CN103145847B - 一种抗cd20抗体-海兔毒素偶联物及其制备方法和应用 - Google Patents
一种抗cd20抗体-海兔毒素偶联物及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种抗CD20抗体-海兔毒素偶联物及其制备方法和应用。所述抗CD20抗体-海兔毒素偶联物由海兔毒素通过连接臂偶联抗CD20抗体构成。其中,抗CD20抗体的轻链氨基酸序列如SEQ ID No.1所示,重链氨基酸序列如SEQ ID No.2所示。所述应用为抗CD20抗体-海兔毒素偶联物在制备抗肿瘤药物中的应用。本发明偶联物具有抗CD20抗体和MMAE两者的生物学功能,在二者的协同下,抗肿瘤效果得到显著增强。
Description
技术领域
本发明属于生物技术和药物领域,尤其涉及一种抗CD20抗体-海兔毒素偶联物及其制备方法和应用。
背景技术
CD20是B淋巴细胞和B淋巴瘤细胞表面特有的膜表面蛋白,在造血干细胞、原始B细胞、正常血浆细胞以及其它正常组织不表达(Bubien J K等,J Cell Biol,1993,121(5):1121-1132)。因此研制抗CD20抗原的单克隆抗体就可以专一性地杀伤B细胞和B淋巴瘤细胞,达到良好的治疗效果。
Ofatumumab(2F2)是全人源化靶向抗CD20单克隆抗体,2009年被FDA批准用于氟达拉滨和阿仑珠单抗治疗无效的顽固性慢性淋巴细胞白血病(CLL)(CASTILLO J.Expert Opin Investing Drμgs,2009,18(4):491-500.)。2F2通过与小型和大型的CD20分子上的小环抗原靶向结合,促使细胞溶解,特异性的诱导CD20阳性细胞凋亡,从而专一性地杀灭B淋巴瘤细胞(BEUM P V,Immuuol,2008,181(1):822-832.),对其他正常组织无不良影响。
但是同时,由于2F2抗体本身的局限性,使得其杀伤CD20阳性B淋巴瘤细胞的能力非常有限,经过一段时间的治疗没有杀尽的肿瘤细胞又会重新生长并对2F2抗体产生耐药性。因此,2F2抗体杀死肿瘤细胞的能力亟需提高,而通过抗体偶联毒性小分子便能达到此效果。
抗体偶联小分子,即为抗体偶联药物,由单抗与“弹头”药物两部分构成。作为“弹头”的抗肿瘤药物主要通过抑制细胞DNA或蛋白质合成、抑制细胞、有丝分裂等方式来杀伤肿瘤细胞。但这些药物对正常的细胞同样有较大杀伤力,从而极大的限制了该类药物的进一步应用和发展。例如化疗药物海兔毒素(Monomethyl auristatin E,MMAE)是一种人工合成抗肿瘤小分子,它通过抑制细胞内微管蛋白二聚化而诱导细胞凋亡。但由于它具有很强的无选择毒性,会对正常细胞造成伤害,所以其本身并不能成药。
单抗是药物良好的靶向性载体,利用药物分子上特殊的功能基团如:羟基、巯基、氨基等可将药物与单抗相连接而组成化学免疫偶联物,单抗的靶向性能将与之相连的药物“精确”地运送到靶细胞,有效地提高了肿瘤局部的药物浓度,极大地降低体内其他组织、器官的药物浓度,从而达到增效减毒的作用。
因此,若能将一些能够强力杀伤肿瘤细胞的毒性药物加以有效利用,将更有利于肿瘤的治疗。
发明内容
本发明提供了一种抗CD20抗体-海兔毒素偶联物,与现有的抗CD20抗体相比,该偶联物对CD20阳性细胞具有更高的杀伤力。
一种抗CD20抗体-海兔毒素偶联物,由海兔毒素通过连接臂偶联本发明所述抗CD20抗体构成。
其中,所述抗CD20抗体的轻链的氨基酸序列如SEQ ID No.1所示,重链的氨基酸序列如SEQ ID No.2所示。
本发明所述抗CD20抗体是抗CD20抗体2F2的突变体,突变发生在重链氨基酸序列的恒定区,第123位的丙氨酸(Ala)突变为半胱氨酸(Cys)。
