CN108404138B - 一种靶向cd24单克隆抗体与二乙胺偶氮鎓二醇盐的偶联物及其应用 - Google Patents
一种靶向cd24单克隆抗体与二乙胺偶氮鎓二醇盐的偶联物及其应用 Download PDFInfo
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- CN108404138B CN108404138B CN201810192838.3A CN201810192838A CN108404138B CN 108404138 B CN108404138 B CN 108404138B CN 201810192838 A CN201810192838 A CN 201810192838A CN 108404138 B CN108404138 B CN 108404138B
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Abstract
本发明属生物工程抗体技术领域,具体涉及一种抗CD24抗体药物偶联物的制备方法及用途。本发明利用化学偶联技术将一种全新一氧化氮供体二乙胺偶氮鎓二醇盐分子通过马来酰亚胺‑双硫键偶联到抗CD24单克隆抗体G7mAb重链恒定区,制备得到抗体药物偶联物HN‑01,利用抗体的特异性靶向作用,将一氧化氮供体分子富集到肿瘤细胞表面并内化至细胞内定向释放一氧化氮,提高瘤内治疗指数的同时降低对正常组织的毒副作用。体内和体外实验结果证明,抗体药物偶联物HN‑01充分利用了抗体的靶向性和一氧化氮抗肿瘤的特异性,解决了一氧化氮供体的肿瘤靶向递释问题,具有良好的临床应用价值。
Description
本发明属于生物工程领域,具体涉及一种新的可与肿瘤细胞表面过表达的白细胞分化抗原CD24分子特异性结合的抗体药物偶联物HN-01,其由单克隆抗体G7mAb和全新的一氧化氮供体二乙胺偶氮鎓二醇盐分子通过马来酰亚胺-双硫键偶联而成。其利用G7mAb的特异性靶向作用将二乙胺偶氮鎓二醇盐分子富集到肿瘤细胞表面,提高了该一氧化氮供体分子的治疗指数,避免了其单独使用时的低靶向性所造成的对正常组织细胞的毒副作用,解决了一氧化氮供体的靶向递释问题,为肝癌的治疗提供了新的选择,同时也可推广到其他CD24分子过表达的恶性肿瘤治疗中。
背景技术
目前,恶性肿瘤的发病率和死亡率一直呈现显著的上升趋势,而化疗一度是肿瘤治疗的标准疗法。但高毒性的小分子药物在杀伤肿瘤细胞的同时会误杀正常细胞,引起严重的毒副作用,因此在临床上的广泛应用受到限制。在过去的20年里,单克隆抗体逐渐成为肿瘤治疗的支柱,但是大多数的抗体单独使用的临床效果却不尽人意。抗体药物偶联物(Antibody drug conjugate,ADC)通过连接子将具有高细胞毒性的毒素分子和抗体完美地结合在一起,通过抗体这一“智能炸弹”(smart bomb)将毒素靶向递呈到肿瘤细胞表面,充分利用抗体选择性强、毒素活性高的特性,提高治疗指数的同时消除了抗体疗效低、小分子毒副作用大的缺陷,实现了高效低毒的效果。
抗体药物偶联物(ADC)
ADC的概念源来已久,100多年前德国科学家Paul Ehrlich首次提出要通过单克隆抗体将细胞毒素靶向运输到肿瘤,Paul Ehrlich将这类药物称为“魔术子弹”。ADC药物实现了基于化学药物的化学疗法和基于单克隆抗体的免疫疗法的优势互补,具有高度靶向特异性的同时提高了高毒性细胞毒素的治疗窗。ADC药物整体结构复杂,主要由三部分组成:“导弹”抗体、连接桥Linker以及细胞毒素。其要作用机理是:ADC药物经血液循环运输到肿瘤部位,抗体识别肿瘤细胞表面靶抗原并与之结合,形成的抗原/ADC药物复合物经内化进入靶细胞,在细胞溶酶体酶等作用下Linker断裂,释放出活性小分子杀伤肿瘤。吸收有ADC药物的靶细胞死亡裂解,释放出毒素分子进入附近肿瘤细胞,进一步发挥杀伤作用,即“旁观者效应”。
ADC药物相关理化性质的优化和技术的进步使得ADC药物于近十年取得飞速进展。但目前ADC药物的发展仍面临多种难题及挑战:只有小于百分之一的ADC药物到达标靶细胞,且只有更少的细胞毒药物起到抗肿瘤作用;Linker设计不够合理,在体内发生提前断裂或无法断裂;且ADC药物批次间差异较大,很难制定一个对其进行药物分析以及体内外活性考察的评价标准。此外,小分子药物具有强疏水性,偶联较多数量药物(DAR为4以上)的ADC极易发生聚沉,稳定性降低。惠氏公司研制的第一个ADC药物Mylotarg就因为其作为Linker的腙键未到达肿瘤组织而提前在血液循环过程中裂解而退市。这些难题充分说明ADC药物的理论虽然比较简单,但在实际的研发和生产过程中仍需要克服很多技术壁垒。
