CN112679612B - 抗cd19人源化抗体及其制备方法与应用 - Google Patents
抗cd19人源化抗体及其制备方法与应用 Download PDFInfo
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Abstract
本发明提供了一种抗CD19人源化抗体及其制备方法与应用,该抗体含有人源化重链可变区VH和人源化轻链可变区VL,所述VH和VL均由决定簇互补区和框架区组成;所述VH和所述VL的决定簇互补区均由CDR1、CDR2和CDR3组成;VH的CDR1的氨基酸序列选自SEQ ID NO.1‑4;VH的CDR2的氨基酸序列选自SEQ ID NO.5‑8;VH的CDR3的氨基酸序列选自SEQ ID NO.9‑12;VL的CDR1的氨基酸序列选自SEQ ID NO.13‑16;VL的CDR2的氨基酸序列选SEQ ID NO.17‑20;VL的CDR3的氨基酸序列选自SEQ ID NO.21‑24;人源化程度高,亲和力高。
Description
技术领域
本发明属于生物技术领域,涉及一种抗CD19人源化抗体及其制备方法与应用。
背景技术
CD19是淋巴细胞表面的糖蛋白,从骨髓祖B淋巴细胞开始表达,直至B细胞成熟期,在浆细胞时消失。与B细胞活化,信号传导和生长调节有密切关系。广泛分布于B淋巴细胞的表面,是一种功能型的受体分子。此外,CD19还表达于恶性B细胞肿瘤,滤泡状树突细胞,急性髓系白血病细胞,非何杰金式淋巴瘤中。临床上常用CD19作为B淋巴细胞白血病及淋巴瘤的免疫分型,为临床诊断提供依据。急性B淋巴细胞性白血病中CD19高表达率达到100%,CD19蛋白作为治疗的靶标,在体外实验,动物模型,以及早期的临床阶段获得了较为理想的结果。随着CD19分子研究的深入,CD19蛋白的抗体研究在抗体药物及嵌合受体研究领域有广阔的应用前景。
鼠单克隆抗体被誉为神奇的子弹,从1975年问世以来,在诊断和治疗方面有广泛的应用。然而鼠源抗体在人体内会引起人抗鼠反应,产生毒性反应,同时也加快了抗体清除,影响抗体药效。为了解决这一问题,从上世纪80年代开始,先后发展了嵌合抗体,CDR区移植人源化抗体,全人源抗体的制备技术。(1)由于鼠源抗体的免疫反应约有90%是针对恒定,要降低鼠源抗体的抗原性,必须对抗体的恒定区进行人源化。将鼠源抗体的可变区与人源抗体的恒定区融合,获得人鼠的嵌合抗体。这一技术可以解决恒定区的抗原性问题,但仍然保留了鼠源抗体的可变区,仍然会有较强的抗原性。(2)为了进一步降低抗体的抗原性,将鼠源抗体的CDR区移植到同源的人源抗体骨架区,获得人源化程度更高的抗体。(3)随着噬菌体展示技术和转基因小鼠技术的出现,可以通过抗体库技术和杂交瘤技术获得全人源的抗体。将人的抗体基因插入噬菌体的基因组,可以获得展示人抗体基因的噬菌体抗体库,通过对抗体库的筛选,获得与特定靶分子结合的人源抗体。但由于所建立的抗体库为天然库,抗体缺少了体内亲和力成熟的过程,筛选得到的抗体亲和力较低,后期需要进一步亲和力成熟才能作为治疗应用。将人抗体基因转入小鼠,经过免疫后,可以获得全人源的抗体。但前期需要将大片段的人抗体基因组转入小鼠,小鼠改造周期较长。因此,将鼠源抗体通过人源化的方法,降低抗体的抗原性,同时保留抗体结合的特异性是治疗抗体开发的有效方式。
因此,如何成功开发一种抗CD19人源化抗体,降低抗体的抗原性,同时保留抗体结合的特异性,成为亟待解决的技术问题。
发明内容
为了解决所述技术问题,本发明提供了一种抗CD19人源化抗体及其制备方法与应用,可以特异性识别CD19蛋白,抗原性低,抗体结合的特异性高。
在本发明的第一方面,提供了一种抗CD19人源化抗体,所述抗CD19人源化抗体能特异性结合CD19抗原,所述抗CD19人源化抗体含有人源化重链可变区VH和人源化轻链可变区VL,所述VH和VL均由决定簇互补区和框架区组成;所述VH和所述VL的决定簇互补区均由CDR1、CDR2和CDR3组成;
所述VH的CDR1的氨基酸序列选自SEQ ID NO.1-SEQ ID NO.4中的一条;
所述VH的CDR2的氨基酸序列选自SEQ ID NO.5-SEQ ID NO.8中的一条;
所述VH的CDR3的氨基酸序列选自SEQ ID NO.9-SEQ ID NO.12中的一条;
所述VL的CDR1的氨基酸序列选自SEQ ID NO.13-SEQ ID NO.16中的一条;
所述VL的CDR2的氨基酸序列选自SEQ ID NO.17-SEQ ID NO.20中的一条;
所述VL的CDR3的氨基酸序列选自SEQ ID NO.21-SEQ ID NO.24中的一条。
进一步地,所述抗CD19人源化抗体包括以下CD19人源化抗体1-4中的至少一种:
抗CD19人源化抗体1:所述VH的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ IDNO.1、SEQ ID NO.5和SEQ ID NO.9所示;所述VL的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.13、SEQ ID NO.17和SEQ ID NO.21所示;
抗CD19人源化抗体2:所述VH的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ IDNO.2、SEQ ID NO.6和SEQ ID NO.10所示;所述VL的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.14、SEQ ID NO.18和SEQ ID NO.22所示;
抗CD19人源化抗体3:所述VH的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ IDNO.3、SEQ ID NO.7和SEQ ID NO.11所示;所述VL的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.15、SEQ ID NO.19和SEQ ID NO.23所示;
抗CD19人源化抗体4:所述VH的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ IDNO.4、SEQ ID NO.8和SEQ ID NO.12所示;所述VL的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.16、SEQ ID NO.20和SEQ ID NO.24所示。
进一步地,所述抗CD19人源化抗体包括如下抗CD19人源化抗体的至少一种:
B5抗CD19人源化抗体:所述人源化重链可变区VH的氨基酸序列如SEQ ID NO:25所示,所述人源化轻链可变区VL的氨基酸序列如SEQ ID NO:26所示;
F5抗CD19人源化抗体:所述人源化重链可变区VH的氨基酸序列如SEQ ID NO:27所示,所述人源化轻链可变区VL的氨基酸序列如SEQ ID NO:28所示;
C1抗CD19人源化抗体:所述人源化重链可变区VH的氨基酸序列如SEQ ID NO:29所示,所述人源化轻链可变区VL的氨基酸序列如SEQ ID NO:30所示;
D12抗CD19人源化抗体:所述人源化重链可变区VH的氨基酸序列如SEQ ID NO:31所示,所述人源化轻链可变区VL的氨基酸序列如SEQ ID NO:32所示。
进一步地,所述抗CD19人源化抗体还包括:所述抗CD19人源化抗体的氨基酸序列经取代、缺失和/或增加一个或多个氨基酸得到的具有相同功能的抗体;和/或所述抗CD19人源化抗体的N端和/或C端连接标签得到的抗体。
