CN114181307A - 抗mmae单克隆抗体及其制备方法与应用 - Google Patents
抗mmae单克隆抗体及其制备方法与应用 Download PDFInfo
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- CN114181307A CN114181307A CN202111577428.9A CN202111577428A CN114181307A CN 114181307 A CN114181307 A CN 114181307A CN 202111577428 A CN202111577428 A CN 202111577428A CN 114181307 A CN114181307 A CN 114181307A
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- mmae
- monoclonal antibody
- antibody
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Abstract
本发明提供了一种抗MMAE单克隆抗体及其制备方法与应用,所述抗体包含有重链可变区的抗原互补决定区aaCDR1、aaCDR2和aaCDR3,轻链可变区的抗原互补决定区aaCDR1、aaCDR2和aaCDR3。本发明的所述抗MMAE单克隆抗体可以应用于相关的配体偶联药物中的研究,且能检测出纳克级或更低浓度MMAE。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种抗MMAE单克隆抗体及其制备方法与应用。
背景技术
Monomethyl auristatin E (MMAE;SGD-1010) 是海兔毒素 10 的合成衍生物,通过抑制微管蛋白聚合而起到有效的有丝分裂抑制作用。 MMAE 广泛用作细胞毒性成分制作抗体偶联药物 (ADCs) 用于癌症的研究。在肿瘤的治疗中,具有抗癌活性的小分子化合物,由于其对正常组织的毒性而限制了其使用,而将其与靶向配体(单克隆抗体、多肽、融合蛋白等)结合,将活性小分子带到靶区域,可以有效地控制小分子化合物对于正常细胞产生毒性的问题。因此,配体药物偶联物,如ADC(Antibody Drug Conjugate,ADC),PDC(peptidedrug conjugate)等XDC应运而生。
同时,在现有的配体偶联物中,出现不少MMAE相关偶联药物的报道,但现有小分子MMAE的检测主要通过液质连用的方法,而在组织样本中对MMAE的分析难度较大,且对于微量的MMAE提取分析对人员和仪器要求较高。
发明内容
为了解决现有技术的不足,本发明提供了一种抗MMAE单克隆抗体及其制备与应用。
本发明的目的通过以下技术方案来实现:
抗MMAE单克隆抗体,包含有重链可变区的抗原互补决定区aaCDR1、aaCDR2和aaCDR3,其氨基酸序列分别为SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3;
轻链可变区的抗原互补决定区aaCDR1、aaCDR2和aaCDR3,其氨基酸序列分别为SEQID NO:4、SEQ ID NO:5、SEQ ID NO:6。
优选地,所述抗体具有SEQ ID NO:7所示的重链区可变区氨基酸序列和SEQ IDNO:8所示的轻链区可变区氨基酸序列。
一种核酸分子,所述核酸分子包括编码以上任一所述的抗MMAE单克隆抗体。
一种细胞系,能够产生如以上任一所述的抗MMAE单克隆抗体。
抗MMAE单克隆抗体的制备方法,所述方法包括,培养以上所述的细胞系,并产生所述的抗MMAE单克隆抗体。
抗MMAE单克隆抗体的应用,所述抗MMAE单克隆抗体在相关抗肿瘤配体偶联药物中的应用,所述配体可以是抗体,多肽,融合蛋白等。
抗MMAE单克隆抗体的应用,所述抗体应用于一步竞争法检测MMAE的含量。
抗MMAE单克隆抗体的应用,所述抗体用于检测MMAE相关ADC药物PK研究中检测结合型抗体的含量。
