CN114457110A - 一种敲除fut8基因的方法 - Google Patents
一种敲除fut8基因的方法 Download PDFInfo
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- CN114457110A CN114457110A CN202011244613.1A CN202011244613A CN114457110A CN 114457110 A CN114457110 A CN 114457110A CN 202011244613 A CN202011244613 A CN 202011244613A CN 114457110 A CN114457110 A CN 114457110A
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Abstract
本发明公开一种敲除FUT8基因以提高抗体ADCC活性的方法。利用CRISPR/Cas9技术编码CHO细胞的FUT8基因，得到FUT8基因沉默的CHO细胞。通过对工程化细胞表达抗体的理化性质及生物活性检测：糖型检测结果表明工程化细胞表达的抗体的岩藻糖完全消除，以曲妥珠单抗为对照，工程化细胞表达的完全无岩藻糖抗体与FcγRs抗原亲和力提高20倍，ADCC效应提高19倍。
Description
技术领域
本发明涉及生物领域，特别提供了一种敲除FUT8基因以提高抗体ADCC活性的方法。
背景技术
ADCC(antibody dependent cellular cytotoxicity，抗体依赖的细胞介导的细胞毒性作用)是指由NK(natural killer)细胞引发的一系列免疫反应，NK细胞通过FcγRIII受体与抗体分子恒定区(Fc)结合，然后通过穿孔素和颗粒酶在靶细胞表面引起细胞降解和引发细胞凋亡。已有研究表明在IgG抗体的Fc片段CH2结构域Asn297部位的糖链结构去除岩藻糖时，可以增强NK细胞的FcγRIIIa与IgG抗体的Fc片段之间的结合力，从而增强抗体的ADCC效应。
现有研究也表明，免疫细胞表面的Fc受体具有基因多态性，且这些基因多态性与曲妥珠单抗临床客观反应率(ORR)和无进展生存期(PFS)密切相关。IgG Fc受体最重要的基因多态性为CD16a，根据其158位上氨基酸是苯丙氨酸(Phe，F)或颉氨酸(Val，V)，可以分为158V/V、158V/F、158F/F三种基因型。患者对于曲妥珠单抗的临床反应与患者CD16a的多态性密切相关。三种基因型患者对曲妥珠单抗的ORR比例分别为CD16a 158V/V(82％)、158V/F(42％)、158F/F(35％)，具有显著差异。曲妥珠单抗治疗中，CD16a 158V/V、158V/F与158F/F基因型患者的无进展生存期分别为15.0个月、11.1个月、12.9个月。Fc受体的基因多态性，其PBMC介导的ADCC活性，以及患者对于曲妥珠单抗的临床反应具有一致性。曲妥单抗不能在CD16a 158F/F类型患者体内有效的介导ADCC活性，也成为曲妥珠单抗对某些患者无效的耐药的一个原因。
CHO细胞是目前单克隆抗体常用细胞株，生产抗体95％为岩藻糖基化。岩藻糖通过一个a-1,6糖苷键与抗体Fc端上的氨基葡萄糖结合。在哺乳动物细胞中，这个糖苷键由FUT8基因编码的a-1,6-岩藻糖基转移酶催化形成。通过对抗体表达细胞进行基因工程改造，敲除FUT8基因将完全消除抗体Fc端的岩藻糖，从而提高单抗药物的ADCC作用。
CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats)即成簇出现规律间隔的短回文序列的缩写，此序列和CRISPR相关基因(Cas基因)共同作用，因其特有的RNA介导的核酸内切酶活性引起了生物学界的广泛关注，其中以来源于Streptococcus Pyogenes为代表的II类CRISPR/Cas9系统应用最为广泛，以RNA作为基因组定位工具，用特定序列的sgRNA(small-guide RNA)引导Cas9核酸内切酶识别并切割靶向序列。
与ZFNs/TALENs相比，CRISPR/Cas9更易于操作，只需要设计跟靶序列配对的sgRNA即可，设计原则是在靶序列后紧跟一个PAM位点。CRISPR/Cas9的效率更高，可以在不同的位点同时引入多个突变。
CHO细胞是在工业上真核表达系统中运用最多的来表达抗体的宿主细胞。在本发明中，通过CRISPR/Cas9基因编辑技术对CHO细胞的FUT8基因进行编辑，使细胞中的a-1,6岩藻糖转移酶的功能丧失，产生完全去岩藻糖化的抗体。
专利CN106701823A公开了一种生产无岩藻糖单克隆抗体的CHO细胞系建立及其应用，利用CRISPR/Cas9技术，针对CHO细胞FUT8基因的exon9区域设计两对sgRNA，构建FUT8敲除载体pX330-sgRNAl质粒和pX330-sgRNA2质粒，得到FUT8基因沉默的稳定FUT8基因缺失的CHO细胞，糖型检测结果表明FUT8细胞表达的单克隆抗体的岩藻糖完全消除。
本发明旨在提供一种可替代的利用CRISPR/Cas9技术敲除FUT8基因的方法，针对CHO细胞FUT8基因的exon1和exon7区域各设计一对sgRNA，构建FUT8敲除载体，获得FUT8基因功能失活的细胞，表达完全无岩藻糖的抗体。本发明还提供一种包含FUT8基因缺失的细胞所产生的抗体的药物组合物，及其在制备用于治疗HER2阳性实体瘤中的药物中的应用。
