CN114702593A - 一种抗folr1/vegf的全人双特异性抗体及其筛选方法和应用 - Google Patents
一种抗folr1/vegf的全人双特异性抗体及其筛选方法和应用 Download PDFInfo
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Abstract
本发明公开了一种抗FOLR1/VEGF的全人双特异性抗体及其筛选方法和应用。所述全人双特异性抗体由抗人FRα抗体和抗人VEGF抗体联合构建而成,其筛选方法简单,通过从卵巢癌患者来源的腹水中分选获得单个抗原特异性靶向的B细胞,最终构建了一个全新的双特异性抗体,所述抗体为来自人体的全人抗体,其免疫原性低,靶向性好,可以同时靶向FOLR1和VEGF,具有很好的肿瘤杀伤活性,能够抑制血管生成和肿瘤生成。
Description
技术领域
本发明属于免疫学和生物技术领域,具体涉及一种抗FOLR1/VEGF的全人双特异性抗体及其筛选方法和应用。
背景技术
卵巢癌是导致女性死亡率最高的一种癌症,65-75%的卵巢癌患者在确诊时已经处于晚期(III期和IV期),卵巢癌腹水是卵巢癌晚期的主要症状,卵巢癌腹水是肿瘤发生转移的表现。研究发现卵巢癌患者的腹水中含有多种类型的免疫细胞,腹水有可能是卵巢癌的Tertiary lymphoid structures(TLSs),但目前还没有研究来阐明卵巢癌患者腹水中的免疫细胞在卵巢癌转移和恶化中的作用。
FRα由FOLR1编码,是分子量38-40KD的细胞表面糖蛋白。FRα被证实在实体瘤中有广泛高表达,比如间皮瘤(72-100%),三阴性乳腺癌(35-68%),卵巢癌(76-89%),非小细胞肺癌(14-74%);而非恶性组织,只有肺部顶端支气管的上皮细胞,有一定比例的表达。FRα在恶性肿瘤的特异性表达,使之成为一个良好的抗肿瘤药物开发靶点。因而小分子药物、抗体药物、双特异性抗体、CAR-T、ADC、叶酸-细胞毒性药物偶联药物等不同形式的药物都在开发中。进展较快的如:Mirvetuximab soravtansine、Farletuzumab等均已经处于临床试验阶段。
血管内皮生长因子(vascular endothelial growth factor,VEGF)是最重要的促血管生成因子,可在体内诱导血管的新生。VEGF在肿瘤生成和湿性黄斑变性等眼科疾病发生中扮演重要的角色。在多数恶性肿瘤如结肠癌、乳腺癌、肺癌、前列腺癌、肾癌、神经胶质瘤、子宫癌、食管癌、胃癌、卵巢癌等组织中均检测到了VEGF及其受体的明显表达,且其表达水平与恶性肿瘤的发生、发展存在密切关系。VEGF是维持恶性肿瘤快速生长、促进其播散转移、预测患者生存预后的关键细胞因子。靶向VEGF的贝伐珠单抗在多重恶性肿瘤的临床治疗显示疗效,在妇科肿瘤治疗方面也已广泛应用。
与单克隆抗体不同,双特异性抗体具有同时靶向2个不同表位的能力,并能起到特殊的生物学功能,例如免疫细胞召集、受体共刺激或共抑制、多价病毒中和等。双特异性抗体根据结构左右对称性分为对称结构和不对称结构,根据IgG分子完整性分为类完整抗体和类抗体片段,以及根据抗原结合区域的数量构型分为两价、三价、四价或更多价的构型等。不同的双特异性抗体设计各有利弊,但是以临床治疗为目的的双特异性抗体设计都要解决同样的问题:第一,保证两对(或以上)不同轻链与重链的正确偶合或配对;第二,保持每个单克隆抗体各自结合域的独立性,同时结合不同表位的时候互相之间不会产生空间位阻的干扰;第三,抗体分子要易于用哺乳动物细胞进行表达,不需要复杂的蛋白修饰工艺,有较好的成药性。
构建并开发可以应用于临床治疗的双特异性抗体需要亟需解决以下几个问题:一是轻重链的正确配对,二是分子的成药性,三是要做到各个结合域不能互相干扰,最后还要注意免疫原性,尽量减少突变位点和额外的肽链,同时又能够利用现有的细胞及纯化工艺进行高效规模化生产。现在国际上比较主流的双特异性抗体平台有DVDIg(艾伯维)、CrossMab(罗氏)、Duobody(GenMab)等。
发明内容
本发明的目的在于提供一种抗FOLR1/VEGF的全人双特异性抗体,所述全人双特异性抗体免疫原性低,靶向性好。
本发明的另一目的在于提供一种全人双特异性抗体的筛选方法,其步骤简单,成功率高。
本发明的另一目的在于提供抗FOLR1/VEGF的全人双特异性抗体的应用。
为实现上述发明目的,本发明采用以下技术方案予以实现:
一种抗FOLR1/VEGF的全人双特异性抗体,所述全人双特异性抗体由抗人FRα抗体和抗人VEGF抗体联合构建而成。
进一步的,所述抗人FRα抗体含有SEQ ID No.1所示的重链可变区序列和SEQ IDNo.2所示的轻链可变区序列。
进一步的,所述抗人FRα抗体以单链抗体(scFv)形式表达,即重链可变区和轻链可变区表达在同一条多肽链中,中间以(GGGGS)3连接。
进一步的,所述抗人VEGF抗体含有SEQ ID No.6所示的重链可变区序列和SEQ IDNo.5所示的轻链可变区序列。
进一步的,所述VEGF抗体以scFv形式表达,即重链可变区和轻链可变区表达在同一条多肽链中,中间以(GGGGS)3连接。
进一步的,所述抗人FRα的抗体重链可变区由SEQ ID No.3所示的核苷酸序列编码;抗人FRα的抗体轻链可变区由SEQ ID No.4所示的核苷酸序列编码。
进一步的,所述抗人VEGF的抗体重链可变区由SEQ ID No.8所示的核苷酸序列编码;抗人VEGF的抗体轻链可变区由SEQ ID No.7所示的核苷酸序列编码。
进一步的,所述全人双特异性抗体中还含有序列如SEQ ID No.12所示的Fc突变体片段。
进一步的,所述Fc突变体片段由SEQ ID No.17所示的核苷酸序列编码。
进一步的,所述Fc突变体片段的突变位点包括S298A、T307A、E333A、K334A、E380A、N430A。
本发明还提供了所述的全人双特异性抗体的筛选方法,所述筛选方法包括以下步骤:
(1)利用卵巢癌腹水筛选、富集抗原特异性B细胞;
(2)从抗原特异性B细胞中,获取FRα抗体和VEGF抗体;
(3)利用FRα抗体和VEGF抗体建立双特异性抗体平台,得到scFv形式的FRα抗体和VEGF抗体;
(4)拼接scFv形式的FRα抗体和VEGF抗体,将Fc突变体片段Fcmu作为连接子,构建入载体,获取同时含有FRα(scFv)、Fcmu和VEGF(scFv)的质粒;
(5)将质粒转入宿主细胞,筛选得到高表达的细胞株;
(6)对高表达的细胞株培养纯化,得到同时含有FRα抗体、VEGF抗体和Fc突变体的蛋白,即为全人双特异性抗体。
具体的,所述筛选方法包括以下步骤:
(1)利采集卵巢癌患者术后腹水,经筛选、富集抗原特异性B细胞;
(2)提取抗原特异性B细胞RNA,反转录得到cDNA,经巢式PCR扩增获取FRα和VEGF目的基因片段,通过双酶切将FRα和VEGF scFv基因片段连接到PCDNA3.1载体中,获取FRα抗体和VEGF抗体;
(3)将FRα抗体和VEGF抗体进行活性鉴定,以确定FRα抗体和VEGF抗体对抗原均具有特异性的结合特性;
(4)利用FRα抗体和VEGF抗体建立双特异性抗体平台,得到scFv形式的FRα抗体和VEGF抗体,即FRαscFv和VEGF scFv;
(4)利用overlap PCR的方法拼接FRαscFv和VEGF scFv,将Fc突变体片段Fcmu作为连接子,得到FRα(scFv)-Fcmu-VEGF(scFv)蛋白,构建入PCDNA3.1载体中,经测序和双酶切后,得到pFR132-FRα(scFv)-Fcmu-VEGF(scFv)质粒;
(5)将pFR132-FRα(scFv)-Fcmu-VEGF(scFv)质粒利用电穿孔系统转进宿主细胞,得到Bis-FRα-Fcmu-VEGF,筛选高表达的细胞株;
(6)扩增高表达的细胞株,经培养纯化得到Bis-FRα-Fcmu-VEGF蛋白,即为全人双特异性抗体Bis-FRα-Fcmu-VEGF,并同时获得了高表达全人双特异性抗体Bis-FRα-Fcmu-VEGF的细胞株。
进一步的,所述FRα(scFv)-Fcmu-VEGF(scFv)蛋白中包含序列如SEQ ID No.9所示的FRα(scFv)、序列如SEQ ID No.10所示的linker1、序列如SEQ ID No.11所示的linker2、序列如SEQ ID No.12所示的Fcmu、序列如SEQ ID No.13所示的VEGF(scFv)。
进一步的,所述Fcmu的突变位点包括S298A、E333A、K334A、T307A、E380A、N430A。
本发明还提供了所述的全人双特异性抗体在制备用于抑制血管生成的药物中应用。
本发明还提供了所述的全人双特异性抗体在制备用于抗肿瘤药物中的应用。
进一步的,所述肿瘤包括肺癌、前列腺癌、乳腺癌、卵巢癌、肠癌、淋巴瘤、鼻咽癌、胃癌、肝癌、肾癌、子宫颈癌和子宫内膜癌、骨肉瘤。
本发明还提供了一种药物组合物,所述药物组合物包含抗FOLR1/VEGF的全人双特异性抗体。
本发明与现有技术相比,具有以下优点和有益效果:
1、目前靶向卵巢癌治疗的抗体均为鼠源或者是人源化的单克隆抗体,免疫原性较强,副作用大。但本发明通过从卵巢癌患者来源的腹水中分选获得单个抗原特异性靶向的B细胞,克隆获得了全人的FRα抗体和VEGF的抗体,为了提高抗体治疗的效果,构建了一个全新的双特异性抗体,该抗体为来自人体的全人抗体,其免疫原性低,靶向性好,可以同时靶向FOLR1和VEGF。
2、本发明构建的双特异性抗体的结构与当前已有的双特异性结构的相比结构简单,易于表达和开发,并且对抗体的恒定区做了特别的设计,抗肿瘤活性更强。
3、本发明中的抗体筛选方法由于是直接获得全人抗体,其筛选的周期短,获得的抗体为全人抗体。与目前由于的杂交瘤筛选,噬菌体或者是酵母展示筛选方法相比,更有技术优势。且所述双抗具有很好的肿瘤杀伤活性,能够抑制血管生成和肿瘤生成。
附图说明
图1为FOLR1抗体和VEFG抗体ELISA结果。
图2为ADCC增强型双特异性抗体平台的结构图。
图3为Bis-FRα-Fcmu-VEGF双抗的稳定细胞株筛选的蛋白表达点杂交图。
图4为Bis-FRα-Fcmu-VEGF双抗的纯化后SDS-PAGE电泳结果。
图5为Bis-FRα-Fcmu-VEGF双抗的亲和力检测结果,其中A为对FOLR1抗体亲和力检测结果;B为对VEFG抗体亲和力检测结果。
图6为Bis-FRα-Fcmu-VEGF对不同类型肿瘤细胞增殖的抑制活性结果。
具体实施方式
结合以下具体实例对本发明的技术方案作进一步详细的说明。
下述实施例中,如无特殊说明,所使用的实验方法均为常规方法,所用材料、试剂等均可从生物或化学试剂公司购买。
