CN108350411B - 口服肿瘤疫苗 - Google Patents
口服肿瘤疫苗 Download PDFInfo
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- CN108350411B CN108350411B CN201680047072.9A CN201680047072A CN108350411B CN 108350411 B CN108350411 B CN 108350411B CN 201680047072 A CN201680047072 A CN 201680047072A CN 108350411 B CN108350411 B CN 108350411B
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Abstract
本发明提供一种能够通过可表达或呈递WT1蛋白质的转化双歧杆菌口服给药的肿瘤疫苗;不同于被限制为某一特定HLA的WT1肽疫苗,被表达或呈递于转化双歧杆菌上的WT1蛋白质是将WT1蛋白质的大部分覆盖的蛋白质;将该转化双歧杆菌作为有效成分的肿瘤疫苗能够适用于各种HLA类型的患者。
Description
技术领域
本法明涉及能够表达或呈递作为抗原的WT1蛋白质的转化双歧杆菌,进一步涉及将该转化双歧杆菌作为有效成分的肿瘤疫苗。
本申请请求通过参考援引于此的日本申请特愿2015-128034号作为优先权。
背景技术
WT1(Wilms tumor 1)基因是作为儿童肾肿瘤、即肾母细胞肿瘤的致病基因之一而分离出的基因。在肾母细胞肿瘤中,存在该基因的缺失或突变,在源自肾母细胞肿瘤的细胞株中导入正常的WT1基因时,细胞增殖被抑制,因而认为WT1基因是抑癌基因。但是,通过之后的研究,发现WT1蛋白质在白血病或各种固体癌中的表达量很高,与其说WT1基因是抑癌基因,不如说其发挥了致癌基因的功能(非专利文献1:Jpn J Clin Oncol 2010;40:377-387)。
癌的免疫疗法始于1980年代通过自然免疫进行的LAK疗法,目前进行NK细胞疗法等的自然免疫疗法和利用获得性免疫的治疗,其中,利用获得性免疫的治疗有:将构成癌抗原的蛋白质的片段的肽作为靶的肽疗法、使树状细胞识别癌肽并导回体内的树状细胞疫苗疗法等。
发现在利用WT1肽对小鼠进行免疫、或者利用WT1肽对于从人的外周血单核细胞分化出的树状细胞进行刺激时,WT1肽可以作为能够诱导WT1特异性细胞毒性T细胞的树状细胞疫苗进行使用。另外,也使用WT1肽进行了临床试验。由于现有的WT1肽以适于某一特定的人白细胞抗原(HLA)的方式进行制备,因而需要通过DNA分型来确认患者的HLA等位基因(专利文献1:日本专利特许第5714619号公报)。通过之后的研究,尝试使用将WT1蛋白质的全部序列覆盖的全序列型疫苗治疗癌症。该疫苗也能够适用于各种HLA类型的患者。该疫苗也将癌抗原特异性的杀伤性T细胞和促进免疫反应的辅助性T细胞激活。
作为WT1疫苗的给药途径,最常见的有皮下或皮内注射,除此之外,还尝试通过各种给药途径,例如经皮给药、颊给药、经鼻给药、舌下给药等的黏膜给药进行免疫诱导。但是,目前尚未有关于口服给药的报告。
细胞的细胞膜是将细胞内外分离的生物膜,细胞膜表面上存在大量具有提供细胞信息的功能和输送细胞内外物质的功能的膜蛋白质。提出了使特定的抗原与膜蛋白融合并呈递于微生物的细胞表层,作为人为诱导抗原抗体反应的口服疫苗进行使用这一概念。例如,存在使用具有对聚-γ-谷氨酸合成酶等酶蛋白的膜结合部进行编码的基因的载体,呈递于宿主微生物的细胞表层的例子(专利文献2:日本专利特表2005-50054号公报)。另外,关于将源自引起传染病的细菌的鞭毛蛋白质作为疫苗使用的技术,存在关于作为胶囊内容物而含有表达鞭毛蛋白的转化微生物的口服疫苗的报告(专利文献3:日本专利特许5187642号公报)。在此,有报告称作为生成鞭毛蛋白的细菌而使用属于双歧杆菌(Bifidobacterium)属的微生物(将其总称为“双歧杆菌”)、或者乳酸菌等所有被称为有益菌的肠内细菌来制备转化微生物。
双歧杆菌是在人或其他动物的小肠下游或大肠中发现的正常细菌,且是专性厌氧性的革兰氏阳性菌,因而培养的选择性高。而且,由于其生物相容性也高,且不存在革兰氏阴性菌中存在的内毒素,因而安全性高。因此,双歧杆菌根据有关食品安全性的审查制度的基准得到了GRAS认证。另外,也有报告称双歧杆菌具有与由覆盖肠管的粘液素构成的黏液的结合性,因而认为其在肠内的肠壁上的附着性高于其他细菌。有报告称已开发出使蛋白质或肽表达、呈递至该双歧杆菌的表层上的技术、以及关于通过使用该技术的双歧杆菌制造新疫苗的技术(专利文献4:日本专利特许第5561681号公报)。
【现有技术文献】
【非专利文献】
非专利文献1:Jpn J Clin Oncol 2010;40:377-387
【专利文献】
专利文献1:日本专利特许第5714619号公报
专利文献2:日本专利特表2005-50054号公报
专利文献3:日本专利特许第5187642号公报
专利文献4:日本专利特许第5561681号公报
发明内容
本发明的课题在于,提供一种能够口服给药的肿瘤疫苗。进而,本发明的课题在于,提供一种并不限定于特定的HLA类型的肿瘤疫苗。
本发明人们经过不断专心研究后发现,在使用能够表达和呈递WT1蛋白质的转化双歧杆菌的情况下,能够口服给药肿瘤疫苗,并完成了本发明。不同于被限定为某一特定HLA的WT1肽疫苗,被表达和呈递于转化双歧杆菌上的WT1蛋白质将WT1蛋白质的几乎全部序列覆盖,并且,将该转化双歧杆菌作为有效成分的肿瘤疫苗能够适用于各种HLA类型的患者。
即,本发明如下所示。
1.一种转化双歧杆菌,其包含编码WT1蛋白质的DNA、和编码源自双歧杆菌的GNB/LNB基质结合膜蛋白质的DNA,且被设计为将作为抗原的WT1蛋白质呈递于双歧杆菌表层上。
2.如前项1所述的转化双歧杆菌,其中,被呈递于细胞表层上的蛋白质是WT1蛋白质与GNB/LNB基质结合膜蛋白质的融合蛋白质、即GL-BP-WT1融合蛋白质。
3.如前项1或2所述的转化双歧杆菌,其中,WT1蛋白质是以下(1)至(3)中的任意一种;
(1)根据以序列号1确定的氨基酸序列而确定的蛋白质;
(2)根据以序列号1确定的氨基酸序列中的、一个或多个氨基酸被取代、缺失、附加、导入而成的氨基酸序列确定的蛋白质,且是作为疫苗具有免疫原性的蛋白质;
(3)根据与以序列号1确定的氨基酸序列具有60%以上同源性的氨基酸序列而确定的蛋白质,且是作为疫苗具有免疫原性的蛋白质。
4.如前项1至3中任一项所述的转化双歧杆菌,其中,编码WT1蛋白质的DNA是以下(1)至(4)中的任意一种;
(1)由以序列号2确定的碱基序列构成的DNA;
(2)对于根据以序列号1确定的氨基酸序列信息得到的蛋白质进行编码的DNA;
(3)与由上述(1)或(2)中确定的碱基序列构成的DNA在严格条件下杂交的DNA;
(4)由与上述(1)至(3)的任意一个中确定的碱基序列具有60%以上同源性的碱基序列构成的DNA。
5.如前项1至4中任一项所述的转化双歧杆菌,其包含编码WT1蛋白质的DNA、和编码源自双歧杆菌的GNB/LNB基质结合膜蛋白质的DNA。
6.如前项1至5中任一项所述的转化双歧杆菌,其中,在编码WT1蛋白质的DNA与编码源自双歧杆菌的GNB/LNB基质结合膜蛋白质的DNA之间,含有编码具有辅助功能的蛋白质的DNA。
7.一种制剂,其作为疫苗的有效成分而含有前项1至6中任一项所述的转化双歧杆菌。
8.如前项7所述的制剂,其中,该制剂为肿瘤疫苗制剂。
9.如前项8所述的制剂,其中,该制剂还含有佐剂。
10.如前项7至9中任一项所述的制剂,其中,该制剂为口服制剂。
11.一种肿瘤的预防或治疗方法,其包含对患者给药前项1至6中任一项所述的转化双歧杆菌、或者前项7至10中任一项所述的制剂。
12.一种双歧杆菌表层呈递蛋白质,其由前项1至4中任一项所述的转化双歧杆菌生成。
13.一种肿瘤疫苗,其作为有效成分而含有前项12所述的双歧杆菌表层呈递蛋白质。
14.一种质粒或穿梭载体,其包含编码WT1蛋白质的DNA、和编码源自双歧杆菌的GNB/LNB基质结合膜蛋白质的DNA,且被设计为将作为抗原的WT1蛋白质呈递于双歧杆菌表层上。
(发明效果)
根据本发明的转化双歧杆菌,能够将WT1蛋白质表达或呈递于双歧杆菌的细胞表层上。通过使WT1抗原蛋白质呈递于双歧杆菌的表层上,能够作为对于表达WT1蛋白质的肿瘤有效的口服疫苗进行使用。在口服疫苗的情况下,儿童或老年人均易于吸收,而且也不会有通常通过注射接种疫苗时的疼痛。尤其是,本发明的口服疫苗使用已有食用经验的双歧杆菌,因而安全性很高。另外,不同于被限定为某一特定HLA的WT1肽疫苗,本发明的口服疫苗是能够表达将WT1蛋白质的几乎全部序列覆盖的蛋白质的双歧杆菌,其HLA的限制性低。将该转化双歧杆菌作为有效成分的肿瘤疫苗能够适用于各种HLA类型的患者。
附图说明
图1是表示在GL-BP基因的下游具有WT1基因的穿梭载体和表达在双歧杆菌表面上的GL-BP-WT1融合蛋白质的示意图。(实施例1)
图2是表示对于被导入转化双歧杆菌中的WT1进行编码的DNA的确认结果的照片图。(实施例1)
图3是表示通过蛋白质印迹法确认本发明的转化双歧杆菌的表面上表达的WT1蛋白质的结果(图3中的A)、和通过免疫荧光染色进行确认的结果(图3中的B)的照片图。(实施例2)
图4是表示利用小鼠确认本发明的转化双歧杆菌的抗肿瘤效果的实验步骤的图。(实施例3)
图5是表示利用小鼠确认本发明的转化双歧杆菌的抗肿瘤效果的实验步骤的图。(实施例3)
图6是表示本发明的转化双歧杆菌的抗肿瘤效果的确认结果的照片图。(实施例3)
图7是表示经时性确认本发明的转化双歧杆菌的抗肿瘤效果的结果的图。(实施例3)
图8是表示本发明的转化双歧杆菌的抗肿瘤效果的确认结果的图。(实施例3)
图9是表示利用小鼠确认本发明的转化双歧杆菌的细胞免疫应答诱导效果的实验步骤的图。(实施例4)
图10是表示给药本发明的转化双歧杆菌时对小鼠体重的影响的确认结果的图。(实施例4)
图11是表示本发明的转化双歧杆菌对于细胞因子产生能力的影响的确认结果的图。(实施例4)
图12是表示本发明的转化双歧杆菌对于活性T细胞诱导能力的影响的确认结果的图。(实施例4)
图13是表示本发明的转化双歧杆菌的WT1特异性CTL诱导能力的确认结果的图。(实施例4)
图14是表示本发明的转化双歧杆菌的WT1特异性细胞毒性激活能力的确认结果的图。
图15是表示利用小鼠确认本发明的转化双歧杆菌的抗肿瘤效果的实验步骤的图。(实施例5)
图16是表示本发明的转化双歧杆菌的抗肿瘤效果的确认结果的图。(实施例5)
图17是表示利用小鼠确认同时使用佐剂时本发明的转化双歧杆菌的抗肿瘤效果的实验步骤的图。(实施例6)
图18是表示同时使用佐剂时本发明的转化双歧杆菌的抗肿瘤效果的确认结果的图。(实施例6)
图19是表示利用小鼠确认同时使用佐剂时本发明的转化双歧杆菌的抗肿瘤效果的实验步骤的图。(实施例7)
图20是表示同时使用佐剂时本发明的转化双歧杆菌的抗肿瘤效果的确认结果的图。(实施例7)
图21是表示在本发明的转化双歧杆菌中,根据基因水平确认对WT1蛋白质进行编码的DNA的结果的图。(实施例8)
图22是表示通过蛋白质印迹法确认本发明的转化双歧杆菌的表面上表达的WT1蛋白质的结果(图22中的A)、和通过免疫荧光染色进行确认的结果(图22中的B)的照片图。(实施例9)
具体实施方式
(WT1蛋白质)
WT1蛋白质是通过作为儿童肾肿瘤即肾母细胞(Wilms)肿瘤的致病基因之一而分离出的WT1基因进行编码的蛋白质。在WT1蛋白质中,相对于多种HLA类型而发现多个T细胞表位。本发明的WT1蛋白质只要至少包含两个以上(优选为三个以上,更优选为四个以上)的T细胞表位即可,其既可以为全长,也可以为N末端或C末端缺失的部分肽。作为WT1蛋白质的全长,可以举出以下示例。
WT1蛋白质的全长基因库登记号P22561.1(序列号22):
MGSDVRDLNALLPAVSSLGGGGGGCGLPVSGARQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTLHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPTIRNQGYSTVTFDGAPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGMAAGSSSSVKWTEGQSNHGIGYESENHTAPILCGAQYRIHTHGVFRGIQDVRRVSGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWHSCQKKFARSDELVRHHNMHQRNMTKLHVAL
WT1蛋白质的全长基因库编号P19544.2(序列号23):
MGSDVRDLNALLPAVPSLGGGGGCALPVSGAAQWAPVLDFAPPGASAYGSLGGPAPPPAPPPPPPPPPHSFIKQEPSWGGAEPHEEQCLSAFTVHFSGQFTGTAGACRYGPFGPPPPSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARSDELVRHHNMHQRNMTKLQLAL
在本说明书中,作为能够呈递于双歧杆菌上的抗原的WT1蛋白质,可以为如下确定的任意一种。
(1)根据以序列号1确定的氨基酸序列而确定的蛋白质。
(2)根据以序列号1确定的氨基酸序列中的下述氨基酸序列、即一个或多个氨基酸、例如1个~120个、优选为1个~60个、更优选为1个~10个、进一步优选为1个~9个氨基酸被取代、缺失、附加、导入而成的氨基酸序列确定的蛋白质,且是作为疫苗具有免疫原性的蛋白质。
(3)根据与以序列号1确定的氨基酸序列具有60%以上、优选为80%以上、更优选为90%以上、进一步优选为95%以上、更进一步优选为97%以上、最优选为98%以上同源性的氨基酸序列而确定的蛋白质,且是作为疫苗具有免疫原性的蛋白质。
WT1蛋白质(序列号1):
PSQASSGQARMFPNAPYLPSCLESQPTIRNQGYSTVTFDGAPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGMAAGSSSSVKWTEGQSNHGIGYESENHTAPILCGAQYRIHTHGVFRGIQDVRRVSGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWHSCQKKFARSDELVRHHNMHQ
上述WT1蛋白质(序列号1)中所含的T细胞表位如下述表1所示。本发明的WT1蛋白质优选含有两个以上的与表1所示的np332、np126、np187、np235相当的T细胞表位。更优选含有三个以上,进一步优选含有全部四个。这些T细胞表位只要能够通过T细胞识别并能够诱导细胞免疫应答即可,也可以具有氨基酸序列中的一个或多个、例如1个~5个、优选为1个~3个、更优选为1个~2个、最优选为1个氨基酸被取代、缺失、附加、导入而成的氨基酸序列。
【表1】
另外,作为本说明书中的WT1蛋白质,还含有根据以下的序列号14确定的WT1蛋白质。
WT1蛋白质(序列号14):
PSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECMTWNQMNLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARSDELVRHHNMHQ
另外,在上述WT1蛋白质(序列号14)中,具有在HLA-A*2402限制性CTL表位中导入M236Y的取代后的氨基酸序列的变异型WT1蛋白质如下所示。本说明书中的WT1蛋白质还含有以下的变异型WT1蛋白质。
变异型WT1蛋白质(序列号16):
PSQASSGQARMFPNAPYLPSCLESQPAIRNQGYSTVTFDGTPSYGHTPSHHAAQFPNHSFKHEDPMGQQGSLGEQQYSVPPPVYGCHTPTDSCTGSQALLLRTPYSSDNLYQMTSQLECYTWNQMNLGATLKGVAAGSSSSVKWTEGQSNHSTGYESDNHTTPILCGAQYRIHTHGVFRGIQDVRRVPGVAPTLVRSASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRKHTGEKPYQCDFKDCERRFSRSDQLKRHQRRHTGVKPFQCKTCQRKFSRSDHLKTHTRTHTGKTSEKPFSCRWPSCQKKFARSDELVRHHNMHQ
本说明书中的WT1蛋白质还含有改造型WT1蛋白质。在此,改造型WT1蛋白质是指通过取代或修饰等对上述确定的WT1蛋白质的全部或者一部分氨基酸进行改造后的蛋白质。改造型WT1蛋白质包含例如上述1)至3)中确定的氨基酸序列中全部或者一部分氨基酸、例如一个或多个、例如一个、两个、三个、四个、五个、六个、七个、八个、九个、十个、十一个或者十二个氨基酸被修饰后的氨基酸序列所构成的蛋白质。作为改造型WT1蛋白质所具有的氨基酸的“修饰”,并不限定于此,例如可以举出:乙酰化;甲基化等的烷基化;糖基化;羟基化;羧基化;醛基化;磷酸化;磺胺化;甲酰化;豆蔻酰化、棕榈酰化以及硬脂酰化等的脂肪链附加修饰;辛酰化;酯化;酰胺化;脱酰胺化;胱氨酸修饰、谷胱甘肽修饰或者氢硫基乙酸修饰等的二硫化物键形成修饰;糖化;泛素化;琥珀酰亚胺形成;谷氨酰胺化;异戊烯化等。改造型WT1蛋白质也可以是组合包含一个以上的氨基酸的取代、缺失或附加与一个以上的氨基酸的修饰的蛋白质。
(双歧杆菌)
在本说明书中,“双歧杆菌”是指属于双歧杆菌(Bifidobacterium)属的微生物。作为双歧杆菌,例如可以举出:青春双歧杆菌(Bifidobacterium adolescentis)、角状双歧杆菌(B.angulatum)、动物双歧杆菌动物亚种(B.animalis subsp.animalis)、动物双歧杆菌乳亚种(B.animalis subsp.lactis)、星状双歧杆菌(B.asteroides)、两歧双歧杆菌(B.bifidum)、布姆双歧杆菌(B.boum)、短双歧杆菌(B.breve)、串珠双歧杆菌(B.catenulatum)、小猪双歧杆菌(B.choerinum)、棒形双歧杆菌(B.coryneforme)、兔双歧杆菌(B.cuniculi)、龋齿双歧杆菌(B.denticolens)、齿双歧杆菌(B.dentium)、高卢氏双歧杆菌(B.gallicum)、鸡胚双歧杆菌(B.gallinarum)、球双歧杆菌(B.globosum)、印度双歧杆菌(B.indicum)、婴儿双歧杆菌(B.infantis)、异形双歧杆菌(B.inopinatum)、乳双歧杆菌(B.lactis)、长双歧杆菌(B.longum)、巨大双歧杆菌(B.magnum)、瘤胃双歧杆菌(B.merycicum)、最小双歧杆菌(B.minimum)、小双歧杆菌(B.parvulorum)、假链状双歧杆菌(B.pseudocatenulatum)、假长双歧杆菌球形亚种(B.pseudolongumsubsp.globosum)、假长双歧杆菌假长亚种(B.pseudolongumsubsp.pseudolongum)、小鸡双歧杆菌(B.pullorum)、瘤胃双歧杆菌(B.ruminale)、反刍双歧杆菌(B.ruminantium)、波伦亚双歧杆菌(B.saeculare)、世纪双歧杆菌(B.scardovii)、细长双歧杆菌(B.subtile)、猪双歧杆菌(B.suis)、嗜酸双歧杆菌(B.thermacidophilum)、以及嗜热双歧杆菌(B.thermophilum)。
其中,优选使用青春双歧杆菌(Bifidobacterium adolescentis)、动物双歧杆菌动物亚种(B.animalissubsp.animalis)、动物双歧杆菌乳亚种(B.animalis subsp.lactis)、两歧双歧杆菌(B.bifidum)、短双歧杆菌(B.breve)、乳双歧杆菌(B.lactis)、长双歧杆菌(B.longum)以及假长双歧杆菌假长亚种(B.pseudolongum subsp.pseudolongum)。
另外,也可以使用上述双歧杆菌的抗性菌株或变异菌株。这些菌株均在市场上有销售,或者能够容易地从保藏机构等获得。例如可以举出B.longum JCM1217(ATCC15707)、B.bifidum ATCC11863等。
(GNB/LNB基质结合膜蛋白质)
GNB/LNB基质结合膜蛋白质(GL-BP:Galacto-n-biose-lacto-n-bioseI-bindingProtein)是属于输送双歧杆菌所具有的乳糖-N-乙糖(即,N-Acetyl-3-O-β-D-galactopyranosyl-D-glucosamine)和半乳糖-N-乙糖(即,N-Acetyl-3-O-(β-D-galactopyranosyl)-α-D-galactosamine)的ABC蛋白质(ATP Binding Cassette protein)家族的膜蛋白质。以下,将GNB/LNB基质结合膜蛋白质仅称为“GL-BP”。ABC蛋白质是作为能量而使用ATP(腺嘌呤核苷三磷酸),在所有生物的细胞膜上主动地进行特异性物质的输送的重要的膜蛋白质,并且,细胞膜上存在多种ABC蛋白质。因此,作为ABC蛋白质的一种的GL-BP,在具备GL-BP表层表达用的细胞功能的双歧杆菌中,只要利用适当的启动子便能够普遍进行表达。在本说明书中,GL-BP的结构并不限定于天然存在的GL-BP,只要具有在双歧杆菌的细胞表层进行表达的能力,则也可以在构成该GL-BP的氨基酸中具有一个以上的取代、插入或者缺失。
(双歧杆菌表层呈递融合蛋白质)
在本发明中,表达/呈递于双歧杆菌的表层上的WT1蛋白质作为与GL-BP的融合蛋白质进行表达。该融合蛋白质从N末端起按GL-BP和WT1蛋白质的顺序连接。也可以根据需要在GL-BP与WT1蛋白质之间含有具有辅助功能的蛋白质。
(转化双歧杆菌的制备)
按照操作顺序对于将WT1蛋白质作为融合蛋白质表达/呈递于双歧杆菌表层上的转化双歧杆菌的制备顺序进行说明。
1.编码各蛋白质的DNA的获得
编码GL-BP的DNA和编码WT1蛋白质的DNA能够根据各自公知的基因信息或氨基酸序列信息获得。例如,可以将从任意的双歧杆菌制备的基因组DNA或cDNA作为模板,使用根据该双歧杆菌的GL-BP的结构基因的基因组信息制成的引物对,并通过聚合酶链式反应(PCR)进行扩增而获得。通常相对于一个氨基酸存在多种遗传密码,因此,也可以是具有与公知的碱基序列或者基于公知的氨基酸序列的碱基序列不同的碱基序列的基因。编码长双岐杆菌(B.longum)的GL-BP的DNA,例如可以根据Acta Crystallographica Section F.,2007年,F63卷,p.751中确定的长双岐杆菌(B.longum)的GL-BP的基因信息而获得。可以将长双岐杆菌(B.longum)的染色体DNA或cDNA作为模板,使用根据基因信息制成的引物对并通过PCR进行扩增,从而获得编码长双岐杆菌(B.longum)的GL-BP的DNA。编码WT1蛋白质的DNA,可以根据针对上述WT1蛋白质确定的氨基酸序列信息,并通过本身公知的方法或今后开发出的所有方法进行制作而获得。编码上述WT1蛋白质以外的各蛋白质的DNA,同样也可以通过本身公知的方法或今后开发出的所有方法进行制作而获得。
上述编码各蛋白质的DNA也可以是与如上述那样获得的DNA在严格条件下杂交的DNA。能够在严格条件下杂交的DNA是指:将上述DNA作为探针,并通过菌落杂交法、噬菌斑杂交法或核酸印迹杂交法等能够获得的DNA。具体而言,可以举出:在使用将源自菌落或噬菌斑的DNA固定化的过滤器,在约0.7M~1.0M氯化钠的存在下以约65℃进行杂交之后,使用约0.1倍~2倍浓度的SSC溶液(1倍浓度的SSC溶液由150mM氯化钠、15mM柠檬酸钠组成)在约65℃的条件下对过滤器进行清洗,从而能够进行鉴定的DNA。作为上述能够杂交的DNA,具体可以举出与编码上述根据公知的碱基序列信息或氨基酸序列信息得到的各蛋白质的DNA的碱基序列至少具有约80%以上同源性的DNA,优选为具有约90%以上同源性的DNA,更优选为具有约95%以上同源性的DNA。编码根据氨基酸序列信息得到的各蛋白质的DNA,只要是编码氨基酸的DNA,则也可以是不同的密码子。
更为具体而言,编码WT1蛋白质的DNA可以为如下确定的任意一种。
(1)由根据序列号2确定的碱基序列构成的DNA。
(2)编码根据以序列号1确定的氨基酸序列信息而得到的蛋白质的DNA。
(3)与由上述(1)或(2)中确定的碱基序列构成的DNA在严格条件下杂交的DNA。
(4)与上述(1)至(3)的任意一项中确定的碱基序列具有60%以上、优选为80%以上同源性的DNA。
编码WT1蛋白质的DNA(序列号2):
CTCGAGCCGTCCCAGGCGTCGTCGGGCCAGGCGAGGATGTTCCCGAACGCGCCCTACCTGCCCAGCTGCCTGGAGTCCCAGCCGACGATCCGCAACCAGGGCTACTCCACCGTGACGTTCGACGGCGCCCCGTCCTACGGCCACACGCCCAGCCACCACGCCGCCCAGTTCCCGAACCACAGCTTCAAGCACGAAGACCCCATGGGCCAGCAGGGCAGCCTCGGCGAACAGCAGTACAGCGTGCCGCCGCCGGTCTACGGCTGCCACACCCCGACCGACTCCTGCACGGGCTCCCAGGCCCTGCTCCTGCGTACGCCGTACTCCTCCGACAACCTCTACCAGATGACCTCCCAGCTGGAGTGCATGACCTGGAACCAGATGAACCTGGGCGCCACGCTGAAGGGAATGGCCGCGGGGTCGTCGAGCTCCGTCAAGTGGACCGAAGGCCAGTCCAACCACGGCATCGGCTACGAGTCCGAGAACCACACCGCGCCGATCCTGTGCGGAGCCCAGTACCGCATCCACACGCACGGCGTCTTCCGCGGCATCCAGGACGTCCGGCGCGTCTCCGGCGTCGCGCCGACCCTGGTGCGGTCCGCCTCCGAGACCTCCGAGAAGCGCCCGTTCATGTGCGCCTACCCGGGCTGCAACAAGCGCTACTTCAAGCTCTCGCACCTGCAGATGCACTCCCGGAAGCACACCGGCGAGAAGCCGTACCAGTGCGACTTCAAGGACTGCGAACGCCGCTTCTCGCGCAGCGACCAGCTGAAGCGCCACCAGCGTAGGCACACCGGCGTGAAGCCCTTCCAGTGCAAGACCTGCCAGCGCAAGTTCTCCCGCAGCGACCACCTCAAGACGCACACCCGCACCCACACCGGCAAGACGTCCGAGAAGCCGTTCTCGTGCCGCTGGCACAGCTGCCAGAAGAAGTTCGCCCGCAGCGACGAGCTCGTGCGCCACCACAACATGCACCAGTGAAGCATGC
另外,作为编码WT1蛋白质的DNA,可以列举出由根据以下的序列号15或序列号16确定的碱基序列构成的DNA。
编码WT1蛋白质的DNA(序列号15):
CCGTCCCAGGCGTCGTCGGGCCAGGCGAGGATGTTCCCGAACGCGCCCTACCTGCCCAGCTGCCTGGAGTCCCAGCCGGCGATCCGCAACCAGGGCTACTCCACCGTGACGTTCGACGGCACCCCGTCCTACGGCCACACGCCCAGCCACCACGCCGCCCAGTTCCCGAACCACAGCTTCAAGCACGAAGACCCCATGGGCCAGCAGGGCAGCCTCGGCGAACAGCAGTACAGCGTGCCGCCGCCGGTCTACGGCTGCCACACCCCGACCGACTCCTGCACGGGCTCCCAGGCCCTGCTCCTGCGTACGCCGTACTCCTCCGACAACCTCTACCAGATGACCTCCCAGCTGGAGTGCATGACCTGGAACCAGATGAACCTGGGCGCCACGCTGAAGGGAGTCGCCGCGGGGTCGTCGAGCTCCGTCAAGTGGACCGAAGGCCAGTCCAACCACTCCACCGGCTACGAGTCCGACAACCACACCACGCCGATCCTGTGCGGAGCCCAGTACCGCATCCACACGCACGGCGTCTTCCGCGGCATCCAGGACGTCCGGCGCGTCCCCGGCGTCGCGCCGACCCTGGTGCGGTCCGCCTCCGAGACCTCCGAGAAGCGCCCGTTCATGTGCGCCTACCCGGGCTGCAACAAGCGCTACTTCAAGCTCTCGCACCTGCAGATGCACTCCCGGAAGCACACCGGCGAGAAGCCGTACCAGTGCGACTTCAAGGACTGCGAACGCCGCTTCTCGCGCAGCGACCAGCTGAAGCGCCACCAGCGTAGGCACACCGGCGTGAAGCCCTTCCAGTGCAAGACCTGCCAGCGCAAGTTCTCCCGCAGCGACCACCTCAAGACGCACACCCGCACCCACACCGGCAAGACGTCCGAGAAGCCGTTCTCGTGCCGCTGGCCCAGCTGCCAGAAGAAGTTCGCCCGCAGCGACGAGCTCGTGCGCCACCACAACATGCACCAGTGAA
编码WT1蛋白质的DNA(序列号17):
