CN105296433B - 一种ctla4抗体、其药物组合物及其用途 - Google Patents
一种ctla4抗体、其药物组合物及其用途 Download PDFInfo
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- CN105296433B CN105296433B CN201410377352.9A CN201410377352A CN105296433B CN 105296433 B CN105296433 B CN 105296433B CN 201410377352 A CN201410377352 A CN 201410377352A CN 105296433 B CN105296433 B CN 105296433B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
- G01N2333/70521—CD28, CD152
Abstract
本发明属于肿瘤治疗和分子免疫学领域,涉及一种CTLA4抗体、其药物组合物及其用途。具体地,本发明涉及一种CTLA4的单克隆抗体。本发明的单克隆抗体能够很好地特异性与CTLA4结合,并且能够十分有效地阻断CLTA4与B7的结合,特异地解除CTLA4对机体免疫抑制,激活T淋巴细胞。
Description
技术领域
本发明属于肿瘤治疗和分子免疫学领域,涉及一种CTLA4抗体、其药物组合物及其用途。具体地,本发明涉及一种CTLA4的单克隆抗体。
背景技术
细胞毒性T淋巴细胞相关抗原-4(cytotoxic T lymphocyte sociated antigen4,亦简称为CTLA4)与CD28分子在基因结构、染色体定位、序列的同源性及基因表达具有十分相近的关系,都是共刺激分子B7的受体,主要表达于被激活T细胞表面。但是作为淋巴细胞激活的共刺激信号,CTLA4与CD28分子的功能是相反的,CTLA4与B7结合后能抑制小鼠和人T细胞的激活,在T细胞活化中起负调节作用。
CTLA4mAb或CTLA4配体可以阻止CTLA4与其天然配体结合,从而封闭CTLA4对T细胞负性调节信号的传导,增强T细胞对各种抗原的反应性,在这方面体内与体外研究结果基本一致。目前已有CTLA4mAb处于临床试验阶段用于治疗前列腺癌,膀胱癌,结肠直肠癌,胃肠道癌,肝癌,恶性黑色素瘤等(Grosso JF.,Jure-Kunkel MN.,CTLA-4blockade in tumormodels:an overview of preclinical and translational research.CancerImmun.2013;13:5.Epub 2013Jan 22)。
白细胞介素2(IL-2)由T细胞产生,是调节T细胞亚群的生长因子,也是调控免疫应答的重要因子,并可促进活化B细胞增殖,参与抗体反应、造血和肿瘤监视。重组的人IL-2已经被美国FDA批准用于治疗恶性肿瘤(包括黑色素瘤、肾肿瘤等),同时正在进行治疗慢性病毒感染的临床研究(Chavez,A.R.,et al.,Pharmacologic administration ofinterleukin-2.Ann N Y Acad Sci,2009.1182:p.14-27)。体外实验中, CTLA4mAb可特异地解除CTLA4对机体免疫抑制,激活T细胞,诱导IL-2产生,在抗肿瘤及寄生虫等疾病的基因治疗中有广泛应用前景。
CTLA4及CTLA4mAb作为T细胞功能状况的重要影响因素,通过干预机体免疫微环境,可对疾病产生特异性治疗作用,并发挥较高疗效,补充传统用药的不足,由此开辟基因治疗的新途径。CTLA4及CTLA4mAb应用于试验及临床各阶段:如在自身免疫性疾病中有效抑制哮喘动物模型的气道高反应性、阻止风湿性疾病的发展以及在同种异体移植中介导机体免疫耐受等。但同时,尽管生物基因治疗在短期临床试验研究中未发现不良反应,我们亦应注意到其长期应用所存在的潜在影响,如CTLA4mAb过度阻断CTLA4-B7信号则可导致自身免疫性疾病的发生。由于抗体可以特异结合其配体并导致靶细胞溶解或阻断病理进程,所以抗体尤其人源性抗体药物的开发利用对人类恶性肿瘤及其他免疫性疾病临床治疗有重要意义。
目前,尚需要开发新的阻断CTLA4与B7的结合的抗体及其人源化抗体。
发明内容
本发明人经过深入的研究和创造性的劳动,利用哺乳动物细胞表达系统表达出重组的CTLA4作为抗原免疫小鼠,经小鼠脾脏细胞与骨髓瘤细胞融合获得杂交瘤细胞。发明人通过进行对大量样本的筛选,得到了一种杂交瘤细胞株能够分泌产生与CTLA4特异性结合的特异性单克隆抗体,并且该单克隆抗体能够十分有效地阻断CTLA4与B7的结合。并进一步制得了人源化抗体。由此提供了下述发明:
本发明的一个方面涉及一种杂交瘤细胞株LT001(CTLA4-8D2),其于2014年6月18日保藏于中国典型培养物保藏中心(CCTCC),保藏编号为CCTCC NO:C2014113,保藏地址为中国.武汉.武汉大学,邮编:430072。
本发明的另一方面涉及本发明的杂交瘤细胞株产生的单克隆抗体。
本发明的再一方面涉及选自如下的单克隆抗体:
(1)重链可变区的氨基酸序如SEQ ID NO:6所示,轻链可变区的氨基酸序列如SEQID NO:8所示;
(2)重链可变区的氨基酸序如SEQ ID NO:10所示,轻链可变区的氨基酸序列如SEQID NO:12所示;
(3)重链可变区的氨基酸序如SEQ ID NO:14所示,轻链可变区的氨基酸序列如SEQID NO:16所示;和
(4)重链可变区的氨基酸序如SEQ ID NO:18所示,轻链可变区的氨基酸序列如SEQID NO:20所示。
根据本发明任一项所述的单克隆抗体,其中,其重链恒定区为小鼠抗体的重链恒定区或人抗体的重链恒定区,其轻链恒定区为小鼠抗体的轻链恒定区或人抗体的轻链恒定区。
本发明的再一方面涉及一种分离的核酸序列或者核酸序列组合,其编码本发明任一项所述的单克隆抗体的重链可变区和轻链可变区;具体地,所述核酸序列或核酸序列组合选自如下的(1)至(4):
(1)SEQ ID NO:5(编码重链可变区),SEQ ID NO:7(编码轻链可变区);
