Syngeneic Models - Oncodesign · Syngeneic Models at Oncodesign Model Organ Host rodent species Injection site ... A20 B cell lymphoma Model Mice were SC injected with A20 tumor cells

Syngeneic Models

for Drug-Host Immune System Interactions Study

December 2015

About Syngeneic Models…

Rodent tumor cells

• Express the mouse/rat homologues of the targets

• Tumor tend to grow fast

Long history of use

• Strong baseline of drug response data

• Wide variety of tumor types

Easy study

• Drug-host immune system interaction study

• Relatively low cost

• Reproducible

page 2 Confidential 2/9/2016

SYNGENEIC MODELS AT

ONCODESIGN



Syngeneic Models at Oncodesign

Model Organ Host rodent

species

Injection

site Tested drugs

Metastatic

Dissemination

4T1 Breast carcinoma Balb/c mouse OT Radiotherapy, Doxorubicin, PD-1 mAb,

CTLA-4 mAb

A20 B cell lymphoma Balb/c mouse SC IV CTLA-4 mAb, PD-1 mAb

AB12 Mesothelioma Balb/c mouse SC

AY27 Bladder transitional

carcinoma Ficher rat OT BCG, CDDP

B16-F10 Malignant melanoma C57Bl/6 mouse SC IV IC Paclitaxel, Doxorubicin, TMZ, PD-1 mAb,

Cyclophosphamide, CTLA-4 mAb, PD-L1 mAb

C1498 AML C57Bl/6 mouse SC IV

C26 Colon adenocarcinoma Balb/c mouse IP SC CDDP

C38 Colon carcinoma C57Bl/6 mouse SC Radiotherapy, CPT-11, L-OHP, CTLA-4

C-51 Colon carcinoma Balb/c mouse SC CPT-11, LOHP

C6 Glioma Wistar rat OT SC BCNU

CT26 Colon carcinoma Balb/c mouse OT SC 5-FU, CPT-11, PD-1 mAb, CTLA-4 mAb,

PD-L1 mAb

*DHD/K12/TRb =

PROb Colon adenocarcinoma BDIX rat IP SC

CDDP, Doxorubicin, BCG, 5-FU, L-OHP,

Mitoxantrone

*DHD/K12/TSb =

REGb Colon adenocarcinoma BDIX rat SC

EMT6 Breast carcinoma Balb/c mouse OT SC Doxorubicin, Paclitaxel, Radiotherapy, PD-1

mAb, CTLA-4 mAb

GS-9L Gliosarcoma Ficher rat OT SC BCNU, TMZ

Good characterization

Characterization not complete

Limited characterization

* Ask for rat availability

Syngeneic Models at Oncodesign

Model Organ Host rodent

species

Injection

site Tested drugs

Metastatic

Dissemination

*GV1A1 Glioma BDIX rat OT SC

Hepa1-6 Hepatocarcinoma C57Bl/6 mouse OT Sorafenib, CTLA-4 mAb

L1210 Lymphocytic leukemia DBA/2 mouse IP BCNU, CDDP, Doxorubicin, 5-FU

L-OHP, Mitoxantrone

L1210/CDDP Lymphocytic leukemia DBA/2 mouse IP CDDP, L-OHP

LLC1 Lewis lung carcinoma C57Bl/6 mouse IV SC Vinorelbine, Cyclophosphamide

CTLA-4 mAb, PD-1 mAb

MAT-LyLu Prostate adenocarcinoma Cop rat SC

MBT-2 Bladder carcinoma C3H mouse OT SC BCG, Mitomycin C, PD-1

MPC-11 Plasmacytoma Balb/c mouse SC

NBT-II Bladder carcinoma Wistar rat OT

P388 Leukemia DBA/2 mouse IP BCNU, Doxorubicin,

Mitoxantrone

P388/ADR Leukemia DBA/2 mouse IP BCNU, Doxorubicin,

Vinblastine

R3327-AT3 Prostate adenocarcinoma Cop rat SC

R3327H Prostate adenocarcinoma Cop rat OT SC

Paclitaxel, Mitoxantrone, Trastuzumab,

Leuroprolin, Castration, Decapeptyl,

Bicalutamide, Radiotherapy

Renca Kidney carcinoma Balb/c mouse OT SC CDDP, Paclitaxel, Radiotherapy,

Gemcitabine, Carboplatin, Sorafenib

Good characterization

Characterization not complete

Limited characterization

page 5 Confidential 2/9/2016 * Ask for rat availability

Chemically Induced Tumor Models

Understanding the progression of cancer and determining the most appropriate

treatment periods

Determination of efficacy when using chronic treatment approaches - preventive or

curative


Model Organ Species Tested drugs

DMH-induced Colon Rat

DMBA-induced Breast Rat Tamoxifen, Bromocriptine, Ovariectomy, Letrozole, Faslodex

Skin painting Skin Mice

DEN/AAF-induced Liver Rat

MNNG-induced Colon Rat

AOM-induced Colon Rat

BenzoPyrene-induced Stomach Mice

BenzoPyrene-induced Lung Mice

NNK-induced Lung Mice

Chemically Induced Tumor Models

DEN/AAF-induced hepatic lesions

Initiation: 1 IP injection of DEN

Promotion: hepatectomy followed by repeated

oral administrations of 2-AAF for 6 weeks

Tumor uptake: 30%

Skin painting: cutaneous tumor model in

hairless SKH-1 mice

Initiation: 1 topical application of DMBA

Promotion: 2 applications of TPA/week, for 5

consecutive weeks

Progression in carcinoma: application of 4-

NQO


Histology of the 2 steps of papilloma

development in skin-painting tumor models.

