CN110914421A - 选择性细胞死亡诱导酶系统 - Google Patents

选择性细胞死亡诱导酶系统 Download PDF

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CN110914421A
CN110914421A CN201880031362.3A CN201880031362A CN110914421A CN 110914421 A CN110914421 A CN 110914421A CN 201880031362 A CN201880031362 A CN 201880031362A CN 110914421 A CN110914421 A CN 110914421A
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西尔万·图雷尔
塔贝·克拉夫特
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Abstract

本发明涉及用于人和动物的癌症和肿瘤的疗法和/或治疗的包含选择性细胞死亡诱导酶系统的融合蛋白、方法及其用途。

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选择性细胞死亡诱导酶系统
描述
本发明涉及用于人和动物的癌症和肿瘤的疗法和/或治疗的含有选择性细胞死亡诱导酶系统的融合蛋白、方法及其用途。
癌症是一类疾病，其特征在于不受控制的细胞生长和变性细胞(degeneratecell)在体内的扩散并且在转移的情况下，最终导致患者死亡。肿瘤和癌症疾病的治疗在很大程度上取决于出现的肿瘤类型，如今，除了侵入性手术以外，通常还涉及放射疗法或化学疗法的使用。癌症疾病既由外部因素(吸烟、传染性生物或病毒、诱变剂和电离辐射)触发，也由内部因素(遗传易感性、激素、免疫系统因子和自发性体细胞突变)触发。癌症也可通过免疫疗法、激素疗法以及靶向疗法来治疗。使用化学疗法杀伤肿瘤细胞的有利方面在于其通过对细胞DNA或RNA施加破坏性破坏性作用来阻断细胞分裂的能力。一旦肿瘤细胞不再分裂，它们就会死亡。细胞分裂得越快，它们可被化学治疗剂杀伤以及肿瘤因细胞死亡的诱导而缩小的可能性就越高。因此，化学疗法对迅速分裂的细胞作用最有效。然而，化学疗法无法区分癌细胞/肿瘤细胞与身体的快速生长的正常细胞，因此会出现诸如脱发、疲劳、疼痛、血细胞计数变化和恶心的副作用。根据作用机理，化学疗法分为五大类：烷化剂、植物生物碱、抗肿瘤抗生素和抗代谢物。
所谓的靶向疗法利用了我们对癌细胞与正常健康细胞的差异的了解。靶向疗法旨在通过利用这些癌细胞的特定特征来消除癌细胞，从而不会损害正常健康的细胞。此类靶向疗法的活性成分尤其包括特异性识别并结合癌细胞的单克隆抗体，以及特异性抑制供应肿瘤的血管的生长的血管生成抑制剂。在大多数情况下，靶向疗法使用可穿透癌细胞膜并阻止细胞代谢，尤其是触发细胞凋亡，杀死细胞的有机小分子。已经描述了许多靶向触发此类细胞凋亡的细胞内信号通路的活性成分。其他活性成分识别并结合细胞表面上的肿瘤特异性受体。
然而，这些疗法给免疫系统带来了巨大负担，并且在许多情况下只能有限地使用。另外，在大多数情况下，这些形式的疗法在各个治疗之间需要长时间的停顿，以再生免疫系统。因此，近年来，尤其是基因治疗方法或基因疫苗接种已被证明在治疗或支持这些经典措施方面是很有前途的。
基因疗法和基因疫苗接种是分子医学方法，其在治疗和预防疾病中的一般用途对医学实践具有相当大的影响。两种方法都是基于将核酸或肽引入患者的细胞或组织，然后基于这些细胞或组织，处理由引入的核酸编码的信息，即基于所需多肽的表达。
现有基因疗法和基因疫苗接种方法的常用方法是使用DNA将所需的遗传信息引入细胞。关于这一点上，已经描述了将DNA引入细胞的各种程序，诸如磷酸钙转染、
Figure BDA0002269361060000021
转染、原生质体融合、电穿孔、显微注射和脂转染。
已提出的特别是用于基因疫苗接种的另一种方法，是将DNA病毒用作DNA媒介物。此类病毒的有利方面在于其感染特性使它们可以实现非常高的转染率。
通过凋亡的生理过程消除疾病相关细胞对患者非常有益。与其他形式的细胞死亡相反，凋亡是受到高度调节和控制的过程，其赋予多种有利方面。凋亡细胞死亡非常快，并且所产生的称为凋亡小体的细胞碎片可被吞噬细胞清除。从而保护周围细胞不受任何损害(Kreitman RJ:Immunotoxins in cancer therapy.Current Opinion in Immunology1999,11:570-578)。
此外，其他死亡诱导物，诸如植物或细菌来源的免疫毒素，经常面临中和抗体产生的问题。这些试剂的免疫原性被认为是临床应用的主要障碍。
然而，已知癌细胞可以抵抗凋亡性损伤，这使得肿瘤能够发生和发展。有缺陷或低效的凋亡信号传导通常是由导致促存活蛋白上调或促凋亡蛋白被抑制的突变引起的。
细胞的凋亡可通过各种促凋亡机制和蛋白质来诱导。这些机制和蛋白质的共同点在于它们激活了针对细胞的蛋白水解性半胱氨酸蛋白酶(称为胱天蛋白酶)的级联。该级联涉及最初活化的胱天蛋白酶(诸如胱天蛋白酶8和胱天蛋白酶9)，激活效应子级联，诸如，例如胱天蛋白酶3和6或7。这些胱天蛋白酶继而裂解一系列细胞底物，从而导致受影响的细胞凋亡。
在本发明的上下文中，术语“程序性细胞死亡”可用作“凋亡”的同义词。如本发明所定义的，“诱导的细胞死亡”是其中活性物质优选通过胱天蛋白酶触发细胞凋亡或程序性细胞死亡的细胞死亡。
然而，已知胱天蛋白酶可用于肿瘤治疗，例如，如US20030054000A1中所公开的。
通过递送胱天蛋白酶3、7、8或10诱导凋亡可以避免这些酶上游的凋亡途径的所有功能障碍。胱天蛋白酶，特别是胱天蛋白酶3、胱天蛋白酶7、胱天蛋白酶8或胱天蛋白酶10在凋亡途径中起主要作用，它们在细胞中提供一百多种蛋白质靶标。与其他胱天蛋白酶相反，胱天蛋白酶3、胱天蛋白酶7、胱天蛋白酶8或胱天蛋白酶10单独地或一起在递送至癌细胞时能够诱导凋亡。
然而，也存在几种胱天蛋白酶3、7、8或10抗性癌细胞。这些细胞表达水平升高的凋亡蛋白抑制剂(IAP)，其为抑制胱天蛋白酶依赖性凋亡的天然存在的细胞内蛋白。
存在一系列仅对具有特定识别序列的底物蛋白具有酶促活性的蛋白酶。下表列出了一些实例。P1表示在其后发生裂解的氨基酸的位置，P4、P3和P2是限制性位点P1之前的N-端位置。P1'和P2'是在P1之后的C端位置。这意味着蛋白酶在P1与P1'之间切割多肽链。
Figure BDA0002269361060000041
使用单字母代码表示氨基酸
特别有效和特异性的胱天蛋白酶(见表1)是胱天蛋白酶3、7、8和10。
从该现有技术出发，发明人的目标是通过活性成分引起癌细胞或肿瘤细胞的诱导的细胞死亡。
胱天蛋白酶被表达为无活性的酶原，需要特定的裂解来激活。为了特异性地激活根据本发明的胱天蛋白酶3、7、8、10，用被烟草刻蚀病毒(在下文中简称为“TEV”)蛋白酶独特地裂解的氨基酸序列(识别位点)替代其天然裂解位点。
通常，胱天蛋白酶3、7、8、10被翻译成例如被颗粒酶B天然裂解的无活性酶原。胱天蛋白酶原3、7、8、10基因由N端前结构域组成，其后是两个组分，即所谓的小亚基和大亚基(Earnshaw WC,Martins LM,Kaufmann SH:Mammalian caspases:structure,activation,substrates,and functions during apoptosis.Annu Rev Biochem 1999,68:383-424)。加工位点位于前结构域与大亚基的交界处，以及两个亚基之间的亚基间接头处。胱天蛋白酶成熟所需的所有天然裂解均发生在天冬氨酸残基(称为P1'残基，请参见表1)的羧基侧。
据报道，亚单位间接头的裂解是必需的，并且足以诱导活性。因此，根据本发明使用的优选胱天蛋白酶3、7、8、10变体在优选呈现丝氨酸(S)的所述胱天蛋白酶的小亚基与大亚基之间的P1'位置配备了TEV蛋白酶裂解位点。由此，去除了对于被颗粒酶B裂解至关重要的内源性P1'天冬氨酸(D)，并且天然地提供TEV蛋白酶偏好的丝氨酸(S)作为P1'。
此外，将识别位点置于两个域之间确保了有利的表面可及性。
令人惊讶的是，肿瘤细胞有可能通过细胞死亡诱导性酶系统死亡，所述酶系统包含融合蛋白，所述融合蛋白包含胱天蛋白酶3和/或胱天蛋白酶7和/或胱天蛋白酶8和/或胱天蛋白酶10的无活性形式，或编码其的核酸，以及TEV(SEQ ID NO.1或SEQ ID NO.2)或编码它的核酸，其中胱天蛋白酶3、7、8、10以包含至少一种识别位点(识别序列)ENLYFQS(SEQ IDno.3)和/或ENLYFQG(SEQ ID no.4)(对于TEV而言)的改变的形式提供。
根据本发明，TEV识别胱天蛋白酶3和/或胱天蛋白酶7和/或胱天蛋白酶8和/或胱天蛋白酶10的改变的形式中的识别位点(识别序列)ENLYFQS(SEQ ID no.3)或ENLYFQG(SEQID no.4)，其中优选在胱天蛋白酶3和/或胱天蛋白酶7和/或胱天蛋白酶8和/或胱天蛋白酶10的大亚基与小亚基之间联接(连接)所述识别位点(识别序列)。
这样的序列可以定义如下：
改变的胱天蛋白酶3，其中TEV的识别位点以粗体表示(SEQ ID NO.5):
Figure BDA0002269361060000061
改变的胱天蛋白酶7，其中TEV的识别位点以粗体表示(SEQ ID NO.6):
ADDQGCIEEQGVEDSANEDSVDAKPDRSSFVPSLFSKKKKNVTMRSIKTTRDRVPTYQYNMNFEKLGKCIIINNKNFDKVTGMGVRNGTDKDAEALFKCFRSLGFDVIVYNDCSCAKMQDLLKKASEEDHTNAACFACILLSHGEENVIYGKDGVTPIKDLTAHFRGDRCKTLLEKPKLFFIQACRGTELDDGIQAENLYFQSGPINDTDANPRYKIPVEADFLFAYSTVPGYYSWRSPGRGSWFVQALCSILEEHGKDLEIMQILTRVNDRVARHFESQSDDPHFHEKKQIPCVVSMLTKELYGFSQ
改变的胱天蛋白酶8，其中TEV的识别位点以粗体表示(SEQ ID NO.7):
MDSESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKLHSIRDRNGTHLDAGALTTTFEELHFEIKPHDDCTVEQIYEILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPIYELTSQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPVETDSENLYFQGMDLSSPQTRYIPDEADFLLGMATVNNCVSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNYEVSNKDDKKNMGKQMPQPTFTLRKKLVFPSD
改变的胱天蛋白酶10，其中TEV的识别位点以粗体表示(SEQ ID NO.8)：
MDVKTFLEALPQESWQNKHAGSNGNRATNGAPSLVSRGMQGASANTLNSETSTKRAAVYRMNRNHRGLCVIVNNHSFTSLKDRQGTHKDAEILSHVFQWLGFTVHIHNNVTKVEMEMVLQKQKCNPAHADGDCFVFCILTHGRFGAVYSSDEALIPIREIMSHFTALQCPRLAEKPKLFFIQACQGEEIQPSVSIEAENLYFQGQAPTSLQDSIPAEADFLLGLATVPGYVSFRHVEEGSWYIQSLCNHLKKLVPRMLKFLEKTMEIRGRKRTVWGAKQISATSLPTAISAQTPRPPMRRWSSVS
因此，该目的在由拟定的权利要求所规定的其全部范围内得以实现。
一旦将胱天蛋白酶3和/或胱天蛋白酶7和/或胱天蛋白酶8和/或胱天蛋白酶10的无活性形式和TEV一起引入肿瘤细胞并表达(如果适用的话)，TEV就会释放胱天蛋白酶3和/或胱天蛋白酶7和/或胱天蛋白酶8和/或胱天蛋白酶10的活性形式，通过凋亡或程序性细胞死亡诱导细胞死亡。
本发明中所用的胱天蛋白酶3和/或胱天蛋白酶7和/或胱天蛋白酶8和/或胱天蛋白酶10的创造性选择，以及所使用的将胱天蛋白酶3和/或胱天蛋白酶7和/或胱天蛋白酶8和/或胱天蛋白酶10的无活性形式暴露成活性形式的手段(即TEV)是特别有利的。一旦这两种多肽存在于肿瘤细胞中，就可以以完全特异性和高效的方式进行暴露。在此特别有利的是，人或哺乳动物中都没有胱天蛋白酶原和TEV。
在文件Kapust等，The P1′specificity of tobacco etch virus protease,Biochemical and Biophysical Research Communications,294(2002)949-955中提到了TEV。TEV是指来自烟草腐蚀病毒的核包涵体(NIa)蛋白酶的具有催化活性的27kDa C末端结构域(Dougherty WG,Parks TD,Cary SM,Bazan JF,Fletterick RJ:Characterization ofthe catalytic residues of the tobacco etch virus 49-kDa proteinase.Virology1989,172:302-310)。该蛋白酶识别七个残基的靶序列ENLYFQ/S，其中“/”表示裂解的肽键。丝氨酸P1'残基可被几种其他氨基酸取代，而对加工效率的影响相对较小。其高度严格的序列特异性使TEV蛋白酶成为用于体外和体内融合蛋白的受控细胞内加工的有用试剂。识别的序列不存在于人蛋白质组中，这使得其在体内的应用相对无毒(Kapust RB,Waugh DS:Controlled intracellular processing of fusion proteins by TEVprotease.Protein Expr Purif 2000,19(2):312-318)。
因此，本发明涉及药物或融合蛋白，其包含胱天蛋白酶3和/或胱天蛋白酶7和/或胱天蛋白酶8和/或胱天蛋白酶10的无活性形式)或编码其的核酸以及TEV(例如，SEQ IDno.1或SEQ ID no.2)或编码其的核酸。TEV识别改变的胱天蛋白酶3和/或改变的胱天蛋白酶7和/或改变的胱天蛋白酶8和/或改变的胱天蛋白酶10的无活性形式(如SEQ ID no.5或SEQ ID no.6或SEQ ID no.7或SEQ ID no.8)中的识别位点(识别序列)ENLYFQS(SEQ IDno.3)或ENLYFQG(SEQ ID no.4)。
在本发明的一个优选实施方案中，胱天蛋白酶3和/或胱天蛋白酶7和/或胱天蛋白酶8和/或胱天蛋白酶10的无活性形式是改变的“胱天蛋白酶原”(SEQ ID No.5或SEQ IDNo.6或SEQ ID No.7或SEQ ID No.8)或编码其的核酸。
在本发明的另一个优选实施方案中，胱天蛋白酶3和/或胱天蛋白酶7和/或胱天蛋白酶8和/或胱天蛋白酶10的无活性形式是融合蛋白或编码其的核酸，其中通过TEV(例如，SEQ ID no.1或SEQ ID no.2)在ENLYFQ(SEQ ID no.3)或ENLYFQG(SEQ ID no.4)处的裂解释放或获得选自SEQ ID no.9、10、11、12、13、14、15、16的至少一个序列。
因此，本发明涉及胱天蛋白酶3和/或胱天蛋白酶7和/或胱天蛋白酶8和/或胱天蛋白酶10的无活性形式，即包含SEQ ID no.5、6、7或8的融合蛋白或编码其的核酸，其中通过TEV(例如，SEQ ID no.1或SEQ ID no.2)在识别序列ENLYFQS(SEQ ID no.3)或ENLYFQG(SEQID no.4)处的裂解释放选自SEQ ID no.9、10、11、12、13、14、15或16的至少一个序列。
因此，本发明涉及胱天蛋白酶3和/或胱天蛋白酶7和/或胱天蛋白酶8和/或胱天蛋白酶10的无活性形式(即包含选自包含ENLYFQS(SEQ ID no.3)或ENLYFQG(SEQ ID no.4)的SEQ ID no.5、6、7或8的至少一个序列的融合蛋白)或编码其的序列。可以以相应的方式(例如，借助于HIS标签等)制备任何其他融合蛋白，其中样品标签可以被任何肽(例如50至100个氨基酸)替代。
本领域技术人员能够产生和设计合适的融合蛋白(Ausubel等(编辑),(1989).Preparation of Genomic DNA from Mammalian Tissue.In:Short Protocols inMolecular Biology:A Compendium of Methods from CURRENT PROTOCOLS IN MOLECULARBIOLOGY.John Wiley&Sons)，另参考实施例。
在本发明的另外一个优选实施方案中，癌细胞的凋亡抗性应通过同时递送融合蛋白和抗凋亡蛋白抑制剂(特别地选自Smac/DIABLO(SEQ ID no.17)和XAF1(SEQ ID no.18))来克服。
Smac/DIABLO是一种细胞内蛋白，其起着拮抗，即抑制IAP的活性的作用(同上)。此外，Smac/DIABLO可以促进胱天蛋白酶原的蛋白水解活化，然而也可以促进成熟胱天蛋白酶的酶促活性。在凋亡刺激下，Smac/DIABLO通常从线粒体释放。无论如何，这种释放通常在癌细胞中被Bcl-2阻断。在本发明的另一个方面，去除了Smac/DIABLO的线粒体靶向序列，以确保在胞质溶胶中直接表达。
XAF1在正常组织中普遍表达，但在许多癌症中以低水平或无法检测到的水平存在。其可降解IAP，并诱导Bax表达。另外，XAF1可结合已知抑制胱天蛋白酶的锌。
在另外一个优选实施方案中，根据本发明的融合蛋白可包含抗凋亡蛋白，诸如但不限于Smac/DIABLO(SEQ ID no.17)或XAF1(SEQ ID no.18)。
仅仅为了释放此类抗凋亡蛋白，融合蛋白可包含一个或多个如图1中示例性描述的TEV识别位点。这种融合蛋白的一个合适实例示于SEQID No.19中，其中例如胱天蛋白酶3、胱天蛋白酶7、SMAC、XAF1和TEV排列在一个质粒中，其中在不同蛋白质之间具有插入的TEV识别位点和接头序列。
本发明的融合蛋白或组合制剂和药物可具有添加至其中的合适的赋形剂和添加剂。合适的添加剂和/或赋形剂的实例是例如生理盐水溶液、稳定剂、蛋白酶抑制剂、核酸酶抑制剂等。
因此，本发明还涉及如上针对在治疗和/或预防人和动物(尤其是哺乳动物)的癌症或肿瘤疾病方面的应用或用途所描述的组合制剂或药物。
在另一个优选实施方案中，本发明的组合制剂或药物通过基因治疗方法施用。
基因治疗方法可以例如通过将本发明的核酸与脂质体复合获得。适用于该目的的脂质混合物由Felgner,P.L.等(1987)Proc.Nat l.Acad.Sci,USA 84,7413；Behr,J.P.等(1989)Proc.Nat l.Acad.Sci.USA 86,6982；Felgner,J.H.等(1994)J.Biol.Chem.269,2550或Gao,X.&Huang,L.(1991)Biochim.Biophys.Acta 1189,195进行了描述。生产脂质体时，DNA以离子形式结合到脂质体的表面，并以保持净正电荷的比率存在，DNA完全被脂质体复合。具有聚乙二醇(PEG)壳的在空间上稳定化的脂质体显示出明显减少的通过单核吞噬细胞系统(MPS)的摄入，并且还大大延长了血液循环时间，减少了PEG化囊泡的聚集，并改善了脂质体制剂的稳定性。类似于PEG，直链和超支化的聚甘油(lPG和hbPG)显示出极好的生物相容性，但允许通过添加官能团形成更多的衍生物。通过使用亲脂性引发剂(诸如胆固醇或1,2-双-n-烷基甘油基醚将各种环氧单体进行阴离子组合聚合，来制备基于超支化聚甘油、直链-超支化PEG-hbPG嵌段共聚物和统计PEG-PG共聚物的新型脂质。已使用1,2-二油酰基-sn-甘油-3-磷酸胆碱(DOPC)作为共脂质(colipid)将新型两亲性结构成功地引入到脂质体膜中。
因此，本发明还涉及基因治疗方法，该方法涉及通过使用媒介物递送至靶细胞(优选肿瘤细胞)中。
在另一个实施方案中，该媒介物可选自脂质体、纳米颗粒或微粒、病毒、脂质复合物等(Gene delivery by lipoplexes and polyplexes.Tros de Ilarduya C,Sun Y,
Figure BDA0002269361060000111
Eur J Pharm Sci.2010年1月14日；40(3):159-70.doi:10.1016/j.ejps.2010.03.019.Epub 2010Mar 30；Efficient gene delivery by EGF-lipoplexesin vitro and in vivo,
