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Design, synthesis and validation of Axl-targeted monoclonal antibody probe for microPET imaging of human lung cancer
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Design, synthesis and validation of Axl-targeted monoclonal antibody probe for microPET imaging of human lung cancer
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DESIGN, SYNTHESIS AND V ALIDATION OF AXL-TARGETED
MONOCLONAL ANTIBODY PROBE FOR MICROPET IMAGING OF
HUMAN LUNG CANCER
By
Jiacong Guo
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)
May 2013
Copyright 2013 Jiacong Guo
ii
Acknowledgements
First and foremost, I would like to thank my parents for all their support, without
which my graduate study wouldn’t have been possible.
I would like to take this opportunity to thank a few members who have been crucial at
every stage in my work. I am extremely thankful to Dr. Zibo Li, for his invaluable
guidance and immense support in this project. I would also like to thank Dr. Scott Liu and
Ms. Dan Li for their tutoring and day-to-day guidance all along, which enlightened me to
finish my work.
In addition, I want to convey my sincere gratitude to Dr. Parkash S. Gill and his lab
members for kindly providing us with all the antibodies without which I can hardly get
my project done.
Last but not the least, I wish to express my sincere appreciation to Dr. Joseph R.
Landolph and Dr. Axel H. Schö nthal for their extensive motivation and great advice.
iii
Table of Contents
Acknowledgements .......................................................................................................................... ii
List of Figures .................................................................................................................................. v
Abstract .......................................................................................................................................... vii
1. Introduction ................................................................................................................................. 1
1.1 Receptor tyrosine kinase.................................................................................................. 1
1.2 Axl is a receptor tyrosine kinase ..................................................................................... 1
1.3 Monoclonal antibody, MAb173, was developed for Axl ................................................ 4
1.4 Characterization of MAb173 therapeutic effects ............................................................. 6
1.5 MicroPET imaging .......................................................................................................... 7
2. Materials and Methods ................................................................................................................ 9
2.1 Materials .......................................................................................................................... 9
2.2 DOTA conjugation and radiolabeling ............................................................................. 9
2.3 MicroPET Imaging Study.............................................................................................. 10
2.4 Western Blot .................................................................................................................. 11
2.5 Immunofluorescence. .................................................................................................... 11
2.6 Cell uptake assay. .......................................................................................................... 11
2.7 Flow Cytometry. ............................................................................................................ 12
2.8 Murine tumor xenograft models. ................................................................................... 13
2.9 Data processing and statistics. ....................................................................................... 13
3. Results ....................................................................................................................................... 14
3.1 Cell uptake of
64
Cu-DOTA-mAb173 on A549 cells ..................................................... 14
3.2 Fluorescence-activated cell sorting analysis of A549 cells: Axl expression level ........ 15
3.3 Western blot analysis of mAb173 in eight cell lines ..................................................... 17
iv
3.4 In Vivo microPET imaging study .................................................................................. 18
3.5 MAb173 distribution in A549 tumors ........................................................................... 23
4. Discussion ................................................................................................................................. 25
Bibliography .................................................................................................................................. 29
v
List of Figures
Figure 1:The extracellular domain of 4 RTK subfamilies. Axl is similar to platelet-derived
growth receptor and EphA receptors in that it shares similar FNIII and Ig domains (Hafizi,
S. and B. Dahlback et al. 2006). .................................................................................... 3
Figure 2: TAM subfamily is composed of Sky, Mer and Axl and has two ligands Protein S
and Gas6. Gas6 has been identified as the ligand for Axl (Hafizi, S. and B. Dahlback et al.
