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Hormone Molecular Biology and Clinical Investigation

Editor-in-Chief: Chetrite, Gérard S.

Editorial Board: Alexis, Michael N. / Baniahmad, Aria / Beato, Miguel / Bouillon, Roger / Brodie, Angela / Carruba, Giuseppe / Chen, Shiuan / Cidlowski, John A. / Clarke, Robert / Coelingh Bennink, Herjan J.T. / Darbre, Philippa D. / Drouin, Jacques / Dufau, Maria L. / Edwards, Dean P. / Falany, Charles N. / Fernandez-Perez, Leandro / Ferroud, Clotilde / Feve, Bruno / Flores-Morales, Amilcar / Foster, Michelle T. / Garcia-Segura, Luis M. / Gastaldelli, Amalia / Gee, Julia M.W. / Genazzani, Andrea R. / Greene, Geoffrey L. / Groner, Bernd / Hampl, Richard / Hilakivi-Clarke, Leena / Hubalek, Michael / Iwase, Hirotaka / Jordan, V. Craig / Klocker, Helmut / Kloet, Ronald / Labrie, Fernand / Mendelson, Carole R. / Mück, Alfred O. / Nicola, Alejandro F. / O'Malley, Bert W. / Raynaud, Jean-Pierre / Ruan, Xiangyan / Russo, Jose / Saad, Farid / Sanchez, Edwin R. / Schally, Andrew V. / Schillaci, Roxana / Schindler, Adolf E. / Söderqvist, Gunnar / Speirs, Valerie / Stanczyk, Frank Z. / Starka, Luboslav / Sutter, Thomas R. / Tresguerres, Jesús A. / Wahli, Walter / Wildt, Ludwig / Yang, Kaiping / Yu, Qi

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Volume 30, Issue 2

Issues

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

Junichiro Sonoda / Mark Z. Chen / Amos Baruch
Published Online: 2017-05-19 | DOI: https://doi.org/10.1515/hmbci-2017-0002

Abstract

Fibroblast growth factor 21 (FGF21) analogs and FGF21 receptor agonists (FGF21RAs) that mimic FGF21 ligand activity constitute the new “FGF21-class” of anti-obesity and anti-diabetic molecules that improve insulin sensitivity, ameliorate hepatosteatosis and promote weight loss. The metabolic actions of FGF21-class proteins in obese mice are attributed to stimulation of brown fat thermogenesis and increased secretion of adiponectin. The therapeutic utility of this class of molecules is being actively investigated in clinical trials for the treatment of type 2 diabetes and non-alcoholic steatohepatitis (NASH). This review is focused on various FGF21-class molecules, their molecular designs and the preclinical and clinical activities. These molecules include modified FGF21 as well as agonistic antibodies against the receptor for FGF21, namely the complex of FGF receptor 1 (FGFR1) and the obligatory coreceptor βKlotho (KLB). In addition, a novel approach to increase endogenous FGF21 activity by inhibiting the FGF21-degrading protease fibroblast activation protein (FAP) is discussed.

Keywords: monoclonal antibodies; NAFLD; obesity; therapeutics; type 2 diabetes

Introduction

A chronic positive energy balance promoted by a sedentary life style and excess consumption of calories drives the global obesity epidemic. Veritably, the excess consumption of calorie-rich foods leading to obesity-associated ailments is now recognized as the leading cause of disability around the world [1]. Mechanistically, ingestion of calories that exceed the storage capacity of the adipose tissue has been linked to the accumulation of ectopic lipids in non-adipose organs, and the induction of low-grade tissue inflammation, endoplasmic reticulum stress and insulin resistance [2], [3]. These metabolic defects increase the risk of developing serious medical conditions including type 2 diabetes, non-alcoholic steatohepatitis (NASH), cardiovascular diseases and various forms of cancer [4]. Currently, treatment options for obesity and related co-morbidities are limited thus making this an urgent area of unmet medical need. In particular, pharmaceutical agents that correct energy imbalance and improve insulin sensitivity by promoting energy expenditure (EE) without significant adverse effects may provide a new approach to combat obesity and its pathological sequela [5], [6], [7].

Fibroblast growth factors (FGFs) are a family of signaling proteins that regulate reproduction, development and metabolism by activating the FGF receptor (FGFR) family of membrane spanning receptor tyrosine kinases [8]. Two endocrine FGFs – FGF19 (FGF15 in rodents) and FGF21 – have emerged as attractive therapeutic targets to treat obesity-related metabolic disorders due to their beneficial effects on lipids and carbohydrate metabolism [9], [10], [11]. FGF19 and FGF21 exhibit similar ability to increase EE and induce weight loss, reduce hepatic triglyceride and other cardiovascular risk factors and improve insulin sensitivity in obese mice. This was first reported for FGF19 in 2002 [12], [13] and soon thereafter for FGF21 [14], [15], [16]. The similarity in the metabolic effects of FGF21 and FGF19 was further substantiated by the subsequent discovery that they act through common receptors, namely the “c” splice isoform of FGFR1, 2 and 3, bound by the obligatory coreceptor βKlotho (KLB) (Figure 1) [17], [18], [19], [20].

