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FGF19 Analogue as a Surgical Factor Mimetic That Contributes to Metabolic Effects

Beyond Glucose Homeostasis

Alex M. DePaoli1, Mei Zhou1, Daniel D. Kaplan1, Steven C. Hunt2,3, Ted D. Adams2,4, R. Marc

Learned1, Hui Tian1 and Lei Ling1*

1NGM Biopharmaceuticals, 333 Oyster Point Blvd., South San Francisco, CA, USA

2Division of Cardiovascular Genetics, Department of Internal Medicine, University of Utah

School of Medicine, Salt Lake City, UT, USA

3Department of Genetic Medicine, Weill Cornell Medicine, Doha, Qatar

4Intermountain LiveWell Center, Intermountain Healthcare, Salt Lake City, UT, USA

*Correspondence: Lei Ling, NGM Biopharmaceuticals, Inc., 333 Oyster Point Blvd., South San

Francisco, CA 94080, USA; Telephone: 650-243-5546; Email: [email protected]

Tweet (275 characters including spaces):

“Reprograming of intestinal architecture through bariatric surgery increases circulating levels of

gut hormone FGF19. While its effect on the regulation of glucose homeostasis may be limited in

T2D patients, FGF19 analogue can rapidly reduce liver fat content and improve NASH”

(Fig. 5L)

Running title: FGF19 as a surgical factor mimetic

1

Diabetes Publish Ahead of Print, published online March 12, 2019 Diabetes Page 2 of 70

ABSTRACT

Bariatric surgery has proven to be the most effective treatment for controlling hyperglycemia in severely obese diabetic patients. Here we show that FGF19, a gut hormone, is rapidly induced by bariatric surgery in rodents and humans. Administration of FGF19 achieves diabetes remission independent of weight loss in animal models of diabetes, supporting a role for

FGF19 in the hormonal remodeling that restores metabolic function following the surgery.

Through an unbiased, systematic screen in diabetic mice, we identified selective, safe and effective FGF19 analogues. Unexpectedly, a lead FGF19 analogue, NGM282, did not correct hyperglycemia in patients with type 2 diabetes. In contrast, administration of NGM282 resulted in rapid, robust and sustained reduction in liver fat content and histology in patients with non- alcoholic steatohepatitis, faithfully replicating another key benefit of bariatric surgery. Our work identifies a strategy for replacing the surgery by an equally effective, but less invasive, treatment for non-alcoholic steatohepatitis (This trial is registered with clinicaltrials.gov, number

NCT01943045).

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INTRODUCTION

The growing incidence of obesity and type 2 diabetes mellitus globally is widely

recognized as one of the most challenging contemporary threats to public health (1; 2). Bariatric

surgery, including Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy, is the most

effective treatment for severe obesity and type 2 diabetes (3-5). Interestingly, the marked

improvement in glucose homeostasis occurs early after the RYGB procedure, before any

appreciable weight loss (6). The underlying molecular mechanisms contributing to these benefits

remains an area of active investigation. There remains a significant need for pharmacological

mimetics of these surgeries.

The resolution of type 2 diabetes following gastric bypass attests to the important role of

the gastrointestinal tract in glucose homeostasis. Among the changes to gut physiology observed

following gastric bypass surgery is altered enterohepatic circulation of bile acids, and significant

increases in circulating total bile acids were observed in humans and in rodent models (7-10). In

this report, we show that levels of fibroblast growth factor 19 (FGF19), a gut hormone that has a

crucial role in controlling bile acid, carbohydrate, protein and energy homeostasis (11), are

elevated as early as 7 days post-surgery in humans. Using a large-scale, unbiased, in vivo screen,

we have discovered safe and efficacious FGF19 analogues. One of these FGF19 analogues,

NGM282, has recently demonstrated clinical efficacy in patients with chronic liver diseases,

including non-alcoholic steatohepatitis (NASH) (12), primary sclerosing cholangitis (PSC) (13)

and primary biliary cholangitis (PBC) (14). Furthermore, we present here data from the first

randomized, double-blind, placebo-controlled trial of an FGF19 analogue in patients with type 2

diabetes.

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RESEARCH DESIGN AND METHODS

NGM282 Clinical Trial in Patients with Type 2 Diabetes. In this multicenter, randomized, double-blind, placebo-controlled study in patients with type 2 diabetes inadequately controlled on metformin therapy, NGM282 was administered once daily via subcutaneous injection for 28 consecutive days. The trial protocol was approved by the ethics committees and institutional review boards at each participating site (3 sites in Australia and 6 sites in New Zealand) prior to study initiation. The study was conducted according to the provisions of the Declaration of

Helsinki and in compliance with International Conference on Harmonization Good Clinical

Practice guidelines. All patients provided written informed consent before participation in the trial. The primary endpoint was absolute change in fasting plasma glucose from baseline to day

28. Secondary endpoints included the assessment of HbA1c, fructosamine, glucose tolerance, insulin resistance, -cell function, body weight, safety, and tolerability. Exploratory endpoints included the assessment of serum levels of 7-hydroxy-4-cholesten-3-one (C4). Inclusion criteria were: type 2 diabetes male and female participants with inadequate glycemic control; 18–

70 years of age with a stable body weight and a BMI of 24-40 kg/m2; participants on metformin monotherapy with a fasting plasma glucose between 126-240 mg/dL; participants on metformin combination therapy with a fasting plasma glucose between 126-210 mg/dL. Patients remain on their standard dose/regimen of anti-hyperglycemic medications (metformin alone or metformin plus other anti-diabetic therapies) during the study. Exclusion criteria were: type 1 diabetes; history of bariatric surgery; active acute coronary heart disease; clinically significant clinical laboratory abnormalities at screening; acute electrocardiogram findings or significant variants at screening. A sample size of 16 completed participants per treatment group would provide 80% power to detect a difference in mean glucose changes from baseline of 41 mg/dL assuming an

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SD of 40 mg/dL using a 2-sided significance level of 0.05. To accommodate a dropout rate of up

to 20% from randomization to study completion, a sample size of 20 randomized participants per

treatment group (80 participants in total) was planned.

From November 2013 to July 2014, a total of 81 eligible subjects were randomized in a

1:1:1:1 ratio to receive either NGM282 2 mg (n=21), NGM282 5 mg (n=20), NGM282 10 mg

(n=20) or placebo (n=20). One subject in the NGM282 5 mg dose group was withdrawn from the

study following randomization prior to NGM282 dosing for taking prednisone and was excluded

from the analysis. Participants self-administered the first dose under the supervision of site staff,

and returned to the study site on days 7, 14, 21 and 28 (end of treatment) for assessment.

Participants returned to the study site on day 42 for a follow-up visit. AEs were assessed using

the Common Terminology Criteria for Adverse Events v4.03. This trial is registered with

clinicaltrials.gov (NCT01943045).

An oral glucose tolerance test (GTT) was conducted on day 1 and day 28. Blood samples

were collected at pre-glucose load, and at 30, 60, and 120 minutes post-glucose load. HOMA-IR

and HOMA-bcf were assessed from fasting glucose and insulin levels on day 1 and day 28 pre-

glucose load. HbA1c, fructosamine, and body weight were measured on day 1 and day 28 in all

participants. Serum levels of C4 were only analyzed in the 2 mg and 5 mg groups.

Patients for Gastric Bypass Surgery. From February 2010 through June 2011, 29 patients with

severe obesity from a single bariatric surgical center (Rocky Mountain Associated Physicians,

Salt Lake City) seeking Roux-en-Y gastric bypass surgery were enrolled. All patients provided

written informed consent to participate in the study. At baseline, 7 and 21 days post surgery, a

250-mL standard liquid meal (Isosource, Novartis, 1.5 calories/mL,18% protein, 44%

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carbohydrate and 38% fat) was administered, and blood was drawn just before meal ingestion and 30 and 60 minutes following the meal.

Animal Experiments. All animal studies were approved by the Institutional Animal Care and

Use Committee at NGM. Animals were housed in a pathogen-free animal facility at 22ºC under controlled 12 hour light/12 hour dark cycle. All animals were kept on standard chow diet (Harlan

Laboratories, Teklad 2918) and autoclaved water ad libitum unless otherwise specified. Male mice and rats were used. Animals were randomized into the treatment groups based on body weight and blood glucose. All injections and tests were performed during the light cycle.

C57BL/6J, db/db, TALLYHO, NONNZO and Fxr-deficient mice were purchased from Jackson

Laboratory. Zucker fa/fa rats were purchased from Envigo.

Duodenal-jejunal bypass (DJB) surgery in Zucker fa/fa rats: RYGB causes rapid and dramatic weight loss in rodents to a much greater degree than in humans, which makes interpretation of early gene expression changes difficult. In contrast, DJB, a surgical procedure that preserves the normal transit of nutrients through the stomach, does not result in substantial weight reduction, allowing us to focus on transcriptome change solely derived from the altered intestinal architecture. Therefore, we chose to use DJB instead of RYGB as the bariatric surgery procedure in the rodent studies. Surgical procedures were performed on 20-week old Zucker fa/fa rats (Envigo). DJBs were carried out as previously described (8; 15). In brief, the abdominal cavity was exposed through a ventral midline incision, at 0.75-1 cm of the starting point of duodenum, a suture was tightly tied the duodenum vertically, and a 0.4 cm horizontal incision was performed at this area, and bowel continuity was interrupted at the level of the distal jejunum (10 cm from the ligament of Treitz). The distal limb was directly connected to the horizontal duodenum incision (duodenal-jejunal anastomosis) and the proximal limb carrying the

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biliopancreatic juices was reconnected downward to the alimentary limb at a distance of the

same length of bypassed entire duodenum and proximal jejunum from the duodenal-jejunal

anastomosis. For sham operations, rats were undergo a similar surgical procedure as DJB,

however all resections were re-anastomosed to maintain the physiologic circuit of food through

the bowel. Rats were sacrificed 14 days after surgery and intestines were harvested for RNA

preparation and transcriptome profiling.

db/db mice (BKS.Cg-Dock7m +/+ Leprdb/J, #000642): For the unbiased, in vivo screen in

db/db mice, 10-12 week old db/db mice were injected intravenously with 3 x 1011 vector genome

of AAV carrying either FGF19 mutants or a control gene GFP. During the 24-week study

periods with continuous exposure to FGF19 mutant transgenes, db/db mice were subjected to

glucose measurements at various time points, and euthanized at the end of the studies for liver

tumor quantification.

Fxr-deficient mice (B6.129X1(FVB)-Nr1h4tm1Gonz/J, #007214): Fxr-deficient mice were

fed a high-fat, high-fructose, high-cholesterol diet (HFFCD) (Research Diets #D09100301i) to

induce NASH. Treatment was initiated after 16 weeks of HFFCD feeding by injecting mice with

3x1011 vector genome AAV-FGF19, AAV-NGM282 or a control virus through tail veins.

HFFCD feeding was subsequently maintained for an additional 34 weeks, at which time mice

were euthanized for histological and gene expression analysis.

Statistics. For studies in animals, results are expressed as the mean+standard error of the mean

(SEM). One-way analysis of variance (ANOVA) followed by Dunnett’s post-test was used to

compare data from multiple groups (GraphPad Prism). Two-way ANOVA followed by Sidak’s

post-test was used to compare data when multiple groups and time points are involved

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(GraphPad Prism). When indicated, an unpaired Student’s t-test was used to compare two treatment groups. Circulating FGF19 levels in patients before and after RYGB were evaluated by a paired, two-tailed, t-test. For NGM282 clinical trial in patients with type 2 diabetes, the change from baseline to day 28 was compared between treatment groups using an analysis of covariance

(ANCOVA) model with treatment group as factor and baseline HbA1c and baseline endpoint value as covariates. Difference in least-squares means, 95% confidence intervals (CI) for the difference and corresponding P values were presented. Within each treatment group, changes from baseline to day 28 were evaluated by a paired, two-tailed, t-test. Statistical analyses for clinical trials were carried out using SAS version 9.4 (SAS Institute, Cary, NC).

Additional materials are provided in the Supplemental Information file.