MRI performed at D120. Lesions are ~200 µm

to 1mm in diameter

VALIDATED SYNGENEIC MODELS

AND ASSOCIATED DATA


0

25

50

75

100

125

150

175

Lung metastasis Liver metastasis

Media

n a

nd m

eta

stasi

s num

ber

repart

itio

n (

D28)

per

mouse

4T1Balb/c Female MiceMfp engrafment at D0

Sacrifice: D28

(C)

4T1 Breast Carcinoma Model

Positive control: Doxorubicin

Other tested drugs: Radiotherapy,

CTLA-4 mAb, PD-1 mAb


(X5)

(X10)

Blood vessel

Metastases at

the vessel periphery

0

200

400

600

800

1000

1200

1400

1600

1800

2000

0 5 10 15 20 25 30

Mean t

um

or

volu

me (

mm

3)

Time post tumor cell injection (days)

Vehicle

Doxorubicin 4 mg/kg IV Q7Dx3


4T1Balb/c Female MiceMfp engrafment at D0Treatment starts at D7

Mice were OT injected with 4T1 murine breast tumor

cells at D0. Mice were randomized based on tumor

volume at D11 and treated IV with vehicle or

Doxorubicin at 4 or 8 mg/kg/inj (Q7Dx3).

Histological analysis of the liver of 4T1-bearing mice

confirms the presence of macro and micro metastases.

Liver sections were stained with hematoxylin-eosine

(HE, magnification x5 or x10).


4T1 OT mouse model, CTLA-4 mAb and PD-1 mAb pilot efficacy study

Lung metastases number at D25 and D32


Nu

mb

er o

f lu

ng

met

ast

ass

e (D

25

)

0

2

4

6

8

0

20

40

60

80

100

Nu

mb

er o

f lu

ng

met

ast

ass

e (D

32

)

Gp1 Not treated

Gp2 Anti-CTLA4 10 mg/kg IP TWx2

Gp3 Anti-PD1 10 mg/kg IP TWx2



Mice were OT injected with 4T1 murine breast tumor cells

at D0. Mice were randomized based on tumor volume at

D13 and treated IP with mAb against CTLA-4 (clone 9H10,

Syrian Hamster IgG1) or PD-1 (clone RMP1-14, Rat IgG2a) at

10 mg/kg/inj (Q5Dx3).

Immune checkpoint inhibitors

0

200

400

600

800

1 000

1 200

1 400

1 600

1 800

2 000

0 5 10 15 20 25 30

Tum

or

volu

me (

mm

3)

Time (Days)

Control

0

200

400

600

800

1 000

1 200

1 400

1 600

1 800

2 000

0 5 10 15 20 25 30

Tum

or

volu

me (

mm

3)

Time (Days)

CTLA4 mAb

0200400600800

1 0001 2001 4001 6001 8002 000

0 5 10 15 20 25 30

Tum

or

volu

me (

mm

3)

Time (Days)

PD1 mAb

T/C % (D21)

Control 100

CTLA4 mAb 66

PD1 mAb 68


Mice were OT injected with 4T1 murine breast tumor cells

at D0. Mice were randomized based on tumor volume at

D13 and treated IP with mAb against CTLA-4 (clone 9H10,

Syrian Hamster IgG1) or PD-1 (clone RMP1-14, Rat IgG2a) at

10 mg/kg/inj (Q5Dx3).


0

200

400

600

800

1 000

1 200

1 400

1 600

1 800

2 000

0 5 10 15 20 25 30

Tum

or

volu

me (

mm

3)

Time (Days)

Control

0

200

400

600

800

1 000

1 200

1 400

1 600

1 800

2 000

0 5 10 15 20 25 30

Tum

or

volu

me (

mm

3)

Time (Days)

CTLA4 mAb

0200400600800

1 0001 2001 4001 6001 8002 000

0 5 10 15 20 25 30

Tum

or

volu

me (

mm

3)

Time (Days)

PD1 mAb


CD4+ CD8a+ CD4+FoxP3+

(n=8)

Mean 52.3 16.8 18.2

SD 5.8 8.7 5.1

Median 52.9 13.9 19.3

Mean 49.3 17.0 17.9

SD 3.9 4.3 4.1

Median 47.8 17.0 17.0

Mean 49.9 18.7 16.3

SD 3.7 6.8 2.8

Median 51.4 16.1 17.4

(% of CD3+)

Control

CTLA-4

mAb

PD-1

mAb

4T1 Breast Carcinoma Model, Treg quantification

TDLN (ID3213 untreated)


Tumor (ID3213 untreated)

Quantification of tumor infiltrating T cells at sacrifice

(D30) by FACS.



FACS analysis of CD45+, CD11b+, Gr1+, Ly6C+, Ly6G+ MDSC cells

Tumor MDSCs

G-MDSC

M-MDSC

Ly-6G

Ly-6

C

CD11b

Gr1

0

500

1 000

1 500

2 000

0 5 10 15 20 25 30 35 40 45

Tum

or

volu

me (

mm

3)

Time (Days)

1.10e6 cells

A20 B cell lymphoma Model

Ongoing development

SC model


IV model

A20 B cell lymphoma Model

Mice were SC injected with A20 tumor cells at

D0. Mice were randomized based on tumor

volume at D9 and treated with IP injections of

vehicle or of mAb against CTLA-4 (clone 9H10,

Syrian Hamster IgG1) or PD-1 (clone RMP1-14,

Rat IgG2a) at 10 mg/kg/inj (Q4Dx3).

Immune checkpoint inhibitor: CTLA-4 and PD-1 mAbs


T/C % (D23)

Control 100

CTLA4 mAb 25

PD1 mAb 37

0

500

1 000

1 500

2 000

2 500

0 10 20 30 40

Tum

or

volu

me (

mm

3)

Time (Days)

Vehicle

0

500

1 000

1 500

2 000

2 500

0 10 20 30 40Tum

or

volu

me (

mm

3)

Time (Days)

PD-1 mAb

0

500

1 000

1 500

2 000

2 500

0 10 20 30 40

Tum

or

volu

me (

mm

3)

Time (Days)

CTLA-4 mAb

AY-27 Bladder Carcinoma Model

Positive control: CDDP

Other tested drugs: BCG


Rat were OT injected with AY-27 rat

tumor cells at D0.

