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. 2014 Feb;34(4):606-18.
doi: 10.1128/MCB.01307-13. Epub 2013 Dec 2.

An antibody blocking activin type II receptors induces strong skeletal muscle hypertrophy and protects from atrophy

Estelle Lach-Trifilieff  1 , Giulia C Minetti, KellyAnn Sheppard, Chikwendu Ibebunjo, Jerome N Feige, Steffen Hartmann, Sophie Brachat, Helene Rivet, Claudia Koelbing, Frederic Morvan, Shinji Hatakeyama, David J Glass
Affiliations

An antibody blocking activin type II receptors induces strong skeletal muscle hypertrophy and protects from atrophy

Estelle Lach-Trifilieff et al. Mol Cell Biol. 2014 Feb.

Abstract

The myostatin/activin type II receptor (ActRII) pathway has been identified to be critical in regulating skeletal muscle size. Several other ligands, including GDF11 and the activins, signal through this pathway, suggesting that the ActRII receptors are major regulatory nodes in the regulation of muscle mass. We have developed a novel, human anti-ActRII antibody (bimagrumab, or BYM338) to prevent binding of ligands to the receptors and thus inhibit downstream signaling. BYM338 enhances differentiation of primary human skeletal myoblasts and counteracts the inhibition of differentiation induced by myostatin or activin A. BYM338 prevents myostatin- or activin A-induced atrophy through inhibition of Smad2/3 phosphorylation, thus sparing the myosin heavy chain from degradation. BYM338 dramatically increases skeletal muscle mass in mice, beyond sole inhibition of myostatin, detected by comparing the antibody with a myostatin inhibitor. A mouse version of the antibody induces enhanced muscle hypertrophy in myostatin mutant mice, further confirming a beneficial effect on muscle growth beyond myostatin inhibition alone through blockade of ActRII ligands. BYM338 protects muscles from glucocorticoid-induced atrophy and weakness via prevention of muscle and tetanic force losses. These data highlight the compelling therapeutic potential of BYM338 for the treatment of skeletal muscle atrophy and weakness in multiple settings.

