CN116789822A - Claudin18.2人源化抗体及其应用 - Google Patents
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Abstract
本发明提出了一种Claudin18.2人源化抗体及其应用,该抗体含有选自下列至少之一的CDR序列或与其具有至少90%同一性的氨基酸序列:轻链可变区CDR序列:SEQ ID NO:1‑3和7‑9;重链可变区CDR序列:SEQ ID NO:4‑6和10‑12,所述抗体或其抗原结合片段具有人源化修饰。根据本发明实施例的上述人源化抗体能够特异性的靶向结合Claudin18.2,具有与人鼠嵌合抗Claudin18.2单克隆抗体一致的体外活性,满足成药性要求,且其体内半衰期得到显著延长。
Description
技术领域
本发明涉及生物技术领域,具体地,本发明涉及Claudin18.2人源化抗体及其应用,更具体地,本发明涉及能够特异性识别Claudin18.2的抗体或其抗原结合片段、核酸分子、表达载体、重组细胞、药物组合物、制药用途以及检测Claudin18.2的试剂盒。
背景技术
Claudins(CLDN)是一个蛋白质家族,其作用是维持控制细胞间分子交换的紧密连接。广泛分布于胃、胰腺和肺组织,可用于诊断和治疗。Claudin18.2亚型是一种胃特异性亚型,自从发现Claudin18.2是一种高度选择性的分子,并且只在癌细胞中广泛表达,它就成为一种理想的靶点。Claudin18.2通常埋藏在胃粘膜中,正常组织中的单克隆抗体基本上接触不到,恶性肿瘤的发生会导致紧密连接的破坏,使肿瘤细胞表面的Claudin18.2表位暴露出来,成为特定的靶点,因此,Claudin18.2赋予靶向治疗的特异性。
目前,已存在采用免疫小鼠的、杂交瘤技术制备出的鼠源抗Claudin18.2单克隆抗体,但是鼠源的抗Claudin18.2单克隆抗体进入人体存在较高的免疫原性,从而影响在人体内的药代动力学。因此,仍需进一步开发效果更优的针对Claudin18.2的治疗性抗体。
发明内容
本申请是基于发明人对下列问题和事实的发现而提出的:
Claudins广泛分布于胃、胰腺和肺组织,恶性肿瘤发生时,肿瘤细胞表面的Claudin18.2表位暴露出来,因此,Claudin18.2可以作为开发新型治疗药物的有效靶点,目前已存在鼠源Claudin18.2单克隆抗体,但存在较高的免疫原性;因此,本申请的发明人将抗Claudin18.2单克隆抗体的框架区(FR区)和恒定区替换为人源的,保留鼠源抗Claudin18.2单克隆抗体可变区的CDR区,得到一系列的抗Claudin18.2人源化抗体。发明人发现,本申请所获得的这些人源化抗体,不仅能够特异性的靶向结合Claudin18.2,具有与嵌合抗体一致的结合活性,而且具有更长的体内半衰期。
因此,在本发明的的第一方面,本发明提出了一种能够特异性识别Claudin18.2的抗体或其抗原结合片段。根据本发明的实施例,所述抗体含有选自下列至少之一的CDR序列或与其具有至少90%同一性的氨基酸序列:轻链可变区CDR序列:X1SSQSLFNSGNQKNYLT(SEQID NO:1),WASTRES(SEQ ID NO:2),QNDYSYPLT(SEQ ID NO:3),X4SSQSLLNSGNQKNYLT(SEQID NO:7),WASTRES(SEQ ID NO:8),和QNDYSYPFT(SEQ ID NO:9);重链可变区CDR序列:DYNMH(SEQ ID NO:4),YINPNNGGTSYX2QKFX3G(SEQ ID NO:5),TRYLAV(SEQ ID NO:6),SYWMH(SEQ ID NO:10),MIHPNSGSTNYX5X6KFX7X8(SEQ ID NO:11),和RYYGSISPDY(SEQ ID NO:12);其中,X1为K或R,X2为N或S,X3为K或Q,X4为K或R,X5为N或A,X6为E或Q,X7为K或Q,X8为S或G,所述抗体或其抗原结合片段具有人源化修饰。需要说明的是,本申请所述的“人源化修饰”是指能够使得所述抗体或其抗原结合片段的免疫原性降低的氨基酸的改变,包括氨基酸的突变、插入、缺失、化学基团的赘合等。根据本发明实施例的上述抗体不仅能够特异性的靶向结合Claudin18.2,具有与嵌合抗体一致的结合活性,而且具有更长的体内半衰期。
根据本发明的实施例,上述抗体或其抗原结合片段还可以包括如下附加技术特征中的至少之一:
根据本发明的实施例,所述抗体具有如SEQ ID NO:57-59或者SEQ ID NO:65-67所示氨基酸序列的轻链可变区,具有如SEQ ID NO:60-64或者SEQ ID NO:68-72所示氨基酸序列的重链可变区。
根据本发明的实施例,所述抗体含有重链恒定区和轻链恒定区的至少之一,所述重链恒定区和轻链恒定区的至少之一的至少一部分来自于人源抗体;所述抗体的轻链恒定区和重链恒定区均来自于人源IgG抗体或其突变体;进一步地,所述抗体的轻链恒定区来自于人源的Kappa轻链恒定区;重链恒定区来自于人源IgG4或IgG1,或,IgG4或IgG1的突变体。更进一步地,所述抗体恒定区的全长序列如SEQ ID NO:73、74或75所示。
根据本发明的实施例,所述抗体具有SEQ ID NO:76-78任一项所示的轻链,和,SEQID NO:79-88任一项所示的重链。
根据本发明的实施例,所述抗体具有SEQ ID NO:89-91任一项所示的轻链,和,SEQID NO:92-101任一项所示的重链。
在本发明的第二方面,本发明提出了一种核酸分子。根据本发明的实施例,所述核酸分子编码第一方面所述的抗体或其抗原结合片段。根据本发明实施例的核酸分子获得的抗体不仅能够特异性的靶向结合Claudin18.2,具有与嵌合抗体一致的结合活性,而且具有更长的体内半衰期,降低抗体的内化比例。
在本发明的第三方面,本发明提出了一种表达载体。根据本发明的实施例,所述表达载体携带前面所述的核酸分子。根据本发明实施例的表达载体导入合适的受体细胞后,可在调控系统的介导下,有效实现前面所述的特异性识别Claudin18.2的抗体或其抗原结合片段表达,进而实现所述抗体或抗原结合片段的体外大量获得。
在本发明的第四方面,本发明提出了一种免疫细胞。根据本发明的实施例,所述免疫细胞携带前面所述的核酸分子,或者表达前面所述的抗体或其抗原结合片段。根据本发明实施例的免疫细胞可大量表达前面所述的特异性识别Claudin18.2的抗体或其抗原结合片段,以体外大量获得所述抗体或其抗原结合片段,更进一步地,可以将所述免疫细胞进行体内回输,以治疗Claudin18.2相关性疾病。
在本发明的第五方面,本发明提出了一种重组细胞。根据本发明的实施例,所述重组细胞携带前面所述的核酸分子,或者表达前面所述的抗体或其抗原结合片段。根据本发明实施例的重组细胞可用于前面所述的特异性识别Claudin18.2的抗体或其抗原结合片段体外表达和大量获得。
在本发明的第六方面,本发明提出了一种CAR-T细胞。根据本发明的实施例,包括嵌合抗原受体,其中,所述嵌合抗原受体包括:胞外区,所述胞外区包括单链抗体的重链可变区和轻链可变区,所述轻链可变区具有SEQ ID NO:57-59或者SEQ ID NO:65-67所示氨基酸序列,所述重链可变区具有SEQ ID NO:60-64或者SEQ ID NO:68-72所示氨基酸序列;跨膜区,所述跨膜区与所述胞外区相连,并且嵌入到所述T淋巴细胞的细胞膜中;以及胞内区,所述胞内区与所述跨膜区相连。表达该嵌合抗原受体的CAR-T细胞可以有杀伤表达Claudin18.2的肿瘤细胞。
在本发明的第七方面,本发明提出了一种药物组合物。根据本发明的实施例,所述药物组合物含有前面所述的抗体,核酸分子,表达载体,免疫细胞,重组细胞或CAR-T细胞。如前所述,所述抗体,以及表达后能够获得所述抗体的核酸分子、表达载体、免疫细胞、重组细胞或CAR-T细胞不仅能够特异性的靶向结合Claudin18.2,具有与嵌合抗体一致的结合活性,而且具有更长的体内半衰期,因此,根据本发明实施例的药物组合物中所包含的抗体或表达的抗体能够特异性的靶向结合Claudin18.2,特异性强,发挥较好的靶向作用,进而实现药物组合物中其他药物的生物学作用,如Claudin18.2分子活性抑制,表达Claudin18.2分子的细胞杀伤等作用。此外,根据本发明实施例的药物组合物可以发挥诊断作用,依赖于本发明第一方面所提出的能够特异性的靶向结合Claudin18.2的抗体,与诊断试剂进行组合,进而发挥对生物体异常表达Claudin18.2的部位进行诊断,如与诊断性核素、纳米材料等进行组合,实现对生物体异常表达CLDN18.2的细胞、组织、器官进行可视化观察,进而辅助医疗工作者或科研工作者对病灶进行更准确的判断。
在本发明的第八方面,本发明提出了前面所述的抗体、前面所述的核酸分子、前面所述的表达载体、免疫细胞、重组细胞、CAR-T细胞、或前面所述的药物组合物在制备药物中的用途,所述药物用于治疗或者预防Claudin18.2相关疾病。如前所述,所述抗体,以及表达后能够获得所述抗体的核酸分子、表达载体、免疫细胞、重组细胞或CAR-T细胞不仅能够特异性的靶向结合Claudin18.2,具有与嵌合抗体一致的结合活性,而且具有更长的体内半衰期,因此,包含上述物质的药物同样能够有效靶向结合Claudin18.2,且具有较长的体内半衰期,可以有效杀伤表达Claudin18.2分子的细胞。
在本发明的第九方面,本发明提出了一种检测Claudin18.2的试剂盒。根据本发明的实施例,所述试剂盒包括前面任一所述的抗体。前面所述的Claudin18.2抗体能够特异性靶向结合Claudin18.2,根据本发明实施例的试剂盒可以实现Claudin18.2的特异性检测,如当抗体结合有荧光基团时,可以采用荧光检测装置实现对Claudin18.2的定位或实时检测;当抗体结合有生物素等标记物时,可以通过显色试剂显色实现对Claudin18.2的定性或定量检测;抗体也可以结合抗抗体,实现夹心或者双夹心的方法,进而实现信号逐级放大,对Claudin18.2进行检测。
在本发明的第十方面,本发明提出了前面所述的抗体、前面所述的核酸分子、前面所述的表达载体或前面所述的重组细胞在制备试剂盒中的用途,所述试剂盒用于检测Claudin18.2或者诊断Claudin18.2相关的疾病。根据本发明的实施例,所述试剂盒可以通过直接检测Claudin18.2的表达量,如高表达、低表达、不表达,进而实现对疾病的诊断,也可以与其他诊断试剂进行组合,获取生物体或者组织、细胞的状态,如与诊断性核素结合,可以可视化体内表达Claudin18.2细胞的数量、组织的大小及部位等。
附图说明
图1是根据本发明实施例的对经ProteinA柱获取的蛋白样品1B6-mVH-HC(IgG1)mVL、1B6-huVH1-HC(IgG1)huVL1、1B6-huVH1-HC(IgG1)huVL2、1B6-huVH2-HC(IgG1)huVL1、1B6-huVH2-HC(IgG1)huVL2、1B6-huVH3-HC(IgG1)huVL1进行Non-Reduced(非还原性)、Reduced(还原性)SDS-PAGE鉴定的结果图,其中,Samples表示样品;
图2是根据本发明实施例的对经ProteinA柱获取的蛋白样品1B6-huVH3-HC(IgG1)huVL2、1B6-huVH3-HC(IgG1)huVL3、1B6-huVH4-HC(IgG1)huVL2、1B6-huVH4-HC(IgG1)huVL3、1B6-huVH5-HC(IgG1)huVL2、1B6-huVH5-HC(IgG1)huVL3进行Non-Reduced(非还原性)、Reduced(还原性)SDS-PAGE鉴定的结果图,其中,Samples表示样品;
图3是根据本发明实施例的对经ProteinA柱获取的蛋白样品1E7-mVH-HC(IgG1)mVL、1E7-huVH1-HC(IgG1)huVL1、1E7-huVH1-HC(IgG1)huVL2、1E7-huVH2-HC(IgG1)huVL1、1E7-huVH2-HC(IgG1)huVL2、1E7-huVH3-HC(IgG1)huVL1进行Non-Reduced(非还原性)、Reduced(还原性)SDS-PAGE鉴定的结果图,其中,Samples表示样品;
图4是根据本发明实施例的对经ProteinA柱获取的蛋白样品1E7-huVH3-HC(IgG1)huVL2、1E7-huVH3-HC(IgG1)huVL3、1E7-huVH4-HC(IgG1)huVL2、1E7-huVH4-HC(IgG1)huVL3、1E7-huVH5-HC(IgG1)huVL2、1E7-huVH5-HC(IgG1)huVL3进行Non-Reduced(非还原性)、Reduced(还原性)SDS-PAGE鉴定的结果图,其中,Samples表示样品;
图5是根据本发明实施例的对经ProteinA柱获取的蛋白样品1B6-mVHmVL、1B6-huVH1huVL1、1B6-huVH1huVL2、1B6-huVH2huVL1、1B6-huVH2huVL2、1B6-huVH3huVL1进行Non-Reduced(非还原性)、Reduced(还原性)SDS-PAGE鉴定的结果图,其中,Samples表示样品;
图6是根据本发明实施例的对经ProteinA柱获取的蛋白样品1B6-huVH3huVL2、1B6-huVH3huVL3、1B6-huVH4huVL2、1B6-huVH4huVL3、1B6-huVH5huVL2、1B6-huVH5huVL3进行Non-Reduced(非还原性)、Reduced(还原性)SDS-PAGE鉴定的结果图,其中,Samples表示样品;
图7是根据本发明实施例的对经ProteinA柱获取的蛋白样品1E7-mVHmVL、1E7-huVH1huVL1、1E7-huVH1huVL2、1E7-huVH2huVL1、1E7-huVH2huVL2、1E7-huVH3huVL1进行Non-Reduced(非还原性)、Reduced(还原性)SDS-PAGE鉴定的结果图,其中,Samples表示样品;
图8是根据本发明实施例的对经ProteinA柱获取的蛋白样品1E7-huVH3huVL2、1E7-huVH3huVL3、1E7-huVH4huVL2、1E7-huVH4huVL3、1E7-huVH5huVL2、1E7-huVH5huVL3进行Non-Reduced(非还原性)、Reduced(还原性)SDS-PAGE鉴定的结果图,其中,Samples表示样品;
图9是根据本发明实施例的人源化抗体1B6-huVH1huVL1、1B6-huVH1huVL2、1B6-huVH2huVL1、1B6-huVH2huVL2、1B6-huVH3huVL1、1B6-huVH3huVL2、1B6-huVH3huVL3、1B6-huVH4huVL2、1B6-huVH4huVL3、1B6-huVH5huVL2、1B6-huVH5huVL3的ELISA检测结果分析图;
图10是根据本发明实施例的人源化抗体1E7-huVH1huVL1、1E7-huVH1huVL2、1E7-huVH2huVL1、1E7-huVH2huVL2、1E7-huVH3huVL1、1E7-huVH3huVL2、1E7-huVH3huVL3、1E7-huVH4huVL2、1E7-huVH4huVL3、1E7-huVH5huVL2、1E7-huVH5huVL3的ELISA检测结果分析图;
图11是根据本发明实施例的人源化抗体1E7-huVH1-HC(IgG1)huVL1、1E7-huVH1-HC(IgG1)huVL2、1E7-huVH2-HC(IgG1)huVL1、1E7-huVH2-HC(IgG1)huVL2、1E7-huVH3-HC(IgG1)huVL1、1E7-huVH3-HC(IgG1)huVL2、1E7-huVH3-HC(IgG1)huVL3、1E7-huVH4-HC(IgG1)huVL2、1E7-huVH4-HC(IgG1)huVL3、1E7-huVH5-HC(IgG1)huVL2、1E7-huVH5-HC(IgG1)huVL3的ELISA检测结果分析图;
图12是根据本发明实施例的人源化抗体1B6-huVH1-HC(IgG1)huVL1、1B6-huVH1-HC(IgG1)huVL2、1B6-huVH2-HC(IgG1)huVL1、1B6-huVH2-HC(IgG1)huVL2、1B6-huVH3-HC(IgG1)huVL1、1B6-huVH3-HC(IgG1)huVL2、1B6-huVH3-HC(IgG1)huVL3、1B6-huVH4-HC(IgG1)huVL2、1B6-huVH4-HC(IgG1)huVL3、1B6-huVH5-HC(IgG1)huVL2、1B6-huVH5-HC(IgG1)huVL3的ELISA检测结果分析图;
图13是根据本发明实施例的嵌合抗体抗体1B6-mVH-HC(IgG1)mVL、人源化抗体1B6-huVH2-HC(IgG1)huVL1、1B6-huVH2-HC(IgG1)huVL2、1B6-huVH4-HC(IgG1)huVL3的ADCC活性评估结果分析图,其中,横坐标表示抗体浓度(μg/mL)的Log值,纵坐标表示发光信号的强度;
图14是根据本发明实施例的嵌合抗体1E7-mVH-HC(IgG1)mVL、人源化抗体1E7-huVH1-HC(IgG1)huVL1、1E7-huVH1-HC(IgG1)huVL2、1E7-huVH2-HC(IgG1)huVL1、1E7-huVH3-HC(IgG1)huVL2的ADCC活性评估结果分析图;
图15是根据本发明实施例的嵌合抗体抗体1B6-mVH-HC(IgG1)mVL、人源化抗体1B6-huVH2-HC(IgG1)huVL1、1B6-huVH3-HC(IgG1)huVL1的CDC活性评估结果分析图,其中,横坐标表示抗体浓度(μg/mL)的Log值,纵坐标表示OD450;
图16是根据本发明实施例的嵌合抗体抗体1E7-mVH-HC(IgG1)mVL、人源化抗体1E7-huVH1-HC(IgG1)huVL1、1E7-huVH1-HC(IgG1)huVL2、1E7-huVH2-HC(IgG1)huVL1、1E7-huVH3-HC(IgG1)huVL2的CDC活性评估结果分析图,其中,横坐标表示抗体浓度(μg/mL)的Log值,纵坐标表示OD450。
