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Pasman, Yfke, Eva Nagy, and Azad K. Kaushik. "Enhanced Bovine Herpesvirus Type 1 Neutralization by Multimerized Single-Chain Variable Antibody Fragments Regardless of Differential Glycosylation." Clinical and Vaccine Immunology 19, no. 8 (June 13, 2012): 1150–57. http://dx.doi.org/10.1128/cvi.00130-12.
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ABSTRACTSingle-chain variable antibody fragments (scFvs) with a 2-amino-acid linker capable of multimerization as di-, tri-, or tetrabodies that neutralize bovine herpesvirus type 1 (BoHV-1)in vitrowere constructed and expressed inPichia pastoris. In contrast to the monomeric form, multimeric scFvs had a higher virus neutralization potency, as evidenced by a 2-fold increase in their ability to neutralize BoHV-1 due to avidity effects. Mass spectrum (quadrupole time of flight [Q-TOF]) analyses of multimeric scFv demonstrated extensive heterogeneity due to differential cleavage, variable glycosylation (1 to 9 mannose residues), and the incorporation of minor unidentified adducts. Regardless of the differential glycosylation patterns, the scFvs recognized non-gB or -gE target viral epitopes in the BoHV-1 envelope fraction in a Western blot and also neutralized BoHV-1 in infected Madin-Darby kidney (MDBK) cellsin vitro. Indirect evidence for the noncovalent multimerization of scFv was the presence of a major peak of multimerized scFv without a His tag (due to differential cleavage) in the Q-TOF profile, unlike monomeric scFv, which copurified with normally His-tagged scFv and recognized the target antigen. Overall, differentially glycosylated recombinant scFvs against BoHV-1 with a short linker (2 amino acids) are capable of assembly into functional multimers that confer high avidity, resulting in increased virus neutralizationin vitrocompared to that of monovalent scFv with a long (18-amino-acid) flexible linker. Overall, recombinant multimerized scFv5-2L potentially provides a high-potency therapeutic and immunodiagnostic reagent against BoHV-1, which is suitable for passive immunization and topical application.
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Škerlová, Jana, Vlastimil Král, Milan Fábry, Juraj Sedláček, Václav Veverka, and Pavlína Řezáčová. "Optimization of the crystallizability of a single-chain antibody fragment." Acta Crystallographica Section F Structural Biology Communications 70, no. 12 (November 14, 2014): 1701–6. http://dx.doi.org/10.1107/s2053230x1402247x.
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Single-chain variable antibody fragments (scFvs) are molecules with immense therapeutic and diagnostic potential. Knowledge of their three-dimensional structure is important for understanding their antigen-binding mode as well as for protein-engineering approaches such as antibody humanization. A major obstacle to the crystallization of single-chain variable antibody fragments is their relatively poor homogeneity caused by spontaneous oligomerization. A new approach to optimization of the crystallizability of single-chain variable antibody fragments is demonstrated using a representative single-chain variable fragment derived from the anti-CD3 antibody MEM-57. A Thermofluor-based assay was utilized to screen for optimal conditions for antibody-fragment stability and homogeneity. Such an optimization of the protein storage buffer led to a significantly improved ability of the scFv MEM-57 to yield crystals.
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Gallo, Eugenio, Sophia Wienbar, Avin C. Snyder, Kalin V. Vasilev, Bruce A. Armitage, and Jonathan W. Jarvik. "A Single-Chain-Variable-Fragment Fluorescence Biosensor Activates Fluorogens from Dissimilar Chemical Families." Protein & Peptide Letters 21, no. 12 (November 5, 2014): 1289–94. http://dx.doi.org/10.2174/0929866521666140616121800.
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Current advancements in biological protein discovery utilize bi-partite methods of fluorescence detection where chromophore and scaffold are uncoupled. One such technology, called fluorogen-activating proteins (FAPs), consists of single-chain-variable-fragments (scFvs) selected against small organic molecules (fluorogens) that are non-fluorescent in solution, but highly fluorescent when bound to the scFv. In unusual circumstances a scFv may activate similar fluorogens from a single chemical family. In this report we identified a scFv biosensor with fluorescence activity against multiple fluorogens from two structurally dissimilar families. In-vitro analysis revealed highly selective scFv-ligand interactions at sub-micromolar ranges. Additionally, each scFv-fluorogen complex possesses unique excitation and emission spectra, which allows broader detection limits from the biosensor. Further analysis indicated that ligand activation, regardless of chemical family, occurs at a common scFv binding region that proves flexible, yet selective for fluorogen binding. As a protein reporter at the surface of mammalian cells, the scFv revealed bright signal detection and minimal background. Additionally, when tagged to a G-protein-coupled receptor, we observed agonist dependent signaling leading to protein traffic from cell surface to endosomes via multi-color fluorescence tracking. In summary, this report unveils a noncanonical scFv biosensor with properties of high ligand affinity and multi-channel fluorescence detection, which consequently offers expanded opportunities for cellular protein discovery.
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Kubelkova, Klara, and Ales Macela. "Development of tularemic scFv antibody fragments using phage display." Open Life Sciences 5, no. 3 (June 1, 2010): 310–17. http://dx.doi.org/10.2478/s11535-010-0015-3.
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AbstractPolyclonal antibodies, as well as monoclonal antibodies are efficacious in providing protective immunity against Francisella tularensis. This study demonstrates the application of phage display libraries for the construction of monoclonal antibodies against F. tularensis. Novel single-chain fragment variable (scFv) antibodies were generated against a whole bacterial lysate of F. tularensis live vaccine strain using the human single fold scFv libraries I (Tomlinson I + J). A total of 20 clones reacted with the bacterial cell lysate. Further, the library contains two clones responsive to recombinant lipoprotein FTT1103Δsignal (F. tularensis subsp. tularensis Schu S4), which was constructed without a signal sequence. These positively-binding scFvs were evaluated by scFv-phage enzyme-linked immunosorbent assay (ELISA). Then, positive scFvs were expressed in a soluble form in Escherichia coli HB2151 and tested for positive scFvs by using scFv-ELISA.
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Brettschneider, Kerstin, Anja Naumann, Sonja Neimanis, Joerg Kahle, Christine Heller, Thomas Klingebiel, Dirk Schwabe, and Christoph Koenigs. "Functional Analysis of Phage Display Selected Single-Chain Variable Antibody Fragments (scFvs) Specific for Anti-FVIII Antibodies." Blood 124, no. 21 (December 6, 2014): 1499. http://dx.doi.org/10.1182/blood.v124.21.1499.1499.
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Abstract The development of inhibitory antibodies against coagulation factor VIII (FVIII) is currently the most serious complication for hemophilia A patients that undergo FVIII replacement therapy. In addition, non-hemophilia A patients can spontaneously develop inhibitory auto-antibodies to FVIII, which results in acquired haemophilia A. The control of the allo- or autoimmune response to FVIII apparently includes the elicitation of anti-idiotypic antibodies. In this study the capacity of anti-idiotypic single-chain variable antibody fragments (scFvs) for neutralization of inhibitory anti-FVIII antibodies (FVIII inhibitors) was evaluated in vitro and in vivo. Anti-idiotypic scFvs were selected from phage-displayed libraries against murine monoclonal FVIII-specific inhibitors. As the majority of inhibitory antibodies is directed against the A2 or C2 domain of FVIII, strongly inhibitory A2- and C2-specific antibodies served as targets. Selected scFvs were expressed as scFv-Fc fusion proteins. Analysis of the scFv-Fcs by ELISA confirmed the specific binding to the cognate targets and binding studies via surface plasmon resonance revealed high affinities within the nanomolar range. Further characterization showed that binding of inhibitors to immobilized FVIII was blocked by specific scFv-Fcs in vitro. The ability of scFv-Fcs to neutralize their corresponding inhibitors was analyzed in a functional clotting assay. By adding scFv-Fcs to plasma spiked with inhibitors, FVIII activity was restored to 80% in a concentration dependent manner. FVIII knockout mice served as model organism for testing the capacity of scFv-Fcs to restore coagulation in vivo. Subsequent injection of FVIII following the injection of the inhibitors resulted in a largely reduced FVIII activity. However, FVIII activity was recovered in a concentration dependent manner by adding cognate anti-idiotypes. The scFv-Fcs were either preincubated with the corresponding inhibitor or added to the FVIII mixture without preincubation. The latter represents an adaption to a therapeutic setting. In conclusion, phage display selected anti-idiotypic scFvs are able to bind and effectively neutralize their target inhibitors in vitro and in vivo. Based on these promising results the potential of anti-idiotypic scFvs for the development of specific cell based immunotherapies for hemophilia A patients with inhibitors is currently under investigation. Disclosures No relevant conflicts of interest to declare.
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Naumann, A., A. K. Scherger, J. Neuwirth, A. Orlowski, J. Kahle, D. Schwabe, and C. Königs. "Selection and characterisation of FVIII-specific single chain variable fragments." Hämostaseologie 33, S 01 (2013): S39—S45. http://dx.doi.org/10.1055/s-0037-1619801.
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SummaryThe development of inhibitory anti-FVIII antibodies is currently the most severe complication in the treatment of haemophilia A patients. Inhibitor eradication can be achieved by immune tolerance induction (ITI). Recent findings suggest a correlation between the FVIII-specific IgG subclass distribution and the duration or outcome of ITI. To quantify FVIII-specific IgG subclasses in patients’ plasma FVIII-specific IgG standards are required. Here, the isolation of FVIII-specific single chain variable fragments (scFvs) from synthetic phage display libraries and the characterisation of their FVIII domain specificity are described. The isolated scFv 1G10, which binds to the FVIII A2 domain, was cloned into the context of the four human IgG (hIgG) subclasses and expressed in mammalian cells. Purified 1G10-hIgG1, -hIgG2, -hIgG3 and -hIgG4 are used as standards to determine the absolute amounts and relative contribution of the different FVIII-specific IgG subclasses in future studies. The results from these studies will eventually add to understanding the role of the FVIII-specific IgG subclass distribution as prognostic factor for the outcome of ITI.
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Pasman, Yfke, Eva Nagy, and Azad Kaushik. "Construction of multimerized scFv that neutralize Bovine Herpes Virus-1 (52.12)." Journal of Immunology 184, no. 1_Supplement (April 1, 2010): 52.12. http://dx.doi.org/10.4049/jimmunol.184.supp.52.12.
