[Construction and screening of phage antibody libraries against epidermal growth factor receptor and soluble expression of single chain Fv].

PubMed

Sheng, Wei-Jin; Miao, Qing-Fang; Zhen, Yong-Su

2009-06-01

Recent studies have shown that epidermal growth factor receptor (EGFR) is an important target for cancer therapy. The present study prepared single chain Fv (scFv) directed against EGFR. Balb/c mice were immunized by human carcinoma A431 cells, and total RNA of the splenic cells was extracted. VH and VL gene fragments were amplified by RT-PCR and further joined into scFv gene with a linker, then scFv gene fragments were ligated into the phagemid vector pCANTAB 5E. The phagemid containing scFv were transformed into electro-competent E. coli TG1 cells. The recombinant phage antibody library was constructed through rescuing the transformed cells with help phage M13K07. The specified recombinant phages were enriched through 5 rounds of affinity panning and the anti-EGFR phage scFv clones were screened and identified with ELISA. A total of 48 clones from the library were selected randomly and 45 clones were identified positive. After infecting E. coli HB2151 cells with one positive clone, soluble recombinant antibodies about 27 kD were produced and located in the periplasm and the supernatant. The result of sequencing showed that the scFv gene was 768 bp, which encoded 256 amino acid residues. VH and VL including 3 CDRs and 4 FRs, respectively, were all homologous to mouse Ig. The soluble scFv showed the specific binding activity to purified EGFR and EGFR located in carcinoma cell membrane. The successful preparation of anti-EGFR scFv will provide an EGFR targeted molecule for the development of antibody-based drugs and biological therapy of cancer.

Development of single chain variable fragment (scFv) antibodies against surface proteins of ‘Ca. Liberibacter asiaticus’

USDA-ARS?s Scientific Manuscript database

‘Ca. Liberibacter asiaticus’ is the causal agent of citrus huanglongbing, the most serious disease of citrus worldwide. We have developed and applied immunization and affinity screening methods to develop a primary library of recombinant single chain variable fragment (scFv) antibodies in an M13 vec...

IG and TR single chain fragment variable (scFv) sequence analysis: a new advanced functionality of IMGT/V-QUEST and IMGT/HighV-QUEST.

PubMed

Giudicelli, Véronique; Duroux, Patrice; Kossida, Sofia; Lefranc, Marie-Paule

2017-06-26

IMGT®, the international ImMunoGeneTics information system® ( http://www.imgt.org ), was created in 1989 in Montpellier, France (CNRS and Montpellier University) to manage the huge and complex diversity of the antigen receptors, and is at the origin of immunoinformatics, a science at the interface between immunogenetics and bioinformatics. Immunoglobulins (IG) or antibodies and T cell receptors (TR) are managed and described in the IMGT® databases and tools at the level of receptor, chain and domain. The analysis of the IG and TR variable (V) domain rearranged nucleotide sequences is performed by IMGT/V-QUEST (online since 1997, 50 sequences per batch) and, for next generation sequencing (NGS), by IMGT/HighV-QUEST, the high throughput version of IMGT/V-QUEST (portal begun in 2010, 500,000 sequences per batch). In vitro combinatorial libraries of engineered antibody single chain Fragment variable (scFv) which mimic the in vivo natural diversity of the immune adaptive responses are extensively screened for the discovery of novel antigen binding specificities. However the analysis of NGS full length scFv (~850 bp) represents a challenge as they contain two V domains connected by a linker and there is no tool for the analysis of two V domains in a single chain. The functionality "Analyis of single chain Fragment variable (scFv)" has been implemented in IMGT/V-QUEST and, for NGS, in IMGT/HighV-QUEST for the analysis of the two V domains of IG and TR scFv. It proceeds in five steps: search for a first closest V-REGION, full characterization of the first V-(D)-J-REGION, then search for a second V-REGION and full characterization of the second V-(D)-J-REGION, and finally linker delimitation. For each sequence or NGS read, positions of the 5'V-DOMAIN, linker and 3'V-DOMAIN in the scFv are provided in the 'V-orientated' sense. Each V-DOMAIN is fully characterized (gene identification, sequence description, junction analysis, characterization of mutations and amino

Efficient production of soluble recombinant single chain Fv fragments by a Pseudomonas putida strain KT2440 cell factory.

PubMed

Dammeyer, Thorben; Steinwand, Miriam; Krüger, Sarah-C; Dübel, Stefan; Hust, Michael; Timmis, Kenneth N

2011-02-21

Recombinant antibody fragments have a wide range of applications in research, diagnostics and therapy. For many of these, small fragments like single chain fragment variables (scFv) function well and can be produced inexpensively in bacterial expression systems. Although Escherichia coli K-12 production systems are convenient, yields of different fragments, even those produced from codon-optimized expression systems, vary significantly. Where yields are inadequate, alternative production systems are needed. Pseudomonas putida strain KT2440 is a versatile biosafety strain known for good expression of heterologous genes, so we have explored its utility as a cell factory for production of scFvs. We have generated new broad host range scFv expression constructs and assessed their production in the Pseudomonas putida KT2440 host. Two scFvs bind either to human C-reactive protein or to mucin1, proteins of significant medical diagnostic and therapeutic interest, whereas a third is a model anti-lysozyme scFv. The KT2440 antibody expression systems produce scFvs targeted to the periplasmic space that were processed precisely and were easily recovered and purified by single-step or tandem affinity chromatography. The influence of promoter system, codon optimization for P. putida, and medium on scFv yield was examined. Yields of up to 3.5 mg/l of pure, soluble, active scFv fragments were obtained from shake flask cultures of constructs based on the original codon usage and expressed from the Ptac expression system, yields that were 2.5-4 times higher than those from equivalent cultures of an E. coli K-12 expression host. Pseudomonas putida KT2440 is a good cell factory for the production of scFvs, and the broad host range constructs we have produced allow yield assessment in a number of different expression hosts when yields in one initially selected are insufficient. High cell density cultivation and further optimization and refinement of the KT2440 cell factory will achieve

Bovine single chain Fv antibody inhibits bovine herpesvirus-1 infectivity by targeting viral glycoprotein D.

PubMed

Xu, Jian; Wu, Jing; Jiang, Bo; He, Houjun; Zhang, Xixi; Li, Xiaoyang; Yang, Dawei; Huang, Xiufen; Sealy, Joshua E; Iqbal, Munir; Li, Yongqing

2017-12-01

Glycoprotein D (gD) of bovine herpesvirus-1 (BoHV-1) is essential for attachment and penetration of cells during infection and is a major target for neutralizing antibodies during an adaptive immune response. Currently there are no recombinant antibodies capable of binding gD epitopes for use in treating BoHV-1 infection. In this study, a bovine scFv gene derived from a hybridoma secreting monoclonal antibodies (McAbs) against the amino acid motif MEESKGYEPP of gD was expressed in E. coli. Molecular modeling, western blot and ELISA analysis showed that this scFv had a high affinity for BoHV-1 gD, with a Kd of 161.2 ± 37.58 nM and for whole BoHV-1 virus, with a Kd of 67.44 ± 16.99 nM. In addition, this scFv displayed a high affinity for BoHV-1 antigen in an ELISA and competed with BoHV-1 anti-serum in a competitive ELISA. Immunofluorescence assay (IFA) and laser confocal microscopy showed that this scFv could efficiently bind to and be internalized by BoHV-1 infected Madin-Darby bovine kidney (MDBK) cells. Importantly, this scFv was shown to inhibit BoHV-1 infectivity and to reduce the number of viral plaques by blocking viral attachment to MDBK cells. Our study suggests that this bovine single-chain antibody could be developed for use as a diagnostic and therapeutic agent against BoHV-1 infection in cattle.

Bacterial Expression of a Single-Chain Variable Fragment (scFv) Antibody against Ganoderic Acid A: A Cost-Effective Approach for Quantitative Analysis Using the scFv-Based Enzyme-Linked Immunosorbent Assay.

PubMed

Yusakul, Gorawit; Nuntawong, Poomraphie; Sakamoto, Seiichi; Ratnatilaka Na Bhuket, Pahweenvaj; Kohno, Toshitaka; Kikkawa, Nao; Rojsitthisak, Pornchai; Shimizu, Kuniyoshi; Tanaka, Hiroyuki; Morimoto, Satoshi

2017-01-01

Due to the highly specific binding between an antibody and its target, superior analytical performances was obtained by immunoassays for phytochemical analysis over conventional chromatographic techniques. Here, we describe a simple method for producing a functional single-chain variable fragment (scFv) antibody against ganoderic acid A (GAA), a pharmacologically active metabolite from Ganoderma lingzhi. The Escherichia coli BL21(DE3) strain produced a large amount of anti-GAA scFv. However, in vitro refolding steps, which partially recovered the reactivity of the scFv, were required. Interestingly, the functional scFv was expressed as a soluble and active form in the cytoplasm of an engineered E. coli SHuffle ® strain. Purified anti-GAA scFv, which yielded 2.56 mg from 1 L of culture medium, was obtained from simple and inexpensive procedures for expression and purification. The anti-GAA scFv-based indirect competitive enzyme-linked immunosorbent assay (icELISA) exhibited high sensitivity (linearity: 0.078-1.25 µg/mL) with precision (CV: ≤6.20%) and reliability (recovery: 100.1-101.8%) for GAA determination. In summary, the approach described here is an inexpensive, simple, and efficient expression system that extends the application of anti-GAA scFv-based immunoassays. In addition, when in vitro refolding steps can be skipped, the cost and complexity of scFv antibody production can be minimized.

Engineering a Single Chain Fv Antibody to αvβ6 Integrin using the Specificity-Determining Loop of a Foot-and-Mouth Disease Virus

PubMed Central

Kogelberg, Heide; Tolner, Berend; Thomas, Gareth J.; Di Cara, Danielle; Minogue, Shane; Ramesh, Bala; Sodha, Serena; Marsh, Dan; Lowdell, Mark W.; Meyer, Tim; Begent, Richard H.J.; Hart, Ian; Marshall, John F; Chester, Kerry

2010-01-01

Summary The αvβ6 integrin is a promising target for cancer therapy. Its expression is up-regulated de novo on many types of carcinoma where it may activate transforming growth factor-β1 and transforming growth factor-β3, interact with the specific extracellular matrix proteins and promote migration and invasion of tumour cells. The viral protein 1 (VP1) coat protein of the O1 British field strain serotype of foot-and-mouth disease virus is a high-affinity ligand for αvβ6, and we recently reported that a peptide derived from VP1 exhibited αvβ6-specific binding in vitro and in vivo. We hypothesized that this peptide could confer binding specificity of an antibody to αvβ6. A 17-mer peptide of VP1 was inserted into the complementary-determining region H3 loop of MFE-23, a murine single-chain Fv (scFv) antibody reactive with carcinoembryonic antigen (CEA). The resultant scFv (B6-1) bound to αvβ6 but retained residual reactivity with CEA. This was eliminated by point mutation (Y100bP) in the variable heavy-chain domain to create an scFv (B6-2) that was as structurally stable as MFE-23 and reacted specifically with αvβ6 but not α5β1, αvβ3, αvβ5, αvβ8 or CEA. B6-2 was internalized into αvβ6-expressing cells and inhibited αvβ6-dependent migration of carcinoma cells. B6-2 was subsequently humanized. The humanized form (B6-3) was obtained as a non-covalent dimer from secretion in Pichia pastoris (115 mg/l) and was a potent inhibitor of αvβ6-mediated cell adhesion. Thus, we have used a rational stepwise approach to create a humanized scFv with therapeutic potential to block αvβ6-mediated cancer cell invasion or to deliver and internalize toxins specifically to αvβ6-expressing tumours. PMID:18656482

Single chain variable fragment displaying M13 phage library functionalized magnetic microsphere-based protein equalizer for human serum protein analysis.

PubMed

Zhu, Guijie; Zhao, Peng; Deng, Nan; Tao, Dingyin; Sun, Liangliang; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

2012-09-18

Single chain variable fragment (scFv) displaying the M13 phage library was covalently immobilized on magnetic microspheres and used as a protein equalizer for the treatment of human serum. First, scFv displaying M13 phage library functionalized magnetic microspheres (scFv@M13@MM) was incubated with a human serum sample. Second, captured proteins on scFv@M13@MM were eluted with 2 M NaCl, 50 mM glycine-hydrochloric acid (Gly-HCl), and 20% (v/v) acetonitrile with 0.5% (v/v) trifluoroacetic acid in sequence. Finally, the tightly bonded proteins were released by the treatment with thrombin. The eluates were first analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with silver staining. Results indicated that the difference of protein concentration was reduced obviously in NaCl and Gly-HCl fractions compared with untreated human serum sample. The eluates were also digested with trypsin, followed by online 2D-strong cation exchange (SCX)-RPLC-ESI-MS/MS analysis. Results demonstrated that the number of proteins identified from an scFv@M13@MM treated human serum sample was improved 100% compared with that from the untreated sample. In addition, the spectral count of 10 high abundance proteins (serum albumin, serotransferrin, α-2-macroglobulin, α-1-antitrypsin, apolipoprotein B-100, Ig γ-2 chain C region, haptoglobin, hemopexin, α-1-acid glycoprotein 1, and α-2-HS-glycoprotein) decreased evidently after scFv@M13@MM treatment. All these results demonstrate that scFv@M13@MM could efficiently remove high-abundance proteins, reduce the protein concentration difference of human serum, and result in more protein identification.

Development of a Novel Human Single Chain Antibody Against EGFRVIII Antigen by Phage Display Technology.

PubMed

Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Akbari, Bahman; Ahdi Khosroshahi, Shiva

2016-12-01

Purpose: EGFRvIII as the most common mutant variant of the epidermal growth factor receptor is resulting from deletion of exons 2-7 in the coding sequence and junction of exons 1 and 8 through a novel glycine residue. EGFRvIII is highly expressed in glioblastoma, carcinoma of the breast, ovary, and lung but not in normal cells. The aim of the present study was identification of a novel single chain antibody against EGFRvIII as a promising target for cancer therapy. Methods: In this study, a synthetic peptide corresponding to EGFRvIII protein was used for screening a naive human scFv phage library. A novel five-round selection strategy was used for enrichment of rare specific clones. Results: After five rounds of screening, six positive scFv clones against EGFRvIII were selected using monoclonal phage ELISA, among them, only three clones had expected size in PCR reaction. The specific interaction of two of the scFv clones with EGFRvIII was confirmed by indirect ELISA. One phage clone with higher affinity in scFv ELISA was purified for further analysis. The purity of the produced scFv antibody was confirmed using SDS-PAGE and Western blotting analyses. Conclusion: In the present study, a human anti- EGFRvIII scFv with high affinity was first identified from a scFv phage library. This study can be the groundwork for developing more effective diagnostic and therapeutic agents against EGFRvIII expressing cancers.

Method for preparation of single chain antibodies

DOEpatents

Cheung, Nai-Kong V [New York, NY; Guo, Hong-fen [New York, NY

2012-04-03

This invention provides a method for identifying cells expressing a target single chain antibody (scFv) directed against a target antigen from a collection of cells that includes cells that do not express the target scFv, comprising the step of combining the collection of cells with an anti-idiotype directed to an antibody specific for the target antigen and detecting interaction, if any, of the anti-idiotype with the cells, wherein the occurrence of an interaction identifies the cell as one which expresses the target scFv. This invention also provides a method for making a single chain antibody (scFv) directed against an antigen, wherein the selection of clones is made based upon interaction of those clones with an appropriate anti-idiotype, and heretofore inaccessible scFv so made. This invention provides the above methods or any combination thereof. Finally, this invention provides various uses of these methods.

Production of recombinant scFv against p24 of human immunodeficiency virus type 1 by phage display technology.

PubMed

Mohammadzadeh, Sara; Rajabibazl, Masoumeh; Fourozandeh, Mehdi; Rasaee, Mohammad Javad; Rahbarizadeh, Fatemeh; Mohammadi, Mohammad

2014-02-01

Phage display has a fundamental role in protein isolation and engineering. Isolated proteins produced with this method can be modified for specific binding and affinity. P24 is the most produced protein during human immune deficiency virus (HIV) replication; especially in the early steps of HIV-1 infection, its evaluation may have diagnostic values. To test the HIV-1 infection, p24 antigen assay appears to be a very promising alternative to RNA assays. In this study, we have generated a recombinant mouse single chain antibody fragment against p24 of the HIV-1 with the use of phage display technology. After isolation of antibody variable-region (V) gene of B cells extracted from the spleen of an immunized mouse, a library of single chain Fv fragments (scFv) was constructed. The library was used in a series of bio-panning processes against recombinant p24 protein expressed from Escherichia coli. The isolated scFv antibody specifically recognizes the HIV-1 capsid protein p24. The affinity constant of the isolated scFv antibody (MF85) was found to be 2×10(-9) M. Our studies showed that the MF85 scFV antibody has similar properties as that of monoclonal antibodies produced by the hybridoma technology.

Development of sugar chain-binding single-chain variable fragment antibody to adult T-cell leukemia cells using glyco-nanotechnology and phage display method.

PubMed

Muchima, Kaname; Todaka, Taro; Shinchi, Hiroyuki; Sato, Ayaka; Tazoe, Arisa; Aramaki, Rikiya; Kakitsubata, Yuhei; Yokoyama, Risa; Arima, Naomichi; Baba, Masanori; Wakao, Masahiro; Ito, Yuji; Suda, Yasuo

2018-04-01

Adult T-cell leukemia (ATL) is an intractable blood cancer caused by the infection of human T-cell leukemia virus type-1, and effective medical treatment is required. It is known that the structure and expression levels of cell surface sugar chains vary depending on cell states such as inflammation and cancer. Thus, it is expected that the antibody specific for ATL cell surface sugar chain would be an effective diagnostic tool and a strong candidate for the development of an anti-ATL drug. Here, we developed a stable sugar chain-binding single-chain variable fragment antibody (scFv) that can bind to ATL cells using a fibre-type Sugar Chip and phage display method. The fiber-type Sugar Chips were prepared using O-glycans released from ATL cell lines. The scFv-displaying phages derived from human B cells (diversity: 1.04 × 108) were then screened using the fiber-type Sugar Chips, and an O-glycan-binding scFv was obtained. The flow cytometry analysis revealed that the scFv predominantly bound to ATL cell lines. The sugar chain-binding properties of the scFv was evaluated by array-type Sugar Chip immobilized with a library of synthetic glycosaminoglycan disaccharide structures. Highly sulphated disaccharide structures were found to have high affinity to scFv.

Isolation and characterization of a novel human scFv inhibiting EGFR vIII expressing cancers.

PubMed

Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Dariushnejad, Hassan; Hosseini, Mohammad Kazem

2016-12-01

EGFRvIII, a mutant form of epidermal growth factor receptor is highly expressed in glioblastoma, carcinoma of the breast, ovary, and lung but not in normal cells. This tumor specific antigen has emerged as a promising candidate for antibody based therapy of several cancers. The aim of the present study was isolation and characterization of a human single chain antibody against EGFRvIII as a promising target for cancer therapy. For this, a synthetic peptide corresponding to EGFRvIII protein was used for screening the naive human scFv phage library. Selection was performed using a novel screening strategy for enrichment of rare specific clones. After five rounds of screening, six positive scFv clones against EGFRvIII were selected using monoclonal phage ELISA, among them, a clone with an amber mutation in VH CDR2 coding sequence showed higher reactivity. The mutation was corrected through site directed mutagenesis and then scFv fragment was expressed after subcloning into the bacterial expression vector. Expression in BL21 pLysS resulted in a highly soluble scFv appeared in soluble fraction of E. coli lysate. Bioinformatic in silico analysis between scFv and EGFRvIII sequences confirmed specific binding of desired scFv to EGFRvIII in CDR regions. The specific reactivity of the purified scFv with native EGFRvIII was confirmed by cell based ELISA and western blot. In conclusion, human anti- EGFRvIII scFv isolated from a scFv phage library displayed high reactivity with EGFRvIII. The scFv isolated in this study can be the groundwork for developing more effective diagnostic and therapeutic agents against EGFRvIII expressing cancers. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Novel avian single-chain fragment variable (scFv) targets dietary gluten and related natural grain prolamins, toxic entities of celiac disease.

PubMed

Stadlmann, Valerie; Harant, Hanna; Korschineck, Irina; Hermann, Marcela; Forster, Florian; Missbichler, Albert

2015-12-01

Celiac disease (CD) is a chronic, small intestinal inflammatory disease mediated by dietary gluten and related prolamins. The only current therapeutic option is maintenance of a strict life-long gluten-free diet, which implies substantial burden for CD patients. Different treatment regimes might be feasible, including masking of toxic celiac peptides with blocking antibodies or fragments thereof. The objective of this study was therefore to select and produce a recombinant avian single-chain fragment variable (scFv) directed against peptic-tryptic digested gliadin (PT-Gliadin) and related celiac toxic entities. Gluten-free raised chicken of same age were immunized with PT-Gliadin. Chicken splenic lymphocytes, selected with antigen-coated magnetic beads, served as RNA source for the generation of cDNA. Chicken VH and VL genes were amplified from the cDNA by PCR to generate full-length scFv constructs consisting of VH and VL fragments joined by a linker sequence. ScFv constructs were ligated in a prokaryotic expression vector, which provides a C-terminal hexahistidine tag. ScFvs from several bacterial clones were expressed in soluble form and crude cell lysates screened for binding to PT-Gliadin by ELISA. We identified an enriched scFv motif, which showed reactivity to PT-Gliadin. One selected scFv candidate was expressed and purified to homogeneity. Polyclonal anti-PT-Gliadin IgY, purified from egg yolk of immunized chicken, served as control. ScFv binds in a dose-dependent manner to PT-Gliadin, comparable to IgY. Furthermore, IgY competitively displaces scFv from PT-Gliadin and natural wheat flour digest, indicating a common epitope of scFv and IgY. ScFv was tested for reactivity to different gastric digested dietary grain flours. ScFv detects common and khorasan wheat comparably with binding affinities in the high nanomolar range, while rye is detected to a lesser extent. Notably, barley and cereals which are part of the gluten-free diet, like corn and rice, are

Production of a germline-humanized cetuximab scFv and evaluation of its activity in recognizing EGFR- overexpressing cancer cells.

PubMed

Banisadr, Arsham; Safdari, Yaghoub; Kianmehr, Anvarsadat; Pourafshar, Mahdieh

2018-04-03

The aim of this study was to produce a humanized single chain antibody (scFv) as a potential improved product design to target EGFR (Epidermal Growth Factor Receptor) overexpressing cancer cells. To this end, CDR loops of cetuximab (an FDA-approved anti-EGFR antibody) were grafted on framework regions derived from type 3 (VH3 and VL3 kappa) human germline sequences to obtain recombinant VH and VL domainslinked together with a flexible linker [(Gly 4 Ser) 3 ] to form a scFv. Codon optimized synthetic gene encoding the scFv (with NH2-VH-linker-VL-COOH orientation) was expressed in E. coli Origami™ 2(DE3) cells and the resultant scFv purified by using Ni-NTA affinity chromatography. The scFv, called cet.Hum scFv, was evaluated in ELISA and immunoblot to determine whether it can recognize EGFR. The scFv was able to recognize EGFR over-expressing cancer cells (A-431) but failed to detect cancer cells with low levels of EGFR (MCF-7 cells). Although the affinity of the scFv forA-431 cells was 9 fold lower than that of cetuximab, it was strong enough to recognize these cells. Considering its ability to bind EGFR molecules, the scFv may exhibit a potential application for the detection of EGFR-overexpressing cancer cells.

Efficient refolding and immobilization of PMMA-tag-fused single-chain Fv antibodies for sensitive immunological detection on a PMMA plate.

PubMed

Kumada, Yoichi; Ishikawa, Yasuyuki; Fujiwara, Yusuke; Takeda, Rui; Miyamoto, Ryosuke; Niwa, Daisuke; Momose, Shun; Kang, Bongmun; Kishimoto, Michimasa

2014-09-01

In this study, we investigated the efficient refolding and site-specific immobilization of single-chain variable fragments (scFvs) genetically fused with a poly(methylmethacrylate)-binding peptide (PMMA-tag). According to the results of an aggregation test of a scFv-PM in the presence of 0.5 M urea, aggregation was hardly detectable at a weak-alkaline pH (8.5) with lower concentrations of NaCl. Consequently, more than 93% recovery of the anti-RNase scFv-PM model was attained, when it was refolded by dialysis against 50 mM TAPS (pH8.5). These results suggested that the apparent isoelectric point (pI) of a target scFv was decreased to a great extent by the genetic fusion of a PMMA-tag containing 5 acidic amino acids, and, thus, the solubility of the scFv-PM in its semi-denatured form was considerably improved. We also designed alternative peptide-tags composed of plural aspartic acid residues (D5, D10 and D15-tags) to decrease the apparent pI value of the fusion protein. As a consequence, scFv-D5, scFv-D10 and scFv-D15 were also efficiently refolded with yields of more than 95%. It is noteworthy that even scFv-PS-D15, which had both a positively charged polystyrene-binding peptide (PS-tag) and a negatively charged D15-tag, was serially connected at the C-terminal region of scFvs, and also refolded with a yield of 96.1%. These results clearly indicate that controlling the apparent pI value of scFvs by the fusion of oligo-peptides composed of acidic amino acids at the C-terminus resulted in a high degree of recovery via dialysis refolding. According to the results of a sandwich ELISA using scFv-PMs, scFv-D15 and scFv-PS-D15 as ligands, high antigen-binding signals were detected from both the PMMA and phi-PS plates immobilized with scFv-PMs. Furthermore, the high antigen-binding activity of scFv-PMs was maintained in an adsorption state when it was immobilized on the surface of not only PMMA, but also hydrophilic PS (phi-PS) and polycarbonate (PC). These results

Development of single chain variable fragment (scFv) antibodies against Xylella fastidiosa subsp. pauca by phage display.

PubMed

Yuan, Qing; Jordan, Ramon; Brlansky, Ronald H; Istomina, Olga; Hartung, John

2015-10-01

Xylella fastidiosa is a member of the gamma proteobacteria. It is fastidious, insect-vectored and xylem-limited and causes a variety of diseases, some severe, on a wide range of economically important perennial crops, including grape and citrus. Antibody based detection assays are commercially available for X. fastidiosa, and are effective at the species, but not at the subspecies level. We have made a library of scFv antibody fragments directed against X. fastidiosa subsp. pauca strain 9a5c (citrus) by using phage display technology. Antibody gene repertoires were PCR-amplified using 23 primers for the heavy chain variable region (V(H)) and 21 primers for the light chain variable region (V(L)). The V(H) and V(L) were joined by overlap extension PCR, and then the genes of the scFv library were ligated into the phage vector pKM19. The library contained 1.2×10(7) independent clones with full-length scFv inserts. In each of 3cycles of affinity-selection with 9a5c, about 1.0×10(12) phage were used for panning with 4.1×10(6), 7.1×10(6), 2.1×10(7) phage recovered after the first, second and third cycles, respectively. Sixty-six percent of clones from the final library bound X. fastidiosa 9a5c in an ELISA. Some of these scFv antibodies recognized strain 9a5c and did not recognize X. fastidiosa strains that cause Pierce's disease of grapevine. Published by Elsevier B.V.

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment.

PubMed

Austerberry, James I; Dajani, Rana; Panova, Stanislava; Roberts, Dorota; Golovanov, Alexander P; Pluen, Alain; van der Walle, Christopher F; Uddin, Shahid; Warwicker, Jim; Derrick, Jeremy P; Curtis, Robin

2017-06-01

The aggregation propensities for a series of single-chain variable fragment (scFv) mutant proteins containing supercharged sequences, salt bridges and lysine/arginine-enriched motifs were characterised as a function of pH and ionic strength to isolate the electrostatic contributions. Recent improvements in aggregation predictors rely on using knowledge of native-state protein-protein interactions. Consistent with previous findings, electrostatic contributions to native protein-protein interactions correlate with aggregate growth pathway and rates. However, strong reversible self-association observed for selected mutants under native conditions did not correlate with aggregate growth, indicating 'sticky' surfaces that are exposed in the native monomeric state are inaccessible when aggregates grow. We find that even though similar native-state protein-protein interactions occur for the arginine and lysine-enriched mutants, aggregation propensity is increased for the former and decreased for the latter, providing evidence that lysine suppresses interactions between partially folded states under these conditions. The supercharged mutants follow the behaviour observed for basic proteins under acidic conditions; where excess net charge decreases conformational stability and increases nucleation rates, but conversely reduces aggregate growth rates due to increased intermolecular electrostatic repulsion. The results highlight the limitations of using conformational stability and native-state protein-protein interactions as predictors for aggregation propensity and provide guidance on how to engineer stabilizing charged mutations. Copyright © 2017. Published by Elsevier B.V.

Methods of preparing and using single chain anti-tumor antibodies

DOEpatents

Cheung, Nai-Kong; Guo, Hong-Fen

2010-02-23

This invention provides a method for identifying cells expressing a target single chain antibody (scFv) directed against a target antigen from a collection of cells that includes cells that do not express the target scFv, comprising the step of combining the collection of cells with an anti-idiotype directed to an antibody specific for the target antigen and detecting interaction, if any, of the anti-idiotype with the cells, wherein the occurrence of an interaction identifies the cell as one which expresses the target scFv. This invention also provides a method for making a single chain antibody (scFv) directed against an antigen, wherein the selection of clones is made based upon interaction of those clones with an appropriate anti-idiotype, and heretofore inaccessible scFv so made. This invention provides the above methods or any combination thereof. Finally, this invention provides various uses of these methods.

Blocking monocyte transmigration in in vitro system by an anti-CD99 human antibody in single chain fragment variable (scFv) format. Efficient large scale purification of biological active scFv from inclusion bodies in E. coli expression system

PubMed Central

Moricoli, Diego; Muller, William A.; Carbonella, Damiano Cosimo; Balducci, Maria Cristina; Dominici, Sabrina; Fiori, Valentina; Watson, Richard; Weber, Evan; Cianfriglia, Maurizio; Scotlandi, Katia; Magnani, Mauro

2015-01-01

Migration of leukocytes into a site of inflammation involves several steps mediated by various families of adhesion molecules. CD99 play a significant role in transendothelial migration (TEM) of leukocytes. Inhibition of TEM by specific monoclonal antibody (mAb) can provide a potent therapeutic approach to treating inflammatory conditions. However, the therapeutic utilization of whole IgG can lead to an inappropriate activation of Fc receptor-expressing cells inducing serious adverse side effects due to cytokine release. In this regard, specific recombinant antibody in single chain variable fragments (scFvs) originated by phage library may offer a solution by affecting TEM function in a safe clinical context. However, this consideration requires large scale production of functional scFv antibodies under GMP conditions and hence, the absence of toxic reagents utilized for the solubilization and refolding steps of inclusion bodies that may discourage industrial application of these antibody fragments. In order to apply the scFv anti-CD99 named C7A in a clinical setting we herein describe an efficient and large scale production of the antibody fragments expressed in E.coli as insoluble protein avoiding gel filtration chromatography approach, and laborious refolding step pre- and post-purification. Using differential salt elution which is a simple, reproducible and effective procedure we are able to separate scFv in monomer format from aggregates. The purified scFv antibody C7A exhibits inhibitory activity comparable to an antagonistic conventional mAb, thus providing an excellent agent for blocking CD99 signalling. Thanks to the original purification protocol that can be extended to other scFvs that are expressed as inclusion bodies in bacterial systems, the scFv anti-CD99 C7A herein described represents the first step towards the construction of new antibody therapeutic. PMID:24798881

Engineered Recombinant Single-Chain Fragment Variable Antibody for Immunosensors

PubMed Central

Shen, Zhihong; Mernaugh, Raymond L.; Yan, Heping; Yu, Lei; Zhang, Ying; Zeng, Xiangqun

2008-01-01

A recombinant single-chain fragment variable (scFv) antibody (designated A10B) was engineered to contain two histidines within the linker peptide used to join the scFv heavy and light chains. A piezoimmunosensor using the scFv was successfully developed. A10B scFv bound to the gold piezoimmunosensor surface were correctly oriented, retained antigen-binding activity, and coupled at high surface concentration. These results, and results obtained from an earlier study using an scFv containing a linker cysteine, suggest that the location on the linker sequence in which the amino acids were incorporated was well tolerated by the scFv and did not interfere with scFv antigen-binding activity. The scFv-modified QCM sensor was thoroughly characterized and used to specifically detect antigen in crude serum sample and had a sensitivity of 2.3 ± 0.15 nM (n = 4) with a linear range over 2.3 × 10−9–3.3 × 10−8 M. The piezoimmunosensor was also used to study the kinetics and thermodynamics of antigen/scFv antibody binding. PMID:16255580

A conjugate of an anti-midkine single-chain variable fragment to doxorubicin inhibits tumor growth

PubMed Central

Zhao, Shuli; Zhao, Guangfeng; Xie, Hao; Huang, Yahong; Hou, Yayi

2012-01-01

Doxorubicin (DOX) was conjugated to a single-chain variable fragment (scFv) against human midkine (MK), and the conjugate (scFv-DOX) was used to target the chemotherapeutic agent to a mouse solid tumor model in which the tumor cells expressed high levels of human MK. The His-tagged recombinant scFv was expressed in bacteria, purified by metal affinity chromatography, and then conjugated to DOX using oxidative dextran (Dex) as a linker. The molecular formula of this immunoconjugate was scFv(Dex)1.3(DOX)20. In vitro apoptosis assays showed that the scFv-DOX conjugate was more cytotoxic against MK-transfected human adenocarcinoma cells (BGC823-MK) than untransfected cells (55.3 ± 2.4 vs 22.4 ± 3.8%) for three independent experiments. Nude mice bearing BGC823-MK solid tumors received scFv-DOX or equivalent doses of scFv + DOX for 2 weeks and tumor growth was more effectively inhibited by the scFv-DOX conjugate than by scFv + DOX (51.83% inhibition vs 40.81%). Histological analysis of the tumor tissues revealed that the highest levels of DOX accumulated in tumors from mice treated with scFv-DOX and this resulted in more extensive tumor cell death than in animals treated with the equivalent dose of scFv + DOX. These results show that the scFv-DOX conjugate effectively inhibited tumor growth in vivo and suggest that antigen-specific scFv may be competent drug-carriers. PMID:22267001

Symmetry of Fv architecture is conducive to grafting a second antibody binding site in the Fv region.

PubMed Central

Keck, P C; Huston, J S

1996-01-01

Molecular modeling studies on antibody Fv regions have been pursued to design a second antigen-binding site (chi-site) in a chimeric single-chain Fv (chi sFv) species of about 30 kDa. This analysis has uncovered an architectural basis common to many Fv regions that permits grafting a chi-site onto the Fv surface that diametrically opposes the normal combining site. By using molecular graphics analysis, chimeric complementarity-determining regions (chi CDRs) were defined that comprised most of the CDRs from an antibody binding site of interest. The chain directionality of chi CDRs was consistent with that of specific bottom loops of the sFv, which allowed for grafting of chi CDRs with an overall geometry approximating CDRs in the parent combining site. Analysis of 10 different Fv crystal structures indicates that the positions for inserting chi CDRs are very highly conserved, as are the corresponding chi CDR boundaries in the parent binding site. The results of this investigation suggest that it should be possible to generally apply this approach to the development of chimeric bispecific antibody binding site (chi BABS) proteins. Images FIGURE 2 FIGURE 3 PMID:8889174

An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification

PubMed Central

Sotelo, Pablo H.; Collazo, Noberto; Zuñiga, Roberto; Gutiérrez-González, Matías; Catalán, Diego; Ribeiro, Carolina Hager; Aguillón, Juan Carlos; Molina, María Carmen

2012-01-01

Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene. In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles. PMID:22692130

Single-chain antibody-delivered Livin siRNA inhibits human malignant melanoma growth in vitro and in vivo.

PubMed

Wang, Hao; Yang, Yifei; Wang, Wei; Guan, Bing; Xun, Meng; Zhang, Hai; Wang, Ziling; Zhao, Yong

2017-05-01

Although gene therapy has brought new insights into the treatment of malignant melanoma, targeting delivery of nucleic acid which targets critical oncogene/anti-oncogene in vivo is still a bottleneck in the therapeutic application. Our previous in vitro studies have found that the oncogene Livin could serve as a potential molecular target by small interfering RNA for gene therapy of malignant melanoma. However, how to transport Livin small interfering RNA into malignant melanoma cells specifically and efficiently in vivo needs further investigation. Cumulative evidence has suggested that single-chain antibody-mediated small interfering RNA targeted delivery is an effective way to silence specific genes in human cancer cells. Indeed, this study designed a protamine-single-chain antibody fusion protein, anti-MM scFv-tP, to deliver Livin small interfering RNA into LiBr cells. Further experiments confirmed the induction of cell apoptosis and suppression of cell proliferation by anti-MM scFv-tP in LiBr cells, along with efficient silence of Livin gene both in vitro and in vivo. Altogether, our findings provide a feasible approach to transport Livin small interfering RNA to malignant melanoma cells which would be a new therapeutic strategy for combating malignant melanoma.

Genetic modification of mesenchymal stem cells to express a single-chain antibody against EGFRvIII on the cell surface.

PubMed

Balyasnikova, Irina V; Franco-Gou, Rosa; Mathis, J Michael; Lesniak, Maciej S

2010-06-01

Human adult mesenchymal stem cells (hMSCs) are under active investigation as cellular carriers for gene therapy. hMSCs possess natural tropism toward tumours; however, the targeting of hMSCs to specific cell populations within tumours is unexplored. In the case of glioblastoma multiforme (GBM), at least half of the tumours express EGFRvIII on the cell surface, an ideal target for antibody-mediated gene/drug delivery. In this study, we investigated the feasibility of genetically modifying hMSCs to express a single-chain antibody (scFv) to EGFRvIII on their surfaces. Nucleofection was used to transfect hMSCs with cDNA encoding scFv EGFRvIII fused with PDGFR or human B7-1 transmembrane domains. The expression of scFv EGFRvIII on the cell surface was assessed by FACS. A stable population of scFv EGFRvIII-expressing hMSCs was selected, based on antibiotic resistance, and enriched using FACS. We found that nucleofection allows the efficient expression of scFv EGFRvIII on the cell surface of hMSCs. hMSCs transfected with the construct encoding scFv EGFRvIII as a fusion with PDGFRtm showed scFv EGFRvIII expression in up to 86% of cells. Most importantly, human MSCs expressing scFv against EGFRvIII demonstrated enhanced binding to U87-EGFRvIII cells in vitro and significantly increased retention in human U87-EGFRvIII-expressing tumours in vivo. In summary, we provide the first conclusive evidence of genetic modification of hMSCs with a single-chain antibody against an antigen expressed on the surface of tumour cells, thereby opening up a new venue for enhanced delivery of gene therapy applications in the context of malignant brain cancer. Copyright 2009 John Wiley & Sons, Ltd.

Heterodimeric bispecific single chain variable fragments (scFv) killer engagers (BiKEs) enhance NK-cell activity against CD133+ colorectal cancer cells

PubMed Central

JU, Schmohl; MK, Gleason; PR, Dougherty; JS, Miller; DA, Vallera

2015-01-01

Background Natural killer (NK) cells are potent cytotoxic lymphocytes that play a critical role in tumor immunosurveillance and control. Cancer stem cells (CSC) initiate and sustain tumor cell growth, mediate drug refractory cancer relapse and express the well-known surface marker CD133. Methods DNA fragments from two fully humanized single chain fragment variable (scFv) antibody recognizing CD16 on NK-cells and CD133 on CSC were genetically spliced forming a novel drug, 16 × 133 BiKE that simultaneously recognizes these antigen to facilitate an immunologic synapse. The anti-CD133 was created using a fusion protein prepared by fusing DNA fragments encoding the two extracellular domains of CD133. Immunization of mice with the resulting fusion protein generated an unique antibody that recognized the molecular framework and was species cross-reactive. Results In vitro 51chromium release cytotoxicity assays at both high and low effector:target ratios demonstrated the ability of the heterodimeric biological drug to greatly enhance NK-cell killing of human Caco-2 colorectal carcinoma cells known to overexpress CD133. The tumor associated antigen specificity of the drug for CD133 even enhanced NK-cell cytotoxicity against the NK-resistant human Burkitt's lymphoma Daudi cell line, which has less than 5% CD133 surface expression. Flow cytometry analysis revealed increases in NK-cell degranulation and Interferon-γ production upon co-culture with Caco-2 targets in the presence of the drug. Conclusion These studies demonstrate that the innate immune system can be effectively recruited to kill CSC using bispecific antibodies targeting CD133, and that this anti-CD133 scFv may be useful in this bispecific platform or, perhaps, in the design of more complex trispecific molecules for carcinoma therapy. PMID:26566946

Anti-CTGF single-chain variable fragment dimers inhibit human airway smooth muscle (ASM) cell proliferation by down-regulating p-Akt and p-mTOR levels.

PubMed

Gao, Wei; Cai, Liting; Xu, Xudong; Fan, Juxiang; Xue, Xiulei; Yan, Xuejiao; Qu, Qinrong; Wang, Xihua; Zhang, Chen; Wu, Guoqiu

2014-01-01

Connective tissue growth factor (CTGF) contributes to airway smooth muscle (ASM) cell hyperplasia in asthma. Humanized single-chain variable fragment antibody (scFv) was well characterized as a CTGF antagonist in the differentiation of fibroblast into myofibroblast and pulmonary fibrosis in our previous studies. To further improve the bioactivity of scFv, we constructed a plasmid to express scFv-linker-matrilin-6×His fusion proteins that could self-assemble into the scFv dimers by disulfide bonds in matrilin under non-reducing conditions. An immunoreactivity assay demonstrated that the scFv dimer could highly bind to CTGF in a concentration-dependent manner. The MTT and EdU assay results revealed that CTGF (≥10 ng/mL) promoted the proliferation of ASM cells, and this effect was inhibited when the cells were treated with anti-CTGF scFv dimer. The western blot analysis results showed that increased phosphorylation of Akt and mTOR induced by CTGF could be suppressed by this scFv dimer. Based on these findings, anti-CTGF scFv dimer may be a potential agent for the prevention of airway remodeling in asthma.

Anti-CTGF Single-Chain Variable Fragment Dimers Inhibit Human Airway Smooth Muscle (ASM) Cell Proliferation by Down-Regulating p-Akt and p-mTOR Levels

PubMed Central

Xu, Xudong; Fan, Juxiang; Xue, Xiulei; Yan, Xuejiao; Qu, Qinrong; Wang, Xihua; Zhang, Chen; Wu, Guoqiu

2014-01-01

Connective tissue growth factor (CTGF) contributes to airway smooth muscle (ASM) cell hyperplasia in asthma. Humanized single-chain variable fragment antibody (scFv) was well characterized as a CTGF antagonist in the differentiation of fibroblast into myofibroblast and pulmonary fibrosis in our previous studies. To further improve the bioactivity of scFv, we constructed a plasmid to express scFv-linker-matrilin-6×His fusion proteins that could self-assemble into the scFv dimers by disulfide bonds in matrilin under non-reducing conditions. An immunoreactivity assay demonstrated that the scFv dimer could highly bind to CTGF in a concentration-dependent manner. The MTT and EdU assay results revealed that CTGF (≥10 ng/mL) promoted the proliferation of ASM cells, and this effect was inhibited when the cells were treated with anti-CTGF scFv dimer. The western blot analysis results showed that increased phosphorylation of Akt and mTOR induced by CTGF could be suppressed by this scFv dimer. Based on these findings, anti-CTGF scFv dimer may be a potential agent for the prevention of airway remodeling in asthma. PMID:25478966

Gladiolus plants transformed with single-chain variable fragment antibodies to Cucumber mosaic virus

USDA-ARS?s Scientific Manuscript database

Transgenic plants of Gladiolus ‘Peter Pears’ or ‘Jenny Lee’ were developed that contain single-chain variable fragments (scFv) to Cucumber mosaic virus (CMV) subgroup I or II. The CMV subgroup I heavy and light chain scFv fragments were placed under control of either the duplicated CaMV 35S or suga...

A novel anti-alpha-fetoprotein single-chain variable fragment displays anti-tumor effects in HepG2 cells as a single agent or in combination with paclitaxel.

PubMed

Ji, Xiaonan; Shen, Yanli; Sun, Hao; Gao, Xiangdong

2016-08-01

Human hepatocellular carcinoma (HCC) has a high rate of tumor recurrence and metastasis, resulting in shortened survival time. The function of alpha-fetoprotein (AFP) as a regulatory factor in the growth of HCC cells has been well defined. The aim of this study was to investigate the use of a novel AFP-specific single-chain variable fragment that blocked AFP and inhibited HCC cell growth. The results indicated that the anti-AFP single-chain variable fragment (scFv) induced growth inhibition of AFP-expressing HCC cell lines in vitro through induction of G1 cell cycle arrest and apoptosis. The mechanism of apoptosis probably involved with blocking AFP internalization and regulation of the PTEN/PI3K/Akt signaling network. Moreover, the anti-AFP-scFv also effectively sensitized the HepG2 cells to paclitaxel (PTX) at a lower concentration. The combination effect of PTX and anti-AFP-scFv displayed a synergistic effect on HepG2 cells both in vitro and in vivo. Our results demonstrated that targeting AFP by specific antibodies has potential immunotherapeutic efficacy in human HCC.

Peptide docking of HIV-1 p24 with single chain fragment variable (scFv) by CDOCKER algorithm

NASA Astrophysics Data System (ADS)

Karim, Hana Atiqah Abdul; Tayapiwatana, Chatchai; Nimmanpipug, Piyarat; Zain, Sharifuddin M.; Rahman, Noorsaadah Abdul; Lee, Vannajan Sanghiran

2014-10-01

In search for the important residues that might have involve in the binding interaction between the p24 caspid protein of HIV-1 fragment (MET68 - PRO90) with the single chain fragment variable (scFv) of FAB23.5, modern computational chemistry approach has been conducted and applied. The p24 fragment was initially taken out from the 1AFV protein molecule consisting of both light (VL) and heavy (VH) chains of FAB23.5 as well as the HIV-1 caspid protein. From there, the p24 (antigen) fragment was made to dock back into the protein pocket receptor (antibody) by using the CDOCKER algorithm to conduct the molecular docking process. The score calculated from the CDOCKER gave 15 possible docked poses with various docked ligand's positions, the interaction energy as well as the binding energy. The best docked pose that imitates the original antigen's position was determined and further processed to the In Situ minimization to obtain the residues interaction energy as well as to observe the hydrogen bonds interaction in the protein-peptide complex. Based on the results demonstrated, the specific residues in the complex that have shown immense lower interaction energies in the 5Å vicinity region from the peptide are from the heavy chain (VH:TYR105) and light chain (VL: ASN31, TYR32, and GLU97). Those residues play vital roles in the binding mechanism of Antibody-Antigen (Ab-Ag) complex of p24 with FAB23.5.

In vivo near-infrared fluorescence imaging of FAP-expressing tumors with activatable FAP-targeted, single-chain Fv-immunoliposomes.

PubMed

Rüger, Ronny; Tansi, Felista L; Rabenhold, Markus; Steiniger, Frank; Kontermann, Roland E; Fahr, Alfred; Hilger, Ingrid

2014-07-28

Molecular and cellular changes that precede the invasive growth of solid tumors include the release of proteolytic enzymes and peptides in the tumor stroma, the recruitment of phagocytic and lymphoid infiltrates and alteration of the extracellular matrix. The reactive tumor stroma consists of a large number of myofibroblasts, characterized by high expression of fibroblast activation protein alpha (FAP). FAP, a type-II transmembrane sialoglycoprotein is an attractive target in diagnosis and therapy of several pathologic disorders especially cancer. In the underlying work, a fluorescence-activatable liposome (fluorescence-quenched during circulation and fluorescence activation upon cellular uptake), bearing specific single-chain Fv fragments directed against FAP (scFv'FAP) was developed, and its potential for use in fluorescence diagnostic imaging of FAP-expressing tumor cells was evaluated by whole body fluorescence imaging. The liposomes termed anti-FAP-IL were prepared via post-insertion of ligand-phospholipid-conjugates into preformed DY-676-COOH-containing liposomes. The anti-FAP-IL revealed a homogeneous size distribution and showed specific interaction and binding with FAP-expressing cells in vitro. The high level of fluorescence quenching of the near-infrared fluorescent dye sequestered in the aqueous interior of the liposomes enables fluorescence imaging exclusively upon uptake and degradation by cells, which results in fluorescence activation. Only FAP-expressing cells were able to take up and activate fluorescence of anti-FAP-IL in vitro. Furthermore, anti-FAP-IL accumulated selectively in FAP-expressing xenograft models in vivo, as demonstrated by blocking experiments using free scFv'FAP. The local tumor fluorescence intensities were in agreement with the intrinsic degree of FAP-expression in different xenograft models. Thus, anti-FAP-IL can serve as a suitable in vivo diagnostic tool for pathological disorders accompanied by high FAP

Construction and characterization of a highly reactive chicken-derived single-chain variable fragment (scFv) antibody against Staphylococcus aureus developed with the T7 phage display system.

PubMed

Li, Jingquan; Xu, Yongping; Wang, Xitao; Li, Yuan; Wang, Lili; Li, Xiaoyu

2016-06-01

The purpose of this study was to construct a single-chain variable fragment (scFv) antibody from chicken egg yolk immunoglobulin (IgY) by means of genetic engineering and subsequent panning for a specific antibody against Staphylococcus aureus. We amplified the scFv using blood and spleen obtained from 100-day-old Roman chickens immunized with inactivated S. aureus and subsequently constructed a T7 phage display antibody library using phage display technology. Four non-repeated blood scFv and 6 spleen scFv were obtained following 3 rounds of panning of the T7 phage display antibody library, enzyme-linked immunosorbent assay and sequencing. These 10 scFv were cloned into the prokaryotic expression vector pCold I with expression induced at a low temperature. Four soluble proteins were obtained. Among them, soluble protein SFV6 derived from the spleen showed good reactivity against S. aureus using indirect ELISA and produced a particularly strong antibacterial effect in vitro. We were successful in isolating a highly specific scFv antibody against S. aureus from the spleen phage display library. This study provides a simple and rapid method for the quick preparation of a large number of antibodies against S. aureus and provides the foundation for the positioning of antibodies in the organism and the study of the antibacterial mechanism through which the antibody functions. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression of anti-tumor necrosis factor alpha (TNFα) single-chain variable fragment (scFv) in Spirodela punctata plants transformed with Agrobacterium tumefaciens.

PubMed

Balaji, Parthasarathy; Satheeshkumar, P K; Venkataraman, Krishnan; Vijayalakshmi, M A

2016-05-01

Therapeutic antibodies against tumor necrosis factor alpha (TNFα) have been considered effective for some of the autoimmune diseases such as rheumatoid arthritis, Crohn's diseases, and so on. But associated limitations of the current therapeutics in terms of cost, availability, and immunogenicity have necessitated the need for alternative candidates. Single-chain variable fragment (scFv) can negate the limitations tagged with the anti-TNFα therapeutics to a greater extent. In the present study, Spirodela punctata plants were transformed with anti-TNFα through in planta transformation using Agrobacterium tumefaciens strain, EHA105. Instead of cefotaxime, garlic extract (1 mg/mL) was used to remove the agrobacterial cells after cocultivation. To the best of our knowledge, this report shows for the first time the application of plant extracts in transgenic plant development. 95% of the plants survived screening under hygromycin. ScFv cDNA integration in the plant genomic DNA was confirmed at the molecular level by PCR. The transgenic protein expression was followed up to 10 months. Expression of scFv was confirmed by immunodot blot. Protein expression levels of up to 6.3% of total soluble protein were observed. β-Glucuronidase and green fluorescent protein expressions were also detected in the antibiotic resistant plants. The paper shows the generation of transgenic Spirodela punctuata plants through in planta transformation. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

Novel Immunocytokine IL12-SS1 (Fv) Inhibits Mesothelioma Tumor Growth in Nude Mice

PubMed Central

Kim, Heungnam; Gao, Wei; Ho, Mitchell

2013-01-01

Mesothelin is a glycosylphosphatidylinositol-anchored glycoprotein that is highly expressed on the cell surface of malignant mesothelioma. Monoclonal antibodies against mesothelin are being evaluated for the treatment of mesothelioma. Immunocytokines represent a novel class of armed antibodies. To provide an alternative approach to current mesothelin-targeted antibody therapies, we have developed a novel immunocytokine based on interleukin-12 (IL12) and the SS1 Fv specific for mesothelin. IL12 possesses potent anti-tumor activity in a wide variety of solid tumors. The newly-developed recombinant immunocytokine, IL12-SS1 (Fv), was produced in insect cells using a baculovirus-insect cell expression system. The SS1 single-chain Fv was fused to the C terminus of the p35 subunit of IL12 through a short linker (GSADGG). The single-chain IL12-SS1 (Fv) immunocytokine bound native mesothelin proteins on malignant mesothelioma (NCI-H226) and ovarian (OVCAR-3) cells as well as recombinant mesothelin on A431/H9 cells. The immunocytokine retained sufficient bioactivity of IL12 and significantly inhibited human malignant mesothelioma (NCI-H226) grown in the peritoneal cavity of nude mice and showed comparable anti-tumor activity to that of the SS1P immunotoxin. IL12-SS1 (Fv) is the first reported immunocytokine to mesothelin-positive tumors and may be an attractive addition to mesothelin-targeted cancer therapies. PMID:24260587

Structural and functional characterization of an anti-West Nile virus monoclonal antibody and its single-chain variant produced in glycoengineered plants

PubMed Central

Lai, Huafang; He, Junyun; Hurtado, Jonathan; Stahnke, Jake; Fuchs, Anja; Mehlhop, Erin; Gorlatov, Sergey; Loos, Andreas; Diamond, Michael S.; Chen, Qiang

2014-01-01

Previously, our group engineered a plant-derived monoclonal antibody (MAb pE16) that efficiently treated West Nile virus (WNV) infection in mice. In this study, we developed a pE16 variant consisting of a single-chain variable fragment (scFv) fused to the heavy chain constant domains (CH) of human IgG (pE16scFv-CH). pE16 and pE16scFv-CH were expressed and assembled efficiently in Nicotiana benthamiana ΔXF plants, a glycosylation mutant lacking plant specific N-glycan residues. Glycan analysis revealed that ΔXF plant-derived pE16scFv-CH (ΔXFpE16scFv-CH) and pE16 (ΔXFpE16) both displayed a mammalian glycosylation profile. ΔXFpE16 and ΔXFpE16scFv-CH demonstrated equivalent antigen binding affinity and kinetics, and slightly enhanced neutralization of WNV in vitro compared to the parent mammalian cell-produced E16 (mE16). A single dose of ΔXFpE16 or ΔXFpE16scFv-CH protected mice against WNV-induced mortality even 4 days after infection at equivalent rates as mE16. This study provides a detailed tandem comparison of the expression, structure and function of a therapeutic MAb and its single-chain variant produced in glycoengineered plants. Moreover, it demonstrates the development of anti-WNV MAb therapeutic variants that are equivalent in efficacy to pE16, simpler to produce, and likely safer to use as therapeutics due to their mammalian N-glycosylation. This platform may lead to a more robust and cost effective production of antibody-based therapeutics against WNV infection and other infectious, inflammatory, or neoplastic diseases. PMID:24975464

Generation of high-affinity, internalizing anti-FGFR2 single-chain variable antibody fragment fused with Fc for targeting gastrointestinal cancers.

PubMed

Borek, Aleksandra; Sokolowska-Wedzina, Aleksandra; Chodaczek, Grzegorz; Otlewski, Jacek

2018-01-01

Fibroblast growth factor receptors (FGFRs) are promising targets for antibody-based cancer therapies, as their substantial overexpression has been found in various tumor cells. Aberrant activation of FGF receptor 2 (FGFR2) signaling through overexpression of FGFR2 and/or its ligands, mutations, or receptor amplification has been reported in multiple cancer types, including gastric, colorectal, endometrial, ovarian, breast and lung cancer. In this paper, we describe application of the phage display technology to produce a panel of high affinity single chain variable antibody fragments (scFvs) against the extracellular ligand-binding domain of FGFR2 (ECD_FGFR2). The binders were selected from the human single chain variable fragment scFv phage display libraries Tomlinson I + J and showed high specificity and binding affinity towards human FGFR2 with nanomolar KD values. To improve the affinity of the best binder selected, scFvF7, we reformatted it to a bivalent diabody format, or fused it with the Fc region (scFvF7-Fc). The scFvF7-Fc antibody construct presented the highest affinity for FGFR2, with a KD of 0.76 nM, and was selectively internalized into cancer cells overexpressing FGFR2, Snu-16 and NCI-H716. Finally, we prepared a conjugate of scFvF7-Fc with the cytotoxic drug monomethyl-auristatin E (MMAE) and evaluated its cytotoxicity. The conjugate delivered MMAE selectively to FGFR2-positive tumor cells. These results indicate that scFvF7-Fc-vcMMAE is a highly potent molecule for the treatment of cancers with FGFR2 overexpression.

Control of Established Colon Cancer Xenografts Using a Novel Humanized Single Chain Antibody-Streptococcal Superantigen Fusion Protein Targeting the 5T4 Oncofetal Antigen

PubMed Central

Patterson, Kelcey G.; Dixon Pittaro, Jennifer L.; Bastedo, Peter S.; Hess, David A.; Haeryfar, S. M. Mansour; McCormick, John K.

2014-01-01

Superantigens (SAgs) are microbial toxins that cross-link T cell receptors with major histocompatibility class II (MHC-II) molecules leading to the activation of large numbers of T cells. Herein, we describe the development and preclinical testing of a novel tumor-targeted SAg (TTS) therapeutic built using the streptococcal pyrogenic exotoxin C (SpeC) SAg and targeting cancer cells expressing the 5T4 tumor-associated antigen (TAA). To inhibit potentially harmful widespread immune cell activation, a SpeC mutation within the high-affinity MHC-II binding interface was generated (SpeCD203A) that demonstrated a pronounced reduction in mitogenic activity, yet this mutant could still induce immune cell-mediated cancer cell death in vitro. To target 5T4+ cancer cells, we engineered a humanized single chain variable fragment (scFv) antibody to recognize 5T4 (scFv5T4). Specific targeting of scFv5T4 was verified. SpeCD203A fused to scFv5T4 maintained the ability to activate and induce immune cell-mediated cytotoxicity of colorectal cancer cells. Using a xenograft model of established human colon cancer, we demonstrated that the SpeC-based TTS was able to control the growth and spread of large tumors in vivo. This required both TAA targeting by scFv5T4 and functional SAg activity. These studies lay the foundation for the development of streptococcal SAgs as ‘next-generation’ TTSs for cancer immunotherapy. PMID:24736661

T-cell immunotherapy for human MK-1-expressing tumors using a fusion protein of the superantigen SEA and anti-MK-1 scFv antibody.

PubMed

Ueno, Aruto; Arakawa, Fumiko; Abe, Hironori; Matsumoto, Hisanobu; Kudo, Toshio; Asano, Ryutaro; Tsumoto, Kohei; Kumagai, Izumi; Kuroki, Motomu; Kuroki, Masahide

2002-01-01

The bacterial superantigen staphylococcal enterotoxin A (SEA) is an extremely potent activator of T lymphocytes when presented on major histocompatibility complex (MHC) class II molecules. To develop a tumor-specific superantigen for cancer therapy, we constructed a recombinant fusion protein of SEA and the single-chain variable fragment (scFv) of the FU-MK-1 antibody, which recognizes a glycoprotein antigen (termed MK-1 antigen) present on most carcinomas. We employed recombinant DNA techniques to fuse recombinant mutant SEA to an scFv antibody derived from FU-MK-1 and the resulting fusion protein (SEA/FUscFv) was produced by a bacterial expression system, purified with a metal-affinity column, and characterized for its MK-1-binding specificity and its antitumor activity. The SEA/FUscFv fusion protein retained the reactivity with MK-1-expressing tumor cells, introduced a specific cytotoxicity of lymphokine-activated killer T-cells to the tumor cells, and consequently suppressed the tumor growth in a SCID mouse xenograft model. This genetically engineered SEA/FUscFv fusion protein may serve as a potentially useful immunotherapeutic reagent for human MK-1-expressing tumors.

Screening of a ScFv Antibody With High Affinity for Application in Human IFN-γ Immunoassay

PubMed Central

Yang, Hang; Zhong, Yanfang; Wang, Juncheng; Zhang, Qinghong; Li, Xiulan; Ling, Sumei; Wang, Shihua; Wang, Rongzhi

2018-01-01

Interferon gamma (IFN-γ), a signal proinflammatory cytokine secreted by immune cell, and plays a critical role in the pathogenesis and progression of many diseases. It has been regarded as an important marker for determination of disease-specific immune responses. Therefore, it is urgent to develop a feasible and accurate method to detect IFN-γ in clinic real blood samples. Until now, the immunoassay based on singe chain variable fragment (scFv) antibody for human IFN-γ is still not reported. In the present study, an scFv antibody named scFv-A8 with high specificity was obtained by phage display and biopanning, with the affinity 2.6 × 109 L/mol. Maltose binding protein (MBP) was used to improve the solubility of scFv by inserting an linker DNA between scFv and MBP tag, and the resulted fusion protein (MBP-LK-scFv) has high solubility and antigen biding activity. The expressed and purified MBP-LK-scFv antibody was used to develop the indirect competitive enzyme-linked immunosorbent assay (ELISA) (ic-ELISA) for detection of human IFN-γ, and the result indicated that the linear range to detect IFN-γ was 6–60 pg/mL with IC50 of 25 pg/mL. The limit of detection was 2 pg/mL (1.3 fm), and the average recovery was 85.05%, further demonstrating that the detection method based on scFv has higher recovery and accuracy. Hence, the developed ic-ELISA can be used to detect IFN-γ in real samples, and it may be further provided a scientific basis for disease diagnosis. PMID:29563896

Intracellular Acidosis Promotes Mitochondrial Apoptosis Pathway: Role of EMMPRIN Down-regulation via Specific Single-chain Fv Intrabody

PubMed Central

Thammasit, Patcharin; Sangboonruang, Sirikwan; Suwanpairoj, Supattara; Khamaikawin, Wannisa; Intasai, Nutjeera; Kasinrerk, Watchara; Tayapiwatana, Chatchai; Tragoolpua, Khajornsak

2015-01-01

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a human leukocyte surface molecule that is enriched on the surface of many cancer cells, and it plays an important role in proliferation and metastasis. In this study, we utilized the chimeric adenoviral vector Ad5/F35 carrying gene encoding scFv against EMMPRIN (scFv-M6-1B9) to down-regulate EMMPRIN cell surface expression and investigated programmed cell death response in colorectal cancer (CRC) cell, Caco-2. The scFv-M6-1B9 intrabody exhibits robust activity in reducing EMMPRIN cell surface expression. This approach led to the inducing of apoptosis, which was relative to the increasing of apoptotic bodies in sub-G1 peak, phosphatidylserine externalization, as well as TUNEL-positive cells. In addition, real-time RT-PCR and western blotting analysis indicated that apoptosis was enhanced through the mitochondrial pathway, a marked reduction of Bcl-2, leading to the translocation of cytochrome c and also the dramatic activation of caspase-3. Moreover, carcinoembryonic antigen (CEA), a tumor marker for CRC, was found to have significantly diminished in both secreted protein and mRNA levels. In conclusion, these findings suggest that EMMPRIN down-regulation by scFv-M6-1B9 intrabody has great potential in enhancing the efficacy of apoptosis induction through the mitochondrial pathway and in effecting a decline in the CEA level. Thus, its benefits could be applied to project the future prospects for targeted gene therapy and therapeutic application in monitoring colorectal cancer. PMID:25663946

A Recombinant Human Anti-Platelet scFv Antibody Produced in Pichia pastoris for Atheroma Targeting

PubMed Central

Vallet-Courbin, Amelie; Larivière, Mélusine; Hocquellet, Agnès; Hemadou, Audrey; Parimala, Sarjapura-Nagaraja; Laroche-Traineau, Jeanny; Santarelli, Xavier; Clofent-Sanchez, Gisèle; Jacobin-Valat, Marie-Josée; Noubhani, Abdelmajid

2017-01-01

Cells of the innate and adaptive immune system are key factors in the progression of atherosclerotic plaque, leading to plaque instability and rupture, potentially resulting in acute atherothrombotic events such as coronary artery disease, cerebrovascular disease and peripheral arterial disease. Here, we describe the cloning, expression, purification, and immunoreactivity assessment of a recombinant single-chain variable fragment (scFv) derived from a human anti-αIIbβ3 antibody (HuAb) selected to target atheromatous lesions for the presence of platelets. Indeed, platelets within atheroma plaques have been shown to play a role in inflammation, in platelet-leucocyte aggregates and in thrombi formation and might thus be considered relevant biomarkers of atherosclerotic progression. The DNA sequence that encodes the anti-αIIbβ3 TEG4 scFv previously obtained from a phage-display selection on activated platelets, was inserted into the eukaryote vector (pPICZαA) in fusion with a tag sequence encoding 2 cysteines useable for specific probes grafting experiments. The recombinant protein was expressed at high yields in Pichia pastoris (30 mg/L culture). The advantage of P. pastoris as an expression system is the production and secretion of recombinant proteins in the supernatant, ruling out the difficulties encountered when scFv are produced in the cytoplasm of bacteria (low yield, low solubility and reduced affinity). The improved conditions allowed for the recovery of highly purified and biologically active scFv fragments ready to be grafted in a site-directed way to nanoparticles for the imaging of atherosclerotic plaques involving inflammatory processes and thus at high risk of instability. PMID:28125612

Anti-Aβ single-chain variable fragment antibodies exert synergistic neuroprotective activities in Drosophila models of Alzheimer's disease

PubMed Central

Fernandez-Funez, Pedro; Zhang, Yan; Sanchez-Garcia, Jonatan; de Mena, Lorena; Khare, Swati; Golde, Todd E.; Levites, Yona; Rincon-Limas, Diego E.