图17是根据本发明实施例的嵌合抗体的内化情况检测结果图,其中:
17-A表示1E7-mVH-HC(IgG1)mVL、1E7-huVH3huVL1、1E7-huVH3huVL2、1E7-huVH1huVL1和1E7-huVH2huVL2抗体的内化检测结果图;
17-B表示1B6-mVH-HC(IgG1)mVL、1B6-huVH4huVL3抗体的内化检测结果图;
17-C表示1B6-mVH-HC(IgG1)mVL、1B6-huVH3huVL1、1B6-huVH4-HC(IgG1)huVL3、1B6-huVH3-HC(IgG1)huVL1、1B6-mVHmVL和1B6-huVH3-HC(IgG1)huVL3抗体的内化检测结果图;
图18是人源化claudin18.2抗体CAR-T体外效果研究;
图19是人源化claudin18.2抗体CAR-T体内效果研究。
具体实施方式
下面详细描述本发明的实施例,所述实施例的示例在附图中示出,其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的,旨在用于解释本发明,而不能理解为对本发明的限制。
在对本发明描述的过程中,对于本文中有关的术语进行了解释和说明,这些解释和说明仅仅是为了方便对于方案的理解,并不能看做是对本发明保护方案的限制。
抗体
本文中,术语“抗体”是能够与特异性抗原结合的免疫球蛋白分子。包括两条分子量较轻的轻链和两条分子量较重的重链,重链(H链)和轻链(L链)由二硫键连接形成一个四肽链分子。其中,肽链的氨基端(N端)氨基酸序列变化很大,称为可变区(V区),羧基端(C端)相对稳定,变化很小,称为恒定区(C区)。L链和H链的V区分别称为VL和VH。
在可变区中某些区域氨基酸组成和排列顺序具有更高的变化程度,称为高变区(Hypervariable region,HVR),高变区为抗原和抗体结合的位置,因此也称为决定簇互补区(complementarity-determining region,CDR)。重链可变区和轻链可变区上均有三个CDR区。
需要说明的是,“免疫原性”是指能引起免疫应答的性能,即抗原能刺激特定的免疫细胞,使免疫细胞活化、增殖、分化,最终产生免疫效应物质抗体和致敏淋巴细胞的特性。本申请的发明人进一步对筛选得到的鼠源单克隆抗体序列进行人源化改造(人源化修饰),得到人源化抗体,该人源化抗体既能保留亲本鼠单克隆抗体的亲和力和特异性,又大幅降低了其免疫原性,提高了其安全性,延长了其半衰期。
在一些实施方案中,本发明提出了一种能够特异性识别Claudin18.2的抗体或其抗原结合片段,所述抗体含有选自下列至少之一的CDR序列或与其具有至少90%同一性的氨基酸序列:轻链可变区CDR序列:X1SSQSLFNSGNQKNYLT(SEQ ID NO:1),WASTRES(SEQ IDNO:2),QNDYSYPLT(SEQ ID NO:3),X4SSQSLLNSGNQKNYLT(SEQ ID NO:7),WASTRES(SEQ IDNO:8),和QNDYSYPFT(SEQ ID NO:9);重链可变区CDR序列:DYNMH(SEQ ID NO:4),YINPNNGGTSYX2QKFX3G(SEQ ID NO:5),TRYLAV(SEQ ID NO:6),SYWMH(SEQ ID NO:10),MIHPNSGSTNYX5X6KFX7X8(SEQ ID NO:11),和RYYGSISPDY(SEQ ID NO:12);其中,X1为K或R,X2为N或S,X3为K或Q,X4为K或R,X5为N或A,X6为E或Q,X7为K或Q,X8为S或G,所述抗体或其抗原结合片段具有人源化修饰。需要说明的是,本申请所述的“人源化修饰”是指能够使得所述抗体或其抗原结合片段的免疫原性降低的氨基酸的改变,包括氨基酸的突变、插入、缺失、化学基团的赘合等。根据本发明实施例的上述抗体具有较高的ADCC活性和CDC活性,EC50和IC50值较小,不仅能够特异性的靶向结合Claudin18.2,具有与嵌合抗体一致的结合活性,而且具有更长的体内半衰期。在这些实施方案中,本发明所提供的抗体或者抗原结合片段中具有保守氨基酸取代。“抗原结合片段”是指保持特异性结合抗原(ROR2)能力的抗体片段。“保守氨基酸取代”指的是氨基酸被另一氨基酸发生生物学上、化学上或者结构上相似的残基所取代。生物学上相似的指的是该取代不破坏Claudin18.2抗体或者与Claudin18.2抗原的生物学活性。结构上相似指的是氨基酸具有相似长度的侧链,如丙氨酸、甘氨酸或丝氨酸,或具有相似大小的侧链。化学相似性指的是氨基酸具有相同的荷电或者都是亲水或者疏水的。例如疏水残基异亮氨酸、缬氨酸、亮氨酸或者甲硫氨酸相互取代。或者用极性氨基酸例如用精氨酸取代赖氨酸、谷氨酸取代天冬氨酸、谷氨酰胺取代天冬酰胺,丝氨酸取代苏氨酸等等。
本发明所述的“1B6-mVH-HC(IgG1)mVL”可以理解为包含轻链和重链的嵌合抗体,其中,轻链、重链可变区均为鼠源抗体1B6的可变区,恒定区来自于人源IgG1;“1B6-mVHmVL”可以理解为包含轻链和重链的嵌合抗体,其中,轻链、重链可变区均为鼠源抗体1B6的可变区,恒定区来自于人源IgG4;“1E7-mVH-HC(IgG1)mVL”可以理解为包含重链和轻链的嵌合抗体,其中,轻链、重链可变区均为鼠源抗体1E7可变区,重链恒定区来自于人源IgG1;“1E7-mVHmVL”可以理解为包含轻链和重链的嵌合抗体,其中,轻链、重链可变区均为鼠源抗体1E7可变区,恒定区来自于人源IgG4;“1B6”、“1E7”可以理解为由VH-HC-VL-LC形成的双链抗体。
本申请中人源化抗体的命名方式为“鼠抗名称-重链可变区名称-重链恒定区(来源抗体名称)-轻链可变区”,其中,轻链恒定区默认为Kappa链恒定区,未进行标记释出,当所述重链恒定区来源于人源IgG1抗体时,重链恒定区表示为HC(IgG1),当所述重链恒定区来源于人源IgG4抗体时,重链恒定区则不进行释出。例如,人源抗体“1B6-huVH1-HC(IgG1)huVL1”可以理解为将鼠源抗体1B6的框架区、恒定区均替换为人源IgG1抗体的框架区、恒定区获得的人源化抗体,优选地,抗体的轻链可变区氨基酸序列可以如SEQ ID NO:57所示,重链可变区氨基酸序列可以如SEQ ID NO:60所示,抗体的轻链氨基酸序列可以如SEQ ID NO:76所示,抗体的重链氨基酸序列可以如SEQ ID NO:79所示;人源抗体“1B6-huVH1huVL1”可以理解为将鼠源抗体1B6的框架区、恒定区均替换为人源IgG4抗体的框架区、恒定区获得的人源化抗体,优选地,抗体的轻链可变区氨基酸序列可以如SEQ ID NO:57所示,重链可变区氨基酸序列可以如SEQ ID NO:60所示,抗体的轻链氨基酸序列可以如SEQ ID NO:76所示,抗体的重链氨基酸序列可以如SEQ ID NO:84所示;
人源抗体“1E7-huVH1-HC(IgG1)huVL1”可以理解为将鼠源抗体1E7的框架区、恒定区均替换为人源IgG1抗体的框架区、恒定区获得的人源化抗体,优选地,抗体的轻链可变区氨基酸序列可以如SEQ ID NO:65所示,重链可变区氨基酸序列可以如SEQ ID NO:68所示,抗体的轻链氨基酸序列可以如SEQ ID NO:89所示,抗体的重链氨基酸序列可以如SEQ IDNO:92所示;人源抗体“1E7-huVH1huVL1”可以理解为将鼠源抗体1E7的框架区、恒定区均替换为人源IgG4抗体的框架区、恒定区获得的人源化抗体,优选地,抗体的轻链可变区氨基酸序列可以如SEQ ID NO:65所示,重链可变区氨基酸序列可以如SEQ ID NO:68所示,抗体的轻链氨基酸序列可以如SEQ ID NO:89所示,抗体的重链氨基酸序列可以如SEQ ID NO:97所示。
根据本发明的一些具体实施方案,上述抗体或抗原结合片段还可以进一步包括如下附加技术特征至少之一:
根据本发明的一些具体实施方案,所述抗体包括:分别如SEQ ID NO:1、2和3或者与SEQ ID NO:1、2和3具有至少90%同一性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列;或者分别如SEQ ID NO:7、8和9或者与SEQ ID NO:7、8和9具有至少90%同一性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列。
根据本发明的一些具体实施方案,所述抗体包括:分别如SEQ ID NO:4、5和6或者与SEQ ID NO:4、5和6具有至少90%同一性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列;或者分别如SEQ ID NO:10、11和12或者与SEQ ID NO:10、11和12具有至少90%同一性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列。
根据本发明的一些具体实施方案,所述抗体含有重链框架区序列和轻链框架区序列的至少之一,所述重链框架区序列和轻链框架区序列的至少之一的至少一部分来自于人源抗体或其突变体。根据本发明的一些具体实施例,发明人对鼠源单克隆抗体进行了轻链框架区和/或重链框架区的人源化修饰。
根据本发明的一些具体实施方案,所述抗体包括:分别如SEQ ID NO:13-16所示的轻链框架区FRL1、FRL2、FRL3、FRL4序列,或者分别如SEQ ID NO:21-24所示的轻链框架区FRL1、FRL2、FRL3、FRL4序列,
DIX9MTQSPSSLX10X11X12X13GX14X15VTX16SX17C(SEQ ID NO:13),
WYQQKPGX18X19PKLLIY(SEQ ID NO:14),
GVPX20DRFX21GSGSGTDFTLTISSX22QX23EDX24AX25YX26C(SEQ ID NO:15),
FGX27GTKLEX28K(SEQ ID NO:16);
DIX53MTQSPSSLSX54X55AX56GX57X58VTX59X60C(SEQ ID NO:21),
WYQQKPGX61X62PKLLIY(SEQ ID NO:22),
GVPX63RFX64GSGSGTDFTLTISSX65QX66EDX67AX68YYC(SEQ ID NO:23),
FGX69GTKLEIK(SEQ ID NO:24),
其中,X9为V或Q,X10为S或T,X11为V或A,X12为T或S,X13为A或V,X14为E或D,X15为K或R,X16为M或I,X17为S或T,X18为Q或K,X19为P或A,X20为D或S,X21为T或S,X22为V或L,X23为A或P,X24为L或F,X25为V或T,X26为F或Y,X27为A或G,X28为L或I,X53为V或Q,X54为V或A,X55为T或S,X56为A或V,X57为E或D,X58为K或R,X59为M或I,X60为S或T,X61为Q或K,X62为P或A,X63为D或S,X64为T或S,X65为V或L,X66为A或P,X67为L或F,X68为V或T,X69为S或G。条件是,当所述抗体包括:分别如KSSQSLFNSGNQKNYLT(SEQ ID NO:29)、WASTRES(SEQ ID NO:30)、QNDYSYPLT(SEQ IDNO:31)所示的轻链可变区CDR1、CDR2、CDR3序列时,X9为V,X10为T,X11为V,X12为T,X13为A,X14为E,X15为K,X16为M,X17为S,X18为Q,X19为P,X20为D,X21为T,X22为V,X23为A,X24为L,X25为V,X26为F,X27为A,X28为L不同时成立;当所述抗体包括:分别如KSSQSLLNSGNQKNYLT(SEQ ID NO:35)、WASTRES(SEQ ID NO:36)、QNDYSYPFT(SEQ ID NO:37)所示的轻链可变区CDR1、CDR2、CDR3序列时,X53为V,X54为V,X55为T,X56为A,X57为E,X58为K,X59为M,X60为S,X61为Q,X62为P,X63为D,X64为T,X65为V,X66为A,X67为L,X68为V,X69为S不同时成立。
根据本发明的一些具体实施方案,所述抗体包括:分别如SEQ ID NO:41-44所示的轻链框架区FRL1、FRL2、FRL3、FRL4序列,或者分别如SEQ ID NO:49-52所示的轻链框架区FRL1、FRL2、FRL3、FRL4序列,
DIX90MTQSPSSLSX91TAGEKVTX92SC(SEQ ID NO:41),
WYQQKPGQPPKLLIY(SEQ ID NO:42),
GVPX93DRFTGSGSGTDFTLTISSX94QAEDLAVYX95C(SEQ ID NO:43),
FGAGTKLELK(SEQ ID NO:44);或者
分别如SEQ ID NO:49-52所示的轻链框架区FRL1、FRL2、FRL3、FRL4序列,
DIX107MTQSPSSLSX108TAGEKVTX109SC(SEQ ID NO:49),
WYQQKPGX110X111PKLLIY(SEQ ID NO:50),
GVPX112RFTGSGSGTDFTLTISSX113QAEDLAVYYC(SEQ ID NO:51),
FGSGTKLEIK(SEQ ID NO:52),其中,X90为V或Q,X91为V或A,X92为M或I,X93为D或S,X94为V或L,X95为F或Y,X107为V或Q,X108为V或A,X109为M或I,X110为Q或K,X111为P或A,X112为D或S,X113为V或L。
根据本发明的一些具体实施方案,所述抗体包括:分别如SEQ ID NO:17-20所示的重链框架区FRH1、FRH2、FRH3和FRH4序列,或者分别如SEQ ID NO:25-28所示的重链框架区FRH1、FRH2、FRH3和FRH4序列,
X29VQLX30QSGX31EX32X33X34PGASVKX35SCKASGYTFT(SEQ ID NO:17),
WVX36QX37X38GX39X40LEWX41G(SEQ ID NO:18),
X42X43TX44TX45X46X47SX48STAYMELX49SLX50SEDX51AVYYCVT(SEQ ID NO:19),
WGX52GTTVTVSS(SEQ ID NO:20);
QVQLX70QX71GX72EX73X74KPGASVKX75SCKASGYTFT(SEQ ID NO:25),
WVX76QX77PGQGLEWX78G(SEQ ID NO:26),
X79X80X81TX82TVDX83SX84STX85YMX86LSSLX87SEDX88AVYYCAR(SEQ ID NO:27),
WGQGTTX89TVSS(SEQ ID NO:28),其中,X29为E或Q,X30为Q或V,X31为P或A,X32为L或V,X33为V或K,X34为R或K,X35为M或V,X36为K或R,X37为S或A,X38为H或P,X39为K或Q,X40为S或R,X41为I或M,X42为K或R,X43为A或V,X44为L或I,X45为V或R,X46为N或D,X47为K或T,X48为S或A,X49为R或S,X50为T或R,X51为S或T,X52为T或Q,X70为Q或V,X71为P或S,X72为S或A,X73为L或V,X74为V或K,X75为L或V,X76为K或R,X77为R或A,X78为I或M,X79为S或G,X80为R或K,X81为A或V,X82为L或M,X83为K或T,X84为S或T,X85为A或V,X86为Q或E,X87为T或R,X88为S或T,X89为L或V;条件是,当所述抗体包括:分别如DYNMH(SEQ ID NO:32)、YINPNNGGTSYNQKFKG(SEQ ID NO:33)、TRYLAV(SEQ ID NO:34)所示的重链可变区CDR1、CDR2、CDR3序列时,X29为E,X30为Q,X31为P,X32为L,X33为V,X34为R,X35为M,X36为K,X37为S,X38为H,X39为K,X40为S,X41为I,X42为K,X43为A,X44为L,X45为V,X46为N,X47为K,X48为S,X49为R,X50为T,X51为S,X52为T不同时成立;当所述抗体包括:分别如SYWMH(SEQ ID NO:38)、MIHPNSGSTNYNEKFKS(SEQ ID NO:39)、RYYGSISPDY(SEQID NO:40)所示的重链可变区CDR1、CDR2、CDR3序列时,X70为Q,X71为P,X72为S,X73为L,X74为V,X75为L,X76为K,X77为R,X78为I,X79为S,X80为K,X81为A,X82为L,X83为K,X84为S,X85为A,X86为Q,X87为T,X88为S,X89为L不同时成立。
根据本发明的一些具体实施方案,所述抗体包括:分别如SEQ ID NO:45-48所示的重链框架区FRH1、FRH2、FRH3和FRH4序列,或者分别如SEQ ID NO:53-56所示的重链框架区FRH1、FRH2、FRH3和FRH4序列,
X96VQLQQSGPELVRPGASVKX97SCKASGYTFT(SEQ ID NO:45),
WVKQSHGX98X99LEWX100G(SEQ ID NO:46),
X101X102TX103TVX104X105SX106STAYMELRSLTSEDSAVYYCVT(SEQ ID NO:47),
WGTGTTVTVSS(SEQ ID NO:48),
QVQLQQPGSELVKPGASVKX114SCKASGYTFT(SEQ ID NO:53),
WVKQX115PGQGLEWX116G(SEQ ID NO:54),
X117X118TX119TVDX120SSSTX121YMQLSSLTSEDSAVYYCAR(SEQ ID NO:55),
WGQGTTLTVSS(SEQ ID NO:56),其中,X96为E或Q,X97为M或V,X98为K或Q,X99为S或R,X100为I或M,X101为K或R,X102为A或V,X103为L或I,X104为N或D,X105为K或T,X106为S或A,X114为L或V,X115为R或A,X116为I或M,X117为R或K,X118为A或V,X119为L或M,X120为K或T,X121为A或V。
