TABLE 2

SMAD-responsive luciferase reporter gene assay

Recombinant FS315-hFc variants with either myostatin or activin A were incubated with A204 rhabdomyosarcoma cells stably expressing a SMAD-responsive luciferase reporter construct. The IC50 for each of the variants was determined by fitting the inhibition curve using GraphPad Prism software and is represented as mean ± S.D. (n = 3).

FS315-hFc variantIC50 for Myostatin StimulationIC50 for Activin A Stimulation
nM
Wild type0.6 ± 0.031.0 ± 0.02
Heparin-binding variants
 ΔHBS0.4 ± 0.021.4 ± 0.1
 del75-86>10*>10*
 K82E0.6 ± 0.011.1 ± 0.02
 K(81,82)E0.6 ± 0.091.0 ± 0.2
 R78E/K82E0.5 ± 0.11.0 ± 0.1
 K(82,84)E0.7 ± 0.061.1 ± 0.01
 K(76,82,84)E0.4 ± 0.031.0 ± 0.1
 K(76,81,82)A0.8 ± 0.071.1 ± 0.1
 K(76,81,82)E0.6 ± 0.030.8 ± 0.01
 K(76,81,82)D0.7 ± 0.031.1 ± 0.1
Hyperglycosylation variants
 K82T0.9 ± 0.041.2 ± 0.04
 G74N/K76S0.9 ± 0.061.3 ± 0.06
 K75N/C77T/K82T1.8 ± 0.13.7 ± 0.6
 C66A/K75N/C77T1.5 ± 0.012.1 ± 0.05
 C66S/K75N/C77T1.9 ± 0.094.4 ± 1.2
  • * The IC50 curve was not saturated because of the weak binding.