CN110669770B - 人源化单克隆抗体、其制备方法及其用途 - Google Patents
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Abstract
本发明公开一种抗人CD20人源化单克隆抗体、编码该抗体的核酸分子、包含该核酸分子的重组载体、包含所述重组载体的重组细胞以及所述抗人CD20人源化单克隆抗体的制备方法及其医药用途。其中，编码所述抗体的核酸分子包含如SEQ ID NO:1所示的编码轻链的核苷酸序列和如SEQ ID NO:2所示的编码重链的核苷酸序列；并分别在所述两个序列上设计信号肽和终止密码子。本发明由于对密码子进行优化，转基因后的CHO细胞表达抗人CD20人源化抗体的表达量高；且本发明提供的发酵方法，特别是在添加补料批培养基后，延长细胞生长时间，提高表达水平，降低生产成本，获得高纯度的目的蛋白。
Description
技术领域
本发明属于生物技术领域，尤其涉及一种抗CD20人源化单克隆抗体SH006-2、编码该抗体的核酸分子、包含该核酸分子的重组载体、包含所述重组载体的重组细胞以及所述抗CD20人源化单克隆抗体SH006-2的制备方法及其医药用途。
背景技术
CD20抗原是前B细胞和成熟的B淋巴细胞上的疏水性跨膜蛋白，其对B淋巴细胞的增殖和分化具有非常重要的调节作用。CD20在早期前B细胞发育过程中表达，并且持续表达至浆细胞分化时。研究发现，超过90％的非霍奇金淋巴瘤患者、慢性淋巴瘤患者及大B细胞淋巴瘤患者B细胞表面高表达CD20抗原。抗CD20单克隆抗体可以与肿瘤表面的CD20分子结合，通过抗体依赖性细胞介导的细胞毒作用(antibody-dependent cell-mediatedcytotoxicity，ADCC)和补体依赖的细胞毒性作用(complement-dependent cytotoxicity，CDC)杀死肿瘤细胞。
自1997年第一个抗CD20治疗性单抗利妥昔单抗(rituximab，
美罗华)被FDA批准上市至今，利妥昔单抗一直位列全球畅销药物前列，同时也推动了该类药物的迅速发展。根据抗体不同的结构、人源化程度以及Fc段修饰等大致经历了3代研发：第一代抗体为鼠源抗体或嵌合型抗体(-ximab，以利妥昔单抗最具代表性)；第二代抗体为人源化抗体(-zumab)或全人源抗体(-mumab，以奥法木单抗(ofatumumab，
)为代表)，其相较于第一代抗体免疫原性大大降低；第三代抗体则是在第一代和第二代的基础上通过糖基化等工程对抗体Fc段进行改造修饰从而增加抗体对FcγIIIa结合亲和力而增强效应功能，最终提高治疗活性(以奥奴珠单抗(obinutuzumab，
GA101)为代表)。
现有的利妥昔单抗属于人鼠嵌合抗体，分子中含有较多的鼠源氨基酸序列，可能存在一定的输液反应和免疫原性而导致安全性方面的隐患。目前的临床实践已表明，利妥昔单抗的常见不良反应之一即为输液反应性。而且已有大量临床数据表明，对于低度恶性淋巴瘤，
的单药疗效约为50％，另外50％左右病人无效，60％初治有效病人再次治疗无效；对于恶性度较高的弥漫大B细胞淋巴瘤、CD20表达低下的慢性淋巴细胞性白血病患者，即使与CHOP方案(环磷酰胺+多柔比星+长春新碱+泼尼松)联合应用，仍然有半数或以上患者治疗无效或复发。
此外，目前世界上抗体药物的产能远低于需求量。抗体的产量除了受制于培养规模、生产工艺以及纯化工艺等因素外，从很大程度上还依赖于抗体基因的偏好性及其在哺乳动物细胞内的表达水平。
发明内容
为了克服现有技术的不足、获得具有更高表达水平和生物学活性的新抗体，本发明公开了一种通过信号肽及密码子优化和基因敲除技术获得的抗人CD20人源化单克隆抗体SH006-2的核酸分子，继而通过载体构建、细胞转染、细胞株筛选、细胞培养和活性物质生产、纯化等手段获得的岩藻糖完全敲除的重组抗人CD20人源化单克隆抗体SH006-2。本发明具体包括以下几个方面：
本发明的第一方面涉及编码抗人CD20人源化单克隆抗体SH006-2的核酸分子，其特征在于包含编码轻链的核苷酸序列和编码重链的核苷酸序列。
编码轻链的核苷酸序列为：
GATATTGTGATGACTCAGACTCCACTGTCACTGCCCGTGACACCTGGCGAGCCCGCCTCTATCTCCTGTAGGAGCTCTAAGTCCCTGCTGCATTCCAACGGCATCACCTACCTGTATTGGTACCTGCAGAAGCCTGGCCAGTCTCCTCAGCTGCTGATCTACCAGATGTCCAACCTGGTGTCTGGCGTGCCTGATAGGTTTTCCGGCTCTGGCTCCGGCACAGACTTTACCCTGAAGATCTCCAGAGTGGAGGCTGAGGATGTGGGCGTGTATTACTGCGCCCAGAATCTGGAGCTGCCATATACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGAGAACCGTGGCTGCCCCAAGCGTGTTTATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGCACAGCTAGCGTGGTGTGCCTGCTGAATAACTTCTACCCCAGAGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCTCTGCAGTCTGGCAACTCCCAGGAGTCTGTGACAGAGCAGGATTCCAAGGACAGCACATACTCCCTGTCTAGCACCCTGACACTGAGCAAGGCTGACTACGAGAAGCACAAGGTGTACGCTTGCGAGGTCACTCATCAGGGACTGTCATCTCCTGTCACTAAGAGTTTTAATCGCGGCGAGTGT(SEQ ID NO:1)；
编码重链的核苷酸序列为：