CCGTCCCAGGCGTCGTCGGGCCAGGCGAGGATGTTCCCGAACGCGCCCTACCTGCCCAGCTGCCTGGAGTCCCAGCCGGCGATCCGCAACCAGGGCTACTCCACCGTGACGTTCGACGGCACCCCGTCCTACGGCCACACGCCCAGCCACCACGCCGCCCAGTTCCCGAACCACAGCTTCAAGCACGAAGACCCCATGGGCCAGCAGGGCAGCCTCGGCGAACAGCAGTACAGCGTGCCGCCGCCGGTCTACGGCTGCCACACCCCGACCGACTCCTGCACGGGCTCCCAGGCCCTGCTCCTGCGTACGCCGTACTCCTCCGACAACCTCTACCAGATGACCTCCCAGCTGGAGTGCTACACCTGGAACCAGATGAACCTGGGCGCCACGCTGAAGGGAGTCGCCGCGGGGTCGTCGAGCTCCGTCAAGTGGACCGAAGGCCAGTCCAACCACTCCACCGGCTACGAGTCCGACAACCACACCACGCCGATCCTGTGCGGAGCCCAGTACCGCATCCACACGCACGGCGTCTTCCGCGGCATCCAGGACGTCCGGCGCGTCCCCGGCGTCGCGCCGACCCTGGTGCGGTCCGCCTCCGAGACCTCCGAGAAGCGCCCGTTCATGTGCGCCTACCCGGGCTGCAACAAGCGCTACTTCAAGCTCTCGCACCTGCAGATGCACTCCCGGAAGCACACCGGCGAGAAGCCGTACCAGTGCGACTTCAAGGACTGCGAACGCCGCTTCTCGCGCAGCGACCAGCTGAAGCGCCACCAGCGTAGGCACACCGGCGTGAAGCCCTTCCAGTGCAAGACCTGCCAGCGCAAGTTCTCCCGCAGCGACCACCTCAAGACGCACACCCGCACCCACACCGGCAAGACGTCCGAGAAGCCGTTCTCGTGCCGCTGGCCCAGCTGCCAGAAGAAGTTCGCCCGCAGCGACGAGCTCGTGCGCCACCACAACATGCACCAGTGAA
2.双歧杆菌转化用载体的制备
对于具有上述1.中制备的编码各蛋白质的DNA的重组体DNA的制备进行说明。在本说明书中,重组体DNA可以使用表达载体或染色体整合型载体(例如同源重组型载体)。作为用于制备上述载体的质粒,只要是能够利用双歧杆菌表达的质粒便没有特别限制,也可以是本身公知的质粒或今后开发出的所有质粒。例如,作为源自双歧杆菌的质粒,可以使用pTB6、pBL67、pBL78、pNAL8H、pNAL8M、pNAC1、pBC1、pMB1、pGBL8b等。也可以使用上述质粒与大肠杆菌的质粒的复合质粒,例如可以使用pBLES 100、pKKT427、pRM2等。从表达的稳定性和用于制备转化株的DNA的制备容易度的观点来看,在上述质粒中,优选为由长双岐杆菌(B.longum)的质粒和大肠杆菌的质粒合成的复合质粒。
从选择转化株的观点来看,表达载体优选通过本身公知的方法具有抗生素抗性、氨基酸需要量等的选择标记。表达载体优选为附加有调节序列的载体,以便于表达、或者有利于表达GL-BP与WT1蛋白质的融合蛋白质。作为调节序列,例如可以举出:启动子序列、前导序列、前肽序列、增强子序列、信号序列、终止子序列等。上述调节序列只要是利用双歧杆菌表达的序列,则其来源便没有特别限制。作为启动子序列,只要是利用双歧杆菌表达的序列,便没有特别限制。从表达效率的观点来看,优选使用长双岐杆菌(B.longum)的组蛋白样蛋白(HU)的启动子序列、LDH启动子等。另外,从提高表达效率的观点来看,优选具有终止子序列。作为终止子序列,优选使用上述HU基因的终止子序列。另外,也可以在编码GL-BP的DNA与编码WT1蛋白质的DNA之间配置适当长度的编码衔接物(linker)的DNA。
由此,根据需要在上述质粒中导入启动子序列、终止子序列等的调节序列和选择标记基因,从而制备克隆载体。作为选择标记,可以举出壮观霉素(SPr)、氨苄青霉素(Ampr)、四环素(TETr)、卡那霉素(KMr)、链霉素(STr)、新霉素(NEOr)等的抗生素抗性标记;绿色荧光蛋白质(GFP)、红色荧光蛋白质(REP)等的荧光标记;以及LacZ等的酶。优选在克隆载体的启动子的下游具备具有多克隆位点的衔接物等。通过使用上述衔接物,编码上述融合蛋白质的DNA以能够在框内(in-frame)表达融合蛋白质的方式被整合在启动子的下游。作为克隆载体用的质粒,可以使用代表性的pBLES100、pBLEM100等。
通过将上述获得的HU启动子序列、编码GL-BP的DNA以及编码WT1蛋白质的DNA在框内整合至该质粒pBLES100,能够制成在双歧杆菌的表层表达融合蛋白质的载体。通过上述方法制成的表达载体被用于转化双歧杆菌。
3.表达融合蛋白质的转化双歧杆菌的制备
将重组体DNA、例如表达载体导入作为宿主的双歧杆菌中。转化方法可以使用本身公知的方法或今后开发出的所有方法。具体而言,例如可以举出电穿孔法(electroporation method)、磷酸钙法、脂质体法、钙离子法、原生质体法、显微注射法以及基因枪法等。在本发明中,优选使用电穿孔法。在使用电穿孔法的情况下,能够以0.5kV/cm~2kV/cm、0.5μsec~10msec的条件进行。更优选以2kV/cm~10kV/cm、50μsec~5msec的条件进行。
转化株可以选择融合蛋白质表达载体所具有的选择标记作为指标。作为培养转化株的培养基,可以举出适于各宿主微生物的培养基、例如葡萄糖血肝(BL)琼脂培养基、MRS(De Man Rogosa Sharpe)琼脂培养基、GAM琼脂培养基、改良GAM(TGAM)琼脂培养基、Briggs琼脂培养基以及酵母提取物葡萄糖蛋白胨(YGP)琼脂培养基。
转化体的培养优选在能够培养双歧杆菌的厌氧培养条件下进行。通过在厌氧性条件下进行培养,能够防止好氧菌的增殖。厌氧性条件可以举出能够在保持为双歧杆菌可增殖的程度的厌氧度的密闭容器中、例如厌氧室或厌氧箱等中进行培养的条件。培养温度只要是能够培养双歧杆菌的温度即可,通常为4℃~45℃,优选为15℃~40℃,更优选为24℃~37℃。
所得到的转化双歧杆菌的融合蛋白质的表达,可以通过基因重组技术中适用的本身公知的方法或今后开发出的各种方法进行确认,例如可以通过蛋白质印迹法进行确认。蛋白质印迹法也可以通过本身公知的方法进行。尤其是WT1蛋白质被呈递于双歧杆菌表层上的情况,相对于转化双歧杆菌而言,例如可以通过使用相对于WT1蛋白质的抗体和FITC标记抗IgG抗体的免疫抗体法容易地进行确认。另外,在表达GL-BP、具有辅助功能的蛋白质以及WT1蛋白质的融合蛋白质时,由于具有辅助功能的蛋白质和WT1蛋白质被呈递于双歧杆菌的表层上,因而确认中使用的抗体可以是相对于任意一种蛋白质的抗体。
已经确认了WT1蛋白质的表层呈递的转化双歧杆菌,也可以通过本领域技术人员常用的方法进行培养、回收,并直接用于制造制剂。得到的双歧杆菌也可以通过加热杀菌处理或放射线照射等进行灭活后使用。转化双歧杆菌也可以通过公知的方法进行后处理。例如,也可以通过离心分离等进行部分纯化(partial purification)。另外,也可以根据希望在部分纯化之后,使其溶解或悬浮于生理盐水、磷酸缓冲生理盐水(PBS)或者乳酸林格氏液等本领域目前使用的溶剂中。另外,也可以根据希望进行冷冻干燥或喷雾干燥,从而形成为粉状物或粒状物。
(含有转化双歧杆菌的制剂)
本发明的转化双歧杆菌,在为了治疗或预防疾病的目的而给药呈递于其表层上的WT1蛋白质为佳时,可以以任意的制剂形态进行给药。给药途径并无特别限定,可以口服给药或非消化道给药(parenteral administration),但根据本发明的目的,优选口服给药。
作为适用于口服给药的制剂的例子,例如可以举出片剂、颗粒剂、细粒剂、散剂、糖浆剂、液剂、胶囊剂或者混悬剂等。作为适于非消化道给药的制剂的例子,例如可以举出注射剂、滴剂、吸入剂、喷雾剂、栓剂、经皮吸收剂、经黏膜吸收剂等。
口服给药用的液体制剂的制造中,可以使用例如水、蔗糖、山梨糖醇、果糖等的糖类;聚乙二醇、丙二醇等的二醇类;芝麻油、橄榄油、大豆油等的油类;对羟基苯甲酸酯类等的防腐剂等的制剂用添加剂。另外,胶囊剂、片剂、散剂或颗粒剂等固体制剂的制造中,可以使用例如乳糖、葡萄糖、蔗糖、甘露醇等的赋形剂;淀粉、海藻酸钠等的崩解剂;硬脂酸镁、滑石粉等的润滑剂;聚乙烯醇、羟丙基纤维素、明胶等的黏合剂;脂肪酸酯等的表面活性剂;甘油等的增塑剂。
(口服疫苗)
本发明的呈递有WT1蛋白质的转化双歧杆菌适合作为口服疫苗进行利用。例如,WT1蛋白质在肠管壁上被识别为抗原,从而产生抗体。因此,成为有效的口服疫苗。例如,以下所述的耐酸性胶囊制剂(无缝胶囊制剂、软胶囊制剂以及硬胶囊制剂)在口服给药时,不会在pH为1~3的胃内溶解,而是通过胃内到达肠中,并在肠内溶解。通过胶囊的溶解而从制剂中释放出的转化双歧杆菌,即使在肠内环境中也保持大部分的蛋白质结构,并将WT1蛋白质呈递于其表层上。
通过口服给药转化双歧杆菌,该双歧杆菌的表层上表达的WT1蛋白质被摄入肠相关淋巴组织(GALT)中,并通过GALT内的抗原呈递细胞(APC)在适当的表位进行处理。进而,认为在GALT内被加工处理后的肽与MHCII类或MHC I类一同被呈递于APC,并诱导具有相对于该肽呈特异性的T细胞受体的CTL。通过APC而使CD8阳性T细胞、CD4阳性T细胞被激活,在从CD4阳性T细胞中释放出的IL-2等的各种细胞因子的作用下,相对于肿瘤细胞呈特异性的CD8阳性T细胞(细胞毒性T细胞(CTL))增殖。本发明的WT1蛋白质,由于CD8阳性T细胞和CD4阳性T细胞均被激活,因而相对于WT1表达肿瘤细胞能够有效地发挥抗肿瘤效果。
另外,本发明的作为有效成分而含有转化双歧杆菌的口服疫苗,也可以含有佐剂。佐剂具有增强疫苗效果的作用。作为本发明的口服疫苗中可使用的佐剂,优选为能够增强黏膜免疫的诱导的佐剂,可以举出氢氧化铝及其无机盐类、鲨烯或油等碳化氢类、霍乱毒素、大肠杆菌不耐热性肠毒素的B亚单位(LTB)、来自沙门氏菌的类脂A(MPLA)等的细菌毒素、壳聚糖或菊糖等的多糖类、以及上述物质的组合,但并不限定于此。
本发明还涉及包含向目标人员给药本发明的口服疫苗这一工序的、目标人员的肿瘤预防或治疗方法。有效成分的给药量根据目标人员的体重、年龄、症状、给药方法等而变动,本领域技术人员可以适当地进行选择。本发明的口服疫苗能够预防或治疗的肿瘤,只要能够表达WT1蛋白质便可以是任意一种,例如包括白血病、骨髓增生异常综合症、多发性骨髓瘤、恶性淋巴瘤等的造血系统肿瘤;胃癌、大肠癌、肺癌、乳癌、生殖细胞癌、肝癌、皮肤癌、膀胱癌、前列腺癌、子宫癌、宫颈癌、卵巢癌、脑癌等的固体癌。
(含有转化双歧杆菌的耐酸性胶囊制剂的制造)
本发明的口服疫苗优选为胶囊制剂的形态。在本说明书中,将内部含有内容物的胶囊称为“胶囊制剂”。本发明的胶囊制剂由胶囊包衣和表层上表达WT1蛋白质的转化双歧杆菌构成,该胶囊包衣呈耐酸性。由呈耐酸性的胶囊包衣和表层上表达WT1蛋白质的转化双歧杆菌构成的胶囊制剂,只要具有耐酸性的胶囊包衣,而且作为胶囊内容物而含有表层上表达WT1蛋白质的转化双歧杆菌,便可以采用任意构成和形状,但该胶囊制剂不排除还含有其他构成要素。因此,表层上表达WT1蛋白质的转化双歧杆菌被耐酸性的胶囊包衣包覆或封装于内部(即,被包含在由耐酸性的包衣形成的胶囊的内部区域中)。能够适用于本发明的转化双歧杆菌的胶囊制剂,可以使用本身公知的方法或今后开发出的所有方法进行制造。
为了使表层上表达WT1蛋白质的转化双歧杆菌作为口服疫苗发挥作用,必须使该转化双歧杆菌通过胃部并到达肠中,而且在肠内也保持有WT1抗原蛋白质和双歧杆菌细胞壁的蛋白质。然而,胃的pH为1~3,在如此明显很低的pH下,经口摄取的双歧杆菌中的大部分蛋白质会发生变性。因此,为了使本发明所使用的转化双歧杆菌以保持各种蛋白质结构的状态到达人的肠内,从而呈递WT1蛋白质,尽量避免转化双歧杆菌受到胃酸的影响为佳。
因此,在本发明中,优选形成为通过耐酸性的胶囊包衣包覆或封装转化双歧杆菌、即将转化双歧杆菌包含在耐酸性包衣的胶囊内侧的胶囊制剂。关于胶囊制剂的构成、形状等,只要包衣相对于胃酸具有抗性便没有特别限制。即,最好构成为胃酸不会进入胶囊内与转化双歧杆菌接触。胶囊包衣也可以是pH4以下、优选为pH1~3下不会溶解的包衣。胶囊化的方法也没有特别限制。
实施例
以下,根据实施例对本发明具体进行说明,但本发明并不限定于以下的实施例。
(实施例1:GL-BP-WT1表层呈递双歧杆菌的制备)
A.GL-BP基因的分离
从长双岐杆菌(Bifidobacterium longum)JCM1217(ATCC15707)基因组(Accession:EU193949),使用引物glt-f:5’-ggggtgctgatatattggtttg-3’(序列号3)和终止密码子被替代为XhoI的glt-r:5’-gctcgagctcggaaacagacaggccgaagtt-3’(序列号4)和KOD-Plus-(TOYOBO公司制)进行PCR反应,从而使GL-BP基因扩增。对含有扩增后的GL-BP基因的PCR产物进行琼脂糖凝胶电泳,切出1989bp的PCR产物,并使用Wizard SV凝胶和PCR清理系统(Promega公司制)仅分离出GL-BP基因扩增片段并进行纯化。
B.具有分离出的GL-BP基因的pMW118质粒的构建
将分离并纯化后的GL-BP基因扩增片段导入具有氨苄青霉素抗性基因(Ampr)的pMW118(株式会社NIPPON GENE制)的SmaI位点,从而构建质粒。另外,使用DNA连接试剂盒Ver.2(TAKARA BIO株式会社制)进行连接。通过热休克法(42℃、30秒)将构建好的质粒导入大肠杆菌DH5α(TAKARA BIO株式会社制)中,并涂敷在含有氨苄青霉素100μg/mL的LB琼脂培养基(Difco公司制)上,在37℃下培养一晚,得到保持有含有GL-BP基因的质粒的转化大肠杆菌。使用Quantum Prep质粒小量提取试剂盒(Bio-Rad公司制)从转化大肠杆菌中提取出质粒并进行纯化,并通过定序确认序列,从而得到被导入GL-BP基因的重组质粒。将得到的重组质粒命名为pJT101。
C.WT1基因的分离
对编码小鼠WT1的第117个至第439个氨基酸序列的DNA(序列号2)进行全合成(FUNAKOSHI株式会社)。另外,合成时使用双歧杆菌中使用频率高的密码子。另外,在N末端侧附加XhoI识别序列(CTCGAG:序列号5),在C末端侧附加终止密码子,接着在该终止密码子上附加SphI识别序列(GCATGC:序列号6)。将其导入pUC18载体的SmaI位点,从而构建质粒。使用DNA连接试剂盒Ver.2(TAKARA BIO株式会社制)进行连接。通过热休克法(42℃、30秒)将构建好的质粒导入大肠杆菌DH5α(TAKARA BIO株式会社制)中,并涂敷在含有氨苄青霉素100μg/mL的LB琼脂培养基(Difco公司制)上,在37℃下培养一晚,从而得到保持有含有编码小鼠WT1(117~439)的DNA的质粒的转化大肠杆菌。使用Quantum Prep质粒小量提取试剂盒(Bio-Rad公司制)从转化大肠杆菌中提取出质粒并进行纯化,并通过定序确认序列。将得到的重组质粒命名为pTK2875-1。
合成的小鼠WT1基因的序列(序列号2)
