1 5 NGM Biopharmaceuticals, Inc., South San Francisco, CA...
Transcript of 1 5 NGM Biopharmaceuticals, Inc., South San Francisco, CA...
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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 TianNGM Biopharmaceuticals, Inc., South San Francisco, CA 94080, USA
.
Serum concentrations of alanine aminotransferase (ALT) and aspartateaminotransferase (AST), markers of hepatocellular injury, were determinedon Cobas Integra I400 clinical analyzer
NGM282 and FGF19 improve the biochemical and histologicalfeatures 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 atleast some of the anti-fibrotic actions of NGM282 occurindependent of FXR
The effect on fibrosis was observed in the absence of significantreduction in hepatic steatosis, indicating that at least some of theanti-fibrotic actions of NGM282 occur independent of changes inliver fat content
Prolonged exposure to FGF19, but not NGM282, induces theformation of hepatocellular carcinomas in a FXR-deficient, diet-induced mouse model of NASH
These data support further evaluation of NGM282 in clinicalstudies of patients with NASH
References
1. Kliewer et al., Dig Dis 2015; 33:327-3312. Degirolamo et al., Nat Rev Drug Discov 2016; 15:51-693. Zhou et al., Cancer Res 2014; 74:3306-33164. Luo et al., Sci Transl Med 2014; 6:247ra100 5. Zhou et al., Hepatology 2016; 63:914-9296. Luo et al., EASL 20157. Ling et al., EASL 20158. Harrison et al., Lancet 2018; 391:11749. Harrison et al., EASL 201810. 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 toassess hepatic steatosis, inflammation, hepatocellular degeneration,and liver fibrosis10.11 Quantitative RT-PCR analysis of hepatic expression of markers of
macrophage/monocyte infiltration
Fibroblast growth factor 19 (FGF19) is an endocrine hormone producedin the ileum in an Farnesoid X receptor (FXR)-dependent manner 1-2
NGM282 is an engineered analogue of FGF19 which retains CYP7A1inhibition via FGFR4-bKlotho binding, retains insulin-sensitizing andbody weight-reduction metabolic activities, but lacks the potentialtumorigenic activity of FGF193
• CYP7A1 catalyzes the first and rate-limiting step in bile acidsynthesis
NGM282 is non-tumorigenic analogue of human FGF19 demonstratingrapid and significant improvements in NASH-related histology andfibrosis 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 tofurther investigate the underlying mechanism for the anti-inflammatoryand 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 acidbiosynthetic 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 stellatecell activation and fibrogenesis
Frozen livers were embedded in OCT, sectioned, and stained withosmium tetraoxide. Hepatocellular lipids were shown as blackstaining (top panels). Hematoxylin and eosin (H&E)-stained sectionswere 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 (onHFFC 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 treatedwith Dnase. qRT-PCR was conducted using premade Taqman GeneExpression 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-glutaminesynthetase (GS) and DAB substrates (brown color)
