CN105473616B - Egfr抗体缀合物 - Google Patents
Egfr抗体缀合物 Download PDFInfo
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- CN105473616B CN105473616B CN201480038307.9A CN201480038307A CN105473616B CN 105473616 B CN105473616 B CN 105473616B CN 201480038307 A CN201480038307 A CN 201480038307A CN 105473616 B CN105473616 B CN 105473616B
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- egfr
- antibody
- cells
- cetuximab
- immunoconjugate
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Abstract
将美登素类通过不可切割的接头与作为完全EGFR拮抗物的EGFR抗体,诸如西妥昔单抗或帕尼单抗共价连接。结果获得具有在癌细胞中增强但在正常细胞中不增强的细胞毒性的抗癌剂。在作为部分拮抗物的EGFR抗体中或未被溶酶体加工的毒素中没有观察到这种益处。
Description
发明领域
本发明涉及一种免疫缀合物,其靶向表达EGFR的癌细胞群体并包括与微管损伤剂诸如美登素类(maytansinoid)缀合的抗EGFR抗体,诸如帕尼单抗(panitumumab)或西妥昔单抗(cetuximab)。
发明背景
细胞结合蛋白(诸如抗体)与有效的细胞杀伤剂的缀合增强它们的抗癌活性,并提供所谓的“魔术子弹(magic bullets)”,具有混合的临床结果。
相对于裸露抗体,免疫缀合物通常显示增强的细胞杀伤功效,这增加它们抗击表达抗体靶向抗原的癌细胞的活性。但是,在表达相同抗原的正常细胞中也观察到功效的相同增加。特别关注的是增加的抗快速增殖组织(诸如皮肤)的细胞毒性。例如,由美登素类和CD44v6抗体组成的CD44v6靶向免疫缀合物非常活跃的抗击癌细胞,但因为严重的皮肤毒性(诸如毒性表皮坏死溶解,其作为增强免疫缀合物抗击也表达CD44v6的皮肤细胞的活性的结果而发生(Tijinket al.,Clin Cancer Res,2006,12:6064))而被中止。
另一种细胞表面蛋白,表皮生长因子受体或EGFR,是开发抗癌免疫缀合物的有吸引力的靶标,因为许多肿瘤表达该抗原及其快速的内化。但是,因为皮肤组织也表达EGFR,EGFR靶向药剂,诸如抗体西妥昔单抗和帕尼单抗,也显示皮肤毒性水平,这或者要求剂量降低或者在一些情况下是如此严重导致治疗的中止。
通过免疫缀合物,这些抗体的毒性利用缀合有效的细胞毒素得到扩大。这加剧了对正常细胞已经具有固有毒性的抗体的问题。例如,当通过CD44v6抗体递送时,与毒性美登素类的缀合造成严重的针对皮肤细胞的毒性。因此由于对这类免疫缀合物增强的皮肤毒性的顾虑,还没有进行基于批准的抗EGFR抗体的抗EGFR免疫缀合物的开发。正在使用其他策略来开发抗EGFR免疫缀合物。
现在正在设计抗EGFR免疫缀合物来专门解决这些安全性顾虑。这些缀合物基于针对EGFR的突变但天然存在的形式(称为EGFRvIII)或EGFR的构象形式的抗体,这两种都在肿瘤细胞而非皮肤细胞上占优势(分别见US7628986和US 7589180)。例如,抗EGFR抗体MAb806是靶向只在癌细胞上发现的EGFR表位的抗体,有可能提供超过现有EGFR抗体的优点,这些现有EGFR抗体对正常器官诸如人的皮肤都显示显著的结合。关于这种特异性,应该意识到“与现有EGFR抗体相比MAb 806最重要的优点是MAb 806可以直接缀合到细胞毒性剂”,该方法不适用于其他EGFR抗体,因为“细胞毒性缀合几乎是必然诱导严重的毒性”(US7589180)。包括与抗微管负载连接的EGFR MAb 806的免疫缀合物目前正处于晚期实体瘤患者的临床I期试验中。
还在尝试筛选裸露抗EGFR抗体及其免疫缀合物,以鉴定具有抗EGFR的部分拮抗活性和降低的抗角质形成细胞活性的那些抗体(参见US2012/0156217),基于“掩蔽”的抗EGFR抗体(其优先在肿瘤微环境中活化)的免疫偶联物(WO 2009/025846),和基于中等亲和力的抗体(其优先在肿瘤而非正常组织中积聚)的免疫缀合物(WO 2012/100346)。所有这些策略的目的在于降低针对皮肤和表达EGFR的其他器官的毒性,因为目前批准的抗EGFR抗体被认为不适合开发成免疫缀合物。
本发明的目的是提供一种用于治疗EGFR+疾病细胞包括EGFR+癌细胞和包括它们的肿瘤的免疫缀合物。本发明的另一个目的是提供一种选择性增强EGFR抗体针对疾病细胞的细胞毒性的方法。通过这种方法,基本上避免了针对正常细胞的毒性的增强。
发明概述
本发明源自以下预料不到的最初发现:抗EGFR抗体西妥昔单抗与毒性美登素类通过不可切割的接头的缀合产生一种免疫缀合物,其显示增强的抗癌细胞的细胞毒性而抗皮肤细胞的细胞毒性没有相应增加。本发明人证明,一些抗EGFR抗体针对癌症和角质形成细胞的活性通过连接美登素类和其他细胞杀伤剂而显著增强,但是美登素类缀合的西妥昔单抗的活性只针对癌细胞但不针对角质形成细胞增强。在这些研究中发现,与预期一致,西妥昔单抗与除抗微管毒素以外的细胞杀伤剂(诸如皂草素)的缀合具有伴随的和显著增强的针对角质形成细胞的毒性。
还证明另一种具有针对EGFR的完全拮抗物活性的抗体,即帕尼单抗,当与抗微管负载诸如美登素类缀合时,也显示针对EGFR+细胞的选择性增强,显示对EGFR+癌细胞的毒性而不影响EGFR+正常细胞诸如角质形成细胞。
基于本发明公开的这些和其他发现,本发明能够筛选产生免疫缀合物必需的组分,所述免疫缀合物包括EGFR抗体和毒性负载,所述毒性负载增强针对癌细胞但不针对正常细胞诸如角质形成细胞的抗体活性。具有所述特性的免疫缀合物要求筛选作为完全拮抗物的EGFR抗体,作为抗微管物质的毒素,和最优选不可切割的接头。通过使用这些标准,提供一种基于EGFR抗体的免疫缀合物,其具有显著的抗EGFR+癌细胞的治疗活性,但抗EGFR+正常细胞包括皮肤细胞诸如角质形成细胞的毒性没有相应增加。缺乏抗皮肤细胞的毒性的额外增强是至关重要的,因为裸露EGFR靶向抗体的特征在于高发生率的皮肤毒性,不增强这些副作用是有益的。