2015-01-01

Both active and passive immunotherapy protocols decrease insoluble amyloid-ß42 (Aß42) peptide in animal models, suggesting potential therapeutic applications against the main pathological trigger in Alzheimer's disease (AD). However, recent clinical trials have reported no significant benefits from humanized anti-Aß42 antibodies. Engineered single-chain variable fragment antibodies (scFv) are much smaller and can easily penetrate the brain, but identifying the most effective scFvs in murine AD models is slow and costly. We show here that scFvs against the N- and C-terminus of Aß42 (scFv9 and scFV42.2, respectively) that decrease insoluble Aß42 in CRND mice are neuroprotective in Drosophila models of Aß42 and amyloid precursor protein neurotoxicity. Both scFv9 and scFv42.2 suppress eye toxicity, reduce cell death in brain neurons, protect the structural integrity of dendritic terminals in brain neurons and delay locomotor dysfunction. Additionally, we show for the first time that co-expression of both anti-Aß scFvs display synergistic neuroprotective activities, suggesting that combined therapies targeting distinct Aß42 epitopes can be more effective than targeting a single epitope. Overall, we demonstrate the feasibility of using Drosophila as a first step for characterizing neuroprotective anti-Aß scFvs in vivo and identifying scFv combinations with synergistic neuroprotective activities. PMID:26253732

Combinatorial Libraries of Arrayable Single-Chain Antibodies

NASA Astrophysics Data System (ADS)

Benhar, Itai

Antibodies that bind their respective targets with high affinity and specificity have proven to be essential reagents for biological research. Antibody phage display has become the leading tool for the rapid isolation of single-chain variable fragment (scFv) antibodies in vitro for research applications, but there is usually a gap between scFv isolation and its application in an array format suitable for high-throughput proteomics. In this chapter, we present our antibody phage display system where antibody isolation and scFv immobilization are facilitated by the design of the phagemid vector used as platform. In our system, the scFvs are fused at their C-termini to a cellulose-binding domain (CBD) and can be immobilized onto cellulose-based filters. This made it possible to develop a unique filter lift screen that allowed the efficient screen for multiple binding specificities, and to directly apply library-derived scFvs in an antibody spotted microarray.

Targeting nanodisks via a single chain variable antibody--apolipoprotein chimera.

PubMed

Iovannisci, David M; Beckstead, Jennifer A; Ryan, Robert O

2009-02-06

Nanodisks (ND) are nanometer scale complexes of phospholipid and apolipoprotein that have been shown to function as drug delivery vehicles. ND harboring significant quantities of the antifungal agent, amphotericin B, or the bioactive isoprenoid, all trans retinoic acid, have been generated and characterized. As currently formulated, ND possess limited targeting capability. In this study, we constructed a single chain variable antibody (scFv).apolipoprotein chimera and assessed the ability of this fusion protein to form ND and recognize the antigen to which the scFv is directed. Data obtained revealed that alpha-vimentin scFv.apolipoprotein A-I is functional in ND formation and antigen recognition, opening the door to the use of such chimeras in targeting drug-enriched ND to specific tissues.

Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain

PubMed Central

Liu, Rui; Liang, Xiao; Xiang, Dandan; Guo, Yirong; Liu, Yihua; Zhu, Guonian

2016-01-01

Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv) antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb) against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL) to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10−10 M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples. PMID:27338340

Development and Evaluation of an Optimal Human Single-Chain Variable Fragment-Derived BCMA-Targeted CAR T Cell Vector.

PubMed

Smith, Eric L; Staehr, Mette; Masakayan, Reed; Tatake, Ishan J; Purdon, Terence J; Wang, Xiuyan; Wang, Pei; Liu, Hong; Xu, Yiyang; Garrett-Thomson, Sarah C; Almo, Steven C; Riviere, Isabelle; Liu, Cheng; Brentjens, Renier J

2018-06-06

B cell maturation antigen (BCMA) has recently been identified as an important multiple myeloma (MM)-specific target for chimeric antigen receptor (CAR) T cell therapy. In CAR T cell therapy targeting CD19 for lymphoma, host immune anti-murine CAR responses limited the efficacy of repeat dosing and possibly long-term persistence. This clinically relevant concern can be addressed by generating a CAR incorporating a human single-chain variable fragment (scFv). We screened a human B cell-derived scFv phage display library and identified a panel of BCMA-specific clones from which human CARs were engineered. Despite a narrow range of affinity for BCMA, dramatic differences in CAR T cell expansion were observed between unique scFvs in a repeat antigen stimulation assay. These results were confirmed by screening in a MM xenograft model, where only the top preforming CARs from the repeat antigen stimulation assay eradicated disease and prolonged survival. The results of this screening identified a highly effective CAR T cell therapy with properties, including rapid in vivo expansion (>10,000-fold, day 6), eradication of large tumor burden, and durable protection to tumor re-challenge. We generated a bicistronic construct including a second-generation CAR and a truncated-epithelial growth factor receptor marker. CAR T cell vectors stemming from this work are under clinical investigation. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Enhanced transport of plant-produced rabies single-chain antibody-RVG peptide fusion protein across an in cellulo blood-brain barrier device.

PubMed

Phoolcharoen, Waranyoo; Prehaud, Christophe; van Dolleweerd, Craig J; Both, Leonard; da Costa, Anaelle; Lafon, Monique; Ma, Julian K-C

2017-10-01

The biomedical applications of antibody engineering are developing rapidly and have been expanded to plant expression platforms. In this study, we have generated a novel antibody molecule in planta for targeted delivery across the blood-brain barrier (BBB). Rabies virus (RABV) is a neurotropic virus for which there is no effective treatment after entry into the central nervous system. This study investigated the use of a RABV glycoprotein peptide sequence to assist delivery of a rabies neutralizing single-chain antibody (ScFv) across an in cellulo model of human BBB. The 29 amino acid rabies virus peptide (RVG) recognizes the nicotinic acetylcholine receptor (nAchR) at neuromuscular junctions and the BBB. ScFv and ScFv-RVG fusion proteins were produced in Nicotiana benthamiana by transient expression. Both molecules were successfully expressed and purified, but the ScFv expression level was significantly higher than that of ScFv-RVG fusion. Both ScFv and ScFv-RVG fusion molecules had potent neutralization activity against RABVin cellulo. The ScFv-RVG fusion demonstrated increased binding to nAchR and entry into neuronal cells, compared to ScFv alone. Additionally, a human brain endothelial cell line BBB model was used to demonstrate that plant-produced ScFv-RVG P fusion could translocate across the cells. This study indicates that the plant-produced ScFv-RVG P fusion protein was able to cross the in celluloBBB and neutralize RABV. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Analysis of the relationship between end-to-end distance and activity of single-chain antibody against colorectal carcinoma.

PubMed

Zhang, Jianhua; Liu, Shanhong; Shang, Zhigang; Shi, Li; Yun, Jun

2012-08-22

We investigated the relationship of End-to-end distance between VH and VL with different peptide linkers and the activity of single-chain antibodies by computer-aided simulation. First, we developed (G4S)n (where n = 1-9) as the linker to connect VH and VL, and estimated the 3D structure of single-chain Fv antibody (scFv) by homologous modeling. After molecular models were evaluated and optimized, the coordinate system of every protein was built and unified into one coordinate system, and End-to-end distances calculated using 3D space coordinates. After expression and purification of scFv-n with (G4S)n as n = 1, 3, 5, 7 or 9, the immunoreactivity of purified ND-1 scFv-n was determined by ELISA. A multi-factorial relationship model was employed to analyze the structural factors affecting scFv: rn=ABn-ABO2+CDn-CDO2+BCn-BCst2. The relationship between immunoreactivity and r-values revealed that fusion protein structure approached the desired state when the r-value = 3. The immunoreactivity declined as the r-value increased, but when the r-value exceeded a certain threshold, it stabilized. We used a linear relationship to analyze structural factors affecting scFv immunoreactivity.

Fusion Peptide Improves Stability and Bioactivity of Single Chain Antibody against Rabies Virus.

PubMed

Xi, Hualong; Zhang, Kaixin; Yin, Yanchun; Gu, Tiejun; Sun, Qing; Shi, Linqing; Zhang, Renxia; Jiang, Chunlai; Kong, Wei; Wu, Yongge

2017-04-28

The combination of rabies immunoglobulin (RIG) with a vaccine is currently effective against rabies infections, but improvements are needed. Genetic engineering antibody technology is an attractive approach for developing novel antibodies to replace RIG. In our previous study, a single-chain variable fragment, scFv57R, against rabies virus glycoprotein was constructed. However, its inherent weak stability and short half-life compared with the parent RIG may limit its diagnostic and therapeutic application. Therefore, an acidic tail of synuclein (ATS) derived from the C-terminal acidic tail of human alpha-synuclein protein was fused to the C-terminus of scFv57R in order to help it resist adverse stress and improve the stability and halflife. The tail showed no apparent effect on the preparation procedure and affinity of the protein, nor did it change the neutralizing potency in vitro. In the ELISA test of molecular stability, the ATS fusion form of the protein, scFv57R-ATS, showed an increase in thermal stability and longer half-life in serum than scFv57R. The protection against fatal rabies virus challenge improved after fusing the tail to the scFv, which may be attributed to the improved stability. Thus, the ATS fusion approach presented here is easily implemented and can be used as a new strategy to improve the stability and half-life of engineered antibody proteins for practical applications.

Kinetic analysis of a monoclonal therapeutic antibody and its single-chain homolog by surface plasmon resonance.

PubMed

Patel, Rekha; Andrien, Bruce A

2010-01-01

Monoclonal antibodies (mAbs) and antibody fragments have become an emerging class of therapeutics since 1986. Their versatility enables them to be engineered for optimal efficiency and decreased immunogenicity, and the path to market has been set by recent regulatory approvals. One of the initial criteria for success of any protein or antibody therapeutic is to understand its binding characteristics to the target antigen. Surface plasmon resonance (SPR) has been widely used and is an important tool for ligand-antigen binding characterization. In this work, the binding kinetics of a recombinant mAb and its single-chain antibody homolog, single-chain variable fragment (scFv), was analyzed by SPR. These two proteins target the same antigen. The binding kinetics of the mAb (bivalent antibody) and scFv (monovalent scFv) for this antigen was analyzed along with an assessment of the thermodynamics of the binding interactions. Alternative binding configurations were investigated to evaluate potential experimental bias because theoretically experimental binding configuration should have no impact on binding kinetics. Self-association binding kinetics in the proteins' respective formulation solutions and antigen epitope mapping were also evaluated. Functional characterization of monoclonal and single-chain antibodies has become just as important as structural characterization in the biotechnology field.

[Targeted detecting HER2 expression with recombinant anti HER2 ScFv-GFP fusion antibody].

PubMed

Gao, Guohui; Chen, Chong; Yang, Yanmei; Yang, Han; Wang, Jindan; Zheng, Yi; Huang, Qidi; Hu, Xiaoqu

2012-08-01

To verify the reliability of targeted detecting HER2 positive cancer cells and clinical pathological tissue specimens with a recombinant anti HER2 single chain antibody in single chain Fv fragment (scFv) format, we have constructed the fusion variable regions of the ScFv specific for HER2/neu. labeled a green-fluorescent protein(GFP). The humanized recombinant Anti HER2 ScFv-GFP gene was inserted into pFast Bac HT A, and expressed in insect cells sf9. Then the recombinant fusion protein Anti HER2 ScFv-GFP was properly purified with Ni2+-NTA affinity chromatography from the infected sf9 cells used to test the specificity of the fusion antibody for HER2 positive cancer cells. Firstly, the purified antibody incubated with HER2 positive breast cancer cells SKBR3, BT474 and HER2 negative breast cancer cells MCF7 for 12 h/24 h/48 h at 37 degrees C, in order to confirm targeted detecting HER2 positive breast cancer cells by Laser Confocal Microscopy. Furthermore, the same clinical pathological tissue samples were assessed by immunohistochemistry (IHC) and the fusion antibody Anti HER2 ScFv-GFP in the meanwhile. The data obtained indicated that the recombinant eukaryotic expression plasmid pFast Bac HT A/Anti HER2 ScFv-GFP was constructed successfully In addition, obvious green fluorescent was observed in insect cells sf9. When the purified fusion antibody was incubated with different cancer cells, much more green fluorescent was observed on the surface of the HER2 positive cancer cells SKBR3 and BT474. In contrast, no green fluorescent on the surface of the HER2 negative cancer cells MCF7 was detected. The concentration of the purified fusion antibody was 115.5 microg/mL, of which protein relative molecular weight was 60 kDa. The analysis showed the purity was about 97% and the titer was about 1:64. The detection results of IHC and fusion antibody testing indicated the conformity. In summary, the study showed that the new fusion antibody Anti HER2 ScFv-GFP can test HER2

Generation of a Highly Reactive Chicken-Derived Single-Chain Variable Fragment against Fusarium verticillioides by Phage Display

PubMed Central

Hu, Zu-Quan; Liu, Jin-Long; Li, He-Ping; Xing, Shu; Xue, Sheng; Zhang, Jing-Bo; Wang, Jian-Hua; Nölke, Greta; Liao, Yu-Cai

2012-01-01

Fusarium verticillioides is the primary causal agent of Fusarium ear and kernel rot in maize, producing fumonisin mycotoxins that are toxic to humans and domestic animals. Rapid detection and monitoring of fumonisin-producing fungi are pivotally important for the prevention of mycotoxins from entering into food/feed products. Chicken-derived single-chain variable fragments (scFvs) against cell wall-bound proteins from F. verticillioides were isolated from an immunocompetent phage display library. Comparative phage enzyme-linked immunosorbant assays (ELISAs) and sequencing analyses identified four different scFv antibodies with high sensitivity. Soluble antibody ELISAs identified two highly sensitive scFv antibodies, FvCA3 and FvCA4, with the latter being slightly more sensitive. Three-dimensional modeling revealed that the FvCA4 may hold a better overall structure with CDRH3, CDRL1 and CDRL3 centered in the core region of antibody surface compared with that of other scFvs. Immunofluorescence labeling revealed that the binding of FvCA4 antibody was localized to the cell walls of conidiospores and hyphae of F. verticillioides, confirming the specificity of this antibody for a surface target. This scFv antibody was able to detect the fungal mycelium as low as 10−2 μg/mL and contaminating mycelium at a quantity of 10−2 mg/g maize. This is the first report that scFv antibodies derived from phage display have a wide application for rapid and accurate detection and monitoring of fumonisin-producing pathogens in agricultural samples. PMID:22837678

A fully human anti-Ep-CAM scFv-beta-glucuronidase fusion protein for selective chemotherapy with a glucuronide prodrug.

PubMed

de Graaf, M; Boven, E; Oosterhoff, D; van der Meulen-Muileman, I H; Huls, G A; Gerritsen, W R; Haisma, H J; Pinedo, H M

2002-03-04

Monoclonal antibodies against tumour-associated antigens could be useful to deliver enzymes selectively to the site of a tumour for activation of a non-toxic prodrug. A completely human fusion protein may be advantageous for repeated administration, as host immune responses may be avoided. We have constructed a fusion protein consisting of a human single chain Fv antibody, C28, against the epithelial cell adhesion molecule and the human enzyme beta-glucuronidase. The sequences encoding C28 and human enzyme beta-glucuronidase were joined by a sequence encoding a flexible linker, and were preceded by the IgGkappa signal sequence for secretion of the fusion protein. A CHO cell line was engineered to secrete C28-beta-glucuronidase fusion protein. Antibody specificity and enzyme activity were retained in the secreted fusion protein that had an apparent molecular mass of 100 kDa under denaturing conditions. The fusion protein was able to convert a non-toxic prodrug of doxorubicin, N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate to doxorubicin, resulting in cytotoxicity. A bystander effect was demonstrated, as doxorubicin was detected in all cells after N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-beta-glucuronyl carbamate administration when only 10% of the cells expressed the fusion protein. This is the first fully human and functional fusion protein consisting of an scFv against epithelial cell adhesion molecule and human enzyme beta-glucuronidase for future use in tumour-specific activation of a non-toxic glucuronide prodrug. Copyright 2002 Cancer Research UK

Generation of a rabbit single-chain fragment variable (scFv) antibody for specific detection of Bradyrhizobium sp. DOA9 in both free-living and bacteroid forms

PubMed Central

Srila, Witsanu; Yuttavanichakul, Watcharin; Teamtisong, Kamonluck; Teaumroong, Neung; Boonkerd, Nantakorn; Tittabutr, Panlada

2017-01-01

A simple and reliable method for the detection of specific nitrogen-fixing bacteria in both free-living and bacteroid forms is essential for the development and application of biofertilizer. Traditionally, a polyclonal antibody generated from an immunized rabbit was used for detection. However, the disadvantages of using a polyclonal antibody include limited supply and cross-reactivity to related bacterial strains. This is the first report on the application of phage display technology for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. Bradyrhizobium sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of Aeschynomene americana in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism of plant–microbe interactions in the future. PMID:28654662

Generation of a rabbit single-chain fragment variable (scFv) antibody for specific detection of Bradyrhizobium sp. DOA9 in both free-living and bacteroid forms.

PubMed

Vu, Nguyen Xuan; Pruksametanan, Natcha; Srila, Witsanu; Yuttavanichakul, Watcharin; Teamtisong, Kamonluck; Teaumroong, Neung; Boonkerd, Nantakorn; Tittabutr, Panlada; Yamabhai, Montarop

2017-01-01

A simple and reliable method for the detection of specific nitrogen-fixing bacteria in both free-living and bacteroid forms is essential for the development and application of biofertilizer. Traditionally, a polyclonal antibody generated from an immunized rabbit was used for detection. However, the disadvantages of using a polyclonal antibody include limited supply and cross-reactivity to related bacterial strains. This is the first report on the application of phage display technology for the generation of a rabbit recombinant monoclonal antibody for specific detection and monitoring of nitrogen-fixing bacteria in both free-living form and in plant nodules. Bradyrhizobium sp. DOA9, a broad host range soil bacteria, originally isolated from the root nodules of Aeschynomene americana in Thailand was used as a model in this study. A recombinant single-chain fragment variable (scFv) antibody library was constructed from the spleen of a rabbit immunized with DOA9. After three rounds of biopanning, one specific phage-displayed scFv antibody, designated bDOA9rb8, was identified. Specific binding of this antibody was confirmed by phage enzyme-linked immunosorbent assay (phage ELISA). The phage antibody could bind specifically to DOA9 in both free-living cells (pure culture) and bacteroids inside plant nodules. In addition to phage ELISA, specific and robust immunofluorescence staining of both free-living and bacteroid forms could also be observed by confocal-immunofluorescence imaging, without cross-reactivity with other tested bradyrhizobial strains. Moreover, specific binding of free scFv to DOA9 was also demonstrated by ELISA. This recombinant antibody can also be used for the study of the molecular mechanism of plant-microbe interactions in the future.

Escherichia coli surface display of single-chain antibody VRC01 against HIV-1 infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Lin-Xu; School of Biological Sciences, University of Nebraska—Lincoln, Lincoln, NE 68583; Mellon, Michael

Human immunodeficiency virus type 1 (HIV-1) transmission and infection occur mainly via the mucosal surfaces. The commensal bacteria residing in these surfaces can potentially be employed as a vehicle for delivering inhibitors to prevent HIV-1 infection. In this study, we have employed a bacteria-based strategy to display a broadly neutralizing antibody VRC01, which could potentially be used to prevent HIV-1 infection. The VRC01 antibody mimics CD4-binding to gp120 and has broadly neutralization activities against HIV-1. We have designed a construct that can express the fusion peptide of the scFv-VRC01 antibody together with the autotransporter β-barrel domain of IgAP gene frommore » Neisseria gonorrhoeae, which enabled surface display of the antibody molecule. Our results indicate that the scFv-VRC01 antibody molecule was displayed on the surface of the bacteria as demonstrated by flow cytometry and immunofluorescence microscopy. The engineered bacteria can capture HIV-1 particles via surface-binding and inhibit HIV-1 infection in cell culture. - Highlights: • Designed single-chain VRC01 antibody was demonstrated to bind HIV-1 envelope gp120. • Single-chain VRC01 antibody was successfully displayed on the surface of E. coli. • Engineered bacteria can absorb HIV-1 particles and prevent HIV-1 infection in cell culture.« less

Fast conversion of scFv to Fab antibodies using type IIs restriction enzymes.

PubMed

Sanmark, Hanna; Huovinen, Tuomas; Matikka, Tero; Pettersson, Tiina; Lahti, Maria; Lamminmäki, Urpo

2015-11-01

Single chain variable fragment (scFv) antibody libraries are widely used for developing novel bioaffinity reagents, although Fab or IgG molecules are the preferred antibody formats in many final applications. Therefore, rapid conversion methods for combining multiple DNA fragments are needed to attach constant domains to the scFv derived variable domains. In this study we describe a fast and easy cloning method for the conversion of single framework scFv fragments to Fab fragments using type IIS restriction enzymes. All cloning steps excluding plating of the Fab transformants can be done in 96 well plates and the procedure can be completed in one working day. The concept was tested by converting 69 scFv clones into Fab format on 96 well plates, which resulted in 93% success rate. The method is particularly useful as a high-throughput tool for the conversion of the chosen scFv clones into Fab molecules in order to analyze them as early as possible, as the conversion can significantly affect the binding properties of the chosen clones. Copyright © 2015 Elsevier B.V. All rights reserved.

PET Imaging of 64Cu-DOTA-scFv-Anti-PSMA Lipid Nanoparticles (LNPs): Enhanced Tumor Targeting over Anti-PSMA scFv or Untargeted LNPs

PubMed Central

Wong, Patty; Li, Lin; Chea, Junie; Delgado, Melissa K.; Crow, Desiree; Poku, Erasmus; Szpikowska, Barbara; Bowles, Nicole; Channappa, Divya; Colcher, David; Wong, Jeffrey Y.C.; Shively, John E.; Yazaki, Paul J.

2017-01-01

Introduction Single chain (scFv) antibodies are ideal targeting ligands due to their modular structure, high antigen specificity and affinity. These monovalent ligands display rapid tumor targeting but have limitations due to their fast urinary clearance. Methods An anti-prostate membrane antigen (PSMA) scFv with a site-specific cysteine was expressed and evaluated in a prostate cancer xenograft model by Cu-64 PET imaging. To enhance tumor accumulation, the scFv-cys was conjugated to the co-polymer DSPE-PEG-maleimide that spontaneously assembled into a homogeneous multivalent lipid nanoparticle (LNP). Results The targeted LNP exhibited a 2-fold increase in tumor uptake compared to the scFv alone using two different thiol ester chemistries. The anti-PSMA scFv-LNP exhibited a 1.6 fold increase in tumor targeting over the untargeted LNP. Conclusions The targeted anti-PSMA scFv-LNP showed enhanced tumor accumulation over the scFv alone or the untargeted DOTA-micelle providing evidence for the development of this system for drug delivery. Advances in Knowledge and implications for patient care Anti-tumor scFv antibody fragments have not achieved their therapeutic potential due to their fast blood clearance. Conjugation to a LNP enables multivalency to the tumor antigen as well as increased molecular size for chemotherapy drug delivery. PMID:28126683

Structural Basis of Neutralization of the Major Toxic Component from the Scorpion Centruroides noxius Hoffmann by a Human-derived Single-chain Antibody Fragment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Canul-Tec, Juan Carlos; Riaño-Umbarila, Lidia; Rudiño-Piñera, Enrique

2011-08-09

It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 {angstrom} resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complexmore » reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of {beta}-toxins to its Na+ channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs.« less

A dual chain chimeric antigen receptor (CAR) in the native antibody format for targeting immune cells towards cancer cells without the need of an scFv.

PubMed

Faitschuk, E; Nagy, V; Hombach, A A; Abken, H

2016-10-01

Adoptive cell therapy with chimeric antigen receptor (CAR)-modified T cells showed remarkable therapeutic efficacy in the treatment of leukaemia/lymphoma. However, the application to a variety of cancer entities is often constricted by the non-availability of a single chain antibody (scFv), which is usually the targeting domain in a CAR, while antibodies in the natural format are often available. To overcome the limitation, we designed a CAR that uses an antibody in its natural configuration for binding. Such CAR consists of two chains, the immunoglobulin light and heavy chain with their constant regions, whereby the heavy chain is anchored to the membrane and linked to an intracellular signalling domain for T-cell activation. The two chains form a stable heterodimer, a so-called dual chain CAR (dcCAR), and bind with high affinity and in a specific manner to their cognate antigen. By specific binding, the dcCAR activates engineered T cells for the release of pro-inflammatory cytokines and for target cell lysis. We provide evidence by three examples that the dcCAR format is universally applicable and thereby broadens the CAR cell therapy towards a larger variety of targets for which an scFv antibody is not available.

Reversible dimer formation and stability of the anti-tumour single-chain Fv antibody MFE-23 by neutron scattering, analytical ultracentrifugation, and NMR and FT-IR spectroscopy.

PubMed

Lee, Yie Chia; Boehm, Mark K; Chester, Kerry A; Begent, Richard H J; Perkins, Stephen J

2002-06-28

MFE-23 is a single chain Fv (scFv) antibody molecule used to target colorectal cancer through its high affinity for the tumour marker carcinoembryonic antigen (CEA). ScFv molecules are formed from peptide-linked antibody V(H) and V(L) domains, and many of these form dimers. Our recent crystal structure for MFE-23 showed that this formed an unusual symmetric back-to-back association of two monomers that is consistent with a domain-swapped diabody structure. Neutron scattering and modelling fits showed that MFE-23 existed as compact V(H)-V(L)-linked monomers at therapeutically relevant concentrations below 1 mg/ml. Size-exclusion gel chromatography showed that the monomeric and dimeric forms of MFE-23 could be separated, and that the proportions of these two forms depended on the starting MFE-23 concentration. Sedimentation equilibrium experiments by analytical ultracentrifugation at nine concentrations of MFE-23 indicated a reversible monomer-dimer self-association equilibrium with an association constant of 1.9x10(3)-2.2x10(3) M(-1). Sedimentation velocity experiments using the time derivative g(s(*)) method showed that MFE-23-His has a concentration-dependent weight average sedimentation coefficient that increased from 1.8 S for the monomer to about 3-6 S for the dimer. Both values agreed with those calculated from the MFE-23 crystal structure. In relation to the thermal stability of MFE-23, denaturation experiments by (1)H NMR and FT-IR spectroscopy showed that the molecule is stable up to 47 degrees C, after which denaturation was irreversible. MFE-23 dimerisation is discussed in terms of a new model for diabody structures, in which the V(H) and V(L) domains in the monomer are able to dissociate and reassociate to form a dimer, or diabody, but in which symmetric back-to-back contacts between the two monomers are formed. This dimerisation in solution is attributed to the complementary nature of the C-terminal surface of the MFE-23 monomer. Crystal structures for

Production of a soluble single-chain variable fragment antibody against okadaic acid and exploration of its specific binding.

PubMed

He, Kuo; Zhang, Xiuyuan; Wang, Lixia; Du, Xinjun; Wei, Dong

2016-06-15

Okadaic acid is a lipophilic marine algal toxin commonly responsible for diarrhetic shellfish poisoning (DSP). Outbreaks of DSP have been increasing and are of worldwide public health concern; therefore, there is a growing demand for more rapid, reliable, and economical analytical methods for the detection of this toxin. In this study, anti-okadaic acid single-chain variable fragment (scFv) genes were prepared by cloning heavy and light chain genes from hybridoma cells, followed by fusion of the chains via a linker peptide. An scFv-pLIP6/GN recombinant plasmid was constructed and transformed into Escherichia coli for expression, and the target scFv was identified with IC-CLEIA (chemiluminescent enzyme immunoassay). The IC15 was 0.012 ± 0.02 μg/L, and the IC50 was 0.25 ± 0.03 μg/L. The three-dimensional structure of the scFv was simulated with computer modeling, and okadaic acid was docked to the scFv model to obtain a putative structure of the binding complex. Two predicted critical amino acids, Ser32 and Thr187, were then mutated to verify this theoretical model. Both mutants exhibited significant loss of binding activity. These results help us to understand this specific scFv-antigen binding mechanism and provide guidance for affinity maturation of the antibody in vitro. The high-affinity scFv developed here also has potential for okadaic acid toxin detection. Copyright © 2016. Published by Elsevier Inc.

Engineering of a recombinant trivalent single-chain variable fragment antibody directed against rabies virus glycoprotein G with improved neutralizing potency.

PubMed

Turki, Imène; Hammami, Akil; Kharmachi, Habib; Mousli, Mohamed

2014-02-01

Human and equine rabies immunoglobulins are currently available for passive immunization against rabies. However, these are hampered by the limited supply and some drawbacks. Advances in antibody engineering have led to overcome issues of clinical applications and to improve the protective efficacy. In the present study, we report the generation of a trivalent single-chain Fv (scFv50AD1-Fd), that recognizes the rabies virus glycoprotein, genetically fused to the trimerization domain of the bacteriophage T4 fibritin, termed 'foldon' (Fd). scFv50AD1-Fd was expressed as soluble recombinant protein in bacterial periplasmic space and purified through affinity chromatography. The molecular integrity and stability were analyzed by polyacrylamide gradient-gel electrophoresis, size-exclusion chromatography and incubation in human sera. The antigen-binding properties of the trimeric scFv were analyzed by direct and competitive-ELISA. Its apparent affinity constant was estimated at 1.4 ± 0.25 × 10(9)M(-1) and was 75-fold higher than its monovalent scFv (1.9 ± 0.68 × 10(7)M(-1)). The scFv50AD1-Fd neutralized rabies virus in a standard in vitro and in vivo neutralization assay. We showed a high neutralization activity up to 75-fold compared with monovalent format and the WHO standard serum. The gain in avidity resulting from multivalency along with an improved biological activity makes the trivalent scFv50AD1-Fd construct an important reagent for rabies protection. The antibody engineering approach presented here may serve as a strategy for designing a new generation of anti-rabies for passive immunotherapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

A fully human anti-Ep-CAM scFv-beta-glucuronidase fusion protein for selective chemotherapy with a glucuronide prodrug

PubMed Central

de Graaf, M; Boven, E; Oosterhoff, D; van der Meulen-Muileman, I H; Huls, G A; Gerritsen, W R; Haisma, H J; Pinedo, H M

2002-01-01

Monoclonal antibodies against tumour-associated antigens could be useful to deliver enzymes selectively to the site of a tumour for activation of a non-toxic prodrug. A completely human fusion protein may be advantageous for repeated administration, as host immune responses may be avoided. We have constructed a fusion protein consisting of a human single chain Fv antibody, C28, against the epithelial cell adhesion molecule and the human enzyme β-glucuronidase. The sequences encoding C28 and human enzyme β-glucuronidase were joined by a sequence encoding a flexible linker, and were preceded by the IgGκ signal sequence for secretion of the fusion protein. A CHO cell line was engineered to secrete C28-β-glucuronidase fusion protein. Antibody specificity and enzyme activity were retained in the secreted fusion protein that had an apparent molecular mass of 100 kDa under denaturing conditions. The fusion protein was able to convert a non-toxic prodrug of doxorubicin, N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-β-glucuronyl carbamate to doxorubicin, resulting in cytotoxicity. A bystander effect was demonstrated, as doxorubicin was detected in all cells after N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-β-glucuronyl carbamate administration when only 10% of the cells expressed the fusion protein. This is the first fully human and functional fusion protein consisting of an scFv against epithelial cell adhesion molecule and human enzyme β-glucuronidase for future use in tumour-specific activation of a non-toxic glucuronide prodrug. British Journal of Cancer (2002) 86, 811–818. DOI: 10.1038/sj/bjc/6600143 www.bjcancer.com © 2002 Cancer Research UK PMID:11875747

Design and construction of immune phage antibody library against Tetanus neurotoxin: Production of single chain antibody fragments.

PubMed

Sadreddini, Sanam; Seifi-Najmi, Mehrnosh; Ghasemi, Babollah; Kafil, Hossein Samadi; Alinejad, Vahideh; Sadreddini, Sevil; Younesi, Vahid; Jadidi-Niaragh, Farhad; Yousefi, Mehdi

2015-12-23

Tetanus neurotoxin (TeNT) is composed of a light (LC) and heavy chain (HC) polypeptides, released by anaerobic bacterium Clostridium tetani and can cause fatal life-threatening infectious disease. Toxin HC and LC modules represents receptor binding and zinc metalloprotease activity, respectively. The passive administration of animal-derived antibodies against tetanus toxin has been considered as the mainstay therapy for years. However, this treatment is associated with several adverse effects due to the presence of anti-isotype antibodies. In the present study, we have produced the fully human single chain antibody fragments (HuScFv) from two human antibody phage display libraries. Twenty-four different HuscFvs were isolated from two anti TeNT immune libraries. Our produced human ScFv (HuScFv) were converted to IgG platform and analyzed regarding their specific reactivity to TeNT. All of the selected scFvs have the same VL but different VH. Three HuscFvs from the first library (TTX15, 51, 75) and two HuscFvs from the second library (TTX16, 20) were chosen to convert to IgG1 using pOptiVEC and pcDNA3.3 systems. Production of IgG1 from transfected DG44 and binding capacity of them to tetanus toxin and toxoid were measured by ELISA. ELISA results showed no detectable production of TTX16 and TTX20 IgG1. Although, TTX51 and TTX75 were converted and produced as IgG1, no reactivity to tetanus toxin and toxoid was observed. However, TTX15 was successfully produced as whole IgG1 platform with reactivity to both tetanus toxin and toxoid. The latter would be an appropriate replacement for conventional polyclonal antibodies if would meet the further characterization including specificity determination, affinity measurement and toxin neutralizing assays. Our results demonstrated production of functional IgG1 derived from TTX15 scFv and might be an appropriate replacement for polyclonal Tetabulin but it needs further characterization.

Aβ-oligomer uptake and the resulting inflammatory response in adult human astrocytes are precluded by an anti-Aβ single chain variable fragment in combination with an apoE mimetic peptide.

PubMed

Montoliu-Gaya, Laia; Mulder, Sandra D; Herrebout, Maaike A C; Baayen, Johannes C; Villegas, Sandra; Veerhuis, Robert

2018-06-01

An imbalance between production and clearance of soluble amyloid-β (Aβ) initiates the pathological process in sporadic Alzheimer's disease (AD). Aβ-specific antibodies seemed promising as therapeutic option in AD mouse models. In patients, however, vascular side-effects and Aβ-antibody complex-induced microglial and/or perivascular macrophage inflammatory responses were encountered. To prevent inflammatory reactions, we designed a single chain variable fragment (scFv-h3D6), based on monoclonal antibody bapineuzumab (mAb-h3D6), but lacking the Fc region. ScFv-h3D6 reduced Aβ-oligomer burden and prevented AD-associated behavioral and cellular changes in 3xTg-AD mice. As scFv-h3D6 lacks the Fc-tail, it cannot enhance Fc-receptor mediated Aβ clearance by microglia and probably exerts its beneficial effects in 3xTg-AD mice through other mechanisms. ScFv-h3D6 restored the increased apoE and apoJ levels in 3xTg-AD brains back to normal. ApoE and apoJ influence cholesterol transport, Aβ aggregation and clearance, and their genetic variants are risk factors for sporadic AD. Astrocytes are constitutive scavengers of soluble Aβ from the CNS. We previously found apoE and apoJ to inhibit Aβ uptake by adult human astrocytes, in vitro, and thus to potentially protect astrocytes from Aβ cytotoxicity. In the present study, scFv-h3D6 and mAb-h3D6 inhibited Aβ-oligomer uptake by adult human astrocytes. ApoE- and apoJ- mimetic peptides (MP) affected Aβ uptake as well as Aβ-induced cytokine release similar to intact apoE and apoJ, without interfering with the strong inhibitory effects of scFv-h3D6 on Aβ-oligomer uptake. These results suggest that combining Aβ-specific scFv and apoE-MP, that inhibits Aβ oligomer-induced cytokine release by astrocytes, could offer advantages over currently used therapeutics. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Genetically engineered multivalent single chain antibody constructs for cancer therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Surinder Batra, Ph D

2006-02-27

increase its tumor: normal tissue ratio for improved therapeutic index, we engineered a variety antibody constructs. These constructs were evaluated using novel approaches like special radionuclides, pretargeting and optimization. Due to the smaller size, the engineered antibody molecules should penetrate better throughout a tumor mass, with less dose heterogeneity, than is the case with intact IgG. Multivalent scFvs with an appropriate radionuclide, therefore, hold promising prospects for cancer therapy and clinical imaging in MAb-based radiopharmaceuticals. In addition, the human anti-mouse antibodies (HAMA) responses in patients against antibody-based therapy are usually directed against the immunoglobulin constant regions; however, anti-idiotypic responses can also be detected. The HAMA responses reduce the efficacy of treatment by removing the circulating antibody molecules, fragments, and possibly scFvs by altering the pharmacokinetic properties of the antibody. HAMA responses against divalent IgG, divalent Ig fragments, and possibly multimeric scFvs could cause immune complex formation with hypersensitivity or allergic reactions that could be harmful to patients. The use of small molecules, such as scFvs (monomeric as well as multimeric), with their shorter biological half-lives and the lack of the constant regions and humanized variable (binding regions) performed in our studies should reduce the development of HAMA. The generation of humanized and fully human scFvs should further reduce the development of HAMA. Specific accomplishments on the project are the production of large amounts of recombinant antibodies as they are required in large amounts for cancer diagnosis and therapy. A variety of single-chain Fv (scFv) constructs were engineered for the desired pharmacokinetic properties. Tetrameric and dimeric scFvs showed a two-fold advantage: (1) there was a considerable gain in avidity as compared to smaller fragments, and (2) the biological half-life was more

Retargeting of adenovirus vectors through genetic fusion of a single-chain or single-domain antibody to capsid protein IX.

PubMed

Poulin, Kathy L; Lanthier, Robert M; Smith, Adam C; Christou, Carin; Risco Quiroz, Milagros; Powell, Karen L; O'Meara, Ryan W; Kothary, Rashmi; Lorimer, Ian A; Parks, Robin J

2010-10-01

Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions.

Bispecific single-chain diabody-immunoliposomes targeting endoglin (CD105) and fibroblast activation protein (FAP) simultaneously.

PubMed

Rabenhold, Markus; Steiniger, Frank; Fahr, Alfred; Kontermann, Roland E; Rüger, Ronny

2015-03-10

Liposomes are well-established drug delivery systems with cancer chemotherapy as main focus. To increase the cellular drug delivery, liposomes can be endowed with ligands, e.g. recombinant antibody fragments, which ensure specific cell interaction. Multispecific immunoliposomes can be prepared to improve the liposome to cell interaction by targeting multiple different targets at the same time, for instance by coupling two or more different ligands to the liposomal surface, resulting in a synergistic or additive increase in binding. An alternative approach is the use of bispecific ligands to address at least two different targets. For this purpose we cloned a single-chain diabody fragment (scDb`), a bispecific molecule targeting two antigens, endoglin (CD105) and fibroblast activation protein (FAP), expressed on cells of the tumor microenvironment. As model cell system, a human fibrosarcoma cell line was used expressing endoglin and FAP simultaneously. Monospecific immunoliposomes directed either against endoglin or FAP were compared in vitro for cell binding and cytotoxic activity with bispecific dual-targeted scFv`-IL (bispecific scFv`FAP/CD105-IL) and bispecific single-chain diabody`-IL (scDb`CD105/FAP-IL) targeting endoglin and FAP simultaneously. In the underlying study, bispecific scFv`FAP/CD105-IL interacted stronger with cells expressing FAP and endoglin (both targets simultaneously) compared to the monospecific immunoliposomes. Furthermore, bispecific scDb`-immunoliposomes increased the cell interaction massively and showed enhanced cytotoxicity against target cells using doxorubicin-loaded immunoliposomes. The use of recombinant bispecific ligands as scDb`-molecules facilitates the generation of bispecific immunoliposomes by using the established post-insertion technique, enabling an extension of the ligand specificity spectrum via genetic modification. Copyright © 2015 Elsevier B.V. All rights reserved.

Selection of Single-Chain Antibodies against the VP8* Subunit of Rotavirus VP4 Outer Capsid Protein and Their Expression in Lactobacillus casei

PubMed Central

Monedero, Vicente; Rodríguez-Díaz, Jesús; Viana, Rosa; Buesa, Javier; Pérez-Martínez, Gaspar

2004-01-01

Single-chain antibodies (scFv) recognizing the VP8* fraction of rotavirus outer capsid and blocking rotavirus infection in vitro were isolated by phage display. Vectors for the extracellular expression in Lactobacillus casei of one of the scFv were constructed. L. casei was able to secrete active scFv to the growth medium, showing the potential of probiotic bacteria to be engineered to express molecules suitable for in vivo antirotavirus therapies. PMID:15528568

Synergistic capture of Clostridium botulinum Type A neurotoxin by scFv antibodies to novel epitopes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gray, Sean A.; Barr, John R.; Kalb, Suzanne R.

2011-10-01

A non-immune library of human single chain fragment variable (scFv) antibodies displayed on Saccharomyces cerevisiae was screened for binding to the Clostridium botulinum neurotoxin serotype A binding domain [BoNT/A (Hc)] with the goal of identifying scFv to novel epitopes. To do this, an antibody-mediated labeling strategy was used in which antigen-binding yeast clones were selected after labeling with previously characterized monoclonal antibodies (MAbs) specific to the Hc. Twenty unique scFv clones were isolated that bound Hc. Of these, three also bound to full-length BoNT/A toxin complex with affinities ranging from 5 nM to 170 nM. Epitope binning showed that themore » three unique clones recognized at least two epitopes that were distinct from one another and from the detection MAbs. After production in E. coli, the scFv were coupled to magnetic particles and tested for their ability to capture BoNT/A holotoxin using an Endopep-MS assay. In this assay, toxin captured by scFv coated magnetic particles was detected by incubation of the complex with a peptide containing a BoNT/A-specific cleavage sequence. Mass spectrometry was used to detect the ratio of intact peptide to cleavage products as evidence for toxin capture. When tested individually, each of the scFv showed a weak positive Endopep-MS result. However, when the particles were coated with all three scFv simultaneously, they exhibited significantly higher Endopep-MS activity, consistent with synergistic binding. These results demonstrate novel approaches toward the isolation and characterization of scFv antibodies specific to unlabeled antigen. They also provide evidence that distinct scFv antibodies can work synergistically to increase the efficiency of antigen capture onto a solid support.« less

Duplex microfluidic SERS detection of pathogen antigens with nanoyeast single-chain variable fragments.

PubMed

Wang, Yuling; Rauf, Sakandar; Grewal, Yadveer S; Spadafora, Lauren J; Shiddiky, Muhammad J A; Cangelosi, Gerard A; Schlücker, Sebastian; Trau, Matt

2014-10-07

Quantitative and accurate detection of multiple biomarkers would allow for the rapid diagnosis and treatment of diseases induced by pathogens. Monoclonal antibodies are standard affinity reagents applied for biomarkers detection; however, their production is expensive and labor-intensive. Herein, we report on newly developed nanoyeast single-chain variable fragments (NYscFv) as an attractive alternative to monoclonal antibodies, which offers the unique advantage of a cost-effective production, stability in solution, and target-specificity. By combination of surface-enhanced Raman scattering (SERS) microspectroscopy using glass-coated, highly purified SERS nanoparticle clusters as labels, with a microfluidic device comprising multiple channels, a robust platform for the sensitive duplex detection of pathogen antigens has been developed. Highly sensitive detection for individual Entamoeba histolytica antigen EHI_115350 (limit of detection = 1 pg/mL, corresponding to 58.8 fM) and EHI_182030 (10 pg/mL, corresponding 453 fM) with high specificity has been achieved, employing the newly developed corresponding NYscFv as probe in combination with SERS microspectroscopy at a single laser excitation wavelength. Our first report on SERS-based immunoassays using the novel NYscFv affinity reagent demonstrates the flexibility of NYscFv fragments as viable alternatives to monoclonal antibodies in a range of bioassay platforms and paves the way for further applications.

Structural and functional characterization of a novel scFv anti-HSP60 of Strongyloides sp.

PubMed Central

Levenhagen, Marcelo Arantes; de Almeida Araújo Santos, Fabiana; Fujimura, Patrícia Tiemi; Caneiro, Ana Paula; Costa-Cruz, Julia Maria; Goulart, Luiz Ricardo

2015-01-01

Phage display is a powerful technology that selects specific proteins or peptides to a target. We have used Phage Display to select scFv (single-chain variable fragment) clones from a combinatorial library against total proteins of Strongyloides venezuelensis. After scFv characterization, further analysis demonstrated that this recombinant fragment of antibody was able to bind to an S. venezuelensis antigenic fraction of ~65 kDa, present in the body periphery and digestive system of infective larvae (L3), as demonstrated by immunofluorescence. Mass spectrometry results followed by bioinformatics analysis showed that this antigenic fraction was a heat shock protein 60 (HSP60) of Strongyloides sp. The selected scFv was applied in serodiagnosis by immune complexes detection in serum samples from individuals with strongyloidiasis using a sandwich enzyme-linked immunosorbent assay (ELISA), showing sensitivity of 97.5% (86.84–99.94), specificity of 98.81 (93.54–99.97), positive likelihood ratio of 81.60 and an area under the curve of 0.9993 (0.9973–1.000). Our study provided a novel monoclonal scFv antibody fragment which specifically bound to HSP60 of Strongyloides sp. and was applied in the development of an innovative serodiagnosis method for the human strongyloidiasis. PMID:25994608

Generation of a mouse scFv library specific for porcine aminopeptidase N using the T7 phage display system.

PubMed

Sun, Dongbo; Shi, Hongyan; Chen, Jianfei; Shi, Da; Zhu, Qinghe; Zhang, Hong; Liu, Shengwang; Wang, Yunfeng; Qiu, Huaji; Feng, Li

2012-06-01

Porcine aminopeptidase N (pAPN) is a common cellular receptor for swine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV). To investigate single-chain fragment variable (scFv) repertoire against pAPN, the genes encoding the immunoglobulin light chain variable region (VL) and heavy chain variable region (VH) were amplified by reverse transcript polymerase chain reaction (RT-PCR) using a series of degenerate primers from the spleen of BABL/c mice immunized with native pAPN. The VL and VH amplicons were combined randomly by a 12 amino acid flexible linker by splicing by overlap extension PCR (SOE-PCR), which produced the scFv gene repertoire. After ligation of the scFv gene repertoire into the T7Select10-3b vector, a mouse scFv phage library specific for pAPN was produced through in vitro packaging. The primary scFv library against pAPN contained 2.0×10(7) recombinant phage clones, and the titer of the amplified library was 3.6×10(9)pfu/mL. BstNI restriction analysis and DNA sequencing revealed that 28 phage clones from the primary pAPN scFv library showed excellent diversity. The effectiveness of the scFv library against pAPN was verified further by phage ELISA using the recombinant protein of the pAPN C subunit as coating antigen. The construction and evaluation of a murine scFv library against the common receptor pAPN of porcine coronaviruses TGEV and PEDV using the T7 phage display system are described. Copyright © 2012 Elsevier B.V. All rights reserved.

Production and characterization of single-chain antibody (scFv) against 3ABC non-structural protein in Escherichia coli for sero-diagnosis of Foot and Mouth Disease virus.

PubMed

Sharma, Gaurav K; Mahajan, Sonalika; Matura, Rakesh; Subramaniam, Saravanan; Mohapatra, Jajati K; Pattnaik, Bramhadev

2014-11-01

Differentiation of Foot-and-Mouth Disease infected from vaccinated animals is essential for effective implementation of vaccination based control programme. Detection of antibodies against 3ABC non-structural protein of FMD virus by immunodiagnostic assays provides reliable indication of FMD infection. Sero-monitoring of FMD in the large country like India is a big task where thousands of serum samples are annually screened. Currently, monoclonal or polyclonal antibodies are widely used in these immunodiagnostic assays. Considering the large population of livestock in the country, an economical and replenishable alternative of these antibodies was required. In this study, specific short chain variable fragment (scFv) antibody against 3B region of 3ABC poly-protein was developed. High level of scFv expression in Escherichia coli system was obtained by careful optimization in four different strains. Two formats of enzyme immunoassays (sandwich and competitive ELISAs) were optimized using scFv with objective to differentiate FMD infected among the vaccinated population. The assays were statistically validated by testing 2150 serum samples. Diagnostic sensitivity/specificity of sandwich and competitive ELISAs were determined by ROC method as 92.2%/95.5% and 89.5%/93.5%, respectively. This study demonstrated that scFv is a suitable alternate for immunodiagnosis of FMD on large scale. Copyright © 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

Crystal structure of the anti-(carcinoembryonic antigen) single-chain Fv antibody MFE-23 and a model for antigen binding based on intermolecular contacts.

PubMed

Boehm, M K; Corper, A L; Wan, T; Sohi, M K; Sutton, B J; Thornton, J D; Keep, P A; Chester, K A; Begent, R H; Perkins, S J

2000-03-01

MFE-23 is the first single-chain Fv antibody molecule to be used in patients and is used to target colorectal cancer through its high affinity for carcinoembryonic antigen (CEA), a cell-surface member of the immunoglobulin superfamily. MFE-23 contains an N-terminal variable heavy-chain domain joined by a (Gly(4)Ser)(3) linker to a variable light-chain (V(L)) domain (kappa chain) with an 11-residue C-terminal Myc-tag. Its crystal structure was determined at 2.4 A resolution by molecular replacement with an R(cryst) of 19.0%. Five of the six antigen-binding loops, L1, L2, L3, H1 and H2, conformed to known canonical structures. The sixth loop, H3, displayed a unique structure, with a beta-hairpin loop and a bifurcated apex characterized by a buried Thr residue. In the crystal lattice, two MFE-23 molecules were associated back-to-back in a manner not seen before. The antigen-binding site displayed a large acidic region located mainly within the H2 loop and a large hydrophobic region within the H3 loop. Even though this structure is unliganded within the crystal, there is an unusually large region of contact between the H1, H2 and H3 loops and the beta-sheet of the V(L) domain of an adjacent molecule (strands DEBA) as a result of intermolecular packing. These interactions exhibited remarkably high surface and electrostatic complementarity. Of seven MFE-23 residues predicted to make contact with antigen, five participated in these lattice contacts, and this model for antigen binding is consistent with previously reported site-specific mutagenesis of MFE-23 and its effect on CEA binding.

Purification and refolding of anti-T-antigen single chain antibodies (scFvs) expressed in Escherichia coli as inclusion bodies.

PubMed

Yuasa, Noriyuki; Koyama, Tsubasa; Fujita-Yamaguchi, Yoko

2014-02-01

T-antigen (Galβ1-3GalNAcα-1-Ser/Thr) is an oncofetal antigen that is commonly expressed as a carbohydrate determinant in many adenocarcinomas. Since it is associated with tumor progression and metastasis, production of recombinant antibodies specific for T-antigen could lead to the development of cancer diagnostics and therapeutics. Previously, we isolated and characterized 11 anti-T-antigen phage clones from a phage library displaying human single-chain antibodies (scFvs) and purified one scFv protein, 1G11. More recently, we purified and characterized 1E8 scFv protein using a Drosophila S2 expression system. In the current study, four anti-T-antigen scFv genes belonging to Groups 1-4 were purified from inclusion bodies expressed in Escherichia coli cells. Inclusion bodies isolated from E. coli cells were denatured in 3.5 M Gdn-HCl. Solubilized His-tagged scFv proteins were purified using Ni(2+)-Sepharose column chromatography in the presence of 3.5 M Gdn-HCl. Purified scFv proteins were refolded according to a previously published method of step-wise dialysis. Two anti-T-antigen scFv proteins, 1E6 and 1E8 that belong to Groups 1 and 2, respectively, were produced in sufficient amounts, thus allowing further characterization of their binding activity with T-antigen. Specificity and affinity constants determined using enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), respectively, provided evidence that both 1E8 and 1E6 scFv proteins are T-antigen specific and suggested that 1E8 scFv protein has a higher affinity for T-antigen than 1E6 scFv protein.

Engineering of a novel zipFv using leucine zipper motif against rabies virus glycoprotein G with improved protection potency in vivo.

PubMed

Xi, Hualong; Zhang, Kaixin; Yin, Yanchun; Gu, Tiejun; Sun, Qing; Li, Zhuang; Cheng, Yue; Jiang, Chunlai; Kong, Wei; Wu, Yongge

2017-06-01

Rabies is an acute zoonotic infectious disease with a high fatality rate but is preventable with vaccination and rabies immunoglobulin (RIG). The single-chain Fv fragment (scFv), a small engineered antigen-binding protein derived from antibody variable heavy (V H ) and light (V L ) chains connected by a peptide linker, can potentially be used to replace RIG. Here, we produced two peptides V H -JUN-HIS and V L -FOS-HA separately in Escherichia coli and assembled them to form zipFv successfully in vitro. The new zipFv utilizes FOS and JUN leucine zippers to form an antibody structure similar to the IgG counterpart with two free N-terminal ends of V H and V L . The zipFv protein showed notable improvement in binding ability and affinity over its corresponding scFv. The zipFv also demonstrated greater stability in serum and the same protective rate as RIG against challenge with a standard rabies virus (CVS-24) in mice. Our results indicated zipFv as a novel and efficient antibody form with enhanced neutralizing potency. Copyright © 2017. Published by Elsevier B.V.

A novel anti-PSMA human scFv has the potential to be used as a diagnostic tool in prostate cancer

PubMed Central

Han, Yueheng; Wei, Ming; Han, Sen; Lin, Ruihe; Sun, Ziyong; Yang, Fa; Jiao, Dian; Xie, Pin; Zhang, Lingling; Yang, An-Gang; Zhao, Aizhi; Wen, Weihong; Qin, Weijun

2016-01-01

Prostate cancer (PCa) is the most commonly diagnosed malignancy and the second leading cause of cancer related death in men. The early diagnosis and treatment of PCa are still challenging due to the lack of efficient tumor targeting agents in traditional managements. Prostate specific membrane antigen (PSMA) is highly expressed in PCa, while only has limited expression in other organs, providing an ideal target for the diagnosis and therapy of PCa. The antibody library technique has opened the avenue for the discovery of novel antibodies to be used in the diagnosis and therapy of cancer. In this paper, by screening a large yeast display naive human single chain antibody fragment (scFv) library, we obtained a high affinity scFv targeting PSMA, called gy1. The gy1 scFv was expressed in E.coli and purified via a C terminal 6His tag. The binding affinity of gy1 was shown to be at the nanomolar level and gy1 can specifically bind with PSMA positive cancer cells, and binding triggers its rapid internalization through the endosome-lysosome pathway. The specific targeting of gy1 to PSMA positive tumor tissues was also evaluated in vivo. We showed that the IRDye800CW labeled gy1 can efficiently target and specifically distribute in PSMA positive tumor tissues after being injected into xenograft nude mice. This study indicated that the novel antibody gy1 could be used as a great tool for the development of PSMA targeted imaging and therapy agents for PCa. PMID:27448970

Detection of Metallothionein in Javanese Medaka (Oryzias javanicus), Using a scFv-Immobilized Protein Chip

PubMed Central

Lee, Euiyeon; Jeon, Hyunjin; Kang, Chungwon; Woo, Seonock; Yum, Seungshic; Kwon, Youngeun

2018-01-01

Environmental pollution by various industrial chemicals and biological agents poses serious risks to human health. Especially, marine contamination by potentially toxic elements (PTEs) has become a global concern in recent years. Many efforts have been undertaken to monitor the PTE contamination of the aquatic environment. However, there are few approaches available to assess the PTE exposure of aquatic organisms. In this research, we developed a strategy to evaluate the heavy metal exposure of marine organisms, by measuring the expression levels of metallothionein protein derived from Oryzias javanicus (OjaMT). OjaMT is a biomarker of heavy metal exposure because the expression level increases upon heavy metal exposure. The developed assay is based on a real-time, label-free surface plasmon resonance (SPR) measurement. Anti-OjaMT antibody and anti-OjaMT single-chain fragment of variable region (scFv) were used as detection probes. Two types of SPR sensor chips were fabricated, by immobilizing antibody or Cys3-tagged scFv (scFv-Cys3) in a controlled orientation and were tested for in situ label-free OjaMT detection. Compared to the antibody-presenting sensor chips, the scFv-presenting sensor chips showed improved performance, displaying enhanced sensitivity and enabling semi-quantitative detection. The portable SPR system combined with scFv-immobilized sensor chips is expected to provide an excellent point-of-care testing system that can monitor target biomarkers in real time. PMID:29614840

Development of anti-bovine IgA single chain variable fragment and its application in diagnosis of foot-and-mouth disease

PubMed Central

Sridevi, N. V.; Shukra, A. M.; Neelakantam, B.; Anilkumar, J.; Madhanmohan, M.; Rajan, S.; Dev Chandran

2014-01-01

Recombinant antibody fragments like single chain variable fragments (scFvs) represent an attractive yet powerful alternative to immunoglobulins and hold great potential in the development of clinical diagnostic/therapeutic reagents. Structurally, scFvs are the smallest antibody fragments capable of retaining the antigen-binding capacity of whole antibodies and are composed of an immunoglobulin (Ig) variable light (VL) and variable heavy (VH) chain joined by a flexible polypeptide linker. In the present study, we constructed a scFv against bovine IgA from a hybridoma cell line IL-A71 that secretes a monoclonal antibody against bovine IgA using recombinant DNA technology. The scFv was expressed in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). The binding activity and specificity of the scFv was established by its non-reactivity toward other classes of immunoglobulins as determined by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis. Kinetic measurement of the scFv indicated that the recombinant antibody fragment had an affinity in picomolar range toward purified IgA. Furthermore, the scFv was used to develop a sensitive ELISA for the detection of foot and mouth disease virus (FMDV) carrier animals. PMID:24678404

Effects of Environmental Factors on Soluble Expression of a Humanized Anti-TNF-α scFv Antibody in Escherichia coli

PubMed Central

Sina, Mohammad; Farajzadeh, Davoud; Dastmalchi, Siavoush

2015-01-01

Purpose: The bacterial cultivation conditions for obtaining anti-TNF-α single chain variable fragment (scFv) antibody as the soluble product in E. coli was investigated. Methods: To avoid the production of inclusion bodies, the effects of lactose, IPTG, incubation time, temperature, shaking protocol, medium additives (Mg+2, sucrose), pH, osmotic and heat shocks were examined. Samples from bacterial growth conditions with promising results of soluble expression of GST-hD2 scFv were affinity purified and quantified by SDS-PAGE and image processing for further evaluation. Results: The results showed that cultivation in LB medium under induction by low concentrations of lactose and incubation at 10 °C led to partial solubilization of the expressed anti-TNF-α scFv (GST-hD2). Other variables which showed promising increase in soluble expression of GST-hD2 were osmotic shock and addition of magnesium chloride. Furthermore, addition of sucrose to medium suppressed the expression of scFv completely. The other finding was that the addition of sorbitol decreased the growth rate of bacteria. Conclusion: It can be concluded that low cultivation temperature in the presence of low amount of inducer under a long incubation time or addition of magnesium chloride are the most effective environmental factors studied for obtaining the maximum solubilization of GST-hD2 recombinant protein. PMID:26819916

Effects of Environmental Factors on Soluble Expression of a Humanized Anti-TNF-α scFv Antibody in Escherichia coli.

PubMed

Sina, Mohammad; Farajzadeh, Davoud; Dastmalchi, Siavoush

2015-11-01

The bacterial cultivation conditions for obtaining anti-TNF-α single chain variable fragment (scFv) antibody as the soluble product in E. coli was investigated. To avoid the production of inclusion bodies, the effects of lactose, IPTG, incubation time, temperature, shaking protocol, medium additives (Mg+2, sucrose), pH, osmotic and heat shocks were examined. Samples from bacterial growth conditions with promising results of soluble expression of GST-hD2 scFv were affinity purified and quantified by SDS-PAGE and image processing for further evaluation. The results showed that cultivation in LB medium under induction by low concentrations of lactose and incubation at 10 °C led to partial solubilization of the expressed anti-TNF-α scFv (GST-hD2). Other variables which showed promising increase in soluble expression of GST-hD2 were osmotic shock and addition of magnesium chloride. Furthermore, addition of sucrose to medium suppressed the expression of scFv completely. The other finding was that the addition of sorbitol decreased the growth rate of bacteria. It can be concluded that low cultivation temperature in the presence of low amount of inducer under a long incubation time or addition of magnesium chloride are the most effective environmental factors studied for obtaining the maximum solubilization of GST-hD2 recombinant protein.

Human scFv antibody fragments specific for hepatocellular carcinoma selected from a phage display library.

PubMed

Yu, Bing; Ni, Ming; Li, Wen-Han; Lei, Ping; Xing, Wei; Xiao, Dai-Wen; Huang, Yu; Tang, Zhen-Jie; Zhu, Hui-Fen; Shen, Guan-Xin

2005-07-14

To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DNA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in E.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M(r) value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.

Rapid optical imaging of EGF receptor expression with a single-chain antibody SNAP-tag fusion protein.

PubMed

Kampmeier, Florian; Niesen, Judith; Koers, Alexander; Ribbert, Markus; Brecht, Andreas; Fischer, Rainer; Kiessling, Fabian; Barth, Stefan; Thepen, Theo

2010-10-01

The epidermal growth factor receptor (EGFR) is overexpressed in several types of cancer and its inhibition can effectively inhibit tumour progression. The purpose of this study was to design an EGFR-specific imaging probe that combines efficient tumour targeting with rapid systemic clearance to facilitate non-invasive assessment of EGFR expression. Genetic fusion of a single-chain antibody fragment with the SNAP-tag produced a 48-kDa antibody derivative that can be covalently and site-specifically labelled with substrates containing 0 (6)-benzylguanine. The EGFR-specific single-chain variable fragment (scFv) fusion protein 425(scFv)SNAP was labelled with the near infrared (NIR) dye BG-747, and its accumulation, specificity and kinetics were monitored using NIR fluorescence imaging in a subcutaneous pancreatic carcinoma xenograft model. The 425(scFv)SNAP fusion protein accumulates rapidly and specifically at the tumour site. Its small size allows efficient renal clearance and a high tumour to background ratio (TBR) of 33.2 +/- 6.3 (n = 4) 10 h after injection. Binding of the labelled antibody was efficiently competed with a 20-fold excess of unlabelled probe, resulting in an average TBR of 6 +/- 1.35 (n = 4), which is similar to that obtained with a non-tumour-specific probe (5.44 +/- 1.92, n = 4). When compared with a full-length antibody against EGFR (cetuximab), 425(scFv)SNAP-747 showed significantly higher TBRs and complete clearance 72 h post-injection. The 425(scFv)SNAP fusion protein combines rapid and specific targeting of EGFR-positive tumours with a versatile and robust labelling technique that facilitates the attachment of fluorophores for use in optical imaging. The same approach could be used to couple a chelating agent for use in nuclear imaging.

Systemic p53 gene therapy of cancer with immunolipoplexes targeted by anti-transferrin receptor scFv.

PubMed Central

Xu, L.; Tang, W. H.; Huang, C. C.; Alexander, W.; Xiang, L. M.; Pirollo, K. F.; Rait, A.; Chang, E. H.

2001-01-01

BACKGROUND: A long-standing goal in genetic therapy for cancer is a systemic gene delivery system that selectively targets tumor cells, including metastases. Here we describe a novel cationic immunolipoplex system that shows high in vivo gene transfer efficiency and anti- tumor efficacy when used for systemic p53 gene therapy of cancer. MATERIALS AND METHODS: A cationic immunolipoplex incorporating a biosynthetically lipid-tagged, anti-transferrin receptor single-chain antibody (TfRscFv), was designed to target tumor cells both in vitro and in vivo. A human breast cancer metastasis model was employed to evaluate the in vivo efficacy of systemically administered, TfRscFv-immunolipoplex-mediated, p53 gene therapy in combination with docetaxel. RESULTS: The TfRscFv-targeting cationic immunolipoplex had a size of 60-100 nm, showed enhanced tumor cell binding, and improved targeted gene delivery and transfection efficiencies, both in vitro and in vivo. The p53 tumor suppressor gene was not only systemically delivered by the immunolipoplex to human tumor xenografts in nude mice but also functionally expressed. In the nude mouse breast cancer metastasis model, the combination of the p53 gene delivered by the systemic administration of the TfRscFv-immunolipoplex and docetaxel resulted in significantly improved efficacy with prolonged survival. CONCLUSIONS: This is the first report using scFv-targeting immunolipoplexes for systemic gene therapy. The TfRscFv has a number of advantages over the transferrin (Tf) molecule itself: (1) scFv has a much smaller size than Tf producing a smaller immunolipoplex giving better penetration into solid tumors; (2) unlike Tf, the scFv is a recombinant protein, not a blood product; (3) large scale production and strict quality control of the recombinant scFv, as well as scFv-immunolipoplex, are feasible. The sensitization of tumors to chemotherapy by this tumor-targeted and efficient p53 gene delivery method could lower the effective dose of

Expression and purification of a novel therapeutic single-chain variable fragment antibody against BNP from inclusion bodies of Escherichia coli.

PubMed

Bu, Dawei; Zhou, Yuwei; Tang, Jian; Jing, Fang; Zhang, Wei

2013-12-01

Abnormal brain natriuretic peptide (BNP) secretion is regarded as the dominating mechanism of cerebral salt wasting syndrome (CSW), which results from a renal loss of sodium and water during intracranial disease leading to hyponatremia. Scale preparation of therapeutic single-chain variable fragment (scFv) that can neutralize elevated circulating BNP may have potential value for clinical use. In this report, we used a recently isolated humanized anti-BNP scFv fragment (3C1) as model antibody (Ab) to evaluate the potential of scale production of this therapeutic protein. The truncated gene encoding for scFv fragment cloned in pET22b (+) was mainly overexpressed as inclusion bodies in Escherichia coli (E. coli) Rosetta (DE3) pLysS cells. The insoluble fragment was solubilized and purified by Ni-NTA agarose resin under denaturation conditions, and recovered via an effective refolding buffer containing 50 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM EDTA, 0.5 M arginine, 2 mM GSH, 1 mM GSSG, and 5% glycerol. The refolded scFv fragment was concentrated by PEG20000, and dialyzed in PBS (containing 5% glycerol, pH 7.4). The final yield was approximately 10.2 mg active scFv fragment per liter of culture (3.4 g wet weight cells). The scFv fragment was more than 95% pure assessed by SDS-PAGE assay. Recombinant scFv fragment with His tag displayed its immunoreactivity with anti-His tag Ab by western blotting. ELISA showed the scFv fragment specifically bound to BNP, and it displayed similar activity as the traditional anti-BNP monoclonal Ab (mAb). Thus, the current strategy allows convenient small-scale production of this therapeutic protein. Copyright © 2013 Elsevier Inc. All rights reserved.

Rejection of syngeneic colon carcinoma by CTLs expressing single-chain antibody receptors codelivering CD28 costimulation.

PubMed

Haynes, Nicole M; Trapani, Joseph A; Teng, Michele W L; Jackson, Jacob T; Cerruti, Loretta; Jane, Stephen M; Kershaw, Michael H; Smyth, Mark J; Darcy, Phillip K

2002-11-15

A new strategy to improve the therapeutic utility of redirected T cells for cancer involves the development of novel Ag-specific chimeric receptors capable of stimulating optimal and sustained T cell antitumor activity in vivo. Given that T cells require both primary and costimulatory signals for optimal activation and that many tumors do not express critical costimulatory ligands, modified single-chain Ab receptors have been engineered to codeliver CD28 costimulation. In this study, we have compared the antitumor potency of primary T lymphocytes expressing carcinoembryonic Ag (CEA)-reactive chimeric receptors that incorporate either TCR-zeta or CD28/TCR-zeta signaling. Although both receptor-transduced T cell effector populations demonstrated cytolysis of CEA(+) tumors in vitro, T cells expressing the single-chain variable fragment of Ig (scFv)-CD28-zeta chimera had a far greater capacity to control the growth of CEA(+) xenogeneic and syngeneic colon carcinomas in vivo. The observed enhanced antitumor activity of T cells expressing the scFv-CD28-zeta receptor was critically dependent on perforin and the production of IFN-gamma. Overall, this study has illustrated the ability of a chimeric scFv receptor capable of harnessing the signaling machinery of both TCR-zeta and CD28 to augment T cell immunity against tumors that have lost expression of both MHC/peptide and costimulatory ligands in vivo.

