Development of Peritoneal Tumor-Targeting Vector by In Vivo Screening with a Random Peptide-Displaying Adenovirus Library
et al. (2012) Development of Peritoneal Tumor-Targeting Vector by In Vivo Screening with
a Random Peptide-Displaying Adenovirus Library. PLoS ONE 7(9): e45550. doi:10.1371/journal.pone.0045550
Development of Peritoneal Tumor-Targeting Vector by In Vivo Screening with a Random Peptide-Displaying Adenovirus Library
Takeshi Nishimoto 0
Yuki Yamamoto 0
Kimiko Yoshida 0
Naoko Goto 0
Shumpei Ohnami 0
Kazunori Aoki 0
Ilya Ulasov, University of Chicago, United States of America
0 1 Division of Gene and Immune Medicine, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan, 2 Central Radioisotope Division, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan, 3 Department of Neurosurgery, Graduate School of Medicine, Hiroshima University , Minami-ku, Hiroshima , Japan
The targeting of gene transfer at the cell-entry level is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success, because the proper targeting ligands on the cells of interest are generally unknown. To overcome this limitation, we have constructed a random peptide library displayed on the adenoviral fiber knob, and have successfully selected targeted vectors by screening the library on cancer cell lines in vitro. The infection of targeted vectors was considered to be mediated by specific receptors on target cells. However, the expression levels and kinds of cell surface receptors may be substantially different between in vitro culture and in vivo tumor tissue. Here, we screened the peptide display-adenovirus library in the peritoneal dissemination model of AsPC-1 pancreatic cancer cells. The vector displaying a selected peptide (PFWSGAV) showed higher infectivity in the AsPC-1 peritoneal tumors but not in organs and other peritoneal tumors as compared with a non-targeted vector. Furthermore, the infectivity of the PFWSGAV-displaying vector for AsPC-1 peritoneal tumors was significantly higher than that of a vector displaying a peptide selected by in vitro screening, indicating the usefulness of in vivo screening in exploring the targeting vectors. This vector-screening system can facilitate the development of targeted adenovirus vectors for a variety of applications in medicine.
Funding: This work was supported in part by a grant-in-aid for the 3rd Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health,
Labour and Welfare of Japan, grants-in-aid for Research from the Ministry of Health, Labour and Welfare of Japan, by the program for promotion of Foundation
Studies in Health Science of the National Institute of Biomedical Innovation (NIBIO) and by the National Cancer Center Research and Development Fund (23-A-9).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Recombinant adenovirus vectors have been widely used for
gene delivery to a range of cell types and employed in a number of
gene therapy approaches . On the other hand, a selective
delivery of a therapeutic gene to target cells by adenovirus vectors
is precluded by the widespread distribution of the primary cellular
receptors for adenoviruses [2,3]. The suppression of nave viral
tropism is needed to reduce the undesirable infection of non-target
normal tissues, whereas the antitumor effect of adenovirus vector is
determined by the capacity to infect tumor cells. Thus, the
addition of a tumor-targeting potential to an adenovirus vector
ablated for nave tropism is important to enhance its therapeutic
Recently, several strategies have been developed to redirect the
tropism of the adenovirus vector to permit efficient target gene
delivery to specific cell types [4,5]. In particular, targeting has been
achieved by direct genetic modifications of the capsid proteins:
targeting ligands can be incorporated into the C-terminal and
HIloop of fiber proteins ablated for native tropism, and these vectors
provide an important platform for evaluating the targeting
potential of selected peptide ligands . However, cell-type
specific ligands for targeted adenovirus vectors are generally
unknown, which impedes the wider application of fiber-modified
adenovirus vectors for targeted therapies. Although a phage
display library has been used to identify targeting peptide motifs,
the incorporation of the peptides selected by phage display into the
adenoviral capsid has not been successful in developing targeted
vectors except for a few cases , possibly due to the
peptideinduced conformational change of the virus capsid and the loss of
specificity and affinity of ligand-receptor binding . To
overcome this limitation, we have developed a system for
producing adenoviral libraries displaying a variety of peptides on
the fiber by Cre-lox mediated in vitro recombination between an
adenoviral fiber-modified plasmid library and an adenoviral
DNAterminal protein complex, and have established a procedure to
select an adenoviral vector with high infectivity in target cells in
vitro [14,15]. Another group also reported a method for generating
libraries displayed on an adenovirus fiber in which modified-fiber
genes were directly shuttled into a replicating virus genome in
helper cells . Since the binding affinity might be determined by
the overall conformation of a modified fiber and not by inserted
peptides alone, the selection of targeting peptides is highly useful in
the context of the adenoviral capsid.
We were able to successfully screen adenovirus vectors
displaying targeted peptide sequences after several rounds of
amplification of viruses on cultured cancer cell lines [14,15]. The
infection of targeted vectors was considered to be mediated by
specific receptors on target cells. However, the expression levels
and kinds of cell surface receptors may be substantially different
between in vitro culture and in vivo tumor tissue. Therefore, an in
vivo screening of the peptide display adenovirus library may be
useful to develop vectors which specifically transduce certain
tumors in vivo. To this aim, here we examined whether the
tumortargeting vectors could be selected from the adenovirus library by
in vivo screening in a murine peritoneal dissemination model of
pancreatic cancer cells. A particular sequence was observed after 2
rounds of selection, and the adenovirus displaying the selected
peptide showed a high infectivity preferentially for peritoneal
tumors but not organs. The results demonstrated that an in vivo
screening with an adenovirus library is a promising strategy for the
development of targeted vectors, for which this is the first report of
a targeted adenovirus developed by an in vivo approach alone.
