Design and characterization of FGFR1c/β-Klotho bispecific Avimers.
(A) Cartoon representation of FGFR1c monomer C2987 and β-Klotho dimer C2240 and orientation of these component Avimers in the bispecific Avimers C3201 and C3209. (B) Purified Avimers C2987, C2240, C3201 and C3209 analyzed by SDS-PAGE. 1 µg of protein was run per lane, with samples either reduced (R) or not reduced (NR). The upward shift on reduction is characteristic of Avimer proteins. (C) ELISA analysis of purified Avimers showing the specificity for both FGFR1c and β-Klotho is retained in the bispecific Avimers C3201 and C3209. Binding is measured by optical density (OD) at 450 nm. (D) Bispecific Avimers C3201 and C3209 induce luciferase activity in an FGF21-responsive engineered reporter cell line following four hour stimulation of serum-starved cells. Luciferase activity is measured in relative luminescence units (RLU). (E) FGF21 and bispecific Avimers C3201 and C3209 induce ERK phosphorylation in cultured primary human adipocytes. Data are expressed as % ERK protein that is phosphorylated, as measured by MSD. (F) Monospecific Avimers C2987 and C2240 were titrated on FGF21-responsive reporter cells +/−2 nM FGF21. The FGFR1c monomer C2987 antagonizes FGF21. Luciferase activity is measured in relative luminescence units (RLU). Data are represented as mean +/− SEM (n = 2).