CN1406137A - 天冬氨酸特异性半胱氨酸蛋白酶活化的前体药物治疗 - Google Patents
天冬氨酸特异性半胱氨酸蛋白酶活化的前体药物治疗 Download PDFInfo
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- CN1406137A CN1406137A CN01805618A CN01805618A CN1406137A CN 1406137 A CN1406137 A CN 1406137A CN 01805618 A CN01805618 A CN 01805618A CN 01805618 A CN01805618 A CN 01805618A CN 1406137 A CN1406137 A CN 1406137A
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Abstract
本发明提供了药物定位送递的新方法,所述方法是通过给予靶向目的细胞类型的天冬氨酸特异性半胱氨酸蛋白酶偶联物,并额外给予一种前体药物,所述前体药物在天冬氨酸特异性半胱氨酸蛋白酶的存在下可在局部转变为活性药物。本发明进一步提供新的靶向药物(其包含天冬氨酸特异性半胱氨酸蛋白酶)和前体药物,所述前体药物包含天冬氨酸特异性半胱氨酸蛋白酶可裂解的前体药物组分。本发明也提供包含本发明天冬氨酸特异性半胱氨酸蛋白酶偶联物和前体药物的药物组合物以及治疗方法。
Description
发明背景
发明领域
本发明涉及药物定位送递的新方法,所述方法是通过给予靶向目的细胞类型的天冬氨酸特异性半胱氨酸蛋白酶(caspase)偶联物,并额外给予一种前体药物,所述前体药物在caspase的存在下可在局部转变为活性药物。具体的实施方案中,本发明涉及前体药物(如用于癌症治疗中的药物)的靶向给予,其给予以不同细胞类型(如赘生性细胞)为特征的区域,并且该前体药物由caspase在该特定细胞类型所属区域局部转化为活性药物。本发明提供新的靶向药物,其包含caspase和前体药物,所述前体药物包含可裂解的前体药物组分。本发明还涉及包含本发明caspase偶联物和前体药物的药物组合物以及治疗方法。
相关文献说明
一些文献中已有关于在癌症的治疗中使用抗体偶联物将细胞毒性药物局部送递到肿瘤细胞的报道(Syrigos和Epenetos(1999)Anticancer Research19:605-614;Niculescu-Duvaz和Springer(1997)Adv.Drg.Del.Rev.26:151-172;美国专利4975278)。当细胞毒性药物的系统性给予不但使需要消除的肿瘤细胞死亡,还会导致正常细胞死亡时,就需要将这些药物局部给予肿瘤。根据一种抗癌药物送递系统,细胞毒性药物偶联于一种肿瘤特异性抗体上形成免疫偶联物,所述免疫偶联物结合肿瘤细胞,并因此将该细胞毒性药物“送递”到肿瘤位点。在这种靶向系统中使用的免疫偶联物包括抗体-药物偶联物(参见,如Baldwin等,(1986)Lancet pp.(Mar.15,1986):603-05)和抗体-毒素偶联物(Thorpe,“Antibody Carriers Of Cytotoxic Agents InCancer Therapy:A Review,”in Monoclonal Antibodies’84:Biological AndClinical Applications,A.Pinchera等(ed.s),pp.475-506(1985))。有报道单克隆抗体和多克隆抗体均可用于这些策略中(Rowland等,(1986)CancerImmunol.Immunother.,21:183-87)。这些方法中使用的药物包括柔红菌素、阿霉素、氨甲蝶呤和长春花碱(Rowland等,(1986),
同上)。在抗体-毒素偶联物中使用的毒素包括细菌毒素如白喉毒素,植物毒素如蓖麻蛋白以及小分子毒素如美旦辛苷(Liu等,(1996)Proc.Natl.Acad.Sci.USA 93:8618-8623)和加利车霉素(Lode等,(1998)Cancer Res.58:2928;Hinman等,(1993)CancerRes.53:3336-3342)。
ADEPT是一种两步的药物送递方法,其中将抗体-酶融合蛋白或偶联物给予受试者,然后给药前体药物(Syrigos和Epenetos(1999),
同上;Niculescu-Duvaz和Springer(1997),
同上)。使该抗体偶联物定位于肿瘤靶。从循环中将未结合的融合蛋白清除后,给予一种无活性的前体药物。该前体药物被局部的酶偶联物在该肿瘤内或其周围活化。
在鼠异体移植模型中已证明ADEPT是一种有效的抗肿瘤方法(Syrigos和Epenetos(1999),
同上;Niculescu-Duvaz和Springer(1997),
同上)。但是,一般在ADEPT模型中使用的细菌性酶和早期临床实验中使用的啮齿动物来源的抗体对于哺乳动物系统可能有免疫原性(Sharma(1992)Cell Biophysics21:109-120)。使用人源化抗体-人β-葡萄糖醛酸酶融合蛋白的ADEPT在小鼠中有效(Bosslet等,(1994)Cancer Res.54:2151-2159)。然而,由于人β-葡萄糖醛酸酶分子较大(150kDa),其不是ADEPT中的优选酶。同样,在人体系统使用人源化的酶可能导致内源酶使前体药物意外活化,以及来自内源底物或抑制剂的干扰。人羧肽酶A1被工程化以便其活化一种前体药物(Smith等,(1997)J.Biol.Chem.272:15804-15816),所述前体药物不是野生型酶的底物。其在
体内无效(Wolfe等,(1999)Bioconjugate Chemistry 10:38-48)。
caspase是一个胞内半胱氨酸蛋白酶家族,其在细胞因子的成熟和细胞凋亡中起作用(Talamian等,(1997)J.Biol.Chem.272:9677-9682)。caspase以单链酶原的形式产生,需经蛋白酶解活化(Stennick & Salvesen(1998)Biochimica et Biophysica Acta 1378:17-31)。caspase 3(先前被称为Yama,apopain和CPP32)是一种相对小(57kDa)的哺乳动物蛋白酶。其裂解序列Asp-Glu-Val-Asp(SEQ ID NO:3)和Asp-Glu-Ile-Asp(SEQ ID NO:4)后的位点,这种底物特异性只被其它caspase如caspase 7所共有(Thornberry等,(1997)J.Biol.Chem.272:17907-17911)。内源性caspase 3和7受严谨调节,被认为其仅在经历细胞凋亡的细胞中具有活性。
HER2/neu原癌基因(也被称为c-erbB2)在20-30%的人类原发性乳腺癌和卵巢癌中扩增和/或过表达,并且是总存活率降低和复发时间的强烈预警(Slamon等,(1987)Science 235:177-182;Slamon等,(1989)Science 244:707-712)。发展了许多基于抗体的策略作为癌症的潜在治疗方法,所述癌症过表达HER2/neu基因的p1 85HER2产物(Shalaby等,(1992)J.Exp.Med.175:217-225;Baselga等,(1996)J.Clin.Onc.14:737-744;Pegram等,J.Clin.Onc.(1998)16:2659-2671)。
人源化的抗-p185HER2抗体,humAb4D5-8(Herceptin)(Carter等,(1992a)Proc.Natl.Acad.Sci.USA 89:4285-4289)在治疗转移性乳腺癌的二期临床实验中,作为单独药物(Basegla等,(1996)J.Clin.Onc.14:737-744)以及与细胞毒化学治疗组合(Pegram等,(1998)J.Clin.Onc.16:2659-71)都表现出抗肿瘤的活性。在两次关键性的三期实验后(Cobleigh等,91999)J.Clin.Onc.17:2639-2648),Herceptin于1998年9月通过美国食品和药品管理局的认可用于治疗转移的乳腺癌。
Herceptin被用做基本材料以设计其它潜在的更有效的免疫治疗。这些包括用于细胞毒T细胞的重新靶向的人源化双特异性F(ab’)2和二价抗体(diabody)片段(Shalaby等,(1992)J.Exp.Med.172:217-225;Zhu等,(1995)Intern.J.Cancer 62:319-324;Zhu等,(1996)Bio/Technology 14:192-196)用于定向药物送递的隐形(stealth)免疫脂质体(Park等,(1995)Proc.Natl.Acad.Sci.USA 92:1327-1331)和用于前体药物活化的二硫键稳定的Fv-β内酰胺酶融合蛋白(Rodrigues等,(1995)Chemistry and Biology 2:223-227;Kirpotin(1997)Biochemistry 36:66-75)。
发明简述
本发明提供了用于诊断、预后和治疗多种疾病的新的方法和组合物。本发明包括药物定位送递的新方法,所述方法是通过给予靶向目的细胞类型的caspase,并额外给予一种前体药物,所述前体药物由caspase局部转变为活性药物。具体的实施方案中,本发明提供了将细胞毒性药物送递到目的细胞类型的方法,所述方法包含以下步骤:给予有效剂量的靶向细胞的caspase偶联物并给予该caspase可转变的前体药物,所述偶联物将一种caspase可转变的细胞毒性前体药物转变为活性的细胞毒性药物。
本发明提供包含caspase的组合物,特别是药物组合物。优选实施方案中,该caspase是作为一种靶向的caspase偶联物提供。本发明的caspase偶联物包括caspase/靶向药物复合物,特别是caspase-抗体偶联物,其中组成型活化的caspase,或者通过化学交联键或者通过重组融合连接于一种靶向药物,如抗体。
根据本发明,该caspase偶联物靶向或定向于一种目的细胞类型。因此,根据本发明,caspase连接于一种靶向药物,优选是通过融合或化学偶联。优选的靶向药物包括天然产生的和工程化的受体配体,肽和肽类(peptidometic)配体,抗体,特别是单克隆抗体,包括如Fab,Fab’,F(ab’)2和Fv片段的抗体片段,二价抗体,线性抗体,单链抗体分子,由多个抗体片段形成的多特异性抗体等。在众多靶向药物中优选抗体。
本发明优选的caspase是哺乳动物的caspase,包括人caspase 1-10中任何一种,特别是组成型活化的caspase如反向(reverse)caspase。优选实施方案中,所述方法和组合物利用一种前凋亡型(proapoptotic)组成型活化的caspase。根据本发明这一方面优选的caspase选自:caspase 2,caspase 3和caspase 7,尤其优选caspase 3。
本发明进一步提供了治疗各种疾病的方法,所述疾病尤其是以出现或存在特定细胞类型为特征的疾病。这些细胞包括被细菌或病毒感染并表达该感染所特征性的细胞表面表位的细胞,赘生性细胞和恶性细胞(如肿瘤细胞)以及因其在炎症区域的存在或表现而被鉴定的细胞。本发明提供了一种治疗疾病的方法,所述方法包含将本发明的caspase偶联物给予有需要的受试者的步骤。在具体的实施方案中,本发明提供了一种治疗以赘生性细胞或恶性细胞类型的表达为特征的疾病的方法,所述方法利用靶向该赘生性细胞或恶性细胞类型的抗体。优选的实施方案中,本发明提供了一种治疗疾病的方法,所述疾病特征是存在表达以下因子的细胞类型,包括如Apo2,CD20,CD40,muc-1,前列腺特异性膜抗原(PSMA),前列腺干细胞抗原(PSCA),表皮生长因子受体(EGFR),CD33,CD19,加速腐烂的因子(decay accelerating factor,DAF),EpCAM,CD52,癌胚抗原(CEA),TAG72抗原,c-MET,前列腺的六-跨膜表皮抗原(STEAP)或ErbB2。根据本发明的具体方面,这些方法包含给予caspase-抗体偶联物,其中所述抗体是抗-CD20,抗-CD40,抗-ErbB2或抗Apo2抗体,尤其是单克隆抗体或抗体片段。
本发明进一步提供了将活性药物(如细胞毒性药物)送递到特定细胞类型的方法,所述方法包含给予前体药物的步骤,所述前体药物在caspase的存在下可转变为活性药物。合适的前体药物包含caspase可裂解的前体药物组分,如Asp-Xaa-Xaa-Asp,Asp-Glu-Xaa-Asp,Asp-Glu-Val-Asp(SEQ ID NO:3),或Asp-Glu-Ile-Asp(SEQ ID NO:4)肽序列。优选的前体药物包括前-细胞毒性药物。本发明上下文本中优选的前体药物包括细胞毒性前体药物,选自美旦辛苷(maytansinoids)、加利车霉素、阿霉素(doxorubicin)、柔红菌素(daunorubicin)、表阿霉素(epirubicin)、他克唑(taxol)、泰索帝(taxotere)、长春新碱(vincristine)、长春花碱(vinblastine),丝裂霉素C(mitomycin C)、依托泊苷(etoposide),氨甲蝶呤(methotrexate)、顺铂(cisplatin)、环磷酰胺(cyclophosphamide)、左旋苯丙氨酸氮芥(melphalan)、哈乐泰斯停(Halotestin)、环磷酰胺(cyclophosphamide)、噻替派(Thio-TEPA)、苯丁酸氮芥(chlorambucil),5-FU和癌得星(cytoxan),其中所述前体药物包含一个caspase可裂解的组分。
本发明包括组合物,包括包含前体药物和靶向的caspase偶联物(如caspase-抗体融合蛋白)的药物组合物,所述组合物用于治疗多种疾病,本发明还包括试剂盒和制品。试剂盒和制品优选包括:
(a)容器;
(b)所述容器上的标签;和
(c)包含所述容器中所装靶向的caspase偶联物的组合物;
其中所述组合物可有效治疗疾病,所述容器上的可选标签显示该组合物可用于治疗特定的疾病。所述试剂盒优选包含其它组分,如caspase可活化的前体药物或药物,以及附加组件,如包含一种可药用缓冲液的容器以及使用所述组合物治疗疾病的方法。
附图说明
图1、caspase可裂解的前体药物Ac-DEVD-PABC-阿霉素在SK-BR-3和MCF7细胞中的积累。根据标准曲线估计阿霉素的摄入,该标准曲线是利用加入先前未经处理的细胞中的阿霉素的已知量制备的。
图2、有或无caspase 3的情况下,SK-BR-3和MCF7乳腺癌细胞中caspase可裂解的前体药物Ac-DEVD-PABC-阿霉素的体外细胞毒性。
图3、人肺癌细胞(H460)和结肠癌细胞(HCT116)中Ac-DEVD-PABC-阿霉素的体外细胞毒性。
图4、人肺癌细胞(H460)和结肠癌细胞(HCT116)中Ac-DEVD-PABC-他克唑的体外细胞毒性。
图5、caspase 3在人血浆中的稳定性。
图6、质粒pLCrC3.HCrC3中,抗-HER2 Fab反向caspase 3偶联物的核酸(SEQ ID NO:1)和氨基酸(SEQ ID NO:2和25)序列。SEQ ID NO:2由SEQ ID NO:1的439-1977位核苷酸编码。SEQ ID NO:25由SEQID NO:1的2025-3605位核苷酸编码。
图7、抗-HER2 Fab反向caspase 3偶联物pLCrC3.HCrC3,以及其构建中使用的质粒pLCr3和pHCrC3的图示。
图8、Ac-DEVD-阿霉素前体药物的制备:(i)阿霉素的氢氯化物,DCC,HOSu,DIPEA,DMF,0-23℃和(ii)Pd(PPh3)4,Bu3SnH,AcOH,DEM,23℃。
图9、Ac-DEVD-PABC(Asp-Glu-Val-对氨基苯甲基氧羰基)前体药物组分的制备。在此实施例中DEVD是所述caspase可裂解的前体药物组分,PABC是自我牺牲型连接物;(iii)4-氨基苯甲基醇,EEDQ,DMF,23℃以及(iv)4-硝基苯氯甲酸酯,2,6-二甲基吡啶,DCM,DMF,23℃.
