ChipCytometryClinical Trial Support in Immunology

Immunology: Clinical Trial Support

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Conventional Flow Cytometry ChipCytometry

Sample Stability 1-3 days > 24 months

Markers/Sample 8-16 > 70

Re-interrogate SampleX

NEW!

Cell Storage (fixed cells)

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Product ZellSafe™ Cells ZellSafe™ Rare ZellSafe™ TissueSpecimen cell suspension rare cells (<0.02%) sectionsLoading capacity 40-100µl 40-300µl 6 biopsies or 2x1cm sectionTotal cell number typically 250,000 typically 1,000,000 tissue-dependent

Sample inlet

Outlet

++++++- - - - - -

Validated Stability for >20 Months

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Compatible Specimens

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Cell Suspensions:• PBMC / whole blood• bone marrow• CSF• bronchoalveolar lavage (BAL)• cell lines• sorted cells• digested tissues (lung, gut, tonsil, spleen, liver)

Tissue / Sections• lung• gut• bone• cancer biopsy• skin• bone sections

Sample Analysis Workflow

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Load Chip

1

2 3

ZellScanner™ Instrument

StartInstrument

Stain

Image

Switch-offLong-term

Storage

Data Processing >> FCS

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Stain same cell again and again …

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Source:Maroz et al.Leukemia, 2014

Central Lab Services

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- Fully automated analyzer- 24/7 operation- 4,000 data points/week- “Spirit of GLP”

Global Footprint

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Sponsor

Central Lab BioBank ZellSafe™Box World Courier

Predictive Biomarkers

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Outcome:- Predictive biomarker

candidate identified- Prospective study

pending

- Disease: Neonate Sepsis

- Study: Clinical

- Patients: 370

- Sample: PBMC

Respiratory

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CD3 CD4 CD8 CD45R0 CD45RACD19 CD56 HLA-DR CD16 CD14

Alveolar macrophages

Neutrophils

T cells

Th cells

B cells

- Disease: Primary Immunodeficiency

- Study: Exploratory

- Sample: BAL

Immuno-Oncology

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Outcome:Infiltrating cell types identified and quantified

- Disease: Colon Carcinoma

- Study: Translational

- Sample: Cryosections

By courtesy of

Translational Immunology

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PX4: Germinal center B-cells: CD19(+), CD86+, CD10+, CD38+, CD11c+/-CD14(+)

PX4DC

MZ: Marginal zone B-cells: CD19++, CD86-, CD10+, CD38-, CD11c-,IgD+

MZ

PX4

Outcome:- CD11c+ cells identified

and localized By courtesy of

Validated Markers

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CCR3 * CD25 CD68 IgMCCR4 * CD27 CD69 * IL10CCR5 * CD272 (BTLA) CD71 IL12CCR6 * CD279 (PD-1) CD8 IL17ACCR7 * CD3 CD86 IL17FCD10 CD30 CTLA-4 IL1bCD115 CD31 CXCR3 * IL8CD11b CD38 CXCR4 * kappa light chainCD11c CD4 CXCR5 * lambda light chainCD14 CD40 FoxP3 pan CytokeratinCD141 CD45 GM-CSF PD-1 (CD279)CD16 CD45RA Helios ROR gtCD163 CD45RO HLA-DR T-betCD19 CD5 IFNy TNFaCD20 CD54 IgA Zap-70CD206 CD56 IgD  *= Markers to be analyzed on live 

cells only.CD24 CD64 IgG

Deep Immunophenotyping

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LEVEL 1 Phenotyping• T-cells (CD3+CD56-) • B-cells (CD19+) • NK-cells (CD56+CD3-) • NKT-cells (CD3+CD56+) • Monocytes (CD14+CD4dimCD3-) • Neutrophils (CD16+CD14-) • Basophils (CD123+HLA-DR-CD3-

CD4-CD8,CD14-,CD19-CD56-) • mDC (CD11c+HLA-DR+CD123-CD3-

CD4-CD8,CD14-CD19-CD56-) • pDC (CD11c+HLA-DR+CD123-CD3-

CD4-CD8,CD14-CD19-CD56-)

LEVEL 2 Advanced Phenotyping• T-cells: CD4/CD8/DN/DP (CD4, CD8) • T-cells: Central/Peripheral Effector/Memory

(CD27, CD45RO, CD45RA) • B-cells: Memory, Naive, Transitional (IgD,

CD27, CD38)

LEVEL 3 Functional Phenotyping• T-cells: activation Status (CD25,MHC2) • B-cells: activation Status (CD86, CD25, AID,

CD27, CD38) • Monocyte/Neutrophil activation Status

(MHC2 CD64) • Upon request: Cell-Cycle analysis, Cytokine

production, Homing markers

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Preparing Clinical Trials

Candidate Sites 1, 2, 3, 4,..

2. Site Qualification Kit

1. SOP, Training Video

Typical Milestone-based Project

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2. Load- Load sample, wait 5‘- Fix (4% PFA), wait 15‘- rinse, seal chip

1. Prep- 1 ml blood- CPT, Ficoll or RBC lysis- total time: 40’

3. Store- store on-site- temperature 4°C- up to several months

4. ShipGDP Logistics (Good Distribution Practice)

5. Bioanalysis“Spirit of GLP” clinical trial support (SOP, QA)

6. Re-interrogateTest additional markers over >24 months

Selected Primary Publications

• Happle, Science Translational, 2014

• Dijkstra, Nature Medicine 2012

• Hennig, Br. J. Hematology 2010

• Woelbeling, Immunobiology 2011

• Polte, JACI 2008

System Validation according to:

• O'Hara, JIM 2011

• Green, JIM 2011

• Cunliffe, Pharm. Res. 2009

• Lee, Pharm. Res. 2006

• Bioanalytical Method Validation, FDA, 2013

Thank you for your attention!

“Precision Immunology” References

A B C Control Diseased Groups Group

Focus on:- Translational Immunology- Mode of Action(MoA)- Drug Safety- Patient Stratification