Improving the productivity of single-chain Fv antibody against c-Met by rearranging the order of its variable domains

Single-chain Fv (scFv) antibody against c-Met is expected to be employed in clinical treatment or imaging of cancer cells owing to the important biological roles of c-Met in the proliferation of malignancies. Here, we show that the productivity of scFv against c-Met in Escherichia coli is significantly influenced by the orientation of its variable domains. We generated anti-c-Met scFv antibodies with two different domain orders (i.e., VL-linker-VH and VHlinker- VL), expressed them in the cytoplasm of E. coli trx/ gor deleted mutant, and compared their specific activities as well as their productivities. Productivity of total and functional anti-c-Met scFv with VH/VL orientation was more than five times higher than that with VL/VH format. Coexpression of DsbC enhanced the yield of soluble amounts of anti-c-Met scFv protein for both constructs. The purified scFv antibodies of the two different formats exhibited almost the same antigen-binding activities. We also compared the productivities and specific activities of anti-c-Met diabodies with VH/VL or VL/VH formats and obtained similar results to the case of scFv antibodies.     

肝癌细胞特异性结合单链抗体三维结构的同源模建

用MSI公司开发的计算机辅助分子设计系统模建肝癌细胞表面抗原特异性单链抗体三维结构。先分别模建VH(variable region of the heave chain)和VL(variable region of the light chain)两个结构域,然后搭建出scFv(single chain variable fragment)片段的整体三维结构,并对模建的结构进行分子力学和动力学优化;对结构的合理性验证显示模建结构是合理的。文章可为预测该特异性单链抗体的生物活性以及研制高亲和力、高特异性的双价抗体提供结构信息。     

Bacterial single-chain antibody fragments, specific for carcinoembryonic antigen.

We have produced single-chain antibody (scFv) fragments in bacteria specific for carcinoembryonic antigen (CEA). Polymerase chain reaction (PCR) was used for the cloning and modification of the heavy and light variable regions (VH and VL) of the mouse monoclonal antibody (MAb) CB-CEA.1. A 14-amino acid linker was used in the synthesis of the scFv gene. The VH and VL regions were amplified from cDNA by PCR using 5' end FR1 and 3' end constant region primers, and then sequenced. VH was then amplified by PCR using an exact 5' end FR1 primer, and a phosphorylated (PP) 3' end primer for J2 that also encoded the first 7 amino acids of the linker. VL was amplified with a PP 5' end primer for FR1, also encoding the remaining 7 amino acids of the linker, and a 3' end primer for J5, plus a stop codon and a BglII restriction site. The fragments were ligated and reamplified with the PP VH 5' and VL 3' end primers. The VH-linker-VL structure was blunt-cloned into expression vectors bearing the tryptophan promoter and pelB or ompA signal peptide sequences. Culture supernatant, bacteria pellet and periplasm preparations were assayed in Western blot and a protein of about 27 kDa was identified with rabbit antibodies specific for the Fab of CB-CEA.1. Bacterial supernatant and periplasm preparations also inhibited the recognition of CEA by HRP-labeled CB-CEA.1 in enzyme-linked immunosorbent assay (ELISA). Periplasm preparations were purified by affinity chromatography with specific anti-idiotypic MAbs. The Western blot of the eluates identified a protein of approximately 27 kDa that blocked the recognition of CEA by HRP-labeled CB-CEA.1 in ELISA. The VH-linker-VL structure was cloned into a vector bearing the lacZ promoter and the pelB signal peptide. The recombinant bacterial clones also expressed about 27 kDa scFv, specific for CEA.     

