CN116920112A - Icam1或其编码基因在制备胃癌治疗或诊断产品中的用途 - Google Patents
Icam1或其编码基因在制备胃癌治疗或诊断产品中的用途 Download PDFInfo
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- CN116920112A CN116920112A CN202210332176.1A CN202210332176A CN116920112A CN 116920112 A CN116920112 A CN 116920112A CN 202210332176 A CN202210332176 A CN 202210332176A CN 116920112 A CN116920112 A CN 116920112A
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Abstract
本发明涉及医药领域,特别是涉及ICAM1或其编码基因在制备胃癌治疗产品或诊断产品中的用途,ICAM1或其编码基因能够作为胃癌治疗产品或胃癌诊断产品的分子靶标,具体的,特异性结合ICAM1的物质例如抗体偶联药物或其药学上可接受的盐,或ICAM1抑制剂能够用于制备胃癌治疗产品中,以及特异性结合ICAM1的MRI分子探针,用于制备ICAM1阳性胃癌的分子诊断产品中或用于制备预测胃癌ICAM1相关药物的治疗效果产品中。利用本发明ICAM1或其编码基因制备的抗体偶联药物在对胃癌细胞杀伤效果显著的情况下,不杀伤正常上皮细胞,克服了传统化疗药物在杀伤肿瘤细胞的同时损伤正常组织、细胞的缺点。
Description
技术领域
本发明涉及医药领域,特别是涉及ICAM1或其编码基因在制备胃癌治疗或诊断产品中的用途。
背景技术
胃癌是最常见的恶性肿瘤之一,发病率和死亡率均较高,分别位于恶性肿瘤的第5位和第4位,严重威胁人类的生命健康。胃癌是一种高度异质性肿瘤,不同亚型之间呈现出较大的差异化。目前已有多种对胃癌的分型方法,传统分型方法基于肿瘤的外观形态,对临床指导意义有限,不能有效指导个体化治疗的靶向用药。2014年,肿瘤基因组计划(TheCancer Genome Atlas,TCGA)在Nature上提出了一种全新的分型方法——TCGA分型,将胃癌分为染色体不稳定型(CIN型)、微卫星不稳定型(MSI型)、染色体稳定型(GS型)和人类四型孢疹病毒阳性型(EBV型),使肿瘤分类从传统的形态学转向以分子特征为基础的分子分型,使个体化精准治疗成为可能。TCGA分型中,染色体不稳定型(CIN)胃癌发病比例最高,约占49.8%,发病部位多位于胃食管结合部,预后较差。
CIN是指染色体区域中DNA发生拷贝数改变或结构异常,从而导致染色体发生不可逆的易位、扩增或缺失的现象,最常扩增的区域包括染色体1q、5p、6p、7p、7q、8q、13q、19p、20p、20q,最常丢失的区域包括染色体3p、4p、4q、5q、6q、9p、14q、18q、21q。CIN亚型胃癌多属于Lauren分型中的肠型,具有TP53基因频繁突变和RTKs基因扩增的特点。研究显示,CIN能够驱动肿瘤中异质性的克隆进化,进而获得耐药性。引起CIN的分子机制复杂多样,目前已知的可能性有:(1)有丝分裂过程中检查点缺陷、动粒障碍、姐妹染色单体凝聚和分离缺陷、中心体扩增和端粒功能障碍等导致的染色体分离异常。(2)细胞周期检查点缺陷:CDK激酶过度活跃会使DNA损伤的细胞细胞周期延续,含受损DNA的细胞增殖;TP53突变消除G1-S检查点会导致基因扩增、缺失和基因组重排。(3)致癌基因诱导的有丝分裂应激:致癌基因RAS过表达导致中心体过度复制、后期桥的产生和多极纺锤体的形成;MET过表达可通过PI3K-AKT途径诱导多中心体形成;Raf癌基因中BRAF突变可诱导异常纺锤体、多中心体、染色体错误分离和非整倍体。(4)复制压力:有丝分裂前错误和受损的复制叉进程导致复制压力。18q号染色体上3个CIN抑制基因PIGN(MCD4)、RKHD2(MEX3C)、ZNF516(KIAA0222),18q丢失与非整倍体在腺瘤-癌转变时相关。CIN抑制基因沉默导致DNA复制压力、结构染色体异常和染色体错误分离。在病理检查中,恶性肿瘤的增殖活性常用有丝分裂细胞比例或有丝分裂指数来表示,因此靶向有丝分裂被认为是癌症治疗的有力手段。但靶向有丝分裂具有挑战性,因为它会损伤正常增殖的细胞,导致严重不良反应,因此鉴定分子缺陷、降低药物不良反应是开发抑制有丝分裂的抗肿瘤药物的关键所在。
GS亚型胃癌(GSGC)患者约占胃癌总体患者的20%,初诊年龄偏低(中位年龄59岁),约73%属于Lauren分型中的弥漫型,该型CDH1、RHOA基因突变率高,且常见CLDN18-ARHGAP26(GRAF)融合现象。随后,Bo Hwa Sohn等研究者通过分析TCGA项目的基因表达数据并基于4种亚型的基因表达特征建立预测模型,利用该模型分别检测了两个独立队列中各亚型与预后的相关性,结果均表明EBV亚型的预后最佳,MSI和CIN亚型患者预后次之,而GS亚型的预后最差。可见,GSGC多数是弥漫型胃癌,转移较快且预后最差,这类致死性较高的胃癌亚型具有重要未满足的医学需求。
抗体偶联药物(antibody drug conjugate,ADC)将小分子细胞毒药物与靶向特异性抗原的单克隆抗体通过连接子进行偶联,使药物能够选择性地靶向肿瘤,抗体药物以其高特异性、安全性和有效性的特点成为全球抗肿瘤药物研发的热点。ADC药物利用单克隆抗体的靶向性,特异性识别肿瘤细胞表面抗原,内化后进入细胞,转运至溶酶体中,释放细胞毒药物,发挥DNA酶抑制或微管抑制作用,诱导细胞死亡。ADC药物既具有小分子细胞毒药物的抗肿瘤效应,又具有高选择性、较好的稳定性和良好的药代动力学优点。ADC药物自提出至今一直在创新优化,目前已经过三次变革,第三代ADC药物通过对抗体进行改造,在特定位点插入非天然氨基酸实现定点偶联,实现了更多新靶点的发现、载药值的提高和连接子的优化的目标。全球目前已有14个ADC药物已获批,在血液瘤和少数实体瘤中取得了良好的治疗效果。目前,除了HER2和CD靶点外,c-Met、EGFR、BCMA、Trop2和CD20也是ADC药物研发的热门靶点。尽管ADC类药物在实体瘤中展现出了可观的治疗潜力,但是目前适应于胃癌的ADC类药物屈指可数。目前已批准上市的抗体偶联药物T-DM1、T-Dxd和RC48可用于胃癌治疗,这3种ADC药物采用的靶蛋白均为HER2,在乳腺癌中取得良好的治疗效果,在胃癌中生存获益较小,治疗效果有限,可能是因为HER2过表达在不同癌种中功能不同。Trastuzumabderuxtecan(T-DXd)(DS-8201;)是首个获FDA批准用于治疗胃癌的ADC药物。早在2019年时,由第一三共和阿斯利康合作开发的新型靶向HER2的ADC类药物DS-8201获批用于治疗转移性HER2阳性乳腺癌。DS-8201由人源化抗HER2抗体trastuzumab/>可裂解的四肽连接体和DNA拓扑异构酶I抑制剂DXd组成,药物/抗体比值(DAR)高达8。随后,2021年1月15日,FDA批准了DS-8201用于治疗局部晚期或转移性HER2阳性胃癌或胃食管交界处腺癌的成年患者,这些患者先前已接受过以曲妥珠单抗为基础的治疗方案。另一款用于胃癌治疗的DS-8201获批用于治疗转移性HER2阳性乳腺癌ADC类药物是2021年6月获批上市的Disitamab vedotin(RC48,/>)。RC48由荣昌生物制药(烟台)有限公司自主研发,是国内第一款上市的国产ADC药物,适应症为HER2阳性晚期或转移性胃癌。RC48-ADC的抗体是我国自主研发的全新人源化抗HER2抗体——迪西妥单抗,相比曲妥珠单抗抗原识别位点略有不同,与HER2受体的亲和力更强,在肿瘤细胞的内吞效果好;毒性分子MMAE分子较小并且对多种肿瘤细胞具有毒性作用;RC48-ADC连接子可裂解,可以更好地发挥旁杀效应。然而,HER2阳性患者仅占总体胃癌患者的17-20%,且常见于肠型胃癌和位于胃近端或胃食管交界处癌症患者,因此GSGC和CIN患者未能从靶向HER2的ADC药物中明显获益,开发针对不同分子分型的新靶点和及其靶向的ADC药物对GSGC或CIN患者这一群体具有重大意义。随着抗体技术的成熟,ADC药物应用范围也逐渐扩大,越来越多新的靶点用于药物研发当中,开发HER2外的新靶点对胃癌的免疫治疗十分必要。
