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Genzyme Corporation and
Genzyme Molecular Oncology, Framingham, MA 01701
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DCs are potent Ag presenters that express high levels of costimulatory molecules and are capable of activating both CD4+ and CD8+ naive T lymphocytes (6, 7, 8). Results obtained in several animal models have shown that DCs pulsed with defined tumor-associated peptides or with peptides eluted from the surface of tumor cells are capable of inducing an Ag-specific CTL response resulting in protection from tumor challenge and, in some instances, regression of established tumors (9, 10, 11, 12). The same type of approach has also been tested in human clinical trials with encouraging results. For example, Hsu et al. have reported that four B cell lymphoma patients infused with autologous DCs pulsed with tumor-specific Id protein all developed an Id-specific proliferative response, accompanied by complete tumor regression in two patients and partial regression in a third (13). More recently, Nestle et al. reported that melanoma patients treated with autologous DCs pulsed with tumor lysate or a mixture of CTL peptide epitopes developed cell-mediated immunity with objective clinical responses in 5 of 16 patients evaluated (14).
We have favored a gene-based rather than a peptide/protein-based approach to DC immunization, because transduction of DCs with a TAA-encoding transgene offers several potential advantages over peptide-pulsing. First, expression of an entire TAA gene circumvents the need for the identification of specific CTL epitopes within the protein since it allows for processing and presentation of all natural CTL and, possibly, helper epitopes in the context of the host’s MHC type. In addition, TAA expression within DCs provides the cell with a renewable supply of Ag for presentation, as opposed to a single pulse of peptide(s), which eventually decays from the cell surface. As a result, the Ag-presenting activity of genetically modified DCs shows greater persistence (G. Linetie, S. Shankara, R. Doll, L. Eaton, B. Roberts, and C. Nicolette, manuscript in preparation).
The introduction of genetic material into DCs can be achieved with varying levels of efficacy, using techniques such as electroporation, lipid-mediated transfection, calcium phosphate precipitation, and virally mediated gene transfer (15, 16, 17). Adenoviral vectors were selected in this study since we and others have found adenovirus (Ad) to be a highly efficient and reproducible method of gene transfer into DCs (15, 17, 18, 19, 20).
It has been reported that immunization of mice with DCs transduced with Ad vector encoding a model Ag (e.g., OVA and ß-galactosidase (ß-gal)) gives rise to a specific CTL response and provides protective and/or therapeutic immunity against tumor cells stably expressing the same Ag (17, 18, 19, 20). In this study, we investigated the ability of Ad vector-transduced DCs to induce protective immunity against endogenous tumor Ags, as opposed to foreign model Ags introduced exogenously.
The B16 melanoma tumor model was used as a test system to evaluate and characterize the immunizing activity of DCs transduced with Ad vectors encoding melanoma-associated Ags (MAAs). The B16 tumor cell line expresses the murine homologues of human MAAs such as gp100, tyrosinase-related protein (TRP)-1, TRP-2, and melanoma Ag recognized by T cells 1 (MART-1) (21, 22, 23). Accordingly, DCs derived from murine bone marrow were transduced with Ad vector encoding the human and/or murine version of two known MAAs, gp100 and TRP-2, and were tested for their ability to induce a CTL response and provide immunity against B16 melanoma tumor cells. The impact of various factors, such as the dose of DCs, nature of the MAA, CD4 activity, and preexisting immunity to Ad, on the development of antitumor immunity was investigated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female wt C57BL/6 mice and C57BL/6 CD4 knockout (KO) mice were
purchased from Taconic (Germantown, NY) and were used at 8–12 wk of
age. Syngeneic SV40-transformed fibroblasts (SVB6KHA) have been
described elsewhere (24) and were a gift from Dr. Linda
Gooding (Emory University, Atlanta, GA). The YAC-1 NK cell target
derived from the A/Sn mouse strain and the C57BL/6-derived EL4 lymphoma
cell line were both purchased from the American Type Culture Collection
(ATCC, Manassas, VA). The B16.F10 melanoma cell line syngeneic to
C57BL/6 mice was obtained from the National Cancer Institute (Bethesda,
MD). For injection, B16.F10 cells (1.5–2 x
104 cells) were resuspended in PBS and delivered
to the abdomen s.c. in a 100-µl volume. Tumor size was measured with
electronic digital calipers three times per week, starting around day
10. Tumors 
3 mm2 in size were scored as
positive.
