By
From the * Division of Pulmonary and Critical Care Medicine, Department of Dermatology, § Division of Hematology-Oncology, The New York Hospital-Cornell Medical Center, New York
10021; and
James Ewing Laboratory of Developmental Hematopoiesis, Memorial Sloan-Kettering
Cancer Center, New York 10021
Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in the initiation
of antitumor immune responses. In this study, we show that genetic modifications of a murine
epidermis-derived DC line and primary bone marrow-derived DCs to express a model antigen
-galactosidase (
gal) can be achieved through the use of a replication-deficient, recombinant
adenovirus vector, and that the modified DCs are capable of eliciting antigen-specific, MHC-restricted CTL responses. Importantly, using a murine metastatic lung tumor model with syngeneic colon carcinoma cells expressing
gal, we show that immunization of mice with the
genetically modified DC line or bone marrow DCs confers potent protection against a lethal
tumor challenge, as well as suppression of preestablished tumors, resulting in a significant survival advantage. We conclude that genetic modification of DCs to express antigens that are also
expressed in tumors can lead to antigen-specific, antitumor killer cells, with a concomitant resistance to tumor challenge and a decrease in the size of existing tumors.
Many tumor cells express epitopes on their surface that
provide a potential target for therapeutic vaccine
strategies that boost natural immune-mediated tumor defense mechanisms (1). One of the challenges to developing antitumor vaccines is to develop strategies that focus
the immune response to the relevant tumor antigen. The
present study approaches this challenge by using replication-deficient, recombinant adenovirus (Ad)1 vectors to
transfer a gene coding for a model antigen ( In this context, there is emerging evidence that, in some
circumstances, Ad vectors can act as adjuvants to boost
CTL response against heterologous transgenes (4). This
evidence, together with the knowledge that genes expressed by Ad vectors are expressed through the class I
pathway (10, 11), and that DCs are the most potent members of the general class of antigen presenting cells (12),
led us to the hypothesis that genetically modifying DCs
with an Ad vector coding for a tumor antigen might help
focus the immune response to generating antigen-specific
CTLs.
With this background, the objectives of this study are to
assess the ability of Ad vectors to genetically modify DCs
ex vivo, and to assess the ability of DCs modified with
model antigens transferred to a syngeneic host to (a) induce
antigen-specific CTLs, (b) prevent tumors from developing
in animals challenged with tumor cells expressing the
model antigen, and (c) suppress the growth of preexisting
tumors composed of the same tumor cells. To accomplish
this, we used the XS52 BALB/c DC line (16, 17), as well
as freshly isolated DCs from bone marrow of BALB/c
mice. By using an Ad vector expressing Mice and Cell Lines.
6-8-wk-old male BALB/c (H-2d) mice
were obtained from the Jackson Lab. (Bar Harbor, ME). Animals
were housed under specific pathogen-free conditions and treated
according to National Institutes of Health guidelines.
-galactosidase;
gal) to dendritic cells (DCs) ex vivo, and administering
the genetically modified DCs to a syngeneic host. We
choose this approach to capitalize on the biologic properties of Ad vectors and DCs that are relevant to inducing antigen-specific CTLs directed against specific antigens.
gal and the
BALB/c syngeneic colon carcinoma cell line CT26.CL25
expressing
gal (18), the data dramatically demonstrate that
administration of DCs modified to express
gal induces
gal-specific CTLs, prevents tumor growth, and suppresses
the growth of preexisting tumors.
gal gene (both cell lines provided
by N.P. Restifo, National Cancer Institute, Bethesda, MD; reference 18). E22 cells, a clone of mouse thymoma EL4 (H-2b) cell
line transduced with the
gal gene, was used as a negative control
in the CTL assay (E22 was provided by Y. Paterson, University of Pennsylvania, Philadelphia, PA; reference 18). The SVBalb (H-2d) fibroblast cell line was used as in vitro stimulator cells in the CTL assay; this cell line is syngeneic to BALB/c mice (provided by L. Gooding, Emory University, Atlanta, GA; reference
19). CT26.WT and SVBalb were grown in complete RPMI media. CT26.CL25 and E22 were grown in complete RPMI containing 400 µg/ml G418 (GIBCO BRL, Gaithersburg, MD).
Generation of Dendritic Cells In Vitro from Bone Marrow. Primary bone marrow DCs were obtained from mouse bone marrow precursors as described by Inaba et al. (20). In brief, lymphocyte- and erythrocyte-depleted murine bone marrow cells harvested from femurs and tibias were plated in completed RPMI media supplemented with recombinant murine GM-CSF (100 U/ml) and recombinant murine interleukin 4 (20 ng/ml; Genzyme, Farmington, MA). On days 2 and 4, nonadherent granulocytes were gently removed and fresh media was added. On day 6, loosely adherent proliferating DC aggregates were dislodged and replated. On day 8 of culture, released, mature, nonadherent cells with the typical morphological features of DCs were used for in vitro phenotypic and functional analysis as well as for the immunization of mice. For phenotypic analysis, the nonadherent cells from the bone marrow culture were stained with a panel of antibodies characteristic of DCs, including CD11b (No. 01714D, PharMingen, San Diego, CA), CD44 (No. 01225A, PharMingen), and IAd (No. 06274D, PharMingen) and then quantified by flow cytometry. Isotype-matched monoclonal antibodies were used as controls. Bone marrow DCs were also tested for their ability to activate naive allogeneic T cells in vitro using a mixed lymphocyte reaction assay (20).
