CN105175527B - 一种乳腺癌特异性的热休克蛋白复合物及其应用 - Google Patents
一种乳腺癌特异性的热休克蛋白复合物及其应用 Download PDFInfo
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Abstract
本发明公开了一种乳腺癌相关的Her‑2、EGFR及uPar蛋白表达的长度为9个氨基酸的肽段,所述肽段的氨基酸序列见序列表中的序列1至序列7中的任意一个。本发明还提供了这些抗原肽与热休克蛋白gp96和HSP78的复合物及其制备方法,复合物包括gp96和HSP78与抗原多肽以非共价键结合的复合体,和二者以共价键连接形成的融合蛋白。这些肽段单独或形成的复合物可以诱导针对乳腺癌的特异性的CTL。这种复合物可用于制备乳腺癌的治疗和预防的疫苗。
Description
本发明属于生物技术领域,更具体地,本发明公开了一类能有效激活乳腺癌特异性T细胞的热休克蛋白复合物以及制备方法及其在治疗和预防乳腺癌中的应用。
技术背景
肿瘤尤其是恶性肿瘤是当今世界严重危害人类健康的疾病,乳腺癌对妇女的生命健康的威胁尤为严重。现全世界每年乳腺癌发病已达到130 万例,乳腺癌与宫颈癌一起被称为女性“双癌”疾病。近年来随着分子生物学、细胞生物学、遗传学和免疫学等研究的不断进展,乳腺癌治疗已形成在传统的手术和放化疗基础上,结合内分泌治疗和生物治疗的综合治疗体系。
人源化抗体Trastuzumab(赫塞汀)靶向Her-2,通过介导细胞毒作用杀伤肿瘤细胞、阻断信号通路、抑制肿瘤细胞生长,取得了很好的治疗效果。目前赫塞汀己成为乳腺癌等治疗的一线药物。uPAR(Urokinase plasminogen activator receptor),表达于细胞表面,能够引起胞外基质的降解,从而在肿瘤的转移中发挥重要作用。68%的侵染性乳腺癌组织表达uPAR,并且抑制uPAR的表达可以增强HER-2抗体的抑增殖效果。EGFR(表皮生长因子受体)在多种恶性肿瘤组织中表达显著增高,在临床上应用EGFR的抗体可以显著减少癌细胞表面EGFR的水平发挥治疗效果。在乳腺癌患者中有接近一半的癌患者肿瘤组织过表达EGFR。现在已经出现针对这些乳腺癌相关抗原的靶向治疗方案。但是T细胞活化是肿瘤治疗最关键的因素,目前却没有很好的针对这些靶点的乳腺癌T细胞治疗方案。
gp96是热休克蛋白HSP90家族中的重要成员,主要定位于内质网腔(EndoplasmicReticulum),在正常组织和肿瘤中均有广泛表达。正常情况下gp96在细胞中呈低水平表达,而热休克、葡萄糖缺乏、细菌和病毒感染等均可增加其表达。gp96分子具有多肽结合的能力,能结合5-25个氨基酸的多肽序列。热休克蛋白HSP78是细胞质中HSP70家族中的成员,分子量约78kDa。HSP78分子作为分子伴侣能与细胞中各种短肽结合。目前已证实gp96和HSP78分子可以结合多种组织特异性抗原多肽,包括泡状口炎病毒抗原区肽、小鼠H-2Kb限制的卵清蛋白抗原表位肽、H-2La限制的白血病表位肽、乙肝病毒抗原肽等。
在细胞外,gp96可作为呈递分子将结合的短肽呈递给I类MHC分子,激活特异性和记忆性T细胞,引发机体细胞免疫反应。因此HSP本身不具备抗原性,所结合的短肽决定了HSP复合物的抗原性。gp96还可以活化树突细胞(DC)促进MHC I类和MHC II类分子以及共刺激因子的表达,从而增强免疫反应。
肿瘤自体gp96复合物因为携带有患者自身肿瘤的抗原肽,可以激活患者个体特异性的T细胞反应杀伤肿瘤细胞,目前已成功应用于黑色素瘤、神经胶质瘤等的临床治疗。而乳腺癌相关的临床应用未见报道。
发明内容
