CN112451657A - 免疫疗法疫苗的制备方法 - Google Patents
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Abstract
本文提供用于癌症免疫疗法的疫苗的制备方法。外泌体在常温放置一定时间以上时,膜结构发生崩溃,不能与树突细胞融合。因此,需要在促进蛋白质转化为肽并且维持外泌体膜结构的条件下进行加热处理,该条件为56℃以下并且30分钟以下。在其以上的温度条件,蛋白质发生变性,不能保持抗原性。因此,通过将从癌症患者的癌细胞或血液等体液分离的外泌体(Exosome)在56℃以下并且30分钟以下的条件进行加热处理,促进外泌体内的蛋白质酶失活,使蛋白质降解酶及脂质降解酶失活。然后将该外泌体导入从癌症患者或健康人的血液等诱导的树突细胞或与所述树突细胞共培养。
Description
技术领域
本发明涉及在使用癌细胞的外泌体和树突细胞的免疫疗法中使用的树突细胞疫苗的制备方法。
背景技术
对免疫疗法而言,存在以癌细胞产生的特定的蛋白质作为标志提呈特异性抗体,产生攻击癌细胞的免疫细胞(CTL)的疗法(获得性免疫),以及识别异物蛋白质而进行排除异物(自然免疫)两种疗法。
在至今为止的免疫疗法中,使从患者的血液单核细胞等分离的树突细胞(DC:dendritic cell)与从患者得到的癌细胞(也包括癌前细胞)融合,将该融合细胞单独或与刺激细胞毒性T细胞应答、体液免疫应答的细胞因子一起通过皮下注射等进行施用。
在该以往的免疫疗法的情况下,树突细胞和癌细胞的融合效率低,为10%左右,因此,施用的疫苗的抗原呈递效率差。另一方面,使用外泌体而被DC摄入时,相对于105个DC,能够获得外泌体的数量为108~109个,在大部分的DC中摄入外泌体。
另外,正常的成熟树突细胞来源的外泌体和NK细胞来源的外泌体等导致癌细胞增殖的阻碍,调节性T细胞(Treg)的阻碍,MDSC的阻碍,血管生成的阻碍,而在破坏癌组织的方向发挥功能。因此,成熟树突细胞来源的外泌体和NK细胞来源的外泌体等正用于癌症治疗中。
另一方面,癌细胞来源的外泌体存在使癌细胞恶化而使其基因发生突变的倾向。另外,造成阻止癌细胞增殖的免疫细胞的失活。在这种情况下,癌细胞会从构成疫苗的树突细胞的抗原呈递功能下逃出,即使融合细胞作为抗原呈递细胞(APC)发挥作用,诱导细胞毒性T细胞(CTL),自然杀伤细胞(NK),CTL的活性、NK活性也丧失,失去抗癌活性。即,由于癌细胞从免疫疗法下逃出,释放癌细胞来源的外泌体,频繁使其基因发生突变,且使免疫细胞的活性失活。尚没有建立对此有效的应对方法。
作为解决如树突细胞和癌细胞的融合效率较低这样的现有问题的现有技术,提出专利文献1。在该专利文献1中,公开了将从癌细胞释放的外泌体(exosome)通过电穿孔处理(electroporation)插入树突细胞、T细胞等免疫细胞中,将经该电穿孔处理的细胞(疫苗)施用于患者。
根据专利文献1,癌细胞的基因经常变化,癌细胞使基因突变则癌细胞表达的蛋白质也发生变化,从抗原呈递中逃出。细胞来源的外泌体维持与作为分泌来源的细胞相同的蛋白质和功能。不仅正常的成熟树突细胞来源的或NK细胞来源的外泌体,从癌细胞释放的外泌体也携带与分泌来源的癌细胞相同的蛋白质,即抗原。即,在使用癌细胞来源的外泌体的情况下,即使癌细胞的基因发生突变,在发生突变的癌细胞来源的外泌体中,也携带与发生突变的癌细胞相同的抗原。
