Advanced Human Immune System Mouse Models · I-O: immune check point inh., ADCC; macrophage polarisation, oncolyicbacteriaand viruses, vaccination, CRS (cytokine release syndrome)

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+33 456 44 81 [email protected]

Biopark, Le Forum 1 & 2 260, av. Marie Curie, FR-74160 Archamps, France

Advanced Human Immune System Mouse ModelsTMIn vivo pharmacology services

TODAY: INFLAMMATIONSébastien Tabruyn, PHDCSO

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Advanced Human Immune System MouseTM

NOD-Prkdcem26Cd52 ll2rgemCd22

NCG/NOG/NSG

Life-long reconstitution of full HUMAN immune system

Immuno-deficientMOUSE

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Highest Engraftment Rate and Quality Control

1) chemoablation 2) hCD34+ engraftment

Time(Week)O 14

4-week old animal

Customer Study

Monthly production:450 hu-NCG

QC hCD45+ ≥ 25%Average in 2019: >50%Ø 8/10 hu-mice (total

2450) pass

Quality control:complete flow cytometry panel of

human immune cells

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All Identified Human Immune Cell MarkersImmune populations Flow cytometry markersT cells CD3, CD4, CD8

Central and effector memory T cells CD45RO, CD45RA, CD62L, CCR7

Activated / Exhausted T cells CD69, CD25, TIM3, Lag3, PD-1, 41BB, Ox40, Granzyme B, IFNG, Ki67

Regulatory T cells CD25, FOXP3, CD127

Gamma-Delta T cells Vd2 TCR

Tetramer specific T cells Mart-1, NYESO-1, PRAME, WT-1

NK cells CD56, CD16, NKp46

Myeloid cells CD11b, CD33

Monocytes / macrophages CD14, CD16, CD163, CD86

Myeloid dendritic cells CD11c, CD141, CD209, CD86, HLADR

Plasmacytoid dendritic cells CD123, CD303, CD304, CD86, HLADR

Neutrophils, eosinophils, basophils, mastcells

SSC, CD15, CD16, CD24, CD123, HLADR

B cells CD19, CD20, CD22, IgD, IgM, IgG

Plasma cells, Plasmablasts CD27, CD38, CD138

Tumor cells CD146, PDL1, PDL2

Can’t find your favored?

Let us know and we will check it

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Transient expression for 48-72 hours of selected human cytokines by liver cells:

• Physiological level of expression of the chosen human cytokines• No over-stimulation (not constitutive nor deleterious)• Stable differentiation: up to 3 months downstream effect• Adaptable

For enhanced differentiation/boosting of:

Myeloid/Dendritic Cells: hGM-CSF + hIL3 + hIL4 + FLT3LNK Cells: hIL15

• via hydrodynamic DNA deliveryPlasmid DNA in 2 ml of saline for 10 sec. i.v.

Transient Human Cytokine Boost

-7 3 141

10

100

1000

10000

100000

FLT3L

Days after hydrodynamic boost

pg/m

L

-7 3 1410

100

1000

IL3


pg/m

L

-7 3 141

10

100

IL4


pg/m

L

-7 3 140

20

40

60

GMCSF


pg/m

L

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NOG-EXL: NK cells (1.5%), Monocytes (6.5%), Granulocytes (5%) B-NCG: NK cells (2%), Monocytes (6%), Granulocytes (0.5%)

Level of human cells in peripheral blood (D7 after boost) hu-NCG

T cells

B cells

NK cells

Monocytes

Granulocy

tes

Dendriti

c cell

s0

2

4

6

820406080

% o

f hum

an c

ell

GM-CSF IL3 IL4 FLT3L hu-NCG

T cells

B cells

NK cells

Monocytes

Granulocy

tes

Dendriti

c cell

s0

2

4

6

820406080

% o

f hum

an c

ell

hu-NCG

T cells

B cells

NK cells

Monocytes

Granulocy

tes

Dendriti

c cell

s0

2

4

6

820406080

% o

f hum

an c

ell

IL15 hu-NCG

Myeloid lineage, DC and NK cells Boosting

Non BoostednoBoost: hGM-CSF + hIL3 + hIL4 + FLT3L hIL15none

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Myeloid and DC Boost in hu-NCG