肿瘤标志物CD24分子
白细胞分化抗原CD24(cluster of differentiation 24)是一种高度糖基化的黏附分子,具有整合素类特性。研究表明,CD24分子作为一种潜在的致癌因子,在结直肠癌、乳腺癌、肝癌、小细胞肺癌等多种肿瘤组织中过表达,而在正常组织中不表达或低表达。CD24分子作为肝癌细胞表面重要的标记物,对肿瘤的存活、增殖及致癌性存在着重要的影响,具有被开发为分子靶标的潜力。目前使用的抗CD24抗体SWA11是运用杂交瘤技术研制出的。研究表明,SWA11具有良好的靶向性,在体内和体外均可抑制肿瘤细胞的增殖、侵袭与转移。Barbara A.Froesch团队利用SWA11的靶向作用将阿霉素运输至人小细胞肺癌肿瘤病灶。2011年,Shiran Shapira团队将这种设计用于新的肿瘤组织类型,该团队设计的免疫毒素SWA11-ZZ-PE38可以靶向CD24抗原,同时PE38基团可以诱导肿瘤细胞的凋亡,能有效抑制裸鼠模型CD24阳性移植瘤的生长。单克隆抗体SWA11的ADC设计佐证了CD24靶标可以用于开发ADC类药物。
NO供体型药物
NO供体型药物一般是指由NO供体及相关药物或某种活性化合物通过各种连接基团而形成的前药。现已发现多种结构类型的NO供体,如亚硝基硫醇、硝酸酯、NO-金属复合物(硝普盐)、呋咱N-氧化物、偶氮鎓二醇盐等;其中,偶氮鎓二醇盐(diazeniumdiolates)在选择性和靶向性释放NO方面具有明显优势。一方面是偶氮鎓二醇盐在生理条件下极易不稳定,能自动释放1~2分子NO,其半衰期从几秒钟到几小时不等。另一方面,将偶氮鎓二醇盐的O2位(与氮鎓离子相连的O称之为O1,与烯键氮原子相连的O为O2)烷基化后,可转化成在生理条件下稳定的前药;通过体内某些特定酶识别或特殊生理环境作用下,去除O2保护基团,转化为不稳定的偶氮鎓二醇盐阴离子,从而达到选择性和(或)靶向性释放NO。迄今为止,已发展出多种O2保护新策略。
NO与癌症
一氧化氮(nitric oxide,NO)是生物体内一种水溶性的、携带自由基的气体,具有氧化还原特性,在生理、病理方面扮演重要角色。研究表明NO是一种潜在抗肿瘤剂,可以调节肿瘤相关过程,包括血管生成、凋亡、侵袭与转移、细胞周期等。NO对肿瘤细胞的直接作用机制尚不清楚,目前报道的主要有3个方面:(1)NO与胞内超氧阴离子作用形成过氧亚硝酸根,质子化分解为NO2和羟基自由基,羟基自由基可与肿瘤细胞的多种分子结合,从而引起肿瘤细胞损伤,如脂质过氧化、蛋白质、氨基酸交联反应;(2)NO极易与含有Fe-S中心的蛋白质Fe形成Fe-NO,从而引起线粒体呼吸链上Fe-S辅基和顺乌头酸酶的降解,因而阻止细胞内能量的合成、诱导细胞凋亡;(3)NO可以直接作用于核糖核酸还原酶,影响肿瘤细胞DNA的复制,同时还可以使DNA发生硝基化,抑制肿瘤细胞增殖。NO抗肿瘤的另一主要机制是NO介导的巨噬细胞对肿瘤细胞的杀伤性,具体机理是:T细胞识别抗原→T细胞分泌细胞因子→细胞因子刺激产生NO→NO杀伤肿瘤细胞。另外,有研究证明NK细胞毒性也是NO依赖性的。因此,设计和研究NO前体药物成为抗肿瘤药物创新的重要策略之一。美国国立癌症研究所(NCI)、一些世界知名医药企业如Merk、Pfizer、NicOx、NitroMed等均投资支持NO供体型药物开发。近年来,NO供体型药物的研发也取得了很大进展,NCI已将抗肿瘤药物JS-K列入快速研发计划。
基于上述理论基础与科研实践,本发明将实验室自制靶向CD24单克隆抗体G7mAb与一氧化氮供体二乙胺偶氮鎓二醇盐分子(NO供体)通过马来酰亚胺-双硫键相偶联,设计出一种新型抗体药物偶联物HN-01。HN-01的设计将抗体的靶向性与一氧化氮供体分子的抗肿瘤作用结合起来,在发挥一氧化氮供体分子对肿瘤细胞抑制和杀伤作用的同时显著减低其所附带产生的对正常组织细胞的毒副作用。因此,抗体药物偶联物HN-01的开发,将为肿瘤免疫治疗提供了新的候选ADC药物。
发明内容
发明目的
本发明提供一种具有抗肿瘤疗效的抗CD24抗体药物偶联物HN-01。本发明抗体药物偶联物的特征为单克隆抗体G7mAb和一氧化氮供体二乙胺偶氮鎓二醇盐分子通过马来酰亚胺-双硫键偶联而成,可以同时发挥单克隆抗体G7mAb的靶向性和一氧化氮供体二乙胺偶氮鎓二醇盐分子的细胞毒性,在体外可以抑制CD24阳性的肝癌细胞Huh-7和BEL-7402的增殖。
