Tumor-Immune System Interactioncontent.stockpr.com/.../3904/pdf/Dr.+Samir+Khleif.pdf · 2015 Dr. Samir Khleif aPD-1 n=10 E7 n=9 Non-treated n=10 E7+ aPD-1 n=8 cm3 Days after tumor

2015 Dr. Samir Khleif

Dr. Samir KhleifDirector of the State of Georgia Cancer Center,

Georgia Regents University Cancer Center and

The Cancer Service Line

21

Tumor-Immune System Interaction


TumorCo-I Ligand

T cell

T cellT cell

Co-inhibitoryTreg

Treg

IL-4

IL-13

TAM

M2

TAM

M2

DC

IDOCTLA-4

CD80

ARG1

iNOS

TGFβ

IDO,

IL-10

IL-10

GM-CSF,

VEGF,

IL-1β

MDSC

MDSC

Tumor-Immune System Interaction

23


Potential Immunotherapy Combinations

• Enhancing the Immune effector

• VACCINE

• Inhibition of immune suppression effect

24


Induction of

immune response

Inhibition of

suppression

Idea adopted from Bhardwaj N, 2007

Tumor Eradication

Immunotherapy Strategy Platform

25


PD-1/PD-L1 Background

Keir et al, Annu Rev Immunol, 2008

25


E7+aPD-1

Days 0 8 15 22

TC-1

Groups:

1. Non-treated2. aPD-13. E74. E7 + aPD-1

E7+aPD-1 E7+aPD-1

Monitoring of tumor growth and survival

Day 7after implantation of

50,000 TC-1 cell

Vaccine/Anti-PD-1 Combination

26


aPD-1n=10

E7n=9

Non-treatedn=10

E7+aPD-1n=8

Tum

or

Vo

lum

e, c

m3

Days after tumor implantation

Pe

rce

nt

Surv

ival


0

20

40

60

80

100

120

10 15 20 25 30 35 40 45

Non-immunized

E7

E7+CT011

CT-011

Non-immunized

E7

E7+aPD-1

aPD-1

Vaccine/Anti-PD-1 Combination

27


Tumor-Immune Interaction

TumorPD-L1

T cell

T cellT cell

PD-1Treg

Treg

IL-4

IL-13

TAM

M2

TAM

M2

DC

IDOCTLA-4

CD80

ARG1

iNOS

TGFβ

IDO,

IL-10

IL-10

GM-CSF,

VEGF,

IL-1β

MDSC

MDSC

29


Tumor-Immune Interaction

TumorPD-L1

T cell

T cellT cell

PD-1Treg

Treg

IL-4

IL-13

TAM

M2

TAM

M2

DC

IDOCTLA-4

CD80

ARG1

iNOS

TGFβ

IDO,

IL-10

IL-10

GM-CSF,

VEGF,

IL-1β

MDSC

MDSC

30


Potential Immunotherapy Combinations

• Enhancing the Immune effector

• VACCINE

• Inhibition of immune suppression effect

• Anti PD1

• CPM

31


Treg Cell Inhibitor-Cyclophosphamide (CPM)

E7+aPD-1

CPM

Days 0 7 8 15 22

TC-1

Monitoring of tumor growth and survival

E7+aPD-1 E7+aPD-1

31


0

0.3

0.6

0.9

1.2

1.5

5 8 15 22 26 29 31 35 380

0.3

0.6

0.9

1.2

1.5

5 8 15 22 26 29 31 35 38

0

0.3

0.6

0.9

1.2

1.5

5 8 15 22 26 29 31 35 38

0

0.3

0.6

0.9

1.2

1.5

5 8 15 22 26 29 31 35 38

0

0.3

0.6

0.9

1.2

1.5

5 8 15 22 26 29 31 35 38

0

0.3

0.6

0.9

1.2

1.5

5 8 15 22 26 29 31 35 38

0

0.3

0.6

0.9

1.2

1.5

5 8 15 22 26 29 31 35 38

0

0.3

0.6

0.9

1.2

1.5

5 8 15 22 26 29 31 35 38


Tum

or

Vo

lum

e,

cm3

E7+CPM+aPD-1

Non-immunized E7 aPD-1

CPM

E7+CPM

aPD-1+CPM

E7+aPD-1

Tumor Growth

32


Vaccine/Anti-PD-1/CPM Combination Promotes

Tumor Rejection

CPM (n=15)

aPD-1 (n=15)

