A fresh approach to Immuno-oncology: Ex vivo analysis of drug efficacy in fresh patient tumortissue
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1 A fresh approach to Immuno-oncology: Ex vivo analysis of drug efficacy in fresh patient tumortissue Soner Altiok, MD, PhD Chief Scientific Officer Nilogen Oncosystems Tampa, Florida
2 - Nilogen Oncosystems, a CLIA-certified laboratory, is dedicated to the improvement of cancer drug development for pharmaceutical and biotechnology companies. - Nilogen s proprietary drug testing platforms utilize fresh patient tumor tissue with intact tumor immune microenvironment to accurately assess the efficacy of immunotherapeutics.
3 Cancer immunotherapy delivers treatment of high specificity, low toxicity and prolonged activity in subsets of patients. Courtesy of Scott Antonia, MD, PhD, Chief Medical Officer
4 Elements of Inhibition of Immune Responses in the Tumor Microenvironment p38mapk T reg PI3K AKT mtor IDO CD8 CD86 GITRL CD4 HVEM Chemokines CAF ICOS-L OX4L 4.1BBL PD-L2 PD-L1 MHC CD28 CTLA4 GITR CD4L BTLA PD1 PD1 TCR ICOS 4.1BB OX4L PD1 Cytotoxic Tcell LAG3 PD1 PD1 TIM3 A2aR MHC-II PD-L1 PD-L2 Galec>n9 Adenosine PGE2 TGFβ Arg1 IDO STAT 3 MDSC JA K PD-L1 Cytokines p38mapk Nilogen s drug testing platforms allow analysis of different components of intact tumor microenvironment
5 1 5 3D-EX: Ex -vivo drug testing and biomarker discovery using fresh tumor microspheroids TUMOR MICROENVIRONMENT ANALYSIS IFNγ (pg/ml) FRESH TUMOR TISSUE TUMOR MICROSPHEROIDS (EX VIVO TREATMENT) DRUG RESPONSE PROFILING & BIOMARKERDISCOVERY Nilogen s 3D-Ex proprietary ex vivo platform to rapidly andaccurately analyze tumor response to oncology and immuno-oncology drugs in fresh patient tumor tissue with intact tumor immune microenvironment
6 Comparison of original patient tumor and microspheroids used in the ex vivo assays CAF Tumor cell 1µm Original Patient tumor: Adenocarcinoma of the lung TIL 3D microspheroid prepared from patient s fresh tumor tissue for the ex vivoassay 1µm Nilogen s ex vivo assays utilize 3D tumor microspheroids prepared from fresh patent tumors with intact tumor immune microenvironment
7 H&E Original patient tumor: Squamous cell carcinoma of the lung 1µm H&E CD8 1µm 3D microspheroids prepared from patient s fresh tumor tissue for the ex vivo assay PD1 PD-L1
8 3D-EX: Ex vivo drug testing and biomarker discovery using fresh tumor microspheroids TUMOR MICROENVIRONMENT ANALYSIS FRESHTUMOR TISSUE - TIL activation and composition - Multiplex cytokine/chemokine analysis - Enzymatic activity (IDO) and checkpoints - Gene expression/epigenetic analysis TUMOR TUMOR MICROSPHEROIDS MIC IDS (EX VIVO TREATMENT) - Macrophage polarization - Tumor cellviability - Pharmacodynamicstudies
9 Assessment of drug response to ex vivo treatment in fresh tumor samples Ki6 7 Ki6 7 Control (44.7%) CD3 Nivolumab (79.9%) CD3 K y n /T r p (m M /M ) r a tio MULTIPLEX CYTOKINE ANALYSIS Analysis of IDO ac/vity T u m o r 1 T u m o r 2 T u m o r 3 T u m o r 4 T u m o r 5 IFNG CTLA4 DPP4 LCP1 STAT4 CXCR3 TBX21 LAG3 MAF CD8A CD3E CD2 PTPRC TNFSF18 CD8 CD7 IRF4 CD86 LCK CD5 LILRB1 MS4A1 TIGIT CD8B CXCR4 CD3G IL13 CCR1 IL12RB1 IL12RB2 IL12A IL18RAP CD79B CD7 CCR5 ITGA1 SOCS1 FLOWCYTOMETRY MASS SPECTRA ANALYSIS NANOSTRINGANALYSIS
