Flow cytometry and Phenotypic/Functional Analysis in Immuno-Oncology. Adoptive T cell Therapies

Transcription

1 Flow cytometry and Phenotypic/Functional Analysis in ImmunoOncology Adoptive T cell Therapies Yoav Peretz, Ph.D. Scientific Director Caprion Biosciences/ImmuneCarta Adjunct Professor McGill University

2 Outline Flow Cytometry Platform Overview Backround on Adoptive Cell Therapy and T cell Retargeting Strategies Autologous transfer of NYESO1 TCRtransduced T cells Phenotypic and Functional Flow Cytometry Assay Development Monitoring NYESO1 T cells (Persistence, Memory, Maturation, Immune Exhaustion markers) Functional profile of NYESO1 specific T cells (ICS) Clinical study Flow Cytometry Analysis of the Manufactured Product Immune Monitoring of patient PBMC (Clinical Progression vs NonProgression) TIL analysis Conclusions

3 CAPRION S PROPRIETARY CORE TECHNOLOGIES PROTEOCARTA, is a gelfree, labelfree mass spectrometry platform for comprehensive, quantitative and robust measurements of proteins across large sets of samples for biomarker discovery and validation Bestinclass, industrialized, and quantitative mass spectrometry technology for unbiased protein biomarker discovery Leading expertise in development and deployment of targeted proteomics using highly multiplexed MRM assays for biomarker development, validation Greater reproducibility, throughput, and multiplexing capabilities than competing technologies Expanded offering of proprietary biomarker assays IMMUNECARTA is a proprietary immune monitoring platform offering multiparametric flow cytometry for comprehensive functional and phenotypical analyses of complex innate and adaptive immune responses Differentiated expertise in multiparametric flow cytometry (up to 1 parameters) Provides advanced immune monitoring for preclinical and clinical development of vaccines, biologics and immunotherapeutics Differentiated ability to rapidly develop, validate and deploy customized complex assays for clinical immune monitoring studies Deep expertise in immuneoncology Differentiated service offering from 2 proprietary core technologies Balanced mix between discovery, preclinical and clinical development studies GLP operation with rigorous QA/QC

4 Technologies and Applications ü Technology: 1color Mul5parametric Single Cell Analysis (cell surface, intracytoplasmic, intranuclear) ü Enumerate: Discrete cellular subsets in whole blood (ex: CD34, TBNK using TruCount) ü Phenotype: Cellular Differen5a5on, Apoptosis, Cell subsetspecific panels (MDSC, Treg, T memory, B cells, DCs ) ü Func,onality: Cell Signaling (PhosFlow TM ), Cytokine Secre,on Profile (ICS), Cell prolifera5on (CFSE), Degranula5on (BAT) ü An,genspecific response: Mul5mer detec5on and iden5fica5on of HLArestricted s5mulatory epitopes by ELISPOT and flow cytometry ü Comprehensive T Cell Epitope Mapping: Breadth, Magnitude & Specificity of HLArestricted s5mulatory epitopes by ELISPOT ü Serological profiling: Mul5plexed detec5on of soluble inflammatory mediators in response to immune modula5ng agents (MSD, ELISA)

5 Flow Cytometers LSR Fortessa equipped with 5 lasers Up to 1 unique fluorescent markers can be monitored simultaneously plus with FSC and SSC parameters Enables redistribution of fluorescent markers onto other lasers in order to minimize fluorescence spillover between channels Flow panel customization Selection of Clone/Fluorochrome combinations Anticipated Antigen Expression Intensity Assay conditions (i.e Perm/no perm) Panel setup and qualification is performed before Immune monitoring Titrations Instrument settings and compensation FMO controls Precision on critical readouts Laser Name Wavelength Power Detector Array Blue 4 nm 5 mw Octagon Violet 45 nm 5 mw Octagon Yellow Green 561 nm 5 mw Octagon Red 64 nm 4 mw Trigon UV 355 nm 2 mw Trigon Mirror Filter 63 LP 695/4 BP 55 LP 53/3 BP 4/1 BP 75 LP 7/6 BP 69 LP 71/5 BP 63 LP 66/2 BP 595 LP 61/2 BP 55 LP 525/5 BP 45/5 BP 735 LP 7/6 BP 635 LP 67/3 BP 6 LP 61/2 BP 55/15 BP 735 LP 7/6 BP 71 LP 73/45 BP 67/14 BP 69 LP 74/35 BP 45 LP 515/3 BP 379/25 BP Laser, detector and filter combinations for LSR cytometers at Caprion

