Gut-based immunotherapy - implications for NASHTargeting the gut immune system for alleviation of the systemic

inflammatory response without immune suppression

Yaron Ilan, M.D.

Gastroenterology and Liver Units, Department of Medicine

Hebrew University-Hadassah Medical Center

Jerusalem, Israel

July 2018

Disclosure I have financial relationships with the companies below and the content of my presentation does include a discussion of the investigative use of products being developed by the companies below. The studies described were supported in part by several of these companies.

Consultant:

Teva; Abbott; ENZO; Protalix; Therapix; Betalin Therapeutics; Immunepharma; JTI; Immuron; Chiasma Pharma; Nasvax; Alcobra; One Day Pharma; Cure Tech; Lutea; Kamedis; MedWell; Accelmed; Medial; SciM; Tiziana Pharma;

Medical Director:

Exalenz Bioscience; Natural Shield; Oberon Sciences; Plantylight;

Forward-looking statement Please be advised that the information and projections provided in this presentation may include forward-looking statements with respect to plans, projections or future performance of the compounds and companies presented, the occurrence of which involves certain risks and uncertainties and is not under the control of these companies, including, but not limited to, changes in regulatory environment and success in implementing its research, development, sales, marketing and manufacturing plans, protection and validity of patents and other intellectual property rights, the impact of currency exchange rates and the effect of competition by other companies.

Therapy for NASH: The next decade

a. Target multiple mechanisms

b. Long term safety

c. Target the whole spectrum of the disease: from prevention to cirrhosis

d. A combination therapy

e. Alleviates concomitant disorders (e.g. diabetes, hyperlipidemia)

Ilan, Y Dig Dis Sci. 2018 Apr 20.Ilan, y Ann Gastroenterol. 2018 May-Jun;31(3):296-304.)

Oral immune therapy Examples Advantages

Picasso Stadel Museum, Frankfurt

How does our environment blends into us

Oral immune therapy

Immune system

Metabolic syndrome = low grade chronic inflammation

Based on: Hotamisligil GS. Nature 2006;444:860–7Tilg H, et al. Gastroenterology 2006;131:934–45

Disruption of the interface between inflammatory

and metabolic pathways is central to the

pathogenesis of the metabolic syndrome

Oral immune therapy

Inflammation: Fueling the growing fireDiverse immune pathways at different disease stages

https://seekingalpha.com/article/3546436-revisiting-nash-therapeutic-targets-competitive-landscape

https://imarketsmart.com/why-you-should-add-fuel-to-the-fire/

http://oncologypro.esmo.org/Education-Library/Essentials-for-Clinicians/Lymphomas/Chapter-1-The-Immune-System

Presented to the gut-associated lymphoid tissue /dendritic cells

Antigen / Antibody / adjuvant

Target organs• Liver• Pancreas• Adipose tissue• Bowel• Muscle

Promotion of Tregs in

mesenteric lymph nodes

Image from Fagarasan S & Honjo T. Nat Rev Immunol 2003;3:63–72

Oral immunotherapy: modulation of the systemic immune response via alteration of the gut immune system without immune suppression

Ilan Y, et al. Immunol Cell Biol 2009;87:514–24Ilan Y, Hum Immunol. 10:768-76, 2009

Oral immune therapy

Tregs: Regulatory T cells

Adapted from: Ilan, Y 2016 . Alimentary Pharmacology & TherapeuticsIlan Y, Clinical & Translational Immunology; 2016

Oral immunotherapy: Re-educating the immune system

Gut-associated lymphoid tissue

Liver

Oral immunotherapy compounds

Microbiome

DC

NKT

T cell

MLN

Regulatory cells

Immune signal

• Immune modulation without immunosuppression

• Oral immunotherapy using non-absorbable compounds

are being evaluated in pre-clinical & phase I/II NASH trials

DC: Dendritic cellsMLN: Mesenteric lymph nodesNKT: Natural killer lymphocytes

Induction of oral tolerance towards fatty liver-extracted proteins

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Elinav E J Pathol 2006; 208: 74–81

Control

Fed with liver extracts

Reduction of hepatic fat content by MRI

Reduction of hepatic triglycerides content

Improved glucose intolerance

1911 Wells

Jewish sages wrote:

“If one is bitten by a mad dog, he may

eat his liver and be cured.”