即,将癌细胞来源的外泌体作为抗原使用时,即使癌细胞基因发生变化而使表达的蛋白质发生变化,其蛋白质也与癌细胞来源的外泌体具有的蛋白质相同,因此,癌细胞不能从抗原呈递下逃脱。
另外,在非专利文献1及非专利文献2中公开了通过将癌细胞来源的外泌体进行脉冲导入树突细胞中,由此该树突细胞对患者的T细胞诱导自身肿瘤细胞的细胞毒活性。
进一步,在非专利文献3中记载了经过用白血病细胞来源的外泌体刺激的树突细胞诱导针对白血病细胞的细胞毒性T淋巴细胞免疫反应。
现有技术文献
专利文献
专利文献1:日本特表2013-523824号公报
非专利文献
非专利文献1:日本外科学会杂志,2005年,vol.106,临时增刊号,p.195,摘要No.SF2309-2
非专利文献2:Biotherapy,2004年,vol.18,Suppl.1,p.116,摘要No.p-27
非专利文献3:PLOS ONE,2004年,vol.9,no.3,e91463,p.1-7
发明内容
发明所要解决的问题
如果使从癌细胞释放的外泌体与树突细胞(DC:dendritic cell)融合,将该融合的细胞作为疫苗施用于患者,则即使癌细胞基因发生突变,该疫苗也不丧失作为抗原呈递细胞(APC)的功能。从而,现有技术文献公开的方法有效。
已判明抗原呈递的机理为与癌细胞特异性的蛋白质在细胞内被分解为肽,该肽与存在于癌细胞表面的MHC分子结合而作为抗原肽被提呈,细胞毒性T细胞(CTL)识别该抗原肽而攻击癌细胞。
然而,外泌体内的蛋白质分解为肽需要时间,免疫反应并不容易发生。即,存在即使从癌细胞释放的外泌体与树突细胞融合,到作为疫苗发挥效果也需要时间的问题。
另外,存在外泌体不能长期保存的问题。与树突细胞融合的外泌体必需是维持膜结构的颗粒。然而,由于在常温中经过一定时间膜结构崩溃,因此无法保存。
如果在极低温度例如-96℃进行冻存则能够长期保存,但在制作疫苗时必须进行融解,如果反复融解,则此时存在膜发生损坏而不再保有作为颗粒的结构的情况。也考虑不进行冻存而是在4℃左右的低温进行保存。然而,与具有膜结构的病毒(流感,副流感,疱疹病毒)等颗粒同样,在1个月左右失活(99%),颗粒被破坏,抗原呈递效率变差。无法制成蛋白质。
解决问题的手段
对外泌体而言,可在癌细胞恶化的同时与其母细胞具有相同的抗原。因此,具有相对于其突变有效的抗原呈递。
本发明涉及的癌症免疫疗法疫苗的制备方法从癌细胞(包括癌干细胞)的培养上清液、癌症患者的血液、腹水等体液分离外泌体,将该分离的外泌体通过加热处理而失活,将该失活的外泌体导入树突细胞进行融合。
如上所述,外泌体在常温放置一定时间以上时,膜结构发生崩溃,不能与树突细胞融合。因此,需要利用促进蛋白质转化为肽并且维持外泌体膜结构的条件进行加热处理,该条件为56℃以下并且30分钟以下。在其以上的温度条件,蛋白质发生变性,不能保持抗原性。
本发明涉及的用于癌症免疫疗法的疫苗的制备方法中,作为使从癌细胞、患者的体液等分离的外泌体导入树突细胞的手段,可以使用电穿孔法,PEG(聚乙二醇)法,仙台病毒法。
在使用电穿孔法的情况下,认为由于在瞬间摄入,另外,在1ml的末期癌症患者的血清中存在109个以上的外泌体颗粒,因此被树突细胞的摄入率为90%以上。因此,认为与以往的使树突细胞和癌细胞融合的情况相比,抗原呈递细胞(APC)大幅增加。
另外,外泌体不仅能从癌症患者的血液分离,也能够从尿、腹水等体液分离。
发明的效果
