CN112979827A - 一种抗EGFR scFv::FTH1/FTH1蛋白纳米粒子的应用 - Google Patents
一种抗EGFR scFv::FTH1/FTH1蛋白纳米粒子的应用 Download PDFInfo
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Abstract
本发明公开了一种抗EGFR scFv::FTH1/FTH1蛋白纳米粒子在制备抗三阴性乳腺癌的制剂中的应用,所述抗EGFR scFv::FTH1/FTH1蛋白由抗EGFRscFv::FTH1蛋白和FTH1蛋白复合而成。与商业化抗EGFR单克隆西妥昔单抗相比,在相同的给药剂量下能够更优地下调EGFR及其信号通路,对表达EGFR的肿瘤细胞的增殖以及荷瘤小鼠肿瘤的生长有更显著的抑制效果,因而表现出更好的抗肿瘤功效,并且在生物体内具有较高的安全性,其为基于EGFR靶点的蛋白类纳米药物的应用提供了广阔的前景。
Description
技术领域
本发明属于纳米生物医学领域,涉及一种抗EGFR scFv::FTH1/FTH1蛋白纳米粒子的应用,特别涉及一种抗EGFR scFv::FTH1/FTH1蛋白纳米粒子在制备抗三阴性乳腺癌的制剂中的应用。
背景技术
乳腺癌(Breast Cancer,BC)是最常见的恶性肿瘤之一,也是全世界女性因癌症而致死的主要原因。三阴性乳腺癌(TNBC)约占所有乳腺恶性肿瘤的10-20%,这种组织学亚型缺乏孕激素受体(PR)和雌激素受体(ER)的表达,并且也不表达人表皮生长因子2(HER2)。鉴于缺乏这些靶向受体,化学疗法仍是TNBC的标准治疗方法,其他可以选择的治疗方案非常有限。在乳腺癌患者中,TNBC患者通常预后不良,因为它具有高度的转移性和侵袭性,患者的5年存活率非常低。
近年来有研究表明,表皮生长因子受体(epidermal growth factor receptor,EGFR)作为ErbB家族成员之一,在响应细胞外信号和通过效应通路启动下游信号级联中发挥重要作用,并可促进肿瘤发生和癌症进展。大约50%的TNBC患者中EGFR受体表达上调。与其他乳腺癌亚型相比,对TNBC特异的EGFR蛋白的过表达,通常会增加这类癌细胞对常规疗法的耐药性。EGFR在结构上由细胞外配体结合结构域,跨膜区,酪氨酸激酶结构域和含有受体自磷酸化位点的无结构C末端尾巴组成。通常表皮生长因子(EGF)等配体与EGFR的胞外结构域结合,引起构象变化,从而促进与ErbB家族成员的同二聚和异二聚化。二聚化诱导受体的酪氨酸激酶活性的激活,导致EGFR细胞内部分C末端的五个特定酪氨酸(Tyr)残基(Tyr1173、1148、1086、1068和992)自磷酸化,而Tyr 1068是主要的自磷酸化位点;磷酸化的酪氨酸残基充当衔接蛋白的停靠位点,衔接蛋白将受体连接到下游信号通路,包括Ras/Raf/MEK/ERK1/2,以实现细胞迁移,增殖和存活等的调控。因此,提示EGFR途径的激活仍然是TNBC中的主要药物靶标,抑制这种受体可能会增强TNBC治疗的功效。