图10、Ac-DEVD-PABC-阿霉素前体药物的制备:(v)阿霉素的盐酸盐,DIPEA,DMF,23℃和(vi)Pd(PPh3)4,Bu3SnH,AcOH,DMF,23℃。
图11、Ac-DEVD-PABC-紫杉醇(paclitaxel)前体药物的制备:(vii)紫杉醇(paclitexel),DMAP,MeCN,23℃和(viii)Pd(PPh3)4,Bu3SnH,AcOH,DMF,23℃。
优选实施方案的详细说明
定义
本发明中所述术语“氨基酸’’以其最广义的方式表达,包括天然产生的L-氨基酸或残基。本文使用通常所用的表示天然产生的氨基酸的单字母和三字母缩写(Lehninger,A.L.,Biochemistry,2d ed.,pp.71-92,(1975),Worth Publishers,New York)。该术语包括D-氨基酸以及化学修饰的氨基酸,如氨基酸类似物,通常不存在于蛋白中的天然氨基酸(如正亮氨酸),以及具有本领域已知的氨基酸特征的化学合成的化合物。例如,氨基酸的定义也包括如苯丙氨酸或脯氨酸的类似物或模拟物,其和天然苯丙氨酸和脯氨酸同样地限制肽化合物的构象。本文中也将这种类似物和模拟物称为氨基酸的“功能等价物”。其它氨基酸的例子由Roberts和Vellaccio等列出(The Peptides:Analysis,Synthesis,Biology,Eds.Gross and Meiehofer,Vol.5p 341,Academic Press,Inc,N.Y.1983),在此引用作为参考。
术语抗体和免疫球蛋白可互换使用,用于表示具有特定结构特征的糖蛋白。术语“抗体”以其最广泛的意义使用,特定地包括单克隆抗体(包括激动剂抗体和拮抗剂抗体),以及具有多表位特异性的抗体组合物。术语“抗体”特异地包括单克隆抗体(包括全长的单克隆抗体),多克隆抗体,多特异性抗体(如双特异性抗体)和抗体片段。
定义抗体或免疫球蛋白时,通常以人IgG1一般性说明免疫球蛋白,并特指免疫球蛋白的结构域结构(Kabat E. A.(1978)Adv.Protein Chem.32:1-75)。相应地,免疫球蛋白通常是约150000道尔顿的异四聚体糖蛋白,由两个相同的轻链(L)和两个相同的重链(H)组成。每个轻链通过一个共价二硫键连接到一个重链上,而不同免疫球蛋白同种型的重链间二硫键数量各有不同。每条重链和轻链还具有规律地间隔的链间二硫键。每条重链具有一个氨基末端可变区(VH),接着排列的是羧基末端恒定区。每条轻链有一个N-末端可变区(VL)和一个C-末端恒定区;轻链的恒定区与重链的第一个恒定区(CH1)对应排列,轻链可变区与重链可变区对应排列。根据免疫球蛋白多肽链的区的定义,轻链(L)有两个构象相似的区VL和CL;重链有四个区(VH,CH1,CH2和CH3),各具有一个链内二硫键。
基于重链恒定区(C)的氨基酸序列,免疫球蛋白可被分为不同类。主要有5类免疫球蛋白:IgA,IgD,IgE,IgG,和IgM。相应于免疫球蛋白不同类的重链恒定区分别被称为:α,δ,ε,γ和μ。序列研究表明IgM的μ链包含5个区:VH,CHμ1,CHμ2,CHμ3和CHμ4。IgE(ε)的重链也包含5个区,但IgA(α)的重链只有4个区。免疫球蛋白类可进一步地分为亚类(同种型),如IgG1,IgG2,IgG3,IgG4,IgA1和IgA2。
不同类免疫球蛋白的亚单位结构和三维构象已为人熟知。其中IgA和IgM是多聚体,并且其每个亚单位包含两条轻链和两条重链。IgG(γ)的重链在CH1和CH2区之间包含一段多肽链,其被称为铰链区。IgA的α链具有含一个O-连接糖基化位点的铰链区,μ和ε链不具有与γ和α链的铰链区类似的序列,但其包含在其它免疫球蛋白中不存在的第四个恒定区。免疫球蛋白链的区的组成可归纳如下:
轻链λ=VλCλ
κ=VκCκ
重链IgG(γ)=VHCHγ1,铰链区CHγ2CHγ
IgM(μ)=VHCHμ1 CHμ2CHμ3CHμ4
IgA(α)=VHCHα1铰链区CHα2CHα3
IgE(ε)=VHCHε1CHε2CHε3CHε4
IgD(δ)=VHCHδI铰链区CHδ2CHδ3
“铰链区”通常被定义为人IgG1中从Glu216到Pro230的区域(Burton,Molec.Immunol.22:161-206(1985))。其它IgG同种型的铰链区可与IgG1的序列对比排列,即对齐在相同位置形成重链之链间二硫键的第一个和最后一个半胱氨酸残基。
木瓜蛋白酶消化抗体可产生两个相同的各带有单个抗原结合位点的抗原结合片段(称为“Fab”片段)和残余的“Fc”’片段,Fc段的名称反应了其易于结晶的能力。经胃蛋白酶处理可产生具有两个抗原结合位点并仍然能交联抗原的F(ab’)2片段。
Fab段还包括λ轻链恒定区和重链的第一个恒定区(CH1)。Fab’与Fab的差别在于Fab’在重链CH1的羧基末端多出几个残基,包括抗体铰链区的一个或多个半胱氨酸。Fab’-SH在本文中是指恒定区半胱氨酸残基中有一个游离巯基的Fab’。F(ab’)2抗体片段在最初产生为Fab’片段对,在它们之间具有铰链区半胱氨酸。抗体片段的其它化学偶联是众所周知的。
“Fv”段是含有完整的抗原识别和抗原结合位点的最小抗体片段。此区由紧密地非共价连接的一个重链可变区与一个轻链可变区的二聚体组成。。
“抗体片段”包括完整抗体的一部分,优选包括其抗原结合区或可变区。抗体片段的实例包括Fab、Fab’、F(ab’)2和Fv片段;二价抗体;线性抗体;单链抗体分子;和由多个抗体片段形成的多特异性抗体。
“单链Fv”或“scFv”抗体片段包括抗体的VH和VL结构域,这些结构域存在于单个多肽链上。优选Fv多肽在VH和VL结构域之间还包含一个多肽接头,它能使scFv形成抗原结合所需的结构。有关sFv的综述参见Pluckthun in The Pharmacology of Monoclonal Antibodies,vol.113,Rosenburgand Moore eds.Springer-Verlag,New York,pp.269-315(1994)。
术语“二价抗体”是指具有两个抗原结合位点的小分子抗体片段,这些片段在一条多肽链(VH-VL)上含有相连的一个重链可变区(VH)和一个轻链可变区(VL)。利用一种非常短的接头,其使得同一条链上的两个结构域无法配对,不得不与另一条链上的互补结构域配对,从而形成两个抗原结合位点。二价抗体的详细说明参见,如EP404,097;WO 93/11161;以及Hollinger等,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)。
本文中“线性抗体”是指在Zapata等,Protein Eng.8(10):1057-1062(1995)中所述抗体。简而言之,这些抗体包含一对串联的Fd片段(VH-CH1-VH-CH1),其形成一对抗原结合区。线性抗体可以是双特异性,也可以是单特异性的。
术语“癌症”和“癌性的”是指或描述哺乳动物中以细胞生长失控为典型特点的病理状态。癌症的实例包括但不限定于癌(carcinoma),淋巴瘤,母细胞瘤,肉瘤和白血病。这类癌更具体的例子包括鳞状细胞癌,小细胞肺癌、非小细胞肺癌,胃肠癌,胰腺癌,胶质母细胞瘤,宫颈癌,卵巢癌,肝癌(liver cancer),膀胱癌,肝细胞瘤(hepatoma),乳腺癌,结肠癌,结直肠癌,子宫内膜癌,唾液腺癌,肾癌,前列腺癌,外阴癌,甲状腺癌,肝癌(hepatic crcinoma),以及各种头、颈部癌。
本发明的“caspase”是结构相关的半胱氨酸蛋白酶家族的任何成员,其主要对裂解Asp残基后面的肽链有显著特异性(Stennicke,H.和Salvesen,G.(1998)Biochimica et Biophysica Acta 1387:17-31),并包括天然产生的caspase以及其变体,如本文中详细说明的。已知产生了一系列天然caspase(Stennicke和Salvesen(1998)
同上)。该系列成员的氨基酸序列不是完全同源。但该系列的caspase表现了相同或相似的蛋白水解活性。通常,caspase具有以下相同的特性:i)它们是属于Barrett和Rawlings分类法中C14家族的同源半胱氨酸蛋白酶(Barrett,A.J.,(1997)Eur.J.Biochem.250:1-6);它们优选在肽底物中Asp残基后裂解;它们存在于动物细胞的细胞溶胶中;它们包含保守的QACXG(SEQ ID NO:5),其中X是Arg,Gln或Gly,五肽活性位点基元。
通常caspase在“P1”底物位置必需Asp,该术语的定义见Schecter,I和Berger,A.,(1967)Biochem.Biophys.Res.Commun.27:157-162。caspase对肽底物有特异性,并且底物的一级序列对于caspase的裂解是必要的。caspase可分为三组。I组caspase(caspase 1,4和5)优选P4位置是疏水氨基酸,并具有最佳序列Trp-Glu-His-Asp(SEQ ID NO:6)(P4-P3-P2-P1)。II组caspase(caspase 2,3,7和CED-3)在P4只能是Asp,优选序列是Asp-Glu-X-Asp。III组caspase(caspase6,8,9和10)在P4处可以是多种氨基酸,但优选具有分支的脂肪族侧链的氨基酸,最佳序列为Val/Leu-Glu-X-Asp。所有的caspase都优选P3是Glu。I组caspase经常被称为炎症反应的介质,I组caspase被称为细胞凋亡的效应物,III组被称为细胞凋亡的激活物。
表I
参考Thornberry等((1997)J.Biol.Chem.272:17907-17911)及以下内容:caspase组别 最佳序列I组
caspase 1 WEHD(SEQ ID NO:6)
Caspase 4 W/LEHD
Caspase 5 W/LEHDII组
caspase 3 DEVD(SEQ ID NO:3)
caspase 7 DEVD(SEQ ID NO:3)
caspase 2 DEHD(SEQ ID NO:8)
CED-3 DETD(SEQ ID NO:9)III组
Caspase 6 VEHD(SEQ ID NO:10)
Caspase 8 LETD(SEQ ID NO:11)
Caspase 9 LEHD(SEQ ID NO:7)
caspase 10 LE(Nle)D(SEQ ID NO:12)
根据本发明,II组和III组的caspase被称为“前凋亡型caspase(proapoptotic caspases)”。
术语“caspase”和“野生型caspase”用来指具有与天然caspase,或含天然caspase氨基酸序列的重组产生的caspase多肽相应的氨基酸序列的多肽。天然caspase包括来源于人,以及其它动物物种,如兔、大鼠、猪、非人灵长类动物、马、鼠和绵羊的caspase。哺乳动物caspase的氨基酸序列是众所周知的,或可通过传统技术获得(Stennicke和Salvesen(1998)
同上)。caspase 1-10的氨基酸序列以及氨基酸的编号参见Cohen((1997)Biochem.J.326:1-16)。
“caspase变体”等是指非天然存在,但却来源于或可衍生于前体野生型caspase的caspase。该caspase变体具有与前体caspase相同的底物特异性,但在野生型caspase氨基酸序列中有氨基酸的取代。因此根据本发明,caspase包括caspase变体,其中编码前体caspase的DNA序列被修饰,从而产生一种突变的DNA序列,该突变DNA序列编码对天然caspase的氨基酸序列的一个或以上的氨基酸取代,只要该caspase符合本文所述的活性和结构限制。
本发明中“caspase可转化的前体药物”或“前体药物”是指一种药物(如化疗药物),其最佳活性需要caspase对其的裂解,这种药物包含一个“caspase可裂解的前体药物组分”或“前体药物组分”,如本文列出的作为caspase底物的肽基组分。前体药物通常比亲本药物活性低10倍。优选实施方案中该前体药物比亲本药物活性低10-100倍以上。更优选的实施方案中该前体药物比亲本药物的活性低100倍以上,更优选比亲本药物低1000倍以上。
本发明caspase偶联物“靶向”特定细胞类型的情况是,在体外或优选在体内,该靶向分子以足够的亲和力和特异性结合特定细胞类型并特异性“引导”至,“结合”于或“靶向”一种特定细胞类型(参见,如在Pasqualini和Ruoslahti((1996)Nature,380:364-366),Arap等((1998)Science 279:377-380)中使用的术语“定向”或“靶向”)。通常该靶向分子将以低于约1μM,优选低于约100nM更优选低于约10nM的亲和力结合一种特定细胞类型或其上的表面分子。但是与细胞表位的亲和力小于约1nM,或优选在约1pM和1nM之间的靶向分子也在本发明靶向分子的范围之内。
本发明中使用的术语“靶向分子”或“药物”包括能结合特定细胞表位或能作为细胞表位的配体的蛋白、肽、糖蛋白(如抗体)、糖肽、糖脂、多糖、寡糖、核酸等。本发明的靶向药物包括针对细胞相关分子(如特定细胞类型中表达的细胞受体或细胞分布(cell distribution,CD)抗原)的配体(如抗体),如:
i)针对内皮细胞表面的器官选择性定位(address)分子的配体,如那些被鉴定为将淋巴细胞引导至各种淋巴器官以及引导至经历炎症反应的组织的分子(Belivaqua等(1989)Science,243:1160-1165;Siegelman等,(1989)Science 243:1165-1171;Cepek等.(1994)Nature 372:190-193和Rosen和Bertozzi(1994)Curr.Opin.Cell Biol.6:663-673)。
ii)针对内皮细胞标记的配体,如负责将肿瘤引导至各种器官的Erb2(Johnson等(1993)J.Cell.Biol.121:1423-1432),包括“遗传调节蛋白(Heregulin)”(HRG),其在本文中使用时指由遗传调节蛋白的基因(见美国专利5641869或Marchionni等Nature,362:312-318(1993))编码的多肽。遗传调节蛋白包括遗传调节蛋白-α,遗传调节蛋白-β1,遗传调节蛋白-β2和遗传调节蛋白-β3(Holmes等,Science,256:1205-1210(1992);美国专利5641869);neu分化因子(NDF)(Peles等.Cell 69:205-216(1992));乙酰胆碱受体-诱导活性(ARIA)(Falls等,Cell 72:801-815(1993));胶质细胞生长因子(GGFs)(Marchionni等,Nature,362:312-318(1993));感觉和运动神经元衍生因子(SMDF)(Ho等,J.Biol.Chem.270:14523-14532(1995));-遗传调节蛋白(Schaefer等,Oncogene 15:1385-1394(1997))。该术语包括天然序列HRG多肽的生物活性片段和/或氨基酸序列变体,如其EGF-样功能域片段(如HRG 1177-244)。
iii)肿瘤细胞抗原或“肿瘤抗原”,其作为是否存在前赘生性细胞或赘生性细胞的标记。
肽类靶向分子药物或配体的实例包括,如:
i)可介导对各种器官(如大脑和肾脏)的选择性定位的肽(Pasqualini和Ruoslohti(1996)Nature 380:364-366)。这些肽经常包含主导性的(dominant)氨基酸基元(如在定位于大脑的肽中经常发现Ser-Arg-Leu基元)(Pasqualini和Ruoslahti(1996)同上)。
ii)包含识别结构相关受体(如整联蛋白)的氨基酸序列的肽。例如氨基酸序列Arg-Gly-Asp(RGD)存在于细胞外基质蛋白中,所述基质蛋白如已知结合各种存在于血小板、内皮细胞、白细胞、淋巴细胞、单核细胞和粒细胞上的整联蛋白的纤维蛋白原,纤连蛋白,冯维勒布兰德(von Willibrand)因子以及thrombospondin。包含RGD基元的肽可用于调节鉴别RGD的整联蛋白的活性(Gurrath等,(1992)Eur.J.Biochem.210:911-921;Koivunen等,(1995)Bio/Technology 13:265-270;O’Neil等,(1992)Proteins 14:509-515)。例如,可特异性引导至肿瘤血管的肽(如通过体内噬菌体选择所识别的肽),在其肽结构中包含Arg-Gly-Asp(RGD)基元,并选择性地结合v3和v5整联蛋白(Arap等,(1998)Science 279:377-380)。