ControlFed with liver

extracts

Control

Ilan, Y. Am J of Physiology, 2016

Fig. 1

Journal of Hepatology .2014.11.002

Circulating phospholipid profiling identifies portal contribution to NASH signature in obesity

Prevalence of overweight, insulin resistance and type II diabetes before and after enzyme therapy

Before enzyme therapy

Cross-sectional study (after enzyme therapy, median 11

years)

Prevalence of being overweight (%) 16 56

Prevalence of insulin resistance (%) Not known 6

Prevalence of type II diabetes (%) 0 8.2

Langeveld M, et al. Blood Cells Mol Dis 2008;40:428–32Ilan Y. Immunolgy Cell Biology 2008

Increase in BMI during treatmentwith ERT (n=32)

Bo

dy

mas

s in

dex

(kg

/m2)

Evolutionary advantage of patients with Gaucher’s disease: An immune protective effect of b-glucosylceramide (GC)

GC-treatedControl

Control GC-treated

GC improves glucose intolerance and decreases hepatic fat accumulation in the ob/ob model

Margalit M, et al. J Pharmacol Exp Ther 2006

GC-treated Control

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400

GLU

COSE

TIME

Lalazar Mole immunology 2008Ilan Y, et al. Immunol Cell Biol 2009;87:514–24

http://acseenotes.wordpress.com/2011/03/07/cytology

GC

Tregs

NKT

T cell

Altered lipid rafts

Altered expression of raft membrane proteins

NFkB

DC

STAT

Flotilin 2

Cross talk

Oral administration of glycosphingolipids alters lipid rafts on cells membranes

GC: b-glycosylceramideLC: b-lactosylceramideIGL: b-glycosylceramide+b-lactosylceramide

Primary endpoint - HbA1c Secondary endpoint - % Fat by MRI

Week 0 Week 40N=2340 weeks

Safety and Effect of Oral Administration of GC (EGS21) in Subjects with Diabetes and NASH: Results of a double blind placebo controlled trial

Lalazar, J Med Food 2017 20(5:)458-464.

0,00

2,00

4,00

6,00

8,00

Placebo Drug

NK

T ly

mp

ho

cyte

(%

)

Oral administration of DR6MP: A minimally absorbed delayed-release 6-mercaptopurine

-2

-1

0

1

2

3Purinethol DR-6MP

0

20

40

60

80

100

BASELINE WEEK 12

% Patientswith leukopenia

-10

0

10

20

30

Baseline Week 12CHANGE

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150

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Baseline Week 2 Week 4 Week 6 Week 8 Week 10 Week 12

CD

AI

SC

OR

E DR-6MP

Phase II double blind placebo controlled trialN=70

Ilan, E. Clin Exp Immunol. 2015 181(2):362-72

No Leukopenia in DR6MP-treated CD

Reduction in IFN-gamma ELISPOT assay

Reduction in CDAI in DR6MP-treated CD

Alteration of systemic Immune system in DR6MP-treated

PLP-EAE in SJL

0 5 10 15 20 25 30 35 40 45Rx

0

1

2

3

4

5

CD3 50 gCD3 5 g Control IgG

CD3 500 g

Mea

n cl

inic

al s

core

Days post-immunization

Ochi H, et al. Nat Med 2006;12:627-35

0 0.5 hr 1 hr 3 hr

iv 3hr

Ora

l

IV

Induction of oral immunotherapy using oral anti-CD3

Weiner et al. Immunol Rev; Brain. 2016; Immunotherapy. 2016.

Biologically active anti-CD3 recovered from the gut

Ilan Y, et al. Proc Natl Acad Sci U S A; 2010

Induction of oral immunotherapy using oral anti-CD3 for NASH

TGF-b IL-10

IL-2 IFN-g

* *

*

*

IL-2 IFNg

An adjuvant effect on the innate immune system in the gut

TregTGF-bIL10

macrophage

Adipose tissue

Treg

Treg

DCmacrophage

Deactivates innate immune cells

Promotes Treg

Downregulation of inflammation in adipose tissue

T cell

TCR/CD3

NKT

CD1d

Oral anti-CD3 + GCAdaptive immunity Innate immunity

anti-CD3 GC

liver

pancreas

Amelioration of fatty liver &islet hypertrophy in the pancreas

Ilan Y, et al. Proc Natl Acad Sci U S A; 2010 107:9765–70

macrophage

DC=Dendritic cellsTregs=regulatory T cellsNKT=natural killer lymphocytes

Oral anti-CD3 in NASH: promotes regulatory T cells, decreases liver enzymes, and alleviates insulin resistance