Adenoviral vectors
All recombinant Ad vectors used were derived from Ad serotype 2, from which the E1 region was deleted and replaced with an expression cassette containing a CMV promoter driving expression of the transgene. The vectors encoding ß-galactosidase (Ad2/ß-gal-4) and hugp100 (Ad2/hugp100v1) contained intact E3 and E4 regions (25, 26). A second version of Ad2/hugp100 (Ad2/hugp100v2), as well as the vectors encoding enhanced green fluorescent protein (Ad2/EGFP) and murine gp100 (Ad2/mgp100) or vector lacking a transgene (Ad2/EV), possessed an intact E3 region with an E4 region modified by removal of all open reading frames and replacement with the E4 open reading frame 6 and protein IX moved from its original location (27). Finally, the Ad vector encoding murine TRP-2 (Ad2/mTRP-2) contained an intact E4 region but was deleted for E3. The E2 region was left intact in all vectors.
Adenoviral particles were gradient purified as previously described (27), and titers were determined by end-point dilution on 293 cells using FITC-conjugated anti-hexon Ab (28).
Preparation of bone marrow-derived DCs
DCs were prepared from bone marrow essentially as described by Inaba et al. (29). Briefly, bone marrow was flushed from the tibias and femurs of C57BL/6 mice and depleted of erythrocytes with commercial lysis buffer (Sigma, St.Louis, MO). Bone marrow cells were then treated with a mixture of Abs (PharMingen, San Diego, CA) directed against CD8 (clone 53-6.7), CD4 (clone GK1.5), CD45R/B220 (clone RA3-6B2), Ly-6G/Gr-1 (clone RB6-8C5), and Ia (clone KH74), followed by rabbit complement (Accurate Chemical and Scientific, Westbury, NY) to deplete lymphocytes, granulocytes, and Ia+ cells. The remaining cells were cultured for 6 days in six-well plates in RPMI 1640 medium (Life Technologies, Grand Island, NY), supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 10% FCS, and 100 ng/ml recombinant mouse GM-CSF (Genzyme, Cambridge, MA). Loosely adherent DCs were then collected, replated in 100-mm dishes, and cultured in the same medium for another 24 h after removal of contaminating nonadherent cells. This final DC population was then collected for FACS analysis and transduction with Ad vector.
For analysis of surface markers, DCs were first incubated with unlabeled Abs (PharMingen) specific for the MHC class I (clone AF6-88.5) and class II (clone AF6-120.1) molecules, the costimulatory molecules B7.1 (CD80; clone IG10) and B7.2 (CD86; clone GL-1), the adhesion molecule ICAM-1 (CD54; clone 3E2), the integrin CD11c (clone 3E2), and the myeloid surface marker CD13 (clone R3-242). The cells were then counterstained with FITC-conjugated Abs specific for the primary Ab. FACS analysis of the stained cells was performed on an EPICS Profile Analyzer from Coulter (Palo Alto, CA).
Transduction of DCs with Ad vector was conducted in six-well plates with 4 x 106 DCs/well in a 3-ml volume of RPMI 1640 medium containing 10% FCS and 100 ng/ml GM-CSF. Virus was added to the wells at a multiplicity of infection (MOI) of 500, and the DCs were collected after 18–24 h of incubation. For injection, transduced DCs were washed and resuspended in a 100-µl volume of PBS and delivered either s.c. to the abdomen or i.v. into the tail vein as specified in the text.
