Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3730-3736
Immunostimulatory CpG Oligodeoxynucleotides Enhance the Immune
Response to Vaccine Strategies Involving Granulocyte-Macrophage
Colony-Stimulating Factor
By
Hsin-Ming Liu,
Sally E. Newbrough,
Sudershan K. Bhatia,
Christopher E. Dahle,
Arthur M. Krieg, and
George J. Weiner
From the University of Iowa Interdisciplinary Graduate Program in
Immunology, University of Iowa Department of Internal Medicine,
University of Iowa Cancer Center, and Iowa City Veterans Affairs
Medical Center, Iowa City, IA.
 |
ABSTRACT |
Immunostimulatory oligodeoxynucleotides containing the CpG motif
(CpG ODN) can activate various immune cell subsets and induce production of a number of cytokines. Prior studies have demonstrated that both CpG ODN and granulocyte-macrophage colony-stimulating factor
(GM-CSF) can serve as potent vaccine adjuvants. We used the 38C13
murine lymphoma system to evaluate the immune response to a combination
of these two adjuvants. Immunization using antigen, CpG ODN, and
soluble GM-CSF enhanced production of antigen-specific antibody and
shifted production towards the IgG2a isotype, suggesting an enhanced
TH1 response. This effect was most pronounced after repeat
immunizations with CpG ODN and antigen/GM-CSF fusion protein. A single
immunization with CpG ODN and antigen/GM-CSF fusion protein 3 days
before tumor inoculation prevented tumor growth. CpG ODN enhanced the
production of interleukin-12 by bone marrow-derived dendritic cells and
increased expression of major histocompatibility complex
class I and class II molecules, particularly when cells were pulsed
with antigen/GM-CSF fusion protein. We conclude that the use of CpG ODN
in combination with strategies involving GM-CSF enhances the immune
response to antigen and shifts the response towards a TH1 response and
that this approach deserves further evaluation in tumor immunization
approaches and other conditions in which an antigen-specific TH1
response is desirable.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
A NUMBER OF bacterial products, such as
lipopolysaccaride, are known to stimulate mammalian immune responses.
Recently, bacterial DNA itself has been reported to be one such
molecule.1-9 One of the major differences between bacterial
DNA, which has potent immunostimulatory effects, and vertebrate DNA,
which does not, is that bacterial DNA contains a higher frequency of
unmethylated CpG dinucleotides than does vertebrate DNA.1
Select synthetic oligodeoxynucleotides containing unmethylated CpG
motifs (CpG ODN) have immunologic effects similar to those seen with
bacterial DNA. CpG ODN can induce activation of B cells, NK cells, and
antigen-presenting cells (APCs), such as monocytes and
macrophages.1-6 It can also enhance production of cytokines
known to participate in the development of an active immune response,
including tumor necrosis factor-
, interleukin-12 (IL-12), and
IL-6.7-9
The molecular mechanisms responsible for CpG ODN-induced immunologic
effects are still under investigation. Nevertheless, the
immunostimulatory effects of CpG ODN may be useful therapeutically. We
have demonstrated that CpG ODN can enhance antibody-dependent cellular
cytotoxicity and improve the in vivo efficacy of monoclonal antibody
therapy in a syngeneic murine lymphoma model.10 In addition, mice immunized with tumor antigen using CpG ODN as an adjuvant were protected from tumor challenge to a degree similar to
that seen in mice immunized using complete Freund's adjuvant (CFA).11 CpG ODN was as effective as CFA at enhancing
production of anti-idiotype (Id) antibody after
immunization and was more effective at inducing production of IgG2a
anti-Id, suggesting a TH1 response. Evaluation of CpG ODN in other
systems has yielded similar findings, including an enhanced cellular
response.12-16
Soluble granulocyte-macrophage colony-stimulating factor (GM-CSF) has
also been shown to function as an immune adjuvant in tumor antigen
immunization.17-20 Other promising approaches based on this
cytokine include immunization with antigen/GM-CSF fusion proteins,21 immunization using tumor cells transfected with GM-CSF,22 immunization using dendritic cells cultured in
GM-CSF,23,24 and immunization using dendritic cells pulsed
with antigen/GM-CSF fusion proteins.25 Although some of
these studies have reported enhanced cellular immunity, most describe
development of a humoral response. A reagent capable of enhancing the
adjuvant effect of GM-CSF and directing it toward a TH1 response would
be very useful in tumor antigen immunization and other strategies for
which an antigen-specific TH1 response is desirable. We therefore
assessed whether CpG ODN and immunization approaches using GM-CSF are
synergistic and capable of inducing a pronounced antigen-specific TH1
response.
| |
MATERIALS AND METHODS |
Tumor model and tumor antigens.
