CN116120456B - 一种针对her2的双特异性单域抗体及其编码序列和应用 - Google Patents
一种针对her2的双特异性单域抗体及其编码序列和应用 Download PDFInfo
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Abstract
一种针对HER2的双特异性单域抗体及其编码序列和应用,本发明靶向HER2的特异性抗体FBXT212及靶向IgG的特异性抗体FBXT311,通过人源化改造,获得人源化后的纳米抗体蛋白;两组纳米抗体选出优化分子后组合成双特异性抗体;本发明的方法获得了一系列靶向HER2和IgG的双特异性纳米抗体,其VHH链的框架区FR和互补决定区CDR的氨基酸序列,以及编码该纳米抗体或其VHH链的基因序列;本发明的纳米抗体基因序列,为靶向HER2的研究,包括抗体偶联药物及多特异性抗体药物开发提供了研发基础。
Description
技术领域
本发明涉及生物医学或生物技术应用领域,尤其涉及一种针对HER2的双特异性抗体及编码序列和潜在应用方向。
背景技术
HER2属于酪氨酸激酶受体家族,家族有四个成员:HER1(也称为EGFR)、HER2、HER3和HER4。当被激活时,HER蛋白发生同源二聚或异源二聚并随后激活复杂的细胞信号级联,包括PI3K-AKT和RAS-MAPK(ERK)通路,这些通路调节细胞增殖和生存,以及肿瘤细胞的转移。HER2阳性肿瘤患者大都具有更加恶性的表型,肿瘤病灶的转移与侵袭能力较强,且对多数化疗药物治疗抵抗,术后复发机率较高。HER2在多种人类癌症中过表达,包括卵巢癌、结肠直肠癌和尿路上皮癌等。HER2已经成为治疗癌症的重要靶标。
在实际诊疗中,一方面由于存在HER家族受体的不完全抑制,如膜相关粘蛋白4(Membrane-associated mucin 4,MUC4)对HER2结合表位的遮蔽等,导致下游的存活和增殖信号通路重新被激活,另一方面,肿瘤内的异质性导致的对HER2信号传导具有生存依赖的细胞被清除,而没有HER2过表达的细胞则会继续生长,最终表现为治疗抗性。
单域抗体(Single domain antibody,sdAb,VHH)是具有抗原结合能力的最小抗体片段,VHH的本质是一个由110多个氨基酸组成的肽链,空间结构单一,却可以高强度地与特定的抗原结合,并且具有极强的热稳定性以及化学稳定性(例如清洁剂与尿素)。研究人员在1999年的一项研究中指出,一些特殊的VHH在90℃的高温下依旧保持很好的抗原结合能力,而几乎所有抗体在这一条件下都失去了活性。VHH也具有很强的抗胃酸以及胃蛋白酶能力,一些特殊序列的VHH可以通过口服的方法进入体内发挥活性。此外,VHH的抗原互补决定区(Complementarity determining regions,CDR)具有较大的环形结构,可以遮盖住通常与轻链结合的脂溶性位点,因此VHH脂溶性较低,容易在水溶液中溶解。此外,其结构简单,易于进行蛋白质的化学修饰。
但是,VHH实际应用中也存在缺陷,如在静脉给药后,单域抗体是具有抗原结合能力的最小的抗体片段。相较于抗体具有更好的组织和血管渗透性,在肿瘤中能有更为均质的分布。然而,单域抗体其本身分子量只有~15kDa,小于肾脏过滤蛋白质尺寸60kDa的阈值。在静脉给药后,这些单域抗体放射性示踪剂会通过肾小球滤过迅速从血液循环中清除,然后在近端小管中重新吸收,导致在肾皮质滞留时间较长,造成了非靶器官的毒性。目前,以单域抗体为靶向分子的放射靶向治疗研究中,常用的降低肾脏摄取,减小非靶器官毒性的策略是在治疗过程中共注射血浆扩容剂鱼精蛋白(gelofusin)或带正电荷的氨基酸(如精氨酸),去除掉单域抗体C端组氨酸标签以降低极性等方。但是,由于单域抗体本身的血池代谢很快,所以肿瘤的绝对摄取值不高,单次给药的治疗效果较为一般,往往需要多次重复给药才能得到一定的治疗效果。