RESULTS

FGF19 is Induced by Bariatric Surgery in Rodents and in Humans

In an effort to identify potential gut-derived factors that could contribute to the beneficial effects of bariatric surgery, we examined gene expression changes in various segments of the intestine before and after surgery using an established rodent model, the duodenal-jejunal bypass

(DJB) surgery in Zucker fa/fa (ZF) rats (8) (Figure 1A). DJB, a surgical procedure that preserves the normal transit of nutrients through the stomach, does not result in substantial weight reduction as seen with RYGB, allowing us to focus on transcriptome change solely derived from the altered intestinal architecture. Among the genes whose expression are differentially regulated by DJB versus sham procedures, FGF15 (the rodent orthologue of human FGF19) was one of the gut factors that showed the greatest increase in expression. Elevated levels of FGF15 mRNA

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were detected as early as 14 days post-surgery,and likely resulted from the accelerated delivery

of bile to the hind gut (Figure 1B and 1C). Induction of Nr1h4 (also known as FXR) expression

was also observed (Figure 1B). Ingenuity pathway analysis of transcriptome profiling data

revealed that several biological pathways, and transcriptional programs associated with

cholesterol metabolism in particular, were enriched in the DJB group when compared to the

sham procedure group (Figure 1D).

To determine whether these observations in animal models are recapitulated in human

patients, we measured FGF19 concentrations in serum samples from obese patients who had

undergone RYGB surgery (Figure 1E). Serum FGF19 concentrations increased significantly

following RYGB in the majority of patients, either under fasting conditions or after a

standardized meal (Figure 1F). The increase in serum FGF19 was observed as early as seven

days after surgery, and persisted 21 days after surgery. Patient characteristics at baseline and

post-surgery are shown in Table S1.

In summary, we observed significantly elevated levels of circulating FGF19 in rodents

and in humans as early as 7 days after bariatric surgery, supporting a role for FGF19 in the

hormonal remodeling that restores metabolic function following the surgery.

Unbiased, Systematic, In vivo Screen for the Identification of Efficacious, Non-tumorigenic

FGF19 Variants in db/db Mice

Despite the demonstration of profound anti-diabetic effects following administration of

FGF19 in a variety of mouse models of diabetes (including the monogenic db/db mice, the

polygenic TALLYHO and NONNZO mice, and the -cell-deficient mice; Figure S1), potential

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safety concerns have hindered its development as a therapeutic for treatment of type 2 diabetes.

Although FGF19 has minimal mitogenic activity on fibroblasts and hepatocytes in vitro, ectopic expression of FGF19 in skeletal muscle of transgenic mice promotes hepatocyte proliferation, hepatocellular dysplasia and neoplasia, and by 10-12 months of age, leads to the development of hepatocellular carcinomas (16). Multiple groups have endeavored to engineer safe and effective variants of FGFs, including FGF19 (17-20). Although the FGF19 mutants described in these reports exerted modest glucose-lowering in diet-induced obese mice, possibly secondary to the observed weight reduction in this model, they failed to improve the severe hyperglycemic condition when tested in db/db mice (Figure S2A-D). Moreover, significant safety concerns on these engineered FGF molecules, previously unrecognized from cell-based assays, were uncovered upon prolonged exposure of mice to these molecules (Figure S2E). Given the limitations of biochemical or cell-based assays for modeling complex, multi-system diseases, such as diabetes and cancer, we employed a systematic, unbiased, in vivo screen to simultaneously assess glucose-lowering and tumorigenicity of FGF19 mutants in db/db mice.

During the course of this effort, we injected recombinant AAV carrying over 150 FGF19 mutant transgenes intravenously into db/db mice for evaluation, monitoring blood glucose, body weight, and liver tumor formation over a 24-week study period. Detailed sequences of select mutants are included in Tables S2-S5 (and data not shown).

To establish a structure-activity relationship (SAR) and dissect FGF19 functional domains, we systematically changed predicted secondary structural elements in the FGF19 protein, including -sheets, loops between -sheets, and -helices (Figure S3A-B). Among these

FGF19 mutants, mutation of the 8-9 loop region resulted in a marked reduction in the number of liver tumors when compared with wild-type FGF19, without compromising the anti-diabetic

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efficacy (Figure 2A-B). This FGF19 variant also did not promote the increase in liver weight

associated with prolonged exposure to wild type FGF19 (Figure 2C). To gain insight into the

molecular basis for the differentiated profile of the FGF19 variant carrying the 8-9 loop

mutation, we superimposed the crystal structures of FGF19 (PDB 2P23) (21) and Klotho (PDB

5VAQ) (22) onto the X-ray structure for the ternary FGF23-Klotho-FGFR1 complex (PDB

5W21) (23) (Figure 2D). Strikingly, the 8-9 loop, a surface-exposed region in FGF19

(indicated as red spheres in Figure 2E), makes direct contact with the highly conserved linker

region between Ig-like domain 2 (D2) and D3 in FGFR1. In this complex, multiple hydrogen

bonds are predicted to form between Gly-130 and Asn-132 residues in the 8-9 loop of FGF19

and Arg-250 in FGFR1 (Figure 2F), an invariant arginine residue conserved between the FGFR1,

FGFR2, FGFR3, and FGFR4 receptors (Figure S3C-D).

With an absence of electron density for the N-terminus of FGF19 in published crystal

structures (PDB 2P23 and 1PWA) (21; 24), this domain of FGF19 remains poorly characterized,

with respect to both structure and function. Moreover, a lack of homology between the N-

terminus of FGF19 and FGF23 limited the utility of modeling this region to the FGF23-Klotho-

FGFR1 ternary structure. As a means towards better understanding the role this region plays in

mediating the biological activities of FGF19, we used a systematic mutational analysis to

precisely dissect this domain and identify the key residues involved in mediating these functions.

As shown in Figures 2G-H, several FGF19 mutants entirely lack the tumorigenic potential of

wild type FGF19, even while maintaining robust glucose-lowering activity in db/db mice.

Relative to wild type FGF19, liver weights were also reduced by these non-tumorigenic mutants

(Figure 2I). Based on these fine mapping analysis, amino acids 30-33 appear to be essential for

both the metabolic and proliferative activities of FGF19. A close inspection of the FGF23-FGFR

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(Figure 2J-K, and Figure S3E-F) and the FGF8-FGFR (Figure 2L and Figure S3G) structures revealed that residues located in similar N-terminal positions make close contacts with the receptor. Therefore, changes in amino acids 30-33 in FGF19 may directly impact interactions with the receptor and influence downstream signaling. Additional in vivo SAR analyses are included in Figures S4 and S5.

In summary, through an extensive SAR analysis, we identified distinct regions in FGF19 that are essential for its metabolic and proliferative features.

A Randomized, Double-Blind, Placebo-Controlled Trial of an FGF19 Analogue, NGM282, in Patients with Type 2 Diabetes

The identification of non-tumorigenic FGF19 analogues with robust anti-diabetic efficacy has ignited renewed interest in exploiting this class of hormones for therapeutic purpose.

However, it has remained unproven whether these results from animal models represent a relevant glucose-lowering mechanism in humans. To functionally validate these findings in humans, we conducted a multicenter, randomized, double-blind, placebo-controlled study of the

FGF19 variant NGM282 [N1-15 in Figure 2G-I, also known as M70 (25)], in patients with type

2 diabetes.

A total of 81 patients were randomly assigned to receive daily doses of either NGM282 2 mg (n=21), NGM282 5 mg (n=20), NGM282 10 mg (n=20), or placebo (n=20) (Figure 3). The patient characteristics of the study population are summarized in Table 1. NGM282 was administered subcutaneously daily for 28 consecutive days. Following randomization but prior to

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NGM282 dosing, one patient in the NGM282 5 mg dose group withdrew from the study and was

excluded from the analysis.

In contrast to results obtained from animal models, administration of NGM282 at any of

the tested dose levels failed to relieve hyperglycemia in patients with type 2 diabetes, as

evidenced by the lack of significant change in plasma glucose or HbA1c levels (Figure 4A-C).

Improvements in insulin resistance based on HOMA-IR were observed in patient cohorts treated

with 5 mg and 10 mg of NGM282, with a significant decrease at day 28 in subjects treated with

NGM282 10 mg (P<0.001 versus baseline; Figure 4D). Levels of fasting insulin and HOMA-bcf,

a surrogate measure of insulin secretion, were decreased from baseline in the NGM282 10 mg

group (Figure 4E-F). Mean body weight and fructosamine levels decreased in patients treated

with NGM282 10 mg versus those administered placebo (Table 2). No significant changes in

oral glucose tolerance tests were observed in any of the cohorts (Table 2).

Of note, serum levels of liver enzymes, and those of alanine aminotransferase (ALT) and

aspartate aminotransferase (AST) in particular, were significantly decreased in all NGM282

treatment groups as early as day 7 and remained lowered at day 28 (Figure 4G). Elevated ALT

(>40 U/L) in patients with type 2 diabetes is highly predictive for non-alcoholic steatohepatitis

(NASH), a progressive form of non-alcoholic fatty liver disease with risk of cirrhosis and liver

failure (26). A dose-dependent decrease in ALT was observed with NGM282 in patients with

presumptive NASH (ALT >40 U/L at baseline) (Figure 4H). No changes in alkaline phosphatase

or gamma-glutamyl transpeptidase were observed in NGM282-treated patients.

Given that FGF19 has been shown to control bile acid metabolism through actions on

CYP7A1 (11), the first and rate-limiting enzyme in the classic pathway of bile acid synthesis, we

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conducted exploratory analysis of serum levels of 7-hydroxy-4-cholesten-3-one (C4), a biomarker of hepatic CYP7A1 activity, in patients treated with the 2 and 5 mg doses of

NGM282. Serum C4 concentrations were suppressed by 70% and 91% in patients treated with

NGM282 2 mg and 5 mg, respectively, indicative of profound target engagement (Figure 4I).

Overall, NGM282 appears to be well-tolerated in the type 2 diabetes patient population.

Whereas 85% (68/80) of patients experienced at least one adverse event (AE), the majority of which were limited to grade 1 events (78%; 62/80). The most commonly (>10%) reported AEs include diarrhea (32%, 26/80), nausea (32%, 26/80), injection site bruising (18% of patients,

14/80), injection site pruritus (16% of patients, 13/80), and headache (15%, 12/80) (Table S6).

These AEs were reported more frequently in the NGM282 groups compared with placebo. Nine participants withdrew from the study early, mostly due to gastrointestinal symptoms: two in the

NGM282 2 mg group, three in the NGM282 5 mg group, three in the NGM282 10 mg group and one in the placebo group. No serious adverse events (SAE), life-threatening events (grade 4), or patient deaths (grade 5) were reported in any treatment group during the course of the study.

In summary, administration of NGM282 for 28 days in patients with type 2 diabetes did not produce a significant glucose-lowering effect. However, the observed reductions in ALT,

AST, HOMA-IR, and marked suppression of C4, are indicative of potent target engagement.

FGF19 and NGM282 Ameliorates Liver Inflammation and Fibrosis Associated with Non-

Alcoholic Steatohepatitis

The lack of glucose-lowering activity of NGM282 in patients with type 2 diabetes suggests that FGF19 signaling may not be responsible for the rapid diabetes remission following

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gastric bypass surgery in humans. However, gastric bypass surgery’s clinical benefits are not

limited to diabetes remission, but include correction of a wide range of metabolic dysfunctions,

including NASH (27-29). The observation that C4, ALT and AST, key parameters relevant to the

pathogenesis of NASH, decrease with NGM282 treatment spurred interest in evaluating the

FGF19 pathway in patients with NASH.

Strikingly, hepatic CYP7A1 expression was significantly elevated in patients with NASH

compared to healthy subjects in multiple microarray datasets (Figure 5A). Of note, lower FGF19

levels, as well as elevated hepatic Klotho and FGFR4 expression were observed in NASH

patients, suggesting that the FGF19- Klotho-FGFR4 pathway may be modulated in a clinically

and physiologically relevant manner in this population (Figure 5B).

We examined the effect of FGF19 and NGM282 on disease progression, and liver

fibrosis in particular, in a mouse model of severe NASH (Figure 5C). Treatment of high-fat,

high-fructose, high-cholesterol diet (HFFCD)-fed, Fxr-deficient mice with FGF19 or NGM282

significantly improved serum levels of ALT and AST, and reduced circulating concentrations of

total bile acids (TBA) (Figure 5D). Hepatic mRNA levels of bile acid synthetic enzymes,

markers of monocyte/macrophage infiltration, markers of stellate cell/myofibroblast activation

and fibrosis, were notably decreased by FGF19 or NGM282 treatment (Figure 5E-H).

Morphometric quantification of Sirius red-stained collagen area revealed evidently less fibrosis

in FGF19- or NGM282-treated animals (Figure 5I-J). Mice administered with FGF19 and

NGM282 had lower circulating insulin, suggestive of improved insulin resistance (Figure 5K).

Overall, FGF19 and NGM282 demonstrate anti-inflammatory and anti-fibrotic activities in a

mouse model of NASH in an Fxr-independent manner.

