Date: September 20th, 2012

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)


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


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 1Day 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




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




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


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)


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


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?


The Balance Between Co-stimulation and Inhibition is Critical to Maintaining T Cell Homeostasis and Function

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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


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



Intracellular Cytokine Staining Measuring Degranulation (CD107a), IFNγ and TNFα Secretion



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.


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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


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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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


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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


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


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


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


Freq

uenc

y of

CD4

(%)

Deconvolute

Total IL-2 secretion

4 3 2 1 0# of Functions

Polyfunctional

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Monofunctional


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Freq

uenc

y of

CD8

(%)

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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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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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

Date: September 20th, 2012

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Transcript of Date: September 20th, 2012