The 38C13 murine B-cell lymphoma model has been used extensively in
studies of antibody-based therapy and active immunization of
lymphoma.20,26-29 The Id of the 38C13 surface IgM serves as a highly specific tumor-associated antigen.30 Id was
obtained from the supernatant of a cell line that secretes 38C13 IgM as described31 and purified by protein A affinity
chromatography. Purified Id was conjugated to keyhole limpet hemocyanin
(KLH) using gluteraldehyde and used as the immunogen. Id-negative 38C13 (38C13 T1A) is a variant of 38C13 that contains cytoplasmic 38C13 heavy
chain but produces no light chain and fails to express surface Id.27 The cell line that produces 38C13 Id/murine GM-CSF
fusion protein (Id/GM-CSF) was kindly provided by Dr Ronald Levy
(Stanford University, Palo Alto, CA). This cell line was
cultured in a hollow fiber reactor (Unisyn Technologies, Hopkinton,
MA), and fusion protein was obtained by protein A affinity
chromatography. The fusion protein consists of the 38C13 Id heavy and
light chain variable regions, the human IgG1 heavy and light chain
constant regions, and murine GM-CSF sequences.21
Bifunctional reactivity was confirmed by enzyme-linked immunosorbent
assay (ELISA) before use. Plates were coated with anti-Id, serial
dilutions of fusion protein were added, and the presence of bound
GM-CSF moieties was assessed by probing with anti-GM-CSF antibodies.
38C13 Id/human GM-CSF fusion protein was obtained in a similar manner
and used as a control.
Immunization.
Three phosphorothioate CpG ODNs were purchased commercially and
produced under GMP conditions (Oligos Etc, Wilsonville, OR). Two of
these ODN sequences (1758 and 1826) were immunostimulatory and had
similar effects in all assays. CpG ODN 1758 was used unless stated
otherwise. ODN 1758 had the sequence
TCTCCCAGCGTGCGCCAT and ODN 1826 had the sequence
TCCATGACGTTCCTGACGTT. ODN 1758 and ODN 1826 were
unmethylated. Prior studies demonstrated that nonimmunostimulatory ODN
has little adjuvant effect.11 Nevertheless, ODN 1812 was used as a control in select studies. ODN 1812 is identical to ODN 1758 but contains CpG motifs with methylated cytosines. No detectable
endotoxin was present in the CpG ODN preparations by LAL assay. Murine
GM-CSF for in vitro production of dendritic cells was purchased
commercially (PeproTech, Rocky Hill, NJ). GM-CSF for in vivo studies
was kindly supplied by Immunex (Seattle, WA).
Female C3H/HeN mice, obtained from Harlan-Sprague-Dawley, were housed
in the University of Iowa Animal Care Unit and used at 6 to 9 weeks of
age. Each mouse was immunized subcutaneously with indicated antigen and
adjuvant in a total volume of 200 µL, using phosphate-buffered saline
(PBS) as a vehicle.
ELISA determination of anti-Id levels.
Serum was obtained by retroorbital puncture from mice after inhalation
anesthesia with metophane. Microtiter plates were coated with 5 µg/mL
38C13 IgM or irrelevant IgM overnight. IgM-coated plates were blocked
with 5% milk, and serial dilutions of serum were added. Serum from
naive mice to which a known concentration of monoclonal anti-Id was
added served as a standard. Plates were washed, and alkaline
phosphatase-labeled heavy chain-specific goat antimouse IgG, IgG1, or
IgG2a (Southern Biotechnology Associates, Birmingham, AL) was added,
followed by the colorimetric substrate p-nitrophenylphosphate. Plates
were evaluated using a microplate reader. Test curves were
compared with standard curves to determine the concentration of
anti-Id. Values were considered valid only if the standard curves and
the sample curves had the same shape. Reactivity of serum with a
control, irrelevant murine IgM was evaluated in parallel and was
negative in all assays, confirming that the immune response was not due
to development of an isotypic response.