Ad Vector-mediated Gene Transfer and Expression in DCs In Vitro.
The replication-deficient Ad vectors used in this study are E1a,
partially E1b
, and partially E3
vectors based on human Ad5
(21, 22). The construction of these Ad vectors have been described previously, including vectors expressing no transgene
(AdNull) and the E. coli
gal gene (Ad
gal; reference 23). All Ad
vectors were propagated in 293 cells, purified by two rounds of
CsCl density centrifugation, dialyzed, and stored at
70°C as
previously described (21, 22). The titer of viral stock was determined by plaque-forming assay using 293 cells. All vector preparations were demonstrated to be free of replication competent adenovirus (24).
Induction of Antigen-specific CTLs.
To determine if the administration of DCs by in vitro infection with Adgal would evoke
gal-specific CTLs, the XS52 DC line or primary bone marrow
DCs were infected with Ad
gal or AdNull (moi 100) as described above. BALB/c mice were immunized once by subcutaneous injection of 3 × 105 modified DCs suspended in 100 µl
HBSS. Spleens were removed 14 d after DC inoculation, the in
vivo primed splenocytes were pooled and cocultured (2 × 106/
ml) with irradiated (5,000 rad) syngeneic SVBalb cells (2 × 105/
ml) pulsed with the synthetic
gal peptide (1 µM) in a 6-well plate (5 ml/well). After 5 d of coculture, the in vitro restimulated viable splenocytes were assayed in a 51Cr-release assay for specific
cell lysis against a variety of target cells, including syngeneic cell
lines CT26.WT,
gal-expressing CT26.CL25, and
gal peptide-pulsed CT26.WT, as well as a MHC-mismatched,
gal-expressing
E22 (H-2b) cell line. The
gal peptide used in the CTL studies is
the nanomer TPHPARIGL (residues 876-885) that is naturally
presented by the H-2 Ld molecule (26). The peptide was synthesized (Biosynthesis, Lewisville, TX) to a purity >99% as determined by high pressure liquid chromatography and amino acid
analysis. The 51Cr-release assay was performed as previously described (9, 27).
Protection against Lethal Tumor Challenge after XS52 DC Immunization.
To evaluate the ability of the XS52 DC line infected
with Adgal to protect against a tumor challenge, 6-8-wk-old
BALB/c mice were immunized with 3 × 105 Ad
gal-modified
(moi 100, 24 h, 37°C) XS52 (referred to as XS52-Ad
gal) in 100 µl HBSS injected subcutaneously in the left lower quadrant of
the ventral abdominal wall (n = 4). Control animals received either no XS52 (n = 5), XS52 infected with AdNull (XS52-AdNull; n = 2), or XS52-Ad
gal that were frozen and thawed four times before injection to lyse the cells (n = 5). 2 wk later, naive and
XS52-immunized mice were challenged with syngeneic 105
CT26.CL25 murine colon carcinoma cells injected intravenously through the jugular vein. This model generated multiple diffuse pulmonary metastases that were highly lethal. 12-16 d after tumor challenge, the mice were killed, their lungs harvested, fixed in 4% paraformaldehyde, and stained for
gal expression with
X-Gal (5-bromo-4-chloro-3-indolyl-
-D-galactopyranoside). To
quantify the tumor burden, the number of blue staining lung metastases was carefully counted in a blinded fashion under a dissecting microscope. Only metastatic deposits
250 could be reliably
enumerated; lungs with >250 deposits were assigned an empirical
number of 250.
Suppression of Preestablished Lung Metastases by XS52 DC Line
Immunization.
To mimic the clinical scenario more closely, we
evaluated whether preexisting lung metastases could be suppressed after immunization with XS52 cell line modified with
Adgal (n = 4) or AdNull (n = 4). BALB/c mice were first injected with 3 × 104 CT26.CL25 tumor cells intravenously to
generate multiple lung metastases. 3 d later, tumor-bearing mice
were either left untreated, or treated with subcutaneous injections
of XS52-AdNull (n = 4), XS52-Ad
gal (n = 4), or XS52-Ad
gal that were frozen and thawed four times to lyse the cells (n = 3). 20 d after tumor implantation, the animals were killed and the
lungs evaluated for the presence of metastases in a similar fashion
as described in the prevention experiment.
Protection against Lethal Tumor Challenge after Bone Marrow DC
Immunization.
To evaluate the ability of bone marrow DCs infected with Adgal to protect against a tumor challenge, 6-8-wk-old
BALB/c mice (n = 8/group) were immunized with 3 × 105
bone marrow DC-Ad
gal in 100 µl HBSS injected subcutaneously in the left lower quadrant of the ventral abdominal wall.
