Fusion of fibroblast growth factor 21 to a thermally responsive biopolymer forms an injectable depot with sustained anti-diabetic action

Abstract

Fibroblast growth factor 21 (FGF21) is under investigation as a type 2 diabetes protein drug, but its efficacy is impeded by rapid in vivo clearance and by costly production methods. To improve the protein's therapeutic utility, we recombinantly expressed FGF21 as a fusion with an elastin-like polypeptide (ELP), a peptide polymer that exhibits reversible thermal phase behavior. Below a critical temperature, ELPs exist as miscible unimers, while above, they associate into a coacervate. The thermal responsiveness of ELPs is retained upon fusion to proteins, which has notable consequences for the production and in vivo delivery of FGF21. First, the ELP acts as a solubility enhancer during E. coli expression, yielding active fusion protein from the soluble cell lysate fraction and eliminating the protein refolding steps that are required for purification of FGF21 from inclusion bodies. Second, the ELP's phase transition behavior is exploited for facile chromatography-free purification of the ELP-FGF21 fusion. Third, the composition and molecular weight of the ELP are designed such that the ELP-FGF21 fusion undergoes a phase transition triggered solely by body heat, resulting in an immiscible viscous phase upon subcutaneous (s.c.) injection and thereby creating an injectable depot. Indeed, a single s.c. injection of ELP-FGF21 affords up to five days of sustained glycemic control in ob/ob mice. The ELP fusion partner massively streamlines production and purification of FGF21, while providing a controlled release method for delivery that reduces the frequency of injection, thereby enhancing the pharmacological properties of FGF21 as a protein drug to treat metabolic disease.

Keywords: Drug delivery; Elastin-like polypeptide; Fibroblast growth factor 21; Recombinant fusion protein; Subcutaneous depot; Type 2 diabetes.

Fig. 1

Schematic demonstrating the principles underlying the injectable FGF21 drug depot. An ELP consisting of 120 repeats of the [Val-Pro-Gly-X_aa-Gly] pentapeptide, with a Val:Ala ratio of 4:1 at the X_aa position, is genetically fused to the N-terminus of FGF21. At temperatures below the tunable transition temperature (T_t) of the ELP, the fusion exists as miscible unimers, while above the T_t it forms an immiscible coacervate. Tuning the T_t below 37°C allows body heat to trigger the phase transition in vivo, forming a depot from which unimers dissolve over time.

Fig. 2

Purification of ELP-FGF21 by inverse transition cycling (ITC). (A) ITC purification schematic. Addition of salt (typically sodium chloride or ammonium sulfate) to the soluble fraction of the bacterial cell lysate triggers the LCST phase transition of the ELP fusion protein. Centrifugation at room temperature (“warm spin”) results in pelleting of the ELP fusion and separation from soluble contaminants. The ELP fusion pellet is then dissolved in cold buffer. Subsequent centrifugation at 4°C (“cold spin”) pellets the insoluble contaminants, while the ELP fusion remains in solution. The process is repeated by triggering the phase transition via salt addition at room temperature. Three to five rounds of ITC are typically sufficient to purify an ELP fusion protein from cell lysate. (B) SDS-PAGE analysis of ELP-FGF21 purification by ITC. ELP_Depot-FGF21 was purified by ITC, and then evaluated for purity on a coomassie-stained SDS-PAGE gel. 1: MW ladder (kDa). 2: Cell lysate. 3: Insoluble fraction following DNA precipitation. 4: Soluble fraction following DNA precipitation. 5: Supernatant after first warm spin. 6: ITC round 1. 7: ITC round 2. 8: ITC round 3.

Fig. 3

ELP-FGF21 stimulates ERK1/2 phosphorylation. (A) Qualitative FGF21 activity assay in adipocytes. 3T3-L1 murine fibroblasts were differentiated into adipocytes, serum starved, then stimulated with 100 nM ELP-tev-FGF21 or 100 nM FGF21 for indicated times. Cell lysates were probed with antibodies against phosphorylated ERK1/2, as well as total ERK1/2 as a loading control. (B) HEK293 cell line stably expresses the FGF21 receptor complex. HEK293 cells were transfected with genes encoding murine FGFR1 and βKlotho (KLB), selected for stably expressing clones, and cell lysates were probed with antibodies against the respective receptors. (C) Qualitative FGF21 activity assay in transfected HEK293 cell line. A HEK293 cell line stably transfected with FGFR1 and KLB was serum starved, then stimulated with 100 nM ELP-tev-FGF21 for indicated times. Cell lysates were probed with antibodies against phosphorylated ERK1/2, as well as total ERK1/2 as a loading control. (D–E) Dose-response curves for ELP-FGF21 fusion proteins. A HEK293 cell line stably expressing the FGF21 receptor complex was serum starved then stimulated for 5 minutes with increasing concentrations of ELP-tev-FGF21, FGF21 cleaved from ELP-tev-FGF21, or ELP_Depot-FGF21. Cells were lysed and assayed for phosphorylated and total ERK1/2 content. Data are presented as mean % phosphorylated ERK1/2 ± SEM, n=3. EC₅₀s were determined by fitting a three-parameter dose-response curve.

