CN108129564B - 一种全人源的抗vegf单链抗体及其应用 - Google Patents
一种全人源的抗vegf单链抗体及其应用 Download PDFInfo
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Abstract
本发明公开了一种全人源的抗VEGF单链抗体,所述单链抗体具有独特的CDR区,对抗原具有高度特异性的识别能力。所述单链抗体又具有优异的肿瘤结合效果,其对不同类型的胰腺癌细胞系均有特异性结合。本发明还公开了偶联了指示剂的所述抗体在制备体外肿瘤诊断试剂和体内生物成像剂中的应用。当所述成像剂应用在肿瘤动物模型上,标记了近红外荧光的该单链抗体可以清楚地显示出小鼠肿瘤的大小和位置,具有极高的体内成像能力。
Description
技术领域
本发明涉及一种抗体,更为具体地,本发明涉及一种单链抗体。
背景技术
肿瘤是威胁人类生存最大的疾病。世界卫生组织研究显示,2012年中国癌症发病人数为306.5万,约占全球发病的五分之一;癌症死亡人数为220.5万,约占全球癌症死亡人数的四分之一。
肿瘤的临床诊断技术也在逐渐提高。从最初的肿瘤标记物的检测、超声、透视等手段,发展到现在更多的肿瘤相关因子的蛋白水平和分子水平的检测、CT、核磁、PET、SPET,以及肿瘤微创的活检等等技术都已在临床上广泛应用。
肿瘤的诊断分为体外和体内诊断。在体外,肿瘤组织的免疫组织化学染色是确诊肿瘤的一个重要指标。体内诊断,一般是利用一些显像剂再辅助一些仪器进行肿瘤成像。肿瘤体内成像不仅能够早期诊断肿瘤,而且能够实时监测抗肿瘤治疗的效果以及肿瘤的进展情况。因为它是无创检查,给病人带来的痛苦小,已经成为临床确诊和***的必不可少的诊断手段。目前医院常用的肿瘤体内显像使用的是放射性核素显像,以及核磁。
近年来,光学成像以其非侵袭性、实时、分辨率高等优势广泛应用于肿瘤研究领域,可对肿瘤进行早期诊断,反映肿瘤解剖学结构及代谢情况。近红外荧光成像是目前光学分子成像领域研究的热点,其光谱范围为700-1000nm,在此波段范围内被监测的生物体与组织的自发荧光干扰较小,穿透组织距离可高达数厘米,提高了成像的准确度和灵敏度。
用来确诊肿瘤的靶点通常都是细胞表面蛋白。而我们发现血管内皮生长因子(VEGF)也可以作为一个肿瘤诊断的靶标,VEGF是分泌型可溶性蛋白,能直接作用于血管内皮细胞促进血管内皮细胞增殖,增加血管通透性。研究表明,良性肿瘤血管生成稀少,血管生长缓慢;而大多数恶性肿瘤的血管生成密集且生长迅速。因此,血管生成在肿瘤的发展转移过程中起到重要作用,抑制这一过程将能明显阻止肿瘤组织的发展和扩散转移。抗血管内皮生长因子的抗体可以使用在组织切片染色和confocal染色,也同样可以使用在体内作为肿瘤成像剂。
随着肿瘤靶点不断增多,利用特异性针对肿瘤靶抗原的抗体作为指示剂的研究也在逐渐深入。但是传统的全分子抗体分子量较大,并不能很快分布到全身,也不能很好地结合一些被遮蔽的靶位,所以抗体作为指示剂的可行性大打折扣。但是基因工程技术创造出了仅有抗原结合功能的分子量小的抗体片段,使得这一设想得以实现。但是这种小分子量抗体片段在应用中也出现了诸多问题,例如稳定性差,特异性不高,存在异源性免疫反应、以及抗体与指示剂的偶联适应度不高等问题而限制了这一诊断手段的进一步应用。
发明内容
基于现有技术存在的问题,本发明的目的是研发出一种具有特异性识别能力的抗血管内皮生长因子(VEGF)的单链抗体,进而提供一种指示剂与该抗体偶联的生物偶联物,并提供该偶联物在制备体外诊断试剂及体内成像剂中的应用。