目前较为常见的抗体片段放射靶向治疗过程中增效减毒的策略是融合表达白蛋白结合域(Albumin-binding domain,ABD),利用白蛋白能参与新生儿Fc受体(Neonatal Fcreceptor,FcRn)介导的回收过程,从而产生延长的血液循环时间。同时,由于治疗药物非共价结合于白蛋白之后,其分子量超过了肾过滤阈值,使得其在肾脏的摄取降低。许多研究探究了非共价结合白蛋白分子并不会影响单域抗体本身对于肿瘤组织的渗透性。与白蛋白类似,IgG在血清中含量也十分丰富,占据血浆蛋白的10%~20%。IgG的血浆半衰期(~23天)较之白蛋白(~19天)的半衰期更长一些。此外,FcRn介导的IgG回收效率高于白蛋白。并且有研究发现IgG结合域(IgG-binding domain,IgBD)可以介导一定的抗体依赖性细胞介导的细胞毒作用(Antibody dependent cellular cytotoxicity,ADCC)、补体依赖的细胞毒性(Complement dependent cytotoxicity,CDC)、抗体依赖的细胞介导的吞噬作用(Antibody-dependent cellular phagocytosis,ADCP),有利于单域抗体治疗过程中细胞免疫参与。所以利用内源性IgG进行药物控释研究是一个具有潜力且值得探索的方向。虽然目前已有研究展示了IgBD在延长血液半衰期方面的良好效果,但基于该策略制备的药物全身药代特性及各种参数并未被测定,另外其在肿瘤放射靶向治疗的研究中还未有应用。
发明内容
为克服现有技术中VHH的临床应用缺陷,本发明通过非共价结合IgG的方式延长单域抗体的血液半衰期,获得了更好的体内药代动力学的特性,通过融合表达靶向HER2的单域抗体FBXT212和特异性结合IgG的单域抗体FBXT311延长血液循环时间,基于靶向HER2的研究,为包括抗体偶联药物及多特异性抗体药物开发提供了研发基础。
本发明的目的是提供一种针对HER2和IgG的双特异性单域抗体,同时提供该单域抗体的编码序列及该单域抗体在检测及治疗中的应用。
本发明所采用的技术方案是:
本发明的第一方面是提供一种靶向HER2的VHH FBXT212,其特征在于,所述FBXT212的CDR 1-3分别为SEQ ID NO:3-5所示。在一个具体的实施例中,所述的VHHFBXT212的氨基酸序列如SEQ ID NO:1所示。
本发明的第二个方面是提供一种靶向IgG的VHH FBXT311,其特征在于,所述VHHFBXT311的CDR 1-3分别为SEQ ID NO:6-8所示;在一个具体的实施例中,所述VHH FBXT311的氨基酸序列如SEQ ID NO:2所示。
本发明的第三个方面是提供一种经人源化改造后能够特异性结合HER2的单域抗体,所述单域抗体的氨基酸序列选自SEQ ID NOs:9-21中的任意一条。
本发明的第四个方面是提供一种经人源化改造后能够特异性结合IgG的单域抗体,所述单域抗体的氨基酸序列选自SEQ ID NOs:22-32中的任意一条。
本发明的第五个方面是提供一种人源化的靶向HER2和IgG双特异性单域抗体,其由靶向IgG的纳米单域抗体FBXT311与靶向HER2的纳米单域抗体FBXT212通过linker连接后,对其进行人源化修饰改造而成,其特征在于,所述FBXT212的CDR 1-3分别为SEQ ID NO:3-5所示;所述FBXT311的CDR 1-3分别为SEQ ID NO:6-8所示;在一个具体的实施例中,所述的靶向HER2的单域抗体选自SEQ ID NOs:9-21中的任意一条;在另外一个具体的实施例中,所述的靶向IgG的单域抗体选自SEQ ID NOs:22-32中的任意一条;在另外一个具体的实施例中,其中所述的linker为(G4S);在另外一个具体的实施例中,其中所述的双特异性单域抗体选自SEQ ID NOs:33-42任一所示的氨基酸序列。
本发明的第六个方面提供一种核酸分子,其特征在于,所述的核酸分子编码本发明第一个方面、第三个方面所述的靶向HER2 VHH的氨基酸序列。
本发明的第七个方面提供一种核酸分子,其特征在于,所述的核酸分子编码本发明第二个方面和第四个方面所述的靶向IgG的VHH的氨基酸序列。
本发明的第八个方面提供一种编码人源化的靶向HER2和IgG双特异性单域抗体的核酸分子,其编码本发明第五个方面项所述的双特异性单域抗体;在一个具体的实施例中所述的核酸分子如SEQ ID NOs:67-76任一项所示。
本发明的第九个方面提供本发明第五个方面所述人源化的靶向HER2和IgG双特异性单域抗体或第八方面所述的核酸分子在制备检测HER2蛋白的产品中的应用;在一个具体的实施例中,其中所述的抗体为放射性核素偶联物。
本发明的第十个方面提供本发明第五个方面所述人源化的靶向HER2和IgG双特异性单域抗体或本发明第八个方面所述的核酸分子在制备治疗HER2异常表达相关疾病的制剂中的用途;在一个具体的实施例中,其中所述的抗体为抗体偶联药物或放射性核素偶联药物。
