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Immunology |
Laboratory of Tumor Immunology and Biology, National Cancer Institute, NIH, Bethesda, Maryland 20892 [E. K., J. S., J. W. G.], and Therion Biologics Corporation, Cambridge, Massachusetts 02142 [D. L. P., G. M.]
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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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Recombinant avipox virus-based vector systems are known to infect a broad spectrum of mammalian cells with a high efficiency of foreign gene expression in vivo (21) , and their restrictive capacity for replication to avian species ensures their overall safety (22 , 23) . The present study was designed to evaluate two recombinant avipox viruses, fowl pox and canary pox, engineered to express murine GM-CSF. Those two recombinant avipox viruses were examined for their ability to produce biologically active GM-CSF in vivo and function as biological adjuvants in vaccine protocols designed to generate host immunity to a self tumor antigen. The evaluation of whether either recombinant avipox virus produces biologically active GM-CSF in vivo was based on the same criteria used previously to characterize vaccinia-GM-CSF (18) . Specifically, after injection of either recombinant virus, regional lymph nodes draining the injection site were examined for cellular content and phenotype and, more specifically, for the accumulation of professional APCs. Those cellular and phenotypic changes were used to assess both the magnitude as well as the temporal aspects of the changes occurring within the regional nodes.
CEA, a Mr 180,000–200,000 glycoprotein that is overexpressed in a high percentage of human adenocarcinomas (colon, pancreatic, breast, and lung), has been shown to be an attractive target for immunotherapy (24 , 25) . Because no CEA homologue has been identified in rodents, mice expressing human CEA as a transgene (26, 27, 28) are being used to study different vaccine strategies. The present study also examined whether the avipox-GM-CSF viruses were compatible with an recombinant avipox-CEA vaccine and capable of augmenting the generation of host immunity to CEA in the CEA.Tg murine model. The findings demonstrate that recombinant avian poxviruses can deliver GM-CSF to an immunization site that, in turn, augmented host and antitumor immunity directed at a self tumor antigen.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The description of the MC-38 (H-2b) cells expressing the human CEA gene and designated MC-38-CEA-2 has been published (29) . The cells were examined for CEA expression by flow cytometry using a murine anti-CEA monoclonal antibody, COL-1 (30) . MC-38 and MC-38-CEA-2 cell lines were maintained in DMEM supplemented with high glucose and 10% heat-inactivated FBS. FDCP-1 cells were provided by Dr. Jim Ihle (St. Jude’s Hospital, Memphis, TN) and grown as described previously (18) . Lyophilized recombinant murine GM-CSF (PeproTech, Inc., Rock Hill, NJ) was reconstituted in saline containing 1% mouse serum and stored at -20°C. The biological activity was checked periodically using FDCP-1 cells (31) .
Recombinant Avian Poxviruses.
Recombinant avipox viruses used were fowlpox and canarypox (ALVAC) and
are collectively referred to as recombinant avipox viruses in this
report. The specific recombinant avipox viruses used to generate the
data are listed for each table and figure and identified as avipox(F)
and avipox(A) for fowlpox and canarypox (ALVAC)-based vectors,
respectively.
Avipox(F)-GM-CSF.
The parental virus used for the generation of avipox(F)-GM-CSF was
plaque-purified from a tissue culture-adapted vaccine strain of fowlpox
virus. Avipox(F)-GM-CSF was constructed via homologous recombination
in vivo between the parental fowlpox DNA and a plasmid
vector that contains the murine GM-CSF gene. The recombinant
virus was isolated and purified as described previously
(32)
. A seed stock was generated and characterized by
genomic and protein expression analysis. Wild-type fowlpox
[avipox(F)-WT] served as the control virus.
Avipox(A)-Recombinants.