(2)SEQ ID NO:9(编码重链可变区),SEQ ID NO:11(编码轻链可变区);
(3)SEQ ID NO:13(编码重链可变区),SEQ ID NO:15(编码轻链可变区);和
(4)SEQ ID NO:17(编码重链可变区),SEQ ID NO:19(编码轻链可变区)。
本发明的再一方面涉及一种单克隆抗体偶联物,包括单克隆抗体和偶联部分,其中,所述单克隆抗体为本发明中任一项所述的单克隆抗体,所述偶联部分为选自放射性核素、药物、毒素、细胞因子、酶、荧光素、 和生物素中的一种或多种;具体地,所述偶联部分为蓖麻毒素;更具体地,所述偶联部分为蓖麻毒素A链。
本发明的再一方面涉及一种药物组合物,其含有本发明中任一项所述的单克隆抗体或者本发明的单克隆抗体偶联物;可选地,其还包括药学上可接受的载体或辅料。
本发明的再一方面涉及本发明中任一项所述的单克隆抗体或者本发明的单克隆抗体偶联物在制备预防和/治疗和/或辅助治疗肿瘤的药物中的用途;具体地,所述肿瘤选自黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠直肠癌、胃肠道癌和肝癌。
本发明的再一方面涉及本发明中任一项所述的单克隆抗体或者本发明的单克隆抗体偶联物在制备如下药物中的用途:
阻断CTLA4与B7结合的药物、调节(例如下调)CTLA4活性或CTLA4水平的药物、解除CTLA4对机体免疫抑制的药物、激活T淋巴细胞的药物或者提高T淋巴细胞中IL-2表达的药物。
本发明的再一方面涉及一种预防和/治疗和/或辅助治疗肿瘤的方法,包括使用有效量的本发明中任一项所述的单克隆抗体或者本发明的单克隆抗体偶联物的步骤;具体地,所述肿瘤选自黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠直肠癌、胃肠道癌和肝癌。
本发明的再一方面涉及一种在体内或体外方法,包括使用有效量的本发明中任一项所述的单克隆抗体或者本发明任一项所述的单克隆抗体偶联物的步骤,所述方法选自如下:
阻断CTLA4与B7结合的方法、调节(下调)CTLA4活性或CTLA4水平的方法物、解除CTLA4对机体免疫抑制的方法、激活T淋巴细胞的方法或者提高T淋巴细胞中IL-2表达的方法。
在本发明的一个实施方案中,所述方法是非治疗目的的。
在本发明中,术语EC50是指半最大效应浓度(concentration for 50%of maximaleffect),是指能引起50%最大效应的浓度。
本发明中,如果没有特别说明,所述B7为B7-1和/或B7-2;其具体蛋白序列为现有技术中已知序列,可以参考现有文献或者GenBank中公开的序列。例如,B7-1(CD80,NCBIGene ID:941);B7-2(CD86,NCBI Gene ID:942)。
发明的有益效果
本发明的单克隆抗体8D2能够很好地特异性与CTLA4结合,并且能够十分有效地阻断CLTA4与B7的结合,特异地解除CTLA4对机体免疫抑制,激活T淋巴细胞。
附图说明
图1:CTLA4ECD-mFc融合蛋白的SDS-PAGE检测结果。从左至右的4个泳道的样品及其上样量依次为:M:marker 10μL;CTLA4ECD-mFc融合蛋白1μg;CTLA4ECD-mFc融合蛋白2μg;CTLA4ECD-mFc融合蛋白3μg。
图2:8D2抗体的SDS-PAGE检测结果。从左至右的4个泳道的样品及其上样量依次为:M:marker 10μL;还原型蛋白电泳上样缓冲液样品抗体0.3μg;非还原型蛋白电泳上样缓冲液2μL;非还原型蛋白电泳上样缓冲液样品抗体0.3μg。
图3:8D2重组抗体(8D2(Re))的SDS-PAGE检测结果。从左至右的4个泳道的样品及其上样量依次为:M:marker 10μL;还原型蛋白电泳上样缓冲液样品抗体1μg;非还原型蛋白电泳上样缓冲液2μL;非还原型蛋白电泳上样缓冲液样品抗体1μg。
图4:8D2的人源化抗体8D2H1L1的SDS-PAGE检测结果。从左至右的4个泳道的样品及其上样量依次为:M:marker 10μL;还原型蛋白电泳上样缓冲液样品抗体1μg;非还原型蛋白电泳上样缓冲液2μL;非还原型蛋白电泳上样缓冲液样品抗体1μg。
图5:8D2的人源化抗体8D2H2L2的SDS-PAGE检测结果。从左至右的4个泳道的样品及其上样量依次为:M:marker 10μL;还原型蛋白电泳上样缓冲液样品抗体1μg;非还原型蛋白电泳上样缓冲液2μL;非还原型蛋白电泳上样缓冲液样品抗体1μg。
图6:8D2的人源化抗体8D2H3L3的SDS-PAGE检测结果。从左至右的4个泳道的样品及其上样量依次为:M:marker 10μL;还原型蛋白电泳上样缓冲液样品抗体1μg;非还原型蛋白电泳上样缓冲液2μL;非还原型蛋白电泳上样缓冲液样品抗体1μg。
图7:单抗8D2的动力学特征参数检测结果。
图8:8D2H1L1的动力学特征参数检测结果。
图9:8D2H2L2的动力学特征参数检测结果。
图10:8D2H3L3的动力学特征参数检测结果。
图11:流式细胞仪检测293F细胞无标记,同型对照,阳性对照的CTLA4表达直方图(细胞数-荧光(FITC))。
图12:流式细胞仪检测293F细胞无标记,同型对照,阳性对照的CTLA4表达的平均荧光强度(MFI)。
图13:单抗8D2与标记的表达CTLA4抗原的宿主细胞293F的结合的EC50结果。
图14:8D2(Re)抗体与标记的表达CTLA4抗原的宿主细胞293F的结合的EC50结果。
图15:8D2H1L1与标记的表达CTLA4抗原的宿主细胞293F的结合的EC50结果。
图16:8D2H2L2与标记的表达CTLA4抗原的宿主细胞293F的结合的EC50结果。
图17:8D2H3L3与标记的表达CTLA4抗原的宿主细胞293F的结合的EC50结果。
图18:采用间接ELISA方法检测8D2、8D2H1L1、8D2重组抗体与CTLA4的结合。
图19:采用间接ELISA方法检测8D2H2L2、8D2H3L3抗体与人CTLA4结合。
图20:采用间接ELISA方法检测8D2H2L2、8D2H3L3抗体与猴CTLA4结合。
图21:8D2、8D2H1L1、8D2重组抗体与B7-1竞争ELISA结果。
图22:8D2、8D2H1L1、8D2重组抗体与B7-2竞争ELISA结果。