本发明通过从卵巢癌患者来源的腹水中分选获得单个抗原特异性靶向的B细胞,克隆获得了全人的FRα抗体和VEGF抗体,为了提高抗体治疗的效果,构建了一个全新的双特异性抗体,其中的两个特异性的抗体片段为自主筛选获得,采用了全新的连接组合方式,通过FcR亲和力增强型的Fc段为linker,提高双抗募集免疫细胞和杀伤的能力。
实施例1:FRα和VEGF的特异性B细胞的筛选和克隆
收集临床病例中已经确诊为卵巢高级别浆液性癌的患者术后腹水液体,取腹水800*g离心后收集细胞沉淀,采用RPMI-1640培养液重悬,离心后最终使用PBS缓冲液重悬细胞,此时细胞活力超过85%。采用如下步骤扩增抗原特异性B细胞。
1、CD27+记忆B细胞富集
根据EasySepTM人类记忆B细胞分离试剂盒,STEMCELL,通过免疫磁性阳性选择从纯化的B细胞中分离CD27+记忆B细胞。简而言之,CD27+记忆B细胞用结合了CD27抗体的磁珠进行标记,并使用EasySepTM磁体进行分离。纯化的CD27+B细胞在含有2%(v/v)胎牛血清(FBS)和1mM EDTA的PBS中洗脱和洗涤。使用0.4%(w/v)台盼蓝染色剂和Countess自动细胞计数器对CD27+B细胞进行计数。
2、抗原结合B细胞富集
生物素化的FRα和VEGF重组蛋白购自Sino Biological Inc。每次在B细胞富集前制备新鲜抗原/链霉亲和素M-280Dynabeads(Thermofisher)复合物。简而言之,将含有6.5×107个珠子的100μL M-280珠子涡旋30秒,并在使用前置于室温。珠子在磁架上用1mL 1×PBS洗涤两次,并在100μL 1×PBS中洗脱。将珠子(100μL)与20μg生物素化FRα和VEGF重组蛋白混合,并在室温下孵育30分钟。孵育后,在磁性架上使用500μL 1×PBS将复合物洗涤3次。洗涤后的复合物在100μL 1×PBS中洗脱,并储存在冰上直至使用。在抗原富集之前,复合物在室温下平衡。使用的珠子复合物的数量是根据与纯化B细胞数量的1:1比例计算的。将FRα和VEGF磁珠复合物直接加入B细胞混合物中,混合并在4℃下在热混合器上孵育30分钟。孵育后,将混合物置于磁力架上,去除上清液。通过总共混合四次磁体来洗涤珠子。最终的抗原富集B细胞在含有2%(v/v)胎牛血清(FBS)和1mM EDTA的1×PBS中洗脱。使用0.4%(w/v)台盼蓝染色剂和Countess自动细胞计数器对富集的B细胞进行计数。
3、单B细胞抗体序列的克隆
用Trizol(Thermo)分离B细胞的总RNA,使用反转录酶(Invitrogen)以获取cDNA。上述操在均按照厂家说明书进行。用巢式PCR的方法,通过2次PCR反应,所用扩增酶为KODplus(Thermo)确保扩增过程中减少可能的突变,扩增获得的酶切产物委托测序公司测序确认,PCR获得FRα和VEGF目的基因片段,通过双酶切将FRα和VEGFscFv基因片段连接到PCDNA3.1载体中。
PCR扩增体系:
cDNA | 1μl |
dNTP | 0.5μl |
MgCl<sub>2</sub> | 2μl |
Buffer | 2μl |
Primer F/R | 0.5μl |
KOD plus | 0.5μl |
H<sub>2</sub>O | 13μl |
总计 | 20μl |
PCR反应条件:
扩增不同类型的B细胞使用到的引物:
表1:第一轮PCR上游引物
表2:第一轮PCR下游引物
表3:第二轮PCR上游引物
表4:第二轮PCR下游引物
经过测序获得的FRα和VEGF抗体的序列如下:
FRα(VH-VL)(简称为:FRαscFv)的氨基酸序列:
EVQLVESGGGLVQPGGSLRLSCAASGFPFSNHWMNWVRQAPGKGLEWVGEIRSKSMNSATHYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARNYYGSTYDHWGQGTLVTVSS(SEQ ID No.1)
EIVLTQSPDFQSVTPKEKVTITCRASQFVGYSIHWYQQKPDQSPKLLIKYASESRSGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQSHSWHFTFGQGTKLEIK(SEQ ID No.2)
FRα(VH-VL)的核苷酸序列:
GAAATTGTGCTCACACAGTCACCAGACTTTCAGTCTGTCACCCCTAAGGAGAAAGTGACCATCACTTGCAGGGCCTCTCAGTTCGTCGGCTATAGTATCCACTGGTACCAGCAGAAACCCGATCAGTCCCCTAAACTGCTGATCAAGTACGCCTCTGAATCAAGGTCAGGTGTCCCCAGTCGATTTTCTGGATCAGGATCTGGTACCGACTTCACCCTCACCATCAATAGCTTGGAGGCCGAGGACGCTGCTACCTACTACTGCCAACAAAGCCACAGCTGGCACTTTACTTTTGGCCAGGGGACCAAGCTT(SEQ ID No.3)
GAAGTCCAGCTGGTCGAGAGCGGTGGCGGGCTGGTGCAACCCGGTGGATCACTGCGGCTCAGCTGCGCTGCTAGTGGCTTTCCCTTCTCTAACCACTGGATGAATTGGGTCCGGCAGGCTCCAGGAAAGGGTCTGGAGTGGGTGGGTGAGATCAGGAGTAAGTCTATGAACTCCGCCACACACTATGCTGAAAGCGT(SEQ ID No.4)
VEGF(VL-VH)(简称为:VEGF scFv)的氨基酸序列:
VMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGIYFCSQSTHVPPWTFGGGTKLEIKT(SEQ ID No.5)
QLLESGPELVKPGASVKISCKTSGFTFTDYNMHWVKQSHGKSLEWIGYIYPYNGATGYNQNFKNKATLTVDSSSSTAYMELRSLTSEDSAVYYCASSLLRVGGFDYWGQGTTLTVSSA(SEQ ID No.6)
VEGF(VL-VH)的核苷酸序列:
GTGATGACCCAGACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAACAGAGTGGAGGCTGAGGATCTGGGAATTTATTTCTGCTCTCAAAGTACACATGTTCCTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAACC(SEQ ID No.7)
CAGCTGCTCGAGTCAGGACCTGAGCTGGTGAAACCTGGGGCCTCAGTGAAGATTTCCTGCAAGACTTCTGGATTCACATTCACTGACTACAACATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTTATCCTTACAATGGTGCTACTGGCTACAACCAGAACTTCAAGAACAAGGCCACATTGACTGTAGACAGTTCCTCCAGTACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGTTCATTACTACGGGTGGGGGGTTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCC(SEQ ID No.8)
实施例2:FRα和VEGF抗体的表达和活性鉴定
1、FRα和VEGF抗体的瞬时表达
细胞瞬时转染:293细胞转染按LipofectamineTM 2000操作说明书进行,以6孔板中的1孔为例,步骤如下:向该孔接种4×105细胞,37℃、5%CO2培养18h后换液1次,12h后开始转染,贴壁细胞在转染时的理想汇合度为90-95%。将4μg质粒DNA和10μl LipofectamineTM2000分别用无抗生素、无血清的培养基稀释至250μl,温和混匀,室温静置15min形成脂质体复合物。连续培养三天后收取上清采用间接ELISA方法检测目的蛋白的特异性。
2、FRα和VEGF抗体与抗原的特异性结合ELISA试验
(1)抗原包被(设置空白对照,、阴性对照):将FRα或者VEGF抗原使用包被稀释液稀释到适当2ug/ml,每孔抗原加入100μl,4℃,24h过夜包被;弃去孔中液体(为避免蒸发,板上应加盖或将板平放在底部有湿纱布的金属湿盒中)。
(2)封闭酶标反应孔:5%小牛血清置37℃封闭40min,封闭时将封闭液加满各反应孔,并去除各孔中的气泡,封闭结束后用洗涤液满孔洗涤3遍,每遍3min。
洗涤方法:吸干孔内反应液,将洗涤液注满板孔,放置2min略作摇动,吸干孔内液,倾去液体后在吸水纸上拍干,洗涤次数3次。
(3)加入FRα或者VEGF抗体(建立合适的浓度梯度):采用1:100-1:100000的稀释度,将20μl稀释好的样品加入酶标反应孔中,每样品至少加双孔,每孔100μl,置于37℃,40-60min;用洗涤液满孔洗涤3遍,每遍3min。
(4)加入酶标抗体:加入HRP标记的羊抗人抗体,稀释比例为1:1000,37℃,孵育30-60min。
(5)加入底物液(现用现配):每孔加入100μl TMB底物,置37℃避光放置3-5分钟,加入2ml硫酸终止液显色。
(6)结果判断:450nm波长来检测,与空白对照对确定抗体的特异性和效价。
从ELISA结果可知(图1),FRα和VEGF抗体对抗原均有特异性的结合。
实施例3:ADCC增强型双特异性抗体平台的建立
ADCC增强型双特异性抗体平台的结构如图2所示。构建得到双特异性抗体FRαscFv-linker1-CH2-CH3-linker2-VEGF scFv,其氨基酸序列如下:
FRα(scFv):
EVQLVESGGGLVQPGGSLRLSCAASGFPFSNHWMNWVRQAPGKGLEWVGEIRSKSMNSATHYAESVKGRFTISRDDSKNSLYLQMNSLKTEDTAVYYCARNYYGSTYDHWGQGTLVTVSS(GGGGS)3EIVLTQSPDFQSVTPKEKVTITCRASQFVGYSIHWYQQKPDQSPKLLIKYASESRSGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQSHSWHFTFGQGTKLEIK(SEQ ID No.9)
linker1:
EPKSCDKTHTCPPCPA(SEQ ID No.10)
linker2:
(GGGGS)3(SEQ ID No.11)
CH2-CH3:
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLAVLHQDWLNGKEYKCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVAWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHAHYTQKSLSLSPGK(SEQID No.12)
其中下划线为突变位点,具体为S298A、T307A、E333A、K334A、E380A、N430A
VEGF(scFv):
VMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGIYFCSQSTHVPPWTFGGGTKLEIKT(GGGGS)3QLLESGPELVKPGASVKISCKTSGFTFTDYNMHWVKQSHGKSLEWIGYIYPYNGATGYNQNFKNKATLTVDSSSSTAYMELRSLTSEDSAVYYCASSLLRVGGFDYWGQGTTLTVSSA(SEQ ID No.13)
FRαscFv-linker1-CH2-CH3-linker2-VEGF scFv的核苷酸序列为:
FRα(scFv):
GAAATTGTGCTCACACAGTCACCAGACTTTCAGTCTGTCACCCCTAAGGAGAAAGTGACCATCACTTGCAGGGCCTCTCAGTTCGTCGGCTATAGTATCCACTGGTACCAGCAGAAACCCGATCAGTCCCCTAAACTGCTGATCAAGTACGCCTCTGAATCAAGGTCAGGTGTCCCCAGTCGATTTTCTGGATCAGGATCTGGTACCGACTTCACCCTCACCATCAATAGCTTGGAGGCCGAGGACGCTGCTACCTACTACTGCCAACAAAGCCACAGCTGGCACTTTACTTTTGGCCAGGGGACCAAGCTTGAGATCAAAGGAGGAGGAGGATCAGGAGGAGGAGGATCAGGAGGAGGAGGATCAGAAGTCCAGCTGGTCGAGAGCGGTGGCGGGCTGGTGCAACCCGGTGGATCACTGCGGCTCAGCTGCGCTGCTAGTGGCTTTCCCTTCTCTAACCACTGGATGAATTGGGTCCGGCAGGCTCCAGGAAAGGGTCTGGAGTGGGTGGGTGAGATCAGGAGTAAGTCTATGAACTCCGCCACACACTATGCTGAAAGCGTGAAAGGGCGCTTCACAATCTCTAGAGACGATTCAAAGAACTCTCTGTACCTGCAGATGAACAGTCTGAAAACAGAGGACACCGCTGTGTATTACTGTGCTCGGAACTACTACGGTTCAACTTACGACCACTGGGGCCAAGGTACACTGGTCACCGTCTCGAGT(SEQ ID No.14)
linker 1:
GAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCA(SEQ ID No.15)
linker 2:
GAGATCAAAGGAGGAGGAGGATCAGGAGGAGGAGGATCAGGAGGAGGAGGATCA(SEQ ID No.16)
CH2-CH3:
CCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACGCCACGTACCGGGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGCCGCAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA(SEQ ID No.17)
VEGF(scFv):
GTGATGACCCAGACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAACAGAGTGGAGGCTGAGGATCTGGGAATTTATTTCTGCTCTCAAAGTACACATGTTCCTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAACCGAGATCAAAGGAGGAGGAGGATCAGGAGGAGGAGGATCAGGAGGAGGAGGATCACAGCTGCTCGAGTCAGGACCTGAGCTGGTGAAACCTGGGGCCTCAGTGAAGATTTCCTGCAAGACTTCTGGATTCACATTCACTGACTACAACATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTTATCCTTACAATGGTGCTACTGGCTACAACCAGAACTTCAAGAACAAGGCCACATTGACTGTAGACAGTTCCTCCAGTACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGTTCATTACTACGGGTGGGGGGTTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCC(SEQ IDNo.18)
将实施例1中筛选获得的FRα(scFv)和VEGF(scFv)与CH2-CH3采用overlap PCR的方法拼接获得FRα(scFv)-Fc突变体(Fcmu)-VEGF(scFv);其中Fc突变体(Fcmu)采用突变后的序列(即CH2-CH3),根据已知文献报道,Fc突变体中S298A、E333A、K334A的突变可以增加亲和力,T307A、E380A、N430A的突变可以提高半衰期。选用突变后的Fc片段做FRα(scFv)和VEGF(scFv)的连接子,将FRα(scFv)-Fcmu-VEGF(scFv)构建入PCDNA3.1载体中,经测序和双酶切后,确认获得pFR132-FRα(scFv)-Fcmu-VEGF(scFv),采用无内毒素质粒大抽试剂盒抽提表达质粒。
实施例4:FRα和VEGF抗体双特异性抗体的制备和活性鉴定
1、Bis-FRα-Fcmu-VEGF双抗的电转和稳定细胞株的筛选
使用Bio-Rad电穿孔系统将pFR132-FRα(scFv)-Fcmu-VEGF(scFv)转进宿主细胞(CHO细胞),利用本细胞筛选标记DHFR,进行MTX压力筛选(终压力为200nM),标记每孔为:Bis-FRα-Fcmu-VEGF,以DOT Blotting评价克隆池的表达水平,逐步获得高表达的克隆池细胞。
结果如图3和表5所示,最终选择2株P2P261和P2P3472株细胞株进行发酵表达培养。
表5:Bis-FRα-Fcmu-VEGF双抗克隆池的表达水平检测
克隆池名称 | 表达量 |
132F180103Bis-FRα-Fcmu-VEGF-P2P25 | 0 |
132F171229Bis-FRα-Fcmu-VEGF-P1P183 | 57.3 |
132F171225Bis-FRα-Fcmu-VEGF-P1P232 | 70.8 |
132F171229Bis-FRα-Fcmu-VEGF-P1P122 | 20.5 |
132F180103Bis-FRα-Fcmu-VEGF-P2P261 | 712 |
132F180103Bis-FRα-Fcmu-VEGF-P2P322 | 285 |
132F171225Bis-FRα-Fcmu-VEGF-P2P193 | 165 |
132F171225Bis-FRα-Fcmu-VEGF-P1P137 | 过低 |
132F171225Bis-FRα-Fcmu-VEGF-P2P170 | 72.6 |
132F171225Bis-FRα-Fcmu-VEGF-P2P218 | 268 |
132F171208Bis-FRα-Fcmu-VEGF-P2P347 | 750 |
132F171201Bis-FRα-Fcmu-VEGF-P2P85 | 40.1 |
132F171205Bis-FRα-Fcmu-VEGF-P2P89 | 过低 |
132F180103Bis-FRα-Fcmu-VEGF-P2P350 | 33 |
132F180103Bis-FRα-Fcmu-VEGF-P2P50 | 41.2 |
132F-180125Bis-FRα-Fcmu-VEGF-P2P293 | 270.9 |
2、Bis-FRα-Fcmu-VEGF双抗的发酵和纯化
选用表达量最高的Bis-FRα-Fcmu-VEGF-P2P3472细胞株进行扩增,转到200ml摇瓶中培养,带细胞生长密度达到80%时,离心收集细胞,转入5L的发酵罐中进行连续培养发酵。
接种密度 | 接种体积 | 接种培养基 | 补料方式 |
1×10<sup>6</sup>cells/mL | 30mL/60mL | CD01N+INS | 20%CDF+Gluc(4g/mL) |
每天检测细胞密度,待胞密度达到90%时,收罐,离心出去细胞陈沉淀,对上清进行纯化。
所获得的发酵液首先使用Protein A(耐碱型ProteinA介质BXK26/20柱管,上海博格隆)进行捕获,然后进行阳离子中纯(SP-5PW,TOSOH)分离酸碱峰,最后以WCX、SEC以及SDS-PAGE对所获得的样品进行纯度分析,纯化过程中分段收集的样本采用SDS-PAGE电泳分析。
结果如图4所示,F1,F2,F3为纯化获得的Bis-FRα-Fcmu-VEGF双抗,蛋白的条带清晰单一,分子量正确。
实施例5:Bis-FRα-Fcmu-VEGF双抗的生物活性
1、Bis-FRα-Fcmu-VEGF双抗的亲和力检测
分子互作仪是利用生物膜干涉技术(Biolayer-interferometry,BLI)进行动力学测定或浓度测定。可见光在传感器末端的光学膜层的两个界面会形成两束反射光谱,叠加形成一束干涉光谱,而分子的结合导致膜层厚度变化,并导致原有干涉光谱的位移,通过检测和分析干涉光谱的位移值而实现检测功能。Bis-FRα-Fcmu-VEGF双抗可以与FRα和VEGF同时结合,通过检测抗体的亲和力,评价待测样品的抗体性能。
利用分子互作仪,将Bis-FRα-Fcmu-VEGF双抗待测样品固定至ProteinA传感器,再与FRα和VEGF蛋白进行结合和解离,通过软件分析拟合曲线的数据,评价待测样品的亲和力。
如图5和表6所示,Bis-FRα-Fcmu-VEGF双抗对FRα和VEGF抗原的结合常数分别为:2.99×105和2.25×105,解离常数分别为:2.45×10-4和1.87×10-4。
表6:Bis-FRα-Fcmu-VEGF双抗的亲和力检测试验
2、Bis-FRα-Fcmu-VEGF双抗抗体抑制血管内皮细胞的生长和和体外血管形成
