CN104628866B - 一种靶向vegfr2的抗体融合蛋白的制备及其用途 - Google Patents
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Abstract
本发明属基因工程抗体技术领域,具体涉及肿瘤血管内皮生长因子受体2(VEGFR2/KDR3)抗体和MICA的融合蛋白的制备方法及用途。本发明利用基因工程技术将VEGFR2的全长抗体与NK细胞的活化型受体(NKG2D)的配体MHC I类分子相关蛋白A(MICA)通过柔性肽连接并通过CHO细胞进行表达,形成的融合蛋白一方面能够作用于肿瘤细胞表面的VEGFR达到抑制或杀死肿瘤肿瘤及抑制或破坏肿瘤新生血管的目的,另一方面能够增加肿瘤细胞表面MICA的含量,通过NK细胞表面NKG2D的识别,刺激NK细胞杀伤肿瘤细胞,重建由NKG2D途径激活机体自身的免疫监视作用,并且增强抗体Fc段介导的依赖细胞介导的细胞毒性(antibody‑dependent cell‑mediated cytotoxicity,ADCC)效应等杀伤肿瘤细胞。
Description
技术领域
本发明属于生物工程领域,具体涉及一种可与肿瘤血管内皮生长因子受体(VEGFR/KDR)及NK细胞的活化型受体(NKG2D)特异结合的高亲和力全人源的融合蛋白,能抑制血管内皮生长因子(KDR)受体的活化,可抑制高表达血管内皮生长因子受体人血管内皮细胞HUVEC的生长,并且可增强NK细胞对人乳腺癌细胞的抗体依赖性的细胞介导的细胞毒作用(ADCC),是一种具有靶向VEGFR及NKG2D的抗肿瘤及血管生成活性的高特异性基因工程融合蛋白。
背景技术
如何在杀死肿瘤细胞的同时不伤害病人是我们一直面临的难题。目前抗肿瘤药物设计的关键是不仅要杀死肿瘤细胞,而且要恢复机体的免疫监视作用,让免疫系统来杀死肿瘤细胞。肿瘤和免疫系统之间的相互作用可以分为:早期免疫介导的肿瘤“清除”、“平衡”和肿瘤的“逃逸”三个阶段。免疫系统免疫监护作用的一个重要的机制就是肿瘤细胞表面MHC-I相关抗原分子A和B(MHC class I-related chain molecules A and B,MICA/B)的表达,MICA/B是表达于自然杀伤细胞(Natural killer cells,NK细胞)的活化型受体NKG2D的配体。NK细胞借MICA/B与NKG2D的相互作用而与肿瘤细胞靠近、结合,从而通过释放细胞因子等诱导肿瘤细胞的溶解,进而杀伤肿瘤细胞。但研究发现血清可溶性MICA(sMIC)水平的显著升高,导致NK细胞功能缺陷,并进一步指出可溶性MICA由肿瘤细胞表面MIC-A的脱落产生,肿瘤细胞逃脱免疫即发生免疫逃逸。据此,有研究者设计可溶性肽和抗MICA的抗体阻止MICA的脱落,可能发展成为一种新的抗肿瘤治疗方法。
激活和重建机体肿瘤免疫作用的一种常规的药物是抗体,比如通过阻断CTLA4的抗体已经被批准上市。抗肿瘤抗体除了人们熟知的中和或抑制某种因子诱导的细胞增殖从而抑制肿瘤生长以外,它的免疫效应功能的主要机制是抗体通过其Fc段与NK或巨噬细胞表面的Fc受体(Fc gamma receptor,FcγR),FcγRIIIa结合,免疫细胞接近结合有抗体的肿瘤细胞,激活免疫细胞以抗体依赖细胞介导的细胞毒性(antibody-dependent cell-mediated cytotoxicity,ADCC)。
抗血管内皮生长因子受体抗体(VEGFR2 antibody)
