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。     

抗人P185^erbB2嵌合抗体的CHO细胞中的高效表达及其活性分析

为降低人抗鼠抗体（HAMA）反应并在CHO细胞中高效表达抗人P185^erbB2人／鼠嵌合抗体,将抗人P185^erbB2单抗C25的轻、重链可变区基因分别克隆入具有人抗体恒定区基因组序列和弱化启动子驱地劝的选择标志基因的真核表达载体中,共转染CHO－dhfr^-细胞,经G418及氨甲喋呤（MTX）梯度加压筛选进行了嵌合抗体的高效表达,采用RT－PCR、ELISA、细胞ELISA、免疫荧光细胞化等实验证实了所表达的抗人P185^erbB2嵌合抗体的人源性及抗原特异性。培养上清中的抗体产量可达100mg/L,所表达的嵌合抗体具有抑制P185^erbB2高表达肿瘤细胞增殖的作用。     

人-鼠嵌合Fab片段的温度诱导表达

构建了具有λＰＲＰＬ启动子高效表达人鼠嵌合Ｆａｂ片段的温度诱导表达型载体ｐＨＺ０１,并在大肠杆功中表达了三种嵌合Ｆａｂ片段：抗前列腺特异抗原（ＰＳＡ）的嵌合Ｆａｂ,抗溶菌酶（ＨＥＬ）的嵌合Ｆａｂ和抗破伤风类毒素（ＴＴ）的嵌合Ｆａｂ,三种表达的可溶性嵌合Ｆａｂ都具有特异结合抗原的能力,嵌合Ｆａｂ的ＣＨ１和ＣＫ区均为人源的,较之鼠源Ｆａｂ具有更好的应用前景。     

抗CD3基因工程嵌合抗体IgG在哺乳动物细胞中的表达和活性测定

利用基因工程方法将鼠源性抗CD3抗体HIT3a的可变区和人源抗体(IgG)的完整的恒定区连接起来,构建全抗型抗CD3嵌合抗体,该型抗体具有较低的免疫源性可作为免疫抑制剂应用于器官移植,减少受体产生免疫排斥,提高移植器官的存活率。利用PCR方法从抗CD3 ScFv重组噬菌体表达载体pCANTAB 5E上扩增抗CD₃抗体的轻链和重链可变区,将轻链和重链可变区组装到含有人抗体（IgG）恒定区的表达载体中,构建抗CD3嵌合抗体IgG的轻链和重链表达载体PKN100和PG1D105,并用脂质体法共转染CHO细胞。结果证明,抗CD₃嵌合抗体的V_L和V_H与HIT3a抗体的V_L和V_H完全相符,ELISA和Western blot检测结果证实转染细胞的培养上清中含有抗CD₃嵌合抗体IgG的表达,表达产物能与Jurkat细胞结合,并能竞争性抑制HIT3a抗体和Jurkat细胞结合活性,³H-TdR掺入实验表明, 抗CD3嵌合抗体与亲代抗体HIT3a一样,具有促进外周血单核细胞增殖的作用。我室构建的全抗型抗CD3嵌合抗体分子表达载体可在CHO细胞中稳定表达,表达产物有较好生物活性,具有潜在的临床应用价值。     

乙肝表面抗原专一的单链嵌合抗体在大肠杆菌中的表达

利用重组ＰＣＲ技术将乙肝表面抗原专一单抗的Ｖｋ与人源的Ｃ基因拼接成轻链嵌合抗体Ｖ－Ｃ,再通过编码柔性肽段（Ｇｌｙ４Ｓｅｒ）的Ｌｉｎｋｅｒ与Ｖ连接成单链嵌合抗体ＳｃＦＶ－Ｃ,并分别在大肠杆菌的热敏与分泌型表达系统中进行表达。表达产物的Ｗｅｓｔｅｒｎ－ｂｌｏｔ与间接ＥＬＩＳＡ检测结果表明,ＳＣＦｖ－Ｃ产两种系统中的表达产物均具有抗原结合活性,并已在分泌肽的指导下ＳｃＦｖ－Ｃｋ能被Ｅ．ｃｏｌｉ分泌出胞外。     