将人乳腺癌细胞MDA-MB-231、肺癌细胞A549、人脐静脉内皮细胞HUVEC和前列腺癌PC-3以5×106种植于96孔板内,待细胞贴壁后,培养液内加入100μg/ml HAI-178,设置PBS对照,采用MTT方法,检测Bis-FRα-Fcmu-VEGF抗体对上述细胞体外培养生长情况的影响。
(1)Bis-FRα-Fcmu-VEGF抑制血管内皮细胞生长
如图6所示,Bis-FRα-Fcmu-VEGF双抗可以有效抑制HUVEC细胞的生长,抑制率达20%左右。血管内皮细胞的生长是血管新生的一个重要环节,Bis-FRα-Fcmu-VEGF双抗抗体可以通过抑制血管内皮细胞的生长起到抑制新生血管生长的作用。
另外,采用Matrigel血管枝形成实验,观察Bis-FRα-Fcmu-VEGF双抗对HUVEC细胞体外血管形成影响作用。观察发现HUVEC细胞在Bis-FRα-Fcmu-VEGF双抗的作用下,血管枝形成显著减少,其作用与作为阳性对照的血管抑素Angiostatin 2相当。
(2)Bis-FRα-Fcmu-VEGF对肿瘤细胞的杀伤活性
结果显示(如图6),100μg/ml Bis-FRα-Fcmu-VEGF抗体能显著抑制肿瘤细胞与内皮细胞的增殖。作用48小时后,对MDA-MB-231细胞、A549细胞、PC3细胞和HUVEC细胞的抑制率分别为77.6%、52.1%、17.7%和18.4%;作用72h后,抑制率分别达到83.2%、53.1%、12.5%和20.7%,而对作为对照的CHO细胞无抑制作用。
综上,本发明成功获得了一株高表达Bis-FRα-Fcmu-VEGF双抗的细胞株,通过纯化获得了高纯度的Bis-FRα-Fcmu-VEGF双抗,并且生物学验证了其具有很好的肿瘤杀伤活性,能够抑制血管生成和肿瘤生成。
以上实施例仅用以说明本发明的技术方案,而非对其进行限制;尽管参照前述实施例对本发明进行了详细的说明,对于本领域的普通技术人员来说,依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或替换,并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
序列表
<110> 苏州思萃免疫技术研究所有限公司
<120> 一种抗FOLR1/VEGF的全人双特异性抗体及其筛选方法和应用
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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Asn His
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Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
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Gly Glu Ile Arg Ser Lys Ser Met Asn Ser Ala Thr His Tyr Ala Glu
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Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser
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Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
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Tyr Cys Ala Arg Asn Tyr Tyr Gly Ser Thr Tyr Asp His Trp Gly Gln
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Gly Thr Leu Val Thr Val Ser Ser
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Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Phe Val Gly Tyr Ser
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Lys Tyr Ala Ser Glu Ser Arg Ser Gly Val Pro Ser Arg Phe Ser Gly
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Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala
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gaaattgtgc tcacacagtc accagacttt cagtctgtca cccctaagga gaaagtgacc 60
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gatcagtccc ctaaactgct gatcaagtac gcctctgaat caaggtcagg tgtccccagt 180
cgattttctg gatcaggatc tggtaccgac ttcaccctca ccatcaatag cttggaggcc 240
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<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gaagtccagc tggtcgagag cggtggcggg ctggtgcaac ccggtggatc actgcggctc 60
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ccaggaaagg gtctggagtg ggtgggtgag atcaggagta agtctatgaa ctccgccaca 180
cactatgctg aaagcgt 197
<210> 5
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<212> PRT
<213> 人工序列(Artificial Sequence)
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Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln
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Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly
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Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg
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Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Asn Arg
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Val Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Thr
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<212> PRT
<213> 人工序列(Artificial Sequence)
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Gln Leu Leu Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val
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Lys Ile Ser Cys Lys Thr Ser Gly Phe Thr Phe Thr Asp Tyr Asn Met
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His Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Tyr
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Ile Tyr Pro Tyr Asn Gly Ala Thr Gly Tyr Asn Gln Asn Phe Lys Asn
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Lys Ala Thr Leu Thr Val Asp Ser Ser Ser Ser Thr Ala Tyr Met Glu
65 70 75 80
Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Ser
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Ser Leu Leu Arg Val Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr
100 105 110
Leu Thr Val Ser Ser Ala
115
<210> 7
<211> 336
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gtgatgaccc agactccact ctccctgcct gtcagtcttg gagatcaagc ctccatctct 60
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cagaagccag gccagtctcc aaagctcctg atctacaaag tttccaaccg attttctggg 180