CAGGTCCAGCTGGTCCAGAGTGGTGCAGAAGTGAAGAAGCCAGGCTCCAGCGTGAAGGTGTCCTGTAAGGCCAGCGGCTACGCCTTTAGCTACTCCTGGATCAATTGGGTGCGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCAGAATCTTCCCTGGCGATGGCGACACCGATTACAACGGCAAGTTCAAGGGCAGAGTGACCATCACAGCCGATAAGAGCACCTCCACAGCCTACATGGAGCTGTCTAGCCTGAGATCCGAGGACACCGCCGTGTATTACTGCGCTAGAAACGTGTTCGACGGCTATTGGCTGGTGTATTGGGGCCAGGGCACACTGGTGACAGTGTCTAGCGCCTCTACAAAGGGCCCCAGCGTGTTTCCACTGGCTCCCTCCTCTAAGAGCACAAGCGGCGGCACCGCTGCCCTGGGCTGTCTGGTGAAGGACTACTTTCCAGAGCCTGTGACAGTGAGCTGGAATTCCGGCGCTCTGACCTCTGGCGTGCACACCTTTCCAGCCGTGCTGCAGTCTTCCGGCCTGTACTCCCTGTCTAGCGTGGTGACCGTGCCCAGCTCCTCTCTGGGCACCCAGACATATATCTGCAACGTGAATCACAAGCCTTCCAATACAAAGGTGGACAAGAAGGTGGAGCCAAAGTCCTGTGACAAGACCCATACATGCCCCCCATGTCCTGCTCCCGAGCTGCTGGGCGGCCCTTCCGTGTTCCTGTTTCCCCCAAAGCCCAAGGATACCCTGATGATCAGCAGAACCCCAGAGGTGACATGCGTGGTGGTGGACGTGTCCCATGAGGATCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAACGCCAAGACAAAGCCTAGAGAGGAGCAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTGTCTAATAAGGCCCTGCCTGCTCCAATCGAGAAGACAATCTCTAAGGCTAAGGGCCAGCCTCGGGAGCCCCAGGTGTATACCCTGCCTCCATCCAGAGACGAGCTGACCAAGAATCAGGTGTCTCTGACATGCCTGGTGAAGGGCTTCTATCCATCCGATATCGCTGTGGAGTGGGAGAGCAATGGCCAGCCTGAGAACAATTATAAGACAACCCCACCTGTGCTGGATTCTGACGGCAGCTTTTTCCTGTATTCCAAGCTGACCGTGGATAAGTCTAGATGGCAGCAGGGCAACGTGTTCTCCTGTAGCGTGATGCACGAGGCACTGCATAATCACTACACCCAGAAGTCACTGTCACTGAGTCCAGGCAAA(SEQ ID NO:2)。
进一步地，可以将此类核苷酸序列与编码对于初始抗体而言天然的信号肽或异源信号肽的多核苷酸融合。具体而言，所述核酸分子可在编码其轻链的核苷酸序列和编码其重链的核苷酸序列的5’端分别进一步包含编码信号肽的核苷酸序列，所述信号肽可以是天然的信号肽，也可以是异源信号肽；在编码轻链的核苷酸序列和编码重链的核苷酸序列的3’端分别进一步包含终止密码子。
更进一步地，所述信号肽由氨基酸序列：MGWSCIILFLVATATGVHS(SEQ ID NO:6)组成。
编码所述信号肽的核苷酸序列为：
ATGGGATGGTCCTGTATTATCCTGTTCCTGGTCGCAACCGCAACTGGTGTCCACTCA(SEQ ID NO:5)。
所述轻链上可以包含的终止密码子为TGA，所述重链上可以包含的终止密码子为TAA。
本发明的第二方面涉及一种载体，例如表达载体，其包含编码抗人CD20人源化单克隆抗体SH006-2的重链或轻链或二者的核苷酸序列。在此类载体中，本发明的核苷酸序列可以与一种或多种调节元件可操作连接。其中，所述调节元件选自表达调控序列，如启动子、增强子等。
本发明的载体包括一段与编码抗人CD20人源化单克隆抗体SH006-2重链或轻链的核酸序列可操作性连接的调控元件(例如启动子或增强子)。“可操作性连接”是指所构成的核酸序列的布置使得可执行其正常功能。因此，与编码多肽的核苷酸序列可操作性连接的调控元件能够指导转录、复制和/或翻译而得到所述抗体。在一种实施方案中，该载体编码人源化的单克隆抗体SH006-2的轻链或重链氨基酸序列。
在本发明中，所述表达载体例如为原核表达载体、真核表达载体、噬菌体载体或病毒载体。进一步地，所述载体选自真核载体。更进一步地，所述载体选自市售载体pcDNA3.4-DHFR和pcDNA3.4-G418。抗体的重链和轻链可以分别在pcDNA3.4-DHFR载体和pcDNA3.4-G418中表达。pcDNA3.4-DHFR上还含有真核筛选标记DHFR标签和原核筛选标签Ampicilline，可通过氨甲喋呤加压筛选高表达重链细胞株，pcDNA3.4-G418上含有真核筛选标记G418标签和原核筛选标签Ampicilline，可以通过新霉素加压筛选高表达轻链细胞株。
在本发明的一个具体实施方案中，在编码轻链的核苷酸序列(SEQ ID NO:1)的5’端分别依次加上kozak序列、HindIII和信号肽序列，在3’端加上终止密码子及XhoI酶切位点,通过酶切连接插入到pcDNA3.4-G418；在编码重链的核苷酸序列(SEQ ID NO:2)的5’端分别依次加上kozak序列、HindIII和信号肽序列，在3’端加上终止密码子和XhoI酶切位点，通过酶切连接插入到pcDNA3.4-DHFR载体中后，将最终获得的含SH006-2全长重链和轻链基因的重组质粒命名分别命名为pcDNA3.4-DHFR-SH006-2和pcDNA3.4-G418-SH006-2。pcDNA3.4-G418-SH006-2和pcDNA3.4-DHFR-SH006-2的质粒图谱参见附图2。
