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抗体产业关键技术之一:用于重组抗体生产的细胞构建技术

已有 12431 次阅读 2013-7-14 16:56 |系统分类:论文交流| 筛选, 载体, 重组抗体, 细胞系构建, 转染

 

   摘要:构建生产细胞系是重组抗体产业化制备的第一步。目前国际上用于重组抗体生产的工程细胞系表达水平可达20-70pg/cell/daypcd。多种动物细胞根据其特性的不同,在抗体药物生产中有着不同的应用。近年来,工程细胞系构建技术的研究进展主要集中在以下三个方面:利用细胞工程技术提高宿主的生长、表达能力,新型载体功能元件和定点整合技术克服载体随机整合时发生的“位置效应”,以及应用流式细胞分选术和高通量筛选机器提高了重组细胞的筛选通量和效率。

关键词:重组抗体;细胞构建;载体元件;筛选策略

 

近年,随着宿主细胞遗传特性的改造,载体功能元件的优化,以及高通量克隆筛选技术的发展,国际上用于抗体生产的工程细胞系表达水平可达20~70 PCD(pg/cell/day)。业内完成稳定细胞系的构建时间也缩短至3~6个月[1]。生产细胞系的构建是重组抗体产业化制备的第一步。作为抗体产业上游关键技术,如何快速、高效建立生产细胞系,是我国抗体产业发展中急需解决的共性问题[2]

1        用于抗体生产的工程细胞系构建流程

抗体分子具备四聚体糖蛋白的复杂分子结构,其胞外表达又遵循着“分泌蛋白”的一般规律:全抗体分子的轻、重链各自翻译后,经折叠、组装,糖基化修饰后才具生物活性。通过对已上市抗体的临床研究也表明发现,宿主细胞对重组抗体的“翻译后修饰”,直接影响到抗体药物的临床疗效和免疫原性[3][4]

在常用的异源蛋白表达系统中:大肠杆菌不适合表达全抗体分子,只能表达单链抗体或者抗体片段;酵母细胞、昆虫细胞、转基因植物等由于缺乏翻译后修饰能力,其所表达的重组抗体与人天然抗体存在着显著的质量差异。只有哺乳动物细胞适宜重组抗体的异源人源抗体的表达。因此,目前已上市的治疗性抗体中,除了少数抗体片段(如:LucentisTM)外,均由哺乳动物细胞生产[5, 6]

1.1 用于抗体药物生产的宿主细胞

哺乳动物细胞中,CHO、骨髓瘤细胞、杂交瘤细胞、PerC.6、HEK293等均有用于重组抗体生产的实例(见表1)[6],这其中CHO、NS0及sp2/0细胞由于具备相对成熟“平台化开发技术”,在重组抗体生产中应用较为广泛。上述不同动物细生长、代谢特性以及对重组抗体的表达、修饰能力也不尽均不相同。CHO、NS0,sp2/0均可产生非人源的糖基化修饰。如:sp2/0生产的Cetuximab因含非人源“α(1,3)半乳糖结构造成临床上出现过敏反应[7]

表1 用于表达抗体的宿主细胞特性与应用

Table1 the characteristics of various animal cells and cases in commercial antibody production

细胞系及来源

优点

缺点

在抗体生产中的应用[6]

CHO DHFR–  

ATCC CRL-9096

CHOK1

遗传背景清晰,产业化应用广泛。

非人源糖基化修饰(如:NeuGc型唾液酸,高甘露聚糖等)

上市抗体:AvastiTMn, CampathTM, Herceptin, TM HumiraTM,RaptivaTM,RituxanTM, TMVectivbix®, XolairTM, ZevalinTMTocillizumabTM

NS0

ATCC CRL-1827,-2695, -2696

缺乏内源性谷氨酰胺合成酶,更适合GS筛选体系

培养过程需添加外源胆固醇;非人源糖基化修饰如:α(1,3)半乳糖结构

上市抗体:MylotargTM, SolirisTM, SynagiTMs, TysabrTMi,ZenapaxTM

Sp2/0

ATCC CRL-2016

能够无血清悬浮培养

非人源糖基化修饰:如(α(1,3)半乳糖结构

上市抗体:ErbituxTM, RemicadeTM, ReoproTM, SimulectTM

Murine Hybridomas

鼠杂交瘤技术成熟

多用于表达鼠源抗体

上市抗体:BexaarTM, OthocloneTM

Perc 6

Crucell公司)

