Browse
Submit Data
Databases
API
Help

Dataset Information

23 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Characterization of a novel mCH3 conjugated anti-PcrV scFv molecule.

ABSTRACT: Pseudomonas aeruginosa (PA) is a leading cause of nosocomial infections and death in cystic fibrosis patients. The study was conducted to evaluate the physicochemical structure, biological activity and serum stability of a recombinant anti-PcrV single chain variable antibody fragment genetically attached to the mCH3cc domain. The stereochemical properties of scFv-mCH3 (YFL001) and scFv (YFL002) proteins as well as molecular interactions towards Pseudomonas aeruginosa PcrV were evaluated computationally. The subcloned fragments encoding YFL001 and YFL002 in pET28a were expressed within the E. coli BL21-DE3 strain. After Ni-NTA affinity chromatography, the biological activity of the proteins in inhibition of PA induced hemolysis as well as cellular cytotoxicity was assessed. In silico analysis revealed the satisfactory stereochemical quality of the models as well as common residues in their interface with PcrV. The structural differences of proteins through circular dichroism spectroscopy were confirmed by NMR analysis. Both proteins indicated inhibition of ExoU positive PA strains in hemolysis of red blood cells compared to ExoU negative strains as well as cytotoxicity effect on lung epithelial cells. The ELISA test showed the longer serum stability of the YFL001 molecule than YFL002. The results were encouraging to further evaluation of these two scFv molecules in animal models.

SUBMITTER: Komijani S

PROVIDER: S-EPMC8010009 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Quantum dot-conjugated anti-GRP78 scFv inhibits cancer growth in mice.

Project description:Semiconductor quantum dots (Qdots) have recently been shown to offer significant advantages over conventional fluorescent probes to image and study biological processes. The stability and low toxicity of QDs are well suited for biological applications. Despite this, the potential of Qdots remains limited owing to the inefficiency of existing delivery methods. By conjugating Qdots with small antibody fragments targeting membrane-bound proteins, such as GRP78, we demonstrate here that the Quantum dot- Anti-GRP78 scFv (Qdot-GRP78) retains its immunospecificity and its distribution can be monitored by visualization of multi-color fluorescence imaging both in vitro and in vivo. Moreover we demonstrate here for the first time that Qdot-GRP78 scFv bioconjugates can be efficiently internalized by cancer cells, thus upregulate phophosphate-AKT-ser473 and possess biological anti-tumour activity as shown by inhibition of breast cancer growth in a xenograft model. This suggests that nanocarrier-conjugated scFvs can be used as a therapeutic antibody for cancer treatment.

| S-EPMC6268310 | biostudies-literature

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

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

| S-EPMC4440035 | biostudies-literature

Generation and characterization of novel recombinant anti-hERG1 scFv antibodies for cancer molecular imaging.

Project description:Modern molecular imaging techniques have greatly improved tumor detection and post-treatment follow-up of cancer patients. In this context, antibody-based imaging is rapidly becoming the gold standard, since it combines the unique specificity of antibodies with the sensitivity of the different imaging technologies. The aim of this study was to generate and characterize antibodies in single chain Fragment variable (scFv) format directed to an emerging cancer biomarker, the human ether-à-go-go-related gene-1 (hERG1) potassium channel, and to obtain a proof of concept for their potential use for in vivo molecular imaging. The anti-hERG1scFv was generated from a full length monoclonal antibody and then mutagenized, substituting a Phenylalanine residue in the third framework of the VH domain with a Cysteine residue. The resulting scFv-hERG1-Cys showed much higher stability and protein yield, increased affinity and more advantageous binding kinetics, compared to the "native" anti-hERG1scFv. The scFv-hERG1-Cys was hence chosen and characterized: it showed a good binding to the native hERG1 antigen expressed on cells, was stable in serum and displayed a fast pharmacokinetic profile once injected intravenously in nude mice. The calculated half-life was 3.1 hours and no general toxicity or cardiac toxic effects were detected. Finally, the in vivo distribution of an Alexa Fluor 750 conjugated scFv-hERG1-Cys was evaluated both in healthy and tumor-bearing nude mice, showing a good tumor-to-organ ratio, ideal for visualizing hERG1-expressing tumor masses in vivo. In conclusion, the scFv-hERG1-Cys possesses features which make it a suitable tool for application in cancer molecular imaging.

| S-EPMC6201861 | biostudies-literature

Anti-ROR1 scFv-EndoG as a Novel Anti-Cancer Therapeutic Drug

Project description:Aim: Immunotoxins are proteins that consist of an antibody fragment linked to a toxin, used as agents for targeted

| S-EPMC5844643 | biostudies-literature

Anti-PcrV antibody in cystic fibrosis: a novel approach targeting Pseudomonas aeruginosa airway infection.

