A Sensitive and Selective Immunoassay for the Quantitation of Serum Latent Myostatin after In Vivo Administration of SRK-015, a Selective Inhibitor of Myostatin Activation

Abstract

Myostatin, a member of the transforming growth factor β (TGFβ) superfamily, is a key regulator of skeletal muscle mass and a therapeutic target for muscle wasting diseases. We developed a human monoclonal antibody, SRK-015, that selectively binds to and inhibits proteolytic processing of myostatin precursors, thereby preventing growth factor release from the latent complex. As a consequence of antibody binding, latent myostatin accumulates in the circulation of animals treated with SRK-015 or closely related antibodies, suggesting that quantitation of latent myostatin in serum may serve as a biomarker for target engagement. To accurately measure SRK-015 target engagement, we developed a sensitive plate-based electrochemiluminescent immunoassay to quantitate latent myostatin in serum samples. The assay selectively recognizes latent myostatin without cross-reactivity to promyostatin, mature myostatin, or closely related members of the TGFβ superfamily. To enable use of the assay in samples from animals dosed with SRK-015, we incorporated a low-pH step that dissociates SRK-015 from latent myostatin, improving drug tolerance of the assay. The assay meets inter- and intra-assay accuracy and precision acceptance criteria, and it has a lower limit of quantitation (LLOQ) of 10 ng/mL. We then tested serum samples from a pharmacology study in cynomolgus monkeys treated with SRK-015. Serum latent myostatin increases after treatment with SRK-015, reaches a dose-dependent plateau approximately 20 days after dosing, and trends back toward baseline after cessation of antibody dosing. Taken together, these data suggest that this assay can be used to accurately measure levels of the primary circulating form of myostatin in population-based or pharmacodynamic studies.

Keywords: cynomolgus monkey; immunoassay; myostatin.

Figure 1.

Processing of the promyostatin complex into the active growth factor. (A) Promyostatin is cleaved by a furin protease to generate (B) latent myostatin. (C) An additional cleavage by a tolloid protease allows subsequent release of the active growth factor. (D) Summary of the relative binding affinities of antimyostatin antibodies for the pro-, latent, and mature myostatin forms.

Figure 2.

Optimization of the serum concentration and sample diluent pH. (A) Similar standard curves are generated from standards prepared in 0%, 1%, or 10% cynomolgus monkey matrix. (B) The background signal of the assay, however, shows an increase in signal with 10% serum, while including 1% serum is comparable to buffer alone.

Figure 3.

Comparison of assay recovery in the presence of SRK-015 after dilution into sample buffers at pH 4.0, 5.0, and 7.4. Serum samples containing (A) 10,000 ng/mL and (B) 100 ng/mL of latent myostatin were incubated with varying concentrations of SRK-015. These samples were assayed to calculate the percentage of recovery of the nominal concentration. Accurate quantitation is achieved only when using a sample buffer at pH 4.0.

Figure 4.

The optimized assay is highly selective toward latent myostatin. After optimization of the assay, each myostatin form and GDF11 were spiked into the assay at (A) 10 µg/mL or (B) 1 µg/mL, and analyzed for detection. Only latent myostatin shows robust signals in the assay at a high and low concentration.

Figure 5.

Group-averaged latent myostatin concentration (reported as mean and percentage of coefficient of variation, n = 6). Cynomolgus monkeys were dosed with SRK-015 weekly for a total of eight doses. Serum samples were collected throughout the dosing phase and through a 4-week washout period. There is a dose-dependent (but not dose-proportional) response to the amount of accumulated latent myostatin. After the last dose, latent myostatin trends back toward baseline.