技术方案
一种抗CD24单克隆抗体与二乙胺偶氮鎓二醇盐分子偶联物,其特征在于:该偶联物以抗CD24单克隆抗体和一氧化氮供体分子为基础,通过化学偶联方法,利用马来酰亚胺-双硫键将具有一氧化氮供体分子和抗体连接起来,制成抗体药物偶联物;所述的一氧化氮供体分子为二甲胺偶氮鎓二醇盐,二乙胺偶氮鎓二醇盐,N-甲基乙醇胺偶氮鎓二醇盐,二乙醇胺偶氮鎓二醇盐,吡咯偶氮鎓二醇盐,哌啶偶氮鎓二醇盐,哌嗪偶氮鎓二醇盐;抗CD24单克隆抗体的轻链氨基酸序列表为SEQ NO.1,其重链氨基酸序列表为SEQ NO.2。
一种化合物HL-2,该化合物由二乙胺偶氮鎓二醇盐分子与马来酰亚胺-双硫键反应制得,其结构如下(Z)-1-((4-((4-(((4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl)oxy)methyl)phenyl)disulfanyl)benzyl)oxy)-1-(N,N-diethylamino)diazen-1-iu-m-1,2-diolate。
所述的化合物HL-2的制备方法,其特征在于按如下步骤实现:
步骤a:将0.1M的碳酸氢钠水溶液435mL经滴液漏斗缓慢加入到80mL浓度为0.55mol/L的化合物1水溶液中。室温反应20min;经油泵拉干,然后在冷冻干燥装置上进行最终干燥,得到化合物2;
步骤b:将12.4g化合物3加至250mL反应瓶中,加入100mL苯,搅拌,缓慢加入26.6gNBS,室温下反应5h,TLC检测反应完全,得到化合物4;
步骤c:在化合物4的反应瓶中加入0.82g偶氮二异丁腈,并补加26.6g NBS,80℃下回流反应20h;旋除溶剂,然后用乙酸乙酯萃取,饱和食盐水洗,无水Na2SO4干燥,浓缩并柱层析,得到白色固体状的化合物5;
步骤d:将202mg化合物5溶于3.5mL DMF中,加入69.75mg二乙胺偶氮鎓二醇盐,氮气保护,-5℃下反应4h,TLC检测反应完全。倾入50mL乙酸乙酯萃取,饱和食盐水洗,无水Na2SO4干燥,浓缩柱层析,得到无色油状的化合物6。
步骤e:将455mg化合物6溶于10mL丙酮:DMF(2:1)的混合溶液中。将205mg化合物5缓慢加入反应瓶中,氮气保护,45℃回流3.5h;TLC检测反应完全。旋除丙酮,倾入100mL乙酸乙酯萃取,饱和食盐水洗,无水Na2SO4干燥,浓缩柱层析,得到无色油状的化合物7,该化合物的分子式为(Z)-1-((4-((4-(((4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl)oxy)methyl)phenyl)disulfanyl)benzyl)oxy)-1-(N,N-diethylamino)diazen-1-iu-m-1,2-diolate。
所述的方法,其特征在于:步骤c、步骤d和步骤e中采用乙酸乙酯萃取,饱和食盐水洗,无水Na2SO4干燥和浓缩柱层析的方法来纯化反应产物。
一种化学偶联方法,将HL-2分子偶联到抗CD24单克隆抗体重链恒定区呈还原状态的半胱氨酸巯基上。
一种由抗CD24单克隆抗体与HL-2分子制备而成的抗体药物偶联物,其制备方法包括如下步骤:
步骤一:抗CD24单克隆抗体经过Protein A柱亲和层析纯化和葡糖凝胶G25FF脱盐柱分子筛层析替换抗体溶液体系后,BCA法测定抗体浓度,将还原剂TCEP与抗体按摩尔比2.5:1混合,加入后缓慢搅拌均匀,于4℃反应1h,4000rpm,低温超滤去除残余的TCEP,更换为含有1M抗氧化剂二乙烯三胺五乙酸的PBS,pH 7.0体系准备偶联反应;
步骤二:将还原后的抗CD24单克隆抗体与权利要求2所述的化合物按摩尔比1:15进行偶联,用DMSO溶解权利要求2所述的化合物、缓慢加入1mg/mL抗CD24单克隆抗体中,轻柔搅拌均匀,置于4℃反应1h,超滤;即得抗CD24单克隆抗体与二乙胺偶氮鎓二醇盐分子偶联物;
步骤三:8%的非还原性SDS-PAGE凝胶电泳初步鉴定偶联物结构的完整性,BCA法测定偶联物浓度后,置于-70℃保存。
抗CD24抗体药物偶联物HN-01的应用方式:单克隆抗体G7mAb发挥靶向性作用将具有细胞毒性的二乙胺偶氮鎓二醇盐分子特异性地展示于CD24高表达的肿瘤细胞表面。二乙胺偶氮鎓二醇盐分子裂解释放出NO分子,发挥对肿瘤细胞抑制与毒性作用。
