CN111087465A - 一种针对密蛋白6的抗体偶联药物及应用 - Google Patents
一种针对密蛋白6的抗体偶联药物及应用 Download PDFInfo
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- CN111087465A CN111087465A CN201911360909.7A CN201911360909A CN111087465A CN 111087465 A CN111087465 A CN 111087465A CN 201911360909 A CN201911360909 A CN 201911360909A CN 111087465 A CN111087465 A CN 111087465A
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- C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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Abstract
本发明公开了一种针对密蛋白6的抗体偶联药物及应用,本发明涉及用于制备密蛋白6(CLDN6)抗体的抗原序列,其对应的核苷酸序列以及用于表达所述核苷酸序列的基因工程载体、工程菌或细胞系;通过所述抗原多肽进行动物免疫,可产生具有高特异性密蛋白6抗体;进一步通过免疫动物细胞制备杂交瘤细胞可得到对应的单克隆抗体;将所述抗密蛋白6的抗体与连接结构、药物结合,可组成抗体偶联药物;所述抗体偶联药物可根据密蛋白6的靶向作用,将药物运载至密蛋白6表达阳性的肿瘤细胞中,从而达到特异杀伤肿瘤细胞的功效,对肿瘤的治疗具有重要的意义。
Description
技术领域
本发明属于生物医药领域,涉及一种抗原及其对应的抗体应用,具体涉及 针对密蛋白6的抗体偶联药物及应用。
背景技术
20世纪原发性肝癌已上升为我国第二位癌症杀手,全世界每年就有100余 万患者死与肝癌,而我国肝癌的发病率占全世界的40%。肝癌的治疗以手术为 主,但临床上90%的肝癌患者在被诊断为肝癌的时候,已经失去手术机会。而 且,肝切除术后5年内复发率高达80%,长期存活率仍然不高。其他肝癌治疗 方法的例子包括组织移植、***化疗、放疗和电灼疗法。但是这些手段都表现 出高复发率,且引起严重的副作用,如移植排斥反应。即便是成功的切除手术, 也有25%的复发率。
随着生物技术的迅猛发展及人类基因组计划(HGP)全基因组测序的完成,人 类进入了后基因组时代,其中蛋白质组学的研究越来越受到人们的关注。用蛋 白质组学技术来检测、分析和确定标志蛋白和靶蛋白,阐明肿瘤蛋白表达水平 的变化与肿瘤发生发展不同阶段的相互关系及其规律,是目前解决肿瘤早期诊 断难题的最有利的途径;同时,也是肿瘤及其药物筛选研究中最新并且最强有 力的手段。
近年来,利用组织特异性基因启动子建立的基因治疗方法,为肿瘤靶向性 治疗开辟了新的思路。基因的表达受其启动子的调控,启动子的转录活性与基 因转录水平直接相关。某些启动子具有严格的组织特异性,即特定的启动子仅 在特定的组织中存在活性。因此,某些基因仅表达于特定组织。利用肿瘤组织 特异性基因的启动子,可以靶向性地在肿瘤细胞中表达某些抗肿瘤基因,从而 实现针对肿瘤细胞的干预,减轻甚至避免对正常细胞的影响。
发明内容
针对上述问题,本发明的目的是提供用于肝癌的、靶向性高、疗效好的抗 体偶联药物。
一种抗原多肽,所述多肽的氨基酸序列为:
(1a)如SEQ ID NO.1所示的氨基酸序列;或者
