CN103254317B - 一种抗cd20单克隆抗体-海兔毒素偶联物及其制备方法和应用 - Google Patents
一种抗cd20单克隆抗体-海兔毒素偶联物及其制备方法和应用 Download PDFInfo
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明公开了一种抗CD20单克隆抗体-海兔毒素偶联物及其制备方法和应用。所述制备方法包括:将抗CD20单克隆抗体溶于缓冲液中,加入三氯乙基磷酸酯,进行还原反应;将被还原的抗CD20单克隆抗体与带连接臂的海兔毒素混合,进行偶联反应;反应完成后,分离纯化得到所述抗CD20单克隆抗体-海兔毒素偶联物。与现有技术相比,本发明偶联物中最多偶联有8个海兔毒素分子,具有抗CD20单克隆抗体和MMAE两者的生物学功能,抗肿瘤效果得到显著增强;且具有较强的肿瘤靶向性,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。
Description
技术领域
本发明属于生物技术和药物领域,具体涉及一种抗CD20单克隆抗体-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(简称OFA)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.)。OFA单抗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,特异性地诱导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.),而对其他正常组织无不良影响。
但是同时,由于OFA单抗本身的局限性,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OFA单抗产生耐药性。因此,OFA单抗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。
申请号为201310046396.9的中国专利文献公开了一种抗CD20抗体-海兔毒素偶联物的制备方法,将OFA单抗重链恒定区第123位的丙氨酸突变为半胱氨酸,以获得OFA单抗与海兔毒素的偶联位点。
由于该制备方法需要对OFA单抗进行定点突变,操作步骤繁琐,并且突变后获得的抗CD20抗体只能偶联1~2个海兔毒素分子,对肿瘤细胞的杀伤力还有待提高。
发明内容
本发明提供了一种抗CD20单克隆抗体-海兔毒素偶联物的制备方法,利用该制备方法获得的抗CD20单克隆抗体-海兔毒素偶联物对CD20阳性细胞具有更高的杀伤力。
一种抗CD20单克隆抗体-海兔毒素偶联物的制备方法,包括:
(1)将抗CD20单克隆抗体溶于缓冲液中,加入三氯乙基磷酸酯,进行还原反应;
所述抗CD20单克隆抗体优选为Ofatumumab单抗。Ofatumumab单抗能够特异结合CD20分子,从而专一地杀伤B淋巴瘤细胞,而对其他正常组织无不良影响。
由于抗CD20单克隆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;因此在与海兔毒素偶联之前,需使抗CD20单克隆抗体形成链间二硫键的半胱氨酸巯基处于游离状态。
三氯乙基磷酸酯可使抗CD20单克隆抗体链间二硫键断开,优选地,所述缓冲液为pH7.4的PBS缓冲液;三氯乙基磷酸酯与抗CD20单克隆抗体的摩尔比为5.5~10:1;所述还原反应的条件优选为:25~37℃水浴反应1~3h。
(2)将被还原的抗CD20单克隆抗体与带连接臂的海兔毒素混合,进行偶联反应;
作为优选,所述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.Bioconjμgate Chem..13(4)855–869(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 of potentmonoclonal antibody auristatin conjμgates for cancer therapy[J].NatureBiotechnology..21(7)778-784(2003).)。
所述偶联反应的温度优选为4℃以下,时间为30~60min。
偶联时,vcMMAE通过缬氨酸上的马来酰亚胺与抗CD20单克隆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;
(3)反应完成后,分离纯化得到所述抗CD20单克隆抗体-海兔毒素偶联物;
由于本发明抗CD20单克隆抗体-海兔毒素偶联物的分子量均大于150kDa,而反应体系中存在的其他分子的分子量均小于2kDa,因此,用HiPrepTM26/10脱盐柱脱盐即可除去体系中的小分子,将所得抗体流出液过滤除菌,即为所述抗CD20单克隆抗体-海兔毒素偶联物。
本发明还提供了由所述制备方法获得的抗CD20单克隆抗体-海兔毒素偶联物。
由于抗CD20单克隆抗体的链间二硫键有四对,经还原后产生8个游离的半胱氨酸巯基,因此可偶联多达8个海兔毒素分子。一般地,每个抗CD20单克隆抗体分子偶联3~8个海兔毒素分子。偶联8个海兔毒素分子获得的偶联物诱导肿瘤细胞WIL2-S凋亡最为明显,早凋/晚凋为23.5%/38.4%。