根据本发明的一些具体实施方案,所述抗体具有如SEQ ID NO:57-59或者SEQ IDNO:65-67所示氨基酸序列的轻链可变区。
1B6人源化抗体轻链可变区1(1B6-huVL1):
DIVMTQSPSSLSVSVGDRVTMTCKSSQSLFNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDF TLTISSVQPEDFATYFCQNDYSYPLTFGGGTKLEIK(SEQ ID NO:57)。
1B6人源化抗体轻链可变区2(1B6-huVL2):
DIQMTQSPSSLSASVGDRVTMTCKSSQSLFNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQNDYSYPLTFGGGTKLEIK(SEQ ID NO:58)。
1B6人源化抗体轻链可变区3(1B6-huVL3):
DIQMTQSPSSLSASVGDRVTITCRSSQSLFNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQNDYSYPLTFGGGTKLEIK(SEQ ID NO:59)。
1E7人源化抗体轻链可变区1(1E7-huVL1):
DIVMTQSPSSLSVSVGDRVTMTCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDF TLTISSVQPEDFATYYCQNDYSYPFTFGGGTKLEIK(SEQ ID NO:65)。
1E7人源化抗体轻链可变区2(1E7-huVL2):
DIQMTQSPSSLSASVGDRVTMTCKSSQSLLNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQNDYSYPFTFGGGTKLEIK(SEQ ID NO:66)。
1E7人源化抗体轻链可变区3(1E7-huVL3):
DIQMTQSPSSLSASVGDRVTITCRSSQSLLNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQNDYSYPFTFGGGTKLEIK(SEQ ID NO:67)。
根据本发明的一些具体实施方案,所述抗体具有如SEQ ID NO:60-64或者SEQ IDNO:68-72所示氨基酸序列的重链可变区。
1B6人源化抗体重链可变区1(1B6-huVH1):
EVQLVQSGAEVKKPGASVKMSCKASGYTFTDYNMHWVRQAPGKSLEWIGYINPNNGGTSYNQKFKGKATLTVNK SSSTAYMELSSLRSEDTAVYYCVTTRYLAVWGQGTTVTVSS(SEQ ID NO:60)。
1B6人源化抗体重链可变区2(1B6-huVH2):
QVQLVQSGAEVKKPGASVKMSCKASGYTFTDYNMHWVRQAPGQRLEWIGYINPNNGGTSYNQKFKGKATLTVDT SASTAYMELSSLRSEDTAVYYCVTTRYLAVWGQGTTVTVSS(SEQ ID NO:61)。
1B6人源化抗体重链可变区3(1B6-huVH3):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLEWIGYINPNNGGTSYNQKFKGRVTITVDTS ASTAYMELSSLRSEDTAVYYCVTTRYLAVWGQGTTVTVSS(SEQ ID NO:62)。
1B6人源化抗体重链可变区4(1B6-huVH4):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLEWMGYINPNNGGTSYNQKFQGRVTITVDT SASTAYMELSSLRSEDTAVYYCVTTRYLAVWGQGTTVTVSS(SEQ ID NO:63)。
1B6人源化抗体重链可变区5(1B6-huVH5):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLEWMGYINPNNGGTSYSQKFQGRVTITRDT SASTAYMELSSLRSEDTAVYYCVTTRYLAVWGQGTTVTVSS(SEQ ID NO:64)。
1E7人源化抗体重链可变区1(1E7-huVL1):
QVQLVQSGAEVKKPGASVKLSCKASGYTFTSYWMHWVRQRPGQGLEWIGMIHPNSGSTNYNEKFKSKATLTVDKS TSTAYMELSSLRSEDTAVYYCARRYYGSISPDYWGQGTTVTVSS(SEQ ID NO:68)。
1E7人源化抗体重链可变区2(1E7-huVL2):
QVQLVQSGAEVKKPGASVKLSCKASGYTFTSYWMHWVRQAPGQGLEWIGMIHPNSGSTNYNEKFKSRATLTVDTS TSTAYMELSSLRSEDTAVYYCARRYYGSISPDYWGQGTTVTVSS(SEQ ID NO:69)。
1E7人源化抗体重链可变区3(1E7-huVL3):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGMIHPNSGSTNYNEKFKSRVTMTVDT STSTAYMELSSLRSEDTAVYYCARRYYGSISPDYWGQGTTVTVSS(SEQ ID NO:70)。
1E7人源化抗体重链可变区4(1E7-huVL4):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGMIHPNSGSTNYNEKFKSRVTMTVD TSTSTVYMELSSLRSEDTAVYYCARRYYGSISPDYWGQGTTVTVSS(SEQ ID NO:71)。
1E7人源化抗体重链可变区5(1E7-huVL5):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGMIHPNSGSTNYAQKFQGRVTMTRD TSTSTVYMELSSLRSEDTAVYYCARRYYGSISPDYWGQGTTVTVSS(SEQ ID NO:72)。
根据本发明的一些具体实施方案,所述抗体含有重链恒定区和轻链恒定区的至少之一,所述重链恒定区和轻链恒定区的至少之一的至少一部分来自于人源抗体。
根据本发明的一些具体实施方案,所述抗体的轻链恒定区和重链恒定区均来自于人源IgG抗体或其突变体。
根据本发明的一些具体实施方案,所述抗体的轻链恒定区来自于人源的Kappa轻链恒定区;重链恒定区来自于人源IgG4或IgG1,或,IgG4或IgG1的突变体。
根据本发明的一些具体实施方案,所述抗体恒定区的全长序列如SEQ ID NO:73、74或75所示。
轻链恒定区LC(Kappa):
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:73)。
IgG1重链恒定区HC:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:74)。
IgG4重链恒定区HC:
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:75)。
根据本发明的一些具体实施方案,所述抗体具有SEQ ID NO:76-78任一项所示的轻链,和,SEQ ID NO:79-88任一项所示的重链。
1B6人源化抗体轻链1B6-huVL1-LC(Kappa):
DIVMTQSPSSLSVSVGDRVTMTCKSSQSLFNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDF TLTISSVQPEDFATYFCQNDYSYPLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:76)。
1B6人源化抗体轻链1B6-huVL2-LC(Kappa):
DIQMTQSPSSLSASVGDRVTMTCKSSQSLFNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQNDYSYPLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:77)。
1B6人源化抗体轻链1B6-huVL3-LC(Kappa):
DIQMTQSPSSLSASVGDRVTITCRSSQSLFNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQNDYSYPLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:78)。
1B6人源化抗体重链1B6-huVH1-HC(IgG1):
EVQLVQSGAEVKKPGASVKMSCKASGYTFTDYNMHWVRQAPGKSLEWIGYINPNNGGTSYNQKFKGKATLTVNKSSSTAYMELSSLRSEDTAVYYCVTTRYLAVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO:79)。
1B6人源化抗体重链1B6-huVH2-HC(IgG1):
QVQLVQSGAEVKKPGASVKMSCKASGYTFTDYNMHWVRQAPGQRLEWIGYINPNNGGTSYNQKFKGKATLTVDTSASTAYMELSSLRSEDTAVYYCVTTRYLAVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO:80)。
1B6人源化抗体重链1B6-huVH3-HC(IgG1):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLEWIGYINPNNGGTSYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCVTTRYLAVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO:81)。
1B6人源化抗体重链1B6-huVH4-HC(IgG1):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLEWMGYINPNNGGTSYNQKFQGRVTITVDTSASTAYMELSSLRSEDTAVYYCVTTRYLAVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO:82)。
1B6人源化抗体重链1B6-huVH5-HC(IgG1):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLEWMGYINPNNGGTSYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCVTTRYLAVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO:83)。
1B6人源化抗体重链1B6-huVH1-HC(IgG4):
EVQLVQSGAEVKKPGASVKMSCKASGYTFTDYNMHWVRQAPGKSLEWIGYINPNNGGTSYNQKFKGKATLTVNKSSSTAYMELSSLRSEDTAVYYCVTTRYLAVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO:84)。
1B6人源化抗体重链1B6-huVH2-HC(IgG4):
QVQLVQSGAEVKKPGASVKMSCKASGYTFTDYNMHWVRQAPGQRLEWIGYINPNNGGTSYNQKFKGKATLTVDTSASTAYMELSSLRSEDTAVYYCVTTRYLAVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO:85)。
1B6人源化抗体重链1B6-huVH3-HC(IgG4):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLEWIGYINPNNGGTSYNQKFKGRVTITVDTSASTAYMELSSLRSEDTAVYYCVTTRYLAVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO:86)。
1B6人源化抗体重链1B6-huVH4-HC(IgG4):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLEWMGYINPNNGGTSYNQKFQGRVTITVDTSASTAYMELSSLRSEDTAVYYCVTTRYLAVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO:87)。
1B6人源化抗体重链1B6-huVH5-HC(IgG4):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLEWMGYINPNNGGTSYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCVTTRYLAVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ IDNO:88)。
根据本发明的一些具体实施方案,所述抗体具有SEQ ID NO:89-91任一项所示的轻链,和,SEQ ID NO:92-101任一项所示的重链。
1E7人源化抗体轻链1E7-huVL1-LC(Kappa):
DIVMTQSPSSLSVSVGDRVTMTCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDF TLTISSVQPEDFATYYCQNDYSYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:89)。
1E7人源化抗体轻链1E7-huVL2-LC(Kappa):
DIQMTQSPSSLSASVGDRVTMTCKSSQSLLNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQNDYSYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:90)。
1E7人源化抗体轻链1E7-huVL3-LC(Kappa):
DIQMTQSPSSLSASVGDRVTITCRSSQSLLNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQNDYSYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:91)。
1E7人源化抗体重链1E7-huVH1-HC(IgG1):
QVQLVQSGAEVKKPGASVKLSCKASGYTFTSYWMHWVRQRPGQGLEWIGMIHPNSGSTNYNEKFKSKATLTVDKSTSTAYMELSSLRSEDTAVYYCARRYYGSISPDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:92)。
1E7人源化抗体重链1E7-huVH2-HC(IgG1):
QVQLVQSGAEVKKPGASVKLSCKASGYTFTSYWMHWVRQAPGQGLEWIGMIHPNSGSTNYNEKFKSRATLTVDTSTSTAYMELSSLRSEDTAVYYCARRYYGSISPDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:93)。
1E7人源化抗体重链1E7-huVH3-HC(IgG1):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGMIHPNSGSTNYNEKFKSRVTMTVDTSTSTAYMELSSLRSEDTAVYYCARRYYGSISPDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:94)。
1E7人源化抗体重链1E7-huVH4-HC(IgG1):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGMIHPNSGSTNYNEKFKSRVTMTVDTSTSTVYMELSSLRSEDTAVYYCARRYYGSISPDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:95)。
1E7人源化抗体重链1E7-huVH5-HC(IgG1):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGMIHPNSGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARRYYGSISPDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:96)。
1E7人源化抗体重链1E7-huVH1-HC(IgG4):
QVQLVQSGAEVKKPGASVKLSCKASGYTFTSYWMHWVRQRPGQGLEWIGMIHPNSGSTNYNEKFKSKATLTVDKSTSTAYMELSSLRSEDTAVYYCARRYYGSISPDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:97)。
1E7人源化抗体重链1E7-huVH2-HC(IgG4):
QVQLVQSGAEVKKPGASVKLSCKASGYTFTSYWMHWVRQAPGQGLEWIGMIHPNSGSTNYNEKFKSRATLTVDTSTSTAYMELSSLRSEDTAVYYCARRYYGSISPDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:98)。