ICAM1(细胞间黏附分子-1,CD54)是一种跨膜糖蛋白,是免疫球蛋白超家族成员之一,是一种广泛分布的细胞黏附分子,介导了细胞-细胞和细胞-细胞外基质的相互作用、细胞信号转导、免疫炎性反应和血管生成等生理过程。目前已发现其在多种癌症中过表达,如胰腺癌、三阴性乳腺癌、黑色素瘤和甲状腺癌等,过表达的ICAM1一方面可脱落进入血液,成为可溶型的sICAM1,sICAM1与淋巴细胞相关抗原-1(LFA-1)或Mac-1相互作用,竞争性抑制ICAM1-LFA-1依赖的非主要组织相容性复合体(MHC)限制性的免疫识别,另一方面与肿瘤细胞附近高表达LFA-1的浸润淋巴细胞结合增强,降低肿瘤细胞之间的黏附,介导了肿瘤的发生、侵袭和转移。
发明内容
鉴于以上所述现有技术的缺点,本发明的目的在于提供ICAM1或其编码基因在制备胃癌治疗或诊断产品中的用途,用于解决现有技术中的问题。
为实现上述目的及其他相关目的,本发明提供ICAM1或其编码基因作为分子识别靶标在制备染色体不稳定型胃癌诊断或治疗产品中的用途。
本发明还提供特异性结合ICAM1的物质在制备染色体不稳定型胃癌治疗或诊断产品中的用途。
本发明提供ICAM1或其编码基因作为分子识别靶标在制备基因组稳定型胃癌诊断或治疗产品中的用途。
本发明还提供特异性结合ICAM1的物质在制备基因组稳定型胃癌治疗或诊断产品中的用途。
优选的,所述特异性结合ICAM1的物质选自抗体偶联药物或其药学上可接受的盐或溶剂化合物。
本发明还提供一种抗体偶联药物或其药学上可接受的盐,所述抗体偶联药物的结构如下所示:Ab-(L-D)n,其中Ab为抗ICAM1抗体,L为连接子,D为细胞毒剂,n为1-40的整数。
优选的,所述抗ICAM1抗体的重链和轻链氨基酸序列分别如SEQ ID NO.7和8所示。
优选的,所述抗体偶联药物选自anti-ICAM1 mAb-MC-VC-PAB-MMAE(简称为ICAM1-MMAE)或anti-ICAM1 mAb-MC-GGFG-Dxd(简称为ICAM1-Dxd)。
本发明还提供所述抗体偶联药物的制备方法,包括如下步骤:
1)将二硫键还原剂与抗ICAM1抗体混合,使抗ICAM1抗体半胱氨酸中的二硫键至少部分被还原成巯基;
2)将连接子、细胞毒剂与步骤1)的产物混合,使抗ICAM1抗体与连接子偶联得到所述抗体偶联药物。
如上所述,本发明的ICAM1或其编码基因在制备胃癌治疗或诊断产品中的用途,具有以下有益效果:
第一,ADC药物在胃癌中已上市的药物较少,主要以Her2为靶点进行设计,相较于乳腺癌等适应症,在胃癌中治疗效果不够理想。本发明所使用的ICAM1靶点是一个新颖且有效的胃癌ADC药物设计的靶点。
第二,ICAM1靶点广泛分布于CIN亚型的胃癌细胞中,在正常细胞中不表达,这种蛋白表达的差异可以有效介导ADC药物准确靶向肿瘤组织而不杀伤正常组织,安全性高,克服了目前临床常见化疗药物严重不良反应的缺点。
第三,anti-ICAM1 mAb-MC-VC-PAB-MMAE治疗效果更好,连接子和细胞毒剂更佳,且优于目前临床常见的胃癌化疗药物5-氟尿嘧啶和奥沙利铂,有望成为具有临床价值的药物。
附图说明
图1-1显示为免疫组化法观察ICAM1在CIN亚型肿瘤组织和癌旁组织表达差异的染色结果图。
图1-2显示为免疫组化法观察ICAM1在CIN亚型肿瘤组织和癌旁组织表达差异的统计图。
图2显示为四株CIN亚型胃癌细胞和人肾上皮细胞ICAM1膜蛋白表达(红色峰为对照组IgG峰,蓝色峰为实验组ICAM1峰)。
图3显示为ICAM1介导的抗体内吞效率,其中A为AGS细胞ICAM1抗体内吞效率。B为NUGC3细胞ICAM1抗体内吞效率。
图4显示为免疫荧光法检测ICAM1膜蛋白定位。
图5显示为免疫荧光法观察ICAM1介导的抗体内吞。
图6显示为抗体偶联药物anti-ICAM1 mAb-MC-VC-PAB-MMAE(A)和anti-ICAM1mAb-MC-GGFG-Dxd(B)的结构示意图。
图7显示为抗体偶联药物anti-ICAM1 mAb-MC-VC-PAB-MMAE、anti-ICAM1 mAb-MC-GGFG-Dxd和anti-ICAM1 mAb的IC50值。
图8-1显示为弥漫型胃癌患者肿瘤组织及癌旁组织中ICAM1的IHC染色。
图8-2显示为弥漫型胃癌患者胃癌组织及癌旁组织IHC染色的ICAM1表达情况分析。
图8-3显示为弥漫型胃癌患者术后生存期与ICAM1表达情况的关系。
图9显示为GSGC细胞ICAM1表达的FCM检测结果。
图10显示为IF染色可视化GSGC细胞的ICAM1膜蛋白表达情况。
图11-1显示为IF染色定性评估GSGC细胞对ICAM1抗体的内吞效率。
图11-2显示为FCM定量评估GSGC细胞对ICAM1抗体的内吞效率。
图12显示为ADC及对照药物的体外对GSGC细胞毒性检测。
具体实施方式
本发明通过免疫组化法在组织水平观察到CIN和GSGC胃癌患者肿瘤组织ICAM1表达水平显著高于癌旁组织。流式细胞术和免疫荧光法实验进一步验证,ICAM1在染色体不稳定型和基因组稳定型胃癌细胞中过表达,能够有效介导抗体内吞。在本发明中,研发了两种基于ICAM1靶点的ADC药物,将anti-ICAM1 mAb分别与微管抑制剂Monomethyl auristatinE(MMAE)和拓扑异构酶抑制剂Exatecan derivative for ADC(Dxd)通过可裂解连接子进行偶联,用CIN亚型和GSGC的胃癌细胞进行体外实验验证,结果显示ADC药物anti-ICAM1 mAb-MC-VC-PAB-MMAE比临床常见的细胞毒性药物5-氟尿嘧啶和奥沙利铂有更好的治疗效果和更高的安全性,具有较高的临床价值。
本发明首先提供ICAM1或其编码基因作为靶标在制备胃癌治疗或诊断产品中的用途。
所述ICAM1或其编码基因作为靶标在制备胃癌治疗或诊断产品具体是指:将ICAM1蛋白或其编码基因作为识别对象,能够降低ICAM1水平或杀伤胃癌细胞的物质。
本发明还提供特异性结合ICAM1的物质在制备胃癌治疗或诊断产品中的用途。
在一种实施方式中,所述特异性结合ICAM1的物质选自抗体偶联药物或其药学上可接受的盐或溶剂化合物。
在一种实施方式中,所述抗体偶联药物的结构如下所示:Ab-(L-D)n,其中Ab为抗ICAM1抗体,L为连接子,D为细胞毒剂,n为1-40的整数。
本发明中,抗ICAM1抗体选自单克隆抗体(包括具有免疫球蛋白Fc区的全长抗体)、多特异性抗体(例如,双特异性抗体)以及抗体片段(例如,Fab,F(ab’)2,Fv,scFv)。术语“免疫球蛋白”(Ig)和“抗体”可互换使用。
所述抗ICAM1抗体可以选自现有技术中任意能够与ICAM1结合的抗体或其抗原结合片段,例如MabPlex公司的ICAM1抗体。
在一种实施方式中,所述抗ICAM1抗体的重链和轻链氨基酸序列分别如SEQ IDNO.1和2所示。
所述抗ICAM1抗体经连接子偶联至细胞毒剂。连接子分为两类:不可断裂连接子和可断裂连接子。可断裂连接子可以在目标细胞内断裂并释放出药物毒剂。可断裂连接子可分为两个主要的类别:化学不稳定连接子和酶不稳定连接子。化学不稳定连接子可以由于血浆和细胞质性质的不同而选择性的断裂。这样的性质包括pH值、谷胱甘肽浓度等。对pH值敏感的连接子,通常又称为酸断裂连接子,这样的连接子在血液的中性环境下相对稳定(pH7.3-7.5),但是在弱酸性的内涵体(pH5.0-6.5)和溶酶体(pH4.5-5.0)内将会被水解。对于谷胱甘肽敏感的连接子,又称二硫键连接子。酶不稳定连接子,如肽连接子,能够更好地控制药物释放。肽连接子能够被溶酶体内蛋白酶,如组织蛋白酶(Cathepsin B)或纤溶酶(在一些肿瘤组织中此类酶含量增加)有效地切断。这种肽连接在血浆循环中非常稳定,这是因为细胞外不合宜的pH值及血清蛋白酶抑制剂导致蛋白酶通常不具备活性。鉴于较高的血浆稳定性和良好的细胞内断裂选择性和有效性,酶不稳定连接子被广泛用做抗体药物偶联物的可断裂连接子。典型的酶不稳定连接子包括Val-Cit(vc)、Phe-Lys、Gly-Gly-Phe-Gly等。