CTCGAGCCGTCCCAGGCGTCGTCGGGCCAGGCGAGGATGTTCCCGAACGCGCCCTACCTGCCCAGCTGCCTGGAGTCCCAGCCGACGATCCGCAACCAGGGCTACTCCACCGTGACGTTCGACGGCGCCCCGTCCTACGGCCACACGCCCAGCCACCACGCCGCCCAGTTCCCGAACCACAGCTTCAAGCACGAAGACCCCATGGGCCAGCAGGGCAGCCTCGGCGAACAGCAGTACAGCGTGCCGCCGCCGGTCTACGGCTGCCACACCCCGACCGACTCCTGCACGGGCTCCCAGGCCCTGCTCCTGCGTACGCCGTACTCCTCCGACAACCTCTACCAGATGACCTCCCAGCTGGAGTGCATGACCTGGAACCAGATGAACCTGGGCGCCACGCTGAAGGGAATGGCCGCGGGGTCGTCGAGCTCCGTCAAGTGGACCGAAGGCCAGTCCAACCACGGCATCGGCTACGAGTCCGAGAACCACACCGCGCCGATCCTGTGCGGAGCCCAGTACCGCATCCACACGCACGGCGTCTTCCGCGGCATCCAGGACGTCCGGCGCGTCTCCGGCGTCGCGCCGACCCTGGTGCGGTCCGCCTCCGAGACCTCCGAGAAGCGCCCGTTCATGTGCGCCTACCCGGGCTGCAACAAGCGCTACTTCAAGCTCTCGCACCTGCAGATGCACTCCCGGAAGCACACCGGCGAGAAGCCGTACCAGTGCGACTTCAAGGACTGCGAACGCCGCTTCTCGCGCAGCGACCAGCTGAAGCGCCACCAGCGTAGGCACACCGGCGTGAAGCCCTTCCAGTGCAAGACCTGCCAGCGCAAGTTCTCCCGCAGCGACCACCTCAAGACGCACACCCGCACCCACACCGGCAAGACGTCCGAGAAGCCGTTCTCGTGCCGCTGGCACAGCTGCCAGAAGAAGTTCGCCCGCAGCGACGAGCTCGTGCGCCACCACAACATGCACCAGTGAAGCATGC
具有编码小鼠WT1的第117个至第350个氨基酸序列的DNA的质粒如下那样进行制备。即,以如上所述得到的pTK2875为模板,使用引物WT1-f(5’-CGCTCGAGCCGTCCCAGGCGTCGT-3’:序列号7)和引物WT1-r2(5’-GCGCATGCTCACTCGCCGGTGTGCTTCCGG-3’:序列号8)以及KOD-Plus-(TOYOBO公司制)进行PCR反应,从而使编码小鼠WT1(117~350)的DNA片段扩增。另外,在C末端侧附加终止密码子,接着在该终止密码子上附加SphI识别序列(GCATGC:序列号6)。对扩增后的PCR产物进行琼脂糖凝胶电泳,切出721bp的PCR产物,并使用Wizard SV凝胶和PCR清理系统(Promega公司制)进行分离纯化。将其导入pUC18载体的SmaI位点,从而构建质粒。使用DNA连接试剂盒Ver.2(TAKARA BIO株式会社制)进行连接。通过热休克法(42℃、30秒)将构建好的质粒导入大肠杆菌DH5α(TAKARA BIO株式会社制)中,并涂敷在含有氨苄青霉素100μg/mL的LB琼脂培养基(Difco公司制)上,在37℃下培养一晚,从而得到保持有含有编码小鼠WT1(117~350)的DNA的质粒的转化大肠杆菌。使用Quantum Prep质粒小量提取试剂盒(Bio-Rad公司制)从转化大肠杆菌中提取出质粒并进行纯化,并通过定序确认序列。将得到的重组质粒命名为pTK2875-2。
D.在GL-BP基因的下游具有WT1基因的质粒的构建
使用限制酶XhoI和SphI对上述C.中得到的保持有WT1基因的质粒pTK2875-1和pTK2875-2进行处理,并进行琼脂糖凝胶电泳,分别切出986bp和718bp的DNA片段,再使用Wizard SV凝胶和PCR清理系统(Promega公司制)进行分离纯化。使用DNA连接试剂盒Ver.2,将上述WT1基因扩增片段分别导入同样利用限制酶XhoI和SphI处理后的上述pJT101质粒中,从而构建质粒。通过热休克法将构建好的质粒分别导入大肠杆菌DH5α中,并涂敷在含有氨苄青霉素100μg/ml的LB琼脂培养基上,在37℃下培养一晚,从而得到保持有含有GL-BP基因与WT1基因的融合基因(图1)的质粒的转化大肠杆菌。使用Quantum Prep质粒小量提取试剂盒从得到的转化大肠杆菌中提取出质粒并进行纯化,并通过定序确认序列,从而得到在GL-BP基因的下游连接有WT1基因的重组质粒。将得到的重组质粒分别命名为pTK2895(GLBP-WT1(117~439))和pTK2896(GLBP-WT1(117~350))。
E.大肠杆菌-双歧杆菌穿梭载体的构建
作为大肠杆菌-双歧杆菌穿梭载体,使用Vaccine.28:6684-6691(2010)中公开的大肠杆菌-双歧杆菌穿梭载体pJW241。
F.由GL-BP基因与WT1基因连接而成的基因向大肠杆菌-双歧杆菌穿梭载体pJW241的嵌入
将具有由GL-BP基因与WT1基因连接而成的融合基因(以下称为“该融合基因”)的载体pTK2895(GLBP-WT1(117~439))和pTK2896(GLBP-WT1(117~350))分别作为模板,并使用引物Infusion-F(5’-ggaaaactgtccatagatggcgaggcgaacgccacg-3’:序列号9)和引物Infusion-R(5’-tttcatctgtgcatagtgctgcaaggcgattaagtt-3':序列号10)进行PCR。对PCR扩增产物进行琼脂糖凝胶电泳而切出该融合基因,并使用Wizard SV凝胶和PCR清理系统(Promega公司制)进行分离纯化。另外,与之分开另外使用限制酶NdeI对大肠杆菌-双歧杆菌穿梭载体pJW241(Vaccine.28:6684-6691(2010))进行处理。使用In-Fusion HD克隆试剂盒(Clontech公司制)将纯化后的该融合基因与pJW241加以连接,通过热休克法将得到的质粒导入大肠杆菌DH5α中,并涂敷在含有壮观霉素70μg/mL的LB琼脂培养基中,在37℃下培养一晚,从而得到保持有具有大肠杆菌复制起点ori区域、壮观霉素抗性基因(SPr)、双歧杆菌的复制起点ori区域以及该融合基因的质粒的转化大肠杆菌。使用Quantum Prep质粒小量提取试剂盒从转化大肠杆菌中提取出质粒并进行纯化,并对该融合基因的序列的存在情况进行了确认。将得到的重组质粒分别命名为pTK2897(GLBP-WT1(aa117~439))和pTK2898(GLBP-WT1(aa117~350))。
G.宿主双歧杆菌液的制备
将长双歧杆菌(Bifidobacterium longum)105-A(Matsumura H.等,Biosci.Biotech.Biochem.,1997年,61卷,pp.1211-1212:由东京大学名誉教授光冈知足先生提供)接种至50mL的GAM培养基(日水制药株式会社制)中,并使用AnaeroPackTM厌氧罐(三菱瓦斯化学株式会社制)在37℃下进行了培养。在培养期间,测量了波长600nm下的吸光度,当吸光度达到0.4~0.8时停止培养。在培养结束后,使用高速离心分离机进行离心分离(6000×g、10分钟),并收集菌体。向收集到的菌体中加入10%(v/v)甘油溶液10mL并使菌体悬浮于溶液中,使用高速离心分离机进行离心分离,并将菌体清洗2~3次。
H.通过使重组质粒pTK2897和pTK2898向双歧杆菌的转化而形成的GL-BP-WT1融合蛋白质表层呈递双歧杆菌的制备
向上述G.中得到的宿主双歧杆菌液中加入10%(v/v)甘油溶液500μL并使菌液悬浮于溶液中。在其他试管中取200μL该悬浊液,加入5μL分别含有上述F.中得到的重组质粒pTK2897和pTK2898的溶液并进行混合,在冰上放置5分钟。接着,在0.2cm的电穿孔管(Bio-Rad公司制)中加入混合液,并使用Gene Pulser Xcell电穿孔系统(Bio-Rad公司制)在2kV、2.5μF、200Ω的条件下进行电穿孔。电穿孔后立即加入预先保持为37℃的GAM培养基0.8mL,并使用AnaeroPackTM厌氧罐在37℃下培养3小时。接着,涂敷在含有70μg/mL壮观霉素的GAM琼脂培养基(日水制药株式会社制)上,并使用AnaeroPackTM厌氧罐在37℃下进行培养,从而得到转化双歧杆菌。将得到的转化双歧杆菌接种至含有70μg/mL壮观霉素的GAM培养基上,使用AnaeroPackTM厌氧罐在37℃下进行培养。在培养结束后,将培养液分注至1.5mL试管中,加入等量的50%(v/v)甘油溶液并使其培养液悬浮于溶液上。将得到的悬浊液在-80℃下进行保存从而制成冻存液(freezestock),并将其作为GL-BP-WT1融合蛋白质表层呈递双歧杆菌(有时也称为“转化双歧杆菌”)的种细胞(master cell)(分别为TK2900(GLBP-WT1(aa117~439))和TK2903(GLBP-WT1(aa117~350)))。
图2是表示通过电泳确认扩增片段的长度的结果的图,其中,该扩增片段是使用下述引物并通过PCR使重组双歧杆菌的基因(DNA)扩增后得到的扩增片段。
正向引物410、420:ACGATCCGCAACCAGGGCTACTC(序列号11)
反向引物410:ggtgcgagagcttgaagtagcgc(序列号12)
反向引物420:gtcgctgcgggcgaacttcttc(序列号13)
410是长双岐杆菌(B.longum)410,且是利用插入了编码小鼠WT1(aa170~350)的DNA的穿梭载体进行转化后的双歧杆菌,相当于上述TK2903(GLBP-WT1(aa117~350))。420是长双岐杆菌(B.longum)420,且是利用插入了编码小鼠WT1(aa117~439)的DNA的穿梭载体进行转化后的双歧杆菌,相当于上述TK2900(GLBP-WT1(aa117~439))。2012是长双岐杆菌(B.longum)2012,且是利用未插入编码小鼠WT1的DNA而仅插入了GLBP基因的穿梭载体进行转化后的双歧杆菌。显示为410的引物使编码小鼠WT1(aa117~350)的DNA扩增,显示为420的引物使编码小鼠WT1(aa117~439)的DNA扩增。
从图2的结果可知,编码WT1的DNA确实被导入重组双歧杆菌中。
(实施例2:转化双歧杆菌的GL-BP-WT1融合蛋白质表层呈递的确认)
(1)使用高速离心机对上述实施例1中得到的转化双歧杆菌进行离心,并收集菌体。在收集到的菌体中加入PBS并使菌体悬浮于PBS中,利用高速离心分离机进行离心分离而将菌体清洗三次。在菌体中加入含有PBS、1M的Tris-HCl(pH8.0)(株式会社Nippon Gene制)以及Triton X-100(和光纯药工业株式会社制)的溶液,并在冰上放置30分钟。在该溶液中加入等量的2×SDS凝胶电泳缓冲液,在95℃下放置5分钟,得到电泳用样本。接着,将8%(w/v)丙烯酰胺凝胶设置在电泳装置(ATTO株式会社制)中,并涂上所得到的样本,与分子量标准参照物(molecular weight marker)一同以20mA的电流进行1.5小时电泳。将电泳后的凝胶放置在硝酸纤维素膜(ATTO株式会社制)上,并向印迹装置(Bio-Rad公司制)施加20mA的电流进行印迹。在印迹之后,使硝酸纤维素膜在含有4%(w/v)脱脂乳(BD公司制)的缓冲液TBS(株式会社Nippon Gene制)中浸泡1小时进行阻断。在阻断之后,使用TBS将硝酸纤维素膜清洗两次。在清洗之后,将硝酸纤维素膜在添加了0.5%(w/v)的一级抗体(WT1抗体(C-19):sc-192:SANTA CRUZ BIOTECHNOLOGY公司制)的TBS中浸泡1.5小时,并使用TBS清洗三次。接着,将硝酸纤维素膜在添加了0.5%(w/v)的二级抗体(山羊抗兔IgG-HRP:sc-2004:SANTA CRUZ BIOTECHNOLOGY公司制)的TBS中浸泡3小时。接着,使用TBS将硝酸纤维素膜清洗三次,使用1-Steptm NBT/BCIP plus Suppressor kit(PIERCE公司制)在遮光条件下生色30分钟,在利用纯水清洗后,根据生色确认GL-BP-WT1融合蛋白质的表层表达。
蛋白质印迹的结果如图3中的A所示。从图3中的A可知,在长双歧杆菌(B.longum)420中存在相当于WT1(aa117~439)与GL-BP的融合蛋白质的分子量总和的82.9kDa的明确的条带(band)。由此可知,转化双歧杆菌(B.longum 420)表达了GL-BP-WT1融合蛋白质。
(2)使用高速离心分离机对于培养后的上述实施例1中得到的转化双歧杆菌进行离心分离,并收集菌体。在收集到的菌体中加入缓冲液PBS(株式会社Nippon Gene制)并使菌体悬浮于PBS中,通过利用高速离心分离机进行离心分离而将菌体清洗三次。接着,在含有1%(w/v)BSA的PBS中加入一级抗体(WT1抗体(C-19):sc-192:SANTA CRUZBIOTECHNOLOGY公司制),再将其加入双歧杆菌液中并使菌液悬浮于该PBS中,并在37℃下放置30分钟。使用高速离心分离机对放置30分钟后的菌液进行离心分离,并收集菌体。在收集到的菌体中加入PBS并使菌体悬浮于PBS中,通过利用高速离心分离机进行离心分离而将菌体清洗两次。接着,在含有1%(w/v)BSA的PBS中加入二级抗体Alexa FluorTM 488兔抗小鼠IgG抗体(Molecular Probes公司制),再将其加入双歧杆菌液中并使菌液悬浮于该PBS中,并在37℃下放置30分钟。使用高速离心分离机对放置30分钟后的菌液进行离心分离,并收集菌体。在收集到的菌体中加入PBS并使菌体悬浮于PBS中,通过利用高速离心机进行离心分离而将菌体清洗两次,然后利用荧光显微镜(KEYENCE公司制)进行观察。
使用荧光显微镜观察的结果如图3中的B所示。图3中的B的左图是上述实施例1中得到的转化双歧杆菌、即长双歧杆菌(B.longum)420的荧光显微镜照片,图3中的B的右图是长双歧杆菌(B.longum)2012的荧光显微镜照片。从荧光显微镜照片可知,在长双歧杆菌(B.longum)420的细胞表面上存在WT1。
(实施例3:口服给药转化双歧杆菌的GL-BP-WT1融合蛋白质的抗肿瘤效果的确认)
对于向小鼠口服给药上述实施例1中得到的冷冻保存的转化双歧杆菌时的抗肿瘤效果进行了确认。实验步骤如图4和图5所示。
将小鼠WT1表达C1498细胞(小鼠白血病细胞)皮下移植至C57BL/6(雌性、6~8周龄)的右侧腹部。细胞按每只小鼠1×106cells/200μLRPMI1640和基质胶进行移植,并将移植当天设为Day0。每隔四天确认肿瘤的大小。
接种肿瘤第19天(Day19)的代表性的皮下肿瘤的照片如图6所示。另外,肿瘤大小的日变化(daily variation)如图7所示,接种肿瘤第29天(Day29)的肿瘤大小如图8所示。可知在长双歧杆菌(B.longum)420的单独给药组中,相比PBS给药组能够显著地抑制肿瘤增殖(*:p<0.05、**:p<0.001)。另外可知,通过同时使用长双歧杆菌(B.longum)420和IL-2,与PBS给药组相比,从Day19起能够显著地抑制肿瘤增殖(p<0.01),且在Day29,与长双歧杆菌(B.longum)420的单独给药组相比,能够显著地抑制肿瘤增殖(p<0.01)。通过同时使用IL-2,增强了长双歧杆菌(B.longum)420的抗肿瘤效果。
(实施例4:口服给药转化双歧杆菌的GL-BP-WT1融合蛋白质的细胞免疫应答诱导效果的确认)
对于向小鼠口服给药上述实施例1中得到的冷冻保存的转化双歧杆菌时的细胞免疫应答诱导效果进行了确认。实验步骤如图9所示。另外,在Day0至Day29的观察期间中,长双歧杆菌(B.longum)420给药组的平均体重与其他组同样地变化(图10),并未发现因为给药长双歧杆菌(B.longum)420而引起腹泻、行动不良等副作用。