图23:8D2H2L2、8D2H3L3抗体与B7-1竞争ELISA结果。
图24:8D2H2L2、8D2H3L3抗体与B7-2竞争ELISA结果。
图25:分别为将外周血单核细胞(PBMC),Raji细胞和人源化抗体8D2H1L1、8D2H2L2、8D2H3L3共培养72小时后采用ELISA方法检测对共孵育培养的细胞的IL-2分泌水平的影响。结果显示单抗8D2的人源化抗体通过阻止CTLA4的受体提高T淋巴细胞的IL-2的分泌。
关于生物材料保藏的说明:
一种杂交瘤细胞株LT001(CTLA4-8D2),其于2014年6月18日保藏于中国典型培养物保藏中心(CCTCC),保藏编号为CCTCC NO:C2014113,保藏地址为中国.武汉.武汉大学,邮编:430072。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解,下面的实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著,黄培堂等译的《分子克隆实验指南》,第三版,科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市场购买获得的常规产品。
在本发明的下述实施例中,使用的BALB/C小鼠购自广东省医学实验动物中心。
在本发明的下述实施例中,使用的T细胞来自中山康方生物医药有限公司。
实施例1:CTLA4-8D2杂交瘤细胞株LT001的获得以及单克隆抗体8D2的制备
利用哺乳动物细胞表达系统表达出重组的CTLA4作为抗原免疫小鼠,经小鼠脾脏细胞与骨髓瘤细胞融合获得杂交瘤细胞。通过大量的样本筛选,得到了一种杂交瘤细胞株(CTLA4-8D2杂交瘤细胞株LT001),该细胞株能够分泌产生与CTLA4特异性结合的单克隆抗体8D2。具体方法如下:
1.基因CTLA4ECD-mFc的合成:
对基因CTLA4的胞外片段CTLA4ECD(Cytotoxic T-Lymphocyte Antigen 4,NCBIGene ID:1493,SEQ ID NO:1)所对应的氨基酸(SEQ ID NO:2)与小鼠IgG的Fc蛋白片段(mFc)进行融合设计(SEQ ID NO:3),其中,mFc是指鼠IgG的Fc蛋白片段,其氨基酸序列如SEQ ID NO:3的下划线部分所示。
为提高目的基因在293f细胞表达系统中的表达效率,委托金斯瑞公司对SEQ IDNO:3蛋白序列相对应的核酸序列进行优化,优化主要考虑密码子的偏好性、GC含量、mRNA的二级结构、重复序列等因素。最终CTLA4ECD-mFc融合蛋白基因优化后序列如下(SEQ ID NO:4),并委托金斯瑞公司合成。
基因CTLA4ECD的序列:(375bp)
GCAATGCACGTGGCCCAGCCTGCTGTGGTACTGGCCAGCAGCCGAGGCATCGCCAGCTTTGTGTGTGAGTATGCATCTCCAGGCAAAGCCACTGAGGTCCGGGTGACAGTGCTTCGGCAGGCTGACAGCCAGGTGACTGAAGTCTGTGCGGCAACCTACATGATGGGGAATGAGTTGACCTTCCTAGATGATTCCATCTGCACGGGCACCTCCAGTGGAAATCAAGTGAACCTCACTATCCAAGGACTGAGGGCCATGGACACGGGACTCTACATCTGCAAGGTGGAGCTCATGTACCCACCGCCATACTACCTGGGCATAGGCAACGGAACCCAGATTTATGTAATTGATCCAGAACC GTGCCCAGATTCTGAC(SEQ IDNO:1)
CTLA4ECD编码的蛋白序列:(125aa)
AMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSD(SEQ ID NO:2)
CTLA4ECD-mFc融合蛋白序列:(364aa)
其中,带波浪下划线的为CTLA4ECD部分,带实线下划线的为mFc部分。
AMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVN LTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDENLYFQGPRGPTIKPCPPCKCPAPNLLGG PSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWM SGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNY KNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK(SEQ ID NO:3)
CTLA4ECD-mFc融合蛋白相对应的基因编码序列:(1092bp)
其中,带波浪下划线的为CTLA4ECD部分,带实线下划线的为mFc部分。
GCAATGCATGTCGCACAGCCTGCAGTGGTCCTGGCAAGCTCCAGGGGAATCGCTAGCTTCGTGTGCGAATACGCTTC CCCAGGCAAGGCAACCGAGGTCCGGGTGACAGTCCTGAGACAGGCCGACAGCCAGGTGACAGAAGTCTGCGCCGCTA CTTATATGATGGGCAACGAGCTGACCTTTCTGGACGATAGCATTTGTACCGGGACATCTAGTGGAAACCAAGTGAAT CTGACCATCCAGGGCCTGCGCGCTATGGACACAGGGCTGTACATTTGTAAAGTGGAGCTGATGTATCCCCCTCCATA CTATCTGGGAATCGGCAACGGGACCCAGATCTACGTGATTGATCCTGAACCATGCCCCGACTCCGATGAGAATCTGT ATTTCCAGGGACCACGAGGCCCCACAATTAAGCCATGTCCCCCTTGCAAATGTCCTGCACCAAACCTGCTGGGAGGA CCAAGCGTGTTCATCTTTCCACCCAAGATCAAGGACGTGCTGATGATCTCACTGAGCCCCATTGTGACCTGCGTGGT