发明内容
本发明的目的在于提供一种可替代的利用CRISPR/Cas9技术敲除FUT8基因的方法，获得FUT8基因功能失活的细胞，表达完全无岩藻糖的抗体。
本发明的目的主要通过以下技术方案实现的：
本发明涉及一种敲除FUT8基因的方法，所述方法包括：
利用CRISPR/Cas9技术，针对CHO细胞FUT8基因的exon1和exon7区域各设计一对sgRNA，构建FUT8敲除载体质粒1和质粒2；
将质粒1和质粒2转染进CHO细胞，敲除CHO细胞中的FUT8基因，得到FUT8基因沉默的稳定工程化细胞。
优选的，所述CHO细胞表达抗HER2抗体、抗CD20抗体、抗CD19抗体、抗EGFR抗体。
优选的，所述CHO细胞表达的抗HER2抗体为曲妥珠单抗。
更优选的，所述曲妥珠单抗具有SEQ ID N0.1所示的重链和SEQ ID N0.2所示的轻链。
优选的，所述FUT8基因沉默的稳定工程化细胞的两条等位基因都发生了基因突变。
更优选的，所述FUT8基因功能失活。
本发明还涉及一种FUT8基因缺失的细胞，根据上述方法获得FUT8基因沉默的CHO细胞。
本发明还涉及一种药物组合物，包含上述FUT8基因缺失的细胞所产生的抗体。
优选的，所述的药物组合物还包含海藻糖、组氨酸或组氨酸盐、聚山梨酯20。
本发明还涉及上述药物组合物在制备用于治疗HER2阳性实体瘤中的药物中的应用。
优选的，所述药物组合物在制备用于治疗乳腺癌、胆管癌、胃癌、胰腺癌、直肠癌中的药物中的应用。
更优选的，所述药物组合物在制备用于治疗158F/F、158V/V型肿瘤中的药物中的应用。
与现有技术相比，本发明具有如下有益效果：
1、与之前应用的基因编辑技术相比，本发明利用CRISPR/Cas9基因编辑技术，提供一种可替代的敲除FUT8基因的方法，操作简便，时间成本低。
2、现有CRISPR/Cas9技术进行基因编辑时，针对某个外显子区域需要设计两对甚至更多的sgRNA，本发明针对外显子1区域只需设计一对sgRNA，对外显子7区域也只需设计一对sgRNA。
3、检测完全去岩藻糖化的抗Her2抗体、曲妥珠单抗与人FcγRs结合的亲和力强弱，可以看出完全去岩藻糖化的抗Her2抗体比曲妥珠单抗对CD16a(158F/F)结合的亲和力提高了20倍，对CD16a(158V/V)结合的亲和力提高了10倍。
4、检测完全去岩藻糖化的抗Her2抗体、曲妥珠单抗对SKBR3、BT474细胞的ADCC效应，可以看出完全去岩藻糖化的抗体比曲妥珠单抗对158V/V型SKBR3细胞的ADCC效应提高6倍，对158F/F型SKBR3细胞的ADCC效应提高3倍，对158V/V型BT474细胞的ADCC效应提高19倍。
附图说明
下面结合附图及实施方式对本发明作进一步详细的说明：
图1为CHO细胞配套载体pCHO1.0-L-H结构图；
图2为完全去岩藻糖化的抗Her2抗体、曲妥珠单抗与人CD16a(158V/V)结合的亲和力；
图3为完全去岩藻糖化的抗Her2抗体、曲妥珠单抗与人CD16a(158F/F)结合的亲和力；
图4为完全去岩藻糖化的抗Her2抗体和曲妥珠单抗通过NKCD16a(158V/V)对SKBR3细胞的ADCC活性量效曲线；
图5为完全去岩藻糖化的抗Her2抗体和曲妥珠单抗通过NKCD16a(158F/F)对SKBR3细胞的ADCC活性量效曲线；
图6为完全去岩藻糖化的抗Her2抗体和曲妥珠单抗通过NKCD16a(158V/V)对BT474细胞的ADCC活性量效曲线；
图7为完全去岩藻糖化的抗Her2抗体和曲妥珠单抗通过NKCD16a(158F/F)对BT474细胞的ADCC活性量效曲线。
图8为完全去岩藻糖化的抗Her2抗体在人乳腺癌细胞系BT474移植瘤模型上的体内药效。
具体实施方式
本发明利用CRISPR/Cas9技术，针对CHO细胞FUT8全基因组序列的第一外显子和第七外显子编码功能区设计gRNA序列，分别将gRNA插入到相应载体中，构建CRISPR-CAS9表达载体质粒1和质粒2，将质粒1和质粒2转染进CHO细胞，敲除CHO细胞中的FUT8基因，得到了FUT8基因沉默的稳定FUT8基因缺失细胞；根据测序结果，得到的基因敲除的稳定细胞两条等位基因均发生了基因突变。
构建可以表达抗体的质粒1和质粒2，转染野生型及FUT8缺失的CHO细胞，表达产生正常的岩藻糖化抗体及完全去岩藻糖化的抗体。通过对表达抗体的理化性质及生物活性进行检测，发现去岩藻糖化的抗体与野生型相比，理化性质稳定，糖型检测结果表明工程化细胞表达的抗Her2抗体的岩藻糖完全消除。
检测分析完全去岩藻糖化的抗Her2抗体、曲妥珠单抗与人FcγRs结合的亲和力强弱，可以得到完全去岩藻糖化的抗Her2抗体比曲妥珠单抗对CD16a(158F/F)结合的亲和力提高了20倍，对CD16a(158V/V)结合的亲和力提高了10倍。LDH法检测细胞毒性，从而检测抗体对癌细胞的ADCC作用。我们采用HER2高表达SKBR3细胞模型、BT474细胞模型，结果显示完全去岩藻糖化的抗体比曲妥珠单抗对158V/V型SKBR3细胞的ADCC效应提高6倍，对158F/F型SKBR3细胞的ADCC效应提高3倍，对158V/V型BT474细胞的ADCC效应提高19倍。
下面结合实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干调整和改进。这些都属于本发明的保护范围。
