CN1541109A - Her3活性抑制剂 - Google Patents
Her3活性抑制剂 Download PDFInfo
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- CN1541109A CN1541109A CNA028156153A CN02815615A CN1541109A CN 1541109 A CN1541109 A CN 1541109A CN A028156153 A CNA028156153 A CN A028156153A CN 02815615 A CN02815615 A CN 02815615A CN 1541109 A CN1541109 A CN 1541109A
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- her3
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Abstract
本发明涉及包含HER3活性抑制剂、特别是抗HER3抗体为活性剂的药物组合物。本发明进一步公开了该组合物在诊断、预防或治疗过度增殖性疾病、特别是肿瘤疾病中的用途。
Description
发明描述
本发明涉及包含HER3活性抑制剂、特别是抗HER3抗体为活性剂的药物组合物。本发明进一步公开了该组合物在诊断、预防或治疗过度增殖性疾病、特别是肿瘤疾病中的用途。
已知蛋白质酪氨酸激酶是介导可调节细胞生长和分化的信号转导过程的酶。受体蛋白质酪氨酸激酶通过配体刺激的底物酪氨酸磷酸化而起作用。HER3(也称为ErbB3)是受体蛋白质酪氨酸激酶表皮生长因子受体(EGFR)亚家族的成员(Plowman等人,Proc.Natl.Acad.Sci.U.S.A.87(1990),4905-4909;Kraus等人,Proc.Natl.Acad.Sci.U.S.A.86(1989),9193-9197以及Kraus等人,Proc.Natl.Acad.Sci.U.S.A.90(1993),2900-2904)。
已经发现HER3在若干癌症类型、例如乳腺、胃肠道和胰腺癌中过表达。若HER3与受体蛋白质酪氨酸激酶EGFR亚家族的另一成员HER2共表达,则形成活性异二聚体信号复合物。
针对HER3的单克隆抗体(Rajkumar等人,Br.J.Cancer 70(1994),459-456)对表达HER3的细胞系的无贴壁依赖性生长具有激动效应。另一方面,据报道美国专利5,968,511(对应于WO 97/35885)所述抗HER3抗体可降低Heregulin诱导的HER2/HER3异二聚体形成。然而,只是对提高Heregulin与HER3结合的抗体证明了这种活性。因此,尚不清楚哪种类型的抗HER3抗体-如果存在的话-具有供治疗性应用的潜能。
Vadlamudi等人(Oncogenes 18(1999),305-314)描述了通过HER2受体调节环加氧酶(COX-2)途径。发现COX-2的特异性抑制剂能够抑制结肠直肠癌细胞的促有丝分裂及侵袭作用。进一步发现,与单克隆抗HER3抗体温育可致HER2/HER3异二聚体水平下降,但是只部分降低COX-2表达。
WO 00/31048公开了可用作受体酪氨酸激酶(例如EGFR、HER2和HER4)抑制剂的喹唑啉(quinazoline)衍生物。但未公开HER3的抑制。
WO 00/78347公开了阻止或抑制细胞生长的方法,其中包含防止或降低ErbB-2/ErbB-3异二聚体形成。例如,该药剂可以是抗HER2胞外域抗体与抗HER3抗体(例如购自Neomarkers的HER3抗体H3.105.5)的组合。然而尚不清楚需要何种类型的抗HER3抗体,以获得所需治疗效果。
美国专利5,804,396描述了用于治疗增殖性疾病的药剂的鉴定方法,其中包含对一种潜在的药剂通过HER2/HER3或HER2/HER4或HER3/HER4异二聚体抑制信号转导的活性进行分析的步骤。并未公开特异性的HER3抑制剂。
在表达不同HER2∶HER3比例的侵袭性乳癌细胞系MCR-7(DKFZHeidelberg)、MDA-MB-468(ATCC HTB-132)和MDA-MB231(ATCCHTB-26)中,我们比较了一种激动性的抗HER2单克隆抗体Herceptin与抗HER3抗体的生物学特性,即(i)小鼠单克隆抗体IgG1,α-HER3-ECD,Upstate Biotechnology,目录号#05-471,针对HER3的Heregulin结合位点,(ii)我们实验室的抗体1B4C3以及(iii)同样来自我们实验室的抗体2D102。我们提供了证据,证明利用抗HER3抗体在α/β-Heregulin(α/β-HRG)刺激之前预处理乳癌细胞系,与Herceptin相比,可减少HER2/HER3酪氨酸磷酸化程度。此外,抗HER3抗体取消了HER2/HER3的异二聚体化,还降低了PI3-激酶的p85亚基和连接(adaptor)蛋白质SHC与HER3的复合物形成,分别导致PI3-激酶和c-jun-末端激酶活性(JNK)下降。与Herceptin相比,抗HER3抗体也能够在α/β-HRG刺激之后下调细胞外信号调节的激酶2(ERK2)。此外我们证明,在利用抗HER3抗体预处理之后,乳癌细胞系的迁移和增殖特性显著下降。我们的数据清楚表明,在HRG刺激之后,抗HER3抗体比Herceptin更有效降低信号转导过程。此外,在特定癌症类型例如黑色素瘤中,抗HER3抗体可有效降低迁移和增殖特性,而抗HER2抗体根本没有任何显著作用。这些数据证明,抗HER3抗体或其它HER3抑制剂具有治疗乳癌及其它以通过HER3及其异二聚体化配偶体的超级信号为特征的恶性肿瘤的巨大潜能。