Construction of an Immunized Rabbit Phage Display Library for Selecting High Activity against Bacillus thuringiensis Cry1F Toxin Single-Chain Antibodies.

PubMed

Xu, Chongxin; Zhang, Cunzheng; Zhong, Jianfeng; Hu, Hui; Luo, Shimin; Liu, Xiaoqin; Zhang, Xiao; Liu, Yuan; Liu, Xianjin

2017-07-26

In the present study, a Cry1F-immunized rabbit phage display library (6.96 × 10 8 cfu/mL) was constructed for selecting high activity of anti-Cry1F toxin single-chain antibody (a single-chain variable fragment, scFv) by biopanning. A total of 16 positive monoclonal phage scFv's were obtained after 4 rounds of panning, which were identified by enzyme-linked immunosorbent assay (ELISA), polymerized chain reaction, and DNA sequencing. The most positive phage scFv (named RF4) was expressed in Escherichia coli HB2151, and a soluble protein of approximately 30 kDa was purified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An indirect competitive ELISA (IC-ELISA) was established on the basis of purified soluble RF4-scFv for Cry1F toxin. It indicated the 50% inhibition of the control (IC 50 ) was 11.56 ng/mL and the detection limit (IC 10 ) was 0.18 ng/mL and showed weak cross-reactivities for Cry1Ab (2.8%), Cry1Ac (1.3%), and Cry1B, Cry1C, Cry1Ie, and Cry2A (less than 0.1%). It was found that IC-ELISA detected Cry1F toxin spiked in rice, wheat, corn, and soil samples with good accuracy, stability, and repeatability. The recoveries were in the range of 80.2-99.6%, and the coefficients of variation were in the range of 2.5-10.0%. These results showed that IC-ELISA based on scFv from the immunized rabbit phage display library was promising for specific detection of Cry1F toxin in agroproducts and environmental samples.

Modular Construction of Large Non-Immune Human Antibody Phage-Display Libraries from Variable Heavy and Light Chain Gene Cassettes.

PubMed

Lee, Nam-Kyung; Bidlingmaier, Scott; Su, Yang; Liu, Bin

2018-01-01

Monoclonal antibodies and antibody-derived therapeutics have emerged as a rapidly growing class of biological drugs for the treatment of cancer, autoimmunity, infection, and neurological diseases. To support the development of human antibodies, various display techniques based on antibody gene repertoires have been constructed over the last two decades. In particular, scFv-antibody phage display has been extensively utilized to select lead antibodies against a variety of target antigens. To construct a scFv phage display that enables efficient antibody discovery, and optimization, it is desirable to develop a system that allows modular assembly of highly diverse variable heavy chain and light chain (Vκ and Vλ) repertoires. Here, we describe modular construction of large non-immune human antibody phage-display libraries built on variable gene cassettes from heavy chain and light chain repertoires (Vκ- and Vλ-light can be made into independent cassettes). We describe utility of such libraries in antibody discovery and optimization through chain shuffling.

Development of a Novel Human scFv Against EGFR L2 Domain by Phage Display Technology.

PubMed

Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Veisi, Kamal; Khosroshahi, Shiva Ahdi; Tanomand, Asghar

2017-01-01

Epidermal growth factor receptor (EGFR) as a transmembrane tyrosine kinase receptor frequently overexpresses in tumors with epithelial origin. The L2 domain from extracellular part of EGFR is involved in ligand binding and the blockage of this domain prevents activation of related signaling pathways. This study was aimed to develop a novel human scFv against EGFR L2 domain as a promising target for cancer therapy. The L2 recombinant protein was purified and used for panning a human scFv phage library (Tomlinson I). In this study, a novel screening strategy was applied to select clones with high binding and enrichment of rare specific phage clones of the L2 protein. After five biopanning rounds several specific clones were isolated which among them one phage clone with high binding was purified for further analysis. The specific interaction of selected clone against target antigen was confirmed by ELISA and western blotting. Immunofluorescence staining showed that purified scFv binds to A431 cells surface, displaying EGFR surface receptor. In the present study, we isolated for the first time a novel human scFv against EGFR L2 domain. This study can be the groundwork for developing more effective diagnostic and therapeutic agents against EGFR overexpressing cancers using this novel human anti-L2 ScFv. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Single-chain antigen recognition receptors that costimulate potent rejection of established experimental tumors.

PubMed

Haynes, Nicole M; Trapani, Joseph A; Teng, Michèle W L; Jackson, Jacob T; Cerruti, Loretta; Jane, Stephen M; Kershaw, Michael H; Smyth, Mark J; Darcy, Phillip K

2002-11-01

Tumor cells are usually weakly immunogenic as they largely express self-antigens and can down-regulate major histocompatability complex/peptide molecules and critical costimulatory ligands. The challenge for immunotherapies has been to provide vigorous immune effector cells that circumvent these tumor escape mechanisms and eradicate established tumors. One promising approach is to engineer T cells with single-chain antibody receptors, and since T cells require 2 distinct signals for optimal activation, we have compared the therapeutic efficacy of erbB2-reactive chimeric receptors that contain either T-cell receptor zeta (TCR-zeta) or CD28/TCR-zeta signaling domains. We have demonstrated that primary mouse CD8(+) T lymphocytes expressing the single-chain Fv (scFv)-CD28-zeta receptor have a greater capacity to secrete Tc1 cytokines, induce T-cell proliferation, and inhibit established tumor growth and metastases in vivo. The suppression of established tumor burden by cytotoxic T cells expressing the CD28/TCR-zeta chimera was critically dependent upon their interferon gamma (IFN-gamma) secretion. Our study has illustrated the practical advantage of engineering a T-cell signaling complex that codelivers CD28 activation, dependent only upon the tumor's expression of the appropriate tumor associated antigen.

Production and characterization of a single-chain variable fragment linked alkaline phosphatase fusion protein for detection of O,O-diethyl organophosphorus pesticides in a one-step enzyme-linked immunosorbent assay

USDA-ARS?s Scientific Manuscript database

A single-chain variable fragment (scFv) and alkaline phosphatase (AP) fusion protein for detection of O, O-diethyl organophosphorus pesticides (OPs) was produced and characterized. The scFv gene was prepared by cloning VL and VH genes from a hybridoma cell secreting monoclonal antibody with broad-s...

ESCRT-mediated Uptake and Degradation of Brain-targeted α-synuclein Single Chain Antibody Attenuates Neuronal Degeneration In Vivo

PubMed Central

Spencer, Brian; Emadi, Sharareh; Desplats, Paula; Eleuteri, Simona; Michael, Sarah; Kosberg, Kori; Shen, Jay; Rockenstein, Edward; Patrick, Christina; Adame, Anthony; Gonzalez, Tania; Sierks, Michael; Masliah, Eliezer

2014-01-01

Parkinson's disease and dementia with Lewy bodies are neurodegenerative disorders characterized by accumulation of α-synuclein (α-syn). Recently, single-chain fragment variables (scFVs) have been developed against individual conformational species of α-syn. Unlike more traditional monoclonal antibodies, these scFVs will not activate or be endocytosed by Fc receptors. For this study, we investigated an scFV directed against oligomeric α-syn fused to the LDL receptor-binding domain from apolipoprotein B (apoB). The modified scFV showed enhanced brain penetration and was imported into neuronal cells through the endosomal sorting complex required for transport (ESCRT) pathway, leading to lysosomal degradation of α-syn aggregates. Further analysis showed that the scFV was effective at ameliorating neurodegenerative pathology and behavioral deficits observed in the mouse model of dementia with Lewy bodies/Parkinson's disease. Thus, the apoB modification had the effect of both increasing accumulation of the scFV in the brain and directing scFV/α-syn complexes for degradation through the ESCRT pathway, leading to improved therapeutic potential of immunotherapy. PMID:25008355

ESCRT-mediated uptake and degradation of brain-targeted α-synuclein single chain antibody attenuates neuronal degeneration in vivo.

PubMed

Spencer, Brian; Emadi, Sharareh; Desplats, Paula; Eleuteri, Simona; Michael, Sarah; Kosberg, Kori; Shen, Jay; Rockenstein, Edward; Patrick, Christina; Adame, Anthony; Gonzalez, Tania; Sierks, Michael; Masliah, Eliezer

2014-10-01

Parkinson's disease and dementia with Lewy bodies are neurodegenerative disorders characterized by accumulation of α-synuclein (α-syn). Recently, single-chain fragment variables (scFVs) have been developed against individual conformational species of α-syn. Unlike more traditional monoclonal antibodies, these scFVs will not activate or be endocytosed by Fc receptors. For this study, we investigated an scFV directed against oligomeric α-syn fused to the LDL receptor-binding domain from apolipoprotein B (apoB). The modified scFV showed enhanced brain penetration and was imported into neuronal cells through the endosomal sorting complex required for transport (ESCRT) pathway, leading to lysosomal degradation of α-syn aggregates. Further analysis showed that the scFV was effective at ameliorating neurodegenerative pathology and behavioral deficits observed in the mouse model of dementia with Lewy bodies/Parkinson's disease. Thus, the apoB modification had the effect of both increasing accumulation of the scFV in the brain and directing scFV/α-syn complexes for degradation through the ESCRT pathway, leading to improved therapeutic potential of immunotherapy.

A functional role for CD28 costimulation in tumor recognition by single-chain receptor-modified T cells.

PubMed

Moeller, Maria; Haynes, Nicole M; Trapani, Joseph A; Teng, Michele W L; Jackson, Jacob T; Tanner, Jane E; Cerutti, Loretta; Jane, Stephen M; Kershaw, Michael H; Smyth, Mark J; Darcy, Phillip K

2004-05-01

T cells engineered to express single-chain antibody receptors that incorporate TCR-zeta and cluster designation (CD)28 signaling domains (scFv-alpha-erbB2-CD28-zeta) can be redirected in vivo to cancer cells that lack triggering costimulatory molecules. To assess the contribution of CD28 signaling to the function of the scFv-CD28-zeta receptor, we expressed a series of mutated scFv-CD28-zeta receptors directed against erbB2. Residues known to be critical for CD28 signaling were mutated from tyrosine to phenylalanine at position 170 or proline to alanine at positions 187 and 190. Primary mouse T cells expressing either of the mutant receptors demonstrated impaired cytokine (IFN-gamma and GM-CSF) production and decreased proliferation after antigen ligation in vitro and decreased antitumor efficacy in vivo compared with T cells expressing the wild-type scFv-CD28-zeta receptor, suggesting a key signaling role for the CD28 component of the scFv-CD28-zeta receptor. Importantly, cell surface expression, binding capacity and cytolytic activity mediated by the scFv-CD28-zeta receptor were not diminished by either mutation. Overall, this study has definitively demonstrated a functional role for the CD28 component of the scFv-CD28-zeta receptor and has shown that incorporation of costimulatory activity in chimeric scFv receptors is a powerful approach for improving adoptive cancer immunotherapy.

Targeting foreign major histocompatibility complex molecules to tumors by tumor cell specific single chain antibody (scFv).

PubMed

Li, Jinhua; Franek, Karl J; Patterson, Andrea L; Holmes, Lillia M; Burgin, Kelly E; Ji, Jianfei; Yu, Xianzhong; Wagner, Thomas E; Wei, Yanzhang

2003-11-01

Down-regulation of the major histocompatibility complex (MHC) is one of the major mechanisms that tumor cells adopted to escape immunosurveillance. Therefore, specifically coating tumor cells with foreign MHC may make tumor cells a better target for immune recognition and surveillance. In this study, we designed and generated a fusion protein, H2Kd/scPSMA, consisting of a single chain antibody against human prostate specific membrane antigen (PSMA) and the extracellular domain of mouse H-2Kd. The expression of this fusion protein in B16F0 mouse melanoma cells was confirmed by RT-PCR and fluorescent activated cell sorting (FACS). Our animal study showed that the expression of H2Kd/scPSMA in B16F0/PSMA5, a B16F0 cell line expressing human PSMA, significantly inhibited tumor growth as demonstrated in the pulmonary metastasis assay and tumor growth study and improved overall survival.

Construction, expression, and characterization of a single-chain variable fragment antibody against 2,4-dichlorophenoxyacetic acid in the hemolymph of silkworm larvae.

PubMed

Sakamoto, Seiichi; Pongkitwitoon, Benyakan; Nakamura, Seiko; Sasaki-Tabata, Kaori; Tanizaki, Yusuke; Maenaka, Katsumi; Tanaka, Hiroyuki; Morimoto, Satoshi

2011-07-01

A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly(4)Ser)(3) between two domains. The yield of functional 2,4-D-scFv after purification was 640 μg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.

A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity

PubMed Central

Ordóñez, Adriana; Pérez, Juan; Tan, Lu; Dickens, Jennifer A.; Motamedi-Shad, Neda; Irving, James A.; Haq, Imran; Ekeowa, Ugo; Marciniak, Stefan J.; Miranda, Elena; Lomas, David A.

2015-01-01

Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin.—Ordóñez, A., Pérez, J., Tan, L., Dickens, J. A., Motamedi-Shad, N., Irving, J. A., Haq, I., Ekeowa, U., Marciniak, S. J., Miranda, E., Lomas, D. A. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity. PMID:25757566

Kinetic Characterisation of a Single Chain Antibody against the Hormone Abscisic Acid: Comparison with Its Parental Monoclonal

PubMed Central

Badescu, George O.; Marsh, Andrew; Smith, Timothy R.; Thompson, Andrew J.; Napier, Richard M.

2016-01-01

A single-chain Fv fragment antibody (scFv) specific for the plant hormone abscisic acid (ABA) has been expressed in the bacterium Escherichia coli as a fusion protein. The kinetics of ABA binding have been measured using surface plasmon resonance spectrometry (BIAcore 2000) using surface and solution assays. Care was taken to calculate the concentration of active protein in each sample using initial rate measurements under conditions of partial mass transport limitation. The fusion product, parental monoclonal antibody and the free scFv all have low nanomolar affinity constants, but there is a lower dissociation rate constant for the parental monoclonal resulting in a three-fold greater affinity. Analogue specificity was tested and structure-activity binding preferences measured. The biologically-active (+)-ABA enantiomer is recognised with an affinity three orders of magnitude higher than the inactive (-)-ABA. Metabolites of ABA including phaseic acid, dihydrophaseic acid and deoxy-ABA have affinities over 100-fold lower than that for (+)-ABA. These properties of the scFv make it suitable as a sensor domain in bioreporters specific for the naturally occurring form of ABA. PMID:27023768

Anti-HER2 antibody and ScFvEGFR-conjugated antifouling magnetic iron oxide nanoparticles for targeting and magnetic resonance imaging of breast cancer

PubMed Central

Chen, Hongwei; Wang, Liya; Yu, Qiqi; Qian, Weiping; Tiwari, Diana; Yi, Hong; Wang, Andrew Y; Huang, Jing; Yang, Lily; Mao, Hui

2013-01-01

Antifouling magnetic iron oxide nanoparticles (IONPs) coated with block copolymer poly(ethylene oxide)-block-poly(γ-methacryloxypropyltrimethoxysilane) (PEO-b-PγMPS) were investigated for improving cell targeting by reducing nonspecific uptake. Conjugation of a HER2 antibody, Herceptin®, or a single chain fragment (ScFv) of antibody against epidermal growth factor receptor (ScFvEGFR) to PEO-b-PγMPS-coated IONPs resulted in HER2-targeted or EGFR-targeted IONPs (anti-HER2-IONPs or ScFvEGFR-IONPs). The anti-HER2-IONPs bound specifically to SK-BR-3, a HER2-overexpressing breast cancer cell line, but not to MDA-MB-231, a HER2-underexpressing cell line. On the other hand, the ScFvEGFR-IONPs showed strong reactivity with MDA-MB-231, an EGFR-positive human breast cancer cell line, but not with MDA-MB-453, an EGFR-negative human breast cancer cell line. Transmission electron microscopy revealed internalization of the receptor-targeted nanoparticles by the targeted cancer cells. In addition, both antibody-conjugated and non-antibody-conjugated IONPs showed reduced nonspecific uptake by RAW264.7 mouse macrophages in vitro. The developed IONPs showed a long blood circulation time (serum half-life 11.6 hours) in mice and low accumulation in both the liver and spleen. At 24 hours after systemic administration of ScFvEGFR-IONPs into mice bearing EGFR-positive breast cancer 4T1 mouse mammary tumors, magnetic resonance imaging revealed signal reduction in the tumor as a result of the accumulation of the targeted IONPs. PMID:24124366

Antiproliferative and Apoptotic Effects of Novel Anti-ROR1 Single-Chain Antibodies in Hematological Malignancies.

PubMed

Aghebati-Maleki, Leili; Younesi, Vahid; Baradaran, Behzad; Abdolalizadeh, Jalal; Motallebnezhad, Morteza; Nickho, Hamid; Shanehbandi, Dariush; Majidi, Jafar; Yousefi, Mehdi

2017-04-01

Receptor tyrosine kinase-like orphan receptor (ROR) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including cell survival, differentiation, cell migration, cell communication, cell polarity, proliferation, metabolism, and angiogenesis. ROR1 has recently been shown to be expressed in various types of cancer cells but not normal cells. Pharmacokinetics and pharmacodynamics of single-chain Fragment variable (scFv) antibodies provide potential therapeutic advantages over whole antibody molecules. In the present study, scFvs against a specific peptide from the extracellular domain of ROR1 were selected using phage display technology. The selected scFvs were further characterized using polyclonal and monoclonal phage enzyme-linked immunosorbent assay (ELISA), soluble monoclonal ELISA, colony PCR, and sequencing. Antiproliferative and apoptotic effects of selected scFv antibodies were also evaluated in lymphoma and myeloma cancer cell lines using MTT and annexin V/PI assays. The results of ELISA indicated specific reactions of the isolated scFvs against the ROR1 peptide. Colony PCR confirmed the presence of full-length V H and Vκ inserts. The percentages of cell growth after 24 h of treatment of cells with individual scFv revealed that the scFv significantly inhibited the growth of the RPMI8226 and chronic lymphocytic leukemia (CLL) cells in comparison with the untreated cells ( p < 0.05). Interestingly, 24-h treatment with specific scFv induced apoptosis cell death in the RPMI8226 and CLL cells. Taken together, our results demonstrate that targeting of ROR1 using peptide-specific scFv can be an effective immunotherapy strategy in hematological malignancies.

Characterization of single chain antibody targets through yeast two hybrid

PubMed Central

2010-01-01

Background Due to their unique ability to bind their targets with high fidelity, antibodies are used widely not only in biomedical research, but also in many clinical applications. Recombinant antibodies, including single chain variable fragments (scFv), are gaining momentum because they allow powerful in vitro selection and manipulation without loss of function. Regardless of the ultimate application or type of antibody used, precise understanding of the interaction between the antibody's binding site and its specific target epitope(s) is of great importance. However, such data is frequently difficult to obtain. Results We describe an approach that allows detailed characterization of a given antibody's target(s) using the yeast two-hybrid system. Several recombinant scFv were used as bait and screened against highly complex cDNA libraries. Systematic sequencing of all retained clones and statistical analysis allowed efficient ranking of the prey fragments. Multiple alignment of the obtained cDNA fragments provided a selected interacting domain (SID), efficiently narrowing the epitope-containing region. Interactions between antibodies and their respective targets were characterized for several scFv. For AA2 and ROF7, two conformation-specific sensors that exclusively bind the activated forms of the small GTPases Rab6 and Rab1 respectively, only fragments expressing the entire target protein's core region were retained. This strongly suggested interaction with a non-linear epitope. For two other scFv, TA10 and SF9, which recognize the large proteins giantin and non-muscle myosin IIA, respectively, precise antibody-binding regions within the target were defined. Finally, for some antibodies, secondary targets within and across species could be revealed. Conclusions Our method, utilizing the yeast two-hybrid technology and scFv as bait, is a simple yet powerful approach for the detailed characterization of antibody targets. It allows precise domain mapping for linear

[Construction of a phage antibody library and screening of anti-epidermal growth factor receptor variant III single chain antibody].

PubMed

Han, Dong-gang; Duan, Xiao-yi; Guo, You-min; Zhou, Qi; Wang, Quan-ying; Yang, Guang-xiao

2010-01-01

To obtain specific anti-epidermal growth factor receptor variant III (EGFRvIII) single chain antibody (ScFv) by phage antibody library display system. The total RNA was extracted from the spleen B cells of BALB/c mice immunized with pep-3-OVA protein, and the first-strand cDNA was synthesized by reverse transcription. Antibody VH and VL gene fragments were amplified and joined to a ScFv gene with the linker. The ScFv gene was ligated into the phagemid vector pCANTAB5E, which was transformed into competent E. coli TG1. The transformed cells were then infected with M13KO7 helper phage to yield the recombinant phage to construct the phage ScFv library. Pep-3-BSA protein was used to screen the phage antibody library and ELISA carried out to characterize the activity of the antibody. The VH and VL gene fragments of the antibody were about 350 bp and 320 bp in length as analyzed by agarose gel electrophoresis. The ScFv gene was 780 bp, consistent with the expected length. The recombinant phagemid with ScFv gene insert was rescued, and an immune phage ScFv library with the content of 5.0x10(6) was constructed. The recombinant ScFv phage had a titer of 3.0x10(4) cfu/ml, and the fourth phage harvest yielded 56 times as much as that of the first one. SDS-PAGE demonstrated a molecular mass of the soluble ScFv of about 28 kD. ELISA results indicated good specificity of the ScFv to bind EGFRvIII. An immune phage ScFv library is successfully constructed, and the ScFv antibody fragment is capable of specific binding to EGFRvIII.

Human scFv antibodies (Afribumabs) against Africanized bee venom: Advances in melittin recognition.

PubMed

Pessenda, Gabriela; Silva, Luciano C; Campos, Lucas B; Pacello, Elenice M; Pucca, Manuela B; Martinez, Edson Z; Barbosa, José E

2016-03-15

Africanized Apis mellifera bees, also known as killer bees, have an exceptional defensive instinct, characterized by mass attacks that may cause envenomation or death. From the years 2000-2013, 77,066 bee accidents occurred in Brazil. Bee venom comprises several substances, including melittin and phospholipase A2 (PLA2). Due to the lack of antivenom for bee envenomation, this study aimed to produce human monoclonal antibody fragments (single chain fragment variable; scFv), by using phage display technology. These fragments targeted melittin and PLA2, the two major components of bee venom, to minimize their toxic effects in cases of mass envenomation. Two phage antibody selections were performed using purified melittin. As the commercial melittin is contaminated with PLA2, phages specific to PLA2 were also obtained during one of the selections. Specific clones for melittin and PLA2 were selected for the production of soluble scFvs, named here Afribumabs: prefix: afrib- (from Africanized bee); stem/suffix: -umab (fully human antibody). Afribumabs 1 and 2 were tested in in vitro and in vivo assays to assess their ability to inhibit the toxic actions of purified melittin, PLA2, and crude bee venom. Afribumabs reduced hemolysis caused by purified melittin and PLA2 and by crude venom in vitro and reduced edema formation in the paws of mice and prolonged the survival of venom-injected animals in vivo. These results demonstrate that Afribumabs may contribute to the production of the first non-heterologous antivenom treatment against bee envenomation. Such a treatment may overcome some of the difficulties associated with conventional immunotherapy techniques. Copyright © 2016 Elsevier Ltd. All rights reserved.

Hydrogel Tethering Enhances Interdomain Stabilization of Single-Chain Antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiong, Yijia; Ford, Nicole R.; Hecht, Karen A.

Self-assembly of recombinant proteins within the biosilica of living diatoms represents a means to construct functional materials in a reproducible and scalable manner that enable applications that harness the inherent specificities of proteins to sense and respond to environmental cues. Here we describe the use of a silaffin-derived lysine-rich 39 amino-acid targeting sequence (Sil3T8) to direct a single chain fragment variable (scFv) antibody or an enhanced green fluorescent protein (EGFP) to assemble within the biosilica frustule, resulting in abundances in excess of 200,000 proteins per frustule. The fluorescence of either a derivative of trinitrotoluene (TNT) bound to the scFv ormore » the endogenous fluorescence of EGFP was used to monitor pro-tein conformational dynamics, accessibility to external quenchers, binding affinity, and conformational stability. We find that proteins within isolated frustules undergo isotropic rotational motions with two-fold increases in rotational correlation times, which are indicative of weak macromolecular associations within the biosilica. Solvent accessibilities and high-affinity (pM) binding are comparable to those in solution. In contrast to solution conditions, scFv antibod-ies within the biosilica matrix retain their binding affinity in the presence of chaotropic agents (i.e., 8 M urea). These results argue that dramatic increases in protein conforma-tional stability within the biosilica frustule matrices arise through molecular crowding, acting to retain native protein folds and associated functionality to allow the utility of engineered proteins under a range of harsh environmental conditions associated with environmental sensing and industrial catalytic transformations.« less

Making cell-permeable antibodies (Transbody) through fusion of protein transduction domains (PTD) with single chain variable fragment (scFv) antibodies: potential advantages over antibodies expressed within the intracellular environment (Intrabody).

PubMed

Heng, Boon Chin; Cao, Tong

2005-01-01

Over the past decade, there has been growing interest in the use of antibodies against intracellular targets. This is currently achieved through recombinant expression of the single chain variable fragment (scFv) antibody format within the cell, which is commonly referred to as an intrabody. This possesses a number of inherent advantages over RNA interference (iRNA). Firstly, the high specificity and affinity of intrabodies to target antigens is well-established, whereas iRNA has been frequently shown to exert multiple non-specific effects. Secondly, intrabodies being proteins possess a much longer active half-life compared to iRNA. Thirdly, when the active half-life of the intracellular target molecule is long, gene silencing through iRNA would be slow to yield any effect, whereas the effects of intrabody expression would be almost instantaneous. Lastly, it is possible to design intrabodies to block certain binding interactions of a particular target molecule, while sparing others. There is, however, various technical challenges faced with intrabody expression through the application of recombinant DNA technology. In particular, protein conformational folding and structural stability of the newly-synthesized intrabody within the cell is affected by reducing conditions of the intracellular environment. Also, there are overwhelming safety concerns surrounding the application of transfected recombinant DNA in human clinical therapy, which is required to achieve intrabody expression within the cell. Of particular concern are the various viral-based vectors that are commonly-used in genetic manipulation. A novel approach around these problems would be to look at the possibility of fusing protein transduction domains (PTD) to scFv antibodies, to create a 'cell-permeable' antibody or 'Transbody'. PTD are short peptide sequences that enable proteins to translocate across the cell membrane and be internalized within the cytosol, through atypical secretory and

Fv1-like restriction of N-tropic replication-competent murine leukaemia viruses in mCAT-1-expressing human cells.

PubMed

Aagaard, Lars; Mikkelsen, Jacob Giehm; Warming, Søren; Duch, Mogens; Pedersen, Finn Skou

2002-02-01

To study the replication of murine leukaemia viruses in human cells we have used full-length as well as EGFP-tagged ecotropic viruses in combination with mCAT-1-expressing human cells. We present results showing that N-tropic murine leukaemia viruses are restricted in both infection and replication in such cells while B-tropic viruses, modified at capsid position 110, escape restriction. These results support a recently reported Fv1-like restriction in mammalian cells. We extend the analysis of Fv1-like restriction by demonstrating that NB-tropic viruses also escape restriction and human mCAT-1-expressing cells are thus similar to murine Fv1(b) cells with respect to infection though the ecotropic receptor pathway.

Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation.

PubMed

Tillotson, Benjamin J; Goulatis, Loukas I; Parenti, Isabelle; Duxbury, Elizabeth; Shusta, Eric V

2015-01-01

The equilibrium binding affinity of receptor-ligand or antibody-antigen pairs may be modulated by protonation of histidine side-chains, and such pH-dependent mechanisms play important roles in biological systems, affecting molecular uptake and trafficking. Here, we aimed to manipulate cellular transport of single-chain antibodies (scFvs) against the transferrin receptor (TfR) by engineering pH-dependent antigen binding. An anti-TfR scFv was subjected to histidine saturation mutagenesis of a single CDR. By employing yeast surface display with a pH-dependent screening pressure, scFvs having markedly increased dissociation from TfR at pH 5.5 were identified. The pH-sensitivity generally resulted from a central cluster of histidine residues in CDRH1. When soluble, pH-sensitive, scFv clone M16 was dosed onto live cells, the internalized fraction was 2.6-fold greater than scFvs that lacked pH-sensitive binding and the increase was dependent on endosomal acidification. Differences in the intracellular distribution of M16 were also observed consistent with an intracellular decoupling of the scFv M16-TfR complex. Engineered pH-sensitive TfR binding could prove important for increasing the effectiveness of TfR-targeted antibodies seeking to exploit endocytosis or transcytosis for drug delivery purposes.

Engineering an Anti-Transferrin Receptor ScFv for pH-Sensitive Binding Leads to Increased Intracellular Accumulation

PubMed Central

Tillotson, Benjamin J.; Goulatis, Loukas I.; Parenti, Isabelle; Duxbury, Elizabeth; Shusta, Eric V.

2015-01-01

The equilibrium binding affinity of receptor-ligand or antibody-antigen pairs may be modulated by protonation of histidine side-chains, and such pH-dependent mechanisms play important roles in biological systems, affecting molecular uptake and trafficking. Here, we aimed to manipulate cellular transport of single-chain antibodies (scFvs) against the transferrin receptor (TfR) by engineering pH-dependent antigen binding. An anti-TfR scFv was subjected to histidine saturation mutagenesis of a single CDR. By employing yeast surface display with a pH-dependent screening pressure, scFvs having markedly increased dissociation from TfR at pH 5.5 were identified. The pH-sensitivity generally resulted from a central cluster of histidine residues in CDRH1. When soluble, pH-sensitive, scFv clone M16 was dosed onto live cells, the internalized fraction was 2.6-fold greater than scFvs that lacked pH-sensitive binding and the increase was dependent on endosomal acidification. Differences in the intracellular distribution of M16 were also observed consistent with an intracellular decoupling of the scFv M16-TfR complex. Engineered pH-sensitive TfR binding could prove important for increasing the effectiveness of TfR-targeted antibodies seeking to exploit endocytosis or transcytosis for drug delivery purposes. PMID:26713870

Phage display vectors for in vivo recombination of immunoglobulin heavy and light chain genes to make large combinatorial libraries.

PubMed

Tsurushita, N; Fu, H; Warren, C

1996-06-12

New phage display vectors for in vivo recombination of immunoglobulin (Ig) heavy (VH) and light (VL) chain variable genes, to make single-chain Fv fragments (scFv), were constructed. The VH and VL genes of monoclonal antibody (mAb) EP-5C7, which binds to both human E- and P-selectin, were cloned into a pUC19-derived plasmid vector, pCW93, and a pACYC184-derived phagemid vector, pCW99, respectively. Upon induction of Cre recombinase (phage P1 recombinase), the VH and VL genes were efficiently recombined into the same plasmid via the two loxP sites (phage P1 recombination sites), one located downstream from a VH gene in pCW93 and another upstream from a VL gene in pCW99. In the resulting phagemid, the loxP sequence also encodes a polypeptide linker connecting the VH and VL domains to form a scFv of EP-5C7. Whether expressed on the phage surface or as a soluble form, the EP-5C7 scFv showed specific binding to human E- and P-selectin. This phagemid vector system provides a way to recombine VH and VL gene libraries efficiently in vivo to make extremely large Ig combinatorial libraries.

Construction, expression and in vitro biological behaviors of Ig scFv fragment in patients with chronic B cell leukemia.

PubMed

Zhu, Lijuan; Liao, Wenjun; Zhu, Huifen; Lei, Ping; Wang, Zhihua; Shao, Jingfang; Zhang, Yue; Shen, Guanxin

2006-01-01

The expression vector of SmIg scFv fragment was constructed in patient with B cell chronic lymphocyte leukemia (B-CLL) and expressed in E. coli to obtain scFv fragment, and the effect of the protein on the proliferation of stimulated peripheral blood mononuclear cells (PBMC) was investigated in vitro. Two pairs of primers were designed, and variable region genes of light chain and heavy chain were amplified by PCR respectively from the pGEM-T vectors previously constructed in our laboratory which containing light chain gene or Fd fragment of heavy chain gene. The PCR product was digested, purified and inserted into pHEN2 vector to construct the soluble expression vector pHEN2-scFv. After the induction by IPTG, the scFv protein was identified by SDS-PAGE electrophoresis and purified by Ni-NTA-Chromatography. MTT was used to determine the effect of purified protein on the proliferation of stimulated PBMC in vitro. Plasmid PCR and restriction enzyme digestion of pHEN2-scFv revealed the pHEN2-scFv vector was constructed successfully. Id-scFv protein was expressed in positive clone after induced by IPTG. SDS-PAGE analysis showed that the relative molecular weight of fusion protein was about 30 kD (1 kD= 0.9921 ku), which was consistent with the theoretically predicted value. Proliferation of PBMC could be induced by purified Id-scFv. It was suggested that the expression vector of SmIg scFv fragment was constructed successfully, and scFv protein was expressed and secreted from E. coli, which could induce proliferation of PBMC. This may lay an experimental foundation for further research of Id-HSP complex vaccine for B-CLL.

Production and characterization of recombinant scFv against digoxin by phage display technology.

PubMed

Alirezapour, Behruz; Rajabibazl, Masoumeh; Rasaee, Mohhamad Javad; Omidfar, Kobra

2013-06-01

The cardiac glycoside digoxin is widely used for the treatment of congestive heart failure and cardiac arrhythmias. Digoxin is a highly toxic drug and consequently is routinely measured in sera of treated patients. In such cases, antibodies are required against digoxin for detection as well as detoxification purposes. To obtain recombinant single chain antibody against digoxin, RNA was extracted from spleen of BALB/c mice immunized with digoxin-BSA and converted to cDNA. The gene fragment corresponding to the variable regions of the repertoire of antibody genes were amplified by PCR. ScFv construct was generated by randomly joining individual heavy- and light-chain variable domains through gene splicing by overlapping extension PCR. Recombinant phage library expressing scFv polypeptides were produced. Phages with higher affinity toward digoxin were selected in the biopanning process. Sensitivity of produced recombinant MAb (AR85) was determined to be about 100 pg/well, while intact MAb (BBA) produced by hybridoma technology (data not shown) was reported to be around 100 pg/well too. The saturation value for recombinant scFv MAb was found to be 1000 ng/well while that for hybridoma MAb was reported to be 10 ng/well. The affinity constant of recombinant MAb (AR85) towards digoxin was also found to be around ka=3.8×10(7) M(-1) while that for hybridoma MAb (BBA) was reported to be ka=2.6×10(8) M(-1).

Human anti-CD30 recombinant antibodies by guided phage antibody selection using cell panning

PubMed Central

Klimka, A; Matthey, B; Roovers, R C; Barth, S; Arends, J-W; Engert, A; Hoogenboom, H R

2000-01-01

In various clinical studies, Hodgkin’s patients have been treated with anti-CD30 immunotherapeutic agents and have shown promising responses. One of the problems that appeared from these studies is the development of an immune response against the non-human therapeutics, which limits repeated administration and reduces efficacy. We have set out to make a recombinant, human anti-CD30 single-chain variable fragment (scFv) antibody, which may serve as a targeting moiety with reduced immunogenicity and more rapid tumour penetration in similar clinical applications. Rather than selecting a naive phage antibody library on recombinant CD30 antigen, we used guided selection of a murine antibody in combination with panning on the CD30-positive cell line L540. The murine monoclonal antibody Ki-4 was chosen as starting antibody, because it inhibits the shedding of the extracellular part of the CD30 antigen. This makes the antibody better suited for CD30-targeting than most other anti-CD30 antibodies. We have previously isolated the murine Ki-4 scFv by selecting a mini-library of hybridoma-derived phage scFv-antibodies via panning on L540 cells. Here, we report that phage display technology was successfully used to obtain a human Ki-4 scFv version by guided selection. The murine variable heavy (VH) and light (VL) chain genes of the Ki-4 scFv were sequentially replaced by human V gene repertoires, while retaining only the major determinant for epitope-specificity: the heavy-chain complementarity determining region 3 (CDR3) of murine Ki-4. After two rounds of chain shuffling and selection by panning on L540 cells, a fully human anti-CD30 scFv was selected. It competes with the parental monoclonal antibody Ki-4 for binding to CD30, inhibits the shedding of the extracellular part of the CD30 receptor from L540 cells and is thus a promising candidate for the generation of anti-CD30 immunotherapeutics. © 2000 Cancer Research Campaign PMID:10901379

Production of anti-amoxicillin ScFv antibody and simulation studying its molecular recognition mechanism for penicillins.

PubMed

Liu, Jing; Zhang, Hui C; Duan, Chang F; Dong, Jun; Zhao, Guo X; Wang, Jian P; Li, Nan; Liu, Jin Z; Li, Yu W

2016-11-01

The molecular recognition mechanism of an antibody for its hapten is very interesting. The objective of this research was to study the intermolecular interactions of an anti-amoxicillin antibody with penicillin drugs. The single chain variable fragment (ScFv) antibody was generated from a hybridoma cell strain excreting the monoclonal antibody for amoxicillin. The recombinant ScFv antibody showed similar recognition ability for penicillins to its parental monoclonal antibody: simultaneous recognizing 11 penicillins with cross-reactivities of 18-107%. The three-dimensional structure of the ScFv antibody was simulated by using homology modeling, and its intermolecular interactions with 11 penicillins were studied by using molecular docking. Results showed that three CDRs are involved in antibody recognition; CDR L3 Arg 100, CDR H3 Tyr226, and CDR H3 Arg 228 were the key contact amino acid residues; hydrogen bonding was the main antibody-drug intermolecular force; and the core structure of penicillin drugs was the main antibody binding position. These results could explain the recognition mechanism of anti-amoxicillin antibody for amoxicillin and its analogs. This is the first study reporting the production of ScFv antibody for penicillins and stimulation studying its recognition mechanism.

Potent Inhibition of Human Immunodeficiency Virus Type 1 Replication by an Intracellular Anti-Rev Single-Chain Antibody

NASA Astrophysics Data System (ADS)

Duan, Lingxun; Bagasra, Omar; Laughlin, Mark A.; Oakes, Joseph W.; Pomerantz, Roger J.

1994-05-01

Human immunodeficiency virus type 1 (HIV-1) has a complex life cycle, which has made it a difficult target for conventional therapeutic modalities. A single-chain antibody moiety, directed against the HIV-1 regulatory protein Rev, which rescues unspliced viral RNA from the nucleus of infected cells, has now been developed. This anti-Rev single-chain construct (SFv) consists of both light and heavy chain variable regions of an anti-Rev monoclonal antibody, which, when expressed intracellularly within human cells, potently inhibits HIV-1 replication. This intracellular SFv molecule is demonstrated to specifically antagonize Rev function. Thus, intracellular SFv expression, against a retroviral regulatory protein, may be useful as a gene therapeutic approach to combat HIV-1 infections.

Immunoassay for wheat processing quality: utilization of a sandwich assay incorporating an immobilized single-chain fragment.

PubMed

Hill, A S; Giersch, T M; Loh, C S; Skerritt, J H

1999-10-01

A single-chain fragment (scFv) was engineered from a monoclonal antibody to high molecular weight glutenin subunits (HMW-GS), wheat flour polypeptides that play a major role in determining the mixing- and extension strength-related properties of dough and its subsequent baking performance. The scFv was expressed in a thioredoxin mutant Escherichia coli strain that allows disulfide bond formation in the cytoplasm and incorporated into a diagnostic test for wheat quality. Although the scFv lacks the more highly conserved antibody constant regions usually involved with immobilization, it was able to be directly immobilized to a polystyrene microwell solid phase without chemical or covalent modification of the protein or solid phase and utilized as a capture antibody in a double-antibody (two-site) immunoassay. In the sandwich assay, increasing HMW-GS concentrations produced increasing assay color, and highly significant correlations were obtained between optical densities obtained in the ELISA using the scFv and the content of large glutenin polymers in flours as well as measures of dough strength as measured by resistance to dough extension in rheological testing. The assay using the scFv was able to be carried out at lower flour sample extract dilutions than that required for a similar assay utilizing a monoclonal capture antibody. This research shows that engineered antibody fragments can be utilized to provide superior assay performance in two-site ELISAs over monoclonal antibodies and is the first application of an engineered antibody to the analysis of food processing quality.

Complementarity determining regions and frameworks contribute to the disulfide bond independent folding of intrinsically stable scFv

PubMed Central

Gąciarz, Anna

2017-01-01

CyDisCo is a system facilitating disulfide bond formation in recombinant proteins in the cytoplasm of Escherichia coli. Previously we screened for soluble expression of single chain antibody fragments (scFv) in the cytoplasm of E. coli in the presence and absence of CyDisCo, with >90% being solubly expressed. Two scFv, those derived from natalizumab and trastuzumab, were solubly produced in high amounts even in the absence of folding catalysts i.e. disulfide bond formation is not critical for their folding. Here we investigate the contribution of the framework and the complementarity determining regions (CDRs) of scFv to the disulfide-independence of folding. We swapped CDRs between four scFv that have different properties, including two scFv that can efficiently fold independently from disulfide bonds and two more disulfide-dependent scFv. To confirm disulfide-independence we generated cysteine to alanine mutants of the disulfide-independent scFv. All of the scFv were tested for soluble expression in the cytoplasm of E. coli in the presence and absence of the oxidative folding catalysts Erv1p and PDI. Eight of the hybrid scFv were solubly produced in the presence of CyDisCo, while seven were solubly produced in the absence of CyDisCo, though the yields were often much lower when CyDisCo was absent. Soluble expression was also observed for scFv natalizumab and trastuzumab containing no cysteines. We compared yields, thermal stability and secondary structure of solubly produced scFv and undertook binding studies by western blotting, dot blotting or surface plasmon resonance of those produced in good yields. Our results indicate that both the CDRs and the framework contribute to the disulfide-dependence of soluble production of scFv, with the CDRs having the largest effect. In addition, there was no correlation between thermal stability and disulfide-dependence of folding and only a weak correlation between the yield of protein and the thermal stability of the

Complementarity determining regions and frameworks contribute to the disulfide bond independent folding of intrinsically stable scFv.

PubMed

Gąciarz, Anna; Ruddock, Lloyd W

2017-01-01

CyDisCo is a system facilitating disulfide bond formation in recombinant proteins in the cytoplasm of Escherichia coli. Previously we screened for soluble expression of single chain antibody fragments (scFv) in the cytoplasm of E. coli in the presence and absence of CyDisCo, with >90% being solubly expressed. Two scFv, those derived from natalizumab and trastuzumab, were solubly produced in high amounts even in the absence of folding catalysts i.e. disulfide bond formation is not critical for their folding. Here we investigate the contribution of the framework and the complementarity determining regions (CDRs) of scFv to the disulfide-independence of folding. We swapped CDRs between four scFv that have different properties, including two scFv that can efficiently fold independently from disulfide bonds and two more disulfide-dependent scFv. To confirm disulfide-independence we generated cysteine to alanine mutants of the disulfide-independent scFv. All of the scFv were tested for soluble expression in the cytoplasm of E. coli in the presence and absence of the oxidative folding catalysts Erv1p and PDI. Eight of the hybrid scFv were solubly produced in the presence of CyDisCo, while seven were solubly produced in the absence of CyDisCo, though the yields were often much lower when CyDisCo was absent. Soluble expression was also observed for scFv natalizumab and trastuzumab containing no cysteines. We compared yields, thermal stability and secondary structure of solubly produced scFv and undertook binding studies by western blotting, dot blotting or surface plasmon resonance of those produced in good yields. Our results indicate that both the CDRs and the framework contribute to the disulfide-dependence of soluble production of scFv, with the CDRs having the largest effect. In addition, there was no correlation between thermal stability and disulfide-dependence of folding and only a weak correlation between the yield of protein and the thermal stability of the

[Preparation of monoclonal antibody against 4-amylphenol and homology modeling of its Fv fragment].

PubMed

Cheng, Lei; Wu, Haizhen; Fei, Jing; Zhang, Lujia; Ye, Jiang; Zhang, Huizhan

2017-03-01

Objective To prepare and characterize a monoclonal antibody (mAb) against 4-amylphenol (4-AP), clone its cDNA sequence and make homology modeling for its Fv fragment. Methods A high-affinity anti-4-AP mAb was generated from a hybridoma cell line F10 using electrofusion between splenocytes from APA-BSA-immunized mouse and Sp2/0 myeloma cells. Then we extracted the mRNA of F10 cells and cloned the cDNA of mAb. The homology modeling and molecular docking of its Fv fragment was conducted with biological software. Results Under the optimum conditions, the ic-ELISA equation was y=A 2 +(A 1 -A 2 )/(1+(x/x 0 ) p ) (A 1 =1.28; A 2 =-0.066; x 0 =12560.75; p=0.74) with a correlation coefficient (R 2 ) of 0.997. The lowest detectable limit was 0.65 μg/mL. The heavy and light chains of mAb respectively belonged to IgG1 and Kappa. The homology modeling and molecular docking studies revealed that the binding of 4-Ap and mAb was attributed to the hydrogen bond and hydrophobic interactions. Conclusion The study successfully established a stable 4-AP mAb-secreting hybridoma cell line. The study on spatial structure of Fv fragment using homology modeling provided a reference for the development and design of single chain variable fragments.

Crystal structures of a therapeutic single chain antibody in complex with two drugs of abuse—Methamphetamine and 3,4-methylenedioxymethamphetamine

PubMed Central

Celikel, Reha; Peterson, Eric C; Owens, S Michael; Varughese, Kottayil I

2009-01-01

Methamphetamine (METH) is a major drug threat in the United States and worldwide. Monoclonal antibody (mAb) therapy for treating METH abuse is showing exciting promise and the understanding of how mAb structure relates to function will be essential for future development of these important therapies. We have determined crystal structures of a high affinity anti-(+)-METH therapeutic single chain antibody fragment (scFv6H4, KD= 10 nM) derived from one of our candidate mAb in complex with METH and the (+) stereoisomer of another abused drug, 3,4-methylenedioxymethamphetamine (MDMA), known by the street name “ecstasy.” The crystal structures revealed that scFv6H4 binds to METH and MDMA in a deep pocket that almost completely encases the drugs mostly through aromatic interactions. In addition, the cationic nitrogen of METH and MDMA forms a salt bridge with the carboxylate group of a glutamic acid residue and a hydrogen bond with a histidine side chain. Interestingly, there are two water molecules in the binding pocket and one of them is positioned for a C—H⋯O interaction with the aromatic ring of METH. These first crystal structures of a high affinity therapeutic antibody fragment against METH and MDMA (resolution = 1.9 Å, and 2.4 Å, respectively) provide a structural basis for designing the next generation of higher affinity antibodies and also for carrying out rational humanization. PMID:19760665

Crystal structures of a therapeutic single chain antibody in complex with two drugs of abuse-Methamphetamine and 3,4-methylenedioxymethamphetamine.

PubMed

Celikel, Reha; Peterson, Eric C; Owens, S Michael; Varughese, Kottayil I

2009-11-01

Methamphetamine (METH) is a major drug threat in the United States and worldwide. Monoclonal antibody (mAb) therapy for treating METH abuse is showing exciting promise and the understanding of how mAb structure relates to function will be essential for future development of these important therapies. We have determined crystal structures of a high affinity anti-(+)-METH therapeutic single chain antibody fragment (scFv6H4, K(D)= 10 nM) derived from one of our candidate mAb in complex with METH and the (+) stereoisomer of another abused drug, 3,4-methylenedioxymethamphetamine (MDMA), known by the street name "ecstasy." The crystal structures revealed that scFv6H4 binds to METH and MDMA in a deep pocket that almost completely encases the drugs mostly through aromatic interactions. In addition, the cationic nitrogen of METH and MDMA forms a salt bridge with the carboxylate group of a glutamic acid residue and a hydrogen bond with a histidine side chain. Interestingly, there are two water molecules in the binding pocket and one of them is positioned for a C--H...O interaction with the aromatic ring of METH. These first crystal structures of a high affinity therapeutic antibody fragment against METH and MDMA (resolution = 1.9 A, and 2.4 A, respectively) provide a structural basis for designing the next generation of higher affinity antibodies and also for carrying out rational humanization.

Biocompatible coupling of therapeutic fusion proteins to human erythrocytes

PubMed Central

Villa, Carlos H.; Pan, Daniel C.; Johnston, Ian H.; Greineder, Colin F.; Walsh, Landis R.; Hood, Elizabeth D.; Cines, Douglas B.; Poncz, Mortimer; Siegel, Don L.

2018-01-01

Carriage of drugs by red blood cells (RBCs) modulates pharmacokinetics, pharmacodynamics, and immunogenicity. However, optimal targets for attaching therapeutics to human RBCs and adverse effects have not been studied. We engineered nonhuman-primate single-chain antibody fragments (scFvs) directed to human RBCs and fused scFvs with human thrombomodulin (hTM) as a representative biotherapeutic cargo (hTM-scFv). Binding fusions to RBCs on band 3/glycophorin A (GPA; Wright b [Wrb] epitope) and RhCE (Rh17/Hr0 epitope) similarly endowed RBCs with hTM activity, but differed in their effects on RBC physiology. scFv and hTM-scFv targeted to band 3/GPA increased membrane rigidity and sensitized RBCs to hemolysis induced by mechanical stress, while reducing sensitivity to hypo-osmotic hemolysis. Similar properties were seen for other ligands bound to GPA and band 3 on human and murine RBCs. In contrast, binding of scFv or hTM-scFv to RhCE did not alter deformability or sensitivity to mechanical and osmotic stress at similar copy numbers bound per RBCs. Contrasting responses were also seen for immunoglobulin G antibodies against band 3, GPA, and RhCE. RBC-bound hTM-scFv generated activated protein C (APC) in the presence of thrombin, but RhCE-targeted hTM-scFv demonstrated greater APC generation per bound copy. Both Wrb- and RhCE-targeted fusion proteins inhibited fibrin deposition induced by tumor necrosis factor-α in an endothelialized microfluidic model using human whole blood. RhCE-bound hTM-scFv more effectively reduced platelet and leukocyte adhesion, whereas anti-Wrb scFv appeared to promote platelet adhesion. These data provide a translational framework for the development of engineered affinity ligands to safely couple therapeutics to human RBCs. PMID:29365311

[Cloning of VH and VL Gene of Human anti-IL1RAP McAb and Construction of Recombinant Chimeric Receptor].

PubMed

Yin, Ling-Ling; Ruan, Su-Hong; Tian, Yu; Zhao, Kai; Xu, Kai Lin

2015-10-01

To clone the variable region genes of human anti-IL1RAP (IL-1 receptor accessory protein) monoclonal antibodies (McAb) and to construct IL1RAP chimeric antigen receptors (CARs). The VH and VL DNA of IL1RAP single chain antibodies were amplified by RACE and overlap extension PCR from total RNA extracted from 3H6E10 and 10D8A7 hybridoma and ligated into specific IL1RAP single-chain variable fragments (scFv). CD8α transmembrane domain, CD137 intracellular domain, TCR ζ chain, human CD8α signal peptide and scFv-anti-IL1RAP were cloned into plasmid LV-lac. Recombinant lentiviruses were generated by co-transfection of recombinant plasmid LV-lac, pMD2. G, and psPAX2 helper vectors into 293FT packing cells. The VH and VL genes of 2 human anti-IL1RAP McAb were acquired. The 3H6E10 VH and VL genes consisted of 402 bp and 393 bp encoding 134 and 131 aminoacid residues, respectively; 10D8A7 VH and VL genes consisted of 423 bp and 381 bp encoding 141 and 127 amine acid residues, respectively. Recombinant expression vertors LV-3H6E10 scFv-ICD and LV-10D8A7 scFv-ICD (ICD: CD8α transmembrane domain-CD137 intracellular domain-TCR ζ chain) were constructed. The target fragments were demonstrated by sequencing analysis. Recombinant plasmids were transfected into 293FT cells and lentiviral particles were acquired. Human anti-IL1RAP recombinant receptors are constructed successfully and lay a good foundation for the construction of IL1RAP-CAR killer T cell vaccine.

The catalytic activity of a recombinant single chain variable fragment nucleic acid-hydrolysing antibody varies with fusion tag and expression host.

PubMed

Lee, Joungmin; Kim, Minjae; Seo, Youngsil; Lee, Yeonjin; Park, Hyunjoon; Byun, Sung June; Kwon, Myung-Hee

2017-11-01

The antigen-binding properties of single chain Fv antibodies (scFvs) can vary depending on the position and type of fusion tag used, as well as the host cells used for expression. The issue is even more complicated with a catalytic scFv antibody that binds and hydrolyses a specific antigen. Herein, we investigated the antigen-binding and -hydrolysing activities of the catalytic anti-nucleic acid antibody 3D8 scFv expressed in Escherichia coli or HEK293f cells with or without additional amino acid residues at the N- and C-termini. DNA-binding activity was retained in all recombinant forms. However, the DNA-hydrolysing activity varied drastically between forms. The DNA-hydrolysing activity of E. coli-derived 3D8 scFvs was not affected by the presence of a C-terminal human influenza haemagglutinin (HA) or His tag. By contrast, the activity of HEK293f-derived 3D8 scFvs was completely lost when additional residues were included at the N-terminus and/or when a His tag was incorporated at the C-terminus, whereas a HA tag at the C-terminus did not diminish activity. Thus, we demonstrate that the antigen-binding and catalytic activities of a catalytic antibody can be separately affected by the presence of additional residues at the N- and C-termini, and by the host cell type. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Identification of Fusarium virguliforme FvTox1-Interacting Synthetic Peptides for Enhancing Foliar Sudden Death Syndrome Resistance in Soybean

PubMed Central

Wang, Bing; Swaminathan, Sivakumar; Bhattacharyya, Madan K.

2015-01-01

Soybean is one of the most important crops grown across the globe. In the United States, approximately 15% of the soybean yield is suppressed due to various pathogen and pests attack. Sudden death syndrome (SDS) is an emerging fungal disease caused by Fusarium virguliforme. Although growing SDS resistant soybean cultivars has been the main method of controlling this disease, SDS resistance is partial and controlled by a large number of quantitative trait loci (QTL). A proteinacious toxin, FvTox1, produced by the pathogen, causes foliar SDS. Earlier, we demonstrated that expression of an anti-FvTox1 single chain variable fragment antibody resulted in reduced foliar SDS development in transgenic soybean plants. Here, we investigated if synthetic FvTox1-interacting peptides, displayed on M13 phage particles, can be identified for enhancing foliar SDS resistance in soybean. We screened three phage-display peptide libraries and discovered four classes of M13 phage clones displaying FvTox1-interacting peptides. In vitro pull-down assays and in vivo interaction assays in yeast were conducted to confirm the interaction of FvTox1 with these four synthetic peptides and their fusion-combinations. One of these peptides was able to partially neutralize the toxic effect of FvTox1 in vitro. Possible application of the synthetic peptides in engineering SDS resistance soybean cultivars is discussed. PMID:26709700

Enzyme-linked immunosorbent assay with monoclonal and single-chain variable fragment antibodies selective to coplanar polychlorinated biphenyls.

PubMed

Inui, Hideyuki; Takeuchi, Tetsuya; Uesugi, Akari; Doi, Fumito; Takai, Mikio; Nishi, Kosuke; Miyake, Shiro; Ohkawa, Hideo

2012-02-22

Coplanar polychlorinated biphenyls (Co-PCBs) consisting of non-ortho and mono-ortho-chlorinated PCBs are dioxin-like compounds and cause wide contamination in the environment. To monitor Co-PCB residues, it was attempted to establish an enzyme-linked immunosorbent assay (ELISA) with monoclonal and recombinant antibodies selective to Co-PCBs. When 3,3',5,5'-tetrachlorobiphenoxybutyric acid (PCBH)-keyhole limpet hemocyanin conjugate was immunized into mice, two monoclonal antibodies, Mab-0217 and Mab-4444, were obtained. 3,3',5,5'-Tetrachlorobiphenyl (PCB80) was determined with an IC(50) value of 2.6 and 0.46 ng mL(-1) in ELISA based on Mab-0217 and Mab-4444, respectively. Mab-4444 cross-reacted with Co-PCB congeners, except for PCB77 and PCB81. Mab-0217 reacted with PCB80 and cross-reacted with PCB111. A single-chain variable fragment (scFv) antibody derived from Mab-4444 was produced in recombinant Escherichia coli cells. The scFv antibody showed nearly the same sensitivity toward PCBH as the parent monoclonal antibody in ELISA. These results clearly suggested that Mab-4444 and its scFv antibodies were suitable for monitoring the representative congeners of Co-PCBs.

Conversion of scFv peptide-binding specificity for crystal chaperone development

PubMed Central

Pai, Jennifer C.; Culver, Jeffrey A.; Drury, Jason E.; Motani, Rakesh S.; Lieberman, Raquel L.; Maynard, Jennifer A.

2011-01-01

In spite of advances in protein expression and purification over the last decade, many proteins remain recalcitrant to structure determination by X-ray crystallography. One emerging tactic to obtain high-quality protein crystals for structure determination, particularly in the case of membrane proteins, involves co-crystallization with a protein-specific antibody fragment. Here, we report the development of new recombinant single-chain antibody fragments (scFv) capable of binding a specific epitope that can be introduced into internal loops of client proteins. The previously crystallized hexa-histidine-specific 3D5 scFv antibody was modified in the complementary determining region and by random mutagenesis, in conjunction with phage display, to yield scFvs with new biochemical characteristics and binding specificity. Selected variants include those specific for the hexa-histidine peptide with increased expression, solubility (up to 16.6 mg/ml) and sub-micromolar affinity, and those with new specificity for the EE hexa-peptide (EYMPME) and nanomolar affinity. Complexes of one such chaperone with model proteins harboring either an internal or a terminal EE tag were isolated by gel filtration. The 3.1 Å resolution structure of this chaperone reveals a binding surface complementary to the EE peptide and a ∼52 Å channel in the crystal lattice. Notably, in spite of 85% sequence identity, and nearly identical crystallization conditions, the engineered scFv crystallizes in a different space group than the parent 3D5 scFv, and utilizes two new crystal contacts. These engineered scFvs represent a new class of chaperones that may eliminate the need for de novo identification of candidate chaperones from large antibody libraries. PMID:21217145

Single Chain Antibodies as Tools to Study transforming growth factor-β-Regulated SMAD Proteins in Proximity Ligation-Based Pharmacological Screens.

PubMed

Blokzijl, Andries; Zieba, Agata; Hust, Michael; Schirrmann, Thomas; Helmsing, Saskia; Grannas, Karin; Hertz, Ellen; Moren, Anita; Chen, Lei; Söderberg, Ola; Moustakas, Aristidis; Dübel, Stefan; Landegren, Ulf

2016-06-01

The cellular heterogeneity seen in tumors, with subpopulations of cells capable of resisting different treatments, renders single-treatment regimens generally ineffective. Accordingly, there is a great need to increase the repertoire of drug treatments from which combinations may be selected to efficiently target sets of pathological processes, while suppressing the emergence of resistance mutations. In this regard, members of the TGF-β signaling pathway may furnish new, valuable therapeutic targets. In the present work, we developed in situ proximity ligation assays (isPLA) to monitor the state of the TGF-β signaling pathway. Moreover, we extended the range of suitable affinity reagents for this analysis by developing a set of in-vitro-derived human antibody fragments (single chain fragment variable, scFv) that bind SMAD2 (Mothers against decapentaplegic 2), 3, 4, and 7 using phage display. These four proteins are all intracellular mediators of TGF-β signaling. We also developed an scFv specific for SMAD3 phosphorylated in the linker domain 3 (p179 SMAD3). This phosphorylation has been shown to inactivate the tumor suppressor function of SMAD3. The single chain affinity reagents developed in the study were fused tocrystallizable antibody fragments (Fc-portions) and expressed as dimeric IgG-like molecules having Fc domains (Yumabs), and we show that they represent valuable reagents for isPLA.Using these novel assays, we demonstrate that p179 SMAD3 forms a complex with SMAD4 at increased frequency during division and that pharmacological inhibition of cyclin-dependent kinase 4 (CDK4)(1) reduces the levels of p179SMAD3 in tumor cells. We further show that the p179SMAD3-SMAD4 complex is bound for degradation by the proteasome. Finally, we developed a chemical screening strategy for compounds that reduce the levels of p179SMAD3 in tumor cells with isPLA as a read-out, using the p179SMAD3 scFv SH544-IIC4. The screen identified two kinase inhibitors, known inhibitors

Human IP10-scFv and DC-induced CTL synergistically inhibit the growth of glioma in a xenograft model.

PubMed

Wang, Xuan; Zhang, Fang-Cheng; Zhao, Hong-Yang; Lu, Xiao-Ling; Sun, Yun; Xiong, Zhi-Yong; Jiang, Xiao-Bing

2014-08-01

The epidermal growth factor receptor (EGFR) mutant of EGFRvIII is highly expressed in glioma cells, and the EGFRvIII-specific dendritic cell (DC)-induced tumor antigen-specific CD8(+) cytotoxic T lymphocytes (CTLs) may hold promise in cancer immunotherapy. Interferon (IFN)-γ-inducible protein (IP)-10 (IP-10) is a potent inhibitor of angiogenesis and can recruit CXCR3(+) T cells, including CD8(+) T cells, which are important for the control of tumor growth. In this study, we assessed if the combination of IP10-EGFRvIIIscFv with DC-induced CTLs would improve the therapeutic antitumor efficacy. IP10-scFv was generated by linking the human IP-10 gene with the DNA fragment for anti-EGFRvIIIscFv with a (Gly4Ser)3 flexible linker, purified by affinity chromatography, and characterized for its anti-EGFRvIII immunoreactivity and chemotactic activity. DCs were isolated from human peripheral blood monocyte cells and pulsed with EGFRvIII-peptide, then co-cultured with autologous CD8(+) T cells. BALB/c-nu mice were inoculated with human glioma U87-EGFRvIII cells in the brain and treated intracranially with IP10-scFv and/or intravenously with DC-induced CTLs for evaluating the therapeutic effect. Treatment with both IP10-scFv and EGFRvIII peptide-pulsed, DC-induced CTL synergistically inhibited the growth of glioma and prolonged the survival of tumor-bearing mice, which was accompanied by the inhibition of tumor angiogenesis and enhancement of cytotoxicity, thereby increasing the numbers of brain-infiltrating lymphocytes (BILs) and prolonging the residence time of CTLs in the tumor.

Screening of synthetic phage display scFv libraries yields competitive ligands of human leptin receptor.

PubMed

Molek, Peter; Vodnik, Miha; Strukelj, Borut; Bratkovič, Tomaž

2014-09-26

Initially considered the main endogenous anorexigenic factor, fat-derived leptin turned out to be a markedly pleiotropic hormone, influencing diverse physiological processes. Moreover, hyperleptinemia in obese individuals has been linked to the onset or progression of serious disorders, such as cancer, autoimmune diseases, and atherosclerosis, and antagonizing peripheral leptin's signalization has been shown to improve these conditions. To develop an antibody-based leptin antagonist we have devised a tailored panning procedure and screened two phage display libraries of single chain variable antibody fragments (scFvs) against recombinant leptin receptor. One of the scFvs was expressed in Escherichia coli and its interaction with leptin receptor was characterized in more detail. It was found to recognize a discontinuous epitope and to compete with leptin for receptor binding with IC50 and Kd values in the nanomolar range. The reported scFv represents a lead for development of leptin antagonists that may ultimately find use in therapy of various hyperleptinemia-related disorders. Copyright © 2014 Elsevier Inc. All rights reserved.

Single-step colony assay for screening antibody libraries.

PubMed

Kato, Mieko; Hanyu, Yoshiro

2017-08-10

We describe a method, single-step colony assay, for simple and rapid screening of single-chain Fv fragment (scFv) libraries. Colonies of Escherichia coli expressing the scFv library are formed on a hydrophilic filter that is positioned in contact with a membrane coated with an antigen. scFv expression is triggered upon treatment of colonies with an induction reagent, following which scFvs are secreted from the cells and diffused to the antigen-coated membrane. scFvs that exhibit binding affinity for the antigen are captured by the membrane-immobilized antigen. Lastly, detection of scFv binding of the antigen on the membrane allows identification of the clones on the filter that express antigen-specific scFvs. We tested this methodology by using an anti-rabbit IgG scFv, scFv(A10B), and a rat immune scFv library. Experiments conducted using scFv(A10B) revealed that this method improves scFv expression during the colony assay. By using our method to screen an immune library of 3×10 3 scFv clones, we established several clones exhibiting affinity for the antigen. Moreover, we tested 7 other antigens, including peptides, and successfully identified positive clones. We believe that this simple procedure and controlled scFv expression of the single-step colony assay could make the antibody screening both rapid and reliable and lead to successful isolation of positive clones from antibody libraries. Copyright © 2017 Elsevier B.V. All rights reserved.

Enzymatic single-chain antibody tagging: a universal approach to targeted molecular imaging and cell homing in cardiovascular disease.

PubMed

Ta, H T; Prabhu, S; Leitner, E; Jia, F; von Elverfeldt, D; Jackson, Katherine E; Heidt, T; Nair, A K N; Pearce, H; von Zur Muhlen, C; Wang, X; Peter, K; Hagemeyer, C E

2011-08-05

Antibody-targeted delivery of imaging agents can enhance the sensitivity and accuracy of current imaging techniques. Similarly, homing of effector cells to disease sites increases the efficacy of regenerative cell therapy while reducing the number of cells required. Currently, targeting can be achieved via chemical conjugation to specific antibodies, which typically results in the loss of antibody functionality and in severe cell damage. An ideal conjugation technique should ensure retention of antigen-binding activity and functionality of the targeted biological component. To develop a biochemically robust, highly reproducible, and site-specific coupling method using the Staphylococcus aureus sortase A enzyme for the conjugation of a single-chain antibody (scFv) to nanoparticles and cells for molecular imaging and cell homing in cardiovascular diseases. This scFv specifically binds to activated platelets, which play a pivotal role in thrombosis, atherosclerosis, and inflammation. The conjugation procedure involves chemical and enzyme-mediated coupling steps. The scFv was successfully conjugated to iron oxide particles (contrast agents for magnetic resonance imaging) and to model cells. Conjugation efficiency ranged between 50% and 70%, and bioactivity of the scFv after coupling was preserved. The targeting of scFv-coupled cells and nanoparticles to activated platelets was strong and specific as demonstrated in in vitro static adhesion assays, in a flow chamber system, in mouse intravital microscopy, and in in vivo magnetic resonance imaging of mouse carotid arteries. This unique biotechnological approach provides a versatile and broadly applicable tool for procuring targeted regenerative cell therapy and targeted molecular imaging in cardiovascular and inflammatory diseases and beyond.