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On the basis of results described above, as well as studies in HFFCD-fed C57BL6 mice

(30), we conducted a multicenter, randomized, double-blind, placebo-controlled study to assess

effects of NGM282 in patients with biopsy-confirmed NASH. As we reported recently (12), a

12-week treatment with NGM282 produced rapid and significant reductions in liver fat content,

ALT, AST, and biomarkers of fibrosis in patients with NASH. Importantly, treatment with

NGM282 improved NASH-related histology, including NAFLD activity score (NAS) and

fibrosis stage, in 12 weeks in patients with NASH (31).

Thus, NGM282 demonstrated remarkable efficacy in improving the histological and

biochemical features of NASH, mimicking the beneficial effect of bariatric surgery on

alleviating NASH.

DISCUSSION

Initially considered as a surgical procedure to promote nutrient malabsorption, a growing

body of evidence indicates that restriction and malabsorption are not the primary mechanisms

driving the metabolic improvements associated with bariatric surgery. We identify FGF19 as a

gut hormone rapidly (i.e., within 7 days) induced by bariatric surgery and moreover, demonstrate that pharmacological administration of FGF19 or its analogue NGM282 mimics many of the beneficial metabolic effects of this procedure in animal models and in human patients. These findings have important implications for therapies aimed at simulating bariatric surgery in humans.

Although bile acids and FXR signaling are implicated as the molecular underpinnings for the beneficial effects of bariatric surgery (32; 33), and that the elevated FGF19 concentrations

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were observed in patients who underwent the surgery (34-37), most of these studies examined

FGF19 levels in small number of patients or at time points too late to implicate a causative role

of hormonal change in the metabolic effects. Our study is the first report that has convincingly

showed that FGF19 levels are elevated as early as 7 days post-surgery in humans, addressing an

important knowledge gap in understanding the mechanisms for metabolic improvements

observed shortly after bariatric surgery.

Despite the impressive metabolic efficacy demonstrated in preclinical models, as shown

in this report and previously (38; 39), safety and ethical concerns have prevented researchers

from testing FGF19 in the clinic, since mice expressing an FGF19 transgene develop

hepatocellular carcinomas (16). To address these concerns, we used a systematic, unbiased, in

vivo screen to engineer and characterize FGF19 analogues that lack tumorigenic potential and

thereby enable the first human testing of an FGF19-based therapy. As shown in the current

study, FGF19 dramatically improves glycemic control in multiple rodent models of diabetes of

differing etiology, recapitulating the rapid, weight loss-independent impact on glycemic control

following gastric bypass surgery in diabetic human patients. The anti-diabetic effect of FGF19 in

these models is likely mediated by the FGFR1c-Klotho receptor complex through its action on

the nervous system (40). These results are also consistent with previous studies demonstrating

that FGF19 increases metabolic rate and reduces adiposity in mice (38), acts as a postprandial,

insulin-independent activator of hepatic glycogen and protein synthesis (11), regulates hepatic

glucose metabolism by inhibiting the CREB-PGC1 pathway (11), and improves glucose

effectiveness through the hypothalamus-pituitary-adrenal axis (41; 42). Given that FGF19 can

stimulate adiponectin expression and production by adipose tissue (43), and that circulating

adiponectin levels are increased after RYGB (44), it is possible that one of the mechanisms by

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which FGF19 and NGM282 improve NAFLD and NASH is the induction of adiponectin.

Indeed, we have recently reported that selective activation of the FGFR1-βKlotho complex with an agonistic antibody increased serum adiponectin, and high molecular weight form of adiponectin in particular, in obese patients (45).

Contrary to expectations from the field, we report here that the FGF19 analogue

NGM282 lacks the glucose-lowering activity so compellingly demonstrated in animal models when tested in patients with type 2 diabetes. However, NGM282 proved highly effective in

NASH patients, significantly reducing liver fat content (12) and NASH-related histological features (31). The scale of the effect of NGM282, and the ability to elicit robust response in nearly 90% of patients, indicate that FGF19 may be a key driver of improvements in fatty liver and NASH by gastric bypass surgery. Furthermore, NGM282 achieved reductions in liver fat content more rapidly (−60% in liver fat content after 12-week treatment) than RYGB (−73% in liver fat content 1 year after surgery) (28) or FXR agonist OCA (−17% in liver fat content after

72-week treatment) (46). We hypothesize that endogenously mounted FGF19 induction by either

RYGB or FXR agonists require longer time to inhibit NASH progression, but that a pharmacological intervention could achieve greater exposure and faster, more robust effect.

Whereas NASH is considered a manifestation of metabolic syndrome in the liver, whether

NASH is a cause or a consequence of type 2 diabetes has long been the subject of debate. The pronounced anti-NASH activity without associated anti-hyperglycemic activity of NGM282 is consistent with genetic studies suggesting that excess hepatic fat is associated with progressive liver disease, but does not always increase the risk of type 2 diabetes. Perhaps the deficit in ß- cell/islet function is a predominant underlying factor in hyperglycemia which is not corrected by lowering intra-hepatic fat and inflammation. The current trial reminds us the need for a careful

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re-examination of previous conclusions drawn from animal studies regarding a role for FGF19 in

glucose regulation, and that the clinical development of FGF19 analogues should focus instead

on liver diseases.

The discovery of NGM282 through an in vivo SAR screen represents the culmination of

years of careful work that yielded the first FGF19 analogue for testing in humans. Large-scale in

vitro biochemical or cell-based screens with sufficient throughput have been routinely used to

identify new compounds. However, maintaining relevance to complex disease pathophysiology

is challenging. No single in vitro assay can predict outcomes in systemic diseases such as

diabetes and cancer that involve multiple organs. Advances in gene delivery using adeno-

associated virus enabled us to efficiently evaluate a large number of engineered FGF19

molecules for prolonged period of time in mice. The results of our efforts to apply an in vivo

approach as a means of investigating the metabolic and cancer dependencies of FGF19

demonstrate the feasibility of systematically dissecting a complex structure-activity relationship

in great detail, representing new avenues for the development of potential therapeutics. Results

from our in vivo SAR analysis and structural modeling revealed novel mechanistic insights,

consistent with a model that the N-terminus of FGF19 is intrinsically disordered when unbound,

but upon binding to FGFR, makes close contact with the D3 domain of FGFR. Changes in amino

acid sequence in the N-terminal region may result in conformational shifts in the ternary FGF19-

FGFR-Klotho complex that lead to diverse downstream signaling. This may explain why,

unlike wild type FGF19, NGM282 does not activate signal transducer and activator of

transcription 3 (STAT3), a signaling pathway essential for FGF19-mediated

hepatocarcinogenesis. Moreover, we identified a novel region (loop 8-9) in FGF19 that is

crucial for both the gluco-regulatory and tumorigenic activities.

19 Diabetes Page 20 of 70

The underlying molecular mechanism contributing to the benefits of bariatric surgery remains an area of active investigation. Several potential mechanisms have been proposed, including an increased secretion of gut hormone GLP-1 (47). Given the well-established glucose- lowering effect of GLP-1 analogues in humans, GLP-1 could be the surgical factor for diabetes remission. However, GLP-1 analogues have failed to demonstrate a robust reduction in liver fat content to levels that are seen in patients who had undergone gastric bypass (48; 49). In contrast, while its effect on the regulation of glucose hemostasis may be limited in humans, FGF19 analogue can rapidly and markedly reduce liver fat content and improve NASH-related histology. Together, FGF19 and GLP-1 may be the long-sought-after “surgical factors” that contribute to the improvement in fatty liver, NASH and diabetes by gastric bypass surgery

(Figure 5L). Loss-of-function studies, such as those requiring the use of FGF15 (the murine orthologue of FGF19)-deficient mice, are needed but challenging due to species-specific, divergent regulation of metabolism and carcinogenesis by FGF19 and FGF15 (50). Unlike

FGF19, FGF15 does not exert glucose-lowering, HbA1c-normalizing effects in diabetic animals.

A further complication arises from the fact that FGF15-deficient mice are healthy only when bred on a mixed background, and crossing to C57BL6 strain, the gold standard in mouse genetic studies, resulted in progressive loss of homozygous mice. These difficulties pose substantial challenges for the accurate and timely characterization of loss of function in animal models.

In summary, our results demonstrated that the gut hormone FGF19 likely plays a direct role in the metabolic improvements observed after gastric bypass surgery, although its effects on the regulation of glucose homeostasis may be limited in humans. The translation of preclinical findings is an inherently unpredictable and time-consuming process; great care must be exercised before testing new drugs in humans. NGM282 has cleared the rigorous safety testing in multiple

20 Page 21 of 70 Diabetes

preclinical species including the non-human primate models. As clinical data are accumulating, it

is emerging that the regulation of metabolism by the FGF19 pathway may be more complex than

initially assumed. The present work has important clinical implications and advances our

understanding of the biological processes that underlie bariatric surgery. Exploitation of the

FGF19 signaling pathway by informed engineering of selective modulators of the FGF receptors

could represent a pharmacological approach to replace bariatric procedure by equally effective,

but less invasive, treatments.

ACKNOWLEDGEMENTS

We thank all of the patients who participated in the clinical studies, and the investigators, study

coordinators, and staff of all of the participating clinical centers for their support and assistance.

We thank Drs. Jin-Long Chen and Darrin Lindhout for advice and insightful discussions. We

thank Hong Yang, Mark Humphrey, Van Phung, Josh Lichtman, Yi Fritz, Yarong Lu and

Andrew Yan for technical assistance, and NGM vivarium staff for the care of the animals used in

the studies. Study conceptualization: AMD, HT, LL; study design: AMD, MZ, DDK, SCH,

TDA, RML, HT, LL; study investigation and data collection: AMD, MZ, DDK, SCH, TDA, LL;

manuscript writing: AMD, MZ, DDK, SCH, TDA, RML, HT, LL. LL is the guarantor of this

work and, as such, had full access to all the data in the study and takes responsibility for the

integrity of the data and the accuracy of the data analysis. AMP, MZ, DDK, RML, HT, and LL

report being employees and stockholders of NGM Biopharmaceuticals. Other authors declare no

competing interests. This study was funded by NGM Biopharmaceuticals. Data and resource

availability: The datasets generated during and/or analyzed during the current study are available

21 Diabetes Page 22 of 70

in public repositories (http://www.ncbi.nlm.nih.gov/gds for the Gene Expression Omnibus database; https://www.rcsb.org for the PDB database). Graphic artwork was created using Biorender software. The resource generated during the current study is available from the corresponding author on reasonable request under a material transfer agreement.