(1)在Day27从小鼠回收脾脏,并制备脾细胞进行培养,并且测量了脾细胞培养上清液中的细胞性免疫系统各种细胞因子的浓度。作为刺激脾细胞的抗原,使用为了表达小鼠WT1蛋白质而导入了小鼠WT1基因的C1498小鼠白血病细胞株(C1498-WT1细胞),作为对照而使用导入了未插入小鼠WT1基因的空载体的C1498细胞(C1498-Mock细胞)。
使用经丝裂霉素C处理的C1498-WT1细胞或C1498-Mock细胞(各4×104cells/well),并利用96孔板将4×105cells/well的小鼠脾细胞在37℃下刺激培养三天(n=5)。培养后回收细胞培养液,并通过酶联免疫吸附测定(ELISA)法测量了各种细胞因子(干扰素γ(IFN-γ)、白介素2(IL-2)、肿瘤坏死因子α(TNF-α))的浓度。各种细胞因子的浓度是使用小鼠干扰素γ(IFN-γ)ELISA试剂盒(R&D Systems,Minneapolis,MN)、小鼠肿瘤坏死因子α(TNF-α)(R&D Systems)以及小鼠白介素2(IL-2)ELISA试剂盒(Thermo Scientific,Waltham,MA),并按照试剂盒的使用手册中的方法进行测量。
结果如图11所示。在长双歧杆菌(B.longum)420给药组中,通过利用C1498-WT1细胞进行再刺激,与非刺激组相比,IFN-γ、IL-2、TNF-α产生量显著增加(*:p<0.01)。与PBS给药组和长双歧杆菌(B.longum)2012给药组相比,长双歧杆菌(B.longum)420给药组中的IFN-γ产生量显著增加(*:p<0.01)。由此可知,通过口服给药转化双歧杆菌的GL-BP-WT1融合蛋白质,对于WT1特异性抗肿瘤免疫重要的各种细胞因子的产生在WT1蛋白质的刺激下得到了增强。
(2)对于与上述(1)同样地制备的脾细胞进行细胞内细胞因子染色(ICCS),并确认了CD4阳性T细胞或CD8阳性T细胞中的细胞因子产生T细胞的比率。
将小鼠脾细胞(2×106cells/well、n=5)与2×105cells/well的C1498-WT1细胞混合,并利用24孔板在37℃、5%CO2条件下培养42小时。在此,在各孔中添加GolgiStop或GolgiPlug(BD公司制),进一步培养6小时。回收细胞,并使用BD/Cytofix/Cytoperm增强型固定破膜试剂盒(BD公司制)进行细胞内细胞因子染色。添加FITC标记抗CD3单克隆抗体、FITC标记抗CD8单克隆抗体或者FITC标记抗CD4单克隆抗体,并进行混合。利用染色用缓冲液清洗细胞。在向细胞加入各种抗细胞因子抗体并使其稳定悬浮之后,静置于呈室温的暗处。在对细胞进行清洗之后,再次悬浮于染色用缓冲液中。然后,使用流式细胞仪并利用附属的分析软件对细胞进行了分析。具体的方法按照试剂盒的使用手册进行。
结果如图12所示。与其他的给药组相比,在长双歧杆菌(B.longum)420给药组中,脾细胞中产生IFN-γ、IL-2以及TNF的CD4+T细胞、CD8+T细胞均显著增加(*:p<0.05)。由此可知,通过口服给药转化双歧杆菌的GL-BP-WT1融合蛋白质,使得产生参与WT1特异性细胞性免疫的细胞因子的CD4+T细胞和CD8+T细胞增加。
(3)对于与上述(1)同样地制备的脾细胞,使用WT1四聚物对CD8阳性T细胞中的WT1(Db126肽)特异性CD8+T细胞的比率进行了确认。
将2×106cells/well的小鼠脾细胞(n=5)与2×105cells/well的C1498-WT1细胞混合,并利用24孔板在37℃、5%CO2条件下培养7天。在培养的第一天、第三天添加20Iu/mL的IL-2诱导CTL。培养后,使用FITC标记抗CD3单克隆抗体、FITC标记抗CD8单克隆抗体检测CD8阳性T细胞,并使用H-2Db WT1Tetramer-RMFPNAPYL(MBL公司制)检测WT1肽特异性CD8+T细胞(CTL)。使用流式细胞仪并利用附属的分析软件对细胞进行了分析。
结果如图13所示。通过口服给药转化双歧杆菌,该双歧杆菌的表层上表达的GL-BP-WT1融合蛋白质被摄入肠相关淋巴组织(GALT)中,通过GALT内的抗原呈递细胞(APC)在适当的表位被处理。进而,认为在GALT内被加工处理后的肽与MHC II类分子一同被呈递于APC,从而诱导具有相对于该肽呈特异性的T细胞受体的CTL。由于H-2Db WT1 Tetramer-RMFPNAPYL与相对于WT1蛋白质中所含的表位之一、即CD8表位(a.a.126-134:RMFPNAPYL(序列号19))呈特异性的T细胞受体相结合而发出荧光,因此,可以确认相对于该CD8表位呈特异性的CTL的诱导。从图13的结果可知,与其他给药组相比,长双歧杆菌(B.longum)420给药组的脾细胞中的WT1四聚物阳性CTL的比率显著增加(*;p<0.05)。由此可知,口服给药的转化双歧杆菌的GL-BP-WT1融合蛋白质被适当地处理,诱导出在抗肿瘤效果中起到重要作用的WT1肽特异性CTL。
(4)对于与上述(1)同样地制备的脾细胞,测量了WT1特异性细胞毒性T细胞(CTL)的活性。
将3×107cells/well的小鼠脾细胞(n=5)与3×106cells/well的C1498-WT1细胞混合,并利用6孔板在37℃、5%CO2条件下培养6天。在培养的第一天、第三天添加20Iu/mL的IL-2诱导CTL。回收脾细胞,并利用96孔板将脾细胞与1×104cells/well的C1498-WT1细胞或C1498-Mock细胞以20:1、10:1或者5:1的比率混合后,在37℃、5%CO2条件下培养8小时。回收培养上清液,使用Cytotox 96非放射性细胞毒性检测试剂盒(Promega公司制)测量了培养上清液中的乳酸脱氢酶活性,并据此计算出细胞毒活性。乳酸脱氢酶是存在于细胞质中的酶,通常不会透过细胞膜,但在细胞膜受到损伤时会释放到培养基中,因而可以用作细胞毒活性的指标。
结果如图14所示。在所有的细胞混合比率中,长双歧杆菌(B.longum)420给药组中的WT1特异性细胞毒活性显著上升(p<0.01)。由此可知,通过口服给药转化双歧杆菌的GL-BP-WT1融合蛋白质,诱导出具有WT1特异性细胞毒活性的CTL。
(实施例5:口服给药转化双歧杆菌的GL-BP-WT1融合蛋白质的抗肿瘤效果的确认2)
对于向小鼠口服给药上述实施例1中得到的冷冻保存的转化双歧杆菌时的抗肿瘤效果进行了确认。实验步骤如图15所示。
对C57BL/6N小鼠(雌性、6周龄)(n=25)皮下接种1×106cellsC1498-WT1细胞或C1498-Mock细胞。两天后将小鼠分为三组(n=5),并开始口服给药转化双歧杆菌。每次口服给药时,对长双歧杆菌(B.longum)420给药组给药1×109CFU in 100μL PBS,对长双歧杆菌(B.longum)2012给药组给药1×109CFU in 100μL PBS,对PBS给药组给药100μL PBS。另外,抗肿瘤效果的评价根据下述肿瘤体积的计算值进行。
(式)肿瘤体积(mm3)=长径×短径2×1/2
结果如图16所示。在Day25,与PBS给药组和长双歧杆菌(B.longum)2012给药组相比,长双歧杆菌(B.longum)420给药组的肿瘤体积被显著抑制。另外,在长双歧杆菌(B.longum)420给药组中,未发现对于C1498-Mock细胞具有抗肿瘤效果,因而可知口服给药转化双歧杆菌的GL-BP-WT1融合蛋白质的抗肿瘤效果在WT1表达细胞中呈特异性。
(实施例6:佐剂对于口服给药转化双歧杆菌的GL-BP-WT1融合蛋白质的抗肿瘤效果的影响的确认)
对于将霍乱霉素作为黏膜免疫佐剂使用,并向小鼠口服给药上述实施例1中得到的冷冻保存的转化双歧杆菌时的抗肿瘤效果进行了确认。实验步骤如图17所示。
将1×106cells C1498-WT1细胞皮下接种至6周龄雌性C57BL/6N小鼠的右背侧。7天后确认肿瘤形成情况,将小鼠分为三组(n=3),并开始口服给药转化双歧杆菌。每次口服给药时,对长双歧杆菌(B.longum)420给药组给药6.4×109CFU in 200μL PBS,对长双歧杆菌(B.longum)420+霍乱霉素给药组给药6.4×109CFU+10μg霍乱霉素(Wako)in 200μL PBS,对PBS给药组给药200μL PBS。疫苗是将肿瘤接种日设为Day0,并在Day7、Day14、Day21总计给药三次。在Day0至Day27期间,测量了肿瘤直径。抗肿瘤效果的评价与实施例5同样地根据肿瘤体积的计算值进行。
结果如图18所示。一周给药一次,从Day18开始,通过同时使用长双歧杆菌(B.longum)420和霍乱霉素,发现与PBS给药组相比更为明显的抗肿瘤效果(p<0.05)。另外,对于未接种肿瘤的小鼠同样进行了口服给药,其结果是,同时使用霍乱霉素组的平均体重与其他组同样地变化(图18右侧)。未发现因为给药而引起腹泻、行动不良等副作用。由此可知,通过同时使用霍乱霉素,通过低频率的给药能够安全地增强抗肿瘤效果。
(实施例7:各种黏膜免疫佐剂对于口服给药转化双歧杆菌的GL-BP-WT1融合蛋白质的抗肿瘤效果的影响的确认)
对于使用各种黏膜免疫佐剂,并向小鼠口服给药上述实施例1中得到的冷冻保存的转化双歧杆菌时的抗肿瘤效果进行了确认。实验步骤如图19所示。
将6周龄雌性C57BL/6N小鼠分成以下五个组(n=3),并开始口服给药转化双歧杆菌。即:长双歧杆菌(B.longum)420+20μg/dose LTB(大肠杆菌不耐热肠毒素B亚单位(Sigma公司))、长双歧杆菌(B.longum)420+20μg/dose MPLA(单磷酰脂质A(Sigma公司)、长双歧杆菌(B.Longum)420+100μg/dose壳聚糖(壳聚糖低分子量(Sigma公司)、长双歧杆菌(B.longum)420+10μg/dose CpG 1585(Invivogen公司)、PBS。
使用探针对小鼠口服给药各双歧杆菌给药液6.4×109CFU/200μL或PBS 200μL。在Day49,将C1498-WT1细胞皮下接种至右背侧,并在截止Day76为止的期间内测量了肿瘤直径。抗肿瘤效果的评价与实施例6同样地根据肿瘤体积的计算值进行。
结果如图20所示。在Day67,与PBS给药组相比较,所有的同时使用佐剂组的肿瘤增殖均被明显抑制(p<0.05)。在Day70,分别同时使用LTB和壳聚糖组与PBS组相比肿瘤被明显抑制,在Day73,分别同时使用LTB、壳聚糖以及CpG1585组与PBS组相比肿瘤被明显抑制(p<0.05)。由此可知,通过口服给药转化双歧杆菌的GL-BP-WT1融合蛋白质,具有预防性的抗肿瘤效果。另外,在Day0至Day49的口服给药期间,所有的同时使用佐剂组的平均体重与PBS组同样地变化。未发现因为给药而引起腹泻、行动不良等副作用。
(实施例8:GL-BP-WT1表层呈递双歧杆菌的制备2)
通过与实施例1相同的方法进行A.GL-BP基因的分离、B.具有分离出的GL-BP基因的pMW118质粒的构建。
C.WT1基因的分离
对于编码人WT1的第117个至第439个氨基酸序列的DNA(序列号15)进行全合成(FUNAKOSHI株式会社)。另外,合成时使用双歧杆菌中使用频率高的密码子。另外,在N末端侧附加XhoI识别序列(CTCGAG:序列号5),在C末端侧附加终止密码子,接着在该终止密码子上附加SphI识别序列(GCATGC:序列号6)。将其导入pUC18载体的SmaI位点,从而构建质粒。使用DNA连接试剂盒Ver.2(TAKARA BIO株式会社制)进行连接。通过热休克法(42℃、30秒)将构建好的质粒导入大肠杆菌DH5α(TAKARA BIO株式会社制)中,并涂敷在含有氨苄青霉素100μg/mL的LB琼脂培养基(Difco公司制)上,在37℃下培养一晚,从而得到保持有含有编码人WT1蛋白质(117~439)的DNA的质粒的转化大肠杆菌。使用Quantum Prep质粒小量提取试剂盒(Bio-Rad公司制)从转化大肠杆菌中提取出质粒并进行纯化,并通过定序确认序列。
合成的人WT1基因的序列(序列号15)
CCGTCCCAGGCGTCGTCGGGCCAGGCGAGGATGTTCCCGAACGCGCCCTACCTGCCCAGCTGCCTGGAGTCCCAGCCGGCGATCCGCAACCAGGGCTACTCCACCGTGACGTTCGACGGCACCCCGTCCTACGGCCACACGCCCAGCCACCACGCCGCCCAGTTCCCGAACCACAGCTTCAAGCACGAAGACCCCATGGGCCAGCAGGGCAGCCTCGGCGAACAGCAGTACAGCGTGCCGCCGCCGGTCTACGGCTGCCACACCCCGACCGACTCCTGCACGGGCTCCCAGGCCCTGCTCCTGCGTACGCCGTACTCCTCCGACAACCTCTACCAGATGACCTCCCAGCTGGAGTGCATGACCTGGAACCAGATGAACCTGGGCGCCACGCTGAAGGGAGTCGCCGCGGGGTCGTCGAGCTCCGTCAAGTGGACCGAAGGCCAGTCCAACCACTCCACCGGCTACGAGTCCGACAACCACACCACGCCGATCCTGTGCGGAGCCCAGTACCGCATCCACACGCACGGCGTCTTCCGCGGCATCCAGGACGTCCGGCGCGTCCCCGGCGTCGCGCCGACCCTGGTGCGGTCCGCCTCCGAGACCTCCGAGAAGCGCCCGTTCATGTGCGCCTACCCGGGCTGCAACAAGCGCTACTTCAAGCTCTCGCACCTGCAGATGCACTCCCGGAAGCACACCGGCGAGAAGCCGTACCAGTGCGACTTCAAGGACTGCGAACGCCGCTTCTCGCGCAGCGACCAGCTGAAGCGCCACCAGCGTAGGCACACCGGCGTGAAGCCCTTCCAGTGCAAGACCTGCCAGCGCAAGTTCTCCCGCAGCGACCACCTCAAGACGCACACCCGCACCCACACCGGCAAGACGTCCGAGAAGCCGTTCTCGTGCCGCTGGCCCAGCTGCCAGAAGAAGTTCGCCCGCAGCGACGAGCTCGTGCGCCACCACAACATGCACCAGTGAA
另外,与上述同样地对编码变异型WT1蛋白质的DNA进行了全合成,从而制成重组质粒,其中,该变异型WT1蛋白质是在人WT1的第117个至第439个氨基酸序列中具有在HLA-A*2402限制性CTL表位中导入了M236Y的取代后的氨基酸序列的蛋白质。
合成的人WT1基因的序列(序列号17)