10 Diagram Example of Flow Analysis for Ex Vivo Tumor Response Assessment CD3-/CD68+CD11b+ Macrophages CD3-/CD19+ BCells CD3+/CD8/ CD4 Morph SingleCells SingleCells Live/Dead CD45+/- CD45+/CD3+/- CD3-/CD56+ NKCells CD3-/ CD68+/- CD4+Granzyme B+ CD4+ICOS+ CD4+Ki-67+ CD3-/CD68-CD11c +CD11b+DCs CD4+OX4+ CD4+PD-1+
11 Analysis of drug effect on tumor immune microenvironment (frequently used flow panels to assess immune response exvivo) Immune cell types panel: CD45, CD3, CD4, CD8, CD11b, CD68, CD11c, CD56, CD19 T cell activation & Treg panel: CD3, CD4, CD8, CD25, CD69, FOXP3, OX4, ICOS, PD1, CTLA4 T cell & APCpanel: CD3, CD4, CD8, CD25, Ki67, CD14, CD11c, CD19, CD86, HLA-DR T cell & immune checkpoint panel: CD3, CD4, CD8, CD25, CD127, Lag 3, PD-1, BTLA, CTLA-4, TIM3, A2aR Nilogen can customize the flow cytometry panels for optimal evaluation of tumor immune microenvironment and assessment of drug response
12 Tumor 1: 78 WF Control Nivolumab Tumor 2: 59 WM Control Nivolumab Comparable CD3 cell population in both tumors CD Nivolumab binds to PD1 on CD3 cells in both tumors PD Tumor 1, but not Tumor 2 responses to Nivolumab exvivo CD17 a Nilogen s 3D-EX ex vivo platform allows evaluation of tumor immune microenvironment, cytotoxic efficacy of immuno-oncology drugs and tumor-drug interaction
13 Ex vivo Analysis of Combinatorial Treatment in Immunotherapy IFNγ (pg/ml) C Nivo D1 D2 D3 Nivo Nivo Nivo +D1 +D2 +D3 MIP1b (pg/ml) C Nivo D1 D2 D3 Nivo Nivo Nivo +D1 +D2 +D3 Invasive urothelial carcinoma (bladder)
14 Ex vivo Analysis of Combinatorial Treatment with Chemo-and Immunotherapy 1 T u m o r 2 ( P D - L 1 n e g a t i v e ) P <. 1 T u m o r 4 ( P D - L 1 n e g a t i v e ) T u m o r 6 ( P D - L 1 n e g a t i v e ) P <. 1 8 P =. 3 4 p g / m l 6 4 p g / m l 2 1 p g / m l Mean IFNγ expression levels of ex vivo drug treated 3Dmicrospheroids ASCO 215, Poster #15892
15 Cell-Match: In vitro drug testing and biomarker discovery with autologous patient-derived cell lines TUMOR MICROENVIRONMENT ANALYSIS TUMORCELLS TUMOR INFILTRATING LYMPHOCYTES (TIL) FRESHTUMOR TISSUE CANCERASSOCIATED FIBROBLASTS(CAF) - TIL activationand composition - CAFactivity andviability - Enzymatic activity (IDO) and checkpoints - Pharmacodynamic analysis of drug effect - Tumor cell viability
16 Evaluation of drug effect to overcome CAF-mediated inhibition of TIL activity by Cell-Match Tm1 Tm IL2 (pg/ml) IL2 (pg/ml) Tm3 Tm IL2 (pg/ml) IL2 (pg/ml)
17 Immuno-PDX: In vivo drug testing with autologous patient-derived xenograft models and immune cells FRESHTUMOR TISSUE TUMOR MICROENVIRONMENT ANALYSIS PERIPHERAL BLOOD Optimization of T-CellTherapies: - Engineered T-CellTherapies - Adoptive T-CellTherapy PBMC PBMC TUMOR INFILTRATING LYMPHOCYTES (TIL)
18 THANK YOU
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