This document discusses optical imaging techniques for preclinical pharmaceutical development, including fluorescence and Cerenkov imaging of antibodies, proteins, and peptides. It provides examples of imaging tumor targeting antibodies and peptides, as well as longitudinal imaging of tumor models. The document discusses the methodology for in vivo fluorescent imaging, including labeling, treatment groups, time points, tissue validation, and sensitivity. It also discusses using Cerenkov light to image radioactive probes and correlating this signal with PET imaging.

1. Antibody, Protein & Peptide
in vivo Fluorescence &
Cerenkov Imaging
Alexandra De Lille, DVM, PhD
Director of Technical Applications
alexandra.delille@perkinelmer.com
View this presentation as a movie:
http://www.youtube.com/watch?v=jLFh8VwWncU&feature=g-upl
2 © 2009 PerkinElmer
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2. Optical imaging is valuable for preclinical pharmaceutical development
Low Cost
High throughput
High sensitivity
Translational Research
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3. Examples of Peptide, Protein and Antibody Imaging
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4. Antibody Tumor Targeting
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).
a b c
3 x 107 cells 250 pmol Co-Registration
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5. Antibody Tumor Targeting
Her2sense (fluorescently-labeled humanized antibody (trastuzumab) targets
Her2+ Skov-3 tumors
Quantification
Tumor
3.00E+09
(Avg Radiant Efficiency)
Tumor Fluorescence
Background
2.00E+09
1.00E+09
0.00E+00
3hr 5hr 24hr 48hr
Time
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6. Antibody Tumor Targeting
Herceptin-CF750 targets MDA-MB-231-Luc metastases
BLI FLI
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7. XenoLight Rediject integrin 750
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
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8. NIRF and micro-PET imaging of knottin probes cross validate in murine human
tumor xenograft models
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.
19 Kimura et al., 2010 – Bioconjugate Chem.

9. In Vivo Optical Imaging Methodology:
What does it take?
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10. Easy Fluorescent Dye Labeling
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11. 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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12. Treatment Groups and Views
 5 Treatment Groups
 Dorsal and Ventral Views
Tumor
Dorsal
Ventral
24 Zou et al., 2009 – Molecular Pharmaceutics

13. Longitudinal Time Points 15 min – 96 hrs,
25 Zou et al., 2009 – Molecular Pharmaceutics

14. Longitudinal Tumor and Liver ROI Quantification
26 Zou et al., 2009 – Molecular Pharmaceutics

15. Ex Vivo Validation
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16. ex vivo Tissue Fluorescence Validation
Image tissues ex vivo and quantify fluorescence with in vivo optical imaging
system
29 Zou et al., 2009 – Molecular Pharmaceutics

17. In vivo methodologies still need ex vivo validation
A challenge faced in mab preclinical development:
“Is the binding of an antibody specific to the relevant target in the tumor tissue?”
Nuance LCTF camera
 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
32 section.

18. LabChip GXII – Evaluate Antibody Stability
Determine serum stability of IgG isotypes by electrophoresis on a small,
microfluidic chip with the LabChip GX/GXII
 Stability of IgG isotypes in serum
Electropherograms from 3 separate analysis
of a labeled mAb after 4 and 24 hours in
whole blood.
33 Correia et al., 2010

19. Sensitivity
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20. Fluorescence Sensitivity:
Transillumination Fluorescence Imaging offers picomole sensitivity at depth
EPI NTF EPI NTF
S/B: 0.81 S/B: 6.69 S/B: 1.18 S/B: 2.47
CF680 dye CF680 dye
250 25
picomole picomole
S/B: 1.11 S/B: 9.19 S/B: 1.11 S/B: 4.27
CF750 dye CF750 dye
250 25
picomole picomole
Detect 0.4 picomole
Qdots in the lungs
S/B: 1.31 S/B: 8.32 S/B: 1.05 S/B: 2.57
QD800 QD800
4 0.4
picomole picomole
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Epi Illumination Fluorescence (EPI) versus Normalized Transmission Fluorescence (NTF) Deep In Lung Of Mouse

21. Translational Imaging:
Optical Imaging Of Radioactive Probes By Cerenkov Light
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22. Image PET and SPECT Probes with the IVIS
Cerenkov Light : low energy window of light emission resulting from radiation (400–1000 nm)
  p  n  e  e e+
e  e  2 ' s
e
18F-FDG Na18F Na131I 90Y-RGD-BBN


PET PET SPECT PET
41 Liu et al., 2010 - Plos

23. Cerenkov luminescence imaging of medical isotopes
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.
42 Ruggiero et al., 2010 – J Nucl Med

24. Deep Tissue Whole Animal 3D FMT Imaging
Biodistribution
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25. Whole Animal 3D - Biodistribution
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.
FMT Correlation to Tissue Homogenate FMT Correlation to Ex Vivo FRI
35 35
Liver
Kidney
30 r² = 0.996 30 Lung r² = 0.969
FMT Imaging (%ID/g)
Heart
FMT Imaging %ID/g)
25 25 Brain
20 20
15 15
10 10
Heart & Carotids
AngioSense680 (IV) 5 5
T=5 min: 350 pmol
0 0
0 10 20 30 0 0.05 0.1 0.15 0.2 0.25 0.3
Lungs (asthma) Tissue Homogenate (%ID/g) Mean Tissue Fluorescence (counts/energy)
ProSense680 (IV)
T=24 h: 130 pmol
Liver Ex Vivo Organ Reflectance Imaging
VivoTag680XL-Albumin (IV) Brain Lungs Heart Kidneys Spleen 0.10
T=24 h: 540 pmol
Kidneys 0.08
ReninSense680 (IV) 0.06
T=24 h: 80 pmol Stomach 0.03
AngioSPARK680 (PO)
0.01
T= 5 min: 1400 pmol Counts/Energy
Stomach Intestines Liver
0.10 0.30
Intestines
AngioSPARK680 (PO) 0.08 0.23
T= 3 h: 1150 pmol Bladder
VivoTag680XL (IV) 0.06 0.17
T=5 min: 560 pmol
0.03 0.10
0.01 0.03
Counts/Energy Counts/Energy
44 Vasquez et al., 2011, PLOS One

26. 3D Cy5.5 biodistribution, co-registered to Quantum FX CT
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27. Testimonials
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
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28. 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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29. 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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30. THANK YOU
For questions, experimental design consulting, custom
presentations, data analysis, please contact:
alexandra.deLille@perkinelmer.com
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