b-Glycosphingolipids as immune
modulators for oral immune therapy
Yaron Ilan, M.D.
Liver and Gastroenterology Units,
Department of Medicine
Hebrew University-Hadassah Medical Center
Jerusalem, Israel
6/2012
EWGGD
Disclosure
I have financial relationships with the companies below and the
content of my presentation does include a discussion of the
investigative use of Imm124, anti-CD3, and glucosylceramide.
Medical Director:
Accelmed; Immuron; Exalenz Biosciences, Adjuvan Pharma
Board member:
Exalenz Biosciences, Plantylight, WAYS.
Consultant:
Abbott, Teva Pharmaceuticals, ENZO Biochem, Chiasma Pharma,
Plantylight, Nasvax, Alcobra, One Day.
b-Glucosylceramide (GC)
You want less
We want more
3
b-Glucosylceramide (GC)
• Oral immune therapy
• GC in oral immune therapy
• GC in animal models
• GC in clinical trials
4
Oral Immune therapy
• An approach to treat autoimmune, infectious,
malignant and inflammatory diseases.
• An active process that uses the inherent ability of
the GI tract's immune system to control unwanted
systemic immune responses, by inducing regulatory
T cells in an antigen-specific manner.
Antigen / Antibody / adjuvant
Presented to the
Gut associated lymphoid tissue
/dendritic cells
Target organs
• Bowel
• Pancreas
• Adipose tissue
• Liver
• Brain
Induction of regulatory T cells
(Tregs) in mesenteric lymph nodes
Oral Immune therapy
• Platform for a wide range of diseases
• No side effects or toxicity
• Not associated with general immune suppression
• No risks of severe infection or malignancy
• Easily tolerated by patients
Hundreds of years ago, sages wrote in the Talmud:
“ If one is bitten by a mad dog,
he may eat his liver and be cured.”
The immune system of the bowel
 The gut mucosal immune system is the largest lymphoid organ.
 It differentiates the antigenic signals against the high “background
noise” of food and bacterial antigens.
 Despite constant antigenic stimulation, suppression of
inflammation is the rule.
Antigen+adjuvant
M
cell
DC
NKT
DC
Macrophage
DC
DC
Interfollicular T cell area
Th2 cell
Tregs
Th3 cell
Tr1
Perifollicular area
B cell follicule
Ilan Y, Immunology
Cell Biology, 2009
NKT lymphocytes





NKT cells co-express CD3/abTCR and NK markers.
Rare in the peripheral blood.
Abundant in the liver and bowel.
Secrete large amounts of IFNg or IL4.
Regulate immune mediated disorders.
Antigen+adjuvant
LSEC
NKT
DC
M
cell
Kupffer cells
Lamina Propria
Tregs
DC
NKT
DC
Macrophage
DC
DC
Liver
Interfollicular T cell area
Th2 cell
Tregs
Th3 cell
Tr1
Perifollicular area
B cell follicule
NKT - DC cross talk
NKT cells recognize exogenous
glycosphingolipids anchored by
the MHC-like CD1d molecule.
Glycosphingolipid
CD1d
NKT
DC
Potential ligands for NKT regulatory lymphocytes
 The endogenous ligands have yet to be identified.
 Alpha-galactosylceramide is a potent activator for type I NKT.
 Sulfatides are potential ligands for type II NKT cells.
Doyle, Nature Reviews Immunology, 2007
b-Glucosylceramide (GC)
•
•
Patients with Gaucher’s disease may have an
altered NKT cells number and function.
GC may activate dendritic cells and/or NKT cells
in the gut.