T1-weighted MRI image at D25

Sagittal orientation

(yellow : bladder, orange: tumor)

AY-27 Bladder Carcinoma Model



0

500

1000

1500

2000

Group medians

Time post tumor induction (days)

TV

(m

m3)

31 41

G5 CDDP 2 mg/kg IP Q7Dx3

G1 Vehicle PO Q1Dx28

0

500

1000

1500

2000



TV

(m

m3)

31 410

500

1000

1500

2000

G5 CDDP 2 mg/kg IP Q7Dx3


TV

(m

m3)

31 41

B16-F10 Malignant Melanoma Model

Positive control: Paclitaxel

Other tested drugs: Doxorubicin, TMZ


0

500

1000

1500

2000

2500

0 3 6 9 12 15 18


Mean t

um

or

volu

me (

mm

3)

Vehicle

Paclitaxel 15 mg/kg IV Q4Dx4

B16F10

C57BL/6 Mouse

Tumor Adm.: Cells - SC

Treatment: Start at D8

Mice were SC injected with B16F10 mouse tumor cells at

D0. Mice were randomized based on tumor volume at D8

and treated with IV injections of vehicle or paclitaxel at

15 mg/kg/injection (Q4Dx4).

Other tested drug: CTLA-4 mAb

Mice were SC injected with B16F10 murine tumor cells

at D0. Mice were randomized based on body weight at D3

and treated IP with mAb against CTLA-4 (clone 9H10) at

100 µg/inj (Q4Dx3)

0

200

400

600

800

1 000

1 200

1 400

1 600

1 800

2 000

0 5 10 15 20 25 30

Tum

or

volu

me (

mm

3)

Time (Days)

Control

0

200

400

600

800

1 000

1 200

1 400

1 600

1 800

2 000

0 5 10 15 20 25 30

Tum

or

volu

me (

mm

3)

Time (Days)

CTLA4 mAb

0

500

1 000

1 500

2 000

2 500

0 5 10 15 20 25 30 35

Tum

or

volu

me (

mm

3)

Time (Days)

Control

0

500

1 000

1 500

2 000

2 500

0 5 10 15 20 25 30 35Tum

or

volu

me (

mm

3)

Time (Days)

PD-L1

0

500

1 000

1 500

2 000

2 500

0 5 10 15 20 25 30 35

Tum

or

volu

me (

mm

3)

Time (Days)

CTLA-4



Mice were SC injected with B16F10 mouse tumor cells at D0. Mice were randomized based on body weight at

D5 and treated IP with mAb against CTLA-4 (clone 9H10) at 10 mg/kg (TWx2) or against PD-L1 (clone 10F.9G2)

at 10 mg/kg/inj (Q2Dx9).

Combined Immune checkpoint inhibitors

0

500

1 000

1 500

2 000

2 500

0 5 10 15 20 25 30 35

Tum

or

volu

me (

mm

3)

Time (Days)

CTLA-4 + PD-L1


Positive control: Temozolomide


Mice were intracranially injected with B16F10 mouse tumor

cells at D0. Mice were randomized based on body weight at

D3 and treated with PO administrations of vehicle or

temozolomide at 50 mg/kg/administration once daily for 5

consecutive days (treatment schedule Q1Dx5).

0

20

40

60

80

100

0 5 10 15 20 25 30


Surv

ival (%

)

Vehicle

Temozolomide 50 mg/kg PO Q1Dx5

B16F10C57BL/6 Mice

Tumor Adm.: Cells - Icran


0

25

50

75

100

125

150

175

200

225

250

275

300

Cyclophosphamide 200 mg/kg IP Q7Dx2

Vehicule

Media

n a

nd L

ung m

eta

stasi

s num

ber

repart

itio

n (

D14)

per

mouse

B16-F10C57BI/6J Female MiceIV injection at D0

Treatment start at DD

Sacrifice: D14

Mice were IV injected with B16-F10 mouse tumor cells at

D0. Mice were randomized based on body weight at D0 and

treated with IP administrations of vehicle or

Cyclophosphamide at 200 mg/kg/administration (Q7Dx2).

Mice were sacrificed at D14 and the number of lung

metastases was evaluated.

Positive control: Cyclophosphamide

C1498 AML Model

Ongoing development

SC model


IV model

0

500

1 000

1 500

2 000

2 500

0 5 10 15 20 25

Tum

or

volu

me (

mm

3)

Time (Days)

2.10e6 cells

C38 Colon Carcinoma Model

Positive control: Oxaliplatin (L-OHP)

Other tested drugs: Radiotherapy, CPT-11, 5-FU, CTLA-4


0

500

1000

1500

2000

2500

3000

10 20 30 40 50

Time post tumor fragment implantation (days)

Mean t

um

or

volu

me (

mm

3)

Vehicle

l-OHP 5 mg/kg IV Q7Dx3

l-OHP 12 mg/kg IV Q7Dx3C38

C57Bl/6 Mice

Tumor Adm.: Fragments - SC


Mice were SC xenografted with C38 mouse colon tumor

fragments at D0. Mice were randomized based on

tumor volume at D11 and treated with IV injections of

l-OHP at 5 or 12 mg/kg/injection (Q7Dx3).


C38 Colon Carcinoma Model

0

500

1 000

1 500

2 000

2 500

0 10 20 30

Tum

or

volu

me (

mm

3)

Time (Days)

Vehicle


(ongoing study)

T/C % (D24)

CTLA-4 mAb 1

PD-1 mAb 11

0

500

1 000

1 500

2 000

2 500

0 10 20 30

Tum

or

volu

me (

mm

3)

Time (Days)

CTLA-4 mAb

0

500

1 000

1 500

2 000

2 500

0 10 20 30

Tum

or

volu

me (

mm

3)

Time (Days)

PD-1 mAb

Mice were SC xenografted with C38 mouse colon tumor fragments at D0. Mice were randomized based on tumor volume

at D6 and treated with IP injection of mAb against CTLA-4 (clone 9H10, Syrian Hamster IgG1) at 10 mg/kg/inj (Q5Dx3),

or mAb against PD-1 (clone RMP1-14, Rat IgG2a) following the schedule TWx2

C6 Glioma Model

Positive control: BCNU IC50 (µM) : 228


NMR Biomed. 2008

Nov;21(10):1043-56.

J Cereb Blood Flow Metab.

