Journal: Frontiers in Immunology
Article Title: A Novel Bifunctional Fusion Protein, Vunakizumab-IL22, for Protection Against Pulmonary Immune Injury Caused by Influenza Virus
doi: 10.3389/fimmu.2021.727941
Figure Lengend Snippet: vmab-mIL22 exhibits dual functionality, and the functional integrity of the cytokine payloads is preserved. (A, B) Biacore sensorgrams of the interaction of vmab with mIL17A and BIAcore sensorgrams of the interaction of vmab-mIL22 with mIL17A. vmab-mIL22 retained a high affinity for the cognate antigen, similar to that of the parental antibody Vmab, which was confirmed by surface plasmon resonance analysis. Sensorgrams display the response values at IL17A concentrations of 0.01 to 0.2 μg/mL, with a CM5 chip and capture with 100 RU vmab or vmab-mIL22. The equilibrium dissociation constant (K D ) was calculated using k d /k a for each measurement. k d , dissociation rate constant or off-rate constant; k a , association rate constant or on-rate constant. Binding was biphasic, and the data were fit with heterogeneous ligand models. (C) In vitro inhibition of functional activity by vmab-mIL22. Inhibition of Groα release from HT-29 cells as a measure of IL-17A inhibition. HT29 cells were stimulated with IL-17A (5 nM) in the presence of various concentrations of vmab-mIL22. vmab was used as a positive control. Groα levels in the supernatants were measured by ELISA from R&D systems. (D) Western blot analysis of STAT3 phosphorylation (p-STAT3) induced by 10 nM vmab-hIL22 in HepG2 cells at different times. (E) Western blot analysis of STAT3 phosphorylation (p-STAT3) induced by 10 nM mIL-22Fc or vmab-mIL22 in HepG2 cells at 1.5 h Serum-starved cells (3×10 6 /mL) were stimulated with PBS, 10 nM mIL22-Fc or Vmab-mIL22 for 1.5 h at 37°C. Whole-cell lysates were resolved on denaturing gels, transferred, and immunoblotted with a polyclonal anti-phospho-STAT3 antibody.
Article Snippet: The mean values of repeated wells were plotted and the standard deviation was calculated using the four parameter dose response inhibition function in Graphpad prism.
Techniques: Functional Assay, SPR Assay, Binding Assay, In Vitro, Inhibition, Activity Assay, Positive Control, Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics
