PD-1 and the Immune Exhaustion Paradigm - Caprion · 3 Advanced Immune Monitoring Services to...

Date: September 20th, 2012

IMMUNECARTATM Services

201 President-Kennedy, Suite PK-3900, Montréal, QC, Canada

[email protected] / T 514-360-3600

www.immunecarta.com

PD-1 and the Immune Exhaustion Paradigm:

Immune Profiling Tools for Drug Discovery and Clinical Monitoring

Yoav Peretz, Ph.D.

Xtalks Webinar

2

OUTLINE

1. Overview of ImmuneCarta Services

2. Technologies and Applications

a. PHENOTYPIC ANALYSES

b. FUNCTIONAL ANALYSES

� Epitope Mapping by ELISPOT

� Intracellular Cytokine Staining

� In Vitro Proliferation

3. Overview of PD-1 and Co-inhibition

� Immune Activation/Inhibition/Exhaustion (Is PD-1 sufficient?)

4. Immune Monitoring applied to the analysis of Co-Inhibition and Exhaustion

a. Vaccine Hyporesponse

b. Analyzing the Immune Inhibitory Profile (PD-1, TIM3, CD160, CTLA-4, etc.)

c. Functional Restoration

� Intracellular Cytokine Staining (ICS)

� CFSE Proliferation

3

Advanced Immune Monitoring Services to Support

Vaccine & Drug Development

• Contract Service Business

• Strategic alliance with Caprion, an exclusive supplier of

ImmuneCarta Services

� Flow-based immune monitoring of subjects enrolled in

Phase I-II Clinical Trials in a GLP/GCLP compliant

environment

� Immunological profiling and biomarker discovery for

the development of:

� Small molecules

� Biologics/Biosimilars

� Vaccines

� In vitro screening of novel immune-modulating drugs

� Development and validation of customized assays

IMMUNECARTATM

Services

4

Flow Cytometry

� Enumeration: Specific immune cells in

whole blood

� Antigen-specific responses, Epitope Mapping: Multimer

detection and identification of HLA-restricted stimulatory epitopes by ELISPOT

and FACS

� Functionality: Cell Signaling, Cytokine Secretion Profile,

Proliferation, Degranulation

� Phenotyping: Cellular Differentiation, Maturation,

Activation, Inhibition, Apoptosis

� Serological profiling: Multiplexed detection of soluble inflammatory mediators

in response to immune modulating agents

� Technology: Multiparametric single

cell analysis (cell surface, intra-

cytoplasmic, intra-nuclear)

IMMUNECARTATM

Services

Functional Cell-Based Assays that Monitor Antigen-

Specific Immune Responses

5

Flow cytometry is a unique technology that gathers phenotypic and functional data on

single cells from a heterogeneous population found in the blood or tissues.

• Relative distribution of phenotypic and functional subsets

• Predictive and/or correlative value with clinical parameters of disease progression

or therapeutic efficacy

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Services

ELISPOT Assay Detection of IFNγ-secreting lymphocytes

Coating with capture antibodies, αIFNγ

Block plates (PBS-BSA 1%)

Peptide stimulation and incubation of cells (O/N)

Add 2nd antibody, αIFN-γ-ALP conjugate

Spot development by adding BCIP/NBT substrate

Spots (IFNγ-secreting T cells) are counted using a CTL

Immunospot analyzer

6

Epitope MappingIMMUNECARTATM

Services

Da

y 1

Da

y 2

7

11 2760 194 108 87 291 44 151 205 44

POL1 POL2 POL3 POL4 POL5 POL6 POL7 POL8 POL9 POL10

65 POL11 pol4254 pol4264 pol4274 pol4284 pol4294 pol4304 pol4314 pol4324 pol4334 pol4344




-20 POL15 pol4258 pol4268 pol4278 pol4288 pol4298 pol4308 pol4318 pol4328 pol4338 pol4348




-9 POL19 pol4262 pol4272 pol4282 pol4292 pol4302 pol4312 pol4322 pol4332 pol4342 pol4352


0

250

500

750

1000

1250

Gag Pol Acc EnvNefMa

gn

itu

de

(S

FC

/10

6P

BM

C)

SLYNTVATL Magnitude, Breadth & Specificity

IMMUNECARTATM

ServicesComprehensive Epitope Mapping using Overlapping Peptide Pools

8

PD-1 and the Family of Coinhibitory Molecules

� Restore/Enhance immune function (Cancer, Chronic Infection)

� Balance inflammation (Autoimmune Disorders)

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� Spontaneous autoimmunity observed in PD-1 knockout mice

� PD-1 is involved in both central (thymus) and peripheral T cell tolerance

� Signaling through PD-1 inhibits CD8 and CD4 T cell effector functions

� PD-1 exerts critical inhibitory functions in settings of persistent antigenic stimulation (Self-

antigens, Chronic viral infections such as HIV, Oncology)

9

PD-1 Regulates the Delicate Balance between Protective

Immunity and Tolerance

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Services

Adapted from Wherry, J et al. Nature immunology. 2011.