6 Adoptive Autologous T cell Therapies ImmTACs (Immune mobilizing monoclonal TCRs against cancer) TIL Target: peptidehla Affinity enhanced TCR ex: NYESO1 in Myeloma Recognizes endogenously processed Tumor Ag Greater number of targets available Target: Surface protein ex: CD19 in NHL Target: peptidehla Biopsy or resection is required Target: peptidehla Target: Surface protein ex: CD19 in NHL Affinity enhanced and fused to anticd3 No cellular manufacturing Ab binding is higher affinity vs TCR Only approx. 1% of targets are surface bound DARTs (DualAffinity retargeting technology) BITEs (Bispecific T cell engager) T cell redirecting Rx Can recruit several arms of the immune system Easier/faster to produce Less costly

7 Increasing the affinity of Tumorspecific TCRs (NYESO1) All T cells undergo Thymic selection (2 45 TCR variants) This has evolved to remove autoreactive T cells from an individuals repertoire Therefore the circulating repertoire to self antigens (tumor antigens) are of low affinity MHC down regulation Local immunosuppressive mechanisms Aleksic, M., et al. (212). Eur. J. Immunol

8 Are NYESO1 specific T cells Persisting? What is the Phenotype of NYESO1 specific T cells with respect to markers of Immune Dysfunction, Memory and Maturation? What is the functionality of NYESO1 specific T cells? What is the Phenotype and Function of the Manufactured product prior to infusion?

9 Hierarchical Loss of T Cell Function leads to Immune Exhaustion Adapted from Wherry, J et al. Nature immunology Hierarchical Loss of T Cell Function is Associated with Duration of Antigenic Exposure, Inflammation and Increased Expression of Inhibitory Molecules (PD1, CD16, 2B4)

10 Method Development of Flow Cytometry Panels Assay Phases Flow Cytometry Panel Setup Identify; Establish Method Development Method Sample Analysis (Prevalidation) Validation (PreValidation) Identify; Establish Confirm Apply; Monitor Antibody cocktail Titration Instrument settings Specificity (Isotype) Spillover (FMOC) Assay Format Matrix Selection Stability LOD/LLOQ Precision Dilution/Linearity Specificity Precision Dilution/ Linearity LLOQ Stability

11 An,body Panels (T cell Panel) Phenotyping An,body Live Dead CD3 CD4 Lineage CD Pentamer NYESO (9C) CD45RA CCR7 Memory CD45RO CD95 CD25 Treg markers CD127 LAG3 PD1 Immune inhibition / exhaustion TIM3 Mahnke, Y., et al. (213). Eur. J. Immunol

12 Boolean Analysis of Checkpoint Marker Expression on T Cell Subsets CD CD4 T REG Bar Chart Legend SUBJECT_ID: L5 SUBJECT_ID: L747 SUBJECT_ID: L77 SEBstimulated PBMC (72h) LAG3 PD1 TIM3. LAG3 PD1 TIM3 CD T Cell Panel (Naïve/memory, PD1, TIM3, LAG3) LAG3 PD1 TIM3 Intraassay CV below 1% for most readouts This is a function of the number of events accumulated in each target gate