因此,本发明涉及包含HER3活性的特定类型抑制剂为活性剂以及药物可接受的载体、稀释剂和/或助剂的药物组合物。本发明的HER3抑制剂的特征在于,该抑制剂与HER3的结合可降低HER3介导的信号转导。在一个实施方案中,通过下调HER3引起HER3分子至少部分从细胞表面消失,从而导致HER3介导的信号转导下降。在本发明另一实施方案中,通过使细胞表面的HER3分子稳定于基本失活的形式,即比非稳定形式显示较低的信号转导的形式,从而导致HER3介导的信号转导下降。
本发明的抑制剂可能影响Heregulin与HER3的结合,特别是降低Heregulin与HER3的结合。但在另一实施方案中,该抑制剂可能并不竞争Heregulin与HER3的结合。
在优选实施方案中,该抑制剂是抗HER3抗体。优选地,该抗体针对HER3的胞外域。然而应当注意,也可能使用其它HER3抑制剂,特别是低分子量抑制剂,例如肽或有机化合物。
根据本发明,术语”抗体”包括单克隆抗体、多克隆抗体、由至少两种抗体形成的多特异性抗体(例如双特异性抗体)以及抗体片段,只要其具有所需活性即可。
该抗体可以是利用Khler等人(Nature 256(1975),495)所述杂交瘤方法或重组DNA方法(例如参见美国专利4,816,567)获得的单克隆抗体。也可以利用Clackson等人(Nature 352(1991),624-628)以及Marks等人(J.Mol.Biol.222(1991),581-597)所述技术,从噬菌体抗体文库中分离单克隆抗体。该抗体可以是IgM、IgG,例如IgG1、IgG2、IgG3或IgG4。
抗体片段包含抗体的一部分,一般是完整抗体的抗原结合区或可变区。抗体片段的例子包括Fab、Fab′、F(ab′)2和Fv片段、双功能抗体(Diabody)、单链抗体分子以及多特异性抗体片段。
特别而言,该抗体可以是重组抗体或抗体片段,更特别而言,选自嵌合抗体或其片段(Morrison等人,Proc.Natl.Acad.Sci.U.S.A.81(1984),6851-6855)、人源化抗体(Jones等人,Nature 321(1986),522-525;Riechmann等人,Nature 332(1988),323-329以及Presta,Curr.Op.Struct.Biol.2(1992),593-596)、单链Fv抗体(Plücktuhn在:ThePharmacology of Monoclonal Antibodies 113,Rosenburg和Moore,EDS,Springer Verlag,N.Y.(1994),pp.269-315)以及双功能抗体(Hollinger等人,Proc.Natl.Acad.Sci.U.S.A.90(1993),6444-6448)。
在特别优选的实施方案中,该抗体选自杂交瘤细胞系DSM ACC2527或DSM ACC 2517产生的抗体1B4C3(IgG2a)和2D1D12(IgG1)、其片段或其重组衍生物。1B4C3是导致HER3内在化的抗体,2D1D12是导致HER3稳定化的抗体。进一步优选与保藏的杂交瘤细胞系所产生的抗体具有基本相同的生物学活性(例如实施例所述)、例如结合到HER3的相同表位的抗体,例如嵌合或人源化抗体或其片段。按照BudapestTreaty for the Deposit of Microorganisms,杂交瘤细胞系DSM ACC 2517于2001年7月24日保藏在Deutsche Sammlung von Mikroorganismenund Zellkulturen GmbH(DSMZ),Mascheroder Weg 1b,38124Braunschweig,Germany。产生抗体1B4C3的杂交瘤细胞系DSM ACC2527于2001年8月7日保藏在DSMZ。
本发明的抗体可以偶联标记基团,特别用于诊断性应用。合适的标记基团的例子,例如放射性基团、荧光基团或其它标记基团为本领域已知。此外,特别对治疗性应用而言,该抗体可能偶联效应基团,例如细胞毒性基团,如放射性基团、毒素或本领域已知的其它效应基团。
在进一步优选的实施方案中,该抑制剂可能选自非抗体来源的结合蛋白质,例如纤连蛋白III型结构域或anticalins(Skerra,″Engineeredprotein scaffolds for molecular recognition″,J.Mol Recog.13(2000),167-187以及在此引用的文献)
