Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 217-225
doi:10.1111/j.1745-7270.2008.00389.x
Identification and
characterization of a novel peptide ligand of Tie2 for targeting gene therapy
Xianghua Wu1,
Zonghai Li2, Ming Yao2, Huamao Wang2, Sumin Qu2,
Xianlian Chen2, Jinjun Li2, Ye Sun3, Yuhong Xu2,3,
and Jianren Gu2*
1 Department of Medical Oncology, Cancer Hospital
of Fudan University, Shanghai 200032, China
2 National Laboratory for Oncogenes and Related
Genes, Shanghai Cancer
Institute, Shanghai
Jiao-Tong University Medical School, Shanghai 200032, China
3 School of Pharmacy, Shanghai Jiao-Tong University,
Shanghai 200030, China
Received: September 4, 2007 Accepted: December
12, 2007
This work was
supported by a grant from the Major State Basic Research Development Program of
China (No. 2004CB518802)
*Corresponding author:
Tel/Fax, 86-21-64177401; E-mail, [email protected]
Tyrosine
kinase with immunoglobulin and epidermal growth factor homology domain-2 (Tie2)
has been considered as a rational target for gene therapy in solid tumors. In
order to identify a novel peptide ligand of Tie2 for targeted gene therapy, we
screened a phage display peptide library and identified a candidate peptide
ligand NSLSNASEFRAPY (designated GA5). Binding assays and Scatchard analysis
revealed that GA5 could specifically bind to Tie2 with a dissociation constant
of 2.1?10–8 M. In
addition, we showed that GA5 was internalized into tumor cells highly
expressing Tie2. In the biodistribution assay, 125I-GA5 was
mainly accumulated in SPC-A1 xenograft tumors that express Tie2. In gene
delivery studies, GA5-conjugated polyethylenimine vector could achieve greater
transgene transduction than non-targeted vectors both in vitro and in
vivo. Tumor growth inhibition was observed in SPC-A1 xenograft-bearing mice
that received eight intratumoral injections of GA5-polyethylenimine/p53
complexes in 3 weeks. The difference in tumor volume between the experiment and
control groups was significant (p<0.05). Our results showed that GA5 is a potentially efficient targeting element for cancer gene or molecular therapy.
Keywords Tie2;
gene therapy; phage display; polyethylenimine; p53 gene
Targeted therapeutic gene delivery into a desired tumor cell or
tissue is of unquestionable importance for improving therapeutic efficacy and minimizing
adverse effects derived from random distribution of therapeutic agents [1,2].
However, gene therapy is currently limited by the difficulty of achieving
efficient gene delivery into defined target cells. To overcome the common
disadvantages of current gene delivery vehicles, many efforts have been made to
design optimal vehicles for efficient gene delivery [3–5]. A receptor-mediated
gene delivery system has been developed as an attractive approach that can
potentially target genes into defined cells over-expressing cellular membrane
receptor. It is particularly interesting because of its potential to circumvent
the main disadvantage of viral vector [6]. Polyethylenimine (PEI) derivatives
are linear (22 kda) or branched
(25 kda) molecules that were shown
to be efficient in gene transfer in vitro and in vivo [7,8]. They
can target genes into desired cells successfully based on recognizable
receptor-mediated gene delivery system. PEI/DNA complexes conjugated with the
cell-binding epidermal growth factor receptor (EGFR) peptide ligand could
achieve 10-fold to 100-fold higher gene expression levels in tumor tissue than
in other tissues [9]. Recently, small molecular peptide ligands have
been pursued as potential tumor targeting agents for selective delivery of
therapeutic genes to tumors [10–13]. When compared with macromolecular natural ligands, these
small-sized peptides have the advantages of readily diffusible ability, less
immunogenicity, and higher target-to-background ratios [14,15]. Tyrosine kinase
with immunoglobulin and epidermal growth factor homology domain-2 (Tie2) has
been described as playing a critical role in the angiogenesis process in
various cancers and becomes a novel marker of microvasculature of solid tumors
[16–18].