Fig. 4

LCST phase transition behavior of a depot-forming ELP-FGF21 fusion protein. (A) Turbidity profiles as a function of solution temperature for a depot-forming ELP, alone and fused to FGF21. The optical density at 350 nm of ELP_Depot or ELP_Depot-FGF21, each prepared at 400 µM in PBS, was measured as a function of temperature, with a temperature ramp up to 37°C then down to 20°C at a rate of 1°C/min. (B) Transition temperature (T_t) as a function of concentration for a depot-forming ELP, alone and fused to FGF21. The optical density at 350 nm of indicated dilutions of ELP_Depot or ELP_Depot-FGF21 was measured as a function of temperature, as described in (A). The T_t was defined as the temperature corresponding to the 50% maximum optical density, and was plotted against ELP concentration. Data are presented as mean ± SEM, n=3.

Fig. 5

Short-term efficacy of ELP_Depot-FGF21 in a diabetic mouse model. (A–D) Fed blood glucose levels following a single injection of FGF21 or ELP_Depot-FGF21. 7–9-week-old ob/ob mice (n=4–5) were injected via s.c. with indicated dose of ELP_Depot-FGF21, FGF21, or vehicle control and blood glucose levels were monitored. Data were analyzed via two-way repeated measures ANOVA followed by uncorrected Fisher’s LSD tests at each discrete time point. Data are presented as mean ± SEM (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ^ indicates significance for 40 mg/kg cohort; NS = treatment effects are not statistically significant). (E–F) Effects of ELP_Depot-FGF21 on blood glucose area under the curve (AUC) and weight gain. AUC was calculated from the blood glucose vs. time data reported in B–D; weights were measured 1 day prior to injection and on day 6 post-injection. Data are presented as mean ± SEM, and were analyzed for statistical significance via one-way ANOVA followed by Dunnett’s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001).

Fig. 6

Fluorescence tomography imaging and quantification of ELP-FGF21 depots. (A) Male ob/ob mice were injected via s.c. with 20 mg/kg fluorescently labeled ELP_Depot-FGF21 or FGF21. Injection sites were imaged by fluorescence molecular tomography every 24 h for the ELP_Depot-FGF21 cohort (top panel) or at indicated time points for FGF21 cohort (bottom panel). (B) Image analysis software was used to quantify fluorescence within assigned regions of interest, and these data were correlated to protein drug residing in the s.c. space for each respective cohort (n=5). Data are presented as means ± SEM.

Fig. 7

Serum concentration vs. time profiles for ELP-fused FGF21. Male ob/ob mice (n=3–4) were injected with radiolabeled FGF21 or ELP_Depot-FGF21 via (A) i.v. at 1 mg/kg or (B) s.c. at 20 mg/kg. Blood samples were collected at indicated times and radiolabeled protein was quantified via gamma counting. Data are presented as mean ± SEM. Solid lines represent a linear regression fit to the terminal portions of the i.v. and s.c. data, as well as early i.v. time points for extrapolation of initial serum concentration. The horizontal dotted line indicates the estimated minimum therapeutically effective concentration for ELP_Depot-FGF21.

Fig. 8

Long-term efficacy of ELP_Depot-FGF21 in a diabetic mouse model. 7-week-old ob/ob mice (n=5) received repeated s.c. injections of FGF21 or ELP_Depot-FGF21 at 20 mg/kg every 5 days for 8 weeks. Fed blood glucose levels (A) and body weights (B) were monitored throughout the treatment period. (C) Blood glucose AUC was calculated from the blood glucose vs. time data reported in A. (D) %HbA1c was measured on day 55, 5 days following the final dose, and is reported as a magnitude change in %HbA1c compared to t=0. (E–F) Serum insulin and triglyceride levels were measured on day 30, prior to a scheduled treatment dose. Data are presented as mean ± SEM, and were analyzed for statistical significance via one-way ANOVA followed by Dunnett’s multiple comparisons tests (**p<0.01).