-7 7 -7 70

20000

40000

60000


Cel

ls/m

L

Vehicle

GM-CSF IL3 IL4 FLT3L

x 6

• Peripheral Blood

-7 7 -7 70

20000

40000

60000


Cel

ls/m

L

Vehicle


x 6

-7 7 -7 70

5000

10000

15000

20000

25000


Cel

ls/m

L

Vehicle


x 4

-7 7 -7 70

50000

100000

150000


Cel

ls/m

L

Vehicle


x 13

-7 7 -7 70

1000

2000

3000


Cel

ls/m

L

Vehicle


x 18

-7 7 -7 70

20000

40000

60000

80000


Cel

ls/m

L

Vehicle


x 51

-7 7 -7 70

1000

2000

3000

4000


Cel

ls/m

L

Vehicle


x 3

Myeloid cells(CD11b+)

Monocytes(CD11b+CD14+)

Conventional Dendritic cells(CD11b+Lin-HLADR+CD11c+)

Dendritic cells type 1(CD11b+Lin-HLADR+CD11c+CD141+)

Dendritic cells type 2(CD11b+Lin-HLADR+CD11c+CD1c+)

Plasmacytoid dendritic cells(CD11b+Lin-HLADR+CD11c+CD123+CD303+)

7

Slide #8 TCS – 2019 – Confidential

NK cells

Monocyte

s

Granulo

cyte

s

Dendrit

ic ce

lls0

1

2

3

4

% o

f hum

an c

ell

Myeloid boosthu-NCG

NK cells

Monocyte

s

Granulo

cyte

s

Dendrit

ic ce

lls0

1

2

3

4

% o

f hum

an c

ell

hu-NCG

Tissues were harvested on Day 14 after boost (n=5)

NK cells

Monocyte

s

Granulo

cyte

s

Dendrit

ic ce

lls0

5

10

15

20

% o

f hum

an c

ell

hu-NCG

NK cells

Monocyte

s

Granulo

cyte

s

Dendrit

ic ce

lls0

5

10

15

20

% o

f hum

an c

ell

Myeloid Boosthu-NCG

Bone marrow Spleen Liver

x2.5x2.5

x2.5

NK cells

Monocyte

s

Granulo

cyte

s

Dendrit

ic ce

lls0

5

10

15

20

25

% o

f hum

an c

ell

hu-NCG

NK cells

Monocyte

s

Granulo

cyte

s

Dendrit

ic ce

lls0

5

10

15

20

25

% o

f hum

an c

ell

Myeloid Boost hu-NCG

x2.5

hCD45 = 2.4 x 105 cells/femur hCD45 = 6.7 x 105 cells/femur hCD45 = 9.9 x 106 cells/g hCD45 = 1.5 x 107 cells/g hCD45 = 4.5 x 106 cells/g hCD45 = 7.3 x 106 cells/g

Myeloid and DC Boost in hu-NCG• Bone Marrow, Spleen, Liver

Slide #9 TCS - Confidential

Major advantages of hu-NCG

4. Human cytokine boosts available for increasingthe number of myeloid – NK – dendritic cells

Hydrodynamic injections of plasmid DNA encoding humanM-CSF, GM-CSF, IL3-4-15, FLT3L

2. Lifelong stability (> 18 months) of the immune system

No irradiation – No AnemiaHumanisation following chemoablation

1. No GvHDhigh purity of hCD34+ with < 2% hCD8+

3. Trusted source of human cord bloodEtablissement Français du Sang: EFS BesanconEthic/legal: informed/signed consent by the parents, anonymization, only for R&D purposes

5. Disease models compatibility:I-O: immune check point inh., ADCC; macrophage polarisation, oncolyic bacteria and viruses, vaccination, CRS (cytokine release syndrome)Infectious: HIV, Dengue, hCMVInflammation: IBD chronic and acute, RA, AsthmaImmunization: 100% human IgG

6. Ample historical data12’750 hu-mice produced last 5 years2020: >450 hu-NCG produced per month

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Cancer Immuno-Oncology• Solid tumors • Liquid tumors • PDX• Cytokines Storm (CRS)

Vaccines & ImmunoSafety

• 100% human IgG • Prophylaxis

Infectious Diseases

• HIV • Dengue

• hCMV

InflammationAutoimmune Diseases

• IBD• Asthma

• GVHD (PBMC)