Mixed lymphocyte reaction and Ag-specific proliferation
To test for the ability of DCs to induce a MLR, varying numbers of bone marrow-derived C57BL/6 DCs (102, 103, 104) were used to stimulate 2 x 105 allogeneic BALB/c T lymphocytes isolated from spleen cells by passage through a commercial T cell purification column (R&D Systems, Minneapolis, MN). Untransduced DCs, as well as Ad2/EGFP-transduced DCs, were tested for MLR induction. In assays of Ag-specific proliferation, 2 x 105 column-purified (R&D Systems) T lymphocytes from naive C57BL/6 mice were incubated with 104 syngeneic DCs that were either untransduced or transduced with wt Ad2, Ad2/EGFP, Ad2/ß-gal-4 or Ad2/mTRP-2. All assay cultures were performed in triplicate in round-bottom 96-well plates in a total volume of 200 µl. The cells were cultured for 5 days at 37°C/ 5% CO2 and pulsed with 1 µCi/well [3H]thymidine (New England Nuclear, Boston, MA) for the last 18 h of incubation. Cells were then harvested onto glass fiber filters with a 96-well plate cell harvester (Skatron Instruments, Sterling, VA), and cell-associated radioactivity was measured by scintillation counting (LS6800 Scintillation Counter from Beckman, Fullerton, CA).
Cytotoxic T cell assay
To evaluate levels of CTL activity, spleen cells from mice in
the same treatment group (three mice/group) were pooled and stimulated
in vitro with syngeneic SVB6KHA fibroblasts transduced with Ad2 vector
at an MOI of 100 for 24 h. Cells were cultured in 24-well plates
containing 5 x 106 spleen cells and
0.8–1.5 x 105 stimulator fibroblasts per
well in a 2-ml volume. Cytolytic activity was assayed after 6 days of
incubation. Target cells consisted of B16 melanoma cells, EL4 lymphoma
cells, YAC cells (NK cell target), and fibroblasts, untransduced or
transduced with virus at an MOI of 100 for 48 h. Targets were
treated with 100 U/ml recombinant mouse IFN-
(Genzyme) for 24 h
(except for YAC cells), labeled with 51Cr (51-Cr;
New England Nuclear) overnight (30 µCi/105
cells) and plated in round-bottom 96-well plates at 5 x
103 cells/well. Effector cells were added at
various E:T cell ratios in triplicate. In specified instances, effector
cells were incubated with a 50-fold excess of unlabeled "cold" YAC
cells for 1 h before the addition of
51Cr-labeled target cells to inhibit nonspecific
lysis by NK cells (30). The total reaction volume was kept
constant at 200 µl/well. After 5 h of incubation of effector and
target cells at 37°C/5% CO2, 25 µl of
cell-free supernatant was collected from each well and counted in a
MicroBeta Trilux Liquid Scintillation Counter (Wallac, Gaithersburg,
MD). The amount of 51Cr spontaneously released
was obtained by incubating target cells in medium alone. Spontaneous
release from target cells was typically below 20%. The total amount of
51Cr incorporated was determined by adding 1%
Triton X-100 in distilled water, and the percentage lysis was
calculated as follows: % lysis = [(sample cpm -
spontaneous cpm)/(total cpm - spontaneous cpm)] x 100.
Enzyme-linked immunospot assay
The presence of gp100-specific effector cells in immunized mice
was also assessed in an enzyme-linked immunospot (ELISPOT) assay
(31). Briefly, spleen cells from mice immunized with DCs
transduced with Ad2/hugp100 or Ad2/EV were stimulated with either a
known MHC class I-restricted CTL peptide epitope from hugp100 (Ref. 32 ;
hUgp10025–33-KVPRNQDWL), the
homologous epitope from mgp100
(mgp10025–33-EGSRNQDWL), or an irrelevant
H-2b-binding CTL epitope from OVA (Ref. 17 ;
OVA257–264-SIINFEKL). The peptides were
synthesized by Quality Controlled Biochemicals (Hopkinton, MA) and were
>90% pure by reverse phase HPLC. Peptide-stimulated spleen cells, as
well as unstimulated spleen cells, were plated in the wells of 96-well
nitrocellulose filter plates (2.5–5 x 104
cells in 100 µl) coated with rat anti-mouse IFN- capture Ab
(clone RMMG-1 from Biosource International, Camarillo, CA) and were
incubated for 
48 h at 37°C/5% CO2. The
cells were then removed by washing with PBS, and the presence of
IFN- produced by spleen cells was detected by the addition of
biotinylated rat anti-mouse IFN- (clone XMG1.2 from PharMingen),
followed by alkaline phosphatase-conjugated streptavidin (Kirkegaard &
Perry Laboratories, Gaithersburg, MD). The number of stained spots
corresponding to IFN--producing cells was enumerated under a
dissecting microscope.