Figure BDA0002269361060000112
M,
Figure BDA0002269361060000113
Zalba S,Garrido MJ,de IlarduyaCT.Nanomedicine(Lond).2011Jan；6(1):89-98.doi:10.2217/nnm.10.100；Geneticnanomedicine:gene delivery by targeted lipoplexes,
Figure BDA0002269361060000114
de IlarduyaCT.Methods Enzymol.2012；509:355-67.doi:10.1016/B978-0-12-391858-1.00018-6)。
在一个特别优选的实施方案中，本发明的媒介物在表面上具有识别肿瘤标志物的配体。此类配体的实例是能够与肿瘤标志物结合的多克隆或单克隆抗体或共价结合物(适体)。
最后，此类呈递肿瘤标志物不能局限于或可以特别包括以下标志物：
癌胚抗原(CEA)、甲胎蛋白(AFP)、碳水化合物抗原19-9(CA19-9)、癌症抗原72-4(CA 72-4)、癌症抗原125、癌症抗原15-3(CA15-3)、神经元特异性烯醇化酶(NSE)、鳞状细胞癌抗原(SCC)、细胞角蛋白片段(CYFRA)、人绒毛膜促性腺激素(HCG)、前列腺特异性抗原(PSA)、人甲状腺球蛋白(HTG)、粘蛋白样癌相关抗原(MCA)等。图2显示了肿瘤标志物及其所适于的癌症的实例。
因此，本发明还涉及用于引入本发明药物或融合蛋白的方法，其中将包含编码SEQID no.5、6、7或8的核酸的胱天蛋白酶3和/或胱天蛋白酶7和/或胱天蛋白酶8和/或胱天蛋白酶10的无活性形式以及编码烟草蚀刻病毒蛋白酶的核酸(例如SEQ ID NO.1或SEQ IDNO.2)，尤其是包含编码含有选自SEQ ID no.9、10、11、12、13、14、15或16的至少一个序列和ENLYFQS(SEQ ID no.3)或ENLYFQG(SEQ ID no.4)的融合蛋白的核酸的胱天蛋白酶3和/或胱天蛋白酶7和/或胱天蛋白酶8和/或胱天蛋白酶10的无活性形式和编码烟草蚀刻病毒蛋白酶的核酸，
i.)引入至少一种媒介物中，
ii.)进入肿瘤细胞并在其中表达，
iii.)产生胱天蛋白酶3和/或胱天蛋白酶7和/或胱天蛋白酶8和/或胱天蛋白酶10的活性形式并诱导肿瘤细胞的细胞死亡。
该方法可以通过其他先前提到的实施方案来相应地调整。可优选地将本发明的药物、融合蛋白，尤其是其媒介物向人和动物局部施用，例如皮下施用。当然，本发明包括在肿瘤治疗中的所有应用。
如本发明中所定义，术语“功能变体”应被理解为意指与本发明的肽在功能上相关的多肽或核酸。术语“变体”也应被理解为意指等位基因变体或源自其他生物体、细胞或组织的多肽和核酸。
更广泛地，其还应被理解为意指与指定的SEQ ID N具有约70％，优选约80％，特别优选约90％，最优选约95％的序列同源性，尤其是序列同一性的多肽或核酸。
这还包括在约1-50个氨基酸，优选约1-30个氨基酸，特别优选约1-15个氨基酸，最优选约1-6个氨基酸范围内的多肽缺失。例如，第一氨基酸可以缺乏甲硫氨酸，而基本上不改变多肽的功能。
另外，这还包括含有上述本发明多肽的融合蛋白，所述融合蛋白本身已经具有相应的SEQ ID的功能，或者仅在消除融合部分后才能够获得特定功能。最重要的是，这包括其尤其是非人序列的组分为约1-50个氨基酸，优选约1-30个氨基酸的融合蛋白。非人肽序列的实例是例如来自大肠杆菌(E.coli)半乳糖苷酶的原核肽序列或具有所谓的组氨酸标签(例如Met-Ala-His6-标签)的那些原核肽序列。具有所谓的组氨酸标签的融合蛋白所适合的特别有利的应用是通过含金属离子的柱子(例如通过Ni2+-NTA柱子)纯化表达的蛋白。此处“NTA”代表螯合剂次氮基三乙酸(Qiagen GmbH,Hilden)。
特别地，还可使用经典肽合成法(Merrifield方法)合成多肽的上述部分。它们尤其适合于获得抗血清，其可用于搜索合适的基因表达文库以获得本发明多肽的其他功能变体。
在一个优选实施方案中，前面在每种情况下提及的本发明的核酸是DNA、cDNA或RNA，优选为双链DNA，然而PNA或类似物也是可能的。
本发明的核酸还可以通过(表达)载体，例如通过具有组成型CMV启动子的载体pcDNATM3.1(Invitrogen)等引入肿瘤细胞。
如本发明中所定义，术语肿瘤、癌症、癌细胞和肿瘤细胞应被理解为同义词，并且包括每个良性或恶性肿瘤，尤其是具有局部限制的组织体积增加的生长，包括由于以下原因引起的每个局部肿胀：水肿、急性和慢性炎症、动脉瘤增大(搏动性肿瘤(pulsatingtumo))等，以及炎症性器官肿胀(例如，如在所谓的脾脏肿瘤的情况下)以及以身体自身组织的自发、自主和不可逆的过度生长的形式存在的不同程度去抑制的组织肿瘤(生长、母细胞瘤、瘤形成)，这通常与不同严重程度的特定细胞和组织功能丧失相关(参见Pschyrembel，(第266版)2014年，de Gruyter,Berlin)。
实施例和附图：
这些实施例仅用于解释本发明，而不是将本发明限制于这些实施例。
实施例：
实施例1
实验数据
将编码胱天蛋白酶3、胱天蛋白酶7、胱天蛋白酶8、胱天蛋白酶10、Smac/DIABOLO和XAF1的基因克隆到商购可得的EGFP质粒(pEGFP-N1)中。荧光蛋白EGFP允许通过荧光显微镜使蛋白质可视化。将TEV识别位点(ENLYFQ/S或ENLYFQ/A)插入不同的蛋白质之间。TEV识别位点两侧是甘氨酸/丙氨酸接头序列，以提供结构灵活性，从而提高裂解效率。胱天蛋白酶的天然裂解位点被TEV识别位点交换，例如，如SEQ ID No.5-8中所示。
使用Fugene6转染试剂(Promega)将所得的质粒转染到不同的细胞系例如HEK和WM35(黑色素瘤)中。转染前一天，将1x 104个细胞接种在96孔板的每个孔中，导致转染当天约80％的汇合。将FuGene6转染试剂添加到装有Opti-MEM I(Invitrogen,Karlsruhe)的试管中，并孵育5分钟。使用3:1的试剂DNA比。将100ng质粒DNA添加到FuGene 6转染试剂/培养基中，并立即混合。在室温下孵育15分钟后，将8μL转染样品添加到细胞培养基中。通过荧光显微镜分析细胞。
通过伪足变圆和收缩、质膜破裂以及凋亡小体形成来证明细胞凋亡。
HEK细胞：
用Cas3-TEV-EGFP、Cas7-TEV-EGFP、Cas3-Cas7-TEV-EGFP、XAF1-Smac-TEV-EGFP和Cas3-Cas7-Smac-TEV-EGFP诱导凋亡。
Smac-TEV-EGFP和XAF1-TEV-EGFP不能诱导健康细胞凋亡。
WM35细胞：
单蛋白TEV构建体可以部分诱导细胞凋亡。
通过组合构建体Cas3-Cas7-TEV-EGFP、XAF1-Smac-TEV-EGFP和Cas3-Cas7-Smac-TEV-EGFP实现更好的凋亡诱导。
附图说明：
图1描述融合肽的实施方案。
图2显示某些癌症疾病的肿瘤标志物。
序列表
<110> SIT Biotech GmbH
<120> 选择性细胞死亡诱导酶系统
<130> SIT126-12WO
<140> 尚未知
<141> 2018-03-13
<150> EP17160694.0
<151> 2017-03-13
<160> 19
<170> PatentIn version 3.5
<210> 1
<211> 243
<212> PRT
<213> 烟草蚀刻病毒
<400> 1
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Ile Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu Val Ser Thr Arg
145 150 155 160
Asp Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe Thr Asn Thr
165 170 175
Asn Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu Leu Leu Thr
180 185 190
Asn Gln Glu Ala Gln Gln Trp Val Ser Gly Trp Arg Leu Asn Ala Asp
195 200 205
Ser Val Leu Trp Gly Gly His Lys Val Phe Met Val Lys Pro Glu Glu
210 215 220
Pro Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn Glu Leu Val
225 230 235 240
Tyr Ser Gln
<210> 2
<211> 262
<212> PRT
<213> 烟草蚀刻病毒
<400> 2
Met Lys His His His His His His Pro Met Ser Gly Leu Val Pro Arg
1 5 10 15
Gly Ser Ala Met Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn
20 25 30
Pro Ile Ser Ser Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His
35 40 45
Thr Thr Ser Leu Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn
50 55 60
Lys His Leu Phe Arg Arg Asn Asn Gly Thr Leu Leu Val Gln Ser Leu
65 70 75 80
His Gly Val Phe Lys Val Lys Asp Thr Thr Thr Leu Gln Gln His Leu
85 90 95
Ile Asp Gly Arg Asp Met Met Ile Ile Arg Met Pro Lys Asp Phe Pro
100 105 110