基于上述发明目的,本发明首先提供了一种抗血管内皮生长因子的单链抗体,所述抗体的轻链的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ ID NO.1、2和3所示,所述抗体的重链的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ ID NO.5、6和7所示。
在一个优选的技术方案中,所述抗体的轻链可变区和重链可变区的氨基酸序列分别如SEQ ID NO.4和8所示。
在一个更为优选的技术方案中,所述抗体的轻链可变区和重链可变区通过连接多肽相连。
在一个最为优选的技术方案中,所述连接多肽的氨基酸序列如SEQ ID NO.9所示。
其次,本发明提供了一种生物大分子偶联物,所述偶联物含有上述任一抗体。
在一个优选的技术方案中,在所述偶联物中,绿色荧光蛋白、近红外荧光蛋白或者辣根过氧化物酶与所述抗体偶联。最后,本发明提供了上述偶联物在制备肿瘤诊断制剂中的应用。
在一个优选的技术方案中,所述绿色荧光蛋白与所述抗体偶联,所述偶联物被制备为免疫荧光诊断试剂。
在另一个优选的技术方案中,所述辣根过氧化物酶与所述抗体偶联,所述偶联物被制备为免疫组化诊断试剂。
在又一个优选的技术方案中,所述近红外荧光蛋白与所述抗体偶联,所述偶联物被制备为体内荧光成像剂。
本发明利用噬菌体抗体库方法筛选高亲和力抗VEGF的ScFv,然后使用绿色荧光蛋白、辣根过氧化物酶或近红外荧光蛋白标记偶联,分别作为体外免疫荧光诊断、免疫组化诊断抗体和体内荧光显像剂。本发明提供的抗VEGF单链抗体能够特异性结合肿瘤组织中的VEGF蛋白,其具有优异的亲和力,亲和力常数为2.14×10-10M。而大多数自身抗体的亲和力常数小于10-5--8M,因此本发明的抗VEGF单链抗体用于体内诊断显像时可以竞争自身抗体与VEGF结合,在经过与适宜的指示剂偶联后可以对肿瘤细胞和组织进行染色和显像,显示了本发明的抗EGFR单链抗体在制备体内和体外诊断试剂应用上的良好前景。由于本发明选用的是抗体小分子片段,该单链抗体的分子量小,可以在原核细胞表达***中表达,因此能够极大地降低抗体药物的生产成本。并且,本发明的抗VEGF单链抗体是利用人抗体基因制备而成的抗体库中筛选得到的,其基因序列为全人源,免疫原性低,可以作为药物应用到人体,这也是可以作为体内显像剂的一个有利因素。
附图说明
图1.VH和VL PCR产物鉴定图;
图2.载体图谱和重组载体鉴定图;
图3.筛选出的VEGF ScFv与VEGF-165蛋白的的结合试验柱状比较图;图4.纯化后的VEFG ScFv聚丙烯酰氨凝胶电泳鉴定图;
图5.VEGFR阻断VEGF ScFv与VEGF-165蛋白的结合试验曲线图;
图6.FITC标记的VEGF ScFv对胰腺癌肿瘤细胞的免疫荧光结果图;
图7.辣根过氧化物酶标记的VEGF ScFv对小鼠胰腺癌原位瘤组织化学显色图;
图8.近红外荧光标记的VEGF ScFv对小鼠胰腺癌原位瘤的体内成像图。
具体实施方式
下面结合具体实施例来进一步描述本发明,本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的,并不对本发明的保护范围构成任何限制。
实施例1抗VEGF单链抗体的制备
1.1高库容量天然抗体库的建立。