在本发明的第二方面,提供了一种编码所述抗CD19人源化抗体的核酸分子。
进一步地,所述核苷酸分子包括:
核酸分子1:编码重链可变区的核苷酸序列如SEQ ID NO:33所示,编码轻链可变区的核苷酸序列为SEQ ID NO:34所示;
核酸分子2:编码重链可变区的核苷酸序列如SEQ ID NO:35所示,编码轻链可变区的核苷酸序列为SEQ ID NO:36所示;
核酸分子3:编码重链可变区的核苷酸序列如SEQ ID NO:37所示,编码轻链可变区的核苷酸序列为SEQ ID NO:38所示;
核酸分子4:编码重链可变区的核苷酸序列如SEQ ID NO:39所示,编码轻链可变区的核苷酸序列为SEQ ID NO:40所示。
在本发明的第三方面,提供了一种含有所述核酸分子的生物材料,所述生物材料包括重组DNA、质粒载体、噬菌体载体、病毒载体、工程菌或转基因细胞系。
在本发明的第四方面,提供了具有所述抗CD19人源化抗体的细胞或偶联物。
进一步地,所述细胞包括嵌合抗原受体T细胞、嵌合抗原受体NK细胞、和经人工编辑的细胞;所述偶联物包括抗CD19人源化抗体-细菌毒素偶联物、抗CD19人源化抗体-细菌毒素变体偶联物、抗CD19人源化抗体-细胞因子偶联物、以及抗CD19人源化抗体-化疗药物偶联物。
在本发明的第五方面,提供了所述的抗人CD19的人源化抗体在制备肿瘤抑制剂或肿瘤细胞抑制剂中的应用。
在本发明的第五方面,提供了一种所述的抗人CD19的人源化抗体的制备方法,所述方法包括:
获得cDNA,用人源抗体库引物对所述cDNA进行扩增,获得所述的人源化重链可变区VH和人源化轻链可变区VL;
合成抗体CDR3和FR4部分;
将所述人源化重链可变区VH的可变区V基因与所述CDR3和所述FR4融合,获得重链基因片段;
将所述人源化轻链可变区VL的可变区V基因与所述CDR3和所述FR4融合,获得轻链基因片段;
将所述重链基因片段和所述轻链基因片段通过linker连接,插入噬菌粒载体,获得以噬菌粒的形式保存在宿主菌中的scfv抗体库;
利用CD19蛋白在所述抗体库中淘选,经筛选后,获得所述抗人CD19的人源化抗体。
本发明实施例中的一个或多个技术方案,至少具有如下技术效果或优点:
本发明将抗体识别最重要的重链和轻链CDR3,移植到人源抗体的可变区V基因上。相对于常规的CDR移植技术,将6个CDR移植到一个固定抗体骨架上。本发明移植两个CDR区,提高了抗体的人源化程度,使抗体可变区的人源化程度达到90%以上。多模板抗体骨架的选择,使抗体的CDR3更好地保留了原始构象。改造前后的抗体在流式检测和ELISA检测中差异较小,部分克隆EC50差异在2倍以内,说明本发明的抗体改造方法,很好地保留了抗体的亲和力。
附图说明
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例描述中所需要使用的附图作一简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其它的附图。
图1是人源化抗体基因序列分析结果;
图2是流式检测FMC63抗体图;
图3是流式检测人源化抗体B5图;
图4是Western blot方法检测B5抗体结果。
具体实施方式
下文将结合具体实施方式和实施例,具体阐述本发明,本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解,这些具体实施方式和实施例是用于说明本发明,而非限制本发明。
在整个说明书中,除非另有特别说明,本文使用的术语应理解为如本领域中通常所使用的含义。因此,除非另有定义,本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾,本说明书优先。
除非另有特别说明,本发明中用到的各种原材料、试剂、仪器和设备等,均可通过市场购买得到或者可通过现有方法制备得到。
本发明实施例提供的技术方案为解决上述技术问题,总体思路如下:
将人源抗体的骨架区分别与鼠源抗体的CDR3融合,获得只保留鼠源抗体重链和轻链CDR3的抗体基因。用此基因构建噬菌体展示抗体文库,通过高通量筛选的方法,获得人源化的抗体基因。相对于常规的CDR移植方案,本发明选择了更多的人源抗体基因作为模板,获得人源化程度更高的抗体,同时保持原始抗体的高亲和力。
所述抗CD19人源化抗体的CDR区的氨基酸序列如下表1所示:
表1
B5抗CD19人源化抗体:所述人源化重链可变区VH的氨基酸序列如SEQ ID NO:25所示,所述人源化轻链可变区VL的氨基酸序列如SEQ ID NO:26所示;所述VH的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.1、SEQ ID NO.5和SEQ ID NO.9所示;所述VL的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.13、SEQ ID NO.17和SEQ ID NO.21所示;
F5抗CD19人源化抗体:所述人源化重链可变区VH的氨基酸序列如SEQ ID NO:27所示,所述人源化轻链可变区VL的氨基酸序列如SEQ ID NO:28所示;所述VH的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.2、SEQ ID NO.6和SEQ ID NO.10所示;所述VL的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.14、SEQ ID NO.18和SEQ ID NO.22所示;
C1抗CD19人源化抗体:所述人源化重链可变区VH的氨基酸序列如SEQ ID NO:29所示,所述人源化轻链可变区VL的氨基酸序列如SEQ ID NO:30所示;所述VH的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.3、SEQ ID NO.7和SEQ ID NO.11所示;所述VL的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.15、SEQ ID NO.19和SEQ ID NO.23所示;
D12抗CD19人源化抗体:所述人源化重链可变区VH的氨基酸序列如SEQ ID NO:31所示,所述人源化轻链可变区VL的氨基酸序列如SEQ ID NO:32所示。所述VH的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.4、SEQ ID NO.8和SEQ ID NO.12所示;所述VL的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.16、SEQ ID NO.20和SEQ ID NO.24所示。
这些抗CD19人源化抗体,经实验验证,保留了原鼠抗体与抗原结合的活性,抗体的骨架区和除CDR3以外的可变区,均用人源化抗体序列替换,相对于CDR区移植获得的抗体,这些抗体具有更高的人源化程度。
下面将结合实施例及实验数据对本申请的效果进行详细说明。
实施例1获得抗CD19人源化抗体
一、噬菌体展示抗体库构建
1、淋巴细胞分离与抗体基因扩增
取一支15ml离心管,先加入与血液样本等量的分离液,小心吸取血液样本加于分离液之液面上,450-650g,离心20-30min;离心后,此时离心管中由上至下分为四层,依次为血浆层,环状乳白色淋巴细胞层,透明分离液层和红细胞层;用吸管小心吸取第二层环状乳白色淋巴细胞层到另一15ml离心管中;向所得离心管中加入10ml清洗液,混匀细胞,250xg,离心10min;离完心看上清液是否澄清,不够澄清加长离心时间;用1xPBS清洗细胞2次,加trizol裂解细胞-80℃保存。