iii)肽的噬菌体展示文库产生了具有清楚的溶液构象的短肽,其可结合,如胰岛素样生长因子结合蛋白-1并产生胰岛素生长因子样活性(Lowman等,(1998)Biochemistry 37:8870-8878。
iv)肽的随机噬菌体展示文库所分离的小肽,其结合并活化细胞受体(如EPO受体),可选地包含全长激动剂肽(如刺激红细胞生成的肽,如Wrighton等所述((1996)Science 273:458-463)或刺激TPO应答细胞增殖的肽(如Cwirla等所述((1997)Science 276:1696-1699))。
“ErbB配体”是指结合于和/或激活ErbB受体的多肽。本文具体的目的ErbB配体是天然序列的人ErbB配体,如表皮生长因子(EGF)(Savage等,J.Biol.Chem.247:7612-7621(1972));转化生长因子α(TGF-a)(Marquardt等,Science 223:1079-1082(1984));双向调节素,又被称为神经鞘瘤或角质化细胞自分泌生长因子(schwanoma or keratinocyte autocrine growth factor)(Shoyab等.Science 243:1074-1076(1989);Kimura等.Nature 348:257-260(1990);和Cook等Mol.Cell.Biol.11:2547-2557(1991));β-细胞素(betacellulin)(Shing等,Science 259:1604-1607(1993);和Sasada等Biochem.Biophys.Res.Commun.190:1173(1993));结合肝素的表皮生长因子(HB-EGF)(Higashiyama等,Science 251:936-939(1991));表调节素(Toyoda等,J.Biol.Chem.270:7495-7500(1995);和Komurasaki等Oncogene 15:2841-2848(1997)),遗传调节蛋白(参见下述);神经调节素-2(NRG-2)(Carraway等,Nature 387:512-516(1997));神经调节素-3(NRG-3)(Zhang等,Proc.Natl.Acad.Sci.94:9562-9567(1997));或cripto(CR-1)(Kannan等J.Biol.Chem.272(6):3330-3335(1997))。结合EGFR的ErbB配体包括EGF,TGF-a,双向调节素,β细胞素,HB-EGF和表调节素。结合ErbB3的ErbB配体包括遗传调节蛋白。可结合ErbB4的ErbB配体包括β细胞素,表调节素,HB-EGF,NRG-2,NRG-3和遗传调节蛋白。
优选的靶向药物包括天然的和工程化的受体配体、肽和肽类配体、抗体(特别是单克隆抗体,包括抗体片段如Fab,Fab′,F(ab′)2,和Fv片段,二价抗体,线性抗体,单链抗体分子,由多个抗体片段形成的多特异性抗体以及其类似物)。优选的靶向药物是抗体。
“化疗药物”是用于治疗癌症的化合物。化疗药物的实例包括关旦辛苷类(Maytansinoids)如美旦辛苷(Maytansinoid)和袢环丝裂霉素(Ansamitocins),以及其合成类似物,Enediyne抗生素包括:加利车霉素,特别是加利车霉素γ1 I和加利车霉素θ1 I(参见Angew,(1994)Chemn.Int.Ed.Engl.,33:183-186),Dynemicins特别是DynemicinA和其合成类似物,以及新制癌菌素(Neocarzinostatin)生色团和相关的生色蛋白enediyne抗生素生色团,Esperamicins(参见美国专利4675187)如Esperamicin A1;羟基柔红霉素(阿霉素)和吗啉基-阿霉素(吗啉基-ADR),氰基吗啉基-阿霉素(氰基吗啉基-ADR),2-吡咯基-阿霉素,也被称为AN-201,脱氧阿霉素,Tichothecenes特别是T-2毒素,Verracurin A,杆苞菌素A和蛇形菌素(Anguidine),Epothilones,Rhizoxin,Acetogenins,特别是Bullatacin和Bullatacinone,Cryptophycins,特别是Cryptophycinl和Cryptophycin 8,Dolastatin,Callystatin,CC-1065以及其合成类似物,特别是阿多来新(Adozelesin),Carzelesin和Bizelesin,Duocarmycins以及其合成类似物,特别是KW-2189和CBI-TMI,Sarcodictyins,Eleutherobin,Spongistatins,薯司他丁(Bryostatins),Pancratistatin,喜树碱(Camptothecin)以及其合成类似物,特别是托泊替堪(Topotecan),表阿霉素,5-氟尿嘧啶,阿糖胞苷(“Ara-C”),环磷酰胺,噻替派,白消安(Busulfan),紫杉烷(Taxoids),如他克唑(TAXOL,Bristol-Myers Squibb Oncology,Princeton,NJ)和泰索帝(Docetaxel)(泰索帝,Rhone-Poulene Rorer,Antony,Rnace),氨甲蝶呤,顺铂,左旋苯丙氨酸氮芥以及其它的相关氮芥,长春花碱,博莱霉素,依托泊苷,异环磷酰胺(Ifosfamide),丝裂霉素如丝裂霉素C,米托蒽醌,长春新碱,长春瑞宾(Vinorelbine),卡铂(Carboplatin),替尼泊苷(Teniposide),道诺霉素(Daunomycin),洋红霉素(Carminomycin),氨基蝶呤,放线菌素D。该定义也包括用于调节或抑制对肿瘤具有激素功能的激素类药物,如他莫昔芬(tamoxifen)和奥那司酮(onapristone)。
“CD20”抗原在早期的前-B细胞发育中表达,可能调节细胞周期的起始和分化所需的细胞活化中的一个步骤。CD20抗原在赘生性B细胞中有高水平的表达,但其也存在于正常B细胞上。识别CD20表面抗原的抗-CD20抗体在临床上被用来靶向并摧毁赘生性B细胞(Maloney等(1994)Blood 84:2457-2466;Press等,(1993)NEJM 329:1219-1224;Kaminski等(1993)NEJM329:459-465;McLaughlin等,(1996)Proc.Am.Soc.Clin.Oncol.15:417)。嵌合的和人源化的抗-CD20抗体介导靶B细胞的补体依赖性裂解(Maloney等,
同上)。单克隆抗体C2B8识别人B细胞限制的分化抗原Bp35(Liu等,(1987)J.Immunol.139:3521;Maloney等,(1994)Blood 84:2457)。”C2B8″在国际申请W094/11026中被定义为抗CD20的单克隆抗体。
“疾病”是将受益于包含本发明caspase偶联物和前体药物的组合物治疗的任何病症。这包括慢性和急性疾病,包括使哺乳动物易患上所讨论疾病的病理状况。本文中需治疗的疾病的非限定实例包括良性和恶性肿瘤;白血病和淋巴样恶性肿瘤;神经元、神经胶质细胞、星形胶质细胞、下丘脑和其它腺体、巨噬细胞、上皮细胞、间质和囊胚腔的疾病;炎性、血管原性和免疫性疾病。
术语“HER2”,“ErbB2”,“c-Erb-B2”可交换使用。除非有其它说明,本文所用术语“ErbB2”,“c-Erb-B2”和“HER2”是指人类蛋白,而“her2”,“erbB2”和“c-erb-B2”是指人类基因。人erbB2基因和ErbB2蛋白在以下文献中有所说明,如Semba等,(1985)PNAS(USA)82:6497-6501和Yamamoto等(1986)Nature 319:230-234(Genebank登录号X03363)。ErbB2包含四个功能域(功能域1-4)。
术语“制剂”,“药物制剂”或“药物”等在本文中与“亲本制剂”或“亲本药物”可互换使用,指在药物学领域具有一定用途的化合物。药物制剂由于在缺乏本发明caspase可裂解的前体药物组分时具有生物活性,如细胞毒性而具有药学活性或“生物活性”。这类分子包括小的生物有机分子,如拟肽、抗体、免疫粘附分子、蛋白、肽、糖蛋白、糖肽、糖脂、多糖、寡糖、核酸、生物有机分子、药物和其代谢物,转录和翻译调控序列等。
“原caspase”是指无活性或只有很小活性酶原的caspase序列,该原caspase内在部分的裂解导致产生具有显著增强活性的“成熟”形式。caspase以酶原形式合成,其活性形式包括一个大亚单位(约17-20kDa)和一个小亚单位(9-12kDa),它们通过蛋白酶解从前体释放。自然界发现的许多蛋白酶是翻译的原酶产物,在没有翻译后加工的情况下,就以这种方式表达。
本文使用的术语“前体药物”用来表示亲本药物的衍生物,其药理上有利的特性(如相对无活性,转运、生物可利用性,药物动力学等)可能得以增强,并需要“生物转化”,即由caspase酶解裂解“前体药物组分”,以释放具有活性的亲本药物。
底物以三字母或单字母(如Pn...P2-P1-P1′-P2′...Pn′)表示。“P1”残基是指底物上易分裂的肽键(即P1和P1′残基之间)之前(即其N-端)的位置,如Schechter和Berger所定义(Schechter,I.and Berger,A.,Biochem.Biophys.Res.Commun.27:157-162(1967))。类似的,术语P1′是指底物上易分裂的肽键之后(即其C-端)的位置。增加的数字是指分离该肽键之前(如P2和P3)和之后(如P2′和P3′)的下一个连续的位置。根据本发明,易分裂的肽键是由本发明caspase所裂解的键。
术语“有效治疗剂量”是指可有效治疗哺乳动物疾病的药物剂量。对于癌症而言,药物的有效治疗剂量可减少癌细胞数;缩小肿瘤体积;抑制(即,一定程度上减缓并优选终止)癌细胞对外周器官的浸润;抑制(即,一定程度上减缓并优选终止)肿瘤转移;在某种程度上抑制肿瘤生长;和/或在某种程度上减轻与该疾病相关的一种或多种症状。在某种程度上该药物可能预防现有的癌细胞的生长和/或将其杀死,它可能能抑制细胞或具有细胞毒性。对于癌症治疗来说,可通过例如评估疾病进展时间(TTP)或测定应答率(RR)来测定其效力。
本文所用术语 “治疗”指治愈性治疗,预防性治疗,和防止性治疗。
本文中术语“哺乳动物”是指属于哺乳动物类的任何哺乳动物,包括人、牛、绵羊、马、狗和猫。本发明的优选实施方案中该哺乳动物是人。
实施本发明的模式
本发明涉及caspase的靶向给予,所述给予用于裂解caspase可裂解的前体药物,还涉及将药物局部地送递的方法,所述方法是通过给予一种靶向目的细胞类型的caspase偶联物并额外给予一种caspase存在下在局部转变为活性形式的前体药物而实现。对于ADEPT方法一般可参考Syrigos和Epenetos(1999)
同上的方法。在具体的实施方案中,本发明涉及前体药物(如那些在癌症治疗中有用的前体药物)的靶向给予,其给予以各种细胞类型(如赘生性细胞)为特征的区域,以及该前体药物通过caspase在该特定细胞类型区域局部向活性形式的转变。本发明提供了包含caspase的新的靶向药物,以及包含caspase可裂解的前体药物组分的新的前体药物。
本发明的caspase组分包括本文中定义的任何caspase。优选人caspase1-10或粒酶B中的任一种。优选的caspase是前凋亡型caspase 2,3,6,7,8,9,10。最优选的caspase是caspase 2,3,7。
caspase具有精确的底物特异性(Xaa-Glu-Xaa-Asp),这与已知的除粒酶B以外的其它蛋白酶不同,因此有可能用于前体药物的活化。前凋亡型caspase作为无活性或具有极小活性的酶原广泛存在,但活性的酶仅局限于进行细胞凋亡的细胞胞内区间。caspase 2,3,7的最佳底物分别是DEHD(SEQ ID NO:8),DEVD(SEQ ID NO:3)和DEVD(SEQ ID NO:3)。这些序列对于粒酶B(其优选底物是IEPD(SEQ ID NO:13))(Thornberry等,(1997)同上)和原炎症性caspase(caspase 1,4,5,11,12,13)(其偏好S4位的大疏水残基(caspase 1 WEHD(SEQ ID NO:6),caspase 4(W/L)EHD,caspase5(W/L)EHD)而言是极难处理的底物。例如发现Ac-EDVD-pna可被重组的商品化caspase 3容易地水解,但粒酶B不对其产生可检测的裂解。
因此,根据本发明,选择一种caspase,将其与特定的靶向分子(即一种定向于或结合目的细胞类型的分子)连接。构建相应的前体药物,使该药物的无活性形式或前体药物形式包含一个可被caspase裂解的组分,如本文所述的肽基前体药物组分。
由于天然的caspase为酶原,因此需要产生组成型活化的caspase。Srinivasula等描述了一种产生组成型活化的caspase的简便方法((1998)J.Biological Chem.273(17):10107-10111)。根据该方法,通过将大小亚单位转换位置产生定名为“反向(reverse)caspase”的caspase,这种位置的转换使该工程化分子模拟了经加工的野生型活性分子所表现的结构。尽管以上提供了产生活性caspase的简便方法,其只作为实例而非限制。
靶向组分
该靶向组分是本文所述的任何结合或定向于目的细胞类型的分子。抗体和肽类分子是优选的靶向分子。
优选实施方案中,该靶向分子是一种抗体。本发明偶联物的抗体组分包括任何特异地结合特定细胞类型的抗体。例如,该抗体可结合肿瘤相关抗原。这种抗体的例子包括但不限于:特异性结合瘤、黑素瘤、淋巴瘤和骨及软组织肉瘤以及其它肿瘤上的抗原的抗体。优选长时间结合于细胞表面或极缓慢内化的抗体。抗体可以是多克隆的,或优选的是单克隆,可以是完整的抗体或其包含活性结合区的片段,如Fab或F(ab’)2,可通过本领域成熟的技术生产。
本发明范围内的抗体包括但不限于:抗-IL-8(St John等,(1993)Chest103:932和国际申请WO95/23865);抗-CD11a(Filcher等,Blood,77:249-256,Steppe等,(1991)Transplant Intl.4:3-7,&Hourmant等,(1994)Transplantation58:377-380);抗-IgE(Presta等,(1993)J.Immunol.151:2623-2632,和国际申请WO95/19181);抗-HER2(Carter等,(1992)Proc.Natl.Acad.Sci.USA89:4285-4289和国际申请92/20798);抗-VEGF(Jin Kim,(1992)GrowthFactors,7:53-64和国际申请96/30046);和抗-CD20(Maloney等,(1994)Blood,84:2457-2466和Liu等,(1987)J.Immunol.139:3521-3526)。同样的,靶向以下肿瘤细胞抗原的抗体或其它分子都可用做本发明合适的靶向药物:Apo2,CD20,CD40,muc-1,前列腺特异性膜抗原(PSMA),前列腺干细胞抗原(PSCA),表皮生长因子受体(EGFR),CD33,CD19,衰退加速因子(DAF),EpCAM,CD52,癌胚抗原(CEA),TAG72抗原,c-MET或前列腺的6跨膜表皮抗原(STEAP)。
本发明的caspase可通过本领域已知的任何方法连接于该靶向分子,以产生本发明的caspase偶联物。例如,该caspase可通过共价键连接于该靶向分子。共价键连接的方式是本领域所熟知的,并且包括如,使用异双功能交联剂,SPDP(N-琥珀酰亚氨基-3-(2-吡啶基二硫基)丙酸酯)或SMCC(琥珀酰亚氨基4-(N-顺丁烯二酰亚氨基甲基)环己烷-1-羧酸酯[参见,如P.E.Thorpe等,“The Preparation And Cytotoxic Properties Of Antibody-ToxinConjugates,”Immunological Rev.,62,pp.119-58(1982);J.M.Lambert等,同上p.12038;G.F.Rowland等,pp.183-84 J.Gallego等,同上pp.737-38.]
