3）反应完成后，分离纯化得到rTRAIL突变体-海兔毒素偶联物；
由于本发明rTRAIL突变体-海兔毒素偶联物单体的分子量为23kDa，而反应体系中存在的其他分子的分子量均小于10kDa，因此，用截留分子量10kDa的超滤管即可除去体系中的小分子，再经离心去沉淀，将所得上清过滤除菌，即得本发明的偶联物。
用于突变的TRAIL分子可来源于人或动物，但为了对人的肿瘤治疗更有帮助，优选为人的天然TRAIL分子。天然rTRAIL单体分子表面也有半胱氨酸巯基，但均用于形成稳定的三聚体，三聚体分子表面因没有游离巯基存在而失去了偶联位点。因此，本发明利用PCR定点突变手段获得了含有一个半胱氨酸突变位点的rTRAIL突变体，步骤为：
（1）提取人组织总RNA，以总RNA为模板进行反转录，获得cDNA文库；
（2）以所述cDNA为模板，利用引物P1和P2进行PCR扩增，得到TRAIL编码序列；
（3）以所述TRAIL编码序列为模板，利用引物P3和P4进行PCR定点突变扩增，获得突变序列；
（4）将突变序列可操作性地连入载体，转化宿主细胞；
（5）诱导经转化的宿主细胞表达融合蛋白，获得所述rTRAIL突变体。
所述引物P1和P2的序列为：
P1：5’-ATGGCTATGATGGAGGTCCAGG-3’；
P2：5’-TTAGCCAACTAAAAAGGCCCCG-3’；
所述引物P3和P4的序列为：
P3：5’-TATACCATGGGCACCTCTGAGGAAACCATT
TCTACAGTTCAAGAAAAGCAACAATGTATTTCT-3’；
P4：5’-TTCTCGAGTTAGCCAACTAAAAAGGCCCC
GAAAAAACTGGCTTCATGGTCCATGTCCATGTC-3’。
本发明还提供了所述rTRAIL突变体-海兔毒素偶联物在制备抗肿瘤药物中的应用。
所述抗肿瘤药物包括有效量的rTRAIL突变体-海兔毒素偶联物，以及至少一种药学上可接受的载体、稀释剂或赋形剂。制备时，通常将活性成分与赋形剂混合，或用赋形剂稀释，或包在可以胶囊或药囊形式存在的载体中。当赋形剂起稀释剂作用时，它可采用固体、半固体或液体材料作为赋形剂、载体或活性成分的介质。因此，组合物可以是片剂、丸剂、粉剂、溶液剂、糖浆剂、灭菌注射溶液等。
合适的赋形剂包括：乳糖、葡萄糖、蔗糖、山梨醇、甘露醇、淀粉、微晶纤维素、聚乙烯吡咯烷酮、纤维素、水等；制剂还可包括：湿润剂、乳化剂、防腐剂（如羟基苯甲酸甲酯和丙酯）、甜味剂等。所述抗肿瘤药物可制成单元或多元剂型，各剂型包含为了产生所期望的疗效而计算出预定量的rTRAIL突变体-海兔毒素偶联物，以及合适的药剂学赋形剂。
所述的抗肿瘤药物可以通过常规途径进行给药，包括（但并不限于）：肌内、腹膜内、静脉内、皮下、皮内、局部给药等。
使用该药物时，是将安全有效量的rTRAIL突变体-海兔毒素偶联物施用于人，其中该安全有效量的范围优选为0.5~50毫克/千克体重，更优选为1~10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是在熟练医师技能范围之内的。
此外，本发明的偶联物还可与其他治疗药物联用，其中包括（但并不限于）：各种细胞因子，如IFN、TNF、IL-2等；各种肿瘤化疗药物，如5-FU、氨甲喋呤等影响核酸生物合成的药物；氮芥、环磷酰胺等烷化剂类药物；阿霉素、放线菌素D等干扰转录过程阻止RNA合成的药物；长春新碱、喜树碱类等影响蛋白质合成的药物及某些激素类药物，等等。
与现有技术相比，本发明的有益效果为：
（1）本发明偶联物具有rTRAIL突变体和MMAE两者的生物学功能，既具有rTRAIL突变体在肿瘤细胞外诱导凋亡的能力，又具有MMAE在细胞内抑制微管蛋白从而诱导凋亡的能力，在二者的协同下，抗肿瘤效果得到显著增强。
（2）本发明偶联物通过rTRAIL突变体与肿瘤细胞表面的死亡受体特异结合，将MMAE定向转运到肿瘤细胞，并在肿瘤细胞内释放发挥作用，既可杀伤对TRAIL敏感性低甚至抵抗的肿瘤细胞，也减少MMAE单独给药产生的毒副作用。
