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 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 2 IMMUNECARTATM Services 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 3 Flow Cytometry IMMUNECARTATM Services Technology: Multiparametric single cell analysis (cell surface, intracytoplasmic, intra-nuclear) Enumeration: Specific immune cells in whole blood Phenotyping: Cellular Differentiation, Maturation, Activation, Inhibition, Apoptosis Functionality: Cell Signaling, 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 IMMUNECARTATM Services Functional Cell-Based Assays that Monitor AntigenSpecific Immune Responses 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 5 Epitope Mapping IMMUNECARTATM Services ELISPOT Assay Detection of IFNγ-secreting lymphocytes Coating with capture antibodies, αIFN Day 1 Block plates (PBS-BSA 1%) Peptide stimulation and incubation of cells (O/N) Add 2nd antibody, αIFN--ALP conjugate Day 2 Spot development by adding BCIP/NBT substrate Spots (IFNγ-secreting T cells) are counted using a CTL Immunospot analyzer 6 Comprehensive Epitope Mapping using Overlapping Peptide Pools 2760 194 108 87 291 44 151 205 44 POL1 POL2 POL3 POL4 POL5 POL6 POL7 POL8 POL9 POL10 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 POL17 pol4260 pol4270 pol4280 pol4290 pol4300 pol4310 pol4320 pol4330 pol4340 pol4350 POL18 pol4261 pol4271 pol4281 pol4291 pol4301 pol4311 pol4321 pol4331 pol4341 pol4351 POL19 pol4262 FRDYVDRFYKTLRAE pol4272 pol4282 pol4292 pol4302 pol4312 pol4322 pol4332 pol4342 pol4352 pol4273 pol4283 pol4293 pol4303 pol4313 pol4323 pol4333 pol4343 pol4353 33 2500 11 65 378 SLYNTVATLYCVHQR 6 6 PBMC)(SFC/10 PBMC) Cumulative Magnitude (SFC/10 Magnitude IMMUNECARTATM Services -9 2250 44 POL20 pol4263 Single IFN-g secretion 2000 Dual IFN-g/IL-2 secretion 1750 1500 Magnitude, Breadth & Specificity SLYNTVATL 1250 1000 750 500 250 0 Gag Pol Nef Acc Env 7 IMMUNECARTATM Services PD-1 and the Family of Coinhibitory Molecules Restore/Enhance immune function (Cancer, Chronic Infection) Balance inflammation (Autoimmune Disorders) 8 IMMUNECARTATM Services PD-1 Regulates the Delicate Balance between Protective Immunity and Tolerance Francisco et al. Page 38 NIH-PA Author Manuscript NIH-PA Author Manuscript Fig. 7. Mechanisms of PD-1-mediated T-cell tolerance PD-L1 ligation of PD-1 following TCR stimulation results in two possible T-cell fates: Spontaneous autoimmunity observed PD-1iTreg knockout mice diminished T-effector responses andin augmented development, thus tipping the balance towards immunologic tolerance. 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 (Selfantigens, Chronic viral infections such as HIV, Oncology) 9 IMMUNECARTATM Services Hierarchical Loss of T Cell Function is Associated with Duration of Antigenic Exposure, Inflammation and Increased Expression of Inhibitory Molecules (PD-1, CD160, 2B4) Outstanding Questions: 1. Is this a reversible process? 2. Can we distinguish between an activated and an exhausted antigen-specific T cell? Adapted from Wherry, J et al. Nature immunology. 2011. 