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)

5


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

6

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


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


ZellScannerChipcytometrytechnology

How does it work and what it can offer over

conventional flow cytometry


ZellScanner Chipcytometry technology

9

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



10

How does it work and what can it offer over conventional flow cytometry


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


11

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


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.

1

•Apply lymph node cells to chip

•scan background

2

•PE-CD20 to identify B cells

•Scan, followed by photobleaching

3

•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


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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• 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.



21

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

Intra Assay Precision

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Inter-Assay Precision

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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


– 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


– 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


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.

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