抗甜菜坏死黄脉病毒单链抗体表达载体的构建及其表达

用ＰＣＲ方法以分泌抗甜菜坏死黄脉病毒（ＢＮＹＶＶ）单克隆抗体的杂交瘤细胞的基因组为模板,扩增了编码ＢＮＹＶＶ单抗的重链可变区（ＶＨ）基因。测序表明,该ＶＨ序列属于小鼠ＩＩ（Ａ）亚类,全长为３６０ｂｐ,编码１２０个氨基酸。将其和先前克隆的轻链基因分别插入到一个含有连接ＶＨ和ＶＬ基因的连接序列的质粒之中,构建成单链抗体（ｓｃＦｖ）基因的表达载体ｐＴＣｓｃＦｖ。将质粒在大肠杆菌中表达,ＥＬＩＳＡ法检测出     

噬菌体抗体库的构建及抗乳腺癌细胞单链抗体的筛选

构建抗人乳腺癌细胞MCF 7的噬菌体单链抗体库 ,从中筛选MCF 7细胞特异性单链抗体。用MCF-7细胞免疫BALB C小鼠 ,取脾脏 ,提取总RNA ,用RT-PCR技术扩增小鼠抗体重链 (V_H)和轻链 (V_L)可变区基因 ,经重叠PCR(SOE-PCR) ,在体外将V_H和V_{L连接成单链抗体 (scFv)基因 ,并克隆到噬菌粒载体pCANTAB5E中 ,电转化至大肠杆菌TG1,经辅助噬菌体超感染 ,构建噬菌体单链抗体库。从该抗体库中筛选特异性识别MCF-7细胞的噬菌体单链抗体 ,将表面展示单链抗体的单克隆噬菌体转化大肠杆菌TOP10进行可溶性表达。成功地构建了库容为12×10⁶ 的抗MCF-7乳腺癌细胞的单链抗体库 ,初步筛选到了与MCF 7细胞特异性结合的scFv,Westernblot检测表明 ,在大肠杆菌TOP10中实现了单链抗体可溶性表达}     

Generation and characterisation of functional recombinant antibody fragments against RNA replicase NIb from plum pox virus

A monoclonal antibody (mAb 2A) able to react against the RNA replicase NIb from plum pox virus (PPV) was obtained and used for generating a specific scFv fragment. The VH and VL coding sequences were cloned and expressed as a fusion scFv protein to alkaline phosphatase. This fusion protein was able to recognise viral NIb in both Western and tissue-print ELISA blots. The affinity and specificity of scFv2A for NIb was similar to that of the parental mAb and the region YLEAFY from PPV-NIb was identified by PEPSCAN assay as the putative epitope. Isolated VH domains from scFv2A were also expressed as fusion to alkaline phosphatase. However, their ability to react against NIb was greatly altered. scFv2A fragments were transiently expressed in the cytosol of Nicotiana benthamiana and although they accumulated to low levels, inhibition-ELISA results indicated that they retained antigen-binding activity.     

Heavy and light chain variable single domains of an anti-DNA binding antibody hydrolyze both double- and single-stranded DNAs without sequence specificity

Anti-DNA antibodies (Abs) are of biomedical interest because they are associated with autoimmune diseases in human and mice. Previously we isolated an anti-DNA monoclonal Ab 3D8 from an autoimmune-prone MRL-lpr/lpr mouse. Here we have characterized DNA binding kinetics and hydrolyzing activities of the recombinant single chain variable fragment (scFv) and the single variable domains of heavy chain (VH) and light chain (VL) using various single-stranded (ss) and double-stranded (ds) DNA substrates. All the Abs bound to both ds- and ssDNAs without significant preferential sequence specificity showing scFv higher affinities (KD = approximately 17-74 nm) than VH (KD = approximately 2.4-8.4 microm) and VL (KD = approximately 3.2-72 microm), and efficiently hydrolyzed both ds- and ssDNAs without sequence specificity in a Mg2+-dependent manner, except for the poor activity of 3D8 scFv for ss-(dT)40. Elucidated crystal structure-based His to Ala mutations on the complementarity determining regions of VH (His-H35 --> Ala) and/or VL (His-L94 --> Ala) of 3D8 scFv significantly inhibited the catalytic activities, indicating that the His residues are involved in the catalytic mechanism of 3D8 scFv. However, the DNA hydrolyzing activities of single domain VH and VL were not affected by the mutations, indicative of their different catalytic mechanisms from that of 3D8 scFv. Our results demonstrate single domain Abs with DNase activities for the first time, which might provide new insights into substrate recognition and catalytic mechanisms of anti-DNA Abs.     