此外,本申请涉及HER3活性抑制剂的用途,用于制备药剂,供诊断、预防和/或治疗过度增殖性疾病,特别是肿瘤疾病,例如乳癌、胃肠癌、胰腺癌、前列腺癌、神经胶质瘤、黑色素瘤或其它HER3表达或过表达的癌症或肿瘤转移形成,其中所述抑制剂结合到HER3可降低HER3介导的信号转导。该疾病可能与增强的HER3信号转导有关,并可能与相伴的HER2表达或缺少HER2表达有关。特别而言,该疾病与增强的HER3磷酸化、和/或增强的HER2/HER3异二聚体化、和/或增强的PI3激酶活性、和/或增强的c-jun末端激酶活性和/或AKT活性、和/或增强的ERK2活性和/或PYK2活性有关。
令人吃惊的是,发现本发明的HER3抑制剂,特别是抗HER3抗体,在降低信号转导过程方面比HER2抑制剂、例如Herceptin更为显著高效。特别而言,在黑色素瘤细胞中,抗HER3抗体有效,虽然黑色素瘤细胞表达HER2,但Herceptin没有显著效果。
优选地,本发明的HER3抑制剂具有至少一种下列特征:
-降低Heregulin(p85)与反式激活的HER3的结合,优选基本上完全抑制p85与HER3的结合,
-抑制GRB2结合到HER2、HER2结合到HER3和/或GRB2结合SHC,
-抑制受体酪氨酸磷酸化,
-抑制AKT磷酸化,
-降低肿瘤侵袭性,特别是乳癌和黑色素瘤,
-抑制PYK2酪氨酸磷酸化以及
-抑制ERK2磷酸化。
本发明进一步涉及诊断、预防或治疗过度增殖性疾病、特别是肿瘤疾病的方法,其包含给予所需受试者(例如人)有效剂量的HER3活性抑制剂,其中所述抑制剂结合到HER3可降低HER3介导的信号转导。
通过活性剂与生理上可接受的载体、稀释剂和/或助剂混合,将该HER3抑制剂、特别是抗HER3抗体配制成例如冻干剂型、含水溶液、悬浮液或固体制剂,例如片剂、糖锭剂或胶囊剂,如Remington′sPharmaceutical Sciences所述。
该剂型可能包含多于一种的活性化合物,例如其它受体蛋白质酪氨酸激酶、例如EGFR、HER2、HER4或血管内皮生长因子(VEGF)的抑制剂。或者或另外,该组合物可能包含细胞毒性剂例如阿霉素(doxorubicin)、顺铂(cisplatin)或卡铂(carboplatin)、或细胞因子。
本发明的抑制剂还适于诊断性应用,例如以便确定HER3的表达和/或对靶细胞的活性。这种诊断性应用可按照已知方法实现。
根据待治疗疾病的类型及严重性,可给予人类患者约1μg/kg-15mg/kg的抗体,例如通过一次或多次分别给予或连续输注。典型的每日剂量可根据上述因素为约1μg/kg至约100mg/kg或更多。对于几天或更长时间的重复给药而言,根据所治疗的疾病持续进行治疗,直至对疾病症状产生所需要的抑制为止。
本发明进一步由以下附图及实施例进行说明。
实施例
1.单克隆抗体α-HER3ECD降低HER3和HER2的受体酪氨酸磷酸化
根据其不同的HER2∶HER3比值及其固有的迁移特性选择乳癌细胞系MCF-7(DKFZ-Heidelberg)、MDA-MB-468(ATCC HTB-132)和MDA-MB-231(ATCC HTB-26),MDA-MB-231是最具侵袭性的细胞系。为了与trastuzumab(Herceptin)比较α-HER3ECD(Upstate Biotechnology,Cat.#05-471)的功能性作用,我们在Heregulin(HRG)刺激之前,分别利用α-HERECD和HC预处理细胞,进行受体免疫沉淀实验,并利用抗磷酸酪氨酸抗体(PY)进行探测(图1)。我们的数据表明,利用α-HER3ECD预处理基本上降低α-HRG刺激后MCF-7(图1a)和MDA-MB-231(图1c)中HER3和HER2的酪氨酸磷酸化程度,但反而提高MDA-MB-468中HER3的酪氨酸磷酸化(图1b)。α-HER3ECD甚至增强了HER2与HER3的结合,尽管酪氨酰磷酸化受体的含量显著下降(图1a、b中上图泳道4和8)。相比之下,所有细胞系在HRG存在与否的情况下,HC上调受体酪氨酰磷酸化并促进HER3与HER2的结合(图1a、b、c上图泳道3、7、11和15)。至于对α-HRG不敏感的MDA-MB-486细胞,使用β-HRG作为刺激物。
2.α-HER3ECD取消SHC和PI3-K与HER3以及GRB2与HER2的结合
我们随后探询α-HER3ECD是否作用于HER3的已知底物,即分别引起MAPK级联活化以及脂质信号的效应蛋白质SHC和磷脂酰-3-OH-激酶(PI3-K)。因此,我们在上述实验条件下免疫沉淀SHC和PI3-K,评估这些效应物的酪氨酸磷酸化。如图2所示,α-HER3ECD显著降低MCF-7和MDA-MB-486细胞系中SHC的酪氨酸磷酸化(图2a、b泳道13和16比较)。引人注意的是,在MCF-7细胞中SHC的结合衰减,而在MDA-MB-486中,α-HER3ECD引起HER3与SHC的结合提高。PI3-K调节亚基的免疫沉淀产生基本相似的结果。酪氨酸磷酸化的HER3与PI3-K的结合在MCF-7中消失,在MDA-MB-486中则观察到提高(图1b、2b)。然而,利用α-HER3ECD预处理MDA-MB-486细胞,导致SHC和PI3-K与HER3的结合提高,而HC再次在所有细胞系中显示交联特性。由于SHC在HRG刺激之后与连接分子GRB2结合,我们通过测定GRB2结合研究了SHC降低的酪氨酸磷酸化的作用(图2c)。为此我们按前述相同的实验设计,使用GST-GRB2融合子在MCF-7细胞中进行GST下拉(GST-pulldown)分析。降低的SHC酪氨酰磷酸化确实导致GRB2与SHC的结合剧烈下降(图2c下图,比较泳道5和8),并完全抑制其结合HER2(图2c中图,比较泳道5和8)。这些数据清楚表明,α-HER3ECD在MCF-7中抑制SHC和PI3-K结合HER3,在MDA-MB-486中则不然,两种蛋白质都结合HER3,而与HER3的磷酸化状态无关。
