WO2023024444A1 - 抗msln单克隆内化抗体及其制备方法和应用 - Google Patents
抗msln单克隆内化抗体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MSLN单克隆内化抗体及其制备方法和应用,所述抗体包括重链可变区和轻链可变区,所述重链可变区包含如SEQ ID NO:1所示的重链可变区CDR1;如SEQ ID NO:3所示的重链可变区CDR2;如SEQ ID NO:5所示的重链可变区CDR3;所述轻链可变区包含如SEQ ID NO:7所示的轻链可变区CDR1;如SEQ ID NO:9所示的轻链可变区CDR2;和如SEQ ID NO:11所示的轻链可变区CDR3。本发明的抗体特异性好,亲合力较高,可结合细胞表面表达,并可被细胞高效转运至胞内,携带毒素分子同步进入细胞从而将细胞杀死,该抗体可广泛应用在肿瘤免疫治疗的药物中。
Description
本发明涉及一种单克隆抗体及其制备方法和应用,尤其涉及一种抗MSLN单克隆内化抗体及其制备方法和应用。
间皮素(mesothelin,MSLN)是位于细胞表面的40KD的糖蛋白,通过糖基磷脂酰肌醇锚定于细胞膜上。间皮素基因编码一种69kDa的前体蛋白,被弗林蛋白酶(成对碱性氨基酸蛋白酶,furin)样转化酶水解为两条链,C端约40KD的膜结合蛋白即为成熟的间皮素,N端约30KD称之为巨核细胞促进因子(MPF)的片断脱落并释放出细胞外。MPF和膜锚MSLN均为N-糖基化,MPF可在体外促进巨核细胞克隆的形成,膜锚MSLN可以与MUC16(亦称CA125)互作,在细胞粘附过程中起重要作用,因此目前靶向治疗中均选择膜锚MSLN做为靶点,故目前MSLN专指MSLN的C端40KD片段,即膜锚MSLN。
在正常情况下,MSLN仅低表达于胸膜、腹膜、心包膜等间皮组织中,在其它组织中不表达,表达谱极窄。但在癌变组织中,MSLN的强表达却极其普遍,目前已在间皮瘤、非小细胞肺癌、卵巢癌、子宫内膜癌、宫颈癌、胰腺癌等多种实体瘤肿瘤组织中检测到很高的表达量,甚至食道癌、转移性三阴性乳腺癌和肾癌中也有MSLN的表达。就癌细胞系而言,已知MSLN高表达于HO-8910、HEY-T30、OVCAR3(特别是HO-8910)三种卵巢癌细胞系、转移胰腺癌细胞系AsPC1和宫颈癌细胞系Hela中,在SKOV3、3AO二种卵巢癌细胞系和肺腺癌细胞系A549极低表达,而在人肝癌细胞系Huh7不表达。
就细胞水平而言,MSLN参与了(癌)细胞增殖、凋亡、粘附等细胞生物学过程,并对化疗的效果产生不利影响。例如,MSLN表达下调的SKOV3细胞用于小鼠移植瘤构建时成瘤能力下降,同时亦可造成该细胞凋亡增加以及癌组织的转移减弱。阻断MSLN与MUC16结合则细胞粘附能力下降。下调MSLN表达后,癌组织对顺铂及紫杉醇的敏感性增加。另外观察到MSLN过表达会引起金属蛋白酶9的过表达,从而促进了肿瘤细胞的迁移和浸润。因此可以推测,MSLN通过与MUC16相互作用引起金属蛋白酶9以及其它蛋白的表达,进而引起细胞增殖、粘附、凋亡等过程的相应改变,在肿瘤腹膜转移的侵袭过程中发挥重要作用,且能够促进肿瘤细胞抗凋亡。
MSLN在正常细胞中几乎不表达(仅低表达于胸膜、腹膜、心包膜)而在多 种癌组织中强烈表达的特点,使其成为靶向治疗的良好靶点,决定了以其做为靶点的靶向治疗天然具有脱靶效应低、适应症广的潜在优势。比如MSLN在60-90%的肺癌组织中表达,其中20%表达量较高,但在正常肺组织中不表达,且MSLN高表达的肺腺癌预后较差,提示MSLN是治疗肺癌的一个潜在靶标。实验证实,以MSLN为靶点的CART经尾静脉回输荷瘤小鼠后,皮下接种高表达MSLN的肺腺癌组织造成的肿瘤体积增长明显被抑制。