抗CD24抗体药物偶联物HN-01在治疗肿瘤药物中的应用。
有益效果
本发明首次合成了新化合物HL-2,并利用其能与抗CD24抗体偶联的特点将其开发成新的抗体药物偶联物具体为:本发明利用杂交瘤技术获取可表达抗CD24单克隆抗体的细胞株G7,腹水接种法制备抗体G7mAb;通过流式细胞技术和近红外成像技术证明G7mAb在体外、体内的抗原特异性靶向作用,所以选择G7mAb作为本发明抗体药物偶联物的“生物弹头”。并且,本发明以HL-2作为小分子细胞毒素。HL-2由一氧化氮供体二乙胺偶氮鎓二醇盐分子和马来酰亚胺-双硫键Linker构成,具有以下几个特性:(1)每个分子裂解后可释放2分子的NO;(2)HL-2的一端是马来酰亚胺结构,可与抗体经还原形成的半胱氨酸巯基反应形成硫醚键;(3)HL-2中间是一个双硫键结构,也是该分子裂解释放NO分子的关键。相比于腙键Linker,该双硫键在循环系统中稳定,在细胞内还原环境下被分解。研究表明,在相对低氧的肿瘤细胞内,谷胱甘肽(glutamine,GSH)的浓度比正常细胞高1000倍以上,可达到毫摩尔数量级,因此,双硫键的HL-2分子可在肿瘤细胞内顺利裂解,释放出NO。本发明利用还原剂TCEP还原抗体链间二硫键的方法实现毒素与抗体的偶联,通过控制TCEP的量选择性地还原抗体铰链区的两对二硫键,计划每个抗体上偶联2~4个小分子。HL-2分子的马来酰亚胺结构与抗体半胱氨酸巯基形成稳定硫醚键,实现小分子与抗体的结合,获取抗体药物偶联物HN-01,结构如如图1所示。HN-01被肿瘤细胞内吞,在胞内谷胱甘肽的作用下双硫键断裂可释放活性效应分子NO,具体机理如下所示:
上面结构式展示的是抗体药物偶联物HN-01效应分子NO释放机理。HN-01被肿瘤细胞内吞,在胞内GSH(谷胱甘肽)作用下双硫键断裂可释放活性效应分子NO。
附图说明
图1展示的是抗体药物偶联物HN-01结构示意图。二乙胺偶氮鎓二醇盐分子通过马来酰亚胺-双硫键结合于G7mAb铰链区处于还原状态的半胱氨酸巯基。
图2是抗体药物偶联物HN-01的非还原SDS-PAGE蛋白电泳图。从图中可以看出,HN-01具有明显的主条带,条带位置与亲本抗体G7mAb齐平,因为偶联的Linker-drug的分子量是558Da,偶联小分子毒素并不能显著增加抗体分子量(150KDa),所以HN-01在电泳图中位置与G7mAb基本相当。且由于抗体的糖基化作用,二者在电泳图中的位置比150KDa marker的位置偏高。图中HN-01的泳道除主条带外,还有两条浅条带,分析可能是TCEP还原了抗体的重、轻链之间的二硫键,致使极少部分偶联物抗体部分轻链或重链的缺失。
图3展示的是采用HIC-HPLC(疏水层析-高效液相色谱法)得到的抗体药物偶联物HN-01色谱峰。以裸抗G7mAb在相同分析条件下的出峰时间为参照(图4A),我们发现图4B在裸抗出峰时间10min左右并没有相应峰,说明分析产物中不存在裸抗,即所有分析产物均偶联有小分子;由于目标是偶联2~4个小分子,对照图4B我们可以判断从左到右峰分别是2-drug、4-drug、6-drug、8-drug对应峰。
图4是HN-01的MTT试验结果图,展示的是HN-01对CD24阳性肿瘤细胞Huh-7和BEL-7402的增殖抑制作用。加药48h后,1000nM HN-01对CD24阳性细胞Huh-7和BEL-7402的增殖抑制率则分别为41.00%和41.16%,与相同浓度母体单抗G7mAb组相比有了显著的提高(1000nM G7mAb作用48h后对两株阳性细胞的抑制率分别是21.96%和24.92%)。综合分析发现,偶联物HN-01对CD24阳性细胞株增值抑制作用随着剂量的增加呈依赖性增长趋势。本实验根据HN-01的摩尔数及单抗与小分子HL-2的偶联比设置对应摩尔数的小分子HL-2为对照组,结果显示,随着HL-2浓度的增高,其对细胞的增殖有一定的抑制作用,但其抑制率显著低于偶联物HN-01,但略高于G7mAb组。