因此,本发明要解决的技术问题是筛选能激活乳腺癌特异性T细胞的肽段。克服肿瘤细胞免疫编辑导致的低免疫原性,能够在健康个体及乳腺癌患者中诱导产生特异性细胞毒性T淋巴细胞(杀伤性T细胞,CTL),而且CTL具有强大的抗乳腺癌作用,对正常细胞及组织无破坏作用。
本发明的一个目的是提供一种与gp96和HSP78结合的乳腺癌抗原的复合物。所述的乳腺癌抗原可以分别是乳腺癌肿瘤抗原Her-2蛋白上第635-643位的氨基酸序列,其序列可以是VVLGVVFGI,或其变异序列;乳腺癌肿瘤抗原EGFR蛋白上第28-36位的氨基酸序列,其序列可以是KLKDPELSL,或其变异序列;乳腺癌肿瘤抗原uPar蛋白上第52-60位的氨基酸序列,其序列可以是RLWEEGEE,或其变异序列;乳腺癌肿瘤抗原uPar蛋白上第80-88位的氨基酸序列,其序列可以是RTGLKITSL,或其变异序列;乳腺癌肿瘤抗原uPar蛋白上第87-95位的氨基酸序列,其序列可以是SLTEVVCGL,或其变异序列;乳腺癌肿瘤抗原uPar蛋白上第107-115位的氨基酸序列,其序列可以是VTYSRSRYL,或其变异序列;乳腺癌肿瘤抗原uPar蛋白上第144-152位的氨基酸序列,其序列可以是CLDVVTHWI,或其变异序列。
本发明的复合物既包括热休克蛋白以非共价键与多肽结合形成的复合物,又包括热休克蛋白以共价键与多肽结合形成的复合物。本发明提供了制备本发明的如上所述抗原多肽与热休克蛋白gp96和HSP78的复合物的方法。
本发明所诉表位肽可以单独或以热休克蛋白复合物形式在HLA-A2转基因小鼠中诱导形成特异性的CTL。
本发明所诉表位肽可以在体外单独或以热休克蛋白复合物形式刺激HLA-A2阳性乳腺癌患者PBMC分泌IFN-γ,且这种作用在自体gp96免疫后显著增强。
本发明所诉表位肽可以单独或以热休克蛋白复合物形式提高HLA-A2阳性乳腺癌患者特异性T细胞反应。
本发明所述特异性CTL的诱导可以被Treg细胞抑制剂增强。
我们首次从五例Her-2阳性乳腺癌肿瘤组织中纯化的热休克蛋白gp96复合物中分离出一特异9肽,经氨基酸序列分析为“VVLGVVFGI”,经查询发现该序列位于乳腺癌特异性抗原Her-2的635-643位,人工合成该序列并与体外表达的gp96蛋白组装,体外合成gp96-9肽复合物,将该gp96-9肽复合物免疫HLA-A2转基因小鼠,能刺激小鼠产生特异性细胞毒性T细胞(CTL),且应用调节性T细胞的抑制剂可以增强gp96-9肽复合物的免疫效果2倍以上。实验结果表明gp96-9肽复合物可开发成为一种新型乳腺癌的治疗性药物。
附图简要说明
图1.小鼠(BALB/cHLA-A2+)的特异性CTL反应。以gp96-9肽复合物按0、1、3周的周期免疫小鼠,最后一次免疫3天后检测特异性细胞裂解率。效应细胞CTL对特异性靶细胞的裂解百分率以4小时标准51Cr释放法测定,效应细胞与靶细胞之比分别是10、25、50和100,图中裂解率为10只小鼠平均值。
图2.gp96-肽段1免疫的小鼠脾脏淋巴细胞可抑制裸鼠皮下接种的乳腺癌细胞的生长。裸鼠腋下皮下接种人乳腺癌细胞SK-BR-3,荷瘤14天后转输不同来源的小鼠脾脏淋巴细胞。抗原肽1组(n=20)接受经过gp96-抗原肽1复合物免疫的BALB/c HLA-A2+小鼠的脾脏淋巴细胞;无关肽对照组(n=20)接受经过gp96-无关肽(HBcAg87-95)复合物免疫的Balb/c HLA-A2+小鼠的脾脏淋巴细胞。