(1b)与SEQID NO.1具有90%同一性且具有相同抗原功能的氨基酸序列。
经过研究发现,密蛋白6(CLDN6)在肝细胞癌病人的早期癌变的癌邻近 组织及癌组织中都有较高的表达,且其在这些组织中的表达阳性是特异的。因 此,密蛋白6可作为肿瘤治疗的靶点。
本发明的抗原多肽为对应密蛋白6的多肽肽段通过一系列生物信息学的分 析与比对得到,其评分最高,与其他紧密连接结构相关蛋白交叉性最小;对应 密蛋白6蛋白序列的第27位到第38位氨基酸。
可在序列的末端添加相应的连接结构。
优选地,用于连接KIH载体时,在序列末端添加氨基酸C。
本发明还要求保护所述抗原多肽的编码基因,所述抗原多肽的编码基因为 编码如SEQ ID NO.1所示氨基酸序列的核苷酸序列。
进一步地,本发明还要求保护将所述抗原多肽的编码基因通过生物工程的 方法可制备得到的相应的表达载体、工程菌或细胞系。
本发明还要求保护所述抗原多肽、编码基因,以及含所述编码基因的表达 载体、工程菌或细胞系在制备抗密蛋白6的抗体中的用途。
本发明还要求保护通过本发明所述抗原多肽制备得到的抗密蛋白6的抗体。
通过本领域一般的技术方法,将所述抗原多肽对进行动物免疫注射,可制 得相应的具有抗密蛋白6的抗体,或通过免疫动物制备杂交瘤细胞后再制备抗 体。
本发明还提供了一株用于表达所述抗密蛋白6抗体的杂交瘤细胞,小鼠杂 交瘤单克隆细胞C6-e11,所述杂交瘤细胞通过所述抗原多肽制得,于2019年11 月12日保藏于中国典型培养物保藏中心,保藏编号为CCTCC NO.C2019282, 地址:中国.武汉.武汉大学,湖北省武汉市武昌区八一路珞珈山。
通过将所述抗原多肽免疫动物,并分离免疫动物的骨髓瘤细胞进行细胞融 合,进一步挑选其中高效价的克隆即得到所述杂交瘤细胞。
本发明还要求保护由所述杂交瘤细胞系产生的抗密蛋白6的单克隆抗体。
本发明还提供了一种抗体偶联药物,所述抗体偶联药物包括抗密蛋白6抗 体、连接结构和药物,所述连接结构分别连接所述抗密蛋白6抗体和药物。
作为本发明的优选实施方式,所述连接结构为SMCC,所述药物为美登素。
SMCC是一类含有N-羟基琥珀酰亚胺(NHS)活性酯和马来酰亚胺的双功能 偶联剂,可以将分别含有巯基和氨基的化合物键接在一起。NHS活性酯与伯胺 (-NH2)在pH7-9的环境形成酰胺键,马来酰胺与巯基(-SH)在pH6.5-7.5 的环境下形成稳定的硫醚键。在水溶液中,NHS活性酯的水解与氨基的反应为 竞争反应。马来酰胺比NHS稳定,但是当pH大于7.5时,马来酰胺会慢慢水 解,失去与巯基反应的特异性。因而,使用SMCC时通常在pH7.2-7.5的环境下 进行,并且先让NHS发生反应。
SMCC的分子量极小,通过化学键连接,效率高,且能够很好的与药物分 子分离,且其连接条件温和(pH=7.0左右),亦不会影响抗体活性。SMCC结构 里的环己烷环可以降低马来酰胺的水解速率,使得蛋白质在用SMCC修饰之后 可以冻干存放一段时间。
由于SMCC连接子要求药物需要有巯基基团,因此需要选择具有巯基键的 药物。美登素作为一种高效、低毒、安全性大的抗癌药物,可与SMCC连接, 研究证明其对实体瘤,卵巢癌、乳腺癌、鼻咽癌、骨癌哈淋巴癌等具有强烈的 抗肿瘤作用,临床效果亦已得到验证。
本发明还要求保护所述抗原多肽,所述抗原多肽的编码基因,含有所述编 码基因的表达载体、工程菌或细胞系,所述单克隆细胞系,所述抗体以及所述 抗体偶联药物在制备治疗密蛋白6表达阳性的肿瘤的药物或药物组合物中的应 用。
进一步地,所述密蛋白6表达阳性的肿瘤为肝脏肿瘤。
本发明还要求保护包括所述的抗体偶联药物的药物组合物。