连接子可以包含一种或多种连接子构件。例示性的连接子构件包括6-马来酰亚胺基己酰基、马来酰亚胺基丙酰基-缬氨酸-瓜氨酸、丙氨酸-苯丙氨酸、对氨基苯甲酰氧基羧基、N-琥珀酰亚胺基4-(2-吡啶基硫代)戊酸酯、N-琥珀酰亚胺基4-(N-马来酰亚胺基甲基)环己烷-1羧酸酯和N-琥珀酰亚胺基(4-碘-乙酰基)氨基苯甲酸酯。其它示例性的连接子构件还可以是包含氨基酸单元以容许蛋白酶切割的连接子,由此便于在暴露于胞内蛋白酶(诸如溶酶体酶)后从抗体偶联药物释放细胞毒剂。示例性的氨基酸单元包括但不限于二肽、三肽、四肽和五肽。示例性的二肽包括:缬氨酸-瓜氨酸;丙氨酸-苯丙氨酸;苯丙氨酸-赖氨酸;或N-甲基-缬氨酸-瓜氨酸。示例性的三肽包括:甘氨酸-缬氨酸-瓜氨酸或甘氨酸-甘氨酸-甘氨酸。示例性的四肽包括:甘氨酸-甘氨酸-苯丙氨酸-甘氨酸。
所述细胞毒剂选自毒素、化疗药物、抗生素、放射性同位素、生长抑制剂。
示例性的细胞毒剂包括:美登素;类美登素;拓扑异构酶Ⅰ抑制剂(如喜树碱衍生物:DX-8951衍生物Dxd);微管蛋白抑制剂(如单甲基耳抑素肽E(MMAE)和单甲基耳抑素肽F(MMAF);卡奇霉素类(如卡奇霉素);阿霉素类(如阿霉素);苯并二吡咯类抗生素(如duocarmycins、CC-1065等)和其它的环丙基吡咯吲哚-4-酮(cyclopropapyrroloind-4-one,CPI)衍生物,如环丙苯并吲哚-4-酮类似物,以及吡咯并苯二氮卓类(PBD)或者PBD二聚体类。
所述抗体偶联药物的结构Ab-(L-D)n中n为药物抗体比值(DAR值),n的范围选自以下任一:1~5、5~10、10~15、15~20、20~25、25~30、30~35、35~40。
在一种实施方式中,所述抗体偶联药物选自anti-ICAM1 mAb-MC-VC-PAB-MMAE(简称为ICAM1-MMAE)或anti-ICAM1 mAb-MC-GGFG-Dxd(简称为ICAM1-Dxd)。
所述ICAM1-MMAE的DAR值为4。
所述ICAM1-DXD的DAR值为8。
所述连接子中的马来酰亚胺基与抗ICAM1抗体中的半胱氨酸共价偶联。
在另一种实施方式中,所述特异性结合ICAM1的物质选自ICAM1抑制剂。
ICAM1抑制剂指对于ICAM1具有抑制效果的分子。对于ICAM1具有抑制效果包括但不限于:抑制ICAM1的水平或活性。
抑制ICAM1活性是指使ICAM1活力下降。优选地,相比抑制前,ICAM1活力下降至少10%,较佳的降低至少30%,再佳的降低至少50%,更佳的降低至少70%,最佳的降低至少90%。
抑制ICAM1水平可以是通过抑制ICAM1基因的转录或翻译,具体的,可以是指:使ICAM1基因不转录,或降低ICAM1基因的转录活性,或者使ICAM1基因不翻译,或降低ICAM1基因的翻译水平。
本领域技术人员可以使用常规方法对ICAM1基因表达进行调节,如基因敲除、同源重组,干扰RNA等。
优选地,与野生型相比,ICAM1基因表达降低至少10%,较佳的降低至少30%,再佳的降低至少50%,更佳的降低至少70%,又佳的降低至少90%,最佳地ICAM1基因完全没有表达。
所述ICAM1抑制剂包括但不限于:核酸分子、碳水化合物、脂类、小分子化学药、抗体药、多肽、蛋白、干扰慢病毒、腺相关病毒、纳米颗粒、脂质体、细胞外囊泡或细胞。所述核酸包括但不限于:反义寡核苷酸、双链RNA(dsRNA)、核酶、核糖核酸内切酶I II制备的小干扰RNA或者短发夹RNA(shRNA)。
所述胃癌治疗或诊断产品必然包括特异性结合ICAM1的物质,并以特异性结合ICAM1的物质作为有效成分。
所述胃癌治疗或诊断产品可以为单成分物质,亦可为多成分物质。
所述胃癌治疗或诊断产品的形式无特殊限制,可以为固体、液体、凝胶、半流质、气雾等各种物质形式。
所述胃癌治疗或诊断产品主要针对的对象为哺乳动物。所述哺乳动物优选为啮齿目动物、偶蹄目动物、奇蹄目动物、兔形目动物、灵长目动物等。所述灵长目动物优选为猴、猿或人。
所述胃癌治疗或诊断产品包括但不限于药物、保健品、食品等。
所述胃癌治疗产品选自:靶向的ICAM1抗体或抗原结合片段,外泌体,脂质体,SLP纳米颗粒,CAR-T细胞,溶瘤病毒,ADC,小分子偶联药物,双特异性抗体,核酸分子、小分子化学药、多肽、蛋白、干扰慢病毒或腺相关病毒等等;所述胃癌诊断产品选自:外泌体,CTC,CTDNA,血液/尿液蛋白或MRI/PET/超声等影像探针等。
所述胃癌选自染色体不稳定型(CIN型)、微卫星不稳定型(MSI型)、染色体稳定型(GS型)和人类四型孢疹病毒阳性型(EBV型)。
本发明还提供一种抗体偶联药物或其药学上可接受的盐所述抗体偶联药物的结构如下所示:Ab-(L-D)n,其中Ab为抗ICAM1抗体,L为连接子,D为细胞毒剂,n为1-40的整数。
本发明中,抗ICAM1抗体选自单克隆抗体(包括具有免疫球蛋白Fc区的全长抗体)、多特异性抗体(例如,双特异性抗体)以及抗体片段(例如,Fab,F(ab’)2,Fv,scFv)。术语“免疫球蛋白”(Ig)和“抗体”可互换使用。
所述抗ICAM1抗体可以选自现有技术中任意能够与ICAM1结合的抗体或其抗原结合片段,例如MabPlex公司的ICAM1抗体。
在一种实施方式中,所述抗ICAM1抗体的重链和轻链氨基酸序列分别如SEQ IDNO.1和2所示。
所述抗ICAM1抗体经连接子偶联至细胞毒剂。连接子分为两类:不可断裂连接子和可断裂连接子。可断裂连接子可以在目标细胞内断裂并释放出药物毒剂。可断裂连接子可分为两个主要的类别:化学不稳定连接子和酶不稳定连接子。化学不稳定连接子可以由于血浆和细胞质性质的不同而选择性的断裂。这样的性质包括pH值、谷胱甘肽浓度等。对pH值敏感的连接子,通常又称为酸断裂连接子,这样的连接子在血液的中性环境下相对稳定(pH7.3-7.5),但是在弱酸性的内涵体(pH5.0-6.5)和溶酶体(pH4.5-5.0)内将会被水解。对于谷胱甘肽敏感的连接子,又称二硫键连接子。酶不稳定连接子,如肽连接子,能够更好地控制药物释放。肽连接子能够被溶酶体内蛋白酶,如组织蛋白酶(Cathepsin B)或纤溶酶(在一些肿瘤组织中此类酶含量增加)有效地切断。这种肽连接在血浆循环中非常稳定,这是因为细胞外不合宜的pH值及血清蛋白酶抑制剂导致蛋白酶通常不具备活性。鉴于较高的血浆稳定性和良好的细胞内断裂选择性和有效性,酶不稳定连接子被广泛用做抗体药物偶联物的可断裂连接子。典型的酶不稳定连接子包括Val-Cit(vc)、Phe-Lys、Gly-Gly-Phe-Gly等。
连接子可以包含一种或多种连接子构件。例示性的连接子构件包括6-马来酰亚胺基己酰基、马来酰亚胺基丙酰基-缬氨酸-瓜氨酸、丙氨酸-苯丙氨酸、对氨基苯甲酰氧基羧基、N-琥珀酰亚胺基4-(2-吡啶基硫代)戊酸酯、N-琥珀酰亚胺基4-(N-马来酰亚胺基甲基)环己烷-1羧酸酯和N-琥珀酰亚胺基(4-碘-乙酰基)氨基苯甲酸酯。其它示例性的连接子构件还可以是包含氨基酸单元以容许蛋白酶切割的连接子,由此便于在暴露于胞内蛋白酶(诸如溶酶体酶)后从抗体偶联药物释放细胞毒剂。示例性的氨基酸单元包括但不限于二肽、三肽、四肽和五肽。示例性的二肽包括:缬氨酸-瓜氨酸;丙氨酸-苯丙氨酸;苯丙氨酸-赖氨酸;或N-甲基-缬氨酸-瓜氨酸。示例性的三肽包括:甘氨酸-缬氨酸-瓜氨酸或甘氨酸-甘氨酸-甘氨酸。示例性的四肽包括:甘氨酸-甘氨酸-苯丙氨酸-甘氨酸。
所述细胞毒剂选自毒素、化疗药物、抗生素、放射性同位素、生长抑制剂。
示例性的细胞毒剂包括:美登素;类美登素;拓扑异构酶Ⅰ抑制剂(如喜树碱衍生物:DX-8951衍生物Dxd);微管蛋白抑制剂(如单甲基耳抑素肽E(MMAE)和单甲基耳抑素肽F(MMAF);卡奇霉素类(如卡奇霉素);阿霉素类(如阿霉素);苯并二吡咯类抗生素(如duocarmycins、CC-1065等)和其它的环丙基吡咯吲哚-4-酮(cyclopropapyrroloind-4-one,CPI)衍生物,如环丙苯并吲哚-4-酮类似物,以及吡咯并苯二氮卓类(PBD)或者PBD二聚体类。
所述抗体偶联药物的结构Ab-(L-D)n中n为药物抗体比值(DAR值),n的范围选自以下任一:1~5、5~10、10~15、15~20、20~25、25~30、30~35、35~40。
