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.

A Randomized Phase 1 Safety, Pharmacokinetic and Pharmacodynamic Study of the Novel Myostatin Inhibitor Apitegromab (SRK-015): A Potential Treatment for Spinal Muscular Atrophy

“…This relationship suggests that apitegromab doses at above 3 mg/kg allow for full engagement of latent myostatin, and higher doses allow for longer duration of full target engagement. These results are consistent with trends seen in both cynomolgus monkeys treated with apitegromab [25] and in an SMND7 mouse model of SMA after treatment with the parental muSRK-015P molecule [21].…”

“…The bioanalytical assay used to measure serum latent myostatin implements an acid-dissociation step to dissociate the drug-antigen complex, allowing for the quantitation of total serum latent myostatin [25]. In part A (SAD) of the study, serum latent myostatin levels increased and peaked at about 2000-3000 ng/ mL by days 14-28.…”

“…While apitegromab binds to and engages both pro- and latent forms of myostatin with similar affinity (2.9 nM, and 2.4 nM, respectively), only latent myostatin is detected in the serum [ 10 ]. The bioanalytical assay used to measure serum latent myostatin implements an acid-dissociation step to dissociate the drug–antigen complex, allowing for the quantitation of total serum latent myostatin [ 25 ]. In part A (SAD) of the study, serum latent myostatin levels increased and peaked at about 2000–3000 ng/mL by days 14–28.…”

Myostatin as a Biomarker of Muscle Wasting and other Pathologies-State of the Art and Knowledge Gaps

“…Their results were partially confirmed by the SomaScan aptamer technology measurement of samples from two large cohort studies in which, however, sarcopenia was not assessed [ 110 ]. Further research investigated various, specific myostatin measurement methods, such as immunoassays, immunoradiometric sandwich assay, antibody-free assay and liquid chromatography-tandem mass spectrometry assay combined with immunoprecipitation [ 111 , 112 , 113 , 114 ]. These new, specific assays could be used to clarify discrepancies in earlier results, as well as serve as a validation tool for commercially available methods.…”

Preclinical Safety Assessment and Toxicokinetics of Apitegromab, an Antibody Targeting Proforms of Myostatin for the Treatment of Muscle-Atrophying Disease

“…Target engagement with apitegromab in cynomolgus monkeys and rats. Target engagement with apitegromab has previously been confirmed in pharmacology studies in rodents 11 and cynomolgus monkeys, 24 wherein apitegromab exposure led to increased circulating levels of latent myostatin. In our current studies, we confirmed the target engagement with apitegromab by measuring serum latent myostatin levels across the dosing and recovery phases (Figure 4).…”