Production and characterization of monoclonal antibody and its recombinant single chain variable fragment specific for a food-born mycotoxin, fumonisin B1.

PubMed

Min, Won-Ki; Cho, Young-Jin; Park, Jun-Bock; Bae, Yi-Hyun; Kim, Eun-Jeong; Park, Kyungmoon; Park, Yong-Cheol; Seo, Jin-Ho

2010-01-01

Fumonisin B(1) (FMB(1)) is a food-born mycotoxin produced by Fusarium moniliforme. Monoclonal antibody against FMB(1) (anti-FMB(1) mAb) was produced in the hybridoma DV9, which was established from a BALB/c mouse immunized with bovine serum albumin conjugated FMB(1) (FMB(1)-BSA). A competitive direct enzyme-linked immunosorbent assay (ELISA) showed that anti-FMB(1) mAb has about 10 ppb of minimum FMB(1) detection concentration and 220 ppb of 50% inhibition concentration (IC(50)). Much lower cross-reactivity of anti-FMB(1) mAb on ochratoxin A, aflatoxin B(1) and deoxynivalenol provided that anti-FMB(1) mAb was specific for FMB(1). The gene coding single chain variable fragment against FMB(1) (anti-FMB(1) scFv) was cloned from the hybridoma DV9 and was expressed in recombinant Escherichia coli. Insoluble anti-FMB(1) scFv required optimization of its refolding condition, and hence functional scFv was obtained. By using indirect ELISA, about 12-fold lower binding activity of anti-FMB(1) scFv on FMB(1)-BSA was obtained in comparison with that of the parental mAb.

Identification of inhibitory scFv antibodies targeting fibroblast activation protein utilizing phage display functional screens

PubMed Central

Zhang, Jiping; Valianou, Matthildi; Simmons, Heidi; Robinson, Matthew K.; Lee, Hyung-Ok; Mullins, Stefanie R.; Marasco, Wayne A.; Adams, Gregory P.; Weiner, Louis M.; Cheng, Jonathan D.

2013-01-01

Fibroblast activation protein (FAP) is a serine protease selectively expressed on tumor stromal fibroblasts in epithelial carcinomas and is important in cancer growth, adhesion, and metastases. As FAP enzymatic activity is a potent therapeutic target, we aimed to identify inhibitory antibodies. Using a competitive inhibition strategy, we used phage display techniques to identify 53 single-chain variable fragments (scFvs) after three rounds of panning against FAP. These scFvs were expressed and characterized for binding to FAP by surface plasmon resonance and flow cytometry. Functional assessment of these antibodies yielded an inhibitory scFv antibody, named E3, which could attenuate 35% of FAP cleavage of the fluorescent substrate Ala-Pro-7-amido-4-trifluoromethylcoumarin compared with nonfunctional scFv control. Furthermore, a mutant E3 scFv was identified by yeast affinity maturation. It had higher affinity (4-fold) and enhanced inhibitory effect on FAP enzyme activity (3-fold) than E3. The application of both inhibitory anti-FAP scFvs significantly affected the formation of 3-dimensional FAP-positive cell matrix, as demonstrated by reducing the fibronectin fiber orientation from 41.18% (negative antibody control) to 34.06% (E3) and 36.15% (mutant E3), respectively. Thus, we have identified and affinity-maturated the first scFv antibody capable of inhibiting FAP function. This scFv antibody has the potential to disrupt the role of FAP in tumor invasion and metastasis.—Zhang, J., Valianou, M., Simmons, H., Robinson, M. K., Lee, H.-O., Mullins, S. R., Marasco, W. A., Adams, G. P., Weiner, L. M., Cheng, J. D. Identification of inhibitory ScFv antibodies targeting fibroblast activation protein utilizing phage display functional screens. PMID:23104982

Successful construction and stable expression of an anti-CD45RA scFv-EGFP fusion protein in Chinese hamster ovary cells.

PubMed

Wang, Zhujun; Chen, Yuanyuan; Li, Sisi; Cheng, Yuping; Zhao, Haizhao; Jia, Ming; Luo, Zebin; Tang, Yongmin

2014-02-01

CD45RA has been found highly expressed on leukemia cells and may be a potential target of the disease. In this study, an anti-CD45RA single-chain antibody fragment (scFv3A4) was genetically linked to the N terminus of the enhanced green fluorescent protein (EGFP) to generate a scFv3A4-EGFP fusion protein. The scFv3A4-EGFP with a molecular weight of 57kDa was stably expressed and secreted from the transfected CHO cells through the ER/Golgi-dependent pathway. The fusion protein was soluble in the culture supernatant and the yield was 1350μg/L. Flow cytometry analysis showed that the scFv3A4-EGFP had the same binding site and a very similar reactivity pattern with its parental murine monoclonal antibody (mAb) 3A4. Furthermore, comparing to conventional labeled 3A4-FITC antibody, the scFv3A4-EGFP was more resistant to illumination and more suitable for immunofluorescence histology (IFH) detection. Therefore, the scFv3A4-EGFP fusion protein can be a powerful tool to investigate the targeting of CD45RA on leukemia cells, biological activity of the target and possibly for the genetic manipulation of the antibody. Copyright © 2013 Elsevier Inc. All rights reserved.

Factor V Has Anticoagulant Activity in Plasma in the Presence of TFPIα: Difference between FV1 and FV2.

PubMed

van Doorn, Peter; Rosing, Jan; Duckers, Connie; Hackeng, Tilman M; Simioni, Paolo; Castoldi, Elisabetta

2018-06-04

 Activated factor V (FVa) is a potent procoagulant cofactor in the prothrombinase complex, whereas its precursor factor V (FV) stimulates the inhibition of factor Xa (FXa) by tissue factor pathway inhibitor-α (TFPIα), presumably by promoting TFPIα binding to phospholipids. Plasma FV comprises two glycosylation isoforms (FV1 and FV2) with low and high phospholipid-binding affinity, respectively. The FV1/FV2 ratio is increased in carriers of the FV R2 haplotype.  This article demonstrates the TFPIα-cofactor function of FV in plasma and compares FV1 and FV2.  Thrombin generation at low TF concentration was measured in FV-depleted plasma reconstituted with 0 to 100% FV, FV1 or FV2, and in 122 individuals genotyped for the R2 haplotype. The TFPIα-cofactor activities of FV1 and FV2 were also investigated in a model system of TFPIα-mediated FXa inhibition.  In the FV titration, thrombin generation first increased (up to 5% FV) and then progressively decreased at higher FV concentrations. This anticoagulant effect of FV, which was also observed with FV2 but not with FV1, was largely abolished by anti-TFPIα antibodies, suggesting that it reflects TFPIα-cofactor activity of FV. In the model system of TFPIα-mediated FXa inhibition, FV2 was a more potent TFPIα-cofactor than FV1, in line with their respective phospholipid affinities. Accordingly, FV R2 carriers had higher thrombin generation than non-carriers, even after correction for demographics and plasma levels of coagulation factors and inhibitors.  FV (and particularly its FV2 isoform) contributes to the TFPIα-dependent down-regulation of thrombin generation in plasma triggered with low TF. Schattauer GmbH Stuttgart.

A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.

PubMed

Ordóñez, Adriana; Pérez, Juan; Tan, Lu; Dickens, Jennifer A; Motamedi-Shad, Neda; Irving, James A; Haq, Imran; Ekeowa, Ugo; Marciniak, Stefan J; Miranda, Elena; Lomas, David A

2015-06-01

Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin. © FASEB.

Anti-CHMP5 single chain variable fragment antibody retrovirus infection induces programmed cell death of AML leukemic cells in vitro.

PubMed

Wang, Hai-rong; Xiao, Zhen-yu; Chen, Miao; Wang, Fei-long; Liu, Jia; Zhong, Hua; Zhong, Ji-hua; Ou-Yang, Ren-rong; Shen, Yan-lin; Pan, Shu-ming

2012-06-01

Over-expressed CHMP5 was found to act as oncogene that probably participated in leukemogenesis. In this study, we constructed the CHMP5 single chain variable fragment antibody (CHMP5-scFv) retrovirus and studied the changes of programmed cell death (PCD) of AML leukemic cells after infection by the retrovirus. The anti-CHMP5 KC14 hybridoma cell line was constructed to generate monoclonal antibody of CHMP5. The protein expression of CHMP5 was studied using immunofluorescence analysis. pMIG-CHMP5 scFv antibody expressible retroviral vector was constructed to prepare CHMP5-scFv retrovirus. AML leukemic U937 cells were infected with the retrovirus, and programmed cell death was studied using confocal microscope, FCM and Western blot. We obtained a monoclonal antibody of CHMP5, and found the expression of CHMP5 was up-regulated in the leukemic cells. After U937 cells were infected with CHMP5-scFv retrovirus, CHMP5 protein was neutralized. Moreover, the infection resulted in a significant increase in apoptosis and necrosis of U937 cells. In U937 cells infected with CHMP5-scFv retrovirus, apoptosis-inducing factor (AIF)-mediated caspase-independent necrotic PCD was activated, but autophagic programmed cell death was not observed. Neither the intrinsic nor extrinsic apoptotic PCD pathway was activated. The granzyme B/perforin-mediated caspase-dependent apoptotic PCD pathway was not activated. CHMP5-scFv retrovirus can neutralize the abnormally high levels of the CHMP5 protein in the cytosol of AML leukemic U937 cells, thereby inducing the programmed cell death of the leukemic cells via AIF-mediated caspase-independent necrosis and apoptosis.

Refolding of autodisplayed anti-NEF scFv through oxidation with glutathione for immunosensors.

PubMed

Bong, Ji-Hong; Song, Hyun-Woo; Kim, Tae-Hun; Kang, Min-Jung; Jose, Joachim; Pyun, Jae-Chul

2018-04-15

In this study, a single-domain antibody against negative regulatory factor (anti-NEF scFv) was autodisplayed on the outer membrane of Escherichia coli and used to detect NEF in an immunoassay based on fluorescence-activated cell sorting, enzyme-linked immunosorbent assay, and surface plasmon resonance biosensors. Next, the autodisplayed single-domain antibody was oxidized to form disulfide bonds by using glutathione, and the change in NEF-binding activity of anti-NEF scFv was analyzed by fluorescence-activated cell sorting-based immunoassay, chromogenic immunoassay, and surface plasmon resonance biosensor. For each type of immunoassays the anti-NEF scFv on the isolated outer membrane showed more NEF binding activity after the disulfide bond formation by glutathione. To determine the role of cysteines in anti-NEF scFv, three mutants were prepared, and the NEF binding activity of mutants was compared with that of wild-type anti-NEF scFv in a competitive immunoassay based on FACS. In these mutant studies, the refolding process of autodisplayed anti-NEF scFv by following oxidation via GSH/GSSG revealed that disulfide bonds formed and increased NEF binding activity. Copyright © 2017 Elsevier B.V. All rights reserved.

Screening for single-chain variable fragment antibodies against multiple Cry1 toxins from an immunized mouse phage display antibody library.

PubMed

Dong, Sa; Bo, Zongyi; Zhang, Cunzheng; Feng, Jianguo; Liu, Xianjin

2018-04-01

Single-chain variable fragment (scFv) is a kind of antibody that possess only one chain of the complete antibody while maintaining the antigen-specific binding abilities and can be expressed in prokaryotic system. In this study, scFvs against Cry1 toxins were screened out from an immunized mouse phage displayed antibody library, which was successfully constructed with capacity of 6.25 × 10 7  CFU/mL. Using the mixed and alternative antigen coating strategy and after four rounds of affinity screening, seven positive phage-scFvs against Cry1 toxins were selected and characterized. Among them, clone scFv-3H9 (MG214869) showing relative stable and high binding abilities to six Cry1 toxins was selected for expression and purification. SDS-PAGE indicated that the scFv-3H9 fragments approximately 27 kDa were successfully expressed in Escherichia coli HB2151 strain. The purified scFv-3H9 was used to establish the double antibody sandwich enzyme-linked immunosorbent assay method (DAS-ELISA) for detecting six Cry1 toxins, of which the lowest detectable limits (LOD) and the lowest quantitative limits (LOQ) were 3.14-11.07 and 8.22-39.44 ng mL -1 , respectively, with the correlation coefficient higher than 0.997. The average recoveries of Cry1 toxins from spiked rice leaf samples were ranged from 84 to 95%, with coefficient of variation (CV) less than 8.2%, showing good accuracy for the multi-residue determination of six Cry1 toxins in agricultural samples. This research suggested that the constructed phage display antibody library based on the animal which was immunized with the mixture of several antigens under the same category can be used for the quick and effective screening of generic antibodies.

Comparison of humanized IgG and FvFc anti-CD3 monoclonal antibodies expressed in CHO cells.

PubMed

Serpieri, Flavia; Inocencio, Andre; de Oliveira, Jose Marcelino; Pimenta, Alécio A; Garbuio, Angélica; Kalil, Jorge; Brigido, Marcelo M; Moro, Ana Maria

2010-07-01

Two humanized monoclonal antibody constructs bearing the same variable regions of an anti-CD3 monoclonal antibody, whole IgG and FvFc, were expressed in CHO cells. Random and site-specific integration were used resulting in similar expression levels. The transfectants were selected with appropriate selection agent, and the surviving cells were plated in semi-solid medium for capture with FITC-conjugated anti-human IG antibody and picked with the robotic ClonePix FL. Conditioned media from selected clones were purified by affinity chromatography and characterized by SDS-PAGE, Western-blot, SEC-HPLC, and isoelectric focusing. Binding to the target present in healthy human mononuclear cells was assessed by flow cytometry, as well as by competition between the two constructs and the original murine monoclonal antibody. The humanized constructs were not able to dislodge the murine antibody while the murine anti-CD3 antibody could dislodge around 20% of the FvFc or IgG humanized versions. Further in vitro and in vivo pre-clinical analyses will be carried out to verify the ability of the humanized versions to demonstrate the immunoregulatory profile required for a humanized anti-CD3 monoclonal antibody.

Recombinant scFv antibodies against infectious pancreatic necrosis virus isolated by flow cytometry.

PubMed

Xu, Li-Ming; Zhao, Jing-Zhuang; Liu, Miao; Cao, Yong-Sheng; Yin, Jia-Sheng; Liu, Hong-Bai; Lu, Tongyan

2016-11-01

Infectious pancreatic necrosis is a significant disease of farmed salmonids in China. In this study, a single chain variable fragment (scFv) antibody library derived from rainbow trout (Oncorhynchus mykiss) and viral protein VP2 of a Chinese infectious pancreatic necrosis virus (IPNV) isolate ChRtm213 were co-expressed by a bacterial display technology. The library was subjected to three rounds of screening by flow cytometry (FCM) to select IPNV specific antibodies. Six antibody clones with different mean fluorescence intensities (MFI) were obtained by picking colonies at random. The antibody clones were expressed and purified. The purified IPNV-specific scFv antibodies were used successfully in Western blotting, enzyme linked immunosorbent assay (ELISA) and an immunofluorescence antibody test (IFAT). This method provides a high throughput means to screen an antibody library by flow cytometry, and isolate a panel of antibody that can be used as potential reagents for the detection and study of IPNV that are prevalent in China. Copyright © 2016 Elsevier B.V. All rights reserved.

A human scFv antibody that targets and neutralizes high molecular weight pathogenic amyloid-β oligomers.

PubMed

Sebollela, Adriano; Cline, Erika N; Popova, Izolda; Luo, Kevin; Sun, Xiaoxia; Ahn, Jay; Barcelos, Milena A; Bezerra, Vanessa N; Lyra E Silva, Natalia M; Patel, Jason; Pinheiro, Nathalia R; Qin, Lei A; Kamel, Josette M; Weng, Anthea; DiNunno, Nadia; Bebenek, Adrian M; Velasco, Pauline T; Viola, Kirsten L; Lacor, Pascale N; Ferreira, Sergio T; Klein, William L

2017-07-03

Brain accumulation of soluble oligomers of the amyloid-β peptide (AβOs) is increasingly considered a key early event in the pathogenesis of Alzheimer's disease (AD). A variety of AβO species have been identified, both in vitro and in vivo, ranging from dimers to 24mers and higher order oligomers. However, there is no consensus in the literature regarding which AβO species are most germane to AD pathogenesis. Antibodies capable of specifically recognizing defined subpopulations of AβOs would be a valuable asset in the identification, isolation, and characterization of AD-relevant AβO species. Here, we report the characterization of a human single chain antibody fragment (scFv) denoted NUsc1, one of a number of scFvs we have identified that stringently distinguish AβOs from both monomeric and fibrillar Aβ. NUsc1 readily detected AβOs previously bound to dendrites in cultured hippocampal neurons. In addition, NUsc1 blocked AβO binding and reduced AβO-induced neuronal oxidative stress and tau hyperphosphorylation in cultured neurons. NUsc1 further distinguished brain extracts from AD-transgenic mice from wild type (WT) mice, and detected endogenous AβOs in fixed AD brain tissue and AD brain extracts. Biochemical analyses indicated that NUsc1 targets a subpopulation of AβOs with apparent molecular mass greater than 50 kDa. Results indicate that NUsc1 targets a particular AβO species relevant to AD pathogenesis, and suggest that NUsc1 may constitute an effective tool for AD diagnostics and therapeutics. © 2017 International Society for Neurochemistry.

An affinity improved single-chain antibody from phage display of a library derived from monoclonal antibodies detects fumonisins by immunoassay.

PubMed

Hu, Zu-Quan; Li, He-Ping; Wu, Ping; Li, Ya-Bo; Zhou, Zhu-Qing; Zhang, Jing-Bo; Liu, Jin-Long; Liao, Yu-Cai

2015-03-31

Fumonisin B analogs, particularly FB1, FB2, and FB3, are major mycotoxins found in cereals. Single-chain fragment variable (scFv) antibodies represent a promising alternative immunoassay system. A phage-displayed antibody library derived from four monoclonal antibodies (mAbs) generated against FB1 was used to screen high binding affinity scFv antibodies; the best candidate was designated H2. Surface plasmon resonance measurements confirmed that the H2 scFv displayed a 82-fold higher binding affinity than its parent mAb. Direct competitive enzyme-linked immunosorbent assay demonstrated that the H2 antibody could competitively bind to free FB1, FB2, and FB3, with an IC50 of 0.11, 0.04, and 0.10 μM, respectively; it had no cross-reactivity to deoxynivalenol, nivalenol and aflatoxin. Validation assays with naturally contaminated samples revealed a linear relationship between the H2 antibody-based assay results and chemical analysis results, that could be expressed as y=1.7072x+5.5606 (R(2)=0.8883). Homology modeling of H2 revealed a favorable binding structure highly complementary to the three fumonisins. Molecular docking analyses suggested that the preferential binding of the H2 scFv to FB2 was due to the presence of a hydrogen radical in its R1 position, leading to a proper electrostatic matching and hydrophobic interaction. The H2 scFv antibody can be used for the rapid, accurate, and specific detection of fumonisin contamination in agricultural samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens.

PubMed

Gray, Sean A; Weigel, Kris M; Ali, Ibne K M; Lakey, Annie A; Capalungan, Jeremy; Domingo, Gonzalo J; Cangelosi, Gerard A

2012-01-01

The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv) in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.

A strategy for high-level expression of a single-chain variable fragment against TNFα by subcloning antibody variable regions from the phage display vector pCANTAB 5E into pBV220.

PubMed

Yang, Tao; Yang, Lijun; Chai, Weiran; Li, Renke; Xie, Jun; Niu, Bo

2011-03-01

A phage display single-chain variable fragment (scFv) library against TNFα was constructed using a recombinant phage antibody system (RPAS). The cloned scFv gene was introduced into the phage display vector pCANTAB 5E and expressed in Escherichia coli (E. coli) with a yield of up to 0.15 mg/l of total protein. With the attempt to improve the expression level of TNF-scFv, a strategy was established for subcloning the scFv gene from pCANTAB 5E into the plasmid pBV220. Under the control of a highly efficient tandem P(R)P(L) promoter system, scFv production was increased to 30% of total protein as inclusion bodies. After extraction from the cell pellet by sonication, the inclusion bodies were solubilized and denatured in the presence of 8M urea. Purification of denatured scFv was performed using nickel column chromatography followed by renaturation. The purity and activity of the refolded scFv were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting and by an enzyme-linked immunoabsorbent assay (ELISA). The results reveal that the overall yield of bioactive TNF-scFv from E. coli flask cultures was more than 45 mg/l culture medium and 15 mg/g wet weight cells. The renatured scFv exhibited binding activity similarly to soluble scFv. In conclusion we developed a method to over-express TNF-scFv, which have biological function after purification and renaturation. Copyright © 2010 Elsevier Inc. All rights reserved.

Isolation of phage-display library-derived scFv antibody specific to Listeria monocytogenes by a novel immobilized method.

PubMed

Nguyen, X-H; Trinh, T-L; Vu, T-B-H; Le, Q-H; To, K-A

2018-02-01

To select Listeria monocytogenes-specific single-chain fragment variable (scFv) antibodies from a phage-display library by a novel simple and cost-effective immobilization method. Light expanded clay aggregate (LECA) was used as biomass support matrix for biopanning of a phage-display library to select L. monocytogenes-specific scFv antibody. Four rounds of positive selection against LECA-immobilized L. monocytogenes and an additional subtractive panning against Listeria innocua were performed. The phage clones selected using this panning scheme and LECA-based immobilization method exhibited the ability to bind L. monocytogenes without cross-reactivity toward 10 other non-L. monocytogenes bacteria. One of the selected phage clones was able to specifically recognize three major pathogenic serotypes (1/2a, 1/2b and 4b) of L. monocytogenes and 11 tested L. monocytogenes strains isolated from foods. The LECA-based immobilization method is applicable for isolating species-specific anti-L. monocytogenes scFv antibodies by phage display. The isolated scFv antibody has potential use in development of immunoassay-based methods for rapid detection of L. monocytogenes in food and environmental samples. In addition, the LECA immobilization method described here could feasibly be employed to isolate specific monoclonal antibodies against any given species of pathogenic bacteria from phage-display libraries. © 2017 The Society for Applied Microbiology.

Cell growth inhibition and apoptotic effects of a specific anti-RTFscFv antibody on prostate cancer, but not glioblastoma, cells

PubMed Central

Nejatollahi, Foroogh; Bayat, Payam; Moazen, Bahareh

2017-01-01

Background: Single chain antibody (scFv) has shown interesting results in cancer immunotargeting approaches, due to its advantages over monoclonal antibodies. Regeneration and tolerance factor (RTF) is one of the most important regulators of extracellular and intracellular pH in eukaryotic cells. In this study, the inhibitory effects of a specific anti-RTF scFv were investigated and compared between three types of prostate cancer and two types of glioblastoma cells.  Methods: A phage antibody display library of scFv was used to select specific scFvs against RTF using panning process. The reactivity of a selected scFv was assessed by phage ELISA. The anti-proliferative and apoptotic effects of the antibody on prostate cancer (PC-3, Du-145 and LNCaP) and glioblastoma (U-87 MG and A-172) cell lines were investigated by MTT and Annexin V/PI assays.  Results: A specific scFv with frequency 35% was selected against RTF epitope. This significantly inhibited the proliferation of the prostate cells after 24 h. The percentages of cell viability (using 1000 scFv/cell) were 52, 61 and 73% for PC-3, Du-145 and LNCaP cells, respectively, compared to untreated cells. The antibody (1000 scFv/cell) induced apoptosis at 50, 40 and 25% in PC-3, Du-145 and LNCaP cells, respectively. No growth inhibition and apoptotic induction was detected for U-87 and A172 glioblastoma cells.  Conclusions: Anti-RTFscFv significantly reduced the proliferation of the prostate cancer cells. The inhibition of cell growth and apoptotic induction effects in PC-3 cells were greater than Du-145 and LNCaP cells. This might be due to higher expression of RTF antigen in PC-3 cells and/or better accessibility of RTF to scFv antibody. The resistance of glioblastoma cells to anti-RTF scFv offers the existence of mechanism(s) that abrogate the inhibitory effect(s) of the antibody to RTF. The results suggest that the selected anti-RTF scFv antibody could be an effective new alternative for prostate cancer

[A novel protein equalizer based on single chain variable fragment display M13 phage library for nephropathy patient urine study].

PubMed

Zhao, Peng; Tao, Dingyin; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

2009-05-01

A novel protein equalizer was developed with single chain variable fragment (scFv) library displaying M13 phage covalently bonded on monolithic cryogel. Due to the great number and various kinds of displayed scFv fragments, as well as strong and specific binding capacity between scFv fragments and proteins, a new protein equalizer technology is preferable in the pretreatment of complex protein samples. After the sample dissolved in phosphate buffer solution (PBS), it was repeatedly loaded onto the equalizer for five times, the bound proteins were in sequence eluted by 2 mol/L NaCl and 50 mmol/L Gly-HC1 (pH 2.5) solution, followed by digestion with thrombin. All proteins or peptides collected from each fraction were further analyzed by high performance liquid chromatography-electrospray tandem mass spectrometry (RPLC-ESI-MS/MS) with a serially coupled long microcolumn. Compared with the untreated samples, the identified protein number was increased from 142 to 396. Furthermore, from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis results, it was found that the protein concentration difference was reduced obviously in the eluant of direct sample loading, and most high abundance proteins were identified in the eluant of NaCl. All these results demonstrate that the novel protein equalizer with scFv display M13 phage library immobilized on cyrogel could effectively reduce the dynamic range of proteins in complex samples, enabling the identification of more low abundance proteins.

Methanol induction optimization for scFv antibody fragment production in Pichia pastoris.

PubMed

Cunha, A E; Clemente, J J; Gomes, R; Pinto, F; Thomaz, M; Miranda, S; Pinto, R; Moosmayer, D; Donner, P; Carrondo, M J T

2004-05-20

Fibronectin splice variant ED B (extracellular domain B) is a promising marker for angiogenesis in growing solid tumors. Currently, recombinant antibodies against ED B are being investigated concerning their potential use, for either therapeutic or diagnostic purposes. Single-chain antibody fragments directed against the ED B can be efficiently expressed in Pichia pastoris; thus, a recombinant strain of the methylotropic yeast P. pastoris was used for this work. Three different forms of scFv antibody fragment are found in the supernatant from this fermentation: covalent homodimer, associative homodimer, and monomer. Both homodimeric forms can be converted to the monomeric form (under reducing conditions) and be efficiently radiolabeled, whereas the monomeric form of scFv already present in the supernatant cannot. It was also found that the fraction of protein in the monomeric form is highly dependent on the mode of induction rather than scFv concentration. This suggests that the monomeric form of the scFv present in the supernatant might be a result of events occurring at the expression, secretion, or folding level. A high cell density fermentation protocol was developed by optimizing methanol induction, yielding the highest scFv antibody fragment production rate and product quality; cell concentration at the induction point and specific methanol uptake rate were found to be the most important control variables. A decrease in specific methanol uptake rate led to a higher specific production rate for the scFv antibody fragment (5.4 microg g(cell) h(-1)). Product quality, i.e., percentage of product in a homodimeric form, also increased with the decrease in methanol uptake rate. Furthermore, the volumetric productivity depended on cell concentration at the induction point, increasing with the increase of cell concentration up to 320 g L(-1) wet cell weight (WCW). The reduction of the methanol feeding rate for induction, and consequently of the oxygen uptake rate

Blocking monocyte transmigration in in vitro system by a human antibody scFv anti-CD99. Efficient large scale purification from periplasmic inclusion bodies in E. coli expression system.

PubMed

Moricoli, Diego; Muller, William Anthony; Carbonella, Damiano Cosimo; Balducci, Maria Cristina; Dominici, Sabrina; Watson, Richard; Fiori, Valentina; Weber, Evan; Cianfriglia, Maurizio; Scotlandi, Katia; Magnani, Mauro

2014-06-01

Migration of leukocytes into site of inflammation involves several steps mediated by various families of adhesion molecules. CD99 play a significant role in transendothelial migration (TEM) of leukocytes. Inhibition of TEM by specific monoclonal antibody (mAb) can provide a potent therapeutic approach to treating inflammatory conditions. However, the therapeutic utilization of whole IgG can lead to an inappropriate activation of Fc receptor-expressing cells, inducing serious adverse side effects due to cytokine release. In this regard, specific recombinant antibody in single chain variable fragments (scFvs) originated by phage library may offer a solution by affecting TEM function in a safe clinical context. However, this consideration requires large scale production of functional scFv antibodies and the absence of toxic reagents utilized for solubilization and refolding step of inclusion bodies that may discourage industrial application of these antibody fragments. In order to apply the scFv anti-CD99 named C7A in a clinical setting, we herein describe an efficient and large scale production of the antibody fragments expressed in E. coli as periplasmic insoluble protein avoiding gel filtration chromatography approach, and laborious refolding step pre- and post-purification. Using differential salt elution which is a simple, reproducible and effective procedure we are able to separate scFv in monomer format from aggregates. The purified scFv antibody C7A exhibits inhibitory activity comparable to an antagonistic conventional mAb, thus providing an excellent agent for blocking CD99 signaling. This protocol can be useful for the successful purification of other monomeric scFvs which are expressed as periplasmic inclusion bodies in bacterial systems. Copyright © 2014 Elsevier B.V. All rights reserved.

Unbiased analysis of TCRα/β chains at the single-cell level in human CD8+ T-cell subsets.

PubMed

Sun, Xiaoming; Saito, Masumichi; Sato, Yoshinori; Chikata, Takayuki; Naruto, Takuya; Ozawa, Tatsuhiko; Kobayashi, Eiji; Kishi, Hiroyuki; Muraguchi, Atsushi; Takiguchi, Masafumi

2012-01-01

T-cell receptor (TCR) α/β chains are expressed on the surface of CD8(+) T-cells and have been implicated in antigen recognition, activation, and proliferation. However, the methods for characterization of human TCRα/β chains have not been well established largely because of the complexity of their structures owing to the extensive genetic rearrangements that they undergo. Here we report the development of an integrated 5'-RACE and multiplex PCR method to amplify the full-length transcripts of TCRα/β at the single-cell level in human CD8(+) subsets, including naive, central memory, early effector memory, late effector memory, and effector phenotypic cells. Using this method, with an approximately 47% and 62% of PCR success rate for TCRα and for TCRβ chains, respectively, we were able to analyze more than 1,000 reads of transcripts of each TCR chain. Our comprehensive analysis revealed the following: (1) chimeric rearrangements of TCRδ-α, (2) control of TCRα/β transcription with multiple transcriptional initiation sites, (3) altered utilization of TCRα/β chains in CD8(+) subsets, and (4) strong association between the clonal size of TCRα/β chains and the effector phenotype of CD8(+) T-cells. Based on these findings, we conclude that our method is a useful tool to identify the dynamics of the TCRα/β repertoire, and provides new insights into the study of human TCRα/β chains.

Selection of Human Antibody Fragments Which Bind Novel Breast Tumor Antigens

DTIC Science & Technology

1998-09-01

chain Fv analogue produced in Escherichia coli. Proc Natl Acad Sci U S A. 85: 5879-83. 11. Adams, G.P., McCartney, J.E., Tai, M.-S., Oppermann, H...antidigoxin single-chain Fv analogue produced in E coil. Proc NailAcadSci Bernard Foundation, the Frank Strick Foundation and the USA 85:5879-5883 CaPCURE...recovery of infectious phage was increased by preincubation of cells with chloroquine . Measurement of phage recovery from within the cytosol as a

EXPERIMENTAL CHALLENGE STUDY OF FV3-LIKE RANAVIRUS INFECTION IN PREVIOUSLY FV3-LIKE RANAVIRUS INFECTED EASTERN BOX TURTLES (TERRAPENE CAROLINA CAROLINA) TO ASSESS INFECTION AND SURVIVAL.

PubMed

Hausmann, Jennifer C; Wack, Allison N; Allender, Matthew C; Cranfield, Mike R; Murphy, Kevin J; Barrett, Kevin; Romero, Jennell L; Wellehan, James F X; Blum, Stella A; Zink, M Christine; Bronson, Ellen

2015-12-01

The Maryland Zoo in Baltimore experienced an outbreak of Frog virus-3 (FV3)-like ranavirus during the summer of 2011, during which 14 of 27 (52%) of its captive eastern box turtles (Terrapene carolina carolina) survived. To assess survival, immunity, and viral shedding, an experimental challenge study was performed in which the surviving, previously infected turtles were reinfected with the outbreak strain of FV3-like ranavirus. Seven turtles were inoculated with virus intramuscularly and four control turtles received saline intramuscularly. The turtles were monitored for 8 wk with blood and oral swabs collected for quantitative polymerase chain reaction (qPCR). During that time, one of seven (14%) inoculated turtles and none of the controls (0%) died; there was no significant difference in survival. Clinical signs of the inoculated turtles, except for the turtle that died, were mild compared to the original outbreak. Quantitative PCR for FV3-like ranavirus on blood and oral swabs was positive for all inoculated turtles and negative for all controls. The turtle that died had intracytoplasmic inclusion bodies in multiple organs. Three inoculated and two control turtles were euthanized at the end of the study. No inclusion bodies were present in any of the organs. Quantitative PCR detected FV3-like ranavirus in the spleen of a control turtle, which suggested persistence of the virus. The surviving five turtles were qPCR-negative for FV3-like ranavirus from blood and oral swabs after brumation. Quantitative PCR for Terrapene herpesvirus 1 found no association between ranavirus infection and herpesvirus loads. In conclusion, previously infected eastern box turtles can be reinfected with the same strain of FV3-like ranavirus and show mild to no clinical signs but can shed the virus from the oral cavity.

A single-chain fragment against prostate specific membrane antigen as a tool to build theranostic reagents for prostate cancer.

PubMed

Frigerio, B; Fracasso, G; Luison, E; Cingarlini, S; Mortarino, M; Coliva, A; Seregni, E; Bombardieri, E; Zuccolotto, G; Rosato, A; Colombatti, M; Canevari, S; Figini, M

2013-06-01

Prostate carcinoma is the most common non-cutaneous cancer in developed countries and represents the second leading cause of death. Early stage androgen dependent prostate carcinoma responds well to conventional therapies, but relatively few treatment options exist for patients with hormone-refractory prostate cancer. One of the most suitable targets for antibody-mediated approaches is prostate specific membrane antigen (PSMA) which is a well known tumour associated antigen. PSMA is a type II integral cell-surface membrane protein that is not secreted, and its expression density and enzymatic activity are increased progressively in prostate cancer compared to normal prostate epithelium, thereby making PSMA an ideal target for monoclonal antibody imaging and therapy. To obtain a small protein that can better penetrate tissue, we have engineered a single-chain variable fragment (scFv) starting from the variable heavy and light domains of the murine anti-PSMA monoclonal antibody D2B. scFvD2B was analysed in vitro for activity, stability, internalisation ability and in vivo for targeting specificity. Maintenance of function and immunoreactivity as well as extremely high radiolabelling efficiency and radiochemical purity were demonstrated by in vitro assays and under different experimental conditions. Despite its monovalent binding, scFvD2B retained a good strength of binding and was able to internalise around 40% of bound antigen. In vivo we showed its ability to specifically target only PSMA expressing prostate cancer xenografts. Due to these advantageous properties, scFvD2B has the potential to become a good theranostic reagent for early detection and therapy of prostate cancers. Published by Elsevier Ltd.

Integration of molecular dynamics simulation and hotspot residues grafting for de novo scFv design against Salmonella Typhi TolC protein.

PubMed

Leong, Siew Wen; Lim, Theam Soon; Ismail, Asma; Choong, Yee Siew

2018-05-01

With the development of de novo binders for protein targets from non-related scaffolds, many possibilities for therapeutics and diagnostics have been created. In this study, we described the use of de novo design approach to create single-chain fragment variable (scFv) for Salmonella enterica subspecies enterica serovar Typhi TolC protein. Typhoid fever is a global health concern in developing and underdeveloped countries. Rapid typhoid diagnostics will improve disease management and therapy. In this work, molecular dynamics simulation was first performed on a homology model of TolC protein in POPE membrane bilayer to obtain the central structure that was subsequently used as the target for scFv design. Potential hotspot residues capable of anchoring the binders to the target were identified by docking "disembodied" amino acid residues against TolC surface. Next, scFv scaffolds were selected from Protein Data Bank to harbor the computed hotspot residues. The hotspot residues were then incorporated into the scFv scaffold complementarity determining regions. The designs recapitulated binding energy, shape complementarity, and interface surface area of natural protein-antibody interfaces. This approach has yielded 5 designs with high binding affinity against TolC that may be beneficial for the future development of antigen-based detection agents for typhoid diagnostics. Copyright © 2017 John Wiley & Sons, Ltd.

FV-MHMM: A Discussion on Weighting Schemes.

NASA Astrophysics Data System (ADS)

Franc, J.; Gerald, D.; Jeannin, L.; Egermann, P.; Masson, R.

2016-12-01

Upscaling or homogenization techniques consist in finding block-equivalentor equivalent upscaled properties on a coarse grid from heterogeneousproperties defined on an underlying fine grid. However, this couldbecome costly and resource consuming. Harder et al., 2013, have developeda Multiscale Hybrid-Mixed Method (MHMM) of upscaling to treat Darcytype equations on heterogeneous fields formulated using a finite elementmethod. Recently, Franc et al. 2016, has extended this method of upscalingto finite volume formulation (FV-MHMM). Although convergence refiningLagrange multipliers space has been observed, numerical artefactscan occur while trapping numerically the flow in regions of low permeability. This work will present the development of the method along with theresults obtained from its classical formulation. Then, two weightingschemes and their benefits on the FV-MHMM method will be presented insome simple random permeability cases. Next example will involve alarger heterogeneous 2D permeability field extracted from the 10thSPE test case. Eventually, multiphase flow will be addressed asan extension of this single phase flow method. An elliptic pressureequation solved on the coarse grid via FV-MHMM will be sequentiallycoupled with a hyperbolic saturation equation on the fine grid. Theimproved accuracy thanks to the weighting scheme will be measuredcompared to a finite volume fine grid solution. References: Harder, C., Paredes, D. and Valentin, F., A family of multiscalehybrid-mixed finite element methods for the Darcy equation with roughcoefficients, Journal of Computational Physics, 2013. Franc J., Debenest G., Jeannin L., Egermann P. and Masson R., FV-MHMMfor reservoir modelling ECMOR XV-15th European Conference on the Mathematicsof Oil Recovery, 2015.

Unbiased Analysis of TCRα/β Chains at the Single-Cell Level in Human CD8+ T-Cell Subsets

PubMed Central

Sun, Xiaoming; Saito, Masumichi; Sato, Yoshinori; Chikata, Takayuki; Naruto, Takuya; Ozawa, Tatsuhiko; Kobayashi, Eiji; Kishi, Hiroyuki; Muraguchi, Atsushi; Takiguchi, Masafumi

2012-01-01

T-cell receptor (TCR) α/β chains are expressed on the surface of CD8+ T-cells and have been implicated in antigen recognition, activation, and proliferation. However, the methods for characterization of human TCRα/β chains have not been well established largely because of the complexity of their structures owing to the extensive genetic rearrangements that they undergo. Here we report the development of an integrated 5′-RACE and multiplex PCR method to amplify the full-length transcripts of TCRα/β at the single-cell level in human CD8+ subsets, including naive, central memory, early effector memory, late effector memory, and effector phenotypic cells. Using this method, with an approximately 47% and 62% of PCR success rate for TCRα and for TCRβ chains, respectively, we were able to analyze more than 1,000 reads of transcripts of each TCR chain. Our comprehensive analysis revealed the following: (1) chimeric rearrangements of TCRδ-α, (2) control of TCRα/β transcription with multiple transcriptional initiation sites, (3) altered utilization of TCRα/β chains in CD8+ subsets, and (4) strong association between the clonal size of TCRα/β chains and the effector phenotype of CD8+ T-cells. Based on these findings, we conclude that our method is a useful tool to identify the dynamics of the TCRα/β repertoire, and provides new insights into the study of human TCRα/β chains. PMID:22792299

Simple, mild, one-step labelling of proteins with gallium-68 using a tris(hydroxypyridinone) bifunctional chelator: a 68Ga-THP-scFv targeting the prostate-specific membrane antigen.

PubMed

Nawaz, Saima; Mullen, Gregory E D; Sunassee, Kavitha; Bordoloi, Jayanta; Blower, Philip J; Ballinger, James R

2017-10-25

Labelling proteins with gallium-68 using bifunctional chelators is often problematic because of unsuitably harsh labelling conditions such as low pH or high temperature and may entail post-labelling purification. To determine whether tris(hydroxypyridinone) (THP) bifunctional chelators offer a potential solution to this problem, we have evaluated the labelling and biodistribution of a THP conjugate with a new single-chain antibody against the prostate-specific membrane antigen (PSMA), an attractive target for staging prostate cancer (PCa). A single-chain variable fragment (scFv) of J591, a monoclonal antibody that recognises an external epitope of PSMA, was prepared in order to achieve biokinetics matched to the half-life of gallium-68. The scFv, J591c-scFv, was engineered with a C-terminal cysteine. J591c-scFv was produced in HEK293T cells and purified by size-exclusion chromatography. A maleimide THP derivative (THP-mal) was coupled site-specifically to the C-terminal cysteine residue. The THP-mal-J591c-scFv conjugate was labelled with ammonium acetate-buffered gallium-68 from a 68 Ge/ 68 Ga generator at room temperature and neutral pH. The labelled conjugate was evaluated in the PCa cell line DU145 and its PSMA-overexpressing variant in vitro and xenografted in SCID mice. J591c-scFv was produced in yields of 4-6 mg/l culture supernatant and efficiently coupled with the THP-mal bifunctional chelator. Labelling yields > 95% were achieved at room temperature following incubation of 5 μg conjugate with gallium-68 for 5 min without post-labelling purification. 68 Ga-THP-mal-J591c-scFv was stable in serum and showed selective binding to the DU145-PSMA cell line, allowing an IC50 value of 31.5 nM to be determined for unmodified J591c-scFv. Serial PET/CT imaging showed rapid, specific tumour uptake and clearance via renal elimination. Accumulation in DU145-PSMA xenografts at 90 min post-injection was 5.4 ± 0.5%ID/g compared with 0.5 ± 0.2%ID/g in DU145

Aptamers, antibody scFv, and antibody Fab' fragments: An overview and comparison of three of the most versatile biosensor biorecognition elements.

PubMed

Crivianu-Gaita, Victor; Thompson, Michael

2016-11-15

The choice of biosensing elements is crucial for the development of the optimal biosensor. Three of the most versatile biosensing elements are antibody single-chain Fv fragments (scFv), antibody fragment-antigen binding (Fab') units, and aptamers. This article provides an overview of these three biorecognition elements with respects to their synthesis/engineering, various immobilization techniques, and examples of their use in biosensors. Furthermore, the final section of the review compares and contrasts their characteristics (time/cost of development, ease and variability of immobilization, affinity, stability) illustrating their advantages and disadvantages. Overall, scFv fragments are found to display the highest customizability (i.e. addition of functional groups, immobilizing peptides, etc.) due to recombinant synthesis techniques. If time and cost are an issue in the development of the biosensor, Fab' fragments should be chosen as they are relatively cheap and can be developed quickly from whole antibodies (several days). However, if there are sufficient funds and time is not a factor, aptamers should be utilized as they display the greatest affinity towards their target analytes and are extremely stable (excellent biosensor regenerability). Copyright © 2016 Elsevier B.V. All rights reserved.

Stability engineering of anti-EGFR scFv antibodies by rational design of a lambda-to-kappa swap of the VL framework using a structure-guided approach.

PubMed

Lehmann, Andreas; Wixted, Josephine H F; Shapovalov, Maxim V; Roder, Heinrich; Dunbrack, Roland L; Robinson, Matthew K

2015-01-01

Phage-display technology facilitates rapid selection of antigen-specific single-chain variable fragment (scFv) antibodies from large recombinant libraries. ScFv antibodies, composed of a VH and VL domain, are readily engineered into multimeric formats for the development of diagnostics and targeted therapies. However, the recombinant nature of the selection strategy can result in VH and VL domains with sub-optimal biophysical properties, such as reduced thermodynamic stability and enhanced aggregation propensity, which lead to poor production and limited application. We found that the C10 anti-epidermal growth factor receptor (EGFR) scFv, and its affinity mutant, P2224, exhibit weak production from E. coli. Interestingly, these scFv contain a fusion of lambda3 and lambda1 V-region (LV3 and LV1) genes, most likely the result of a PCR aberration during library construction. To enhance the biophysical properties of these scFvs, we utilized a structure-based approach to replace and redesign the pre-existing framework of the VL domain to one that best pairs with the existing VH. We describe a method to exchange lambda sequences with a more stable kappa3 framework (KV3) within the VL domain that incorporates the original lambda DE-loop. The resulting scFvs, C10KV3_LV1DE and P2224KV3_LV1DE, are more thermodynamically stable and easier to produce from bacterial culture. Additionally, C10KV3_LV1DE and P2224KV3_LV1DE retain binding affinity to EGFR, suggesting that such a dramatic framework swap does not significantly affect scFv binding. We provide here a novel strategy for redesigning the light chain of problematic scFvs to enhance their stability and therapeutic applicability.

Ophiophagus hannah venom: proteome, components bound by Naja kaouthia antivenin and neutralization by N. kaouthia neurotoxin-specific human ScFv.

PubMed

Danpaiboon, Witchuda; Reamtong, Onrapak; Sookrung, Nitat; Seesuay, Watee; Sakolvaree, Yuwaporn; Thanongsaksrikul, Jeeraphong; Dong-din-on, Fonthip; Srimanote, Potjanee; Thueng-in, Kanyarat; Chaicumpa, Wanpen

2014-05-13

Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagus hannah) is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5'-nucleotidase). N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv) cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins.

Construction of a scFv Library with Synthetic, Non-combinatorial CDR Diversity.

PubMed

Bai, Xuelian; Shim, Hyunbo

2017-01-01

Many large synthetic antibody libraries have been designed, constructed, and successfully generated high-quality antibodies suitable for various demanding applications. While synthetic antibody libraries have many advantages such as optimized framework sequences and a broader sequence landscape than natural antibodies, their sequence diversities typically are generated by random combinatorial synthetic processes which cause the incorporation of many undesired CDR sequences. Here, we describe the construction of a synthetic scFv library using oligonucleotide mixtures that contain predefined, non-combinatorially synthesized CDR sequences. Each CDR is first inserted to a master scFv framework sequence and the resulting single-CDR libraries are subjected to a round of proofread panning. The proofread CDR sequences are assembled to produce the final scFv library with six diversified CDRs.

Gene transfer of Hodgkin cell lines via multivalent anti-CD30 scFv displaying bacteriophage.

PubMed

Chung, Yoon-Suk A; Sabel, Katja; Krönke, Martin; Klimka, Alexander

2008-04-16

The display of binding ligands, such as recombinant antibody fragments, on the surface of filamentous phage makes it possible to specifically attach these phage particles to target cells. After uptake of the phage, their internal single-stranded DNA is processed by the host cell, which allows transient expression of an encoded eukaryotic gene cassette. This opens the possibility to use bacteriophage as vectors for targeted gene therapy, although the transduction efficiency is very low. Here we demonstrate the display of an anti-CD30 single chain variable fragment fused to the major coat protein pVIII on the surface of bacteriophage. These phage particles showed an improved binding and transduction efficiency of CD30 positive Hodgkin-lymphoma cells, compared to bacteriophage with the anti-CD30 single chain variable fragment fused to the minor coat protein pIII. We can conclude from the results that the postulated multivalency of the anti-CD30-pVIII displaying bacteriophage combined with disseminated display of the anti-CD30 scFv on the whole particle surface is responsible for the improved gene transfer rate. These results mark an important step towards the use of phage particles as a cheap and safe gene transfer vehicle for the gene delivery of the desired target cells via their specific surface receptors.

Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

PubMed

Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

2016-09-11

Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

Single Chain Variable Fragments Produced in Escherichia coli against Heat-Labile and Heat-Stable Toxins from Enterotoxigenic E. coli.

PubMed

Ozaki, Christiane Y; Silveira, Caio R F; Andrade, Fernanda B; Nepomuceno, Roberto; Silva, Anderson; Munhoz, Danielle D; Yamamoto, Bruno B; Luz, Daniela; Abreu, Patrícia A E; Horton, Denise S P Q; Elias, Waldir P; Ramos, Oscar H P; Piazza, Roxane M F

2015-01-01

Diarrhea is a prevalent pathological condition frequently associated to the colonization of the small intestine by enterotoxigenic Escherichia coli (ETEC) strains, known to be endemic in developing countries. These strains can produce two enterotoxins associated with the manifestation of clinical symptoms that can be used to detect these pathogens. Although several detection tests have been developed, minimally equipped laboratories are still in need of simple and cost-effective methods. With the aim to contribute to the development of such diagnostic approaches, we describe here two mouse hybridoma-derived single chain fragment variable (scFv) that were produced in E. coli against enterotoxins of ETEC strains. Recombinant scFv were developed against ETEC heat-labile toxin (LT) and heat-stable toxin (ST), from previously isolated hybridoma clones. This work reports their design, construction, molecular and functional characterization against LT and ST toxins. Both antibody fragments were able to recognize the cell-interacting toxins by immunofluorescence, the purified toxins by ELISA and also LT-, ST- and LT/ST-producing ETEC strains. The developed recombinant scFvs against LT and ST constitute promising starting point for simple and cost-effective ETEC diagnosis.

Prevalence of 1691G>A FV mutation in females from Bosnia and Herzegovina - a preliminary report

PubMed Central

Yaljevac, Amina; Mehić, Bakir; Kiseljaković, Emina; Ibrulj, Slavka; Garstka, Agnieszka; Adler, Grazyna

2013-01-01

Factor V is the liver-synthesized multidomain glycoprotein encoded by a gene localised on chromosome 1q23. The point mutation 1691G>A in this gene results in formation of an altered protein of V Factor resistant to activated protein C (APC) cleavage. This mutation alone is the most frequent cause of inborn thrombophilia and the most widely acknowledged genetic risk factor for venous thrombosis in a Caucasian population. This study was designed to provide the first estimate of the frequency of the allele 1691A FV in the Bosnian female population. The 1691G>A FV mutation was examined by polymerase chain reaction-restriction fragment length polymorphism, in a group of 67 women, mean age of 58.6 years with no history of cardiovascural incident. Our findings revealed an absence of the mutated allele 1691A FV in the studied group. This is the first report on the 1691G>A FV mutation in a population from Bosnia and Herzegovina. Further research is needed to establish prevalence of the mutated allele in the population from Bosnia and Herzegovina. PMID:23448608

Anti-CD20 single chain variable antibody fragment-apolipoprotein A-I chimera containing nanodisks promote targeted bioactive agent delivery to CD20-positive lymphomas.

PubMed

Crosby, Natasha M; Ghosh, Mistuni; Su, Betty; Beckstead, Jennifer A; Kamei, Ayako; Simonsen, Jens B; Luo, Bing; Gordon, Leo I; Forte, Trudy M; Ryan, Robert O

2015-08-01

A fusion protein comprising an α-CD20 single chain variable fragment (scFv) antibody, a spacer peptide, and human apolipoprotein (apo) A-I was constructed and expressed in Escherichia coli. The lipid interaction properties intrinsic to apoA-I as well as the antigen recognition properties of the scFv were retained by the chimera. scFv•apoA-I was formulated into nanoscale reconstituted high-density lipoprotein particles (termed nanodisks; ND) and incubated with cultured cells. α-CD20 scFv•apoA-I ND bound to CD20-positive non-Hodgkins lymphoma (NHL) cells (Ramos and Granta) but not to CD20-negative T lymphocytes (i.e., Jurkat). Binding to NHL cells was partially inhibited by pre-incubation with rituximab, a monoclonal antibody directed against CD20. Confocal fluorescence microscopy analysis of Granta cells following incubation with α-CD20 scFv•apoA-I ND formulated with the intrinsically fluorescent hydrophobic polyphenol, curcumin, revealed α-CD20 scFv•apoA-I localizes to the cell surface, while curcumin off-loads and gains entry to the cell. Compared to control incubations, viability of cultured NHL cells was decreased upon incubation with α-CD20 scFv•apoA-I ND harboring curcumin. Thus, formulation of curcumin ND with α-CD20 scFv•apoA-I as the scaffold component confers cell targeting and enhanced bioactive agent delivery, providing a strategy to minimize toxicity associated with chemotherapeutic agents.

A Human Recombinant Autoantibody-Based Immunotoxin Specific for the Fetal Acetylcholine Receptor Inhibits Rhabdomyosarcoma Growth In Vitro and in a Murine Transplantation Model

PubMed Central

Gattenlöhner, S.; Jörißen, H.; Huhn, M.; Vincent, A.; Beeson, D.; Tzartos, S.; Mamalaki, A.; Etschmann, B.; Muller-Hermelink, H. K.; Koscielniak, E.; Barth, S.; Marx, A.

2010-01-01

Rhabdomyosarcoma (RMS) is the most common malignant soft tissue tumor in children and is highly resistant to all forms of treatment currently available once metastasis or relapse has commenced. As it has recently been determined that the acetylcholine receptor (AChR) γ-subunit, which defines the fetal AChR (fAChR) isoform, is almost exclusively expressed in RMS post partum, we recombinantly fused a single chain variable fragment (scFv) derived from a fully human anti-fAChR Fab-fragment to Pseudomonas exotoxin A to generate an anti-fAChR immunotoxin (scFv35-ETA). While scFv35-ETA had no damaging effect on fAChR-negative control cell lines, it killed human embryonic and alveolar RMS cell lines in vitro and delayed RMS development in a murine transplantation model. These results indicate that scFv35-ETA may be a valuable new therapeutic tool as well as a relevant step towards the development of a fully human immunotoxin directed against RMS. Moreover, as approximately 20% of metastatic malignant melanomas (MMs) display rhabdoid features including the expression of fAChR, the immunotoxin we developed may also prove to be of significant use in the treatment of these more common and most often fatal neoplasms. PMID:20204062

The intervening removable affinity tag (iRAT) production system facilitates Fv antibody fragment‐mediated crystallography

PubMed Central

Nomura, Yayoi; Sato, Yumi; Suno, Ryoji; Horita, Shoichiro

2016-01-01

Abstract Fv antibody fragments have been used as co‐crystallization partners in structural biology, particularly in membrane protein crystallography. However, there are inherent technical issues associated with the large‐scale production of soluble, functional Fv fragments through conventional methods in various expression systems. To circumvent these problems, we developed a new method, in which a single synthetic polyprotein consisting of a variable light (VL) domain, an intervening removable affinity tag (iRAT), and a variable heavy (VH) domain is expressed by a Gram‐positive bacterial secretion system. This method ensures stoichiometric expression of VL and VH from the monocistronic construct followed by proper folding and assembly of the two variable domains. The iRAT segment can be removed by a site‐specific protease during the purification process to yield tag‐free Fv fragments suitable for crystallization trials. In vitro refolding step is not required to obtain correctly folded Fv fragments. As a proof of concept, we tested the iRAT‐based production of multiple Fv fragments, including a crystallization chaperone for a mammalian membrane protein as well as FDA‐approved therapeutic antibodies. The resulting Fv fragments were functionally active and crystallized in complex with the target proteins. The iRAT system is a reliable, rapid and broadly applicable means of producing milligram quantities of Fv fragments for structural and biochemical studies. PMID:27595817

Light-chain residue 95 is critical for antigen binding and multispecificity of monoclonal antibody G2.

PubMed

Usui, Daiki; Inaba, Satomi; Kamatari, Yuji O; Ishiguro, Naotaka; Oda, Masayuki

2017-09-02

The monoclonal antibody, G2, specifically binds to the immunogen peptide derived from the chicken prion protein, Pep18mer, and two chicken proteins derived peptides, Pep8 and Pep395; G2 binds with equal affinity to Pep18mer. The amino acid sequences of the three peptides are completely different, and so the recognition mechanism of G2 is unique and interesting. We generated a single-chain Fv (scFv) antibody of G2, and demonstrated its correct folding with an antigen binding function similar to intact G2 antibody. We also generated a Pro containing mutant of G2 scFv at residue 95 of the light chain, and analyzed its antigen binding using a surface plasmon biosensor. The mutant lost its binding ability to Pep18mer, but remained those to Pep8 and Pep395. The results clearly indicate residue 95 as being critical for multispecific antigen binding of G2 at the site generated from the junctional diversity introduced at the joints between the V and J gene segments. Copyright © 2017 Elsevier Inc. All rights reserved.

Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding.

PubMed

Davé, Emma; Adams, Ralph; Zaccheo, Oliver; Carrington, Bruce; Compson, Joanne E; Dugdale, Sarah; Airey, Michael; Malcolm, Sarah; Hailu, Hanna; Wild, Gavin; Turner, Alison; Heads, James; Sarkar, Kaushik; Ventom, Andrew; Marshall, Diane; Jairaj, Mark; Kopotsha, Tim; Christodoulou, Louis; Zamacona, Miren; Lawson, Alastair D; Heywood, Sam; Humphreys, David P

2016-10-01

An antibody format, termed Fab-dsFv, has been designed for clinical indications that require monovalent target binding in the absence of direct Fc receptor (FcR) binding while retaining substantial serum presence. The variable fragment (Fv) domain of a humanized albumin-binding antibody was fused to the C-termini of Fab constant domains, such that the VL and VH domains were individually connected to the Cκ and CH1 domains by peptide linkers, respectively. The anti-albumin Fv was selected for properties thought to be desirable to ensure a durable serum half-life mediated via FcRn. The Fv domain was further stabilized by an inter-domain disulfide bond. The bispecific format was shown to be thermodynamically and biophysically stable, and retained good affinity and efficacy to both antigens simultaneously. In in vivo studies, the serum half-life of Fab-dsFv, 2.6 d in mice and 7.9 d in cynomolgus monkeys, was equivalent to Fab'-PEG.

Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding

PubMed Central

Davé, Emma; Adams, Ralph; Zaccheo, Oliver; Carrington, Bruce; Compson, Joanne E.; Dugdale, Sarah; Airey, Michael; Malcolm, Sarah; Hailu, Hanna; Wild, Gavin; Turner, Alison; Heads, James; Sarkar, Kaushik; Ventom, Andrew; Marshall, Diane; Jairaj, Mark; Kopotsha, Tim; Christodoulou, Louis; Zamacona, Miren; Lawson, Alastair D.; Heywood, Sam; Humphreys, David P.

2016-01-01

ABSTRACT An antibody format, termed Fab-dsFv, has been designed for clinical indications that require monovalent target binding in the absence of direct Fc receptor (FcR) binding while retaining substantial serum presence. The variable fragment (Fv) domain of a humanized albumin-binding antibody was fused to the C-termini of Fab constant domains, such that the VL and VH domains were individually connected to the Cκ and CH1 domains by peptide linkers, respectively. The anti-albumin Fv was selected for properties thought to be desirable to ensure a durable serum half-life mediated via FcRn. The Fv domain was further stabilized by an inter-domain disulfide bond. The bispecific format was shown to be thermodynamically and biophysically stable, and retained good affinity and efficacy to both antigens simultaneously. In in vivo studies, the serum half-life of Fab-dsFv, 2.6 d in mice and 7.9 d in cynomolgus monkeys, was equivalent to Fab'-PEG. PMID:27532598

FvSet2 regulates fungal growth, pathogenicity, and secondary metabolism in Fusarium verticillioides.

PubMed

Gu, Qin; Wang, Zhenzhong; Sun, Xiao; Ji, Tiantian; Huang, Hai; Yang, Yang; Zhang, Hao; Tahir, Hafiz Abdul Samad; Wu, Liming; Wu, Huijun; Gao, Xuewen

2017-10-01

Histone H3 lysine 36 methylation (H3K36me) is generally associated with activation of gene expression in most eukaryotic cells. However, the function of H3K36me in filamentous fungi is largely unknown. Set2 is the sole lysine histone methyltransferase (KHMTase) enzyme responsible for the methylation of H3K36 in Saccharomyces cerevisiae. In the current study, we identified a single ortholog of S. cerevisiae Set2 in Fusarium verticillioides. We report that FvSet2 is responsible for the trimethylation of H3K36 (H3K36me3). The FvSET2 deletion mutant (ΔFvSet2) showed significant defects in vegetative growth, FB 1 biosynthesis, pigmentation, and fungal virulence. Furthermore, trimethylation of H3K36 was found to be important for active transcription of genes involved in FB 1 and bikaverin biosyntheses. These data indicate that FvSet2 plays an important role in the regulation of secondary metabolism, vegetative growth and fungal virulence in F. verticillioides. Copyright © 2017 Elsevier Inc. All rights reserved.

75 FR 29307 - Web Based Supply Chain Management Commodity Offer Form, Paperwork Collection Notice

Federal Register 2010, 2011, 2012, 2013, 2014

2010-05-25

... DEPARTMENT OF AGRICULTURE Agricultural Marketing Service [Doc. No FV10-CP-01, AMS-FV-10-0041] Web... collection request is required for the implementation of a new system named Web Based Supply Chain Management...-2782. Mail: David Tuckwiller, Project Manager, Web Based Supply Chain Management System, Agricultural...

Selection of stable scFv antibodies by phage display.

PubMed

Brockmann, Eeva-Christine

2012-01-01

ScFv fragments are popular recombinant antibody formats but often suffer from limited stability. Phage display is a powerful tool in antibody engineering and applicable also for stability selection. ScFv variants with improved stability can be selected from large randomly mutated phage displayed libraries with a specific antigen after the unstable variants have been inactivated by heat or GdmCl. Irreversible scFv denaturation, which is a prerequisite for efficient selection, is achieved by combining denaturation with reduction of the intradomain disulfide bonds. Repeated selection cycles of increasing stringency result in enrichment of stabilized scFv fragments. Procedures for constructing a randomly mutated scFv library by error-prone PCR and phage display selection for enrichment of stable scFv antibodies from the library are described here.

Production of in vivo biotinylated scFv specific to almond (Prunus dulcis) proteins by recombinant Pichia pastoris.

PubMed

de la Cruz, Silvia; Alcocer, Marcos; Madrid, Raquel; García, Aina; Martín, Rosario; González, Isabel; García, Teresa

2016-06-10

The methylotropic yeast Pichia pastoris has demonstrated its suitability for large-scale production of recombinant proteins. As an eukaryotic organism P. pastoris presents a series of advantages at expression and processing of heterologous proteins when compared with Escherichia coli. In this work, P. pastoris has been used to express a scFv from a human synthetic library previously shown to bind almond proteins. In order to facilitate purification and post processing manipulations, the scFv was engineered with a C-terminal tag and biotinylated in vivo. After purification, biotinylated scFv were bound to avidin conjugated with HRP producing a multimeric scFv. The multimeric scFv showed to maintain their ability to recognize almond protein when assayed in ELISA, reaching a LOD of 470mgkg(-1). This study describes an easy method to produce large quantities of in vivo biotinylated scFv in P. pastoris. By substituting the enzyme or fluorochromes linked to avidin, it will be possible to generate a diverse number of multimeric scFv as probes to suit different analytical platforms in the detection of almond in food products. Copyright © 2016 Elsevier B.V. All rights reserved.

Fluorescent immunolabeling of cancer cells by quantum dots and antibody scFv fragment.

PubMed

Zdobnova, Tatiana A; Dorofeev, Sergey G; Tananaev, Piter N; Vasiliev, Roman B; Balandin, Taras G; Edelweiss, Eveline F; Stremovskiy, Oleg A; Balalaeva, Irina V; Turchin, Ilya V; Lebedenko, Ekaterina N; Zlomanov, Vladimir P; Deyev, Sergey M

2009-01-01

Semiconductor quantum dots (QDs) coupled with cancer-specific targeting ligands are new promising agents for fluorescent visualization of cancer cells. Human epidermal growth factor receptor 2/neu (HER2/neu), overexpressed on the surface of many cancer cells, is an important target for cancer diagnostics. Antibody scFv fragments as a targeting agent for direct delivery of fluorophores offer significant advantages over full-size antibodies due to their small size, lower cross-reactivity, and immunogenicity. We have used quantum dots linked to anti-HER2/neu 4D5 scFv antibody to label HER2/neu-overexpressing live cells. Labeling of target cells was shown to have high brightness, photostability, and specificity. The results indicate that construction based on quantum dots and scFv antibody can be successfully used for cancer cell visualization.

Phage displayed scFv: pIII scaffold may fine tune binding specificity.

PubMed

Goswami, Pooja; Saini, Deepti; Sinha, Subrata

2009-10-01

The fine specificity of antibodies is important for their discriminating powers during diagnostics and in vivo therapy. We have attempted to isolate human scFv antibodies to the oncofetal antigen, the placental isozyme of alkaline phosphatase (PLAP) in which it is important to distinguish between the closely related intestinal alkaline phosphatase (IAP) and bone alkaline phosphatase (BAP) isozymes. As the antibodies are selected in the phage displayed form and might be finally used as different entities, including the soluble scFv form, it may be important to look at the influence of scaffolds in determining specificity. There have been earlier reports of the role of the constant region and other scaffolding proteins in determining specificity. In this paper, we report isolation of one such clone, E6, which showed specificity to PLAP in phage antibody form but lost the specificity when soluble scFv was tested for same, and showed partial cross reactivity to BAP. We suggest that the altered specificity of scFv might be the result of loss of phage pIII scaffold, which is present in phage-displayed antibody and may help the displayed antibody to assume specific conformational structure, which may govern binding characteristics of the same.

Fv-clasp: An Artificially Designed Small Antibody Fragment with Improved Production Compatibility, Stability, and Crystallizability.

PubMed

Arimori, Takao; Kitago, Yu; Umitsu, Masataka; Fujii, Yuki; Asaki, Ryoko; Tamura-Kawakami, Keiko; Takagi, Junichi

2017-10-03

Antibody fragments are frequently used as a "crystallization chaperone" to aid structural analysis of complex macromolecules that are otherwise crystallization resistant, but conventional fragment formats have not been designed for this particular application. By fusing an anti-parallel coiled-coil structure derived from the SARAH domain of human Mst1 kinase to the variable region of an antibody, we succeeded in creating a novel chimeric antibody fragment of ∼37 kDa, termed "Fv-clasp," which exhibits excellent crystallization compatibility while maintaining the binding ability of the original IgG molecule. The "clasp" and the engineered disulfide bond at the bottom of the Fv suppressed the internal mobility of the fragment and shielded hydrophobic residues, likely contributing to the high heat stability and the crystallizability of the Fv-clasp. Finally, Fv-clasp antibodies showed superior "chaperoning" activity over conventional Fab fragments, and facilitated the structure determination of an ectodomain fragment of integrin α6β1. Copyright © 2017 Elsevier Ltd. All rights reserved.

Controlled chain polymerisation and chemical soldering for single-molecule electronics.