10 IMMUNECARTATM Services The Balance Between Co-stimulation and Inhibition is Critical to Maintaining T Cell Homeostasis and Function 11 Accumulation of Inhibitory Molecules during Chronic HIV Infection 40 30 20 10 30 20 10 CD160 EC ST ic on C ni 90 P = 0.0001 80 P = 0.0003 70 60 50 CMV HIV 40 30 20 10 P = 0.0001 100 90 P = 0.008 80 70 P = 0.31 60 CMV HIV 50 40 30 20 10 0 EC on ST ic e ut Ac d te ec nf ct fe ut e 0 Un in hr te d Ac ut e nf ec c EC CD160-PD-1+ (SP-PD-1) 100 ed Frequency of CD160+PD-1- T cells (% of tetramer) CD160+PD-1(SP-CD160) hr 5 CMV HIV 40 C 10 50 U 4 10 Frequency of CD160-PD-1+ T cells (% of tetramer) 3 10 CD160 60 ni 2 P = 0.0001 70 U 10 P = 0.0001 80 EC 0 ST 5 c 10 ni 4 ST ct fe in 10 90 0 Un 3 10 Tetramer P = 0.004 100 0 0 0 Frequency of CD160+PD-1+ T cells (% of tetramer) CMV HIV 50 ed 102 60 ni 0 P = 0.006 ro 3 P = 0.0001 70 e 10 80 ro 3 90 Ch 4 PD-1 10 5 P = 0.0002 Ac 10 PD-1 CD8 104 CD160+PD-1+ (DP) 100 ut 10 5 CD160-PD-1- (DN) P = 0.0001 Frequency of CD160-PD-1- T cells (% of tetramer) 10 B . B*07 CMV B*07 Nef Total CD8 Ch A Ac IMMUNECARTATM Services Antigen persistence shifts the phenotype (SP-PD-1 to DP) of antigen-specific CD8 T cells Peretz, Y et al. PLOS Pathogens (2012) 12 Longitudinal Analysis of CD160 and PD-1 Expression during Gag AcuteA*02 &CMV Chronic A*03 HIV Infection 5 10 5 10 4 10 4 10 3 10 3 PD-1 Acute <6 months . B*07 CMV B*07 Nef Total CD8 10 PD-1 IMMUNECARTATM Services 0 0 0 10 4 3 10 Chronic >12 months 3 0 2 0 0 3 10 Tetramer 10 4 10 5 0 10 2 3 10 CD160 4 10 10 2 3 10 CD160 10 4 10 5 10 5 10 5 10 4 10 4 10 3 0 10 2 10 CD160 3 10 4 10 5 0 10 2 10 CD160 3 10 4 10 5 PD-1 10 10 PD-1 10 5 PD-1 CD8 104 10 PD-1 5 PD-1 10 10 3 0 5 CD160 Peretz, Y et al. PLOS Pathogens (2012) 0 0 10 2 3 10 CD160 10 4 10 5 CD160 13 IMMUNECARTATM Services Intracellular Cytokine Staining Measuring Degranulation (CD107a), IFNγ and TNFα Secretion # 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 Case Studies 15 IMMUNECARTATM Services I - Vaccine Hyporesponse (VHR) in Healthy Elderly Subjects Cohort: 174 healthy subjects of age ≥ 65, HBV seronegative Hepatitis A/B (Twinrix) Clinical Sites: 2 recruiting sites Dukoral (WC/rBS) 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. Tetanus/Diphteria (Td) Visit 1 Visit 2 Visit 3 Visit 4 Visit 5 SCREENING VISIT BASELINE DAY 7 MONTH 1 MONTH 2 16 IMMUNECARTATM Services Serum aliquoting/storage II - Sample Management Ficoll PBMC cryopreservation Other assays PRIMARY ENDPOINTS ELISA (Ab Titers) Other assays ImmuKnow assay Flow cytometry T cell panel Innate panel Cell pellet cryopreservation Paxgene tube storage DNA analysis RNA/mRNA analysis Flow cytometry B cell panel SECONDARY IMMUNOLOGICAL ENDPOINTS 174 subjects; 4 TP/subject; cohorts of 20 subjects/shipment; 11 blood tubes/subject 17 IMMUNECARTATM Services III - Multidimensional Flow Cytometry Analysis Using N Parameters 2N Parameters combinations (512 different populations in CD4+ and CD8+ T cells = 1024 subsets per sample) 18 IMMUNECARTATM Services IV - Reduction of High Dimensionality Immune Markers to Minimal Parameters Boolean analysis of 9 markers in CD4+ and CD8+ T cells (N = 512 subsets) Prediction of vaccine hyporesponse at baseline (N = 174 subjects) Reduce dimensionality: summing 7 parameters on 2 N = 9 parameters T Cell phenotype Ab Fluor. FITC CD38 PerCP-eF710 PD-1 eFluor 450 CD57 V500 CD3 Qdot 605 CD27 eF650 NC CD8 APC CD62L Alexa700 HLA-DR APC-eF780 CD4 PE CD28 ECD CD45RA PE-Cy7 CCR7 Analysis Export New Results for N = 2 parameters PREDICTIVE MODELING(combination of 2 Innate phenotype markers) ! "#$%%"&' $( ) *+&$ No response Ab Fluor. ,. %/ ) 01 Vaccine X: FITC Response CD11c . 