CGTGGACGTGAGCGAGGACGATCCTGATGTGCAGATCAGTTGGTTCGTCAACAATGTGGAAGTCCACACAGCTCAGA CTCAGACCCATAGGGAGGATTACAATAGTACTCTGCGCGTCGTGTCAGCACTGCCCATTCAGCACCAGGACTGGATG AGCGGCAAGGAGTTCAAGTGCAAAGTGAACAACAAGGATCTGCCCGCACCTATCGAGAGAACTATTTCCAAGCCTAA AGGGTCTGTGAGGGCCCCACAGGTGTATGTCCTGCCTCCACCCGAGGAAGAGATGACTAAGAAACAGGTGACACTGA CTTGTATGGTCACCGACTTCATGCCCGAAGATATCTACGTGGAGTGGACTAACAATGGGAAGACCGAACTGAACTAT AAAAATACAGAGCCTGTGCTGGACTCAGATGGAAGCTACTTTATGTATAGCAAGCTGCGAGTGGAAAAGAAAAACTG GGTCGAGCGGAACAGCTACTCTTGTAGTGTGGTCCACGAAGGGCTGCATAATCACCACACCACTAAATCATTCTCCC GAACTCCAGGCAAA(SEQ ID NO:4)
2.pUC57simple-CTLA4ECD-mFc质粒的获得:
由金斯瑞公司将合成的CTLA4ECD-mFc融合基因(SEQ ID NO:4)克隆到pUC57simple(金斯瑞公司提供)表达载体中,获得pUC57simple-CTLA4ECD-mFc质粒。
3.pcDNA3.1-CTLA4ECD-mFc重组质粒的构建:
将质粒pUC57simple-CTLA4ECD-mFc进行酶切(Xba I和BamH I),电泳回收得到的融合基因片段CTLA4ECD-mFc与pcDNA3.1表达载体(购自Invitrogen公司)进行连接反应,获得pcDNA3.1-CTLA4ECD-mFc,转染感受态大肠杆菌细胞DH5a(购自TIANGEN公司),转染和培养按照说明书进行。筛选得到阳性的pcDNA3.1-CTLA4ECD-mFc克隆菌落,按照常规方法扩增大肠杆菌, 然后采用试剂盒(购自天根生化科技(北京)有限公司,DP103-03)并按照试剂盒的说明书提取得到pcDNA3.1-CTLA4ECD-mFc重组质粒。
4.按照lipofectamin转染试剂盒(购自Invitrogen公司)方法将重组质粒pcDNA3.1-CTLA4ECD-mFc转染293F细胞(购自Invitrogen公司)。
5.将重组质粒pcDNA3.1-CTLA4ECD-mFc转染293F细胞7天后,将培养液通过高速离心、微孔滤膜抽真空过滤以及HiTrap protein A HP柱进行纯化CTLA4ECD-mFc融合蛋白,并取纯化后样品加入还原型蛋白电泳上样缓冲液,进行SDS-PAGE电泳检测。如图1所示,目标蛋白大约在45kD处。
6.CTLA4-8D2杂交瘤细胞株LT001的建立
用CTLA4ECD-mFc融合蛋白作为抗原,取免疫BALB/C小鼠(购自广东医学实验动物中心)的脾细胞与小鼠骨髓瘤细胞融合成杂交瘤细胞,参照目前已确立的方法(e.g.,Stewart,S.J.,“Monoclonal Antibody Production”,in Basic Methods in antibodyProduction and Characterization,Eds.G.C.Howard and D.R.Bethell,Boca Raton:CRCPress,2000)。
用CTLA4作为抗原包被酶标板,进行间接ELISA法筛选,得到分泌与CTLA4特异性结合的新的抗体的杂交瘤细胞。对间接ELISA筛选得到的杂交瘤细胞,通过竞争ELISA筛选出能够分泌与配体B7-1(CD80,NCBI Gene ID:941)、B7-2(CD86,NCBI Gene ID:942)竞争结合CTLA4的单克隆抗体的杂交瘤细胞株,并经过有限稀释法得到稳定的杂交瘤细胞株。将该杂交瘤细胞株命名为CTLA4-8D2杂交瘤细胞株,并经过有限稀释法得到CTLA4-8D2稳定细胞株(本发明中亦简称为LT001细胞,其分泌的单克隆抗体命名为8D2)。
杂交瘤细胞株LT001(CTLA4-8D2),其于2014年6月18日保藏于中国典型培养物保藏中心(CCTCC),保藏编号为CCTCC NO:C2014113,保藏地址为中国.武汉.武汉大学,邮编:430072。
7.抗体8D2的制备
用含10%的低IgG胎牛血清对本发明CTLA4-8D2(LT001)细胞株进行培养,7天后收集细胞培养上清进行纯化制备抗体8D2。
8.抗体8D2的SDS-PAGE电泳检测:
将纯化后的样品分别加入还原型蛋白电泳上样缓冲液和非还原型蛋白电泳上样缓冲液,煮沸后进行检测。检测结果显示,还原型蛋白样品目标蛋白大约在50kD和25kD处,非还原型蛋白样品目标蛋白大约在150kD处(图2)。
实施例2:单克隆抗体8D2的轻链和重链序列的获得
按照培养细胞细菌总RNA提取试剂盒(Tiangen,货号DP430)的方法,从实施例1制得的CTLA4-8D2杂交瘤细胞株(LT001细胞)中提取mRNA。
按照InvitrogenIII First-Strand Synthesis System for RT-PCR试剂盒说明书合成cDNA,并进行PCR扩增。PCR扩增产物直接进行TA克隆,具体操作参照pEASY-T1Cloning Kit(Transgen CT101)试剂盒说明书进行。将TA克隆的产物直接进行测序,测序结果如下:
重链可变区的DNA测序结果:(345bp)
GAGGTGAAACTGGACGAAACTGGCGGGGGGCTGGTGCAGCCCGGACGACCTATGAAGCTGTCATGCGTCGCCAGCGGCTTCACCTTTAGCGACAACTGGATGAATTGGGTGAGGCAGAGCCCAGAGAAGGGGCTGGAATGGCTGGCTCAGATCCGCAACAAACCCTACAATTATGAGACCTACTATTCTGACAGTGTGAAGGGCCGGTTCACAATTTCCAGAGACGATTCTAAAAGCTCCGTCTACCTGCAGATGAACAATCTGAGAGGCGAAGATATGGGGATCTACTATTGCACAGCACAGTTCGCTTATTGGGGACAGGGCACTCTGGTCACAGTCTCCGCC(SEQ ID NO:5)
