time-resolved interaction analysis on living cells
TRANSCRIPT
3/4/2017
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Time-resolved interaction
analysis on living cells: Comprehensive data generates new
knowledge and better decisions
Karl Andersson
CEO
Karl Andersson, PhD
• Ridgeview Instruments AB
– Molecular interactions on living cells
– Founder, inventor, CEO
• Uppsala University
– Associate professor & senior lecturer
– Dpt of Immunology, Genetics and Pathology
• In the past
– Biacore R&D (1997-2007)
– PhD bioinformatics
– MSc scientific computing
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The first contact between drug and cell
The first contact between drug and cell
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2
0
100
4 6 8 h
% occupied target
0
• 3 simulated binding traces
– Same affinity 1 nM
– Different interaction dynamics
– First 2 h incubation @ 10 nM
– Then 7 h wash-out
Technology background
• Fast on - fast off
requires continuous
supply
• Slow on - slow off
will occupy fraction of
targets for a long time2
0
100
4 6 8 h
% occupied target
0
Technology background
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• Frequent administration of
low concentration.
• Rare administration of high
concentration.
Affinity alone cannot provide this information
2
0
100
4 6 8 h
% occupied target
0
Technology background
• Curve shape may be
indicative of mode-of-action
• Relative signal may reflect
receptor abundance
Affinity alone cannot provide this information
2 4 6 8 h
% occupied target
Technology background
0
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0
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LigandTracer cell based assay
• Cells in regular Petri dish
• Rotating, sloping support
• Detection at drained
higher end
Petri dish with cells (T) and background (B) position
Sig
nal
Time
Detector
Medium + Ligand LigandTracer Output:Background corrected real-time binding curve
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Our solution: LigandTracer
• Measure interactions
– Often: Protein – cell
– Truly: Any – Any
• Label based detection
– Fluorescense or
radioactivity
The first contact between drug and cell
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The first contact between drug and cell
• What can you learn about your molecules?
• Examples from:
– EGF – EGFR
– Rituximab - CD20
Examples from the EGFR family
• Overexpressed in variety of cancers
• Used to establish our toolbox of methods
• Common trait: Comparison of similar states
– Culturing conditions / cell lines / labels / molecules
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EGF(125I) – EGFR multiple conditions and cell lines
Björkelund H, et al, PLoS One. 2011;6(9):e24739.
A431 cells
Conditions
Red: Regular
Blue: Starving
Green: Gefitinib
Black: Gefitinib +
starvation
3 nM 9 nM 0 nM
3 nM 9 nM 0 nM
3 nM 9 nM 0 nM
3 nM 9 nM 0 nM
Comparing cell lines…
EGFR HER2
A431 +++ +
SKOV3 ++ +++
Conditions
Red: Regular conditions
Blue: Starvation
Green: Gefitinib (Iressa)
Black: Gefitinib + starvationBjörkelund H, et al, PLoS One. 2011;6(9):e24739.
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Unlocking results with Interaction Map
EGFR finding: Complex dimer biology
• Dimerization depends on drug and on cell line
• Altered dimerization pattern affects affinity
Functional evaluation of dimerization mechanisms
Björkelund H et al, Mol Clin Onc. 2013. 1: 343-352
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• Add fluorescently
labeled binder ( )
Evolve into proximity assay…
• Add quencher
labeled binder ( )
• Signal drop =
proximity confirmed
Slide modified due to confidentiality
Slide deleted due to confidentiality
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Extending toolbox beyond EGFR
• Nanoparticles
• Suspension cancer cells
• Tissue samples
• Non-human cell models
– Virus
– Bacteria
Suspension cancer cells
• Challenge: Attachment
• Update a historic
tethering protocol
• Manuscript submitted
(Bondza et al)
BAM
Target cells
in PBS
Cultivate in
cell-culture
medium
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Suspension cancer cells
• Attachment: Verified
• Enables detailed
binding characteristics
within immunology
Jurkat
0 h
6 h
K562Daudi
Suspension cancer cells
• Verification (A): Tethered adherent A431 cells behave as
regularly adhered A431 cells
• Live test (B): Jurkat (T-cells) – anti CD3 antibody works
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Rituximab – CD20 : still biology to unveil
• Robust & Repeatable
• Verified on Daudi and
Ramos cells
• Affinity
– 0.9 ± 0.05 nM (Daudi)
– 0.7 ± 0.01 nM (Ramos)
• Not 1:1 on Daudi
• Our next challenge
All in all…
• We work with time-resolved molecular
interaction analysis on living cells
– Brings new light on biology
– Precise & repeatable
– Ideal for comparisons
– Advanced assays and data evaluation
– Simple technology with low running costs
– Available in all aspects; hardware, software
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But one challenge remains…
• Exploring Biophysical principles in Cell biology:
– Different way of thinking
– Different constraints
– New types of results
But one challenge remains…
• Exploring Biophysical principles in Cell biology:
– Different way of thinking
– Different constraints
– New types of results
• Create learning package!
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1. HOW
2. WHY
3. WHAT
4. TEST
All in all…
• We work with time-resolved molecular
interaction analysis on living cells
– Brings new light on biology
– Precise & repeatable
– Ideal for comparisons
– Advanced assays and data evaluation
– Simple technology with low running costs
– Available in all aspects; hardware, software
– Built-in training
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AcknowledgingFantastic colleagues and
collaborators, former and
current, far too many to list
Karl Andersson, PhD
CEO
Thank you
Karl Andersson, PhD Nils-Fredrik Kaiser, PhD
Sales Manager
[email protected]@ridgeview.eu
CEO