Identification of novel neutralizing single‐chain antibodies against vascular endothelial growth factor receptor 2

Erdağ, Berrin; Balcıoğlu, Bertan Koray; Bahadır, Aylin Özdemir; Serhatlı, Müge; Kaçar, Ömer; Bahar, Aydin; Şeker, Urartu Özgür Şafak; Akgun, Emel; Ozkan, Abdulkadir; Kılıç, Türker; Tamerler, Candan; Baysal, Kemal

doi:10.1002/bab.61

Cited by 15 publications

(10 citation statements)

References 48 publications

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“…The periplasmic protein fraction of E. coli was extracted by cold osmotic shock [25]. Briefly, the harvested bacterial cells were gently suspended in Tris-EDTA-Sucrose (TES) buffer (50 mM Tris buffer pH 7.4, 1 mM EDTA and 20% sucrose w / v ) and were incubated on ice for 30 min.…”

Section: Methodsmentioning

confidence: 99%

QbD Based Media Development for the Production of Fab Fragments in E. coli

Kumar¹,

Batra²,

Komives

et al. 2019

Bioengineering

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Ranibizumab is a biotherapeutic Fab fragment used for the treatment of age-related macular degeneration and macular oedema. It is currently expressed in the gram-negative bacterium, Escherichia coli. However, low expression levels result in a high manufacturing cost. The protein expression can be increased by manipulating nutritional requirements (carbon source, nitrogen source, buffering agent), process parameters (pH, inducer concentration, agitation, temperature), and the genetic make-up of the producing strain. Further, understanding the impact of these factors on product quality is a requirement as per the principles of Quality by Design (QbD). In this paper, we examine the effect of various media components and process parameters on the expression level and quality of the biotherapeutic. First, risk analysis was performed to shortlist different media components based on the literature. Next, experiments were performed to screen these components. Eight components were identified for further investigation and were examined for their effect and interactions using a Fractional Factorial experimental design. Sucrose, biotin, and pantothenate were found to have the maximum effect during Fab production. Furthermore, cyanocobalamin glutathione and biotin-glutathione were the most significant interactions observed. Product identification was performed with Liquid Chromatography–Mass Spectrometry (LC-MS), the expression level was quantified using Bio-layer Interferometry, Reverse Phase-HPLC, and SDS-PAGE, and product quality were measured by RP-HPLC. Overall, a five-fold enhancement of the target protein titer was obtained (from 5 mg/L to 25 mg/L) using the screened medium components vis-a-vis the basal medium, thereby demonstrating the efficacy of the systematic approach purported by QbD.

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Section: Methodsmentioning

confidence: 99%

QbD Based Media Development for the Production of Fab Fragments in E. coli

Kumar¹,

Batra²,

Komives

et al. 2019

Bioengineering

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“…Second, small molecular weight tyrosine kinase inhibitors (TKI's), such as PTK787, Sutent, Sorafinib, Zactima, and Recentin. Last, antibodies or antibody‐like molecules that block the interaction of VEGF‐A with VEGFR‐2, such as IMC1121b and IMC1C11 6–8. The vast majority of clinical competitors for Avastin in oncology, however, are small molecule VEGFR inhibitors, which have multiple intracellular targets accounting for some of the additional observable toxicities not directly related to VEGFR‐2 inhibition.…”

Section: Introductionmentioning

confidence: 99%

Phage‐derived fully human antibody scFv fragment directed against human vascular endothelial growth factor receptor 2 blocked its interaction with VEGF

Zhang

Wang

et al. 2012

Biotechnology Progress

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Vascular endothelial growth factor receptor 2 (VEGFR-2) plays a critical role in tumor angiogenesis. None therapeutic antibodies targeting VEGFR-2 are available in clinical use. Herein, we describe the screening of a new single-chain antibody fragment (scFv) targeting extracellular domain 3 of human VEGFR-2 (kinase insert domain-containing receptor [KDR]3) from Griffin phage display scFv library. A comprehensive sequence analysis was performed to assign the framework and complementary-determining regions. The scFv exerted particular binding sites to KDR3 on molecular docking, and the binding affinity was further convinced by binding analysis both in quantitative ELISA and real-time kinetic determination by biosensors (K(D) = 40 nM). Finally, the scFv was revealed to inhibit VEGF-stimulated proliferation of human umbilical vein endothelial cells (HUVECs; IC(50) = 5 nM) and to inhibit HUVEC migration significantly at 17 nM. Taken together, our results indicate that we have successfully isolated a scFv which differentially recognizes KDR3 and has potential clinical applications in the treatment of angiogenesis related diseases.

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“…The scFvs were prepared according to the RPAS ScFv and Expression Modules protocol (Amersham Biosciences Corp., Piscataway, NJ) and cloned into the SfiI/NotI linearized pDUCK1 phagemid. The ScFv/pDUCK1 phagemid was then introduced into E. coli TG1 cells, and rescued with the helper phage M13KO7 (Amersham Biosceinces Corp., Piscataway, NJ) [18,19].…”

Section: Generation Of the Scfvs And The Display On Filamentous Phagementioning

confidence: 99%

Cloning of anti-HBsAg single-chain variable fragments from hybridoma cells for one-step ELISA

Erdağ

Balcıoğlu

Bahadır

et al. 2017

Biotechnology & Biotechnological Equipment

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Hepatitis B virus (HBV) infection is a worldwide health problem. More than 400 million people are chronic HBV carriers in the world. Infected individuals are at a high risk of developing liver cirrhosis and hepatocellular carcinoma as the main consequences of HBV. The discoveries of fast diagnostic systems and new therapeutic applications are crucial in the fight against viral hepatitis. In this paper we present the generation of a single-chain variable fragment (scFv) from a mouse monoclonal antibody specific to the HBV surface antigen (HBsAg) and demonstrate its expression as a bacterial alkaline phosphatase (AP) fusion protein. In this study, we constructed scFvs from hybridoma cells expressing HBsAg-specific antibody using phage display technology and expressed them in Escherichia coli. The anti-HBsAg scFvs were inserted into pQE-2 vector to produce scFv antibody genetically fused to bacterial AP. Reproducibility of the recombinant HBsAg-scFv fusion protein was tested using Enzyme-linked Immunosorbent Assay (ELISA). Present preliminary findings indicate that the anti-HBsAg-scFv AP conjugate could be further used for the development of one-step ELISA for the detection of HBV.

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Identification of novel neutralizing single‐chain antibodies against vascular endothelial growth factor receptor 2

Cited by 15 publications

References 48 publications

QbD Based Media Development for the Production of Fab Fragments in E. coli

QbD Based Media Development for the Production of Fab Fragments in E. coli

Phage‐derived fully human antibody scFv fragment directed against human vascular endothelial growth factor receptor 2 blocked its interaction with VEGF

Cloning of anti-HBsAg single-chain variable fragments from hybridoma cells for one-step ELISA

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