实施例
本实施例涉及一种敲除FUT8基因以提高抗体ADCC活性的方法，具体步骤如下：
1.抗HER2抗体表达载体构建
目的基因表达载体为CHO配套载体pCHO1.0，采用DHFR扩增体系。目的基因载体序列通过密码子优化合成，克隆至表达载体中。克隆后的质粒，抗体轻重链的启动子分别为CMV/EF1、EF2/CMV杂合启动子，终止子分别为猴空泡毒PloyA(SV40 PA)和巨细胞病毒PloyA(CMVPA)。含有一个嘌呤霉素抗性基因(PuroR)和一个卡那霉素抗性基因(KanR)作为筛选标记。进行表达载体酶切图谱验证和序列验证，结果显示：酶切图谱正确，测序结果与理论序列一致，抗HER2抗体表达载体构建成功。
2.FUT8基因敲除表达载体构建
FUT8基因如SEQ ID N0.3所示，为了去除抗体药物的核心岩藻糖修饰，针对CHO细胞FUT8全基因组序列的第一外显子序列和第七外显子序列设计sgRNA序列(见表1和表2)，选择最优的sgRNA插入载体中，构建CRISPR-CAS9表达载体质粒1和质粒2。
表1针对第一外显子所设计的sgRNA序列
| 编号 | 序列 |
| 1 | GCGGGCATGGACTGGTTCC SEQ ID N0.4 |
| 2 | ATGGACTGGTTCCTGGCGT SEQ ID N0.5 |
| 3 | TATGCTCATTCTTTTTGCC SEQ ID N0.6 |
| 4 | ATGCTCATTCTTTTTGCCT SEQ ID N0.7 |
| 5 | TGCTCATTCTTTTTGCCTG SEQ ID N0.8 |
| 6 | GCTCATTCTTTTTGCCTGG SEQ ID N0.9 |
| 7 | GGACCTTATTGTTTTATAT SEQ ID N0.10 |
| 8 | CCTTATTGTTTTATATAGG SEQ ID N0.11 |
| 9 | TTTTATATAGGTGGTCATT SEQ ID N0.12 |
| 10 | TCCAAGATTCTTGCAAAGC SEQ ID N0.13 |
| 11 | ACAACAAAATGAAGACTTG SEQ ID N0.14 |
| 12 | AATGAAGACTTGAGGAGAA SEQ ID N0.15 |
| 13 | ATGAGCATAATCCAACGCC SEQ ID N0.16 |
| 14 | GAGCATAATCCAACGCCAGG SEQ ID N0.17 |
| 15 | TAAAACAATAAGGTCCCCC SEQ ID N0.18 |
| 16 | CCACCTATATAAAACAATA SEQ ID N0.19 |
| 17 | TCTGCTAGAATGGTCAGGG SEQ ID N0.20 |
| 18 | TTCTCTGCTAGAATGGTCA SEQ ID N0.21 |
| 19 | GTTCTCTGCTAGAATGGTC SEQ ID N0.22 |
| 20 | GGAGAGTTCTCTGCTAGAA SEQ ID N0.23 |
| 21 | TCCAGCTTTGCAAGAATCT SEQ ID N0.24 |
表2针对第七外显子所设计的sgRNA序列
3.pCHO1.0-L-H质粒转染
将pCHO1.0-L-H表达载体转染到E.coliDH5α，划板活化，挑选出单克隆进行培养，抽提质粒，加入乙醇沉淀后，用无菌水溶解并用核酸蛋白定量仪进行浓度测定。
离心收集处于对数生长期的CHO细胞，将120μg pCHO1.0-L-H质粒电击转染入CHO细胞。转染后的细胞置于50ml预热的CD Forti CHO无血清培养基中，摇床培养。
4.抗性筛选与加压筛选
转染48h后，取样计数，将细胞悬液离心后弃上清，接种至嘌呤霉素筛选培养基(HT-)中，筛选出表达载体整合成功的细胞。摇床培养，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。
为了提高目的基因拷贝数，提高细胞目的蛋白表达量，进行MTX梯度加压，每隔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，再进行下一轮压力筛选。获得4个在压力下稳定生长的细胞池。
分别对稳定生长的细胞池1-4进行细胞计数接种至含有MTX的HT-中分批(Batch)培养。摇床培养，细胞计数，细胞上清留样并进行ELISA检测细胞抗体表达量。
根据稳定表达细胞池分批培养数据，选择表达量稍高的细胞池2进行下一步母细胞的筛选工作。
5.母细胞筛选
细胞初步克隆化，进行96孔板筛选，铺板前进行细胞计数，离心后用筛选培养基CDForti CHO(含24μg/ml嘌呤霉素，1000nM MTX，8mM L-Glutamax)重悬细胞，上下颠倒混匀，吸取200μl加入96孔板，使得每个培养孔中有50个细胞。
将孔板置于CO2培养箱中进行培养14天，标记出现细胞团的孔板，吸取上清放入96孔板中，采用ELISA法检测上清表达浓度，逐步培养筛选，选择表达量在30mg/L以上的克隆，在备份之后进行Bacth细胞培养，按照5×105个细胞/ml接种在摇瓶中，当细胞活力降至70％以下或培养周期达到8或者9天进行收样，离心，收集细胞培养上清，采用ELISA方法测定细胞上清抗体表达水平，选择表达量最高的作为下一步FUT8基因敲除CHO细胞。
