fgfr1 fc protein  (R&D Systems)

Journal: Molecular Medicine

Article Title: The cytotoxic conjugate of highly internalizing tetravalent antibody for targeting FGFR1-overproducing cancer cells

doi: 10.1186/s10020-021-00306-2

Figure Lengend Snippet: Conjugation of tetravalent engineered antibody (T-Fc) with a cytotoxic payload. a Hypothetical model of the effect of FGFR1 clustering on the receptor endocytosis. FGFR1 dimerization via FGF1 binding induces receptor activation and clathrin-mediated endocytosis. Clustering of FGFR1 into large structures on the plasma membrane with tetravalent T-Fc largely improves the cellular uptake of FGFR1-antibody complexes. Furthermore, FGFR1 clustering changes the mechanism of the receptor endocytosis by engaging dynamin-2-dependent CIE pathways. b The chemical structure of monomethyl auristatin E bearing the valine-citrulline linker (vcMMAE). c The schematic representation of the conjugation of T-Fc with the cytotoxic compound MMAE. The Fc region of IgG (CH2 and CH3 domains) is labeled in gray, and anti-FGFR1 scFv proteins (VH and VL fusions) are marked in blue. Antibody regions recognizing epitopes within FGFR1 are marked in orange. Thiol groups of reduced cysteines are marked in yellow and attached cytotoxic payloads are marked in red. d , e The efficiency of the conjugation and purity of T-Fc-vcMMAE were analyzed with SDS/PAGE ( d ) and western blotting ( e ) with antibodies recognizing the Fc fragment. f The spectroscopic analysis of DAR parameter for T-Fc-vcMMAE. DAR was calculated through the absorbance measurement for T-Fc and T-Fc-vcMMAE at 248 nm and 280 nm wavelengths according to (Chen ).

Article Snippet: To examine the interaction of antibody alone and its conjugate with murine FGFR1, each compound (40 nM) was injected on the CM4 sensor chip with immobilized murine recombinant FGFR1 (10,135-FR, R&D Systems) at 535 RU for 120 s at 30 μL·min −1 flow rate.

Techniques: Conjugation Assay, Binding Assay, Activation Assay, Clinical Proteomics, Membrane, Labeling, SDS Page, Western Blot

Journal: Molecular Medicine

Article Title: The cytotoxic conjugate of highly internalizing tetravalent antibody for targeting FGFR1-overproducing cancer cells

doi: 10.1186/s10020-021-00306-2

Figure Lengend Snippet: Interaction of T-Fc-vcMMAE with FGFR1. a Evaluation of T-Fc and T-Fc-vcMMAE interaction with FGFR1 by BLI. The extracellular region of FGFR1 was immobilized on BLI sensors and incubated either with T-Fc or T-Fc-vcMMAE. The association and dissociation profiles were measured. b SPR-determined kinetic parameters of the interaction between T-Fc-vcMMAE and FGFR1. The extracellular region of FGFR1 was immobilized on SPR sensors and incubated with various concentrations of T-Fc-vcMMAE. K D value is presented. c SPR results of the interaction between T-Fc and T-Fc-vcMMAE, and murine FGFR1, respectively. The murine recombinant FGFR1 was immobilized on SPR sensors and incubated with T-Fc or T-Fc-vcMMAE. The association and dissociation profiles were measured. d T-Fc and T-Fc-vcMMAE are unable to activate FGFR1. Serum-starved NIH3T3 cells were incubated with FGF1 (positive control) or with different concentrations of T-Fc or T-Fc-vcMMAE. Cells were lysed and activation of FGFR1, and receptor-downstream signaling was assessed with western blotting (WB). The level of tubulin served as a loading control

Article Snippet: To examine the interaction of antibody alone and its conjugate with murine FGFR1, each compound (40 nM) was injected on the CM4 sensor chip with immobilized murine recombinant FGFR1 (10,135-FR, R&D Systems) at 535 RU for 120 s at 30 μL·min −1 flow rate.

Techniques: Incubation, Recombinant, Positive Control, Activation Assay, Western Blot, Control