其编码的蛋白序列:(115aa)
EVKLDETGGGLVQPGRPMKLSCVASGFTFSDNWMNWVRQSPEKGLEWLAQIRNKPYNYETYYSDSVKGRFTISRDDSKSSVYLQMNNLRGEDMGIYYCTAQFAYWGQGTLVTVSA(SEQ ID NO:6)
轻链可变区的DNA测序结果:(318bp)
GACATTCAGATGACACAGAGTCCTGCTTCCCTGAGTGCCTCAGTGGGGGAGACCGTCACAATCACTTGCGGCACCTCTGAAAACATCTACGGCGGGCTGAATTGGTATCAGCGGAAGCAGGGCAAAAGTCCCCAGCTGCTGATCTTCGGAGCAACAAACCTGGCCGACGGCATGAGCTCCCGGTTTAGCGGGTCCGGATCTGGCAGACAGTACAGCCTGAAGATTTCTAGTCTGCACCCAGACGATGTGGCTACTTACTATTGCCAGAATGTCCTGAGGAGTCCCTTCACCTTTGGGTCAGGAACAAAGCTGGAGATC(SEQ ID NO:7)
其编码的蛋白序列:(106aa)
DIQMTQSPASLSASVGETVTITCGTSENIYGGLNWYQRKQGKSPQLLIFGATNLADGMSSRFSGSGSGRQYSLKISSLHPDDVATYYCQNVLRSPFTFGSGTKLEI(SEQ ID NO:8)
实施例3:人源化抗体8D2H1L1、8D2H2L2和8D2H3L3的轻链和重链序列的设计
根据CTLA4蛋白的三维晶体结构(Nat.Struct.Biol.(1997)4p.527)以及实施例2获得的抗体8D2的序列,通过计算机模拟抗体模型,根据模型设计突变,得到抗体8D2H1L1、8D2H2L2和8D2H3L3的可变区序列(抗体恒定区序列,来自NCBI的数据库),可变区序列如下:
1.单克隆抗体8D2H1L1的轻链和重链序列
重链可变区的DNA序列:(345bp)
GAAGTGCAGCTGGTCGAGTCCGGGGGGGGCCTGGTGCAGCCAGGAGGATCAATGCGACTGAGCTGCGCCGCTTCCGGCTTCACCTTC AGCGACAACTGGATGAATTGGGTCAGGCAGGCACCAGGAAAGGGACTGGAGTGGCTGGCACAGATCCGCAACAAACCTTACAACTACGAAACTTACTACAGCGACTCCGTGAAGGGGCGGTTCACCATTTCTAGAGACGATTCTAAAAACAGTGTGTACCTGCAGATGAATAGCCTGAAGACCGAGGATACAGGAGTCTACTATTGTACCGCACAGTTTGCTTATTGGGGGCAGGGCACTCTGGTGACAGTCTCTTCA(SEQ ID NO:9)
其编码的蛋白序列:(115aa)
EVQLVESGGGLVQPGGSMRLSCAASGFTFSDNWMNWVRQAPGKGLEWLAQIRNKPYNYETYYSDSVKGRFTISRDDSKNSVYLQMNSLKTEDTGVYYCTAQFAYWGQGTLVTVSS(SEQ ID NO:10)
轻链可变区的DNA序列:(321bp)
GACATTCAGATGACTCAGAGCCCTTCAAGCCTGTCCGCATCTGTGGGCGACCGAGTCACCATCACATGCAGAACCTCCGAGAACATCTACGGCGGGCTGAATTGGTATCAGCGAAAGCAGGGGAAAAGTCCCAAGCTGCTGATCTACGGGGCAACAAACCTGGCCAGCGGAATGAGCTCCAGATTCAGTGGATCAGGCAGCGGGACAGATTATACTCTGAAAATTTCTAGTCTGCACCCAGACGATGTGGCAACCTACTATTGCCAGAATGTCCTGAGGTCACCCTTCACCTTTGGAAGCGGCACAAAACTGGAGATCAAG(SEQ ID NO:11)
其编码的蛋白序列:(107aa)
DIQMTQSPSSLSASVGDRVTITCRTSENIYGGLNWYQRKQGKSPKLLIYGATNLASGMSSRFSGSGSGTDYTLKISSLHPDDVATYYCQNVLRSPFTFGSGTKLEIK(SEQ ID NO:12)
2.8D2人源化单克隆抗体8D2H2L2的轻链和重链序列
重链可变区的DNA序列:(345bp)
GAAGTGCAGCTGGTCGAGTCCGGGGGGGGCCTGGTGCAGCCAGGAGGATCAATGCGACTGAGCTGCGCCGCTTCCGGCTTCACCTTCAGCGACAACTGGATGAATTGGGTCAGGCAGGCACCAGGAAAGGGACTGGAGTGGCTGGCACAGATCCGCAACAAACCTTACAACTACGAAACTTACTACAGCGCCTCCGTGAAGGGGCGGTTCACCATTTCTAGAGACGATTCTAAAAACAGTGTGTACCTGCAGATGAATAGCCTGAAGACCGAGGATACAGGAGTCTACTATTGTACCGCACAGTTTGCTTATTGGGGGCAGGGCACTCTGGTGACAGTCTCTTCA(SEQ ID NO:13)
其编码的蛋白序列:(115aa)
EVQLVESGGGLVQPGGSMRLSCAASGFTFSDNWMNWVRQAPGKGLEWLAQIRNKPYNYETYYSASVKGRFTISRDDSKNSVYLQMNSLKTEDTGVYYCTAQFAYWGQGTLVTVSS(SEQ ID NO:14)
轻链可变区的DNA序列:(324bp)