gtcccagaca ggttcagtgg cagtggatca gggacagatt tcacactcaa gatcaacaga 240
gtggaggctg aggatctggg aatttatttc tgctctcaaa gtacacatgt tcctccgtgg 300
acgttcggtg gaggcaccaa gctggaaatc aaaacc 336
<210> 8
<211> 354
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
cagctgctcg agtcaggacc tgagctggtg aaacctgggg cctcagtgaa gatttcctgc 60
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aagagccttg agtggattgg atatatttat ccttacaatg gtgctactgg ctacaaccag 180
aacttcaaga acaaggccac attgactgta gacagttcct ccagtacagc ctacatggag 240
ctccgcagcc tgacatctga ggactctgca gtctattact gtgcaagttc attactacgg 300
gtggggggtt ttgactactg gggccaaggc accactctca cagtctcctc agcc 354
<210> 9
<211> 242
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Pro Phe Ser Asn His
20 25 30
Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
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Gly Glu Ile Arg Ser Lys Ser Met Asn Ser Ala Thr His Tyr Ala Glu
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Asn Tyr Tyr Gly Ser Thr Tyr Asp His Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Asp
130 135 140
Phe Gln Ser Val Thr Pro Lys Glu Lys Val Thr Ile Thr Cys Arg Ala
145 150 155 160
Ser Gln Phe Val Gly Tyr Ser Ile His Trp Tyr Gln Gln Lys Pro Asp
165 170 175
Gln Ser Pro Lys Leu Leu Ile Lys Tyr Ala Ser Glu Ser Arg Ser Gly
180 185 190
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
195 200 205
Thr Ile Asn Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln
210 215 220
Gln Ser His Ser Trp His Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu
225 230 235 240
Ile Lys
<210> 10
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
1 5 10 15
<210> 11
<211> 15
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 12
<211> 216
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
1 5 10 15
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
20 25 30
Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
35 40 45
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
50 55 60
Tyr Asn Ala Thr Tyr Arg Val Val Ser Val Leu Ala Val Leu His Gln
65 70 75 80
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
85 90 95
Leu Pro Ala Pro Ile Ala Ala Thr Ile Ser Lys Ala Lys Gly Gln Pro
100 105 110
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
115 120 125
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
130 135 140
Asp Ile Ala Val Ala Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
145 150 155 160
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
165 170 175
Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
180 185 190
Ser Cys Ser Val Met His Glu Ala Leu His Ala His Tyr Thr Gln Lys
195 200 205
Ser Leu Ser Leu Ser Pro Gly Lys
210 215
<210> 13
<211> 245
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln
1 5 10 15
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser Asn Gly
20 25 30
Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys
35 40 45
Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg
50 55 60
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Asn Arg
65 70 75 80
Val Glu Ala Glu Asp Leu Gly Ile Tyr Phe Cys Ser Gln Ser Thr His
85 90 95
Val Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Thr
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
115 120 125
Leu Leu Glu Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys
130 135 140
Ile Ser Cys Lys Thr Ser Gly Phe Thr Phe Thr Asp Tyr Asn Met His
145 150 155 160
Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly Tyr Ile
165 170 175
Tyr Pro Tyr Asn Gly Ala Thr Gly Tyr Asn Gln Asn Phe Lys Asn Lys
180 185 190
Ala Thr Leu Thr Val Asp Ser Ser Ser Ser Thr Ala Tyr Met Glu Leu
195 200 205
Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Ser Ser
210 215 220
Leu Leu Arg Val Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
225 230 235 240
Thr Val Ser Ser Ala
245
<210> 14
<211> 726
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gaaattgtgc tcacacagtc accagacttt cagtctgtca cccctaagga gaaagtgacc 60
atcacttgca gggcctctca gttcgtcggc tatagtatcc actggtacca gcagaaaccc 120
gatcagtccc ctaaactgct gatcaagtac gcctctgaat caaggtcagg tgtccccagt 180
cgattttctg gatcaggatc tggtaccgac ttcaccctca ccatcaatag cttggaggcc 240
gaggacgctg ctacctacta ctgccaacaa agccacagct ggcactttac ttttggccag 300
gggaccaagc ttgagatcaa aggaggagga ggatcaggag gaggaggatc aggaggagga 360
ggatcagaag tccagctggt cgagagcggt ggcgggctgg tgcaacccgg tggatcactg 420
cggctcagct gcgctgctag tggctttccc ttctctaacc actggatgaa ttgggtccgg 480
caggctccag gaaagggtct ggagtgggtg ggtgagatca ggagtaagtc tatgaactcc 540
gccacacact atgctgaaag cgtgaaaggg cgcttcacaa tctctagaga cgattcaaag 600
aactctctgt acctgcagat gaacagtctg aaaacagagg acaccgctgt gtattactgt 660