然而,在自然界中EGFR受体有很多配体,比较典型的有表皮生长因子(EGF)和双调蛋白,它们都可以与EGFR结合,但是由于结合的强度和作用方式等不同,所引起下游的信号通路差别很大,导致其生物效应差别也很大。因此,以EGFR为靶点的药物开发是非常困难的。虽然看似许多药物有着相同的作用机制,但是其药物因为不同的空间结构、与靶点的亲和力、以及作用的方式的不同,其抑制的信号通路差别较大,导致其药效和安全性也不同。例如:西妥昔单抗(Cetuximab)作为研究最广泛、临床最认可的嵌合单抗,可特异性抑制EGFR。许多临床研究显示,在不同的肿瘤类型中,西妥昔单抗均显示出抗肿瘤的效果。其目前已被FDA批准用于治疗转移性结直肠癌和晚期头颈部细胞瘤。对于TNBC的治疗,在临床前研究中,Cetuximab已显示与化学疗法或酪氨酸激酶抑制剂联合,能抑制TNBC肿瘤的生长,但在单独给药Cetuximab时,靶向治疗TNBC并没有明显的疗效。此外,还出现了皮疹和肺炎等副作用,进一步限制了西妥昔单抗等靶向EGFR抗体的临床用药。进一步证明,特异性结合受体的配体,在实际应用过程中可能疗效有限,甚至没有疗效。这也是临床上缺乏有效治疗癌症的药物的主要原因之一,因此有必要进一步发展相关的治疗TNBC的药物。
发明内容
本发明所要解决的问题是为克服现有技术中缺乏特异性靶向三阴性乳腺癌细胞、抑制肿瘤生长且低毒副作用的分子靶向药物的缺陷,从而提供一种抗EGFR scFv::FTH1/FTH1蛋白纳米粒子在制备抗三阴性乳腺癌的制剂中的应用、以及含有抗EGFR scFv::FTH1/FTH1蛋白纳米粒子的药物组合物或套装药盒在制备抗三阴性乳腺癌的产品中的应用。本发明的抗EGFR scFv::FTH1/FTH1蛋白纳米粒子能够抑制EGFR信号通路,并有效抑制三阴性乳腺癌细胞及其生长,从而获得较好的治疗效果,同时呈现出高的生物安全性。
本申请人在前期构建的融合蛋白抗EGFR scFv::FTH1以及抗EGFR scFv::FTH1/FTH1蛋白纳米粒子的基础上,已证明其对表达EGFR的细胞具有很好的靶向特性(专利号:ZL201010239499.3),以及在制备治疗和防治哮喘的药物中的作用(专利号:ZL201810196090.4),但其对肿瘤(尤其是三阴性乳腺癌)治疗效果尚不清楚。
单链抗体(single chain variable fragment,scFv)是抗体衍生物的一种,它是由抗体的重链可变区(VH)和轻链可变区(VL)通过一个弹性多肽(linker)连接起来的,其分子量只有完整抗体的六分之一。虽然其亲和力有所降低,但它的优点如低免疫原性和高特异性,也使得它们成为癌症免疫治疗的候选抗体。基于此,本发明将抗EGFR scFv作为靶向性分子来介导载体蛋白或纳米粒子与细胞特异性的结合用于TNBC的治疗。首先将抗EGFRscFv融合于铁蛋白重链亚基(FTH1)的N端,获得融合蛋白抗EGFR scFv::FTH1,并进一步与FTH1蛋白一起变性复性得到抗EGFR scFv::FTH1/FTH1蛋白。
该抗EGFR scFv::FTH1/FTH1蛋白属于纳米粒子,是由24个亚基自行组装成中空笼状结构的高度对称的生物大分子。该蛋白纳米粒子是本申请人早期构建的一个蛋白,其具体序列及制备方法已在专利号为ZL201010239499.3的专利中公布。但该专利中并未对其肿瘤治疗效果进行评估,故本发明在此基础上,进一步考察该蛋白用于治疗三阴性乳腺癌的效果。
本发明第一方面提供一种抗EGFR scFv::FTH1/FTH1蛋白纳米粒子在制备抗三阴性乳腺癌的制剂中的应用,所述抗EGFR scFv::FTH1/FTH1蛋白由抗EGFR scFv::FTH1蛋白和FTH1蛋白复合而成;其中,所述抗EGFR scFv::FTH1蛋白的氨基酸序列如SEQ ID NO:1所示,且所述FTH1蛋白的氨基酸序列如SEQ ID NO:2所示;所述抗EGFR scFv::FTH1/FTH1蛋白纳米粒子中抗EGFRscFv::FTH1蛋白和FTH1蛋白的摩尔比例的比值≤2。