22 Page 23 of 70 Diabetes

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31. Harrison SA, Rossi SJ, Bashir M, Guy C, Banerjee R, Jaros MJ, Owers S, Baxter B, Ling L, DePaoli AM: NGM282 improves fibrosis and NASH-related histology in 12 weeks in patients with biopsy-confirmed NASH, which is preceded by significant decreases in hepatic steatosis, liver transaminases and fibrosis markers at 6 weeks. Journal of hepatology 2018;68:S65 [SUPPL] 32. Ryan KK, Tremaroli V, Clemmensen C, Kovatcheva-Datchary P, Myronovych A, Karns R, Wilson-Perez HE, Sandoval DA, Kohli R, Backhed F, Seeley RJ: FXR is a molecular target for the effects of vertical sleeve gastrectomy. Nature 2014; 33. Bozadjieva N, Heppner KM, Seeley RJ: Targeting FXR and FGF19 to Treat Metabolic Diseases-Lessons Learned From Bariatric Surgery. Diabetes 2018;67:1720-1728 34. Jansen PL, van Werven J, Aarts E, Berends F, Janssen I, Stoker J, Schaap FG: Alterations of hormonally active fibroblast growth factors after Roux-en-Y gastric bypass surgery. Digestive diseases 2011;29:48-51 35. Pournaras DJ, Glicksman C, Vincent RP, Kuganolipava S, Alaghband-Zadeh J, Mahon D, Bekker JH, Ghatei MA, Bloom SR, Walters JR, Welbourn R, le Roux CW: The role of bile after Roux-en-Y gastric bypass in promoting weight loss and improving glycaemic control. Endocrinology 2012;153:3613-3619 36. Gerhard GS, Styer AM, Wood GC, Roesch SL, Petrick AT, Gabrielsen J, Strodel WE, Still CD, Argyropoulos G: A role for fibroblast growth factor 19 and bile acids in diabetes remission after Roux-en- Y gastric bypass. Diabetes care 2013;36:1859-1864 37. Nemati R, Lu J, Dokpuang D, Booth M, Plank LD, Murphy R: Increased Bile Acids and FGF19 After Sleeve Gastrectomy and Roux-en-Y Gastric Bypass Correlate with Improvement in Type 2 Diabetes in a Randomized Trial. Obesity surgery 2018; 38. Tomlinson E, Fu L, John L, Hultgren B, Huang X, Renz M, Stephan JP, Tsai SP, Powell-Braxton L, French D, Stewart TA: Transgenic mice expressing human fibroblast growth factor-19 display increased metabolic rate and decreased adiposity. Endocrinology 2002;143:1741-1747 39. Fu L, John LM, Adams SH, Yu XX, Tomlinson E, Renz M, Williams PM, Soriano R, Corpuz R, Moffat B, Vandlen R, Simmons L, Foster J, Stephan JP, Tsai SP, Stewart TA: Fibroblast growth factor 19 increases metabolic rate and reverses dietary and leptin-deficient diabetes. Endocrinology 2004;145:2594-2603 40. Lan T, Morgan DA, Rahmouni K, Sonoda J, Fu X, Burgess SC, Holland WL, Kliewer SA, Mangelsdorf DJ: FGF19, FGF21, and an FGFR1/beta-Klotho-Activating Antibody Act on the Nervous System to Regulate Body Weight and Glycemia. Cell metabolism 2017;26:709-718 e703 41. Morton GJ, Matsen ME, Bracy DP, Meek TH, Nguyen HT, Stefanovski D, Bergman RN, Wasserman DH, Schwartz MW: FGF19 action in the brain induces insulin-independent glucose lowering. The Journal of clinical investigation 2013;123:4799-4808 42. Perry RJ, Lee S, Ma L, Zhang D, Schlessinger J, Shulman GI: FGF1 and FGF19 reverse diabetes by suppression of the hypothalamic-pituitary-adrenal axis. Nature communications 2015;6:6980 43. You M, Zhou Z, Daniels M, Jogasuria A: Endocrine Adiponectin-FGF15/19 Axis in Ethanol-Induced Inflammation and Alcoholic Liver Injury. Gene Expr 2018;18:103-113 44. Swarbrick MM, Stanhope KL, Austrheim-Smith IT, Van Loan MD, Ali MR, Wolfe BM, Havel PJ: Longitudinal changes in pancreatic and adipocyte hormones following Roux-en-Y gastric bypass surgery. Diabetologia 2008;51:1901-1911 45. DePaoli AM, Phung V, Yan AZ, Ling L, Baxter BA, Tian H: NGM313, a novel activator of bklotho/FGFR1c: Single dose PK, safety and tolerability, coupled with an increase in adiponectin (a marker of target engagement and insulin sensitivity) support potential for treatment of NASH. NASH- TAG 2019;Jan:3-5 46. Neuschwander-Tetri BA, Loomba R, Sanyal AJ, Lavine JE, Van Natta ML, Abdelmalek MF, Chalasani N, Dasarathy S, Diehl AM, Hameed B, Kowdley KV, McCullough A, Terrault N, Clark JM, Tonascia J, Brunt EM, Kleiner DE, Doo E, Network NCR: Farnesoid X nuclear receptor ligand obeticholic acid for non- cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebo-controlled trial. Lancet 2015;385:956-965

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47. Thaler JP, Cummings DE: Minireview: Hormonal and metabolic mechanisms of diabetes remission after gastrointestinal surgery. Endocrinology 2009;150:2518-2525 48. Smits MM, Tonneijck L, Muskiet MH, Kramer MH, Pouwels PJ, Pieters-van den Bos IC, Hoekstra T, Diamant M, van Raalte DH, Cahen DL: Twelve week liraglutide or sitagliptin does not affect hepatic fat in type 2 diabetes: a randomised placebo-controlled trial. Diabetologia 2016;59:2588-2593 49. Tang A, Rabasa-Lhoret R, Castel H, Wartelle-Bladou C, Gilbert G, Massicotte-Tisluck K, Chartrand G, Olivie D, Julien AS, de Guise J, Soulez G, Chiasson JL: Effects of Insulin Glargine and Liraglutide Therapy on Liver Fat as Measured by Magnetic Resonance in Patients With Type 2 Diabetes: A Randomized Trial. Diabetes care 2015;38:1339-1346 50. Zhou M, Luo J, Chen M, Yang H, Learned RM, DePaoli AM, Tian H, Ling L: Mouse species-specific control of hepatocarcinogenesis and metabolism by FGF19/FGF15. Journal of hepatology 2017;

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Table 1. Baseline Characteristics of Patients with Type 2 Diabetes in a Randomized,

Double-Blind, Placebo-Controlled Trial of NGM282

Placebo NGM282 2 mg NGM282 5 mg NGM282 10 mg

(n=20) (n=21) (n=20) (n=20) Age (years) 57.3±9.4 59.5±6.9 55.9±9.0 61.1±8.4 Male, n (%) 15 (75%) 15 (71%) 12 (60%) 16 (80%) Female, n (%) 5 (25%) 6 (29%) 8 (40%) 4 (20%) Race, n (%) Asian 0 1 (5%) 1 (5%) 0 Black 0 0 0 0 White 14 (70%) 17 (81%) 17 (85%) 15 (75%) Pacific Islander 0 0 0 1 (5%) Other 6 (30%) 3 (14%) 2 (10%) 4 (20%) Metabolic factors Glucose (mg/dL) 169.2±48.6 163.8±27.0 158.4±23.4 153.0±34.2 HbA1c (% [mmol/mol]) 7.7±0.7 7.5±0.7 7.2±0.4 7.2±0.6 (83.9±7.6) (81.8±7.6) (78.5±4.4) (78.5±6.5) Fructosamine (mol/L) 288.4±47.8 277.2±40.0 266.8±35.9 269.7±32.8 GTT glucose AUC 16.0±3.2 15.9±2.4 15.6±2.2 15.5±2.3 GTT insulin AUC 39.5±30.7 37.9±6.2 53.9±29.3 50.2±30.5 Insulin (IU/mL) 13.9±8.2 15.4±6.0 18.9±7.5 17.0±9.2 HOMA-IR 4.8±3.1 6.2±2.5 7.5±3.9 6.8±5.0 HOMA-bcf 58.1±58.0 56.5±26.5 67.4±26.4 75.4±67.4 Weight (kg) 97.6±18.6 95.3±15.5 95.5±14.7 95.7±16.0 BMI (kg/m2) 32.4±4.8 32.0±4.5 32.2±3.5 32.1±4.6 Liver enzymes ALP (U/L) 75.0±34.3 65.3±23.4 65.7±16.6 77.9±30.6 ALT (U/L) 33.9±16.7 27.6±10.6 36.7±25.9 34.4±19.8 AST (U/L) 25.1±11.4 21.5±7.6 27.4±14.9 26.4±11.5 GGT (U/L) 60.6±104.9 32.5±17.3 35.4±13.9 53.0±36.6 Shown are mean±SD or n (%). ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUC, area under curve; BMI, body mass index; GGT, gamma- glutamyl transpeptidase; GTT, glucose tolerance test; HbA1C, glycated hemoglobin; HOMA-

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bcf, homeostasis model assessment–estimated -cell function; HOMA-IR, homeostasis model assessment–estimated insulin resistance; SD, standard deviation

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Table 2. Change in Key Outcome Measures from Baseline to Day 28 in a Randomized,

Double-Blind, Placebo-Controlled Trial of NGM282 in Patients with Type 2 Diabetes

Difference in LS Means (95% CI) (NGM282 vs. Placebo) NGM282 2 mg NGM282 5 mg P NGM282 10 P P (n=21) (n=19) mg (n=20) Key Outcome Measures Glucose (mg/dL) −7.2 (−19.8, 9.0) 0.94 −7.2 (−19.8, 0 (−12.6, 12.6) 1.00 0.94 9.0) HbA1c (%) 0.25 (0, 0.5) 0.19 0.3 (0, 0.5) 0.19 0 (−0.3, 0.4) 0.76 HbA1c 2.7 (0, 5.5) 0.19 3.3 (0, 5.5) 0.19 0 (−3.3, 4.4) 0.76 (mmol/mol) Fructosamine 2.6 (−14.3, −1.7 (−19.3, 0.99 −17.7 (−35.0, 0.97 0.043 (mol/L) 19.4) 15.9) −0.5) GTT glucose 1.0 (−1.0, 3.1) 0.46 0.8 (−1.1, 2.6) 0.66 0.5 (−1.5, 2.5) 0.89 AUC GTT insulin −6.0 (−12.2, −8.3 (−16.7, 0.06 −7.4 (−21.0, 0.22 0.22 AUC 0.8) −2.8) 0.8) Insulin −1.0 (−6.9, 4.8) 0.95 −1.5 (−6.7, 3.6) 0.80 −3.7 (−9.5, 2.0) 0.28 (IU/mL) HOMA-IR 0.9 (−1.3, 3.1) 0.60 0.9 (−1.5, 3.4) 0.66 −0.9 (−3.2, 1.4) 0.64 HOMA-bcf −4.5 (−16.0, −1.5 (−19.0, 1.00 −13.5 (−27.0, 1.00 0.30 11.0) 17.0) 6.0) Weight (kg) −0.7 (−2.0, 0.6) 0.46 −1.5 (−2.8, −0.6 (−1.9, 0.6) 0.50 0.019 −0.2) AUC, area under curve; BMI, body mass index; CI, confidence interval; GTT, glucose tolerance test; HbA1c, glycated hemoglobin; HOMA-bcf, homeostasis model assessment– estimated -cell function; HOMA-IR, homeostasis model assessment–estimated insulin resistance; LS, least-squares

SAS (version 9.4) was used for all analyses. The difference between NGM282 and placebo groups was analyzed using analysis of covariance (ANCOVA) model with treatment group as the factor and baseline values of the outcome as cofactor. All statistical analyses were carried out using two-sided tests at the 5% level of significance. Difference in LS means, 95% CI for the difference and corresponding P values were presented.

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FIGURE LEGENDS

Figure 1. Levels of FGF15/FGF19 are Elevated Following Gastric Bypass Surgery in

Rodents and in Humans.

(A) Schematic drawing of gastric bypass surgery in Zucker fa/fa rats. The duodenal-jejunal bypass (DJB) procedure in rats resembles the Roux-en-Y gastric bypass (RYGB) procedure performed in humans. We hypothesize that, in both DJB and RYGB, the bile, unmixed with food, is directly delivered to the distal jejunum and ileum, where it induces FGF15/FGF19 expression in an FXR-dependent manner. Segments representative of the intestinal anatomy were collected in bypass and sham-operated animals.

(B) Top genes differentially induced by DJB surgery compared with sham procedure in Zucker fa/fa rats. Rats were sacrificed 14 days after surgery and ilea were harvested for transcriptome profiling. FGF15 and Nr1h4 (also known as FXR) are in bold.

(C) qPCR analysis showing that mRNA levels of FGF15 were increased after DJB surgery in the ileum. Duodenum (in 2 segments), jejunum (in 3 segments), ileum, colon and liver were harvested from Zucker fa/fa rats 14 days after sham or DJB procedures (n=3 biological replicates per group).

(D) Ingenuity pathway analysis of transcriptome profiling in Zucker fa/fa rats (DJB surgery versus sham procedure). Top regulated canonical pathways are ranked by –Log (P value) with threshold P value of 0.05. Highest ranking categories are displayed along the x-axis in a decreasing order of significance. Orange bars, pathways with Z-score > 0 (upregulated pathways); blue bars, pathways with Z-score < 0 (downregulated pathways); grey bars, no activity pattern available.

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(E) RYGB surgery in human patients. Serum samples were collected before surgery (baseline)

and at 7 and 21 days post surgery.

(F) Serum concentrations of FGF19 were increased following RYGB surgery in humans (n=29).

Fasting, as well as fed (30 or 60 minutes after meal) FGF19 concentrations were determined.

Values are mean ± SEM. ***P < 0.001 by paired, two-tailed t-test.

Figure 2. A Systematic, Unbiased, in vivo Screen of FGF19 mutants in diabetic db/db mice.

(A) Glucose and body weight results from in vivo screen of FGF19 mutants with systematic

changes in loop regions connecting -sheets. 10-12 week old db/db mice were injected

intravenously with 3 x 1011 vector genome AAV carrying FGF19, FGF19 mutants, or a control

gene GFP. Glucose and body weight were measured before AAV injection (week 0), and at

weeks 1, 4, and 24 post AAV injection. n=5-6 mice per group.

