CN1705489A - 基于树突状细胞的癌症免疫治疗的临床应用 - Google Patents
基于树突状细胞的癌症免疫治疗的临床应用 Download PDFInfo
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Abstract
本发明涉及一种基于树突状细胞的癌症免疫治疗的临床应用,其采用树突状细胞以治疗癌症,尤其涉及一种癌症免疫治疗方法(1)将树突状细胞注入经射线扫描后的肿瘤中,以及将组合物(2)注射至癌症患者体内,该组合物在癌症免疫治疗中使用,通过电穿孔将肿瘤抗原载入至树突状细胞中、细胞融合或刺激的方式使用。
Description
技术领域
本发明涉及一种癌症的免疫疗法,尤其涉及利用树突状细胞的癌症免疫治疗,这是一种新型的基于树突状细胞的癌症免疫治疗,其方法是用放射线扫描肿瘤后,将树突状细胞接种于肿瘤中,其与利用全细胞裂解液作为抗原源的基于已知树突状细胞癌症免疫治疗以及利用电穿孔方法将抗原载入树突状细胞的方法相一致。
背景技术
尽来,由于传统癌症疗法的负作用以及基因不稳定性而引起的耐药性的原因使得癌症很难避免地导致死亡。
因此,需要开发一种基于免疫调控系统的抗肿瘤疫苗。目前为止,已知的有联合肽疫苗、基因疫苗、热冲击蛋白疫苗等。在上述方法中,树突状细胞方法是最有效的方法。树突状细胞是活体内最有效的重要抗原细胞,因此,可以充分利用该细胞开发抗肿瘤疫苗以及治疗与免疫反应有关的大多数疾病。
然而,利用树突状细胞的免疫疗法并没有标准方法。一些研究中心正致力于寻找最有效且易于操作的方法。众所周知,免疫系统能够识别肿瘤并产生抵抗作用。体液免疫和细胞介导免疫都与分解肿瘤有关,但大多数情况下,通过肿瘤抗原的特定识别使肿瘤消亡是与细胞介导的免疫相关的。尤其是,CD8+细胞毒T淋巴细胞(CTL)识别肿瘤细胞并杀死该细胞已通过各种模型得以证明。(1.Boon,T.,J.C.Cerottini,B.Van den Eynde,P.van der Bruggen,and A.VanPel.1994.Tumor antigens recognizedby T lymphocytes.Annu Rev Immunol 12:337.2.Celluzzi,C.M.,J.1.Mayordomo,W.J.Storkus,M.T.Lotze,and L.D.Falo,Jr.1996.Peptide-pulsed dendritic cells induce antigen-specific CTL-mediatedprotective tumor immunity.J Exp Med183,1:283.3.Feltkamp,M.C.,H.L.Smits,M.P.Vierboom,R.P.Minnaar,B.M.de Jongh,J.W.Drijfhout,J.ter Schegget,C.J.Melief,and V.M.Kast.1993.Vaccination with cytotoxic T lymphocyteepitope-containing peptide protects against a tumor induced byhumanpapillomavirus type 16-transformed cells.Eur J Immunol 23,9:2242.3)。树突状细胞是独特的抗原呈递细胞从而诱导免疫反应,该细胞被广泛用于治疗癌症和感染性疾病的抗原特异性免疫疗法中。(Banchereau,J.,F.Briere,C.Caux,J.Davoust,S.Lebecque,Y.J.Liu,B.Pulendran,and K.Palucka.2000.Immunobiology of dendritic cells.Annu Rev Immunol 18:767.)根据肿瘤特异性方法,启动树突状细胞使T细胞识别并杀死肿瘤细胞的能力在多种动物模型中已有报导。此外,由树突状细胞诱导的免疫反应可以诱导免疫记忆从而预防肿瘤的形成。
