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https://www.readbyqxmd.com/read/29727718/preparation-of-single-chain-antibody-against-vp3-protein-of-duck-hepatitis-virus-type-1-by-phage-display-technology
#1
Yongjuan Wang, Pingfu Cui, Shanyuan Zhu, Ting Meng, Fuxing Hao, Guoqiang Zhu, Weiyong Zuo
To construct phage antibody library for VP3 protein of duck hepatitis virus type 1(DHAV-1) and pan the specific single-chain variable fragment antibody (scFv), total RNA was extracted from the protein VP3- immunized mice spleen., vp3 gene encoding VP3 protein was amplified from the genome of DHAV-1 by RT-PCR method for the following recombinant pET-VP3 construction, immunogenic VP3 expression and purification, and combined with SOE-PCR method to complete the assembly of scFv. The scFv gene was cloned into pCANTAB5E vector for phage antibody library construction...
May 1, 2018: Journal of Virological Methods
https://www.readbyqxmd.com/read/29723982/scfvs-as-allosteric-inhibitors-of-vegfr-2-novel-tools-to-harness-vegf-signaling
#2
Kurt Ballmer-Hofer, Caroline A C Hyde, Thomas Schleier, Dragana Avramovic
Vascular Endothelial Growth Factor Receptor 2 (VEGFR-2) is the main mediator of angiogenic signaling in endothelial cells and a primary responder to VEGF. VEGF dependent VEGFR-2 activation regulates endothelial cell migration and proliferation, as well as vessel permeability. VEGF is presented as an antiparallel homodimer, and its binding to VEGFR-2 brings two receptors in close proximity. Downstream signaling is triggered by receptor dimerization, kinase activation, and receptor internalization. Our aim was to further investigate allosteric inhibition using binders targeting extracellular subdomains 4⁻7 of VEGFR-2 as an alternative to existing anti-angiogenic therapies, which rely on neutralizing VEGF or blocking of the ligand-binding site on the receptor...
May 1, 2018: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/29714016/array-in-well-epitope-mapping-of-phage-displayed-antibodies
#3
Urpo Lamminmäki, Gaurav Batra, Petri Saviranta
Novel affinity reagents, such as single chain (scFv) antibody fragments, can be generated by isolating them from recombinant protein libraries using phage display selection. A successful selection process against a target protein can produce a number of binder candidates among which the desired binders are identified by screening and characterization of individual clones. Obtaining information on the binding properties, such as the binding epitope, already during the screening step helps to choose the most useful candidates for further development at early phase saving time and resources...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29694034/site-specific-antibody-functionalization-using-tetrazine-styrene-cycloaddition
#4
Benjamin J Umlauf, Kalie A Mix, Vanessa A Grosskopf, Ronald T Raines, Eric V Shusta
Biologics, such as antibody-drug conjugates, are becoming mainstream therapeutics. Consequently, methods to functionalize biologics without disrupting their native properties are essential for identifying, characterizing, and translating candidate biologics from the bench to clinical practice. Here, we present a method for site-specific, carboxy-terminal modification of single-chain antibody fragments (scFvs). ScFvs displayed on the surface of yeast were isolated and functionalized by combining intein-mediated expressed protein ligation (EPL) with inverse electron-demand Diels-Alder (IEDDA) cycloaddition using a styrene-tetrazine pair...
May 3, 2018: Bioconjugate Chemistry
https://www.readbyqxmd.com/read/29678657/development-and-evaluation-of-an-optimal-human-single-chain-variable-fragment-derived-bcma-targeted-car-t-cell-vector
#5
Eric L Smith, Mette Staehr, Reed Masakayan, Ishan J Tatake, Terence J Purdon, Xiuyan Wang, Pei Wang, Hong Liu, Yiyang Xu, Sarah C Garrett-Thomson, Steven C Almo, Isabelle Riviere, Cheng Liu, Renier J Brentjens
B cell maturation antigen (BCMA) has recently been identified as an important multiple myeloma (MM)-specific target for chimeric antigen receptor (CAR) T cell therapy. In CAR T cell therapy targeting CD19 for lymphoma, host immune anti-murine CAR responses limited the efficacy of repeat dosing and possibly long-term persistence. This clinically relevant concern can be addressed by generating a CAR incorporating a human single-chain variable fragment (scFv). We screened a human B cell-derived scFv phage display library and identified a panel of BCMA-specific clones from which human CARs were engineered...
