CN107812013B - 一种治疗耐药结核的生物制剂及其制备方法 - Google Patents
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Abstract
本发明属于生物制剂技术领域,公开了一种治疗耐药结核的新型生物制剂及其制备方法,所述治疗耐药结核的新型生物制剂为结核特异性淋巴细胞,其是利用IL‑1α、IL‑2,CD3单克隆抗体,结核杆菌特异性抗原(CFP10,ESAT6)制备的。与现有结核病的治疗方法相比,本发明在国内外上属于首次报道利用体外培养的淋巴细胞治疗肺结核患者获得显著疗效,经细胞治疗后患者外周血中淋巴细胞明显上升、痰检结果迅速转阴、肺部阴影快速吸收、病程缩短、精神状态好转、食欲体重增加,显著提高患者在抗结核治疗中的生活质量。
Description
技术领域
本发明属于生物制剂技术领域,尤其涉及一种治疗耐药结核的新型生物制剂及其制备方法。
背景技术
结核分枝杆菌感染引起的结核病是世界范围内主要的公众健康问题之一。WHO在其2016年全球结核病报告中指出:2015年全球新发结核病人数较2014年上升10%,达到1040万例,导致死亡约140万例;耐药结核检测与治疗危机仍然再继续,58万耐药结核患者中,只有12.5万(20%)得到了治疗;而印度、中国和俄罗斯三个国家占所有耐药病例的45%。因此,我国是结核病高发大国之一,也是耐药结核高发国家,尽管人们在结核病诊断和治疗方面已做了坚持不懈的努力,但是由于对发病机制认识的局限性,对耐药结核的早期诊断和有效治疗及控制尚不十分满意,已至死亡率居高不下。因此,结核病防控工作面临巨大挑战。深入研究结核病发生发展中的分子机制,寻找早期检测、有效治疗的新方法,将为患者带来福音,也是国家科技攻关和国家重大科技战略的需要。
目前抗结核治疗主要有抗结核化学药物疗法和免疫治疗两大部分。其中抗结核化学药物疗法是通过化学合成药物的相互联合作用,共同杀灭存在于细胞内外的结核杆菌起到治疗结核病的作用。另一种是运用免疫调节剂:如细胞因子、注射用母牛分枝杆菌,提高患者的免疫力从而起到辅助治疗目的。但是由于长期传代导致BCG的活力下降和免疫调节在临床使用后效果不佳,使得传统免疫治疗和预防渐入窘境。而耐多药结核病的流行使得现有常规化疗手段也显乏力。近年来随着对结核病的重视度提升,各国在新型抗结核药物和新型疫苗的研究均投入了大量资金但进展较少且远远无法满足临床的需求,因此由于耐多药结核病自身的特性使化学合成药物的联合应用的效果大打折扣。
综上所述,现有技术存在的问题是:化学合成药物治疗耐多药结核病有效率低下。其主要原因是耐多药结核杆菌对现有的多种抗结核化学药物发生了耐药突变,同时由于新型抗结核化学药物开发周期过长难度大且投入经费过多,使得利用联合多种化学合成药物治疗耐多药结核病的难度加大。
发明内容
针对现有技术存在的问题,本发明提供了一种治疗耐药结核的新型生物制剂及其制备方法。
本发明是这样实现的,自体免疫细胞治疗是现代生物技术与临床医学等多学科交叉融合而形成的针对人类重大疾病进行治疗研究的新兴领域,它主要是通过现代分子生物学和基因工程技术等手段,调动人体天然防御机制、激发免疫反应、抑制和杀伤病原体或肿瘤细胞的一种生物治疗的方法。自体免疫细胞治疗经过十几年的基础和临床研究,已经发展为包含LAK、NK、CIK、CTL、DC等多种免疫细胞在内的一种复杂的免疫治疗方法,并被临床认为是继手术、放疗和化疗后第四类癌症治疗方法。CIK细胞治疗是自体免疫细胞治疗在临床运用中的一种。CIK是1991年由美国斯坦福大学SchmidtWolf等人首先发现的,主要通过细胞因子和体外培养等手段,获得的一群CD3+CD56+双阳性的T淋巴细胞(NKT)并利用这群细胞非特异性的杀伤肿瘤细胞或者病原体。