Imaging cytometry: the advantages of hybrid technology in support - PowerPoint PPT Presentation

Imaging cytometry: the advantages of hybrid technology in support of drug discovery Richard Hughes EBF Open Meeting 2017

Imaging cytometry: the advantages of hybrid technology in support of drug discovery Overview • Technological advances in cytometry – pros and cons • ZellScanner chipcytometry technology - how does it work and what it can offer over conventional flow cytometry • Examples of chipcytometry to support drug discovery • Validation of chipcytometry for use in clinical application Nov 2017/ EBF 10 th Open Symposium

Advances in flow cytometry – ImageStream/FlowCyte (Amnis, MerckMillipore) http://www.merckmillipore.com/GB/en/life- science-research/cell-analysis/amnis-imaging- flow-cytometers/ – CyTOF Mass Cytometer (Fluidigm) https://www.fluidigm.com/products/cytof 5 Nov 2017/ EBF 10 th Open Symposium

Advances in flow cytometry – ImageStream/FlowCyte – CyTOF Mass Cytometer – Chipcytometry, Zellkraftwerk GmbH ZellScanner ONE™ Cytobot ™ Name System Benchtop instrument Automated system (Tecan) Operation Manual Fully automated, 24/7 Application Exploratory / Phase I trials Phase II/III trials 6 Nov 2017/ EBF 10 th Open Symposium

Advances in flow cytometry – Pros and Cons Manufacturer MerckMillipore Fluidigm/DVS Sony Zellkraftwerk Zellkraftwerk Instrument ImageStream-X MKII CyTOF 2 SP6800 Spectral Analyzer ZellScannerONE Cytobot Technology imaging flow cytometry mass cytometry spectral cytometry Chipcytometry ChipCytometry Technology Features Multiplexing: Max 10 colors 100 25 unlimited unlimited theoretical limit Multiplexing: 12 40 19 100 100 actual limit subcellular localization ++ - - + + sample storage 1-5 days 1-5 days 1-5 days at least 20 months at least 20 months cell-loss / drop out rate 10% 1.00% ? <0.5% <0.5% tissue cytometry - Coming soon - + Coming soon Instrument Features cells/second 5,000 1,000 10,000 2,000 6,000 de-novo setup of a 15-marker not possible 3 month 4 month <1 day <1 day panel Total cost of ownership (USD) ≈400,000 ≈590,000 ≈400,000 Basic instrument 280,000 980,000 ≈2,000 ≈8,000 ≈2,000 Energy supply 2,000 4,000 Argon gas supply not required 60,000 not required not required not required ≈40,000 ≈50,000 ≈35,000 ≈18,000 ≈50,000 Maintenance Contract Pros & Cons statistical microscopy with many many publications by inventor discrimination of fluorescent best instrument for low cell precious samples: option for 20 biggest pros morphological parameters available proteins / fluorochromes numbers and precious samples months storage bench-top instrument has limited to max. 10 colors / when proprietary labels required / medium-low sample throughput // biggest cons more than 6 colors are required cumbersome panel development price dedicated user necessary fully automated system is cumbersome panel development expensive Nov 2017/ EBF 10 th Open Symposium

ZellScanner Chipcytometry technology How does it work and what it can offer over conventional flow cytometry Nov 2017/ EBF 10 th Open Symposium

ZellScanner Chipcytometry technology How does it work and what can if offer over conventional flow cytometry ZellSafe™ Cells ZellSafe™ Rare ZellSafe™ Tissue Product Specimen cell suspension rare cells (<0.02%) sections 6 biopsies or 2x1cm Loading capacity 40-100 µL 40-300 µL section Total cell number typically 250,000 typically 1,000,000 tissue-dependent 9 Nov 2017/ EBF 10 th Open Symposium

ZellScanner Chipcytometry technology How does it work and what can it offer over conventional flow cytometry PBMC Image from ‘Validation of Treg , Th17 and Plasma Cell Assays’ 10 J. Detmers, A. Mirenska, C. Hennig, S. Poelmans, M. Van Roy, T. Van Bogaert Nov 2017/ EBF 10 th Open Symposium AAPS, NBS Poster 2017 conference poster M1025

ZellScanner Chipcytometry technology How does it work and what can it offer over conventional flow cytometry Conventional ChipCytometry Flow Cytometry ≈ 24 months Sample Stability 1-3 days Markers/Sample 8-16 > 100 X Re-interrogate Sample? 11 Nov 2017/ EBF 10 th Open Symposium

ZellScanner Chipcytometry versatility What can it offer over conventional flow cytometry Stain the same cells repeatedly Identify infiltrating cell types in carcinoma tissue courtesy of Definiens AG, Munich Compatible Specimens Cell Suspensions: • PBMC / whole blood • bone marrow • CSF • bronchoalveolar lavage (BAL) • cell lines • sorted cells • digested tissues (lung, gut, tonsil, spleen, liver) • Nasal scrapes Maroz et al.Leukemia, 2014 Tissue / Sections • lung • gut • bone • cancer biopsy • skin • bone sections Insert your date / confidentiality text here 12 Nov 2017/ EBF 10 th Open Symposium Muller, M et al, Fraunhofer ITEM

