Journal articles: 'Phage displayed scFv'

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Zhao, Peng, Guijie Zhu, Lihua Zhang, Zhen Liang, Zonghai Li, and Yukui Zhang. "New method for determination of average scFv fragment number displayed on the M13 phage surface." Pure and Applied Chemistry 82, no. 1 (January 3, 2010): 205–11. http://dx.doi.org/10.1351/pac-con-09-02-03.

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Single-chain-Fv (scFv) display M13 phage library has been regarded as a powerful tool for screening specific antibodies via binding with target proteins. Generally, the library quality is evaluated through detecting gene fragments by molecular biology methods, which is not only time- and labor-consuming, but also impossible to obtain quantitative information about the binding capacity of the phage library. In our recent study, a new method to calculate the average scFv number displayed on the M13 phage surface was proposed by capillary electrophoresis with laser-induced fluorescence (CE-LIF) detection. By this method, enhanced green fluorescent protein (EGFP) and scFv phage clones that could specifically bind with EGFP were mixed with different ratios, followed by analysis by CE-LIF. With the dilution of EGFP by phage solution, the peak areas of scFv phage clones and free EGFP were decreased continuously, while that of the EGFP-M13 phage complex was found to decrease initially, then trend to be stable, and finally decrease further. When the volume ratio of the M13 phage to EGFP reached 660:1, corresponding to the molecule number ratio as 1:2.6, no more EGFP was found to bind with the M13 phage, which demonstrated that, by average, 2.6 scFv fragments that could bind with EGFP were displayed on the M13 phage surface. All these experimental results demonstrated that, by such a method, the quantitative evaluation of the phage library could be achieved with high throughput and accuracy.

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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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Goswami, Pooja, Deepti Saini, and Subrata Sinha. "Phage Displayed scFv: pIII Scaffold May Fine Tune Binding Specificity." Hybridoma 28, no. 5 (October 2009): 327–31. http://dx.doi.org/10.1089/hyb.2009.0008.

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Muller, Bruno H., Florence Lafay, Caroline Demangel, Pierre Perrin, Noël Tordo, Anne Flamand, Pierre Lafaye, and Jean-Luc Guesdon. "Phage-displayed and soluble mouse scFv fragments neutralize rabies virus." Journal of Virological Methods 67, no. 2 (September 1997): 221–33. http://dx.doi.org/10.1016/s0166-0934(97)00099-2.

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Wind, Troels, Brian Stausbøl-Grøn, Svend Kjær, Liselotte Kahns, Kristian H. Jensen, and Brian F. C. Clark. "Retrieval of phage displayed scFv fragments using direct bacterial elution." Journal of Immunological Methods 209, no. 1 (November 1997): 75–83. http://dx.doi.org/10.1016/s0022-1759(97)00151-8.

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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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Naumann, Anja, Jörg Kahle, Nadia Reiss, Dirk Schwabe, Christine Heller, Thomas Klingebiel, and Christoph Königs. "Development of a Factor VIII-Specific Immunotherapy for Hemophilia A." Blood 120, no. 21 (November 16, 2012): 3361. http://dx.doi.org/10.1182/blood.v120.21.3361.3361.

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Abstract Abstract 3361 The development of neutralizing allo-antibodies against coagulation factor VIII (FVIII) is currently the most serious complication for hemophilia A patients that undergo FVIII replacement therapy. Non-hemophiliacs can spontaneously develop inhibitory auto-antibodies to FVIII, which results in a condition called acquired hemophilia A. The control of the allo- or autoimmune response to FVIII apparently includes the elicitation of an anti-idiotypic immune response. Immune tolerance induction (ITI) by frequent administration of high doses of FVIII is successful in most patients. The remaining up to 20% failing ITI face increased morbidity and mortality. To elucidate the capacity of single-chain variable antibody fragments (scFvs) for neutralization of inhibitory anti-FVIII antibodies (FVIII inhibitors), scFvs that specifically interact with strong inhibitory murine monoclonal anti-human FVIII antibodies (anti-hFVIII mAbs) were selected by screening synthetic scFv phage displayed libraries. Since the majority of inhibitory anti-hFVIII mAbs are directed against the FVIII A2 or C2 domain, selection of scFvs was performed for two anti-A2 and two anti-C2 mAbs. Affinity selection identified several specific, potential anti-idiotypic scFvs for each anti-hFVIII mAb, which were further analyzed. ScFvs expressed as IgG fusion protein bind to mAbs with affinities up to 0,1nM. Binding of mAbs to immobilized FVIII was inhibited via specific scFvs by more than 90%. In the functional Bethesda Assay FVIII activity was fully restored when corresponding anti-idiotypic scFvs were added to plasma spiked with FVIII-specific mAbs. In addition, the cross-reactivity of scFvs with heterologous mAbs specific for A2 and C2 was tested and confirmed the exclusive interaction of the selected scFvs with their respective mAb. Overall, anti-idiotypic scFvs specific for anti-hFVIII mAbs can be successfully selected from phage displayed libraries and efficiently neutralize FVIII inhibitors. ScFv anti-idiotypes may therefore facilitate the development of specific immunotherapies for hemophilia patients with inhibitors. Disclosures: No relevant conflicts of interest to declare.

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Stuknytė, Milda, Eeva-Christine Brockmann, Tuomas Huovinen, Simone Guglielmetti, Diego Mora, Valentina Taverniti, Stefania Arioli, Ivano De Noni, and Urpo Lamminmäki. "Lactobacillus helveticus MIMLh5-Specific Antibodies for Detection of S-Layer Protein in Grana Padano Protected-Designation-of-Origin Cheese." Applied and Environmental Microbiology 80, no. 2 (November 15, 2013): 694–703. http://dx.doi.org/10.1128/aem.03057-13.

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ABSTRACTSingle-chain variable-fragment antibodies (scFvs) have considerable potential in immunological detection and localization of bacterial surface structures. In this study, synthetic phage-displayed antibody libraries were used to select scFvs against immunologically active S-layer protein ofLactobacillus helveticusMIMLh5. After three rounds of panning, five relevant phage clones were obtained, of which four were specific for the S-layer protein ofL. helveticusMIMLh5 and one was also capable of binding to the S-layer protein ofL. helveticusATCC 15009. All five anti-S-layer scFvs were expressed inEscherichia coliXL1-Blue, and their specificity profiles were characterized by Western blotting. The anti-S-layer scFv PolyH4, with the highest specificity for the S-layer protein ofL. helveticusMIMLh5, was used to detect the S-layer protein in Grana Padano protected-designation-of-origin (PDO) cheese extracts by Western blotting. These results showed promising applications of this monoclonal antibody for the detection of immunomodulatory S-layer protein in dairy (and dairy-based) foods.

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Schmidt, Anja, Kerstin Brettschneider, Jörg Kahle, Aleksander Orlowski, Karin Becker-Peters, Diana Stichel, Jörg Schulze, et al. "Neutralisation of factor VIII inhibitors by anti-idiotypes isolated from phage-displayed libraries." Thrombosis and Haemostasis 116, no. 07 (January 2016): 32–41. http://dx.doi.org/10.1160/th15-12-0925.