13 Analysis of AntigenSpecific Responses by Intracellular Cytokine Staining (ICS) Quantification of the frequency of antigenspecific T cells through analysis of cytokine expression combined to Pentamer analysis Assay performed on cryopreserved PBMC PBMC are thawed and counted PBMC are stimulated and incubated 6hrs prior to staining for Flow 1 2 Stimulate cells and treat with protein and transport inhibitor 3 4 Fix and permeabilize cells Stain cells Flow Cytometry Analysis SEBstimulated PBMC (6h) An,body Panel Live Dead CD3 CD4 Lineage CD Pentamer NYESO (9C) CD45RA CCR7 Ki67 HLADR IL2 TNFa IFNg Granzyme B CD17a Memory Activation/ Proliferation Effector cytokines

14 Analysis of the Distribution of AntigenSpecific CD4 & CD T Cell Subsets (Boolean Approach) D1 D2 D3 Pie Chart Arc Legend IFN gamma IL2 TNF alpha IL17 Bar Chart Legend TIMEPOINT: Day 1 TIMEPOINT: Day 2 TIMEPOINT: Day 3 TIMEPOINT: Day 1 TIMEPOINT: Day 2 TIMEPOINT: Day Total IL2 secretion Deconvolute Frequency of CD (%) IFN gamma IL2 TNF alpha IL17 Pie Slice Bar Chart Legend TIMEPOINT: Day 1 TIMEPOINT: Day 2 TIMEPOINT: Day IL2 e Slice. IFN gamma IL2 TNF alpha IL17 Pie Slice # of Functions Polyfunctional Monofunctional

15 Flow cytometry Panel Qualification 15K FSCH 25K 2K Ungated Singlets Lymphocytes Viables CD3 9 SSCA 25K 2K 15K 1 3 1K 1K K 5K 1 2 <AquaA>: Viability <Qdot 655A>: CD K 1K 15K 2K 25K FSCA 5K 1K 15K 2K 25K FSCA <V45A>: CD <Qdot 65A>: CD4 25K Ungated 25K Singlets 1 5 Lymphocytes Viables CD3 CD Viables CD3 5.1 FSCH 2K 15K 1K 5K 95.7 SSCA 2K 15K 1K 5K 4.9 <AquaA>: Viability <PEA>: Pentamer <Qdot 655A>: CD <PEA>: Pentamer K 1K 15K 2K 25K FSCA K 1K 15K 2K 25K <Qdot 65A>: CD4 CD4 <Qdot 655A>: CD <V45A>: CD3 FSCA <PECy7A>: CCR CD4 CD CDPentamer Gated on CD FMOC No CD45RA 1 FMOC No CCR7 1 3 Full Cocktail <APCCy7A>: CD45RA <APCCy7A>: CD45RA <APCCy7A>: CD45RA <PECy7A>: CCR <PECy7A>: CCR L25 (SEB) <PECy7A>: CCR <PECy7A>: CCR <PECy7A>: CCR Fluorescence minus One Control Specificity/Sensitivity Spill Over Signal detection threshold <APCCy7A>: CD45RA <APCCy7A>: CD45RA <APCCy7A>: CD45RA e e WAVE (PMAIONO) <PECy7A>: CCR <PECy7A>: CCR <PECy7A>: CCR <APCCy7A>: CD45RA <APCCy7A>: CD45RA <APCCy7A>: CD45RA

16 FMOC IFNγ (Gated on CD) Viables CD3 <Qdot 655A>: CD FMOC No IFNγ CD L25 (SEB) IL2 TNFa IFNg Ki67 HLADR Granzyme B CD17a Full Cocktail CD3 IL2 TNFa IFNg Ki67 HLADR Granzyme B CD17a e WAVE (PMAIONO) FMOC No IFNγ CD3 IL2 TNFa IFNg Ki67 HLADR Granzyme B CD17a Full Cocktail CD3 IL2 TNFa IFNg Ki67 HLADR Granzyme B CD17a