Examples of chipcytometry to support drug discovery Low sample volume Rare events from small cell numbers Sample storage and re-interrogation

Analysis of B cells from Cynomolgus macaque Lymph Node Aspirates 1 Aim: To demonstrate target engagement in • Apply lymph node cells to chip CD20+ B cells from lymph nodes • scan background – Lymph node aspirates were collected by CRO 2 following dosing of the animals with 1 mg/kg, 10 • PE-CD20 to identify B cells mg/kg of drug or Vehicle • Scan, followed by photobleaching – Time points: 24, 48, 96, 168, 672 (4 weeks), 1032 (6 weeks + 1 day) and 1680 hours (10 weeks). 3 – The cells from the aspirates were placed immediately • Add PE-drug into a tube and 25 µl of sterile PBS was added. • Addition of PE-conjugated drug acts in competition to bound dosed drug – Each tube was then shipped to GSK Stevenage on • Scan, followed by photobleaching ice for processing. where samples were centrifuged, the supernatant collected for PK analysis, and the cells re-suspended in 50 µL of Zellkraftwerk wash 4. • PE-non competitive anti-target buffer. • Will bind to target receptor regardless of whether drug is bound, thus gives an indication of whether receptor is internalised or has been shed 14 Nov 2017/ EBF 10 th Open Symposium Joanne Thompson, Exploratory Biomarkers, BIB, IVIVT, GSK

Analysis of B cells from Cynomolgus macaque Lymph Node Aspirates 48 hrs 24 hrs 168 hrs Add storage buffer to the chip - store for up to 2 years at 0-10  C Convert the files to fcs and treat as normal flow data within FlowJo Using GIMP, can visualise overlays of the different stains on the cells Vehicle PE Conjugated Drug 24 hours post dose Vehicle Target 1 mg/kg 10 mg/kg Target 10 mg/kg CD20 15 Nov 2017/ EBF 10 th Open Symposium Joanne Thompson, Exploratory Biomarkers, BIB, IVIVT, GSK

Validation of chipcytometry to support clinical studies Nov 2017/ EBF 10 th Open Symposium

Validation of chipcytometry to support clinical studies – GSK2831781 is a monoclonal antibody being developed by GlaxoSmithKline for the treatment of Psoriasis. The antibody targets the T cell activation marker LAG-3, which is mainly expressed in inflamed tissues. GSK2831781 entered this Phase 1 clinical trial, initially in healthy subjects, in 2014 with the first Psoriasis subjects being dosed in 2016. – Samples from Clinical studies are currently analysed by flow cytometry using two 8-colour panels. Even with a limited number of Clinical Sites this has still proved to be both technically and logistically challenging and would be impossible moving forward into Phase II studies. – To determine if Chipcytometry would offer an alternative solution to measure PD biomarkers for LAG-3, a subset of samples collected from patients enrolled in the Psoriasis cohorts of the study will be collected and stored for analysis using the validated 12-colour Chipcytometry assay. Full Sample Pilot study regulatory analysis validation 17 Nov 2017/ EBF 10 th Open Symposium

LAG-3 Chipcytometry Pilot Study Study objective: To assess feasibility of measuring LAG-3 on PBMCs isolated from whole blood – Deliverables – Compare 2 LAG-3 antibodies (Clone 3DS223H - eBioScience and Clone REA351 - Miltenyi Biotec) – Evaluate LAG-3 expression on unstimulated and IL- 12/18 PBMCs (n=3 donors) – Antibody Panel: CD3, CD4, CD8, CD45RA, LAG-3 – Enumerate percentage of LAG-3+ cells – Direct comparison with flow was not performed 18 Nov 2017/ EBF 10 th Open Symposium

LAG-3 Chipcytometry Pilot Study Study objective: To assess feasibility of measuring LAG-3 on PBMCs isolated from whole blood – Enumerate percentage of LAG-3+ cells (sensitivity) 19 Nov 2017/ EBF 10 th Open Symposium

Validation of chipcytometry to support clinical studies Validation is closely performed in line with validation criteria published by O’Hara et al., 2011. • For all validation experiments, PBMCs from 5 different healthy donors stimulated with IL-12/IL-18 • After stimulation, PBMCs from all tubes per donor will be pooled and loaded onto ZellSafeTM chips • One biological replicate is defined as PBMCs isolated from one healthy donor and pooled after stimulation. • Multiple chips of the same production lot containing the same biological replicate are used for independent repeated measurements (hereafter referred to as technical replicates). • Each chip undergoes a quality inspection and only chips that pass the quality control are used to conduct the experiments. 20 Nov 2017/ EBF 10 th Open Symposium