因此,在一个一般方面,提供一种免疫缀合物,包括具有对EGFR的完全拮抗物活性的抗体和与其通过不可切割接头缀合的毒素,所述免疫缀合物相对于裸露形式的抗体具有对EGFR+角质形成细胞基本上未增强的细胞毒性作用。所述免疫缀合物对EGFR+癌细胞的细胞毒性作用期望是增强的。毒性负载期望是抗微管毒素。
在另一个一般方面,提供一种用于增强EGFR抗体的抗癌活性但不增强其对正常EGFR+细胞的作用的方法,所述方法包括:
(i)选择用于缀合EGFR抗体,所述抗体是完全EGFR拮抗物并与西妥昔单抗竞争结合EGFR;
(ii)选择通过EGFR抗体递送的抗微管毒素;
(iii)选择用于偶联所选EGFR抗体和抗微管毒素的接头;和
产生一种免疫缀合物,其在抗体和毒素之间掺入接头,从而提供具有细胞毒性的免疫缀合物,所述细胞毒性针对EGFR+疾病细胞增强,但针对正常EGFR+角质形成细胞基本上不增强。
在一个具体方面,提供一种包括西妥昔单抗和与其缀合的毒素的免疫缀合物,所述免疫缀合物相对于裸露西妥昔单抗具有细胞毒性作用,所述细胞毒性作用(1)针对EGFR+癌细胞增强,和(2)针对EGFR+角质形成细胞基本上未增强,其中所述免疫缀合物包括西妥昔单抗和通过不可切割接头缀合的抗微管毒素诸如美登素类DM-1。在选择性实施方案中,西妥昔单抗是西妥昔单抗的等同物,诸如西妥昔单抗的EGFR结合片段,或西妥昔单抗的EGFR结合变体,其在抗体恒定区或框架区掺入1个或2个或更多个良性取代,但不影响抗体与受体的结合或抗体缀合介导的细胞杀伤。例如,可以使用标准方法将嵌合的西妥昔单抗进一步人源化以产生更像人形式的抗体。可选的,通过对人Fab文库筛选可以结合并强烈抑制EGFR和与西妥昔单抗竞争受体结合的抗体,开发出完整的人抗EGFR抗体,诸如necitumumab,也称为IMC-11F8,其被认为与西妥昔单抗功能等效(Li S.,Kussie P.,Fergusson KM,Structural basis for EGF receptor inhibition by the therapeuticantibody IMC-11F8.Structure.2008 Feb;16(2):216-27)。
在另一个具体方面,提供另一种包括帕尼单抗和与其缀合的毒素的免疫缀合物,所述免疫缀合物相对于裸露帕尼单抗具有细胞毒性作用,所述细胞毒性作用(1)针对EGFR+癌细胞增强,和(2)针对EGFR+角质形成细胞基本上不改变,其中所述免疫缀合物包括帕尼单抗和通过不可切割接头缀合的抗微管毒素诸如美登素类DM-1。在选择性实施方案中,帕尼单抗是帕尼单抗的EGFR结合片段,或是帕尼单抗的变体,其掺入1个、2个或更多个良性取代但仍维持帕尼单抗母体的EGFR结合和抑制特征。
在优选的实施方案中,使用不可切割接头实现抗体与毒素的缀合。利用不可切割接头,通过溶酶体对药物缀合物的胞内破坏实现细胞毒性负载的释放。不可切割的接头基本上耐受酸诱导的切割、光诱导的切割、肽酶诱导的切割、酯酶诱导的切割或二硫键切割,而可切割接头(其可选择地使用但较不优选)是可以被上述切割剂中的一种或更多种切割的接头。这类不可切割接头的实例包括是或衍生自基于卤代乙酰基(haloacetyl)部分的那些接头,选自N-琥珀酰亚胺-4-(碘代乙酰基)-氨基苯甲酸酯(SIAB),碘乙酸N-羟基琥珀酰亚胺酯(SIA),溴乙酸N-羟基琥珀酰亚胺酯(SBA),和N-琥珀酰亚胺基3-[溴代乙酰基氨基]丙酸酯(SBAP)。可选的,不可切割接头是或衍生自基于马来酰亚胺基的部分,选自N-琥珀酰亚胺基4-(马来酰亚胺甲基)环己烷羧酸酯(SMCC),N-琥珀酰亚胺基-4-(N-马来酰亚胺甲基)-环己烷-1-羧基-(6-氨基己酸酯)(LC-SMCC),κ-马来酰亚胺基十一酸N-琥珀酰亚胺酯(KMUA),γ-马来酰亚胺基丁酸N-琥珀酰亚胺酯(GMBS),ε-马来酰亚胺基己酸N-羟基琥珀酰亚胺酯(EMCS),m-马来酰亚胺苯甲酸-N-羟基琥珀酰亚胺酯(MBS),N-(α-马来酰亚胺乙酸)-琥珀酰亚胺酯(AMAS),琥珀酰亚胺-6-(β-马来酰亚胺丙酰胺基)己酸酯(SMPH),N-琥珀酰亚胺基4-(p-马来酰亚胺苯基)-丁酸酯(SMPB),和N-(p-马来酰亚胺苯基)异氰酸酯(PMPI);另一种不可切割的接头是马来酰亚胺己酰。(更多细节参见US 2005/0169933;,Yoshitake etal,101 Eur.J.Biochem.395-399(1979);Hashida et al,J.Applied Biochem.56-63(1984);和Liu et al,18 690-697(1979),和Doronina et al,.Bioconjugate Chem.,2006Jan-Feb;17(1):114-24)。
在一个优选的实施方案中,使用不可切割的交联剂作为接头诸如琥珀酰亚胺基4-(N-马来酰亚胺甲基)-环己烷-1-羧酸酯(SMCC)实现缀合。不可切割接头的其他有用形式包括N-琥珀酰亚胺基碘乙酸酯(SIA),硫代-SMCC,m-马来酰亚胺苯甲酸-N-羟基琥珀酰亚胺酯(MBS),硫代-MBS和琥珀酰亚胺基-碘乙酸酯,如文献所述,其引入1-10个反应基团。(参见,Yoshitake et al,101 Eur.J.Biochem.395-399(1979);Hashida et al,J.AppliedBiochem.56-63(1984);和Liu et al,18 690-697(1979))。尤其可用于连接作为抗微管毒素的奥里斯他汀(auristatin)的是Doronina等人(Bioconjugate Chem.,2006Jan-Feb;17(1):114-24))描述的不可切割的马来酰亚胺己酰接头。
在另一个方面,提供一种药物组合物,包括对EGFR+癌细胞具有细胞毒性量的所述基于EGFR抗体的免疫缀合物,以及药学上可接受的载体。
在另一个方面,提供所述药物组合物用于治疗EGFR+癌细胞的用途。在一个相关方面,提供一种治疗具有EGFR+癌细胞的受试者的方法,包括给受试者施用对EGFR+癌细胞具有细胞毒性量的所述免疫缀合物。
现在参考附图更详细的描述本发明的这些和其他方面,其中∶
附图简述
图1是显示裸露抗EGFR抗体及其美登素类缀合物针对角质形成细胞的细胞毒活性的图示。将约2-3,000个细胞/孔接种于平底96孔组织培养板,并与溶于培养基的各种浓度的测试物质在37℃温育5天。通过基于WST-8的比色测定确定剩余细胞的生存力。