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Abstract Our laboratory earlier constructed functional antibody fragments, scFv (single chain fragment variable), with an 18 amino acid linker, against bovine herpes virus-1 (BoHV-1) (Koti et al. 2010. Molec. Immunol. in press). With an objective to enhance scFv avidity, the scFv in Vl-linker-VH configuration was constructed with a short flexible amino acid linker. An overlap PCR product comprising Vl-linker-VH was cloned into pPICZα vector and expressed in Pichia pastoris, under the influence of inducible AOX1 promoter. Affinity purified scFvs were analyzed by Western blot and mass spectrometry that confirmed the expected molecular weight of the scFv. Further, the multimerized scFvs, both purified and unpurified, neutralized BoHV-1 infection of MDBK cells in vitro. Taken together with previous studies, the scFvs with varying linker size are capable of effectively neutralizing BoHV-1. The scFv against BoHV-1 will provide protection at respriratory mucosal surfaces and prevent infection. In conclusion, scFvs both in multimerized and non-multimerized form provide a useful diagnostic and therapeutic agent against an important cattle pathogen. (Supported by NSERC Canada)
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Susi, Petri, Angelika Ziegler, and Lesley Torrance. "Selection of Single-Chain Variable Fragment Antibodies to Black Currant Reversion Associated Virus from a Synthetic Phage Display Library." Phytopathology® 88, no. 3 (March 1998): 230–33. http://dx.doi.org/10.1094/phyto.1998.88.3.230.
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Single-chain variable fragment (scFv) antibodies that bind to black currant reversion associated virus (BRAV) were obtained from a synthetic phage display antibody gene library without recourse to animal immunizations. Several different BRAV-specific phage scFv were obtained quickly, after only three rounds of selection against immobilized virus antigen. The phage scFv gave enzyme-linked immunosorbent assay (ELISA) absorbance values that were greater than seven times the control healthy plant extracts. In contrast, comparative tests using a rabbit antiserum failed, because unacceptably high background values were obtained with healthy plant extracts. Two of the scFv were subcloned into the pDAP2 vector for the rapid and efficient production of scFv-alkaline phosphatase fusion proteins. Functional fusion proteins were obtained after expression in Escherichia coli, and preparations from periplasmic extracts detected BRAV in ELISA. The results demonstrate that antibody fragments obtained from a synthetic phage display library are useful research tools, and they proved to be a viable practical alternative when traditional antisera failed to detect BRAV, a weak immunogen. Furthermore, the genetic fusion of antibody fragments to alkaline phosphatase obviates the need for further chemical coupling procedures, and the fusion proteins can be obtained cheaply.
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di Tommaso, Anne, Matthieu O. Juste, Zineb Lakhrif, Marie-Noëlle Mévélec, Coraline Borowczyk, Pierre Hammeni, Guillaume Désoubeaux, et al. "Engineering and Functional Evaluation of Neutralizing Antibody Fragments Against Congenital Toxoplasmosis." Journal of Infectious Diseases 224, no. 4 (August 7, 2021): 705–14. http://dx.doi.org/10.1093/infdis/jiab141.
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Abstract Maternal-fetal transmission of Toxoplasma gondii tachyzoites acquired during pregnancy has potentially dramatic consequences for the fetus. Current reference-standard treatments are not specific to the parasite and can induce severe side effects. In order to provide treatments with a higher specificity against toxoplasmosis, we developed antibody fragments—single-chain fragment variable (scFv) and scFv fused with mouse immunoglobulin G2a crystallizable fragment (scFv-Fc)—directed against the major surface protein SAG1. After validating their capacity to inhibit T. gondii proliferation in vitro, the antibody fragments’ biological activity was assessed in vivo using a congenital toxoplasmosis mouse model. Dams were treated by systemic administration of antibody fragments and with prevention of maternal-fetal transmission being used as the parameter of efficacy. We observed that both antibody fragments prevented T. gondii dissemination and protected neonates, with the scFv-Fc format having better efficacy. These data provide a proof of concept for the use of antibody fragments as effective and specific treatment against congenital toxoplasmosis and provide promising leads.
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Westlund, Karin N., Marena A. Montera, Aleyah E. Goins, Sascha R. A. Alles, Nikita Suri, Sabrina L. McIlwrath, Robyn Bartel, Ravi V. Durvasula, and Adinarayana Kunamneni. "Single-Dose P2 X4R Single-Chain Fragment Variable Antibody Permanently Reverses Chronic Pain in Male Mice." International Journal of Molecular Sciences 22, no. 24 (December 19, 2021): 13612. http://dx.doi.org/10.3390/ijms222413612.
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Non-opioid single-chain variable fragment (scFv) small antibodies were generated as pain-reducing block of P2X4R receptor (P2X4R). A panel of scFvs targeting an extracellular peptide sequence of P2X4R was generated followed by cell-free ribosome display for recombinant antibody selection. After three rounds of bio-panning, a panel of recombinant antibodies was isolated and characterized by ELISA, cross-reactivity analysis, and immunoblotting/immunostaining. Generated scFv antibodies feature binding activity similar to monoclonal antibodies but with stronger affinity and increased tissue penetrability due to their ~30% smaller size. Two anti-P2X4R scFv clones (95, 12) with high specificity and affinity binding were selected for in vivo testing in male and female mice with trigeminal nerve chronic neuropathic pain (FRICT-ION model) persisting for several months in untreated BALBc mice. A single dose of P2X4R scFv (4 mg/kg, i.p.) successfully, completely, and permanently reversed chronic neuropathic pain-like measures in male mice only, providing retention of baseline behaviors indefinitely. Untreated mice retained hypersensitivity, and developed anxiety- and depression-like behaviors within 5 weeks. In vitro P2X4R scFv 95 treatment significantly increased the rheobase of larger-diameter (>25 µm) trigeminal ganglia (TG) neurons from FRICT-ION mice compared to controls. The data support use of engineered scFv antibodies as non-opioid biotherapeutic interventions for chronic pain.
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Pasman, Yfke, and Azad Kaushik. "Elucidating the role of bovine heavy- and light-chain variable domains in polyspecific antigen-binding (P6104)." Journal of Immunology 190, no. 1_Supplement (May 1, 2013): 141.15. http://dx.doi.org/10.4049/jimmunol.190.supp.141.15.
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Abstract The bovine antibody repertoire is generated from a limited germline sequence divergence as well as combinatorial diversity. Generation of an exceptionally long CDR3H (up to 61 amino acids) in the heavy chain variable region (VH) provides an additional mechanism to generate antibody diversity, not found in other species to date. These long CDR3H loops, present in polyspecific IgM, originate from VDJ recombination encoded by a specific VH gene, unusually long single DH-gene (capable of encoding up to 51 codons) and insertion of 15-18 base long conserved short nucleotide sequences (CSNS) specifically at VH-DH junction. In cattle, it is thought that antigen-binding is mainly a function of the variable-region of the heavy chain where light chain provides only structural support. To test this hypothesis, single chain variable fragment (scFv) and single domains (Fd) from polyspecific IgM were constructed. Both purified scFv as well as FdVH showed polyspecific binding to structurally dissimilar antigens in an ELISA. Whether scFv and FdVH bind to multiple epitopes on an antigen or if they recognize the same epitope can now be determined. The structural-functional complexities of these antibody fragments and the role of the heavy- and light-chains in antigen-binding will be discussed. [Supported by NSERC Canada]
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Riaño-Umbarila, Lidia, José Alberto Romero-Moreno, Luis M. Ledezma-Candanoza, Timoteo Olamendi-Portugal, Lourival D. Possani, and Baltazar Becerril. "Full Neutralization of Centruroides sculpturatus Scorpion Venom by Combining Two Human Antibody Fragments." Toxins 13, no. 10 (October 6, 2021): 708. http://dx.doi.org/10.3390/toxins13100708.
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A fundamental issue of the characterization of single-chain variable fragments (scFvs), capable of neutralizing scorpion toxins, is their cross-neutralizing ability. This aspect is very important in Mexico because all scorpions dangerous to humans belong to the Centruroides genus, where toxin sequences show high identity. Among toxin-neutralizing antibodies that were generated in a previous study, scFv 10FG2 showed a broad cross-reactivity against several Centruroides toxins, while the one of scFv LR is more limited. Both neutralizing scFvs recognize independent epitopes of the toxins. In the present work, the neutralization capacity of these two scFvs against two medically important toxins of the venom of Centruroides sculpturatus Ewing was evaluated. The results showed that these toxins are recognized by both scFvs with affinities between 1.8 × 10−9 and 6.1 × 10−11 M. For this reason, their ability to neutralize the venom was evaluated in mice, where scFv 10FG2 showed a better protective capacity. A combination of both scFvs at a molar ratio of 1:5:5 (toxins: scFv 10FG2: scFv LR) neutralized the venom without the appearance of any signs of intoxication. These results indicate a complementary activity of these two scFvs during venom neutralization.
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Singh, Pawan Kumar, Ranu Agrawal, Dev Vrat Kamboj, Garima Gupta, M. Boopathi, Ajay Kumar Goel, and Lokendra Singh. "Construction of a Single-Chain Variable-Fragment Antibody against the Superantigen Staphylococcal Enterotoxin B." Applied and Environmental Microbiology 76, no. 24 (October 15, 2010): 8184–91. http://dx.doi.org/10.1128/aem.01441-10.
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ABSTRACT Staphylococcal food poisoning (SFP) is one of the most prevalent causes of food-borne illness throughout the world. SFP is caused by 21 different types of staphylococcal enterotoxins produced by Staphylococcus aureus. Among these, staphylococcal enterotoxin B (SEB) is the most potent toxin and is a listed biological warfare (BW) agent. Therefore, development of immunological reagents for detection of SEB is of the utmost importance. High-affinity and specific monoclonal antibodies are being used for detection of SEB, but hybridoma clones tend to lose their antibody-secreting ability over time. This problem can be overcome by the use of recombinant antibodies produced in a bacterial system. In the present investigation, genes from a hybridoma clone encoding monoclonal antibody against SEB were immortalized using antibody phage display technology. A murine phage display library containing single-chain variable-fragment (ScFv) antibody genes was constructed in a pCANTAB 5E phagemid vector. Phage particles displaying ScFv were rescued by reinfection of helper phage followed by four rounds of biopanning for selection of SEB binding ScFv antibody fragments by using phage enzyme-linked immunosorbent assay (ELISA). Soluble SEB-ScFv antibodies were characterized from one of the clones showing high affinity for SEB. The anti-SEB ScFv antibody was highly specific, and its affinity constant was 3.16 nM as determined by surface plasmon resonance (SPR). These results demonstrate that the recombinant antibody constructed by immortalizing the antibody genes from a hybridoma clone is useful for immunodetection of SEB.