Targeting Tie2 in gene therapy has been proposed as a potentially powerful
approach for the treatment of cancer [19–21]. Tumor suppressor gene, p53, was found to be mutated in many
solid tumors [22]. In lung cancer, mutated p53 was found in nearly 50% of
cases and was associated with poor prognosis [23]. Because p53 and
PI3K/Akt regulate cell survival and death [24], different approaches for
targeting p53 replacement gene therapies have been explored [25–28]. PEI/wt p53
transfection can inhibit the growth of human head and neck squamous cell
carcinoma xenografts with mutated p53 in mice [29].In this study, we identified a peptide ligand of Tie2 by screening a
phage display peptide library and investigated its targeting effect in vitro
and in vivo. We also conjugated the peptide ligand with PEI to construct
a Tie2-mediated non-viral gene delivery vector and used the vector to transfer
the reporter gene in vitro and in vivo. The targeted gene therapy experiment was carried out in mice
bearing human lung carcinoma xenografts by iteratively transferring the
wild-type p53 gene with the gene vector.
Materials and methods
Cell culture and xenograft
tumor model
SMMC7721, a human hepatic carcinoma cell line that negatively expresses
Tie2 [30], was a gift from the second
university of military medicine (Shanghai, China). SMMC7721-ExTie2 was derived from
SMMC7721 cells transfected with the plasmid pcDNA3.0-ExTie2 (a plasmid
constructed by inserting a cDNA fragment of the extracellular and transmembrane
domain of Tie2 into EcoRI/XhoI sites of plasmid pcDNA3.0) and
selected by G418 (Stratagene, La Jolla, USA) [30]. SPC-A1, a human lung
adenocarcinoma cell line expressing Tie2 [30], was provided by American Type
Culture Collection (Manassas, USA) and was used to establish the xenograft
tumor models for in vivo assay. These cells were incubated at 37 ?C in
Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum
(Gibco BRL, Carlsbad, USA) in a humidified 5% CO2 atmosphere.
Biopanning
The Ph.D-12 phage display peptide library was purchased from New
England Biolabs (Beverly, USA). The procedure for screening the phage display
library was modified according to the manufacturer’s instructions. Briefly, cultured
SMMC7721-ExTie2 cells were washed with phosphate-buffered saline (PBS), then
the phage library (4?1010 pfu/per well) diluted in 1 ml of DMEM containing 1% (w/v) bovine serum albumin (BSA; Sigma-Aldrich, St. Louis,
USA) was added per well. Phages were allowed to incubate with cells for 1 h at
37 ?C. Unbound phages were removed by washing 10 times with Tris-based buffered
saline containing 0.1% (v/v) tween-20 (TBST). Finally, phages bound to Tie2 receptors were
eluted specifically by adding TBS containing 10 mg/ml angiopoietin-2
(R&D Systems, Minneapolis, USA) to each well with gentle agitation for 1 h
at 37 ?C. Eluted phages were titered and amplified in Escherichia coli ER2537
cells then reapplied in subsequent rounds of panning. The elution procedure was
repeated four times and the final elute was used for amplification and
titration. Individual
blue clones were randomly selected and amplified by infecting ER2537 cells.
Phage single-strand DNA was isolated for sequencing.
The candidate peptide sequence was determined by amino acid sequence analysis
displayed on the most enriched phages.
Binding activity evaluation of
enriched phages
SMMC7721-ExTie2 cells, SPC-A1 cells, and SMMC7721 cells were seeded
in 96-well plates at a density of 1?104
cells per well. After blocking with PBS containing 1% BSA, 1?1010 pfu phages were added to each well and incubated
with the cells for 60 min at 37 ?C, then washed 10 times with cold TBST. Bound phages were detected by incubation with a 1:5000 horseradish peroxidase-conjugated
anti-M13 antibody (Amersham Biosciences, Piscataway, USA) for 1 h, followed by
washing and the addition of a peroxidase substrate (o-phenylenediamine,
0.4 mg/ml) in citrate-phosphate buffer (pH 5.0) containing 0.02% (v/v)
H2O2. The reaction was stopped with 50 ml of 12.5% H2SO4. A405 was
determined by using a Bio-Rad model 550 microplate reader (Bio-Rad, Hercules,
USA). In the phage recovery assay, recombined human Tie2/Fc protein (rh-Tie2;
R&D Systems) and BSA were immobilized on 96-well plates with 50 ml NaHCO3 (0.1 M, pH 8.6) per well overnight at 4 ?C. A total of 2?1010 pfu enriched and insertless phages diluted into 50 ml TBS containing
1% BSA were added to each well. After incubation for 1 h at 37 ?C under gentle
agitation, unbound phages were washed 10 times with TBST. Phages
bound to Tie2 receptors were eluted with 100 ml glycine-HCl (0.2 M, pH 2.2) for 10 min at room temperature. Recovery phages were determined
by titrating on X-gal/IPTG agar plates [7].
