Fluobodies: green fluorescent single-chain Fv fusion proteins

An expression system (pSKGFP), which permits the expression of single-chain variable fragments as fusion proteins with modified green fluorescent proteins, was designed. This expression system is comparable to frequently used phage display vectors and allows single-step characterization of the selected recombinant antibodies by flow cytometry or fluorescent cell staining. Two different single-chain variable fragment antibodies, both directed against the lipopolysaccharide of the bacterium Ralstonia solanacearum have been genetically fused to a red-shifted green fluorescent protein and the produced fusion protein tested for usefulness. These fluobodies can be produced in cultures of bacterial cells and purified using immobilized metal affinity chromatography. They function well in flow cytometry and immunofluorescent cell staining, are specific for their target antigens and, unlike FITC-conjugated antibodies, they do not fade upon illumination.     

Efficient display of scFv antibodies on bacteriophage lambda

In the present work we demonstrate the efficient display of functional scFv antibodies on the bacteriophage lambda capsid. A single-chain (scFv) anti-CEA antibody gene was cloned in two different vectors to obtain fusion of the scFv antibody to the N- or C-terminus of the bacteriophage lambda capsid protein D (gpD). Lambda bacteriophage assembly occurs in the reducing environment of the cytoplasm; despite this the lambda-displayed anti-CEA antibody fragments retain the capacity to recognize the antigen, indicating correct single-chain antibody folding.

Efficient production of functional scFv exposed on lambda capsid with viable antigen binding specificity allowed us to study and compare the capacity of display, the stability of recombinant antibody expression and the assembly efficiency of bacteriophage particles decorated with recombinant antibody fused to the amino- or carboxy-terminus of lambda D protein.     

IL-13及其变异体真核表达载体的构建表达与亚细胞定位

目的：体外构建野生型人白细胞介素-13（whIL-13）及变异型人白细胞介素-13（mhIL-13）与增强型绿色荧光蛋白（EGFP）融和蛋白真核表达载体，分析其在COS-7细胞中的表达和亚细胞定位。方法：以RT-PCR方法扩增whIL-13、mhIL-13全长编码基因，构建EGFP-whIL-13、EGFP-mhIL-13融和蛋白真核表达载体，转染COS-7细胞，以激光扫描共聚焦显微镜观察融合蛋白的表达及其在细胞内的分布情况。结果：两种融和蛋白表达载体均构建正确，将其转染COS-7细胞后，在阳性克隆胞浆内均可见明亮的绿色荧光，胞核空虚。结论：成功构建EGFP-whIL-13、EGFP-mhIL-13融和蛋白表达载体，并在COS-7细胞中得到表达，表达的两种融和蛋白细胞内分布特征没有区别，均位于胞浆内。     

Recombinant immunotoxins for the treatment of Hodgkin's disease (Review)

In recent years, substantial experience has been accumulated with tumor-specific immunotherapeutics which seem to be effective against minimal residual disease. The coupling of toxins to monoclonal antibodies has indicated promising results in early clinical trials. Recombinant DNA technology makes it possible to genetically fuse coding regions of V genes or cytokines to modified toxin domains. These recombinant immunotoxins can easily be manipulated to increase the cytotoxic potency or affinity. Binding single-chain variable fragments (scFv) expressed as chimeric fusion proteins on the surface of filamentous bacteriophages were selected on Hodgkin-derived cell lines. This technique was also used to create a new humanized anti-CD30 scFv which exhibits similar binding to the CD30 antigen when compared to its murine predecessor. ScFvs were then inserted into a new bacterial expression vector and thus fused to a deletion mutant of Pseudomonas exotoxin. Anti-CD25(scFv)-ETA' and anti-CD30(scFv)-ETA' were isolated from E. coli periplasm and purified by metal chelate affinity and size exclusion chromatography. All immunotoxins produced showed specific cytotoxicity against Hodgkin lymphoma cell lines as documented by competitive assays. In addition, these constructs were highly efficient in the treatment of disseminated human Hodgkin's disease in SCID mice. These in vivo data indicate a possible clinical impact for patients with relapsed CD25- and/or CD30-positive lymphoma.     