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SummaryFollowing replacement therapy with coagulation factor VIII (FVIII), up to 30 % of haemophilia A patients develop FVIII-specific inhibitory antibodies (FVIII inhibitors). Immune tolerance induction (ITI) is not always successful, resulting in a need for alternative treatments for FVIII inhibitor-positive patients. As tolerance induction in the course of ITI appears to involve the formation of anti-idiotypes specific for anti-FVIII antibodies, such anti-idiotypes might be used to restore haemostasis in haemophilia A patients with FVIII inhibitors. We isolated antiidiotypic antibody fragments (scFvs) binding to murine FVIII inhibitors 2-76 and 2-77 from phage-displayed libraries. FVIII inhibitor/anti-idiotype interactions were very specific as no cross-reactivity with other FVIII inhibitors or isotype controls was observed. ScFvs blocked binding of FVIII inhibitors to FVIII and neutralised their cognate inhibitors in vitro and a monoclonal mouse model. In addition, scFv JkH5 specific for FVIII inhibitor 2-76 stained 2-76-producing hybridoma cells. JkH5 residues R52 and Y226, located in complementary determining regions, were identified as crucial for the JkH5/2-76 interaction using JkH5 alanine mutants. SPR spectroscopy revealed that JkH5 interacts with FVIII inhibitor 2-76 with nanomolar affinity. Thus, FVIII inhibitorspecific, high-affinity anti-idiotypes can be isolated from phagedisplayed libraries and neutralise their respective inhibitors. Furthermore, we show that anti-idiotypic scFvs might be utilised to specifically target inhibitor-specific B cells. Hence, a pool of anti-idiotypes could enable the reestablishment of haemostasis in the presence of FVIII inhibitors in patients or even allow the depletion of inhibitors by targeting inhibitor-specific B cell populations.

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Massion, P. P., T. V. Pedchenko, D. V. Parekh, and R. Mernaugh. "Selection of lung cancer-associated scFv antibodies from cultured cells." Journal of Clinical Oncology 27, no. 15_suppl (May 20, 2009): e22137-e22137. http://dx.doi.org/10.1200/jco.2009.27.15_suppl.e22137.

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e22137 Background: Lung cancer is the most common cause of cancer-related deaths in the world. There is a critical need for new strategies of early lung cancer detection. The identification of tumor-associated antigens and corresponding antibodies is one approach to discovery of diagnostic biomarkers. We used a large phage-displayed recombinant antibody library and normal human lung epithelial and non-small cell lung cancer cell lines to select for and identify recombinant antibodies specific for proteins expressed, or over-expressed, in lung cancer. Methods: The antibody library was used to select for recombinant scFv antibodies reactive with proteins present, or aberrantly expressed, in non-small cell lung cancer cell lines (A549, H549, H157, H23) in comparison to normal lung cell lines (BEAS-2B, 16-HBE, KT). Soluble scFv antibodies were obtained through 2 rounds of phage antibody cross-absorption (on normal cell lines) and selection (on non-small lung cancer cell lines). Soluble scFv were assayed by a high-throughput fluorometric microvolume assay technology (FMAT) against normal and cancer lung cell line proteins. ScFv antibodies selected by FMAT were evaluated further with Western blot-based assays. Results: More than 100 scFv antibodies identified by FMAT bound preferentially to proteins in lung cancer. Of these, 46 scFv were assayed by a high throughput Western slot blot immunoassay against pooled normal lung and lung cancer cell lysates. Eight scFv were assayed in Western blot against individual lung normal and non-small lung cancer cell line lysates. Four of these demonstrated differential binding to normal and cancer cell lysates. Conclusions: In summary, we were able to detect cancer-associated antigens in lung cancer cell lines using a phage display antibody library. In combination with high-throughput fluorescent and Western blot assays, four unique scFv antibodies were selected that differentially bound to normal and lung cancer cell lysates. These scFv will be tested as candidate biomarkers of lung cancer in independent tissue and serum samples from patient with and without lung cancer to determine utility for use in lung cancer diagnosis. No significant financial relationships to disclose.

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Cirino, Nick M., Daniele Sblattero, David Allen, Scott R. Peterson, James D. Marks, Paul J. Jackson, Andrew Bradbury, and Bruce E. Lehnert. "Disruption of Anthrax Toxin Binding with the Use of Human Antibodies and Competitive Inhibitors." Infection and Immunity 67, no. 6 (June 1, 1999): 2957–63. http://dx.doi.org/10.1128/iai.67.6.2957-2963.1999.

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ABSTRACT The protective antigen (PA83) of Bacillus anthracis is integral to the mechanism of anthrax toxicity. We have isolated a human single-chain Fv antibody fragment (scFv) that blocks binding of a fluorescently tagged protective antigen (PA) moiety to cell surface receptors. Several phage-displayed scFv were isolated from a naive library biopanned against PA83. Soluble, monomeric scFv were characterized for affinity and screened for their capacity to disrupt receptor-mediated binding of PA. Four unique scFv bound to PA83, as determined by surface plasmon resonance, the tightest binder exhibiting a Kd of 50 nM. Two scFv had similar affinities for natural PA83 and a novel, recombinant, 32-kDa carboxy-terminal PA fragment (PA32). Binding of scFv to green fluorescent protein fused to the amino-terminal 32-kDa fragment of B. anthracis edema factor, EGFP-EF32, was used to confirm specificity. Fusion of EGFP to PA32 facilitated development of a novel flow cytometric assay that showed that one of the scFv disrupted PA receptor binding. This method can now be used as a rapid assay for small molecule inhibitors of PA binding to cell receptors. The combined data presented suggest the potential utility of human scFv as prophylactics against anthrax poisoning. Moreover, recombinant PA32 may also be useful as a therapeutic agent to compete with anthrax toxins for cellular receptors during active infection.

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Nikfarjam, Sepideh, Mohammad Reza Tohidkia, Tayebeh Mehdipour, Ramin Soleimani, Ali Akbar Rahim Rahimi, and Mohammad Nouri. "Successful Application of Whole Cell Panning for Isolation of PhageAntibody Fragments Specific to Differentiated Gastric Cancer Cells." Advanced Pharmaceutical Bulletin 9, no. 4 (October 24, 2019): 624–31. http://dx.doi.org/10.15171/apb.2019.072.

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Purpose: Generation of antibodies which potentially discriminate between malignant andhealthy cells is an important prerequisite for early diagnosis and treatment of gastric cancer(GC). Comparative analysis of cell surface protein landscape will provide an experimental basisfor biomarker discovery, which is essential for targeted molecular therapies. This study aimedto isolate phage-displayed antibody fragments recognizing cell surface proteins, which weredifferentially expressed between two closely related GC cell lines, namely AGS and MKN-45.Methods: We selected and screened a semisynthetic phage-scFv library on AGS, MKN-45, andNIH-3T3 cell lines by utilizing a tailored selection scheme that was designed to isolate phagescFvsthat not only recognize the differentiated AGS cells but also distinguish them from NIH-3T3 fibroblasts and the poorly differentiated MKN-45 cells.Results: After four rounds of subtractive whole cell panning, 14 unique clones were identifiedby ELISA screening and nucleotide sequencing. For further characterization, we focused on fourphage-scFvs with strong signals in screening, and their specificity was confirmed by cell-basedELISA. Furthermore, the selected phage-scFvs were able to specifically stain AGS cells with38.74% (H1), 11.04% (D11), 76.93% (G11), and 69.03% (D1) in flow cytometry analysis whichsupported the ability of theses phage scFvs in distinguishing AGS from MKN-45 and NIH-3T3cells.Conclusion: Combined with other proteomic techniques, these phage-scFvs can be applied tomembrane proteome analysis and, subsequently, identification of novel tumor-related antigensmediating proliferation and differentiation of cells. Furthermore, such antibody fragments canbe exploited for diagnostic purposes as well as targeted drug delivery of GC.<br />

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Anisimov, R. L., O. A. Ershova, A. V. Ershov, M. A. Filatova, S. A. Katorkin, and V. M. Simonov. "Recombinant β-Glucocerebrosidase specific immunoaffinity ligands selected from phage-displayed combinatorial scFv libraries." Protein Expression and Purification 170 (June 2020): 105573. http://dx.doi.org/10.1016/j.pep.2020.105573.