人源细胞,借助病毒载体可实现重组抗体的高表达。

Crucell公司授权许可使用

14种候选药物处于临床研究阶段

ECo li

技术成熟,培养成本低

只适合表达抗体片段

上市抗体:LucentisTM

GlycoFi

Merck公司)

具备均一的糖基化修饰能力,生产成本低

Merck公司授权许可使用

能够成功表达糖基化高度均一的IgG

HEK-293

ATCC CRL-1573

人源细胞,瞬时转染技术成熟

受培养规模、工艺路线所限,不适合产业化生产

适合快速制备mg级样品。

 

1.2“工程细胞”的概念与构建流程

用于抗体药物生产的工程细胞系需具备以下特性:生长特性良好,无血清悬浮培养密度高(1~2×107cell/ml);异源蛋白表达能力强(20~70 pg/cell/dayPCD),具备正确的翻译后修饰能力;宿主及重组细胞系遗传背景清晰、表型稳定,符合相关法规要求。

目前,业内细胞系构建中常用的筛选体系为Dhfr-和“GS”系统,基于此技术的工程细胞构建流程如下:携带有目的基因和筛选标记(DhfrGS,或其他抗性基因,如:neohygro)的质粒转染至宿主细胞后,使用相应的条件培养基筛选出重组细胞。为提高重组细胞的表达水平,可采用加压多轮筛选的策略,增加目的基因拷贝数。初筛得到的候选克隆,经悬浮、无血清驯化后,在反应器中对其生长能力、表达水平,产物质量综合评价,选择具有产业价值的细胞系冻存[8][9, 10]。最终,建立符合cGMP要求的主细胞库Master Cell Bank、工作细胞库Working Cell Bank

2 细胞工程技术在宿主改造中的应用

胞工程(cell engineering)技术可以通过对宿主细胞进行遗传改造,来提高工程细胞系的生长能力、表达水平,满足抗体药物对于产能、质量的要求[11]。近年来,此方面研究进展日新月异,甚至已经开始改变了上述(表1)宿主细胞的遗传特性。如敲除GS基因的CHO构建完成[12],更适合GS筛选系统;化学诱变筛得到不依赖胆固醇的NS0细胞系[13];导入抗凋亡基因的Sp2/0-Ag14更适合无血清培养表达重组抗体[14],等。

2.1提高细胞的生长能力、抗凋亡能力

为提高细胞的生长能力,可过度表达抗凋亡基因。如:NS0CHO细胞过度表达Bcl-2,可以抑制细胞线粒体凋亡途径[15] CHO 细胞中过度表达P21P27,,可使细胞生长周期停滞在G1 [16];通过过度表达E1B-19KAven基因[17],或热激蛋白[18],牛磺酸转运蛋白[19]等,能显著提高细胞活性、延长培养周期。

为降低细胞培养过程中的副产物,对副产物代谢过程中的关键基因进行操作。如:通过上调细胞的丙酮酸羧化酶基因,下调乳酸脱氢酶基因,来减少乳酸的产生[20, 21];通过过度表达尿素循环酶、氨甲酰磷酸合成酶、鸟氨酸氨甲酸基转移酶等,来减少氨的生成[22]

2.2提高重组抗体的翻译、修饰及分泌能力

多于多数重组细胞而言,翻译、修饰及分泌是抗体的生物合成过程的限速步骤[23]。因此,可以对参与目的蛋白生物合成的分子伴侣进行遗传操作,也可提高宿主细胞的重组抗体表达能力。此类分子伴侣包括:蛋白质二硫键异构酶(protein disulfide isomerase、内质网氧化还原酶(Endoplasmic reticulum oxidoreductase、重链结合蛋白(the heavy chain-binding protein )、ERp57GRP94X-盒结合蛋白1(XBP-1S)[24-26]

抗体的糖基化修饰对其生物活性影响显著[27],利用细胞工程技术对宿主的糖基化修饰能力进行改造,可以提高重组抗体的临床疗效。如:通过敲除岩藻糖转移酶(FUT8),或过度表达乙酰氨基葡萄糖转移酶(GnT-III),可降低重组抗体糖链中的岩藻糖含量,以提高抗体药物的ADCC效应功能[28]通过过度表达唾液酸转移酶,提高抗体的唾液酸含量,以提高抗体的抗炎活性[29]等。

