Targeting c-Met on gastric cancer cells through a fully human fab antibody isolated from a large naive phage antibody library

Zarei, Bahareh; Javidan, Zahra; Fatemi, Elnaz; Jamnani, Fatemeh Rahimi; Khatami, Shohreh; Khalaj, Vahid

doi:10.1007/s40199-020-00334-z

Cited by 7 publications

(4 citation statements)

References 38 publications

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“…To produce soluble scFv antibodies, exponentially growing E. coli HB2151 bacteria were infected with phages purified from 11 phage clones (MEH63, MEH79 and MEH94 from PBMC–plasma biopanning; MEH121, MEH131, MEH169 and MEH178 from blood biopanning; and MEH158, MEH183, MEH188 and MEH199 from biofilm biopanning), exhibiting significant binding in the monoclonal phage assay 104 . The expression of scFv antibodies was induced by isopropyl β‐d‐1‐thiogalactopyranoside (0.1 mM IPTG) (GE Healthcare) as previously described by Eisenhardt et al 106 .…”

Section: Methodsmentioning

confidence: 99%

“…The strains were classified into non-biofilm producers and weak, moderate and strong biofilm producers based on the results. 100 Enrichment of a fully human scFv library against living S. aureus A phage-display human scFv library, with total diversity of 2 × 10 10 , [102][103][104][105] was screened against two MRSA strains grown in human PBMCs with plasma (PBMC-plasma biopanning), in human whole blood (blood biopanning) 100 or as a biofilm (biofilm biopanning). 100 In brief, an overnight culture of bacteria (S. aureus S.a.48 and S.a.124), grown in the medium containing TSB and PBMCs plus plasma (or human whole blood), was diluted 1:50 in fresh medium and incubated for 3 h at 37°C.…”

Section: Biofilm Assaymentioning

confidence: 99%

“…To produce soluble scFv antibodies, exponentially growing E. coli HB2151 bacteria were infected with phages purified from 11 phage clones (MEH63, MEH79 and MEH94 from PBMC-plasma biopanning; MEH121, MEH131, MEH169 and MEH178 from blood biopanning; and MEH158, MEH183, MEH188 and MEH199 from biofilm biopanning), exhibiting significant binding in the monoclonal phage assay. 104 The expression of scFv antibodies was induced by isopropyl β-d-1-thiogalactopyranoside (0.1 mM IPTG) (GE Healthcare) as previously described by Eisenhardt et al 106 Next, the presence and the expression level of scFv fragments in the periplasmic extracts of E. coli HB2151 bacteria, harbouring the selected phagemids (MEH63, MEH158, MEH169, MEH178, MEH183 and MEH188), were analysed by SDS-PAGE and Western blotting. For Western blotting, the blocked polyvinylidene fluoride (PVDF) membrane (GE Healthcare), containing proteins transferred from the 12% SDS-PAGE gel, was incubated with mouse anti-human scFv polyclonal antibody (Supplementary figure 18) for 1 h at RT.…”

Section: Expression Of Soluble Scfv Antibodies Sds-page and Western Blot Analysismentioning

confidence: 99%

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Bactericidal fully human single‐chain fragment variable antibodies protect mice against methicillin‐resistant Staphylococcusaureus bacteraemia

et al. 2021

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Objectives. The increasing prevalence of antibiotic-resistant Staphylococcus aureus, besides the inadequate numbers of effective antibiotics, emphasises the need to find new therapeutic agents against this lethal pathogen. Methods. In this study, to obtain antibody fragments against S. aureus, a human single-chain fragment variable (scFv) library was enriched against living methicillin-resistant S. aureus (MRSA) cells, grown in three different conditions, that is human peripheral blood mononuclear cells with plasma, whole blood and biofilm. The antibacterial activity of scFvs was evaluated by the growth inhibition assay in vitro. Furthermore, the therapeutic efficacy of anti-S. aureus scFvs was appraised in a mouse model of bacteraemia. Results. Three scFv antibodies, that is MEH63, MEH158 and MEH183, with unique sequences, were found, which exhibited significant binding to S. aureus and reduced the viability of S. aureus in in vitro inhibition assays. Based on the results, MEH63, MEH158 and MEH183, in addition to their combination, could prolong the survival rate, reduce the bacterial burden in the blood and prevent inflammation and tissue destruction in the kidneys and spleen of mice with MRSA bacteraemia compared with the vehicle group (treated with normal saline). Conclusion. The combination therapy with anti-S. aureus scFvs and conventional antibiotics might shed light on the treatment of patients with S. aureus infections.

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Section: Methodsmentioning

confidence: 99%

Section: Biofilm Assaymentioning

confidence: 99%

Section: Expression Of Soluble Scfv Antibodies Sds-page and Western Blot Analysismentioning

confidence: 99%

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Bactericidal fully human single‐chain fragment variable antibodies protect mice against methicillin‐resistant Staphylococcusaureus bacteraemia

et al. 2021

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“…Using the peripheral blood of 78 individuals, we constructed a Fab antibody library with a capacity of 4.27×10 However, in the study of Zarei et al (Zarei et al, 2020), the VH4 gene family was the most common. The amino acid length analysis showed that the number of amino acids in the CDR3 region of VL was mainly 9 and in the CDR3 region of VH was mainly 14, which was similar to the report by Kügler et al (Bostrom et al, 2009).…”

Section: Soluble Expression Of Positive Clones and Identificationmentioning

confidence: 99%

Construction of a Natural Human Fab Phage Antibody Library and Screening of Phage Antibody against PD-L1

J¹,

Guo²,

Zhang³

et al. 2022

ijSciences

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Programmed Death Ligand-1(PD-L1) is an important target to drive T cell dysfunction when it connects with Programmed Death-1(PD-1), leading to immune escape of tumor cells, thus anti-PD-L1 antibody shows a promising prospect in the treatment of tumor. In order to construct a large natural antibody library, we collected a large number of lymphocytes of adults and children. The light chain and Fd genes of antibody were amplified by PCR, and the Fab phage antibody library with a library capacity of 4.27×10 9 was constructed. The insertion rates of the light chain library and the Fab library were 90% and 70%, respectively. The cloned sequences identified by PCR showed that all the sequences analyzed were unique, and the amino acid sequences of the CDR regions were diverse, which proved that there was good diversity in the antibody library. The positive clones that bind to PD-L1 were screened by phage ELISA, PCR identification and sequence analysis. In the end, two high-affinity positive clones were determined. The successful construction of this natural phage antibody library provided an effective method for screening human PD-L1 antibodies, which was expected to screen humanized antibodies against various antigens.

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Production of antibodies in SHuffle Escherichia coli strains

Eaglesham

García

Berkmen

2021

Recombinant Protein Expression: Prokaryotic Hosts and Cell-Free Systems

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Targeting c-Met on gastric cancer cells through a fully human fab antibody isolated from a large naive phage antibody library

Cited by 7 publications

References 38 publications

Bactericidal fully human single‐chain fragment variable antibodies protect mice against methicillin‐resistant Staphylococcusaureus bacteraemia

Bactericidal fully human single‐chain fragment variable antibodies protect mice against methicillin‐resistant Staphylococcusaureus bacteraemia

Construction of a Natural Human Fab Phage Antibody Library and Screening of Phage Antibody against PD-L1

Production of antibodies in SHuffle Escherichia coli strains

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