图5是HN-01的凋亡试验流式散点图,展示的是HN-01诱导Huh-7和BEL-7402细胞发生凋亡的作用。如图所示,100nM和500nM的HN-01作用48h后,诱导的Huh-7细胞的凋亡率分别是13.79%和21.43%;相同浓度的G7mAb基本不诱导细胞的凋亡,对应的凋亡率分别是9.78%和11.3%;相当摩尔浓度的小分子HL-2可诱导的凋亡率则分别是13.63%和17.12%;相同条件下的空白对照组对应的凋亡率是8.02%。100nM和500nM的HN-01作用48h后,HN-01诱导的BEL-7402细胞的凋亡率分别是3.77%和25.81%;相同浓度的G7mAb基本不诱导细胞的凋亡,所对应的凋亡率分别是4.58%和5.42%;相当摩尔浓度的小分子HL-2可诱导的凋亡率则分别是5.88%和7.55%;相同条件下的空白对照组对应的凋亡率是2.24%。结果显示,随着HN-01给药浓度的增加,其诱导的细胞凋亡也相应增强;母体抗体G7mAb不能显著引起细胞的凋亡,小分子HL-2具有一定的细胞毒性,高浓度的HL-2在诱导细胞凋亡的同时导致细胞发生坏死现象。
图6是HN-01的凋亡试验结果的统计学分析(统计分析依据三次独立的实验结果以means±SD形式显示;***p<0.001:非常显著)。T-检验结果表明,HN-01高浓度给药剂量(500nM)作用下诱导的Huh-7和BEL-7402细胞凋亡率与G7mAb和HL-2两组之间都存在显著差异。
具体实施方式
实施例中涉及的百分比,其中固定试剂为重量体积百分比,液体试剂为体积百分比。
实施例1 HL-2的合成方法与鉴定:
步骤a:将0.1M的碳酸氢钠水溶液435mL经滴液漏斗缓慢加入到80mL浓度为0.55mol/L的化合物1水溶液中。室温反应20min;经油泵拉干,然后在冷冻干燥装置上进行最终干燥,得到化合物2;
步骤b:将12.4g化合物3加至250mL反应瓶中,加入100mL苯,搅拌,缓慢加入26.6gNBS,室温下反应5h,TLC检测反应完全,得到化合物4;
步骤c:在化合物4的反应瓶中加入0.82g偶氮二异丁腈,并补加26.6g NBS,80℃下回流反应20h;旋除溶剂,然后用乙酸乙酯萃取,饱和食盐水洗,无水Na2SO4干燥,浓缩并柱层析,得到白色固体状的化合物5;
步骤d:将202mg化合物5溶于3.5mL DMF中,加入69.75mg二乙胺偶氮鎓二醇盐,氮气保护,-5℃下反应4h,TLC检测反应完全。倾入50mL乙酸乙酯萃取,饱和食盐水洗,无水Na2SO4干燥,浓缩柱层析,得到无色油状的化合物6。
步骤e:将455mg化合物6溶于10mL丙酮:DMF(2:1)的混合溶液中。将205mg化合物5缓慢加入反应瓶中,氮气保护,45℃回流3.5h;TLC检测反应完全。旋除丙酮,倾入100mL乙酸乙酯萃取,饱和食盐水洗,无水Na2SO4干燥,浓缩柱层析,得到无色油状的化合物7;
具体合成方法如下:
Sodium 4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoate(2).
将碳酸氢钠(8.391g,0.1mmol)溶于1L的水中制备成0.1M的碳酸氢钠水溶液。其中,将0.1M的碳酸氢钠水溶液435mL经滴液漏斗缓慢加入4-马来酰亚胺丁酸13(8.014g,43.75mmol)的水(80mL)溶液中。室温反应20min。经油泵拉干,然后在冷冻干燥装置上进行最终干燥。得灰白色固体化合物2(9.06g;收率100%)。1H NMR(300MHz,D2O)1.76(q,J=7.1Hz,2H,CH2),2.09(t,J=7.38Hz,2H,CH2),3.45(t,J=6.98Hz,2H,CH2),6.76(s,2H,CH×2)。m/z(ESI):182[M-H]-.
1,2‐bis(4‐(bromomethyl)phenyl)disulfane(5).
一锅法制备化合物5:四甲基苯硫酚3(12.4g,100mmol)加至250mL反应瓶中,加入苯(100mL),搅拌,缓慢加入N‐溴代丁二酰亚胺(NBS,26.6g,150mmol),室温下反应5h,TLC检测反应完全,全部转化为中间体4;再加入偶氮二异丁腈(AIBN,0.82g,5mmol)并补加NBS(26.6g,150mmol),80℃下回流反应20h。旋除溶剂,然后用乙酸乙酯萃取,饱和食盐水洗,无水Na2SO4干燥,浓缩并柱层析,得白色固体化合物5(5.06g,收率25%)。1H NMR(300MHz,CDCl3)4.38(s,4H,CH2)7.33(d,J=6Hz,4H)7.45(d,J=6Hz,4H)。
(Z)‐1‐((4‐((4‐(bromomethyl)phenyl)disulfanyl)benzyl)oxy)‐1‐(N,N‐diethylamino)diazen‐1‐iu‐m‐1,2‐diolate(6).
