CN105985435A - 全人源her2抗体的突变抗体及其编码基因和应用 - Google Patents
全人源her2抗体的突变抗体及其编码基因和应用 Download PDFInfo
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Abstract
本发明提供了全人源HER2抗体GB235-019的突变抗体,其中所述突变抗体的重链可变区的氨基酸序列和轻链可变区的氨基酸序列分别为:SEQ ID NO:10,SEQ ID NO:2;SEQ ID NO:11,SEQ ID NO:2;或SEQ ID NO:12,SEQ ID NO:2。所述突变抗体具有与GB235-019抗体相似的人HER2抗原特异性的结合能力,并且也可以与其他HER2阳性肿瘤治疗剂联合用于治疗HER2阳性肿瘤、弱阳性肿瘤或阴性肿瘤。
Description
技术领域
本发明涉及抗体技术领域,具体涉及全人源HER2抗体的突变抗体及其编码基因和应用。
背景技术
乳腺癌是全球女性最常见的恶性肿瘤。HER家族调节正常乳腺的生长和发育,HER2的过度表达与乳腺癌有关。曲妥珠单抗(Trastuzumab),商品名赫赛汀(赫赛汀),是第一个用于治疗人表皮生长因子受体2(HER2)阳性转移性乳腺癌的人源化的单克隆抗体药物。虽然在HER2阳性的恶性肿瘤中,赫赛汀已成为标准的治疗方案,但仍然有40%的患者对赫赛汀没有响应。此外,在抗HER2的治疗方法中,耐药性已成为一个常见的严重问题。生长因子的冗余和细胞内信号转导通路之间的串扰被认为是在乳腺癌患者中促进耐药性的主要原因。帕妥珠单抗(Pertuzumab)是美国Genentech公司与罗氏公司联合近来研制开发出的一种新的抗HER2人源化抗体,与赫赛汀不同,它所针对的抗原表位位于HER2受体的细胞外II区。临床实验研究结果表明单独使用帕妥珠仅产生较弱的抗肿瘤治疗作用,但是已有研究表明帕妥珠与赫赛汀联用可因作用机制的互补而能更完全地阻断HER信号转导,从而更有效地抑制肿瘤细胞的生长。
蛋白糖基化修饰是特异糖链在细胞内质网中添加到蛋白质上形成寡糖链的过程,具有酶定向和位点特异性。根据与蛋白质部分连接方式的不同,蛋白质糖基化修饰分为O-连接和N-连接两种,其N-糖基化保守位点为Asn-X-Thr/Ser,X为除Pro外的任何氨基酸。人IgG在其重链的Fc段的CH2区域内有一个保守的N-连接糖基化位点Asn297。Fc糖链对于抗体和各种受体的最佳结合、抗体对病源物的有效清除以及治疗性抗体临床性质的控制来说都是必需的。人IgG Fab的N-糖基化修饰可对抗体的抗原结合功能有明显促进或抑制作用。糖基化修饰位置上的微小改变即可对糖链的后续加工及抗体的抗原结合活性产生完全不同的影响。
我们之前已利用全人源scFv噬菌体文库筛选技术和基因工程重组表达技术,获得了全人源的抗人HER2(Her-2/neu)单克隆抗体GB235-019(可参见中国专利申请201410705404.0),其可降低输液反应和免疫原性,提高药物安全性,具有更好的药物动力学特征。此外,GB235-019可以与其他HER2阳性肿瘤治疗剂联合用于治疗HER2阳性肿瘤。
发明内容
本发明提供了全人源HER2抗体GB235-019的突变抗体,其中所述突变抗体的重链可变区的氨基酸序列和轻链可变区的氨基酸序列分别为:SEQ ID NO:10,SEQ ID NO:2;SEQ ID NO:11,SEQ ID NO:2;或SEQ ID NO:12,SEQ ID NO:2。
在一个实施方案中,本发明的全人源HER2抗体GB235-019的突变抗体是Fab、Fab’、F(ab’)2、Fv或scFv的形式。所述Fab、Fab’、F(ab’)2、Fv或scFv具有本领域通常所理解的含义。
在一个实施方案中,本发明的上述全人源HER2抗体GB235-019的突变抗体,还可以包括人IgG的重链恒定区和轻链恒定区。在一个具体的实施方案中,所述人IgG为IgG1。在一个具体的实施方案中,所述人IgG重链恒定区的氨基酸序列为SEQ ID NO:5,所述人IgG轻链恒定区的氨基酸序列为SEQ ID NO:6。
本发明提供了编码本发明全人源HER2抗体GB235-019的突变抗体的核苷酸序列。
在一个具体的实施方案中,编码氨基酸序列为SEQ ID NO:10的重链可变区的核苷酸序列为SEQ ID NO:13,编码氨基酸序列为SEQ IDNO:11的重链可变区的核苷酸序列为SEQ ID NO:14,编码氨基酸序列为SEQ ID NO:12的重链可变区的核苷酸序列为SEQ ID NO:15,编码氨基酸序列为SEQ ID NO:2的轻链可变区的核苷酸序列为SEQ IDNO:4。
在一个具体的实施方案中,当本发明的全人源HER2抗体GB235-019的突变抗体为全长抗体时,在本发明的核苷酸序列中,编码重链恒定区的核苷酸序列为SEQ ID NO:7,编码轻链恒定区的核苷酸序列为SEQ ID NO:8。
本发明提供了一种表达载体,其中本发明的核苷酸序列与表达载体的表达控制序列可操作地连接。在具体的实施方案中,所述表达载体是pGEM-T载体或293载体。
本发明提供了一种细胞,其包含本发明的表达载体。所述细胞可以是原核或真核的。在具体的实施方案中,所述细胞可以为哺乳动物细胞,例如FreeStyle 293F细胞。
本发明提供了一种药物组合物,其包含本发明的全人源HER2抗体GB235-019的突变抗体和可药用载体。
本发明提供了一种联合药物,其包含本发明的全人源HER2抗体GB235-019的突变抗体和其他HER2阳性肿瘤治疗剂,所述HER2阳性肿瘤治疗剂为赫赛汀和/或帕妥珠。其中,所述联合药物可以以如下的量施用于受试者:0.001-500mg/kg GB235-019的突变抗体+0.001-500mg/kg赫赛汀和/或帕妥珠;0.001-300mg/kg GB235-019的突变抗体+0.001-300mg/kg赫赛汀和/或帕妥珠;0.001-200mg/kg GB235-019的突变抗体+0.001-200mg/kg赫赛汀和/或帕妥珠;0.01-200mg/kgGB235-019的突变抗体+0.01-200mg/kg赫赛汀和/或帕妥珠;0.01-100mg/kg GB235-019的突变抗体+0.01-100mg/kg赫赛汀和/或帕妥珠;0.1-90mg/kg GB235-019的突变抗体+0.1-90mg/kg赫赛汀和/或帕妥珠;0.1-70mg/kg GB235-019的突变抗体+0.1-70mg/kg赫赛汀和/或帕妥珠;0.1-60mg/kg GB235-019的突变抗体+0.1-60mg/kg赫赛汀和/或帕妥珠;0.1-50mg/kg GB235-019的突变抗体+0.1-50mg/kg赫赛汀和/或帕妥珠;0.1-40mg/kg GB235-019的突变抗体+0.1-40mg/kg赫赛汀和/或帕妥珠;1-40mg/kg GB235-019的突变抗体+1-40mg/kg赫赛汀和/或帕妥珠。本发明的全人源HER2抗体GB235-019的突变抗体与所述其他HER2阳性肿瘤治疗剂可以分开或同时给予受试者。给药途径可以为本领域常用的抗体给药途径。
本发明提供了一种试剂盒,其包含本发明的全人源HER2抗体GB235-019的突变抗体。所述试剂盒可用于检测样品中的HER2蛋白。所述试剂盒还可包含本领域检测HER2试剂盒中的其他常用组分。
本发明提供了本发明的全人源HER2抗体GB235-019的突变抗体用于制备用于治疗受试者中的HER2阳性肿瘤、弱阳性肿瘤或阴性肿瘤的药物的用途。
所述“HER2阳性肿瘤”是指如果IHC〔免疫组化法〕检查结果为3个加号(+++),即,大于30%的肿瘤细胞的胞膜呈现完整的强着色,就表明为HER2阳性;如果是2个加号(++),即,至少10%的肿瘤细胞呈现弱至中度完整的胞膜染色,那么进一步做FISH〔荧光原位杂交法〕或CISH〔显色原位杂交法〕检查,倘若结果为阳性〔发生基因扩增〕,就可以确诊为HER2阳性。优选地,HER2阳性肿瘤检测结果是使用我国食品药品监督管理总局认证的检测试剂盒(IHC,FISH和CISH检测试剂盒)获得的结果。执业医师熟知如何判定肿瘤是否为HER2阳性肿瘤。
所述“HER2弱阳性肿瘤”是指如果IHC“免疫组化法”检查结果是2个加号(++),即,至少10%的肿瘤细胞呈现弱至中度完整的胞膜染色,那么进一步做FISH“荧光原位杂交法”或CISH“显色原位杂交法”检查,倘若结果未发生基因扩增,就表明为HER2弱阳性。相应地,所述“HER2阴性肿瘤”是指如果IHC〔免疫组化法〕检查结果为1个加号(+)或0,就表明为HER2阴性。
所述HER2阳性的肿瘤可以选自HER2阳性的乳腺癌、胃癌、肺癌、非小细胞肺癌、骨癌、胰腺癌、皮肤癌、头或颈癌、皮肤或眼内黑素瘤、子宫癌、卵巢癌、直肠癌、肛门区癌、结肠癌、输卵管癌、子宫内膜癌、宫颈癌、阴道癌、外阴癌、霍奇金病、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织癌、尿道癌、阴茎癌、前列腺癌、膀胱癌、肾或尿道癌、肾细胞癌、肾盂癌、间皮瘤、肝细胞癌、胆囊癌、慢性或急性白血病、淋巴细胞淋巴瘤、中枢神经系统(CNS)癌、脊柱肿瘤、脑干神经胶质瘤、多形性成胶质细胞瘤(glioblastoma multiforme)、星形细胞瘤、神经鞘瘤、室管膜瘤、成神经管细胞瘤、脑膜瘤、鳞状细胞瘤和垂体腺瘤。
优选地,所述受试者是人。
附图说明
图1A显示了重组全长抗人HER2抗体GB235-019重链表达载体(293-VH-CH)的结构示意图;图1B显示了重组全长抗人HER2抗体GB235-019轻链表达载体(293-VL-CL)的结构示意图。以PCR方法,利用相应模板和引物(详见实施例5)分别获得含5’端EcoRI酶切位点的信号肽、重链可变区(VH)、含TGA终止密码子和3’端BamH I酶切位点的重链恒定区(CH)基因片段,并以over-lapping PCR方法将三段联接,获得GB235-019抗体的重链全长基因片段。以相同方法获得GB235-019抗体含信号肽、轻链可变区(VL)和轻链恒定区(CL)的轻链全长基因片段。利用EcoR I和BamH I酶切形成的粘性末端分别将重链和轻链全长基因片段克隆至pGEM-T载体。