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Monnier, Philippe, Robin Vigouroux, and Nardos Tassew. "In Vivo Applications of Single Chain Fv (Variable Domain) (scFv) Fragments." Antibodies 2, no. 4 (April 11, 2013): 193–208. http://dx.doi.org/10.3390/antib2020193.
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Develter, Jan, Maarten Dewilde, Ann Gils, and Paul J. Declerck. "Comparative study of inhibitory antibody derivatives towards thrombin activatable fibrinolysis inhibitor." Thrombosis and Haemostasis 102, no. 07 (2009): 69–75. http://dx.doi.org/10.1160/th08-09-0834.
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SummaryThrombin activatable fibrinolysis inhibitor (TAFI) attenuates fibrinolysis and is considered as an attractive drug target. We generated two different antibody fragments, an antigen-binding fragment (Fab) and a single-chain variable fragment (scFv), derived from three distinct monoclonal antibodies (MAs) that inhibit the activation of TAFI by the thrombin/thrombomodulin complex (T/TM) and plasmin (MA-T1C10 and MA-T94H3) or by T/TM alone (MA-T12D11). The Fabs were obtained by papain digestion of the purified Mas, whereas the scFvs were cloned and subsequently expressed in bacteria. All antibody fragments revealed similar or slightly decreased affinities compared to those of the respective Mas, except scFv-T94H3. In the presence of a 16-fold molar excess of all antibody fragments, activation of TAFI by T/TM was completely blocked. Furthermore, Fab and scFv-derivatives from MA-T1C10 and MA-T94H3 were capable of interfering with the plasmin-mediated activation of TAFI. Addition of 850 nM of MA, Fab or scFv to an in-vitro clot lysis assay caused a significant reduction of clot lysis time (except for scFv-T94H3) and this effect was comparable to that of potato tuber carboxypeptidase inhibitor, a well-known TAFIa inhibitor. Dose-response experiments with the antibody (derivatives) in clot lysis and chromogenic assay revealed that the inhibitory capacity of the Fabs was comparable to that of the Mas, whereas the scFvs had a more reduced potency. In conclusion, these highly specific TAFI inhibitors are interesting tools to further evaluate the concept of TAFI inhibition in various in-vitro and in-vivo models.
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Liu, Mei-Yun, Wei Han, Yan-Li Ding, Tian-Hong Zhou, Rui-Yang Tian, Sheng-Li Yang, Hui Liu, and Yi Gong. "Generation and Characterization of C305, a Murine Neutralizing scFv Antibody That Can Inhibit BLyS Binding to Its Receptor BCMA." Acta Biochimica et Biophysica Sinica 37, no. 6 (June 1, 2005): 415–20. http://dx.doi.org/10.1111/j.1745-7270.2005.00059.x.
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Abstract B-lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor (TNF) family and a key regulator of B cell response. Neutralizing single-chain fragment variable (scFv) antibody against BLyS binding to its receptor BCMA has the potential to play a prominent role in autoimmune disease therapy. A phage display scFv library constructed on pIII protein of M13 filamentous phage was screened using BLyS. After five rounds of panning, their binding activity was characterized by phage-ELISA. Nucleotide sequencing revealed that at least two different scFv gene fragments (C305 and D416) were obtained. The two different scFv gene fragments were expressed to obtain the soluble scFv antibodies, then the soluble scFv antibodies were characterized by means of competitive ELISA and in vitro neutralization assay. The results indicated that C305 is the neutralizing scFv antibody that can inhibit BLyS binding to its receptor BCMA.
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Seo, Haneul, Andre Ditya Maulana Lubis, Tae-Jin Choi, Tae-Sung Jung, Taek-Kyun Lee, and Sukchan Lee. "Development of an Immunoassay Detection System for Koi Herpesvirus Using Recombinant Single-Chain Variable Fragments." Fishes 7, no. 6 (December 2, 2022): 370. http://dx.doi.org/10.3390/fishes7060370.
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Koi herpesvirus (KHV) is a highly contagious virus that causes high mortality in koi and common carp, leading to a reduction in production worldwide. Recent diagnostic tests based on molecular methods alone (nucleic acid amplification) and indirect immunoassay methods (antibody detection) can be confirmed over KHV infections or prior exposure and latent infections. Unfortunately, there is no established method to detect KHV virus particles, especially when virus titers are low. Therefore, we propose an alternative, direct immunoassay method for viral detection using a single-chain variable fragment (scFv), a specific region of IgG antibodies that binds specifically to KHV particles. The results of functional analyses indicated that four putative scFv candidates, C5, F8, F6, and E4, were specific to KHV, but only F6 and C5 had a high binding affinity. The binding characteristics were confirmed by indirect competitive and sandwich enzyme-linked immunosorbent assays, which indicated that F6 and C5 have a broad penetration area to the binding region and share a similar epitope with commercial KHV monoclonal antibodies. These characteristics were further confirmed by their interactions with purified KHV coat protein by indirect ELISA and Western blot analyses. In conclusion, the F6 and C5 scFvs have adequate binding affinity to KHV particles to permit their use in immunoassays.
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ZHOU, HUI-REN, JAMES J. PESTKA, and L. PATRICK HART. "Molecular Cloning and Expression of Recombinant Phage Antibody against Fumonisin B1." Journal of Food Protection 59, no. 11 (November 1, 1996): 1208–12. http://dx.doi.org/10.4315/0362-028x-59.11.1208.
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Bacteriophage expression systems offer promise for the development of novel antibody reagents applicable to detection of microbial agents and their toxins in foods. In this study, fumonisin B1 (FB1)-specific antibodies, composed of a single chain containing a variable heavy (VH) and light (VL) chain fragments (ScFv), were cloned using mRNA from either spleen cells of mice immunized with FB1-BSA conjugate or from an existing hybridoma cell line that produces anti-FB1 antibody. The approach consisted of (i) reverse transcription of isolated mRNA, (ii) polymerase chain reaction amplification of VH and VL cDNAs, (iii) ligation of VL and VH chains, and (iv) expression of ScFv proteins on the surface of a filamentous bacteriophage or as a soluble fragment. The efficacy of using the recombinant ScFv proteins for the detection of FB1 were evaluated in a competitive indirect enzyme-linked immunosorbent assay (CI-ELISA). Compared to polyclonal or monoclonal antibodies for FB1, the recombinant ScFv proteins were 10 to 100 times less sensitive in the CI-ELISA. The secreted ScFv protein did not cross-react with FB2 or FB3. The results indicated that production of a recombinant antibody to a mycotoxin is feasible. However, problems associated with the affinity or avidity of ScFv fragments need to be further addressed before this technology is adaptable to the development of improved immunodiagnostic kits for mycotoxins or other food borne disease hazards.
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Marín-Argany, Marta, Geovanny Rivera-Hernández, Joaquim Martí, and Sandra Villegas. "An anti-Aβ (amyloid β) single-chain variable fragment prevents amyloid fibril formation and cytotoxicity by withdrawing Aβ oligomers from the amyloid pathway." Biochemical Journal 437, no. 1 (June 14, 2011): 25–34. http://dx.doi.org/10.1042/bj20101712.
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Aβ (amyloid β) immunotherapy has been revealed as a possible tool in Alzheimer's disease treatment. In contrast with complete antibodies, the administration of scFvs (single-chain variable fragments) produces neither meningoencephalitis nor cerebral haemorrhage. In the present study, the recombinant expression of scFv-h3D6, a derivative of an antibody specific for Aβ oligomers, is presented, as well as the subsequent proof of its capability to recover the toxicity induced by the Aβ1–42 peptide in the SH-SY5Y neuroblastoma cell line. To gain insight into the conformational changes underlying the prevention of Aβ toxicity by this antibody fragment, the conformational landscape of scFv-h3D6 upon temperature perturbation is also described. Heating the native state does not lead to any extent of unfolding, but rather directly to a β-rich intermediate state which initiates an aggregation pathway. This aggregation pathway is not an amyloid fibril pathway, as is that followed by the Aβ peptide, but rather a worm-like fibril pathway which, noticeably, turns out to be non-toxic. On the other hand, this pathway is thermodynamically and kinetically favoured when the scFv-h3D6 and Aβ1–42 oligomers form a complex in native conditions, explaining how the scFv-h3D6 withdraws Aβ1–42 oligomers from the amyloid pathway. To our knowledge, this is the first description of a conformational mechanism by which a scFv prevents Aβ-oligomer cytotoxicity.
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Wei, Jingyan, Yang Liu, Songchuan Yang, Junjie Xu, Hangtian Kong, Bing Han, Yongli Bao, et al. "Screening of Single-Chain Variable Fragments against TSP50 from a Phage Display Antibody Library and Their Expression as Soluble Proteins." Journal of Biomolecular Screening 11, no. 5 (April 28, 2006): 546–52. http://dx.doi.org/10.1177/1087057106287901.
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A novel gene, testes-specific protease 50 ( TSP50), is abnormally activated and differentially expressed in most patients with breast cancer, suggesting it as a novel biomarker for this disease. The possibility that TSP50 may be an oncogene is presently under investigation. In this study, the single-chain variable fragments (scFvs) against TSP50 were panned from a phage display antibody library using TSP50-specific peptide, pep-50, as a target antigen. After 4 rounds of panning, 3 clones (A1, A11, and C8) from the library were verified to show strong binding affinities for TSP50 by enzyme-linked immunosorbent assay (ELISA) and to contain the variable region genes of the light and heavy chains of scFv antibodies but different complementary determining regions by sequencing. The genes of scFv-A1 and scFv-A11 were cloned into expression vector pPELB and successfully expressed as a soluble protein in Escherichia coli Rosetta. The yields of expressions were about 4.0 to 5.0 mg of protein from 1 L of culture. The expressed proteins were purified by a 2-step procedure consisting of ion-exchange chromatography, followed by immobilized metal affinity chromatography. The purified proteins were shown a single band at the position of 31 KDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Sandwich ELISA demonstrated that the expressed scFv proteins were able to specifically react with pep-50, laying a foundation for the investigation of the function of TSP50 in the development and treatment of breast cancer.
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Gould, L. Hannah, Jianhua Sui, Harald Foellmer, Theodore Oliphant, Tian Wang, Michel Ledizet, Akikazu Murakami, et al. "Protective and Therapeutic Capacity of Human Single-Chain Fv-Fc Fusion Proteins against West Nile Virus." Journal of Virology 79, no. 23 (December 15, 2005): 14606–13. http://dx.doi.org/10.1128/jvi.79.23.14606-14613.2005.