将化合物5(202mg,0.5mmol)溶于3.5mL DMF中,加入(69.75mg,0.45mmol)二乙胺偶氮鎓二醇盐,氮气保护,‐5℃下反应4h,TLC检测反应完全。倾入乙酸乙酯(50mL)萃取,饱和食盐水洗,无水Na2SO4干燥,浓缩柱层析,得无色油状物化合物6(100mg,收率43.8%)。1HNMR(300MHz,CDCl3)0.96(t,J=6Hz,6H,CH3×2),3.04(q,J=6Hz,4H,CH2×2),4.42(s,2H),5.20(S,2H),7.28(d,J=4.5Hz,2H),7.34(d,J=4.5Hz,2H),7.46(d,J=4.5Hz,2H),7.43(d,J=4.5Hz,2H).13C NMR(75MHz,CDCl3)δ130.25,129.87,129.81,128.17,128.02,128.01,127.73,127.41,75.44,49.34,33.55,11.97.
(Z)‐1‐((4‐((4‐(((4‐(2,5‐dioxo‐2,5‐dihydro‐1H‐pyrrol‐1‐yl)butanoyl)oxy)methyl)phenyl)disulfanyl)benzyl)oxy)‐1‐(N,N‐diethylamino)diazen‐1‐iu‐m‐1,2‐diolate(7).
化合物6(455mg,1mmol)溶于丙酮:DMF(2:1)共10mL。将化合物17(205mg,1mmol)缓慢加入反应瓶中,氮气保护,45℃回流3.5h。TLC检测反应完全。旋除丙酮,倾入乙酸乙酯(100mL)萃取,饱和食盐水洗,无水Na2SO4干燥,浓缩柱层析,得无色油状物化合物7(200mg,收率36%)。1H NMR(300MHz,CDCl3)0.99(t,J=7.08Hz,6H,CH3×2),1.94(q,J=7.08Hz,2H,CH2),2.36(t,J=5.43Hz,2H,CH2),3.06(q,J=7.08Hz,4H,NCH2×2),3.58(t,J=7.2Hz,2H,CH2),5.07(s,2H,CH2),5.23(s,2H,CH2),6.68(s,2H,2ArH),6.72(s,2H,2ArH),7.32(m,4H,4ArH),7.46(m,2H,CH×2).13C NMR(75MHz,CDCl3)δ11.45,23.73,30.98,37.00,48.72,65.78,74.99,127.46,127.64,129.04,129.32,134.10,134.14,170.73,170.76,172.33,172.63,177.15.ESI‐MS:[M+Na]+,found 581.1.C26H30N4O6S2requires 558.HRMS m/z calcdfor C26H30N4O6S2[M+Na]+,581.1504.found 581.1515.ppm error 1.9.
实施例2 抗体蛋白偶联物HL-01的制备
1.抗CD24单克隆抗体G7mAb(按文献制备:Ma Z等,Selective targeted
delivery of doxorubicin via conjugating to anti-CD24antibody resultsin enhanced antitumor potency for hepatocellular carcinoma both in vitro andin vivo.J Cancer Res Clin Oncol.2017,143(10):1929-1940)经过Protein A柱亲和层析纯化和葡糖凝胶G25FF脱盐柱分子筛层析替换抗体溶液体系后,BCA法测定抗体浓度,将还原剂TCEP与抗体按摩尔比2.5:1混合,加入后缓慢搅拌均匀,于4℃反应1h,4000rpm,低温超滤去除残余的TCEP,更换为含有1M抗氧化剂二乙烯三胺五乙酸的PBS(pH 7.0)体系准备偶联反应。
2.将还原后的G7mAb与Linker-NO供体分子HL-2按摩尔比1:15进行偶联,将溶解于DMSO的小分子少量多次、缓慢加入1mg/mL抗体中,轻柔搅拌均匀,置于4℃反应1h,超滤去除多余的HL-2分子。即得抗体药物偶联物HN-01。
1.3.8%的非还原性SDS-PAGE凝胶电泳初步鉴定偶联物结构的完整性,BCA
法测定偶联物浓度后,置于-70℃保存。
实施例3 抗体蛋白偶联物HN-01的非还原SDS-PAGE电泳鉴定
1.制样:取若干EP管,加入6μL含250mM Tris-HCl(pH 6.8),10%SDS,0.5%溴酚兰,50%甘油的5×Loading Buffer,在每管中依次加入24μL样品,混匀溶液,沸水浴5min,8000rpm,离心3min,备用。