人VEGFR2称为KDR(kinase inserted domain containing receptor),是血管内皮生长因子(vascular endothelial growth factor,VEGF)发挥刺激内皮细胞的增殖、迁移,促进新生血管形成的主要受体。肿瘤新生血管的形成是肿瘤向恶性肿瘤转变必不可少的条件。此外,VEGFR-2还在乳腺癌、结肠癌、非小细胞肺癌、白血病等多种肿瘤细胞中高表达。因此,VEGF或VEGFR2的抑制剂既可以通过抑制新生血管生成,以“饥饿疗法”阻断肿瘤的营养摄取,又可以抑制肿瘤细胞的增殖或者促进凋亡。
目前临床上使用的VEGF抗体-贝伐单抗(bevacizumab)是的一株抑制VEGF通路的单抗。FDA于2014年4月批准靶向VEGFR2的抗体Ramucirumab(IMC-1121B)上市,用于治疗化疗失败的胃癌、胃食管连接处腺癌。相对于临床伴有高血压和出血等副反应的贝伐单抗,抗VEGFR2抗体有较好的安全性。
肿瘤细胞表面MHC-I相关抗原分子A(MICA)
主要组织相容性复合体(major histocompatibility complex,MHC)在免疫应答过程中参与抗原识别,与免疫系统密切相关。其中包括了对免疫起重要作用的MIC(MHCclass c ainrelated Gene)家族。MIC家族又称PerB11,位于MHCI类区域,具有高度多态性,存在多种等位基因。它包括MICA、MICB、MICC、MICD、MICE、MICF和MICG7个成员,其中只有MICA、MICB具有编码、表达、转录蛋白的功能,其余均为假基因。其中MICA位于HLA-B位点上游约46kb,全长1722bp编码1382bp的转录子。
人体中MICA集中表达在肠道上皮中,其他组织如脑、心脏、肺等中都无表达,但MICA能表达在大多数上皮性肿瘤细胞(乳腺癌、肺癌、肾癌及卵巢癌等)中,被认为是一种肿瘤相关性抗原。因此,MIC-A呈阳性表达的肿瘤细胞在机体的免疫机制中难以逃逸,MICA的脱落则使上述肿瘤细胞虽然呈MICA阳性表达,但依然能逃脱免疫监视。且在上述大多MICA呈阳性的上皮性肿瘤细胞(乳腺癌、肺癌、结肠癌等)中均存在血管内皮生长因子受体2(vascular endothelial growth factor receptor 2,VEGFR2)高表达,故VEGFR-2抗体可作为有效的载体连接MIC-A蛋白,激活NKG2D途径,从而恢复NK细胞免疫监视作用。
发明内容
发明目的
本发明提供一种具有潜在医学和药学价值的一种靶向VEGFR2的抗体融合蛋白。本发明融合蛋白的特征为特异性结合人KDR的胞外3区及NKG2D,在体外能够抑制人乳腺癌细胞MDA-MB-231和人血管内皮细胞HUVEC的生长,且能够增强NK细胞对肿瘤细胞的ADCC效应,其诱导产生的ADCC效应优于VEGFR2抗体诱导产生的效应。
技术方案
一种靶向VEGFR2的抗体融合蛋白,该融合蛋白以全人源的抗VEGFR2的全长抗体和NKG2D的配体MICA为基础,通过基因重组构建成融合蛋白,利用柔性肽将两段蛋白连接起来。
其中一条链由VEGFR2全长抗体重链和MICA蛋白组成,其氨基酸序列表为SEQNO.1;另一条链为VEGFR2全长抗体轻链,其氨基酸序列表为SEQ NO.2。
一种表达纯化法,其用于分离纯化上述的融合蛋白。
一种分离的核酸,其特征在于:该核酸编码权利要求1的靶向VEGFR2的抗体融合蛋白。
一组表达载体,含有权利要求4所述的核酸。
一种重组宿主细胞,含有权利要求5所述的表达载体。
上述任一项的蛋白或蛋白片段的应用,其特征在于选择性地与VEGFR2/NKG2D结合或抑制VEGFR2/NKG2D与VEGFR2配体/NKG2D配体的结合阻断其信号的转导。
上述任一项的蛋白或蛋白片段的偶联物。
靶向VEGFR2的抗体融合蛋白在治疗肿瘤中的应用。
发明进一步说明:
本发明中靶向VEGFR2的抗体融合蛋白由两条链组成,其中一条链由全长抗体和MICA蛋白通过柔性肽(GGGGS)连接,另一条是全长抗体的轻链。