Dendritic cell
NKT cell
Promotion of the DC-NKT interaction
by b-glycosphingolipids
b-glycosphingolipids
DC
GC
NKT
Effector
T cell
Treg
GC inhibits NKT cell proliferation
STIMULATION INDEX
1.2
1
0.8
0.6
0.4
*
NKT+BSA
NKT+GC
NKT+DC+BSA
NKT+DC+GC
0.2
0
Am J Physiol 2005
NKT - DC cross talk can increase or decrease
immune responses
Doyle, Nature Reviews Immunology, 2007
Administration of GC alleviates ConA hepatitis
900
ConA
800
700
600
GC+ConA (2h)
ConA+GC (2h)
ConA
GC
Naïve
500
400
300
Naive
GC+ConA
200
100
0
ALT
AST
GC in naive
Am J Physiol 2005
Administration of GC alleviates colitis
TNBS+GC
TNBS
NAÏVE+GC
NAÏVE
Gut 2007
Administration of GC suppresses melanoma
tumor growth
Control
GC
Oral administration of GC suppresses
hepatocellular carcinoma growth
90
80
*
130
70
110
GC
CONTROL
60
90
*
50
70
40
50
30
Survival (%)
Tumor volume (mm3)
Gut 2007
Administration of GC alleviates GVHD in the bowel
Control
GC-treated
Semi-allogeneic
Chronic
Transplantation, 2007
Th1: acute GVHD, semi-allogeneic: C57BL/6  F1,C57BL/6 x Balb/c
Th2: chronic GVHD : B10.D2  Balb/c
Administration of GC alleviates
GVHD in the liver
Control
GC-treated
Semi-allogeneic
Chronic
Optimization of b-glycolipid structure
b-D-glucosyl-thio-ceramide
14000
aGalCer
GCT
12000
AST
ALT
HO
HO
10000
U/ml
O
OH
HN
O
S
OH
OH
8000
6000
PBS
4000
GC
GCT
aGalCer+GC
2000
0
A
B
C
D
E
F
G
H
Ben Yaakov A, Mol Immunol 2009
Inhibition of STAT1 expression
STAT1
Phospho-STAT1
b-actin
GC
GCT
8.00
6.00
4.00
2.00
0.00
A
B
C
D
E
F
G
Inhibition of NKT lymphocytes
8
PBS
aGalCer
% gated
6
GC
GCT
4
aGalCer+GC
2
0
A
B
C
D
E
F
G
H
Inhibition of apoptosis
PBS
PBS
1mg
a-GalCer
a-GalCer
1.5mg
GC,GC,
IP IP
1.5mg
1.5mg b-D-thiol GC, IP
15mg
GC,
1mg
GC,
IPIP
15mg b-D-thiol GC, IP
150mg b-D-thiol GC, IP
Oral administration of GC delays liver cell
proliferation following partial hepatectomy
Oil-red-O staining 48 hours after partial hepatectomy shows
an increase in fat accumulation in the GC treated mice.
Administration of GC reduces BrdU incorporation after
partial hepatectomy
p=0.017
The frequency of mitotic bodies is lower in GC vs. PBS
treated mice
p=0.047
Oral GC in the metabolic syndrome
•
Disruption of the interface between inflammatory and
metabolic pathways is central to the pathogenesis of
chronic metabolic diseases.
•
Obesity is characterized by chronic activation of
inflammatory pathways in peripheral tissues.
Hotamisligil Nature Reviews Immunolo, 2008 8:943
Organs involved in the pathogenesis
of the metabolic syndrome
Immune system
Based on: Hotamisligil, Nature, 2006
Non alcoholic fatty liver disease
Non alcoholic steatohepatitis
A. Mae Diehl, EASL NASH, 2009
Non alcoholic fatty liver disease
Non alcoholic steatohepatitis
A. Mae Diehl, EASL NASH, 2009
Leptin deficient ObOb mice
• Features the metabolic syndrome
• Non-alcoholic steatohepatitis, diabetes, obesity,
hyperlipidemia
• Altered NKT cell function
b-Glucosylceramide
GC
GC decreases transaminase levels
GC decreases triglyceride levels
3
700
OB/OB+GC
2.5
600
IU
400
LEAN+ GC
1.5
*
500
*
2
*
OB/OB+PBS
300
LEAN+ PBS
200
1
100
AST
ALT
GC improves glucose tolerance test
400
350
GLUCOSE
300
250
200
150
100
50
0
0
15
30
60
90
120
180
TIME
Lalazar, Am. J. Pathol., 2009
GC decreases hepatic fat accumulation
Control Ob/Ob
GC-treated Ob/Ob
20
OB/OB+PBS
15
LEAN+ GC
10
*
CM 2 x SI INDEX
OB/OB+GC
LEAN+ PBS
5
0
Control
Ob/Ob
GC-treated
Ob/Ob
Margalit M, J Pharmacol Exp Ther. 2007
Margalit,
J. JExp.
Therap.
