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Date: September 20th, 2012

PD-1 and the Immune Exhaustion Paradigm: Immune Profiling Tools for Drug Discovery and Clinical Monitoring. Yoav Peretz, Ph.D. Xtalks Webinar. Date: September 20th, 2012. IMMUNE CARTA TM Services 201 President-Kennedy, Suite PK-3900, Montréal, QC, Canada Info@caprion.com / T 514-360-3600

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Date: September 20th, 2012

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  1. PD-1 and the Immune Exhaustion Paradigm: Immune Profiling Tools for Drug Discovery and Clinical Monitoring Yoav Peretz, Ph.D. Xtalks Webinar 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

  2. OUTLINE • Overview of ImmuneCarta Services • Technologies and Applications • PHENOTYPIC ANALYSES • FUNCTIONAL ANALYSES • EpitopeMapping by ELISPOT • Intracellular Cytokine Staining • In Vitro Proliferation • Overview of PD-1 and Co-inhibition • Immune Activation/Inhibition/Exhaustion (Is PD-1 sufficient?) • Immune Monitoring applied to the analysis of Co-Inhibition and Exhaustion • Vaccine Hyporesponse • Analyzing the Immune Inhibitory Profile (PD-1, TIM3, CD160, CTLA-4, etc.) • Functional Restoration • Intracellular Cytokine Staining (ICS) • CFSE Proliferation

  3. Advanced Immune Monitoring Services to Support Vaccine & Drug Development IMMUNECARTATMServices • 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 • Immunologicalprofiling 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

  4. Flow Cytometry IMMUNECARTATMServices • Technology:Multiparametric single cell analysis (cell surface, intra-cytoplasmic, intra-nuclear) • Enumeration:Specific immune cells in whole blood • Phenotyping:Cellular Differentiation, Maturation, Activation, Inhibition, Apoptosis • Functionality: CellSignaling, Cytokine Secretion Profile, Proliferation, Degranulation • Antigen-specific responses, Epitope Mapping:Multimer detection and identification of HLA-restricted stimulatory epitopes by ELISPOT and FACS • Serological profiling: Multiplexed detection of soluble inflammatory mediators in response to immune modulating agents 4

  5. Functional Cell-Based Assays that Monitor Antigen-Specific Immune Responses IMMUNECARTATMServices 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

  6. Epitope Mapping IMMUNECARTATMServices 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 2ndantibody, αIFN--ALP conjugate Spot development by adding BCIP/NBT substrate Spots (IFNγ-secreting T cells) are counted using a CTL Immunospot analyzer Day 1 Day 2

  7. IMMUNECARTATMServices Comprehensive Epitope Mapping using Overlapping Peptide Pools Magnitude, Breadth & Specificity SLYNTVATL Magnitude (SFC/106 PBMC)

  8. PD-1 and the Family of Coinhibitory Molecules IMMUNECARTATMServices • Restore/Enhance immune function (Cancer, Chronic Infection) • Balance inflammation (Autoimmune Disorders) 8

  9. PD-1 Regulates the Delicate Balance between Protective Immunity and Tolerance IMMUNECARTATMServices • 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)

  10. Hierarchical Loss of T Cell Function is Associated with Duration of Antigenic Exposure, Inflammation and Increased Expression of Inhibitory Molecules (PD-1, CD160, 2B4) IMMUNECARTATMServices Outstanding Questions: Is this a reversible process? Can we distinguish between an activated and an exhausted antigen-specific T cell? Adapted from Wherry, J et al. Nature immunology. 2011. 10

  11. The Balance Between Co-stimulation and Inhibition is • Critical to Maintaining T Cell Homeostasis and Function IMMUNECARTATMServices 11

  12. Accumulation of Inhibitory Molecules during Chronic HIV Infection IMMUNECARTATMServices A B CD160-PD-1- (DN) CD160+PD-1+ (DP) PD-1 CD160 CD160+PD-1- (SP-CD160) CD160-PD-1+ (SP-PD-1) Antigen persistence shifts the phenotype (SP-PD-1 to DP) of antigen-specific CD8 T cells 12 Peretz, Y et al. PLOS Pathogens (2012)