含有本发明VEGFR2抗体/MICA融合蛋白基因的表达载体和宿主细胞株均属于本发明的保护范围。
本发明的另一个目的是提供一种可以表达和纯化上述融合蛋白的方法。
本发明利用PCR技术将筛选获得的MICA蛋白与本实验室专利单链抗体(专利号:ZL200910264180.3)构建的VEGFR2全长抗体进行克隆重组,构建VEGFR2全长抗体/MICA融合蛋白重组载体,电转入CHO细胞中,将融合蛋白基因整合到染色体上,利用新霉素G418进行筛选高表达融合抗体的稳定细胞株;对稳定细胞株扩大培养,低温离心取上清,将上清过Protein A柱进行分离纯化;Western Blot鉴定分离纯化得到的抗体;SPR实验分析抗体与抗原的亲和能力;MTT检测融合蛋白对人血管内皮细胞HUVEC和人乳腺癌细胞MDA-MB-231的生长抑制作用;ADCC实验证实融合蛋白能够增强NK细胞对肿瘤细胞的ADCC效应,其诱导产生的ADCC效应优于VEGFR2抗体诱导产生的效应。
附图说明
图1是融合蛋白基因重组分析图,重组基因包括重链基因大小约为2303bp、轻链基因大小约为762bp。
图2是融合蛋白的结构示意图,该蛋白由两条链组成,其中VEGFR2抗体的Fc区和MICA蛋白通过柔性肽(GGGGS)连接,而重链与轻链及重链是在表达细胞中自行通过二硫键结合组装在一起。
图3是SDS-PAGE蛋白质电泳图和Western Blot鉴定融合蛋白图。图3A描述发酵表达的融合蛋白通过镍柱进行分离纯化的结果,图3BC描述Western Blot鉴定分离到的融合蛋白结果。图3B是孵育anti-H+L,图3C是孵育anti-MICA。泳道1为VEGFR2的全长抗体mAb04,泳道2为NKG2D的配体MICA,泳道3为分离得到的融合蛋白mAb04-MICA。
图4是抗原抗体亲和力拟和曲线图,展示融合蛋白mAb04-MICA与抗原VEGFR2(图4A:ka(1/Ms):6.18E+05,kd(1/s):8.00E-04,KD(M):1.29E-09)或配体NKG2D(图4B:ka(1/Ms):2.65E+08,kd(1/s):188.2,KD(M):7.10E-07)的结合测试实验(Biacore)。
图5是流式检测图,描述融合蛋白mAb04-MICA与HUVEC细胞表面VEGFR2或U937细胞表面NKG2D的结合(以正常无表达VEGFR2及NKG2D的HEK293细胞作为阴性对照)。图5A,HUVEC细胞;图5B,U937细胞;图5C,HEK293细胞。
图6是曲线图,描述融合蛋白mAb04-MICA对HUVEC细胞(图6A,以VEGFR2的全长抗体mAb04为阳性对照,以NKG2D的配体MICA作为阴性对照)的生长抑制作用或对人乳腺癌细胞MDA-MB-231的生长抑制作用(图6B,以VEGFR2的全长抗体mAb04为阳性对照,以NKG2D的配体MICA作为阴性对照)
图7是流式检测图,描述融合蛋白mAb04-MICA对MDA-MB-231细胞的凋亡作用。
图8是ADCC实验结果柱形图,描述融合蛋白mAb04-MICA激活NK细胞杀伤人乳腺癌细胞MDA-MB-231的ADCC效应(以VEGFR2的全长抗体mAb04及NKG2D的配体MICA作为阴性对照)。
具体实施方式
实施例1融合蛋白mAb04-MICA的构建
分别以mAb04重轻链基因,MICA基因及pCA puro、pMH3质粒为模板,设计并合成引物进行PCR扩增。融合蛋白mAb04-MICA的构建以mAb04重链基因H’为基础,再进行重叠PCR延伸扩增获得完整的H’-MICA基因。PCR产物用1.0%琼脂糖凝胶电泳检测,琼脂糖凝胶回收试剂盒回收目的基因。PCR扩增终产物及质粒pCApuro、pMH3用限制性内切酶进行双酶切,酶切产物切胶回收后,用T4连接酶16℃过夜连接。连接后转化大肠杆菌HB2151感受态,涂布平板,次日挑取单克隆双酶切及测序鉴定。