Pro Phe Pro Gln Lys Leu Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg
115 120 125
Ile Cys Leu Val Thr Thr Asn Phe Gln Ala Lys Ser Met Ser Ser Met
130 135 140
Val Ser Asp Thr Ser Cys Thr Phe Pro Ser Ser Asp Gly Ile Phe Trp
145 150 155 160
Lys His Trp Ile Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu Val
165 170 175
Ser Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser Ala Ser Asn Phe
180 185 190
Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys Asn Phe Met Glu
195 200 205
Leu Leu Thr Asn Gln Glu Ala Gln Gln Trp Val Ser Gly Trp Arg Leu
210 215 220
Asn Ala Asp Ser Val Leu Trp Gly Gly His Lys Val Phe Met Val Lys
225 230 235 240
Pro Glu Glu Pro Phe Gln Pro Val Lys Glu Ala Thr Gln Leu Met Asn
245 250 255
Glu Leu Val Tyr Ser Gln
260
<210> 3
<211> 7
<212> PRT
<213> 人工的
<220>
<223> TEV识别位点
<400> 3
Glu Asn Leu Tyr Phe Gln Ser
1 5
<210> 4
<211> 7
<212> PRT
<213> 人工的
<220>
<223> TEV识别位点
<400> 4
Glu Asn Leu Tyr Phe Gln Gly
1 5
<210> 5
<211> 272
<212> PRT
<213> 人工的
<220>
<223> 具有TEV识别位点的胱天蛋白酶3
<400> 5
Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu
1 5 10 15
Glu Pro Lys Ile Ile His Gly Ser Glu Ser Met Asp Ser Gly Ile Ser
20 25 30
Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile
35 40 45
Ile Ile Met Thr Ser Arg Ser Gly Thr Asp Val Asp Ala Ala Asn Leu
50 55 60
Arg Glu Thr Phe Arg Asn Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp
65 70 75 80
Leu Thr Arg Glu Glu Ile Val Glu Leu Met Arg Asp Val Ser Lys Glu
85 90 95
Asp His Ser Lys Arg Ser Ser Phe Val Cys Val Leu Leu Ser His Gly
100 105 110
Glu Glu Gly Ile Ile Phe Gly Thr Asn Gly Pro Val Asp Leu Lys Lys
115 120 125
Ile Thr Asn Phe Phe Arg Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys
130 135 140
Pro Lys Leu Phe Ile Ile Gln Ala Cys Arg Cys Thr Glu Leu Asp Cys
145 150 155 160
Gly Ile Glu Thr Glu Asn Leu Tyr Phe Gln Ser Gly Val Asp Asp Asp
165 170 175
Met Ala Cys His Lys Ile Pro Val Glu Ala Asp Phe Leu Tyr Ala Tyr
180 185 190
Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn Ser Lys Asp Gly Ser
195 200 205
Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys Gln Tyr Ala Asp Lys
210 215 220
Leu Glu Phe Met His Ile Leu Thr Arg Val Asn Arg Lys Val Ala Thr
225 230 235 240
Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe His Ala Lys Lys Gln
245 250 255
Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu Leu Tyr Phe Tyr His
260 265 270
<210> 6
<211> 308
<212> PRT
<213> 人工的
<220>
<223> 具有TEV识别位点的胱天蛋白酶7
<400> 6
Ala Asp Asp Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser Ala
1 5 10 15
Asn Glu Asp Ser Val Asp Ala Lys Pro Asp Arg Ser Ser Phe Val Pro
20 25 30
Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile Lys
35 40 45
Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe Glu
50 55 60
Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys Val
65 70 75 80
Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala Leu
85 90 95
Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn Asp
100 105 110
Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu Glu
115 120 125
Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His Gly
130 135 140
Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys Asp
145 150 155 160
Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu Lys
165 170 175
Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp Asp
180 185 190
Gly Ile Gln Ala Glu Asn Leu Tyr Phe Gln Ser Gly Pro Ile Asn Asp
195 200 205
Thr Asp Ala Asn Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe Leu
210 215 220
Phe Ala Tyr Ser Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly
225 230 235 240
Arg Gly Ser Trp Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu His
245 250 255
Gly Lys Asp Leu Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg
260 265 270
Val Ala Arg His Phe Glu Ser Gln Ser Asp Asp Pro His Phe His Glu
275 280 285
Lys Lys Gln Ile Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu Tyr
290 295 300
Gly Phe Ser Gln
305
<210> 7
<211> 265
<212> PRT
<213> 人工的
<220>
<223> 具有TEV识别位点的胱天蛋白酶8
<400> 7
Met Asp Ser Glu Ser Gln Thr Leu Asp Lys Val Tyr Gln Met Lys Ser
1 5 10 15
Lys Pro Arg Gly Tyr Cys Leu Ile Ile Asn Asn His Asn Phe Ala Lys
20 25 30
Ala Arg Glu Lys Val Pro Lys Leu His Ser Ile Arg Asp Arg Asn Gly
35 40 45
Thr His Leu Asp Ala Gly Ala Leu Thr Thr Thr Phe Glu Glu Leu His
50 55 60
Phe Glu Ile Lys Pro His Asp Asp Cys Thr Val Glu Gln Ile Tyr Glu
65 70 75 80
Ile Leu Lys Ile Tyr Gln Leu Met Asp His Ser Asn Met Asp Cys Phe
85 90 95
Ile Cys Cys Ile Leu Ser His Gly Asp Lys Gly Ile Ile Tyr Gly Thr