(B) Liver tumors in db/db mice expressing FGF19 mutants in loop regions. db/db mice were

euthanized at the end of the study (24 weeks after AAV injection) for liver tumor quantification.

n=5-6 mice per group.

(C) Liver weight of db/db mice expressing FGF19 mutants. n=5-6 mice per group.

(D) Molecular modeling of the FGF19-FGFR1-Klotho 2:2:2 ternary complex. Klotho is in

green, FGF19 in red, FGFR in blue. Ribbon structure of the same complex is shown in the lower

panel.

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(E) The 8-9 loop (red spheres) in FGF19 contacts the linker region in FGFR. FGFR1 surface

representation is in blue, with the Ig-like domain 2 (D2) and 3 (D3) indicated; FGF19 (red) and

Klotho (green) are shown in ribbons.

(F) Detailed view of loop 8-9 interaction with FGFR. Hydrogen bonds between the invariant

Arg-250 in FGFR and Gly-130 and Asn-132 in FGF19 are indicated by dashed lines. Loop 8-

9 is shown as yellow ribbon.

(G) Glucose and body weight results from in vivo screen of FGF19 mutants with deletions or

mutations in the N-terminal region. 10-12 week old db/db mice were injected intravenously with

3 x 1011 vector genome AAV carrying FGF19, FGF19 mutants, or a control gene GFP. Glucose

and body weight were measured before AAV injection (week 0), and at weeks 4 and 24 post

AAV injection. n=5-6 mice per group.

(H) Liver tumors in db/db mice expressing FGF19 mutants with deletions or mutations in the N-

terminal region. db/db mice were euthanized at the end of the study (24 weeks after AAV

injection) for liver tumor quantification. n=5-6 mice per group.

(I) Liver weight of db/db mice expressing FGF19 mutants with deletions or mutations in the N-

terminal region. n=5-6 mice per group.

(J) Alignment of amino acid sequences in the N-terminal regions. Note that the N-terminus of

FGF19 shares no homology with FGF23, FGF8 or FGF2. The first -sheet is shaded in blue.

(K) The N-terminus of FGF23 (red spheres) in the FGF23-FGFR1-Klotho ternary complex (PDB

5W21). Note that the N-terminus of FGF23 directly interacts with the D3 domain of FGFR.

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(L) The N-terminus of FGF8 (red spheres) in the FGF8-FGFR2 receptor complex (PDB 2FDB).

Note that the N-terminus of FGF8 directly interacts with the D3 domain of FGFR.

Values are mean ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05. For glucose levels (panels A, G),

we used two-way ANOVA with Sidak’s multiple comparison tests versus GFP; for liver tumors

(panels B, H), we used one-way ANOVA with Dunnett’s multiple comparison tests versus

FGF19.

Figure 3. CONSORT Flow Chart.

In this multicenter, randomized, double-blind, placebo-controlled study in patients with type 2

diabetes, 81 patients were randomized to receive either the FGF19 analogue NGM282 at doses

of 2 mg (n=21), 5 mg (n=20), 10 mg (n=20) or placebo (n=20). One subject in the NGM282 5

mg dose group was withdrawn from the study following randomization prior to NGM282 dosing

for taking prednisone and was excluded from the analysis.

Figure 4. A Randomized, Double-Blind, Placebo-Controlled Trial of the FGF19 Analogue

NGM282 in Patients with Type 2 Diabetes.

(A) Trial design. NGM282 was administered daily for 28 consecutive days.

(B) Fasting blood glucose over time.

(C) Glycated hemoglobin (HbA1c) levels at baseline (pre-dose on day 1) and day 28.

33 Diabetes Page 34 of 70

(D) The index for homeostasis model assessment of insulin resistance (HOMA-IR) at baseline and day 28.

(E) Fasting insulin concentrations at baseline and day 28.

(F) The index for homeostasis model assessment of -cell function (HOMA-bcf) at baseline and day 28.

(G) Levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) over time.

(H) Change in ALT from baseline to day 28 in patients with presumptive non-alcoholic steatohepatitis (ALT >40 U/L at baseline).

(I) Serum concentrations of 7-hydroxy-4-cholesten-3-one (C4) at baseline and day 28 in patients treated with NGM282 2 mg or 5 mg.

Values are mean ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05 by paired, two-tailed, t-test.

Figure 5. FGF19 and NGM282 Ameliorates Liver Inflammation and Fibrosis Associated with Non-Alcoholic Steatohepatitis.

(A) Hepatic CYP7A1 mRNA levels are elevated in patients with non-alcoholic steatohepatitis

(NASH) compared with normal subjects. Results from three independent cohorts (GEO datasets

GSE89632, GSE48452, GSE61260) are shown.

(B) Expression of FGF19 is lower, while expression of receptors FGFR4 and Klotho is higher, in patients with NASH compared with normal subjects. Shown are data from GSE89632.

34 Page 35 of 70 Diabetes

(C) Study design. Fxr-deficient mice were fed a high-fat, high-fructose, high-cholesterol diet

(HFFCD) to induce NASH. Treatment was initiated after 16 weeks of HFFCD feeding, by

injecting mice with AAV-FGF19, AAV-NGM282 or a control virus AAV-GFP through tail

veins. HFFCD feeding was continued for an additional 34 weeks when mice were euthanized for

gene expression and histology analysis.

(D) FGF19 and NGM282 reduce serum concentrations of alanine aminotransferase (ALT),

aspartate aminotransferase (AST), and total bile acids (TBA). n=5 mice per group.

(E) FGF19 and NGM282 suppress hepatic expression of Cyp7a1 and Cyp8b1 by qPCR analysis.

n=10 biologically independent samples per group.

(F) FGF19 and NGM282 inhibit expression of pro-inflammatory genes. n=10 biologically

independent samples per group.

(G) FGF19 and NGM282 inhibit expression of markers of stellate cell activation. n=10

biologically independent samples per group.

(H) FGF19 and NGM282 reduce expression of fibrosis-related genes. n=10 biologically

independent samples per group.

(I) Representative images of livers stained with Sirius Red. Sirius Red stains collagen in red,

indicative of liver fibrosis. Morphometric quantifications of Sirius Red-positive (SR+) area as

percentage of total liver area were also included. Scale bars, 100 m.

(J) Fibrosis scores using Kleiner criteria. n=5 mice per group.

(K) Serum concentrations of insulin. n=5 mice per group.

35 Diabetes Page 36 of 70

(L) Cartoon. Reprograming of intestinal architecture through gastric bypass surgery increases circulating levels of two gut hormones, FGF19 and GLP-1. GLP-1 analogues, however, have previously failed to demonstrate a robust reduction in liver fat content to levels that are seen in patients who had undergone gastric bypass, despite its well-established role in glycemic control.

FGF19 analogue, while its effects on the regulation of glucose hemostasis may be limited in humans, can rapidly and markedly reduce liver fat content and improve NASH-related histology.

Together, FGF19 and GLP-1 may be the long-sought-after “surgical factors” that contribute to the improvement in fatty liver, NASH and diabetes by gastric bypass surgery.

Values are mean ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05 versus GFP by one-way ANOVA with Dunnett’s multiple comparison tests.

36 Page 37 of 70 Diabetes

Figure 1. Levels of FGF15/FGF19 are Elevated Following Gastric Bypass Surgery in Rodents and in Humans. Diabetes Page 38 of 70

Figure 2. A Systematic, Unbiased, in vivo Screen of FGF19 mutants in diabetic db/db mice. Page 39 of 70 Diabetes

Figure 3. CONSORT Flow Chart. Diabetes Page 40 of 70

Figure 4. A Randomized, Double-Blind, Placebo-Controlled Trial of the FGF19 Analogue NGM282 in Patients with Type 2 Diabetes. Page 41 of 70 Diabetes

Figure 5. FGF19 and NGM282 Ameliorates Liver Inflammation and Fibrosis Associated with Non-Alcoholic Steatohepatitis. Diabetes Page 42 of 70

SUPPLEMENTAL INFORMATION

FGF19 Analogue as a Surgical Factor Mimetic That Contributes to Metabolic Effects

Beyond Glucose Homeostasis

Alex M. DePaoli, Mei Zhou, Daniel D. Kaplan, Steven C. Hunt, Ted D. Adams, R. Marc

Learned, Hui Tian, and Lei Ling*

*Correspondence: Lei Ling, NGM Biopharmaceuticals, Inc., 333 Oyster Point Blvd., South San

Francisco, CA 94080; Telephone: 650-243-5546; Email: [email protected]

1 Page 43 of 70 Diabetes

TABLE OF CONTENT

TABLE OF CONTENT...... 2 Supplemental Materials ...... 3 Supplemental Figures ...... 10 Supplementary Figure S1. FGF19 Induces Marked, Rapid and Sustained Diabetes Remission in Diabetic Mice...... 11 Supplementary Figure S2. Long-Term Evaluation of Previously Reported FGF Mutants in db/db Mice...... 14 Supplementary Figure S3. In vivo Structure-Activity Relationship Analysis of FGF19...... 16 Supplementary Figure S4. In vivo Structure-Activity Relationship Analysis of FGF19 in the C- terminal Region...... 18 Supplementary Figure S5. In vivo Structure-Activity Relationship Analysis of FGF19 in the Heparin-Binding Region...... 20 Supplemental Tables...... 21 Supplementary Table S1. Gastric Bypass Subject Characteristics at Baseline, and at Day 7 and Day 21 Post Surgery (N=29)...... 21 Supplementary Table S2. Sequence of FGF19 Mutants in the Loop Regions...... 22 Supplementary Table S3. Sequence of FGF19 Mutants in the -Sheet Regions ...... 24 Supplementary Table S4. Sequence of FGF19 Mutants in the N-terminal Region...... 26 Supplementary Table S5. Sequence of FGF19 Mutants in the C-terminal Region...... 28 Supplementary Table S6. Treatment Emergent Adverse Events in a Double-Blind, Placebo- Controlled trial of NGM282 in Patients with Type 2 Diabetes ...... 29

2 Diabetes Page 44 of 70

Supplemental Materials

Patients for Gastric Bypass Surgery. Participants were 25 to 60 years of age, with a body mass index (BMI) of 35-60 kg/m2, had no history of myocardial infarction, coronary bypass surgery, stroke, alcohol or narcotics abuse, had not undergone bariatric surgery, and had not had gastric or duodenal ulcers, a myocardial infarction (in the previous 6 months), or active cancer (in the previous 5 years). The study was reviewed and approved by the institutional review board at the

University of Utah in accordance with national guidelines and the provisions of the Helsinki

Declaration. All patients provided written informed consent to participate in the study. Patients were evaluated by a multidisciplinary team (including a diabetologist, a dietitian, and a nurse), and serum samples were collected before surgery (baseline) and at 7 and 21 days post surgery.

Animal Experiments. All animal studies were approved by the Institutional Animal Care and

Use Committee at NGM.

db/db mice (BKS.Cg-Dock7m +/+ Leprdb/J, #000642): Beginning at an age of 7 weeks,

most db/db mice developed severe hyperglycemia (non-fasting blood glucose > 400 mg/dL)

accompanied by polyuria, polydipsia, polyphagia. In Figure S1A-D, 11-week old diabetic db/db mice received intravenously a total of 1 x 1010 genome copies of AAV carrying either FGF19 or a control gene GFP. For the unbiased, in vivo screen in db/db mice, we designed over 150 FGF19

mutants covering a diverse range of secondary structures (-sheets, loops, -helices), and serial

deletions at the N- and C-terminus (Table S2-S5, and data not shown). 5 to 6 db/db mice were

grouped as biological replicates for each FGF19 mutant, providing the coverage necessary to

obtain reproducible results, and enabling successful in vivo characterization of metabolic and

carcinogenic phenotypes. 10-12 week old db/db mice were injected intravenously with 3 x 1011

3 Page 45 of 70 Diabetes

vector genome of AAV carrying either FGF19 or a control gene GFP. During the 24-week study

periods with continuous exposure to FGF19 mutant transgenes, db/db mice were subjected to

glucose measurements at various time points, and euthanized at the end of the studies for liver

tumor quantification.

TALLYHO mice (TALLYHO/JngJ, #005314): The TALLYHO mouse is an inbred

polygenic model for type 2 diabetes showing characteristic signs of hyperglycemia,

hyperinsulinemia, hyperleptinemia, but only moderate obesity. 5 week-old TALLYHO mice

received a single intravenous injection of 3 x 1010 vector genome AAV-FGF19 or AAV-GFP via

tail vein. Blood glucose and body weight were measured prior to AAV administration, and at

week 3 and 12 after AAV administration. Mice were euthanized at the end of the study for

HbA1c measurements.

