Efficient production and purification of extracellular domain of human FGFR-Fc fusion proteins from Chinese hamster ovary cells

Sokołowska-Wędzina, Aleksandra; Borek, Aleksandra; Chudzian, Julia; Jakimowicz, Piotr; Zakrzewska, Małgorzata; Otlewski, Jacek

doi:10.1016/j.pep.2014.03.012

Cited by 36 publications

(40 citation statements)

References 21 publications

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“…Fully glycosylated extracellular domains of FGFRs fused to the Fc fragment of human IgG1: FGFR1 IIIc (FGFR1‐Fc), FGFR2 IIIc (FGFR2‐Fc), FGFR3 IIIc (FGFR3‐Fc), and FGFR4 (FGFR4‐Fc), and the extracellular part of FGFR1 lacking the N‐terminal D1 domain (FGFR1 D2‐D3) were produced as described previously by our group [48]. FGFR1 GST‐D1 was expressed in E. coli BL21 CodonPlus (DE3)‐RIL (Agilent Technologies, Santa Clara, CA, USA) and purified with use of Glutathione Sepharose column as described previously [5,6,49].…”

Section: Methodsmentioning

confidence: 99%

FGFR1 clustering with engineered tetravalent antibody improves the efficiency and modifies the mechanism of receptor internalization

et al. 2020

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Fibroblast growth factor receptor 1 (FGFR1) transmits signals through the plasma membrane regulating essential cellular processes like division, motility, metabolism, and death. Overexpression of FGFR1 is observed in numerous tumors and thus constitutes an attractive molecular target for selective cancer treatment. Targeted anti‐cancer therapies aim for the precise delivery of drugs into cancer cells, sparing the healthy ones and thus limiting unwanted side effects. One of the key steps in targeted drug delivery is receptor‐mediated endocytosis. Here, we show that the efficiency and the mechanism of FGFR1 internalization are governed by the spatial distribution of the receptor in the plasma membrane. Using engineered antibodies of different valency, we demonstrate that dimerization of FGFR1 with bivalent antibody triggers clathrin‐mediated endocytosis (CME) of the receptor. Clustering of FGFR1 into larger oligomers with tetravalent antibody stimulates fast and highly efficient uptake of the receptor that occurs via two distinct mechanisms: CME and dynamin‐dependent clathrin‐independent endocytic routes. Furthermore, we show that all endocytic pathways engaged in FGFR1 internalization do not require receptor activation. Our data provide novel insights into the mechanisms of intracellular trafficking of FGFR1 and constitute guidelines for development of highly internalizing antibody‐based drug carriers for targeted therapy of FGFR1‐overproducing cancers.

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Section: Methodsmentioning

confidence: 99%

FGFR1 clustering with engineered tetravalent antibody improves the efficiency and modifies the mechanism of receptor internalization

et al. 2020

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“…Recombinant FGF1 and FGF2 were produced in E. coli, as described previously [37]. The Fc fragment of IgG and the full-length extracellular portion of FGFR1 (IIIc), FGFR2 (IIIc), FGFR3 (IIIc) and FGFR4 (FGFR1ecd-Fc, FGFR2ecd-Fc, FGFR3ecd-Fc, FGFR4ecd-Fc) were expressed in CHO cells and purified using Protein A Sepharose [38].…”

Section: Recombinant Proteinsmentioning

confidence: 99%

“…The extracellular portion of FGFR1 is N-glycosylated at several positions [19,20]. We produced a full-length extracellular part of FGFR1 (isoform IIIc) as a fusion with the Fc region of human IgG (FGFR1ecd-Fc) in the CHO cells, which allow for receptor glycosylation [38]. Biolayer interferometry (BLI) with biosensor-immobilized FGFR1ecd-Fc and recombinant galectin-1 or galectin-3 revealed a direct interaction between these proteins (Fig.…”