Preimmunization with wt adenovirus
To generate a cohort of mice with preexisting immunity against
Ad, animals were instilled intranasally with 109
infectious units (i.u.) of wt Ad2, followed by a second instillation
with 108 i.u. 14 days later. Eyebleeds were
collected 1 day before the administration of DCs, and serum titers of
Ad-specific Abs were assessed by ELISA. Serial 2-fold dilutions of
serum were added to the wells of 96-well plates coated with
heat-inactivated Ad2. Bound virus-specific Abs were detected by the
addition of HRP-conjugated goat anti-mouse IgG, IgM, and IgA
(Cappel, Durham, NC). The titer was defined as the reciprocal of the
highest dilution of serum that produced an OD490
0.1.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DCs derived from mouse bone marrow exhibited the veiled dendrite
morphology typical of DCs (Fig. 1
) and
displayed a characteristic set of DC surface markers (33)
as determined by FACS analysis (Table I).
The cells expressed high levels of the MHC class I and class II
molecules, the costimulatory molecules B7.1 and B7.2, the ICAM-I
adhesion molecule, the integrin CD11c and the CD13 myeloid surface
marker. Exposure of DCs to recombinant Ad2-based vector at a MOI of 500
reproducibly resulted in a transduction efficiency of 90% or greater
as determined by the percentage of DCs exhibiting fluorescence
following transduction with Ad vector encoding EGFP (Ad2/EGFP).
Transduction did not affect the distribution of DC surface markers
significantly except for a reproducible increase in levels of MHC class
I molecules (Table I).
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A). Moreover, DCs transduced
with Ad vectors encoding various transgenes were found to induce
proliferation of naive syngeneic T lymphocytes, most likely due to
processing and presentation of transgene products and/or Ad proteins by
the DCs (Fig. 2B).
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After confirming the functionality of DCs in vitro, their ability to induce a CTL response against an MAA was evaluated in vivo. DCs were transduced with an Ad vector encoding hugp100 (Ad2/hugp100v1), a differentiation Ag that is expressed by most melanomas but is also present in normal melanocytes and pigmented cells of the retina. Ad2/hugp100v1-transduced DCs (5 x 105) were administered i.v. to C57BL/6 mice, and, 15 days later, spleens were collected for assessment of CTL activity. Separate groups of mice were also treated with vehicle as a negative control or with the Ad2/hugp100v1 vector itself for comparison. The vector was delivered under conditions previously determined to be optimal for immunization (3 x 109 i.u., intradermally (i.d.)).
After in vitro restimulation with syngeneic fibroblasts transduced with
Ad2/hugp100v1, effector splenocytes were tested for cytolytic activity
against 51Cr-labeled target fibroblasts that were
either untransduced or transduced with Ad2/hugp100v1 or wt E3-deleted
Ad (Ad2
2.9). The CTLs were also tested against B16 tumor cells, a
cell line originally derived from a spontaneously arising melanoma in
C57BL/6 mice that expresses the murine equivalent of hugp100.
As expected, mice treated with vehicle failed to develop any
significant CTL activity against any of the targets (Fig. 3A). Mice immunized with
transduced DCs developed high levels of CTL activity against target
fibroblasts infected with the Ad2/hugp100v1 vector. Interestingly, the
bulk of the CTL response appeared to be directed against the hugp100
transgene product rather than adenoviral protein(s) since there was
very little lysis of fibroblasts infected with E3-deleted wt Ad (Fig. 3B). A similar bias in the specificity of the CTL response
toward the transgene product was also observed by Wan et al.
(20) and Gong et al. (18), following
immunization of mice with DCs transduced with Ad vector encoding the
polyoma middle T Ag or the DF3/MUC1 tumor-associated Ag,
respectively.