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ABSTRACT West Nile virus has spread rapidly across the United States, and there is currently no approved human vaccine or therapy to prevent or treat disease. Passive immunization with antibodies against the envelope protein represents a promising means to provide short-term prophylaxis and treatment for West Nile virus infection. In this study, we identified a panel of 11 unique human single-chain variable region antibody fragments (scFvs) that bind the envelope protein of West Nile virus. Selected scFvs were converted to Fc fusion proteins (scFv-Fcs) and were tested in mice for their ability to prevent lethal West Nile virus infection. Five of these scFv-Fcs, 11, 15, 71, 85, and 95, protected 100% of mice from death when given prior to infection with virus. Two of them, 11 and 15, protected 80% of mice when given at days 1 and 4 after infection. In addition, four of the scFv-Fcs cross-neutralized dengue virus, serotype 2. Binding assays using yeast surface display demonstrated that all of our scFvs bind to sites within domains I and II of West Nile virus envelope protein. These recombinant human scFvs are potential candidates for immunoprophylaxis and therapy of flavivirus infections.
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Ahmad, Zuhaida Asra, Swee Keong Yeap, Abdul Manaf Ali, Wan Yong Ho, Noorjahan Banu Mohamed Alitheen, and Muhajir Hamid. "scFv Antibody: Principles and Clinical Application." Clinical and Developmental Immunology 2012 (2012): 1–15. http://dx.doi.org/10.1155/2012/980250.
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To date, generation of single-chain fragment variable (scFv) has become an established technique used to produce a completely functional antigen-binding fragment in bacterial systems. The advances in antibody engineering have now facilitated a more efficient and generally applicable method to produce Fv fragments. Basically, scFv antibodies produced from phage display can be genetically fused to the marker proteins, such as fluorescent proteins or alkaline phosphatase. These bifunctional proteins having both antigen-binding capacity and marker activity can be obtained from transformed bacteria and used for one-step immunodetection of biological agents. Alternatively, antibody fragments could also be applied in the construction of immunotoxins, therapeutic gene delivery, and anticancer intrabodies for therapeutic purposes. This paper provides an overview of the current studies on the principle, generation, and application of scFv. The potential of scFv in breast cancer research is also discussed in this paper.
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Oliinyk, O. S. "Recombinant single chain variable fragment antibodies (scFv) against Pro(144)-Leu(155) fragment of human protein C." Ukrainian Biochemical Journal 87, no. 2 (April 27, 2015): 88–94. http://dx.doi.org/10.15407/ubj87.02.088.
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Huber, Georg, Zoltán Bánki, Renate Kunert, and Heribert Stoiber. "Novel Bifunctional Single-Chain Variable Antibody Fragments to Enhance Virolysis by Complement: Generation and Proof-of-Concept." BioMed Research International 2014 (2014): 1–14. http://dx.doi.org/10.1155/2014/971345.
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When bound to the envelope of viruses, factor H (FH), a soluble regulator of complement activation, contributes to the protection against a potent immune defense mechanism, the complement-mediated lysis (CML). Thus, removing FH from the surface renders viruses, such as HIV, susceptible to CML. For a proof of concept, we developed a construct consisting of recombinant bifunctional single-chain variable fragment (scFv) based on a monoclonal antibody against Friend murine leukemia virus (F-MuLV) envelope protein gp70, which was coupled to specific binding domains (short consensus repeats 19-20; SCR1920) of FH. We usedPichia pastorisas expression system in common shake flasks and optimized expression in high density bench top fermentation. Specific binding of recombinant scFv was proven by flow cytometry. The recombinant scFv-SCR significantly enhanced CML of F-MuLVin vitroimplying that FH binding to the viral surface was impaired by the scFv-SCR. This novel concept to enhance virolysis may provide a new approach for antiviral treatment.
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Delcommenne, M., H. Klingemann, H. C. Fung, and S. A. Gregory. "Characterization of functional heparan sulfate motifs overexpressed on multiple myeloma cells using single chain antibody variable fragments generated by phage display." Journal of Clinical Oncology 24, no. 18_suppl (June 20, 2006): 13060. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.13060.
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13060 Background: Multiple myeloma (MM) is the most prevalent plasma cell neoplasm and is characterized by the infiltration of the bone marrow by terminally differentiated B lymphocytes. Although novel drugs have recently been introduced, the disease remains incurable. No therapeutic antibody is as yet available for treatment of MM. One of the key molecules involved in multiple myeloma (MM) tumor progression is syndecan-1, a cell surface heparan sulfate (HS) proteoglycan expressed on both MM cells and normal plasma cells. Syndecan-1 regulates cell interactions with soluble molecules and the extracellular matrix through its heparan sulfate chains. These chains allow it to bind multiple growth factors such as Fibroblast Growth Factor type 2 (FGF-2), WNT, Hepatocyte Growth Factor (HGF), midkine, all of which promote survival and metastasis of MM cells. In addition, syndecan-1 on MM cells binds and internalizes osteoprotegerin, the RANKL antagonist, through its HS chains and depletes it from the bone marrow milieu, which triggers osteoclastogenesis. Methods: Using the phage display technique, we have generated a panel of MM-specific human antibody variable fragments (scFvs) against the MM cell line RPMI-8226. These scFvs were tested against a variety of cell lines and primary normal bone marrow or myeloma cells. The biochemical identity of the targeted antigens was determined. Results: Two of the selected scFvs, D4A4 and D6B10, reacted against heparan sulfate motifs that were highly expressed in MM cell lines and patient MM cells but absent (scFv D6B10) or poorly expressed (scFv D4A4) in normal plasma cells. Binding of scFv D6B10 to MM heparan sulfate chains required NS sulfation, 6-O-sulfation and, to a lesser extent, 2-O-sulfation. Additional experiments indicated that FGF-2, midkine and osteoprotegerin competed with scFv D6B10 for binding to HS. Conclusions: This study indicates that the structure of syndecan-1-associated HS is altered in MM cells and may influence its capacity to bind to growth factors and promote tumor progression. Human therapeutic antibodies derived from scFvs D4A4 and D6B10 may be useful for immune targeting of MM cells and interfering with pro-survival signaling pathways. No significant financial relationships to disclose.
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Chia, Kin Yen, Khuen Yen Ng, Rhun Yian Koh, and Soi Moi Chye. "Single-chain Fv Antibodies for Targeting Neurodegenerative Diseases." CNS & Neurological Disorders - Drug Targets 17, no. 9 (November 2, 2018): 671–79. http://dx.doi.org/10.2174/1871527317666180315161626.
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Background & Objective: Protein misfolding and aggregation have been considered the common pathological hallmarks for a number of neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (HD). These abnormal proteins aggregates damage mitochondria and induce oxidative stress, resulting in neuronal cell death. Prolonged neuronal damage activates microglia and astrocytes, development of inflammation reaction and further promotes neurodegeneration. Thus, elimination of abnormal protein aggregates without eliciting any adverse effects are the main treatment strategies. To overcome this, recent studies have deployed single- chain fragment variable antibodies (scFvs) to target the pathological protein aggregates, such as amyloid-beta (Aβ) peptides, α-synuclein (α-syn) and Huntingtin (Htt). To date scFv has been effective at inhibiting abnormal protein aggregates formation in both in vitro and in vivo model system of AD, PD and HD. Conclusion: Currently active research is still ongoing to improve the scFv gene delivery technology, to further enhance brain penetration, intracellular stability, solubility and efficacy of scFv intrabody.
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Liu, Chenjiang, Yoshihiro Kobashigawa, Soichiro Yamauchi, Yuya Toyota, Manaka Teramoto, Yuka Ikeguchi, Natsuki Fukuda, et al. "Preparation of single-chain Fv antibodies in the cytoplasm of Escherichia coli by simplified and systematic chaperone optimization." Journal of Biochemistry 166, no. 6 (September 6, 2019): 455–62. http://dx.doi.org/10.1093/jb/mvz059.
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Abstract A single-chain variable fragment (scFv) antibody is a recombinant protein in which a peptide linker connects the variable regions of the heavy chain and light chain. Due to its smaller molecular size, an scFv can be expressed using Escherichia coli. The presence of two disulphide bonds in the molecule often prevents expression of correctly folded scFv in the E. coli cytoplasm, making a refolding process necessary to regenerate scFv activity. The refolding process is time-consuming and requires large amounts of expensive reagents, such as guanidine hydrochloride, l-arginine and glutathione. Here, to conveniently obtain scFv proteins, we devised a simple and systematic method to optimize the co-expression of chaperone proteins and to combine them with specially engineered E. coli strains that permit the formation of stable disulphide bonds within the cytoplasm. Several scFv proteins were successfully obtained in a soluble form from E. coli cytoplasm. Thermal denaturation experiments and/or surface plasmon resonance measurements revealed that the thus-obtained scFvs possessed a stable tertiary structure and antigen-binding activity. The combined use of engineered E. coli with the simplified and systematic chaperone optimization can be useful for the production of scFv proteins.
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New, Jaa Yien, Jose Perdomo, Xing-Mai Jiang, and Beng Chong. "Heparin-Induced Thrombocytopenia and Thrombosis: Therapeutic Strategy Using a Single-Chain Variable Fragment (scFv) Antibody." Blood 120, no. 21 (November 16, 2012): 3346. http://dx.doi.org/10.1182/blood.v120.21.3346.3346.
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Abstract Abstract 3346 Introduction and Aim Heparin-Induced Thrombocytopenia and Thrombosis (HIT) is a life threatening disorder that affects 1–5% of patients receiving heparin therapy. A low platelet count is usually recorded (<150,000 per cubic millimetre) with a decrease of 50% or more from the baseline. The occurrence of HIT is due to the presence of an IgG antibody that recognizes the immune complex formed between Platelet Factor 4 (PF4) and heparin. The antibody/PF4/Heparin complex binds to the FcγRIIa receptor on platelets, leading to platelet activation and thrombotic complications in patients receiving heparin. IV.3 is a murine monoclonal antibody that was raised against the FcγRIIa receptor and has been used as an inhibitor in specificity assays to confirm HIT in patients. We have developed a humanized single-chain variable fragment (scFv) antibody based on the IV.3 monoclonal antibody that binds to the FcγRIIa receptor on platelets and prevents platelet aggregation induced by HIT antibodies. Methods The variable heavy chain (VH) and light chain (VL) of the IV.3 antigen binding fragment (Fab) moiety were amplified using polymerase chain reaction (PCR). These two fragments were then coupled with a linker (Glycine4 and Serine)6. This was followed by introduction of several components including fusion tags (FLAG and c-Myc) at both termini for cloning, detection and purification purposes. The construct was transformed into E. coli (strain-BL21) for protein expression of the scFv. The presence of the protein was detected via immunostaining using anti-FLAG and anti-c-Myc antibodies. The scFv was purified by affinity chromatography and the binding activity was detected using flow cytometry and confocal microscopy. The functional activity was determined using Platelet Aggregation Assay. The scFv was then humanized to minimize potential immunogenicity. Humanization was achieved by introducing specific mutations that rendered the molecule human-like but did not affect binding specificity. The humanized scFv was also expressed in E. coli, purified and tested as before. Results The scFv protein (32kDa) was expressed, purified and confirmed via immunostaining. The created humanized scFv exhibits binding activity against the FcγRIIa on human platelets as determined by flow cytometry and confocal microscopy. In addition, the protein successfully inhibits platelet aggregation at micro molar concentrations in aggregation assays conducted in vitro in the presence of HIT antibodies. Conclusions The humanized scFv was successful in recapitulating the properties of the IV.3 murine monoclonal antibody. It demonstrated binding activity against the FcgRIIa on human platelets and exhibited functional activity by inhibiting platelet activation and aggregation in vitro. This implies that our scFv is able to stop binding of the antibody/PF4/Heparin immune complex to platelets, thus hindering one of the critical initial steps in HIT. The scFv described here may be able to ameliorate the unwanted side effects of heparin therapy and could serve as a potential therapeutic drug for HIT patients. Disclosures: No relevant conflicts of interest to declare.