抗人大肠癌单链抗体与绿色荧光蛋白融合基因的构建和表达

目的构建并表达绿色荧光蛋白(GFP)和鼠抗人大肠癌单链抗体ND-1scFv的融合蛋白,产生具有荧光的抗体。方法将鼠抗人大肠癌单链抗体ND-1scFv的基因克隆到pET28a(+)-GFP的表达载体,转化到大肠杆菌BL21中进行融合基因ND-1-scFv/GFP诱导表达。Ni-NTA亲和层析对表达产物进行分离、纯化,免疫印迹和荧光显微镜方法验证融合蛋白的表达。结果 ND-1scFv被克隆到表达载体pET28a(+)-GFP中,诱导表达的融合蛋白以包涵体形式存在,分子量为58kDa。SDS-PAGE灰度扫描结果显示纯化后的蛋白纯度为90%,荧光显微镜检测显示,表达有目的蛋白的大肠杆菌BL21具有明显的绿色荧光。结论成功构建并表达融合基因ND-1-scFv/GFP,为该单抗的肿瘤特异成像研究奠定了基础。E.coli     

Sheep red blood cells armed with anti-CD20 single-chain variable fragments (scFvs) fused to a glycosylphosphatidylinositol (GPI) anchor: a strategy to target CD20-positive tumor cells

Single-chain variable fragment antibodies (scFv) retain antigen specificity and offer advantages over intact antibodies as therapeutic agents. We cloned the cDNA of the V(H) and V(kappa) regions from a mouse hybridoma (HB-9645) directed against human CD20. In addition to the basic scFv construct (V(kappa)-L-V(H)), we genetically engineered a secretory signal, six histidine residues, and a 'Flu' tag to facilitate secretion, purification, and detection. A glycosyl-phosphatidylinositol (GPI) modification signal was added at the C terminus. The GPI-tagged and the non-tagged scFvs were expressed in high yields on the surface of stably transfected insect cells. The CD20-binding properties of purified non-GPI tagged scFv were examined using flow cytometry and immunocytochemistry. The non-GPI-tagged scFv selectively recognizes CD20-positive cells in a concentration-dependent manner. Double-flow cytometry analysis using fresh peripheral blood lymphocytes and WSU-FSCCL cells revealed that our scFv resolves the B-cell population better than the intact antibody. The GPI-tagged scFv was loaded onto the surface of sheep erythrocytes to form rosettes with CD20-positive cells. The genetically engineered anti-CD20 scFv and GPI-tagged derivative have binding specificity for the CD20 antigen. The scFvs described here has potential uses as an in vivo tumor-imaging agent and as a carrier vehicle for targeted delivery of cytocidal agents to CD20-positive cancer cells.     

Expression of single-chain Fv-Fc fusions in Pichia pastoris

Phage display technology makes possible the direct isolation of monovalent single-chain Fv antibody fragments. For many applications, however, it is useful to restore Fc mediated antibody functions such as avidity, effector functions and a prolonged serum half-life. We have constructed vectors for the convenient, rapid expression of a single-chain antibody Fv domain (scFv) fused to the Fc portion of human IgG1 in the methylotrophic yeast Pichia pastoris. The scFv-Fc fusion protein is secreted and recovered from the culture medium as a disulfide-linked, glycosylated homodimer. The increased size of the dimer (approximately 106 kDa vs. approximately 25 kDa for a scFv) results in a prolonged serum half-life in vivo, with t(1/2) of the beta phase of clearance increasing from 3.5 h for a typical scFv to 93 h for a scFv-Fc fusion in mice. The scFv-Fc fusion is capable of mediating antibody-dependent cellular cytotoxicity against tumor target cells using human peripheral blood mononuclear cells as effectors. Finally, the Fc domain is a convenient, robust affinity handle for purification and immunochemical applications, eliminating the need for proteolytically sensitive epitope and/or affinity tags on the scFv.     

Properties of a panel of single chain variable fragments against Potato leafroll virus obtained from two phage display libraries.

