CN103880965A - 被导靶的干扰素显示强的细胞凋亡和抗肿瘤活性 - Google Patents
被导靶的干扰素显示强的细胞凋亡和抗肿瘤活性 Download PDFInfo
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Abstract
本发明提供了表现出明显抗癌效力的新的嵌合部分。某些实施方案中,所述嵌合部分包含附着于干扰素的导靶部分。某些实施方案中,所述嵌合部分包含融合蛋白,其中特异结合癌症标记物的抗体与干扰素α(IFN-α)融合在一起。
Description
本申请是申请日为2008年9月19日,申请号为200880117225.8(国际申请号为PCT/US2008/077074),名称为“被导靶的干扰素显示强的细胞凋亡和抗肿瘤活性”的发明专利申请的分案申请。
相关申请的交叉引用
本申请要求2007年9月21日提交的USSN60/994,717的优先权和权益,所述申请通过引用全文并入本文。
关于政府支持的声明
本发明是在国立卫生学院提供的政府资助No.CA87990的支持下完成的。政府对本发明拥有一定权利。
发明领域
本发明涉及肿瘤学领域。发明提供了具有显著抗癌活性的嵌合构建体。
发明背景
尽管可以检测到抗肿瘤相关抗原的自发免疫反应(TAAs)(Hrouda et al.(1999)Semin.Oncol.26:455-471)(Disis et al.(1997)J.Clin.Oncol.15:3363-3367),但导致疾病的恶性细胞不能引发免疫反应,从而进行排异。许多研究已表明有可能通过向肿瘤细胞中引入诸如细胞因子的免疫刺激分子和协同刺激分子来增强肿瘤细胞的免疫原性(Dranoff and Mulligan(1995)Adv.Immunol.58:417-454;Hrouda et al.(1999)Semin.Oncol.26:455-471;Hurford et al.(1995)Nat.Genet.10:430-435);但高效的基因转移仍然具有挑战性。此外,要根除残存的癌症细胞可能需要针对那些无法直接进行基因转移的大范围内分散的微转移肿瘤沉积。
先天性免疫反应和获得性免疫反应对于提供抗感染性病原菌和肿瘤的保护作用都是必需的。先天性和获得性免疫之间的交流通过细胞和细胞因子间的相互作用进行调节。先天免疫系统中的细胞所产生的细胞因子可以直接或间接地激活获得性免疫反应中的细胞,并且在引发保护性抗肿瘤免疫中发挥着重要作用(Belardelli and Ferrantini(2002)Trends Immunol.23:201-208)。激活先天免疫系统的关键是检测到导致释放促炎症细胞因子(诸如IFN-α、TNF-α和IL-1)的细菌产物或“危险”信号。
IFN-α是具有强大抗病毒和免疫调节活性的促炎症细胞因子,并且是树突状细胞(DCs)分化和活性的刺激剂(Santini et al.(2000)J.Exp.Med.191:1777-1788)。I型IFNs(IFN-α和IFN-β)对免疫反应有多方面影响(Theofilopoulos et al.(2005)Annu.Rev.Immunol.23:307-336)。IFN-α在Th1细胞的分化(Finkelman et al.(1991)J.Exp.Med.174:1179-1188)和应答特异抗原时CD8+T细胞的长期存活(Tough et al.(1996)Science272:1947-1950)中发挥作用。
多项研究显示IFNs在动物模型(Ferrantini et al.(1994)J.Immunol.153:4604-4615)以及癌症患者(14.Gutterman et al.(1980)Ann.Intern.Med.93:399-406)中也能够发挥抗肿瘤效果。除了增强获得性抗肿瘤免疫反应,IFN-α还能够提高肿瘤抑制基因P53的表达(Takaoka et al.(2003)Nature424:516-523)、抑制血管发生(Sidky and Borden(1987)Cancer Res.47:5155-5161)以及在肿瘤细胞中引发细胞凋亡(Rodriguez-Villanueva and McDonnell(1995)Int.J.Cancer61:110-11417)。虽然这些性能提示IFN-α应当是治疗癌症的有效治疗剂,其较短的半衰期和系统毒性限制了它的应用。
发明概述
在多项实施方案中,本发明涉及这样的发现,即将干扰素附着到导靶部分(例如能够特异和/或优先结合细胞上的或者与细胞关联的标记物的分子)上能够显著地提高干扰素的治疗效果,并且似乎能够降低其系统毒性。相应地,在多项实施方案中,本发明提供了包含附着于导靶部分的干扰素的构建体,以及这类构建体在某些靶细胞(例如癌细胞)的生长或分化的特异和/或优先抑制或者甚至杀伤中的用途。
相应地,在某些实施方案中,提供了这样的嵌合构建体,所述构建体包含附着于能够结合肿瘤相关抗原(TAA)的导靶部分的干扰素(例如,干扰素-α、干扰素-β、干扰素-γ等),当所述构建体与肿瘤细胞接触时,导致肿瘤细胞被杀死或者其生长或分化被抑制。在某些实施方案中,提供了这样的嵌合构建体,所述构建体包含附着于能够结合细胞表面标记物或细胞相关标记物的导靶部分的干扰素,其中导靶部分不是通过(Gly4Ser)3(SEQ ID NO:31)接头附着于干扰素的。在多项实施方案中,干扰素是1型干扰素。在多项实施方案中,干扰素是2型干扰素。在多项实施方案中,干扰素是干扰素α、干扰素-β或干扰素-γ。在某些实施方案中,导靶部分是能够结合肿瘤相关抗原的抗体。在某些实施方案中,导靶部分与干扰素化学偶联。在某些实施方案中,导靶部分通过肽接头与干扰素连接。在某些实施方案中,所述肽接头长度低于15、低于14、低于12、低于11、低于10、低于9、低于8、低于7、低于6、低于5、低于4、低于3或低于2个氨基酸。在某些实施方案中,接头长度为15、14、13、12、11、10、9、8、7、6、5、4、3、2或1个氨基酸。在某些实施方案中,接头不是(Gly4Ser)3(SEQ ID NO:31)。在某些实施方案中,接头是抗蛋白质水解或者基本上抗蛋白质水解的接头。某些实施方案中,肽接头是Gly4Ser(SEQ ID NO:32)。在某些实施方案中,接头包含表2中的氨基酸序列或者由其组成。某些实施方案中,构建体是重组表达的融合蛋白。在某些实施方案中,抗体特异结合选自EGFR、HER4、HER3、HER2/neu、MUC-1、G250、间皮素(mesothelin)、gp100、酪氨酸酶和MAGE的标记物。在某些实施方案中,导靶部分是结合CD20的抗体。某些实施方案中,导靶部分是单链抗体,所述单链抗体包含来自选自抗CD20(利妥昔单抗(Rituximab))、替伊莫单抗(Ibritumomab tiuxetan)、托西莫单抗(tositumomab)、AME-133v、Ocrelizumab、Ofatumumab、TRU-015、IMMU-106等的抗体的CDRs和/或可变区。在多项实施方案中,导靶部分是结合HER2的抗体。在某些实施方案中,抗体是C6抗体。在某些实施方案中,抗体包含VH和VL CDRs或C6MH3-B1的VH和VL。在多项实施方案中,抗体是IgG(例如IgG1、IgG3等)、IgE、单链Fv(scFv)、FAB、(Fab’)2、(ScFv)2等。某些实施方案中,抗体是选自Rituxan、IF5、B1、1H4、CD19、B4、B43、FVS191、hLL2、LL2、RFB4、M195、HuM195、AT13/5、赫赛汀4D5、HuCC49、HUCC39ΔCH2B72.3、12C10、IG5、H23、BM-2、BM-7、12H12、MAM-6和HMFG-1的抗体。某些实施方案中,抗体是能够结合EGF受体家族成员的抗体。某些实施方案中,抗体选自C6.5、C6ML3-9、C6MH3-B1、C6-B1D2、F5、HER3.A5、HER3.F4、HER3.H1、HER3.H3、HER3.E12、HER3.B12、EGFR.E12、EGFR.C10、EGFR.B11、EGFR.E8、HER4.B4、HER4.G4、HER4.F4、HER4.A8、HER4.B6、HER4.D4、HER4.D7、HER4.D11、HER4.D12、HER4.E3、HER4.E7、HER4.F8和HER4.C7。在某些实施方案中,构建体包含附着了干扰素的抗HER2IgG1抗体。
发明还提供了药物制剂。在多项实施方案中,所述制剂包含的嵌合构建体含有附着于导靶部分的干扰素。某些实施方案中,嵌合构建体包含以上(和/或下文)描述的构建体(例如抗CD20-干扰素和抗HER2-干扰素等)。在某些实施方案中,制剂是单位剂量的制剂。在某些实施方案中,制剂配制成用于非胃肠道给药。某些实施方案中,制剂配制成经由选自口服、静脉内给药、肌内给药、直接肿瘤给药、吸入、直肠给药、阴道给药、透皮给药以及皮下储库式给药的途径进行给药。
多项实施方案中提供了抑制癌细胞生长和/或增殖的方法。所述方法一般包括将癌细胞与本文描述的嵌合构建体进行接触。某些实施方案中,癌细胞是转移细胞,和/或细胞处于实体瘤中。某些实施方案中,癌细胞是乳腺癌细胞。某些实施方案中,癌细胞是B细胞淋巴瘤。某些实施方案中,所述癌细胞是由选自B细胞淋巴瘤、肺癌、支气管癌、结肠直肠癌、前列腺癌、乳腺癌、胰腺癌、胃癌、卵巢癌、膀胱癌、脑或中枢神经系统癌症、外周神经系统癌症、食道癌、宫颈癌、黑素瘤、子宫或子宫内膜癌、口腔癌或喉癌、肝癌、肾癌、胆管癌、小肠癌或阑尾癌、唾液腺癌、胸腺癌、肾上腺癌、骨肉瘤、软骨肉瘤、脂肪瘤、睾丸癌以及恶性纤维组织细胞瘤的癌症产生的细胞。在多项实施方案中,接触包括将嵌合部分系统地给予哺乳动物。某些实施方案中,接触包括将嵌合部分直接给予肿瘤部位。某些实施方案中,接触包括静脉内给予嵌合部分。某些实施方案中,癌细胞是人或非人哺乳动物中的癌细胞。
某些实施方案中,提供了编码本文描述的嵌合构建体的核酸。在多项实施方案中,核酸编码的融合蛋白包含附着于抗EGFR家族成员抗体、抗HER2抗体、抗C6单链抗体或抗CD20单链抗体的干扰素。在多项实施方案中,核酸所编码的干扰素是I型干扰素。某些实施方案中,干扰素是IFN-α或干扰素-β。在多项实施方案中,核酸编码的抗体包含C6MH3-B1的VH和VLCDRs。在多项实施方案中,核酸编码将抗体附着到干扰素上的肽接头(例如象本文描述的)。某些实施方案中,核酸编码抗CD20(利妥昔单抗)的CDRs和/或可变区。
发明还提供了包含以上描述的编码嵌合构建体的核酸的细胞。某些实施方案中,所述细胞表达嵌合构建体。
在多项实施方案中,发明提供了本文描述的嵌合构建体在制备用于抑制癌细胞生长和/或增殖的药物中的用途。
某些实施方案中,本发明的方法和构建体特别排除利用了美国专利申请公开2002/0193569A1中公开的抗体的构建体。某些实施方案中,本发明的方法和构建体特别排除引入了抗CD20抗体的构建体。某些实施方案中,本发明的方法和构建体特别排除引入了与任何以下靶分子结合的抗体的构建体:CD19、CD20、CD22、CD33、CD38、EGF-R、HM1.24、磷脂酰丝氨酸抗原、HER-2、TAG-72和/或MUC-1。某些实施方案中,本文描述的构建体可以用于治疗诸如多发性硬化症、HCV介导的血管炎等的病变。
定义
术语“多肽”、“肽”和“蛋白质”在本文中可互换用于氨基酸残基的聚合物。这些术语适用于其中的一个或多个氨基酸残基是相应的天然氨基酸的人工化学类似物的那些氨基酸聚合物,也适用于天然的氨基酸聚合物。术语还包括将构成多肽的氨基酸连接起来的常规肽接头的变体。优选的“肽”、“多肽”和“蛋白质”是α碳通过肽键连接的氨基酸链。因此链的一端(氨基端)的末端氨基酸带有游离的氨基,而链另一端(羰基端)的末端氨基酸带有游离羰基。术语“氨基端”(缩写为N端)用于本文是指肽氨基末端的氨基酸上的游离α-氨基基团或者肽内其他位置上的氨基酸的α-氨基基团(参与肽键时是亚氨基基团)。类似地,术语"羰基端"是指肽的羰基端上的游离羰基基团或者肽内其他位置上的氨基酸的羰基基团。肽还包括基本上任何多聚氨基酸,包括但不限于肽模拟物,比如通过醚键而不是酰胺键连接的氨基酸。
“抗体”用于本文是指基本上由免疫球蛋白基因或免疫球蛋白基因的片段编码的一或多个多肽所构成的蛋白质。公认的免疫球蛋白基因包括κ、λ、α、γ、δ、ε和μ恒定区基因,以及无数免疫球蛋白可变区基因。轻链划分为κ或λ。重链划分为γ、μ、α、δ或ε,它们又继而分别定义了免疫球蛋白种类:IgG、IgM、IgA、IgD和IgE。
典型的免疫球蛋白(抗体)结构单元已知包含四体。每个四体由两对相同的多肽链构成,每对含有一个“轻链”(约25kD)和一个“重链”(约50-70kD)。每个链的N端界定了主要负责抗原识别的有大约100到110或更多个氨基酸的可变区。术语可变轻链(VL)和可变重链(VH)分别指轻链和重链的这些区域。
抗体的存在方式包括完整的免疫球蛋白,或者是通过用多种肽酶消化或者重新表达产生的许多已被很好定性的片段。因此,例如胰蛋白酶消化抗体铰链区中的二硫键产生F(ab)'2,Fab二聚体,其自身是通过二硫键与VH-CH1连接的轻链。可以在温和条件下还原F(ab)'2,将铰链区中的二硫键打开从而将(Fab')2二聚体转化为Fab'单体。Fab'单体基本上就是Fab,和部分铰链区(参见Fundamental Immunology,W.E.Paul,ed.,Raven Press,N.Y.(1993)中对其他抗体片段的更详细描述)。虽然多种抗体片段是根据完整抗体的消化来定义的,本领域技术人员理解这类Fab'片段可以通过化学或者利用重组DNA方法重新合成。因此,术语抗体用于本文还包括通过对完整抗体进行修饰或者利用重组DNA方法重新合成所产生的抗体片段包括,但不限于Fab’2、IgG、IgM、IgA、IgE、scFv、dAb、纳米抗体、迷你抗体(unibodies)和二体(diabodies)。在多项实施方案中,优选的抗体包括,但不限于Fab’2、IgG、IgM、IgA、IgE和单链抗体,更优选单链Fv(scFv)抗体,其中可变重链和可变轻链被(直接或经由肽接头)连接在一起形成连续的多肽。
某些实施方案中,用于构建本发明的抗体和片段可以是双特异性的。双特异性抗体或片段可以是多种构型的。例如,双特异性抗体可能类似于单个抗体(或抗体片段),但具有两个不同的抗原结合位点(可变区)。在多项实施方案中,可以通过化学技术(Kranz et al.(1981)Proc.Natl.Acad.Sci.,USA,78:5807)、“polydoma”技术(参见例如U.S.Pat.No.4,474,893)或者重组DNA技术生产双特异性抗体。某些实施方案中,本发明的双特异性抗体可以具有对至少两种不同表位,其中至少一个是肿瘤相关抗原的结合特异性。在多项实施方案中,抗体和片段还可以是异源抗体。异源抗体是连接在一起的两个或多个抗体,或者抗体结合片段(例如,Fab),每个抗体或片段具有不同的特异性。
“抗原结合位点”或“结合部位”是指免疫球蛋白分子中参与抗原结合的部分。抗原结合位点是由重链(H)和轻链(L)的N末端可变区(V)的氨基酸残基形成的。重链和轻链V区内的三个高度分歧的片段称为“高变区”,该区介于更保守的称为“框架区”或“FRs”的旁侧片段之间。因此,术语“FR”是指免疫球蛋白中天然存在于高变区之间和高变区邻近的氨基酸序列。在抗体分子中,轻链的三个高变区和重链的三个高变区在三维空间中相对对方形成抗原结合“表面”。这个表面介导靶抗原的识别和结合。每个重链和轻链的三个高变区被称为“互补决定区”或“CDRs”,并通过例如Kabat et al.Sequences ofproteins of immunological interest,4th ed.U.S.Dept.Health and HumanServices,Public Health Services,Bethesda,MD(1987)进行表征。