2006). 4
Figure 3: KSHV infected endothelial cells treated with IgG and MAb173 were treated with
control IgG and MAb173 for the indicated time points. Cells were then lysed to Western
Blot with antibodies against Axl and pAKT. ................................................................. 5
Figure 4: Co-staining of Axl (red) and Ki67 (green) in two groups of Kaposi sarcoma cells
treated with IgG and Mab173, respectively. .................................................................. 6
Figure 5: KSHV transformed endothelial cells were incubated by 10μg/ml biotinylated
MAb173 at 37° C for 15minutes, 1 hour and at 4° C for 1 hour, respectively. Cells were
fixed and MAb173 (green) and Nuclei (blue) were co-stained. .................................... 7
Figure 6: Cell uptake assay of
64
Cu-MAb173-DOTA and
64
Cu-hIgG-DOTA on A549 and
H249 cells (n=3, mean± SD) ........................................................................................ 14
Figure 7: A. FACS analysis of two tumor cell lines (A549 and H249) using mAb173 as the
primary antibody B. Quantification of mAb173 binding percentage to the two cell lines
Figure 8: Expression of Axl in 4 types of cell lines, with 2 different cell subtypes in each of
these cell lines, was analyzed by western blot of whole-cell lysates. .......................... 17
Figure 9: A. Serial microPET scans of A549 tumor-bearing mice after injection of
64
Cu-DOTA-mAb173,
64
Cu-DOTA-IgG, respectively.
64
Cu-DOTA-mAb173 and
64
Cu-DOTA-IgG were also injected into H249 tumor-bearing mice. All whole-body
coronal images that contain the tumor were decay-corrected. Arrow indicated the tumor
vi
position. B. Heart, liver, tumor, kidney and muscle uptake of
64
Cu-DOTA-mAb173 and
64
Cu-DOTA-IgG at 3h, 16h, 28h, 45h after injection in A549 and H249 tumor bearing mice
(n=3, means± SD). C. Time activity curves of A549 and H249 tumor uptake of
64
Cu-DOTA-mAb173 from 1h to 45h post injection. .................................................. 22
Figure 10: Immuno-staining of IgG and mAb173 staining on A549 tumor sections. . 24
vii
Abstract
Accumulating experimental evidence indicates that overexpression of oncogenic receptor
tyrosine kinase, Axl, plays a key role in the tumorigenesis and metastasis of various types
of cancer. The objective of this study was to design a novel imaging probe based on the
monoclonal antibody, MAb173, for microPET imaging of Axl expression in human lung
cancer. A bifunctional chelator, DOTA, was used to radiolabel MAb173 with
64
Cu. The
receptor binding affinity of
64
Cu was evaluated in vitro with human lung cancer cell line.
In vivo micro-PET imaging of the Axl-positive A549 xenograft model was carried out to
evaluate the Axl targeting probe. In vitro cell uptake assay and flow cytometry analysis
were performed to detect the expression level of Axl on A549 cells and the results
showed that Axl probes were highly immunoreactive with A549 cells. Subsequently, Axl
expression was further analyzed by Western Blot in various liver, breast, lung and
Kaposi-sarcoma (KS) cancer cell lines, respectively. Identical to the two positive controls
KS-SLK and KS-IMM, Axl is highly expressed in A549 cell line. For microPET imaging
in the subcutaneous A549 model, the tumor demonstrated strong uptake compared with
the uptake level of the control cell model, H249. Immuno-fluorescence staining also
supported the in vivo micro-PET imaging results. Thus,
64
Cu-DOTA-mAb173 could be
used as a potential probe for noninvasive imaging of Axl expression, which can help us
predict whether lung tumors will effectively respond to certain Axl-targeted therapeutic
interventions, as well as monitor the corresponding response to therapy.
1
1. Introduction
1.1 Receptor tyrosine kinase
Receptor tyrosine kinases (RTKs) are a class of protein kinases that play a critical
role in the development and progression of various types of cancer. Up to now,
approximately 20 different RTK classes have been identified and TAM, which is
composed of Axl, Sky and Mer, is one out of the 20 RTK subfamilies.(Hafizi and
Dahlback 2006; Hafizi and Dahlback 2006)
1.2 Axl is a receptor tyrosine kinase
The Axl receptor tyrosine kinase was isolated as a transforming gene from primary
human myeloid leukemia cells (O'Bryan, Frye et al. 1991). Axl is implicated in vascular
remodeling, regulation of smooth muscle cells and migration of endothelial cells
(Melaragno, Fridell et al. 1999; Korshunov, Mohan et al. 2006; Collett, Sage et al. 2007).
The extracellular region of Axl is comprised of two immunoglobulin-like (IgL) and dual
fibronection typeIII (FNIII) repeats and a single transmembrane spanning domain.