（3）本发明偶联物中的rTRAIL突变体与MMAE偶联后，其水溶液具有比单纯的rTRAIL分子更佳的稳定性，表现为经反复冻融后不出现沉淀。
附图说明
图1为大肠杆菌表达的rTRAIL突变体N109C电泳图。其中，M代表低分子量Marker；1为未诱导的菌液；2、3为诱导的菌液；4为未诱导的菌液破碎后离心取的上清（可溶部分）；5、6为诱导的菌液破碎后离心取的上清（可溶部分）；7、8、9为诱导和不诱导的沉淀用8M尿素重溶后的样品。
图2为rTRAIL突变体N109C的亲和纯化结果电泳图。其中，M为蛋白分子量marker。1为IPTG诱导后的菌液，2为破菌后的上清；3为Ni柱的流穿液；4为10mM咪唑洗脱液；5为60mM咪唑洗脱液；6为500mM咪唑洗脱液。
图3为rTRAIL突变体N109C的SP强阳离子交换柱纯化结果电泳图。其中，1为10mM咪唑洗脱液脱盐后的蛋白溶液；2和3都是SP柱的洗脱蛋白，即纯化后的N109C，4为N109C非变性电泳的结果。
图4为N109C及其MMAE偶联物的电泳图。其中，1为N109C，2为N109C-vcMMAE偶联物，3为N109C-vcMMAE脱盐后的产物，4、5、6分别为1、2、3样品非变性条件下的电泳结果。
图5为rTRAIL突变体-vcMMAE偶联物各部分连接关系示意图。
图6为各TRAIL元件对不同细胞系的杀伤效果。
图7为N109C-vcMMAE偶联物在荷瘤小鼠体内的分布。
图8为N109C-vcMMAE偶联物的肿瘤细胞内吞实验图。
图9为rTRAIL突变体-vcMMAE偶联物诱导凋亡机制示意图。
具体实施方式
下面结合附图和具体实施方式对本发明作进一步详细说明。
1、建立人***cDNA文库
（1）提取人***mRNA
液氮研磨冻存的人***组织小块（100mg左右）成粉末状，加TRIZOL试剂1mL继续反复研磨，转移至1.5mL无RNA酶EP管中，室温放置5min，加0.2mL氯仿剧烈振荡15s，室温放置3min。4℃12000g离心15min，转移上层水相至另一无RNA酶的EP管中。加入异丙醇0.5mL，室温放置10min，沉淀RNA。4℃12000g离心10min，去上清，75％乙醇洗涤沉淀。4℃12000g离心5min，去上清，干燥后用50μL DEPC水溶解，-70℃保存。
（2）反转录PCR
以oligodT为引物，人***RNA为模板，加AMV逆转录酶进行Reverse-PCR，得到cDNA文库。具体步骤为：
1）RNA预变性：5μg人***RNA+25μg OligodT，用0.1％DEPC补足至10μL；70℃水浴5min，冷却至室温，破坏mRNA二级结构。
2）反转录：合成体系为：
PCR程序为：逆转录过程42℃ 60min；
逆转录酶灭活 70℃ 10min。
获得的cDNA存于-20℃，备用。
2、获取TRAIL编码序列
以上述cDNA为模板，P1和P2为上下游引物，Taq DNA聚合酶催化合成和扩增TRAIL编码序列。经序列分析，所得DNA序列与GenBank登记的序列（NM_003842.4）所显示的TRAIL编码序列一致，即得到了TRAIL编码序列。引物的序列为：
P1：5’-ATGGCTATGATGGAGGTCCAGG-3’；
P2：5’-TTAGCCAACTAAAAAGGCCCCG-3’。
PCR反应体系为：
PCR反应程序：
3、获取TRAIL 95~281（rTRAIL）突变序列
（1）获取rTRAILN109C突变序列
以TRAIL编码序列为模板，P3和P4为上下游引物，Taq DNA聚合酶催化合成和扩增rTRAIL N109C突变体编码序列。经测序分析，所得DNA序列如SEQ ID No.2所示，与预想一致。引物的序列为：
P3：5’-TATACCATGGGCACCTCTGAGGAAACCATT