Journal: Molecular Medicine

Article Title: The cytotoxic conjugate of highly internalizing tetravalent antibody for targeting FGFR1-overproducing cancer cells

doi: 10.1186/s10020-021-00306-2

Figure Lengend Snippet: FGFR1-mediated internalization of T-Fc and T-Fc-vcMMAE. a FGFR1-dependent endocytosis of T-Fc and T-Fc-vcMMAE. U2OS-R1 cells stably expressing FGFR1 were incubated with T-Fc or T-Fc-vcMMAE for 15 min at 37 °C. Nuclei were stained with NucBlue Live, and early endosomes were labeled by CellLight Early Endosomes-RFP. Cells were fixed, and internalized antibodies were visualized with Zenon AF-488 using wide-field fluorescence microscope. Scale bar represents 20 μm. b – e Confocal microscopy analysis of the T-Fc and T-Fc-vcMMAE internalization. B and C . U2OS-R1 cells were briefly incubated with T-Fc and T-Fc-vcMMAE (t = 0 min) and analyzed with quantitative immunofluorescence microscopy using Zenon-AF-488 to label Fc-bearing recombinant proteins. d , e T-Fc and T-Fc-vcMMAE were incubated with U2OS-R1 cells for different time periods (5, 15, 30, 60 min) and internalized antibodies were labeled with Zenon AF-488, and analyzed with confocal microscopy. Scale bar represents 50 μm. Quantification of T-Fc and T-Fc-vcMMAE internalization (expressed as integral fluorescence intensity in arbitrary units, AU) was performed using the HARMONY software. Mean values of three independent experiments of integral intensity of Zenon AF-488 signal ( c ) and integral intensity of Zenon AF-488 vesicles ( e ) ± SEM are shown. T -test was used to assess the statistical significance of measured differences in internalization; * p < 0.05, ** p < 0.01, *** p < 0.0001, n.s.- not significant. f Efficiency and selectivity of T-Fc and T-Fc-vcMMAE internalization studied with flow cytometry. Internalization was analyzed with serum-starved U2OS and U2OS-R1 cells, treated with T-Fc or T-Fc-vcMMAE labeled with DyLight550. After 40 min incubation on ice, cells were transferred to 37 °C for 15 min, and then subsequently analyzed by flow cytometry

Article Snippet: To examine the interaction of antibody alone and its conjugate with murine FGFR1, each compound (40 nM) was injected on the CM4 sensor chip with immobilized murine recombinant FGFR1 (10,135-FR, R&D Systems) at 535 RU for 120 s at 30 μL·min −1 flow rate.

Techniques: Stable Transfection, Expressing, Incubation, Staining, Labeling, Fluorescence, Microscopy, Confocal Microscopy, Immunofluorescence, Recombinant, Software, Flow Cytometry

Journal: Molecular Medicine

Article Title: The cytotoxic conjugate of highly internalizing tetravalent antibody for targeting FGFR1-overproducing cancer cells

doi: 10.1186/s10020-021-00306-2

Figure Lengend Snippet: Cytotoxicity of T-Fc-vcMMAE against FGFR1-overproducing cancer cells. a FGFR1 expression levels in studied cell lines analyzed by western blotting using anti-FGFR1 antibody. Tubulin level assessed with anti-tubulin antibody served as a loading control. b – i Cytotoxic potential of T-Fc and T-Fc-vcMMAE. Negative-FGFR1 cells: U2OS ( b ), HCC15 ( d ) and T47D ( e ), and positive-FGFR1 cells: U2OS-R1 ( c ), JIMT-1 ( f ), COLO-699 ( g ), NCI-H520 ( h ) and NCI-H1581 ( i ) were treated with indicated agents at various concentrations for 96 h and their viability was assessed with the Presto Blue assay. Presented results are mean values from three experiments ± SD. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.0001, not significant differences are not marked. j EC 50 values for T-Fc-vcMMAE for each cell line, respectively. EC50 values were calculated based on the Hill equation using Origin 7 software (Northampton, MA). k The T-Fc internalization into studied cancer cell lines, visualized with fluorescence microscopy. Cell line tested in cytotoxic assays were incubated with 15 µg/mL of T-Fc for 30 min at 37 °C. Nuclei were stained with NucBlue Live, cells were fixed, and internalized antibodies were visualized with Zenon AF-488 using wide-field fluorescence microscopy. Scale bar represents 20 μm

Article Snippet: To examine the interaction of antibody alone and its conjugate with murine FGFR1, each compound (40 nM) was injected on the CM4 sensor chip with immobilized murine recombinant FGFR1 (10,135-FR, R&D Systems) at 535 RU for 120 s at 30 μL·min −1 flow rate.

Techniques: Expressing, Western Blot, Control, Software, Fluorescence, Microscopy, Incubation, Staining

Journal: Scientific Reports

Article Title: Specific inhibition of FGF5-induced cell proliferation by RNA aptamers

doi: 10.1038/s41598-021-82350-w

Figure Lengend Snippet: Inhibition of NIH3T3 cell proliferation by aptamers F5f1–F5f7 and random RNA ( a ) and the truncated aptamers F5f1_56 and F5f3_56 ( b ). Cells were cultured with human FGF5 (“circle”) and human FGF2 (“triangle”). The experiments were performed three times, and the mean value and errors were shown. ( c ) Immunoblotting images of phospho-FGFR1 in FGF5-stimulated NIH3T3 cells in the presence of F5f1 (lanes 3–6) or random RNA (lanes 7–10).