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Muñoz-López, Paola, Rosa María Ribas-Aparicio, Elayne Irene Becerra-Báez, Karla Fraga-Pérez, Luis Fernando Flores-Martínez, Armando Alfredo Mateos-Chávez, and Rosendo Luria-Pérez. "Single-Chain Fragment Variable: Recent Progress in Cancer Diagnosis and Therapy." Cancers 14, no. 17 (August 30, 2022): 4206. http://dx.doi.org/10.3390/cancers14174206.
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Cancer remains a public health problem worldwide. Although conventional therapies have led to some excellent outcomes, some patients fail to respond to treatment, they have few therapeutic alternatives and a poor survival prognosis. Several strategies have been proposed to overcome this issue. The most recent approach is immunotherapy, particularly the use of recombinant antibodies and their derivatives, such as the single-chain fragment variable (scFv) containing the complete antigen-binding domains of a whole antibody that successfully targets tumor cells. This review describes the recent progress made with scFvs as a cancer diagnostic and therapeutic tool, with an emphasis on preclinical approaches and their potential use in clinical trials.
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Safarnejad, Mohammad Reza, Hossein Safarpour, Fatemeh Shahryari, Marzieh Basirat, Meisam Tabatabaei, Alaeddin Kordenaeej, Amir Mohammad Naji, and Mojdeh Kakouienejad. "SELECTION OF SPECIFIC SINGLE CHAIN VARIABLE FRAGMENTS (SCFV) AGAINST POLYMYXA BETAE FROM PHAGE DISPLAY LIBRARIES." Journal of Plant Protection Research 53, no. 4 (October 1, 2013): 357–63. http://dx.doi.org/10.2478/jppr-2013-0054.
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Abstract Sugar beet is one of the most important industrial crops in Iran. For the last two decades it has been mainly affected by a destructive virus, beet necrotic yellow vein virus (BNYVV). The Polymyxa betae is the only natural transmitting agent of the disease among the plants. Developing accurate diagnostic methods may have a major impact on the rising of resistant germplasms. In the present study, specific monoclonal recombinant antibodies in the form of single chain variable fragments (scFv) were obtained from naïve phage display libraries. The fungus specific glutathione-S-transferase (GST) protein was chosen as an antigen for developing antibodies and diagnostic purposes. To generate specific scFv, screening of Tomlinson phage display libraries was performed by applying both recombinant and native fungal GST. Using the recombinant GST in the panning process resulted in the isolation of an antibody only bound to recombinant GST but it failed to detect native GST in the infected plants. Alternatively, the process of panning was carried out by applying native fungal GST trapped to immunotubes through specific polyclonal antibody intermediate. The recent approach resulted in the selection of a specific scFv binding to native GST which was able to detect the presence of the fungi within infected plants. To the best of our knowledge, this is the first report on the generation of recombinant antibodies against Polymyxa betae, fungal vector of sugar beet rhizomania disease.
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Zhu, Wenli, Zhen Wang, Suying Hu, Ye Gong, Yuanchu Liu, Huanhuan Song, Xiaoli Ding, Ying Fu, and Yaping Yan. "Human C5-specific single-chain variable fragment ameliorates brain injury in a model of NMOSD." Neurology - Neuroimmunology Neuroinflammation 6, no. 3 (April 4, 2019): e561. http://dx.doi.org/10.1212/nxi.0000000000000561.
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ObjectiveUsing phage display, we sought to screen single-chain variable fragments (scFvs) against complement C5 to treat neuromyelitis optica spectrum disorder (NMOSD).MethodsAfter 5 rounds of phage display, we isolated individual clones and identified phage clones specifically binding to C5 using ELISA. Using aquaporin-4 (AQP4)-transfected cells in vitro, we confirmed whether these scFvs prevented complement-dependent cytotoxicity (CDC) caused by the serum of patients with NMOSD and human complement (hC). We selected an NMOSD mouse model, in which intracerebral NMOSD immunoglobulin G (IgG) and hC injections induce NMOSD-like lesions in vivo.ResultsWe obtained scFvs to test specificity and blocking efficiency. The scFv C5B3 neutralized C5 in the complement activation pathway, which prevented AQP4-IgG–mediated CDC in AQP4-transfected cells. In an NMOSD mouse model, C5B3 prevented AQP4 and astrocyte loss, decreased demyelination, and reduced inflammatory infiltration and membrane attack complex formation in lesions.ConclusionsWe used phage display to screen C5B3 against C5, which was effective in inhibiting cytotoxicity in vitro and preventing CNS pathology in vivo.
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Lee, Chi-Hsin, Sy-Jye Leu, Yu-Ching Lee, Chia-I. Liu, Liang-Tzung Lin, Pharaoh Mwale, Jen-Ron Chiang, et al. "Characterization of Chicken-Derived Single Chain Antibody Fragments against Venom of Naja Naja Atra." Toxins 10, no. 10 (September 21, 2018): 383. http://dx.doi.org/10.3390/toxins10100383.
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Traditional, horse-derived antivenin is currently the most efficient treatment against snake bites. However, it is costly and has unpredictable side effects. Thus, alternative, cost-effective strategies for producing antivenin are needed. In this study, we immunized hens with inactivated NNA venom proteins from the cobra Naja naja atra (NNA). Purified yolk IgY antibodies showed specific anti-NNA binding activity comparable to that of the equine-derived antivenin. We used phage display technology to generate two antibody libraries containing 9.0 × 108 and 8.4 × 108 clones with a short or long linker, respectively. The phage ELISA indicated that anti-NNA clones displaying single-chain variable fragments (scFv) were significantly enriched after biopanning. The nucleotide sequences of the light and heavy chain genes of 30 monoclonal scFv antibodies were determined and classified into six groups with the short linker and nine groups with the long linker. These scFv clones specifically bound to NNA proteins but not to venom proteins from other snakes. Their binding affinities were further determined by competitive ELISA. Animal model studies showed that anti-NNA IgY antibodies exhibited complete protective effects, while a combination of scFv antibodies raised the survival rates and times of mice challenged with lethal doses of NNA venom proteins.
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Gupta, Vijay, Indulekha P. Sudhakaran, Zeyaul Islam, Nishant N. Vaikath, Issam Hmila, Tamas Lukacsovich, Prasanna R. Kolatkar, and Omar M. A. El-Agnaf. "Expression, purification and characterization of α-synuclein fibrillar specific scFv from inclusion bodies." PLOS ONE 15, no. 11 (November 6, 2020): e0241773. http://dx.doi.org/10.1371/journal.pone.0241773.
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Aggregation of α-synuclein (α-syn) has been implicated in multiple neurodegenerative disorders including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), collectively grouped as synucleinopathies. Recently, recombinant antibody fragments (Fab, scFvs and diabodies) against α-syn have emerged as an alternative to the traditional full-length antibody in immunotherapeutic approaches owing to their advantages including smaller size and higher stability, specificity and affinity. However, most of the recombinant antibody fragments tend to be expressed as inclusion bodies (IBs) making its purification extremely challenging. In the current study, a single-chain variable fragment (scFv-F) antibody, targeting the pathogenic α-syn fibrils, was engineered and expressed in E. coli. Majority of the expressed scFv-F accumulated in insoluble aggregates as IBs. A variety of mild and harsh solubilizing conditions were tested to solubilize IBs containing scFv-F to obtain the active protein. To preserve secondary structure and bioactivity, a mild solubilizing protocol involving 100 mM Tris, pH 12.5 with 2 M urea was chosen to dissolve IBs. Slow on-column refolding method was employed to subsequently remove urea and obtain active scFv-F. A three-dimensional (3D) model was built using homology modeling and subjected to molecular docking with the known α-syn structure. Structural alignment was performed to delineate the potential binding pocket. The scFv-F thus purified demonstrated high specificity towards α-syn fibrils compared to monomers. Molecular modeling studies suggest that scFv-F shares the same structural topology with other known scFvs. We present evidence through structural docking and alignment that scFv-F binds to α-syn C-terminal region. In conclusion, mild solubilization followed by slow on-column refolding can be utilized as a generalized and efficient method for hard to purify disease relevant insoluble proteins and/or antibody molecules from IBs.
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Skora, Andrew D., Jacqueline Douglass, Michael S. Hwang, Ada J. Tam, Richard L. Blosser, Sandra B. Gabelli, Jianhong Cao, et al. "Generation of MANAbodies specific to HLA-restricted epitopes encoded by somatically mutated genes." Proceedings of the National Academy of Sciences 112, no. 32 (July 27, 2015): 9967–72. http://dx.doi.org/10.1073/pnas.1511996112.
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Mutant epitopes encoded by cancer genes are virtually always located in the interior of cells, making them invisible to conventional antibodies. We here describe an approach to identify single-chain variable fragments (scFvs) specific for mutant peptides presented on the cell surface by HLA molecules. We demonstrate that these scFvs can be successfully converted to full-length antibodies, termed MANAbodies, targeting “Mutation-Associated Neo-Antigens” bound to HLA. A phage display library representing a highly diverse array of single-chain variable fragment sequences was first designed and constructed. A competitive selection protocol was then used to identify clones specific for mutant peptides bound to predefined HLA types. In this way, we obtained two scFvs, one specific for a peptide encoded by a common KRAS mutant and the other by a common epidermal growth factor receptor (EGFR) mutant. The scFvs bound to these peptides only when the peptides were complexed with HLA-A2 (KRAS peptide) or HLA-A3 (EGFR peptide). We converted one scFv to a full-length antibody (MANAbody) and demonstrate that the MANAbody specifically reacts with mutant peptide–HLA complex even when the peptide differs by only one amino acid from the normal, WT form.