Materials and Methods
A human embryonic kidney cell line (293), pancreatic cancer
cell lines (AsPC-1 and PSN-1), gastric cancer cell line (MKN45)
and ovarian cancer cell line (SKOV3) were used in this study. All
the cancer cell lines except for PSN-1 were obtained from
American Tissue Culture Collection (ATCC; Rockville, MD), and
the PSN-1 cell line was established by H. Yamada et al. . 293
cells were cultured in Dulbeccos modified eagles medium (Sigma,
St. Louis, MO) with 10% fetal bovine serum (FBS), and cancer cell
lines in an RPMI-1640 medium (Nissui Pharmaceutical, Tokyo,
Japan) with 10% FBS. 293.HissFv.rec cells express an artificial
receptor against six histidine (His) residues, containing an anti-His
single chain antibody (sFv) . The 293-38 is a high-efficiency
virus-producing clone of 293 cells, and the 293-38 cells expressing
an anti-His sFv stably (293-38.HissFv.rec) were generated by
retrovirus-mediated transduction .
Shuttle Plasmids and Recombinant Adenovirus DNA
The adenoviral shuttle plasmids pBHI and pBHIDCAR include
a 76.1100 map unit (mu) of the type 5 adenoviral genome with
a single loxP site at the E3 region deleted (79.484.8 mu) [14,15].
The pBHI has a wild type of fiber. The pBHIDCAR has two
incompatible restriction enzyme sites in the HI-loop to display
random peptides and includes 4-point mutations in the AB-loop of
the fiber knob to abolish CAR binding, and six histidine residues
were incorporated into the carboxy-terminal of the fiber knob, so
that the vector can be propagated in the 293 cells expressing an
anti-His sFv . The pBHIDCAR-PFW and pBHIDCAR-SYE
plasmids have CCTTTTTGGAGTGGGGCTGTT (PFWSGAV)
and TCGTATGAGAATTTTAGTGCG (SYENSFA) sequences
in the HI-loop of the pBHIDCAR plasmid, respectively. The
adenoviral cosmid cAd-WT includes the 079.4 mu of the
adenovirus genome containing a wild-type E1 region and a single
loxP site at 79.4 mu. The cAd-WT was recombined with
pBHIDCAR to generate AdDCAR-WT for preparation of
adenoviral DNA tagged with a terminal protein (DNA-TPC). In
the cAd-LucEGFP, the E1 gene is replaced by the CMV
promoter-driven luciferase-EGFP fusion gene (LucEGFP) in
cAd-WT. The cAd-LucEGFP was recombined with
pBHIDCAR-PFW, pBHIDCAR-SYE, pBHIDCAR and pBHI plasmids
to generate adenovirus vectors AdDCAR-LucEGFP-PFW,
AdDCAR-LucEGFP-SYE, AdDCAR-LucEGFP and Ad-LucEGFP,
respectively. The adenovirus vectors were quantified by optical
absorbance . The infectious units of the viruses were examined
in 293-38.HissFv.rec cells, and the ratio of the viral particle to
infectious unit for each virus was approximately 30.
Construction of a Random Peptide-display Adenovirus
The random peptide-display adenovirus library used in this
study was the same as we previously screened on AsPC-1 cells in
vitro . We used the adenovirus vector ablated for CAR binding
as a backbone construct of adenovirus library to reduce natural
tropism. Briefly, the adenovirus shuttle plasmid
pBHIDCAREGFP has a cytomegalovirus immediate early enhancer/promoter
(CMV promoter), the enhanced green fluorescent protein (EGFP)
gene and a SV40 poly (A) signal downstream of the loxP site at the
deleted E3 region in the pBHIDCAR plasmid. The degenerate
59-AACGGTACACAGGAAACAGGAGACACAACTTTCGAA(NNK)7ACTAGTCCAAGTGCATACTCTATGTCATTTTCATGG-39 (N = A, T, G or C, K = G or T)
served as a template for PCR with the primers
59-CATAGAGTATGCACTTGGACTAGT-39. The PCR product was ligated into
the HI-loop portion of pBHIDCAR-EGFP to construct a
fibermodified shuttle plasmid library, and transfected into Max
Efficiency electrocompetent cells (Invitrogen, Carlsbad, CA) by
electroporation. The fiber-modified shuttle plasmid library was
recombined with equal moles of the left hand of the digested
DNA-TPC by Cre recombinase (Clontech, Madison, WI) in vitro to
produce a full-length adenovirus genomic DNA library. Then, to
generate replication-competent peptide-display adenovirus
libraries, recombined adenoviral DNA was transfected by the lipofection
method (Lipofectamine Reagent; Invitrogen) in 293-38.HissFv.rec
cells. When the cells showed an expansion of the cytopathic effect,
the first generation of the adenovirus library was harvested.