作为本发明的优选实施方式,所述药物组合物还包括索拉菲尼。
新一代的抗体偶联药物是一个热门的研究开发领域。抗体偶联药物(ADC) 由抗体区、连接区与细胞毒性药物区三个重要组分组成,由肿瘤特异性表达的 靶向标记物(如膜蛋白)或抗体在体内追踪靶标并附着于癌细胞的表面。抗体 部分与靶标蛋白结合触发信号,连接区断裂,药物内吞对肿瘤细胞发挥特异性 杀伤作用。由于肝癌细胞中密蛋白6特异性表达,因此密蛋白6可作为良好的 药物靶标,密蛋白6抗原及抗体本身可诱发免疫反应,含有密蛋白6的抗体偶 联药物可将药物导向密蛋白6阳性表达的肿瘤细胞中,特异性地在发挥作用, 而不影响正常细胞,减少副作用。本发明为***,特别是密蛋白6阳性表 达的肿瘤提供了新的思路,具有重要意义。
附图说明
图1为DM1、Ab、Ab-DM1的紫外吸收光谱。
图2为DM1、Ab、Ab-DM1的红外吸收光谱。
图3为抗体偶联药物在不同肝癌细胞系中的抗原表位结合作用。
图4为单克隆抗体及其制备出的抗体偶联药物在肝癌细胞系中的内吞作用 结果。
图5为抗体偶联药物对肝癌细胞系HepG2的杀伤作用。
图6为抗体偶联药物对肝癌细胞系PLC-8024的杀伤作用。
图7为裸鼠皮下成瘤体内实验同型对照偶联药物组与抗体偶联药物组的体 重对比。
图8为裸鼠皮下成瘤体内实验同型对照偶联药物索拉菲尼联用组与抗体偶 联药物索拉菲尼联用组的体重对比。
图9为裸鼠皮下成瘤体内实验同型对照偶联药物组与抗体偶联药物组的肿 瘤生长对比。
图10为裸鼠皮下成瘤体内实验同型对照偶联药物索拉菲尼联用组与抗体偶 联药物索拉菲尼联用组的肿瘤生长对比。
图11为裸鼠皮下成瘤体内实验同型对照偶联药物组与抗体偶联药物组的肿 瘤大小对比。
图12为裸鼠皮下成瘤体内实验同型对照偶联药物索拉菲尼联用组与抗体偶 联药物索拉菲尼联用组的肿瘤大小对比。
图13为五个肝癌病人来源的原代类器官的药敏试验结果。
图14为五个肝癌病人的癌组织中CLDN6的表达升高倍数与其对ADC药物 的敏感性(IC50)的相关性。
具体实施方式
为更好的说明本发明的目的、技术方案和优点,下面将结合附图和具体实 施例对本发明作进一步说明。
实施例1抗原序列及其制备
通过生物信息学的分析与比对,筛选出一段评分最高、与其他紧密连接结 构相关蛋白交叉性最小的多肽序列:
SEQ ID NO.1
LPMWKVTAFIGN
所述多肽序列对应密蛋白6蛋白序列的第27位到第38位氨基酸。
为了便于接入相应的载体KIH,在上述序列末端添加连接结构C,进行纯 度达到90%的多肽片段的如下肽段合成,序列为:
LPMWKVTAFIGNC。
实施例2抗体制备
将合成的多肽序列与载体蛋白(如KLH)进行偶联,并对3只BALB/c小 鼠并通过标准程序加强免疫。选取免疫应答最佳的小鼠,取脾细胞与骨髓瘤细 胞进行细胞融合制备杂交瘤细胞。
得到的杂交瘤细胞通过增殖和ELISA筛选得到高效价的克隆;并通过有限 稀释法对2~8个阳性母细胞进行亚克隆,最后确定一株阳性克隆做亚型鉴定, 所述阳性克隆细胞的保藏编号为CCTCC NO.C2019282,保藏日期为2019年11 月12日。
将该阳性克隆细胞扩大培养后分别对5只小鼠进行腹水生产,并通过蛋白G 纯化后得出对应的单克隆抗体。
实施例3抗体偶联药物的制备和鉴定
1.主要材料
抗体:实施例2制备得到的单克隆抗体(下简称Ab);
连接结构:SMCC,购于AAT Bioquest公司(USA);
药物:美登素(下简称DM1)。
2.制备方法
1)将SMCC溶解于DMSO中,4℃保存;