本发明的第三方面涉及一种重组细胞，其含有本发明第二方面任一项的重组载体。本发明所述重组细胞为敲除负责编码岩藻糖基转移酶8即FUT8的基因的细胞。
进一步地，所述细胞为CHO细胞，所述CHO细胞选自CHO-S、CHO-K1、CHO/DG44细胞。所述敲除是指，利用CRISPR/Cas9基因编辑技术将CHO细胞中负责编码岩藻糖的基因即FUT8基因敲除。
本发明的第四方面涉及抗人CD20人源化单克隆抗体SH006-2，其由本发明第一方面任一项的核酸分子、第二方面任一项的重组载体或者第三方面任一项的重组细胞制备得到。
进一步地，所述抗体轻链的氨基酸序列如SEQ ID NO:3所示，其重链的氨基酸序列如SEQ ID NO:4所示。
进一步地，所述抗体包括CH2结构域；所述CH2结构域中几乎或完全不具有岩藻糖糖基化修饰。
在本发明的一个具体实施方案中，所述抗人CD20人源化单克隆抗体SH006-2具有较利妥昔单抗增强的ADCC活性。
在本发明的一个具体实施方案中，所述抗人CD20人源化单克隆抗体SH006-2的ADCC活性强于GA101单抗。
本发明的第五方面涉及所述抗人CD20人源化单克隆抗体SH006-2的制备方法，其具体包括以下步骤：
1)将本发明第一方面所述核酸分子的序列克隆入表达载体中，得到重组表达载体；
2)将重组表达载体转入宿主细胞，即负责编码岩藻糖基转移酶8即FUT8的基因被敲除的CHO细胞中，得到重组细胞；
3)在筛选培养基上对重组细胞进行加压筛选，得到稳定生长的细胞池；
4)从步骤3)的细胞池中筛选高表达细胞株，然后检测单克隆上清中SH006-2的表达水平，筛选培养得到表达量较高的克隆，扩增培养后冻存细胞备用；
5)取步骤4)获得的克隆细胞于目标培养基中培养，检测抗体表达产量；然后挑选出表达高的克隆进行培养，获得高表达的单克隆细胞株，收获培养上清并纯化，得到本发明第四方面所述的抗人CD20人源化单克隆抗体SH006-2。
在上述步骤1)中，所述重组表达载体可以分别为pcDNA3.4-G418和pcDNA3.4-DHFR。在编码轻链的核苷酸序列(SEQ ID NO:1)的5’端分别依次加上kozak序列、HindIII和信号肽序列，在3’端加上终止密码子及XhoI酶切位点，通过酶切连接插入到pcDNA3.4-G418；在编码重链的核苷酸序列(SEQ ID NO:2)的5’端分别依次加上kozak序列、HindIII和信号肽序列，在3’端加上终止密码子和XhoI酶切位点。通过酶切连接插入到pcDNA3.4-DHFR载体中后，将最终获得的含SH006-2全长重链和轻链基因的重组质粒命名分别命名为pcDNA3.4-DHFR-SH006-2和pcDNA3.4-G418-SH006-2。
在上述步骤2)中，所述敲除是指，利用CRISPR/Cas9基因编辑技术将CHO细胞中负责编码岩藻糖基转移酶8即FUT8的基因敲除。
在上述步骤3)中，所述加压筛选采用甲氨蝶呤(Methotrexate,MTX)梯度加压；所述稳定生长的细胞池是在1000nM MTX压力下稳定生长的细胞池。
在上述步骤4)中，所述检测是指采用ELISA法检测。
在上述步骤5)中，所述目标培养基与步骤3)所述的筛选培养基相同，均为不含次黄嘌呤和胸腺嘧啶的培养基即HT-培养基，优选为含有CD OptiCHO培养基、GlutaMAX(100×)培养基和10％F-68(100×)培养基的混合培养基。并且，对所述挑选出的表达高的克隆进行分批补料式培养至收样。
本发明的第六方面涉及一种本发明第一方面所述的核酸分子、第二方面所述的重组载体或者第三方面所述的重组细胞在制备本发明第四方面所述的抗人CD20人源化单克隆抗体SH006-2中的用途。
本发明的第七方面涉及一种含有所述抗人CD20人源化单克隆抗体SH006-2作为活性成分的药物，所述药物任选含有药学上可接受的载体或赋形剂。
本发明还涉及所述抗人CD20人源化单克隆抗体SH006-2在预防或治疗与CD20抗原有关的疾病或病症如肿瘤、自身免疫性疾病或炎症疾病的药物中的用途；优选地，所述肿瘤选自B细胞型非霍奇金氏淋巴瘤、慢性淋巴细胞白血病；所述自身免疫疾病选自自身免疫溶血性贫血或原发性血小板减少性紫癜；所述炎症疾病选自类风湿性关节炎或多发性硬化症。
本发明还涉及抗人CD20人源化单克隆抗体SH006-2在制备与CD20抗原有关的诊断剂中的用途，该诊断剂用于一种在体内诊断与CD20抗原有关的疾病或病症的方法，所述方法包括对要检查的受试者施用有效量的本发明第四方面所述的抗人CD20人源化单克隆抗体。
本发明还涉及含有如上所述的抗人CD20人源化单克隆抗体SH006-2作为活性成分的试剂、组合物或试剂盒。
发明的有益效果
本研究在GA101抗体的基础上通过密码子优化和分子生物学等技术，设计获得了一种新的抗人CD20人源化单克隆抗体SH006-2的编码基因，其编码的抗体氨基酸序列与GA101相同，但在表达体系中相比较于其它序列表达量更高。同时通过基因敲除技术完全去除了抗体的岩藻糖糖基化修饰，增强了抗体的ADCC活性，达到提升肿瘤治疗效果和/或扩大肿瘤适用范围的目的。
由于本发明所述单克隆抗体SH006-2为全人源化抗体，所以不会发生其他抗体所容易产生的致命免疫应答(HAMA)反应，并且与受体的亲和力大大增强，同时不受血清中正常IgG的竞争抑制，可以在体内更加有效地清除B淋巴细胞，弥补该靶分子可能出现的耐药和无效情况。所述抗体的岩藻糖修饰几乎或完全被敲除，并且具有更高的ADCC活性，这种改变在临床上将有益地降低因患者对抗体发生治疗性抵制的几率。同时，本发明所述制备方法较通过基因工程化改造细胞(如中国专利CN1902231A和CN101291954A中公开)更加便捷，抗体产量也显著增加。