CCGTCCCAGGCGTCGTCGGGCCAGGCGAGGATGTTCCCGAACGCGCCCTACCTGCCCAGCTGCCTGGAGTCCCAGCCGGCGATCCGCAACCAGGGCTACTCCACCGTGACGTTCGACGGCACCCCGTCCTACGGCCACACGCCCAGCCACCACGCCGCCCAGTTCCCGAACCACAGCTTCAAGCACGAAGACCCCATGGGCCAGCAGGGCAGCCTCGGCGAACAGCAGTACAGCGTGCCGCCGCCGGTCTACGGCTGCCACACCCCGACCGACTCCTGCACGGGCTCCCAGGCCCTGCTCCTGCGTACGCCGTACTCCTCCGACAACCTCTACCAGATGACCTCCCAGCTGGAGTGCTACACCTGGAACCAGATGAACCTGGGCGCCACGCTGAAGGGAGTCGCCGCGGGGTCGTCGAGCTCCGTCAAGTGGACCGAAGGCCAGTCCAACCACTCCACCGGCTACGAGTCCGACAACCACACCACGCCGATCCTGTGCGGAGCCCAGTACCGCATCCACACGCACGGCGTCTTCCGCGGCATCCAGGACGTCCGGCGCGTCCCCGGCGTCGCGCCGACCCTGGTGCGGTCCGCCTCCGAGACCTCCGAGAAGCGCCCGTTCATGTGCGCCTACCCGGGCTGCAACAAGCGCTACTTCAAGCTCTCGCACCTGCAGATGCACTCCCGGAAGCACACCGGCGAGAAGCCGTACCAGTGCGACTTCAAGGACTGCGAACGCCGCTTCTCGCGCAGCGACCAGCTGAAGCGCCACCAGCGTAGGCACACCGGCGTGAAGCCCTTCCAGTGCAAGACCTGCCAGCGCAAGTTCTCCCGCAGCGACCACCTCAAGACGCACACCCGCACCCACACCGGCAAGACGTCCGAGAAGCCGTTCTCGTGCCGCTGGCCCAGCTGCCAGAAGAAGTTCGCCCGCAGCGACGAGCTCGTGCGCCACCACAACATGCACCAGTGAA
关于D.在GL-BP基因的下游具有WT1基因的质粒的构建、E.大肠杆菌-双歧杆菌穿梭载体的构建、F.由GL-BP基因与WT1基因连接而成的基因向大肠杆菌-双歧杆菌穿梭载体pJW241的嵌入、以及G.宿主双歧杆菌液的制备,与实施例1同样地制备两种转化双歧杆菌。
图21是表示使用图21右侧所示的特异性引物,并按基因水平识别两种转化双歧杆菌的情况的图。另外,430是长双歧杆菌(B.longum)430,且是利用插入了编码人WT1蛋白质(117~439)的DNA的穿梭载体转化后的双歧杆菌。440是长双歧杆菌(B.longum)440,且是利用插入了编码具有氨基酸取代M236Y的人WT1蛋白质(117~439)的DNA的穿梭载体转化后的双歧杆菌。2012与实施例1同样为长双歧杆菌(B.longum)2012,且是利用未插入编码WT1的DNA而仅插入了GLBP基因的穿梭载体转化后的双歧杆菌。显示为430的引物使编码人WT1蛋白质(117~439)的DNA扩增,显示为440的引物使编码具有氨基酸取代M236Y的人WT1蛋白质(117~439)的DNA扩增。
(实施例9:转化双歧杆菌的GL-BP-WT1融合蛋白质表层呈递的确认)
对于上述实施例8中得到的转化双歧杆菌,通过与实施例2相同的方法,对GL-BP-WT1融合蛋白质的表层表达进行了确认。
蛋白质印迹的结果如图22中的A所示,荧光免疫染色的结果如图22中的B所示。从图22明确可知,与长双歧杆菌(B.longum)420同样地,在长双歧杆菌(B.longum)430和长双歧杆菌(B.longum)440中发现相当于WT1与GL-BP的融合蛋白质的分子量总和的约82.5kDa的条带。另外,从荧光显微镜照片可知,长双歧杆菌(B.longum)430和长双歧杆菌(B.longum)440的细胞表面上存在WT1。由此可知,转化双歧杆菌表达了GL-BP-WT1融合蛋白质。
(工业上的可利用性)
如以上所详述,根据本发明的转化双歧杆菌,能够将WT1蛋白质表达或呈递于双歧杆菌的细胞表层上。通过使WT1抗原蛋白质呈递于双歧杆菌的表层上,能够作为对于表达WT1蛋白质的肿瘤有效的口服疫苗进行使用。在口服疫苗的情况下,儿童或老年人均易于吸收,而且也不会有通常通过注射接种疫苗时的疼痛。尤其是,本发明的口服疫苗使用已有食用经验的双歧杆菌,因而安全性很高。另外,不同于被限制为某一特定HLA的WT1肽,本发明的抗原WT1蛋白质是能够表达将WT1的大部分序列覆盖的WT1蛋白质的双歧杆菌,将该转化双歧杆菌作为有效成分的肿瘤疫苗能够适用于各种HLA类型的患者。
SEQUENCE LISTING
<110> 国立大学法人神户大学(NATIONAL UNIVERSITY CORPORATION KOBEUNIVERSITY)
国立大学法人大阪大学(OSAKA UNIVERSITY)
<120> 口服肿瘤疫苗
<130> GP16-1009PCT
<150> JP2015-128034
<151> 2015-06-25
<160> 26
<170> PatentIn version 3.5
<210> 1
<211> 323
<212> PRT
<213> 小鼠(Mus musculus)
<400> 1
Pro Ser Gln Ala Ser Ser Gly Gln Ala Arg Met Phe Pro Asn Ala Pro
1 5 10 15
Tyr Leu Pro Ser Cys Leu Glu Ser Gln Pro Thr Ile Arg Asn Gln Gly
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Tyr Ser Thr Val Thr Phe Asp Gly Ala Pro Ser Tyr Gly His Thr Pro
35 40 45
Ser His His Ala Ala Gln Phe Pro Asn His Ser Phe Lys His Glu Asp
50 55 60
Pro Met Gly Gln Gln Gly Ser Leu Gly Glu Gln Gln Tyr Ser Val Pro
65 70 75 80
Pro Pro Val Tyr Gly Cys His Thr Pro Thr Asp Ser Cys Thr Gly Ser
85 90 95
Gln Ala Leu Leu Leu Arg Thr Pro Tyr Ser Ser Asp Asn Leu Tyr Gln
100 105 110
Met Thr Ser Gln Leu Glu Cys Met Thr Trp Asn Gln Met Asn Leu Gly
115 120 125
Ala Thr Leu Lys Gly Met Ala Ala Gly Ser Ser Ser Ser Val Lys Trp
130 135 140
Thr Glu Gly Gln Ser Asn His Gly Ile Gly Tyr Glu Ser Glu Asn His
145 150 155 160
Thr Ala Pro Ile Leu Cys Gly Ala Gln Tyr Arg Ile His Thr His Gly
165 170 175
Val Phe Arg Gly Ile Gln Asp Val Arg Arg Val Ser Gly Val Ala Pro
180 185 190
Thr Leu Val Arg Ser Ala Ser Glu Thr Ser Glu Lys Arg Pro Phe Met
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Cys Ala Tyr Pro Gly Cys Asn Lys Arg Tyr Phe Lys Leu Ser His Leu
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Gln Met His Ser Arg Lys His Thr Gly Glu Lys Pro Tyr Gln Cys Asp
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Phe Lys Asp Cys Glu Arg Arg Phe Ser Arg Ser Asp Gln Leu Lys Arg
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His Gln Arg Arg His Thr Gly Val Lys Pro Phe Gln Cys Lys Thr Cys
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Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr His Thr Arg Thr
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His Thr Gly Lys Thr Ser Glu Lys Pro Phe Ser Cys Arg Trp His Ser
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Cys Gln Lys Lys Phe Ala Arg Ser Asp Glu Leu Val Arg His His Asn
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Met His Gln
<210> 2
<211> 985
<212> DNA
<213> 小鼠(Mus musculus)
<400> 2
ctcgagccgt cccaggcgtc gtcgggccag gcgaggatgt tcccgaacgc gccctacctg 60
cccagctgcc tggagtccca gccgacgatc cgcaaccagg gctactccac cgtgacgttc 120
gacggcgccc cgtcctacgg ccacacgccc agccaccacg ccgcccagtt cccgaaccac 180
agcttcaagc acgaagaccc catgggccag cagggcagcc tcggcgaaca gcagtacagc 240
gtgccgccgc cggtctacgg ctgccacacc ccgaccgact cctgcacggg ctcccaggcc 300
ctgctcctgc gtacgccgta ctcctccgac aacctctacc agatgacctc ccagctggag 360
tgcatgacct ggaaccagat gaacctgggc gccacgctga agggaatggc cgcggggtcg 420
tcgagctccg tcaagtggac cgaaggccag tccaaccacg gcatcggcta cgagtccgag 480
aaccacaccg cgccgatcct gtgcggagcc cagtaccgca tccacacgca cggcgtcttc 540
cgcggcatcc aggacgtccg gcgcgtctcc ggcgtcgcgc cgaccctggt gcggtccgcc 600
tccgagacct ccgagaagcg cccgttcatg tgcgcctacc cgggctgcaa caagcgctac 660
ttcaagctct cgcacctgca gatgcactcc cggaagcaca ccggcgagaa gccgtaccag 720
tgcgacttca aggactgcga acgccgcttc tcgcgcagcg accagctgaa gcgccaccag 780
cgtaggcaca ccggcgtgaa gcccttccag tgcaagacct gccagcgcaa gttctcccgc 840
agcgaccacc tcaagacgca cacccgcacc cacaccggca agacgtccga gaagccgttc 900
tcgtgccgct ggcacagctg ccagaagaag ttcgcccgca gcgacgagct cgtgcgccac 960
cacaacatgc accagtgaag catgc 985
<210> 3
<211> 22
<212> DNA
<213> 人工(Artificial)
<220>
<223> GL-BP的引物(glt-r)(Primer for GL-BP (glt-r))
<400> 3
ggggtgctga tatattggtt tg 22
<210> 4
<211> 31
<212> DNA
<213> 人工(Artificial)
<220>
<223> GL-BP的引物(glt-r)(Primer for GL-BP (glt-r))
<400> 4
gctcgagctc ggaaacagac aggccgaagt t 31
<210> 5
<211> 6
<212> DNA
<213> 人工(Artificial)
<220>
<223> XhoI的识别序列(Recognition sequence for XhoI)
<400> 5
ctcgag 6
<210> 6
<211> 6
<212> DNA
<213> 人工(Artificial)
<220>
<223> SphI的识别序列(Recognition sequence for SphI)
<400> 6
gcatgc 6
<210> 7
<211> 24
<212> DNA
<213> 人工(Artificial)
<220>
<223> WT1的引物(WT1-f)(Primer for WT1 (WT1-f))
<400> 7
cgctcgagcc gtcccaggcg tcgt 24
<210> 8
<211> 30
<212> DNA
<213> 人工(Artificial)
<220>
<223> WT1的引物(WT1-r2)(Primer for WT1 (WT1-r2))
<400> 8
gcgcatgctc actcgccggt gtgcttccgg 30
<210> 9
<211> 36
<212> DNA
<213> 人工(Artificial)
<220>
<223> 引物(Infusion-F)(Primer (Infusion-F))
<400> 9
ggaaaactgt ccatagatgg cgaggcgaac gccacg 36
<210> 10
<211> 36
<212> DNA
<213> 人工(Artificial)
<220>
<223> 引物(Infusion-F)(Primer (Infusion-F))
<400> 10
tttcatctgt gcatagtgct gcaaggcgat taagtt 36
<210> 11
<211> 23
<212> DNA
<213> 人工(Artificial)
<220>
<223> 引物(410, 420-F)(Primer (410, 420-F))
<400> 11
acgatccgca accagggcta ctc 23
<210> 12
<211> 23
<212> DNA
<213> 人工(Artificial)
<220>
<223> 引物(410-R)(Primer (410-R))
<400> 12
ggtgcgagag cttgaagtag cgc 23
<210> 13
<211> 22
<212> DNA
<213> 人工(Artificial)
<220>
<223> 引物(420-R)(Primer (420-R))
<400> 13
gtcgctgcgg gcgaacttct tc 22
