CN106957367A - 抗idh1 r132h抗体及其制备方法和用途 - Google Patents
抗idh1 r132h抗体及其制备方法和用途 Download PDFInfo
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- CN106957367A CN106957367A CN201710208613.8A CN201710208613A CN106957367A CN 106957367 A CN106957367 A CN 106957367A CN 201710208613 A CN201710208613 A CN 201710208613A CN 106957367 A CN106957367 A CN 106957367A
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- idh1r132h
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Abstract
本发明涉及抗IDH1 R132H抗体及其制备方法和用途。所述抗体或其功能性衍生物特异性地识别IDH1 R132H的表位，所述单克隆抗体具有：(1)SEQ IDNO：2、6、10中的一个表示的抗体重链可变区的氨基酸序列或其保守型变异序列；和(2)SEQ ID NO：4、8、12中的一个表示的轻链可变区的氨基酸序列或其保守型变异序列。本发明还涉及所述抗体的编码核酸分子、构建载体、表达系统、对应的人源化抗体、制备方法、用途以及药物组合物。本发明的所述抗体对人I型人异柠檬酸脱氢酶(IDHI1)R132H具有免疫特异性，能够特异性地识别与恶性疾病相关的IDH1 R132H的突变位点并与之特异性地结合可用于诊断和治疗IDH1 R132H突变后发挥致病作用的那些疾病，例如胶质瘤及白血病等疾病。
Description
技术领域
本发明涉及抗IDHI R132H抗体和它们在诊断和治疗中的用途。
背景技术
异柠檬酸脱氢酶(isocitrate dehydrogenase，IDH)是三羧酸循环的关键酶，催化异柠檬酸氧化脱羧生成α-酮戊二酸，同时利用烟酰胺腺嘌呤二核苷酸/烟酰胺腺嘌呤二核苷磷酸作为辅助因子生成NADH/NADPH(还原型烟酰胺腺嘌呤二核苷酸/烟酰胺腺嘌呤二核苷磷酸)，在能量代谢、维生素、氨基酸合成的过程中发挥着非常重要的作用。IDH主要分为IDH1、IDH2和IDH3亚型。人类IDH1和IDH2的相似性达到70％，分别由不同基因编码(IDH1基因位于染色体2q33；IDH2基因位于染色体5q26)。IDH1表达于细胞质和过氧化物酶体，而IDH2主要存在于线粒体。
2009年，美国Yan Hai实验室和复旦大学赵世民团队等发现胶质瘤新的生物标记物—异柠檬酸脱氢酶1(Isocitrate dehydrogenase 1，IDH1)R132的点突变。研究发现IDH1第132位的精氨酸突变为组氨酸会显著抑制细胞内IDH1的活力，导致胞内α-酮戊二酸(α-KG)水平明显下降，而α-KG的下降则进一步导致脯氨酸羟基化酶(prolylhdroxylase)活力的降低。仿佛推倒了多米诺骨牌一样，一系列反应导致了细胞缺氧诱导因子(hypoxia-inducing factor，HIF1α)的稳定性增加，从而激活了HIF1α信号通路，最终促进肿瘤生长。大量临床胶质瘤样品筛查发现，IDH1基因R132突变在继发性神经胶质瘤中的突变频率高达75％以上，使得IDH1基因成为潜在的神经胶质瘤的诊断指针和靶向治疗目标。
IDH1在世界卫生组织(WHO)分级II及III级弥散性胶质瘤中发生突变的频率高。93％的IDH1突变特点是氨基酸发生置换R132H。
在急性髓系白血病中，IDH1基因也发生突变，IDH1 R132H是其中一种主要突变。