Carboxyl terminal to the FNIII repeats in Axl is a single transmembrane and a
cytoplasmic domain with tyrosine kinase activity. Gas6 has been identified as the ligand
for Axl and the Gas6/Axl system is implicated in several types of cancer, autoimmune
diseases as well as other vascular and kidney diseases.(Hafizi and Dahlback 2006) In
2
recent years, the significant role of Axl in tumor initiation and metastases has been
reinforced by the fact that Axl is overexpressed in multiple cancer types including
prostate (Sainaghi, Castello et al. 2005), breast (Zhang, Knyazev et al. 2008; Liu, Greger
et al. 2009; Gjerdrum, Tiron et al. 2010), lung (Shieh, Lai et al. 2005; Ishikawa, Sonobe
et al. 2012; Postel-Vinay and Ashworth 2012; Vaughan, Singh et al. 2012; Byers, Diao et
al. 2013), gastric (Wu, Li et al. 2002), glioblastoma (Vajkoczy, Knyazev et al. 2006;
Hutterer, Knyazev et al. 2008) and Kaposi sarcoma (Liu, Gong et al. 2010). Several
studies also indicate that the expression level of Axl is highly correlated with lung tumor
progression (Shieh, Lai et al. 2005) and invasivity of breast cancer cells (Zhang, Knyazev
et al. 2008). In addition, Axl is also identified as a potential therapeutic target for
overcoming EGFR inhibitor resistance (Byers, Diao et al. 2013), and for lapatinib and
trastuzumab resistant breast cancer cells(Liu, Greger et al. 2009).
3
Figure 1: The extracellular domain of 4 RTK subfamilies. Axl is similar to
platelet-derived growth receptor and EphA receptors in that it shares similar FNIII and Ig
domains (Hafizi, S. and B. Dahlback et al. 2006).
4
Figure 2: TAM subfamily is composed of Sky, Mer and Axl and has two ligands Protein S
and Gas6. Gas6 has been identified as the ligand for Axl (Hafizi, S. and B. Dahlback et al.
2006).
1.3 Monoclonal antibody, MAb173, was developed for Axl
In order to hasten the pace of developing anti-Axl based cancer therapy for clinical
trials, it is necessary to apply noninvasive methods to visualize and quantify Axl
5
expression in vivo. Recently, monoclonal antibodies which are specific for Axl have been
developed and one of these antibodies, MAb173, appears to bind to the first fibronection
domain and has been characterized as having the function in reducing the expression of
Axl in Kaposi sarcoma herpesvirus (KSHV) infected endothelial cells in vitro and
inhibiting the growth of Kaposi sarcoma cells in vivo. (Liu, Gong et al. 2010).
Figure 3: KSHV infected endothelial cells treated with IgG and MAb173 were treated
with control IgG and MAb173 for the indicated time points. Cells were then lysed for
Western Blot with antibodies against Axl and pAKT.
6
Figure 4: Co-staining of Axl (red) and Ki67 (green) in two groups of Kaposi sarcoma
cells treated with IgG and Mab173, respectively.
1.4 Characterization of MAb173 therapeutic effects
The therapeutic effects of MAb173 have been identified in that MAb173 can degrade
cell surface Axl to trigger endocytosis to kill Kaposi sarcoma tumor cells(Liu, Gong et al.
2010). If the KS cells were treated with MAb173 at 4° C for only 60 minutes, MAb173
still stayed only on the cell membrane of KS cells. Whereas, if the temperature was
increased to 37° C and cells were for 15mins, MAb173 began to be translocated from the
extracellular membrane to the cytoplasm. Moreover, under the same temperature, if the
treatment time was elongated to 1 hour, almost 80% of MAb173 had been transported
inside of the KS cells, indicating that MAb173 could trigger endocytosis.
7
Figure 5: KSHV transformed endothelial cells were incubated with 10 μg/ml biotinylated
MAb173 at 37° C for 15 minutes, 1 hour and at 4° C for 1 hour, respectively. Cells were
fixed and MAb173 (green) and nuclei (blue) co-staining are shown.
1.5 MicroPET imaging
Preclinical imaging is the visualization of living animals for research purposes. For
anatomical imaging, techniques such as high-frequency micro-ultrasound, magnetic
resonance imaging (MRI) and computed tomography (CT) are usually utilized. Optical
imaging including fluorescence and bioluminescence, positron emission tomography
(PET) and single photon emission computed tomography (SPECT) are often times
8
utilized for molecular visualizations. PET is a nuclear medical imaging technique that can
produce a three-dimensional image of the human body. By detecting gamma rays emitted
by a positron-emitting radionuclide tracer, it can keep track of wherever a tracer goes
after it is injected into a mouse. In this study, we labeled mAb173 with
64
Cu to create an
antibody tracer that could be used for microPET imaging studies to noninvasively
quantify Axl expression in vivo.