取-80℃保存的外周淋巴细胞裂解液在室温中融化;加入0.2ml氯仿,盖上盖子,剧烈振荡15s,室温放置2min后,于12000g、4℃离心15min;将上清小心转至另一离心管中,加入等体积异丙醇,混匀后室温放置10min;于12000g,4℃离心15min;离心后弃上清,沉淀用75%的酒精漂洗,再次于12000g,4℃离心5min;离心后弃上清,将离心管置于室温,待其干燥用无RNase的水溶解RNA;取少许溶解的RNA跑琼脂糖胶,并测定浓度,判断RNA是否降解。
从-80℃取出总RNA于冰上融化;PCR仪上65℃5min打开RNA的二级结构;放置冰上2min;依次加入buffer和逆转录酶及引物37℃15min,98℃5min;用内参引物验证得到的cDNA;-20℃保存(-80℃长期保存)。配制PCR反应体系,用人源抗体库引物(RHuVk1aBACKFV,RHuVk2aBACKFV,RHuVk3aBACKFV,RHuVk4aBACKFV,RHuVk5aBACKFV,RHuVk6aBACKFV,HuVH1aBACKSfi,HuVH2aBACKSfi,HuVH3aBACKSfi,HuVH4aBACKSfi,HuVH5aBACKSfi,HuVH6aBACKSfi,H-R-FR3,L-R-FR3),从cDNA中扩增出抗体重链和轻链可变区可变区V基因;
PCR反应条件94℃4min,(94℃30s,54℃30s,72℃1min)25个循环,72℃4min;将PCR产物进行2%琼脂糖凝胶电泳分析,并切下约300bp左右的目的条带,用胶回收柱回收目的片段扩增产物。
将H-F-CDR3与H-R-FR4混合,L-F-CDR3与L-R-FR4混合;采用PCR反应获得抗体的的CDR3和FR4片段。采用PCR连接抗体的可变区基因片段与CDR3和FR4,获得完整的抗体VH和VL基因,扩增引物为RHuVk1aBACKFV,RHuVk2aBACKFV,RHuVk3aBACKFV,RHuVk4aBACKFV,RHuVk5aBACKFV,RHuVk6aBACKFV,HuVH1aBACKSfi,HuVH2aBACKSfi,HuVH3aBACKSfi,HuVH4aBACKSfi,HuVH5aBACKSfi,HuVH6aBACKSfi,H-F-CDR3,H-R-FR4,L-F-CDR3,L-R-FR4。
表2:人抗体引物设计
2.抗体基因拼接与电转大肠杆菌建库
将重链和轻链回收的抗体基因,分别用PCR方法添加linker(linker序列为:GGTGGAGGCGGTTCAGGCGGAGGTGGTTCTGGCGGTGGCGGATCG),跑胶回收后按照等物质量混合,加入酶切位点引物,通过PCR扩增反应(反应条件同可变区扩增),实现重链和轻链通过linker连接。按如下体系将各试剂加入到PCR管中:SfiⅠ(10U/l),1μl;10xBuffer 5μl;抗体/载体,2μg;加水至30μl。在PCR仪上50℃过夜进行SfiⅠ酶切;取出后冷却至室温,继续向体系中加入以下成分:10xBuffer,5μL;NotI,1μl;加水至50μl,在PCR仪上37℃保温过夜进行NotⅠ酶切;跑1.5%的琼脂糖胶切胶,用天根DNA回收试剂盒回收目的片段,分装后于-20℃冻存待用;配制反应体系,将抗体基因连入酶切后的pCANTAB5E载体。按下列体系配制连接体系:载体,1.5μg;抗体,0.5μg;T4连接酶,2μl;10xbuffer,10μl加水至100μl;在PCR仪上16℃连接过夜。
划线Minimal培养板37℃培养过夜;接种TG1单菌落至5mL2YT培养液中于37℃振荡培养过夜;次日将接种过夜培养的菌液5mL加至300mL的2YT培养液中,振荡培养至OD600达到0.4-0.5;将菌液冰浴30min后,在预冷的离心机中于4℃以4000g离心15min;用300mL预冷的灭菌去离子水于冰水中轻柔重悬沉淀,至沉淀细胞完全均匀地分散于水中;在预冷的离心机中于4℃以4 000g离心15min;再依次用150mL预冷的灭菌去离子水和30mL预冷的10%的甘油(用灭菌去离子水配制)如上所述重悬细胞两次;最后将细胞重悬于1mL预冷的10%的甘油中,置于冰上立即使用或分装;冻存于-80℃中待用;向100uL感受态中加入5uL连接产物,放于冰上预冷,转入预冷后的电转杯中;调节电转仪电压2.5KV电击时间5ms,电击后,迅速加入0.9ml 2YT培养基,37℃振荡培养1.5小时;取10uL梯度稀释,涂布于SOBAG平板上,计算库容,剩余菌液涂布于10个SOBAG平板上,37℃培养过夜。
对梯度稀释的菌落计数,计算出本次建成的抗体库的库容;从SOBAG平板上随机挑取20个克隆,用菌落PCR检测抗体基因插入载体的效率;将随机挑选的20个克隆送测序分析,检测抗体库容,抗体基因的完整性及多样性。
二、人源化抗体筛选
1、制备噬菌体展示抗体库
将构建好的纳米抗体库以噬菌粒的形式保存在宿主菌中,在淘选过程开始前,应当先拯救文库,使其成为噬菌体展示的抗体库。具体方法如下:
将1.5mL带E-tag标签的抗体库接种到300mL2YT-AG培养基中,至OD600nm约0.3-0.4;37℃振荡培养约1.5h至OD600nm=0.5-0.6;按细菌:phage=1:5加入helper phage辅助噬菌体(M13K07),37℃振荡培养约1h;4000rpm 15℃离心15min,去除培养基;加入200mL2YT-AK(100μg/mL Amp,50μg/mL Kan)培养基重悬细菌,37℃培养2h;10000rpm离心20min去除沉淀;上清加入40mL PEG/NaCl沉淀phage,冰浴过夜;10000rpm离心20min,去掉上清;用0.6mL 2YT培养基重悬phage,4℃备用。若需要大量制备phage,更换kan抗性的培养基以后,培养时间从两小时延长至过夜培养。获得的噬菌体经过梯度稀释,感染TG1菌,涂布SOBAG平板,通过菌落计数计算噬菌体库滴度。
本实验利用His Bind Resin结合抗原蛋白,从噬菌体展示的抗体库中淘选抗体,具体过程如下:活化His Bind Resin:取200μL His Bind Resin于1.5mL离心管中,1000g离心1min,去掉保存溶液;加入200μL的ddH2O清洗树脂一次,1000g离心1min,去掉上清,重复此步骤一次;加入200μL离子化缓冲液,重悬树脂,静置10min,离心去掉上清;加入200μL结合缓冲液,重悬树脂,静置10min;取40μL树脂加入1.5mL离心管中,离心去掉上清。
2.淘选识别目的蛋白的单链抗体
取纯化后的CD19蛋白10μg,加入165μL PBS中,混匀后加入装有活化后的树脂的EP管中,旋转混匀1h,1000g离心1min,去掉上清;加入200μL漂洗缓冲液,重悬树脂,1000g离心1min,去掉上清,此步骤重复一次。取拯救后的噬菌体展示抗体库溶液300μL,加入0.3μLTriton X-100,用微量移液器轻轻混匀;加入40μL未活化的树脂,轻微旋转反应1h;1000g离心1min,取上清加入40μL已包被抗原蛋白的树脂,轻微旋转反应2h;1000g离心1min,去掉上清;加入500μL漂洗缓冲液(含0.1%Triton X-100),重悬树脂,轻微振荡漂洗5min,1000g离心1min,去掉上清,此步骤重复5次;加入500μL漂洗缓冲液含(0.1%Tween-20),重悬树脂,轻微振荡漂洗5min,1000g离心1min,去掉上清,此步骤重复5次;最后一次漂洗后,将树脂转移到新的EP管中,1000g离心1min,去掉上清;加入200μL洗脱缓冲液,轻微旋转洗脱20min;1000g离心1min,取上清,加入5mL TG1菌液中,37℃感染1h;将感染的菌液涂布SOBAG平板,30℃倒置培养过夜;次日,用2YT-AG培养基刮下平板上的菌落,并拯救成噬菌体进行下一轮淘选。