附图说明
图1为构建CHO-DG44Fut8-/-细胞株的技术路线图。
图2为含有SH006-2轻链的质粒pcDNA3.4-G418-SH006-2和含有SH006-2重链的质粒pcDNA3.4-DHFR-SH006-2的图谱，其中LC代表SH006-2的轻链，其中HC代表SH006-2的重链。
图3为质粒线性化酶切验证电泳图：泳道1是SH006-2经PvuI-HF酶切电泳图；泳道2和泳道3分别是Maker125和Maker15000。
图4和图5为SH006-2抗体与FcγRIIIa(CD16a,158F)的结合曲线。
图6为SH006-2诱导的NK-92MI-CD16a细胞对Raji细胞和Daudi细胞的ADCC效应曲线。
图7为
SH006-2和
对B淋巴细胞瘤细胞Daudi细胞的抑制率图。
图8为试验各组的小鼠肿瘤照片(Daudi实体瘤模型)：从上到下分别为：SH006-230mg/kg、SH006-2 3mg/kg、SH006-2 0.3mg/kg、
3mg/kg、
3mg/kg、
0.3mg/kg和PBS组。
具体实施方式
以下结合附图与具体实施例对本发明做进一步的描述，本发明的保护内容不局限于以下实施例。还应该理解，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。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求及其任何等同物为本发明的保护范围。在本发明的说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。实施本发明的过程、条件、试剂、实验方法等，除以下专门提及的内容之外，均为本领域技术人员的普遍知识和公知常识，本发明没有特别限制内容。
实施例1核苷酸序列的优化及瞬时表达评价
本发明所述单克隆抗体轻链和重链的氨基酸序列来自WHO Drug Information,Vol.22,No.2,2008。将上述文献报道的氨基酸序列转化为核苷酸序列，并针对可能影响抗体在哺乳动物细胞中表达的一系列参数：密码子偏好性、GC含量(即DNA的4种碱基中鸟嘌呤G和胞嘧啶C所占的比率)、CpG岛(即CpG双核苷酸在基因组中密度较高的区域)、mRNA的二级结构、拼接位点、前成熟PolyA位点、内部Chi位点(基因组中一段短的DNA片段，在该位点附近发生同源重组的几率增加)或者核糖体结合位点、RNA不稳定序列、反向重复序列及可能干扰克隆的限制性酶切位点等进行优化；同时增加了可能会提高翻译效率的相关序列，例如Kozak序列、SD序列，以及终止密码子。设计得到新的编码与GA101相同氨基酸序列的重链基因和轻链基因，另外在重链和轻链的5’端分别设计上根据氨基酸序列优化而得的编码信号肽的核苷酸序列；此外，还对轻链和重链核苷酸序列的3’端分别加上终止密码子。
最终优化获得8对抗体优化核苷酸候选序列(SH006-1、SH006-2、SH006-3、SH006-4、SH006-5、SH006-6、SH006-7、SH006-8)，将所述核苷酸序列克隆至商业化真核表达载体pcDNA3.4(购自Life公司)中，瞬时转染评价发现，SH006-2优化核苷酸序列的表达量远远优于其他分子(参见表1)。
表1抗体优化核苷酸候选序列的瞬转表达量
SH006-2优化核苷酸序列含有与GA101相同氨基酸序列的重链基因(SEQ ID NO:1)和轻链基因(SEQ ID NO:2)，另外在重链和轻链的5’端分别设计上编码信号肽的核苷酸序列(SEQ ID NO:5)，其编码得到SEQ ID NO:6的信号肽；此外，对轻链核苷酸序列的3’端加上终止密码子TGA，对重链核苷酸序列的3’端加上终止密码子TAA。
优化好的编码SH006-2的轻链的核苷酸序列：
ATGGGATGGTCCTGTATTATCCTGTTCCTGGTCGCAACCGCAACTGGTGTCCACTCAGATATTGTGATGACTCAGACTCCACTGTCACTGCCCGTGACACCTGGCGAGCCCGCCTCTATCTCCTGTAGGAGCTCTAAGTCCCTGCTGCATTCCAACGGCATCACCTACCTGTATTGGTACCTGCAGAAGCCTGGCCAGTCTCCTCAGCTGCTGATCTACCAGATGTCCAACCTGGTGTCTGGCGTGCCTGATAGGTTTTCCGGCTCTGGCTCCGGCACAGACTTTACCCTGAAGATCTCCAGAGTGGAGGCTGAGGATGTGGGCGTGTATTACTGCGCCCAGAATCTGGAGCTGCCATATACCTTCGGCGGCGGCACCAAGGTGGAGATCAAGAGAACCGTGGCTGCCCCAAGCGTGTTTATCTTCCCTCCATCTGATGAGCAGCTGAAGTCTGGCACAGCTAGCGTGGTGTGCCTGCTGAATAACTTCTACCCCAGAGAGGCCAAGGTGCAGTGGAAGGTGGATAACGCTCTGCAGTCTGGCAACTCCCAGGAGTCTGTGACAGAGCAGGATTCCAAGGACAGCACATACTCCCTGTCTAGCACCCTGACACTGAGCAAGGCTGACTACGAGAAGCACAAGGTGTACGCTTGCGAGGTCACTCATCAGGGACTGTCATCTCCTGTCACTAAGAGTTTTAATCGCGGCGAGTGTTGA；
优化好的编码SH006-2的重链的核苷酸序列：