17 Protocols for Pentamer Staining combined with ICS We observed that the frequency of NYESO1 TCR detected and backround levels observed by ICS varied depending on the protocol used Following TCR triggering, the complex gets downregulated Pentamer staining triggers the TCR Protocol 1: Pentamer staining BEFORE stimulation Plate cells (1.5X1 6 cells per well) Add pentamer and Incubate for 1 min at RT Wash Add stimulators and CD17a (or DMSO in unstimulated samples) and Golgi Plug/Stop, and incubate for 6h at 37 C Wash Add surface cocktail and incubate 3 min at 4 C Wash Fix Permeabilize Add intracellular cocktail and incubate 3 min at 4 C Wash Acquire data on flow cytometer Protocol 2: Pentamer staining AFTER stimulation Plate cells (1.5X1 6 cells per well) Add stimulators and CD17a (or DMSO in unstimulated samples) and Golgi Plug/Stop, and incubate for 6h at 37 C Wash Add pentamer and incubate for 1 min at RT Wash Add surface cocktail and incubate 3 min at 4 C Wash Fix Permeabilize Add intracellular cocktail and incubate 3 min at 4 C Wash Acquire data on flow cytometer

18 Protocol Comparison (L25, Unstimulated) e Protocol 1 <Qdot 655A>: CD STAIN BEFORE STIM <PEA>: Pentamer <Qdot 65A>: CD4.632 Gated on CD Gated on CDPentamer CD3 CD3 IL2 TNFa IFNg Ki67 HLADR G Staining before stimulation induces backround <Qdot 655A>: CD IL2 TNFa IFNg e Protocol 2 STAIN AFTER STIM <Qdot 655A>: CD <PEA>: Pentamer <Qdot 65A>: CD4.964 Gated on CD Gated on CDPentamer CD3 CD3 IL2 TNFa IFNg Ki67 HLADR G Better pentamer separation and detection frequency Lower backround levels <Qdot 655A>: CD IL2 TNFa IFNg Ki67 HLADR G

19 Protocol Comparison (L25, CEF) e Protocol 1 <Qdot 655A>: CD STAIN BEFORE STIM <PEA>: Pentamer <Qdot 65A>: CD4.535 Gated on CD Gated on CDPentamer CD3 CD3 IL2 TNFa IFNg Ki67 HLADR Gr CMVspecific responses are detected (TNFα and IFNγ) Better pentamer separation and detected frequency <Qdot 655A>: CD IL2 TNFa IFNg e <Qdot 655A>: CD Gated on CD CD3 Protocol 2 STAIN AFTER STIM <PEA>: Pentamer <Qdot 65A>: CD4.65 Gated on CDPentamer CD3 IL2 TNFa IFNg Ki67 HLADR Gr Stimulation with CEF induces TCR downregulation <Qdot 655A>: CD IL2 TNFa IFNg Ki67 HLADR Gr

20 Clinical Study Design Cohorts and Study Design: Phase I/IIa single arm trial that enrolled 2 pa5ents who received Autologous Stem Cell Transplant followed by genemodified T cells (NYESO1 C259 ) Inclusion criteria Pa5ent screening for HLA*21, LAGE1 or NYESO1 posi5ve myeloma (7 screened / 5 excluded) Clinical Sites: 2 clinical sites Objec,ves Primary: Determine the safety and tolerability of autologous gene5cally modified T cells. Secondary: Evaluate correlates of treatment efficacy by measuring immune parameters such as the persistence and func5onality of NYESO1 transduced T cells. Hypothesis: Adop5ve transfer of NYESO1 enhanced TCR s would improve the dura5on of ASCT in advanced MM Rapoport, A.P. et al. July 215. Nature Medicine

21 Investigational Drug Manufacturing Process and Screening Patient conditioning and lymphodepleting chemotherapy T cell Infusion Mean: 2 x 1 9 / donor Patient Screening (NY ESO1/LAGE1 mrna and HLAA*21) Blood Cell Collection (Leukapheresis) 1 day Clinical sites T cell Product and Release Testing Central Manufacturing Site Enrichment/ Selection of CD3 T cells Deplete Monocytes and CD25 cells 1 day 1 days Harvesting/Bead Removal & Formulation Activation/Lentiviral Transduction (NYESO1 TCR) Beadbased activation/expansion (CD3/CD2) T cell Expansion >2% transduction efficiency 12 days