TCTACAGTTCAAGAAAAGCAACAATGTATTTCT-3’；
P4：5’-TTCTCGAGTTAGCCAACTAAAAAGGCCCC
GAAAAAACTGGCTTCATGGTCCATGTCCATGTC-3’。
除引物和模板外，PCR反应体系和反应程序同步骤2。
（2）获取rTRAIL S96C突变序列
以TRAIL编码序列为模板，P5和P6为上下游引物，Taq DNA聚合酶催化合成和扩增rTRAIL N109C突变体编码序列。经测序分析，所得DNA序列如SEQ ID No.4所示，与预想一致。引物的序列为：
P5：5’-TATACCATGGGCACCTGTGAGGAAACCATT
TCTACAGTTCAAGAAAAGCAACAAAATATTTCT-3’；
P6：5’-TTCTCGAGTTAGCCAACTAAAAAGGCCCC
GAAAAAACTGGCTTCATGGTCCATGTCCATGTC-3’。
除引物和模板外，PCR反应体系和反应程序同步骤2。
下面以rTRAIL N109C突变序列为例，对rTRAIL突变体的制备及偶联物的构建作进一步说明。rTRAIL S96C突变体的制备及偶联物的构建与之相同。
4、构建含rTRAIL突变序列的表达载体
rTRAIL N109C突变序列和pET28a（+）分别进行NcoI/XhoI双酶切，然后按照3：1的摩尔比连接，连接酶为T4（Takara）。连接产物转化大肠杆菌DH5a感受态细胞，挑取阳性克隆培养。抽提质粒后NcoI/XhoI双酶切验证连接情况，再经测序验证，得到rTRAIL突变体表达载体：pET28a（+）-rTRAILN109C。
5、表达载体转化大肠杆菌，建立工程菌
将pET28a（+）-rTRAIL N109C表达载体转化大肠杆菌表达宿主BL21（DE3）（购自Novagen），从卡那霉素抗性平板上挑取单克隆于37℃160rpm培养过夜。用菌液PCR的方法确认rTRAIL突变体表达载体已转化表达宿主：以菌液作为模板，P3、P4作为引物进行PCR。经验证为阳性的单克隆即为所需要的工程菌，菌液加10％~15％甘油后-80℃保存。
6、rTRAIL突变体的制备和纯化
将构建好的工程菌接种至200mL LB培养基（含15μg/mL kanamycin）中，37℃160rpm摇床培养至菌液OD600=0.8左右。加入终浓度为1mM的IPTG诱导BL21（DE3）表达融合蛋白，诱导时间为12h。7200g离心10min得到BL21菌体。
用上样缓冲液A重悬菌体，法式压力破碎。7200g离心30min后，取上清过0.22μm水膜。然后用GE公司Ni-NTA亲和柱进行金属亲和层析。10mM咪唑洗脱杂蛋白，60mM咪唑洗脱目的蛋白。60mM咪唑洗脱的组分用GE公司脱盐柱将其缓冲液替换为缓冲液B，然后进行进一步的离子交换层析，选用的柱子为GE公司的SP强阳离子交换柱。缓冲液C用于洗脱离子交换柱上的蛋白。
其中，各缓冲液的组分为：
缓冲液A：50mM NaH2PO4，300mM NaCl，pH 7.4
缓冲液B：20mM NaH2PO4，pH 6.0
缓冲液C：20mM NaH2PO4，1M NaCl，pH 6.0
图1、图2和图3所示的为rTRAIL N109C突变体的原核表达及纯化结果电泳图。由图1可见，rTRAIL N109C突变体可在大肠杆菌内成功地可溶性表达。由图2可见，在10mM咪唑浓度下rTRAIL N109C突变体已经开始洗脱，初步纯化后其纯度在85％以上。由图3可见，经SP柱纯化后，能去掉部分杂蛋白，但还有三条细小的杂带。同时，对基本纯化的rTRAIL N109C突变体进行非变性凝胶电泳发现，获得的rTRAIL N109C突变体中存在二聚体、四聚体等，这是突变蛋白分子间二硫键所导致的。
获得的N109C突变体的氨基酸序列如SEQ ID No.1所示，S96C突变体的氨基酸序列如SEQ ID No.3所示。
7、rTRAIL突变体与MMAE偶联