本发明第十一个方面提供一种药物组合物,其包括本发明第五个方面所述人源化的靶向HER2和IgG的双特异性单域抗体或本发明第八个方面所述的核酸分子及其药学上可接受的载体。
本发明的有益效果:
本发明利用序列SEQ ID NOs:1-2,进行人源化改造,所获得的10条序列由ExpiCHO表达载体通过瞬转表达获得针对HER2蛋白与IgG蛋白的双特异性单域抗体。经SPR(离子共振成像)试验鉴定,所述抗体具有良好的免疫检测效果,为建立快速、准确的检测HER2的抗原方法奠定了关键基础。
附图说明
图1:FBXT212可变区结构模型示意图
图2:FBXT311可变区结构模型示意图
图3:FBXT212驼源纳米抗体序列分析对比图
图4:FBXT311驼源纳米抗体序列分析对比图
图5:FBXT212磷酸化位点预测结果图
图6:FBXT311磷酸化位点预测结果图
图7:人源化分子与抗原human IgG的亲和力ELISA检测结果-1
图8:人源化分子与抗原human IgG的亲和力ELISA检测结果-2
图9:人源化分子与抗原human HER2的亲和力ELISA检测结果-1
图10:人源化分子与抗原human HER2的亲和力ELISA检测结果-2
图11:双抗分子与抗原human IgG的亲和力ELISA检测结果
图12:双抗分子与抗原human HER2的亲和力ELISA检测结果
具体实施方式
下面结合具体实施例和说明书附图对本发明所述作进一步说明,但应该理解,本发明的保护范围并不限于以下实施例。
面结合具体实施例,进一步阐述本发明:
实施例1同源建模及人源化设计
1、同源建模
纳米抗体母本序列FBXT212和FBXT311经数据库查询比对,确定最相似的人源Germline,定义CDR与framework区后,根据framework区的差异位点,设计不同程度人源化的序列,经基因合成,质粒构建,蛋白表达,蛋白纯化及鉴定,获得人源化后的纳米抗体蛋白,然后进行相关活性验证;两组纳米抗体选出优化分子后组合成双抗,再次进行质粒构建,蛋白表达、纯化与检测,选出双特异性抗体。
其中FBXT212的氨基酸序列如下所示:
FBXT311的氨基酸序列如下所示
其中,方框部分为CDRs,下划线部分为预测的低免疫原性肽段。
建模示意图如1-2所示。
2、纳米抗体序列分析
1)驼源位点分析
与人Germline序列比对,FBXT212可变区含13个驼源位点,比对结果图3所示。
与人Germline序列比对,FBXT311可变区含12个驼源位点,比对结果如4所示。
2)翻译后修饰位点分析
分别对FBXT212与FBXT311进行翻译后修饰分析,分析结果如表所示:
表1.翻译后修饰分析结果表
3)磷酸化位点的预测
FBXT212磷酸化位点预测结果:
结果如图5所示。
FBXT311磷酸化位点预测结果:
结果如图6所示。
3、人源化设计
3.1.与人源Germline序列比对及设计
3.1.1.母本分子FBXT212为驼源序列,长度为124个氨基酸,经序列比对分析,与人源序列IGHV3-23,30,64胚系基因Germline同源度较高。
3.1.2.FBXT212的可变区(Framework)与人源序列存在13个差异位点,采用将差异位点突变为人源氨基酸序列的方式,逐步增加突变氨基酸的数量,同时进行各种突变位点组合,人源化程度从低到高设计了13条人源化序列,母本分子初始人源化程度为89.5%,设计后人源化程度为91.9%~99.2%。
3.1.3.FBXT212选择同源度最高的IGHV3-23、30、64为人源化设计模板,设计序列为VHH1-VHH13。母本分子命名为FBXT212-P,13个人源化分子名称为:FBXT212-VHH1、FBXT212-VHH2、FBXT212-VHH3、FBXT212-VHH4、FBXT212-VHH5、FBXT212-VHH6、FBXT212-VHH7、FBXT212-VHH8、FBXT212-VHH9、FBXT212-VHH10、FBXT212-VHH11、FBXT212-VHH12、FBXT212-VHH13。
3.1.4.母本分子FBXT311为驼源序列,长度为115个氨基酸,经序列比对分析,与人源序列IGHV3-64,74胚系基因Germline同源度较高。