在一种实施方式中,所述抗体偶联药物选自anti-ICAM1 mAb-MC-VC-PAB-MMAE(简称为ICAM1-MMAE)或anti-ICAM1 mAb-MC-GGFG-Dxd(简称为ICAM1-Dxd)。
所述ICAM1-MMAE的DAR值为4。
所述ICAM1-DXD的DAR值为8。
所述连接子中的马来酰亚胺基与抗ICAM1抗体中的半胱氨酸共价偶联。
本发明还提供所述抗体偶联药物的制备方法,包括如下步骤:
1)将二硫键还原剂与抗ICAM1抗体混合,使抗ICAM1抗体半胱氨酸中的二硫键至少部分被还原成巯基;
2)将连接子、细胞毒剂与步骤1)的产物混合,使抗ICAM1抗体与连接子偶联得到所述抗体偶联药物。
步骤1)中所述二硫键还原剂选自DTT、三(2-羧乙基)膦或他们的盐。在一种实施方式中,所述二硫键还原剂选自三(2-羧乙基)膦盐酸盐。
在本发明的某些实施方式中,步骤1)和2)的反应在溶剂中进行。本领域技术人员可以选择合适的溶剂种类和用量,以使反应物可以在反应体系中充分分散。所述溶剂可以是缓冲液。更具体的,所述溶剂可以选自硼酸盐缓冲液、磷酸盐缓冲液中的任一种或多种。
在本发明的某些实施方式中,步骤1)或步骤2)的反应温度为0-37℃。在本发明的某些实施方式中,所述反应温度为10-35℃。较佳的,所述反应温度为15-30℃。
步骤1)中,二硫键还原剂的量相对于抗ICAM1抗体按照摩尔量计通常是等量或过量的。在本发明的某些实施方式中,二硫键还原剂与抗ICAM1抗体的摩尔比为1-50:1。在一较佳的实施方式中,二硫键还原剂与抗ICAM1抗体的摩尔比为1-30:1。在一更佳的实施方式中,二硫键还原剂与抗ICAM1抗体的摩尔比为5-20:1。
连接子与细胞毒剂可以是市购的已经连接成功的一种试剂,即为连接子-细胞毒剂;连接子与细胞毒剂也可以自制。
在本发明的某些实施方式中,连接子与细胞毒剂已经连接成功为连接子-细胞毒剂,步骤2)中不同连接子-细胞毒剂的使用量可能不同,但连接子-细胞毒剂的量相对于步骤1)产物按照摩尔量计通常是等量或过量的。在本发明的某些实施方式中,连接子-细胞毒剂与步骤1)产物的摩尔比为1-50:1。在一较佳的实施方式中,连接子-细胞毒剂与步骤1)产物的摩尔比为1-40:1。在一更佳的实施方式中,连接子-细胞毒剂与步骤1)产物的摩尔比为5-25:1。
在本发明的某些实施方式中,步骤2)中还包括去除未反应的连接子、细胞毒剂。
以下通过特定的具体实例说明本发明的实施方式,本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用,本说明书中的各项细节也可以基于不同观点与应用,在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前,应理解,本发明的保护范围不局限于下述特定的具体实施方案;还应当理解,本发明实施例中使用的术语是为了描述特定的具体实施方案,而不是为了限制本发明的保护范围;在本发明说明书和权利要求书中,除非文中另外明确指出,单数形式“一个”、“一”和“这个”包括复数形式。
当实施例给出数值范围时,应理解,除非本发明另有说明,每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义,本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外,根据本技术领域的技术人员对现有技术的掌握及本发明的记载,还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
实施例中主要实验材料如下:
自美国生物标准品收藏中心公司购买人胃癌细胞系(AGS、NUGC4、NUGC3)和人肾上皮细胞293T,自中国科学院(中国上海)的典型培养物保藏委员会细胞库购买人胃癌细胞系MKN7。
RPMI-1640培养基、DMEM/F12培养基(Gibco);胎牛血清(BiologicalIndustries);胰蛋白酶(浙江森瑞生物科技有限公司);hoechst染液、青霉素-链霉素溶液、CCK-8试剂盒(biosharp);BSA(SIGMA);PBS溶液(VISTECH);4%多聚甲醛(Solarbio);PEanti-human CD54、PE anti-mouse IgG1、PE Mouse IgG1、Purified anti-human CD54(BioLegend);5-氟尿嘧啶、奥沙利铂(MedChemExpress);anti-ICAM1一抗(Sigma);反应增强液、增强酶标山羊抗兔IgG、DAB染液、苏木精、山羊血清缓冲液(中杉金桥);柠檬酸三钠、二甲苯(上海凌峰化学试剂有限公司);抗体偶联药物Anti-ICAM1 mAb-MC-VC-PAB-MMAE、Anti-ICAM1 mAb-MC-GGFG-Dxd和Anti-ICAM1 mAb由申请人自制,其中Anti-ICAM1 mAb委托第三方公司合成,Anti-ICAM1 mAb重链和轻链氨基酸序列分别如SEQ ID NO.1和2所示:
SEQ ID NO:1
MGWSCIILFLVATATGVHSQVQLQQSGPELVRPGVSVKISCKGSGYTFIDYAIHWVKESHAKSLEWIGVISAYSGDTNYNQKFKGKATMTVDKSSNTAYLELARLTSEDSAIYYCARGGWLLLSFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*
SEQ ID NO:2
MGWSCIILFLVATATGVHSDVVMTQSPLSLPVSLGDQASISCRSSQSLVHSNGNNYLHWYLQKSGQAPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCSQSTHVPLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
MC-VC-PAB-MMAE、MC-GGFG-Dxd购于陶术生物。
实施例中细胞培养方法如下:
CIN亚型的胃癌细胞(AGS、MKN7、NUGC3和NUGC4):
人胃癌细胞NUGC3、NUGC4和MKN7用RPMI-1640完全培养基(10%胎牛血清、1%青霉素-链霉素溶液),人胃癌细胞AGS用DMEM/F12完全培养基(10%胎牛血清、1%青霉素-链霉素溶液)培养,人肾上皮细胞293T用DMEM完全培养基(10%胎牛血清、1%青霉素-链霉素溶液)培养,培养温度37℃,CO2浓度5%。
4株GSGC细胞(Fu97,GCIY,Hs746T和SNU-1)和1株正常人胃黏膜上皮细胞(GES-1):Fu97细胞使用的培养基为含10%胎牛血清(FBS)、1%青霉素-链霉素混合液(Penicillin-Streptomycin Solution,P/S)混合液和10mg/L胰岛素的Dulbecco’s Modified Eagle’sMedium(DMEM)培养基,GCIY细胞使用的培养基为含15%FBS和1%P/S的DMEM培养基,Hs746T细胞使用的培养基为含10%FBS和1%P/S的DMEM培养基,SNU-1和GES-1细胞使用的培养基为含10%FBS和1%P/S的Roswell Park Memorial Institute(RPMI)-1640培养基。以上细胞均置于含有5%CO2的37℃恒温培养箱中培养。
实施例1至实施例4为针对染色体不稳定型胃癌的实验。实施例5至实施例13为染色体稳定型胃癌的实验。
实施例1至实施例4通过流式细胞术和免疫荧光法实验验证ICAM1在TCGA分型中染色体不稳定型胃癌中也存在过表达的现象,在正常细胞中表达较低。免疫组化结果也显示癌组织ICAM1表达水平显著高于癌旁组织,流式细胞术和免疫荧光法显示该靶点可有效介导抗体内吞。本研究验证了ICAM1是ADC设计的理想靶点,基于此进行ADC药物设计,设计了两种ADC药物:anti-ICAM1 mAb-MC-VC-PAB-MMAE和anti-ICAM1 mAb-MC-GGFG-Dxd,在ICAM1单克隆抗体上分别连接微管抑制剂和拓扑异构酶抑制剂,抑制癌细胞有丝分裂,体外实验验证药物的抗肿瘤活性,发现所设计的anti-ICAM1 mAb-MC-VC-PAB-MMAE药物有良好的抗肿瘤效果,对正常细胞毒性较小,有望成为治疗ICAM1阳性染色体不稳定型胃癌的候选药物。