While glutamine synthetase-positive liver tumors (Tu) were evident inthe FGF19 group, none of these findings occurred in the NGM282 group
Control FGF19 NGM282
S ir iu s re d %
C o n tr o lF G F 1 9M 7 0
0
5
1 0
1 5
Sir
ius
re
d %
15
10
5
0
SR
+ a
rea
(%
)
******
G F P1 1 6 2 2 8 2
0 .0
0 .5
1 .0
1 .5
T ra n s fo rm o f C o l1 a 1
1.5
1.0
0.5
0
******
G F P1 1 6 2 2 8 2
0 .0
0 .5
1 .0
1 .5
T ra n s fo rm o f C o l1 a 2
******
FGF19
FXRRE
FX
R
RX
R
FGF19
G F P1 1 6 2 2 8 2
0 .0
0 .5
1 .0
1 .5
T r a n s fo r m o f L o x l2
Re
lativ
e E
xp
re
ss
ion
G F P1 1 6 2 2 8 2
0 .0
0 .5
1 .0
1 .5
T r a n s fo r m o f T g fb 1
Re
lati
ve
Ex
pre
ss
ion
1.5
1.0
0.5
0
***
***
G F P1 1 6 2 2 8 2
0 .0
0 .5
1 .0
1 .5
T r a n s fo rm o f D e s
Re
lati
ve
Ex
pre
ss
ion
G F P1 1 6 2 2 8 2
0 .0
0 .5
1 .0
1 .5
T ra n s fo r m o f C y p 7 A 1
Re
lativ
e E
xp
re
ss
ion
1.5
1.0
0.5
0R
ela
tive
ex
pre
ss
ion
G F P1 1 6 2 2 8 2
0 .0
0 .5
1 .0
1 .5
T r a n s fo rm o f C y p 8 B 1
Re
lativ
e E
xp
re
ss
ion
******
******
CYP7A1 CYP8B1
RESULTSBACKGROUND 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 MiceNGM282 Does Not Impact Steatosis
in HFFCD-Fed, FXR-Deficient Mice
T B A
Co
ntr
ol
FG
F19
M70
0
1 0 0
2 0 0
3 0 0
To
tal
Bil
e A
cid
(u
M)
** **
300
200
100
0
Bil
e a
cid
s(m
mo
l/L
)
A L T
Co
ntr
ol
FG
F1
9
M7
0
0
1 0 0
2 0 0
3 0 0
4 0 0
AL
T (
U/L
)
******
A S T
Co
ntr
ol
FG
F19
M70
0
1 0 0
2 0 0
3 0 0
4 0 0
AS
T (
U/L
) **
***
400
300
200
100
0
ALT
(U
/L)
400
300
200
100
0
AS
T (
U/L
)
NGM282 Reduces ALT and AST
1.5
1.0
0.5
0
G F P1 1 6 2 2 8 2
0 .0
0 .5
1 .0
1 .5
T r a n s fo r m o f C D 6 8
Re
lativ
e E
xp
re
ss
ion
******
1.5
1.0
0.5
0
G F P1 1 6 2 2 8 2
0 .0
0 .5
1 .0
1 .5
T r a n s fo r m o f C c r 2
Re
lativ
e E
xp
re
ss
ion
***
***
G F P1 1 6 2 2 8 2
0 .0
0 .5
1 .0
1 .5
T r a n s fo rm o f L y 6 c 1
Re
lativ
e E
xp
re
ss
ion
**
**
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
Re
lati
ve
ex
pre
ss
ion
CCL2
G F P1 1 6 2 2 8 2
0 .0
0 .5
1 .0
1 .5
T ra n s fo rm o f C c l2
Re
la
tiv
e E
xp
re
ss
io
n
***
***
1.5
1.0
0.5
0
1.5
1.0
0.5
0
1.5
1.0
0.5
0
Re
lati
ve
ex
pre
ss
ion
Re
lati
ve
ex
pre
ss
ion
Re
lati
ve
ex
pre
ss
ion 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
S p le e n (m g )
Co
ntr
ol
FG
F19
M70
0
1 0 0
2 0 0
3 0 0
4 0 0
Sp
lee
n W
eig
ht
(mg
)
S p le e n % B W
Co
ntr
ol
FG
F19
M70
0 .0
0 .5
1 .0
1 .5
Sp
lee
n %
Bo
dy
We
igh
t
L iv e r % B W
Co
ntr
ol
FG
F19
M70
0
5
1 0
1 5
Liv
er %
Bo
dy
We
igh
t
L iv e r (g )
Co
ntr
ol
FG
F19
M70
0
1
2
3
4
5
Liv
er W
eig
ht
(g)
*****
* **
5
4
3
2
0
Liv
er
(g)
15
10
5
0
Liv
er
% B
W
1
******
******
400
300
200
100
0
Sp
lee
n (
mg
)
1.5
1.0
0.5
0
Sp
lee
n %
BW
Control FGF19 NGM282***p<0.001, **p<0.01
vs control group