NONNZO mice (NONcNZO10/LtJ, #004456): The polygenic NONNZO mouse strain

typically exhibits hyperglycemia, insulin resistance and obesity. 6 month-old NONNZO mice

received a single intravenous injection of 3 x 1010 vector genome AAV-FGF19 or AAV-GFP via

tail vein. Mice were placed on LabDiet 5K20 diet (Jackson Laboratory) throughout the study.

Diets containing metformin (0.2% 1,1-dimethylbiguanide in diet) or pioglitazone (0.05% in diet)

were included as positive controls. Blood glucose, body weight, body composition, and HOMA-

IR were determined 6 weeks after treatment initiation. For body composition measurements, un-

anesthetized mice were placed in “live” probe, and scanned for one minute inside the EcoMRI-

5000 whole body composition analyzer. Fat mass was calculated using EcoMRI software from

multiple primary accumulation numbers.

STZ mice: C57BL6J mice treated with streptozotocin (STZ) is a non-obese, insulin-

deficient mouse model. C57BL/6J (#000664) mice were i.p. injected daily with 50 mg/kg

4 Diabetes Page 46 of 70

streptozotocin (Sigma) for 5 days. Blood glucose was monitored every 3 days after the STZ treatment. STZ mice developed severe diabetes with cachexia, and mice with stable, overt diabetes (glucose >400 mg/dL) received a single intravenous injection of 3 x 1010 vector genome

AAV-FGF19 or AAV-GFP via tail vein. Blood glucose and body weight were measured prior to

AAV administration, and at week 4 and 12 after AAV administration. Mice were euthanized at the end of the study for HbA1c, insulin, glucagon measurements.

Transcriptome Profiling, Pathway Analysis and Databases. RNAs were isolated from ileum of Zucker fa/fa rats 14 days after DJB or sham surgery, and were treated with DNase I (Thermo

Fisher Scientific). RNA integrity and purity were confirmed by Bioanalyzer (Agilent

Technologies) with RIN numbers > 9.0. The raw expression data from Affymetrix Rat Gene 1.0

ST whole-transcript arrays were normalized using robust multi-array average (RMA) approach.

The metadata and matrix tables have been deposited to the Gene Expression Omnibus (GEO) repository (accession number GSE120530). Ingenuity Pathway Analysis (Qiagen), including canonical pathways, upstream analysis, diseases and functions, was conducted on genes differentially represented in DJB versus sham procedures. Top canonical pathways were ranked by –Log (P value) with threshold P value of 0.05. Highest ranking categories were sorted in a decreasing order of significance.

Microarray datasets (GSE89632, GSE48452 and GSE61260) on patients with non- alcoholic steatohepatitis and normal subjects were extracted from OmicSoft DiseaseLand database (Qiagen), which contains datasets retrieved from a variety of public projects including

GEO, SRA (Sequence Read Archive), ArrayExpress, and dbGAP (The Database of Genotypes and Phenotypes). Comparison in gene expression in disease versus normal was conducted using

ArrayStudio software version 10.0 from OmicSoft (Qiagen).

5 Page 47 of 70 Diabetes

Blood parameters. Blood glucose was measured in conscious animals from a hand-held

glucometer (Accu-check, Roche Diagnostics) on tail nip blood. Levels of HbA1c were

determined on COBAS INTEGRA 400 Plus Clinical Analyzer (Roche Diagnostics) using whole

blood. Serum samples were prepared by centrifugation at 4ºC for 10 minutes at 2000g after

clotting at room temperature for 30 minutes, for measurements of FGF19 concentrations by

enzyme-linked immunosorbent assays (ELISA) (Biovendor, RD191107200R). ). Plasma insulin

and glucagon were measured with ELISA kits from ALPCO. All assays were performed

according to the manufacturers’ instructions.

Histology. Tissues were fixed in 10% neutral-buffered formalin and embedded in paraffin. 5 m

sections were deparaffinized in xylenes (5 minutes), rehydrated sequentially in graded ethanol

(100%, 95%, 80%, 70%, 50%, 2 minutes each) and PBS (2 minutes). Stains with hematoxylin

and eosin, periodic acid-Schiff or Sirius Red were performed using standard process. For

osmium tetroxide staining of lipids in the liver, frozen livers were embedded in optimal cutting

temperature compound and directly processed for staining. For immunohistochemistry,

specimens were subjected to antigen retrieval in a citrate-based Antigen Unmasking Solution

(Vector Laboratories, #H-3300), and incubated for 30 minutes with 3% H2O2 at room

temperature to block endogenous peroxidase activity. Sections were blocked in PBST (PBS +

0.1% Tween-20) containing 10% goat serum, stained with primary antibodies against insulin

(Sigma) or glucagon (Sigma) diluted in blocking solution at 4ºC overnight. Specimens were then

washed three times for 5 minutes each in PBST and incubated with biotinylated secondary

antibodies in blocking solution for 1 hour at room temperature. Biotinylated secondary antibody,

ABC-HRP reagent and DAB colorimetric peroxidase substrate (Vector Laboratories) were used

6 Diabetes Page 48 of 70

for detection. Digital imaging microscopy was performed using a Leica DM4000 microscope equipped with DFC500 camera (Leica).

DNA constructs. Human FGF19 cDNA (NM005117) was sub-cloned into pAAV-EF1 vector using SpeI and NotI sites with primers 5’-

CCGACTAGTCACCATGCGGAGCGGGTGTGTGG -3’ (sense) and 5’-

ATAAGAATGCGGCCGCTTACTTCTCAAAGCTGGGACTCCTC-3’ (antisense). FGF19 mutants were generated by Quick-Change site-directed mutagenesis kit (Agilent Technologies). cDNAs for previously reported FGF19 mutants, FGF2 mutants and chimera were chemically synthesized (DNA2.1).

AAV Production. AAV293 cells (Agilent Technologies) were cultured in Dulbeco’s

Modification of Eagle’s Medium (DMEM; Mediatech) supplemented with 10% fetal bovine serum (FBS) and 1x antibiotic antimycotic solution (Mediatech). Cells were cultured in a humidified incubator with 5% CO2 and 95% air at 37ºC, confirmed to be mycoplasma free, and authenticated by short tandem repeat DNA profiling. The cells were transfected with 3 plasmids

(AAV transgene, pHelper (Agilent Technologies) and AAV2/9) for viral production. Viral particles were purified using a discontinued iodixanal (Sigma) gradient and re suspended in phosphate buffered saline (PBS) with 10% glycerol and stored at –80ºC. Viral titer or vector genome number was determined by quantitative PCR using custom Taqman assays specific for

Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequences. Standard curves for WPRE were obtained from serial dilutions over a 6 log range of the corresponding plasmids. AAV-mediated gene delivery provides a means to achieve long-lasting transgene expression without the inflammatory responses that are commonly associated with other viral

7 Page 49 of 70 Diabetes

vectors. When introduced into adult mice, sustained expression of up to one year has been

observed. The major site of transgene expression is the hepatocytes.

Quantitative PCR with Reverse Transcription (qPCR). Tissues were snap-frozen in liquid

nitrogen upon euthanization of animals. Total RNA was extracted using RNeasy Mini kit

(Qiagen) and treated with DNase I (Thermo Fisher Scientific). qPCR assays were performed

using QuantiTect multiplex qRT-PCR master mix (Qiagen) and premade Taqman gene

expression assays (Life Technologies). Samples were loaded into an optical 384-well plate and

qPCR were performed in duplicates on QuantStudio 7 Flex Real-Time PCR System (Applied

Biosystems). After an initial hold at 50ºC for 30 minutes to allow reverse transcription to

complete, HotStart Taq DNA polymerase was activated at 95ºC for 15 minutes. Forty cycles of a

three-step PCR (94ºC for 45 seconds, 56ºC for 45 seconds, and 76ºC for 45 seconds) were

applied and the fluorescence intensity was measured at each change of temperature to monitor

amplification. Target gene expression was determined using the comparative threshold cycle

(Ct) approach and normalized to the expression of housekeeping gene glyceraldehyde 3-

phosphate dehydrogenase (GAPDH).

Structural Analysis. The published crystal structures of FGF23-FGFR1-Klotho (PDB code

5W21), FGF19 (PDB code 2P23), and Klotho (PDB code 5VAQ) were used to build a model of

a ternary complex of FGF19-FGFR1-Klotho using Molecular Operating Environment (MOE)

program (Chemical Computing Group). The FGF19-FGFR1-Klotho 2:2:2 complex was

examined for stereo-chemical quality and further optimized via multiple step energy

minimizations. Interactions between specific residues in FGF23-FGFR1-Klotho (PDB code

5W21), FGF8-FGFR2 (PDB 2FDB), FGF2-FGFR1-heparin (PDB 1FQ9) were also examined

8 Diabetes Page 50 of 70

using MOE program. AMBER10:EHT force-field with reaction field implicit solvation was used during analysis.

9 Page 51 of 70 Diabetes

Supplemental Figures

A B db/db mice db/db mice Glucose (all)

TallyHo mice ) BW (all) AAV-FGF19 NONNZO mice 800 60

dL 800 60 AAV-GFP STZ mice GFPControl Control 600 600 FGF19 FGF19 4040 FGF19 400400 20 200 20 Glucose (mg/dL) Glucose 200 *** *** Body Weight (g) 0 1 2 3 4 5 6 7 8 9 10 11 12 Week *** 0 *** 0 Body Weight (g) Weight Body Glucose (mg/ Glucose 002 24 46 68 810 10 02 24 46 68 810 10 Week Week Weeks Weeks

C db/db mice D db/db mice HbA1c FGF19 ELISA (W10) GFP FGF19 GFP FGF19 1010 3300 88 2200 66

4 4 10 HbA1c (%) 10

*** Insulin 2 (ng/ml) FGF19 2 Glucagon

HbA1c (%) HbA1c 100 m 0 00

0 (ng/mL) FGF19 GFP FGF19 GFP FGF19

Control FGF19 Control FGF19 E TallyHo mice Glucose_line

) BW_line 15 600 60 88 15 dL 600 GFPAAV-GFP 60 AAV-GFP AAV-NPT1162 *** FGF19 AAV-NPT116266 10 400400 4040 44 *** 55 200200 2020 2 HbA1c (%) HbA1c 2 HbA1c (%) HbA1c Glucose (mg/dL) Glucose Body Weight (g) *** (ng/ml) FGF19 0 0 *** 00 00 (ng/mL) FGF19 0 Body Weight (g) Weight Body Glucose (mg/ Glucose GFP FGF19 GFP FGF19 00 36 69 912 12 003 36 69 912 12 WeeksWeek WeeksWeek AAV-GFP AAV-GFP % fat (Week 6) F NONNZO mice AAV-NPT1162 AAV-NPT1162HOMA-IR (Week 6) Glucose (Week 6) BW (Week 6) ) 600600 8800 6060 8 dL

60 *** IR 60 - 6 400400 * 4040 4040 4

*** % Fat

** HOMA-IR 200200 ** 2020 *** 2020 *** HOMA 2 Body Weight (g) Glucose (mg/dL) Glucose *** 0 0 (%) mass Fat 0 *** 0 (g) Weight Body 0 0 0 Glucose (mg/ Glucose

e one GFP GFP anide GFP GFP anide uanide Chow Chow uanide Chow Chow NPT1162 NPT1162 NPT1162 NPT1162 Pioglitaz Pioglitazon Pioglitazone Pioglitazone

Dimethylbig Dimethylbigu Dimethylbig Dimethylbigu

G STZ mice H STZ miceInsulin Glucagon (M5) HbA1c (M5)

) Glucose_line BW_line 1.00 800 1000 dL 800 GFPGFP 30 15 1000 1000

15 /mL) 30 GFP

NPT1162 /mL) 600 FGF19 NPT1162 0.75 750 600 750 pg 750 ** 2200 10 pg 400400 ** 5000.50 500500 10 5 Insulin (ng/ml) Insulin 200 10 (%) HbA1c *** 2500.25 250 200 (mg/dL) Glucose 250 Glucagon (pg/ml) Glucagon Glucose (mg/dL) Glucose

* *** (%) HbA1c 0 ( Insulin 0.000 0 Glucose (mg/ Glucose 0 0 Body Weight (g) Weight Body 0 4 8 12 GFP FGF19 ( Glucagon 0 4 8 12 0 4 8 12 GFP FGF19 GFP FGF19 0 4 8 12 GFP GFP Week GFP Week NPT1162 NPT1162 Weeks Weeks NPT1162