22 Clinical Blood Sample Processing & Storage Capabilities Whole blood samples SST No additive Standard K 2/3 EDTA NaHeparin CytoCHEX K 3 EDTA CPT NaHeparin NaCitrate Leukapheresis ACD FICOLL SPIN FICOLL Frozen serum aliquots Frozen plasma aliquots Smart Tube Fixative PBMC isolation Sample assayed fresh (within a stability window) PBMC isolation Samples assayed in batches PBMC Cryovials All procedures are conducted using approved SOP

23 Thawing Sta,s,cs of the Cohort Viability Recovery N = 26 Viability = 95% (median) Recovery = 61% (median)

24 Phenotyping Assay Hierarchical Ga,ng 253 (Clinical Progressor) Frequency of viable CD (Clinical Responder) Frequency of viable CD L25 L25 WAVE49B WAVE49B _MP CD4 lymphocytes _MP _Day _Day _Day42 CD4 lymphocytes _Day _Day _Day _Day _Day _Day _Day _Day _Day36 Frequency of viable CD3 Frequency of viable CD L25 L25 WAVE49B WAVE49B _MP CD lymphocytes _MP _Day _Day _Day42 CD lymphocytes _Day _Day _Day _Day _Day _Day _Day _Day _Day36 Noted differences in CD4/CD ratios and frequencies of CD4 between both donors for both the MP and the baseline timepoint CD4 frequencies were approx. 3 times higher in donor 21 MP and at baseline CD4/CD Ratio CD4/CD Ratio L25 L25 WAVE49B WAVE49B _MP _MP CD4/CD Ratio _Day _Day _Day42 CD4/CD Ratio _Day _Day _Day _Day _Day _Day _Day _Day _Day36

25 NYESO1 T Cell Persistence Frequency of viable CD L25 PentamerCD WAVE49B _MP _Day5.56%.22% _Day _Day _Day1 Frequency of viable CD L25 PentamerCD4 WAVE49B _MP _Day _Day _Day _Day1 253 (Clinical Progression) Frequency of viable CD L25 PentamerCD WAVE49B _MP _Day _Day _Day _Day _Day _Day _Day _Day36 Frequency of viable CD L25 PentamerCD4 WAVE49B _MP _Day _Day _Day _Day _Day _Day _Day _Day36 Loss of NYESO1 T cell persistence is associated with clinical progression 21 (Clinical Response)

26 Memory Profile of NYESO1 PentamerCD4 Frequencies of parent (% of PentamerCD4) 5, 4, 3, 2, 1,, SUBJECT_ID Subset CCR7 CD45RA CD45RO Pie Slice (Clinical Progression) PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD4 Bar Chart Legend TIMEPOINT: MP TIMEPOINT: Day7 TIMEPOINT: Day21 TIMEPOINT: Day42 TIMEPOINT: Day1 TIMEPOINT: Day1 TIMEPOINT: Day27 Bar Chart Legend TIMEPOINT: MP TIMEPOINT: Day21 TIMEPOINT: Day42 TIMEPOINT: Day1 TIMEPOINT: MP TIMEPOINT: Day7 TIMEPOINT: Day21 TIMEPOINT: Day42 TIMEPOINT: Day1 TIMEPOINT: Day1 TIMEPOINT: Day (Clinical Response) TIMEPOINT: Day36 Bar Chart Legend TIMEPOINT: TIMEPOINT: Day36 MP TIMEPOINT: Day7 TIMEPOINT: Day21 TIMEPOINT: Day42 TIMEPOINT: Day1 TIMEPOINT: Day1 TIMEPOINT: Day27. SUBJECT_ID SUBJECT_ID Subset PentamerCD CCR7 Subset PentamerCD4 CD45RA CCR7 CD45RO CD45RA Pie Slice CD45RO Pie Slice PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD4 PentamerCD PentamerCD4 PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD4 Distinct distribution of memory/maturation profiles within NYESO1 CD4 T cells (MP and Patient sample) Progression associated with Effector phenotypes contrary to TCR persistence associated with a Central memory phenotype

