Syngeneic Models:
Immunology Platform
Joseph Murphy PhD, July 28th 2015
©Copyright, 2015 Charles River Laboratories. All rights reserved
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Today’s Presenter
Joseph Murphy, PhD
Director of R&D, Science Operations, Oncology
Charles River
• Dr. Murphy has more than 20 years' experience in biological research in academia and the
biotechnology industry.
• In 2011, he was appointed Director of Cancer Therapeutics and Immunology at the Southern
Research Institute in Birmingham, Alabama. Prior to that appointment, he was a senior research
scientist and lecturer at Trinity College, University of Dublin, Ireland.
• He also served as founder/managing director of Emmerex Limited, a company focused on
developing an immune-based anticancer therapy.
• Dr. Murphy holds a Bachelor of Science degree in mathematics, experimental physics, and biology
from the National University of Ireland and earned his Doctorate of Philosophy from the Department
of Biological Sciences, University of Salford, United Kingdom.
• Dr. Murphy conducted postdoctoral work at INSERM U331/Institute Pasteur, Lyon, France, and
Stanford University, CA. He has written and contributed to more than 100 scientific abstracts and
articles.
• He sits on the industry committee for the Society for Immunotherapy for Cancer (SITC) and has
served as a reviewer for Clinical Medicine Insights: Oncology; Clinical Medicine Insights: Cardiology,
International Journal of Cancer; Journal of Medical Genetics and Genomics, and MOJ Immunology.
IMMUNO-ONCOLOGY
CANCER IMMUNOTHERAPY IS THE USE OF THE IMMUNE SYSTEM
TO TREAT CANCER
CELLULAR: PD1, CTLA-4
ANTIBODY: BEVACIZUMAB, CETUXIMAB
CYTOKINE: INTERLUKIN-2
CRL Discovery Syngeneic Models
• Response to Immune Check Point Inhibitors: anti-PD1 and anti-CTLA-4 mAbs
• Immune Cell Tumor Infiltrate (TIL): T cells; T regulatory cells (Treg); and
Myeloid Derived Suppressor Cells (MDSC)
Breast
4T1 BALB/c
EMT-6 BALB/c
Colon
Colon26 BALB/c
CT26 BALB/c
MC38 C57BL/6
Leukemia/Lymphoma
A20 BALB/c
L1210 B6D2F1
P388 B6D2F1
Lung
KLN 205 DBA/2
Lewis Lung C57BL/6
Madison109 BALB/c
Melanoma
B16F10 C57BL/6
Renal
Renca BALB/c
Syngeneic Models: T cell activation/priming
Lymph Node
Clonal expansion
Secretion of cytokines
Tumor cell killing
CD80/86PD-L2
MHCCD80
Dendritic Cell
CD28TCR
CD8
CD8 T cell
Expression of negative receptors:
PD1 and CTLA-4
PD1 (CD279)
CTLA-4 (CD152)
CTLA-4
CD28
PD1
CD8/TCR
CD8 T effector
Anti-PD1
Anti-CTLA-4
Block PD1 and CTLA-4 signaling in T effector and Tregs:
• Prevent T effector cell exhaustion and death
• Decrease Treg activity and proliferation
• Induction of Treg death by ADCC
• Create a favorable CD8 T-effector:T reg ratio
Syngeneic Models: Effects of PD1 and CTLA-4 Signaling
ADCC/macrophage
Anti-CTLA-4
Tumor
Lck
ZAP70
class I
PI3KAkt
CD28
TCR
CD4
CTLA-4
Treg
PD-L1
Tumor cell
CD80/86
APC
PD-L2
SHP2
PP2a
SHP2
Syngeneic Models: Efficacy of anti-CTLA-4 therapy.
Days
0 5 10 15 20 25 30
Tu
mo
r vo
lum
e (
mm
3)
0
200
400
600
800
1000
Iso rt (IgG2)
Control IgG
4F10 (hm IgG1)
9D9 (ms IgG2)
9H10 (hm IgG2)
Colon26 Mean DataClones 4F10, 9D9 and 9H10
100mg d8; 50mg d11 and 14
Reduction of Tregs cells in B16-BL6 tumors using an
adoptive transfer + GVAX +/- anti-CTLA approach.
