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

<div><p>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 <em>in vivo</em> proteolytic degradation and its propensity to <em>in vitro</em> 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 <em>in vitro</em> and <em>in vivo</em> 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 <em>in vitro</em> 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.</p> </div

In vitro activity of FGF21 variants.

<p>293T cells stably expressing human βKlotho along with luciferase reporter constructs were treated with FGF21 variants for 6 hours and lysates were collected to measure luciferase activity.</p

Identification of aggregation-resistant FGF21 mutants. (A–B) FGF21 aggregation was concentration-(A), temperature- (B), and time-dependent (A&B).

<p>The % of high molecular weight species over the total was obtained from size exclusion chromatography (SEC) analysis. (C) Homology model of FGF21 (residues 14–155) with the mutated residues shown in magenta. The leucine to arginine mutation is shown for residue 98 (magenta) and all other mutation sites were shown as native sequence. The FGF21 homology model was prepared using Modeler (Fiser and Sali) in Discovery studio 3.1 (Accelrys) with FGF19 (PDB code: 2P23) used as a template. Image was created using PyMol (Schrödinger, LLC). (D) The aggregation rate of FGF21 mutants. Samples were concentrated at 60±10 mg/ml and stored at 4°C for 1, 6 and 10 days before SEC analysis.</p

The N-terminal Fc fusion (Fc-FGF21) was superior to the C-terminal Fc fusion (FGF21-Fc) in retaining the biological activity of FGF21.

<p>(A–B) Stimulation of Elk1-luciferase reporter activity by FGF21, Fc-FGF21 and FGF21-Fc in 293T cells stably expressing human βKlotho (A) or in CHO cells stably expressing both human βKlotho and human FGFR1c (B); (C) Activation of Erk phosphorylation by FGF21, Fc-FGF21 and FGF21-Fc in human primary adipocytes. All <i>in vitro</i> data represent mean ± SEM, n = 4/concentration group. (D-F) 8-9 week old male <i>db/db</i> (C57/BL6 strain) mice were ip administered with various doses of native FGF21 (D), Fc-FGF21 or FGF21-Fc (E). Blood glucose levels were measured at baseline and 6 h after injection (D–E). % change of blood glucose levels from baseline was plotted as a function of FGF21 molar doses (F). Data are mean ± SEM, n = 9–10 animals per group, ̂ p<0.01; # p<0.001 compared with vehicle. (G–J) The binding activity of FGF21 (G), Fc-FGF21 (H) and FGF21-Fc (I) to human βKlotho determined in a solution equilibrium binding assay on a BIAcore instrument. (J) Quantification of βKlotho binding to the biotinylated FGF21 immobilized on the chip. 100% βKlotho binding is the signal obtained with no FGF21 in the solution.</p

Fc-FGF21(RG) was resistant to degradation in cynomolgus monkeys.

<p>LBMS (MALDI-TOF) analysis of native FGF21 (A), Fc-FGF21 (B) or Fc-FGF21(RG) (C) in male cynomolgus monkeys. FGF21 (10 mg/kg) or Fc-FGF21 variants (23.5 mg/kg) were iv administered in cynomolgus monkeys and blood samples were collected at indicated time points. The mass positions of the parent constructs are indicated by vertical hashed lines. The peak marked with a heavy arrow corresponds to the primary metabolite of FGF21 or Fc-FGF21, which is absent in the Fc-FGF21(RG). Peaks marked with asterisks in all of the spectra correspond to sinapinic acid adducts of primary peaks and are an artifact of the ionization process of MALDI. The MALDI-TOF mass spectra are normalized to the most intense peak in the plotted <i>m/z</i> range.</p

In vitro activity of Fc-FGF21(RG) relative to native FGF21 and Fc-FGF21 wild type.

<p>(A) Structural diagram of Fc-FGF21(RG), an E coli-expressed homodimeric Fc-FGF21 fusion protein containing 2 engineered point mutations at positions 98 and 171. 15G indicates the 15 aa polyglycine and polyserine linker. (B) The binding activity of FGF21, Fc-FGF21 and Fc-FGF21(RG) to human βKlotho determined in a solution equilibrium binding assay on a BIAcore instrument. (C) Elk1-luciferase reporter activity in 293T cells treated with FGF1, FGF21, Fc-FGF21 and Fc-FGF21(RG). The cells were stably expressing reporter constructs but no βKlotho. (D) Stimulation of Elk1-luciferase reporter activity by FGF21, Fc-FGF21 and Fc-FGF21(RG) in 293T cells stably expressing human βKlotho along with luciferase reporter constructs. (E) Stimulation of Elk1-luciferase reporter activity by FGF21, Fc-FGF21 and Fc-FGF21(RG) in mouse NIH/3T3 fibroblast cells transiently co-transfected with human βKlotho and human FGFR1c. (F) Stimulation of Erk phosphorylation by FGF21, Fc-FGF21 and Fc-FGF21(RG) in human primary adipocytes. All data represent mean ± SEM, n = 4/concentration group. ND: not detectable. FGF21, closed circle; Fc-FGF21: opened circle; Fc-FGF21(RG): closed triangular and dash line; FGF1: closed square.</p

Fc-FGF21(RG) had a prolonged efficacy in <i>db/db</i> or <i>ob/ob</i> mice.

<p>(A-B) 8-9 week old male <i>db/db</i> mice were ip administered with FGF21 (A) or Fc-FGF21 (B) at various molar equivalent doses. The molar equal dose is defined as an equivalent molar dose of FGF21 as a unit. Blood glucose levels were measure at the indicated time points. (C–D) Effect of vehicle (closed circle), FGF21 (opened circle), Fc-FGF21(closed square) or Fc-FGF21(RG) (opened squared) on blood glucose levels (C) and body weight gains (D) following a single injection at a molar equivalent dose in <i>db/db</i> mice. Blood glucose levels and body weight were measured over a course of 144 h. (E–F) Dose-response and time course effects of Fc-FGF21(RG) on blood glucose levels (E) and body weight gains (F) in <i>ob/ob</i> mice. 8–9 week old male <i>ob/ob</i> mice were ip administered with various doses of Fc-FGF21(RG). Blood glucose levels and body weight were measured over a course of 168 h. All data are mean ± SEM, n = 9–10 animals per group, * p<0.05, ̂ p<0.01, # p<0.001 compared with vehicle at each time point.</p

LC-ESI-MS analysis of metabolites of Fc-FGF21 variants in male C57BL6 mice following i.v. administration.

<p>Each Fc-FGF21 variant was iv administered at 23 mg/kg into male C57BL6 mice. Blood samples were collected at 6, 24 and 48 h post injection and were pooled from 6 mice per time point for ligand binding LC-ESI-MS analysis. Full-length Fc-FGF21 contains 424 aa residues. * Poor resolutions for the quantification of each mass peak; 0 h time point represents reduced standard.</p

Fc-FGF21(RG) was resistant to aggregation.

<p>(A) Size exclusion chromatography (SEC) of native FGF21, Fc-FGF21 wild type and Fc-FGF21(RG) concentrated at 65 mg/ml stored at 25°C for various days. (B-C) Aggregation rate of FGF21 (closed circle), Fc-FGF21 (opened circle) or Fc-FGF21(RG) (closed triangular) at 65 mg/ml stored at either 25°C (B) or 4°C (C). (D–E) Aggregation rate of Fc-FGF21 (closed circle), Fc-FGF21(L98R) (opened circle) or Fc-FGF21(RG) (closed triangular) at 65 mg/ml stored at either 25°C (D) or 4°C (E).</p