较佳地,用于肿瘤细胞EGFR表达呈阳性的肿瘤制剂治疗应用。
更佳地,所述的EGFR阳性肿瘤细胞为EGFR高表达的三阴性乳腺癌细胞。
所述抗EGFR scFv::FTH1蛋白和/或FTH1蛋白优选为重组蛋白。
所述抗EGFR scFv::FTH1/FTH1蛋白纳米粒子的平均粒径优选为18nm±5nm。
在本发明一较佳实施例中,所述抗EGFR scFv::FTH1/FTH1蛋白纳米粒子中抗EGFRscFv::FTH1蛋白和FTH1蛋白的摩尔比例为4:6。
本发明中的所述制剂优选包括抗EGFR scFv::FTH1/FTH1蛋白纳米粒子以及至少一种药用载体。
所述抗EGFR scFv::FTH1/FTH1蛋白纳米粒子优选为所述制剂的唯一活性成分。
本发明第二方面提供一种含有抗EGFR scFv::FTH1/FTH1蛋白纳米粒子的药物组合物或套装药盒在制备抗三阴性乳腺癌的产品中的应用。
对于所述抗EGFR scFv::FTH1蛋白、所述FTH1蛋白以及所述抗EGFR scFv::FTH1/FTH1蛋白纳米粒子的优选限定同本发明第一方面
在本发明某一较佳实施例中,当荷瘤裸鼠模型是三阴性乳腺癌时,在2.5nmol/mouse剂量下每间隔一天给药,治疗5次的肿瘤抑制率达到62±6%;当荷瘤裸鼠模型是三阴性乳腺癌时,尾静脉注射抗EGFR scFv::FTH1/FTH1蛋白纳米粒子,在2.5nmol/mouse剂量下每间隔一天给药,治疗3次,治疗周期内呈现高度生物安全性。
优选地,抗EGFR scFv::FTH1/FTH1蛋白纳米粒子抑制肿瘤细胞生长的最大剂量为3.75×10-7mol/L。
本发明中所述抗EGFR scFv::FTH1/FTH1蛋白纳米粒子在制备抗三阴性乳腺癌的制剂中的应用意指抗EGFR scFv::FTH1/FTH1蛋白纳米粒子具有预防和/或治疗三阴性乳腺癌的作用。
在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较优选实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于:
与商业化抗EGFR单克隆西妥昔单抗相比,抗EGFR scFv::FTH1/FTH1蛋白纳米粒子应用于EGFR阳性肿瘤的治疗,显示出更有效地抑制增殖和存活的信号通路,即在相同的给药剂量下能够更优地下调EGFR及其信号通路,对表达EGFR的肿瘤细胞的增殖以及荷瘤小鼠肿瘤的生长有更显著的抑制效果,因而表现出更为更好的抗肿瘤功效,并且在生物体内具有较高的安全性,其为基于EGFR靶点的蛋白类纳米药物的应用提供了广阔的前景。
附图说明
图1为抗EGFR scFv::FTH1/FTH1蛋白纳米粒子在生物体内的安全性评价图。
图2为抗EGFR scFv::FTH1/FTH1蛋白纳米粒子对MDA-MB-468细胞信号通路中EGFR、p-EGFR的表达水平影响图。
图3为抗EGFR scFv::FTH1/FTH1蛋白纳米粒子对MDA-MB-468细胞信号通路中ERK、p-ERK的表达水平影响图。
图4为抗EGFR scFv::FTH1/FTH1蛋白纳米粒子对EGFR阳性细胞MDA-MB-468和MDA-MB-231的细胞增殖影响图。