The canarypox strain was isolated from a pox lesion on an infected
canary and attenuated by 200 serial passages in chick embryo
fibroblasts and was subjected to four successive rounds of plaque
purification under agarose (33)
. All amplifications and
plaque titrations were performed on primary chick embryo
fibroblasts. Avipox(A)-GM-CSF (vCP319), avipox(A)-rabies glycoprotein G
[designated avipox(A)-RG, vCP65], and avipox(A)-CEA (vCP248) were
kindly provided by Dr. James Tartaglia (Aventis Pasteur, Toronto,
Ontario, Canada). GM-CSF expression was confirmed by a bioassay (see
below), and CEA expression was confirmed by Western blot analysis using
COL-1 (30)
.
In Vitro GM-CSF Production.
The procedure used to measure GM-CSF production by MC-38 cells infected
with the recombinant avipox-GM-CSF viruses has been published
(18)
. Supernatants were harvested from infected cells at
different time points, and the amount of biologically active GM-CSF
present was determined using the GM-CSF-dependent FDCP-1 cell line.
Injections.
CEA.Tg mice were injected (s.c.) with avipox-CEA or avipox-RG in 100
µl of HBSS at the base of the tail. Where indicated, recombinant
avipox-GM-CSF viruses or rGM-CSF were mixed with avipox-CEA and
administered simultaneously. When recombinant avipox viruses expressing
GM-CSF or rGM-CSF were administered alone, they were injected s.c. at
the base of the tail. rGM-CSF (20 µg/injection) was diluted in saline
containing 1% mouse serum and administered for 4 consecutive days.
Regional Lymph Node Analyses.
After the administration of avipox viruses and rGM-CSF, subiliac,
para-aortic, and sacral lymph nodes were isolated, cells were
mechanically dispersed and suspended in ACK lysing buffer (0.15
M NH4Cl, 1.0 mM
KHCO3, and 0.1 mM
Na2EDTA, pH 7.2–7.4) on ice for 5 min. The
supernatants were removed, cells were pelleted by centrifugation
(500 x g) and washed twice in cold
Ca2+-Mg2+-free DPBS. After
two washes, the cells were resuspended in
Ca2+-Mg2+-free DPBS at a
concentration of 0.5–1.0 x 106
cells/ml and incubated with 1 µg of FITC-labeled
anti-I-Ab (BALB/c mouse, IgG2a,
) or
appropriate control antibody (PharMingen, Inc., San Diego, CA) for
1 h at 4°C. One µg of unlabeled 2.2G2 antibody (CD16) was
added to each sample to block Fc receptors. Results were analyzed using
a Becton Dickinson FACScan as described previously (18)
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CD11c+ Cell Isolation.
A detailed explanation of the isolation of CD11c+
cells from the regional lymph nodes has been published
(18)
. Single-cell suspensions from the lymph nodes were
incubated at 4°C for 1 h in cold DPBS containing 1.5
ml/108 cells of biotin-anti-CD11c (clone B-ly6;
PharMingen, Inc.). After washing, the cells were incubated in the
presence of MACS colloidal supra-paramagnetic MicroBeads conjugated to
streptavidin (Miltenyi Biotec, Inc., Gladbach, Germany) at 4°C for 15
min. A MACS LS+ separation column was placed
within the MIDI MACS magnetic separator according to the
manufacturer’s instructions. The cell suspension was applied to the
column, and the nonmagnetic cells passed through. The procedure was
repeated to enrich the MACS+ cell fraction. The
cells in the MACS+ fraction were counted with a
hemocytometer and analyzed by flow cytometry using a double stain
consisting of a biotin-phycoerythrin conjugate and an
anti-I-Ab-FiTc antibody (clone M5/114.15.2,
IgG2b). More than 80% of the MACS+ cell fraction
were CD11c+/I-Ab+,
CD19-, and CD3-
(18)
.
MLC.
Purified splenic BALB/c (H-2d) T cells were grown
in RPMI 1640 containing 10% heat-inactivated FBS in the presence of
irradiated C57BL/6 (H-2b) lymph node cells (1:1
ratio). After incubation for 5 days at 37°C in T-25 flasks, viable T
cells were recovered by density centrifugation over a Ficoll-Hypaque
gradient and used in a unidirectional CTL assay with MC-38
(H-2b) and P815 (H-2d) as
targets.