术语“干扰素”是指全长干扰素,或者基本保持全长野生型干扰素的生物活性(例如保持全长抗体至少80%、优选至少90%、更优选至少95%、98%或99%)的干扰素片段(截短的干扰素)或干扰素突变体。干扰素包括I型干扰素(例如,干扰素-α和干扰素-β)和II型干扰素(例如,干扰素-γ)。所述干扰素(例如IFN-α)可以是来自几乎任何哺乳动物物种。某些优选实施方案中,干扰素来自选自人、马类、牛类、啮齿动物、猪类、兔类、猫类、犬类、鼠、山羊、羊类、非人灵长类等的物种。在多项实施方案中,突变干扰素包含一或多个氨基酸取代、插入和/或缺失。
抗-HER2/neu抗体是特异或优先结合HER2/neu受体的抗体。
术语“个体(subject)”用于本文是指人或非人动物,包括但不限于猫、狗、马、猪、牛、绵羊、山羊、兔、小鼠、大鼠或猴。
术语“C6抗体”用于本文是指由C6.5衍生的抗体,其序列在例如美国专利6,512,097和5,977,322,以及PCT公开WO97/00271中明确提供。C6抗体优选对HER2/neu的结合亲和力在大约1.6x10-8或以上。某些实施方案中,C6抗体来源于筛选噬菌体展示文库(对c-erbB-2/HER2/neu亲和力),所述文库中已知的C6可变重链(VH)与多个可变轻链(VL)联合表达,或者反过来,已知的C6可变轻链与多个可变重链联合表达。C6抗体还包括象例如美国专利6,512,097和5,977,322,以及PCT公开WO97/00271中描述的,通过向可变重链或可变轻链互补决定区(CDR1、CDR2或CDR3)中引入突变而产生的那些抗体。此外,C6抗体包括这些应用于C6.5及其衍生物的修饰方法的任意组合所产生的那些抗体。
“抗-EGFR家族抗体”是指特异结合表皮生长因子受体家族成员的抗体(例如,结合ErbB-1(又称为表皮生长因子受体(EGFR)、ErbB-2(在人中又称为HER2,在啮齿动物中称为neu)、ErbB-3(又称为HER3)和/或ErbB-4(又称为HER4)的抗体)。示范性的抗-EGFR家族抗体包括,但不限于诸如C6.5、C6ML3-9、C6MH3-B1、C6-B1D2、F5、HER3.A5、HER3.F4、HER3.H1、HER3.H3、HER3.E12、HER3.B12、EGFR.E12、EGFR.C10、EGFR.B11、EGFR.E8、HER4.B4、HER4.G4、HER4.F4、HER4.A8、HER4.B6、HER4.D4、HER4.D7、HER4.D11、HER4.D12、HER4.E3、HER4.E7、HER4.F8以及HER4.C7等抗体(参见,例如美国专利申请公开US2006/0099205A1和US2004/0071696A1,这两份文献均通过引用并入本文)。
单链Fv(“sFv”或“scFv”)多肽是共价连接的VH:VL异二聚体,在某些实施方案中可以由包含直接或者通过肽编码接头连在一起的VH-和VL-编码序列的核酸表达产生。Huston,et al.Proc.Nat.Acad.Sci.USA,85:5879-5883(1988)。有多种结构能将天然地聚集在一起但化学结构各自独立的轻链和重链多肽由抗体V区转化为sFv分子,它们能够折叠成与抗原结合位点结构实质上类似的三维结构。参见例如美国专利5,091,513、5,132,405和4,956,778。
“CD20”是成熟B细胞表面上的非糖基化的磷酸蛋白(参见例如Cragg etal.(2005)Curr.Dir.Autoimmun.,8:140-174)。该蛋白还可见于B细胞淋巴瘤、毛细胞白血病、B细胞慢性淋巴细胞白血病、皮肤/黑素瘤癌症干细胞等。
短语“抑制癌细胞的生长和/或增殖”指降低癌细胞的生长速度和/或增殖速度。某些实施方案中,这包括癌细胞的死亡(例如通过细胞凋亡)。某些实施方案中,该术语还指抑制实体瘤的生长和/或增殖,和/或诱发肿瘤体积下降或者消除肿瘤。
术语“癌症标记物”指这样一些诸如蛋白、碳水化合物、糖蛋白等的生物分子,所述分子专门或者优势地或者有区别地在癌细胞上表达,和/或被发现与癌细胞相关,因此可以作为癌症的优选或特异的目标。在多项实施方案中,优势表达可以是与生物体中其他细胞相比的优势表达,或者是生物体中特定区域内的优势表达(例如,在特定器官或组织中)。
具体地,本发明涉及如下各项:
1.嵌合构建体,所述构建体包含附着了能够结合肿瘤相关抗原的导靶部分的干扰素,当所述构建体与肿瘤细胞接触时,导致所述肿瘤细胞被杀死或者其生长或增殖受到抑制。
2.嵌合构建体,所述构建体包含附着了能够结合细胞表面标记物或细胞相关标记物的导靶部分的干扰素,其中所述导靶部分不是通过(Gly4Ser)3(SEQ ID NO:31)接头附着于所述干扰素的。
3.如项1-2中任一项所述的构建体,其中所述干扰素是1型干扰素。
4.如项1-2中任一项所述的构建体,其中所述干扰素是2型干扰素。
5.如项3所述的构建体,其中所述干扰素是干扰素α。
6.如项3所述的构建体,其中所述干扰素是干扰素β。
7.如项1-6中任一项所述的构建体,其中所述导靶部分是能够结合肿瘤相关抗原的抗体。
8.如项1-7中任一项所述的构建体,其中所述导靶部分与所述干扰素是化学偶联的。
9.如项1-7中任一项所述的构建体,其中所述导靶部分是借助肽接头与干扰素连在一起的。
10.如项9所述的构建体,其中所述肽接头长度小于15个氨基酸。
11.如项9所述的构建体,其中所述肽接头不是(Gly4Ser)3。
12.如项9所述的构建体,其中所述肽接头是Gly4Ser。
13.如项9-12中任一项所述的构建体,其中所述构建体是重组表达的融合蛋白。
14.如项7所述的构建体,其中所述抗体特异结合选自HER3、HER2/neu、MUC-1、G250、间皮素、gp100、酪氨酸酶和MAGE的标记物。
15.如项14所述的构建体,其中所述导靶部分是能够结合CD20的抗体。
16.如项15所述的构建体,其中所述导靶部分是单链抗体,其包含抗CD20(利妥昔单抗)的可变区。
17.如项14所述的构建体,其中所述导靶部分是能够结合HER2的抗体。
18.如项17所述的构建体,其中所述抗体是C6抗体。
19.如项17所述的构建体,其中所述抗体包含C6MH3-B1的VH和VL CDRs。
20.如项17所述的构建体,其中所述抗体包含C6MH3-B1的VH和VL结构域。
21.如项7所述的构建体,其中所述抗体是选自单链Fv(scFv)、FAB、(Fab’)2、(ScFv)2和全长IgG的抗体。
22.如项7所述的构建体,其中所述抗体是选自Rituxan、IF5、B1、1H4、CD19、B4、B43、FVS191、hLL2、LL2、RFB4、M195、HuM195、AT13/5、赫赛汀、4D5、HuCC49、HUCC39ΔCH2B72.3、12C10、IG5、H23、BM-2、BM-7、12H12、MAM-6、HMFG-1的抗体。
23.如项7所述的构建体,其中所述抗体是能够结合EGF受体家族成员的抗体。
24.如项23所述的构建体,其中所述抗体选自C6.5、C6ML3-9、C6MH3-B1、C6-B1D2、F5、HER3.A5、HER3.F4、HER3.H1、HER3.H3、HER3.E12、HER3.B12、EGFR.E12、EGFR.C10、EGFR.B11、EGFR.E8、HER4.B4、HER4.G4、HER4.F4、HER4.A8、HER4.B6、HER4.D4、HER4.D7、HER4.D11、HER4.D12、HER4.E3、HER4.E7、HER4.F8和HER4.C7
25.药物制剂,所述制剂在药物可接受的赋形剂中包含如项1-21中任一项所述的构建体。
26.如项25所述的药物制剂,其中所述制剂是单位剂量制剂。
27.如项25所述的药物制剂,其中所述制剂被配制成肠胃外给药的制剂。
28.如项25所述的药物制剂,其中所述制剂被配制成用于通过选自口服、静脉内给药、肌内给药、直接肿瘤给药、吸入、直肠给药、阴道给药、透皮给药和皮下储库给药的途径进行给药的制剂。
29.抑制癌细胞生长和/或增殖的方法,其中所述方法包含将所述癌细胞与如项1-21中任一项所述的嵌合构建体进行接触。
30.如项29所述的方法,其中所述癌细胞是转移细胞。
31.如项29所述的方法,其中所述癌细胞在实体瘤中。
32.如项29所述的方法,其中所述癌细胞是乳腺癌细胞。
33.如项29所述的方法,其中所述癌细胞是B细胞淋巴瘤。
34.如项29所述的方法,其中所述癌细胞是由选自B细胞淋巴瘤、肺癌、支气管癌、结肠直肠癌、前列腺癌、乳腺癌、胰腺癌、胃癌、卵巢癌、膀胱癌、脑或中枢神经系统癌症、外周神经系统癌症、食道癌、宫颈癌、黑素瘤、子宫或子宫内膜癌、口腔癌或喉癌、肝癌、肾癌、胆管癌、小肠癌或阑尾癌、唾液腺癌、胸腺癌、肾上腺癌、骨肉瘤、软骨肉瘤、脂肪瘤、睾丸癌以及恶性纤维组织细胞瘤的癌症产生的细胞。
35.如项29所述的方法,其中所述接触包括将所述嵌合部分系统地给予哺乳动物。
36.如项29所述的方法,其中所述接触包括将所述嵌合部分直接给药到肿瘤部位。
37.如项29所述的方法,其中所述接触包括静脉内给予所述嵌合部分。
38.如项29所述的方法,其中所述癌细胞是人体内的癌细胞。
39.如项29所述的方法,其中所述癌细胞是非人哺乳动物体内的癌细胞。
40.编码融合蛋白的核酸,所述融合蛋白包含附着于C6单链抗体或抗CD20单链抗体的干扰素。
41.如项35所述的核酸,其中所述干扰素是I型干扰素。
42.如项35所述的核酸,其中所述干扰素是IFN-α。
43.如项40-37中任一项所述的核酸,其中所述抗体包含C6MH3-B1的VH和VL CDRs。
44.如项43所述的核酸,其中所述抗体通过肽接头附着于所述IFN-α。
45.如项38所述的核酸,其中所述抗体包含C6MH3-B1的VH和VL结构域。
46.如项40-37中任一项所述的核酸,其中所述抗体包含抗CD20(利妥昔单抗)的可变区。
47.如项46所述的核酸,其中所述抗体通过15个氨基酸或更短的肽接头附着于所述IFN-α。
48.如项46所述的核酸,其中所述抗体通过Gly4Ser肽接头附着于所述IFN-α。
49.含有表达融合蛋白的核酸的细胞,所述细胞包含如项40-48中任一项所述的核酸。
50.如项1-21中任一项所述的构建体在制备用于抑制癌细胞生长和/或增殖的药物中的用途。
附图简述
图1A-图1O显示了本文描述的多种构建体的核酸和氨基酸序列。图1A显示了抗-HER2/neu IgG3重链-IFN-α(SEQ ID NO:1)和抗-HER2/neu IgG3轻链的氨基酸序列(SEQ ID NO:2)。单下划线的是接头,双下划线的是鼠IFN-α,无下划线的是抗-HER2/neu;图1B:αCD20轻链,核酸(SEQ ID NO:3),氨基酸序列(SEQ ID NO:4);图1C:αCD20-IgG3-muIFNαGly4Ser接头,核酸(SEQ ID NO:5),氨基酸序列(SEQ ID NO:6);图1D:αCD20-IgG3-muIFNαα螺旋接头,核酸(SEQ ID NO:7),氨基酸序列(SEQ ID NO:8);图1E:αCD20-IgG3-huIFNαGly4Ser接头,核酸(SEQ ID NO:9),氨基酸序列(SEQID NO:10);图1F:αCD20-IgG3-huIFNαα螺旋接头,核酸(SEQ IDNO:11),氨基酸序列(SEQ ID NO:12);图1G:αCD20-IgG1-muIFNαGly4Ser接头,核酸(SEQ ID NO:13),氨基酸序列(SEQ ID NO:14);图1H:αCD20-IgG1-muIFNαα螺旋接头,核酸(SEQ ID NO:15),氨基酸序列(SEQID NO:16);图1I:αCD20-IgG1-huIFNαGly4Ser接头,核酸(SEQ IDNO:17),氨基酸序列(SEQ ID NO:18);图1J:αCD20-IgG1-huIFNαα螺旋接头,核酸(SEQ ID NO:19),氨基酸序列(SEQ ID NO:20);图1K:αHer2/neu轻链核酸(SEQ ID NO:21),氨基酸序列(SEQ ID NO:22);图1L:αHer2/neu-IgG1-muIFNαGlySer接头,核酸序列(SEQ ID NO:23),氨基酸序列(SEQ ID NO:24);图1M:αHer2/neu-IgG1-muIFNαα螺旋接头,核酸序列(SEQ ID NO:25),氨基酸序列(SEQ ID NO:26);图1N:αHer2/neu-IgG1-huIFNαGlySer接头,核酸序列(SEQ ID NO:27),氨基酸序列(SEQ ID NO:28);图1O:αHer2/neu-IgG1-huIFNαα螺旋接头,核酸序列(SEQ ID NO:29),氨基酸序列(SEQ ID NO:30)。应当理解,虽然本图中显示的构建体带有特定接头,在某些实施方案中,可以用文中描述的其他接头代替。
图2A、图2B、图2C和图2D图示了抗-HER2/neu IgG3-IFN-α的构建和表征。图2A:抗-HER2/neu-IgG3-IFN-α的示意图。实心区域代表抗-HER2/neu可变区。空心区域代表人IgG3和κ恒定区。白色圆形区域代表鼠IFN-α。图2B:纯化的抗-HER2/neu-IgG3(泳道1和4)、IgG3-IFN-α(泳道2和5)和抗-HER2/neu-IgG3-IFN-α(泳道3和6)在非还原(泳道1–3)或还原(泳道4–6)条件下的SDS-PAGE。分子量标记蛋白示于每个凝胶的左侧。图2C:抗-HER2/neu-IgG3和抗-HER2/neu-IgG3-IFN-α结合HER2/neu。表达高水平人HER2/neu的鼠结肠细胞系CT26/HER2与带有或不带有肝素的抗-HER2/neu-IgG3、IgG3-IFN-α或抗-HER2/neu-IgG3-IFN-α反应,然后与PE标记的兔抗人IgG反应。破折号线代表不加入重组蛋白时细胞产生的信号。图2D:IFN-α标准和不同IFN-α融合蛋白抗VSV的保护活性。制备100μl1U IFN-α标准、0.21ng(10pM)抗-HER2/neu-IgG3-IFN-α、0.21ng(10pM)IgG3-IFN-α,或0.17ng(10pM)抗-HER2/neu-IgG3的稀释液,并加给L-929细胞。温育24小时后,加入4000PFU的VSV。48小时后,用结晶紫染料将活细胞染色,经甲醇溶解,用ELISA检测仪于570nm检测溶解的染料。
图3A和图3B显示不同IFN-α融合蛋白和rIFN-α的体内抗肿瘤活性。C3H/HeN小鼠用1x10338C13/HER2细胞s.c.(皮下)攻击,在肿瘤攻击后1、3和5天用2.5μg(图3A)或1μg(图3B)指定蛋白i.p.(静脉)处置。测量每只小鼠的肿瘤体积。观察小鼠,直至s.c.肿瘤直径达到15mm。
图4A和图4B显示IFN-α上融合IgG3提高了它的抗肿瘤活性并且提高了其体内半衰期。图4A:小鼠在肿瘤攻击后1和3天用9600U的rIFN-α或9600U(4μg)的IgG3-IFN-α处置。跟踪动物的存活情况,并在s.c.肿瘤直径达到15mm时处死。图4B:将三只一组的C3H/HeN小鼠i.p.注射66μCi125I-标记的rIFN-α、IgG3-IFN-α或抗-HER2/neu-IgG3-IFN-α。在注射125I-标记的蛋白后的不同间隔用小鼠全身放射性计数器测量残余放射性。结果代表三只小鼠的平均值。条带,SD。