分离人外周血单个核淋巴细胞:随机选取100个健康成年人,抽取每人外周血10ml。用含10%肝素的RPMI-1640培养液1:1稀释,加到装有淋巴细胞分离液的离心管(稀释静脉血与淋巴细胞分离液的体积比为2:1)中,2,000×g,离心17分钟。吸取淋巴细胞分离液界面乳白色单个核细胞层,PBS缓冲液洗两次。
1.2细胞总RNA的提取
按每5×106cells/ml的比例加入Trizol试剂,吹打细胞使之裂解。室温孵育5分钟,移入经DEPC处理的EP管中,加入1/5体积的氯仿,剧烈振荡15秒,室温孵育3分钟。4℃,10,000×g离心15分钟,吸取上层水相至一个新的离心管中,加入1/2体积的异丙醇,冰浴10分钟。4℃,12,000×g离心10分钟,弃上清,加入1ml 75%乙醇清洗沉淀。4℃,7,500×g离心5分钟,弃上清,室温干燥沉淀,溶于无RNase的水或沉淀于无水乙醇内,-80℃保存备用。
1.3逆转录合成cDNA第一链
先用无RNase的DNaseⅠ处理总RNA以消除残存的基因组DNA。取处理过的RNA样品2μg和Oligo(dT)15(500μg/ml)1μl,补加DEPC水至12μl,70℃,加热10分钟。取出后马上置于冰浴中,依次加入5×Buffer5μl;dNTP(10mmol/L)5μl;RNA酶抑制剂1μl,MMLV逆转录酶1μl,补加DEPC水至25μl,42℃,保温60分钟进行逆转录反应,70℃,15分钟灭活酶活性。
1.4 PCR扩增VH和VL基因
以逆转录合成cDNA第一链为模板,采用人ScFv抗体库引物扩增所表达的VH和VL基因。反应体系如下:
加去离子水至终体积为50μl。
引物1:5’GAGCTCCAGATGACCCAGTCTCCT3’
引物2:5’ACTAGTGACGGATGGGCTCTGTGT3’
PCR参数:94℃变性3分钟后,再以94℃30秒;61℃30秒;72℃1分钟,进行PCR,共30个循环,最后72℃延伸10分钟。反应结束后取5μl反应产物进行1%琼脂糖凝胶电泳分析。
PCR产物回收
(1)PCR产物进行1.5%琼脂糖凝胶电泳,从琼脂糖凝胶上切下目的DNA片段,放入1.5ml离心管中。
(2)加入400μl溶胶液A,70℃溶解5分钟,至凝胶全部溶解。
(3)加入200μl溶胶液B,混均后将全部液体吸入回收柱中。
(4)12,000×g离心1分钟,弃废液。
(5)加入500μl中和液,12,000×g离心1分钟,弃废液。
(6)加入700μl洗涤液,12,000×g离心1分钟,弃废液。
重复步骤(6)1次。
(7)12,000×g离心2分钟,弃废液,将回收柱移入新的接收管上,室温干燥5分钟。
(8)加入30μl去离子水,12,000×g离心1分钟,洗脱DNA片段,于-20℃保存备用。
1.5搭桥PCR法进行VH和VL的拼接
将制备的VH和VL片段作为模板,进行VH和VL片段的串联。
PCR反应体系:
RSC-F:5’GCTGGAGATCAAAGGTGGCGGT3’
RSC-B:5’TGTAGCTGCACCTGAGATCCACC3’
PCR反应条件为94℃预变性5分钟,然后按如下参数进行30个循环:94℃变性30秒,60℃退火45秒,72℃延伸1.5分钟,最后72℃延伸10分钟。重复胶回收步骤,PCR产物溶于30μl ddH2O中。图1为VH和VL PCR产物鉴定图,其中右侧泳道为VH+VL串联体。
1.6连接到噬菌体载体上,
Sfi1酶切载体和PCR产物
37℃酶切2小时,用相同的方法回收酶切片段。
连接反应