虽然在肿瘤及白血病中已经发现IDH1 R132H的突变，但是目前并没有关于针对这一位点的抗体尤其是单克隆抗体问世。另一方面，目前对于疾病的诊断以及针对肿瘤的免疫治疗都需要有特异性的抗体。因此，研发新型的抗IDH1 R132H单克隆抗体以用于疾病的诊断及癌症的免疫治疗或者给患者提供更多的药物选择，将成为研究热点和迫切需求。
发明内容
鉴于以上所述的现有技术现状，本发明的目的在于获得高亲和力的抗IDH1 R132H的抗体，并验证其相关功能，以用于解决现有技术中的问题并满足迫切的实际需要。
为实现上述目的及其他相关目的，本发明提供了如下技术方案：
1、一种分离的抗IDH1 R132H抗体或其功能性衍生物，所述抗IDH1 R132H抗体或其功能性衍生物特异性地识别IDH1 R132H的表位，所述抗IDH1 R132H抗体具有：(1)SEQIDNO：2、6、10中的一个表示的抗体重链可变区的氨基酸序列或其保守型变异序列；和(2)SEQ ID NO：4、8、12中的一个表示的轻链可变区的氨基酸序列或其保守型变异序列。
2、如技术方案1所述的抗IDH1 R132H抗体或其功能性衍生物，所述衍生物为所述抗IDH1 R132H抗体的片段、抗体/抗体片段一因子融合蛋白、或抗体/抗体片段一化学偶联物。
3、如技术方案2所述的抗IDH1 R132H抗体或其功能性衍生物，所述抗IDH1 R132H抗体的片段为Fab、Fab'、F(ab)'2、或scFv。
4、一种人源化抗体或其功能性衍生物，所述人源化抗体包含人源化的重链和人源化的轻链，其中所述重链包含：(1)技术方案1中所述的重链可变区的氨基酸序列或其保守型变异序列，和(2)来自人受体抗体重链的骨架；其中所述轻链包含：(1)技术方案1中所述的轻链可变区的氨基酸序列或其保守型变异序列，和(2)来自人受体抗体轻链的骨架。
5、一种分离的核酸分子，所述核酸分子编码技术方案1至4中任一项所述的抗IDH1R132H抗体的重链和/或轻链的可变区或所述抗IDH1 R132H抗体的全长的氨基酸。
6、一种构建载体，所述构建体含有如技术方案1至4中任一项所述的核酸分子；优选的是，所述构建体由所述核酸分子插入到表达载体的多克隆位点构建而成。
7、一种表达系统，所述表达系统由技术方案5所述的构建体转染到宿主细胞构建而成。
8、一种制备技术方案1至4中任一项所述的抗IDH1 R132H抗体的方法，所述方法包括如下步骤：(1)在适合表达所述抗IDH1 R132H抗体的条件下，培养技术方案6所述的表达系统，从而表达出所述抗IDH1 R132H抗体；(2)纯化分离出所述抗IDH1 R132H抗体。
9、如技术方案1至4中任一项所述的抗IDH1 R132H抗体或其功能性衍生物在制备分子阻滞药物上的用途；优选为在制备用于肿瘤治疗或肿瘤诊断的分子阻滞药物中的用途；更优选为在制备用于诊断神经胶质瘤尤其是II及III级弥散性神经胶质瘤或者急性髓系白血病的诊断试剂中的用途。
10、一种药物组合物，所述药物组合物包含治疗有效量的如技术方案1至4中任一项所述的抗IDH1 R132H抗体或其功能性衍生物。
本发明的所述抗体能够特异性地识别与恶性疾病相关的IDHI R132H的突变位点并与之特异性地结合，可用于诊断和治疗IDHI R132H突变后发挥致病作用的那些疾病，例如胶质瘤及白血病等疾病。
附图说明
图1为斑点免疫印迹法(dot blot)检测抗IDH1 R132H抗体特异性的检测结果。
图2为在神经胶质瘤细胞系U87中验证IDH1 R132H抗体特异性的验证结果。
图3为在神经胶质瘤细胞系U251中验证IDH1 R132H抗体特异性的验证结果。
图4为A2E8细胞株分泌单抗功能验证的结果。
图5为A11B3细胞株分泌单抗功能验证的结果。
图6为D4C7细胞株分泌单抗功能验证的结果。
图7为A2E8细胞株分泌单抗在人胶质瘤标本中染色的结果。
具体实施方式
