Junming Yie

FGF21 N- and C-termini play different roles in receptor interaction and activation.

MINT‐6799907, MINT‐6799922: FGF21 (uniprotkb: Q9NSA1) binds (MI:0407) to β‐Klotho (uniprotkb: Q86Z14) by surface plasmon resonance (MI:0107)

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

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.

Understanding the physical interactions in the FGF21/FGFR/β-Klotho complex: structural requirements and implications in FGF21 signaling.

The endocrine fibroblast growth factor 21 (FGF21) requires both fibroblast growth factor receptor (FGFR) and β‐Klotho for signaling. In this study, we sought to understand the inter‐molecular physical interactions in the FGF21/FGFR/β‐Klotho complex by deleting key regions in FGFR1c or FGF21. Deletion of the D1 and the D1‐D2 linker (the D1/linker region) from FGFR1c led to β‐Klotho‐independent receptor activation by FGF21, suggesting that there may be a direct interaction between FGF21 and the D1/linker region‐deficient FGFR1c. Consistent with this, the extracellular portion of FGFR1c lacking the D1/linker region blocked FGF21 action in a reporter assay, presumably by binding to and sequestering FGF21 from acting on cell surface receptor complex. In addition, the D1/linker region‐deficient FGFR1c had enhanced interaction with β‐Klotho. Further, we demonstrated that deletion of the D1/linker region enhanced the formation of the FGF21/β‐Klotho/FGFR1c ternary complex in both Biacore and asymmetrical flow field flow fractionation studies. Finally, we found that the N‐terminus of FGF21 is involved in the interaction with FGFR1c and FGF21/β‐Klotho/FGFR1c ternary complex formation. Taken together, our data suggest that the D1/linker region regulates both the FGF21/FGFR1c and FGFR1c/β‐Klotho interaction, and a direct interaction of FGF21 with FGFR1c may be an important step in receptor‐mediated FGF21 signaling.

Polyethylene glycol modified FGF21 engineered to maximize potency and minimize vacuole formation.

Fibroblast growth factor 21 (FGF21) is involved in regulating energy metabolism, and it has shown significant promise as a treatment for type II diabetes; however, the native protein has a very short circulating half-life necessitating frequent injections to maintain a physiological effect. Polyethylene glycol (PEG) conjugation to proteins has been used as a method for extending the circulating half-life of many pharmaceutical proteins; however, PEG does carry the risk of vacuole formation, particularly in the renal tubular epithelium. Since renal vacuole formation may be particularly problematic for diabetic patients, we engineered site-directed PEGylated variants of FGF21 with sustained potency and minimized vacuole formation. This was accomplished both by probing the site of PEGylation on FGF21 as well as by examining various PEG configurations. While the site of PEGylation has a significant impact on the bioactivity of FGF21, it has only a marginal impact on vacuole formation; however, the configuration and number of PEGs conjugated to the protein has a much more profound effect on vacuologenesis.

A Novel Approach to Improve the Function of FGF21

Background and ObjectiveFibroblast growth factor 21 (FGF21) has potent effects on normalizing glucose, lipid, and energy homeostasis, and represents an attractive novel therapy for type 2 diabetes mellitus and obesity. Approaches to improve the pharmacokinetic properties of FGF21, such as conjugation with polyethylene glycol, have been explored for therapeutic development. However, not only is there room for further pharmacokinetic improvements, additional re-engineering approaches to improve the potency and stability of FGF21 have not been reported. Here, we describe a novel approach to modify and improve the function of FGF21 by altering its C-terminal βKlotho interaction domain.MethodsWe first identified Avimer proteins that are capable of binding βKlotho. Then we explored replacing the C-terminal βKlotho interaction domain of FGF21 with a βKlotho-binding Avimer protein.ResultsSuch a βKlotho-binding Avimer protein was able to fully complement the C-terminal domain function of FGF21. The resulting FGF21-Avimer fusion is functionally indistinguishable from wild type FGF21, and more tolerant of C-terminal modification.ConclusionThese results demonstrate a viable strategy to modulate the affinity, potency, and engineering of FGF21, paving the way for further improvements of FGF21 as a therapeutic.

A Novel Fc-FGF21 With Improved Resistance to Proteolysis, Increased Affinity Toward β-Klotho, and Enhanced Efficacy in Mice and Cynomolgus Monkeys

Fibroblast growth factor (FGF) 21 is a natural hormone that modulates glucose, lipid, and energy metabolism. Previously, we engineered an Fc fusion FGF21 variant with two mutations, Fc-FGF21(RG), to extend the half-life and reduce aggregation and in vivo degradation of FGF21. We now describe a new variant developed to reduce the extreme C-terminal degradation and improve the binding affinity to β-Klotho. We demonstrate, by introducing one additional mutation located at the C terminus of FGF21 (A180E), that the new molecule, Fc-FGF21(RGE), has gained many improved attributes. Compared with Fc-FGF21(RG), Fc-FGF21(RGE) has similar in vitro potency, preserves β-Klotho dependency, and maintains FGF receptor selectivity and cross-species reactivity. In vivo, Fc-FGF21(RGE) showed reduced susceptibility to extreme C-terminal degradation and increased plasma levels of the bioactive intact molecule. The circulating half-life of intact Fc-FGF21(RGE) increased twofold compared with that of Fc-FGF21(RG) in mice and cynomolgus monkeys. Additionally, Fc-FGF21(RGE) exhibited threefold to fivefold enhanced binding affinity to coreceptor β-Klotho across mouse, cynomolgus monkey, and human species. In obese and diabetic mouse and cynomolgus monkey models, Fc-FGF21(RGE) demonstrated greater efficacies to Fc-FGF21(RG), resulting in larger and more sustained improvements in multiple metabolic parameters. No increased immunogenicity was observed with Fc-FGF21(RGE). The superior biophysical, pharmacokinetic, and pharmacodynamic properties, as well as the positive metabolic effects across species, suggest that further clinical development of Fc-FGF21(RGE) as a metabolic therapy for diabetic and/or obese patients may be warranted.