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Pedchenko, T. V., R. Mernaugh, and P. P. Massion. "Tumor specific antibodies for the early diagnosis of lung cancer." Journal of Clinical Oncology 25, no. 18_suppl (June 20, 2007): 18068. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.18068.

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18068 Background: The survival rates in lung cancer vary significantly by stage, the earlier the better. However, only 16% of patients are diagnosed at stage I. This implies an urgent need to identify a panel of biomarkers for the early detection of lung cancer in order to offer an improve chance for cure. We hypothesized that a unique phage-display recombinant antibody library approach will allow us to capture from serum and evaluate cancer antibodies as novel diagnostic biomarkers of non-small cell lung cancer. Methods: Immunoglobulins G (IgG) were purified from 3 groups of pooled serum: 10 diagnosed with adenocarcinoma, 10 squamous carcinomas, and 10 controls. A naïve rodent scFv (single chain fragment variable) phage-displayed recombinant antibody library (∼2.9 x 109 members) was used to select scFv specific for cancer human IgG. Individual bacterial colonies obtained after two rounds of subtractive biopanning on purified normal IgG and cancer IgG were picked and induced to express soluble E-tagged ScFv antibodies. An ELISA was used to screen ScFv for binding activity to pooled normal and cancer serum IgG. Single scFv clones that showed preferential binding to cancer IgG in ELISA were further validated for binding specificity by dot blot. ScFv were assayed against individual serum samples obtained from 120 normal, 60 adenocarcinoma, 56 squamous carcinoma, and 35 other, non-small cell, lung cancers. Dot blot signal intensity was quantified using Typhoon 8600 imaging system. Results: Seventy-five individual bacterial clones produced scFv that, by ELISA, distinguished purified normal from cancer IgG from pooled serum samples. Selected scFv antibodies show significantly higher binding activity to cancer-related IgG as compared to normal IgG. Twenty-five scFv were further validated for binding activity in individual human serum samples by dot blot assays. Ten scFv clearly differentiated cancer immunoglobulins from normal in human serum by dot blot, with preferential binding either to adeno- or squamous carcinoma. Conclusions: In this preliminary dataset, we identified scFv antibodies that interact with immunoglobulins present in human cancer serum samples. We show that this is a feasible and promising approach to discover cancer-specific immunoglobulins as candidate diagnostic biomarkers. No significant financial relationships to disclose.

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Jeong, Sukyo, Hyun Joo Ahn, Kyung Jin Min, Jae Won Byun, Hyun Mi Pyo, Mi Young Park, Bok Kyung Ku, et al. "Phage Display Screening of Bovine Antibodies to Foot-and-Mouth Disease Virus and Their Application in a Competitive ELISA for Serodiagnosis." International Journal of Molecular Sciences 22, no. 9 (April 21, 2021): 4328. http://dx.doi.org/10.3390/ijms22094328.

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For serodiagnosis of foot-and-mouth disease virus (FMDV), monoclonal antibody (MAb)-based competitive ELISA (cELISA) is commonly used since it allows simple and reproducible detection of antibody response to FMDV. However, the use of mouse-origin MAb as a detection reagent is questionable, as antibody responses to FMDV in mice may differ in epitope structure and preference from those in natural hosts such as cattle and pigs. To take advantage of natural host-derived antibodies, a phage-displayed scFv library was constructed from FMDV-immune cattle and subjected to two separate pannings against inactivated FMDV type O and A. Subsequent ELISA screening revealed high-affinity scFv antibodies specific to a serotype (O or A) as well as those with pan-serotype specificity. When BvO17, an scFv antibody specific to FMDV type O, was tested as a detection reagent in cELISA, it successfully detected FMDV type O antibodies for both serum samples from vaccinated cattle and virus-challenged pigs with even higher sensitivity than a mouse MAb-based commercial FMDV type O antibody detection kit. These results demonstrate the feasibility of using natural host-derived antibodies such as bovine scFv instead of mouse MAb in cELISA for serological detection of antibody response to FMDV in the susceptible animals.

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Liu, Chang C., Antha V. Mack, Meng-Lin Tsao, Jeremy H. Mills, Hyun Soo Lee, Hyeryun Choe, Michael Farzan, Peter G. Schultz, and Vaughn V. Smider. "Protein evolution with an expanded genetic code." Proceedings of the National Academy of Sciences 105, no. 46 (November 11, 2008): 17688–93. http://dx.doi.org/10.1073/pnas.0809543105.

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We have devised a phage display system in which an expanded genetic code is available for directed evolution. This system allows selection to yield proteins containing unnatural amino acids should such sequences functionally outperform ones containing only the 20 canonical amino acids. We have optimized this system for use with several unnatural amino acids and provide a demonstration of its utility through the selection of anti-gp120 antibodies. One such phage-displayed antibody, selected from a naïve germline scFv antibody library in which six residues in VH CDR3 were randomized, contains sulfotyrosine and binds gp120 more effectively than a similarly displayed known sulfated antibody isolated from human serum. These experiments suggest that an expanded “synthetic” genetic code can confer a selective advantage in the directed evolution of proteins with specific properties.

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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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Yoo, Duck Kyun, Seung Ryul Lee, Yushin Jung, Haejun Han, Hwa Kyoung Lee, Jerome Han, Soohyun Kim, Jisu Chae, Taehoon Ryu, and Junho Chung. "Machine Learning-Guided Prediction of Antigen-Reactive In Silico Clonotypes Based on Changes in Clonal Abundance through Bio-Panning." Biomolecules 10, no. 3 (March 8, 2020): 421. http://dx.doi.org/10.3390/biom10030421.