FGF19 and FGF21 receptor specificity and function. FGF19 and FGF21 activate homodimeric FGFR tyrosine kinases bound by the obligatory co-receptor KLB. The downstream effects of FGFR1/KLB activation are well characterized, however, a role for FGFR2/KLB and FGFR3/KLB is yet to be defined. FGF19 uniquely activates the FGFR4/KLB complex and elicits distinct downstream effects as compared to FGF21 receptors.
Figure 1:

FGF19 and FGF21 receptor specificity and function. FGF19 and FGF21 activate homodimeric FGFR tyrosine kinases bound by the obligatory co-receptor KLB. The downstream effects of FGFR1/KLB activation are well characterized, however, a role for FGFR2/KLB and FGFR3/KLB is yet to be defined. FGF19 uniquely activates the FGFR4/KLB complex and elicits distinct downstream effects as compared to FGF21 receptors.

Unique to the activity of FGF19 is that, unlike FGF21, it suppresses hepatic bile acid biosynthesis, stimulates gall bladder filling [9] and promotes hepatocyte growth causing spontaneous emergence of hepatocellular carcinoma when overproduced in mice [21], [22]. In contrast, transgenic overproduction of FGF21 suppresses chemically induced hepatic tumorigenesis [23], [24] and extends longevity [25]. The mitogenic activity of FGF19 is attributed to its ability to activate the FGFR4/KLB complex (Figure 1) [17], [19], [22], [26]. The FGFR4-sparing property of FGF21 has made it a more attractive drug candidate as compared with FGF19.

Over the past decade, FGF21 analogs and other forms of agonists that directly activate the FGFR1/KLB receptor complex (i.e. FGF21 receptor agonists or FGF21RAs) have been tested in non-human primates (NHPs) and humans, revealing their potential to ameliorate obesity and obesity-related comorbidities [27], [28], [29], [30], [31], [32]. In addition, generation of “FGF21-like” analogs of FGF19 that lack FGFR4 activity have been reported [33], [34], although none have been tested in primates to date. Understanding of the class-effects of FGF21-related molecules is important in distinguishing on-target effects from molecule-specific off-target side effects, which may arise, among other reasons, from interference of endogenous FGF/FGFR or FGF19/KLB interactions.

The activity signature of FGF21-class molecules

Cellular activities of FGF21

FGF21 was first identified as a metabolic regulator in the Kharitonenkov lab at Eli Lilly on the basis of its ability to increase insulin-independent glucose uptake in 3T3-L1 mouse adipocytes [14]. This activity was attributed to an increase in Glut1 mRNA and protein expression [14]. In addition, FGF21 and FGF21RA induce expression of Uncoupling protein 1 (Ucp1) mRNA in cultured primary adipocytes [27], [35], [36], [37], which may play a role in inducing white adipose tissue “browning” in mice [27], [38]. At the target tissues, receptor activation by FGF21 triggers a transcriptional response of various genes, including Ucp1 upregulation in adipose tissues [15], [16]. While many of the transcriptional responses are tissue-type specific and may be driven by indirect effects, several genes induced by FGF21 are not restricted by cell type and likely represent proximal target downstream of FGFR. These genes include Sprouty 4 (Spry4) and Dual specificity phosphatase 4 and 6 (Dusp4 and Dusp6), the known negative feedback regulators of FGFR [8], [27], [39], [40]. Determining the induction of these genes in tissue biopsies could serve as proximal FGF21 pathway biomarkers in preclinical and clinical settings.

The bulk of the metabolic action of FGF21 is likely mediated by the mitogen-activated protein kinase (MAPK) signaling cascade downstream of FGFR [11], [14]. FGF21 triggers phosphorylation of FGFR1 and the MAPK signaling intermediates, such as FGFR substrate 2 (FRS2), MEK, ERK, p90RSK and the transcription factors STAT3 and CREB in cell lines or mouse tissues expressing FGFR1c, 2c or 3c together with KLB [14], [17], [41], [42]. These signaling events can be blocked by FGFR inhibitors or MEK inhibitors [41]. While ERK phosphorylation can be directly determined to measure receptor activation, in vitro assays that utilize a luciferase reporter in transfected human HEK293 cells or rat L6 cells can provide a wider dynamic range and a better assay reproducibility [34], [43]. In this system, activation of MAPK signaling pathway leads to an ERK-dependent phosphorylation of the transcription factor ELK1 fused to the GAL4 DNA binding domain and the consequential induction of luciferase reporter gene under the control of GAL-response elements [44]. Neither HEK293 nor L6 cells endogenously express KLB protein, therefore exogenous expression of KLB is necessary to measure specific FGF21 activity. The readout in HEK293 cells provides a wide dynamic range, typically yielding more than 20-fold induction of luciferase activity. As HEK293 cells express endogenous FGFRs, this system may not be suitable to study the requirement of specific FGFR isoforms for signaling unless engineered to eliminate endogenous FGFR. The rat L6 cell system provides a lower degree of luciferase induction as compared with HEK293 but is more suitable for measuring receptor specificity due to the lack of endogenous FGFR expression [34]. These reporter assays as well as a direct measurement of ERK phosphorylation have been widely used to test and screen various FGF21RAs as well as FGFR4-sparing FGF19 analogs [27], [33], [34], [41], [45].