Production and characterization of monoclonal and recombinant antibodies against antimicrobial sulfamethazine

A monoclonal antibody (mab) against the antimicrobial sulfamethazine was prepared and characterized by an indirect competitive enzyme-linked immunosorbent assay (IC-ELISA). Sulfamethazine in the range of 0.2 and 45 ng/ml could be determined with the mab by IC-ELISA. cDNAs encoding a variable heavy chain and variable light chain of the mab were cloned to produce recombinant antibodies using phage display technology. Following phage rescue and three rounds of panning, a single-chain variable fragment (scFv) antibody with high sulfamethazine-binding affinity was obtained. ELISA analysis revealed that scFv antibody and parent mab showed similar, but not identical, characteristics. The IC50 value by IC-ELISA with scFv antibody was 4.8 ng/ml, compared with 1.6 ng/ml with the parent mab. Performances of the assays in the presence of milk matrix were compared; the mab-based assay was less affected than the scFv-based assay. Sixty milk samples were analyzed by mab-based IC-ELISA, and four samples were sulfamethazine positive; these results were favorably correlated with those obtained by HPLC.     

Recombinant single-chain Fv antibodies that recognize the p25 protein of the Maedi-Visna virus

Single chain Fv (scFv) antibodies (generated by phage display technology, molecules representing new and efficient tools in the research and diagnostics of infectious diseases) against the capsid protein (p25) of Maedi-Visna virus were selected. Several clones of p25 specific scFv antibodies were identified; one of them was expressed as a soluble scFv molecule, purified by immobilized metal-affinity chromatography and further characterized by sequencing and determination of the kinetic equilibrium association constant. Sequence analysis showed that the rearranged VL and VH domains of the analyzed scFv clone used sequences from the VL3 family (germline DPL16/VL3.1) and VH1 family (germline VH20), respectively. The kinetic equilibrium association constant was determined as KA = 1.12 +/- 0.52 L/mumol.     

Contributions of a highly conserved VH/VL hydrogen bonding interaction to scFv folding stability and refolding efficiency.

The assembly of single-chain Fv (scFv) antibody fragments, consisting of an interconnected variable heavy chain (VH) and variable light chain (VL), is a cooperative process that requires coupled folding and domain association. We report here an initial investigation of VH/VL domain-domain assembly with a site-directed mutagenesis study that probes a highly conserved VH/VL hydrogen bonding interaction. Gln168 of the S5 scFv (Kabat VH 39) is absolutely conserved in 95% of all VH, and Gln44 (Kabat VL 38) is found in 94% of all kappa VL (Glx in 95% of all lambda VL). These side chains form two hydrogen bonds in head-to-tail alignment across the VH/VL interface. Double mutant cycles at Gln168 and Gln44 were constructed to first investigate their contribution to thermodynamic folding stability, second to investigate whether stability can be improved, and third to determine whether refolding efficiencies are affected by mutations at these positions. The results demonstrate that the Gln168-Gln44 interaction is not a key determinant of S5 scFv folding stability, as sequential modification to alanine has no significant effect on the free energy of folding. Several mutations that alter the glutamines to methionine or charged amino acids significantly increase the thermodynamic stability by increasing the m(g) associated with the unfolding isotherm. These effects are hypothesized to arise largely from an increase in the VH/VL association free energy that leads to tighter coupling between domain-domain association and folding. All of the mutants also display a reduced antigen binding affinity. Single and double methionine mutants also displayed significant increases in refolding efficiency of 2.4- to 3-fold over the native scFv, whereas the double alanine/methionine mutants displayed moderate 1.9- to 2.4-fold enhancement. The results suggest that reengineering the VH/VL interface could be useful in improving the stability of single-chain antibodies, as Ala/Met mutations at these conserved positions increase the free energy of folding by 46% while minimally perturbing binding affinity. They also could be useful in improving scFv recovery from inclusion bodies as the mutations increase the refolding efficiency by more than twofold.     