100 105 110
Asp Gly Gln Glu Ala Pro Ile Tyr Glu Leu Thr Ser Gln Phe Thr Gly
115 120 125
Leu Lys Cys Pro Ser Leu Ala Gly Lys Pro Lys Val Phe Phe Ile Gln
130 135 140
Ala Cys Gln Gly Asp Asn Tyr Gln Lys Gly Ile Pro Val Glu Thr Asp
145 150 155 160
Ser Glu Asn Leu Tyr Phe Gln Gly Met Asp Leu Ser Ser Pro Gln Thr
165 170 175
Arg Tyr Ile Pro Asp Glu Ala Asp Phe Leu Leu Gly Met Ala Thr Val
180 185 190
Asn Asn Cys Val Ser Tyr Arg Asn Pro Ala Glu Gly Thr Trp Tyr Ile
195 200 205
Gln Ser Leu Cys Gln Ser Leu Arg Glu Arg Cys Pro Arg Gly Asp Asp
210 215 220
Ile Leu Thr Ile Leu Thr Glu Val Asn Tyr Glu Val Ser Asn Lys Asp
225 230 235 240
Asp Lys Lys Asn Met Gly Lys Gln Met Pro Gln Pro Thr Phe Thr Leu
245 250 255
Arg Lys Lys Leu Val Phe Pro Ser Asp
260 265
<210> 8
<211> 305
<212> PRT
<213> 人工的
<220>
<223> 具有TEV识别位点的胱天蛋白酶10
<400> 8
Met Asp Val Lys Thr Phe Leu Glu Ala Leu Pro Gln Glu Ser Trp Gln
1 5 10 15
Asn Lys His Ala Gly Ser Asn Gly Asn Arg Ala Thr Asn Gly Ala Pro
20 25 30
Ser Leu Val Ser Arg Gly Met Gln Gly Ala Ser Ala Asn Thr Leu Asn
35 40 45
Ser Glu Thr Ser Thr Lys Arg Ala Ala Val Tyr Arg Met Asn Arg Asn
50 55 60
His Arg Gly Leu Cys Val Ile Val Asn Asn His Ser Phe Thr Ser Leu
65 70 75 80
Lys Asp Arg Gln Gly Thr His Lys Asp Ala Glu Ile Leu Ser His Val
85 90 95
Phe Gln Trp Leu Gly Phe Thr Val His Ile His Asn Asn Val Thr Lys
100 105 110
Val Glu Met Glu Met Val Leu Gln Lys Gln Lys Cys Asn Pro Ala His
115 120 125
Ala Asp Gly Asp Cys Phe Val Phe Cys Ile Leu Thr His Gly Arg Phe
130 135 140
Gly Ala Val Tyr Ser Ser Asp Glu Ala Leu Ile Pro Ile Arg Glu Ile
145 150 155 160
Met Ser His Phe Thr Ala Leu Gln Cys Pro Arg Leu Ala Glu Lys Pro
165 170 175
Lys Leu Phe Phe Ile Gln Ala Cys Gln Gly Glu Glu Ile Gln Pro Ser
180 185 190
Val Ser Ile Glu Ala Glu Asn Leu Tyr Phe Gln Gly Gln Ala Pro Thr
195 200 205
Ser Leu Gln Asp Ser Ile Pro Ala Glu Ala Asp Phe Leu Leu Gly Leu
210 215 220
Ala Thr Val Pro Gly Tyr Val Ser Phe Arg His Val Glu Glu Gly Ser
225 230 235 240
Trp Tyr Ile Gln Ser Leu Cys Asn His Leu Lys Lys Leu Val Pro Arg
245 250 255
Met Leu Lys Phe Leu Glu Lys Thr Met Glu Ile Arg Gly Arg Lys Arg
260 265 270
Thr Val Trp Gly Ala Lys Gln Ile Ser Ala Thr Ser Leu Pro Thr Ala
275 280 285
Ile Ser Ala Gln Thr Pro Arg Pro Pro Met Arg Arg Trp Ser Ser Val
290 295 300
Ser
305
<210> 9
<211> 170
<212> PRT
<213> 人工的
<220>
<223> TEV对胱天蛋白酶3的切割部分
<400> 9
Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu
1 5 10 15
Glu Pro Lys Ile Ile His Gly Ser Glu Ser Met Asp Ser Gly Ile Ser
20 25 30
Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile
35 40 45
Ile Ile Met Thr Ser Arg Ser Gly Thr Asp Val Asp Ala Ala Asn Leu
50 55 60
Arg Glu Thr Phe Arg Asn Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp
65 70 75 80
Leu Thr Arg Glu Glu Ile Val Glu Leu Met Arg Asp Val Ser Lys Glu
85 90 95
Asp His Ser Lys Arg Ser Ser Phe Val Cys Val Leu Leu Ser His Gly
100 105 110
Glu Glu Gly Ile Ile Phe Gly Thr Asn Gly Pro Val Asp Leu Lys Lys
115 120 125
Ile Thr Asn Phe Phe Arg Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys
130 135 140
Pro Lys Leu Phe Ile Ile Gln Ala Cys Arg Cys Thr Glu Leu Asp Cys
145 150 155 160
Gly Ile Glu Thr Glu Asn Leu Tyr Phe Gln
165 170
<210> 10
<211> 102
<212> PRT
<213> 人工的
<220>
<223> TEV对胱天蛋白酶3的切割部分
<400> 10
Ser Gly Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala
1 5 10 15
Asp Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg
20 25 30
Asn Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu
35 40 45
Lys Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val
50 55 60
Asn Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr
65 70 75 80
Phe His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys
85 90 95
Glu Leu Tyr Phe Tyr His
100
<210> 11
<211> 202
<212> PRT
<213> 人工的
<220>
<223> TEV对胱天蛋白酶7的切割部分
<400> 11
Ala Asp Asp Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser Ala
1 5 10 15
Asn Glu Asp Ser Val Asp Ala Lys Pro Asp Arg Ser Ser Phe Val Pro
20 25 30
Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile Lys
35 40 45
Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe Glu
50 55 60
Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys Val
65 70 75 80
Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala Leu
85 90 95
Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn Asp
100 105 110
Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu Glu
115 120 125
Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His Gly
130 135 140
Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys Asp