鉴于以MSLN为靶点的靶向治疗展现出的适应症广、可能的脱靶效应低的特点,以MSLN为靶点的治疗方案向多个方向展开研发,包括重组单克隆单特异抗体药、双特异抗体、合成蛋白、抗体偶联药物(毒素或放射性同位素)、疫苗和过继性免疫细胞治疗等12个药物进行临床试验(抗体类药物5个)。其中由美国国立癌症研究所研发的Amatuximab单抗(MORAb-009)已完成治疗恶性间皮瘤的二期临床试验。
就目前研发中的抗体而言,虽然已知的许多间皮素单克隆抗体都是可用的,但是没有一个可以展现出针对于肿瘤细胞的补体依赖的细胞毒作用(complement dependent cytotoxicity,CDC)。抗体特异地与细胞表面的抗原结合,其Fc区则招募C1q,进而激活补体激活的经典途径,最终在表达抗原的细胞表面形成攻膜复合物,引起靶细胞裂解。可见该作用有助于癌症治疗。利用传统杂交瘤技术获得强CDC效应的靶向MSLN的抗体是一件困难的事情。比如传统方法获得的抗体组织穿透力差治疗效果不足、免疫原性强需要人源化改造、稳定性差运输和保存要求高。总之,已有的传统抗体尽管为疾病的治疗/检测起到了极其重要的作用,但目前已有的、以MSLN为靶点的抗体,其不足之处亦很明显。
目前处于临床阶段的MSLN靶点药物共12个,其中包括Amatuximab在内的抗体类药物共5个。这些抗体为人-鼠嵌合抗体或人源化抗体,均来源于小鼠,采用杂交瘤技术获得。目前靶向MSLN的疗法有细胞治疗及裸抗,临床效果有限,特别是对实体瘤来说,这两种方法几乎无效。研发人员普遍认为,抗体药物偶联物(antibody-drug conjugate,ADC)将是未来针对该靶点的癌症治疗的希望之一。另外这些抗体并非全人源抗体,与全人源抗体相比,存在着免疫原性较高、安全性较差、体内稳定性可能较低的问题。
发明内容
发明目的:本发明的目的是提供一种具有细胞内化功能、CDC作用的抗MSLN单克隆内化抗体;本发明的另一目的是提供一种抗MSLN单克隆内化抗体的制备方法;本发明的另一目的是提供一种抗MSLN单克隆内化抗体的应用。
技术方案:本发明的抗MSLN单克隆内化抗体,所述抗体包括重链可变区和轻链可变区,所述重链可变区包含如SEQ ID NO:1所示的重链可变区CDR1;如SEQ ID NO:3所示的重链可变区CDR2;和如SEQ ID NO:5所示的重链可变区CDR3。
所述SEQ ID NO:1为GYTFTSYY;所述SEQ ID NO:3为INPSGGST;所述SEQ ID NO:5为ARDRGTYYYGSGDLGY。
进一步地,所述轻链可变区包含如SEQ ID NO:7所示的轻链可变区CDR1;如SEQ ID NO:9所示的轻链可变区CDR2;和如SEQ ID NO:11所示的轻链可变区CDR3。
所述SEQ ID NO:7为QGISTW;所述SEQ ID NO:9为AAS;所述SEQ ID NO:11为QQANSFPLT。
进一步地,所述抗体是全人源抗体。
进一步地,所述抗体与癌细胞表面MSLN抗原特异性结合。
另一方面,本发明提供了一种核酸分子,所述核酸分子编码上述的单克隆内化抗体。
另一方面,本发明提供了一种表达载体,所述表达载体包括上述的核酸分子。
另一方面,本发明提供了一种宿主细胞,所述宿主细胞包括上述的表达载体。
另一方面,本发明提供了一种抗MSLN单克隆内化抗体的制备方法,所述方法包括通过培养包括上述的宿主细胞。具体的方法如下:
以商品化的重组人MSLN生物素标签蛋白和噬菌体展示抗体库为起始材料;将MSLN生物素标签蛋白与链霉亲和素包被磁珠共孵育,通过链霉亲和素与生物素之间的互作将MSLN固定在磁珠上,用抗体库与其孵育,洗去未结合/结合弱的噬菌体,将与MSLN蛋白结合的噬菌体洗脱下来;如此反复3次,每次改变MSLN生物素标签蛋白的使用量和洗涤条件,逐次淘汰掉结合弱的噬菌体并尽可能保留更多的结合强的噬菌体;用强结合的噬菌体侵染宿主菌,涂板并过夜培养,获得单克隆菌落,即将可结合MSLN的噬菌体抗体进行单克隆化;利用单克隆培养上清进行ELISA筛选,并对单克隆进行核酸序列测定;
将信号肽、抗MSLN抗体、人源IgG1 Fc的编码序列连接,同框阅读,构建哺乳动物表达载体;转染293F细胞,振荡培养5天后收获上清,利用蛋白A磁珠亲和层析从上清中纯化获得融合蛋白,并对其与MSLN结合特性进行鉴定;培养靶细胞,于4℃将抗体与其共孵育,平行做两组;一组经4℃孵育后转移至37℃继续孵育,另一组继续于4℃孵育。孵育结束后,加入荧光标记检测抗体, 4℃孵育并用流式细胞术检测荧光抗体,即获知细胞表面被MSLN捕获的抗MSLN抗体。将靶细胞铺于96孔板中,过夜培养使细胞充分贴壁。加入抗体和/或毒素试剂后进行细胞培养并观察细胞生长特性;培养结束后,检测细胞活率。
另一方面,本发明提供了一种试剂盒,所述试剂盒包括上述的单克隆内化抗体。
另一方面,本发明提供了一种药物组合物,所述药物组合物包含权利要求上述单克隆内化抗体。
上述单克隆内化抗体在制备抗体药物偶联物中的应用。
上述单克隆内化抗体在制备抗癌或癌症检测试剂、产品或药物中的应用。
有益效果:与现有技术相比,本发明具有如下显著优点:本发明的抗体特异性好,亲合力较高,可结合细胞表面表达,并可被细胞高效转运至胞内,携带毒素分子同步进入细胞从而将细胞杀死,该抗体可广泛应用在肿瘤免疫治疗的药物中。
图1为实施例2中phage粗培液与人源MSLN结合ELISA检测结果;
图2为实施例4中phage粗培液与人源MSLN结合FACS检测结果;
图3为实施例3中MSLN抗体表达载体结构示意图;
图4为实施例3中纯化抗体SDS-PAGE电泳;
图5为实施例4中纯化抗体与超表达于细胞表面的MSLN结合性能检测;
图6为实施例4中纯化抗体与Hela细胞表面的MSLN结合性能检测;
图7为实施例4中纯化抗体与Huh7、HEK293细胞结合的脱靶检测;
图8为实施例5中抗体与固相载体表面MSLN分子结合的EC50检测;
图9为实施例6中纯化抗体内化性能检测;
图10为实施例6中纯化抗体相对内化性能;
图11为实施例7中纯化抗体内化介导毒素对细胞的杀伤效能。
下面结合附图对本发明的技术方案作进一步说明。
实施例1:噬菌体展示全人源抗体文库淘选
1)宿主菌TG1活化:制备mini agar培养基平板[1×M9盐,2%葡萄糖,2mM MgSO
4,0.1mM CaCl
2,1mM维生素B1],划线法过夜培养TG1于37℃培养箱。
2)磁珠洗涤及封闭:吸取50ul磁珠(购自Invitrogen),置于磁力架上,吸附后吸去液体,1ml PBS重悬、洗涤两次,用1ml 1.5%脱脂奶粉+1.5%BSA的封 闭剂(第二轮、第三轮淘选时封闭剂浓度逐渐增加)封闭1小时,去除液体。
3)抗原结合:按16ug/ml的浓度将人源MSLN蛋白(购自Acro Biosystem,以后各轮淘选时抗原浓度逐渐降低)用PBS中(pH7.2-7.4)稀释至1ml,重悬磁珠,旋转孵育1小时。
4)库封闭:与抗原结合至磁珠同步,取10
11pfu噬菌体病毒颗粒(来自于原始抗体库或淘选扩增产物),用1ml 1.0%脱脂奶粉+1.0%BSA的封闭剂旋转孵育1小时。