Zygnmod
Em, Am.
Physiology
Endo2006
2009
IGL
GC
b-Glucosylceramide
LC
b-Lactosylceramide
Psammomys obesus
 The desert gerbil Psammomys obesus (sand rat) is a model of a
nutritionally-induced type II diabetes, characterized by insulin
resistance.
 It is adapted to a low energy diet, the Saltbush.
 When transferred to a high energy diet, it develops obesity,
hyperinsulinemia, hyperglycemia, and steatohepatitis.
Tayer-Shiffman; Hepatology 44:4 (S1) 71A, 2006
IGL decreases insulin and glucose
post-prandial levels
p<0.01
p<0.01
100
200
*
*
50
IGL
*
mg/dl
pmol/l
GC
100
*
0
0
GROUPS
Insulin
GROUPS
Glucose
IGL decreases fat accumulation in the liver
PBS
IP
OP
IGL
IP
OP
MRI index of intrahepatic fat
p<0.01
0.3
0.25
0.2
0.15
*
*
0.1
SI – 0.23
SI – 0.15
PBS
IGL
0.05
0
GROUPS
b-glycoshpingolipids improve insulin resistance
in Cohen diabetic rats
Islet
Islet
Control
IGL
Zigmond, Am. J Physiology, 2009
b-glycoshpingolipids improve liver damage
in Cohen diabetic rats
3
A-GC
B-LC
C-IGL
Liver histological score
2.5
2
D-PBS
*
*
1.5
*
1
0.5
0
Experimental1 groups
b-glycoshpingolipids increase TGFb
in Cohen diabetic rats
12000
10000
*
*
*
TGFb pg/ml
8000
6000
4000
2000
0
TGFβgroups
Experimental
b-glycoshpingolipids alter NKT and CD8 cell distribution
in Cohen diabetic rats
NKT
4
3.5
CD8
8
A-GC
*
*
B-LC
7
C-IGL
A-GC
B-LC
C-IGL
D-PBS
2.5
Liver CD8 lymphocytes
Intrahepatic NKT (%)
Liver
3
*
2
1.5
1
0.5
2.5
3
*
2
Expermental1 groups
1.8
A-GC
B-LC
*
*
1.6
C-IGL
D-PBS
1.5
1
A-GC
*
B-LC
C-IGL
1.4
Liver/splleen CD8
Liver/spleen NKT
4
0
Experimental1 groups
Liver/spleen
5
1
0
2
D-PBS
6
1.2
1
D-PBS
*
0.8
0.6
0.4
0.5
0.2
0
0
Expermental1 groups
Experimental1 groups
Zigmond, Am. J Physiology, 2009
Can GC alter the structure of lipid rafts and
cell signaling?
GC alters GM1-patching
behavior on T cells
GC
Altered lipid rafts
Tregs
NKT
DC
T cell
Cross talk
Altered expression of raft membrane proteins
Flotilin 2
NFkB
STAT
Ilan, Immunology and Cell Biology, 2009
Can GC serve as an adjuvant
in the gut immune system?