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Aghebati-Maleki, Leili, Vahid Younesi, Behzad Baradaran, Jalal Abdolalizadeh, Morteza Motallebnezhad, Hamid Nickho, Dariush Shanehbandi, Jafar Majidi, and Mehdi Yousefi. "Antiproliferative and Apoptotic Effects of Novel Anti-ROR1 Single-Chain Antibodies in Hematological Malignancies." SLAS DISCOVERY: Advancing the Science of Drug Discovery 22, no. 4 (January 31, 2017): 408–17. http://dx.doi.org/10.1177/2472555216689659.
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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 VH 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.
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Rafiq, Sarwish, Hollie J. Jackson, Oladapo Yeku, Terence J. Purdon, Dayenne G. van Leeuwen, Kevin J. Curran, Ryan N. Ahmed, et al. "Enhancing CAR T Cell Anti-Tumor Efficacy through Secreted Single Chain Variable Fragment (scFv) Immune Checkpoint Blockade." Blood 130, Suppl_1 (December 7, 2017): 842. http://dx.doi.org/10.1182/blood.v130.suppl_1.842.842.
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Abstract T cell therapies have had valuable clinical responses in patients with cancer. Chimeric antigen receptor (CAR) T cells can be genetically engineered to recognize tumor cells and CAR T cell therapy has shown impressive results in the setting of B cell acute lymphoblastic leukemia but has been less effective in treating other types of hematologic and solid tumors. The inhibitory tumor microenvironment (TME), including expression of ligands that bind inhibitory receptors on T cells, e.g. programmed death receptor 1 (PD-1), can dampen CAR T cell responses. Separately, immune checkpoint blockade therapy involving the disruption of PD-1 and programmed death receptor ligand1 (PD-L1) interaction allows for re-activation of tumor-infiltrating lymphocytes (TIL) to have anti-tumor function. This approach has shown clinical responses in a range of malignancies, but has been less efficacious in poorly immunogenic tumors. To prevent PD-1-mediated dampening of CAR T cell function, we have co-modified CAR T cells to secrete PD-1 blocking single chain variable fragments (scFv). We first designed mouse constructs with which we could investigate the scFv-secreting CAR T cells in the context of a syngeneic immune-competent intact TME. CAR constructs were engineered directed against either human CD19 or MUC-16 (ecto) with mouse signaling domains and a anti-mouse PD-1 scFv. Mouse T cells transduced with these constructs expressed the CAR on the surface and secreted detectable amounts of scFv that bound to mouse PD-1. The scFv-secreting CAR T cells were cytotoxic and produced IFN-g when co-cultured with PD-L1 expressing tumors in vitro . We utilized a syngeneic mouse model to study scFv secreting CAR T cells in a model with an intact TME. In tumor-bearing mice treated with CAR T cells, scFv-secreting CAR T cells enhanced survival as compared to second generation CAR T cells. The survival benefit achieved with scFv-secreting CAR T cells was comparable to that achieved with systemic infusion of PD-1 blocking antibody, but with localized delivery of PD-1 blockade. Mice treated with scFv-secreting CAR T cells had detectable scFv in vivo in the TME. Lastly, long term surviving mice had detectable CAR T cells in the bone marrow by PCR, demonstrating persistence and suggesting an immunological memory. We next aimed to translate PD-1 blocking scFv CAR T cells to a clinically relevant human model utilizing a novel anti-human PD-1 blocking scFv. CAR constructs were engineered with recognition domains directed against human CD19 or MUC-16 (ecto) and human signaling domains. Human T cells modified with the CAR constructs express the CAR on the surface and secrete detectable amounts of PD-1 blocking scFv. The scFv binds to human PD-1 and scFv-secreting CAR T cells are cytotoxic to PD-L1 expressing tumors. Expression of PD-1-blocking scFv enhances CAR T cell function against PD-L1 expressing tumors in xenograft models of hematological and solid tumors by enhancing survival in tumor-bearing mice as compared to second generation CAR T cells. Furthermore, scFv-secreting CAR T cells exhibit in vivo bystander T cell enhancement of function, suggesting scFv-secreting CAR T cells can reactivate endogenous TILs in the TME. These data support the novel concept that localized delivery of scFv by CAR T cells can successfully block PD-1 binding to PD-L1 and work in an autocrine manner to prevent dampening of CAR T cell responses as well as a paracrine manner to activate endogenous tumor infiltrating lymphocytes to enhance the overall anti-tumor efficacy of CAR T cell therapy. Disclosures Curran: Juno Therapeutics: Research Funding; Novartis: Consultancy. Yan: Eureka Therapeutics Inc: Employment. Wang: Eureka Therapeutics Inc.: Employment, Equity Ownership. Xiang: Eureka Therapeutics Inc.: Employment. Liu: Eureka Therpeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Brentjens: Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.
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Bitencourt, André L. B., Raquel M. Campos, Erika N. Cline, William L. Klein, and Adriano Sebollela. "Antibody Fragments as Tools for Elucidating Structure-Toxicity Relationships and for Diagnostic/Therapeutic Targeting of Neurotoxic Amyloid Oligomers." International Journal of Molecular Sciences 21, no. 23 (November 24, 2020): 8920. http://dx.doi.org/10.3390/ijms21238920.
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The accumulation of amyloid protein aggregates in tissues is the basis for the onset of diseases known as amyloidoses. Intriguingly, many amyloidoses impact the central nervous system (CNS) and usually are devastating diseases. It is increasingly apparent that neurotoxic soluble oligomers formed by amyloidogenic proteins are the primary molecular drivers of these diseases, making them lucrative diagnostic and therapeutic targets. One promising diagnostic/therapeutic strategy has been the development of antibody fragments against amyloid oligomers. Antibody fragments, such as fragment antigen-binding (Fab), scFv (single chain variable fragments), and VHH (heavy chain variable domain or single-domain antibodies) are an alternative to full-length IgGs as diagnostics and therapeutics for a variety of diseases, mainly because of their increased tissue penetration (lower MW compared to IgG), decreased inflammatory potential (lack of Fc domain), and facile production (low structural complexity). Furthermore, through the use of in vitro-based ligand selection, it has been possible to identify antibody fragments presenting marked conformational selectivity. In this review, we summarize significant reports on antibody fragments selective for oligomers associated with prevalent CNS amyloidoses. We discuss promising results obtained using antibody fragments as both diagnostic and therapeutic agents against these diseases. In addition, the use of antibody fragments, particularly scFv and VHH, in the isolation of unique oligomeric assemblies is discussed as a strategy to unravel conformational moieties responsible for neurotoxicity. We envision that advances in this field may lead to the development of novel oligomer-selective antibody fragments with superior selectivity and, hopefully, good clinical outcomes.
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van den Brink, Edward N., Ellen A. M. Turenhout, Julian Davies, Niels Bovenschen, Karin Fijnvandraat, Willem H. Ouwehand, Marjolein Peters, and Jan Voorberg. "Human antibodies with specificity for the C2 domain of factor VIII are derived from VH1 germline genes." Blood 95, no. 2 (January 15, 2000): 558–63. http://dx.doi.org/10.1182/blood.v95.2.558.
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A serious complication in hemophilia care is the development of factor VIII (FVIII) neutralizing antibodies (inhibitors). The authors used V gene phage display technology to define human anti-FVIII antibodies at the molecular level. The IgG4-specific, variable, heavy-chain gene repertoire of a patient with acquired hemophilia was combined with a nonimmune, variable, light-chain gene repertoire for display as single-chain variable domain antibody fragments (scFv) on filamentous phage. ScFv were selected by 4 rounds of panning on immobilized FVIII light chain. Sequence analysis revealed that isolated scFv were characterized by VH domains encoded by germline genes DP-10, DP-14, and DP-88, all belonging to the VH1 gene family. All clones displayed extensive hypermutation and were characterized by unusually long CDR3 sequences of 20 to 23 amino acids. Immunoprecipitation revealed that all scFv examined bound to the C2 domain of FVIII. Furthermore, isolated scFv competed with an inhibitory murine monoclonal antibody for binding to the C2 domain. Even though scFv bound FVIII with high affinity, they did not inhibit FVIII activity. Interestingly, the addition of scFv diminished the inhibitory potential of patient-derived antibodies with C2 domain specificity. These results suggest that the epitope of a significant portion of anti-C2 domain antibodies overlaps with that of the scFv isolated in this study.
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Lukosaityte, Deimante, Jean-Remy Sadeyen, Angita Shrestha, Joshua E. Sealy, Sushant Bhat, Pengxiang Chang, Paul Digard, and Munir Iqbal. "Engineered Recombinant Single Chain Variable Fragment of Monoclonal Antibody Provides Protection to Chickens Infected with H9N2 Avian Influenza." Vaccines 8, no. 1 (March 3, 2020): 118. http://dx.doi.org/10.3390/vaccines8010118.
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Passive immunisation with neutralising antibodies can be a potent therapeutic strategy if used pre- or post-exposure to a variety of pathogens. Herein, we investigated whether recombinant monoclonal antibodies (mAbs) could be used to protect chickens against avian influenza. Avian influenza viruses impose a significant economic burden on the poultry industry and pose a zoonotic infection risk for public health worldwide. Traditional control measures including vaccination do not provide rapid protection from disease, highlighting the need for alternative disease mitigation measures. In this study, previously generated neutralizing anti-H9N2 virus monoclonal antibodies were converted to single-chain variable fragment antibodies (scFvs). These recombinant scFv antibodies were produced in insect cell cultures and the preparations retained neutralization capacity against an H9N2 virus in vitro. To evaluate recombinant scFv antibody efficacy in vivo, chickens were passively immunized with scFvs one day before, and for seven days after virus challenge. Groups receiving scFv treatment showed partial virus load reductions measured by plaque assays and decreased disease manifestation. These results indicate that antibody therapy could reduce clinical disease and shedding of avian influenza virus in infected chicken flocks.
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Luken, Brenda M., Paul H. P. Kaijen, Ellen A. M. Turenhout, Rob Fijnheer, and Jan Voorberg. "Multiple B Cell Clones Producing Antibodies Directed to the Spacer and Disintegrin/TSR1 Domains of ADAMTS13 in a Patient with Acquired TTP." Blood 104, no. 11 (November 16, 2004): 257. http://dx.doi.org/10.1182/blood.v104.11.257.257.