Peptide synthesis and binding
assay
Candidate peptide NSLSNASEFRAPY (designated GA5) and an irrelevant
peptide, HY12 (HATGTHGLSLSHY), were synthesized (GL Biochem, shanghai, China). The chloramine-T
procedure was used to radioiodinate GA5 and HY12 with 125I
[31]. For the in vitro binding assay, 1?104 SMMC7721-ExTie2 or SMMC7721 cells per
well were inoculated in 96-well plates, and grown until cells reached 70%
confluence. Cells were then washed three times with PBS, then 1?105 cpm (counts per minute) 125I-GA5 was
added into 100 ml binding buffer [DMEM containing 0.1% (W/V) BSA].
After incubating at 37 ?C for 30 min, cells were washed three times with PBST
to remove unbound radioactivity and lysed in 0.2 M NaOH for 15 min. Then the
lysate was transferred to a test tube and counted in a gamma counter. Binding
activities were also evaluated in the absence or presence of 1 mM unlabeled GA5
or angiopoietin-2. In order to calculate the GA5
binding constant, 1?104
SMMC7721-ExTie2 cells were inoculated in each well and cultured overnight. The next
day, cells were washed three times with PBS and blocked with 200 ml blocking
buffer (PBS containing 10 mg/ml BSA). After washing three times with PBST,
cells were incubated with varying concentrations of serially diluted 125I-GA5
(0–2000
ng/ml) for 30 min at 37 ?C and were washed three times with ice-cold PBST to
remove unbound radioactivity. The cultured cells were digested with 100 ml of 0.25%
trypsin and transferred to a test tube for radioactivity counting.Candidate peptide NSLSNASEFRAPY (designated GA5) and an irrelevant
peptide, HY12 (HATGTHGLSLSHY), were synthesized (GL Biochem, shanghai, China). The chloramine-T
procedure was used to radioiodinate GA5 and HY12 with 125I
[31]. For the in vitro binding assay, 1?104 SMMC7721-ExTie2 or SMMC7721 cells per
well were inoculated in 96-well plates, and grown until cells reached 70%
confluence. Cells were then washed three times with PBS, then 1?105 cpm (counts per minute) 125I-GA5 was
added into 100 ml binding buffer [DMEM containing 0.1% (W/V) BSA].
After incubating at 37 ?C for 30 min, cells were washed three times with PBST
to remove unbound radioactivity and lysed in 0.2 M NaOH for 15 min. Then the
lysate was transferred to a test tube and counted in a gamma counter. Binding
activities were also evaluated in the absence or presence of 1 mM unlabeled GA5
or angiopoietin-2. In order to calculate the GA5
binding constant, 1?104
SMMC7721-ExTie2 cells were inoculated in each well and cultured overnight. The next
day, cells were washed three times with PBS and blocked with 200 ml blocking
buffer (PBS containing 10 mg/ml BSA). After washing three times with PBST,
cells were incubated with varying concentrations of serially diluted 125I-GA5
(0–2000
ng/ml) for 30 min at 37 ?C and were washed three times with ice-cold PBST to
remove unbound radioactivity. The cultured cells were digested with 100 ml of 0.25%
trypsin and transferred to a test tube for radioactivity counting.
In vivo biodistribution assay
Four-week-old female athymic mice (BALB/c) were maintained in the
Shanghai Cancer Institute Isolation Facility (Shanghai Jiao-Tong University
Medical School, Shanghai, China). An athymic mice model bearing SPC-A1
xenograft was established [30]. Either 1 mCi 125I-GA5
or 125I was injected into the lateral tail vein in a total volume of 100 ml PBS. Mice were
killed by cervical dislocation at 0.5 h or 4 h after injection and dissected.
Tumor tissues and other organs were removed and blotted dry on tissue paper.