本发明的有益效果体现在:提供了一种抗MMAE单克隆抗体,并提供了相应的制备方法,本发明的所述抗MMAE单克隆抗体可以应用于在制备治疗和预防癌症的抗体偶联药物中的研究,且能更快度方便的检测出纳克级或更低浓度MMAE。
附图说明
图1:本发明实施例5中抗体与MMAE结合在450 nm波长下的OD值。
图2:通过本发明实施例7中所述的方法检测MMAE含量的示意图。
具体实施方式
本发明揭示了一种抗MMAE单克隆抗体,包含有重链可变区的抗原互补决定区aaCDR1、aaCDR2和aaCDR3,其氨基酸序列分别为SEQ ID NO:1 DYTFNDYE;SEQ ID NO:2VHPGSGDT;SEQ ID NO:3 TRGWD。轻链可变区的抗原互补决定区aaCDR1、aaCDR2和aaCDR3,其氨基酸序列分别为SEQ ID NO:4 QDIGNS;SEQ ID NO:5 ATS;SEQ ID NO:6 LQYASSPYT。
所述抗体具有SEQ ID NO:7所示的重链区可变区氨基酸序列和SEQ ID NO:8所示的轻链区可变区氨基酸序列。
其中,重链可变区,SEQ ID NO:7
QVQLQQSGAELVRPGASVKLSHRALDYTFNDYEMHWVKQTPVHGLEWIGGVHPGSGDTSYNQKFKGKATLTADKSSNTAYLELSNLTSEDSAVYYCTRGWDWGQGTTLTVSS
轻链可变区,SEQ ID NO:8 DIQMTQSPSSLSASLGERVSLTCRASQDIGNSLNWLQQEPDGTIKRLIYATSTLDSGVPKRFSGSRSGSDYSLTISSLESEDFVAYYCLQYASSPYTFGGGTKLEVK
本发明还揭示了一种核酸分子及细胞系,以及以上所述抗MMAE单克隆抗体的制备方法。
所述核酸分子由编码以上任一所述的抗MMAE单克隆抗体获得。所述细胞系能够产生如以上任一所述的抗MMAE单克隆抗体。所述方法包括培养上述的细胞系,并产生所述的抗MMAE单克隆抗体。本发明的所述抗MMAE单克隆抗体可以应用于相关抗肿瘤配体偶联药物中的研究。
为更好的理解本发明,以下进行具体的实施举例说明。
实施例1:抗原制备
公司内部合成MMAE小分子,并标记一个半胱氨酸(MMAE-CYS)以便于偶联载体蛋白,进行免疫或检测用。载体蛋白偶联,免疫抗原制备,由于MMAE分子比较小,毒性大,不适合直接免疫,将MMAE-CYS与载体蛋白进行偶联,选择Thermo Scientific的马来酰胺活化的钥孔血蓝(KLH)蛋白试剂盒,按照试剂盒提供的流程操作,进行与载体蛋白偶联。
实施例2:动物免疫
将实施例1中制备的免疫用抗原(MMAE-CYS-KLH)用弗氏完全佐剂乳化,免疫4~6周龄雌性Balb/c小鼠(购自北京维通利华实验动物技术有限公司),腹部皮下注射,每只小鼠6点,剂量为100µg/只。每14天加强免疫一次,抗原使用弗氏非完全佐剂乳化,剂量为50µg/只。第3次加强免疫后7天,以间接ELISA(波长450nm)检测小鼠血清中抗免疫原的多抗效价,效价最高的小鼠以尾静脉注射冲击免疫,抗原用生理盐水混匀,剂量为50µg/只。
实施例3:杂交瘤细胞制备
1、融合:将实施例2中免疫达标的小鼠采血,取脾脏制备细胞悬液,与小鼠骨髓瘤细胞F0以10:1比例混合,离心1200rpm,5分钟。弃上清后,离心管放入37℃水浴中,在1分钟内缓慢加入1 mL的PEG1500(Roche公司),并混匀细胞。在温水中静置l分钟后,加入10 mL无血清的IMDM(Hyclone公司),混匀,离心1000rpm,5分钟。弃上清后,加入10 mL含10%血清(Excell公司)的IMDM培养基,将细胞轻轻地吹打起来,并加入配好的HAT完全培养基(Sigma公司)混匀,铺96孔板。将铺好细胞的培养板,放入37℃、5% CO2培养箱中培养。
2、筛选及亚克隆:利用ELISA的方法进行初步的筛选,将筛选得到的阳性抗体进行稀释直至得到单个亚克隆细胞。通过ELISA筛选,得到多个阳性克隆,挑选其中一个阳性克隆进行扩增,检测定株。以下以编号04CT2.2.3为挑选的个体举例说明。