本发明还提供了所述抗CD20单克隆抗体-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单克隆抗体-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,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抗CD20单克隆抗体-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,是将安全有效量的抗CD20单克隆抗体-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。
本发明的偶联物对CD20阳性细胞Daudi、WIL2S、Raji的IC50值约为25ng/mL、51ng/mL、28ng/mL,且对CD20阴性细胞K562的IC50值大于5000ng/mL;而Ofatumumab单抗本身对Daudi、WIL2S、Raji的IC50值均大于100000ng/mL。表明本发明的偶联物对CD20阳性细胞具有较高的亲和力和极强的杀伤力。
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单克隆抗体和MMAE两者的生物学功能,既具有抗CD20单克隆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单克隆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;
(3)本发明偶联物的载药量为每个抗CD20单克隆抗体最多可偶联8个海兔毒素分子,最大程度地提高了抗CD20单克隆抗体的抗肿瘤活性。
附图说明
图1a为OFA单抗的紫外吸收图谱;
图1b为OFA-vcMMAE的紫外吸收图谱;
图2为OFA-vcMMAE和OFA单抗对WIL2-S细胞的亲和性检测图;
图3a为OFA-vcMMAE和OFA单抗对WIL2-S细胞的CDC杀伤效果图;
图3b为OFA-vcMMAE和OFA单抗对Raji细胞的CDC杀伤效果图;
图4为OFA-vcMMAE和OFA单抗诱导WIL2-S细胞凋亡图;
图5a为OFA-vcMMAE给药组和对照组Scid鼠体内的肿瘤生长曲线;
图5b为从对照组Scid鼠中剥离的瘤体图。
具体实施方式
下面结合附图和具体实施方式对本发明作进一步详细说明。
实施例一OFA-vcMMAE偶联物的制备
OFA-vcMMAE偶联物的制备方法包括:
(1)在10mg/mL OFA单抗(溶解于PBS,pH7.4)中加入5.5-10倍摩尔量的磷酸三(β-氯乙基)酯(TCEP),37℃水浴2h,置于冰上;
(2)边搅拌边加入用30%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OFA单抗的10倍),4℃反应60min,加入过量的半胱氨酸终止反应;
(3)过HiPrepTM26/10desalting柱,用PBS洗脱,去除小分子,得到偶联物OFA-vcMMAE;所得到的偶联物过0.22μm孔径的水膜除菌,-20℃保存备用。
OFA单抗的半胱氨酸巯基与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OFA单抗和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OFA-vcMMAE在280、248nm处吸收峰进行紫外分光光度法检测,同时以OFA单抗为阴性参照。经过计算,平均每个OFA单抗偶联上7-8个MMAE药物。未反应的抗体和反应后的偶联物(TCEP还原剂下)进HPLC分析,检测波长为248nm(MMAE的最大吸收波长)和280nm(抗体的最大吸收波长)。
如图1a所示,OFA有轻链(L0)和重链(H0)两个峰(判断依据是重链在280nm峰面积高于轻链,且和报道的抗体在同样条件下出峰时间符合);如图1b所示,偶联上MMAE以后,主峰还是两个,出峰时间推迟到L1和H3(分别代表连上1个MMAE分子的轻链和连上3个MMAE分子的重链,因为MMAE偶联会使248nm/280nm的吸收比增强,偶联个数的判断方法参考上述Hamblett等人的报道)。从液相上判断偶联物中海兔毒素的个数近似为8。
实施例2偶联物的生物活性检测
下面以CD20阳性细胞Raji、Daudi和WIL2-S及CD20阴性细胞K562为对象,检测OFA和OFA-vcMMAE偶联物的生物学活性。
1流式亲和性检测
(1)取1×106个WIL2-S细胞与不同浓度(分别为10、3.33、1.11、0.37、0.12μg/mL)的OFA单抗或者OFA-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OFA或者OFA-vcMMAE与CD20阳性细胞WIL2-S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2所示,由于OFA-vcMMAE中OFA单抗被还原后再偶联海兔毒素,因此OFA单抗的结构受到影响,对CD20阳性细胞WIL2-S的亲和力有所下降,但也基本保持OFA单抗对CD20阳性细胞WIL2-S的结合能力。
2补体依赖性细胞毒性(CDC)活性测定