gctcggaact actacggttc aacttacgac cactggggcc aaggtacact ggtcaccgtc 720
tcgagt 726
<210> 15
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagca 48
<210> 16
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gagatcaaag gaggaggagg atcaggagga ggaggatcag gaggaggagg atca 54
<210> 17
<211> 651
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
cctgaactcc tggggggacc gtcagtcttc ctcttccccc caaaacccaa ggacaccctc 60
atgatctccc ggacccctga ggtcacatgc gtggtggtgg acgtgagcca cgaagaccct 120
gaggtcaagt tcaactggta cgtggacggc gtggaggtgc ataatgccaa gacaaagccg 180
cgggaggagc agtacaacgc cacgtaccgg gtggtcagcg tcctcaccgt cctgcaccag 240
gactggctga atggcaagga gtacaagtgc aaggtctcca acaaagccct cccagccccc 300
atcgccgcaa ccatctccaa agccaaaggg cagccccgag aaccacaggt gtacaccctg 360
cccccatccc gggatgagct gaccaagaac caggtcagcc tgacctgcct ggtcaaaggc 420
ttctatccca gcgacatcgc cgtggagtgg gagagcaatg ggcagccgga gaacaactac 480
aagaccacgc ctcccgtgct ggactccgac ggctccttct tcctctacag caagctcacc 540
gtggacaaga gcaggtggca gcaggggaac gtcttctcat gctccgtgat gcatgaggct 600
ctgcacaacc actacacgca gaagagcctc tccctgtctc cgggtaaatg a 651
<210> 18
<211> 744
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
gtgatgaccc agactccact ctccctgcct gtcagtcttg gagatcaagc ctccatctct 60
tgcagatcta gtcagagcct tgtacacagt aatggaaaca cctatttaca ttggtacctg 120
cagaagccag gccagtctcc aaagctcctg atctacaaag tttccaaccg attttctggg 180
gtcccagaca ggttcagtgg cagtggatca gggacagatt tcacactcaa gatcaacaga 240
gtggaggctg aggatctggg aatttatttc tgctctcaaa gtacacatgt tcctccgtgg 300
acgttcggtg gaggcaccaa gctggaaatc aaaaccgaga tcaaaggagg aggaggatca 360
ggaggaggag gatcaggagg aggaggatca cagctgctcg agtcaggacc tgagctggtg 420
aaacctgggg cctcagtgaa gatttcctgc aagacttctg gattcacatt cactgactac 480
aacatgcact gggtgaagca gagccatgga aagagccttg agtggattgg atatatttat 540
ccttacaatg gtgctactgg ctacaaccag aacttcaaga acaaggccac attgactgta 600
gacagttcct ccagtacagc ctacatggag ctccgcagcc tgacatctga ggactctgca 660
gtctattact gtgcaagttc attactacgg gtggggggtt ttgactactg gggccaaggc 720
accactctca cagtctcctc agcc 744
<210> 19
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
atggactgga cctggagcat cc 22
<210> 20
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
atggactgga cctggaggat cctc 24
<210> 21
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
atggactgga cctggagggt cttc 24
<210> 22
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
atggactgga tttggagggt cctcttc 27
<210> 23
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
atggacacac tttgctacac actcctgc 28
<210> 24
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
actttgctcc acgctcctgc 20
<210> 25
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
ggctgagctg ggttttcctt gttg 24
<210> 26
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
ggctccgctg ggttttcctt gttg 24
<210> 27
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
cacctgtggt tcttcctcct gctg 24
<210> 28
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
atgaaacacc tgtggttctt cctcctcc 28
<210> 29
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
acatctgtgg ttcttccttc tcctggtg 28
<210> 30
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
gcctctccac ttaaacccag gctc 24
<210> 31
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
atgtctgtct ccttcctcat cttcctgc 28
<210> 32
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
atggagttgg ggctgagctg g 21
<210> 33
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
atggggtcaa ccgccatcct c 21
<210> 34
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
atgaggctcc ttgctcagct tctgg 25
<210> 35
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
atggaagccc cagctcagct tc 22
<210> 36
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
cccagctcag cttctcttcc tcctg 25
<210> 37
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
tggtgttgca gacccaggtc ttcatttc 28
<210> 38
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
gtcccaggtt cacctcctca gcttc 25
<210> 39
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
gccatcacaa ctcattgggt ttctgctg 28
<210> 40
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
tccctgctca gctcctggg 19
<210> 41
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
cctgggactc ctgctgctct g 21
<210> 42
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
ccctgggtca tgctcctcct gaaatc 26
<210> 43
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
ctctgctgct cctcactctc ctcac 25
<210> 44
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
atggcatgga tccctctctt cctcg 25
<210> 45
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
cctctctggc tcactctcct cactc 25
<210> 46