9
2. Materials and Methods
2.1 Materials
Monoclonal antibody, MAb 173 (humanized monoclonal antibody for microPET
imaging, Cell uptake, Flow Cytometry and Western blot) and all the cell lines were
kindly provided by Vasgene Therapeutics Inc. (Los Angeles, CA). Antibodies against
human Axl (goat polyclonal, for immunostaining) were from R&D Systems. All cells
were grown in RPMI-1640 supplemented with 10% fetal bovine serum in 5% CO2 at
37 ° C. 64CuCl2 was purchased from Washington University (St. Louis).
2.2 DOTA conjugation and radiolabeling
DOTA (macrocyclic ligand 1, 4, 7, 10-tetraazacyclododecane-N, N’, N’’,
N’’’-tetraacetic acid) was activated with EDC (Ethylene Dichloride) and SNHS (Sulfo
N-hydroxysulfosuccinimide) at a molar ratio of 10:5:4 (DOTA/EDC/SNHS) at pH 5.5 for
30 minutes. DOTA-N-hydroxysulfosuccinimidyl (OSSu) was cooled to 4° C and added to
MAb173 and IgG at a molar ratio of 50:1. The reaction mixture was adjusted to pH 8.5
with borate buffer (0.1 M, pH 8.5) and then incubated overnight at 4° C. The
DOTA-MAb173 and DOTA-IgG conjugates were then purified by PD-10 column
chromatography (Millipore, Bedford, MA).
64
CuCl2 (1.8-2 mCi) was diluted in 300 μL of
0.1 mol/L sodium acetate buffer (pH 5.5) and added to the DOTA-MAb173 (200 µ g in
10
PBS) and DOTA-IgG conjugates. The reaction mixture was incubated for 1 hour at 40° C
with constant shaking. The two conjugates were then purified by PD-10 column using
PBS as the mobile phase. The radioactive fractions containing
64
Cu-DOTA-mAb173 was
collected and passed through a 0.2μm syringe filter for further in vitro and in vivo
experiments.
2.3 MicroPET Imaging Study
The tumor-bearing mice were imaged using a microPET R4 rodent model scanner
(Concorde Microsystems, Knoxville, TN). The A549 and H249 tumor-bearing mice (n =
3) were imaged in the prone position in the microPET scanner. The tumor-bearing mice
were injected with 250–300 μCi of
64
Cu labeled tracer via the tail vein, then anesthetized
with 2% isoflurane and put in the center of the field of view. Multiple static scans were
obtained at 3 h, 16 h, 28 h, and 45 h after injection. The images were reconstructed by a
two dimensional ordered subsets expectation maximum algorithm. After each microPET
scan, the regions of interest (ROIs) were drawn over the tumor and major organs on
decay-corrected whole-body coronal images. The average radioactivity concentration
within the tumor or an organ was obtained from mean pixel values within the multiple
ROI volume, which were converted to microcuries per gram using the calibration
constant C. Assuming a tissue density of 1 g/ml, the ROIs and were then divided by the
total decay-corrected administered activity to obtain the imaging ROI-derived percentage
11
administered activity per gram of tissue (%ID/g).
2.4 Western Blot
Cells grown on a 100-mm dish were lysed on ice with 1.2mL lysis buffer (Bio-Rad)
and 20 μg of whole-cell lysates was run on 4% to 20% Tris-glycine gradient gel (Bio-Rad)
and transferred onto nitrocellulose membrane (Bio-Rad). The membrane was blocked
with 5% nonfat dry milk in Tris-buffered saline and washed with 0.05% Tris-buffered
saline–Tween-20 for 40 minutes, and then incubated with 1 μg/mL primary antibody at
4° C overnight.