本发明所使用的连接臂为马来酰亚胺修饰的缬氨酸-瓜氨酸二肽。其合成方法参考文献:Gene M.D.,et al.Cathepsin B-Labile Dipeptide Linkersfor Lysosomal Release of Doxorubicin from Internalizing Immunoconjμgates:Model Studies of Enzymatic Drμg Release and Antigen-Specific In VitroAnticancer Activity.BioconjμgateChem..13(4)855869(2002)。
所述海兔毒素的合成方法参考美国专利文献:Tumer inhibitingtetrapeptide bearing modified phenethyl amides(专利号:5,635,483)。
本发明带连接臂的海兔毒素(vcMMAE)由江阴康诺泰生物技术有限公司代为合成,也可以参考文献(Svetlana O.D.,et al.Development ofpotentmonoclonal antibody auristatin conjμgates for cancer therapy[J].NatureBiotechnology..21(7)778-784(2003).)。
偶联时,vcMMAE通过缬氨酸上的马来酰亚胺与抗CD20抗体的半胱氨酸巯基进行烷基化反应,最终形成本发明的抗CD20抗体-海兔毒素偶联物。其中,每个抗CD20抗体分子连接1~2个海兔毒素分子。
本发明还提供了一种抗CD20抗体-海兔毒素偶联物的制备方法,包括:
(1)将抗CD20抗体与带连接臂的海兔毒素混合,进行偶联反应;偶联反应的温度为4℃以下,时间为60~120min;由于抗CD20抗体的分子呈Y型,因此偶联完成后,每个抗CD20抗体上可以结合1-2个海兔毒素分子;
(2)反应完成后,分离纯化得到所述抗CD20抗体-海兔毒素偶联物;
由于本发明抗CD20抗体-海兔毒素偶联物单体的分子量约为150kDa,而反应体系中存在的其他分子的分子量均小于10kDa,因此,用截留分子量10kDa的超滤管即可除去体系中的小分子,再经离心去沉淀,将所得上清过滤除菌,即为所述抗CD20抗体-海兔毒素偶联物。
由于2F2抗体表面的半胱氨酸巯基均用于形成重链-重链、重链-轻链之间的二硫键,因而不存在游离的半胱氨酸巯基用于与海兔毒素偶联。为此,本发明采用PCR定点突变手段获得了含有一个半胱氨酸突变位点的2F2突变体,即为本发明的抗CD20抗体。具体步骤包括:
(a)人工合成2F2抗体的重链、轻链的可变区DNA编码序列;
(b)构建轻链表达载体和重链表达载体;
(c)以所述重链表达载体为模板,利用引物P1和P2进行PCR定点突变扩增,获得突变重链序列;
所述引物P1和P2的碱基序列为:
P1:5’-GTCTCCTCATGTAGCACCAAGGGCCCA-3’;
P2:5’-TGGGCCCTTGGTGCTACATGAGGAGAC-3’;
(d)将所述突变重链序列可操作性地连入载体,获得重链突变表达载体;
(f)将轻链表达载体和重链突变表达载体转化宿主细胞,宿主细胞表达融合蛋白,获得所述抗CD20抗体。
所述宿主细胞可选细菌、酵母、昆虫细胞或哺乳动物细胞,优选为哺乳动物细胞,最优选为中国仓鼠卵巢细胞(CHO)。
最初从宿主细胞表达出的抗CD20抗体,其半胱氨酸巯基上往往连接有半胱氨酸或谷胱甘肽。因此在与海兔毒素偶联之前,需使抗CD20抗体的半胱氨酸巯基处于游离状态。具体方法包括:
(a)将从宿主细胞表达出的抗CD20抗体溶于PBS缓冲液(pH7.4)中,加入三氯乙基磷酸酯,30~40℃水浴反应1~3h;
三氯乙基磷酸酯可使半胱氨酸或谷胱甘肽与抗CD20抗体之间的二硫键断开;
(b)超滤并用PBS洗涤除去反应液中分子量低于10kDa的物质;
(c)加入脱氢抗坏血酸,30~40℃水浴反应1~3h;
三氯乙基磷酸酯也会使抗CD20抗体自身的链间二硫键断开,因此除去小分子后,在体系中加入脱氢抗坏血酸使链间二硫键恢复;
(d)超滤并用PBS洗涤除去反应液中分子量低于10kDa的物质,获得具有游离半胱氨酸巯基的2F2突变体。
本发明还提供了所述抗CD20抗体-海兔毒素偶联物在制备抗肿瘤药物中的应用。