3.1.5.FBXT311的可变区(Framework)与人源序列存在12个差异位点,采用将差异位点突变为人源氨基酸序列的方式,逐步增加突变氨基酸的数量,同时进行各种突变位点组合,人源化程度从低到高设计了11条人源化序列,母本分子初始人源化程度为89.6%,设计后人源化程度为92.9%~99.2%。
3.1.6.FBXT311选择同源度最高的IGHV3-64,74为人源化设计模板,设计序列为VHH1-VHH11。母本分子命名为FBXT311-P,11个人源化分子命名为:FBXT311-VHH1、FBXT311-VHH2、FBXT311-VHH3、FBXT311-VHH4、FBXT311-VHH5、FBXT311-VHH6、FBXT311-VHH7、FBXT311-VHH8、FBXT311-VHH9、FBXT311-VHH10、FBXT311-VHH11。
表2 FBXT212人源化程度信息汇总表
样品名称 | 回复突变位点个数 | 人源化程度 |
FBXT212-P | 13 | 89.50% |
FBXT212-VHH1 | 10 | 91.90% |
FBXT212-VHH2 | 9 | 92.70% |
FBXT212-VHH3 | 8 | 93.50% |
FBXT212-VHH4 | 7 | 94.40% |
FBXT212-VHH5 | 7 | 94.40% |
FBXT212-VHH6 | 6 | 95.20% |
FBXT212-VHH7 | 5 | 96.00% |
FBXT212-VHH8 | 5 | 96.00% |
FBXT212-VHH9 | 4 | 96.80% |
FBXT212-VHH10 | 4 | 96.80% |
FBXT212-VHH11 | 3 | 97.60% |
FBXT212-VHH12 | 2 | 98.40% |
FBXT212-VHH13 | 1 | 99.20% |
表3 FBXT311人源化程度信息汇总表
样品名称 | 回复突变位点个数 | 人源化程度 |
FBXT311-P | 12 | 89.60% |
FBXT311-VHH1 | 9 | 92.20% |
FBXT311-VHH2 | 8 | 93.70% |
FBXT311-VHH3 | 7 | 94.50% |
FBXT311-VHH4 | 6 | 94.40% |
FBXT311-VHH5 | 6 | 94.40% |
FBXT311-VHH6 | 5 | 95.20% |
FBXT311-VHH7 | 5 | 96.00% |
FBXT311-VHH8 | 4 | 96.90% |
FBXT311-VHH9 | 3 | 97.60% |
FBXT311-VHH10 | 2 | 98.40% |
FBXT311-VHH11 | 1 | 99.40% |
实施例2抗体人源化表达及验证
1.质粒构建
根据人源化设计的结果,经基因合成、同源重组、转化、鉴定、测序、比对、抽提后比对序列,将序列构建到pcDNA3.4载体上,得到纳米抗体表达质粒。
2.表达与纯化
将构建的质粒转染至Expi CHO细胞,进行瞬转表达,表达时间7天,表达体积10mL,并在表达第6天,通过Gator检测表达量,表达结束后进行蛋白纯化、分装。
3.双抗分子的质粒构建与表达纯化
根据单抗分子的实验数据,选择FBXT212-VHH9、FBXT212-VHH11、FBXT212-VHH12、FBXT311-VHH6、FBXT311-VHH8及FBXT311-VHH11组合成双抗分子,共产生9个双抗分子,同时构建母本的双抗分子,使用Expi CHO进行了30mL蛋白瞬转表达,表达结束后进行纯化、分装,进行SDS-PAGE、SEC、DSF、Elisa、亲和力检测。
4.SDS-PAGE鉴定
4.1.方法与步骤
1)纯化样品溶液制备:根据A280测定浓度非还原样品1μg加入4×LDS上样缓冲液,碘代乙酰胺(终浓度40mM),75℃干浴加热10min,还原样品2μg加入4×LDS上样缓冲液,DTT(终浓度5mM),100℃干浴加热10min。
2)电泳:140V,75min。
3)染色、脱色、扫描:凝胶考马斯亮蓝染色,脱色后用EPSON V550彩色扫描仪扫描。
4)计算纯度:用ImageJ按照峰面积归一法计算还原条带纯度,或者还原重链加轻链和的纯度。
4.2.系统适应性
参照品IPI非还原条带分子量150kDa左右,纯度大于90%;还原重链分子量50kDa左右,轻链分子量25kDa左右,重链加轻链纯度大于90%。