2009 Oct;29(10):1714-26.

In vitro cytotoxicity of

BCNU treatment : 96h

1 10 100 1000 100000

50

100

C6

DL50 = 228 M

[BCNU] M

Surv

ivin

g c

ells (%

)

AACR 2006

#4372

AACR 2004

#947

0

20

40

60

80

100

0 20 40 60 80 100 120


Surv

ival (%

)

Vehicle

BCNU 10 mg/kg IV Q14Dx2

C6Wistar Rats

Tumor Adm.: Cells - OT


Rats were randomized based on body weight at D17 and treated with

IV injections of vehicle or BCNU at 10 mg/kg/injection (Q14Dx2).

http://www.ncbi.nlm.nih.gov/pubmed/18615861

http://www.ncbi.nlm.nih.gov/pubmed/19584891

http://www.oncodesign.com/assets/files/posters/2006/AACR2006_Inserm_OncoD.pdf

http://www.oncodesign.com/assets/files/posters/2005-2004/AACR2004_Inserm_OncoD.pdf

C6 Glioma Model

MRI monitoring

Gene expression


D17

C6

Edema:

Intratumoral

Periphery

+

+

Cell density ++

Limits sharp

Necrosis yes

Pseudo-cysts no

Nuclei aspects small-

round

Vessel density

/normal tissue

<

Vessel size

/normal tissue

>

Collagen IV

over-expression

± ?

Vessel

- perfusion

- permeability

most

yes

pSTAT3

(92KD)

EGFR

(170KD)

PTEN

(50KD)

C6 - - +

OT injection of C6 glioma cells

MRI Imaging at 2.35 T

Randomization when tumor

volume = 50-75 mm3 using T2-

weighted SE

CT-26 Colon Carcinoma Model

Positive control: Paclitaxel or CTLA-4 mAb

Other tested drugs: CPT-11, CTLA-4 mAb, PD-1 mAb


Mice were SC injected with CT26 rodent tumor cells at D0. Mice were

randomized based on tumor volume at D10 and treated with

• IV injections of vehicle or 5-FU at 25, 50 and 100 mg/kg/injection (Q7Dx3)

• IP injection of CPT-11 at 25 and 50 mg/kg/injection (Q1Dx5).

Metastases observed after orthotopic

injection of CT-26 fragments. The mean

survival time of mice is 20 +/- 6 days. > 80%

of mice develop metastases in the liver,

spleen & lymph nodes.

Hepatic

metastasis

Splenic

metastasis 0

500

1000

1500

2000

0 5 10 15 20 25

Mean t

um

or

volu

me (

mm

3)


Vehicle IV Q7Dx3

5-FU 25 mg/kg IV Q7Dx3



CPT-11 25 mg/kg IP Q1Dx5

CPT-11 50mg/kg IP Q1Dx5

CT26Balb/c MiceTumor Adm.: Cells - SC



Mice were SC injected with CT-26 murine colon tumor

cells at D0. Mice were randomized based on tumor volume

at D7 and treated IP with mAb against CTLA-4 (clone 9H10)

or PD-1 (clone RMP1-14) at 10 mg/kg/inj (Q5Dx3).

Immune checkpoint inhibitors T/C % (D21)

Control 100

CTLA4 mAb 11

PD1 mAb 95 0

200

400

600

800

1 000

1 200

1 400

1 600

1 800

2 000

0 5 10 15 20 25

Tum

or

volu

me (

mm

3)

Time (Days)

Control

0

200

400

600

800

1 000

1 200

1 400

1 600

1 800

2 000

0 5 10 15 20 25

Tum

or

volu

me (

mm

3)

Time (Days)

CTLA4 mAb

0200400600800

1 0001 2001 4001 6001 8002 000

0 5 10 15 20 25

Tum

or

volu

me (

mm

3)

Time (Days)

PD1 mAb



Immune checkpoint inhibitors : FACS analysis of FoxP3+ CD4+ T cells

Tumor

Median value Median value

Tumor


Control

ID#5711

CTLA-4 mAb

ID#5719

CD45

CD

3

CD

8

CD4

FoxP3

CD4



Tumor



Mice were SC injected with CT-26 murine colon tumor cells at D0. Mice were randomized based on tumor volume

at D7 and treated IP with mAb against CTLA-4 (clone 9H10) or PD-1 (clone RMP1-14) at 10 mg/kg/inj (TWx2).

Combined Immune checkpoint inhibitors

0

500

1 000

1 500

2 000

2 500

0 10 20 30 40 50 60 70

Tum

or

volu

me (

mm

3)

Time (Days)

Controls

0

500

1 000

1 500

2 000

2 500

0 10 20 30 40 50 60 70

Tum

or

volu

me (

mm

3)

Time (Days)

PD-1 mAb

0

500

1 000

1 500

2 000

2 500

0 10 20 30 40 50 60 70

Tum

or

volu

me (

mm

3)

Time (Days)

CTLA-4 mAb

0

500

1 000

1 500

2 000

2 500

0 10 20 30 40 50 60 70Tum

or

volu

me (

mm

3)

Time (Days)

PD-1 + CTLA-4 mAbs

PR: 2

CR: 0

PR: 3

CR: 7 PR: 2

CR: 8



Mice were SC injected with CT-26 murine colon tumor cells at D0. Mice were randomized based on tumor volume at

D7 and treated IP with mAb against CTLA-4 (clone 9H10) or PD-1 (clone RMP1-14) at 10 mg/kg/inj (TWx2). CT-26

tumor challenge was done at D51 on 5 mice per group (cured from the primary tumor).

