Benefits of orthotopic mouse models

1. Jean-François MIRJOLET
Head of In vitro Sciences department
Experimentation Division
Oncodesign
Advantages of orthotopic
mouse models

2. AACR 2018 - Chicago 2
I have the following financial relationships to disclose:
Full type employee of: Oncodesign, Dijon, France
- and -
I will not discuss off label use and/or investigational use in my presentation.
Disclosure Information

3. The skin microenvironment
3
Mouse skin structure
Densely packed hair follicles
Thin epidermis and thin dermis
Mouse skin immune population
Epidermis
Mostly Vγ5+ dendritic epidermal T cells (DETCs)
Dermis
Macrophages, mast cells, conventional αβ T cells and a small
population of innate lymphoid cells (ILCs).
In mouse skin
important contribution from recruited γδ T cells to skin immune
surveillance and interleukin-17 production.
AACR 2018 - Chicago

4. Introduction
4AACR 2018 - Chicago

5. Immune checkpoint inhibitor efficacy depends on tumor volume at start
5
0
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1 500
2 000
0 5 10 15 20 25 30 35 40
Tumorvolume(mm3)
Time (Days)
CTLA-4 mAb
0
500
1 000
1 500
2 000
0 5 10 15 20 25 30 35 40
Tumorvolume(mm3)
Time (Days)
PD-1 mAb
0
500
1 000
1 500
2 000
0 5 10 15 20 25 30 35 40
Tumorvolume(mm3)
Time (Days)
Control
Mice were SC injected with B16F10 mouse tumor cells at D0. Mice were randomized based on body weight or TV and treated
IP with mAb against CTLA-4 (clone 9H10) at 10 mg/kg (TWx2) or against PD-1 (clone RPMI-14) at 10 mg/kg/inj (Q2Dx).
0
500
1 000
1 500
2 000
0 5 10 15 20 25 30 35 40
Tumorvolume(mm3)
Time (Days)
Control
0
500
1 000
1 500
2 000
0 5 10 15 20 25 30 35 40
Tumorvolume(mm3)
Time (Days)
PD-1 mAb
0
500
1 000
1 500
2 000
0 5 10 15 20 25 30 35 40
Tumorvolume(mm3)
Time (Days)
CTLA-4 mAb D0
0 mm342% 29%
Nothing
preexisting
80% 90%
D10
100 mm3
Existing
microenvironment
AACR 2018 - Chicago

6. Immune checkpoint inhibitor efficacy depends on tumor volume at start
6
B16-F10
CT-26
EMT-6
AACR 2018 - Chicago

7. Immune checkpoint inhibitor efficacy depends on tumor volume at start
7
Early Late
CTLA-4 TV at start T/C (%) TV at start T/C (%)
mean 0 20 73 97
SD 0 7 28 4
median 0 22 76 100
n 3 3 3 3
PD-1
mean 1 57 181 115
SD 2 18 77 30
median 0 45 135 105
n 3 3 3 3
PD-L1
mean 17 44 121 121
SD 15 11 9 30
median 17 44 121 121
n 2 2 2 2
AACR 2018 - Chicago
Early Late
TV at start T/C (%) TV at start T/C (%)
69 17 146 40
8 8 43 16
66 19 116 38
7 7 5 5
74 69 124 75
13 16 13 12
73 68 121 73
19 18 9 8
67 57 113 79
14 11 8 8
66 63 116 77
9 9 3 3
B16-F10 CT-26

8. Immune cells are biomarkers of efficacy
8
0
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1 500
2 000
2 500
0 10 20 30 40 50
Tumorvolume(mm3
)
Time (Days)
CTLA-4 mAb
0
500
1 000
1 500
2 000
2 500
0 10 20 30 40 50
Tumorvolume(mm3
)
Time (Days)
PD-1 + CTLA-4 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 CTLA-4 (clone 9H10) at 5 mg/kg/inj, PD-1
(clone RMP1-14) at 10 mg/kg/inj (TWx2) or
combination of both. Mice were terminated at D20
and tumor analyzed by Flow Cytometry for T cells
content.
AACR 2018 - Chicago

9. Immune cells are biomarkers of efficacy
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Vehicle
PD-1 mAb
D9 D16
Cells/mm2Median(Cells/mm2)
EMT6 SC tumor

10. Physiologic behavior of orthotopic models
10
Left
kidney
Primary
tumor
Normal
lung
Lung
+ mets
Primary
tumor
D14D9
Presence of lung metastasis in OT model
(nothing in SC)
RenCa SC OT (tumor weight)
PD-1 TV at start T/C (%) TV at start T/C (%)
mean 112 62 NA 146
SD 23 1 NA 53
median 112 62 NA 146
n 2 2 NA 2
Sensitivity Partially sensitive Resistant
Metastasis No Lung
AACR 2018 - Chicago