5.SEC鉴定
5.1.方法与步骤
1)流动相:0.15M PB+NaCl,pH 6.0。
2)流速:0.8mL/min。
3)上样体积:20μL。
4)检测器参数:检测波长280nm,带宽16nm,参比波长360nm,带宽100nm,峰宽(响应时间)>0.1min(2s);狭缝4nm;负吸光度基线100mAU。
5.2.系统适应性标准
参比品Herceptin单体纯度大于95%,BSA单体与二聚体的分离度大于1.5,基线平稳,则视为系统适应性通过。
6.DSF检测
6.1.方法与步骤
1)测试样品制备:在八连管或者96孔板中,加入用1×PBS(pH 7.4)稀释到0.2mg/mL的样品,然后加入100×SYPRO Orange工作液使其终浓度为5×,终体积20μL,轻弹管壁混匀,2000rpm离心10秒;每个样品制备3个重复。
2)上机:将样品置于ABI7500 Fast Real-Time PCR仪上,实验类型选择溶解曲线,采取连续模式,扫描温度25℃~99℃,25℃平衡5min,升温速率为1%,报告基团ROX,淬灭基团None。
3)结果判定:以溶解曲线导函数的第一个峰谷对应的温度确定为该蛋白质的变性温度Tm1,第二个峰谷对应的温度确定为该蛋白质的变性温度Tm2,第三个峰谷对应的温度确定为该蛋白质的变性温度Tm3。
6.2.系统适应性
参照品Herceptin蛋白Tm1=68.5℃±1.0℃,Tm2=81.0℃±1.0℃。
7.理化性质检测结果
8.ELISA-Binding检测
8.1.方法与步骤
1)包板:用1×PBS稀释抗原至浓度为2μg/mL,按30μL/孔加入到96孔Elisa板中,4℃包被过夜。
2)封闭:用PBST洗板3次,加入封闭液(5% PBS-Milk)室温封闭2h。
3)孵育:洗板,按30μL/孔加入1% Milk稀释的样品,室温孵育60min。
4)二抗孵育:用PBST洗板3次,加入二抗,室温孵育60min。
5)显色:用PBST洗板3次,每孔加入30μL TMB。
6)中止:加入2M终止液终止反应同时检测OD450。
8.2.系统适应性
表5判断标准
编号 | 判定指标 | 放行标准 |
1 | 样品名称准确性 | 完全一致 |
2 | 复孔CV(%) | ≤20% |
3 | R2 | ≥0.98 |
4 | QC1与PC3的比值 | 70%~130% |
5 | QC2与PC4的比值 | 70%~130% |
6 | 阳性抗体上下平台OD比值 | ≥3 |
7 | 阳性抗体S型曲线 | 上下平台完整 |
8 | 阳性抗体板间误差CV(%) | ≤20% |
9 | 空白对照OD值(背景值) | ≤0.25 |
注释:QC1、QC2为质控点值,PC3、PC4为阳性对照第3、4点的值具体检测结果如表6以及图7-图12所示。
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9.亲和动力学
9.1.方法与步骤-Gator
1)选模式:打开GATOR仪器及相关软件,选择Kientics实验模式。
2)上机:分析程序见下表:
表7.亲和动力学检测过程-Gator
分析步骤 | 分析时间(s) | 样品 |
Baseline1 | 30 | Q buffer |
Loading | 120 | 100or 200nM antibody in Qbuffer |
Baseline2 | 60 | Q buffer |
Association | 120 | 2-fold diluted antigen from 2400nM to 4.688nM with Qbuffer |
Dissociation | 180 | Q buffer |
Regeneration | 25 | R buffer&Q buffer |
9.2.系统适应性
非标记生物分子分析仪(Gator)所检测的KD限值为1E-06M。实验结果显示所有抗体在Global拟合模式下的相关系数R2都大于0.95,符合系统适应性要求,结果可靠。
9.3.方法与步骤-Biacore
1)设备为Biacore T200(Cytiva),使用anti-his芯片,将双抗分子梯度稀释后结合到芯片上,流动相为抗原。
2)参数设定
表8.Biacore检测参数设定
步骤 | 时间(s) | 流速(μL/min) | 样品 |
捕获 | 20 | 10 | 30nM抗体 |
