CN102219856B - 一种抗血管内皮细胞生长因子受体2/抗cd3双特异单链抗体 - Google Patents
一种抗血管内皮细胞生长因子受体2/抗cd3双特异单链抗体 Download PDFInfo
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Abstract
本发明涉及一种基因工程抗体,具体地说涉及一种抗血管内皮细胞生长因子受体2/抗CD3双特异单链抗体,该双特异抗体包括两个分别对血管内皮细胞生长因子受体2和CD3抗原特异性的结合位点。本发明还涉及编码所述双特异抗体的核苷酸序列以及含有所述核苷酸序列的载体以及用所述载体转染的宿主细胞。本发明提供的抗VEGFR2/抗CD3双特异单链抗体分子量小、免疫原性小、且易穿透致密的肿瘤屏障,进入实体瘤周围的微循环,在制备肿瘤治疗药物中具有应用前景。
Description
技术领域
本发明涉及一种基因工程双特异抗体,具体地说涉及一种抗血管内皮细胞生长因子受体2/抗CD3双特异单链抗体。本发明还涉及编码所述双特异抗体的核苷酸序列以及含有所述核苷酸序列的载体,用所述载体转染的宿主细胞。
背景技术
双特异单链抗体(bispecific single-chain antibody,bsc-Ab)具有两条抗原结合臂,可同时结合效应细胞和靶细胞表面的触发因子,从而激活静止状态的效应细胞并募集到靶细胞周围,介导靶细胞的裂解。通常介导的效应细胞主要是T细胞,其主要功能是识别并迅速地破坏病变细胞或组织。T细胞的激活是发挥细胞杀伤作用的根本前提。CD3分子是广泛分布于成熟T细胞表面的膜抗原,与T细胞表面膜受体形成复合物,在抗原识别和细胞内信息传递中起着重要作用。抗CD3/抗肿瘤细胞表面抗原组成的bsc-Ab可以激活静态的T细胞,在T细胞和靶细胞之间形成一个免疫连接,可以引发T细胞杀伤和裂解肿瘤细胞。例如,Bargou等构建的抗CD19×CD3双特异单链抗体(MT103)能够将病人体内的细胞毒性T淋巴细胞(CTL)富集至肿瘤细胞,并激活他们溶解靶细胞,低给药剂量即可清除非霍杰金淋巴瘤患者血液中的靶细胞,为恶性肿瘤的治愈带来希望。
相对于完整抗体,bsc-Ab具有诸多优点,能够充分挖掘人体内丰富的细胞毒性T细胞(CTL)的肿瘤杀伤效应,分子量小、免疫原性小、体内半衰期短,且易穿透致密的肿瘤屏障,进入实体瘤周围的微循环,缺乏Fc段,去除Fc介导的受体结合,使得其快速地向肿瘤集中,对靶细胞进行特异杀伤,在肿瘤的生物治疗中有良好的临床应用前景。
血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)及其受体(VEGFR)在肿瘤的生长和转移中起关键性作用,可以通过阻断VEGF/VEGFR信号传导通路来控制肿瘤生长,达到治疗肿瘤的目的。VEGFR2(VascularEndothelial Growth Factor Receptor 2,血管内皮细胞生长因子受体2)作为目前研究较为明确的VEGF受体之一,大量研究结果表明VEGFR2不仅在肿瘤血管内皮细胞中过表达,而且一些肿瘤细胞自身也表达VEGFR2,例如白血病和黑色素瘤,在介导内皮细胞增殖、迁移、分化、微管形成及血管通透性增加等一系列生物学活性中发挥着重要作用,可以通过封闭血管内皮细胞生长因子受体2抑制肿瘤生长。近年来以VEGFR2为靶分子的抗肿瘤新药研发取得了很大进展,尤其是抗VEGFR2单克隆抗体。例如,美国ImClone公司开发的人源化抗VEGFR2抗体IMC-1121B,能够抑制VEGF对VEGFR2的激活,针对乳腺癌的治疗现已进入III期临床试验。但是该抗体不能激活机体T淋巴细胞的免疫杀伤作用,从而丧失了机体对肿瘤的杀伤效果。抗VEGFR2/抗CD3(bscVEGFR2×CD3)双特异单链抗体也许可以有效激活静止状态的T细胞杀伤肿瘤细胞,改善人源化抗VEGFR2抗体的抗肿瘤效果。针对VEGFR2抗原的双特异单链抗体国内外未见报道。
