Imaging cytometry: the - ZELLKRAFTWERK · Imaging cytometry: the advantages of hybrid technology in...
Transcript of Imaging cytometry: the - ZELLKRAFTWERK · Imaging cytometry: the advantages of hybrid technology in...
Imaging cytometry: the
advantages of hybrid
technology in support
of drug discovery
Richard Hughes
EBF Open Meeting 2017
• 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
Imaging cytometry: the advantages of hybrid technology in support of drug discovery
Overview
Nov 2017/ EBF 10th Open Symposium
http://www.merckmillipore.com/GB/en/life-
science-research/cell-analysis/amnis-imaging-
flow-cytometers/
– ImageStream/FlowCyte (Amnis,
MerckMillipore)
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Nov 2017/ EBF 10th Open Symposium
Advances in flow cytometry
https://www.fluidigm.com/products/cytof
– CyTOF Mass Cytometer (Fluidigm)
– ImageStream/FlowCyte
– CyTOF Mass Cytometer
– Chipcytometry,
Zellkraftwerk GmbH
Advances in flow cytometry
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Name ZellScanner ONE™ Cytobot™
System Benchtop instrument Automated system (Tecan)
Operation Manual Fully automated, 24/7
Application Exploratory / Phase I trials Phase II/III trials
Nov 2017/ EBF 10th Open Symposium
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:
theoretical limitMax 10 colors 100 25 unlimited unlimited
Multiplexing:
actual limit12 40 19 100 100
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
panelnot possible 3 month 4 month <1 day <1 day
Total cost of ownership (USD)
Basic instrument ≈400,000 ≈590,000 ≈400,000 280,000 980,000
Energy supply ≈2,000 ≈8,000 ≈2,000 2,000 4,000
Argon gas supply not required 60,000 not required not required not required
Maintenance Contract ≈40,000 ≈50,000 ≈35,000 ≈18,000 ≈50,000
Pros & Cons
biggest prosstatistical microscopy with many
morphological parameters
many publications by inventor
available
discrimination of fluorescent
proteins / fluorochromes
best instrument for low cell
numbers and precious samples
precious samples: option for 20
months storage
biggest cons
limited to max. 10 colors / when
more than 6 colors are required
cumbersome panel development
proprietary labels required /
dedicated user necessarycumbersome panel development
bench-top instrument has
medium-low sample throughput //
fully automated system is
expensive
price
Advances in flow cytometry – Pros and Cons
Nov 2017/ EBF 10th Open Symposium
ZellScannerChipcytometrytechnology
How does it work and what it can offer over
conventional flow cytometry
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ZellScanner Chipcytometry technology
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How does it work and what can if offer over conventional flow cytometry
Product ZellSafe™ Cells ZellSafe™ Rare ZellSafe™ Tissue
Specimen cell suspension rare cells (<0.02%) sections
Loading capacity 40-100 µL 40-300 µL
6 biopsies or 2x1cm
section
Total cell number typically 250,000 typically 1,000,000 tissue-dependent
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ZellScanner Chipcytometry technology
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How does it work and what can it offer over conventional flow cytometry
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PBMC Image from ‘Validation of Treg, Th17 and Plasma Cell Assays’
J. Detmers, A. Mirenska, C. Hennig, S. Poelmans, M. Van Roy, T. Van Bogaert
AAPS, NBS Poster 2017 conference poster M1025
ZellScanner Chipcytometry technology
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How does it work and what can it offer over conventional flow cytometry
Conventional
Flow CytometryChipCytometry
Sample Stability 1-3 days ≈ 24 months
Markers/Sample 8-16 > 100
Re-interrogate Sample? X
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ZellScanner Chipcytometry versatility
Insert your date / confidentiality text here 12
What can it offer over conventional flow cytometry
Cell Suspensions:•PBMC / whole blood•bone marrow•CSF•bronchoalveolar lavage (BAL)•cell lines•sorted cells•digested tissues (lung, gut, tonsil, spleen, liver)•Nasal scrapes
Tissue / Sections•lung•gut•bone•cancer biopsy•skin•bone sections
Stain the same cells repeatedly Identify infiltrating cell types in carcinoma tissue
Maroz et al.Leukemia, 2014
Muller, M et al, Fraunhofer ITEM Nov 2017/ EBF 10th Open Symposium
Compatible Specimens
courtesy of Definiens AG, Munich
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
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Aim: To demonstrate target engagement in
CD20+ B cells from lymph nodes
– Lymph node aspirates were collected by CRO
following dosing of the animals with 1 mg/kg, 10
mg/kg of drug or Vehicle
– Time points: 24, 48, 96, 168, 672 (4 weeks), 1032 (6
weeks + 1 day) and 1680 hours (10 weeks).