Twelve single chain variable fragment (scFv) antibodies that bind to particles of Potato leafroll virus (PLRV) were obtained from two naive phage display libraries. Phages were selected against PLRV particles or dissociated PLRV particles immobilised onto tubes. Individual PLRV-binding scFv were identified by ELISA, after their expression either fused to the surface of phage particles, or as soluble scFv (scFv-c-myc), or as scFv-alkaline phosphatase fusion proteins (scFv-AP), obtained by subcloning into pSKAP/S. These procedures resulted in the isolation of scFv with different properties. For example, some of the scFv reacted strongly with virus particles but not with dissociated capsid protein, which suggests that they had reacted with discontinuous epitopes. Others reacted with dissociated capsid proteins and SDS-denatured protein, which suggests that they had reacted with continuous epitopes. ScFv were also subcloned into pC(L) for expression as fusion proteins with human kappa constant region (scFv-C(L)). Expression of these constructs in Escherichia coli yielded 0.2-1 mg protein per litre of bacterial culture. The different scFv fusion proteins were evaluated in ELISA to detect PLRV in leaf extracts of Physalis floridana. Absorbance values obtained with the fusion proteins were greater than those obtained with the scFv-c-myc, and were similar to those obtained in assays done using monoclonal or polyclonal antibodies.     

Separation of E. coli expressing functional cell-wall bound antibody fragments by FACS

Background: The rapid development of recombinant antibody technology in the last few years has facilitated the generation of antibody libraries in bacteria. Recombinant antibodies against various antigens have been selected from these libraries by presenting each antibody on the surface of a phagemid particle that contains the antibody's gene. An alternative screening system is the display of antibody fragments on bacteria. A major advantage is the possibility to select single cells directly from a large number of bacteria by using fluorescently labeled antigens and fluorescence assisted cell sorting (FACS). Objectives: pAP is an expression vector for the bacterial display of antibody fragments. E. coli transformed with pAP express a single chain antibody (scFv) fused to the peptidoglycan-associated-lipoprotein (PAL). This fusion protein binds strongly to the cell wall. To employ this system for screening, we have investigated the possibility of selecting antigen-specific clones by FACS. Study design and results: Several DNA fragments coding for various scFvs were inserted into the pAP expression vector. E. coli were transformed with these plasmids and immunostained with fluorescent antigens under given conditions. We were able to select stained E. coli expressing a specific scFv from unstained E. coli expressing a non-binding scFv by FACS. The specific selection of the bacteria was demonstrated by amplifying their genes by PCR. Conclusions: Conditions are described for separating E. coli containing scFv bound to their cell wall by FACS using fluorescently labeled antigens. These studies provide a basis for screening libraries of scFv antibodies.     

Controlling complement resistance in cancer by using human monoclonal antibodies that neutralize complement-regulatory proteins CD55 and CD59

Expression of the complement-regulatory proteins (CRP) CD46, CD55 and CD59 represents a strategy used by tumor cells to evade complement-dependent cell cytoxicity stimulated by monoclonal antibodies. We have isolated two single-chain variable fragments (scFv) to CD55 and CD59 from a human phage-display library and from these scFv we have produced two miniantibodies (MB), MB-55 (against CD55) and MB-59 (against CD59), containing the human hinge-CH2-CH3 domains of IgG1. The specificity of the two MB for the corresponding CRP was assessed by ELISA using purified CD46, CD55 and CD59. MB-55 and MB-59 neutralized the inhibitory activity of CD55 and CD59, respectively, restoring the complement-mediated lysis of sheep and guinea pig erythrocytes that was otherwise inhibited by the two CRP. FACS analysis revealed binding of MB-55 and MB-59 to the lymphoma cell line Karpas 422. The two MB induced a two-fold increase in the complement-dependent killing of these cells stimulated by Rituximab, a chimeric anti-CD20 monoclonal antibody. Transfection of HEK293T cells with vectors encoding MB-55 or MB-59 markedly reduced the expression of CD55 and CD59. We conclude that the human antibodies MB-55 and MB-59 may represent a therapeutic tool to increase the complement-dependent killing activity of Rituximab in the treatment of non-Hodgkin's lymphoma.     