ATGGGATGGTCCTGTATTATCCTGTTCCTGGTCGCAACCGCAACTGGTGTCCACTCACAGGTCCAGCTGGTCCAGAGTGGTGCAGAAGTGAAGAAGCCAGGCTCCAGCGTGAAGGTGTCCTGTAAGGCCAGCGGCTACGCCTTTAGCTACTCCTGGATCAATTGGGTGCGGCAGGCCCCCGGCCAGGGCCTGGAGTGGATGGGCAGAATCTTCCCTGGCGATGGCGACACCGATTACAACGGCAAGTTCAAGGGCAGAGTGACCATCACAGCCGATAAGAGCACCTCCACAGCCTACATGGAGCTGTCTAGCCTGAGATCCGAGGACACCGCCGTGTATTACTGCGCTAGAAACGTGTTCGACGGCTATTGGCTGGTGTATTGGGGCCAGGGCACACTGGTGACAGTGTCTAGCGCCTCTACAAAGGGCCCCAGCGTGTTTCCACTGGCTCCCTCCTCTAAGAGCACAAGCGGCGGCACCGCTGCCCTGGGCTGTCTGGTGAAGGACTACTTTCCAGAGCCTGTGACAGTGAGCTGGAATTCCGGCGCTCTGACCTCTGGCGTGCACACCTTTCCAGCCGTGCTGCAGTCTTCCGGCCTGTACTCCCTGTCTAGCGTGGTGACCGTGCCCAGCTCCTCTCTGGGCACCCAGACATATATCTGCAACGTGAATCACAAGCCTTCCAATACAAAGGTGGACAAGAAGGTGGAGCCAAAGTCCTGTGACAAGACCCATACATGCCCCCCATGTCCTGCTCCCGAGCTGCTGGGCGGCCCTTCCGTGTTCCTGTTTCCCCCAAAGCCCAAGGATACCCTGATGATCAGCAGAACCCCAGAGGTGACATGCGTGGTGGTGGACGTGTCCCATGAGGATCCCGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAACGCCAAGACAAAGCCTAGAGAGGAGCAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTGTCTAATAAGGCCCTGCCTGCTCCAATCGAGAAGACAATCTCTAAGGCTAAGGGCCAGCCTCGGGAGCCCCAGGTGTATACCCTGCCTCCATCCAGAGACGAGCTGACCAAGAATCAGGTGTCTCTGACATGCCTGGTGAAGGGCTTCTATCCATCCGATATCGCTGTGGAGTGGGAGAGCAATGGCCAGCCTGAGAACAATTATAAGACAACCCCACCTGTGCTGGATTCTGACGGCAGCTTTTTCCTGTATTCCAAGCTGACCGTGGATAAGTCTAGATGGCAGCAGGGCAACGTGTTCTCCTGTAGCGTGATGCACGAGGCACTGCATAATCACTACACCCAGAAGTCACTGTCACTGAGTCCAGGCAAATAA。
实施例2 CHO-DG44 Fut8-/-细胞株的构建
使用的宿主细胞为CHO-DG44Fut8-/-细胞株，该细胞株为岩藻糖双等位基因敲除(Fut8-/-)的CHO-DG44的细胞系，由南京金斯瑞生物科技股份有限公司开发。具体方法是通过基因工程技术即CRISPR/Cas9技术改造表达系统，定点敲除抗体表达宿主细胞CHO-DG44中负责编码岩藻糖的基因即FUT8基因。具体技术路线如图1所示。
实施例3表达载体的构建
采用pcDNA3.4-G418和pcDNA3.4-DHFR载体(购自invitrogen)作为表达转染CHO-DG44细胞的专用载体。pcDNA3.4-G418含有轻链所使用的启动子CMV Promoter、真核筛选标记G418标签和原核筛选标签Ampicilline。pcDNA3.4-DHFR含有重链所使用的启动子CMVPromoter、真核筛选标记DHFR标签和原核筛选标签Ampicilline。
基因合成得到SH006-2抗体表达轻链和重链的核苷酸序列，并分别连接到pcDNA3.1(+)和pcDNA3.1-Hygro(+)载体(由南京金斯瑞合成)。分别以含有SH006-2轻链的pcDNA3.1(+)载体和含有SH006-2重链的pcDNA3.1-Hygro(+)载体为模板，用HindIII、XhoI两种限制性内切酶进行双酶切，37℃水浴酶切1h，以110V，300mA，40min进行DNA琼脂糖凝胶电泳，回收SH006-2的轻链和重链片段(片段1：约717bp，片段2：约1407bp)。与此同时，将pcDNA3.4-G418和pcDNA3.4-DHFR载体用HindIII、XhoI两种限制性内切酶进行双酶切，37℃水浴酶切1h，以110V，300mA，40min进行DNA琼脂糖凝胶电泳，分别回收pcDNA3.4-G418和pcDNA3.4-DHFR载体大片段(片段3：约6091bp，片段4：约5930bp)。将片段1和片段3、片段2和片段4通过DNA连接酶进行酶连，并转化大肠杆菌感受态细胞DH5α，挑选出阳性克隆并进行质粒提取和酶切验证，对酶切验证正确的质粒进行DNA测序，获得含有SH006-2轻链片段的pcDNA3.4-G418载体以及含有SH006-2重链片段的pcDNA3.4-DHFR载体，即pcDNA3.4-G418-SH006-2载体和pcDNA3.4-DHFR-SH006-2载体。其质粒结构示意图如图2所示。
根据SH006-2的琼脂糖凝胶电泳图，SH006-2酶切后的片段大小与理论值大小相一致。经过美吉公司的DNA测序，SH006-2的轻链和重链的DNA序列与理论的DNA序列相一致。
实施例4 SH006-2质粒的制备
加入200μl SH006-2的质粒菌液到500ml含有50μg/ml的氨苄西林的LB液体培养基。37℃、200rpm，过夜培养。质粒提取按照无内毒素质粒大提试剂盒(天根，货号：DP117，批号：N3125)的操作说明进行操作提取。