3.α-HER3ECD下调JNK1和PI3-K活性
连接蛋白质SHC介导生长因子受体下游的MAPK信号途径,分别活化ERK2和JNK。为研究α-HER3ECD对ERK2和JNK的作用,我们在MCF-7和MB-468中于相同实验条件下进行MAPK激酶分析(图3)。我们在所有细胞系中观察到JNK活性剧烈下降,但只在MCF-7中检测到HC对JNK的等效作用(图3a)。ERK2活性通过α-HER3ECD仅仅略微但显著下降,而HC对ERK2活性没有作用(数据略)。由于最近证明PI3-K与癌侵袭有关,我们研究了α-HER3ECD对PI3-K活性的抑制特性,并进行了PI3-K分析(图3)。在MCF-7和MDA-MB-486中,与HRG处理的细胞相比,PI3-K活性剧烈下降(图3a、b)。在MDA-MB-486中,HC对PI3-K活性的作用甚至强于α-HER3ECD。这些数据提示,α-HER3ECD在MCF-7和MDA-MB-486细胞中分别下调JNK和PI3-K活性。
4.α-HER3ECD增强HER3的内吞下调
在HRG刺激之后,HER2和HER3被内吞并再循环。我们感兴趣确定α-HER3ECD介导的抑制HER3酪氨酸磷酸化是否起源于加速的内吞作用。为进一步理解,我们利用MCF-7细胞,分别在α-HER3ECD或HRG存在与否的情况下,继之以HRG刺激,进行时间过程分析(图4)。然后在膜蛋白质生物素化之后,对HER3进行免疫沉淀。我们观察到利用α-HER3ECD预处理之后,HER3被快速内吞(图4b上图)。给予细胞HRG具有相同作用,差别在于,HER3于2小时以后回输到膜,3小时以后再次被内吞。利用PY探测全细胞裂解物(WCL),作为对照(图4b下图)。与3小时后酪氨酰磷酸化蛋白质含量降低的HRG处理的细胞相比,利用α-HER3ECD预处理1小时后发生HER3的加速内吞作用。为比较α-HER3ECD和HC,我们利用HC和免疫沉淀的HER2进行相同实验(图4a上图)。惊人的是,利用HC预处理之后,HER2在任何时间点均未被内吞,而HRG则引起快速内吞作用。比较利用抗磷酸化酪氨酸(PY)探测的内吞受体及全细胞裂解物,清楚显示利用α-HER3ECD则磷酸化酪氨酸含量下降,利用HC则不然(图4b下图)。我们的数据表明,α-HER3ECD通过加速的内吞作用快速下调HER3,从而致使细胞对HRG刺激不敏感。
5.a-HER3ECD抑制乳癌细胞系的迁移和增殖特性
为评价α-HER3ECD对乳癌细胞系迁移及增殖特性的生物学功能,我们在α-HER3ECD存在与否的情况下继之以HRG刺激,进行BrdU掺入分析。利用α-HER3ECD预处理使MCF-7和MDA-MB-486的增殖分别降低28.7%±6.18%和21.1%±7.62%。观察到的增殖抑制与ERK2分析有关,而HC在这些细胞系中没有作用(数据略)。此外,为研究α-HER3ECD对乳癌细胞迁移特性的作用,我们在α-HER3ECD存在与否的情况下,利用MCF-7和MDA-MB-486进行趋化(chemotaxis)实验。我们观察到MCF-7和MDA-MB-486的迁移率分别剧烈下降59.1%(P=0.018)和55.4%(P=0.00005)。另外,MDA-MB231的迁移也被抑制35%,但显著性略低(P=0.06)。我们的数据清楚表明α-HER3ECD对MCF-7和MDA-468增殖和迁移的抑制作用。
6.产生抗HER3的单克隆抗体
我们然后利用HER3胞外部分和His-Tag C末端的重组人融合蛋白质(HER3-6xHis-CT)免疫Balb/c小鼠,产生抗HER3胞外域的小鼠单克隆抗体。通过在HEK293细胞中转染、G418选择以及稳定表达构建体,获得免疫原,收集最高表达水平克隆的细胞培养上清,于硫酸铵沉淀、透析以及随后的金属离子亲和层析(Ni-NTA)之后纯化蛋白质。利用Western印迹进行质量保证(数据略)。按照厂家规程(Qiagen ImmunEasyMouse Adjuvant)腹膜内注射22μg HER3-6xHis-CT进行免疫。使用标准方法得到产生单克隆抗体的杂交瘤细胞系。
7.抗HER3的单克隆抗体优先结合其蛋白质主链,并对HER3的内吞过程具有不同作用