所述抗肿瘤药物包括有效量的抗CD20抗体-海兔毒素偶联物,以及至少一种药学上可接受的载体、稀释剂或赋形剂。制备时,通常将活性成分与赋形剂混合,或用赋形剂稀释,或包在可以胶囊或药囊形式存在的载体中。当赋形剂起稀释剂作用时,它可采用固体、半固体或液体材料作为赋形剂、载体或活性成分的介质。因此,组合物可以是片剂、丸剂、粉剂、溶液剂、糖浆剂、灭菌注射溶液等。
合适的赋形剂包括:乳糖、葡萄糖、蔗糖、山梨醇、甘露醇、淀粉、微晶纤维素、聚乙烯吡咯烷酮、纤维素、水等;制剂还可包括:湿润剂、乳化剂、防腐剂(如羟基苯甲酸甲酯和丙酯)、甜味剂等。所述抗肿瘤药物可制成单元或多元剂型,各剂型包含为了产生所期望的疗效而计算出预定量的所述抗CD20抗体-海兔毒素偶联物,以及合适的药剂学赋形剂。
所述的抗肿瘤药物可以通过常规途径进行给药,包括(但并不限于):肌内、腹膜内、静脉内、皮下、皮内、局部给药等。
使用该药物时,是将安全有效量的所述抗CD20抗体-海兔毒素偶联物施用于人,其中该安全有效量的范围优选为0.5~50毫克/千克体重,更优选为1~10毫克/千克体重。当然,具体剂量还应考虑给药途径、病人健康状况等因素,这些都是在熟练医师技能范围之内的。
此外,本发明的偶联物还可与其他治疗药物联用,其中包括(但并不限于):各种细胞因子,如TNF、IFN、IL-2等;各种肿瘤化疗药物,如5-FU、氨甲喋呤等影响核酸生物合成的药物;氮芥、环磷酰胺等烷化剂类药物;阿霉素、放线菌素D等干扰转录过程阻止RNA合成的药物;长春新碱、喜树碱类等影响蛋白质合成的药物及某些激素类药物,等等。
与现有技术相比,本发明的有益效果为:
(1)本发明偶联物具有抗CD20抗体和MMAE两者的生物学功能,既具有2F2抗体对肿瘤细胞的杀伤能力,又具有MMAE在细胞内抑制微管蛋白从而诱导凋亡的能力,在二者的协同下,抗肿瘤效果得到显著增强;
(2)本发明偶联物通过抗CD20抗体与肿瘤细胞表面的CD20受体特异结合,将MMAE定向转运到肿瘤细胞,并在肿瘤细胞内释放发挥作用,既可杀伤CD20阳性肿瘤细胞,也减少MMAE单独给药产生的毒副作用。
附图说明
图1为2F2抗体和2F2突变体的电泳图;其中,M代表分子量Marker,1为2F2突变体的还原条带,2为2F2的还原条带,3为2F2突变体的非还原条带,4为2F2的非还原条带;
图2为2F2抗体的分子筛图;
图3为2F2突变体的分子筛图;
图4为2F2突变体-vcMMAE偶联物各部分连接关系示意图;
图5为2F2突变体-vcMMAE偶联物对不同CD20阳性细胞的亲和性图;
图6为2F2突变体-vcMMAE偶联物对不同细胞系的CDC杀伤效果图;
图7为2F2突变体-vcMMAE偶联物对不同细胞系的ADCC杀伤效果图;
图8为2F2突变体-vcMMAE偶联物对不同细胞系的直接杀伤效果图;
图9为2F2抗体和2F2突变体对2F2突变体-vcMMAE偶联物的竞争性杀伤抑制效果图;
图10为2F2突变体-vcMMAE偶联物诱导Ramos细胞凋亡图;
图11为2F2突变体-vcMMAE偶联物诱导细胞凋亡机制示意图。
具体实施方式
实施例12F2突变体-海兔毒素偶联物的构建
12F2抗体重链、轻链表达载体的构建
(1)合成2F2抗体可变区序列
根据公开号为US2004/0167319A15的专利文献合成2F2抗体重链和轻链的可变区DNA序列,分别如下:
轻链可变区(VL)(含信号肽序列和酶切位点,下划线部分为酶切位点,小写字母部分为信号肽序列):
5’-atggaagccccagctcagcttctcttcctcctgctactctggctcccagataccaccggaGAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA 3’,如SEQ ID No.4所示;
重链可变区(VH)(含信号肽序列和酶切位点,下划线部分为酶切位点,小写字母部分为信号肽序列):