A single chain antibody obtained by cell panning of antibody phage inhibits homoaggregation of human leukemia cells

The aim of this study was to establish a method to obtain antibodies against cell-surface molecules of hematopoietic stem and progenitor cells in situ. A phage-displayed scFv antibody library with a size of 2 x 10(6) clones was constructed from spleens of mice immunized with living KG1a human leukemia cells. Living cell panning was used to screen anti-KG1a cell antibody fragments. After four rounds of panning, 27 out of 126 scFv fragments showed detectable binding to KG1a cells without cross-reaction to non-hematopoietic cell lines. Individual clones were analyzed by cell-based ELISA and flow cytometry for their binding specificity. Their sequence analysis showed highest homology to mouse gamma heavy chain subgroup II and mouse Kappa subgroup III genes, with four amino acids difference in VH and identical VL. Further, a fusion protein of scFv 5C1 to core-streptavidin was cloned and produced. It was used to search for cell-surface antigen on immunoblots and to test its effect on KG1a cell growth in cell culture. The scFv5C1::core-streptavidin fusion protein recognized a molecule with MW 85/125 KDa in immunoblots of KG1a cell membrane proteins and inhibited KG1a cell homoaggregation in cell cultures. The results validate an efficient approach of making antibodies against living cells and searching for functional inhibitors of cell-surface molecules.     

Single-chain Fv antibody-alkaline phosphatase fusion proteins produced by one-step cloning as rapid detection tools for ELISA

A system was constructed for the production of alkaline phosphatase (aP)-labeled antibody single-chain Fv (scFv) fragments in Escherichia coli. The expression vector pASK75 was modified by sequentially inserting the E. coli aP coding region and the scFv cloning cassette. Engineering the cloning sites SfiI and NotI located at the 5' and 3' end of the scFv gene provides an easy means to insert scFv fragments. These cloning sites are widely used in recombinant antibody technology and, thus, enable the one-step cloning of scFv fragments derived from corresponding antibody phage libraries into the expression vector. An expressed herbicide-specific scFv aP fusion protein retained both, analyte binding and enzymatic activity, as determined by ELISA. Therefore, this system permits the production of scFv-aP conjugates in E. coli, which can replace conventionally prepared aP-labeled antibodies in immunoassays. These fusion proteins are designed to accelerate the immunochemical detection of analytes, since the assay duration is essentially reduced by omitting the use of enzyme labeled secondary antibodies.     

Ovine dendritic cells transduced with an adenoviral CTLA4eEGFP fusion protein construct induce hyporesponsiveness to allostimulation

CTLA4 (CD152) is a transmembrane molecule expressed on activated T cells and functions as a negative regulator of T cell activation upon binding to the costimulatory molecules CD80/86. In this study, CTLA4eEGFP constructs were engineered by cloning the extracellular domains of ovine and human CTLA4 (CTLA4e) 'in frame' with the enhanced green fluorescent protein (EGFP). Recombinant adenoviral vectors were generated by incorporation of the CTLA4eEGFP sequence into the adenoviral genome using homologous recombination in Esherichia coli. The functional activity of the adenoviral vectors was shown by the secretion of the CTLA4eEGFP upon infection of ovine fibroblasts and the binding of the fusion protein to the target ovine and human dendritic cells expressing CD80/86 receptors by flow cytometry. The EGFP tag facilitated molecular size determinations and quantification of the secreted ovine CTLA4 fusion protein by immunoprecipitation and enzyme-linked immunosorbent assay (ELISA), respectively, using anti-GFP mAbs. Ovine dendritic cells obtained from pseudoafferent lymphatic cannulation of sheep were characterized based on high major histocompatibility complex (MHC) class II expression and cross-reactivity with monoclonal antibodies to the human dendritic cell markers, CD83 and CMRF-56. In addition, ovine dendritic cells (DC) were transfected with the adenoviral CTLA4eEGFP and when used as stimulators in a mixed lymphocyte reaction showed a reduced capacity to induce allogeneic lymphocyte proliferation. This study verifies that the ovine CTLA4eEGFP fusion protein functions similarly to its human homologue and that DC modified with adenoviral CTLA4-EGFP may provide an effective therapeutic approach in targeting alloreactive T cells to prolong allograft acceptance in a preclinical ovine model of renal transplantation.     

