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2016 INCUCYTE™ LIVE-CELL ANALYSIS WEBCASTS Smart cell assays for immuno-oncology Revealing the interplay between cancer and immune cells Tim O’Callaghan Product Manager Webinar contents ✔ Cancer immunotherapy - the promise, the need & challenges ✔ Common immune cell assays ✔ Continuous live-cell imaging & analysis ✔ New assays & reagents for immuno-oncology ✔ Case studies ✔ Summary & perspective 2 Webinar contents ✔ Cancer immunotherapy - the promise, the need & challenges ✔ Common immune cell assays ✔ Continuous live-cell imaging & analysis ✔ New assays & reagents for immuno-oncology ✔ Case studies ✔ Summary & perspective 3 WEBINAR: Smart Cell Assays for Immuno-Oncology Cancer immunotherapy - the promise and need The promise Harnessing the immune system to fight cancer Immune specificity and memory Potential for safer, more effective treatments Recent preclinical and clinical success The need I-O drugs have shown success in the clinic However many patients need next gen treatments Non-responders Relapse 4 WEBINAR: Smart Cell Assays for Immuno-Oncology Immuno-oncology drug development Challenges and complexities Tumor heterogeneity and evolution Variable and complex response kinetics Off target activity Combination therapies Opportunity for in vitro cell based assays Ready access to relevant immune cell types Valuable in vitro & ex vivo studies 5 Webinar Contents ✔ Cancer immunotherapy - the promise, the need & challenges ✔ Common immune cell assays ✔ Continuous live-cell imaging & analysis ✔ New assays & reagents for immuno-oncology ✔ Case studies ✔ Summary & perspective 6 WEBINAR: Smart Cell Assays for Immuno-Oncology Commonly used cell assays for immuno-oncology Boyden chamber SEAP Reporter Assay ELISpot 51Chromium Flow Cytometry Release LDH MTT Typically: • End-point • Non image based • Indirect read outs • Cell processing 7 WEBINAR: Smart Cell Assays for Immuno-Oncology Continuous live-cell imaging and analysis Image-based continuous (or semi-continuous) cellular measurements ✔ Real-time analysis Capture full response kinetics ✔ Image-based measurements Visualize cell interactions, verify metrics ✔ Living non-perturbed cells Environmental control is key ✔ Non-exhaustive Cells are not depleted ✔ Long term as required Hours, days, weeks ✔ Applicable across workflows Culture, manipulation, assay 8 WEBINAR: Smart Cell Assays for Immuno-Oncology Continuous live-cell analysis Comparison verses end-point workflow Culture Manipulate Assay Endpoint Single time point Continuous e.g. Cell Health, Morphology, Growth e.g. Transfection, Plating, Pre-treatments Kinetic assay 9 WEBINAR: Smart Cell Assays for Immuno-Oncology Key features for live-cell analysis systems Capacity, throughput Automated real-time analysis • • • • Run experiments in parallel Multi-vessel, multi-user Measure full kinetic response No sample prep, no end point Environmental control Image based measurements • • • • Critical for longer term studies e.g. within incubator Fits into existing workflows • • Culture, manipulations & assays Cells are not exhausted Characterise cell interactions Validate measurements Flexibility • • Optics, readouts, cells, vessels Multiplex assay readouts 10 WEBINAR: Smart Cell Assays for Immuno-Oncology Reagents for live-cell analysis Non-perturbing to cells Viable cell analysis Research flexibility Protocols for live-cell assays Dead cell analysis Cell clearance Cell counting Cytolysis Apoptosis Phagocytosis Genetically encoded fluorescent proteins Membrane impermeable dyes Enzyme activated/PS probes pH sensitive membrane dye NucLight™ BacMam 3.0 Cytotox Reagents Caspase-3/7 Reagent pHrodo® Cell Labeling Kit NucLight™ Lenti Annexin V Reagents ✔ Multiplex reagents for additional insight 11 Webinar contents ✔ Cancer immunotherapy - the promise, the need & challenges ✔ Common immune cell assays ✔ Continuous live-cell imaging & analysis ✔ New assays & reagents for immuno-oncology ✔ Case studies ✔ Summary & perspective 12 WEBINAR: Smart Cell Assays for Immuno-Oncology Continuous live-cell analysis for I-O research ✔ Tumor cells Growth & metastasis Immune cells Activation & homing Co-cultures Killing & Clearance Proliferation Clustering & Proliferation Immune-cell killing Cell migration & invasion Chemotaxis TEM Phagocytosis Continuously monitor & analyze key stages of immune response to cancer 13 WEBINAR: Smart Cell