Tumor challenge and memory response

0

500

1 000

1 500

2 000

2 500

0 10 20 30 40 50 60 70

Tum

or

volu

me (

mm

3)

Time (Days)

CTLA-4 mAb

0

500

1 000

1 500

2 000

2 500

0 10 20 30 40 50 60 70

Tum

or

volu

me (

mm

3)

Time (Days)

PD-1 + CTLA-4 mAbs

PR: 3

CR: 7

PR: 2

CR: 8

0

100

200

300

400

500

0 5 10 15 20 25

Tum

or

volu

me (

mm

3)

Time (Days)

CTLA-4 mAb

Growth: 2/5

0

100

200

300

400

500

0 5 10 15 20 25Tum

or

volu

me (

mm

3)

Time (Days)

PD-1 + CTLA-4 mAbs

Growth: 0/5

0

500

1 000

1 500

2 000

2 500

0 5 10 15 20 25

Tum

or

volu

me (

mm

3)

Time (Days)

PD-L1 mAb


Immune checkpoint inhibitor: PD-L1 mAb

Mice were SC injected with CT-26 murine colon tumor

cells at D0. Mice were randomized based on tumor volume

at D9 and treated IP with mAb against PD-L1(clone

10F.9G2) at 10 mg/kg/inj (Q2Dx8).

T/C % (D22)

Control 100

PD-L1 mAb 36

0

500

1 000

1 500

2 000

2 500

0 5 10 15 20 25

Tum

or

volu

me (

mm

3)

Time (Days)

Control


DHD/K12/TRb = PROb Colon Adenocarcinoma Model (rat

availability issue)

Positive control: Oxaliplatin (L-OHP), BCG

Other tested drugs: Cisplatin, Doxorubicin, BCG, 5-FU, L-OHP, Mitoxantrone


0

20

40

60

80

100

0 40 80 120 160 200 240

Surv

ival

(%)


Vehicle

l-OHP 1 mg/kg IP

l-OHP 2 mg/kg IP

l-OHP 4 mg/kg IP

BCG 2.5 mg/kg IP

DHD/K12/PRObBDIX RatsTumor Adm.: Cells - IPTreatment start at D3

Rats were IP injected with PROb cells at D0. Rats were

randomized based on body weight at D3 and treated

with IP injections of vehicle, l-OHP at 1, 2 or 4

mg/kg/injection, or BCG at 2.5 mg/kg/injection three

times a week for 3 consecutive weeks.

AACR 2012

#5383

http://www.oncodesign.com/assets/files/posters/2012/ONCODESIGN_Preclinical immune response_AACR2012.pdf

EMT6 Breast Carcinoma Model

Positive control: Doxorubicin

Other tested drugs: Paclitaxel, radiotherapy, CTLA-4 mAb, PD-1 mAb


0

500

1000

1500

2000

0 10 20 30

Mean t

um

or

volu

me (

mm

3)


Vehicle



EMT-6Balb/c MiceTumor Adm.: Cells - SCTreatment: Start at D11

Mice were SC injected with EMT-6

murine breast tumor cells at D0. Mice

were randomized based on tumor volume

at D11 and treated with IV injections of

Doxorubicin (Q4Dx4).



Mice were SC injected with EMT6 murine breast tumor cells

at D0. Mice were randomized based on tumor volume at D7

and treated IP with mAb against CTLA-4 (clone 9H10) or

PD-1 (clone RMP1-14) at 10 mg/kg/inj (Q5Dx3).


0200400600800

1 0001 2001 4001 6001 8002 000

0 5 10 15 20 25 30

Tum

or

volu

me (

mm

3)

Time (Days)

Control

0200400600800

1 0001 2001 4001 6001 8002 000

0 5 10 15 20 25 30

Tum

or

volu

me (

mm

3)

Time (Days)

PD1 mAb

0200400600800

1 0001 2001 4001 6001 8002 000

0 5 10 15 20 25 30

Tum

or

volu

me (

mm

3)

Time (Days)

CTLA4 mAb

0

200

400

600

800

1 000

1 200

1 400

1 600

1 800

0 5 10 15 20 25 30

Tum

or

volu

me (

mm

3)

Time (Days)

Control

CTLA4 mAb

PD1 mAb

T/C % (D27)

Control 100

CTLA4 mAb 4

PD1 mAb 63




TDLN

Control

ID#8923

TV 597 mm3

CTLA mAb

ID#8901

TV 56 mm3

Median value

Median value without non-responding tumors ()

TDLN Tumor

CTLA4 mAb Non-responding tumors



FACS analysis of CD11b+, Ly6C+, Ly6G+ MDSC cells

Tumor MDSCs

G-MDSC

M-MDSC

Ly-6G

Ly-6

C

CD45

CD

11b

GS-9L Gliosarcoma Model



0

20

40

60

80

100

0 2 4 6 8 10 12 14 16


Surv

ival (%

)

Vehicle

BCNU 10 mg/kg IV Q14Dx2

9LF344 Fisher Rats



Rats were OT injected with GS-9L rat tumor cells at

D0. Rats were randomized based on body weight at D3

and treated with IV injections of vehicle or BCNU at


Radiology 2012 Feb;29,

262, 495-502.

Intern. Jour. of Radiation,

2010 Dec1; Vol.78, Issue

5, 1503-1512

AACR 2006

#4372

AACR 2004

#947

Temozolomide (50 mg/kg PO Q1Dx5)

Vehicle D5

D8

D12

Temozolomide

0

10

20

30

40

50

Group medians


TV

(m

m3)

5 8 12

G5 TEMODAL 50mg/kg PO Q1Dx5


0

20

40

60

80

100



TV

(m

m3)

5 8 120

20

40

60

80

100

G5 TEMODAL 50mg/kg PO Q1Dx5


TV

(m

m3)

5 8 12

Vehicle

Temozolomide

http://radiology.rsna.org/content/262/2/495.abstract

http://www.redjournal.org/article/S0360-3016%2810%2900870-9/abstract



GS-9L Gliosarcoma Model

MRI monitoring (D10)

Gene expression


9L

Edema:

Intratumoral

Periphery

+

++

Cell density ++

Limits sharp

Necrosis no

Pseudo-cysts no

Nuclei aspects small-round

small-oblong

Vessel density

/normal tissue

≤ ?