<210> 14
<211> 323
<212> PRT
<213> 人类(Homo sapiens)
<400> 14
Pro Ser Gln Ala Ser Ser Gly Gln Ala Arg Met Phe Pro Asn Ala Pro
1 5 10 15
Tyr Leu Pro Ser Cys Leu Glu Ser Gln Pro Ala Ile Arg Asn Gln Gly
20 25 30
Tyr Ser Thr Val Thr Phe Asp Gly Thr Pro Ser Tyr Gly His Thr Pro
35 40 45
Ser His His Ala Ala Gln Phe Pro Asn His Ser Phe Lys His Glu Asp
50 55 60
Pro Met Gly Gln Gln Gly Ser Leu Gly Glu Gln Gln Tyr Ser Val Pro
65 70 75 80
Pro Pro Val Tyr Gly Cys His Thr Pro Thr Asp Ser Cys Thr Gly Ser
85 90 95
Gln Ala Leu Leu Leu Arg Thr Pro Tyr Ser Ser Asp Asn Leu Tyr Gln
100 105 110
Met Thr Ser Gln Leu Glu Cys Met Thr Trp Asn Gln Met Asn Leu Gly
115 120 125
Ala Thr Leu Lys Gly Val Ala Ala Gly Ser Ser Ser Ser Val Lys Trp
130 135 140
Thr Glu Gly Gln Ser Asn His Ser Thr Gly Tyr Glu Ser Asp Asn His
145 150 155 160
Thr Thr Pro Ile Leu Cys Gly Ala Gln Tyr Arg Ile His Thr His Gly
165 170 175
Val Phe Arg Gly Ile Gln Asp Val Arg Arg Val Pro Gly Val Ala Pro
180 185 190
Thr Leu Val Arg Ser Ala Ser Glu Thr Ser Glu Lys Arg Pro Phe Met
195 200 205
Cys Ala Tyr Pro Gly Cys Asn Lys Arg Tyr Phe Lys Leu Ser His Leu
210 215 220
Gln Met His Ser Arg Lys His Thr Gly Glu Lys Pro Tyr Gln Cys Asp
225 230 235 240
Phe Lys Asp Cys Glu Arg Arg Phe Ser Arg Ser Asp Gln Leu Lys Arg
245 250 255
His Gln Arg Arg His Thr Gly Val Lys Pro Phe Gln Cys Lys Thr Cys
260 265 270
Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr His Thr Arg Thr
275 280 285
His Thr Gly Lys Thr Ser Glu Lys Pro Phe Ser Cys Arg Trp Pro Ser
290 295 300
Cys Gln Lys Lys Phe Ala Arg Ser Asp Glu Leu Val Arg His His Asn
305 310 315 320
Met His Gln
<210> 15
<211> 973
<212> DNA
<213> 人类(Homo sapiens)
<400> 15
ccgtcccagg cgtcgtcggg ccaggcgagg atgttcccga acgcgcccta cctgcccagc 60
tgcctggagt cccagccggc gatccgcaac cagggctact ccaccgtgac gttcgacggc 120
accccgtcct acggccacac gcccagccac cacgccgccc agttcccgaa ccacagcttc 180
aagcacgaag accccatggg ccagcagggc agcctcggcg aacagcagta cagcgtgccg 240
ccgccggtct acggctgcca caccccgacc gactcctgca cgggctccca ggccctgctc 300
ctgcgtacgc cgtactcctc cgacaacctc taccagatga cctcccagct ggagtgcatg 360
acctggaacc agatgaacct gggcgccacg ctgaagggag tcgccgcggg gtcgtcgagc 420
tccgtcaagt ggaccgaagg ccagtccaac cactccaccg gctacgagtc cgacaaccac 480
accacgccga tcctgtgcgg agcccagtac cgcatccaca cgcacggcgt cttccgcggc 540
atccaggacg tccggcgcgt ccccggcgtc gcgccgaccc tggtgcggtc cgcctccgag 600
acctccgaga agcgcccgtt catgtgcgcc tacccgggct gcaacaagcg ctacttcaag 660
ctctcgcacc tgcagatgca ctcccggaag cacaccggcg agaagccgta ccagtgcgac 720
ttcaaggact gcgaacgccg cttctcgcgc agcgaccagc tgaagcgcca ccagcgtagg 780
cacaccggcg tgaagccctt ccagtgcaag acctgccagc gcaagttctc ccgcagcgac 840
cacctcaaga cgcacacccg cacccacacc ggcaagacgt ccgagaagcc gttctcgtgc 900
cgctggccca gctgccagaa gaagttcgcc cgcagcgacg agctcgtgcg ccaccacaac 960
atgcaccagt gaa 973
<210> 16
<211> 323
<212> PRT
<213> 人类(Homo sapiens)
<400> 16
Pro Ser Gln Ala Ser Ser Gly Gln Ala Arg Met Phe Pro Asn Ala Pro
1 5 10 15
Tyr Leu Pro Ser Cys Leu Glu Ser Gln Pro Ala Ile Arg Asn Gln Gly
20 25 30
Tyr Ser Thr Val Thr Phe Asp Gly Thr Pro Ser Tyr Gly His Thr Pro
35 40 45
Ser His His Ala Ala Gln Phe Pro Asn His Ser Phe Lys His Glu Asp
50 55 60
Pro Met Gly Gln Gln Gly Ser Leu Gly Glu Gln Gln Tyr Ser Val Pro
65 70 75 80
Pro Pro Val Tyr Gly Cys His Thr Pro Thr Asp Ser Cys Thr Gly Ser
85 90 95
Gln Ala Leu Leu Leu Arg Thr Pro Tyr Ser Ser Asp Asn Leu Tyr Gln
100 105 110
Met Thr Ser Gln Leu Glu Cys Tyr Thr Trp Asn Gln Met Asn Leu Gly
115 120 125
Ala Thr Leu Lys Gly Val Ala Ala Gly Ser Ser Ser Ser Val Lys Trp
130 135 140
Thr Glu Gly Gln Ser Asn His Ser Thr Gly Tyr Glu Ser Asp Asn His
145 150 155 160
Thr Thr Pro Ile Leu Cys Gly Ala Gln Tyr Arg Ile His Thr His Gly
165 170 175
Val Phe Arg Gly Ile Gln Asp Val Arg Arg Val Pro Gly Val Ala Pro
180 185 190
Thr Leu Val Arg Ser Ala Ser Glu Thr Ser Glu Lys Arg Pro Phe Met
195 200 205
Cys Ala Tyr Pro Gly Cys Asn Lys Arg Tyr Phe Lys Leu Ser His Leu
210 215 220
Gln Met His Ser Arg Lys His Thr Gly Glu Lys Pro Tyr Gln Cys Asp
225 230 235 240
Phe Lys Asp Cys Glu Arg Arg Phe Ser Arg Ser Asp Gln Leu Lys Arg
245 250 255
His Gln Arg Arg His Thr Gly Val Lys Pro Phe Gln Cys Lys Thr Cys
260 265 270
Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr His Thr Arg Thr
275 280 285
His Thr Gly Lys Thr Ser Glu Lys Pro Phe Ser Cys Arg Trp Pro Ser
290 295 300
Cys Gln Lys Lys Phe Ala Arg Ser Asp Glu Leu Val Arg His His Asn
305 310 315 320
Met His Gln
<210> 17
<211> 973
<212> DNA
<213> 人类(Homo sapiens)
<400> 17
ccgtcccagg cgtcgtcggg ccaggcgagg atgttcccga acgcgcccta cctgcccagc 60
tgcctggagt cccagccggc gatccgcaac cagggctact ccaccgtgac gttcgacggc 120
accccgtcct acggccacac gcccagccac cacgccgccc agttcccgaa ccacagcttc 180
aagcacgaag accccatggg ccagcagggc agcctcggcg aacagcagta cagcgtgccg 240
ccgccggtct acggctgcca caccccgacc gactcctgca cgggctccca ggccctgctc 300
ctgcgtacgc cgtactcctc cgacaacctc taccagatga cctcccagct ggagtgctac 360
acctggaacc agatgaacct gggcgccacg ctgaagggag tcgccgcggg gtcgtcgagc 420
tccgtcaagt ggaccgaagg ccagtccaac cactccaccg gctacgagtc cgacaaccac 480
accacgccga tcctgtgcgg agcccagtac cgcatccaca cgcacggcgt cttccgcggc 540
atccaggacg tccggcgcgt ccccggcgtc gcgccgaccc tggtgcggtc cgcctccgag 600
acctccgaga agcgcccgtt catgtgcgcc tacccgggct gcaacaagcg ctacttcaag 660
ctctcgcacc tgcagatgca ctcccggaag cacaccggcg agaagccgta ccagtgcgac 720
ttcaaggact gcgaacgccg cttctcgcgc agcgaccagc tgaagcgcca ccagcgtagg 780
cacaccggcg tgaagccctt ccagtgcaag acctgccagc gcaagttctc ccgcagcgac 840
cacctcaaga cgcacacccg cacccacacc ggcaagacgt ccgagaagcc gttctcgtgc 900
cgctggccca gctgccagaa gaagttcgcc cgcagcgacg agctcgtgcg ccaccacaac 960
atgcaccagt gaa 973
<210> 18
<211> 16
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> T细胞表位np332(T cell epitope np332)
<400> 18
Lys Arg Tyr Phe Lys Leu Ser His Leu Gln Met His Ser Arg Lys His
1 5 10 15
<210> 19
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> T细胞表位np126(T cell epitope np126)
<400> 19
Arg Met Phe Pro Asn Ala Pro Tyr Leu
1 5
<210> 20
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> T细胞表位n187(T cell epitope n187)
<400> 20
Ser Leu Gly Glu Gln Gln Tyr Ser Val
1 5
<210> 21
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> T细胞表位n235(T cell epitope n235)
<400> 21
Cys Met Thr Trp Asn Gln Met Asn Leu
1 5
<210> 22
<211> 449
<212> PRT
<213> 小鼠(Mus musculus)
<400> 22
Met Gly Ser Asp Val Arg Asp Leu Asn Ala Leu Leu Pro Ala Val Ser
1 5 10 15
Ser Leu Gly Gly Gly Gly Gly Gly Cys Gly Leu Pro Val Ser Gly Ala
20 25 30
Arg Gln Trp Ala Pro Val Leu Asp Phe Ala Pro Pro Gly Ala Ser Ala
35 40 45
Tyr Gly Ser Leu Gly Gly Pro Ala Pro Pro Pro Ala Pro Pro Pro Pro
50 55 60