2.5 Immunofluorescence.
Fresh frozen tissue embedded in cryo-embedding media Optical Cutting Temperature
compound (OCT) was sectioned at 5 μm and fixed in phosphate-buffered 4%
paraformaldehyde and washed in PBS. Sections were then incubated with primary
antibodies overnight at 4 ° C. After washing with PBS, antibody binding was localized
with Alexa Fluorconjugated appropriate secondary antibodies (Invitrogen). Nuclei were
counterstained with 6-diamidino-2-phenylindole dihydrochloride hydrate (DAPI). Images
were obtained with an Olympus AX70 fluorescence microscope.
2.6 Cell uptake assay.
12
Cell uptake studies were performed to quantify the uptake levels of the
64
Cu-DOTA-MAb173 and 64Cu- DOTA-IgG. Experiments were performed in triplicates.
A549 and H249 cells were seeded into a 6-well plate at a density of 6 × 104 cells per well
for overnight incubation at 37 ° C. Cells were rinsed three times with PBS, followed by
the addition of
64
Cu-DOTA-IgG,
64
Cu-DOTA-MAb173 to the cultured wells in triplicate
(about 4 μCi/well). After incubation at 37 °C for 1.5 h, cells were rinsed three times with
PBS and lysed with NaOH-sodium dodecyl sulfate (SDS) (0.2 M NaOH, 1% SDS). The
cell lysate was collected in measurement tubes for counting. The cell uptake was
normalized in terms of added radioactivity.
2.7 Flow Cytometry.
Human lung cancer cell lines A549 and H249 were used to assess cell binding and
membrane uptake efficiencies of MAb173 probes. To minimize nonspecific uptake of
peptides by pinocytosis, incubations were performed on ice followed by flow cytometric
analysis for rapid quantification of fluorescence. For flow cytometry (FACScan; Becton
Dickinson), 10,000 cells were counted and viable cells with similar size and granularity
in the forward- and side-scatter plots were analyzed. The fluorescence profiles and the
overall mean fluorescent intensities of the cells within this region were obtained and
analyzed using CellQuest Software (Becton Dickinson).
13
2.8 Murine tumor xenograft models.
A549 and H249 cells were propagated, trypsinized, collected, and resuspended in
serum-free medium. Cells (2× 10
6
) diluted in 100 μl PBS were injected subcutaneously
into the right shoulder of Nude/SCID mice.
2.9 Data processing and statistics.
All of the data are given as means ± SD of three independent measurements. Student’s
t-test was used to analyze the data. Statistical significance was assigned for P values <
0.05. Based on results from three tumor-bearing mice at each time point,
target-to-background ratios were determined as the average plus or minus the standard
deviation.
14
3. Results
3.1 Cell uptake of
64
Cu-DOTA-mAb173 on A549 cells
Cell uptake studies were performed using human A549 and H249 cells. Two control
groups in which
64
Cu-DOTA-IgG was added were established for comparison with the
experimental groups. As shown in Figure 6, the cell uptake percentage of
64
Cu-DOTA-MAb173 (1.96%) by A549 cells was significantly higher than H249 cells
(0.32%) and the other two control groups (0.36% for A549+64Cu-DOTA-IgG and 0.3%
for H249+64Cu-DOTA-IgG, respectively).
Figure 6: Cell uptake assay of
64
Cu-MAb173-DOTA and
64
Cu-hIgG-DOTA on A549 and
H249 cells (n=3, mean ± SD)
15
3.2 Fluorescence-activated cell sorting analysis of A549 cells: Axl
expression level
Fluorescence-activated cell sorting (FACS) analysis using mAb173 as the primary
antibody against Axl clearly showed that A549 cells had a prominently higher uptake of
mAb173 (82.2%) than its control (1.0%) in which only second antibody was added. As
with the control cell line, H249, we hardly saw any immunoreactivity of mAb173 on it
(1.0%) (Figure 7).
16
A.
B
Figure 7: A. FACS analysis of two tumor cell lines (A549 and H249) using mAb173 as
the primary antibody B. Quantification of mAb173 binding percentage to the two cell
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
90.00%
A549 cells H249 cells
mAb173
2nd antibody only
17
lines
3.3 Western blot analysis of mAb173 in eight cell lines
To better confirm our hypothesis that Axl is highly expressed on A549 cells, a
western blot experiment was conducted to detect Axl expression. Two Kaposi-sarcoma
cancer cell lines KS-IMM and KS-SLK were included as the positive controls (Liu, Gong
et al. 2010). Liver cancer cell line Huh7 was used as a negative control.