抗人P185~(erbB2)嵌合抗体在CHO细胞中的高效表达及其活性分析

为降低人抗鼠抗体 (HAMA)反应并在CHO细胞中高效表达抗人P185 erbB2 人 /鼠嵌合抗体 ,将抗人P185 erbB2 单抗C2 5的轻、重链可变区基因分别克隆入具有人抗体恒定区基因组序列和弱化启动子驱动的选择标志基因的真核表达载体中 ,共转染CHO dhfr-细胞 ,经G418及氨甲喋呤 (MTX)梯度加压筛选进行了嵌合抗体的高效表达。采用RT PCR、ELISA、细胞ELISA、免疫荧光细胞化学等实验证实了所表达的抗人P185 erbB2 嵌合抗体的人源性及抗原特异性。培养上清中的抗体产量可达 10 0mg/L ,所表达的嵌合抗体具有抑制P185 erbB2 高表达肿瘤细胞增殖的作用     

基因工程抗体表达系统的研究进展

本文就基因工程抗体几种常用表达系统包括大肠杆菌、昆虫细胞、酵母、植物、哺乳类细胞等系统的研究近况作一综述,并分别介绍了各表达系统的优缺点。     

基因工程抗体的研究进展

基因工程抗体以其独特的优点（免疫原性低、可按人的意愿加以改造等）正逐渐取代动物源性单抗。随着基因工程和蛋白质工程等生物技术在抗体研制领域的广泛应用,适应不同需要的基因工程抗体的种类日趋多样化,构建日趋合理化,在体内的生物学效应与日臻完善,使之较天然单抗的治疗效果更好,范围更广,并在初步临床试用中展示了光辉的前景。     

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.     

人鼠嵌合单克隆抗体c30.6的纯化

采用介体电泳技术对CHO细胞培养液中的人鼠嵌合单克隆抗体c30.6进行纯化,第一步在pH8.6条件下进行,抗体带正电荷而培养液中的大部分杂质带负电荷,抗体和杂质的在电场作用下向两电极运动,结果被分离在分离膜的两侧,SDS－PAGE显示抗体纯度达95％,且剩余杂质的分子量均小于抗体的分子量。第二步是在pH6.0条件下进行,此时抗体和杂质都带正电荷,它们在电场作用下向同一方向运动,但分离膜只允许分子量较小的杂质通过,抗体得以进一步纯化,抗体的回收率为80％,而且生物活性未受影响。     

Construction and characterization of the chimeric antibody 8C11 to the hepatitis E virus

Homodimers of the truncated hepatitis E virus (HEV) capsid proteins, E2 and p239, were conformed to model the dominant antigenic determinants of HEV. Using E2 as an immunogen, two neutralizing monoclonal antibodies (mAbs), namely 8C11 and 8H3, were produced. We constructed a mouse-human chimeric antibody derived from 8C11 and its expression in Chinese hamster ovary (CHO) cells. cDNAs encoding variable regions of heavy and light chains were isolated from hybridoma cells and inserted into mammalian expression vectors containing cDNA of human gamma-1 and kappa constant regions, respectively. The vectors were then cotransfected into CHO cells, and a stable cell line was established. Results from indirect enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showed that the chimeric antibody was assembled correctly to the native IgG molecule and could be secreted from the cells. Similar to the original mAb, the expressed chimeric antibody displayed HEV antigen-binding activity and an enhancement effect on 8H3 binding to HEV antigen. The chimeric antibody could specifically inhibit the binding of p239 to HepG2 cells and compete with HEV IgG in positive serum by antibody-competitive ELISA. The chimeric antibody is expected to be less immunogenic in human and more suitable for antibody therapy of hepatitis E.     

针对猪瘟病毒E2蛋白的嵌合猪源化单克隆抗体的表达及抗病毒活性鉴定

猪瘟(Classical swine fever,CSF)是严重危害养猪业的一种烈性传染病,常造成巨大的经济损失,是世界动物卫生组织要求必须申报的动物疫病之一。猪瘟的病原是猪瘟病毒(Classical swine fever virus,CSFV),CSFV的结构蛋白由衣壳蛋白(C)和囊膜糖蛋白(E~(rns)、E1、E2)构成。E2蛋白是CSFV主要的保护性抗原,可以诱导机体产生中和抗体,从而抵抗CSFV的感染。此前,本团队制备了一株针对CSFV E2蛋白的鼠源单克隆抗体HQ06。文中将HQ06抗体重链和轻链可变区基因与猪源恒定区基因嵌合后克隆至真核表达载体,利用中国仓鼠卵巢(CHO)细胞制备一株针对CSFV E2蛋白的嵌合猪源化单克隆抗体c HQ06。应用ELISA、Western blotting试验证实了c HQ06与CSFV E2蛋白具有良好的反应性;中和试验结果表明c HQ06可以中和CSFV。综上所述,本研究应用CHO细胞稳定表达了具有良好反应性和中和活性的针对CSFV E2蛋白的嵌合猪源化单克隆抗体c HQ06,为研究CSFV E2蛋白结构、功能以及开发新型的CSFV诊断和治疗制剂奠定基础。     

