fluorescence antibody and protein imaging movie
DESCRIPTION
Preclinical in vivo imaging knowledge sharing video forumTRANSCRIPT
2 2 © 2009 PerkinElmer
Antibody, Protein & Peptide in vivo Fluorescence & Cerenkov Imaging
2
Alexandra De Lille, DVM, PhD
Director of Technical Applications
View this presentation as a movie: http://www.youtube.com/watch?v=jLFh8VwWncU&feature=g-upl
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Optical imaging is valuable for preclinical pharmaceutical development
Low Cost
High throughput
High sensitivity
Translational Research
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Examples of Peptide, Protein and Antibody Imaging
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Antibody Tumor Targeting
a b c
3 x 107 cells 250 pmol Co-Registration
Td-Tomato P3CM cells (a) were targeted with an Therapeutic Antibody-750 probe (b). An overlay of Td-tomato expression and fluorescent target can be visualized (c).
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0.00E+00
1.00E+09
2.00E+09
3.00E+09
3hr 5hr 24hr 48hr
Tum
or
Flu
ore
scen
ce
(A
vg R
adia
nt
Eff
icie
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)
Time
Tumor
Background
Quantification
Antibody Tumor Targeting
Her2sense (fluorescently-labeled humanized antibody (trastuzumab) targets
Her2+ Skov-3 tumors
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Herceptin-CF750 targets MDA-MB-231-Luc metastases
Antibody Tumor Targeting
BLI FLI
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Rediject Integrin-750 Probe is a fluorescently labeled cyclic RGD peptide that binds αvβ3
Integrin, enabling noninvasive fluorescence imaging of U-87 MG Luc tumors in nu/nu
Tumor
XenoLight Rediject integrin 750
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NIRF and micro-PET imaging of knottin probes cross validate in murine human tumor xenograft models
Kimura et al., 2010 – Bioconjugate Chem.
Cy5.5- or 64Cu-DOTA-labeled knottin
peptides could be used to image integrin
expression in mouse tumor models using
near-infrared fluorescence (NIRF) imaging or
positron emission tomography (PET). Plots
of tumor- to-background tissue ratios for
Cy5.5 versus 64Cu uptake were well-
correlated over several time points post
injection, demonstrating pharmacokinetic
cross validation of imaging labels.
Mice bearing U87MG tumors were injected
via tail vein with either (A) 1.5 nmol of
DOTA/Cy5.5-2.5D or (D) ∼100 μCi of 64Cu-
DOTA/Cy5.5-2.5D. For blocking experiments
(B,E), mice were coinjected with an excess
(0.5 μmol) of unlabeled c(RGDyK) in addition
to labeled knottin peptides.
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In Vivo Optical Imaging Methodology:
What does it take?
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Easy Fluorescent Dye Labeling
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Label any peptide, protein or antibody with easy to use Kit.
NIR fluorochromes optimized and validated for in vivo imaging.
Amine-reactive NIR fluorochromes for labeling via an NHS ester linkage.
Thiol-reactive NIR fluorochromes for coupling via maleimide chemistry to
label free cysteines or thiol groups.
Non reactive control dyes of same wavelength.
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Treatment Groups and Views
Zou et al., 2009 – Molecular Pharmaceutics
5 Treatment Groups
Dorsal and Ventral Views
Dorsal
Ventral
Tumor
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Longitudinal Time Points 15 min – 96 hrs,
Zou et al., 2009 – Molecular Pharmaceutics
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Longitudinal Tumor and Liver ROI Quantification
Zou et al., 2009 – Molecular Pharmaceutics
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Ex Vivo Validation
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Image tissues ex vivo and quantify fluorescence with in vivo optical imaging
system
ex vivo Tissue Fluorescence Validation
Zou et al., 2009 – Molecular Pharmaceutics
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A challenge faced in mab preclinical development:
“Is the binding of an antibody specific to the relevant target in the tumor tissue?”
In vivo methodologies still need ex vivo validation
See where the fluorophore is inside tumor with the Nuance Spectral Unmixing
Microscope Camera in ex vivo tissue sections.
and co-localize with up to 5 molecular markers simultaneously in a single tissue
section.
Nuance LCTF camera
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Determine serum stability of IgG isotypes by electrophoresis on a small,
microfluidic chip with the LabChip GX/GXII
LabChip GXII – Evaluate Antibody Stability
Stability of IgG isotypes in serum
Electropherograms from 3 separate analysis
of a labeled mAb after 4 and 24 hours in
whole blood.
Correia et al., 2010
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Sensitivity
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Fluorescence Sensitivity: Transillumination Fluorescence Imaging offers picomole sensitivity at depth
CF680 dye
25
picomole
CF680 dye
250
picomole
CF750 dye
250
picomole
QD800
4
picomole
EPI NTF EPI NTF S/B: 0.81 S/B: 6.69 S/B: 1.18 S/B: 2.47
S/B: 1.11 S/B: 9.19 S/B: 1.11 S/B: 4.27
S/B: 1.31 S/B: 8.32 S/B: 1.05 S/B: 2.57
CF750 dye
25
picomole
QD800
0.4
picomole
Detect 0.4 picomole
Qdots in the lungs
Epi Illumination Fluorescence (EPI) versus Normalized Transmission Fluorescence (NTF) Deep In Lung Of Mouse
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Translational Imaging:
Optical Imaging Of Radioactive Probes By Cerenkov Light
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Image PET and SPECT Probes with the IVIS
e
e+
Cerenkov Light : low energy window of light emission resulting from radiation (400–1000 nm)
18F-FDG Na18F Na131I 90Y-RGD-BBN
PET SPECT PET PET
Liu et al., 2010 - Plos
2 '
ep n e
e e s
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89Zr-DFO-J591 for immuno-PET of PSMA expression was used to coregister and correlate
the CLI signal with the immuno-PET images.