Article Snippet: Dissociation constants were determined using a Langmuir (1:1) binding model. To assess the binding specificity of the aptamer, 100 nM of recombinant human FGF1, FGF2, FGF4, FGF6, or FGFR1α (IIIc)-Fc chimera (all proteins purchased from R&D Systems, Inc.) was injected with four equivalents of low-molecular-weight heparin.

Techniques: Inhibition, Cell Culture, Western Blot

Journal: Scientific Reports

Article Title: Specific inhibition of FGF5-induced cell proliferation by RNA aptamers

doi: 10.1038/s41598-021-82350-w

Figure Lengend Snippet: Specificity of F5f1 aptamer binding to FGF5. After immobilization of F5f1 aptamer on the sensor chip, 100 nM human FGF5, other human FGF family members, or extracellular domain of human FGFR1 was injected with four equivalents of low-molecular-weight heparin.

Article Snippet: Dissociation constants were determined using a Langmuir (1:1) binding model. To assess the binding specificity of the aptamer, 100 nM of recombinant human FGF1, FGF2, FGF4, FGF6, or FGFR1α (IIIc)-Fc chimera (all proteins purchased from R&D Systems, Inc.) was injected with four equivalents of low-molecular-weight heparin.

Techniques: Binding Assay, Injection, Molecular Weight

Journal: Scientific Reports

Article Title: Specific inhibition of FGF5-induced cell proliferation by RNA aptamers

doi: 10.1038/s41598-021-82350-w

Figure Lengend Snippet: Competitive inhibition of the binding of FGF5 to FGFR1 by the F5f1 aptamer. After immobilization of the extracellular domain of human FGFR1 on the sensor chip, a mixture of 100 nM human FGF5 and 100 nM low-molecular-weight heparin was injected with or without 200 nM F5f1 aptamer.

Article Snippet: Dissociation constants were determined using a Langmuir (1:1) binding model. To assess the binding specificity of the aptamer, 100 nM of recombinant human FGF1, FGF2, FGF4, FGF6, or FGFR1α (IIIc)-Fc chimera (all proteins purchased from R&D Systems, Inc.) was injected with four equivalents of low-molecular-weight heparin.

Techniques: Inhibition, Binding Assay, Molecular Weight, Injection

Journal: The Journal of Biological Chemistry

Article Title: Heparin potentiates Avastin-mediated inhibition of VEGF binding to fibronectin and rescues Avastin activity at acidic pH

doi: 10.1074/jbc.RA119.009194

Figure Lengend Snippet: Avastin enhances VEGF binding to heparin and heparin binds VEGF–Avastin complexes and enhances VEGF binding to Avastin. A, streptavidin-coated 96-well microtiter plates were coated with biotin (1 μg/ml) or biotin–heparin (5 μg/ml) and incubated with 125I-VEGF (10 ng/ml) alone or with Avastin at the concentrations indicated (0–100 nm) for 2 h at 4 °C. 125I-VEGF bound to the plates was extracted and counted, and the average of triplicate determinations ± S.D. is presented. ANOVA was performed, and the conditions that showed a statistically significant increase in binding are indicated by *. B, Avastin was incubated with biotin (1 μg/ml) or biotin–heparin (5 μg/ml)-coated plates in the presence and absence of VEGF (10 ng/ml) for 2 h at 4 °C, and then an ELISA protocol was used to detect bound Avastin. The average values of triplicate determinations ± S.D. are shown. Only conditions where VEGF and Avastin were incubated with biotin–heparin-coated plates produced a statistically significant signal above background. C, to understand whether heparin influences VEGF binding to Avastin, Avastin (0.5 nm) or Fc-FGFR1 (0.5 nm)-coated protein A plates were incubated with 125I-VEGF (10 ng/ml) and the indicated concentration of heparin (0–100 μg/ml) for 2 h at 4 °C. The 125I-VEGF bound to the surface was extracted and measured. The average of triplicate values ± S.D. is presented. All concentrations of heparin tested produced a statistically significant increase in VEGF binding to Avastin.