1E7人源化抗体重链1E7-huVH3-HC(IgG4):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGMIHPNSGSTNYNEKFKSRVTMTVDTSTSTAYMELSSLRSEDTAVYYCARRYYGSISPDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:99)。
1E7人源化抗体重链1E7-huVH4-HC(IgG4):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGMIHPNSGSTNYNEKFKSRVTMTVDTSTSTVYMELSSLRSEDTAVYYCARRYYGSISPDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:100)。
1E7人源化抗体重链1E7-huVH5-HC(IgG4):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGMIHPNSGSTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARRYYGSISPDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:101)。
根据本发明的一些具体实施方案,所述抗体包括:SEQ ID NO:76所示的轻链,SEQID NO:79所示的重链,或SEQ ID NO:77所示的轻链,SEQ ID NO:79所示的重链,或SEQ IDNO:76所示的轻链,SEQ ID NO:80所示的重链,或SEQ ID NO:77所示的轻链,SEQ ID NO:80所示的重链,或SEQ ID NO:76所示的轻链,SEQ ID NO:81所示的重链,或SEQ ID NO:77所示的轻链,SEQ ID NO:81所示的重链,或SEQ ID NO:78所示的轻链,SEQ ID NO:81所示的重链,或SEQ ID NO:77所示的轻链,SEQ ID NO:82所示的重链,或SEQ ID NO:78所示的轻链,SEQ ID NO82所示的重链,或SEQ ID NO:77所示的轻链,SEQ ID NO:83所示的重链,或SEQID NO:78所示的轻链,SEQ ID NO:83所示的重链,或SEQ ID NO:76所示的轻链,SEQ ID NO:84所示的重链,或SEQ ID NO:77所示的轻链,SEQ ID NO:84所示的重链,或SEQ ID NO:76所示的轻链,SEQ ID NO:85所示的重链,或SEQ ID NO:77所示的轻链,SEQ ID NO:85所示的重链,或SEQ ID NO:76所示的轻链,SEQ ID NO:86所示的重链,或SEQ ID NO:77所示的轻链,SEQ ID NO:86所示的重链,或SEQ ID NO:78所示的轻链,SEQ ID NO:86所示的重链,或SEQID NO:77所示的轻链,SEQ ID NO:87所示的重链,或SEQ ID NO:78所示的轻链,SEQ ID NO:87所示的重链,或SEQ ID NO:77所示的轻链,SEQ ID NO:88所示的重链,或SEQ ID NO:78所示的轻链,SEQ ID NO:88所示的重链。
根据本发明的一些具体实施方案,所述抗体包括:SEQ ID NO:89所示的轻链,SEQID NO:92所示的重链,或SEQ ID NO:90所示的轻链,SEQ ID NO:92所示的重链,或SEQ IDNO:89所示的轻链,SEQ ID NO:93所示的重链,或SEQ ID NO:90所示的轻链,SEQ ID NO:93所示的重链,或SEQ ID NO:89所示的轻链,SEQ ID NO:94所示的重链,或SEQ ID NO:90所示的轻链,SEQ ID NO:94所示的重链,或SEQ ID NO:91所示的轻链,SEQ ID NO:94所示的重链,或SEQ ID NO:90所示的轻链,SEQ ID NO:95所示的重链,或SEQ ID NO:91所示的轻链,SEQ ID NO:95所示的重链,或SEQ ID NO:90所示的轻链,SEQ ID NO:96所示的重链,或SEQID NO:91所示的轻链,SEQ ID NO:96所示的重链,或SEQ ID NO:89所示的轻链,SEQ ID NO:97所示的重链,或SEQ ID NO:90所示的轻链,SEQ ID NO:97所示的重链,或SEQ ID NO:89所示的轻链,SEQ ID NO:98所示的重链,或SEQ ID NO:90所示的轻链,SEQ ID NO:98所示的重链,或SEQ ID NO:89所示的轻链,SEQ ID NO:99所示的重链,或SEQ ID NO:90所示的轻链,SEQ ID NO:99所示的重链,或SEQ ID NO:91所示的轻链,SEQ ID NO:99所示的重链,或SEQID NO:90所示的轻链,SEQ ID NO:100所示的重链,或SEQ ID NO:91所示的轻链,SEQ IDNO:100所示的重链,或SEQ ID NO:90所示的轻链,SEQ ID NO:101所示的重链,或SEQ IDNO:91所示的轻链,SEQ ID NO:101所示的重链。
根据本发明的一些具体实施方案,所述抗体为单链抗体、多聚体抗体、CDR移植抗体或小分子抗体。
根据本发明的一些具体实施方案,所述小分子抗体包括Fab抗体、Fv抗体、单域抗体以及最小识别单位的至少之一。
免疫细胞、CAR-T细胞
术语“嵌合抗原受体(CAR)”,是结合基于抗体的针对期望的抗原(例如,肿瘤抗原)的特异性与T细胞受体-激活细胞内结构域以产生展示特异性抗肿瘤细胞免疫活性的嵌合蛋白的分子。
“CAR-T细胞”是指嵌合抗原受体T细胞,可以理解为能够表达嵌合抗原受体的T细胞,以使T细胞行使特异的杀伤功能。
在一些实施方案中,本发明提出了一种免疫细胞,所述免疫细胞携带前面所述的核酸分子,或者表达前面所述的抗体或其抗原结合片段。根据本发明一些具体实施方案的免疫细胞可大量表达前面所述的特异性识别Claudin18.2的抗体或其抗原结合片段,以体外大量获得所述抗体或其抗原结合片段,更进一步地,可以将所述免疫细胞进行体内回输,以治疗Claudin18.2相关性疾病。
根据本发明的一些具体实施方案,上述免疫细胞还可以进一步包括如下附加技术特征至少之一:
根据本发明的一些具体实施方案,所述免疫细胞包括T淋巴细胞、DC细胞、NK细胞和NKT淋巴细胞的至少之一。根据本发明一些具体实施方案的免疫细胞利用本发明前面所提出的抗体对靶标蛋白、细胞、组织、器官等进行识别、杀伤,进而发挥生物学功能。
在一些实施方案中,本发明提出了一种CAR-T细胞,包括嵌合抗原受体,其中,所述嵌合抗原受体包括:胞外区,所述胞外区包括单链抗体的重链可变区和轻链可变区,所述轻链可变区具有SEQ ID NO:57-59或者SEQ ID NO:65-67所示氨基酸序列,所述重链可变区具有SEQ ID NO:60-64或者SEQ ID NO:68-72所示氨基酸序列;跨膜区,所述跨膜区与所述胞外区相连,并且嵌入到所述T淋巴细胞的细胞膜中;以及胞内区,所述胞内区与所述跨膜区相连。表达该嵌合抗原受体的CAR-T细胞可以有杀伤表达Claudin18.2的肿瘤细胞。
在一些实施方案中,所述跨膜区为OX40跨膜区、ICOS跨膜区或CD8跨膜区,优选地,所述跨膜区为CD8跨膜区。
在一些实施方案中,所述CD8跨膜区trans-membrane(TM)的氨基酸序列如SEQ IDNO:106所示:
IYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO:106)
所述ICOS跨膜区trans-membrane(TM)的氨基酸序列如SEQ ID NO:107所示:
FWLPIGCAAFVVVCILGCILICWL(SEQ ID NO:107)
所述OX40跨膜区trans-membrane(TM)的氨基酸序列如SEQ ID NO:108所示:
VAAILGLGLVLGLLGPLAILLALYLL(SEQ ID NO:108)
在一些实施方案中,所述胞内段为4-1BB胞内段以及ICOS或OX-40胞内段;优选地,胞内段为4-1BB胞内段以及ICOS胞内段。
所述4-1BB免疫共刺激因子(co-stimulator)胞内段的氨基酸序列如SEQ ID NO:109所示:
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:109)
所述ICOS免疫共刺激因子(co-stimulator)胞内段的氨基酸序列如SEQ ID NO:110所示:
CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL(SEQ ID NO:110)
在一些实施方案中,所述胞外区还包含CD8铰链区。CD8铰链区(hinge)的氨基酸序列如SEQ ID NO:111所示:
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO:111)
在一些实施方案中,所述胞内段还包含CD3ζ链。CD3ζ链的氨基酸序列如SEQ IDNO:112所示:
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:112)
在一些实施方案中,所述ICOS胞内段的N端与CD8跨膜区的C端相连,所述,所述ICOS胞内段的C端与4-1BB胞内段的N端相连,所述4-1BB胞内段的C端与所述CD3ζ链的N端相连。
根据本发明的实施例,所述嵌合抗原受体的结构为:信号肽-Anti-Claudin18.2scfv-CD8铰链+CD8 TM-ICOS-4-1BB-CD3ζ。所述Anti-Claudin18.2 scfv包含如SEQ ID NO:57-59或者SEQ ID NO:65-67所示氨基酸序列的轻链可变区和包含如SEQ ID NO:60-64或者SEQ ID NO:68-72所示氨基酸序列的重链可变区。
核酸分子、表达载体、重组细胞
在制备或者获取这些抗体或嵌合抗原受体的过程中,可以利用表达这些抗体或嵌合抗原受体的核酸分子,与不同的载体连接,然后在不同细胞中表达,来获得相应抗体或嵌合抗原受体。
在一些实施方案中,本发明提出了一种核酸分子,所述核酸分子编码第一方面所述的抗体或其抗原结合片段。根据本发明一些具体实施方案的核酸分子获得的抗体不仅能够特异性的靶向结合Claudin18.2,具有与嵌合抗体一致的结合活性,而且具有更长的体内半衰期。
根据本发明的一些具体实施方案,上述核酸分子还可以进一步包括如下附加技术特征至少之一:
根据本发明的一些具体实施方案,所述核酸分子为DNA。
需要说明的是,对于本发明说明书和权利要求书中所提及的核酸,本领域技术人员应当理解,实际包括互补双链的任意一条,或者两条。为了方便,在本说明书和权利要求书中,虽然多数情况下只给出了一条链,但实际上也公开了与之互补的另一条链。另外,本申请中的核酸序列包括DNA形式或RNA形式,公开其中一种,意味着另一种也被公开。
在一些实施方案中,本发明提出了一种表达载体,所述表达载体携带前面所述的核酸分子。在将上述核酸连接到载体上时,可以将所述核酸与载体上的控制元件直接或者间接相连,只要这些控制元件能够控制所述核酸的翻译和表达等即可。当然这些控制元件可以直接来自于载体本身,也可以是外源性的,即并非来自于载体本身。当然,所述核酸与控制元件进行可操作地连接即可。本文中“可操作地连接”是指将外源基因连接到载体上,使得载体内的控制元件,例如转录控制序列和翻译控制序列等等,能够发挥其预期的调节外源基因的转录和翻译的功能。当然用来编码抗体重链和轻链的所述核酸分子,可以分别独立的插入到不同的载体上,常见的是插入到同一载体上。常用的载体例如可以为质粒、噬菌体等等。根据本发明的一些具体实施方案的表达载体导入合适的受体细胞后,可在调控系统的介导下,有效实现前面所述的特异性识别Claudin18.2的抗体或其抗原结合片段表达,进而实现所述抗体或抗原结合片段的体外大量获得。
根据本发明的一些具体实施方案,上述表达载体还可以进一步包括如下附加技术特征至少之一:
根据本发明的一些具体实施方案,所述表达载体为真核表达载体或病毒。进而实现前面所述的特异性识别Claudin18.2的抗体或其抗原结合片段在合适受体细胞中的表达,如CHO细胞。
根据本发明的一些具体实施方案,所述病毒包括慢病毒。
在一些实施方案中,本发明提出了一种重组细胞,所述重组细胞携带前面所述的核酸分子,或者表达前面所述的抗体或其抗原结合片段。根据本发明一些具体实施方案的重组细胞可用于前面所述的特异性识别Claudin18.2的抗体或其抗原结合片段体外表达和大量获得。
根据本发明的一些具体实施方案,上述重组细胞还可以进一步包括如下附加技术特征至少之一:
根据本发明的一些具体实施方案,所述重组细胞是通过将前面所述的表达载体引入至宿主细胞中而获得的。
根据本发明的一些具体实施方案,通过电转导的方法将所述表达载体引入所述宿主细胞中。
根据本发明的一些具体实施方案,所述重组细胞为真核细胞。
根据本发明的一些具体实施方案,所述重组细胞为哺乳动物细胞。
根据本发明的一些具体实施方案,所述真核细胞不包括动物生殖细胞、受精卵或胚胎干细胞。
需要注意的是,本发明所述重组细胞不受特别限制,可以为原核细胞、真核细胞或噬菌体。所述原核细胞可以为大肠杆菌、枯草杆菌、链霉菌或奇异变形菌等。所述真核细胞可以为包括巴斯德毕赤酵母、酿酒酵母、裂殖酵母、木霉等真菌,草地粘虫等昆虫细胞,烟草等植物细胞,BHK细胞、CHO细胞、COS细胞、骨髓瘤细胞等哺乳动物细胞。在一些实施例中,本发明所述重组细胞优选为哺乳动物细胞,包括BHK细胞、CHO细胞、NSO细胞或COS细胞,且不包括动物生殖细胞、受精卵或胚胎干细胞。
需要说明的是,本申请说明书中所述的“适合条件”,是指适合本申请所述重组抗体表达的条件。本领域技术人员容易理解的是,适合重组抗体表达的条件包括但不限于合适的转化或转染方式、合适的转化或转条件、健康的宿主细胞状态、合适的宿主细胞密度、适宜的细胞培养环境、适宜的细胞培养时间。“适合条件”不受特别限制,本领域技术人员可根据实验室的具体环境,优化最适的所述重组抗体表达的条件。
药物组合物、试剂盒及制药用途和在制备试剂盒中的用途
在一些实施方案中,本发明提出了一种药物组合物,所述药物组合物含有前面所述的抗体,核酸分子,表达载体,免疫细胞,重组细胞或CAR-T细胞。如前所述,所述抗体,以及表达后能够获得所述抗体的核酸分子、表达载体、免疫细胞、重组细胞或CAR-T细胞不仅能够特异性的靶向结合Claudin18.2,具有与嵌合抗体一致的结合活性,而且具有更长的体内半衰期,因此,根据本发明的一些具体实施方案的药物组合物中所包含的抗体或表达的抗体能够特异性的靶向结合Claudin18.2,特异性强,发挥较好的靶向作用,进而实现药物组合物中其他药物的生物学作用,如Claudin18.2分子活性抑制,表达Claudin18.2分子的细胞杀伤等作用。
在一些实施例中,这些药物组合物进一步包括药学上可接受的载体,包括任何溶剂、固体赋形剂、稀释剂、粘合剂、崩解剂、或其他液体赋形剂、分散剂、矫味剂或悬浮剂、表面活性剂、等渗剂、增稠剂、乳化剂、防腐剂、固体粘合剂、助流剂或润滑剂,等等,适合于特有的目标剂型。除了任何常规的辅料与本发明的化合物不相容的范围,例如所产生的任何不良的生物效应或与药学上可接受的组合物的任何其他组分以有害的方式产生的相互作用,它们的用途也是本发明所考虑的范围。
例如,本发明的抗体可掺入适用于胃肠外施用(例如静脉内、皮下、腹膜内、肌肉内)的药物组合物中。这些药物组合物可以被制备成各种形式。例如液体、半固体和固体剂型等,包括但不限于液体溶液(例如,注射溶液和输注溶液)、分散剂或悬浮剂、片剂、丸剂、粉末、脂质体和栓剂。典型的药物组合物为注射溶液或输注溶液形式。所述抗体可通过静脉输注或注射或肌肉内或皮下注射来施用。
需要注意的是,所述组合物包括在时间和/或空间上分开的组合,只要其能够共同作用以实现本发明的目的。例如,所述组合物中所含的成分可以以整体施用于受试者,或者分开施用于受试者。当所述组合物中所含的成分分开地施用于受试者时,各个成分可以同时或依次施用于受试者。
在一些实施方案中,本发明提出了前面所述的抗体、前面所述的核酸分子、前面所述的表达载体、免疫细胞、重组细胞、CAR-T细胞、或前面所述的药物组合物在制备药物中的用途,所述药物用于治疗或者预防Claudin18.2相关疾病。如前所述,所述抗体,以及表达后能够获得所述抗体的核酸分子、表达载体、免疫细胞、重组细胞或CAR-T细胞不仅能够特异性的靶向结合Claudin18.2,具有与嵌合抗体一致的结合活性,而且具有更长的体内半衰期,因此,包含上述物质的药物同样能够有效靶向结合Claudin18.2,且具有较长的体内半衰期,可以有效杀伤表达Claudin18.2分子的细胞。
根据本发明的一些具体实施方案,上述用途还可以进一步包括如下附加技术特征至少之一:
根据本发明的一些具体实施方案,所述Claudin18.2相关疾病包括:胃癌、食管癌、胰腺癌、肺癌、结肠癌和直肠癌中的至少之一。本领域技术人员可以理解,本发明所述的药物可以对高表达Claudin18.2的任何组织或器官有效,因此,所述药物可用于治疗或缓解相应组织或器官的病变。
在一些实施方案中,本发明提出了一种检测Claudin18.2的试剂盒,所述试剂盒包括前面任一所述的抗体。应用本发明提供的试剂盒,例如可以用于免疫印迹、免疫沉淀等涉及到利用Claudin18.2抗原和抗体特异性结合性能,来检测的试剂盒等。这些试剂盒可包含下列中的任意一种或多种:拮抗剂、Claudin18.2抗体或者药物参照材料;蛋白纯化柱;免疫球蛋白亲和纯化缓冲剂;细胞的测定稀释剂;说明书或者文献等。Claudin18.2抗体可被用于不同类型的诊断测试,例如可以在体外或者体内检测各种各样的疾病或者药物、毒素或者其他蛋白等的存在。例如可以通过对受试者的血清或者血液进行检测,用来测试相关疾病。例如癌症或肿瘤,这些癌症或者肿瘤可以是任何不受调控的细胞生长。