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c-Met is a promising target in cancer therapy for its intrinsic oncogenic properties. However, there are currently no c-Met-specific inhibitors available in the clinic. Antibodies blocking the interaction with its only known ligand, hepatocyte growth factor, and/or inducing receptor internalization have been clinically tested. To explore other therapeutic antibody mechanisms like Fc-mediated effector function, bispecific T cell engagement, and chimeric antigen T cell receptors, a diverse panel of antibodies is essential. We prepared a chicken immune scFv library, performed four rounds of bio-panning, obtained 641 clones using a high-throughput clonal retrieval system (TrueRepertoireTM, TR), and found 149 antigen-reactive scFv clones. We also prepared phagemid DNA before the start of bio-panning (round 0) and, after each round of bio-panning (round 1–4), performed next-generation sequencing of these five sets of phagemid DNA, and identified 860,207 HCDR3 clonotypes and 443,292 LCDR3 clonotypes along with their clonal abundance data. We then established a TR data set consisting of antigen reactivity for scFv clones found in TR analysis and the clonal abundance of their HCDR3 and LCDR3 clonotypes in five sets of phagemid DNA. Using the TR data set, a random forest machine learning algorithm was trained to predict the binding properties of in silico HCDR3 and LCDR3 clonotypes. Subsequently, we synthesized 40 HCDR3 and 40 LCDR3 clonotypes predicted to be antigen reactive (AR) and constructed a phage-displayed scFv library called the AR library. In parallel, we also prepared an antigen non-reactive (NR) library using 10 HCDR3 and 10 LCDR3 clonotypes predicted to be NR. After a single round of bio-panning, we screened 96 randomly-selected phage clones from the AR library and found out 14 AR scFv clones consisting of 5 HCDR3 and 11 LCDR3 AR clonotypes. We also screened 96 randomly-selected phage clones from the NR library, but did not identify any AR clones. In summary, machine learning algorithms can provide a method for identifying AR antibodies, which allows for the characterization of diverse antibody libraries inaccessible by traditional methods.

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Watkins, Nicholas A., Lily M. Du, J. Paul Scott, Willem H. Ouwehand, and Cheryl A. Hillery. "Single-chain antibody fragments derived from a human synthetic phage-display library bind thrombospondin and inhibit sickle cell adhesion." Blood 102, no. 2 (July 15, 2003): 718–24. http://dx.doi.org/10.1182/blood-2002-11-3497.

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AbstractThe enhanced adhesion of sickle red blood cells (RBCs) to the vascular endothelium and subendothelial matrix likely plays a significant role in the pathogenesis of vaso-occlusion in sickle cell disease. Sickle RBCs have enhanced adhesion to the plasma and extracellular matrix protein thrombospondin-1 (TSP) under conditions of flow in vitro. In this study, we sought to develop antibodies that bind TSP from a highly diverse library of human single-chain Fv fragments (scFvs) displayed on filamentous phage. Following 3 rounds of phage selection of increasing stringency 6 unique scFvs that bound purified TSP by enzyme-linked immunosorbent assay were isolated. Using an in vitro flow adhesion assay, 3 of the 6 isolated scFvs inhibited the adhesion of sickle RBCs to immobilized TSP by more than 40% compared with control scFvs (P < .001). Furthermore, scFv TSP-A10 partially inhibited sickle RBC adhesion to activated endothelial cells (P < .005). Using TSP proteolytic fragments to map the binding site, we showed that 2 of the inhibitory scFvs bound an epitope in the calcium-binding domain or proximal cell-binding domain of TSP, providing evidence for the role of these domains in the adhesion of sickle RBCs to TSP. In summary, we have isolated a panel of scFvs that specifically bind to TSP and differentially inhibit sickle RBC adhesion to surface-bound TSP under flow conditions. These scFvs will be useful reagents for investigating the role of the calcium and cell-binding domains of TSP in sickle RBC adhesion.

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Chang, Seohee, Soohyun Kim, Jerome Han, Suji Ha, Hyunho Lee, Seo Woo Song, Daewon Lee, Sunghoon Kwon, Junho Chung, and Junhoi Kim. "A High-Throughput Single-Clone Phage Fluorescence Microwell Immunoassay and Laser-Driven Clonal Retrieval System." Biomolecules 10, no. 4 (March 29, 2020): 517. http://dx.doi.org/10.3390/biom10040517.

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Phage display is one of the most frequently used platform technologies utilized to screen and select therapeutic antibodies, and has contributed to the development of more than 10 therapeutic antibodies used in the clinic. Despite advantages like efficiency and low cost, it has intrinsic technical limitations, such as the asymmetrical amplification of the library after each round of biopanning, which is regarded as a reason for it yielding a very limited number of antigen binders. In this study, we developed a high-throughput single-clonal screening system comprised of fluorescence immunoassays and a laser-driven clonal DNA retrieval system using microchip technology. Using this system, from a single-chain variable fragment (scFv) library displayed on phages with a complexity of 5.21 × 105 harboring random mutations at five amino acid residues, more than 70,000 clones—corresponding to ~14% of the library complexity—were screened, resulting in 78 antigen-reactive scFv sequences with mutations restricted to the randomized residues. Our results demonstrate that this system can significantly reduce the number of biopanning rounds, or even eliminate the need for this process for libraries with lower complexity, providing an opportunity to obtain more diverse clones from the library.

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Hu, Zu-Quan, He-Ping Li, Jin-Long Liu, Sheng Xue, An-Dong Gong, Jing-Bo Zhang, and Yu-Cai Liao. "Production of a phage-displayed mouse ScFv antibody against fumonisin B1 and molecular docking analysis of their interactions." Biotechnology and Bioprocess Engineering 21, no. 1 (January 2016): 134–43. http://dx.doi.org/10.1007/s12257-015-0495-0.

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Stoyanova, Vishnya, Radoslav Aleksandrov, Maria Lukarska, Deyan Duhalov, Vasil Atanasov, and Svetla Petrova. "Recognition of Vipera ammodytes meridionalis neurotoxin vipoxin and its components using phage-displayed scFv and polyclonal antivenom sera." Toxicon 60, no. 5 (October 2012): 802–9. http://dx.doi.org/10.1016/j.toxicon.2012.06.003.

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Bagheri, Vahid, Foroogh Nejatollahi, Seyed Alireza Esmaeili, Amir Abbas Momtazi, Mohamad Motamedifar, and Amirhossein Sahebkar. "Neutralizing human recombinant antibodies against herpes simplex virus type 1 glycoproteins B from a phage-displayed scFv antibody library." Life Sciences 169 (January 2017): 1–5. http://dx.doi.org/10.1016/j.lfs.2016.11.018.

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Liu, Xin, Yixiang Xu, Wei Xiong, Bingnan Yin, Yuqian Huang, Junjun Chu, Changsheng Xing, et al. "Development of a TCR-like antibody and chimeric antigen receptor against NY-ESO-1/HLA-A2 for cancer immunotherapy." Journal for ImmunoTherapy of Cancer 10, no. 3 (March 2022): e004035. http://dx.doi.org/10.1136/jitc-2021-004035.