实施例5 SH006-2质粒的线性化
按如下步骤对SH006-2质粒进行线性化：
1)酶切：按照以下的酶切体系进行酶切，1000μl SH006-2质粒，80μl PvuI，120μlNEB Buffer加入到1.5ml EP罐中，酶切总体系为1200μl，37℃水浴酶切4h；
2)从EP管中取出酶切产物，转移到15ml的离心管并加入120μl 3M NaAC和3000μl的无水乙醇进行混匀，-20℃过夜；
3)取出离心管进行12000g，4℃离心10min；
4)弃去上清，加入1ml 70％乙醇，12000g，4℃离心5mim，重复一次；
5)将离心管进行室温干燥，然后加入100μl的ddH2O，-20℃冻存；
6)用NANO Drop 2000测定线性化质粒的浓度，并进行琼脂糖凝胶电泳。
实验结果：SH006-2的分子线性化后的琼脂糖凝胶电泳图如图3所示。通过限制性内切酶PvuI-HF酶切后，酶切条带大小符合预期。
实施例6 SH006-2稳转细胞株筛选
使用电转染方法通过MaxCyte将上述获得的SH006-2表达载体电转入CHO-DG44宿主细胞，在筛选培养基(即HT-培养基，含1000nM MTX的CD OptiCHO+GlutaMAX(100×)+F-68(100×))中于37℃，5％CO2，110rpm下培养，每2～3天进行细胞计数和传代，更换新鲜培养基，直至细胞活力恢复至85％以上。然后开始进行MTX梯度加压，每隔2～3天进行细胞计数和传代，更换含有MTX的新鲜培养基，直至细胞活力恢复至85％以上，再进行下一轮压力筛选；最终获得在1000nM MTX压力下稳定生长的细胞池。从细胞池中筛选高表达细胞株，检测单克隆上清中SH006-2的表达水平，进一步筛选培养得到表达量TOP10单克隆，扩增培养后冻存细胞备用。然后在目标培养基即含1000nM MTX的CD OptiCHO+GlutaMAX(100×)+F-68(100×)中对获得的单克隆进行批培养，进一步挑选出表达高的克隆进行培养，获得能够高效表达SH006-2抗体的5株单克隆细胞株。
细胞株经过培养基筛选及小试工艺优化，扩大至15L细胞发酵罐，采用分批补料式培养方式：将细胞进行37℃水浴解冻，接种至含有1000nM MTX的CD OptiCHO+GlutaMAX(100X)+F-68(100X)筛选培养基，传代3次，通过200g，5min进行离心更换新鲜的含有1000nMMTX的CD FortiCHO+GlutaMAX(100X)+F-68(100X)，并进行传代培养3次，细胞培养14天，并于第4、6、8、10天分别添加7.5％Feed-C，葡萄糖浓度低于2.5g/L时，补充葡萄糖。至浓度为5-5.5g/L，在第4、6、8、10、12、14天进行取样计数，检测细胞密度和活力；当细胞活力降至75％以下或培养周期达到14天，离心收集细胞培养上清，采用HPLC方法测定细胞上清抗体表达水平。采用Protein A(Pharmacia公司)亲和层析柱从细胞培养上清中直接分离纯化，并用SDS-PAGE电泳证明，所得产物纯度大于90％，然后将亲和层析的产物再次经过分子筛层析，获得纯度>98％的样品。对这些样品进行以下质量、体外结合活性、细胞生物活性和体内药效学试验分析。
实施例7抗体的质谱肽图分析
对获得的5株单克隆细胞株所产生的抗体进行质谱肽图分析，以确定信号肽的切除比例。结果如表2所示，表明本发明所述编码SH006-2抗体的基因中的信号肽得到了有效且精确地切割。
表2母克隆质谱肽图分析
| 克隆名称 | 重链信号肽未切除比例(％) | 轻链信号肽未切除比例(％) |
| S5C2 | 0.03 | 0.57 |
| S8H4 | 0.04 | 0.85 |
| S9H10 | 0.05 | 0.67 |
| S4C6 | 0.03 | 0.74 |
| S4E10 | 0.04 | 0.77 |
实施例8 SH006-2抗体的N-糖基化分析
将SH006-2的5株母克隆(S5C2、S8H4、S9H10、S4C6、S4E10)样品分别进行糖苷酶PNGaseF脱糖处理，然后分别往样品中先后加入40μL HILIC标记试剂(配制比例为24mg无水醋酸钠、20mg硼酸、30mg邻氨基苯甲酸即2-AA、32mg氰基硼氢化钠溶于1mL甲醇)和60μL甲醇，置于80℃水浴75min，取出后15000rpm、10℃离心20min将蛋白沉淀，取上清真空离心干燥。待干燥后加入20μL50％乙腈复溶，使用HILIC-UPLC-MS进行分离鉴定，相应仪器及条件参数如下：Waters H-Class Bio超高效液相色谱仪；色谱柱：ACQUITY UPLC BEH GlycanColum，1.7μm,2.1mm×150mm；柱温：60℃；流动相A：100mM甲酸铵，流动相B：乙腈；梯度从第3分钟的22％A变化至第33分钟的37％A；激发波长：360nm；发射波长：425nm；后进入质谱Thermo LTQ-Orbitrap Discovery质谱仪；Spray Voltage：3.7KV；Tube Lense：150V；Capillary Temperature：300℃；一级FTMS设定Resolution为30000，Mass Range为600～2000，伴随一个FT二级扫描。
测定2-AA标记寡糖分子量与理论值差别均不超过0.02Da。根据SH006-2的5株母克隆(S5C2、S8H4、S9H10、S4C6、S4E10)样品色谱峰面积计算的寡糖含量比例如表3所示，SH006-2的5株母克隆(S5C2、S8H4、S9H10、S4C6、S4E10)均没有岩藻糖成分。
表3母克隆N-糖基化分析
| 克隆名称 | G0-GlcNAc | G1 | G1-GlcNAc | G2 | Man5 | Man6 | G0 | 未知物 |
