Journal: Cancer Research

Article Title: GP369, an FGFR2-IIIb–Specific Antibody, Exhibits Potent Antitumor Activity against Human Cancers Driven by Activated FGFR2 Signaling

doi: 10.1158/0008-5472.can-10-1489

Figure Lengend Snippet: Figure 1. Characterization of human cancer cell lines with FGFR2 amplification. A, FGFR copy number of human cancer cell lines. Quantitative PCR was carried out using primers specific for FGFRs or a reference gene, transketolase (TKT), and copy number was calculated. B, FGFR expression analysis. Quantitative RT-PCR was carried out using primers specific for FGFRs and HPRT. The expression levels were normalized to HPRT. C, FGFR2 cell surface expression by flow cytometry. The cells were incubated with 100 μL of primary FGFR2 antibodies (5 μg/mL) for 1 h, followed by incubation with 1:100 phycoerythrin (PE)-conjugated secondary antibodies (Jackson ImmunoResearch) for 30 min. Analysis of stained cells was performed on a FC500 flow cytometer (Beckman Coulter).

Article Snippet: These findings rt the hypothesis that dysregulated FGFR2 signaling is the many critical tumorigenesis pathways in human s. Targeted therapy using FGFR2 mAb could be benefir patients with aberrantly activated/amplified FGFR2 female (Taco in 1:1 Tumo calipe formu appro of 10 contro the M mice 0.72 m Resea MFMappro 10 ani by i.p. using Resu Chara FGFR 8/7630.pdf by guest on 20 July 2024 ing. rials and Methods nes and reagents O III, HEC-1-A, AN3 CA, SNU-16, and human lung canll lines were acquired from the American Type Culture tion (ATCC).

Techniques: Amplification, Real-time Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Flow Cytometry, Incubation, Staining

Journal: Cancer Research

Article Title: GP369, an FGFR2-IIIb–Specific Antibody, Exhibits Potent Antitumor Activity against Human Cancers Driven by Activated FGFR2 Signaling

doi: 10.1158/0008-5472.can-10-1489

Figure Lengend Snippet: Figure 2. Epitope mapping of GP369. A, specific binding of GP369 to human and mouse FGFR2-IIIb. The extracellular domains of human FGFR2-IIIb, human FGFR2-IIIc, mouse FGFR2-IIIb, and human Jag-1 (control) were expressed as human Fc fusion proteins and captured on anti-hFc Octet biosensors (ForteBio). Sensograms were recorded when the loaded biosensors were incubated with GP369 in Octet sample diluent (ForteBio). B, epitope mapping. Ten overlapping peptides were biotinylated at their NH2 termini and captured on streptavidin Octet biosensors. Sensograms were recorded when peptide- loaded biosensors were incubated with GP369 in Octet sample diluent. C, GP369 binding epitopes. GP369 binding peptides (from amino acid 309 to 345; yellow) mapped to the structure of FGFR2-IIIb (light blue; D2 on top, D3 at the bottom) in complex with FGF10 (red; PDB structure 1NUN; ref. 35). D, peptide sequences used for epitope mapping. The residues in human FGFR2-IIIb that differ from FGFR2-IIIc are in red. The amino acids involved in binding to FGF10 are highlighted in yellow. The peptides that bind to GP369 are denoted with “+” and those that do not bind are denoted with “−”.

Article Snippet: These findings rt the hypothesis that dysregulated FGFR2 signaling is the many critical tumorigenesis pathways in human s. Targeted therapy using FGFR2 mAb could be benefir patients with aberrantly activated/amplified FGFR2 female (Taco in 1:1 Tumo calipe formu appro of 10 contro the M mice 0.72 m Resea MFMappro 10 ani by i.p. using Resu Chara FGFR 8/7630.pdf by guest on 20 July 2024 ing. rials and Methods nes and reagents O III, HEC-1-A, AN3 CA, SNU-16, and human lung canll lines were acquired from the American Type Culture tion (ATCC).

Techniques: Binding Assay, Control, Incubation

Journal: Cancer Research

Article Title: GP369, an FGFR2-IIIb–Specific Antibody, Exhibits Potent Antitumor Activity against Human Cancers Driven by Activated FGFR2 Signaling

doi: 10.1158/0008-5472.can-10-1489

Figure Lengend Snippet: Figure 3. Characterization of FGFR2 antibodies by proliferation assays. A, GP369 suppresses cell proliferation driven by the WT or the COOH-terminally truncated isoform of FGFR2-IIIb or the mutant variants of FGFR2-IIIb. GP369 mAb was added to FDCP-1 cells expressing FGFR2-IIIb, FGFR2-IIIc, a COOH-terminally truncated IIIb isoform, or the S252W and N550K mutant variants in the presence of FGF1 (8 ng/mL) and heparin (5 μg/mL). MTT assays were carried out after 2 d. B, inhibition of the proliferation of SUM52PE cells by GP369. The cells were incubated in reduced serum medium (0.5% FBS) and were either left untreated (medium) or treated with mIgG or GP369 at various concentrations for 5 d. Cell proliferation was assessed by MTT assays. C, inhibition of the FGF7-induced proliferation of MCF7 cells by GP369. The cells were incubated in serum-free medium and were either left untreated (medium) or treated with mIgG or GP369 at 30 μg/mL for 3 d in the absence or presence of FGF7 (25 ng/mL). Cell proliferation was assessed by MTT assays.

Article Snippet: These findings rt the hypothesis that dysregulated FGFR2 signaling is the many critical tumorigenesis pathways in human s. Targeted therapy using FGFR2 mAb could be benefir patients with aberrantly activated/amplified FGFR2 female (Taco in 1:1 Tumo calipe formu appro of 10 contro the M mice 0.72 m Resea MFMappro 10 ani by i.p. using Resu Chara FGFR 8/7630.pdf by guest on 20 July 2024 ing. rials and Methods nes and reagents O III, HEC-1-A, AN3 CA, SNU-16, and human lung canll lines were acquired from the American Type Culture tion (ATCC).

