RT Journal Article
SR Electronic
T1 Protein Engineering on Human Recombinant Follistatin: Enhancing Pharmacokinetic Characteristics for Therapeutic Application
JF Journal of Pharmacology and Experimental Therapeutics
JO J Pharmacol Exp Ther
FD American Society for Pharmacology and Experimental Therapeutics
SP 291
OP 302
DO 10.1124/jpet.118.248195
VO 366
IS 2
A1 Chuan Shen
A1 Andrea Iskenderian
A1 Dianna Lundberg
A1 Tao He
A1 Kathleen Palmieri
A1 Robert Crooker
A1 Qingwei Deng
A1 Matthew Traylor
A1 Sheng Gu
A1 Haojing Rong
A1 David Ehmann
A1 Brian Pescatore
A1 Bettina Strack-Logue
A1 Alla Romashko
A1 George Baviello
A1 John Gill
A1 Bohong Zhang
A1 Muthuraman Meiyappan
A1 Clark Pan
A1 Angela W. Norton
YR 2018
UL http://jpet.aspetjournals.org/content/366/2/291.abstract
AB Follistatin (FS) is an important regulatory protein, a natural antagonist for transforming growth factor-β family members activin and myostatin. The diverse biologic roles of the activin and myostatin signaling pathways make FS a promising therapeutic target for treating human diseases exhibiting inflammation, fibrosis, and muscle disorders, such as Duchenne muscular dystrophy. However, rapid heparin-mediated hepatic clearance of FS limits its therapeutic potential. We targeted the heparin-binding loop of FS for site-directed mutagenesis to improve clearance parameters. By generating a series of FS variants with one, two, or three negative amino acid substitutions, we demonstrated a direct and proportional relationship between the degree of heparin-binding affinity in vitro and the exposure in vivo. The triple mutation K(76,81,82)E abolished heparin-binding affinity, resulting in ∼20-fold improved in vivo exposure. This triple mutant retains full functional activity and an antibody-like pharmacokinetic profile, and shows a superior developability profile in physical stability and cell productivity compared with FS variants, which substitute the entire heparin-binding loop with alternative sequences. Our surgical approach to mutagenesis should also reduce the immunogenicity risk. To further lower this risk, we introduced a novel glycosylation site into the heparin-binding loop. This hyperglycosylated variant showed a 10-fold improved exposure and decreased clearance in mice compared with an IgG1 Fc fusion protein containing the native FS sequence. Collectively, our data highlight the importance of improving pharmacokinetic properties by manipulating heparin-binding affinity and glycosylation content and provide a valuable guideline to design desirable therapeutic FS molecules.