48
Oral anti-CD3 suppresses EAE
PLP-EAE in SJL
Indirect immune fluorescence
Control IgG
a CD3 5  g
a CD3 50  g
a CD3 500  g
4
0
0.5 hr
1 hr
3 hr
Oral
3
2
1
0
IV
M e a n clin ica l sco re
5
0
Rx
5
10
15
20
25
30
35
40
45
Days post-immunization
iv 3hr
Ochi, Nature Medicine, 2006
Oral anti-CD3 with GC alleviate insulin resistance and NASH
Islet cell area (% of x10/ field)
20
15
10
5
0
GC
aCD3
aCD3+GC
800
600
400
200
GC
aC
D3
+
D3
aC
GC
S
0
PB
Fat area by Oil red (pixelsx1000/ field)
PBS
Ilan, PNAS, 2010
Oral anti-CD3 with GC alters cytokine secretion
by anti-CD3 activated PBLs
TGF-b
IL-10
*
300
250
800
IL-10 (pg/ml)
TGF-b (pg/ml)
*
1000
200
150
100
600
400
200
50
0
0
PBS
GC
aCD3
PBS
aCD3+GC
IL-2
GC
aCD3
aCD3+GC
IFN-g
3
40
2
*
1
IFN-g (ng/ml)
IL-2 (ng/ml)
35
30
25
20
15
10
*
5
0
0
PBS
GC
aCD3
aCD3+GC
PBS
GC
aCD3
aCD3+GC
Oral anti-CD3 with GC increases
TGF-b and IL-10 secretion from dendritic cells
A
PBS
PBS
GC
GC
aCD3
aCD3
*
aCD3+GC
*
aCD3+GC+aTGF
*
aCD3+GC
*
aCD3+GC+aTGF
0
50
100
150
200
TGFb (Relative OD expression)
0
500
1000 1500 2000 2500 3000
IL10 (Relative OD expression)
Ilan Y, PNAS, 2010
Adaptive immunity
TCR/CD3
Oral anti-CD3 + GC
anti-CD3
CD1d
GC
NKT
T cell
Treg
Innate immunity
Promotes Treg
DC
macrophage
TGF-b
IL10
Amelioration of fatty liver &
islet hypertrophy in the pancreas
Deactivates innate
immune cells
Downregulation of
inflammation in adipose tissue
Treg
liver
Treg
macrophage
macrophage
pancreas
Adipose tissue
Ilan Y, PNAS, 2010
Phase I clinical trial:
Oral administration of OKT3+GC
Healthy male volunteers (3 per group) were orally administered mouse
anti-human OKT3 at three doses: 0.2, 1.0, 5.0 mg/feeding.
40
20
30
15
CD8+CD25+ (%)
CD4+CD25+ (%)
Oral OKT3 increases CD4+CD25+ and CD8+CD25+
CD4+CD25+
CD8+CD25+
20
10
0
10
5
0
0
5
Days
10
0
5
10
Days
Ilan Y, Clinical Immunology, 2010
The effect of GC as an adjuvant in the gut
Healthy volunteers were orally administered 7.5 mg of GC alone or in
combination with 0.2 mg or 1.0 mg OKT3.
Oral OKT3 with GC increases CD4+FoxP3+
OKT3+GC
1.5
1.5
1.25
1.25
CD4+FoxP3 (%)
CD4+FoxP3 (%)
OKT3
1
0.75
0.5
1
0.75
0.5
0.25
0.25
0
0
0
5
Days
10
10
5
2
Days
10
3
Safety and Effect of Oral Administration of GC (EGS21) in
Subjects with Diabetes and Fatty Liver Disease
Primary endpoint - HbA1c
Secondary endpoint - % Fat by MRI
Study design:
Double-blind, placebo controlled
Treatment regimen – every day
Treatment duration – 40 weeks
Dose: 7.5 mg of GC in 5 ml PBS
N=40
Week 0
Week 40
Zygmond E, Hepatology 2008, A
Bacterial translocation and NASH
“Leaky Gut”
 Endotoxin is hypothesized to
play a role in the activation of
inflammatory pathways
associated with NASH.
 Endotoxin may induce NASH
in a background of fatty liver.
Bacterial antigens / Endotoxin
Antigen+adjuvant
M
cell
Lamina Propria
DC
NKT
Macrophage
DC
DC
Perifollicular area
DC
Interfollicular T cell area
B cell follicule
Alteration of the immune system
Immune imbalance
Immune-mediated disorders
A synergistic effect between anti-LPS antibodies
and adjuvnats in colostrum
Imm124-E
Anti LPS antibodies
Adjuvants (GC)
 Decrease bacterial translocation
 Increase regulatory suppressor T cells
Suppressing the chronic inflammatory state
in NASH and Type II diabetes
Imm124-E decreased liver enzymes
Imm124-E decreased serum triglycerides
900
800
* P<0.05
700
*
600
ALT levels (u/L)
500
400
300
200
100
0
An adjuvant effect in the gut
Imm124-E decreased Hepatic TGs
* P<0.05; ** P<0.009
* P<0.05
Adar T. Clin Exp Immunol., 2012.