145 150 155 160
Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu Lys
165 170 175
Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp Asp
180 185 190
Gly Ile Gln Ala Glu Asn Leu Tyr Phe Gln
195 200
<210> 12
<211> 106
<212> PRT
<213> 人工的
<220>
<223> TEV对胱天蛋白酶7的切割部分
<400> 12
Ser Gly Pro Ile Asn Asp Thr Asp Ala Asn Pro Arg Tyr Lys Ile Pro
1 5 10 15
Val Glu Ala Asp Phe Leu Phe Ala Tyr Ser Thr Val Pro Gly Tyr Tyr
20 25 30
Ser Trp Arg Ser Pro Gly Arg Gly Ser Trp Phe Val Gln Ala Leu Cys
35 40 45
Ser Ile Leu Glu Glu His Gly Lys Asp Leu Glu Ile Met Gln Ile Leu
50 55 60
Thr Arg Val Asn Asp Arg Val Ala Arg His Phe Glu Ser Gln Ser Asp
65 70 75 80
Asp Pro His Phe His Glu Lys Lys Gln Ile Pro Cys Val Val Ser Met
85 90 95
Leu Thr Lys Glu Leu Tyr Gly Phe Ser Gln
100 105
<210> 13
<211> 167
<212> PRT
<213> 人工的
<220>
<223> TEV对胱天蛋白酶8的切割部分
<400> 13
Met Asp Ser Glu Ser Gln Thr Leu Asp Lys Val Tyr Gln Met Lys Ser
1 5 10 15
Lys Pro Arg Gly Tyr Cys Leu Ile Ile Asn Asn His Asn Phe Ala Lys
20 25 30
Ala Arg Glu Lys Val Pro Lys Leu His Ser Ile Arg Asp Arg Asn Gly
35 40 45
Thr His Leu Asp Ala Gly Ala Leu Thr Thr Thr Phe Glu Glu Leu His
50 55 60
Phe Glu Ile Lys Pro His Asp Asp Cys Thr Val Glu Gln Ile Tyr Glu
65 70 75 80
Ile Leu Lys Ile Tyr Gln Leu Met Asp His Ser Asn Met Asp Cys Phe
85 90 95
Ile Cys Cys Ile Leu Ser His Gly Asp Lys Gly Ile Ile Tyr Gly Thr
100 105 110
Asp Gly Gln Glu Ala Pro Ile Tyr Glu Leu Thr Ser Gln Phe Thr Gly
115 120 125
Leu Lys Cys Pro Ser Leu Ala Gly Lys Pro Lys Val Phe Phe Ile Gln
130 135 140
Ala Cys Gln Gly Asp Asn Tyr Gln Lys Gly Ile Pro Val Glu Thr Asp
145 150 155 160
Ser Glu Asn Leu Tyr Phe Gln
165
<210> 14
<211> 98
<212> PRT
<213> 人工的
<220>
<223> TEV对胱天蛋白酶8的切割部分
<400> 14
Gly Met Asp Leu Ser Ser Pro Gln Thr Arg Tyr Ile Pro Asp Glu Ala
1 5 10 15
Asp Phe Leu Leu Gly Met Ala Thr Val Asn Asn Cys Val Ser Tyr Arg
20 25 30
Asn Pro Ala Glu Gly Thr Trp Tyr Ile Gln Ser Leu Cys Gln Ser Leu
35 40 45
Arg Glu Arg Cys Pro Arg Gly Asp Asp Ile Leu Thr Ile Leu Thr Glu
50 55 60
Val Asn Tyr Glu Val Ser Asn Lys Asp Asp Lys Lys Asn Met Gly Lys
65 70 75 80
Gln Met Pro Gln Pro Thr Phe Thr Leu Arg Lys Lys Leu Val Phe Pro
85 90 95
Ser Asp
<210> 15
<211> 203
<212> PRT
<213> 人工的
<220>
<223> TEV对胱天蛋白酶10的切割部分
<400> 15
Met Asp Val Lys Thr Phe Leu Glu Ala Leu Pro Gln Glu Ser Trp Gln
1 5 10 15
Asn Lys His Ala Gly Ser Asn Gly Asn Arg Ala Thr Asn Gly Ala Pro
20 25 30
Ser Leu Val Ser Arg Gly Met Gln Gly Ala Ser Ala Asn Thr Leu Asn
35 40 45
Ser Glu Thr Ser Thr Lys Arg Ala Ala Val Tyr Arg Met Asn Arg Asn
50 55 60
His Arg Gly Leu Cys Val Ile Val Asn Asn His Ser Phe Thr Ser Leu
65 70 75 80
Lys Asp Arg Gln Gly Thr His Lys Asp Ala Glu Ile Leu Ser His Val
85 90 95
Phe Gln Trp Leu Gly Phe Thr Val His Ile His Asn Asn Val Thr Lys
100 105 110
Val Glu Met Glu Met Val Leu Gln Lys Gln Lys Cys Asn Pro Ala His
115 120 125
Ala Asp Gly Asp Cys Phe Val Phe Cys Ile Leu Thr His Gly Arg Phe
130 135 140
Gly Ala Val Tyr Ser Ser Asp Glu Ala Leu Ile Pro Ile Arg Glu Ile
145 150 155 160
Met Ser His Phe Thr Ala Leu Gln Cys Pro Arg Leu Ala Glu Lys Pro
165 170 175
Lys Leu Phe Phe Ile Gln Ala Cys Gln Gly Glu Glu Ile Gln Pro Ser
180 185 190
Val Ser Ile Glu Ala Glu Asn Leu Tyr Phe Gln
195 200
<210> 16
<211> 102
<212> PRT
<213> 人工的
<220>
<223> TEV对胱天蛋白酶10的切割部分
<400> 16
Gly Gln Ala Pro Thr Ser Leu Gln Asp Ser Ile Pro Ala Glu Ala Asp
1 5 10 15
Phe Leu Leu Gly Leu Ala Thr Val Pro Gly Tyr Val Ser Phe Arg His
20 25 30
Val Glu Glu Gly Ser Trp Tyr Ile Gln Ser Leu Cys Asn His Leu Lys
35 40 45
Lys Leu Val Pro Arg Met Leu Lys Phe Leu Glu Lys Thr Met Glu Ile
50 55 60
Arg Gly Arg Lys Arg Thr Val Trp Gly Ala Lys Gln Ile Ser Ala Thr
65 70 75 80
Ser Leu Pro Thr Ala Ile Ser Ala Gln Thr Pro Arg Pro Pro Met Arg
85 90 95
Arg Trp Ser Ser Val Ser
100
<210> 17
<211> 163
<212> PRT
<213> SMAC
<400> 17
Met Ala Val Pro Ile Ala Gln Lys Ser Glu Pro His Ser Leu Ser Ser
1 5 10 15
Glu Ala Leu Met Arg Arg Ala Val Ser Leu Val Thr Asp Ser Thr Ser
20 25 30
Thr Phe Leu Ser Gln Thr Thr Tyr Ala Leu Ile Glu Ala Ile Thr Glu
35 40 45
Tyr Thr Lys Ala Val Tyr Thr Leu Thr Ser Leu Tyr Arg Gln Tyr Thr
50 55 60
Ser Leu Leu Gly Lys Met Asn Ser Glu Glu Glu Asp Glu Val Trp Gln
65 70 75 80
Val Ile Ile Gly Ala Arg Ala Glu Met Thr Ser Lys His Gln Glu Tyr
85 90 95
Leu Lys Leu Glu Thr Thr Trp Met Thr Ala Val Gly Leu Ser Glu Met
100 105 110
Ala Ala Glu Ala Ala Tyr Gln Thr Gly Ala Asp Gln Ala Ser Ile Thr
115 120 125
Ala Arg Asn His Ile Gln Leu Val Lys Leu Gln Val Glu Glu Val His
130 135 140