PubMed

Okawa, Yuji; Akai-Kasaya, Megumi; Kuwahara, Yuji; Mandal, Swapan K; Aono, Masakazu

2012-05-21

Single functional molecules offer great potential for the development of novel nanoelectronic devices with capabilities beyond today's silicon-based devices. To realise single-molecule electronics, the development of a viable method for connecting functional molecules to each other using single conductive polymer chains is required. The method of initiating chain polymerisation using the tip of a scanning tunnelling microscope (STM) is very useful for fabricating single conductive polymer chains at designated positions and thereby wiring single molecules. In this feature article, developments in the controlled chain polymerisation of diacetylene compounds and the properties of polydiacetylene chains are summarised. Recent studies of "chemical soldering", a technique enabling the covalent connection of single polydiacetylene chains to single functional molecules, are also introduced. This represents a key step in advancing the development of single-molecule electronics.

Xenopus-FV3 host-pathogen interactions and immune evasion.

PubMed

Jacques, Robert; Edholm, Eva-Stina; Jazz, Sanchez; Odalys, Torres-Luquis; Francisco, De Jesús Andino

2017-11-01

We first review fundamental insights into anti-ranavirus immunity learned with the Xenopus laevis/ranavirus FV3 model that are generally applicable to ectothermic vertebrates. We then further investigate FV3 genes involved in immune evasion. Focusing on FV3 knockout (KO) mutants defective for a putative viral caspase activation and recruitment domain-containing (CARD)-like protein (Δ64R-FV3), a β-hydroxysteroid dehydrogenase homolog (Δ52L-FV3), and an immediate-early18kDa protein (FV3-Δ18K), we assessed the involvement of these viral genes in replication, dissemination and interaction with peritoneal macrophages in tadpole and adult frogs. Our results substantiate the role of 64R and 52L as critical immune evasion genes, promoting persistence and dissemination in the host by counteracting type III IFN in tadpoles and type I IFN in adult frogs. Comparably, the substantial accumulation of genome copy numbers and exacerbation of type I and III IFN gene expression responses but deficient release of infectious virus suggests that 18K is a viral regulatory gene. Copyright © 2017 Elsevier Inc. All rights reserved.

Human antibodies against spores of the genus Bacillus: a model study for detection of and protection against anthrax and the bioterrorist threat.

PubMed

Zhou, Bin; Wirsching, Peter; Janda, Kim D

2002-04-16

A naive, human single-chain Fv (scFv) phage-display library was used in bio-panning against live, native spores of Bacillus subtilis IFO 3336 suspended in solution. A direct in vitro panning and enzyme-linked immunosorbent assay-based selection afforded a panel of nine scFv-phage clones of which two, 5B and 7E, were chosen for further study. These two clones differed in their relative specificity and affinity for spores of B. subtilis IFO 3336 vs. a panel of spores from 11 other Bacillus species/strains. A variety of enzyme-linked immunosorbent assay protocols indicated these scFv-phage clones recognized different spore epitopes. Notably, some spore epitopes markedly changed between the free and microtiter-plate immobilized state as revealed by antibody-phage binding. An additional library selection procedure also was examined by constructing a Fab chain-shuffled sublibrary from the nine positive clones and by using a subtractive panning strategy to remove crossreactivity with B. licheniformis 5A24. The Fab-phage clone 52 was improved compared with 5B and was comparable to 7E in binding B. subtilis IFO 3336 vs. B. licheniformis 5A24, yet showed a distinctive crossreactivity pattern with other spores. We also developed a method to directly detect individual spores by using fluorescently labeled antibody-phage. Finally, a variety of "powders" that might be used in deploying spores of B. anthracis were examined for antibody-phage binding. The strategies described provide a foundation to discover human antibodies specific for native spores of B. anthracis that can be developed as diagnostic and therapeutic reagents.

Histone H3 lysine 9 methyltransferase FvDim5 regulates fungal development, pathogenicity and osmotic stress responses in Fusarium verticillioides.

PubMed

Gu, Qin; Ji, Tiantian; Sun, Xiao; Huang, Hai; Zhang, Hao; Lu, Xi; Wu, Liming; Huo, Rong; Wu, Huijun; Gao, Xuewen

2017-10-16

Histone methylation plays important biological roles in eukaryotic cells. Methylation of lysine 9 at histone H3 (H3K9me) is critical for regulating chromatin structure and gene transcription. Dim5 is a lysine histone methyltransferase (KHMTase) enzyme, which is responsible for the methylation of H3K9 in eukaryotes. In the current study, we identified a single ortholog of Neurospora crassa Dim5 in Fusarium verticillioides. In this study, we report that FvDim5 regulates the trimethylation of H3K9 (H3K9me3). The FvDIM5 deletion mutant (ΔFvDim5) showed significant defects in conidiation, perithecium production and fungal virulence. Unexpectedly, we found that deletion of FvDIM5 resulted in increased tolerance to osmotic stresses and upregulated FvHog1 phosphorylation. These results indicate the importance of FvDim5 for the regulation of fungal development, pathogenicity and osmotic stress responses in F. verticillioides. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Using engineered single-chain antibodies to correlate molecular binding properties and nanoparticle adhesion dynamics.

PubMed

Haun, Jered B; Pepper, Lauren R; Boder, Eric T; Hammer, Daniel A

2011-11-15

Elucidation of the relationship between targeting molecule binding properties and the adhesive behavior of therapeutic or diagnostic nanocarriers would aid in the design of optimized vectors and lead to improved efficacy. We measured the adhesion of 200-nm-diameter particles under fluid flow that was mediated by a diverse array of molecular interactions, including recombinant single-chain antibodies (scFvs), full antibodies, and the avidin/biotin interaction. Within the panel of scFvs, we used a family of mutants that display a spectrum of binding kinetics, allowing us to compare nanoparticle adhesion to bond chemistry. In addition, we explored the effect of molecular size by inserting a protein linker into the scFv fusion construct and by employing scFvs that are specific for targets with vastly different sizes. Using computational models, we extracted multivalent kinetic rate constants for particle attachment and detachment from the adhesion data and correlated the results to molecular binding properties. Our results indicate that the factors that increase encounter probability, such as adhesion molecule valency and size, directly enhance the rate of nanoparticle attachment. Bond kinetics had no influence on scFv-mediated nanoparticle attachment within the kinetic range tested, however, but did appear to affect antibody/antigen and avidin/biotin mediated adhesion. We attribute this finding to a combination of multivalent binding and differences in bond mechanical strength between recombinant scFvs and the other adhesion molecules. Nanoparticle detachment probability correlated directly with adhesion molecule valency and size, as well as the logarithm of the affinity for all molecules tested. On the basis of this work, scFvs can serve as viable targeting receptors for nanoparticles, but improvements to their bond mechanical strength would likely be required to fully exploit their tunable kinetic properties and maximize the adhesion efficiency of nanoparticles that

Ligand-induced rapid skeletal muscle atrophy in HSA-Fv2E-PERK transgenic mice.

PubMed

Miyake, Masato; Kuroda, Masashi; Kiyonari, Hiroshi; Takehana, Kenji; Hisanaga, Satoshi; Morimoto, Masatoshi; Zhang, Jun; Oyadomari, Miho; Sakaue, Hiroshi; Oyadomari, Seiichi

2017-01-01

Formation of 43S and 48S preinitiation complexes plays an important role in muscle protein synthesis. There is no muscle-wasting mouse model caused by a repressed 43S preinitiation complex assembly. The aim of the present study was to develop a convenient mouse model of skeletal muscle wasting with repressed 43S preinitiation complex assembly. A ligand-activatable PERK derivative Fv2E-PERK causes the phosphorylation of eukaryotic initiation factor 2α (eIF2α), which inhibits 43S preinitiation complex assembly. Thus, muscle atrophic phenotypes, intracellular signaling pathways, and intracellular free amino acid profiles were investigated in human skeletal muscle α-actin (HSA) promoter-driven Fv2E-PERK transgenic (Tg) mice. HSA-Fv2E-PERK Tg mice treated with the artificial dimerizer AP20187 phosphorylates eIF2α in skeletal muscles and leads to severe muscle atrophy within a few days of ligand injection. Muscle atrophy was accompanied by a counter regulatory activation of mTORC1 signaling. Moreover, intracellular free amino acid levels were distinctively altered in the skeletal muscles of HSA-Fv2E-PERK Tg mice. As a novel model of muscle wasting, HSA-Fv2E-PERK Tg mice provide a convenient tool for studying the pathogenesis of muscle loss and for assessing putative therapeutics.

Hydrogel Tethering Enhances Interdomain Stabilization of Single-Chain Antibodies.

PubMed

Xiong, Yijia; Ford, Nicole R; Hecht, Karen A; Roesijadi, Guritno; Squier, Thomas C

2017-11-15

Here, we identify the importance of molecular crowding agents in the functional stabilization of scFv antibodies. Antibodies were tethered through an engineered calmodulin (CaM)-binding peptide into a stimulus-responsive hydrogel composed of poly(ethylene glycol) (PEG)-functionalized CaM. Macromolecular crowding is modulated by transient heating, which decreases effective pore sizes. Using a fluorescent ligand bound to the scFv, frequency-domain fluorescence spectroscopy was used to assess the structural coupling between the V H and the V L domains and relationships with functional stabilization. There is minimal structural coupling between the V H and the V L domains in solution, as is apparent from the substantial rotational mobility for the bound ligand, that is suggestive of an independent mobility for the V H and the V L domains. In comparison, the hydrogel matrix acts to structurally couple the V H and the V L domains, resulting in a reduction in rotational mobility and a retention of ligand binding in the presence of 8.0 M urea. Under these same conditions, ligand binding is disrupted for scFv antibodies in solution. Increases in the stabilization of scFv antibodies in hydrogels is not simply the result of molecular crowding because decreases in pore size act to destabilize ligand binding. Rather, our results suggest that the functional stabilization of the scFv antibody within the PEG hydrogel matrix includes important factors involving protein solvation that stabilize interdomain interactions between the V H and the V L domains necessary for ligand binding.

Single-Chain Folding of Synthetic Polymers: A Critical Update.

PubMed

Altintas, Ozcan; Barner-Kowollik, Christopher

2015-11-23

The current contribution serves as a critical update to a previous feature article from us (Macromol. Rapid Commun. 2012, 33, 958-971), and highlights the latest advances in the preparation of single chain polymeric nanoparticles and initial-yet promising-attempts towards mimicking the structure of natural biomacromolecules via single-chain folding of well-defined linear polymers via so-called single chain selective point folding and repeat unit folding. The contribution covers selected examples from the literature published up to ca. September 2015. Our aim is not to provide an exhaustive review but rather highlight a selection of new and exciting examples for single-chain folding based on advanced macromolecular precision chemistry. Initially, the discussion focuses on the synthesis and characterization of single-chain folded structures via selective point folding. The second part of the feature article addresses the folding of well-defined single-chain polymers by means of repeat unit folding. The current state of the art in the field of single-chain folding indicates that repeat unit folding-driven nanoparticle preparation is well-advanced, while initial encouraging steps towards building selective point folding systems have been taken. In addition, a summary of the-in our view-open key questions is provided that may guide future biomimetic design efforts. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Isolation of tumor antigen-specific single-chain variable fragments using a chimeric antigen receptor bicistronic retroviral vector in a Mammalian screening protocol.

PubMed

Lipowska-Bhalla, Grazyna; Gilham, David E; Hawkins, Robert E; Rothwell, Dominic G

2013-12-01

The clinical potential of chimeric antigen receptors in adoptive cellular therapy is beginning to be realized with several recent clinical trials targeting CD19 showing promising results in advanced B cell malignancies. This increased efficacy corresponds with improved engineering of the chimeric receptors with the latest-generation receptors eliciting greater signaling and proliferation potential. However, the antigen-binding single-chain variable fragment (scFv) domain of the receptors is critical in determining the activity of the chimeric receptor-expressing T cells, as this determines specificity and affinity to the tumor antigen. In this study, we describe a mammalian T cell line screening protocol employing a 2A-based bicistronic retroviral vector to isolate functional scFvs. This approach involves expression of the scFv library in a chimeric antigen receptor, and is based on selection of clones capable of stimulating CD69 upregulation in a T cell line and has a number of advantages over previously described methods in that the use of a 2A cassette ensures the exclusion of nonexpressing scFvs and the screening using a chimeric receptor in a mammalian T cell line ensures selection in the optimum context for therapeutic use. Proof-of-principle experiments show that the protocol was capable of a 10(5)-fold enrichment of positive clones after three rounds of selection. Furthermore, an antigen-specific clone was successfully isolated from a partially enriched scFv library, confirming the strength of the protocol. This approach has the potential to identify novel scFvs of use in adoptive T cell therapy and, potentially, wider antibody-based applications.

Effects of protein engineering and rational mutagenesis on crystal lattice of single chain antibody fragments

PubMed Central

Kalyoncu, Sibel; Hyun, Jeongmin; Pai, Jennifer C.; Johnson, Jennifer L.; Entzminger, Kevin; Jain, Avni; Heaner, David P.; Morales, Ivan A.; Truskett, Thomas M.; Maynard, Jennifer A.; Lieberman, Raquel L.

2014-01-01

Protein crystallization is dependent upon, and sensitive to, the intermolecular contacts that assist in ordering proteins into a three dimensional lattice. Here we used protein engineering and mutagenesis to affect the crystallization of single chain antibody fragments (scFvs) that recognize the EE epitope (EYMPME) with high affinity. These hypercrystallizable scFvs are under development to assist difficult proteins, such as membrane proteins, in forming crystals, by acting as crystallization chaperones. Guided by analyses of intermolecular crystal lattice contacts, two second-generation anti-EE scFvs were produced, which bind to proteins with installed EE tags. Surprisingly, although non-complementarity determining region (CDR) lattice residues from the parent scFv framework remained unchanged through the processes of protein engineering and rational design, crystal lattices of the derivative scFvs differ. Comparison of energy calculations and the experimentally-determined lattice interactions for this basis set provides insight into the complexity of the forces driving crystal lattice choice and demonstrates the availability of multiple well-ordered surface features in our scFvs capable of forming versatile crystal contacts. PMID:24615866

Construction and Validation of SRA-FV Need Assessment.

PubMed

Thornton, David; Knight, Raymond A

2015-08-01

This article describes the construction and testing of a newly designed instrument to assess psychological factors associated with increased rates of sexual recidivism. The new instrument (Structured Risk Assessment-Forensic Version or SRA-FV) was based on previous research using the SRA framework. This article describes the results of testing SRA-FV with a large sample (N = 566) of sexual offenders being evaluated for an early civil commitment program. SRA-FV was found to significantly predict sexual recidivism for both child molesters and rapists and to have incremental predictive value relative to two widely used static actuarial instruments (Static-99R; Risk Matrix 2000/S). © The Author(s) 2013.

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection

PubMed Central

Goffinet, Marine; Chinestra, Patrick; Lajoie-Mazenc, Isabelle; Medale-Giamarchi, Claire; Favre, Gilles; Faye, Jean-Charles

2008-01-01

Background The Rho GTPases A, B and C proteins, members of the Rho family whose activity is regulated by GDP/GTP cycling, function in many cellular pathways controlling proliferation and have recently been implicated in tumorigenesis. Although overexpression of Rho GTPases has been correlated with tumorigenesis, only their GTP-bound forms are able to activate the signalling pathways implicated in tumorigenesis. Thus, the focus of much recent research has been to identify biological tools capable of quantifying the level of cellular GTP-bound Rho, or determining the subcellular location of activation. However useful, these tools used to study the mechanism of Rho activation still have limitations. The aim of the present work was to employ phage display to identify a conformationally-specific single chain fragment variable (scFv) that recognizes the active, GTP-bound, form of Rho GTPases and is able to discriminate it from the inactive, GDP-bound, Rho in endogenous settings. Results After five rounds of phage selection using a constitutively activated mutant of RhoB (RhoBQ63L), three scFvs (A8, C1 and D11) were selected for subsequent analysis. Further biochemical characterization was pursued for the single clone, C1, exhibiting an scFv structure. C1 was selective for the GTP-bound form of RhoA, RhoB, as well as RhoC, and failed to recognize GTP-loaded Rac1 or Cdc42, two other members of the Rho family. To enhance its production, soluble C1 was expressed in fusion with the N-terminal domain of phage protein pIII (scFv C1-N1N2), it appeared specifically associated with GTP-loaded recombinant RhoA and RhoB via immunoprecipitation, and endogenous activated Rho in HeLa cells as determined by immunofluorescence. Conclusion We identified an antibody, C1-N1N2, specific for the GTP-bound form of RhoB from a phage library, and confirmed its specificity towards GTP-bound RhoA and RhoC, as well as RhoB. The success of C1-N1N2 in discriminating activated Rho in

77 FR 35850 - Safety Zone; F/V Deep Sea, Penn Cove, WA

Federal Register 2010, 2011, 2012, 2013, 2014

2012-06-15

... 1625-AA00 Safety Zone; F/V Deep Sea, Penn Cove, WA AGENCY: Coast Guard, DHS. ACTION: Temporary final rule. SUMMARY: The Coast Guard is establishing a safety zone around the Fishing Vessel (F/V) Deep Sea... with the sunken F/V Deep Sea. B. Basis and Purpose On the evening of May 13, 2012, the F/V Deep Sea...

Single chain Fc-dimer-human growth hormone fusion protein for improved drug delivery.

PubMed

Zhou, Li; Wang, Hsuan-Yao; Tong, Shanshan; Okamoto, Curtis T; Shen, Wei-Chiang; Zaro, Jennica L

2017-02-01

Fc fusion protein technology has been successfully used to generate long-acting forms of several protein therapeutics. In this study, a novel Fc-based drug carrier, single chain Fc-dimer (sc(Fc) 2 ), was designed to contain two Fc domains recombinantly linked via a flexible linker. Since the Fc dimeric structure is maintained through the flexible linker, the hinge region was omitted to further stabilize it against proteolysis and reduce FcγR-related effector functions. The resultant sc(Fc) 2 candidate preserved the neonatal Fc receptor (FcRn) binding. sc(Fc) 2 -mediated delivery was then evaluated using a therapeutic protein with a short plasma half-life, human growth hormone (hGH), as the protein drug cargo. This novel carrier protein showed a prolonged in vivo half-life and increased hGH-mediated bioactivity compared to the traditional Fc-based drug carrier. sc(Fc) 2 technology has the potential to greatly advance and expand the use of Fc-technology for improving the pharmacokinetics and bioactivity of protein therapeutics. Copyright © 2016 Elsevier Ltd. All rights reserved.

Glycosylphosphatidylinositol-Anchored Anti-HIV scFv Efficiently Protects CD4 T Cells from HIV-1 Infection and Deletion in hu-PBL Mice

PubMed Central

Ye, Chaobaihui; Wang, Weiming; Cheng, Liang; Li, Guangming; Wen, Michael; Wang, Qi; Zhang, Qing; Li, Dan

2016-01-01

ABSTRACT Despite success in viral inhibition and CD4 T cell recovery by highly active antiretroviral treatment (HAART), HIV-1 is still not curable due to the persistence of the HIV-1 reservoir during treatment. One patient with acute myeloid leukemia who received allogeneic hematopoietic stem cell transplantation from a homozygous CCR5 Δ32 donor has had no detectable viremia for 9 years after HAART cessation. This case has inspired a field of HIV-1 cure research focusing on engineering HIV-1 resistance in permissive cells. Here, we employed a glycosylphosphatidylinositol (GPI)-scFv X5 approach to confer resistance of human primary CD4 T cells to HIV-1. We showed that primary CD4 T cells expressing GPI-scFv X5 were resistant to CCR5 (R5)-, CXCR4 (X4)-, and dual-tropic HIV-1 and had a survival advantage compared to control cells ex vivo. In a hu-PBL mouse study, GPI-scFv X5-transduced CD4 T cells were selected in peripheral blood and lymphoid tissues upon HIV-1 infection. Finally, GPI-scFv X5-transduced CD4 T cells, after being cotransfused with HIV-infected cells, showed significantly reduced viral loads and viral RNA copy numbers relative to CD4 cells in hu-PBL mice compared to mice with GPI-scFv AB65-transduced CD4 T cells. We conclude that GPI-scFv X5-modified CD4 T cells could potentially be used as a genetic intervention against both R5- and X4-tropic HIV-1 infections. IMPORTANCE Blocking of HIV-1 entry is one of most promising approaches for therapy. Genetic disruption of the HIV-1 coreceptor CCR5 by nucleases in T cells is under 2 clinical trials and leads to reduced viremia in patients. However, the emergence of viruses using the CXCR4 coreceptor is a concern for therapies applying single-coreceptor disruption. Here, we report that HIV-1-permissive CD4 T cells engineered with GPI-scFv X5 are resistant to R5-, X4-, or dual-tropic virus infection ex vivo. In a preclinical study using hu-PBL mice, we show that CD4 T cells were protected and that GPI-scFv X5

Cloning, bacterial expression and crystallization of Fv antibody fragments

NASA Astrophysics Data System (ADS)

E´, Jean-Luc; Boulot, Ginette; Chitarra, V´ronique; Riottot, Marie-Madeleine; Souchon, H´le`ne; Houdusse, Anne; Bentley, Graham A.; Narayana Bhat, T.; Spinelli, Silvia; Poljak, Roberto J.

1992-08-01

The variable Fv fragments of antibodies, cloned in recombinant plasmids, can be expressed in bacteria as functional proteins having immunochemical properties which are very similar or identical with those of the corresponding parts of the parent eukaryotic antibodies. They offer new possibilities for the study of antibody-antigen interactions since the crystals of Fv fragments and of their complexes with antigen reported here diffract X-rays to a higher resolution that those obtained with the cognate Fab fragments. The Fv approach should facilitate the structural study of the combining site of antibodies and the further characterization of antigen-antibody interactions by site-directed mutagenesis experiments.

Effect of Chain Conformation on the Single-Molecule Melting Force in Polymer Single Crystals: Steered Molecular Dynamics Simulations Study.

PubMed

Feng, Wei; Wang, Zhigang; Zhang, Wenke

2017-02-28

Understanding the relationship between polymer chain conformation as well as the chain composition within the single crystal and the mechanical properties of the corresponding single polymer chain will facilitate the rational design of high performance polymer materials. Here three model systems of polymer single crystals, namely poly(ethylene oxide) (PEO), polyethylene (PE), and nylon-66 (PA66) have been chosen to study the effects of chain conformation, helical (PEO) versus planar zigzag conformation (PE, PA66), and chain composition (PE versus PA66) on the mechanical properties of a single polymer chain. To do that, steered molecular dynamics simulations were performed on those polymer single crystals by pulling individual polymer chains out of the crystals. Our results show that the patterns of force-extension curve as well as the chain moving mode are closely related to the conformation of the polymer chain in the single crystal. In addition, hydrogen bonds can enhance greatly the force required to stretch the polymer chain out of the single crystal. The dynamic breaking and reformation of multivalent hydrogen bonds have been observed for the first time in PA66 at the single molecule level.

FvVE1 Regulates Biosynthesis of Fumonisins and Fusarins in Fusarium verticillioides

PubMed Central

MYUNG, KYUNG; LI, SHAOJIE; BUTCHKO, ROBERT A.E.; BUSMAN, MARK; PROCTOR, ROBERT H; ABBAS, HAMED K.; CALVO, ANA M.

2009-01-01

The veA gene positively regulates sterigmatocystin production in Aspergillus nidulans and aflatoxin production in A. parasiticus and A. flavus. Whether veA homologs have a role in regulating secondary metabolism in other fungal genera is unknown. In this study, we examined the role of the veA homolog, FvVE1, on production of two mycotoxin families, fumonisins and fusarins, in the important corn pathogen F. verticillioides. We found that FvVE1 deletion completely suppressed fumonisin production on two natural substrates, corn and rice. Furthermore, our results revealed that FvVE1 is necessary for the expression of the pathway-specific regulatory gene FUM21 and structural genes in the fumonisin biosynthetic gene (FUM) cluster. FvVE1 deletion also blocked production of fusarins. The effects of FvVE1 deletion on the production of these toxins were found to be the same in two separate mating types. Our results strongly suggest that FvVE1 play an important role in regulating mycotoxin production in F. verticillioides. PMID:19382792

Isolation and characterization of anti ROR1 single chain fragment variable antibodies using phage display technique.

PubMed

Aghebati-Maleki, Leili; Younesi, Vahid; Jadidi-Niaragh, Farhad; Baradaran, Behzad; Majidi, Jafar; Yousefi, Mehdi

2017-01-01

Receptor tyrosine kinase-like orphan receptor (ROR1) belongs to one of the families of receptor tyrosine kinases (RTKs). RTKs are involved in the various physiologic cellular functions including proliferation, migration, survival, signaling and differentiation. Several RTKs are deregulated in various cancers implying the targeting potential of these molecules in cancer therapy. ROR1 has recently been shown to be expressed in various types of cancer cells but not in normal adult cells. Hence a molecular inhibitor of extracellular domain of ROR1 that inhibits ROR1-cell surface interaction is of great therapeutic importance. In an attempt to develop molecular inhibitors of ROR1, we screened single chain variable fragment (scFv) phage display libraries, Tomlinson I + J, against one specific synthetic oligopeptide from extracellular domain of ROR1 and selected scFvs were characterized using various immunological techniques. Several ROR1 specific scFvs were selected following five rounds of panning procedure. The scFvs showed specific binding to ROR1 using immunological techniques. Our results demonstrate successful isolation and characterization of specific ROR1 scFvs that may have great therapeutic potential in cancer immunotherapy.

Domain walls in single-chain magnets

NASA Astrophysics Data System (ADS)

Pianet, Vivien; Urdampilleta, Matias; Colin, Thierry; Clérac, Rodolphe; Coulon, Claude

2017-12-01

The topology and creation energy of domain walls in different magnetic chains (called Single-Chain Magnets or SCMs) are discussed. As these domain walls, that can be seen as "defects", are known to control both static and dynamic properties of these one-dimensional systems, their study and understanding are necessary first steps before a deeper discussion of the SCM properties at finite temperature. The starting point of the paper is the simple regular ferromagnetic chain for which the characteristics of the domain walls are well known. Then two cases will be discussed (i) the "mixed chains" in which isotropic and anisotropic classical spins alternate, and (ii) the so-called "canted chains" where two different easy axis directions are present. In particular, we show that "strictly narrow" domain walls no longer exist in these more complex cases, while a cascade of phase transitions is found for canted chains as the canting angle approaches 45∘. The consequence for thermodynamic properties is briefly discussed in the last part of the paper.

Chemotactic Signaling by Single-Chain Chemoreceptors

PubMed Central

Mowery, Patricia; Ames, Peter; Reiser, Rebecca H.; Parkinson, John S.

2015-01-01

Bacterial chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family operate in commingled clusters that enable cells to detect and track environmental chemical gradients with high sensitivity and precision. MCP homodimers of different detection specificities form mixed trimers of dimers that facilitate inter-receptor communication in core signaling complexes, which in turn assemble into a large signaling network. The two subunits of each homodimeric receptor molecule occupy different locations in the core complexes. One subunit participates in trimer-stabilizing interactions at the trimer axis, the other lies on the periphery of the trimer, where it can interact with two cytoplasmic proteins: CheA, a signaling autokinase, and CheW, which couples CheA activity to receptor control. As a possible tool for independently manipulating receptor subunits in these two structural environments, we constructed and characterized fused genes for the E. coli serine chemoreceptor Tsr that encoded single-chain receptor molecules in which the C-terminus of the first Tsr subunit was covalently connected to the N-terminus of the second with a polypeptide linker. We showed with soft agar assays and with a FRET-based in vivo CheA kinase assay that single-chain Tsr~Tsr molecules could promote serine sensing and chemotaxis responses. The length of the connection between the joined subunits was critical. Linkers nine residues or shorter locked the receptor in a kinase-on state, most likely by distorting the native structure of the receptor HAMP domain. Linkers 22 or more residues in length permitted near-normal Tsr function. Few single-chain molecules were found as monomer-sized proteolytic fragments in cells, indicating that covalently joined receptor subunits were responsible for mediating the signaling responses we observed. However, cysteine-directed crosslinking, spoiling by dominant-negative Tsr subunits, and rearrangement of ligand-binding site lesions revealed subunit

A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library.

PubMed

Adler, Adam S; Bedinger, Daniel; Adams, Matthew S; Asensio, Michael A; Edgar, Robert C; Leong, Renee; Leong, Jackson; Mizrahi, Rena A; Spindler, Matthew J; Bandi, Srinivasa Rao; Huang, Haichun; Tawde, Pallavi; Brams, Peter; Johnson, David S

2018-04-01

Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of "randomly paired" scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.

A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library

PubMed Central

Adler, Adam S.; Bedinger, Daniel; Adams, Matthew S.; Asensio, Michael A.; Edgar, Robert C.; Leong, Renee; Leong, Jackson; Mizrahi, Rena A.; Spindler, Matthew J.; Bandi, Srinivasa Rao; Huang, Haichun; Brams, Peter; Johnson, David S.

2018-01-01

ABSTRACT Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of “randomly paired” scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs. PMID:29376776

Prediction of Winter Storm Tracks and Intensities Using the GFDL fvGFS Model

NASA Astrophysics Data System (ADS)

Rees, S.; Boaggio, K.; Marchok, T.; Morin, M.; Lin, S. J.

2017-12-01

The GFDL Finite-Volume Cubed-Sphere Dynamical core (FV3) is coupled to a modified version of the Global Forecast System (GFS) physics and initial conditions, to form the fvGFS model. This model is similar to the one being implemented as the next-generation operational weather model for the NWS, which is also FV3-powered. Much work has been done to verify fvGFS tropical cyclone prediction, but little has been done to verify winter storm prediction. These costly and dangerous storms impact parts of the U.S. every year. To verify winter storms we ran the NCEP operational cyclone tracker, developed at GFDL, on semi-real-time 13 km horizontal resolution fvGFS forecasts. We have found that fvGFS compares well to the operational GFS in storm track and intensity, though often predicts slightly higher intensities. This presentation will show the track and intensity verification from the past two winter seasons and explore possible reasons for bias.

A molecular model for cocaine binding by the immunotherapeutic human/mouse chimeric monoclonal antibody 2E2.

PubMed

Lape, Michael; Paula, Stefan; Ball, William J

2010-06-01

Immunotherapy by cocaine-binding monoclonal antibodies (mAbs) has emerged as a promising strategy for the treatment of cocaine addiction. The human (gamma1 heavy chain)/murine (lambda light chain) chimeric mAb 2E2 has excellent affinity and specificity for cocaine and recent animal studies have demonstrated 2E2's ability in vivo to reduce cocaine levels in the brain as well as alter cocaine self-administration behavior in rats. In this study, we used mAb 2E2 amino acid sequence information to create a homology model for the 3-D structure of its Fv fragment. Subsequent computational docking studies revealed the intermolecular interactions potentially responsible for mAb 2E2's cocaine binding properties. The driving force of cocaine binding was identified as a combination of hydrophobic interactions and a single hydrogen bond between a light chain tyrosine residue and a carbonyl oxygen atom of cocaine. The model also allowed for an in silico evaluation of single/double residue mutations in the heavy and light chain variable regions that might further enhance mAb 2E2's cocaine binding properties. Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.

A Molecular Model for Cocaine Binding by the Immunotherapeutic Human/Mouse Chimeric Monoclonal Antibody 2E2

PubMed Central

Lape, Michael; Paula, Stefan; Ball, William J.

2010-01-01

Immunotherapy by cocaine-binding monoclonal antibodies (mAbs) has emerged as a promising strategy for the treatment of cocaine addiction. The human (γ1 heavy chain)/murine (λ light chain) chimeric mAb 2E2 has excellent affinity and specificity for cocaine and recent animal studies have demonstrated 2E2’s ability in vivo to reduce cocaine levels in the brain as well as alter cocaine self-administration behavior in rats. In this study, we used mAb 2E2 amino acid sequence information to create a homology model for the 3-D structure of its Fv fragment. Subsequent computational docking studies revealed the intermolecular interactions potentially responsible for mAb 2E2’s cocaine binding properties. The driving force of cocaine binding was identified as a combination of hydrophobic interactions and a single hydrogen bond between a light chain tyrosine residue and a carbonyl oxygen atom of cocaine. The model also allowed for an in silico evaluation of single/double residue mutations in the heavy and light chain variable regions that might further enhance mAb 2E2’s cocaine binding properties. PMID:20185210

Anti-EpCAM scFv gadolinium chelate: a novel targeted MRI contrast agent for imaging of colorectal cancer.

PubMed

Khantasup, Kannika; Saiviroonporn, Pairash; Jarussophon, Suwatchai; Chantima, Warangkana; Dharakul, Tararaj

2018-05-08

The development of targeted contrast agents for magnetic resonance imaging (MRI) facilitates enhanced cancer imaging and more accurate diagnosis. In the present study, a novel contrast agent was developed by conjugating anti-EpCAM humanized scFv with gadolinium chelate to achieve target specificity. The material design strategy involved site-specific conjugation of the chelating agent to scFv. The scFv monomer was linked to maleimide-DTPA via unpaired cysteine at the scFv C-terminus, followed by chelation with gadolinium (Gd). Successful scFv-DTPA conjugation was achieved at 1:10 molar ratio of scFv to maleimide-DTPA at pH 6.5. The developed anti-EpCAM-Gd-DTPA MRI contrast agent was evaluated for cell targeting ability, in vitro serum stability, cell cytotoxicity, relaxivity, and MR contrast enhancement. A high level of targeting efficacy of anti-EpCAM-Gd-DTPA to an EpCAM-overexpressing HT29 colorectal cell was demonstrated by confocal microscopy. Good stability of the contrast agent was obtained and no cytotoxicity was observed in HT29 cells after 48 h incubation with 25-100 µM of Gd. Favorable imaging was obtained using anti-EpCAM-Gd-DTPA, including 1.8-fold enhanced relaxivity compared with Gd-DTPA, and MR contrast enhancement observed after binding to HT29. The potential benefit of this contrast agent for in vivo MR imaging of colorectal cancer, as well as other EpCAM positive cancers, is suggested and warrants further investigation.

Single-chain behavior of poly(3-hexylthiophene)

NASA Astrophysics Data System (ADS)

Ivanov, Momchil; Gross, Jonathan; Janke, Wolfhard

2017-03-01

Poly(3-hexylthiophene) (P3HT) has been in the focus of recent studies due to its promising future use in organic photovoltaics, electronics and photonics. Recent publications investigate the melt behavior of P3HT, its interaction with other molecules, mainly various fullerene derivates, and isolated chains interacting with substrates. In this work we lay the focus on the single-chain properties of P3HT in vacuum. We compare structural properties obtained from simulations using two coarse-grained models and an atomistic model of the polymer for various chain lengths and temperatures.

Anti-CDR3 Therapy for B-Cell Malignancies

DTIC Science & Technology

2014-10-01

are happy to summarize substantial completion of Task 3. Fig 3 shows a schematic of an M13 phage displaying a single chain Fv. The Tomlinson I phage...the M13 bacteriophage displaying a single chain Fv fused with one copy of the pIII protein. HC LC T A G CDR3 CDR2 CDR1 CDR2 CDR3 CDR1 p

A fully human chimeric antigen receptor with potent activity against cancer cells but reduced risk for off-tumor toxicity

PubMed Central

Song, De-Gang; Ye, Qunrui; Poussin, Mathilde; Liu, Lin; Figini, Mariangela; Powell, Daniel J.

2015-01-01

Chimeric antigen receptors (CARs) can redirect T cells against antigen-expressing tumors in an HLA-independent manner. To date, various CARs have been constructed using mouse single chain antibody variable fragments (scFvs) of high affinity that are immunogenic in humans and have the potential to mediate “on-target” toxicity. Here, we developed and evaluated a fully human CAR comprised of the human C4 folate receptor-alpha (αFR)-specific scFv coupled to intracellular T cell signaling domains. Human T cells transduced to express the C4 CAR specifically secreted proinflammatory cytokine and exerted cytolytic functions when cultured with αFR-expressing tumors in vitro. Adoptive transfer of C4 CAR T cells mediated the regression of large, established human ovarian cancer in a xenogeneic mouse model. Relative to a murine MOv19 scFv-based αFR CAR, C4 CAR T cells mediated comparable cytotoxic tumor activity in vitro and in vivo but had lower affinity for αFR protein and exhibited reduced recognition of normal cells expressing low levels of αFR. Thus, T cells expressing a fully human CAR of intermediate affinity can efficiently kill antigen-expressing tumors in vitro and in vivo and may overcome issues of transgene immunogenicity and “on-target off-tumor” toxicity that plague trials utilizing CARs containing mouse-derived, high affinity scFvs. PMID:26101914

Anti-Staphylococcus aureus single-chain variable region fragments provide protection against mastitis in mice.

PubMed

Wang, Man; Zhang, Yan; Zhu, Jianguo

2016-03-01

Staphylococcus aureus is a leading causative agent of bovine mastitis, which can result in significant economic losses to the dairy industry. However, available vaccines against bovine mastitis do not confer adequate protection, although passive immunization with antibodies may be useful to prevent disease. Hence, we constructed a bovine single-chain variable region fragment (scFv) phage display library using cDNAs from peripheral blood lymphocytes of cows with S. aureus-induced mastitis. After four rounds of selection, eight scFvs that bound S. aureus antigens with high affinity were obtained. The framework regions of the variable domains (VH and VL) of the eight scFvs were highly conserved, and the complementarity-determining regions (CDRs) displayed significant diversity, especially CDR3 of the VH domain. All eight scFvs inhibited S. aureus growth in culture medium. Lactating mice were challenged by injecting S. aureus into the fourth mammary gland. Histopathological analysis showed that treatment with these scFvs prior to bacterial challenge maintained the structure of the mammary acini, decreased infiltration of polymorphonuclear neutrophils, increased levels of interferon-gamma and interleukin-4, and reduced tumor necrosis factor-alpha levels in mammary tissues, as compared with mice treatment with physiological saline (P < 0.05). These novel bovine scFvs may be suitable candidates for therapeutic agents for the prevention of S. aureus-induced bovine mastitis.

Paired analysis of TCRα and TCRβ chains at the single-cell level in mice

PubMed Central

Dash, Pradyot; McClaren, Jennifer L.; Oguin, Thomas H.; Rothwell, William; Todd, Brandon; Morris, Melissa Y.; Becksfort, Jared; Reynolds, Cory; Brown, Scott A.; Doherty, Peter C.; Thomas, Paul G.

2010-01-01

Characterizing the TCRα and TCRβ chains expressed by T cells responding to a given pathogen or underlying autoimmunity helps in the development of vaccines and immunotherapies, respectively. However, our understanding of complementary TCRα and TCRβ chain utilization is very limited for pathogen- and autoantigen-induced immunity. To address this problem, we have developed a multiplex nested RT-PCR method for the simultaneous amplification of transcripts encoding the TCRα and TCRβ chains from single cells. This multiplex method circumvented the lack of antibodies specific for variable regions of mouse TCRα chains and the need for prior knowledge of variable region usage in the TCRβ chain, resulting in a comprehensive, unbiased TCR repertoire analysis with paired coexpression of TCRα and TCRβ chains with single-cell resolution. Using CD8+ CTLs specific for an influenza epitope recovered directly from the pneumonic lungs of mice, this technique determined that 25% of such effectors expressed a dominant, nonproductively rearranged Tcra transcript. T cells with these out-of-frame Tcra mRNAs also expressed an alternate, in-frame Tcra, whereas approximately 10% of T cells had 2 productive Tcra transcripts. The proportion of cells with biallelic transcription increased over the course of a response, a finding that has implications for immune memory and autoimmunity. This technique may have broad applications in mouse models of human disease. PMID:21135507

Good Practices for Learning to Recognize Actions Using FV and VLAD.

PubMed

Wu, Jianxin; Zhang, Yu; Lin, Weiyao

2016-12-01

High dimensional representations such as Fisher vectors (FV) and vectors of locally aggregated descriptors (VLAD) have shown state-of-the-art accuracy for action recognition in videos. The high dimensionality, on the other hand, also causes computational difficulties when scaling up to large-scale video data. This paper makes three lines of contributions to learning to recognize actions using high dimensional representations. First, we reviewed several existing techniques that improve upon FV or VLAD in image classification, and performed extensive empirical evaluations to assess their applicability for action recognition. Our analyses of these empirical results show that normality and bimodality are essential to achieve high accuracy. Second, we proposed a new pooling strategy for VLAD and three simple, efficient, and effective transformations for both FV and VLAD. Both proposed methods have shown higher accuracy than the original FV/VLAD method in extensive evaluations. Third, we proposed and evaluated new feature selection and compression methods for the FV and VLAD representations. This strategy uses only 4% of the storage of the original representation, but achieves comparable or even higher accuracy. Based on these contributions, we recommend a set of good practices for action recognition in videos for practitioners in this field.

Purification optimization for a recombinant single-chain variable fragment against type 1 insulin-like growth factor receptor (IGF-1R) by using design of experiment (DoE).

PubMed

Song, Yong-Hong; Sun, Xue-Wen; Jiang, Bo; Liu, Ji-En; Su, Xian-Hui

2015-12-01

Design of experiment (DoE) is a statistics-based technique for experimental design that could overcome the shortcomings of traditional one-factor-at-a-time (OFAT) approach for protein purification optimization. In this study, a DoE approach was applied for optimizing purification of a recombinant single-chain variable fragment (scFv) against type 1 insulin-like growth factor receptor (IGF-1R) expressed in Escherichia coli. In first capture step using Capto L, a 2-level fractional factorial analysis and successively a central composite circumscribed (CCC) design were used to identify the optimal elution conditions. Two main effects, pH and trehalose, were identified, and high recovery (above 95%) and low aggregates ratio (below 10%) were achieved at the pH range from 2.9 to 3.0 with 32-35% (w/v) trehalose added. In the second step using cation exchange chromatography, an initial screening of media and elution pH and a following CCC design were performed, whereby the optimal selectivity of the scFv was obtained on Capto S at pH near 6.0, and the optimal conditions for fulfilling high DBC and purity were identified as pH range of 5.9-6.1 and loading conductivity range of 5-12.5 mS/cm. Upon a further gel filtration, the final purified scFv with a purity of 98% was obtained. Finally, the optimized conditions were verified by a 20-fold scale-up experiment. The purities and yields of intermediate and final products all fell within the regions predicted by DoE approach, suggesting the robustness of the optimized conditions. We proposed that the DoE approach described here is also applicable in production of other recombinant antibody constructs. Copyright © 2015 Elsevier Inc. All rights reserved.

Development of an affinity-matured humanized anti-epidermal growth factor receptor antibody for cancer immunotherapy.

PubMed

Nakanishi, Takeshi; Maru, Takamitsu; Tahara, Kazuhiro; Sanada, Hideaki; Umetsu, Mitsuo; Asano, Ryutaro; Kumagai, Izumi

2013-02-01

We showed previously that humanization of 528, a murine anti-epidermal growth factor receptor (EGFR) antibody, causes reduced affinity for its target. Here, to improve the affinity of the humanized antibody for use in cancer immunotherapy, we constructed phage display libraries focused on the complementarity-determining regions (CDRs) of the antibody and carried out affinity selection. Two-step selections using libraries constructed in a stepwise manner enabled a 32-fold affinity enhancement of humanized 528 (h528). Thermodynamic analysis of the interactions between the variable domain fragment of h528 (h528Fv) mutants and the soluble extracellular domain of EGFR indicated that the h528Fv mutants obtained from the first selection showed a large increase in negative enthalpy change due to binding, resulting in affinity enhancement. Furthermore, mutants from the second selection showed a decrease in entropy loss, which led to further affinity maturation. These results suggest that a single mutation in the heavy chain variable domain (i.e. Tyr(52) to Trp) enthalpically contributed for overcoming the energetic barrier to the antigen-antibody interaction, which was a major hurdle for the in vitro affinity maturation of h528. We reported previously that the humanized bispecific diabody hEx3 Db, which targets EGFR and CD3, shows strong anti-tumor activity. hEx3 Db mutants, in which the variable domains of h528 were replaced with those of the affinity-enhanced mutants, were prepared and characterized. In a growth inhibition assay of tumor cells, the hEx3 Db mutants showed stronger anti-tumor activity than that of hEx3 Db, suggesting that affinity enhancement of h528Fv enhances the anti-tumor activity of the bispecific diabody.

Isolation of a high-affinity Bet v 1-specific IgG-derived ScFv from a subject vaccinated with hypoallergenic Bet v 1 fragments.

PubMed

Gadermaier, E; Marth, K; Lupinek, C; Campana, R; Hofer, G; Blatt, K; Smiljkovic, D; Roder, U; Focke-Tejkl, M; Vrtala, S; Keller, W; Valent, P; Valenta, R; Flicker, S

2018-01-09

Recombinant hypoallergenic allergen derivatives have been used in clinical immunotherapy studies, and clinical efficacy seems to be related to the induction of blocking IgG antibodies recognizing the wild-type allergens. However, so far no treatment-induced IgG antibodies have been characterized. To clone, express, and characterize IgG antibodies induced by vaccination with two hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1 in a nonallergic subject. A phage-displayed combinatorial single-chain fragment (ScFv) library was constructed from blood of the immunized subject and screened for Bet v 1-reactive antibody fragments. ScFvs were tested for specificity and cross-reactivity to native Bet v 1 and related pollen and food allergens, and epitope mapping was performed. Germline ancestor genes of the antibody were analyzed with the ImMunoGeneTics (IMGT) database. The affinity to Bet v 1 and cross-reactive allergens was determined by surface plasmon resonance measurements. The ability to inhibit patients' IgE binding to ELISA plate-bound allergens and allergen-induced basophil activation was assessed. A combinatorial ScFv library was obtained from the vaccinated donor after three injections with the Bet v 1 fragments. Despite being almost in germline configuration, ScFv (clone H3-1) reacted with high affinity to native Bet v 1 and homologous allergens, inhibited allergic patients' polyclonal IgE binding to Bet v 1, and partially suppressed allergen-induced basophil activation. Immunization with unfolded hypoallergenic allergen derivatives induces high-affinity antibodies even in nonallergic subjects which recognize the folded wild-type allergens and inhibit polyclonal IgE binding of allergic patients. © 2018 The Authors. Allergy Published by John Wiley & Sons Ltd.

A Three Monoclonal Antibody Combination Potently Neutralizes Multiple Botulinum Neurotoxin Serotype E Subtypes.

PubMed

Garcia-Rodriguez, Consuelo; Razai, Ali; Geren, Isin N; Lou, Jianlong; Conrad, Fraser; Wen, Wei-Hua; Farr-Jones, Shauna; Smith, Theresa J; Brown, Jennifer L; Skerry, Janet C; Smith, Leonard A; Marks, James D

2018-03-01

Human botulism is most commonly caused by botulinum neurotoxin (BoNT) serotypes A, B, and E. For this work, we sought to develop a human monoclonal antibody (mAb)-based antitoxin capable of binding and neutralizing multiple subtypes of BoNT/E. Libraries of yeast-displayed single chain Fv (scFv) antibodies were created from the heavy and light chain variable region genes of humans immunized with pentavalent-toxoid- and BoNT/E-binding scFv isolated by Fluorescence-Activated Cell Sorting (FACS). A total of 10 scFv were isolated that bound one or more BoNT/E subtypes with nanomolar-level equilibrium dissociation constants (K D ). By diversifying the V-regions of the lead mAbs and selecting for cross-reactivity, we generated three scFv that bound all four BoNT/E subtypes tested at three non-overlapping epitopes. The scFvs were converted to IgG that had K D values for the different BoNT/E subtypes ranging from 9.7 nM to 2.28 pM. An equimolar combination of the three mAbs was able to potently neutralize BoNT/E1, BoNT/E3, and BoNT/E4 in a mouse neutralization assay. The mAbs have potential utility as therapeutics and as diagnostics capable of recognizing multiple BoNT/E subtypes. A derivative of the three-antibody combination (NTM-1633) is in pre-clinical development with an investigational new drug (IND) application filing expected in 2018.

Amino acid sequence of the Fv region of a human monoclonal IgM (protein WEA) with antibody activity against 3,4-pyruvylated galactose in Klebsiella polysaccharides K30 and K33.

PubMed Central

Goñi, F; Frangione, B

1983-01-01

We have determined the amino acid sequence of the Fv [variable heavy (VH) and variable light (VL)] region of a human monoclonal IgM-kappa with antibody activity against 3,4-pyruvylated galactose, isolated from the plasma of patient WEA with Waldenström macroglobulinemia. The VH region has 114 residues, belongs to subgroup III, and has a very short third complementarity-determining region (CDR3), probably due to a small D segment/or an unusual D-J rearrangement (D, diversity; J, joining). The VL region has 108 residues and belongs to subgroup V kappa I. Compared to other members of the human VHIII and V kappa I families, WEA Fv does not appear to have significant differences within the framework residues but has unique CDRs that might be responsible for the particular antibody activity. Another IgM-kappa (GAL), which has an as-yet-undetermined antibody activity, shares a striking homology in V kappa with WEA, including an identical CDR1. PMID:6410398

A single-chain TALEN architecture for genome engineering.

PubMed

Sun, Ning; Zhao, Huimin

2014-03-04

Transcription-activator like effector nucleases (TALENs) are tailor-made DNA endonucleases and serve as a powerful tool for genome engineering. Site-specific DNA cleavage can be made by the dimerization of FokI nuclease domains at custom-targeted genomic loci, where a pair of TALENs must be positioned in close proximity with an appropriate orientation. However, the simultaneous delivery and coordinated expression of two bulky TALEN monomers (>100 kDa) in cells may be problematic to implement for certain applications. Here, we report the development of a single-chain TALEN (scTALEN) architecture, in which two FokI nuclease domains are fused on a single polypeptide. The scTALEN was created by connecting two FokI nuclease domains with a 95 amino acid polypeptide linker, which was isolated from a linker library by high-throughput screening. We demonstrated that scTALENs were catalytically active as monomers in yeast and human cells. The use of this novel scTALEN architecture should reduce protein payload, simplify design and decrease production cost.

Formal Verification of the AAMP-FV Microcode

NASA Technical Reports Server (NTRS)

Miller, Steven P.; Greve, David A.; Wilding, Matthew M.; Srivas, Mandayam

1999-01-01

This report describes the experiences of Collins Avionics & Communications and SRI International in formally specifying and verifying the microcode in a Rockwell proprietary microprocessor, the AAMP-FV, using the PVS verification system. This project built extensively on earlier experiences using PVS to verify the microcode in the AAMP5, a complex, pipelined microprocessor designed for use in avionics displays and global positioning systems. While the AAMP5 experiment demonstrated the technical feasibility of formal verification of microcode, the steep learning curve encountered left unanswered the question of whether it could be performed at reasonable cost. The AAMP-FV project was conducted to determine whether the experience gained on the AAMP5 project could be used to make formal verification of microcode cost effective for safety-critical and high volume devices.

Transfection of Fv-1 permissive and restrictive mouse cells with integrated DNA of murine leukemia viruses (host range restriction/Fv-1 gene/DNA transfection)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsu, I.C.; Yang, W.K.; Tennant, R.W.

1978-03-01

Whole-cell DNA preparations isolated from SC-1 cells chronically infected with N- or B-tropic murine leukemia viruses (MuLV) were tested for infectious activity in an Fv-1/sup n/ (NIH-3T3) and two Fv-1/sup b/ (C57BL/6 and SV-A31) cell cultures. Efficiency of transfection for all DNAs was better in the NIH-3T3 cells than in C57BL/6 or SV-A31 cells; and an (N-tropic MuLV)SC-1 cell DNA preparation was slightly more infectious than a (B-tropic MuLV)SC-1 cell DNA preparation in all three cell cultures, regardless of their Fv-1 geonotypes. Progeny viruses from the transfection showed N- or B-tropism corresponding to that of the parent viruses produced bymore » the infected SC-I cells that were used for the DNA preparation. DNA dose-response studies in NIH-3T3 cells revealed a one-hit mechanism for both the (B-tropic MuLV)SC-1 cell DNA and the (N-tropic MuLV)SC-1 cell DNA preparation. These results demonstrate that, in contrast to virion infection, transfection of N- and B-tropic MuLV with DNA preparations from chronically infected cells is not affected by the Fv-1 gene.« less

Equivalence of chain conformations in the surface region of a polymer melt and a single Gaussian chain under critical conditions.

PubMed

Skvortsov, A M; Leermakers, F A M; Fleer, G J

2013-08-07

In the melt polymer conformations are nearly ideal according to Flory's ideality hypothesis. Silberberg generalized this statement for chains in the interfacial region. We check the Silberberg argument by analyzing the conformations of a probe chain end-grafted at a solid surface in a sea of floating free chains of concentration φ by the self-consistent field (SCF) method. Apart from the grafting, probe chain and floating chains are identical. Most of the results were obtained for a standard SCF model with freely jointed chains on a six-choice lattice, where immediate step reversals are allowed. A few data were generated for a five-choice lattice, where such step reversals are forbidden. These coarse-grained models describe the equilibrium properties of flexible atactic polymer chains at the scale of the segment length. The concentration was varied over the whole range from φ = 0 (single grafted chain) to φ = 1 (probe chain in the melt). The number of contacts with the surface, average height of the free end and its dispersion, average loop and train length, tail size distribution, end-point and overall segment distributions were calculated for a grafted probe chain as a function of φ, for several chain lengths and substrate∕polymer interactions, which were varied from strong repulsion to strong adsorption. The computations show that the conformations of the probe chain in the melt do not depend on substrate∕polymer interactions and are very similar to the conformations of a single end-grafted chain under critical conditions, and can thus be described analytically. When the substrate∕polymer interaction is fixed at the value corresponding to critical conditions, all equilibrium properties of a probe chain are independent of φ, over the whole range from a dilute solution to the melt. We believe that the conformations of all flexible chains in the surface region of the melt are close to those of an appropriate single chain in critical conditions, provided

Evaluating the Use of Antibody Variable Region (Fv) Charge as a Risk Assessment Tool for Predicting Typical Cynomolgus Monkey Pharmacokinetics*

PubMed Central

Bumbaca Yadav, Daniela; Sharma, Vikas K.; Boswell, Charles Andrew; Hotzel, Isidro; Tesar, Devin; Shang, Yonglei; Ying, Yong; Fischer, Saloumeh K.; Grogan, Jane L.; Chiang, Eugene Y.; Urban, Konnie; Ulufatu, Sheila; Khawli, Leslie A.; Prabhu, Saileta; Joseph, Sean; Kelley, Robert F.

2015-01-01

The pharmacokinetic (PK) behavior of monoclonal antibodies in cynomolgus monkeys (cynos) is generally translatable to that in humans. Unfortunately, about 39% of the antibodies evaluated for PKs in cynos have fast nonspecific (or non-target-mediated) clearance (in-house data). An empirical model relating variable region (Fv) charge and hydrophobicity to cyno nonspecific clearance was developed to gauge the risk an antibody would have for fast nonspecific clearance in the monkey. The purpose of this study was to evaluate the predictability of this empirical model on cyno nonspecific clearance with antibodies specifically engineered to have either high or low Fv charge. These amino acid changes were made in the Fv region of two test antibodies, humAb4D5-8 and anti-lymphotoxin α. The humAb4D5-8 has a typical nonspecific clearance in cynos, and by making it more positively charged, the antibody acquires fast nonspecific clearance, and making it less positively charged did not impact its clearance. Anti-lymphotoxin α has fast nonspecific clearance in cynos, and making it more positively charged caused it to clear even faster, whereas making it less positively charged caused it to clear slower and within the typical range. These trends in clearance were also observed in two other preclinical species, mice and rats. The effect of modifying Fv charge on subcutaneous bioavailability was also examined, and in general bioavailability was inversely related to the direction of the Fv charge change. Thus, modifying Fv charge appears to impact antibody PKs, and the changes tended to correlate with those predicted by the empirical model. PMID:26491012

High Specific Selectivity and Membrane-Active Mechanism of Synthetic Cationic Hybrid Antimicrobial Peptides Based on the Peptide FV7

PubMed Central

Tan, Tingting; Wu, Di; Li, Weizhong; Zheng, Xin; Li, Weifen; Shan, Anshan

2017-01-01

Hybrid peptides integrating different functional domains of peptides have many advantages, such as remarkable antimicrobial activity, lower hemolysis and ideal cell selectivity, compared with natural antimicrobial peptides. FV7 (FRIRVRV-NH2), a consensus amphiphilic sequence was identified as being analogous to host defense peptides. In this study, we designed a series of hybrid peptides FV7-LL-37 (17–29) (FV-LL), FV7-magainin 2 (9–21) (FV-MA) and FV7-cecropin A (1–8) (FV-CE) by combining the FV7 sequence with the small functional sequences LL-37 (17–29) (LL), magainin 2 (9–21) (MA) and cecropin A (1–8) (CE) which all come from well-described natural peptides. The results demonstrated that the synthetic hybrid peptides, in particular FV-LL, had potent antibacterial activities over a wide range of Gram-negative and Gram-positive bacteria with lower hemolytic activity than other peptides. Furthermore, fluorescent spectroscopy indicated that the hybrid peptide FV-LL exhibited marked membrane destruction by inducing outer and inner bacterial membrane permeabilization, while scanning electron microscopy (SEM) and transmission electron microscopy (TEM) demonstrated that FV-LL damaged membrane integrity by disrupting the bacterial membrane. Inhibiting biofilm formation assays also showed that FV-LL had similar anti-biofilm activity compared with the functional peptide sequence FV7. Synthetic cationic hybrid peptides based on FV7 could provide new models for combining different functional domains and demonstrate effective avenues to screen for novel antimicrobial agents. PMID:28178190

Generation and characterization of high affinity humanized fab against hepatitis B surface antigen.

PubMed

Tiwari, Ashutosh; Dutta, Durgashree; Khanna, Navin; Acharya, Subrat K; Sinha, Subrata

2009-09-01

5S is a mouse monoclonal IgG1 that binds to the 'a' epitope of the Hepatitis B surface antigen (HBsAg) and tested positive in an in vitro test for virus neutralization. We have earlier reported the generation of humanized single chain variable fragment (scFv) from the same. In this article we report the generation of a recombinant Fab molecule by fusing humanized variable domains of 5S with the constant domains of human IgG1. The humanized Fab expressed in E. coli and subsequently purified, retained a high binding affinity (K(D) = 3.63 nmol/L) to HBsAg and bound to the same epitope of HBsAg as the parent molecule. The humanized Fab also maintained antigen binding in the presence of various destabilizing agents like 3 M NaCl, 30% DMSO, 8 M urea, and extreme pH. This high affinity humanized Fab provides a basis for the development of therapeutic molecules that can be safely utilized for the prophylaxis and treatment for Hepatitis B infection.

Downregulation of Extracellular Matrix Metalloproteinase Inducer by scFv-M6-1B9 Intrabody Suppresses Cervical Cancer Invasion Through Inhibition of Urokinase-Type Plasminogen Activator.

PubMed

Panich, Tipattaraporn; Tragoolpua, Khajornsak; Pata, Supansa; Tayapiwatana, Chatchai; Intasai, Nutjeera

2017-02-01

Overexpression of extracellular matrix metalloproteinase inducer (EMMPRIN) accelerates tumor invasion and metastasis via activation of matrix metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA) expression. The authors were interested in whether the scFv-M6-1B9 intrabody against EMMPRIN that retains EMMPRIN in endoplasmic reticulum could be a potential tool to suppress cervical cancer invasion through inhibition of uPA. The chimeric adenoviral vector Ad5/F35-scFv-M6-1B9 was transferred into human cervical carcinoma HeLa cells to produce the scFv-M6-1B9 intrabody against EMMPRIN. Cell surface expression of EMMPRIN, the membrane-bound uPA, the enzymatic activity of secreted uPA, and the invasion ability were analyzed. The scFv-M6-1B9 intrabody successfully diminished the cell surface expression of EMMPRIN and the membrane-bound uPA on HeLa cells. uPA activity from tissue culture media of EMMPRIN-downregulated HeLa cells was decreased. The invasion ability of HeLa cells harboring scFv-M6-1B9 intrabody was also suppressed. These results suggested that the scFv-M6-1B9 intrabody might represent a potential approach for invasive cervical cancer treatment. The application of scFv-M6-1B9 intrabody in animal experiments and preclinical studies would be investigated further.

Improved Brain Expression of Anti-Amyloid β scFv by Complexation of mRNA Including a Secretion Sequence with PEG-based Block Catiomer.

PubMed

Perche, Federico; Uchida, Satoshi; Akiba, Hiroki; Lin, Chin-Yu; Ikegami, Masaru; Dirisala, Anjaneyulu; Nakashima, Toshihiro; Itaka, Keiji; Tsumoto, Kohei; Kataoka, Kazunori

2017-01-01

resulted in 40 % Aβ decrease in an acute amyloidosis model; with no decrease detected with control scFv mRNA nor DNA polyplexes. However, no Aβ decrease was detected in a more challenging transgenic model of Alzheimer's disease. Our results introduce a concerted approach not only for Alzheimer's disease treatment but also for immunotherapy against neurological diseases. The effectivity of our platform required the intracranial delivery of anti-Aβ scFv as mRNA not DNA, as mRNA with an IL2ss secretion sequence to favor engagement of Aβ in the amyloidosis model, complexation with a smart copolymer for efficient transfection of primary neurons and to achieve detectable mRNA expression in the brain during 48h. Amyloid burden decrease in an acute amyloidosis model was only achieved when these three factors (mRNA coding scFv, smart copolymer, IL2ss) were integrated into a single formulation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Effect of chain stiffness on the structure of single-chain polymer nanoparticles

NASA Astrophysics Data System (ADS)

Moreno, Angel J.; Bacova, Petra; Lo Verso, Federica; Arbe, Arantxa; Colmenero, Juan; Pomposo, José A.

2018-01-01

Polymeric single-chain nanoparticles (SCNPs) are soft nano-objects synthesized by purely intramolecular cross-linking of single polymer chains. By means of computer simulations, we investigate the conformational properties of SCNPs as a function of the bending stiffness of their linear polymer precursors. We investigate a broad range of characteristic ratios from the fully flexible case to those typical of bulky synthetic polymers. Increasing stiffness hinders bonding of groups separated by short contour distances and increases looping over longer distances, leading to more compact nanoparticles with a structure of highly interconnected loops. This feature is reflected in a crossover in the scaling behaviour of several structural observables. The scaling exponents change from those characteristic for Gaussian chains or rings in θ-solvents in the fully flexible limit, to values resembling fractal or ‘crumpled’ globular behaviour for very stiff SCNPs. We characterize domains in the SCNPs. These are weakly deformable regions that can be seen as disordered analogues of domains in disordered proteins. Increasing stiffness leads to bigger and less deformable domains. Surprisingly, the scaling behaviour of the domains is in all cases similar to that of Gaussian chains or rings, irrespective of the stiffness and degree of cross-linking. It is the spatial arrangement of the domains which determines the global structure of the SCNP (sparse Gaussian-like object or crumpled globule). Since intramolecular stiffness can be varied through the specific chemistry of the precursor or by introducing bulky side groups in its backbone, our results propose a new strategy to tune the global structure of SCNPs.

Forced unfolding of single-chain polymeric nanoparticles.

PubMed

Hosono, Nobuhiko; Kushner, Aaron M; Chung, Jaeyoon; Palmans, Anja R A; Guan, Zhibin; Meijer, E W

2015-06-03

Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is applied to single-chain polymeric nanoparticles (SCPNs) to acquire information about the internal folding structure of SCPNs and inherent kinetic parameters of supramolecular self-assembling motifs embedded into the SCPNs. The SCPNs used here are polyacrylate-based polymers carrying 2-ureido-4-[1H]-pyrimidinone (UPy) or benzene-1,3,5-tricarboxamide (BTA) pendants that induce an intramolecular chain collapse into nanoparticles consisting of one polymer chain only via internal supramolecular cross-linking. The SCPN is stretched by an AFM cantilever to unfold mechanically, which allows measuring of force-extension profiles of the SCPNs. Consecutive peaks observed in the force profiles are attributed to rupture events of self-assembled UPy/BTA units in the SCPNs. The force profiles have been analyzed statistically for a series of polymers with different UPy/BTA incorporation densities. The results provide insights into the internal conformation of SCPNs, where the folding structure can be changed with the incorporation density of UPy/BTA. In addition, dynamic loading rate analysis allows the determination of kinetic parameters of BTA self-assembly, which has not been accessible by any other method. This study offers a rational tool for understanding the folding structure, kinetics, and pathway of two series of SCPNs.

Dynamics of Apis mellifera Filamentous Virus (AmFV) Infections in Honey Bees and Relationships with Other Parasites.

PubMed

Hartmann, Ulrike; Forsgren, Eva; Charrière, Jean-Daniel; Neumann, Peter; Gauthier, Laurent

2015-05-22

Apis mellifera filamentous virus (AmFV) is a large double stranded DNA virus of honey bees, but its relationship with other parasites and prevalence are poorly known. We analyzed individual honey bees from three colonies at different times post emergence in order to monitor the dynamics of the AmFV gut colonization under natural conditions. Prevalence and loads of microsporidia and trypanosomes were also recorded, as well as five common honey bee RNA viruses. The results show that a high proportion of bees get infected with AmFV during the first week post-emergence (75%) and that AmFV DNA levels remained constant. A similar pattern was observed for microsporidia while trypanosomes seem to require more time to colonize the gut. No significant associations between these three infections were found, but significant positive correlations were observed between AmFV and RNA viruses. In parallel, the prevalence of AmFV in France and Sweden was assessed from pooled honey bee workers. The data indicate that AmFV is almost ubiquitous, and does not seem to follow seasonal patterns, although higher viral loads were significantly detected in spring. A high prevalence of AmFV was also found in winter bees, without obvious impact on overwintering of the colonies.

Dynamics of Apis mellifera Filamentous Virus (AmFV) Infections in Honey Bees and Relationships with Other Parasites

PubMed Central

Hartmann, Ulrike; Forsgren, Eva; Charrière, Jean-Daniel; Neumann, Peter; Gauthier, Laurent

2015-01-01

Apis mellifera filamentous virus (AmFV) is a large double stranded DNA virus of honey bees, but its relationship with other parasites and prevalence are poorly known. We analyzed individual honey bees from three colonies at different times post emergence in order to monitor the dynamics of the AmFV gut colonization under natural conditions. Prevalence and loads of microsporidia and trypanosomes were also recorded, as well as five common honey bee RNA viruses. The results show that a high proportion of bees get infected with AmFV during the first week post-emergence (75%) and that AmFV DNA levels remained constant. A similar pattern was observed for microsporidia while trypanosomes seem to require more time to colonize the gut. No significant associations between these three infections were found, but significant positive correlations were observed between AmFV and RNA viruses. In parallel, the prevalence of AmFV in France and Sweden was assessed from pooled honey bee workers. The data indicate that AmFV is almost ubiquitous, and does not seem to follow seasonal patterns, although higher viral loads were significantly detected in spring. A high prevalence of AmFV was also found in winter bees, without obvious impact on overwintering of the colonies. PMID:26008705

Isolation and expression of recombinant antibody fragments to the biological warfare pathogen Brucella melitensis.

