Journal: Frontiers in Immunology

Article Title: Physicochemical and biological characterization of a bispecific antibody in a CrossMab/KIH format that targets EGFR and VEGF-A

doi: 10.3389/fimmu.2025.1659966

Figure Lengend Snippet: Physiochemical characterization of anti-EGFR/VEGF-A CrossMab/KIH BsAb. (A) The schematic representation of anti-EGFR/VEGF-A BsAb with CrossMab/KIH format illustrates how the BsAb was constructed from four antibody fragments derived from faricimab and cetuximab and was designed to target EGFR and VEGF-A. (B) Structural integrity was evaluated via SDS-PAGE analysis of 2 µg anti-EGFR/VEGF-A BsAb, 2 µg faricimab (FAR), and 2 µg cetuximab (CET) protein samples under non-reducing and reducing conditions. (C) Representative chromatograms of anti-EGFR/VEGF-A BsAb (15 µg) and faricimab (15 µg) protein samples were generated using SEC-HPLC method (D) Representative electropherograms of anti-EGFR/VEGF-A BsAb and faricimab protein samples were generated using non-reducing and reducing CE-SDS method. Data presented in this figure represented at least three independent experiments.

Article Snippet: Biotinylated human EGFR protein, His,AvitagTM was obtained from Acro Biosystems (Newark, DE, USA).

Techniques: Construct, Derivative Assay, SDS Page, Generated

Journal: Frontiers in Immunology

Article Title: Physicochemical and biological characterization of a bispecific antibody in a CrossMab/KIH format that targets EGFR and VEGF-A

doi: 10.3389/fimmu.2025.1659966

Figure Lengend Snippet: Potency characterization of anti-EGFR/VEGF-A BsAb. (A) Dose-dependent binding activity of anti-EGFR/VEGF-A BsAb, bevacizumab, faricimab, and cetuximab was evaluated using an ELISA binding assay. (B) Simultaneous binding activity of anti-EGFR/VEGF-A BsAb to its targets, biotinylated EGFR and VEGF-A was evaluated using an ELISA binding assay. (C) Dose-dependent inhibition of VEGF-A/VEGFR2 activation by anti-EGFR/VEGF-A BsAb, bevacizumab, faricimab, and ramucirumab was performed using a VEGF activity bioassay.

Article Snippet: Biotinylated human EGFR protein, His,AvitagTM was obtained from Acro Biosystems (Newark, DE, USA).

Techniques: Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Activation Assay, Bioassay

Journal: Frontiers in Immunology

Article Title: Physicochemical and biological characterization of a bispecific antibody in a CrossMab/KIH format that targets EGFR and VEGF-A

doi: 10.3389/fimmu.2025.1659966

Figure Lengend Snippet: Thermal stability evaluation of anti-EGFR/VEGF-A BsAb. Anti-EGFR/VEGF-A BsAb (15 µg) was stressed at 42°C for 2 weeks. The protein samples were collected at seven different time points: 0 h (Day 0), 24 h (Day 1), 48 h (Day 2), 72 h (Day 3), 168 h (Day 7), 240 h (Day 10), and 336 h (Day 14). (A) Overlaid chromatograms of thermal stressed and unstressed anti-EGFR/VEGF-A BsAb protein samples was generated using a SEC-HPLC method. (B) SDS-PAGE analysis showed the structural integrity of thermal stressed and unstressed anti-EGFR/VEGF-A BsAb protein samples under non-reducing and reducing conditions. (C) Dose-dependent binding activity of anti-EGFR/VEGF-A BsAb protein samples to EGFR and VEGF-A was evaluated using the ELISA binding assay. (D) Dose-dependent inhibition of VEGF-A/VEGFR2 activation by thermal stressed and unstressed anti-EGFR/VEGF-A BsAb protein samples was performed using a VEGF activity bioassay.

Article Snippet: Biotinylated human EGFR protein, His,AvitagTM was obtained from Acro Biosystems (Newark, DE, USA).

Techniques: Generated, SDS Page, Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Inhibition, Activation Assay, Bioassay

Journal: Frontiers in Immunology

Article Title: Physicochemical and biological characterization of a bispecific antibody in a CrossMab/KIH format that targets EGFR and VEGF-A

doi: 10.3389/fimmu.2025.1659966

Figure Lengend Snippet: Inhibition of ligand-induced activation of EGFR by anti-EGFR/VEGF-A BsAb. (A) Western blot analysis was performed to measure EGFR expression levels in HUVEC and OC cell lines: PA-1, CaOV3, OVCAR3, and SKOV3 cells. Whole cell lysates were prepared from each cell line, and Western blotting was performed to measure relative EGFR protein levels in these cell lines. (B) Phospho-EGFR and EGFR levels were measured using Western blot analysis of serum-starved WCL collected from CaOV3 and SKOV3 cells. Cells were pre-treated with 10 µg indicated monoclonal antibodies and BsAb for 1 hour followed by 100 ng/mL ligand stimulation for 15 minutes.