树突状细胞数量在全部的循环白细胞中只占约0.3%,其由各种不同于巨噬细胞的具有特定表达类型的细胞形成。树突状细胞是一种强抗原呈递细胞,其与B细胞截然不同,B细胞或巨噬细胞具有较弱的抗原呈递能力。单核细胞通过IL-4和GM-CSF或单细胞前体(CD34+细胞)的刺激而诱导产生树突状细胞。未成熟树突状细胞从水溶性蛋白中提取到凋亡或坏死细菌和某抗原或抗原表位。抗原通过各种途径进入树突状细胞中,这些途径有捕获凋亡体、巨胞饮和甘露糖受体或胞吞作用等,这些途径是通过CD32或CD64与主要组织复合体通过这一系列的步骤形成复合物而完成。
捕获的抗原被MHC II类分子所承载,然后作为肽-MHC复合体进行处理并由此诱导CD4+T细胞反应。近来,以证明树突状细胞具有呈递能力,其依赖于从外界导入抗原,利用蛋白降解酶得到MHC I分子和TAP。因此,树突状细胞通过外界抗原的特定处理程序能够刺激T细胞和B细胞。基于上述描述,树突状细胞能被广泛用于癌症免疫治疗中。
在树突状细胞癌症免疫治疗中最常用的抗原是表位肽。然而,必须检查肿瘤患者体内是否含有上述抗原。该抗原只能用于具有能形成复合体的特定HLA型的患者中。而且,在美国和欧洲也没有太多的肽。在韩国,没有被确定的肿瘤抗原。而且,消化系统癌症和消化器官癌症的抗原是本发明研究的对象,这些抗原还没有过报导。肿瘤抗原的研究需要长期时间。在韩国不可能利用肽抗原进行树突状细胞癌症免疫治疗。
为了避免上述问题,尝试将未经鉴定的TAA(全细胞裂解液、凋亡/死亡细胞、全部mRNA、与DC融合的肿瘤细胞等)作为抗原。但此时则存在一个问题,即将未鉴定的TAA如何导入树突状细胞中的问题。已知全细胞裂解液能够刺激CD4+辅助性T细胞。将mRNA直接从肿瘤组织或肿瘤细胞中分离出不是件简单的事。将肿瘤细胞分离出并进行培养之后,还将凋亡细胞与树突状细胞一起培养是不利的,这样会造成凋亡。癌细胞和树突状细胞的融合需要将癌细胞从肿瘤组织中分离而培养。本研究中,上述不利因素的研究是在与韩国科学基金的卫生和福利部的合作下所进行的。研究结果得到一种树突状细胞癌症免疫治疗的新方法,如在放射线的扫描下,将树突状细胞注入肿瘤中,以及利用电穿孔将抗原导入树突状细胞的方法。
根据卫生和福利部和国家癌症中心所报导的2000年癌症调查结果,发展中国家的典型癌症胃癌和宫颈癌在所有癌症中的比例已逐渐降低,而发达国家的典型癌症大肠癌、乳腺癌和肺癌逐渐增加。2000年统计的全部癌症人数为83,846,与1999年相比增加了1.9%。在这些癌症中,消化器官的癌症数量最多。对于男性,胃癌、肝癌和大肠癌分别为24.5%,16.3%和10.2%,占所有癌症的一半。对于女性,胃癌和大肠癌分别为15.8%和10.5%。呼吸系统癌症肺癌数目显著升高,其在所有癌症中位居第二(11.5%),仅次于胃癌。在癌症死亡率中,肺癌位居首位。
尽管由于诊断技术的发展使癌症能被更早地发现,加上增长的癌症预防趋势和手术、放射性治疗和抗癌化学治疗的发展,但这些都没有使癌症发生率和由其导致的死亡率得以降低,因此,迫切需要开发一种改进的传统癌症治疗方法或一种新型的治疗技术。在外科手术或放疗这些典型的癌症治疗方法中,仍可能存在远端微转移灶。还可能产生副作用,即由于骨髓系统得到损害而导致造血功能受限。在这种情况下,会产生对肿瘤细胞中抗癌物质的耐受性,从而不能达到所需要的治疗效果。因此,最大程度降低治疗耐受和副作用并能防止最小程度的残余病变的方法能够显著降低由于癌症复发而导致的死亡率。在这个意义上,免疫治疗,即基于树突状细胞的癌症免疫治疗为成为最有效的抗肿瘤疫苗提供了可能。
本发明意欲介绍一种基于树突状细胞的癌症免疫治疗方法并依据该新方法治愈患者。
发明内容
本发明的目的是利用树突状细胞作为癌症免疫治疗剂并将该治疗剂用于临床上以治愈患者。
本发明开发了一种将树突状细胞注入经放射线的扫描的肿瘤中的方法,以及一种基于电穿孔将抗原导入树突状细胞的方法,由此进行一种新型的基于树突状细胞的癌症免疫治疗。在前一方法中,可能准确地将树突状细胞注入肿瘤组织中,该组织由于放射线而造成凋亡,但不必分离肿瘤组织。后一种方法,由于有可能直接将蛋白抗原注入细胞质中,因此,更能够刺激与杀伤癌细胞直接相关的CD8+细胞毒T细胞。本发明的一个目的在于引入了利用全细胞裂解液作为抗原源的常规基于树突状细胞的癌症免疫治疗的原理,并将该治疗方法应用于临床中治疗癌症。