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
acactcctgc tcccactcct caac 24
<210> 47
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
atggcctgga tccctctact tctcc 25
<210> 48
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 48
atggcctggg tctccttcta cc 22
<210> 49
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 49
atggcctgga ctcctctctt tctgttc 27
<210> 50
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 50
atggcctgga tgatgcttct cctc 24
<210> 51
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
gtcccctctc ttcctcaccc tcatc 25
<210> 52
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
ctcctcgctc actgcacagg 20
<210> 53
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
cctctcctcc tcaccctcct c 21
<210> 54
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 54
ctcctcctca ccctcctcac tc 22
<210> 55
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 55
atggcctgga cccctctcc 19
<210> 56
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 56
atggcctgga ccccactcc 19
<210> 57
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 57
gcttcgttag aacgcggcta c 21
<210> 58
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 58
atggagtcgg gaaggaagtc 20
<210> 59
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 59
ccgacgggga attctcacag 20
<210> 60
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 60
aggtgtgcac gccgctggtc 20
<210> 61
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 61
gttcggggaa gtagtccttg ac 22
<210> 62
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 62
gtttctcgta gtctgctttg ctca 24
<210> 63
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 63
gtgctgtcct tgctgtcctg ct 22
<210> 64
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 64
caccagtgtg gccttgttgg cttg 24
<210> 65
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 65
ctcctcactc gagggyggga acagagtg 28
<210> 66
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 66
aactgcacct cggttctatc gattgaattc atggactgga cctggagcat cc 52
<210> 67
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 67
aactgcacct cggttctatc gattgaattc atggactgga cctggaggat cctc 54
<210> 68
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 68
aactgcacct cggttctatc gattgaattc atggactgga cctggagggt cttc 54
<210> 69
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 69
aactgcacct cggttctatc gattgaattc atggactgga tttggagggt cctcttc 57
<210> 70
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 70
aactgcacct cggttctatc gattgaattc atggacacac tttgctacac actcctgc 58
<210> 71
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 71
aactgcacct cggttctatc gattgaattc atggacacac tttgctccac gctcctgc 58
<210> 72
<211> 64
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 72
aactgcacct cggttctatc gattgaattc atggagtttg ggctgagctg ggttttcctt 60
gttg 64
<210> 73
<211> 64
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 73
aactgcacct cggttctatc gattgaattc atggaactgg ggctccgctg ggttttcctt 60
gttg 64
<210> 74
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 74
aactgcacct cggttctatc gattgaattc atgaaacacc tgtggttctt cctcctgctg 60
<210> 75
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 75
aactgcacct cggttctatc gattgaattc atgaaacacc tgtggttctt cctcctcc 58
<210> 76
<211> 63
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 76
aactgcacct cggttctatc gattgaattc atgaaacatc tgtggttctt ccttctcctg 60
gtg 63
<210> 77
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 77
aactgcacct cggttctatc gattgaattc atgcaagtgg gggcctctcc acttaaaccc 60
aggctc 66
<210> 78
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 78
aactgcacct cggttctatc gattgaattc atgtctgtct ccttcctcat cttcctgc 58
<210> 79
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 79
aactgcacct cggttctatc gattgaattc atggagttgg ggctgagctg g 51
<210> 80
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 80
aactgcacct cggttctatc gattgaattc atggggtcaa ccgccatcct c 51
<210> 81
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 81
aactgcacct cggttctatc gattgaattc atgaggctcc ttgctcagct tctgg 55
<210> 82
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 82
aactgcacct cggttctatc gattgaattc atggaagccc cagctcagct tc 52
<210> 83
<211> 63
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 83
aactgcacct cggttctatc gattgaattc atggaaaccc cagctcagct tctcttcctc 60
ctg 63
<210> 84
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 84
aactgcacct cggttctatc gattgaattc atggtgttgc agacccaggt cttcatttc 59
<210> 85
<211> 60
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 85
aactgcacct cggttctatc gattgaattc atggggtccc aggttcacct cctcagcttc 60
<210> 86
<211> 63