5’-atggagttgggactgagctggattttccttttggctattttaaaaggtgtccagtgtGAAGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGCAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAATGATTATGCCATGCACTGGGTCCGGCAAGCTCCAGGGAAGGGCCTGGAGTGGGTCTCAACTATTAGTTGGAATAGTGGTTCCATAGGCTATGCGGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAAGTCCCTGTATCTGCAAATGAACAGTCTGAGAGCTGAGGACACGGCCTTGTATTACTGTGCAAAAGATATACAGTACGGCAACTACTACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA-3’;如SEQ ID No.5所示。
(2)构建重链、轻链表达载体
1)将重链可变区(VH)、带重链恒定区(碱基序列如SEQ ID No.8所示)的载体pFUSE-CHIg-hG1(phIgG1)用Nhe I(N)酶切4.5h;获得的VH(N)、phIgG1(N)再用EcoR I(E)酶切5h;
2)将轻链可变区(VL)、带轻链恒定区(碱基序列如SEQ ID No.6所示)的载体pFUSE2-CLIg-hk(phk)用Age I(A)酶切4.5h;获得的VL(A)、phk(A)再用BsiW I(B)酶切5h;
3)分别将酶切过的可变区和恒定区通过T4连接酶连接,转化感受态细菌,挑取阳性克隆培养,提取质粒测序验证,得到重链表达载体H和轻链表达载体L;
4)将测序验证正确的质粒对应菌种摇大瓶,提取质粒H、L各600μL备用。
(2)重链突变表达载体的构建
1)点突变PCR
以重链表达载体H为模板,引物P1和P2为上下游引物,进行点突变PCR,引物P1和P2的序列为:
P1:5’-GTCTCCTCATGTAGCACCAAGGGCCCA-3’;
P2:5’-TGGGCCCTTGGTGCTACATGAGGAGAC-3’;
PCR反应体系为:
PCR反应条件为:98℃变性3min;98℃变性30s,64.7℃退火15s,72℃延伸5min,33个循环;72℃延伸10min。
2)DpnI消化
PCR完成后,直接在PCR管中加入1~3μL的DpnI,37℃水浴2小时,利用DpnI消化PCR产物中的模板DNA,只剩下含突变位点的子代DNA。
酶消化体系如下:
3)转化E.coli DH5α感受态细胞
取约5μLDpnI反应液转化至200μL的感受态细胞中;挑取阳性单克隆测序,验证选出阳性重组子克隆扩增的质粒命名为H’;对H’进行酶切(酶切位点为EcoR I、BspH I),回收带突变位点的DNA;带恒定区的载体IgG1也进行酶切保留大部分载体部分,连接回收的DNA酶切产物,转化感受态细胞,挑选阳性单个克隆测序,获得突变表达载体Thio-H,该载体中含有突变后的重链恒定区,其碱基序列如SEQ ID No.7所示。
32F2抗体和2F2突变体的表达
(1)转染
1)选对数生长期的CHO细胞接种到6孔培养板上,约达到90%的融合,加入8μL lipofectamine2000、242μL Nutrient mixture F-12ham’skaighn’s modification(F12k),25℃室温孵育5min;
2)将轻链表达载体L、重链表达载体H(或轻链表达载体L、重链突变表达载体Thio-H)各2μg与246μL混合(共250μL),25℃室温孵育5min,
3)混合步骤1)和2)中的液体,室温下静置20min;
4)将6孔板中的细胞用F12k冲洗两遍后,加入0.5mL F12k;
5)将DNA与脂质体的混合液逐滴加入孔中,摇动培养板,轻轻混匀,在37℃、5%CO2培养箱中孵育4小时;