抗EGFRvIII噬菌体抗体库的构建和筛选

目的：应用噬菌体展示技术筛选针对表皮生长因子受体突变体Ш (epidermal growth factor receptor variant type Ⅲ, EGFRvIII)的单链抗体 (single chain Fv, scFv)。方法：利用原核表达纯化的人EGFRvIIIex蛋白和高表达EGFRvIIIex的小鼠成纤维细胞系NIH3T3免疫小鼠,扩增VH和VL片段并拼装成scFv 基因,连接至噬菌粒pCANTAB 5E,电击转化Hpd3cells,构建噬菌体单链抗体库,并进行3轮富集筛选。在第4轮筛选时,采用了降低抗原浓度的方法。然后将筛选得到的阳性克隆测序分析,转化E.coli HB2151,IPTG 诱导可溶性scFv 的表达。结果：构建了库容为7.9×107 的噬菌体单链抗体库。经过第4轮低浓度抗原筛选,得到了较高亲和力的克隆。取单个阳性克隆测序分析结果表明,该抗EGFRvIII scFv 基因序列长807 bp,编码268个氨基酸。IPTG诱导后表达的可溶性scFv 可分别与纯化的EGFRvIIIex抗原以及细胞表面的EGFRvIIIex结合。结论：利用噬菌体抗体库筛选得到了高亲和力的抗EGFRvIII scFv,为开发针对EGFRvIII的抗体药物提供了靶向载体分子。     

Recombinant shark natural antibodies to thyroglobulin

As cartilaginous fish are the vertebrates most distal from man to produce antibodies, fundamental information regarding conservation and variation of the antigen binding site should be gained by comparing the properties of antibodies directed against the same antigen from the two species. Since monoclonal cell lines cannot be generated using shark B cells, we isolated antigen binding recombinant single chain Fv antibodies (scFv) comprising of the complete variable regions from shark light and heavy chains. Thyroglobulin was used as the selecting antigen as both sharks and humans express natural antibodies to mammalian thyroglobulin in the absence of purposeful immunization. We report that recombinant sandbar shark (Carcharhinus plumbeus) scFvs that bind bovine thyroglobulin consist of heavy chain variable regions (VH) homologous to those of the human VHIII subset and light chain variable regions (VL) homologous to those of the human Vlambda6 subgroup. The homology within the frameworks is sufficient to enable the building of three-dimensional models of the shark VH/VL structure using established human structures as templates. In natural antibodies of both species, the major variability lies in the third complementarity determining region (CDR3) of both VH and VL.     

基于核糖体展示技术进行抗狂犬病毒人源抗体的制备

构建了核糖体展示人源抗狂犬病毒单链抗体(scFv)库,筛选制备特异抗狂犬病毒糖蛋白(RVGp)的稳定性人源抗体.应用核糖体抗体库技术,从经狂犬病毒Vero疫苗免疫的志愿者外周血淋巴细胞中分离、构建核糖体展示scFv基因库.体外转录翻译后,以RVGp重组蛋白作筛选抗原,采用亲和富集法淘选RVGp特异性scFv抗体基因.在原核系统pET22b(+)/BL21(DE3)中实现scFv抗体片段的可溶性表达,ELISA鉴定阳性克隆.然后对筛选的scFv进行稳定性改构,构建VH-Lc-VK稳定性抗体,并对其生物学活性进行初步研究.成功构建了库容量约为6.2×1012的核糖体展示scFv抗体基因库.在180个筛选克隆中,克隆RB24、RB71、RB109和RB156显示出较高的ELISA值,其基因序列分析结果显示,它们是全新的人源抗RVGp抗体.改构后的抗RVGp VH-Lc-VK抗体的稳定性明显改进,可特异识别RVGp并有效中和狂犬病毒,抑制狂犬病毒对靶细胞的感染.以上结果表明,人源抗RVGp特异性抗体的获得,为狂犬病的有效预防、诊断和治疗提供了新的途径,而且将为其他人源抗体的制备提供理论依据和技术基础.     