发明内容
本发明所要解决的技术问题是用基因工程技术开发一种表达效率高、活性好的抗VEGFR2/抗CD3双特异单链抗体。
本发明的抗VEGFR2/抗CD3双特异单链抗体是由抗人VEGFR2单链抗体和抗人CD3单链抗体连接而成的,可以同时发挥抗VEGFR2抗体和抗CD3抗体的作用,能同时结合VEGFR2阳性细胞和CD3阳性细胞;
本发明的抗VEGFR2/抗CD3双特异单链抗体中,两个单链抗体采用柔性短肽Gly4Ser连接,单链抗体可变区采用柔性短肽(Gly4Ser)3连接,其中丝氨酸Ser是亲水性最强的氨基酸,可增加连接肽的亲水性,甘氨酸Gly是分子量最小,侧链最短的氨基酸,可增加侧链的柔性,并且能够减少抗原性,连接肽Gly4-Ser和(Gly4-Ser)3能保持双特异抗体分子的柔韧性及分子的正确折叠;
本发明的抗VEGFR2/抗CD3双特异单链抗体中,功能域的排列顺序为VLVEGFR2-(Gly4Ser)3-VHVEGFR2-Gly4Ser-VHCD3-(Gly4Ser)3-VLCD3;
其中VLVEGFR2表示抗人血管内皮细胞生长因子受体2单链抗体的轻链可变区;
其中VHVEGFR2表示抗人血管内皮细胞生长因子受体2单链抗体的重链可变区;
其中VHCD3表示抗人CD3单链抗体的重链可变区;
其中VLCD3表示抗人CD3单链抗体的轻链可变区;
本发明的抗VEGFR2/抗CD3双特异单链抗体中,抗人VEGFR2单链抗体的轻链可变区具有序列表SEQ ID NO:1所示氨基酸序列;
本发明的抗VEGFR2/抗CD3双特异单链抗体中,抗人血管内皮细胞生长因子受体2单链抗体的重链可变区具有序列表SEQ ID NO:2所示氨基酸序列;
本发明的抗VEGFR2/抗CD3双特异单链抗体中,抗人CD3单链抗体的重链可变区具有序列表SEQ ID NO:3所示氨基酸序列;
本发明的抗VEGFR2/抗CD3双特异单链抗体中,抗人CD3单链抗体的轻链可变区具有序列表SEQ ID NO:4所示氨基酸序列;
本发明的抗VEGFR2/抗CD3双特异单链抗体中连有分泌信号肽;
本发明的抗VEGFR2/抗CD3双特异单链抗体中的分泌信号肽具有如SEQID NO:5所示氨基酸序列;
本发明提供一种编码以上所述bsc-Ab的核苷酸序列;
本发明提供一种含有所述核苷酸序列的载体,所述载体为真核表达载体pcDNA3.1(+),pcDNA3.1的选择标记基因neo前为弱启动子,目的基因前为强启动子CMV,所以下游neo基因表达量远远低于上游bscVEGFR2×CD3的表达量。neo基因编码的磷酸转移酶能使G418失活,当细胞表达了这种neo抗性基因以后就会在含有G418的选择培养基中存活;
本发明提供一种所述表达载体转染的宿主细胞,所述细胞为CHO-K1细胞,CHO具有一套完整的合成、组装和分泌蛋白质的细胞装置,因此,产生的抗体分子能维持正确的蛋白构象以及翻译后糖基化加工,成为功能性抗体分子,并分泌到细胞外,便于分离纯化;
本发明提供一种制备所述bsc-Ab的方法,包括转染宿主细胞、筛选及培养稳定高效表达bsc-Ab的宿主细胞并从培养物中分离所述的bsc-Ab。
本发明与现有针对VEGFR2抗原的其他形式的抗体相比有以下优点:抗VEGFR2/抗CD3双特异单链抗体,可以同时发挥抗VEGFR2抗体和抗CD3抗体的作用,可以同时结合VEGFR2阳性细胞和CD3阳性细胞,可引导T细胞募集在靶细胞周围,为进一步细胞毒性实验和体内实验奠定基础;利用中国仓鼠细胞表达的重组bsc-Ab,产量高,活性好,适合进行产业化。抗VEGFR2/抗CD3双特异单链抗体分子量小、免疫原性小、且易穿透致密的肿瘤屏障,进入实体瘤周围的微循环,在制备肿瘤治疗药物中具有应用前景。
