CN117586407A - 靶向人磷脂酰肌醇蛋白聚糖3的单域抗体及融合蛋白 - Google Patents
靶向人磷脂酰肌醇蛋白聚糖3的单域抗体及融合蛋白 Download PDFInfo
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Abstract
本发明提供了靶向人磷脂酰肌醇蛋白聚糖3的单域抗体及融合蛋白。所述融合蛋白包括单域抗体RYPE247或其人源化抗体;所述的单域抗体RYPE247重链互补决定区CDR1序列为SEQ ID NO.1,CDR2序列为SEQ ID NO.2,CDR3序列为SEQ ID NO.3。本发明提供的单域抗体构建的融合蛋白具有高亲和力、细胞结合强、内吞效果好的特点,对于肝癌的治疗具有较好的应用前景。
Description
技术领域
本发明属于抗体领域,具体涉及靶向人磷脂酰肌醇蛋白聚糖3的单域抗体及其融合蛋白。
背景技术
人磷脂酰肌醇蛋白聚糖3(Glypican3,GPC3)是一种膜性硫酸乙酰肝素糖蛋白,GPC3在细胞生长、分化和迁移过程中发挥重要的作用,其基因在不同的组织中表达差异很大,在肝细胞癌(HCC)组织中高表达,而在正常组织中不表达或低表达,可作为潜在HCC治疗靶点和早期诊断的生物标记物。近年来针对该靶点治疗性抗体类药物的开发研究越来越多。
如中国专利202010826784.9中公开的能够结合于磷脂酰肌醇蛋白聚糖3的特定区域的抗体及其人源化抗体;中国专利201810582099.9则提供了抗磷脂酰肌醇聚糖蛋白3的抗体药物偶联体,所采用的通过一类新型双取代马来酰亚胺连接子将强细胞毒活性物质和生物大分子进行偶联;中国专利201610290837.3以高亲和力结合GPC3或GPC3上硫酸类肝素(HS)链的人单克隆抗体的鉴定;PCT/CN2014/076913公开了编码GPC3嵌合抗原受体蛋白的核酸及表达GPC3嵌合抗原受体蛋白的T淋巴细胞。
但现有技术中对于GPC3抗体的研究尚存在不足:
Roche开发识别GPC3的C端多肽表位的人源化抗体GC33,主要通过结合肝癌肿瘤组织后由Fc端介导ADCC效应来发挥杀伤肿瘤细胞功能,但是在二期临床研究中没有达到预设终点,临床研究失败;
NCI开发的识别GPC3的C端多肽表位的人源化抗体YP7,以及识别GPC-3的N端空间表位的人单克隆单域抗体HN3都处于临床前研究阶段,但这两抗体的细胞结合相对较弱,具体药效还有待考究;
在CAR-T方面,科济生物医药(上海)有限公司有多款靶向GPC-3的CAR-T设计,其中CT011处在临床一期,但CAR-T技术治疗过程中常发生的细胞因子风暴等副作用和价格相对高昂。
为了使GPC3抗体更好地应用于肝癌治疗,开发新的抗体类型十分必要。
发明内容
本发明所涉及的氨基酸序列为结合人磷脂酰肌醇蛋白聚糖3的人源化重链免疫球蛋白单可变结构域(单域抗体,single-domain antibody,SdAb)。构建中依据GPC-3蛋白的自然特性:体内会被furin酶切割产生N端40kd可溶性部分以及30kd部分通过GPI分子锚定于细胞膜的C端。
一方面,本发明提供了一种融合蛋白。
所述的融合蛋白中包括单域抗体RYPE247或其人源化抗体。
所述的单域抗体RYPE247重链互补决定区CDR1序列为SEQ ID NO.1,CDR2序列为SEQ ID NO.2,CDR3序列为SEQ ID NO.3。
所述的融合蛋白中还包括单域抗体RYPE246或其人源化抗体。
所述的单域抗体RYPE246重链互补决定区CDR1序列为SEQ ID NO.4,CDR2序列为SEQ ID NO.5,CDR3序列为SEQ ID NO.6。
优选地,所述的单域抗体RYPE246人源化抗体重链互补决定区CDR1序列为SEQ IDNO.7,CDR2序列为SEQ ID NO.8,CDR3序列为SEQ ID NO.9。
优选地,所述的单域抗体RYPE247的重链可变区氨基酸序列为SEQ ID NO.10。