6.CHO细胞FUT8基因敲除载体转染
将质粒1和质粒2表达载体转染到E.coliDH5α，划板活化，挑选出单克隆进行培养，抽提质粒，加入乙醇沉淀后，用无菌水溶解并用核酸蛋白定量仪进行浓度测定。
离心收集处于对数生长期的CHO细胞，将6.5μg质粒1和2.5μg质粒2混合电击转染入CHO细胞。转染后的细胞置于2ml预热的CD Forti CHO(含24μg/ml嘌呤霉素，1000nM MTX，8mM L-Glutamax)中进行培养。培养48h后，收集细胞提取基因组DNA测序，验证FUT8基因敲除效果。结果显示：针对第一外显子和第七外显子的sgRNA测序图谱均出现双峰，提示所获得的基因敲除细胞池存在FUT8基因序列的突变，可用于进一步的克隆纯化。
表3引物序列表
7.FUT8基因突变细胞单克隆化
采用FUT8基因敲除载体转染稳定表达抗体母克隆，转染24h后进行两轮有限稀释铺板，进行单克隆化。综合分析细胞表达量及目的蛋白产品质量，最终选择生产细胞。
8.对工程化细胞表达的抗Her2抗体、曲妥珠单抗的糖型进行检测
研究工程化细胞表达的抗Her2抗体的糖型，进行N糖谱分析和单糖分析。通过糖苷酶将糖基与糖蛋白分离，然后进行2-AB标记，LC-MS分析N糖谱；在酸性环境下寡糖水解成单糖，单糖释放后，2-AA标记，液相分离检测单糖含量。寡糖的含量和组成见表4。结果显示，工程化细胞表达的抗Her2抗体的主要糖型为G0、G1、G1”，非唾液酸糖苷的比例为100％，单唾液酸糖苷的比例为0.57％，岩藻糖苷比例为0％。
表4工程化细胞表达的抗Her2抗体的N糖类型和含量分析结果
9.检测完全去岩藻糖化的抗Her2抗体、曲妥珠单抗与人FcγRs结合的亲和力
利用生物膜层干涉(BLI)实时监测分子间相互作用。这种技术通过生物传感器来实现，光通过传感器的生物膜层后会发生透射与反射，反射光的频率受到生物膜层厚度的影响。当结合在传感器生物膜上的分子A与溶液中的分子B结合，使生物膜层厚度增加，因而产生相对位移，这个相对位移随着分子B结合量的增加而增加，最后达到一个平衡状态，从而能实时记录分子间相互作用的过程，包括结合速度、解离速度、亲和力等测定。配置缓冲液，传感器预湿，在96孔板中稀释加样，加样完成后，设置程序并检测。
结果如表5所示，完全去岩藻糖化的抗Her2抗体比曲妥珠单抗对CD16a(158F/F)结合的亲和力提高了20倍，对CD16a(158V/V)结合的亲和力提高了10倍。
表5完全去岩藻糖化的抗Her2抗体、曲妥珠单抗与人FcγRs结合的亲和力
| 人FcγRs | 完全去岩藻糖化的抗Her2抗体 | 曲妥珠单抗 |
| CD16a(158F/F) | 79.3nM | 1560nM |
| CD16a(158V/V) | 25.9nM | 275nM |
10.LDH法检测完全去岩藻糖化的抗Her2抗体、曲妥珠单抗对SKBR3、BT474细胞的ADCC效应
人乳腺癌细胞系SKBR3采用含10％FBS的DMEM培养基贴壁培养；BT474采用含10％FBS、1.5g/LNaHCO3、2.5g/L葡萄糖、0.11g/L丙酮酸钠的RPMI1640培养基贴壁培养，将这些细胞都置于37℃、5％CO2培养箱中，每三天左右进行一次传代。
分别取对数生长期的人乳腺癌细胞SKBR3和BT474每孔1×104个细胞铺于96孔板，采用含5％FBS的RPMI 1640培养基重悬，加入96孔U型细胞培养板，然后每孔加入5×104个(50μl)NK-92MI-CD16a(158V/V和158F/F)细胞。将去岩藻糖化的抗Her2抗体和曲妥珠单抗进行系列稀释，三重复，抗CD20单抗为无关抗体。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)、体积校正空白孔(BV)和培养基背景空白孔(BM)。离心培养，对细胞上清LDH进行检测。振荡混匀，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(BV)本底，其它各孔扣除培养基补偿孔(BM)，计算细胞死亡率(％)。
以抗体浓度为横坐标，细胞死亡率(％)为纵坐标，采用非线性回归分析方法，拟合曲线，得到相应的拟合参数。
表6完全去岩藻糖化的抗Her2抗体与曲妥珠单抗对肿瘤细胞的ADCC活性试验结果
结果显示，完全去岩藻糖化的抗Her2抗体比曲妥珠单抗对158V/V型SKBR3细胞的ADCC效应提高6倍，对158F/F型SKBR3细胞的ADCC效应提高3倍，对158V/V型BT474细胞的ADCC效应提高19倍。
11.完全去岩藻糖化的抗Her2抗体处方制剂组成
完全去岩藻糖化的抗Her2抗体处方制剂如表7所示。
表7完全去岩藻糖化的抗Her2抗体处方制剂组成(pH 6.0)
| 组分 | 含量(g/L) |
| 完全去岩藻糖化的抗Her2抗体 | 20 |
| 一水合盐酸组氨酸 | 0.4714 |
| 组氨酸 | 0.3048 |
| 聚山梨酯20 | 0.0857 |
| 二水合海藻糖(α，α) | 19.0476 |
12.完全去岩藻糖化的抗Her2抗体在人乳腺癌细胞系BT474移植瘤模型上的体内药效学
试验选择了1mg/kg、5mg/kg、30mg/kg共3个剂量组，并纳入5mg/kg曲妥珠单抗作为对照。