实施例1验证ICAM1在胃癌组织及癌旁组织中的表达差异
1.免疫组化分析ICAM1胃癌组织及癌旁组织中的表达和定位
由于CIN亚型病人多为Lauren分型中肠型患者,选取Lauren分型为肠型的196例患者进行统计学分析,其中癌组织196例,159例癌旁组织作为对照。病人样本来自国科大肿瘤医院,免疫组化实验由国科大肿瘤医院完成。
(1)脱蜡和水化:石蜡切片置于新鲜二甲苯中,浸泡10min,3次;去除多余液体后,置于无水乙醇中,浸泡3min,3次;去除多余液体后,置于95%乙醇中,浸泡3min,2次;去除多余的液体后,置于75%乙醇中,浸泡3min,2次;蒸馏水冲洗1min,置于PBS缓冲液中。
(2)抗原修复:脱蜡后在清水中冲洗一段时间,用3%H2O2/甲醇溶液室温处理10min。去除液体,在清水中洗2次,再加入柠檬酸盐缓冲液,放入微波炉中蒸煮3min,冷却至室温,再蒸煮1次,冷却至室温,PBS洗3次。
(3)血清封闭和滴加一抗:用5%正常山羊血清缓冲液于37℃封闭30min,4℃与一抗孵育过夜。
(4)滴加反应增强液:滴加100μl反应增强液,室温孵育20min,PBS缓冲液洗3min,3次。
(5)加二抗:样品与增强酶标山羊抗兔IgG在37℃孵育1h,PBS缓冲液清洗3min,3次。
(6)显色:加入适量DAB显色液,室温孵育5-8min。
(7)复染:DAB染色后,所有组织用苏木精复染,脱水后封闭。
(8)观察:用200倍放大的倒置显微镜对结果进行分析和拍照。
(9)分析:国科大肿瘤医院病理科医生用单盲法给免疫组化结果做定性评分。
免疫组化结果统计
用Graph Pad Prism 9软件进行统计学分析,卡方(χ2)检验分析ICAM1蛋白的表达量表达的差异,以P﹤0.05为统计学显著性差异。
结果显示,ICAM1抗体着色于细胞膜上,如图1-1所示。实验结果证明ICAM1定位在细胞膜上,在癌组织中,ICAM1阴性有166例,H score<6有21例,H score≥6有9例。在癌旁组织中,ICAM1阴性有157例,阳性有2例。ICAM1表达在癌、癌旁组织中的差异表达χ2值为21.12,p<0.001***,差异有统计学意义,如图1-2所示。
实施例2验证ICAM1靶蛋白在CIN亚型胃癌细胞中的表达
1.流式细胞术定量检测CIN亚型的胃癌细胞ICAM1膜蛋白表达
收集4×106个细胞,分为2组,1ml PBS重悬,1000r/min-1离心5min。冰浴条件下,用含1%BSA的PBS封闭30min。封闭后,1000r/min-1离心5min,细胞沉淀加800μl含1%BSA的PBS,混悬,分为两组:IgG组(2μl PE Mouse IgG1+200μl细胞悬液);ICAM1组(2μl PE anti-human CD54+200μl细胞悬液)。室温条件下100r/min摇床上避光孵育45min。1ml PBS洗细胞1次,洗去未结合抗体,细胞重悬于0.5ml PBS中,流式细胞仪分析。
通过流式细胞仪对AGS、NUGC3、NUGC4和MKN7人胃癌细胞ICAM1膜蛋白表达进行定量分析,人肾上皮细胞293T作为对照,Flowjo软件分析平均荧光强度。从图2可以看出,AGS、NUGC3和MKN7细胞有较强的ICAM1表达,NUGC4细胞有较弱的ICAM1表达,人肾上皮细胞293T为ICAM1阴性。以上实验说明ICAM1是一个潜在的ADC药物设计的靶点,可以使ADC药物有效靶向肿瘤细胞而不杀伤正常细胞。
2.流式细胞术定量检测CIN亚型的胃癌细胞ICAM1介导的抗体内吞效率
收集1×107个细胞,分为5组(0min、30min、60min、120min和240min),1ml PBS重悬,1000r/min-1离心5min。冰上孵育一抗Purified anti-human CD54 30min,1ml PBS洗细胞1次,除去未结合的一抗,0min组立即加入1ml PBS和2μl二抗PE anti-mouse IgG1,冰上孵育30min后,1ml PBS洗细胞1次,除去未结合的二抗,4%多聚甲醛固定细胞10min,1mlPBS洗1次,重悬于500μl PBS中。其余4组在1ml PBS中37℃内吞相应的时间后,加二抗,后续同0min组。流式细胞仪检测荧光下降值,t时间点的内吞百分比=100-t min样品的MFI(mean fluorescence intensity,平均荧光强度)×100/0min样品的MFI。
流式细胞术定量检测AGS细胞和NUGC3细胞抗体内吞效率,通过细胞表面结合抗体荧光强度的下降水平得出不同时间点0.5、1、2、4h时抗体内吞的效率。如图3所示,两株细胞随着时间推移,抗体内吞呈逐渐上升趋势,AGS细胞在4h后内吞效率达到12.336%,NUGC3细胞在4h后内吞效率达到65.130%,说明ICAM1靶点可有效介导抗体内吞,是较为理想的药物设计的靶点。
3.免疫荧光法观察ICAM1膜蛋白定位
1×106个细胞/共聚焦皿,加1ml培养基,37℃过夜,分为PBS组、IgG组和ICAM1组。去除培养基后,冰浴条件下,用含1%BSA的PBS封闭细胞15min。37℃下:PBS组于1ml PBS中孵育细胞1h;IgG组于2μl PE Mouse IgG1和1ml PBS中孵育细胞1h;ICAM1组于2μl PEanti-human CD54和1ml PBS中孵育细胞1h。PBS洗一次,去除未结合抗体。hoechst染液37℃染色20-30min,PBS洗2次,激光共聚焦显微镜578nm处观察。
免疫荧光法对流式细胞术鉴定出的ICAM1阳性细胞进行进一步可视化验证,设置IgG组和ICAM1组,激光共聚焦下观察。如图4所示,可以看到2组细胞细胞核均染上DNA特异染料hoechst,细胞核呈现蓝色,ICAM1阳性的细胞株细胞膜上染上带红色荧光的ICAM1-PEanti-human CD54抗体,证明ICAM1是一种在CIN亚型胃癌细胞中分布广泛的膜蛋白。
4.免疫荧光法观察ICAM1介导的抗体内吞
1×106个细胞/共聚焦皿,加1ml培养基,37℃过夜,共4组。去除培养基,加入1mlPBS和2μl PE anti-human CD54,冰浴条件下,染色30min。PBS洗一次,加1ml PBS,37℃中使其内吞0、60、120和240min。去除PBS,加1ml hoechst染液,37℃染色20-30min。PBS洗2次,4%多聚甲醛固定10min,PBS洗2次,激光共聚焦显微镜下观察。
免疫荧光法观察0-4h肿瘤细胞NUGC3和AGS细胞ICAM1靶蛋白介导的抗体内吞效果,如图5所示,0h时,带红色荧光的PE anti-human CD54抗体主要分布在细胞膜上,随着时间推移,膜上荧光强度随着孵育时间的延长而减弱,细胞质内荧光强度增强,抗体逐渐向细胞内分布,发生抗体内吞。可以看出ICAM1靶点能够有效地介导抗体的内吞,是较为理想的抗体偶联药物设计靶点。
实施例3抗体偶联药物的制备
将ICAM1单克隆抗体与通过马来酰亚胺基己酰基-缬氨酸-瓜氨酸-对氨基苯甲酰氧基羧基(MC-VC-PAB)连接子与微管抑制剂Monomethyl auristatin E(MMAE)偶联制备获得anti-ICAM1 mAb-MC-VC-PAB-MMAE(ICAM1-MMAE)药物;将ICAM1单克隆抗体与通过马来酰亚胺-GGFG四肽(MC-GGFG)连接子与拓扑异构酶Ⅰ抑制剂喜树碱衍生物(DX-8951衍生物Dxd)偶联制备获得anti-ICAM1 mAb-MC-GGFG-Dxd(ICAM1-Dxd)药物。具体制备过程如下:
将ICAM1抗体在硼酸盐缓冲液中与三(2-羧乙基)膦盐酸盐反应在25℃反应2h,把半胱氨酸中的二硫键部分还原成巯基。再加入过量的MC-VC-PAB-MMAE(摩尔比值10:1)或MC-GGFG-DXD(摩尔比值20:1),在pH=6.5的PBS缓冲液中通过巯基反应将ICAM1单克隆抗体上的巯基与MC-VC-PAB-MMAE或MC-GGFG-DXD连接子在25℃进行反应2h,生成最终产物ICAM1-MMAE或ICAM1-DXD,并通过超滤方法去除未反应的MC-VC-PAB-MMAE或MC-GGFG-DXD。通过控制投料比方法将ICAM1-MMAE的药物抗体比值(DAR值)控制为4;ICAM1-DXD的DAR值控制为8。