图2是显示裸露抗EGFR抗体及其美登素类缀合物针对癌细胞系的细胞毒活性的图示。将约2-3,000个细胞/孔接种于平底96孔组织培养板,并与溶于培养基的各种浓度的测试物质在37℃温育5天。通过基于WST-8的比色测定确定剩余细胞的生存力。
本文的图1和2是US 2012/0156217中附图的复制。
图3是显示裸露抗EGFR抗体西妥昔单抗及其美登素类缀合物针对H226癌细胞系的细胞毒活性的图示。将约2-3,000个细胞/孔接种于平底96孔组织培养板,并与溶于培养基的各种浓度的测试物质在37℃温育72h。通过阿尔玛蓝(alamar blue)细胞生存力测定确定剩余细胞的生存力。
图4是显示裸露抗EGFR抗体西妥昔单抗及其美登素类缀合物针对A431癌细胞系的细胞毒活性的图示。将约2-3,000个细胞/孔接种于平底96孔组织培养板,并与溶于培养基的各种浓度的测试物质在37℃温育72h。通过阿尔玛蓝细胞生存力测定确定剩余细胞的生存力。
图5是显示裸露抗EGFR抗体西妥昔单抗及其美登素类缀合物针对正常角质形成细胞系HaCaT的细胞毒活性的图示。将约2-3,000个细胞/孔接种于平底96孔组织培养板,并与溶于培养基的各种浓度的测试物质在37℃温育5天。通过阿尔玛蓝细胞生存力测定确定剩余细胞的生存力。
图6是显示裸露抗EGFR抗体西妥昔单抗及其美登素类缀合物针对原代角质形成细胞的细胞毒活性的图示。将2-3,000个细胞/孔接种于平底96孔组织培养板,并与溶于培养基的各种浓度的测试物质在37℃温育72h或5天。通过阿尔玛蓝细胞生存力测定确定剩余细胞的生存力。
图7是显示裸露抗EGFR抗体帕尼单抗及其美登素类缀合物针对原代角质形成细胞的细胞毒活性的图示。将2-3,000个细胞/孔接种于平底96孔组织培养板,并与溶于培养基的各种浓度的测试物质在37℃温育72h或5天。通过阿尔玛蓝细胞生存力测定确定剩余细胞的生存力。
图8是显示裸露抗EGFR抗体帕尼单抗及其美登素类缀合物针对原代角质形成细胞的细胞毒活性的图示。将2-3,000个细胞/孔接种于平底96孔组织培养板,并与溶于培养基的各种浓度的测试物质在37℃温育72h或5天。通过阿尔玛蓝细胞生存力测定确定剩余细胞的生存力。
图9-13显示掺入不是根据本文规定的标准所选元件的免疫缀合物的结果,如实施例4所述。
图9显示帕尼单抗和西妥昔单抗是非常强的EGFR活化阻断剂,因此不会通过缀合对正常角质形成细胞增强,而部分拮抗性抗体诸如J2898A是增强的(参见图10和11)。
图10显示通过缀合(IMGN289),经由IMGN(J2989A)的部分拮抗性抗EGFR抗体在正常角质形成细胞中得到增强(还可参见Setiady et al,Proceedings of the 104thAnnual Meeting of the American Association for Cancer Research;2013 Apr 6-10;Washington,DC.Philadelphia(PA):AACR;Cancer Res 2013;73(8 Suppl):Abstract nr5463)。
图11显示通过不可切割的接头与负载缀合(6LC-DM1),增强了西妥昔单抗突变抗体6-LC(具有降低的亲和力,使其成为针对EGFR的部分拮抗物)的毒性。
图12显示帕尼单抗和帕尼单抗-DM1对角质形成细胞的作用,并显示通过不可切割的接头将帕尼单抗与DM1缀合(Avid300-DM1)不增加其针对正常细胞的活性;2C9-DM1是用作对照的西妥昔单抗-DM1缀合物。
图13显示通过可切割接头(缬氨酸-瓜氨酸)将西妥昔单抗与MMAE抗微管负载的缀合(Cetux 2C9-MMAE)增强其针对正常细胞和MDA-MB-468癌细胞的毒性,而通过不可切割接头(SMCC)的缀合(Cetux2C9-DM1)只增强抗癌活性,因此显示良好的治疗窗口。
优选实施方案的详细说明
本发明的免疫缀合物基于结合人表皮生长因子受体(hEGFR)的抗体,所述hEGFR是存在于许多不同细胞类型,尤其包括皮肤细胞诸如角质形成细胞表面的蛋白。如本文使用的,术语“hEGFR”是指包括人her-1基因的表达和加工产物的任意蛋白,其中所述蛋白被指定为UniProtKB/Swiss-Prot P00533。术语EGFR在本文中被宽泛使用,是指野生型蛋白及其所有天然存在的变体。术语“wtEGFR”更特定地用于野生型形式的人EGFR。术语“EGFRvIII”是指EGFR变体蛋白,包括缺乏外显子2-7的her-1基因的变体的表达和加工产物,因此包括仅由her-1的外显子1和8编码的多肽序列。术语“结构域III”与EGFRvIII无关,而是指EGFR内的位置,表示对EGF配体结合以及高拮抗性抗体西妥昔单抗和帕尼单抗的结合非常关键的胞外位点(Voigt et al,2012 November;14(11):1023–1031)。
本发明的免疫缀合物包括EGFR抗体,其是EGFR的完全拮抗物。作为“完全拮抗物”的EGFR抗体是完全或接近完全阻断常规情况下被EGF配体刺激通过wtEGFR到wtEGFR缀合的酪氨酸激酶的信号传导的抗体。作为完全拮抗物的EGFR抗体尤其是直接结合EGFR结构域III的EGFR抗体。本发明的免疫缀合物尤其优选包括具有这些特性并且EGFR结合亲和力为5纳摩(nM)或更低的EGFR抗体。通过这种标准,并且如图9所示,至少下列已知的抗体不适用于本发明的免疫缀合物:J2898A,intellimab6-LC(WO 2012/100346教导的西妥昔单抗变体),尼妥珠单抗(nimotuzumab)和马妥珠单抗(matuzumab)。
发现当选择完全拮抗物EGFR抗体来递送毒性负载时,杀伤作用只对EGFR+癌细胞增强,但对EGFR+正常细胞诸如角质形成细胞不增强。当EGFR抗体是部分拮抗物,即允许一些EGF介导信号传导的抗体时,情况不是这样。当缀合毒素时,这些部分拮抗物抗体对EGFR+正常细胞和EGFR+癌细胞均显示杀伤作用的增强。
在一个实施方案中,本发明的免疫缀合物更具体的基于称为西妥昔单抗的hEGFR抗体,目前可商购自礼来公司,商品名为西妥昔单抗是重组的人/小鼠嵌合IgG1抗体,其特异性结合wtEGFR的胞外结构域。本文列出西妥昔单抗重链(SEQ ID No.1-3)和西妥昔单抗轻链(SEQ ID No.4-6)的CDR的氨基酸序列。还列出西妥昔单抗的重链可变区(SEQID No.7)和轻链可变区(SEQ ID No.8)的氨基酸序列,以及西妥昔单抗的完整重链(SEQ IDNo.9)和完整轻链(SEQ ID No.10)的氨基酸序列。