取0.5mg rTRAIL N109C突变体溶解于0.8mL PBS（pH 6.0，含10μMZnCl2）中，加入6μL磷酸三（β-氯乙基）酯（TCEP），37℃水浴2h。边搅拌边加入用50μL 30％乙腈/水溶解的10倍过量vcMMAE（由江阴康诺泰生物技术有限公司代为合成，10倍过量即反应体系中vcMMAE的摩尔量大于等于N109C的10倍），4℃反应40min，加入过量的半胱氨酸终止反应。用Millipore截留分子量为10kDa的超滤管除去反应体系中的小分子。所得到的偶联物过0.22μm孔径的水膜除菌，-20℃保存备用。
图4为rTRAIL N109C与MMAE偶联的电泳结果图。由图可见，偶联后的蛋白条带发生明显偏移，表明小分子MMAE已经偶联到蛋白上，使其电泳行为发生变化。其中，泳道1和4对比可见N109C由于突变了一个半胱氨酸残基，所以有多聚体存在；而泳道3与6的对比表明，偶联时，rTRAIL N109C的多聚体在TCEP的作用下解聚，锌离子促使N109C单体形成稳定的三聚体，其半胱氨酸巯基与vcMMAE的马来亚酰胺基团发生烷基化反应，变成了偶联不同个数MMAE的偶联物。rTRAIL突变体-海兔毒素偶联物各部分的连接关系如图5所示。
8、偶联物的体外抗肿瘤活性测定
选择四个类型的细胞：TRAIL敏感型（NCI-H460）、不敏感型(Hela)、耐受型（MCF-7）、和普通细胞（HEK293）对rTRAIL突变体与MMAE偶联物的体外抗肿瘤活性进行测定。这四种细胞表面皆有TRAIL死亡受体。
下面以TRAIL敏感型细胞NCI-H460为例对试验步骤作详细说明，包括：
（1）细胞NCI-H460消化成为单个细胞后，稀释为1×104个/mL，铺96孔板（每孔100μL），正常条件下继续培养24小时。
（2）将N109C、N109C-vcMMAE、S96C-vcMMAE分别用培养基稀释，加入细胞板作为实验组，使终浓度为：32、63、125、250、500、1000ng/mL；以标准品TRAIL（114~281）为阳性对照，以溶解实验品的缓冲液为阴性对照，并用培养基以同样方式稀释。空白对照照为不加任何溶液的培养基。实验中对照组和实验组皆做三个复孔。
（3）将对照组和实验组加入细胞板中，37℃继续培养96h，观察实验组和对照组对目的细胞的杀伤能力。
（4）培养结束后，每孔加入10μLCCK-8显色液，置于培养箱中孵育显色1h。取出以450nm、630nm双波长检测。
（5）结果计算：以实验组中OD值高于同等稀释度的阴性对照的样品为阳性（t检验P<0.01）。计算结果如图6所示。
由图6可见，对四种不同的细胞，rTRAIL突变体-vcMMAE偶联物都比rTRAIL突变体的肿瘤杀伤力强。
对于NCI-H460细胞，rTRAIL突变体及其MMAE偶联物都不能比TRAIL（114~281）的活性更好。
对Hela和MCF-7细胞，rTRAIL突变体-vcMMAE在较高浓度时表现出更强的杀伤效果，而在低浓度（＜250ng/mL）时稍微低于TRAIL（114~281）。其原因可能是低浓度时，被细胞内吞的rTRAIL突变体-vcMMAE偶联物并未积累足够量的MMAE来诱导细胞凋亡，所以在高浓度时杀伤效果更好。然而不管是低浓度还是高浓度下，N109C-vcMMAE比S96C-vcMMAE的杀伤力更强。
对于正常细胞HEK293，TRAIL（114~281）、rTRAIL突变体及其MMAE偶联物都毒性较小。
9、偶联物在荷瘤动物模型中的分布
以N109C-vcMMAE为例，采用荧光标记法实时跟踪偶联物在小鼠体内的走向，观察偶联物的肿瘤靶向能力。
（1）荷瘤动物模型的建立
选用体重约20g的无胸腺小鼠，在其前腿一侧腋下皮下注射107个NCI-H460细胞，三天后即可见到明显出瘤现象。
（2）偶联物的荧光标记
荧光标记物采用Cy5这一近红外激发的染料，方便荧光穿透动物皮肤，达到实时活体观测的目的。Cy5标记的具体步骤参考厂家（Lumiprobe）的说明书。