First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers

This first-in-human study examined the safety and pharmacokinetics of ch-mAb7F9, an anti-methamphetamine monoclonal antibody, in healthy volunteers. Single, escalating doses of ch-mAb7F9 over the range of 0.2 to 20 mg/kg were administered to 42 subjects who were followed for 147 d. Safety was measured by physical examinations, adverse events, vital signs, electrocardiograms, and clinical laboratory testing. Serum ch-mAb7F9 concentration and immunogenicity analyses were performed. There were no serious adverse reactions or discontinuations from the study due to adverse events. No trends emerged in the frequency, relatedness, or severity of adverse events with increased dose or between active and placebo treated subjects. Ch-mAb7F9 displayed expected IgG pharmacokinetic parameters, including a half-life of 17–19 d in the 3 highest dose groups and volume of distribution of 5–6 L, suggesting the antibody is confined primarily to the vascular compartment. Four (12.5%) of the 32 subjects receiving ch-mAb7F9 were confirmed to have developed a human anti-chimeric antibody response by the end of the study; however, this response did not appear to be dose related. Overall, no apparent safety or tolerability concerns were identified; a maximum tolerated dose was not reached in this Phase 1 study. Ch-mAb7F9 therefore appears safe for human administration.     

HSV-IgM人-鼠嵌合抗体制备及稳定细胞株构建工艺开发

为实现体外大规模制备单纯疱疹病毒HSV-IgM(HSV1,HSV2)人鼠嵌合抗体,本研究通过RNA连接酶介导的cDNA末端快速扩增(RNA ligase-mediated rapid amplification of cDNA ends,RLM-RACE)技术获取其对应杂交瘤细胞基因序列,构建嵌合抗体至真核表达载体,在CHO-S细胞中稳定表达所需目的蛋白。同时优化稳定细胞株筛选工艺,对细胞池构建阶段和单克隆筛选阶段的加压条件进行摸索与探究,最后目的抗体采用蛋白L亲和纯化法进行纯化并进行生物活性检测;最终成功制备899 kDa和909 kDa的稳定高表达重组IgM抗体(HSV1,HSV2)细胞株。结果表明,最适筛选压力为20P200M(一轮加压)和50P1000M(二轮加压);使用加压培养基进行单克隆筛选抗体表达量较高,HSV1-IgM和HSV2-IgM单克隆最终表达量分别为1620 mg/L和623 mg/L。本研究为HSV1和HSV2的IgM系列重组抗体质控品开发以及体外高表达分泌IgM亚型抗体提供理论与实践基础。     

First human study of a chimeric anti-methamphetamine monoclonal antibody in healthy volunteers

This first-in-human study examined the safety and pharmacokinetics of ch-mAb7F9, an anti-methamphetamine monoclonal antibody, in healthy volunteers. Single, escalating doses of ch-mAb7F9 over the range of 0.2 to 20 mg/kg were administered to 42 subjects who were followed for 147 d. Safety was measured by physical examinations, adverse events, vital signs, electrocardiograms, and clinical laboratory testing. Serum ch-mAb7F9 concentration and immunogenicity analyses were performed. There were no serious adverse reactions or discontinuations from the study due to adverse events. No trends emerged in the frequency, relatedness, or severity of adverse events with increased dose or between active and placebo treated subjects. Ch-mAb7F9 displayed expected IgG pharmacokinetic parameters, including a half-life of 17–19 d in the 3 highest dose groups and volume of distribution of 5–6 L, suggesting the antibody is confined primarily to the vascular compartment. Four (12.5%) of the 32 subjects receiving ch-mAb7F9 were confirmed to have developed a human anti-chimeric antibody response by the end of the study; however, this response did not appear to be dose related. Overall, no apparent safety or tolerability concerns were identified; a maximum tolerated dose was not reached in this Phase 1 study. Ch-mAb7F9 therefore appears safe for human administration.     