可用于本文的西妥昔单抗变体是高度拮抗性的EGFR结合剂,其与西妥昔单抗竞争结合人EGFR。有用的西妥昔单抗变体已经在上文提及,包括西妥昔单抗的片段,其包括西妥昔单抗的EGFR结合位点,诸如本文描述的全部轻链和重链CDR。可用于本文的其他西妥昔单抗变体是在抗原结合结构域之外,诸如框架区或恒定区(Fc)掺入1个、2个或更多个取代的西妥昔单抗变体。鉴于它们相对于西妥昔单抗自身不降低细胞毒性,这类取代是良性的。
在另一个实施方案中,本发明的免疫缀合物基于称为帕尼单抗的EGFR抗体,其现在是可商购的,并以商品名出售。帕尼单抗是重组的完全人IgG2抗体,其特异性结合wtEGFR的胞外结构域。US 6,235,883和US 7,807,798列出了帕尼单抗的重链和轻链的氨基酸序列。帕尼单抗的有效替代物是与帕尼单抗竞争结合EGFR的其EGFR结合变体,诸如包括其抗原结合位点但具有其他缺失或改变的恒定区的帕尼单抗片段。
本发明的免疫缀合物还可以基于其他EGFR抗体,只要它们显示如上所述的完全拮抗物活性,诸如选择性结合EGFR的结构域III的EGFR抗体,以及与EGF竞争并完全或接近完全阻断EGF刺激的下游信号传导的任意其他EGFR抗体。
在本发明的免疫缀合物中,完全拮抗物EGFR抗体,诸如西妥昔单抗或帕尼单抗缀合到抗微管毒素诸如美登素类毒素。“抗微管毒素”表示具有细胞毒性的物质,所述细胞毒性通过干扰对细胞有丝分裂重要的微管结构来介导,诸如通过抑制微管蛋白的形成或抑制其组建。
该毒素家族包括美登素类和奥里斯他汀,以及近年来开发的具有相同作用机制的许多其他物质。奥里斯他汀具体来说通过抑制微管蛋白的聚合来阻断细胞复制,因此是抗有丝分裂的。可用于本文并称为MMAE或vedotin的奥里斯他汀的结构如下所示∶
存在美登素类的各种有效形式。这些全部基于天然分子美登素(maytansine)的复杂结构。
尤其有用的是包括DM-1和DM-4的美登素类。在一个具体实施方案中,偶联至EGFRMAb的毒素是具有下文所示结构的DM-1。
还可作为抗微管毒素使用的是dolostatin、奥里斯他汀、tubulysin和cryptophycin。各类有效物质的具体实例包括dolostatin 10、单甲基dolostatin10、奥里斯他汀E、单甲基奥里斯他汀E(MMAE)、奥里斯他汀F、单甲基奥里斯他汀F、HTI-286、tubulysin M以及微管蛋白结合剂诸如tubulysin IM-1、tubulysin IM-2、tubulysin IM-3、秋水仙碱DA和美登素类AP-3、DM-1和DM-4。
可以使用目前已知的或今后发展的偶联“不可切割”的接头的任何技术形成完全拮抗物EGFR抗体,诸如西妥昔单抗或帕尼单抗和抗微管毒素,诸如美登素类或奥里斯他汀的缀合物。在给受试者施用后通常遇到的条件下(包括胞外环境),这些接头保持完整,并保留抗体和毒素的共价结合。更具体地,不可切割的接头产生ADC构建体,通过胞内溶酶体破坏抗体来实现细胞毒性负载的释放。
接头整合的方法例如描述于US 5,208,020;US 8,088,387和US 6,441,163。一种优选的方法是利用琥珀酰亚胺基4-(N-马来酰亚胺甲基)-环己烷-1-羧酸酯(SMCC)修饰EGFR抗体,例如西妥昔单抗,以引入马来酰亚胺基,然后将修饰抗体与包含硫醇的美登素类反应以获得硫醚连接的缀合物。获得的化学结构如下所示。每抗体分子缀合1到10个药物分子。
不可切割接头的其他有用形式包括N-琥珀酰亚胺基碘乙酸酯(SIA)、硫代-SMCC、m-马来酰亚胺苯甲酸-N-羟基琥珀酰亚胺酯(MBS)、硫代-MBS和琥珀酰亚胺基-碘乙酸酯,如文献所述,以引入1-10个反应基团。(参见,Yoshitake et al,101 Eur.J.Biochem.395-399(1979);Hashida et al,J.Applied Biochem.56-63(1984);和Liu et al,18 690-697(1979))。尤其可用于连接作为抗微管毒素的奥里斯他汀的是Doronina等人描述的不可切割的马来酰亚胺己酰接头(Bioconjugate Chem.,2006 Jan-Feb;17(1):114-24)。
在一个具体实施方案中,免疫缀合物包括通过SMCC接头连接至DM-1的西妥昔单抗。
在另一个具体实施方案中,免疫缀合物包括通过SMCC接头连接至DM-1的帕尼单抗。
因此,在一个一般方面,本发明提供一种用于增强EGFR抗体的抗癌活性但不增强其对正常EGFR+细胞的作用的方法,所述方法包括∶
(i)选择用于缀合的EGFR抗体,所述抗体是完全EGFR拮抗物;
(ii)选择通过EGFR抗体递送的抗微管毒素;
(iii)选择用于将所选EGFR抗体偶联到抗微管毒素的不可切割的接头;和
在抗体和毒素之间掺入接头以提供具有细胞毒活性的免疫缀合物,所述细胞毒活性针对EGFR+疾病细胞增强,但针对正常EGFR+细胞不增强。
根据前述内容可以理解,完全拮抗物EGFR抗体优选的是西妥昔单抗或帕尼单抗。抗微管毒素优选的是奥里斯他汀或美登素类,最优选的是DM-1。不可切割接头优选的是SMCC。
在另一个具体实施方案中,存在一个附加条件:完全拮抗物EGFR抗体不是西妥昔单抗。在另一个具体实施方案中,存在一个附加条件:完全拮抗物EGFR抗体不是帕尼单抗。
通过将具有所需纯度的缀合物与可选的药学上可接受的载体、赋形剂或稳定剂混合(Remington’s Pharmaceutical Sciences,第16版,Osol,A.Ed.[1980]),采用冻干制剂或水溶液的形式,制备用于直接治疗用途或用于保存的缀合物的治疗制剂。可接受的载体、赋形剂或稳定剂在所用剂量和浓度对受者来说是无毒的,并包括缓冲液诸如磷酸盐、柠檬酸盐和其他有机酸;抗氧化剂包括抗坏血酸和甲硫氨酸;防腐剂(诸如十八烷基二甲基苄基;氯化六烃季铵;氯化苯甲烃铵,氯化苄乙氧铵;苯酚、丁醇或苯甲醇;烷基对羟苯甲酸酯诸如对羟苯甲酸甲酯或对羟苯甲酸丙酯;儿茶酚;间苯二酚;环己醇;3-戊醇;和间甲酚);低分子量(低于约10个残基)多肽;蛋白诸如血清、白蛋白、明胶或免疫球蛋白;亲水聚合物诸如聚乙烯吡咯烷酮;氨基酸诸如甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、精氨酸或赖氨酸;单糖、二糖和其他碳水化合物包括葡萄糖、甘露糖或糊精;螯合剂诸如EDTA;糖诸如蔗糖、甘露醇、海藻糖或山梨糖醇;盐形成反离子诸如钠;金属复合物(例如,锌-蛋白复合物);和/或非离子型表面活性剂诸如Tween、PLURONICS或聚乙二醇(PEG)。