Control animals received either no bone marrow DCs, bone
marrow DCs infected with AdNull (bone marrow DC-AdNull),
or bone marrow DC-Ad
gal that were frozen and thawed four
times before injection to lyse the cells. 2 wk later, naive and immunized mice were challenged with syngeneic 105 CT26.CL25
murine colon carcinoma cells injected intravenously through the
jugular vein. The mice were then followed for survival. In addition, to evaluate if the antitumor immunity was
gal specific, another group of mice (n = 8) immunized with bone marrow DC-
Ad
gal was challenged with CT26.WT tumor cells that did not
express
gal and followed for survival.
Suppression of Preestablished Lung Metastases by Bone Marrow DC
Immunization.
To mimic the clinical scenario more closely, we
evaluated whether preexisting lung metastases could be suppressed after immunization with gal-expressing bone marrow
DCs. BALB/c mice were first injected with 3 × 104 CT26.CL25
tumor cells intravenously to generate multiple lung metastases. 3 d
later, tumor-bearing mice (n = 8/group) were either left untreated, or treated with subcutaneous injections of bone marrow
DC-AdNull, bone marrow DC-Ad
gal, or bone marrow DC-
Ad
gal that were frozen and thawed four times to lyse the cells.
The mice were then followed for survival. To evaluate if the
treatment effect was
gal specific, a group of mice (n = 8) with
preestablished CT26.WT tumors that did not express
gal was
treated with bone marrow DC-Ad
gal and followed for survival.
Statistical Analysis. All animal experiments used a minimum of four animals except for the XS52 prevention experiment in which the XS52-AdNull group had only two animals (inadvertently, two animals in this group were left out of the study; in a repeat study, this group had five animals). All studies were repeated at least twice with similar results. Quantitative results are expressed as mean ± standard error of the mean. Statistical analysis was performed using the unpaired two-tailed Student's t test, with the exception of the survival data which was analyzed using the log-rank test (28).
To evaluate
the ability of Ad vectors to transfer and express genes in
XS52 DCs, XS52 DCs were infected with Adgal, and the cells were quantified for
gal activity at 24 h after infection by flow cytometry. Although naive XS52 (Fig. 1, A) and
AdNull-infected XS52 (B) showed only background levels
of
gal activity, expression of
gal was readily detected in
the majority (73%) of XS52 infected with Ad
gal at moi of
100 (C), demonstrating that successful gene transfer and expression can be achieved in DCs using an Ad vector. Parallel studies carried out using other moi demonstrated 14% XS52 cells expressing
gal with a moi of 30 and 62% at a
moi of 300 (not shown). On the basis of these studies, all
subsequent studies were carried out with a moi of 100.
Induction of CTLs Using Ad Vector-transduced XS52 Cells.
To determine if Ad-mediated gene expression in
DCs would induce an antigen-specific CTL response in
vivo, BALB/c mice were first immunized with 3 × 105
XS52 cells that had been modified in vitro with AdNull- or
Adgal-infection. Splenocytes from immunized mice were
then harvested and stimulated in vitro with
gal peptide
and were then assayed for specific cell lysis against a panel
of target cells in a standared 51Cr-release assay. Although
immunization of naive XS52 or XS52-AdNull infected
cells did not result in a measurable
gal-specific CTL response (Fig. 2, A), significant lysis of
gal-expressing
CT26.CL25 target cells or
gal peptide-pulsed CT26.WT
target cells were observed in mice immunized with XS52-Ad
gal (B). Only negligible lysis of non-
gal-expressing
CT26.WT target cells was detected in mice immunized
with XS52-Ad
gal, indicating that the cellular immune response induced by XS52-Ad
gal was
gal specific. This specific cytotoxicity was mediated predominantly by MHC-restricted CTLs rather than by natural killer cells since E22,
an MHC-mismatched (H-2b),
gal-expressing murine tumor cell line, was not significantly lysed in the same assay.
Protection against Lethal Tumor Challenge after Immunization with the Ad
Between 12 and
16 d after an intravenous tumor challenge, tumor-bearing
mice which were not treated showed signs of respiratory distress and developed hemorrhagic pleural effusion and
diffuse lung metastases (Fig. 3, A). Similarly, mice which
were previously immunized with XS52-AdNull developed
numerous lung metastases (B). In marked contrast, mice
which were previously immunized with XS52-Adgal
showed very few visible lung metastases under the dissecting microscope (C and D), indicating that immunization of
mice with DCs genetically modified to express the
gal
model antigen could effectively protect against a lethal tumor challenge. Quantitative analysis of the number of lung
metastases confirmed the significant difference between XS52-Ad
gal-immunized mice and untreated mice (P <0.0001)
or XS52-AdNull-immunized mice (E). Further, immunization with XS52-Ad
gal that had been frozen and thawed
did not confer protection when compared to untreated and XS52-AdNull-immunized groups (P >0.1; not shown),
i.e., live DCs were a prerequisite to generate an effective
antitumor immunity.