Assays for Immuno-Oncology Tumor cell growth and proliferation Treatment 2 100 Control 80 (%) (%) confluence Cell Phase object confluence Assay principle Treatment 1 60 Control 40 Treatment 2 20 0 24 48 72 Time (h) Images and data generated with the IncuCyte ZOOM® live cell analysis system ✔ Label-free measures of growth/growth inhibition ✔ Rapidly profile multiple donor cells, isogenics cell lines etc 14 WEBINAR: Smart Cell Assays for Immuno-Oncology Tumor cell growth and proliferation NucLight™ Red labeled tumor cells Assay principle SK-BR-3 Co-culture + Lapatinib SK-BR-3 Monoculture + Lapatinib Images and data generated with the IncuCyte ZOOM® live cell analysis system ✔ Real-time viable cell counting in mono-/co-cultures ✔ Investigate the effect of micro-environment on tumor cell viability 15 WEBINAR: Smart Cell Assays for Immuno-Oncology Tumor cell migration and invasion Assay principle SEED CELLS ImageLock™ 96-well Plates For precision repeat imaging CREATE WOUNDS ECM OVERLAY WoundMaker™ 96-pin wound making tool LIVE CELL ANALYSIS ✔ Create scratch wounds in seconds in 96-well plates ✔ No labels required, amenable to wide range of cells 16 WEBINAR: Smart Cell Assays for Immuno-Oncology Tumor cell migration and invasion Assay principle Migration SEED CELLS CREATE WOUNDS Invasion (Matrigel) ECM OVERLAY LIVE CELL ANALYSIS Images and data generated with the IncuCyte ZOOM® live cell analysis system ✔ Compare migration and invasion profiles in one 96-well plate ✔ Investigate how treatments alter metastatic potential 17 WEBINAR: Smart Cell Assays for Immuno-Oncology Continuous live-cell analysis for I-O research ✔ Tumor cells Growth & metastasis Immune cells Activation & homing Co-cultures Killing & Clearance Proliferation Clustering & Proliferation Immune-cell killing Cell migration & invasion Chemotaxis TEM Phagocytosis Continuously monitor & analyze key stages of immune response to cancer 18 WEBINAR: Smart Cell Assays for Immuno-Oncology Immune cell activation: clustering and proliferation Activated: Anti-CD3/IL2 Assay principle Non-Activated Images and data generated with the IncuCyte ZOOM® live cell analysis system ✔ Monitor proliferation and clustering in real-time ✔ Label free, kinetic measurements 19 WEBINAR: Smart Cell Assays for Immuno-Oncology Chemotaxis and transendothelial migration Assay principle COAT MEMBRANE ADD CELLS & TREATMENT ADD CHEMOATTRACTANT ClearView™ 96-well chemotaxis plate ✔ Visualize and quantify migration toward chemoattractants ✔ No labels required, low cell usage 20 WEBINAR: Smart Cell Assays for Immuno-Oncology Chemotaxis and transedothelial migration Assay principle T Cell TEM to SDF-1α Neutrophil Chemotaxis to IL-8 COAT MEMBRANE ADD CELLS & TREATMENT 100 C o n tr o l 1 .5 n M 4 .6 n M 1 3 .8 n M 4 1 .5 n M 1 2 4 .4 n M 0 .4 3 7 3 .3 3 n M % TEM (6h) [IL-8] 0 .8 % M ig ra tio n (6 h r) ADD CHEMOATTRACTANT ce e C o n flu e nt0h p S u r fa cNormalized d T oSurface) a lis e(Top o r mArea NCell Images and data generated with the IncuCyte ZOOM® live cell analysis system 1 .2 75 50 25 EC50 17.5 nM 1120 nM 0 .0 0 2 4 T im e ( h ) 6 0 -1 0 -9 -8 -7 -6 lo g [S D F -1 ] (M ) ✔ Automatically monitor entire time course of chemotaxis ✔ Investigate migration across ECM or through an endothelial monolayer 21 WEBINAR: Smart Cell Assays for Immuno-Oncology Continuous live-cell analysis for I-O research ✔ Tumor cells Growth & metastasis Immune cells Activation & homing Co-cultures Killing & Clearance Proliferation Clustering & Proliferation Immune-cell killing Cell migration & invasion Chemotaxis TEM Phagocytosis Continuously monitor & analyze key stages of immune response to cancer 22 WEBINAR: Smart Cell Assays for Immuno-Oncology Immune cell killing - T cell killing, ADCC Assay principle Caspase-3/7 Reagent NucLight™ Red Reagent Activated: Anti-CD3/IL-2 175 2500 Activated: Anti-CD3/IL-2 ADD TREATMENTS Non-Activated 2 e rmm2 )m ) leei rpm ll coobujenct t(rceodu nnut c(p V ia b le s pmaosre c-3e/7 C atu 150 SEED TARGET CELLS 2000 125 1500 100 75 Non-Activated 1000 50 500 25 ADD IMMUNE CELLS 00 00 3 30 0 60 0 6 T im im e e (h (h )) T 99 00 11 22 00 Images and data generated with the IncuCyte ZOOM® live cell analysis system (SK-OV-3 cells) ✔ Direct measurements of tumor cell death and viability ✔ Highly flexible format – use your choice of effector/target cells 23 WEBINAR: Smart Cell Assays for Immuno-Oncology Cell clearance - efferocytosis