两种ADC药物的反应原理是首先用还原剂将ICAM1抗体半胱氨酸中的二硫键还原为巯基,再将巯基与连接子中的马来酰亚胺基进行共价偶联反应,形成ADC复合结构,制备完成的ADC药物以1mg/mL的浓度溶解于PBS中,分装后冻存于-80℃。
ICAM1单克隆抗体与MC-VC-PAB-MMAE或MC-GGFG-Dxd偶联制备的两种ADC药物的化学结构如图6所示。
实施例4抗体偶联药物和常见化疗药物5-氟尿嘧啶和奥沙利铂体外药效测定
设置空白组、对照组和实验组,空白组100μl完全培养基,对照组和实验组在96孔板中接种细胞悬液(90μl/孔),肿瘤细胞AGS细胞3000个/孔,NUGC3细胞10000个/孔,NUGC4细胞10000个/孔,MKN7细胞12000个/孔;正常人肾上皮细胞293T细胞5000个/孔。细胞贴壁生长24h后,实验组给予10μl抗体偶联药物或化疗药物,设置3个复孔,给药组化疗药物终浓度为0.004、0.02、0.1、0.5、1、5、20和100μg/ml,抗体偶联药物终浓度为0.000001、0.00001、0.0001、0.001、0.01、0.1、1、10μg/ml。继续培养72h,后,每孔加10%的CCK-8试剂,37℃、5%CO2条件下孵育1-4h,酶标仪测定450nm处吸光值。
CCK-8法测定了CIN亚型的胃癌细胞(AGS、MKN7、NUGC3和NUGC4)和1株正常人肾上皮细胞(293T)抗体偶联药物anti-ICAM1 mAb-MC-VC-PAB-MMAE、anti-ICAM1 mAb-MC-GGFG-Dxd和临床常用药物5-FU和奥沙利铂半数抑制浓度(IC50),并研究了ICAM1裸抗的杀伤作用。化疗药物5-FU和奥沙利铂对肿瘤细胞和正常胃黏膜上皮细胞毒性如表1所示。
表1.常见化疗药物5-FU和奥沙利铂细胞毒性
如图7所示,anti-ICAM1 mAb-MC-VC-PAB-MMAE对肿瘤细胞AGS、MKN7、NUGC3和NUGC4的IC50值分别为0.04813、0.01175、0.006682和0.03852μg/ml,对正常胃黏膜上皮细胞毒性较弱;anti-ICAM1 mAb-MC-GGFG-Dxd对肿瘤细胞AGS和NUGC3的IC50值分别为0.1375μg/ml和0.1230μg/m,对肿瘤细胞MKN7、NUGC3、NUGC4和正常人肾上皮细胞293T杀伤作用较弱;anti-ICAM1 mAb对肿瘤细胞和正常细胞几乎无杀伤作用。以上结果说明本发明所设计的抗体偶联药物anti-ICAM1 mAb-MC-VC-PAB-MMAE对CIN亚型的胃癌细胞有较好的杀伤效果,且优于目前临床广泛使用的化疗药物5-FU和奥沙利铂,对正常细胞毒性较小,具有良好的临床开发价值。
以下实施例为针对GSGC分子分型的胃癌的实验,主要内容为:
(1)GSGC中ICAM1靶点的验证:首先,通过对弥漫型胃癌患者的胃癌组织及其癌旁组织进行免疫组化(IHC)染色,验证ICAM1是膜蛋白,并证实ICAM1在胃癌组织中高度过表达,而在正常胃壁组织中无明显表达。其次,通过流式细胞术(FCM)对GSGC细胞系的ICAM1膜蛋白进行定量分析,同时对比正常人胃黏膜上皮细胞的ICAM1表达情况,筛选出过表达ICAM1膜蛋白的GSGC细胞株。接下来,对筛选出的GSGC细胞株进行免疫荧光(IF)染色实验,进一步验证ICAM1在GSGC细胞系中过表达。最后,通过IF定性分析结合FCM定量分析来评估筛选出的GSGC细胞对ICAM1抗体的内吞效率。
(2)靶向ICAM1的ADC药物的设计、制备和表征:靶向ICAM1的ADC药物的抗体部分选用半胱氨酸偶联、IgG1亚型的人源化靶向ICAM1的单克隆抗体;连接子部分选用可裂解连接子或不可裂解连接子;弹头部分选用微管抑制剂或DNA拓扑异构酶I抑制剂。DAR值基于弹头的毒性和偶联方式选择4或8。通过巯基共价偶联方法完成ADC药物的制备与表征。
(3)靶向ICAM1的ADC药物的体外药效评价:使用筛选出的GSGC细胞株,通过CCK8实验测定靶向ICAM1的ADC药物的IC50值,对比测定人源性ICAM1抗体、临床使用的化药、获批上市的靶向HER2的ADC药物的体外药效。同时,测定上述药物对正常人胃黏膜上皮细胞的体外药效,证实药物对正常人胃黏膜上皮细胞是否有影响。通过体外实验筛选出疗效最好的靶向ICAM1的ADC药物。
实施例8弥漫型胃癌患者肿瘤组织及癌旁组织中ICAM1的IHC染色
选取在浙江省肿瘤医院接受诊治的胃癌患者的110例弥漫型胃癌组织及102例癌旁组织样本。本研究已通过医院伦理委员会批准。通过IHC染色法对筛选的弥漫型胃癌组织及癌旁组织中ICAM1的表达水平进行检测,并由肿瘤医院病理医生对染色结果进行评分。采用H-score进行组织学评分,具体计算公式为H-score=∑(IS×AP),其中IS代表染色强度,数值范围为0-3,AP代表阳性细胞数量占切片中所有细胞数量的百分比,数值范围为0-4。H-score≥1表示ICAM1阳性,而H-score=0则表示ICAM1阴性。
IHC染色结果(图8-1)显示,ICAM1主要分布于细胞膜上,证实了ICAM1是一种膜蛋白靶点。前文提及GSGC患者中约73%属于Lauren分型中的弥漫型,故实验选取弥漫型胃癌患者的胃癌组织及癌旁组织进行IHC染色。结合IHC染色的ICAM1表达情况的统计结果(图8-2),110例弥漫型胃癌组织中ICAM1阳性的病例共计36例,所占比例高达32.73%,并且在102例癌旁组织中仅有1例为ICAM1阳性,故ICAM1表达水平在胃癌组织与正常组织中存在显著差异(p<0.0001)。由以上结果可以看出,ICAM1在胃癌组织中高度过表达,而在正常组织中无明显表达,符合ADC靶点选择的要素。
此外,本发明还研究了ICAM1与弥漫型胃癌患者术后生存期的关系(图8-3),结果表明ICAM1表达水平与患者预后无显著性差异。
实施例9 FCM定量分析细胞ICAM1膜蛋白含量
收集2×106个处于对数生长期的细胞,PBS洗涤一次后,冰浴条件下,细胞于含1%牛血清白蛋白(BSA)的PBS中封闭30min,离心后用800μL的含1%BSA的PBS重悬细胞。设三个实验组,各加入200μL细胞悬液,分别加入5μL PBS、PE Mouse IgG1和phycoerythrin(PE)/anti-ICAM1抗体,在室温下避光孵育45min。孵育结束后用PBS洗涤两次,再重悬于500μLPBS中,用CytoFLEX LX流式细胞仪检测样本的荧光信号。按照生产商提供的实验方案,参照Quantum Simply Cellular磁珠绘制的标准曲线,将样本的荧光信号强度换算成细胞表面ICAM1抗原的密度。
通过FCM对3株GSGC细胞(Fu97,GCIY和Hs746T)的ICAM1膜蛋白含量进行分析(图9)。定量结果如表2所示,Fu97和GCIY细胞高表达ICAM1膜蛋白,Hs746T细胞低表达ICAM1膜蛋白。
表2 GSGC细胞ICAM1表达的FCM检测定量结果
实施例10 IF染色可视化ICAM1膜蛋白
取处于对数生长期的6×105个Fu97细胞、各3×106个GCIY和Hs746T细胞,分别重悬于6mL完全培养基中,每株细胞各设三个组别,接种至共聚焦培养皿。置于含有5%CO2的37℃恒温培养箱中培养过夜。吸去培养基,用PBS洗涤两次。室温下,用4%组织细胞固定液固定10min,固定完成后用PBS洗涤两次。冰浴条件下,样本于含1%BSA的PBS中封闭30min。封闭结束后,分别加入2μL PBS、PE Mouse IgG1和PE/anti-ICAM1抗体,在室温下避光孵育1h。孵育完成后,用PBS洗涤两次。加入DAPI染色液覆盖住样品,室温避光孵育5min,吸除DAPI染色液,再用PBS洗涤两次。加1mL PBS,于4℃避光保存样品。用单光子共聚焦显微镜采集和分析荧光图像。