Axl expression in breast cancer tumor is closely related to the invasiveness of breast
cancer cells. In the highly invasive cancer cell line, MDA-MB231, Axl has a widespread
expression, whereas in the less invasive MCF cells, Axl can hardly be found. In
comparison to HepG2 cells, there is a significant elevation of Axl expression in A549
lung cancer cell lines. The expression of Axl in H249 cells in the western blot experiment
is also very low, which is consistent with the previous in vitro results (Figure 3).
Figure 8: Expression of Axl in 4 types of cell lines, with two different cell subtypes in
each of these cell lines, was analyzed by western blot of whole-cell lysates.
18
3.4 In Vivo microPET imaging study
The microPET imaging study was performed on
64
Cu-DOTA-mAb173 and
64
Cu-DOTA-IgG using athymic nude mice bearing A549, H249 human lung cancer cell
at multiple time points (3h, 16h, 28h, 45h), respectively. Figure 1A shows representative
decay-corrected coronal images and radioactivity quantification in several typical organs
of the A549 and H249 tumor-bearing mice after administration of 121 μCi and 107 μCi
of
64
Cu-DOTA-mAb173. For the control group, we injected 103 μCi and 114 μCi of
64
Cu-DOTA-IgG into the A549 and H249 xenografts. At early time points, both
conjugates exhibited high uptake into the heart because of normal blood pooling activity.
Similarly, liver also has relatively high uptake due to the nonspecific binding of tracers
through the reticular-endothelial circulation system. As for other organs, their uptake was
not significantly different from the background level.
Figure 4A indicates that the tumor uptake of
64
Cu-DOTA-mAb173 by A549 tumor
xenograft was significantly higher than that for
64
Cu-DOTA-mAb173 by another lung
cancer cell line, H249 xenograft and also higher than that of the control A549
tumor-bearing mice into which
64
Cu-DOTA-IgG was injected.
Figure 4B reveals that the A549 tumor uptake of increased over time after 45-hour
post-injection. The uptake of
64
Cu-DOTA-mAb173 into A549 tumors was
3.63± 1.38%ID/g, 8.26± 1.14%ID/g, 9.67± 0.5% ID/g and 11.1± 0.27% ID/g at 3, 16, 28
19
and 45-hour post-injection, all of which were 8-11 times higher than that in H249 tumors
(corresponding uptake is 0.35± 0.53%ID/g, 0.74± 0.75%ID/g, 1.16± 0.35%ID/g and
1.19± 0.18%ID/g, respectively). As with other organs (heart, liver, kidney and muscle)
and the A549 xenograft, the levels of
64
Cu-DOTA-mAb173 decreased significantly in
these tissues as time increased; however, for H249, the tracer uptake by the tumor was
essentially no different from muscle and other organs.
20
A.
A549
H249
A549
H249
64
Cu-DOTA-IgG
64
Cu-DOTA-MAb173
21
B.
0.00%
5.00%
10.00%
15.00%
20.00%
25.00%
Heart Liver Tumor Kidney Muscle
%ID/g
64
Cu-DOTA-MAb173 on A549
3h
16h
28h
45h
0.00%
5.00%
10.00%
15.00%
20.00%
25.00%
Heart Liver Tumor Kidney Muscle
%ID/g
64
Cu-DOTA-MAb173 on H249
3h
16h
28h
45h
22
C.
Figure 9: A. Serial microPET scans of A549 tumor-bearing mice after injection of
64
Cu-DOTA-mAb173,
64
Cu-DOTA-IgG, respectively.
64
Cu-DOTA-mAb173 and
64
Cu-DOTA-IgG were also injected into H249 tumor-bearing mice. All whole-body
coronal images that contain the tumor were decay-corrected. Arrow indicates the tumor
position. B. Heart, liver, tumor, kidney and muscle uptake of
64
Cu-DOTA-mAb173 and
64
Cu-DOTA-IgG at 3h, 16h, 28h, 45h after injection in A549 and H249 tumor bearing
0.00%
5.00%
10.00%
15.00%
20.00%
25.00%
Heart Liver Tumor Kidney Muscle
%ID/g
64
Cu-DOTA-IgG on A549
3h
16h
28h
45h
-2.00%
0.00%
2.00%
4.00%
6.00%
8.00%
10.00%
12.00%
3h 16h 28h 45h
ID%/g
Time after injection
64
Cu-DOTA-MAb173 uptake
A549 tumor
H249 tumor
23
mice (means± SD, n=3). C. Time course of A549 and H249 tumor uptake of
64
Cu-DOTA-mAb173 from 1 h to 45 h post injection.