Production of human-mouse chimeric antibody by high cell density perfusion culture

Two mouse myeloma cell lines which were transfected with chimeric mouse variable-human constant immunoglobulin heavy and light chain genes have been cultured at high cell density in a settling perfusion culture vessel to produce chimeric antibody specific for human common acute lymphocytic leukemia antigen (cALLA).J558L transfectant proliferated well in a serum-free medium (ITES-eRDF) to a viable cell density of 3.7×10⁷ cells/ml and produced chimeric antibody to a maximum value of 60 g/ml in 120 ml scale vessel. X63Ag8.653 transfectant reached a density of 1.9×10⁷ cells/ml in 1.2 I scale vessel in serum supplemented medium (10% FCS-eRDF) and produced chimeric antibody which consisted of chimeric gamma and chimeric kappa chains to a maximum value of 5.8 g/ml.     

The in vitro antitumor effect and in vivo tumor-specificity distribution of human-mouse chimeric antibody against transferrin receptor

Transferrin receptor (TfR/CD71) deserves attention as a selective target for cancer therapy due to its higher expression in tumors versus normal tissues. Also, it has been shown the mouse-derived monoclonal antibody against TfR can significantly inhibit the proliferation of tumor cells. Through constructing the chimeric antibody against TfR, the antigenicity of antibody can be weakened, and most importantly, the antitumor effect of antibody can be strengthened by the introduction of the human Fc fragment. In previous studies, we successfully constructed the human-mouse chimeric antibody against TfR (D2C) and demonstrated that its Fab fragment could specially recognize the TfR on the surface of target cells. In this study, through labeling the chimeric antibody D2C with ¹²⁵I, we calculated the affinity constant (Ka) of 9.34–9.62×10⁹ l/mol for this antibody according to the Scatchard drawing method. Moreover, in vivo studies in nude mice-bearing human liver cancer (SMMC-7721) xenografts have shown that the radioactivity distribution ratio of ¹³¹I-D2C on T/NT was 2–14:1 or 3–21:1 on the seventh day after intraperitoneal or intratumoral injection of ¹³¹I-labeled D2C (¹³¹I-D2C). These evidences indicated that the in vivo distribution of D2C display the characteristics of certain tumor-specificity localization. In vitro studies, D2C can induce the apoptosis of K562 through the mitochondria death pathway and arrest the cell at G₁ phase, as determined by cell cycle analysis. Using the human tumor cells (K562, CEM, and SMMC-7721) expressing TfR as target cells, and normal human PBMC as effector cells, Fc fragment of D2C can perform both the antibody-dependent cell-mediated cytotoxicity and the complement-dependent cytotoxicity. Together, it was demonstrated that the D2C display a tumor-specificity distribution, and has a strong antitumor effect. Thus, it has the potential therapeutic significance.Ye Qing and Wang Shuo contributed equally to this work.     

Attack on neoplastic cell membranes by therapeutic antibody

Mouse monoclonal antibody is not well fitted to destroying tumour cell targets. Complement and cellular effectors are inefficiently recruited, the cells can undergo antigenic modulation, antigen-negative mutants can arise, and the tumour-bearing subject can amount an immune response against the therapeutic antibody.This paper describes the preparation of two chimeric antibody derivatives designed to cirvumvent some of these problems. The first derivative is FabFc, prepared by linking Fab' from monoclonal antibody to Fc from human IgG. The bismaleimide linking agent forms a thioether bond with an SH group released by reduction of SS bonds in the hinge of each constituent. The second derivative is bisFabFc, formed by a bismaleimide in this case joining two FabFc molecules via a free SH in the Fc hinge of each. As regards antibody activity against target cells bisFabFc can be univalent (one active, one inactive Fab arm), bivalent, or bispecific (with each Fab arm directed against a different cell surface antigen). Its juxtaposed dual Fc regions are designed to promote cooperative binding of effectors.Some preliminary characterization in vitro has employed antibodies of anti-idiotypic specificity directed against guinea-pig L₂C leukaemic B lymphocytes. The parent mouse IgG1 antibody failed to invoke complement cytotoxicity or antibody-dependent cellular cytotoxicity, while the chimeric derivatives yielded good killing in both systems. In complement lysis bivalent bicFabFc outperformed univalent, which in turn outperformed the FabFc monomer.