2.配胶:将双层玻璃板固定于配胶模具,用单蒸水检漏,分别配置下层分离胶和上层积层胶。先将分离胶加入到双层玻璃板中,立即用1mL无水乙醇压平。大约25min下层的分离胶凝固,随后加入积层胶,并插入梳齿。待积层胶完全凝固后,拆开配胶模具,取出双层玻璃板,将其安装于电泳槽内,并向电泳槽中倒入一定体积含Tris,Glycine,SDS,pH 8.0的电泳缓冲液,以没过最低水平线。
3.上样:用微量进样针上样,Marker的上样量为5μL,HN-01和G7mAb样品的上样量为25μL,样品上完后,接通电极,开始电泳。
4.电泳:恒压电流,初始电压为80V,当蓝色条带前沿迁移到积层胶与分离胶交界处时,将电压调为120V。待蓝色条带跑出分离胶时,停止电泳,剥胶。
5.染色脱色:将胶剥至盒子中,加染色液,煮沸,放置在脱色摇床上染色30min。回收染色液,自来水冲洗。在盒子中加入脱色液,煮沸,之后放置在脱色摇床上振摇20min,脱色2次,直至条带清晰可见。
6.成像:用胶小心放置在白色瓷板上,放置在凝胶成像仪下观察Marker及HN-01和G7mAb的条带。实验结果如图2所示。
实施例4 HIC-HPLC检测HN-01的DAR(药物抗体偶联比)
许多偶联到抗体上的小分子毒素是疏水性的,因此不同偶联比的偶联物的疏水性有较大差异,可以用疏水层析色谱(HIC)串联紫外检测器进行分析,通过色谱峰的分布可以判断小分子药物偶联到抗体上的个数,通过峰面积可以判断偶联有不同化学计量数小分子药物的偶联物所占总混合物的比例。
HPLC:岛津LC-20AT
HIC色谱柱:TSKgel Butyl-NPR(2.5μm粒径,4.6mm内径,长10cm)
流动相A:1.5M(NH4)2SO4+25mM PBS,pH 7.0
流动相B:25mM PBS,pH 7.0(75%)+异丙醇(25%)
洗脱梯度:
流速:0.5mL/min
柱温:24℃
进样量:10μL
检测吸光度:280nm
样品:母体单抗G7mAb(4m/mL);抗体药物偶联物HN-01(6mg/mL)
检测结果:见表1和图3
表1 HIC-HPLC分析加权平均DAR
表1是采用HIC-HPLC法分析得到的加权平均DAR(药物抗体偶联比)。通过不同DAR对应峰峰面积按以下公式计算加权平均DAR:DAR=,结果如表1所示,计算所得加权平均DAR为3.327。
实施例5 抗体药物偶联物HN-01抑制细胞增殖
ADC类药物利用抗体的亲和性和靶向性将细胞毒素富集到肿瘤细胞表面,提高小分子治疗效果的同时可减低其系统毒性。本研究选用MTT比对法检测偶联物HN-01对CD24阳性细胞增殖的抑制作用,具体方法如下:
(1)实验组设置:空白对照组(不含细胞)、阴性对照组(含细胞,不加药)、实验组(G7mAb、HN-01组、HL-2组),每组设3个复孔。
(2)细胞铺板:取对数期阳性细胞Huh-7、BEL-7402细胞,消化、用含5%FBS的培养基重悬、计数,调整细胞悬液浓度为3×104cells/mL,每孔加入细胞悬液100μL,板的四周加入100μL无菌PBS消除边缘效应,将板置于细胞培养箱中培养24h。每种细胞铺设一块96孔板,检测药物作用48h对肿瘤细胞增殖的抑制情况。
(3)药物配制:用含2%FBS的培养基配制药物母液,倍比稀释药物G7mAb、HN-01和NO供体分子HL-2各6个浓度。G7mAb和HN-01设置0.8、4、20、100、500、1000nM六个浓度梯度,HL-2以偶联比3.0为基础,配制对应摩尔浓度给药浓度:2.4、12、60、300、1500、3000nM六种浓度。
(4)加药:用1mL注射器吸出实验孔和对照组孔内培养基,PBS清洗后各组对应加入含对应药物的培养基100μL,对照组加入100μL含2%FBS的培养基。37℃继续培养,待检测。
(5)检测:加药作用48h后,取出96孔板,避光条件下每孔加入11μL的MTT(5mg/mL),继续37℃培养4h。4h后取出板离心,平板离心机3000rpm离心5min,用1mL注射器吸除孔中上清液,每孔加入150μL的DMSO,微量振荡仪振动10min。酶标仪检测波长570nm和630nm处的吸光值。
(6)数据处理:A570-A630作为每孔净吸光值,药物抑制率计算如下:%抑制率=(阴性对照组平均值-实验组值)/(阴性对照组平均值-空白对照组平均值)*100。
实验结果如图4A,4B所示。
实施例6 抗体药物偶联物HN-01诱导肿瘤细胞凋亡
本实验选用Annexin V/PI双染色法检测HN-01诱导的肿瘤细胞凋亡作用,实验操作参照细胞凋亡检测试剂盒(购于Vazyme)说明书,具体流程如下:
(1)铺板:选择对数期状态良好的Huh-7、BEL-7402细胞,消化,重悬,计数,调整细胞悬液浓度为2x105cells/mL,吸取1.5mL悬液加入到六孔板中,37℃培养过夜。
(2)加药:吸去细胞培养上清,无菌PBS清洗3次。加入不同浓度的HN-01和G7mAb(100nM、500nM)以及小分子HL-2(35nM、170nM)作用细胞,37℃培养48h。