Metabolic effects in obese mice

Mice have been the preferred model organism used to test the in vivo metabolic effects of human FGF21 protein and engineered analogs [14], [15], [16]. In addition, one FGF21-receptor binding agonist (i.e. bispecific anti-FGFR1/KLB antibody) with an activity toward the mouse co-receptor has been reported [27]. Some of the most evident metabolic changes commonly elicited by these FGF21-class molecules in high-fat-diet-induced obese mice include a significant lowering of body weight and adiposity that is not accompanied by a reduction in calorie intake [15], [16]. In fact, mice treated with FGF21-class molecules tend to consume more food as compared with the control group, especially after significant weight loss is achieved [46], [47]. Weight loss induction by FGF21-class molecules in diet-induced obese mice is associated with an improvement in hepatic and whole body insulin sensitivity, normalization of glycemia, an alleviation of hepatosteatosis and improvements in various circulating metabolic markers [15], [16], [27]. FGF21-induced glucose lowering is not accompanied by excessive hypoglycemia, supporting a mechanism driven by insulin sensitization, rather than insulin secretion [14]. The most commonly observed changes in circulating metabolic markers after treatment with FGF21-class molecules include a decrease in insulin, total triglycerides, total cholesterol and total free fatty acids, and an increase in total and high molecular weight adiponectin [15], [16], [27], [48], [49], [50].

One of the primary mechanisms implicated in the chronic effects of FGF21-class proteins and the related FGF19 protein on obesity, insulin sensitivity and metabolic parameters is an increase in whole body EE [12], [13], [15], [16]. A concomitant increase in uptake of glucose tracer into interscapular brown adipose tissue (BAT) as well an increase in resting core body temperature suggests that EE is predominantly driven by BAT thermogenesis [12], [13], [15], [16], [27], [51]. The insulin sensitizing effects of FGF21 are adipose tissue dependent as shown by an inability of FGF21 to improve insulin sensitivity in lipodystrophic transgenic mice [41], [52]. In contrast, surgical resection of the interscaplar BAT does not abolish the response to FGF21-analogs, suggesting that small BAT depots distributed throughout the mouse may be sufficient to mediate the FGF21 effects in mouse [46], [51], [53], [54].

The mechanisms through which FGF21 stimulates BAT thermogenesis are not fully understood. However, several independent studies have demonstrated that some of the FGF21-mediated metabolic effects are preserved in Ucp1-deficient mice, suggesting that FGF21 is likely to stimulate BAT thermogenesis via an Ucp1-independent mechanism [55], [56], [57]. In addition, mouse genetic studies employing tissue-specific disruption of the Klb gene suggested that activation of FGFR/KLB complexes in the central nervous system (CNS) and the subsequent stimulation of sympathetic nerves that innervate the BAT play a critical role in this process [28], [39]. This was rather surprising as KLB is abundantly expressed in liver, adipose tissues and pancreas [19], [58], and downstream FGFR signaling activation is primarily observed in these metabolic organs following administration of FGF21 analogs and FGF21RA [17], [27]. While the expression of Klb mRNA in localized compartments within the mouse brain has been reported, the nature of the Klb-expressing neurons is still elusive [59], [60].

In addition to the regulation of BAT thermogenesis, recent studies indicate that FGF21 can influence central reward circuits and reduce sweet and alcohol consumption via activating KLB complexes expressed in the CNS [59], [61], [62]. Reduction in saccharin-containing water consumption by a FGF21-immunoglobulin G (IgG) fusion protein is conserved in NHPs [61]. In addition, a genome-wide association study in humans identified KLB locus as a gene that regulates alcohol consumption [62]. These reports together suggest that the regulation of the central reward circuits is an evolutionarily conserved function of FGF21.

Another mechanism by which FGF21-class molecules improve metabolic homeostasis in diet-induced obese mice appears to be through an increased secretion of high molecular weight adiponectin. Studies using adiponectin-deficient mice suggested that adiponectin may not be absolutely necessary for the effect of FGF21-class molecules, but may potentiate their effects following long-term treatment [27], [48], [49]. In addition to adiponectin, at least two other hormones, insulin and leptin, also mediate the metabolic action of FGF21. A genetic study indicated that FGF21 improves hepatic lipid metabolism by improving insulin action in the liver [46]. Leptin involvement was suggested by the inability of FGF21-class molecules to effectively induce weight loss in ob/ob or db/db mice that lack intact leptin signaling [27], [34], [63]. The ability of FGF21-class proteins to improve insulin sensitivity and lower blood glucose remains intact in ob/ob and db/db mice, indicating that insulin sensitization and weight loss diverge downstream of FGF21 signaling [14], [63].