24# 23, #5 67 #5 < #5 => #5 =; 23B DE, F5 G 235 ! ==; 23K #5 6; 89: ; 8: ; ; ? 5 ) *@; : $. @: ; "A# , C# , %$HI ">; ; CJ CJ F4HG CJ F#+: CJ F#+> CD1c CD16 CD3 CD19 CD4 CD303 CD56 HLA-DR CD141 CD14 CD40 PerCP-eF710 + V450 V500 V500 eF650 NC # + # + 8 0 2 0 5 4 APC 3 Alexa 700 2 APC-CY7 1 PE ECD PE-Cy7 0 5 7 + + P D+ + ( B a r # ) S h o w n v a lu e s :V is it= V 2 ;A g e = 6 5 + ;C D = 4 #c and +: Stat Significant compared to group E x lu d e d v a lu e s :D i= ;D i= N A /p ;H = N A /p ;T = N A /p “HepB+ Vaccine Response” A v e r a g e d o v e r :V is it;A g e ;D i;T ;S u b je c tI D ;C D S u m m e d o v e r :R 7 ;2 7 ;2 8 ;3 8 ;R A ;6 2 L ;D R p<0.05, Wilcoxon-Rank and Student’s t-tests T h r e s h o ld = 0 + ( T te s t)o r# ( W ilc o x o n ) :p < 0 . 0 5 ;c o m p a r e d to H = H _ V A C C + 19 I - Phenotypic Characterization of Inhibition/Activation/Exhaustion IMMUNECARTATM Services • METHOD: 16-parameter, 14-color phenotyping cocktail of immune inhibitory markers on viral-specific CD8+ T cells 20120319_BI_QUALIFICATION • Layout Hierarchical gating scheme identifying the main CD4 and CD8 naïve/memory T cell subsets Experiment ID: SUBJECT ID: Group ID DATE OF FACS RUN: Timepoint 95.7 250K 250K 105 105 4 104 27.2 105 1.68 200K 150K 100K 100K 50K 50K 3 10 77.5 CD8 Viability SSC-A FSC-H 10 150K 4 A*0201 CMV pp65 10 CD45RA 200K 68.8 103 103 0 0 0 68.7 0 0 0 50K 100K 150K 200K 250K 0 50K FSC-A 100K 150K 200K 250K 0 FSC-A 250K 103 104 10 5 104 200K 150K 150K 100K 100K 50K 50K 103 104 105 105 4 79.1 105 0 3 10 0 39.1 0 103 104 105 CD27 CD4 Memory Subsets 4.2 28.9 105 105 6.34 0 103 CD27 104 10 5 40.1 10 3 10 0 14.3 0.474 4 10 CCR7 3 10 104 4 10 CCR7 CCR7 CCR7 0.242 4 10 103 CD45RA CCR7 43.4 14.8 67 0 CD45RA CD27 Phenotyping Cocktail 0 0 2.75 10 5 FSC FSC FSC 200K 0 105 104 Pentamer 250K 33 CD45RA 10 3 0 105 CD4 43 57 103 0 CD3 Live/Dead Tetramer/Pentamer CD3 CD4 CD8 CTLA4/CD152 CD45RA CD27 CCR7 Tim-3/CD PD-1/CD279 CD160 2B4/CD244 Lag-3/CD223 Negati CMV te CMV te HIV Tet HIV Tet HIV Tet HIV Tet 3 10 0 12.7 54.1 0 103 104 29.4 105 CD27 30.1 0 103 104 10 5 CD27 CD8 Memory Subsets 20 100K II - Phenotypic Characterization of Inhibition/Activation/Exhaustion 50K 50K 0 0 0 103 104 105 CD45RA 4.79 20.1 4 105 4 105 3.33 8.75 3.01 105 4 4 10 10 Boolean analysis of 6 parameters quantifying the relative distribution of 64 (26) subsets with various patterns of inhibitory receptor expression 3 10 0 3 10 0 36.2 3 10 0 38.9 7.96 19.8 104 105 103 0 104 10 5 55.8 103 0 104 PD-1 2B4 CTLA-4 103 0 CD27 CD8 Memory Subsets LAG-3 TIM-3 CD45RA CD27 21.5 78.5 C 66 12.7 30.1 3.35 1.24 CD4 0.517 48.8 105 CD27 CD4 Memory Subsets CD160 3 10 0 32.2 CD27 CD27 Analysis20120319_BI_QUALIFICATION of CD4, CD8, Pentamer, and Memory/Naive subsets 103 0 8.34 0 2 10 10 3 10 4 10 5 0 10 3 10 4 10 5 0 10 3 10 4 5 10 0 3 10 4 5 10 10 0 10 3 10 4 10 5 0 10 3 10 4 