利用线性表达框高通量表达人源单克隆抗体

目的:建立不通过克隆步骤高通量表达来源于人单个B细胞的抗体轻、重链基因的方法。方法和结果:PCR扩增3个末端重叠的DNA片段,即1CMV启动子和编码抗体引导区序列的片段;2抗体Ig G1重链恒定区序列和牛生长激素(BGH)poly(A)信号序列,轻链Igκ恒定区序列和BGH poly(A)信号序列,轻链Igλ恒定区序列和BGH poly(A)信号序列;以及3抗体基因可变区序列V_H、V_κ或V_λ。3个片段通过重叠延伸PCR构建全长线性片段即线性表达框,将此来源于人单个B细胞的配对的抗体轻、重链线性表达框共转染293E细胞,72 h收集上清检测到表达的抗体。结论:构建的抗体基因线性表达框是无须克隆,快速高通量表达抗体基因进行筛选分析的策略。     

人癌胚抗原单链抗体基因的构建和筛选

从分泌抗癌胚抗原(carcinoembryoni antigen, CEA)单抗的杂交瘤细胞株C50中提取总RNA, 逆转录成cDNA, PCR扩增分别得到抗体轻、重链可变区基因, 再利用两对PCR引物合成和扩增得到全单链抗体基因. 将含轻、重链可变区序列的DNA片段克隆于含噬菌体基因Ⅲ的噬菌粒pCANTAB5. 重组克隆在噬菌体表面表达基因Ⅲ与单链抗体的融合蛋白. 表达具抗原结合活性的单链抗体的重组噬菌体可以通过亲和筛选的方法筛选得到并富集. 利用该方法我们可以从许多分泌不同抗体的杂交瘤细胞RNA中快速克隆和筛选功能性抗体可变区基因.     

抗TNF-α单链抗体的表达及其纯化

克隆抗人肿瘤坏死因子(TNF-α)鼠源单抗的可变区基因以构建其单链抗体(ScFv)表达载体,实现在大肠杆菌的表达,并进行ScFv的可溶性纯化与鉴定。采用RT-PCR技术,以前导肽序列的引物从1个分泌抗人TNF-α的鼠单抗杂交瘤细胞系中克隆抗体轻链、重链可变区基因(VL,VH),构建ScFv基因,将ScFv基因片段与pGEX-4T-1表达载体连接,在大肠杆菌中表达并采用十二烷基肌氨酸钠(Sarkosyl)进行可溶性纯化,最后鉴定其生物活性。结果显示,得到了功能性重排的轻、重链可变区基因,分别构建了VH和VL不同连接顺序的HLL(VH-Linker-VL)和LLH(VL-Linker-VH)两种ScFv,LLH的表达量较HLL的高,但亲和力不及HLL。采用Sarkosyl溶解包涵体,对目的蛋白进行可溶性纯化,蛋白纯度达到90%,纯化后的蛋白经ELISA和WB证明ScFv维持了亲本抗体与TNF-α特异性结合的能力,且具有细胞毒中和活性。实验中研究探索了一种新颖的,操作简单,省时的裂解、纯化方案,实现了单链抗体经原核系统的表达后得以可溶性纯化。     

抗HEV嵌合抗体的构建及在CHO细胞中的表达

通过RT-PCR方法从分泌戊型肝炎(戊肝)病毒中和性鼠源单克隆抗体(单抗)8C11的杂交瘤细胞中克隆出抗体基因的重链可变区(VH)、轻链可变区(VK)序列,并分别克隆到含有人gamma 1重链和kappa轻链恒定区序列的pcDNA3.1/Hygro和pcDNA3.1( )质粒中,共转染中华仓鼠卵巢癌细胞(CHO)细胞.RT-PCR结果表明,转染的CHO细胞转录了嵌合重链及轻链基因,间接ELISA及Western blot结果表明:翻译出的两种多肽在细胞内正确组装成嵌合抗体分子,并可分泌至细胞外,表达的嵌合抗体保留了原鼠单抗的抗原结合特异性及对8H3结合抗原的增强作用.8C11嵌合抗体的成功表达可降低鼠源性,为探讨戊肝抗体治疗的可能性奠定了基础.     