优选地,所述的单域抗体RYPE246的重链可变区氨基酸序列为SEQ ID NO.11。
优选地,所述的单域抗体RYPE246人源化抗体的重链可变区氨基酸序列为SEQ IDNO.12。
具体地,所述的融合蛋白还包括人IGg的Fc段;优选为人IGg1的Fc段。
优选地,所述的融合蛋白中还包括SEQ ID NO.16-20中的一种或多种。
另一方面,本发明提供了一种单域抗体RYPE247。
所述的单域抗体重链互补决定区CDR1序列为SEQ ID NO.1,CDR2序列为SEQ IDNO.2,CDR3序列为SEQ ID NO.3。
优选地,所述的单域抗体的重链可变区氨基酸序列为SEQ ID NO.10。
再一方面,本发明还提供了一种单域抗体RYPE246。
所述的单域抗体重链互补决定区CDR1序列为SEQ ID NO.4,CDR2序列为SEQ IDNO.5,CDR3序列为SEQ ID NO.6。
优选地,所述的单域抗体的重链可变区氨基酸序列为SEQ ID NO.11。
又一方面,本发明提供了前述单域抗体的人源化抗体。
优选地,所述的人源化抗体为单域抗体RYPE246的人源化抗体。
进一步优选地,所述的人源化抗体重链互补决定区CDR1序列为SEQ ID NO.7,CDR2序列为SEQ ID NO.8,CDR3序列为SEQ ID NO.9。
更进一步地,所述的人源化抗体重链可变区为SEQ ID NO.12。
又一方面,本发明提供了一种核酸分子。
所述的核酸分子用于表达权利前述的单域抗体,或其人源化抗体,或融合蛋白。
优选地,所述的核酸分子核苷酸序列包括SEQ ID NO.13-15中的一种或多种。
又一方面,本发明提供了一种表达载体。
所述的表达载体上包括前述的核酸分子。
优选地,所述的表达载体骨架为pcDNA3.1。
又一方面,本发明提供了一种基因工程细胞。
所述的基因工程细胞中包括前述的表达载体。
优选地,所述的基因工程细胞为HEK293或CHO。
又一方面,本发明提供了前述的单域抗体和/或人源化抗体和/或融合蛋白和/或核酸分子和/或表达载体和/或基因工程细胞在制备肝癌药物中应用。
又一方面,本发明提供了一种治疗肝癌的药物。
所述的药物中包括前述的单域抗体和/或人源化抗体和/或融合蛋白和/或核酸分子和/或表达载体和/或基因工程细胞。
所述的药物中还包括其他药学上可接受的载体或赋形剂。
本发明的有益效果:
本发明通过免疫羊驼,筛选获得具有高亲和力、细胞结合强、内吞效果好的单域抗体;并通过融合Fc或串联等技术构建双抗,这样的结构赋予抗体更优化的结构及功能上的特性,包括:更好的亲和力,更好的细胞结合,更好的稳定性,更少的聚体存在,更容易评价其生物性功能等等。
附图说明
图1为实施例中构建的融合蛋白的结构。
图2为SDS-PAGE分析蛋白纯度的结果。
图3为融合蛋白310-311的SEC-HPLC检测结果。
图4为融合蛋白319的SEC-HPLC检测结果。
图5为融合蛋白458的SEC-HPLC检测结果。
图6为融合蛋白483-484的SEC-HPLC检测结果。
图7为融合蛋白493-484的SEC-HPLC检测结果。
图8为抗HepG2抗体的FACS结合结果。
图9为流式细胞仪检测HepG2细胞的内吞试验结果。
具体实施方式
下面结合具体实施例,对本发明作进一步详细的阐述,下述实施例不用于限制本发明,仅用于说明本发明。以下实施例中所使用的实验方法如无特殊说明,实施例中未注明具体条件的实验方法,通常按照常规条件,下述实施例中所使用的材料、试剂等,如无特殊说明,均可从商业途径得到。
基础实验例
(1)抗人GPC-3蛋白单域抗体文库的构建
将人GPC-3蛋白与弗氏佐剂混合后免疫两只美洲驼,每两周一次共免疫四次。免疫结束后提取骆驼外周血细胞分离淋巴细胞进行单域抗体片段的提取分离。单域抗体片段提取分离的步骤为:提取总RNA,逆转录合成cDNA后利用巢式PCR扩增单域抗体片段,限制性内切酶酶切后连接进入噬菌体展示载体,电转进入感受态细胞。成功构建了两个独立的抗人GPC-3蛋白的单域抗体噬菌体展示文库。通过梯度稀释文库涂于平板上计算单克隆数以测定库容,所得两个文库的大小均为>108。从每个文库中随机挑取30个单克隆进行菌落PCR检测,所建文库空载率均为0。
