CN103463633B - 一种靶向的嵌合乙肝核心抗原治疗性疫苗及其用途 - Google Patents
一种靶向的嵌合乙肝核心抗原治疗性疫苗及其用途 Download PDFInfo
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Abstract
本发明属于生物医药领域,涉及一种靶向表皮生长因子受体突变体3的嵌合乙肝核心抗原治疗型疫苗及其用途,本发明的治疗型疫苗由表皮生长因子受体突变体3表位抗原和乙肝核心抗原病毒样颗粒制备成嵌合抗原,该治疗型疫苗中EGFRvIII表位和乙肝核心抗原HBc能同时诱导抗EGFRvIII和HBc的抗体及相应的细胞免疫,对病毒性乙肝病人及肝癌具有协同治疗作用,可用于临床治疗肝癌及乙肝携带者肝癌病人。对预防和减低我国原发性肝癌的发生率具有显著地临床意义。
Description
技术领域
本发明属于生物医药领域,涉及一种靶向表皮生长因子受体突变体3的嵌合乙肝核心抗原治疗性疫苗及其用途,尤其是表皮生长因子受体突变体3EGFRvIII和乙肝核心抗原HBc病毒颗粒及其在治疗原发性肝癌及乙肝病毒引起的肝癌药物中的用途。
背景技术
表皮生长因子受体III突变体3(EGFRvIII)是表皮生长因子受体(EGFR)的一种最多见,也是近年受本领域研究关注的突变体。研究显示,该突变体除了在胶质瘤细胞中广泛分布,还在乳腺癌,卵巢癌,非小细胞肺癌等肿瘤细胞中均有发现,更重要的是到目前为止还未见有EGFRvIII在正常组织中表达的报道,这意味着以该突变体作为靶点而发展的抗肿瘤药物将能避免靶向野生型EGFR所带来的副作用,增加临床使用的安全性,显示了EGFRvIII作为完全的肿瘤特异性的受体,可成为新的理想的肿瘤特异性靶点(MarcH,GanesanV,PhilW,etal,NuclMedBiol,2009,36:117-128;WangS,GuoP,WangX.M,MolCancerTher.2008,7:407-417;FarnazY,VidyaB,ShayE.P,etal,MolCancerTher.2008,7:3586-3597;JamesM.R,JoshuaO.S,KimM.C,etal,BreastCancerResTr,2004,87:87-95;SintoS,JeffreyS,StephanJ.R,etal,BBA-RevCancer,2006,1766:120-139)。胶质细胞瘤作为最早发现EGFRvIII存在的肿瘤细胞,基于该突变体的用于治疗脑瘤(胶质细胞瘤)的EGFRvIII多肽疫苗取得了极大的成功,已经进入III期临床试验;EGFRvIII在其他肿瘤如乳腺癌、卵巢癌等的研究也不断深入。基于上述的发展,本申请拟提供EGFRvIII对于其它高表达该突变体的肿瘤是有希望的靶点。
中国科研人员分别2005,2006年报道在EGFRvIII肝癌及癌旁组织中高表达(欧超,吴飞翔,罗元等,癌症,2005,24:166-169;徐立,张昌卿,冯凯涛等,中山大学学报(医学科学版),2006,27:467-471.),以及EGFRvIII参与了肝癌细胞的转化及肿瘤的生成的报道,这些研究结果显示了我国肝癌发病机理及类型与国外肝癌是有差异的。
科学界越来越认同HBV慢性感染是肝癌形成的主要病因之一,HBV慢性感染能极大的增加肝癌的患病风险,相对非感染者,其患肝癌危险性甚至达到几十倍。众所周知,中国是HBV患者最多的国家,有超过1亿的HBV感染者,每年其中0.4-0.6%的慢性HBV感染者被诊断为肝癌,而肝癌患者治疗过程中都存在着免疫耐受及耐药等导致治疗失败的情况,这意味着在中国HBV感染与肝癌的发生密切相关。同时提示,仅仅针对肝癌的治疗,并不能改变患者HBV感染引发的肝癌高危险性。
EGFRvIII的表位抗原制作多肽疫苗尚存在如下缺陷:由于其缺乏天然蛋白的三维结构,免疫原性差,半衰期短,机体免疫应答低,还可能引起免疫耐受等,也不能激活抗HBV效应。