2)将Ab与SMCC在共轭缓冲液(50mm磷酸钾,50mm氯化钠,2mm EDTA, pH 7.2)中以1:10的摩尔比混合,在4℃下搅拌反应4~6h;
3)反应后将混合液置于透析袋中(M=5000),在4℃下透析三天;
4)收集透析液,真空冷冻干燥机冷冻干燥;
5)用BCA对蛋白样品进行定量分析;
6)修饰后抗体与DM1分子以摩尔比为1:10在PBS缓冲液(pH=7.0)中混 合,在4℃下搅拌反应4~6h;
7)将混合液加入透析袋中(M=5000),在4℃下透析三天;
8)收集透析液,真空冷冻干燥机冷冻干燥;
9)在252nm处检测抗Ab-DM1偶联物的吸光度,并通过BCA对抗体进行 定量。
3.复合物检测
1)紫外可见光吸收图谱
分别检测DM1、Ab、Ab-DM1紫外吸收光谱。检测结果如图1。
由图1可得,DM1最高吸收峰为252nm,Ab的最高吸收峰为280nm,Ab-DM1 最高吸收峰为268nm。
2)分析药物抗体比
建立DM1浓度-光度值标准曲线;根据标准曲线计算出Ab-DM1中DM1质 量。通过BCA定量得到抗体质量。根据摩尔定律n=m/M计算个分子摩尔数; 实验重复三次,并取平均值。
通过抗体与药物摩尔比值,计算得到每个抗体上约共价结合3.6个DM1。
3)红外吸收光谱
分别检测SMCC、Ab、Ab-SMCC、DM1、Ab-DM1红外吸收光谱,结果如 图2。
由图2结果显示:SMCC在1660nm、1676nm处有特异性-C=O键,1485nm 处存在-C=C-键;Ab在1006nm处存在-CNH2-键;Ab-SMCC在1632nm处-C=O 键,3375nm处特异性-NH2键;DM1在3113nm处有-NH-键。从Ab-DM1的吸 收光谱图可见各分子基团特异性的化学键,说明构建成功。
4)粒径和电位分析
分别检测Ab、Ab-SMCC、Ab-DM1粒径及Zeta电势,结果如表1。由表1 可得,Ab-SMCC粒径发生明显变化,可能是由于SMCC强极性使得与抗体间发 生聚合;Ab-SMCC的Zeta电势也发生变化,说明抗体修饰成功。
表1粒径及Zeta电位
Ab | Ab-SMCC | Ab-DM1 | |
粒径(±SD nm) | 33.12±7.61 | 338.4±9.33 | 29.89±7.55 |
Zeta电势(±SD mV) | -14.51±1.32 | -18.60±1.46 | -5.48±1.42 |
实施例4细胞实验
(一)流式细胞膜表面染色实验
通过流式细胞膜表面染色实验分别检测实施例3制备的抗体偶联药物与 HepG2细胞和PLC-8024细胞的结合作用,过程如下:
1.用0.5~1mL 1×PBS重悬细胞,将细胞分装进流式管中,离心;
2.用1×PBS重悬细胞,离心去上清液;重复本步骤两次;
3.用100μL稀释好的抗体或抗体偶联药物重悬细胞;
4.在冰上孵育15~30分钟;离心后去上清;
5.用1×PBS重悬,离心去上清,重复本步骤两次;
6.在100μL稀释的荧光染料标记的二抗中重悬细胞;
7.在冰上孵育30分钟,离心后去上清;
8.用1×PBS重悬,离心去上清,重复操作;
9.用1×PBS重悬细胞,通过流式细胞分析仪进行分析。
流式结果如图3:本发明制备的抗体偶联药物在不同肝癌细胞系中的抗原表 位结合作用有明显差异,在密蛋白6表达阳性的HepG2细胞系膜表面抗原结合 能力强,在密蛋白6表达阴性的PLC-8024细胞膜表面抗原结合能力弱。
(二)抗体偶联药物在肝癌细胞系中的内吞作用
进一步检测实施例3制备的抗体偶联药物在HepG2细胞中的内吞作用,将细胞 分成两组:CLDN6-Ab组和CLDN6-DM1组;其中,CLDN6-Ab使用实施例2 制备的单克隆抗体,CLDN6-DM1使用实施例3制备的抗体偶联药物。实验过程 如下:
1.培养HepG2细胞至细胞密度约为3X104个/孔;
2.在细胞培养液里加入终浓度为1μM单克隆抗体或抗体偶联药物;
3.分别在37℃细胞培养箱里培养1~8小时;
4.终止培养,吸除培养基,用PBS清洗两次;
5.加入甲醇溶液室温固定20min;
6.去除甲醇溶液,用PBS在室温漂洗三次,每次5min;
7.滴加二抗室温避光孵育1h;
8.PBS冲洗3次,每次5min;
9.共聚焦荧光显微镜下观察并拍摄。
结果如图4,单克隆抗体(CLDN6-Ab)及其相应的抗体偶联药物 (CLDN6-DM1)都能在与肝癌细胞系膜表面抗原结合被内吞至细胞内后进行特 异性杀伤作用。
(三)抗体偶联药物的药效作用:
分别对密蛋白6表达阳性的肝癌细胞系HepG2与密蛋白6表达阴性的肝癌 细胞系PLC-8024进行药效作用评价。分成3组,单药组:直接使用DM1,同 型对照偶联药物组:使用IgG-DM1,以及抗体偶联药物组:CLDN6-DM1,使用 实施例3制备的抗体偶联药物。
实验过程如下:
将细胞接种于96孔板中,每孔1000细胞。经过24小时的细胞培养后,用 不同浓度(0~172μM)的药物(DM1、CLDN6-DM1、IgG-DM1)分别加入到各 孔板中。药物处理72小时后测定细胞生长抑制率。
结果显示,在密蛋白6表达阳性的肝癌细胞系HepG2中(如图5),抗体偶 联药物的杀灭作用比单药更加敏感;而在密蛋白6表达阴性的肝癌细胞系PLC-8024中(如图6),抗体偶联药物基本没有发挥作用,由此可以说明此抗体 偶联药物在体外具有特异的杀伤作用。
实施例5动物实验
选择密蛋白6表达阳性的肝癌细胞系HepG2在裸鼠背侧皮下注射106个细 胞,观察成瘤体积达到1000mm3后取出瘤块,分成相同体积(1-5mm3左右)的 小瘤块接种至多只裸鼠背侧皮下,观察成瘤体积达到50-150mm3后进行给药。
荷瘤小鼠被随机分为四组(每组5只小鼠):同型对照偶联药物组(使用 lgG-DM1)、密蛋白6抗体偶联复合物组(使用CLDN6-DM1)、同型对照偶联药 物索拉菲尼联用组(使用索拉菲尼+lgG-DM1)与抗体偶联药物索拉菲尼联用组 (使用索拉菲尼+CLDN6-DM1)。
其中,索拉菲尼的给药方式为腹腔注射,给药浓度为20mg/kg;同型对照偶 联药物与抗体偶联药物给药方式为尾静脉注射,给药浓度为4mg/kg。在后面两 组(同型对照偶联药物索拉菲尼联用组与抗体偶联药物索拉菲尼联用组)的给 药实验中,一开始先给单药索拉菲尼,观察测量,待肿瘤的生长速率开始增大 时,(图12中的箭头处)再加上同型对照偶联药物或抗体偶联药物联用,给药 后观察皮下肿瘤生长情况与小鼠体重变化,一周测量两次,体重测量结果如图7、 8,肿瘤生长曲线如图9、10。体重变化结果显示各组体重无明显差异,说明各 组药物都无明显的体内毒性。
实验结束后,取各组裸鼠皮下成瘤体,结果如图11、12所示。
由图9~12所示,各组对肿瘤生长均有抑制作用,但抗体偶联药物索拉菲尼 联用组>密蛋白6抗体偶联药物组>同型对照偶联药物索拉菲尼联用组>同型对 照偶联药物组。
实施例6体外原代药物效果评价
通过收集了五例肝癌患者的癌组织进行类器官培养,培养约2周后进行传 代且在96孔培养板中铺板,铺板24小时后分别加入不同浓度的药物(同型对 照偶联药物或抗体偶联药物),72小时后分别测定细胞活性,并绘制出药效曲线, 结果如图13。
通过分别检测五例肝癌患者的密蛋白6在癌组织与对应正常肝组织中的表 达差异,并分析,结果如图14。由图14可发现:癌组织相对应正常肝组织的 CLDN6表达倍数与其药物IC50成负相关。
