Rationale-Based Engineering of a Potent Long-Acting FGF21 Analog for the Treatment of Type 2 Diabetes

Abstract

Fibroblast growth factor 21 (FGF21) is a promising drug candidate for the treatment of type 2 diabetes. However, the use of wild type native FGF21 is challenging due to several limitations. Among these are its short half-life, its susceptibility to in vivo proteolytic degradation and its propensity to in vitro aggregation. We here describe a rationale-based protein engineering approach to generate a potent long-acting FGF21 analog with improved resistance to proteolysis and aggregation. A recombinant Fc-FGF21 fusion protein was constructed by fusing the Fc domain of human IgG1 to the N-terminus of human mature FGF21 via a linker peptide. The Fc positioned at the N-terminus was determined to be superior to the C-terminus as the N-terminal Fc fusion retained the βKlotho binding affinity and the in vitro and in vivo potency similar to native FGF21. Two specific point mutations were introduced into FGF21. The leucine to arginine substitution at position 98 (L98R) suppressed FGF21 aggregation at high concentrations and elevated temperatures. The proline to glycine replacement at position 171 (P171G) eliminated a site-specific proteolytic cleavage of FGF21 identified in mice and cynomolgus monkeys. The derived Fc-FGF21(RG) molecule demonstrated a significantly improved circulating half-life while maintaining the in vitro activity similar to that of wild type protein. The half-life of Fc-FGF21(RG) was 11 h in mice and 30 h in monkeys as compared to 1-2 h for native FGF21 or Fc-FGF21 wild type. A single administration of Fc-FGF21(RG) in diabetic mice resulted in a sustained reduction in blood glucose levels and body weight gains up to 5-7 days, whereas the efficacy of FGF21 or Fc-FGF21 lasted only for 1 day. In summary, we engineered a potent and efficacious long-acting FGF21 analog with a favorable pharmaceutical property for potential clinical development.

FGF21-receptor agonists: an emerging therapeutic class for obesity-related diseases

“…Several pharmaceutical companies thus focused on improving various properties of human FGF21 protein to make it suitable for therapeutic development. The native mature form of FGF21 is a 19.4 kDa protein that is cleared rapidly from circulation most likely through the kidney, and has an estimated half-life of ∼1 h in rodents and 0.5-2 h in NHPs [31], [68]. In addition to the rapid renal clearance, human FGF21 protein undergoes rapid proteolysis upon injection into animals so that the 4 N-terminal amino acid residues [69] and the C-terminal 10 amino acid residues are cleaved off [68].…”

“…The native mature form of FGF21 is a 19.4 kDa protein that is cleared rapidly from circulation most likely through the kidney, and has an estimated half-life of ∼1 h in rodents and 0.5-2 h in NHPs [31], [68]. In addition to the rapid renal clearance, human FGF21 protein undergoes rapid proteolysis upon injection into animals so that the 4 N-terminal amino acid residues [69] and the C-terminal 10 amino acid residues are cleaved off [68]. In vitro studies using recombinant truncated proteins showed that while the 4 N-terminal amino acid residues are not necessary for the signaling activity, the C-terminal 10 amino acid residues are essential for binding to KLB and signaling activity [43], [70].…”

Mechanistic Investigation of the Preclinical Pharmacokinetics and Interspecies Scaling of PF-05231023, a Fibroblast Growth Factor 21–Antibody Protein Conjugate

“…Site-specific conjugation of 30-kDa polyethylene glycol to FGF21 demonstrated 10-to 50-fold improvements in CL/F compared with rFGF21, albeit at the expense of 5-to 10-fold reductions in in vitro potency (Mu et al, 2012). Fusion of NT (Fc-FGF21) or CT of FGF21 (FGF21-Fc) to the Fc fragment of hIgG 1 right-shifted in vitro potency 2-to 4-fold and 1000-fold, respectively (Hecht et al, 2012). The slightly reduced in vitro potency of Fc-FGF21 was offset by a 30-fold increase in serum persistence.…”

Biotransformation and In Vivo Stability of Protein Biotherapeutics: Impact on Candidate Selection and Pharmacokinetic Profiling

“…Native FGF21 has a very short half-life and is cleared predominantly by renal excretion after administration (Hager et al, 2013). To attempt to create a longer-acting therapeutic, FGF21 was initially recombinantly fused to the C terminus of human Fc (Hecht et al, 2012). However, by using differential enzyme-linked immunosorbent assay coupled with ligand-binding MS, it was found that the C terminus underwent very fast peripheral catabolism at a proline residue 10 amino acids upstream of the terminus.…”

“…Since the C terminus of the construct had to remain intact to retain potency, protein engineering efforts were undertaken to stabilize this catabolic liability. After numerous constructs were generated, the construct with this proline mutated to a glycine residue showed retained potency with complete stability against biotransformation in this region (Hecht et al, 2012;Hager et al, 2013). These efforts demonstrated the necessity of understanding biotransformation, using this information to stabilize the molecule for retained potency and decreased clearance, and selecting the proper LBA to track only the bioactive entities during PK profiling.…”