27 Memory Profile of NYESO1 PentamerCD 6, 4, 253 (Clinical Progression) Bar Chart Legend TIMEPOINT: MP TIMEPOINT: Day21 TIMEPOINT: Day42 TIMEPOINT: Day1 Frequencies of parent (% of PentamerCD) 2,, SUBJECT_ID Subset CCR7 CD45RA CD45RO Pie Slice PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD TIMEPOINT: Day1 TIMEPOINT: MP TIMEPOINT: Day7 TIMEPOINT: Day21 TIMEPOINT: Day42 TIMEPOINT: Day1 TIMEPOINT: Day1 TIMEPOINT: Day27 TIMEPOINT: Day36 TIMEPOINT: Day SUBJECT_ID Subset SUBJECT_ID PentamerCD PentamerCD PentamerCD PentamerCD CCR7 Subset PentamerCD PentamerCD PentamerCD CD45RA CCR7 CD45RO CD45RA Pie Slice CD45RO Pie Slice 21 (Clinical Response) PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD Bar Chart Legend TIMEPOINT: MP TIMEPOINT: Day7 TIMEPOINT: Day21 TIMEPOINT: Day42 TIMEPOINT: Day1 TIMEPOINT: Day1 TIMEPOINT: Day PentamerCD Bar TIMEPOINT: Chart Legend Day36 TIMEPOINT: MP TIMEPOINT: Day7 TIMEPOINT: Day21 TIMEPOINT: Day42 TIMEPOINT: Day1 TIMEPOINT: Day1 TIMEPOINT: Day27 TIM Distinct distribution of memory/maturation profiles within NYESO1 CD T cells (MP and Patient sample) Progression associated with Effector phenotypes contrary to TCR persistence associated with a Central memory phenotype

28 Checkpoint Molecule Expression (NYESO1 PentamerCD), 4, 253 (Clinical Progression) Bar Chart Legend TIMEPOINT: MP TIMEPOINT: Day21 TIMEPOINT: Day42 2, TIMEPOINT: Day1 15, Frequencies of parent (% of PentamerCD) 1, 5,, SUBJECT_ID Subset LAG3 PD1 TIM PentamerCD 21 (Clinical Response) PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD Bar Chart Legend TIMEPOINT: MP TIMEPOINT: Day7 TIMEPOINT: Day21 TIMEPOINT: Day42 TIMEPOINT: Day1 TIMEPOINT: Day1 TIMEPOINT: Day27 TIMEPOINT: Day SUBJECT_ID Subset LAG3 PD1 TIM PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD PentamerCD No accumulation of checkpoints on NYESO1 CD T cells in both donors Switch in phenotype during progression (TIM3 to PD1 switch)

29 ICS Assay IFNγ (NYESO1 PentamerCD), 253 (Clinical Progression) 6, Bar Chart Legend TIMEPOINT: MP 4, TIMEPOINT: Day21 Frequencies of parent (% of PentamerCD) 2,, SUBJECT_ID STIMULATION Subset IFNg T2 PentamerCD T2NYESO PentamerCD PMAIONO PentamerCD TIMEPOINT: Day42 TIMEPOINT: Day1 21 (Clinical Response) Bar Chart Legend TIMEPOINT: MP TIMEPOINT: Day7 TIMEPOINT: Day21 TIMEPOINT: Day42 TIMEPOINT: Day1 TIMEPOINT: Day36 TIMEPOINT: Day1. SUBJECT_ID STIMULATION Subset IFNg T2 PentamerCD T2NYESO PentamerCD PMAIONO PentamerCD TIMEPOINT: Day27 Presence of NYESO1 specific IFNγ secretion in both patients