The metastatic lung tumor model in this study was
highly lethal, such that mice immunized with the negative
control DCs (XS52-AdNull) succumbed to advanced pulmonary malignancy an average of two wk after tumor
implantation (Fig. 4). In contrast, mice which had been preimmunized with XS52-Adgal exhibited a markedly prolonged survival (P <0.0001, Fig. 4), consistent with the
findings that XS52-
gal-immunized mice had far fewer
lung metastases than the control treatment groups after a
tumor challenge. Importantly, no protection was conferred
by XS52-Ad
gal immunization against CT26.WT tumor
cells which did not express
gal (P >0.06; not shown), suggesting that the antitumor immunity generated by administration of the Ad
gal-modified DCs was antigen (
gal)-
specific.
Suppression of Preestablished Lung Metastases after XS52-Ad
To evaluate if tumor protection after
DC immunization could be extended to a more stringent
and clinically relevant model, Adgal-modified XS52 were
used to treat mice with preexisting tumors. In this experiment, diffuse lung metastases were first generated in
BALB/c mice by intravenous administration of 3 × 104
CT26.CL25 tumor cells. 3 d after tumor implantation, the
mice were either left untreated or were treated with subcutaneous administration of 3 × 105 XS52-AdNull modified
cells or XS52-Ad
gal-modified cells. Tumor-bearing mice
that were not immunized (Fig. 5, A) or those that were immunized with the control DCs (XS52-AdNull; B) exhibited multiple lung metastases 20 d after tumor implantation.
In contrast, tumor-bearing mice which received XS52-Ad
gal treatment demonstrated a striking reduction in the
number of metastases (C and D), demonstrating that DCs
genetically modified to express a tumor antigen could effectively suppress preexisting tumors. Quantitative analysis of the number of metastatic nodules confirmed the significant treatment differences between XS52-Ad
gal-treated
group and the control groups (P <0.009; E). In contrast,
XS52-Ad
gal cells that had been lysed by repeated freezing
and thawing did not confer any therapeutic effect when compared to untreated or XS52-AdNull-treated animals (P >0.4;
not shown).
In cancer treatment, any observed treatment response
becomes even more meaningful if it translates into a survival advantage. To evaluate if the reduction in the number
of lung metastases in mice treated with XS52-Adgal conferred a survival advantage, a group of animals were followed for survival. Tumor-bearing mice that were treated
with XS52-Ad
gal lived significantly longer than the animals in the negative control groups (P <0.002, Fig. 6).
Consistent with the observation in the prevention experiment, XS52-Ad
gal treatment did not suppress preestablished non-
gal-expressing CT26.WT tumors (not shown),
again suggesting that the therapeutic effect is antigen
(
gal)-specific (P >0.2). Further, lysed XS52-Ad
gal cells
did not confer a survival advantage as compared to untreated mice or mice treated with XS52-AdNull (P >0.8;
not shown).
Adenovirus Vector Modification of Primary Bone Marrow DCs.
To evaluate if the observations made using XS52 cell line could also be reproduced using primary DCs, DC modification, tumor prevention, and tumor treatment experiments were carried out similar to that with the XS52 DC line. Bone marrow DC from BALB/c mice were isolated and expanded ex vivo for 7 d, followed by their characterization using composite criteria of typical morphology, cell surface markers, and functional mixed lymphocyte reaction assay. Cell surface phenotypic analysis by flow cytometry revealed high frequency of expression of CD11b (two separate preparations, 72, 73%), CD44 (98, 99%), and MHC class II antigen IAd (75, 80%; not shown). Consistent with the known function of DCs, as few as 100 DCs induced an allogenic T lymphocyte proliferation in a mixed lymphocyte reaction with stimulation indices of >20 (not shown).
To evaluate the ability of Ad vectors to transfer and express genes in primary bone marrow DCs, Adgal was used
to infect bone marrow DCs an moi of 100, and the cells
were quantified for
gal activity at 24 h after infection by
flow cytometry. Similar to the findings with XS52-Ad
gal
(Fig. 1, C), expression of
gal was readily detectable (95%)
in bone marrow DCs infected with Ad
gal (Fig. 7, C), but
not in naive bone marrow DCs (A) or bone marrow DCs
infected with AdNull (B), demonstrating that Ad vectors can transfer and express genes in primary DCs. Parallel
studies carried out using other moi demonstrated 17% bone
marrow DC cells expressing
gal with an moi of 30 and
96% at an moi of 300 (not shown). Based on these studies,
all subsequent studies were carried out at an moi 100.
Induction of CTL Response In Vivo Using Ad Vector -transduced Primary Bone Marrow DCs.
To evaluate if genetically modified primary bone marrow DCs would induce an
antigen-specific CTL response in vivo, splenocytes from mice
immunized with subcutaneous administration of bone marrow DCs transduced with Adgal were stimulated in vitro
with
gal peptide and were then evaluated for specific cell
lysis against a panel of target cells in a standard 51Cr-release
assay. Immunization with bone marrow DC-Ad
gal cells induced a
gal-specific CTL response (Fig. 8, B), whereas
the negative control bone marrow DC-AdNull cells did
not (A). Only negligible lysis of non-
gal-expressing
CT26.WT target cells were detected in mice immunized
with bone marrow DC-Ad
gal cells, indicating that the
cellular immune response induced was
gal specific. This specific cytotoxicity was mediated predominately by MHC-restricted CTLs rather than by natural killer cells since E22,
an MHC-mismatched (H-2b)
gal-expressing murine tumor cell line, was not significantly lysed in the same assay.