In vivo survival studies.
Three days after a single subcutaneous immunization using the indicated
antigen and adjuvant, mice were inoculated
intraperitoneally with 1,000 viable 38C13 cells. Cells
were growing in log phase for at least 4 days before inoculation. Mice
that developed tumor displayed inguinal and abdominal masses, ascites,
and cachexia. All mice that developed tumor died. Survival was
determined, and significance with respect to time to death was assessed
using Cox regression analysis. For statistical purposes, survival of 60 days was assigned for mice that remained tumor free. All such mice
remained tumor free indefinately and were monitored for a minimum of
100 days.
Dendritic cell production and stimulation.
Dendritic cells were obtained using a modification of the approach
previously described.23,32 Briefly, bone marrow cells were
obtained by flushing the femurs and tibias of naive 6- to 8-week-old
C3H/HeN mice. Red blood cells were lysed and T cells were removed by
complement-mediated lysis using a mixture of anti-CD3 (145.2C11),
anti-CD4 (GK1.5), and anti-CD8 (53.6.7) antibodies. B cells were then
removed by panning using a flask coated with anti-B220. The remaining
cells were allowed to adhere overnight. Nonadherent cells were cultured
in media supplemented with 1,000 U/mL GM-CSF and 1,000 U/mL murine IL-4
(muIL-4; PeproTech) at a concentration of 1.25 × 105
cells/mL. Media was changed after 4 days, and dendritic cells were
harvested 7 days after bone marrow harvest. Dendritic cell phenotype
and morphology were confirmed by flow cytometric analysis and scanning
electron microscopy. Dendritic cells were washed and counted, and 1 × 105 were cultured for 18 hours in a total volume of
200 µL with antigen at a final concentration of 100 µg/mL and CpG
ODN at a final concentration of 5 µg/mL. Immunophenotyping was
performed by flow cytometric analysis using fluorescein isothiocyanate
(FITC)-labeled anti-class I (anti-H-2Kb), anti-class II (anti-I-Ak),
anti-B7-1 (1G10), and anti-B7-2 (GL1; PharminGen, San Diego CA). For
measurement of cytokine levels, all samples were run in quadruplicate.
Supernatant was harvested and assayed by ELISA for the presence of IL-6
and IL-12, as described.9,33
| |
RESULTS |
CpG ODN further enhances development of an antibody response to Id-KLH
immunization when using GM-CSF as an adjuvant.
Although CpG ODN is known to induce production by APCs of a number of
cytokines including GM-CSF,34 it is unclear whether the
addition of CpG ODN to GM-CSF would enhance the immune response further. We therefore assessed the response in mice immunized with a
single subcutaneous injection of 50 µg of Id-KLH in PBS mixed in
aqueous solution with 50 µg of CpG ODN, 10 µg of GM-CSF, or a
combination of CpG ODN and GM-CSF. Serum was obtained weekly and
evaluated by ELISA for the presence of antigen-specific IgG (anti-Id
IgG). As shown in Fig 1, mice immunized
using both CpG ODN and GM-CSF developed the highest levels of anti-Id
IgG. The effect of these two adjuvants appeared to be additive.
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| Fig 1.
Production of anti-Id following immunization using a
combination of CpG ODN and soluble GM-CSF. Mice were immunized with 50 µg of Id-KLH as a single subcutaneous dose mixed in aqueous solution
with GM-CSF, CpG ODN, or both. Blood was obtained weekly, and serum was
evaluated for the presence of anti-Id IgG by ELISA. Normal mouse serum
supplemented with a known concentration of monoclonal anti-Id was used
as a standard. Three mice were included in each group. ( ) Id-KLH + GM-CSF; ( ) Id-KLH + CpG ODN; ( ) Id-KLH + GM-CSF + CpG
ODN.
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CpG ODN enhances production of anti-Id antibodies after immunization
with Id/GM-CSF fusion protein.