我们利用FACS分析鉴定了可特异性识别MCF-7细胞表面的天然HER3的3株单克隆抗体(数据略)。1B4C2和1B4C3是IgG2a同型抗体,而2D1D12是IgG1同型抗体。未检测到与EGFR家族其它成员有交叉反应性(数据略)。然后我们试图确定该抗体结合HER3的糖基化结构还是结合HER3的蛋白质主链,及其对受体内吞调节的影响。为此,我们利用已知可防止细胞表面蛋白质N-连接糖基化的抗生素Tunicamycin,在其存在与否的情况下预处理MCF-7细胞16小时。裂解细胞之后,我们利用单克隆抗体2F1 2(针对HER3的胞内部分)、α-HER3ECD(HER3的胞外部分)、1B4C2、1B4C3和2D1D12免疫沉淀HER3。我们的数据表明,α-HER3ECD、1B4C3和2D1D12均优先结合HER3的蛋白质主链,而1B4C3对HER3的糖基化形式也具有亲和性(图6A)。
为研究1B4C3和2D1D12对HER3内吞过程的作用,我们分别利用1B4C3或2D1D12温育MCF-7细胞不同时间,进行时间过程实验。将细胞表面蛋白质生物素化,利用抗HER3胞内域的抗体沉淀,利用链亲和素检测。我们观察到1B4C3类似于α-HER3ECD加速HER3的内吞作用,2D1D12则有稳定作用,从而在细胞表面积聚HER3(图6B)。
8.单克隆抗体1B4C3和2D1D12抑制HER3和HER2的下游信号
然后我们探询1B4C3和2D1D12是否可以抑制HER2和HER3底物SHC的酪氨酸磷酸化。由于GRB2与HRG刺激的HER2结合,并按与SHC相同的方式传递促分裂信号到MAPK途径,我们在未处理或经抗体预处理并随后经HRG刺激的MCF-7细胞中免疫沉淀SHC,同时进行GST-GRB2下拉分析(图6C)。该实验表明,两种抗体均抑制SHC的酪氨酸磷酸化以及GRB2与SHC的结合。此外,该抗体抑制SHC和HER3的结合以及HER2和HER3的异二聚体化。这些数据表明,下游信号受1B4C3和2D1D12抑制,虽然2D1D12比1B4C3的抑制作用更强。
9.单克隆抗体2D1D12抑制乳癌细胞系MDA-MB-435S、ZR-75-1和黑色素瘤细胞系Mel Gerlach的增殖
我们然后着手研究1B4C3和2D1D12在两株乳癌细胞系MDA-MB-435S(ATCC HTB-129)、ZR-75-1(ATCC CRL-1500)以及黑色素瘤细胞系Mel Gerlach(Klinikum Groβ hadern,Munich)中的生物学活性。我们因其在裸鼠中的致瘤性及其HER3高表达水平而选择这些细胞系。应当注意,黑色素瘤细胞过表达HER3,因为HER3在黑色素细胞(melanocytes)以及少突胶质细胞(oligodendrocytes)的发育过程中是非常关键的。为了检验我们有关1B4C3和2D1D12取消促细胞分裂信号、因而取消癌细胞的增殖特性的假说,我们在抗体存在与否的情况下进行BrdU掺入分析(图7)。2D1D12极大降低所有细胞系的增殖,而1B4C3只在Mel Gerlach中具有抑制作用。总之,我们的数据构成了第一个证据,证明抗HER3单克隆抗体可潜在视作抗癌的新治疗武器。
产生抗体1B4C3和2D1D12的杂交瘤细胞系分别于2001年8月7日和2001年7月24日保藏在DSMZ。
10.HER3抗体对信号转导的作用
10.1.方法
MDA-MB-435S得自ATCC(HTB-129),Mel-Juso得自Cell LinesServive(CLS)(0282-HU)。GST-p85(a.a 333-430)得自Santa Cruz。GST-GRB2如前所述纯化。Phospho-AKT(P-Ser 473)来自New EnglandBiolabs(NEB)。HER2抗体按所述纯化自杂交瘤培养上清(ATCCCRL-10463)。GST下拉分析中使用1.25μg诱饵(bait)蛋白质。如前所述进行BrdU掺入及侵袭分析。所有实验至少进行两次。
10.2.结果与讨论
为了检测HER2和HER3受体在MDA-MB-435S和Mel-Juso中的表面表达,我们另外利用FACS分析测定其表达水平(图8A、B)。我们观察了HER2和HER3在这些细胞系中的表达,继而仔细分析HER3抗体作用于Heregulin(HRG)介导的信号转导的分子机制。为此,我们在人乳癌细胞系MDA-MB-435S和黑色素瘤细胞系Mel-Juso中进行GST下拉分析(图8)。休眠细胞经HER3抗体1B4C3,2D1D12、对照抗HER2抗体以及PI(3)K抑制剂LY294002预处理,然后经β-HRG刺激。细胞裂解以后,归一化蛋白质水平,由于HER3具有6个潜在的p85结合位点,利用GST-p85(a.a.333-430)为诱饵进行GST下拉分析。针对磷酸酪氨酸(PY)的Western印迹表明,抗HER2和1B4C3同样降低p85与反式活化的HER3的结合,而LY294002(阴性对照)对MDA-MB-435S中p85的结合没有抑制作用(图8C)。然而,2D1D12几乎完全取消MDA-MB-435S中p85与HER3的结合(图8C)