Prevention against Lethal Tumor Challenge after Bone Marrow DC Immunization.
Consistent with the studies using XS52-Adgal modified DCs, BALB/c mice that were immunized
with bone marrow DC-Ad
gal before challenge with tumor cells were effectively protected against the tumor challenge, as evidenced by a prolonged survival in the bone
marrow DC-Ad
gal group compared to the bone marrow
DC-AdNull group (P <0.0001, Fig. 9). In contrast, mice
immunized with bone marrow DC-Ad
gal were not protected against a challenge by CT26.WT cells (P >0.9; not
shown), indicating that the immune response was
gal specific.
Suppression of Preestablished Lung Metastases by Bone Marrow DC Immunization.
Mice with preexisting lung metastases
that received bone marrow DC-Adgal treatment lived
significantly longer than the bone marrow DC-AdNull
control group (P <0.002, Fig. 10), demonstrating that primary DCs that are genetically modified to express the tumor antigen could suppress preestablished tumors. As in the prevention experiment, bone marrow DC-Ad
gal immunization had no therapeutic effect on CT26.WT tumors
that did not express
gal (P >0.5; not shown), indicating
that the antitumor effect was tumor antigen specific. Similarly, lysed bone marrow DCs were ineffective in prolonging survival compared to control groups (P >0.1; not
shown).
This study evaluates the use of an adenovirus gene transfer vector to deliver a model antigen into DCs in vitro, and
the ability of subsequent in vivo administration of these genetically modified DCs to induce tumor antigen-specific
antitumor immunity. The data demonstrate that an Ad
vector encoding gal as a model antigen can effectively
transfer and express the transgene in a DC line and primary
bone marrow DCs. In vivo administration of these modified DCs elicits an MHC-restricted, antigen-specific CTL
response, protects syngeneic mice against tumor challenge,
and suppresses preexisting lethal tumors. Importantly, a reduction in tumor burden in all studies was translated into
significant survival advantage. These observations underscore the ability of genetically modified DCs to effectively
present tumor antigens and induce immunity against tumors. With the discovery and definition of more tumor antigens, this strategy of genetic manipulation of DCs to express specific tumor antigens may provide an effective
treatment for an increasing number of malignancies.
DCs are the most potent professional antigen presenting cells that are capable of initiating T cell-dependent immune responses (12). Strategies that have been used to adapt this biologic potential to tumor therapy includes manipulating DCs by coculturing (pulsing) DCs with whole tumor antigens, defined tumor antigenic peptides, or total RNA from tumor cells (29). Using such approaches, it has been shown that effective antigen-specific immunity and antitumor responses can be elicited (29). Genetically modifying DCs by genes coding for specific tumor antigens to DCs has potential advantages. First, the genetically modified DCs may present previously unknown epitopes in association with different MHC molecules. Second, gene transfer to DCs ensures that the gene product is endogenously processed, leading to the generation of MHC class I-restricted CTLs which is the effector arm of cell-mediated immune response against tumor cells. Third, by using viral vectors to modify DCs in vitro, there is far less administration of viral proteins, in contrast to direct in vivo vaccination with viral vectors, thus minimizing the generation of neutralizing antibodies that prevent repeat treatment (27, 39).
Theoretically, direct gene transfer to DCs has advantages in generating CTLs toward the protein coded by the transgene, in that the gene will be transferred to the DC nucleus, expressed, and presented in the context of class I MHC for the immune system to recognize as self or nonself (10, 11). In this regard, there is emerging evidence that when Ad vectors expressing heterologous transgenes are administered in vivo the Ad functions as an "adjuvant" to enhance cellular immune responses against the heterologous transgene product, including the generation of transgene product-specific CTLs (4). It is not known whether or not this is secondary to in vivo transfer of the transgene to DCs in the local milieu, or is by other mechanisms. However, independent of the process by which Ad vectors induce CTLs against heterologous transgenes, the data in the present study clearly demonstrate that when an Ad vector expressing a heterologous transgene is used to transfer the transgene to DCs in vitro, subsequent transfer of the genetically modified DCs to a syngeneic host will result in the induction of CTLs against the heterologous transgene product.
Different gene transfer approaches have been explored to
use genetically modified DCs in antitumor therapy (40-
44). Several groups have reported the successful transduction of human DCs in vitro by retrovirus vectors encoding
a model antigen (gal) and tumor antigens including the
melanoma MART-1 antigen and the human epithelial tumor antigen mucin (40, 41, 43). Brossart et al. (44) has recently shown that adenovirus and vaccinia virus vectors
could transfer a model antigen (ovalbumin) into murine
immortalized and primary DCs, and that the virus-infected DCs elicited stronger antigen-specific CTL response in
vivo than immunization with DCs which were pulsed with
ovalbumin peptide or protein. Importantly, immunization
of mice twice with the virus-infected immortalized DCs
protected the mice from a lethal challenge with tumor cells
expressing ovalbumin. Consistent with these observations, the present study demonstrated that a single immunization
of genetically modified DC line and primary DCs elicited
antigen-specific CTL responses in vivo that were highly effective in protecting the mice from a lethal tumor challenge. Importantly, our study also shows that treatment of
tumor-bearing mice with a single immunization of genetically modified DCs led to a marked suppression of the growth of established tumors. To further improve the therapeutic efficacy of this strategy, the codelivery of cytokine
gene and tumor antigen gene into DCs and/or repeat immunization of DCs may enhance the antigen-specific antitumor immunity.