在一些实施方案中,本发明提出了前面所述的抗体、前面所述的核酸分子、前面所述的表达载体或前面所述的重组细胞在制备试剂盒中的用途,所述试剂盒用于检测Claudin18.2或者诊断Claudin18.2相关的疾病。所述Claudin18.2抗体可以与任何检测试剂或治疗制剂联用,例如与诊断性核素、纳米材料等联合使用,通过核素的放射性对靶标部位进行探测,进而获取靶标部位的信息;也可以与治疗性核素联用,利用核素的放射性,特异性杀伤靶标细胞、组织等。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解,下面的实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件的,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1人源化抗体序列设计及分析
申请人对1B6鼠源抗体序列(SEQ ID NO:102、103)、1E7鼠源抗体序列(SEQ ID NO:104、105)分别使用Discovery Studio和Antibody Modeling,使用PDB BLAST调取序列最接近的10个抗体晶体结构模型(结构分辨率高于2.5埃),对比自动建模模型,选取最优的结构模型。并使用IgBLAST将1B6、1E7鼠源抗体序列与人GermLine序列比对,选择同源度最高的GermLine。结合建模的结构模型、人GermLine序列比对结果及不同亚型的特性(IgG1、IgG4),将鼠源FR区替换成人源FR区,并且对人源FR区某些关键残基回复突变成鼠源,可变区人源化程度(除去CDR区)>90%即为人源化设计完成,获得的人源抗体1B6具体序列如SEQ ID NO:57-64所示,获得的人源抗体1E7具体序列如SEQ ID NO:65-72所示。此外,发明人将鼠源轻链恒定区替换为人源轻链恒定区(Kappa)(SEQ ID NO:73),以及,将鼠源重链恒定区替换为人源IgG1抗体的重链恒定区(SEQ ID NO:74)或人源IgG4抗体的重链恒定区(SEQ ID NO:75)。
鼠源抗体1B6轻链可变区(1B6-VL):
DIVMTQSPSSLTVTAGEKVTMSCKSSQSLFNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDF TLTISSVQAEDLAVYFCQNDYSYPLTFGAGTKLELK(SEQ ID NO:102)。
鼠源抗体1B6重链可变区(1B6-VH):
EVQLQQSGPELVRPGASVKMSCKASGYTFTDYNMHWVKQSHGKSLEWIGYINPNNGGTSYNQKFKGKATLTVNKS SSTAYMELRSLTSEDSAVYYCVTTRYLAVWGTGTTVTVSS(SEQ ID NO:103)。
鼠源抗体1E7轻链可变区(1E7-VL):
DIVMTQSPSSLSVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDF TLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIK(SEQ ID NO:104)。
鼠源抗体1E7重链可变区(1E7-VH):
QVQLQQPGSELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGMIHPNSGSTNYNEKFKSKATLTVDKS SSTAYMQLSSLTSEDSAVYYCARRYYGSISPDYWGQGTTLTVSS(SEQ ID NO:105)。
人源化1B6抗体轻链可变区1(1B6-huVL1):
DIVMTQSPSSLSVSVGDRVTMTCKSSQSLFNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPDRFSGSGSGTDF TLTISSVQPEDFATYFCQNDYSYPLTFGGGTKLEIK(SEQ ID NO:57)。
人源化1B6抗体轻链可变区2(1B6-huVL2):
DIQMTQSPSSLSASVGDRVTMTCKSSQSLFNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQNDYSYPLTFGGGTKLEIK(SEQ ID NO:58)。
人源化1B6抗体轻链可变区3(1B6-huVL3):
DIQMTQSPSSLSASVGDRVTITCRSSQSLFNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQNDYSYPLTFGGGTKLEIK(SEQ ID NO:59)。
人源化1B6抗体重链可变区1(1B6-huVH1):
EVQLVQSGAEVKKPGASVKMSCKASGYTFTDYNMHWVRQAPGKSLEWIGYINPNNGGTSYNQKFKGKATLTVNK SSSTAYMELSSLRSEDTAVYYCVTTRYLAVWGQGTTVTVSS(SEQ ID NO:60)。
人源化1B6抗体重链可变区2(1B6-huVH2):
QVQLVQSGAEVKKPGASVKMSCKASGYTFTDYNMHWVRQAPGQRLEWIGYINPNNGGTSYNQKFKGKATLTVDT SASTAYMELSSLRSEDTAVYYCVTTRYLAVWGQGTTVTVSS(SEQ ID NO:61)。
人源化1B6抗体重链可变区3(1B6-huVH3):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLEWIGYINPNNGGTSYNQKFKGRVTITVDTS ASTAYMELSSLRSEDTAVYYCVTTRYLAVWGQGTTVTVSS(SEQ ID NO:62)。
人源化1B6抗体重链可变区4(1B6-huVH4):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLEWMGYINPNNGGTSYNQKFQGRVTITVDT SASTAYMELSSLRSEDTAVYYCVTTRYLAVWGQGTTVTVSS(SEQ ID NO:63)。
人源化1B6抗体重链可变区5(1B6-huVH5):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMHWVRQAPGQRLEWMGYINPNNGGTSYSQKFQGRVTITRDT SASTAYMELSSLRSEDTAVYYCVTTRYLAVWGQGTTVTVSS(SEQ ID NO:64)。
人源化1E7抗体轻链可变区1(1E7-huVL1):
DIVMTQSPSSLSVSVGDRVTMTCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDF TLTISSVQPEDFATYYCQNDYSYPFTFGGGTKLEIK(SEQ ID NO:65)。
人源化1E7抗体轻链可变区2(1E7-huVL2):
DIQMTQSPSSLSASVGDRVTMTCKSSQSLLNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDF TLTISSLQPEDFATYYCQNDYSYPFTFGGGTKLEIK(SEQ ID NO:66)。
人源化1E7抗体轻链可变区3(1E7-huVL3):
DIQMTQSPSSLSASVGDRVTITCRSSQSLLNSGNQKNYLTWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQNDYSYPFTFGGGTKLEIK(SEQ ID NO:67)。
人源化1E7抗体重链可变区1(1E7-huVH1):
QVQLVQSGAEVKKPGASVKLSCKASGYTFTSYWMHWVRQRPGQGLEWIGMIHPNSGSTNYNEKFKSKATLTVDKS TSTAYMELSSLRSEDTAVYYCARRYYGSISPDYWGQGTTVTVSS(SEQ ID NO:68)。
人源化1E7抗体重链可变区2(1E7-huVH2):
QVQLVQSGAEVKKPGASVKLSCKASGYTFTSYWMHWVRQAPGQGLEWIGMIHPNSGSTNYNEKFKSRATLTVDTS TSTAYMELSSLRSEDTAVYYCARRYYGSISPDYWGQGTTVTVSS(SEQ ID NO:69)。
人源化1E7抗体重链可变区3(1E7-huVH3):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGMIHPNSGSTNYNEKFKSRVTMTVDT STSTAYMELSSLRSEDTAVYYCARRYYGSISPDYWGQGTTVTVSS(SEQ ID NO:70)。
人源化1E7抗体重链可变区4(1E7-huVH4):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGMIHPNSGSTNYNEKFKSRVTMTVD TSTSTVYMELSSLRSEDTAVYYCARRYYGSISPDYWGQGTTVTVSS(SEQ ID NO:71)。
人源化1E7抗体重链可变区5(1E7-huVH5):
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGMIHPNSGSTNYAQKFQGRVTMTRD TSTSTVYMELSSLRSEDTAVYYCARRYYGSISPDYWGQGTTVTVSS(SEQ ID NO:72)。
人源轻链恒定区LC(Kappa):
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA DYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:73)。
人源IgG1重链恒定区HC:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:74)。
人源IgG4重链恒定区HC:
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:75)。
实施例2人源化抗体制备及鉴定
根据实施例1人源化设计的结果,将序列构建到两个亚型的pcDNA3.4载体上,经PCR、酶切、连接、转化、鉴定、测序、比对、抽提后得到质粒。根据实施例1中人源化设计后推荐的表达组合(表1),运用ExpiCHO-S表达系统,将上述质粒按照要求进行在CHO细胞中为期7天的表达,并在表达的第6天,通过Gator非标记生物分析仪测出表达量。第7天收集CHO细胞表达液,离心后,用滤器过滤,取上清,随后使用Protein-A亲和层析纯化重组抗体。经ProteinA柱获取的蛋白样品进行SDS-PAGE鉴定,具体的实验结果如图1-8所示。通过ImageJ软件按照峰面积归一法计算还原条带纯度。与参照品IPI对比,非还原条带分子量150kDa左右,纯度大于90%;还原重链分子量55kDa左右,轻链分子量25kDa左右,重链加轻链纯度大于90%,所有人源化设计的抗体均符合抗体分子量的特性且纯度良好,满足成药性要求。
表1:
样品名称 | 重链氨基酸序列 | 轻链氨基酸序列 |
1B6-huVH1-HC(IgG1)huVL1 | SEQ ID NO:79 | SEQ ID NO:76 |
1B6-huVH1-HC(IgG1)huVL2 | SEQ ID NO:79 | SEQ ID NO:77 |
1B6-huVH2-HC(IgG1)huVL1 | SEQ ID NO:80 | SEQ ID NO:76 |
1B6-huVH2-HC(IgG1)huVL2 | SEQ ID NO:80 | SEQ ID NO:77 |
1B6-huVH3-HC(IgG1)huVL1 | SEQ ID NO:81 | SEQ ID NO:76 |
1B6-huVH3-HC(IgG1)huVL2 | SEQ ID NO:81 | SEQ ID NO:77 |
1B6-huVH3-HC(IgG1)huVL3 | SEQ ID NO:81 | SEQ ID NO:78 |
1B6-huVH4-HC(IgG1)huVL2 | SEQ ID NO:82 | SEQ ID NO:77 |
1B6-huVH4-HC(IgG1)huVL3 | SEQ ID NO:82 | SEQ ID NO:78 |
1B6-huVH5-HC(IgG1)huVL2 | SEQ ID NO:83 | SEQ ID NO:77 |
1B6-huVH5-HC(IgG1)huVL3 | SEQ ID NO:83 | SEQ ID NO:78 |
1E7-huVH1-HC(IgG1)huVL1 | SEQ ID NO:92 | SEQ ID NO:89 |
1E7-huVH1-HC(IgG1)huVL2 | SEQ ID NO:92 | SEQ ID NO:90 |
1E7-huVH2-HC(IgG1)huVL1 | SEQ ID NO:93 | SEQ ID NO:89 |
1E7-huVH2-HC(IgG1)huVL2 | SEQ ID NO:93 | SEQ ID NO:90 |
1E7-huVH3-HC(IgG1)huVL1 | SEQ ID NO:94 | SEQ ID NO:89 |
1E7-huVH3-HC(IgG1)huVL2 | SEQ ID NO:94 | SEQ ID NO:90 |
1E7-huVH3-HC(IgG1)huVL3 | SEQ ID NO:94 | SEQ ID NO:91 |
1E7-huVH4-HC(IgG1)huVL2 | SEQ ID NO:95 | SEQ ID NO:90 |
1E7-huVH4-HC(IgG1)huVL3 | SEQ ID NO:95 | SEQ ID NO:91 |
1E7-huVH5-HC(IgG1)huVL2 | SEQ ID NO:96 | SEQ ID NO:90 |
1E7-huVH5-HC(IgG1)huVL3 | SEQ ID NO:96 | SEQ ID NO:91 |
1B6-huVH1huVL1 | SEQ ID NO:84 | SEQ ID NO:76 |
1B6-huVH1huVL2 | SEQ ID NO:84 | SEQ ID NO:77 |
1B6-huVH2huVL1 | SEQ ID NO:85 | SEQ ID NO:76 |
1B6-huVH2huVL2 | SEQ ID NO:85 | SEQ ID NO:77 |
1B6-huVH3huVL1 | SEQ ID NO:86 | SEQ ID NO:76 |
1B6-huVH3huVL2 | SEQ ID NO:86 | SEQ ID NO:77 |
1B6-huVH3huVL3 | SEQ ID NO:86 | SEQ ID NO:78 |
1B6-huVH4huVL2 | SEQ ID NO:87 | SEQ ID NO:77 |
1B6-huVH4huVL3 | SEQ ID NO:87 | SEQ ID NO:78 |
1B6-huVH5huVL2 | SEQ ID NO:88 | SEQ ID NO:77 |
1B6-huVH5huVL3 | SEQ ID NO:88 | SEQ ID NO:78 |
1E7-huVH1huVL1 | SEQ ID NO:97 | SEQ ID NO:89 |
1E7-huVH1huVL2 | SEQ ID NO:97 | SEQ ID NO:90 |
1E7-huVH2huVL1 | SEQ ID NO:98 | SEQ ID NO:89 |
1E7-huVH2huVL2 | SEQ ID NO:98 | SEQ ID NO:90 |
1E7-huVH3huVL1 | SEQ ID NO:99 | SEQ ID NO:89 |
1E7-huVH3huVL2 | SEQ ID NO:99 | SEQ ID NO:90 |
1E7-huVH3huVL3 | SEQ ID NO:99 | SEQ ID NO:91 |
1E7-huVH4huVL2 | SEQ ID NO:100 | SEQ ID NO:90 |
1E7-huVH4huVL3 | SEQ ID NO:100 | SEQ ID NO:91 |
1E7-huVH5huVL2 | SEQ ID NO:101 | SEQ ID NO:90 |
1E7-huVH5huVL3 | SEQ ID NO:101 | SEQ ID NO:91 |
实施例3人源化抗体聚体含量评估(SEC)
发明人采用Agilent HPLC 1100仪器,XBridgeSEC 3.5μm,7.8×300mm(批号:ZL20200823)色谱柱,在流动相为0.15M PB(pH 7.4)的条件下,以0.8mL/min的流速上样20μL,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)采集数据,具体的数据如表2所示。除抗体1E7-huVH5huVL3(单体比例为67.67%)外,其余抗体的单体的比例均大于90%,绝大部分人源化抗体单体比例比嵌合抗体更好或相当,说明人源化设计的抗体均一性良好,满足成药性要求。
表2:
样品名称 | 单体保留时间(min) | 高分子聚合物(%) | 单体(%) | 低分子量物质(%) |
1B6-mVH-HC(IgG1)mVL | 8.64 | 1.94 | 98.06 | / |
1B6-huVH1-HC(IgG1)huVL1 | 8.61 | 1.96 | 97.34 | 0.70 |
1B6-huVH1-HC(IgG1)huVL2 | 8.59 | 1.06 | 98.94 | / |
1B6-huVH2-HC(IgG1)huVL1 | 8.66 | 1.01 | 98.56 | 0.43 |
1B6-huVH2-HC(IgG1)huVL2 | 8.64 | 1.13 | 98.87 | / |
1B6-huVH3-HC(IgG1)huVL1 | 8.66 | 1.42 | 98.17 | 0.41 |
1B6-huVH3-HC(IgG1)huVL2 | 8.64 | 1.41 | 98.08 | 0.50 |