最后应当说明的是,以上实施例仅用以说明本发明的技术方案,而非对本 发明保护范围的限制,尽管参照较佳实施例对本发明作了详细地说明,本领域 的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换, 而不脱离本发明技术方案的实质和范围。
SEQUENCE LISTING
<110> 广州医科大学
<120> 一种针对密蛋白6的抗体偶联药物及应用
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 13
<212> PRT
<213> Homo sapiens
<400> 1
Leu Pro Met Trp Lys Val Thr Ala Phe Ile Gly Asn
1 5 10
Claims (13)
1.一种抗原多肽,其特征在于,所述多肽为:
(1a)如SEQIDNO.1所示的氨基酸序列;或者
(1b)与SEQIDNO.1具有90%同源性且具有相同抗原功能的氨基酸序列。
2.如权利要求1所述抗原多肽的编码基因,其特征在于,所述抗原多肽的编码基因为编码如SEQIDNO.1所示的核苷酸序列。
3.包含如权利要求2所述抗原多肽编码基因的表达载体、工程菌或细胞系。
4.如权利要求1所述抗原多肽、如权利要求2所述的编码基因、如权利要求3所述含有所述编码基因的表达载体、工程菌或细胞系在制备抗密蛋白6的抗体中的应用。
5.通过如权利要求1所述抗原多肽制备得到的抗密蛋白6的抗体。
6.小鼠杂交瘤单克隆细胞C6-e11,其特征在于,所述杂交瘤细胞通过如权利要求1所述抗原多肽制得,保藏编号为CCTCC NO.C2019282,保藏日期为2019年11月12日。
7.一种抗密蛋白6的单克隆抗体,其特征在于,所述抗体由如权利要求6所述的杂交瘤细胞产生。
8.一种抗体偶联药物,其特征在于,包括如权利要求5或7所述的抗密蛋白6抗体、连接结构和药物,所述连接结构分别连接所述抗密蛋白6抗体和药物。
9.如权利要求8所述抗体偶联药物,其特征在于,所述连接结构为SMCC,所述药物为美登素。
10.如权利要求1所述的抗原多肽、如权利要求2所述的编码基因、如权利要求3所述的表达载体、工程菌或细胞系、如权利要求6所述的单克隆细胞系、如权利要求5或7所述的抗体、或如权利要求8或9所述的抗体偶联药物在制备治疗密蛋白6表达阳性的肿瘤的药物或药物组合物中的应用。
11.如权利要求10所述的应用,其特征在于,所述密蛋白6表达阳性的肿瘤为肝脏肿瘤。
12.一种药物组合物,其特征在于,包括如权利要求8所述的抗体偶联药物。
13.如权利要求12所述的药物组合物,其特征在于,还包括索拉菲尼。
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WO2009028663A1 (ja) * | 2007-08-30 | 2009-03-05 | Kyowa Hakko Kirin Co., Ltd. | 抗Claudin-3抗体 |
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WO2024022512A1 (en) * | 2022-07-29 | 2024-02-01 | Nanjing Legend Biotech Co., Ltd. | Claudin-6 binding moieties and uses thereof |
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