5)噬菌体结合:将磁珠置于磁力架上,去除液体。将封闭后的库加入磁珠,重悬并旋转孵育1小时,去除液体。
6)洗涤:用1ml PBST[0.01M PBS(pH7.4),0.1%Tween-20(第二、三轮Tween-20浓度分别用0.2%、0.3%)]洗涤,再用0.01M PBS(pH7.4)洗涤。
7)洗脱:吸去液体,用300ul 0.2M甘氨酸-盐酸(pH2.2)洗脱10分钟,加入20ul中和液[1M Tris-Cl(pH9.0)]混匀,暂时保存于4℃。
8)测滴度:取2ul、0.2ul(原液用2×YT培养基稀释10倍后取2ul)、0.02ul(原液用2×YT培养基稀释100倍后取2ul)洗脱液,与0.2ml对数中期(OD600=0.5)的TG1混匀,室温孵育30分钟,均匀涂于2×YT-GA100[含2%葡萄糖,100ug/ml氨苄青霉素]平板上,37℃过夜培养,计数约50个克隆的平板上的克隆数,根据稀释倍数计算滴度。
9)噬菌体扩增:在淘选进行的同时,挑取mini agar平板上的TG1单克隆,接种于10ml 2×YT培养液中,37℃下,250rpm振荡培养至对数中期(OD600=0.5)。加入200ul淘选获得的洗脱产物,37℃孵育30分钟。加入辅助噬菌体M13KO7,37℃再孵育30分钟,37℃下,250rpm振荡培养1小时。离心去上清,用20ml含工作浓度100ug/ml氨苄青霉素和工作浓度50ug/ml卡那霉素的2×YT重悬,30℃,220rpm振荡培养过夜。
10)噬菌体沉淀:10000rpm离心15分钟去除菌体,上清中加入1/5体积的2.5M NaCl/20%PEG8000,冰浴2小时。10000rpm离心10分钟得到噬菌体沉淀,将残液去除干净,加入0.2ml 0.01M PBS(pH7.4)液重悬沉淀,如上文测滴度。
11)重复步骤2)-10)两或三次,获得结合力强的噬菌体展示抗体。
实施例2:单克隆ELISA
1)0.3ug/ml链霉亲和素4℃过夜包被酶标板,2%BSA/PBS封闭液处理2h,并用PBS洗涤3次。
2)从2×YT-GA100平板上挑取单克隆菌落,振荡培养至对数中期,加入辅 助噬菌体M13KO7,37℃孵育30分钟。37℃,220rpm振荡培养1小时,4000rpm离心15分钟。用400ul含工作浓度100ug/ml氨苄青霉素和工作浓度50ug/ml卡那霉素的2×YT重悬,30℃,220rpm振荡培养过夜。4000rpm离心15分钟,沉淀菌体。
3)取50ul 4%BSA/PBS加入到酶标板中,同时取50ul噬菌体上清,混匀,孵育1小时。
4)去除液体,0.1%PBST洗5次,PBS洗3次,去除液体。
5)将HRP标记抗M13噬菌体抗体(购自北京义翘神州)用2%BSA稀释3000倍,取100ul加入到酶标板中,孵育1小时,去除液体,0.1%PBST洗3次,拍干残液。
6)加入100ul TMB显色液,37℃孵育10min或至蓝色充分显现,加入100ul 1M硫酸终止反应,在酶标仪上读取OD450。数据见图1和表1,如图所示,在噬菌体淘选过程中,用含有phage病毒颗粒的2×YT培养基50ul做检测。其原始数据如表1所示,其中数值为450nm处的吸光值及其比值。
表1 phage粗培液与人源MSLN结合ELISA检测
7)挑选阳性单克隆测序,对单克隆内化抗体No.ID 1的V区基因进行核酸序列测定。
单克隆内化抗体No.ID 1的重链和轻链的序列如下:
抗体重链可变区CDR1氨基酸序列SEQ ID NO:1为GYTFTSYY;