29338.HissFv.rec cells were infected with the crude viral lysate (CVL)
again, and the second generation of the library was harvested. The
library was estimated to display more than 16104 peptides on the
fiber per 60-mm dish . Since the library used in the screening
was collected from twenty 60-mm dishes, the complexity of the
peptide sequences displayed in the library was estimated to be
approximately at a 26105 level, and the final concentration of the
virus library was prepared as 16109 plaque forming unit (PFU)/
Screening of a Random Peptide-display Adenovirus
Library in Mice with Peritoneal Dissemination
Four to 5-week-old female BALB/c nude mice were purchased
from Charles River Japan, Inc. (Kanagawa, Japan), and were
housed under sterilized conditions. Animal studies were carried
out according to the Guideline for Animal Experiments of the
National Cancer Center Research Institute and approved by the
Institutional Committee for Ethics in Animal Experimentation.
AsPC-1 (56106) cells were intraperitoneally injected into the mice,
which resulted in the peritoneal dissemination and the formation
of tumor nodules in pancreatic regions within 14 days. Eighteen
days after the injection of tumor cells, 200 ml of an adenovirus
library solution (16108 PFU) was intraperitoneally injected into
the mice. Seven days following the injection, peritoneal tumors
were harvested, and after the pulverization of the tumors, the CVL
was prepared from the tumors by freezing and thawing 3 times. To
expand the selected viruses, 293-38.HissFv.rec cells were infected
with the CVL from the peritoneal tumors. Seven days after the
infection, the CVL from 293-38.HissFv.rec cells was
intraperitoneally reapplied in the mice with peritoneal tumors of AsPC-1 cells
as a second round of selection. Seven days later the replicated
adenoviruses were harvested from the peritoneal tumors again.
PCR and Sequencing of Adenovirus Library Clones
DNA was extracted from the CVL of the peritoneal tumors by
the 1st and 2nd selections, and then served as a template for a PCR
with the primers containing upstream and downstream sequences
of the HI-loop: 59-GAAACAGGAGACACAACTTTCGAA-39
and 59-CATAGAGTATGCACTTGGACTAGT-39. PCR
products were cloned into the pBHIDCAR plasmid. Randomly
assigned clones were sequenced using the primer 59-
Assay for Luciferase Activity in vitro
The cells were seeded at 16104 per well in 96-well plates
(Optilux multiplate; BD Biosciences, Franklin Lakes, NJ) and
infected with adenoviruses at various moi (1, 3, 10, 30 and 100).
Twenty-four hours after the infection, 100 ml of luciferase assay
substrate (Bright-GloTM luciferase assay system; Promega,
Madison, WI) was added to each well. The light units of luciferase
activity were measured using a luminometer (EnVision multilabel
plate reader; Parkin Elmer, Shelton, CT). The assays (carried out
in 8 wells) were repeated a minimum of two times and the mean 6
standard deviation was plotted.
Assay for Luciferase Activity in vivo
To examine the in vivo infectivity of a targeted adenovirus
vector, AsPC-1 cells (56106 cells) were injected intraperitoneally
into the BALB/c nude mice. Fourteen days later, 200 ml of a viral
solution (16107, 36107 and 16108 PFU) of
AdDCAR-LucEGFPPFW was intraperitoneally injected into the mice with a 29-gauge
hypodermic needle. The luciferase assay was performed as
described previously .
Detection of Adenovirus DNA from the Peritoneal
Tumors and Organs
The peritoneal tumors and organs such as the liver, spleen,
pancreas and small intestine were collected 2 days after the
intraperitoneal injection of an adenovirus solution (16108 PFU),
and DNA was extracted from the tumors and organs using
Sepagene (Sanko Junyaku Co. Ltd., Tokyo, Japan). The viral
DNA in 1 mg total DNA was analyzed by a real-time PCR using
EcoTM Real-Time PCR system (Illumina Inc., San Diego, CA).
The primers to detect a 68-bp region in E4 were utilized as
described previously . Briefly, the sequences of the upstream
and the downstream E4 primers were
59-GGAGTGCGCCGAGACAAC-39 and 59-ACTACGTCCGGCGTTCCAT-39,
respectively. The sequence of the TaqMan probe (6-FAM-labeled probe)
was TGGCATGACACTACGACCAACACGATCT. A final
volume of 10 ml/reaction containing 16Gene Expression Master
Mix (Applied Biosystems, Foster City, CA), 100 nM upstream
primer, 100 nM downstream primer, 1.1 mM probe and extracted
DNA was applied to the real-time PCR. For the standard curve to
quantify the E4 copy numbers, E4 template DNA with a known
copy number (2.461062.4610) was also analyzed. Thermal
cycling conditions were as follows: initial denaturation at 95uC for
10 min, and then 40 cycles at 95uC for 15 s and at 60uC for
Comparative analysis of luciferase activity was performed by the
Students t-test, and differences were considered statistically
significant when the P value was ,0.05.