开始给药后42天，溶剂对照组荷瘤鼠的瘤体积达到857mm3。与溶剂对照组相比，受试物完全去岩藻糖化的抗Her2抗体各组均具有显著的抑瘤作用，肿瘤体积分别为300mm3(T/C＝34.98％，TGI＝83.59％，p＝0.006)，148mm3(T/C＝17.25％，TGI＝106.39％，p＝0.000)和177mm3(T/C＝20.67％，TGI＝102.00％，p＝0.001)；完全去岩藻糖化的抗Her2抗体5mg/kg的抑瘤作用与30mg/kg近似，均好于1mg/kg，但均无统计学差异。曲妥珠单抗5mg/kg组显示出温和的抑瘤作用，但未表现出显著性差异，肿瘤体积为517mm3(T/C＝60.38％，TGI＝50.93％，p＝0.103)。在相同剂量条件下，完全去岩藻糖化的抗Her2抗体的抑瘤作用显著优于曲妥珠单抗(p＝0.028)。瘤重数据与肿瘤生长曲线基本保持一致。
所有给药组，受试药物完全去岩藻糖化的抗Her2抗体和曲妥珠单抗对荷瘤鼠无明显的体重降低现象发生。
综上所述，完全去岩藻糖化的抗Her2抗体在1mg/kg、5mg/kg和30mg/kg剂量下对动物耐受并对BT474皮下异种移植瘤模型显示出显著性抗肿瘤作用，相同剂量下完全去岩藻糖化的抗Her2抗体的抑瘤效果显著优于曲妥珠单抗。
序列表
<110> 盛禾（中国）生物制药有限公司
<120> 一种敲除FUT8基因的方法
<160> 44
<170> SIPOSequenceListing 1.0
<210> 1
<211> 450
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 2
<211> 214
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 3
<211> 2825
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
tttcaggttg ctgctctggc ttaggccatc tatgaccctg gtggtgtttt cattcactat 60
aagtccttcc catctttatt aactgagcaa gttcagctag taattttaga gaccgaggtt 120
caagcaataa cacctatctc tgcaataccg tgtggctttc ttcaatgtct tacatcctaa 180
ggaaaggaag catgtagagc ccaggaagca caggacaaga aagctgcctc cttgtatcac 240
caggaagatc tttttgtaag agtcatcaca gtataccaga gagactaatt ttgtctgaag 300
catcatgtgt tgaaacaaca gaaacttatt ttcctgtgtg gctaactaga accagagtac 360
aatgtttcca attctttgag ctccgagaag acagaaggga gttgaaactc tgaaaatgcg 420
ggcatggact ggttcctggc gttggattat gctcattctt tttgcctggg ggaccttatt 480
gttttatata ggtggtcatt tggttcgaga taatgaccac cctgaccatt ctagcagaga 540
actctccaag attcttgcaa agctggagcg cttaaaacaa caaaatgaag acttgaggag 600
aatggctgag tctctccgaa taccagaagg ccctattgat caggggacag ctacaggaag 660
agtccgtgtt ttagaagaac agcttgttaa ggccaaagaa cagattgaaa attacaagaa 720
acaagctagg aatgatctgg gaaaggatca tgaaatctta aggaggagga ttgaaaatgg 780
agctaaagag ctctggtttt ttctacaaag tgaattgaag aaattaaaga aattagaagg 840
aaacgaactc caaagacatg cagatgaaat tcttttggat ttaggacatc atgaaaggtc 900
tatcatgaca gatctatact acctcagtca aacagatgga gcaggtgagt ggcgggaaaa 960
agaagccaaa gatctgacag agctggtcca gcggagaata acatatctgc agaatcccaa 1020
ggactgcagc aaagccagaa agctggtatg taatatcaac aaaggctgtg gctatggatg 1080
tcaactccat catgtggttt actgcttcat gattgcttat ggcacccagc gaacactcat 1140
cttggaatct cagaattggc gctatgctac tggaggatgg gagactgtgt ttagacctgt 1200
aagtgagaca tgcacagaca ggtctggcct ctccactgga cactggtcag gtgaagtgaa 1260
ggacaaaaat gttcaagtgg tcgagctccc cattgtagac agcctccatc ctcgtcctcc 1320