March 28, 2018: Molecular Therapy: the Journal of the American Society of Gene Therapy
https://www.readbyqxmd.com/read/29669811/linker-engineering-in-anti-tag-72-antibody-fragments-optimizes-biophysical-properties-serum-half-life-and-high-specificity-tumor-imaging
#6
Nicholas E Long, Brandon J Sullivan, Haiming Ding, Stephanie R Doll, Michael A Ryan, Charles L Hitchcock, Edward W Martin, Krishan Kumar, Michael F Tweedle, Thomas J Magliery
Antibody (Ab) fragments have great clinical potential as cancer therapeutics and diagnostics. Their small size allows for fast clearance from blood, low immunoreactivity, better tumor penetration, and simpler engineering and production. The smallest fragment derived from a full-length IgG that retains binding to its antigen, the single-chain variable fragment (scFV), is engineered by fusing the variable light and variable heavy domains with a peptide linker. Along with switching the domain orientation, altering the length and amino acid sequence of the linker can significantly affect scFV binding, stability, quaternary structure, and other biophysical properties...
April 18, 2018: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/29575477/ribosome-display-and-selection-of-scfvs-effectively-inhibit-growth-and-progression-of-microspheres-in-vitro-and-in-vivo
#7
Shangke Huang, Lu Feng, Gaili An, Xiaojin Zhang, Zixuan Zhao, Rui Han, Fuxi Lei, Yujiao Zhang, Anqi Luo, Xin Jing, Lin Zhao, Shanzhi Gu, Xinhan Zhao, Lingxiao Zhang
Distinguishing the surface markers of cancer stem cells (CSCs) is a useful method for early diagnosis and treatment of tumours, since CSCs may participate in tumourigenesis and metastasis by migrating into the circulatory system. However, the potential targets of CSCs are expressed at low levels in the natural state and are always changing. Thus, dynamic screening has been demonstrated to be an effective measure for exploring CSCs markers. In recent years, diverse scFvs have been widely used in immunotherapy...
March 25, 2018: Cancer Science
https://www.readbyqxmd.com/read/29507671/vegfr2-specific-fncar-effectively-redirects-the-cytotoxic-activity-of-t-cells-and-yt-nk-cells
#8
Sergey V Kulemzin, Andrey A Gorchakov, Anton N Chikaev, Valeriya V Kuznetsova, Olga Y Volkova, Daria A Matvienko, Alexey V Petukhov, Andrey Y Zaritskey, Alexandr V Taranin
T and NK cells armed with chimeric antigen receptors (CAR) are promising tools for the specific elimination of cancer cells. In most CAR designs implemented to date, the recognition of target cells is mediated by single-chain variable fragments (scFvs) derived from murine monoclonal antibodies. This format, however, has a number of limitations, including its relatively large size and potential immunogenicity in humans. In this study, we explored the feasibility of using human fibronectin type III domains (Fn3) as the antigen recognition domain in CARs...
February 6, 2018: Oncotarget
https://www.readbyqxmd.com/read/29494481/a-three-monoclonal-antibody-combination-potently-neutralizes-multiple-botulinum-neurotoxin-serotype-e-subtypes
#9
Consuelo Garcia-Rodriguez, Ali Razai, Isin N Geren, Jianlong Lou, Fraser Conrad, Wei-Hua Wen, Shauna Farr-Jones, Theresa J Smith, Jennifer L Brown, Janet C Skerry, Leonard A Smith, James D Marks
Human botulism is most commonly caused by botulinum neurotoxin (BoNT) serotypes A, B, and E. For this work, we sought to develop a human monoclonal antibody (mAb)-based antitoxin capable of binding and neutralizing multiple subtypes of BoNT/E. Libraries of yeast-displayed single chain Fv (scFv) antibodies were created from the heavy and light chain variable region genes of humans immunized with pentavalent-toxoid- and BoNT/E-binding scFv isolated by Fluorescence-Activated Cell Sorting (FACS). A total of 10 scFv were isolated that bound one or more BoNT/E subtypes with nanomolar-level equilibrium dissociation constants (KD )...
March 1, 2018: Toxins
https://www.readbyqxmd.com/read/29491415/rational-design-of-a-trispecific-antibody-targeting-the-hiv-1-env-with-elevated-anti-viral-activity
#10
James J Steinhardt, Javier Guenaga, Hannah L Turner, Krisha McKee, Mark K Louder, Sijy O'Dell, Chi-I Chiang, Lin Lei, Andrey Galkin, Alexander K Andrianov, Nicole A Doria-Rose, Robert T Bailer, Andrew B Ward, John R Mascola, Yuxing Li
HIV-1 broadly neutralizing antibodies (bNAbs) are being explored as passively administered therapeutic and preventative agents. However, the extensively diversified HIV-1 envelope glycoproteins (Env) rapidly acquire mutations to evade individual bNAbs in monotherapy regimens. The use of a "single" agent to simultaneously target distinct Env epitopes is desirable to overcome viral diversity. Here, we report the use of tandem single-chain variable fragment (ScFv) domains of two bNAbs, specific for the CD4-binding site and V3 glycan patch, to form anti-HIV-1 bispecific ScFvs (Bi-ScFvs)...