In vivo Selection of Library Clones Targeting Peritoneal
To examine whether an in vivo screening could select peptides
displayed on the fiber knob that produces higher transduction
efficiency to peritoneal tumors, the adenovirus display library was
intraperitoneally injected into the mice with peritoneal
dissemination of AsPC-1 cells (Fig. 1). In the initial phase of the screening,
many low-affinity or nonspecific viruses might bind and internalize
into peritoneal tumor cells; however, the use of a
replicationcompetent type of adenovirus could allow for the rapid
amplification and spreading of the most efficient viruses present
in the library, leading to an effective enrichment of such viruses.
Amplified adenoviruses in peritoneal tumors were recovered and
subjected to a second round of selection. The DNA region
containing the oligonucleotide insert of adenoviruses recovered
from the first and second rounds of selection was then amplified by
DNA sequencing of the PCR products revealed enrichment of
various peptides after the first round of selection, whereas 31 of 32
clones (96.9%) showed the same sequence PFWSGAV after the
second round of selection (Table 1). The PFWSGAV sequence
was recognized in 2 of 41 clones (4.9%) at the first round. The
screening was repeated to confirm the reproducibility of the
results, and the 1st screening also enriched the same PFWSGAV
motif (8 of 32 clones; 25.0%) in peritoneal tumors from the same
library. None of the selected sequences was found in the 100
randomly isolated clones from the unselected adenovirus library.
The fact that the identical sequence was repeatedly enriched in the
2 independent screenings potentially indicates the feasibility and
reliability of an in vivo screening procedure to select targeted
vectors to tumors. The selected motif PFWSGAV was different
from a sequence (SYENFSA) selected by in vitro screening on
AsPC-1 cells with the same adenovirus library . The peptide
motifs selected from the same library may be different according to
the environment in which tumor cells grow. To prove that the
selected peptides are a result of binding-mediated selection and not
due to a low complexity bias in the library, PSN-1 peritoneal
tumors were also screened using the same library. All peptide
sequences selected from the library after 2 rounds of screenings in
PSN-1 peritoneal tumors were different from those selected from
AsPC-1 peritoneal tumors (data not shown).
Infectivity of Adenovirus Displaying a Selected Peptide
for AsPC-1 Peritoneal Tumors
To test the infectivity of the adenovirus vector displaying
a selected peptide, various amounts of a luciferase-expressing
targeted vector ablated for CAR binding
(AdDCAR-LucEGFPPFW) were intraperitoneally injected into the mice with peritoneal
dissemination of AsPC-1 cells. The peritoneal tumors were
harvested 2 days after the injection, and the luciferase activity in
the tumors was measured. In this assay, the
replication-incompetent adenoviruses were employed to eliminate the possibility
LQTSLGC(5) ASIGFLV(1) LQASLGC(1)
PFWSGAV PFWSGAV PFWSGAV
CWVRECR(3) GASVFPT(1) WLTSSLN(1)
PFWSGAV PFWSGAV PFWSGAV
LGTVCDL(3) WGTVCGL(1) YMVIWLG(1
PFWSGAV PFWSGAV PFWSGAV
RLDGGAV(2) GVVWYMA(1) IRQLRFG(1)
PFWSGAV PFWSGAV PFWSGAV
GVQWLNG(2) GGGRGFA(1) VRLRGGE(1)
PFWSGAV PFWSGAV PFWSGAV
ARGSGVC(2) TECVPPG(1) VPVVWWR(1) PFWSGAV PFWSGAV PFWSGAV
WLGWAVS(2) VLSRVCY(1) FWRCGVD(1) PFWSGAV PFWSGAV PFWSGAV
PFWSGAV(2) FCNGRAF(1) SVGCFLG(1)
PFWSGAV PFWSGAV PFWSGAV
WVVVFSF(1) GSVVGRY(1) EGWVGCD(1) PFWSGAV PFWSGAV PFWSGAV
GARTVVR(1) TSYGAYC(1) *G*LEHS(1)
PFWSGAV PFWSGAV PFWSGAV /42 (): Numbers of repetition. *: Stop codon.
PFWSGAV PFWSGAV VRLRGGE PFWSGAV PFWSGAV /32
that the high luciferase activity was due to the efficient replication
of the adenovirus in the tumors. The luciferase activity assay
showed that the gene transduction efficiency of a targeted vector
was 1.6-fold higher at 16107 PFU level, 4.1-fold higher at 36107
PFU level and 6.9-fold higher at 16108 PFU level than that of an
untargeted adenovirus vector ablated for CAR binding
(AdDCARLucEGFP) for AsPC-1 peritoneal tumors (Fig. 2a), indicating the
selected peptide significantly enhanced the adenovirus infectivity
for target tumors.