10 5 0 10 3 10 4 10 5 0 10 3 10 4 5.7 36.9 14.9 15.5 1.29 10 5 0 55.7 43.3 56.7 CD8 5.11 0 102 10 3 10 4 10 5 0 10 3 10 4 10 5 0 10 3 10 4 105 0 103 104 105 0 10 3 10 4 10 5 0 10 3 10 4 10 5 0 10 3 10 4 10 5 0 10 3 10 4 10 5 0 2.26 Pentamer • 69.3 10 CCR7 CCR7 • 2.94 105 10 104 CD45RA CCR7 105 103 0 CCR7 IMMUNECARTATM Services 100K 8.19 18.1 2 0 10 10 3 10 4 10 5 0 10 3 10 4 10 5 0 10 3 10 4 5 10 14.7 87.3 26.6 85 1.41 0 3 10 4 10 5 10 0 10 3 10 4 10 5 0 10 3 10 4 10 5 0 10 3 10 4 10 5 0 10 3 10 4 10 5 0 21 III - Graphical Presentation of a Phenotypic Analysis Frequency (% of CD8) IMMUNECARTATM Services # Markers 4 3 2 1 0 Following SEB-stimulation, the relative distribution of CD8 subsets expressing various combinations of immune inhibitory markers shifts 22 IMMUNECARTATM Services I - Intracellular Cytokine Staining Gating of T cells 17.6 150K 100K 100K 50K 50K 0 0 10 CD8 85.9 0 50K 100K 150K FSC-A 200K 250K 105 0 50K 100K 150K FSC-A 200K 250K 5 10 4 10 3 CD27 150K SSC-A FSC-H 200K 97.1 60.2 10 4 10 3 10 2 21.5 CD4+ T Cell Subsets 67.1 CM 10 Naive 5 43 48.9 CM Naive EM LD 104 CD27 250K 200K CD8+ T cell Subsets CD4+ and CD8+ T cells CD8+ 5 T10cells Live CD3+ T cells 250K 103 10 4 10 3 <Aqua-A>: Viability <eFluor 650-A>: CD8 99.9 CD4+ T cells 0 0 69.3 0 EM 7.21 0 10 2 3 4 10 10 <Qdot 605-A>: CD27 10 5 <V450-A>: CD3 0 10 3 10 4 10 5 0 2 10 3 10 4 10 2 0 7.17 4.18 10 5 0 0.869 102 103 104 105 CD45RA CD45RA CD4 Aggr- Gate Event Count: 215893 10 LD CD4+ T cells 1.53 4.85 62.3 83.7 2.55 71.6 2.68 LD CD107a 0.766 15.7 80.2 Granzyme B 0.32 27.8 0.0129 55.2 0.142 8.41 0.341 CM CD107a 0.645 99.5 0.129 Granzyme B 0.806 9.85 2.37 74 59.1 67.2 2.43 EM CD107a 1.07 32 64.8 Granzyme B 0.252 20.2 6.81e-3 13.9 0.0839 0.361 0.606 Naive CD107a 0.0681 99.6 0.093 Granzyme B IL-2 Granzyme B IL-17 IFNg CD107a TNFa 23 II – Analysis of the Distribution of Functional Antigen-Specific CD4 & CD8 T Cell Subsets IMMUNECARTATM Services Deconvolute Frequency of CD4 (%) Total IL-2 secretion # of Functions 4 Polyfunctional 3 2 1 0 Monofunctional 24 IMMUNECARTATM Services III – Analysis of the Distribution of Functional Antigen-Specific CD4 & CD8 T Cell Subsets Deconvolute Frequency of CD8 (%) Total IFNγ secretion # of Functions 4 Polyfunctional 3 2 1 0 Monofunctional 25 In vitro Rescue of Proliferation in the Presence of Compound IMMUNECARTATM Services NS NS NS Pep de alone Peptide Pep de alone Isotype Control Control Isotype Control B*08 Nef B*08 Nef FLKEKGGL FLKEKGGL + + αPD-L1 αPD-L1 αPD-L1 αHVEM αHVEM αHVEM αHVEM + αHVEM + αPD-L1 + αHVEM αPD-L1 αPD-L1 Antigen-specific CD8 T cell proliferation is restored following in vitro blockade of inhibitory molecule interaction 26 IMMUNECARTATM Services Summary Our Mission is to Accelerate the Development of Vaccines & Immune-modulating Therapeutics 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 Acknowledgements IMMUNECARTATM Services Martin Leblanc Claire Landry Marylène Fortin Lina Palmaccio Benoit Houle Salim Ahmed Khan David Favre Jean-Francois Poulin Carey Sheu John Kamins Geneviève Lévesque Gilbert Croteau Nathalie Saha Caroline Hébert-Benoit Sasan Ziaie Karyne Savard Phyla Kay Valérie Hébert Dominic Gagnon Dominike Sauvé Thank you! 28