GACATTCAGATGACTCAGAGCCCTTCAAGCCTGAGTGCCTCAGTGGGAGACCGGGTCACCATCACATGCAGAACCAGCGAGAACATCTACGGCGGCCTGAACTGGTATCAGCGAAAGCCAGGCAAGAGCCCCAAGCTGCTGATCTACGGGGCAACCAACCTGGCCTCTGGAGTGAGCTCCAGATTCAGCGGCAGCGGCTCTGGGACCGACTATACTCTGACCATTTCTAGTCTGCAGCCTGAAGATGTGGCAACATACTATTGCCAGAATGTCCTGAGGTCCCCATTCACCTTTGGATCTGGCACCAAGCTGGAGATCAAGCGC(SEQ ID NO:15)
其编码的蛋白序列:(108aa)
DIQMTQSPSSLSASVGDRVTITCRTSENIYGGLNWYQRKPGKSPKLLIYGATNLASGVSSRFSGSGSGTDYTLTISSLQPEDVATYYCQNVLRSPFTFGSGTKLEIKR(SEQ ID NO:16)
3.8D2人源化单克隆抗体8D2H3L3的轻链和重链序列
重链可变区的DNA序列:(345bp)
GAGGTGCAGCTGGTCGAGTCTGGAGGCGGCCTGGTGCAGCCCGGCGGGTCACTGCGACTGAGCTGCGCCGCTTCCGGCTTCACCTTCAGCGACAACTGGATGAATTGGGTGAGGCAGGCACCCGGGAAGGGGCTGGAGTGGGTCGCTCAGATCCGCAACAAACCTTACAATTATGAGACAGAATACGCAGCCTCTGTGAAGGGGCGGTTCACTATTAGTAGAGACGATAGCAAGAACAGCGCCTATCTGCAGATGAATAGCCTGAAGACCGAAGATACAGCCGTCTACTATTGTACAGCTCAGTTTGCATACTGGGGCCAGGGAACTCTGGTGACCGTCAGCTCC(SEQ ID NO:17)
其编码的蛋白序列:(115aa)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDNWMNWVRQAPGKGLEWVAQIRNKPYNYETEYAASVKGRFTISRDDSKNSAYLQMNSLKTEDTAVYYCTAQFAYWGQGTLVTVSS(SEQ ID NO:18)
轻链可变区的DNA序列:(321bp)
GACATTCAGATGACTCAGAGCCCTTCTTCTCTGTCCGCATCTGTGGGAGACCGGGTCACCATCACATGCAGAGCCAGCGAGAACATCTACGGCGGCCTGAACTGGTATCAGCAGAAGCCAGGCAAAGCTCCCAAGCTGCTGATCTACGGAGCAACCTCCCTGGCATCTGGAGTGCCATCCCGGTTCAGTGGATCAGGCAGCGGGACCGACTATACTCTGACCATTAGCTCCCTGCAGCCTGAAGACTTCGCCACATACTATTGCCAGAACGTGCTGAGGTCCCCATTCACCTTTGGATCTGGCACCAAGCTGGAGATCAAG(SEQ ID NO:19)
其编码的蛋白序列:(107aa)
DIQMTQSPSSLSASVGDRVTITCRASENIYGGLNWYQQKPGKAPKLLIYGATSLASGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQNVLRSPFTFGSGTKLEIK(SEQ ID NO:20)
实施例4:8D2重组抗体8D2(Re)以及8D2人源化抗体8D2H1L1、8D2H2L2和8D2H3L3的
制备和SDS-PAGE电泳检测
1.8D2重组抗体即8D2(Re)的制备和SDS-PAGE电泳检测
将8D2的重链cDNA序列(其可变区序列如SEQ ID NO:5所示)和轻链的cDNA序列(其可变区序列如SEQ ID NO:7所示)分别克隆到pUC57simple(金斯瑞公司提供)载体中,分别获得pUC57simple-8D2H和pUC57simple-8D2L质粒。
分别将质粒pUC57simple-8D2H和pUC57simple-8D2L进行酶切(HindIII&EcoRI),电泳回收得到的重链轻链分别亚克隆到pcDNA3.1载体中,提取重组质粒共转染293F细胞。细胞培养7天后,将培养液通过高速离心、微孔滤膜抽真空过滤以及HiTrap protein A HP柱进行纯化,并将纯化后的样品分别加入还原型蛋白电泳上样缓冲液和非还原型蛋白电泳上样缓冲液,煮沸后进行SDS-PAGE电泳检测。如图3所示,还原型蛋白样品目标蛋白大约在50kD和25KD处,非还原型蛋白样品目标蛋白大约在150kD处。
2.8D2人源化抗体8D2H1L1、8D2H2L2和8D2H3L3的制备和SDS-PAGE电泳检测
将8D2H1L1、8D2H2L2和8D2H3L3的重链cDNA(其可变区序列分别如SEQ ID NO:9、SEQ ID NO:13、SEQ ID NO:17所示)和轻链的cDNA(其可变区序列分别如SEQ ID NO:11、SEQID NO:15、SEQ ID NO:19所示)分别克隆到pUC57simple(金斯瑞公司提供)载体中,获得pUC57simple-8D2H1L1、pUC57simple-8D2H2L2和pUC57simple-8D2H3L3质粒,并分别亚克隆到pcDNA3.1载体中,方法同前述8D2(Re)。
将重组质粒转染293F细胞,将培养液纯化后进行检测(同上所述8D2(Re)的方法),结果分别如图4、图5和图6所示,还原型蛋白样品目标蛋白大约在50kD和25KD处,非还原型蛋白样品目标蛋白大约在 150kD处。
实施例5:抗体的动力学参数测定
使用Fortebio分子相互作用仪测定抗体8D2及人源化8D2H1L1、8D2H2L2、8D2H3L3与抗原CTLA4(NCBI Gene ID:1493,编码核酸序列为SEQ ID NO:21,所编码的氨基酸序列为SEQ ID NO:22)结合的动力学参数。
1.用TEV蛋白酶酶切CTLA4-mFc蛋白(CTLA4-mFc合成方法同实施例1中所述CTLA4ECD-mFc的合成),并过柱纯化获得CTLA4抗原。
基因CTLA4的序列:(636bp)