ttacttaccc ttggctgtac cagaagacct tgcagatcga ctcctgagag tccatggtga 1380
tcctgcagtg tggtgggtat cccagtttgt caaatacttg atccgtccac aaccttggct 1440
ggaaagggaa atagaagaaa ccaccaagaa gcttggcttc aaacatccag ttattggagt 1500
ccatgtcaga cgcactgaca aagtgggaac agaagcagcc ttccatccca ttgaggaata 1560
catggtacac gttgaagaac attttcagct tctcgaacgc agaatgaaag tggataaaaa 1620
aagagtgtat ctggccactg atgacccttc tttgttaaag gaggcaaaga caaagtactc 1680
caattatgaa tttattagtg ataactctat ttcttggtca gctggactac acaaccgata 1740
cacagaaaat tcacttcggg gcgtgatcct ggatatacac tttctctccc aggctgactt 1800
ccttgtgtgt actttttcat cccaggtctg tagggttgct tatgaaatca tgcaaacact 1860
gcatcctgat gcctctgcaa acttccattc tttagatgac atctactatt ttggaggcca 1920
aaatgcccac aaccagattg cagtttatcc tcaccaacct cgaactaaag aggaaatccc 1980
catggaacct ggagatatca ttggtgtggc tggaaaccat tggaatggtt actctaaagg 2040
tgtcaacaga aaactaggaa aaacaggcct gtacccttcc tacaaagtcc gagagaagat 2100
agaaacagtc aaatacccta catatcctga agctgaaaaa tagagatgga gtgtaagaga 2160
ttaacaacag aatttagttc agaccatctc agccaagcag aagacccaga ctaacatatg 2220
gttcattgac agacatgctc cgcaccaaga gcaagtggga accctcagat gctgcactgg 2280
tggaacgcct ctttgtgaag ggctgctgtg ccctcaagcc catgcacagt aaaataatgt 2340
actcacacat aacatacaaa tggattattt tctactttgc cctttaaata ttctgtcccc 2400
atgaaacaaa cactgccaca ttatgtaatt taagtgacac agacgttttg tgtgagactt 2460
caaacatggt gcctatatct gagagacctc tgtgatttac tgagaagatg agaacagctc 2520
ccttctgtgg ggaagttggt tcttagtcag tggtggactg gccactgaat tcactgcaat 2580
caacagattc agaatgagaa tggatgtttt tcctttatat ggttgtctgg atttttttta 2640
aagtaatttc atcagttcag ttcatccacc tcattaataa atgaaggaat ataccaataa 2700
aatcaaatga aatattcact gtccattagg aagttttata aaacaatgcc atgaacaaaa 2760
aattctttag tactcaatgt ttctggacat tctctttgat aacaaaaata aattttaaaa 2820
aggaa 2825
<210> 4
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gcgggcatgg actggttcc 19
<210> 5
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggactggt tcctggcgt 19
<210> 6
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tatgctcatt ctttttgcc 19
<210> 7
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atgctcattc tttttgcct 19
<210> 8
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
tgctcattct ttttgcctg 19
<210> 9
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gctcattctt tttgcctgg 19
<210> 10