11. MBT-2 bladder model: subcutaneous versus orthotopic models
11
0
500
1 000
1 500
2 000
2 500
0 10 20 30 40 50
Tumorvolume(mm3)
Time (Days)
Untreated
OT injection with MBT-2 tumor cells at D0
Randomization based on body weight at D5
Treatment with IP injection at 10 mg/kg/inj (TWx2)
- mAb against CTLA-4 (clone 9H10)
- mAb against PD-1 (clone RMP1-14)
- Isotype (clone 2A3)
T/C : >260%
0
20
40
60
80
100
120
0 10 20 30 40 50 60 70
Survival(%)
Time (Days)
Vehicle
CTLA4 mAb
0
500
1 000
1 500
2 000
2 500
0 5 10 15 20 25 30 35 40
Tumorvolume(mm3)
Time (Days)
CTLA-4 mAb
0
500
1 000
1 500
2 000
2 500
0 5 10 15 20 25 30 35 40
Tumorvolume(mm3)
Time (Days)
PD-1 mAb
T/C : 73%
T/C : 83%
T/C : 148%
0
20
40
60
80
100
120
0 10 20 30 40 50 60 70
Survival(%)
Time (days)
Untreated
Isotype
PD-1 mAb
SC injection with MBT-2 tumor cells at D0
Randomization based on tumor volume at D14
Treatment with IP injection at 10 mg/kg/inj (TWx2)
- mAb against CTLA-4 (clone 9H10)
- mAb against PD-1 (clone RMP1-14)
TV=60 mm3
Tumor
Bladder
Pelvic fat
Thigh muscle
Abdominal fat
AACR 2018 - Chicago

12. MBT-2 bladder model: subcutaneous versus orthotopic models
12
MBT-2 SC OT (survival)
CTLA-4 TV at start T/C (%) TV at start T/C (%)
mean 100 66 NA 285
SD 12 18 NA 25
median 100 66 NA 285
n 2 2 NA 2
PD-1 TV at start T/C (%) TV at start T/C (%)
mean 97 68 NA 142
SD 16 13 NA 11
median 99 72 NA 148
n 9 9 NA 7
AACR 2018 - Chicago

13. N
aive
m
ice
D
10
D
17
D
23
0
500
1000
1500
KC
pg/mL
Development of OT PAN02 model
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N=18 mice.
Tumor fragment implanted OT at D0
horizontal bar : median of the group
D
9
D
16
D
22
0
500
1000
1500
Days after fragment implantation
Tumorvolume(mm3
)/MRI
N
aive
m
ice
D
10
D
17
D
23
0
200
400
600
800
1000
IP-10
pg/mL
N
aive
m
ice
D
10
D
17
D
23
0
2
4
6
8
10
IFN-g
pg/mL
N
aive
m
ice
D
10
D
17
D
23
0
10
20
30
RANTES
pg/mL
White arrow head: tumor
N
aive
m
ice
D
10
D
17
D
23
0
100
200
300
IL-6
pg/mL
N
aive
m
ice
D
10
D
17
D
23
0
20
40
60
80
100
GM-CSF
pg/mL

14. PAN02 model : OT versus SC
AACR 2018 – Chicago 14
D
9
D
16
D
22
0
500
1000
1500
Days after fragment implantation
Tumorvolume(mm3
)/MRI

15. Conclusion
AACR 2018 – Chicago 15
•Reproducible
•Cost and time effective tumor
measurements
•Applicable to many tumor cell types
•Assess influences of immune
surveillance and evasion
•Limited histological and phenotypic
similarities to primary cancers
•Loss of tumor heterogeneity
•Low metastatic rates
•Lack of native tumor
microenvironment
•High metastatic rates
•Correct tumor microenvironment
•Assessment of tumor-stromal
interactions
•Assess anti-tumor efficacy-of primary
and metastases
•Assess influence on immune
surveillance and evasion
•More time and labor intensive
•In vitro artificial selection of cell lines
•Histological dissimilarities with
human tumors
•Loss of tumor heterogeneity
•Imaging modalities needed for in situ
assessment of tumor development
• Cell lines based
• Ectopic implantation
• Non-physiologic growth
location
• Cell lines based
• Implantation into organ of
origin
• Reconstitutes organ
microenvironment
• Local and metastatic spread
Strenghths & Advantages Weaknesses & caveats
Ectopic models
Orthotopic models
Modified from Ruggeri et al. Biochemical Pharmacology 87 (2014) 150–161

16. However…
16
Genomic responses in mouse models poorly mimic human
inflammatory diseases Seok J. et al, PNAS 2013, 110(9):3507–3512
Of Mice and Not Men: Differences between Mouse and Human
Immunology Modified from Javier Mestas and Christopher C. W. Hughes,
J Immunol 2004; 172:2731-2738
Mutational heterogeneity in cancer and the search for new
cancer-associated genes
0
250
500
750
1000
1250
1500
B16-F10
4T1
LLC
EMT-6
A20
RenCa
CT-26
MBT-2
Totalnumberofmutations
MS Lawrence et al. (2013) Nature 499:214-8
A Mouse is not a Human
size
lifespan
immune system (ecological
niches)
metabolic liver function
…
carcinogenesis mechanisms
AACR 2018 - Chicago

17. In vitro sciences (Dijon)
Vincent Blancheteau
Josselin Caradec
Anne-Lucie Nugues
Guillaume Serin
In vivo sciences (Les Ulis)
Edwige Nicodeme
In vivo sciences (Dijon)
Cyril Berthet
Damien France
Marc Hillairet De Boisferon
Sylvie Maubant
Caroline Mignard
Philippe Slos
David Vallerand
In vivo sciences (Synergie MTL)
Jill Rocchetti
Contacts:
MIRJOLET JF: jfmirjolet@oncodesign.com