(3)悬浮细胞制备:收集上清,用不含EDTA的胰酶消化细胞后300g,4℃离心5min收集细胞,加入所收集对应上清液重悬细胞。
(4)清洗:加入预冷的PBS,300g,4℃离心5min洗涤。重复两次。用PBS重悬细胞,计数,调整每组细胞数为5x105个。
(5)细胞重悬:300g,4℃离心5min,加入100μL 1x的Binging Buffer重悬细胞。
(6)染色:加入5μL的Annexin V-FITC和5μL的PI staining Solution,轻轻吹打混匀。
(7)反应:避光,室温反应10min。
(8)检测:加入400μL 1x Binging Buffer,轻轻混匀,流式细胞仪检测,激发波长为488nm,FITC的绿色荧光在FL1通道检测,PI的红色荧光在FL2或FL3检测,我们选择FL3通道。
实验结果如图5A,5B所示,统计学分析结果如图6A,6B所示。
序列表
<110> 中国药科大学
<120> 一种靶向CD24单克隆抗体与二乙胺偶氮鎓二醇盐的偶联物及其应用
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Claims (6)
1.一种抗CD24单克隆抗体与二乙胺偶氮鎓二醇盐分子偶联物,其特征在于:该偶联物以抗CD24单克隆抗体和一氧化氮供体分子为基础,通过化学偶联方法,利用马来酰亚胺-双硫键将一氧化氮供体分子和抗体连接起来,制成抗体药物偶联物;所述的一氧化氮供体分子为二甲胺偶氮鎓二醇盐,二乙胺偶氮鎓二醇盐,N-甲基乙醇胺偶氮鎓二醇盐,二乙醇胺偶氮鎓二醇盐,吡咯偶氮鎓二醇盐,哌啶偶氮鎓二醇盐,哌嗪偶氮鎓二醇盐中任意一种;抗CD24单克隆抗体的轻链氨基酸序列如SEQ NO.1所示,其重链氨基酸序列如SEQ NO.2所示。
步骤a:将0.1M的碳酸氢钠水溶液435mL经滴液漏斗缓慢加入到80mL浓度为0.55mol/L的化合物1水溶液中;室温反应20min;经油泵拉干,然后在冷冻干燥装置上进行最终干燥,得到化合物2;
步骤b:将12.4g化合物3加至250mL反应瓶中,加入100mL苯,搅拌,缓慢加入26.6g NBS,室温下反应5h,TLC检测反应完全,得到化合物4;
步骤c:在化合物4的反应瓶中加入0.82g偶氮二异丁腈,并补加26.6g NBS,80℃下回流反应20h;旋除溶剂,然后用乙酸乙酯萃取,饱和食盐水洗,无水Na2SO4干燥,浓缩并柱层析,得到白色固体状的化合物5;
步骤d:将202mg化合物5溶于3.5mL DMF中,加入69.75mg二乙胺偶氮鎓二醇盐,氮气保护,-5℃下反应4h,TLC检测反应完全;倾入50mL乙酸乙酯萃取,饱和食盐水洗,无水Na2SO4干燥,浓缩柱层析,得到无色油状的化合物6;
步骤e:将455mg化合物6溶于10mL丙酮:DMF(2:1)的混合溶液中;将205mg化合物5缓慢加入反应瓶中,氮气保护,45℃回流3.5h;TLC检测反应完全;旋除丙酮,倾入100mL乙酸乙酯萃取,饱和食盐水洗,无水Na2SO4干燥,浓缩柱层析,得到无色油状的化合物7,该化合物的化学名称为(Z)-1-((4-((4-(((4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl)oxy)methyl)phenyl)disulfanyl)benzyl)oxy)-1-(N,N-diethylamino)diazen-1-iu-m-1,2-diolate。
4.根据权利要求3所述的方法,其特征在于:步骤c、步骤d和步骤e中采用乙酸乙酯萃取,饱和食盐水洗,无水Na2SO4干燥和浓缩柱层析的方法来纯化反应产物。
5.根据权利要求1所述的一种抗CD24单克隆抗体与二乙胺偶氮鎓二醇盐分子偶联物的制备方法,其特征在于包括如下步骤:
步骤一:抗CD24单克隆抗体经过Protein A柱亲和层析纯化和葡糖凝胶G25FF脱盐柱分子筛层析替换抗体溶液体系后,BCA法测定抗体浓度,将还原剂TCEP与抗体按摩尔比2.5:1混合,加入后缓慢搅拌均匀,于4℃反应1h,4000rpm,低温超滤去除残余的TCEP,更换为含有1M抗氧化剂二乙烯三胺五乙酸的PBS,pH 7.0体系准备偶联反应;
步骤二:将还原后的抗CD24单克隆抗体与权利要求2所述的化合物按摩尔比1:15进行偶联,用DMSO溶解权利要求2所述的化合物、缓慢加入1mg/mL抗CD24单克隆抗体中,轻柔搅拌均匀,置于4℃反应1h,超滤;即得抗CD24单克隆抗体与二乙胺偶氮鎓二醇盐分子偶联物;
步骤三:8%的非还原性SDS-PAGE凝胶电泳初步鉴定偶联物结构的完整性,BCA法测定偶联物浓度后,置于-70℃保存。
6.如权利要求1所述的一种抗CD24单克隆抗体与二乙胺偶氮鎓二醇盐分子偶联物在制备抗肿瘤药物中的应用。
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