基因突变技术在抗体亲和力体外成熟中的应用

刘媛,林曼曼,张霄,徐重新,焦凌霞,仲建锋,武爱华,刘贤金(江苏省食品质量安全重点实验室和农业部食用农产品安全监控重点开放实验室,南京210014)

基因突变技术在抗体亲和力体外成熟中的应用

刘媛,林曼曼,张霄,徐重新,焦凌霞,仲建锋,武爱华,刘贤金*
(江苏省食品质量安全重点实验室和农业部食用农产品安全监控重点开放实验室,南京210014)

摘要从天然抗体库中筛选得到的抗体通常亲和力较低,在微摩尔水平;同时,经过多次免疫获得的天然抗体也存在100 pmol/L的亲和力极限.然而,以抗体亲和力体内成熟为理论基础的亲和力体外成熟技术,可以模拟体细胞高频突变和克隆选择过程.该技术可以解决库来源抗体亲和力不高、实际应用难的问题,也可以帮助天然抗体突破其亲和力极限.抗体基因体外突变技术可以模拟自体高频突变过程,是抗体亲和力体外成熟的重要手段,常见的抗体基因体外突变技术可以分为易错聚合酶链式反应(polymerase chain reaction,PCR)、DNA改组、突变株、定点突变和链替换等方法.易错PCR可以在抗体基因全长或部分区域随机引入突变,但随着突变率的增加,阳性克隆数量呈指数性递减.DNA改组包括抗体片段随机化切割和重组步骤,可以加快抗体的体外进化速度.突变株法易于构建超大容量的抗体库,但有害突变和突变率难以控制.定点突变的区域通常选择与抗原直接接触的互补决定区(complementary determination region,CDR)或自体突变热点进行操作.链替换通常保留母体抗体的一条重链或轻链,而对另一条链进行随机化组合.这些基因突变方法在提高抗体亲和力中获得了不同程度的应用,但也存在效率不高,且方法选择较为盲目等问题.可是,采用抗体X射线晶体衍射和计算机模拟等方法,却可以帮助预测抗体结合部位,为突变位点的理性设计和方法选择提供有效信息,因而成为亲和力体外成熟技术未来发展的趋势之一.

关键词抗体;亲和力成熟;易错PCR;DNA改组;突变株;定点突变;链替换

浙江大学学报(农业与生命科学版) 42(1):1～7,2016

Journal of Zhejiang University(Agric.&Life Sci.)

http://www.journals.zju.edu.cn/agr

E-mail:zdxbnsb@zju.edu.cn

第一作者联系方式:刘媛(http://orcid.org/0000-0003-3842-7571)，Tel:+86-025-84390401,E-mail:zeranol@163.com

URL:http://www.cnki.net/kcms/detail/33.1247.S.20151215.1739.002.html

Applications of mutagenesis methods on affinity maturation of antibodies in vitro.Journal of Zhejiang University(Agric.&Life Sci.),2016,42(1):1-7

LIU Yuan,LIN Manman,ZHANG Xiao,XU Chongxin,JIAO Linxia,ZHONG Jianfeng,WU Aihua,LIU Xianjin*(Key Laboratory of Food Safety of Jiangsu Province and Key Laboratory of Food Safety Monitoring and Management of Ministry of Agriculture,Nanjing 210014,China)

Summary Antibodies selected from naive antibody libraries usually have low affinity at the micromolar level.Meanwhile,the affinities of natural antibodies obtained by repeated immunization were also observed to have a limit around 100 picomole because of the inability to discriminate slower dissociation kinetics relative to intrinstric B cell receptor internalization rate.As an essential determinant for antibody function for therapeutic and diagnostics use,a lots of antibodies,either isolated from antibody libraries or cloned from monoclonal antibodies,have been targeted to generate high affinity by directed evolution.