用于体内施用的活性成分必须是无菌的。这可以通过无菌膜的过滤轻易地实现。
可以制备缓释制剂。缓释制剂的合适实例包括包含缀合物的固态疏水多聚体的半渗透基质,所述基质采用成形产品的形式,例如膜或微胶囊。缓释基质的实例包括聚酯、水凝胶(例如,聚(2-羟乙基-异丁烯酸)、聚乳酸(参见美国专利号3,773,919)、L-谷氨酸和乙基-L-谷氨酸的共聚物、不可降解的亚乙基-醋酸乙烯酯、可降解的乳酸-羟基乙酸共聚物诸如由乳酸-羟基乙酸共聚物和醋酸亮丙瑞林组成的注射用微球,以及聚-D-(-)-3-羟丁酸。尽管多聚体诸如亚乙基-醋酸乙烯酯和乳酸-羟基乙酸能够释放分子超过100天,一些水凝胶释放蛋白的时间更短。
缀合物可用于治疗EGFR+疾病细胞。这类治疗导致存在这类疾病细胞的受试者中所述疾病细胞的数目、大小或分布降低。在实施方案中,缀合物用于治疗EGFR+疾病细胞,所述EGFR+疾病细胞是EGFR+癌细胞和包括它们的肿瘤。这类治疗优选的导致这类癌细胞和包括它们的肿瘤的数目、大小、体积或分布降低,或至少在存在它们的受试者中这类细胞和肿瘤的数目、大小、体积或分布的增加速率降低。
在生理样品诸如活检切片组织中借助检测作为蛋白或作为核酸前体(DNA或RNA)的受体的测定,可以鉴定存在EGFR+癌细胞的受试者。EGFR蛋白的合适检验是商品化供应的,FDA批准的Dako EGFR 检测试剂盒。
为了治疗存在EGFR+癌细胞的受试者,缀合物的合适剂量将取决于上文定义的待治疗的疾病类型、疾病的严重性和病程、施用物质是用于预防还是治疗目的、先前的治疗、患者临床史和对物质的应答以及主治医师的判断。物质适于一次性或者在一系列治疗中施用给患者。
例如,根据疾病的类型和严重性,约1μg/kg到15mg/kg(例如,0.1-20mg/kg)的缀合物是给患者施用的候选剂量,无论例如是通过一次或更多次单独施用还是通过连续的输注。一个典型的每日剂量范围可以从大约1μg/kg到500mg/kg或者更多,取决于如上所述的因素。对于在数天或者更长时间内重复给药,根据病症,维持治疗直到发生期望的疾病症状抑制。但是,可以使用其他给药方案。可以方便的通过常规技术和测定监测这类疗法的进展。
因此应理解,免疫缀合物的有效量是单独的或作为治疗方式一部分的有效量,其迟滞或抑制EGFR+疾病细胞的生长或增殖速率。
EGFR+疾病细胞是在其表面存在可检测的EGFR(例如通过EGFR抗体结合,或通过检测编码her-1的胞内mRNA)的疾病细胞。特定的EGFR+疾病细胞包括在其表面具有异常高密度和/或活性的EGFR分子,或存在EGFR的EGFRvIII变体的那些细胞。
以下是有效的,首先通过静脉内输注施用作为起始剂量的本发明的缀合物,继之以维持剂量,诸如90分钟内4mg/kg的初始剂量,然后30分钟内2mg/kg,每周一次,多达52周,根据需要进行随访。在帕尼单抗缀合物的特定情况下,给药方案可以基于使用帕尼单抗自身的方案,包括每两周给予一次6mg/kg,1小时的输注。
缀合物可用于治疗各种癌症,抑制EGFR+癌细胞和包括它们的肿瘤(包括造血细胞癌和实体瘤)的生长或增殖。待治疗的疾病或病症包括良性或恶性肿瘤(例如,肾(renal)、肝、肾(kidney)、膀胱、乳腺、胃、卵巢、结肠直肠、前列腺、胰腺、肺、外阴和甲状腺);肝癌;肉瘤;恶性胶质瘤;和各种头颈部肿瘤;白血病和淋巴恶性肿瘤。在具体实施方案中,使用抗体或二价片段治疗表达EGFRvIII(通过本文描述的筛选测定来确定)的这类癌细胞。在具体实施方案中,癌细胞是存在EGFR+的癌细胞,包括头颈癌,尤其是头颈部的鳞状细胞癌,结肠直肠癌,胃肠癌,包括恶性胶质瘤的脑瘤,和包括非小细胞肺癌的肺部肿瘤,以及乳腺、胰、食管、肾、卵巢、子宫颈和前列腺的肿瘤。在具体实施方案中,EGFR+癌是西妥昔单抗已针对其获得FDA上市许可的癌症,诸如头颈部的鳞状细胞癌和结肠直肠的。
应理解可以受益于所述方法的受试者包括哺乳动物,包括人以及家畜和宠物。
其他治疗方式可以与本发明缀合物的施用进行组合。例如,待治疗的患者还可以接受放射治疗,诸如外线束放射。可选的,或此外,可以给患者施用化疗剂。可以根据制造商的说明书或由熟练从业人员通过经验确定这类化疗剂的制备和给药方案。这类化疗的制备和给药方案还描述于Chemotherapy Service Ed.,M.C.Perry,Williams&Wilkins,Baltimore,Md.(1992)。化疗剂可以在缀合物之前或之后施用,或可以与其同时给予。缀合物可以与任意抗癌毒素或任意其他合适的药物组合,尤其包括依立替康(CPT-11)、顺铂、环磷酰胺、苯丙氨酸氮芥、氮烯唑胺、多柔比星、道诺红菌素和托泊替康,以及酪氨酸激酶抑制剂,尤其包括EGFR激酶抑制剂诸如AG1478((4-(3-氯苯胺-6,7-二甲氧基喹唑啉)、吉非替尼()、厄洛替尼()、拉帕替尼()、卡奈替尼(PD183805,Pfizer)、PKI-166(Novartis)、PD158780和培利替尼。
还希望施用针对其他肿瘤相关抗原或它们的配体的抗体或缀合物,诸如结合以下物质的抗体:ErbB2(包括市售为的曲妥单抗,和市售为的帕妥珠单抗)、ErbB3、ErbB4,或血管内皮因子(VEGF),和/或结合EGF或TGFα的抗体。
在本发明的另一个实施方案中,提供一种包含可用于治疗本文所述病症的量的缀合物的制品。所述制品包括容器和标签。合适的容器包括,例如,瓶子、小瓶、注射器和试管。容器可以由各种材料形成,诸如玻璃或塑料。容器包含可有效治疗疾病的组合物,并具有无菌进入孔(例如,容器可以是具有可被皮下注射针头刺穿的塞子的静脉内溶液袋或小瓶)。容器上的或粘贴的标签说明组合物用于治疗癌症疾病。所述制品还可以包含第二容器,其包含药学上可接受的缓冲液,诸如磷酸盐缓冲液、Ringer's溶液和右旋糖溶液。它还可以包括从商品化和使用观点来看所希望的其他物质,包括其他缓冲液、稀释剂、滤器、针头、注射器以及含使用说明书的装箱单。
本发明的抗癌免疫缀合物可以与药学上可接受的稀释剂、载体或赋形剂在单位剂型中施用。缀合物的单位剂量合适地是50mg、100mg、150mg、200mg、250mg、300mg和400mg。可以在单次使用小瓶中配制药物,浓度诸如20mg/mL,例如100mg溶于5mL赋形剂诸如0.9%盐水,200mg溶于10mL或400mg溶于20mL。