图3.小鼠(BALB/cHLA-A2+)的特异性CTL反应。以Her-2阳性或者Her-2阴性的乳腺癌患者肿瘤组织来源gp96抗原复合物按0、1、3周的周期免疫小鼠,最后一次免疫3天后检测9肽特异性T细胞的变化。以IFN-γ的ELISPOT方法检测小鼠脾脏淋巴细胞中9肽特异性T细胞。无关肽HBcAg82-90肽为阴性对照。结果显示只有Her-2阳性乳腺癌患者来源的gp96抗原复合物能激活9肽特异性的T细胞,表明复合物中包含相关的抗原。
图4.乳腺癌患者免疫自体肿瘤gp96复合物后9肽特异性T细胞的变化。以IFN-γ的ELISPOT方法检测患者外周血中9肽特异性T细胞。无关肽HBcAg82-90肽为阴性对照。
具体实施方式:
下面用实施例来进一步说明本发明,但本发明并不受其限制。下列实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂商所建议的条件。
实施例1. 从乳腺组织中纯化gp96蛋白
将Her-2+ 乳腺癌组织或者Her-2- 乳腺癌组织匀浆后离心,用30%/70%饱和度(NH4)2SO4沉淀,沉淀溶解后采用ConA Sepharose(GE公司)进行亲和层析,用8%的α-甲基葡萄糖苷洗脱结合的蛋白,糖苷洗脱液以Hitrap-Q (GE公司层析系统)进行阴离子层析,经过这三步纯化获得>95%纯度的gp96蛋白。gp96蛋白用gp96/grp94单克隆抗体(Santa Cruz公司)进行Western鉴定。其纯度用SDS-PAGE、银染和反相HPLC鉴定。
实施例2.从gp96蛋白释放非共价结合的多肽
将纯化的gp96蛋白加入三氟乙酸(TFA)使其终浓度达到0.2%(pH约2.0),然后用超滤法(分子截留为30 kDa, Millipore公司)分离多肽,将多肽混合物进行MALDI-TOF质谱(ABI 4700)分析,多肽分子量大多在600-1200 Da之间。
实施例3.反相HPLC分析多肽
多肽混合物冻干后溶于溶液A(0.065% TFA,2%乙腈), 反相层析柱C18上样(Sephasil peptide C18;5 um粒度;4.6 X 250mm,GE公司),从0至65%溶液B(0.05% TFA,100%乙腈)进行梯度洗脱,流速为1 mL/min,用214nm波长检测。比较Her-2+与Her-2- 肿瘤组织HPLC图谱,发现一个Her-2+ 组织共有而Her-2- 组织中没有的肽峰。
实施例4.多肽微量测序与序列分析
收集该特异肽峰用MALDI-TOF质谱(ABI公司 4700系统)鉴定其纯度,只发现分子量为1015 Da的单一峰。对该肽微量测序(Procise 491.蛋白测序仪,ABI公司),其氨基酸序列为“VVLGVVFGIL”,5份肿瘤组织得到的序列一致。在蛋白数据库中查询该序列(NCBI),发现它位于Her-2蛋白的635-643位。
实施例5. gp96蛋白与人工合成的多肽体外快速组装
人工合成9肽“VVLGVVFGI”(委托吉尔生化有限公司合成),将gp96蛋白与多肽进行体外组装,体外结合反应体系:
2.7 mmol/L KCl,1.47 mmol/L KH2PO4,8.1 mmol/L Na2HPO4,138mmo1/L NaCl,10%(V/V)甘油,3.0 mmol/L 9肽,0.421 mmol/L gp96蛋白,60℃反应10分钟,超滤法(截留分子量30 kDa,Millipore公司)去除未结合的多肽。
实施例6表达乳腺癌抗原肽1和热休克蛋白gp96的融合蛋白