Serum Antibody Responses.
Serum samples were collected from CEA.Tg mice and analyzed by ELISA for
the presence of antibodies to the appropriate antigen. Microtiter
plates were sensitized overnight at 4°C with 100 ng/well CEA (Vitro
Diagnostics, Littleton, CO), OVA (Sigma Chemicals, St. Louis, MO),
murine rGM-CSF, or 5 x 105
pfu/well ALVAC, and the ELISA assay was carried out as described
previously (18)
. Triplicates of positive and negative
controls and serum samples were included in all assays. Positive
controls for CEA and ALVAC were COL-1 and a rabbit polyclonal
anti-ALVAC IgG, both developed in the laboratory. A commercially
available rat antimouse GM-CSF monoclonal antibody (clone MP1–22E9;
PharMingen) was the positive control in the anti-GM-CSF assays.
Antibody titers are the reciprocal of the serum dilution that resulted
in an A490 nm of 0.5.
T-Cell Proliferation Assay.
Mouse splenocytes were enriched for T cells by magnetic murine pan B
(B220) Dynabeads (Dynal, A.S., Oslo, Norway). The isolated T
lymphocytes were resuspended in RPMI 1640 containing 15 mM
HEPES (pH 7.4), 10% heat-inactivated FBS, 2 mM
L-glutamine, 0.1 mM nonessential amino acids, 1
mM sodium pyruvate, 50 units/ml gentamicin, and 50
µM ß-mercaptoethanol (T-cell medium). The assay
consisted of coincubating 5 x 105
irradiated splenocytes from naive, syngeneic B6 mice (APC) and
1.5 x 105 purified splenic T
lymphocytes in the presence of CEA, OVA, or medium in each well of
flat-bottomed, 96-well plates. Cells were pulsed with
[3
H]thymidine (1 µCi/well; Amersham Corp.,
Arlington Heights, IL) after 4–5 days, and radioactivity was measured
24 h later by liquid scintillation spectroscopy (Wallac, Inc.,
Gaithersburg, MD).
CTLs and Cytotoxicity Assay.
Spleens from immune mice were pooled and 25 x 106 splenocytes were added in 10 ml of T-cell
medium to T-25 flasks along with 10 µg/ml of
CEA526–533 (EAQNTTYL).
CEA526–533 is a CEA-specific murine T-cell
epitope capable of inducing a CD8+ CTL response
restricted by the H-2Db
allele.3
T-cell cultures were stimulated twice at weekly intervals by harvesting
the T cells over a Ficoll-Hypaque gradient and incubating 2 x 105 T cells in the presence of
5 x 106 irradiated syngeneic
splenocytes, 10 µg of CEA526–533/ml, and 10
units/ml recombinant human IL-2 (Proleukin; Chiron Corp., Emeryville,
CA). After two in vitro stimulations, cytotoxicity was
assessed using EL-4, a murine lymphoma cell line, pulsed with either
CEA526–533 or Flu H3N2 influenza A virus
nucleoprotein epitope (NP366–374), which is also
an H-2Db epitope (34)
.
CTL activity was assessed by making modifications as described
previously (35)
. EL-4 cells (4 x 106) were radiolabeled with 50 µCi in
111In-labeled oxyquinoline (Amersham, Chicago,
IL) for 30 min at 37°C. Peptide-pulsed target cells were incubated
with 1 µg of peptide/ml after labeling. Target and effector cells
were mixed at predetermined ratios and incubated for 18 h at
37°C. The amount of 111In released was measured
in a gamma counter (Cobra Autogamma; Packard Instruments, Downers
Grove, IL), and the percentage of specific lysis and LUs were
calculated as follows:
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Cytokine Production Assays.