Finally, FGF21 was recently described as a secretagogue of digestive enzymes by acting directly on the FGFR/KLB receptor complex expressed in pancreatic acinar cells [64]. The specific FGFRs mediating this effect is unknown; however, anti-FGFR1/KLB agonist antibody induces ERK phosphorylation in pancreatic acinar cells in mice, thus FGFR1 is likely involved [27]. The contribution of this activity to the overall FGF21-induced metabolic effects is unknown; however, such modulation of digestive enzymes may influence intestinal absorption of nutrients. Indeed, chronic FGF21 administration in high-fat-fed obese mice modestly increased fecal fat content [15].

Metabolic effects in NHPs and humans

To date, at least 12 FGF21-class molecules have been tested in NHPs and/or in humans [27], [28], [29], [30], [31], [65], [66]. The reported results highlight substantial species differences in response to FGF21-class molecules. For example, while FGF21-class molecules readily normalize blood glucose levels in mouse models of obesity-induced insulin resistance [14], [15], [16], only a modest glucose lowering effect was observed in human patients with type 2 diabetes [28], [29]. In addition, FGF21-analogs strongly suppress food intake in NHPs [28], [32], while a mild increase in food consumption occurs in mice [15], [16]. Hence, although FGF21-class molecules induced robust weight loss both in NHPs and in diet-induced obese mice, the mechanism of weight loss in NHPs appears to be related to reduction in food consumption [28], [67], while thermogenesis primarily drives weight loss in mice. The weight-loss induced by FGF21-class molecules in NHPs is accompanied by improvements in various cardiometabolic lipid factors [28], [30], [31], [32]. Whether or not FGF21 activates BAT thermogenesis in NHPs has yet to be determined.

Anorexia and weight loss can be attributed to a number of factors (e.g. toxicity, food aversion, etc.), therefore weight loss independent markers are useful in determining whether the observed pharmacological effects are due to FGF21 pathway activation or other molecule-specific mechanism (e.g. disruption of endogenous FGF signaling pathways). This aspect is particularly important when testing FGF21RAs that do not cross-react with the rodent receptors, thus cannot be studied thoroughly in rodents. In this respect, circulating adiponectin represents an important pathway-specific biomarker. Circulating adiponectin increases after treatment with native FGF21 protein in mice [48], [49], [50] and in spontaneously obese NHPs [31]. In addition, several engineered FGF21-class molecules increase circulating adiponectin in NHPs and humans [27], [28], [29], [32]. One exception to this FGF21-class effect was demonstrated by a FGF21RA anti-KLB antibody, named mimAb1. Although mimAb1 activates the FGFR1/KLB receptor complex in cell-based assays and induces weight loss in NHPs, the treatment did not result in an increase in circulating adiponectin [66]. This distinctive activity suggests that mimAb1 may induce weight loss via a separate mechanism (discussed below).

At least nine different FGF21-class molecules have been tested in phase 1 or phase 2 clinical studies. Seven of them are modified FGF21 proteins: LY2405319, LY3025876, LY3084077, BMS986036, BMS986171, PF05231023 and AMG876. The other two are antibody-based receptor agonists: BFKB8488A and NGM313 (Figure 2). So far, clinical trial data has been reported for LY2405319, an engineered form of human FGF21 [29], and PF05231023, a bivalent fusion of human FGF21 to human IgG [28]. These phase 1, placebo-controlled, blinded studies were performed in obese or overweight subjects with type 2 diabetes treated for 4 weeks. Despite the small sample size, several consistent effects were observed including weight loss, decreases in fasting insulin, triglycerides and LDL cholesterol and increases in adiponectin and HDL cholesterol. Fasting plasma glucose levels appeared to be decreasing during treatment but this effect could not be statistically substantiated. The mechanisms contributing to weight loss and other metabolic effects were not clear as food intake or energy expenditure were not measured nor reported in either study. Based on preclinical data, it is likely that FGF21 analogs will affect both “energy in” and “energy out”, however, the relative contribution of these pathways to weight loss in human subjects is still unknown.

Examples of FGF21-receptor agonists. Various approaches were employed to mimic the activity of FGF21 while introducing drug-like properties to increase stability and duration of action. Several of these molecules were tested in humans (bold black). The FGF21 C-terminal peptide (red) is critical for activity and is susceptible to processing by FAP. Fc-FGF21(RG) fusion protein possesses stabilizing mutations that were introduced to prevent FAP-mediated proteolysis and extend terminal half-life of the active molecule (asterisks). Molecules are ordered by their predicted duration of action.
Figure 2:

Examples of FGF21-receptor agonists. Various approaches were employed to mimic the activity of FGF21 while introducing drug-like properties to increase stability and duration of action. Several of these molecules were tested in humans (bold black). The FGF21 C-terminal peptide (red) is critical for activity and is susceptible to processing by FAP. Fc-FGF21(RG) fusion protein possesses stabilizing mutations that were introduced to prevent FAP-mediated proteolysis and extend terminal half-life of the active molecule (asterisks). Molecules are ordered by their predicted duration of action.