上述反应物充分混匀并离心使其沉于管底后,4℃连接过夜。酶切后的片段与pComb3XSS载体片段连接,构建成重组质粒。图2为重组载体电泳鉴定图。其中,右侧泳道为重组体片段pComb3XSS载体酶切图谱,左侧泳道为分子量标记。
1.7转化和表达
将构建好的载体(质粒)转化(电转)到大肠杆菌内(特定的大肠杆菌),扩增大肠杆菌并加辅助噬菌体,收集重组后的噬菌体就是噬菌体抗体库。
经检测百人天然ScFv抗体库的库容量为2*1010,完全满足抗体筛选的需要。
1.8VEGF抗体的筛选
从抗体库中取出10ul在大肠杆菌中扩增,收集扩增后的抗体库,VEGF A亚型蛋白包被ELISA板后加入抗体库,孵育后冲洗非特异噬菌体,消化特异结合噬菌体,富集后将消化的噬菌体感染大肠杆菌涂板,挑取单克隆菌团并小量诱导,小量诱导后的单克隆菌上清做ELISA筛选阳性克隆,经过多次验证,选取亲和力和特异性都高(亲和力达到10-8-10-9KD)的阳性克隆进行抗体表达。图3为筛选出的VEGF ScFv与VEGF-165蛋白的结合试验。其中5号克隆的OD值最高,显示出其具有的高度的特异性亲和力,选取该克隆进行进一步的序列分析。
经过序列分析,该ScFv克隆的轻链的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQID NO.1、2和3所示,所述抗体的重链的CDR1、CDR2和CDR3区的氨基酸序列分别如SEQ IDNO.5、6和7所示。
所述抗体的轻链可变区和重链可变区的氨基酸序列分别如SEQ ID NO.4和8所示。
本发明提供的单链抗体来源于人源的噬菌体抗体库,因此其序列是全人源的,这为该抗体在人体内作为治疗药物或者体内成像诊断试剂的使用奠定了低免疫原性基础。对该抗体进行进一步改构,在轻链和重链之间添加如SEQ ID NO.9所示的连接多肽。将编码该单链抗体的核苷酸序列(SEQ ID NO.10)克隆入pGEM-T Easy,并转化入大肠杆菌DH5α保存。制备该单链抗体时,将编码该单链抗体的核苷酸序列克隆入表达载体pET30,再转化到相应表达菌BL21(DE3)中,筛选最佳表达条件进行大量诱导表达,最后选择最佳的纯化方法及缓冲液,获得抗体蛋白。图4为纯化后的VEFG ScFv聚丙烯酰氨凝胶电泳鉴定图。
大肠杆菌BL21(DE3),基因型为hsdS gal(λcIts857ind1Sam7nin5lacUV5-T7),用于重组蛋白质的表达。
pGEM-T Easy:用于PCR产物的克隆,购自Promega公司。
原核表达质粒载体pET30,pET42,pGEX-4t-1均为本室保存。
VEGF单链抗体的纯化
将含有六个组氨酸标记的融合蛋白VEGF ScFv-GH-His6以0.45μm滤膜过滤,准备过柱。
HisTrap kit亲和柱用Binding Buffer 10ml平衡后,加入已经准备好的待纯化样品。调节流速约为8-10滴/分钟。
使用Binding Buffer洗柱。
使用6ml Elution Buffer洗脱柱子。分管收集洗脱液。取少量进行SDS-PAGE鉴定,结果见图4所示,目的条带在25KD左右,富含目的蛋白的各管于-70℃保存。
实施例2.VEGFR对VEGF单链抗体和VEGF-165蛋白结合的阻断试验
通过竞争ELISA试验验证VEGF单链抗体能够阻断VEGF-165蛋白与其受体VEGFR2的结合。将不同稀释度的VEGFR与VEGF单链抗体混合,加入包被了VEGF-165蛋白的孔中,孵育1小时。进行常规ELISA操作。结果见图5所示。由图5可见,随着VEGFR2浓度不断升高,OD值越小,意味着VEGFR2对VEGF单链抗体和VEGF-165结合的阻断效应越强。即该VEGF单链抗体可以阻断机体中VEGF-165与其受体VEGFR2的结合。经计算其IC50值为0.002mg/ml。