6)更换含有10%血清的F12k,在37℃、5%CO2培养箱中孵育24h,用800μg/mL Zeocin、8μg/mL Blasticidin筛选,48h后换液一次,筛选14天后铺96孔板挑选单克隆。
(2)单克隆挑选
单克隆生长至一定数量后,将其扩大到24孔板培养,收集上清。ELISA检测表达量:
1)山羊抗人k链抗体用包被液稀释至1μg/mL包被于96孔酶标板中,每孔加100μL,在37℃下温育2h;
2)倒去板上所有孔中的液体,用PBST洗4次,每孔加入100μL细胞表达两天的上清,在37℃下温育1h;倒去板上所有孔中的液体,用PBST洗4次;
3)每孔中加入100μL标记了HRP的山羊抗人IgG抗体,在37℃下温育1h;倒去板上所有孔中的液体,用PBST洗5次;
4)每孔中加入100μL TMB显色液,在37℃下避光温育10min;
5)每孔中加入100μL终止缓冲液,轻轻拍动ELISA板,确保每个孔混合均匀;
6)尽快在酶标仪上读取各孔OD值(测定波长:450nm);
7)挑出ELISA吸收高的单克隆,换成无血清培养基EXCELL302培养2天,上清跑SDS-PAGE,挑出高表达2F2或者2F2突变体的单克隆细胞株。
(3)2F2突变体的表达纯化
1)将挑出的高表达单克隆细胞株扩大培养,在大方瓶里长满90%后换成无血清培养基EX-CELL302,于37℃、5%CO2孵箱培养4天;
2)收集培养基1L,离心取上清,加入100mL0.5M Tris-HCl(pH7.4),用于上样;
3)proteinA层析柱用结合缓冲液(50mM Tris-HCl,pH7.4)平衡,流速1mL/min上样;
4)样品流完后用80mL结合缓冲液流洗柱子,洗脱缓冲液(1M醋酸钠,pH3.0)洗脱,紫外检测,收集第一峰,即为纯化的2F2突变体,经SDS-PAGE分析(图1),2F2突变体的分子量为150KD。
2F2抗体的获取方法与上述方法相同。2F2抗体与2F2突变体的分子筛分析结果分别如图2和图3所示。
经测序,2F2抗体与2F2突变体轻链氨基酸序列如SEQ ID No.1所示,2F2突变体的重链氨基酸序列如SEQ ID No.2所示,2F2抗体的重链氨基酸序列如SEQ ID No.3所示。
(4)2F2突变体与MMAE偶联
1)取3.5mg2F2突变体溶解于0.4mL PBS(pH7.4)中,加入10μL磷酸三(β-氯乙基)酯(TCEP),37℃水浴3h;
2)用Millipore截留分子量为10kDa的超滤管除去反应体系中的小分子;
3)加入10μL去氢抗坏血酸(DHAA),37℃水浴3h;
4)用Millipore截留分子量为10kDa的超滤管除去反应体系中的小分子;
5)边搅拌边加入用80μL30%乙腈/水溶解的10倍过量vcMMAE(由江阴康诺泰生物技术有限公司代为合成,10倍过量即反应体系中vcMMAE的摩尔量大于等于2F2突变体的10倍),4℃反应60min,加入过量的半胱氨酸终止反应;
6)用Millipore截留分子量为10kDa的超滤管除去反应体系中的小分子;所得到的偶联物过0.22μm孔径的水膜除菌,-20℃保存备用。
2F2突变体的半胱氨酸巯基与vcMMAE的马来酰亚胺基团发生烷基化反应,变成了偶联不同个数MMAE的偶联物。
由于2F2突变体和MMAE的最大吸收波长不同,可参考文献Hamblettet al.Effects of Drug Loading on the Antitumor Activity of a Monoclonal.Clinical Cancer Research,10:7063-7070(2004),对2F2突变体-vcMMAE在280、248nm处吸收峰进行检测,同时以2F2突变体为阴性参照。经过计算,平均每个2F2突变体偶联上1-2个MMAE药物。2F2突变体-vcMMAE偶联物各部分的连接关系如图4所示。
实施例2偶联物的生物活性
下面以CD20阳性细胞Ramos、Raji、Daudi和WIL2-S为对象,检测2F2突变体和2F2突变体-vcMMAE偶联物的生物学活性。