在人黑色素瘤细胞系Mel-Juso中,1B4C3和2D1D12同样降低p85与HER3的结合,而抗HER2显示更显著降低p85的受体结合(图8D)。LY294002再次显示对p85的结合没有抑制作用(图8D)。此外,我们在磷酸酪氨酸印迹中观察到某些显著的酪氨酸磷酸化条带,在MDA-MD-435S中为125kDa和66kDA,而在Mel-Juso中只有125kDA的一条主带。已知PI(3)K物理上结合粘着斑激酶(FAK),因而我们利用FAK抗体重新探测该印迹(图8C、D下图)。我们的数据表明,在两株细胞系中,只有2D1D12和PI(3)K抑制剂LY294002可取消FAK与p85的结合。利用HER2和HER3抗体重新探测该印迹,确认捕获的HER3的量减少,其与HER2的异二聚体化降低(图8C、D中图)。总之,我们的数据表明,虽然HER3和HER2抗体降低受体酪氨酸磷酸化水平,但它们能够在受体结合效应蛋白质的次级水平调节不同的应答。
由于GRB2只直接结合HER2,并通过SHC间接结合HER3,我们在相同的实验条件下利用GST-GRB2为诱饵以及上述人肿瘤细胞系进行另外的GST下拉实验(图9A,B)。我们观察到1B4C3和2D1D12减少160和185kDa之间的受体酪氨酸磷酸化,而LY294002没有抑制作用(图9A、B上图)。然而,利用抗HER2抗体预处理细胞导致受体酪氨酸磷酸化提高,这可由β-HRG进一步加强。利用HER2、HER3和SHC抗体重新探测表明,1B4C3和2D1D12基本上抑制GRB2结合HER2及其间接结合HER3(图9A、B中图),及其在两株细胞系中与SHC的结合(图9A、B下图)。另一方面,抗HER2抗体提高GRB2与HER2和SHC的结合。
为更加了解抗体介导的下游信号,我们还分析了上述实验的全细胞裂解物(WCL)(图10)。当我们关注总细胞蛋白质的磷酸蛋白质含量时,我们观察到在两株细胞系中,抗HER2组成型活化受体,而1B4C3和2D1D12抑制受体酪氨酸磷酸化(图10A上图)。LY294002仍没有作用。
已经非常确定,HRG可活化促细胞分裂剂活化的蛋白质激酶(MAPK)途径,引起细胞增殖、细胞存活并增强各种基因的转录。为检测HER2和HER3抗体对HRG诱导的MAPK活化的作用,利用phospho-ERK(T202/Y204)抗体探测MDA-MB435S和Mel-Juso全细胞提取物的免疫印迹(图10)。MAPK ERK1/2(p44/p42)的磷酸化表明,虽然活化受体,但抗HER2轻微降低ERK1磷酸化,而1B4C3和2D1D12对ERK1磷酸化没有抑制作用(图10A中图)。进一步重新探测该印迹,确认上样了等量蛋白质(图10A下图)。
另外,我们研究了AKT的活化状态,AKT是PI(3)K的下游靶分子,在细胞存活中具有重要作用。我们观察到抗HER2、1B4C3和2D1D12显著抑制Mel-Juso黑色素瘤细胞中AKT的磷酸化(图10B上图)。在MDA-MB-435S乳癌细胞中,HER2和HER3抗体均显著降低AKT磷酸化(图10C)。LY294002作为阳性对照。此观察结果极为重要,因为活化的AKT表达显著提高的乳癌患者更易复发远端转移,而致临床结果较差(Perez-Tenorio G等人British Journal of Cancer,86,540-545(2002)。
单克隆抗体1B4C3和2D1D12抑制乳癌细胞系MDA-MB-435S和黑色素瘤细胞系Mel-Juso的增殖和迁移
为评价1B4C3和2D1D12对细胞周期进程及肿瘤侵袭的抑制功能,我们进行了BrdU掺入及侵袭分析(图11)。
我们发现在2D1D12预处理的MDA-MB-435S和Mel-Juso细胞中,β-HRG刺激的BrdU掺入剧烈下降(图11A)。侵袭分析显示,抗HER3抗体2D1D12和1B4C3基本降低MDA-MB-435S乳癌和Mel-Juso黑色素瘤细胞的侵袭力。令人惊讶的是,HER2抗体4D5只在MDA-MB-435S中显示抑制作用,在黑色素瘤细胞系Mel-Juso中则不然,尽管在细胞表面表达受体(图11B、C及图8A、B)。我们的结果提示,可以使用抗HER3抗体治疗乳癌和黑色素瘤。
单克隆抗体2D1d12抑制Heregulin刺激的PYK2磷酸化
我们先前证明,胞内酪氨酸激酶PYK2可结合HER3并由HER3磷酸化,提示PYK2具有HER3活性介导剂的功能。与此相一致,显性失活的(dominant-negative)PYK2可抑制HRG介导的神经胶质瘤细胞侵袭。为此,我们试图研究抗HER3抗体对HRG诱导的PYK2酪氨酸磷酸化的作用。我们利用抗HER2、1B4C3和2D1D12预处理休眠的SF767人神经胶质瘤细胞,然后利用α-HRG刺激细胞。裂解后按相同蛋白质数量归一化,我们免疫沉淀了PYK2,并针对磷酸酪氨酸(PY)进行印迹。我们观察到抗HER2和1B4C3对PYK2的酪氨酸磷酸化没有抑制作用,而2D1D12显著降低PYK2的酪氨酸磷酸化(图12A)。因此,抗HER3抗体可有效抑制HRG诱导的PYK2酪氨酸磷酸化。
通过利用phospho-ERK抗体探测WCL的免疫印迹,我们观察到利用抗HER2、1B4C3和2D1D12预处理细胞可抑制α-HRG活化的ERK2磷酸化(图12B中图)。利用ERK抗体重新探测,确认上样了等量蛋白质(图12B下图)。我们的数据再次显示,HER3抗体可下调MDA-MB-435S、Mel-Juso和SF767中HRG介导的信号事件。另外,我们的分析提示,针对HER2和HER3胞外域的抗体可调节不同的信号,引起下游效应蛋白质的不同响应。