实施例3.绿色荧光蛋白(FITC)标记的VEGF ScFv的细胞免疫荧光染色(confocal)
利用胰腺癌细胞系panc-1验证VEGF ScFv对肿瘤细胞的结合。
首先FITC标记VEGF ScFv,步骤如下:
(1)将待交联的抗体(浓度≥1mg/ml)对交联反应液透析三次(4℃),至pH=9.0。交联反应液配制方法:7.56g NaHCO3,1.06g Na2CO3,7.36g NaCl,加水定容至1L。
(2)将FITC溶于DMSO中,浓度为1mg/ml。每次交联使用的FITC均应新鲜配制,避光。
(3)按P:F(抗体:FITC)=1mg:150μg的比例将FITC缓慢加入于抗体溶液中,边加边轻轻晃动使其与抗体混合均匀,暗处4℃反应8小时。
(4)加入5mol/L的NH4Cl至终浓度50mmol/L,4℃终止反应2小时。
(5)将交联物在PBS中透析四次以上,至透析液清亮。
(6)交联物的鉴定。
蛋白浓度(mg/ml)=[A280–0.31×A495]/1.4,F/P比例:3.1×A495/[A280 0.31×A495],该值应介于2.5~6.5之间。FITC交联的抗体应置于pH7.4的磷酸盐缓冲液中,加入0.1%NaN3、1%BSA,4℃暗处保存。
然后进行细胞染色,步骤如下:
1)细胞种于共聚焦专用培养皿里,冰PBS洗三遍,每次5分钟。
2)细胞半干时,覆盖以4%冷的多聚甲醛固定15分钟,避光。
3)吸去多聚甲醛后,用冰PBS洗三遍,每次5分钟。
4)0.5%Triton X-100覆盖细胞10分钟,冰PBS洗三遍,每次5分钟。
5)进口胎牛血清室温封闭30分钟。
6)配制一抗VEGF ScFv:FBS=1:200。
7)加入一抗覆盖细胞,锡纸包裹4度避光过夜。
8)次日,取出细胞复温至室温约1小时。
9)冰1‰Tween洗两次,每次5分钟于摇床。冰PBS洗一次,5分钟于摇床。
10)DAPI染核,每皿1滴,完全覆盖住细胞即可。
11)冰1‰Tween洗两次,每次5分钟于摇床。冰PBS洗一次,5分钟于摇床。
12)加入防荧光淬灭封片剂,避光。
13)上机confocol。
如图6所示,标记绿色荧光蛋白的VEGF ScFv可以结合panc-1细胞胞浆中的VEGF,使得整个细胞显绿色,DAPI将细胞核染成蓝色,两种颜色叠加相互吻合,说明该绿色荧光显示的是细胞形态,该VEGF ScFv可对细胞进行成像。
实施例4.VEGF ScFv对三种胰腺癌细胞的免疫组化染色
利用三种胰腺癌细胞制作小鼠原位癌。取肿瘤组织进行冰冻切片,使用辣根过氧化物酶标记的VEGF ScFv进行组织切片染色,观察VEGF ScFv对肿瘤组织的显色效果。
首先将VEGF ScFv进行辣根过氧化物酶标记。步骤如下:
(1)称取5mg HRP溶解于1ml蒸馏水中。
(2)于上液中加入0.2ml新配的0.1M NaIO4溶液,室温下避光搅拌20分钟。
(3)将上述溶液装入透析袋中,对1mM PH4.4的醋酸钠缓冲液透析,4℃过夜。
(4)加20μl 0.2M PH9.5碳酸盐缓冲液,使以上醛化桯RP的PH升高到9.0~9.5,然后立即加入10mg IgG(抗体,或SPA5mg)在1ml 0.01M碳酸盐缓冲液中,室温避光轻轻搅拌2小时。