可以使用任何适当的施用途径,例如,肠胃外、静脉内、皮下、肌内、颅内、眶内、眼、心室内、囊内、脊柱内、脑池内、腹腔内、鼻内、雾化、肺部、或口服施用。
实施例1:西妥昔单抗-SMCC-DM1缀合物的制备(A-H)
A.西妥昔单抗抗体的制备和测量
西妥昔单抗获自开放市场,或按照WO 2012/100346所述产生,用于利用不可切割异型双功能交联剂SMCC来缀合DM1。
然后将西妥昔单抗抗体缓冲液置换为50mM磷酸钾,50mM氯化钠,2mM EDTA;pH 6.5缓冲液(缓冲液A)。使用生色鲎属阿米巴细胞溶解产物(LAL)方法(Cambrex)来验证本实验的所有缓冲液不含内毒素。使用280nm的1.45mL/mg/cm消光系数和145,781g的分子量测量抗体浓度。
B.SMCC储液的制备和测量
在DMSO中制备20mM SMCC(6.69mg/mL)(Concortis Biosystems Corp.)溶液。该溶液用测定缓冲液进行1/40稀释,测量样品在302nm的吸光度。使用602/M/cm的摩尔消光系数计算储液浓度。
C.DM1储液的制备和测量
在DMA(7.37mg/mL)中制备10mM DM1(游离巯基形式;Concortis BiosystemsCorp.)溶液。在280nm测量储液在乙醇中的稀释液的吸光度。使用280nm处5700/M/cm的摩尔消光系数计算储液DM1的浓度。使用Elman's试剂(DTNB)测量储液DM1制品中游离-SH的浓度。在测定缓冲液中制备储液的稀释液到3%(v/v)DMA,然后添加溶于DMSO的100mM DTNB(1/100体积)。针对空白试剂测量412nm处吸光度的增加,并使用14150/M/cm的消光系数计算浓度。使用源自Elman's测定的-SH浓度表示DM1储液浓度来计算缀合条件。
D.利用SMCC交联剂修饰西妥昔单抗
以20mg/ml抗体使用7.5倍摩尔过量的SMCC对抗体进行修饰。在室温搅拌条件下,在含DMSO(5%v/v)的缓冲液A(95%v/v)中进行反应。
E.G25层析以去除过量的SMCC
利用缓冲液A平衡的1.5×4.9cm Sephadex G25树脂的预填充柱凝胶过滤西妥昔单抗-SMCC反应混合物。加载和洗脱体积参照制造商的说明书(Amersham Biosciences)。使用如上所述的消光系数通过分光光度法测定修饰抗体溶液的浓度。
F.西妥昔单抗-SMCC与DM1的缀合
将修饰抗体与比接头(假定5个接头每抗体)1.7倍过量的DM1反应。在含DMA(6%v/v)的缓冲液A中以10mg/ml抗体浓度(94%v/v)进行反应。在添加DM1后,反应在室温搅拌条件下温育16.5小时。
G.通过G25层析的缀合纯化
利用1×磷酸缓冲盐溶液(PBS),pH 6.5(缓冲液B)平衡的1.5×4.9cm SephadexG25树脂的预填充柱凝胶过滤缀合反应混合物。加载和洗脱体积参照制造商的说明书(Amersham Biosciences)。通过在252nm和280nm测量洗脱材料的吸光度,确定每摩尔西妥昔单抗连接的DM1分子数目。发现DM1/抗体比例是2和4。分析所获缀合物的结合和细胞毒性。
H.西妥昔单抗-SMCC-DM1的检测
用于这些研究的细胞系具有下列特征∶
A431:人上皮外阴癌细胞系;获自ATCC;在DMEM-10%FBS中按照4000细胞/孔,100μl/孔接种于96孔板。
H226:肺鳞状细胞癌细胞系;获自ATCC;在RPMI-10%FBS中按照4000细胞/孔,100μl/孔接种于96孔培养板。
MDA-MB-468:乳腺(mammary gland)/乳房(breast);来源于转移部位:胸膜积液;获自ATCC;在添加胎牛血清至10%终浓度的ATCC配制的Leibovitz's L-15培养基(CatalogNo.30-2008)中按照4000细胞/孔,100μl/孔接种于96孔板。
HaCaT:来自组织学正常皮肤的体外自发转化的角质形成细胞;获自武汉大学的中国典型培养物保藏中心;在DMEM-10%FBS中按照2000细胞/孔,100μl/孔接种于96孔培养板。
HEKa:正常人原代表皮角质形成细胞,成人;获自PromoCell GmbH;在EpiLife培养基中按照4000细胞/孔接种,100μl/孔,EpiLife培养基#MEP1500CA,Invitrogen+HKGS人角质形成细胞生长补充剂(#S-001-5)
结合研究显示,抗体与DM1的缀合不影响KD;通过表面等离子体共振法(~0.3nM,表1),裸露西妥昔单抗抗体和西妥昔单抗-SMCC-DM1缀合物具有相同的对EGFR的结合亲和力。样品体外细胞毒性的鉴定显示,与西妥昔单抗相比,西妥昔单抗-SMCC-DM1针对癌细胞系的细胞毒性显著更高(图3&4),但出人意料的,西妥昔单抗-SMCC-DM1和裸露西妥昔单抗具有相同的针对角质形成细胞的细胞毒活性(图5和6)。
表1∶与DM1的缀合对西妥昔单抗KD的影响
样品 | 实验的# | EGFR ecd KD(nM) |
商品化西妥昔单抗 | 3 | 0.27+/-0.02 |
西妥昔单抗-SMCC-DM1 | 3 | 0.28+/-0.02 |
在ImmunoGen's公开的US专利申请2012/0156217中显示DM1缀合对不同EGFR MAbs(huML66和huEGFR-7R)的影响,本文的图1和2是该申请中附图的复制。更具体的,如图1所示,抗EGFR抗体huML66-SMCC-DM1和huEGFR-7R与美登素类的缀合产生huML66-SMCC-DM1和huEGFR-7R-SMCC-DM1免疫缀合物,增加针对正常角质形成细胞的细胞毒活性。类似地,与预期一致,抗EGFR抗体huML66和huEGFR-7R与美登素类的缀合产生huML66-SMCC-DM1和huEGFR-7R-SMCC-DM1免疫缀合物,显著增强所述抗体针对H226癌细胞系的细胞毒活性(图2)。
如图3和4所示,抗EGFR抗体西妥昔单抗(HC-LC)的DM1缀合产生西妥昔单抗-SMCC-DM1免疫缀合物(HC-LC/DM1),显著增强所述抗体针对癌细胞的细胞毒活性,与预期一致,并显示于图3。如图4所示,并且与预期一致,抗EGFR抗体西妥昔单抗(HC-LC)与美登素类的缀合产生西妥昔单抗-SMCC-DM1免疫缀合物(HC-LC/DM1),显著增强所述抗体针对癌细胞的细胞毒活性。