平衡 | 60 | 30 | 1×HBS-EP+buffer |
结合 | 120 | 30 | 梯度稀释供试品 |
解离 | 300 | 30 | 1×HBS-EP+buffer |
再生 | 30 | 30 | 甘氨酸1.5 |
9.4.实验结果
亲和动力学检测结果:
表9.FBXT311人源化分子亲和力动力学检测结果(Gator,抗原human IgG)
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备注:KD:解离平衡常数;Ka:结合速率常数;Kd:解离速率常数;R2:线性拟合常数;Rmax:曲线拟合最高响应值。
表10.FBXT212人源化分子亲和力动力学检测结果(Gator,抗原human HER2)
表11.双抗及部分单抗人源化分子亲和力动力学检测结果(Gator,抗原humanIgG)
表12.双抗人源化分子亲和力动力学检测结果(Biacore,抗原human HER2)
备注:KD:解离平衡常数;Ka:结合速率常数;Kd:解离速率常数;R2:线性拟合常数;Rmax:曲线拟合最高响应值。
以上显示和描述了本发明的基本原理和主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (17)
1.一种靶向HER2的单域抗体VHH FBXT212,其特征在于,所述VHH FBXT212的CDR 1-3分别为SEQ ID NO:3-5所示。
2.根据权利要求1所述的单域抗体VHH FBXT212,其特征在于,所述的VHH FBXT212的氨基酸序列如SEQ ID NO:1所示。
3.一种靶向IgG的单域抗体VHH FBXT311,其特征在于,所述VHH FBXT311的 CDR 1-3分别为SEQ ID NO:6-8所示。
4.根据权利要求3所述的靶向IgG的单域抗体VHH FBXT311,其特征在于,所述VHHFBXT311的氨基酸序列如SEQ ID NO:2所示。
5.一种经人源化改造后能够特异性靶向HER2的单域抗体,所述单域抗体的氨基酸序列选自SEQ ID NOs:9-21中的任意一条。
6.一种经人源化改造后能够特异性靶向IgG的单域抗体,所述单域抗体的氨基酸序列选自SEQ ID NOs:22-32中的任意一条。
7.一种人源化的靶向 HER2和IgG的双特异性单域抗体,其由靶向IgG的纳米单域抗体FBXT311与靶向HER2的纳米单域抗体FBXT212通过linker 连接后,对其进行人源化修饰改造而成,其特征在于,
所述的靶向 HER2的纳米单域抗体选自权利要求5中的任意一条;
所述的靶向IgG的纳米单域抗体选自权利要求6中的任意一条。
8.根据权利要求7所述的靶向 HER2和IgG的双特异性单域抗体,其特征在于,该双特异性单域抗体选自SEQ ID NOs:33-42任一所示的氨基酸序列。
9.一种核酸分子,其特征在于,所述的核酸分子编码权利要求1、2或5所述的靶向HER2的单域抗体的氨基酸序列。
10.一种核酸分子,其特征在于,所述的核酸分子编码权利要求3、4或6所述的靶向IgG的单域抗体的氨基酸序列。
11.一种编码人源化的靶向HER2和IgG的双特异性单域抗体的核酸分子,其特征在于,其编码权利要求7-8任一项所述的双特异性单域抗体。
12.根据权利要求11所述的核酸分子,其特征在于,所述的核酸分子序列如SEQ IDNOs:67-76任一项所示。
13.权利要求7-8任一项所述的人源化的靶向 HER2和IgG的双特异性单域抗体或权利要求11-12任一项所述的核酸分子在制备检测 HER2蛋白的产品中的应用。
14.权利要求7-8任一项所述的人源化的靶向 HER2和IgG的双特异性单域抗体或权利要求11-12任一项所述的核酸分子在制备治疗HER2异常表达相关疾病的制剂中的用途,所述的HER2异常表达相关疾病为HER2高表达的癌症。
15.一种药物组合物,其包括权利要求7-8任一项所述的人源化的靶向 HER2和IgG的双特异性单域抗体或权利要求11-12任一项所述的核酸分子及其药学上可接受的载体。
16.根据权利要求13所述的应用,其特征在于,其中所述的抗体为放射性核素偶联物。
17.根据权利要求14所述的用途,其特征在于,其中所述的抗体为抗体偶联药物或放射性核素偶联物。
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