30 ICS Assay IFNγ, TNFα, and IL2 (NYESO1 PentamerCD) 3, 25, 2, 253 (Clinical Progression) Bar Chart Legend TIMEPOINT: MP 15, TIMEPOINT: Day21 Frequencies of parent (% of PentamerCD) 1, 5,, SUBJECT_ID STIMULATION Subset IFNg IL2 TNFa T2NYESO PentamerCD 21 (Clinical Response) T2NYESO PentamerCD T2NYESO PentamerCD T2NYESO PentamerCD T2NYESO PentamerCD T2NYESO PentamerCD T2NYESO PentamerCD T2NYESO PentamerCD TIMEPOINT: Day42 TIMEPOINT: Day1 Bar Chart Legend TIMEPOINT: MP TIMEPOINT: Day7 TIMEPOINT: Day21 TIMEPOINT: Day42 TIMEPOINT: Day1 TIMEPOINT: Day36 2. TIMEPOINT: Day1 TIMEPOINT: Day SUBJECT_ID STIMULATION Subset IFNg IL2 TNFa T2NYESO PentamerCD T2NYESO PentamerCD T2NYESO PentamerCD T2NYESO PentamerCD T2NYESO PentamerCD T2NYESO PentamerCD T2NYESO PentamerCD T2NYESO PentamerCD Persistence and functional diversification of responses are apparent only in patient 21 Presence of IL2 secretion capacity is also present only for patient 21

31 Clinical Immune Monitoring of Tumor Infiltrating Lymphocytes (TIL) ELISpot Multiparametric Flow Cytometry Network of clinical and academic collaborators Drafting protocol and consent forms for submission to the Research Ethics Board Clinical sample procurement Clinical data and pathology report available Sample shipment in temperaturecontrolled containers for immune monitoring and/or proteomics analysis Sample preparation Mechanical disruption of tumor sample / enzymatic digestion into a single cell suspension Immune monitoring and/or proteomic analysis Phenotypic and functional single cell profiling

32 Tumor Microenvironment and Frequency Regulatory T cells Healthy donor PBMC control CD45 CD4 FoxP3 CD CD25 TIL from NSCLC tumor samples CD45 CD4 FoxP3 CD CD25 Inverted CD4/CD ratio in situ versus PBMC Increased frequency of intratumoral T REG as compared to PBMC samples

33 Phenotypic Characterization of Lymphocytes from Tumor and Adjacent Tissues (n=1) % of CD4 or CD T cells expressing marker of interest # # # # # # # Increased expression of several checkpoint markers on TIL as compared to lymphocytes from healthy adjacent tissue. CD GITR 4. CD CD PD1 4. CD TIM3 4 % of CD T cells expressing marker of interest Coexpression of PD1 and TIM3 on TIL as compared to lymphocyte from healthy adjacent tissue. CD GITR 137 PD1 TIM3 Bar Chart Legend SAMPLE_TYPE: Adjacent Tissue SAMPLE_TYPE: Tumor

34 Assessment of the Functionality of Tumor Infiltrating Lymphocytes Healthy Donor PBMC control NSCLC Tumor Sample CD4 CD CD3 IFNγ Significant decrease in the frequency of IFNγproducing CD and CD4 TILs compared to PBMC following ex vivo anticd3/anticd2 stimulation

35 Conclusion Flow cytometry is a unique technology that gathers phenotypic and functional data on single cells from heterogeneous mixtures such as blood and tissues. Relative distribution of phenotypic and functional subsets Predictive and/or correlative value with markers of clinical disease progression Several factors must be considered when developing a Flow Cytometry panel Sample Matrix, Integrity and Stability Clone/Fluorophore Combinations Anticipated Antigen Expression Levels Signal Specificity (Isotype and FMO Controls) Defining optimal duration of stimulation and staining procedures Trending Control

36 For additional information, please visit our website: or contact us: Poster presentation by Laetitia Cortes Title: Metaproteomic analysis of the infant fecal microbiome Booth 535