Welcome. Nanostring Immuno-Oncology Summit. September 21st, FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
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1 Welcome Nanostring Immuno-Oncology Summit September 21st, FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
2 Agenda 4:00-4:30 PM Guest registration and reception 4:30-5:00 PM Niro Ramachandran, Sr. Director, Product Management, NanoString Capturing the Complexity of Immuno-Oncology Research with Digital Barcodes 5:00-5:30 PM Terri McClanahan, Executive Director, Profiling & Expression, Merck Research Labs Translational Oncology & Biomarker Development for the Era of Combination Cancer Immunotherapy 5:30-6:00 PM Alessandra Cesano, Chief Medical Officer, NanoString Bringing the Next Generation of Immuno-Oncology Biomarkers to the Clinic 6:00-6:30 PM Justin Balko, Assistant Professor, Vanderbilt University Quantitative Protein Profiling in FFPE to Characterize Toxicities Associated with Immune Checkpoint Inhibitors 6:30-7:00 PM Networking and close 2 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
3 Capturing the complexity of immuno-oncology research with optical barcodes Niro Ramachandran PhD September 21st, FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
4 Integrating multiple analytical methodologies and cancer models 4 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
5 A Growing Suite of Applications Protein SNV Gene Expression Single Cell Expression Copy Number Variation Gene Fusions Precisely profile up to 800 targets from a sample in a single tube 5 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
6 3D Biology: Chemistry New Suite of 3D Biology Optical Barcode Reagents 6 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
7 Simple, integrated workflow 3D Biology Single sample Low input requirements Simultaneous, single-platform digital counting of all analyte classes 7 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
8 3D Biology assays built upon foundational PanCancer 770-plex Hallmarks of Cancer gene expression series How the tumor starts How the body responds How the tumor progresses PanCancer Pathways PanCancer Immune Profiling PanCancer Progression RNA (770-plex) Originally published in Cell, 144, Hanahan D & Weinberg RA, Hallmarks of Cancer: The Next Generation, , Copyright Elsevier FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
9 PanCancer IO 360 Gene Expression Panel 770 human genes total Including 20 reference genes Tumor Microenvironment Immune Response 16 Pathways and Processes Enriched with known and potentially clinically relevant genes Includes overlapping reference genes from PanCancer panel collection for cross-panel analysis Compatible with Plus Products and Protein for 3D 15+ Signatures measuring Immune and Tumor Activity 14 Cell type abundance scores Merck 18-gene Tumor Inflammation Signature (TIS) 9 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
10 Vantage 3D RNA:Protein Immune Cell Profiling and Signaling RNA + Protein = Targeted I/O Research Targeted I/O Profiling for Cell Suspensions þ 770 RNA + 30 cell surface or 26 intracellular proteins þ As few as 20,000 cells þ Custom protein spike Chen & Mellman (2013) Immunity 10 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
11 3D Flow : Deeply profile rare T Cells Advancing T Cell biology requires more biological information from rare sub-populations Current methods for deep genomic and proteomic analysis of defined T Cell populations are complex, inefficient and costly 3D Flow expands on existing flow cytometry methods Co-stain with oligo-tagged NanoString and fluorescent flow sorting antibodies Streamlined workflow for high plex RNA and protein analysis Measure up to 30 proteins and 770 RNA 11 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
12 Elucidate activated/inhibited states of your T Cells Biological question (What) Insight (Why) Markers (How) T Cell Profiling Elucidate T Cell Biology Analyze extracellular markers across T Cell subtypes High-plex protein data to probe differences between cell populations Quantify T Cell Activation Profile the reactivity of specific T Cell populations Analyze levels of activation with T Cell specific activation markers Measure T Cell Inhibition Measure inhibitory proteins and T Cell exhaustion markers Observed balance between markers of activation and inhibition Assess Cellular Purity No sort is perfect- quantify the presence of possible contaminating immune cells Validate with Internal Controls Check data quality- Ensure immune content is present and consistent across samples 4-1BB, BTLA, CD27, CD28, CD3, CD40, CD40L, CD45RO, CD8, CD152, GITR, ICOS, IL2RA, KIR3DL1, NT5E, OX40, CD279, CD274, CD BB, CD27, CD28, CD40, CD40L, CD45RO, CD8, ICOS, IL2RA, KIR3DL1, OX40 CTLA-4, PD-1, PD-L1, PD-L2, NT5E, BTLA, GITR CD127,CD14, CD163, CD33, CD3, CD68, HLA-DRA, CD158E1, NCAM, NKP46 CD4, CD9, CD45 12 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
