H U B R E C H T O R G A N O I D T E C H N O L O G Y

HUB Organoids: a patient in the labA Paradigm-shifting Technology

Drug Discovery & Development, Companion Diagnostics and

(pre) Clinical Patient Stratification

Drug Development – Inefficient, Unpredictable, Expensive

2

Bridge the Gap Between

Lab & Clinic

Patient in the Lab

Drug Development Focus:

Highly Innovative Drugs

Developed on

>50y Old Models

Small Molecules

Antibodies

IO DrugsPre-Clinical

Development

Clinical Phase I-III

Post Registration

Adult Stem Cells

3 Hans Clevers

Hubrecht Institute

LGR5 Stem Cell Derived Organoids

4

1 2 3 4 5 6 7 8 9

10 11 12 13 14

day

day

Organoids Recapitulate

Organ Function

Organoids Represent

Tumor Heterogeneity

In vitro Models Generated From Any Patient and Most Organs

Sato et al., Nature 2009; Gastoenterology 2011

Why Organoids: Credible Models for Drug Development

5

Mimic PatientResponse.

All Patients.

Represent Tumor

Heterogeneity.Combination Therapy.

Biomarker.

Patient Relevant Model.

(Pre)-clinical Identification of

Clinical ResponsePatient Stratification

Companion Diagnostic.

Organoid Technology: Personalized Medicine

6

HUB Organoid Biobanks:

Pre-clinical Patient Population

7

Co

ntr

ol

Org

an

oid

Pati

en

t

Org

an

oid

Organoid growth

Tumor growth

Pre-treatment

Organoid death

Post-treatment

Pati

en

t

CT

Scan

A living biobank of breast cancer organoids capture disease heterogeneity

Sachs et al., 2018, Cell 172, 1–14

Patient-derived organoids model treatment response of metastaticgastrointestinal cancers

Vlachogiannis et al. Science 2018;359:920-926

Organoid Profiling identifies common responders to chemotherapy in pancreatic cancer

Tuveson et al. Cancer Discov; 8(9); 1112–29

Organoid Predict Clinical Response

Organoids Are A Patient in the Lab

HUB Organoid Platform

9

Human Organ Human Disease Animal (Species)

Breast

Colon

Kidney

Lung

Liver

Pancreas

Small intestine

Stomach

Head & Neck

Ovary

…

Cystic Fibrosis

Genetic diseases (AAT, etc)

IBD

COPD

Cancer

Infectious Diseases (Virology,

Malaria, etc.)

…

Dog

Mouse

Mini pig

Rat

Monkey

Replacing

Animals.Reducing

Time & Costs.

Patient Relevant

Model.