Ilan Y J Clin Immunology 2015

Safety:

I. No treatment-related adverse events

II. Normal blood cell counts: No change in CD3+ counts

Efficacy biomarkers:

I. Liver enzymes ↓

II. TG ↓

III. Glucose ↓ Insulin ↓

• Phase II, randomized single blinded clinical trial• 36 subjects with biopsy-proven NASH with type II diabetes• 0.2, 1.0, 5 mg or placebo daily for 30 days

0

5

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15

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25

30

35

40

0 14 30 60

CD

4 L

AP

+ (

% o

f to

tal C

D4

)

Placebo

0.2 mg

1 mg

5 mg

Day

**

*

Oral anti-CD3 increased Tregs

PBS anti-CD3+GC

Decrease CD11b+F4/80 macrophages increase in foxp3 expression in CD4+ T cells

A decrease in cell infiltration into adipose tissue

Decrease TNF

Increase in IL10 & TGFb

Effect of oral administration of anti-CD3 with glycosphingolipids on adipose tissue in a NASH model

Ilan Y, PNAS, 2010

IL10 TNF TGFb

Oral administration of Foralumab

Foralumab: Humanized Oral anti-CD3 in NASH

4

T

5

adipose tissue

pancreas

1

Foralumab survives digestion in the stomach

2

Foralumab remains localized in gut wall 3Antigen presented to innate immune system in mesenteric lymph nodes

Dendritic cells induce regulatory T cells

TT

Anti-inflammatory cytokines downregulate inflammation in target organs

Increase in Tregs

Does not pass into blood stream

liver

muscle

Ilan, Shailubhai, Sanyal, Clin Exp Immun 2018 (In Press)

IMM124E

Liver damage

Regulatory T cells

Anti LPS Ab

LPS contributes to activation of inflammatory pathways associated with inducing NASH in a background of fatty liver

Adopted from AASLD 2010; J Hepatol 2007

Adjuvants: Glycosphingolipids

Oral administration of anti-LPS antibodies with glycosphingolipids:An adjuvant effect on the innate immune system in the gut

Yaʼacov AB. BMC Gastroenterol. 2015

* P<0.05; ** P<0.009

0

100

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900

ALT

leve

ls (

u/L

)

*

Decreased liver enzymes Decreased serum triglycerides

Adar T. Clin Exp Immunol., 2012

Pre clinical: Leptin-deficient model of NASH

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5,0

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25,0

TNBS,water

TNBS,colostrum

cont

TNBS, 50µganti LPS

TNBS,500µg anti

LPS

We

igh

t lo

ss (

%)

**

An adjuvant effect on the innate immune system in the gut in NASH &colitis

Mizrahi M. J Inflamm Res. 2012

Day 1 Day 30

Increased GLP1 and adiponectin Increased CD4+CD25+Foxp3+ Tregs

Oral administration of IMM124E in NASH: Results of a phase I/IIA

IMM124E :Results of Phase IIA clinical trial

Mizrahi M. J Inflamm Res. 2012

Improved insulin resistance Decrease in liver enzymes

Alleviation of hyperlipidemia

Oral administration of a BY-2 plant cell-expressing recombinant anti-TNF fusion protein (PRX-106), which consists of soluble form of human TNF receptor fused

to the Fc component of a human antibody IgG1 domain

Ilan Y Immunobiology 222:544-551, 2017Khury T, World J Gastroenterol. 2016

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Saline BY2 (-) PRX-1060.5µg

PRX-10610µg

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len

ic/h

ep

ati

c

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4+

CD

25

+F

ox

P3

+ r

ati

o

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350

Saline BY2-(MOCK)

PRX-1060.5µg

PRX-10610µgS

eru

m T

G (

mg

/dL

)

0

200

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600

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1000

Saline BY2 (-) PRX-1060.5µg

PRX-10610µg

AST

(IU

)