本实例提供了乳腺癌抗原肽1(VVLGVVFGI)与热休克蛋白gp96以共价键形成的融合蛋白的方法。我们人工合成互补的核酸序列(5‘ GATCC GTGGTCTTGGGGGTGGTCTTTGGGATCA 3’及5‘ GATCT GATCCCAAAGACCACCCCCAAGACCAC G 3‘),并通过复性获得双链DNA片段,核酸序列对应于该肽的氨基酸序列,同时还在序列的5‘端引入限制性酶切位点BamH I粘性末端,3端引入限制性酶切位点Bgl II的粘性末端,获得粘性片段1。我们通过PCR在gp96的基因两端分别引入限制性酶切位点Bgl II和Xho I,限制性酶切获得粘性片段2。我们酶切真核表达载体pSecTag2/Hygro A获得BamH I、XhoI双切的粘性片段3。将3个DNA片段用T4连接酶连接,将抗原肽-gp96表达框插入到表达载体pSecTag2/Hygro A。将基因工程载体pSecTag2 /HygroB-Seq1-hgp96通过电穿孔的方式转染CHO细胞,以潮霉素B 抗生素筛选阳性克隆,并通过ELISA检测培养上清中的gp96获得高产细胞株。
实施例7. 免疫小鼠
选择出生8-10周的HLA-A2转基因的BALB/c小鼠(HLA-A2+)用于本实验。
免疫采用皮下免疫方式,部位为颈部,体积100uL,溶剂为PBS。同时在每次免疫前1天腹腔注射0.4 mg的环磷酰胺。低剂量环磷酰胺可以抑制在gp96免疫中起抑制作用的Treg细胞从而能增强免疫效果。
gp96蛋白-9肽复合物的最适免疫剂量为0.1 nmol。
免疫时间为0、1、3周进行三次的强化免疫,优于免疫一次或二次的效果。
免疫操作:将乳腺癌组织来源的gp96复合物及gp96-9肽复合物溶于PBS中,注射前充分混匀。免疫剂量, gp96-9肽复合物剂量为0.10nmol。肿瘤gp96的免疫剂量为25ug。采用颈背皮下注射,第一次免疫1周后进行第二次免疫,2周后加强免疫,3天后进行T细胞活性检测。
实施例8. 细胞毒性(CTL)分析
小鼠加强免疫3天后,从每只小鼠收获得约3 X 107脾脏淋巴细胞悬于含有10 mMHEPES缓冲液、5 X 10-5 M巯基乙醇,抗生素和10% (V/V) FCS培养液中,于培养瓶中与经幅射(4500 Rad)的T2细胞(3:1)和1ug/mL肽在完全培养基中37℃培养。6天后收集脾细胞进行4小时标准51Cr释放实验(具体方法见Kuhrober, A, et al. 1997.InternationalImmunology, 9(8):1203-1212)测定细胞毒性活性。简而言之,靶细胞用10ug/mL抗原肽或无关肽于37℃致敏30分钟后加入不同数量的效应细胞,反应体系为100uL的完全培养基。37℃共培养4小时后收集上清测定特异裂解率。
从51Cr释放实验可以看出gp96-9肽复合物可刺激小鼠产生特异性细胞毒性T细胞,细胞毒性测定靶细胞的裂解率在50%以上,通过注射环磷酰胺的预处理可以明显的提高gp96-9肽复合物的免疫效果,且这种细胞毒效应是表位肽特异性的(图1)。实验结果表明gp96-9肽复合物可开发成为一种新型抗乳腺癌的治疗药物。
实施例9细胞转输体内抑瘤实验
以gp96-乳腺癌抗原肽1免疫HLA-A2转基因小鼠获得抗原肽1特异性的脾脏淋巴细胞。将SK-BR3(HLA-A2+/HER-2+)细胞接种于Balb/c(nu/nu)腋窝皮下准备乳腺癌荷瘤小鼠,在接种第14天时开始接受小鼠脾脏淋巴细胞转输。实验组荷瘤小鼠接受肽特异性淋巴细胞转输(◆)(n=20);对照组小鼠转输免疫gp96-无关肽的小鼠淋巴细胞(■)(n=20),每周转输一次,共3周。检测肿瘤大小,如图所示转输gp96-乳腺癌抗原肽1复合物免疫小鼠的淋巴细胞可明显抑制人乳腺癌细胞的生长(图2)。
实施例10 乳腺癌患者免疫自体gp96复合物治疗肿瘤
该研究中入组的乳腺癌患者需完成10个疗程的gp96免疫治疗。一周为一个疗程,每个疗程注射一次。试验组在术后8周内开始自体gp96免疫治疗。
操作流程