3.5 MAb173 distribution in A549 tumors
Following microPET imaging of mAb173 distribution in vivo and after the majority
of radioactivity decayed 72 hours post-injection, the mice were sacrificed and their
respective tumors were harvested to be sectioned. Frozen tumor sections were stained
with secondary antibodies for IgG and mAb173. After sample mounting in buffer
containing DAPI, we examined the tumor sections under a fluorescence microscope and
found that A549 cells had a significantly higher uptake of mAb173 than the control IgG.
This experiment provided us with strong evidence that mAb173 can specifically bind to
A549 cells in vivo.
24
Figure 10: Immuno-staining of IgG and mAb173 staining on A549 tumor sections.
25
4. Discussion
Up until now, 20 distinct subfamilies of RTKs have been found and categorized
according to their identities in amino-acid sequence and structural similarities in their
extracellular regions. One of these is the subfamily, TAM, which is composed of Axl,
Sky and Mer. This RTK subfamily is characterized by a combination of dual
immunoglobulin (Ig)-like repeats and dual fibronectin type III domains in the
extracellular region. (Robinson, Wu et al. 2000)
Axl and its ligand, Gas6, have been found to play a pivotal role in the survival and
metastasis of multiple cancers and other diseases(Wu, Li et al. 2002; Sun, Fujimoto et al.
2004; Sainaghi, Castello et al. 2005; Gustafsson, Bostrom et al. 2009; Gustafsson,
Martuszewska et al. 2009; Ekman, Jö nsen et al. 2011; Ammoun, Provenzano et al. 2013).
We are the first to synthesize
64
Cu-labeled MAb173, a humanized monoclonal antibody
against Axl and found it to have high Axl specificity in vitro and in vivo.
In this study, the novel
64
Cu-labeled MAb173 tracer was synthesized and
characterized to demonstrate that imaging Axl expression with PET is possible. Stable
attachment of
64
Cu to a targeting molecule required the use of a bifunctional chelator
(BFC) and in our experiment, the chelator we used was the macrocyclic ligand 1, 4, 7,
10-tetraazacyclododecane-N, N’, N’’, N’’’- tetraacetic acid (DOTA).
In our study, we chose two human lung cancer cell lines, A549 and H249. The two
26
main types of lung cancer are small-cell lung carcinoma (SCLC) and non-small-cell lung
carcinoma (NSCLC). A549 cells are adenocarcinomic alveolar basal epithelial cells and
can be categorized as non-small-cell lung carcinoma type. H249, on the other hand,
belongs to small-cell lung carcinoma.
The in vitro tumor uptake experiment indicated that only A549 had a high uptake of
the monoclonal antibody tracer,
64
Cu-DOTA-MAb173, whereas H249 had a very low
level of uptake. This result led us to conduct other in vitro experiments to further confirm
our uptake assay result. In the fluorescence-activated cell sorting analysis, the uptake
percentage of Axl by A549 cells was 82.2%, significantly higher than the other cancer
cell line, H249, with the uptake level of only 1%.
In order to further confirm the FACS result, we went on to do another in vitro
experiment, western blot, to detect the expression level of Axl in these two cancer cell
lines. Two Kaposi-sarcoma cell lines, KS-IMM and KS-SLK were included as the
positive controls because they were previously found to be Axl-positive.(Liu, Gong et al.
2010) The highly invasive breast cancer cell line, MDA-MB231 and less invasive cell
line MCF7 were also investigated for Axl expression. Previously Axl was found to be
only expressed in highly invasive breast cancer cells, rather than in breast cancers of low
invasivity; our western blot experiment confirmed this finding .(Zhang, Knyazev et al.
2008) Two liver cancer cell lines were also included for analysis because Axl is
implicated in hepatic regeneration from oval cells in the rat and indentified as a mediator
of YAP-dependent oncogenic functions in hepatocellular carcinoma. Our results showed
27
that Axl was expressed in HepG2, a liver cancer cell line. Consistent with the cell uptake
and FACS results, immunoblot analysis confirmed that Axl could only be found in A549
cells, but not in H249.