(1)调整Raji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OFA单抗或者OFA-vcMMAE,使终浓度分别为10、3.33、1.11、0.37、0.12μg/mL,阴性对照孔加0.05mLRPMI-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。
检测结果如图3a和图3b所示,Raji和WIL2-S都能够在OFA和OFA-vcMMAE介导下产生明显的CDC作用,OFA-vcMMAE的CDC功能略低于OFA单抗。
3细胞毒性检测
(1)CD20阳性的Raji、Daudi和WIL2-S细胞与CD20阴性的K562细胞分别铺板(含10%小牛血清的RPMI-1640培养液0.1mL);
(2)加入不同浓度10%小牛血清的RPMI-1640培养液稀释的OFA、OFA-vcMMAE或者2F2突变体-vcMMAE(申请号为201310046396.9的中国专利文献公开),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4天;
(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;并计算抗体和偶联物对各种细胞的IC50值。计算结果如表1所示。
表1抗体和偶联物对各细胞的IC50值(ng/mL)
细胞系 | OFA | OFA-vcMMAE | 2F2突变体-vcMMAE |
Daudi | >100,000 | 25±4.2 | >100 |
WIL2S | >100,000 | 51±2.3 | >100 |
Raji | >100,000 | 28±10.2 | >100 |
K562 | / | >5,000 | / |
由表1可见,与OFA单抗相比,OFA-vcMMAE对所有CD20阳性细胞的杀伤力都有明显提高,也高于2F2突变体-vcMMAE对CD20阳性细胞的杀伤力;并且OFA-vcMMAE只对CD20阳性细胞表现高毒性,对CD20阴性细胞K562毒性很低,表明OFA-vcMMAE偶联物只特异性杀伤CD20阳性靶细胞。
4细胞凋亡分析
分别取5×104个/mL WIL2-S细胞在培养基、5μg/mL OFA、5μg/mLOFA-vcMMAE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(膜联蛋白V),10μL PI(碘化丙啶),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4所示。
由图4可见,OFA单抗处理的细胞凋亡率很低,和空白不加药组几乎没有差异;而OFA-vcMMAE诱导细胞凋亡的现象非常明显,早凋/晚凋率分别为23.5%和38.4%;显示OFA-vcMMAE比OFA单抗对CD20阳性细胞具有更强的杀伤能力。
5体内抗肿瘤实验
体外培养的Daudi细胞处于对数生长期时,离心并用RPMI-1640洗涤两次,再加适量无血清的RPMI-1640细胞培养液配成浓度为5×107/mL的细胞悬浮液;
用75%的酒精消毒Scid鼠前肢右腋下皮肤,每只注射0.2mL即1×107个肿瘤细胞;待Scid鼠体内肿瘤的平均大小为260mm3以上时,将18只Scid鼠随机分成5组,除OFA-vcMMAE组6只,其他4组对照各3只;
对5组Scid鼠经尾静脉分别给予PBS、OFA(3mg/kg)、OFA-vcMMAE(3mg/kg)、MMAE(等3mg/kg OFA-vcMMAE中含的MMAE量)、Herceptin-vcMMAE(3mg/kg,作为阴性对照,偶联数约为7.3),3天一次,共3次。停药一天后处死4组对照组Scid鼠,剥离瘤体,如图5b所示;OFA-vcMMAE给药组继续养着,检测肿瘤生长情况;检测结果如图5a所示。
由图5a可见,只有OFA-vcMMAE组在第一次给药时抑制肿瘤生长,第二、三次给药后已经使肿瘤完全消退并且保持到第29天没有复发;到第40天只有一只出现复发症状,复发率仅为16.7%;
而其他对照组(PBS、OFA单抗、游离的MMAE、非CD20靶向性的Herceptin-vcMMAE)均不能抑制肿瘤的生长,在给药7天后(第八天)安乐死并解剖取肿瘤;实验过程中动物状态良好,未表现明显毒副作用。
表明OFA-vcMMAE具有高效的抗肿瘤活性,且具有良好的靶向性,能将小分子毒性药物聚集到肿瘤部位。
Claims (4)
1.一种抗CD20单克隆抗体-海兔毒素偶联物的制备方法,包括:
(1)在含有10mg/mL Ofatumumab单抗的PBS缓冲液中加入5.5-10倍摩尔量的磷酸三(β-氯乙基)酯,37℃水浴2h,置于冰上;
所述PBS缓冲液的pH值为7.4;
(2)边搅拌边加入用30%乙腈/水溶解的10倍过量带连接臂的海兔毒素,4℃反应60min,加入过量的半胱氨酸终止反应;
(3)过HiPrepTM26/10desalting柱,用PBS洗脱,去除小分子,得到所述抗CD20单克隆抗体-海兔毒素偶联物。
2.如权利要求1所述的制备方法,其特征在于,所述连接臂为马来酰亚胺修饰的缬氨酸-瓜氨酸二肽。
3.如权利要求1~2任一所述的制备方法获得的抗CD20单克隆抗体-海兔毒素偶联物,其特征在于,每个抗CD20单克隆抗体分子偶联8个海兔毒素分子。
4.如权利要求3所述的抗CD20单克隆抗体-海兔毒素偶联物在制备抗肿瘤药物中的应用;其特征在于,所述抗肿瘤药物为抗B淋巴瘤药物。
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