Article Snippet: Recombinant human VEGF165 and Fc-FGFR1 chimeric protein were purchased from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Produced, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Heparin potentiates Avastin-mediated inhibition of VEGF binding to fibronectin and rescues Avastin activity at acidic pH

doi: 10.1074/jbc.RA119.009194

Figure Lengend Snippet: Avastin's capacity to inhibit VEGF binding to FN is reduced at acidic pH due to decreased Avastin–VEGF binding. A, 125I-VEGF (10 ng/ml) ± Avastin was incubated with FN-coated plates in binding buffer at the indicated pH values (5–8) for 2 h at 4 °C. After the incubation, 125I-VEGF bound to FN was extracted and counted. The average of triplicate values ± S.D. is shown. * indicates a statistically significant difference between VEGF binding in the presence and absence of Avastin; N.S. means not significant. B, values of 125I-VEGF bound in the presence and absence of Avastin were used calculate % VEGF binding inhibition at each pH using the equation: % VEGF binding inhibition = (VEGF bound (absence of Avastin) − (presence of Avastin)/VEGF bound in the absence of Avastin) × 100. C, 125I-VEGF (10 ng/ml) binding to Avastin (0.5 nm) bound to protein A plates was measured at various pH values after a 2-h incubation at 4 °C. 125I-VEGF association to Fc-FGFR1 (0.5 nm)-coated plates was also measured to control for nonspecific interactions. The average 125I-VEGF bound (fmol/well) ± S.D. of triplicate determinations is shown. Statistically significant reduction in VEGF binding compared with the value at pH 8 is noted (*).

Article Snippet: Recombinant human VEGF165 and Fc-FGFR1 chimeric protein were purchased from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Incubation, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Heparin potentiates Avastin-mediated inhibition of VEGF binding to fibronectin and rescues Avastin activity at acidic pH

doi: 10.1074/jbc.RA119.009194

Figure Lengend Snippet: Avastin enhances VEGF binding to heparin and heparin binds VEGF–Avastin complexes and enhances VEGF binding to Avastin. A, streptavidin-coated 96-well microtiter plates were coated with biotin (1 μg/ml) or biotin–heparin (5 μg/ml) and incubated with 125I-VEGF (10 ng/ml) alone or with Avastin at the concentrations indicated (0–100 nm) for 2 h at 4 °C. 125I-VEGF bound to the plates was extracted and counted, and the average of triplicate determinations ± S.D. is presented. ANOVA was performed, and the conditions that showed a statistically significant increase in binding are indicated by *. B, Avastin was incubated with biotin (1 μg/ml) or biotin–heparin (5 μg/ml)-coated plates in the presence and absence of VEGF (10 ng/ml) for 2 h at 4 °C, and then an ELISA protocol was used to detect bound Avastin. The average values of triplicate determinations ± S.D. are shown. Only conditions where VEGF and Avastin were incubated with biotin–heparin-coated plates produced a statistically significant signal above background. C, to understand whether heparin influences VEGF binding to Avastin, Avastin (0.5 nm) or Fc-FGFR1 (0.5 nm)-coated protein A plates were incubated with 125I-VEGF (10 ng/ml) and the indicated concentration of heparin (0–100 μg/ml) for 2 h at 4 °C. The 125I-VEGF bound to the surface was extracted and measured. The average of triplicate values ± S.D. is presented. All concentrations of heparin tested produced a statistically significant increase in VEGF binding to Avastin.

Article Snippet: Recombinant human VEGF165 and Fc-FGFR1 chimeric protein were purchased from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Produced, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Heparin potentiates Avastin-mediated inhibition of VEGF binding to fibronectin and rescues Avastin activity at acidic pH

doi: 10.1074/jbc.RA119.009194

Figure Lengend Snippet: Avastin's capacity to inhibit VEGF binding to FN is reduced at acidic pH due to decreased Avastin–VEGF binding. A, 125I-VEGF (10 ng/ml) ± Avastin was incubated with FN-coated plates in binding buffer at the indicated pH values (5–8) for 2 h at 4 °C. After the incubation, 125I-VEGF bound to FN was extracted and counted. The average of triplicate values ± S.D. is shown. * indicates a statistically significant difference between VEGF binding in the presence and absence of Avastin; N.S. means not significant. B, values of 125I-VEGF bound in the presence and absence of Avastin were used calculate % VEGF binding inhibition at each pH using the equation: % VEGF binding inhibition = (VEGF bound (absence of Avastin) − (presence of Avastin)/VEGF bound in the absence of Avastin) × 100. C, 125I-VEGF (10 ng/ml) binding to Avastin (0.5 nm) bound to protein A plates was measured at various pH values after a 2-h incubation at 4 °C. 125I-VEGF association to Fc-FGFR1 (0.5 nm)-coated plates was also measured to control for nonspecific interactions. The average 125I-VEGF bound (fmol/well) ± S.D. of triplicate determinations is shown. Statistically significant reduction in VEGF binding compared with the value at pH 8 is noted (*).

Article Snippet: Recombinant human VEGF165 and Fc-FGFR1 chimeric protein were purchased from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Incubation, Inhibition