Transcript Cell Imaging Technology

Molecular Imaging of
Therapeutic Cells: Pre-Clinical
and Clinical Studies
Shahriar Yaghoubi, Ph.D.
Chief Scientific Officer
CellSight Technologies, Inc.
1
Monday, July 06, 2015
Outline
• Background
• Non-invasive Cell Imaging Techniques
• Cell Transplantation Imaging
• Cell Trafficking Imaging
• Clinical Reporter Gene Imaging of
Adoptively Transferred Therapeutic Cells
Cellular Imaging Wish List
• Repeatedly image the location(s) of one or more
specific population of cells of interest and determine
cell quantity at all locations in living subjects
• Repeatedly image the “status” of populations of
cells in living subjects including potential
interactions with other cells
• Do not significantly perturb the cells while
repeatedly imaging them
• Allow smooth transition from pre-clinical to clinical
models
Methods for Imaging Therapeutic
Cells in Living Subjects
Direct Labeling with an Imaging Probe: 111In-Oxine,
64Cu-PTSM,
Ferridex, Nanoparticles
Incorporation of a Constitutively Expressed
Imaging Reporter Gene Through Transduction,
Transfection, Electroporation, etc …, then
Imaging at Multiple Time Points with a Reporter
Probe
Using a Highly Specific Probe to Image
Products of a Gene Solely Expressed by the
Target Cells: mRNA, Protein (Enzyme, Surface Receptor)
Direct Labeling Strategies
Imaging Trafficking of Cytokine Induced
Killer Cells with 111In-Oxine
Liver
Spleen
Pre-CIK
18F
FDG PET
111In
24 hours
CIK SPECT
42 hours
Reporter Gene Based Therapeutic Cell Imaging
Reporter Gene Strategies
Green Fluorescent Protein (Fluorescence)
Red Fluorescent Protein (Fluorescence)
Firefly Luciferase (Bioluminescence)
Renilla Luciferase (Bioluminescence)
Gaussia Luciferase (Bioluminescence)
Herpes Simplex Virus Type 1 Thymidine Kinase (PET/SPECT)
Dopamine Type 2 Receptor (PET)
Sodium-Iodide Symporter (PET/SPECT)
Somatostatin type 2 Receptor (PET/SPECT)
Transferrin Receptor (MRI)
Reporter Gene
Imaging with
Bioluminescence
Optical Imaging
Positron Emission Tomography
e+
e+
positron scatters in
tissue losing energy
nucleus
e+
511 keV
e+
yes
e– e+
annihilation
511 keV
coincidence ?
Enzyme Based PET Reporter Gene Imaging
[18F]-FHBG
Ad-CMV-HSV1-sr39tk
Reporter Gene
Imaging with
microPET
Gambhir S.S. et. al.,
JNM 1998, PNAS 2000, 2001
Tri-fusion Multimodality Reporters
CMV
fl/hrl
ttk/wttk
egfp/rfp/mrfp1
LENSHASAGYQAST
TAGPGSAT
Reporter Genes:
Bioluminescence:
Fluorescence:
PET:
 Firefly luciferase (fl)
 Synthetic renilla
luciferase (hrl)
 Enhanced Green
Fluorescence protein (egfp)
 Red Fluorescence Protein (rfp)
 Monomeric Red Fluorescence
Protein (mrfp1)

Deletion mutant of
HSV1-sr39tk (ttk)
 Deletion mutant of
Wild type
HSV1-tk (wttk)
Ray et. al., Cancer Research (March 2004)
Tri-fusion Multimodality Reporters
Bioluminescence
Cell
Fluorescence
microPET
Ray et. al., Cancer Research (March 2004)
Clinical
PET
Imaging
Trafficking of
T Cell
Hybridomas
Stably
Expressing a
Triple Fusion
Reporter
Gene in
Collagen
Induced
Arthritic Mice
Trafficking of
Yaghoubi et al. J Biomed Optics 12(6):064025-1 (2007)
Cell Transplantation
Imaging
Cell Transplantation
Histology
MicroPET Imaging
Autoradiography
Wu et. al., Circulation, Sept. 2003
Imaging ES Cell Survival & Proliferation
P/s/cm2/s
r
10
P/s/cm2/s
r
10
8
Control
P/s/cm2/s
r
10
8
P/s/cm2/s
r
10
8
P/s/cm2/s
r
10
8
8
6 ×1
6 ×1
03
6 ×1
05
6 ×1
05
06
6 ×1
06
4
4
4
4
4
2
2
2
2
2
0
Day
5
0
0
Day
10
Day
15
0.5%ID/
g
0
0
Day
20
1%ID/g
0%ID/g
0%ID/g
6
TK activity
2.5E+09
2.0E+09
4
1.5E+09
3
1.0E+09
2
5.0E+08
1
1.0E+06
0
4
7
3.0E+09
5
14
21
BMI(phontons/sec/cm^2)
firefly activity
microPET(%ID/g)
3.0E+09
BMI (photons/sec/cm^2)
A
y = 5E+08x
R2 = 0.924
2.5E+09
2.0E+09
1.5E+09
1.0E+09
5.0E+08
1.0E+03
0
days after cell transplantaion
Cao F, et al. Circulation 2006;113:1005-1014
1
2
3
m icroPET(%ID/g)
4
5
Ablation of Cellular “Misbehavior” with
TK Suicide Gene Therapy
Thymidine kinase serves as a PET reporter gene when [18F]-FHBG PET reporter
probe is used in pico-nanomolar concentration. Thymidine kinase serves as
suicide gene when ganciclovir is administered in milligram dosages.