(2)抗人GPC-3蛋白的单域抗体筛选过程
将人GPC-3蛋白偶联在酶标板上包被过夜,封闭液封闭后加入前步构建的噬菌体文库,PBST多次洗涤后用TEA洗脱液将与人GPC-3蛋白特异结合的噬菌体解离,并用来感染对数生长期的大肠杆菌细胞,扩大培养噬菌体用于下一轮筛选。每个文库经过三轮Biopanning富集的特异性VHH抗体,达到了利用噬菌体展示技术筛取抗体库中结合人GPC-3蛋白特异性抗体的目的。
(3)用噬菌体的酶联免疫方法(ELISA)筛选特异性单个阳性克隆
从两文库中随机选取60个单菌落接种培养,生长至对数期后,IPTG诱导表达,离心收集菌体后利用渗透冲击法从周质中获得粗提抗体,加入已包被人GPC-3蛋白的酶标板中孵育,PBST洗板后分别以mouse Anti-HA tag抗体为一抗,羊抗小鼠碱性磷酸酶标记抗体(goat anti-mouse alkaline phosphatase conjugate)为二抗结合,加入碱性磷酸酶显色并读取吸光度值,将OD值大于对照孔3倍以上的样品孔判定为阳性对照孔。将所有阳性克隆培养,提取质粒并进行测序,获得多个单域抗体序列,其中一条命名为RYPE247,其氨基酸序列如SEQ ID NO.10所示;另一条命名为RYPE246,其氨基酸序列如SEQ ID NO.11所示。
采用单域抗体人源化通用框架移植法,对RYPE246单域抗体进行人源化,获得多个RYPE246单域抗体的人源化变体,其中一株命名为RYPE246-33,其氨基酸序列如SEQ IDNO.12所示。
实施例1靶向人磷脂酰肌醇蛋白聚糖3融合蛋白的制备方法
RYPE246、RYPE246-33、RYPE247分别与IgG1 Fc段进行融合,然后克隆到pcDNA3.1表达载体酶切位点NheI和EcoRI,RYPE247、RYPE246、RYPE246-33对应核苷酸序列为SEQ IDNO.13-15,融合蛋白的构建计划如图1所示,其中319、320、458、459所示的融合蛋白各部分核苷酸序列亚克隆至表达载体进行转染表达;其他融合蛋白是分别将Fc knob侧和Fc hole侧亚克隆至不同表达载体,然后共转染表达。
图1中:
sFc的序列为SEQ ID NO.16;
Fc knob的序列为SEQ ID NO.17;
Fc hole的序列为SEQ ID NO.18;
G4S linker的序列为SEQ ID NO.19;
IgG铰链区的序列为SEQ ID NO.20。
构建成功的载体分别转染哺乳动物细胞(如:HEK293、CHO等),继续培养表达一定时间(7到10天),离心收集上清。通过protein A亲和层析(以20mMPB,150mM NaCl,pH7.2缓冲液平衡Mabselect Sure Lx自装柱;然后进行上样,上样结束后以20mM PB,1M NaCl,pH6.5进行洗杂;再以20mM Cit-Na3Cit,pH3.5进行洗脱,并用1M Tris pH9.0调节样品pH至6.5),阴、阳离子或/和疏水层析方法进行精细纯化(以阳离子为例:以buffer A:20mMPB,pH6.5平衡Capto S impact自装柱;然后进行上样,上样结束后;以0-30%bufferB:20mMPB,1M NaCl,pH6.5,20CV进行洗脱,并收集洗脱样品)。
实施例2融合蛋白检测分析
实施例1得到的纯化产物采用SDS-PAGE 4%-12%梯度胶分析蛋白的纯度;结果如图2。
SEC-HPLC检测结果如下:
分子名称 | 聚体% | 主峰% | 碎片% |
310-311 | 2.176 | 95.77 | 2.054 |
319 | 8.672 | 89.143 | 2.185 |
320 | 7.742 | 90.181 | 2.076 |