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BackgroundThe current therapeutic antibodies and chimeric antigen receptor (CAR) T cells are capable of recognizing surface antigens, but not of intracellular proteins, thus limiting the target coverage for drug development. To mimic the feature of T-cell receptor (TCR) that recognizes the complex of major histocompatibility class I and peptide on the cell surface derived from the processed intracellular antigen, we used NY-ESO-1, a cancer-testis antigen, to develop a TCR-like fully human IgG1 antibody and its derivative, CAR-T cells, for cancer immunotherapy.MethodsHuman single-chain variable antibody fragment (scFv) phage library (~10∧11) was screened against HLA-A2/NY-ESO-1 (peptide 157–165) complex to obtain target-specific antibodies. The specificity and affinity of those antibodies were characterized by flow cytometry, ELISA, biolayer interferometry, and confocal imaging. The biological functions of CAR-T cells were evaluated against target tumor cells in vitro. In vivo antitumor activity was investigated in a triple-negative breast cancer (TNBC) model and primary melanoma tumor model in immunocompromised mice.ResultsMonoclonal antibody 2D2 identified from phage-displayed library specifically bound to NY-ESO-1157-165 in the context of human leukocyte antigen HLA-A*02:01 but not to non-A2 or NY-ESO-1 negative cells. The second-generation CAR-T cells engineered from 2D2 specifically recognized and eliminated A2+/NY-ESO-1+tumor cells in vitro, inhibited tumor growth, and prolonged the overall survival of mice in TNBC and primary melanoma tumor model in vivo.ConclusionsThis study showed the specificity of the antibody identified from human scFv phage library and demonstrated the potential antitumor activity by TCR-like CAR-T cells both in vitro and in vivo, warranting further preclinical and clinical evaluation of the TCR-like antibody in patients. The generation of TCR-like antibody and its CAR-T cells provides the state-of-the-art platform and proof-of-concept validation to broaden the scope of target antigen recognition and sheds light on the development of novel therapeutics for cancer immunotherapy.

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Zhikui, Li, Guo Changcun, Nie Yongzhan, He Fengtian, Ren Xingling, Li Shujun, Han Zheyi, Han Ying, Wang Xin, and Fan Daiming. "Screening and Identification of Recombinant Anti-Idiotype Antibodies against Gastric Cancer and Colon Cancer Monoclonal Antibodies by a Phage-Displayed Single-Chain Variable Fragment Library." Journal of Biomolecular Screening 15, no. 3 (February 11, 2010): 308–13. http://dx.doi.org/10.1177/1087057109360252.

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Several monoclonal antibodies (McAbs) have been developed that show high sensitivity and specificity to gastric cancer and colorectal cancer. However, few of the antigens recognized by these antibodies have been identified. The authors now report the selection of anti-idiotype (anti-id) antibodies of MGb1 McAb against gastric cancer and MC5 McAb against colorectal cancer using phage-displayed single-chain variable fragment (ScFv) libraries. After purification, the anti-id antibodies were approximately 30 kd and could be recognized by MGb1/MC5 McAb. Anti-id antibodies significantly blocked the binding of MGb1 and MC5 to gastric cancer/colorectal cancer cells, respectively, suggesting that the antibodies were specific to MGb1 and MC5. Antibodies against gastric and colorectal cancer could be detected in mice at 6 weeks after immunization with the anti-id antibodies. At week 8, antibody titers reached 1:400. The anti-id antibodies may be useful as novel reagents for developing vaccines against gastric cancer and colorectal cancer.

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Planque, Stephanie, Xiaochang Peng, Sangeeta Karle, Yasuhiro Nishiyama, Yukie Mitsuda, Hiroaki Taguchi, Keri C. Smith, et al. "Adaptive development of catalytic antibodies to the gp120 superantigenic site in lupus and HIV infected patients (44.43)." Journal of Immunology 178, no. 1_Supplement (April 1, 2007): S56—S57. http://dx.doi.org/10.4049/jimmunol.178.supp.44.43.

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Abstract The B cell superantigenic site of the HIV protein gp120 (gp120SAg) is important in viral entry into host cells and gp120SAg binding IgG in the preimmune repertoire reduce the risk of infection. Like the gp120SAg binding function, the innate catalytic function of antibody (Ab) variable domains is subject to deterioration during B cell maturation, and gp120SAg binding Abs are not produced appreciably following infection. We report the selection of catalytic single chain Fv (scFv) and light chain (L chain) clones from the Ab repertoire of patients with lupus, a condition involving increased production of gp120SAg binding Abs. The Ab fragments were isolated by covalent phage selection using electrophilic gp120 analogs. Several selected fragments cleaved gp120 via a nucleophilic mechanism coordinated with noncovalent recognition of gp120(421–433), determined by electrophoretic assays. Certain clones neutralized HIV infection in cultured peripheral blood mononuclear cells (low μg/ml potency), including two scFv clones that neutralized primary clade B, C and D strains. In contrast to lupus patients, studies on polyclonal IgG and IgA from HIV infected patients did not support the presence of an amplified Ab response to gp120SAg. IgAs from a minority of infected subjects with no progression to AIDS 5.5 years following seroconversion displayed increased gp120 hydrolysis compared to rapid progressors (P<0.0001). These data suggest catalytic Abs as a protective anti-viral mechanism and open the way to considerations of immunogens that stimulate catalytic immunity as candidate vaccines.

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ZHANG, Jin-Yong. "ISOLATION AND CHARACTERIZATION OF SPECIFIC CLONES REACTED WITH MYXOBOLUS ROTUNDUS RELATED ANTIGEN FROM A COMBINATORIAL PHAGE DISPLAYED SCFV LIBRARY." Acta Hydrobiologica Sinica 32, no. 4 (November 20, 2008): 568–78. http://dx.doi.org/10.3724/sp.j.1035.2008.00568.

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de la Cruz, Silvia, Raquel Madrid, Aina García-García, Marcos Alcocer, Rosario Martín, Isabel González, and Teresa García. "Identification and characterisation of the proteins bound by specific phage-displayed recombinant antibodies (scFv) obtained against Brazil nut and almond extracts." Journal of the Science of Food and Agriculture 98, no. 5 (September 25, 2017): 1685–95. http://dx.doi.org/10.1002/jsfa.8640.

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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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Kala, Mrinalini, Kiran Bajaj, and Subrata Sinha. "Magnetic Bead Enzyme-Linked Immunosorbent Assay (ELISA) Detects Antigen-Specific Binding by Phage-Displayed scFv Antibodies That Are Not Detected with Conventional ELISA." Analytical Biochemistry 254, no. 2 (December 1997): 263–66. http://dx.doi.org/10.1006/abio.1997.2378.

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Guchhait, Prasenjit, Corie Shrimpton, Kochi Honke, Perumal Thiagarajan, and Jose A. Lopez. "A Critical Role for Membrane Sulfatide in Platelet Aggregation." Blood 104, no. 11 (November 16, 2004): 626. http://dx.doi.org/10.1182/blood.v104.11.626.626.

