Post on 01-Dec-2018
Date: September 20th, 2012
IMMUNECARTATM Services
201 President-Kennedy, Suite PK-3900, Montréal, QC, Canada
Info@caprion.com / 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
IMMUNECARTATM
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
302 POL12 pol4255 pol4265 pol4275 pol4285 pol4295 pol4305 pol4315 pol4325 pol4335 pol4345
399 POL13 pol4256 pol4266 pol4276 pol4286 pol4296 pol4306 pol4316 pol4326 pol4336 pol4346
1725 POL14 pol4257 pol4267 pol4277 pol4287 pol4297 pol4307 pol4317 pol4327 pol4337 pol4347
-20 POL15 pol4258 pol4268 pol4278 pol4288 pol4298 pol4308 pol4318 pol4328 pol4338 pol4348
22 POL16 pol4259 pol4269 pol4279 pol4289 pol4299 pol4309 pol4319 pol4329 pol4339 pol4349
378 POL17 pol4260 pol4270 pol4280 pol4290 pol4300 pol4310 pol4320 pol4330 pol4340 pol4350
33 POL18 pol4261 pol4271 pol4281 pol4291 pol4301 pol4311 pol4321 pol4331 pol4341 pol4351
-9 POL19 pol4262 pol4272 pol4282 pol4292 pol4302 pol4312 pol4322 pol4332 pol4342 pol4352
44 POL20 pol4263 pol4273 pol4283 pol4293 pol4303 pol4313 pol4323 pol4333 pol4343 pol4353
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)
IMMUNECARTATM
Services
� 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
IMMUNECARTATM
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
13Peretz, Y et al. PLOS Pathogens (2012)
IMMUNECARTATM
Services
Intracellular Cytokine Staining Measuring
Degranulation (CD107a), IFNγ and TNFα Secretion
14Peretz, Y et al. PLOS Pathogens (2012)
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
IMMUNECARTATM
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
IMMUNECARTATM
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
IMMUNECARTATM
Services
Fre
qu
en
cy o
f C
D4
(%
)
Deconvolute
Total IL-2 secretion
4 3 2 1 0# of Functions
Polyfunctional
24
Monofunctional
IMMUNECARTATM
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