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ggaccttatt gttttatat 19
<210> 11
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ccttattgtt ttatatagg 19
<210> 12
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ttttatatag gtggtcatt 19
<210> 13
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
tccaagattc ttgcaaagc 19
<210> 14
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
acaacaaaat gaagacttg 19
<210> 15
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
aatgaagact tgaggagaa 19
<210> 16
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
atgagcataa tccaacgcc 19
<210> 17
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gagcataatc caacgccagg 20
<210> 18
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
taaaacaata aggtccccc 19
<210> 19
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
ccacctatat aaaacaata 19
<210> 20
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
tctgctagaa tggtcaggg 19
<210> 21
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
ttctctgcta gaatggtca 19
<210> 22
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
gttctctgct agaatggtc 19
<210> 23
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
ggagagttct ctgctagaa 19
<210> 24
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
tccagctttg caagaatct 19
<210> 25
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
gtcagacgca ctgacaaag 19
<210> 26
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
tcagacgcac tgacaaagt 19
<210> 27
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
gcagccttcc atcccattg 19
<210> 28
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
catcccattg aggaataca 19
<210> 29
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
ctcgaacgca gaatgaaag 19
<210> 30
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
gataaaaaaa gagtgtatc 19
<210> 31
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
gatgaccctt ctttgttaa 19
<210> 32
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
gacccttctt tgttaaagg 19
<210> 33
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
gaacgcagaa tgaaagtgg 19
<210> 34
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
tattcctcaa tgggatgga 19
<210> 35
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
catgtattcc tcaatggga 19
<210> 36
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