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Abstract Thrombotic thrombocytopenic purpura (TTP) is associated with a severe deficiency of the von Willebrand factor-cleaving protease ADAMTS13. In plasma of the majority of patients with acquired TTP, antibodies that inhibit ADAMTS13 activity have been detected. In this study we have used phage display to isolate anti-ADAMTS13 antibodies from the total immunoglobulin repertoire of a patient with acquired TTP. The immunoglobulin G variable heavy chain (VH) gene repertoire was amplified from CD19 positive B cells and combined with a variable light chain (VL) gene repertoire. The resulting library consisted of 3.4 x 108 individual clones. Combined VH and VL segments, expressed as single-chain Fv fragments (scFv) on the surface of filamentous phage, were selected for binding to an ADAMTS13 fragment consisting of the disintegrin/ first thrombospondin type 1 repeat (TSR1)/ cysteine-rich/ spacer domains. After three rounds of selection, six different scFv antibody clones were identified that were assigned to four groups based on the use of VH germline gene segments. ScFv 9, 10, and 27, all use the VH1–69 germline gene segments, possess the same CDR3, and show a similar pattern of somatic hypermutation. These results suggest that scFv 9, 10 and 27 are derived from a common B cell precursor. ScFv 26 also belongs to the VH1 family, but uses germline gene segment VH1–02 instead. Clones 16 and 41 are both part of the VH3 family and are derived from germline gene segments VH3–07 and VH3–09 respectively. The affinity of the scFv for the ADAMTS13 disintegrin/ TSR1/ cysteine-rich/ spacer fragment was determined by surface plasmon resonance analysis. Purified disintegrin/ TSR1/ cysteine-rich/ spacer fragment was immobilized on an activated CM5-sensor chip and subsequently different concentrations of purified scFv were added. Binding and dissociation of scFv was followed for 2 minutes and the affinity of the different scFv for the immobilized ADAMTS13 fragment was calculated from the obtained binding curves. ScFv 9 binds with high affinity to the disintegrin/ TSR1/ cysteine-rich/ spacer fragment (Kd 3.6 ± 0.8 nM); whereas scFv 10, 16, 26, 27 and 41 displayed a somewhat lower affinity (Kd ranging from 20–200 nM). Epitope mapping using several smaller ADAMTS13 fragments in the disintegrin/ TSR1/ cysteine-rich/ spacer region revealed that scFv 9, 10, and 41, bind specifically to the ADAMTS13 spacer domain. The epitope of scFv 16 however, resides in the disintegrin/ TSR1 domains. Our results indicate that multiple B cell clones that produce antibodies recognizing epitopes in the spacer and disintegrin/ TSR1 domains of ADAMTS13 are present in the immunoglobulin repertoire of a patient with acquired TTP.
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Zhang, Fanqing, Yuxue Chen, Yong Ke, Lei Zhang, Bo Zhang, Liang Yang, and Jianguo Zhu. "Single Chain Fragment Variable (scFv) Antibodies Targeting the Spike Protein of Porcine Epidemic Diarrhea Virus Provide Protection against Viral Infection in Piglets." Viruses 11, no. 1 (January 14, 2019): 58. http://dx.doi.org/10.3390/v11010058.
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Porcine epidemic diarrhea virus (PEDV) is a highly contagious coronavirus that causes severe diarrhea and death in neonatal piglets. Passive immunization with neutralizing antibodies against PEDV is an effective prevention measure. In this study, single chain fragment variable (scFv) antibodies against PEDV were screened from the porcine scFv phage display library. After four rounds of biopanning, scFvs that showed higher affinity to the PEDV antigen were selected for further study. The scFv genes were cloned into the expression plasmid for recombinant protein expression. These scFvs were shown to inhibit PEDV infectivity by the plaque reduction neutralization assay. Immunofluorescence assay (IFA) revealed that the epitopes recognized by these scFvs were in the S1 region of the spike protein. The potential of scFvs to provide prevention against PEDV infections in piglets was further investigated. Piglets orally administered scFvs showed no to mild clinical symptoms, significantly less viral shedding, no mortality and no intestinal lesions. The field application also revealed that the survival rate of piglets was significantly increased by oral administration of scFvs. Our data support the potential role of scFvs in the prevention and treatment of PEDV infection.
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Brichta, J., H. Vesela, and M. Franek. "Production of scFv recombinant fragments against 2,4-dichlorophenoxyacetic acid hapten using naďve phage library." Veterinární Medicína 48, No. 9 (March 30, 2012): 237–47. http://dx.doi.org/10.17221/5776-vetmed.
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Three single chain variable fragment (scFv) antibodies against 2,4-dichlophenoxyacetic acid (2,4-D) herbicide were produced by the Griffin1.library. The selection of the scFv from the phage library was carried out by 2,4-D-protein coated tubes with different levels of hapten substitution in the conjugate. The scFv phage clones were isolated within the five round library panning and the antibodies were expressed in Escherichia coli HB2151. The recombinant products were purified by metal affinity chromatography yielding 200 g of pure scFv per 1 liter of bacterial culture. The antibody fragments provided steep curves in conventional indirect ELISA having the IC<sub>50</sub> values from 10.2 to 14.5 ng/ml established for 2,4-D standard. Interestingly enough, the recombinant ScFv E1 antibody exhibited 68% cross-reactivity with 2,4-dichlorphenol (2,4-D = 100%), and 38.0% with methylchlorophenoxyacetic acid (MCPA) whereas reaction with other phenoxyacetic compounds was low. Similar characteristics were obtained for other two recombinant products. Low stability for the isolated scFv antibodies was found in storage buffer even in the presence of stabilizers and protease inhibitors. Factors influencing stability of the recombinant antibodies are discussed.
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Kim, Chol-Jin, Sunll Choe, Kum-Chol Ri, Chol-Ho Kim, Hyon-Gwang Li, and Un-Hui Yun. "Selection of a Single Chain Variable Fragment Antibody (scFv) against Subtilisin BRC and its Interaction with Subtilisin BRC." Current Biotechnology 8, no. 1 (September 25, 2019): 24–31. http://dx.doi.org/10.2174/2211550108666190417113342.
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Background: The focus of this study was the selection of a single chain variable fragment antibody (scFv) against subtilisin BRC, a fibrinolytic enzyme using phage display, and to characterize the interaction between the antibody and subtilisin BRC. Methods: The subtilisin BRC-specific phage clones were selected using Griffin.1 scFv phage library and sequenced. The gene of subtilisin BRC-specific scFv (scFv-BRC) from selected phage clone was expressed in E. coli and scFv-BRC was characterized. Molecular modeling of the three-dimensional (3D) structures of scFv-BRC was performed using MODELLER 9.19 modeling software and assessed by PROCHEK. Molecular docking of subtilisin BRC with scFv-BRC was carried out using PATCHDOCK. Results: The size of scFv-BRC gene is 635bp and it consists of 54bp of heavy chain region (VH), 336bp of light chain region (VL), 45bp of a linker. scFv-BRC was actively expressed by E. coli expression vector pET28a-scFv in E. coli BL21 (DE3), and the amount of expressed scFv-BRC was about 50 mg/L. Its molecular weight is ~26kDa. The CDR domain of scFv-BRC consists of 6 amino acids in CDR L1, 3 amino acids in CDR L2 and 9 amino acids in CDR L3. Docking results of subtilisin BRC and scFv-BRC showed global energy of - 56.29 kJ/mol. Furthermore, the results showed that amino acid residues in subtilisin BRC for binding with scFv-BRC are Tyr6, Ser182, Ser204, and Gln206. Conclusion: scFv against subtilisin BRC selected using phage display showed relatively strong binding energy with subtilisin BRC. The amino acid residues in subtilisin BRC for binding with scFv-BRC are not relevant to that in subtilisin BRC for binding with its substrates. These results suggested that scFv-BRC can be used as a ligand for detection and affinity purification of subtilisin BRC.
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Liu, Han-Lin, Wei-Fang Lin, Wen-Chi Hu, Yung-An Lee, and Ya-Chun Chang. "A Strategy for Generating a Broad-Spectrum Monoclonal Antibody and Soluble Single-Chain Variable Fragments against Plant Potyviruses." Applied and Environmental Microbiology 81, no. 19 (July 24, 2015): 6839–49. http://dx.doi.org/10.1128/aem.01198-15.
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ABSTRACTPotyviruses are major pathogens that often cause mixed infection in calla lilies. To reduce the time and cost of virus indexing, a detection method for the simultaneous targeting of multiple potyviruses was developed by generating a broad-spectrum monoclonal antibody (MAb) for detecting the greatest possible number of potyviruses. The conserved 121-amino-acid core regions of the capsid proteins ofDasheen mosaic potyvirus(DsMV),Konjak mosaic potyvirus(KoMV), andZantedeschia mild mosaic potyvirus(ZaMMV) were sequentially concatenated and expressed as a recombinant protein for immunization. After hybridoma cell fusion and selection, one stable cell line that secreted a group-specific antibody, named C4 MAb, was selected. In the reaction spectrum test, the C4 MAb detected at least 14 potyviruses by indirect enzyme-linked immunosorbent assay (I-ELISA) and Western blot analysis. Furthermore, the variable regions of the heavy (VH) and light (VL) chains of the C4 MAb were separately cloned and constructed as single-chain variable fragments (scFvs) for expression inEscherichia coli. Moreover, the pectate lyase E (PelE) signal peptide ofErwinia chrysanthemiS3-1 was added to promote the secretion of C4 scFvs into the medium. According to Western blot analysis and I-ELISA, the soluble C4 scFv (VL-VH) fragment showed a binding specificity similar to that of the C4 MAb. Our results demonstrate that a recombinant protein derived from fusion of the conserved regions of viral proteins has the potential to produce a broad-spectrum MAb against a large group of viruses and that the PelE signal peptide can improve the secretion of scFvs inE. coli.
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Shestopal, Svetlana A., Leonid A. Parunov, Mikhail V. Ovanesov, Timothy K. Lee, and Andrey G. Sarafanov. "Characterization of Interaction of Factor VIII with Engineered Variants of a Single-Chain Variable Antibody Fragment." Blood 132, Supplement 1 (November 29, 2018): 1170. http://dx.doi.org/10.1182/blood-2018-99-115455.