0

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50

60

Saline BY2 (-) PRX-1060.5µg

PRX-10610µg

mg

TG/g

r Li

v (%

)

Saline BY2(-) PRX-106 0.5 PRX-106 10g

Oral administration of a recombinant anti-TNF fusion protein is biologically active

in the gut promoting regulatory T cells: Results of a phase I clinical trial

No effect on white blood cells and lymphocytes counts were noted in any of the dosages

0%

200%

400%

600%

2 mg 8 mg 16 mg

% in

cre

ase

CD4+CD25+

0%

200%

400%

600%

2 mg 8 mg 16 mg

% in

cre

ase CD8+CD25+

Oral administration of all three dosages was associated with an increase in CD4+CD25+ and CD8+CD25+ subset of suppressor lymphocytes

Almon E, J Immunol . Methods. 446:21-29, 2017

• OPRX-106 was not absorbed systemically.

• Oral administration using OPRX-106 was safe and well tolerated

• OPRX-16 was not associated with immune suppression.

• OPRX-106 was effective as demonstrated by clinical response and improvement in various disease parameters.

29

Orally administered recombinant anti-TNF alpha fusion protein for the treatment of ulcerative colitis: Phase 2a clinical trial

Ilan, y Gastroenterology 2018;154:S-153; Abst 739

Mechanism of oral immunotherapy: Promotion of regulatory T cells

Lamina propria

Mesenteric lymph nodes

Regulatory T cell

NKT cell

M cell

DC

Bacterial/viral/ fungal products

Food derived antigens

Systemic immune system

Glycosphingolipids

Anti CD3

IMM124E

DR6MP

PRX106

Macrophage

Ilan Y, Clinical & Translational Immunology; 2016Adopted from: Weiner et al. 2011. Immunol Rev; Brain. 2016; Immunotherapy. 2016

Soy-derived fractions

IL-10 Treg TGF-b Treg

Oral Immunotherapy for NASH: Advantages

Mechanism o An ability of the gut immune system to deliver signals to control the systemic immune response

Target o Not dependent on a specific molecular pathway

o Promotes regulatory T cellsSafety o Non-absorbable

o No immune suppression

o Low dose is sufficient to achieve a clinically meaningful effect

Spectrum of disease o Treatment for early and late stages of diseaseo Induction of remission and maintenance

Associated disorders o Treats type 2 diabetes and hyperlipidemia

Adjuvant o An adjuvant for other metabolic and anti-fibrotic drugs

Ilan, Y 2016. Alimentary Pharmacology & Therapeutics

Ingram Pinn

SummaryOral immunotherapy-based compounds: A new class of drugs for NAFLD

a.Oral immunotherapy using non-absorbable immune modulators redirect the immune system towards an anti-inflammatory pathway.

b.It provides a platform for a long-term safe therapy which targets chronic inflammation in NASH in patients of all disease stages.

Brigham and Women’s Hospital

Harvard Medical School, Boston, MA

Howard Weiner Samia J. Khoury

Ruth Maron Francisco Quintana

Queens College, City University of NY

Robert Bittman

Bonn University, Germany

Gustav Schwarzmann

UC Davis, California

Eric Gershwin

Sharee Zedek, Medical CenterAri Zimran Deborah Elstein

Tel Hashomer, Medical CenterArnon Nagler Meir OhanaBen Gurion UniversitySmadar Cohen

Faculty of MedicineArie Dagan Roni Kalman

Liver Unit

Oren Shibolet

Gadi Lalazar

Eyal Shteyer

Alla Milhem

Ehud Zigmond

Meir Mizrahi

Tomer Adar

Yuval Horwitz

Efrat Orenbuch

Madi El-Haj

Dori Rotnemer

Diabetes Unit

Itamar Raz

Ehud Ziv

Sarah Zangen

Endocrinology

Gil Leibowitz

Gastroenterology

Eran Israeli

Tiberiu Hershcovici

Hebrew University-Hadassah Medical Center, Jerusalem

Menahem Hareati

Nila Hemed

Mina Rowe

Tawfik Huri

Ariel Drori

Yuval Ishay

Dean Nachman

Neurology

Adi Dembinsky

Collaborators

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

Northwestern University, Chicago

Richard Silveramn

Thank You