Tackling some of the key mechanistic questions around FGF21-class drugs will be critical for maximizing their therapeutic potential in humans. Some of these questions can be answered via measuring the effect on resting metabolic rate by indirect calorimetry, on food intake and preference by direct measurement of food consumed or patient questionnaires, and on hepatic lipid contents in individuals with hepatosteatosis by magnetic resonance imaging or other quantitative methods. In addition, it is important to determine whether FGF21-induced insulin sensitization as suggested by reduced fasting insulin levels would drive overall improvement in glycemic control and yield a significant lowering of HbA1c. These effects will likely require longer treatment duration and will benefit from the use of long-acting FGF21 agonists.

Turning FGF21 into a drug

While the observed pharmacological effects of recombinant human FGF21 in preclinical disease models clearly highlighted its therapeutic potential, FGF21 in its native form was not suitable for commercialization for a variety of reasons. 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]. 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]. Fibroblast activation protein (FAP) was recently identified as the protease responsible for the proteolytic cleavage and inactivation of human FGF21 in blood [71], [72], [73].

In addition to the poor pharmacokinetics, native FGF21 is unstable and prone to aggregation in solution [68], [69]. Various engineering approaches have been undertaken to enable a stable liquid formulation at high concentration with a low viscosity. These characteristics are important for both providing extended shelf life and enabling small injection volumes for safe and convenient subcutaneous delivery. Achieving good bioavailability via subcutaneous injections is also an important consideration.

When engineering various mutations into FGF21 to improve bioavailability and stability, one should consider the co-introduction of potential immunogenic epitopes. Immunogenic responses can lead to the production of anti-drug antibodies that can mediate rapid clearance or neutralization of the drug’s activity [74]. Such immunogenic reactions not only lead to loss of drug efficacy, but also can cause severe adverse reactions. For example, anti-drug antibodies against a mutant FGF21 have the potential to cross-react and neutralize the endogenous FGF21 protein leading to autoimmune FGF21-deficiencies. FGF21 deficient mice are viable, but exhibit a heightened pathology when acute pancreatitis or non-alcoholic steatohepatitis is experimentally induced [64], [75], [76], [77]. Thus, the immunogenicity of each FGF21-analog should be carefully monitored during clinical studies. The identification of FGF21 receptors has enabled the discovery of various binding proteins and monoclonal antibodies that activate the FGF21 receptor complexes, specifically, the FGFR1/KLB complex (FGF21RAs). FGF21RAs offer an approach to generating long-acting protein therapeutics with FGF21-like activity without the concern of cross-reacting immunogenicity to the endogenous FGF21 protein.

Short-acting FGF21 analog LY2405319

LY2405319 is a FGF21 analog, designed to enable large-scale production in the yeast Pichia pastoris and to reduce aggregation in solution [69]. This modified protein lacks the N-terminal four amino acid residues, His-Pro-Ile-Pro, which are functionally dispensable and prone to proteolysis in yeast as well as in human serum. In addition, it was engineered with a stabilizing disulfide bond through mutating Leu118 and Ala134 to Cys residues. An additional mutation, Ser167 to Ala, prevents the post-translational modification of O-glycosylation in yeast. Despite these modifications, LY2405319 exhibits potency and efficacy that are almost identical to the native human FGF21 [29], [32], [69].

As described above, LY2405319 provided the first proof of concept example for the biological activity of FGF21-class molecule in humans [29]. The highest dose group was 20 mg daily delivered via subcutaneous injection into subjects with an average body mass index of 32 kg/m2 [29]. This was a substantially lower dose compared with the LY2405319 doses used in a previously reported NHP study (3–50 mg/kg) [32]. Nonetheless, this dose was sufficient to maintain average steady-state circulating plasma drug concentrations at 150 ng/mL in plasma and alter a number of metabolic parameters following 28 days of treatment [29]. Treatment with LY2405319 was generally well tolerated in human subjects although injection-site-related adverse events and anti-drug immunogenicity was also frequently observed. No further studies involving this molecule have been reported.

Engineered FGF21 analogs with an improved circulating half-life

The native form of FGF21 and LY2405319 are rapidly cleared via the kidney due to their low molecular weight (∼19.4 kDa) [68], [69]. Several FGF21 analogs were designed to increase their molecular weight to lengthen the circulating half-life and drug exposure. One approach commonly used for this purpose is to fuse polyethylene glycol (PEG) moieties to a recombinant protein (e.g. pegfilgrastim, pegvisomant, peginterferon α-2a, methoxy PEG-epoetin β). PEG30-Q108 is a PEGylated form of FGF21 that is conjugated to a 30 kDa PEG via a non-natural amino acid residue at the position 108, and has molecular weight of approximately 50 kDa [78], [79] (Figure 2). Although this variant has a ∼6-fold lower potency compared to native FGF21, due to its superior pharmacokinetic profile, its in vivo efficacy in rodents with metabolic disease is comparable to more frequently administered native FGF21. The PEGylation approach carries a risk of tissue vacuole formation particularly in the renal tubular epithelium, which may be a significant safety concern for the treatment of diabetic patients with renal insufficiency [80]. PEGylated FGF21-analogs have been tested in clinical trials [BMS986036 (also named ARX618) and BMS986171], but data from these studies have not yet been disclosed.