Claims (29)
1.药物组合物,其包含HER3活性抑制剂为活性剂以及药物可接受的载体、稀释剂和/或助剂,其中所述抑制剂与HER3的结合可降低HER3介导的信号转导。
2.权利要求1的组合物,其中通过下调HER3导致所述信号转导降低。
3.权利要求1的组合物,其中通过使HER3稳定于基本失活的形式而导致所述信号转导降低。
4.权利要求1-3中任一项的组合物,其中该抑制剂并不竞争Heregulin与HER3的结合。
5.权利要求1-4中任一项的组合物,其中所述抑制剂是抗HER3抗体。
6.权利要求5的组合物,其中所述抗体是单克隆抗体或其片段。
7.权利要求5的组合物,其中所述抗体是重组抗体或抗体片段。
8.权利要求7的组合物,其中所述重组抗体或抗体片段选自嵌合抗体、人源化抗体、单链抗体及其片段。
9.权利要求5-8的组合物,其中所述抗体偶联标记基团或效应基团。
10.权利要求5-9的组合物,其中该抗体选自杂交瘤细胞系DSMACC 2527或DSM ACC 2517产生的抗体1B4C3和2D1 D12、其片段或其重组衍生物。
11.权利要求1-10中任一项的组合物,其包含另一活性剂。
12.权利要求1-11中任一项的组合物,其用于诊断性应用。
13.权利要求1-11中任一项的组合物,其用于治疗性应用。
14.权利要求1-10中任一项定义的HER3活性抑制剂的用途,用于制备供诊断、预防或治疗过度增殖性疾病、特别是肿瘤疾病的药剂。
15.权利要求14的用途,其中所述抑制剂是抗HER3抗体。
16.权利要求15的用途,其中该抗体选自杂交瘤细胞系DSM ACC2527或DSM ACC 2517产生的抗体1B4C3和2D1 D12、其片段或其重组衍生物。
17.权利要求14-16中任一项的用途,用于诊断、预防或治疗乳癌、胃肠癌、胰腺癌、前列腺癌、神经胶质瘤、黑色素瘤或肿瘤转移形成。
18.权利要求14-17中任一项的用途,其中所述疾病与HER3的磷酸化提高有关。
19.权利要求14-18中任一项的用途,其中所述疾病与HER2/HER3的异二聚体化提高有关。
20.权利要求14-19中任一项的用途,其中所述疾病与PI3激酶、c-jun末端激酶、AKT、ERK2和/或PYK2的活性提高有关。
21.诊断、预防或治疗过度增殖性疾病、特别是肿瘤疾病的方法,其包含给予有此需要的受试者有效量的权利要求1-10中任一项定义的HER3活性抑制剂。
22.权利要求21的方法,其中所述抑制剂是抗HER3抗体。
23.权利要求22的方法,其中该抗体选自杂交瘤细胞系DSM ACC2527或DSM ACC 2517产生的抗体1B4C3和2D1 D12、其片段或其重组衍生物。
24.权利要求21-23中任一项的方法,其中所述受试者是人。
25.用于诊断、预防或治疗过度增殖性疾病、特别是肿瘤疾病的新型药剂的鉴定方法,其包含分析候选化合物是否能够抑制HER3活性,其中所述抑制剂与HER3的结合可降低HER3介导的信号转导。
26.权利要求25的方法,其中将HER3抑制剂或其衍生化合物配制成药物组合物。
27.杂交瘤细胞系DSM ACC 2517以及其衍生的细胞系。
28.杂交瘤细胞系DSM ACC 2527以及其衍生的细胞系。
29.权利要求27或28的细胞系产生的抗体。
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CN102822201A (zh) * | 2009-12-22 | 2012-12-12 | 罗切格利卡特公司 | 抗her3抗体及其用途 |
CN105209493A (zh) * | 2013-03-14 | 2015-12-30 | 德克萨斯州大学系统董事会 | 用于诊断和治疗用途的her3特异性单克隆抗体 |
CN105264382A (zh) * | 2013-04-05 | 2016-01-20 | 美国控股实验室公司 | 基于检测her3活化辅助癌症的诊断、预后和治疗的系统和方法 |
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2001
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2002
- 2002-08-09 DK DK02794590T patent/DK1414494T3/da active
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- 2002-08-09 AT AT02794590T patent/ATE424219T1/de active
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- 2002-08-09 US US10/486,113 patent/US9011851B2/en active Active
- 2002-08-09 PT PT02794590T patent/PT1414494E/pt unknown