Section: Galectin-1 and 3 Directly Bind To The Glycosylated Extracellmentioning

confidence: 99%

Differential regulation of fibroblast growth factor receptor 1 trafficking and function by extracellular galectins

et al. 2019

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Fibroblast growth factor receptors (FGFRs) are integral membrane proteins that transmit signals through the plasma membrane. FGFRs signaling needs to be precisely adjusted as aberrant FGFRs function is associated with development of human cancers or severe metabolic diseases. The subcellular localization, trafficking and function of FGFRs rely on the formation of multiprotein complexes. In this study we revealed galectins, lectin family members implicated in cancer development and progression, as novel FGFR1 binding proteins. We demonstrated that galectin-1 and galectin-3 directly bind to the sugar chains of the glycosylated extracellular part of FGFR1. Although both galectins compete for the same binding sites on FGFR1, these proteins elicit different impact on FGFR1 function and cellular trafficking. Galectin-1 mimics fibroblast growth factor as it efficiently activates FGFR1 and receptor-downstream signaling pathways that result in cell proliferation and apoptotic evasion. In contrast, galectin-3 induces extensive clustering of FGFR1 on the cell surface that inhibits constitutive internalization of FGFR1. Our data point on the interplay between extracellular galectins and FGFRs in the regulation of cell fate.

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“…Fully glycosylated extracellular domain (ECD) of selected FGFRs fused to the Fc domain of human IgG 1 , FGFR1 IIIc (ECD_FGFR1-Fc), FGFR2 IIIc (ECD_FGFR2-Fc), FGFR3 IIIc (ECD_FGFR3-Fc), and FGFR4 (ECD_FGFR4-Fc), was produced as described previously by our group (33). Briefly, FGFRs coding sequences were cloned into pLEV113 vector, expressed in CHO-S cells and purified using Protein A Sepharose Fast Flow Resin (GE Healthcare).…”

Section: Proteinsmentioning

confidence: 99%

“…FGFR1 expression level in all cell lines was assessed in wholecell lysates by Western blotting using anti-FGFR1 antibody (9740, Cell Signaling Technology). CHO-S cells were purchased from Thermo Fisher Scientific and cultured as described previously (33).…”

Section: Cell Linesmentioning

confidence: 99%

High-Affinity Internalizing Human scFv-Fc Antibody for Targeting FGFR1-Overexpressing Lung Cancer

Sokołowska-Wędzina

Chodaczek

Chudzian

et al. 2017

Molecular Cancer Research

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Targeted delivery of anticancer drugs using antibodies specific for tumor-associated antigens represents one of the most important approaches in current immuno-oncology research. Fibroblast growth factor receptor 1 (FGFR1) has been demonstrated to be a high-frequency targetable oncogene specific for smokingassociated lung cancers, present in over 20% of lung squamous cell carcinoma cases. This report describes the generation of a potent, fully human antibody fragment in scFv-Fc format efficiently targeting FGFR1. Antibody phage display was used to select high-affinity scFv antibody fragments against the extracellular domain of FGFR1(IIIc). Enzyme immunoassay (ELISA) and surface plasmon resonance (SPR) analysis were used for antibody screening and characterization. The best binder (named D2) was cloned to diabody and Fc fusion formats. All D2 antibodies demonstrated high affinity for FGFR1 with dissociation constants of 18 nmol/L (scFvD2), 0.82 nmol/L (scFvD2 diabody), and 0.59 nmol/L (scFvD2-Fc). scFvD2 was found to be exquisitely selective for FGFR1 versus other FGFR family members and bound FGFR1 even in the presence of its natural ligand FGF2, as shown by competitive analysis. Confocal microscopy revealed that scFvD2-Fc was specifically and rapidly internalized by a panel of cell lines overexpressing FGFR1. Finally, it was demonstrated that scFvD2-Fc mediated specific delivery of a cytotoxic payload into lung cancer cells harboring oncogenic FGFR1 gene amplifications.Implications: This study reports a highly specific internalizing antibody fragment that can serve as a therapeutic targeting agent for efficient delivery of cytotoxic drugs into FGFR1-positive lung cancer cells. Mol Cancer Res; 15(8); 1040-50. Ó2017 AACR.

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Efficient production and purification of extracellular domain of human FGFR-Fc fusion proteins from Chinese hamster ovary cells

Cited by 36 publications

References 21 publications

FGFR1 clustering with engineered tetravalent antibody improves the efficiency and modifies the mechanism of receptor internalization

FGFR1 clustering with engineered tetravalent antibody improves the efficiency and modifies the mechanism of receptor internalization

Differential regulation of fibroblast growth factor receptor 1 trafficking and function by extracellular galectins

High-Affinity Internalizing Human scFv-Fc Antibody for Targeting FGFR1-Overexpressing Lung Cancer

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