Expression and purification of a single-chain variable fragment antibody derived from a polyol-responsive monoclonal antibody

A previously described polyol-responsive monoclonal antibody (PR-mAb) was converted to a single-chain variable fragment (scFv). This antibody, PR-mAb NT73, reacts with the beta' subunit of Escherichia coli RNA polymerase and has been used for the immunoaffinity purification of polymerase. mRNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) were used as the template for cDNA synthesis. The sequences were joined by the addition of a "linker" sequence and then cloned into several expression vectors. A variety of expression plasmids and E. coli hosts were used to determine the optimal expression system. Expression was highest with the pET22b(+) vector and the Rosetta(DE3)pLysS host strain, which produced approximately 60 mg purified His-tagged scFv per liter of culture (3.3 g wet weight cells). Although the production of soluble scFv was preferred, overproduced scFv formed inclusion bodies under every expression condition. Therefore, inclusion bodies had to be isolated, washed, solubilized, and refolded. The FoldIt protein refolding kit and enzyme-linked immunosorbent assay were sequentially used to determine the optimal refolding conditions that would produce active His-tagged scFv. Immobilized metal affinity chromatography was used for the final purification of the refolded active scFv. The polyol-responsiveness of the scFv was determined by an ELISA-elution assay. Although the scFv loses considerable affinity for its antigen, it maintains similar polyol-responsiveness as the parent monoclonal antibody, PR-mAb NT73.     

天然兔源噬菌体单链抗体库的构建与鉴定

目的:构建天然兔源噬菌体单链抗体库。方法:采用RT-PCR法从未免疫的兔子脾脏中克隆得到抗体重链可变区(VH)与轻链可变区(VL)基因,重叠PCR将VH和VL拼接成scFv片段,将scFv连接到噬菌粒pComb3XSS上,电转入XL1-Blue菌中,得到单链抗体库,并用此抗体库筛选抗肌酸激酶抗体。结果:构建了容量为4×108,基因重组率95%的单链抗体库,DNA指纹图谱显示抗体库多样性良好。以肌酸激酶为抗原,从该库中筛到3株抗肌酸激酶的抗体。结论:分析表明构建的天然兔源单链抗体库质量良好,可用于快速筛选、制备多种单链抗体。     

抗TNF-α单链抗体噬菌体展示文库的建立和鉴定

应用噬菌体展示技术构建抗肿瘤坏死因子α(tumornecrosis factor α,TNF-α)单链抗体(single chain Fv,scFv)文库,从中筛选抗TNF-αscFv并进行鉴定.利用重组人TNF-α(rhTNF-α)免疫小鼠,分别扩增小鼠VH和VL基因,经重叠延伸反应将VH和VL基因拼接成scFv基因,以SfiⅠ/NotⅠ位点定向插入pCANTAB 5E噬菌粒载体,转化E.coli TG1,构建了库容为4.6×108的抗TNF-α单链抗体库.对抗体库进行3轮富集筛选后,ELISA检测阳性克隆的抗原特异性,取1株阳性克隆进行测序分析.结果表明,抗TNF-αscFv基因序列长774bp,编码258个氨基酸.将此阳性克隆转化E.coliHB2151,IPTG诱导可溶性scFv的表达,经SDS-PAGE和Western印迹分析,scFv的分子量约为28kD.经亲和纯化后的scFv可与rhTNF-α结合,并可中和由rhTNF-α引起的L929细胞毒性.本文利用噬菌体抗体库筛选到了高亲和力的抗TNF-αscFv,为研制临床免疫治疗的新型抗体奠定了实验基础.     