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- 2002-08-09 DE DE60231407T patent/DE60231407D1/de not_active Expired - Lifetime
- 2002-08-09 CN CN2010105512370A patent/CN102078613A/zh active Pending
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- 2002-08-09 WO PCT/EP2002/008938 patent/WO2003013602A1/en active Application Filing
- 2002-08-09 CN CN028156153A patent/CN1541109B/zh not_active Expired - Lifetime
- 2002-08-09 EP EP02794590A patent/EP1414494B1/en not_active Revoked
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CN102822201A (zh) * | 2009-12-22 | 2012-12-12 | 罗切格利卡特公司 | 抗her3抗体及其用途 |
CN102822201B (zh) * | 2009-12-22 | 2014-09-24 | 罗切格利卡特公司 | 抗her3抗体及其用途 |
CN108424456A (zh) * | 2011-11-23 | 2018-08-21 | 医学免疫有限责任公司 | 特异于her3的结合分子及其用途 |
CN105209493A (zh) * | 2013-03-14 | 2015-12-30 | 德克萨斯州大学系统董事会 | 用于诊断和治疗用途的her3特异性单克隆抗体 |
CN105209493B (zh) * | 2013-03-14 | 2019-05-03 | 德克萨斯州大学系统董事会 | 用于诊断和治疗用途的her3特异性单克隆抗体 |
US10358501B2 (en) | 2013-03-14 | 2019-07-23 | The Board Of Regents Of The University Of Texas System | HER3 specific monoclonal antibodies for diagnostic and therapeutic use |
US11174320B2 (en) | 2013-03-14 | 2021-11-16 | The Board Of Regents Of The University Of Texas System | HER3 specific monoclonal antibodies for diagnostic and therapeutic use |
CN105264382A (zh) * | 2013-04-05 | 2016-01-20 | 美国控股实验室公司 | 基于检测her3活化辅助癌症的诊断、预后和治疗的系统和方法 |
Also Published As
Publication number | Publication date |
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PT1414494E (pt) | 2009-06-12 |
CY1110322T1 (el) | 2015-01-14 |
ATE424219T1 (de) | 2009-03-15 |
EP1283053A1 (en) | 2003-02-12 |
EP1414494B1 (en) | 2009-03-04 |
CA2456723A1 (en) | 2003-02-20 |
CN102078613A (zh) | 2011-06-01 |
WO2003013602A1 (en) | 2003-02-20 |
AU2002333384B2 (en) | 2007-11-15 |
JP2005504044A (ja) | 2005-02-10 |
CA2456723C (en) | 2016-02-09 |
DK1414494T3 (da) | 2009-07-06 |
DE60231407D1 (de) | 2009-04-16 |
EP1414494A1 (en) | 2004-05-06 |
CN1541109B (zh) | 2013-07-10 |
US9011851B2 (en) | 2015-04-21 |
EP2067792A2 (en) | 2009-06-10 |
ES2323772T3 (es) | 2009-07-24 |
JP5226926B2 (ja) | 2013-07-03 |
US20040197332A1 (en) | 2004-10-07 |
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