Enhancement of the Pharmacological Efficacy of FGF-21 by Genetic Modification and PEGylation

Abstract

FGF-21 is a potential candidate for the treatment of type 2 diabetes mellitus. However, the clinical application of wild-type human FGF-21 is challenging due to some limitations, such as its poor hypoglycemic potency and short in vivo half-life. In this paper, we have produced an FGF-21 mutant (ahmFGF-21) by exchanging the functional domain of hFGF-21 with that of mFGF-21 to improve the potency of FGF-21. Results showed that the ahmFGF-21 protein was more potent than wild-type hFGF-21 in stimulating glucose uptake in vitro and lowering blood glucose levels of diabetic animals. To decrease its immunogenicity and increase its biostability, the N-terminus of ahmFGF-21 was modified in a sitespecific manner with 20 kDa mPEG-propionaldehyde (mPEG-ALD). We found that the preservation time of ahmFGF-21 in vitro was significantly prolonged after PEGylation. The serum antibody levels against ahmFGF-21 in immunized rabbits with the PEGylated ahmFGF-21 were significantly reduced than those with the unmodified ahmFGF-21, and the target protein concentration in the rabbits administrated with the PEGylated ahmFGF-21 increased 9.5-fold higher than that of the unmodified ahmFGF-21. The animal experimental results showed that PEGylation of ahmFGF-21 enhanced the hypoglycemic effect in diabetic mice. These results suggest that the in vitro and in vivo hypoglycemic effects of FGF-21 are significantly enhanced by genetic modification and the metabolic pharmacology of FGF-21 in type 2 diabetic mice is improved by PEGylation at a specific site.

Pilot-scale production and characterization of PEGylated human FGF-21 analog

“…Unfortunately, the low biological activity and short half-life in vivo of wild type hFGF-21 (human FGF-21) influence its therapeutic efficacy. In our previous study, we optimized the gene of hFGF-21 to improve its biological activity, and then site-specifically PEGylated FGF-21 mutant for prolonging its pharmacokinetics and decreasing its immunogenicity (Ye et al, 2013). Up to now, it has not been reported about the pilot-scale production of PEGylated FGF-21, which seriously restricts its clinical application.…”

“…The engineering bacteria expressing pSUMO-ahmFGF-21 protein were prepared as our previous study (Ye et al, 2013). A frozen glycerol seed strain was inoculated into a 50 mL flask containing 10 mL Luria-Bertani (LB) medium supplemented with 100 g/mL ampicillin (Sigma-Aldrich) in a shaking incubator at 37 • C/220 rpm.…”

“…Then, the N-terminus of ahmFGF-21 was modified with 20 kD mPEG-ALD in a site-specific manner. Our previous results showed that the long lasting anti-diabetic effect of FGF-21 was significantly improved by genetic modification and PEGylation (Ye et al, 2013).…”

“…So far, some researches have indicated that PEGylated FGF-21 could become a new long-lasting hypoglycemic drug to potentially treat type 2 diabetes (Huang et al, 2011;Ye et al, 2013Ye et al, , 2015a. However, the pilot-scale production of PEGylated FGF-21 with biological activity is considered as a rate-limiting step.…”

Potent long-acting rhFGF21 analog for treatment of diabetic nephropathy in db/db and DIO mice

“…Recent clinical trials of an FGF21 variant did not significantly reduce the glucose level, probably due to its short serum half-life in vivo [13]. Polyethylene glycol modification (PEGylation) is regarded as one of the most successful strategies to improve pharmacokinetic properties of protein drugs by increasing their in-vivo circulation half-life, which implies prolonged therapeutic effects and reduced dosage [14–17].…”