附图说明
结合附图更有助于理解本发明,附图只是用于图示说明,并不是用于对本发明的限定。
图1显示了治疗效果。从图中可以看出,只对大腿右侧区域的肿瘤进行射线扫描,没有观察到治疗效果;观察到治疗效果的是IR组和(IR+DCs)组,前者是用放射线对肿瘤进行扫描,后者是放射线对肿瘤扫描后,再将同源的树突状细胞接种于肿瘤中。对于右侧大腿区域的肿瘤进行放射线扫描后,再将树突状细胞接种入左后侧,可观察到显著的治疗效果。IR组是只进行放射线扫描,IR+DCs组是对肿瘤进行放射扫描后,再将同源的树突状细胞引入相对一侧。IR组并没有产生显著的效果。右侧大腿区域的肿瘤在IR+DCs处理之后显著缩减。通过将树突状细胞采用肿瘤内注射的方式注入射线扫描后的肿瘤中来诱导抗肿瘤免疫从而治疗远端肿瘤。照片(B)是最后一次免疫后拍摄的。
图2中,采用不同浓度形成实体瘤并对实体瘤的大小进行了比较(蓝色)。在实验组中,形成实体瘤,然后用放射线进行处理。给予未成熟的树突状细胞。肿瘤生长受到抑制,测量其大小(红色)。结果是得到了预想效果。除肿瘤浓度最大的一个样本外,实验组的所有样本在6周后被治愈,没有观察到实体瘤。对于仍具有肿瘤的一个样本,肿瘤平均大小从250mm2减小到50mm2。
图3中,将IR+DCs试验组分成无肿瘤(Tm(-))组、肿瘤比较组(Tm(+))、树突状细胞处理组(DCs)、射线扫描组(IR)、以及射线扫描后树突状细胞处理组,然后进行试验。在IR+DCs组中显示了最有效的细胞毒性,可见,癌症免疫治疗方法的高效性。5∶1、10∶1、20∶1表示脾细胞∶肿瘤细胞的比值。通过将树突状细胞(DC)采用病变部位内注射方式注射到射线扫描后(IR)的肿瘤中可增强抗肿瘤免疫性。
本发明的最佳实施方式
根据本发明研究者的试验结果,在利用树突状细胞的细胞毒试验中,为使成熟的树突状细胞刺激免疫性,将未成熟树突状细胞准确地接种于已经射线扫描后的肿瘤中。试验结果是由于免疫性得到刺激,使细胞毒性显著提高。而且,通过对成熟的树突细胞中外源载入肿瘤抗原使得白细胞介素-2的显著增加成为可能。此外,通过对实体瘤进行试验,还获得了上述效果。
实施例
对本发明的实际试验进行详细描述。
利用树突状细胞作为癌症免疫治疗进行下述实验。试验结果是对于实体瘤而言,抗肿瘤效果和白细胞介素-2都得到提高。
实施例1
将MCA102纤维瘤细胞接种于同系C57BL/6小鼠(6-8周,雌性)的右大腿处的皮肤下,肿瘤生长至直径8mm大小。将具有肿瘤的大腿置于直线射线扫描仪的(Semens mevatron 67)的射线扫描区域内,然后15Gy射线对肿瘤中的同源性树突状细胞内扫描。上述过程每周进行一次,共进行三次。一周后,从无肿瘤对照组、含肿瘤组、肿瘤未经射线扫描而被注入同源性树突状细胞的组、对肿瘤进行了射线扫描的组以及经射线扫描且同源性树突状细胞也经扫描的组的小鼠中分别分离出脾细胞,然后测量其抗肿瘤细胞的细胞毒性。分别对细胞数为脾细胞的5、10和20倍的肿瘤细胞进行试验,分别观察产生的细胞毒性。
结果是,只含有肿瘤的试验组,其细胞毒性与无肿瘤的对照组不同。肿瘤未经射线扫描,但注入同源性树突状细胞的组显示了低细胞毒性。但对于经射线扫描同时同源性树突状细胞也被扫描的组,其细胞毒性与其它组显著不同。因此,通过在经射线扫描的肿瘤中接种树突状细胞可以显著提高抗肿瘤免疫性(见图3)。
此外,对射线扫描肿瘤的组和经射线扫描并接种同源性树突状细胞的组,测定其对侧的治疗效果。射线扫描到右侧大腿区的肿瘤内,没有观察到治疗效果。对射线扫描到右侧大腿肿瘤内、然后接种树突状细胞的组可以观察到显著的治疗效果(见图1)。采用不同浓度形成实体瘤并对实体瘤的大小进行比较(蓝色)。在实验组中,形成实体瘤,然后用放射线进行扫描。然后接种未成熟的树突状细胞,肿瘤生长受到抑制,测量其大小(红色)。结果是得到了预想效果。除接种肿瘤浓度最大的一个样本外,所有样本在6周后被治愈,没有观察到实体瘤。对于仍具有肿瘤的小鼠,肿瘤平均大小从250mm2减小到50mm2(见图2)。
实施例2