Project description:Pseudomonas aeruginosa (Pa) airway infection is associated with increased morbidity and mortality in cystic fibrosis (CF). The type III secretion system is one of the factors responsible for the increased virulence and pro-inflammatory effects of Pa. KB001 is a PEGylated, recombinant, anti-Pseudomonas-PcrV antibody Fab' fragment that blocks the function of Pa TTSS. We studied the safety, pharmacokinetic (PK), and pharmacodynamic properties of KB001 in CF subjects with chronic Pa infection. Twenty-seven eligible CF subjects (?12 years of age, FEV1 ?40% of predicted, and sputum Pa density >10(5) ?CFU/g) received a single intravenous dose of KB001 (3?mg/kg or 10?mg/kg) or placebo. Safety, PK, Pa density, clinical outcomes, and inflammatory markers were assessed. KB001 had an acceptable safety profile and a mean serum half-life of 11.9 days. All subjects had Pa TTSS expression in sputum. There were no significant differences between KB001 and placebo for changes in Pa density, symptoms, or spirometry after a single dose. However, compared to baseline, at Day 28 there was a trend towards a dose-dependent reduction in sputum myeloperoxidase, IL-1, and IL-8, and there were significant overall differences in change in sputum neutrophil elastase and neutrophil counts favoring the KB001 10?mg/kg group versus placebo (-0.61?log(10) and -0.63?log(10) , respectively; P?<?0.05). These results support targeting Pa TTSS with KB001 as a nonantibiotic strategy to reduce airway inflammation and damage in CF patients with chronic Pa infection. Repeat-dosing studies are necessary to evaluate the durability of the anti-inflammatory effects and how that may translate into clinical benefit. (NCT00638365).

| S-EPMC4079258 | biostudies-literature

Anti-PcrV antibody strategies against virulent Pseudomonas aeruginosa.

Project description:Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes fatal acute lung infections in critically ill individuals. Its pathogenesis is associated with bacterial virulence conferred by the type III secretion system (TTSS), through which P. aeruginosa causes necrosis of the lung epithelium and disseminates into the circulation, resulting in bacteremia, sepsis, and mortality. TTSS allows P. aeruginosa to directly translocate cytotoxins into eukaryotic cells, inducing cell death. The P. aeruginosa V-antigen PcrV, a homolog of the Yersinia V-antigen LcrV, is an indispensable contributor to TTS toxin translocation. Vaccination against PcrV ensures the survival of challenged mice and decreases lung inflammation and injury. Both the rabbit polyclonal anti-PcrV antibody and the murine monoclonal anti-PcrV antibody, mAb166, inhibit TTS toxin translocation. mAb166 IgG was cloned, and a molecular engineered humanized anti-PcrV IgG antigen-binding fragment, KB001, was developed for clinical use. KB001 is currently undergoing Phase-II clinical trials for ventilator-associated pneumonia in France and chronic pneumonia in cystic fibrosis in USA. In these studies, KB001 has demonstrated its safety, a favorable pharmacokinetic profile, and promising potential as a nonantibiotic strategy to reduce airway inflammation and damage in P. aeruginosa pneumonia.

| S-EPMC5443083 | biostudies-literature

Production of scFv-conjugated affinity silk film and its application to a novel enzyme-linked immunosorbent assay.