图5A、图5B、图5C和图5D显示IFN-α融合蛋白抑制了细胞增殖并体外诱发38C13/HER2的细胞凋亡。IFN-α融合蛋白抑制肿瘤细胞增殖。与不同剂量的不同融合蛋白温育48小时后,利用MTS检测来测量活的38C13/HER2(图5A)或38C13(图5B)细胞。这些试验一式三份进行了三次;误差条,测量值的SD。图5C:IFN-α融合蛋白诱导38C13/HER2细胞的细胞凋亡。简而言之,将1x10638C13/HER2细胞与1nM指定蛋白温育72h。然后将细胞清洗,用Alexa Fluor488、膜联蛋白(annexin)V和PI染色,并通过流式细胞术分析。角落处显示了位于每个象限中的细胞的百分比。图5D:IFN-α融合蛋白抑制了存活的38C13/HER2细胞的增殖。简单来说,将1x10638C13/HER2细胞用2.5μM CFSE标记,并立即固定(破折号线),或者用PBS(细黑线)、或1nM抗-HER2/neu IgG3(细黑线、与PBS对照重叠)、IgG3-IFN-α(粗黑线)或抗-HER2/neu-IgG3-IFN-α(黑色区域)处理48h。然后将细胞清洗,通过流式细胞术分析。通过选通活细胞群获得图谱。
图6A、6B和6C显示IFN-α融合蛋白在38C13/HER2细胞中诱导了STAT1活化。简单来说,1x10738C13/HER2细胞用1000U/ml抗-HER2/neu-IgG3-IFN-α(图6A)或IgG3-IFN-α(图6B)处理指定的时间。细胞裂解物经SDS-PAGE分离,并利用多克隆兔抗磷STAT1通过Western印迹分析。为了证实蛋白样品上样量相同,用抗GAPDH的偶联HRP的兔多克隆抗体检测印迹。图6C:对每个指定时间点,用抗GAPDH的强度将抗磷STAT1的强度归一化,得到的数值除以0时间点处的数值得到STAT1的活化倍数。这些试验进行了两次;误差条,测量值的SD。﹡,出现两组差异p<0.05的唯一点。
图7IFN-α融合蛋白抑制了已建立的肿瘤的生长。将C3H/HeN小鼠s.c.注射1x10338C13/HER2细胞。12天后,连续三天用5μg指定蛋白将小鼠i.p.处理。测量每只小鼠的肿瘤体积。当s.c.肿瘤直径达到15mm,将动物处死。
图8显示重组抗体与表达CD20的人细胞发生结合。将Daudi细胞与重组IgG3或利妥昔单抗温育,随后与生物素化的大鼠抗人IgG和PE-标记的链霉亲和素温育,并通过流式细胞术分析。A,仅与二抗温育的细胞;B,与重组IgG3温育的细胞;C,与利妥昔单抗温育的细胞。
图9显示了抗体-IFN-α融合蛋白的重链的示意图。特别是该图显示了将(Gly4Ser)3(SEQ ID NO:31)缩短为Gly4Ser(SEQ ID NO:32)接头产生了全长αCD20-IgG3-mIFNα。
图10显示了从蛋白A Sepharose洗脱下来的级分的SDS-PAGE分析。表达带有(Gly4Ser)3(SEQ ID NO:31)接头的抗-CD-20-IgG3-IFNa的细胞的培养物上清流过蛋白A Sepharose,融合蛋白在洗脱前结合。A.蛋白未经还原跑胶。泳道1,IgG3;泳道2-6,从蛋白A Sepharose洗脱下来的级分。B.蛋白在分析前被还原。泳道2,IgG3;泳道3-7,从蛋白A Sepharose洗脱下来的级分。
图11显示了通过在HEK293T细胞中瞬时表达产生的蛋白的SDS-PAGE分析。泳道1,带有延长的(Gly4Ser)3(SEQ ID NO:31)接头的抗-CD20-IgG3-泳道2,带有缩短的Gly4Ser(SEQ ID NO:32)接头的抗-CD20-IgG3泳道3,带有延长的(Gly4Ser)3(SEQ ID NO:31)接头的抗泳道4,带有缩短的Gly4Ser(SEQ ID NO:32)接头的抗泳道5,抗-CD20IgG3。
图12显示了利用流式细胞术对与Daudi细胞结合的蛋白进行的分析。1x106Daudi细胞用1μg含有人IFN-α或Rituxan的融合蛋白染色。
图13显示了通过流式细胞术对与38C13/CD20结合的蛋白进行的分析。
图14.Daudi细胞与不同浓度的IFN-α、抗体或融合蛋白温育72小时。利用CellTiter96AQueous细胞增殖分析法评价细胞的生长抑制情况。
图15.Daudi细胞用10pM指定蛋白处理72小时。用膜联蛋白V和PI染色,进行流式细胞术分析,确定细胞活力和细胞凋亡情况。
图16.38C13/CD20细胞用10pM指定蛋白处理48小时。用膜联蛋白V和PI染色,进行流式细胞术分析,确定细胞活力和细胞凋亡情况。
图17.用各种浓度的不同蛋白处理后细胞增殖的抑制情况。38C13-CD20细胞用各种浓度的指定蛋白处理48小时。处理后,利用MTS分析法监测增殖程度。
图18.38C13/CD20细胞用不同浓度的指定蛋白处理48小时。用膜联蛋白V和PI染色,进行流式细胞术分析,确定细胞活力和细胞凋亡情况。
图19.Daudi细胞与不同浓度的融合蛋白温育72小时。用膜联蛋白V和PI染色,进行流式细胞术分析,确定细胞活力和细胞凋亡情况。
图20.Daudi细胞用各种浓度的融合蛋白处理72小时。加入MTS溶液定量细胞活力。
图21.Daudi细胞与1pM带有Gly4Ser接头(32)(Gly-Ser接头)的抗-CD20-IgG3-hIFNα或者1pM带有α螺旋接头(Alpha helix Linker)的抗-CD20-IgG3-hIFNα温育72小时。用膜联蛋白V和PI染色,进行流式细胞术分析,确定细胞活力和细胞凋亡情况。
图22显示了接种5000个38C13-CD20细胞,并在第1、2和3天用HBSS或者指定量的抗-CD20-IFN-α融合蛋白处理过的小鼠的存活情况。
图23显示了接种5000个38C13-CD20细胞,并在第5、6和6天用10μg抗-CD20-IgG1、抗-CD20-IgG3、利妥昔单抗或抗-CD20-IgG3-mIFNα处理过的小鼠的存活情况。
图24.接种5000个38C13-CD20细胞,并在第5、6和6天用10μg抗-CD20-IgG3、抗-CD20-IgG3+IFNα、抗-DNS-IgG3或抗-CD20-IgG3-mIFFNα处理过的小鼠的存活情况。
图25.8只一组的小鼠在第0天注射5000个38C13-CD20细胞。第8、9和10天,它们用HBSS或100μg抗-CD20-IgG3-mIFNα处理。持续监测肿瘤生长情况。
图26.8只一组的小鼠在第0天注射5000个38C13-CD20细胞。第8、9和10天,它们用HBSS或100μg抗-CD20-IgG3-mIFNα处理。持续监测存活情况。
发明详述
干扰素α(IFN-α)是启动先天性免疫反应的一种重要细胞因子,它还表现出广泛的抗肿瘤活性。但是干扰素(例如IFN-α)在临床上作为抗癌药物的应用却受到严重影响其治疗效果的较短的半衰期所限制。某些实施方案中,本发明涉及这样的发现,即通过将干扰素附着于能够特异/优先结合靶细胞(例如肿瘤细胞)上或者与靶细胞相关的标记物的导靶部分可以提高干扰素的治疗指数。这使得可以向靶位点递送更高剂量的干扰素,而系统并发症更少。这一点在一个实施方案中通过由抗-HER2/neu IgG3和IFN-α(抗-HER2/neu-IgG3-IFN-α)构成的融合蛋白的构建和使用,在另一个实施方案中由抗-CD20-IFN-α融合蛋白的构建和使用予以展示。
HER2/neu-IgG3-IFN-α构建体的效力在用人HER2/neu转导的鼠B-细胞淋巴瘤38C13上进行了测试。抗-HER2/neu-IgG3-IFN-α融合蛋白表现出强大的抑制38C13/HER2体内肿瘤生长的效果,甚至在肿瘤攻击后,仅给予1μg抗-HER2/neu IgG3-IFN-α即导致88%的长期存活。
不同寻常的是,抗-HER2/neu IgG3-IFN-α显示出强大的抗已建立的38C13/HER2肿瘤的活性,在88%处理小鼠中观察到肿瘤完全消失。这样巨大的抗肿瘤活性是由IFN-α诱导的细胞凋亡介导的,通过抗-HER2/neu IgG3抗体将IFN-α导靶到38C13/HER2肿瘤细胞对增强这些效果是关键的。
抗-CD20-IgG3-IFN-α构建体(参见实施例2)中观察到了类似的结果。这些结果表明干扰素(例如,IFN-α)附着(例如融合)于导靶部分(例如附着于肿瘤特异性抗体)产生了可用于抑制靶细胞的生长和/或增殖甚至将其杀死的有效治疗剂。因此,例如本文描述的示例性构建体可以方便地用于临床上治疗B细胞淋巴瘤和其他癌症。
因此,某些实施方案中,本发明提供了这样的构建体(例如嵌合部分),所述构建体包含附着于导靶部分(例如附着于特异结合癌细胞上的癌症特异标记物的抗体)的干扰素(例如IFN-α)。这类构建体包括化学偶联体和融合蛋白。还提供了编码融合蛋白的核酸,和转染了所述核酸从而表达融合蛋白的细胞。还提供了抑制癌细胞生长和增殖的方法,以及用于治疗各种癌症的包含例如本文描述的嵌合部分的试剂盒。
I.包含附着了干扰素的导靶部分的嵌合构建体。
我们惊奇地发现,包含附着了导靶部分的天然(野生型)或修饰的IFN(例如IFN-α)的嵌合构建体可以有效地用于抑制靶癌细胞生长和/或增殖,所述癌细胞表达导靶部分所针对的标记物或者与所述标记物相关。某些实施方案中,导靶部分与干扰素化学偶联,而在其他实施方案中,导靶部分与IFN-α表达为融合蛋白。当以融合蛋白产生时,导靶部分(例如抗体)成分与IFN-α可以是直接融合或者借助肽接头(例如,(Gly4Ser)3(SEQ ID NO:31)接头、GlyGlyGlyGlySer(SEQ ID NO:32)接头、AEAAAKEAAAKA(SEQ ID NO:33)等)附着。
A)导靶部分(targetting moieties)。
在多项实施方案中,导靶部分是这样的分子,其与靶细胞所表达的(例如表达在靶细胞表面的)标记物或者和靶细胞相关的标记物特异地或优先地结合。虽然靶细胞几乎可以是任何细胞,某些优选的细胞包括那些与特征在于细胞过度增殖的病态(即过度增殖病)相关的细胞。示范性的过度增殖病包括,但不限于银屑病、中性白细胞增多症、红细胞增多症、血小板增多症和癌症。
定性为癌症的过度增殖病包括,但不限于实体瘤,诸如发生在乳腺、呼吸道、脑、生殖器官、消化道、尿道、眼、肝脏、皮肤、头颈、甲状腺、甲状旁腺的癌症,以及它们的远端转移。这类疾病还包括淋巴瘤、肉瘤和白血病。乳腺癌的实例包括,但不限于浸润性导管癌、浸润性小叶癌、乳腺导管原位癌和乳腺小叶原位癌。呼吸道癌症的实例包括,但不限于肺小细胞癌和非小细胞肺癌,以及支气管腺瘤和胸膜肺母细胞瘤。脑癌的实例包括,但不限于脑干和下丘脑角质瘤、小脑和大脑星状细胞瘤、髓母细胞瘤、室管膜瘤,以及神经外胚层和松果体肿瘤。男性生殖器肿瘤包括,但不限于前列腺和睾丸癌。女性生殖器肿瘤包括,但不限于子宫内膜、宫颈、卵巢、阴道和外阴癌,以及子宫瘤。消化道肿瘤包括,但不限于肛门、结肠、结肠直肠、食管、胆囊、胃、胰腺、直肠、小肠和唾液腺癌。尿道肿瘤包括,但不限于膀胱、阴茎、肾、肾盂、输尿管和尿道癌。眼癌包括,但不限于眼球内黑素瘤和视网膜母细胞瘤。肝癌的实例包括,但不限于肝细胞癌(有或没有纤维板层变异的肝细胞瘤)、胆管癌(肝内胆管癌)和混合型肝细胞胆管细胞癌。皮肤癌包括,但不限于鳞状细胞癌、卡波西(Kaposi's)肉瘤、恶性黑素瘤、默克(Merkel)细胞皮肤癌和非黑素瘤型皮肤癌。头颈癌包括,但不限于喉/下咽/鼻咽/口咽癌,和嘴唇和口腔癌。淋巴癌包括,但不限于AIDS相关的淋巴癌、非霍奇金(Hodgkin's)淋巴癌、皮肤T-细胞淋巴癌、霍奇金氏病,以及中枢神经系统淋巴癌。肉瘤包括,但不限于软组织肉瘤、骨肉瘤、恶性纤维组织细胞瘤、淋巴肉瘤和横纹肌肉瘤。白血病包括,但不限于急性髓样白血病、急性淋巴细胞白血病、慢性淋巴细胞白血病、慢性粒细胞白血病和毛细胞白血病。
这些疾病在人类中已有成熟的研究,但它们也以类似的病原学存在于其他哺乳动物中,可以通过给予本发明的药物组合物来治疗。
某些实施方案中,导靶部分是能够结合癌症标记物(例如,肿瘤相关抗原)的部分。有大量癌症标记物是本领域技术人员已知的。这些标记物不需要是癌细胞特有的,但在以下情况中也是有效的,即标记物在癌细胞中的表达提高(相比正常的健康细胞)或者周边组织不存在可比水平的标记物(特别是当嵌合部分用于局部递送)。
示范性的癌症标记物包括,例如ND4单克隆抗体识别的肿瘤标记物。该标记物可见于低分化的结肠直肠癌,以及胃神经内分泌肿瘤(参见例如Tobi et al.(1998)Cancer Detection and Prevention,22(2):147-152)。其他重要的癌症免疫疗法目标是膜结合补体调控糖蛋白:CD46、CD55和CD59,这些蛋白已被发现体内和体外在多数肿瘤细胞上都有表达。人粘蛋白(例如MUC1)与在黑素瘤中发现的gp100、酪氨酸酶和MAGE都是已知的肿瘤标记物。野生型Wilms'瘤基因WT1不仅在多数急性髓细胞、急性淋巴细胞和慢性髓细胞白血病中,而且在包括肺癌的多种类型的实体瘤中高水平表达。
急性淋巴细胞白血病已通过TAAs HLA-Dr、CD1、CD2、CD5、CD7、CD19和CD20表征。急性髓细胞白血病已通过TAAs HLA-Dr、CD7、CD13、CD14、CD15、CD33和CD34表征。乳腺癌已通过标记物EGFR、HER2、MUC1和Tag-72表征。多种癌通过标记物MUC1、TAG-72和CEA进行了表征。慢性淋巴细胞白血病已通过标记物CD3、CD19、CD20、CD21、CD25和HLA-DR进行了表征。毛细胞白血病通过标记物CD19、CD20、CD21、CD25进行了表征。霍奇金症已通过Leu-M1标记物进行了表征。多种黑素瘤已通过HMB45标记物进行了表征。非霍奇金淋巴瘤通过CD20、CD19和Ia标记物进行了表征。多种前列腺癌已通过PSMA和SE10标记物进行了表征。
此外,许多类型的肿瘤细胞展示罕见的抗原,这些抗原或者是对于该细胞类型和/或其环境不相宜的,或者正常情况下只在生物体发育过程中出现(例如胚胎抗原)。这类抗原的实例包括鞘糖脂GD2,这种双唾液酸神经节苷脂通常只在神经元细胞的外表面膜上有显著水平的表达,其与免疫系统的接触受到血脑屏障的限制。GD2在包括成神经细胞瘤、髓母细胞瘤、星形细胞瘤、黑素瘤、肺小细胞癌、骨肉瘤和其他软组织肉瘤的许多种肿瘤细胞表面都有表达。因此GD2是免疫疗法的一个方便的肿瘤特异性目。
其他类型的肿瘤细胞展示一些是健康细胞表面罕见或者不存在的细胞表面受体,这些受体负责细胞信号传导途径的激活,导致肿瘤细胞不受控制的生长和分裂。实例包括(ErbB2).HER2/neu,这种组成性活化细胞表面受体在乳腺癌肿瘤细胞表面上异常高水平地产生。
其他有用的目标包括,但不限于CD20、CD52、CD33、表皮生长因子受体等。
表1中提供了合适的肿瘤标记物的说明性而非限制性列表。这些以及其他癌症标记物的抗体是本领域技术人员已知的,可以购买到或者利用例如噬菌体展示技术很容易地制备。
表1.说明性的癌症标记物和相关的参考文献,为了便于确认提及的肿瘤标记物,这些文献全部通过引用并入本文。
以上任何标记物都可以作为包含本发明的干扰素-导靶部分构建体的导靶部分的目标。某些实施方案中,靶标记物包括,但不限于表皮生长因子家族成员(例如HER2、HER3、EGF、HER4)、CD1、CD2、CD3、CD5、CD7、CD13、CD14、CD15、CD19、CD20、CD21、CD23、CD25、CD33、CD34、CD38、5E10、CEA、HLA-DR、HM1.24、HMB45、1a、Leu-M1、MUC1、PMSA、TAG-72、磷脂酰丝氨酸抗原(phosphatidyl serine antigen)等。
以上标记物仅用于示范而非限制。其他肿瘤相关抗原对于本领域技术人员也是已知的。
肿瘤标记物是细胞表面受体的情况中,该受体的配体可以作为导靶部分。类似的,这类配体的模拟物也可以作为导靶部分。
抗体。
某些实施方案中,导靶部分可以包含能够特异或优势结合肿瘤标记物的抗体、迷你抗体或affybodies。能够特异或优势结合肿瘤标记物的抗体是本领域技术人员所熟知的。例如结合在人B细胞上表达的CD22抗原的抗体包括HD6、RFB4、UV22-2、Tol5、4KB128、人源化抗CD22抗体(hLL2)(参见例如Li et al.(1989)Cell.Immunol.111:85-99;Mason et al.(1987)Blood69:836-40;Behr et al.(1999)Clin.Cancer Res.5:3304s-3314s;Bonardi et al.(1993)Cancer Res.53:3015-3021)。