本发明在第一方面提供了一种抗IDH1 R132H抗体尤其是抗IDH1 R132H抗体或其功能性衍生物，所述抗IDH1 R132H抗体的重链可变区的氨基酸序列为SEQ ID NO：2、6、10或其保守型变异序列，所述抗IDH1 R132H抗体的轻链可变区的氨基酸序列为SEQ ID NO：4、8、12或其保守型变异序列。
在一些实施方式中，重链和轻链的保守保守型变异序列相应地包括下表中所示的序列：
| A2E8重链CDR1 | GYVFSKFW | SEQ ID NO:13 |
| A2E8重链CDR2 | STLEMVTL | SEQ ID NO:14 |
| A2E8重链CDR3 | KIDGGRLVCILPPGTLVTVSA | SEQ ID NO:15 |
| A2E8轻链CDR1 | KSVSTSGYSY | SEQ ID NO:16 |
| A2E8轻链CDR2 | LVS | - |
| A2E8轻链CDR3 | QHIRELTRSEGGPSWK | SEQ ID NO:17 |
| A11B3重链CDR1 | GYAFSSSW | SEQ ID NO:18 |
| A11B3重链CDR2 | IYPGDGDT | SEQ ID NO:19 |
| A11B3重链CDR3 | AREGSYYTYDVRAYVVYGMDY | SEQ ID NO:20 |
| A11B3轻链CDR1 | QSLLYGSNQKNY | SEQ ID NO:21 |
| A11B3轻链CDR2 | WAS | - |
| A11B3轻链CDR3 | QQCYRYPLT | SEQ ID NO:22 |
| D4C7重链CDR1 | GYTFTSFW | SEQ ID NO:23 |
| D4C7重链CDR2 | INPHDGYT | SEQ ID NO:24 |
| D4C7重链CDR3 | SIPYPGMDY | SEQ ID NO:25 |
| D4C7轻链CDR1 | KSVSTSGYSY | SEQ ID NO:26 |
| D4C7轻链CDR2 | LAS | - |
| D4C7轻链CDR3 | QHIRELTRSEGGPSWKYN | SEQ ID NO:27 |
本发明进一步提供了所述抗IDH1 R132H抗体的功能性衍生物，所述衍生物为抗IDH1 R132H抗体的片段、抗体/抗体片段-因子融合蛋白、抗体/抗体片段-化学偶联物。
所述抗IDH1 R132H抗体的片段可以为Fab、Fab'、F(ab)'2、或scFv等。
所述抗体/抗体片段-因子融合蛋白具体可以为抗体-因子融合蛋白、或抗体片段-因子融合蛋白。
所述抗体/抗体片段-化学偶联物具体可以为抗体-化学偶联物、或抗体片段-化学偶联物。
在一些优选的实施方式中，所述抗IDH1 R132H抗体由细胞系A2E8、A11B3或D4C7所生成。在另外一些优选的实施方式中，所述IDH1 R132H的表位被细胞系A2E8、A11B3或D4C7所生成的抗IDH1 R132H抗体所结合。
本发明在第二方面提供了一种分离的核酸分子(例如DNA分子)，该核酸分子编码所述抗IDH1 R132H抗体的重链和/或轻链的可变区或编码所述抗IDH1 R132H抗体的全长氨基酸序列。
本发明在第三方面提供了一种构建体，该构建体包含所述分离的核酸分子。
优选的是，所述构建体通过将所述分离的核酸分子插入到表达载体的多克隆位点来构建制得。
本发明中的表达载体指本领域熟知的噬菌体、细菌质粒、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒表达载体或其他表达载体。
更优选的是，所述表达载体选自由pcDNA3.1+、pEE14.4和pHLX101组成的组。
本发明在第四方面提供了一种抗IDH1 R132H抗体的表达系统，该表达系统通过将所述构建体转染到宿主细胞来构建制得。