可通过如顺丁烯二酰亚氨基-羟基琥珀酰亚胺酯等异双功能交联剂获得更有选择性的连接。所述酯与酶的反应将在该酶上衍生氨基,这种衍生物然后可与,如具有自由巯基基团的抗体Fab片段(或其上附着有巯基基团的更大的片段或完整的免疫球蛋白,这种附着可通过如Traut’s试剂实现)反应。
Arpicco等((1997)Bioconj.Chem.8:327-337)和Dosio等((1998)Bioconj.Chem.9:372-381)描述了优选的二硫键连接。
将酶与靶向分子(如抗体)中远离抗原结合位点的位点连接是有利的。这可通过如上所述连接于被裂解的链内巯基得以实现。另外的方法包括将抗体与酶反应,所述抗体的碳水化合物部分已被氧化,而所述酶至少具有一个自由的氨基基团。这产生一个初始的Schiff碱(亚胺)连键,其优选通过还原仲胺,如通过氢硼化物还原而稳定,以形成最终偶联物。
对于抗体分子及其类似物,可通过本领域已知的重组DNA技术构建偶联物,其包含抗体的至少一个抗原结合区域,该区域与本发明caspase的至少一个活性功能部分连接。基于连键的类型,caspase可通过其N-或C-末端连接于靶向分子的N-或C-末端。例如,编码caspase的核酸分子可操作地连接到编码该靶向分子序列的核酸,可选是通过一个接头区。典型地,该构建体编码一种融合蛋白,该融合蛋白包含一个靶向结构域,如抗体或抗体片段,其中caspase的N-或C-末端连接于该抗体或抗体片段的N-末端。不过也可以将caspase的C-或N-末端与该靶向结构域的N-或C-末端连接。
优选的靶向结构域是抗体和抗体片段。典型的,这种融合形式使编码的融合蛋白将至少保留免疫球蛋白重链恒定区的CH1和铰链区,在某些实施方案中,至少保留CH2和CH3区。融合也可在如恒定区Fc部分的C末端,或紧邻重链CH1的N末端或轻链的相应区域发生。
caspase与免疫球蛋白结构域融合的确切氨基酸位点不是关键;已熟知特定的位点,对其的选择要使生物活性、分泌或结合特性最优。
由于偶联物的大小,通常优选将一个抗体连接于一个酶分子。但是,单个的酶分子上连接多个抗体片段(如Fab或F(ab’)2片段),以增加其与靶抗原的结合亲和力或结合效率是有益的。或者,如果该酶的体积不太大,可将多个该酶分子连接于一个抗体或抗体片段,以增加该偶联物的周转数,并增强诊断或治疗药物在靶点的沉积速率。可使用一种以上的caspase和抗体的偶联物,只要它们可到达靶点并且不会被很快清除。在同样的前提条件下,可使用不同大小的偶联物的混合,或包含积聚体的偶联物。
该靶向分子-caspase偶联物可进一步用放射性同位素或核磁共振成像增强试剂进行标记,或与它们偶联,或进行调整以便与它们偶联,以监测其从哺乳动物循环系统中的清除,并确保在给予前体药物之前,其充分定位于靶位点。替代的,该偶联物可带有一种标签,如放射性标签,荧光标签或类似物,其许可对体液(如血液和尿液)中该偶联物的检测和定量,以便于检测和/或推证其靶向和/或清除。
任何适于体内标记蛋白的传统放射性标记方法通常都适合标记靶向药物/caspase偶联物,经常也适合标记底物-药物偶联物,正如以下所述。这可以通过直接用如I-131,I-121标记,用如Tc-99m或Cu离子等通过传统方式或通过结合放射性金属或顺磁离子的螯合剂使其金属化。这种螯合剂以及其结合抗体的模式是普通技术人员所熟知的,其本身公开于上述Goldenberg专利和Childs等(J.Nuc.Med.,26:293(1985))中。
药物细分
适用于本发明的合适药物包括任何在治疗特定疾病中指明的药物。通过使用一种或多种传统方法,本领域技术人员能容易地确定哪些分子合适特定的用途。例如,细胞毒药物或化疗药物适用于各种癌症的治疗方案,而且可能只有当以前体药物的形式给予时有效,这种前体药物是能在特定位点转变为更具活性的药物的那些。化疗药物的实例包括美旦辛苷类如美旦辛苷和袢环丝裂霉素,以及其合成类似物,Enediyne抗生素包括:加利车霉素,特别是加利车霉素γ1 I和加利车霉素θ1 I(参见Angew,(1994)Chemn.Int.Ed.Engl.,33:183-186),Dynemicins特别是DynemicinA和其合成类似物,以及新制癌菌素生色团和相关的生色蛋白enediyne抗生素生色团,Esperamicins(参见美国专利4675187)如Esperamicin A1;羟基柔红霉素(阿霉素)和吗啉基-阿霉素(吗啉基-ADR),氰基吗啉基-阿霉素(氰基吗啉基-ADR),2-吡咯基-阿霉素,也被称为AN-201,脱氧阿霉素,Tichothecenes特别是T-2毒素,Verracurin A,杆苞菌素A和蛇形菌素,Epothilones,Rhizoxin,Acetogenins,特别是Bullatacin和Bullatacinone,Cryptophycins,特别是Cryptophycinl和Cryptophycin 8,Dolastatin,Callystatin,CC-1065以及其合成类似物,特别是阿多来新,Carzelesin和Bizelesin,Duocarmycins以及其合成类似物,特别是KW-2189和CBI-TMI,Sarcodictyins,Eleutherobin,Spongistatins,薯司他丁,Pancratistatin,喜树碱以及其合成类似物,特别是托泊替堪,表阿霉素,5-氟尿嘧啶,阿糖胞苷(“Ara-C”),环磷酰胺,噻替派,白消安,紫杉烷,如他克唑(TAXOL,Bristol-Myers Squibb Oncology,Princeton,NJ)和泰索帝(泰索帝,Rhone-Poulene Rorer,Antony,Rnace),氨甲蝶呤,顺铂,左旋苯丙氨酸氮芥以及其它的相关氮芥,长春花碱,博莱霉素,依托泊苷,异环磷酰胺,丝裂霉素如丝裂霉素C,米托蒽醌,长春新碱,长春瑞宾,卡铂,替尼泊苷,道诺霉素,洋红霉素,氨基蝶呤,放线菌素D。该定义也包括用于调节或抑制对肿瘤具有激素功能的激素类药物,如他莫昔芬和奥那司酮。
前体药物组分的设计
本发明包括包含caspase可裂解的前体药物组分的新的前体药物。因此,根据本发明,活性药物以前体药物的形式给予,该前体药物需要本发明caspase的作用才能达到最佳活性。通常药物的选择是基于待处理的疾病。caspase可裂解的前体药物组分连接于该药物。连接位点基于药物的不同而不同,但典型的是发挥高水平功能潜力所必需的位点。前体药物组分的连接将产生活性变弱或活性最低的药物。
该前体药物组分通常将至少包含4个氨基酸,并在P1的位置上是Asp。因此,本发明优选具有通式P4-P3-P2-Asp的前体药物组分。对前体药物组分的选择要根据所使用的特定的caspase。已有关于10种已知caspase的特异性的说明。关于合适的前体药物组分的设计和构建,本领域技术人员可参考Thornbery等(1997)
同上的方法。例如,具有通式Asp-Xaa-Xaa-Asp的前体药物组分对于caspase 3,7和2是优选的,Asp-Glu-Val-Asp(SEQ ID NO:3)对于caspase 3和7是优选的,Asp-Glu-His-Asp(SEQ ID NO:4)对于caspase2是优选的。
优选的前体药物具有以下通式:
X-S4-S3-S2-Asp-药物或X-S4-S3-S2-Asp-接头-药物,其中可选择缺失X,或如本文详细描述的,如酰基如乙酰基是可选的接头结构域。
接头结构域
根据本发明,接头结构域是在两个或更多活性结构域之间提供空间桥梁的任何分子集团,如本文以下要详细说明的。根据本发明这一方面,活性结构域,如化疗药物,和一种caspase可裂解的前体药物组分通过如化学偶联而连接。本发明这种杂合分子的接头组分不一定参与该杂合分子的功能,但其有助于这种功能。因此,根据本发明,该接头结构域是提供肽结构域形式的前体药物组分与药物结构域之间空间桥梁的任何分子集团。
接头结构域可以具有多种长度和组成。技术人员应考虑接头分子的长度,其组成,包括血浆稳定性,与caspase活性位点的相容性,自我裂解的能力(Carl,Chakravarty和Katzenellenbogen(1981)J.Medicinal Chem.24(5):479-480);可溶性以及这种经修饰的药物为细胞所摄入的能力。这种接头结构域优选可使该杂合分子的肽结构域与对应的caspase分子相互作用,而基本上没有空间/构象上的限制。因此,接头结构域的长度是基于两个功能域(如杂合分子的肽和药物结构域)的特性。构建合适的接头结构域时应考虑到,优选的接头结构域是当不存在caspase可裂解的前体药物组分时,提供一种与亲本药物不稳定的连键,使得当前体药物被裂解后,可快速地丢失该接头,从而释放游离的活性亲本药物。因此,优选的接头结构域是“自我牺牲型(self-immolative)”。优选接头结构域的描述见于Dubowchik等,(1998)Bioorg.Med.Chem.Letts.8:3341-3346和Dubowchik等,(1998)Bioorg.Med.Chem.Letts.8:347-3352。化学合成
产生本发明化合物的一种方法涉及化学合成。这可通过本领域已知方法实现(参见Kelley,R.F.&Winkler,M.E in Genetic Engineering Principles andMethods,Setlow,J.K.,ed.,Plenum Press,N.Y.,vol.12,pp1-19(1990),Stewart,J.M.Young,J.D.,Solid Phase Peptide Synthesis,Pierce Chemical Co.,Rockford,IL(1984);也参见美国专利4105603;3972859;3842067;和3862925。
本发明的前体药物可使用混合固相肽合成容易地制备(Merrifield,(1964)J.Am.Chem.Soc.,85:2149;Houghten,(1985)Proc.Natl.Acad.Sci.USA,82:5132)一种有机化学或重组合成。固相合成从假定肽的羧基末端,通过在惰性固相支持物上偶联一个被保护的氨基酸起始。这种惰性固相支持物是可用做启始氨基酸C末端的锚的任何大分子。典型的,这种大分子支持物是交联的聚合树脂(如聚酰胺或聚苯乙烯树脂),如Stewart和Young(
同上)的第2和4页图1-1和1-2所示。一个实施方案中,该C末端氨基酸偶联于一种聚苯乙烯树脂,形成一种苄型酯。选择大分子支持物使该肽-锚连接体在肽合成中使封闭氨基酸的α氨基去保护的条件下稳定。如果使用遇碱不稳定的α保护基团,则最好在该肽和固体支持物之间使用遇酸不稳定的连接。例如一种遇酸不稳定酯树脂对于遇碱不稳定的Fmoc-氨基酸肽合成有效,如Stewart和Young(
同上)的第16页所述。替代的,可使用对酸裂解具有不同易变性的肽锚连接和α保护基团。例如,氨甲基树脂,如苯乙酰氨甲基(Pam)树脂在Boc-氨基酸肽合成中有效,如Stewart和Young(
同上)的第11-12页所述。
起始氨基酸与惰性固相支持物偶联之后,起始氨基酸的α氨基保护基团用如三氟乙酸(TFA)的二氯甲烷溶液去除,并用如三乙胺(TEA)中和。在合成中,起始氨基酸的α氨基去保护之后,加入后续α氨基和侧链被保护的氨基酸。然后通过缩合,将剩下的α氨基酸(如需要也包括侧链被保护的氨基酸)以所需顺序依次偶联,以获得连接于固相支持物的中间化合物。替代的,一些氨基酸可能相互偶联以形成所需肽的片段,接着将该肽片段加入到正在延伸的固相肽链上。
两个氨基酸、一个氨基酸与一个肽或两个肽之间的缩合反应可根据通常的缩合方法进行,如氮化物法,混合酸酐法,DCC(N,N′-二环己基碳二亚胺)或DIC(N,N′-二异丙基碳二亚胺)法,活性酯法,对硝基苯酯法,BOP(苯并三唑-1-基-氧基-三[二甲氨基]鏻六氟磷酸盐)法,N-羟基琥珀酸亚氨基酯法等。Woodward试剂K法,HBTU(O-[苯并三唑-1-基]-1,1,3,3-四甲基脲鎓六氟磷酸盐)法,HATU(O-[7-氮杂苯并三唑-1-基]-1,1,3,3-四甲基脲鎓六氟磷酸)法和PyBOP(苯并三唑-1-基-氧基-三吡咯烷鏻六氟磷酸盐)法。
肽的化学合成中,通常用合适保护基团保护的氨基酸的任何具有反应活性的侧链基团。最终当需要的肽链顺序地装配完成后,去除这些保护基团。也通常保护氨基酸或肽段的α氨基基团,而该氨基酸或肽段的C-末端羧基基团与正在延伸的固相肽链的自由N-末端氨基基团反应,接着选择性地移去该α氨基基团,使下一个氨基酸或肽段能加入该固相多肽链。相应地,多肽合成中,通常产生一种中间化合物,其包含以需要的顺序定位于该肽链的各氨基酸残基,其中的残基单元仍带有侧链保护基团。这些保护基团可基本在同时被去除,以便从固相分离后可产生需要的多肽产物。
α和∈氨基侧链可用以下基团保护:苄氧基羰基(缩写为Z)、异烟酰氧基羰基(iNOC)、对氯苄氧基羰基[Z(2Cl)]、对硝基苄氧基羰基[Z(NO2)]、对甲氧基苄氧基羰基[Z(OMe)]、叔丁氧羰基(Boc)、叔戊氧羰基(Aoc)、异冰片氧基羰基、金刚烷基氧羰基、2-(4-联苯)-2-丙氧羰基(Bpoc)、9-芴基甲氧羰基(Fmoc)、甲磺酰基乙氧羰基(Msc)、三氟乙酰基、邻苯甲二酰基、甲酰基、2-硝基苯基亚磺酰基(NPS)、二苯基膦硫代酰基(thioyl)(Ppt)和二甲基膦硫代酰基(Mpt)基团等。
羧基功能基团的保护性基团如苄酯(OBzl),环己基酯(OChx),4-硝基苄酯(ONb),叔丁基酯(Ot-Bu),4-吡啶基甲酯(Opic),烯丙基酯(OAII)等。经常需要对具有除氨基和羧基以外的基团的特定氨基酸(如精氨酸,半胱氨酸和丝氨酸)用合适的保护基团进行保护。例如,精氨酸的胍基可用以下基团保护:硝基、对甲苯磺酰基、苄氧羰基、金刚烷基氧羰基、对甲氧苯磺酰基、4-甲氧-2,6-二甲基苯磺酰基(Nds)、1,3,5-三甲基苯磺酰基(Mts)等。半胱氨酸的巯基可用对-甲氧基苄基,三苯甲基进行保护。
上述多种封闭氨基酸可来自商业来源,如Novabiochem(San Diego,CA),Bachem CA(Torrence,CA)或Peninsula Labs(Belmont,CA)。
Stewart和Young(
同上)提供了关于肽制备方法的详细资料。第14-18页描述了α氨基基团的保护,第18-28页描述了侧链的封闭。第149-151页提供了保护胺、羟基和巯基功能的保护基团的列表。
完成需要的氨基酸序列后,可将该肽从固相支持物上分离,回收并纯化。通过一种可破坏肽-固相连接的试剂将该肽从固相支持物上分离,并且可选的将该肽上封闭的侧链功能基团去保护。一个实施方案中,该肽是通过液态氢氟酸(HF)的酸解从固相上分离,HF也去除任何残留的侧链保护基团。为了避免肽中残基的烷基化(如甲硫氨酸,半胱氨酸和酪氨酸残基的烷基化),优选该酸解反应混合物包含巯基-甲酚和甲酚清除剂。HF裂解后,用醚清洗该树脂,并用乙酸溶液顺序地润洗从固相中提取游离肽。将合并的洗液冻干,并纯化该肽。
重组合成
本发明包含一种组合物,其包含编码本文所述caspase偶联物的分离的核酸,优选是DNA。编码本发明偶联物的DNA可通过本领域已知方法制备。这些方法包括但不限于Engels等((1989)Agnew.Chem.Int.Ed.Engl.,28:716-734,在此为参考目的全文引用)所述的化学合成,如三酯、亚磷酸盐、亚磷酰胺(phosphoramidite)和H-膦酸酯方法。一个实施方案中,宿主细胞中优选表达的密码子被用于设计该编码DNA。替代的,通过使用重组DNA技术,编码该偶联物的DNA可被改变,从而编码一种或多种DNA变体,所述技术如定点诱变(Kunkel等,(1991)Methods Enzymol.204:125-139;Carter,P.,等(1986)Nucl.Acids.Res.13:4331;Zoller,M.J.等,(1982)Nucl.Acids Res.10:6487),盒式诱变(Wells,J.A.等(1985)Gene 34:315),限制性选择诱变(Wells,J.A.等(1986)Philos.Trans,R.Soc.London SerA 317,415)等。
本发明进一步包括表达调控序列和表达载体,该调控序列可操作地连接编码本发明偶联物的DNA分子,该载体(如质粒)包含该DNA分子,其中该调控序列可被转化有该质粒的宿主细胞所识别。通常,质粒载体包含复制和调控序列,所述序列来源于和该宿主细胞相容的物种。该载体通常带有一个复制位点,以及编码序列,其编码在转化细胞中可提供表型选择的蛋白。
表达该DNA的合适宿主细胞包括原核、酵母或更高等的真核细胞。合适的原核细胞包括但不限于,真细菌,如革兰氏阴性或革兰氏阳性生物,如肠杆菌科(如大肠杆菌)。各种大肠杆菌菌株都是公众可获得的,如大肠杆菌K12株MM294(ATCC 31446);大肠杆菌X1776(ATCC 31537);大肠杆菌W3110株(ATCC 27325)和K5 772(ATCC 53635)。
除原核生物外,真核生物如酵母或来源于多细胞生物的细胞都可用做宿主细胞。在酵母宿主细胞(如普通的面包酵母或酿酒酵母)中表达的合适载体包括:基于2μ质粒的附加型复制载体,整合载体和酵母人工染色体(YAC)载体。用于表达的合适宿主细胞也来源于多细胞生物。无脊椎动物细胞实例包括如果蝇S2和草地夜蛾Sf9,以及植物细胞。昆虫宿主细胞(如Sf9细胞)中表达的合适载体包括杆状病毒载体。植物宿主,尤其是双子叶植物宿主(如烟草)细胞中表达的合适载体包括来源于根癌农杆菌Ti质粒的载体。
有用的哺乳动物宿主细胞包括如SV40转化的猴肾CV1细胞系(COS-7,ATCC CRL 1651);人胚肾细胞系(293或293细胞,经亚克隆可在悬浮培养物中生长)(Graham等,(1977)J.Gen Virol.,36:59);幼仓鼠肾细胞(BHK,ATCC CCL 10);中国仓鼠卵巢细胞/-DHFR(CHO,Urlaub and Chasin,(1980)Proc.Natl.Acad.Sci.USA,77:4216);鼠滋养细胞(sertoli cell)(TM4,Mather,(1980)Biol.Repro.,23:243-25 1);猴肾细胞(CV I ATCC CCL 70);非洲绿猴肾细胞(VERO-76,ATCC CRL-1587);人宫颈癌细胞(HELA,ATCCCCL 2);猫肾脏细胞(MDCK,ATCC CCL 34);buffalo大鼠肝细胞(BRL3A,ATCCCRL 1442);人肺细胞(W138,ATCC CCL 75);人肝细胞(Hep G2,HB8065);鼠乳腺瘤(MMT 060562,ATCC CCL51);TRI细胞(Mather等,(1982)Annals N.Y.Acad.Sci.,383:44-68);MRC 5细胞;FS4细胞;和人肝癌细胞系(Hep G2)。