In addition to demonstrating that Ad vector-transduced DCs could generate an antigen-specific CTL response that protected immunized animals against a tumor challenge, this study also showed that the genetically modified DCs effectively suppress preestablished, rapidly growing metastatic tumors. The latter observation is clinically relevant since patients have preexisting tumors at the time of presentation and the Achilles heel of conventional cancer treatment today remains the failure to effectively control metastatic disease. In this regard, one clinical scenario to which this study is particularly relevant is one of micrometastases which are often present at the time of primary detection of many malignancies such as breast, colon, and lung cancer (45, 46). A possible clinical strategy to approach this problem would be to modify autologous DCs from such patients using an Ad vector encoding the relevant tumor antigen, and then administering the genetically modified DCs as adjuvant treatment after primary therapy.
Address correspondence to Ronald G. Crystal, Division of Pulmonary and Critical Care Medicine, The New York Hospital-Cornell Medical Center, 520 East 70th Street, ST505, New York, New York 10021. Phone: 212-746-2258; FAX: 212-748-8383; E-mail: rgcryst{at}mail.med.cornell.edu
Received for publication 3 July 1997 and in revised form 7 August 1997.
These studies were supported, in part, by GenVec, Inc. (Rockville, MD) and the Will Rogers Memorial Fund (White Plains, NY). M.A.S. Moore is supported by the Gar Reichman Fund of the Cancer Research Institute (New York), S. Rafii is supported by a grant from the National Institutes of Health (K08-HL 02926), and Dorothy Rodbell Foundation for Sarcoma Research, (New York), R. Granstein is supported by a grant from the National Institutes of Health (R01-AR 40667) and a grant from the Edith C. Blum Foundation (New York), H.-L. Kong is supported, in part, by the Ministry of Health of Singapore, the Singapore National Medical Research Council, and the National University Hospital (Singapore, Republic of Singapore).We would like to thank Susan Cho and Philip L. Leopold for helpful advice and assistance in carrying out these studies, and N. Mohamed for help in preparation of the manuscript.
1. | Pardoll, D.M.. 1993. Cancer vaccines. Immunol. Today. 14: 310-316 [Medline]. |
2. | Houghton, A.N.. 1994. Cancer antigens: immune recognition of self and altered self. J. Exp. Med. 180: 1-4 [Medline]. |
3. | Boon, T., and P. van der Bruggen. 1996. Human tumor antigens recognized by T lymphocytes. J. Exp. Med. 183: 725-729 [Medline]. |
4. | Juillard, V., P. Villefroy, D. Godfrin, A. Pavirani, A. Venet, and J.G. Guillet. 1995. Long-term humoral and cellular immunity induced by a single immunization with replication-defective adenovirus recombinant vector. Eur. J. Immunol. 25: 3467-3473 [Medline]. |
5. | Chen, P.W., M. Wang, V. Bronte, Y. Zhai, S.A. Rosenberg, and N.P. Restifo. 1996. Therapeutic antitumor response after immunization with a recombinant adenovirus encoding a model tumor-associated antigen. J. Immunol. 156: 224-231 [Abstract]. |
6. | Xiang, Z.Q., Y. Yang, J.M. Wilson, and H.C. Ertl. 1996. A replication-defective human adenovirus recombinant serves as a highly efficacious vaccine carrier. Virology. 219: 220-227 [Medline]. |
7. | Flanagan, B., C.R. Pringle, and K.N. Leppard. 1997. A recombinant adenovirus expressing the simian immunodeficiency virus Gag antigen can induce long-lived immune responses in mice. J. Gen. Virol. 78: 991-997 [Abstract]. |
8. | Rodriques, E.G., F. Zavala, D. Eichinger, J.M. Wilson, and M. Tsuji. 1997. Single immunizing dose of recombinant adenovirus efficiently induces CD8+ T cell-mediated protective immunity against malaria. J. Immunol. 158: 1268-1274 [Abstract]. |
9. | Song, W., H. Kong, P. Traktman, and R.G. Crystal. 1997. Cytotoxic T lymphocyte responses to proteins encoded by heterologous transgenes transferred in vivo by adenoviral vectors. Hum. Gene Ther. 8: 1207-1217 [Medline]. |
10. | Germain, R.N.. 1994. MHC-dependent antigen processing and peptide presentation: providing ligands for T lymphocyte activation. Cell. 76: 287-299 [Medline]. |
11. | Bevan, M.J.. 1995. Antigen presentation to cytotoxic T lymphocytes in vivo. J. Exp. Med. 182: 639-641 [Medline]. |
12. | Steinman, R.M.. 1991. The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9: 271-296 [Medline]. |
13. | Grabbe, S., S. Beissert, T. Schwarz, and R.D. Granstein. 1995. Dendritic cells as initiators of tumor immune responses: a possible strategy for tumor immunotherapy? Immunol. Today. 16: 117-121 [Medline]. |
14. | Young, J.W., and K. Inaba. 1996. Dendritic cells as adjuvants for class I major histocompatibility complex-restricted antitumor immunity. J. Exp. Med. 183: 7-11 [Medline]. |
15. | Steinman, R.M.. 1997. Dendritic cells and immune-based therapies. Exp. Hematol. (NY). 24: 859-862 . |
16. | Xu, S., P.R. Bergstresser, and A. Takashima. 1995. Phenotypic and functional heterogeneity among murine epidermal-derived dendritic cell clones. J. Invest. Dermatol. 105: 831-836 [Abstract]. |