抗人CD3单链抗体/p53四聚功能域融合基因的构建及表达

目的：构建抗人CD3单链抗体(scFv)／p53四聚功能域融合基因，并进行真核表达及活性测定。方法：在已经构建抗人CD3 scFv和人IgG3上游铰链区／p53四聚功能域融合基因基础上，将抗人CD3 scFv克隆入载体pUC18／IgG3／p53中，构建抗人CD3 scFv／p53四聚功能域融合基因。经酶切鉴定及序列测定证实后，将融合基因克隆入真核表达载体pSecTag2-B中，并转染Hela细胞进行表达。表达产物纯化后，利用流式细胞仪进行活性测定。结果：获得了抗人CD3 scFv／p53四聚功能域融合基因，基因全长为882 bp，可编码294个氨基酸，与已发表的抗人CD3 scFv、人IgG3上游铰链区和人p53四聚功能域基因cDNA序列相一致。表达产物经SDS-PAGE和Western blot证实，为Mr约35000的特异蛋白条带。纯化后经流式细胞仪检测，可特异性地结合人外周血单个核细胞(PBMC)，亲和力高于scFv。结论：获得了可与PBMc特异性结合的抗人CD3 scFv四聚体，为进一步临床应用奠定了基础。     

抗人CD33单链抗体的基因构建、表达及其生物活性检测

目的：构建及表达抗人CD33单链抗体（抗CD33-scFv）基因，并检测其生物活性。方法：采用RT—PCR方法从分泌抗人类白细胞表面分化抗原CD33单克隆抗体（mAb）的杂交瘤细胞中克隆出VL和VH可变区基因，再通过重叠延伸拼接（splice—overlap extension）PCR方法在VH和VL可变区基因之间引入柔性连接肽（Gly4Ser）3，体外构建抗人CD33-scFv基因。将其克隆至原核表达载体PET-28a（＋）并在大肠杆菌Rosetta（DE3）中表达。结果：SDS-PAGE和Western blot分析结果表明，抗CD33-scFv在Rosetta（DE3）菌中获得高效表达，重组蛋白的相对分子质量（Mr）为30000，表达产物以不溶性包涵体形式存在，经过溶解包涵体，镍柱亲和层析纯化和体外复性过程，获得了高纯度的scFv片段。流式细胞术（FCM）分析结果证实抗CD33-scFv可与人类白细胞表面的分化抗原CD33结合，保留了鼠源性mAb的与CD33结合的活性。结论：重组抗人CD33-scFv基因构建与表达成功，并且通过复性得到有生物活性的scFv，为下一步针对髓系恶性肿瘤的靶向治疗奠定了基础。     

Modified HIV envelope proteins with enhanced binding to neutralizing monoclonal antibodies

The target for neutralizing antibodies against human immunodeficiency virus (HIV) is the trimeric Env protein on the native virion. Conserved neutralizing epitopes of receptor binding sites are located in the recessed core of the Env protein, partially masked by glycosylations and variable loops. In this study, we have investigated the effects of modifications of the HIV Env protein by glycosylation site mutations, deletions of variable loops, or combinations of both types of mutations on their protein functions and reactivities with neutralizing antibodies. Modified Env proteins were expressed in insect or mammalian cells, and their reactivity with epitope-specific broadly neutralizing monoclonal antibodies (Mabs) was determined by flow cytometry. A unique mutant designated 3G with mutations in three glycosylation motifs within the V3/C3 domains surrounding the CD4 binding site showed higher levels of binding to most broadly neutralizing Mabs (b12 and 2F5) in both insect and mammalian expression systems. Mutants with a deletion of both V1 and V2 loop domains or with a unique combination of both types of mutations also bound to most neutralizing Mabs at higher levels compared to the wild-type control. Most mutants maintained the ability to bind CD4 and to induce syncytium formation at similar or higher levels as compared to that of the wild-type Env protein, except for a mutant with a combination of variable loop deletions and deglycosylation mutations. Our study suggests that modified HIV Env proteins with reduced glycosylation in domains surrounding the CD4 binding site or variable loop-deleted mutants expose important neutralizing epitopes at higher levels than wild type and may provide novel vaccine immunogens.     