3.挑选识CD19蛋白的阳性克隆
从SOBAG平板上随机挑取单菌落接种到96孔细菌培养板中,每孔加入200μL2YT-AG,37℃培养过夜;吸取25μL菌液到新的细菌培养板中,加入175μL 2YT-AG培养基,37℃培养3h;3500rpm离心10min,去上清,菌沉淀重悬于200μL 2YT-AI(100μg/mL Amp,1mM IPTG)培养基中,30℃诱导过夜;3500rpm离心10min,上清4℃保存备用;将纯化的的目的蛋白加入到ELISA板中,4℃包被过夜;倾掉包被液后,以PBS洗3次,4%PBSM(PBS含4%脱脂牛奶)封闭1h;以PBS洗1次后,每孔加入50μL以上制备好的单链抗体上清和50μL 4%PBSM,于37℃反应1h;用PBS和PBST各洗3次后,每孔加入100μL anti-E/HRP conjugation(以4%PBSM按1:5000稀释),37℃保温1h;用PBST和PBS洗涤三次,加入100μL TMB底物溶液,避光反应15min,加入25μL 2mol/L H2SO4终止反应,用酶标仪测定OD450nm值判定目的蛋白的浓度。
4.阳性克隆测序和序列分析
将上述淘选得到的OD450nm值较大的目的蛋白经ELISA鉴定,将鉴定为阳性的单克隆送金开瑞测序,测序的通用引物为S1:5’-GACCATGATTACGCCAAGC-3’,用DNAstar和Clustalw1.8分析抗体的重链与轻链可变区序列。
实施例2人源化抗体基因抗体表达
转染前将所有试剂置于室温10分钟,以下操作使用6孔培养皿;将3μg质粒DNA稀释到250μL无血清的DMEM培养基,用移液枪吹吸3-4次;将5μL PEI试剂稀释到250μL无血清的DMEM培养基,用移液枪吹吸3-4次;注意:无血清的DMEM培养基是稀释液,不能使用含血清的培养基进行DNA和将稀释好的PEI转染试剂一次性全部加入到已稀释好的质粒DNA中,立即用移液枪吹吸3-4次;室温放置10-15分钟,以形成PEI-DNA复合物;转染前18-24小时进行细胞的计数并铺板,以便在转染时,使贴壁细胞的汇合度大约80%左右;弃去孔中原培养基,加入1ml新鲜的DMEM完全培养基;制备PEI-DNA复合物;将PEI-DNA混合液均匀滴入细胞培养基中,轻轻做十字运动,让PEI-DNA复合物分散均匀;培养皿放于5%CO2,37℃恒温培养箱中,72h后收集细胞,检测蛋白表达量。按照相同的比例转化100ml细胞,纯化重组表达的抗体。取上清进行亲和层析纯化和SDS-PAGE电泳分析,纯化的重组抗体用间接竞争ELISA初步测定其活性。通过优化诱导表达条件(如宿主菌、表达载体、诱导培养时间、温度以及IPTG浓度等),可以进一步提高目的蛋白表达量,为大量制备重组提供了途径。
实验例1
一、人源化抗体基因序列分析
通过软件分析,重链和轻链的Z-score分别为-1.5和-1.0。通过人源化改造以后,B5的重链和轻链Z-score分别为1.6和0.4,D12的重链和轻链Z-score分别为1.2和0.7。通过人源化以后得到的两株抗体,Z-score都有较大幅度的提升,从图中的结果可知,这两株抗体基本上是全人源的抗体。
FMC63重链分析结果如图1(A)所示,表明未改造的抗体是鼠源序列,序列经软件分析的Z-score分布在鼠源抗体的范围。
FMC63轻链分析结果如图1(B)所示,表明未改造的抗体是鼠源序列,序列经软件分析的Z-score分布在鼠源抗体的范围。
人源化抗体B5重链分析结果如图1(C)所示,表明人源化以后,抗体的序列与人源序列相似较高,Z-score分布在人源抗体的范围内。
人源化抗体B5轻链分析结果如图1(D)所示,表明表明人源化以后,抗体的序列与人源序列相似较高,Z-score分布在人源抗体的范围内。
二、流式细胞仪检测抗体
取Raji细胞,1000rpm/min,3min离心细胞850-1000rpm,弃上清;用PBS重悬,1000rmp/min,3min离心850-1000rpm,弃上清。10%正常非免疫山羊血清(PBS稀释)重悬,4℃孵育45min,(在静音混合器上孵育);经1000rpm/min,3min离心,弃上清。用5%正常山羊血清稀释一抗,至终浓度为0.01ug/ul;加入一抗稀释液100ul,4℃孵育45min,(在静音混合器上孵育);经1000rpm/min,3min离心,弃上清;用PBS重悬后,静置2min。经1000rpm/min,3min离心,弃上清,重复两次。用5%正常山羊血清稀释二抗,稀释比例为1:100;加入二抗稀释液50μL,4℃孵育30min,(在静音混合器上孵育,注意避光);经1000rpm/min,3min离心,弃上清;用PBS重悬,1000rpm/min,3min离心,弃上清.根据细胞量的多少加入400μL或者200μL的PBS重悬细胞,转移液体至流式上样管,立即上流式仪检测。
流式检测FMC63抗体如图2所示,表明未改造的CD19抗体可以识别细胞表面的CD19蛋白。
流式检测人源化抗体B5如图3所示,表明人源化以后的抗体保留了识别CD19蛋白的活性,检测的阳性率略低于原始抗体,人源化以后的抗体亲和力略微降低。
三、SDS-PAGE检测B5抗体
人源化以后的B5抗体,经重组表达后,用SDS-PAGE分析,其结果如图4所示。人源化以后的抗体可以在哺乳细胞中表达,产生完整的重链和轻链。
实验例2抗CD19人源化抗体与CD19蛋白结合情况
将实施例1筛选获得的4株抗CD19人源化抗体,经过实施例2表达后,分别与CD19结合,检测抗体与CD19结合的EC50值,CD19蛋白按照2μg/ml包被微孔板;检测结果如表3所示。
表3
组别 | EC50 |
B5重组抗体 | 0.15ng/ml |
F5重组抗体 | 2.01ng/ml |
C1重组抗体 | 5.03ng/ml |
D12重组抗体 | 0.21ng/ml |
鼠抗体 | 0.10ng/ml |
由表2可知,4株抗CD19人源化抗体的EC50分别为如表3所示:改造后的抗体与原鼠源抗体相比,亲和力略有下降,其中亲和力较高的B5和D12差异较小。用此方法改造的人源化抗体,很好地保留了原抗体的高亲和力。
最后,还需要说明的是,尽管已描述了本发明的优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
序列表
<110> 武汉华美生物工程有限公司
<120> 抗CD19人源化抗体及其制备方法与应用
<160> 58
<170> SIPOSequenceListing 1.0
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<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Gly Phe Thr Phe Ser Ser Tyr Gly
1 5
<210> 2
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Gly Phe Thr Leu Gly Asp Tyr Pro
1 5
<210> 3
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Ser Gly Phe Thr Phe Ser Ser Tyr Ala