gtaccatgta ttcctcaat 19
<210> 37
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
tgtaccatgt attcctcaa 19
<210> 38
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
aacaaagaag ggtcatcag 19
<210> 39
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
tgcctccttt aacaaagaa 19
<210> 40
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
ttgcctcctt taacaaaga 19
<210> 41
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
caacagaaac ttattttcct gtgtg 25
<210> 42
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
cactgacttt taaaaagtct gctac 25
<210> 43
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
gctatgttaa agtatttgtg aagtg 25
<210> 44
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
gacttcttaa tttgcatcag ataca 25
Claims (10)
1.一种敲除FUT8基因的方法，其特征在于，所述方法包括：
利用CRISPR/Cas9技术，针对CHO细胞FUT8基因的exon1和exon7区域各设计一对sgRNA，构建FUT8敲除载体质粒1和质粒2；
将质粒1和质粒2转染进CHO细胞，敲除CHO细胞中的FUT8基因，得到FUT8基因沉默的稳定工程化细胞。
2.根据权利要求1所述的敲除FUT8基因的方法，其特征在于，所述CHO细胞表达抗体为抗HER2抗体、抗CD20抗体、抗CD19抗体、抗EGFR抗体。
3.根据权利要求2所述的敲除FUT8基因的方法，其特征在于，所述CHO细胞表达的抗HER2抗体为曲妥珠单抗。
4.根据权利要求3所述的敲除FUT8基因的方法，其特征在于，所述曲妥珠单抗具有SEQID N0.1所示的重链和SEQ ID N0.2所示的轻链。
5.根据权利要求1至4中任一项所述的敲除FUT8基因的方法，其特征在于，所述FUT8基因沉默的稳定工程化细胞的两条等位基因都发生了基因突变。
6.根据权利要求5所述的敲除FUT8基因的方法，其特征在于，所述FUT8基因功能失活。
7.一种FUT8基因缺失的细胞，其特征在于，根据权利要求1至6中任一项所述方法获得FUT8基因沉默的CHO细胞。
8.一种药物组合物，其特征在于，包含权利要求7所述的细胞所产生的抗体。
9.根据权利要求8所述的药物组合物，其特征在于，还包含海藻糖、组氨酸或组氨酸盐、聚山梨酯20。
10.根据权利要求8或9所述的药物组合物在制备用于治疗HER2阳性实体瘤中的药物中的应用，优选的，所述药物组合物在制备用于治疗乳腺癌、胆管癌、胃癌、胰腺癌、直肠癌中的药物中的应用，更优选的，所述药物组合物在制备用于治疗158F/F、158V/V型肿瘤中的药物中的应用。
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| CN114652687A (zh) * | 2020-12-23 | 2022-06-24 | 盛禾(中国)生物制药有限公司 | 一种去岩藻糖基化的抗Her2抗体冻干粉针剂的制备方法 |
| CN114934053A (zh) * | 2022-06-30 | 2022-08-23 | 澳斯康生物(南通)股份有限公司 | 岩藻糖基转移酶8缺陷型cho细胞系及其制备方法和应用 |
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Cited By (4)
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| CN114652687A (zh) * | 2020-12-23 | 2022-06-24 | 盛禾(中国)生物制药有限公司 | 一种去岩藻糖基化的抗Her2抗体冻干粉针剂的制备方法 |
| CN114652687B (zh) * | 2020-12-23 | 2024-02-20 | 盛禾(中国)生物制药有限公司 | 一种去岩藻糖基化的抗Her2抗体冻干粉针剂的制备方法 |
| CN114934053A (zh) * | 2022-06-30 | 2022-08-23 | 澳斯康生物(南通)股份有限公司 | 岩藻糖基转移酶8缺陷型cho细胞系及其制备方法和应用 |
| CN114934053B (zh) * | 2022-06-30 | 2024-02-06 | 澳斯康生物(南通)股份有限公司 | 岩藻糖基转移酶8缺陷型cho细胞系及其制备方法和应用 |
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