附图说明
图1为bscVEGFR2×CD3基因片段的设计和表达载体的构建;
图2为直接竞争ELISA法筛选表达bscVEGFR2×CD3的阳性克隆株;
图3为纯化bscVEGFR2×CD3的SDS-PAGE(泳道1)和Western blot结果(泳道2);
图4为FCM分析bscVEGFR2×CD3与VEGFR2+细胞系A375(A)、CD3+细胞系Jurkat(B)、VEGFR2和CD3双阴性细胞系K562(C)的结合活性。
具体实施方式
1、bscVEGFR2×CD3基因片段的设计和表达载体的构建
根据VLVEGFR2、VHVEGFR2、VHCD3、VLCD3的氨基酸序列和功能域的排列顺序VLVEGFR2-(Gly4Ser)3-VHVEGFR2-Gly4Ser-VHCD3-(Gly4Ser)3-VLCD3。在序列的5’端依次添加HindIII酶切位点、KOZAK序列和分泌信号肽序列,在起始密码子ATG前设计KOZAK序列GCCACCATG,可提高翻译的起始效率,在ATG后加入分泌信号肽序列(SEQ ID NO:5),以利于在哺乳动物细胞中分泌表达。在序列的3’端依次添加六个组氨酸标签和EcoR I酶切位点,组氨酸标签便于重组双特异抗体的纯化和鉴定。bscVEGFR2×CD3的全基因序列示意图为:KOZAK-Secretary signal sequence-VLVEGFR2-(Gly4Ser)3-VHVEGFR2-Gy4Ser-VHCD3-(Gly4Ser)3-VLCD3-(His)6。参照哺乳动物细胞常用密码子,使用OptimumGeneTM软件对设计的bsc-Ab基因片段进行密码子优化,编码双特异单链抗体的核苷酸序列如SEQ ID NO:6所示。由公司合成编码bscVEGFR2×CD3的全基因序列(共1548bp)并用Hind III/EcoR I亚克隆入真核表达载体pcDNA3.1(+),命名为pcDNA3.1/bsc-Ab(图1)。pcDNA3.1/bsc-Ab转化大肠杆菌感受态细胞DH5α,挑取阳性克隆菌落进行测序分析。
2、bscVEGFR2×CD3在CHO细胞中的稳定表达
将测序正确的pcDNA3.1/bsc-Ab菌液扩大培养,取过夜培养菌液150ml提取纯化pcDNA3.1/bsc-Ab,提取的pcDNA3.1/bsc-Ab浓度为730mg/L,使用Lipofectamine 2000(Invitrogen公司)转染pcDNA3.1/bsc-Ab至CHO细胞中,同时设立转染空载体pcDNA3.1的CHO细胞为对照组,用浓度为800mg/L的G418加压筛选两周,待阳性克隆长出后,以有限稀释法进行克隆化,取96孔板中CHO细胞培养上清于1.5mL离心管中,1500r/min离心10min,取100μL上述的培养上清,以转染空载体的CHO细胞培养上清为阴性对照组,以带His标签的CD20重组蛋白为阳性对照组,包被酶标板,4℃孵育过夜,含0.5mL/LTween20的磷酸盐缓冲液(PBST),洗涤后加入200∶1 30g/L牛血清白蛋白(BSA)封闭2h,PBST洗涤后加入100∶1鼠抗His-tag mAb(浓度为1mg/L),37℃反应1h,PBST洗涤5次,加入100∶1 HRP标记羊抗鼠IgG多克隆抗体后PBST洗涤,TMB显色,酶标仪测A450吸光度值。筛选到6株高效分泌表达bsc-Ab的克隆株(图2),分别命名为18D11、18E11、12G11、1A4、24E10、24D8。取成功筛选的单克隆细胞株各2×106个细胞置于25cm2培养瓶中培养,待细胞长至80%汇合度,换成无血清DMEM/F12培养液培养3d,取细胞培养的上清液,以转染空载体的CHO细胞上清液为阴性对照,ELISA法检测bsc-Ab的分泌量,18D11分泌量最高,为2.5mg/L。