458 | 2.613 | 94.797 | 2.59 |
459 | 3.046 | 94.527 | 2.427 |
477-478 | 3.014 | 95.013 | 1.973 |
479-480 | 2.957 | 94.077 | 2.967 |
481-482 | 5.649 | 91.235 | 3.116 |
483-484 | 10.366 | 87.726 | 1.908 |
485-486 | 7.973 | 89.089 | 2.937 |
487-488 | 9.08 | 88.135 | 2.785 |
473-474 | 5.802 | 92.566 | 1.632 |
475-476 | 8.089 | 90.67 | 1.241 |
493-484 | 12.074 | 85.473 | 2.454 |
494-486 | 5.663 | 91.614 | 2.723 |
图3-7分别为融合蛋白310-311、319、458、483-484、493-484的SEC-HPLC检测结果图片。
实施例3使用表面等离振子共振测定亲和性及种属交叉
使用在Biacore8K仪器上的表面等离振子共振,进行抗GPC-3单域抗体融合构建体与人和食蟹猴GPC-3的动力学分析。其中人GPC-3购自ACRO,货号:GP3-H52H4;食蟹猴GPC-3购自ACRO,货号:GP3-C5225。
以HBS-EP+(购自GE,货号为BR100669)为实验缓冲液进行,在25℃条件下进行,将配体捕获到Anti-His Antibody芯片(购自GE,货号为28995056)表面,待芯片表面稳定之后,将梯度稀释的分析物以一定流速流经固定了配体的芯片表面。以实验缓冲液为空白对照,并且设定一个重复浓度。结果如下:
实施例4细胞结合实验
1、将待测样品(融合蛋白)稀释至终浓度为100nM,由于用量大于150μL,以不小于200μL去稀释。用1%BSA梯度(2.5倍)稀释。
2、培养用的细胞(HepG2,中国科学院细胞库,SCSP-510)离心(230g 5min)后去上清,用DPBS冲洗细胞。加入2-3mL StemPro Accutase Cell dissociation reagent(购自Gibco,货号为A1110501)或者0.25%胰酶(购自Gibco,货号为25200-056)来分散细胞,显微镜下观察细胞呈分散状态即可。再加入6-8mLMEM完全培养基(购自Gibco,货号为11090081)将细胞吹打混匀计数。
3、将细胞离心(RT 1000rpm 5min),通过细胞计数仪进行计数,用1%BSA调整细胞密度至3×106cells/mL。
4、在96孔圆底板内加入稀释好的细胞,每孔100μL。
5、将96孔圆底板离心(4℃,1500rpm,5min)去除上清,再加100μL/well预先稀释好的样品,然后孵育(4℃1h)。
6、将96孔圆底板离心(4℃,1500rpm,5min)去除上清,用1%BSA悬浮细胞再离心(4℃,1500rpm,5min)进行清洗,清洗2次。
7、用1%BSA将二抗抗体进行200倍稀释至5μg/mL,计算好所需量。
8、再加入100g/m/well预先稀释的二抗抗体FITC,再孵育(4℃,0.5h)。注意此步骤中BlanK是不需要加二抗的。
实施例5细胞内吞实验
收集HepG2细胞,每个样品加入2×105个细胞;在4℃条件下,2000rpm离心5分钟去上清;每孔加入目标抗体(100nM,100μL),2-8℃孵育1小时后,离心去除未结合的抗体;每孔加入100μL 1%BSA,分别于4℃和37℃孵育0、1、2和4小时;1:1000加入FACS二抗(Goat antihuIgG购自Abcam,货号为Ab97224),避光冰上孵育30分钟;离心去除上清,以4%PFA固定细胞;离心去除上清,每孔加入100μL 1%BSA重悬细胞,用流式细胞仪进行分析。