本发明中的宿主细胞可以是原核细胞(如细菌细胞)、低等真核细胞(如酵母细胞)、或高等真核细胞(如哺乳动物细胞)。
优选的是，所述宿主细胞选自由中国仓鼠卵巢细胞系(CHO)，多种COS细胞系、Hela细胞系、骨髓细胞系(如SP2/0细胞系、NSO细胞系、YB2/0细胞系等)、和转化的细胞或杂交瘤细胞组成的组。
本发明在第五方面提供了一种制备所述抗IDH1 R132H抗体的方法，所述方法包括如下步骤：(1)在适合于表达所述抗体的条件下，培养本发明第四方面所述的表达系统，从而表达出所述抗IDH1 R132H抗体；和(2)纯化分离出所述的抗IDH1 R132H抗体。
本发明中所用的宿主细胞均为现有技术，可通过商业途径购买获取，培养中所用的培养基亦为各种常规培养基，本领域技术人员可根据经验选择适用的培养基，在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用例如温度转换或化学诱导法诱导选择的启动子，将细胞再培养一段时间。在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于常规的复性处理、用蛋白沉淀剂处理盐析方法、离心、渗透破菌、超处理、超离心、分子筛层析凝胶过滤、吸附层析、离子交换层析、高效液相层析和其它各种液相层析技术及这些方法的结合。
本发明从单克隆培养的细胞株中筛选获取目的抗体的基因序列，用以构建真核表达载体，表达后即可重建抗体的活性，获得抗IDH1 R132H抗IDH1 R132H抗体。
本发明在第六方面提供了一种人源化抗体，其包含人源化重链和人源化轻链，其中：
(1)所述人源化重链的可变区可以包含来自小鼠A2E8、A11B3或D4C7重链的互补决定区和来自人受体抗体重链的骨架，所述来自人受体抗体重链的骨架可任选地具有一个或多个人骨架残基取代；以及
(2)人源化轻链的可变区可以包含三个来自小鼠A2E8、A11B3或D4C7轻链的互补决定区和来自人受体抗体轻链的骨架，所述来自人受体抗体轻链的骨架可任选地具有一个或多个人骨架残基取代；并且
(3)人源化抗体特异性结合到人IDH1 R132H的表位。
本发明在第七方面提供了所述抗IDH1 R132H抗体在制备分子阻滞药物中的用途。
优选的是，所述用途可以为制备用于肿瘤治疗或肿瘤诊断的分子阻滞药物中的用途。
本发明第八方面提供了所述抗IDH1 R132H抗体在制备诊断试剂的用途，所述诊断试剂优选为用于诊断肿瘤及白血病的诊断试剂，更优选为用于诊断神经胶质瘤尤其是II及III级弥散性神经胶质瘤或者急性髓系白血病的诊断试剂。
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
在进一步描述本发明具体实施方式之前，应理解的是，本发明的保护范围不局限于下述特定的具体实施方案。还应当理解的是，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。在本发明说明书和权利要求书中，除非文中另外明确指出，否则诸如“一个”、“一”和“这个”或“一种”等包括复数形式。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组技术及相关领域的常规技术。这些技术在现有文献中已有完善说明。
实施例1
1.抗IDH1 R132H抗体的生产
抗原的制备
人工合成含有IDH1 R132H的短肽(由上海强耀生物技术公司合成)，与载体偶联后免疫小鼠。
杂交瘤的制备
我们使用标准的体内免疫方式和融合方法来制备抗IDH1 R132H抗体。该方法的简要过程如下：
小鼠免疫在上述得到IDH1 R132H抗原，任选一个型别的与弗氏完全佐剂等体积混合乳化，进行四肢肌肉多点注射，每只每次注射200微升。首次免疫后，分别用同样剂量的另外任一型别的加弗氏不完全佐剂进行加强免疫。在第四次加强免疫后，采血，检测其与的反应滴度。当滴度达到106以上时，取小鼠脾脏细胞用于融合。融合前72hr再次进行加强免疫经尾静脉注射1次，50ul/只。制备60块融合板。