I STZ mice Kidney Kidney%BW GFP FGF19 600600 2.0

1.5 400 1.0

2mm 1.0 200 ** ** 0.5 Kidney % Kidney Kidney Weight (mg) Weight Kidney Body w Body eight Kidney (mg) Kidney Kidney % Body Weight 00 0.00 GFP FGF19 GFP FGF19 GFP GFP

NPT1162 NPT1162

10 Diabetes Page 52 of 70

Supplementary Figure S1. FGF19 Induces Marked, Rapid and Sustained Diabetes Remission in Diabetic Mice. (A) Study design. Diabetic db/db mice, TALLYHO mice, NONNZO mice, or streptozotocin (STZ)-treated mice received a single dose of adeno-associated virus (AAV) carrying FGF19 or green fluorescent protein (GFP) through tail veins. Blood glucose levels were evaluated at various time points. (B) Blood glucose and body weight over time in db/db mice (n=5 mice per group). Note that levels of plasma glucose were significantly reduced from baseline values within 7 days of AAV- FGF19 delivery, and further decreased over time, with no changes in body weight observed, in db/db mice (C) Glycated hemoglobin (HbA1c) and FGF19 levels at the end of the study (12 weeks after AAV administration) in db/db mice (n=5 mice per group). Long-term glycemic control was observed with FGF19 treatment, as evidenced by reductions in glycated hemoglobin (HbA1c) levels. Circulating FGF19 levels were 21.5±2.9 ng/mL at the end of the study in these db/db mice. (D) Representative images of pancreatic islets in db/db mice. Pancreata were stained with anti- insulin or anti-glucagon antibodies, and developed using DAB substrates (brown color). Slides were counterstained with hematoxylin. n=5 mice per group. FGF19 administration improved - cell mass and -cell organization. (E) Blood glucose and body weight over time, as well as HbA1c and FGF19 levels at the end of the study, in the polygenic, diabetic TALLYHO mice (n=5 mice per group). Note that plasma concentrations of glucose were significantly reduced following AAV-FGF19 injection without weight loss. FGF19 treatment significantly lowered HbA1c levels at the end of the study. Circulating FGF19 levels were 9.9±2.7 ng/mL in these mice. (F) Glucose, body weight, fat mass and index for homeostasis model assessment of insulin resistance (HOMA-IR) in NONNZO mice (n=5 mice per group). Metformin and pioglitazone, drugs approved for treatment of type 2 diabetes, were included for comparison. Note that comparing with FGF19, treatment with metformin or pioglitazone either resulted in modest reductions in glucose levels or excess weight gain, respectively. FGF19 administration was also accompanied by a marked improvement in insulin sensitivity. (G) Blood glucose and body weight over time, as well as HbA1c levels at the end of the study, in STZ mice (n=5 mice per group). In contrast to AAV-GFP-injected STZ mice that developed severe hyperglycemia, mice treated with FGF19 remained normoglycemic, with significantly reduced HbA1c levels at the end of the study. (H) Serum concentrations of insulin and glucagon in STZ mice (n=5 mice per group). Note that administration of FGF19 resulted in suppression of diabetic hyperglucagonemia in STZ mice. (I) Kidney histology and kidney weight in STZ mice. Scale bars, 2 mm. Magnifications of boxed regions show glomerulosclerosis and an increase in Bowman’s capsular space, features of nephropathy, in control STZ mice but not in FGF19-treated STZ mice. Representative Periodic acid–Schiff stained kidney sections were shown. n=5 mice per group.

11 Page 53 of 70 Diabetes

Values are mean ± SEM. ***P < 0.001, **P < 0.01, *P < 0.05 versus GFP, by two-way analysis of variance (ANOVA) with Sidak’s multiple comparisons adjusting for multiple testing (glucose and body weight in panels B, E, G), or one-way ANOVA with Dunnett’s multiple comparison tests (panel F), or unpaired, two-tailed t-test (HbA1c in panels C, E, G; insulin and glucagon in panel H; kidney weight in panel I).

12 Diabetes Page 54 of 70

A GLUCOSE 800

) 800 dL 600600 *** 400400

200200 Glucose (mg/dL) Glucose Glucose (mg/ 0 0 0 1 4 24 0 1 4 24 0 1 4 24 0 1 4 24 0 1 4 9 0 1 4 9 0 1 4 24 0 1 4 0 1 4 0 1 4 0 1 4 0 1 4 0 1 4 0 1 4 GFP24 FGF1924 19-424 GQV24 FGF2 24 FGF224 FGF2-1924 Weeks afterBW injection (K128D/ chimera 80 R129Q/ B 80 K134V) 6060

4040

20 Body Weight(g) 20 Body weight (g) 0 0 0 1 4 24 0 1 4 24 0 1 4 24 0 1 4 24 0 1 4 9 0 1 4 9 0 1 4 24 0 1 4 0 1 4 0 1 4 0 1 4 0 1 4 0 1 4 0 1 4 GFP24 FGF1924 19-424 GQV24 FGF224 FGF224 FGF2-2419 Weeks after injection (K128D/ chimera Tumor Nodule R129Q/ K134V)Liver Weight C 88 D 66

66 44 44

22 22 Liver Liver tumors Liver Weight (g) Liver Liver weight (g) Tumor Nodule Per Liver Per Nodule Tumor 00 00

) ) 3) 3) GFP 1162 GFP 1162

NPT1162Amg 19-4Amg GQV NPT1162Amg 19-4Amg GQV FGF2s(wtFGF2s(m FGF2s(wtFGF2s(m FGF2-NPT FGF2-NPT Survival E 100 FGF19 FGF2 FGF2 (K128D/ (3 mutation) R129Q/K134V) 5050 Survival Survival (%) Percent survival Percent P=0.008 Log rank test 00 00 5050 100 150 200

DaysDays

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Supplementary Figure S2. Long-Term Evaluation of Previously Reported FGF Mutants in db/db Mice. (A) Glucose results of FGF mutants previously reported (Goetz et al., 2012; Suh et al., 2014; Wu et al., 2011a; Wu et al., 2010). 10-12 week old db/db mice were injected intravenously with 3 x 1011 vector genome AAV carrying FGF19, FGF mutants, or a control gene GFP. Glucose and body weight were measured before AAV injection (week 0), and at weeks 1, 4, and 24 post AAV injection. n=5-6 mice per group. Although the FGF mutants described in these reports exerted modest glucose-lowering in diet-induced obese mice, possibly secondary to weight reduction in this model, they failed to improve the severe hyperglycemic condition when tested in db/db mice. Note that db/db mice injected with the FGF2 mutants did not reach the 24-week time point due to reduced survival. n=5-6 mice per group. (B) Body weights in db/db mice expressing FGF mutants previously reported. n=5-6 mice per group. (C) Liver tumors in db/db mice expressing FGF mutants previously reported. db/db mice were euthanized at the end of the study (24 weeks after AAV injection) for liver tumor quantification. n=5-6 mice per group. (D) Liver weight of db/db mice expressing FGF mutants previously reported. n=5-6 mice per group. (E) Kaplan-Meier survival curve. Significant safety concerns (i.e., reduced survival) on these engineered FGF molecules, previously unrecognized from limited cell-based assays, were uncovered upon prolonged exposure in the db/db mouse model. Values are mean ± SEM. ***P < 0.001 by two-way ANOVA with Sidak’s multiple comparison tests versus GFP.

14 Diabetes Page 56 of 70

A B

C D Asp-125 Ionic interaction energy -24.230 kJ/mol 8-9 8 loop 9 Arg-250 FGF19 ---AFEEEIRPDGYNVYRS--- FGF23 ---RFQHQTLENGYDVYHS--- FGF21 ---SFRELLLEDGYNVYQS--- FGF1 ---LFLERLEENHYNTYIS--- FGF2 ---FFFERLESNNYNTYRS---

E F Lys-278 N-terminus 1 Tyr-25 Leu-261 Asn-27 FGF23 YPNASPLLGSSWGGLIHLYT FGF8 FTQHVREQSLVTDQLSRRLIRTYQLYS Pro-26 FGF19 RPLAFSDAGPHVHYGWGDPIRLRHLYT N1-15 MR-----DSSPLVHYGWGDPIRLRHLYT Met-276

G His-2387 Phe-2352 Gln-2389 Leu-2343 Asp-44 Glu-2325 Phe-32 Thr-2341 Arg-48 His-35 Leu-2309

Ionic interaction energy Hydrogen bond energy Hydrogen bond energy VdW energy -26.574 kJ/mol -5.993 kJ/mol -5.865 kJ/mol -3.162 kJ/mol -1.728 kJ/mol -1.79 kJ/mol

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Supplementary Figure S3. In vivo Structure-Activity Relationship Analysis of FGF19. (A) Crystal structure of FGF19 used for mutant design. Shown is PDB 2P23, with -sheets in FGF19 represented as yellow ribbons, the loops between -sheets in blue, and the -helices in red. (B) Amino acid sequence of FGF19. -sheets in FGF19 are indicated with yellow ribbons above amino acid sequence, the loops between -sheets in blue, and the -helices in red. (C) Alignment of amino acid sequences in the 8- 9 region. Note that Asp-125 in FGF23 is colored in orange. The -sheets 8 and 9 are shaded in blue. (D) A two-dimensional view of interaction between Asp-125 in loop 8-9 of FGF23 and the invariant Arg-250 in FGFR. An ionic interaction energy of −24.230 kJ/mol was calculated in the FGF23-FGFR1-Klotho structure (PDB 5W21). (E) Alignment of amino acid sequences in the N-terminal region. Note that key residues directly interact with FGFR in FGF23 (Tyr-25, Pro-26, Asn-27) and FGF8 (Phe-32, His-35, Asp-44, Arg- 48) are colored in orange. The changes in the non-tumorigenic FGF19 mutant N1-15 (also known as NGM282 or M70) are colored in blue. (F) Detailed view of interaction between N-terminus of FGF23 and the D3 domain of FGFR. Left panel, Tyr-25 in FGF23 (yellow ribbon) interacts with Leu-261 and Met-276 in FGFR1 (blue ribbon). Right panel, Pro-26 and Asn-27 in FGF23 interact with Lys-278 in FGFR1. (G) Detailed view of interaction between N-terminus of FGF8 and the D3 domain of FGFR. Arg- 48 in FGF8 (yellow ribbon) interacts with Glu-2325 in FGFR2 (blue ribbon), with an ionic interaction energy of −26.574 kJ/mol; Asp-44 in FGF8 interacts with His-2387 in FGFR2, with a hydrogen bond energy of −5.993 kJ/mol; His-35 in FGF8 interacts with Gln-2389 in FGFR2, with a hydrogen bond energy of −5.865 kJ/mol; Phe-32 in FGF8 forms van der Waals (VdW) interactions with multiple residues in FGFR2. Values are mean ± SEM. ***P < 0.001 by two-way ANOVA with Sidak’s multiple comparison tests versus GFP.