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C). Importantly, CTLs from mice immunized with
transduced DCs and, to a lesser extent, with Ad vector were both able
to lyse B16 tumor cells, suggesting that the CTLs induced by
immunization with Ad2/hugp100 also recognized the endogenous mouse
gp100 expressed by the tumor cells (Fig. 3, B and
C). A similar cross-reactivity between the human and murine
Ag was observed by Overwijk et al. following immunization of C57BL/6
mice with recombinant vaccinia virus encoding hugp100
(32).
An ELISPOT assay was used to confirm the presence of CTLs specific for
gp100 since CTLs against culture medium components could potentially be
present that may participate in the in vitro lysis of B16 cells. In
these studies, mice were immunized with DCs transduced with
Ad2/hugp100v2, or Ad2/EV as a negative control, and spleen cells were
stimulated in vitro with peptide corresponding to either a known
H-2b-binding CTL epitope from hugp100
(32), the homologous sequence from mgp100
(32), or a known H-2b-binding CTL
epitope from OVA as a negative control (17). The number of
class I-restricted CTLs that produced IFN- upon specific peptide
recognition was measured after 48 h. Results shown in Fig. 4 confirm the presence of CTLs specific
for hugp100 peptide in mice immunized with Ad2/hugp100v2-transduced DCs
and demonstrate the extensive cross-reactivity between the human and
murine epitope. As expected, spleen cells from mice that received DCs
transduced with Ad2/EV show little or no reactivity against gp100
peptides, and neither group of mice shows any significant reactivity
against the negative control OVA peptide.
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, mice
immunized with Ad2/hugp100v1-transduced DCs delivered s.c. or i.v
developed similar levels of CTL activity against B16 melanoma cells
with comparatively little lysis of syngeneic gp100-negative EL4
lymphoma cells. Significant lysis of the YAC NK cell target was also
observed. However, NK cells could not account entirely for the lysis of
B16 tumor cells since cold target inhibition with an excess of
unlabeled YAC cells successfully prevented lysis of labeled YAC cells
without significantly affecting specific lysis of B16 cells. These
results indicate that the s.c. and i.v. routes of immunization with
transduced DCs elicit equivalent levels of CTL activity against B16
tumor cells.
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The ability of Ad2/hugp100v1-transduced DCs to induce effector
CTLs capable of lysing B16 tumor cells in vitro suggested that DC
immunization may also provide antitumor protection in vivo. This was
first tested in a pretreatment model whereby mice were immunized with
an i.v. injection 5 x 105
Ad2/hugp100v1-transduced DCs and challenged 15 days later with a lethal
s.c. injection of 2 x 104 B16 tumor cells.
Animals in negative control groups that were pretreated with vehicle or
untransduced DCs developed rapidly growing tumors leading to death of
the animals within 30 days (Fig. 6). In
contrast, mice preimmunized with transduced DCs showed significant
resistance to tumor growth, and only one of five animals developed a
tumor with delayed kinetics. To test for the presence of immunological
memory, the remaining tumor-free mice were given a second B16
challenge, 50 days after the first B16 cell injection. Three of the
four mice preimmunized with transduced DCs remained tumor-free upon
rechallenge, indicating that a single administration of DCs was
sufficient to induce long-term antitumor immunity.
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Nature of the Ag.
The Ad2/hugp100v1 vector expresses the hugp100 Ag, which, upon
presentation by DCs, was found to elicit a protective immune response
against murine B16 melanoma cells. To determine whether immunization
against a homologous murine MAA would be as effective in inducing
protective antitumor immunity, mice were preimmunized i.v. with DCs
transduced with Ad vectors encoding the murine MAAs gp100 (Ad2/mgp100)
or tyrosinase-related protein 2 (Ad2/mTRP-2). Protection from B16 tumor
cell challenge administered 15 days later was compared with that
obtained following immunization with Ad2/hugp100v1-transduced DCs, or
untransduced DCs as a negative control. As observed previously, mice
pretreated with untransduced DCs developed rapidly growing tumors
whereas mice preimmunized with Ad2/hugp100v1-transduced DCs showed
resistance to tumor challenge so that only two of five mice developed
tumors (Fig. 7A). In contrast,
four of five mice treated with DCs transduced with Ad vector encoding
the murine homologue of gp100 showed progressive tumor growth,
indicating that protective immunity failed to develop in these animals.