（3）给药并实时观测
将200μL含有约50μg Cy5标记的偶联物尾静脉注射入荷瘤小鼠体内。空白组注射等量的生理盐水。
利用小动物活体成像仪（型号：NightOWLN320）每隔24小时观察小鼠体内荧光分布。观察前，小鼠禁食12小时，用***麻醉。连续观察4天，直至小鼠全身荧光全部消失。第4天后处死小鼠，分离心脏、肝脏、脾脏、肺、肾脏、肿瘤组织，利用小动物活体荧光成像仪对偶联物在实验组小鼠体内器官的具体分布进行验证。成像结果如图7所示。图中白色箭头所指的为肿瘤部位。
由图7可见，第三天大部分的荧光已经聚集到肿瘤部位，而第四天更是只剩肿瘤部位有荧光。处死小鼠后对各组织的荧光检测结果也与活体实时检测相一致。表明N109C-vcMMAE具有良好的肿瘤靶向效应。
10、偶联物抗肿瘤活性的分子机理研究
下面以NCI-H460细胞为例，以激光共聚焦手段观察rTRAIL突变体-vcMMAE偶联物在肿瘤细胞内的行为。操作步骤为：
①用1M HCl和70％乙醇浸泡玻片，超声30分钟，双蒸水洗涤5~6次。然后灭菌待用；
②将灭好菌的玻片置入24孔板中，细胞以104个/孔的密度铺板，37℃过夜培养；
③给药：N109C-vcMMAE、S96C-vcMMAE分别稀释至终浓度1μg/mL，不同时间点（1h，4h，8h，12h）按步骤4处理细胞了，空白对照为不给药的培养基；
④玻片用冷的PBS洗涤三次，然后用4％多聚甲醛常温固定10min；
⑤再用PBS洗涤三次；
⑥加0.1％Triton X-100室温透化10min；
⑦PBS洗涤三次后，加2％BSA封闭30min；
⑧一抗孵育：将兔抗人rTRAIL多抗用1％BSA/PBS稀释至1μg/mL，室温孵育45min。
⑨二抗孵育：将FITC标记的羊抗兔二抗用1％BSA/PBS稀释500倍，室温孵育45min。
⑩DAPI细胞核染色：PBS洗涤三次后，每个玻片上滴加用PBS稀释1000倍的DAPI，以刚好覆盖细胞为宜，25℃孵育2min左右；封片：PBS洗涤后，玻片上加一滴抗荧光淬灭剂，倒扣在载玻片上，然后用指甲油封住玻片四周；
共聚焦检测。检测结果见图8。
图8中显蓝色荧光的为细胞核，显绿色荧光的是rTRAIL突变体-vcMMAE偶联物。在给药一个小时后，大量N109C-vcMMAE已经进入细胞内；4小时后，越来越多的N109C-vcMMAE进入到细胞内，并开始慢慢聚集；8小时后，细胞内已经有更多的聚集点；12小时后，细胞内的聚集点逐渐减少，细胞开始出现凋亡现象，表现为细胞核出现异质化。
推断rTRAIL突变体-vcMMAE偶联物的作用机理为：偶联物通过rTRAIL突变体与肿瘤细胞表面的死亡受体结合，并被肿瘤细胞内吞，形成吞噬体；吞噬体进一步与溶酶体融合，溶酶体中的组织蛋白酶Cathepsin水解偶联物中的二肽连接臂，释放出MMAE；MMAE在肿瘤细胞中发挥作用，抑制微管蛋白二聚化，从而诱导细胞凋亡。其诱导凋亡机制如图9所示。
Claims (10)
1.一种rTRAIL突变体，其特征在于，氨基酸序列如SEQ ID NO.1所示。
2.一种编码如权利要求1所述rTRAIL突变体的基因，其特征在于，碱基序列如SEQ ID NO.2所示。
3.一种含有如权利要求2所述基因的表达单元、重组载体或表达***。
4.一种rTRAIL突变体-海兔毒素偶联物，其特征在于，由海兔毒素通过连接臂偶联rTRAIL突变体三聚体构成，rTRAIL突变体的氨基酸序列如SEQ ID NO.1所示。
5.如权利要求4所述的rTRAIL突变体-海兔毒素偶联物，其特征在于，所述连接臂为马来酰亚胺修饰的缬氨酸-瓜氨酸二肽。
6.如权利要求4～5任一所述rTRAIL突变体-海兔毒素偶联物在制备抗肿瘤药物中的应用。
7.一种rTRAIL突变体-海兔毒素偶联物的制备方法，包括：
(1)将rTRAIL突变体多聚体解聚，重新聚合得到rTRAIL突变体三聚体；
(2)将rTRAIL突变体三聚体与带连接臂的海兔毒素混合，进行偶联反应；