To compare the infectivity of AdDCAR-LucEGFP-PFW with
AdDCAR-LucEGFP-SYE and Ad-LucEGFP, which has a wild
type of fiber, in AsPC-1 peritoneal tumors, 16108 PFU of
adenovirus vectors were intraperioneally injected into the mice
with AsPC-1 peritoneal dissemination, and peritoneal tumors were
harvested 2 days after the injection. The luciferase assay showed
that the infectivity of AdDCAR-LucEGFP-PFW was significantly
higher than AdDCAR-LucEGFP-SYE for AsPC-1 peritoneal
tumors (Fig. 2b). Although the fiber knob of
AdDCARLucEGFP-PFW was ablated for CAR binding, its infectivity in
AsPC-1 peritoneal tumors was compatible with Ad-LucEGFP
(Fig. 2b), demonstrating the high infectivity of the adenovirus
vector displaying PFWSGAV for AsPC-1 tumors.
Next, to examine whether gene transduction is also enhanced
by the selected peptide in vitro, several cancer cells were infected
with AdDCAR-LucEGFP-PFW at various moi. The gene
transduction efficiency of AdDCAR-LucEGFP-PFW was higher in
AsPC-1 (2.5-fold at moi 100) and PSN-1 cells (1.8-fold at moi 100)
compared with AdDCAR-LucEGFP, whereas the luciferase
activity of AdDCAR-LucEGFP-PFW-infected cells was lower than
those of AdDCAR-LucEGFP infection in SKOV-3 (0.35-fold at
moi 100) and MKN45 cells (0.94-fold at moi 100) (Fig. 3a). The
infectivity of AdDCAR-LucEGFP-PFW was lower than
AdDCARLucEGFP-SYE in AsPC-1 cells in culture (Fig. 3a), whereas
AdDCAR-LucEGFP-PFW showed a higher luciferase activity
compared with AdDCAR-LucEGFP-SYE in AsPC-1 peritoneal
tumors (Fig. 2b). The results suggest that the in vivo screening is also
a promising strategy in developing the targeted adenovirus vectors.
To validate that the targeting is mediated by the selected
peptide, the transduction efficiency of AdDCAR-LucEGFP-PFW
in AsPC-1 cells was determined in the presence or absence of the
cognate peptide. A substantial competitive inhibition by the
cognate peptide but not by a control unrelated peptide was
observed at moi 30 in a dose-dependent manner (Fig. 3b),
confirming that the enhanced transduction of the targeted vector
in the cells is mediated by the insertion of the selected peptide.
Infectivity of Adenovirus Displaying a Selected Peptide
for Other Peritoneal Tumors and Organs
To examine whether the adenovirus vector displaying the
selected peptide could effectively transduce the gene in various
peritoneal tumors, the luciferase activity was assessed in AsPC-1,
PSN-1, SKOV3 and MKN45 peritoneal tumors 2 days after the
intraperitoneal injection of the vectors. The
AdDCAR-LucEGFPPFW showed a higher luciferase activity in AsPC-1 peritoneal
tumors than did AdDCAR-LucEGFP as shown in Fig. 2a, whereas
the luciferase activities in 3 other tumors of
AdDCAR-LucEGFPPFW-injected mice were not significantly elevated compared with
those of AdDCAR-LucEGFP-injected mice (PSN-1; 1.6-fold,
SKOV3; 0.32-fold, MKN45; 1.0-fold) (Fig. 4), suggesting the
specificity of a selected peptide for AsPC-1 peritoneal tumors.
Although the AdDCAR-LucEGFP-PFW showed different
infectivity between in vitro culture and peritoneal tumors for
AsPC1 cells, its infectivity in peritoneal tumors was compatible with
those in in vitro culture for PSN-1, SKOV3 and MKN45 cells
(Figs. 3a and 4), demonstrating the importance of in vivo screening
with target cancer cells.
Finally, to confirm the AsPC-1 tumor-targeting effect of the
selected vector, various organs such as liver, spleen, pancreas and
small intestine were also harvested 2 days after the intraperitoneal
injection of vectors at a dose of 16108 PFU. The luciferase
activities in the organs were markedly lower than those in AsPC-1
tumors in the AdDCAR-LucEGFP-PFW-injected mice. The
luciferase activities in organs of
AdDCAR-LucEGFP-PFW-injected mice were generally similar to those of
AdDCARFigure 3. Infectivity of cancer cells with the adenovirus displaying the selected peptide. (a) Luciferase activities following infection of
adenovirus vectors in vitro. The AsPC-1, PSN-1, SKOV3 and MKN45 cells were infected with adenovirus vectors (AdDCAR-LucEGFP-PFW,
AdDCARLucEGFP-SYE, AdDCAR-LucEGFP or Ad-LucEGFP), and 24 h later the luciferase activities were measured. (b) Competitive inhibition of transduction of
the adenovirus vector with selected peptide. Transduction efficiencies of AdDCAR-LucEGFP-PFW in AsPC-1 cells were evaluated at moi 30 in the
presence of the cognate or a control unrelated peptide at 0.21.5 mmol/L. The data are expressed as the relative luciferase activity (luciferase activity
in the presence of peptide/that in the absence of peptide). Control peptide: AQGQWAL.
LucEGFP-SYE- and AdDCAR-LucEGFP-injected mice, which
were lower than those of Ad-LucEGFP-injected mice (Fig. 5a),
indicating that the selected peptide does not enhance the
infectivity in the organs.
Furthermore, we analyzed the distribution of adenovirus DNA
by a real-time quantitative PCR method in peritoneal tumors and
various organs 2 days after the injection of adenovirus vectors.