1流式亲和性测定
(1)取1×106个Ramos细胞与不同浓度(分别为10、3.33、1.11、0.37、0.12μg/mL)的2F2抗体、2F2突变体(Thio-2F2)、2F2突变体-vcMMAE(Thio-2F2-vcMMAE)在1%BSA(PBS溶解)溶液里4℃孵育30min;
(2)PBS洗涤两次后,加入FITC标记的山羊抗人IgG(H+L)多抗(1:200稀释)在4℃孵育30min;PBS洗后用流式细胞仪检测细胞的平均荧光强度(MFI)。
流式细胞仪检测2F2抗体、2F2突变体、2F2突变体-vcMMAE对CD20阳性细胞Ramos的结合,以二抗标记后FITC的平均荧光强度来显示结合力的强弱。
检测结果如图5所示,2F2突变体及2F2突变体-vcMMAE偶联物与2F2抗体有相似的结合曲线,且均呈浓度依赖性,表明2F2突变体及2F2突变体-vcMMAE偶联物保持了2F2抗体对CD20阳性细胞的高亲和性。
2补体依赖性细胞毒性(CDC)活性测定
(1)调整Ramos、Raji、Daudi和WIL2-S细胞浓度至4-6×105个/mL,0.1mL/孔(含10%小牛血清的RPMI-1640培养液);
(2)每孔中加入0.05mL不同浓度的2F2抗体、2F2突变体、2F2突变体-vcMMAE,使终浓度分别为10、3.33、1.11、0.37、0.12μg/mL,阴性对照孔加0.05mL RPMI-1640培养液,37℃、5%CO2的饱和水汽二氧化碳孵箱中培养20min;
(3)取稀释好的补体,50μL/孔加样,室温震荡混匀约1min,置37℃、5%CO2培养箱中培养120min;
(4)CCK检测活性,测定450nm OD值。
CDC实验结果如图6显示,Ramos、Raji、Daudi和WIL2-S细胞都能够在2F2抗体、2F2突变体、2F2突变体-vcMMAE介导下产生明显的CDC作用,突变和偶联并没有干扰2F2抗体的CDC功能。
3抗体依赖性细胞毒性(ADCC)活性测定
(1)外周血单个核细胞(PBMC)的分离
1)无菌采集静脉血到含有肝素的离心管中,轻轻混匀,加入等体积PBS;
2)取10mL离心管,每管加入4mL室温的淋巴细胞分离液,倾斜离心管,沿管壁缓慢加入稀释后的抗凝外周血4mL/管,不要破坏界面,室温1800r/min离心15min,管内分为四层(从上至下依次为血浆层、环状乳白色淋巴细胞和单个核细胞层、透明分离液层及红细胞层);
3)收集第二层细胞加入等体积PBS,充分混匀后1800r/min离心20min,弃上清,沉淀细胞用PBS再洗两次,用无酚红的RPMI-1640培养液调整细胞密度为5×106个/mL;
4)置于37℃、5%CO2细胞培养箱备用。
(2)ADCC活性测定
1)取对数生长期的Ramos和Raji细胞悬液,离心,弃上清,用PBS洗2次,重悬于无酚红RPMI-1640培养液,密度为1×105个/mL;
2)设定无细胞的培养基孔(背景空白对照),未经药物处理的对照细胞孔(样品对照),未经药物处理的用于后续裂解的细胞孔(样品最大酶活性对照),及药物处理的细胞孔(实验组),每种情况都设3个平行孔;
3)用无酚红RPMI-164O培养基将2F2抗体、2F2突变体、2F2突变体-vcMMAE分别稀释至浓度为20μg/mL,然后分别以3倍比稀释共获得5个浓度(每个浓度300μL),每管加入调整好细胞密度的靶细胞300μL,4℃作用30min;
4)1500r/min离心5min,PBS洗2次,悬于300μL无酚红RPMI-1640培养液中,分加到96孔板上,100μL/孔。每孔加入100μL效应细胞,37℃,5%CO2细胞培养箱5-10h后显色;
5)将96孔板1500r/min离心5min后,每孔吸取120μL上清至另一96孔板相应孔中,按照乳酸脱氢酶细胞毒性检测试剂盒中的方法加入混合好的显色液60μL/孔至96孔板中;
6)室温下避光作用30min,酶标仪读490nm的光吸收值。