(3)反应完成后，分离纯化得到rTRAIL突变体-海兔毒素偶联物；
rTRAIL突变体的氨基酸序列如SEQ ID NO.1所示；
所述连接臂为马来酰亚胺修饰的缬氨酸-瓜氨酸二肽。
8.如权利要求7所述的制备方法，其特征在于，所述rTRAIL突变体三聚体制备方法为：
将rTRAIL突变体溶于含锌离子的缓冲液中，加入磷酸三(β-氯乙基)酯，30～40℃水浴反应1～3h。
9.如权利要求7所述的制备方法，其特征在于，所述偶联反应的温度为4℃以下，时间为30～60min。
10.如权利要求7所述的制备方法，其特征在于，所述分离纯化方法为：利用超滤除去反应液中分子量低于10kDa的物质，再经离心去沉淀，将所得上清过滤除菌，即为rTRAIL突变体-海兔毒素偶联物。
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| CN2012104166488A CN102936281B (zh) | 2012-10-25 | 2012-10-25 | 一种rTRAIL突变体及其海兔毒素偶联物 |
| JP2015538259A JP6209616B2 (ja) | 2012-10-25 | 2013-06-20 | ｒＴＲＡＩＬ突然変異体およびそのモノメチルアウリスタチンＥコンジュゲート |
| PCT/CN2013/077566 WO2014063495A1 (zh) | 2012-10-25 | 2013-06-20 | 一种rTRAIL突变体及其海兔毒素偶联物 |
| KR1020157013621A KR102050621B1 (ko) | 2012-10-25 | 2013-06-20 | Rtrail 변이체 및 이의 모노메틸 오리스타틴 e 접합체 |
| US14/437,987 US9617326B2 (en) | 2012-10-25 | 2013-06-20 | RTRAIL mutant and monomethyl auristatin E conjugate thereof |
| EP13848770.7A EP2924048B1 (en) | 2012-10-25 | 2013-06-20 | Rtrail mutant and monomethyl auristatin e conjugate thereof |
| AU2013334334A AU2013334334B2 (en) | 2012-10-25 | 2013-06-20 | rTRAIL mutant and monomethyl auristatin E conjugate thereof |
| HK15111182.8A HK1210478A1 (zh) | 2012-10-25 | 2015-11-12 | 種 突變體及其海兔毒素偶聯物 |
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| CN103408654A (zh) * | 2013-08-07 | 2013-11-27 | 浙江大学 | 一种rTRAIL突变体-单甲基化聚乙二醇马来酰亚胺偶联物及其应用 |
| WO2014063495A1 (zh) * | 2012-10-25 | 2014-05-01 | 浙江大学 | 一种rTRAIL突变体及其海兔毒素偶联物 |
| WO2014141094A1 (en) * | 2013-03-14 | 2014-09-18 | Adamed Sp. Z O.O. | Anticancer conjugate |
| CN104744584A (zh) * | 2015-03-25 | 2015-07-01 | 浙江大学 | 一种聚乙二醇-rTRAIL突变体三聚体-海兔毒素偶联物及其制备方法和应用 |