IncuCyte™ pHrodo® Red Cell Labeling Kit Assay principle Apoptotic Target Cells 1 .5 Apoptotic Target Cells T o ta l R e d O b je c t A r e a x 1 0 4 (µ m ²/Im a g e ) 2 .0 1 .0 Non-Apoptotic Target Cells 0 .5 Non-Apoptotic Target Cells 0 .0 0 6 12 18 24 T im e (h ) Images and data generated with the IncuCyte ZOOM® live cell analysis system (Jurkats) ✔ Quantify & validate phagocytosis using time-lapse imaging ✔ Simple labeling of your choice of target cells 24 WEBINAR: Smart Cell Assays for Immuno-Oncology Cell clearance - antibody mediated cell phagocytosis IncuCyte™ pHrodo® Red Cell Labeling Kit Assay principle CD47 blockade - uncloaking tumor cells Images and data generated with the IncuCyte ZOOM® live cell analysis system Bone marrow derived macrophages CCRF-CEM T lymphoblasts ✔ Visualize and measure antibody mediated phagocytosis in real time ✔ Anti-CD47 and antibody dependent cellular phagocytosis (ADCP) 25 Webinar contents ✔ Cancer immunotherapy - the promise, the need & challenges ✔ Common immune cell assays ✔ Continuous live-cell imaging & analysis ✔ New assays & reagents for immuno-oncology ✔ Case studies ✔ Summary & perspective 26 WEBINAR: Smart Cell Assays for Immuno-Oncology Case study 1: Probing the mechanisms of T cell activation Automated analysis of T cell clustering kinetics Visual analysis of cluster formation Single cell clustering Zumwalde, N. et al. J. Immunol. 191, 3681–93 (2013). Cluster aggregation Zumwalde, N. A. PhD Dissertation, University of Minnesota (2013) Images and data generated with the IncuCyte ZOOM® live cell analysis system • • ICAM-1 KO and anti–LFA-1 integrin blockade inhibit the formation of T cell activation clusters Long-term, automated, image-based analysis enable direct phenotypic measurements challenging by other technologies 27 WEBINAR: Smart Cell Assays for Immuno-Oncology Case study 2: Tumor cell killing by redirected T cells Bispecific T cell engagers induce dose dependent & selective killing Donor T cell variability influences killing kinetics McCormack, E. et al. Cancer Immunol. Immunother. 62, 773–85 (2013). Images and data generated with the IncuCyte ZOOM® live cell analysis system • • Adams, K. PhD Dissertation, Cardiff University (2013) Images and data generated with the IncuCyte ZOOM® live cell analysis system Selective analysis of target cell apoptosis to determine ImmTAC potency and specificity Directed killing can be missed in short-term assays e.g. 51Cr- and extended LDH-release assays 28 WEBINAR: Smart Cell Assays for Immuno-Oncology Case study 3: Tumor cell killing by CAR T cells Data provided by an immunotherapy biopharma company Live-cell analysis correlates with flow cytometry Data provided by an academic IncuCyte® customer Kinetic monitoring of T cell exhaustion No modulation of CAR T cell ‘serial killing’ CTRL TD1 TD2 TD3 TD4 No T cells No ligand Ligand modulation of CAR T cell ‘serial killing’ CTRL TD1 TD2 TD3 TD4 No T cells With ligand Images and data generated with the IncuCyte ZOOM® live cell analysis system 29 WEBINAR: Smart Cell Assays for Immuno-Oncology Value of continuous live-cell imaging and analysis Real time observation and measurement Full time-course – capture response kinetics Visualise cell interactions -make direct measurements Multiplex readouts & cell morphology build confidence Images & movies verify biology BIOLOGICAL INSIGHT Workflow enhancements Automated real time image gathering & analysis 96 & 384-well assays (> 1000 data points per hour) Cells still viable for follow on studies e.g. Flow, ELISAs Multi-vessel, multi-user PRODUCTIVITY 30 Wrap up and perspective ✔ I-O drug development requires an understanding of immune and tumor cell response kinetics ✔ Common cell based assays lack temporal resolution of cellular events & visualization of cell activity ✔ Continuous live-cell analysis enables real-time and direct cell based measurements providing valuable biological insight and enhanced productivity for I-O drug researchers around the world ✔ Novel non-perturbing, live-cell reagents enable real-time multiplexed measures of immune cell response to cancer 31 Further information and next steps ✔ Follow up e-mail: Webcast Recording Copy of Webinar Slides (pdf) ✔ Learn more: essenbioscience.com/communications/immuno-oncology/ ✔ Live cell imaging & analysis publications essenbioscience.com/publications 32 Questions and comments? ✔Contact us at: [email protected] 33