Vessel size

/normal tissue

>

Collagen IV

over-expression

+

Vessel

- perfusion

- permeability

most

yes

pSTAT3

(92KD)

EGFR

(170KD)

PTEN

(50KD)

9L + + +

OT injection of GS-9L glioma cells


Randomization when tumor

volume = 50-75 mm3 using T2-

weighted SE

GV1A1 Glioma Model (rat availability issue)



0

20

40

60

80

100

0 5 10 15 20 25

Surv

ival

(%)

Time post tumor cell injection(days)

Vehicle

Carmustine 10 mg/kg/inj

GV1A1BDIX RatsTumor Adm.: Cells - OTTreatment: Start at D3

Female BDIX rats were OT injected with glioma cells. Rats were ranked at D3 according to body

weight and received 2 IV injections of 10 mg/kg BCNU (Q14Dx2).

pSTAT3

(92KD)

EGFR

(170KD)

PTEN

(50KD)

GV1A1 - ++ +

In vitro cytotoxicity of

BCNU treatment : 96h

1 10 100 1000 100000

50

100

GV1-A1

IC50 = 78 M

[BCNU] M

Surv

ivin

g c

ells (%

)

AACR 2004

#947

AACR 2006

#4372



GV1A1 Glioma Model (rat availability issue)


High level of expression of TGFβ

MRI monitoring

Markers Expression

Level

CD4 ±

CD8 +++

CD25 +

CD45 +

D9

GV1-A1

Edema:

Intratumoral

Periphery

+

+

Cell density ++

Limits sharp

Necrosis no

Pseudo-cysts yes

Nuclei aspects small-round

large-round

Vessel density

/normal tissue

<

Vessel size

/normal tissue

>

Collagen IV

over-expression

-

Vessel

- perfusion

- permeability

all

yes

OT injection of GV1A1 glioma cells


Randomization when tumor volume = 50-75 mm3

using T2-weighted SE

Intratumoral immune infiltrate

Hepa1-6 Hepatocarcinoma Model

Positive control: sorafenib


liver aorta stomach spine gallbladder tumor

Axial MRI of the liver

Mice were IS injected with Hepa1-6 tumor cells at D0.

Mice were randomized based on body weight at D5 and

treated with PO administrations of vehicle or Sorafenib

at 100 mg/kg/adm (Q1Dx21). Mice were terminated at

D23 and liver collected and weighed.

Median value






treated with PO administrations of vehicle or Sorafenib

at 100 mg/kg/adm (Q1Dx21). Mice were terminated at

D23 and liver collected and weighed.

Median value

Axial MRI of the liver

1.10e6 cells, mouse #3033 D8, 10% tumor in liver D16, 30% tumor in liver

2.10e6 cells, mouse #3083 D8, 30% tumor in liver D16, 90% tumor in liver

Circulating AFP level at D20 in mice bearing OT Hepa1-6

tumors. AFP was quantified by ELISA. Values upper than the

HLOQ were set at 400 µg/ml and values lower than the

LLOQ were set at 6.24 µg/ml.




Median value

Linear correlation between serum AFP concentration and

liver weight in mice bearing OT Hepa1-6 tumors.


Immune checkpoint inhibitor: CTLA-4 mAb




treated with vehicle or IP with mAb against CTLA-4

(clone 9H10) at 10 mg/kg/inj (TWx2). Mice were

terminated at D23 and liver collected and weighed.

L1210 & L1210/CDDP Lymphocytic leukemia Model


Other tested drugs: BCNU, Doxorubicin, 5-FU, L-OHP, Mitoxantrone


AACR 2010

#571

0

20

40

60

80

100

0 2 4 6 8 10 12


Surv

ival (%

)

Untreated

CDDP 2 mg/kg IV Q4Dx2

L1210DBA/2J Mice

Tumor Adm.: Cells - IP


Mice were IP injected with L1210/CDDP mouse leukemia

cells at D0. Mice were randomized based on body weight and

treated at D1 with IV injections of CDDP at 2 mg/kg/injection

(Q4Dx2).

0

20

40

60

80

100

0 2 4 6 8 10 12


Surv

ival (%

)

Untreated

CDDP 2 mg/kg IV Q4Dx2

L1210/CDDPDBA/2J Mice

Tumor Adm.: Cells - IP


Mice were IP injected with L1210 mouse leukemia cells at

D0. Mice were randomized based on body weight and

treated at D1 with IV injections of CDDP at


http://www.oncodesign.com/assets/files/posters/2010/AACR2010_OncoD_Debiopharm.pdf

L1210 & L1210/CDDP Lymphocytic leukemia Model


Other tested drugs: BCNU, Doxorubicin, 5-FU, L-OHP, Mitoxantrone


Mice were IP injected with L1210/CDDP mouse leukemia

cells at D0. Mice were randomized based on body weight

and treated at D1 with a single IV injection of CDDP at

10 mg/kg or with IP injections of l-OHP at 4 mg Pt/kg/inj

following the schedule Q7Dx3.

Mice were IP injected with L1210 mouse leukemia cells

at D0. Mice were randomized based on body weight and

treated at D1 with a single IV injection of CDDP at

10 mg/kg or with IP injections of l-OHP at 4 mg Pt/kg/inj

following the schedule Q7Dx3.

L1210 L1210/CDDP

LLC1 Lewis Lung Carcinoma Model

Positive control: Cyclophosphamide

Other tested drugs: Vinorelbine


0

500

1000

1500

2000

0 3 6 9 12 15 18 21


Mean t

um

or

volu

me (

mm

3)

Vehicle


LLC

C57BL/6 mice

Tumor Adm.: Cells - SC


Mice were SC injected with LLC tumor cells at D0. Mice were

randomized based on body weight at D4 and treated with IP

injections of vehicle or Cyclophosphamide at 100

mg/kg/injection (Q4Dx3).

G1 G4

0

15

30

45

60

Vehicle


Mean

an

d i

nd

ivid

ual

nu

mb

er

of

LL

Clu

ng

meta

sta

sis

Mice were IV injected with LLC tumor cells at D0. Mice

were randomized based on body weight at D3 and treated

with IV injections of vehicle or control IgG at 1

mg/kg/injection (Q7Dx3), or IP injections of

Cyclophosphamide at 100 mg/kg/injection (Q4Dx3). Mice

were terminated at D14 and their lungs were collected

for counting of metastases.