原核宿主中表达的合适载体包括pBR322(ATCC 37017),phGH107(ATCC 40011),pBO475,pS0132,pRIT5,属于pRIT20或pRIT30系列的任何载体(Nilsson和Abrahmsen,(1990)Meth.Enzymol.,185:144-161),pRIT2T,pKK233-2,pDR540和pPL-λ。包含本发明表达载体的原核宿主细胞包括大肠杆菌K12菌株294(ATCC 31446),大肠杆菌菌株JM101(Messing等,(1981)Nucl.Acid Res.,9:309),大肠杆菌菌株B,大肠杆菌菌株1776(ATCC 3 1537),大肠杆菌c600(Appleyard,Genetics,39:440(1954)),大肠杆菌W3110(F-,γ-,原养型,ATCC27325),大肠杆菌菌株27C7(W3110,tonA,phoA E15,(argF-lac)169,ptr3,degP41,ompT,kanr)(美国专利5288931,ATCC 55244),枯草芽孢杆菌,鼠伤寒沙门氏菌,粘质沙雷氏菌(Serratiamarcesans)和假单胞菌属。
哺乳动物宿主细胞中表达的有用载体包括来源于SV40的载体,来源于巨细胞病毒的载体,如pRK载体,包括pRK5和pRK7(Suva等,(1987)Science,237:893-896;EP 307247(3/15/89),EP 278776(8/17/88)),来源于痘苗病毒或其它痘病毒的载体,以及反转录病毒载体,如来源于Moloney鼠白血病病毒(MoMLV)的载体。
可选的,编码目的偶联物的DNA可操作地连接于一段分泌引导序列,其可导致宿主细胞将表达产物分泌到培养基。分泌引导序列实例包括如stII,丝氨酸蛋白酶抑制物(ecotin),lamB,疱疹GD,Ipp,碱性磷酸酶,转化酶和α因子。蛋白A的36个氨基酸的引导序列也适用于本文(Abrahmsen等,(1985)EMBO J.,4:3901)。
宿主细胞被转染,并优选用本发明上述表达或克隆载体转化,并在传统的营养培养基中培养,该培养基适于诱导启动子,筛选转化子或扩增编码所需序列的基因。
转染是指宿主细胞摄入表达载体,无论编码序列是否实际表达。技术人员熟知多种的转染方法,如CaPO4沉淀和电穿孔。当宿主细胞内有任何迹象表明该载体的操作时,则表明成功转染。
转化是指将DNA引入生物以便该DNA可复制,或者作为染色体外元件的形式或者作为染色体的整合体。基于所用宿主细胞的不同,使用适合这些细胞的标准方法进行转化。如Sambrook等所述(Molecular Cloning(2nded.),Cold Spring Harbor Laboratory,NY(1989),第1.82节),利用氯化钙的钙处理法通常被用于原核细胞或其它包含细胞壁实体屏障的细胞。根癌农杆菌感染用于某些植物细胞的转化,如Shaw等((1983)Gene,23:315)和1989年6月29日公布的WO 89/05859所述。对于没有所述细胞壁的哺乳动物细胞,优选Sambrook等第16.30-16.37节(同上)所述的磷酸钙沉淀法。哺乳动物细胞宿主系统转化的一般方面在Axel的1983年8月16日公布的美国专利4399216中有所说明。酵母的转化典型地是根据Van Solingen等((1977)J.Bact.,130:946)和Hsiao等((1979)Proc.Natl.Acad.Sci.(USA),76:3829)所述方法。但也可使用将DNA引入细胞中的其它方法,如核注射、电穿孔、或通过原生质体融合。
治疗方案
本发明的方法通常通过胃肠外注射实施。胃肠外注射的各种类型包括但不限于:腔内(如腹膜内)、静脉内、动脉内、胸膜内、鞘内、肌肉内、淋巴内和局部动脉内、病灶内、皮下、导管灌注等。
对于癌症的成像和/或治疗,静脉内、动脉内、或胸膜内给药通常用于肺癌、乳腺癌和白血病。腹膜内给药对于卵巢肿瘤是有益的。鞘内给药对于脑瘤和白血病有益。皮下给药对于何杰金氏(Hodgkin)病、淋巴瘤和乳腺癌有益。导管灌注用于转移性肺癌、乳腺癌或肝脏的干细胞癌。病灶内给药用于肺癌和乳腺癌。
以上举例说明了本发明的靶向药物-caspase偶联物给药的一般方法。应理解由于偶联物的清除途径和生物学分布通常不同,这两种偶联物(即该caspase偶联物和该前体药物)的给药模式可能不同。例如,抗体-酶偶联物的腹膜内给药可能对于靶向卵巢肿瘤有利,但可能希望的是前体药物偶联物静脉内给药,因为对其的沉积速率能更好地控制以及容易监测清除率。
该靶向药物-caspase偶联物通常以适合于体内给药的无菌载体中的水溶液给予。该靶向药物-caspase偶联物给药的剂量单位优选是约50μg到约5mg,无论其是单次给予或多次给予,尽管一些特殊情况时需要更大或更小的剂量。可能需要降低剂量和/或使用来自不同种属的抗体和/或低过敏原性抗体(如片段或杂合的人或灵长类抗体),以降低患者敏感度,特别是对于治疗以及特别当治疗过程需要重复给药或额外的诊断步骤而言更是如此。
IgG抗体通常约需要2到14天时间才可定位于靶点并在给予前体药物偶联物前从哺乳动物循环系统中基本清除。F(ab’)2抗体片段的相应定位和清除时间是约2到7天,Fab和Fab′抗体片段的是约1到3天。其它抗体可能需要不同的时间段以定位靶点,而且上述时间段可能受偶联的酶的影响。注意标记抗体-酶偶联物以监测其定位和清除。
IgG通常在肝中代谢,其次在消化系统中代谢。F(ab’)2通常主要在肾脏中代谢,但在肝脏和消化系统中也有代谢。Fab和Fab’通常主要在肾脏中代谢,但在肝脏和消化系统中也有代谢。
通常在给予该底物-药物偶联物之前,至少需要0.0001%注射剂量的抗体-酶偶联物定位于靶点。为使偶联物达到更高的靶向效率,该百分比可能更高,而且给予较小的剂量。
其结果是,抗体-酶偶联物的有效剂量使得足以将该偶联物靶向该靶点的抗原,从而结合足量的酶,其使得足量的可溶性底物-药物偶联物转化为产物,导致靶点处累积的药物量足以进行诊断或治疗。
该底物-治疗或诊断药物偶联物通常以PBS的水溶液给予。如果用于人,应为无菌溶液。底物-药物偶联物的给予应该在一定时间之后,该时间足以使抗体-酶偶联物定位于靶点并从该哺乳动物的循环系统中基本清除。
药物组合物
本发明化合物的药物组合物是通过将caspase偶联物或包含具有需要纯度的化合物的前体药物与任选的可药用载体、赋形剂、或稳定剂(Remington′sPharmaceutical Sciences 16th edition,Osol,A.Ed.[1980])混合而制成冻干剂型或水溶液形式的储存物。可用的载体,赋形剂或稳定剂在所使用的剂量和浓度时对受体无毒,包括缓冲液(如磷酸、柠檬酸和其它有机酸);抗氧化剂(包括抗坏血酸和甲硫氨酸);防腐剂(如氯化十八烷基甲基苄基氯化铵;氯化己烷双胺;苯扎氯铵,苯索氯铵;酚,丁基或苄基醇;烷基对羟基苯甲酸酯(如甲基或丙基对羟基苯甲酸酯);儿茶酚;间苯二酚;环己醇;3-戊醇和间-甲酚);低分子量(小于约10个残基)多肽;蛋白(如血清白蛋白,明胶,或免疫球蛋白);亲水聚合物(如聚乙烯吡咯烷酮);氨基酸(如甘氨酸,谷氨酰胺,天冬酰胺,组氨酸或赖氨酸);单糖,二糖和其它碳水化合物(包括葡萄糖,甘露糖或糊精);螯合剂(如EDTA);糖(如蔗糖,甘露糖醇,海藻糖或山梨醇);成盐反离子(如钠);金属复合物(如锌-蛋白复合物);和/或非离子表面活性剂(如TWEENTM,PLURONICSTM或聚乙二醇(PEG))。
以下实施例是为说明而非限制目的给出。本说明中引述的所有文献为参考目的引用。实施例
实施例I Ac-DEVD-阿霉素的制备(图8)
方法:
(i)肽[1](38μmol),1,3-环己基碳二亚胺(40μmol)和N-羟基琥珀酰亚胺(57μmol)的无水DMF溶液(1.5ml)在0℃用乙基二异丙胺(98μmol)处理10分钟。逐滴加入阿霉素氢氯化物(32μmol)和乙基二异丙胺(98μmol)的无水DMF溶液(3.0ml),使该混合物升温至23℃ 72小时并避光。真空浓缩,残余物质通过制备性HPLC纯化,产生一种橘红色不定形固体[2](8.9μmol,28%)。
[HPLC:C-18反向21mm i.d.×250mm柱;流速10ml/min;以60分钟内40-60%(乙腈+0.1%TFA)在(水+0.1%TFA)中的线性梯度洗脱;滞留时间为28分钟。]
(ii)[2](4.7μmol)和四(三苯膦)钯(0.3μmol)的脱气无水DMF溶液(1.5ml)在23℃用乙酸(70μmol)和氢氧化三丁基锡(41μmol)处理,避光搅动。混合物进一步用乙酸(87μmol)和氢氧化三丁基锡(45μmol)处理1.5小时,在第34小时(0.3μmol)和第72小时(0.6μmol)加入四(三苯膦)钯。91小时后真空浓缩并用制备性HPLC纯化残留物,获得一种橘红色不定形固体[3](2.1μmol,44%)。
[HPLC:以60分钟内0-60%的线性梯度;其它条件如前;滞留时间为43分钟。]
实施例II Ac-DEVD-PABC前体药物组分的制备(图9)
方法:
(iii)肽[1](88μmol),4-氨基苄醇(179μmol)和2-乙氧基-1-乙氧羰基-1,2-二氢喹啉(178μmol)的无水DMF溶液(1.0ml)在23℃反应24小时。真空浓缩并用制备性HPLC纯化残余物质,获得一种白色不定形固体[4](63μmol,72%)。
[HPLC:以60分钟内0-60%的线性梯度洗脱,其它条件如前;滞留时间48分钟。]
(iv)肽[4](181μmol)和4-硝基氯甲酸酯(216μmol)的无水二氯代甲烷溶液(6.0ml)在23℃加入2,6-二甲基吡啶(541μmol)。2小时后用无水DMF(2.0ml)稀释该混合物,并进一步用2,6-二甲基吡啶(360μmol)处理。在24小时,27小时和46小时再加入2,6-二甲基吡啶(860μmol)和4-硝基氯甲酸酯(175μmol)。84小时后将混合物用碳酸氢钠饱和水溶液处理,并用乙酸乙酯(总体积为150ml)抽提三次。将有机相合并,用柠檬酸水溶液(80ml,0.5M),碳酸氢钠饱和水溶液和盐水洗,用无水硫酸钠干燥并真空浓缩。通过制备性HPLC纯化残余物质,产生一种白色不定形固体[5](131μmol,72%)。
[HPLC:以15分钟内0-40%,45分钟内40-60%进行洗脱,其它条件如前;滞留时间为46分钟。]
实施例III Ac-DEVD-PABC-阿霉素的制备(图10)
方法:
(v)碳酸盐[5](74μmol)和阿霉素氢氯化物(86μmol)的无水DMF溶液(10ml)在23℃逐滴加入乙基二异丙胺(402μmol),避光搅动16小时。真空浓缩,残余物质用制备性HPLC纯化,获得一种橘红色不定形固体[6](45μmol,61%)。
[HPLC:以15分钟内0-40%,45分钟内40-60%洗脱,其它条件如前;滞留时间为45分钟。]
(vi)[6](12μmol)和四(三苯膦)钯(O)(1.5μmol)的脱气无水DMF溶液(2.0ml)在23℃加入乙酸(245μmol)和氢氧化三丁基锡(123μmol)。避光搅动该混合物16小时,然后真空浓缩。残余物质通过制备性HPLC纯化,获得一种深橘红色不定形固体[7](5.7μmol,47%)。
[HPLC:以60分钟内0-50%的线性梯度洗脱,其它条件如前;滞留时间为52分钟;使用30%等度洗脱进一步纯化,滞留时间为35分钟。]
实施例IV Ac-DEVD-PABC-紫杉醇的制备(图10)
方法:
(vii)碳酸盐[5](57μmol),紫杉醇(58μmol)和4-二甲基氨基吡啶(176μmol)的无水乙腈溶液(10ml)在23℃反应20小时。真空浓缩,残余物质用制备性HPLC纯化,产生一种白色不定形固体[8](47μmol,83%)。
[HPLC:以15分钟内0-50%,45分钟内50-70%洗脱,其它条件如前;滞留时间为38分钟]
(viii)在23℃向[8](46μmol)和四(三苯膦)钯(0)(5.1μmol)的脱气无水DMF溶液(6.0ml)中添加乙酸(926μmol)和氢氧化三丁基锡(457μmol)。混合物避光搅动18小时,然后真空浓缩。残余物质通过制备性HPLC纯化,产生一种深橘红色不定形固体[9](31μmol,68%)。
[HPLC:以15分钟内0-40%洗脱,45分钟内40-60%进行洗脱,其它条件如前;滞留时间34分钟。]
缩写:
Ac=乙酰基
A11=烯丙基(2-丙烯-1-基)
Bu=正-丁基
DCC=1,3-二环己基碳二亚胺
DCM=二氯代甲烷
DIPEA=二异丙基乙胺
DMAP=4-(二甲基氨基)吡啶
DMF=N,N-二甲基甲酰胺
EEDQ=2-乙氧基-1-乙氧羰基-1,2-二氢喹啉
HPLC=高效液相色谱
Me=甲基
Ph=苯基
Su=N-琥珀酰亚氨基
TFA=三氟乙酸
实施例V 阿霉素和Ac-DEVD-PABC-阿霉素的细胞内累积
SK-BR-3和MCF7乳腺癌细胞(美国典型培养物保藏中心(ATCC),Rockville,MD)在37℃,5%CO2下培养于Dulbecco′s修饰的Eagle′s培养基中:Ham′s营养培养基F-12(50∶50),其中添加了2μM谷氨酰胺,100单位/mL青霉素,100μg/mL链霉素(Gibco BRL,Grand Island,NY),和10%(w/v)胎牛血清(Hyclone,Logan,UT)(细胞培养基)。用包含0.05%胰蛋白酶,0.6mM EDTA的磷酸缓冲盐溶液处理(5分钟)使贴壁生长的细胞脱壁,以106细胞/mL浓度用新鲜细胞培养基重悬。细胞可直接使用(″未处理的″)或添加阿霉素或Ac-DEVD-PABC-阿霉素(其终浓度为10μM)。细胞在37℃孵育0-2小时,然后离心(5分钟,500g,4℃)沉淀。弃上清,将细胞温和重悬于10ml冰冷的磷酸缓冲盐溶液中。重复细胞的沉淀和重悬步骤。在先前未处理的细胞中加入阿霉素(12.5nmol,1.25nmol或0.125nmol),以制备标准曲线。细胞沉淀溶于200μL 0.3M HCl的50%(v/v)乙醇溶液,并转移到1.5mL Eppendorf管中。微离心以沉淀碎片(5min,14000rpm,25℃)。将150μL的上清转移到96孔微滴定平板的一个孔中。用荧光平板读取仪(Fluoroskan,Helsinki,Finland)检测荧光,吸收和发射波长分别为485和590nm。根据标准曲线估计阿霉素的摄入量,该标准曲线利用加入先前未处理细胞中的阿霉素浓度而制备。发现阿霉素在MCF7和SKBR3细胞中有显著积累,而Ac-DEVD-PABC-阿霉素则不积累(图1)。
实施例VI 前体药物的细胞毒性分析1
SK-BR-3和MCF7乳腺癌细胞(ATCC)于Dulbecco′s修饰的Eagle′s培养基:Ham′s营养培养基F-12(50∶50),其中添加有2mM谷氨酰胺,100单位/mL青霉素,100μg/mL链霉素(Gibco BRL),和10%(w/v)胎牛血清(Hyclone)(细胞培养液)中,37℃,5%CO2培养。细胞以10000细胞/孔(SK-BR-3),或3000细胞/孔(MCF7)接种于96孔组织培养板(Falcon,Becton-Dickinson,Franklin Lakes,NJ)使其附着24小时。吸去细胞培养基并换为包含2mM PIPES,1mM DTT,0.1mM EDTA,0.01%CHAPS,10mM NaCl和1%蔗糖的新鲜细胞培养基(100μL/孔),期间存在0到10-4μM的Ac-DEVD-PABC-阿霉素或阿霉素,并存在或不存在13ng重组的人caspase 3(Calbiochem,San Diego,CA)。孵育3小时后,用细胞培养基洗平板两次(37℃),进一步孵育使总实验时间达到120小时。用0.25%(w/v)结晶紫的50%(v/v)乙醇溶液染色以终止反应。然后用水润洗平板,用50mM柠檬酸钠的50%(v/v)乙醇溶液(pH4.5)溶解残余的结晶紫。使用微滴板读取仪(SpectraMax 340,Molecular Devices,Sunnyvale,CA)读取540nm的吸收值。
实施例VII Ac-DEVD-PABC-阿霉素被caspase 3活化
Ac-DEVD-PABC-阿霉素(60μM)与1ng重组的人caspase 3(Calbiochem)在存在或不存在caspase 3的酶抑制剂,Z-DEVD-FMK(400μM)(Calbiochem)的含5%(v/v)二甲亚砜和45mM DTT的磷酸缓冲盐溶液中孵育,总反应体积为700mL。进行一个对照反应,其中不包含caspase 3和其抑制剂。37℃下反应进行0-2小时,然后用干冰冷冻。用Microsorb-MV C 18反向柱(4.6mm内径×250mm长度,5μm粒径,100孔径)(Rainin,Emeryville,CA)对该反应在等度洗脱条件下进行反向HPLC分析:0.1%(v/v)TFA酸,35%(v/v)乙腈,流速为1.5mL/min,监测254nm的吸收值。Ac-DEVD-PABC-阿霉素和阿霉素的滞留时间分别为7.7分钟和5.1分钟。发现AC-DEVD-PABC-阿霉素对于MCF7和SK-BR-3细胞的毒性比阿霉素的毒性低100倍还多。用caspase 3处理后,Ac-DEVD-PABC-阿霉素与阿霉素的毒性相同(图2)。Ac-DEVD-PABC-阿霉素可被caspase 3有效激活,这表现为其转变为阿霉素(表II)。
实施例VIII Ac-DEVD-PABC-他克唑被caspase 3活化
Ac-DEVD-PABC-他克唑(35μM)与1ng的重组人caspase 3(Calbiochem)在存在或不存在caspase 3的抑制剂Z-DEVD-FMK(400μM)(Calbiochem)的含5%(v/v)二甲亚砜和45mM DTT的磷酸缓冲盐溶液中孵育,总反应体积为700μL。进行一个对照反应,其中不包含caspase 3和其抑制剂。37℃将反应溶液孵育0-2小时,然后干冰冷冻。用Microsorb-MVC18反向柱(4.6mm内径×250mm长,5μm粒径,100孔径)(Rainin)对反应物在等度洗脱条件下进行反向HPLC分析:0.1%(v/v)TFA,46%(v/v)乙腈,流率1.5mL/min,监测254nm的吸收值。他克唑和Ac-DEVD-PABC-他克唑的滞留时间分别是13.3分钟和10.4分钟。AcDEVD-PABC-他克唑被caspase 3有效活化,表现为其转化为他克唑(表III)。
实施例IX 前体药物的细胞毒性分析2
人肺癌细胞(H460,SK-MES-1),结肠癌细胞(HCT116),乳腺癌细胞系(BT-474,MCF7,SK-BR-3)和正常的肺成纤维细胞(WI-38)购自ATCC并维持在高葡萄糖DMEM中:Ham′s F-12(50∶50),添加有10%(v/v)热灭活的FBS(Gibco,BRL)和2mM L-谷氨酰胺。正常的人乳腺上皮细胞(HMEC)购自Clonetics/Biowhittaker(Walkersville,MD)并维持在乳腺上皮细胞培养基(MEGM,Clonetics)中。用包含0.05%(w/v)胰蛋白酶和0.6mM EDTA(5min)的磷酸缓冲盐溶液处理使细胞与培养瓶分离,并以104细胞/孔(WI-38和HMEC),1.5×104细胞/孔(H460,SK-MES-1和HCT116),或2×104细胞/孔(MCF7,BT-474,SK-BR-3)的密度将细胞接种于96-孔微滴定平板。使其过夜附着,加入药物或前体药物,各自终浓度为:阿霉素或Ac-DEVD-PABC-阿霉素,0到1μM;他克唑或Ac-DEVD-PABC-他克唑,0到0.04μM。处理72小时后,从孔中轻缓地移去培养液,用0.5%(w/v)结晶紫的20%(v/v)甲醇溶液对细胞单层染色。平板用水冲洗,并使其干燥。用50mM柠檬酸钠缓冲液的50%(v/v)乙醇溶液(pH4.2)(200μL/每孔)溶解染料,平板在25℃摇动30分钟,使用340ATC微滴定平板读取仪(SLT LabInstruments,Salzburg,Austria)读取540nm的光吸收。
