L45SA; Meso Scale Discovery) were blocked with the assay diluent [2.5% bovine serum albumin (BSA) in tris-buffered saline, pH 7.4, with 0.1% Tween-20] for 1 h at room temperature (RT). 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 ST16 the TGF 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 Purmorphamine 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: myostatin, immunoassay, cynomolgus monkey Introduction The transforming growth factor beta (TGF) superfamily protein myostatin Purmorphamine [growth differentiation factor 8 (GDF8)] is a potent negative regulator of skeletal muscle mass. Myostatin is expressed primarily in skeletal muscle,1 with low levels of messenger RNA (mRNA) reported in adipose2 and cardiac3 tissues. Mutations that attenuate myostatin signaling lead to a hypermuscular phenotype in multiple species,4C9 and, conversely, genetic overexpression in rodents induces muscle wasting and fat loss.10 As a consequence of its impact on muscle mass, myostatin is an attractive drug target for diseases involving muscle atrophy, and agents that target the myostatin signaling pathway have entered the clinic for such disorders. Myostatin is initially expressed as promyostatin, a proprotein in which the active C-terminal growth factor is held in a dimeric latent complex through association with a large prodomain. Release of the Purmorphamine mature growth factor from the unprocessed precursor is regulated through two discrete protease cleavage events ( Fig. 1 ): First, promyostatin is cleaved Purmorphamine at a conserved RXXR motif between the prodomain and mature growth factor by proprotein convertases such as furin/PACE3 (paired basic amino acid cleaving enzyme 3) or PCSK5 (proprotein convertase subtilisin/kexin type 5).10C12 This initial cleavage produces latent myostatin, an inactive complex in which the prodomains remain associated with the growth factor.13 A second prodomain cleavage by members of the BMP/tolloid family, such as TLL2 (tolloid-like protein 2) or BMP1 (bone morphogenetic protein 1), activates latent myostatin by releasing the mature growth factor from the latent complex, allowing it to bind to ActRIIB (activin receptor type IIB) receptors, and thereby activating downstream signaling.11C14 Open in a separate window 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. We recently described the identification and characterization of SRK-015, a monoclonal antibody that inhibits the tolloid-mediated proteolysis of latent myostatin.15 SRK-015 binds pro- and latent myostatin but does not bind the mature growth factor or any forms of GDF11, myostatins closest paralog.15 Furthermore, SRK-015 shows no.