1B6-huVH3-HC(IgG1)huVL3 | 8.64 | 1.62 | 98.38 | / |
1B6-huVH4-HC(IgG1)huVL2 | 8.68 | 1.65 | 98.35 | / |
1B6-huVH4-HC(IgG1)huVL3 | 8.69 | 1.29 | 98.71 | / |
1B6-huVH5-HC(IgG1)huVL2 | 8.68 | 1.54 | 97.89 | 0.57 |
1B6-huVH5-HC(IgG1)huVL3 | 8.69 | 1.13 | 98.46 | 0.41 |
1E7-mVH-HC(IgG1)mVL | 8.69 | 1.35 | 98.65 | / |
1E7-huVH1-HC(IgG1)huVL1 | 8.68 | 0.39 | 99.14 | 0.47 |
1E7-huVH1-HC(IgG1)huVL2 | 8.66 | 0.47 | 99.53 | / |
1E7-huVH2-HC(IgG1)huVL1 | 8.72 | 0.32 | 99.68 | / |
1E7-huVH2-HC(IgG1)huVL2 | 8.70 | 1.16 | 98.55 | 0.30 |
1E7-huVH3-HC(IgG1)huVL1 | 8.73 | 0.40 | 99.26 | 0.34 |
1E7-huVH3-HC(IgG1)huVL2 | 8.71 | 0.37 | 99.43 | 0.20 |
1E7-huVH3-HC(IgG1)huVL3 | 8.72 | 0.58 | 99.42 | / |
1E7-huVH4-HC(IgG1)huVL2 | 8.73 | 0.39 | 99.21 | 0.40 |
1E7-huVH4-HC(IgG1)huVL3 | 8.74 | 0.69 | 98.79 | 0.52 |
1E7-huVH5-HC(IgG1)huVL2 | 8.77 | 4.16 | 90.37 | 5.46 |
1E7-huVH5-HC(IgG1)huVL3 | 8.77 | 0.52 | 98.44 | 1.04 |
1B6-mVHmVL | 8.74 | 1.22 | 98.78 | / |
1B6-huVH1huVL1 | 8.69 | 0.97 | 99.03 | / |
1B6-huVH1huVL2 | 8.68 | 0.98 | 99.02 | / |
1B6-huVH2huVL1 | 8.74 | 1.00 | 99.00 | / |
1B6-huVH2huVL2 | 8.73 | 0.66 | 99.34 | / |
1B6-huVH3huVL1 | 8.74 | 1.07 | 98.93 | / |
1B6-huVH3huVL2 | 8.73 | 0.41 | 99.59 | / |
1B6-huVH3huVL3 | 8.74 | 1.60 | 98.40 | / |
1B6-huVH4huVL2 | 8.77 | 1.70 | 98.30 | / |
1B6-huVH4huVL3 | 8.78 | 1.94 | 98.06 | / |
1B6-huVH5huVL2 | 8.77 | 0.90 | 99.10 | / |
1B6-huVH5huVL3 | 8.78 | 4.18 | 95.82 | / |
1E7-mVHmVL | 8.79 | 1.43 | 98.57 | / |
1E7-huVH1huVL1 | 8.75 | 1.53 | 98.47 | / |
1E7-huVH1huVL2 | 8.73 | 1.07 | 98.93 | / |
1E7-huVH2huVL1 | 8.79 | 0.78 | 99.22 | / |
1E7-huVH2huVL2 | 8.77 | 1.19 | 98.81 | / |
1E7-huVH3huVL1 | 8.80 | 5.34 | 94.66 | / |
1E7-huVH3huVL2 | 8.78 | 1.89 | 98.11 | / |
1E7-huVH3huVL3 | 8.79 | 2.33 | 97.67 | / |
1E7-huVH4huVL2 | 8.79 | 1.04 | 98.96 | / |
1E7-huVH4huVL3 | 8.80 | 1.05 | 98.95 | / |
1E7-huVH5huVL2 | 8.83 | 3.72 | 96.28 | / |
1E7-huVH5huVL3 | 8.84 | 32.33 | 67.67 | / |
实施例4人源化抗体热稳定性评估(DSF)
在八连管或者96孔板中,加入用1XPBS(pH7.4)稀释到0.2mg/mL的样品,然后加入100X SYPRO Orange工作液使其终浓度为5X,终体积为20μL,轻弹管壁混匀,2000rpm离心10秒;每个样品制备3个重复。将样品置于ABI7500Fast 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。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。具体的实验结果如表3所示,所有抗体的最小Tm值均大于64℃,说明人源化设计的抗体热稳定性良好,满足成药性要求。
表3:
样品名称 | Tm 1(℃) | Tm 2(℃) |
1B6-mVH-HC(IgG1)mVL | 77.2 | N/A |
1B6-huVH1-HC(IgG1)huVL1 | 68.95 | 77.04 |
1B6-huVH1-HC(IgG1)huVL2 | 69.19 | 77.26 |
1B6-huVH2-HC(IgG1)huVL1 | 68.76 | 77.94 |
1B6-huVH2-HC(IgG1)huVL2 | 68.92 | 77.79 |
1B6-huVH3-HC(IgG1)huVL1 | 69.23 | 79.8 |
1B6-huVH3-HC(IgG1)huVL2 | 69.28 | 78.88 |
1B6-huVH3-HC(IgG1)huVL3 | 69.08 | 77.76 |
1B6-huVH4-HC(IgG1)huVL2 | 68.77 | 78.39 |
1B6-huVH4-HC(IgG1)huVL3 | 69.27 | 77.61 |
1B6-huVH5-HC(IgG1)huVL2 | 74.41 | N/A |
1B6-huVH5-HC(IgG1)huVL3 | 73.91 | N/A |
1E7-mVH-HC(IgG1)mVL | 75.35 | N/A |
1E7-huVH1-HC(IgG1)huVL1 | 69.05 | 75.60 |
1E7-huVH1-HC(IgG1)huVL2 | 73.75 | N/A |
1E7-huVH2-HC(IgG1)huVL1 | 69.05 | 76.01 |
1E7-huVH2-HC(IgG1)huVL2 | 72.92 | N/A |
1E7-huVH3-HC(IgG1)huVL1 | 69.08 | 76.08 |
1E7-huVH3-HC(IgG1)huVL2 | 74.22 | N/A |
1E7-huVH3-HC(IgG1)huVL3 | 73.29 | N/A |
1E7-huVH4-HC(IgG1)huVL2 | 73.85 | N/A |
1E7-huVH4-HC(IgG1)huVL3 | 72.95 | N/A |
1E7-huVH5-HC(IgG1)huVL2 | 71.22 | N/A |
1E7-huVH5-HC(IgG1)huVL3 | 70.38 | N/A |
1B6-mVHmVL | 65.16 | 75.31 |
1B6-huVH1huVL1 | 65.43 | 75.50 |
1B6-huVH1huVL2 | 65.40 | 75.65 |
1B6-huVH2huVL1 | 65.37 | 76.09 |
1B6-huVH2huVL2 | 65.16 | 76.18 |
1B6-huVH3huVL1 | 65.09 | 77.02 |
1B6-huVH3huVL2 | 64.79 | 76.18 |
1B6-huVH3huVL3 | 64.72 | 75.37 |
1B6-huVH4huVL2 | 65.06 | 76.46 |
1B6-huVH4huVL3 | 65.40 | 76.09 |
1B6-huVH5huVL2 | 65.56 | 73.64 |
1B6-huVH5huVL3 | 65.68 | 73.01 |
1E7-mVHmVL | 65.19 | 74.57 |
1E7-huVH1huVL1 | 65.13 | 74.04 |
1E7-huVH1huVL2 | 65.19 | 72.48 |
1E7-huVH2huVL1 | 64.75 | 74.35 |
1E7-huVH2huVL2 | 72.45 | N/A |
1E7-huVH3huVL1 | 65.06 | 74.50 |
1E7-huVH3huVL2 | 64.69 | 73.42 |
1E7-huVH3huVL3 | 64.20 | 72.67 |
1E7-huVH4huVL2 | 65.53 | 73.11 |
1E7-huVH4huVL3 | 65.22 | 72.49 |
1E7-huVH5huVL2 | 71.28 | N/A |
1E7-huVH5huVL3 | 65.19 | 74.57 |
实施例5人源化抗体ELISA亲和力评估
用claudin-18.2抗原(厂家:金斯瑞,批号:R50092011)包被ELISA板,2μg/mL,每孔30μL,4℃孵育过夜。用PBST清洗板3次,并在室温下用5%PBS-牛奶封闭板2小时。用PBST清洗板3次,并将稀释的抗体添加到ELISA板中,每孔30μL,室温孵育60分钟。用PBST洗板3次,并在室温下用抗人IgG-Fc-HRP(abcam,ab97225)孵育50分钟。用PBST洗板12次后,添加TMB,每孔30μL,用终止溶液终止反应并读取OD450,具体的实验结果如图9-12所示。计算ELISA亲和力EC50,具体实验数据如表4所示,其中,所有抗体ELISA亲和力均在1.25μg/mL以下,说明人源化设计的抗体满足成药性要求。
表4:
样品名称 | EC50(μg/mL) |
1E7-huVH1-HC(IgG1)huVL1 | 0.04788 |
1E7-huVH1-HC(IgG1)huVL2 | 0.03047 |
1E7-huVH2-HC(IgG1)huVL1 | 0.1114 |
1E7-huVH2-HC(IgG1)huVL2 | 0.05551 |
1E7-huVH3-HC(IgG1)huVL1 | 0.06634 |
1E7-huVH3-HC(IgG1)huVL2 | 0.05757 |
1E7-huVH3-HC(IgG1)huVL3 | 0.07069 |
1E7-huVH4-HC(IgG1)huVL2 | 0.03966 |
1E7-huVH4-HC(IgG1)huVL3 | 0.04997 |
1E7-huVH5-HC(IgG1)huVL2 | 0.1406 |
1E7-huVH5-HC(IgG1)huVL3 | 0.1301 |
1E7-huVH1huVL1 | 0.3093 |
1E7-huVH1huVL2 | 0.156 |
1E7-huVH2huVL1 | 0.4359 |
1E7-huVH2huVL2 | 0.2617 |
1E7-huVH3huVL1 | 0.6061 |
1E7-huVH3huVL2 | 0.5605 |
1E7-huVH3huVL3 | 0.5089 |
1E7-huVH4huVL2 | 0.3091 |
1E7-huVH4huVL3 | 0.4412 |
1E7-huVH5huVL2 | 0.4965 |
1E7-huVH5huVL3 | 0.09306 |
1B6-huVH1-HC(IgG1)huVL1 | 0.02478 |
1B6-huVH1-HC(IgG1)huVL2 | 0.01621 |
1B6-huVH2-HC(IgG1)huVL1 | 0.04025 |
1B6-huVH2-HC(IgG1)huVL2 | 0.03015 |
1B6-huVH3-HC(IgG1)huVL1 | 0.05919 |
1B6-huVH3-HC(IgG1)huVL2 | 0.06137 |
1B6-huVH3-HC(IgG1)huVL3 | 0.07251 |
1B6-huVH4-HC(IgG1)huVL2 | 0.04313 |
1B6-huVH4-HC(IgG1)huVL3 | 0.05188 |
1B6-huVH5-HC(IgG1)huVL2 | 0.02978 |
1B6-huVH5-HC(IgG1)huVL3 | 0.03496 |
1B6-huVH1huVL1 | 0.08392 |
1B6-huVH1huVL2 | 0.07751 |
1B6-huVH2huVL1 | 0.09779 |
1B6-huVH2huVL2 | 0.1918 |
1B6-huVH3huVL1 | 0.0569 |
1B6-huVH3huVL2 | 0.245 |
1B6-huVH3huVL3 | 0.2094 |
1B6-huVH4huVL2 | 0.2101 |
1B6-huVH4huVL3 | 0.3457 |
1B6-huVH5huVL2 | 0.2018 |
1B6-huVH5huVL3 | 0.1744 |
实施例6人源化抗体亲和动力学评估
打开GATOR仪器及相关软件,选择Kientics实验模式。按照表5所示的分析程序进行人源化抗体亲和动力学评估,数据采集后进行Kientics拟合,计算亲和动力学参数。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。人源化抗体亲和动力学检测的实验数据如表6所示,其中,所有抗体KD值都在E-08至E-11之间,说明人源化设计的抗体满足成药性要求,部分人源化抗体KD值与鼠源抗体相当甚至更好。
表5:
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表6:
样品名称 | KD(M) | ka(1/Ms) | kd(1/s) | R2 |
1B6-mVH-HC(IgG1)mVL | 4.44E-08 | 1.78E+04 | 7.92E-04 | 0.992 |
1B6-huVH1-HC(IgG1)huVL1 | 6.33E-08 | 1.94E+04 | 1.23E-03 | 0.990 |
1B6-huVH1-HC(IgG1)huVL2 | 8.67E-08 | 1.97E+04 | 1.71E-03 | 0.990 |
1B6-huVH2-HC(IgG1)huVL1 | 9.84E-09 | 1.03E+04 | 1.01E-04 | 0.996 |
1B6-huVH2-HC(IgG1)huVL2 | 1.18E-08 | 1.10E+04 | 1.30E-04 | 0.996 |
1B6-huVH3-HC(IgG1)huVL1 | 7.65E-09 | 1.02E+04 | 7.83E-05 | 0.996 |
1B6-huVH3-HC(IgG1)huVL2 | 1.09E-08 | 1.11E+04 | 1.20E-04 | 0.996 |
1B6-huVH3-HC(IgG1)huVL3 | 9.64E-09 | 1.21E+04 | 1.16E-04 | 0.996 |
1B6-huVH4-HC(IgG1)huVL2 | 2.05E-08 | 1.01E+04 | 2.07E-04 | 0.996 |
1B6-huVH4-HC(IgG1)huVL3 | 4.39E-08 | 1.82E+04 | 7.99E-04 | 0.99 |
1B6-huVH5-HC(IgG1)huVL2 | 5.97E-08 | 2.16E+04 | 1.29E-03 | 0.987 |
1B6-huVH5-HC(IgG1)huVL3 | 7.68E-08 | 2.27E+04 | 1.75E-03 | 0.989 |
1B6-mVHmVL | 8.42E-08 | 2.23E+04 | 1.88E-03 | 0.989 |
1B6-huVH1huVL1 | 9.88E-08 | 2.24E+04 | 2.22E-03 | 0.989 |
1B6-huVH1huVL2 | 1.06E-08 | 1.55E+04 | 1.64E-04 | 0.996 |
1B6-huVH2huVL1 | 4.57E-09 | 1.39E+04 | 6.36E-05 | 0.995 |
1B6-huVH2huVL2 | 4.86E-09 | 1.39E+04 | 6.78E-05 | 0.996 |
1B6-huVH3huVL1 | 9.80E-10 | 1.30E+04 | 1.27E-05 | 0.996 |
1B6-huVH3huVL2 | 1.27E-08 | 9.48E+03 | 1.21E-04 | 0.997 |
1B6-huVH3huVL3 | 8.11E-09 | 1.06E+04 | 8.56E-05 | 0.996 |
1B6-huVH4huVL2 | 7.06E-09 | 9.99E+03 | 7.06E-05 | 0.996 |
1B6-huVH4huVL3 | 7.24E-09 | 1.11E+04 | 8.07E-05 | 0.996 |
1B6-huVH5huVL2 | 1.06E-08 | 1.36E+04 | 1.44E-04 | 0.996 |
1B6-huVH5huVL3 | 7.51E-09 | 1.30E+04 | 9.78E-05 | 0.996 |
1E7-mVHmVL | 2.66E-09 | 1.02E+04 | 2.71E-05 | 0.995 |
1E7-huVH1huVL1 | 3.58E-09 | 1.06E+04 | 3.81E-05 | 0.995 |
1E7-huVH1huVL2 | 5.08E-09 | 1.09E+04 | 5.56E-05 | 0.995 |
1E7-huVH2huVL1 | 2.77E-10 | 9.55E+03 | 2.65E-06 | 0.995 |