Affinity maturation in vitro could mimic the mutation and selection process of affinity maturation in vivo,it couldhelp to improve the affinity of the antibodies derived from antibody libraries and make the natural antibodies to break through affinity ceiling.At the mutagenesis level,strategies to affinity maturation in vitro can be grouped in five categories:error prone PCR,DNA shuffling,mutator strains,site directed mutagenesis and chain shuffling.Error PCR could generate random mutations into antibodies throughout the whole antibodies gene.But it is notable that when the mutation rate increases,the active clones decreases exponentially.DNA shuffling includes random fragmentation and reassembly steps;it can be used with longer DNA sequences,and also allows for the selection of clones with mutations outside the binding or active site of the antibodies.By mutator strains,the mutations are produced after the transformation,which allows the randomly mutated libraries of even 1012_1014clones be produced.However,by mutator strains,mutations are also produced in the vector part to cause deleterious effects.In site-directed mutagenesis,selected residues are targeted to be mutated.The most common sites for mutation are the CDR regions which directly contact with antigen.However,mutations that do not directly contact antigen can also result in enhanced affinity.And the mutation hotspots in affinity maturation in vivo can also be targeted to the mutation sites.In chain shuffling,a variable heavy or light chain of a specific antibody is recombined with a complementary variable domain library.In a lot of experiments,light chains were used to be shuffling,because heavy chain is crucial for the antigen binding.But there are also some reports on producing the affinity-matured antibodies by heavy chain shuffling.

These mutagenesis methods have been used with various degrees of success.However,the efficiency of these methods was relatively low and there was a lack of a rational guidance to choose these methods.Knowledge gained on antibody structure through X-ray crystallography or computer-aided design could help to predict the paratope and mutation sites,which is one of the development trend of affinity maturation in vitro.

Key words antibodies;affinity maturation;error PCR;DNA shuffling;mutator strains;site-directed mutagenesis;chain shuffling

抗体制备技术的发展,已经历了多克隆抗体技术、单克隆抗体技术和抗体库技术3个发展阶段[1].单克隆抗体技术的出现,解决了多克隆抗体亲和力、特异性相对较低和难以重复制备的问题.而抗体库技术的出现,则能以体外建库和筛选代替单克隆抗体复杂的免疫和杂交瘤筛选过程,被誉抗体制备创新性的换代技术,随着相关行业的发展迅猛,国内外已有大量抗体库筛选的报道[2 5].

然而,抗体库来源的抗体与传统的单、多克隆抗体相比,实际应用非常少见,究其原因是由于库来源抗体与天然抗体相比,亲和力偏低,难以满足多种实际应用需求.特别是来自于未经免疫的天然抗体库中的抗体,抗体的亲和力通常在微摩尔水平,这仅与动物在初次免疫应答后获得的抗体亲和力水平相当[6].与此同时,天然抗体的亲和力通常存在一个“天花板效应”,通过免疫获得的天然抗体亲和力通常低于100 pmol/L,这是由于B细胞受体难以区分更低的解离速率造成的.可是,抗体的亲和力体外成熟技术却可以帮助解决这些问题.

1　抗体的亲和力体外成熟的理论基础

要想实现抗体的亲和力体外成熟,就必须充分地了解天然抗体的亲和力体内成熟原理;设计模拟体内可能出现和存在的变化,从而促进抗体的体外进化[7].

天然抗体的亲和力成熟可以分为体细胞高频突变和克隆选择2个过程.机体接受抗原刺激后,体细胞发生高频突变,突变产生的各种亲和力不同的B细胞克隆,经过滤泡树突细胞捕获,使得后代B细胞及其产生的抗体对抗原的平均亲和力得到了提升,天然抗体从而实现了亲和力的体内成熟.

在天然抗体亲和力成熟的过程中,抗原刺激下的体细胞高频突变有着举足轻重的作用,因此亲和力体外成熟的策略也多在抗体基因突变水平上,即采用各种突变方法来模拟体内的高频突变.本文将对抗体亲和力体外成熟技术中常用的突变方法加以介绍.

2　抗体基因的体外突变技术

2.1易错PCR

易错PCR技术是最常用的抗体基因突变技术,可以在抗体基因的全长或部分区域随机引入突变.该技术可以通过提高镁离子浓度、加入锰离子、失衡4种脱氧核苷三磷酸(deoxyribonucleoside triphosphate,d NTPs)浓度、使用低保真DNA聚合酶等方法,来提高抗体基因的突变率[8].

除此之外,突变率的高低也可以采用改变模板DNA的复制次数进行控制.复制次数越多,突变率将累积得越高.因此,要想获得高突变率,可以采用低浓度的DNA模板同时增加PCR循环数来实现.但是,随着突变率的提高也将造成大量突变抗体失活,从而影响突变库库容.表1中列出了一些易错PCR的应用实例,DAUGHERTY等[9]对易错PCR的突变率与抗体活性保留关系进行了研究.通过对地高辛单链抗体(single-chain variable fragment,scFv)进行低(0.22%)、中(0.5%)和高(3%)3个水平的随机突变研究,表明随着突变率的提高,突变库中活性克隆所占比例大幅下降,在3种突变率下对应的活性克隆比例分别为40%、6.7%和0.17%.但是,亲和力提高最多的克隆(提高1.9倍)仍然来自于高频突变库.