可列举根据本发明而抗原呈递功能提高的效果。如上所述,癌细胞特有的蛋白质(外泌体内的蛋白质)被分解为肽,其与MHC分子结合而作为抗原肽提呈给细胞毒性T细胞(CTL),一般而言,蛋白质为了保持生理活性,而以不成为蛋白酶、脂肪酶等蛋白质降解酶、脂质降解酶的攻击目标的形式采取三维立体结构。然而,通过本发明的加热处理,蛋白质的三维结构没有发生损坏,而仅在活性基团中发生变化,酶活性丧失。而且,癌细胞特有的蛋白质容易分解为肽而与MHC分子结合。因此,对于细胞毒性T细胞(CTL)的抗原呈递功能提高。
作为次要效果,可列举蛋白质降解酶、脂质降解酶失活,作为外泌体的颗粒能够维持稳定性,能够实现长期保存。进一步,也考虑通过加热外泌体而蛋白质(热休克蛋白:HeatShock Protein,HSP)的表达量发生增加的可能。认为这些HSP具有强化免疫的功能。
上述HSP为将免疫全部活化并且具有耐热性的蛋白质,通过热处理不被失活而被活化。这一点对于将热处理的外泌体作为抗原使用而言也有意义。
附图说明
[图1]将加热条件设为56℃并且30分钟的情况下的外泌体的显微镜照片。
[图2]将加热条件设为56℃并且30分钟的情况下的外泌体的显微镜照片。
[图3]将加热条件设为56℃并且30分钟的情况下的外泌体的显微镜照片。
[图4]示出将经热处理的外泌体的热处理后的时期和细胞毒活性(裂解%)进行比较的结果的图。
[图5]为导入外泌体的树突细胞(DC)的蛋白质的荧光(绿色荧光和红色荧光)及明场的显微镜照片,上图示出了将未进行热处理的外泌体利用电穿孔处理(electroporation)导入的树突细胞(DC),中图为将利用本发明的条件进行了热处理的外泌体通过共培养(co-culture)导入的树突细胞(DC),下图为将本发明的条件进行了热处理的外泌体通过电穿孔处理(electroporation)导入的树突细胞(DC)。
具体实施方式
下文中对本发明的实施例进行说明。本发明的权利范围并不受该实施例限制。
(A)从细胞培养上清提取外泌体
步骤1:利用去除外泌体的FCS添加培养基培养2天之后,从培养上清进行提取。在提取中,作为分离试剂盒使用miRCURY Exosome Cell/Urine/CSF试剂盒(TAKARA BioCat.300102)。
步骤2:测量步骤1中提取的外泌体颗粒数。
步骤3:通过进行加热处理(56℃,30分钟),将外泌体灭活。
外泌体容易通过加热而细胞膜崩解。由于细胞膜崩解则不能与树突细胞融合,通过改变加热条件而确定加热处理的上限温度及上限时间。确定方法根据每个条件的显微镜照片,观察外泌体的膜结构而进行。
图1~3均选定56℃、30分钟作为加热条件。在各图中,圆形、接近黑色部分为膜结构,膜结构能够保持的外泌体,比接近黑色部分更大的圆形、接近白色部分为膜结构发生崩解的外泌体。在膜结构能够保持的外泌体内观察不到核,可确认外泌体从细胞质分泌出来。在膜结构发生崩解的外泌体内也存在核消失的情况。
在作为温度条件设为不到56℃,例如37℃(体温)的情况下不能称为加热,是没有发生酶的活性基团的变性的普通外泌体。另外,超过56℃则膜结构发生崩解的外泌体增加。对处理时间而言也是一样。
在56℃×30分钟的情况下,从显微镜照片也可知,膜结构发生了一定程度崩解的外泌体也夹杂其中。这反而是证明存在膜结构不发生崩解的外泌体,外泌体内的酶变性处于进行中的证据,因此,优选在56℃以下并且30分钟以下的条件内,尽可能在接近56℃并且30分钟的条件下进行加热处理。
(B)由活化细胞毒性T细胞(CTL)引起的肿瘤细胞的细胞毒性