T-cell lines were incubated in flat-bottomed, 96-well plates at a cell
density of 2 x 104
cells/well,
5 x 105 irradiated (2000 rad)
syngeneic CEA.Tg mouse splenocytes/well with different CEA peptides
concentrations. Supernatants were harvested after 48 h, and
IFN-
and IL-4 levels were measured using the appropriate ELISA assay
(Endogen, Inc., Cambridge, MA).
Tumor Therapy Studies.
Six- to 8-week-old male and female CEA.Tg mice received injections i.p.
of 2 mg of cyclophosphamide, and 4 days later, 3 x 105 MC-38-CEA-2 tumor cells (in 100 µl) were
injected s.c. in the right flank. At the time of tumor inoculation,
cytometric analysis showed that >85% of the MC-38-CEA-2 cells
expressed CEA, strong MHC class I, and no MHC class II
(I-Ab) expression. Four to 5 days later (tumor
volumes, 30–50 mm3
), mice received the primary
vaccination of 108 pfu avipox-CEA/RG alone or in
combination with either 108 pfu avipox-GM-CSF or
20 µg of rGM-CSF. rGM-CSF was injected at the vaccination site for
the next 4 days. Booster vaccinations were given 2 weeks later. Tumors
were measured twice/week, and the volumes were calculated as: [(mm,
short axis)2
x (mm, long axis)]/2.
Mice bearing tumors of >2 cm3
were sacrificed
for humane reasons, and the day of death was recorded. Reductions in
tumor volume were presented as: (a) tumor regression, a
measured decrease in tumor volume; and (b) tumor
eradication, the complete disappearance of tumor. Tumor-free CEA.Tg
mice received a second injection of 3 x 105 MC-38-CEA-2 tumor cells in the opposite flank
(s.c.).
Statistical Analysis.
Statistical significance of T-cell proliferation/lysis data were based
on Student’s two-tailed t test. Differences in the growth
rate of the MC-38-CEA-2 tumors as measured by changes in tumor volume
for each treatment group were compared using the Mann-Whitney
U test. Differences in survival (see Fig. 7, C and D
) were analyzed by Kaplan Meier. All Ps
reported are two-sided and have not been adjusted for the multiplicity
of evaluation performed on the data. P < 0.05 was considered significant.
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RESULTS |
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Cellular/Functional Changes in Regional Lymph Nodes after
Avipox-GM-CSF or rGM-CSF
Seven days after an injection of 107 or
108 pfu of the recombinant avipox-GM-CSF viruses
and, to a lesser extent, the appropriate control viruses, significant
regional lymphadenopathy was observed in the B6 mice (Table 1)
. The most dramatic increase in the total number of cells/node was in
those mice that received 108 pfu of either of the
GM-CSF-expressing recombinant avipox viruses. For example, in mice
injected with 108 pfu avipox(F)-GM-CSF, the total
number of cells/node rose 
12-fold (P < 0.05) when compared with the HBSS-treated mice. In addition, increases
in the total number of cells/node was significantly
(P < 0.05) higher in mice treated with
recombinant avipox viruses expressing GM-CSF than with the appropriate
control viruses administered at the same pfu. Accompanying this
increase in lymph node cellularity was a selective increase in the
percentage of class II-expressing cells in mice treated with the
recombinant avipox viruses expressing GM-CSF (Table 1)
. In the
HBSS-treated mice or mice that received injections of the control
avipox viruses, 25–29% of the lymph node cells expressed MHC class II
antigens. However, injection of either of the recombinant avipox-GM-CSF
viruses (107 or 108 pfu)
increased the percentage of class II-positive cells to 43–51% by day
7 (P < 0.05), with an accompanying boost in
their MFI (Table 1)
. A single injection of rGM-CSF did enhance lymph
node cellularity, but with no accompanying change in class II
expression (Table 1)
. Four injections of rGM-CSF significantly
(P < 0.05) enhanced lymph node cellularity
and MHC class II expression when compared with the HBSS-treated mice
(Table 1)
. Regional lymph nodes from all of the groups of mice were
again analyzed 21 days after treatment. Interestingly, elevations in
the total number of cells, percentage of class
II+ lymph node cells, and the MFI were found in
the regional lymph nodes of those mice that were treated with the
recombinant avipox-GM-CSF viruses. For comparison, both the total
number of cells and the class II expression levels within the lymph
nodes of the mice that were treated previously with rGM-CSF returned to
baseline levels by day 21 (Table 1)
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, the total number of class
II+ cells/node were increased 3-fold
(P < 0.05) when compared with HBSS-treated
or mice receiving injections of the appropriate control virus. The
number of class II-expressing cells remained elevated for 21–28 days
in the lymph nodes of mice treated with either recombinant
avipox-GM-CSF virus. For comparison, the time course of the increases
in class II-expressing lymph node cells in mice treated for 4 days with
rGM-CSF is also presented (Fig. 1A)
.