MicroPET imaging was conducted to characterize Axl binding affinity with A549 and
H249 cells in vivo and the results showed that PET quantification correlated well with the
Western Blot and FACS analysis. Notably, despite the good in vitro specificity of
64
Cu-DOTA-MAb173, compared to mice injected with
64
Cu-DOTA-IgG, mice injected
with the former had a significant kidney uptake in vivo. Thus, further in vitro
investigations are required to explain this phenomenon. Although it is a common
phenomenon for antibody probes to have relatively high kidney uptake, the injection dose
should perhaps be limited when
64
Cu-DOTA-MAb173 is applied to potential clinical
therapies.
After 72 hours, when most of the radioactivity in mice had decayed, we confirmed the
in vivo microPET imaging data for the A549 xenograft mice and took out their tumors
for immune-staining. Compared to the group treated with only control IgG antibody, the
experimental group in which we added MAb173 as the primary antibody, demonstrated
high level expression of Axl. Both the microPET imaging studies and the
immunofluorescence staining confirmed that MAb173 was specific for A549 cells.
Therefore, the difference in tumor uptake is a good reflection of the Axl expression levels
of different lung tumor cells.
28
In summary, we have developed a prototype Axl targeted tracer that may serve as a
candidate for microPET imaging Axl-positive tumors. The possible correlation between
Axl expression level and tumor invasiveness in multiple cancer types makes this
MAb173 imaging probe potentially useful for detecting human lung cancer.
This study shows that
64
Cu-DOTA-mAb173, a humanized MAb against Axl, exhibits
high binding affinity with Axl in A549 lung cancer cell both in vivo and in vitro.
Whereas, as with another lung cancer cell model, H249, the uptake level of
64
Cu-DOTA-mAb173 is relatively very low.
29
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Abstract (if available)
Abstract
Accumulating experimental evidence indicates that overexpression of oncogenic receptor tyrosine kinase, Axl, plays a key role in the tumorigenesis and metastasis of various types of cancer. The objective of this study was to design a novel imaging probe based on the monoclonal antibody, MAb173, for microPET imaging of Axl expression in human lung cancer. A bifunctional chelator, DOTA, was used to radiolabel MAb173 with ⁶⁴Cu. The receptor binding affinity of ⁶⁴Cu was evaluated in vitro with human lung cancer cell line. In vivo micro-PET imaging of the Axl-positive A549 xenograft model was carried out to evaluate the Axl targeting probe. In vitro cell uptake assay and flow cytometry analysis were performed to detect the expression level of Axl on A549 cells and the results showed that Axl probes were highly immunoreactive with A549 cells. Subsequently, Axl expression was further analyzed by Western Blot in various liver, breast, lung and Kaposi-sarcoma (KS) cancer cell lines, respectively. Identical to the two positive controls KS-SLK and KS-IMM, Axl is highly expressed in A549 cell line. For microPET imaging in the subcutaneous A549 model, the tumor demonstrated strong uptake compared with the uptake level of the control cell model, H249. Immuno-fluorescence staining also supported the in vivo micro-PET imaging results. Thus, ⁶⁴Cu-DOTA-mAb173 could be used as a potential probe for noninvasive imaging of Axl expression, which can help us predict whether lung tumors will effectively respond to certain Axl-targeted therapeutic interventions, as well as monitor the corresponding response to therapy.
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Asset Metadata
Creator
Guo, Jiacong
(author)
Core Title
Design, synthesis and validation of Axl-targeted monoclonal antibody probe for microPET imaging of human lung cancer
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Molecular Microbiology and Immunology
Publication Date
05/03/2013
Defense Date
03/08/2013
Publisher
University of Southern California
(original),
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Tag
Axl,lung cancer,microPET imaging,monoclonal antibody,OAI-PMH Harvest
Language
English
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Electronically uploaded by the author
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Advisor
Landolph, Joseph R., Jr. (
committee member
), Schonthal, Axel H. (
committee member
), Tahara, Stanley M. (
committee member
)
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batista.guo@gmail.com
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Tags
Axl
lung cancer
microPET imaging
monoclonal antibody