This finding is in agreement with the results of Overwijk et al., who
reported that recombinant vaccinia virus encoding murine gp100 was
nonimmunogenic in C57BL/6 mice (32). The failure to induce
antitumor protection may have been attributed to the difficulty in
breaking immunological tolerance against a self Ag as opposed to the
heterologous human protein. However, mice immunized against mTRP-2 with
Ad2/mTRP-2-transduced DCs did develop a protective immune response
against B16 cells, and five of five mice remained tumor-free (Fig. 7A). This result indicates that it is in fact possible to
generate an effective immune response against a tumor self Ag, but that
not all tumor-associated self Ags can be expected to be equally
potent.
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B underscore the additional importance of
CD4+ T cells, and consequently class
II-restricted epitopes, in the development of an optimal antitumor
response. In the experiment shown, wt and CD4 KO C57BL/6 mice were
immunized in parallel with Ad2/mTRP-2-transduced DCs and challenged
with B16 cells 14 days later. As observed above, preimmunization of wt
mice with Ad2/mTRP-2-transduced DCs offered 100% protection from B16
challenge (eight of eight mice tumor-free). In contrast, only three of
ten CD4 KO mice were able to inhibit tumor growth, indicating that,
despite the presence of CD8+ cells, the
development of antitumor immunity was severely impaired in these
animals (Fig. 7B).
Dose dependence.
The level of antitumor protection achieved by preimmunization with
Ad-transduced DCs was also found to be dependent on the dose of DCs
administered. As shown in Fig. 7C, maximal 100% protection
from B16 challenge was achieved with s.c. administration of 5 x
105 Ad2/mTRP-2-infected DCs, with an observed
decrease in levels of antitumor protection as the dose was reduced to
5 x 104 and 5 x
103 transduced DCs. Increasing the dose to 5
x 106 DCs did not provide any additional benefit
but also failed to induce any discernible toxicity.
Immunization with Ad vector-transduced DCs in Ad-immune mice
Most individuals in the general population have been preexposed to
wt Ad and are expected to possess some level of preexisting immunity
against Ad. To mimic the expected clinical situation and evaluate the
impact of Ad immunity on the activity of Ad-transduced DCs, mice were
preimmunized intranasally with wt virus until they developed high
titers of Ad-specific Abs (Fig. 8) and,
as documented previously, virus-specific CTLs (34).
Ad-immune and naive mice were then immunized s.c. with 5 x
105 Ad2/mTRP-2-transduced DCs and were challenged
15 days later with B16 tumor cells. As shown in Fig. 8, naive and
Ad-immune mice developed comparable levels of tumor protection with
twelve of twelve and ten of twelve tumor-free mice, respectively.
Similar results were also obtained with Ad2/hugp100v1-transduced DCs
delivered via the i.v. route of immunization (not shown). As expected,
negative control animals, which received DCs transduced with Ad2/EV
lacking a transgene, developed tumors whether the mice were naive (zero
of eight tumor-free) or preimmunized against Ad (one of eight
tumor-free). These results suggest that immunization with Ad
vector-transduced DCs is unlikely to be impaired significantly in
individuals previously exposed to Ad.
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Up to 100% protection against a lethal challenge of B16
tumor cells was achieved by preimmunization with DCs transduced with Ad
vector-expressing MAAs. To extend these findings to a more clinically
relevant model, Ad-transduced DCs were also tested in an active
treatment setting. In the study shown in Fig. 9A, mice received a lethal s.c
injection of B16 tumor cells, which were allowed to establish
themselves for 4 days before treatment with Ad-transduced DCs. As
expected, negative control animals that were untreated or treated with
Ad2/EV-transduced DCs were unable to control tumor growth. Mice treated
with Ad2/hugp100v1-transduced DCs, which provided significant antitumor
protection in a preimmunization setting, showed little or no protection
from tumor growth (one of five tumor-free) in an active treatment
setting. Treatment with Ad2/mTRP-2-transduced DCs, which provided 100%
protection in a pretreatment setting, gave rise to partial antitumor
protection with three of five mice remaining tumor-free. Therefore, an
overall reduction in efficacy was seen in the more stringent active
treatment model, which requires rapid mobilization of an immune
response against aggressive tumor cell growth.
