Antibodies and protein reagents

REGN1033 is a fully human monoclonal antibody specific to myostatin derived by immunizing with the mature human myostatin using Regeneron’s VelocImmune® mice [17, 18] in which the myostatin gene was also homozygously ablated, so as to decrease immunotolerance to this protein. The selected anti-myostatin antibody contains an IgG4 constant region. Soluble human ActRIIB-hFc (ActRIIB-hFc) was produced in Chinese hamster ovary (CHO) cells and contains the extracellular domain (1-133) of the human ActRIIB receptor (ACVR2B) made as an in-frame fusion with human IgG1 Fc-domain. A second human monoclonal antibody (REGN647) which was shown to be specific for myostatin in binding and bioassay studies was used for immunoblotting; to visualize myostatin propeptide, an anti-propeptide sheep polyclonal antibody (AF1539; R&D Systems: Minneapolis, MN) was used. Human myostatin, growth and differentiation factor 11 (GDF11), and activin A were purchased from R&D Systems.

Surface plasmon resonance

The affinities of REGN1033 and ActRIIB-hFc for human myostatin, GDF11, and activin A were measured in surface plasmon resonance biacore experiments performed on a Biacore T200 instrument (GE Healthcare, Pittsburgh, PA) using a dextran-coated (CM5) chip at 25 °C. The running buffer was filtered HEPES-buffered Steinberg’s solution (HBS-T) containing 10 mM HEPES, 150 mM NaCl, 3 mM ethylenediaminetetraacetic acid (EDTA), 0.05 % (v/v) Surfactant P20, 10 mg/ml carboxymethyldextran sodium salt, and 0.5 mg/ml bovine serum albumin (pH 7.4). A capture sensor surface was prepared by covalently immobilizing goat anti-human Fcγ antibody (Jackson ImmunoResearch, West Grove, PA) to the chip surface using 1 ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling chemistry. Following surface activation, anti-human Fc antibody in 0.1 M acetate buffer (pH 4.5) was injected over the activated chip surface until a resonance unit (RU) signal of about 4000 RU (anti-human Fc antibody) was reached. The activated coupled chip surfaces were then washed and treated with 10 mM glycine-HCl (pH 1.5) to remove uncoupled residual proteins. REGN1033 and ActRIIB-hFc were captured through their Fc regions between 32 and 39 RU by an anti-human Fc antibody immobilized on the sensor chips and were tested for binding to the human myostatin, GDF11, and activin A. Myostatin, GDF11, and activin A proteins were prepared at concentrations between 40 and 0.78 nM and individually injected over captured REGN1033 or ActRIIB-hFc surface. All capture surfaces were regenerated with one 30-s pulse of 10 mM glycine-HCl (pH 1.5). Kinetic parameters were obtained by globally fitting the data to a 1:1 binding model using Biacore T200 Evaluation Software. The equilibrium dissociation constant (K D ) was calculated by dividing the dissociation rate constant (k d ) by the association rate constant (k a ).

Smad2/3 cell-based activity assay

A cell-based assay for determining Smad2/3 activation was established in A204 cells stably expressing Smad-dependent (CAGA12) luciferase. Ligands were serially diluted and added to 20,000 A204/Smad2/3/Luc cells per well plated in McCoy’s 5A medium supplemented with 10 % fetal calf serum (FCS)/G418 to generate a dose-response curve. Inhibition of ligand-mediated ActRIIB signaling by REGN1033, ActRIIB-hFc, or control antibody was tested by serially diluting the antibodies or soluble receptor and incubating with a constant concentration of 1 nM myostatin, 0.4 nM GDF11, or 0.4 nM activin A and cells for 6 h at 37 °C. Luminescence was measured using One Glo (Promega, Madison, WI).

Human myoblast treatment and immunoblot analysis

Human skeletal myoblasts (Lonza Group Ltd, Anaheim, CA) were cultured in complete growth media (Lonza). Cells (6 × 105 per well in a six-well plate) were cultured for 24 h, and serum starved for 4 h before treated with myostatin, GDF-11, or activin A at 4 nM in presence of 40 nM of REGN1033 or ActRIIB-hFc for 30 min. Cells were lysed in NP-40 buffer containing 1 % NP-40, 100 mM KCL, 20 mM Tris/HCL (pH 7.6), 1 mM EGTA, and 1 mM NaF with various protease and phosphatase inhibitors and cleared by centrifugation. Soluble fractions were separated in 4–20 % Invitrogen Precast Tris-Glycine gels using SDS-PAGE followed by transfer to polyvinylidene fluoride (PVDF) membranes. Total levels of Smad2/3 and phosphorylated Smad2 were determined with rabbit anti-Smad2/3 and phospho-Smad2 (Ser465/467) antibodies (Cell Signaling Technology, Danvers, MA).