Gln Leu Ser Arg Lys Ala Glu Thr Lys Leu Ala Glu Ala Gln Ile Glu
145 150 155 160
Glu Leu Arg
<210> 18
<211> 301
<212> PRT
<213> XAF1
<400> 18
Met Glu Gly Asp Phe Ser Val Cys Arg Asn Cys Lys Arg His Val Val
1 5 10 15
Ser Ala Asn Phe Thr Leu His Glu Ala Tyr Cys Leu Arg Phe Leu Val
20 25 30
Leu Cys Pro Glu Cys Glu Glu Pro Val Pro Lys Glu Thr Met Glu Glu
35 40 45
His Cys Lys Leu Glu His Gln Gln Val Gly Cys Thr Met Cys Gln Gln
50 55 60
Ser Met Gln Lys Ser Ser Leu Glu Phe His Lys Ala Asn Glu Cys Gln
65 70 75 80
Glu Arg Pro Val Glu Cys Lys Phe Cys Lys Leu Asp Met Gln Leu Ser
85 90 95
Lys Leu Glu Leu His Glu Ser Tyr Cys Gly Ser Arg Thr Glu Leu Cys
100 105 110
Gln Gly Cys Gly Gln Phe Ile Met His Arg Met Leu Ala Gln His Arg
115 120 125
Asp Val Cys Arg Ser Glu Gln Ala Gln Leu Gly Lys Gly Glu Arg Ile
130 135 140
Ser Ala Pro Glu Arg Glu Ile Tyr Cys His Tyr Cys Asn Gln Met Ile
145 150 155 160
Pro Glu Asn Lys Tyr Phe His His Met Gly Lys Cys Cys Pro Asp Ser
165 170 175
Glu Phe Lys Lys His Phe Pro Val Gly Asn Pro Glu Ile Leu Pro Ser
180 185 190
Ser Leu Pro Ser Gln Ala Ala Glu Asn Gln Thr Ser Thr Met Glu Lys
195 200 205
Asp Val Arg Pro Lys Thr Arg Ser Ile Asn Arg Phe Pro Leu His Ser
210 215 220
Glu Ser Ser Ser Lys Lys Ala Pro Arg Ser Lys Asn Lys Thr Leu Asp
225 230 235 240
Pro Leu Leu Met Ser Glu Pro Lys Pro Arg Thr Ser Ser Pro Arg Gly
245 250 255
Asp Lys Ala Ala Tyr Asp Ile Leu Arg Arg Cys Ser Gln Cys Gly Ile
260 265 270
Leu Leu Pro Leu Pro Ile Leu Asn Gln His Gln Glu Lys Cys Arg Trp
275 280 285
Leu Ala Ser Ser Lys Gly Lys Gln Val Arg Asn Phe Ser
290 295 300
<210> 19
<211> 1147
<212> PRT
<213> 人工的
<220>
<223> 融合蛋白
<400> 19
Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu
1 5 10 15
Glu Pro Lys Ile Ile His Gly Ser Glu Ser Met Asp Ser Gly Ile Ser
20 25 30
Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile
35 40 45
Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg
50 55 60
Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn
65 70 75 80
Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile
85 90 95
Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser
100 105 110
Ser Phe Val Cys Val Leu Leu Ser His Gly Glu Glu Gly Ile Ile Phe
115 120 125
Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg
130 135 140
Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile
145 150 155 160
Gln Ala Cys Arg Cys Thr Glu Leu Asp Cys Gly Ile Glu Thr Glu Asn
165 170 175
Leu Tyr Phe Gln Ser Gly Val Asp Asp Asp Met Ala Cys His Lys Ile
180 185 190
Pro Val Glu Ala Asp Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr
195 200 205
Tyr Ser Trp Arg Asn Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu
210 215 220
Cys Ala Met Leu Lys Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile
225 230 235 240
Leu Thr Arg Val Asn Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser
245 250 255
Phe Asp Ala Thr Phe His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser
260 265 270
Met Leu Thr Lys Glu Leu Tyr Phe Tyr His Gly Ala Gly Ala Gly Asp
275 280 285
Tyr Lys Asp Asp Asp Asp Lys Gly Asp Tyr Lys Asp Asp Asp Asp Lys
290 295 300
Ala Ala Ala Gly Gly Glu Asn Leu Tyr Phe Gln Ala Gly Ala Gly Ala
305 310 315 320
Met Gly Ala Gly Ala Gly Glu Asn Leu Tyr Phe Gln Ala Asp Asp Gln
325 330 335
Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser Ala Asn Glu Asp Ser
340 345 350
Val Asp Ala Lys Pro Asp Arg Ser Ser Phe Val Pro Ser Leu Phe Ser
355 360 365
Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile Lys Thr Thr Arg Asp
370 375 380
Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe Glu Lys Leu Gly Lys
385 390 395 400
Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys Val Thr Gly Met Gly
405 410 415
Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala Leu Phe Lys Cys Phe
420 425 430
Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn Asp Cys Ser Cys Ala
435 440 445
Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu Glu Asp His Thr Asn
450 455 460
Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His Gly Glu Glu Asn Val
465 470 475 480
Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys Asp Leu Thr Ala His
485 490 495
Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu Lys Pro Lys Leu Phe
500 505 510
Phe Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp Asp Gly Ile Gln Ala
515 520 525
Glu Asn Leu Tyr Phe Gln Ser Gly Pro Ile Asn Asp Thr Asp Ala Asn
530 535 540
Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe Leu Phe Ala Tyr Ser
545 550 555 560
Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly Ser Trp
565 570 575
Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu His Gly Lys Asp Leu
580 585 590
Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg Val Ala Arg His
595 600 605
Phe Glu Ser Gln Ser Asp Asp Pro His Phe His Glu Lys Lys Gln Ile
610 615 620
Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu Tyr Gly Phe Ser Gln
625 630 635 640
Gly Gly Gly Ala Ala Ala Gly Ala Gly Ala Gly Asp Tyr Lys Asp Asp
645 650 655
Asp Asp Lys Gly Asp Tyr Lys Asp Asp Asp Asp Lys Ala Ala Ala Gly
660 665 670
Gly Glu Asn Leu Tyr Phe Gln Ala Gly Ala Gly Ala Met Ala Val Pro
675 680 685
Ile Ala Gln Lys Ser Glu Pro His Ser Leu Ser Ser Glu Ala Leu Met
690 695 700
Arg Arg Ala Val Ser Leu Val Thr Asp Ser Thr Ser Thr Phe Leu Ser
705 710 715 720
Gln Thr Thr Tyr Ala Leu Ile Glu Ala Ile Thr Glu Tyr Thr Lys Ala
725 730 735
Val Tyr Thr Leu Thr Ser Leu Tyr Arg Gln Tyr Thr Ser Leu Leu Gly
740 745 750
Lys Met Asn Ser Glu Glu Glu Asp Glu Val Trp Gln Val Ile Ile Gly
755 760 765
Ala Arg Ala Glu Met Thr Ser Lys His Gln Glu Tyr Leu Lys Leu Glu
770 775 780
Thr Thr Trp Met Thr Ala Val Gly Leu Ser Glu Met Ala Ala Glu Ala
785 790 795 800
Ala Tyr Gln Thr Gly Ala Asp Gln Ala Ser Ile Thr Ala Arg Asn His
805 810 815
Ile Gln Leu Val Lys Leu Gln Val Glu Glu Val His Gln Leu Ser Arg
820 825 830
Lys Ala Glu Thr Lys Leu Ala Glu Ala Gln Ile Glu Glu Leu Arg Gly
835 840 845
Ala Gly Ala Gly Asp Tyr Lys Asp Asp Asp Asp Lys Gly Asp Tyr Lys
850 855 860
Asp Asp Asp Asp Lys Ala Ala Ala Gly Gly Glu Asn Leu Tyr Phe Gln
865 870 875 880
Ala Gly Ala Gly Ala Met Glu Asn Leu Tyr Phe Gln Ser Ala Gly Ala
885 890 895
Gly Ala Gly Glu Ser Leu Phe Lys Gly Pro Arg Asp Tyr Asn Pro Ile
900 905 910
Ser Ser Thr Ile Cys His Leu Thr Asn Glu Ser Asp Gly His Thr Thr
915 920 925
Ser Leu Tyr Gly Ile Gly Phe Gly Pro Phe Ile Ile Thr Asn Lys His
930 935 940
Leu Phe Arg Arg Asn Asn Gly Thr Leu Leu Val Gln Ser Leu His Gly
945 950 955 960
Val Phe Lys Val Lys Asp Thr Thr Thr Leu Gln Gln His Leu Ile Asp
965 970 975
Gly Arg Asp Met Met Ile Ile Arg Met Pro Lys Asp Phe Pro Pro Phe
980 985 990
Pro Gln Lys Leu Lys Phe Arg Glu Pro Gln Arg Glu Glu Arg Ile Cys
995 1000 1005
Leu Val Thr Thr Asn Phe Gln Ala Lys Ser Met Ser Ser Met Val
1010 1015 1020
Ser Asp Thr Ser Cys Thr Phe Pro Ser Ser Asp Gly Ile Phe Trp
1025 1030 1035
Lys His Trp Ile Gln Thr Lys Asp Gly Gln Cys Gly Ser Pro Leu
1040 1045 1050
Val Ser Thr Arg Asp Gly Phe Ile Val Gly Ile His Ser Ala Ser
1055 1060 1065
Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser Val Pro Lys Asn
1070 1075 1080
Phe Met Glu Leu Leu Thr Asn Gln Glu Ala Gln Gln Trp Val Ser
1085 1090 1095
Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly His Lys
1100 1105 1110
Val Phe Met Val Lys Pro Glu Glu Pro Phe Gln Pro Val Lys Glu
1115 1120 1125
Ala Thr Gln Leu Met Asn Glu Leu Val Tyr Ser Gln Gly Ala Gly
1130 1135 1140
Ala Gly Ala Gly
1145

Claims (12)

1.一种用于治疗癌症或肿瘤疾病的药物，其包含融合蛋白，所述融合蛋白包含选自以下组中的至少一个序列：
源自胱天蛋白酶3的SEQ ID no.9、10和/或
源自胱天蛋白酶7的SEQ ID no.11、12和/或
源自胱天蛋白酶8的SEQ ID no.13、14和/或
源自胱天蛋白酶10的SEQ ID no.15、16和/或
或编码其的核酸，或者二者之一的功能变体，
和
至少一种烟草刻蚀病毒蛋白酶或编码其的核酸，或者二者之一的功能变体，
和
至少一个识别位点ENLYFQS(SEQ ID no.3)或ENLYFQG(SEQ ID no.4)或编码其的核酸，
其特征在于
SEQ ID no.9、10、11、12、13、14、15或16或其功能变体通过由烟草蚀刻病毒蛋白酶进行的裂解而释放。
2.根据权利要求1的用于治疗癌症或肿瘤疾病的药物，其特征在于所述融合蛋白包含SEQ ID no.5、6、7或8或编码其的核酸，或者二者之一的功能变体。
3.根据权利要求1的用于治疗癌症或肿瘤疾病的药物，其特征在于所述融合蛋白包含SEQ ID no.1或2或编码其的核酸，或者二者之一的功能变体。
4.根据权利要求1的用于治疗癌症或肿瘤疾病的药物，其特征在于所述烟草蚀刻病毒蛋白酶识别胱天蛋白酶3和/或胱天蛋白酶7和/或胱天蛋白酶8和/或胱天蛋白酶10的改变形式中的识别位点ENLYFQS(SEQ ID no.3)或ENLYFQG(SEQ ID no.4)，其中所述识别位点在胱天蛋白酶3和/或胱天蛋白酶7和/或胱天蛋白酶8和/或胱天蛋白酶10的大亚基与小亚基之间联接(连接)。
5.根据权利要求1至3中任一项的用于治疗癌症或肿瘤疾病的药物，其中所述融合蛋白包含抗凋亡蛋白，特别是Smac/DIABLO(SEQ ID no.17)或XAF1(SEQ ID no.18)。
6.根据权利要求1至5中任一项的用于治疗癌症或肿瘤疾病的药物，以及可能的赋形剂和添加剂。
7.根据权利要求6的用于治疗癌症或肿瘤疾病的药物，其用于治疗人和动物。
8.根据前述权利要求中任一项的用于治疗癌症或肿瘤疾病的药物，其特征在于其通过基因治疗方法施用。
9.根据前述权利要求中任一项的用于治疗癌症或肿瘤疾病的药物，其特征在于该基因治疗方法通过媒介物来进行。
10.根据前述权利要求中任一项的用于治疗癌症或肿瘤疾病的药物，其特征在于该媒介物选自脂质体、纳米颗粒或微粒、病毒和脂质复合物。
11.根据前述权利要求中任一项的用于治疗癌症或肿瘤疾病的药物，其特征在于这些媒介物包含识别肿瘤标志物的配体。
12.如前述权利要求中任一项所述的引入药物的方法，其中将包含编码SEQ ID no.5、6、7或8的核酸以及编码烟草蚀刻病毒蛋白酶的核酸(例如，SEQ ID no.1或SEQ ID no.2)的胱天蛋白酶3和/或胱天蛋白酶7和/或胱天蛋白酶8和/或胱天蛋白酶10的无活性形式，尤其是包含编码含有选自SEQ ID no.9、10、11、12、13、14、15或16的至少一个序列和ENLYFQS(SEQ ID no.3)或ENLYFQG(SEQ ID no.4)的融合蛋白的核酸以及编码烟草蚀刻病毒蛋白酶的核酸的胱天蛋白酶3和/或胱天蛋白酶7和/或胱天蛋白酶8和/或胱天蛋白酶10的无活性形式，
i.)引入至少一种媒介物中，
ii.)进入肿瘤细胞并在其中表达，
iii.)产生胱天蛋白酶3和/或胱天蛋白酶7和/或胱天蛋白酶8和/或胱天蛋白酶10的活性形式，并在所述肿瘤细胞中诱导细胞死亡。
CN201880031362.3A 2017-03-13 2018-03-13 选择性细胞死亡诱导酶系统 Pending CN110914421A (zh)

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