融合：取血清滴度最高的3只小鼠的脾脏细胞，与小鼠骨髓瘤细胞相融合。先将脾脏研磨，得到脾细胞悬液，然后将其与细胞数目低十倍的处于对数生长期的SP2/0小鼠骨髓瘤细胞混合，并经PEG1500作用1min，将两种细胞融合一起；然后把融合细胞液分装到60块96孔板中培养。融合培养基为含HAT和20％FBS的RPMI 1640完全筛选培养基。抗原广谱性克隆通过ELISA及中和实验筛选得到，并且经3次克隆化后，得到稳定的抗IDH1 R132H抗体细胞株。
杂交瘤的筛选：融合细胞在96孔板中培养10天后，吸取细胞上清，进行ELISA及中和检测。将阳性孔继续克隆化，直至细胞株所分泌的抗体能够稳定结合抗原为止。
筛选结果获得三株分泌抗IDH1 R132H抗体的细胞株：A2E8、A11B3和D4C7。
杂交瘤的培养：将稳定的杂交瘤细胞株在二氧化碳培养箱中扩增培养：从96孔板转移至24孔板，再转移至50ml细胞培养瓶。然后，收集细胞培养瓶内的细胞，将其注射到小鼠腹腔内，并在一天后从小鼠腹腔中吸取腹水。
单克隆抗体的纯化
腹水先用50％的硫酸铰沉淀处理，然后用pH7.2的PBS进行透析，然后用DEAE柱进行纯化，最终得到经纯化的单克隆抗体。
实施例2
抗IDH1 R132H抗体特异性的检测。
斑点杂交验证：
利用带Flag标签表达IDH1全长的过表达载体和表达IDH1 R132H的过表达载体转染HEK293FT细胞，72hr后裂解细胞，取裂解液30ul点在NC膜上，室温自然风干，用含5％脱脂奶粉的PBS室温封闭1hr，加入TBST 1：5000稀释的单克隆抗体，室温作用1hr，TBST漂洗膜10min×3；加入稀释后的二抗(均为1：4000稀释)，水平摇床室温孵育1h。吸净二抗，TBST漂洗膜，10min×4，混合ECL(A：B＝100：1液)，充分覆盖湿润NC膜表面，静置1min；于暗室中用保鲜膜将NC膜封好，将X光片平放于NC膜上显影并记录数据。结果可见，筛选到的3株单克隆抗体可以特异性识别IDH1 R132H表位(见图1)。Flag标签的IDH1野生型(WT)和IDH1 R132H突变的表达产物可以用Flag抗体(Sigma，M2，1：5000)识别反应(最右边两个点)。
Western blot法检测：利用表达IDH1全长的过表达载体和表达IDH1 R132H的过表达载体转染胶质瘤细胞系U87MG和U251MG，感染72hr后收集细胞蛋白样品进行电泳。电泳时，按每孔50μg蛋白上样量等量上样。以400mA恒压转膜100min。将膜放入放入1％酪蛋白/TBS中于室温下振荡封闭1h。加入TBST稀释后的IDHI抗体(1：5000稀释)，4℃振荡孵育过夜。吸净一抗，TBST漂洗膜10min×3；加入稀释后的二抗(均为1：4000稀释)，水平摇床室温孵育1h。吸净二抗，TBST漂洗膜，10min×4，混合ECL(A：B＝100：1液)，充分覆盖湿润NC膜表面，静置1min；于暗室中用保鲜膜将NC膜封好，将X光片平放于NC膜上显影并记录数据。结果可见，IDH1 R132H抗体可以特异性识别胶质瘤细胞中的IDH1 R132H表位(见图2-3)。
实施例3
单克隆抗体的轻链基因和重链基因可变区的分离
采用聚合酶链式反应法分离抗体基因可变区，其中VLF与VLR为用于轻链可变区基因扩增的下游引物，VLF和VLR用于重链可变区基因扩增的下游引物。所使用的模板为利用Trizol法提取的杂交瘤细胞株总RNA，消化DNA后，利用反转录试剂盒反转录成cDNA。条件为PCR反应条件为：95℃预变性5min；然后95℃15s，55℃30s，72℃50s，反应40个循环。回收目的片段，并克隆到T-载体中，然后进行测序(英骏公司)。对测序序列进行比对，以确定抗体可变区的核普酸序列，并进而确定相应的氨基酸序列。