Pro Pro Pro Pro His Ser Phe Ile Lys Gln Glu Pro Ser Trp Gly Gly
65 70 75 80
Ala Glu Pro His Glu Glu Gln Cys Leu Ser Ala Phe Thr Leu His Phe
85 90 95
Ser Gly Gln Phe Thr Gly Thr Ala Gly Ala Cys Arg Tyr Gly Pro Phe
100 105 110
Gly Pro Pro Pro Pro Ser Gln Ala Ser Ser Gly Gln Ala Arg Met Phe
115 120 125
Pro Asn Ala Pro Tyr Leu Pro Ser Cys Leu Glu Ser Gln Pro Thr Ile
130 135 140
Arg Asn Gln Gly Tyr Ser Thr Val Thr Phe Asp Gly Ala Pro Ser Tyr
145 150 155 160
Gly His Thr Pro Ser His His Ala Ala Gln Phe Pro Asn His Ser Phe
165 170 175
Lys His Glu Asp Pro Met Gly Gln Gln Gly Ser Leu Gly Glu Gln Gln
180 185 190
Tyr Ser Val Pro Pro Pro Val Tyr Gly Cys His Thr Pro Thr Asp Ser
195 200 205
Cys Thr Gly Ser Gln Ala Leu Leu Leu Arg Thr Pro Tyr Ser Ser Asp
210 215 220
Asn Leu Tyr Gln Met Thr Ser Gln Leu Glu Cys Met Thr Trp Asn Gln
225 230 235 240
Met Asn Leu Gly Ala Thr Leu Lys Gly Met Ala Ala Gly Ser Ser Ser
245 250 255
Ser Val Lys Trp Thr Glu Gly Gln Ser Asn His Gly Ile Gly Tyr Glu
260 265 270
Ser Glu Asn His Thr Ala Pro Ile Leu Cys Gly Ala Gln Tyr Arg Ile
275 280 285
His Thr His Gly Val Phe Arg Gly Ile Gln Asp Val Arg Arg Val Ser
290 295 300
Gly Val Ala Pro Thr Leu Val Arg Ser Ala Ser Glu Thr Ser Glu Lys
305 310 315 320
Arg Pro Phe Met Cys Ala Tyr Pro Gly Cys Asn Lys Arg Tyr Phe Lys
325 330 335
Leu Ser His Leu Gln Met His Ser Arg Lys His Thr Gly Glu Lys Pro
340 345 350
Tyr Gln Cys Asp Phe Lys Asp Cys Glu Arg Arg Phe Ser Arg Ser Asp
355 360 365
Gln Leu Lys Arg His Gln Arg Arg His Thr Gly Val Lys Pro Phe Gln
370 375 380
Cys Lys Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr
385 390 395 400
His Thr Arg Thr His Thr Gly Lys Thr Ser Glu Lys Pro Phe Ser Cys
405 410 415
Arg Trp His Ser Cys Gln Lys Lys Phe Ala Arg Ser Asp Glu Leu Val
420 425 430
Arg His His Asn Met His Gln Arg Asn Met Thr Lys Leu His Val Ala
435 440 445
Leu
<210> 23
<211> 449
<212> PRT
<213> 人类(Homo sapiens)
<400> 23
Met Gly Ser Asp Val Arg Asp Leu Asn Ala Leu Leu Pro Ala Val Pro
1 5 10 15
Ser Leu Gly Gly Gly Gly Gly Cys Ala Leu Pro Val Ser Gly Ala Ala
20 25 30
Gln Trp Ala Pro Val Leu Asp Phe Ala Pro Pro Gly Ala Ser Ala Tyr
35 40 45
Gly Ser Leu Gly Gly Pro Ala Pro Pro Pro Ala Pro Pro Pro Pro Pro
50 55 60
Pro Pro Pro Pro His Ser Phe Ile Lys Gln Glu Pro Ser Trp Gly Gly
65 70 75 80
Ala Glu Pro His Glu Glu Gln Cys Leu Ser Ala Phe Thr Val His Phe
85 90 95
Ser Gly Gln Phe Thr Gly Thr Ala Gly Ala Cys Arg Tyr Gly Pro Phe
100 105 110
Gly Pro Pro Pro Pro Ser Gln Ala Ser Ser Gly Gln Ala Arg Met Phe
115 120 125
Pro Asn Ala Pro Tyr Leu Pro Ser Cys Leu Glu Ser Gln Pro Ala Ile
130 135 140
Arg Asn Gln Gly Tyr Ser Thr Val Thr Phe Asp Gly Thr Pro Ser Tyr
145 150 155 160
Gly His Thr Pro Ser His His Ala Ala Gln Phe Pro Asn His Ser Phe
165 170 175
Lys His Glu Asp Pro Met Gly Gln Gln Gly Ser Leu Gly Glu Gln Gln
180 185 190
Tyr Ser Val Pro Pro Pro Val Tyr Gly Cys His Thr Pro Thr Asp Ser
195 200 205
Cys Thr Gly Ser Gln Ala Leu Leu Leu Arg Thr Pro Tyr Ser Ser Asp
210 215 220
Asn Leu Tyr Gln Met Thr Ser Gln Leu Glu Cys Met Thr Trp Asn Gln
225 230 235 240
Met Asn Leu Gly Ala Thr Leu Lys Gly Val Ala Ala Gly Ser Ser Ser
245 250 255
Ser Val Lys Trp Thr Glu Gly Gln Ser Asn His Ser Thr Gly Tyr Glu
260 265 270
Ser Asp Asn His Thr Thr Pro Ile Leu Cys Gly Ala Gln Tyr Arg Ile
275 280 285
His Thr His Gly Val Phe Arg Gly Ile Gln Asp Val Arg Arg Val Pro
290 295 300
Gly Val Ala Pro Thr Leu Val Arg Ser Ala Ser Glu Thr Ser Glu Lys
305 310 315 320
Arg Pro Phe Met Cys Ala Tyr Pro Gly Cys Asn Lys Arg Tyr Phe Lys
325 330 335
Leu Ser His Leu Gln Met His Ser Arg Lys His Thr Gly Glu Lys Pro
340 345 350
Tyr Gln Cys Asp Phe Lys Asp Cys Glu Arg Arg Phe Ser Arg Ser Asp
355 360 365
Gln Leu Lys Arg His Gln Arg Arg His Thr Gly Val Lys Pro Phe Gln
370 375 380
Cys Lys Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr
385 390 395 400
His Thr Arg Thr His Thr Gly Lys Thr Ser Glu Lys Pro Phe Ser Cys
405 410 415
Arg Trp Pro Ser Cys Gln Lys Lys Phe Ala Arg Ser Asp Glu Leu Val
420 425 430
Arg His His Asn Met His Gln Arg Asn Met Thr Lys Leu Gln Leu Ala
435 440 445
Leu
<210> 24
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物F:430(Primer F:430)
<400> 24
atgacctccc agctggagtg catgacctgg 30
<210> 25
<211> 30
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物F:440(Primer F:440)
<400> 25
atgacctccc agctggagtg ctacacctgg 30
<210> 26
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 引物R: 430, 440(Primer R: 430, 440)
<400> 26
ccagctgcca gaagaagttc gcccgcagcg ac 32
Claims (7)
1.一种转化双歧杆菌,其特征在于,
包含编码以序列号1、序列号14以及序列号16所表示的氨基酸序列中的任意一种氨基酸序列确定的WT1蛋白质的DNA、和编码源自双歧杆菌的GNB/LNB基质结合膜蛋白质的DNA,且被设计为将作为抗原的WT1蛋白质与GNB/LNB基质结合膜蛋白质的融合蛋白质, 即GL-BP-WT1融合蛋白质呈递于双歧杆菌表层上。
2.如权利要求1所述的转化双歧杆菌,其特征在于,
编码WT1蛋白质的DNA是以序列号2、15以及序列号17所表示的碱基序列中的任意一种确定的DNA。
3.如权利要求1或2所述的转化双歧杆菌,其特征在于,
在编码WT1蛋白质的DNA与编码源自双歧杆菌的GNB/LNB基质结合膜蛋白质的DNA之间,含有编码具有辅助功能的蛋白质的DNA。
4.一种制剂,其特征在于,作为疫苗的有效成分而含有权利要求1至3中任一项所述的转化双歧杆菌。
5.如权利要求4所述的制剂,其特征在于,所述制剂为肿瘤疫苗制剂。
6.如权利要求4或5所述的制剂,其特征在于,所述制剂为口服制剂。
7.一种质粒或穿梭载体,其特征在于,
包含编码以序列号1、序列号14以及序列号16所表示的氨基酸序列中的任意一种氨基酸序列确定WT1蛋白质的DNA、和编码源自双歧杆菌的GNB/LNB基质结合膜蛋白质的DNA,且被设计为将作为抗原的WT1蛋白质呈递于双歧杆菌表层上。
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CA2626238C (en) | 2005-10-17 | 2015-10-06 | Sloan Kettering Institute For Cancer Research | Wt1 hla class ii-binding peptides and compositions and methods comprising same |
US9265816B2 (en) | 2006-04-10 | 2016-02-23 | Sloan Kettering Institute For Cancer Research | Immunogenic WT-1 peptides and methods of use thereof |
US20150104413A1 (en) | 2012-01-13 | 2015-04-16 | Memorial Sloan Kettering Cancer Center | Immunogenic wt-1 peptides and methods of use thereof |
EP3769782A1 (en) | 2013-01-15 | 2021-01-27 | Memorial Sloan Kettering Cancer Center | Immunogenic wt-1 peptides and methods of use thereof |
US10815273B2 (en) | 2013-01-15 | 2020-10-27 | Memorial Sloan Kettering Cancer Center | Immunogenic WT-1 peptides and methods of use thereof |
EP3560513A4 (en) | 2016-12-26 | 2020-10-14 | National University Corporation Kobe University | ANTICANCER THERAPY USING A COMBINATION OF AN ORAL TUMOR VACCINE AND AN IMMUNOSUPPRESSION INHIBITOR |
CN116134131A (zh) | 2019-12-31 | 2023-05-16 | 伊利克斯根治疗公司 | 核酸和蛋白质向细胞和组织的基于温度的瞬时递送 |
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Also Published As
Publication number | Publication date |
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WO2016208332A1 (ja) | 2016-12-29 |
US10695385B2 (en) | 2020-06-30 |
JPWO2016208332A1 (ja) | 2018-06-28 |
JP6770269B2 (ja) | 2020-10-14 |
CN108350411A (zh) | 2018-07-31 |
US20180169156A1 (en) | 2018-06-21 |
EP3315599A4 (en) | 2019-03-06 |
EP3315599A1 (en) | 2018-05-02 |
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