步骤1:从健康人采血之后,由末梢血单核细胞诱导树突细胞。具体而言,采末梢血(50ml)。然后进行离心(c.f.g.1800xg 15min),收集中间层的细胞。这之后,用PBS洗涤2次(c.f.g.1200r.p.m.5min),测量细胞数,以2×106/ml再悬浮于培养基(CTS AIM V培养基(Gibco,Thermo Fisher Scientific)。
以1ml/孔接种于24孔板中,于37℃,5%CO2培养箱中培养2小时。然后,除去悬浮细胞,加入1ml/孔树突细胞用培养基(在AIM V培养基中添加了细胞因子(最终10ng/ml GM-CSF及IL-4,Miltenyi Biotec)和2%自身血浆)并开始培养。
之后,将500ul新鲜树突细胞用培养基添加到孔中,另外添加成熟用的细胞因子(最终10ng/ml TNFα,Miltenyi Biotec)。
步骤2:作为树突细胞的抗原刺激,进行以下测试区(1)~(3)。
(1)由DC和肿瘤细胞的PEG(聚乙二醇)引起的细胞融合
(2)DC和外泌体的电穿孔
(3)DC和外泌体的共培养
步骤3:进行利用上述测试区(1)~(3)进行活化的树突细胞与细胞毒性T细胞(CTL)的共培养。
步骤4:进行上述共培养的细胞毒性T细胞(CTL)与肿瘤细胞的共培养。
通过MTT测定观察上述细胞毒性T细胞(CTL)的细胞毒性。将结果示于图4,图5。
该图4为示出靶标:效应器(Target:Effector)的比率与细胞毒性T细胞的细胞毒性(裂解)的关系的图,可知通过采用本发明方法进行加热处理的外泌体以电穿孔处理(electroporation)法导入DC细胞中并将其作为抗原的细胞毒性T细胞(CTL)显示出与现在使用的、采用由PEG(聚乙二醇)引起的细胞融合得到的细胞毒性T细胞(CTL),及共培养物(co-culture)同等的细胞毒性。
特别是在靶标:效应器(横轴)中,当使效应器增加时,与PEG细胞融合及共培养物(co-culture)相比,细胞毒性(裂解)升高。
另外,根据图5可知,在经热处理的外泌体的蛋白质表达量增加。
因此,根据本发明方法得到的疫苗对免疫疗法而言有效。
对本发明的疫苗即通过将经过热处理的外泌体采用电穿孔等导入DC(树突细胞)中的那些而言,作为抗原呈递细胞(APC)发挥功能。在将该抗原呈递细胞(APC)通过皮下注射等施用至患者的体内时,抗原呈递细胞(APC)移动至淋巴结,培育淋巴结中的T细胞变为细胞毒性T细胞(CTL)。
如上所述,由于与DC相比,外泌体的数量极多,因此,摄入外泌体的DC的数量也变多。这样的DC数量变多,则抗原呈递细胞(APC)的数量也增加,细胞毒性T细胞(CTL)也增加而免疫效果提高。
另外,也可以将导入外泌体的DC(树突细胞)进一步与T细胞共培养得到的细胞毒性T细胞(CTL)作为疫苗。
Claims (3)
1.免疫疗法疫苗的制备方法,其特征在于将从癌症患者的癌细胞或血液等体液分离的外泌体(Exosome)在56℃并且30分钟的条件下加热处理,促进外泌体内的蛋白质降解酶失活,然后将该外泌体导入从癌症患者或健康人血液等诱导的树突细胞或与所述树突细胞共培养作为抗原呈递细胞。
2.免疫疗法疫苗的制备方法,其特征在于在权利要求1所述的免疫疗法疫苗的制备方法中,通过所述加热处理使蛋白质降解酶及脂质降解酶失活,维持作为外泌体的颗粒的稳定性。
3.免疫疗法疫苗的制备方法,其特征在于在权利要求1或权利要求2所述的免疫疗法疫苗的制备方法中,将所述树突细胞与T细胞进行共培养作为细胞毒性T细胞。
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