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Abstract Introduction Replacement therapy for Hemophilia A requires frequent infusions of Factor VIII (FVIII) due to its relatively short half-life of ~12 h in plasma. Previous attempts to extend this half-life by genetic and chemical modification of FVIII met the barrier of ~20 h, which is a half-life of von Willebrand factor (VWF), a carrier of FVIII in plasma. A single-chain variable antibody fragment (scFv) KM33 was shown to inhibit FVIII activity, and interactions with VWF and the low-density lipoprotein receptor-related protein 1 (LRP), the major clearance receptor of FVIII (Bovenschen et al, 2005, Blood, 106:906-12). A study indicated that scFv KM33 may prolong the half-life of FVIII in mice to the level exceeding that of VWF half-life (Mertens et al, US Patent 2008, 20080219983A1). This would make scFv KM33 a promising tool for new designs of the longer-acting FVIII products. Study objective We aimed to generate a scFv KM33 variant that can delay FVIII clearance but can be removed from FVIII during its activation by thrombin. Such antibody fragment may extend the half-life of FVIII above that of VWF. Experimental design We generated three scFv KM33 variants with different linkers connecting the subunits VL and VH of the antibody fragment. The linkers contained variants of thrombin cleavage sites identical to those on FVIII. The proteins were expressed using a baculovirus system, purified by Ni-affinity and size exclusion chromatography (SEC), and tested for their properties. Results The engineered scFv variants, along with the unmodified KM33, were tested for binding to FVIII by surface plasmon resonance (SPR). All scFv versions demonstrated similar affinity for FVIII (~1 nM). In addition, a selected variant of scFv inhibited FVIII binding to LRP. These showed that the modifications of scFv did not affect its binding to FVIII. Thrombin treatment of the engineered scFv variants resulted in dissociation of their VL and VH domains, verified by SEC. However, the respective rates of thrombin cleavage were slower than that of FVIII. The preparation of a thrombin-cleaved scFv still inhibited the interaction of FVIII with LRP by SPR, similarly to that observed for the unmodified KM33. All variants of scFv inhibited FVIII activity in a thrombin generation assay suggesting that their moiety remained in complex with FVIII upon its activation. Conclusions The rate of thrombin cleavage of sites within FVIII is higher than that of the identical sites within the scFv. This suggests that additional determinants of FVIII (e. g. sulfated tyrosines adjacent to the sites) contribute to the higher rate of cleavage. The cleavage of the linker between the VL and VH subunits of scFv KM33 results in dissociation of the subunits and breakdown of the antibody fragment. This mechanism is likely applicable to any scFv, and may be useful in a broad range of applications involving such ligands. Both subunits of thrombin-cleaved scFv KM33, most likely, re-assemble on FVIII and form a tertiary complex FVIII/VL/VH. In turn, thrombin cleavage of the scFv, complexed with FVIII, does not result in its dissociation from FVIII. These indicate that in such design, an scFv should have lower affinity for FVIII to ensure its release from the complex. Disclosures No relevant conflicts of interest to declare.
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Shibata, Yasuko, Kimiyo Kurihara, Hisashi Takiguchi, and Yoshimitsu Abiko. "Construction of a Functional Single-Chain Variable Fragment Antibody against Hemagglutinin from Porphyromonas gingivalis." Infection and Immunity 66, no. 5 (May 1, 1998): 2207–12. http://dx.doi.org/10.1128/iai.66.5.2207-2212.1998.
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ABSTRACT Hemagglutinin is a major glycoprotein of Porphyromonas gingivalis vesicles and likely confers the ability to adsorb and penetrate into host tissue cells. To protect this bacterial invasion, murine monoclonal antibody (MAb) Pg-vc, which inhibited the hemagglutinating activity, was prepared by using P. gingivalis vesicles as an antigen. Western blot analysis revealed that when both MAb Pg-vc and anti-HA-Ag2 antibody raised against theP. gingivalis hemagglutinin adhesin (M. Deslauriers and C. Mouton, Infect. Immun. 60:2791–2799, 1992) were allowed to react with protein blots from P. gingivalis vesicles, a superimposable profile was observed. To obtain a recombinant antibody, cDNAs coding for the variable domains of the L and H chains of MAb Pg-vc were cloned by PCR, and a plasmid specifying a single-chain variable fragment (ScFv) was constructed. Following transformation of Escherichia coli cells, a recombinant ScFv protein was successfully expressed. The immunological properties of this protein were identical to those of the parental murine MAb, specifically recognizing the two proteins (43 and 49 kDa) originating from P. gingivalisvesicles. In addition, the ScFv antibody inhibited theP. gingivalis vesicle-associated hemagglutinating activity. The amino acid sequences deduced from nucleotide sequencing experiments confirmed that variable heavy-chain and variable light-chain regions belonged to VH1 and Vκ12/13 families, respectively. Since the expression system used in this study can readily provide large quantities of single-chain recombinant antibody, it may be a useful in developing a therapeutic agent for passive immunization in humans.
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Knight, Jennifer B., Scott A. Halperin, Kenneth A. West, and Song F. Lee. "Expression of a Functional Single-Chain Variable-Fragment Antibody against Complement Receptor 1 in Streptococcus gordonii." Clinical and Vaccine Immunology 15, no. 6 (April 2, 2008): 925–31. http://dx.doi.org/10.1128/cvi.00500-07.
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ABSTRACT Streptococcus gordonii, an oral commensal organism, is a candidate vector for oral-vaccine development. Previous studies have shown that recombinant S. gordonii expressing heterologous antigens was weakly immunogenic when delivered intranasally. In this study, antigen was specifically targeted to antigen-presenting cells (APC) in order to potentiate antigen-APC interactions and increase the humoral immune response to the antigen. To achieve this goal, a single-chain variable-fragment (scFv) antibody against complement receptor 1 (CR1) was constructed. Anti-CR1 scFv purified from Escherichia coli was able to bind to mouse mixed lymphocytes and bone marrow-derived dendritic cells. The in vivo function of the anti-CR1 scFv protein was assessed by immunizing mice intranasally with soluble scFv and determining the immune response against the hemagglutinin (HA) peptide located on the carboxy terminus of the scFv. The serum anti-HA immunoglobulin G (IgG) immune response was dose dependent; as little as 100 ng of anti-CR1 scFv induced a significant IgG immune response, while such a response was minimal when the animals were given an unrelated scFv. The anti-CR1 scFv was expressed in S. gordonii as a secreted protein, which was functional, as it bound to dendritic cells. Mice orally colonized by the anti-CR1-secreting S. gordonii produced an anti-HA IgG immune response, indicating that such an approach can be used to increase the immune response to antigens produced by this bacterium.
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Dewi, Kartika Sari, and Asrul Muhamad Fuad. "Comparison of Gene Expression Between Two Types of Anti-EGFRvIII ScFv Antibodies Having Different Variable Domain Orders in Escherichia coli." ANNALES BOGORIENSES 21, no. 1 (June 22, 2017): 29. http://dx.doi.org/10.14203/ann.bogor.2017.v21.n1.29-37.
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Several studies reported that the expression of various kinds of Single-chain variable fragment (scFv) antibodies in Escherichia coli are significantly influenced by the order of their variable domains. To date, the effect of the order of variable domains in the expression of scFv antibodies against epidermal growth factor receptor variant III (EGFRvIII) has not been reported. This study aimed to compare the expression between VH-linker-VL and VL-linker-VH domain orders of the anti-EGFRvIII scFv antibodies in E. coli expression system. Recombinant plasmids inserted with DNA encoding scFv proteins were transformed into E. coli NiCo21(DE3) competent cells and characterized by colony PCR. The expression of scFv proteins was done by using optimum concentration of inducer. Total proteins, soluble periplasmic and cytoplasmic proteins, also extracellular proteins were isolated, subsequently characterized by SDS-PAGE, Slot Blot, and ImageJ software analyses. The antigen-binding activity of both scFvs proteins against EGFRvIII was observed. The results showed that the relative percentage of scFv expression with VH-linker-VL domain order is higher than that of VL-linker-VH in each compartment. Moreover, both of scFvs proteins have antigen-binding activity against EGFRvIII.
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Chen, Weifeng, Li Hu, Aiping Liu, Jinquan Li, Fusheng Chen, and Xiaohong Wang. "Expression and characterization of single-chain variable fragment antibody against staphylococcal enterotoxin A in Escherichia coli." Canadian Journal of Microbiology 60, no. 11 (November 2014): 737–43. http://dx.doi.org/10.1139/cjm-2014-0468.
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The staphylococcal enterotoxins (SEs) are potent gastrointestinal exotoxins synthesized by Staphylococcus aureus, which is responsible for various diseases including septicemia, food poisoning, and toxic shock syndrome, as well as bovine mastitis. Among them, staphylococcal enterotoxin A (SEA) is one of the most commonly present serotypes in staphylococcal food poisoning cases. In this study, the stable hybridoma 3C12 producing anti-SEA monoclonal antibody was established with an equilibrium dissociation constant (KD) of 1.48 × 10−8 mol·L−1, its ScFv-coding genes were obtained and then the anti-SEA single chain variable fragment (ScFv) protein was expressed in Escherichia coli. Characterization of the expressed target ScFv protein was analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis, Western blot, and enzyme-linked immunosorbent assay. The results demonstrated that the recombinant anti-SEA ScFv protein retained a specific binding activity for SEA, and the KD value of the soluble ScFv was about 3.75 × 10−7 mol·L−1. The overall yield of bioactive anti-SEA ScFv in E. coli flask culture was more than 10 mg·L−1.
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Kim, So-Hee, Seung-Young Hwang, Yong-Seok Lee, In-Hak Choi, Sae-Gwang Park, and Weon-Gyu Kho. "Single-Chain Antibody Fragment Specific for Plasmodium vivax Duffy Binding Protein." Clinical and Vaccine Immunology 14, no. 6 (April 25, 2007): 726–31. http://dx.doi.org/10.1128/cvi.00456-06.
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ABSTRACT Phage display of single-chain variable fragment (scFv) antibodies is a powerful tool for selecting important, useful, and specific human antibodies. We constructed a library from three patients infected with Plasmodium vivax. Panning on recombinant PvRII enriched a population of scFvs that recognized region II of the P. vivax Duffy binding protein (DBP). Three clones of scFvs that reacted with PvRII were selected, and their biological functions were analyzed. These scFvs inhibited erythrocyte binding to DBP. Clone SFDBII92 had the greatest affinity (dissociation constant = 3.62 × 10−8 M) and the greatest inhibition activity (50% inhibitory concentration ≈ 2.9 μg/ml) to DBP. Thus, we demonstrated that human neutralizing antibody could be made from malaria patients using phage display and that these neutralizing scFvs should prove valuable for developing both passive and active immunization strategies based on DBP.