13 Characterization of sorted T cell populations Quantify up to 30 extracellular proteins? CD28 CD27 ICOS What is the underlying biology of rare T cells? Exhaustion? activation? What is the state of T cell populations both before and after immunotherapy? Can engagement of key regulatory pathways explain the underlying biology differentiating T cell populations? PD-1 CTLA-4 BTLA 13 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
14 Vantage 3D Portfolio. Powered by 3D Biology Technology. 14 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
15 Are we there yet? Protein SNV Gene Expression Single Cell Expression Copy Number Variation Gene Fusions What is missing? 15 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
16 but wait, there is more! Protein SNV Gene Expression Single Cell Expression Copy Number Variation Gene Fusions Spatial information 16 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
17 Design of a digital spatial profiling microscope What the Data Looks Like How it Works H i Expression Label FFPE Slide with Antibody Mix UV Light Lo CD9 CD4 PD-L1 IL2R CD28 Illuminate Region of Interest Analyze Optical Barcodes on ncounter 17 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
18 Digital Spatial Profiling: Overall workflow 18 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
19 Digital Spatial Profiling: Instrument setup 19 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
20 Single-molecule counting of photocleaved oligos Lymphoid Tissue Region 5 Region 8 Region 9 Region µM 400µM ncounter Counts Single-molecule digital counts Ki67 CD Ki-67 CD Region 5 Region 8 There are 135,211 Ki67 antibodies located here 20 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Region 9 Region 10
21 Single-molecule counting of photocleaved oligos Region 5 Region 8 Region 9 Region µM 400µM Single-molecule digital counts Digital counts for all of Lymphoid Tissue the invisible barcoded antibodies Ki CD3 ncounter Counts Ki-67 CD Region 5 Region 8 There are 135,211 Ki67 antibodies located here 21 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Region 9 Region 10
22 Infinite control and precision Geometric Phenotypic Single Cell CD3 PanCK DNA PanCK DNA GZMB Ki67 DNA Profile tumor and immune cells within a circular region Profile tumor cells only (yellow) in a colon cancer sample Profile GranzymeB+ cells (Red) only, in a defined region of tissue 22 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
23 Automated selection of phenotypic regions of interest PanCK + DNA Overlay 23 Mask generated automatically 100%-Tumor Profiling FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
24 Deep Profiling. Deep Questions. Deep Insights. Biological Questions (What) Insights (Why) Targets (How) Characterize GLOBAL IMMUNE RESPONSE Determine T-CELL ACTIVATION LEVEL Immune Microenvironment Quantify NK Cells, T-Regs, Cytotoxic T- Cells, B-Cells, Macrophages, and Monocytes Assess Antigen Presentation Characterize T-cell proliferation & differentiation Balance between T-Cell activation & inhibition Assess cytotoxic response or supportive potential Quantify TUMOR REACTIVITY Spatial awareness of hot/cold immune signature Localization of cellular proliferation Contextualized tumor heterogeneity CD8A, CD68, CD14, CD4, FOXP3, CD56, CD19, CD45, MS4A1(CD20), CD3, CD45RO CD45RO, CD8A, CD4, CD3, CD45, FOXP3, CD56, PD1, PD- L1, STAT3, STAT3-P, B7-H3 (CD276), PTEN, GZMB, CD14, VISTA, CD44, Beta-Catenin, Pan-Cytokeratin, B7-H3 (CD276), PTEN, GZMB, Ki67, Beta-2-microglobulin, AKT, P-AKT, VISTA, PD1, PD-L1, CD44, STAT3-P, Bcl-2 24 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
25 Immune characterization of excisional biopsies Colorectal cancer Four colorectal cancer samples was analyzed (one shown) fluorescent staining for PanCK, CD45, and DNA 23-plex cocktail of oligo-labeled antibodies 200-μm diameter ROIs in PanCK+, CD45+, and stroma Distinct protein expression profiles from different regions are observed with clustering by sample of origin 25 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
26 Multiplexed mrna and protein: Digital Spatial Profiling in breast cancer 26 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
27 Technology Access Program: Started November 2016 Multiplex many analytes on one tissue slice in a single pass Quantitation based on linear singlemolecule counting: up to 6 logs Single-cell limit of detection Non-destructive: sample is only touched by light Single antigen retrieval without effects from order-ofaddition Join the Technology Access Program. TAP@nanostring.com 27 FOR RESEARCH USE ONLY. Not for use in diagnostic procedures.
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