Although the quantitative PCR showed that the adenovirus DNA
was more detected in the peritoneal tumors when compared with
organs in AdDCAR-LucEGFP-PFW-injected mice, the difference
was not statistically significant (Fig. 5b). Considering that the
luciferase assay showed much lower expression of the gene in
organs than in peritoneal tumors (Fig. 5a), it is plausible that DNA
detected by quantitative PCR analysis might be the partially
degraded fragments of adenoviral DNA.
The redirection of virus tropism is one of the most rational
approaches to targeted vector development. We have shown that
Figure 4. Infectivity of various peritoneal tumors with the adenovirus displaying the selected peptide. The mice were injected
intraperitoneally with 16108 PFU of adenoviruses (AdDCAR-LucEGFP-PFW or AdDCAR-LucEGFP) (AsPC-1; n = 14, others; n = 4), and 2 days after the
injection, peritoneal tumors were harvested and the luciferase activity was measured.
the in vitro screening of a random peptide display adenovirus
library on a cell type of interest allows the selection of targeted
adenovirus vectors as previously reported [14,15]. Here, we
screened pancreatic peritoneal dissemination with the adenovirus
library and identified a candidate targeting ligand sequence, which
was not isolated from the same library by in vitro screening on
AsPC-1 cells. The in vivo library screening is also useful to explore
the targeting vectors as well as screening on culture cells, and it
may be important to employ the experimental models in
accordance with an intended disease condition of target cancer
cells in the library screening for developing a targeted vector.
The infectivity of AdDCAR-LucEGFP-PFW for AsPC-1
peritoneal tumors was similar with that of Ad-LucEGFP, whereas the
infectivity of AdDCAR-LucEGFP-PFW for organs was much
lower than Ad-LucEGFP (Fig. 5a). The relative luciferase activity
of AdDCAR-LucEGFP-PFW for peritoneal tumor compared with
the liver was 61.5-fold, whereas that of Ad-LucEGFP was 9.8-fold,
demonstrating that the insertion of PFWSGAV in the fiber knob
enhanced the in vivo tumor-specificity of adenovirus vector. The
results suggest that the combination of a targeting peptide with
suppression of nave viral tropism improves the safety compared
with the wild type of fiber. In case we do not need to consider the
infection of adenovirus vectors in organs, the incorporation of
targeting peptides into the capsid of wild fiber may be useful to
more enhance the infectivity for target tumors.
In our previous manuscript, we showed that approximately 10%
of clones in the shuttle plasmid libraries converted successfully into
adenovirus virions, whereas 90% of the peptide insertions into the
HI-loop disturbed virus production, possibly due to the
conformational change of the fiber and disturbance of the fiber
trimerization . Therefore, we estimate that the maximum
complexity of the 7 amino acids displayed on the fiber knob is at
the16108 level. The 26105 complexity level used in this study
represents only a small fraction of the possible vector clones.
Although the selected peptide would be the most efficient sequence
in this adenovirus library, more efficient peptide motifs can be
isolated by a scaling up of the library size. Since the complexity of
an adenovirus library depends on the number of helper cells
transfected with recombined DNA, the simple scaling up of the
number and size of the plates could increase the complexity of the
library. We intend to employ a high complexity of library in
comprehensively exploring targeted vectors for various tumors.
A CAR-binding region in the backbone adenovirus construct is
ablated to reduce the natural tropism. However, since
CARbinding ablation alone does not strongly reduce the in vivo natural
tropism of the adenovirus vector, the additional ablation of
binding sites with integrin and heparan sulfate proteoglycans from
the adenoviral capsid should be useful to further reduce nave
tropism . Furthermore, it was reported that coagulation factor
(F) X directly binds the hypervariable regions (HVR) of the hexon
surface in an adenovirus, leading to liver infection . A targeted
adenovirus constructed on a mutant HVR backbone to suppress
a liver transduction might effectively allow for the development of
vectors to specifically transduce certain tumors even through
Although there was a possibility to isolate peptide sequences
that can generally enhance gene transduction in various tumors
and organs, a selected peptide showed AsPC-1 peritoneal tumor
type-directed infection (Figs. 4 and 5a), which was consistent with
our previous in vitro results [14,15], suggesting that library
screening based on the binding to target tumor cells may enable
identification of tumor specific-targeting ligands. However, it may
be useful to incorporate a negative selection method in the early
screening phase in addition to the positive selection. We are
developing 2 strategies for negative selection: one is the absorption
of vectors, which show high infectivity for many cells, upon the
Figure 5. Infectivity of organs with the adenovirus displaying the selected peptide. (a) Infectivity of organs with adenovirus vectors. The
mice were injected intraperitoneally with 16108 PFU of adenovirus vectors (AdDCAR-LucEGFP-PFW, AdDCAR-LucEGFP-SYE, AdDCAR-LucEGFP or
AdLucEGFP)(n = 3,5), and 2 days after the injection, organs such as the liver, spleen, pancreas and small intestine were harvested and the luciferase
activities were measured. (b) Distribution of adenovirus DNA after intraperitoneal injection of adenovirus vectors. DNAs from AsPC-1 tumors and
organs (n = 3) were analyzed by a real-time quantitative PCR for the adenovirus E4 copy number. The results are shown as adenoviral copy number
per 1 mg of tissue DNA.
mixture of various normal cells before the screening on the target
cells. The other strategy is in vivo systemic screening of the library
to specifically transduce tumors using animal models, since normal
tissues may absorb the vectors with generally high infectivity. In
fact, Wu et al. isolated an adenovirus vector targeting to
prostatespecific membrane antigen (PSMA) through virus-displayed
semirandom peptide display screening by counter and positive
selections with PSMA-expressing cancer cells and a systemic
injection in a tumor mouse model .