针对CD33的抗体包括例如HuM195(参见例如Kossman et al.(1999)Clin.Cancer Res.5:2748-2755)和CMA-676(参见例如Sievers et al.,(1999)Blood93:3678-3684)。
针对CD38的抗体包括例如AT13/5(参见例如Ellis et al.(1995)J.Immunol.155:925-937)、HB7等。
某些实施方案中,导靶部分包含抗HER2抗体。ergB2基因(更常用的名称是Her-2/neu)是编码跨膜受体的原癌基因。已经开发的几种抗Her-2/neu的抗体包括曲妥珠单抗(trastuzumab)(例如,Fornier et al.(1999)Oncology(Huntingt)13:647-58)、TAB-250(Rosenblum et al.(1999)Clin.Cancer Res.5:865-874)、BACH-250(Id.)、TA1(Maier et al.(1991)Cancer Res.51:5361-5369)和美国专利5,772,997、5,770,195(mAb4D5;ATCC CRL10463)和5,677,171中描述的mAbs。
说明性的抗MUC-1抗体包括,但不限于Mc5(参见例如Peterson et al.(1997)Cancer Res.57:1103-1108;Ozzello et al.(1993)Breast Cancer Res.Treat.25:265-276)和hCTMO1(参见例如Van Hof et al.(1996)Cancer Res.56:5179-5185)。
说明性的抗TAG-72抗体包括,但不限于CC49(参见例如Pavlinkova etal.(1999)Clin.Cancer Res.5:2613-2619)、B72.3(参见例如Divgi et al.(1994)Nucl.Med.Biol.21:9-15)和美国专利5,976,531中公开的那些抗体。
说明性的抗HM1.24抗体包括,但不限于小鼠单克隆抗HM1.24IgG2a/κ和人源化抗HM1.24IgG1/κ.抗体(参见例如,Ono et al.(1999)Mol.Immuno.36:387-395)。
已经开发了许多特异结合HER2的抗体,其中一些已用于临床。它们包括例如曲妥珠单抗(例如,Fornier et al.(1999)Oncology(Huntingt)13:647-658)、TAB-250(Rosenblum et al.(1999)Clin.Cancer Res.5:865-874),BACH-250(Id.)、TA1(参见例如Maier et al.(1991)Cancer Res.51:5361-5369)和美国专利5,772,997、5,770,195和5,677,171中描述的抗体。
其他人抗HER2/neu抗体是本领域技术人员公知的。这类抗体包括,但不限于C6抗体,比如C6.5、DPL5、G98A、C6MH3-B1、B1D2、C6VLB、C6VLD、C6VLE、C6VLF、C6MH3-D7、C6MH3-D6、C6MH3-D5、C6MH3-D3、C6MH3-D2、C6MH3-D1、C6MH3-C4、C6MH3-C3、C6MH3-B9、C6MH3-B5、C6MH3-B48、C6MH3-B47、C6MH3-B46、C6MH3-B43、C6MH3-B41、C6MH3-B39、C6MH3-B34、C6MH3-B33、C6MH3-B31、C6MH3-B27、C6MH3-B25、C6MH3-B21、C6MH3-B20、C6MH3-B2、C6MH3-B16、C6MH3-B15、C6MH3-B11、C6MH3-B1、C6MH3-A3、C6MH3-A2和C6ML3-9。美国专利6,512,097和5,977,322、PCT公开WO97/00271、Schier et al.(1996)J Mol Biol255:28-43、Schier et al.(1996)J Mol Biol263:551-567等中描述了这些和其他的抗HER2/neu抗体。
更普遍来说,针对表皮生长因子受体家族不同成员的抗体非常适合作为本发明的构建体的导靶部分。这类抗体包括,但不限于美国专利5,844,093和5,558,864,以及欧洲专利706,799A中描述的抗EGF-R抗体。其他说明性抗EGFR家族抗体包括,但不限于诸如C6.5、C6ML3-9、C6MH3-B1、C6-B1D2、F5、HER3.A5、HER3.F4、HER3.H1、HER3.H3、HER3.E12、HER3.B12、EGFR.E12、EGFR.C10、EGFR.B11、EGFR.E8、HER4.B4、HER4.G4、HER4.F4、HER4.A8、HER4.B6、HER4.D4、HER4.D7、HER4.D11、HER4.D12、HER4.E3、HER4.E7、HER4.F8和HER4.C7等(参见例如美国专利申请公开US2006/0099205A1和US2004/0071696A1,这两份文献通过引用并入本文)等抗体。
正如美国专利6,512,097和5,977,322中所述,其他抗EGFR家族成员抗体可以通过轻链和/或重链的重排,随后进行一或多轮的亲和选择而容易地制备得到。因此某些实施方案中,本发明考虑了在VL和/或VH区中使用一个、两个或三个CDR,所述CDRs在以上确定的抗体中和/或以上确定的出版物中进行了描述。
在多项实施方案中,导靶部分包含特异或优势结合CD20的抗体。抗CD20抗体是本领域技术人员已知的,包括但不限于利妥昔单抗、替伊莫单 抗(Ibritumomab tiuxetan),和托西莫单抗、AME-133v(Applied Molecular Evolution)、Ocrelizumab(Roche)、Ofatumumab(Genmab)、TRU-015(Trubion)和IMMU-106(Immunomedics)。
本发明不必限于以上描述的抗体的用途,其他这类本领域技术人员已知的抗体也可以用于本文描述的组合物和方法。
虽然以上讨论涉及的是抗体,可以用亲和体(affybodies)和/或迷你抗体代替抗体使用。
迷你抗体(unibodies)。
迷你抗体技术是能够产生比某些小的抗体形式预期具有更长的治疗窗的稳定的、较小的抗体形式。某些实施方案中,通过删除IgG4抗体的铰链区制备迷你抗体。与全长IgG4抗体不同的是该半分子片段非常稳定,称为迷你抗体。将IgG4分子一分为二仅留下迷你抗体上能够结合目标分子的一个区域。PCT公开WO2007/059782中详细描述了制备迷你抗体的方法,该文献通过引用全文并入本文(还可以参见Kolfschoten et al.(2007)Science317:1554-1557)。
亲和体(affibodies)。
亲和体分子是基于有58个氨基酸残基的蛋白结构域的一类亲和蛋白,所述蛋白结构域来源于链球菌蛋白A的一个IgG结合结构域。这个有三个螺旋束的结构域已被用做构建组合噬粒文库的支架,由此文库可以利用噬菌体展示技术挑选针对所需分子的亲和体变体(参见例如Nord et al.(1997)Nat.Biotechnol.15:772-777;Ronmark et al.(2002)Eur.J.Biochem.,269:2647-2655.)。亲和体的详细情况和制备方法是本领域技术人员已知的(参见例如美国专利5,831,012,该文献通过引用全文并入本文)。
可以意识到,以上描述的抗体可以利用本领域技术人员熟知的方法提供为完整的抗体(例如IgG)、抗体片段或者单链抗体。此外,虽然抗体可以来自基本上任何哺乳动物物种,为了减少免疫原性,最好使用来自与构建体(例如抗HER2/neu-IFN-α嵌合分子)将要用在的物种相同的物种的抗体。换句话说,在人中使用时,最好用人、人源化或者嵌合人抗体。
B)IFN-α和经过修饰的IFN-α
在多项实施方案中,本发明的嵌合部分包含连接了导靶部分(例如,抗HER2/neu抗体)的干扰素(例如,IFN-α)。干扰素可以是全长野生型干扰素(例如IFN-α、IFN-β、IFN-γ等)、干扰素片段(例如IFN-α片段),和/或突变的干扰素。一般来说,干扰素片段是优选具有野生型干扰素的至少80%,更优选至少90%或95%,最优选至少98%、99%、100%,或者更高的内源性活性的片段。
鉴定这类修饰过的干扰素分子的手段对本领域技术人员来说是常规的。在一个说明性过程中,制备了截短的和/或突变的IFN-α的文库,并进行了IFN-α活性筛选。制备多肽变体文库的方法是本领域技术人员熟知的。因此,可以利用例如易错PCR来产生突变的和/或截短的IFN-α的文库(参见例如美国专利6,365,408)。
然后可以根据本领域技术人员已知的标准技术来筛选这样得到的文库成员。因此,可以通过例如测量抗特定测试病毒的抗病毒活性来检测IFN-α活性。检测IFN-α活性的试剂盒可以购买到(参见例如,Neutekbio,Ireland的iLiteTM alphabeta试剂盒)。
这些方法意在说明而非限制。利用本文提供的教导,可以容易地鉴定并生产其他合适的修饰过的干扰素(例如,经过修饰的IFN-α、IFN-β、IFN-γ等)。
C.抗体(例如,抗HER2/neu)与IFN-α的附着。
一般来说,导靶部分(例如,抗HER2/neu抗体和抗CD20抗体等)可以按照任何顺序连接在一起。因此,例如可以将抗体连接到干扰素的氨基或羧基端。只要附着不会影响到抗体与靶标记物(例如,HER2/neu受体)的结合,也可以将抗体连接到干扰素的内部区域,或者反过来,可以将干扰素连接到抗体的内部或者任一端。
可以通过本领域技术人员熟知的任何手段将抗体(例如,C6抗HER2/neu)与干扰素(例如IFN-α)附着。某些实施方案中,干扰素是直接或者通过接头(间隔臂)与抗体偶联。但是,某些实施方案中优选将嵌合部分重组表达为融合蛋白。
i)导靶部分与干扰素的化学偶联。
某些实施方案中,导靶部分(例如,诸如C6.5、C6MH3-B1、G98A、ML3-9、H3B1、B1D2等的抗HER2/neu抗体)与干扰素分子(例如IFN-α)化学偶联。将分子化学地偶联的手段是本领域技术人员已知的。
用于偶联两个分子的程序根据试剂的化学结构而不同。多肽一般含有各种功能基团(例如羧酸(COOH)或游离氨基(-NH2))可供与另一个肽上的或者连接这两个分子的接头上的合适功能基团进行反应。
替代的,可以将抗体和/或IFN-α衍生从而暴露或者附着上其他反应性功能基团。衍生反应可以包括附着上诸如Pierce Chemical Company(Rockford,Illinois)销售的多种接头中的任意一种。
“接头”用于本文一般是指用于将抗体与IFN-α连在一起的分子。在多项实施方案中,接头能够与抗体和IFN-α均形成共价键。合适的接头是本领域技术人员已知的,包括但不限于,直连或支链碳接头、杂环碳接头或者肽接头。某些实施方案中,接头可以与抗体和/或IFN-α的氨基酸通过其侧链基团(例如通过与半胱氨酸的二硫键)连接。某些优选实施方案中,接头被连接到抗体和/或IFN-α末端氨基酸的α碳氨基和/或羧基基团上。
可以使用双功能接头,即带有一个能与抗体上的基团反应的功能基团和另一个能够与IFN-α反应的基团,来形成所需的偶联物。替代地,衍生反应可以包括导靶部分的化学处理。用于产生例如多肽(比如抗体或抗体片段)上的游离巯基的程序是已知的(参见美国专利4,659,839)。
用于将包括放射性核素金属螯合剂、毒素和药物的各种化合物附着于蛋白质的许多程序和接头是已知的。参见例如,欧洲专利申请188,256;美国专利4,671,958、4,659,839、4,414,148、4,699,784、4,680,338、4,569,789和4,589,071;以及Borlinghaus et al.(1987)Cancer Res.47:4071-4075。尤其是各种免疫毒素的制备是本领域公知的,可以在例如“MonoclonalAntibody-Toxin Conjugates:Aiming the Magic Bullet,”Thorpe et al.,Monoclonal Antibodies in Clinical Medicine,Academic Press,pp.168-190(1982);Waldmann(1991)Science,252:1657;美国专利4,545,985和4,894,443等中找到。
ii)融合蛋白的产生。
某些实施方案中,利用重组DNA技术合成嵌合导靶部分-干扰素融合蛋白。通常,这包括产生编码所述融合蛋白的DNA序列,将DNA置于受到特定启动子调控的表达盒中,在宿主中表达蛋白,分离表达产生的蛋白,以及如果需要将蛋白复性。
编码本发明的融合蛋白的DNA可以通过任何合适的方法制备,包括例如合适的序列的克隆和限制性消化,或者通过诸如Narang et al.(1979)Meth.Enzymol.68:90-99的磷酸三酯法;Brown et al.(1979)Meth.Enzymol.68:109-151的磷酸二酯法;Beaucage et al.(1981)Tetra.Lett.,22:1859-1862)的二乙基亚磷酸酰胺方法;美国专利4,458,066的固体支持物方法等直接化学合成。
化学合成产生单链寡核苷酸。这可以通过与互补序列杂交,或者将该单链作为模板用DNA聚合酶通过聚合转化为双链DNA。本领域技术人员会认识到,虽然DNA化学合成限于大约100碱基的序列,通过较短序列的连接可以得到更长的序列。
替代地,可以克隆亚序列,并用合适的限制酶将适当的亚序列切割。然后可以将片段连接产生所需的DNA序列。
某些实施方案中,利用DNA扩增方法,比如聚合酶链式反应(PCR)克隆编码本发明融合蛋白的DNA。因此,例如利用含有例如NdeI限制位点的有义引物和含有HindIII限制位点的反义引物PCR扩增IFN-α的基因。这样可以产生编码成熟IFN-α序列并带有末端限制位点的核酸。带有“互补”限制位点的抗体可以类似地克隆,然后连接到IFN-α上,和/或连接到附着在IFN-α上的接头上。核酸序列的连接和插入载体产生编码与抗HER2/neu抗体相连的IFN-α的载体。
虽然可以将两个分子直接连在一起,本领域技术人员明白,可以通过由一或多个氨基酸构成的肽间隔臂将分子隔开。通常间隔臂除了将蛋白连接起来或者保持它们之间的某个最小距离或其他空间关系之外,没有具体的生物活性。但是,在某些实施方案中,可以选择间隔臂的组成氨基酸能够影响分子的某些属性,比如折叠、净电荷或疏水性。
但是,令人惊讶的是某些接头不适合用于制备本发明的融合蛋白。因此,例如(Gly4Ser)3(SEQ ID NO:31)接头不太适合用于制备抗CD20-IFN-α构建体。不受到具体理论的限制,据信干扰素通过蛋白水解被从融合蛋白上去除。利用抗Fc和抗干扰素进行的Western印迹分析证实上面的条带均为重链,但只有最大条带含有干扰素。
相应地,在某些优选实施方案中,希望使用抗蛋白水解的接头。某些优选接头是(Gly4Ser)3(SEQ ID NO:31)接头以外的接头。某些优选接头是长度低于15个氨基酸的接头,或者是低于14、13、12、11、10、9、8、7、6、5、4、3或2个氨基酸的接头。某些实施方案中,接头是最多约12或13或14个氨基酸长的α螺旋接头。
某些非常适合用于本发明的构建体的示范性蛋白水解抗性接头如表2所示。
表2.示范性的抗蛋白水解的接头。
接头序列 | SEQ ID NO |
GGGGS | 32 |
AEAAAKEAAAKA | 33 |
A(EAAAK)nA,其中n=1、2、3、4或5 | 34 |
GGGGG | 35 |
GGGGGGGG | 36 |
GGAGG | 37 |
GAGAGAGAGA | 38 |
RPLSYRPPFPFGFPSVRP | 39 |
YPRSIYIRRRHPSPSLTT | 40 |