在CIK被发现后其对肿瘤的杀伤机制亦被不断揭示。通过大量研究已经证实CIK细胞是通过刺激肿瘤细胞的裂解、激活激活细胞凋亡信号、分泌抗肿瘤细胞因子等三条途径来实现对肿瘤的杀伤。随着CIK细胞杀伤肿瘤机制和效果的不断揭示,不少学者已将其运用在临床肿瘤的研究和治疗中。2000年Takayama等人在《柳叶刀》上报道了体外扩增活化自体淋巴细胞预防肝癌术后肿瘤复发的临床试验研究,从而第一次在临床上证实手术和细胞治疗联合使用可以有效降低肿瘤复发与转移。2008年HunderNN等在《新英格兰医学杂志》刊登对一名52岁男性恶性黑色素瘤患者在进行免疫治疗的报道,患者经自体免疫细胞治疗后病灶消失,两年内没有肿瘤的复发,这是医学界首例利用复制免疫细胞的方法成功治愈癌症的个案,是癌症治疗的一个里程碑。同时我国学者还将CIK与DC及化疗联合运用在肿瘤的治疗中。除了对肿瘤的治疗CIK在治疗感染性疾病方面也取得了重大突破,有临床研究在利用CIK治疗肝癌合并乙肝的病例时发现CIK细胞治疗组的HBV-DNA含量在治疗后显著下降,并有部分病例乙型肝炎表面抗原转阴。而在治疗白血病的临床研究中也发现CIK细胞可以明显抑制甚至清除白血病患者体内丙型肝炎病毒(HCV),患者肝功能不但没有加重,反而明显改善。除此之外经CIK治疗后的患者T细胞、NK细胞和NKT细胞在淋巴细胞所占的比例明显升高,CD8+/CD3+、CD4+/CD8+、Th1/Th2的比值也明显升高并且IFN-γ等抗感染细胞因子分泌增加而Treg比例随着治疗次数的增加不断降低
通过总结肿瘤治疗和病毒性肝炎治疗,发现CIK细胞在直接杀伤肿瘤细胞的同时还能分泌大量的细胞因子,其中IFN-γ和TNF–α是M1型巨噬细胞活化的关键而M1型巨噬细胞则是机体抗结核杆菌的关键细胞,同时TNF–α还能通过调控结核杆菌结构的形成与完整性而参与了抗结核免疫,除了分泌细胞因子外CIK细胞治疗还能提高患者CD4+/CD8+、Th1/Th2的比值同时降低Treg的比例,极大的刺激了Th1型细胞的特异性免疫应答而这正好抑制了结核杆菌得免疫逃逸机制,使机体能够重新获得杀伤结核杆菌的能力。由此推测在CIK细胞治疗培养方法的基础上利用已知的结核抗原(CFP10,ESAT6)培养结核杆菌特异性T淋巴细胞,再利用这种新型的生物制剂对结核病进行治疗。
本发明是一种治疗耐药结核的新型生物制剂,其特征在于,所述治疗耐药结核的新型生物制剂为结核特异性淋巴细胞,其是利用IL-1α、IL-2,CD3单克隆抗体,结核杆菌特异性抗原(CFP10,ESAT6)制备的。
本发明的另一目的在于提供一种所述治疗耐药结核的新型生物制剂的制备方法,所述治疗耐药结核的新型生物制剂的制备方法包括以下步骤:
步骤一,取正常人外周血,与PBS按照1:2的比例稀释后进行Ficoll密度梯度离心。先将提前置于室温条件的Ficoll加入华氏管中,然后按照Ficoll:稀释血液=1:2的比例,沿管壁缓慢加入稀释血液(华氏管倾斜45°,切忌Ficoll与稀释血液的界面发生强烈震荡),然后于控温离心机中离心(1800rpm/min,30min,20~24℃)。离心结束后,所需要的PBMC来自中间云雾层。用移液管仔细吸取云雾层细胞,然后用10倍体积的PBS洗涤离心,最后得到PBMC;
步骤二,将PBMC细胞用RMPI1640完全培养基(含10%胎牛血清)配成细胞悬液,然后于各组细胞培养液中加入相应细胞因子:第0天加入抗CD3mAb(100ng/ml),IFN-γ(1000U/ml),IL-1α(10ng/ml),CFP10(10ng/ml),ESAT6(10ng/ml),将细胞置于37℃、5%CO2、饱和湿度95%的CO2培养箱内培养。