小鼠PDL1-Red2融合蛋白真核表达载体的构建、表达及其效应

目的:小鼠跨膜型PD-L1与红色荧光蛋白(RFP)融合基因真核表达载体的构建、表达与鉴定, 及其效应初步研究.方法:应用基因工程技术构建重组载体, 脂质体转染NIT细胞株, 以流式细胞术(FCM)和倒置相差荧光显微镜检测融合蛋白的表达;采用混合淋巴细胞培养, 以FCM测定脾细胞的增殖反应.结果:融合基因在转染的真核细胞中获得表达, 并呈膜分布, 转染PD-L1/pDsRed2-N1的细胞对淋巴细胞增殖反应有一定的抑制作用, 表明PD-L1/pDsRed2-N1融合蛋白中分子标签未影响PD-L1的结构及其生物学活性.结论:PD-L1与DsRed2融合基因载体构建成功, 表达的融合蛋白具有生物学活性.     

融合蛋白anti-HER2-ScFv-GFP在昆虫细胞中的表达及其靶向结合乳腺癌细胞的功能分析

目的:利用昆虫细胞-杆状病毒表达系统表达获得携带绿色荧光蛋白(GFP)的抗人表皮生长因子受体2(HER2)的单链抗体可变区片段(ScFv),分析其靶向结合乳腺癌细胞表面受体HER2的功能。方法: 构建融合基因anti-HER2-ScFv-GFP,重组获得真核表达载体重组子pFAST Bac-to-Bac HT A/anti-HER2-ScFv-GFP,转化DH10Bac,收集重组病毒bacmid,分别转染、感染粉纹夜蛾细胞Tn-5B1-4,SDS-PAGE及Western blotting分析融合蛋白表达产物。Ni²⁺-NTA 亲和层析法纯化anti-HER2-ScFv-GFP融合蛋白后分别滴入乳腺癌细胞HER2阳性的SKBR3和HER2阴性的MCF7,对比携带绿色荧光的抗HER2单链抗体靶向结合乳腺癌细胞表面HER2受体情况。结果: 获得长度约1 539 bp的融合基因anti-HER2-ScFv-GFP,感染重组病毒的Tn-5B1-4细胞膜附近有明显绿色荧光,SDS-PAGE、Western blotting法检测到60 kD的特异性条带。 结合实验显示HER2阳性的SKBR3细胞表面有明显绿色荧光,而HER2阴性的MCF7细胞表面荧光易被洗脱。结论: 在Tn-5B1-4中成功表达携带绿色荧光的融合蛋白anti-HER2-ScFv-GFP,该携带绿色荧光的抗HER2单链抗体具有靶向结合乳腺癌细胞表面HER2受体的效能。     

Systematic improvement of lentivirus transduction protocols by antibody fragments fused to VSV-G as envelope glycoprotein

Lentiviral vectors (LV) are widely used to successfully transduce cells for research and clinical applications. Lentiviral vectors pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) can be produced to high titers and mediate high transduction efficiencies in vitro. For clinical applications the need for optimized transduction protocols and the limited activity of retronectin as LV enhancer, results in the application of a high multiplicity of infection (MOI) to achieve effective transduction efficiencies for a number of therapeutically relevant cells, e.g. CD34⁺ hematopoietic stem cells, T- and B-cells. Our study describes an optimized LV infection protocol including a non-toxic poloxamer-based adjuvant combined with antibody-retargeted lentiviral particles, improving transduction efficiency at low MOI. Cell specificity of lentiviral vectors was increased by displaying different ratios of scFv-fused VSV-G glycoproteins on the viral envelope. The system was validated with difficult to transduce human CD30⁺ lymphoma cells, and EGFR⁺ tumor cells. Highly efficient transduction of lymphoma cells was achieved, >50% of cells were transduced when MOI 1 was used. The scFv displaying lentiviral particles gained relative specificity for transduction of target cells. Preferential gene delivery to CD30⁺ or EGFR⁺ cells was increased 4-fold in mixed cell cultures by presenting scFv antibody fragments binding to respective surface markers. A combination of spinoculation, poloxamer-based chemical adjuvant, and LV displaying scFv fragments increases transduction efficiencies of hard-to-transduce suspension lymphoma cells, and promises new chances for the future development of improved clinical protocols.