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Abstract Sulfatide (galactocylceramide 3′-sulfate) is a sulfated glycosphingolipid expressed on the surfaces of erythrocytes, leukocytes, platelets and a variety other cells, that is known to interact with several cell adhesion molecules involved in hemostasis, including von Willebrand factor (VWF), laminin, thrombospondin, P-selectin and β2-glycoprotein I. Because these ligands are involved in many platelet adhesive interactions, we hypothesize that membrane sulfatide plays an important role in these processes. To examine this, we have cloned and purified a sulfatide-specific single-chain variable fragment (scFv) antibody from a phage-display library constructed from mRNA taken from the lymphocytes of patients with systemic lupus erythematosis. This scFv, PA38, specifically bound sulfatide, and did not react with the related sphingolipids cerebroside, ceramide, or sphingomyelin, or the phospholipids phosphatidylserine, phosphatidylcholine, or phosphatidylethanolamine. Using this tool, we examined the role of sulfatide in platelet function. We observed that PA38 dose-dependently (at 5 and 10 μg/ml) inhibited the aggregation of human platelets induced by either collagen or ADP. A control scFv produced in a similar manner had no effect. Furthermore, PA38 delayed platelet plug formation by 23 sec (with collagen-ADP agonist) and 46 sec (with collagen-epinephrine) in whole blood from normal human donors, as measured in a platelet function analyzer, PFA-100 (Dade Behring). Further, to verify that this was a sulfatide-specific effect, we compared collagen-induced platelet aggregation in normal mice to that of mice deficient in cerebroside sulfotransferase (CST)—a critical enzyme in the sulfatide synthetic pathway. The CST−/− mice fail to express sulfatide on the cell surface, and displayed defective platelet aggregation. Consistent with this, the PA38 also significantly inhibited collagen-induce platelet aggregation in wild-type mice. Given the importance of lipid rafts in signaling and adhesive processes, we looked for the localization of sulfatide in these membrane microdomains. Indeed, we found that sulfatide is enriched in lipid rafts suggesting a role for sulfatide in lipid-raft mediated events. Thus, we provide evidence for a key role of a membrane lipid, sulfatide in the adhesive interactions involved in platelet function. With one notable exception, the key adhesive roles in platelet-platelet interaction have all previously been assigned to proteins.

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Hennig, Ewa E., Ray Mernaugh, Jennifer Edl, Ping Cao, and Timothy L. Cover. "Heterogeneity among Helicobacter pylori Strains in Expression of the Outer Membrane Protein BabA." Infection and Immunity 72, no. 6 (June 2004): 3429–35. http://dx.doi.org/10.1128/iai.72.6.3429-3435.2004.

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ABSTRACT The BabA adhesin of Helicobacter pylori is an outer membrane protein that binds to the fucosylated Lewis b histo-blood group antigen on the surface of gastric epithelial cells. We screened a phage-displayed ScFv (single-chain fragment variable) recombinant antibody library for antibodies reactive with a recombinant BabA fragment and identified two such antibodies. Each antibody recognized an ∼75-kDa protein present in wild-type H. pylori strain J99 but absent from an isogenic babA mutant strain. An immunoreactive BabA protein was detected by at least one of the antibodies in 18 (46%) of 39 different wild-type H. pylori strains and was detected more commonly in cagA-positive strains than in cagA-negative strains. Numerous amino acid polymorphisms were detected among BabA proteins expressed by different strains, with the greatest diversity occurring in the middle region of the proteins. Among the 18 strains that expressed a detectable BabA protein, there was considerable variation in the level of binding to Lewis b in vitro. Heterogeneity among H. pylori strains in expression of the BabA protein may be a factor that contributes to differing clinical outcomes among H. pylori-infected humans.

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Tohidkia, Mohammad R., Maryam Sepehri, Shirin Khajeh, Jaleh Barar, and Yadollah Omidi. "Improved Soluble ScFv ELISA Screening Approach for Antibody Discovery Using Phage Display Technology." SLAS DISCOVERY: Advancing the Science of Drug Discovery 22, no. 8 (March 27, 2017): 1026–34. http://dx.doi.org/10.1177/2472555217701059.

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

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Chahboun, Siham, Michael Hust, Yvonne Liu, Thibaut Pelat, Sebastian Miethe, Saskia Helmsing, Russell GA Jones, Dorothea Sesardic, and Philippe Thullier. "Isolation of a nanomolar scFv inhibiting the endopeptidase activity of botulinum toxin A, by single-round panning of an immune phage-displayed library of macaque origin." BMC Biotechnology 11, no. 1 (2011): 113. http://dx.doi.org/10.1186/1472-6750-11-113.

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KERMANI, POUNEH, LUC PÉLOQUIN, and JACQUELINE LAGACÉ. "Production of ScFv Antibody Fragments Following Immunization with a Phage-Displayed Fusion Protein and Analysis of Reactivity to Surface-Exposed Epitopes of the Protein F ofPseudomonas aeruginosaby Cytofluorometry." Hybridoma 14, no. 4 (August 1995): 323–28. http://dx.doi.org/10.1089/hyb.1995.14.323.

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Griep, Remko A., Marcel Prins, Charlotte van Twisk, Hans J. H. G. Keller, Randolf J. Kerschbaumer, Richard Kormelink, Rob W. Goldbach, and Arjen Schots. "Application of Phage Display in Selecting Tomato spotted wilt virus-Specific Single-Chain Antibodies (scFvs) for Sensitive Diagnosis in ELISA." Phytopathology® 90, no. 2 (February 2000): 183–90. http://dx.doi.org/10.1094/phyto.2000.90.2.183.

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A panel of recombinant single-chain antibodies (scFvs) against structural proteins of Tomato spotted wilt virus (TSWV) was retrieved from a human combinatorial scFv antibody library using the novel phage display technique. After subcloning the encoding DNA sequences in the expression vector pSKAP/S, which allowed the scFvs to be expressed as alkaline phosphatase fusion proteins, 17 different scFv antibodies were obtained. Of these, 12 scFvs were directed against the nucleoprotein (N) and 5, putatively, against the glycoproteins (G1 and G2). Five of the N-specific antibodies cross-reacted with two other tospoviruses (Tomato chlorotic spot virus and Groundnut ringspot virus), but none recognized the more distantly related tospoviruses Impatiens necrotic spot virus, Watermelon silverleaf mottle virus, Iris yellow spot virus, or Physalis severe mottle virus. The successful use of one of the antibodies as coating and detection reagent in a double-antibody sandwich enzyme-linked immunosorbent assay showed the potential of the phage display system in obtaining antibodies for routine TSWV diagnosis.

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Li, Wei, Chuan Chen, Aleksandra Drelich, David R. Martinez, Lisa E. Gralinski, Zehua Sun, Alexandra Schäfer, et al. "Rapid identification of a human antibody with high prophylactic and therapeutic efficacy in three animal models of SARS-CoV-2 infection." Proceedings of the National Academy of Sciences 117, no. 47 (November 2, 2020): 29832–38. http://dx.doi.org/10.1073/pnas.2010197117.

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Effective therapies are urgently needed for the SARS-CoV-2/COVID-19 pandemic. We identified panels of fully human monoclonal antibodies (mAbs) from large phage-displayed Fab, scFv, and VH libraries by panning against the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. A high-affinity Fab was selected from one of the libraries and converted to a full-size antibody, IgG1 ab1, which competed with human ACE2 for binding to RBD. It potently neutralized replication-competent SARS-CoV-2 but not SARS-CoV, as measured by two different tissue culture assays, as well as a replication-competent mouse ACE2-adapted SARS-CoV-2 in BALB/c mice and native virus in hACE2-expressing transgenic mice showing activity at the lowest tested dose of 2 mg/kg. IgG1 ab1 also exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection. The mechanism of neutralization is by competition with ACE2 but could involve antibody-dependent cellular cytotoxicity (ADCC) as IgG1 ab1 had ADCC activity in vitro. The ab1 sequence has a relatively low number of somatic mutations, indicating that ab1-like antibodies could be quickly elicited during natural SARS-CoV-2 infection or by RBD-based vaccines. IgG1 ab1 did not aggregate, did not exhibit other developability liabilities, and did not bind to any of the 5,300 human membrane-associated proteins tested. These results suggest that IgG1 ab1 has potential for therapy and prophylaxis of SARS-CoV-2 infections. The rapid identification (within 6 d of availability of antigen for panning) of potent mAbs shows the value of large antibody libraries for response to public health threats from emerging microbes.