步骤三,第一天加IL-2(1000U/ml),之后视细胞状态每隔3天补一次细胞因子液,补液细胞因子为IL-2(500U/ml)。
步骤四,在培养的第十天,进行细菌培养,内毒素检测,流式细胞术检测亚群变化,完成结核特异性T细胞的制备。
进一步,所述结核特异性淋巴细胞是指将人外周血单个核细胞在体外用多种细胞因子抗CD3单克隆抗体、IL-2、IFN-γ和结核杆菌特异性抗原(CFP10,ESAT6)共同培养获得的一群特异性淋巴细胞。
进一步,培养基为淋巴细胞培养基;
淋巴细胞培养基包括药用级的人类白蛋白,CD3单克隆抗体、IL-2、IFN-γ和结核杆菌特异性抗原(CFP10,ESAT6)。
进一步,pH8.0。
本发明的优点及积极效果为:与现有结核病的治疗方法相比,本发明在国内外上属于首次报道利用体外培养的结核特异性淋巴细胞治疗肺结核患者并取得100%有效性,耐多药结核病患者经细胞治疗后外周血中CD4+T淋巴细胞比例由36%上升到44%,CD8+T淋巴细胞比例由24%上升到33%、100%的患者经治疗后痰涂片转阴,88.9%患者治疗后的痰培养转阴、100%的患者肺部病灶发生了缩小、100%的患者咳嗽咳痰症状消失、100%患者的乏力状况消失、食欲体重增加,显著提高患者在抗结核治疗中的生活质量。
附图说明
图1是本发明实施例提供的治疗耐药结核的新型生物制剂的制备方法流程图。
图2是本发明实施例提供的细胞治疗前后外周血淋巴细胞亚群的变化示意图。
图3是本发明实施例提供的结核特异性淋巴细胞治疗前后CT检测病灶明显吸收示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
自体细胞免疫治疗是现代生物技术与临床医学等多学科交叉融合而形成的针对人类重大疾病进行治疗研究的新兴领域,通过总结前期肿瘤治疗和病毒性肝炎治疗的多篇研究报道,发现淋巴细胞在直接杀伤肿瘤细胞的同时还能分泌大量的细胞因子,其中IL-12、IFN-γ和TNF-α是巨噬细胞向M1型极化的关键,同时TNF-α还能通过调控结核杆菌的结构形成与完整性而参与抗结核免疫,由此推测细胞治疗可以通过极化患者巨噬细胞从而对结核病的治疗起到积极作用。
下面结合附图对本发明的应用原理作详细的描述。
本发明实施例提供的治疗耐药结核的新型生物制剂为IL-1α、IL-2,CD3单克隆抗体,结核杆菌特异性抗原(CFP10,ESAT6)。
如图1所示,本发明实施例提供的治疗耐药结核的新型生物制剂的制备方法包括以下步骤:
S101:取正常人外周血,与PBS按照1:2的比例稀释后进行Ficoll密度梯度离心。先将提前置于室温条件的Ficoll加入华氏管中,然后按照Ficoll:稀释血液=1:2的比例,沿管壁缓慢加入稀释血液(华氏管倾斜45°,切忌Ficoll与稀释血液的界面发生强烈震荡),然后于控温离心机中离心(1800rpm/min,30min,20~24℃)。离心结束后,所需要的PBMC来自中间云雾层。用移液管仔细吸取云雾层细胞,然后用10倍体积的PBS洗涤离心,最后得到PBMC;
S102:将PBMC细胞用RMPI1640完全培养基(含10%胎牛血清)配成细胞悬液,然后于各组细胞培养液中加入相应细胞因子:第0天加入抗CD3mAb(100ng/ml),IFN-γ(1000U/ml),IL-1α(10ng/ml),CFP10(10ng/ml),ESAT6(10ng/ml),将细胞置于37℃、5%CO2、饱和湿度95%的CO2培养箱内培养;
S103:第一天加IL-2(1000U/ml),之后视细胞状态每隔3天补一次细胞因子液,补液细胞因子为IL-2(500U/ml);
S104:在培养的第十天,进行细菌培养,内毒素检测,流式细胞术检测亚群变化,完成结核特异性T细胞的制备。