图2显示了重组全长抗人HER2抗体GB235-019的SDS-PAGE电泳结果图。将纯化得到的GB235-019和赫赛汀对照样品在50mM二硫苏糖醇还原条件下经10%聚丙烯酰氨凝胶电泳解析,结果显示GB235-019抗体和赫赛汀均呈现分子量为50KDa和25KDa的两条带,分别为抗体的重链和轻链。
图3A和图3B显示了GB235-019的还原分子量分析结果,GB235-019抗体在二硫苏糖醇还原条件下,经Waters H-Class Bio超高效液相色谱仪分析,原始质谱生成文件经PROMASS软件去卷积后得到相应实测分子量。重链理论分子量(Fc含G0F糖型)经GPMAW6.0软件计算为50416.7Da,轻链理论分子量为23120.8Da。图3A的的结果显示,实测GB235-019抗体实测轻链分子量与理论分子量一致,轻链未发生糖基化。图3B的结果显示,实测GB235-019抗体实测重链分子量与理论分子量有较大偏差(>1500Da),比对理论序列,发现除了在Fc区域之外,在Fab框架区域也存在理论N-糖基化位点(Asn-Thr-Ser)。
图4显示了GB235-019突变抗体的SDS-PAGE电泳结果图。将纯化得到的GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体和赫赛汀对照样品在50mM二硫苏糖醇还原条件下经10%聚丙烯酰氨凝胶电泳解析,结果显示,GB235-019突变抗体和赫赛汀均呈现分子量为50KDa和25KDa的两条带,分别为抗体的重链和轻链。
图5A、图5B和图5C显示了GB235-019突变抗体的还原分子量分析结果。GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体在二硫苏糖醇还原条件下,经Waters H-Class Bio超高效液相色谱仪分析,原始质谱生成文件经PROMASS软件去卷积后得到相应实测分子量。重链理论分子量(Fc含G0F糖型)经GPMAW6.0软件计算为50416.7Da。图5A,GB235-019N73D实测重链分子量与理论分子量一致,重链未发生糖基化。图5B,图5C显示GB235-019N73Q、GB235-019S75A突变抗体实测重链分子量与理论分子量非常一致(偏差<1Da),证实在Fab框架区域的N-糖基化位点已被去除。
图6显示了重组全长抗人HER2GB235-019突变抗体与人HER2抗原的结合结果图。以人HER2抗原包被ELISA板,以不同浓度的GB235-019WT,GB235-019N73D、GB235-019N73Q、GB235-019S75A抗体和赫赛汀,帕妥珠与包被于板上的抗原分子结合,并以HRP标记的羊抗人IgG Fc抗体测定结合的抗体。图6的结果显示,GB235-019WT抗体和GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体具有与人HER2抗原特异性的结合能力。
图7A显示了重组全长抗人HER2GB235-019突变抗体体外抑制BT-474细胞增殖活性的实验结果。将P-HER2高表达的HER2阳性BT-474乳腺癌细胞在完全培养基中添加Heregulin-α,不同浓度的GB235-019WT抗体和GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体和赫赛汀单独给药,孵育6天后用AlarmarBlue测定细胞活性。结果显示,完全培养基中添加Heregulin-α,Heregulin-α诱导了BT-474细胞的增殖。BT-474细胞对赫赛汀单独给药变得耐药,GB235-019WT抗体和GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体单独给药作用不明显。
图7B显示了重组全长抗人HER2抗体体外逆转Heregulin-α诱导BT-474细胞对赫赛汀耐药作用的实验结果。将P-HER2高表达的HER2阳性BT-474乳腺癌细胞在完全培养基中添加Heregulin-α,不同浓度的GB235-019WT抗体和GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体分别和赫赛汀联合给药,孵育6天后用Alarmar Blue测定细胞活性。结果显示,完全培养基中添加Heregulin-α,Heregulin-α诱导了BT-474细胞的增殖。BT-474细胞对赫赛汀单独给药变得耐药,而GB235-019WT抗体和GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体分别和赫赛汀联合给药抑制了Heregulin-α诱导的增殖作用,并明显抑制到低于Heregulin-α诱导前的水平,并呈现出浓度依赖性,GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体与GB235-019WT抗体的作用相当。
图8显示了重组全长抗人HER2抗体对乳腺癌BT-474细胞信号转导的抑制作用。将P-HER2高表达的HER2阳性BT-474乳腺癌细胞在0.1%胎牛血清培养基饥饿培养24小时后,加入GB235-019WT抗体和GB235-019N73D突变抗体各20μg/ml,以及赫赛汀20μg/ml,帕妥珠20μg/ml单独给药,抗体处理BT-474细胞6小时后,添加终浓度为100ng/ml的Heregulin-α诱导10分钟后取样。以细胞裂解液做免疫印迹,以相应抗体分别探测全部和磷酸化的HER3、Akt和ERK。图8的结果显示,相比较不加Heregulin-α的对照组,Heregulin-α引起BT-474细胞HER3磷酸化的上调作用。GB235-019WT抗体,GB235-019N73D突变抗体单独用药显著抑制了Heregulin-α对BT-474细胞中HER3磷酸化的上调作用,完全逆转了Heregulin-α对HER3磷酸化的上调作用,GB235-019N73D突变抗体与GB235-019WT抗体的作用相似。帕妥珠也完全抑制了Heregulin-α对BT-474细胞中HER3磷酸化的上调作用,赫赛汀也显著抑制了Heregulin-α对BT-474细胞中HER3磷酸化的上调作用。同时,GB235-019WT抗体,GB235-019N73D突变抗体单独用药,没有抑制Heregulin-α对Akt磷酸化的上调作用。帕妥珠单独用药显著抑制了Heregulin-α对Akt磷酸化的上调作用。GB235-019N73D突变抗体,GB235-019WT抗体单独用药显著抑制了抑制了Heregulin-α对ERK1/2磷酸化的上调作用,GB235-019N73D突变抗体与GB235-019WT抗体的作用相似。
图9显示了重组全长抗人HER2抗体对乳腺癌MCF 7细胞信号转导的抑制作用。将表达较低水平的HER2和高水平HER3,但不表达P-HER2和P-HER3的HER2阴性MCF 7乳腺癌细胞在0.1%胎牛血清培养基饥饿培养24小时后,加入GB235-019WT抗体和GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体各20μg/ml,以及赫赛汀20μg/ml,帕妥珠20μg/ml单独给药,抗体处理MCF7细胞6小时后,添加终浓度为100ng/ml的Heregulin-α诱导10分钟后取样。以细胞裂解液做免疫印迹,以相应抗体分别探测全部和磷酸化的HER3、Akt和ERK。图9的结果显示,相比较不加Heregulin-α的对照组,Heregulin-α引起MCF 7细胞HER3磷酸化的上调作用。GB235-019WT抗体和GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体各自单独用药明显抑制了Heregulin-α对MCF 7细胞中HER3磷酸化的上调作用。赫赛汀和帕妥珠单独用药也显著抑制了Heregulin-α对MCF 7细胞中HER3磷酸化的上调作用。GB235-019WT抗体和GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体各自单独用药显著抑制了Heregulin-α对Akt磷酸化的上调作用,帕妥珠单独用药完全逆转了Heregulin-α对Akt磷酸化的上调作用。GB235-019N73D、GB235-019N73Q突变抗体各自单独用药显著抑制了Heregulin-α对MCF 7细胞中Heregulin-α对ERK1/2磷酸化的上调作用,GB235-019WT抗体,GB235-019S75A突变抗体单独用药微弱的抑制了Heregulin-α对ERK1/2磷酸化的上调作用。帕妥珠单独用药完全逆转了Heregulin-α对ERK1/2磷酸化的上调作用。
图10显示了GB235-019N73D、GB235-019N73Q、GB235-019S75A三种突变抗体的分子排阻色谱图。三种突变抗体各30μg分别经分子排阻色谱(SEC-HPLC)确定相应纯度,GB235-019N73D抗体的主峰纯度为88.3%,GB235-019N73Q抗体的主峰纯度为89.7%,GB235-019S75A的主峰纯度为93.1%。。
图11显示了GB235-019N73D、GB235-019N73Q、GB235-019S75A三种突变抗体的成像毛细管等电聚焦电泳图。三种突变抗体分别经成像毛细管等电聚焦(iCIEF)确定相应主峰等电点(pI)及电荷异构体纯度。三种突变抗体的实测等电点都相对较高,在9.4-9.6左右,GB235-019N73D的等电点相对GB235-019N73Q、GB235-019S75A两个分子约低0.1左右,且主峰纯度相对最高。