LLC1 Lewis Lung Carcinoma Model

Mice were SC injected with LLC tumor cells at D0. Mice were randomized based on tumor volume at D8 and treated with

IP injections of vehicle or of mAb against CTLA-4 (clone 9H10, Syrian Hamster IgG1) or PD-L1 (clone 10F.9G2, Rat IgG2b)

at 10 mg/kg/inj (TWx2).

Immune checkpoint inhibitor: CTLA-4 and PD-L1 mAbs

0

500

1 000

1 500

0 5 10 15 20 25

Tum

or

volu

me (

mm

3)

Time (Days)

Control

0

500

1 000

1 500

0 5 10 15 20 25

Tum

or

volu

me (

mm

3)

Time (Days)

Cyclophosphamide

0

500

1 000

1 500

0 5 10 15 20 25

Tum

or

volu

me (

mm

3)

Time (Days)

PD-L1 mAb

0

500

1 000

1 500

0 5 10 15 20 25

Tum

or

volu

me (

mm

3)

Time (Days)

CTLA-4 mAb


T/C % (D19)

Cyclophosphamide 16

CTLA-4 mAb 97

PD-L1 mAb 88

MBT-2 Bladder carcinoma Model

Positive control: Mitomycin C

Other tested drugs: BCG


Mice were OT injected with MBT-2 mouse tumor cells at D0.


treated with repeated intravesical instillations of vehicle or

MMC at 1 mg/kg/inj or BCG at 1.35 mg/mouse/inj following

the schedule Q7Dx3. Mice were terminated at D21 for tumor

measurement.



treated with repeated intravesical instillations of vehicle or

MMC at 1 mg/kg/inj following the schedule (Q2Dx3)x15 or

left untreated.


Immune checkpoint inhibitors: CTLA-4 and PD-1 mAbs




treated with repeated intravesical instillations of BCG at

1.35 mg/kg/inj following the schedule (Q2Dx3)x3, with IP

injection of isotype mAb or mAb against PD-1 (clone RMP1-

14, Rat IgG2a) following the schedule TWx2 or left

untreated.



treated with repeated intravesical instillations of BCG at

1.35 mg/kg/inj or vehicle following the schedule (Q2Dx3)x3,

or treated with IP injection of mAb against CTLA-4 (clone

9H10, Syrian Hamster IgG1) following the schedule TWx2.

T/C %

CTLA4 mAb > 260

PD1 mAb 148


MBT-2 OT model: Imaging of the bladder

page 53 Confidential 9-Feb-16

TV=18 mm3 TV=0 mm3

TV=60 mm3

Tumor Bladder

Pelvic fat Thigh muscle

Abdominal fat


Immune checkpoint inhibitor: PD-1 mAb


Mice were OT injected with MBT-2 mouse tumor cells at D0. Mice

were randomized based on body weight at D3 and treated with

repeated intravesical instillations of BCG at 1.35 mg/kg/inj

following the schedule (Q2Dx3)x3, with IP injection of isotype mAb

or mAb against PD-1 (clone RMP1-14, Rat IgG2a) following the

schedule TWx2 or left untreated.

MBT-2 OT model: Imaging

TV=60 mm3

Tumor Bladder

Pelvic fat Thigh muscle

Abdominal fat

TV=18 mm3 TV=0 mm3


SC model


Mice were SC injected with MBT-2 mouse tumor cells at D0. Mice were randomized based on tumor volume at D14 and

treated with IP injection of mAb against CTLA-4 (clone 9H10, Syrian Hamster IgG1) at 10 mg/kg/inj (TWx2), or mAb

against PD-1 (clone RMP1-14, Rat IgG2a) following the schedule TWx2, or mAb against PD-L1 (clone 10F.9G2, Rat IgG2b)

at 10 mg/kg/inj (Q2Dx) or left untreated.

0

500

1 000

1 500

2 000

2 500

0 10 20 30 40 50

Tum

or

volu

me (

mm

3)

Time (Days)

Untreated

0

500

1 000

1 500

2 000

2 500

0 5 10 15 20 25 30 35 40

Tum

or

volu

me (

mm

3)

Time (Days)

CTLA-4 mAb

T/C % (D27)

CTLA-4 mAb 83

PD-1 mAb 73

PD-L1 mAb 74

0

500

1 000

1 500

2 000

2 500

0 5 10 15 20 25 30 35 40

Tum

or

volu

me (

mm

3)

Time (Days)

PD-1 mAb

0

500

1 000

1 500

2 000

2 500

0 5 10 15 20 25 30 35 40Tum

or

volu

me (

mm

3)

Time (Days)

PD-L1 mAb

MPC-11 plasmocytoma model

Positive control: -


Mice were SC injected with MPC-11 syngeneic

plasmacytoma cells at D0.

0

500

1000

1500

2000

2500

0 5 10 15 20 25

Mean t

um

or

volu

me (

mm

3)


Untreated

MPC-11Balb/c MiceTumor Adm.: Cells - SC

Mouse IL6 concentration were quantified in

sera of MPC-11 tumor bearing mice at D15,

D20 and D25 using ELISA.

P388 Leukemia Model

Positive control: BCNU

Other tested drugs: Doxorubicin, Mitoxantrone


0

20

40

60

80

100

0 10 20 30 40

Surv

ival (%

)


Control

Carmustin IP 10mg/kg Q1Dx3

P388MiceTumor Adm.: Cells - IPTreatment: Start at D0

Mice were IP injected with P388 mouse tumor

cells at D0. Mice were randomized based on

body weight at D0 and treated with IP

administrations of 10 mg/kg of Carmustine

following the schedule Q1Dx3 (D1 to D3).

R3327H Prostate adenocarcinoma Model

Positive control: Castration, Bicalutamide

Other tested drugs: Paclitaxel, Mitoxantrone, Trastuzumab, Leuroprolin,

Decapeptyl


Rats were SC xenografted with R3327H tumor

fragments at D0. Rats were treated with

administration of vehicle or Bicatulamide 30mg/kg

(Q1Dx70).

High expression of PSA in R-3327H tumors

at 250X magnification.