The wet weight of all samples was recorded, and the radioactivity in each
sample was measured with an automated gamma counter. The percentage of injected
dose per gram (%ID/g) was calculated according to the injection dose standard
curve.
Internalization assay
Fluorescein-isothiocyanate (FITC; Pierce, Rockford, USA) was
conjugated to the NH2 terminus of GA5 according to previous report [7].
FITC-labeled peptide was purified by gel filtration with Sephadex G-25 (GE
Healthcare Bio-Sciences Corp, Piscataway, USA). The SPC-A1 cells and SMMC7721
cells were cultured on cover slides, then incubated with FITC-labeled GA5 at 37
?C for 10 min and washed three times with PBS. The cells were visualized under
a Zeiss Axioskop 2 fluorescence microscope (Zeiss, Oberkochen, Germany).
Preparation of GA5-PEI/DNA
complexes
PEI of 22 kDa (Exgen 500; Fermentas, Hanover, USA) was conjugated
with GA5 at the molar ratio of 1:1. The conjugation procedure of GA5-PEI was
largely the same as that described previously [32]. Briefly,
dithiobis(succinimidylpropionate) (Sigma-Aldrich) was conjugated first with
PEI, then with GA5 peptide. The reaction mixture was incubated for 2 h at room
temperature, then reaction by-products and DMSO were removed by dialysing.
GA5-PEI and plasmid DNA were sterilized by filtration through 0.22 mm filters
(Millipore, Billerica, USA). Plasmid DNA was dissolved in small aliquot of
distilled water. The PEI cation to DNA anion ratio is presented as the molar
ratio of PEI nitrogen to DNA phosphate. The DNA/GA5-PEI complex was prepared by
mixing DNA with GA5-PEI for 20 min. The resulting polyplexes were subjected to
electrophoresis in 1% agarose containing 0.5 mg/ml ethidium bromide, and
approximately 0.5 mg plasmid DNA was loaded into each well.
Targeted reporter gene
delivery in vitro and in vivo
In the in vitro gene delivery assay, 2?104 SMMC7721 and SPC-A1 cells were seeded into 0.5 ml
medium in each well of a 24-well plate (Falcon, St. Louis, USA). After cells
reached a confluence of approximately 50%, the medium was removed and washed
with 0.5 ml PBS, then replaced with 0.5 ml serum-free media containing
GA5-PEI/pBK-CMV (pCMV)-luciferase polyplexes or PEI/DNA with the quantity
equivalent to 1 mg DNA. Cells were cultured at 37 ?C for 4 h. The incubation media
were removed and cells were rinsed with 0.5 ml PBS, followed by the addition of
0.5 ml fresh media containing antibiotics and 10% fetal bovine serum. The cells
were incubated for another 24 h, and the activity of luciferase was measured in
terms of relative light units per milligram protein (RLU/mg). In the in vivo
assay, GA5-PEI/pCMV-luciferase polyplexes in a volume of 200 ml were
intratumorally injected into athymic mice with SPC-A1 tumor xenograft at a dose
equivalent to 50 mg DNA per mouse. The mice were killed 24 h after injection and tumor
xenografts, heart, liver, spleen, lung, kidney, and brain were removed and
washed three times with 0.1 M PBS (pH 7.4). The expression of luciferase was
determined in the tumor as well as in other tissues according to methods
reported previously [7]. All experiments were carried out in experimental
groups containing at least six mice bearing 500 mm3 tumor.
PEI/pCMV-luciferase complexes were used as controls.
Tumor growth inhibition
experiments
Human lung cancer SPC-A1 cells (5?106) were inoculated subcutaneously into 4-week-old
female athymic mice (BALB/c). When the tumor size reached 500 mm3,
mice were randomly divided into the following five groups with six mice in each
group: GA5; PEI/wt p53; GA5-PEI/wt p53; wt p53; and NS.
Mice were intratumorally injected with GA5-PEI/wt p53 complexes and
other agents every 2 d for a total of eight times. Tumor growth was monitored
by measuring the tumor dimensions with a Vernier caliper three times weekly
until necrobiosis appeared in tumor xenograft. Tumor volume was calculated
according to the formula (V=pab2/6).
Statistical analysis
One-way ANOVA followed by the two-tailed Student’s t-test was
used for statistical evaluation of differences.