图12显示了GB235-019N73D、GB235-019N73Q、GB235-019S75A三种突变抗体的还原法毛细管凝胶电泳图。三种突变抗体分别经还原法毛细管凝胶电泳分析(rCE-SDS)确定相应纯度(轻重链百分比之和),三种突变抗体相应的轻重链之和纯度结果。三种突变抗体都存在一定的低分子量和高分子量杂质,GB235-019N73D的杂质含量相对最少,轻重链之和(LC+HC)纯度最高。
图13显示了GB235-019N73D、GB235-019N73Q、GB235-019S75A三种突变抗体的差示扫描量热图。三种突变抗体分别经差示扫描量热分析(DSC)确定相应Tm值(相变温度,代表在该温度时,50%的生物分子为去折叠状态)。三种分子的Tm1有所不同,GB235-019N73Q的Tm1相对最小,GB235-019N73Q、GB235-019S75A相近。Tm值越高表示热稳定性越好,Tm1的不同也一定程度上预示突变对CH2结构域存在一定影响。GB235-019N73D和GB235-019S75A的热稳定性优于GB235-019N73Q。
具体实施方式
下面以具体的实施例,对本发明的技术方案做进一步的说明;但本发明并不限于这些实施例。
实施例1从全人源scFv噬菌体文库中筛选和人HER2-Fc特异性结合的克隆
利用ELISA技术的抗原抗体结合专一性,将人HER2(胞外域)-Fc融合蛋白(简称为hHER2-Fc)抗原包被到酶标板,通过洗涤和淘选特异性地粘附于包被抗原上的噬菌体。人HER2-Fc(购自Sino Biological公司,货号:10004-H02H)抗原用PBS(0.01M Na2HPO4·12H2O+0.002M KH2PO4+0.14M NaCl+0.002M KCl,pH=8.6)稀释至5μg/ml,按照100μl/孔加入酶标板中,4℃包被过夜。PBST(含0.05%吐温20的PBS缓冲液)洗板4次后加入5%BSA(购自Amresco公司,美国,货号:0332-100g,溶液为PBS)300μl/孔,37℃封闭1小时。再用PBST洗板2次。将含有7×1010独立克隆的全人源scFv噬菌体抗体文库(此抗体库由优瑞科(北京)生物技术有限公司以多个健康人淋巴细胞的抗体可变区基因与人工合成的重链CDR3基因组合构建而成)的悬液按照100μl/孔加入酶标板中,在37℃条件下孵育2小时。孵育结束后,吸出酶标板孔中的噬菌体悬液,然后在每孔中加入PBST 300μl/孔,充分吹打一遍,每遍5分钟,以去除与包被抗原的非特异性结合的噬菌体。加入含0.1%BSA(购自Amresco,美国,货号:0332-100g,溶液为PBS)的0.2M甘氨酸-盐酸(pH=2.2)洗脱液,室温孵育10分钟后,充分地吹打,洗脱特异性地粘附于包被抗原上的噬菌体。将洗脱下的噬菌体悬液用1M Tris-HCl(pH9.1)缓冲液中和。将洗脱的噬菌体感染加入1ml对数期的TG1(OD600约0.3~04.)(购自Lucigen,美国,货号:60500-0)进行感染,37℃,静置1小时。将感染后菌液取10μl进行10倍梯度稀释,进行10倍、100倍、1000倍稀释后涂平板计数。取90μl感染后菌液保存甘油菌,甘油终浓度为10%,放-80℃保存。剩余侵染后菌液全部涂于150mm 2×YT-A固体平板(17g/L胰化蛋白胨,10g/L酵母提取物,5g/L氯化钠,15g/L琼脂,100μg/ml氨苄青霉素),37℃过夜培养。加入5ml 2×YT-A-10%甘油培养基到150mm平板菌上过夜培养,并用无菌涂布棒轻轻刮下,到平板上无残留菌液即可。第二轮扩增,取适量刮取得菌液到5ml 2×YT-AMP-glucose液体培养基(17g/L胰化蛋白胨,10g/L酵母提取物,5g/L氯化钠,2%葡萄糖,100μg/ml氨苄青霉素)中(OD600约0.05~0.1即可),37℃、200rpm培养到对数期(OD600约0.3~0.4),加入20倍菌体总数的M13K07辅助噬菌体(购自NEB公司,美国,货号:N0315S)侵染,37℃,1小时。侵染完后1500g、5分钟收集菌体重悬于2×YT-AMP-Kana培养基中(17g/L胰化蛋白胨,10g/L酵母提取物,5g/L氯化钠,50μg/ml卡那霉素,100μg/ml氨苄青霉素),30℃、200rpm过夜培养,完成重组噬菌体的扩增制备。进行相同的第二轮淘选,第三轮扩增和淘选。挑取菌落到5ml(2×YT-AMP-glucose液体培养基(17g/L胰化蛋白胨,10g/L酵母提取物,5g/L氯化钠,2%葡萄糖,100μg/ml氨苄青霉素),37℃、200rpm过夜培养。质粒提取试剂盒(购自Qiagen公司,美国,货号:12943)抽提质粒,通过测序鉴定,质粒-80℃保存。
实施例2用酶联免疫吸附法(ELISA)鉴定特异性结合人HER2-Fc噬菌体的免疫反应性
用酶联免疫吸附法(ELISA)进一步鉴定实施例1中得到的特异性结合人HER2-Fc噬菌体的免疫反应性。人HER2-Fc抗原(购自SinoBiological公司,货号:10004-H02H)用PBS(pH=8.6)稀释2μg/ml,按照100μl/孔加入酶标板中,4℃包被过夜。PBST洗板4次后加入5%BSA(购自Amresco,美国,货号:0332-100g,溶液为PBS)300μl/孔,37℃封闭1小时。再用PBST洗板2次,加入100μl/孔噬菌体克隆悬液,在37℃条件下孵育2小时。PBST洗板4次。加入HRP标记的抗M13K07噬菌体抗体(购自GE公司,美国,货号:27-9421-01,PBST 1:5000稀释,100μl/孔),室温孵育1小时。PBST洗板4次,加入100μl/孔可溶型单组分3,3',5,5'-四甲基联苯胺底物溶液(购自Tiangen公司,货号:PA107-01)。室温孵育15分钟显色,加入50μl/孔终止液(1M硫酸),在多功能酶标仪(Bio-Rad,Model 680Micro reader,美国)上450/570nm波长下读出吸光值。
结果显示,经过三轮重复筛选,共获得1312个可与人HER2-Fc抗原结合的scFv噬菌体克隆,其中499个可与人HER2-Fc抗原特异性结合的scFv噬菌体克隆数。经DNA测序,这些克隆中有102种DNA/氨基酸序列都不相同的scFv(如表1所示)。
表1
实施例3用ELISA法检测102个人HER2-Fc特异性scFv的种属交叉反应和HER家族成员分子间的交叉反应
用ELISA法检测102个人HER2-Fc特异性scFv的种属交叉反应和与HER家族成员分子间的交叉反应。方法同实施例2,将包被的人HER2-Fc抗原分别换成猴HER2-Fc(购自Sino Biological公司,货号:90295-C02H)、小鼠HER2-Fc(购自Sino Biological公司,货号:50714-M02H)、人HER1-Fc(购自Sino Biological公司,货号:10001-H02H)、人HER3-Fc(购自Sino Biological公司,货号:10201-H05H)和人HER4-Fc(购自Sino Biological公司,货号:10363-H02H)。抗原用PBS(pH=8.6)稀释2μg/ml,按照100μl/孔加入酶标板中,4℃包被过夜。PBST洗板4次后加入5%BSA(购自Amresco,美国,货号:0332-100g,溶液为PBS)300μl/孔,37℃封闭1小时。再用PBST洗板2次,加入100μl/孔102个ScFv噬菌体克隆悬液,在37℃条件下孵育2小时。PBST洗板4次。加入HRP标记的抗M13K07噬菌体抗体(购自GE公司,美国,货号:27-9421-01,PBST1:5000稀释,100μl/孔),室温孵育1小时。PBST洗板4次,加入100μl/孔可溶型单组分3,3',5,5'-四甲基联苯胺底物溶液(购自Tiangen,货号:PA107-01)。室温孵育15分钟显色,加入50μl/孔终止液(1M硫酸),在多功能酶标仪(Bio-Rad,Model 680Micro reader,美国)上450/570nm波长下读出吸光值。
结果显示,有96个ScFv噬菌体克隆与猴HER2-Fc抗原有交叉反应,其中有20个ScFv噬菌体克隆与鼠HER2-Fc抗原有交叉反应。所有的102个克隆与人HER1-Fc、人HER3-Fc和人HER4-Fc抗原均无交叉反应(如表2所示)。
表2
抗原 | HER2-Fc | 小鼠HER2-Fc | 人HER1-Fc | 人HER3-Fc | 人HER4-Fc |
克隆数 | 96 | 20 | 0 | 0 | 0 |
实施例4 102个ScFv噬菌体克隆的亲和力排序
102个ScFv噬菌体克隆通过ELISA法亲和力排序,将人HER2-Fc抗原用PBS缓冲液从25μg/ml开始十倍比稀释共8个梯度,分别与102个ScFv噬菌体克隆的噬菌体悬液在室温孵育4小时以达到平衡;将所得到的混合液加入到2μg/ml人HER2-Fc抗原(pH=8.6PBS,4℃过夜,100μl/孔)事先包被的酶标板中,并以5%BSA(购自Amresco公司,美国,货号:0332-100g,溶液为PBS)封闭好的酶标板,以结合未被捕获的ScFv抗体。加入HRP标记的抗M13噬菌体抗体(购自GE公司,美国,货号:27-9421-01,以PBST 1:5000稀释,100μl/孔),以与实施例2相同的方法检测。以IC50值对102个阳性克隆的亲和力进行排序(IC50值越低,亲和力越高)。
结果显示了102个ScFv噬菌体克隆的IC50值分布范围,其中有4个克隆的亲和力高于赫赛汀。
表3
IC50(nM) | ≤2.0 | 2.0-10.0 | 10.0-100.0 | >100.0 | 未测出 |
克隆数 | 4 | 21 | 37 | 25 | 15 |
实施例5 GB235-019重组全长IgG1型抗体的真核表达载体的构建