E7 (n=14)

Non-treated (n=15)

E7 + aPD-1 + CPM (n=20)

aPD-1+CPM (n=15)

E7+CPM (n=14)

E7+aPD-1 (n=15)

8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 760

20

40

60

80

100

Pe

rce

nt

Surv

ival

Days after tumor implantation8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76

0

20

40

60

80

100

8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 760

20

40

60

80

100

8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 760

20

40

60

80

100

33


0

5

10

15

20

25

Tumor (days) 7 8 11 14 18 21 26

CPM (days) 0 +1 +4 +7 +11 +14 +19

CPM

NT

aPD-1+CPM

% o

f Tre

gs

wit

hin

CD

4+T

cell p

op

ula

tio

n

Anti-PD-1/CPM Synergize to Decrease and

Maintain Low Level of Tregs in Periphery

** ***

***

aPD-1

NS NS

34


***P<0.001

0

20

40

60

80

100

120

140

160

E7 E7

+aPD-1

aPD-1

+CPM

NTE7

+aPD-1

+CPM

E7

+CPM

E7+aPD-1 or PD-L2-Ig

CPM TERMINATION

Days 0 7 8 15 21

TC-1 tumor

Both Vaccine/aPD-1/CPM and Vaccine/PD-L2-Ig/CPM

Combinations Induce Potent Antigen-Specific Immune

Responses in Tumor Bearing Mice

IFN

spo

ts p

er

10

e6

sple

no

cyte

s

***

Mkrtichyan et al. Eur J Immunol 2011

Anti-PD-1 mAb

35


0.E+00

1.E+03

2.E+03

3.E+03

4.E+03

5.E+03

6.E+03

7.E+03

8.E+03

9.E+03

E7 E7

+aPD-1aPD-1

+CPM

NTE7+

aPD-1

+CPM

E7

+CPM

CD8+ cells***

****

CD

8 p

er 1

0e6

tu

mo

r ce

lls

Anti-PD-1 mAb Synergizes with Vaccine/CPM to

Increase CD8 T Cell Infiltration Into the Tumor, While

PD-L2-Ig Does Not Affect CD8 T Cell Numbers

*P<0.05, ***P<0.001

Mkrtichyan et al. Eur J Immunol 2011

Anti-PD-1 mAb

36


*P<0.05 and **P<0.01

E7 E7

+aPD-1

aPD-1

+CPM

NTE7

+aPD-1

+CPM

E7

+CPM

**

*

Tre

g-

% o

f C

D4

+ce

lls

0

5

10

15

20

25

30

35

40Day 21

0.0E+00

5.0E+02

1.0E+03

1.5E+03

2.0E+03

2.5E+03

E7 E7

+aPD-1

aPD-1

+CPM

NTE7+

aPD-1

+CPM

E7

+CPM

Treg cells

***

***

***

Ab

solu

te n

um

be

rs p

er

10

e6

tum

or

cells

Anti-PD-1 Ab and CPM Synergize to Decrease

the Level Splenic and Tumor Infiltrated Treg Cells

37


*P<0.05, ***P<0.001

Vaccine/anti-PD-1/CPM Combination Increases

the CD8/Treg Ratio in Tumor Microenvironment

0

2

4

6

8

10

12C

D8

+/T

reg

Rati

o***

*****

*

E7 E7

+aPD-1

aPD-1

+CPM

NTE7+

aPD-1

+CPM

E7

+CPM

CD8/ Treg ratio

38


Clinical Trial Translation

A Phase II Clinical Trial of Anti-PD1 and CPM with

Provenge® in Prostate Cancer Patients

A Phase II Clinical Trial of Anti-PD1 and

Chemotherapy in Pancreatic Cancer Patients as

an Adjuvant Therapy

Provenge® is a registered trademark of Dendreon Corporation

39


PD-1/PD-L1 Engagement Suppresses Effector T

Cells

TCR

CD28

PD-1

Activate

d

MHC/

peptide

B7

APCTreg

Tumor cell

PD-L1

Suppressed/Apoptotic

T cell

MDSC

41


Anti-PD-1

PD-1/PD-L1 Engagement Suppresses Effector T

Dells

TCR

CD28

PD-1

Activate

d

MHC/

peptide

B7

APCTreg

Tumor cell

PD-L1

Suppressed/Apoptotic

T cell

MDSC

Activated

T cell

42


Combinational Listeria-Based Immunotherapy

Despite all the advantages of Listeria-based vaccines, it has been

demonstrated that infection with listeria lead to up-regulation of PD-Ls on

immune cells (Rowe JH et al. 2008 J Immunol).