GRAM等[10]从非免疫鼠组合抗体库中获得的Fab抗体,对黄体酮-牛血清白蛋白(bovine serum albumin,BSA)的亲和力仅为10-4～10-5mol/L,作者随后采用易错PCR法对4个抗体基因模板重、轻链可变区随机突变,PCR体系采用了系列浓度的模板DNA和优化的MgCl2、MnCl2以及d NTP浓度,筛选到的最佳克隆较母体抗体亲和力提高了30 倍,突变率为1.5%.作者观察到的最主要突变形式是A/G和T/C的替换,同时发现突变热点的存在.PERSSON等[11]采用Ampli Taq聚合酶,对将睾丸激素抗体结合片段(fragment antibody-binding,Fab)进行随机突变,结果表明构建的2个突变库CTep1和CTep2的突变率分别是0.77%和0.87%,其中89%的突变含有插入碱基,而80%突变存在于抗体的框架结构.作者随后采用了逐轮减少抗原浓度、改变和洗脱捕获条件等方法,获得的最佳克隆亲和力为2 nmol/L,亲和力提高了200倍.

2.2DNA改组

DNA改组技术是对同源的抗体基因,采用脱氧核糖核酸酶Ⅰ将其切割成不超过50 bp的片段,再随机组合后进行PCR扩增成完整的抗体基因.它包含了抗体片段随机化切割、重组和筛选的过程,一定程度上模拟了天然抗体的亲和力成熟过程,并加快了体外定向进化速度[18 19].采用连续多轮的DNA改组具有去除有害突变优势[20].另外,DNA改组获得的突变体,突变位点可以位于非抗原直接接触的区域,有扩大库容的作用[7].

为了获得足够多的点突变,DNA改组技术还常与易错PCR技术联用.BODER等[15]以荧光素单链抗体4-4-20为研究对象,其亲和力为0.3 nmol/L,已接近于天然抗体所能获得的最大亲和力,但是通过DNA改组和易错PCR的组合应用对其进行了四轮突变及筛选后,获得的最佳克隆4M5.3在PBS的半数解离常数达到了5 d,比母体抗体延长了4个数量级,解离时间超过了生物素与链霉亲和素的解离时间.在LSB溶液中与生物素化荧光素的亲和力可达48 fmol/L,比4-4-20提高了208倍,是目前公开报道的亲和力最高的非共价结合物.

2.3突变株

Escherichia coli MutD5是最常用的大肠埃希菌突变株,该菌株具有校对和复制后错配修复缺陷,可以产生高频率的单个碱基替换,突变率高于普通菌株105倍.突变株法的优势是突变发生在转化宿主菌后,而其他方法的突变均发生于转化之前,而转化效率是限制大容量突变库构建的关键因素之一.使用突变株法可以构建库容为1012～1014个克隆的超大容量抗体库,但是该方法较难对突变率进行控制,且获得的随机突变也可能发生于载体部分,造成有害突变.

LOW等[17]采用E.coli MutD5对半抗原3-苯基-噁唑酮抗体进行突变,获得的突变体亲和力可达3.2 nmol/L,亲和力提高了100倍.MUTEEB等[21]也报道了采用E.coli XL1-red作为突变菌株的研究.

2.4定点突变

由于天然抗体在亲和力成熟过程中,体细胞高频突变发生区域并非均匀分布,而是主要集中在与抗原直接接触的CDR区.这样既可以获得足够的序列多样性,又不会破坏蛋白质结构.因此在抗体的亲和力体外成熟中,CDR区是最常选用的定点突变区域.

在表2所列出的一系列定点突变中,有平行对多个CDR进行突变或对多个CDR进行逐步优化,即CDR步行法(CDR walking).STEIDL等[22]报道了对CDR-H2区的突变,将母体抗体亲和力提高近300 倍.除了CDR区的点突变外,LAMMINMAKI[23]和PARHAMI-SEREN[24]通过增加CDR-H2 loop环长度的方法提高半抗原特异性抗体的亲和力.KOBAYASHI[25]则对半抗原17β-雌二醇母体抗体进行了2步法突变,首先是在抗体的CDR H2,H3,L1 和L3进行了CDR改组,对该突变库中筛选出亲和力提高的克隆,再进行重、轻链可变区的易错PCR,多轮筛选获得的最佳克隆亲和力提高了10倍.