Organoid Drug Development

10

First Organoid Based Drug in Clinical Trial

11

Biclonics®

Fe

atu

re M

ea

su

rem

en

ts

High content screening on tumor

and normal organoidsBiclonics® targeting CRC

Organoids – ex vivo patient-

derived tumor tissue

HUB, Merus develop New CRC Treatment Using Organoids

12

Identify effective compound on

patient derived relevant>500 Bispecific antibodies

3 Organoid Lines

52 Hits screened24 CRC organoids lines

1 BispecificOptimization

CT

Pre-clinical identification of target

population

Mechanism and Lead development

Clinical Trial start Q1 2018

Identification of Specific Target Pair: LGR5/EGFR

13

Frac

tio

n a

po

pto

tic

nu

clei

MCLA-158

Cetuximab

LGR

4

LGR

5

ZNR

F3

RN

F43

LGR

4

LGR

5

ZNR

F3

RN

F43

LGR

4

LGR

5

ZNR

F3

RN

F43

LGR

5

TT Mock Control RNF43

1st Arm

Ce

tuxi

mab

PI3

Ki

Biclonics®

Dose

10 μg/ml

2 μg/ml

Normal

Tumor

Lead Antibody Differentiates Between Normal & Tumor Organoid

14

+

Strong Inhibition Weak Inhibition

KRASmut MCLA-158Lgr5 mAb +

EGFR mAbCetuximab

G12D

G12D

G12V

WT

G12C

WT

WT

WT

WT

WT

WT

Tum

or

Org

an

oid

s

Norm

al

Org

anoid

s

Organoid Growth

Organoids (Ex vivo): From Tumor and Normal Colon

Tissue from CRC Patients

MCLA-158

CetuximabVehicle

PDX (In vivo): Mice Transplanted with Tumor Organoids

and Treated with Cetuximab and MCLA-158

1-3

10-20

10-50

>100

Primary Screen

Diseased/Normal Control

Patients

Mechanism and Lead

Selection

Lead and Patient

Stratification

<50K Compound Library

100-200 Primary Hits

5-10 Candidate Leads

1 Lead

# Organoid Biobank

Used for Screening

Patient Derived Organoid Based Drug Development

Human Organoid Models

Intestinal Organoids

Characterization & Assays (IBD &Tox)

16

17

Wistar WU

Minipig

Beagle

Animal Derived Organoid Models

•Organoids derived from different species recapitulate discrepancies in toxicity.

•Species: • Rat (Wistar WU)

• Dog (Beagle)

• Mouse

• Mini pig

• Monkey

• More in pipeline…

18

Rat

Dog

Human

OTX, Bromodomain InhibitorGefetinib HDM2 Antagonist

Organoids Predict Efficacy and Safety

μMμMμM

0.001 0.01 0.1 1 10 1000

50

100

150

Gefitinib (μM)

rela

tive

via

bili

ty %

Gefitinib 72 hrs

Rat_Gefitinib

Human_Gefitinib

Dog_Gefitinib

0.01 0.1 1 10 100

0

50

100

150

200

JNJ-233 (µM)

rela

tive

viab

ility

%

JNJ-233 EM

HDM2 antagonist

DuodenumToxicity screen 168h

Dog duodenum_JNJ-233-AAA (EM)

Rat duodenum_JNJ-233-AAA (EM)

Human duodenum_JNJ-233-AAA (EM)

Z-score: Rat duodenum: 0.551101Human duodenum: -0.087418Dog duodenum: 0.643017

quadruplicate

0.01 0.1 1 10 100 10000

50

100

150

Metabolism in human GI tract organoids

10 60 120

0

500

1000

1500

t (min)

Meta

bolit

e (

ng/m

l)

BIBR0951_CES2_organoids

10 60 120

0

500

1000

1500

t (min)

Meta

bolit

e (

ng/m

l)

Estradiol-3-glucoronide_UGT1A1_organoids

10 60 120

0

20

40

60

80

t (min)

Meta

bolit

e (

ng/m

l)

7-Hydroxycoumarin-sulfate_SULT_organoids

10 60 120

0

50

100

150

t (min)

Meta

bolit

e (

ng/m

l)

5-NAcetylASA_NAT1_organoids

pIEC: Pooled Human Enterocytes

Metmax™: Pooled Human Enterocytes

Intestinal Organoids to Test Barrier Function and Transporters

20Pourfazad et al., in preparation.

Side view

Top view

Monolayer in Expansion

WENR

Enterocyte Differentiation

(iWNT)

Trans Epitelial Electerical Resistance (TEER)

Monolayer Transport: Basal to Apical

21

PSC833

Rhodamine 123Cell permeable

P-g

p

RFU in Top Compartment

Basal to Apical Transport• Rhodamine 123: transport via P-gp (MDR1, ABCB1,

apical )• PSC833: P-gp inhibitor

Monolayer Transport: Apical to Basal

22

Apical to Basal Permeability, Basal to Apical Transport• Rhodamine 123: transport via P-gp (MDR1, ABCB1,

