An Engineered Human Follistatin Variant: Insights into the Pharmacokinetic and Pharmocodynamic Relationships of a Novel Molecule with Broad Therapeutic Potential

Abstract

Human follistatin is a regulatory glycoprotein with widespread biologic functions, including antiinflammatory activities, woundhealing properties, and muscle-stimulating effects. The role of follistatin in a wide range of biologic activities shows promise for potential clinical application, which has prompted considerable interest in the investigation of the protein as a potential diseasemodifying agent. In spite of this potential, the development of follistatin as a broad use biotherapeutic has been severely hindered by a poor understanding and characterization of its pharmacokinetic/pharmacodynamic (PK/PD) relationships. Therefore, to better define these relationships, we performed in-depth analyses of the PK/PD relationships of native follistatin-315 (FST315). Our data indicate that the intrinsic PK/PD properties of native FST315 are poorly suited for acting as a parentally administered biotherapeutic with broad systemic effects. Here, we leveraged protein engineering to modify the PK characteristics of the native molecule by fusing FST315 to a murine IgG 1 Fc and removing the intrinsic heparan sulfate-binding activity of follistatin. The engineered variant molecule had~100-and 1600-fold improvements in terminal half-life and exposure, respectively. In contrast to the native FST315, the variant showed a robust, dose-dependent pharmacological effect when administered subcutaneously on a weekly basis in mouse models of muscle atrophy and degeneration. These studies highlight the underappreciated and critical relationship between optimizing multiple physical and chemical properties of follistatin on its overall PK/PD profile. Moreover, our findings provide the first documented strategy toward the development of a follistatin therapeutic with potential use in patients affected with skeletal muscle diseases.

Insights into the Impact of Heterogeneous Glycosylation on the Pharmacokinetic Behavior of Follistatin-Fc–Based Biotherapeutics

“…In our previously published work, we showed that fusion of FST315 to a Fc fragment of IgG4 and removal of its intrinsic heparin sulfate binding activity resulted in a PK/PD profile that supported parenteral delivery (Datta-Mannan et al, 2013). Though these changes were sufficient to optimize the PK/PD relationship, several of the other molecular characteristics of FST-DHBS-Fc, such as poor yields after protein purification, were found to be incompatible with continued for therapeutic development (data not shown).…”

“…In summary, our previous work (Datta-Mannan et al, 2013) and current studies highlight the exquisite importance of understanding the interplay between heparin binding and glycosylation on the overall PK properties of FST-based therapies. The current studies in this report indicate that the residue differences between MV9 and MV12 within the HBS to reduce heparin binding impacted the PK similarly, and the observed PK differences between the two molecular platforms were predominantly driven by differences in glycosylation (sialylation) and not primary amino acid differences.…”

“…In these studies, we fused FST315 to an IgG-derived Fc and removed the protein's intrinsic heparin-binding activity to generate FST-DHBS-Fc. After protein engineering, FST-DHBS-Fc exhibited ;1600-fold and ;90-fold improvements in exposure and half-life, respectively, in mice relative to unmodified FST315 (Datta-Mannan et al, 2013). This molecule, in contrast to FST315, also displayed robust, dose-dependent pharmacologic effects when administered subcutaneously on a weekly basis in a mouse model of muscle atrophy (Datta-Mannan et al, 2013).…”

“…Indeed, several groups have reported potent beneficial effects of FST administration in various indications, such as inflammation, liver repair, fibrosis, wound healing, hair regrowth, and muscle disorders, including muscular dystrophy (Kogure et al, 1995;Wankell et al, 2001;Fuwii et al, 2005;Fumagalli et al, 2007;Tsuchida 2008;Zimber et al, 2011;de Kretser et al, 2012;Datta-Mannan et al, 2013).…”

“…In our previous studies, we reported the first published example of the successful engineering of a FST315 variant, deemed follistatin 315 heparan sulfate-binding deficient mutant human IgG 4 Fc fusion (FST-DHBS-Fc), which has the potential to serve as a platform for developing a parenterally administered biotherapeutic (Datta-Mannan et al, 2013) in the treatment of diseases where muscle atrophy is implicated (Yaden et al, 2014). In these studies, we fused FST315 to an IgG-derived Fc and removed the protein's intrinsic heparin-binding activity to generate FST-DHBS-Fc.…”