实施例2融合蛋白mAb04-MICA的表达、纯化、鉴定
将重组质粒H’-MICA-pCA puro、H’-MICA-pMH3、L-pCA puro、L-pMH3电转进入CHOs细胞株,通过三轮挑取单克隆,获得稳定表达融合蛋白的高产细胞株。将高产细胞株扩大培养,取上清,8000rpm离心15min,0.22μm滤膜过滤样品用Protein A柱进行纯化,使用洗脱液进行洗脱,获得纯化后蛋白。A.8%(12%)SDS-PAGE分析收集样品,挑选高纯度蛋白做鉴定。B.将高纯度蛋白样品进行Western Blot鉴定。4℃,250mA恒流转印1.5h,将蛋白转印到PVDF膜(购自Millipore);转印结束,将膜在5%脱脂牛奶中室温封闭2h;PBS洗3遍,按1∶2000的比例加入anti-H+L,anti-Fc,anti-MICA抗体,37℃孵育1h,用含0.05%吐温的PBS(TPBS)洗3遍,再按1∶5000比例加入HRP偶联的羊抗鼠IgG多克隆抗体,37C孵育1h,TBS洗3遍,滴加ECL发光显色液,凝胶成像仪曝光拍照。将最终纯化并鉴定后的样品透析或超滤至PBS中,液氮速冻,-70℃保存。
实施例3融合蛋白mAb04-MICA的SPR实验
该实验中融合蛋白mAb04-MICA和各自抗原结合使用Biacore X100作为SPR-依赖的生物传感器来检测融合蛋白与VEGFR2或NKG2D之间的相互作用:A.使用CM5芯片,连接带有Fc片段的重组融合蛋白分子。分别检测浓度为0.84375、1.6875、3.375、6.25、12.5、25、50、100、200nmol/L的VEGFR2抗原,获得结合与解离曲线,用BIA评估软件分析ka(1/Ms)为6.18E+05,kd(1/s)为8.00E-04计算获得平衡解离常数KD(M)值为1.29E-09。B.使用CM5芯片,连接带有Fc片段的重组融合蛋白分子。分别检测3.906、7.8125、15.625、31.25、62.5、125、250nmol/L的NKG2D,获得结合与解离曲线,用BIA评估软件分析ka(1/Ms)为2.65E+08,kd(1/s)为188.2计算获得平衡解离常数KD(M)值为7.1E-07。
实施例4融合蛋白mAb04-MICA的流式结合实验
该实验中将融合蛋白mAb04-MICA与高表达VEGFR2的HUVEC细胞或高表达NKG2D的U937细胞作用后用流式进行检测,用于检测融合蛋白与抗原VEGFR2及配体NKG2D的结合以分析融合蛋白的结合能力。A.HUVCE细胞经胰酶消化后悬浮于PBS-0.5%BSA,细胞与抗体冰育30min。抗体与细胞表面天然VEGFR2结合后,先加入anti-IgG抗体冰育60min,PBS洗3遍后用FACs Calibur流式细胞仪分析抗原抗体结合以分析融合蛋白与抗原的结合能力。B.U937细胞离心后悬浮于PBS-0.5%BSA,细胞与抗体冰育30min。抗体与细胞表面天然VEGFR2结合后,先加入anti-IgG抗体冰育60min,PBS洗3遍后用FACs Calibur流式细胞仪分析抗原抗体结合以分析融合蛋白与抗原的结合能力。
实施例5融合蛋白mAb04-MICA对人乳腺癌细胞MDA-MB-231/血管内皮细胞HUVEC的生长抑制作用