ADCC实验结果如图7所示,Ramos和Raji细胞能够在2F2抗体、2F2突变体、2F2突变体-vcMMAE介导下产生明显的ADCC作用,突变和偶联并没有对2F2抗体的ADCC功能产生较大影响。
4细胞毒性检测
1)CD20阳性的Ramos、Raji、Daudi和WIL2-S细胞与CD20阴性的HepG2细胞分别铺板,0.8-1.5×104个/孔(含10%小牛血清的RPMI-1640培养液0.1mL);
2)加入不同浓度(分别为60、20、6.6、……、0.003μg/mL)的2F2抗体、2F2突变体、2F2突变体-vcMMAE偶联物(含10%小牛血清的RPMI-1640培养液稀释),0.1mL/孔,阴性对照孔加0.1mL的RPMI-1640培养液,37℃、5%CO2的饱和水汽二氧化碳孵箱中培养92h;
3)每孔加10μL CCK,37℃、5%CO2的饱和水汽二氧化碳孵箱中培养4h,测定450nm OD值。
检测结果如图8所示,2F2突变体-vcMMAE偶联物对所有细胞的杀伤力相比2F2抗体和2F2突变体都有明显提高,而且2F2突变体-vcMMAE偶联物只对CD20阳性细胞表现高毒性,对CD20阴性细胞HepG2毒性很低,表明2F2突变体-vcMMAE偶联物只特异性杀伤CD20阳性靶细胞。
5竞争性抑制
竞争性抑制试验是为了间接观察偶联物是否通过结合CD20抗原然后发挥活性。细胞在1μg/mL的2F2突变体-vcMMAE偶联物作用下,添加不同浓度(分别是60、20、……0.25μg/mL)的2F2抗体或者2F2突变体,37℃、5%CO2的饱和水汽二氧化碳孵箱中培养72h,CCK检测活性,测定450nm OD值。与
如图9所示,2F2抗体和2F2突变体能够竞争性的抑制2F2突变体-vcMMAE偶联物(1μg/mL)对CD20阳性细胞Ramos的杀伤作用。并且,随着2F2抗体和2F2突变体浓度的增加,抑制效果逐渐明显,而且2F2抗体和2F2突变体对偶联物的抑制效果很接近,间接说明了突变对2F2突变体亲和性的影响较小。2F2突变体-vcMMAE偶联物的特异性在竞争性的杀伤性实验中也得到了体现。
结果表明,未结合的2F2抗体和2F2突变体能抑制细胞对2F2突变体-vcMMAE偶联物的内吞作用而抑制偶联物的活性。
6细胞凋亡分析
分别将1×105个/mL Ramos细胞在培养基、5μg/mL的2F2抗体、2F2突变体、2F2突变体-vcMMAE偶联物作用下培养72h;细胞离心,去培养基,用PBS洗涤,取2×105个细胞,加500μL结合缓冲液,5μL AnnexinV,10μL PI,室温避光孵育5min;流式检测凋亡细胞的百分比。检测结果如图10所示。
Ramos细胞的Annexin V/PI双染结果显示,2F2抗体和2F2突变体在24h时对细胞的影响和空白不加药组几乎没有差异,而2F2突变体-vcMMAE偶联物和MMAE诱导细胞凋亡的现象非常明显。
2F2突变体-vcMMAE偶联物早凋/晚凋24.5%/52.6%,MMAE7.5%/89.9%。显示2F2突变体-vcMMAE偶联物比2F2抗体对CD20阳性细胞具有更强的杀伤能力。
推断2F2突变体-vcMMAE偶联物的作用机理为:
偶联物通过2F2突变体与肿瘤细胞表面的CD20结合,并被肿瘤细胞内吞进入溶酶体,溶酶体中的组织蛋白酶Cathepsin水解偶联物中的二肽连接臂,释放出MMAE;MMAE在肿瘤细胞中发挥作用,抑制微管蛋白二聚化,从而诱导细胞凋亡。其诱导凋亡机制如图11所示。
Claims (4)
1.一种抗CD20抗体-海兔毒素偶联物,其特征在于,由海兔毒素通过连接臂偶联抗CD20抗体构成,所述抗CD20抗体的轻链氨基酸序列如SEQ ID No.1所示,重链氨基酸序列如SEQ ID No.2所示,所述连接臂为马来酰亚胺修饰的缬氨酸-瓜氨酸二肽,每个抗CD20抗体分子偶联1~2个海兔毒素分子。
2.一种抗CD20抗体-海兔毒素偶联物的制备方法,包括:
(1)将抗CD20抗体与带连接臂的海兔毒素混合,进行偶联反应;