1 5
<210> 4
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Gly Tyr Ser Phe Thr Asp Tyr Trp
1 5
<210> 5
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Ile Ser Gly Ser Gly Gly Arg Thr
1 5
<210> 6
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
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Ile Ser Gly Ser Gly Gly Ser Thr
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<210> 7
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Ile Ser Gly Ser Gly Gly Ser Thr
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<210> 8
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
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Ile Tyr Pro Asp Asp Ser Asp Thr
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<210> 9
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
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Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp
1 5 10
<210> 10
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
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Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp
1 5 10
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<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
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Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp
1 5 10
<210> 12
<211> 12
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp
1 5 10
<210> 13
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
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Gln Ser Ile Ser Ser Tyr
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<210> 14
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
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Gln Ser Val Ser Ser Tyr
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<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
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Gln Ser Ile Ser Ser His
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<212> PRT
<213> 人工序列(Artificial Sequence)
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Gln Gly Ile Ser Ser Asn
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<210> 17
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<212> PRT
<213> 人工序列(Artificial Sequence)
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Ala Ala Ser
1
<210> 18
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<213> 人工序列(Artificial Sequence)
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Asp Ala Ser
1
<210> 19
<211> 3
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Gly Ala Ser
1
<210> 20
<211> 3
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Thr Ala Ser
1
<210> 21
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
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Gln Gln Gly Asn Thr Leu Pro Tyr Thr
1 5
<210> 22
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 22
Gln Gln Gly Asn Thr Leu Pro Tyr Thr
1 5
<210> 23
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Gln Gln Gly Asn Thr Leu Pro Tyr Thr
1 5
<210> 24
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 24
Gln Gln Gly Asn Thr Leu Pro Tyr Thr
1 5
<210> 25
<211> 121
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Gly Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 26
<211> 109