(5)加0.1ml新配的4mg/ml NaBH 4液,混匀,再置4℃2小时。
(6)将上述液装入透析袋中,对0.15M PH7.4PBS透析,4℃过夜。
(7)10000×g 4℃离心30分钟;
(8)将上清移入另一管中,按体积1:1加入甘油,使终浓度达50%;
(9)置-20℃保存。
然后直接法进行免疫组化染色,简便、快速,用特异性标记的VEGFScFv与组织细胞内抗原结合。
操作流程:
(1)冰冻切片经固定,凉干PBS洗;石蜡切片脱蜡至水,消化30分钟,PBS洗。
(2)适当稀释荧光抗体滴加在组织切片上,湿盒内37℃温育1小时,PBS洗3×3分钟。
(3)0.01%伊文氏蓝衬染1~3分钟,PBS洗3×3分钟,蒸馏水洗2次,除去NaCl结晶。
(4)pH9.0缓冲甘油封片。
(5)镜检。
如图7所示,分别使用Aspc1,Panc1,Bxpc3这三种细胞建立小鼠肿瘤模型,取部分肿瘤组织进行冰冻组织切片,然后进行抗体染色,可见肿瘤细胞均被VEGF ScFv单链抗体染成褐色。着色均匀,呈特异性染色。因此该VEGF ScFv单链抗体可以作为体外活检组织化学染色剂应用。
实施例5.VEGF ScFv作为体内荧光成像剂的应用
用近红外荧光(IRDye800)标记VEGF ScFv,然后尾静脉注射肝癌原位肿瘤模型小鼠,观察该荧光标记单链抗体在肿瘤模型鼠体内的分布。
标记探针方法同FITC标记方法。如图8所示,A:采集不同时间点,荧光标记探针在小鼠体内的分布。荧光颜色有蓝色、绿色和红色三种,红色为特异性着色,其他的为背景光色。看见尾静脉注射10分钟后小鼠肝脏肿瘤组织就呈现红色荧光,探针已经集聚在肿瘤部位。20分钟后肝脏红色荧光开始减少,1小时后基本代谢干净,只有散在红色斑点,考虑是***显色。B:解剖肿瘤模型小鼠,取出多个部位组织,如肠、***、肝癌组织、气管、肺、肾等器官,比较它们残留的荧光强度,发现只有肝癌组织还有特异性荧光着色,进一步证明该荧光标记探针对肿瘤组织的特异性着色。
序 列 表
<110> 北京格根生物科技有限公司
亦康(北京)医药科技有限公司
<120> 一种全人源抗VEGF单链抗体及其应用
<160> 10
<170> PatentIn version 3.3
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Claims (8)
1.一种全人源的抗VEGF单链抗体,其特征在于,所述单链抗体的轻链可变区和重链可变区的氨基酸序列分别如SEQ ID NO.4和8所示。
2.根据权利要求1所述的单链抗体,其特征在于,所述单链抗体的轻链可变区和重链可变区通过连接多肽相连。
3.根据权利要求2所述的单链抗体,其特征在于,所述连接多肽的氨基酸序列如SEQ IDNO.9所示。
4.一种生物大分子偶联物,其特征在于,在所述偶联物中,绿色荧光蛋白、近红外荧光或者辣根过氧化物酶与权利要求1-3任一所述单链抗体偶联。
5.权利要求4所述的偶联物在制备肿瘤诊断制剂中的应用。
6.根据权利要求5所述的应用,其特征在于,所述绿色荧光蛋白与所述单链抗体偶联,所述偶联物被制备为免疫荧光诊断试剂。
7.根据权利要求5所述的应用,其特征在于,所述辣根过氧化物酶与所述单链抗体偶联,所述偶联物被制备为免疫组化诊断试剂。
8.根据权利要求5所述的应用,其特征在于,所述近红外荧光与所述单链抗体偶联,所述偶联物被制备为体内荧光成像剂。
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