Results
Enzyme-linked immunosorbent
assay and phage recovery assay
After five rounds of screening, 20 phage clones were picked out
randomly and amplified for DNA sequencing. Of these, only 17 clones had
efficiently inserted peptides, and approximately 35% (6/17) of recovered clones
expressed the consensus amino acid sequence NSLSNASEFRAP. Another enriched
phage clone (No. 3) displayed a peptide XXGTHGHCQLSH. To assess the specificity
of the selected phage, the enriched phage clones No. 46, bearing NSLSNASEFRAP,
and No. 3 were amplified for further characterization. Fig. 1(A)
illustrates the binding affinity of No. 46 phage clones on various targets by
enzyme-linked immunosorbent assay. In phage recovery assay, the binding
activity of No. 46 phage clone could be inhibited by Ang-2. However, the
insertless phage clone and clone No. 3 did not have such binding specificity [Fig.
1(B–D)]. Based on these data, the peptide
clone No. 46 was chosen as our candidate ligand.
Specific binding assay of 125I-labeled GA5 in vitro
and in vivo
In order to label with 125I, tyrosine
(Y) was added to the C-terminal of the peptide that was displayed by phage
clone No. 46 and was designated GA5 (NSLSNASEFRAPY). As shown in Fig. 2(A),
the bound 125I-GA5 radioactivities appeared mainly in SMMC7721-ExTie2 cells, but
not in the parent Tie2-negative SMMC7721 cells. In the absence of competitors,
approximately 60% of 125I-GA5 was found to localize in the
SMMC7721-ExTie2 cells. In the presence of 100-fold molar excess of either Ang-2
or GA5, the bound radioactivity was reduced dramatically to the background
level. The dose-response curves of total, specific, and non-specific binding of
125I-GA5 to Tie2 are shown in Fig. 2(B). The specific binding of
125I-GA5 reached a plateau, indicating that it was saturated. The
binding constant of radiolabeled GA5 was calculated using Scatchard analysis.
The Scatchard plot of GA5 binding to SMMC7721-ExTie2 cells is shown in Fig.
2(C). The Kd value
was determined to be (2.10±0.15)?10–8 M. The number of binding sites for the labeled peptide (receptor
density) was estimated as (4.52±0.15)?105
per SMMC7721-ExTie2 cell. The biodistribution assay showed that the
radioiodine activity level in kidney peaked at 30 min after injection, but
declined to one-eighth of its peak by 4 h. However, the tumor uptake of 125I-GA5
was in a different pattern, as shown in Fig. 2(D). The uptake of 125I-GA5
in SPC-A1 xenografts at 0.5 h after injection was up to (8.31±0.46)%ID/g, and
declined to (2.41±0.13)%ID/g 4 h later. With the exception of brain, the ratios
of radioactivity of tumors to different normal tissues were approximately 3:1.
Blocking studies revealed that 100 mg non-radioactive GA5 co-injected with the
radiolabeled peptide solution significantly reduced the tumor uptake of
radioiodinated GA5 (p<0.05). These data showed that the radioactivity (%ID/g) of tumors was significantly higher than that of other tissues at 4 h (p<0.05) or 0.5 h (p<0.05) after injection except in kidney and blood.
Internalization experiments
To examine whether GA5 can be internalized into Tie2-expressing
cells, SPC-A1 and SMMC-7721 cells were incubated with FITC-labeled GA5
peptides, and it was found that the peptides were taken up efficiently by
SPC-A1 cells but not SMMC-7721 cells (Fig. 3).
In vitro and in vivo gene
delivery assay
To better understand the effect of GA5 peptide on targeted tumor
cells, GA5/PEI conjugate was prepared and pCMV-luciferase was used as a
reporter to test its capability for gene delivery. As shown in Fig. 4(A),
GA5-PEI/pCMV-luciferase and PEI/pCMV-luciferase could efficiently mediate gene
delivery into SPC-A1 and SMMC7721 cells. There was no significant difference in
gene delivery in SPC-A1 cells by either GA5-conjugated PEI or PEI alone.
However, the luciferase activity in SPC-A1 cells treated with
GA5-PEI/pCMV-luciferase was significantly higher than that in Tie2-negative
SMMC7721 cells (p<0.05). By in vivo gene delivery assay, the luciferase activity was higher in
SPC-A1 tumor xenografts transfected with GA5-PEI/DNA complex than that with
PEI/DNA (P<0.05) [Fig. 4(B)]. These data indicated that
GA5-PEI vector could mediate gene-targeted delivery.