| S5C2 | 9.43 | 8.96 | 0.72 | 0.63 | 5.27 | 0.33 | 71.11 | 3.55 |
| S8H4 | 10.35 | 8.97 | 0.86 | 0.68 | 5.32 | 0.67 | 69.77 | 3.38 |
| S9H10 | 10.66 | 9.02 | 0.91 | 0.62 | 5.48 | 0.52 | 69.4 | 3.39 |
| S4C6 | 5.32 | 11.71 | 0.5 | 0.81 | 2.69 | 0.23 | 74.72 | 4.01 |
| S4E10 | 10.16 | 12.87 | 1.43 | 1.15 | 6.02 | 1.16 | 63.42 | 3.79 |
实施例9 SH006-2抗体的活性检测
9.1SH006-2与FcγRIIIa(CD16a,158F)的结合活性分析：
采用pH 7.4的PBS缓冲液将His-Tag抗体稀释至1μg/mL，每孔100μL加入到96孔ELISA板中，4℃包被过夜。用含1％BSA的PBS封闭液封闭1小时后，用PBST洗板4次。将CD16a(158F)用含1％BSA的PBS稀释至0.25μg/mL，每孔100μL加入96孔ELISA板中，室温孵育1小时。同时将SH006-2抗体、
稀释至100μg/mL，进行3倍系列稀释后，按摩尔比2:1分别与山羊抗人IgG F(ab’)2的F(ab’)2片段蛋白共孵育1小时。PBST洗板4次后，将预孵育的混合抗体加入96孔ELISA板中，采用双复孔加样，每孔100μL。采用两块微孔板，微孔板1检测抗体
S5C2、S8H4、S9H10和S4C6六个样品；微孔板2检测
S8H4和S4E10四个抗体。抗体和CD16a(158F)室温孵育1小时后，用PBST洗板4次，将HRP标记的山羊抗人IgG Fc用样品稀释液按1:10000稀释，每孔加入100μL，室温孵育1小时。PBST洗板4次后，每孔加入100μLTMB底物，室温避光孵育8分钟，每孔加入50μL1M H2SO4液终止显色反应。在多功能酶标仪上选择波长450nm，参比波长570nm测定96孔板中各孔的吸光值，每孔吸光值(OD)＝OD450nm-OD570nm。将抗体浓度取对数后作为横坐标，测得的每孔吸光值为纵坐标，选用Sigmoidal dose-response(Variable Slope)方式(GraphPadPrism软件，GraphPad Software，San Diego，California)进行非线性回归，得到不同抗体与CD16a(158F)的结合曲线(图4和图5)。
实验结果显示所有抗体呈浓度依赖性地与CD16a(158F)结合。
与CD16a结合的EC50明显大于SH006-2样品或
与CD16a结合的EC50。表明SH006-2和
与CD16a的结合显著强于
与CD16a的结合，并且SH006-2与CD16a(158F)结合活性也强于
9.2SH006-2的ADCC活性分析：
将SH006-2、
和
用含5％FBS的RPMI1640培养基从20μg/mL起进行10倍系列稀释9个浓度至2×10-7μg/mL，每孔100μL加入2×样品稀释液到96孔U型细胞培养板中。Raji细胞、Daudi细胞和NK-92MI-CD16a细胞均置于37℃、5％CO2孵箱中培养，每2-3天传代一次。取对数生长期的Raji细胞和Daudi细胞分别作为靶细胞，采用含5％FBS的RPMI1640培养基重悬，分别以每孔1×104个(50μL)加入96孔U型细胞培养板，然后每孔加入5×104个(50μL)NK-92MI-CD16a细胞作为效应细胞，三复孔。同时设靶细胞最大裂解孔(M)、靶细胞自发释放孔(ST)、效应细胞自发释放孔(SE)、体积补偿孔(B-V)和培养基补偿孔(B-M)。离心(250g，4分钟)，于37℃、5％CO2培养箱中孵育4小时。采用乳糖脱氢酶(LDH)试剂盒，参照说明书检测细胞上清中LDH。在多功能酶标仪上振荡5秒混匀后，于490nm测定各孔的吸光值。将靶细胞最大裂解孔(M)扣除体积补偿孔(B-V)本底，其它各孔扣除培养基补偿孔(B-M)，计算杀伤率(％)。杀伤率ADCC％＝[(实验孔－靶细胞自发(ST)－效应细胞自发(SE))/(靶细胞最大(M)－靶细胞自发(ST))]×100％。
以抗体浓度对数值为横坐标，以每孔细胞杀伤率为纵坐标，选用Sigmoidal Dose-response(Variable Slope)分析方法(GraphPad Prism软件，GraphPad Software，SanDiego，California)进行非线性回归得SH006-2诱导的NK细胞对Raji细胞和Daudi细胞的ADCC效应曲线(158F/F，A和B；158V/V，C和D)(参见图6)。
图6的A和B或者C和D均显示，
和SH006-2抗体介导的NK细胞对Raji细胞和Daudi细胞的杀伤作用都呈浓度依赖性增加，可达最高杀伤率平台，曲线呈S型。SH006-2抗体的最高杀伤率均高于
和
的最高杀伤率；从最佳拟合曲线参数列表可以得出，SH006-2的EC50值小于
和
的EC50值，以上均表明相较于原研药
SH006-2的ADCC活性更强，且远远优于第一代CD20单克隆抗体
实施例10 SH006-2抗体对Raji细胞的直接杀伤活性分析
首先在96孔培养板中，将SH006-2、
三种抗体药物以10ug/mL作为初始浓度进行3倍倍比稀释，并进行3个复孔重复，另设阴性对照组(不加药物)和空白对照组(不含细胞)。在以50μL/孔加入已经将对数生长期的人B细胞淋巴瘤Daudi和Raji细胞计数，以2x104/孔的细胞加入到已有抗体稀释的96孔培养板中，50μL/孔。将加有抗体和细胞的96孔板放在细胞培养箱中孵育24小时。然后向每个孔中加入10μL的CCK8溶液，震荡后将培养板放在培养箱内孵育4小时，测定OD450。