ATGGGCGTCCTGCTGACTCAGAGAACCCTGCTGTCCCTGGTGCTGGCACTGCTGTTTCCTTCAATGGCTTCAATGGCTATGCATGTGGCTCAGCCAGCAGTGGTCCTGGCAAGCTCCAGGGGGATCGCCAGTTTCGTGTGCGAGTACGCCTCACCTGGAAAGGCTACAGAAGTCCGGGTGACTGTCCTGAGACAGGCTGACTCTCAGGTGACCGAGGTCTGCGCCGCTACATATATGATGGGCAACGAACTGACCTTTCTGGACGATTCCATTTGTACTGGCACCTCTAGTGGGAACCAAGTGAATCTGACTATCCAGGGACTGCGAGCAATGGACACCGGACTGTACATTTGCAAAGTGGAGCTGATGTATCCCCCTCCATACTATCTGGGCATCGGGAATGGAACACAGATCTACGTGATTGATCCCGAACCTTGTCCAGACAGCGATTTCCTGCTGTGGATTCTGGCAGCCGTGTCAAGCGGCCTGTTCTTTTATAGCTTTCTGCTGACTGCCGTCTCCCTGTCTAAGATGCTGAAGAAACGATCCCCCCTGACCACAGGGGTGGTCGTGAAAATGCCACCTACCGAGCCCGAGTGCGAAAAACAGTTCCAGCCATACTTTATCCCTATCAAT(SEQ ID NO:21)
编码对应的氨基酸序列:(212aa)
MGVLLTQRTLLSLVLALLFPSMASMAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVVVKMPPTEPECEKQFQPYFIPIN(SEQ ID NO:22)
2.分别将抗体8D2及其人源化抗体8D2H1L1、8D2H2L2、8D2H3L3采用氨基偶联的方式固定于AR2G传感器表面,经乙醇胺封闭,于PBST中平衡后,与抗原CTLA4结合,CTLA4用PBST两倍稀释,浓度为300、150、75、37.5、18.75、9.38、4.69、0nM,于PBST中解离。人源化8D2H1L1、H2L2、H3L3的检测方法与8D2相同,抗原浓度为180、90、45、22.5、11.25、5.625、2.813、0nM。
抗体8D2及其人源化抗体8D2H1L1、8D2H2L2、8D2H3L3动力学参数见表1,动力学特征参数检测结果分别如图7-10所示。
表1:抗体8D2、8D2H1L1、8D2H2L2、8D2H3L3动力学参数
KD为亲和力常数;kon为抗原抗体结合速率;kdis为抗原抗体解离速率;KD=kdis/kon。
结果表明,四种抗体均与抗原有较好的亲和力,其中抗体8D2、8D2H1L1与抗原的亲和力强于8D2H2L2、8D2H3L3。
实施例6:流式细胞仪方法检测抗体与表达CTLA4抗原的宿主细胞293F的表面抗原
CTLA4的结合活性
首先构建表达CTLA4抗原的宿主细胞293F;然后用本发明中制备 的单克隆抗体8D2(见实施例1)以及制备的8D2(Re)及8D2人源化抗体8D2H1L1、8D2H2L2和8D2H3L3(见实施例4)对改宿主细胞进行标记。然后采用流式细胞术分析验证抗体8D2,8D2(Re)及8D2人源化抗体8D2H1L1、8D2H2L2和8D2H3L3对细胞表面具有天然构象的抗原具有特异性的结合。
具体步骤如下:
1.表达CTLA4抗原的宿主细胞293F的构建
按照lipofectamin转染试剂盒(购自Invitrogen公司)方法将包含CTLA4的载体pLenti6.3-CTLA4(载体pLenti6.3购自Invitrogen公司)转染293F细胞,经筛选获得稳定表达CTLA4的克隆群体。
2.抗体标记和流式细胞仪检测
采用常规胰酶消化方法上述步骤获得的表达CTLA4抗原的宿主细胞293F,并使每个收集管细胞数为2×105,用PBS(1%BSA)配制浓度分别为20nM,10nM,5nM,1nM,0.1nM,0.01nM,0nM的8D2抗体稀释液,冰上与表达CTLA4的293F细胞孵育2小时,每管加入100μLFITC-Goat-Anti-Mouse IgG(1:500)冰上孵育1小时并加入300μL PBS后,在流式细胞仪上用FITC通道检测荧光信号。其它抗体的检测参照8D2抗体。
3.实验结果
表达CTLA4抗原的宿主细胞293F的CTLA4表达验证结果如图11和图12所示。检测8D2,8D2(Re)抗体及3个人源化抗体与293F细胞的结合结果分别如图13-17所示。由图可见,8D2抗体及其人源化抗体能有效地结合宿主细胞293F表面的靶标CTLA4蛋白,并且其结合效率呈剂量依赖关系,各剂量的荧光强度见表2。
通过对结合的8D2及其人源化抗体进行荧光定量分析,曲线模拟8D2及其人源化抗体的结合效率EC50,如下面的表3。
表2:流式细胞仪检测8D2,8D2(Re)及8D2人源化抗体8D2H1L1,8D2H2L2和8D2H3L3结合CTLA4宿主细胞293F表面抗原CTLA4后荧光强度分析
表3:流式细胞仪检测分析曲线模拟8D2,8D2(Re)及8D2人源化抗体8D2H1L1、8D2H2L2、8D2H3L3与CTLA4宿主细胞293F表面抗原CTLA4的结合效率EC50
8D2 | 8D2(Re) | 8D2H1L1 | 8D2H2L2 | 8D2H3L3 | |
EC50(nM) | 3.84 | 1.38 | 5.06 | 4.37 | 4.54 |
实施例7:间接ELISA方法检测抗体与抗原CTLA4的结合活性
采用间接ELISA方法分别测定杂交瘤细胞产生的单克隆抗体8D2,8D2(Re)及8D2人源化抗体8D2H1L1,8D2H2L2和8D2H3L3与CTLA4的结合活性。
检测8D2抗体及其人源化抗体与抗原CTLA4结合结果分别如图18-20所示。由图可见,8D2,8D2(Re)抗体及8D2人源化抗体能有效地结合CTLA4蛋白,并且其结合效率呈剂量依赖关系,各剂量的荧光强度见表4-表7。通过对结合的8D2,8D2(Re)及人源化抗体进行荧光定量分析,曲线模拟8D2,8D2(Re)及人源化抗体的结合效率EC50(表8)。