PubMed

Hayhurst, Andrew; Happe, Scott; Mabry, Robert; Koch, Zephyr; Iverson, Brent L; Georgiou, George

2003-05-01

Brucella melitensis is a highly infectious animal pathogen able to cause a recurring debilitating disease in humans and is therefore high on the list of biological warfare agents. Immunoglobulin genes from mice immunized with gamma-irradiated B. melitensis strain 16M were used to construct a library that was screened by phage display against similarly prepared bacteria. The selected phage particles afforded a strong enzyme-linked immunosorbent assay (ELISA) signal against gamma-irradiated B. melitensis cells. However, extensive efforts to express the respective single chain antibody variable region fragment (scFv) in soluble form failed due to: (i) poor solubility and (ii) in vivo degradation of the c-myc tag used for the detection of the recombinant antibodies. Both problems could be addressed by: (i) fusing a human kappa light chain constant domain (Ck) chain to the scFv to generate single chain antibody fragment (scAb) antibody fragments and (ii) by co-expression of the periplasmic chaperone Skp. While soluble, functional antibodies could be produced in this manner, phage-displaying scFvs or scAbs were still found to be superior ELISA reagents for immunoassays, due to the large signal amplification afforded by anti-phage antibodies. The isolated phage antibodies were shown to be highly specific to B. melitensis and did not recognize Yersinia pseudotuberculosis in contrast to the existing diagnostic monoclonal YST 9.2.1.

Annual parallax and a dimming event of a Mira variable star, FV Bootis

NASA Astrophysics Data System (ADS)

Kamezaki, Tatsuya; Nakagawa, Akiharu; Omodaka, Toshihiro; Inoue, Kan-ichiro; Chibueze, James O.; Nagayama, Takumi; Ueno, Yuji; Matsunaga, Noriyuki

2016-10-01

We present the first measurement of the trigonometric parallax of water masers associated with a Mira star, FV Bootis (FV Boo) using VLBI Exploration of Radio Astrometry (VERA). Based on our multi-epoch VERA observations, we derived the parallax to be 0.97 ± 0.06 mas, which corresponds to a distance of 1.03^{+0.07}_{-0.06} kpc. The water masers around FV Boo were spatially distributed over an area of 41 au × 41 au, and their internal motions indicate the presence of an outflow. Using the Kagoshima University 1 m optical/infrared telescope, we determined the period to be 305.6 d and the mean apparent magnitude to be +2.91 mag in the K'-band. On the period-luminosity plane, the obtained period and K'-band magnitude puts FV Boo slightly below the sequence of Miras, possibly due to circumstellar reddening. Combining our photometric data with COBE and 2MASS datasets spanning over 20 years, we found in the near infrared that FV Boo was significantly fainter in 2005 compared with preceding and later phases. Its color, however, did not show a large variation through this change. We infer that the dimming could be caused by an eclipse due to a cloud in a binary system.

A highly functional synthetic phage display library containing over 40 billion human antibody clones.

PubMed

Weber, Marcel; Bujak, Emil; Putelli, Alessia; Villa, Alessandra; Matasci, Mattia; Gualandi, Laura; Hemmerle, Teresa; Wulhfard, Sarah; Neri, Dario

2014-01-01

Several synthetic antibody phage display libraries have been created and used for the isolation of human monoclonal antibodies. The performance of antibody libraries, which is usually measured in terms of their ability to yield high-affinity binding specificities against target proteins of interest, depends both on technical aspects (such as library size and quality of cloning) and on design features (which influence the percentage of functional clones in the library and their ability to be used for practical applications). Here, we describe the design, construction and characterization of a combinatorial phage display library, comprising over 40 billion human antibody clones in single-chain fragment variable (scFv) format. The library was designed with the aim to obtain highly stable antibody clones, which can be affinity-purified on protein A supports, even when used in scFv format. The library was found to be highly functional, as >90% of randomly selected clones expressed the corresponding antibody. When selected against more than 15 antigens from various sources, the library always yielded specific and potent binders, at a higher frequency compared to previous antibody libraries. To demonstrate library performance in practical biomedical research projects, we isolated the human antibody G5, which reacts both against human and murine forms of the alternatively spliced BCD segment of tenascin-C, an extracellular matrix component frequently over-expressed in cancer and in chronic inflammation. The new library represents a useful source of binding specificities, both for academic research and for the development of antibody-based therapeutics.

A Highly Functional Synthetic Phage Display Library Containing over 40 Billion Human Antibody Clones

PubMed Central

Weber, Marcel; Bujak, Emil; Putelli, Alessia; Villa, Alessandra; Matasci, Mattia; Gualandi, Laura; Hemmerle, Teresa; Wulhfard, Sarah; Neri, Dario

2014-01-01

Several synthetic antibody phage display libraries have been created and used for the isolation of human monoclonal antibodies. The performance of antibody libraries, which is usually measured in terms of their ability to yield high-affinity binding specificities against target proteins of interest, depends both on technical aspects (such as library size and quality of cloning) and on design features (which influence the percentage of functional clones in the library and their ability to be used for practical applications). Here, we describe the design, construction and characterization of a combinatorial phage display library, comprising over 40 billion human antibody clones in single-chain fragment variable (scFv) format. The library was designed with the aim to obtain highly stable antibody clones, which can be affinity-purified on protein A supports, even when used in scFv format. The library was found to be highly functional, as >90% of randomly selected clones expressed the corresponding antibody. When selected against more than 15 antigens from various sources, the library always yielded specific and potent binders, at a higher frequency compared to previous antibody libraries. To demonstrate library performance in practical biomedical research projects, we isolated the human antibody G5, which reacts both against human and murine forms of the alternatively spliced BCD segment of tenascin-C, an extracellular matrix component frequently over-expressed in cancer and in chronic inflammation. The new library represents a useful source of binding specificities, both for academic research and for the development of antibody-based therapeutics. PMID:24950200

Comparison of chain sampling plans with single and double sampling plans

NASA Technical Reports Server (NTRS)

Stephens, K. S.; Dodge, H. F.

1976-01-01

The efficiency of chain sampling is examined through matching of operating characteristics (OC) curves of chain sampling plans (ChSP) with single and double sampling plans. In particular, the operating characteristics of some ChSP-0, 3 and 1, 3 as well as ChSP-0, 4 and 1, 4 are presented, where the number pairs represent the first and the second cumulative acceptance numbers. The fact that the ChSP procedure uses cumulative results from two or more samples and that the parameters can be varied to produce a wide variety of operating characteristics raises the question whether it may be possible for such plans to provide a given protection with less inspection than with single or double sampling plans. The operating ratio values reported illustrate the possibilities of matching single and double sampling plans with ChSP. It is shown that chain sampling plans provide improved efficiency over single and double sampling plans having substantially the same operating characteristics.

Coupled SPH-FV method with net vorticity and mass transfer

NASA Astrophysics Data System (ADS)

Chiron, L.; Marrone, S.; Di Mascio, A.; Le Touzé, D.

2018-07-01

Recently, an algorithm for coupling a Finite Volume (FV) method, that discretize the Navier-Stokes equations on block structured Eulerian grids, with the weakly-compressible Lagrangian Smoothed Particle Hydrodynamics (SPH) was presented in [16]. The algorithm takes advantage of the SPH method to discretize flow regions close to free-surfaces and of the FV method to resolve the bulk flow and the wall regions. The continuity between the two solutions is guaranteed by overlapping zones. Here we extend the algorithm by adding the possibility to have: 1) net mass transfer between the SPH and FV sub-domains; 2) free-surface across the overlapping region. In this context, particle generation at common boundaries is required to prevent depletion or clustering of particles. This operation is not trivial, because consistency between the Lagrangian and Eulerian description of the flow must be retained to ensure mass conservation. We propose here a new coupling paradigm that extends the algorithm developed in [16] and renders it suitable to test cases where vorticity and free surface significantly pass from one domain to the other. On the SPH side, a novel technique for the creation/deletion of particle was developed. On the FV side, the information recovered from the SPH solver are exploited to improve free surface prediction in a fashion that resemble the Particle Level-Set algorithms. The combination of the two new features was tested and validated in a number of test cases where both vorticity and front evolution are important. Convergence and robustness of the algorithm are shown.

Potent inhibition of OKT3-induced T cell proliferation and suppression of CD147 cell surface expression in HeLa cells by scFv-M6-1B9.

PubMed

Intasai, Nutjeera; Tragoolpua, Khajornsak; Pingmuang, Prakitnavin; Khunkaewla, Panida; Moonsom, Seangdeun; Kasinrerk, Watchara; Lieber, André; Tayapiwatana, Chatchai

2008-01-01

CD147, a multifunctional type I transmembrane glycoprotein, has been implicated in various physiological and pathological processes. It is involved in signal transduction pathways and also plays a crucial role in the invasive and metastatic activity of malignant tumor cells. Diminished expression of this molecule has been shown to be beneficial in suppression of tumor progression. In a previous study, we generated and characterized a recombinant antibody fragment, scFv, which reacted specifically to CD147. In the present study, we further investigated the biological properties, function and the effect of generated scFv on CD147 expression. The in vitro study showed that soluble scFv-M6-1B9 produced from E. coli HB2151 bound to CD147 surface molecule and inhibited OKT3-induced T cell proliferation. Furthermore, soluble lysate of scFv-M6-1B9 from 293A cells, transduced with a scFv-M6-1B9 expressing adenovirus vector, recognized both recombinant and native CD147. These results indicate that scFv-M6-1B9 binds with high efficiency and specificity. Importantly, scFv-M6-1B9 intrabody reduced the expression of CD147 on the cell surface of HeLa cells suggesting that scFv-M6-1B9 is biologically active. In conclusion, our present study demonstrated that scFv-M6-1B9 has a great potential to target both the intracellular and the extracellular CD147. The generated scFv-M6-1B9 may be an effective agent to clarify the cellular function of CD147 and may aid in efforts to develop a novel treatment in various human carcinomas.

Improving the affinity of an antibody for its antigen via long-range electrostatic interactions.

PubMed

Fukunaga, Atsushi; Tsumoto, Kouhei

2013-12-01

To address how long-range electrostatic force can affect antibody-antigen binding, we focused on the interactions between human cardiac troponin I and its specific single-chain antibodies (scFvs). We first isolated two scFvs against two linear epitopes with distinct isoelectric points. For the scFv against the acidic epitope (A1scFv), we mutated five residues of framework region 3 of the light chain to Lys or Arg, designated as the K- or R-mutant, respectively. For the scFv against the basic epitope (A2scFv), we mutated four or three residues in framework region 3 of the light or heavy chain to Asp, to generate the VL- and VH-mutant, respectively. Surface plasmon resonance analyses showed that the kon values of all of the mutants were greater than that of wild type, even though framework region 3 does not make direct contact with the epitope. The affinity of the K-mutant was pM range, and that of the R-mutant improved further by more than two orders of magnitude due to a decrease in the dissociation rate constant. For the A2scFv mutants, the affinity of the VL-mutant for its target improved through an increase in the kon value without a decrease in the koff value. The stability slightly decreased in all mutants. These results suggest that introducing electrostatic interaction can improve the affinity of an antibody for its target, even if the mutation reduces stability of the antibody.

Covalent bond force profile and cleavage in a single polymer chain

NASA Astrophysics Data System (ADS)

Garnier, Lionel; Gauthier-Manuel, Bernard; van der Vegte, Eric W.; Snijders, Jaap; Hadziioannou, Georges

2000-08-01

We present here the measurement of the single-polymer entropic elasticity and the single covalent bond force profile, probed with two types of atomic force microscopes (AFM) on a synthetic polymer molecule: polymethacrylic acid in water. The conventional AFM allowed us to distinguish two types of interactions present in this system when doing force spectroscopic measurements: the first interaction is associated with adsorption sites of the polymer chains onto a bare gold surface, the second interaction is directly correlated to the rupture process of a single covalent bond. All these bridging interactions allowed us to stretch the single polymer chain and to determine the various factors playing a role in the elasticity of these molecules. To obtain a closer insight into the bond rupture process, we moved to a force sensor stable in position when measuring attractive forces. By optimizing the polymer length so as to fulfill the elastic stability conditions, we were able for the first time to map out the entire force profile associated with the cleavage of a single covalent bond. Experimental data coupled with molecular quantum mechanical calculations strongly suggest that the breaking bond is located at one end of the polymer chain.

Radioiodination and biodistribution of the monoclonal antibody TU-20 and its scFv fragment

NASA Astrophysics Data System (ADS)

Kubaštová, H.; Kleinova, V.; Seifert, D.; Fišer, M.; Kranda, K.

2006-01-01

The ability of the monoclonal antibody TU-20 and its scFv fragment to specifically bind to the C-end of the class III beta-tubulin makes these preparations useful as potential diagnostics for in vivo determination of neurodegenerative diseases that entail degradation of neuronal cytoskeleton. To examine this hypothesis, TU-20 and its scFv were labelled with 125I and their properties were extensively investigated. TU-20 and its scFv were labelled via chloramine-T with the yield 90 95% and 64 78%, respectively. Their quality control, performed by an ELISA and gel electrophoresis, determined adequate properties for further studies. The in vitro experiment, involving autoradiography and immunohistochemistry of mice’ brain slices, enabled confirmation of preserved immunospecificity of the radiolabelled substances. Finally, the in vivo biodistribution proved differences in elimination of either TU-20, scFv TU-20, or iodide from the mice.

Molecular dynamics simulation of the folding of single alkane chains with different lengths on single-walled carbon nanotubes and graphene.

PubMed

Liu, Yan Fang; Yang, Hua; Zhang, Hui

2018-05-31

Chain folding is an important step during polymer crystallization. In order to study the effects of the surface on chain folding, molecular dynamics simulations of the folding of different alkane chains on three kinds of single-walled carbon nanotubes (SWCNTs) and graphene were performed. The folding behaviors of the single alkane chains on these surfaces were found to be different from their folding behaviors in vacuum. The end-to-end distances of the chains were calculated to explore the chain folding. An increasing tendency to fold into two or more stems with increasing alkane chain length was observed. This result indicates that the occurrence and the stability of chain folding are related to the surface curvature, the diameter of the SWCNT, and surface texture. In addition, the angle between the direction of the alkane chain segment and the direction of the surface texture was measured on different surfaces.

Toward Single Atom Chains with Exfoliated Tellurium.

PubMed

Churchill, Hugh O H; Salamo, Gregory J; Yu, Shui-Qing; Hironaka, Takayuki; Hu, Xian; Stacy, Jeb; Shih, Ishiang

2017-08-10

We demonstrate that the atom chain structure of Te allows it to be exfoliated as ultra-thin flakes and nanowires. Atomic force microscopy of exfoliated Te shows that thicknesses of 1-2 nm and widths below 100 nm can be exfoliated with this method. The Raman modes of exfoliated Te match those of bulk Te, with a slight shift (4 cm -1 ) due to a hardening of the A 1 and E modes. Polarized Raman spectroscopy is used to determine the crystal orientation of exfoliated Te flakes. These experiments establish exfoliation as a route to achieve nanoscale trigonal Te while also demonstrating the potential for fabrication of single atom chains of Te.

Cloning and characterization of an adenoviral vector for highly efficient and doxycycline – suppressible expression of bioactive human single – chain interleukin 12 in colon cancer

PubMed Central

Wulff, Holger; Krieger, Thorsten; Krüger, Karen; Stahmer, Ingrid; Thaiss, Friedrich; Schäfer, Hansjörg; Block, Andreas

2007-01-01

Background Interleukin-12 (IL-12) is well characterized to induce cellular antitumoral immunity by activation of NK-cells and T-lymphocytes. However, systemic administration of recombinant human IL-12 resulted in severe toxicity without perceptible therapeutic benefit. Even though intratumoral expression of IL-12 leads to tumor regression and long-term survival in a variety of animal models, clinical trials have not yet shown a significant therapeutic benefit. One major obstacle in the treatment with IL-12 is to overcome the relatively low expression of the therapeutic gene without compromising the safety of such an approach. Our objective was to generate an adenoviral vector system enabling the regulated expression of very high levels of bioactive, human IL-12. Results High gene expression was obtained utilizing the VP16 herpes simplex transactivator. Strong regulation of gene expression was realized by fusion of the VP16 to a tetracycline repressor with binding of the fusion protein to a flanking tetracycline operator and further enhanced by auto-regulated expression of its fusion gene within a bicistronic promoter construct. Infection of human colon cancer cells (HT29) at a multiplicity of infection (m.o.i.) of 10 resulted in the production of up to 8000 ng/106 cells in 48 h, thus exceeding any published vector system so far. Doxycycline concentrations as low as 30 ng/ml resulted in up to 5000-fold suppression, enabling significant reduction of gene expression in a possible clinical setting. Bioactivity of the human single-chain IL-12 was similar to purified human heterodimeric IL-12. Frozen sections of human colon cancer showed high expression of the coxsackie adenovirus receptor with significant production of human single chain IL-12 in colon cancer biopsies after infection with 3*107 p.f.u. Ad.3r-scIL12. Doxycycline mediated suppression of gene expression was up to 9000-fold in the infected colon cancer tissue. Conclusion VP16 transactivator-mediated and

Single-copy entanglement in critical quantum spin chains

NASA Astrophysics Data System (ADS)

Eisert, J.; Cramer, M.

2005-10-01

We consider the single-copy entanglement as a quantity to assess quantum correlations in the ground state in quantum many-body systems. We show for a large class of models that already on the level of single specimens of spin chains, criticality is accompanied with the possibility of distilling a maximally entangled state of arbitrary dimension from a sufficiently large block deterministically, with local operations and classical communication. These analytical results—which refine previous results on the divergence of block entropy as the rate at which maximally entangled pairs can be distilled from many identically prepared chains—are made quantitative for general isotropic translationally invariant spin chains that can be mapped onto a quasifree fermionic system, and for the anisotropic XY model. For the XX model, we provide the asymptotic scaling of ˜(1/6)log2(L) , and contrast it with the block entropy.

Development of a microplate coagulation assay for Factor V in human plasma.

PubMed

Tilley, Derek; Levit, Irina; Samis, John A

2011-06-28

Factor V (FV) in its activated form, FVa, is a critical regulator of thrombin generation during fibrin clot formation. There is a need of a simple, fast, and inexpensive microplate-based coagulation assay to measure the functional activity of FV in human plasma. The objective of this study was to develop a microplate-based assay that measures FV coagulation activity during clot formation in human plasma, which is currently not available. The FV assay requires a kinetic microplate reader to measure the change in absorbance at 405nm during fibrin formation in human plasma. The FV assay accurately measures the time, initial rate, and extent of fibrin clot formation in human plasma. The FV microplate assay is simple, fast, economical, sensitive to approx 24-80pM, and multiple samples may be analyzed simultaneously. All the required materials are commercially available. Standard curves of time or initial rate of fibrin clot formation vs FV activity in the 1-stage assay (Without activation by thrombin) may be used to measure FV activity in samples of human plasma. The assay was used to demonstrate that in nine patients with disseminated intravascular coagulation (DIC), the FV 1-stage, 2-stage (With activation by thrombin), and total (2-stage activity - 1-stage activity) activities were decreased, on average, by approximately 54%, 44%, and 42%, respectively, from prolonged clot times when compared to normal pooled human reference plasma (NHP). The results indicate that the FV in the DIC patient plasmas supported both a delayed and slower rate of fibrin clot formation compared with NHP; however, the extent of fibrin clot formation in the DIC patients remained largely unchanged from that observed with NHP. The FV microplate assay may be easily adapted to measure the activity of any coagulation factor using the appropriate factor-deficient plasma and clot initiating reagent. The microplate assay will find use in both research and clinical laboratories to provide measurement

Development of a microplate coagulation assay for Factor V in human plasma

PubMed Central

2011-01-01

Background Factor V (FV) in its activated form, FVa, is a critical regulator of thrombin generation during fibrin clot formation. There is a need of a simple, fast, and inexpensive microplate-based coagulation assay to measure the functional activity of FV in human plasma. The objective of this study was to develop a microplate-based assay that measures FV coagulation activity during clot formation in human plasma, which is currently not available. Methods The FV assay requires a kinetic microplate reader to measure the change in absorbance at 405nm during fibrin formation in human plasma. The FV assay accurately measures the time, initial rate, and extent of fibrin clot formation in human plasma. Results The FV microplate assay is simple, fast, economical, sensitive to approx 24-80pM, and multiple samples may be analyzed simultaneously. All the required materials are commercially available. Standard curves of time or initial rate of fibrin clot formation vs FV activity in the 1-stage assay (Without activation by thrombin) may be used to measure FV activity in samples of human plasma. The assay was used to demonstrate that in nine patients with disseminated intravascular coagulation (DIC), the FV 1-stage, 2-stage (With activation by thrombin), and total (2-stage activity - 1-stage activity) activities were decreased, on average, by approximately 54%, 44%, and 42%, respectively, from prolonged clot times when compared to normal pooled human reference plasma (NHP). The results indicate that the FV in the DIC patient plasmas supported both a delayed and slower rate of fibrin clot formation compared with NHP; however, the extent of fibrin clot formation in the DIC patients remained largely unchanged from that observed with NHP. Conclusions The FV microplate assay may be easily adapted to measure the activity of any coagulation factor using the appropriate factor-deficient plasma and clot initiating reagent. The microplate assay will find use in both research and

Exploring the folding pattern of a polymer chain in a single crystal by combining single-molecule force spectroscopy and steered molecular dynamics simulations.

PubMed

Song, Yu; Feng, Wei; Liu, Kai; Yang, Peng; Zhang, Wenke; Zhang, Xi

2013-03-26

Understanding the folding pattern of a single polymer chain within its single crystal will shed light on the mechanism of crystallization. Here, we use the combined techniques of atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) and steered molecular dynamics (SMD) simulations to study the folding pattern of a polyethylene oxide (PEO) chain in its single crystal. Our results show that the folding pattern of a PEO chain in the crystal formed in dilute solution follows the adjacent re-entry folding model. While in the crystal obtained from the melt, the nonadjacent folding with large and irregular loops contributes to big force fluctuations in the force-extension curves. The method established here can offer a novel strategy to directly unravel the chain-folding pattern of polymer single crystals at single-molecule level.

Improved Soluble ScFv ELISA Screening Approach for Antibody Discovery Using Phage Display Technology.

PubMed

Tohidkia, Mohammad R; Sepehri, Maryam; Khajeh, Shirin; Barar, Jaleh; Omidi, Yadollah

2017-09-01

Phage display technology (PDT) is a powerful tool for the isolation of recombinant antibody (Ab) fragments. Using PDT, target molecule-specific phage-Ab clones are enriched through the "biopanning" process. The individual specific binders are screened by the monoclonal scFv enzyme-linked immunosorbent assay (ELISA) that may associate with inevitable false-negative results. Thus, in this study, three strategies were investigated for optimization of the scFvs screening using Tomlinson I and J libraries, including (1) optimizing the expression of functional scFvs, (2) improving the sensitivity of ELISA, and (3) preparing different samples containing scFvs. The expression of all scFv Abs was significantly enhanced when scFv clones were cultivated in the terrific broth (TB) medium at the optimum temperature of 30 °C. The protein A-conjugated with horseradish peroxidase (HRP) was found to be a well-suited reagent for the detection of Ag-bound scFvs in comparison with either anti-c-myc Ab or the mixing procedure. Based on our findings, it seems there is no universal media supplement for an improved expression of all scFvs derived from both Tomlinson I and J libraries. We thus propose that expression of scFv fragments in a microplate scale is largely dependent on a variety of parameters, in particular the scFv clones and relevant sequences.

Generation and characterization of protective antibodies to Marburg virus.

PubMed

Froude, Jeffrey W; Pelat, Thibaut; Miethe, Sebastian; Zak, Samantha E; Wec, Anna Z; Chandran, Kartik; Brannan, Jennifer Mary; Bakken, Russell R; Hust, Michael; Thullier, Philippe; Dye, John M

Marburg virus (MARV) and Ebola virus (EBOV) have been a source of epidemics and outbreaks for several decades. We present here the generation and characterization of the first protective antibodies specific for wild-type MARV. Non-human primates (NHP), cynomolgus macaques, were immunized with viral-replicon particles expressing the glycoproteins (GP) of MARV (Ci67 isolate). An antibody fragment (single-chain variable fragment, scFv) phage display library was built after four immunogen injections, and screened against the GP 1-649 of MARV. Sequencing of 192 selected clones identified 18 clones with distinct V H and V L sequences. Four of these recombinant antibodies (R4A1, R4B11, R4G2, and R3F6) were produced in the scFv-Fc format for in vivo studies. Mice that were challenged with wild-type Marburg virus (Ci67 isolate) receiving 100 µg of scFv-Fc on days -1, 1 and 3 demonstrated protective efficacies ranging from 75-100%. The amino-acid sequences of the scFv-Fcs are similar to those of their human germline counterparts, sharing an identity ranging between 68 and 100% to human germline immunoglobulin. These results demonstrate for the first time that recombinant antibodies offer protection against wild-type MARV, and suggest they may be promising candidates for further therapeutic development especially due to their human homology.

Single chain technology: Toward the controlled synthesis of polymer nanostructures

NASA Astrophysics Data System (ADS)

Lyon, Christopher

A technique for fabricating advanced polymer nanostructures enjoying recent popularity is the collapse or folding of single polymer chains in highly dilute solution mediated by intramolecular cross-linking. We term the resultant structures single-chain nanoparticles (SCNP). This technique has proven particularly valuable in the synthesis of nanomaterials on the order of 5 -- 20 nm. Many different types of covalent and non-covalent chemistries have been used to this end. This dissertation investigates the use of so-called single-chain technology to synthesize nanoparticles using modular techniques that allow for easy incorporation of functionality or special structural or characteristic features. Specifically, the synthesis of linear polymers functionalized with pendant monomer units and the subsequent intramolecular polymerization of these monomer units is discussed. In chapter 2, the synthesis of SCNP using alternating radical polymerization is described. Polymers functionalized with pendant styrene and stilbene groups are synthesized via a modular post-polymerization Wittig reaction. These polymers were exposed to radical initiators in the presence (and absence) of maleic anhydride and other electron deficient monomers in order to form intramolecular cross-links. Chapter 3 discusses templated acyclic diene metathesis (ADMET) polymerization using single-chain technology, starting with the controlled ring-opening polymerization of a glycidyl ether functionalized with an ADMET monomer. This polymer was then exposed to Grubbs' catalyst to polymerize the ADMET monomer units. The ADMET polymer was hydrolytically cleaved from the template and separated. Upon characterization, it was found that the daughter ADMET polymer had a similar degree of polymerization, but did not retain the low dispersity of the template. Chapter 4 details the synthesis of aldehyde- and diol-functionalized polymers toward the synthesis of SCNP containing dynamic, acid-degradable acetal cross

Photometry and brightening of Galactic transient ASASSN-18fv

NASA Astrophysics Data System (ADS)

Brown, M. J. I.; Upjohn, J.; Rol, E.; Obradovic, A.; Galloway, D. K.; Ackley, K.; Davies, J.; Lane, C.; Olofsson, S.; Reader, N.

2018-03-01

We report follow-up B and V photometry of Galactic transient ASASSN-18fv (Stanek et al. ATEL #11454). The photometry was taken with the 14-inch telescope at Hutton-Westfold Observatory, located at Monash University's Clayton campus.

Earlinet single calculus chain: new products overview

NASA Astrophysics Data System (ADS)

D'Amico, Giuseppe; Mattis, Ina; Binietoglou, Ioannis; Baars, Holger; Mona, Lucia; Amato, Francesco; Kokkalis, Panos; Rodríguez-Gómez, Alejandro; Soupiona, Ourania; Kalliopi-Artemis, Voudouri

2018-04-01

The Single Calculus Chain (SCC) is an automatic and flexible tool to analyze raw lidar data using EARLINET quality assured retrieval algorithms. It has been already demonstrated the SCC can retrieve reliable aerosol backscatter and extinction coefficient profiles for different lidar systems. In this paper we provide an overview of new SCC products like particle linear depolarization ratio, cloud masking, aerosol layering allowing relevant improvements in the atmospheric aerosol characterization.

Functional Consequences of Complementarity-determining Region Deactivation in a Multifunctional Anti-nucleic Acid Antibody*

PubMed Central

Lee, Jiyeon; Kim, Hye-Jin; Roh, Jooho; Seo, Youngsil; Kim, Minjae; Jun, Hye-Ryeong; Pham, Chuong D.; Kwon, Myung-Hee

2013-01-01

Many murine monoclonal anti-DNA antibodies (Abs) derived from mice models for systemic lupus erythematosus have additional cell-penetration and/or nucleic acid-hydrolysis properties. Here, we examined the influence of deactivating each complementarity-determining region (CDR) within a multifunctional anti-nucleic acid antibody (Ab) that possesses these activities, the catalytic 3D8 single chain variable fragment (scFv). CDR-deactivated 3D8 scFv variants were generated by replacing all of the amino acids within each CDR with Gly/Ser residues. The structure of 3D8 scFv accommodated single complete CDR deactivations. Different functional activities of 3D8 scFv were affected differently depending on which CDR was deactivated. The only exception was CDR1, located within the light chain (LCDR1); deactivation of LCDR1 abolished all of the functional activities of 3D8 scFv. A hybrid Ab, HW6/3D8L1, in which the LCDR1 from an unrelated Ab (HW6) was replaced with the LCDR1 from 3D8, acquired all activities associated with the 3D8 scFv. These results suggest that the activity of a multifunctional 3D8 scFv Ab can be modulated by single complete CDR deactivation and that the LCDR1 plays a crucial role in maintaining Ab properties. This study presents a new approach for determining the role of individual CDRs in multifunctional Abs with important implications for the future of Ab engineering. PMID:24155236

Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allows their intratumoral delivery and an improved tumor-growth inhibition.

PubMed

Kleinpeter, Patricia; Fend, Laetitia; Thioudellet, Christine; Geist, Michel; Sfrontato, Nathalie; Koerper, Véronique; Fahrner, Catherine; Schmitt, Doris; Gantzer, Murielle; Remy-Ziller, Christelle; Brandely, Renée; Villeval, Dominique; Rittner, Karola; Silvestre, Nathalie; Erbs, Philippe; Zitvogel, Laurence; Quéméneur, Eric; Préville, Xavier; Marchand, Jean-Baptiste

2016-01-01

We report here the successful vectorization of a hamster monoclonal IgG (namely J43) recognizing the murine Programmed cell death-1 (mPD-1) in Western Reserve (WR) oncolytic vaccinia virus. Three forms of mPD-1 binders have been inserted into the virus: whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv). MAb, Fab and scFv were produced and assembled with the expected patterns in supernatants of cells infected by the recombinant viruses. The three purified mPD-1 binders were able to block the binding of mPD-1 ligand to mPD-1 in vitro . Moreover, mAb was detected in tumor and in serum of C57BL/6 mice when the recombinant WR-mAb was injected intratumorally (IT) in B16F10 and MCA 205 tumors. The concentration of circulating mAb detected after IT injection was up to 1,900-fold higher than the level obtained after a subcutaneous (SC) injection (i.e., without tumor) confirming the virus tropism for tumoral cells and/or microenvironment. Moreover, the overall tumoral accumulation of the mAb was higher and lasted longer after IT injection of WR-mAb1, than after IT administration of 10 µg of J43. The IT injection of viruses induced a massive infiltration of immune cells including activated lymphocytes (CD8 + and CD4 + ). Interestingly, in the MCA 205 tumor model, WR-mAb1 and WR-scFv induced a therapeutic control of tumor growth similar to unarmed WR combined to systemically administered J43 and superior to that obtained with an unarmed WR. These results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs.

Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allows their intratumoral delivery and an improved tumor-growth inhibition

PubMed Central

Kleinpeter, Patricia; Fend, Laetitia; Thioudellet, Christine; Geist, Michel; Sfrontato, Nathalie; Koerper, Véronique; Fahrner, Catherine; Schmitt, Doris; Gantzer, Murielle; Remy-Ziller, Christelle; Brandely, Renée; Villeval, Dominique; Rittner, Karola; Silvestre, Nathalie; Erbs, Philippe; Zitvogel, Laurence; Quéméneur, Eric; Préville, Xavier; Marchand, Jean-Baptiste

2016-01-01

ABSTRACT We report here the successful vectorization of a hamster monoclonal IgG (namely J43) recognizing the murine Programmed cell death-1 (mPD-1) in Western Reserve (WR) oncolytic vaccinia virus. Three forms of mPD-1 binders have been inserted into the virus: whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv). MAb, Fab and scFv were produced and assembled with the expected patterns in supernatants of cells infected by the recombinant viruses. The three purified mPD-1 binders were able to block the binding of mPD-1 ligand to mPD-1 in vitro. Moreover, mAb was detected in tumor and in serum of C57BL/6 mice when the recombinant WR-mAb was injected intratumorally (IT) in B16F10 and MCA 205 tumors. The concentration of circulating mAb detected after IT injection was up to 1,900-fold higher than the level obtained after a subcutaneous (SC) injection (i.e., without tumor) confirming the virus tropism for tumoral cells and/or microenvironment. Moreover, the overall tumoral accumulation of the mAb was higher and lasted longer after IT injection of WR-mAb1, than after IT administration of 10 µg of J43. The IT injection of viruses induced a massive infiltration of immune cells including activated lymphocytes (CD8+ and CD4+). Interestingly, in the MCA 205 tumor model, WR-mAb1 and WR-scFv induced a therapeutic control of tumor growth similar to unarmed WR combined to systemically administered J43 and superior to that obtained with an unarmed WR. These results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs. PMID:27853644

Established a new double antibodies sandwich enzyme-linked immunosorbent assay for detecting Bacillus thuringiensis (Bt) Cry1Ab toxin based single-chain variable fragments from a naïve mouse phage displayed library.

PubMed

Zhang, Xiao; Xu, Chongxin; Zhang, Cunzheng; Liu, Yuan; Xie, Yajing; Liu, Xianjin

2014-04-01

ScFvs are composed of the variable regions of the heavy and light chains via a short linker that maintain the specific antigen binding abilities of antibodies. In this study, we constructed a naïve mouse phage displayed library to generate scFvs against Cry1Ab toxin. After affinity panning, positive phage-scFvs were isolated, sequenced and characterized by ELISA. The best binding ability scFv-G9 was expressed and purified. SDS-PAGE indicated that the relative molecular mass of scFv was estimated at 28 kDa. The purified scFv-G9 was used to develop a new DAS-ELISA for detecting Cry1Ab toxin, within minimum detection limit of 0.008 μg mL(-1), a working range 0.018-6.23 μg mL(-1), and the linear curve displayed an acceptable correlation coefficient of 0.98. The cross-reactivity showed that scFv-G9 had strongly binding ability to Cry1Ac toxin, but not to Cry1B, Cry1C and Cry1F toxin. The average recoveries of Cry1Ab toxin from spiked leaf and rice samples were in the range 92.1-94.8%, and 91.6-98.6%, respectively, with a coefficient of variation (C.V) less than 5.0%. These results showed promising applications of scfv-G9 for detecting Cry1Ab toxin with new DAS-ELISA. Copyright © 2014 Elsevier Ltd. All rights reserved.

Construction and evaluation of a novel humanized HER2-specific chimeric receptor

PubMed Central

2014-01-01

Introduction The human epidermal growth factor receptor 2 (HER2) represents one of the most studied tumor-associated antigens (TAAs) for cancer immunotherapy. The monoclonal antibody (mAb) trastuzumab has improved the outcomes of patients with HER2+ breast cancer. However, a large number of HER2+ tumors are not responsive to, or become resistant to, trastuzumab-based therapy, and thus more effective therapies targeting HER2 are needed. Methods HER2-specific T cells were generated by the transfer of genes that encode chimeric antigen receptor (CAR). Using a multistep overlap extension PCR method, we constructed a novel, humanized HER2 CAR-containing, chA21 single-chain variable fragment (scFv) region of antigen-specific mAb and T-cell intracellular signaling chains made up of CD28 and CD3ζ. An interferon γ and interleukin 2 enzyme-linked immunosorbent assay and a chromium-51 release assay were used to evaluate the antitumor immune response of CAR T cells in coculture with tumor cells. Furthermore, SKBR3 tumor–bearing nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice were treated with HER2 CAR T cells to evaluate antitumor activity. Human CD3+ T cell accumulation in tumor xenograft was detected by immunohistochemistry. Results chA21-28z CAR was successfully constructed, and both CD4+ and CD8+ T cells were transduced. The expanded HER2 CAR T cells expressed a central memory phenotype and specifically reacted against HER2+ tumor cell lines. Furthermore, the SKBR3 tumor xenograft model revealed that HER2 CAR T cells significantly inhibited tumor growth in vivo. Immunohistochemical analysis showed robust accumulation of human CD3+ T cells in regressing SKBR3 lesions. Conclusions The results of this study show that novel chA21 scFv-based, HER2-specific CAR T cells not only recognized and killed HER2+ breast and ovarian cancer cells ex vivo but also induced regression of experimental breast cancer in vivo. Our data support further exploration of the

Single cell digital polymerase chain reaction on self-priming compartmentalization chip.

PubMed

Zhu, Qiangyuan; Qiu, Lin; Xu, Yanan; Li, Guang; Mu, Ying

2017-01-01

Single cell analysis provides a new framework for understanding biology and disease, however, an absolute quantification of single cell gene expression still faces many challenges. Microfluidic digital polymerase chain reaction (PCR) provides a unique method to absolutely quantify the single cell gene expression, but only limited devices are developed to analyze a single cell with detection variation. This paper describes a self-priming compartmentalization (SPC) microfluidic digital polymerase chain reaction chip being capable of performing single molecule amplification from single cell. The chip can be used to detect four single cells simultaneously with 85% of sample digitization. With the optimized protocol for the SPC chip, we first tested the ability, precision, and sensitivity of our SPC digital PCR chip by assessing β-actin DNA gene expression in 1, 10, 100, and 1000 cells. And the reproducibility of the SPC chip is evaluated by testing 18S rRNA of single cells with 1.6%-4.6% of coefficient of variation. At last, by detecting the lung cancer related genes, PLAU gene expression of A549 cells at the single cell level, the single cell heterogeneity was demonstrated. So, with the power-free, valve-free SPC chip, the gene copy number of single cells can be quantified absolutely with higher sensitivity, reduced labor time, and reagent. We expect that this chip will enable new studies for biology and disease.

Single cell digital polymerase chain reaction on self-priming compartmentalization chip

PubMed Central

Zhu, Qiangyuan; Qiu, Lin; Xu, Yanan; Li, Guang; Mu, Ying

2017-01-01

Single cell analysis provides a new framework for understanding biology and disease, however, an absolute quantification of single cell gene expression still faces many challenges. Microfluidic digital polymerase chain reaction (PCR) provides a unique method to absolutely quantify the single cell gene expression, but only limited devices are developed to analyze a single cell with detection variation. This paper describes a self-priming compartmentalization (SPC) microfluidic digital polymerase chain reaction chip being capable of performing single molecule amplification from single cell. The chip can be used to detect four single cells simultaneously with 85% of sample digitization. With the optimized protocol for the SPC chip, we first tested the ability, precision, and sensitivity of our SPC digital PCR chip by assessing β-actin DNA gene expression in 1, 10, 100, and 1000 cells. And the reproducibility of the SPC chip is evaluated by testing 18S rRNA of single cells with 1.6%–4.6% of coefficient of variation. At last, by detecting the lung cancer related genes, PLAU gene expression of A549 cells at the single cell level, the single cell heterogeneity was demonstrated. So, with the power-free, valve-free SPC chip, the gene copy number of single cells can be quantified absolutely with higher sensitivity, reduced labor time, and reagent. We expect that this chip will enable new studies for biology and disease. PMID:28191267

Internal friction of single polypeptide chains at high stretch.

PubMed

Khatri, Bhavin S; Byrne, Katherine; Kawakami, Masaru; Brockwell, David J; Smith, D Alastair; Radford, Sheena E; McLeish, Tom C B

2008-01-01

Experiments that measure the viscoelasticity of single molecules from the Brownian fluctuations of an atomic force microscope (AFM) have provided a new window onto their internal dynamics in an underlying conformational landscape. Here we develop and apply these methods to examine the internal friction of unfolded polypeptide chains at high stretch. The results reveal a power law dependence of internal friction with tension (exponent 1.3 +/- 0.5) and a relaxation time approximately independent of force. To explain these results we develop a frictional worm-like chain (FWLC) model based on the Rayleigh dissipation function of a stiff chain with dynamical resistance to local bending. We analyse the dissipation rate integrated over the chain length by its Fourier components to calculate an effective tension-dependent friction constant for the end-to-end vector of the chain. The result is an internal friction that increases as a power law with tension with an exponent 3/2, consistent with experiment. Extracting the intrinsic bending friction constant of the chain it is found to be approximately 7 orders of magnitude greater than expected from solvent friction alone; a possible explanation we offer is that the underlying energy landscape for bending amino acids and/or peptide bond is rough, consistent with recent results on both proteins and polysaccharides.

Pre-Discovery Detection of ASASSN-18fv by Evryscope

NASA Astrophysics Data System (ADS)

Corbett, H.; Law, N.; Goeke, E.; Ratzloff, J.; Howard, W.; Fors, O.; del Ser, D.; Quimby, R. M.

2018-03-01

We have identified pre-discovery imaging of the probable classical nova ASASSN-18fv by Evryscope-South (http://evryscope.astro.unc.edu/), an array of 6-cm telescopes continuously monitoring 8000 square degrees of sky at 2-minute cadence from CTIO, Chile.

Design of an Active Ultrastable Single-chain Insulin Analog

PubMed Central

Hua, Qing-xin; Nakagawa, Satoe H.; Jia, Wenhua; Huang, Kun; Phillips, Nelson B.; Hu, Shi-quan; Weiss, Michael A.

2008-01-01

Single-chain insulin (SCI) analogs provide insight into the inter-relation of hormone structure, function, and dynamics. Although compatible with wild-type structure, short connecting segments (<3 residues) prevent induced fit upon receptor binding and so are essentially without biological activity. Substantial but incomplete activity can be regained with increasing linker length. Here, we describe the design, structure, and function of a single-chain insulin analog (SCI-57) containing a 6-residue linker (GGGPRR). Native receptor-binding affinity (130 ± 8% relative to the wild type) is achieved as hindrance by the linker is offset by favorable substitutions in the insulin moiety. The thermodynamic stability of SCI-57 is markedly increased (ΔΔGu = 0.7 ± 0.1 kcal/mol relative to the corresponding two-chain analog and 1.9 ± 0.1 kcal/mol relative to wild-type insulin). Analysis of inter-residue nuclear Overhauser effects demonstrates that a native-like fold is maintained in solution. Surprisingly, the glycine-rich connecting segment folds against the insulin moiety: its central Pro contacts ValA3 at the edge of the hydrophobic core, whereas the final Arg extends the A1-A8 α-helix. Comparison between SCI-57 and its parent two-chain analog reveals striking enhancement of multiple native-like nuclear Overhauser effects within the tethered protein. These contacts are consistent with wild-type crystal structures but are ordinarily attenuated in NMR spectra of two-chain analogs, presumably due to conformational fluctuations. Linker-specific damping of fluctuations provides evidence for the intrinsic flexibility of an insulin monomer. In addition to their biophysical interest, ultrastable SCIs may enhance the safety and efficacy of insulin replacement therapy in the developing world. PMID:18332129

Crosslinked polymer nanoparticles containing single conjugated polymer chains

NASA Astrophysics Data System (ADS)

Ponzio, Rodrigo A.; Marcato, Yésica L.; Gómez, María L.; Waiman, Carolina V.; Chesta, Carlos A.; Palacios, Rodrigo E.

2017-06-01

Conjugated polymer nanoparticles are widely used in fluorescent labeling and sensing, as they have mean radii between 5 and 100 nm, narrow size dispersion, high brightness, and are photochemically stable, allowing single particle detection with high spatial and temporal resolution. Highly crosslinked polymers formed by linking individual chains through covalent bonds yield high-strength rigid materials capable of withstanding dissolution by organic solvents. Hence, the combination of crosslinked polymers and conjugated polymers in a nanoparticulated material presents the possibility of interesting applications that require the combined properties of constituent polymers and nanosized dimension. In the present work, F8BT@pEGDMA nanoparticles composed of poly(ethylene glycol dimethacrylate) (pEGDMA; a crosslinked polymer) and containing the commercial conjugated polymer poly(9,9-dioctylfluorene-alt-benzothiadiazole) (F8BT) were synthesized and characterized. Microemulsion polymerization was applied to produce F8BT@pEDGMA particles with nanosized dimensions in a ∼25% yield. Photophysical and size distribution properties of F8BT@pEDGMA nanoparticles were evaluated by various methods, in particular single particle fluorescence microscopy techniques. The results demonstrate that the crosslinking/polymerization process imparts structural rigidity to the F8BT@pEDGMA particles by providing resistance against dissolution/disintegration in organic solvents. The synthesized fluorescent crosslinked nanoparticles contain (for the most part) single F8BT chains and can be detected at the single particle level, using fluorescence microscopy, which bodes well for their potential application as molecularly imprinted polymer fluorescent nanosensors with high spatial and temporal resolution.

Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41.

PubMed

Sabin, Charles; Corti, Davide; Buzon, Victor; Seaman, Mike S; Lutje Hulsik, David; Hinz, Andreas; Vanzetta, Fabrizia; Agatic, Gloria; Silacci, Chiara; Mainetti, Lara; Scarlatti, Gabriella; Sallusto, Federica; Weiss, Robin; Lanzavecchia, Antonio; Weissenhorn, Winfried

2010-11-18

The human monoclonal antibody (mAb) HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1) of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool.

Effects of Water on the Single-Chain Elasticity of Poly(U) RNA.

PubMed

Luo, Zhonglong; Cheng, Bo; Cui, Shuxun

2015-06-09

Water, the dominant component under the physiological condition, is a complicated solvent which greatly affects the properties of solute molecules. Here, we utilize atomic force microscope-based single-molecule force spectroscopy to study the influence of water on the single-molecule elasticity of an unstructured single-stranded RNA (poly(U)). In nonpolar solvents, RNA presents its inherent elasticity, which is consistent with the theoretical single-chain elasticity calculated by quantum mechanics calculations. In aqueous buffers, however, an additional energy of 1.88 kJ/mol·base is needed for the stretching of the ssRNA chain. This energy is consumed by the bound water rearrangement (Ew) during chain elongation. Further experimental results indicate that the Ew value is uncorrelated to the salt concentrations and stretching velocity. The results obtained in an 8 M guanidine·HCl solution provide more evidence that the bound water molecules around RNA give rise to the observed deviation between aqueous and nonaqueous environments. Compared to synthetic water-soluble polymers, the value of Ew of RNA is much lower. The weak interference of water is supposed to be the precondition for the RNA secondary structure to exist in aqueous solution.

Population pharmacokinetics of recombinant coagulation factor VIII-SingleChain in patients with severe hemophilia A.

PubMed

Zhang, Y; Roberts, J; Tortorici, M; Veldman, A; St Ledger, K; Feussner, A; Sidhu, J

2017-06-01

Essentials rVIII-SingleChain is a unique recombinant factor VIII (FVIII) molecule. A population pharmacokinetic model was based on FVIII activity of severe hemophilia A patients. The model was used to simulate factor VIII activity-time profiles for various dosing scenarios. The model supports prolonged dosing of rVIII-SingleChain with intervals of up to twice per week. Background Single-chain recombinant coagulation factor VIII (rVIII-SingleChain) is a unique recombinant coagulation factor VIII molecule. Objectives To: (i) characterize the population pharmacokinetics (PK) of rVIII-SingleChain in patients with severe hemophilia A; (ii) identify correlates of variability in rVIII-SingleChain PK; and (iii) simulate various dosing scenarios of rVIII-SingleChain. Patients/Methods A population PK model was developed, based on FVIII activity levels of 130 patients with severe hemophilia A (n = 91 for ≥ 12-65 years; n = 39 for < 12 years) who had participated in a single-dose PK investigation with rVIII-SingleChain 50 IU kg -1 . PK sampling was performed for up to 96 h. Results A two-compartment population PK model with first-order elimination adequately described FVIII activity. Body weight and predose level of von Willebrand factor were significant covariates on clearance, and body weight was a significant covariate on the central distribution volume. Simulations using the model with various dosing scenarios estimated that > 85% and > 93% of patients were predicted to maintain FVIII activity level above 1 IU dL -1 , at all times with three-times-weekly dosing (given on days 0, 2, and 4.5) at the lowest (20 IU kg -1 ) and highest (50 IU kg -1 ) doses, respectively. For twice weekly dosing (days 0 and 3.5) of 50 IU kg -1 rVIII-SingleChain, 62-80% of patients across all ages were predicted to maintain a FVIII activity level above 1 IU dL -1 at day 7. Conclusions The population PK model adequately characterized rVIII-SingleChain PK, and the model

Mice deficient in LMAN1 exhibit FV and FVIII deficiencies and liver accumulation of α1-antitrypsin

PubMed Central

Zheng, Chunlei; Zhu, Min; Tao, Jiayi; Vasievich, Matthew P.; Baines, Andrea; Kim, Jinoh; Schekman, Randy; Kaufman, Randal J.; Ginsburg, David

2011-01-01

The type 1-transmembrane protein LMAN1 (ERGIC-53) forms a complex with the soluble protein MCFD2 and cycles between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment (ERGIC). Mutations in either LMAN1 or MCFD2 cause the combined deficiency of factor V (FV) and factor VIII (FVIII; F5F8D), suggesting an ER-to-Golgi cargo receptor function for the LMAN1-MCFD2 complex. Here we report the analysis of LMAN1-deficient mice. Levels of plasma FV and FVIII, and platelet FV, are all reduced to ∼ 50% of wild-type in Lman1−/− mice, compared with the 5%-30% levels typically observed in human F5F8D patients. Despite previous reports identifying cathepsin C, cathepsin Z, and α1-antitrypsin as additional potential cargoes for LMAN1, no differences were observed between wild-type and Lman1−/− mice in the levels of cathepsin C and cathepsin Z in liver lysates or α1-antitrypsin levels in plasma. LMAN1 deficiency had no apparent effect on COPII-coated vesicle formation in an in vitro assay. However, the ER in Lman1−/− hepatocytes is slightly distended, with significant accumulation of α1-antitrypsin and GRP78. An unexpected, partially penetrant, perinatal lethality was observed for Lman1−/− mice, dependent on the specific inbred strain genetic background, suggesting a potential role for other, as yet unidentified LMAN1-dependent cargo proteins. PMID:21795745

Synthesis and circularization of N- and B-tropic retroviral DNA in Fv-1 permissive and restrictive mouse cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, W.K.; Kiggans, J.O.; Yang, D.M.

1980-05-01

Production of various forms of nonintegrated viral DNA was measured in cultured mouse cells carrying different Fv-1 alleles early after infection with N-tropic or B-tropic retroviruses. Quantitative analyses were performed by agarose gel electrophoresis, transfer to diazobenzyloxymethyl-paper, and molecular hybridization. In permissive infection of Fv-1/sup n/ cells (NIH Swiss and DBA mouse strains) with N-tropic virus and of Fv-1/sup b/ cells (BALB/c and C57BL/6 strains) with B-tropic virus, form III (double-stranded linear) DNA first appeared at 3 to 4 hr and reached a maximum at 8 to 10 hr; two form I (closed circle) DNAs appeared at 7 to 8more » hr and reached a maximum at or beyond 12 hr. In the two Fv-1/sup b/ cells infected with N-tropic virus and in DBA (Fv-1/sup n/) cells infected with B-tropic virus, formation of the two form I DNAs was quantitatively restricted but formation of form III DNA was unaltered. In Fv-1/sup n/ NIH Swiss mouse embryo cells infected with B-tropic virus, the level of form III DNA was markedly depressed and hence the two form I DNAs were not detectable. In C57BL/6 cells as well as in DBA/2 cells 12 hr after infection, the quantity of form III DNA varied directly with the amount of restricted virus, whereas the quantity of form I DNA varied according to the square of the amount of restricted virus. The significance of these results for understanding the molecular basis of retrovirus replication and its restriction by the Fv-1 gene is discussed.« less

Anti-CDR3 Therapy for B-Cell Malignancies

DTIC Science & Technology

2013-10-01

1-5 in the report). The "Tomlinson" human antibody phage library will be used to pan for antibodies that bind these target CDR3s and not the parent...selection of phage to a test antigen. Successful expression will lead directly to the selection of CDR3-specific antibody-encoding phage : from which we... phage that bind to the purified candidate antibody and not to irrelevant antibodies. Phage are from single chain Fv library. Sequence phage that

High Electrical Conductivity of Single Metal-Organic Chains.

PubMed

Ares, Pablo; Amo-Ochoa, Pilar; Soler, José M; Palacios, Juan José; Gómez-Herrero, Julio; Zamora, Félix

2018-05-01

Molecular wires are essential components for future nanoscale electronics. However, the preparation of individual long conductive molecules is still a challenge. MMX metal-organic polymers are quasi-1D sequences of single halide atoms (X) bridging subunits with two metal ions (MM) connected by organic ligands. They are excellent electrical conductors as bulk macroscopic crystals and as nanoribbons. However, according to theoretical calculations, the electrical conductance found in the experiments should be even higher. Here, a novel and simple drop-casting procedure to isolate bundles of few to single MMX chains is demonstrated. Furthermore, an exponential dependence of the electrical resistance of one or two MMX chains as a function of their length that does not agree with predictions based on their theoretical band structure is reported. This dependence is attributed to strong Anderson localization originated by structural defects. Theoretical modeling confirms that the current is limited by structural defects, mainly vacancies of iodine atoms, through which the current is constrained to flow. Nevertheless, measurable electrical transport along distances beyond 250 nm surpasses that of all other molecular wires reported so far. This work places in perspective the role of defects in 1D wires and their importance for molecular electronics. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A chimeric antigen receptor for TRAIL-receptor 1 induces apoptosis in various types of tumor cells.

PubMed

Kobayashi, Eiji; Kishi, Hiroyuki; Ozawa, Tatsuhiko; Hamana, Hiroshi; Nakagawa, Hidetoshi; Jin, Aishun; Lin, Zhezhu; Muraguchi, Atsushi

2014-10-31

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and its associated receptors (TRAIL-R/TR) are attractive targets for cancer therapy because TRAIL induces apoptosis in tumor cells through TR while having little cytotoxicity on normal cells. Therefore, many agonistic monoclonal antibodies (mAbs) specific for TR have been produced, and these induce apoptosis in multiple tumor cell types. However, some TR-expressing tumor cells are resistant to TR-specific mAb-induced apoptosis. In this study, we constructed a chimeric antigen receptor (CAR) of a TRAIL-receptor 1 (TR1)-specific single chain variable fragment (scFv) antibody (TR1-scFv-CAR) and expressed it on a Jurkat T cell line, the KHYG-1 NK cell line, and human peripheral blood lymphocytes (PBLs). We found that the TR1-scFv-CAR-expressing Jurkat cells killed target cells via TR1-mediated apoptosis, whereas TR1-scFv-CAR-expressing KHYG-1 cells and PBLs killed target cells not only via TR1-mediated apoptosis but also via CAR signal-induced cytolysis, resulting in cytotoxicity on a broader range if target cells than with TR1-scFv-CAR-expressing Jurkat cells. The results suggest that TR1-scFv-CAR could be a new candidate for cancer gene therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

Biodiversity on island chains: Neutral model simulations

NASA Astrophysics Data System (ADS)

Warren, Patrick B.

2010-11-01

A neutral ecology model is simulated on an island chain, in which neighboring islands can exchange individuals but only the first island is able to receive immigrants from a metacommunity. It is found by several measures that α -diversity decreases along the chain. Subtle changes in taxon abundance distributions can be detected when islands in the chain are compared to diversity-matched single islands. The island chain is found to have unexpectedly rich dynamics. Significant β -diversity correlations are found between islands in the chain, which are absent between diversity-matched single islands. The results potentially apply to human microbial biodiversity and biogeography and suggest that measurements of interindividual and intraindividual β -diversity may give insights into microbial community assembly mechanisms.

Assimilation of MODIS and VIIRS AOD to improve aerosols forecasts with FV3-GOCART

NASA Astrophysics Data System (ADS)

Pagowski, M.

2017-12-01

In 2016 NOAA chose the FV3 dynamical core as a basis for its future global modeling system. We present an implementation of aerosol module in the FV3 model and its assimilation framework. The parameterization of aerosols is based on the GOCART scheme. The assimilation methodology relies on hybrid 3D-Var and EnKF methods. Aerosol observations include aerosol optical depth at 550 nm from VIIRS satellite. Results and evaluation of the system against independent observations and NASA's MERRA-2 is shown.

CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

PubMed Central

Alonso-Camino, Vanesa; Sánchez-Martín, David; Compte, Marta; Nuñez-Prado, Natalia; Diaz, Rosa M; Vile, Richard; Alvarez-Vallina, Luis

2013-01-01

A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells. PMID:23695536

Crystal Structure and Size-Dependent Neutralization Properties of HK20, a Human Monoclonal Antibody Binding to the Highly Conserved Heptad Repeat 1 of gp41

PubMed Central

Seaman, Mike S.; Lutje Hulsik, David; Hinz, Andreas; Vanzetta, Fabrizia; Agatic, Gloria; Silacci, Chiara; Mainetti, Lara; Scarlatti, Gabriella; Sallusto, Federica; Weiss, Robin; Lanzavecchia, Antonio; Weissenhorn, Winfried

2010-01-01

The human monoclonal antibody (mAb) HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1) of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool. PMID:21124990

Primitive-path statistics of entangled polymers: mapping multi-chain simulations onto single-chain mean-field models

NASA Astrophysics Data System (ADS)

Steenbakkers, Rudi J. A.; Tzoumanekas, Christos; Li, Ying; Liu, Wing Kam; Kröger, Martin; Schieber, Jay D.

2014-01-01

We present a method to map the full equilibrium distribution of the primitive-path (PP) length, obtained from multi-chain simulations of polymer melts, onto a single-chain mean-field ‘target’ model. Most previous works used the Doi-Edwards tube model as a target. However, the average number of monomers per PP segment, obtained from multi-chain PP networks, has consistently shown a discrepancy of a factor of two with respect to tube-model estimates. Part of the problem is that the tube model neglects fluctuations in the lengths of PP segments, the number of entanglements per chain and the distribution of monomers among PP segments, while all these fluctuations are observed in multi-chain simulations. Here we use a recently proposed slip-link model, which includes fluctuations in all these variables as well as in the spatial positions of the entanglements. This turns out to be essential to obtain qualitative and quantitative agreement with the equilibrium PP-length distribution obtained from multi-chain simulations. By fitting this distribution, we are able to determine two of the three parameters of the model, which govern its equilibrium properties. This mapping is executed for four different linear polymers and for different molecular weights. The two parameters are found to depend on chemistry, but not on molecular weight. The model predicts a constant plateau modulus minus a correction inversely proportional to molecular weight. The value for well-entangled chains, with the parameters determined ab initio, lies in the range of experimental data for the materials investigated.

The story of a unique molecule in hemophilia A: recombinant single-chain factor VIII.

PubMed

Pabinger-Fasching, Ingrid

2016-05-01

For patients with hemophilia A, replacement of deficient factor VIII (FVIII) using plasma-derived or recombinant FVIII (rFVIII) products to restore hemostatic control can reduce bleeding complications and preserve musculoskeletal function. Despite the clinical availability of several of these products, challenges remain in the treatment of hemophilia A, the most notable of which are the risk of inhibitor development and the limited half-life of existing FVIII concentrates, which can make prophylaxis burdensome for patients. The use of recombinant protein technology may lead to novel FVIII products with improved properties. This article describes the story of a unique recombinant FVIII protein, rVIII-SingleChain, which is currently in development. In contrast to native FVIII and other commercially available rFVIII preparations, rVIII-SingleChain uses a strong, covalent bond to connect the light and heavy chains, thereby creating a stable, single-chain rFVIII. It has enhanced intrinsic stability, better integrity after reconstitution, and a higher binding affinity to von Willebrand factor. The physicochemical profile of rVIII-SingleChain and preclinical data on its activity and phamacokinetics strengthened the rationale for its clinical investigation. Available data from the AFFINITY clinical trial program are promising; indicating that it has good hemostatic efficacy when used on demand, for prophylaxis, and in the surgical setting, and is also very well tolerated. A pediatric study and an extension study are ongoing as part of the AFFINITY program. © 2016 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Development of a new high-affinity human antibody with antitumor activity against solid and blood malignancies.

PubMed

Sioud, Mouldy; Westby, Phuong; Vasovic, Vlada; Fløisand, Yngvar; Peng, Qian

2018-04-16

mAbs have emerged as a promising strategy for the treatment of cancer. However, in several malignancies, no effective antitumor mAbs are yet available. Identifying therapeutic mAbs that recognize common tumor antigens could render the treatment widely applicable. Here, a human single-chain variable fragment (scFv) antibody library was sequentially affinity selected against a panel of human cancer cell lines and an antibody fragment (named MS5) that bound to solid and blood cancer cells was identified. The MS5 scFv was fused to the human IgG1 Fc domain to generate an antibody (MS5-Fc fusion) that induced antibody-dependent cellular cytotoxicity and phagocytosis of cancer cells by macrophages. In addition, the MS5-Fc antibody bound to primary leukemia cells and induced antibody-dependent cellular cytotoxicity. In the majority of analyzed cancer cells, the MS5-Fc antibody induced cell surface redistribution of the receptor complexes, but not internalization, thus maximizing the accessibility of the IgG1 Fc domain to immune effector cells. In vitro stability studies showed that the MS5-Fc antibody was stable after 6 d of incubation in human serum, retaining ∼60% of its initial intact form. After intravenous injections, the antibody localized into tumor tissues and inhibited the growth of 3 different human tumor xenografts (breast, lymphoma, and leukemia). These antitumor effects were associated with tumor infiltration by macrophages and NK cells. In the Ramos B-cell lymphoma xenograft model, the MS5-Fc antibody exhibited a comparable antitumor effect as rituximab, a chimeric anti-CD20 IgG1 mAb. These results indicate that human antibodies with pan-cancer abilities can be generated from phage display libraries, and that the engineered MS5-Fc antibody could be an attractive agent for further clinical investigation.-Sioud, M., Westby, P., Vasovic, V., Fløisand, Y., Peng, Q. Development of a new high-affinity human antibody with antitumor activity against solid and

Rapid Isolation of a Single-Chain Antibody against the Cyanobacterial Toxin Microcystin-LR by Phage Display and Its Use in the Immunoaffinity Concentration of Microcystins from Water

PubMed Central

McElhiney, Jacqui; Drever, Mathew; Lawton, Linda A.; Porter, Andy J.

2002-01-01

A naïve (unimmunized) human semisynthetic phage display library was employed to isolate recombinant antibody fragments against the cyanobacterial hepatotoxin microcystin-LR. Selected antibody scFv genes were cloned into a soluble expression vector and expressed in Escherichia coli for characterization against purified microcystin-LR by competition enzyme-linked immunosorbent assay (ELISA). The most sensitive single-chain antibody (scAb) isolated was capable of detecting microcystin-LR at levels below the World Health Organization limit in drinking water (1 μg liter−1) and cross-reacted with three other purified microcystin variants (microcystin-RR, -LW, and -LF) and the related cyanotoxin nodularin. Extracts of the cyanobacterium Microcystis aeruginosa were assayed by ELISA, and quantifications of microcystins in toxic samples showed good correlation with analysis by high-performance liquid chromatography. Immobilized scAb was also used to prepare immunoaffinity columns, which were assessed for the ability to concentrate microcystin-LR from water for subsequent analysis by high-performance liquid chromatography. Anti-microcystin-LR scAb was immobilized on columns via a hexahistidine tag, ensuring maximum exposure of antigen binding sites, and the performance of the columns was evaluated by directly applying 150 ml of distilled water spiked with 4 μg of purified microcystin-LR. The procedure was simple, and a recovery rate of 94% was achieved following elution in 1 ml of 100% methanol. Large-scale, low-cost production of anti-microcystin-LR scAb in E. coli is an exciting prospect for the development of biosensors and on-line monitoring systems for microcystins and will also facilitate a range of immunoaffinity applications for the cleanup and concentration of these toxins from environmental samples. PMID:12406716

Sequence-Based Discovery Demonstrates That Fixed Light Chain Human Transgenic Rats Produce a Diverse Repertoire of Antigen-Specific Antibodies.

PubMed

Harris, Katherine E; Aldred, Shelley Force; Davison, Laura M; Ogana, Heather Anne N; Boudreau, Andrew; Brüggemann, Marianne; Osborn, Michael; Ma, Biao; Buelow, Benjamin; Clarke, Starlynn C; Dang, Kevin H; Iyer, Suhasini; Jorgensen, Brett; Pham, Duy T; Pratap, Payal P; Rangaswamy, Udaya S; Schellenberger, Ute; van Schooten, Wim C; Ugamraj, Harshad S; Vafa, Omid; Buelow, Roland; Trinklein, Nathan D

2018-01-01

We created a novel transgenic rat that expresses human antibodies comprising a diverse repertoire of heavy chains with a single common rearranged kappa light chain (IgKV3-15-JK1). This fixed light chain animal, called OmniFlic, presents a unique system for human therapeutic antibody discovery and a model to study heavy chain repertoire diversity in the context of a constant light chain. The purpose of this study was to analyze heavy chain variable gene usage, clonotype diversity, and to describe the sequence characteristics of antigen-specific monoclonal antibodies (mAbs) isolated from immunized OmniFlic animals. Using next-generation sequencing antibody repertoire analysis, we measured heavy chain variable gene usage and the diversity of clonotypes present in the lymph node germinal centers of 75 OmniFlic rats immunized with 9 different protein antigens. Furthermore, we expressed 2,560 unique heavy chain sequences sampled from a diverse set of clonotypes as fixed light chain antibody proteins and measured their binding to antigen by ELISA. Finally, we measured patterns and overall levels of somatic hypermutation in the full B-cell repertoire and in the 2,560 mAbs tested for binding. The results demonstrate that OmniFlic animals produce an abundance of antigen-specific antibodies with heavy chain clonotype diversity that is similar to what has been described with unrestricted light chain use in mammals. In addition, we show that sequence-based discovery is a highly effective and efficient way to identify a large number of diverse monoclonal antibodies to a protein target of interest.

Choice between Single and Multiple Reinforcers in Concurrent-Chains Schedules

ERIC Educational Resources Information Center

Mazur, James E.

2006-01-01

Pigeons responded on concurrent-chains schedules with equal variable-interval schedules as initial links. One terminal link delivered a single reinforcer after a fixed delay, and the other terminal link delivered either three or five reinforcers, each preceded by a fixed delay. Some conditions included a postreinforcer delay after the single…

Effects of developer exhaustion on DFL Contrast FV-58 and Kodak Insight dental films.

