Syngeneic Models:

Immunology Platform

Joseph Murphy PhD, July 28th 2015

©Copyright, 2015 Charles River Laboratories. All rights reserved

Webinar Logistics

• Technical difficulties– To join the webinar via the Telephone:

• Dial the phone number listed

• Enter the Access Code when prompted

• Enter the Audio Pin when prompted

– Alternatively, you join via Mic & Speakers

• Your participation is encouraged

• Q&A following presentation

• Webinar recording will be available

to all attendees

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

askcharlesriver@crl.com

www.criver.com