实施例X caspase 3的血浆稳定性
离心新鲜的用肝素处理的血液,以沉淀细胞和血小板(5min,1500g,4℃)。在微型离心管中重新对上清(血浆)离心(5min,14000rpm,25℃)。将重组的caspase 3(500μg)加入200μL血浆或200μL磷酸缓冲盐,37℃孵育0-24小时后取等份试样,在液氮中迅速冷冻并储藏于-70℃。将血浆样品解冻,用包含75μg/mL生色底物(乙酰基-L-Asp-L-Glu-L-Val-L-Asp-对-硝基苯胺)(Galbiochem)的caspase缓冲液(20mM PIPES,10mM DTT,1mM EDTA,0.1%CHAPS,10%蔗糖,100mM NaCI,pH7.2)将其稀释5-25倍。通过25℃时使用微滴定平板读取仪(SpectraMax 340,Molecular Devices)跟踪410nm的吸收值,从而监测底物的水解。
实施例XI
编码抗体片段与反向caspase 3融合蛋白的质粒的构建
1)
质粒说明
质粒pLCrC3编码HuMab4D5-8 Fab的轻链(Carter等,1992a,
同上;Carter等,1992b,Bio/Technology 10:163-167),该轻链与编码被称为反向caspase 3的组成型活化形式caspase 3的基因(Srinivasula等,1998同上)通过一个编码(Gly4Ser)3的接头实现融合(示于图7)。
质粒pHCrC3编码HuMab4D5-8 Fab的重链Fd片段(Carter等,1992a,b
同上),该片段与编码反向caspase 3的基因(Srinivasula等,1998
同上)通过一个编码(Gly4Ser)3的接头实现融合(示于图7)。
质粒pLCrC3.HCrC3包含编码HuMab4D5-8 Fab的轻链和重链Fd片段的基因(Carter等,1992a,b
同上),这两个基因分别通过一个编码(Gly4Ser)3的接头与一个编码组成活化形式caspase 3(被称为反向caspase 3)的基因(Srinivasula等,1998
同上)融合。编码HuMAb4D5-8 Fab-反向caspase 3的pLCrC3.HCrC3中的双顺反子型(biscistronic)操纵子图示于图7中,图6中标注了DNA和蛋白序列。该操纵子处于phoA启动子的转录控制之下(C.W.Chang等(1986)Gene 44:121-125),可由缺磷酸盐诱导。huMAb4D5-8的人源化可变区(VL和VH)在其5′末端精确地融合了编码热稳定性肠毒素II(stII)的信号序列(RN Picken等(1983)Infect.Immun.42:269-275),以指导该多肽分泌到大肠杆菌的胞质外周区。反向caspase 3的每个拷贝后均有一段编码8个组氨酸的序列,以便于通过固定化金属亲和层析将产生的融合蛋白纯化。
质粒pLCrC3.HCrC3s与pLCrC3.HCrC3的差异在于,huMAb4D5-8轻链和重链Fd片段分别在其第214和223位密码子编码丝氨酸残基而非半胱氨酸残基。
质粒pET21b.rC3包含一个在载体pET21b(Novagen,Madison,WI)中编码反向caspase 3的基因(Srinivasalu等,(1998)
同上)。
质粒pLCrC3的构建
质粒pLCrC3通过重组PCR装配(Rashtenian(1995)Curr.Opin.Biotech.6:30-36),从编码HuMab4D5-8 Fab′片段的质粒pAK19(Carter等(1992a,b)同上)和编码pET21b(Novagen,Madison,WI)中反向caspase 3的质粒pET21b.rC3起始。编码HuMab4D5-8轻链的基因利用以下引物,从质粒pAK19中进行第一步PCR扩增:
P1:5′GCTACAAACGCGIACGCTGAIATCCAGATGACCCAGTCCCCGAGCTCCCTG 3′(SEQ ID NO:14)
P2:5′CCCCCACCTCCGCTACCTCCCCCGCCACACTCTCCCCTGTTGAAGCTCTTTGTGACG 3′(SEQ ID NO:15)
类似的,编码反向caspase 3的基因利用以下引物从pET21b.rC3中用PCR扩增:
P3:5 ′CGGGGGAGGTAGCGGAGGTGGGGGCTCTGGTGGAGGCGGTTCAAGTGGTGTTGATG 3′(SEQ ID NO:16)
P4:5′GCCGTCGCATGCTTAGTGATGGTGATGGTGATGGTGATGTGTCTCAATGCCACAGTC 3′(SEQ ID NO:17)。
PCR反应条件(″PCR1条件″)如下:50-100ng DNA模板溶于20mMTris-HCI(pH8.8),10mM KCI,10mM(NH4)2SO4,2mM MgSO4,0.1%Triton X-100,0.1mg/mL牛血清白蛋白(BSA)中,四种dNTP各200μM,每种引物25pmol,2.5 U Pfu Turbo(Stratagene,La Jolla,CA),总体积为50μL。热循环条件(″热循环1条件″)如下:95℃ 5分钟,接着以95℃ 20秒,55C20秒,72℃ 90秒进行30个循环,最后72℃ 10分钟。
在1%琼脂糖凝胶(Gibco BRL)中对这些PCR产物进行凝胶纯化。切除具有合适分子量(分别为约690bp和约840bp)的条带,并使用QIAquick Gel抽提试剂盒(Qiagen,Valencia,CA)抽提DNA。接着将这两种DNA片段以1∶1的比率混合,用P1和P4引物,在PCR1条件下进行第二轮的PCR,热循环条件(“热循环2条件”)为:95℃ 5分钟,然后以95℃ 20秒,50℃ 20秒,72℃90秒进行30个循环,最后72℃ 10分钟。利用MluI和SphI位点将该PCR产物克隆于pAK19中,得到pLCrC3,然后通过双脱氧核苷酸测序证实。
质粒pHCrC3的构建
质粒pHCrC3通过重组PCR装配(A.Rashtchian(1995)
同上),由编码HuMab4D5-8 Fab′片段的质粒pAK19和质粒pET21b.rC3起始。编码HuMab4D5-8重链的基因用以下引物从质粒pAK19中进行第一次PCR扩增:
P5 5′TGCTACAAACGCGTACGCTGAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTG3′(SEQ ID NO:18)
P6 5′CCCCACCTCCGCTACCTCCCCCGCCTGTGTGAGTTTTGTCACAAGATTTGGGC 3′(SEQ ID NO:19)
类似的,编码反向caspase 3的基因也利用以下引物从pET21b.rC3中PCR扩增:
P3:5′CGGGGGAGGTAGCGGAGGTGGGGGCTCTGGTGGAGGCGGTTCAAGTGGTGTTGATG 3′(SEQ ID NO:16),
P4:5′GCCGTCGCATGCTTAGTGATGGTGATGGTGATGGTGATGTGTCTCAATGCCACAGTC 3′(SEQ ID NO:17)
PCR1条件和热循环1条件
在1%琼脂糖凝胶(Gibco BRL)中对这些PCR产物进行凝胶纯化。切除具有合适分子量(分别是约730bp和约840bp)的条带,利用QIAquick Gel抽提试剂盒(Qiagen)抽提DNA。接着将这两种DNA以1∶1的比率混合,使用引物P5和P4进行第二轮PCR,条件为PCR1条件和热循环2条件。利用MluI和SphI位点将PCR产物克隆进pAK19,获得pHCrC3,然后用双脱氧核苷酸测序证实。
质粒pLCrC3.HCrC3的构建
质粒pLCrC3.HCrC3通过连接以下三种DNA片段构建:来自pAK19的约4914bp MluI/SphI片段,来自pLCrC3的约1489bp MluI/AflII PCR片段以及来自pHCrC3的约1623bp AflII/SphI PCR片段。
来自pLCrC3的MluI/AflII片段是利用引物P7(5′GCTACAAACGCGTACGCTGATATCCAGATGACCCAGTCCCCGAGCTCCCTG 3′(SEQ IDNO:14))和P1在PCR1条件和热循环1条件下进行PCR扩增,然后用MluI和AflII消化获得。
类似的,来自pHCrC3的AflII/SphI片段是通过使用引物P4和P8(5′TAAGCGGCCTTAAGGCTAAGGGATCCTCTAGAGGTTGAGGTGATTTTATG 3′(SEQ ID NO:20)),在PCR1条件和热循环1条件下进行PCR扩增,然后用AflII和SphI消化获得。质粒pLCrC3.HCrC3通过双脱氧核苷酸测序证实。
质粒pLCrC3.HCrC3s的构建
质粒pLCrC3.HCrC3s来自pLCrC3.HCrC3,是由于huMAb4D5-8轻链和重链Fd片段上分别在第214和223位的密码子的突变,从而其编码丝氨酸残基而非半胱氨酸残基。使用QuikChange定点诱变试剂盒(Stratagene)可实现对轻链和重链的顺序诱变。编码C214S的轻链突变是通过使用两个合成的DNA片段:P9 5′CTTCAACAGGGGAGAGTCTGGCGGG 3′(SEQ IDNO:21)和P10 5′CCCGCCAGACTCTCCCCTGTTGAAG 3′(SEQ ID NO:22)实现,而编码C223S的重链突变是通过使用两个合成DNA片段P115′GCCCAAATCTTCTGACAAAACTCAC 3′(SEQ ID NO:23),和P125′GTGAGTTTTGTCAGAAGATTTGGGC 3′(SEQ ID NO:24)实现的。
实施例XII huMAb4D5-8 Fab-反向caspase 3融合蛋白的摇瓶表达
质粒pLCrC3.HCrC3和pLCrC3.HCrC3s转化入大肠杆菌(E.Coli)菌株25F2(Carter等,(1992b)
同上),并于包含50μg/mL羧苄青霉素的5mLLuria-Bertani(LB)培养液中,37℃过夜摇动培养。过夜培养物取1毫升接种250mL包含50μg/mL羧苄青霉素的完全CRAP培养基,30℃摇动培养过夜。(完全CRAP培养基的制备如下:3.57g(NH4)2SO4,0.71g柠檬酸钠-2H2O,1.07g KCl,5.36g酵母提取物,5.36g Hycase SF-Sheffield,用KOH将pH调整到7.3,用去离子水将体积调整到872mL。高压灭菌并冷却到55℃。加入110mL 1M MOPS pH7.3,11mL 50%葡萄糖,7.0mL 1M MgSO4)。离心沉淀细胞(3000g,15min,4℃),重悬于25mL 10mM Tris-HCI pH 7.6,1mMEDTA。样品在4℃轻柔搅动30分钟,然后离心(27000g,20min,4℃)。上清(″schockates″)调整到100mM磷酸钠(pH8.0),300mM NaCl,20mM咪唑,10mM MgCl2和10mM 3-巯基乙醇。使用Ni-NTA superflow agarose(Qiagen)经固定化金属亲和层析(IMAC)纯化融合蛋白。用包含300mM NaCl,250mM咪唑和10mM β-巯基乙醇的1mL 100mM磷酸钠(pH8.0)洗脱结合的蛋白。″Shockates″和IMAC纯化的样品利用定量的抗-HER2 Fab ELISA,抗多聚组氨酸ELISA分析,并使用生色底物乙酰-L-Asp-L-Glu-L-Val-L-Asp-对-硝基苯胺分析反向caspase 3的活性。
实施例XIII 定量的抗-HER2 Fab ELISA
96-孔ELISA平板(Maxisorp,Nunc)用1μg/mL HER2胞外结构域的Na2CO3溶液(pH9.6)以每孔100μL包被(16小时,4℃)。用平板清洗仪(Skanwasher 300,Skatron Instruments)使用PBST(磷酸缓冲盐,含0.05%Tween 20)洗平板,用包含3%脱脂乳(Carnation)的280μL PBST (PBST-SM)封闭平板(1小时,25℃)。平板再用PBST清洗两次,然后与系列稀释的样品和标准物在PBST-SM中孵育(1小时,25℃)。使用的标准物是huMAb4D5-8Fab(Carter等(1992a,b)
同上),(R.F.Kelley等.(1992)Biochemistry 31:5434-5441),在1-400ng/mL范围内的两倍系列稀释。平板用PBST润洗,然后与抗人κ轻链-辣根过氧化物酶偶联物(Catalog#55233,ICNPharmaceuticals,Aurora,Ohio)一起保温:每孔100μL,为偶联物在PBST-SM中的1∶5000稀释液。润洗平板,然后与每孔100μL新鲜混合的TMB底物(Kirkegaard and Perry Laboratories,Gaithersburg,MD)孵育(2-15min,25℃)。每孔加入100μL 1M磷酸淬灭反应。利用微滴定平板读取仪(SpectraMax 340,Molecular Devices)检测,将450nm的光吸收值减去650nm的光吸收值。数据根据背景校正,然后进行非线性开方(Kaleidagraph version3.0.5,Synergy Software,Reading,PA):A450-A650=(c*A)/(c+B),其中c是标准物的浓度,A和B是常量。计算出的适合度(fit)用来估计样品中huMAb4D5-8 Fab-反向caspase 3融合蛋白的浓度。
实施例XIV 抗多聚组氨酸ELISA
96-孔ELISA平板(Maxisorp,Nunc)用1μg/mL HER2细胞外结构域的Na2CO3溶液(pH9.6)以每孔100μL包被(16小时,4℃)。用平板清洗仪(Skanwasher 300,Skatron Instruments)使用PBST(磷酸盐缓冲液,含0.05%Tween 20)清洗平板,然后使用包含3%脱脂乳(Carnation)的280μL PBST(PBST-SM)封闭平板(1小时,25℃)。该平板用PBST润洗两次,然后与系列稀释的样品和阳性对照在ELISA缓冲液(包含0.5%(w/v)牛血清白蛋白和0.01%硫柳汞的磷酸缓冲盐溶液)中孵育(1小时,25℃)。使用的阳性对照是具有His6标签的huMAb4D5-8(Carter等(1992a)
同上)的scFv片段,在1-400ng/ml的范围的2倍系列稀释。平板用PBST润洗,然后与生物素标记的五-His抗体(Qiagen)孵育:每孔加入100μL在ELISA分析缓冲液中以1∶5000稀释的抗体(1小时,25℃)。润洗平板,然后与链亲和素-辣根过氧化物酶偶联物孵育:每孔加入100μL在ELISA分析缓冲液中以1∶5000稀释的偶联物(1小时,25℃)。润洗平板,然后与每孔100μL的新鲜混合的TMB底物(Kirkegaard和Perry Laboratories)孵育(2-15min,25℃)。每孔加入100μL 1M磷酸终止反应。利用微滴定平板读取仪(SpectraMax 340,MolecularDevices)测定,将450nm的吸收值减去650nm的吸收值。
实施例XV 反向caspase 3的活性分析
用caspase缓冲液(20mM PIPES,10mM DTT,ImM EDTA,0.1%CHAPS,10%蔗糖,100mM NaCl,pH7.2)在96-孔ELISA平板中以2倍系列稀释样品和独立重组的人caspase 3(Calbiochem)。所用的caspase 3标准物的最高浓度是每孔125ng。终体积是250μl包含250μM生色底物,乙酰-L-Asp-L-Glu-L-Val-L-Asp-对-硝基苯胺(Calbiochem)的caspase缓冲液。利用微滴定平板读取仪(SpectraMax 340,Molecular Devices)测定405nm的吸收值,每30秒测定一次,共测定30分钟。
Ac-DEVD-PABC-阿霉素的裂解分析
表II
峰面积未被标准化。Ac-DEVD-PABC-他克唑的裂解分析
样品 | 添加物 | 孵育时间(分钟) | 转化率(%) |
Ac-DEVD-PABC-阿霉素 | caspase 3 | 0 | 1.8 |
Ac-DEVD-PABC-阿霉素 | caspase 3 | 30 | 17 |
Ac-DEVD-PABC-阿霉素 | caspase 3 | 120 | 76 |
Ac-DEVD-PABC-阿霉素 | caspase 3+抑制剂 | 0 | 0 |
Ac-DEVD-PABC-阿霉素 | caspase 3+抑制剂 | 30 | 0 |
Ac-DEVD-PABC-阿霉素 | caspase 3+抑制剂 | 120 | 0 |
表III
样品 | 添加物 | 孵育时间(分钟) | 转化率(%) |
Ac-DEVD-PABC-他克唑 | caspase 3 | 0 | 0 |
Ac-DEVD-PABC-他克唑 | caspase 3 | 30 | 65 |
Ac-DEVD-PABC-他克唑 | caspase 3 | 120 | 100 |
Ac-DEVD-PABC-他克唑 | caspase 3+抑制剂 | 0 | 0 |
Ac-DEVD-PABC-他克唑 | caspase 3+抑制剂 | 30 | 0 |
Ac-DEVD-PABC-他克唑 | caspase 3+抑制剂 | 120 | 0 |
峰面积未被标准化。
huMAb4D-8 Fab-反向caspase融合蛋白的鉴定
pLCrC3.HCrC3和pLCrC3.HCrC3s在大肠杆菌25F2中复制后,通过对相应shockate的定量抗-HER2 Fab ELISA,估计huMAb4D-8 Fab-反向caspase3融合蛋白的表达效价为约200ng/mL和约0.6ng/mL。在两种情况时都通过定量抗-多聚组氨酸ELISA确认了Fab和反向caspase存在于同一分子中。这两项ELISA分析也确认了Fab片段能结合HER2。反向caspase 3的功能通过其水解生色底物乙酰-L-Asp-L-Glu-L-Val-L-Asp-对-硝基苯胺的能力来证实。
1.Carter,P.,et al.,High level Escherichia coli expression and productionof a bivalent humanized antibody fragment.Bio/Technology,1992.10(2):p.163-7.
2.Srinivasula,S.M.,et al.,Generation of constitutively active recombinantcaspases-3 and-6 by rearrangement of their subunits.Journal of BiologicalChemistry,1998.273(17):p.10107-11.
3.Carter,P.,et al.,Humanization of an anti-p 185HER2 antibody for humancancer therapy.Proceedings of the National Academy of Sciences of the UnitedStates of America,1992.89(10):p.4285-9.