  13. Longitudinal Analysis of CD160 and PD-1 Expression during Acute & Chronic HIV Infection IMMUNECARTATMServices Peretz, Y et al. PLOS Pathogens (2012) 13

  14. Intracellular Cytokine Staining Measuring Degranulation (CD107a), IFNγ and TNFα Secretion IMMUNECARTATMServices # sign represents p < 0.05 when compared to DP Co-expression of CD160 and PD-1 identifies CD8 T cells at an advanced stage of dysfunction during chronic HIV infection Peretz, Y et al. PLOS Pathogens (2012) 14

  15. CaseStudies

  16. IMMUNECARTATMServices I - Vaccine Hyporesponse (VHR) in Healthy Elderly Subjects 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. Visit 2 Visit 3 Visit 4 Visit 5 Visit 1 DAY 7 MONTH 1 MONTH 2 BASELINE SCREENING VISIT Hepatitis A/B (Twinrix) Dukoral (WC/rBS) Tetanus/Diphteria (Td)

  17. Serum aliquoting/storage Ficoll ImmuKnow assay Flow cytometry T cell panel Innate panel Cell pellet cryopreservation Paxgene tube storage PBMC cryopreservation Flow cytometry B cell panel PRIMARY ENDPOINTS ELISA (Ab Titers) DNA analysis RNA/mRNA analysis Other assays Other assays II - Sample Management IMMUNECARTATMServices SECONDARY IMMUNOLOGICAL ENDPOINTS 174 subjects; 4 TP/subject; cohorts of 20 subjects/shipment; 11 blood tubes/subject 17

  18. III - Multidimensional Flow Cytometry Analysis IMMUNECARTATMServices Using N Parameters 2N Parameters combinations (512 different populations in CD4+ and CD8+ T cells = 1024 subsets per sample)

  19. IV - Reduction of High Dimensionality Immune Markers to Minimal Parameters IMMUNECARTATMServices Analysis • 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) Reduce dimensionality: summing 7 parameters on 2 Vaccine X: N = 9 parameters N = 2 parameters No response Response

  20. I - Phenotypic Characterization of Inhibition/Activation/Exhaustion IMMUNECARTATMServices • 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 FSC CD45RA CCR7 CD27

  21. II - Phenotypic Characterization of Inhibition/Activation/Exhaustion IMMUNECARTATMServices • 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

  22. IMMUNECARTATMServices III - Graphical Presentation of a Phenotypic Analysis Frequency (% of CD8) 4 3 2 1 0 # Markers Following SEB-stimulation, the relative distribution of CD8 subsets expressing various combinations of immune inhibitory markers shifts

  23. I - Intracellular Cytokine Staining IMMUNECARTATMServices

  24. II – Analysis of the Distribution of Functional Antigen-Specific CD4 & CD8 T Cell Subsets IMMUNECARTATMServices Total IL-2 secretion Deconvolute Frequency of CD4 (%) 4 3 2 1 0 # of Functions Polyfunctional Monofunctional

  25. III – Analysis of the Distribution of Functional Antigen-Specific CD4 & CD8 T Cell Subsets IMMUNECARTATMServices Total IFNγ secretion Deconvolute Frequency of CD8 (%) 4 3 2 1 0 # of Functions Polyfunctional Monofunctional

  26. In vitroRescue of Proliferation in the Presence of Compound IMMUNECARTATMServices NS Peptide Control αPD-L1 αHVEM αPD-L1 + αHVEM Antigen-specific CD8 T cell proliferation is restored following in vitro blockade of inhibitory molecule interaction 26

  27. Summary IMMUNECARTATMServices Our Mission is to Accelerate the Development of Vaccines & Immune-modulatingTherapeutics • 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

  28. Acknowledgements IMMUNECARTATMServices • Martin Leblanc • Claire Landry • Marylène Fortin • LinaPalmaccio • Benoit Houle • Salim Ahmed Khan • David Favre • Jean-Francois Poulin • Carey Sheu • John Kamins • Geneviève Lévesque • Gilbert Croteau • Nathalie Saha • Caroline Hébert-Benoit • SasanZiaie • KaryneSavard • Phyla Kay • Valérie Hébert • Dominic Gagnon • Dominike Sauvé Thankyou!

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