从102个ScFv噬菌体克隆序列中,构建重组全长IgG1型抗体GB235-019(重组全长抗体序列019克隆命名为GB235-019)的真核表达载体。经全人源ScFv噬菌体文库筛选得到WG1-019(ScFv噬菌体文库筛选中得到单链抗体序列克隆命名为WG1-019)单链抗体克隆的核苷酸序列为SEQ ID NO:9,其包含的重链可变区序列和轻链可变区的核苷酸序列分别为SEQ ID NO:3(编码的氨基酸序列为SEQ ID NO:1)和SEQ ID NO:4(编码的氨基酸序列为SEQ ID NO:2)。信号肽氨基酸序列为:MELGLSWIFLLAILKGVQC;核苷酸序列为:ATGGAGTTGGGACTGTCTTGGATTTTCCTGTTGGCTATTCTGAAAGGTGTGCAGTGT(由上海捷瑞生物工程有限公司合成)。
GB235-019重组全长抗体的重链恒定区和轻链恒定区的核苷酸序列分别为SEQ ID NO:7(编码的氨基酸序列为SEQ ID NO:5)和SEQ IDNO:8(编码的氨基酸序列为SEQ ID NO:6)(由上海捷瑞生物工程有限公司合成)。
设计引物用于构建GB235-019重组全长IgG1型抗体重链和轻链的真核表达载体,引物序列如下:
1-1:5’-GAATTCGCGGCCGCATGGAGTTGGGACTG-3’
2-3:5’-CTGGGTCATCTGGATGTCACACTGCACACCTTTC-3’
3-3:5’-GAAAGGTGTGCAGTGTGACATCCAGATGACCCAG-3’
4-4:5’-GATGGTGCAGCCACAGTACGTTTGATCTCCACCTTG-3’
5-2:5’-ATCAAACGTACTGTGGCTGCACCATC-3’
6-1:5’-GTTTAAACGGATCCCTAACACTCTCCCCTGTTG-3’
7-7:5’-GTACCAGCTGGACCTC ACACTGCACACCTTTC-3’
8-7:5’-GAAAGGTGTGCAGTGTGAGGTCCAGCTGGTAC-3’
9-1:5’-GATGGGCCCTTGGTGGAGGCTGAGGAGACGGTCAC-3’
10-1:5’-ACCGTCTCCTCAGCCTCCACCAAGGGCCCATC-3’
11-1:5’-GTTTAAACGGATCCTCATTTACCGGGAGACAGGGAG-3’
以合成的信号肽序列为模板,1-1和2-3为引物,由PCR方法扩增获得含EcoR I酶切位点的基因片段,命名为“SPL-GB235-019”;以合成的轻链可变区序列SEQ ID NO:4为模板,3-3和4-4为引物,PCR法扩增获得轻链可变区基因片段,命名为“VL-GB235-019”;同时以合成的轻链恒定区序列SEQ ID NO:8为模板,5-2和6-1为引物,PCR法扩增获得含TGA终止密码子和BamH I酶切位点的重链恒定区基因片段,命名为“CL-GB235-019”。以SPL-GB235-019、VL-GB235-019、CL-GB235-019基因片段为模板,1-1和6-1为引物,通过over-lappingPCR方法(Higuchi R,et al.A general method of in vitro preparationand specific mutagenesis of DNA fragments:study of protein and DNAinteractions.Nucleic Acids Research,1988,16(15):7351-67.)扩增获得GB235-019抗体的轻链全长基因片段。
同样的方法,以合成的信号肽序列为模板,1-1和7-7为引物,由PCR(聚合酶链反应)方法扩增获得含EcoR I酶切位点的基因片段,命名为“SPH-GB235-019”;以合成的重链可变区序列SEQ ID NO:3为模板,8-7和9-1为引物,PCR法扩增获得重链可变区基因片段,命名为“VH-GB235-019”;同时以合成的重链恒定区序列SEQ ID NO:7为模板,10-1和11-1为引物,PCR法扩增获得含TGA终止密码子和BamHI酶切位点的重链恒定区基因片段,命名为“CH-GB235-019”。以SPH-GB235-019、VH-GB235-019、CH-GB235-019基因片段为模板,1-1和11-1为引物,通过over-lapping PCR方法扩增获得GB235-019抗体的重链全长基因片段。
将以上重链和轻链全长基因片段克隆至pGEM-T载体(购自Promega公司,美国,货号:A3600),使所述基因片段5’端含有EcoRI酶切位点,3’端含有TGA终止密码子和BamH I酶切位点。经DNA测序后,将测序正确的克隆用EcoR I(购自NEB公司,美国,货号:R0101S)和BamH I(购自NEB公司,美国,货号:R0136S)双酶切消化(37℃,4小时),回收目的基因片段。将上述酶切获得的抗体重链全长基因片段和轻链全长基因片段克隆至293载体(购自Invitrogen公司,美国,货号:K8300-01),经DNA测序鉴定后,得到含有构建成功的全长抗体重链真核表达载体或全长抗体轻链真核表达载体的克隆。
图1A为重组全长抗人HER2抗体重链(293-VH-CH)表达载体的结构示意图;图1B为重组全长抗人HER2抗体轻链(293-VL-CL)表达载体的结构示意图。
实施例6 GB235-019抗体的真核细胞瞬时转染表达及纯化
实施例5中所构建的GB235-019抗体的重组载体的表达,可采用共转染FreeStyle 293F细胞(购自Invitrogen,美国,货号:R790-07)的方法。转染前24小时,将FreeStyle 293F细胞按6×105个细胞/ml传代,于恒温摇床135转/分,37℃,8%CO2条件下培养,使得转染当天的细胞密度(血球板计数法)为1.2-1.5×106个细胞/ml。用FreeStyle 293培养基(购自Invitrogen公司,美国,货号:12338-018)稀释细胞,至密度为1×106个细胞/ml。为确保最佳转染效果,细胞活力(台盼蓝染色法)应大于95%。
将转染用试剂FreeStyle Max Reagent(购自Invitrogen公司,美国,货号:16447-500)轻度颠倒混匀4次。将各315μg重链和轻链表达载体质粒分别加入转染用培养液OptiPRO SFM(购自Invitrogen公司,美国,货号:12309-050)中,并用OptiPRO SFM补充体积至10ml,混匀。另取一支离心管,用OptiPRO SFM稀释625μl FreeStyle MaxReagent至10ml,轻度颠倒混匀。将稀释的质粒与稀释的FreeStyle MaxReagent混匀,室温孵育15分钟。将所得的20ml混合液缓慢加入装有500ml FreeStyle 293F培养基(购自Invitrogen公司,美国,货号:12338-018)的摇瓶中。摇瓶于恒温摇床培养7天(135转/分,37℃,8%CO2)。冷冻离心机9000转/分离心20分钟,收集上清液进行下一步蛋白纯化。
上述含GB235-019抗体的FreeStyle 293F细胞上清液,经离心后使用蛋白A(Protein A)柱(购自GE Healthcare Bio-Sciences公司,美国,货号:17-5080-02)捕获IgG1型抗体,用50mM柠檬酸-柠檬酸钠缓冲液(pH3.3)洗脱,收集洗脱物(0.5ml),加入100μl 1M三羟甲基氨基甲烷-盐酸(Tris-HCl)缓冲液(pH11.0)中和至中性,经10K透析膜(购自上海捷瑞生物工程有限公司,货号:M1915)在磷酸盐缓冲液PBS(0.01M Na2HPO4·12H2O+0.002M KH2PO4+0.14MNaCl+0.002M KCl,pH=7.2)中透析后,在OD280nm下测定蛋白含量。经0.22μm滤器(购自Millipore公司,德国,货号:GVHP01300)过滤除菌后-80℃保存。将纯化得到的GB235-019抗体在终浓度为50mM的二硫苏糖醇还原条件下,经10%聚丙烯酰氨凝胶电泳检测其纯度和分子量大小。
图2的结果显示,在完全还原的条件下,GB235-019抗体呈现分子量为50KDa和25KDa的两条带,其分别为抗体的重链和轻链条带(赫赛汀为阳性对照,购自Roche公司)。这些结果表明,我们所构建的GB235-019抗体结构正确,其分子量大小与理论值一致。
实施例7 GB235-019抗体的还原分子量分析
在10μg GB235-019抗体中分别加入终浓度为20mM的二硫苏糖醇,37℃水浴30分钟以打开所有链间的二硫键。采用反相色谱与质谱联用方法分析分开的轻、重链。采用Waters H-Class Bio超高效液相色谱仪(Waters公司,美国);色谱柱:3.0μm,2.1×150mm(购自安捷伦公司,美国,货号:1912-3301);流动相为:A(水),B(乙腈),C(1%三氟乙酸),梯度从第4分钟的35%B变化至第20分钟的42%B,C相保持为10%,流速为0.3mL/分钟;进样量为20μg;采用Thermo LTQ-Orbitrap Discovery质谱仪(赛默飞,美国);喷雾电压为3.7KV;管镜片为230V;毛细管温度为300℃;分辨率为30000;质量与电荷比范围为1000-3000。重链理论分子量(Fc含G0F糖型)经GPMAW6.0软件计算为50416.7Da,轻链理论分子量为23120.8Da。原始质谱生成文件经PROMASS软件去卷积后得到相应实测分子量。
图3A的的结果显示,实测GB235-019抗体实测轻链分子量与理论分子量一致,轻链未发生糖基化。图3B的结果显示,实测GB235-019抗体实测重链分子量与理论分子量有较大偏差(>1500Da),比对理论序列,发现除了在Fc区域之外,在Fab框架区域也存在理论N-糖基化位点(Asn-Thr-Ser),增加了额外的分子量。
实施例8 GB235-019 Fab端重链N-连接聚糖特征序列突变的真核表达载体的构建