apical )• PSC833: P-gp inhibitor

RFU in Bottom Compartment

PSC833

Rhodamine 123Cell permeable

P-g

p

Human Organoid Models

Liver Organoids

23

Human Hepatocyte Organoids for Toxicology

24 DAPI / AFP / MRP2 / F-actin

Anna Rios, Hans Clevers

Prinses Maxima Center, Netherlands

Genetic Diseases Platform

25

Acute Liver Failure

Alpha-1 antitrypsinA1AT mutation

Cirrohsis

COPDAlpha-1 antitrypsin

Huch et al. Cell 2015

Wilson’s DiseaseATP7B mutation

Cupper overload

Vomiting

Brain, eye, kidney damage

Crigler NajjarUGT1A1 gene

UDP glucuronosyltransf.

Metabolism of bilirubin

Brain damage

Alagille SyndromeJAG1 or NOTCH2

bile ducts development

Hepatocyte maturation in differentiated liver organoids7

da

y d

iff

7 da y s d

if f.

1 1 da y s d

if f

1 5 da y s d

if f.

0

11 0 6

21 0 6

31 0 6

41 0 6

Lu

min

es

ce

nc

e/m

l/1

x1

0^

6 c

ell

C Y P 3 A 4

7 da y s d

if f.

1 1 da y s d

if f

1 5 da y s d

if f.

0

21 0 5

41 0 5

61 0 5

81 0 5

11 0 6

Lu

min

es

ce

nc

e/m

l/1

x1

0^

6 c

ell

C Y P 2 C 9CYP2C9 activity

CYP3A4 activityP450-GloTM

CYP3A4

KR7

DNA

CYP3A4

KR7

DNA

CYP3A4

KR7

DNA

11

da

y d

iff

15

da

y d

iff

0

50

100

150

200

250

300

350

0 5 10 15 20 25

OH

-Mid

ng/m

l

Time (hrs)

Hepatocyte

OrganoidsMidazolam metabolism

-4

1×104

1×105

1×106

1×107

CYP3A4P450-Glo™ Assays

lum

inesence/1

0^6

cells

/ml

Organoids diff. day 15

Hepatocytes

27

1 1 0 1 0 0 1 0 0 00

2 0

4 0

6 0

8 0

1 0 0

1 2 0

1 4 0

M

re

lati

ve

via

bil

ity

%

1 4 d a y -re fre s h e d

Immune system

dependent

toxicity

Human Liver Organoids Predict Known Clinical Liver Toxicity

Clinical trial failure

due to

Human liver toxicity

Human Organoid Models

Co-Culture Immune System

28

Modeling Immuno-Oncology

29

T cellsOrganoidsDeath

Anna Rios, Hans Clevers

Prinses Maxima Center, Netherlands

Engineered T-Cells Induce Tumor Specific Cell Death

30

Patient 1

Patient 2

Normal OrganoidsTumor Organoids

Mock T-cells

IFN-γ Measurements

Show a Wide Range of

T-Cell Response

Engineered

T-cellsMock T-cells Engineered

T-cells

Organoid – T-Cell Co-Culture

31

Dijkstra, Voest, Clevers, Cell 2018

Au

tolo

go

us

TIL

De

rive

d C

D8

An

d T

-

Ce

ll R

ea

cti

vit

y t

o O

rga

no

ids

Develop Drugs for Patients on Patients

Patient in the Lab

Pre-clinical Patient

Stratification

Post Registrations

Population

Organoid Characterization

Preclinical Disc Dev

Clinical Trial

32

Pre-Clinical Development

Clinical Phase I-III

Post Registration

Contact Us

33

Hubrecht Organoid Technology is anot-for-profit organization founded in2013 by the Hubrecht Institute, KNAWand the University Medical CenterUtrecht (NL).

Based on the work of Prof. Dr. Hans

Clevers, who discovered the HUBorganoid technology, HUB has adedicated team acting as a global

expertise & reference center

Contact:

Dr. Bahar Ramezanpourb.ramezanpour@hub4organoids.nl+31 611957039

ABOUT HUB