The Id/GM-CSF fusion protein consisting of the 38C13 variable regions,
human IgG constant regions, and murine GM-CSF (Id/GM-CSF) has been
shown to be an excellent immunogen.21 We evaluated whether
CpG ODN can further enhance the specific antibody response induced by
Id/GM-CSF. Mice were immunized with Id-KLH or Id/GM-CSF with and
without CpG ODN as an adjuvant. Serum was obtained weekly and anti-Id
IgG levels were determined. No toxicity was observed in any mice. As
shown in Fig 2, CpG ODN enhanced production
of anti-Id antibodies in response to Id/GM-CSF. This occurred at a
variety of doses of Id/GM-CSF. Anti-Id levels induced by immunization with Id/GM-CSF and control ODN were indistinguishable from those induced by immunization Id/GM-CSF alone (data not shown). In a separate
experiment, mice were immunized on day 0 and boosted on day 14 with the
same antigen and adjuvant. The combination of Id/GM-CSF and CpG ODN
induced remarkably high levels of anti-Id IgG after two immunizations
(Fig 3). Serum obtained 1 week after the
final immunization contained greater than 2.5 mg/mL anti-Id IgG. A
fusion protein consisting of 38C13 Id and human GM-CSF (Id/human
GM-CSF) was included as a control, because human GM-CSF is not active
in the murine system. Id/human GM-CSF was identical to Id/GM-CSF,
except that the murine GM-CSF sequences were replaced with human GM-CSF
sequences. Levels of anti-Id produced after immunization using Id/human
GM-CSF with or without CpG ODN were significantly lower than those seen
after Id/GM-CSF and similar to those seen with Id-KLH (data not shown),
demonstrating that biologically active GM-CSF was important for the
observed effects.
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| Fig 2.
Immunization using a combination of Id/GM-CSF fusion
protein and CpG ODN enhances production of antigen-specific IgG. Mice
were immunized with 50 µg of Id/GM-CSF as a single subcutaneous dose
with or without CpG ODN. Blood was obtained weekly, and serum was
evaluated for the presence of anti-Id IgG by ELISA. Normal mouse serum
supplemented with a known concentration of monoclonal anti-Id was used
as a standard. Three mice were included in each group. (A) Anti-Id
response was measured at various time points after immunization. ( )
Id-KLH; () Id-KLH; ( ) Id/GM-CSF; ( ) Id/GM-CSF + CpG ODN. (B) Anti-Id response was measured 4 weeks after immunization
with various doses of Id/GM-CSF. ( ) Control CpG ODN; () CpG
ODN.
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| Fig 3.
Repeated immunizations with a combination of Id/GM-CSF
fusion protein and CpG ODN induce high levels of antigen-specific IgG.
Mice were immunized with 50 µg of Id/GM-CSF as a subcutaneous dose
with or without CpG ODN on week 0 and again on week 2. Blood was
obtained weekly, and serum was evaluated for the presence of anti-Id
IgG by ELISA. Normal mouse serum supplemented with a known
concentration of monoclonal anti-Id was used as a standard. Three mice
were included in each group. () Id-KLH; () Id-KLH + CpG ODN;
() Id/GM-CSF; () Id/GM-CSF + CpG ODN.
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CpG ODN enhances production of antigen-specific antibody of IgG2a
isotype.
Enhanced production of IgG1 reflects a TH2 response, whereas
predominant IgG2a production indicates a TH1 response.35
Moreover, murine IgG2a is more effective than murine IgG1 at mediating
antibody-dependent cellular cytotoxicity, and monoclonal IgG2a works
better than monoclonal IgG1 with the identical variable region at
preventing tumor growth.36 We therefore performed isotype
analysis on anti-Id IgG and assessed for the presence of anti-Id IgG1
and IgG2a after immunization (Fig 4).
Immunization included various combinations of Id-KLH or Id/GM-CSF with
GM-CSF or CpG ODN. Serum was sampled 4 weeks after a single
immunization. CpG ODN induced enhanced production of anti-Id IgG2a
compared with that seen under the corresponding conditions without CpG
ODN. Similar IgG1/IgG2a ratios were seen at other time points (data not
shown).
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| Fig 4.