Another approach to increase the molecular weight of FGF21 and extend the circulating half-life was through conjugation to IgG molecule. PF05231023 (also named CVX343) was developed by conjugating two molecules of modified FGF21 (ΔHis1, Ala129Cys) to an antibody scaffold via the mutated Cys129 residue of FGF21, resulting in an approximately 190 kDa bivalent molecule [81] (Figure 2). Immunoassays that can detect the intact N-terminus and C-terminus of FGF21 demonstrated that PF05231023 was subjected to rapid proteolysis near the C-terminus, which inactivated the molecule in monkeys and humans [28], [82]. PF05231023 was administered intravenously in obese and diabetic subjects with an approximate body mass index of 30 kg/m2 [28]. The most efficacious doses, 100 mg and 140 mg, were administered twice a week for 4 weeks and achieved similar biological effects to the once daily dosed 20 mg LY2405319, a short acting FGF21-analog described above [29]. PF05231023 was well tolerated at effective doses, with the most frequent adverse event being diarrhea and nausea.

While modifications to increase the molecular weight of FGF21 reduce rapid renal clearance, they do not prevent the FAP-mediated proteolysis and protein inactivation [71], [72], [73]. To mitigate FAP mediated cleavage, a Pro171Gly mutation at the FAP-dependent proteolytic cleavage site was engineered into an Fc-fused FGF21 [68] (Figure 2). This conjugate called Fc-FGF21(RG) is an approximately 90 kDa dimeric protein, and carries an additional mutation Leu98Arg, which improves formulation stability. It is ∼4-fold less potent in cell-based assays as compared to native FGF21, however, it has shown comparable activity in mice and NHPs with less frequent dosing [30], [56], [68]. A variant of this protein called AMG876 has been tested in humans, but the result of the clinical study has not been made available to the public.

FGFR1/KLB receptor agonists

FGF21 can bind and activate 3 FGFR isoforms (1c, 2c and 3c) in the presence of the coreceptor KLB (Figure 1) [17], [18]. Other FGFR isoforms, FGFR1b, 2b and 3b do not form a complex with KLB, therefore do not act as a receptor for FGF21. Several lines of evidence suggest that of the FGFR receptors that bind FGF21, FGFR1 plays a dominant role in mediating FGF21 activity. For example, aP2-CRE-induced deletion of the Fgfr1 gene in mice abrogates the response to recombinant FGF21 protein [66], [83]. In addition, antibody-mediated activation of FGFR1 or the FGFR1/KLB complex is sufficient to recapitulate almost all the beneficial metabolic effects of FGF21 in mice [27], [41]. In particular, the bispecific anti-FGFR1/KLB antibody bFKB1 represents how this mode of receptor activation can be successful in mimicking all of the beneficial metabolic effects and molecular signature of FGF21 pathway activation [27].

One functional difference between FGF21-analogs and FGFR1/KLB agonists is that FGF21 activates FGFR2/KLB and FGFR3/KLB complex that are expressed in the liver, whereas the latter group is liver-sparing due to the paucity of FGFR1 in the liver [17], [27]. In theory, the narrowed receptor selectivity may yield a superior long-term safety profile. On the other hand, activation of FGFR2/KLB and FGFR2/KLB may enhance hepatic benefits. To date, it is not yet clear whether FGFR1/KLB agonists have a therapeutic advantage over FGF21 analogs.

In designing FGF21-mimetic FGFR1 agonist, it is important to restrict FGFR1 activation to tissues that also express KLB protein to avoid undesired effects such as hypophosphatemia [41], [45]. This can be achieved by designing an agonist molecule that selectively acts on the FGFR1/KLB complex. In addition, it is important to avoid interference of endogenous FGF/FGFR or FGF19/KLB interactions by a high affinity FGFR1 or KLB binding protein. A functional interference of ligand-receptor interaction could occur via physical obstruction (e.g. competitive binding, steric hindrance, etc.) or via induction of receptor internalization and degradation. One such example occurred when a neutralizing anti-FGF19 antibody caused severe diarrhea, liver toxicity and weight loss in cynomolgus monkeys [84]. Due to species differences, rodent studies may not reveal toxicity of FGF19/FGFR4 blocking agents [22], [85]. Therefore, when generating a therapeutic candidate, the investigator must be cognizant that the final product not only activates the FGF21 pathway but also does so without overtly interfering with the endogenous FGFR pathways, in particular the FGF19/FGFR4/KLB axis.