4.Kelley,R.F.,et al.,Antigen binding thermodynamics andantiproliferative effects of chimeric and humanized anti-p!85HER2 antibodyFab fragments.Biochemistry,1992.31(24):p.5434-41.antibody Fab fragments.Biochemistry,1992.31(24):p.5434-41.
序列表
序列表<110>杰南技术公司(GENENTECH,INC.)
保罗.J.卡特(Carter,Paul J.)
刘易斯.加扎德(Gazzard,Lewis)<120>天冬氨酸特异性半胱氨酸蛋白酶活化的前体药物治疗<130>P1814R1PCT<141>2001-02-22<150>US 60/184,779<151>2000-02-24<160>25<210>1<211>3614<212>DNA<213>人(Homo sapiens)<400>1gaattcaact tctccatact ttggataagg aaatacagac atgaaaaatc 50tcattgctga gttgttattt aagcttgccc aaaaagaaga agagtcgaat 100gaactgtgtg cgcaggtaga agctttggag attatcgtca ctgcaatgct 150tcgcaatatg gcgcaaaatg accaacagcg gttgattgat caggtagagg 200gggcgctgta cgaggtaaag cccgatgcca gcattcctga cgacgatacg 250gagctgctgc gcgattacgt aaagaagtta ttgaagcatc ctcgtcagta 300aaaagttaat cttttcaaca gctgtcataa agttgtcacg gccgagactt 350atagtcgctt tgtttttatt ttttaatgta tttgtaacta gaattcgagc 400tcggtacccg gggatcctct agaggttgag gtgattttat gaaaaagaat 450atcgcatttc ttcttgcatc tatgttcgtt ttttctattg ctacaaacgc 500gtacgctgat atccagatga cccagtcccc gagctccctg tccgcctctg 550tgggcgatag ggtcaccatc acctgccgtg ccagtcagga tgtgaatact 600gctgtagcct ggtatcaaca gaaaccagga aaagctccga aactactgat 650ttactcggca tccttcctct actctggagt cccttctcgc ttctctggat 700ccagatctgg gacggatttc actctgacca tcagcagtct gcagccggaa 750gacttcgcaa cttattactg tcagcaacat tatactactc ctcccacgtt 800cggacagggt accaaggtgg agatcaaacg aactgtggct gcaccatctg 850tcttcatctt cccgccatct gatgagcagt tgaaatctgg aactgcctct 900gttgtgtgcc tgctgaataa cttctatccc agagaggcca aagtacagtg 950gaaggtggat aacgccctcc aatcgggtaa ctcccaggag agtgtcacag 1000agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 1050agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca 1100tcagggcctg agctcgcccg tcacaaagag cttcaacagg ggagagtgtg 1150gcgggggagg tagcggaggt gggggctctg gtggaggcgg ttcaagtggt 1200gttgatgatg acatggcgtg tcataaaata ccagtggagg ccgacttctt 1250gtatgcatac tccacagcac ctggttatta ttcttggcga aattcaaagg 1300atggctcctg gttcatccag tcgctttgtg ccatgctgaa acagtatgcc 1350gacaagcttg aatttatgca cattcttacc cgggttaacc gaaaggtggc 1400aacagaattt gagtcctttt cctttgacgc tacttttcat gcaaagaaac 1450agattccatg tattgtttcc atgctcacaa aagaactcta tttttatcac 1500ggtggaggcg gttcatctgg aatatccctg gacaacagtt ataaaatgga 1550ttatcctgag atgggtttat gtataataat taataataag aattttcata 1600aaagcactgg aatgacatct cggtctggta cagatgtcga tgcagcaaac 1650ctcagggaaa cattcagaaa cttgaaatat gaagtcagga ataaaaatga 1700tcttacacgt gaagaaattg tggaattgat gcgtgatgtt tctaaagaag 1750atcacagcaa aaggagcagt tttgtttgtg tgcttctgag ccatggtgaa 1800gaaggaataa tttttggaac aaatggacct gttgacctga aaaaaataac 1850aaactttttc agaggggatc gttgtagaag tctaactgga aaacccaaac 1900ttttcattat tcaggcctgc cgtggtacag aactggactg tggcattgag 1950acacatcacc atcaccatca ccatcactaa gcggccttaa ggctaaggga 2000tcctctagag gttgaggtga ttttatgaaa aagaatatcg catttcttct 2050tgcatctatg ttcgtttttt ctattgctac aaacgcgtac gctgaggttc 2100agctggtgga gtctggcggt ggcctggtgc agccaggggg ctcactccgt 2150ttgtcctgtg cagcttctgg cttcaacatt aaagacacct atatacactg 2200ggtgcgtcag gccccgggta agggcctgga atgggttgca aggatttatc 2250ctacgaatgg ttatactaga tatgccgata gcgtcaaggg ccgtttcact 2300ataagcgcag acacatccaa aaacacagcc tacctgcaga tgaacagcct 2350gcgtgctgag gacactgccg tctattattg ttctagatgg ggaggggacg 2400gcttctatgc tatggactac tggggtcaag gaaccctggt caccgtctcc 2450tcggcctcca ccaagggccc atcggtcttc cccctggcac cctcctccaa 2500gagcacctct gggggcacag cggccctggg ctgcctggtc aaggactact 2550tccccgaacc ggtgacggtg tcgtggaact caggcgccct gaccagcggc 2600gtgcacacct tcccggctgt cctacagtcc tcaggactct actccctcag 2650cagcgtggtg accgtgccct ccagcagctt gggcacccag acctacatct 2700gcaacgtgaa tcacaagccc agcaacacca aggtcgacaa gaaagttgag 2750cccaaatctt gtgacaaaac tcacacaggc gggggaggta gcggaggtgg 2800gggctctggt ggaggcggtt caagtggtgt tgatgatgac atggcgtgtc 2850ataaaatacc agtggaggcc gacttcttgt atgcatactc cacagcacct 2900ggttattatt cttggcgaaa ttcaaaggat ggctcctggt tcatccagtc 2950gctttgtgcc atgctgaaac agtatgccga caagcttgaa tttatgcaca 3000ttcttacccg ggttaaccga aaggtggcaa cagaatttga gtccttttcc 3050tttgacgcta cttttcatgc aaagaaacag attccatgta ttgtttccat 3100gctcacaaaa gaactctatt tttatcacgg tggaggcggt tcatctggaa 3150tatccctgga caacagttat aaaatggatt atcctgagat gggtttatgt 3200ataataatta ataataagaa ttttcataaa agcactggaa tgacatctcg 3250gtctggtaca gatgtcgatg cagcaaacct cagggaaaca ttcagaaact 3300tgaaatatga agtcaggaat aaaaatgatc ttacacgtga agaaattgtg 3350gaattgatgc gtgatgtttc taaagaagat cacagcaaaa ggagcagttt 3400tgtttgtgtg cttctgagcc atggtgaaga aggaataatt tttggaacaa 3450atggacctgt tgacctgaaa aaaataacaa actttttcag aggggatcgt 3500tgtagaagtc taactggaaa acccaaactt ttcattattc aggcctgccg 3550tggtacagaa ctggactgtg gcattgagac acatcaccat caccatcacc 3600atcactaagc atgc 3614<210>2<211>513<212>PRT<213>人(Homo sapians)<400>2Met Lys Lys Asn Ile Ala Phe Leu Leu Ala Ser Met Phe Val Phe1 5 10 15Ser Ile Ala Thr Asn Ala Tyr Ala Asp Ile Gln Met Thr Gln Ser
20 25 30Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr
35 40 45Cys Arg Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln
50 55 60Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser
65 70 75Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Arg Ser
80 85 90Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp
95 100 105Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro Thr
110 115 120Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
125 130 135Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
140 145 150Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
155 160 165Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
170 175 180Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
185 190 195Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
200 205 210Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
215 220 225Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly
230 235 240Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Val
245 250 255Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala Asp Phe
260 265 270Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn
275 280 285Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu
290 295 300Lys Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg
305 310 315Val Asn Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp
320 325 330Ala Thr Phe His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met
335 340 345Leu Thr Lys Glu Leu Tyr Phe Tyr His Gly Gly Gly Gly Ser Ser
350 355 360Gly Ile Ser Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met
365 370 375Gly Leu Cys Ile Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr
380 385 390Gly Met Thr Ser Arg Ser Gly Thr Asp Val Asp Ala Ala Asn Leu
395 400 405Arg Glu Thr Phe Arg Asn Leu Lys Tyr Glu Val Arg Asn Lys Asn
410 415 420Asp Leu Thr Arg Glu Glu Ile Val Glu Leu Met Arg Asp Val Ser
425 430 435Lys Glu Asp His Ser Lys Arg Ser Ser Phe Val Cys Val Leu Leu
440 445 450Ser His Gly Glu Glu Gly Ile Ile Phe Gly Thr Asn Gly Pro Val
455 460 465Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg Gly Asp Arg Cys Arg
470 475 480Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile Gln Ala Cys Arg
485 490 495Gly Thr Glu Leu Asp Cys Gly Ile Glu Thr His His His His His
500 505 510His His His<210>3<211>4<212>PRT<213>人工序列<220><223>Act_Site<400>3Asp Glu Val Asp1<210>4<211>4<212>PRT<213>人工序列<220><223>Act_Site<400>4Asp Glu Ile Asp1<210>5<211>5<212>PRT<213>人工序列<220><223>Xaa=Arg,Gln或Gly<400>5Gln Ala Cys Xaa Gly1 5<210>6<211>4<212>PRT<213>人工序列<220><223>第I组天冬氨酸特异性半胱氨酸蛋白酶的最佳序列<400>6Trp Glu His Asp1<210>7<211>4<212>PRT<213>人工序列<220><223>天冬氨酸特异性半胱氨酸蛋白酶9的最佳序列<400>7Leu Glu His Asp1<210>8<211>4<212>PRT<213>人工序列<220><223>天冬氨酸特异性半胱氨酸蛋白酶2的最佳序列<400>8Asp Glu His Asp1<210>9<211>4<212>PRT<213>人工序列<220><223>CED-3的最佳序列<400>9Asp Glu Thr Asp1<210>10<211>4<212>PRT<213>人工序列<220><223>天冬氨酸特异性半胱氨酸蛋白酶6的最佳序列<400>10Val Glu His Asp1<210>11<211>4<212>PRI<213>人工序列<220><223>天冬氨酸特异性半胱氨酸蛋白酶8的最佳序列<400>11Leu Glu Thr Asp1<210>12<211>4<212>PRT<213>人工序列<220><223>Xaa=Nle<400>12Leu Glu Xaa Asp1<210>13<211>4<212>PRT<213>人工序列<220><223>粒酶B的最佳序列<400>13Ile Glu Pro Asp1<210>14<211>51<212>DNA<213>人工序列<220><223>引物结合<400>14gctacaaacg cgtacgctga tatccagatg acccagtccc cgagctccct 50g 51<210>15<211>57<212>DNA<213>人工序列<220><223>引物结合<400>15cccccacctc cgctacctcc cccgccacac tctcccctgt tgaagctctt 50tgtgacg 57<210>16<211>56<212>DNA<213>人工序列<220><223>引物结合<400>16cgggggaggt agcggaggtg ggggctctgg tggaggcggt tcaagtggtg 50ttgatg 56<210>17<211>57<212>DNA<213>人工序列<220><223>引物结合<400>17gccgtcgcat gcttagtgat ggtgatggtg atggtgatgt gtctcaatgc 50cacagtc 57<210>18<211>52<212>DNA<213>人工序列<220><223>引物结合<400>18tgctacaaac gcgtacgctg aggttcagct ggtggagtct ggcggtggcc 50tg 52<210>19<211>53<212>DNA<213>人工序列<220><223>引物结合<400>19ccccacctcc gctacctccc ccgcctgtgt gagttttgtc acaagatttg 50ggc 53<210>20<211>50<212>DNA<213>人工序列<220><223>引物结合<400>20taagcggcct taaggctaag ggatcctcta gaggttgagg tgattttatg 50<210>21<211>25<212>DNA<213>人工序列<220><223>引物结合<400>21cttcaacagg ggagagtctg gcggg 25<210>22<211>25<212>DNA<213>人工序列<220><223>引物结合<400>22cccgccagac tctcccctgt tgaag 25<210>23<211>25<212>DNA<213>人工序列<220><223>引物结合<400>23gcccaaatct tctgacaaaa ctcac 25<210>24<211>25<212>DNA<213>人工序列<220><223>引物结合<400>24gtgagttttg tcagaagatt tgggc 25<210>25<211>527<212>PRT<213>人(Homo sapiens)<400>25Met Lys Lys Asn Ile Ala Phe Leu Leu Ala Ser Met Phe Val Phe1 5 10 15Ser Ile Ala Thr Asn Ala Tyr Ala Glu Val Gln Leu Val Glu Ser