February 28, 2018: Nature Communications
https://www.readbyqxmd.com/read/29484477/screening-for-single-chain-variable-fragment-antibodies-against-multiple-cry1-toxins-from-an-immunized-mouse-phage-display-antibody-library
#11
Sa Dong, Zongyi Bo, Cunzheng Zhang, Jianguo Feng, Xianjin Liu
Single-chain variable fragment (scFv) is a kind of antibody that possess only one chain of the complete antibody while maintaining the antigen-specific binding abilities and can be expressed in prokaryotic system. In this study, scFvs against Cry1 toxins were screened out from an immunized mouse phage displayed antibody library, which was successfully constructed with capacity of 6.25 × 107  CFU/mL. Using the mixed and alternative antigen coating strategy and after four rounds of affinity screening, seven positive phage-scFvs against Cry1 toxins were selected and characterized...
April 2018: Applied Microbiology and Biotechnology
https://www.readbyqxmd.com/read/29453518/chimeric-antigen-receptors-with-human-scfvs-preferentially-induce-t-cell-anti-tumor-activity-against-tumors-with-high-b7h6-expression
#12
Albert T Gacerez, Casey K Hua, Margaret E Ackerman, Charles L Sentman
B7H6 is emerging as a promising tumor antigen that is known to be expressed on a wide array of tumors and is reported to stimulate anti-tumor responses from the immune system. As such, B7H6 presents a good target for tumor-specific immunotherapies. B7H6-specific chimeric antigen receptors (CAR) based on a murine antibody showed successful targeting and elimination of tumors expressing B7H6. However, mouse single chain variable fragments (scFvs) have the potential to induce host anti-CAR responses that may limit efficacy, so human scFvs specific for B7H6 were selected by yeast surface display...
May 2018: Cancer Immunology, Immunotherapy: CII
https://www.readbyqxmd.com/read/29420662/generation-of-high-affinity-internalizing-anti-fgfr2-single-chain-variable-antibody-fragment-fused-with-fc-for-targeting-gastrointestinal-cancers
#13
Aleksandra Borek, Aleksandra Sokolowska-Wedzina, Grzegorz Chodaczek, Jacek Otlewski
Fibroblast growth factor receptors (FGFRs) are promising targets for antibody-based cancer therapies, as their substantial overexpression has been found in various tumor cells. Aberrant activation of FGF receptor 2 (FGFR2) signaling through overexpression of FGFR2 and/or its ligands, mutations, or receptor amplification has been reported in multiple cancer types, including gastric, colorectal, endometrial, ovarian, breast and lung cancer. In this paper, we describe application of the phage display technology to produce a panel of high affinity single chain variable antibody fragments (scFvs) against the extracellular ligand-binding domain of FGFR2 (ECD_FGFR2)...
2018: PloS One
https://www.readbyqxmd.com/read/29411237/cell-growth-inhibition-and-apoptosis-in-breast-cancer-cells-induced-by-anti-fzd7-scfvs-involvement-of-bioinformatics-based-design-of-novel-epitopes
#14
Neda Zarei, Mehdi Fazeli, Mozafar Mohammadi, Foroogh Nejatollahi
BACKGROUND: FZD7 has a critical role as a surface receptor of Wnt/β-catenin signaling in cancer cells. Suppressing Wnt signaling through blocking FZD7 is shown to decrease cell viability, metastasis and invasion. Bioinformatic methods have been a powerful tool in epitope designing studies. Small size, high affinity and human origin of scFv antibodies have provided unique advantages for these recombinant antibodies. METHODS: Two epitopes from extracellular domain of FZD7 were designed using bioinformatic methods...
February 6, 2018: Breast Cancer Research and Treatment
https://www.readbyqxmd.com/read/29384367/tuning-the-hydrolytic-stability-of-next-generation-maleimide-cross-linkers-enables-access-to-albumin-antibody-fragment-conjugates-and-tri-scfvs
#15
Nafsika Forte, Maria Livanos, Enrique Miranda, Maurício Morais, Xiaoping Yang, Vineeth S Rajkumar, Kerry A Chester, Vijay Chudasama, James R Baker
We describe investigations to expand the scope of next generation maleimide cross-linkers for the construction of homogeneous protein-protein conjugates. Diiodomaleimides are shown to offer the ideal properties of rapid bioconjugation with reduced hydrolysis, allowing the cross-linking of even sterically hindered systems. The optimized linkers are exploited to link human serum albumin to antibody fragments (Fab or scFv) as a prospective half-life extension platform, with retention of antigen binding and robust serum stability...