The first reported KLB-dependent FGFR1 agonist protein is a bispecific FGFR1/KLB binding protein based on a non-antibody scaffold called Avimer [65]. The approximately 18 kDa protein C3201 combines a high affinity FGFR1-binding protein and a high affinity KLB-binding protein in tandem. C3201 specifically activates FGFR1 in the presence of human KLB in cell-based assays with high potency. Because C3201 does not cross-react with mouse KLB protein, its activity in vivo could not be assessed in mice. C3201 was tested in obese NHPs as a fusion to human serum albumin (C3201-HSA) in order to improve its pharmacokinetic profile. While C3201-HSA lowered body weight in obese NHPs, no evidence for FGF21 pathway activation, such as changes in gene expression or circulating adiponectin, was provided, and thus the mechanism by which C3201-HSA induced weight loss is not clear. Blocking anti-FGFR1 antibodies induce anorexia and weight loss in NHPs [86]. Because C3201 binds to the ligand-binding site of FGFR1 with high affinity [65], it is possible that C3201 acts as an anti-FGFR1 blocking agent in vivo, in addition to acting as an agonist for the FGFR1/KLB complex.

Development of agonistic antibodies directed against FGF21 receptors is yet another approach to yield a sustained activation of the FGF21 pathway (Figure 2). As mentioned above, mimAb1 is a KLB-dependent FGFR1 agonistic antibody generated by immunizing mice with human FGFR1/KLB complex [66]. Although it exhibits high affinity to KLB and no detectable affinity to FGFR1, this antibody specifically activates FGFR1, but not other FGFR, in a KLB-dependent manner. It is not clear how this antibody with no detectable FGFR1 affinity harbors specificity to FGFR1. However, because mimAb1 was generated by immunizing with FGFR1/KLB complex, it is possible that mimAb1 interacts directly with FGFR1 with an affinity that is too low to measure or binds a unique conformation of KLB that is present only when bound to FGFR1 but not to other FGFRs. Similar to C3201, this antibody does not cross-react with the mouse receptor complex, so it was only tested in NHPs. mimAb1 induces weight loss and lowers blood glucose and triglycerides, but interestingly, is different from other FGF21-class molecules in that it does not affect adiponectin [66]. mimAb1 has an ability to compete with FGF21 for KLB binding. Since FGF21 and FGF19 bind to a common site on KLB [87], mimAb1 likely interferes with endogenous FGF19’s ability to bind and activate the KLB/FGFR4 complex. This may explain how mimAb1 induced weight loss without affecting adiponectin.

Another antibody molecule that can selectively activate the FGFR1/KLB complex is a humanized effector-less bispecific anti-FGFR1/KLB antibody called bFKB1 that combines a Fab arm of a low affinity anti-FGFR1 antibody with a Fab arm of a high affinity anti-KLB antibody [27]. This antibody was designed to activate the FGFR1/KLB complex with potency and efficacy similar to recombinant human FGF21, and importantly, without significantly interfering with endogenous FGF binding to FGFR1 or KLB. bFKB1 cross-reacts with the mouse receptor complex, which has allowed its activity to be thoroughly characterized in mice. As describe earlier, a single dose of bFKB1 into obese mice led to a number of molecular and metabolic events that are anticipated from a continuous infusion of recombinant FGF21 [27]. Similar to other FGF21 analogs, bFKB1 induced weight loss and increased circulating adiponectin in NHPs. This antibody thus provides the first evidence that activation of the FGFR1/KLB complex with a non-FGF21 protein is sufficient to mimic the metabolic effects of FGF21 both in mice and NHPs.

An anti-FGFR1/KLB bispecific agonist antibody called BFKB8488A (also named RG7992) is currently being tested in humans. In addition, a monoclonal antibody engineered to selectively activate the FGFR1/KLB complex called NGM313 is also in clinical testing. Data from the clinical studies have not been available to date for either therapeutic antibody. Antibody-based therapies in general exhibit excellent pharmacokinetic profiles enabling long-term and sustained drug exposure, although certain caveats do apply. Thus, testing the outcome of sustained FGFR/KLB receptor activation by treatment with these antibody-based FGF21RAs may yield new insights into the full potential of FGF21-class drugs in humans.

Approach to augmenting endogenous FGF21 activity

In addition to the approaches discussed above, increasing the levels or activity of endogenous FGF21 may also be a potential therapeutic avenue. Transgenic mice overexpressing FGF21 are resistant to diet-induced weight gain [14], [88] and live 34% longer than their non-transgenic counterparts [25]. The discovery of proliferator-activated receptor α (PPARα) as a transcriptional regulator of FGF21 [89], [90], [91], and the recent discovery of FAP as the protease responsible for cleavage and inactivation of FGF21 [71], [72], [73] opens a novel therapeutic approach to increase the level of endogenous FGF21 activity by simultaneously activating PPARα and inhibiting FAP (Figure 3).