根据FCM定量结果,选择高表达ICAM1膜蛋白的Fu97和GCIY细胞以及低表达ICAM1膜蛋白的Hs746T细胞,通过单光子共聚焦显微镜观察上述细胞的ICAM1表达情况(图10)。由IF染色的实验结果可见,ICAM1的红色荧光信号主要定位在细胞膜上,再次验证ICAM1是膜蛋白。Fu97和GCIY细胞的红色荧光信号强度明显强于Hs746T细胞,说明Fu97和GCIY细胞的ICAM1膜蛋白表达量显著高于Hs746T细胞,这与FCM定量结果是一致的。
实施例11 GSGC细胞对ICAM1抗体的内吞效率分析
IF染色定性评估细胞对ICAM1抗体的内吞效率:各收集2×107个处于对数生长期的Fu97和GCIY细胞,PBS洗涤一次,用1.2mL的完全培养基重悬细胞。每株细胞各设五个组别,各加入200μL细胞悬液,再各加5μL PE/anti-ICAM1抗体,置于37℃恒温孵育箱中摇床孵育10min,使荧光标记的抗体结合于细胞表面。孵育10min后用PBS洗涤两次,以去除未结合的抗体,再重悬于完全培养基中,继续置于37℃恒温孵育箱中摇床孵育到特定时间点(设五个时间点,分别累积孵育10min/30min/60min/120min/240min),使结合于细胞表面的抗体内吞。孵育结束后,用PBS洗涤两次。室温下,用4%组织细胞固定液固定10min,固定完成后用PBS洗涤两次。样品重悬于200μL PBS中,再加入待染色样品体积3倍(600μL)的DAPI染色液并混匀,室温避光孵育5min,吸除DAPI染色液,再用PBS洗涤两次。样品加1mL PBS重悬,转移至共聚焦培养皿中,并于4℃避光保存。用单光子共聚焦显微镜采集和分析荧光图像。
FCM定量评估细胞对ICAM1抗体的内吞效率:各收集107个处于对数生长期的Fu97和GCIY细胞,PBS洗涤一次,用2.2mL的完全培养基重悬细胞。两株细胞各设十个组别,各加入200μL细胞悬液,其中五个对照组各加入5μL IgG,另外五个实验组各加入5μL纯化抗人类CD54抗体,置于37℃恒温孵育箱中摇床孵育10min,使一抗结合于细胞表面。孵育10min后用PBS洗涤两次,以去除未结合的抗体,再重悬于完全培养基中,继续置于37℃恒温孵育箱中摇床孵育到特定时间点(对照组和实验组均设五个时间点,分别累积孵育10min/30min/60min/120min/240min),使结合于细胞表面的抗体内吞。孵育结束后,用预冷的PBS洗涤两次。在冰浴条件下,重悬于200μL预冷的PBS中,分别加入5μL PE抗小鼠IgG1,避光孵育30min,使荧光标记的二抗结合于细胞表面未内吞的一抗。二抗孵育完成后,用预冷的PBS洗涤两次,再重悬于500μL预冷的PBS中,用CytoFLEX LX流式细胞仪检测样本的荧光信号。按照生产商提供的实验方案,参照Quantum Simply Cellular磁珠绘制的标准曲线,将样本的荧光信号强度换算成细胞表面ICAM1抗原的密度。
ADC的靶点选择需要满足的要素之一是靶点能有效介导抗体内吞,因此通过IF染色定性分析(图11-1)结合FCM定量分析(图11-2)来评估所筛选的两株高表达ICAM1的GSGC细胞(Fu97和GCIY)对ICAM1抗体的内吞效率。以Fu97细胞为例,从IF染色结果可以直观看出,细胞与抗体共孵育10min时红色荧光染料标记的ICAM1抗体已经结合在细胞膜上,随着孵育时间的延长,ICAM1抗体逐渐内吞进入胞质,60min时可以明显看到被内吞ICAM1抗体的红色荧光信号聚集分布于胞质中。这与FCM定量分析的结果是一致的,即在Fu97细胞与ICAM1抗体共孵育开始阶段,抗体的内吞比率随孵育时间的延长而上升,并在共孵育60min后抗体的内吞比率逐渐进入平台期。
FCM定量结果如表3所示,最长测试时间点4h时Fu97和GCIY细胞对ICAM1抗体的内吞效率分别达到33.22%和30.05%,可见所筛选的两株高表达ICAM1的GSGC细胞的膜蛋白靶点能有效的介导抗体的内吞,实验结果再次证实了ICAM1作为ADC靶点的可行性。
表3细胞对ICAM1抗体的内吞效率定量结果
实施例12靶向ICAM1的ADC药物的制备
1.靶向ICAM1的ADC的制备
ICAM1-MMAE ADC的制备
在37℃条件下,将ICAM1抗体在含50mM硼酸盐的PBS中(pH 8.0)用二硫苏糖醇(10mM)还原30min。然后,将ICAM1抗体(0.5mg/mL)和MC-VC-PAB-MMAE(9当量/抗体)混合在冷的PBS中(pH 7.4,5%DMSO v/v),于4℃条件下混旋1h。用离心过滤装置去除游离的MC-VC-PAB-MMAE。制备的ICAM1-MC-VC-PAB-MMAE ADC药物用PBS(pH 7.4)洗涤,再重悬于PBS中。
ICAM1-Dxd ADC的制备
在37℃条件下,将ICAM1抗体在含50mM硼酸盐的PBS中(pH 8.0)用二硫苏糖醇(10mM)还原30min。然后,将还原后的ICAM1抗体(0.5mg/mL)和MC-GGFG-Dxd混合在冷的PBS中(pH 7.4,5%DMSO v/v),于4℃条件下混旋1h。用离心过滤装置去除游离的MC-GGFG-Dxd。制备的ICAM1-MC-GGFG-Dxd ADC药物用PBS(pH 7.4)洗涤,再重悬于PBS中。
2.靶向ICAM1的ADC药物的表征
二喹啉甲酸法测定靶向ICAM1的ADC药物的浓度
制备蛋白标准品:在一管蛋白标准固体(30mg BSA)中加入1.2ml蛋白标准配制液,溶解配制成25mg/mL的蛋白标准溶液。取适量的25mg/mL蛋白标准溶液用PBS稀释为0.5mg/mL的蛋白标准品。
配制BCA工作液:将BCA试剂A和BCA试剂B按照50:1的体积比混匀。
靶向ICAM1的ADC药物的浓度检测:将蛋白标准品按0、1、2、4、8、12、16、20μl加到96孔板的标准品孔,加入PBS补足至20μl,相当于蛋白标准品浓度分别为0、0.025、0.05、0.1、0.2、0.3、0.4、0.5mg/mL。在96孔板的样品孔中加入适当体积的待测ADC药物,并用PBS补足至20μl。向各孔中加入200μl的BCA工作液,于37℃条件下放置20-30min。用酶标仪检测562nm波长处的吸光度。根据绘制的标准曲线和加入的待测ADC药物的样品体积计算出ADC药物的浓度。
用PBS稀释ADC药物,通过二喹啉甲酸法测定2种靶向ICAM1的ADC药物ICAM1-MMAE和ICAM1-Dxd的浓度均为1mg/mL。
3.疏水作用色谱法测定ADC药物的DAR值
采用疏水色谱柱在HPLC上检测ADC药物的DAR值。流动相A为pH 7.0的磷酸钠(25mM)和硫酸铵水溶液(1.5M),流动相B混合溶液为20%异丙醇和80%pH 7.0的磷酸钠水溶液(25mM)。在20min内流动相B在0%-100%范围内梯度洗脱。以1ml/min的流速进行洗脱。在280nm波长下收集色谱峰信号。色谱峰的峰面积百分比表征特定弹头数量连接的ADC药物的相对分布。根据色谱峰的峰面积百分比乘以偶联的弹头药物数量得到加权峰面积,加权峰面积求和后除以100得到平均DAR值,具体计算方程为:
平均DAR值=Σ(加权峰面积)/100。
2种靶向ICAM1的ADC药物ICAM1-MMAE和ICAM1-Dxd的DAR值分别4和8。
实施例13 ADC及对照药物的体外对GSGC细胞毒性检测
将细胞悬液以每孔100μl的体积接种于96孔板中,确保Fu97细胞、Hs746T细胞密度为每孔5×103个,GCIY细胞密度为每孔3×103个,GES-1细胞密度为每孔2×103个。用PBS填充96孔板周围一圈的孔以达到保湿和消除边缘效应的目的。将96孔板置于含有5%CO2的37℃恒温培养箱中培养过夜。化药5-氟尿嘧啶(5-FU)和奥沙利铂分别溶解于DMSO配制储备液,使用时用培养基按照十倍梯度稀释成八个浓度梯度。ICAM1-MMAE、ICAM1-Dxd、ICAM1单抗、DS8201及RC48使用时用培养基按照十倍梯度稀释成八个浓度梯度。向96孔板中分别加入100μl培养基或不同浓度的药物,其中两组分别为只含培养基、含有细胞和培养基的对照组别,其余八组为不同药物浓度的实验组别,每个组别设置6个平行孔。其中,5-FU和奥沙利铂最高浓度设为100μg/mL,其余药物最高浓度设为10μg/mL。加药后将96孔板置于含有5%CO2的37℃恒温培养箱中培养5天。每孔加入20μl CCK-8溶液,放于细胞培养箱中继续孵育0.5-4h,每隔0.5h用酶标仪检测在450nm处的吸光值。