Immunoblot analysis of mouse serum following REGN1033 administration

Serum was collected following 4-week treatment of male CB17-severe combined immunodeficiency (SCID) mice with REGN1033 or control antibody (10 mg/kg by subcutaneous (s.c.) dosing; twice in the first week and once a week for three weeks). Serum (60 μl per mouse) was diluted tenfold in phosphate-buffered saline (PBS) containing 0.2 % Nonidet P-40 and 1X protease and phosphatase inhibitors. Immune complexes were captured with Protein A-Sepharose beads and eluted with non-reducing SDS-PAGE buffer. The equivalent of 2.4 μl of serum was then run on a 4–20 % gradient Tris-glycine gel and immunoblotted with a monoclonal antibody against mature myostatin, produced at Regeneron, or with a myostatin propeptide polyclonal antibody. CHO cells stably expressing furin protease were transiently transfected with an expression plasmid encoding human myostatin precursor, and conditioned media was collected after three days; 5 μl was loaded onto the gel to serve as a positive control. To demonstrate myostatin antibody specificity, 2 μl of serum from wild-type and Mstn −/− mice was probed by immunoblot.

Animal studies

Mice (Taconic, Hudson, NY) were housed four to five per cage in a controlled environment (12-h light/dark cycle, 23 ± 1 °C, 60–70 % humidity) and fed ad libitum with standard chow (Purina Laboratory Rodent Diet 5001, LabDiet, St. Louis, MO). All animal studies were conducted in accordance with the Regeneron Pharmaceuticals Institutional Animal Care and Use Committee.

Muscle hypertrophy studies

The effect of REGN1033 on muscle mass was determined after 4-week treatment of varying doses of REGN1033 (0.1–30 mg/kg) or control antibody (30 mg/kg) in 9-week-old male CB17-SCID mice (n = 5/group). Animals were grouped by body weight and dosed via s.c. injection twice the first week and once a week for the following 3 weeks. At the end of the fourth week, tibialis anterior (TA) and gastrocnemius (GA) complex muscle groups were harvested and weighed.

Ex vivo force measurements

REGN1033 or control antibody (10 mg/kg) was administered to C57BL/6 male mice (n = 6/group) twice a week for 3 weeks via s.c. injection. At the end of 3 weeks of treatment, ex vivo force measurements of the TA muscle were obtained. Briefly, mice were anesthetized under isoflurane (4.5 %), and the right TA muscle was excised by cutting the femur just proximal to the femoral head above the knee and the tibia and fibula proximal to the ankle. The muscle was then placed in an oxygenated bath containing Krebs solution with 10 mM glucose at 27 °C. The femoral head was secured to a stanchion while the distal tendon was tied to the arm of a 305C Muscle Lever System (Aurora Scientific, Aurora, ON, Canada). Optimal length was achieved by increasing the length of the muscle by small increments followed by a single 1-Hz stimulation until a maximum twitch force was achieved. Maximal isometric tetanic force was then determined by stimulating each muscle at 10-Hz intervals (from 40 to 100 Hz) with 90-s rest periods prior to each stimulation.

Casting immobilization

Two groups of 12-week-old C57BL/6 male mice (n = 5/group) were anesthetized, and the right ankle joint was immobilized at a 90° angle with casting material for 14 days. During immobilization, mice were injected subcutaneously twice a week with 30 mg/kg of REGN1033 or control antibody. GA muscle weights were compared to a separate, age-matched, non-immobilized group treated with control antibody (30 mg/kg).

Dexamethasone-induced atrophy

Dexamethasone was administered at a rate of 23.0 μg/day by micro-osmotic pump (DURECT Corporation, Cupertino, CA) for 2 weeks in 11-week-old C57BL/6 male mice (n = 5/group). During dexamethasone treatment, animals were given either 10 mg/kg of REGN1033 or isotype control antibody by s.c. injection twice a week. A separate group, implanted with osmotic pumps delivering saline and given 10 mg/kg of control antibody, served as a negative control. At the end of 2 weeks, TA and GA muscles were collected and weighed.