Project description:Bombyx mori (silkworm) silk proteins have been utilized as unique biomaterials for various medical applications. To develop a novel affinity silk material, we generated a transgenic silkworm that spins silk protein containing the fibroin L-chain linked with the single-chain variable fragment (scFv) as a fusion protein. Previously, the scFv-conjugated "affinity" silk powder specifically immunoprecipitated its target protein, Wiskott-Aldrich syndrome protein. To expand the applicability of affinity silk materials, we processed the scFv-conjugated silk protein into a thin film by dissolving it in lithium bromide, then drying it in the wells of 96-well plates. Enzyme-linked immunosorbent assay demonstrated specific detection of Wiskott-Aldrich syndrome protein, both as a recombinant protein and in its native form extracted from mouse macrophages. These findings suggest that this scFv-conjugated silk film serves as the basis for an alternative immunodetection system.

| S-EPMC3921627 | biostudies-other

Characterization of an anti-Bla g 1 scFv: epitope mapping and cross-reactivity.

Project description:Bla g 1 is a major allergen from Blatella germanica and one of the primary allergens used to assess cockroach allergen exposure. The epitope of an anti-Bla g 1 scFv was mapped in order to better understand cross reactivity with other group 1 cockroach allergens and patient IgE epitopes. X-ray crystallography was used to determine the structure of the scFv. The scFv epitope on Bla g 1 was located by alanine scanning site-directed mutagenesis and ELISA. Twenty-six rBla g 1-GST alanine mutants were evaluated for variations in binding to the scFv compared to the wild type allergen. Six mutants showed a significant difference in scFv binding affinity. These mutations clustered to form a discontinuous epitope mainly comprising two helices of Bla g 1. The allergen-scFv complex was modeled based on the results, and the epitope region was found to have low sequence similarity with Per a 1, especially among the residues identified as functionally important for the scFv binding to Bla g 1. Indeed, the scFv failed to bind Per a 1 in American cockroach extract. The scFv was unable to inhibit the binding of IgE antibodies from a highly cockroach allergic patient to Bla g 1. Based on the surface area of Bla g 1 occluded by the scFv, putative regions of patient IgE-Bla g 1 interactions can be inferred. This scFv could be best utilized as a capture antibody in an IgE detection ELISA, or to differentiate Bla g 1 from Per a 1 in environmental exposure assays.

| S-EPMC4097036 | biostudies-literature

Inhibition of Francisella tularensis phagocytosis using a novel anti-LPS scFv antibody fragment.

Project description:Francisella tularensis (Ft), the causative agent of lethal tularemia, is classified as a category A biological warfare threat agent. While Ft infection is treatable by antibiotics, many failed antibiotic treatments were reported, highlighting the need for effective new treatments. It has been demonstrated that binding of antibody-coated bacteria to the Fc receptor located on phagocytic cells is a key process needed for efficient protection against Ft. Yet, Ft utilizes the same receptor to enter the phagocytic cells in order to escape the immune system. To address the question whether an anti-Ft LPS antibody lacking the ability to bind the Fc receptor may inhibit the entry of Ft into host cells, a soluble scFv (TL1-scFv) was constructed from an anti Ft-LPS antibody (TL1) that was isolated from an immune single-chain (scFv) phage-display library. Bacterial uptake was assessed upon infection of macrophages with Ft live attenuated strain (LVS) in the presence of either TL1 or TL1-scFv. While incubation of LVS in the presence of TL1 greatly enhanced bacterial uptake, LVS uptake was significantly inhibited in the presence of TL1-scFv. These results prompt further experiments probing the therapeutic efficacy of TL1-scFv, alone or in combination with antibiotic treatment.

| S-EPMC6684794 | biostudies-literature

Production of scFv-conjugated affinity silk powder by transgenic silkworm technology.

Project description:Bombyx mori (silkworm) silk proteins are being utilized as unique biomaterials for medical applications. Chemical modification or post-conjugation of bioactive ligands expand the applicability of silk proteins; however, the processes are elaborate and costly. In this study, we used transgenic silkworm technology to develop single-chain variable fragment (scFv)-conjugated silk fibroin. The cocoons of the transgenic silkworm contain fibroin L-chain linked with scFv as a fusion protein. After dissolving the cocoons in lithium bromide, the silk solution was dialyzed, concentrated, freeze-dried, and crushed into powder. Immunoprecipitation analyses demonstrate that the scFv domain retains its specific binding activity to the target molecule after multiple processing steps. These results strongly suggest the promise of scFv-conjugated silk fibroin as an alternative affinity reagent, which can be manufactured using transgenic silkworm technology at lower cost than traditional affinity carriers.

| S-EPMC3319607 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data