抗EGFRvⅢ噬菌体抗体库的构建和筛选

目的:应用噬菌体展示技术筛选针对表皮生长因子受体突变体Ш(epidermal growth factorreceptor variant typeⅢ,EGFRvIII)的单链抗体(single chain Fv,scFv)。方法:利用原核表达纯化的人EGFRvIIIex蛋白和高表达EGFRvIIIex的小鼠成纤维细胞系NIH3T3免疫小鼠,扩增VH和VL片段并拼装成scFv基因,连接至噬菌粒pCANTAB5E,电击转化Hpd3cells,构建噬菌体单链抗体库,并进行3轮富集筛选。在第4轮筛选时,采用了降低抗原浓度的方法。然后将筛选得到的阳性克隆测序分析,转化E.coliHB2151,IPTG诱导可溶性scFv的表达。结果:构建了库容为7.9×107的噬菌体单链抗体库。经过第4轮低浓度抗原筛选,得到了较高亲和力的克隆。取单个阳性克隆测序分析结果表明,该抗EGFRvIII scFv基因序列长807bp,编码268个氨基酸。IPTG诱导后表达的可溶性scFv可分别与纯化的EGFRvⅢex抗原以及细胞表面的EGFRvⅢex结合。结论:利用噬菌体抗体库筛选得到了高亲和力的抗EGFRvⅢ scFv,为开发针对EGFRvⅢ...     

Gene cloning, bacterial expression, in vitro refolding, and characterization of a single-chain Fv antibody against PreS1(21-47) fragment of HBsAg

The murine monoclonal antibody 125E11 is an IgG which recognizes PreS1(21-47) fragment of large hepatitis B surface antigen. It has been successfully used for clinical detection of HBV virion in serum of hepatitis B patients. In present study, the genes of variable region in heavy chain (VH) and light chain (VL) of 125E11 have been cloned. Sequence analysis of cloned VH gene and VL gene showed that they had general characterization of immunoglobin variable region genes. According to Kabat classification, VH gene and VL gene belong to VH10 family, subgroup IIID and Vkappa family subgroup I, respectively. An expression vector of 125E11 single-chain Fv antibody fusion protein, in which VH and VL peptide were connected by a flexible linker (Gly(4)Ser)(3), was constructed. The scFv fusion protein was highly expressed in Escherichia coli mainly in inclusion body form. Using urea and pH gradient gel filtration method, the refolding of scFv was efficiently achieved. The refolding efficiency reached about 11% and 2.7 mg refolded scFv was obtained from 1L of culture. The binding activity and specificity of 125E11 scFv against PreS1(21-47)-containing antigen were also analyzed.     

Effect of domain order on the activity of bacterially produced bispecific single-chain Fv antibodies

Bispecific single-chain Fv antibodies comprise four covalently linked immunoglobulin variable (VH and VL) domains of two different specificities. Depending on the order of the VH and VL domains and on the length of peptides separating them, the single-chain molecule either forms two single-chain Fv (scFv) modules from the adjacent domains of the same specificity, a so-called scFv-scFv tandem [(scFv)(2)], or folds head-to-tail with the formation of a diabody-like structure, a so-called bispecific single-chain diabody (scBsDb). We generated a number of four-domain constructs composed of the same VH and VL domains specific either for human CD19 or CD3, but arranged in different orders. When expressed in bacteria, all (scFv)(2) variants appeared to be only half-functional, binding to CD19 and demonstrating no CD3-binding activity. Only the diabody-like scBsDb could bind both antigens. Comparison of the scBsDb with a structurally similar non-covalent dimer (diabody) demonstrated a stabilizing effect of the linker in the middle of the scBsDb molecule. We demonstrated that the mechanism of inactivation of CD19xCD3 diabody under physiological conditions is initiated by a dissociation of the weaker (anti-CD3) VH/VL interface followed by domain swapping with the formation of non-active homodimers. The instability of one homodimer makes the process of diabody dissociation/reassociation irreversible, thus gradually decreasing the fraction of active molecules. The structural parameters influencing the formation of functional bispecific single-chain antibodies are indicated and ways of making relatively stable bispecific molecules are proposed.