制备组E,其中OVA(4mg/ml)被电穿孔,清洗OVA,然后培养16个小时;组P中OVA(4mg/ml)被刺激达16个小时,组E+P,其中OVA被电穿孔,并刺激16个小时。在RF33.70(MHC I类)或DO.11.10(MHC II类)细胞中测量IL-2,所述细胞利用了上述三组的树突状细胞。测量结果表明组E有效地提供了基于MHC I类的OVA。组E+P比组E提供了更多的对于MHC I类的OVA(图1A)。正如所预料的,组P比组E提供了更多的基于MHC II类的OVA,组E+P比组P提供了更多的基于MHC II类的OVA(图1B)。因此,通过将抗原经电穿孔导入树突状细胞中,会更有效地提供基于MHC I类的抗原。可能提供更多基于MHC II类的抗原。因此,可预见电穿孔能够有效提高免疫性。
图1.电穿孔(E)、刺激(P)和二者结合(E+P)后,通过树突状细胞MHCI类(A)和II类(B)呈递的OVA。
实施例3
当OVA经刺激通过电穿孔被导入到同源性树突状细胞中,观察EG7细胞(转染了OVA cDNA的EL4)对脾细胞的细胞毒性,当C57BL/6小鼠由于电穿孔OVA而减少时,检测与基于OVA刺激的DC相比较时是否会产生特异性免疫。制备组E,其中OVA被电穿孔,清洗OVA,然后培养16个小时;组中OVA被刺激达16个小时;组E+P,其中OVA被电穿孔,并刺激16个小时。在C57BL/6小鼠右腹侧皮下接种树突状细胞(1×106细胞),每周接种一次,共接种三次。一周后,分离脾细胞,观察EG7细胞(转染了OVA cDNA的EL4)的细胞毒性。观察结果为,组E比组P的细胞毒性更强。组E+P比组E细胞毒性强(图2A)。当针对EL4细胞进行上述实验,所有组都没有观察到细胞毒性(图2B)。EG7具有针对OVA的特异性细胞毒性。电穿孔增加了基于MHC I类的抗原呈递,因此可有效提高免疫性。
图2.通过电穿孔(E)、刺激(P)以及二者结合(E+P)将OVA载入树突状细胞中,可由该树突状细胞诱导OVA特异性细胞毒活性。
采用上述方式处理小鼠。一周后,将EG7和EL4细胞接种于左后大腿中,其后可检测到抑制肿瘤的作用。该结果表明有可能获得类似于组E和P的肿瘤抑制作用。在组E+P中,抑制肿瘤产生的作用很强(图3)。针对EL4细胞进行上述实验,所有组都没有观察到抑制肿瘤产生的作用。
图3.通过电穿孔(E)、刺激(P)或二者的结合(E+P)将OVA载入DCs中,用该DCs进行免疫,可诱导产生OVA特异性的预防性抗肿瘤免疫性。
工业实用性
如上所述,利用了树突状细胞的本发明癌症免疫治疗可有效用于治疗癌症。本发明所提供的新型基于树突状细胞的癌症免疫治疗(通过电穿孔将抗原载入树突状细胞中的方法)可以被应用于所有实体瘤治疗中。在本发明中,对还没形成癌症组织的患者中的肿瘤进行射线扫描,可准确地在肿瘤病变部位内诱导未成熟的树突状细胞。
本发明能够在不背离实质特征的精神下以多种形式体现,可以理解,除非特别说明,上述实施例并不被任何前述描述细节所限制,因而应该在权利要求所定义的精神和范围下得到广泛解释,所有在权利要求范围内的改变和修饰或等同替换都是权利要求所包含的内容。
Claims (4)
1.一种包含树突状细胞的产品和一种包含该产品的基于树突状细胞的癌症免疫治疗剂,其特征在于,癌症组织杀伤剂被载入具有癌组织的癌症患者体内的未成熟的树突状细胞中,并因此治疗癌症。
2.一种用于治疗癌症而制备的产品和含有该产品的基于树突状细胞的癌症免疫治疗剂,其特征在于,浓缩未成熟的树突状细胞,对不具有癌组织的癌症患者进行射线扫描,将浓缩的树突状细胞接种于病变部位内,从而使之凋亡。
3.一种为联合应用被载入到癌细胞的树突状细胞而制备的产品和含有该产品的基于树突状细胞的癌症免疫治疗剂,其特征在于,将载入到癌细胞中的树突状细胞通过将树突状细胞载入到癌细胞中的方法和基于刺激方法的电穿孔方法而结合。
4.一种用于载入癌细胞的树突状细胞产品和一种含有上述产品的基于树突状细胞的癌症免疫治疗剂,所述二产品是通过电穿孔方法对载入癌细胞中的树突状细胞进行刺激而使用。
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