A substantial competitive inhibition of
AdDCAR-LucEGFPPFW by the cognate peptide suggests that the infection of targeted
vectors was mediated by receptors on target cells. The
corresponding target receptor of the isolated ligand may be a specific cell
surface molecule or its expression may be significantly higher on
the AsPC-1 tumors. The identification of the receptors would be
useful to understand the molecular characteristics of tumors and
can be applied for diagnosis, such as the detection of a relapse, and
for therapy of the disease. However, database searches (BLAST)
did not reveal sequence homology of the selected peptide with
known human proteins. Thus, the candidate receptor responsible
for the PFWSGAV-mediated infection in AsPC-1 tumors is
unknown. Additional work is necessary to identify the
Recently, promising preclinical and clinical data on the
treatment of cancer using a conditionally replication-competent
adenovirus (CRAd) have been reported [24,25]. In this study, we
screened a peptide-display adenovirus library on pancreatic
tumors to explore targeting ligands for pancreatic peritoneal
dissemination, which is often recognized at advanced stages and
may be a clinical target of local oncolytic virus therapy. Although
the infection and spreading of such CRAds are restricted to tumor
tissue in theory, several levels of safety devices are definitely
required in the clinical setting. Therefore, the combination of
CRAds with target specificity is a highly encouraging strategy for
gene therapy. Furthermore, we previously reported that the
intratumoral injection of a replication-competent adenovirus
displaying a selected ligand showed a higher oncolytic potency
when compared with the untargeted adenovirus vector in
subcutaneous tumor models . The antitumor effect of an
oncolytic virus is determined by the capacity to infect tumor cells
[26,27]. Therefore, we expect that the insertion of the PFWSGAV
sequence into the replication-competent adenovirus vector could
enhance the oncolytic activity for AsPC-1 peritoneal tumors. A
library approach with a replication-competent adenovirus may be
highly useful to isolate a targeted oncolytic adenovirus, because the
most efficient adenovirus should be selected from the library based
on its high infectivity and replication capacity through the process
of virus amplification and its spread through target tumors.
Future development will be a screening on the biopsy and
surgical materials of the tumor, thereby leading to a generation of
individualized targeted viruses. On the other hand, extensive
exploration of target peptides with the in vitro and in vivo screening
of an adenovirus library may allow the making of a list of targeting
peptides for each cancer, and it may be more convenient to select
a targeted vector suitable for an individual patient from the list.
This library-based technology for a specific adenovirus vector
selection may have broad implications for a variety of applications
in medicine and medical sciences.
T Nishimoto is an awardee of a Research Resident Fellowship from the
Foundation (Japan) for the Promotion of Science.
Conceived and designed the experiments: KA SO. Performed the
experiments: TN YY KY NG. Analyzed the data: TN YY KY NG KA.
Contributed reagents/materials/analysis tools: TN YY KY. Wrote the
paper: YY KA.
1. Volpers C , Kochanek S ( 2004 ) Adenoviral vectors for gene transfer and therapy . J Gene Med 6 Suppl 1 : S164 - 171 .
2. Bergelson JM , Cunningham JA , Droguett G , Kurt-Jones EA , Krithivas A , et al. ( 1997 ) Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5 . Science 275 : 1320 - 1323 .
3. Tomko RP , Xu R , Philipson L ( 1997 ) HCAR and MCAR: the human and mouse cellular receptors for subgroup C adenoviruses and group B coxsackieviruses . Proc Natl Acad Sci U S A 94 : 3352 - 3356 .
4. Khare R , Chen CY , Weaver EA , Barry MA ( 2011 ) Advances and future challenges in adenoviral vector pharmacology and targeting . Curr Gene Ther 11 : 241 - 258 .
5. Yao XL , Nakagawa S , Gao JQ ( 2011 ) Current targeting strategies for adenovirus vectors in cancer gene therapy . Curr Cancer Drug Targets 11 : 810 - 825 .
6. Krasnykh V , Dmitriev I , Mikheeva G , Miller CR , Belousova N , et al. ( 1998 ) Characterization of an adenovirus vector containing a heterologous peptide epitope in the HI loop of the fiber knob . J Virol 72 : 1844 - 1852 .
7. Dmitriev I , Krasnykh V , Miller CR , Wang M , Kashentseva E , et al. ( 1998 ) An adenovirus vector with genetically modified fibers demonstrates expanded tropism via utilization of a coxsackievirus and adenovirus receptor-independent cell entry mechanism . J Virol 72 : 9706 - 9713 .