Targeted gene therapy assay
In vivo gene therapy experiments showed
that no difference was observed in tumor growth between untreated mice and
control mice treated with irrelevant PEI/DNA complex. Compared with the NS
group, the inhibition rate of the GA5-PEI/p53 group was 62.54%. As shown
in Fig. 5, intratumoral injection with GA5-PEI/wt p53 complexes
could inhibit tumor xenograft growth.
Discussion
Receptor-mediated non-viral gene delivery vectors have been developed
to target therapeutic genes into tumor cells through surface receptor. Because
of its over-expression in both endothelial cells and certain tumor cells, Tie2
has emerged as a promising target for cancer therapy [21]. To identify a small
peptide ligand of Tie2 for targeted gene therapy, we screened a phage display
peptide library by competitive biopanning. After five rounds of biopanning, we
identified a small peptide, NSLSNASEFRAP, then tyrosine (Y) was added for the
purpose of iodination; the peptide NSLSNASEFRAPY was designated GA5. GA5 showed
specific binding capability to Tie2 and could be internalized into
Tie2-expressing cells. Our previous study revealed that SPC-A1 cells express
Tie2 [30]. Therefore, the biodistribution assay was carried out in nude mice
bearing human lung adenocarcinoma cell line SPC-A1 xenograft to examine the
peptide’s targeting ability. After vein injection, we observed higher
accumulation of radiolabeled GA5 in tumors than in other organs except kidney.
GA5 showed greater specificity for tumor xenografts and higher ratios of tumors
to normal tissues at equivalent time points after injection (Fig. 4).
Our data showed that GA5 might be an efficient ligand of Tie2 and a novel agent
for tumor targeting.One of the most promising non-viral vectors that has been developed
is the polycation PEI. Goula et al used linear low molecular weight PEI
(22 kDa)/DNA complexes for systemic application and showed efficient gene
delivery, with high gene expression in lung and lower expression in a variety
of organs including heart, liver, spleen, and kidney [33]. Li et al
reported that GE11, a small peptide ligand of EGFR, conjugated PEI vector can
efficiently transfer genes into EGFR over-expressing cells and tumor xenografts
[7]. Our results showed that high luciferase activity was observed in SPC-A1
cells expressing Tie2 when transfected with reporter gene pCMV-luciferase by
GA5-PEI, but lower luciferase activity was detected in SMMC-7721 cells. In the in
vivo gene delivery assay, it was shown that luciferase activity was higher
in SPC-A1 tumor xenografts transfected with GA5-PEI/DNA complex than that with
PEI/DNA (P<0.05) [Fig. 4(B)]. These results indicated that
GA5-PEI could mediate specific delivery of DNA complexes into tumor cells.In this study, the interesting phenomenon is that the novel vector,
GA5-PEI, could successfully deliver reporter gene into SPC-A1 cells but less
efficiently into SMMC7721 cells in vitro. Both PEI and GA5-PEI can
efficiently mediate reporter gene delivery into SPC-A1 cells, however, in
vivo gene delivery assay showed that GA5-PEI, not PEI, could transfer
reporter gene into SPC-A1 xenografts. This is most likely due to the generally
rapid diffusion and clearance of PEI/DNA polyplexes from circulation and most
normal organs that barely express Tie2. In addition, PEI-conjugated GA5
contributed to its stabilization and was specifically accumulated in
Tie2-positive cells. In the in vivo gene therapy assay, the
inhibition rate of the GA5-PEI/p53 group was 62.54% compared with the
normal saline group. GA5-PEI/p53 complexes transferring inhibited tumor
xenograft growth and GA5 could be an ideal agent targeting cells that express tie2 receptor.
Acknowledgements
We thank
Mrs. Yuyan Zhang for helpful advice in fluorescence microscopy and excellent
technical assistance, Dr. Dafang Wan for helpful suggestions, Dr. Wenxin Qin
for offering pCDNA3.0 plasmid, Dr. Rong Wang (medical
school of Oregon state University, Corvallis, USA) for
giving correction highlights.
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