N-糖基化保守位点为Asn-X-Thr/Ser,X为除Pro外的任何氨基酸。N-连接聚糖连接到Asn-X-Ser/Thr特征序列中的Asn残基上。(ImperialiB,O'Connor SE.Effect of N-linked glycosylation on glycopeptide andglycoprotein structure.Curr Opin Chem Biol 3(6):643–649.)。从实施例5得到的全长抗体GB235-019的Fab框架3区Asn73位为N-糖基位点。通过突变Asn-X-Ser/Thr的专一序列,以去除N-糖基化保守位点(Walsh G.Biopharmaceutical benchmarks-2003.Nat Biotechnol,2003,21:865-870)。通过IgBLAST比对,在Germline基因里有Asp-Thr-Ser组合。比对核苷酸序列可得Asn对应密码子“AAC”,可将其突变为“GAC”,以达到将Asn突变为Asp的目的。同样,Asn和Gln都为酰胺类氨基酸,Gln侧链基团比Asn多一个甲基,属保守性替换。比对核苷酸序列Asn对应密码子“AAC”,可将其突变为“CAG”,以达到将Asn突变为Gln的目的。进行IgBLAST,在Germline基因里有Ser处氨基酸残基为Ala。比对核苷酸序列Ser对应密码子“TCC”,可将其突变为“GCC”,以达到将Ser突变为Ala的目的。
以从实施例5得到的全长抗体重链真核表达载体为模板,以点突变的方法(Kunkel,T.A,et al."Rapid and efficient site-specificmutagenesis without phenotypic selection.Proc.Natl.Acad.Sci,1985(82):488-492.)构建GB235-019抗体Fab端突变N-连接聚糖特征序列的突变抗体重链真核表达载体。设计三个突变方案,分别为将GB235-019抗体重链Asn73(N73)突变为Asp73(D73);将GB235-019抗体重链Asn73(N73)突变为Gln73(Q73),GB235-019抗体重链Ser75(S75)突变为Ala75(A75)。设计引物用于构建GB235-019Fab端N-连接聚糖特征序列点突变的重链表达载体,引物序列如下:
12-1:5’-GTCACCATGACCAGGGACACCTCCAT-3’
12-2:5’-ATGGAGGTGTCCCTGGTCATGGTGAC-3’
13-1:5’-GTCACCATGACCAGGCAGACCTCCATAAGC-3’
13-2:5’-GCTTATGGAGGTCTGCCTGGTCATGGTGAC-3’
14-1:5’-CATGACCAGGAACACCGCCATAAGCAC-3’
14-2:5’-GTGCTTATGGCGGTGTTCCTGGTCATG-3’
以实施例5得到的293-VH-CH表达载体为模板,12-1和12-2为引物,由PCR方法扩增获得PCR产物。取DpnⅠ(购自NEB公司,美国,货号:1235A)2μl到20μl PCR产物中37℃酶切1小时。PCR产物用PCR产物纯化试剂盒(购自Axygen公司,美国,货号:AP-PCR-50)纯化,将纯化得到的PCR产物热休克法(42℃,90秒)转化DH5α大肠杆菌感受态细胞(购自天根公司,货号:CB101),经DNA测序鉴定后,获得突变抗体重链真核表达载体命名为“293-VH-CH-N73D”(其包含的重链可变区的氨基酸序列和核苷酸序列分别为SEQ ID NO:10和SEQ ID NO:13)。同样的以13-1和13-2为引物,由PCR方法扩增获得PCR产物。取DpnⅠ(购自NEB公司,美国,货号:1235A)2μl到20μl PCR产物中37℃酶切1小时。PCR产物用PCR产物纯化试剂盒(购自Axygen公司,美国,货号:AP-PCR-50),将纯化得到的PCR产物热休克法(42℃,90秒)转化DH5α大肠杆菌感受态细胞(购自天根公司,货号:CB101),经DNA测序鉴定后,获得突变抗体重链真核表达载体命名为“293-VH-CH-N73Q”(其包含的重链可变区的氨基酸序列和核苷酸序列分别为SEQ ID NO:11和SEQ ID NO:14)。同样的以14-1和14-2为引物,由PCR方法扩增获得PCR产物。取Dpn Ⅰ(购自NEB公司,美国,货号:1235A)2μl到20μl PCR产物中37℃酶切1小时。PCR产物用PCR产物纯化试剂盒(购自Axygen公司,美国,货号:AP-PCR-50),将纯化得到的PCR产物热休克法(42℃,90秒)转化DH5α大肠杆菌感受态细胞(购自天根公司,货号:CB101),经DNA测序鉴定后,获得突变抗体重链真核表达载体命名为“293-VH-CH-S75A”(其包含的重链可变区的氨基酸序列和核苷酸序列分别为SEQ ID NO:12和SEQ ID NO:15)。
实施例9 GB235-019突变抗体的真核细胞瞬时转染表达及纯化
实施例8中所构建的突变抗体的重组载体的表达,方法同实施例6。293-VH-CH-N73D、293-VH-CH-N73Q、293-VH-CH-S75A分别与293-VL-CL共转染FreeStyle 293F细胞(购自Invitrogen,美国,货号:R790-07)。
将转染用试剂FreeStyle Max Reagent(购自Invitrogen公司,美国,货号:16447-500)轻度颠倒混匀4次。将各315μg重链和轻链表达载体质粒分别加入转染用培养液OptiPRO SFM(购自Invitrogen公司,美国,货号:12309-050)中,并用OptiPRO SFM补充体积至10ml,混匀。另取一支离心管,用OptiPRO SFM稀释625μl FreeStyle MaxReagent至10ml,轻度颠倒混匀。将稀释的质粒与稀释的FreeStyle MaxReagent混匀,室温孵育15分钟。将所得的20ml混合液缓慢加入装有500ml FreeStyle 293F培养基(购自Invitrogen公司,美国,货号:12338-018)的摇瓶中。摇瓶于恒温摇床培养7天(135转/分,37℃,8%CO2)。冷冻离心机9000转/分离心20分钟,收集上清液进行下一步蛋白纯化。突变抗体纯化方法同实施例6。获得的突变抗体经0.22μm滤器(购自Millipore公司,货号:GVHP01300)过滤除菌后-80℃保存。将纯化得到的突变抗体分别命名为GB235-019N73D、GB235-019N73Q和GB235-019S75A。突变抗体在终浓度为50mM的二硫苏糖醇还原条件下,经10%聚丙烯酰氨凝胶电泳检测其纯度和分子量大小。
图4的结果显示,在完全还原的条件下,GB235-019N73D、GB235-019N73Q、GB235-019S75A抗体各呈现分子量为50KDa和25KDa的两条带,其分别为抗体的重链和轻链条带(赫赛汀为阳性对照,购自Roche公司)。这些结果表明,我们所构建的GB235-019N73D、GB235-019N73Q、GB235-019S75A抗体结构正确,其分子量大小与理论值一致。
实施例10重组全长抗体GB235-019突变抗体的还原分子量分析
实施例9中所获得GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体还原分子量分析,方法同实施例7。在各10μg的GB235-19突变抗体GB235-019N73D、GB235-019N73Q、GB235-019S75A中分别加入终浓度为20mM的二硫苏糖醇,37℃水浴30分钟以打开所有链间的二硫键。采用反相色谱与质谱联用方法分析分开的轻、重链。采用Waters H-Class Bio超高效液相色谱仪(Waters公司,美国);色谱柱:3.0μm,2.1×150mm(购自安捷伦公司,美国,货号:1912-3301);流动相为:A(水),B(乙腈),C(1%TFA),梯度从第4分钟的35%B变化至第20分钟的42%B,C相保持为10%,流速为0.3mL/分钟;进样量20μg;采用ThermoLTQ-Orbitrap Discovery质谱仪(赛默飞公司,美国);喷雾电压为3.7KV;管镜片为230V;毛细管温度为300℃;分辨率为30000;质量与电荷比范围为1000-3000。三种突变抗体重链理论分子量(Fc含G0F糖型)经GPMAW6.0软件算,GB235-019S75A的重链理论分子量为50400.7Da,GB235-019N73D的重链理论分子量为50417.7Da,GB235-019N73Q的重链理论分子量为50430.7Da。原始质谱生成文件经PROMASS软件去卷积后得到相应实测分子量。
图5A,GB235-019N73D实测重链分子量与理论分子量一致,重链未发生糖基化。图5B,图5C显示GB235-019N73Q、GB235-019S75A突变抗体实测重链分子量与理论分子量非常一致(偏差<1Da),证实在Fab框架区域的N-糖基化位点已被去除。
人IgG在其重链的Fc段的CH2区域内有一个保守的N-连接糖基化位点Asn297。连接在Asn297的糖链可维持抗体的四级结构以及Fc段的热稳定性,并通过影响IgG分子与FcRs、C1q以及FcRn的结合而分别调节IgG分子的抗体依赖细胞毒作用(ADCC)、补体依赖的细胞毒作用(CDC)以及半衰期。
人IgG Fab的N-糖基化修饰可对抗体的抗原结合功能有明显促进或抑制作用。糖基化修饰位置上的微小改变即可对糖链的后续加工及抗体的抗原结合活性产生完全不同的影响,在抗体的生产工艺中带来质量控制的复杂性。GB235-019野生型抗体经点突变后得到GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体。三种突变抗体改变了GB235-019野生型抗体重链Fab的N-连接聚糖的特征序列(Asn-Thr-Ser)。三种突变抗体经还原分子量测定,重链Fab未发生糖基化,这将有助于生产工艺中的质量控制。通过生物活性分析和理化分析进一步验证这三种突变抗体。
实施例11重组全长抗体GB235-019野生型抗体和突变抗体的免疫学活性鉴定