PubMed

de Carvalho, Fabiano Pachêco; da Silveira, M M F; Frazão, M A G; de Santana, S T; dos Anjos Pontual, M L

2011-09-01

The aim of this study was to compare the properties of the DFL Contrast FV-58 F-speed film (DFL Co., Rio de Janerio, Brazil) with the Kodak Insight E/F speed film (Eastman Kodak, Rochester, NY) in fresh and exhausted processing solutions. The parameters studied were the speed, average gradient and latitude. Five samples of each type of film were exposed under standardized conditions over 5 weeks. The films were developed in fresh and progressively exhausted processing solutions. Characteristic curves were constructed from values of optical density and radiation dose and were used to calculate the parameters. An analysis of variance was performed separately for film type and time. DFL Contrast FV-58 film has a speed and average gradient that is significantly higher than Insight film, whereas the values of latitude are lower. Exhausted processing solutions were not significant in the parameters studied. DFL Contrast FV-58 film has stable properties when exhausted manual processing solutions are used and can be recommended for use in dental practice, contributing to dose reduction.

Effects of developer exhaustion on DFL Contrast FV-58 and Kodak Insight dental films

PubMed Central

de Carvalho, FP; da Silveira, MMF; Frazão, MAG; de Santana, ST; dos Anjos Pontual, ML

2011-01-01

Objectives The aim of this study was to compare the properties of the DFL Contrast FV-58 F-speed film (DFL Co., Rio de Janerio, Brazil) with the Kodak Insight E/F speed film (Eastman Kodak, Rochester, NY) in fresh and exhausted processing solutions. The parameters studied were the speed, average gradient and latitude. Methods Five samples of each type of film were exposed under standardized conditions over 5 weeks. The films were developed in fresh and progressively exhausted processing solutions. Characteristic curves were constructed from values of optical density and radiation dose and were used to calculate the parameters. An analysis of variance was performed separately for film type and time. Results DFL Contrast FV-58 film has a speed and average gradient that is significantly higher than Insight film, whereas the values of latitude are lower. Exhausted processing solutions were not significant in the parameters studied. Conclusion DFL Contrast FV-58 film has stable properties when exhausted manual processing solutions are used and can be recommended for use in dental practice, contributing to dose reduction. PMID:21831975

Fermi-LAT Bright Gamma-ray Detection of Nova ASASSN-18fv

NASA Astrophysics Data System (ADS)

Jean, P.; Cheung, C. C.; Ojha, R.; van Zyl, P.; Angioni, R.

2018-04-01

The Large Area Telescope (LAT), one of two instruments on the Fermi Gamma-ray Space Telescope, has observed bright gamma-ray emission from a source positionally consistent with the bright optical nova ASASSN-18fv (ATel #11454, #11456, #11460, #11467, #11508).

Stretching of Single Polymer Chains Using the Atomic Force Microscope

NASA Astrophysics Data System (ADS)

Ortiz, C.; van der Vegte, E. W.; van Swieten, E.; Robillard, G. T.; Hadziioannou, G.

1998-03-01

A variety of macroscopic phenomenon involve "nanoscale" polymer deformation including rubber elasticity, shear yielding, strain hardening, stress relaxation, fracture, and flow. With the advent of new and improved experimental techniques, such as the atomic force microscope (AFM), the probing of physical properties of polymers has reached finer and finer scales. The development of mixed self-assembling monolayer techniques and the chemical functionalization of AFM probe tips has allowed for mechanical experiments on single polymer chains of molecular dimensions. In our experiments, mixed monolayers are prepared in which end-functionalized, flexible polymer chains of thiol-terminated poly(methacrylic acid) are covalently bonded, isolated, and randomly distributed on gold substrates. The coils are then imaged, tethered to a gold-coated AFM tip, and stretched between the tip and the substrate in a conventional force / distance experiment. An increase in the attractive force due to entropic, elastic resistance to stretching, as well as fracture of the polymer chain is observed. The effect of chain stiffness, topological constraints, strain rate, mechanical hysteresis, and stress relaxation were investigated. Force modulation techniques were also employed in order to image the viscoelastic character of the polymer chains. Parallel work includes similar studies of biological systems such as wheat gluten proteins and polypeptides.

Docking and free energy simulations to predict conformational domains involved in hCG-LH receptor interactions using recombinant antibodies.

PubMed

Majumdar, Ritankar; Railkar, Reema; Dighe, Rajan R

2011-11-01

Single chain fragment variables (ScFvs) have been extensively employed in studying the protein-protein interactions. ScFvs derived from phage display libraries have an additional advantage of being generated against a native antigen, circumventing loss of information on conformational epitopes. In the present study, an attempt has been made to elucidate human chorionic gonadotropin (hCG)-luteinizing hormone (LH) receptor interactions by using a neutral and two inhibitory ScFvs against hCG. The objective was to dock a computationally derived model of these ScFvs onto the crystal structure of hCG and understand the differential roles of the mapped epitopes in hCG-LH receptor interactions. An anti-hCG ScFv, whose epitope was mapped previously using biochemical tools, served as the positive control for assessing the quality of docking analysis. To evaluate the role of specific side chains at the hCG-ScFv interface, binding free energy as well as residue interaction energies of complexes in solution were calculated using molecular mechanics Poisson-Boltzmann/surface area method after performing the molecular dynamic simulations on the selected hCG-ScFv models and validated using biochemical and SPR analysis. The robustness of these calculations was demonstrated by comparing the theoretically determined binding energies with the experimentally obtained kinetic parameters for hCG-ScFv complexes. Superimposition of hCG-ScFv model onto a model of hCG complexed with the 51-266 residues of LH receptor revealed importance of the residues previously thought to be unimportant for hormone binding and response. This analysis provides an alternate tool for understanding the structure-function analysis of ligand-receptor interactions. Copyright © 2011 Wiley-Liss, Inc.

An unusual cysteine VL87 affects the antibody fragment conformations without interfering with the disulfide bond formation.

PubMed

Attallah, Carolina; Aguilar, María Fernanda; Garay, A Sergio; Herrera, Fernando E; Etcheverrigaray, Marina; Oggero, Marcos; Rodrigues, Daniel E

2017-10-01

The Cys residues are almost perfectly conserved in all antibodies. They contribute significantly to the antibody fragment stability. The relevance of two natural contiguous Cys residues of an anti-recombinant human-follicle stimulation hormone (rhFSH) in a format of single-chain variable fragment (scFv) was studied. This scFv contains 5 Cys residues: V H 22 and V H 92 in the variable heavy chain (V H ) and V L 23, V L 87 and V L 88 in the variable light chain (V L ). The influence of two unusual contiguous Cys at positions V L 87 and V L 88 was studied by considering the wild type fragment and mutant variants: V L -C88S, V L -C87S, V L -C87Y. The analysis was carried out using antigen-binding ability measurement by indirect specific ELISA and a detailed molecular modeling that comprises homology methods, long molecular dynamics simulations and docking. We found that V L -C87 affected the antibody fragment stability without interfering with the disulfide bond formation. The effect of mutating the V L -C87 by a usual residue at this position like Tyr caused distant structural changes at the V H region that confers a higher mobility to the V H -CDR2 and V H -CDR3 loops improving the scFv binding to the antigen. Copyright © 2017 Elsevier Ltd. All rights reserved.

Evaluation of glycodendron and synthetically-modified dextran clearing agents for multi-step targeting of radioisotopes for molecular imaging and radioimmunotherapy

PubMed Central

Cheal, Sarah M.; Yoo, Barney; Boughdad, Sarah; Punzalan, Blesida; Yang, Guangbin; Dilhas, Anna; Torchon, Geralda; Pu, Jun; Axworthy, Don B.; Zanzonico, Pat; Ouerfelli, Ouathek; Larson, Steven M.

2014-01-01

A series of N-acetylgalactosamine-dendrons (NAG-dendrons) and dextrans bearing biotin moieties were compared for their ability to complex with and sequester circulating bispecific anti-tumor antibody (scFv4) streptavidin (SA) fusion protein (scFv4-SA) in vivo, to improve tumor to normal tissue concentration ratios for targeted radioimmunotherapy and diagnosis. Specifically, a total of five NAG-dendrons employing a common synthetic scaffold structure containing 4, 8, 16, or 32 carbohydrate residues and a single biotin moiety were prepared (NAGB), and for comparative purposes, a biotinylated-dextran with average molecular weight (MW) of 500 kD was synthesized from amino-dextran (DEXB). One of the NAGB compounds, CA16, has been investigated in humans; our aim was to determine if other NAGB analogs (e.g. CA8 or CA4) were bioequivalent to CA16 and/or better suited as MST reagents. In vivo studies included dynamic positron-emission tomography (PET) imaging of 124I-labelled-scFv4-SA clearance and dual-label biodistribution studies following multi-step targeting (MST) directed at subcutaneous (s.c.) human colon adenocarcinoma xenografts in mice. The MST protocol consists of three injections: first, a bispecific antibody specific for an anti-tumor associated glycoprotein (TAG-72) single chain genetically-fused with SA (scFv4-SA); second, CA16 or other clearing agent; and third, radiolabeled biotin. We observed using PET imaging of 124I-labelled-scFv4-SA clearance that the spatial arrangement of ligands conjugated to NAG (i.e. biotin) can impact the binding to antibody in circulation and subsequent liver uptake of the NAG-antibody complex. Also, NAGB CA32-LC or CA16-LC can be utilized during MST to achieve comparable tumor- to-blood ratios and absolute tumor uptake seen previously with CA16. Finally, DEXB was equally effective as NAGB CA32-LC at lowering scFv4-SA in circulation, but at the expense of reducing absolute tumor uptake of radiolabeled biotin. PMID:24219178

Chain-Length-Dependent Exciton Dynamics in Linear Oligothiophenes Probed Using Ensemble and Single-Molecule Spectroscopy.

PubMed

Kim, Tae-Woo; Kim, Woojae; Park, Kyu Hyung; Kim, Pyosang; Cho, Jae-Won; Shimizu, Hideyuki; Iyoda, Masahiko; Kim, Dongho

2016-02-04

Exciton dynamics in π-conjugated molecular systems is highly susceptible to conformational disorder. Using time-resolved and single-molecule spectroscopic techniques, the effect of chain length on the exciton dynamics in a series of linear oligothiophenes, for which the conformational disorder increased with increasing chain length, was investigated. As a result, extraordinary features of the exciton dynamics in longer-chain oligothiophene were revealed. Ultrafast fluorescence depolarization processes were observed due to exciton self-trapping in longer and bent chains. Increase in exciton delocalization during dynamic planarization processes was also observed in the linear oligothiophenes via time-resolved fluorescence spectra but was restricted in L-10T because of its considerable conformational disorder. Exciton delocalization was also unexpectedly observed in a bent chain using single-molecule fluorescence spectroscopy. Such delocalization modulates the fluorescence spectral shape by attenuating the 0-0 peak intensity. Collectively, these results provide significant insights into the exciton dynamics in conjugated polymers.

Generation and evaluation of antibody agents for molecular imaging of CD44v6-expressing cancers

PubMed Central

Haylock, Anna-Karin; Nilvebrant, Johan; Mortensen, Anja; Velikyan, Irina; Nestor, Marika; Falk, Ronny

2017-01-01

Aim The aim of this study was to generate and characterize scFv antibodies directed to human CD44v6, as well as to radiolabel and evaluate top candidates in vitro and in vivo for their potential use in CD44v6-targeted molecular imaging in cancer patients. Materials and methods Phage display selections were used to isolate CD44v6-specific scFvs. A chain shuffling strategy was employed for affinity maturation based on a set of CD44v6-specific first-generation clones. Two second-generation scFv clones were then chosen for labeling with 111In or 125I and assessed for CD44v6-specific binding on cultured tumor cells. In vivo uptake and distribution was evaluated in tumor-bearing mice using a dual tumor model. Finally, a proof-of-concept small animal PET-CT study was performed on one of the candidates labeled with 124I. Results Two affinity-matured clones, CD44v6-scFv-A11 and CD44v6-scFv-H12, displayed promising binding kinetics. Seven out of eight radiolabeled conjugates demonstrated CD44v6-specific binding. In vivo studies on selected candidates demonstrated very advantageous tumor-to-organ ratios, in particular for iodinated conjugates, where 125I-labeled scFvs exhibited favorable kinetics and tumor-to-blood ratios above five already at 24 hours p.i.. The small animal PET-CT study using 124I-labeled CD44v6-scFv-H12 was in line with the biodistribution data, clearly visualizing the high CD44v6-expressing tumor. Conclusion The single chain fragments, CD44v6-scFv-A11 and CD44v6-scFv-H12 specifically bind to CD44v6, and the radiolabeled counterparts provide high tumor-to-blood ratios and fast clearance from organs and blood. We conclude that radioiodinated CD44v6-scFv-A11 and CD44v6-scFv-H12 possess features highly suitable for stringent molecular imaging. PMID:29029420

Differentiation between Shallow and Deep Charge Trap States on Single Poly(3-hexylthiophene) Chains through Fluorescence Photon Statistics.

PubMed

Grußmayer, Kristin S; Steiner, Florian; Lupton, John M; Herten, Dirk-Peter; Vogelsang, Jan

2015-12-01

Blinking of the photoluminescence (PL) emitted from individual conjugated polymer chains is one of the central observations made by single-molecule spectroscopy (SMS). Important information, for example regarding excitation energy transfer, can be extracted by evaluating dynamic quenching. However, the nature of trap states, which are responsible for PL quenching, often remains obscured. We present a detailed investigation of the photon statistics of single poly(3-hexylthiophene) (P3HT) chains obtained by SMS. The photon statistics provide a measure of the number and brightness of independently emitting areas on a single chain. These observables can be followed during blinking. A decrease in PL intensity is shown to be correlated with either 1) a decrease in the average brightness of the emitting sites; or 2) a decrease in the number of emitting regions. We attribute these phenomena to the formation of 1) shallow charge traps, which can weakly affect all emitting areas of a single chain at once; and 2) deep traps, which have a strong effect on small regions within the single chains. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Dynamical behavior of a single polymer chain under nanometric confinement

NASA Astrophysics Data System (ADS)

Lagrené, K.; Zanotti, J.-M.; Daoud, M.; Farago, B.; Judeinstein, P.

2010-10-01

We address the dynamical behavior of a single polymer chain under nanometric confinement. We consider a polymer melt made of a mixture of hydrogenated and deuterated high molecular mass Poly(Ethylene Oxide) (PEO). The confining material is a membrane of Anodic Aluminum Oxide (AAO), a macroscopically highly ordered confining system made of parallel cylindrical channels. We use Neutron Spin-Echo (NSE) under the Zero Average Contrast (ZAC) condition to, all at once, i) match the intense porous AAO detrimental elastic SANS (Small Angle Neutron Scattering) contribution to the total intermediate scattering function I(Q,t) and ii) measure the Q dependence of the dynamical modes of a single chain under confinement. The polymer dynamics is probed on an extremely broad spacial ([2.2 10-2 Å-1, 0.2 Å-1]) and temporal ([0.1 ns, 600 ns]) ranges. We do not detect any influence of confinement on the polymer dynamics. This result is discussed in the framework of the debate on the existence of a "corset effect" recently suggested by NMR relaxometry data.

Interfacial free energy governs single polystyrene chain collapse in water and aqueous solutions.

PubMed

Li, Isaac T S; Walker, Gilbert C

2010-05-12

The hydrophobic interaction is significantly responsible for driving protein folding and self-assembly. To understand it, the thermodynamics, the role of water structure, the dewetting process surrounding hydrophobes, and related aspects have undergone extensive investigations. Here, we examine the hypothesis that polymer-solvent interfacial free energy is adequate to describe the energetics of the collapse of a hydrophobic homopolymer chain at fixed temperature, which serves as a much simplified model for studying the hydrophobic collapse of a protein. This implies that changes in polymer-solvent interfacial free energy should be directly proportional to the force to extend a collapsed polymer into a bad solvent. To test this hypothesis, we undertook single-molecule force spectroscopy on a collapsed, single, polystyrene chain in water-ethanol and water-salt mixtures where we measured the monomer solvation free energy from an ensemble average conformations. Different proportions within the binary mixture were used to create solvents with different interfacial free energies with polystyrene. In these mixed solvents, we observed a linear correlation between the interfacial free energy and the force required to extend the chain into solution, which is a direct measure of the solvation free energy per monomer on a single chain at room temperature. A simple analytical model compares favorably with the experimental results. This knowledge supports a common assumption that explicit water solvent may not be necessary for cases whose primary concerns are hydrophobic interactions and hydrophobic hydration.

Efficacy and safety of a clinically relevant foamy vector design in human hematopoietic repopulating cells.

PubMed

Everson, Elizabeth M; Hocum, Jonah D; Trobridge, Grant D

2018-06-23

Previous studies have shown that foamy viral (FV) vectors are a promising alternative to gammaretroviral and lentiviral vectors and insulators can improve FV vector safety. However, in a previous analysis of insulator effects on FV vector safety, strong viral promoters were used to elicit genotoxic events. Here we developed and analyzed the efficacy and safety of a high-titer, clinically relevant FV vector driven by the housekeeping promoter elongation factor-1α and insulated with an enhancer blocking A1 insulator (FV-EGW-A1). Human CD34 + cord blood cells were exposed to an enhanced green fluorescent protein expressing vector, FV-EGW-A1, at a multiplicity of infection of 10 and then maintained in vitro or transplanted into immunodeficient mice. Flow cytometry was used to measure engraftment and marking in vivo. FV vector integration sites were analyzed to assess safety. FV-EGW-A1 resulted in high-marking, multi-lineage engraftment of human repopulating cells with no evidence of silencing. Engraftment was highly polyclonal with no clonal dominance and a promising safety profile based on integration site analysis. An FV vector with an elongation factor-1α promoter and an A1 insulator is a promising vector design for use in the clinic. This article is protected by copyright. All rights reserved.

Pricing and ordering policies for price-dependent demand in a supply chain of a single retailer and a single manufacturer

NASA Astrophysics Data System (ADS)

Kim, Jungkyu; Hong, Yushin; Kim, Taebok

2011-01-01

This article discusses joint pricing and ordering policies for price-dependent demand in a supply chain consisting of a single retailer and a single manufacturer. The retailer places orders for products according to an EOQ policy and the manufacturer produces them on a lot-for-lot basis. Four mechanisms with differing levels of coordination are presented. Mathematical models are formulated and solution procedures are developed to determine the optimal retail prices and order quantities. Through extensive numerical experiments, we analyse and compare the behaviours and characteristics of the proposed mechanisms, and find that enhancing the level of coordination has important benefits for the supply chain.

Wheat cultivars selected for high Fv /Fm under heat stress maintain high photosynthesis, total chlorophyll, stomatal conductance, transpiration and dry matter.

PubMed

Sharma, Dew Kumari; Andersen, Sven Bode; Ottosen, Carl-Otto; Rosenqvist, Eva

2015-02-01

The chlorophyll fluorescence parameter Fv /Fm reflects the maximum quantum efficiency of photosystem II (PSII) photochemistry and has been widely used for early stress detection in plants. Previously, we have used a three-tiered approach of phenotyping by Fv /Fm to identify naturally existing genetic variation for tolerance to severe heat stress (3 days at 40°C in controlled conditions) in wheat (Triticum aestivum L.). Here we investigated the performance of the previously selected cultivars (high and low group based on Fv /Fm value) in terms of growth and photosynthetic traits under moderate heat stress (1 week at 36/30°C day/night temperature in greenhouse) closer to natural heat waves in North-Western Europe. Dry matter accumulation after 7 days of heat stress was positively correlated to Fv /Fm . The high Fv /Fm group maintained significantly higher total chlorophyll and net photosynthetic rate (PN ) than the low group, accompanied by higher stomatal conductance (gs ), transpiration rate (E) and evaporative cooling of the leaf (ΔT). The difference in PN between the groups was not caused by differences in PSII capacity or gs as the variation in Fv /Fm and intracellular CO2 (Ci ) was non-significant under the given heat stress. This study validated that our three-tiered approach of phenotyping by Fv /Fm performed under increasing severity of heat was successful in identifying wheat cultivars differing in photosynthesis under moderate and agronomically more relevant heat stress. The identified cultivars may serve as a valuable resource for further studies to understand the physiological mechanisms underlying the genetic variability in heat sensitivity of photosynthesis. © 2014 Scandinavian Plant Physiology Society.

The NASA Human Space Flight Supply Chain, Current and Future

NASA Technical Reports Server (NTRS)

Zapata, Edgar

2007-01-01

The current NASA Human Space Flight transportation system, the Space Shuttle, is scheduled for final flight in 2010. The Exploration initiative will create a new capability with a combination of existing systems and new flight and ground elements. To fully understand and act on the implications of such change it is necessary to understand what, how, when and where such changes occur and more importantly, how all these interact. This paper presents Human Space Flight, with an emphasis on KSC Launch and Landing, as a Supply Chain of both information and materials. A supply chain methodology for understanding the flow of information and materials is presented. Further, modeling and simulation projects funded by the Exploration initiative to understand the NASA Exploration Supply Chain are explained. Key concepts and their purpose, including the Enterprise, Locations, Physical and Organizational Functional Units, Products, and Resources, are explained. It is shown that the art, science and perspective of Supply Chain Management is not only applicable to such a government & contractor operation, it is also an invaluable approach for understanding, focusing improvement and growth. It is shown that such commercial practice applies to Human Space Flight and is invaluable towards one day creating routine, affordable access to and from space.

Enantiopure heterobimetallic single-chain magnets from the chiral Ru(III) building block.

PubMed

Ru, Jing; Gao, Feng; Wu, Tao; Yao, Min-Xia; Li, Yi-Zhi; Zuo, Jing-Lin

2014-01-21

A pair of one-dimensional enantiomers based on the versatile chiral dicyanoruthenate(III) building block have been synthesized and they are chiral single-chain magnets with the effective spin-reversal barrier of 28.2 K.

Transfection of Fv-1 permissive and restrictive mouse cells with integrated DNA of murine leukemia viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsu, I.C.; Yang, W.K.; Tennant, R.W.

1978-03-01

Whole-cell DNA preparations isolated from SC-1 cells chronically infected with N- or B-tropic murine leukemia viruses (MuLV) were tested for infectious activity in an Fv-I/sup n/ (NIH-3T3) and two Fv-I/sup b/ (C57BL/6 and SV-A31) cell cultures. Efficiency of transfection for all DNAs was better in the NIH-3T3 cells than in C57BL/6 or SV-A31 cells; and an (N-tropic MuLV)SC-1 cell DNA preparation was slightly more infectious than a (B-tropic MuLV)SC-1 cell DNA preparation in all three cell cultures, regardless of their Fv-1 genotypes. Progeny viruses from the transfection showed N- or B-tropism corresponding to that of the parent viruses produced bymore » the infected SC-1 cells that were used for the DNA preparation. DNA dose-response studies in NIH-3T3 cells revealed a one-hit mechanism for both the (B-tropic MuLV)SC-1 cell DNA and the (N-tropic MuLV)SC-1 cell DNA preparation. These results demonstrate that, in contrast to virion infection, transfection of N- and B-tropic MuLV with DNA preparations from chronically infected cells is not affected by the Fv-1 gene.« less

A novel lentiviral scFv display library for rapid optimization and selection of high affinity antibodies.

PubMed

Qudsia, Sehar; Merugu, Siva B; Mangukiya, Hitesh B; Hema, Negi; Wu, Zhenghua; Li, Dawei

2018-04-30

Antibody display libraries have become a popular technique to screen monoclonal antibodies for therapeutic purposes. An important aspect of display technology is to generate an optimization library by changing antibody affinity to antigen through mutagenesis and screening the high affinity antibody. In this study, we report a novel lentivirus display based optimization library antibody in which Agtuzumab scFv is displayed on cell membrane of HEK-293T cells. To generate an optimization library, hotspot mutagenesis was performed to achieve diverse antibody library. Based on sequence analysis of randomly selected clones, library size was estimated approximately to be 1.6 × 10 6 . Lentivirus display vector was used to display scFv antibody on cell surface and flow cytometery was performed to check the antibody affinity to antigen. Membrane bound scFv antibodies were then converted to secreted antibody through cre/loxP recombination. One of the mutant clones, M8 showed higher affinity to antigen in flow cytometery analysis. Further characterization of cellular and secreted scFv through western blot showed that antibody affinity was increased by three fold after mutagenesis. This study shows successful construction of a novel antibody library and suggests that hotspot mutagenesis could prove a useful and rapid optimization tool to generate similar libraries with various degree of antigen affinity. Copyright © 2018 Elsevier Inc. All rights reserved.

Interaction of S17 Antibody with the Functional Binding Region of the Hepatitis B Virus Pre-S2 Epitope.

PubMed

Chang, Chang-Yu; Chang, Fu-Ling; Chiang, Chen-Wei; Lo, Yan-Ni; Lin, Tsai-Yu; Chen, Wang-Chuan; Tsai, Keng-Chang; Lee, Yu-Ching

2018-05-30

To understand the mechanism for inhibition of hepatitis B virus (HBV) infection is important. In this study, single-chain variable fragment (scFv) antibodies were generated and directed to the pre-S2 epitope of HBV surface antigen (HBsAg). These human scFvs were isolated from a person with history of HBV infection by phage display technology. An evaluation of panning efficiency revealed that the eluted phage titer was increased, indicating that specific clones were enriched after panning. Selected scFvs were characterized with the recombinant HBsAg through Western blotting and enzyme-linked immunosorbent assay to confirm the binding ability. Flow cytometry analysis and immunocytochemical staining revealed that one scFv, S17, could recognize endogenous HBsAg expressed on the HepG2215 cell membrane. Moreover, the binding affinity of scFv S17 to the pre-S2 epitope was determined to be 4.2 × 10 -8 M. Two ion interactions were observed as the major driving forces for scFv S17 interacting with pre-S2 by performing a rational molecular docking analysis. This study provides insights into the structural basis to understand the interactions between an antibody and the pre-S2 epitope. The functional scFv format can potentially be used in future immunotherapeutic applications.

FV-100 versus valacyclovir for the prevention of post-herpetic neuralgia and the treatment of acute herpes zoster-associated pain: A randomized-controlled trial.

PubMed

Tyring, Stephen K; Lee, Patricia; Hill, Gordon T; Silverfield, Joel C; Moore, Angela Yen; Matkovits, Theresa; Sullivan-Bolyai, John

2017-07-01

This prospective, parallel-group, randomized, double-blind, multicenter study compared the efficacy and safety of FV-100 with valacyclovir for reducing pain associated with acute herpes zoster (HZ). Patients, ≥50 years of age, diagnosed with HZ within 72 h of lesion appearance who had HZ-associated pain, were randomized 1:1:1 to a 7-day course of either FV-100 200 mg QD (n = 117), FV-100 400 mg QD (n = 116), or valacyclovir 1000 mg TID (n =117). Efficacy was evaluated on the basis of the burden of illness (BOI; Zoster Brief Pain Inventory scores); incidence and duration of clinically significant pain (CSP); pain scores; incidence and severity of post-herpetic neuralgia (PHN); and times to full lesion crusting and to lesion healing. Safety was evaluated on the basis of adverse event (AE)/SAE profiles, changes in laboratory and vital signs values, and results of electrocardiograms. The burden of illness scores for pain through 30 days were 114.5, 110.3, and 118.0 for FV-100 200 mg, FV-100 400 mg, and valacyclovir 3000 mg, respectively. The incidences of PHN at 90 days for FV-100 200 mg, FV-100 400 mg, and valacyclovir 3000 mg were 17.8%, 12.4%, and 20.2%, respectively. Adverse event and SAE profiles of the two FV-100 and the valacyclovir groups were similar and no untoward signals or trends were evident. These results demonstrate a potential for FV-100 as an antiviral for the treatment of shingles that could both reduce the pain burden of the acute episode and reduce the incidence of PHN compared with available treatments. © 2016 Wiley Periodicals, Inc.

Primary alcohols activate human TRPA1 channel in a carbon chain length-dependent manner.

PubMed

Komatsu, Tomoko; Uchida, Kunitoshi; Fujita, Fumitaka; Zhou, Yiming; Tominaga, Makoto

2012-04-01

Transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable non-selective cation channel that is mainly expressed in primary nociceptive neurons. TRPA1 is activated by a variety of noxious stimuli, including cold temperatures, pungent compounds such as mustard oil and cinnamaldehyde, and intracellular alkalization. Here, we show that primary alcohols, which have been reported to cause skin, eye or nasal irritation, activate human TRPA1 (hTRPA1). We measured intracellular Ca(2+) changes in HEK293 cells expressing hTRPA1 induced by 1 mM primary alcohols. Higher alcohols (1-butanol to 1-octanol) showed Ca(2+) increases proportional to the carbon chain length. In whole-cell patch-clamp recordings, higher alcohols (1-hexanol to 1-octanol) activated hTRPA1 and the potency increased with the carbon chain length. Higher alcohols evoked single-channel opening of hTRPA1 in an inside-out configuration. In addition, cysteine at 665 in the N terminus and histidine at 983 in the C terminus were important for hTRPA1 activation by primary alcohols. Furthermore, straight-chain secondary alcohols increased intracellular Ca(2+) concentrations in HEK293 cells expressing hTRPA1, and both primary and secondary alcohols showed hTRPA1 activation activities that correlated highly with their octanol/water partition coefficients. On the other hand, mouse TRPA1 did not show a strong response to 1-hexanol or 1-octanol, nor did these alcohols evoke significant pain in mice. We conclude that primary and secondary alcohols activate hTRPA1 in a carbon chain length-dependent manner. TRPA1 could be a sensor of alcohols inducing skin, eye and nasal irritation in human.

The Complete Nucleotide Sequence of the Human Immunoglobulin Heavy Chain Variable Region Locus

PubMed Central

Matsuda, Fumihiko; Ishii, Kazuo; Bourvagnet, Patrice; Kuma, Kei-ichi; Hayashida, Hidenori; Miyata, Takashi; Honjo, Tasuku

1998-01-01

The complete nucleotide sequence of the 957-kb DNA of the human immunoglobulin heavy chain variable (VH) region locus was determined and 43 novel VH segments were identified. The region contains 123 VH segments classifiable into seven different families, of which 79 are pseudogenes. Of the 44 VH segments with an open reading frame, 39 are expressed as heavy chain proteins and 1 as mRNA, while the remaining 4 are not found in immunoglobulin cDNAs. Combinatorial diversity of VH region was calculated to be ∼6,000. Conservation of the promoter and recombination signal sequences was observed to be higher in functional VH segments than in pseudogenes. Phylogenetic analysis of 114 VH segments clearly showed clustering of the VH segments of each family. However, an independent branch in the tree contained a single VH, V4-44.1P, sharing similar levels of homology to human VH families and to those of other vertebrates. Comparison between different copies of homologous units that appear repeatedly across the locus clearly demonstrates that dynamic DNA reorganization of the locus took place at least eight times between 133 and 10 million years ago. One nonimmunoglobulin gene of unknown function was identified in the intergenic region. PMID:9841928

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

PubMed Central

Tilley, Derek; Levit, Irina; Samis, John A.

2012-01-01

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase 1, 2. Manual FV assays have been described 3, 4, but they are time consuming and subjective. Automated FV assays have been reported 5-7, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput 8, 9. Microplate assays have been reported for clot lysis 10, platelet aggregation 11, and coagulation Factors 12, but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma. This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1) 13. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity). Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections 14. DIC is associated with a poor prognosis and increases mortality above the pre

Measurement of factor v activity in human plasma using a microplate coagulation assay.

PubMed

Tilley, Derek; Levit, Irina; Samis, John A

2012-09-09

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase (1, 2). Manual FV assays have been described (3, 4), but they are time consuming and subjective. Automated FV assays have been reported (5-7), but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput (8, 9). Microplate assays have been reported for clot lysis (10), platelet aggregation (11), and coagulation Factors (12), but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma. This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405 nm during fibrin formation in human plasma (Figure 1) (13). The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80 pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity). Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections (14). DIC is associated with a poor prognosis and increases mortality

Tailoring Thermal Conductivity of Single-stranded Carbon-chain Polymers through Atomic Mass Modification

PubMed Central

Liao, Quanwen; Zeng, Lingping; Liu, Zhichun; Liu, Wei

2016-01-01

Tailoring the thermal conductivity of polymers is central to enlarge their applications in the thermal management of flexible integrated circuits. Progress has been made over the past decade by fabricating materials with various nanostructures, but a clear relationship between various functional groups and thermal properties of polymers remains to be established. Here, we numerically study the thermal conductivity of single-stranded carbon-chain polymers with multiple substituents of hydrogen atoms through atomic mass modification. We find that their thermal conductivity can be tuned by atomic mass modifications as revealed through molecular dynamics simulations. The simulation results suggest that heavy homogeneous substituents do not assist heat transport and trace amounts of heavy substituents can in fact hinder heat transport substantially. Our analysis indicates that carbon chain has the biggest contribution (over 80%) to the thermal conduction in single-stranded carbon-chain polymers. We further demonstrate that atomic mass modifications influence the phonon bands of bonding carbon atoms, and the discrepancies of phonon bands between carbon atoms are responsible for the remarkable drops in thermal conductivity and large thermal resistances in carbon chains. Our study provides fundamental insight into how to tailor the thermal conductivity of polymers through variable substituents. PMID:27713563

From single magnetic adatoms on superconductors to coupled spin chains

NASA Astrophysics Data System (ADS)

Franke, Katharina J.

Magnetic adsorbates on conventional s-wave superconductors lead to exchange interactions that induce Yu-Shiba-Rusinov (YSR) states inside the superconducting energy gap. Here, we employ tunneling spectroscopy at 1.1 K to investigate magnetic atoms and chains on superconducting Pb surfaces. We show that individual Manganese (Mn) atoms give rise to a distinct number of YSR-states. The single-atom junctions are stable over several orders of magnitude in conductance. We identify single-electron tunneling as well as Andreev processes. When the atoms are brought into sufficiently close distance, the Shiba states hybridize, thus giving rise to states with bonding and anti-bonding character. It has been shown that the Pb(110) surface supports the self-assembly of Fe chains, which exhibit fingerprints of Majorana bound states. Using superconducting tips, we resolve a rich subgap structure including peaks at zero energy and low-energy resonances, which overlap with the putative Majorana states. We gratefully acknowledge funding by the Deutsche Forschungsgemeinschaft through collaborative research Grant Sfb 658, and through Grant FR2726/4, as well by the European Research Council through Consolidator Grant NanoSpin.

High-throughput microfluidic single-cell digital polymerase chain reaction.

PubMed

White, A K; Heyries, K A; Doolin, C; Vaninsberghe, M; Hansen, C L

2013-08-06

Here we present an integrated microfluidic device for the high-throughput digital polymerase chain reaction (dPCR) analysis of single cells. This device allows for the parallel processing of single cells and executes all steps of analysis, including cell capture, washing, lysis, reverse transcription, and dPCR analysis. The cDNA from each single cell is distributed into a dedicated dPCR array consisting of 1020 chambers, each having a volume of 25 pL, using surface-tension-based sample partitioning. The high density of this dPCR format (118,900 chambers/cm(2)) allows the analysis of 200 single cells per run, for a total of 204,000 PCR reactions using a device footprint of 10 cm(2). Experiments using RNA dilutions show this device achieves shot-noise-limited performance in quantifying single molecules, with a dynamic range of 10(4). We performed over 1200 single-cell measurements, demonstrating the use of this platform in the absolute quantification of both high- and low-abundance mRNA transcripts, as well as micro-RNAs that are not easily measured using alternative hybridization methods. We further apply the specificity and sensitivity of single-cell dPCR to performing measurements of RNA editing events in single cells. High-throughput dPCR provides a new tool in the arsenal of single-cell analysis methods, with a unique combination of speed, precision, sensitivity, and specificity. We anticipate this approach will enable new studies where high-performance single-cell measurements are essential, including the analysis of transcriptional noise, allelic imbalance, and RNA processing.

Human genetic mapping studies using single sperm typing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hubert, R.S.

1993-01-01

Sperm typing is a powerful technique that uses the polymerase chain reaction (PCR) to analyze DNA sequences within single sperm cells in order to construct genetic maps. This methodology was used to estimate the recombination fraction between D3S2 and D3S2 which was found to be 0.28 (95% CI = 0.20-0.36). Pedigree analysis was unable to determine genetic distance between these two markers due to their low informativeness. We also showed that dinucleotide and tetranucleotide repeat polymorphisms can be analyzed in single cells without using radioactivity or denaturing gels. This provides a rich new source of DANA polymorphisms for genetic mappingmore » by sperm typing. In addition, an approach that uses the sperm typing methodology is described that can define the physical boundaries of meiotic recombination hotspots. The hotspot at 4p16.3 near the Huntington disease gene was localized to an interval between D4S10 and D4S126. These studies demonstrated the usefulness of sperm typing as a tool for the study of human genetic.« less

FvSTR1, a striatin orthologue in Fusarium virguliforme, is required for asexual development and virulence.

PubMed

Islam, Kazi T; Bond, Jason P; Fakhoury, Ahmad M

2017-08-01

The soil-borne fungus Fusarium virguliforme causes sudden death syndrome (SDS), one of the most devastating diseases of soybean in North and South America. Despite the importance of SDS, a clear understanding of the fungal pathogenicity factors that affect the development of this disease is still lacking. We have identified FvSTR1, a F. virguliforme gene, which encodes a protein similar to a family of striatin proteins previously reported to regulate signalling pathways, cell differentiation, conidiation, sexual development, and virulence in filamentous fungi. Striatins are multi-domain proteins that serve as scaffolding units in the striatin-interacting phosphatase and kinase (STRIPAK) complex in fungi and animals. To address the function of a striatin homologue in F. virguliforme, FvSTR1 was disrupted and functionally characterized using a gene knock out strategy. The resulting Fvstr1 mutants were largely impaired in conidiation and pigmentation, and displayed defective conidia and conidiophore morphology compared to the wild-type and ectopic transformants. Greenhouse virulence assays revealed that the disruption of FvSTR1 resulted in complete loss of virulence in F. virguliforme. Microtome studies using fluorescence microscopy showed that the Fvstr1 mutants were defective in their ability to colonize the vascular system. The Fvstr1 mutants also showed a reduced transcript level of genes involved in asexual reproduction and in the production of secondary metabolites. These results suggest that FvSTR1 has a critical role in asexual development and virulence in F. virguliforme.

Optical Properties of a Single Carbon Chain-Doped Silicene Nanoribbon

NASA Astrophysics Data System (ADS)

Lu, Dao-Bang; Song, Yu-Ling; Huang, Xiao-yu; Wang, Chong

2018-05-01

Using first-principles spin polarization density function theory calculations, we have studied the electronic and optical properties of zigzag-edge silicene nanoribbons (ZSiNRs) doped with a single carbon chain. Because of the doped carbon chain, there are several defect states in the band structures of ZSiNRs across the Fermi level, and the ferromagnetic ground state is metallic. The dielectric functions in all three dimensions are completely different from each other, and thus the system exhibits strong optical anisotropism. The carbon chain influenced the dielectric functions most at low energy. The first peak in the E//x direction of the dielectric spectrum exhibits a significant blueshift, and its value has changed as well. The main absorption wavelength depends on the polarization direction of the incident light, but occurs within the UV region for all polarization directions. The peaks of the energy loss spectra correspond to the trailing edges in the reflectivity spectrum, and the highest peak corresponds to a plasmon frequency. Our results could be useful for investigating nanodevices based on silicene nanoribbons.

An optimized single chain TCR scaffold relying on the assembly with the native CD3-complex prevents residual mispairing with endogenous TCRs in human T-cells

PubMed Central

Knies, Diana; Klobuch, Sebastian; Xue, Shao-An; Birtel, Matthias; Echchannaoui, Hakim; Yildiz, Oezlem; Omokoko, Tana; Guillaume, Philippe; Romero, Pedro; Stauss, Hans; Sahin, Ugur; Herr, Wolfgang; Theobald, Matthias; Thomas, Simone; Voss, Ralf-Holger

2016-01-01

Immunotherapy of cancer envisions the adoptive transfer of T-cells genetically engineered with tumor-specific heterodimeric α/β T-cell receptors (TCRα/β). However, potential mispairing of introduced TCRα/β-chains with endogenous β/α-ones may evoke unpredictable autoimmune reactivities. A novel single chain (sc)TCR format relies on the fusion of the Vα-Linker-Vβ-fragment to the TCR Cβ-domain and coexpression of the TCR Cα-domain capable of recruiting the natural CD3-complex for full and hence, native T-cell signaling. Here, we tested whether such a gp100(280-288)- or p53(264-272) tumor antigen-specific scTCR is still prone to mispairing with TCRα. In a human Jurkat-76 T-cell line lacking endogenous TCRs, surface expression and function of a scTCR could be reconstituted by any cointroduced TCRα-chain indicating mispairing to take place on a molecular basis. In contrast, transduction into human TCRα/β-positive T-cells revealed that mispairing is largely reduced. Competition experiments in Jurkat-76 confirmed the preference of dcTCR to selfpair and to spare scTCR. This also allowed for the generation of dc/scTCR-modified cytomegalovirus/tumor antigen-bispecific T-cells to augment T-cell activation in CMV-infected tumor patients. Residual mispairing was prevented by strenghtening the Vα-Li-Vβ-fragment through the design of a novel disulfide bond between a Vα- and a linker-resident residue close to Vβ. Multimer-stainings, and cytotoxicity-, IFNγ-secretion-, and CFSE-proliferation-assays, the latter towards dendritic cells endogenously processing RNA-electroporated gp100 antigen proved the absence of hybrid scTCR/TCRα-formation without impairing avidity of scTCR/Cα in T-cells. Moreover, a fragile cytomegalovirus pp65(495-503)-specific scTCR modified this way acquired enhanced cytotoxicity. Thus, optimized scTCR/Cα inhibits residual TCR mispairing to accomplish safe adoptive immunotherapy for bulk endogenous TCRα/β-positive T-cells. PMID:27028870

A unique substituted Co(II)-formate coordination framework exhibits weak ferromagnetic single-chain-magnet like behavior.

PubMed

Zhao, Jiong-Peng; Yang, Qian; Liu, Zhong-Yi; Zhao, Ran; Hu, Bo-Wen; Du, Miao; Chang, Ze; Bu, Xian-He

2012-07-04

A magnetic isolated chain-based substituted cobalt-formate framework was obtained with isonicotine as a spacer. In the chain, canted antiferromagnetic interactions exist in between the Co(II) ions, and slow magnetic relaxation is detected at low temperature. For the block effects of the isonicotine ligands, the complex could be considered as a peculiar example of a weak ferromagnetic single-chain-magnet.

Analysis of p53 gene mutations in human gliomas by polymerase chain reaction-based single-strand conformation polymorphism and DNA sequencing.

PubMed

Sarkar, F H; Kupsky, W J; Li, Y W; Sreepathi, P

1994-03-01

Mutations in the p53 gene have been recognized in brain tumors, and clonal expansion of p53 mutant cells has been shown to be associated with glioma progression. However, studies on the p53 gene have been limited by the need for frozen tissues. We have developed a method utilizing polymerase chain reaction (PCR) for the direct analysis of p53 mutation by single-strand conformation polymorphism (SSCP) and by direct DNA sequencing of the p53 gene using a single 10-microns paraffin-embedded tissue section. We applied this method to screen for p53 gene mutations in exons 5-8 in human gliomas utilizing paraffin-embedded tissues. Twenty paraffin blocks containing tumor were selected from surgical specimens from 17 different adult patients. Tumors included six anaplastic astrocytomas (AAs), nine glioblastomas (GBs), and two mixed malignant gliomas (MMGs). The tissue section on the stained glass slide was used to guide microdissection of an unstained adjacent tissue section to ensure > 90% of the tumor cell population for p53 mutational analysis. Simultaneously, microdissection of the tissue was also carried out to obtain normal tissue from adjacent areas as a control. Mutations in the p53 gene were identified in 3 of 17 (18%) patients by PCR-SSCP analysis and subsequently confirmed by PCR-based DNA sequencing. Mutations in exon 5 resulting in amino acid substitution were found in one thalamic AA (codon 158, CGC > CTT: Arg > Leu) and one cerebral hemispheric GB (codon 151, CCG > CTG: Pro > Leu).(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of the efficiency of antibody selection from semi-synthetic scFv and non-immune Fab phage display libraries against protein targets for rapid development of diagnostic immunoassays.

PubMed

Chan, Conrad E Z; Chan, Annie H Y; Lim, Angeline P C; Hanson, Brendon J

2011-10-28

Rapid development of diagnostic immunoassays against novel emerging or genetically modified pathogens in an emergency situation is dependent on the timely isolation of specific antibodies. Non-immune antibody phage display libraries are an efficient in vitro method for selecting monoclonal antibodies and hence ideal in these circumstances. Such libraries can be constructed from a variety of sources e.g. B cell cDNA or synthetically generated, and use a variety of antibody formats, typically scFv or Fab. However, antibody source and format can impact on the quality of antibodies generated and hence the effectiveness of this methodology for the timely production of antibodies. We have carried out a comparative screening of two antibody libraries, a semi-synthetic scFv library and a human-derived Fab library against the protective antigen toxin component of Bacillus anthracis and the epsilon toxin of Clostridium botulinum. We have shown that while the synthetic library produced a diverse collection of specific scFv-phage, these contained a high frequency of unnatural amber stops and glycosylation sites which limited their conversion to IgG, and also a high number which lost specificity when expressed as IgG. In contrast, these limitations were overcome by the use of a natural human library. Antibodies from both libraries could be used to develop sandwich ELISA assays with similar sensitivity. However, the ease and speed with which full-length IgG could be generated from the human-derived Fab library makes screening this type of library the preferable method for rapid antibody generation for diagnostic assay development. Copyright © 2011 Elsevier B.V. All rights reserved.

Fab is the most efficient format to express functional antibodies by yeast surface display.

PubMed

Sivelle, Coline; Sierocki, Raphaël; Ferreira-Pinto, Kelly; Simon, Stéphanie; Maillere, Bernard; Nozach, Hervé

2018-04-30

Multiple formats are available for engineering of monoclonal antibodies (mAbs) by yeast surface display, but they do not all lead to efficient expression of functional molecules. We therefore expressed four anti-tumor necrosis factor and two anti-IpaD mAbs as single-chain variable fragment (scFv), antigen-binding fragment (Fab) or single-chain Fabs and compared their expression levels and antigen-binding efficiency. Although the scFv and scFab formats are widely used in the literature, 2 of 6 antibodies were either not or weakly expressed. In contrast, all 6 antibodies expressed as Fab revealed strong binding and high affinity, comparable to that of the soluble form. We also demonstrated that the variations in expression did not affect Fab functionality and were due to variations in light chain display and not to misfolded dimers. Our results suggest that Fab is the most versatile format for the engineering of mAbs.

Dispersion relations for circular single and double dusty plasma chains

NASA Astrophysics Data System (ADS)

Tkachenko, D. V.; Sheridan, T. E.; Misko, V. R.

2011-10-01

We derive dispersion relations for a system of identical particles confined in a two-dimensional annular harmonic well and which interact through a Yukawa potential, e.g., a dusty plasma ring. When the particles are in a single chain (i.e., a one-dimensional ring), we find a longitudinal acoustic mode and a transverse optical mode which show approximate agreement with the dispersion relation for a straight configuration for large radii of the ring. When the radius decreases, the dispersion relations modify: there appears an anticrossing of the modes near the crossing point resulting in a frequency gap between the lower and upper branches of the modified dispersion relations. For the double chain (i.e., a two-dimensional zigzag configuration), the dispersion relation has four branches: longitudinal acoustic and optical and transverse acoustic and optical.

Structural basis for autoantibody recognition of phosphatidylserine-β2 glycoprotein I and apoptotic cells

PubMed Central

Cocca, Brian A.; Seal, Samarendra N.; D'Agnillo, Paolo; Mueller, Yvonne M.; Katsikis, Peter D.; Rauch, Joyce; Weigert, Martin; Radic, Marko Z.

2001-01-01

Apoptotic cells contain nuclear autoantigens that may initiate a systemic autoimmune response. To explore the mechanism of antibody binding to apoptotic cells, 3H9, a murine autoantibody with dual specificity for phospholipids and DNA, was used. H chain mutants of 3H9 were constructed, expressed as single-chain Fv (scFv) in Escherichia coli, and assessed for binding to phosphatidylserine, an antigen expressed on apoptotic cells. Both 3H9 and its germline revertant bound to dioleoyl phosphatidylserine in ELISA, and binding was enhanced by β2 glycoprotein I (β2GPI), a plasma protein that selectively binds to apoptotic cells. Higher relative affinity for DOPS-β2GPI was achieved by the introduction of Arg residues into the 3H9 H chain variable region at positions previously shown to mediate DNA binding. Specificity of the two structurally most diverse scFv for apoptotic cells was shown by flow cytometry, and two populations of scFv-bound cells were identified by differences in propidium iodide staining. The results suggest that, in autoimmunity, B cells with Ig receptors for apoptotic cells and DNA are positively selected, and that the antibodies they produce have the potential to affect the clearance and processing of apoptotic cells. PMID:11717440

Spin canting in a Dy-based single-chain magnet with dominant next-nearest-neighbor antiferromagnetic interactions

NASA Astrophysics Data System (ADS)

Bernot, K.; Luzon, J.; Caneschi, A.; Gatteschi, D.; Sessoli, R.; Bogani, L.; Vindigni, A.; Rettori, A.; Pini, M. G.

2009-04-01

We investigate theoretically and experimentally the static magnetic properties of single crystals of the molecular-based single-chain magnet of formula [Dy(hfac)3NIT(C6H4OPh)]∞ comprising alternating Dy3+ and organic radicals. The magnetic molar susceptibility χM displays a strong angular variation for sample rotations around two directions perpendicular to the chain axis. A peculiar inversion between maxima and minima in the angular dependence of χM occurs on increasing temperature. Using information regarding the monomeric building block as well as an ab initio estimation of the magnetic anisotropy of the Dy3+ ion, this “anisotropy-inversion” phenomenon can be assigned to weak one-dimensional ferromagnetism along the chain axis. This indicates that antiferromagnetic next-nearest-neighbor interactions between Dy3+ ions dominate, despite the large Dy-Dy separation, over the nearest-neighbor interactions between the radicals and the Dy3+ ions. Measurements of the field dependence of the magnetization, both along and perpendicularly to the chain, and of the angular dependence of χM in a strong magnetic field confirm such an interpretation. Transfer-matrix simulations of the experimental measurements are performed using a classical one-dimensional spin model with antiferromagnetic Heisenberg exchange interaction and noncollinear uniaxial single-ion anisotropies favoring a canted antiferromagnetic spin arrangement, with a net magnetic moment along the chain axis. The fine agreement obtained with experimental data provides estimates of the Hamiltonian parameters, essential for further study of the dynamics of rare-earth-based molecular chains.

Efficient recovery of the functional IP10-scFv fusion protein from inclusion bodies with an on-column refolding system.

PubMed

Guo, Jun-Qing; Li, Qing-Mei; Zhou, Ji-Yong; Zhang, Gai-Ping; Yang, Yan-Yan; Xing, Guang-Xu; Zhao, Dong; You, Shang-You; Zhang, Chu-Yu

2006-01-01

A functional IP10-scFv fusion protein retaining the antibody specificity for acidic isoferritin and chemokine function was produced at high level in Esherichia coli (E. coli). IP10-scFv gene from the recombinant plasmid pc3IP104c9 was subcloned into pET28a fused to N-terminal His-tag sequence in frame and overexpressed in E. coli BL21(DE3). With an on-column refolding procedure based on Ni-chelating chromatography, the active fusion protein was recovered efficiently from inclusion bodies with a refolding yield of approximate 45% confirmed by spectrophotometer. The activity of refolded IP10-scFv was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting and enzyme-linked immunosorbent assay. The results showed the fusion protein retains the specific binding activity to AIF with an affinity constant of 4.48x10(-8) M as well as the chemokine function of IP-10. The overall yield of IP10-scFv with bioactivity in E. coli flask culture was more than 40 mg/L.

Dynamics of Single Chains of Suspended Ferrofluid Particles

NASA Technical Reports Server (NTRS)

Cutillas, S.; Liu, J.

1999-01-01

We present an experimental study of the dynamics of isolated chains made of super-paramagnetic particles under the influence of a magnetic field. The motivation of this work is to understand if the chain fluctuations exist and, if it does, how does the fluctuation affect chain aggregation. We find that single chains strongly fluctuate and that the characteristic frequency of their fluctuations is inversely proportional to the magnetic field strength. The higher the field the lower the characteristic frequency of the chain fluctuations. In the high magnetic field limit, chains behave like rigid rods without any internal motions. In this work, we used ferrofluid particles suspended in water. These particles do not have any intrinsic magnetization. Once a magnetic field is applied, a dipole moment is induced in each particle, proportional to the magnetic field. A dipolar magnetic interaction then occurs between particles. If dipole-dipole magnetic energy is higher than the thermal energy, the result is a structure change inside the dipolar fluid. The ratio of these two energies is expressed by a coupling constant lambda as: lambda = (pi(a(exp 3))(chi(exp 2))(mu(sub 0))(H(sub 0))(exp 2))/18kT Where a is the particle radius, mu(sub 0) is the vacuum magnetic permeability, H(sub 0) the applied magnetic field, k the Boltzmann constant and T the absolute temperature. If lambda > 1, magnetic particles form chains along the field direction. The lateral coalescence of several chains may form bigger aggregates especially if the particle volume fraction is high. While many studies and applications deal with the rheological properties and the structural changes of these dipolar fluids, this work focuses on the understanding of the chain dynamics. In order to probe the chain dynamics, we used dynamic light scattering (DLS) in self-beating mode as our experimental technique. The experimental geometry is such that the scattering plane is perpendicular to the magnetic field

Alternative types of molecule-decorated atomic chains in Au–CO–Au single-molecule junctions

PubMed Central

Balogh, Zoltán; Makk, Péter

2015-01-01

Summary We investigate the formation and evolution of Au–CO single-molecule break junctions. The conductance histogram exhibits two distinct molecular configurations, which are further investigated by a combined statistical analysis. According to conditional histogram and correlation analysis these molecular configurations show strong anticorrelations with each other and with pure Au monoatomic junctions and atomic chains. We identify molecular precursor configurations with somewhat higher conductance, which are formed prior to single-molecule junctions. According to detailed length analysis two distinct types of molecule-affected chain-formation processes are observed, and we compare these results to former theoretical calculations considering bridge- and atop-type molecular configurations where the latter has reduced conductance due to destructive Fano interference. PMID:26199840

Alternative types of molecule-decorated atomic chains in Au-CO-Au single-molecule junctions.

PubMed

Balogh, Zoltán; Makk, Péter; Halbritter, András

2015-01-01

We investigate the formation and evolution of Au-CO single-molecule break junctions. The conductance histogram exhibits two distinct molecular configurations, which are further investigated by a combined statistical analysis. According to conditional histogram and correlation analysis these molecular configurations show strong anticorrelations with each other and with pure Au monoatomic junctions and atomic chains. We identify molecular precursor configurations with somewhat higher conductance, which are formed prior to single-molecule junctions. According to detailed length analysis two distinct types of molecule-affected chain-formation processes are observed, and we compare these results to former theoretical calculations considering bridge- and atop-type molecular configurations where the latter has reduced conductance due to destructive Fano interference.

Force Induced Globule-to-Coil Transition of Single Polymer Chains.

NASA Astrophysics Data System (ADS)

Gunari, Nikhil; Walker, Gilbert

2008-03-01

Force induced structural transitions of individual homopolymer chains have been studied in different solvent conditions using single molecule force spectroscopy. Single molecule mechanics in the ``fly-fishing'' mode showed a first-order like transition for polystyrene (PS) in water exhibiting a characteristic three regime force extension curve. In contrast, poly methylmethacrylate (PMMA) showed a characteristic saw-tooth pattern reminiscent of multidomain disassembly behavior similar to that seen in modular protein mechanics. The plateau force for PS and the saw-tooth pattern for PMMA disappear when measured in aqueous guanidine hydrochloride solution and in other non-solvents showing that the characteristic deformational behavior observed for the two polymers in water may be due to hydrophobic interactions .

Finite-size effects on the static properties of a single-chain magnet

NASA Astrophysics Data System (ADS)

Bogani, L.; Sessoli, R.; Pini, M. G.; Rettori, A.; Novak, M. A.; Rosa, P.; Massi, M.; Fedi, M. E.; Giuntini, L.; Caneschi, A.; Gatteschi, D.

2005-08-01

We study the role of defects in the “single-chain magnet” CoPhOMe by inserting a controlled number of diamagnetic impurities. The samples are analyzed with unprecedented accuracy with the particle induced x-ray emission technique, and with ac and dc magnetic measurements. In an external applied field the system shows an unexpected behavior, giving rise to a double peak in the susceptibility. The static thermodynamic properties of the randomly diluted Ising chain with alternating g values are then exactly obtained via a transfer matrix approach. These results are compared to the experimental behavior of CoPhOMe, showing qualitative agreement.

Pharmacological efficacy of anti-IL-1β scFv, Fab and full-length antibodies in treatment of rheumatoid arthritis.

PubMed

Qi, Jianying; Ye, Xianlong; Ren, Guiping; Kan, Fangming; Zhang, Yu; Guo, Mo; Zhang, Zhiyi; Li, Deshan

2014-02-01

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that mainly causes the synovial joint inflammation and cartilage destruction. Interleukin-1β (IL-1β) is an important proinflammatory cytokine involved in the pathogenesis of RA. In this study, we constructed and expressed anti-IL-1β-full-length antibody in CHO-K1-SV, anti-IL-1β-Fab and anti-IL-1β-scFv in Rosetta. We compared the therapeutic efficacy of three anti-IL-1β antibodies for CIA mice. Mice with CIA were subcutaneously injected with humanized anti-IL-1β-scFv, anti-IL-1β-Fab or anti-IL-1β-full-length antibody. The effects of treatment were determined by arthritis severity score, autoreactive humoral, cellular immune responses, histological lesion and cytokines production. Compared with anti-IL-1β-scFv treatments, anti-IL-1β-Fab and anti-IL-1β-full-length antibody therapy resulted in more significant effect in alleviating the severity of arthritis by preventing bone damage and cartilage destruction, reducing humoral and cellular immune responses, and down-regulating the expression of IL-1β, IL-6, IL-2, IFN-γ, TNF-α and MMP-3 in inflammatory tissue. The therapeutic effects of anti-IL-1β-Fab and anti-IL-1β-full-length antibodies on CIA mice had no significant difference. However, production of anti-IL-1β-full-length antibody in eukaryotic system is, in general, time-consuming and more expensive than that of anti-IL-1β-Fab in prokaryotic systems. In conclusion, as a small molecule antibody, anti-IL-1β-Fab is an ideal candidate for RA therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

BRITE nanosatellite serendipitously captures oscillatory rise and fall of ASASSN-18fv

NASA Astrophysics Data System (ADS)

Kuschnig, R.; Pigulski, A.; Moffat, A. F. J.; Matthews, J. M.; Zwintz, K.; Baade, D.; Handler, G.; Weiss, W. W.; Wade, G. A.; Rucinski, S. M.; Pablo, H.; Koudelka, O.; Smolec, R.; Popowicz, A.; Neiner, C.; Daszynska-Daszkiewicz, J.; Lovekin, C.; St-Louis, N.; Pamyatnykh, A. A.; Rowe, J.; Orleanski, P.; Mochnacki, S.; Schwarzenberg-Czerny, A.

2018-04-01

One of the five satellites in BRITE-Constellation (http://www.brite-constellation.at/) was obtaining time-series optical photometry of the star HD 92063, only 2 arcmin from the 'Possible, Very Bright Galactic Nova ASASSN-18fv' reported on 21 March 2018 (2018-03-20.32) by ATel #11454.

From single Debye-Hückel chains to polyelectrolyte solutions: Simulation results

NASA Astrophysics Data System (ADS)

Kremer, Kurt

1996-03-01

This lecture will present results from simulations of single weakly charged flexible chains, where the electrostatic part of the interaction is modeled by a Debye-Hückel potential,( with U. Micka, IFF, Forschungszentrum Jülich, 52425 Jülich, Germany) as well as simulations of polyelectrolyte solutions, where the counterions are explicitly taken into account( with M. J. Stevens, Sandia Nat. Lab., Albuquerque, NM 87185-1111) ( M. J. Stevens, K. Kremer, JCP 103), 1669 (1995). The first set of the simulations is meant to clear a recent contoversy on the dependency of the persistence length LP on the screening length Γ. While the analytic theories give Lp ~ Γ^x with either x=1 or x=2, the simulations find for all experimentally accessible chain lengths a varying exponent, which is significantly smaller than 1. This causes serious doubts on the applicability of this model for weakly charged polyelectrolytes in general. The second part deals with strongly charged flexible polyelectrolytes in salt free solution. These simulations are performed for multichain systems. The full Coulomb interactions of the monomers and counterions are treated explicitly. Experimental measurements of the osmotic pressure and the structure factor are reproduced and extended. The simulations reveal a new picture of the chain structure based on calculations of the structure factor, persistence length, end-to-end distance, etc. Even at very low density, the chains show significant bending. Furthermore, the chains contract significantly before they start to overlap. We also show that counterion condensation dramatically alters the chain structure, even for a good solvent backbone.

Dispersion relations for circular single and double dusty plasma chains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tkachenko, D. V.; Misko, V. R.; Sheridan, T. E.

2011-10-15

We derive dispersion relations for a system of identical particles confined in a two-dimensional annular harmonic well and which interact through a Yukawa potential, e.g., a dusty plasma ring. When the particles are in a single chain (i.e., a one-dimensional ring), we find a longitudinal acoustic mode and a transverse optical mode which show approximate agreement with the dispersion relation for a straight configuration for large radii of the ring. When the radius decreases, the dispersion relations modify: there appears an anticrossing of the modes near the crossing point resulting in a frequency gap between the lower and upper branchesmore » of the modified dispersion relations. For the double chain (i.e., a two-dimensional zigzag configuration), the dispersion relation has four branches: longitudinal acoustic and optical and transverse acoustic and optical.« less

scFv-based “grababody” as a general strategy to improve recruitment of immune effector cells to antibody-targeted tumors

PubMed Central

Cai, Zheng; Fu, Ting; Nagai, Yasuhiro; Lam, Lian; Yee, Marla; Zhu, Zhiqiang; Zhang, Hongtao

2013-01-01

Recruitment of immune cells to tumor cells targeted by a therapeutic antibody can heighten the antitumor efficacy of the antibody. For example, p185her2/neu-targeting antibodies not only downregulate the p185her2/neu kinase (ERBB2) but also trigger complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) through the antibody Fc region. Here we describe a generalized strategy to improve immune cell recruitment to targeted cancer cells, using a modified scFv antibody we call a “grababody” that binds the target protein and endogenous immunoglobulins. The model system we used to illustrate the utility of this platform recognizes p185her2/neu and includes an IgG binding domain. The recombinant scFv grababody that was created recruited circulating human IgGs and attracted immune cells carrying Fc receptors to tumor cells that expressed p185her2/neu. The presence of the IgG binding domain significantly enhanced CDC and ADCC activity and improved anti-tumor activity in vivo. Our results illustrate a novel general approach to improve antibody-like proteins for therapeutic applications. PMID:23396586

Pichia pastoris secretes recombinant proteins less efficiently than Chinese hamster ovary cells but allows higher space-time yields for less complex proteins

PubMed Central

Maccani, Andreas; Landes, Nils; Stadlmayr, Gerhard; Maresch, Daniel; Leitner, Christian; Maurer, Michael; Gasser, Brigitte; Ernst, Wolfgang; Kunert, Renate; Mattanovich, Diethard

2014-01-01

Chinese hamster ovary (CHO) cells are currently the workhorse of the biopharmaceutical industry. However, yeasts such as Pichia pastoris are about to enter this field. To compare their capability for recombinant protein secretion, P. pastoris strains and CHO cell lines producing human serum albumin (HSA) and the 3D6 single chain Fv-Fc anti-HIV-1 antibody (3D6scFv-Fc) were cultivated in comparable fed batch processes. In P. pastoris, the mean biomass-specific secretion rate (qp) was 40-fold lower for 3D6scFv-Fc compared to HSA. On the contrary, qp was similar for both proteins in CHO cells. When comparing both organisms, the mean qp of the CHO cell lines was 1011-fold higher for 3D6scFv-Fc and 26-fold higher for HSA. Due to the low qp of the 3D6scFv-Fc producing strain, the space-time yield (STY) was 9.6-fold lower for P. pastoris. In contrast, the STY of the HSA producer was 9.2-fold higher compared to CHO cells because of the shorter process time and higher biomass density. The results indicate that the protein secretion machinery of P. pastoris is much less efficient and the secretion rate strongly depends on the complexity of the recombinant protein. However, process efficiency of the yeast system allows higher STYs for less complex proteins. PMID:24390926

Technology of compact MAb and its application for medicinal plant breeding named as missile type molecular breeding.

PubMed

Putalun, Waraporn

2011-03-01

Single chain fragment-variable (scFv) enhanced solasodine glycoside accumulation in Solanum khasianum hairy root cultures transformed by the ScFv solamargine (As)-scFv gene. The scFv protein was expressed at a high level in inclusion bodies of E. coli. After being renatured, the scFv protein was purified in a one-step manner by metal chelate affinity chromatography. The yield of refolded and purified scFv was 12.5 mg per 100 ml of cell culture. The characteristics of the As-scFv expressed in E. coli and transgenic hairy roots were similar to those of the parent monoclonal antibody (MAb). The expression of scFv protein provides a low cost and a high yield of functional scFv antibody against solamargine. The full linear range of the ELISA assay using scFv was extended from 1.5-10 µg/ml. The expressed anti-solamargine scFv protein could be useful for determination of total solasodine glycoside content in plant samples by ELISA. Solasodine glycoside levels in the transgenic hairy root were 2.3-fold higher than that in the wild-type hairy root based on the soluble protein level and binding activities. The As-scFv expressed in S. khasianum hairy roots enhanced solasodine glycosides accumulation and provide a novel medicinal plant breeding methodology that can produce a high yield of secondary metabolites.

Temporal variation in community composition, pigmentation, and Fv/Fm of desert cyanobacterial soil crusts

USGS Publications Warehouse

Bowker, M.A.; Reed, S.C.; Belnap, J.; Phillips, S.L.

