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Photo H&E GS Tu Tu NGM282 DEMONSTRATES ANTI-INFLAMMATORY AND ANTI-FIBROTIC ACTIVITIES THAT ARE INDEPENDENT OF STEATOSIS REDUCTION IN A FARNESOID X RECEPTOR (FXR)-DEFICIENT, DIET-INDUCED ANIMAL MODEL OF NON-ALCOHOLIC STEATOHEPATITIS (NASH) Lei Ling, Mei Zhou, Marc Learned, Stephen Rossi, Alex DePaoli, Hui Tian NGM Biopharmaceuticals, Inc., South San Francisco, CA 94080, USA . Serum concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), markers of hepatocellular injury, were determined on Cobas Integra I400 clinical analyzer NGM282 and FGF19 improve the biochemical and histological features in FXR-deficient mice fed a high-fat, high-fructose, high- cholesterol diet • improve fibrosis scores • normalize serum ALT and AST concentrations • reduce hepatic expression of pro-inflammatory and pro- fibrotic genes These effects occur in FXR-deficient mice suggesting that that at least some of the anti-fibrotic actions of NGM282 occur independent of FXR The effect on fibrosis was observed in the absence of significant reduction in hepatic steatosis, indicating that at least some of the anti-fibrotic actions of NGM282 occur independent of changes in liver fat content Prolonged exposure to FGF19, but not NGM282, induces the formation of hepatocellular carcinomas in a FXR-deficient, diet- induced mouse model of NASH These data support further evaluation of NGM282 in clinical studies of patients with NASH References 1. Kliewer et al., Dig Dis 2015; 33:327-331 2. Degirolamo et al., Nat Rev Drug Discov 2016; 15:51-69 3. Zhou et al., Cancer Res 2014; 74:3306-3316 4. Luo et al., Sci Transl Med 2014; 6:247ra100 5. Zhou et al., Hepatology 2016; 63:914-929 6. Luo et al., EASL 2015 7. Ling et al., EASL 2015 8. Harrison et al., Lancet 2018; 391:1174 9. Harrison et al., EASL 2018 10. Kleiner et al., Hepatology 2005; 41:1313-1321. 11. Brunt et al., Hepatology 2011; 53:810-820 NAFLD Disease Activity Score (NAS) and fibrosis stage were evaluated to assess hepatic steatosis, inflammation, hepatocellular degeneration, and liver fibrosis 10.11 Quantitative RT-PCR analysis of hepatic expression of markers of macrophage/monocyte infiltration Fibroblast growth factor 19 (FGF19) is an endocrine hormone produced in the ileum in an Farnesoid X receptor (FXR)-dependent manner 1-2 NGM282 is an engineered analogue of FGF19 which retains CYP7A1 inhibition via FGFR4-bKlotho binding, retains insulin-sensitizing and body weight-reduction metabolic activities, but lacks the potential tumorigenic activity of FGF19 3 • CYP7A1 catalyzes the first and rate-limiting step in bile acid synthesis NGM282 is non-tumorigenic analogue of human FGF19 demonstrating rapid and significant improvements in NASH-related histology and fibrosis in multiple murine models and in patients with biopsy- confirmed NASH 3-9 We conducted a study in FXR-deficient mice with diet-induced NASH to further investigate the underlying mechanism for the anti-inflammatory and anti-fibrotic effects of NGM282 Disclosure This study was funded by NGM Biopharmaceuticals, Inc. Lei Ling, Mei Zhou, Marc Learned, Stephen Rossi, Alex DePaoli, and Hui Tian are employees and stockholders of NGM Biopharmaceuticals, Inc. NGM282 represses hepatic expression of enzymes in bile acid biosynthetic pathway and reduces serum levels of bile acids NGM282 reduces hepatocellular fibrosis, as demonstrated by Sirius Red (SR) staining NGM282 reduces collagen mRNA expression in the liver NGM282 decreases hepatic mRNA levels