Techniques: Mutagenesis, Expressing, Inhibition, Incubation

Journal: Cancer Research

Article Title: GP369, an FGFR2-IIIb–Specific Antibody, Exhibits Potent Antitumor Activity against Human Cancers Driven by Activated FGFR2 Signaling

doi: 10.1158/0008-5472.can-10-1489

Figure Lengend Snippet: Figure 4. Inhibition of FGFR2 signaling by GP369. A, GP369 suppresses FGF7-induced FGFR2 tyrosine phosphorylation and ERK1/2 phosphorylation in Ba/F3 cells overexpressing FGFR2-IIIb. After 3 h of serum starvation, the cells were treated with PBS (mock) or 5 μg/mL of either mIgG or GP369 for 1 h at 37°C before stimulation by FGF7 (20 ng/mL) and heparin (5 μg/mL) for 15 min. The protein lysates were analyzed by Western blot. B, GP369 blocks FGF7-induced FGFR2 and FRS2 tyrosine phosphorylation and ERK1/2 phosphorylation in SNU-16 cells. The cells were incubated with PBS (mock) or 5 μg/mL of either mIgG or GP369 for 1 h at 37°C before treatment with either heparin (20 μg/mL) or heparin plus FGF7 (30 ng/mL) for 15 min. Western blotting analysis was then carried out. C, downregulation of the FGFR2 protein levels by GP369. SNU-16 cells were treated with 10 μg/mL of either mIgG or GP369. The cells were harvested at various time points. Total levels of FGFR2 and β-tubulin were determined by Western blotting, and densitometric units quantified using Scion Image software.

Article Snippet: These findings rt the hypothesis that dysregulated FGFR2 signaling is the many critical tumorigenesis pathways in human s. Targeted therapy using FGFR2 mAb could be benefir patients with aberrantly activated/amplified FGFR2 female (Taco in 1:1 Tumo calipe formu appro of 10 contro the M mice 0.72 m Resea MFMappro 10 ani by i.p. using Resu Chara FGFR 8/7630.pdf by guest on 20 July 2024 ing. rials and Methods nes and reagents O III, HEC-1-A, AN3 CA, SNU-16, and human lung canll lines were acquired from the American Type Culture tion (ATCC).

Techniques: Inhibition, Phospho-proteomics, Western Blot, Incubation, Software

Journal: Cancer Research

Article Title: GP369, an FGFR2-IIIb–Specific Antibody, Exhibits Potent Antitumor Activity against Human Cancers Driven by Activated FGFR2 Signaling

doi: 10.1158/0008-5472.can-10-1489

Figure Lengend Snippet: Figure 5. Effect of GP369 on SNU-16 xenografts in vivo. A, GP369 inhibits the in vivo growth of SNU-16. SCID mice inoculated with SNU-16 cells received mIgG control at 20 mg/kg or GP369 at 2, 5, 10 or 20 mg/kg by i.p. injection twice weekly. B, analysis of total and phospho-FGFR2 in SNU-16 tumors treated with either mIgG or GP369. β-Tubulin was used as a loading control. C, Phospho-RTK status of individual tumors collected at the end of study from mice treated with mIgG (top images from mouse nos. 1 and 3 in the mIgG-treated group) or GP369 (bottom images from mouse nos. 1 and 5 in the GP369-treated group). Each RTK array was probed with 250 μg of protein lysate from an individual tumor.

Article Snippet: These findings rt the hypothesis that dysregulated FGFR2 signaling is the many critical tumorigenesis pathways in human s. Targeted therapy using FGFR2 mAb could be benefir patients with aberrantly activated/amplified FGFR2 female (Taco in 1:1 Tumo calipe formu appro of 10 contro the M mice 0.72 m Resea MFMappro 10 ani by i.p. using Resu Chara FGFR 8/7630.pdf by guest on 20 July 2024 ing. rials and Methods nes and reagents O III, HEC-1-A, AN3 CA, SNU-16, and human lung canll lines were acquired from the American Type Culture tion (ATCC).

Techniques: In Vivo, Control, Injection

Journal: Cancer Research

Article Title: GP369, an FGFR2-IIIb–Specific Antibody, Exhibits Potent Antitumor Activity against Human Cancers Driven by Activated FGFR2 Signaling

doi: 10.1158/0008-5472.can-10-1489

Figure Lengend Snippet: Figure 6. Effect of GP369 on MFM-223 xenografts in vivo. A, treatment of MFM-223 xenografts with GP369 results in tumor stasis. Nude mice bearing MFM-223 cells received either mIgG or GP369 at 20 mg/kg by i.p. injection twice weekly. B, analysis of total and phospho-FGFR2 in MFM-223 tumors treated with either the control IgG or GP369. β-Tubulin was used as a loading control.

Article Snippet: These findings rt the hypothesis that dysregulated FGFR2 signaling is the many critical tumorigenesis pathways in human s. Targeted therapy using FGFR2 mAb could be benefir patients with aberrantly activated/amplified FGFR2 female (Taco in 1:1 Tumo calipe formu appro of 10 contro the M mice 0.72 m Resea MFMappro 10 ani by i.p. using Resu Chara FGFR 8/7630.pdf by guest on 20 July 2024 ing. rials and Methods nes and reagents O III, HEC-1-A, AN3 CA, SNU-16, and human lung canll lines were acquired from the American Type Culture tion (ATCC).

Techniques: In Vivo, Injection, Control