Hierarchical Loss of T Cell Function is Associated with Duration of

Antigenic Exposure, Inflammation and Increased Expression of

Inhibitory Molecules (PD-1, CD160, 2B4)

10

Outstanding Questions:

1. Is this a reversible process?

2. Can we distinguish between an activated

and an exhausted antigen-specific T cell?

IMMUNECARTATM

Services

The Balance Between Co-stimulation and Inhibition is

Critical to Maintaining T Cell Homeostasis and Function

11

IMMUNECARTATM

Services

Uni

nfec

ted

Acu

te

Chr

onic ST

EC

0

10

20

30

40

50

60

70

80

90

100

CMV

HIV

P = 0.0001

P = 0.008

P = 0.31

Fre

qu

en

cy o

f C

D1

60

- PD

-1+ T

ce

lls(%

of te

tram

er)

CD160

PD

-1

.

Total CD8

B*07 Nef

B*07 CMV

0 102

103

104

105

CD160

0

103

104

105

PD

-1

A B CD160-PD-1- (DN) CD160+PD-1+ (DP)

CD160-PD-1+

(SP-PD-1)

Uni

nfec

ted

Acu

te

Chr

onic ST

EC

0

10

20

30

40

50

60

70

80

90

100

CMV

HIV

P = 0.0001 P = 0.006

P = 0.0002

Fre

qu

en

cy o

f C

D1

60

- PD

-1- T

ce

lls(%

of

tetr

am

er)

Uni

nfec

ted

Acu

te

Chr

onic ST

EC

0

10

20

30

40

50

60

70

80

90

100

CMV

HIV

P = 0.0003

P = 0.0001F

req

ue

nc

y o

f C

D1

60

+P

D-1

- T

ce

lls(%

of

tetr

am

er)

CD160+PD-1-

(SP-CD160)

Uni

nfec

ted

Acute

Chr

onic ST

EC

0

10

20

30

40

50

60

70

80

90

100

CMVHIV

P = 0.0001

P = 0.0001

P = 0.004

P = 0.0001

Fre

quency o

f C

D160

+P

D-1

+ T

cells

(% o

f te

tram

er)

Accumulation of Inhibitory Molecules during

Chronic HIV Infection

IMMUNECARTATM

Services

Antigen persistence shifts the phenotype (SP-PD-1 to DP) of antigen-specific

CD8 T cells

0 103

104

105

Tetramer

0

102

103

104

105

CD

8

12Peretz, Y et al. PLOS Pathogens (2012)

Longitudinal Analysis of CD160 and PD-1 Expression during

Acute & Chronic HIV Infection

.

Total CD8

B*07 Nef

B*07 CMV

0 102

103

104

105

CD160

0

103

104

105

PD

-1

CD160

PD

-1

0 103

104

105

Tetramer

0

102

103

104

105

CD

8


IMMUNECARTATM

Services

Intracellular Cytokine Staining Measuring

Degranulation (CD107a), IFNγ and TNFα Secretion


IMMUNECARTATM

Services

Co-expression of CD160 and PD-1 identifies CD8 T cells at an advanced stage of dysfunction

during chronic HIV infection

# sign represents p < 0.05

when compared to DP

15

Case Studies

I - Vaccine Hyporesponse (VHR) in Healthy Elderly

Subjects

16

Hepatitis A/B (Twinrix)

Dukoral (WC/rBS)

Tetanus/Diphteria (Td)

SCREENING

VISIT

Visit 1

BASELINE

Visit 2

DAY 7

Visit 3 Visit 4

MONTH 1

Visit 5

MONTH 2

Cohort: 174 healthy subjects of age ≥ 65, HBV seronegative

Clinical Sites: 2 recruiting sites

Objective: Exploratory study aiming to develop a statistical model to predict VHR

(antibody titers) in the elderly based on a set of phenotypic markers measured by

Flow cytometry.

IMMUNECARTATM

Services

17

II - Sample Management

174 subjects; 4 TP/subject; cohorts of 20 subjects/shipment; 11 blood tubes/subject

Flow cytometry

T cell panel

Innate panel

aliquoting/storage

Serum

aliquoting/storage

PRIMARY

ENDPOINTS

ELISA (Ab Titers)Other assays

cryopreservation

Cell pellet

cryopreservation

DNA analysis

storage

Paxgene tube

storage

RNA/mRNA analysis

ImmuKnow

assay

Ficoll

Other assays

Flow cytometry

B cell panel

PBMC

cryopreservation

SECONDARY

IMMUNOLOGICAL

ENDPOINTS

IMMUNECARTATM

Services

2N Parameters combinations

(512 different populations in CD4+

and CD8+ T cells = 1024 subsets per

sample)

Using N Parameters

III - Multidimensional Flow Cytometry Analysis

18

IMMUNECARTATM

Services

19

Ab

CD38

PD-1

CD57

CD3

CD27

CD8

CD62L

HLA-DR

CD4

CD28

CD45RA

CCR7

N = 9 parameters

Reduce dimensionality:

summing 7 parameters on 2

N = 2 parameters

� Boolean analysis of 9 markers in CD4+ and CD8+ T cells (N = 512 subsets)

� Prediction of vaccine hyporesponse at baseline (N = 174 subjects)

Export New Results for

PREDICTIVE

MODELING(combination of 2

markers)

Analysis

Vaccine X:

+ +#

+#

0

1

2

3

4

5

20

80

57

PD

+

+

+

-

-

+

-

-

# and +: Stat Significant compared to group

“HepB+ Vaccine Response”

p<0.05, Wilcoxon-Rank and Student’s t-tests

No response

Response

IV - Reduction of High Dimensionality Immune Markers to

Minimal Parameters IMMUNECARTATM

Services

0 50K 100K 150K 200K 250K

FSC-A

0

50K

100K

150K

200K

250K

FS

C-H

95.7

0 50K 100K 150K 200K 250K

FSC-A

0

50K

100K

150K

200K

250K

SS

C-A

68.7

0 103

104

105

CD3

0

103

104

105

Via

bili

ty

77.5

0 103

104

105

CD4

0

103

104

105

CD

8

68.8

27.2

0 103

104

105

Pentamer

0

103

104

105

CD

45

RA

1.68

0 103

104

105

CD27

0

103

104

105

CC

R7

0.242 79.1

6.3414.3

0 103

104

105

CD27

0

103

104

105

CC

R7

2.75 43.4

39.114.8

0 103

104

105

CD27

0

103

104

105

CC

R7

0.474 40.1

30.129.4

0 103

104

105

CD27

0

103

104

105

CC

R7

4.2 28.9

54.112.7

0 103

104

105

CD45RA

0

50K

100K

150K

200K

250K

FS

C

6733

CD4 Memory Subsets CD8 Memory Subsets

0 103

104

105

CD45RA

0

50K

100K

150K

200K

250K

FS

C

4357

20

I - Phenotypic Characterization of

Inhibition/Activation/Exhaustion

• METHOD: 16-parameter, 14-color phenotyping cocktail of immune inhibitory markers on

viral-specific CD8+ T cells

• Hierarchical gating scheme identifying the main CD4 and CD8 naïve/memory T cell subsets

Phenotyping Cocktail

Live/Dead

Tetramer/Pentamer

CD3

CD4

CD8

CTLA4/CD152

CD45RA

CD27

CCR7

Tim-3/CD

PD-1/CD279

CD160

2B4/CD244

Lag-3/CD223

A*0201 CMV pp65

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Services

CD45RA

FS

C

CD27

CC

R7

21

CD160 PD-1 2B4 CTLA-4 LAG-3 TIM-3

CD

4C

D8

Pen

tam

er

0 102

103

104

105

0.517

0 103

104

105

30.1

0 103

104

105

3.35

0 103

104

105

8.34

0 103

104

105

1.24

0 103

104

105

12.7

0 102

103

104

105

5.11

0 103

104

105

14.9

0 103

104

105

36.9

0 103

104

105

5.7

0 103

104

105

1.29

0 103

104

105

15.5

0 102

103

104

105

18.1

0 103

104

105

8.19

0 103

104

105

85

0 103

104

105

2.26

0 103

104

105

1.41

0 103

104

105

26.6

• Boolean analysis of 6 parameters quantifying the relative distribution of 64 (26) subsets

with various patterns of inhibitory receptor expression

• Analysis of CD4, CD8, Pentamer, and Memory/Naive subsets

II - Phenotypic Characterization of

Inhibition/Activation/ExhaustionIMMUNECARTATM

Services

III - Graphical Presentation of a Phenotypic Analysis

22

Following SEB-stimulation, the relative distribution of CD8 subsets expressing various combinations

of immune inhibitory markers shifts

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Services

4 3 2 1 0# Markers

Fre

qu

en

cy (

% o

f C

D8

)