1E7-huVH2huVL2 | 2.16E-08 | 1.24E+04 | 2.69E-04 | 0.992 |
1E7-huVH3huVL1 | 1.20E-08 | 1.10E+04 | 1.32E-04 | 0.995 |
1E7-huVH3huVL2 | 2.02E-09 | 1.03E+04 | 2.08E-05 | 0.995 |
1E7-huVH3huVL3 | 1.35E-08 | 1.10E+04 | 1.49E-04 | 0.995 |
1E7-huVH4huVL2 | 4.60E-09 | 1.06E+04 | 4.86E-05 | 0.995 |
1E7-huVH4huVL3 | 6.19E-09 | 1.01E+04 | 6.27E-05 | 0.995 |
1E7-huVH5huVL2 | 5.24E-09 | 1.01E+04 | 5.29E-05 | 0.996 |
1E7-huVH5huVL3 | 3.21E-09 | 9.61E+03 | 3.09E-05 | 0.995 |
1E7-mVH-HC(IgG1)mVL | 3.82E-09 | 1.17E+04 | 4.48E-05 | 0.995 |
1E7-huVH1-HC(IgG1)huVL1 | 4.90E-09 | 1.22E+04 | 5.97E-05 | 0.995 |
1E7-huVH1-HC(IgG1)huVL2 | 4.85E-09 | 1.21E+04 | 5.87E-05 | 0.995 |
1E7-huVH2-HC(IgG1)huVL1 | 1.82E-09 | 1.09E+04 | 1.97E-05 | 0.995 |
1E7-huVH2-HC(IgG1)huVL2 | 2.89E-09 | 1.10E+04 | 3.18E-05 | 0.995 |
1E7-huVH3-HC(IgG1)huVL1 | 3.50E-10 | 1.03E+04 | 3.60E-06 | 0.995 |
1E7-huVH3-HC(IgG1)huVL2 | 2.89E-10 | 1.02E+04 | 2.94E-06 | 0.995 |
1E7-huVH3-HC(IgG1)huVL3 | 1.16E-08 | 1.13E+04 | 1.32E-04 | 0.994 |
1E7-huVH4-HC(IgG1)huVL2 | 1.07E-08 | 1.18E+04 | 1.26E-04 | 0.994 |
1E7-huVH4-HC(IgG1)huVL3 | 2.64E-09 | 1.13E+04 | 2.99E-05 | 0.995 |
1E7-huVH5-HC(IgG1)huVL2 | 1.31E-08 | 1.28E+04 | 1.67E-04 | 0.993 |
1E7-huVH5-HC(IgG1)huVL3 | 9.29E-10 | 1.06E+04 | 9.83E-06 | 0.995 |
实施例7人源化抗体ADCC活性评估
将KATOⅢ-Claudin18.2细胞(靶细胞)培养于含10% FBS(Gibco,10099-141C)、0.5μg/mL嘌呤霉素(Gibco,A11138-03)的RPMI1640(Gibco,22400-089)培养液中,在对数生长期收集细胞,用PBS(Hyclone,SHS30256.01)彻底清洗细胞,再用含1% FBS和1%双抗(Hyclone,SV30010)的RPMI1640培养基重悬,调整细胞浓度至每毫升4×105个细胞,以5000个细胞每孔铺到白色底不透的384微孔板(Corning,3570)中。人源化抗体由3.33μg/mL进行4倍梯度稀释,制备10个浓度,25μL每孔加入细胞孔中。Jurkat-NFAT-Luc2/CD16(效应细胞)培养于含10% FBS、1%双抗的RPMI1640培养基中,在对数生长期收集细胞,用PBS彻底清洗细胞,再用含1% FBS和1%双抗的RPMI1640培养基重悬,调整细胞浓度至每毫升2×106个,以20000个每孔加入细胞孔中。将384微孔板置于7℃,5%CO2培养箱中培养12h,每孔加入30μL One-GloTM Luciferase检测试剂(Promega,E6120),使用多功能酶标仪(SpectraMax)读取光学luminescence信号。
1B6部分人源化抗体与鼠源抗体ADCC活性评估实验结果如图13所示,其中,人源化抗体的ADCC活性EC50与鼠源抗体相差不大,部分人源化抗体ADCC活性最高值与嵌合抗体相当,甚至更好。1E7部分人源化抗体、嵌合抗体ADCC活性评估结果如图14所示,其中,人源化抗体ADCC活性EC50与嵌合抗体相差不大,部分人源化抗体ADCC活性最高值与嵌合抗体相当,甚至更好。
实施例8人源化抗体CDC活性评估
将KATOⅢ-claudin18.2细胞培养于含10% FBS(Gibco,10099-141C)、0.5μg/mL嘌呤霉素(Gibco,A11138-03)的RPMI1640(Gibco,22400-089)培养液中,在对数生长期收集细胞,用PBS(Hyclone,SHS30256.01)彻底清洗细胞,再用含2.5%人血清补体成份(Quidel,A113)和1%双抗(Hyclone,SV30010)的RPMI1640培养基重悬,调整细胞浓度至每毫升3×105个,以100μL每孔铺在96孔细胞培养板(Corning,3599)中。用含2.5%人血清补体成份和1%双抗的RPMI1640培养基,将人源化抗体由270μg/mL进行4倍梯度稀释,制备10个浓度,以100μL每孔加入细胞孔中。将384微孔板置于37℃,5%CO2培养箱中孵育4h,每孔加入50μL40%的CCK-8检测试剂(日本同仁,CK04),继续孵育3h,用多功能酶标仪(SpectraMax)读取OD450。
1B6部分人源化抗体与嵌合抗体CDC活性评估实验结果如图15所示,其中,人源化抗体的CDC活性EC50与嵌合抗体相差不大,部分人源化抗体CDC活性最高值与嵌合抗体相当,甚至更好。1E7部分人源化抗体、嵌合抗体CDC活性评估结果如图16所示,其中,人源化抗体CDC活性EC50与嵌合抗体相差不大,部分人源化抗体CDC活性最高值与嵌合抗体相当,甚至更好。
实施例9应用FACS方法采用ZenonTMpHrodoTMiFL IgG Labeling Reagent评价人源化claudin18.2抗体内化情况
构建Claudin18.2稳定表达细胞系A20~18.2(靶细胞),采用含10% FBS(Gibco,10099-141C)、0.05mMβ-巯基乙醇(阿拉丁,M301574)和1μg/ml的嘌呤霉素(Gibco,A11138-03)的1640完全培养基(Gibco,22400-089)培养,待细胞生长到整个培养皿的80%左右,细胞离心,台盼蓝测定活力,确保细胞活力在95%以上,全细胞自动计数仪进行计数,调整细胞密度为2*10^6cells/mL。采用1640完全培养基配制待测抗体,待测抗体终浓度为2μg/mL。采用1640完全培养基配制ZenonTMpHrodoTMiFL IgG Labeling Reagent(Invitrogen,Z25611,Z25612),其终浓度为40nM。将配制好的待测抗体和ZenonTMpHrodoTMiFL IgGLabeling Reagent各25μL加入96孔板中,室温条件下共孵育5min。每孔中再加入50μL A20~18.2细胞,每孔细胞量为1*10^5cells.将96孔板置于37℃,5% CO2孵育箱中孵育4h。PBS洗去剩余抗体或试剂,流式检测抗体内化情况,统计出发生内化的细胞比例。
结果如表7和图17A-C显示,Claudin18.2鼠源单抗1B6发生了明显的抗体内化,最大内化细胞比例达到92.83%。相同条件下,1B6抗体人源化后,抗体内化的比例有不同程度的降低,降低程度与抗体的IgG亚型,不同人源化程度有关。鼠源单抗1E7无明显抗体内化表现,人源化后依旧不发生抗体内化。
表7:Claudin18.2人源化单抗内化检测荧光强度以及内化细胞比例
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实施例10人源化抗体小鼠体内药代动力学评估
雄性Babl/c小鼠18只,随机平均分为6组,3只/组,按2mg/kg分别皮下注射给予(1)嵌合抗体1B6单抗和其人源化抗体P04891(1B6-huVH4huVL3),P04887(1B6-huVH3huVL1);(2)嵌合抗体1E7单抗和其人源化抗体P04898(1E7-huVH2huVL2),P04902(1E7-huVH4huVL2)。分别于给药后0.25、2、7、24、48、72、120、168、240、336、456、504h采血并分离血浆(EDTA-K2抗凝)。采用FACS方法分析各样本中嵌合抗体和人源化抗体的浓度。在稳定表达Claudin18.2的A20细胞中加入梯度浓度的供试品和血浆样品,随后加入FITC偶联的二抗(羊抗人IgG)结合供试品,用流式细胞仪检测细胞表面荧光强度并绘制抗体浓度-荧光强度的拟合曲线,计算出血浆中药物浓度。
根据血浆药物浓度计算药代动力学参数,主要PK参数结果见表8。结果显示,Babl/c小鼠皮下注射嵌合抗体鼠1B6单抗(1B6-mVHmVL)和其人源化抗体P04891(1B6-huVH4huVL3),P04887(1B6-huVH3huVL1)后,平均血浆半衰期分别为70h和76h,160h;嵌合抗体鼠1E7单抗(1E7-mVHmVL)和其人源化抗体P04898(1E7-huVH2huVL2),P04902(1E7-huVH4huVL2)的平均血浆半衰期为111h和177h,196h。与各自分别的嵌合抗体相比,人源化抗体能显著延长抗体在体内的半衰期。
表8:Claudin18.2嵌合单抗及其人源化单抗在小鼠体内半衰期(h)
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实施例11人源化Claudin18.2抗体CAR-T体外效果研究
稳定表达Claudin18.2-ΔC5的MDA-MB-468-18.2细胞,以DMEM完全培养基(含10%FBS、1%Pen/Strep双抗)培养,并置于37℃、5%二氧化碳培养箱中培养。
待肿瘤细胞生长至所需数量时,取对数生长期细胞,弃原培养基,胰酶消化3min。后以含有10%FBS的DMEM培养基终止消化,收集细胞,400rpm离心5min。细胞计数后,用10%FBS的DMEM将细胞稀释铺板平底96孔板,每孔200μL,设置2E+04cells每孔。肿瘤细胞贴壁6h后,设置效靶比E/T=0.2、0.5、1.0,即每孔对应的CAR阳性细胞数量分别为4E+03、1E+04、2E+04个,进行CART细胞与靶细胞共孵育。杀伤孵育时使用CTS完全培养,每孔培养体系均为200μL,每组设置三个复孔,每个效靶比还设置3个仅T细胞的平行孔。
共孵育铺板完成后将细胞置于37℃、5%二氧化碳培养箱中培养。培养20h后使用LDH法进行杀伤检测,490nm测吸光值。
数据处理:阳性对照(PS)=(靶+lysis)-(靶+NC)
实验组杀伤(EFF)=(E/T=n)-(onlyT,E/T=n)
Cytotoxicity=100%*(EFF)/(PS)
结果如图18所示。其中,G1:阴性对照组、G2:LH2组、G3:HL2组,共3组。G1为T细胞;G2、G3组分别为表达靶向Claudin18.2分子的CAR的T细胞,G2与G3的CAR分子中scFv序列的VH和VL方向相反,其中G2组以VL-GS linker-VH相连,G3组以VH-GS linker-VL相连,均为人源化的1E7序列(VL是IE7-huVL2,氨基酸序列如SEQ ID NO:66所示,VH是IE7-huVH4,氨基酸序列如SEQ ID NO:71所示)。
由图可知,G2、G3组的体外杀伤效果显著优于G1对照组。
实施例12人源化Claudin18.2抗体CAR-T体内效果研究
稳定表达Claudin18.2-ΔC5的MDA-MB-468-18.2细胞,以DMEM完全培养基(含10%FBS、1%Pen/Strep双抗)培养,并置于37℃、5%二氧化碳培养箱中培养。待细胞生长至所需数量时,取对数生长期细胞,弃原培养基,并加入1/2体积的PBS冲洗,再弃PBS,然后加入适量0.05%胰酶消化3分钟。然后用DMEM完全培养基终止消化。收集细胞,1000rpm离心5分钟。取0.5ml培养基提取基因组通过PCR法测定支原体为阴性。细胞计数后,用无血清的DMEM培养基和Matrigel混合液(按1:1比例)调整细胞密度为5E+07/mL。抓取固定NCG雌性小鼠,将细胞悬液注射入小鼠右侧背部皮下,100μL/鼠。
MDA-MB-468-18.2模型中,肿瘤生长至100mm3左右时,进行动物分组给药,共有G1:Vehicle组、G2:LH2组、G3:HL2组,共3组。其中G1为阴性对照组,注射PBS;G2、G3分别表达靶向Claudin18.2分子的CAR,G2与G3的CAR分子scFv序列的VH和VL方向相反,其中G2组以VL-VH相连,G3组以VH-VL相连,均为原1E7序列人源化后所得序列(VL是IE7-huVL2,氨基酸序列如SEQ ID NO:66所示,VH是IE7-huVL4,氨基酸序列如SEQ ID NO:71所示)。
收集LH2 CAR-T以及HL2 CAR-T细胞,以PBS溶液调整细胞密度为CAR阳性细胞密度为1.25E+07/ml,每只小鼠尾静脉给药200μL,即2.5E+06个CAR阳性细胞,G1组尾静脉注射200μL PBS。给药结束后,每周2次测量肿瘤的长度(L)与宽度(W),并计算肿瘤体积(V),V=L*W*W*0.5;再计算肿瘤生长抑制率(TGI)。若TX>T0,则TGI=[1-TX/CX]*100%;若TX<T0,TGI=[1-(TX-T0)/T0]*100%,其中TX、CX为测量日实验组与对照组的肿瘤体积,T0、C0为给药当天实验组与对照组的肿瘤体积,以GraphPad Prism 6进行统计学分析,结果如图19所示。
结果可知,LH2 CAR-T以及HL2 CAR-T在NCG小鼠建立的移植瘤模型上,经静脉给药后,均可显著抑制肿瘤的生长。实验结束时,LH2 CAR-T的肿瘤生长抑制率为131%,HL2CAR-T的肿瘤生长抑制率为197%,且具有明显的统计学差异(P<0.01)。
在本说明书的描述中,参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中,对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且,描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外,在不相互矛盾的情况下,本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对本发明的限制,本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (34)
1.一种能够特异性识别Claudin18.2的抗体或其抗原结合片段,其特征在于,所述抗体含有选自下列至少之一的CDR序列或与其具有至少90%同一性的氨基酸序列:
轻链可变区CDR序列:
X1SSQSLFNSGNQKNYLT(SEQ ID NO:1),
WASTRES(SEQ ID NO:2),
QNDYSYPLT(SEQ ID NO:3),
X4SSQSLLNSGNQKNYLT(SEQ ID NO:7),
WASTRES(SEQ ID NO:8),和
QNDYSYPFT(SEQ ID NO:9);
重链可变区CDR序列:
DYNMH(SEQ ID NO:4),
YINPNNGGTSYX2QKFX3G(SEQ ID NO:5),
TRYLAV(SEQ ID NO:6),
SYWMH(SEQ ID NO:10),
MIHPNSGSTNYX5X6KFX7X8(SEQ ID NO:11),和
RYYGSISPDY(SEQ ID NO:12);
其中,X1为K或R,X2为N或S,X3为K或Q,X4为K或R,X5为N或A,X6为E或Q,X7为K或Q,X8为S或G,
所述抗体或其抗原结合片段具有人源化修饰。
2.根据权利要求1所述的抗体或其抗原结合片段,其特征在于,所述抗体包括:
分别如SEQ ID NO:1、2和3或者与SEQ ID NO:1、2和3具有至少90%同一性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列;或者
分别如SEQ ID NO:7、8和9或者与SEQ ID NO:7、8和9具有至少90%同一性的氨基酸序列所示的轻链可变区CDR1、CDR2、CDR3序列。
3.根据权利要求1所述的抗体或其抗原结合片段,其特征在于,所述抗体包括:
分别如SEQ ID NO:4、5和6或者与SEQ ID NO:4、5和6具有至少90%同一性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列;或者
分别如SEQ ID NO:10、11和12或者与SEQ ID NO:10、11和12具有至少90%同一性的氨基酸序列所示的重链可变区CDR1、CDR2、CDR3序列。
4.根据权利要求1所述的抗体或其抗原结合片段,其特征在于,所述抗体含有重链框架区序列和轻链框架区序列的至少之一,所述重链框架区序列和轻链框架区序列的至少之一的至少一部分来自于人源抗体或其突变体。
5.根据权利要求4所述的抗体或其抗原结合片段,其特征在于,所述抗体包括:
分别如SEQ ID NO:13-16所示的轻链框架区FRL1、FRL2、FRL3、FRL4序列,
DIX9MTQSPSSLX10X11X12X13GX14X15VTX16SX17C(SEQ ID NO:13),
WYQQKPGX18X19PKLLIY(SEQ ID NO:14),
GVPX20DRFX21GSGSGTDFTLTISSX22QX23EDX24AX25YX26C(SEQ ID NO:15),
FGX27GTKLEX28K(SEQ ID NO:16);或者
分别如SEQ ID NO:21-24所示的轻链框架区FRL1、FRL2、FRL3、FRL4序列,
