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Transcript of Date: September 20th, 2012 IMMUNECARTA TM Services 201 President-Kennedy, Suite PK-3900, Montréal,...
Date: September 20th, 2012
IMMUNECARTATM Services201 President-Kennedy, Suite PK-3900, Montréal, QC, [email protected] / T 514-360-3600www.immunecarta.com
PD-1 and the Immune Exhaustion Paradigm:Immune Profiling Tools for Drug Discovery and Clinical Monitoring
Yoav Peretz, Ph.D.
Xtalks Webinar
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OUTLINE
1. Overview of ImmuneCarta Services
2. Technologies and Applicationsa. 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 Exhaustiona. Vaccine Hyporesponse
b. Analyzing the Immune Inhibitory Profile (PD-1, TIM3, CD160, CTLA-4, etc.)
c. Functional Restoration
Intracellular Cytokine Staining (ICS)
CFSE Proliferation
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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
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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
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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, αIFN
Block plates (PBS-BSA 1%)
Peptide stimulation and incubation of cells (O/N)
Add 2nd antibody, αIFN--ALP conjugate
Spot development by adding BCIP/NBT substrate
Spots (IFNγ-secreting T cells) are counted using a CTL Immunospot analyzer
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Epitope MappingIMMUNECARTATM Services
Day 1
Day 2
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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
Mag
nitu
de (S
FC/1
06 PBM
C)
SLYNTVATL Magnitude, Breadth & Specificity
IMMUNECARTATM Services Comprehensive Epitope Mapping using Overlapping Peptide Pools
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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)
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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
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IMMUNECARTATM Services
CD160
PD
-1
A B CD160-PD-1- (DN) CD160+PD-1+ (DP)
CD160-PD-1+
(SP-PD-1)CD160+PD-1-
(SP-CD160)
Accumulation of Inhibitory Molecules during Chronic HIV Infection
IMMUNECARTATM Services
Antigen persistence shifts the phenotype (SP-PD-1 to DP) of antigen-specificCD8 T cells
12Peretz, Y et al. PLOS Pathogens (2012)
Longitudinal Analysis of CD160 and PD-1 Expression during Acute & Chronic HIV Infection
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 < 0.05when compared to DP
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Case Studies
I - Vaccine Hyporesponse (VHR) in Healthy Elderly Subjects
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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
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II - Sample Management
174 subjects; 4 TP/subject; cohorts of 20 subjects/shipment; 11 blood tubes/subject
Flow cytometryT cell panel
Innate panel
Serum aliquoting/storage
PRIMARY ENDPOINTS
ELISA (Ab Titers)Other assays
Cell pellet cryopreservation
DNA analysis
Paxgene tube storage
RNA/mRNA analysis
ImmuKnow assay
Ficoll
Other assays
Flow cytometryB 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
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IMMUNECARTATM Services
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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:No responseResponse
IV - Reduction of High Dimensionality Immune Markers to Minimal Parameters
IMMUNECARTATM Services
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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
A*0201 CMV pp65
IMMUNECARTATM Services
CD45RA
FSC
CD27
CCR7
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• 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/Exhaustion
IMMUNECARTATM Services
III - Graphical Presentation of a Phenotypic Analysis
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Following SEB-stimulation, the relative distribution of CD8 subsets expressing various combinationsof immune inhibitory markers shifts
IMMUNECARTATM Services
4 3 2 1 0# Markers
Freq
uenc
y (%
of C
D8)
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I - Intracellular Cytokine StainingIMMUNECARTATM Services
II – Analysis of the Distribution of Functional Antigen-Specific CD4 & CD8 T Cell Subsets
IMMUNECARTATM Services
Freq
uenc
y of
CD
4 (%
)
Deconvolute
Total IL-2 secretion
4 3 2 1 0# of Functions
Polyfunctional
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Monofunctional
IMMUNECARTATM Services
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Freq
uenc
y of
CD
8 (%
)
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 thePresence of Compound
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IMMUNECARTATM Services
NS Peptide Control αPD-L1 αHVEM αPD-L1 + αHVEM
Antigen-specific CD8 T cell proliferation is restored following in vitro blockadeof 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
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IMMUNECARTATM Services
Our Mission is to Accelerate the Development of Vaccines & Immune-modulating Therapeutics
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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 KhanDavid FavreJean-Francois PoulinCarey SheuJohn Kamins
Geneviève LévesqueGilbert CroteauNathalie SahaCaroline Hébert-
BenoitSasan Ziaie