病毒样颗粒(Virus-LikeParticles,VLPs)这一表位展示载体的进展为肝癌和HBV合并治疗方案带来了可能性及可行性。与活病毒疫苗相比,病毒样颗粒无感染性,因此安全性大大提高。目前,VLPs用于制备的预防性疫苗有,人类乳头瘤病毒样颗粒、人类免疫缺陷病毒样颗粒、流感病毒样颗粒、肝炎病毒样颗粒等。默克公司的预防和治疗宫颈癌的VLP疫苗在2006-2007年已经分别上市,意味着VLPs的安全性及有效性都获得了认可。采用HBV核心颗粒(HBc)作为表位载体的疟疾疫苗也在2004-2005年进入临床一期,显示HBc的安全性及强佐剂样作用都已经获得了实验证实。因此,寻找一种既提高多肽疫苗与抗体的亲和力及增加免疫原性,又能激活机体抗病毒作用的靶向的嵌合乙肝核心抗原治疗原发性肝癌及乙肝病毒引起的肝癌药物势在必行。
与本发明相关的现有技术还有:
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发明内容
本发明的目的是提供新的靶向表皮生长因子受体突变体3的嵌合乙肝核心抗原治疗性疫苗及其用途,尤其是表皮生长因子受体突变体3EGFRvIII和乙肝核心抗原HBc病毒颗粒及其在治疗原发性肝癌及乙肝病毒引起的肝癌药物中的用途。
本发明的靶向表皮生长因子受体突变体3的嵌合乙肝核心抗原治疗性疫苗,采用乙肝病毒核心颗粒作为EGFRvIII多肽表位的疫苗载体,利用其强大的佐剂样作用,激活高效的机体免疫反应,以期打破肿瘤和病毒慢性感染所产生的免疫耐受,同时引起针对EGFRvIII表位肽和HBc载体的免疫反应,达到主要针对肝癌治疗及附带乙肝病毒感染进行双重免疫治疗的效果。
本发明的靶向表皮生长因子受体突变体3的嵌合乙肝核心抗原治疗性疫苗其特征在于,包含乙肝核心抗原HBc重组嵌合病毒样颗粒,其嵌合抗原表位是EGFRvIII表位抗原。
本发明中,EGFRvIII表位抗原为一段14个氨基酸残基组成的多肽片段:Leu-Glu-Glu-Lys-Lys-Gly-Asn-Tyr-Val-Val-Thr-Asp-His-Cys。
本发明中,EGFRvIII表位抗原的嵌合位点可以是HBc分子的N端,C端及HBc优势免疫环中的一个或者多个。
本发明通过以下方法制备靶向表皮生长因子受体突变体3的嵌合乙肝核心抗原治疗性疫苗:
1,制备嵌合乙肝病毒核心抗原,
将编码表皮生长因子突变体3(PEP-3)的DNA片段,通过常规的基因工程手段整合入乙肝病毒核心抗原的N末段,C末端或免疫优势区中的一个或者多个;
2,重组整合的HBc-EGFRvIII基因片段导入大肠杆菌,酵母或其他系统的表达质粒中进行表达优化,制得嵌合乙肝病毒核心抗原(HBc);
3,制得的嵌合嵌合HBc抗原纯化后与其他佐剂如GM-CSF等制成完整的治疗性疫苗,所述疫苗可制成溶液,悬液,气溶胶,乳液,固体或脱水物形式,例如作为冻干粉在无菌管形瓶中保存。
给药途径为皮下注射给药、粘膜给药,剂型包括注射液、气雾剂等。
本发明中,其他佐剂包括细胞因子,氢氧化铝等,但是不限定上述范围。
本发明的治疗型疫苗中,EGFRvIII表位和乙肝核心抗原HBc能同时诱导抗EGFRvIII和HBc的抗体及相应的细胞免疫,对病毒性乙肝病人及肝癌具有协同治疗作用,可用于临床中的肝癌及乙肝携带者肝癌病人。对预防和减低我国原发性肝癌的发生率具有显著地临床意义。
附图说明
图1是HBc-EGFRvIII融合蛋白发酵及纯化产物的SDS-PAGE和dot-blot分析,其中,泳道1-2为发酵产物,泳道3为低分子量蛋白标准,泳道4为纯化产物。
图2是抗EGFRvIII及HBc抗体滴度检测,
其中,融合蛋白疫苗免疫2次后一周,用ELISA方法检测抗EGFRvIII及HBc抗体滴度,与生理盐水对照组相比,本发明疫苗组能激发高强度的体液免疫效果。
图3是IL4和IFNγ的检测,
其中,融合蛋白疫苗免疫3次后一周,采集脾脏,用流式细胞仪检测IL4和IFNγ阳性的细胞丰度,结果显示,发现融合蛋白免疫3次后,与生理盐水组相比,细胞免疫被明显激活。
具体实施方式
实施例1制备嵌合HBc重组菌