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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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Liew, Pei Xiong, Feng Ge, Charles Gullo, Gerrard KH Teoh, and William YK Hwang. "Use of Phage Display to Isolate Specific Human Monoclonal Antibody Fragments Against a Potential Target for Multiple Myeloma." Annals of the Academy of Medicine, Singapore 38, no. 7 (July 5, 2009): 621–29. http://dx.doi.org/10.47102/annals-acadmedsg.v38n7p621.

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Introduction: Multiple myeloma (MM), a malignancy of plasma cells, accounts for 10% of all haematological malignancies and is currently incurable. Although it can be treated, the disease tends to relapse after several years and becomes increasingly resistant to conventional therapy. Investigations into using humoral therapy for MM are now underway with a view that novel therapeutic agents may provide a more targeted therapy for MM. Materials and Methods: Here, phage display, a faster and more efficient method compared to classical hybridoma fusion technology, was used as a proof-of-concept to isolate several single-chain Fragment variables (scFv) against Ku86. Results: Anti-Ku86 polyclonal scFvs biopanning was successful where third round scFvs (A450~1.1) showed a 1/3 increase in binding as compared to the first round scFvs (A450~0.4) with 100ug/mL of antigen (purified human Ku86). Subsequent selection and verification of monoclonal antibodies using third round biopanning revealed 4 good affinity binding clones ranging from A450~0.1 to A450~0.15 on 12.5ug/mL of antigen as compared to low binders (A450~0.07) and these antibodies bind to Ku86 in a specific and dose-dependent manner. Comparative studies were also performed with commercially available murine antibodies and results suggest that 2 of the clones may bind close to the following epitopes aa506-541 and aa1-374. Conclusions: These studies using phage display provide an alternative and viable method to screen for antibodies quickly and results show that good affinity antibodies against Ku86 have been successfully isolated and they can be used for further studies on MM and form the basis for further development as anti-cancer therapeutic agents. Key words: Antibody isolation, Ku86, Phage display, ScFv

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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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Mendoza-Salazar, Ivette, Keyla M. Gómez-Castellano, Edith González-González, Ramsés Gamboa-Suasnavart, Stefany D. Rodríguez-Luna, Giovanni Santiago-Casas, María I. Cortés-Paniagua, Sonia M. Pérez-Tapia, and Juan C. Almagro. "Anti-SARS-CoV-2 Omicron Antibodies Isolated from a SARS-CoV-2 Delta Semi-Immune Phage Display Library." Antibodies 11, no. 1 (February 10, 2022): 13. http://dx.doi.org/10.3390/antib11010013.

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This report describes the discovery and characterization of antibodies with potential broad SARS-CoV-2 neutralization profiles. The antibodies were obtained from a phage display library built with the VH repertoire of a convalescent COVID-19 patient who was infected with SARS-CoV-2 B.1.617.2 (Delta). The patient received a single dose of Ad5-nCoV vaccine (Convidecia™, CanSino Biologics Inc.) one month before developing COVID-19 symptoms. Four synthetic VL libraries were used as counterparts of the immune VH repertoire. After three rounds of panning with SARS-CoV-2 receptor-binding domain wildtype (RBD-WT) 34 unique scFvs, were identified, with 27 cross-reactive for the RBD-WT and RBD Delta (RBD-DT), and seven specifics for the RBD-WT. The cross-reactive scFvs were more diverse than the RBD-WT specific ones, being encoded by several IGHV genes from the IGHV1 and IGHV3 families combined with short HCDR3s. Six cross-reactive scFvs and one RBD-WT specific scFv were converted to human IgG1 (hIgG1). Out of the seven antibodies, six blocked the RBD-WT binding to angiotensin converting enzyme 2 (ACE2), suggesting these antibodies may neutralize the SARS-CoV-2 infection. Importantly, one of the antibodies also recognized the RBD from the B.1.1.529 (Omicron) isolate, implying that the VH repertoire of the convalescent patient would protect against SARS-CoV-2 Wildtype, Delta, and Omicron. From a practical viewpoint, the triple cross-reactive antibody provides the substrate for developing therapeutic antibodies with a broad SARS-CoV-2 neutralization profile.

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Zhang, Yixuan, Yue Song, Haojie Ren, Quan Zeng, Yixin Yuan, Lu Xia, and Zhanyong Wei. "Preparation of a Single-Chain Antibody against Nucleocapsid Protein of Porcine Deltacoronavirus by Phage Display Technology." Viruses 14, no. 4 (April 8, 2022): 772. http://dx.doi.org/10.3390/v14040772.

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Porcine deltacoronavirus (PDCoV) mainly causes severe diarrhea and intestinal pathological damage in piglets and poses a serious threat to pig farms. Currently, no effective reagents or vaccines are available to control PDCoV infection. Single-chain fragment variable (scFv) antibodies can effectively inhibit virus infection and may be a potential therapeutic reagent for PDCoV treatment. In this study, a porcine phage display antibody library from the peripheral blood lymphocytes of piglets infected with PDCoV was constructed and used to select PDCoV-specific scFv. The library was screened with four rounds of biopanning using the PDCoV N protein, and the colony with the highest affinity to the PDCoV N protein was obtained (namely, N53). Then, the N53-scFv gene fragment was cloned into plasmid pFUSE-hIgG-Fc2 and expressed in HEK-293T cells. The scFv-Fc antibody N53 (namely, scFv N53) was purified using Protein A-sepharose. The reactive activity of the purified antibody with the PDCoV N protein was confirmed by indirect enzyme-linked immunosorbent assay (ELISA), western blot and indirect immunofluorescence assay (IFA). Finally, the antigenic epitopes that the scFv N53 recognized were identified by a series of truncated PDCoV N proteins. The amino acid residues 82GELPPNDTPATTRVT96 of the PDCoV N protein were verified as the minimal epitope that can be recognized by the scFv-Fc antibody N53. In addition, the interaction between the scFv-Fc antibody N53 and the PDCoV N protein was further analyzed by molecule docking. In conclusion, our research provides some references for the treatment and prevention of PDCoV.

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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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Lee, Myung-Shin, Myung-Hee Kwon, Sun Park, Ho-Joon Shin, and Hyung-Il Kim. "Terminal Protein-specific scFv Production by Phage Display." Immune Network 3, no. 2 (2003): 126. http://dx.doi.org/10.4110/in.2003.3.2.126.

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Noronha, Elvyra J., Xinhui Wang, Smruti A. Desai, Toshiro Kageshita, and Soldano Ferrone. "Limited Diversity of Human scFv Fragments Isolated by Panning a Synthetic Phage-Display scFv Library with Cultured Human Melanoma Cells." Journal of Immunology 161, no. 6 (September 15, 1998): 2968–76. http://dx.doi.org/10.4049/jimmunol.161.6.2968.