Selby et al., Cancer Immunol Res; 1(1) 2013
clone RMP1-14, rat IgG2aanti-PD1 (200mg/ms: d 4,7,11,14)
anti-PD1 (100mg/ms: d 4,7,11,14)
Days
0 5 10 15 20 25 30 35 40
Tu
mo
r vo
lum
e (
mm
3)
0
200
400
600
800
1000Iso rat (IgG2)
anti-PD1 (200 g)
anti-PD1 (100 g)
Colon26-e231 Mean Data: Anti-PD1 Therapy
Syngeneic Models: Efficacy of anti-PD1 therapy.
Syngeneic Models: Immunotherapy Responsive
Colon26-e245 Group Median Data – Model Development
PBS, control
anti-CTLA-4 (9H10-25mg: d8); (12.5mg: d11,14)
anti-CTLA-4 (9H10100mg: d8); (50mg: d11,14)
Days
0 10 20 30 40 50 60
Tu
mo
r v
olu
me
(m
m3
)
0
200
400
600
800
1000
PBS, control
anti-PD1 (100mg: d16,19,23,26)
anti-CTLA-4 (9H10-100mg: d16); (50mg: d19,21)
anti-CTLA-4 + anti-PD1
Days
0 10 20 30 40 50 60T
um
or
vo
lum
e (
mm
3)
0
200
400
600
800
1000
Upstaged Start
Days
0 10 20 30 40 50 60
Tu
mo
r V
olu
me (
mm
3)
0
500
1000
1500
2000
2500
Syngeneic Models: Immunotherapy ResponsiveA20-e215 Individual Animal Data: Responsive to anti-PD1
Days
0 10 20 30 40 50 60
Tu
mo
r V
olu
me
(m
m3)
0
500
1000
1500
2000
2500PBS, control anti-PD1 (100mg: d 3,6,10,13) - 30% TFS
Days
0 10 20 30 40 50 60
Tu
mo
r V
olu
me
(m
m3)
0
500
1000
1500
2000
2500
anti-CTLA-4 (9H10-100mg: d8;
50mg: d 11,14) - 40% TFS
Syngeneic Models: Immunotherapy Responsive
Days
0 10 20 30 40 50 60
Tu
mo
r V
olu
me
(m
m3)
0
500
1000
1500
2000
2500PBS, control
Days
0 10 20 30 40 50 60
Tu
mo
r V
olu
me
(m
m3)
0
500
1000
1500
2000
2500
anti-PD1 (d8,11,15,18) + anti-CTLA-4 (9H10-d8,11,14)
80% TFS
Days
0 10 20 30 40 50 60
Tu
mo
r V
olu
me
(m
m3)
0
500
1000
1500
2000
2500
10% TFS
anti-PD1 (d16,19,23,26) + anti-CTLA-4 (d16,19,22)
m=70mm3
Syngeneic Models: Immunotherapy ResponsiveMC38-e015 Individual Animal Data: Responsive to anti-PD1
Days
0 10 20 30 40 50 60 70 80
Tu
mo
r V
olu
me (
mm
3)
0
500
1000
1500
2000
2500PBS, control – 90% TP
Days
0 10 20 30 40 50 60 70 80
Tu
mo
r V
olu
me (
mm
3)
0
500
1000
1500
2000
2500
anti-PD1 (100mg: d 3,6,10,13) - 70% CR
Days
0 10 20 30 40 50 60 70 80
Tu
mo
r V
olu
me (
mm
3)
0
500
1000
1500
2000
2500
anti-CTLA-4 (4F10-100mg: d8;
50mg: d 11,14) - 30% CR
Days
0 10 20 30 40 50 60 70 80
Tu
mo
r V
olu
me (
mm
3)
0
500
1000
1500
2000
2500
anti-PD1 (100mg: d 3,6,10,13) + anti-CTLA-4
(clone 4F10, 100mg: d8; 50mg: d 11,14) – 70% CR
Syngeneic Models: Immunotherapy Partial Response
PBS, control
anti-PD1 (100mg: d 2,5,9,12)
anti-CTLA-4 (9H10-100mg: d 5); (50mg: d 8,11)
anti-PD1 (100mg: d 2,5,9,12) + anti-CTLA-4 (100mg: d 5); (50mg: d 8,11)
Days
1 2 14 16 18 20 22 24 26 28 30 32 34
% R
em
ain
ing
0
10
20
30
40
50
60
70
80
90
100Control
