CN104211796A - 胰腺癌相关肽和热休克蛋白形成的复合物及其应用 - Google Patents
胰腺癌相关肽和热休克蛋白形成的复合物及其应用 Download PDFInfo
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- CN104211796A CN104211796A CN201310550992.0A CN201310550992A CN104211796A CN 104211796 A CN104211796 A CN 104211796A CN 201310550992 A CN201310550992 A CN 201310550992A CN 104211796 A CN104211796 A CN 104211796A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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Abstract
本发明公开了一种胰腺癌相关的MUCIN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5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胰腺癌的特异性的CTL。这种复合物可用于制备治疗胰腺癌的治疗性疫苗。
Description
技术领域
本发明属于生物技术领域,特别涉及胰腺癌抗原多肽与热休克蛋白gp96和HSP78的复合物以及它们抗胰腺癌的用途。
背景技术
胰腺癌是一种消化系统危害严重的恶性肿瘤。由于胰腺解剖位置特殊,症状缺乏特异性,早期诊断困难,80%的胰腺癌患者在确诊时已经是中晚期,够进行根治性手术者仅10%-15%,而且它的发病率逐年升高,在欧美居常见恶性肿瘤的第4位,在我国位于恶性肿瘤发病率的第7位。
手术切除是目前可能治愈胰腺癌的唯一手段,但仅有极少的胰腺癌患者肿瘤能获得手术切除。并且由于胰腺癌根治术后仍有极高的复发与转移发生率,即便是R0切除的患者其预后仍然很差,3年生存率仅为27% (95% 可信区间23-32%),中位生存时间只有15-19个月。在我国死亡率位于所有恶性肿瘤的第6位。为了使胰腺癌患者根治术后能够获得长期存活,术后辅助治疗仍然是目前的研究重点之一。
近年来研究发现自体肿瘤组织中分离纯化的gp96和HSP78可以激活肿瘤特异性T细胞,起到抑制肿瘤生长的作用。热休克蛋白gp96自体免疫治疗胰腺癌的I期临床试验中位生存期达2.2年,中位无复发生存期达0.9年;治疗过程中未见严重不良事件报告。自体gp96复合物免疫治疗可以在胰腺癌术后辅助治疗中发挥作用。
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(Endoplasmic Reticulum),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。在细胞内gp96作为分子伴侣蛋白,可阻止蛋白质聚集,协助蛋白质折叠、伸展、组装和转运,抑制错折叠蛋白质的分泌。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。
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,从而提高免疫反应。
目前已证实gp96和hsp78分子可以结合多种结合抗原多肽,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。但目前为止未见从胰腺癌患者肿瘤组织中分离鉴定与HSP结合的抗原多肽。
胰腺癌是严重的消化系统恶性肿瘤。前期的动物试验和I期临床试验显示,与目前的胰腺癌的标准治疗方法相比,自体gp96免疫治疗显著延长中位生存期(6月 vs 2年),具有延缓胰腺癌复发的效果。然而,由于胰腺组织特殊的解剖结构,限于这种治疗方法对肿瘤组织大小的要求和疫苗制备的一次性,能够完成有效疗程的患者有限,胰腺癌从总体上仍然不能治愈。
发明内容
因此,本发明要解决的技术问题是筛选能激活胰腺癌特异性T细胞的肽段。克服肿瘤细胞免疫编辑导致的低免疫原性,能够在健康个体及胰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具有强大的抗胰腺癌作用,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结合的胰腺癌抗原的复合物。所述的胰腺癌抗原可以分别是胰腺癌肿瘤抗原MUC1蛋白上第200-209位的氨基酸序列,其序列可以是TLVHNGTSA,或其变异序列;胰腺癌肿瘤抗原MUC1蛋白上第391-399位的氨基酸序列,其序列可以是ALLVLVCVL,或其变异序列;胰腺癌肿瘤抗原MUC1蛋白上第379-387位的氨基酸序列,其序列可以是CVLVALAIV,或其变异序列;胰腺癌肿瘤抗原MUC1蛋白上第119-207位的氨基酸序列,其序列可以是STTPPAHDV,或其变异序列;胰腺癌肿瘤抗原MUC1蛋白上第335-343位的氨基酸序列,其序列可以是AFREGTINV,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,又包括热休克蛋白以共价键与多肽结合。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。
本发明所诉的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。
本发明所诉的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阳性胰腺癌患者PBMC分泌IFN-g,且这种作用在自体gp96免疫后显著增强。
本发明所诉的表位肽可以单独或以热休克蛋白复合物形式提高HLA-A2阳性胰腺癌患者特异性T细胞反应。
本发明所述的特异性CTL的诱导可以被Treg细胞的抑制剂增强。
首次从五例胰腺癌肿瘤组织中与热休克蛋白gp96结合的多肽中分离出一特异9肽,经氨基酸序列分析为“TLVHNGTSA”,经查询发现该序列位于胰腺癌特异性抗原MUC1 的200-209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胰腺癌的治疗性药物。
附图简要说明
图1.小鼠(BALB/cHLA-A2+)的特异性CTL反应。以9肽和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胰腺癌细胞的生长。裸鼠皮下接种人胰腺癌细胞Panc-1,荷瘤7天后转输不同来源的小鼠脾脏淋巴细胞。抗原肽1组(n=20)的接受经过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.胰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-g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. 从胰腺组织中纯化gp96蛋白