0

500

1000

1500

2000

2500

3000

3500

4000

4500

100 120 140 160 180 200 220

Mean t

um

or

volu

me (

mm

3)

Time post tumor fragments implantation (days)

Vehicle

Bicalutamide 30 mg/kg

Castration

R3327H Cop/Ncrl RatsTumor Adm.: Fragments - SCTreatment: Start at D115

0

500

1 000

1 500

2 000

0 10 20 30 40 50

Time (Days)

TLFRT (2Gy/day)

Renca Kidney Carcinoma Model

Tested treatment: TLFRT, Proleukin


Mice were subcutaneously injected with RENCA

human tumor cells at D0. Mice were randomized

based on tumor volume at D25 and treated with

vehicle or TLFRT (Tumor Local Fractioned

RadioTherapy) at 2 Gy (treatment schedule

[Q2Dx3]x2).

0

500

1 000

1 500

2 000

0 10 20 30 40 50 60

Time (Days)

Proleukin SC 3 mg/kg

Mice were subcutaneously injected with RENCA

human tumor cells at D0. Mice were randomized

based on tumor volume at D18 and treated with

vehicle or Proleukin at 3 mg/kg SC (Q1Dx5)x2.

0

500

1 000

1 500

2 000

0 10 20 30 40 50

Tum

or

volu

me (

mm

3)

Time (Days)

Vehicle

0

500

1 000

1 500

2 000

0 10 20 30 40 50 60

Tum

or

volu

me (

mm

3)

Time (Days)

Untreated


Positive control: Sorafenib

Other tested drugs: CDDP, Paclitaxel, Gemcitabine, PD-1 mAb


0

20

40

60

80

100

0 10 20 30 40


Surv

ival (%

)

Vehicle

Sorafenib 100 mg/kg PO Q1Dx21

RENCA

Balb/c mice



Left

kidney

Lymph nodes

metastasis Hepatic

metastasis

Splenic

mets.

Metastases observed at D22 after

orthotopic injection of 5x105 RenCa cells.

Mice were OT injected with RENCA mouse kidney tumor cells at D0.

Mice were randomized based on body weight at D4 and treated with

PO administrations of sorafenib at 100 mg/kg/administration

(Q1Dx21).



0

25

50

75

100

125

150

175

200

Gemcitabine Untreated

Media

n a

nd L

ung m

eta

stasi

s num

ber

repart

itio

n (

D23)

per

mouse

RencaBalb/c MiceOT injection at D0Treatment start at D7

Sacrifice: D23

Mice were OT injected with RENCA mouse kidney tumor cells at D0. Mice were randomized based on body

weight at D7 and treated with IV administrations of Gemcitabine at 120 mg/kg/administration (Q1Dx1).

0

1

1

2

2

3

3

4

4

Gemcitabine 120 mg/kg IV Q1Dx1

Untreated

Tum

or

weig

ht

repart

itio

n (

g)

per

mouse

RencaBalb/c MiceOT injection at D0Treatment start at D7

Sacrifice: D23



Imaging of lung metastasis

T2-weighted Magnetic Resonance Imaging

Left

kidney

Primary

tumor

Normal

lung Lung

+ mets

Primary

tumor

D14 D9 D9

D14

D18


Tumor associated macrophages in untreated and treated tumor bearing

mice


4x

60x

IgG Treated Anti-CSF1 Treated

Cytokines release quantification

ELISA / CBA / Luminex

Immune cells phenotyping and quantification

FACS, IHC

Immune cells functionality

Cell preparation and/or sorting using FACS or magnetic beads

Cytotoxic activity based on Cr51 release assay

Proliferation ability based on H3-Thd incorporation assay

Specific cytokine release based on ELISPOT assay

CFU

Immunological endpoints




Immune response

0

2

4

6

8

10

12Control

Vaccination

7 days 10 days

p = 0.02

p = 0.1

Time post-vaccination

Percen

tag

e o

f

KD

3-s

pec

ific

CD

8 a

mon

g

CD

8 T

cel

ls

Evaluation of specific T cell response to vaccination using FACS-Pentamer analysis. Figure

represents individual and mean percentage of specific CD8+ cells in splenocyte samples from

C57BL/6 mice receiving repeated injections of vaccine. Statistical analysis was performed

using Bonferroni test.



Evaluation of T-cell mediated immune response against antigen-expressing syngeneic tumor grafted

in mice treated with antigen-specific vaccine. Total splenocytes were restimulated with antigen-

specific peptides divided in peptide-pool #1 and #2 and IFNγ secretion was monitored using ELISpot

assay.

0

10

20

30

40

50

Control Vaccine

Mouse #1 Mouse #2 Mouse #1 Mouse #2 Mouse #3 Mouse #4 Mouse #5 Mouse #6

Peptide-pool #1

Peptide-pool #2

IFN

secre

tion

exp

res

sed

in

sp

len

ocy

tes

The results show that antigen-specific T cell

mediated immune response could be detected

in mice therapeutically vaccinated.

Peptide-pool #1 was more potent than peptide-

pool #2 at re-stimulating antigen-specific IFNγ

producing cells.

Immune response

Immune checkpoint inhibitors : Teff/treg ratio

page 67 Confidential 9-Feb-16

CT26 EMT6 Control

CTLA4 mAb

4T1 Control

CTLA4 mAb

Control

CTLA4 mAb

Checkpoint inhibitors

Model Checkpoint Inhibitors NGS

Name Type Site Strain CTLA-4 PD-1 PD-L1

4T1 Breast OT Balb/C X

A20 BCL SC Balb/C X

B16-F10 Melanoma SC C57Bl/6 X

C38 Colon SC C57Bl/6

CT26 Colon SC Balb/C X

EMT6 Breast SC Balb/C X

Hepa1-6 Liver OT C57Bl/6

LLC Lung SC C57Bl/6 X

MBT-2 Bladder OT

C3H

X SC

Renca Kidney OT Balb/C X


TC < 42%

42% < TC < 80%

TC > 80%

Checkpoint inhibitors


Immune cell phenotyping using FACS

Documents

Syngeneic Models - Oncodesign · Syngeneic Models at Oncodesign Model Organ Host rodent species Injection site ... A20 B cell lymphoma Model Mice were SC injected with A20 tumor cells