Results of Phase 1/2 Clinical Trial in NASH and
Metabolic Syndrome
Oral administration of Imm124-E is:
 Safe
 Improves liver enzyme levels
 Improves markers for type 2 diabetes
 Improves hyperlipidemia
 Promotes regulatory cells and corrects some
abnormalities associated with metabolic syndrome
Mizrahi M. Hepatology, 52:163A 2010
Administration of Imm 124-E improved
liver enzyme levels
(7/10 Pt. p<0.002)
Mizrahi M. Hepatology, 52:163A 2010
Administration of Imm124-E improved insulin resistance
and Type 2 diabetes
(9/10 Pt. p<0.001)
Administration of Imm124-E improved hyperlipidemia
(9/10 Pt. p<0.005)
Administration of Imm124-E promoted
CD4+CD25+ regulatory lymphocytes
Day 1
CD4 - FITC
10
10
10
10
-10
5
Day 30
49.01%
5.66%
CD4
4
3
2
1
44.94%
2 0 2
-10 10 10
0.39%
10
3
10
4
CD25 - PE
10
5
Administration of Imm124-E promoted
CD4+CD25+FOXp3 regulatory lymphocytes
Day 1
Day 30
+
Antibody / disease associated antigen directed antibody / antigen
Bowel mucosa
Adjuvant
M cell
Lamina Propria
DC
NKT
DC
Macrophage
Perifollicular area
DC
DC
Interfollicular T cell area
B cell follicule
Tregs
Ilan Y, Elstein D, Zimran A, Immunology Cell Biology, 2009
+
Antibody / disease associated antigen directed antibody / antigen
Bowel mucosa
M cell
Lamina Propria
MLN
MLNs
Adjuvant
DC
NKT
DC
Macrophage
DC
Perifollicular area
DC
DC
Macrophage
Tregs
Tregs
LLN
Liver
LSEC
Macrophage
DC
Tregs
Adipose tissue
DC
Kupffer cells
NKT
ATLN
Macrophage
Tregs
B cell follicule
Interfollicular T cell area
DC
Tregs
Effector cells
DC
PLN
Macrophage
Pancreas
DC
Tregs
Tregs
Tregs
Muscle
Ilan Y, Elstein D, Zimran A, Immunology Cell Biology, 2009
Summary
 Oral administration of b-glycosphingolipids skews
the immune profile and exerts an immune
modulatory beneficial effect.
 The effect of b-glycosphingolipids may be
associated with promotion of the DC - NKT
interaction, and/or by alteration of lipid rafts and
intracellular signaling.
 b-glycosphingolipids can serve as potent adjuvants
for oral immune therapy.
Collaborators
Brigham and Women’s Hospital
Harvard Medical School, Boston, MA
Howard Weiner Samia J. Khoury
Ruth Maron
Francisco Quintana
Faculty of Medicine
Ofer Mandelboim
Chamutal Gur
Arie Dagan
Roni Kalman
Queens College, City University of NY
Robert Bittman
Sharee Zedek, Medical Center
Ari Zimran
Deborah Elstein
Bonn University, Germany
Gustav Schwarzmann
UC Davis, California
Eric Gershwin
Tel Hashomer, Medical Center
Arnon Nagler
Meir Ohana
Ben Gurion University
Smadar Cohen Alex Fisch
Hebrew University-Hadassah Medical Center, Jerusalem
Liver Unit
Oren Shibolet
Gadi Lalazar
Eyal Shteyer
Eran Elinav
Alla Milhem
Maya Margalit
Ehud Zigmond
Meir Mizrahi
Tomer Adar
Yuval Horwitz
Efrat Orenbuch
Madi El-Haj
Ron Cialic
Dan Livovsky
Ami Ben Ya’acov
Lidya Zolotarov
Dimitri Kanovich
Elizabeth Axelrod
Sarah Preston
Shivti Trop
Roslana Alper
Yehudit Shabat
Yoav Lichtenstein
Ibrahim Kasis
Athalia Klein
Menahem Hareati
Nila Hemed
Mina Rowe
Diabetes Unit
Itamar Raz
Ehud Ziv
Sarah Zangen
Endocrinology
Gil Leibowitz
Gastroenterology
Eran Goldin
Eran Israeli
Tiberiu Hershcovici
Neurology
Adi Dembinsky
Pathology
Orit Pappo
Thank you