然而,由于单链抗体或Fab抗体具有6个CDR 区,抗体与抗原结合过程中又涉及多个CDR.因此在确定对抗原结合起作用氨基酸残基,以及CDR边缘氨基酸残基对亲和力起作用方面相当复杂.为了解决这一问题,近年来最突出的进展是利用抗体晶体结构信息或计算机辅助设计,来分析与抗原直接接触的氨基酸位点.BARDERAS等[26]采用了胃泌激素抗体可变区的3D结构模型来辅助预测抗原结合部位的氨基酸位点,设计采用的CDR步行法将胃泌激素单链抗体的亲和力提高了454倍.

除了CDR区突变外,天然抗体亲和力成熟中的突变热点(hotspot)也是常见的定点突变位点.这些热点通常包含或接近AGY和RGYW(R=A or G;Y=C or T;W=A or T)序列[32].

2.5链替换

链替换是保留某个特定抗体的重链或轻链,并与一个随机化的互补链进行组合,从中筛选更高活性的突变株(表3).由于通常认为抗体的重链CDR3区对抗体的结合能力至关重要,因此轻链的替换较为常见.FITZGERALD等[33]将提取第5轮筛选后的溴氯哌喹酮scFv重链可变区基因与原始抗体库的轻链基因随机组合,构建了库容1.25×107的轻链替换库,从中筛选到最佳克隆建立的ELISA检测方法,灵敏度可达80 pg/m L,比母体抗体提高了185倍.

链替换技术除了可以用于提高抗体亲和力外,也有对抗体特异性、稳定性提高方面的报道.该技术可以用于鼠源抗体的人源化改造[40],帮助解决杂交瘤来源的抗体转变为单链抗体形式后,在原核表达中出现失活的问题[34].

除了轻链替换外,也有重链替换或两者组合应用的成功报道.PARKER等[37]通过对母体抗体结构分析,认为轻链在抗原识别中起到决定性作用,因此采用了重链替换的方法对肝炎B病毒scFv进行改造,获得的最佳克隆比母体抗体亲和力提高了6.5倍.KRAMER[38]则对阿特拉津单链抗体的重链和轻链依次进行置换,亲和力成熟抗体的检测灵敏度由最初的5.1μg/L提高到0.2μg/L,抗体的特异性也有所改善.

3　问题与展望

近年来学术界虽然在应用基因突变技术实现抗体亲和力体外成熟中取得了一些进展,但由于人们对亲和力体外成熟的分子基础方面的认知不够明确,抗体的突变策略选择仍然较为盲目,获得成熟抗体的效率不高.

在现有的抗体亲和力成熟分子基础研究中,通过对成熟抗体与母体抗体序列比对分析,可以证明抗体的CDR3氨基酸对于亲和力成熟演化过程非常重要,然而主要用于维持抗体结构保守性、折叠功能和稳定性的框架结构序列,对抗原结合作用也存在影响[41-42].氨基酸序列的差异对抗体功能性影响,在不同区域呈现的相关性又会不同,因此仅采用氨基酸序列分析的方法,难以精确预测出哪些位点的突变会对抗体亲和力的改变产生影响[43].

为了解决这些问题,可以采用X射线晶体衍射或是核磁共振的方法对亲和力成熟抗体及其抗原复合物的三维结构进行分析[44 45].或采用计算机模型来模拟“抗原-抗体”的互作过程,用于预测亲和力成熟的关键位点.对范德华力、氢键、亲水性、构型、电荷互补、变构效应、可塑性和协调性等物化因子进行测定,也可对“抗原-抗体”复合物的形成做定量分析,以增加对亲和力成熟过程的理解,并为突变位点的理性设计提供有效的信息[46].

随着人们对天然抗体亲和力成熟分子基础认知的不断加深,以及突变技术应用于抗体亲和力体外成熟实践的积累,抗体基因体外突变技术将日趋发展成熟,成为更为有效的抗体体外定向进化手段.

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收稿日期(Received):2015-03-25;接受日期(Accepted):2015-06-26;网络出版日期(Published online):2015-12-15

*通信作者(

Corresponding author):刘贤金(http://orcid.org/0000-0003-0184-7978),E-mail:jaasliu@163.com

基金项目:国家自然科学基金(31201535);江苏省社会发展项目(BE2012750);江苏省农业自主创新基金(CX(12)5042).

DOI:10.3785/j.issn.1008-9209.2015.03.251

中图分类号Q789

文献标志码A