该实验中将融合蛋白mAb04-MICA与高表达VEGFR2的HUVEC细胞或高表达VEGFR2的MDA-MB-231细胞作用,并利用SPSS软件对实验数据进行分析,以评估该蛋白的抗新生血管和抗肿瘤活性。A.将3×103~5×103个HUVEC细胞接种96孔细胞培养板,100μl/孔,在37℃5%CO2培养箱中培养。用含20ng/ml VEGF的1%ECM培养基将样品组、阳性对照mAb04、阴性对照MICA分别稀释至10组不同浓度梯度(0、2.5、5、10、20、40、80、160、200nmol/L),24h后加入100μl梯度稀释的抗体,每个浓度设置三个平行孔,继续培养72h。B.将1×104~2×104个A431细胞接种96孔细胞培养板,100μl/孔,在37℃5%CO2培养箱中培养。用1%胎牛血清培养基将样品组Db、阳性对照E10、阴性对照AK404分别稀释至10组不同浓度梯度(0、2.5、5、10、20、40、80、160、200nmol/L),24h后加入100μl梯度稀释的抗体,每个浓度设置三个平行孔,继续培养72h。
观察细胞生长状况后每孔加入11μL的MTT,37℃继续培养4h,小心倾去上清,每孔加入150μLDMSO,在酶标仪570nm/630nm波长处测定光吸收值(OD值),计算细胞增殖。融合蛋白的生物活性由细胞生长的抑制率评价,抑制率=(1-实验组OD值/对照组OD值)×100%。
实施例6融合蛋白mAb04-MICA对人乳腺癌细胞MDA-MB-231的凋亡实验
融合蛋白mAb04-MICA对MDA-MB-231的凋亡实验证明蛋白对细胞的增殖抑制作用是通过细胞程序性死亡而非细胞毒死亡。取对数生长期MDA-MB-231细胞4×105个接种于6孔细胞培养板,1ml/孔,另补充1ml/孔完全培养基,在37℃5%CO2培养箱中培养。用培养基将样品组融合蛋白、阳性对照mAb04、阴性对照MICA分别稀释至4组不同浓度梯度(0、、10、50、250nmol/L),48h后加入1.5ml梯度稀释的抗体,继续培养36h。
观察细胞生长状况后,吸弃培养基,PBS洗2遍,0.25%胰酶消化孔板细胞,冷PBS温和洗涤细胞两次;用195μL的1×Binding Buffer重悬细胞,使细胞密度为2~5×105个/ml;加5μL的Annexin V-FITC至195μL细胞重悬液,混匀后避光,室温孵育10分钟;200μL的1×Binding Buffer洗涤细胞一次,然后将细胞重悬于190μL的1×Binding Buffer;加入10μLPropidium Iodie,利用流式细胞仪分析。结果显示细胞凋亡率与抗体浓度成正相关,证明抗体可诱导血管内皮细胞及表皮癌肿瘤细胞的凋亡。
实施例7融合蛋白mAb04-MICA对人乳腺癌细胞MDA-MB-231的ADCC实验
该实验中将融合蛋白mAb04-MICA与高表达VEGFR2的MDA-MB-231细胞及NK92细胞混合后作用4h后,利用细胞毒性检测试剂盒进行检测。并利用SPSS软件对实验数据进行分析,以评估该蛋白的刺激产生的ADCC效应。将MDA-MB-231细胞、NK92细胞按比例加入96孔板中,再将融合蛋白或阳性对照mAb04或阴性对照MICA按100nM加入孔板中作用4小时,250x g离心平板4分钟,将50μl上清液转移到酶分析板中,把配好的底物50μl/孔加到酶分析板中,盖好平板,室温孵育30分钟,避光,向每孔中加入50μl终止液,在490nm记录吸光值。按
公式计算细胞毒性,利用软件分析结果。
Claims (5)
1.一种VEGFR2的全长抗体与MICA的融合蛋白,其特征在于所述的融合蛋白的一条链由VEGFR2全长抗体重链和MICA蛋白组成,其氨基酸序列为SEQ NO.1;另一条链为VEGFR2全长抗体轻链,其氨基酸序列为SEQ NO.2。
2.一种核酸,其特征在于其编码权利要求1所述的融合蛋白。
3.一种表达载体,含有权利要求2所述的核酸。
4.一种重组宿主细胞,含权利要求3所述的表达载体。
5.权利要求1所述的融合蛋白在制备治疗肿瘤药物中的应用。
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