TPSHLSHILPSFGLPTFN | 41 |
RPVSPFTFPRLSNSWLPA | 42 |
SPAAHFPRSIPRPGPIRT | 43 |
APGPSAPSHRSLPSRAFG | 44 |
PRNSIHFLHPLLVAPLGA | 45 |
MPSLSGVLQVRYLSPPDL | 46 |
SPQYPSPLTLTLPPHPSL | 47 |
NPSLNPPSYLHRAPSRIS | 48 |
LPWRTSLLPSLPLRRRP | 49 |
PPLFAKGPVGLLSRSFPP | 50 |
VPPAPVVSLRSAHARPPY | 51 |
LRPTPPRVRSYTCCPTP | 52 |
PNVAHVLPLLTVPWDNLR | 53 |
CNPLLPLCARSPAVRTFP | 54 |
可以在多种宿主细胞中表达编码融合蛋白的核酸序列,包括大肠杆菌、其他细菌宿主、酵母和各种高等真核细胞,比如COS、CHO和HeLa细胞系和骨髓瘤细胞系。重组蛋白基因通常可操纵地连接有适合各个宿主的表达调控序列。对于大肠杆菌,表达调控序列包括诸如T7、trp或λ启动子的启动子,核糖体结合位点以及优选转录终止信号。对于真核细胞,调控序列包括启动子,优选来源于免疫球蛋白基因、SV40、巨细胞病毒等的增强子,和多聚腺苷酸化序列,还可以包括剪接供体和受体序列。
本发明的质粒可以通过公知技术转移到所选的宿主细胞中,比如用于大肠杆菌的氯化钙转化,用于哺乳动物细胞的磷酸钙处理或电穿孔。可以通过由质粒所含基因(比如amp、gpt、neo和hyg基因)赋予的抗生素抗性来挑选转化了质粒的细胞。
表达后,重组融合蛋白可以按照本领域的常规程序进行纯化,包括硫酸铵沉淀、亲和柱、柱层析、凝胶电泳等(参见,R.Scopes(1982)ProteinPurification,Springer-Verlag,N.Y.:Deutscher(1990)Methods in EnzymologyVol.182:Guide to Protein Purification.,Academic Press,Inc.N.Y.等)。对于药物用途,优选至少约90到95%同质性,最优选98%到99%或以上同质性的基本纯的成分。一旦部分纯化,或者达到所需同质性纯化后,多肽可以用于治疗。
本领域技术人员会认识到,化学合成、生物表达或纯化后,融合蛋白(例如抗HER2/neu-IFN-α,抗CD20-IFN-α等)具有的构象可能与组成多肽的天然构象非常不同。这种情况下,可能需要将多肽变性和还原,然后使多肽重新折叠成优选的构象。蛋白的还原和变性方法以及诱导重新折叠的方法是本领域技术人员已知的(参见例如,Debinski et al.(1993)J.Biol.Chem.,268:14065-14070;Kreitman and Pastan(1993)Bioconjug.Chem.,4:581-585;和Buchner,et al.(1992)Anal.Biochem.,205:263-270)。例如Debinski et al.描述了内含体蛋白在胍-DTE中的变性和还原。然后蛋白在含有氧化型谷胱甘肽和L-精氨酸的氧化还原缓冲液中重新折叠。
某些实施方案中,可以利用瞬时表达系统来表达本文描述的嵌合构建体。虽然有许多细胞系可能可以使用,一种非常适合瞬时表达的细胞系是293T。进行293T瞬时表达时,在第0天将25ml中9百万个细胞接种到每个150mm组织培养板中。用无菌水制备1mg/ml PEI(聚乙烯亚胺)。为了表达完整抗体或抗体融合蛋白,每个板中用H和L各25μg(共50μg)。每个150mm板的转染使用5ml体积。将DNA与DMEM混合,然后加入PEI,混合物在室温下温育10分钟。每μg DNA使用1.75μg PEI。要进行转染时,移出旧培养基,丢弃并换上20ml新鲜培养基(Iscoves+5%小牛血清)。加入转染混合物,旋转培养板。第2天,将培养基换成30ml含有1%FBS(胎牛血清)的Iscoves培养基以便尽量减少存在的牛Ig的量。第4、6和13天,通过移去培养基并用30ml含有1%FBS的新鲜Iscover代替来由细胞收集上清。
实施例1中阐述了抗HER2/neu-IFN-α融合蛋白的克隆和表达,而实施例2中显示了抗CD20-IFN-α融合蛋白的克隆和表达。
本领域技术人员能够认识到这些表达方法是说明性的而非限制性的。可以对本文描述的融合蛋白进行修饰而不会使它们的活性/效力消失。可以进行某些修饰以便协助克隆、表达或者将导靶分子引入融合蛋白。这类修饰是本领域技术人员熟知的,包括例如在氨末端添加甲硫氨酸从而提供起始位点,或者在任意一端添加其他氨基酸从而产生便于使用的限制位点或终止密码子。
可以进行其他修饰以提供血清半衰期和/或生物利用率。这类修饰包括,但不限于引入D-氨基酸(特别是在接头中)、使用非天然的氨基酸、融合蛋白的聚乙二醇化等。
D.其他多价导靶部分。
某些实施方案中,本发明考虑到了利用多价,优选三价、四价、五价或更高价导靶部分(例如,抗HER2/neu抗体、抗CD20抗体等)将干扰素导靶到靶细胞。
例如,多价抗HER2/neu部分可以通过多种方法中的任何一种来产生。例如,具有三个、四个或更多反应位点的接头可以与抗HER2/neu抗体反应以便形成三聚体或更大的偶联体。
在某些实施方案中,可以利用噬菌体展示、酵母展示、细菌展示或其他展示系统来表达和展示多拷贝(例如,至少3个、至少4个、至少5个、至少6个拷贝等)导靶(例如,抗HER2/neu、抗CD20等)抗体,从而有效提供多价导靶部分。
II.组合使用。
本发明的嵌合构建体可以用于抑制靶细胞(例如癌细胞)的生长和/或增殖。在多项实施方案中,可以使用嵌合部分来抑制疾病的发展、缩小肿瘤大小和/或稳定病情的衰退/缓解。
尤其是在癌症的治疗中,本发明的组合物和方法还可以包括其他治疗上和/或药物学可接受的试剂。例如,所述组合物和方法可以包括其他治疗癌症的试剂。这类试剂包括,但不限于烷化剂(例如,二氯甲基二乙酸(氮芥(Mustargen))、环磷酰胺(Cytoxan、Neosar)、异环磷酰胺(Ifex)、苯丙氨酸氮芥、美法仑(melphalen)(爱克兰(Alkeran))、苯丁酸氮芥(chlorambucol)(瘤可宁(Leukeran))、尿嘧啶氮芥、雌氮芥(estramustine)(Emcyt)、噻替派(thiotepa)(Thioplex)、白消安(busulfan)(Myerlan)、罗氮芥(lomustine)(CeeNU)、卡氮芥(carmustine)(BiCNU,BCNU)、链脲霉素(streptozocin)(Zanosar)、达卡巴嗪(dacarbazine)(DTIC-Dome)、顺铂(Platinol,Platinol AQ)、卡铂(伯尔定Paraplatin)、六甲蜜胺(altretamine)(Hexalen)等);抗代谢物(例如,甲氨蝶呤(Amethopterin、Folex、Mexate、Rheumatrex)、5-氟尿嘧啶(Adrucil、Efudex、Fluoroplex)、氟脱氧尿苷(floxuridine)、5-氟脱氧尿苷(fluorodeoxyuridine)(FUDR)、卡培他滨(capecitabine)(希罗达(Xeloda))、氟达拉滨(fludarabine)(福达华(Fludara))、阿糖胞苷(cytosine arabinoside)(Cytaribine、Cytosar、ARA-C)、6-巯基嘌呤(Purinethol)、6-硫鸟嘌呤(Thioguanine)、吉西他滨(gemcitabine)(Gemzar)、克拉屈滨(cladribine)(Leustatin)、脱氧考福霉素(deoxycoformycin)、喷司他丁(pentostatin)(Nipent)等);抗生素(例如多柔比星(doxorubicin)(亚德里亚霉素(Adriamycin)、Rubex、Doxil、柔红霉素脂质体制品)、正定霉素(daunorubicin)(道诺霉素(Daunomycin),Cerubidine)、伊达比星(idarubicin)(Idamycin)、戊柔比星(valrubicin)(Valstar)、米托蒽醌(诺肖林(Novantrone))、更生霉素(dactinomycin)(放线菌素D、Cosmegen)、光辉霉素(mithramycin)、普卡霉素(plicamycin)(Mithracin)、丝裂霉素(mitomycin)C(突变霉素Mutamycin)、博来霉素(Blenoxane)、丙卡巴肼(procarbazine)(Matulane)等);有丝分裂抑制剂(例如太平洋紫杉醇(paclitaxel)(Taxol)、多烯紫杉醇(docetaxel)(Taxotere)、硫酸长春碱(vinblatine sulfate)(Velban、Velsar、VLB)、硫酸长春新碱(vincristine sulfate)(Oncovin、Vincasar PFS、Vincrex)、硫酸长春瑞滨(vinorelbine sulfate)(Navelbine)等);染色质功能抑制剂(例如拓扑替康(topotecan)(Camptosar)、伊立替康(irinotecan)(Hycamtin)、依托泊苷(etoposide)(VP-16、VePesid、Toposar)、替尼泊苷(teniposide)(VM-26、Vumon)等);激素和激素抑制剂(例如,己烯雌酚(diethylstilbesterol)(Stilbesterol、Stilphostrol)、雌二醇(estradiol)、雌激素、酯化雌激素(esterified estrogens)(Estratab、Menest)、雌氮芥(estramustine)(Emcyt)、它莫西芬(tamoxifen)(Nolvadex)、托瑞米芬(toremifene)(Fareston)、瑞宁得(anastrozole)(Arimidex)、来曲唑(letrozole)(Femara)、17-OH-孕酮(progesterone)、甲羟孕酮(medroxyprogesterone)、醋酸甲地孕酮(megestrol acetate)(Megace)、戈舍瑞林(goserelin)(Zoladex)、亮脯利特(leuprolide)(Leupron)、睾丸酮(testosteraone)、甲基睾丸酮(methyltestosterone)、氟甲睾酮(fluoxymesterone)(Android-F、Halotestin)、氟他米特(flutamide)(Eulexin)、比卡鲁胺(bicalutamide)(Casodex)、尼鲁米特(nilutamide)(Nilandron)等);合成抑制剂(例如,氨鲁米特(aminoglutethimide)(Cytadren)、酮康唑(ketoconazole)(Nizoral)等);免疫调节剂(例如,利妥昔单抗(Rituxan)、曲妥珠单抗(trastuzumab)(赫赛汀)、denileukin diftitox(Ontak)、左旋咪唑(levamisole)(Ergamisol)、卡介苗BCG(TheraCys,TICE BCG)、干扰素α-2a、α-2b(罗扰素(Roferon)-A,Intron A)、白介素-2、阿地白介素(aldesleukin)(普留净ProLeukin)等)以及其他试剂,比如l-天冬酰胺酶(Elspar、Kidrolase)、培门冬酶(pegaspasgase)(Oncaspar)、羟基脲(hydroxyurea)(Hydrea、Doxia)、甲酰四氢叶酸(leucovorin)(Wellcovorin)、米托坦(mitotane)(Lysodren)、卟吩姆(porfimer)(Photofrin)、维A酸(tretinoin)(Veasnoid)等。
III.药物组合物。
为了实施本发明的方法,将一或多种本发明的活性试剂(嵌合部分)给予例如被诊断为患有癌症的个体。活性制剂可以以天然形式给予,或者如果需要的话,只要其盐、酯、胺、前药、衍生物适合用于药物(即在所述方法中是有效的),可以给予盐、酯、胺、前药、衍生物等形式。活性制剂的盐、酯、胺、前药和其他衍生物可以利用有机合成化学领域技术人员已知的和例如March(1992)Advanced Organic Chemistry;Reactions,Mechanisms andStructure,4th Ed.N.Y.Wiley-Interscience中描述的常规程序来制备。
例如,酸加成盐可以由游离碱,利用一般涉及到与适宜的酸进行反应的常规方法来制备。通常,将药物的碱形式溶解在诸如甲醇或乙醇的极性有机溶剂中,然后向其中加入酸。将得到的盐沉淀或者通过再加入极性较弱的溶剂使它从溶液中萃取出来。适合制备酸加成盐的酸包括有机酸(例如乙酸、丙酸、羟基乙酸、丙酮酸、草酸、苹果酸、丙二酸、琥珀酸、马来酸、富马酸、酒石酸、柠檬酸、苯甲酸、肉桂酸、杏仁酸、甲基磺酸、乙磺酸、p-甲苯磺酸、水杨酸等)和无机酸(例如盐酸、氢溴酸、硫酸、硝酸、磷酸等)。可以通过用合适的碱处理将酸加成盐转化为游离碱。本文中特别优选的活性试剂的酸加成盐是利用诸如氯化酸或溴化酸制备的卤化盐。发过来,本发明的活性试剂的碱性盐制品是利用药物可接受的碱,比如氢氧化钠、氢氧化钾、氢氧化铵、氢氧化钙、三甲胺等以类似的方式制备的。特别优选的碱性盐包括碱性金属盐,例如钠盐和铜盐。
酯类的制备一般包括药物分子结构中可能存在的羟基和/或羧基的功能化。酯通常是游离醇基的酰基取代衍生物,即来源于通式RCOOH羧酸的部分,其中R是烷基,并且优选是低等烷基。如果需要,可以通过利用常规氢解或水解过程将酯重新转化为游离酸。
酰胺和前药也可以利用本领域技术人员已知的或者相关文献中描述过的技术制备。例如,可以利用合适的胺反应物由酯制备酰胺,或者可以由无水或酸性氯化物通过与氨或低等烷基胺反应来制备。前药的制备通常是通过共价附着上一个部分,得到直至被个体的代谢系统修饰才具备治疗活性的化合物。
本文中确认的活性试剂可以用于胃肠外、外用、口腔、鼻(或者吸入)、直肠给药,或者通过诸如气雾剂或跨膜的局部给药,对本文描述的一或多种病态/指征(例如动脉粥样硬化和/或其症状)进行预防和/或治疗性处置。根据给药方法,可以以多种单位剂量形式给予药物组合物。合适的单位剂量形式包括,但不限于粉末、片剂、药丸、胶囊、锭剂、栓剂、贴片、鼻腔喷雾、注射剂、植入式缓释制剂、脂类复合体等。
本发明的活性试剂通常与药物可接受的载体(赋形剂)组合形成药物组合物。药物可接受的载体含有一种或多种生理可接受的化合物,所述化合物可以例如稳定药物组合物或者增加或减少活性试剂的吸收。生理可接受的化合物可以包括例如,诸如葡萄糖、蔗糖或右旋糖的碳水化合物;诸如抗坏血酸或谷胱甘肽的抗氧化剂;螯合剂;低分子量蛋白;诸如脂类的保护剂和摄取增强剂;减少活性试剂的清除或水解的组合物;或者赋形剂或其他稳定剂和/或缓冲液。
其他生理可接受的化合物包括湿润剂、乳化剂、分散剂,或者对防止微生物的生长或作用特别有用的防腐剂。各种防腐剂是已知的,包括例如酚和抗坏血酸。本领域技术人员明白,药物可接受载体,包括生理可接受化合物的选择取决于例如活性试剂的给药途径和活性试剂的具体生理-化学特性。
赋形剂优选是无菌的,并且通常不含不需要的物质。这类组合物可以通过常规的已知灭菌技术来灭菌。
在治疗应用中,将本发明的组合物以能够有效地防止和/或治愈或者至少部分防止或遏制疾病和/或其并发症的量给予患有例如癌症或者有患癌症风险(例如在手术切除原发肿瘤)的病人。足够实现这一点的量定义为“治疗有效剂量”。这类用途的有效量取决于疾病的严重程度和患者的整体健康情况。根据患者需要的并且能够耐受的剂量和频率,可以将组合物单次或多次给药。任何情况中,组合物都应当提供足够量的发明所述制剂中的活性试剂,以便有效治疗(缓解一种或多种症状)患者。