抗FGF2人鼠嵌合抗体在HEK293T细胞中的表达优化

目的：FGF2是肿瘤血管新生过程中最重要的因子之一,因此通过制备抗FGF2人鼠嵌合抗体中和其发挥作用,以达到抑制肿瘤生长的目的。方法：利用分泌抗FGF2抗体的杂交瘤细胞株IgG9B9和人B淋巴细胞,分别克隆抗体轻链可变区V_L、重链可变区V_H和人重链恒定区C_H基因,从pComb3λ载体中扩增出人λ 链恒定区C_L基因,通过重叠PCR,将V_L,V_H和C_L,C_H片段分别连接形成嵌合抗体的轻链L和重链H,将L/H链以单独构建或串联于同一载体的方式,构建抗FGF2嵌合抗体表达载体,并通过调控元件WPRE优化载体、共转染促生长因子aFGF以及调整表达温度等方式提高嵌合抗体在真核细胞中的表达。结果：成功构建了优化表达载体PLexm-WPRE、PLexm-aFGF;L、H链基因也成功构建,并以L、H或L-F2A-H（2A连接肽将L和H连接起来）的方式分别成功连接到PLexm,PLexm-WPRE载体中。转染细胞上清的ELISA鉴定结果表明,L/H链单独构建要比串联构建的方式具有更高的表达水平,WPRE能有效促进抗体的表达而aFGF并不能促进其表达,与31、37℃相比,33℃时抗体的表达量最高,同时嵌合抗体表现出了很好的结合活性及中和活性,竞争IC50=6.25μg/ml。通过亲和层析获得了高纯度的抗FGF2嵌合抗体。结论：在33℃下,人鼠嵌合抗体基因在WPRE存在下表达量最高,且与抗原FGF2有很好的结合活性及中和活性,为临床应用奠定了基础。     

抗人黑色素瘤单链抗体基因的克隆和分泌型表达

应用ＲＴ－ＰＣＲ技术从分泌抗人黑色素瘤单克隆抗体的杂交瘤细胞ＨＢ８７６０中克隆了抗体轻、重链可变区基因,然后用（Ｇｌｙ４Ｓｅｒ）３连接肽基因将ＶＨ、ＶＬ连接成ＳｃＦｖ基因,并进行了序列测定．计算机分析表明ＶＨ,ＶＬ均符合小鼠抗体可变区的特征,为功能性重排的抗体可变区基因．ＶＨ、ＶＬ、ｌｉｎｋｅｒ拼接正确．ＳｃＦｖ基因全长７２９ｂｐ,其中ＶＨ基因长３６０ｂｐ,编码１２０个氨基酸,ＶＬ基因长３２４ｂｐ,编码１０８个氨基酸．在噬菌粒表达载体ｐＣＡＮＴＡＢ５Ｅ中表达了可溶性的ＳｃＦｖ蛋白,表达产物经流式细胞仪检测可特异地与黑色素瘤细胞结合,不与肝癌、胃癌及良性黑痣细胞结合     

BjαIT的表达、抗血清制备及scFv基因库的构建

根据成熟昆虫神经毒素BjαIT的氨基酸序列,人工合成毒素基因,并克隆至大肠杆菌表达载体pPET-30a（＋）。在IPTG的诱导下,神经毒素在大肠杆菌中融合表达,表达产物经镍亲和层析纯化,纯化蛋白免疫BALB／c小鼠,制备了特异性较高的抗血清,抗体滴度达1：512,000。提取免疫小鼠脾细胞总RNA,通过RT-PCR分别扩增重链可变区（VH）及轻链可变区（VL）基因片段,在连接肽linker的连接下成功构建了包含抗BjαIT的全长scFv基因库。本研究为新型蝎昆虫神经毒素BjαIT的检测奠定了基础。     