实施例4:腹水诱生法制备单克隆抗体
1、腹水制备
对数生长期细胞用无血清培养基洗涤并悬起,计数~5x105/mL,1mL悬浮的细胞腹腔注射事先用石蜡油致敏的小鼠。小鼠腹部鼓胀起来后开始收集腹水,可多次收集。取出的腹水于4℃下离心4000rpm,10分钟。小心吸出中间的腹水收集于离心管中,4℃或-20℃保存。
2、单克隆抗体纯化
用Protein G(GE公司)亲和层析法按说明书从腹水中纯化抗体。SDS-PAGE胶鉴定纯度,Bradford法测定浓度。纯化的抗体保存于-20℃。
实施例5:单克隆抗体特性鉴定
1、亚型检测:用100 mL MPBS(pH 7.4)稀释包被羊抗鼠IgG至0.5 µg/mL,每孔加100 µL,4℃,过夜。倾空液体,用含0.05% Tween的PBS洗3次,每孔加入200 µL封闭液(含2%BSA和3%蔗糖的PBS),37℃下孵育1小时,倾空液体,用PBST清洗3次。每孔加入0.1mL杂交瘤上清,37℃下孵育1小时,倾空液体,用PBST清洗3次。用封闭液1:1000稀释HRP标记的羊抗鼠(κ,λ)抗体或1:2000稀释HRP标记的羊抗鼠(IgM、IgG1、IgG2a、IgG2b、IgG3、IgA)抗体,0.1mL每孔分别加入适当的孔中,37℃下孵育1小时,倾空液体,用PBST清洗3次。每孔加100µLTMB显色液进行显色反应,10-20分钟内测定450 nm波长下的OD值。结果显示,克隆04CT2.2.3为IgG1 κ型鼠源单克隆抗体。
2.抗体效价检测
用PBS(pH 7.2)稀释包被BSA-MMAE,1µg/mL,每孔加100 µL,4℃,过夜。倾空液体,用含0.05% Tween的PBS洗3次,每孔加入200 µL封闭液(含3% BSA的PBS),37℃下孵育1小时,倾空液体,备用。
每孔加入稀释好的抗体溶液,1:3做梯度稀释,每孔100 µL反应液,37℃下孵育1小时,倾空液体,用PBST清洗3次。用封闭液1:20000稀释HRP标记的羊抗鼠抗体,每孔分别加入100 µL,37℃下孵育1小时,倾空液体,用PBST清洗4次。每孔加100µLTMB显色液进行显色反应,10-20分钟内测定450 nm波长下的OD值。结合图1所示,显示挑选的单抗具有与MMAE较好的结合活性。
实施例6:抗体可变区序列测定
取培养的杂交瘤细胞系1x106,离心取上清,将细胞沉淀委托苏州金唯智生物科技有限公司进行抗体重链和轻链可变区序列测定,如下示出示例性的抗体重链可变区序列和抗体轻链可变区序列。
抗体CDR氨基酸序列
样品编号 | 链型 | aaCDR1 | aaCDR2 | aaCDR3 |
04CT2.2.3-VH | IGH | DYTFNDYE | VHPGSGDT | TRGWD |
04CT2.2.3-VL | IGK | QDIGNS | ATS | LQYASSPYT |
抗体可变区氨基酸序列
重链可变区,SEQ ID NO:7
QVQLQQSGAELVRPGASVKLSHRALDYTFNDYEMHWVKQTPVHGLEWIGGVHPGSGDTSYNQKFKGKATLTADKSSNTAYLELSNLTSEDSAVYYCTRGWDWGQGTTLTVSS
轻链可变区,SEQ ID NO:8 DIQMTQSPSSLSASLGERVSLTCRASQDIGNSLNWLQQEPDGTIKRLIYATSTLDSGVPKRFSGSRSGSDYSLTISSLESEDFVAYYCLQYASSPYTFGGGTKLEVK
编码抗体可变区的核酸序列
重链可变区,SEQ ID NO:9
CAGGTTCAACTGCAGCAATCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAGCTGTCCCACAGGGCTTTGGACTACACATTTAATGACTATGAAATGCACTGGGTGAAGCAGACACCTGTGCATGGCCTGGAATGGATTGGAGGTGTTCATCCAGGAAGTGGTGATACTTCCTACAATCAGAAATTCAAGGGCAAGGCCACACTGACTGCAGACAAGTCCTCCAACACAGCCTACCTGGAACTCAGCAACCTGACGTCTGAAGACTCTGCTGTCTATTACTGTACAAGAGGCTGGGACTGGGGCCAGGGCACCACTCTCACAGTCTCCTCA
轻链可变区,SEQ ID NO:10
GACATCCAGATGACCCAGTCTCCATCCTCCTTATCTGCCTCTCTGGGAGAAAGGGTCAGTCTCACTTGTCGGGCAAGTCAGGACATTGGTAATAGCTTGAACTGGCTTCAGCAGGAACCAGATGGAACTATTAAACGCCTGATCTACGCCACATCCACTTTAGATTCTGGTGTCCCCAAAAGGTTCAGTGGCAGTAGGTCTGGGTCAGATTATTCTCTCACCATCAGCAGCCTTGAGTCTGAAGATTTTGTAGCCTATTACTGTCTACAATATGCTAGTTCTCCATACACATTCGGAGGGGGGACCAAGCTGGAAGTAAAA
实施例7:抗体应用
MMAE含量检测方法
一步竞争法:
用PBS(pH 7.2)稀释包被04CT2.2.3抗体,1µg/mL,每孔加100 µL,4℃,过夜。倾空液体,用含0.05% Tween的PBS洗3次,每孔加入200 µL封闭液(含3% BSA的PBS),37℃下孵育1小时,倾空液体,备用。
每孔加入梯度稀释好的MMAE标准溶液50 µL和1:2000稀释MMAE-HRP 50ul,37℃下孵育1小时,倾空液体,用PBST清洗4次。每孔加100µLTMB显色液进行显色反应,10-20分钟内测定450 nm波长下的OD值,得到标准曲线。由图2所示,用此方法可以检测样本中纳克级甚至更低浓度的MMAE。一般ADC为混合物,其中包括有带毒素和不带毒素的抗体,通过本发明中抗MMAE单克隆抗体可以进一步分析结合了毒素的结合型抗体,即相当于有毒素的ADC分子,因此其也可以应用于检测MMAE相关ADC药物PK研究中检测结合型抗体的含量。
且以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
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Claims (8)
1.抗MMAE单克隆抗体,其特征在于:包含有重链可变区的抗原互补决定区aaCDR1、aaCDR2和aaCDR3,其氨基酸序列分别为SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3;
轻链可变区的抗原互补决定区aaCDR1、aaCDR2和aaCDR3,其氨基酸序列分别为SEQ IDNO:4、SEQ ID NO:5、SEQ ID NO:6。
2.如权利要求1所述的抗MMAE单克隆抗体,其特征在于:所述抗体具有SEQ ID NO:7所示的重链区可变区氨基酸序列和SEQ ID NO:8所示的轻链区可变区氨基酸序列。
3.一种核酸分子,其特征在于:所述核酸分子包括编码如权利要求1-2任一所述的抗MMAE单克隆抗体。
4.一种细胞系,其特征在于:能够产生如权利要求1-2任一所述的抗MMAE单克隆抗体。
5.抗MMAE单克隆抗体的制备方法,其特征在于:所述方法包括,培养如权利要求4所述的细胞系,并产生所述的抗MMAE单克隆抗体。
6.抗MMAE单克隆抗体的应用,其特征在于:所述抗MMAE单克隆抗体在相关抗肿瘤配体偶联药物中的应用。
7.抗MMAE单克隆抗体的应用,其特征在于:所述抗体应用于一步竞争法检测出纳克级或更低浓度的MMAE。
8.抗MMAE单克隆抗体的应用,其特征在于:所述抗体用于检测MMAE相关ADC药物PK研究中检测结合型抗体的含量。
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