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Ile Leu Ile
35 40 45
Ser Ala Ala Ser Ser Leu Glu Arg Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Thr Asn Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 27
<211> 121
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Gly Asp Tyr
20 25 30
Pro Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Thr Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 28
<211> 121
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Gly Asp Tyr
20 25 30
Pro Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Thr Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 29
<211> 121
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Gly Asp Tyr
20 25 30
Pro Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Thr Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 30
<211> 109
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 30
Lys Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Thr Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Leu Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 31
<211> 121
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Gly Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu
1 5 10 15
Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Asp Tyr
20 25 30
Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Ala Ile Ile Tyr Pro Asp Asp Ser Asp Thr Lys Tyr Ser Pro Ser Phe
50 55 60
Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr
65 70 75 80
Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Leu Tyr Ser Cys
85 90 95
Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 32
<211> 109
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Phe Leu Ile
35 40 45
Tyr Thr Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 33
<211> 363
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
ggggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60
tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt ccgccaggct 120
ccagggaagg ggctggaatg ggtctcagct attagtggta gtggtggtcg cacatactac 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtat 240
ctgcaaatga acagtctgag agccgaggac accgccttgt attactgtgc aaaacattat 300
tactacggtg gtagctatgc tatggactac tggggccagg gcaccctggt caccgtctcc 360
tca 363
<210> 34
<211> 327
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgttggaga caaagtcacc 60
atcacttgcc gggcaagtca gagtattagc agctatttaa attggtatca gcagaaacca 120
gggaaagccc ctaagatcct gatctctgct gcatccagtt tggaacgtgg ggtcccatct 180
aggttcagtg gcagtggatc tgggacagat ttcagtctca ccattacgaa tctgcaacct 240
gaagattttg caacttatta ctgtcaacag ggtaatacgc ttccgtacac gacttttggc 300
caggggacca aggtggaaat caaacgt 327
<210> 35
<211> 363
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
caggtgcagc tggtgcagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60
tcctgtgcag cctctggatt cacccttggt gattatccta tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
acagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agctgaggac accgccttgt attactgtgc aaaacattat 300
tactacggtg gtagctatgc tatggactac tggggccagg gcaccctggt caccgtctcc 360
tca 363
<210> 36
<211> 327
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
aaaattgtgc tgactcagtc tccagccacc ctgtctttgt ctcccgggga aagagccacc 60
ctctcctgca ggaccagtca gagtgttagc agctacttag cctggtacca acagaaacct 120
ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180
aggttcagtg gcagtgggtc tgggacagac ctcactctca ccatcagcag cctagagcct 240
gaagattttg caacttatta ctgtcaacag ggtaatacgc ttccgtacac gacttttggc 300
caggggacca aggtggaaat caaacgt 327
<210> 37
<211> 363
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
caggtgcagc tggggcagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac 180
gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac accgccttgt attactgtgc aaaacattat 300
tactacggtg gtagctatgc tatggactac tggggccagg gcaccctggt caccgtctcc 360
tca 363
<210> 38
<211> 327
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
gtcacttgcc ggacaaggca gagtattagt agtcatttaa attggtacca gcagaaacct 120
ggccaggctc ccaggctcct catctatggt gcatccacca gggccactgg tatcccagcc 180
aggttcagcg gcagtggata tgggacagag ttcactctca ccatcagcag cctgcagcct 240
gaagattttg caacttatta ctgtcaacag ggtaatacgc ttccgtacac gacttttggc 300
caggggacca aggtggaaat caaacgt 327
<210> 39
<211> 363
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
ggggtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaagatc 60
tcctgtaagg gttctggata cagctttacc gactactgga tcgcctgggt gcgccagatg 120
cccgggaaag gcctggagtg gatggcgatc atctatcctg atgactctga caccaaatac 180
agcccgtcct tccaaggcca ggtcaccatc tcagccgaca agtccatcag caccgcctac 240
ctgcagtgga gcagcctgaa ggcctcggac accgccttgt attcctgtgc aaaacattat 300
tactacggtg gtagctatgc tatggactac tggggccagg gcaccctggt caccgtctcc 360
tca 363
<210> 40
<211> 327
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggccagtca gggcatcagc agtaatttag cctggtatca gcaaaaacca 120
gggaaagccc ctaagttcct gatctatact gcatccactt tgcagagtgg ggtcccatca 180
aggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaaccc 240
gaagattttg caacttatta ctgtcaacag ggtaatacgc ttccgtacac gacttttggc 300
caggggacca aggtggaaat caaacgt 327
<210> 41
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
ggagactggg tcatctggat gtccgatccg cc 32
<210> 42
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
ggagactgag tcatcacaac atccgatccg cc 32
<210> 43
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
ggagactgcg tcaacacaat ttccgatccg cc 32
<210> 44
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
ggagactggg tcatcacgat gtccgatccg cc 32
<210> 45
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
ggagactgcg tgagtgtcgt ttccgatccg cc 32
<210> 46
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
ggagactgag tcagcacaat ttccgatccg cc 32
<210> 47
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
gtcctcgcaa ctgcggccca gccggccatg gcccaggtgc agctggtgca gtctgg 56
<210> 48
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
gtcctcgcaa ctgcggccca gccggccatg gcccaggtca acttaaggga gtctgg 56
<210> 49
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
gtcctcgcaa ctgcggccca gccggccatg gccgaggtgc agctggtgga gtctgg 56
<210> 50
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
gtcctcgcaa ctgcggccca gccggccatg gcccaggtgc agctgcagga gtcggg 56
<210> 51
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
gtcctcgcaa ctgcggccca gccggccatg gcccaggtgc agctgttgca gtctgc 56
<210> 52
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
gtcctcgcaa ctgcggccca gccggccatg gcccaggtac agctgcagca gtcagg 56
<210> 53
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
acagtaatac aaggcggtgt cctc 24