图5为在MDA-MB-231荷瘤小鼠模型上,不同浓度的抗EGFR scFv::FTH1/FTH1蛋白纳米粒子的体内药效研究。其中,A:经尾静脉注射不同浓度的抗EGFR scFv::FTH1/FTH1蛋白纳米粒子的肿瘤体积变化。B:经尾静脉注射相同浓度的抗EGFR scFv::FTH1/FTH1蛋白纳米粒子和Cetuximab后荷瘤小鼠肿瘤体积变化;数据表示为Mean±SEM(n=3),*p<0.05。
图6为复性后的抗EGFR scFv::FTH1/FTH1蛋白纳米粒子的NATIVE-PAGE示意图,其中泳道1为去铁铁蛋白,泳道2为抗EGFR scFv::FTH1/FTH1蛋白纳米粒子。
图7为抗EGFR scFv::FTH1/FTH1蛋白纳米粒子的TEM示意图。
具体实施方式
下面结合具体实施例对本发明做出进一步地详细阐述,所述实施例只用于解释本发明,并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明,均为常规方法;所使用的材料、试剂等,如无特殊说明,为可从商业途径得到的试剂和材料。
实施例1抗EGFR ScFv::FTH1/FTH1蛋白纳米粒子的制备与表征
两种质粒pET-28(+)-FTHI,pET-28a(+)-抗EGFR scFV-FTH1于本实验室现存(具体制备方法详见ZL201010239499.3)。其中,所述抗EGFR scFv::FTH1蛋白的氨基酸序列如SEQID NO:1所示,所述FTH1蛋白的氨基酸序列如SEQ ID NO:2所示。大肠杆菌感受态细胞E.coli.BL21(DE3)和E.coli.DH5a购于北京天根生化科技公司。将上述两种质粒转化入E.coli.DH5a感受态细胞内,以菌落PCR、酶切鉴定法筛选阳性克隆。之后,通过测序将序列正确的表达载体转化入Ecoil.BL.21(DE3)感受态细胞,以获得含有FTH1蛋白基因目标工程表达菌株细胞和抗EGFR scFv::FTH1蛋白基因目标工程表达菌株细胞。
接着,在抗EGFR scFv::FTH1蛋白基因目标工程表达菌株细胞中表达抗EGFRsCFv::FTH1蛋白。将所述菌株细胞培养在37℃含有卡那霉素的LB培养基中。当培养液OD值为0.4~0.6时(卡那霉素浓度为50ug/ml),加入1mM IPTG诱导表达3小时,温度为37℃。诱导后,通过5000转(20分钟)离心得到细胞沉淀,利用Buffer A(50mM Tris-HC1,pH 7.9)洗一次,然后超声破碎。将破碎得到的细胞裂解液经5000转(4℃,15分钟)离心回收沉淀,用Buffer B(50mM Tris-HC1、50mM NaCl,1mM EDTA,1%Triton X-100,pH 7.9)洗四次,得到抗EGFR scFv::FTH1的包涵体。然后将抗EGFR scFv::FTH1孵育在含8M尿素的Buffer C(50mM Tris-HC1、8M尿素、1mM EDTA,10mM DTT,pH 7.9)里,28℃过夜使得蛋白充分变性。
利用FTH1蛋白基因目标工程表达菌株细胞表达FTH1蛋白,表达条件与上述表达抗EGFR scFv::FTH1蛋白相同。不同的是所获得的FTH1为可溶蛋白。在此,采用Superose 6尺寸排阻柱AKTA(购自GE)得到纯化的FTH1蛋白。然后将FTH1蛋白溶于含8M尿素的Buffer C里,28℃过夜使得蛋白充分变性。然后,将洗涤与变性后的抗EGFR scFv::FTH1蛋白与FTHI蛋白以4:6的摩尔比混合,蛋白总质量为5mg,体积为50ml。在含4.3.2.1和0M尿素Buffer D中4℃依次透析,其中Buffer D(50mM Tris-HC1、0.5mM EDTA、50mM Nac1、10%glycerol、0.1%polyethylene glycol(PEG)、0.5mM CuSO4、pH 7.9),然后再转入Buffer E(50mMTris-HC1、50mM NaCl、10%gl ycerol、pH 7.9)透析。最后,将得到的蛋白利用50KD超滤装置过滤浓缩得到高浓度抗EGFR scFv::FTH1/FTH1杂合蛋白。