抗体重链可变区CDR1核苷酸序列SEQ ID NO:2为GGA TAC ACC TTC ACC AGC TAC TAT;
抗体重链可变区CDR2氨基酸序列SEQ ID NO:3为INPSGGST;
抗体重链可变区CDR2核苷酸序列SEQ ID NO:4为ATC AAC CCT AGT GGT GGT AGC ACA;
抗体重链可变区CDR3氨基酸序列SEQ ID NO:5为ARDRGTYYYGSGDLGY;
抗体重链可变区CDR3核苷酸序列SEQ ID NO:6为GCG AGA GAT CGG GGA ACG TAT TAC TAT GGT TCG GGG GAC TTG GGC TAC;
抗体轻链可变区CDR1氨基酸序列SEQ ID NO:7为QGISTW;
抗体轻链可变区CDR1核苷酸序列SEQ ID NO:8为CAG GGT ATT AGC ACC TGG;
抗体轻链可变区CDR2氨基酸序列SEQ ID NO:9为AAS;
抗体轻链可变区CDR2核苷酸序列SEQ ID NO:10为GCT GCA TCC;
抗体轻链可变区CDR3氨基酸序列SEQ ID NO:11为QQANSFPLT;
抗体轻链可变区CDR3核苷酸序列SEQ ID NO:12为CAA CAG GCC AAC AGT TTC CCG CTC ACC。
实施例3:人源MSLN单抗制备
1)从2×YT-GA100平板上挑取的单克隆菌落,液体振荡培养过夜,按质粒提取法提取噬菌粒(phagemid)。
2)合成引物,PCR扩增噬菌体所展示的抗体基因编码区。
3)将以上核酸片段按序插入到真核表达载体Abexp-uIgG1的MCS区(图3),以编码N端为信号肽、中段为抗体、C端为Fc标签的融合蛋白。
4)振荡培养15ml菌液,使用质粒提取试剂盒(购自康为世纪)制备无菌无内毒素的质粒。
5)转染复合物制备:取23ug,用0.75mL的稀释液(如OPM-293 CD05培养基)稀释,同时取70μL的转染试剂(如PEI溶液)加入到0.75mL的稀释液中(如OPM-293 CD05培养基)轻柔混匀。将PEI稀释液加入质粒稀释液中,立即用枪轻柔混匀,室温静置15min,避免扰动。
6)加入到25ml 293F细胞及其培养液中,80rpm,37℃,5%CO
2条件下培养24小时,再加入25mL新鲜生长培养基(如OPM-293 CD05),80rpm,37℃,5%CO
2继续培养72小时。
7)10000rpm离心10分钟,取上清。与平衡后的proteinA亲和磁珠旋转孵育1小时,置于磁力架上,去除上清。
8)用30ml PBS洗杂3次,加入5ml 0.1M甘氨酸(pH 3.0)洗脱10分钟,置于磁力架上,吸取上清立即用1M Tris-HCl缓冲液(pH 8.5)至中性,即获得纯化的抗体。
9)SDS-PAGE检测纯化抗体,同时进行浓度测定,如图4所示,采用还原电泳,抗体分子中的二硫键被打开,分子以伸展的单肽链状态进行电泳迁移。
实施例4:phage或抗体细胞结合能力的流式检测(FACS)
1)将MSLN表达(细胞结合检测,如Hela、超表达MSLN的CHOK1)或不表达(脱靶检测,如超表达Huh7、HEK293)用0.25%的胰酶充分消化,血清终止消化,离心收集细胞,PBS轻轻吹打制备单细胞悬液。
2)10ml PBS洗涤细胞1次,1000rpm离心5min,再用1ml PBS悬浮细胞, 细胞计数。
3)取2.5×10
5个细胞于96孔细胞培养板中,离心收集细胞。
4)加入100μl实施例2步骤2)中的phage上清或100ul 10ug/ml实施例3步骤8)中的纯化抗体,混匀,室温孵育30分钟至1小时。
