Design, expression and evaluation of a novel humanized single chain antibody against epidermal growth factor receptor (EGFR)

Akbari, Bahman; Farajnia, Safar; Zarghami, Nosratollah; Mahdieh, Nejat; Rahmati, Mohammad; Khosroshahi, Shiva Ahdi; Rahbarnia, Leila

doi:10.1016/j.pep.2016.06.001

Cited by 15 publications

(12 citation statements)

References 27 publications

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“…These manipulated phagemid vectors were capable of penetrating the bacteria, for easy cloning and rapid proliferation. 16,31 The scFvs could be extracted from both the periplasmic and cytoplasmic spaces using the correct extraction method. In the periplasmic space, the protein could have the correct folding and high sustainability.…”

Section: Discussionmentioning

confidence: 99%

“…In other studies, reported that used 0.2-1 mM IPTG and 25-30°C temperature. [31][32][33][34][35][36] Affinity chromatography with a stabilized biomolecule, such as protein A, had a high potential for absorbing antibodies. It was found that the efficiency of this purification method was high, but it was uneconomical due to the costly nature of these columns at the industrial scale.…”

Section: Discussionmentioning

confidence: 99%

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Isolation of a novel Anti-KDR3 Single-chain Variable Fragment Antibody from a Phage Display Library

Kordi

Rahmati-Yamchi

Vostakolaei

et al. 2019

IJAAI

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Vascular endothelial growth factor receptor 2 (VEGFR-2) is known as one of the important antigens playing a vital role in angiogenesis. In this study, phage display technology (PDT) was used to produce a single-chain variable fragment (scFv) antibody against a region of the domain 3 in VEGFR-2 called kinase insert domain receptor 3 (KDR3). After designing the KDR3 peptide and biopanning, a colony was chosen for scFv antibody expression. Following expression and purification; western blotting, dot blotting and immunofluorescence (IF) were used to evaluate the antibody function. Surface plasmon resonance (SPR) was also employed to measure affinity of produced antibody. Once a colony was selected and transferred to the expression host, the scFv antibody was expressed in the expected range of 28 kDa. Using a designed chromatography column, antibody purification was found to be about 95%. In this study, a novel scFv with the capability of binding to KDR3 was isolated and purified and its intracellular function was investigated and verified.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Isolation of a novel Anti-KDR3 Single-chain Variable Fragment Antibody from a Phage Display Library

Kordi

Rahmati-Yamchi

Vostakolaei

et al. 2019

IJAAI

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“…Self-assembly of amphiphilic BSA-PCL conjugate; (9,(13)(14)(15). The amphiphilic BSA-PCL conjugate was synthesized as previously described (16), and the nanosized self-assembly of the amphiphilic BSA-PCL conjugate was obtained via emulsion-solvent evaporation method.…”

Section: Methodsmentioning

confidence: 99%

“…Confocal microscopy; (10,15,20). As previously mentioned, cells were planted in the confocal small dish (5x10 3 cells/dish).…”

mentioning

confidence: 99%

Radioiodine-labeled anti-epidermal growth factor receptor binding bovine serum albumin-polycaprolactone for targeting imaging of glioblastoma

Tan

Chang

et al. 2017

Oncology Reports

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The aim of this study was to look for a new medicine in diagnosing and treating of glioblastoma. Radioiodine-labeled anti-epidermal growth factor receptor (EGFR) binding nanoparticles were constructed. In vitro cell-binding assays were confirmed by confocal microscopy and flow cytometry. Cell cytotoxicity assays were evaluated by MTT assay; radionuclide uptake assays were performed by γ-counter. Radioiodine-imaging studies were conducted using a xenograft nude mouse model in vivo. The results showed that EGFR significantly enhanced the uptake and accumulation of BSA-PCL in the experimental model of xenografts in nude mice, suggesting improved specific nanoparticle-based delivery. In conclusion, the data showed 131I-EGFR-BSA-PCL leading radioiodine therapy for U251 and U87 cells had a good effect in vitro and in vivo. Thus, 131I-EGFR-BSA-PCL may provide a new method for glioblastoma treatment.

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“…Production of the small generation of the antibodies like single chain fragment variable (scFv) is used to overcome the size-related problems(8). Also, humanization of the antibodies by replacing murine framework parts of the antibodies with the human counterpart is used as an alternative solution for the problem associated with the immunogenicity(20). It has also been shown that the efficiency of anti-EGFR antibodies is affected by various mutations in EGFR and K-ras genes (20).…”

Section: Introductionmentioning

confidence: 99%

Expression and Evaluation of HuscFv Antibody -PE40 Immunotoxin for Target Therapy of EGFR-Overexpressing Cancers

et al. 2018

Self Cite

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Background Epidermal growth factor receptor (EGFR) plays an important role in the progression and tumorigenesis of the various cancers. In this regards, anti-EGFR antibodies are valuable approved therapeutics for the EGFR over-expressing cancers. However, the occurrence of mutations in the EGFR and/or KRAS genes; a common phenomenon which is seen in many cancers, lead to the resistance to the EGFR-directed antibodies. EGFR based immunotoxins are capable of overcoming this limitation by directing the toxin moieties to the cancer cells resulting in cell death. Objectives In the present study, a novel immunotoxin consisting of the truncated Pseudomonas exotoxin A (PE-40) and anti-EGFR huscFv was developed and evaluated for the induction of cell death in EGFR positive A431tumoral cells. Materials and Methods PE-40 fragment of the exotoxin A was amplified by using PCR and ligated to pET22b-huscFv. The reaction was confirmed by PCR and restriction digestion. The immunotoxin was expressed in E. coli BL21 (plysS) and then was purified by Ni-NTA affinity column. Subsequently, the toxicity of the purified immunotoxin was evaluated on EGFR over-expressing epidermoid carcinoma of skin, A431 cell line. Results PCR and restriction digestion experiments have verified the integrity of the immunotoxin construct. Purification by affinity column resulted in a highly purified recombinant immunotoxin. MTT assay revealed the growth inhibitory effect of the huscFv-PE40 immunotoxin on EGFR-over-expressing A431 cells with an IC50 value of 250 ng.mL -1 . Conclusion In conclusion, the results indicated that the immunotoxin developed in this study has a high toxicity on the EGFR-over-expressing tumor cells and could be considered as a promising candidate for the treatment of the EGFR positive cancers.

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Design, expression and evaluation of a novel humanized single chain antibody against epidermal growth factor receptor (EGFR)

Cited by 15 publications

References 27 publications

Isolation of a novel Anti-KDR3 Single-chain Variable Fragment Antibody from a Phage Display Library

Isolation of a novel Anti-KDR3 Single-chain Variable Fragment Antibody from a Phage Display Library

Radioiodine-labeled anti-epidermal growth factor receptor binding bovine serum albumin-polycaprolactone for targeting imaging of glioblastoma

Expression and Evaluation of HuscFv Antibody -PE40 Immunotoxin for Target Therapy of EGFR-Overexpressing Cancers

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