编码乙肝核心抗原HBc的基因片段由乙肝病毒颗粒经PCR方法获得,截取1-149个氨基酸残基,5’和3’端分别加上BamHI和PstI的酶切位点。编码EGFRvIII的表位抗原PEP-3(Leu-Glu-Glu-Lys-Lys-Gly-Asn-Tyr-Val-Val-Thr-Asp-His-Cys)的DNA片段用重叠PCR的方法插入HBc免疫优势区78-79位氨基酸残基中间。将嵌合的HBc片段通过BamHI和PstI的酶切,连接导入大肠杆菌表达质粒PcoldIV,重组大肠杆菌表达质粒导入大肠杆菌表达宿主菌JM109。
实施例2嵌合HBc-EGFRvIII融合蛋白的发酵和纯化
将鉴定阳性的重组菌落接入5mLLB培养液中,37℃振荡培养至培养液的A600达0.4-0.6。各取2mL培养菌液加入200mL氨苄青霉素的LB培养液中,振荡培养3h,然后加入IPTG至终浓度为1mmol/L,低温培养16h,诱导蛋白表达。收集细菌用裂解缓冲液和超声细胞粉碎机裂解细菌,分别收集上清和沉淀。将沉淀(包涵体)充分溶解于8mol/L尿素中,室温16h,收集上清,在含复性液的透析袋中透析48h,收集上清即为复性后的表达蛋白,经离子交换柱和分子量,纯化后经SDS-PAGE鉴定为17kD左右,用dot-blot方法,使用HBc抗体检测,发现表达和纯化的蛋白呈阳性反应,结果如图1所示。
实施例3疫苗诱导EGFRvIII特异性抗体及细胞免疫应答实验
在6个雌性的BALB/c小鼠中试验,每小鼠在0,21,42天皮下注射10μg融合蛋白和10μg佐剂。另取6个雌性小鼠作为阴性对照,注射生理盐水。在第二次免疫后一周,检测抗EGFRvIII的抗体,结果如图2所示,有超过105的抗EGFRvIII表位的抗体滴度,及超过103滴度的针对HBc的抗体滴度。在第三次免疫后一周,检测脾脏中细胞免疫反应(IL4和IFNγ),结果如图3所示,3次免疫以后,HBc-EGFRvIII融合蛋白疫苗能诱导大量的IL4及IFNγ细胞因子分泌,引发有效的细胞免疫效应。
Claims (6)
1.一种靶向的嵌合乙肝核心抗原治疗性疫苗,其特征在于,该治疗性疫苗包含乙肝核心抗原HBcAg重组嵌合病毒样颗粒,其嵌合抗原表位是EGFRvIII表位抗原;
所述的EGFRvIII表位抗原为一段14个氨基酸残基组成的多肽片段:
Leu-Glu-Glu-Lys-Lys-Gly-Asn-Tyr-Val-Val-Thr-Asp-His-Cys。
2.根据权利要求1所述的靶向的嵌合乙肝核心抗原治疗性疫苗,其特征在于,所述的EGFRvIII表位抗原的嵌合位点是HBcAg分子的N端,C端或免疫优势区。
3.权利要求1的靶向的嵌合乙肝核心抗原治疗性疫苗的制备方法,其特征在于,其包括:
1)制备嵌合乙肝病毒核心抗原,
将编码表皮生长因子突变体3的DNA片段,通过基因工程手段整合入乙肝病毒核心抗原的N末段,C末端或免疫优势区;
2)重组整合的HBc-EGFRvIII基因片段导入大肠杆菌,酵母或其他系统的表达质粒中进行表达优化,制得嵌合乙肝病毒核心抗原;
3)制得的嵌合乙肝病毒核心抗原纯化后与细胞因子或氢氧化铝制成完整的治疗性疫苗。
4.根据权利要求3的方法,其特征在于,所述疫苗制成溶液,悬液,气溶胶,乳液,固体或脱水物形式。
5.权利要求1的靶向的嵌合乙肝核心抗原治疗性疫苗在制备治疗肝癌及乙肝携带者肝癌制剂中的用途。
6.根据权利要求5的用途,所述的治疗性疫苗的给药途径为皮下注射给药或粘膜给药。
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Growth Suppression of Human Hepatocellular Carcinoma Xenografts by a Monoclonal Antibody CH12 Directed to Epidermal Growth Factor Receptor Variant III;Hua jiang,et al.;《The journal of biological chemistry》;20110218;第286卷(第7期);第5913-5920页,尤其是摘要 * |
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