– The cells from the aspirates were placed immediately
into a tube and 25 µl of sterile PBS was added.
– Each tube was then shipped to GSK Stevenage on
ice for processing. where samples were centrifuged,
the supernatant collected for PK analysis, and the
cells re-suspended in 50 µL of Zellkraftwerk wash
buffer.
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•Apply lymph node cells to chip
•scan background
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•PE-CD20 to identify B cells
•Scan, followed by photobleaching
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•Add PE-drug
•Addition of PE-conjugated drug acts in competition to bound dosed drug
•Scan, followed by photobleaching
4.
•PE-non competitive anti-target
•Will bind to target receptor regardless of whether drug is bound, thus gives an indication of whether receptor is internalised or has been shed
Nov 2017/ EBF 10th Open SymposiumJoanne Thompson, Exploratory Biomarkers, BIB, IVIVT, GSK
Analysis of B cells from Cynomolgus macaque
Lymph Node Aspirates
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Target
Target
24 hours post doseVehicle
10 mg/kg
Vehicle
1 mg/kg
10 mg/kg
24 hrs 48 hrs 168 hrs
Nov 2017/ EBF 10th Open SymposiumJoanne Thompson, Exploratory Biomarkers, BIB, IVIVT, GSK
PE
Co
nju
ga
ted
Dru
gCD20
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
Validation of chipcytometry to support clinical studies
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– 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.
Sample analysis
Full regulatory validation
Pilot study
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LAG-3 Chipcytometry Pilot Study
Study objective: To assess feasibility of measuring LAG-3 on PBMCs isolated from whole blood
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– 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
LAG-3 Chipcytometry Pilot Study
Study objective: To assess feasibility of measuring LAG-3 on PBMCs isolated from whole blood
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– Enumerate percentage of LAG-3+ cells (sensitivity)
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Validation of chipcytometry to support clinical studies
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• 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.
Validation is closely performed in line with validation criteria published by O’Hara et al., 2011.
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Validation of chipcytometry to support clinical studies
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Validation is closely performed in line with validation criteria published by O’Hara et al., 2011.
Parameter Details
Intra Assay Precision %CV of all reportable parameters when the assay is performed on 3 technical replicates, and
this will be repeated for 5 different donors. A %CV <25% for all 5 donors is acceptable
Inter Assay Precision Not performed
Stability Baseline values at time point 0 are identical with the values calculated for intra-assay
precision validation. The 4 ‘stability time point’ chips per donor were stored at 4°C in the dark.
After 2, 6 and 12 months, one sample per donor is assayed, respectively. The 4th is
biobanked. 80% of the donors the target cell count is within 25% from the baseline mean
Drug Interference Cells are incubated with drug at room temperature for 30 min (eg. Cmax, Ctrough). Interference
was analysed by comparing results of spiked samples with 3 unspiked replicates of the same
volunteer and calculating the % difference.
Cross-comparison
Chipcytometry -
FACS
Stability and cross-comparison
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Poster 59,
SUNDAY, OCTOBER 8, 2017
ICCS Meeting 2017, Arizona
% of CD3+
Chip cytometry demonstrated good
concordance with standard flow for both
functional and phenotypic markers
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– Low sample volume
– Rare events from small cell numbers
– Long term sample storage and re-interrogation
The advantages of Chip Cytometry in support of drug
discovery
Nov 2017/ EBF 10th Open Symposium
– Low sample volume
– Rare events from small cell numbers
– Long term sample storage and re-interrogation
The advantages of Chip Cytometry in support of drug
discovery
Nov 2017/ EBF 10th Open Symposium
Acknowledgements
Joanne Thompson
Fiona Germaschewski
Karen Leavens
Jan Detmers
www.zellkraftwerk.com
All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures)
Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Animals.