根据检测结果计算药物对细胞的直接抑制率，计算公式为：
抑制率＝(1-(加药组OD450-空白组OD450)/(对照组OD450-空白组OD450))×100％。
试验结果：如图7所示，
SH006-2在0.1-10μg/mL浓度下对Raji细胞和Daudi细胞的生长具有直接杀伤作用，且在不同浓度下抑制率相同。
对Raji细胞和Daudi细胞的直接杀伤作用不大。
实施例11 SH006-2抗体体内活性评价
人B细胞淋巴瘤细胞系Daudi于T175方瓶中悬浮培养，置于37℃、5％CO2孵箱中，每三天左右进行一次传代。收集对数生长期的Daudi细胞，于800rpm，离心3分钟，采用PBS重悬，以4×106个细胞接种于SCID Beige小鼠皮下。待肿瘤体积长至约260mm3时，随机分为7组，每组8只，并分别给予0.3mg/kg、3mg/kg、30mg/kg的SH006-2、3mg/kg
和PBS，腹腔注射(i.p.)，每周两次(biw)，连续两周(参见表4)。每周测量肿瘤大小两次，计算肿瘤体积(V,mm3)：V(mm3)＝(L×W2)/2，L表示肿瘤长度，W表示肿瘤宽度，绘制肿瘤生长曲线。在首次接种细胞后的第38天以安乐死方式处死小鼠，取肿瘤，称量后计算肿瘤抑制率：肿瘤抑制率(％)＝(V对照组－V给药组)×100/V对照组。采用GraphPad Prism软件(Graphpad Prism5Demo，San Diego，California)中的Two-Way ANOVA方式进行统计分析。结果如表5和图8所示。
表4 Daudi实体瘤模型分组和给药方案
表5试验各组小鼠肿瘤体积(Daudi实体瘤模型)
结果发现，与PBS组比较，在接种细胞后的第32天SH006-2 30mg/kg组可显著抑制小鼠体内肿瘤的生长，差异有显著性统计学意义(P＜0.05)；在接种细胞后的第36天，SH006-2的30mg/kg和3mg/kg组以及
3mg/kg组对小鼠体内肿瘤的生长抑制作用非常明显，差异有极显著性统计学意义(P＜0.01)，并在接种细胞后的第38天呈现同样的统计结果。对相同剂量(3mg/kg)的SH006-2、
和
进行比较，三种抗体在该给药剂量下对肿瘤的抑制活性基本相似，未见显著性差异。
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。本发明的全部保护范围由所附的权利要求书及其任何等同物给出。
序列表
<110> 盛禾（中国）生物制药有限公司
<120> 人源化单克隆抗体、其制备方法及用其途
<130> 2017
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tataccttcg gcggcggcac caaggtggag atcaagagaa ccgtggctgc cccaagcgtg 360
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Claims (11)
1.编码抗人CD20人源化单克隆抗体SH006-2的核酸分子，其特征在于包含编码轻链的核苷酸序列和编码重链的核苷酸序列；编码轻链的核苷酸序列如SEQ ID NO:1所示，编码重链的核苷酸序列如SEQ ID NO:2所示。
2.权利要求1的核酸分子，其在编码轻链的核苷酸序列和编码重链的核苷酸序列的5’端分别包含编码信号肽的核苷酸序列；在编码轻链的核苷酸序列和编码重链的核苷酸序列的3’端分别包含终止密码子。
3.权利要求2的核酸分子，其中所述信号肽由氨基酸序列SEQ ID NO:6组成。
4.重组载体，其包含权利要求1-3任一项的核酸分子，其中所述核酸分子与一种或多种调节元件可操作连接。
5.重组细胞，其含有权利要求4所述的重组载体。
6.根据权利要求5所述的重组细胞，所述重组细胞为敲除负责编码岩藻糖基转移酶8即FUT8的基因的CHO细胞。
7.根据权利要求6所述的重组细胞，所述敲除是指利用CRISPR/Cas9基因编辑技术将CHO细胞中编码FUT8的基因敲除。
8.一种抗人CD20人源化单克隆抗体SH006-2的制备方法，其特征在于，所述单克隆抗体SH006-2由权利要求1-3任一项所述的核酸分子、权利要求4所述的重组载体或者权利要求5所述的重组细胞制备得到，并且所述抗体的轻链氨基酸序列如SEQ ID NO:3所示，其重链的氨基酸序列如SEQ ID NO:4所示，所述制备方法包括在使得所述抗人CD20人源化单克隆抗体SH006-2表达的条件下培养权利要求5所述的重组细胞，并收集所表达的抗体SH006-2。
9.如权利要求8所述的制备方法，其特征在于，包括以下步骤：
1)将权利要求1-3任一项所述的核苷酸序列克隆入表达载体中，得到重组表达载体；
2)将重组表达载体转入宿主细胞，即负责编码岩藻糖基转移酶8即FUT8的基因被敲除的CHO细胞中，得到重组细胞；
3)在筛选培养基上对重组细胞进行加压筛选，得到稳定生长的细胞池；
4)从步骤3)的细胞池中筛选高表达细胞株，然后检测单克隆上清中SH006-2的表达水平，筛选培养得到表达量较高的克隆，扩增培养后冻存细胞备用；
5)取步骤4)获得的克隆细胞于目标培养基中培养，检测抗体表达产量；然后挑选出表达高的克隆进行培养，获得高表达的单克隆细胞株，收获培养上清并纯化，得权利要求8所述的抗人CD20人源化单克隆抗体SH006-2。
10.如权利要求9所述的制备方法，其特征在于，步骤2)中所述敲除是指，利用CRISPR/Cas9基因编辑技术将CHO细胞中负责编码岩藻糖的基因即FUT8基因敲除。
11.权利要求1-3任一项所述的核酸分子或权利要求4所述的重组载体或者权利要求5-7任一项所述的重组细胞在权利要求8所述的抗人CD20人源化单克隆抗体SH006-2的制备方法中的用途。
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