按照上述方法，从杂交瘤细胞株A2E8、A11B3和D4C7中克隆出各细胞株所分泌的单抗的可变区基因，并确定了相应的氨基酸序列。表1显示了所使用的引物的序列。表2显示了株单克隆抗体的重链和轻链可变区的核甘酸序列和氨基酸序列的序列编号。
表1：用于扩增单抗可变区基因的引物的序列
表2：3株单克隆抗体的可变区的序列编号
用上述鉴定的序列，通过已知的抗体工程技术，可以制备各种基因工程抗体，例如嵌合抗体，人源化抗体，单链抗体，双抗体等，并保留其所源自的单克隆抗体的生物学特性。因此，凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。
实施例4
抗IDH1 R132H抗体功能鉴定
免疫荧光细胞化学染色观察DH1 R132H抗体与表达IDH1 R132H抗原细胞的结合能力。利用表达IDH1全长的过表达载体和表达IDH1 R132H的过表达载体转染HEK293FT细胞，接种细胞爬片，转染72hr后，用4％多聚甲醛室温固定15min，15％驴血清封闭1h，一抗4℃过夜，PBS洗涤后加入荧光二抗，室温孵育1h，PBS洗涤后封片，荧光显微镜观察并拍照。结果显示获得的单克隆抗体可以特异性识别表达在细胞中的IDH1 R132H表位。(见图4-6)
实施例5
为了验证IDH1 R132H在肿瘤标本中的特异性，我们利用A2E8细胞株分泌单抗对人胶质瘤组织进行染色验证其特异性。脑胶质瘤石蜡切片置于60℃烤片10分钟，二甲苯脱蜡：二甲苯1(10min)，二甲苯2(10min)；梯度水化：无水乙醇1(10min)，无水乙醇2(10min)，95％乙醇(5min)，80％乙醇(5min)，70％乙醇(5min)，50％乙醇(5min)，双蒸水(5min)；1×PBS洗(3min×3次)；抗原修复：预热EDTA抗原修复液，将切片完全浸入修复液，注意微波过程中避免修复液蒸发过多，暴露组织，注意补液，微波过程中应将微波调整至中火或小火，保持温度(92℃-98℃，20min)；自然冷却至室温，组画笔距离组织0.5cm圈出组织轮廓，15％驴血清稀释液室温封闭30min；1×PBS洗(3min×3次)；一抗4℃过夜；1×PBS洗(3min×3次)洗涤后加入荧光二抗，置于37℃孵育2小时。使用含DAPI的甘油封片，并于荧光显微镜下观察拍照。结果显示获得的单克隆抗体可以特异性识别表达在胶质瘤细胞中的IDH1 R132H表位。(见图7)
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公开的所有教导，可以对细节进行各种修改和变动，并且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。
序列表
<110> 北京爱仁医疗科技有限公司
<120> 抗IDHI R132H抗体及其制备方法和用途
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<211> 55
<212> DNA
<213> 人工序列
<400> 28
ggcggtggcg gctcgggtgg aggcggatct gt(cg)(ac)a(ag)c tgcag(cg)agt c(at)gg 55
<210> 29
<211> 55
<212> DNA
<213> 人工序列
<400> 29
ggcggtggcg gctcgggtgg aggcggatct gt(cg)(ac)a(ag)c tgcag(cg)agt c(at)gg 55
<210> 30
<211> 44
<212> DNA
<213> 人工序列
<400> 30
gcgcggccgc tgaggagacg gtgaccgtgg tcccttggcc ccag 44
<210> 31
<211> 41