Cao F, et al. Circulation 2006;113:1005-1014
Cell Trafficking Imaging
SYSTEMICALLY ADMINISTERED T-CELLS
HOME TO INTRACRANIAL GLIOMA
U251T-rLuc+ GBM INTRACRANIAL XENOGRAFT
TUMOR SIGNAL
T-CELLS IMAGED SERIALLY
48hrs
72hrs
T-CELL
SIGNAL
HUMAN T-CELLS
Administered I.V.
M. Jensen
(City of Hope)
96hrs
Towards Clinical Imaging of Cell Transfer
PRG/PRP System
(Enzyme Based)
[18F]FHBG
[18F]FHBG
HSV1-sr39TK
9-[4-[18F]Fluoro-3(hydroxymethyl)butyl]guanine
Imaging hMSC Transduced with
Adenoviral Vectors Carrying HSV1-sr39tk
L
R
L
PET/CT
0.007
18F-FHBG
0.006
0.005
% ID/g
18F-FHBG
0.004
0.003
0.002
0.001
0
Transduced hMSC
18F-FDG
Non-transduce hMSC
Human HSV1-tk Reporter Gene Imaging
Yaghoubi et al. Nature Clinical Practice Oncology 6(1): 53-58 (2009)
Penuelas . et. al. Gastroenterology 128:1787-95 (2005)
Imaging Cytolytic T Cells in Glioma
Patients
Leukapheresis
Separation of
Mononuclear Cells
Activation of
Autologous T Cells
Expand with
IL-2
Electroporation
hCMV
Infusions
12 sessions
107  108
cells
HyTK
hEF1
IL-13
Selection
Of Clones
With
Hygromycin
Ex Vivo
Expansion
with
IL-13+, CD8+,
IL-2
TCR/+, Single
copy of plasmid DNA,
IL-2 dependent,
Susceptible to GCV
induced cell death
Reporter Gene Based Imaging of Therapeutic Cells
(First Clinical Demonstration)
Yaghoubi et al. Nature Clinical Practice Oncology 6(1):53-58 (2009)
[18F]FHBG Activity at CTL Infusion Site,
Before and After Infusions in the Same Patient
[18F]FHBG Brain PET
Superimposed Over Brain MRI
Post-Cell
Pre-Cell
Standard Uptake Values (SUV)
Max Intensity in Regions of Interest (ROI)
0.8
0.7
Max SUV
0.6
0.6
0.5
0.5
0.4
0.70.7
Pre-CTL
PostCTL
0.4
0.4
0.3
0.3
0.2 0.2
0.2
0.2
0.1
0
Tumor
Brain
BKGD
Meninges
Neck
Shoulder
Max SUV
Tumor to Background Ratios
3.5
3.3
3.2
Pre-CTL
Post-CTL
Max SUV Ratios
3
2.5
2
2.0
1.5
1
0.8
0.9
0.8
0.7
0.5
0.5
0
Tumor/Brain
BKGD
Tumor/Meninges
Tumor/Neck
Tumor/Shoulder
Percent Change in [18F]FHBG
Accumulation and Tumor/BKGD
[18F]FHBG Post CTL Infusion
FHBG Accumulation:
Tumor
58%
Brain BKGD
-2%
Meninges
-63%
Tumor/Tissue Ratios:
Tumor/Brain BKGD
Tumor/Meninges
61%
423%
Neck
23%
Tumor/Neck
28%
Shoulder
-3%
Tumor/Shoulder
62%
Summary & Future Directions
• Multimodality Imaging Approaches Should Help
Transition from Pre-Clinical to Clinical Non-invasive Cell
Imaging Studies
• Direct Labeling and Reporter Gene Based Imaging
Strategies are Available for Monitoring Therapeutic Cell
Biodistribution in Patients
• Reporter Gene Based Techniques Allow Imaging
Biodistribution as Well as Survival and Status
• Incorporating Non-Invasive Imaging Into Cell Therapy
Trials Should Help Optimize Protocols and Predict
Efficacy or Potential Adverse Effects
Acknowledgements
Stanford University
Sanjiv Gambhir Multimodality
Imaging Group
Nuclear Medicine Clinic
Cyclotron
City of Hope National
Medical Center
Michael Jensen Group
UCLA Medical Center
Dept of Mol Med
Pharmacology
Nuclear Medicine Clinic
Cyclotron
Funding
National Cancer Institute
ICMIC p50
R01
Patient Volunteers
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7/6/2015