i 患者入选:初发可根治性切除的乳腺癌患者。
ii 肿瘤组织中gp96-肿瘤抗原复合物的纯化
按照孟颂东等发表的三步法(Meng S, Song J, Rao Z, Tien P, Gao G. 2002.Three-step purification of gp96 from human liver tumor tissues suitable forisolation of gp96-bound peptides. Journal of Immunological Methods, 264(1-2):29-35.)对肿瘤组织中gp96蛋白进行提取纯化,通过ConA柱亲和层析结合Hitrap Q柱离子交换方法纯化得到纯度95%以上gp96蛋白。
考马斯亮蓝显色法测定蛋白浓度,每份样本的蛋白提取量应能满足10个疗程的治疗需要。
微生物检测,应满足对蛋白疫苗制品的要求(参照生物制品规程)。
内毒素检测,应满足对蛋白疫苗制品的内毒素含量要求(鲎试剂法)。
iii gp96-肿瘤抗原复合物的使用
患者在手术后8周内接受第一次免疫治疗。每次免疫前1天,静脉注射环磷酰胺,然后三角肌部位皮下或腹部皮下注射自体gp96复合物。每周1次,共注射10次。
iv 免疫学指标检查
以自体肿瘤裂解液、自体gp96复合物抗原以及乳腺癌抗原9肽、gp96-9肽复合物为刺激物检测自体肿瘤特异性T细胞活性。比较乳腺癌患者免疫前后特异性T细胞活性,发现自体gp96复合物免疫显著激活了患者特异性T细胞,预示了良好的预后。
v 随访
注射完成后访视患者,直至患者死亡。
1)2年内每3个月复查1次:
B超(包括乳腺、腋窝、术侧胸壁、腋窝,腹部脏器和妇科检查)
肿瘤标志物(CA-153、CEA、CA23);
2) 2年后每6个月复查1次,复查内容:
B超(包括乳腺、腋窝、术侧胸壁、腋窝,腹部脏器和妇科检查)
肿瘤标志物(CA-153、CEA、CA23);
3) 乳腺钼靶摄片:一年一次
实施例11 ELISPOT法检测特异性T细胞
ELISPOT检测小鼠脾脏淋巴细胞或者乳腺癌患者PBMC中表位特异性CTL。按照ELISPOT试剂盒的说明书操作。
小鼠第3次免疫肿瘤gp96 3天后检测T细胞激活情况。先用含10%胎牛血清的PRMI1640培养基封闭ELISPOT预包被板一小时,临用前倒掉封闭液,加入100uL含1 x 105的小鼠脾脏淋巴细胞。实验组中加入10ug/mL的9肽、阳性对照加入4ug/mL的PHA进行刺激,阴性对照加入无关肽。在细胞培养箱内培养26-36小时后,检测特异性斑点的形成,并进行统计分析。我们选取Her-2阳性及Her-2阴性的乳腺癌患者来源的gp96复合物作为研究对象,结果发现只有Her-2阳性患者来源的gp96可以激活9肽1特异性的T细胞(图3)。
对于人PBMC先用含10%胎牛血清的PRMI 1640培养基封闭ELISPOT预包被板一小时,临用前倒掉封闭液,加入100uL含1 x 105的人的PBMC(直接用新鲜的PBMC或者将PBMC体外扩增培养7天)。实验组中加入10ug/mL的9肽、阳性对照加入4ug/mL的PHA进行刺激,阴性对照加入无关肽。在细胞培养箱内培养26-36小时后,检测特异性斑点的形成,并进行统计分析。我们选取HLA-A2阳性及Her-2阳性的乳腺癌患者作为研究对象,通过ELISPOT方法检测患者新鲜血液中表位特异性CTL,特异性CTL的频率通过计数斑点数量来确定。在4例经自体gp96复合物免疫的HLA-A2阳性及Her-2阳性的乳腺癌患者中均检测到高频率的VVLGVVFGI特异性CTL,且比免疫前有显著的提高,同时并没有检测到无关肽HBcAg82-90(阴性对照)特异性T细胞,说明ELISPOT结果是表位特异的(图4)。