将五份胰腺癌组织和一份正常胰腺组织匀浆后离心,用30%/70%饱和度(NH4)2SO3沉淀,沉淀溶解后采用ConA Sepharose(GE公司)进行亲和层析,用8%的a-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 mm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。比较肿瘤组织和正常组织HPLC图谱,发现一个五份胰腺癌组织共有而正常胰腺组织中没有的肽峰。
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1112 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“TLVHNGTSA”,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MUC1蛋白的200-209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“TLVHNGTSA”(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表达胰腺癌抗原肽1和热休克蛋白gp96的融合蛋白
本实例提供了胰腺癌抗原肽1(TLVHNGTSA)与热休克蛋白gp96以共价键形成的融合蛋白的方法。我们人工合成对应于该肽的核酸序列(ACCCTGGTGCATAACGGCACCAGCGCG),引入限制性酶切位点Bgl II将该9肽的核酸序列与gp96基因5’端用T4连接酶连接,连接产物的5’和3’端引入2个限制性酶切位点EcoR I和Xho I,连接到表达载体pPICZaA中在毕赤酵母中分泌表达,表达产物是gp96与该9肽的融合蛋白。
实施例7. 免疫小鼠
选用生长8-10周的HLA-A2转基因的BALB/c小鼠(HLA-A2+)用于本实验。
免疫方式采用将样品溶于100mL的PBS中颈背皮下注射。皮下注射操作相对简单,要求免疫剂量适中,故采用该种免疫方式。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。
因此采用皮下注射免疫,将9肽溶于缓冲液(90% PBS 10% DMSO 0.1% TFA) gp96-9肽复合物溶于PBS中,注射前剧烈振荡1分钟,每只小鼠免疫剂量9肽分别为0.2 nmol, 2 nmol和20 nmol,将肽与弗氏佐剂乳化后免疫小鼠。gp96-9肽复合物免疫剂量分别是0.01 nmol,0.05 nmol, 0.10 nmol和0.50 nmol,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天后进行细胞毒性分析。每一处理使用10只小鼠。
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脾细胞悬于含有10 mM HEPES缓冲液、5 X 10-5 M琉基乙醇,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1mg/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10mg/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100 mL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,每只小鼠免疫0.1 nmol(约10mg)的gp96-9肽复合物即能诱发机体产生强烈的细胞免疫[Y1] 细胞毒性测定靶细胞的裂解率在60%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胰腺癌的治疗药物。
实施例9细胞转输体内抑瘤实验
以gp96-胰腺癌抗原肽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。将获得的淋巴细胞转输Panc-1皮下接种14天的裸鼠作为实验组(◆)(n=20);对照组的裸鼠传输的是免疫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-胰腺癌抗原肽1复合物免疫小鼠的淋巴细胞可明显抑制人胰腺癌细胞的生长(图2)。
实施例10 胰腺癌患者免疫自体gp96复合物治疗肿瘤
该研究中入组的gp96免疫治疗的胰腺癌患者需完成6个疗程的治疗。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胰腺癌患者。
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 for isolation 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6个疗治的治疗需要。
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6次。
iv 免疫学指标检查
以自体肿瘤裂解液、自体gp96复合物抗原以及胰腺癌抗原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。比较胰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,于3个月、6个月、1年、1年半、2年时各访视1次;之后每半年访视1次,直至患者复发或死亡。
①2年内每3个月复查1次:
a) 第3、9、15、21月复查内容:
肿瘤标志物(CA-199、CEA、AFP);
腹、盆腔B超;
胸部X线;
b) 第6、12、18、24月复查内容:
肿瘤标志物(CA-199、CEA、AFP);
胸、腹、盆腔增强CT;
②2年后每6个月复查1次,复查内容:
肿瘤标志物(CA-199、CEA、AFP);
胸、腹、盆腔增强CT;
实施例11 ELISPOT法检测特异性T细胞
ELISPOT检测胰腺癌患者PBMC中表位特异性CTL。按照ELISPOT试剂盒的说明书操作.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100mL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10mg/mL的9肽、阳性对照加入4mg/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阳性的胰腺癌患者作为研究组,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阳性胰腺癌患者中均检测到高频率的TLVHNGTSA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3)。