Advanced Human Immune System Mouse ModelsTM

NEW Platform: hu-liver

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Inflammatory Bowel Disease (IBD)

TCS - Confidential

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Current IBD Models Have Limitations

• Chemically induced : DSS, TNBS, oxazolone,…

• Gene-manipulated : IL-2-/-, IL-2R-/-, and IL-10-/-

• Bacteria-infected models

• These models on immunocompetent mice : C57BL/6, BALB/c,…

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NOT APPLICABLE for drug targeting human-specific cells/cytokines/targets

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Human Leukocyte Infiltration in hu-IBD Colitis

Human leukocytes infiltration at the site of inflammation (ALU staining) and human TNF-α secretion (mAb staining)

TCS - Confidential

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hCD4 Infiltration in hu-IBD Colon

TCS - Confidential

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Acute hu-IBD Colitis Protocol

Endpoints or Disease Progression Scores Clinical + Histology + Endoscopy

TCS - Confidential

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Acute hu-IBD Colitis : Peripheral blood

Human Immune cells in Peripheral blood

VehicleProduct-1Product-2

n=10

D0

Vehicle

Nicotine

PMI001

Vehicle

Nicotine

PMI001

0

2×105

4×105

6×105

hCD45/ml

*

D12

A

D7

D0

Vehicle

Nicotine

PMI001

Vehicle

Nicotine

PMI001

0

2×103

4×103

6×103

8×103

Treg/ml

B

D12D7

TCS - Confidential

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Clinical hu-IBD Colitis Scales and Scores

TCS - Confidential

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Scoring hu-IBD Colitis Symptoms with Humira

Body WeightStool consistency

Colon length(D13-Sacrifice)

Bleeding

TCS - Confidential

0 2 4 6 8 10 12 140

1

2

3

4

5

time (days)

body

wei

ght l

oss

scor

e humiraisotype control

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Diminution of hu-IBD Colitis Symptoms with anti-huTNFa

Therapeutic human mAb reduces inflammation in hu-IBD model

TCS - Confidential

0 2 4 6 8 10 12 14012345678910

time (days)

glob

al c

linic

al s

core

humiraisotype control

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Reduction of T-cell Infiltration by Cyclosporin-A in hu-IBD

TCS - Confidential

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3 % DSS

1 2 3 4 5 6 7 8 9 10 11 120

1

2

3

4

5

time (days)

Bod

y W

eigh

t Los

s S

core

A

1 2 3 4 5 6 7 8 9 10 11 120

1

2

3

4

5

time (days)

Dia

rrhe

a S

core

3 % DSS

B

****

***

***

1 2 3 4 5 6 7 8 9 10 11 120

1

2

3

time (days)

Rec

tal B

leed

ing

Sco

re

3 % DSS

C

*1 2 3 4 5 6 7 8 9 10 11 12

0

2

4

6

8

10

12

time (days)

Glo

bal C

linic

al S

core

3 % DSS

****

*

D


n=10

Acute hu-IBD Colitis – Case Study with Nicotin Derivatives

Clinical Scores

TCS - Confidential

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Acute hu-IBD Colitis – Colon Shortening


n=10

Vehicle Nicotine PMI00140

60

80

100

120

Col

on L

engt

h (m

m)

A

Vehicle Nicotine PMI00140

60

80

100

120

Col

on L

engt

h (m

m)

*

BD7 D12

TCS - Confidential

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Histopathology hu-IBD Colitis Scales and Scores

TCS - Confidential

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Inflammation and Ulceration in DSS induced hu-IBD

TCS - Confidential

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Acute hu-IBD Colitis – Case Study

Histological Score (D7) – Distal Part


n=10

Vehicle Nicotine PMI0010.0

0.5

1.0

1.5

Infla

mm

atio

n (S

core

)

A

*


0.5

1.0

1.5

Tiss

ue d

amag

e (S

core

)

B

**


0.5

1.0

1.5

2.0

Mon

onuc

lear

infil

tratio

n (S

core

)

C

*

TCS - Confidential

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hu-IBD Colitis MEICS (Murine Endoscopic Index of Colitis Severity) Scale

Advantages:

• Detailed description of colonic mucosa • Mucosal lesion follow-up over time • Early and accurate appearance of colitis compared to the clinical score alone. • Real time treatment efficacy on evolution of the lesions after drug treatment• Good correlation with histological analysis

days after induction

0

5

10

15

0

3

6

9

0 2 4 6

051015

0369

02

46

clin

ical

sco

reen

dosc

opic

sco

re

051015

0369

02

46

clin

ical

sco

reen

dosc

opic

sco

re

Acute DSS model

Martin-Rodriguez et al., in preparation

TCS - Confidential


Thickening of the colon

MEICS 0 1 2 3

Vascular Pattern

Fibrin visible

Granularity of the

mucosa surface

Stool consistence

/3

/3

/3

/3

/3

Murine EndoscopicIndex of Colitis Severity

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Acute hu-IBD Colitis – Case StudyEndoscopy scoring

TCS - Confidential

0 3 6 9 120

2

4

6

8

10

Day

Global Endoscopy Score

*

0 3 6 9 120

1

2

3

Day

Colon thickening

**

*

0 3 6 9 120

1

2

3

Day

Vascular pattern

*

0 3 6 9 120

1

2

3

Day

Visible Fibrin

0 3 6 9 120

1

2

3

Day

Granulosity of mucosal surface

*

0 3 6 9 120

1

2

3

Day

Stool consistency

*

Ab-1Vehicle

n=10

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Chronic hu-IBD Colitis Protocol

Endpoints or Disease Progression Scores Clinical + Histology + Endoscopy

TCS - Confidential

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Diminution of Chronic hu-IBD Colitis with CyclosporinA

0 10 20 30 400

2

4

6

8

10

time (days)

glob

al c

linic

al s

core

Cyclosporin ACtrl

3%DSS 3%DSS 3%DSS

0 10 20 30 400

1

2

3

4

time (days)di

arrh

ea s

core

3%DSS 3%DSS 3%DSS

0 10 20 30 400

1

2

3

4

5

6

time (days)

body

wei

ght s

core

3%DSS 3%DSS 3%DSS

Body WeightStool consistency

Global Clinical Score

Reduction of symptoms by administration of cyclosporin-A

TCS - Confidential

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Microbiome/MicrobiotaMicrobiome manipulations:

• fecal microbiota Transfer (FMT)• oral administration of in vitro expanded bacteria (pups/adults)• antibiotic treatments

• IBD (conventional and hu-mouse)• GVHD (engraftment of PBMC)• Immuno-oncology (Check-point inh. efficacy in hu-mouse)• Immuno-modulation (via flow cytometry)

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General Guidelines for IBD models

• Study size: at least 10 hu-IBD mice per group• Gender: female animals are recommended• Drug validation: each batch of DSS is calibrated• Animal randomization by: humanization rate, CD34+ donor• Positive controls and route of administration: CSA, anti-human TNFa

TCS - Confidential

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Cancer Immuno-Oncology• Solid tumors • Liquid tumors • PDX• Cytokines Storm (CRS)

Vaccines & ImmunoSafety

• 100% human IgG • Prophylaxis

Infectious Diseases

• HIV • Dengue

• hCMV

InflammationAutoimmune Diseases

• IBD• Asthma

• GVHD (PBMC)

Advanced Human Immune System Mouse ModelsTM

NEW Platform: hu-liver

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Involvement of Immune Cells in Asthma

1. Allergen captured by DC 2. DC present antigen to T cells to induce Th23. Th2 promote IgE production by B cells4. IgE bind to mast cells and basophils à local allergic

response5. Recruitment of eosinophils6. Induction of airway hyper-responsiveness, mucus

production, tissue damage

TCS - Confidential

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Presence of human DC, Mast Cells, Eosinophils, Basophils, Neutrophilsin Lungs

TCS - Confidential

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Asthma Induction Model in hu-mouse Models

Boosted IL-3 / GM-CSF hu-Mouse

TCS - Confidential

Study Design 1

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OVA challenge in Asthmatic hu-mouse Models

hCD45 – Total human leukocytes

PBSOVA

0

500

1000

1500

hCD45 in BALF

hCD

45 /

milli

on c

ells

BALF Lungs

TCS - Confidential

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OVA challenge increases neutrophils, Mast Cells and Basophils in lungs of hu-mouse