2002-01-01

Summers on the Colorado Plateau (USA) are typified by harsh conditions such as high temperatures, brief soil hydration periods, and high UV and visible radiation. We investigated whether community composition, physiological status, and pigmentation might vary in biological soil crusts as a result of such conditions. Representative surface cores were sampled at the ENE, WSW, and top microaspects of 20 individual soil crust pedicels at a single site in Canyonlands National Park, Utah, in spring and fall of 1999. Frequency of cyanobacterial taxa, pigment concentrations, and dark adapted quantum yield (Fv/Fm) were measured for each core. The frequency of major cyanobacterial taxa was lower in the fall compared to spring. The less-pigmented cyanobacterium Microcoleus vaginatus showed significant mortality when not in the presence of Nostoc spp. and Scytonema myochrous (Dillw.) Agardh. (both synthesizers of UV radiation-linked pigments) but had little or no mortality when these species were abundant. We hypothesize that the sunscreen pigments produced by Nostoc and Scytonema in the surface of crusts protect other, less-pigmented taxa. When fall and spring samples were compared, overall cyanobacterial frequency was lower in fall, while sunscreen pigment concentrations, chlorophyll a concentration, and Fv/Fm were higher in fall. The ratio of cyanobacterial frequency/chlorophyll a concentrations was 2-3 times lower in fall than spring. Because chlorophyll a is commonly used as a surrogate measure of soil cyanobacterial biomass, these results indicate that seasonality needs to be taken into consideration. In the fall sample, most pigments associated with UV radiation protection or repair were at their highest concentrations on pedicel tops and WSW microaspects, and at their lowest concentrations on ENE microaspects. We suggest that differential pigment concentrations between microaspects are induced by varying UV radiation dosage at the soil surface on these different

Hybrid DG/FV schemes for magnetohydrodynamics and relativistic hydrodynamics

NASA Astrophysics Data System (ADS)

Núñez-de la Rosa, Jonatan; Munz, Claus-Dieter

2018-01-01

This paper presents a high order hybrid discontinuous Galerkin/finite volume scheme for solving the equations of the magnetohydrodynamics (MHD) and of the relativistic hydrodynamics (SRHD) on quadrilateral meshes. In this approach, for the spatial discretization, an arbitrary high order discontinuous Galerkin spectral element (DG) method is combined with a finite volume (FV) scheme in order to simulate complex flow problems involving strong shocks. Regarding the time discretization, a fourth order strong stability preserving Runge-Kutta method is used. In the proposed hybrid scheme, a shock indicator is computed at the beginning of each Runge-Kutta stage in order to flag those elements containing shock waves or discontinuities. Subsequently, the DG solution in these troubled elements and in the current time step is projected onto a subdomain composed of finite volume subcells. Right after, the DG operator is applied to those unflagged elements, which, in principle, are oscillation-free, meanwhile the troubled elements are evolved with a robust second/third order FV operator. With this approach we are able to numerically simulate very challenging problems in the context of MHD and SRHD in one, and two space dimensions and with very high order polynomials. We make convergence tests and show a comprehensive one- and two dimensional testbench for both equation systems, focusing in problems with strong shocks. The presented hybrid approach shows that numerical schemes of very high order of accuracy are able to simulate these complex flow problems in an efficient and robust manner.

Conductance of single microRNAs chains related to the autism spectrum disorder

NASA Astrophysics Data System (ADS)

Oliveira, J. I. N.; Albuquerque, E. L.; Fulco, U. L.; Mauriz, P. W.; Sarmento, R. G.; Caetano, E. W. S.; Freire, V. N.

2014-09-01

The charge transport properties of single-stranded microRNAs (miRNAs) chains associated to autism disorder were investigated. The computations were performed within a tight-binding model, together with a transfer matrix technique, with ionization energies and hopping parameters obtained by quantum chemistry method. Current-voltage (I× V) curves of twelve miRNA chains related to the autism spectrum disorders were calculated and analysed. We have obtained both semiconductor and insulator behavior, and a relationship between the current intensity and the autism-related miRNA bases sequencies, suggesting that a kind of electronic biosensor can be developed to distinguish different profiles of autism disorders.

Chimeric antigen receptors with human scFvs preferentially induce T cell anti-tumor activity against tumors with high B7H6 expression.

PubMed

Gacerez, Albert T; Hua, Casey K; Ackerman, Margaret E; Sentman, Charles L

2018-05-01

B7H6 is emerging as a promising tumor antigen that is known to be expressed on a wide array of tumors and is reported to stimulate anti-tumor responses from the immune system. As such, B7H6 presents a good target for tumor-specific immunotherapies. B7H6-specific chimeric antigen receptors (CAR) based on a murine antibody showed successful targeting and elimination of tumors expressing B7H6. However, mouse single chain variable fragments (scFvs) have the potential to induce host anti-CAR responses that may limit efficacy, so human scFvs specific for B7H6 were selected by yeast surface display. In this study, we validate the functionality of these human scFvs when formatted into chimeric antigen receptors. The data indicate that T cells expressing these B7H6-specific human scFvs as CARs induced potent anti-tumor activity in vitro and in vivo against tumors expressing high amounts of B7H6. Importantly, these human scFv-based CARs are sensitive to changes in B7H6 expression which may potentially spare non-tumor cells that express B7H6 and provides the foundation for future clinical development.

Enzymes involved in branched-chain amino acid metabolism in humans.

PubMed

Adeva-Andany, María M; López-Maside, Laura; Donapetry-García, Cristóbal; Fernández-Fernández, Carlos; Sixto-Leal, Cristina

2017-06-01

Branched-chain amino acids (leucine, isoleucine and valine) are structurally related to branched-chain fatty acids. Leucine is 2-amino-4-methyl-pentanoic acid, isoleucine is 2-amino-3-methyl-pentanoic acid, and valine is 2-amino-3-methyl-butanoic acid. Similar to fatty acid oxidation, leucine and isoleucine produce acetyl-coA. Additionally, leucine generates acetoacetate and isoleucine yields propionyl-coA. Valine oxidation produces propionyl-coA, which is converted into methylmalonyl-coA and succinyl-coA. Branched-chain aminotransferase catalyzes the first reaction in the catabolic pathway of branched-chain amino acids, a reversible transamination that converts branched-chain amino acids into branched-chain ketoacids. Simultaneously, glutamate is converted in 2-ketoglutarate. The branched-chain ketoacid dehydrogenase complex catalyzes the irreversible oxidative decarboxylation of branched-chain ketoacids to produce branched-chain acyl-coA intermediates, which then follow separate catabolic pathways. Human tissue distribution and function of most of the enzymes involved in branched-chain amino acid catabolism is unknown. Congenital deficiencies of the enzymes involved in branched-chain amino acid metabolism are generally rare disorders. Some of them are associated with reduced pyruvate dehydrogenase complex activity and respiratory chain dysfunction that may contribute to their clinical phenotype. The biochemical phenotype is characterized by accumulation of the substrate to the deficient enzyme and its carnitine and/or glycine derivatives. It was established at the beginning of the twentieth century that the plasma level of the branched-chain amino acids is increased in conditions associated with insulin resistance such as obesity and diabetes mellitus. However, the potential clinical relevance of this elevation is uncertain.

Helper-Dependent Properties of Friend Spleen Focus-Forming Virus: Effect of the Fv-1 Gene on the Late Stages in Virus Synthesis

PubMed Central

Eckner, Robert J.

1973-01-01

Co-infection of neonatal BALB/c mice with Friend virus (FV) complex (containing defective spleen focus-forming virus [SFFV] and endogenous N-tropic leukemia-inducing helper virus [LLV-F]) and B-tropic Tennant leukemia virus (TenLV) resulted in the inhibition of LLV-F by the Fv-1b gene and recovery of a TenLV pseudotype of SFFV, abbreviated SFFV(TenLV). The host range of this pseudotype was B-tropic, since SFFV(TenLV) was 10 to 100 times more infectious for B-type (Fv-1bb) than for N-type (Fv-1nn) mice. The similar patterns of neutralization of N-tropic and B-tropic SFFV by type-specific murine antisera suggested that the difference in infectivity between these two SFFV preparations did not reside in envelope determinants. Rather, helper control of SFFV's host range was only apparent and dependent upon the ability of associated virus to provide a helper function for late stages in SFFV synthesis. Early stages in SFFV's infectious cycle were shown to be helper independent. The Fv-1 gene did not act at the level of the cell membrane to effectively restrict SFFV infection, since SFFV-induced transformed cells could be detected in the absence of spleen focus formation and SFFV synthesis. Further, the generation of these transformed cells by SFFV followed a one-hit, dose-response pattern, suggesting that SFFV-induced cell transformation is helper independent. Finally, restriction of helper function by Fv-1 may be an intracellular event, because both SFFV and its associated LLV-F helper share common envelope determinants and presumably adsorb onto and penetrate target cells with equal efficiency. PMID:4127030

Cloning and sequence analysis of complementary DNA encoding an aberrantly rearranged human T-cell gamma chain.

PubMed Central

Dialynas, D P; Murre, C; Quertermous, T; Boss, J M; Leiden, J M; Seidman, J G; Strominger, J L

1986-01-01

Complementary DNA (cDNA) encoding a human T-cell gamma chain has been cloned and sequenced. At the junction of the variable and joining regions, there is an apparent deletion of two nucleotides in the human cDNA sequence relative to the murine gamma-chain cDNA sequence, resulting simultaneously in the generation of an in-frame stop codon and in a translational frameshift. For this reason, the sequence presented here encodes an aberrantly rearranged human T-cell gamma chain. There are several surprising differences between the deduced human and murine gamma-chain amino acid sequences. These include poor homology in the variable region, poor homology in a discrete segment of the constant region precisely bounded by the expected junctions of exon CII, and the presence in the human sequence of five potential sites for N-linked glycosylation. Images PMID:3458221

Recombinant anti-podoplanin (NZ-1) immunotoxin for the treatment of malignant brain tumors

PubMed Central

Chandramohan, Vidyalakshmi; Bao, Xuhui; Kaneko, Mika Kato; Kato, Yukinari; Keir, Stephen T.; Szafranski, Scott E.; Kuan, Chien-Tsun; Pastan, Ira H.; Bigner, Darell D.

2013-01-01

Current study demonstrates the glioma tumor antigen podoplanin to be present at very high levels (>90%) in both glioblastoma (D2159MG, D08-0308MG, and D08-0493MG) and medulloblastoma (D283MED, D425MED, and DAOY) xenografts and cell line. We constructed a novel recombinant single-chain antibody variable region fragment (scFv), NZ-1, specific for podoplanin from the NZ-1 hybridoma. NZ-1-scFv was then fused to Pseudomonas exotoxin A, carrying a C-terminal KDEL peptide (NZ-1-PE38KDEL). The immunotoxin was further stabilized by a disulfide (ds) bond between the heavy-chain and light-chain variable regions as the construct NZ-1-(scdsFv)-PE38KDEL. NZ-1-(scdsFv)-PE38KDEL exhibited significant reactivity to glioblastoma and medulloblastoma cells. The affinity of NZ-1-(scdsFv), NZ-1-(scdsFv)-PE38KDEL and NZ-1 antibody, for podoplanin peptide was 2.1×10−8 M, 8.0×10−8 M, and 3.9×10−10 M, respectively. In a protein stability assay, NZ-1-(scdsFv)-PE38KDEL retained 33-98% of its activity while that of NZ-1-PE38KDEL declined to 13% of its initial levels after incubation at 37°C for 3 days. In vitro cytotoxicity of the NZ-1-(scdsFv)-PE38KDEL was measured in cells isolated from glioblastoma xenografts, D2159MG, D08-0308MG, D08-0493MG, and in the medulloblastoma D283MED, D425MED, and DOAY xenografts and cell line. The NZ-1-(scdsFv)-PE38KDEL immunotoxin was highly cytotoxic, with an IC50 in the range of 1.6–29 ng/mL. Significantly, NZ-1-(scdsFv)-PE38KDEL demonstrated tumor-growth delay, averaging 24 days (P<0.001) and 21 days (P<0.001) in D2159MG and D283MED in vivo tumor models, respectively. Crucially, in the D425MED intracranial tumor model, NZ-1-(scdsFv)-PE38KDEL caused a 41% increase in survival (P≤0.001). In preclinical studies, NZ-1-(scdsFv)-PE38KDEL exhibited significant potential as a targeting agent for malignant brain tumors. PMID:23115013

Recombinant anti-podoplanin (NZ-1) immunotoxin for the treatment of malignant brain tumors.

PubMed

Chandramohan, Vidyalakshmi; Bao, Xuhui; Kato Kaneko, Mika; Kato, Yukinari; Keir, Stephen T; Szafranski, Scott E; Kuan, Chien-Tsun; Pastan, Ira H; Bigner, Darell D

2013-05-15

Our study demonstrates the glioma tumor antigen podoplanin to be present at very high levels (>90%) in both glioblastoma (D2159MG, D08-0308MG and D08-0493MG) and medulloblastoma (D283MED, D425MED and DAOY) xenografts and cell line. We constructed a novel recombinant single-chain antibody variable region fragment (scFv), NZ-1, specific for podoplanin from the NZ-1 hybridoma. NZ-1-scFv was then fused to Pseudomonas exotoxin A, carrying a C-terminal KDEL peptide (NZ-1-PE38KDEL). The immunotoxin (IT) was further stabilized by a disulfide (ds) bond between the heavy-chain and light-chain variable regions as the construct NZ-1-(scdsFv)-PE38KDEL. NZ-1-(scdsFv)-PE38KDEL exhibited significant reactivity to glioblastoma and medulloblastoma cells. The affinity of NZ-1-(scdsFv), NZ-1-(scdsFv)-PE38KDEL and NZ-1 antibody for podoplanin peptide was 2.1 × 10(-8) M, 8.0 × 10(-8) M and 3.9 × 10(-10) M, respectively. In a protein stability assay, NZ-1-(scdsFv)-PE38KDEL retained 33-98% of its activity, whereas that of NZ-1-PE38KDEL declined to 13% of its initial levels after incubation at 37°C for 3 days. In vitro cytotoxicity of the NZ-1-(scdsFv)-PE38KDEL was measured in cells isolated from glioblastoma xenografts, D2159MG, D08-0308MG and D08-0493MG, and in the medulloblastoma D283MED, D425MED and DOAY xenografts and cell line. The NZ-1-(scdsFv)-PE38KDEL IT was highly cytotoxic, with an 50% inhibitory concentration in the range of 1.6-29 ng/ml. Significantly, NZ-1-(scdsFv)-PE38KDEL demonstrated tumor growth delay, averaging 24 days (p < 0.001) and 21 days (p < 0.001) in D2159MG and D283MED in vivo tumor models, respectively. Crucially, in the D425MED intracranial tumor model, NZ-1-(scdsFv)-PE38KDEL caused a 41% increase in survival (p ≤ 0.001). In preclinical studies, NZ-1-(scdsFv)-PE38KDEL exhibited significant potential as a targeting agent for malignant brain tumors. Copyright © 2012 UICC.

"Quenchbodies": quench-based antibody probes that show antigen-dependent fluorescence.

PubMed

Abe, Ryoji; Ohashi, Hiroyuki; Iijima, Issei; Ihara, Masaki; Takagi, Hiroaki; Hohsaka, Takahiro; Ueda, Hiroshi

2011-11-02

Here, we describe a novel reagentless fluorescent biosensor strategy based on the antigen-dependent removal of a quenching effect on a fluorophore attached to antibody domains. Using a cell-free translation-mediated position-specific protein labeling system, we found that an antibody single chain variable region (scFv) that had been fluorolabeled at the N-terminal region showed a significant antigen-dependent fluorescence enhancement. Investigation of the enhancement mechanism by mutagenesis of the carboxytetramethylrhodamine (TAMRA)-labeled anti-osteocalcin scFv showed that antigen-dependency was dependent on semiconserved tryptophan residues near the V(H)/V(L) interface. This suggested that the binding of the antigen led to the interruption of a quenching effect caused by the proximity of tryptophan residues to the linker-tagged fluorophore. Using TAMRA-scFv, many targets including peptides, proteins, and haptens including morphine-related drugs could be quantified. Similar or higher sensitivities to those observed in competitive ELISA were obtained, even in human plasma. Because of its versatility, this "quenchbody" is expected to have a range of applications, from in vitro diagnostics, to imaging of various targets in situ.

Platelet-Derived Short-Chain Polyphosphates Enhance the Inactivation of Tissue Factor Pathway Inhibitor by Activated Coagulation Factor XI.

PubMed

Puy, Cristina; Tucker, Erik I; Ivanov, Ivan S; Gailani, David; Smith, Stephanie A; Morrissey, James H; Gruber, András; McCarty, Owen J T

2016-01-01

Factor (F) XI supports both normal human hemostasis and pathological thrombosis. Activated FXI (FXIa) promotes thrombin generation by enzymatic activation of FXI, FIX, FX, and FV, and inactivation of alpha tissue factor pathway inhibitor (TFPIα), in vitro. Some of these reactions are now known to be enhanced by short-chain polyphosphates (SCP) derived from activated platelets. These SCPs act as a cofactor for the activation of FXI and FV by thrombin and FXIa, respectively. Since SCPs have been shown to inhibit the anticoagulant function of TFPIα, we herein investigated whether SCPs could serve as cofactors for the proteolytic inactivation of TFPIα by FXIa, further promoting the efficiency of the extrinsic pathway of coagulation to generate thrombin. Purified soluble SCP was prepared by size-fractionation of sodium polyphosphate. TFPIα proteolysis was analyzed by western blot. TFPIα activity was measured as inhibition of FX activation and activity in coagulation and chromogenic assays. SCPs significantly accelerated the rate of inactivation of TFPIα by FXIa in both purified systems and in recalcified plasma. Moreover, platelet-derived SCP accelerated the rate of inactivation of platelet-derived TFPIα by FXIa. TFPIα activity was not affected by SCP in recalcified FXI-depleted plasma. Our data suggest that SCP is a cofactor for TFPIα inactivation by FXIa, thus, expanding the range of hemostatic FXIa substrates that may be affected by the cofactor functions of platelet-derived SCP.

Modelling magnetic anisotropy of single-chain magnets in |d/J| ≥ 1 regime

NASA Astrophysics Data System (ADS)

Haldar, Sumit; Raghunathan, Rajamani; Sutter, Jean-Pascal; Ramasesha, S.

2017-11-01

Single-molecule magnets (SMMs) with single-ion anisotropies comparable to exchange interactions J between spins have recently been synthesised. Here, we provide theoretical insights into the magnetism of such systems. We study spin chains with site-spins, s = 1, 3/2 and 2 and strength of on-site anisotropy comparable to the exchange constants between the spins. We find that large on-site anisotropies lead to crossing of the states with different MS values in the same spin manifold to which they belong in the absence of anisotropy. When on-site anisotropy is increased further, we also find that the MS states of the higher energy spin states descend below the MS states of the ground spin manifold. Giant spin in this limit is no longer conserved and describing the axial and rhombic anisotropies of the molecule, DM and EM, respectively, is not possible. However, the giant spin of the low-lying large MS states is very nearly an integer and, using this spin value, it is possible to construct an effective spin-Hamiltonian and compute the molecular magnetic anisotropy constants DM and EM. We report effect of finite sizes, rotations of site anisotropies and chain dimerisation on the effective anisotropy of the spin chains.

IL-2 receptor γ-chain molecule is critical for intestinal T-cell reconstitution in humanized mice.

PubMed

Denton, P W; Nochi, T; Lim, A; Krisko, J F; Martinez-Torres, F; Choudhary, S K; Wahl, A; Olesen, R; Zou, W; Di Santo, J P; Margolis, D M; Garcia, J V

2012-09-01

Intestinal immune cells are important in host defense, yet the determinants for human lymphoid homeostasis in the intestines are poorly understood. In contrast, lymphoid homeostasis has been studied extensively in mice, where the requirement for a functional common γ-chain molecule has been established. We hypothesized that humanized mice could offer insights into human intestinal lymphoid homeostasis if generated in a strain with an intact mouse common γ-chain molecule. To address this hypothesis, we used three mouse strains (non-obese diabetic (NOD)/severe-combined immunodeficient (SCID) (N/S); NOD/SCID γ-chain(-/-) (NSG); and Rag2(-/-) γ-chain(-/-) (DKO)) and two humanization techniques (bone marrow liver thymus (BLT) and human CD34(+) cell bone marrow transplant of newborn mice (hu)) to generate four common types of humanized mice: N/S-BLT, NSG-BLT, NSG-hu, and DKO-hu mice. The highest levels of intestinal human T cells throughout the small and large intestines were observed in N/S-BLT mice, which have an intact common γ-chain molecule. Furthermore, the small intestine lamina propria T-cell populations of N/S-BLT mice exhibit a human intestine-specific surface phenotype. Thus, the extensive intestinal immune reconstitution of N/S-BLT mice was both quantitatively and qualitatively better when compared with the other models tested such that N/S-BLT mice are well suited for the analysis of human intestinal lymphocyte trafficking and human-specific diseases affecting the intestines.

Allergy immunotherapy with a hypoallergenic recombinant birch pollen allergen rBet v 1-FV in a randomized controlled trial.

PubMed

Klimek, Ludger; Bachert, Claus; Lukat, Karl-Friedrich; Pfaar, Oliver; Meyer, Hanns; Narkus, Annemie

2015-01-01

Pollen extracts and chemically modified allergoids are used successfully in allergen immunotherapy (AIT). Recombinant extracts offer potential advantages with respect to pharmaceutical quality, standardization and dosing. A hypoallergenic recombinant folding variant of the major birch pollen allergen (rBet v 1-FV) was compared with an established native birch preparation. A pre-seasonal, randomized, actively controlled phase II study was performed in birch pollen allergic rhino-conjunctivitis with or without asthma, GINA I/ II. 51 patients (24 rBet v 1-FV, 27 native extract) started therapy with subcutaneous allergen immunotherapy (SCIT). Primary end-point was a combined symptom medication score (SMS), changes in nasal provocation test, visual rating score and specific antibody responses secondary end-points. After one pre-seasonal treatment course the combined SMS was 5.86 (median; IQR: 14.02) for the rBet v 1-FV group versus 12.40 (median; IQR: 9.32) for the comparator during the three weeks pollen season (p = 0.330). After treatment in the second year, scores were 3.00 (median; IQR: 6.50) and 2.93 (4.86) respectively. Allergen tolerance in a nasal provocation test improved to a comparable extent in both groups. Significant increases in birch pollen-specific IgG1 and IgG4 were observed in both treatment groups following the first treatment phase and remained significantly raised until the end of the study. In this first in man, proof of concept phase II trial no statistical difference between rBet v 1-FV and an established natural pollen extract could be observed. rBet v 1-FV could be administered in higher doses than the native protein with no increase in adverse effects. The study was registered in clinicalTrials.gov (NCT00266526).

Surface water retardation around single-chain polymeric nanoparticles: critical for catalytic function?

PubMed

Stals, Patrick J M; Cheng, Chi-Yuan; van Beek, Lotte; Wauters, Annelies C; Palmans, Anja R A; Han, Songi; Meijer, E W

2016-03-01

A library of water-soluble dynamic single-chain polymeric nanoparticles (SCPN) was prepared using a controlled radical polymerisation technique followed by the introduction of functional groups, including probes at targeted positions. The combined tools of electron paramagnetic resonance (EPR) and Overhauser dynamic nuclear polarization (ODNP) reveal that these SCPNs have structural and surface hydration properties resembling that of enzymes.

Obligate multivalent recognition of cell surface tomoregulin following selection from a multivalent phage antibody library.

PubMed

Heitner, Tara; Satozawa, Noboru; McLean, Kirk; Vogel, David; Cobb, Ronald R; Liu, Bing; Mahmoudi, Mithra; Finster, Silke; Larsen, Brent; Zhu, Ying; Zhou, Hongxing; Müller-Tiemann, Beate; Monteclaro, Felipe; Zhao, Xiao-Yan; Light, David R

2006-12-01

A therapeutic antibody candidate (AT-19) isolated using multivalent phage display binds native tomoregulin (TR) as a mul-timer not as a monomer. This report raises the importance of screening and selecting phage antibodies on native antigen and reemphasizes the possibility that potentially valuable antibodies are discarded when a monomeric phage display system is used for screening. A detailed live cell panning selection and screening method to isolate multivalently active antibodies is described. AT-19 is a fully human antibody recognizing the cell surface protein TR, a proposed prostate cancer target for therapeutic antibody internalization. AT-19 was isolated from a multivalent single-chain variable fragment (scFv) antibody library rescued with hyperphage. The required multivalency for isolation of AT-19 is supported by fluorescence activated cell sorting data demonstrating binding of the multivalent AT-19 phage particles at high phage concentrations and failure of monovalent particles to bind. Pure monomeric scFv AT-19 does not bind native receptor on cells, whereas dimeric scFv or immunoglobulin G binds with nanomolar affinity. The isolation of AT-19 antibody with obligate bivalent binding activity to native TR is attributed to the use of a multivalent display of scFv on phage and the method for selecting and screening by alternate use of 2 recombinant cell lines.

A putative OTU domain-containing protein 1 deubiquitinating enzyme is differentially expressed in thyroid cancer and identifies less-aggressive tumours

PubMed Central

Carneiro, A P; Reis, C F; Morari, E C; Maia, Y C P; Nascimento, R; Bonatto, J M C; de Souza, M A; Goulart, L R; Ward, L S

2014-01-01

Background: This study aimed to identify novel biomarkers for thyroid carcinoma diagnosis and prognosis. Methods: We have constructed a human single-chain variable fragment (scFv) antibody library that was selected against tumour thyroid cells using the BRASIL method (biopanning and rapid analysis of selective interactive ligands) and phage display technology. Results: One highly reactive clone, scFv-C1, with specific binding to papillary thyroid tumour proteins was confirmed by ELISA, which was further tested against a tissue microarray that comprised of 229 thyroid tissues, including: 110 carcinomas (38 papillary thyroid carcinomas (PTCs), 42 follicular carcinomas, 30 follicular variants of PTC), 18 normal thyroid tissues, 49 nodular goitres (NG) and 52 follicular adenomas. The scFv-C1 was able to distinguish carcinomas from benign lesions (P=0.0001) and reacted preferentially against T1 and T2 tumour stages (P=0.0108). We have further identified an OTU domain-containing protein 1, DUBA-7 deubiquitinating enzyme as the scFv-binding antigen using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. Conclusions: The strategy of screening and identifying a cell-surface-binding antibody against thyroid tissues was highly effective and resulted in a useful biomarker that recognises malignancy among thyroid nodules and may help identify lower-risk cases that can benefit from less-aggressive management. PMID:24937664

Affinity improvement of a therapeutic antibody to methamphetamine and amphetamine through structure-based antibody engineering

PubMed Central

Thakkar, Shraddha; Nanaware-Kharade, Nisha; Celikel, Reha; Peterson, Eric C.; Varughese, Kottayil I.

2014-01-01

Methamphetamine (METH) abuse is a worldwide threat, without any FDA approved medications. Anti-METH IgGs and single chain fragments (scFvs) have shown efficacy in preclinical studies. Here we report affinity enhancement of an anti-METH scFv for METH and its active metabolite amphetamine (AMP), through the introduction of point mutations, rationally designed to optimize the shape and hydrophobicity of the antibody binding pocket. The binding affinity was measured using saturation binding technique. The mutant scFv-S93T showed 3.1 fold enhancement in affinity for METH and 26 fold for AMP. The scFv-I37M and scFv-Y34M mutants showed enhancement of 94, and 8 fold for AMP, respectively. Structural analysis of scFv-S93T:METH revealed that the substitution of Ser residue by Thr caused the expulsion of a water molecule from the cavity, creating a more hydrophobic environment for the binding that dramatically increases the affinities for METH and AMP. PMID:24419156

Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody

PubMed Central

Honey, Denise M.; Best, Annie; Qiu, Huawei

2018-01-01

ABSTRACT Metelimumab (CAT192) is a human IgG4 monoclonal antibody developed as a TGFβ1-specific antagonist. It was tested in clinical trials for the treatment of scleroderma but later terminated due to lack of efficacy. Subsequent characterization of CAT192 indicated that its TGFβ1 binding affinity was reduced by ∼50-fold upon conversion from the parental single-chain variable fragment (scFv) to IgG4. We hypothesized this result was due to decreased conformational flexibility of the IgG that could be altered via engineering. Therefore, we designed insertion mutants in the elbow region and screened for binding and potency. Our results indicated that increasing the elbow region linker length in each chain successfully restored the isoform-specific and high affinity binding of CAT192 to TGFβ1. The crystal structure of the high binding affinity mutant displays large conformational rearrangements of the variable domains compared to the wild-type antigen-binding fragment (Fab) and the low binding affinity mutants. Insertion of two glycines in both the heavy and light chain elbow regions provided sufficient flexibility for the variable domains to extend further apart than the wild-type Fab, and allow the CDR3s to make additional interactions not seen in the wild-type Fab structure. These interactions coupled with the dramatic conformational changes provide a possible explanation of how the scFv and elbow-engineered Fabs bind TGFβ1 with high affinity. This study demonstrates the benefits of re-examining both structure and function when converting scFv to IgG molecules, and highlights the potential of structure-based engineering to produce fully functional antibodies. PMID:29333938

cDNA cloning of rat and human medium chain acyl-CoA dehydrogenase (MCAD)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsubara, Y.; Kraus, J.P.; Rosenberg, L.E.

MCAD is one of three mitochondrial flavoenzymes which catalyze the first step in the ..beta..-oxidation of straight chain fatty acids. It is a tetramer with a subunit Mr of 45 kDa. MCAD is synthesized in the cytosol as a 49 kDa precursor polypeptide (pMCAD), imported into mitochondria, and cleaved to the mature form. Genetic deficiency of MCAD causes recurrent episodes of hypoglycemic coma accompanied by medium chain dicarboxylic aciduria. Employing a novel approach, the authors now report isolation of partial rat and human cDNA clones encoding pMCAD. mRNA encoding pMCAD was purified to near homogeneity by polysome immunoadsorption using polyclonalmore » monospecific antibody. Single-stranded (/sup 32/P)labeled cDNA probe was synthesized using the enriched mRNA as template, and was used to screen directly 16,000 colonies from a total rat liver cDNA library constructed in pBR322. One clone (600 bp) was detected by in situ hybridization. Hybrid-selected translation with this cDNA yielded a 49 kDa polypeptide indistinguishable in size from rat pMCAD and immunoprecipitable with anti-MCAD antibody. Using the rat cDNA as probe, 43,000 colonies from a human liver cDNA library were screened. Four identical positive clones (400 bp) were isolated and positively identified by hybrid-selected translation and immunoprecipitation. The sizes of rat and human mRNAs encoding pMCAD were 2.2 kb and 2.4 kb, respectively, as determined by Northern blotting.« less

Extending the half-life of a fab fragment through generation of a humanized anti-human serum albumin Fv domain: An investigation into the correlation between affinity and serum half-life.

PubMed

Adams, Ralph; Griffin, Laura; Compson, Joanne E; Jairaj, Mark; Baker, Terry; Ceska, Tom; West, Shauna; Zaccheo, Oliver; Davé, Emma; Lawson, Alastair Dg; Humphreys, David P; Heywood, Sam

2016-10-01

We generated an anti-albumin antibody, CA645, to link its Fv domain to an antigen-binding fragment (Fab), thereby extending the serum half-life of the Fab. CA645 was demonstrated to bind human, cynomolgus, and mouse serum albumin with similar affinity (1-7 nM), and to bind human serum albumin (HSA) when it is in complex with common known ligands. Importantly for half-life extension, CA645 binds HSA with similar affinity within the physiologically relevant range of pH 5.0 - pH 7.4, and does not have a deleterious effect on the binding of HSA to neonatal Fc receptor (FcRn). A crystal structure of humanized CA645 Fab in complex with HSA was solved and showed that CA645 Fab binds to domain II of HSA. Superimposition with the crystal structure of FcRn bound to HSA confirmed that CA645 does not block HSA binding to FcRn. In mice, the serum half-life of humanized CA645 Fab is 84.2 h. This is a significant extension in comparison with < 1 h for a non-HSA binding CA645 Fab variant. The Fab-HSA structure was used to design a series of mutants with reduced affinity to investigate the correlation between the affinity for albumin and serum half-life. Reduction in the affinity for MSA by 144-fold from 2.2 nM to 316 nM had no effect on serum half-life. Strikingly, despite a reduction in affinity to 62 µM, an extension in serum half-life of 26.4 h was still obtained. CA645 Fab and the CA645 Fab-HSA complex have been deposited in the Protein Data Bank (PDB) with accession codes, 5FUZ and 5FUO, respectively.

Selection of single chain antibody fragments binding to the extracellular domain of 4-1BB receptor by phage display technology.

PubMed

Bagheri, Salman; Yousefi, Mehdi; Safaie Qamsari, Elmira; Riazi-Rad, Farhad; Abolhassani, Mohsen; Younesi, Vahid; Dorostkar, Ruhollah; Movassaghpour, Ali Akbar; Sharifzadeh, Zahra

2017-03-01

The 4-1BB is a surface glycoprotein that pertains to the tumor necrosis factor-receptor family. There is compelling evidence suggesting important roles for 4-1BB in the immune response, including cell activation and proliferation and also cytokine induction. Because of encouraging results of different agonistic monoclonal antibodies against 4-1BB in the treatment of cancer, infectious, and autoimmune diseases, 4-1BB has been suggested as an attractive target for immunotherapy. In this study, single chain variable fragment phage display libraries, Tomlinson I+J, were screened against specific synthetic oligopeptides (peptides I and II) designed from 4-1BB extracellular domain. Five rounds of panning led to selection of four 4-1BB specific single chain variable fragments (PI.12, PI.42, PII.16, and PII.29) which showed specific reaction to relevant peptides in phage enzyme-linked immunosorbent assay. The selected clones were successfully expressed in Escherichia coli Rosetta-gami 2, and their expression was confirmed by western blot analysis. Enzyme-linked immunosorbent assay experiments indicated that these antibodies were able to specifically recognize 4-1BB without any cross-reactivity with other antigens. Flow cytometry analysis demonstrated an acceptable specific binding of the single chain variable fragments to 4-1BB expressed on CCRF-CEM cells, while no binding was observed with an irrelevant antibody. Anti-4-1BB single chain variable fragments enhanced surface CD69 expression and interleukin-2 production in stimulated CCRF-CEM cells which confirmed the agonistic effect of the selected single chain variable fragments. The data from this study have provided a rationale for further experiments involving the biological functions of anti-4-1BB single chain variable fragments in future studies.

Generation of anti-idiotype scFv for pharmacokinetic measurement in lymphoma patients treated with chimera anti-CD22 antibody SM03.

PubMed

Zhao, Qi; Wong, Pui-Fan; Lee, Susanna S T; Leung, Shui-On; Cheung, Wing-Tai; Wang, Jun-Zhi

2014-01-01

Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen.

Generation of Anti-Idiotype scFv for Pharmacokinetic Measurement in Lymphoma Patients Treated with Chimera Anti-CD22 Antibody SM03

PubMed Central

Zhao, Qi; Wong, Pui-Fan; Lee, Susanna S. T.; Leung, Shui-On; Cheung, Wing-Tai; Wang, Jun-Zhi

2014-01-01

Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated. The anti-idiotype scFv recognized the idiotype of anti-CD22 antibody and inhibited binding of SM03 to CD22 on Raji cell surface. The anti-idiotype scFv was subsequently classified as Ab2γ type. Moreover, our results also demonstrated firstly that the anti-idiotype scFv could be used for pharmacokinetic measurement of circulating residual antibody in lymphoma patients treated with chimera anti-CD22 monoclonal antibody SM03. Of important, the present approach could be easily adopted to generate anti-idiotype antibodies for therapeutic antibodies targeting membrane proteins, saving the cost and time for producing a soluble antigen. PMID:24816427

A novel anti-CD22 scFv-apoptin fusion protein induces apoptosis in malignant B-cells.

PubMed

Agha Amiri, Solmaz; Shahhosseini, Soraya; Zarei, Najmeh; Khorasanizadeh, Dorsa; Aminollahi, Elahe; Rezaie, Faegheh; Zargari, Mehryar; Azizi, Mohammad; Khalaj, Vahid

2017-12-01

CD22 marker is a highly internalizing antigen which is located on the surface of B-cells and is being used as a promising target for treatment of B cell malignancies. Monoclonal antibodies targeting CD22 have been introduced and some are currently under investigation in clinical trials. Building on the success of antibody drug conjugates, we developed a fusion protein consisting of a novel anti-CD22 scFv and apoptin and tested binding and therapeutic effects in lymphoma cells. The recombinant protein was expressed in E. coli and successfully purified and refolded. In vitro binding analysis by immunofluorescence and flow cytometry demonstrated that the recombinant protein specifically binds to CD22 positive Raji cells but not to CD22 negative Jurkat cells. The cytotoxic properties of scFv-apoptin were assessed by an MTT assay and Annexin V/PI flow cytometry analysis and showed that the recombinant protein induced apoptosis preferentially in Raji cells with no detectable effects in Jurkat cells. Our findings indicated that the recombinant anti-CD22 scFv-apoptin fusion protein could successfully cross the cell membrane and induce apoptosis with high specificity, make it as a promising molecule for immunotherapy of B-cell malignancies.

Direct observation of backbone planarization via side-chain alignment in single bulky-substituted polythiophenes

NASA Astrophysics Data System (ADS)

Raithel, Dominic; Simine, Lena; Pickel, Sebastian; Schötz, Konstantin; Panzer, Fabian; Baderschneider, Sebastian; Schiefer, Daniel; Lohwasser, Ruth; Köhler, Jürgen; Thelakkat, Mukundan; Sommer, Michael; Köhler, Anna; Rossky, Peter J.; Hildner, Richard

2018-03-01

The backbone conformation of conjugated polymers affects, to a large extent, their optical and electronic properties. The usually flexible substituents provide solubility and influence the packing behavior of conjugated polymers in films or in bad solvents. However, the role of the side chains in determining and potentially controlling the backbone conformation, and thus the optical and electronic properties on the single polymer level, is currently under debate. Here, we investigate directly the impact of the side chains by studying the bulky-substituted poly(3-(2,5-dioctylphenyl)thiophene) (PDOPT) and the common poly(3-hexylthiophene) (P3HT), both with a defined molecular weight and high regioregularity, using low-temperature single-chain photoluminescence (PL) spectroscopy and quantum-classical simulations. Surprisingly, the optical transition energy of PDOPT is significantly (˜2,000 cm‑1 or 0.25 eV) red-shifted relative to P3HT despite a higher static and dynamic disorder in the former. We ascribe this red shift to a side-chain induced backbone planarization in PDOPT, supported by temperature-dependent ensemble PL spectroscopy. Our atomistic simulations reveal that the bulkier 2,5-dioctylphenyl side chains of PDOPT adopt a clear secondary helical structural motif and thus protect conjugation, i.e., enforce backbone planarity, whereas, for P3HT, this is not the case. These different degrees of planarity in both thiophenes do not result in different conjugation lengths, which we found to be similar. It is rather the stronger electronic coupling between the repeating units in the more planar PDOPT which gives rise to the observed spectral red shift as well as to a reduced calculated electron‑hole polarization.

Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity

PubMed Central

Shen, Yang; Zeng, Lin; Zhu, Aiping; Blanc, Tim; Patel, Dipa; Pennello, Anthony; Bari, Amtul; Ng, Stanley; Persaud, Kris; Kang, Yun (Kenneth); Balderes, Paul; Surguladze, David; Hindi, Sagit; Zhou, Qinwei; Ludwig, Dale L.; Snavely, Marshall

2013-01-01

Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies. PMID:23567210

Developing and sustaining human resources in the health supply chain in Ethiopia: barriers and enablers.

PubMed

Kälvemark Sporrong, Sofia; Traulsen, Janine M; Damene Kabtimer, Woynabeba; Mekasha Habtegiorgis, Bitsatab; Teshome Gebregeorgise, Dawit; Essah, Nana Am; Khan, Sara A; Brown, Andrew N

2016-01-01

The health supply chain is often the weakest link in achieving the health-related Millennium Development Goals and universal health coverage, requiring trained professionals who are often unavailable. In Ethiopia there have been recent developments in the area of health supply chain management. The aim of this study was to explore the current status of the development of human resources in health supply chain management in Ethiopia and to identify important factors affecting this development. A series of face-to-face interviews with key stakeholders was carried out in 2014. The interviews were conducted using a semi-structured interview guide. The interview guide comprised 51 questions. A qualitative analysis of transcripts was made. A total of 25 interviews were conducted. Three themes were identified: General changes: recognition, commitment and resources, Education and training, and Barriers and enablers. Results confirm the development of human resources in health supply chain management in many areas. However, several problems were identified including lack of coordination, partly due to the large number of stakeholders; reported high staff mobility; and a lack of overall strategy regarding the job/career structures necessary for maintaining human resources. Rural areas have a particular set of problems, including in transportation of goods and personnel, attracting and keeping personnel, and in communication and access to information. Ethiopia is on the way to developing a nationwide viable system for health supply chain management. However, there are still challenges. Short-term challenges include the importance of highlighting strategies and programs for human resources in health supply chain management. In the long term, commitments to financial support must be obtained. A strategy is needed for the further development and sustainability of human resources in the health supply chain in Ethiopia.

Novel Monoclonal Antibodies for Studies of Human and Rhesus Macaque Secretory Component and Human J-Chain

PubMed Central

Zhang, Ruijun; Alam, S. Munir; Yu, Jae-Sung; Scearce, Richard; Lockwood, Bradley; Hwang, Kwan-Ki; Parks, Robert; Permar, Sallie; Brandtzaeg, Per; Haynes, Barton F.

2016-01-01

Immunoglobulin A (IgA) antibodies exist in monomeric, dimeric, and secretory forms. Dimerization of IgA depends on a 15-kD polypeptide termed “joining (J) chain,” which is also part of the binding site for an epithelial glycoprotein called “secretory component (SC),” whether this after apical cleavage on secretory epithelia is ligand bound in secretory IgA (SIgA) or in a free form. Uncleaved membrane SC, also called the “polymeric Ig receptor,” is thus crucial for transcytotic export of SIgA to mucosal surfaces, where it interacts with and modulates commensal bacteria and mediates protective immune responses against exogenous pathogens. To evaluate different forms of IgA, we have produced mouse monoclonal antibodies (MAbs) against human J-chain and free SC. We found that J-chain MAb 9A8 and SC MAb 9H7 identified human dimeric IgA and SIgA in enzyme-linked immunoassay and western blot analysis, as well as functioning in immunohistochemistry to identify cytoplasmic IgA of intestinal lamina propria plasmablasts/plasma cells and crypt epithelium of distal human intestine. Finally, we demonstrated that SC MAb 9H7 cross-reacted with rhesus macaque SIgA. These novel reagents should be of use in the study of the biology of various forms of IgA in humans and SIgA in macaques, as well as in monitoring the production and/or isolation of these forms of IgA. PMID:27386924

Single step, pH induced gold nanoparticle chain formation in lecithin/water system.

PubMed

Sharma, Damyanti

2013-07-01

Gold nanoparticle (AuNP) chains have been formed by a single step method in a lecithin/water system where lecithin itself plays the role of a reductant and a template for AuNP chain formation. Two preparative strategies were explored: (1) evaporating lecithin solution with aqueous gold chloride (HAuCl4) at different pHs and (2) dispersing lecithin vesicles in aqueous HAuCl4 solutions of various pHs in the range of 2.5-11.3. In method 1, at initial pH 2.5, 20-50 nm AuNPs are found attached to lecithin vesicles. When pH is raised to 5.5 there are no vesicles present and 20 nm monodisperse particles are found aggregating. Chain formation of fine nanoparticles (3-5 nm) is observed from neutral to basic pH, between 6.5-10.3 The chains formed are hundreds of nanometers to micrometer long and are usually 2-3 nanoparticles wide. On further increasing pH to 11.3, particles form disk-like or raft-like structures. When method (ii) was used a little chain formation was observed. Most of the nanoparticles formed were found either sitting together as raft like structures or scattered on lecithin structures. Copyright © 2013 Elsevier B.V. All rights reserved.

Massively parallel single-molecule and single-cell emulsion reverse transcription polymerase chain reaction using agarose droplet microfluidics.

PubMed

Zhang, Huifa; Jenkins, Gareth; Zou, Yuan; Zhu, Zhi; Yang, Chaoyong James

2012-04-17

A microfluidic device for performing single copy, emulsion Reverse Transcription Polymerase Chain Reaction (RT-PCR) within agarose droplets is presented. A two-aqueous-inlet emulsion droplet generator was designed and fabricated to produce highly uniform monodisperse picoliter agarose emulsion droplets with RT-PCR reagents in carrier oil. Template RNA or cells were delivered from one inlet with RT-PCR reagents/cell lysis buffer delivered separately from the other. Efficient RNA/cell encapsulation and RT-PCR at the single copy level was achieved in agarose-in-oil droplets, which, after amplification, can be solidified into agarose beads for further analysis. A simple and efficient method to graft primer to the polymer matrix using 5'-acrydite primer was developed to ensure highly efficient trapping of RT-PCR products in agarose. High-throughput single RNA molecule/cell RT-PCR was demonstrated in stochastically diluted solutions. Our results indicate that single-molecule RT-PCR can be efficiently carried out in agarose matrix. Single-cell RT-PCR was successfully performed which showed a clear difference in gene expression level of EpCAM, a cancer biomarker gene, at the single-cell level between different types of cancer cells. This work clearly demonstrates for the first time, single-copy RT-PCR in agarose droplets. We believe this will open up new possibilities for viral RNA detection and single-cell transcription analysis.

The Use of Monoclonal Antibodies in Human Prion Disease

NASA Astrophysics Data System (ADS)

Bodemer, Walter

Detection of PrP and its pathological isoform(s) is the key to understanding the etiology and pathogenesis of transmissible spongiform encephalopathy. There is ample evidence that PrP isoforms constitute a major component of an unknown and perhaps unconventional infectious agent. An etiological relationship between human and zoonotic transmissible spongiform encephalopathies may be revealed with monoclonal antibodies. Knowledge of the conformational transition rendering a nonpathogenic, almost ubiquitous cellular protein into a pathogenic one is crucial to defining pathomechanisms. The stepwise or even continuous formation of pathogenic molecules can be monitored. Any improvement in the early diagnosis could help to conceive new therapeutic measures which are not currently available. Determination of PrP isoforms in tissue, cells, or body fluids may be of prognostic value. Many experimental approaches in molecular medicine and molecular biology of the prion protein already rely on monoclonal antibodies. Recombinant antibodies such as the single-chain Fv may soon replace traditional hybridoma techniques. Binding affinity can easily be manipulated by a number of techniques, including in vitro mutagenesis - a step which could never be carried out using the traditional hybridoma technology. Monoclonal antibodies are and will remain an essential support for ongoing research on the prion protein in general and on the unconventional infectious prions.

Sweeter and stronger: enhancing sweetness and stability of the single chain monellin MNEI through molecular design

NASA Astrophysics Data System (ADS)

Leone, Serena; Pica, Andrea; Merlino, Antonello; Sannino, Filomena; Temussi, Piero Andrea; Picone, Delia

2016-09-01

Sweet proteins are a family of proteins with no structure or sequence homology, able to elicit a sweet sensation in humans through their interaction with the dimeric T1R2-T1R3 sweet receptor. In particular, monellin and its single chain derivative (MNEI) are among the sweetest proteins known to men. Starting from a careful analysis of the surface electrostatic potentials, we have designed new mutants of MNEI with enhanced sweetness. Then, we have included in the most promising variant the stabilising mutation E23Q, obtaining a construct with enhanced performances, which combines extreme sweetness to high, pH-independent, thermal stability. The resulting mutant, with a sweetness threshold of only 0.28 mg/L (25 nM) is the strongest sweetener known to date. All the new proteins have been produced and purified and the structures of the most powerful mutants have been solved by X-ray crystallography. Docking studies have then confirmed the rationale of their interaction with the human sweet receptor, hinting at a previously unpredicted role of plasticity in said interaction.

Analysis of B-cell epitopes from the allergen Hev b 6.02 revealed by using blocking antibodies.

PubMed

Pedraza-Escalona, Martha; Becerril-Luján, Baltazar; Agundis, Concepción; Domínguez-Ramírez, Lenin; Pereyra, Ali; Riaño-Umbarila, Lidia; Rodríguez-Romero, Adela

2009-02-01

Hev b 6.02 (hevein), identified as a major allergen from natural rubber latex (NRL), is involved in the latex-fruit syndrome and also acts as a pathogenesis defense-related protein. Its 3D structure has been solved at high resolution, and its linear epitopes have already been reported. However, information about conformational epitopes is still controversial, even though it is relevant for an accurate diagnosis and treatment, as well as for the study of allergen-antibody molecular interactions. We sought to analyze the B-cell epitopes of Hev b 6.02 at a molecular and structural level, using specific recombinant antibodies. We obtained a murine monoclonal antibody (mAb 6E7) and three human single chain fragments (scFvs A6, H8, and G7) anti-Hev b 6.02 that were able to compete for hevein binding with serum IgEs from latex allergic patients. In vitro assays showed that the mAb 6E7 and scFv H8 recognized the area of Hev b 6.02 where the aromatic residues are exposed; while the scFv G7 defined the amino and carboxy-terminal regions that lie close to each other, as a different epitope. The structural modeling of the Hev b 6.02-scFv H8 and Hev b 6.02-scFv G7 complexes revealed the putative regions of two conformational epitopes. In one of these, the aromatic residues, as well as polar side chains are important for the interaction, suggesting that they are part of a dominant conformational epitope also presented on the Hev b 6.02-IgE interactions. Antibodies recognizing this important allergen have potential to be used to diagnose and ultimately treat latex allergy.

Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose

PubMed Central

Abdolalizadeh, Jalal; Majidi Zolbanin, Jafar; Nouri, Mohammad; Baradaran, Behzad; Movassaghpour, AliAkbar; Farajnia, Safar; Omidi, Yadollah

2013-01-01

Purpose: Recombinant tumor necrosis factor-alpha (TNF-α) has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods:In this study, we examined the potential of our produced anti-TNF-α scFv fragments for purification of TNF-α produced by Raji cells. The Raji cells were induced by lipopolysaccharides (LPS) to express TNF-α. Western blotting and Fluorescence-activated cell sorting (FACS) flow cytometry analyses were used to evaluate the TNF-α expression. The anti-TNF-α scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-α and the purity of the protein was assessed by SDS-PAGE. Results: Western blot and FACS flow cytometry analyses showed the successful expression of TNF-α with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-α protein with purity over 95%. Conclusion: These findings confirm not only the potential of the produced scFv antibody fragments but also this highly pure recombinant TNF-α protein can be applied for various in vitro and in vivo applications. PMID:24312807

Numerical analysis of ossicular chain lesion of human ear

NASA Astrophysics Data System (ADS)

Liu, Yingxi; Li, Sheng; Sun, Xiuzhen

2009-04-01

Lesion of ossicular chain is a common ear disease impairing the sense of hearing. A comprehensive numerical model of human ear can provide better understanding of sound transmission. In this study, we propose a three-dimensional finite element model of human ear that incorporates the canal, tympanic membrane, ossicular bones, middle ear suspensory ligaments/muscles, middle ear cavity and inner ear fluid. Numerical analysis is conducted and employed to predict the effects of middle ear cavity, malleus handle defect, hypoplasia of the long process of incus, and stapedial crus defect on sound transmission. The present finite element model is shown to be reasonable in predicting the ossicular mechanics of human ear.

Efficient sampling of reversible cross-linking polymers: Self-assembly of single-chain polymeric nanoparticles

NASA Astrophysics Data System (ADS)

Oyarzún, Bernardo; Mognetti, Bortolo Matteo

2018-03-01

We present a new simulation technique to study systems of polymers functionalized by reactive sites that bind/unbind forming reversible linkages. Functionalized polymers feature self-assembly and responsive properties that are unmatched by the systems lacking selective interactions. The scales at which the functional properties of these materials emerge are difficult to model, especially in the reversible regime where such properties result from many binding/unbinding events. This difficulty is related to large entropic barriers associated with the formation of intra-molecular loops. In this work, we present a simulation scheme that sidesteps configurational costs by dedicated Monte Carlo moves capable of binding/unbinding reactive sites in a single step. Cross-linking reactions are implemented by trial moves that reconstruct chain sections attempting, at the same time, a dimerization reaction between pairs of reactive sites. The model is parametrized by the reaction equilibrium constant of the reactive species free in solution. This quantity can be obtained by means of experiments or atomistic/quantum simulations. We use the proposed methodology to study the self-assembly of single-chain polymeric nanoparticles, starting from flexible precursors carrying regularly or randomly distributed reactive sites. We focus on understanding differences in the morphology of chain nanoparticles when linkages are reversible as compared to the well-studied case of irreversible reactions. Intriguingly, we find that the size of regularly functionalized chains, in good solvent conditions, is non-monotonous as a function of the degree of functionalization. We clarify how this result follows from excluded volume interactions and is peculiar of reversible linkages and regular functionalizations.

Single-fiber myosin heavy chain polymorphism during postnatal development: modulation by hypothyroidism

NASA Technical Reports Server (NTRS)

di Maso, N. A.; Caiozzo, V. J.; Baldwin, K. M.

2000-01-01

The primary objective of this study was to follow the developmental time course of myosin heavy chain (MHC) isoform transitions in single fibers of the rodent plantaris muscle. Hypothyroidism was used in conjunction with single-fiber analyses to better describe a possible linkage between the neonatal and fast type IIB MHC isoforms during development. In contrast to the general concept that developmental MHC isoform transitions give rise to muscle fibers that express only a single MHC isoform, the single-fiber analyses revealed a very high degree of MHC polymorphism throughout postnatal development. In the adult state, MHC polymorphism was so pervasive that the rodent plantaris muscles contained approximately 12-15 different pools of fibers (i.e., fiber types). The degree of polymorphism observed at the single-fiber level made it difficult to determine specific developmental schemes analogous to those observed previously for the rodent soleus muscle. However, hypothyroidism was useful in that it confirmed a possible link between the developmental regulation of the neonatal and fast type IIB MHC isoforms.

Construction and Self-Assembly of Single-Chain Polymer Nanoparticles via Coordination Association and Electrostatic Repulsion in Water.

PubMed

Zhu, Zhengguang; Xu, Na; Yu, Qiuping; Guo, Lei; Cao, Hui; Lu, Xinhua; Cai, Yuanli

2015-08-01

Simultaneous coordination-association and electrostatic-repulsion interactions play critical roles in the construction and stabilization of enzymatic function metal centers in water media. These interactions are promising for construction and self-assembly of artificial aqueous polymer single-chain nanoparticles (SCNPs). Herein, the construction and self-assembly of dative-bonded aqueous SCNPs are reported via simultaneous coordination-association and electrostatic-repulsion interactions within single chains of histamine-based hydrophilic block copolymer. The electrostatic-repulsion interactions are tunable through adjusting the imidazolium/imidazole ratio in response to pH, and in situ Cu(II)-coordination leads to the intramolecular association and single-chain collapse in acidic water. SCNPs are stabilized by the electrostatic repulsion of dative-bonded block and steric shielding of nonionic water-soluble block, and have a huge specific surface area of function metal centers accessible to substrates in acidic water. Moreover, SCNPs can assemble into micelles, networks, and large particles programmably in response to the solution pH. These unique media-sensitive phase-transformation behaviors provide a general, facile, and versatile platform for the fabrication of enzyme-inspired smart aqueous catalysts. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Isolation and characterisation of Ebolavirus-specific recombinant antibody fragments from murine and shark immune libraries.

PubMed

Goodchild, Sarah A; Dooley, Helen; Schoepp, Randal J; Flajnik, Martin; Lonsdale, Stephen G

2011-09-01

Members of the genus Ebolavirus cause fulminating outbreaks of disease in human and non-human primate populations with a mortality rate up to 90%. To facilitate rapid detection of these pathogens in clinical and environmental samples, robust reagents capable of providing sensitive and specific detection are required. In this work recombinant antibody libraries were generated from murine (single chain variable domain fragment; scFv) and nurse shark, Ginglymostoma cirratum (IgNAR V) hosts immunised with Zaire ebolavirus. This provides the first recorded IgNAR V response against a particulate antigen in the nurse shark. Both murine scFv and shark IgNAR V libraries were panned by phage display technology to identify useful antibodies for the generation of immunological detection reagents. Two murine scFv were shown to have specificity to the Zaire ebolavirus viral matrix protein VP40. Two isolated IgNAR V were shown to bind to the viral nucleoprotein (NP) and to capture viable Zaire ebolavirus with a high degree of sensitivity. Assays developed with IgNAR V cross-reacted to Reston ebolavirus, Sudan ebolavirus and Bundibugyo ebolavirus. Despite this broad reactivity, neither of IgNAR V showed reactivity to Côte d'Ivoire ebolavirus. IgNAR V was substantially more resistant to irreversible thermal denaturation than murine scFv and monoclonal IgG in a comparative test. The demonstrable robustness of the IgNAR V domains may offer enhanced utility as immunological detection reagents in fieldable biosensor applications for use in tropical or subtropical countries where outbreaks of Ebolavirus haemorrhagic fever occur. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

One-Shot In Vitro Evolution Generated an Antibody Fragment for Testing Urinary Cotinine with More Than 40-Fold Enhanced Affinity.

PubMed

Oyama, Hiroyuki; Morita, Izumi; Kiguchi, Yuki; Banzono, Erika; Ishii, Kasumi; Kubo, Satoshi; Watanabe, Yoshiro; Hirai, Anna; Kaede, Chiaki; Ohta, Mitsuhiro; Kobayashi, Norihiro

2017-01-03

Immunoassays for cotinine, a major nicotine metabolite, in the urine are useful for monitoring the degree of tobacco smoke exposure. However, hybridoma-based anti-cotinine antibodies lack sufficient binding affinity to perform practically sensitive measurements, and thus most cotinine assays still rely on polyclonal antibodies. Here, we describe the generation of a mutant single-chain Fv fragment (scFv) that was used in an enzyme-linked immunosorbent assay (ELISA) to determine urinary cotinine levels in passive smokers. A "wild-type" scFv (scFv-wt) with a K a value of 2.7 × 10 7 M -1 (at 4 °C) was prepared by linking the V H and V L domains in a mouse anti-cotinine antibody. "One-shot" random mutagenesis on the scFv-wt gene by error-prone PCR generated mutant scFv genes, which were expressed on phage particles. Repeated panning directed toward mutants with slower off-rates selected scFv clones that showed improved sensitivity in an ELISA system. One of these mutants (scFv#m1-54) with five amino acid substitutions showed more than a 40-fold enhanced K a (1.2 × 10 9 M -1 at 4 °C) and, thus, was used to monitor human urinary cotinine. A limited amount of soluble scFv was reacted with urine specimens (or cotinine standards) at 4 °C for 120 min in microwells on which cotinine residues had been immobilized. The midpoint of the dose-response curves under optimized conditions (0.27 ng/assay) was more than 100-fold lower than the ELISA results obtained using scFv-wt. The limit of detection (8.4 pg/assay) corresponded to 0.17 ng/mL urinary cotinine, which was satisfactorily low for testing the threshold levels for passive smoke exposure. The assay values for volunteers correlated with the values determined using a commercial assay kit. This study evidently showed the potential of a molecular breeding approach, in which simple in vitro evolution might generate superior antibody reagents as cloned proteins, overcoming the limited molecular diversity inherent to

SPM for functional identification of individual biomolecules

NASA Astrophysics Data System (ADS)

Ros, Robert; Schwesinger, Falk; Padeste, Celestino; Plueckthun, Andreas; Anselmetti, Dario; Guentherodt, Hans-Joachim; Tiefenauer, Louis

1999-06-01

The identification of specific binding molecules is of increasing interest in the context of drug development based on combinatorial libraries. Scanning Probe Microscopy (SPM) is the method of choice to image and probe individual biomolecules on a surface. Functional identification of biomolecules is a first step towards screening on a single molecule level. As a model system we use recombinant single- chain Fv fragment (scFv) antibody molecules directed against the antigen fluorescein. The scFv's are covalently immobilized on a flat gold surface via the C-terminal cysteine, resulting in a high accessibility of the binding site. The antigen is immobilized covalently via a long hydrophilic spacer to the silicon nitride SPM-tip. This arrangement allows a direct measurement of binding forces. Thus, closely related antibody molecules differing in only one amino acid at their binding site could be distinguished. A novel SPM-software has been developed which combines imaging, force spectroscopic modes, and online analysis. This is a major prerequisite for future screening methods.

Internalization of exogenous cystatin F supresses cysteine proteases and induces the accumulation of single-chain cathepsin L by multiple mechanisms.

PubMed

Colbert, Jeff D; Matthews, Stephen P; Kos, Janko; Watts, Colin

2011-12-09

Cystatin F is an unusual member of the cystatin family of protease inhibitors, which is made as an inactive dimer and becomes activated by proteolysis in the endo/lysosome pathway of the immune cells that produce it. However a proportion is secreted and can be taken up and activated by other cells. We show here that cystatin F acquired in this way induces a dramatic accumulation of the single-chain form of cathepsin L (CatL). Cystatin F was observed in the same cellular compartments as CatL and was tightly complexed with CatL as determined by co-precipitation studies. The observed accumulation of single-chain CatL was partly due to cystatin F-mediated inhibition of the putative single-chain to two-chain CatL convertase AEP/legumain and partly to general suppression of cathepsin activity. Thus, cystatin F stabilizes CatL leading to the dramatic accumulation of an inactive complex composed either of the single-chain or two-chain form depending on the capacity of cystatin F to inhibit AEP. Cross-transfer of cystatin F from one cell to another may therefore attenuate potentially harmful effects of excessive CatL activity while paradoxically, inducing accumulation of CatL protein. Finally, we confirmed earlier data (Beers, C., Honey, K., Fink, S., Forbush, K., and Rudensky, A. (2003) J. Exp. Med. 197, 169-179) showing a loss of CatL activity, but not of CatL protein, in macrophages activated with IFNγ. However, we found equivalent loss of CatL activity in wild type and cystatin F-null macrophages suggesting that an inhibitory activity other than cystatin F quenches CatL activity in activated macrophages.

Mechanical desorption of a single chain: unusual aspects of phase coexistence at a first-order transition.

PubMed

Skvortsov, Alexander M; Klushin, Leonid I; Polotsky, Alexey A; Binder, Kurt

2012-03-01

The phase transition occurring when a single polymer chain adsorbed at a planar solid surface is mechanically desorbed is analyzed in two statistical ensembles. In the force ensemble, a constant force applied to the nongrafted end of the chain (that is grafted at its other end) is used as a given external control variable. In the z-ensemble, the displacement z of this nongrafted end from the surface is taken as the externally controlled variable. Basic thermodynamic parameters, such as the adsorption energy, exhibit a very different behavior as a function of these control parameters. In the thermodynamic limit of infinite chain length the desorption transition with the force as a control parameter clearly is discontinuous, while in the z-ensemble continuous variations are found. However, one should not be misled by a too-naive application of the Ehrenfest criterion to consider the transition as a continuous transition: rather, one traverses a two-phase coexistence region, where part of the chain is still adsorbed and the other part desorbed and stretched. Similarities with and differences from two-phase coexistence at vapor-liquid transitions are pointed out. The rounding of the singularities due to finite chain length is illustrated by exact calculations for the nonreversal random walk model on the simple cubic lattice. A new concept of local order parameter profiles for the description of the mechanical desorption of adsorbed polymers is suggested. This concept give evidence for both the existence of two-phase coexistence within single polymer chains for this transition and the anomalous character of this two-phase coexistence. Consequences for the proper interpretation of experiments performed in different ensembles are briefly mentioned.

Dynamics of Cancer Cell near Collagen Fiber Chain

NASA Astrophysics Data System (ADS)

Kim, Jihan; Sun, Bo

Cell migration is an integrated process that is important in life. Migration is essential for embryonic development as well as homeostatic processes such as wound healing and immune responses. When cell migrates through connective extracellular matrix (ECM), it applies cellular traction force to ECM and senses the rigidity of their local environment. We used human breast cancer cell (MDA-MB-231) which is highly invasive and applies strong traction force to ECM. As cancer cell applies traction force to type I collage-based ECM, it deforms collagen fibers near the surface. Patterns of deforming collagen fibers are significantly different with pairs of cancer cells compared to a single cancer cell. While a pair of cancer cells within 60 um creates aligned collagen fiber chains between them permanently, a single cancer cell does not form any fiber chains. In this experiment we measured a cellular response and an interaction between a pair of cells through the chain. Finally, we analyzed correlation of directions between cancer cell migration and the collagen chain alignment.

A fusion-protein approach enabling mammalian cell production of tumor targeting protein domains for therapeutic development.

PubMed

Hu, Jia; Chen, Xiang; Zhang, Xuhua; Yuan, Xiaopeng; Yang, Mingjuan; Dai, Hui; Yang, Wei; Zhou, Qinghua; Wen, Weihong; Wang, Qirui; Qin, Weijun; Zhao, Aizhi

2018-05-01

A single chain Fv fragment (scFv) is a fusion of the variable regions of heavy (V H ) and light (V L ) chains of immunoglobulins. They are important elements of chimeric antigen receptors for cancer therapy. We sought to produce a panel of 16 extracellular protein domains of tumor markers for use in scFv yeast library screenings. A series of vectors comprising various combinations of expression elements was made, but expression was unpredictable and more than half of the protein domains could not be produced using any of the constructs. Here we describe a novel fusion expression system based on mouse TEM7 (tumor endothelial marker 7), which could facilitate protein expression. With this approach we could produce all but one of the tumor marker domains that could not otherwise be expressed. In addition, we demonstrated that the tumor associated antigen hFZD10 produced as a fusion protein with mTEM7 could be used to enrich scFv antibodies from a yeast display library. Collectively our study demonstrates the potential of specific fusion proteins based on mTEM7 in enabling mammalian cell production of tumor targeting protein domains for therapeutic development. © 2018 The Protein Society.

First-order reversal curves of single domain particles: diluted random assemblages and chains

NASA Astrophysics Data System (ADS)

Egli, R.

2009-04-01

Exact magnetic models can be used to calculate first-order reversal curves (FORC) of single domain (SD) particle assemblages, as shown by Newell [2005] for the case of isolated Stoner-Wohlfarth particles. After overcoming experimental difficulties, a FORC diagram sharing many similarities to Newell's model has been measured on a lake sediment sample (see A.P. Chen et al., "Quantification of magnetofossils using first-order reversal curves", EGU General Assembly 2009, Abstracts Vol. 11, EGU2009-10719). This sample contains abundant magnetofossils, as shown by coercivity analysis and electron microscopy, therefore suggesting that well dispersed, intact magnetosome chains are the main SD carriers. Subtle differences between the reversible and the irreversible contributions of the measured FORC distribution suggest that magnetosome chains might not be correctly described by the Stoner-Wohlfarth model. To better understand the hysteresis properties of such chains, a simple magnetic model has been implemented, taking dipole-dipole interactions between particles within the same chain into account. The model results depend on the magnetosome elongation, the number of magnetosomes in a chain, and the gap between them. If the chain axis is subparallel to the applied field, the magnetic moment reverses by a pseudo-fanning mode, which is replaced by a pseudo-coherent rotation mode at greater angles. These reversal modes are intrinsically different from coherent rotation assumed Stoner-Wohlfarth model, resulting in FORC diagrams with a smaller reversible component. On the other hand, isolated authigenic SD particles can precipitate in the sediment matrix, as it might occur for pedogenic magnetite. In this case, an assembly of randomly located particles provides a possible model for the resulting FORC diagram. If the concentration of the particles is small, each particle is affected by a random interaction field whose statistical distribution can be calculated from first