8. Yoshida Y , Sadata A , Zhang W , Saito K , Shinoura N , et al. ( 1998 ) Generation of fiber-mutant recombinant adenoviruses for gene therapy of malignant glioma . Hum Gene Ther 9 : 2503 - 2515 .
9. Douglas JT , Miller CR , Kim M , Dmitriev I , Mikheeva G , et al. ( 1999 ) A system for the propagation of adenoviral vectors with genetically modified receptor specificities . Nat Biotechnol 17 : 470 - 475 .
10. Nicklin SA , Von Seggern DJ , Work LM , Pek DC , Dominiczak AF , et al. ( 2001 ) Ablating adenovirus type 5 fiber-CAR binding and HI loop insertion of the SIGYPLP peptide generate an endothelial cell-selective adenovirus . Mol Ther 4 : 534 - 542 .
11. Joung I , Harber G , Gerecke KM , Carroll SL , Collawn JF , et al. ( 2005 ) Improved gene delivery into neuroglial cells using a fiber-modified adenovirus vector . Biochem Biophys Res Commun 328 : 1182 - 1187 .
12. Nicklin SA , White SJ , Nicol CG , Von Seggern DJ , Baker AH ( 2004 ) In vitro and in vivo characterisation of endothelial cell selective adenoviral vectors . J Gene Med 6 : 300 - 308 .
13. Muller OJ , Kaul F , Weitzman MD , Pasqualini R , Arap W , et al. ( 2003 ) Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors . Nat Biotechnol 21 : 1040 - 1046 .
14. Miura Y , Yoshida K , Nishimoto T , Hatanaka K , Ohnami S , et al. ( 2007 ) Direct selection of targeted adenovirus vectors by random peptide display on the fiber knob . Gene Ther 14 : 1448 - 1460 .
15. Nishimoto T , Yoshida K , Miura Y , Kobayashi A , Hara H , et al. ( 2009 ) Oncolytic virus therapy for pancreatic cancer using the adenovirus library displaying random peptides on the fiber knob . Gene Ther 16 : 669 - 680 .
16. Lupold SE , Kudrolli TA , Chowdhury WH , Wu P , Rodriguez R ( 2007 ) A novel method for generating and screening peptides and libraries displayed on adenovirus fiber . Nucleic Acids Res 35 : e138 .
17. Yamada H , Yoshida T , Sakamoto H , Terada M , Sugimura T ( 1986 ) Establishment of a human pancreatic adenocarcinoma cell line (PSN-1) with amplifications of both c-myc and activated c-Ki-ras by a point mutation . Biochem Biophys Res Commun 140 : 167 - 173 .
18. Mittereder N , March KL , Trapnell BC ( 1996 ) Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy . J Virol 70 : 7498 - 7509 .
19. Aoki K , Furuhata S , Hatanaka K , Maeda M , Remy JS , et al. ( 2001 ) Polyethylenimine-mediated gene transfer into pancreatic tumor dissemination in the murine peritoneal cavity . Gene Ther 8 : 508 - 514 .
20. Sagawa T , Takahashi M , Sato T , Sato Y , Lu Y , et al. ( 2004 ) Prolonged survival of mice with multiple liver metastases of human colon cancer by intravenous administration of replicable E1B-55K-deleted adenovirus with E1A expressed by CEA promoter . Mol Ther 10 : 1043 - 1050 .
21. Mizuguchi H , Koizumi N , Hosono T , Ishii-Watabe A , Uchida E , et al. ( 2002 ) CAR- or alphav integrin-binding ablated adenovirus vectors, but not fibermodified vectors containing RGD peptide, do not change the systemic gene transfer properties in mice . Gene Ther 9 : 769 - 776 .
22. Waddington SN , McVey JH , Bhella D , Parker AL , Barker K , et al. ( 2008 ) Adenovirus serotype 5 hexon mediates liver gene transfer . Cell 132 : 397 - 409 .
23. Wu P , Kudrolli TA , Chowdhury WH , Liu MM , Rodriguez R , et al. ( 2010 ) Adenovirus targeting to prostate-specific membrane antigen through virusdisplayed, semirandom peptide library screening . Cancer Res 70 : 9549 - 9553 .
24. Pesonen S , Kangasniemi L , Hemminki A ( 2011 ) Oncolytic adenoviruses for the treatment of human cancer: focus on translational and clinical data . Mol Pharm 8 : 12 - 28 .
25. Eager RM , Nemunaitis J ( 2011 ) Clinical development directions in oncolytic viral therapy . Cancer Gene Ther 18 : 305 - 317 .
26. Hemminki A , Dmitriev I , Liu B , Desmond RA , Alemany R , et al. ( 2001 ) Targeting oncolytic adenoviral agents to the epidermal growth factor pathway with a secretory fusion molecule . Cancer Res 61 : 6377 - 6381 .
27. Bauerschmitz GJ , Lam JT , Kanerva A , Suzuki K , Nettelbeck DM , et al. ( 2002 ) Treatment of ovarian cancer with a tropism modified oncolytic adenovirus . Cancer Res 62 : 1266 - 1270 .