February 21, 2018: Bioconjugate Chemistry
https://www.readbyqxmd.com/read/29377386/effect-of-linkers-on-immobilization-of-scfvs-with-biotin-streptavidin-interaction
#16
Svetlana P Ikonomova, Megan T Le, Neha Kalla, Amy J Karlsson
Single-chain variable fragment antibodies (scFvs) are attractive for use in applications that require high specificity and binding to a target, such as biosensors. Previously, we demonstrated that a variety of scFvs can be immobilized onto a streptavidin surface through in vivo biotinylation of the biotin carboxyl carrier protein (BCCP) or smaller AviTag fused to the scFvs. However, the BCCP constructs showed better immobilization than the AviTag constructs. In this work, we investigated whether the discrepancy between the biotinylation tags could be alleviated by incorporating a flexible (G4 S)n linker of varying lengths or a rigid (EA3 K)3 linker between the biotinylation tags and the scFvs scFv13R4 and scFv5...
January 29, 2018: Biotechnology and Applied Biochemistry
https://www.readbyqxmd.com/read/29374508/re-engineering-and-evaluation-of-anti-dna-autoantibody-3e10-for-therapeutic-applications
#17
Zahra Rattray, Valentina Dubljevic, Nicholas J W Rattray, Deanne L Greenwood, Caroline H Johnson, James A Campbell, James E Hansen
A key challenge in the development of novel chemotherapeutics is the design of molecules capable of selective toxicity to cancer cells. Antibodies have greater target specificity compared to small molecule drugs, but most are unable to penetrate cells, and predominantly target extracellular antigens. A nuclear-penetrating anti-DNA autoantibody isolated from the MRL/lpr lupus mouse model, 3E10, preferentially localizes to tumors, inhibits DNA repair, and selectively kills cancer cells with defects in DNA repair...
February 12, 2018: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/29365311/biocompatible-coupling-of-therapeutic-fusion-proteins-to-human-erythrocytes
#18
Carlos H Villa, Daniel C Pan, Ian H Johnston, Colin F Greineder, Landis R Walsh, Elizabeth D Hood, Douglas B Cines, Mortimer Poncz, Don L Siegel, Vladimir R Muzykantov
Carriage of drugs by red blood cells (RBCs) modulates pharmacokinetics, pharmacodynamics, and immunogenicity. However, optimal targets for attaching therapeutics to human RBCs and adverse effects have not been studied. We engineered nonhuman-primate single-chain antibody fragments (scFvs) directed to human RBCs and fused scFvs with human thrombomodulin (hTM) as a representative biotherapeutic cargo (hTM-scFv). Binding fusions to RBCs on band 3/glycophorin A (GPA; Wright b [Wrb ] epitope) and RhCE (Rh17/Hr0 epitope) similarly endowed RBCs with hTM activity, but differed in their effects on RBC physiology...
February 13, 2018: Blood Advances
https://www.readbyqxmd.com/read/29321330/glycosyl-phosphatidylinositol-anchored-anti-hiv-env-single-chain-variable-fragments-interfere-with-hiv-1-env-processing-and-viral-infectivity
#19
Anisha Misra, Emile Gleeson, Weiming Wang, Chaobaihui Ye, Paul Zhou, Jason T Kimata
In previous studies, we demonstrated that single-chain variable fragments (scFvs) from anti-human immunodeficiency virus (HIV) Env monoclonal antibodies act as entry inhibitors when tethered to the surface of target cells by a glycosyl-phosphatidylinositol (GPI) anchor. Interestingly, even if a virus escapes inhibition at entry, its replication is ultimately controlled. We hypothesized that in addition to functioning as entry inhibitors, anti-HIV GPI-scFvs may also interact with Env in an infected cell, thereby interfering with the infectivity of newly produced virions...
April 1, 2018: Journal of Virology
https://www.readbyqxmd.com/read/29315611/isolation-of-a-high-affinity-bet-v-1-specific-igg-derived-scfv-from-a-subject-vaccinated-with-hypoallergenic-bet-v-1-fragments
#20
E Gadermaier, K Marth, C Lupinek, R Campana, G Hofer, K Blatt, D Smiljkovic, U Roder, M Focke-Tejkl, S Vrtala, W Keller, P Valent, R Valenta, S Flicker
BACKGROUND: Recombinant hypoallergenic allergen derivatives have been used in clinical immunotherapy studies, and clinical efficacy seems to be related to the induction of blocking IgG antibodies recognizing the wild-type allergens. However, so far no treatment-induced IgG antibodies have been characterized. OBJECTIVE: To clone, express, and characterize IgG antibodies induced by vaccination with two hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1 in a nonallergic subject...
January 9, 2018: Allergy
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