用ELISA结合实验验证GB235-019野生型抗体(GB235-019WT)和GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体与人HER2抗原的结合能力。方法如下:将人HER2抗原(购自Sino Biological公司,货号:10004-H08H)抗原用PBS缓冲液稀释至1μg/ml,按照100μl/孔加入酶标板中,4℃包被过夜。PBST洗板4次后加入5%BSA300μl/孔(购自Amresco,美国,货号:0332-100g,溶液为PBS),室温封闭1小时。PBST洗板4次后,将GB235-019WT抗体、GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体、帕妥珠(购自Roche公司)和赫赛汀(购自Roche公司)分别从5μg/ml开始五倍比稀释共7个梯度,按照100μl/孔加入酶标板中,在室温条件下孵育1小时。PBST洗板4次,将HRP标记的羊抗人IgG Fc抗体(购自CalBiochem公司,美国,货号:AP113A-K)用PBS缓冲液以1:10000稀释,按照100μl/孔加入酶标板中,室温孵育1小时。PBST洗板4次,加入100μl/孔3,3',5,5'-四甲基联苯胺底物溶液(购自Tiangen公司,货号:PA107-01),室温孵育15分钟显色,加入50μl/孔终止液(1M硫酸),在M5多功能酶标仪(Molecular Devices公司,美国)上450/630nm波长下读出吸光值。
图6的结果显示,GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体具有与人HER2抗原特异性的结合能力,并呈现出浓度依赖性和可饱和性。GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体具有和GB235-019WT抗体相似的结合能力,没有显著差异。
实施例12重组全长GB235-019突变抗体体外抑制乳腺癌BT-474细胞增殖活性
乳腺癌BT-474细胞表达中等水平的HER2和中等水平的HER3,同时表达高水平的P-HER2,但不表达P-HER3(Richard M.Neve.Acollectionof breast cancer cell lines for the study of functionallydistinct cancer subtypes.CANCER CELL,2006,515-527),基于前文所定义的,所述乳腺癌BT-474细胞属于HER2阳性肿瘤细胞。完全培养基中添加Heregulin-α(购自R&D公司,美国,货号:296-HR)的增殖抑制试验中,将处于对数生长期的BT-474细胞以5000细胞/孔于RPMI1640(购自Invitrogen公司,美国,货号:A10491)含10%胎牛血清(购自Invitrogen公司,美国,货号:10099-141)完全培养基中培养到96孔培养板,37℃,5%CO2中培养24小时。GB235-019WT抗体和GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体单独用药,以及与赫赛汀联合用药抑制试验。单独用药组分别加入GB235-019WT抗体和GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体,帕妥珠和赫赛汀(工作终浓度为75、18.8、4.7、1.2、0.29、0.07、0.018、0.005、0.0011、0μg/ml);联合用药组同时加入以上述每个剂量的GB235-019WT、GB235-019N73D、GB235-019N73Q、GB235-019S75A、帕妥珠分别和赫赛汀联合用药。在上述抗体处理2小时后,加入工作终浓度为100ng/ml的Heregulin-α溶液,设置未加Heregulin-α溶液的孔,37℃,5%CO2中继续培养6天。加入AlamarBlue(购自Invitrogen公司,美国,货号:DAL1100)检测BT-474细胞活性,在M5多功能酶标仪(Molecular Devices公司,美国)上544/590nm波长下读取荧光值。
结果显示了重组全长抗人HER2GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体体外逆转Heregulin-α诱导HER2阳性BT-474细胞对赫赛汀耐药作用的实验结果。图7A结果显示,完全培养基中添加Heregulin-α,Heregulin-α诱导了BT-474细胞的增殖。BT-474细胞对赫赛汀单独给药变得不敏感,GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体单独给药也没有抑制作用,同GB235-019WT抗体作用相似。图7B结果显示,GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体分别和赫赛汀联合给药抑制了Heregulin-α诱导的增殖作用。三个突变抗体分别与赫赛汀联合给药使得不仅抑制了Heregulin-α诱导BT-474细胞增殖的作用,并明显抑制到低于Heregulin-α诱导前的水平,并呈现出浓度依赖性。GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体与GB235-019WT抗体的作用相似,没有显著差异。
实施例13重组全长抗人HER2GB235-019突变抗体体外对乳腺癌BT-474细胞信号转导的抑制作用
将处于对数生长期的BT-474细胞以1.8×105细胞/孔于RPMI1640(购自Invitrogen公司,美国,货号:A10491)含10%胎牛血清(购自Invitrogen公司,美国,货号:10099-141)完全培养基中培养到6孔培养板,37℃,5%CO2中培养24小时。第二天,弃去培养基换成0.1%胎牛血清(购自Invitrogen公司,货号:10099-141)的低血清培养基培养24小时。
随后添加GB235-019WT 20μg/ml,GB235-019N73D突变抗体20μg/ml,赫赛汀20μg/ml,及帕妥珠20μg/ml单独给药,抗体处理BT-474细胞6小时后,添加终浓度为100ng/ml的Heregulin-α(购自R&D公司,美国,货号:296-HR)诱导15分钟,设置未加Heregulin-α的空白对照孔。4℃预冷的PBS洗涤一次后终止反应,添加120μl LDS(购自Invitrogen公司,美国,货号:NP0007),冰上放置并迅速收集细胞裂解液,-80℃保存备用。
收集的细胞裂解液在终浓度为50mM的二硫苏糖醇(购自Sangon公司,货号:D0281)还原条件下,经Western-blot分析检测抗体对Heregulin-α(购自R&D公司,货号:296-HR)诱导SK-BR-3细胞中HER-3,Akt和ERK1/2磷酸化的影响。Western-blot方法如下:将电泳后凝胶上的蛋白通过电转移(300mA,80分钟)的方法转移至NC膜上(购自Pall公司,美国,货号:S80209),5%脱脂奶粉(购自Sangon公司,货号:NB0669)封闭后分别加入以1:1000稀释的兔一抗P-HER3Y1289(购自Cell Signaling Technology公司,美国,货号:8017),1:1000稀释的兔一抗HER3(购自Cell Signaling Technology公司,美国,货号:12708),1:1000稀释的兔一抗P-AktS473(购自Cell SignalingTechnology公司,美国,货号:4060),1:1000稀释的兔一抗Akt(购自Cell Signaling Technology公司,美国,货号:4691),1:500稀释的兔一抗P-ERK1/2(购自Cell Signaling Technology公司,美国,货号:4370),1:1000稀释的兔一抗ERK1/2(购自Cell Signaling Technology公司,美国,货号:4695),1:5000稀释的兔一抗GAPDH(Cell SignalingTechnology公司,美国,货号:5174),4℃孵育过夜。用1×TBST洗涤NC膜三遍后再加入1:10000稀释的HRP标记的羊抗兔抗体(购自MERCK公司,美国,货号:401315),用1×TBST洗涤三遍后,再加入ECL(购自PerkinElmer公司,美国,货号:NEL104001EA)显示,胶片曝光记录信号(购自Kodak公司,货号:FF057)。
乳腺癌BT-474细胞表达高水平的P-HER2,但不表达P-HER3,是对赫赛汀敏感的细胞株。图8的结果显示,与不加Heregulin-α的对照组相比,Heregulin-α引起BT-474细胞HER3磷酸化的上调作用。GB235-019WT抗体、GB235-019N73D突变抗体单独用药显著抑制了Heregulin-α对BT-474细胞中HER3磷酸化的上调作用,完全逆转了Heregulin-α对HER3磷酸化的上调作用,GB235-019N73D突变抗体与GB235-019WT抗体的作用相似。帕妥珠也完全抑制了Heregulin-α对BT-474细胞中HER3磷酸化的上调作用,赫赛汀也显著抑制了Heregulin-α对BT-474细胞中HER3磷酸化的上调作用。
同时,GB235-019WT抗体、GB235-019N73D突变抗体单独用药没有抑制Heregulin-α对Akt磷酸化的上调作用。帕妥珠单独用药显著抑制了Heregulin-α对Akt磷酸化的上调作用。GB235-019N73D突变抗体、GB235-019WT抗体单独用药显著抑制了抑制了Heregulin-α对ERK1/2磷酸化的上调作用,GB235-019N73D突变抗体与GB235-019WT抗体的作用相似。