非变性聚丙烯酰胺凝胶电泳NATIVE-PAGE(如图6)和透射电子显微镜TEM(如图7)结果显示抗EGFR scFv::FTH1/FTH1蛋白纳米粒子正确折叠组装且纳米粒子的平均直径为18nm±5nm。
荧光显微镜检测抗EGFR scFv::FTHI/FTH1蛋白纳米粒子与内源性表达EGFR于细胞表面的MDA-MB-468乳腺癌细胞(购自中国科学院细胞库)的结合,如图3所示,说明抗EGFRsCFv::FTHI/FTHI蛋白具有与细胞表面EGFR受体结合的活性。
本实验结果说明,抗EGFR scFv::FTHI/FTHI蛋白纳米粒子制备成功,并在细胞实验上成功表征,得到了有活性的抗EGFR ScFy::FTH1/FTH1蛋白纳米粒子。
实施例2抗EGFR scFv::FTH1/FTH1蛋白纳米粒子在生物体内的安全性评价
试剂配制:
4%多聚甲醛:40g多聚甲醛溶于1000mL PBS,0.22μm滤膜过滤。
表1.1实验分组
实验方法:
如表1.1所示,将健康的Balb/c小鼠(中国科学院上海实验动物中心(上海斯莱克实验动物有限公司))随机分为3组,每组3只,腹腔注射,1次/2天,连续给药3次。在第7天,采用摘眼球取血收集小鼠全血至含EDTA-K2的抗凝管中。随后测定小鼠血液指标,主要指标包括:白细胞数目(WBCs)、淋巴细胞数目(Lymphs)、单核细胞数目(Mons)、中性粒细胞数目(Grans)、红细胞数目(RBCs)、血小板数目(PLTs)、血红蛋白(HGB)、红细胞积压(HCT)、平均红细胞体积(MCV)、平均红细胞血红蛋白含量(MCH)、平均红细胞血红蛋白浓度(MCHC)等。
采用脊椎脱臼法处死小鼠,收集主要器官,包括心脏,肝脏,脾脏,肺脏和肾脏,并将其储存在4%多聚甲醛溶液固定24小时。随后室温下将组织依次用50%乙醇,70%乙醇,85%乙醇脱水各1小时,95%乙醇脱水1.5小时,再放入100%乙醇中脱水1小时。脱水后进行透明,将组织浸泡于无水乙醇与二甲苯以体积比为1:1的比例配制成的混合溶液中1小时,再移至二甲苯溶液中浸泡1小时。透明后进行浸蜡与包埋,将组织置于石蜡与二甲苯体积比为1:1的混合溶液浸泡2小时。随后将组织转移至石蜡液中浸泡3小时,再转入新鲜石蜡液中浸泡3小时。浸蜡结束后,将组织包埋、切片。
采用HE染色试剂盒(碧云天生物技术公司)将组织切片HE染色,并拍照、分析。
实验结果:
Cetuximab的常规使用浓度为6.5nmol/mouse,但使用2.5nmol/mouse的抗EGFRscFv::FTH1/FTH1时即可见明显的治疗效果,所以选取该浓度进行安全性评价。结果如图1A所示,与对照组相比,抗EGFR scFv::FTH1/FTH1蛋白纳米粒子治疗7天后,体重没有明显的变化,表明不会引起明显的全身毒性。如图1B所示,治疗7天后,抗EGFR scFv::FTH1/FTH1蛋白纳米粒子在小鼠中未观察到明显的组织学水肿或炎性浸润现象。血常规检测结果如图1C。根据小鼠血液生化检测参考值,对抗EGFR scFv::FTH1/FTH1蛋白纳米粒子治疗组的白细胞、血小板、红细胞等指标评估显示,在正常范围之内。