活性剂的浓度可能有很大的不同,主要根据所选的具体给药方式和患者的需求,基于液体体积、粘性和体重等来选择。但是一般选择的浓度能够提供约0.1或1mg/kg/天到约50mg/kg/天,或者有时更高的剂量。典型的剂量在约3mg/kg/天到约3.5mg/kg/天的范围内,优选约3.5mg/kg/天到约7.2mg/kg/天,更优选约7.2mg/kg/天到约11.0mg/kg/天,最优选约11.0mg/kg/天到约15.0mg/kg/天的范围内。某些优选实施方案中,剂量处于约10mg/kg/天到约50mg/kg/天的范围内。某些实施方案中,剂量在约20mg到约50mg,每天口服两次。应当明白,这些剂量可以变动,从而优化具体个体或个体组的治疗方案。
某些优选实施方案中,本发明的活性试剂是(例如以片剂)口服的,或者是按照本领域技术人员已知的标准方法注射的。其他优选实施方案中,肽还可以利用常规的跨膜药物递送系统,即跨膜“贴片”(通常其中的活性试剂含在层压结构中,该结构作为药物递送装置固着到皮肤上)进行递送。在这类结构中,药物组合物一般含在位于上衬层下面的层或“储库”中。应当理解,术语“储库”在该语境中是指最终可供递送到皮肤表面的“活性成分”的量。因此,例如“储库”可能包括贴片衬层上粘着剂中的活性成分,或者本领域技术人员已知的多种不同基质制剂中任一种中的活性成分。贴片可以含有单个储库,或者可以含有多个储库。
在一个实施方案中,储库包含药物可接受的接触粘合材料构成的聚合物基质,其将系统在药物递送过程中固着到皮肤上。合适的皮肤接触粘合材料实例包括,但不限于聚乙烯、聚硅氧烷、聚异丁烯、聚丙烯酸酯、聚氨酯等。替代地,含有药物的储库和皮肤接触粘合以单独和分开的层次存在,其中粘合剂位于储库之下,这种情况中,储库可以是上面描述过的聚合物基质,或者可以是液体或水凝胶储库,或者采取其他形式。这些板层中作为装置上表面的衬层,优选是“贴片”中的主要结构单元,为装置提供大部分的柔性。选择作为衬层的材料优选对活性试剂和其他存在的材料是基本通透的。
某些实施方案中,可以通过使用缓释蛋白“包装”系统来维持提高的血清半衰期。这类缓释系统是本领域技术人员已知的。在一个优选实施方案中,用于蛋白质和肽的ProLeaseTM生物可降解微球递送系统(参见,例如Tracy(1998)Biotechnol.Prog.14:108;Johnson et al.(1996),Nature Med.2:795;Herbert et al.(1998),Pharmaceut.Res.15,357)是由生物可降解聚合微球构成的干粉,其中所述微球含有在聚合物基质中的活性试剂,该系统可以在有或没有其他试剂的情况下复合成干制剂。
ProLeaseTM微球制造过程经过特别设计,能够在维持活性试剂的完整性的同时达到高的封装效率。所述过程包括(i)通过将药物溶液与稳定赋形剂喷雾冻干来大量制备冻干的药物颗粒,(ii)制备药物聚合物悬浮液,然后超声处理或均质化来减小药物颗粒的大小,(iii)通过喷雾到液氮中产生冷冻的药物聚合物微球,(iv)用乙醇萃取聚合物溶剂,以及(v)过滤和真空干燥产生最终的干粉产品。这样得到的粉末含有固体形式的活性试剂,活性试剂是均质的,僵硬地分散在多孔聚合物颗粒中。该过程中最常用的聚合物-丙交酯乙交酯共聚物(lactide-co-glycolide)(PLG)是生物相容的和生物可降解的。
封装(encapsulation)可以在低温(例如-40℃)实现。在封装过程中,蛋白质在没有水的情况下保持固态,从而使得蛋白质被水诱导的构象变化最小化,防止那些包括水作为反应物的蛋白降解反应,并且避免形成有机相-水相界面使蛋白在那里发生变性。优选的过程使用多数蛋白不能溶于其中的溶剂,从而达到高封装效率(例如,高于95%)。
另一个实施方案中,可以以“浓缩物”来提供溶液中的一或多种成分,例如在可即时稀释的存储容器中(例如,预先量好的体积),或者以可加入一定体积的水中的可溶性胶囊。
以上制剂和给药方法意在说明而非限制。应当明白,利用本文提供的教导,可以容易地设计其他合适的制剂和给药模式。
IV.试剂盒。
在某些实施方案中,本发明提供了用于治疗原发性癌症和/或用在辅助疗法中的试剂盒。试剂盒一般包含含有本发明的嵌合部分(例如,抗HER2/neu-IFN-α、抗CD20-IFN-α等)的容器。嵌合部分可以存在于药物可接受的赋形剂中。
此外,试剂盒可以任选包含说明材料,公开如何使用嵌合部分(例如治疗癌症和/或作为联合疗法)的方法。说明材料任选还可以教导优选剂量、计数器显示等。
试剂盒还可以包含其他成分以协助试剂盒所预期的具体应用。因此,例如还包含用于给伤口消毒、止痛、附着上敷料等的装置。
虽然说明材料一般包含书写或打印的材料,但并不局限于此。任何能够存储这类说明并将它们传达给终端使用者的媒介都是本发明考虑的。这类媒介包括,但不限于电子存储媒介(例如,磁盘、磁带、盒式磁带、芯片)、光学媒介(例如CD ROM)等。这类媒介可以包含提供说明材料的网址。
实施例
以下实施例仅供对发明进行阐述,而非限制。
实施例1
抗Her2/Neu IgG3和IFN-α融合蛋白显示抗B细胞淋巴瘤的强细胞凋亡和抗肿瘤活性
在本项研究中,我们构建了由带有C6MH3-B1(20)可变区的抗HER2/neu-IgG3和IFN-α构成的融合蛋白,并研究了它对表达人HER2/neu的鼠B细胞淋巴瘤38C13(38C13/HER2)的效果。考虑到该B细胞淋巴瘤对IFN-α(21)的反应性,我们选择在这个模型中评估IFN-α靶向肿瘤的能力。IFN-α与Ab的融合显著提高了它的体内半衰期。我们发现抗HER2/neu-IgG3-IFN-α有效地抑制了小的和已建立的38C13/HER2肿瘤的体内生长,同时在有效剂量没有观察到系统毒性的迹象。抗HER2/neu-IgG3-IFN-α抑制了38C13/HER2细胞的生长并诱导了其细胞凋亡。这些结果表明IFN-α与肿瘤特异性Ab的融合产生了能够有效治疗B细胞淋巴瘤的药剂。
材料与方法
细胞系和培养条件
38C13是来源于C3H/HeN小鼠的高度恶性鼠B细胞淋巴瘤。表达人HER2/neu的38C13(38C13/HER2)的构建和表征已有描述(6)。38C13和38C13/HER2均培养在补充了2mM L-谷氨酰胺、10U/ml盘尼西林、10微克/ml链霉素(GPS;Sigma-Aldrich)和10%小牛血清(Atlanta Biologicals)的IMDM(Irvine Scientific)中。鼠骨髓瘤P3X63Ag8.653(美国典型培养物保藏中心)及其表达抗HER2IgG3-IFN-α或IgG3-IFN-α的衍生物生长在补充了10%小牛血清和GPS的IMDM中。L929成纤维细胞(美国典型培养物保藏中心)培养在含有5%小牛血清和GPS的IMDM中。过表达人HER2/neu的鼠结肠腺癌细胞系CT26/HER2的构建和表征已有描述(6)。CT26/HER2培养在含有5%小牛血清和GPS的IMDM中。
质粒构建。
将抗人HER2/neu scFv:C6MH3-B1的H和L链可变区分别插入人γ3H链(pAH4802)和κL链(pAG4622)表达载体(22),并用于产生该特异性的嵌合IgG3。为了构建抗人HER2/neu-IgG3(C6MH3-B1)-IFN-α融合蛋白,首先利用PCR给成熟小鼠IFN-α基因上游导入BamH1限制酶位点,下游导入XbaI限制酶位点,其中IFN-α基因是用正向引物5'-CGC GGA TCC TGT GAC CTGCCT CAG ACT C-3(SEQ ID NO:55)和反向引物5'-GCT CTA GAT CAT TTCTCT TCT CTC AGT CTT C-3(SEQ ID NO:56)由BALB/c小鼠的基因组DNA经PCR扩增的。将最终的PCR产物连接到TA载体中。得到的载体在测序后,用BamH1和XbaI消化从而释放出插入载体pAH9612中的DNA片段,pAH9612含有IgG3恒定区,其中C6MH3-B1H链可变区和GGGGSGGGGSGGGGS(SEQ ID NO:57)肽接头位于CH3末端。最后的PCR产物pAH9616含有抗HER2/neu-IgG3,之后是GGGGSGGGGSGGGGS(SEQID NO:57)肽接头和小鼠IFN-α。
重组蛋白的生产和纯化
将编码融合了IFN-α的含有C6MH3-B1可变区的IgG3H链的质粒,以960μFd电容和0.2V的脉冲经电穿孔转染到P3X63Ag8.653细胞中,所述细胞或者表达含有C6MH3-B1可变区(23)的L链,从而产生抗HER2/neu-IgG3-IFN-α;或者表达非特异性的L链(4D5;Genentech)(6),从而产生IgG3-IFN-α。挑选产生抗HER2/neu(C6MH3-B1)-IgG3、抗HER2/neu(C6MH3-B1)-IgG3-IFN-α或IgG3-IFN-α的转染体,并按照以前的描述(6)进行表征。利用固定在Sepharose4B fast flow(Sigma-Aldrich)上的蛋白G从培养物上清中纯化抗HER2/neu(C6MH3-B1)-IgG3,利用固定在Sepharose4B fast flow(Sigma-Aldrich)上的蛋白A从培养物上清中纯化抗HER2/neu(C6MH3-B1)-IgG3-IFN-α和IgG3-IFN-α。将SDS-PAGE分离的蛋白进行考马斯蓝染色来评价蛋白的纯度和完整性。用国立卫生院提供的小鼠IFN-α的国际参考标准来确定融合蛋白的IFN活性。rIFN-α从PBL BiomedicalLaboratories获得。
IgG3-IFN-α融合蛋白的FPLC分析
为了确定融合蛋白在溶液中是以单体和/或聚合物存在的,将混合了作为内部对照的400μg OVA的100μg IgG3-IFN-α在30x1.5-cm Superose6柱上通过凝胶过滤进行分析,其中所述Superose6柱子连接快速蛋白液相层析(FPLC),分析使用PBS,以0.5ml/分钟流速进行。在同一柱子上进行IgA2m(主要以分子量350kDa的二体Ab存在)、由Miles IgG(分子量150kDa)和OVA(分子量45kDa)组成的混合物的凝胶过滤以提供分子量标准。
HER2/neu结合活性的流式细胞术分析
为了检测各种抗HER2/neu融合蛋白与CT26/HER2细胞的反应性,将1x106细胞与10pM融合蛋白在4℃温育1小时。某些试验中,融合蛋白在与CT26/HER2细胞温育前,先与900U肝素于4℃温育17小时。然后细胞与生物素化的1/100稀释的大鼠抗人IgG(BD Biosciences)反应。用1/1500稀释的PE标记链霉亲和素(BD Biosciences)检测结合的生物素化Abs,通过流式细胞术用FACScan(BD Biosciences)分析细胞。
IFN-α抗病毒活性
对水泡性口炎病毒(VSV)感染敏感的L-929成纤维细胞系被用于定量IFN-α的生物活性。将L-929细胞以4x104细胞/孔的密度接种在96孔组织培养板(Falcon;BD Biosciences)中,于37℃在5%CO2气中温育过夜。之后,加入不同IFN-α融合蛋白或标准IFN-α(小鼠IFN-α的国际参考标准;National Institutes of Health,Bethesda,MD)的梯度稀释液,培养板在37℃温育24小时。然后向每个孔中加入四千PFU的VSV,于37°C再温育48小时。存活的贴壁细胞用50μl结晶紫(溶于20%乙醇的0.05%溶液)染色10分钟。将培养板水洗,加入100μl100%甲醇将存留的染料溶解。用ELISA检测仪在595nm读培养板。
抗HER2/neu-IgG3-IFN-α抗增殖效果的检测法
简单来说,将38C13或38C13/HER2细胞以1.25x104细胞/孔的密度接种在96孔组织培养板中,加入不同融合蛋白的梯度稀释液。然后将培养板在5%CO2气中于37℃温育48小时。加入20μl MTS溶液(Promega)使培养板显色,并用ELISA检测仪在490nm进行分析。增殖抑制率(百分数)计算为:
100x[(OD试验–OD空白)/(OD培养基-OD空白)]x100。
细胞凋亡的检测法
简单来说,将1x106细胞用不同融合蛋白处理72小时。然后用冰冷的PBS清洗细胞。使用Vybrant Apoptosis Assay Kit2(Molecular Probes),按照制造商建议的步骤进行膜联蛋白V/碘化丙啶(PI)检测。
标记CFSE的38C13/HER2肿瘤细胞的增殖
简单来说,将1x106细胞与2.5μM CFSE(Molecular Probes)于37℃温育10分钟。然后细胞用1nM不同融合蛋白处理48小时,使用CellTrace CFSECell Proliferation Kit(Molecular Probes),按照制造商建议的步骤进行流式细胞术。
小鼠
使用从Taconic Farms得到的6–8周龄的雌性C3H/HeN小鼠。将动物用带有木刨花床垫的高压灭菌的聚碳酸酯笼子关在实验室。动物可随意取食和水。以12/12小时的昼夜循环给予人工光照。实验室的温度为20℃,每小时进行10-15次换气。
半衰期
将小鼠rIFN-α(PBL Biomedical Laboratories)、IgG3-IFN-α和抗HER2/neu-IgG3-IFN-α用Iodo-Beads(Pierce),按照制造商的试验方案用125I碘化为10μCi/μg。小鼠i.p.注射66μCi of125I标记的蛋白。在注射125I标记的rIFN-α、IgG3-IFN-α或抗HER2/neu-IgG3-IFN-α后的不同间隔,用小鼠全身放射性计数器(Wm.B.Johnson and Associates)测量存留的放射性。
肿瘤攻击和Ab疗法
C3H/HeN小鼠经s.c.接受100038C13/HER2肿瘤细胞。在肿瘤攻击后的第1、3和5天或者12、13和14天,通过i.p.注射给予治疗。每隔一天对肿瘤进行测量,使用以下公式估计肿瘤体积(立方毫米):[长(mm)x宽(mm)x高(mm)]/2(24)。观察动物直至s.c.肿瘤的长度达到15mm或者直至观察到任何小鼠遭受折磨或似乎频临死亡。达到这些条件的动物按照研究所政策人道地处死。
Western印迹分析和Ab
简单来说,38C13/HER2细胞用不同融合蛋白处理指定的时间,用冰冷的PBS清洗,在裂解缓冲液(0.125%Nonidet P-40、0.875%Brij97、10mMTris-HCl(pH7.5)、2mM EDTA、0.15M NaCl、0.4mM Na3VO4、0.4mM NaF、1mM PMSF、2.5μM亮肽素(leupeptin)和2.5μM抑酶肽(aprotinin))中在冰上裂解10分钟。细胞裂解物于4℃以10,000x g澄清10分钟。然后将蛋白样品在样品缓冲液中煮沸,随后在8%SDS-PAGE胶上分离,并转移至聚偏氟乙烯微孔膜(Millipore)。用溶于150mM NaCl、50mM Tris-HCl(pH7.6;TBS)的3%BSA在室温下封闭1小时后,印迹用指定的第一Abs在4℃过夜检测。然后印迹在室温下用溶于TBS的0.05%Tween20清洗3次,与合适的偶联了HRP的二抗温育,并借助过氧化物酶催化的ECL检测系统(ECL;Pierce)检测。多克隆兔抗磷酸化STAT1购买自Cell Signaling Technology。多克隆偶联HRP的驴抗兔IgG购买自Amersham Biosciences。多克隆兔抗GAPDH购买自Abcam。
统计分析
体外研究用双尾Student’s t检验,动物存活曲线用对数秩检验(Mantel-Cox)分析进行统计分析。
结果
抗HER2/neu-IgG3-IFN-α的生产和表征