Qualitative and quantitative interpretation of the CLI data was found to give a strong
correlation with immuno-PET and biodistribution studies.
Ruggiero et al., 2010 – J Nucl Med
Cerenkov luminescence imaging of medical isotopes
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Deep Tissue Whole Animal 3D FMT Imaging
Biodistribution
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Quantitative Whole Body Biodistribution in 3D of Fluorescent-Labeled Agents by Non-
Invasive Tomographic Imaging. Strong correlation of in vivo and ex vivo tissue fluorescence.
Whole Animal 3D - Biodistribution
Heart & CarotidsAngioSense680 (IV)T=5 min: 350 pmol
Lungs (asthma)ProSense680 (IV)T=24 h: 130 pmol
LiverVivoTag680XL-Albumin (IV)T=24 h: 540 pmol
KidneysReninSense680 (IV)
T=24 h: 80 pmol
BladderVivoTag680XL (IV)T=5 min: 560 pmol
StomachAngioSPARK680 (PO)T= 5 min: 1400 pmol
IntestinesAngioSPARK680 (PO)
T= 3 h: 1150 pmol
FM
T Im
ag
ing
%ID
/g
)
Mean Tissue Fluorescence (counts/energy)
FMT Correlation to Ex Vivo FRI
r² = 0.996
FM
T Im
ag
ing
(%
ID/g
)
Tissue Homogenate (%ID/g)
FMT Correlation to Tissue Homogenate
r² = 0.969
Liver
Kidney
Lung
Heart
Brain
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15
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30
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0 10 20 300
5
10
15
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0 0.05 0.1 0.15 0.2 0.25 0.3
Counts/Energy
0.03
0.10
0.17
0.23
0.30
Counts/Energy0.01
0.03
0.06
0.08
0.10Brain Lungs Heart Kidneys
Stomach Intestines Liver
Spleen
Ex Vivo Organ Reflectance Imaging Organ Fluorescence
Counts/Energy
0.01
0.03
0.06
0.08
0.10
0.00
0.05
0.10
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bra
in
he
art
lun
gs
live
r
kid
ne
ys
sto
ma
ch
inte
stin
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sple
en
Me
an
Org
an
Flu
ore
sce
nc
e (
co
un
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)
BSA-VT680XL
No Probe
Vasquez et al., 2011, PLOS One
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3D Cy5.5 biodistribution, co-registered to Quantum FX CT
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dual-modality NIRF/PET imaging agent are promising for further development in clinical
applications such as detection of tumors located deep within the body and image-guided
surgical resection.
Kimura et al., 2010 – Bioconjugate Chem.
64Cu-DOTA/Cy5.5 knottin peptide: Plots of tumor to-background tissue ratios for Cy5.5 versus 64Cu uptake were well-correlated over several time points post injection, demonstrating
pharmacokinetic cross validation of imaging labels.
Kimura et al., 2010 – Bioconjugate Chem.
To investigate the in vivo distribution of MAb 92-13, we applied two methods;
one based on the radionuclide modalities using 111In-labeled antibody, and the other based
on the fluorescence imaging using near-infrared-labeled antibodies. The results obtained by
two approaches were very concordant .
Fukukawa et al., Cancer Sci 2008
Testimonials
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We labeled two breast cancer binding antibodies, anti-ErbB2 and anti-EpCAM, with infrared
fluorescence dyes of different wavelengths and determined their in vivo distribution in a breast
cancer xenograft model using a near-infrared (NIR) fluorescence imaging system. Our data
show that these two antibodies can be readily assessed simultaneously in mouse xenograft
model. This will help guide design of dosing strategies for multiple antibodies and identify
potential interaction that could affect pharmacokinetics and possible side effects.
Sun et al., 2012 - Biotechniques
The high-throughput nature of the optical screening strategy enabled high-throughput
screening (up to 60 animals per hour is possible), whereas only a few animals could be
screened by PET/CT imaging in a comparable time frame. Furthermore, screening the animals
with optical imaging was accomplished at a fraction of the cost of PET/CT imaging, without
the added complications of lengthy anesthesia or for the introduction of ionizing radiation.
Deane et al., 2007 – Mol Can Res
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There is a statistically significant correlation between PET and Cerenkov Light Imaging.
CLI provides up to a 10-fold increase in throughput capability for radio-labeled compound
screening in vivo (CLI 5 animals 5 min, Pet 2 animals 20 min).
Robertson, Millenium ,WPC 2012
The ability to simultaneously measure time-dependent changes in tumor uptake of
radiotracers in multiple different tumor models or chemotherapeutic treatment regimes
means that optical CLI offers the potential to conduct rapid, low-cost, high-throughput
screening of novel radiotracers in vivo.
Ruggiero et al., 2010 – J Nucl Med
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