| CN104342444B (zh) * | 2013-07-23 | 2017-11-17 | 四川大学华西医院 | 一种重组trail蛋白及其制备方法和用途 |
| CN107375941A (zh) * | 2017-07-17 | 2017-11-24 | 中国药科大学 | 一种抗人dll4单克隆抗体与海兔毒素衍生物mmae的偶联物 |
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| WO2022080865A2 (ko) * | 2020-10-13 | 2022-04-21 | 신동준 | 반려견의 항암용 재조합 단백질 및 이를 포함하는 반려견을 위한 항암용 조성물 |
| BR112023023525A2 (pt) * | 2021-05-19 | 2024-01-30 | Biohaven Therapeutics Ltd | Conjugados de anticorpo-fármaco usando tecnologia mates para distribuir agentes citotóxicos |
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| WO2014063495A1 (zh) * | 2012-10-25 | 2014-05-01 | 浙江大学 | 一种rTRAIL突变体及其海兔毒素偶联物 |
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| CN104342444B (zh) * | 2013-07-23 | 2017-11-17 | 四川大学华西医院 | 一种重组trail蛋白及其制备方法和用途 |
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| CN104744584A (zh) * | 2015-03-25 | 2015-07-01 | 浙江大学 | 一种聚乙二醇-rTRAIL突变体三聚体-海兔毒素偶联物及其制备方法和应用 |
| CN104744584B (zh) * | 2015-03-25 | 2018-09-28 | 浙江大学 | 一种聚乙二醇-rTRAIL突变体三聚体-海兔毒素偶联物及其制备方法和应用 |
| CN107375941A (zh) * | 2017-07-17 | 2017-11-24 | 中国药科大学 | 一种抗人dll4单克隆抗体与海兔毒素衍生物mmae的偶联物 |
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| US9617326B2 (en) | 2017-04-11 |
| AU2013334334B2 (en) | 2017-10-19 |
| KR102050621B1 (ko) | 2019-11-29 |
| EP2924048A4 (en) | 2016-05-25 |
| US20150307587A1 (en) | 2015-10-29 |
| EP2924048B1 (en) | 2018-02-14 |
| JP2015533838A (ja) | 2015-11-26 |
| KR20150079771A (ko) | 2015-07-08 |
| WO2014063495A1 (zh) | 2014-05-01 |
| HK1210478A1 (zh) | 2016-04-22 |
| AU2013334334A1 (en) | 2015-05-14 |
| CN102936281B (zh) | 2013-12-25 |
| EP2924048A1 (en) | 2015-09-30 |
| JP6209616B2 (ja) | 2017-10-04 |
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