(2)反应完成后,分离纯化得到所述抗CD20抗体-海兔毒素偶联物;
所述抗CD20抗体的轻链氨基酸序列如SEQ ID No.1所示,重链氨基酸序列如SEQ ID No.2所示;
所述连接臂为马来酰亚胺修饰的缬氨酸-瓜氨酸二肽;
与海兔毒素偶联之前,需使抗CD20抗体的半胱氨酸巯基处于游离状态,具体方法包括:
(a)将从宿主细胞表达出的抗CD20抗体溶于pH7.4的PBS缓冲液中,加入三氯乙基磷酸酯,30~40℃水浴反应1~3h;
(b)超滤并用PBS洗涤除去反应液中分子量低于10kDa的物质;
(c)加入脱氢抗坏血酸,30~40℃水浴反应1~3h;
(d)超滤并用PBS洗涤除去反应液中分子量低于10kDa的物质,获得具有游离半胱氨酸巯基的抗CD20抗体。
3.如权利要求2所述的制备方法,其特征在于,所述偶联反应的温度为4℃以下,时间为60~120min。
4.如权利要求2所述的制备方法,其特征在于,所述分离纯化方法为:利用超滤除去反应液中分子量低于10kDa的物质,再经离心去沉淀,将所得上清过滤除菌,即为所述抗CD20抗体-海兔毒素偶联物。
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CN104341503A (zh) * | 2013-07-29 | 2015-02-11 | 西藏海思科药业集团股份有限公司 | 对蒙古人种和高加索人种低免疫原性的、抗cd20的人抗体 |
EP3165237B1 (en) * | 2015-11-03 | 2018-12-19 | Industrial Technology Research Institute | Antibody-drug conjugate (adc) and method for forming the same |
CN106237341B (zh) * | 2016-07-12 | 2019-07-30 | 浙江大学 | 一种抗体偶联药物及其制备方法和应用 |
CN106674347B (zh) * | 2016-08-16 | 2019-12-13 | 滨州医学院 | 一种新型耳抑素抗体 |
CN107915770B (zh) * | 2016-10-11 | 2020-08-25 | 联宁(苏州)生物制药有限公司 | 一种抗体药物偶联物中间体及其制备方法 |
CN107744592B (zh) * | 2017-09-15 | 2020-04-28 | 四川大学 | 抗cd56抗体与海兔毒素偶联复合物及其制备方法和用途 |
CN107875398B (zh) * | 2017-09-27 | 2021-03-23 | 浙江大学 | 一种抗体偶联药物的制备方法、抗体偶联药物及应用 |
LT3691692T (lt) * | 2017-10-14 | 2021-05-10 | Abbvie Inc. | Aktyvuojami anti-cd71 antikūno-vaisto konjugatai ir jų panaudojimo būdai |
CN108084267B (zh) * | 2017-11-24 | 2021-03-30 | 浙江大学 | 一种抗体的抗原结合片段-海兔毒素偶联物及其制备方法和应用 |
US20230293709A1 (en) * | 2020-05-03 | 2023-09-21 | Shanghai Miracogen Inc. | Antibody-drug conjugate and preparation thereof |
CN113398242B (zh) * | 2021-06-04 | 2022-04-29 | 浙江大学 | 一种抗体耦联药物及其应用 |
CN115975030B (zh) * | 2021-09-30 | 2023-09-26 | 杭州邦顺制药有限公司 | 抗cd39抗体-药物偶联物及其用途 |
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