抗HepG2抗体的FACS结合结果如图8。
将96孔圆底板离心(RT 1500rpm 5min)去除上清,用1%BSA清洗3次(此过程仍然是避光操作),再将细胞重悬在100μL 1%BSA中,再通过FACS检测。
结果见图9和下表:
实施例6热稳定性检测
对实施例1获得的融合蛋白进行热稳定性测试,具体方法如下:
目标蛋白经离心浓缩至一定浓度。上样UNCLE设备进行TM、Tonset、Tagging266和粒径分布检测。
结果如下:
Claims (20)
1.一种融合蛋白,其特征在于,包括单域抗体RYPE247或其人源化抗体;所述的单域抗体RYPE247重链互补决定区CDR1序列为SEQ ID NO.1,CDR2序列为SEQ ID NO.2,CDR3序列为SEQ ID NO.3。
2.根据权利要求1所述的融合蛋白,其特征在于,还包括单域抗体RYPE246或其人源化抗体;所述的单域抗体RYPE246重链互补决定区CDR1序列为SEQ ID NO.4,CDR2序列为SEQID NO.5,CDR3序列为SEQ ID NO.6。
3.根据权利要求1所述的融合蛋白,其特征在于,所述的单域抗体RYPE246人源化抗体重链互补决定区CDR1序列为SEQ ID NO.7,CDR2序列为SEQ ID NO.8,CDR3序列为SEQ IDNO.9。
4.根据权利要求1所述的融合蛋白,其特征在于,所述的单域抗体RYPE247的重链可变区氨基酸序列为SEQ ID NO.10。
5.根据权利要求2所述的融合蛋白,其特征在于,所述的单域抗体RYPE246的重链可变区氨基酸序列为SEQ ID NO.11。
6.根据权利要求3所述的融合蛋白,其特征在于,所述的单域抗体RYPE246人源化抗体重链可变区为SEQ ID NO.12。
7.根据权利要求1-6任一项所述的融合蛋白,其特征在于,还包括人IGg的Fc段。
8.根据权利要求7所述的融合蛋白,其特征在于,包括人IGg1的Fc段。
9.一种单域抗体,其特征在于,重链互补决定区CDR1序列为SEQ ID NO.1,CDR2序列为SEQ ID NO.2,CDR3序列为SEQ ID NO.3。
10.一种单域抗体,其特征在于,重链互补决定区CDR1序列为SEQ ID NO.4,CDR2序列为SEQ ID NO.5,CDR3序列为SEQ ID NO.6。
11.权利要求9或10所述的单域抗体的人源化抗体。
12.根据权利要求11所述的人源化抗体,其特征在于,重链互补决定区CDR1序列为SEQID NO.7,CDR2序列为SEQ ID NO.8,CDR3序列为SEQ ID NO.9。
13.一种核酸分子,其特征在于,用于表达权利要求1-8任一项所述的融合蛋白或者权利要求9-12任一项所述的单域抗体。
14.根据权利要求13所述的核酸分子,其特征在于,核苷酸序列包括SEQ ID NO.13-15。
15.根据权利要求8所述的核酸分子,其特征在于,核苷酸序列还包括SEQ ID NO.16。
16.一种表达载体,其特征在于,包括权利要求13-15任一项所述的核酸分子。
17.一种基因工程细胞,其特征在于,包括权利要求16所述的表达载体。
18.根据权利要求17所述的基因工程细胞,其特征在于,为HEK293或CHO。
19.权利要求1-8任一项所述的融合蛋白和/或权利要求9-10任一项所述的单域抗体和/或权利要求11-12任一项所述的人源化抗体和/或权利要求13-15所述的核酸分子和/或权利要求16所述的表达载体和/或权利要求17所述的基因工程细胞在制备肝癌药物中应用。
20.一种治疗肝癌的药物,其特征在于,包括权利要求1-8任一项所述的融合蛋白和/或权利要求9-10任一项所述的单域抗体和/或权利要求11-12任一项所述的人源化抗体和/或权利要求13-15所述的核酸分子和/或权利要求16所述的表达载体和/或权利要求17所述的基因工程细胞。
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