Article Snippet: Biotinylated human EGFR protein, His,AvitagTM was obtained from Acro Biosystems (Newark, DE, USA).

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Bioprocessing

Journal: Frontiers in Immunology

Article Title: Physicochemical and biological characterization of a bispecific antibody in a CrossMab/KIH format that targets EGFR and VEGF-A

doi: 10.3389/fimmu.2025.1659966

Figure Lengend Snippet: Inhibition of ligand-induced activation of VEGFR2 by anti-EGFR/VEGF-A BsAb. (A) Western blot analysis was performed to measure VEGFR2 expression levels in HUVEC and OC cell lines: PA-1, CaOV3, OVCAR3, and SKOV3 cells. WCL were prepared from each cell line, and Western blot was performed to measure relative VEGFR2 protein levels in these cell lines. (B) Phospho-VEGFR2 and VEGFR2 levels were measured using western blot analysis of serum-starved WCL collected from CaOV3 and PA-1 cells. Cells were pre-treated with 10 µg indicated monoclonal antibodies and BsAb for 1 hour followed by 100 ng/mL ligand stimulation for 15 minutes.

Article Snippet: Biotinylated human EGFR protein, His,AvitagTM was obtained from Acro Biosystems (Newark, DE, USA).

Techniques: Inhibition, Activation Assay, Western Blot, Expressing, Bioprocessing

Journal: Frontiers in Immunology

Article Title: Physicochemical and biological characterization of a bispecific antibody in a CrossMab/KIH format that targets EGFR and VEGF-A

doi: 10.3389/fimmu.2025.1659966

Figure Lengend Snippet: Inhibition of paracrine VEGFR2 activation in HUVECs by anti-EGFR/VEGF-A BsAb. (A) The CellTiter-Glo luminescent cell viability assays were performed in HUVEC cells. 10,000 cells were seeded in white bottom of 96-well plates and allowed to adhere overnight in media with 1% FBS. After treatments with 10 μg/mL cetuximab (B) , bevacizumab (C) , or anti-EGFR/VEGF-A BsAb (D) , CellTiter-Glo reagent was added into the plates and luminescence (i.e., viability) was measured using a Promega GloMax Discover plate reader. (E) Western blot analysis was performed to measure the inhibition of ligand-induced activation of VEGFR2 and its downstream pathways (Akt, and FAK) by bevacizumab (BEV) and anti-EGFR/VEGF-A BsAb. (F) The levels of VEGF-A were determined by ELISA assay in supernatants of CaOV3, SKOV3, OVCAR3, and PA-1 cells after serum-starving the cells in a 6-well-plate for 48 h (G) Inhibition of the conditional media (CM)-mediated VEGFR2 activity in HUVEC cells by cetuximab (CET), bevacizumab (BEV), and anti-EGFR/VEGF-A BsAb. The conditional media (CM) samples were collected from SKOV3 cell culture after 48 h serum-starvation. HUVEC cells were serum-starved for 24 h CM samples were collected from the SKOV3 cells and pre-incubated with 10 μg/mL cetuximab (CET), bevacizumab (BEV), or anti-EGFR/VEGF-A BsAb. HUVEC media was removed from the cells and replaced with the CM for 2 h before WCL was harvested. Whole cell lysates were then subjected to western blot analysis.

Article Snippet: Biotinylated human EGFR protein, His,AvitagTM was obtained from Acro Biosystems (Newark, DE, USA).

Techniques: Inhibition, Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Activity Assay, Cell Culture, Incubation

Journal: Nanomedicine : nanotechnology, biology, and medicine

Article Title: Targeted Extracellular Nanoparticles Enable Intracellular Detection of Activated Epidermal Growth Factor Receptor in Living Brain Cancer Cells

doi: 10.1016/j.nano.2011.05.002

Figure Lengend Snippet: Schematics of direct labeling of active EGF-R with Qdots. A. Qdots functionalized with biotinylated anti-eEGF-R antibodies are incubated with the cells on ice, allowing the binding of the functionalized Qdots to the cell-surface EGF-R; B. Cells are stimulated with 1 mg/ml EGF at 37°C; C. EGF activates EGF-R, inducing the intra-cellular internalization of the labeled active receptors; D. Qdot∷eEGF-R Ab complexes bound to the active receptors are internalized in endosomes.

Article Snippet: Antibodies and Immunocytochemistry Cells grown on coverslips were fixed with paraformaldehyde (Sigma, St. Louis, MO) and labeled with biotinylated mouse anti-external-EGF-R (eEGF-R;1:500) – recognizing an external epitope (Meridian Life Sciences, Saco, ME), mouse anti-phosphorylated EGF-R (pEGF-R;1:500) (Meridian Life Sciences, Saco, ME), rabbit anti-phosphorylated-Akt (1:200) (Cell Signaling, Danvers, MO), goat anti-mouse and anti-rabbit AlexaFluor® 488 antibodies (Invitrogen Molecular Probes, Eugene, OR).

Techniques: Labeling, Incubation, Binding Assay