20 25 30Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys
35 40 45Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val
50 55 60Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr
65 70 75Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg
80 85 90Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln
95 100 105Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser
110 115 120Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
125 130 135Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
140 145 150Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
155 160 165Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
170 175 180Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
185 190 195Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
200 205 210Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
215 220 225Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
230 235 240Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Gly Gly Gly Gly
245 250 255Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Gly Val Asp
260 265 270Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala Asp Phe Leu
275 280 285Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn Ser
290 295 300Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys
305 310 315Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val
320 325 330Asn Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala
335 340 345Thr Phe His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu
350 355 360Thr Lys Glu Leu Tyr Phe Tyr His Gly Gly Gly Gly Ser Ser Gly
365 370 375Ile Ser Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly
380 385 390Leu Cys Ile Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly
395 400 405Met Thr Ser Arg Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg
410 415 420Glu Thr Phe Arg Asn Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp
425 430 435Leu Thr Arg Glu Glu Ile Val Glu Leu Met Arg Asp Val Ser Lys
440 445 450Glu Asp His Ser Lys Arg Ser Ser Phe Val Cys Val Leu Leu Ser
455 460 465His Gly Glu Glu Gly Ile Ile Phe Gly Thr Asn Gly Pro Val Asp
470 475 480Leu Lys Lys Ile Thr Asn Phe Phe Arg Gly Asp Arg Cys Arg Ser
485 490 495Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile Gln Ala Cys Arg Gly
500 505 510Thr Glu Leu Asp Cys Gly Ile Glu Thr His His His His His His
515 520 525His His
Claims (26)
1、一种将活性药物送递到目的细胞类型的方法,其包含以下步骤:
a)给予有效剂量的靶向细胞类型的偶联物,该偶联物包含一种天冬氨酸特异性半胱氨酸蛋白酶,该酶可将天冬氨酸特异性半胱氨酸蛋白酶可转变的前体药物转变为活性药物,并
b)给予一种可为天冬氨酸特异性半胱氨酸蛋白酶所转变的前体药物。
2、权利要求1的方法,其中该天冬氨酸特异性半胱氨酸蛋白酶是哺乳动物的天冬氨酸特异性半胱氨酸蛋白酶。
3、权利要求2的方法,其中该天冬氨酸特异性半胱氨酸蛋白酶是人的天冬氨酸特异性半胱氨酸蛋白酶。
4、权利要求3的方法,其中该天冬氨酸特异性半胱氨酸蛋白酶是前凋亡型天冬氨酸特异性半胱氨酸蛋白酶。
5、权利要求4的方法,其中该天冬氨酸特异性半胱氨酸蛋白酶选自天冬氨酸特异性半胱氨酸蛋白酶2,天冬氨酸特异性半胱氨酸蛋白酶3,和天冬氨酸特异性半胱氨酸蛋白酶7。
6、权利要求5的方法,其中该天冬氨酸特异性半胱氨酸蛋白酶是天冬氨酸特异性半胱氨酸蛋白酶3。
7、权利要求1的方法,其中该目的细胞类型是肿瘤细胞。
8、权利要求1的方法,其中该靶向细胞类型的偶联物是抗体偶联物。
9、权利要求8的方法,其中该抗体是一种多克隆抗体。
10、权利要求8的方法,其中该抗体是一种单克隆抗体。
11、权利要求8的抗体,其中该抗体是一种抗体片段。
12、权利要求11的抗体,其中该抗体片段是F(ab’)2。
13、权利要求1的方法,其中该活性药物是一种细胞毒性药物。
14、权利要求13的方法,其中该细胞毒性药物选自:阿霉素,柔红菌素,表阿霉素,他克唑,泰索帝,长春新碱,长春花碱,丝裂霉素C,依托泊苷,氨甲蝶呤,顺铂,环磷酰胺,左旋苯丙氨酸氮芥,哈乐泰斯停,环磷酰胺,噻替派,苯丁酸氮芥,5-FU和癌得星。
15、权利要求14的方法,其中该细胞毒性药物是阿霉素。
16、一种药物组合物,其包含天冬氨酸特异性半胱氨酸蛋白酶的靶向细胞类型的偶联物。
17、一种药物组合物,其包含与抗体偶联的天冬氨酸特异性半胱氨酸蛋白酶。
18、一种前体药物,其包含一个天冬氨酸特异性半胱氨酸蛋白酶可裂解的前体药物组分。
19、权利要求18的前体药物,其中该前体药物组分具有Asp-Xaa-Xaa-Asp序列。
20、权利要求19的前体药物,其中该前体药物组分具有Asp-Glu-Val-Asp序列。
21、权利要求20的前体药物,其包含阿霉素。
22、权利要求20的前体药物,其包含紫杉醇。
23、一种试剂盒,其包含与抗体偶联的天冬氨酸特异性半胱氨酸蛋白酶。
24、权利要求23的试剂盒,其进一步包含一种前体药物,该前体药物可被与抗体偶联的天冬氨酸特异性半胱氨酸蛋白酶转变为一种更具活性的药物。
25、一种治疗哺乳动物的方法,其包含给予该哺乳动物以治疗上有效剂量的前体药物的步骤,该前体药物可被天冬氨酸特异性半胱氨酸蛋白酶转变为一种活性药物。
26、一种治疗哺乳动物的方法,其包含给予该哺乳动物以治疗上有效剂量的前体药物和一种靶向细胞类型的天冬氨酸特异性半胱氨酸蛋白酶的步骤,该前体药物可被天冬氨酸特异性半胱氨酸蛋白酶转变为活性药物。
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US18477900P | 2000-02-24 | 2000-02-24 | |
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US (1) | US20070104719A1 (zh) |
EP (1) | EP1257296A2 (zh) |
JP (1) | JP2003523407A (zh) |
KR (1) | KR20020082227A (zh) |
CN (1) | CN1406137A (zh) |
AU (1) | AU783679B2 (zh) |
BR (1) | BR0108930A (zh) |
CA (1) | CA2399255A1 (zh) |
HU (1) | HUP0300024A2 (zh) |
IL (1) | IL150992A0 (zh) |
MX (1) | MXPA02007939A (zh) |
NZ (1) | NZ520458A (zh) |
PL (1) | PL358187A1 (zh) |
WO (1) | WO2001062300A2 (zh) |
ZA (1) | ZA200206105B (zh) |
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CN106456799A (zh) * | 2014-06-03 | 2017-02-22 | 江苏佳瑞生物技术有限公司 | 肽‑药物偶联物 |
CN109310780A (zh) * | 2016-05-04 | 2019-02-05 | 纳维格蛋白质有限公司 | 包含肽接头的用于化学部分位点-特异性偶联的靶向化合物 |
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JP2013537415A (ja) | 2010-08-03 | 2013-10-03 | アッヴィ・インコーポレイテッド | 二重可変ドメイン免疫グロブリンおよびその使用 |
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US10125105B2 (en) | 2014-06-11 | 2018-11-13 | Arizona Board Of Regents On Behalf Of The University Of Arizona | Triazabutadienes as cleavable cross-linkers |
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TW201710286A (zh) | 2015-06-15 | 2017-03-16 | 艾伯維有限公司 | 抗vegf、pdgf及/或其受體之結合蛋白 |
US10954195B2 (en) | 2015-08-11 | 2021-03-23 | Arizona Board Of Regents On Behalf Of The University Of Arizona | Substituted triazenes protected from degradation by carboxylation of N1 |
WO2018023130A1 (en) | 2016-07-29 | 2018-02-01 | The Arizona Board Of Regents On Behalf Of The University Of Arizona | Triazabutadienes as cleavable cross-linkers |
JP7404252B2 (ja) | 2017-11-08 | 2023-12-25 | ヤフェイ シャンハイ バイオロジー メディスン サイエンス アンド テクノロジー カンパニー リミテッド | 生体分子のコンジュゲートおよびその使用 |
TW202126334A (zh) * | 2019-09-19 | 2021-07-16 | 美商西雅圖遺傳學股份有限公司 | 從生物活性化合物的內化共軛物選擇性釋出藥物 |
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2001
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- 2001-02-22 HU HU0300024A patent/HUP0300024A2/hu unknown
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- 2001-02-22 EP EP01912935A patent/EP1257296A2/en not_active Withdrawn
- 2001-02-22 PL PL01358187A patent/PL358187A1/xx not_active Application Discontinuation
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- 2001-02-22 BR BR0108930-7A patent/BR0108930A/pt not_active IP Right Cessation
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- 2001-02-22 KR KR1020027011059A patent/KR20020082227A/ko not_active Application Discontinuation
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106456799A (zh) * | 2014-06-03 | 2017-02-22 | 江苏佳瑞生物技术有限公司 | 肽‑药物偶联物 |
CN106456799B (zh) * | 2014-06-03 | 2020-05-22 | 江苏佳瑞生物技术有限公司 | 肽-药物偶联物 |
CN106170303A (zh) * | 2014-11-20 | 2016-11-30 | 法罗斯根有限公司 | 通过半胱天冬酶激活的前体药物 |
CN106170303B (zh) * | 2014-11-20 | 2019-10-25 | 法罗斯根有限公司 | 通过半胱天冬酶激活的前体药物 |
CN109310780A (zh) * | 2016-05-04 | 2019-02-05 | 纳维格蛋白质有限公司 | 包含肽接头的用于化学部分位点-特异性偶联的靶向化合物 |
Also Published As
Publication number | Publication date |
---|---|
BR0108930A (pt) | 2002-12-10 |
KR20020082227A (ko) | 2002-10-30 |
CA2399255A1 (en) | 2001-08-30 |
HUP0300024A2 (en) | 2003-05-28 |
ZA200206105B (en) | 2003-07-31 |
NZ520458A (en) | 2005-02-25 |
AU4166701A (en) | 2001-09-03 |
WO2001062300A3 (en) | 2002-04-25 |
EP1257296A2 (en) | 2002-11-20 |
US20070104719A1 (en) | 2007-05-10 |
WO2001062300A2 (en) | 2001-08-30 |
MXPA02007939A (es) | 2003-02-10 |
IL150992A0 (en) | 2003-02-12 |
JP2003523407A (ja) | 2003-08-05 |
AU783679B2 (en) | 2005-11-24 |
PL358187A1 (en) | 2004-08-09 |
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