17. |
Xu, S.,
K. Ariizumi,
G. Caceres-Dittmar,
D. Edelbaum,
K. Hashimoto,
P.R. Bergstresser, and
A. Takashima.
1995.
Successive generation of antigen-presenting, dendritic cell lines
from murine epidermis.
J. Immunol.
154:
2697-2705
|
18. |
Wang, M.,
V. Bronte,
P.W. Chen,
L. Gritz,
D. Panicali,
S.A. Rosenberg, and
N.P. Restifo.
1995.
Active immunotherapy
of cancer with a nonreplicating recombinant fowlpox virus
encoding a model tumor-associated antigen.
J. Immunol.
154:
4685-4692
|
19. |
Rawle, F.C.,
B.B. Knowles,
R.P. Ricciardi,
V. Brahmacheri,
P. Duerksen-Hughes,
W.S. Wold, and
L.R. Gooding.
1991.
Specificity of the mouse cytotoxic T lymphocyte response to
adenovirus 5. E1a is immunodominant in H-2b, but not in
H-2d or H-2k mice.
J. Immunol.
146:
3977-3984
|
20. | Inaba, K., M. Inaba, M. Deguchi, K. Hagi, R. Yasumizu, S. Ikehara, S. Muramatsu, and R.M. Steinman. 1993. Granulocytes, macrophages, and dendritic cells arise from a common major histocompatibility complex class II-negative progenitor in mouse bone marrow. Proc. Natl. Acad. Sci. USA. 90: 3038-3042 [Abstract]. |
21. | Rosenfeld, M.A., W. Siegfried, K. Yoshimura, K. Yoneyama, M. Fukayama, L.E. Stier, P.K. Paakko, P. Gilardi, L.D. Stratford-Perricaudet, M. Perricaudet, et al . 1991. Adenovirus-mediated transfer of a recombinant alpha 1-antitrypsin gene to the lung epithelium in vivo. Science (Wash. DC). 252: 431-434 [Medline]. |
22. | Rosenfeld, M.A., K. Yoshimura, B.C. Trapnell, K. Yoneyama, E.R. Rosenthal, W. Dalemans, M. Fukayama, J. Bargon, L.E. Stier, L. Stratford-Perricaudet, et al . 1992. In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium. Cell. 68: 143-155 [Medline]. |
23. | Hersh, J., R.G. Crystal, and B. Bewig. 1995. Modulation of gene expression after replication-deficient, recombinant adenovirus-mediated gene transfer by the product of a second adenovirus vector. Gene Ther. 2: 124-131 [Medline]. |
24. | Crystal, R.G., N.G. McElvaney, M.A. Rosenfeld, C.S. Chu, A. Mastrangeli, J.G. Hay, S.L. Brody, H.A. Jaffe, N.T. Eissa, and C. Danel. 1994. Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis. Nat. Genet. 8: 42-51 [Medline]. |
25. | MacGregor, G.R., G.P. Nolan, S. Fiering, M. Roederer, and
L.A. Herzenberg. 1991. Use of E. coli lacZ (![]() |
26. |
Gavin, M.A.,
M.J. Gilbert,
S.R. Riddell,
P.D. Greenberg, and
M.J. Bevan.
1993.
Alkali hydrolysis of recombinant proteins allows for the rapid identification of class I MHC-restricted
CTL epitopes.
J. Immunol.
151:
3971-3980
|
27. | Mack, C.A., W.R. Song, H. Carpenter, T.J. Wickham, I. Kovesdi, B.G. Harvey, C.J. Magovern, O.W. Isom, T. Rosengart, E. Falck-Pedersen, et al . 1997. Circumvention of anti-adenovirus neutralizing immunity by administration of an adenoviral vector of an alternate serotype. Hum. Gene Ther. 8: 99-109 [Medline]. |
28. | Peto, R., M.C. Pike, P. Armitage, N.E. Breslow, D.R. Cox, S.V. Howard, N. Mantel, K. McPherson, J. Peto, and P.G. Smith. 1977. Design and analysis of randomized clinical trials requiring prolonged observation of each patient. II. Analysis and examples. Br. J. Cancer. 35: 1-39 [Medline]. |
29. |
Grabbe, S.,
S. Bruvers,
R.L. Gallo,
T.L. Knisely,
R. Nazareno, and
R.D. Granstein.
