Innovating Preclinical Drug Development & Human Cell Therapy. Stephen Minger Chief Scientist, Cellular Sciences Life Sciences, GE Healthcare

Transcription

1 Innovating Preclinical Drug Development & Human Cell Therapy Stephen Minger Chief Scientist, Cellular Sciences Life Sciences, GE Healthcare

2 GE Healthcare 53,000 employees $17 billion revenue $1 billion R&D Medical diagnostic X-ray, CT, MRI, ultrasound and PET imaging Imaging contrast agents and molecular diagnostics Integrated healthcare IT systems Drug discovery and biopharmaceutical manufacturing technologies Reduce drug discovery cycles bring new, more effective products to market

3 Life Sciences - Enabling Development & Production of Future Medicines RESEARCH INSTRUMENTS & CONSUMABLE S REGENERATIVE MEDICINE & CELL TECHNOLOGIES BIOPROCESSING CAPACITY & CAPABILITY MOLECULAR & IN-VITRO DIAGNOSTICS CLINICAL IMAGING DISCOVERY PRE-CLINICAL & CLINICAL DEVELOPMENT APPROVAL & COMPANION DIAGNOSTICS THERAPY SELECTION & MONITORING

4 Stem Cells Types, Flavours & Sources Pluripotent Multipotent Multipotent Multipotent

5 Drug Cardiotoxicity Drug Class Withdrawn Terfenadine Antihistamine 1998 Sertindole Antipsychotic 1998 Astemizole Antihistamine 1999 Grepafloxacin Antibiotic 1999 Cisapride Prokinetic 2000 Droperidol Tranquilizer 2001 Levomethadyl Opiate Dependence 2003 Rofecoxib NSAID 2004 Tegaserod Prokinetic 2007 Sibutramine Appetite Suppressant 2010 Rosiglitazone Antidiabetic 2010

6 Toxicology: Current issues Toxicology has evolved through addition and replacement without integration Using animal models to reflect human responses Testing toxicity using maximum chemical dosage Testing multiple endpoints leading to false-positives Hartung 2009 Nature 460 (7252):208-12

7 Cardiomyocyte production H7 hesc Cardiomyocytes Expansion 5 x 10 9 hes cells Differentiation Media 1 Media 2 Growth Factors Feed

8 Cardiomyocytes in Drug Safety Stem cell expansion & differentiation Electrophysiology High Content Analysis & Image Cytometry

9 Cardiomyocytes

10 Cytiva Plus cardiomyocytes Troponin I 10 GE Title or job number Hoechst 1/23/2015

11 Structural Proteins Transcription Factors Cardiomyocyte phenotyping Mef 2C (muscle-specific gene reg n) Nkx 2.5 (myocardial commitment/diff n) Gata 4 (& Troponin I) (myocardial commitment/diff n) Connexin 43 (gap junction) Troponin I (myofilament component) a-actinin (myofilament component)

12 Cardiomyocyte Electrophysiology Ion channel currents and action potentials Planar patch clamping Voltage clamped whole cell currents Manual perforated patch clamping Current clamped action potentials Nodal Na + 0.8% K + Ventricular APD ±16.8ms 81.6% Ca 2+ Atrial APD ±6.9ms 17.6%

13 HERG Blockers AP prolongation and torsadogenicity

14 Electrophysiology Comparative Pharmacology of APD 90 Prolongation Compound Rabbit PF Canine PF hesc-vm HEK-hERG IC 50 (µm) Terfenadine Positive at 1.0µM False Negative Positive at 0.03µM Quinidine Positive at 1.0µM Positive at 1.0µM Positive at 0.3µM Cisapride Positive at 0.1µM Positive at 0.1µM Positive at 0.01µM Sotalol Positive at 10µM Positive at 100µM Positive at 10µM 268 Verapamil Mixed effect at 10µM Mixed effect at 1.0µM Mixed effect at 1.0 µm Chromanol 293B False Negative False Negative Positive at 300µM 10.7 E-4031 N/A Positive at 0.1µM Positive at 0.1µM Nifedipine N/A Positive at >10µM Positive at 0.03µM N/A The Action Potential and Comparative Pharmacology of Stem Cell-Derived Human Cardiomyocytes Peng, S. et.al., Journal of Pharmacological and Toxicological Methods 61 (2010)

15 IN Cell Analyzer GE Healthcare HCA systems IN Cell Analyzer 2200 Flexible widefield HCA system with on-board image restoration (deconvolution) IN Cell Analyzer 6000 Laser-based Confocal system with scmos detection and variable aperture technology