这些结果表明抗EGFR scFv::FTH1/FTH1蛋白纳米粒子在该治疗剂量不会引起炎症等急性损伤,有潜在的临床应用价值。
实施例2抗EGFR scFv::FTH1/FTH1蛋白纳米粒子对MDA-MB-468细胞信号通路的影响
细胞株:
人三阴性乳腺癌细胞株MDA-MB-468(中国科学院上海实验动物中心(上海斯莱克实验动物有限公司))。其完全培养基为:含10%胎牛血清的L-15培养基,培养条件:100%空气,无CO2,37℃恒温。
试剂配制:
(1)细胞裂解液:将PMSF、磷酸酶抑制剂A、磷酸酶抑制剂B、RIPA细胞裂解液以体积比0.5:1:1:50混合。
(2)10×TBS:12.1g Tris-OH和43.8g NaCl溶于500mL ddH2O中,用0.22μm滤膜过滤,pH 7.6。
(3)TBST:含0.1%Tween-20的1×TBS溶液。
(4)蛋白封闭液:含5%脱脂奶粉的TBST溶液。
(5)磷酸化蛋白封闭液:含5%BSA的TBST溶液。
所有试剂均采用双蒸水配制获得。
实验方法:
将贴壁培养的MDA-MB-468细胞用胰蛋白酶消化并计数,以1.5×106个/瓶的密度铺板于T25细胞培养瓶中,100%空气,无CO2、37℃恒温条件下过夜培养,使细胞贴壁。待细胞融合度达到70%-80%时,更换饥饿培养基(0.5%胎牛血清的L-15培养基),培养24小时。各组在细胞饥饿的同时分别加入不同的药物,其中AG1478(西格玛奥德里奇公司)组仅在细胞饥饿结束前1小时加入AG1478,加药量如表1.2所示。其中AG1478的常规使用量为1×10- 5mol/L,24小时后,以终浓度为20ng/mL的EGF或10%胎牛血清刺激细胞10分钟。
表1.2实验分组
EGF或含10%血清的培养基刺激后,用预冷的1×PBS缓冲液洗涤细胞2次。再加入1×PBS缓冲液,用细胞刮刀将细胞刮下,收集至EP管中。离心4℃,12000rpm,3分钟,弃去上清,收集细胞沉淀。每个EP管中加入50μL细胞裂解液,吹打混匀。冰上孵育30分钟。离心4℃,12000rpm,10分钟,收集上清,即为细胞总蛋白溶液,-80℃保存备用。
用BCA法测量每个样品蛋白含量。
SDS-PAGE:采用8%浓度的聚丙烯酰胺凝胶,蛋白上样量50μg,恒压120V,至看到明显的彩色Maker条带分开,溴酚蓝指示剂距玻璃板下缘约1cm时,即可停止。
Western blot:恒流350mA,湿转3小时。封闭液室温下封闭1小时,TBST洗膜4次,每次7分钟。一抗按表1.3的稀释比例在4℃孵育过夜,TBST洗膜4次,每次7分钟。二抗室温下孵育30分钟,TBST洗膜4次,每次7分钟。ECL显色后曝光、拍照、分析。上述一抗、二抗,如表1.3所示。
表1.3用于检测信号蛋白的抗体
实验结果:
EGFR是TNBC患者中始终被发现过表达或扩增的主要生长因子之一。配体结合后,这种生长因子激活下游信号传导,从而促进肿瘤生长、转移,细胞周期进程,细胞增殖,分化,凋亡和血管生成。EGFR受体酪氨酸激酶可以通过Ras/Raf/MEK/ERK1/2信号通路调节细胞增殖与存活。如图2和3所示,抗EGFR scFv::FTH1/FTH1蛋白纳米粒子能够显著抑制三阴性乳腺癌细胞MDA-MB-468中EGFR、ERK总蛋白的表达,同时下调EGFR和ERK的磷酸化,从而使Ras/Raf/MEK/ERK1/2信号通路受到明显的抑制,且效果优于等同浓度的Cetuximab。