<210> 54
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
agcatagcta ccaccgtagt aataatgttt tgcacagtaa tacaaggcgg tgtcctc 57
<210> 55
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
tgaggagacg gtgaccaggg tgccctggcc ccagtagtcc atagcatagc taccaccg 58
<210> 56
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
tgttgacagt aataagttgc 20
<210> 57
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
ctggccaaaa gtcgtgtacg gaagcgtatt accctgttga cagtaataag ttgc 54
<210> 58
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
acgtttgatt tccaccttgg tcccctggcc aaaagtcgtg tacggaagc 49
Claims (9)
1.一 种抗CD19人源化抗体,其特征在于,所述抗CD19人源化抗体能特异性结合CD19抗原,所述抗CD19人源化抗体含有人源化重链可变区VH和人源化轻链可变区VL,所述VH和VL均由决定簇互补区和框架区组成;所述VH和所述VL的决定簇互补区均由CDR1、CDR2和CDR3组成;
所述抗CD19人源化抗体包括以下CD19人源化抗体1-4中的至少一种:
抗CD19人源化抗体1:所述VH的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.1、SEQ ID NO.5和SEQ ID NO.9所示;所述VL的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ IDNO.13、SEQ ID NO.17和SEQ ID NO.21所示;
抗CD19人源化抗体2:所述VH的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.2、SEQ ID NO.6和SEQ ID NO.10所示;所述VL的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ IDNO.14、SEQ ID NO.18和SEQ ID NO.22所示;
抗CD19人源化抗体3:所述VH的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.3、SEQ ID NO.7和SEQ ID NO.11所示;所述VL的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ IDNO.15、SEQ ID NO.19和SEQ ID NO.23所示;
抗CD19人源化抗体4:所述VH的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ ID NO.4、SEQ ID NO.8和SEQ ID NO.12所示;所述VL的CDR1、CDR2和CDR3的氨基酸序列分别如SEQ IDNO.16、SEQ ID NO.20和SEQ ID NO.24所示。
2.根据权利要求1所述的一种抗CD19人源化抗体,其特征在于,所述抗CD19人源化抗体包括如下抗CD19人源化抗体的至少一种:
B5抗CD19人源化抗体:所述人源化重链可变区VH的氨基酸序列如SEQ ID NO:25所示,所述人源化轻链可变区VL的氨基酸序列如SEQ ID NO:26所示;
F5抗CD19人源化抗体:所述人源化重链可变区VH的氨基酸序列如SEQ ID NO:27所示,所述人源化轻链可变区VL的氨基酸序列如SEQ ID NO:28所示;
C1抗CD19人源化抗体:所述人源化重链可变区VH的氨基酸序列如SEQ ID NO:29所示,所述人源化轻链可变区VL的氨基酸序列如SEQ ID NO:30所示;
D12抗CD19人源化抗体:所述人源化重链可变区VH的氨基酸序列如SEQ ID NO:31所示,所述人源化轻链可变区VL的氨基酸序列如SEQ ID NO:32所示。
3.根据权利要求1所述的一种抗CD19人源化抗体,其特征在于,所述抗CD19人源化抗体还包括:所述抗CD19人源化抗体的N端和/或C端连接标签得到的抗体。
4.一种编码权利要求1-3任一所述抗CD19人源化抗体的核酸分子。
5.根据权利要求4所述的核酸分子,其特征在于,所述核酸分子包括:
核酸分子1:编码重链可变区的核酸序列如SEQ ID NO:33所示,编码轻链可变区的核酸序列为SEQ ID NO:34所示;
核酸分子2:编码重链可变区的核酸序列如SEQ ID NO:35所示,编码轻链可变区的核酸序列为SEQ ID NO:36所示;
核酸分子3:编码重链可变区的核酸序列如SEQ ID NO:37所示,编码轻链可变区的核酸序列为SEQ ID NO:38所示;
核酸分子4:编码重链可变区的核酸序列如SEQ ID NO:39所示,编码轻链可变区的核酸序列为SEQ ID NO:40所示。
6.一种含有权利要求4-5任一所述核酸分子的生物材料,其特征在于,所述生物材料包括重组DNA、质粒载体、噬菌体载体、病毒载体、工程菌或转基因细胞系。
7.具有权利要求1-3任一项所述抗CD19人源化抗体的细胞或偶联物,其特征在于,所述细胞包括嵌合抗原受体T细胞、嵌合抗原受体NK细胞、和经人工编辑的细胞;所述偶联物包括抗CD19人源化抗体-细菌毒素偶联物、抗CD19人源化抗体-细菌毒素变体偶联物、抗CD19人源化抗体-细胞因子偶联物、以及抗CD19人源化抗体-化疗药物偶联物。
8.权利要求1-3任一所述的抗人CD19的人源化抗体在制备肿瘤抑制剂或肿瘤细胞抑制剂中的应用。
9.一种权利要求1-3任一所述的抗人CD19的人源化抗体的制备方法,其特征在于,所述方法包括:
获得cDNA,用人源抗体库引物对所述cDNA进行扩增,获得权利要求1-3任一所述的人源化重链可变区VH和人源化轻链可变区VL;
合成抗体CDR3和FR4;
将所述人源化重链可变区VH的可变区V基因与所述CDR3和所述FR4融合,获得重链基因片段;
将所述人源化轻链可变区VL的可变区V基因与所述CDR3和所述FR4融合,获得轻链基因片段;
将所述重链基因片段和所述轻链基因片段通过linker连接,后转染进噬菌粒,获得以噬菌粒的形式保存在宿主菌中的scfv抗体库;
利用CD19蛋白在所述抗体库中淘选,经筛选后,获得所述抗人CD19的人源化抗体。
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Denomination of invention: Anti CD19 humanized antibody and its preparation method and application Granted publication date: 20220701 Pledgee: Guanggu Branch of Wuhan Rural Commercial Bank Co.,Ltd. Pledgor: CUSABIO BIOTECH Co.,Ltd. Registration number: Y2024980009781 |