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A 11 C-terminus

FGF19 --FLPMLPMV PEEPEDLRGH LESDMFSSPL ETDSMDPFGL VTGLEAVRSP SFEK FGF23 --FLSRRNEI PLIHFNTPIP RRHTQSAEDD SERDPLNVLK PRARMTPAPA SCSQ FGF21 --FLPLPGLP PALPEPPGIL APQPPDVGSS DPLSMVG------PSQGRSP SYAS

B FGF19 C-terminus

Lys-387

Asp-212

C FGF23 C-terminus Trp-417 Arg-187

Asp-820 Lys-429

D FGF21 C-terminus

Leu-194

Glucose HCC nodule

E ) 800800 3030 dL 600600 ** 2020 400400 *** *** 1010 Liver Nodule Liver 200200 Tumors Glucose (mg/dL) Glucose

Glucose (mg/ Glucose 0 0 0 0 0 4 24 0 4 24 0 4 24 0 4 24 0 4 24 0 4 24 0 4 24 0 4 0 4 0 4 0 4 0 4 0 4 0 4 24 24 24 24 24 24 24 GFP GFP FGF19 C1 C2 C3 C4 C5 DC10 DC20 DC30 DC41 DC50 Weeks after injection NPT1162 body weight Liver weight 8080 8 8 6060 6 6

40 40 4 4

2020 Body Weight (g) 2 2 Liver Weight (g) Body w(g) Body eight 0 0 w(g) Liver eight 0 0 0 4 24 0 4 24 0 4 24 0 4 24 0 4 24 0 4 24 0 4 24 0 4 0 4 0 4 0 4 0 4 0 4 0 4 24 24 24 24 24 24 24 GFP FGF19 C1 C2 C3 C4 C5 GFP DC10 DC20 DC30 DC41 DC50 Weeks after injection NPT1162

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Supplementary Figure S4. In vivo Structure-Activity Relationship Analysis of FGF19 in the C-terminal Region. (A) Alignment of amino acid sequences in the C-terminal region. Note that Asp-212 in FGF19 is colored in red, and Arg-187 in FGF23 is colored in orange. The -sheet 11 is shaded in blue. Molecular modeling of the FGF19- Klotho-FGFR1 complex positions the C-terminus of FGF19 to insert into the KL1-KL2 cleft of Klotho and further dip into the central barrel of KL2, similar to the FGF23-Klotho and FGF21- Klotho interactions. By gripping the outreaching C-terminal tail, Klotho anchors the FGF19-FGFR complex. (B) Interaction between FGF19 C-tail and Klotho. Left panel, energy of interactions in ascending order; right panel, detailed view of ionic interaction between Asp-212 in FGF19 (red) and Lys-387 in Klotho (green). Modeling also suggests that FGF19 C-tail forms stronger interaction with Klotho than FGF21, or contacts between FGF23C and Klotho. This increase in affinity is mainly attributable to the unique Asp-212 residue in FGF19 that forms ionic interaction with Lys-387 of Klotho. (C) Interaction between FGF23 C-tail and Klotho. Left panel, energy of interactions in ascending order; right panel, detailed view of contacts between Arg-187 in FGF23 (yellow) and multiple residues in Klotho (green). (D) Interaction between FGF21 C-tail and Klotho. Left panel, energy of interactions in ascending order; note that the interaction between FGF21C and Klotho is weaker than FGF19C-Klotho or FGF23C-Klotho. Right panel, lack of contact between Leu-194 in FGF21 (grey) and Klotho (green). (E) in vivo screen of FGF19 mutants with deletions in the C-terminal region. 10-12 week old db/db mice were injected intravenously with 3 x 1011 vector genome AAV carrying FGF19, FGF19 mutants, or a control gene GFP. Blood glucose was measured before AAV injection (week 0), and at weeks 4 and 24 post AAV injection. Mice were euthanized at the end of the study (24 weeks after AAV injection) for liver tumor and liver weight quantification. n=5-6 mice per group. Consistent with the molecular interactions depicted in panel B, deletion of 30 amino acids from the C-terminus completely abolished FGF19’s ability to lower glucose in db/db mice. Values are mean ± SEM. ***P < 0.001 by two-way ANOVA with Sidak’s multiple comparison tests versus GFP.

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A B Lys-149 Arg-151 90º His-53

Arg-157

C GlucoseGlucose 800800 ) 800 dL 600600600

400400400 *** *** *** *** *** *** 200200200 Glucose (mg/dL) Glucose Glucose (mg/dL) Glucose Glucose (mg/

0 0 0 0 1 5 14 0 1 5 14 0 1 5 14 0 1 5 14 0 1 5 14 0 1 5 14 0 1 5 14 0 1 5 14 0 1 5 14 (Weeks) 00 1 5 00 11 55 00 11 55 0 0 1 15 5 0 01 15 5 0 01 15 5 0 10 51 5 0 10 51 5 0 1 0 5 1 5 C 14 FGF191414 T89A1414 K149A1414 R151A1414 K155A14 14 R157A14 14 K149A14- 14 H53A14- 14 WeeksBody Weeksafter weight Injection after Injection R151A K149A- R151A D 8080 6060

4040

20 Body Weight(g) 20 Body weight (g) 00 0 1 5 14 0 1 5 14 0 1 5 14 0 1 5 14 0 1 5 14 0 1 5 14 0 1 5 14 0 1 5 14 0 1 5 14 (Weeks) 0 1 5 0 1 5 0 1 5 0 1 5 0 1 5 0 1 5 0 1 5 0 1 5 0 1 5 C 14 FGF1914 T89A14 K149A14 R151A14 K155A14 R157A14 K149A14- H53A14- Weeks after Injection R151A K149A- Liver tumor Liver weight R151A E 2525 F 6 6 2020 1515 4 4 1010

Liver Tumors Liver 2 2

Liver Liver tumors 5 5 Liver (g) weight Liver Liver weight (g) 0 0 0 0

1A GFP T89A 151A 1A K149AR151AK155AR157A GFP T89A NPT1162 K149AR151AK155AR157AR151A 49A-R15 NPT1162 K149A-R 149A-R15 K149A- H53A-K1 H53A-K

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Supplementary Figure S5. In vivo Structure-Activity Relationship Analysis of FGF19 in the Heparin-Binding Region. (A) Molecular model of FGF19-FGFR1-heparin 2:2:2 ternary complex. Crystal structure of FGF19 (PDB 2P23) was superimposed onto the FGF2-FGFR1-heparin complex (PDB 1FQ9). FGF19 and FGFR1 are shown in the surface representation in red and blue, respectively; heparin is shown in yellow sticks. The model is rotated 90º between the left and right panels. (B) Expanded view of interaction site between FGF19 and heparin. Key FGF19 residues that are in contact with heparin are labeled in cyan. (C) Glucose results from in vivo screen of FGF19 mutants in heparin-binding region. 10-12 week old db/db mice were injected intravenously with 3 x 1011 vector genome AAV carrying FGF19, FGF19 mutants, or a control gene GFP. Glucose and body weight were measured before AAV injection (week 0), and at weeks 1, 5 and 14 post AAV injection. In summary, we found that FGF19 mutants containing single amino acid substitutions (i.e. Lys-149, Arg-151, Lys-155, or Arg-157) retained anti-diabetic activity, while the combination of these mutations compromised the glucoregulatory activity of the corresponding FGF19 variants. (D) Body weights of db/db mice expressing FGF19 mutants in heparin-binding region. (E) Liver tumors in db/db mice expressing FGF19 mutants in heparin-binding region. db/db mice were euthanized at the end of the study (24 weeks after AAV injection) for liver tumor quantification. Notably, evidence for hepatic tumorigenesis was clearly observed in db/db mice expressing FGF19 with mutations in heparin-binding region. Collectively, these studies suggest that the residual heparin binding exhibited by FGF19 does not appear to play a significant role in FGF19-associated hepatocarcinogenesis. (F) Liver weight of db/db mice expressing FGF19 mutants in heparin-binding region. Values are mean ± SEM. ***P < 0.001 by two-way ANOVA with Sidak’s multiple comparison tests versus GFP.

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Supplemental Tables

Supplementary Table S1. Gastric Bypass Subject Characteristics at Baseline, and at Day 7 and Day 21 Post Surgery (N=29)

Variable Baseline Day 7 Day 21 Sex (female%) 69% ------Age (year) 47.5±8.5 ------Fasting glucose (mg/dL) 113±43 112±40 100±36 HbA1c (%) 7.1±1.2 6.7±1.0*** 6.3±0.8*** Total cholesterol 167±40 173±32 145±35** (mg/dL) 42.0±10.3 33.8±6.3*** 34.8±6.9*** HDL-C (mg/dL) 110±34 123±30* 98±33 LDL-C (mg/dL) 167±53 169±64 139±54* Triglycerides (mg/dL) 130±29 125±28*** 119±27*** Weight (kg) 167±9 ------Height (cm) 46.2±9.8 44.2±9.7*** 42.4±9.8*** BMI (kg/m2) 135±18 129±19*** 127±19*** Waist circumference 48.8±12.4 --- 45.9±12.1*** (cm) 122±15 120±16 113±18** Body Fat (%) 72±8 69±9 67±8** SBP (mm Hg) 52% ------DBP (mm Hg) 59% ------Treated hypertension, % 5.7±5.4 ------Treated dyslipidemia, % Diabetes duration (year)

BMI, body mass index; DBP, diastolic blood pressure; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; SBP, systolic blood pressure

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Supplementary Table S2. Sequence of FGF19 Mutants in the Loop Regions.

Name Amino Acid Sequence FGF19 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 1- 2 RPLAFSDAGPHVHYGWGDPIRLRHLYTDDAQ-QTSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 2- 3 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIREDGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 3- 4 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAA DQSPHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 4- 5 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVKPGVVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 5- 6 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVKTVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 6- 7 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGPDGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 7- 8 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGSLHFDEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 8- 9 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEILEDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 9- 10 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEAHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 10- H RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF

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EEEIRPDGYNVYRSEKHRLPLHLPGNKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK H- 11 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGPARFLPLSHFLPML PMVPEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK Amino acids that differ from wild type FGF19 are shown in red. 1- 2 indicates mutations in the loop region between -sheets 1 and 2; 10- H indicates mutations in the loop region between -sheet 10 and the -helix located immediately after 10.

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Supplementary Table S3. Sequence of FGF19 Mutants in the -Sheet Regions

Name Amino Acid Sequence FGF19 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 1 RPLAFSDAGPHVHYGWGDPIRQRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 2 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSEAHLEIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 3 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGTVGGAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 4 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAESLLQLKALALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 5 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTIQILGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 6 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSSRFLCQRADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 7 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGALYGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 8 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSPEACSF RELLRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 9 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYQSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 10 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF

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EEEIRPDGYNVYRSEKHGLPLHLPGAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK 11 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHLPPALPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK H RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSNKSPHRDPAPRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK Amino acids that differ from wild type FGF19 are shown in red. 1 indicates mutations in - sheet 1. H indicates mutations in the -helix located immediately after 10. None of the FGF19 mutants in the -sheet region were non-tumorigenic while retaining the anti-diabetic effects (data not shown).

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Supplementary Table S4. Sequence of FGF19 Mutants in the N-terminal Region

Name Amino Acid Sequence FGF19 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK N1 R-----DAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK N2 R------VHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK N3 R------GDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK N1-1 R-----DEDPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK N1-2 R-----DEGPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK N1-3 R-----DEDPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK N1-4 R-----DEGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK N1-5 R-----DQGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK N1-6 R-----DQGLHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK N1-7 R-----DQSPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF

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EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK N1-8 R-----DESPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK N1-9 R-----DQSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK N1-10 R-----DESPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK N1-11 R-----DASPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK N1-12 R-----DSSPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK N1-13 R-----DSGPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK N1-14 R-----DSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK N1-15 MR----DSSPLVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK N1-16 M-----DSSPLLQ--WGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK Amino acids that differ from wild type FGF19 are shown in red. Red dash indicates deletion.

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Supplementary Table S5. Sequence of FGF19 Mutants in the C-terminal Region.

Name Sequence FGF19 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGLEAVRSPSFEK C1 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDSMDPFGLVTGL------C2 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESDMFSSPLETDS------C3 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEEPEDLRGHLESD------C4 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFLPMLPMV PEE------C5 RPLAFSDAGPHVHYGWGDPIRLRHLYTSGPHGLSSCFLRIRADGVVDCAR GQSAHSLLEIKAVALRTVAIKGVHSVRYLCMGADGKMQGLLQYSEEDCAF EEEIRPDGYNVYRSEKHRLPVSLSSAKQRQLYKNRGFLPLSHFL------Amino acids that differ from wild type FGF19 are shown in red. Red dash indicates deletion.

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Supplementary Table S6. Treatment Emergent Adverse Events in a Double-Blind, Placebo- Controlled trial of NGM282 in Patients with Type 2 Diabetes

Placebo NGM282 2 mg NGM282 5 NGM282 10 mg (n=20) (n=21) mg (n=20) (n=19)

Adverse Events by Severity Grade, n (%) Grade 1 11 (55%) 16 (76%) 18 (95%) 17 (85%) Grade 2 6 (30%) 7 (33%) 9 (47%) 10 (50%) Grade 3 1 (5%) 1 (5%) 2 (10%) 0 (0%) Grade 4 0 (0%) 0 (0%) 0 (0%) 0 (0%) Most Common (>10%) Adverse Events, n (%) Diarrhea 3 (15%) 6 (29%) 9 (47%) 8 (40%) Nausea 3 (15%) 5 (24%) 9 (47%) 9 (45%) Injection site 2 (10%) 4 (19%) 5 (26%) 3 (15%) bruising Injection site 0 (0%) 3 (14%) 5 (26%) 5 (25%) pruritus Headache 6 (30%) 2 (10%) 2 (10%) 2 (10%) Injection site 0 (0%) 0 (0%) 3 (16%) 6 (30%) erythema Frequent bowel 0 (0%) 2 (10%) 2 (10%) 3 (15%) movements Vomiting 0 (0%) 1 (5%) 3 (16%) 1 (5%) Abdominal 0 (0%) 1 (5%) 0 (0%) 3 (15%) distension Serious Adverse Events, n (%) At least one 0 (0%) 0 (0%) 0 (0%) 0 (0%) serious adverse event

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