anti-PD1
anti-CTLA4
anti-PD1+CTLA4
Days
0 5 10 15 20 25 30
Tu
mo
r vo
lum
e (
mm
3)
0
200
400
600
800
1000
1200
1400
1600
1800
2000
EMT-6-e201 Group Median Data
Syngeneic Models: Immunotherapy Non-Responsive
Days
0 10 20 30 40
Tu
mo
r v
olu
me
(m
m3
)
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
Lewis Lung-e216 Group Median Data
PBS, control
anti-PD1 (100mg: d 3,6,10,13)
anti-CTLA-4 (100mg: d 8); (50mg: d 11,14)
anti-PD1 (100mg: d 3,6,10,13) + anti-CTLA-4 (100mg: d 8); (50mg: d 11,14)
Days
0 5 10 15 20 25
Tu
mo
r v
olu
me
(m
m3
)
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
B16F10-e248 Melanoma Group Median Data
Syngeneic Models: Summary Responses
Responsive
A20
Colon26
CT26
MC38
Moderate Response
EMT-6
Renca
Refractory
4T1
B16F10
Lewis Lung
Madison109
Syngeneic Models: Immune Cell Infiltrate
Perc
en
tag
e o
f to
tal T
IL
0
10
20
30
40
50
60 Colon26
MC38
LL
4T1
B16F10
CD3+/8
+CD3
+/4
+Treg MDSC
CD11b+/Gr1
+
Pe
rce
nta
ge
of
tota
l T
IL
0
10
20
30
40
50
60
70
Colon26
LL
MC38
g-MDSCCD11b+/Ly6G+
m-MDSCCD11b+/Ly6C+
Syngeneic Models : Colon26-e269 Immunotherapy/TIL
mm3 CD8:Treg
486 6.10
320 10.08
500 4.73
mm3 CD8:Treg
196 12.32
228 9.05
288 6.37
mm3 CD8:Treg
352 14.97
320 19.29
288 7.05
anti-PD1: 100mg, d1, d4
anti-CTLA-4: 9H10-100mg, d1; 50mg d4
mm3 CD8:Treg
405 4.50
550 3.49
600 3.37
1 2 3 4 5 6
75
100
125
150
175
200
225 Control
anti-PD1
anti-CTLA4
anti-CTLA4+PD1
Tu
mo
r vo
lum
e (
mm
3)
Days
anti-PD1: 100mg, d1, d4
anti-CTLA-4: 9H10-100mg, d1; 50mg d4
TregCD3+/CD4+CD3+/CD8+
Perc
en
tag
e o
f to
tal T
IL
0
10
20
30
40
50
60
Control
anti-PD-1
anti-CTLA-4 (9H10)
anti-PD-1+CTLA-4
Syngeneic Models : MC38-e025 Immunotherapy/TIL
mm3 CD8:Treg
196 11.54
126 4.46
88 12.45
mm3 CD8:Treg
144 183.8
270 120.3
162 205.7
mm3 CD8:Treg
144 111.9
162 142.6
162 635.0
mm3 CD8:Treg
88* 32.1
144 3.42
245 3.66
Days
0 5 10 15 20 25 30
Tu
mo
r vo
lum
e (
mm
3)
0
100
200
300
400
500
600
• Profiling responses to immune check point inhibitors permits for the rational
decision making of syngeneic models to use in combination with other therapies.
• Different potency and dosing of anti-CTLA-4 mAbs +/- anti-PD1 in specific models
allows for the design of combination therapies to evaluate therapeutic interactions.
• Base line knowledge of TILs in tumor models assists in the selection of therapies
based upon the presence of T effector and immuno-regulatory cells.
• Anti-PD1 and anti-CTLA-4 treatment may modulate the T effector:T regulatory cell
ratio in favor a CTL based anti-tumor response.
Syngeneic Models
Questions?
Call with questions: 1-877-274-8371
www.criver.com