带有C6MH3-B1(20)可变区的抗HER2/neu-IgG3的构建和表达以前已有描述(23)。成熟小鼠IFN-α的氨基末端被融合到抗HER2/neu-IgG3的羧基末端,之间被柔性的[(Gly4)Ser]3(SEQ ID NO:31)接头(图2A)隔开。通过将C6MH3-B1L链代替为4D5(rhuMab HER2,赫赛汀;Genentech)L链构建得到缺乏HER2/neu特异性的相同融合蛋白IgG3-IFN-α。利用蛋白G从培养物上清中纯化得到的蛋白在非还原和还原性条件下通过SDS-PAGE分析(图2B)。在没有还原剂的情况下,抗HER2/neu-IgG3(图2B,泳道1)以170kDa的分子量迁移,而抗HER2/neu-IgG3-IFN-α(图2B,泳道2)和IgG3-IFN-α(图2B,泳道3)是210kDa,这是附着了两个分子小鼠IFN-α的完整IgG3所预期的大小(图2A)。用还原剂处理后,在这些蛋白中可以看到以25kDa分子量迁移的L链(图2B,泳道4-6)。但是,抗HER2/neu-IgG3带有分子量为60kDa的H链(图2B,泳道4),而IgG3-IFN-α(图2B,泳道5)和抗HER2/neu-IgG3-IFN-α(图2B,泳道6)正如预期的,带有分子量80kDa的H链。泳道1下方的条带(图2B)是同样会与蛋白G柱子结合的牛IgG;牛H和L链在泳道4中也可看到(图2B),在泳道5和6中较浅(图2B)。FPLC分析显示IgG3-IFN-α融合蛋白在溶液中以单体存在(数据未显示)。
抗HER2/neu-IgG3-IFN-α的Ag结合和抗病毒活性
抗HER2/neu-IgG3和抗HER2/neu-IgG3-IFN-α均能结合表达高水平人HER2/neu的CT26/HER2细胞,而IgG3-IFN-α仅能微弱结合CT26/HER2(图2C)。多种细胞因子包括IL-1、IL-2、IL-6(25)和IFN-α(26)曾显示与肝素有相互作用。为了确定IgG3-IFN-α和CT26/HER2之间的弱相互作用是否由于肝素结合,在添加给CT26/HER2之前,将蛋白与肝素一起温育。肝素抑制IgG3-IFN-α结合到CT26/HER2细胞上,但是没有抑制抗HER2/neu-IgG3和抗HER2/neu-IgG3-IFN-α的结合(图2C)。
这些结果显示抗HER2/neu-IgG3-IFN-α保留了它结合Ag的能力,IgG3-IFN-α不识别HER2/neu。对VSV感染敏感的L-929成纤维细胞系用于定量融合蛋白与IFN-α标准相比的IFN-α生物活性。抗HER2/neu-IgG3-IFN-α和IgG3-IFN-α在L-929细胞中均表现出~2400U/μg的抵抗VSV诱导型细胞毒性的IFN-α活性,而抗HER2/neu-IgG3未表现抗病毒活性(图2D)。
融合蛋白的体内抗肿瘤活性
为了确定抗HER2/neu-IgG3-IFN-α的体内抗肿瘤活性,同种小鼠s.c.接种1x10338C13/HER2肿瘤细胞,并在肿瘤攻击后第1、3和5天通过i.p.给予不同剂量的蛋白进行处理(图3A-图3B)。用2.5μg IgG3-IFN-α处理过的小鼠显示出肿瘤生长的某些消退,8只小鼠中的一只(13%)在50天后仍存活(图3A)。但是,利用肿瘤特异性Ab将IFN-α体内导靶到肿瘤显著地提高了它的抗肿瘤效果。所有用2.5μg(图3A)抗HER2/neu-IgG3-IFN-α处理过的小鼠在肿瘤攻击后,50天没有肿瘤(与PBS对照相比p=0.0048),并且处理的小鼠均未显示毒性。因此,与连接了非特异性Ab的IFN-α相比,IFN-α导靶到肿瘤细胞表面导致明显的抗肿瘤活性(p=0.007)。当使用较低剂量时,被定靶的抗HER2/neu-IgG3-IFN-α继续表现出强的抗肿瘤活性。用1μg(图3B)抗HER2/neu-IgG3-IFN-α处理过的8只小鼠中的7只(88%)在50天后仍然没有肿瘤。形成强烈对比的是,IgG3-IFN-α处理过的小鼠在这个较低剂量表现出与用PBS处理的小鼠类似的肿瘤生长(p=0.183),8只小鼠中只有1只(13%)存活。当处理增加到三个5μg剂量,抗HER2/neu-IgG3-IFN-α和IgG3-IFN-α均能有效防止肿瘤生长(数据未显示),明确地反映了38C13细胞对IFN-α处理敏感的事实(21,27,28)。用5μg抗HER2/neu-IgG3Ab处理过的小鼠的肿瘤生长情况与PBS对照相同,表明Ab自身没有体内抗肿瘤效果(数据未显示)。这些结果表明通过肿瘤特异性Ab将IFN-α导靶到肿瘤细胞可以显著强化其效力,这一点在给予低剂量时可以明显地看出。重要的是,可以在没有明显毒性的情况下,达到该抗肿瘤活性。
融合了Ab的IFN-α与游离IFN-α相比,抗肿瘤活性提高
正如上文所述,我们发现融合了非肿瘤特异性Ab的IFN-α展示出抗肿瘤活性。为了将它与可溶性rIFN-α的抗肿瘤活性比较,给小鼠s.c接种1x10338C13/HER2肿瘤细胞,并在肿瘤攻击后第1和3天通过i.p.给予9600U(4μg)IgG3-IFN-α或9600U rIFN-α进行处理(图4A)。所有用9600U的IgG3-IFN-α处理过的小鼠均显示延迟的肿瘤生长,75%的小鼠在肿瘤攻击后50天没有肿瘤(p=0.027)。反之,用相同数量单位的rIFN-α处理过的小鼠与PBS对照在它们的肿瘤生长模式方面没有显著不同。
IFN-α的体内半衰期非常短(29)。在以前的研究中,Abs与细胞因子的融合显示能够提高细胞因子的半衰期(6)。125I标记的rIFN-α、IgG3-IFN-α或抗HER2/neu-IgG3-IFN-α的清除在C3H/HeN小鼠中进行检验。小鼠i.p.注射66μCi的125I标记的蛋白,并用小鼠全身计数器测量残存的放射性。rIFN-α被快速清除,到~2.5小时时50%已被除掉(图图4B)。相反,抗HER2/neu-IgG3-IFN-α和IgG3-IFN-α显示出体内半衰期的显著提高,清除50%所注射的放射性需要8小时。IFN-α融合蛋白的抗肿瘤效力可能得益于这个提高的半衰期。因此,IFN-α上融合了IgG3Ab可以显著提高IFN-α的体内抗肿瘤活性。但是,通过将IFN-α导靶至肿瘤可以进一步提高该抗肿瘤活性,使它在较低剂量也有效果。
抗HER2/neu-IgG3-IFN-α体外抑制肿瘤细胞的增殖
IFN-α具备多种活性,其中包括免疫应答和抗肿瘤的直接细胞毒性。为了研究使用抗HER2/neu-IgG3-IFN-α或IgG3-IFN-α时看到的抗肿瘤效果的潜在机制,用1x10338C13/HER2肿瘤细胞攻击一直无肿瘤的8只小鼠(参见图3A)。令人惊讶地,所有小鼠和未处理小鼠一样,快速发展出庞大的肿瘤(数据未显示)。这些结果暗示,在低肿瘤负荷的试验条件下,IFN-α融合蛋白没有启动保护性的获得性免疫反应,相反,其强大的抗肿瘤活性是由先天性免疫系统或者作用于肿瘤细胞的直接细胞毒效果介导的。
为了确定IFN-α融合蛋白是否对肿瘤细胞是直接细胞毒性的,将38C13/HER2或亲代38C13肿瘤细胞与不同蛋白温育48小时,利用MTS检测法测量细胞增殖情况。用抗HER2/neu-IgG3进行的处理未能显著抑制38C13/HER2或亲代38C13肿瘤细胞的增殖(图5A和图5B)。尽管抗HER2/neu-IgG3-IFN-α和IgG3-IFN-α均能抑制38C13/HER2肿瘤细胞的增殖,抗HER2/neu-IgG3-IFN-α比IgG3-IFN-α更有效,两者的IP50值分别为10和100pM(图5A)。相反,抗HER2/neu-IgG3-IFN-α和IgG3-IFN-α对亲代38C13肿瘤细胞表现出类似的抗增殖活性。这些结果提供证据表明IFN-α融合蛋白可以直接抑制B细胞淋巴瘤38C13的增殖,IFN-α被导靶至肿瘤细胞强化了这一效果。
抗HER2/neu-IgG3-IFN-α体外诱导肿瘤细胞中的细胞凋亡
IFN-α信号传导可以在某些肿瘤细胞系中诱发细胞凋亡。为了确定我们观察到的抗增殖效应是否归因于细胞凋亡,利用膜联蛋白V亲和分析法(30)分析用不同蛋白处理过的38C13/HER2细胞将脑磷脂从细胞质膜内层转位到外片层的情况。利用PI将死细胞染色,该染料破坏质膜进入细胞并与DNA结合。与PBS对照相比,用抗HER2/neu-IgG3处理后,死细胞(膜联蛋白V/PI明亮,2%)或早期凋亡细胞(膜联蛋白V明亮,3%)的数量没有增加(图5C)。相反,当用IgG3-IFN-α处理细胞时,死细胞(21%)和早期凋亡细胞(6%)的数量有明显增加。用抗HER2/neu-IgG3-IFN-α进行的处理使得死细胞(33%)和早期凋亡细胞(16%)的数量进一步增加。这些结果表明,IFN-α可以在38C13/HER2肿瘤细胞中诱发细胞凋亡,IFN-α被导靶至肿瘤细胞可以显著提高这一效果。
除了诱发细胞凋亡,IFN-α可以直接抑制肿瘤细胞的增殖(31)。为了确定抑制增殖和细胞凋亡在被处理的肿瘤细胞中是否均有发生,将CFSE标记的8C13/HER2细胞用不同蛋白处理48小时,选通活细胞,通过流式细胞术确定CFSE的水平。抗HER2/neu-IgG3处理过的细胞(图5D,细线)与PBS处理过的细胞的CFSE信号重叠,比标记CFSE后立即固定的细胞的信号明显低(图5D,虚线),表明抗HER2/neu-IgG3未能抑制38C13/HER2的增殖。相反,IgG3-IFN-α明显抑制存活的38C13/HER2细胞的增殖(图5D,粗线),通过HER2/neu-IgG3-IFN-α将IFN-α导靶至38C13/HER2细胞强化了这一效果(图5D,黑色区域)。这些结果表明尽管抗HER2/neu-IgG3-IFN-α处理未能在48小时导致全部细胞死亡,存活细胞的增殖能力降低。
IFN-α融合蛋白在肿瘤细胞中诱发了STAT1激活
尽管结合IFN-α受体能够启动多个STAT蛋白的激活,STAT1在介导IFN-α依赖性的信号传导中起着专门的作用(32)。为了研究IFN-α融合蛋白能否在38C13/HER2中启动IFN-α信号传导,将IFN-α导靶至肿瘤细胞是否增强该效果,我们检验了处理后STAT1的磷酸化。如图6A-6C所示,抗HER2/neu-IgG3-IFN-α和IgG3-IFN-α均能在38C13/HER2内启动强STAT1磷酸化,至10分钟时,STAT1磷酸化提高8倍。但是,抗HER2/neu-IgG3-IFN-α诱发的STAT1磷酸化持续时间更长,抗HER2/neu-IgG3-IFN-α处理过的细胞在30、60和90分钟时观察到更高的STAT1磷酸化。这些结果表明,IFN-α融合蛋白能够在38C13淋巴瘤细胞中诱发IFN-α信号传导,IFN-α被导靶至肿瘤细胞强化了这一效果。
抗HER2/neu-IgG3-IFN-α表现出抗已有肿瘤的强活性
因为抗HER2/neu-IgG3-IFN-α表现出抗38C13/HER2肿瘤细胞的强细胞毒性,我们考察了抗HER2/neu-IgG3-IFN-α能否有效抗已建立的38C13/HER2肿瘤。给同种小鼠s.c.接种1x10338C13/HER2肿瘤细胞,肿瘤攻击后第12、13和14天用5μg(图7)指定蛋白进行i.p.处理。第12天,肿瘤平均大小是100mm3,用PBS或10μg抗HER2/neu-IgG3进行的处理未能抑制肿瘤生长(数据未显示)。用5μg IgG3-IFN-α进行的处理显示抑制肿瘤生长的某些效果;但是,所有小鼠都发展出大块的肿瘤,在肿瘤攻击32天后,没有小鼠存活。相反,用5μg抗HER2/neu-IgG3-IFN-α处理过的全部小鼠的肿瘤生长都得以延迟,8只小鼠中的3只肿瘤完全消退,肿瘤攻击后50天仍没有肿瘤(抗HER2/neu-IgG3-IFN-α相对PBS,p=0.0001;抗HER2/neu-IgG3-IFN-α相对IgG3-IFN-α,p=0.063)。因此,IgG3-IFN-α和抗HER2/neu-IgG3-IFN-α均显示抗肿瘤活性,但抗HER2/neu-IgG3-IFN-α能够更有效地延迟肿瘤生长,只有抗HER2/neu-IgG3-IFN-α处理过的小鼠中观察到了肿瘤的完全缓解。当处理剂量增加到10μg融合蛋白时,几乎所有用抗HER2/neu-IgG3-IFN-α或IgG3-IFN-α处理过的小鼠都发生了肿瘤的完全消退,50天后仍然没有肿瘤。
用三个10μg剂量的融合蛋白处理后仍然没有肿瘤的小鼠在第50天再次用1x10338C13/HER2肿瘤细胞攻击。全部小鼠保持没有肿瘤(数据未显示)。这些结果表明,当用融合了Ab的IFN-α处理较大的已建立肿瘤时,伴有免疫记忆的获得性免疫反应被启动。
讨论
尽管rIFN-α已显示出抗B细胞淋巴瘤和多种骨髓瘤的活性,但效果不稳定及其系统毒性限制了它的应用(33)。本研究显示,将IFN-α与Ab融合提高了其抗肿瘤效力,当IFN-α通过肿瘤特异性Ab导靶至肿瘤细胞时,该效力可以进一步提高。这一抗肿瘤效力未发现有任何明显的毒性相伴。这些研究表明,IFN-α与肿瘤特异性Ab融合可产生治疗B细胞淋巴瘤的高效生物药剂。
为了验证利用Ab将IFN-α导向肿瘤部位能够使效力提高的猜想,我们选择了已被详细研究过的小鼠B细胞淋巴瘤,该淋巴瘤经工程化能够表达常见TAA:HER2/neu,其有Abs可供使用。抗HER2/neu-IgG3-IFN-α看来能比以前描述过的免疫治疗剂更有效地治疗38C13B细胞淋巴瘤,虽然在本研究中通过基因转导引入的外来Ag是目标物。在肿瘤攻击1天后开始的三个1μg剂量抗HER2/neu-IgG3-IFN-α处理看来与肿瘤攻击1天后开始的10μg抗Id IgG1-IL-2融合蛋白处理5天(34)同样有效地抑制肿瘤生长。此外,抗HER2/neu-IgG3-IFN-α能够有效地抗已建立的肿瘤(图7),而抗Id IgG1-IL-2处理如果是在肿瘤攻击3或7天后开始,其抗肿瘤活性很低(34)。治愈已建立肿瘤的能力也表明Ab导靶的IFN-α是比GM-CSF(35)、CTLA-4(36)或融合了Id Ag的CD40配体(37)更强的治疗剂,因为这些疫苗都不能有效地抗已建立的肿瘤。因此,将IFN-α导靶至肿瘤细胞可以是很有前景的治疗B细胞淋巴瘤的方法。
利用肿瘤特异性Ab将IFN-α导靶至肿瘤细胞提高了IFN-α的抗肿瘤效力。抗HER2/neu-IgG3-IFN-α能够比IgG3-IFN-α更有效地在38C13/HER2中抑制增殖和诱发细胞凋亡(图5A-图5D),用2.5或1μg抗HER2/neu-IgG3-IFN-α进行的处理比相同剂量的IgG3-IFN-α能够更有效地抑制体内小肿瘤的生长(图3A和图3B)。这些结果表明肿瘤特异性Ab将IFN-α定向到肿瘤,从而提高了其治疗指数,而系统毒性下降。
不同寻常的是,IgG3-IFN-α表现出比rIFN-α更强的抗肿瘤活性(图4A)。虽然rIFN-α能够有效治疗多种肿瘤(38–40),但需要用高剂量做长时间治疗才能看到抗肿瘤活性,这部分是因为该细胞因子的半衰期非常短。在本研究中,我们展示了给IFN-α融合上IgG3Ab显著提高了它的半衰期(图4B),融合蛋白体内抗肿瘤活性的提高可得益于该延长的半衰期(图4A)。此外,IgG3-IFN-α的Fc区可能帮助IFN-α导靶到B淋巴瘤细胞上存在的Fc受体,从而提高了抗肿瘤活性。因此,IFN-α与IgG3Ab的融合提供了改进IFN-α的抗肿瘤效力的多方面优势。
虽然IFN-α具备包括激活免疫反应在内的多种活性,但看来直接的细胞毒性在抗HER2/neu-IgG3-IFN-α的强抗肿瘤活性中起着重要作用。IFN-α融合蛋白均显示针对38C13/HER2的细胞凋亡和抗增殖活性,而导靶到肿瘤显著提高了这些效果(图5A-图5D)。虽然IFN-α融合蛋白能够非常有效地治疗小肿瘤(图3A和图3B),存活的小鼠均未发展出能够保护它们免于第二次肿瘤攻击的免疫反应,这表明IFN-α融合蛋白的直接细胞毒性非常有效地杀灭了肿瘤细胞,当仅有小的肿瘤负荷时,获得性免疫没有发挥作用。因为38C13是一个极度恶性的B淋巴瘤细胞系,仅注射200个细胞的小鼠在20天内就会发展出大块的肿瘤(36),IFN-α融合蛋白一定要非常有效地杀死多数接种的肿瘤细胞才能有长期存活者。包括下调NF-κB(41)、通过激活半胱天冬酶(caspase)-3诱发细胞凋亡(42)以及上调TRAIL和TRAIL受体(43)的多种机制都曾被显示参与IFN-α介导的抗肿瘤细胞的细胞毒性,我们预计在Ab-IFN-α融合蛋白中看到的抗肿瘤细胞的直接细胞毒性也得益于这些机制。与此一致的,用融合蛋白处理肿瘤细胞后,我们观察到了STAT1的活化(图6A-6C)。