表4:8D2、8D2重组抗体与鼠CTLA4的结合(间接ELISA)
表5:8D2、8D2H1L1、8D2重组抗体与人CTLA4的结合(间接ELISA)
表6:8D2H2L2、8D2H3L3抗体与人CTLA4结合(间接ELISA)
表7:8D2H2L2、8D2H3L3抗体与猴CTLA4结合(间接ELISA)
表8:间接ELISA分析曲线模拟8D2,8D2(Re)及8D2人源化抗体8D2H1L1、8D2H2L2、8D2H3L3与杂交瘤细胞株表面抗原的结合效率EC50
实施例8:竞争ELISA方法检测抗体与抗原CTLA4的结合活性
采用竞争ELISA方法分别测定杂交瘤细胞产生的单克隆抗体8D2,8D2(Re)及8D2人源化抗体8D2H1L1,8D2H2L2和8D2H3L3与B7的竞争结合抗原CTLA4。
检测8D2,8D2(Re)抗体及其人源化抗体与抗原CTLA4结合结果分别如图21-24所示。由图可见,8D2,8D2(Re)抗体及8D2人源化抗体能有效地结合CTLA4蛋白,并且其结合效率呈剂量依赖关系,各剂量的荧光强度见表9-表13。通过对结合的8D2,8D2(Re)及人源化抗体进行荧光定量分析,曲线模拟8D2,8D2(Re)及人源化抗体的结合效率EC50(表14)。
表9:8D2、8D2(Re)与B7-1竞争ELISA
表10:8D2、8D2H1L1、8D2(Re)与B7-1竞争ELISA
表11:8D2、8D2H1L1、8D2重组抗体与B7-2竞争ELISA
表12:8D2H2L2、8D2H3L3抗体与B7-1竞争ELISA
表13:8D2H2L2、8D2H3L3抗体与B7-2竞争结合CTLA4ELISA
表14:竞争ELISA分析曲线模拟8D2,8D2(Re)及8D2人源化抗体8D2H1L1、8D2H2L2、8D2H3L3与B7竞争结合抗原CTLA4的结合效率EC50
实施例9:单克隆抗体8D2及人源化抗体8D2H1L1、8D2H2L2和8D2H3L3的细胞生物学
活性分析
为检测单克隆抗体8D2及人源化抗体8D2H1L1,8D2H2L2和8D2H3L3对外周血单核细胞(Peripheral blood mononuclear cell:PBMC)的IL-2表达的影响,用含肝素钠的血液采集管采集健康者外周血,经PBS稀释和分离液离心(2550rpm,20分钟)后得到细胞悬液即PBMC,向细胞悬液加入PHA并置于饱和湿度37℃、5%CO2的培养箱中继续培养,并加入淋巴细胞Raji细胞和抗体进行共孵育培养。
共孵育培养72小时后收集细胞培养上清,采用ELISA方法(参照试剂盒说明书:DAKEW达科为,DKW12-1020-096)检测细胞共孵育培养上清IL-2表达情况。
实验结果经统计分析,与T cells和with Raji cells组相比,单克隆抗体8D2以及人源化抗体能有效地阻断CTLA4与B7的结合并提高T淋巴细胞中IL-2的表达(图25)。
尽管本发明的具体实施方式已经得到详细的描述,本领域技术人员 将会理解。根据已经公开的所有教导,可以对那些细节进行各种修改和替换,这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。
Claims (16)
1.一种杂交瘤细胞株LT001,其于2014年6月18日保藏于中国典型培养物保藏中心(CCTCC),保藏编号为CCTCC NO:C2014113。
2.权利要求1所述的杂交瘤细胞株产生的单克隆抗体。
3.选自如下的单克隆抗体:
(1)重链可变区的氨基酸序如SEQ ID NO:6所示,轻链可变区的氨基酸序列如SEQ IDNO:8所示;
(2)重链可变区的氨基酸序如SEQ ID NO:10所示,轻链可变区的氨基酸序列如SEQ IDNO:12所示;
(3)重链可变区的氨基酸序如SEQ ID NO:14所示,轻链可变区的氨基酸序列如SEQ IDNO:16所示;和
(4)重链可变区的氨基酸序如SEQ ID NO:18所示,轻链可变区的氨基酸序列如SEQ IDNO:20所示。
4.根据权利要求3所述的单克隆抗体,其中,其重链恒定区为小鼠抗体的重链恒定区或人抗体的重链恒定区,其轻链恒定区为小鼠抗体的轻链恒定区或人抗体的轻链恒定区。
5.一种分离的核酸序列或核酸序列组合,其编码权利要求3或4所述的单克隆抗体的重链可变区和轻链可变区。
6.根据权利要求5所述的核酸序列或核酸序列组合,其选自如下的(1)至(4):
(1)SEQ ID NO:5,SEQ ID NO:7;
(2)SEQ ID NO:9,SEQ ID NO:11;
(3)SEQ ID NO:13,SEQ ID NO:15;和
(4)SEQ ID NO:17,SEQ ID NO:19。
7.一种单克隆抗体偶联物,包括单克隆抗体和偶联部分,其中,所述单克隆抗体为权利要求2至4中任一项所述的单克隆抗体,所述偶联部分为选自放射性核素、药物、毒素、细胞因子、酶、荧光素、和生物素中的一种或多种。
8.根据权利要求7所述的单克隆抗体偶联物,其中,所述偶联部分为蓖麻毒素。
9.根据权利要求7所述的单克隆抗体偶联物,其中,所述偶联部分为蓖麻毒素A链。
10.一种药物组合物,其含有权利要求2至4中任一项所述的单克隆抗体或者权利要求7至9中任一项所述的单克隆抗体偶联物。
11.根据权利要求10所述的药物组合物,其还包括药学上可接受的载体。
12.根据权利要求10所述的药物组合物,其还包括药学上可接受的辅料。
13.权利要求2至4中任一项所述的单克隆抗体或者权利要求7至9中任一项所述的单克隆抗体偶联物在制备预防或治疗肿瘤的药物中的用途。
14.根据权利要求13所述的用途,其中,所述肿瘤选自黑色素瘤、肾肿瘤、前列腺癌、膀胱癌、结肠直肠癌、胃肠道癌和肝癌。
15.权利要求2至4中任一项所述的单克隆抗体或者权利要求7至9中任一项所述的单克隆抗体偶联物在制备如下药物中的用途:
阻断CTLA4与B7结合的药物、调节CTLA4活性或CTLA4水平的药物、解除CTLA4对机体免疫抑制的药物、激活T淋巴细胞的药物或者提高T淋巴细胞中IL-2表达的药物。
16.根据权利要求15所述的用途,其中,所述调节为下调。
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