3高表达载体的构建与优化

在用于重组抗体的表达的载体构建过程中,目的基因的简化[30]、轻重链比例优化[31],顺式作用元件的应用等,都能提高重组抗体的表达水平。这其中载体的构建策略与载体的功能元件,对于细胞的表达能力、遗传稳定性影响最为显著,也是此领域研究的热点。

3.1用于全抗体表达的载体构建策略

生物合成四聚体IgG结构,需要协调表达抗体分子的轻链、重链基因。以下三种载体构建策略均有用于抗体表达的报道

3.1.1单顺反子(mon-cistronic)载体[32][33]

轻链、重链基因分别使用不同的载体,共转染至宿主细胞。该策略对质粒要求不高,且转染效率较高,是表达重组抗体最为常用的载体构建策略。也可将轻链基因、重链基因串联到同一载体,分别使用各自的启动子、终止子转录翻译,该方法理论上可实现轻链、重链基因的等摩尔表达。

3.1.2双顺反子(bi-cistronic)载体[34]

在载体构建中使用核糖体内部进入位点IRES,在其两端串联轻链、重链基因,可实现轻、重链基因同时转录。也可在载体中使用2A肽结构,在同一阅读框架内表达氢、重链基因。

3.1.3反式互补(trans-complementing)载体[35]

表达轻链、重链的两个载体分别含有编码筛选标记Dhfr-DHFR基因的不同序列。只有当含有轻链、重链的两个载体同时转染至宿主细胞时,筛选标记DHFR才能正确表达。以此提高重组细胞的筛选效率。

3.2 载体功能元件的最新进展

    常用的表达载体依靠随机整合至宿主基因组中来表达目的基因。整合位点的“位置效应”往往会造成目的基因的低表达或沉默。近年,出现的多种商品化的染色质开放原件(STAR™EASE™UCOEs™MARs™ ),人工染色体(ACE ™)、逆转录病毒载体(GPEx™),定点整合技术(FlpInRMCETMCompoZrTM)等,能够克服上述随机整合的缺点。载体构建中引入这些功能元件,可大幅提高高表达克隆的比例,缩短工程细胞系的构建周期。(见表2

表2  用于重组抗体表达的载体功能元件

Table2 The vector element for recombinant antibody expression

 

作用原理

STAR™ (Stabilising and antirepressor elements)

Crucell

富含A/T碱基,起到绝缘子效果,克服整合位点的位置效应。

EASE™ (Expression Augmenting Sequence Element)

Amgen

3.6kb顺式表达作用元件,促进目的基因整合至染色体

UCOEs™ (Universal chromatin opening elements)

Millipore

2.5–8 kb,配合启动子hCMV使用,防止异染色质的形成

MARs™ (Scaffold- matrix associatedregions)

Selexis

富含AT核苷酸序列,促使染色质开放

FlpIn

Invitrogen

目的基因定点整合至转录活跃区,适用于CHO细胞

CompoZrTM

sigma

基于锌指酶的定点整合技术

RMCETM(recombinase-mediated cassette exchange)

 

重组酶介导的定点整合技术

GPEx

GALA Biotech

基于逆转录病毒的新型载体

ACE ™            Chromos molecular systems inc              人工染色体[36]

Promosome TEE ™             Promosome technology             翻译增强子

      

3.3大规模瞬时转染技术的发展

瞬时转染技术由于目的基因不需要整合到宿主的基因组中,目的基因拷贝数和转染效率均较稳定转染明显提高。该技术可短时间内内(一般为数周可积累mg~g级的样品,更适用于抗体药物研发的早期阶段。随着大规模瞬时转染技术的发展,瞬时转染的最大生产规模可达为100L。[37, 38]瞬时转染的大规模培养工艺中对质粒需求量很大(1~1.25ug/ml)。由于其工艺复杂、成本较高等原因,限制了该技术在抗体药物的产业化生产中的应用。

4 克隆筛选技术的发展趋势

   克隆筛选环节需要筛选出重组细胞并将其单克隆化,传统的有限稀释法耗时、费力,筛选通量低,往往是稳定细胞系构建的限速步骤。近年来,在克隆筛选手段上出现了标记高表达克隆和高通量筛选机器等技术。[39]借助流式细胞仪进行细胞分选[40],或使用自动机器挑选克隆[41][42],都能显著提高克隆筛选的通量和效率。此外,在克隆筛选策略上,利用微型反应器和毛细管电泳技术,可以在克隆筛选环节,对细胞系的表达水平、生长能力[10][43],产物质量进行综合评价。这样就避免了单纯以目的蛋白表达量为依据,忽视候选克隆细胞系在反应器中的表现、产物质量等问题。