鼠抗HFRSV衣壳蛋白McAb F3株可变区基因的获取及特性分析

培养鼠抗肾综合征出血热病毒衣壳蛋白Ｆ３杂交瘤细胞株,提取总ＲＮＡ,根据鼠源ＩｇＧ抗体基因家族可变区基因碱基序列的特点,设计简并引物,通过逆转录聚合酶链反应,获得抗体轻链可变区和重链可变区基因。分别将其克隆入载体ＰＴ７ＢｌｕｅＴ Ｖｅｃｔｏｒ,选取阳性重组克隆各两个,分别测定了所载重链可变区和轻链可变区基因的碱基序列,比较了不同克隆轻链可变区基因之间和重链可变区基因之间碱基序列的差异;分析了各自的氨基酸框架及其对应蛋白的亲水性。结果显示,两个重链可变区基因碱基序列有４处不同,同源性为９７９％;其中重组克隆ＺＧ３６４ ５Ｆ所载重链可变区基因有完整的开放阅读框架,对应的蛋白含有丰富的亲水基因,第１１２氨基酸处亲水性最高;另一重组克隆ＺＧ３６４ ４Ｆ所载重链可变区基因不能通读。两个轻链基因碱基序列有４处不同,同源性为９９１％,重组克隆ＺＧ３６５ ５Ｆ和ＺＧ３６５ ７Ｆ所载轻链可变区基因均有完整的开放阅读框架,对应的蛋白均含有丰富的亲水基因,ＺＧ３６５ ５Ｆ所载基因对应蛋白第６７氨基酸亲水性最高,ＺＧ３６５ ７Ｆ所载基因对应蛋白第３４氨基酸亲水性最高。     

鼠源E型肉毒毒素免疫噬菌体单链抗体库的构建及筛选

目的:构建鼠源E型肉毒毒素(BoNT/E)免疫噬菌体单链抗体库,筛选BoNT/E特异性抗体。方法:从E型肉毒类毒素免疫小鼠的脾细胞中提取总RNA,反转录成cDNA,分别扩增出小鼠重链可变区基因和轻链可变区基因;通过重叠延伸PCR将重链可变区基因和轻链可变区基因组装成scFv基因,重组于噬粒pS100中,电转化大肠杆菌TG_1,合并所有克隆成初级库;随机挑取克隆进行核苷酸序列测定,对初级库序列多样性进行分析;在辅助噬菌体M_(13)K_(07)的拯救下,构建成scFv噬菌体抗体库;用纯化的BoNT/E对鼠源BoNT/E免疫噬菌体单链抗体库进行3轮富集筛选,制备单克隆的噬菌体抗体颗粒进行酶联免疫吸附试验,阳性克隆进行核苷酸序列测定。结果:鼠源BoNT/E免疫噬菌体单链抗体库的库容为7.09×10~7,随机挑取的20个克隆序列各不相同,序列正确率为85%,基本覆盖了IgHV、IgKV、IgLV的优势家族;纯化的BoNT/E作为抗原通过3轮筛选,噬菌体抗体富集了66倍,第3轮筛选后随机挑取90个克隆制备噬菌体抗体颗粒,酶联免疫吸附试验分析有88个呈现阳性反应,序列比对得到了24个不同序列的BoNT/E特异性抗体。结论:构建了库容量达7.09×10~7的鼠源BoNT/E免疫噬菌体单链抗体库,筛选得到了24个不同序列的BoNT/E特异性抗体。     