16 High Content Analysis information Nuclei Nuc/Cell Intensity Nuc Area Nuc cg X Nuc cg Y Nuc Elongation Nuc 1/(Form Factor) Nuc Displacement Nuc Intensity Nuc Intensity CV Cell Intensity Compactness Light Flux (relative) Chord ratio Intensity (N+C) Integrated Intensity (Nuc) Integrated Intensity (Cell) Integrated Intensity (Whole Cell) Nuclei coordination Spacing (SOI) Neighbor count (SOI) Spacing (MIN) Neighbor count (MIN) Spacing (Gabriel) Neighbor count (Gabriel) Spacing (Lune) Neighbor count (Lune) Example data from multitarget analysis module Organelles Count Spacing Neighbor Count Mean Area 1/(Form Factor) Elongation Distance to Nuc Inclusion/Cell Intensity Intensity Total Area Inclusion/Bckg Intensity Cells Nuc/Cell Intensity Cell/Bckg Intensity Cell Area Nuc/Cell Area Nuc Intensity Nuc Intensity CV Cell Gyration Radius Cell Intensity Cell Elongation Cell 1/(Form Factor) Intensity Spreading Cell cg X Cell cg Y Cell Intensity CV Background Intensity Light Flux (relative) Intensity (N+C) Integrated Intensity (Nuc) Integrated Intensity (Cell) Integrated Intensity (Whole Cell)

17 DNA Mitochondria Ca 2+ hesc-cardiomyocyte Toxicity Assay - Control

18 DNA Mitochondria α-actinin Ca 2+ hesc-cardiomyocyte Toxicity Assay - Amiodarone

19 Cardiotoxicity & Anticancer Drugs Drug Pipelines Toxicity in Drug Development Hepatotoxicity Nephrotoxicity Cardiotoxicity Rhabdomyolysis Other Data from: Drug pipeline: Q411. Mak H.C Nature Biotechnol. 30,15 Data from; Wilke RA et al. Nature Reviews Drug Discovery ,

20 Cardiotoxicity HCA Workflow Cardiomyocytes Compounds* Fluorescent probes Imaging GEHC Cardiomyocytes, live-cell format assay in 384-well plates 7 doses; n=3 replicate wells; 24h, 48h, 72h pre-incubations 4 Fluors: Hoechst, TMRM, TOTO-3, Fluo-4 IN Cell Analyzer 2000, large-chip CCD camera, 40x/0.6NA objective Image Analysis IN Cell Investigator MTA & Developer : 19 Parameters Data Management IN Cell Miner 2 o Analysis & Visualization IN Cell Investigator Spotfire DecisionSite TM *Compounds blinded until data analysis completed

21 Amiodarone (+ve control) 33.0μM Mitochondrial count, morphology & viability Mitochondrial Integrity Calcium Homeostasis Viability

22 Tyrosine kinase inhibitors Axitinib Tascotinib Vandetanib Sunitinib Imatinib Erlotinib 1.24μM 3.70μM 11.0μM 33.0μM

23 Entinostat (HDAC) 33.0μM mitochondrial count and Δ[Ca 2+ ] PD (MEK1) 33.0μM Δ[Ca 2+ ] only Lapatinib (TK EGFR/HER2) 33.0μM Mitochondrial count, morphology & viability Imatinib (TK PDGFR/KIT) 33.0μM Δ[Ca 2+ ], mitochondrial count, morphology & viability

24 Compound 33μM Cluster Compound 33μM Cluster Nifedipine 1 Entinostat 4 Tasocitinib 1 LY Axinitib 2 NVP-BEZ235 4 Mubritinib 2 PIK 90 4 Pazopanib (Votrient) 2 SB PD Dasatinib (Sprycell) 5 Tyrphostin 2 PD U Amiodarone* 6 VX Imatinib (Gleevec)* 6 GECT-Y 3 Lapatinib (Tyverb)* 6 PD Rapamycin 6 Pimecrolimus 3 Sorafenib (Nexavar) 6 Vatalanib 3 Sunitinib (Sutent)* 6 Temsirolimus 6 Vandetanib (Zactima) 6 *Reported Clinical Cardiotoxicity