4.1基于流式细胞仪的细胞分选术

流式细胞仪细胞分选术利用根据流式细胞仪通过对单个细胞所激发的荧光强定量检测来实现细胞分选。它可以对单个细胞进行多参数测定,筛选通量也提高至106。为了标记出高表达克隆,该方法必须与指示蛋白、荧光标记等技术联合使用[40]。如,重组抗体的轻、重链基因分别连接不同的荧光蛋白(eYFPeGFP),共转染细胞后,利用流式细胞仪根据两种荧光强度进行细胞分选。选取荧光强度最高的1%的细胞,其抗体的平均表达水平提高44[44];利用荧光标记的氨甲喋呤与胞内四氢叶酸还原酶的定量结合,可以筛选出dhfr基因拷贝数较高的细胞[45]也可利用“俘获抗体”将细胞分泌的抗体固定在细胞表面。再使用标记有荧光的检测抗体,借助流式细胞仪分选出高产细胞株[46]

4.2 用于克隆筛选的自动筛选机器

 激光分析过程系统(Laser-enabled analysis and processing利用细胞分泌蛋白形成的图像确定克隆表达水平,并使用脉冲激光清除低表达克隆。其他自动筛选机器(如ClonePixTMCellCelectorTMCelloTM)根据细胞在半固定培养基中形成的斑点大小,对细胞的表达能力与生长特性进行评价实现自动分选[41]

5 展望

上世纪九十年代发展起来“基因共扩增”技术,已经在稳定细胞系构建中得到广泛的应用。但是,在此后的近年的产业化实践发现,基于此原理的的DhfrGS表达系统普遍存在着重组克隆差异性大、表型稳定性差等问题。通过对重组细胞的目的基因拷贝数、mRNA、基因整合位点进一步发现:重组抗体的表达水平与基因拷贝数并不总是正相关。过多的外源基因拷贝数会导致重组细胞代谢负荷加大,生长能力减弱。此外,基因插入位点的“位置效应”,宿主的转录、翻译能力,也会影响重组抗体的表达水平。[30, 47, 48]目前,最新的克隆筛选策略开始尝试以轻、重链mRNA水平为指标,预估重组细胞的表达水平、质量差异[49]。。

此外,近年来对高产细胞系的转录组、蛋白组、代谢组的研究发现:重组细胞表达异源蛋白能力的高低,不能简单的归结为若干个关键基因的作用。成就一株高产细胞系,往往是细胞的能量代谢、氧化还原电位、生长能力,蛋白加工能力等诸多特性,以网络的形式综合作用的结果[50]。对高产细胞的组学研究,加深了业内对动物细胞表达异源蛋白机制的认识。可以预见:组学技术的前瞻性指引,结合宿主细胞遗传改造,以及高通量筛选技术的应用,将是未来高产细胞系构建工作的发展方向。

 

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The Technology Progress of Antibody-producing Cell Line Development

Liu Bo-ning

(New Drug Reaserch and Development Center, North China Pharmaceutical Group Corporation and State Key Laboratory of Antibody drug Reaserch and Development, Shijiazhuang, China, 050015)

Abstract:The commercial therapeutic antibodies manufacturing began with construction of antibody-producing cell line, and the productivity of engineering cell line for antibody expression has reached 20-70pg/cell/day at present.  Many industrial cases of various animal cell lines as host cell were summarized, accounting to their different attributes. Furthermore, the latest technology progression of cell line developments was introduced, as follows: Cell engineering technology ensured the host cell with robust growth and high productivity; The New vector element and site-directed integration technology contribute to overcoming the “position effection” by random integration. Lastly, the application of cell-sorting by flow cytometer and automatic screening device have remarkable improved the efficient and throughout of recombinant cell line screening, etc. Additionally, the phenotypic variation and instability of cell line and the technological trend in this field are also discussed.

Keywords:Recombinant antibody; Cell line development; Vector construction; Screening strategy


*国家973项目(2012CB724502)资助

 



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