抗破伤风类毒素单抗可变区基因的克隆及在大肠杆菌中表达的三种工程抗体

我们采用RT-PCR,从小鼠杂交瘤细胞中扩增并克隆了抗破伤风类毒素(TT)抗体轻、重链可变区,重链Fd区基因,测定了其VH、Vk序列。并在大肠杆菌中表达了Fd片段,ELISA分析的结果表明Fd片段具有抗原结合的能力,但特异性很差。进一步采用SOE,和PCR技术,将VH、VK基因与ScFv连接片段组装成单链抗体(ScFv)基因片段,以及将人重链CH1和Fab基因连接片段组装成Fab基因片段。将它们分别插入含噬菌体fd外壳蛋白3基因的phagem-id pHEN 1中,在辅助噬菌体M 13-VCS作用下,噬菌体表面表达了抗TT的噬菌体单链抗体(phage-ScFv)与噬菌体Fab(phage-Fab),经ELISA检测,表明它们都能与TT特异结合。     

治疗性单克隆抗体研究进展

杂交瘤技术使鼠源单克隆抗体（鼠单抗）被广泛用于人类疾病的诊断和研究,建立了治疗性抗体的第一个里程碑。但随后出现的人抗鼠抗体等副作用极大地限制了鼠单抗的临床应用。随着生物学技术的发展和抗体基因结构的阐明,应用DNA重组技术和抗体库技术对鼠单抗进行人源化改造,先后出现了嵌合抗体、改型抗体和全人抗体,同时也涌现了各种单抗衍生物,它们从不同角度克服了鼠单抗临床应用的不足,未人类疾病治疗带来新的曙光。我们就上述治疗性抗体人源化的研究进展做简要综述。     

基因工程抗体研究进展

临床治疗中人抗鼠抗体反应的出现使鼠源性单克隆抗体的应用受到了极大的限制。为降低其免疫原性,人们利用基因工程技术对鼠源抗体进行改造,以减少其鼠源成分。简要概述了目前研究比较多的几种基因工程抗体及其临床应用。     

Monoclonal antibody therapeutics with up to five specificities: Functional enhancement through fusion of target-specific peptides

The recognition that few human diseases are thoroughly addressed by mono-specific, monoclonal antibodies (mAbs) continues to drive the development of antibody therapeutics with additional specificities and enhanced activity. Historically, efforts to engineer additional antigen recognition into molecules have relied predominantly on the reformatting of immunoglobulin domains. In this report we describe a series of fully functional mAbs to which additional specificities have been imparted through the recombinant fusion of relatively short polypeptides sequences. The sequences are selected for binding to a particular target from combinatorial libraries that express linear, disulfide-constrained, or domain-based structures. The potential for fusion of peptides to the N- and C- termini of both the heavy and light chains affords the bivalent expression of up to four different peptides. The resulting molecules, called zybodies, can gain up to four additional specificities, while retaining the original functionality and specificity of the scaffold antibody. We explore the use of two clinically significant oncology antibodies, trastuzumab and cetuximab, as zybody scaffolds and demonstrate functional enhancements in each case. The affect of fusion position on both peptide and scaffold function is explored, and penta-specific zybodies are demonstrated to simultaneously engage five targets (ErbB2, EGFR, IGF-1R, Ang2 and integrin αvβ3). Bispecific, trastuzumab-based zybodies targeting ErbB2 and Ang2 are shown to exhibit superior efficacy to trastuzumab in an angiogenesis-dependent xenograft tumor model. A cetuximab-based bispecific zybody that targeting EGFR and ErbB3 simultaneously disrupted multiple intracellular signaling pathways; inhibited tumor cell proliferation; and showed efficacy superior to that of cetuximab in a xenograft tumor model.     