<212> DNA
<213> 人工序列
<400> 31
cgagccgcca cgcccgagcc gcctcctggt gggaagatgg a 41
Claims (10)
1.一种分离的抗IDH1R132H抗体或其功能性衍生物，其特征在于，所述抗IDH1R132H抗体或其功能性衍生物特异性地识别IDH1R132H的表位，所述抗IDH1R132H抗体具有：(1)SEQIDNO：2、6、10中的一个表示的抗体重链可变区的氨基酸序列或其保守型变异序列；和(2)SEQ ID NO：4、8、12中的一个表示的轻链可变区的氨基酸序列或其保守型变异序列。
2.如权利要求1所述的抗IDH1R132H抗体或其功能性衍生物，其特征在于，所述衍生物为所述抗IDH1R132H抗体的片段、抗体/抗体片段一因子融合蛋白、或抗体/抗体片段一化学偶联物。
3.如权利要求2所述的抗IDH1R132H抗体或其功能性衍生物，其特征在于，所述抗IDH1R132H抗体的片段为Fab、Fab'、F(ab)'2、或scFv。
4.一种人源化抗体或其功能性衍生物，其特征在于，所述人源化抗体包含人源化的重链和人源化的轻链，其中所述重链包含：(1)权利要求1中所述的重链可变区的氨基酸序列或其保守型变异序列，和(2)来自人受体抗体重链的骨架；其中所述轻链包含：(1)权利要求1中所述的轻链可变区的氨基酸序列或其保守型变异序列，和(2)来自人受体抗体轻链的骨架。
5.一种分离的核酸分子，其特征在于，所述核酸分子编码权利要求1至4中任一项所述的抗IDH1R132H抗体的重链和/或轻链的可变区或所述抗IDH1R132H抗体的全长的氨基酸。
6.一种构建载体，其特征在于，所述构建体含有如权利要求1至4中任一项所述的核酸分子；优选的是，所述构建体由所述核酸分子插入到表达载体的多克隆位点构建而成。
7.一种表达系统，其特征在于，所述表达系统由权利要求5所述的构建体转染到宿主细胞构建而成。
8.一种制备权利要求1至4中任一项所述的抗IDH1R132H抗体的方法，其特征在于，所述方法包括如下步骤：(1)在适合表达所述抗IDH1R132H抗体的条件下，培养权利要求6所述的表达系统，从而表达出所述抗IDH1R132H抗体；(2)纯化分离出所述抗IDH1R132H抗体。
9.如权利要求1至4中任一项所述的抗IDH1R132H抗体或其功能性衍生物在制备分子阻滞药物上的用途；优选为在制备用于肿瘤治疗或肿瘤诊断的分子阻滞药物中的用途；更优选为在制备用于诊断神经胶质瘤尤其是II及III级弥散性神经胶质瘤或者急性髓系白血病的诊断试剂中的用途。
10.一种药物组合物，其特征在于，所述药物组合物包含治疗有效量的如权利要求1至4中任一项所述的抗IDH1R132H抗体或其功能性衍生物。
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| CN108948205A (zh) * | 2018-07-13 | 2018-12-07 | 北京金岱生物科技有限公司 | 抗idh1r132h单克隆抗体及其应用 |
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| CN108727500A (zh) * | 2018-05-03 | 2018-11-02 | 南京基诺米医疗科技有限公司 | 鼠抗人idh1 r132h突变蛋白单克隆抗体制备及其免疫组化用途 |
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| CN108948205B (zh) * | 2018-07-13 | 2021-09-28 | 北京金岱生物科技有限公司 | 抗idh1r132h单克隆抗体及其应用 |
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