Neutrophils

PBSOVA

1000

10000

100000

mast cells/100 mg lung

Cel

ls /

100

mg

lung

Mast cells

PBSOVA

10

100

1000

basophils/100 mg lung

Cells

/ 10

0 m

g lun

g

Basophils

PBSOVA

10

100

1000

10000

eosinophils/100 mg lung

Cel

ls /

100

mg

lung

Eosinophils

TCS - Confidential


0 16 70

IMMUNIZATION PHASEOVA/alumPBS

77

Boost IL-3, IL-4, GM-CSF, FLT3-L

93

CHALLENGE PHASEOVA acuteOVA chronicPBS

7 23 30 37 44 51 65days

Asthma Induction Model in hu-mouse ModelsStudy Design 2

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IgE Production

TCS - Confidential

D14 D28 D42 D56 D70 D9310

100

1000

total IgE in plasma

tota

l IgE

, ng/

mL

PBS OVA acute OVA chronic

immunizations challenges

***

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H&E Staining

TCS - Confidential

bronchiole

blood vessel

alveolar duct

alveoli

terminal bronchiole

respiratory epithelium(ciliated cells, gobletcells…)

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Respiratory epithelium thickening

TCS - Confidential

PBS OVA chronic OVA acute

• Cellular Density• Globlet Cells – Mucus Production

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Patrick NEFPhD. CEO

Romain GRETDirector Business

Development

Sébastien TABRUYNPhD. CSO

Kiave-Yune HO WANG YIN, PhD. Director Innovation

Catherine VERHAEGHE, PhD.Director Operations

Hendrik CLAEYSGlobal Sales Manager

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Expertise• Full reconstitution of the human immune

system/functions in hu-mouse models • Protocol designs, tailor-made• Flow cytometry, histology, small animal

imaging, cell biology state-of-the-art• Data collection, analysis, reports

Application• Preclinical candidate selection• Efficacy models for cancer (I-O, PDX), infectious

(HIV, Dengue), inflammation (IBD), Vaccines (100% human IgG), immuno-Safety/Toxicity

• Combination strategy

About us• Bio Safety Level -2 and -3 animal and lab. certified

facilities (740 m2) + offices (200 m2) • High quality and flexibility• Complete Fee for Services• Attractive Pricing

European CRO located in France Near Geneva (Switzerland) 42 FTE, 11 PhD

Who We Are & What We Do

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Laboratory Analytical Platforms• Flow cytometry

• immuno-histochemistry (IHC)

• in vivo imaging for small animals

• TP1020 Automatic Tissue Processor• Microtome: HistoCore Arcadia H• Leica Aperio Versa (8 slides)• paraffin inclusion; cryosection; H&E staining; Histopathology; Immunohistology:• Chromogenic and Fluorescence; Digitalization and image Quantification

• Vilber Newton 7.0 (Bioluminescence – up to 5 animals)• Luciferase and autobioluminescence tumor growth monitoring

• Attune NxT Flow Cytometer (n=2)• Complete tissue and blood sample analysis• 14 channels each for complex human immune cell panel detection• Cell Sorter: BD FACSMelody TM Cell sorter

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One-Stop-Shop Fee-for-ServicesStudyDesign

Protocol Execution

Data Analysis Deliverables

§ Tailor-made protocols (budget, timeline, experimental plan)

§ Quotes§ SOP driven§ Designated Project

Officer§ Clear Role and

Responsibility

§ Raw data transfer§ Final Reporting with

expert analysis & discussion

§ Feedback

§ BSL 2 & 3 animal facility

§ State-of-the-art flow cytometry, histology, imaging, cellular biology

§ Direct access to your Project Officer

§ Frequent interim reporting + draft reports

§ Raw data collection§ Data analysis

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Our Next Webinar: 2020 SeriesOur Next Webinar 2020 SeriesJune 25th 2020: Infectious diseases: full human system mouse models for HIV, Dengue, CMV and Corona virus.

Slide #48

NOW: Q&A Session

Type your question

Thank you for your attention

Documents

Advanced Human Immune System Mouse Models · I-O: immune check point inh., ADCC; macrophage polarisation, oncolyicbacteriaand viruses, vaccination, CRS (cytokine release syndrome)