Hindlimb suspension

Prevention of hindlimb suspension (HLS)-induced atrophy was assessed in 10-week-old C57BL/6 male mice (n = 8/group). Sixteen mice were tail-suspended for 7 days to prevent weight bearing by the hind limbs; forelimbs were unaffected. Mice were housed in special cages (Techshot Inc., Greenville, IN) with free access to food and water. Another group of mice was left unperturbed to serve as negative controls. Animals were treated with 10 mg/kg of REGN1033 or control antibody by s.c. injection 2 days prior to HLS, on the day of HLS, and 4 days into HLS. At the end of 7 days, muscles were collected, weighed, and stored for further analysis. Similarly, the effect of REGN1033 during recovery from 7 days of HLS-induced atrophy was examined in 11-month-old C57BL/6 male mice (n = 6/group). A total of eighteen mice were suspended for 7 days, and an unperturbed group was included in the study as a negative control. On day 7 of HLS, the animals were released from suspension and randomized into three groups based on body weight loss. TA and GA muscles were collected and weighed from one HLS group and the unperturbed control group. The remaining HLS animals were allowed to recover for 1 week, receiving either REGN1033 or control antibody at 10 mg/kg twice a week during recovery, at which time TA and GA muscles were collected and weighed.

Running endurance in aged mice

Male C57BL/6 at 19 months of age were randomized into four groups (n = 6-8/group): a sedentary or exercise group receiving either REGN1033 or control antibody. All mice were dosed via s.c. injection twice per week for 3 weeks at 10 mg/kg. During treatment, mice in the exercise group were placed on an exercise regimen involving one training session a day, consisting of 20 min on an Exer 6 M treadmill (Columbus Instruments, Columbus, OH) at 10 m/min with a 5° incline, 5 days a week for three consecutive weeks. At the end of 3 weeks of treatment, endurance was measured in all four groups using a treadmill exhaustion test. Briefly, mice ran on a treadmill at 10 m/min with a 5° incline for 4 min, and the speed was increased by 2 m/min every subsequent 4 min until 16 m/min was reached. At 30 min, the speed was increased to 18 m/min and this speed was maintained until the mice reached exhaustion. Exhaustion was defined as the inability of the mouse to remain on the treadmill despite mechanical prodding and an electrical shock stimulus.

Histology

Ten-week-old male SCID mice were treated with s.c. injection of REGN1033 or control antibody for 4 weeks (10 mg/kg; n = 6/group). TA muscles were collected and sectioned, and muscle fibers were stained for laminin with polyclonal rabbit anti-LAMA1 antibody (Sigma-Aldrich, St. Louis, MO). Fiber cross-sectional area was measured using MetaMorph software (Molecular Devices, Sunnyvale, CA). Fiber types were measured in sections of GA muscle that were dried and washed with PBS, quenched in methanol and 1 % H 2 O 2 solution, blocked with 4 % goat serum and 1 % BSA, and incubated with the following primary antibodies overnight at 4 °C; (1) laminin (1:2000; Sigma), (2) myosin heavy chain slow (1:400; Novocastra, Leica Microsystems Inc., Buffalo Grove, IL), (3) myosin heavy chain 2A (1:500; DSHB, Iowa City, IA), and (4) myosin heavy chain 2B (1:500; DSHB, Iowa City, IA). After a PBS wash, the slides are incubated with the following secondary antibodies from Vector Laboratories (Burlingame, CA); (1) biotinylated goat α-rabbit IgG (1:1200), (2) biotinylated goat α-mouse IgG (1:1000), and (3) biotinylated goat α-mouse IgM (1:1000) for 1 h at 21 °C followed by detection with the ABC kit (1:500; Vector Laboratories). After diaminobenzidine (DAB)/peroxidase brown visualized reaction (0.4 mg/ml DAB, 0.0003 % H 2 O 2 ), slides were scanned by the Aperio Scanscope AT. Images were analyzed by HALO software (Indica Labs, Corrales, NM).

RNA sequencing

Messenger RNA was prepared, sequenced, and analyzed as previously described [19].

Statistical analysis

Data are presented as mean ± standard error, and values of P < 0.05 were considered statistically significant. Statistical significance was measured through unpaired, two-tailed Student’s t test for comparisons between two groups, one-way or two-way ANOVA with Tukey’s post hoc analysis for studies with group of three or more or two-way repeated measures ANOVA with Bonferroni’s post hoc analysis for studies where time was a factor using Prism software (GraphPad Software).