选择2株高表达ICAM1的细胞系Fu97和GCIY,1株低表达ICAM1的细胞系Hs746T以及1株正常人胃黏膜上皮细胞GES-1进行体外细胞毒性实验(图12)。实验数据见表4。
表4 ADC及对照药物的体外细胞毒性检测结果
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由实验结果可以看出,2种靶向ICAM1的ADC药物(ICAM1-MMAE和ICAM1-Dxd)对ICAM1表达含量最高的Fu97细胞的抑制活性最好,药效优于ICAM1表达含量次之的GCIY细胞和低表达ICAM1的Hs746T细胞,且对于正常人胃黏膜上皮细胞GES-1基本无细胞毒性。以上结果说明靶向ICAM1的ADC药物对GSGC细胞的抑制活性与ICAM1表达含量呈正相关,也证实了靶向ICAM1的ADC药物对高表达ICAM1的GSGC细胞具有较好的靶向性,同时对正常人胃黏膜上皮细胞无明显的毒性副作用。
此外,从实验数据可以看出,ICAM1单抗对GSGC细胞活力基本无抑制作用,说明靶向ICAM1的ADC药物较单抗具有明显的治疗优势。无论是与临床上治疗胃癌常用的化药5-FU和奥沙利铂疗效相比,还是相较于获批治疗HER2阳性胃癌的ADC药物DS8201和RC48,本发明的2种靶向ICAM1的ADC药物对GSGC细胞均具有更加显著的体外毒性。就2种靶向ICAM1的ADC药物对GSGC细胞的体外毒性检测的整体结果而言,ICAM1-MMAE药物对GSGC细胞的细胞活力抑制作用更强,ICAM1-Dxd药物疗效较弱。
理想的ADC药物的靶点需要在肿瘤组织中表达,在正常组织中较少或无表达,或者至少限定于特定的组织中表达。此外,该靶蛋白还应广泛分布于细胞表面,以便ADC在血液循环中结合到特定病变部位。本发明通过免疫组化实验证明,ICAM1在染色体不稳定型和染色体稳定型胃癌组织中高表达,在癌旁组织中几乎不表达,流式细胞术进一步证明ICAM1广泛分布于CIN亚型和GSGC胃癌细胞表面,在正常细胞中不表达。其次,本发明通过流式细胞术和免疫荧光实验观察到ICAM1靶蛋白可以有效介导抗体内吞,是一个良好的药物递送靶点。该靶点作为一种可内化的抗原,在药物结合后内化转运至细胞中,释放细胞毒药物,发挥细胞毒作用。
除了靶点选择,连接子和细胞毒剂的选择和优化对于ADC药物的设计也至关重要。本发明设计了两种ADC药物,一种细胞毒剂为拓扑异构酶抑制剂,另一种细胞毒剂为微管抑制剂MMAE,两者均可抑制有丝分裂,起抗肿瘤活性。通过体外细胞实验发现MMAE细胞毒剂的ADC药物明显优于Dxd细胞毒剂的ADC药物,具有更好的治疗效果。通过与胃癌临床常见的化疗药物5-氟尿嘧啶和奥沙利铂进行细胞毒性比较,发现所设计的ADC药物对肿瘤细胞有更低的IC50,治疗效果更好,对正常细胞杀伤较弱,很好地诠释了抗体偶联药物靶向性和高效性结合的特点,有望成为一种具有临床开发潜力的肿瘤靶向药物,实验结果为临床转化提供了依据。
综上所述,本发明所设计的ADC药物为胃癌ADC药物的开发提供了一个新的靶点,治疗效果理想的同时克服了传统化疗药物严重不良反应的缺陷,扩大了ADC药物的适应症,有望成为具有临床使用价值的ADC药物,使ADC药物惠及更多患者。
以上的实施例是为了说明本发明公开的实施方案,并不能理解为对本发明的限制。此外,本文所列出的各种修改以及发明中方法的变化,在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述,但应当理解,本发明不应仅限于这些具体实施例。事实上,各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。
序列表
<110> 浙江省肿瘤医院
中国科学院基础医学与肿瘤研究所(筹)
<120> ICAM1或其编码基因在制备胃癌治疗或诊断产品中的用途
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Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
165 170 175
Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
180 185 190
Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
195 200 205
Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly
210 215 220
Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
Claims (14)
1.一种抗体偶联药物,其特征在于,所述抗体偶联药物的结构如下所示:Ab-(L-D)n,其中Ab为抗ICAM1抗体,L为连接子,D为细胞毒剂,n为1-40的整数。
2.根据权利要求1所述的抗体偶联药物,其特征在于,抗ICAM1抗体选自全长抗体或抗体片段;优选的,所述抗体片段选自Fab,F(ab’)2,Fv或scFv。
3.根据权利要求1所述的抗体偶联药物,其特征在于,所述抗ICAM1抗体的重链和轻链氨基酸序列分别如SEQ ID NO.1和2所示。
4.根据权利要求1所述的抗体偶联药物,其特征在于,所述连接子选自不可断裂连接子或可断裂连接子;优选的,所述可断裂连接子选自化学不稳定连接子或酶不稳定连接子。
5.根据权利要求1所述的抗体偶联药物,其特征在于,所述连接子包含一种或多种连接子构件,所述连接子构件选自马来酰亚胺基丙酰基、马来酰亚胺基己酰基、对氨基苯甲酰氧基羧基、缬氨酸-瓜氨酸、丙氨酸-苯丙氨酸、甘氨酸-甘氨酸-甘氨酸、甘氨酸-缬氨酸-瓜氨酸、甘氨酸-甘氨酸-苯丙氨酸-甘氨酸中的任一个或多个;优选的,所述连接子选自马来酰亚胺基己酰基-缬氨酸-瓜氨酸-对氨基苯甲酰氧基羧基或马来酰亚胺-GGFG四肽。
6.根据权利要求1所述的抗体偶联药物,其特征在于,所述细胞毒剂选自毒素、化疗药物、抗生素、放射性同位素或生长抑制剂。
7.根据权利要求1所述的抗体偶联药物,其特征在于,所述细胞毒剂选自以下中的任一个或多个:美登素、类美登素、拓扑异构酶Ⅰ抑制剂、微管蛋白抑制剂、卡奇霉素及其衍生物、阿霉素及其衍生物;优选的,所述细胞毒剂选自DX-8951衍生物Dxd、MMAE或MMAF。
8.权利要求1-7任一所述的抗体偶联药物的制备方法,其特征在于,所述制备方法包括如下步骤:
1)将二硫键还原剂与抗ICAM1抗体混合,使抗ICAM1抗体半胱氨酸中的二硫键至少部分被还原成巯基;
2)将连接子、细胞毒剂与步骤1)的产物混合,使抗ICAM1抗体与连接子偶联得到所述抗体偶联药物。
9.权利要求8所述的制备方法,其特征在于,还包括如下特征中的一项或多项:
A.步骤1)中所述二硫键还原剂选自DTT、三(2-羧乙基)膦或它们的盐;
B.步骤1)和2)的反应在溶剂中进行;优选的,所述溶剂是缓冲液;更优选的,所述溶剂选自硼酸盐缓冲液、磷酸盐缓冲液中的任一种或多种;
C.步骤1)或步骤2)的反应温度为0-37℃;
D.步骤1)中二硫键还原剂与抗ICAM1抗体的摩尔比为1-50:1。
10.ICAM1或其编码基因作为靶标在制备染色体不稳定型或基因组稳定型胃癌治疗或诊断产品中的用途。
11.特异性结合ICAM1的物质在制备染色体不稳定型基因组稳定型胃癌治疗或诊断产品中的用途。
12.根据权利要求11所述的用途,其特征在于,所述特异性结合ICAM1的物质选自抗体偶联药物或其药学上可接受的盐。
13.根据权利要求12所述的用途,其特征在于,所述抗体偶联药物选自权利要求1-7任一所述的抗体偶联药物。
14.根据权利要求11所述的用途,其特征在于,所述特异性结合ICAM1的物质为ICAM1抑制剂;优选的,所述ICAM1抑制剂选自核酸、小分子化学药、多肽、蛋白、干扰慢病毒、腺相关病毒、纳米颗粒、脂质体、细胞外囊泡或细胞。
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