实施例12 其它gp96-多肽复合物的免疫活性测定
除上述抗原9肽之外,我们又选取其它6个乳腺癌抗原多肽与gp96体外组装并测定其免疫活性,这6个抗原多肽分别是1)EGFR抗原多肽“KLKDPELSL”,2) uPAR抗原多肽“RLWEEGEEL”, 3 ) uPAR抗原多肽“SLTEVVCGL”,4)uPAR抗原多肽“RTGLKITSL”,5)uPAR抗原多肽“VTYSRSRYL”,6)uPAR抗原多肽“CLDVVTHWI”。
分别人工合成这6种多肽,采用实施例5中所述的反应体系在体外与gp96结合,这6种多肽与gp96均有较高的亲和力,通过测定结合反应平衡常数K,6种肽与gp96结合反应中K值均在5.2以上。采用实施例7中所述的方法用上述6种乳腺癌抗原多肽与gp96形成的复合物,和上述gp96与6种乳腺癌抗原多肽的融合蛋白分别免疫小鼠,并采用实施例7中所述的方法分别对这6种复合物进行CTL分析,从51Cr释放实验可以看出这6种乳腺癌抗原多肽与gp96形成的复合物均可刺激HLA-A2转基因小鼠产生特异性细胞毒性T细胞。每只小鼠免疫剂量为0.1 nmol(约10ug)时即能诱发机体产生强烈的细胞免疫反应,通过对这6种多肽与gp96形成的复合物的细胞毒性测定发现靶细胞的裂解率在55%-75%之间。调节性T细胞抑制剂的应用可以增强复合物的免疫效果。采用实施例9的方法表明这6种肽与gp96形成的复合物免疫在体内也可以抑制乳腺癌细胞的生长。
以上实验结果表明gp96与乳腺癌抗原多肽体外组装合成的复合物可开发成为一种新型乳腺癌的治疗或预防药物。由于实验条件所限本发明专利不可能对每一种乳腺癌抗原多肽进行体外组装及免疫活性测定,但我们通过选取7种有代表性的肿瘤抗原肽作研究对象,在体外与gp96结合并进行免疫活性测定,大量实验表明gp96与这些抗原多肽形成的复合物均能刺激小鼠产生强烈的免疫反应,可开发成为一种新型治疗和预防性的疫苗。因此,除上述7种抗原多肽之外,其它任何乳腺癌抗原多肽与gp96结合作为新型疫苗也应当在本发明的保护范围之内。
实施例13. HSP78-多肽免疫原性测定
将HSP78与7种多肽体外组装形成的复合物,反应体系同gp96(见实施例5),将体外合成的复合物和HSP78与7种多肽的融合蛋白(见实施例6)免疫小鼠。小鼠免疫方式、免疫剂量以及免疫程序同gp96(见实施例7)细胞毒性(CTL)分析方法同gp96(见实施例8)。从51Cr释放实验可以看出HSP78-9肽复合物可刺激小鼠产生特异性细胞毒性T细胞,每只小鼠免疫0.1 nmol(约10ug)即能诱发机体产生强烈的细胞免疫反应,细胞毒性测定靶细胞的裂解率在50%以上。实验表明HSP78-9肽复合物可开发成为一种新型乳腺癌治疗和预防的药物。
序列表
<110> 深圳市康尔诺生物技术有限公司
<120> 一种乳腺癌特异性的热休克蛋白复合物及其应用
<130> 一种乳腺癌特异性的热休克蛋白复合物及其应用
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<170> PatentIn version 3.3
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Claims (1)
1.表位肽与gp96或HSP78的共价或非共价连接的复合物与环磷酰胺联用在制备免疫人体诱导生成杀伤性T细胞的药物中的应用,其特征在于,表位肽VVLGVVFGI氨基酸序列如序列表中SEQ ID NO.1所示。
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