5)离心收集细胞,用300ul PBS洗涤细胞1次。
6)若为phage检测,加入100ul用PBS稀释1000倍的抗M13噬菌体抗体(购自北京义翘神州),孵育30min,PBS洗一次,加入100ul用PBS稀释100倍的荧光标记(如FITC、APC)抗体,室温下避光反应20min,流式细胞仪检测,结果数据如表2和图2所述;
表2:phage粗培液与人源MSLN结合FACS检测结果
如图2和表2所示,图中左侧、右侧柱形图分别代表与野生型CHO-K1、MSLN超表达CHO-K1细胞株结合信号,表中数值为荧光强度中值(MFI)及其比值。
若为纯化抗体检测,加入100μl用PBS稀释200倍的荧光标记(如FITC、APC)抗体,室温下避光反应20min,流式细胞仪检测,数据如表3和图5所示。
表3:纯化抗体与超表达于细胞表面的MSLN结合性能检测
如图5和表3所示,流式细胞术运用于抗体与超表达MSLN的CHO-K1结合检测,其中Amatuximab为阳性对照,hIgG1为阴性对照,每个抗体均用野生型细胞(灰色曲线)和MSLN超表达细胞(黑色曲线)检测,表中数值为荧光强度中值(MFI)。
表4:纯化抗体与Hela细胞表面MSLN的结合性能检测
如图6和表4所示,流式细胞术运用于纯化与癌细胞表面的MSLN结合检测,其中Amatuximab为阳性对照,hIgG1为阴性对照,三种抗体分别与Hela细胞结合。表中数值为荧光强度中值(MFI),或抗体的MFI与hIgG1的MFI值的 比值。
表5:纯化抗体与Huh7(左)、HEK293(右)细胞结合脱靶检测
如图7和表5所示,流式细胞术运用于脱靶检测,其中hIgG1为阴性对照CK,待测抗体和hIgG1均重复检测2次。表中数值为荧光强度中值(MFI),或抗体的MFI与hIgG1的MFI值的比值。
实施例5:纯化抗体ELISA水平(酶联免疫吸附法)结合检测
1)将抗原用包被缓冲液稀释,4℃包被过夜。
2)次日去掉包被液,PBS洗涤。
3)加入封闭液于37℃下封闭1h,PBS洗涤。同时将抗体从10ug/ml开始倍比稀释,获得16个梯度浓度的抗体溶液。
4)将其加入到封闭后的酶标板中,30℃下结合30min,PBST洗涤。
5)按1:10000稀释倍数,加入HRP标记抗人IgG的二抗(购自Biolegend),30℃下结合30min,PBST洗涤。
6)加入100ul TMB显色液,37℃孵育10min或至蓝色充分显现,加入100ul 1M硫酸终止反应,在酶标仪上读取OD450。数据如图8和表6所示,抗体按2倍梯度稀释。hIgG1做为阴性对照设置为平行检测,因其不结合因而无线性关系所以无EC50值,表中数值为450nm处的吸光值。
表6:抗体与固相载体表面MSLN分子结合的EC50检测
实施例6:抗体内化功能检测
1)0.25%Typsin-EDTA消化OVCAR3细胞,离心弃上清,用完全培养基重悬。
2)将75ul细胞悬液加至96孔板中,并使得每孔有1.5×10
5个细胞。
3)按EC80的工作浓度(即饱和浓度的80%)加入25ul抗体,混匀。每个抗体分两板,每板各做一孔。4℃孵育1小时。
4)加入PBS至终体积250ul,500g常温离心5min,甩去上清保留细胞沉淀,用吸水纸吸净残液,轻拍实验板使细胞分散,PBS再洗一次,分散细胞。此步去掉多余未结合的抗体。
5)用100ul基础培养基重悬细胞,其中一板于37℃孵育2小时,另一板于4℃孵育2小时。37℃孵育过程中抗体会转入细胞,而4℃则抑制了内化。
6)加入100ul用PBS稀释的荧光二抗(加入前二抗浓度为工作浓度的2倍),4℃孵育0.5小时。