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summarizes the time course of the increase in the absolute number of
APCs in the regional nodes of mice treated with either recombinant
avipox-GM-CSF virus, control viruses, rGM-CSF, or HBSS. Approximately
1–2% of lymph node cells isolated from control, HBSS-treated mice
were APCs, as defined by their surface phenotype. Administration of
108 pfu of either avipox-GM-CSF increased that
percentage 3-fold (data not shown) and the absolute number of
CD11c+/I-Ab+ cells 12-fold
after 7 days (Fig. 2)
. The time course was virtually identical to that
for class II-expressing cells, a sustained increase in the number of
APCs/node for 21–28 days. Lymph nodes from mice treated with avipox-WT
and avipox-RG did not contain higher numbers of APCs (Fig. 2)
, although
higher numbers of B and T cells were found.
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. When tested for allospecific lysis, significantly higher
(P < 0.05) lytic activity was found in those
cultures that contained lymph node cells from avipox-GM-CSF-treated
mice.
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. Seven days after the first injection of avipox-GM-CSF, the total
number of class II+ cells was increased
10-fold, from 0.5 to 4.9 x 106/node (Fig. 4A)
. By day 28, that
number had fallen to 1.7 x 106
but rose to 4.8 x 106 cells/node
after the second avipox-GM-CSF injection (day 35). A third injection
(day 56) of avipox-GM-CSF once again increased the number of class
II+ cells/node from 2.8 to 5.7 x 106 (Fig. 4A)
. Injection of
avipox(A)-RG resulted in no observable change in the number of class
II+ lymph node cells.
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. After subsequent
injections of avipox-GM-CSF, anti-avipox IgG serum titers rose to
>200,000. No serum IgG titers to GM-CSF were detected (Fig. 4B)
.
Adjuvant Effects of Avipox-GM-CSF on Antigen-specific Immunity
Anti-CEA Antibody Responses in CEA.Tg Mice.
CEA.Tg mice received two monthly vaccinations with avipox-CEA alone or
combined with either avipox-GM-CSF or rGM-CSF, as outlined previously.
Detectable anti-CEA IgG serum titers were present in 60% of mice
vaccinated with either avipox-CEA alone (Fig. 5B)
or avipox-CEA and rGM-CSF (Fig. 5C)
. All 10 CEA.Tg mice
vaccinated with avipox-CEA and avipox-GM-CSF (Fig. 5D)
developed CEA-specific serum IgG titers. No CEA antibody titers were
detected in the sera of naive or CEA.Tg mice vaccinated with the
control virus, avipox-RG (Fig. 5A)
.
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. That proliferative response to CEA was boosted when CEA.Tg mice
received avipox-CEA combined with either avipox-GM-CSF or rGM-CSF
(P < 0.05). No CEA-specific
lymphoproliferation was found using splenic T cells isolated from
avipox-RG-vaccinated CEA.Tg mice (Table 2)
.
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. The highest levels of CEA peptide-specific cytolysis
(P < 0.05 versus either
avipox-CEA or avipox-CEA + rGM-CSF-vaccinated mice), as
measured by LUs, was obtained for T cells from CEA.Tg mice vaccinated
with avipox-CEA in combination with avipox-GM-CSF (Fig. 6A)
.
No appreciable lysis of the Flu NP366–374-pulsed
EL-4 target cells was found. T-cell lines generated from CEA.Tg mice
vaccinated with avipox-CEA combined with either avipox-GM-CSF or
rGM-CSF also produced higher IFN- levels than the T-cell lines
generated from CEA.Tg mice vaccinated with avipox-CEA alone (Fig. 6B)
. No IL-4 was found in any of those cultures.
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. Avipox-CEA vaccination resulted in a transient slowing of tumor
growth in some CEA.Tg mice; however, survival was not prolonged (Fig. 7C)
. Vaccination with avipox-CEA combined with avipox-GM-CSF
induced measurable reductions in tumor volume of 6 of 16 CEA.Tg mice
(Fig. 7A)
. By day 35, the average tumor volume of the
avipox-CEA + avipox-GM-CSF treatment group was significantly
smaller (P < 0.05) than that of untreated,
avipox-RG + avipox(A)-GM-CSF or avipox-CEA-vaccinated CEA.Tg
mice. In fact, five tumor-bearing CEA.Tg mice vaccinated with
avipox-CEA and avipox-GM-CSF became tumor free (Table 3
; Fig. 7A
) by day 28 and remained so for 14 weeks (Fig. 7C)
. At that time, the five tumor-free CEA.Tg mice were
challenged with MC-38-CEA-2 tumor cells, and all were protected (Fig. 7D)
. Four of 14 CEA.Tg mice vaccinated with avipox-CEA and
rGM-CSF also became tumor free (Table 3
; Fig. 7B
), and three
of those four mice rejected tumor at challenge (Fig. 7D)
.
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DISCUSSION |
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; the
percentage (Table 1)
, MFI (Table 1)
, and absolute number of class
II-expressing cells (Fig. 1)
; and the number of
CD11c+/I-Ab+ cells (Fig. 2)
within the regional draining lymph nodes were observed, changes that
are consistent with the local production of biologically active GM-CSF.
Moreover, recombinant fowlpox and canarypox viruses performed equally
well in producing similar amounts of GM-CSF in vitro and
inducing cellular and phenotypic changes in the regional lymph nodes.
Therefore, these recombinant avipox viruses may be useful as delivery
systems for GM-CSF.
The use of recombinant avipox viruses to deliver GM-CSF may have
several advantages over the current use of rGM-CSF. One advantage would
be the ability of a single injection of avipox-GM-CSF to evoke changes
within the draining lymph nodes that usually require multiple
injections of rGM-CSF. In fact, the magnitudes of the increases in the
absolute number of
CD11c+/I-Ab+ cells in the
regional lymph nodes were significantly greater in mice injected with
the avipox-GM-CSF viruses than with rGM-CSF (Fig. 2)
. For example, a
single injection of either avipox-GM-CSF virus boosted the absolute
number of lymph node APCs
(CD11c+/I-Ab+ cells)
14-fold, from 0.1 to 1.4 x 106/node (Fig. 2)
, and 7-fold (0.1 to
0.7 x 106/node) after rGM-CSF
administration (Fig. 2)
. Another advantage of using recombinant avipox
viruses to deliver GM-CSF may be the duration of the enrichment of APCs
within the regional nodes. Upon cessation of rGM-CSF, the lymph nodes
enriched with APCs return to pretreatment levels within 4–5 days (Fig. 2)
. As shown in Figs. 1
and 2
, the increase in the number of class
II+ and
CD11c+/I-Ab+ cells/node
that were present at 7 days after injection of either avipox-GM-CSF
virus were sustained for 21–28 days. In fact, lymph node cells
isolated 21 days after avipox-GM-CSF injection generated a more robust
allospecific CTL response in vitro, indicating their
functional integrity (Fig. 3)
. One might argue that the recombinant
avipox viruses produce a depot of GM-CSF, and the prolonged changes in
the regional nodes represent a release of the cytokine. That seems
unlikely because of the relatively short in vivo half-life
of GM-CSF. Avipox viruses are known to infect mammalian cells without
causing cytolysis and are extremely efficient at expressing inserted
genes. A more plausible explanation is that the sustained increase of
APCs in the regional lymph nodes is attributable to the continuous
production of GM-CSF by the recombinant avipox viruses, which remain at
the injection site. The continual local release of GM-CSF stimulates
the prolonged migration of APCs into the injection site and the
draining regional nodes. Reasons for the loss of the GM-CSF effects
after 28 days remain to be determined.
If recombinant avipox viruses may be used as delivery vehicles for
GM-CSF or other agents, several questions need to be addressed. From an
anticancer vaccine standpoint, an important consideration is whether
coadministration of recombinant avipox viruses engineered to express a
tumor antigen and a cytokine are compatible. Vaccination of CEA.Tg mice
with a recombinant vaccinia-CEA virus (37)
was known to
induce CEA-specific humoral and cell-mediated immunity that was
relatively weak but did confer some protection from tumor challenge.
The present findings demonstrate that avipox-CEA vaccination of CEA.Tg
mice can also generate CEA-specific host immunity that, in turn,
mediates a transient slowing of the growth of CEA-positive tumors
(Table 3)
. Incorporating GM-CSF, either as a recombinant avipox virus
or protein, increased the CEA-specific antibody (Fig. 3)
,
CD4+ proliferation (Table 2)
, and
CD8+-mediated lysis (Fig. 6)
in
avipox-CEA-vaccinated CEA.Tg mice. In fact, the anti-CEA-specific
cellular immune responses were significantly more potent in those
CEA.Tg mice in which avipox-GM-CSF, not rGM-CSF, was the
immunoadjuvant. Thus, the data argue that recombinant avipox viruses
expressing a tumor antigen and GM-CSF are compatible and can be
coadministered to prime and augment antigen-specific host immunity.
Moreover, if the recombinant avipox-CEA virus produces CEA continuously
for 21–28 days, then the coexistence of antigen with elevated local
GM-CSF might result in a continuous loading of dendritic cells with
tumor antigen. Although that may explain the improved cellular response
to CEA when avipox-GM-CSF was the immunoadjuvant, one is left to
speculate why the increase in CEA-specific host immunity did not
translate into more potent antitumor responses in those CEA.Tg mice.
Inherent within those expectations are the pitfalls involved in
comparing immune function in ex vivo assays with in
vivo antitumor immunity. In the case of the CEA.Tg mice, host
immunity was generated against a self antigen that may introduce
factors (i.e., tolerance) that might dampen the expected
antitumor response. Furthermore, the rapid growth of the MC-38-CEA-2
s.c. tumors necessitated that the prime/boost vaccinations be given
biweekly, an interval that, most probably, did not allow for the
optimal development of CEA-specific host immunity. Therefore, a direct
comparison between host and antitumor immunity would be better served
in experimental models using spontaneous tumors that develop over an
extended time period that would permit these cancer vaccines to be
administered at monthly intervals. Such experimental models are
currently under development within the laboratory. Finally, the optimal
use of those avipox viruses expressing GM-CSF, particularly when
combined with different types of immunogens, remains to be determined.
Indeed, preliminary data using a protein or a recombinant fowlpox
vector containing a tumor antigen and costimulatory molecules
(38)
as immunogens show that 107 pfu
of avipox-GM-CSF may be superior than 108 pfu.
Ongoing studies on the dose and dose schedules of avipox-GM-CSF when
used with different immunogens are addressing these issues.
Recombinant avipox viruses are attractive candidates for cancer vaccines because of their ability to express a wide variety of gene products (21, 22, 23) and their documented safety in clinical trials (39, 40, 41) . Previous exposure to vaccinia does not alter the immune response to recombinant avipox viruses (42) ; in diversified prime-and-boost protocols, the two viruses induced antitumor immunity in murine models (35) . The present findings broaden the use of recombinant avipox viruses to include GM-CSF delivery to enrich a vaccination site with APCs, thereby augmenting the generation of host immunity. Another finding was the ability of avipox(A)-GM-CSF to enrich the regional lymph nodes with APCs after repeated injections, despite the presence of serum IgG anti-avipox titers. Nonneutralizing anti-avipox serum titers have been seen in other studies (21 , 43) . In a recent clinical trial,4 multiple injections of avipox-CEA administered to patients with advanced CEA-positive tumors led to an ongoing increase in the CEA-specific T-cell precursor frequencies. Another advantage of using a recombinant avipox-GM-CSF virus would be the ease of mixing it with an immunogen, such as avipox-CEA, and administering the vaccine as a single injection that would simplify vaccine design while, possibly, maximizing the immunoadjuvant effects of GM-CSF.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Laboratory of Tumor Immunology and Biology, Building 10,
Room 8B09, MSC 1750, Bethesda, MD 20892. 
2 The abbreviations used are: GM-CSF,
granulocyte/macrophage-colony stimulating factor; rGM-CSF, recombinant
GM-CSF; avipox(F), recombinant fowlpox virus; avipox(A), recombinant
canarypox (ALVAC) virus; CEA, carcinoembryonic antigen; CEA.Tg, CEA
transgenic; APC, antigen-presenting cell; FBS, fetal bovine serum;
DPBS, Dulbecco’s phosphate buffered saline; MFI, mean fluorescence
intensity; pfu, plaque-forming unit; FACS, fluorescence-activated cell
sorting; MLC, mixed lymphocyte culture; IL, interleukin; LU, lytic
unit.
3 J. Schmitz, J. Hodge, J. Schlom, and J. Greiner.
Generation of a CEA peptide-specific T-cell line that exhibits
antitumor properties in CEA transgenic mice, manuscript in
preparation.
4 J. L. Marshall, R. J. Hoyer, M. A. Toomey, K.
Faraguna, P. Chang, E. Richmond, J. E. Pdeicano, E. Gehan, R. A.
Peck, P. Arlen, K. Y. Tsang, and J. Schlom. Phase I study in cancer
patients of a diversified prime and boost immunization protocol using
recombinant virus and recombinant nonreplicating avian pox virus to
elicit anti-carcinoembryonic antigen immune response, submitted for
publication.
Received 5/ 8/00. Accepted 11/ 1/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and accumulation of dendritic cells at vaccination sites in vivo. Cancer Immunol. Immunother., 48: 123-131, 1999.[Medline]
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) or rGM-CSF (
).
Untreated CEA.Tg mice (dashed line) received HBSS.
Vaccination with avipox(A)-RG alone or combined with either
avipox(A)-GM-CSF or rGM-CSF did not alter overall survival (data not
shown). D, CEA.Tg mice that rejected MC-38-CEA-2 tumors
after being vaccinated with avipox(A)-CEA and avipox(A)-GM-CSF
(n = 5; 
). In C and D, B6 mice
received avipox(A)-GM-CSF (•) or avipox(A)-RG (
, all panels) received 100 µl of
HBSS. After sacrifice, inguinal lymph nodes were removed, and the total
number of cells multiplied by the percentage of class II+
cells/node was used to calculate the total number of class
II+ cells/node. Data are the means of three to four
experiments in which each time point was examined at least twice;
bars, SE.
).
Recombinant GM-CSF (20 µg, •) was given to a cohort of mice
(n = 15) for 4 consecutive days
(solid horizontal line). Control mice (dashed
line) received HBSS. Lymph nodes were removed, and the total
number of CD11c+/I-Ab+ cells was determined as
summarized in "Materials and Methods." Data are the means of a
composite of findings from three to four separate experiments in which
each time point was examined two to three times; bars,
SE.
) or anti-GM-CSF IgG (