25 Multi-parameter Profiling HCA Mitochondrial Morphology, Calcium & ATP 0.05μM 100μM

26 Disposable & flexible technologies: the future is more cost-effective, faster, safer & greener

27 Cell Therapy Generic Workflow Cell Isolation Cell Expansion Cell Analysis Administration Monitoring Enrichment of cells from tissue source (cord blood, bone marrow, adipose) Cell specific separation of cells (positive & negative selection) Ex vivo expansion of cell populations Harvesting and concentration of cells Characterisation of cells to determine viability, potency and batch variation enabling QC and process optimisation Administration and guided delivery technologies for accurate, and efficient cell delivery Biomarkers to identify and track implanted cells; cell viability; and differentiation Imaging for post-implantation assessment enabling direct comparisons for efficacy evaluation Integrated portfolio enables interaction within workflow. Setting standards and providing bench to bedside scalability accelerating development of cell therapies

28 WAVE Bioreactor 2/10 system with automated perfusion system Cellbag bioreactor - Functionally closed, disposable, and presterilized - Feed and harvest lines plus sampling port - Gas exchange via filters to control atmosphere - Two bag sizes: Cellbag 2L: 0.2 to 1 L Cellbag 10L: 0.5 to 5 L Inlet/outlet air filters Feed/harvest lines and sample port NOTE: In the US the WAVE Bioreactor system is sold for research use and for use under IND or IDE cleared by the FDA

29 Workflow for culture in Cellbag 2L From 1 10⁸ to T cells in 11 days Analysis Coulter) Cell size Cell size Cell size Cell size CS FACS FACS Cytokines Day Activate cells w. CD3/CD28 beads Static culture in gas-permeable bag Bioreactor culture at ⁶ cells/ml until 1 L is reached Perfusion rate: 350 ml/day Perfusion rate: 750 ml/day End of culture Daily measurements of: Cell density and cell viability Glucose, lactate, L-glutamine, and ph Perfusion rate: 1000 ml/day Harvest cells Re-activate w CD3/CD28 QC analysis

30 Process parameters Cell concentration gradually increases during expansion in the Cellbag bioreactor whereas ph and levels of metabolites and nutrients are kept constant by increasing the perfusion rate.

31 Cell-based immunotherapy with GE technology

32 Targets for Stem Cell Therapy plus multiple applications to progress conventional drug therapy

33 Cell Therapy - One GEHC Solutions Workflow Application GE Offering Patient selection Pre-planning for intracoronary or intra-myocardial administration Tissue characterisation Diagnostic Imaging Cell sourcing Cell quality Stem cell bespoke separation and expansion Bedside closed loop automated adipose stem cell source In vitro imaging for cell viability, characterisation; potency Wave Bioreactor Ficoll IN Cell Analyzer 2000 IN Cell 6000 Cell delivery Improved cell delivery with intracardiac cell administration and guided delivery Cobra Catheter Echo ICE & TEE Innova 3D/ fluro fusion Cell Assessment Functional Assessment Cell tracking for engraftment, and cell differentiation Cardiac functional assessment using MR, Ultrasound, SPECT and PET Biomarkers and Diagnostic Imaging Diagnostic Imaging

34 Strategic Blue Skies Technology Assessment Develop Next Generation Big Bets/Big Wins Integrated technology & biology Implantable Electronic- Biologic Interfaces Microbiomic Diagnostics Neural prosthetics In vivo Dx - regenerative medicine 3D bio-printing Proprietary and Confidential for GE use only

35 Drivers for organ and tissue beyond 1:1 donation Donor organ demand outstrips supply Areas considered number of procedures: Essential to life organ transplant data Tissue grafts Not included at the moment: Health economic analysis Data from optn.transplant.hrsa.gov and OPTN/SRTR Annual Report. ** Data include deceased and living donors. Graph from Title or Job Number XX Month 201X See tutorial regarding confidentiality disclosures. 35

36 GE/3DBP collaboration model Field of use: regenerative medicine Initial focus: tissue for implantation (cardiac, meniscus (spine, knee), bone repair). Long term: other vascularized tissue and organs. Why use 3DBP? Access to complex, personalized (shape and cells) multi-material constructs. Not accessible by other methods. GE portfolio: multiple touch-points Where are the technology gaps? High resolution CAD modelling. GMP manufacturing steps (cell bio-processing, bioinks, maturation, bio-reactors and QC release). High resolution matrix 3DP and cell positioning. Pre-clinical: ex-vivo imaging Image acquisition CAD model Fabrication step Maturation QC release In vivo transplant Follow up Injection mould or 3DP Clinical: in vivo imaging See tutorial regarding confidentiality disclosures. Delete if not needed.

37 How to design a tissue Title or Job 3

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