在本发明的优选实施例中:结核特异性淋巴细胞是指将人外周血单个核细胞在体外用多种细胞因子如抗CD3单克隆抗体、IL-2、IFN-γ和结核杆菌特异性抗原(CFP10,ESAT6)等共同培养一段时间后获得的一群特异性淋巴细胞。这群淋巴细胞兼具有T淋巴细胞的抗结核杆菌和NK细胞的非MHC限制性杀结核优点。
在本发明的优选实施例中:培养基为淋巴细胞培养基。
在本发明的优选实施例中:培养基由下述组分构成:包括药用级的人类白蛋白,CD3单克隆抗体、IL-2、IFN-γ和结核杆菌特异性抗原(CFP10,ESAT6)。
在本发明的优选实施例中:pH8.0。
下面结合具体实施例对本发明的应用效果作详细的描述。
1)细胞治疗前后外周血淋巴细胞亚群的变化分析,见图2。
2)细胞治疗前后结核杆菌痰培养、痰涂片结果阳性率比较分析见表1。
表1细胞治疗前后结核杆菌痰培养、痰涂片结果阳性率比较
#确切概率法
表1显示:在细胞治疗后,治疗对象痰涂片的阳性率明显下降,从38.7%逐渐下降到0.00%,差异有统计学意义(P值均小于0.05);痰培养的阳性率随着治疗次数的增多逐渐下降,从54.8%下降到20.0%,但无统计学意义。
3)细胞治疗前后的CT检查对比,见图3。
4)37例结核病患者细胞治疗前后影像变化综合分析见表2。
表237例结核病患者细胞治疗前后影像变化的比较*
#包括耐药结核12例;肺结核10例;非结核分枝杆菌病12例;骨结核2例;泌尿系统结核1例
*等级资料非参数统计z=5.50,p<0.0001
表2显示,37例结核病患者细胞治疗后影像改变明显好转,等级资料非参数统计z=5.50,p<0.0001,有统计学差异。
5)9例耐药肺结核患者细胞治疗组与对照组的临床症状汇总分析,见表3。
表3
注:yes=有,%=占总数的百分数
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (1)
1.一种治疗耐药结核的生物制剂,其特征在于,所述治疗耐药结核的生物制剂为结核特异性淋巴细胞,利用IL-1α、IL-2,CD3单克隆抗体,结核杆菌特异性抗原CFP10,ESAT6制备;
所述治疗耐药结核的生物制剂的制备方法包括以下步骤:
步骤一,取正常人外周血,与PBS按照1:2的比例稀释后进行Ficoll密度梯度离心;将提前置于室温条件的Ficoll加入华氏管中,按照Ficoll:稀释血液=1:2的比例,沿管壁缓慢加入稀释血液,于控温离心机中离心1800rpm/min,30min,20~24℃;离心结束后;用移液管仔细吸取云雾层细胞,用10倍体积的PBS洗涤离心,得到PBMC;
步骤二,将PBMC细胞用RMPI1640完全培养基配成细胞悬液,然后于各组细胞培养液中加入相应细胞因子:第0天加入抗CD3mAb 100ng/ml,IFN-γ1000U/ml,IL-1α10ng/ml,CFP1010ng/ml,ESAT6 10ng/ml,将细胞置于37℃、5%CO2、饱和湿度95%的CO2培养箱内培养;
步骤三,第一天加IL-2 1000U/ml,之后视细胞状态每隔3天补一次细胞因子液,补液细胞因子为IL-2 500U/ml;
步骤四,在培养的第十天,进行细菌培养,内毒素检测,流式细胞术检测亚群变化,完成结核特异性T细胞的制备;
所述结核特异性淋巴细胞是指将人外周血单个核细胞在体外用多种细胞因子,CD3单克隆抗体、IL-2、IFN-γ和结核杆菌特异性抗原CFP10,ESAT6共同培养获得的一群特异性淋巴细胞;
培养基为淋巴细胞培养基;
淋巴细胞培养基包括药用级的人类白蛋白,CD3单克隆抗体、IL-2、IFN-γ和结核杆菌特异性抗原CFP10,ESAT6。
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