7)500g常温离心5min,甩去上清保留细胞沉淀,用吸水纸吸净残液,轻拍实验板使细胞分散,PBS再洗一次,分散细胞。
8)加入100ul PBS重悬,流式细胞仪上机检测,Graphpad Prism处理数据。数据如图9、表7、图10所示。
表7:纯化抗体内化性能检测
如图9和表7所示,抗体与细胞结合后,通过荧光二抗检测结合于细胞表面的待测抗体,表中数值为荧光强度中值(MFI)。
如图10和表7所示,将检测样品即hIgG1、Amatuximab及seq No.ID1于37℃测得的MFI平均值除以各自4℃测得的MFI平均值定义为相对内化,该值越高表明内化能力越弱。表中数值为荧光强度中值(MFI)。
实施例7:抗体内化介导毒素对细胞的杀伤效能检测
1)0.25%Typsin-EDTA消化786-0细胞,离心弃上清,用完全培养基重悬。
2)将90ul细胞悬液加至96孔板中,并使得每孔有2500个细胞。
3)将抗体稀释至10倍的EC80值,向其中加入浓度为45nM的FabFc-ZAP human试剂(其工作浓度为4.5nM;购自Advanced Targeting Systems),取该混合液10ul,加入到步骤2)中的细胞中,混匀。
4)37℃,5%CO
2培养箱培养72小时。
5)将CCK8试剂盒室温平衡15min。取出96孔细胞培养板,加入CCK8试剂,于37℃培养箱避光孵育1至3小时,酶标仪读取OD450值。数据如图11和表8所示。
表8:纯化抗体内化介导毒素对细胞的杀伤效能
如图11和表8所示,抗体、毒素和细胞共孵育后,CCK8法测得的细胞活性低于仅抗体和细胞共孵育,从图中亦可以看出,毒素本身的杀伤并不明显,ADC的作用明显。表中数值为450nm的吸光值(A450)。将抗体Amatuximab或抗体No.ID 1与毒素同时处理细胞后测得的450nm吸光值除以仅用抗体Amatuximab或抗体No.ID 1处理细胞后测得的450nm吸光值定义为数值A,将毒素处理细胞后测得的450nm吸光值除以未处理的细胞测得的450nm吸光值定义为数值B,则A/B值即为相对杀伤。
Claims (10)
- 一种抗MSLN单克隆内化抗体,所述抗体包括重链可变区和轻链可变区,其特征在于,所述重链可变区包含如SEQ ID NO:1所示的重链可变区CDR1;如SEQ ID NO:3所示的重链可变区CDR2;和如SEQ ID NO:5所示的重链可变区CDR3。
- 根据权利要求1所述的单克隆内化抗体,其特征在于,所述轻链可变区包含如SEQ ID NO:7所示的轻链可变区CDR1;如SEQ ID NO:9所示的轻链可变区CDR2;和如SEQ ID NO:11所示的轻链可变区CDR3。
- 根据权利要求1所述的单克隆内化抗体,其特征在于,所述抗体与癌细胞表面MSLN抗原特异性结合。
- 一种核酸分子,其特征在于,所述核酸分子编码权利要求1-3任一所述的单克隆内化抗体。
- 一种表达载体,其特征在于,所述表达载体包括权利要求4所述的核酸分子。
- 一种宿主细胞,其特征在于,所述宿主细胞包括权利要求5所述的表达载体。
- 一种抗MSLN单克隆内化抗体的制备方法,其特征在于,所述方法包括通过培养包括权利要求6所述的宿主细胞。
- 一种试剂盒,其特征在于,所述试剂盒包括权利要求1-3任一所述的单克隆内化抗体。
- 一种药物组合物,其特征在于,所述药物组合物包含权利要求1-3任一所述的单克隆内化抗体。
- 权利要求1-3任一项所述的单克隆内化抗体制备抗癌或癌症检测试剂、产品或药物中的应用。
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