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4个胰腺癌抗原多肽与gp96体外组装并测定其免疫活性,这4个抗原多肽分别是1)MUC1抗原多肽“ALLVLVCVL”,2) MUC1抗原多肽“CVLVALAIV”, 3 ) MUC1抗原多肽“STTPPAHDV”,4)MUC1抗原多肽“AFREGTINV”。
分别人工合成这4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4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,4种肽与gp96结合反应中K值均在5.5以上。采用实施例7中所述的方法用上述4种胰腺癌抗原多肽与gp96形成的复合物,和上述gp96与4种胰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4种复合物进行CTL分析,从51Cr释放实验可以看出这4种胰腺癌抗原多肽与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10mg)时即能诱发机体产生强烈的细胞免疫反应,通过对这4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60%-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的方法表明这四种肽与gp96形成的复合物免疫在体内也可以抑制胰腺癌细胞的生长。
以上实验结果表明gp96与胰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胰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胰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5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疫苗。因此,除上述5种抗原多肽之外,其它任何胰腺癌抗原多肽与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5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5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,HSP78-9肽复合物的免疫原性为单独9肽的150倍以上,每只小鼠免疫0.1 nmol(约10mg)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胰腺癌的治疗药物。
序列表
<110> 深圳市康尔诺生物技术有限公司
<120> 胰腺癌相关肽和热休克蛋白形成的复合物及其应用
<130>
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 表位肽1
<400> 1
Thr Leu Val His Asn Gly Thr Ser Ala
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<211> 9
<212> PRT
<213> 人工序列
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<223> 表位肽2
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Ala Leu Leu Val Leu Val Cys Val Leu
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<210> 3
<211> 9
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<213> 人工序列
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<223> 表位肽3
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Cys Val Leu Val Ala Leu Ala Ile Val
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<211> 9
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<213> 人工序列
<220>
<223> 表位肽4
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Ser Thr Thr Pro Pro Ala His Asp Val
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<212> PRT
<213> 人工序列
<220>
<223> 表位肽5
<400> 5
Ala Phe Arg Glu Gly Thr Ile Asn Val
1 5
Claims (8)
1.一种与热休克蛋白结合的表位肽TLVHNGTSA,其特征在于,其氨基酸序列如序列表中SEQ ID NO.1所示。
2.一种与热休克蛋白结合的表位肽ALLVLVCVL,其特征在于,其氨基酸序列如序列表中SEQ ID NO.2所示。
3.一种与热休克蛋白结合的表位肽CVLVALAIV,其特征在于,其氨基酸序列如序列表中SEQ ID NO.3所示。
4.一种与热休克蛋白结合的表位肽STTPPAHDV,其特征在于,其氨基酸序列如序列表中SEQ ID NO.4所示。
5.一种与热休克蛋白结合的表位肽AFREGTINV,其特征在于,其氨基酸序列如序列表中SEQ ID NO.5所示。
6.如权利要求1-5任一项所述的表位肽与gp96或HSP78的共价或非共价连接的复合物。
7.如权利要求1-5任一项所述的表位肽在体外单独或与热休克蛋白形成如权利要求6所述的复合物单独或共同免疫人体诱导生成杀伤性T细胞中用途。
8.如权利要7所述的免疫诱导杀伤性T细胞在预防或治疗胰腺癌中的用途。
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