1991.
Tumor antigen presentation
by murine epidermal cells.
J. Immunol.
146:
3656-3661
|
30. | Mayordomo, J.I., T. Zorina, W.J. Storkus, L. Zitvogel, C. Celluzzi, L.D. Falo, C.J. Melief, S.T. Ildstad, W.M. Kast, A.B. DeLeo, et al . 1995. Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity. Nat. Med. 1: 1297-1302 [Medline]. |
31. | Ossevoort, M.A., M.C. Feltkamp, K.J. van Veen, C.J. Melief, and W.M. Kast. 1995. Dendritic cells as carriers for a cytotoxic T-lymphocyte epitope-based peptide vaccine in protection against a human papillomavirus type 16-induced tumor. J. Immunother. Emphasis Tumor Immunol. 18: 86-94 [Medline]. |
32. | Boczkowski, D., S.K. Nair, D. Snyder, and E. Gilboa. 1996. Dendritic cells pulsed with RNA are potent antigen-presenting cells in vitro and in vivo. J. Exp. Med. 184: 465-472 [Abstract]. |
33. | Celluzzi, C.M., J.I. Mayordomo, W.J. Storkus, M.T. Lotze, and L.D. Falo. 1996. Peptide-pulsed dendritic cells induce antigen-specific, CTL-mediated protective tumor immunity. J. Exp. Med. 183: 283-287 [Abstract]. |
34. | Hsu, F.J., C. Benike, F. Fagnoni, T.M. Liles, D. Czerwinski, B. Taidi, E.G. Engleman, and R. Levy. 1996. Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells. Nat. Med. 2: 52-58 [Medline]. |
35. | Mayordomo, J.I., D.J. Loftus, H. Sakamoto, C.M. De Cesare, P.M. Appasamy, M.T. Lotze, W.J. Storkus, E. Appella, and A.B. De Leo. 1996. Therapy of murine tumors with p53 wild-type and mutant sequence peptide-based vaccines. J. Exp. Med. 183: 1357-1365 [Abstract]. |
36. | Paglia, P., C. Chiodoni, M. Rodolfo, and M.P. Colombo. 1996. Murine dendritic cells loaded in vitro with soluble protein prime cytotoxic T lymphocytes against tumor antigen in vivo. J. Exp. Med. 183: 317-322 [Abstract]. |
37. | Porgador, A., D. Snyder, and E. Gilboa. 1996. Induction of antitumor immunity using bone marrow-generated dendritic cells. J. Immunol. 156: 2918-2926 [Abstract]. |
38. | Zitvogel, L., J.I. Mayordomo, T. Tjandrawan, A.B. DeLeo, M.R. Clarke, M.T. Lotze, and W.J. Storkus. 1996. Therapy of murine tumors with tumor peptide-pulsed dendritic cells: dependence on T cells, B7 costimulation, and T helper cell 1-associated cytokines. J. Exp. Med. 183: 87-97 [Abstract]. |
39. | Yang, Y., Q. Li, H.C.J. Ertl, and J.M. Wilson. 1995. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J. Virol. 69: 2004-2015 [Abstract]. |
40. | Henderson, R.A., M.T. Nimgaonkar, S.C. Watkins, P.D. Robbins, E.D. Ball, and O.J. Finn. 1996. Human dendritic cells genetically engineered to express high levels of the human epithelial tumor antigen mucin (MUC-1). Cancer Res. 56: 3763-3770 [Abstract]. |
41. | Reeves, M.E., R.E. Royal, J.S. Lam, S.A. Rosenberg, and P. Hwu. 1996. Retroviral transduction of human dendritic cells with a tumor-associated antigen gene. Cancer Res. 56: 5672-5677 [Abstract]. |
42. | Wan, Y., J. Bramson, R. Cater, F. Graham, and J. Gauldie. 1996. Dendritic cells transfected with adenovirus vectors encoding a model tumor-associated antigen for tumor immunotherapy. Cold Spring Harbor-Gene Therapy Meeting, Cold Spring Harbor, NY. 316. (Abstr.). |
43. | Aicher, A., J. Westermann, S. Cayeux, G. Willimsky, K. Daemen, T. Blankenstein, W. Uckert, B. Dorken, and A. Pezzutto. 1997. Successful retroviral mediated transduction of a reporter gene in human dendritic cells: feasibility of therapy with gene-modified antigen presenting cells. Exp. Hematol. (NY). 25: 39-44 . |
44. | Brossart, P., A.W. Goldrath, E.A. Butz, S. Martin, and M.J. Bevan. 1997. Virus-mediated delivery of antigenic epitopes into dendritic cells as a means to induce CTL. J. Immunol. 158: 3270-3276 [Abstract]. |
45. | Fisher, B.. 1992. The evolution of paradigms for the management of breast cancer: a personal perspective. Cancer Res. 52: 2371-2383 [Medline]. |
46. | Fidler, I.J. 1995. Invasion and metastasis. In Clinical Oncology. M.D. Abeloff, J.O. Armitage, A.S. Lichter, and J.E. Niederhuber, editors. Churchill Livingstone, New York. 55-76. |