3、bscVEGFR2×CD3的纯化
18D11细胞株细胞调整浓度为106/ml传至75cm2培养瓶中培养,待细胞长至80%汇合度,换成无血清DMEM/F12培养液培养3d,收集约1L无血清上清液,5000r/min,4℃离心30min,取离心后的上清超滤浓缩至15ml,采用NI-NTA柱分离纯化,用咪唑浓度为500mmol/L的洗脱液洗脱目的蛋白,收集的洗脱液行12%(V/V)的SDS-PAGE蛋白电泳,可见在Mr约为56000的区域出现明显的蛋白条带,与bsc-Ab的Mr一致,纯度达到90%,Western-blot分析,证明Anti-His抗体能与这条蛋白条带发生特异性结合,说明洗脱液中包含目的蛋白(图3)。收集特定蛋白的洗脱液,置于PBS中4℃透析过夜后,BCA法测得的蛋白浓度为1.59g/L,为以后的体内外实验创造了条件。
4、纯化bscVEGFR2×CD3生物学活性的初步分析
生长状态良好的Jurkat(CD3阳性细胞系)、A375(VEGFR2阳性细胞系)、K562(CD3和VEGFR2双阴性细胞系),经PBS洗涤后,106个细胞重悬于PBS(含3%胎牛血清和0.1%叠氮钠)中,4℃孵育30min,PBS洗涤两次,加入100μl bsc-Ab(200mg/L,用含3%胎牛血清和0.1%叠氮钠的PBS稀释),4℃孵育30min,PBS洗涤两次,为了检测Bsc-Ab,使用一种FITC标记抗His-tag多克隆抗体。单独使用FITC标记抗His-tag抗体作为阴性对照,加适量固定液,上流式细胞仪检测。结果表明bscVEGFR2×CD3能与CD3阳性细胞Jurkat和VEGFR2阳性细胞A375特异性结合,而与CD3和VEGFR2双阴性细胞系K562没有结合(图4),说明bscVEGFR2×CD3具有与CD3和VEGFR2特异性结合能力。
Claims (8)
1.一种抗血管内皮细胞生长因子受体2/抗CD3双特异单链抗体,其特征在于:它是由抗人血管内皮细胞生长因子受体2单链抗体和抗人CD3单链抗体连接而成的,两个单链抗体采用柔性短肽Gly4Ser连接,单链抗体可变区采用柔性短肽(Gly4Ser)3连接,双特异单链抗体功能域的排列顺序为VLVEGFR2-(Gly4Ser)3-VHVEGFR2-Gly4Ser-VHCD3-(Gly4Ser)3-VLCD3;
其中,VLVEGFR2的氨基酸序列为SEQ ID NO:1所示;VHVEGFR2的氨基酸序列为SEQ ID NO:2所示;VHCD3的氨基酸序列为SEQ ID NO:3所示;VLCD3的氨基酸序列为SEQ ID NO:4所示。
2.根据权利要求1所述的双特异单链抗体,其特征在于:所述双特异单链抗体连有分泌信号肽。
3.根据权利要求2所述的双特异单链抗体,其特征在于:所述双特异单链抗体的分泌信号肽为SEQ ID NO:5所示氨基酸序列。
4.根据权利要求2或3所述的双特异单链抗体,其特征在于:编码所述双特异单链抗体的序列为SEQ ID NO:6所示核苷酸序列。
5.编码权利要求1至4中任何一项所述的双特异单链抗体的核苷酸序列。
6.根据权利要求5所述的核苷酸序列,其特征在于:所述的核苷酸序列如SEQ ID NO:6所示。
7.一种载体,其特征在于:所述载体含有权利要求5或6所述的核苷酸序列。
8.一种宿主细胞,其特征在于:含有权利要求7所述载体的转染宿主细胞。
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