Hypothesis

Combination of Listeria-based vaccine with PD-1/PD-L interaction blocking

antibody will improve the overall anti-tumor efficacy of immunotherapy

43


Second Generation of Listeria-Based Immunotherapy

Results in Significant Inhibition of TC-1 Tumor Growth

and Prolonged Survival

Chen Z et al., CIR 2014

43


Lm-LLO and Lm-LLO-E7 Infection Upregulates

PD-L1 Expression on Mouse DC Surface

Mkrtichyan et al., JITC 2013

45


Lm-LLO Infection Upregulates PD-L1 Expression

on Monocyte-Derived Human DC Surface


46


Combination of Lm-LLO-E7 with Anti-PD-1 mAb

Significantly Improves Therapeutic Potency of

Immunotherapy

Lm-LLO-E7 (5x10e6 CFU)+aPD-1 mAb (50ug)

Monitoring of tumor growth

Days 0 8 15

TC-1 tumor

Tu

mo

r V

olu

me

, c

m3


Pe

rce

nt

Su

rviv

al

Days after tumor implantationMkrtichyan et al., JITC 2013

46



Significantly Increases Antigen-Specific Immune

Response

Lm-LLO-E7 (5x10e6 CFU)+aPD-1 mAb (50ug)

TERMINATION

Days 0 8 15 21

TC-1 tumor

*

***

***

***

**

Nu

mb

er

of

IFN

spo

ts p

er

10

e6

sple

no

cyte

s

*P<0.05, **P<0.01, ***P<0.001 Mkrtichyan et al., JITC 2013

47


Treatment with Listeria Significantly Decreases Splenic

MDSCs Regardless of E7 or Anti-PD-1 mAb Presence

*P<0.05

Tumor free 2.5% 6.2%Tumor-bearing

Non Treated

18.2% Tumor-bearing

Lm-LLO

CD

11

b-A

PC

Gr-1 - FITC

**

*

% o

f M

DS

C i

n s

ple

en


48


Treatment with Listeria Significantly Decreases Splenic

Treg Cells Regardless of E7 or Anti-PD-1 mAb Presence

*P<0.05

% o

f Tre

g w

ith

in C

D4 T

cells

Fo

xP

3-F

ITC

CD4 - APC

* * *


49


Treatment with Listeria Significantly Decreases Tumor-

Infiltrated MDSCs Regardless of E7 or Anti-PD-1 mAb

Presence

*P<0.05

Nu

mb

er

of

MD

SC

s p

er

10e6 t

um

or

cells

CD

11

b-A

PC

Gr-1 - FITCCD45- PE

CD45+

** *


50


0.00E+00

5.00E+02

1.00E+03

1.50E+03

2.00E+03

2.50E+03

3.00E+03

3.50E+03

Treatment with Listeria Significantly Decreases Tumor-

Infiltrated Treg Cells Regardless of E7 or Anti-PD-1 mAb

Presence

*P<0.05

Nu

mb

er

of

Tre

g c

ell

s

pe

r 1

0e

6 t

um

or

ce

lls

CD

4-A

PC

CD45 - PE FoxP3- FITC

CD45+CD4+FoxP3+

* * *


51



Significantly Increases Number of Tumor-Infiltrated

CD8 T Cells

*P<0.05, ***P<0.001C

D8

-Pe

rCP

CD45 - PE

*

***

****

Nu

mb

er

of

CD

8 T

cells p

er

10

e6

tu

mo

r ce

lls


52



While in Absence of LLO Listeria Treatment by Itself

Results in Treg Decrease, Lm-LLO Treatment Leads to

More Significant Reduction

53


Lm Decreases Treg Frequency by Preferentially Inducing

CD4+FoxP3- T Cell and CD8 T Cell Expansions


54


Episomal Expression of a Truncated LLO in LmddA

Induces Expansion of CD4+FoxP3- T cells and CD8 T

Cells to a Higher Level

Proliferation


55

Documents

Tumor-Immune System Interactioncontent.stockpr.com/.../3904/pdf/Dr.+Samir+Khleif.pdf · 2015 Dr. Samir Khleif aPD-1 n=10 E7 n=9 Non-treated n=10 E7+ aPD-1 n=8 cm3 Days after tumor