CD4+ T cells

LD

CM

E

MN

aiv

e

IL-2 Granzyme B IL-17 IFNg CD107a TNFa

4.85

83.70.766

62.3

2.68

71.6

27.8

0.1420.645 8.41 0.341

55.2

9.85

67.21.07

59.12.43

74

20.2

0.08390.0681 0.606 0.361

13.9

Granzyme B

CD

107a

1.53 2.55

80.215.7

Granzyme B

CD

10

7a

0.129

0.01290.32

99.5

Granzyme B

CD

107a

0.806 2.37

64.832

Granzyme B

CD

107a

0.252 6.81e-3

0.09399.6

0 102

103

104

105

CD45RA

0

102

103

104

105

CD

27

43 48.9

0.8697.17

0 102

103

104

105

CD45RA

0

102

103

104

105

CD

27

21.5 67.1

4.187.21

0 103

104

105

CD4

0

103

104

105

CD

8

60.2

17.6

Aggr- GateEvent Count: 215893

0 50K 100K 150K 200K 250K

FSC-A

0

50K

100K

150K

200K

250K

FS

C-H

97.1

0 50K 100K 150K 200K 250K

FSC-A

0

50K

100K

150K

200K

250K

SS

C-A

85.9

0 102

103

104

105

<Qdot 605-A>: CD27

0

103

104

105

<e

Flu

or

65

0-A

>:

CD

8

99.9

<V450-A>: CD3

<A

qua

-A>

: V

iabili

ty

69.3

Gating of T cells

Live CD3+ T cells CD4+ and CD8+ T cells

CD4+ T cells

CD8+ T cells

NaiveCM

LDEM

CM Naive

LDEM

CD4+ T Cell SubsetsCD8+ T cell Subsets

23

I - Intracellular Cytokine StainingIMMUNECARTATM

Services

II – Analysis of the Distribution of Functional Antigen-Specific

CD4 & CD8 T Cell Subsets

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Services

Fre

qu

en

cy o

f C

D4

(%

)

Deconvolute

Total IL-2 secretion

4 3 2 1 0# of Functions

Polyfunctional

24

Monofunctional

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Services

25

Fre

qu

en

cy o

f C

D8

(%

)

Deconvolute

Total IFNγ secretion

4 3 2 1 0# of Functions

MonofunctionalPolyfunctional

III – Analysis of the Distribution of Functional Antigen-Specific

CD4 & CD8 T Cell Subsets

In vitro Rescue of Proliferation in the

Presence of Compound

26

IMMUNECARTATM

Services

αHVEM +

B*08 Nef

FLKEKGGL

+

αPD-L1NS Peptide Control αPD-L1 αHVEM αPD-L1 + αHVEM

Antigen-specific CD8 T cell proliferation is restored following in vitro blockade

of inhibitory molecule interaction

Summary

� In settings of persistent antigenic stimulation and chronic immune activation, there is a hierarchical loss

of immune effector cell function.

� Functional responses can be restored and enhanced following in vitro blockade of inhibitory molecules

� Applications:

� Mutiparametric flow cytometry identifies and distinguishes between activated and exhausted

effector subsets

� Functional restoration of cytokine secretion and proliferation can be measured in vitro in response

to compounds as well as ex vivo in a clinical setting

� Therapeutic areas of interest:

� Oncology

� Infectious Diseases

� Autoimmunity

� Immunosenescence

� Transplantation

27

IMMUNECARTATM

Services

Our Mission is to Accelerate the Development

of Vaccines & Immune-modulating Therapeutics

28

AcknowledgementsIMMUNECARTATM

Services

Thank you!

� Martin Leblanc

� Claire Landry

� Marylène Fortin

� Lina Palmaccio

� Benoit Houle

� Karyne Savard

� Phyla Kay

� Valérie Hébert

� Dominic Gagnon

� Dominike Sauvé

� Salim Ahmed Khan

� David Favre

� Jean-Francois Poulin

� Carey Sheu

� John Kamins

� Geneviève Lévesque

� Gilbert Croteau

� Nathalie Saha

� Caroline Hébert-Benoit

� Sasan Ziaie

PD-1 and the Immune Exhaustion Paradigm - Caprion · 3 Advanced Immune Monitoring Services to...

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