实施例14重组全长抗人HER2 GB235-019突变抗体体外对乳腺癌MCF 7细胞信号转导的抑制作用
乳腺癌MCF 7细胞表达较低水平的HER2和高水平HER3,但不表达P-HER2和P-HER3(Richard M.Neve.A collection of breastcancer cell lines for the study of functionally distinct cancer subtypes.CANCER CELL,2006:515-527.),基于前文所定义的,所述乳腺癌MCF 7细胞属于HER2阴性肿瘤细胞。将处于对数生长期的MCF 7细胞以1.8×105细胞/孔于RPMI1640(购自Invitrogen公司,美国,货号:A10491)含10%胎牛血清(购自Invitrogen公司,美国,货号:10099-141)完全培养基中培养到6孔板中培养24小时。第二天,弃去培养基换成含0.1%胎血清的低血清培养基饥饿培养24小时。GB235-019WT抗体和GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体,帕妥珠和赫赛汀各20μg/ml的RPMI-0.1%胎血清的培养基溶液单独用药处理或者联合用药处理MCF 7细胞6小时,随后加入工作终浓度为100ng/ml的Heregulin-α(购自R&D公司,美国,货号:296-HR)诱导10分钟。设置未加Heregulin-α的空白对照孔。4℃预冷的PBS洗涤一次后终止反应,添加120μl LDS(购自Invitrogen公司,货号:NP0007),冰上放置迅速收集细胞裂解液,-80℃保存备用。
收集的细胞裂解液在终浓度为50mM的二硫苏糖醇还原条件下,经Western-blot分析检测抗体对Heregulin-α诱导MCF 7细胞HER3磷酸化的影响,以及对HER3下游Akt和ERK1/2磷酸化的影响。Western-blot方法同实施例13。
图9显示了GB235-019突变抗体对HER2阴性乳腺癌MCF 7细胞信号转导的抑制作用。结果显示,与不加Heregulin-α的对照组相比,Heregulin-α引起MCF 7细胞HER3磷酸化的上调作用。GB235-019WT抗体和GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体各自单独给药明显抑制了Heregulin-α对MCF 7细胞中HER3磷酸化的上调作用,GB235-019N73D完全逆转了Heregulin-α对HER3磷酸化的上调作用,赫赛汀单独用药也明显抑制了Heregulin-α对MCF 7细胞中HER3磷酸化的上调作用,帕妥珠完全逆转了Heregulin-α对HER3磷酸化的上调作用。GB235-019WT抗体和GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体各自单独给药明显抑制了Heregulin-α对MCF 7细胞中Akt磷酸化的上调作用,帕妥珠完全逆转了Heregulin-α对Akt磷酸化的上调作用。GB235-019WT抗体和GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体各自单独给药明显抑制了Heregulin-α对MCF 7细胞中ERK1/2磷酸化的上调作用,帕妥珠完全逆转了Heregulin-α对ERK1/2磷酸化的上调作用。因此,本发明的抗体还可用于治疗HER2阴性肿瘤。
实施例15 GB235-019突变抗体的分子排阻色谱分析
GB235-19突变抗体GB235-019N73D、GB235-019N73Q、GB235-019S75A分别经分子排阻色谱(SEC-HPLC)确定相应纯度,实验条件如下:Waters 2695液相色谱仪(Waters公司,美国);TSKgelG3000SWXL串联色谱柱(2根)(购自Waters公司,美国,);流动相为0.1M磷酸盐缓冲液,0.1M氯化钠,pH7.0,1.0mL/分钟,等度保持30分钟,进样量为各30μg,检测波长为280nm。
图10显示了三种突变抗体的分子排阻色谱图。三种分子都存在微量的聚合物和片段分子,但含量较低。表4汇总了相应的分子排阻色谱纯度结果,GB235-019N73D抗体的主峰纯度为88.3%,GB235-019N73Q抗体的主峰纯度为89.7%,GB235-019S75A的主峰纯度为93.1%,三种突变抗体的主峰纯度均高于85%。
表4 GB235-019突变抗体分子排阻色谱纯度结果
实施例16 GB235-019突变抗体的成像毛细管等电聚焦分析
GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体分别经成像毛细管等电聚焦(iCIEF)确定相应主峰等电点(pI)及电荷异构体纯度,实验条件如下:ProteinSimple iCE280毛细管等电聚焦仪(Protein Simple公司,美国);iCIEF cartridge(购自Protein Simple公司,美国);两性电解溶液由12μl Pharmalyte 3-10(购自GE公司,美国,货号:17045601),0.5μl pI Marker 5.85(购自GE公司,美国,货号:17-0472-01),0.5μl pI Marker 9.77(购自GE公司,美国,货号:17-0473-01),2μl 500mM L-精氨酸(购自Sangon公司,货号:AB0205-100g),2μl 200mM亚氨基二乙酸(购自Sangon公司,货号:IB0530-100g),70μl 1%甲基纤维素(购自Sangon公司,货号:MB0616-250g),113μl 5.3M尿素(购自Sangon公司,货号:UB0148-500g)混合组成。进样溶液由180μl两性电解溶液和20μl 2.5mg/mL的蛋白溶液混合而成。样品体系在1,500V预聚焦1分钟,3,000V聚焦10分钟。CCD相机采集聚焦图谱,检测波长为280nm。
图11显示了三种突变抗体的成像毛细管等电聚焦电泳图。表5汇总了三种突变抗体相应的主峰等电点和电荷异构体纯度结果。三种突变抗体的实测等电点在9.4-9.6左右,GB235-019N73D的等电点相对GB235-019N73Q、GB235-019S75A两个分子约低0.1左右,且主峰纯度相对最高。
表5 GB235-019突变抗体主峰等电点及电荷异构体纯度结果
实施例17 GB235-019突变抗体的毛细管凝胶电泳分析
GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体分别经还原法毛细管凝胶电泳分析(rCE-SDS)确定相应纯度(轻重链百分比之和),实验条件如下:使用SDS-MW Analysis Kit(购自Beckman公司,美国,货号:390953),毛细管裸管(购自Micro solv公司)进行分析。取100mM Tris-HCl,pH9.0,1%SDS(购自Sangon公司,货号:SB0485-100g)溶液与β-巯基乙醇(购自Sigma公司,美国,货号:M6250)按55:5比例混合,取60μl以上混合液和40μl 2.5mg/mL样品溶液混合,70℃水浴10分钟。5KV进样20s,15KV分离30分钟。使用UV检测器,波长设定为214nm。
图12显示了三种突变抗体的还原法毛细管凝胶电泳图。表6汇总了三种突变抗体相应的轻重链之和纯度结果。三种突变抗体都存在少量的低分子量和高分子量杂质,GB235-019N73D的杂质含量最少,轻重链之和(LC+HC)纯度最高。
表6 GB235-019突变抗体还原法毛细管凝胶电泳纯度结果
实施例18 GB235-019突变抗体的的差示扫描量热分析
GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体分别经差示扫描量热分析(DSC)确定相应Tm值(相变温度,代表在该温度时,50%的生物分子为去折叠状态),实验条件如下:MicroCalVP-Capillary DSC差示扫描热量计(GE公司,美国),测量池体积为130μl,三种突变抗体分别用PBS(0.01M Na2HPO4·12H2O+0.002MKH2PO4+0.14M NaCl+0.002M KCl,pH=8.6)稀释到0.9mg/mL,以PBS作为空白对照,变温设定为从30℃到95℃,扫描速度为60℃/小时。
图13显示了GB235-019三种突变抗体的差示扫描量热图。三种分子的Tm1有所不同,GB235-019N73Q的Tm1相对最小,GB235-019N73Q、GB235-019S75A相近。Tm值越高表示热稳定性越好,Tm1的不同也一定程度上预示突变对CH2结构域存在一定影响。GB235-019N73D和GB235-019S75A的热稳定性优于GB235-019N73Q。
我们利用全人源scFv噬菌体文库筛选技术和基因工程重组表达技术,获得了全人源的抗人HER2(Her-2/neu)单克隆抗体GB235-019(可参见中国专利申请201410705404.0)。GB235-019可结合于人HER2,猴HER2、小鼠HER2,并呈现出浓度依赖性和可饱和性,但不能结合人HER1、HER3、HER4抗原。GB235-019和赫赛汀联合给药可以翻转HER3配体Heregulin-α引起BT-474细胞对赫赛汀的耐药作用,同时GB235-019单独用药抑制了HER3配体Heregulin-α引起BT-474细胞HER3磷酸化的上调作用。同时GB235-019和赫赛汀联合给药也可以逆转HER3配体Heregulin-α引起SK-BR-3细胞对赫赛汀的耐药作用,与帕妥珠类似。GB235-019和赫赛汀联合给药可以显著抑制人乳腺癌(KPL-4)细胞小鼠移植瘤的生长抑制作用。GB235-019的Fab框架3区Asn73为N-糖基位点,通过还原分子量测定发现GB235-019Fab上存在复杂的糖链。人IgG Fab的N-糖基化修饰可对抗体的抗原结合功能有明显促进或抑制作用。糖基化修饰位置上的微小改变即可对糖链的后续加工及抗体的抗原结合活性产生完全不同的影响,在抗体的生产工艺中带来质量控制的复杂性。GB235-019野生型抗体经点突变后得到了GB235-019N73D、GB235-019N73Q、GB235-019S75A三种突变抗体。三种突变抗体改变了GB235-019野生型抗体重链Fab的N-连接聚糖的特征序列(Asn-Thr-Ser)。三种突变抗体经还原分子量测定,确认重链Fab未发生糖基化。