但是,相当出人意料的,如图5和6所示,抗EGFR抗体西妥昔单抗(HC-LC)与美登素类的缀合产生西妥昔单抗-SMCC-DM1免疫缀合物(HC-LC/DM1),不增加针对角质形成细胞的细胞毒性,反映在这些物质基本上相同的IC50值(缀合物的IC50为0.8269vs.裸露抗体的IC50为0.4324;图5,HaCaT-正常人角质形成细胞系&缀合物的IC50为2.210vs.裸露抗体的IC50为0.9348,图6;HEKa-原代人角质形成细胞)。如本文使用的,IC50是指杀伤50%细胞所需的药物浓度。
利用阿尔玛蓝细胞生存力测定(每次实验在96孔板中重复3次),通过比较IC50值测定细胞毒性是否增加。在一个log数级范围内的差异被认为是不显著的,被看作不显示细胞毒性的变化,而大于一个log数级范围的差异显示细胞毒性的显著变化。
实施例2:灵长类动物中的评估
猕猴属先前已被证明是鉴定抗EGFR抗体毒性,包括皮肤副作用的有价值和高度预测性的模型。关于EGFR靶向抗体的猕猴和人毒性数据之间高水平的相关性部分归因于猴和人EGFR受体之间的高度同源性,导致针对抗体的非常类似的KD值:
表1:西妥昔单抗和帕尼单抗对人和猕猴EGFR的结合亲和力是跨种属一致的。表观抗体亲和力被定义为,在利用人EGFR(huEGFR)和猕猴EGFR(cyEGFR)的重组胞外结构域的ELISA中实现最大结合的一半需要的抗体浓度(皮摩尔,pM)。改编自Koefoed et al,MABs.2011Nov-Dec;3(6):584-595。
尤其在灵长类动物中检验西妥昔单抗-SMCC-DM1免疫缀合物通过缀合毒素对正常细胞的任意显著增强的抗体副作用。具体来说,对于皮肤副作用给与密切关注,其是相对于裸露完全拮抗物抗EGFR抗体的预期,性质不同和/或显著加重。
研究最初利用2只猕猴恒河猴,均为雄性,用氯胺酮镇静并用异氟烷麻醉,以便静脉内施用抗体-药物缀合物,按照10mg/kg施用。然后观察动物5天,随后处死以检查全身急性毒性症状,并做进一步检查。
除了利用裸露西妥昔单抗治疗预期的那些副作用以外,详细的组织病理学分析未显示严重的皮肤毒性或其他严重的和性质上新的皮肤副作用。此外,在这些动物中没有观察到其他严重的对靶标的毒性,证明本发明ADC的安全性和没有裸露抗体毒性的显著加重。
在成功完成急性灵长类动物研究后,已经在其他动物中开始重复-剂量的安全性研究。按照10mg/kg剂量,每三周施用一次ADC,共4个周期。这种剂量和方案的选择是基于灵长类动物和人类中利用其他ADC经常使用的给药方案(Poon KA et al,Preclinicalsafety profile of trastuzumabemtansine(T-DM1):mechanism of action of itscytotoxic component retained with improved tolerability.Toxicol ApplPharmacol.2013 Dec 1;273(2):298-313;FDA Package Insert for KadcylaTM,accessedFebruary 24,2014:http://www.accessdata.fda.gov/drugsatfda_docs/label/2013/125427lbl.pdf)。
除了可逆的肝酶升高(其是ADCs的预期副作用类型)外,在目前进行的研究中没有观察到利用裸露西妥昔单抗治疗已经预期的那些副作用之外的其他严重的和/或性质上新的皮肤或其他对靶标的副作用(西妥昔单抗猕猴数据获自:http://www.accessdata.fda.gov/drugsatfda_docs/bla/2004/125084_ERBITUX_PHARMR_P3.PDF)。在本研究中没有观察到先前报道的利用抗CD44v6 ADC的严重的皮肤毒性和毒性表皮坏死溶解(其也利用SMCC-DM1负载技术,并靶向正常角质形成细胞表达的抗原)(Tijink et al.,Clin Cancer Res,2006,12:6064)。这些安全性数据进一步证实体外结果,完全拮抗物裸露抗EGFR抗体对靶标的毒性不因为缀合细胞毒性负载而显著增强。
实施例3:帕尼单抗-SMCC-DM1缀合物的制备(A-H)
基本上按照如上关于西妥昔单抗对应物的描述制备缀合DM-1的帕尼单抗。更具体的,
A.帕尼单抗抗体的制备和测量
帕尼单抗获自开放市场,或按照US 6,235,883或US 7,807,798所述产生,用于利用不可切割异型双功能交联剂SMCC来缀合DM1。
然后将帕尼单抗抗体缓冲液置换为50mM磷酸钾,50mM氯化钠,2mM EDTA;pH 6.5缓冲液(缓冲液A)。使用生色鲎属阿米巴细胞溶解产物(LAL)方法(Cambrex)来验证本实验的所有缓冲液不含内毒素。使用280nm的1.45mL/mg/cm消光系数和145,781g的分子量测量抗体浓度。
B.SMCC储液的制备和测量
在DMSO中制备20mM SMCC(6.69mg/mL)(Concortis Biosystems Corp.)溶液。该溶液用测定缓冲液进行1/40稀释,测量样品在302nm的吸光度。使用602/M/cm的摩尔消光系数计算储液浓度。
C.DM1储液的制备和测量
在DMA(7.37mg/mL)中制备10mM DM1(游离巯基形式;Concortis BiosystemsCorp.)溶液。在280nm测量储液在乙醇中的稀释液的吸光度。使用280nm处5700/M/cm的摩尔消光系数计算储液DM1的浓度。使用Elman's试剂(DTNB)测量储液DM1制品中游离-SH的浓度。在测定缓冲液中制备储液的稀释液到3%(v/v)DMA,然后添加溶于DMSO的100mM DTNB(1/100体积)。针对空白试剂测量412nm处吸光度的增加,并使用14150/M/cm的消光系数计算浓度。使用源自Elman's测定的-SH浓度表示DM1储液浓度来计算缀合条件。
D.利用SMCC交联剂修饰帕尼单抗
以20mg/ml抗体使用7.5倍摩尔过量的SMCC对抗体进行修饰。在室温搅拌条件下,在含DMSO(5%v/v)的缓冲液A(95%v/v)中进行反应。
E.G25层析以去除过量的SMCC
利用缓冲液A平衡的1.5×4.9cm Sephadex G25树脂的预填充柱凝胶过滤帕尼单抗-SMCC反应混合物。加载和洗脱体积参照制造商的说明书(Amersham Biosciences)。使用如上所述的消光系数通过分光光度法测定修饰抗体溶液的浓度。