虽然在接种肿瘤1天后开始对小鼠进行处理的情况下,IFN-α融合蛋白未能启动记忆性免疫反应,当小鼠在接种肿瘤后12天开始接受处理时,IFN-α融合蛋白启动的免疫反应能够保护小鼠抗第二次肿瘤攻击。因此,在有较大肿瘤负荷时,IFN-α融合蛋白会激活保护性获得性免疫。因为IFN-α能够借助刺激DC的分化和成熟而激活获得性免疫(9),有可能在有IFN-α的情况下,已建立的肿瘤提供了更多TAA用于DC活化。此外,在这个模型中,外来的Ag-人HER2/neu可能通过提高肿瘤细胞的免疫原性而促进了抗肿瘤免疫力。
CD20作为B细胞表达的Ag在多数B细胞淋巴瘤中都有表达(44),抗CD20(利妥昔单抗,Genentech;)是最成功的癌症治疗剂之一,其抗淋巴瘤的效力强而毒性很小(45)。尽管抗HER2/neu IgG3-IFN-α能够非常有效地抗38C13/HER2,正常情况下HER2/neu不会在淋巴瘤细胞中表达,因此它可能在治疗淋巴瘤方面只有有限的治疗应用,但应当能够有效地治疗表达HER2/neu的癌症。相反,IFN-α与抗CD20的融合预计能够产生有效抗淋巴瘤的融合蛋白,通过在一种蛋白中结合了抗CD20的抗淋巴瘤活性和IFN-α的强免疫刺激和细胞毒活性得到更高的抗肿瘤活性。此外,正如在B细胞淋巴瘤患者中观察到的(46),IFN-α处理后,IFN-α可进一步上调CD20的表达。目前,我们正在研究抗CD20-IFN-α融合蛋白在B细胞淋巴瘤的小鼠模型中的效果。
总之,我们构建了新的融合蛋白并对其进行了表征,所述融合蛋白中IFN-α连接着识别TAA的抗体。我们的结果表明IFN-α与肿瘤特异性抗体的融合强烈地提高了IFN-α的抗肿瘤活性而未观察到任何明显的毒性。不寻常的是,Ab-IFN-α融合蛋白能够有效地抗已建立的肿瘤。因此,融合了肿瘤特异性抗体的IFN(例如,IFN-α)显示出治疗B细胞淋巴瘤的前景。
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实施例2
抗CD20-IFNα融合蛋白
前言
我们的初期研究表明抗HER2/neu与IFN-α连接形成的融合蛋白是治疗表达HER2/neu的淋巴瘤的有效治疗剂。我们试图进一步扩展这些研究来证明IFN-α和CD20的融合蛋白是治疗表达CD20之淋巴瘤的有效治疗剂。CD20在几乎所有淋巴瘤中都有。但是应当注意的是,HER2/neu在许多癌症中都有表达,可以预期抗HER2/neu融合蛋白应当能够有效治疗这些癌症。在抗CD20融合蛋白中,我们预计融合蛋白中的IFN-α既能够发挥抗肿瘤细胞的直接细胞毒性效应,也能有助引发抗肿瘤免疫反应。
产生CD20的特异性重组抗体。
扩增抗CD20(利妥昔单抗)的可变区并将其克隆到用于产生带有人κ轻链和γ3重链的嵌合抗体的表达载体中。产生蛋白并利用流式细胞术和人B细胞系Daudi检验其识别CD20的能力。如图8所示,重组蛋白能够和重组IgG1-利妥昔单抗一样好地结合CD20。
制备连接着CD20特异性抗体的人干扰素的抗体融合蛋白
a.融合蛋白的设计
当我们最初尝试制备融合蛋白时,我们利用由(Gly4Ser)3构成的柔性甘氨酸-丝氨酸接头(SEQ ID NO:31)将IFN-α与人IgG3基因的羧基端连在一起。图9中图示了重链。
验证融合蛋白载体带有正确的核苷酸序列后,将它与嵌合抗CD20轻链一起转染至NS0细胞中。通过ELISA筛选产生IgG的转染子。将给出最强信号的克隆扩增,随后的亚克隆生长在转瓶中。然后将上清过蛋白ASepharose柱,洗脱结合的蛋白,并在未还原和还原后通过SDS-PAGE分析(参见图10)。尽管分离的蛋白组装成了H2L2分子,多数分离的蛋白比预期的小。进行还原后,多数重链比预期的小,与不含融合蛋白的γ3重链迁移到相同的位置。干扰素看来被蛋白水解从融合蛋白中去除了。使用抗Fc和抗干扰素进行的Western印迹分析证实,两个上方的条带都是重链,但只有最大条带含有干扰素。
柔性接头可能成为蛋白水解切割的目标物。因此,我们将接头缩短到只有一个拷贝的Gly4Ser(SEQ ID NO:32)。将这些载体和带有延长的接头的载体与合适的轻链一起瞬时转染到HEK293T细胞中。细胞通过在35S-甲硫氨酸中生长被同位素标记,用蛋白A沉淀免疫球蛋白,经SDS-PAGE进行分析(图11)。带有延长的接头的融合蛋白发生了明显的切割,而当只有一个Gly4Ser(SEQ ID NO:32)构成的接头时,没有发生切割。因此,制备融合蛋白使用的接头很重要,可影响到蛋白的稳定性。
b.融合蛋白对CD20的识别
为了确定融合蛋白能否识别CD20,将表达CD20的人细胞系Daudi与Rituxan、抗DNS/IgG3-hu-IFN-α或抗CD20/IgG3-hu-IFN-α一起温育。抗CD20/IgG3-hu-IFN-α比Rituxan结合地好(图12)。抗DNS/IgG3-hu-IFN-α融合蛋白也显示出一定的结合,虽然比任一种CD20特异性蛋白都弱。我们提出假设认为,抗DNS/IgG3-hu-IFN-α的结合以及与Rituxan相比,抗CD20/IgG3-hu-IFN-α的结合提高是因为hu-IFN-α部分结合到Daudi细胞上表达的IFN受体。
c.融合蛋白的抗病毒活性
为了评估hu-IFN-α融合蛋白的抗病毒活性,以2x105细胞/ml接种HeLa细胞,并用融合蛋白或罗扰素(重组人干扰素2a)的两倍梯度稀释液处理24小时。然后用4000pfu/100μl的VSV(水泡性口炎病毒)感染细胞。72小时后,细胞用0.1%结晶紫染色。为了确定抗病毒感染的保护作用,可以通过用0.1%结晶紫染色,并用光点密度计测量每孔染料的量来定量感染后存活下来的细胞,或者计数噬斑数量。两种分析法中,融合蛋白都有显著的IFN-α活性,但与罗扰素相比,活性下降了大约100倍。
融合蛋白对Daudi淋巴瘤细胞的生长抑制和杀灭
使用两种方法来评价融合蛋白对表达CD20的淋巴瘤细胞的生长抑制/杀灭。应当提到的是这些试验中使用的是天然表达CD20的人细胞系Daudi。第一种方法中,将Daudi细胞与不同浓度的IFN-α、抗体或融合蛋白一起温育72小时,利用CellTiter96Aqueous细胞增殖检测法评价生长抑制情况(图14)。尽管抗CD20/IgG3-hu-IFN-α在抗病毒分析法中表现出较低的IFN-α活性,它和罗扰素显示出类似的抑制淋巴瘤生长的能力,这表明对IFN-α的导靶增强了其细胞毒效果。与单独的罗扰素相比,抗CD20/IgG3+罗扰素未显示更高的活性。抗DNS/IgG3-hIFN-α、Rituxan和抗CD20/IgG3只在最高使用浓度处表现出某些生长抑制。值得提到的是在这项检测法中,融合蛋白比Rituxan更能防止细胞的生长。
在第二种方法中,将Daudi细胞与不同浓度的IFN-α、抗体或融合蛋白温育72小时,然后用膜联蛋白V和碘化丙啶(PI)染色并通过流式细胞术分析。图15显示了使用10pM各种蛋白时得到的结果。处于细胞凋亡早期的细胞是膜联蛋白V+PI-;凋亡晚期细胞和死细胞是膜联蛋白V+PI+。
这些试验说明了这样几点。即使在最高测试浓度,Rituxan和抗CD20/IgG3都仅能诱发很少甚至不能诱发细胞凋亡。正如预期,小鼠IFN-α抗人细胞系不如人重IFN-α(罗扰素)有效,不能导靶到肿瘤细胞的抗与重组小鼠IFN-α效果类似。但是,利用抗CD20/IgG3-mIFNα将小鼠IFN-α导靶至肿瘤细胞导致能够有效地诱发细胞死亡。抗CD20/IgG3-hIFNα比抗更有效,再次显示了细胞导靶在细胞杀伤中的作用。在该体外检测法中,罗扰素和抗CD20/IgG3-hIFNα表现出类似的活性,即使低至1pM也可导致细胞死亡(数据未显示)。但是,应当指出的是,在体内CD20/IgG3-hIFNα会导靶至肿瘤部位并在此积累,而罗扰素表现的是全身的活性。
融合蛋白对38C13-CD20淋巴瘤细胞的生长抑制和杀灭
正如上文简单提到的,John Timmerman博士的实验室建立了一个表达人CD20的小鼠淋巴瘤38C13-CD20,该细胞系可以在同种C3H/HeJ小鼠中生长。这一细胞系使得有可能检验我们的融合蛋白的体内效力。将38C13-CD20细胞与各种抗体和融合蛋白温育48小时。然后通过用膜联蛋白V和PI将细胞染色并用流式细胞术检验来确定细胞杀伤和凋亡。当使用浓度为100pM的蛋白时(数据未显示),重组mIFN-α和抗CD20-IgG3-mIFN-α都非常高效地导致细胞凋亡,其中抗CD20-IgG3-mIFN-α比重组mIFN-α略胜一筹。用抗DNS-IgG3-mIFN-α或Rituxan处理38C13-CD20细胞诱发了某些凋亡。用该浓度的抗CD20/IgG3进行的处理未影响细胞活力。当处理浓度降低到10pM(图16),重组mIFN-α和抗CD20/IgG3-mIFN-α仍然有效地引起细胞凋亡,其中抗CD20/IgG3-mIFN-α比重组mIFN-α更有效。用抗DNS-IgG3-mIFN-α处理后,只观察到少量的凋亡,这表明利用抗CD20-IgG3-mIFN-α将导靶产生了更有效的治疗剂。在这个浓度,Rituxan仅引起很少的凋亡,表明抗CD20-IgG3/mIFN-α融合蛋白优于未融合的抗CD20抗体。用抗CD20/IgG3进行的处理对细胞活力还是没有影响。处理剂量为1pM时,只有抗CD20-IgG3-mIFN-α在38C13-CD20中诱发了细胞凋亡(数据未显示)。剂量为0.1pM时,所有处理都不能诱发凋亡(数据未显示)。
作为一种替代方法,用不同浓度的各种蛋白处理38C13-CD20细胞并利用MTS检测法监测对细胞生长的抑制(图17)。抗CD20/IgG3-mIFN-α能够最有效地抑制细胞生长,其次是重组在抗中观察到了某些生长抑制。抗CD20/IgG3和Rituxan对细胞生长影响很小。因此,这些检测中得到的结果反映了(mirror)监测细胞凋亡时观察到的结果。
其他IgG-IFNα融合蛋白的制备和表征
a.抗CD20-IgG1-mIFNα和抗CD20-IgG1-hIFNα
将IFN-α与人IgG3骨架融合在一起制备初始的蛋白。Rituxan是一种IgG1。为了确定免疫球蛋白骨架是否影响融合蛋白的性能,制备了m-IFN-α和hu-IFN-α与IgG1融合的融合蛋白。它们具有预期的分子量。
评估了抗CD20/IgG1-mIFNα诱发38C13-CD20细胞凋亡的能力(图18)。研究显示该蛋白有效,甚至可能比IgG3融合蛋白更有效。
评估了抗CD20/IgG1-hIFNα诱发Daudi细胞凋亡的能力。研究显示该蛋白与抗CD20/IgG3-hIFNα的活性类似(图19)。
如图20所示,对融合蛋白抑制Daudi细胞生长的能力进行了评估。IgG1与小鼠和人IFNα的融合具有IgG3融合体抑制Daudi细胞生长方面的能力。
b.通过α螺旋接头将IFN-α与IgG骨架连在一起的融合蛋白。
制备融合蛋白,其中GlySer接头被具有A(EAAAK)2A(SEQ ID NO:33)序列的接头代替。所述序列预期会折叠成α螺旋。
通过在293T细胞中瞬时表达制备蛋白,并通过SDS-PAGE进行评价。蛋白发生了组装,分子量与预期相同。没有观察到接头的切割。
抗CD20-IgG3-hIFNα(α-螺旋接头)融合蛋白在与含有Gly4Ser(SEQ IDNO:32)接头的融合蛋白相同浓度使用时,能够有效地诱发Daudi细胞的凋亡(图21)。
肿瘤的体内治疗
经Timmerman实验室转导从而表达人CD20的38C13淋巴瘤被用于这项研究。38C13是生长在同种C3H/HeJ小鼠中的侵略性的淋巴瘤。转导体38C13-CD20表现出相同的生长特性。因此有可能在免疫力完全的动物中考察融合蛋白介导的保护作用。
a.早期肿瘤的治疗
在第0天,给小鼠(4只一组)皮下注射5000个38C13-CD20细胞。在第1、2和3天,用hepes缓冲的盐溶液(HBSS),或者0.4μg、2μg或10μg抗CD20-m-IFN-α对小鼠静脉处理,并监测肿瘤生长情况。至第所有HBSS处理的动物都有大的肿瘤,不得不被处死。相反,用10μg融合蛋白处理的动物中没有发现肿瘤生长;20天后在用0.4μg融合蛋白处理的4只小鼠中有3只开始肿瘤生长,2μg融合蛋白处理的小鼠中有1只开始肿瘤生长。这些结果显示,抗CD20/IFN-α融合蛋白能够非常有效地抑制体内肿瘤生长并提高存活率(参见例如,图22).
b.抗CD20-mIFNα融合蛋白比利妥昔单抗或抗CD20/IgG3能更有效地治疗中等大小的肿瘤
C3H/HeJ小鼠在第0天接种5000个38C13-CD20细胞。第5、6和7天,用HBSS或10μg抗CD20-IgG1(在293T细胞中产生的)、抗CD20-IgG3、利妥昔单抗或抗CD20-IgG3-mIFNα进行处理。监测它们的肿瘤生长和存活情况(参见,例如图图23)。在防止中等大小的肿瘤的生长方面,抗CD20/IgG3-mIFNα比利妥昔单抗、抗CD20/IgG3或抗CD20/IgG1有效得多。
融合蛋白的肿瘤导靶能力显著地增强了其体内效力。
C3H/HeJ小鼠在第0天接种5000个38C13-CD20细胞。第5、6和7天,用10μg抗CD20-IgG3、10μg抗CD20-IgG3+mIFN-α(选择的剂量与融合蛋白摩尔数相同)、抗DNS-IgG3-IFNα或抗CD20-IgG3-mIFNα进行处理并跟踪肿瘤生长和存活情况(参见例如,图24)。抗CD20-IgG3-IFNα显著地延迟了肿瘤生长并提高了存活率,表明利用抗体结合位点将IFNα导靶至肿瘤产生的治疗剂,比不能导靶IFNα的融合蛋白(抗)或者比将抗CD20和未共价关联的IFNα一起注射(抗)更有效。
融合蛋白处理能够有效抗已建立的肿瘤
8只一组的C3H/HeJ小鼠接种5000个38C13-CD20细胞,并在第8、9和10天用100μg抗CD20-mIFNα或HBSS处理。监测小鼠的肿瘤生长情况(参见图25)和存活情况(参见图26)。接种了抗CD20-mIFNα的小鼠显示提高的存活率(图26)。
应当理解的是,本文中描述的实施例和实施方案仅用于说明的目的,本领域技术人员根据这些教导可以进行各种改动或变化而都符合本申请的精神和范围,处于以下所附权利要求的范围内。文中提及的所有出版物、专利和专利申请均通过引用全文并入本文。
Claims (13)
1.嵌合构建体,所述构建体包含附着了能够结合CD20的完整抗体的干扰素,其中所述抗体是通过肽接头附着于所述干扰素的,其中所述接头的氨基酸序列是SGGGGS,且其中当所述构建体与肿瘤细胞接触时,导致所述肿瘤细胞被杀死或者其生长或增殖受到抑制。
2.如权利要求1所述的构建体,其中所述干扰素是干扰素α。
3.如权利要求1所述的构建体,其中所述干扰素是干扰素β。
4.如权利要求1所述的构建体,其中所述干扰素是1型干扰素。
5.如权利要求1所述的构建体,其中所述干扰素是2型干扰素。
6.如权利要求1-5之一所述的构建体,其中所述抗体是Rituxan。
7.如权利要求1-6之一所述的构建体,其中所述构建体与游离干扰素相比,显示抗肿瘤活性增强和/或体内半衰期延长。
8.药物制剂,所述制剂在药物可接受的赋形剂中包含如权利要求1-7中任一项所述的构建体。
9.如权利要求8所述的药物制剂,其中所述制剂是单位剂量制剂。
10.如权利要求8所述的药物制剂,其中所述制剂被配制成肠胃外给药的制剂。
11.如权利要求8所述的药物制剂,其中所述制剂被配制成用于通过选自口服、静脉内给药、肌内给药、直接肿瘤给药、吸入、直肠给药、阴道给药、透皮给药和皮下储库给药的途径进行给药的制剂。
12.如权利要求1-7中任一项所述的构建体或权利要求8-11任一项所述的药物制剂在制备抑制淋巴癌细胞生长和/或增殖的药物中的用途。
13.如权利要求12所述的用途,其中所述淋巴癌细胞是转移细胞。
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