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Figures

FIG 1
FIG 1
Affinity and potency of BYM338 at inhibiting myostatin (Mst) and activin A (Act A) signaling in a Smad2/3 CAG-luciferase reporter gene assay. (A) Affinity of BYM338 for human ActRIIA (hActRIIA) and human ActRIIB (hActRIIB) determined by solution equilibrium titration. Shown are representative examples of the results from 4 or 5 independent experiments. (B) Myostatin- and activin A-induced dose-dependent increases in Smad2/3 activity were measured with a CAGA-luciferase reporter stably expressed in HEK293 cells. (C) The ActRII-dependent response was determined upon addition of myostatin (10 ng/ml) or activin A (10 ng/ml) in the presence of increasing concentrations of BYM338. Shown are means ± SEMs from 3 to 4 independent experiments. RGA, reporter gene assay; RLU, relative light units.
FIG 2
FIG 2
ActRII inhibition relieves myostatin and activin A inhibition of differentiation. (A) Human primary myoblasts differentiated for 4 days in the absence (control [ctrl]) and presence of myostatin (30 ng/ml) or activin A (30 ng/ml) alone and in combination with BYM338 (10 mg/ml) were stained for MyHC and with DAPI. Shown are representative pictures. (B) Analysis of myotube diameters and the fusion index performed as described in Materials and Methods. Data are expressed as a percentage of the values for the control. Shown are means ± SEMs from 4 independent experiments. Asterisks indicate P values versus the control; pound signs indicate P values versus samples without BYM338 treatment. ****, P < 0.0001; *** or ###, P < 0.001; ** or ##, P < 0.01; *, P < 0.05. (C) BYM338 (mg/ml) reverses myostatin-induced Smad2/3 phosphorylation after 1 h of stimulation (myostatin was used at 10 ng/ml). At 24 h poststimulation, BYM338 prevents a reduction in AKT phosphorylation resulting from myostatin stimulation. Shown are representative immunoblots. Tot, total. (D) Immunoblotting of myogenin versus tubulin (as a loading control) from human primary myoblasts differentiated for 2 or 3 days in the absence or presence of myostatin (30 ng/ml) or activin A (30 ng/ml) alone and in combination with BYM338 (10 mg/ml). (E) Analysis of creatine kinase (CK) activity from myotubes that had been differentiated for 3 or 4 days and treated with either myostatin (10 ng/ml) or activin A (10 ng/ml) in the presence of increasing concentration of BYM338. Shown are means ± SEMs from 3 or 4 independent experiments.
FIG 3
FIG 3
ActRII inhibition relieves myostatin- and activin A-induced atrophy, (A) Human primary myotubes treated for 72 h in the absence (control) and presence of myostatin (100 ng/ml) or activin A (100 ng/ml) alone and in combination with BYM338 (30 μg/ml) were stained with anti-MyHC antibody and DAPI. Shown are representative pictures. (B) Analysis of myotube diameters was performed as described in Materials and Methods. Data are expressed as a percentage of the value for the control, as indicated in Material and Methods. Shown are means ± SEMs from 4 independent experiments, Asterisks indicate P values versus controls; pound signs indicate P values versus samples without BYM338 treatment. **** or ####, P < 0.0001; *** or ###, P < 0.001; ** or ##, P < 0.01; #, P < 0.05. (C) The level of Smad3 phosphorylation in myotubes treated for 2 h in the absence (control) and presence of myostatin (100 ng/ml) or activin A (100 ng/ml) alone and in combination with BYM338 (30 μg/ml) was quantitated using the AlphaScreen SureFire protocol. Shown are means ± SEMs from 4 independent experiments. Asterisks indicate P values versus the control; pound signs indicate P values versus samples without BYM338 treatment. (D) Analysis of AKT phosphorylation was performed using a MesoScale Discovery reader in myotubes treated for 24 h. Shown are means ± SEMs from 6 or 7 independent experiments. (E) Immunoblotting of MyHC from samples of myotubes incubated for 72 h. A blot representative of blots from three independent experiments is shown.
FIG 4
FIG 4
Dose-dependent efficacy of BYM338 in naive SCID mice. (A) Body weight over 4 weeks of treatment with vehicle or BYM338 dosed weekly at 6 or 20 mg/kg. Absolute body weight change values are expressed as means ± SEMs (n = 9 or 10). (B) The weights of the tibialis anterior, EDL, gastrocnemius, and soleus muscles were normalized by the initial body weight (BW). Values are expressed as the mean percent change from the value for the control ± SEM (n = 9 or 10). *, P < 0.05 versus the control; **, P < 0.01 versus the control (Student's t test). (C) Fiber cross-sectional area of the gastrocnemius muscle with the plantaris muscle. The frequency distributions of the fiber cross-sectional area are plotted. Values are expressed as means ± SEMs (n = 4 or 5). (D) Fiber type distribution in the gastrocnemius muscle from control mice and mice treated with BYM338 at 20 mg/kg. Values are expressed as the percentage of the fiber type ± SEM (n = 5 or 6). (E) Fiber number (indicated on the y axis) in the gastrocnemius muscle from vehicle-treated (white bar) and BYM2338 (20 mg/kg)-treated (striped bar) mice expressed as means ± SEMs (n = 5 or 6).
FIG 5
FIG 5
Comparison of efficacy of anti-ActRII antibody treatment versus pharmacological or genetic myostatin inhibition. (A) Inhibition of CAGA-luciferase reporter gene activity induced with 10 ng/ml myostatin with various concentrations of mutant D76A propeptide. (B) Body weight. (C and D) Weight of the gastrocnemius muscle with the plantaris muscle (C) and cross-sectional area of the gastrocnemius muscle with the plantaris muscle (D) from naive SCID mice administered BYM338 (10 mg/kg; striped bar), myostatin propeptide D76A (30 mg/kg; gray bar), or PBS (white bar) i.p. weekly for 5 weeks with an additional administration on day 3. Muscle weight was normalized to the initial body weight measured on day 0. Absolute body weight change values are expressed as means ± SEMs (n = 9 or 10). *, P < 0.05 versus the group treated with PBS; **, P < 0.01 versus the group treated with PBS (Dunnett's test following ANOVA). (E to G) Body weight (E), lean mass (F), and tibialis anterior muscle weight (G) of wild-type mice (n = 10/group) and MstnLn/Ln mice (n = 8/group) administered CDD866, murinized BYM338 (20 mg/kg), or PBS s.c. weekly on days 0, 7, 14, 21, and 28. Body weight and lean mass were measured on day 0 and day 21 using a mouse body composition NMR analyzer. Data are presented as the mean ± SEM and were analyzed with a one-way ANOVA with a Bonferroni post hoc test. *, P < 0.05 compared with wt with the same treatment; #, P < 0.05 compared with vehicle-treated mice with the same genotype. Muscle weights are expressed as the percent difference from the results for wt mice treated with vehicle.
FIG 6
FIG 6
Anti-ActRII antibody efficacy at preventing (A to D) and reversing (E) glucocorticoid-induced atrophy. (A) Weights of the gastrocnemius/plantaris (Gastroc/Plantaris) and tibialis cranialis muscles expressed as the percent difference relative to the weights for the water-treated control group; (B) evoked peak tetanic force of the tibialis cranialis muscle; (C) mean cross-sectional area (CSA) of muscle fibers in the tibialis cranialis muscle; (D) MAFbx and MuRF1 mRNA expression in the tibialis cranialis muscle relative to that in the control group after 14 days of treatment with DEX at ∼2.4 mg/kg/day in drinking water coadministered with BYM338 at 5 and 20 mg/kg or the IgG1-LALA isotype at 20 mg/kg i.p. at days 0, 2, and 7. (E) Weights of the gastrocnemius/plantaris and tibialis cranialis muscles, expressed as the percent difference relative to the water-treated control group, from mice that received DEX daily in the drinking water at ∼2.4 mg/kg/day for 21 days and administered BYM338 at 5 and 20 mg/kg or the IgG1-LALA isotype at 20 mg/kg i.p. on days 21, 23, and 28. Data are presented as means ± SEMs (n = 7 to 10). Group means were compared by one-way ANOVA followed by Tukey's or Dunnett's multiple-comparison test, as appropriate. Differences were considered significant at P values of ≤0.05 (*, **, ***, and ****, P < 0.05, 0.01, 0.001, and 0.0001 versus the group treated with vehicle, respectively; #, ##, ###, and ####, P < 0.05, 0.01, 0.001, and 0.0001 versus the group treated with DEX, respectively).
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