通过免疫学活性鉴定,GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体具有与人HER2抗原特异性的结合能力,并呈现出浓度依赖性和可饱和性。GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体具有和GB235-019WT抗体相似的结合能力,没有显著差异。GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体分别和赫赛汀联合给药抑制了Heregulin-α诱导的增殖作用。三个突变抗体分别与赫赛汀联合给药使得不仅抑制了Heregulin-α诱导BT-474细胞增殖的作用部分,并明显抑制到低于Heregulin-α诱导前的水平,并呈现出浓度依赖性。同时三个突变抗体与GB235-019WT抗体的作用相似,没有显著差异。GB235-019N73D突变抗体单独用药显著抑制了Heregulin-α对BT-474细胞中HER3磷酸化的上调作用,GB235-019N73D突变抗体与GB235-019WT抗体的作用相似。GB235-019N73D突变抗体,GB235-019WT抗体单独用药显著抑制了Heregulin-α对BT-474细胞中ERK1/2磷酸化的上调作用,GB235-019N73D突变抗体与GB235-019WT抗体的作用相似。GB235-019WT抗体和GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体各自单独给药明显抑制了Heregulin-α对MCF 7细胞中HER3磷酸化的上调作用,GB235-019N73D完全逆转了Heregulin-α对MCF 7细胞中HER3磷酸化的上调作用。GB235-019WT抗体和GB235-019N73D、GB235-019N73Q,GB235-019S75A突变抗体各自单独给药明显抑制了Heregulin-α对MCF 7细胞中Akt磷酸化的上调作用。GB235-019WT抗体和GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体各自单独给药明显抑制了Heregulin-α对MCF 7细胞中ERK1/2磷酸化的上调作用,进一步验证了三种突变抗体的生物学活性。综上所述,本发明的抗体不仅能用于治疗HER2阳性肿瘤,还能用于治疗HER2阴性肿瘤。
我们分析了GB235-019N73D、GB235-019N73Q、GB235-019S75A突变抗体的理化性质。分子排阻色谱图分析三种突变抗体,GB235-019N73D抗体的主峰纯度为88.3%,GB235-019N73Q抗体的主峰纯度为89.7%,GB235-019S75A的主峰纯度为93.1%,这三种突变抗体的主峰纯度均高于85%。成像毛细管等电聚焦(iCIEF)分析了这三种突变抗体相应主峰等电点(pI)及电荷异构体纯度。这三种突变抗体的实测等电点在9.4-9.6左右,GB235-019N73D的等电点相对相对GB235-019N73Q、GB235-019S75A两个分子约低0.1左右,且主峰纯度相对最高。还原法毛细管凝胶电泳分析(rCE-SDS)确定三种突变抗体的相应纯度(轻重链百分比之和)。三种突变抗体都存在少量的低分子量和高分子量杂质,GB235-019N73D的杂质含量相对最少,轻重链之和(LC+HC)纯度最高。差示扫描量热分析(DSC)确定三种突变抗体的相应Tm值(相变温度,代表在该温度时,50%的生物分子为去折叠状态)。三种分子的Tm1有所不同,GB235-019N73Q的Tm1相对最小,GB235-019N73Q、GB235-019S75A相近。Tm值越高表示热稳定性越好,Tm1的不同也一定程度上预示突变对CH2结构域存在一定影响。GB235-019N73D和GB235-019S75A的热稳定性优于GB235-019N73Q。
Claims (10)
1.一种全人源HER2抗体,其重链可变区的氨基酸序列和轻链可变区的氨基酸序列分别为:SEQ ID NO:10,SEQ ID NO:2;SEQ ID NO:11,SEQ ID NO:2;或SEQ ID NO:12,SEQ ID NO:2。
2.权利要求1的全人源HER2抗体,其是Fab、Fab’、F(ab’)2、Fv或scFv的形式。
3.权利要求1的全人源HER2抗体,还包括人IgG的重链恒定区和轻链恒定区;优选地,所述人IgG为IgG1;更优选地,所述人IgG重链恒定区的氨基酸序列为SEQ ID NO:5,所述人IgG轻链恒定区的氨基酸序列为SEQ ID NO:6。
4.编码权利要求1-3任一项的全人源HER2抗体的核苷酸序列;优选地,在所述核苷酸序列中,编码所述重链可变区的核苷酸序列为SEQ IDNO:13、SEQ ID NO:14或SEQ ID NO:15,编码所述轻链可变区的核苷酸序列为SEQ ID NO:4。
5.一种包含权利要求4的核苷酸序列的表达载体,所述核苷酸序列与所述表达载体的表达控制序列可操作地连接;优选地,所述表达载体为pGEM-T载体或293载体。
6.一种药物组合物,其包含权利要求1-3任一项的全人源HER2抗体和可药用载体。
7.一种联合药物,其包含权利要求1-3任一项的全人源HER2抗体和其他HER2阳性肿瘤治疗剂,所述其他HER2阳性肿瘤治疗剂为赫赛汀或帕妥珠。
8.一种检测人HER2的试剂盒,其包含权利要求1-3任一项的全人源HER2抗体。
9.权利要求1-3任一项的全人源HER2抗体用于制备用于治疗受试者中的HER2阳性肿瘤、弱阳性肿瘤或阴性肿瘤的药物的用途;优选地,所述HER2阳性的肿瘤选自HER2阳性的乳腺癌、胃癌、肺癌、非小细胞肺癌、骨癌、胰腺癌、皮肤癌、头或颈癌、皮肤或眼内黑素瘤、子宫癌、卵巢癌、直肠癌、肛门区癌、结肠癌、输卵管癌、子宫内膜癌、宫颈癌、阴道癌、外阴癌、霍奇金病、食道癌、小肠癌、内分泌系统癌、甲状腺癌、甲状旁腺癌、肾上腺癌、软组织癌、尿道癌、阴茎癌、前列腺癌、膀胱癌、肾或尿道癌、肾细胞癌、肾盂癌、间皮瘤、肝细胞癌、胆囊癌、慢性或急性白血病、淋巴细胞淋巴瘤、中枢神经系统(CNS)癌、脊柱肿瘤、脑干神经胶质瘤、多形式成胶质细胞瘤(glioblastoma multiforme),星形细胞瘤,神经鞘瘤,室管膜瘤,成神经管细胞瘤,脑膜瘤、鳞状细胞瘤和垂体腺瘤。
10.权利要求9的用途,其中所述受试者是患有HER2阳性肿瘤、弱阳性肿瘤或阴性肿瘤的人。
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EP (1) | EP3115377B1 (zh) |
JP (1) | JP6263778B2 (zh) |
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CN112513094A (zh) * | 2018-08-01 | 2021-03-16 | 三生国健药业(上海)股份有限公司 | 结合人her2的抗体、其制备方法和用途 |
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WO2016164477A1 (en) * | 2015-04-06 | 2016-10-13 | Meso Scale Technologies, Llc. | High throughput system for performing assays using electrochemiluminescence including a consumable shaking apparatus |
EP3509622A4 (en) | 2016-09-08 | 2020-06-17 | Regenerative Research Foundation | BI-FUNCTIONAL ANTI-TAU POLYPEPTIDES AND THEIR USE |
CA3134363A1 (en) * | 2019-03-22 | 2020-10-01 | Olivia Newton-John Cancer Research Institute | Anti-her2 binding molecules |
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EP3115377A4 (en) | 2017-11-15 |
US20170037146A1 (en) | 2017-02-09 |
US10253108B2 (en) | 2019-04-09 |
KR101886772B1 (ko) | 2018-08-08 |
EP3115377B1 (en) | 2020-02-26 |
EP3115377A1 (en) | 2017-01-11 |
KR20160145185A (ko) | 2016-12-19 |
CN105985435B (zh) | 2019-10-15 |
WO2016119523A1 (zh) | 2016-08-04 |
JP6263778B2 (ja) | 2018-01-24 |
JP2017518038A (ja) | 2017-07-06 |
RU2639531C1 (ru) | 2017-12-21 |
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