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B, combination therapy with a
mixture of two DC populations transduced with Ad2/hugp100v2 or
Ad2/mTRP-2 did in fact result in enhanced inhibition of tumor growth,
compared with administration of either DC population alone. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Transduced DCs were
also functionally active in vitro as assessed in a standard mixed
lymphocyte reaction and as demonstrated by their ability to induce
primary Ag-specific proliferation of syngeneic T lymphocytes (Fig. 2).
In vivo testing of Ad2/hugp100v1-transduced DCs demonstrated their
ability to induce a specific CTL response following i.v or s.c.
delivery. The appearance of CTLs specific for gp100 peptide and capable
of lysing B16 tumor cells in vitro correlated with the development of
protective immunity against B16 tumor challenge in vivo.
Immunization with DCs transduced with Ad vectors encoding murine MAAs
(mgp100, mTRP-2), as opposed to a heterologous human Ag (hugp100),
indicated that it was possible to break immunological tolerance and
induce effective immunity against a melanoma self Ag using a DC-based
approach. However, the two murine Ags tested differed markedly in their
ability to induce antitumor immunity. Ad2/mTRP-2-transduced DCs
typically provided complete protection from B16 tumor challenge whereas
Ad2/mgp100-transduced DCs gave rise to little or no protective
activity. It is likely that multiple factors underlie such differences
in activity. For example, the number and potency of CTL
(32) and/or Th epitopes within a particular protein, as
well as the density of target epitopes expressed by the tumor cells,
are two variables that could influence the development of an effective
antitumor response. In fact, whereas much emphasis has been placed on
the identification of MHC class I-restricted CTL epitopes for induction
of antitumor immunity, our results indicate that epitopes recognized by
CD4+ T cells also play an important role.
Although the murine TRP-2 Ag is known to contain an
H-2Kb-restricted CTL epitope (22),
the impaired ability of CD4 KO mice to develop protective immunity
following immunization with Ad2/mTRP-2-transduced DCs (Fig. 7B) underscores the involvement of
CD4+ T cells and suggests that presentation of
MHC class II-restricted epitopes, either by transduced DCs or through
secondary processing of the expressed TRP-2 protein, is essential to
the development of an effective antitumor response. The exact
contribution of CD4+ T lymphocytes in the
afferent or efferent phase of the immune response remains to be
determined, but the lack of class II expression by B16 cells, even
after treatment with IFN- (not shown), suggests that
CD4+ T lymphocytes are more likely to function as
helper cells in the induction of antitumor responses rather than as
effector cells against the largely class II-negative tumor cells. In
any case, the results suggest that optimal immunization protocols
should incorporate the inclusion of class II-restricted epitopes in
addition to class I-restricted CTL epitopes, a criterion likely
fulfilled by delivery of a complete TAA gene to DCs.
The induction of antitumor immunity by Ad vector-transduced DCs was
found to be dose dependent. A single dose of 5 x
103 Ad2/mTRP-2-transduced DCs was sufficient to
provide a benefit compared with vehicle-treated animals, and complete
protection from tumor challenge was attained with 5 x
105 transduced DCs (Fig. 7C). These
results suggest that, in a therapeutic setting, a clinical benefit may
be achievable even with relatively low doses of transduced
DCs.