F.帕尼单抗-SMCC与DM1的缀合
将修饰抗体与比接头(假定5个接头每抗体)1.7倍过量的DM1反应。在含DMA(6%v/v)的缓冲液A的10mg/ml抗体浓度(94%v/v)进行反应。在添加DM1后,反应在室温搅拌条件下温育16.5小时。
G.通过G25层析的缀合纯化
利用1×磷酸缓冲盐溶液(PBS),pH 6.5(缓冲液B)平衡的1.5×4.9cm SephadexG25树脂的预填充柱凝胶过滤缀合反应混合物。加载和洗脱体积参照制造商的说明书(Amersham Biosciences)。通过在252nm和280nm测量洗脱材料的吸光度,确定每摩尔帕尼单抗连接的DM1分子数目。发现DM1/抗体比例是2和4。分析所获缀合物的结合和细胞毒性。
H.帕尼单抗-SMCC-DM1的检测
用于这些研究的细胞系具有下列特征∶
MDA-MB-468:乳腺(mammary gland)/乳房(breast);来源于转移部位:胸膜积液;获自ATCC;在添加胎牛血清至10%终浓度的ATCC配制的Leibovitz's L-15培养基(CatalogNo.30-2008)中按照4000细胞/孔,100μl/孔接种于96孔板。
HaCaT:来自组织学正常皮肤的体外自发转化的角质形成细胞;获自武汉大学的中国典型培养物保藏中心;在DMEM-10%FBS中按照2000细胞/孔,100μl/孔接种于96孔培养板。
利用角质形成细胞的体外试验显示,帕尼单抗与抗微管毒素通过不可切割接头的缀合不增强抗体的毒性。获得的ADC具有与裸露帕尼单抗以及基于完全拮抗性抗体西妥昔单抗的另一个ADC类似的对角质形成细胞的安全性模式。作为抗体与抗微管毒素通过不可切割接头缀合的结果,观察到帕尼单抗显著增强的针对MDA-MB-468癌细胞的抗癌活性。与毒性观察类似,基于帕尼单抗的ADC与基于西妥昔单抗的ADC活性类似。
实施例4:部分拮抗性EGFR Mab缀合物的结果
选择不同于本文那些推荐的免疫缀合物组分的效果在附图中显示。图9显示当缀合DM-1时,部分拮抗性EGFR抗体(称为J2989A)具有增强的对正常角质形成细胞的活性。类似地,图10显示,当缀合DM-1时,另一个部分拮抗物抗体(称为6-LC(西妥昔单抗的取代变体,具有更低的对EGFR的相对亲和力))也具有增强的对正常角质形成细胞的活性。图11显示,当缀合DM1时,帕尼单抗对角质形成细胞的作用不增强,而缀合物的抗癌作用显著增强(图12)。并且,图13显示,西妥昔单抗通过可切割接头(缬氨酸-瓜氨酸)与MMAE的缀合增强其针对正常细胞和MDA-MB-468癌细胞的毒性,而通过不可切割接头(SMCC)的缀合增强抗癌活性。
因此,在这些研究中,只有通过不可切割接头的负载缀合的完全拮抗性抗EGFR抗体不对正常细胞增强,而通过缀合,部分拮抗物EGFR抗体针对正常细胞的毒性增强。此外,我们随后检测了可切割vs.不可切割接头对该活性模式的影响,并维持负载的作用机制相同(即抗微管物质)。将抗体通过可切割接头缀合到抗微管负载,如Doronina等人描述的MMAE(Nature Biotechnology Nov 7,2003,Vol 21:pp 778-784)。可切割接头数据显示,当完全拮抗性抗体通过可切割接头缀合它们的负载时,它们针对正常细胞的毒性被增强。因此,安全的抗EGFR ADC应当包括通过不可切割接头连接抗微管负载的强拮抗性抗EGFR抗体。
权利要求的范围不应受到实施例所述的优选实施方案的限制,但应当给出作为整体符合说明书的最宽泛的解释。
参照序列
SEQ ID No.描述
1 西妥昔单抗重链CDR1
NYGVH
2 西妥昔单抗重链CDR2
VIWSGGNTDYNTPFTS
3 西妥昔单抗重链CDR3
ALTYYDYEFAY
4 西妥昔单抗轻链CDR1
RASQSIGTNIH
5 西妥昔单抗轻链CDR2
ASESIS
6 西妥昔单抗轻链CDR3
QQNNNWPTT
7 西妥昔单抗重链可变区(VH)
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA
8 西妥昔单抗轻链可变区(VL)
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK
9 西妥昔单抗完整重链
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
10 西妥昔单抗完整轻链
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE
Claims (8)
1.一种免疫缀合物,包括(i)具有SEQ ID NO.9的重链和SEQ ID NO.10的轻链的完全拮抗物EGFR抗体,和(ii)与所述抗体通过不可切割接头缀合的抗微管毒素,其中所述接头是SMCC且其中所述毒素是DM-1,所述免疫缀合物相对于所述抗体的裸露形式具有细胞毒性作用,所述细胞毒性作用(1)针对EGFR+癌细胞增强,和(2)针对EGFR+角质形成细胞基本上不改变。
2.一种药物组合物,包括对EGFR+疾病细胞具有细胞毒性量的权利要求1的免疫缀合物,以及药学上可接受的载体。
3.一种用于产生抗癌组合物的方法,包括将药学上可接受的载体与通过SMCC接头缀合于DM-1以形成免疫缀合物的EGFR抗体组合的步骤,所述免疫缀合物相对于裸露抗体对癌细胞和角质形成细胞的作用来说,具有对癌细胞增强的作用和对角质形成细胞基本上未增强的作用,其中所述抗体具有SEQ ID NO.9的重链和SEQ ID NO.10的轻链。
4.权利要求2的药物组合物在制备用于治疗EGFR+疾病细胞的药物中的用途。
5.权利要求1的免疫缀合物在制备用于治疗具有EGFR+疾病细胞的受试者的药物中的用途,所述免疫缀合物对EGFR+疾病细胞具有细胞毒性。
6.权利要求5的用途,其中所述EGFR+疾病细胞是EGFR+癌细胞。
7.权利要求6的用途,其中所述EGFR+癌细胞是头颈癌细胞或结肠直肠癌细胞。
8.一种用于增强完全拮抗物EGFR抗体对EGFR+疾病细胞的作用但不增强其对正常EGFR+细胞的作用的方法,包括将所述抗体与DM-1通过不可切割的接头SMCC进行连接,其中所述抗体具有SEQ ID NO.9的重链和SEQ ID NO.10的轻链。
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