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Abstract To broaden the specificity of the Abs recognizing human melanoma-associated Ags (MAAs), we have isolated human single-chain fragment of the V region (scFv) fragments by panning the synthetic phage Ab library (#1) with the human melanoma cell lines S5 and SK-MEL-28. All of the isolated scFv fragments reacted with the mouse mAb defined high molecular weight melanoma-associated Ag (HMW-MAA). scFv #70 immunoprecipitates the two characteristic subunits of HMW-MAA, while scFv #28 only immunoprecipitates its large subunit. These results challenge the current view regarding the structure of HMW-MAA and indicate that it consists of two independent subunits. The human scFv fragments share some similarities with the mouse anti-HMW-MAA mAb. Like mAb 149.53 and 225.28, scFv #28 reacts with rat B49 neural cells that express a homologue of HMW-MAA. scFv #70 reacts with a determinant that is spatially close to the one identified by mAbs 149.53, VT68.2, and VT86. Besides suggesting similarities in the recognition of human melanoma cells by the mouse and human Ab repertoire, these results indicate that the Abs isolated from synthetic Ab libraries resemble those that are found in natural Ab repertoires. The restricted diversity of the scFv fragments that were isolated by panning synthetic Ab libraries with different melanoma cell lines suggests that certain Ags, like HMW-MAA, are immunodominant in vitro. This phenomenon, which parallels the in vivo immunodominance of certain Ags, implies that the antigenic profile of the cells used for panning determines the specificity of the preponderant population of isolated Abs.

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Japolla, Greice, Ana Flávia Batista Penido, Greyciele Rodrigues Almeida, Luiz Artur Mendes Bataus, Jair Pereira Cunha Junior, Cristina Ribeiro, Sinji Borges Ferreira Tauhata, and Guilherme Rocha Lino de Souza. "Characterization of Monoclonal Antibodies against Bovine herpesvirus type 1 selected by Phage Display Technology." Semina: Ciências Agrárias 38, no. 6 (November 23, 2017): 3915. http://dx.doi.org/10.5433/1679-0359.2017v38n6p3915.

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The specificity of monoclonal antibodies (mAbs) to desired targets makes these molecules suitable for therapeutic and diagnostic uses against a wide range of pathogens. Phage display antibody libraries offer one method by which mAbs can be selected for, without the use of conventional hybridoma technology. In this work, phage display technology was used to construct, select and characterize a combinatorial single chain fragment variable (scFv) antibody library against bovine herpesvirus type 1 (BoHV-1) from the immune repertoire of chickens immunized with the virus. In silico analysis of the hypervariable domains of the antibody heavy chains revealed a high frequency of scFv fragments with low variability, suggesting that selection had probably been carried out and favored by a few im-munogenic viral antigens. The reactivity of the scFv fragments selected against BoHV-1 was demon-strated by Phage-ELISA. A significant increase in antibody reactivity to the target was observed after six rounds of library selection, showing its potential use as a molecule for BoHV-1 diagnosis. The strategy described here opens up a field for the use of phage display as a tool for selection of mono-clonal antibodies that could be used for theranostic applications against infectious and parasitic dis-eases of veterinary interest.

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Costa, Lourena E., Patrícia T. Alves, Ana Paula Carneiro, Ana C. S. Dias, Patrícia T. Fujimura, Galber R. Araujo, Grasiele S. V. Tavares, et al. "Leishmania infantum β-Tubulin Identified by Reverse Engineering Technology through Phage Display Applied as Theranostic Marker for Human Visceral Leishmaniasis." International Journal of Molecular Sciences 20, no. 8 (April 12, 2019): 1812. http://dx.doi.org/10.3390/ijms20081812.

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Two Leishmania infantum mimotopes (B10 and C01) identified by phage display showed to be antigenic and immunogenic for visceral (VL) and tegumentary (TL) leishmaniasis; however, their biological targets in the parasites have not been identified. The aim of the present study was to investigate the native antigens expressing both mimotopes, and to use them in distinct immunological assays. For this, a subtractive phage display technology was used, where a combinatorial library of single-chain variable fragments (scFv) was employed and the most reactive monoclonal antibodies for each target were captured, being the target antigens identified by mass spectrometry. Results in immunoblotting and immunoprecipitation assays showed that both monoclonal scFvs antibodies identified the β-tubulin protein as the target antigen in L. infantum. To validate these findings, the recombinant protein was cloned, purified and tested for the serodiagnosis of human leishmaniasis, and its immunogenicity was evaluated in PBMC derived from healthy subjects and treated or untreated VL patients. Results showed high diagnostic efficacy, as well as the development of a specific Th1 immune response in the cell cultures, since higher IFN-γ and lower IL-10 production was found.

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Hawlisch, Heiko, Ronald Frank, Meike Hennecke, Melanie Baensch, Bettina Sohns, Lubomir Arseniev, Wilfried Bautsch, Axel Kola, Andreas Klos, and Jörg Köhl. "Site-Directed C3a Receptor Antibodies from Phage Display Libraries." Journal of Immunology 160, no. 6 (March 15, 1998): 2947–58. http://dx.doi.org/10.4049/jimmunol.160.6.2947.

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Abstract Recent cloning of the human C3a receptor (C3aR) revealed that this receptor belongs to the large family of rhodopsin-type receptors. A unique feature of the C3aR is the large second extracellular loop comprising about 175 amino acid residues. We constructed combinatorial phage Ab libraries expressing single chain Fv Abs from BALB/c mice immunized with the affinity-purified second extracellular loop of the C3aR, fused to glutathione-S-transferase. A panel of anti-C3aR single chain Fv fragments (scFvs) was selected after four rounds of panning using the second extracellular loop of the C3aR, fused to the maltose binding protein as Ag. Sequencing of the clones obtained revealed three different groups of scFvs, the epitopes of which were mapped to two distinct regions within the loop, i.e., positions 185 to 193 and 218 to 226, representing the immunodominant domains of the loop. By flow cyotmetric analyses, the scFvs bound to RBL-2H3 cells transfected with the C3aR, but not to cells transfected with the C5aR or to nontransfected RBL-2H3 cells. In addition, the scFvs bound to the human mast cell line HMC-1. Immunofluorescence studies showed C3aR expression on polymorphonuclear granulocytes and monocytes, but not on lymphocytes. In addition, no C3aR expression was observed on human erythrocytes or platelets. Surprisingly, none of the scFvs alone or in combination inhibited C3a-induced Ca2+ mobilization from RBL-2H3 cells transfected with the C3aR. In addition, C3a did not displace binding of the scFvs to the receptor, strongly suggesting that the N-terminal part of the second extracellular loop is not involved in ligand binding.

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Kennel, Stephen J., Trish Lankford, Linda Foote, Melissa Wall, and Sandra Davern. "Phage Display Selection of scFv to Murine Endothelial Cell Membranes." Hybridoma and Hybridomics 23, no. 4 (August 2004): 205–11. http://dx.doi.org/10.1089/1536859041651295.

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Xia, Jinsong, Hao Bi, Qin Yao, Shen Qu, and Yiqiang Zong. "Construction of human ScFv phage display library against ovarian tumor." Journal of Huazhong University of Science and Technology 26, no. 5 (October 2006): 497–99. http://dx.doi.org/10.1007/s11596-006-0502-y.

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Journal articles on the topic 'Phage displayed scFv'

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