An important issue to consider in the context of a clinical setting is
that of preexisting immunity against Ad since most individuals in the
general population have been exposed to the wt virus. However, as
shown in Fig. 8, immunity to Ad appears unlikely to interfere with
DC-based immunization since mice preimmunized with wt Ad2 were not
significantly impaired in their ability to develop antitumor immunity
following injection of Ad2/mTRP-2-transduced DCs. This finding is in
agreement with the results of Brossart et al., who reported that DCs
transduced with an Ad vector encoding OVA were able to induce an
OVA-specific CTL response in mice that had been previously immunized
with two injections of Ad vector and had developed Ad-neutralizing Abs
(17). Virus neutralizing Abs were not expected to
interfere with DC-based immunization, but Ad-specific CTLs, which are
also present in Ad-immune mice (34), have the potential to
destroy Ad-transduced DCs. Even though the "half-life" of
transduced DCs administered to Ad-immune mice was not determined in
this study, the observed development of antitumor protection in these
animals indicates that the transduced DCs were present long enough to
induce effective immunity against tumor cells. The observation that Ad
vector-transduced DCs appear to induce a CTL response largely directed
against the transgene product rather than Ad proteins (Refs. 18 and 20 ;
Fig. 3) raises the possibility that limited presentation of Ad Ags by
Ad/TAA-transduced DCs may provide some level of protection from lysis
by Ad-specific CTLs.
The efficacy of Ad vector-transduced DCs was also tested in a
therapeutic setting against established B16 s.c. tumor cells. The level
of antitumor protection achieved was reduced, compared with that
obtained in a preimmunization model. Nevertheless, a single injection
of Ad2/mTRP-2-transduced DCs resulted in complete inhibition of tumor
growth in an average of three of five mice (Fig. 9A).
Several approaches are being considered to improve this outcome
further. For example, simultaneous immunization against two MAAs, as
opposed to a single Ag, was tested as a means to potentiate the immune
response and prevent the escape of tumor variants that may express
insufficient levels of a target Ag for recognition by CTLs. The results
obtained support the validity of this type of approach since
administration of a mixture of DCs transduced separately with Ad vector
encoding hugp100 or murine TRP-2 resulted in greater levels of tumor
growth inhibition, compared with either DC population alone (Fig. 9B). In addition, preliminary results suggest that the
therapeutic efficacy of Ad vector-transduced DCs can also be enhanced
by multiple administrations of DCs (40% increase in day 40 survival
with three doses compared with a single dose) or by coadministration of
low dose IL-2 (not shown). In agreement with the latter observation,
Shimizu et al. (35) have reported recently that low dose
IL-2 dramatically enhanced the antitumor response elicited by DCs
pulsed with tumor lysate in a murine sarcoma model.
Overall, the data obtained in this study provide supporting evidence for the concept of melanoma immunotherapy based on the administration of DCs transduced with Ad vectors encoding MAAs. This type of approach is considered feasible in humans since protocols have been established that allow for the expansion of large numbers of DCs from peripheral blood monocytes cultured in the presence of GM-CSF and IL-4 (36, 37). The DCs obtained can be effectively transduced with Ad vector and can induce a primary CTL response against the transgene product in vitro (G. Linette, S. Shankara, R. Doll, L. Eaton, B. Roberts, and C. Nicolette; manuscript in preparation). These observations, in conjunction with the protective antitumor activity elicited by transduced DCs in the B16 melanoma model, suggest that immunization of melanoma patients with autologous DCs transduced with Ad vector expressing human MAAs may provide a therapeutic benefit.
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: TAA, tumor-associated Ag; DC, dendritic cell; ß-gal, ß-galactosidase; EGFP, enhanced green fluorescent protein; wt, wild type; TRP, tyrosinase-related protein; mTRP, murine TRP; EV, empty vector; hugp100, human gp100 melanoma Ag; mgp100, murine gp100 melanoma Ag; KO, knockout; Ad, adenovirus; MAA, melanoma-associated Ag; MOI, multiplicity of infection; ELISPOT, enzyme-linked immunospot; i.u., infectious unit; i.d., intradermal.
Received for publication November 10, 1998. Accepted for publication April 30, 1999.
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) or Ad2/mTRP-2 (
). Two
groups received a mixture of DCs transduced separately with
Ad2/hugp100v2 or Ad2/mTRP-2 in the amount of 2.5 x
105 (
) or 5 x 105 (
) of each DC
population. An additional group was treated with vehicle (
) as a
negative control. All groups contained eight animals, except for the
vehicle control group, which was limited to six mice. Results are
presented as the mean tumor size over time. Similar results were
obtained in two separate studies.