of genes associated with hepatic stellate cell activation and fibrogenesis Frozen livers were embedded in OCT, sectioned, and stained with osmium tetraoxide. Hepatocellular lipids were shown as black staining (top panels). Hematoxylin and eosin (H&E)-stained sections were shown in the bottom panels Control FGF19 NGM282 Control FGF19 NGM282 CD68 Ly6C CCR2 Collagen 1a1 TGF-b1 Desmin Group Score (mean±SD) Control FGF19 NGM282 NAFLD Activity Score (NAS) 7.5±0.5 5.8±0.2 6.4±0.7 Ballooning degeneration 2.0±0.0 1.8±0.4 1.8±0.4 Inflammation, lobular 2.7±0.5 1.8±0.4 2.0±0.7 Steatosis 2.9±0.4 2.4±0.6 2.6±0.6 Fibrosis Score 3.8±0.2 1.4±0.2*** 1.4±0.2*** Collagen 1a2 LoxL2 FXR-deficient mice (B6.129X1[FVB]-Nr1h4tm1Gonz/J, Jackson Laboratories, #007214) were placed on a high-fat, high-fructose, high- cholesterol diet (HFFCD) (40% fat, 22% fructose, 2% cholesterol) throughout the study FGF19, NGM282, or a control gene GFP was delivered to HFFCD-fed FXR-deficient mice (on HFFCD for 16 weeks) using adeno-associated viral vector (AAV), which enables stable long-term transgene expression after a single tail vein injection 34 weeks after AAV delivery, mice were euthanized for analysis (on HFFC diet for 50 weeks) Livers were stained for H&E, osmium tetrachloride, sirius red, and anti- glutamine synthetase for histopathological analysis Total RNAs were extracted from livers using Trizol reagent and treated with Dnase. qRT-PCR was conducted using premade Taqman Gene Expression Assay primers (Life Technologies) Top panels: representative liver photos Middle panels: hematoxylin and eosin (H&E)-stained liver sections Bottom panels: immuno-histochemical staining with anti-glutamine synthetase (GS) and DAB substrates (brown color) While glutamine synthetase-positive liver tumors (Tu) were evident in the FGF19 group, none of these findings occurred in the NGM282 group Control FGF19 NGM282 15 10 5 0 SR+ area (%) *** *** 1.5 1.0 0.5 0 *** *** *** *** FGF19 FXRRE FXR RXR FGF19 1.5 1.0 0.5 0 *** *** 1.5 1.0 0.5 0 Relative expression *** *** *** *** CYP7A1 CYP8B1 RESULTS BACKGROUND AND AIMS METHODS CONCLUSION NGM282 Reduces Liver Weight and Spleen Weight in HFFCD-Fed, FXR- Deficient Mice NAS and Fibrosis Scores FGF19, But Not NGM282, Induces Liver Tumor Formation in HFFCD-Fed, FXR-Deficient Mice NGM282 Demonstrates Anti-Inflammatory Activity in HFFCD-fed, FXR-Deficient Mice NGM282 Demonstrates Anti-Fibrotic Activity in HFFCD-fed, FXR-Deficient Mice NGM282 Does Not Impact Steatosis in HFFCD-Fed, FXR-Deficient Mice ** ** 300 200 100 0 Bile acids (mmol/L) *** *** ** *** 400 300 200 100 0 ALT (U/L) 400 300 200 100 0 AST (U/L) NGM282 Reduces ALT and AST 1.5 1.0 0.5 0 *** *** 1.5 1.0 0.5 0 *** *** ** ** High-fat, high-fructose, high- cholesterol diet 0 16 50 Weeks AAV-FGF19, i.v. AAV-NGM282, i.v. FXR-deficient mice Histology, NAS and fibrosis scores, serum chemistry, QPCR SERUM BILE ACIDS ALT AST Control FGF19 NGM282 ***p<0.001, **p<0.01 vs control group Control FGF19 NGM282 ***p<0.001, **p<0.01 vs control group Relative expression CCL2 *** *** 1.5 1.0 0.5 0 1.5 1.0 0.5 0 1.5 1.0 0.5 0 Relative expression Relative expression Relative expression 1.5 1.0 0.5 0 *** *** 1.5 1.0 0.5 0 *** *** Control FGF19 NGM282 ***p<0.001, **p<0.01 vs control group ***p<0.001 vs control group ** *** * ** 5 4 3 2 0 Liver (g) 15 10 5 0 Liver % BW 1 *** *** *** *** 400 300 200 100 0 Spleen (mg) 1.5 1.0 0.5 0 Spleen % BW Control FGF19 NGM282 ***p<0.001, **p<0.01 vs control group
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