Strategies to “boost” endogenous FGF21 levels. Similar to the effect of metformin and DPPIV inhibitors on levels of active GLP-1 (top), fibrate and FAP inhibitors may elevate endogenous levels of active FGF21 by stimulating its production and inhibiting its degradation, respectively (bottom).
Figure 3:

Strategies to “boost” endogenous FGF21 levels. Similar to the effect of metformin and DPPIV inhibitors on levels of active GLP-1 (top), fibrate and FAP inhibitors may elevate endogenous levels of active FGF21 by stimulating its production and inhibiting its degradation, respectively (bottom).

Transcriptional induction of FGF21

Circulating FGF21 is produced primarily by the liver in mice [92] and upregulated during fasting or protein deficiency [89], [90], [91], [93]. In humans, elevated blood FGF21 levels are associated with obesity and high hepatic lipid content [94], [95]. Various transcription factors have been identified as a direct regulator of FGF21 gene transcription with the bulk of the data surrounding PPARα [89], [90], [91]. Loss of the Ppara gene in mice abolishes FGF21 expression in the liver while small molecule PPARα agonists increase Fgf21 mRNA level and circulating FGF21 protein [89], [90], [91]. The PPARα agonist fenofibrate increased circulating FGF21 protein in humans after 3 weeks of treatment, which was sustained after 5-years of chronic treatment [96], [97], [98]. Similar to some of the FGF21-class effects, fibrate-class PPARα agonists are known for their ability to improve circulating triglyceride and HDL-cholestrol profiles [99], and may also mildly increase adiponectin [100]. However, chronic treatment with fibrates does not result in other FGF21-induced metabolic effects in human such as weight loss or reduction of insulin. Thus PPARα-mediated upregulation of FGF21 alone does not appear to sufficiently augment the activity of endogenous FGF21 to improve metabolic health in humans.

Stabilization of circulating FGF21

As discussed earlier, the 10 amino acid residues at the C-terminus of human FGF21 protein is rapidly removed by the endopeptidase FAP upon injection into animals [68], [71], [72], [73]. These amino acid residues are essential for binding to KLB and for downstream signaling activity [43], [70], [71]. The discovery of FAP as a negative regulator of FGF21 suggests that a portion of the circulating FGF21 in humans exists in an inactive form, and inhibiting FAP may preserve endogenous FGF21 activity. Indeed, administration of a FAP-specific inhibitor acutely increases the level of the intact FGF21 protein in NHPs [71].

FAP possesses a 51% amino acid identity to dipeptidyl peptidase IV (DPPIV), the target of the gliptin-class of anti-diabetic drugs that function by stabilizing endogenously produced incretin hormone, glucagon-like peptide-1 (GLP-1) [101]. Despite their similarity, FAP does not inactivate GLP-1 and conversely, DPPIV does not inactivate FGF21 near the C-terminus [71]. The commonly used anti-diabetic drug metformin increases the secretion of total GLP-1 from the intestinal L cell via an unknown mechanism [102]. Thus, DPPIV inhibitors synergize with metformin to increase the level of active GLP-1 [102]. In an analogous fashion to DPPIV inhibition, metformin and GLP-1, we propose that FAP inhibition can synergize with fenofibrate or other PPARα agonists to increase active FGF21 levels in humans (Figure 3) [102]. Further preclinical and clinical investigation is necessary to substantiate this therapeutic hypothesis.

FGF21 therapeutics: outlook

As of February 2017, at least nine FGF21-class molecules have been tested in humans, and several are still active in different stages of clinical development as a type 2 diabetes or NASH therapeutic. The initial results from small short-term clinical trials have been encouraging, although more studies are needed to unmask the full potential as well as limitations of FGF21-class molecules. Long-acting FAP-resistant FGF21 analogs and FGFR1/KLB agonist antibodies with a superior pharmacokinetic profile present an exciting opportunity to expand the therapeutic potential of FGF21-class molecules. In addition, a combination therapy utilizing fibrate and a FAP inhibitor may offer an oral therapy to augment endogenous FGF21 action. Further preclinical and clinical studies should determine whether any of these approaches will be viable to combat the global obesity and type 2 diabetes epidemic.

Acknowledgment

We thank Genentech colleagues for comments on the manuscript. JS, MZC and AB are employed by Genentech, Inc., a wholly owned subsidiary of F. Hoffmann-La Roche AG, and hold stock and options.

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About the article

Received: 2017-01-26

Accepted: 2017-02-13

Published Online: 2017-05-19


Author Statement

Research funding: Authors state no funding involved.

Conflict of interest: Authors state no conflict of interest.

Informed consent: Informed consent is not applicable.

Ethical approval: The conducted research is not related to either human or animal use.


Citation Information: Hormone Molecular Biology and Clinical Investigation, Volume 30, Issue 2, 20170002, ISSN (Online) 1868-1891, ISSN (Print) 1868-1883, DOI: https://doi.org/10.1515/hmbci-2017-0002.

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©2017, Junichiro Sonoda et al., published by De Gruyter, Berlin/Boston. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0

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