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 86
aactgcacct cggttctatc gattgaattc atgttgccat cacaactcat tgggtttctg 60
ctg 63
<210> 87
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 87
aactgcacct cggttctatc gattgaattc atgaggctcc ctgctcagct cctggg 56
<210> 88
<211> 71
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 88
aactgcacct cggttctatc gattgaattc atgagggtcc ccgctcagct cctgggactc 60
ctgctgctct g 71
<210> 89
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 89
aactgcacct cggttctatc gattgaattc atgccctggg tcatgctcct cctgaaatc 59
<210> 90
<211> 65
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 90
aactgcacct cggttctatc gattgaattc atggcctggg ctctgctgct cctcactctc 60
ctcac 65
<210> 91
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 91
aactgcacct cggttctatc gattgaattc atggcatgga tccctctctt cctcg 55
<210> 92
<211> 67
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 92
aactgcacct cggttctatc gattgaattc atggcctgga cccctctctg gctcactctc 60
ctcactc 67
<210> 93
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 93
aactgcacct cggttctatc gattgaattc atggcatggg ccacactcct gctcccactc 60
ctcaac 66
<210> 94
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 94
aactgcacct cggttctatc gattgaattc atggcctgga tccctctact tctcc 55
<210> 95
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 95
aactgcacct cggttctatc gattgaattc atggcctggg tctccttcta cc 52
<210> 96
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 96
aactgcacct cggttctatc gattgaattc atggcctgga ctcctctctt tctgttc 57
<210> 97
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 97
aactgcacct cggttctatc gattgaattc atggcctgga tgatgcttct cctc 54
<210> 98
<211> 63
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 98
aactgcacct cggttctatc gattgaattc atggcctggt cccctctctt cctcaccctc 60
atc 63
<210> 99
<211> 77
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 99
aactgcacct cggttctatc gattgaattc atggcctggt ctcctctcct cctcactctc 60
ctcgctcact gcacagg 77
<210> 100
<211> 63
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 100
aactgcacct cggttctatc gattgaattc atggccggct tccctctcct cctcaccctc 60
ctc 63
<210> 101
<211> 67
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 101
aactgcacct cggttctatc gattgaattc atggccagct tccctctcct cctcaccctc 60
ctcactc 67
<210> 102
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 102
aactgcacct cggttctatc gattgaattc atggcctgga cccctctcc 49
<210> 103
<211> 49
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 103
aactgcacct cggttctatc gattgaattc atggcctgga ccccactcc 49
<210> 104
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 104
gggtgccagg gggaagaccg atgggccctt ggtcgaggc 39
<210> 105
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 105
ggggaagacc gatgggccct tggtcgaggc tgaggagacg gtgaccaggg t 51
<210> 106
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 106
ggggaagacc gatgggccct tggtcgaggc tgaagagacg gtgaccattg t 51
<210> 107
<211> 51
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 107
ggggaagacc gatgggccct tggtcgaggc tgaggagacg gtgaccgtgg t 51
<210> 108
<211> 48
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 108
ctcatcagat ggcgggaaga tgaagacaga tggtgcagcc accgtacg 48
<210> 109
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 109
gaagctcctc actcgagggy gggaacagag tg 32
Claims (14)
1.一种抗FOLR1/VEGF的全人双特异性抗体,其特征在于,所述全人双特异性抗体由抗人FRα抗体和抗人VEGF抗体联合构建而成。
2.根据权利要求1所述的全人双特异性抗体,其特征在于,所述抗人FRα抗体含有SEQID No.1所示的重链可变区序列和SEQ ID No.2所示的轻链可变区序列。
3.根据权利要求1所述的全人双特异性抗体,其特征在于,所述抗人VEGF抗体含有SEQID No.6所示的重链可变区序列和SEQ ID No.5所示的轻链可变区序列。
4.根据权利要求1所述的全人双特异性抗体,其特征在于,所述抗人FRα抗体和所述抗人VEGF抗体均以scFv形式表达。
5.根据权利要求2所述的全人双特异性抗体,其特征在于,所述抗人FRα的抗体重链可变区由SEQ ID No.3所示的核苷酸序列编码;所述抗人FRα的抗体轻链可变区由SEQ IDNo.4所示的核苷酸序列编码。
6.根据权利要求3所述的全人双特异性抗体,其特征在于,所述抗人VEGF的抗体重链可变区由SEQ ID No.8所示的核苷酸序列编码;所述抗人VEGF的抗体轻链可变区由SEQ IDNo.7所示的核苷酸序列编码。
7.根据权利要求1所述的全人双特异性抗体,其特征在于,所述全人双特异性抗体中还含有序列如SEQ ID No.12所示的Fc突变体片段。
8.根据权利要求7所述的全人双特异性抗体,其特征在于,所述Fc突变体片段由SEQ IDNo.17所示的核苷酸序列编码。
9.权利要求1-8任一项所述的全人双特异性抗体的筛选方法,其特征在于,所述筛选方法包括以下步骤:
(1)利用卵巢癌腹水筛选、富集抗原特异性B细胞;
(2)从抗原特异性B细胞中,获取FRα抗体和VEGF抗体;
(3)利用FRα抗体和VEGF抗体建立双特异性抗体平台,得到scFv形式的FRα抗体和VEGF抗体;
(4)拼接scFv形式的FRα抗体和VEGF抗体,将Fc突变体片段Fcmu作为连接子,构建入载体,获取同时含有FRα(scFv)、Fcmu和VEGF(scFv)的质粒;
(5)将质粒转入宿主细胞,筛选得到高表达的细胞株;
(6)对高表达的细胞株培养纯化,得到同时含有FRα抗体、VEGF抗体和Fc突变体的蛋白,即为全人双特异性抗体。
10.根据权利要求9所述的全人双特异性抗体的筛选方法,其特征在于,所述Fc突变体片段的突变位点包括S298A、T307A、E333A、K334A、E380A、N430A。
11.权利要求1-8任一项所述的全人双特异性抗体在制备用于抑制血管生成的药物中应用。
12.权利要求1-8任一项所述的全人双特异性抗体在制备用于抗肿瘤药物中的应用。
13.根据权利要求12所述的应用,其特征在于,所述肿瘤包括肺癌、前列腺癌、乳腺癌、卵巢癌、肠癌、淋巴瘤、鼻咽癌、胃癌、肝癌、肾癌、子宫颈癌和子宫内膜癌、骨肉瘤。
14.一种药物组合物,其特征在于,所述药物组合物包含抗FOLR1/VEGF的全人双特异性抗体。
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