DIX53MTQSPSSLSX54X55AX56GX57X58VTX59X60C(SEQ ID NO:21),
WYQQKPGX61X62PKLLIY(SEQ ID NO:22),
GVPX63RFX64GSGSGTDFTLTISSX65QX66EDX67AX68YYC(SEQ ID NO:23),
FGX69GTKLEIK(SEQ ID NO:24),
其中,X9为V或Q,X10为S或T,X11为V或A,X12为T或S,X13为A或V,X14为E或D,X15为K或R,X16为M或I,X17为S或T,X18为Q或K,X19为P或A,X20为D或S,X21为T或S,X22为V或L,X23为A或P,X24为L或F,X25为V或T,X26为F或Y,X27为A或G,X28为L或I,X53为V或Q,X54为V或A,X55为T或S,X56为A或V,X57为E或D,X58为K或R,X59为M或I,X60为S或T,X61为Q或K,X62为P或A,X63为D或S,X64为T或S,X65为V或L,X66为A或P,X67为L或F,X68为V或T,X69为S或G;
条件是,当所述抗体包括:分别如KSSQSLFNSGNQKNYLT(SEQ ID NO:29)、WASTRES(SEQID NO:30)、QNDYSYPLT(SEQ ID NO:31)所示的轻链可变区CDR1、CDR2、CDR3序列时,X9为V,X10为T,X11为V,X12为T,X13为A,X14为E,X15为K,X16为M,X17为S,X18为Q,X19为P,X20为D,X21为T,X22为V,X23为A,X24为L,X25为V,X26为F,X27为A,X28为L不同时成立;
当所述抗体包括:分别如KSSQSLLNSGNQKNYLT(SEQ ID NO:35)、WASTRES(SEQ ID NO:36)、QNDYSYPFT(SEQ ID NO:37)所示的轻链可变区CDR1、CDR2、CDR3序列时,X53为V,X54为V,X55为T,X56为A,X57为E,X58为K,X59为M,X60为S,X61为Q,X62为P,X63为D,X64为T,X65为V,X66为A,X67为L,X68为V,X69为S不同时成立。
6.根据权利要求4所述的抗体或其抗原结合片段,其特征在于,所述抗体包括:
分别如SEQ ID NO:41-44所示的轻链框架区FRL1、FRL2、FRL3、FRL4序列,
DIX90MTQSPSSLSX91TAGEKVTX92SC(SEQ ID NO:41),
WYQQKPGQPPKLLIY(SEQ ID NO:42),
GVPX93DRFTGSGSGTDFTLTISSX94QAEDLAVYX95C(SEQ ID NO:43),
FGAGTKLELK(SEQ ID NO:44);或者
分别如SEQ ID NO:49-52所示的轻链框架区FRL1、FRL2、FRL3、FRL4序列,
DIX107MTQSPSSLSX108TAGEKVTX109SC(SEQ ID NO:49),
WYQQKPGX110X111PKLLIY(SEQ ID NO:50),
GVPX112RFTGSGSGTDFTLTISSX113QAEDLAVYYC(SEQ ID NO:51),
FGSGTKLEIK(SEQ ID NO:52),
其中,X90为V或Q,X91为V或A,X92为M或I,X93为D或S,X94为V或L,X95为F或Y,X107为V或Q,X108为V或A,X109为M或I,X110为Q或K,X111为P或A,X112为D或S,X113为V或L。
7.根据权利要求4所述的抗体或其抗原结合片段,其特征在于,所述抗体包括:
分别如SEQ ID NO:17-20所示的重链框架区FRH1、FRH2、FRH3和FRH4序列,
X29VQLX30QSGX31EX32X33X34PGASVKX35SCKASGYTFT(SEQ ID NO:17),
WVX36QX37X38GX39X40LEWX41G(SEQ ID NO:18),
X42X43TX44TX45X46X47SX48STAYMELX49SLX50SEDX51AVYYCVT(SEQ ID NO:19),
WGX52GTTVTVSS(SEQ ID NO:20);或者
分别如SEQ ID NO:25-28所示的重链框架区FRH1、FRH2、FRH3和FRH4序列,
QVQLX70QX71GX72EX73X74KPGASVKX75SCKASGYTFT(SEQ ID NO:25),
WVX76QX77PGQGLEWX78G(SEQ ID NO:26),
X79X80X81TX82TVDX83SX84STX85YMX86LSSLX87SEDX88AVYYCAR(SEQ ID NO:27),
WGQGTTX89TVSS(SEQ ID NO:28),
其中,X29为E或Q,X30为Q或V,X31为P或A,X32为L或V,X33为V或K,X34为R或K,X35为M或V,X36为K或R,X37为S或A,X38为H或P,X39为K或Q,X40为S或R,X41为I或M,X42为K或R,X43为A或V,X44为L或I,X45为V或R,X46为N或D,X47为K或T,X48为S或A,X49为R或S,X50为T或R,X51为S或T,X52为T或Q,X70为Q或V,X71为P或S,X72为S或A,X73为L或V,X74为V或K,X75为L或V,X76为K或R,X77为R或A,X78为I或M,X79为S或G,X80为R或K,X81为A或V,X82为L或M,X83为K或T,X84为S或T,X85为A或V,X86为Q或E,X87为T或R,X88为S或T,X89为L或V;
条件是,当所述抗体包括:分别如DYNMH(SEQ ID NO:32)、YINPNNGGTSYNQKFKG(SEQ IDNO:33)、TRYLAV(SEQ ID NO:34)所示的重链可变区CDR1、CDR2、CDR3序列时,X29为E,X30为Q,X31为P,X32为L,X33为V,X34为R,X35为M,X36为K,X37为S,X38为H,X39为K,X40为S,X41为I,X42为K,X43为A,X44为L,X45为V,X46为N,X47为K,X48为S,X49为R,X50为T,X51为S,X52为T不同时成立;
当所述抗体包括:分别如SYWMH(SEQ ID NO:38)、MIHPNSGSTNYNEKFKS(SEQ ID NO:39)、RYYGSISPDY(SEQ ID NO:40)所示的重链可变区CDR1、CDR2、CDR3序列时,X70为Q,X71为P,X72为S,X73为L,X74为V,X75为L,X76为K,X77为R,X78为I,X79为S,X80为K,X81为A,X82为L,X83为K,X84为S,X85为A,X86为Q,X87为T,X88为S,X89为L不同时成立。
8.根据权利要求4所述的抗体或其抗原结合片段,其特征在于,所述抗体包括:
分别如SEQ ID NO:45-48所示的重链框架区FRH1、FRH2、FRH3和FRH4序列,
X96VQLQQSGPELVRPGASVKX97SCKASGYTFT(SEQ ID NO:45),
WVKQSHGX98X99LEWX100G(SEQ ID NO:46),
X101X102TX103TVX104X105SX106STAYMELRSLTSEDSAVYYCVT(SEQ ID NO:47),
WGTGTTVTVSS(SEQ ID NO:48);或者
分别如SEQ ID NO:53-56所示的重链框架区FRH1、FRH2、FRH3和FRH4序列,
QVQLQQPGSELVKPGASVKX114SCKASGYTFT(SEQ ID NO:53),
WVKQX115PGQGLEWX116G(SEQ ID NO:54),
X117X118TX119TVDX120SSSTX121YMQLSSLTSEDSAVYYCAR(SEQ ID NO:55),
WGQGTTLTVSS(SEQ ID NO:56),
其中,X96为E或Q,X97为M或V,X98为K或Q,X99为S或R,X100为I或M,X101为K或R,X102为A或V,X103为L或I,X104为N或D,X105为K或T,X106为S或A,X114为L或V,X115为R或A,X116为I或M,X117为R或K,X118为A或V,X119为L或M,X120为K或T,X121为A或V。
9.根据权利要求1所述的抗体或其抗原结合片段,其特征在于,所述抗体具有如SEQ IDNO:57-59或者SEQ ID NO:65-67所示氨基酸序列的轻链可变区。
10.根据权利要求1所述的抗体或其抗原结合片段,其特征在于,所述抗体具有如SEQID NO:60-64或者SEQ ID NO:68-72所示氨基酸序列的重链可变区。
11.根据权利要求1所述的抗体或其抗原结合片段,其特征在于,所述抗体含有重链恒定区和轻链恒定区的至少之一,所述重链恒定区和轻链恒定区的至少之一的至少一部分来自于人源抗体;
任选地,所述抗体的轻链恒定区和重链恒定区均来自于人源IgG抗体或其突变体;
任选地,所述抗体的轻链恒定区来自于人源的Kappa轻链恒定区;重链恒定区来自于人源IgG4或IgG1,或,IgG4或IgG1的突变体。
12.根据权利要求9所述的抗体或其抗原结合片段,其特征在于,所述抗体恒定区的全长序列如SEQ ID NO:73、74或75所示。
13.根据权利要求9所述的抗体或其抗原结合片段,其特征在于,所述抗体具有SEQ IDNO:76-78任一项所示的轻链,和,SEQ ID NO:79-88任一项所示的重链。
14.根据权利要求9所述的抗体或其抗原结合片段,其特征在于,所述抗体具有SEQ IDNO:89-91任一项所示的轻链,和,SEQ ID NO:92-101任一项所示的重链。
15.根据权利要求9所述的抗体或其抗原结合片段,其特征在于,所述抗体包括:
SEQ ID NO:76所示的轻链,SEQ ID NO:79所示的重链,或
SEQ ID NO:77所示的轻链,SEQ ID NO:79所示的重链,或
SEQ ID NO:76所示的轻链,SEQ ID NO:80所示的重链,或
SEQ ID NO:77所示的轻链,SEQ ID NO:80所示的重链,或
SEQ ID NO:76所示的轻链,SEQ ID NO:81所示的重链,或
SEQ ID NO:77所示的轻链,SEQ ID NO:81所示的重链,或
SEQ ID NO:78所示的轻链,SEQ ID NO:81所示的重链,或
SEQ ID NO:77所示的轻链,SEQ ID NO:82所示的重链,或
SEQ ID NO:78所示的轻链,SEQ ID NO82所示的重链,或
SEQ ID NO:77所示的轻链,SEQ ID NO:83所示的重链,或
SEQ ID NO:78所示的轻链,SEQ ID NO:83所示的重链,或
SEQ ID NO:76所示的轻链,SEQ ID NO:84所示的重链,或
SEQ ID NO:77所示的轻链,SEQ ID NO:84所示的重链,或
SEQ ID NO:76所示的轻链,SEQ ID NO:85所示的重链,或
SEQ ID NO:77所示的轻链,SEQ ID NO:85所示的重链,或
SEQ ID NO:76所示的轻链,SEQ ID NO:86所示的重链,或
SEQ ID NO:77所示的轻链,SEQ ID NO:86所示的重链,或
SEQ ID NO:78所示的轻链,SEQ ID NO:86所示的重链,或
SEQ ID NO:77所示的轻链,SEQ ID NO:87所示的重链,或
SEQ ID NO:78所示的轻链,SEQ ID NO:87所示的重链,或
SEQ ID NO:77所示的轻链,SEQ ID NO:88所示的重链,或
SEQ ID NO:78所示的轻链,SEQ ID NO:88所示的重链。
16.根据权利要求9所述的抗体或其抗原结合片段,其特征在于,所述抗体包括:
SEQ ID NO:89所示的轻链,SEQ ID NO:92所示的重链,或
SEQ ID NO:90所示的轻链,SEQ ID NO:92所示的重链,或
SEQ ID NO:89所示的轻链,SEQ ID NO:93所示的重链,或
SEQ ID NO:90所示的轻链,SEQ ID NO:93所示的重链,或
SEQ ID NO:89所示的轻链,SEQ ID NO:94所示的重链,或
SEQ ID NO:90所示的轻链,SEQ ID NO:94所示的重链,或
SEQ ID NO:91所示的轻链,SEQ ID NO:94所示的重链,或
SEQ ID NO:90所示的轻链,SEQ ID NO:95所示的重链,或
SEQ ID NO:91所示的轻链,SEQ ID NO:95所示的重链,或
SEQ ID NO:90所示的轻链,SEQ ID NO:96所示的重链,或
SEQ ID NO:91所示的轻链,SEQ ID NO:96所示的重链,或
SEQ ID NO:89所示的轻链,SEQ ID NO:97所示的重链,或
SEQ ID NO:90所示的轻链,SEQ ID NO:97所示的重链,或
SEQ ID NO:89所示的轻链,SEQ ID NO:98所示的重链,或
SEQ ID NO:90所示的轻链,SEQ ID NO:98所示的重链,或
SEQ ID NO:89所示的轻链,SEQ ID NO:99所示的重链,或
SEQ ID NO:90所示的轻链,SEQ ID NO:99所示的重链,或
SEQ ID NO:91所示的轻链,SEQ ID NO:99所示的重链,或
SEQ ID NO:90所示的轻链,SEQ ID NO:100所示的重链,或
SEQ ID NO:91所示的轻链,SEQ ID NO:100所示的重链,或
SEQ ID NO:90所示的轻链,SEQ ID NO:101所示的重链,或
SEQ ID NO:91所示的轻链,SEQ ID NO:101所示的重链。
17.根据权利要求9所述的抗体或其抗原结合片段,其特征在于,所述抗体为单链抗体、多聚体抗体、CDR移植抗体或小分子抗体;
任选地,所述小分子抗体包括Fab抗体、Fv抗体、单域抗体以及最小识别单位的至少之一。
18.一种核酸分子,其特征在于,所述核酸分子编码权利要求1~17任一项所述的抗体或其抗原结合片段。
19.一种表达载体,其特征在于,携带权利要求18~23任一项所述的核酸分子。
20.根据权利要求19所述的表达载体,其特征在于,所述表达载体为真核表达载体或者病毒;
任选地,所述病毒包括慢病毒。
21.一种免疫细胞,其特征在于,所述免疫细胞携带权利要求18所述的核酸分子,权利要求19-20任一项所述的表达载体或者表达权利要求1~17任一项所述的抗体或其抗原结合片段;
任选地,所述免疫细胞包括T淋巴细胞、DC细胞、NK细胞和NKT淋巴细胞的至少之一。
22.一种重组细胞,其特征在于,所述重组细胞携带权利要求18所述的核酸分子,权利要求19-20任一项所述的表达载体或者表达权利要求1~17任一项所述的抗体或其抗原结合片段。
23.根据权利要求22所述的重组细胞,其特征在于,所述重组细胞是通过将权利要求19-20任一项所述的表达载体引入至宿主细胞中而获得的;
任选地,通过电转导的方法将所述表达载体引入所述宿主细胞中。
24.根据权利要求22所述的重组细胞,其特征在于,所述重组细胞为真核细胞;
任选地,所述重组细胞为哺乳动物细胞。
25.一种CAR-T细胞,其特征在于,包括嵌合抗原受体,其中,
所述嵌合抗原受体包括:
胞外区,所述胞外区包括单链抗体的重链可变区和轻链可变区,所述轻链可变区具有SEQ ID NO:57-59或者SEQ IDNO:65-67所示氨基酸序列,所述重链可变区具有SEQ ID NO:60-64或者SEQ ID NO:68-72所示氨基酸序列;
跨膜区,所述跨膜区与所述胞外区相连,并且嵌入到所述T淋巴细胞的细胞膜中;以及
胞内区,所述胞内区与所述跨膜区相连。
26.根据权利要求25所述的CAR-T细胞,其特征在于,所述跨膜区为ICOS跨膜区、CD8跨膜区或OX40跨膜区;
优选地,所述跨膜区为CD8跨膜区;
更优选地,所述跨膜区氨基酸序列如SEQ ID NO:106所示。
27.根据权利要求25所述的CAR-T细胞,其特征在于,所述胞内段为4-1BB胞内段以及ICOS或OX-40胞内段;
优选地,胞内段为4-1BB胞内段和ICOS胞内段;
更优选地,所述4-1BB胞内段氨基酸序列如SEQ ID NO:109所示;所述ICOS胞内段氨基酸序列如SEQ ID NO:110所示。
28.根据权利要求25所述的CAR-T细胞,其特征在于,所述胞外区还包括CD8铰链区;
优选地,所述CD8铰链区氨基酸序列如SEQ ID NO:111所示。
29.根据权利要求25所述的CAR-T细胞,其特征在于,所述胞内段还包含CD3ζ链;
优选地,所述CD3ζ链氨基酸序列如SEQ ID NO:112所示。
30.根据权利要求28所述的CAR-T细胞,其特征在于,所述ICOS胞内段的N端与CD8跨膜区的C端相连,所述,所述ICOS胞内段的C端与4-1BB胞内段的N端相连,所述4-1BB胞内段的C端与所述CD3ζ链的N端相连。
31.一种药物组合物,其特征在于,含有权利要求1~17任一项所述的抗体,权利要求18所述的核酸分子,权利要求19-20任一项所述的表达载体,权利要求21所述的免疫细胞,权利要求22~24任一项所述的重组细胞,或权利要求25-30任一项所述的CAR-T细胞。
32.权利要求1~17任一项所述的抗体,权利要求18所述的核酸分子,权利要求19~20任一项所述的表达载体,权利要求21所述的免疫细胞,权利要求22~24任一项所述的重组细胞,权利要求25-30任一项所述的CAR-T细胞,或权利要求31所述的药物组合物在制备药物中的用途,所述药物用于治疗或者预防Claudin18.2相关疾病;
任选地,所述Claudin18.2相关疾病包括:胃癌、食管癌、胰腺癌、肺癌、结肠癌和直肠癌中的至少之一。
33.一种检测Claudin18.2的试剂盒,其特征在于,包括权利要求利要求1~17任一项所述的抗体。
34.权利要求1~17任一项所述的抗体,权利要求18所述的核酸分子,权利要求19-20任一项所述的表达载体,或权利要求22~24任一项所述的重组细胞在制备试剂盒中的用途,所述试剂盒用于检测Claudin18.2或者诊断Claudin18.2相关的疾病。
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