本实验结果表明,抗EGFR scFv::FTH1/FTH1蛋白纳米粒子能够与EGFR特异性结合,有效抑制EGFR及其下游信号通路。
实施例3抗EGFR scFv::FTH1/FTH1蛋白纳米粒子对EGFR阳性细胞的存活率的影响
细胞株:
人三阴性乳腺癌细胞株MDA-MB-231和MDA-MB-468(中国科学院上海实验动物中心(上海斯莱克实验动物有限公司))。其完全培养基为:含10%胎牛血清的L-15培养基,培养条件:100%空气,无CO2,37℃恒温。
试剂配制:
5mg/mL MTT溶液:称取250mg MTT溶于50mL PBS中,60℃助溶使其充分溶解,用0.22μm无菌滤膜过滤除菌,pH7.4,-20℃避光保存。
MTT法测定细胞的存活率
实验方法:
将贴壁生长的MDA-MB-231和MDA-MB-468细胞用胰蛋白酶消化并计数,分别以2×103和5×103个细胞每孔的密度接种于96孔板,每孔100μL。次日,加入含有不同浓度抗EGFRscFv::FTH1/FTH1蛋白纳米粒子或Cetuximab继续孵育48小时,每一个浓度设置3个复孔,同时设置3个调零孔和3个非加药对照孔。孵育结束后,每孔加入20μL 5mg/mL MTT工作液,37℃继续孵育4个小时。孵育完成后吸去所有上清液,随后加入150μL DMSO,置于水平摇床上轻摇10分钟使得蓝紫色甲臜结晶彻底溶解。使用酶标仪测定各孔在OD490nm处的吸光度值,计算细胞存活率。细胞存活率=(待测组吸光值-空白对照组吸光值)/非加药组吸光值×100%。最后绘制细胞存活率曲线。
实验结果:
本实验选取两株细胞作为细胞模型,EGFR高表达的三阴性乳腺癌细胞MDA-MB-468,和EGFR表达量低于MDA-MB-468的三阴性乳腺癌细胞MDA-MB-231。如图4中的A所示,抗EGFR scFv::FTH1/FTH1蛋白纳米粒子对MDA-MB-468细胞生长的最大抑制率约为44%,Cetuximab对细胞生长的最大抑制率约为26%,与阴性对照相比,能明显的抑制肿瘤细胞的生长。如图4中的B所示,为EGFR表达量稍低的MDA-MB-231三阴性乳腺癌细胞,抗EGFRscFv::FTH1/FTH1蛋白纳米粒子对于细胞生长的最大抑制率约为25%,Cetuximab对于细胞生长的抑制率约为10%。
本实验结果表明,抗EGFR scFv::FTH1/FTH1蛋白纳米粒子能显著抑制三阴性乳腺癌细胞,其抑制效果优于Cetuximab。
实施例4抗EGFR scFv::FTH1/FTH1蛋白纳米粒子的体内药效研究
实验动物:
SPF级Balb/c雌性裸鼠,体重20±2克,2-3周龄。
实验方法:
MDA-MB-231乳腺癌裸鼠皮下移植瘤模型的建立:将贴壁培养的MDA-MB-231乳腺癌细胞用胰蛋白酶消化,并重悬于PBS缓冲液中,最终细胞密度为1×108cells/mL。取2-3周龄Balb/c雌性裸鼠,在每只腋下皮下注射2×107个MDA-MB-231细胞,至肿瘤体积长至100mm3。
实验分组及给药:当裸鼠体内的肿瘤长至100-300mm3时,选择肿瘤生长良好,无自发性出血坏死、瘤周无感染病灶的荷瘤裸鼠为实验模型。将小鼠随机分成6组,每组3只。如表1.4所示,尾静脉注射,1次/2天,连续给药5次。
表1.4实验分组
裸鼠体重和肿瘤体积生长变化:每天测量荷瘤小鼠的体重,绘制裸鼠的体重随时间变化曲线。给药后每天用游标卡尺对裸鼠的肿瘤体积大小进行测量,并按照公式:肿瘤体积V=1/2×肿瘤长径(L)×肿瘤短径(W)2,计算肿瘤体积变化,绘制肿瘤体积随时间变化的曲线。
实验结果:
如图5中的A所示,不同药物治疗10天后,观察到随着抗EGFR scFv::FTH1/FTH1蛋白纳米粒子浓度的增加,对肿瘤体积的抑制作用也逐渐增强。此外,我们对比了抗EGFRscFv::FTH1/FTH1蛋白纳米粒子和商业化抗EGFR单抗Cetuximab对于肿瘤生长的抑制效果,注射蛋白纳米粒子的量和单抗的量及其他注射条件均一致。如图5中的B所示,与对照组相比,抗EGFR scFv::FTH1/FTH1蛋白纳米粒子显著的抑制了肿瘤的生长,其肿瘤抑制率为62±6%,但是Cetuximab在该剂量下,几乎没有明显的抑制效果。
本实验结果表明,抗EGFR scFv::FTH1/FTH1蛋白纳米粒子能够显著抑制Balb/c小鼠肿瘤生长,其对肿瘤的治疗效果远好于Cetuximab。
最后所应当说明的是,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,对于本领域的普通技术人员来说,在上述说明及思路的基础上还可以做出其它不同形式的变化或变动,这里无需也无法对所有的实施方式予以穷举。凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明权利要求的保护范围之内。
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Claims (7)
1.一种抗EGFR scFv::FTH1/FTH1蛋白纳米粒子在制备抗三阴性乳腺癌的制剂中的应用,其特征在于,所述抗EGFR scFv::FTH1/FTH1蛋白由抗EGFR scFv::FTH1蛋白和FTH1蛋白复合而成;其中,所述抗EGFR scFv::FTH1蛋白的氨基酸序列如SEQ ID NO:1所示,且所述FTH1蛋白的氨基酸序列如SEQ ID NO:2所示;所述抗EGFR scFv::FTH1/FTH1蛋白纳米粒子中抗EGFRscFv::FTH1蛋白和FTH1蛋白的摩尔比例的比值≤2。
2.如权利要求1所述的抗EGFR scFv::FTH1/FTH1蛋白纳米粒子在制备抗三阴性乳腺癌的制剂中的应用,其特征在于,所述抗EGFR scFv::FTH1蛋白和/或FTH1蛋白为重组蛋白。
3.如权利要求1或2所述的抗EGFR scFv::FTH1/FTH1蛋白纳米粒子在制备抗三阴性乳腺癌的制剂中的应用,其特征在于,所述抗EGFR scFv::FTH1/FTH1蛋白纳米粒子的平均粒径为18nm±5nm。
4.如权利要求1所述的抗EGFR scFv::FTH1/FTH1蛋白纳米粒子在制备抗三阴性乳腺癌的制剂中的应用,其特征在于,所述抗EGFR scFv::FTH1/FTH1蛋白纳米粒子中抗EGFRscFv::FTH1蛋白和FTH1蛋白的摩尔比例为4:6。
5.如权利要求1所述的抗EGFR scFv::FTH1/FTH1蛋白纳米粒子在制备抗三阴性乳腺癌的制剂中的应用,其特征在于,所述制剂包括抗EGFR scFv::FTH1/FTH1蛋白纳米粒子和至少一种药用载体。
6.如权利要求5所述的抗EGFR scFv::FTH1/FTH1蛋白纳米粒子在制备抗三阴性乳腺癌的制剂中的应用,其特征在于,所述抗EGFR scFv::FTH1/FTH1蛋白纳米粒子为所述制剂的唯一活性成分。
7.含有抗EGFR scFv::FTH1/FTH1蛋白纳米粒子的药物组合物或套装药盒在制备抗三阴性乳腺癌的产品中的应用。
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