嵌合抗体研究进展

抗体在疾病诊断,治疗和预防中发挥着重要作用,近几年来将基因工程应用到抗体生产的基因工程抗体技术发展迅速,本就基因工程抗体中的重要组成部分－嵌合抗体的研究进展加以综述。     

Isolation and optimization of camelid single-domain antibodies: Dirk Saerens' work on nanobodies

It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies from all other species, these special antibodies are devoid of light chains, and are composed of a heavy chain homodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V-D-J rearrangement and require dedicated constant gamma genes. An immune response is raised in these HCAbs following a classical immunization protocol. These HCAbs are easily purified from serum, and their antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies. The antigen binding site of the dromedary HCAb comprises one single domain, referred to as VHH or nanobody (Nb), therefore, a strategy was designed to clone the Nb repertoire of an immunized dromedary and to select the Nb with specificity for our target antigens. The monoclonal Nb is produced well in bacteria, is very stable and highly soluble, and it binds the antigen with high affinity and specificity. Currently, the recombinant Nb has been developed successfully for research purposes, as a probe in biosensors, to diagnose infections, or to treat diseases such as cancer or trypanosomiasis.     

Monoclonal antibodies: technologies for early discovery and engineering

Antibodies are essential in modern life sciences biotechnology. Their architecture and diversity allow for high specificity and affinity to a wide array of biochemicals. Combining monoclonal antibody (mAb) technology with recombinant DNA and protein expression links antibody genotype with phenotype. Yet, the ability to select and screen for high affinity binders from recombinantly-displayed, combinatorial libraries unleashes the true power of mAbs and a flood of clinical applications. The identification of novel antibodies can be accomplished by a myriad of in vitro display technologies from the proven (e.g. phage) to the emerging (e.g. mammalian cell and cell-free) based on affinity binding as well as function. Lead candidates can be further engineered for increased affinity and half-life, reduced immunogenicity and/or enhanced manufacturing, and storage capabilities. This review begins with antibody biology and how the structure and genetic machinery relate to function, diversity, and in vivo affinity maturation and follows with the general requirements of (therapeutic) antibody discovery and engineering with an emphasis on in vitro display technologies. Throughout, we highlight where antibody biology inspires technology development and where high-throughput, “big data” and in silico strategies are playing an increasing role. Antibodies dominate the growing class of targeted therapeutics, alone or as bioconjugates. However, their versatility extends to research, diagnostics, and beyond.     

Structural understanding of stabilization patterns in engineered bispecific Ig‐like antibody molecules

Bispecific immunoglobulin‐like antibodies capable of engaging multiple antigens represent a promising new class of therapeutic agents. Engineering of these molecules requires optimization of the molecular properties of one of the domain components. Here, we present a detailed crystallographic and computational characterization of the stabilization patterns in the lymphotoxin‐beta receptor (LTβR) binding Fv domain of an anti‐LTβR/anti‐TNF‐related apoptosis inducing ligand receptor‐2 (TRAIL‐R2) bispecific immunoglobulin‐like antibody. We further describe a new hierarchical structure‐guided approach toward engineering of antibody‐like molecules to enhance their thermal and chemical stability. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

治疗性抗体的研究进展

作为一种具有靶向性的生物大分子,单克隆抗体始终是人们关注的热点之一,被广泛用于治疗肿瘤、病毒感染和抗移植排斥等。但鼠源单克隆抗体的临床应用受限于诱导产生人抗鼠抗体、肿瘤渗入量低、亲和力低和半衰期短等。随着分子生物学技术的发展及其向各学科的渗透,通过基因操作技术对抗体进行改造,可使其适用于多种疾病的治疗。抗体人源化已经成为治疗性抗体的发展趋势,同时各种抗体衍生物也不断涌现,它们从不同角度克服了抗体本身的应用局限,也为治疗人类疾病提供了利器。本文简要介绍上述技术的基本原理、特点和治疗性抗体的研究进展。     

噬菌体抗体库技术及其应用研究进展

噬菌体呈现抗体库是近年发展的一项分子生物学新技术,它的建立是抗体技术领域中的一次革命性进展。它以其独特的构建和筛选系统,彻底改变了抗体制备的传统途径,使抗体工程技术进入了一个新的发展阶段,并对生物学领域中许多技术的发展起到了巨大的推动作用。该技术是迄今发展最成熟、应用最广泛的制备抗体技术。我们简要综述此项技术的研究应用进展。