CpG ODN enhances production of antigen-specific antibody
of IgG2a isotype. Mice were immunized with a single dose using various
combinations of Id-KLH, GM-CSF, Id/GM-CSF fusion protein, and CpG ODN.
Serum was obtained 4 weeks after a single immunization. Anti-Id IgG1
and IgG2a was determined by ELISA. Three mice were included in each
group. () Anti-Id IgG2a; () anti-Id IgG1.
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Immunization using CpG ODN as an adjuvant along with Id/GM-CSF fusion
protein led to further protection of mice from tumor growth.
Tao and Levy21 found that the Id/GM-CSF fusion protein was
superior to Id-KLH at inhibiting growth of the 38C13 tumor. Our previous investigations demonstrated that the addition of CpG ODN to
Id-KLH enhanced protection in this same model.11 We
therefore evaluated whether CpG ODN can also serve as an effective
adjuvant with Id/GM-CSF immunization. Mice were challenged with tumor 3 days after a single immunization with Id/GM-CSF with or without CpG
ODN. Immunization using this schedule was only minimally effective in
our prior studies with Id-KLH (unpublished data). CpG ODN
1758 and CpG ODN 1826 were equally effective at prolonging survival when used alone or in combination with Id/GM-CSF. The data shown in
Fig 5 represent the combined results of
mice treated with CpG ODN 1758 and CpG ODN 1826. All unimmunized mice
and mice treated with CpG ODN without antigen developed tumor and died
within 50 days. Thirty percent of mice immunized with Id/GM-CSF alone
remained disease free, whereas 70% of the group immunized with
Id/GM-CSF and CpG ODN remained disease free. Mice immunized with
Id/GM-CSF and CpG ODN had survival that was statistically superior to
that seen with no immunization or treatment with CpG ODN alone
(P < .001). The difference between those immunized with
Id/GM-CSF alone versus those immunized with CpG ODN plus Id/GM-CSF
approached statistical significance (P = .072). To assess
whether the effect of immunization was specific, mice were immunized as
described above and challenged with either wild-type 38C13 cells or
Id-negative 38C13. Mice challenged with WT 38C13 were protected from
tumor growth, whereas no protection was seen for those challenged with Id-negative 38C13 (Table 1). This confirmed
that the protection was Id specific.
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| Fig 5.
CpG ODN enhances the protective effect of Id/GM-CSF. Mice
were immunized with a single injection of Id/GM-CSF and/or CpG
ODN and challenged with tumor 3 days later. Survival was followed for
100 days. All mice that were alive after 51 days remained tumor-free
for the entire observation period. Twenty mice were included in each
group. () No immunization; () CpG ODN; (×) Id/GM-CSF; ()
Id/GM-CSF + CpG ODN.
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Table 1.
Protection From Tumor Growth After Immunization With
Id/GM-CSF CpG ODN Is Limited to Tumors Expressing the Target
Antigen
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CpG ODN effects on dendritic cell phenotype.
The synergistic effects of CpG ODN and GM-CSF suggested the possibility
that these agents together may enhance expression of costimulatory
molecules or major histocompatibility complex (MHC) by
APCs. We therefore evaluated expression of these molecules by bone
marrow-derived dendritic cells. Flow cytometric analysis of dendritic
cells pulsed with Id/GM-CSF and/or CpG ODN demonstrated a
modest increase in expression of class I MHC in response to the
combination of Id/GM-CSF and CpG ODN. Baseline expression of CD80 and
CD86 expression by dendritic cells was high and was increased only
slightly by CpG ODN (CD86) or CpG ODN plus Id/GM-CSF (CD80;
Fig 6).
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| Fig 6.
Expression of MHC class I, MHC class II, CD80, and CD86
after pulsing of bone marrow-derived dendritic cells with Id/GM-CSF
and/or CpG ODN. () Id/GM-CSF + CpG ODN; () CpG ODN;
( ) Id/GM-CSF; ( ) no treatment.
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CpG ODN enhances production of IL-12 by dendritic cells pulsed with
Id/GM-CSF.
The enhanced TH1 response to antigen could be explained by the ability
of CpG ODN to enhance production of IL-12 by APCs such as dendritic
cells. We therefore assessed whether CpG ODN is capable of enhancing
the production of IL-12 by bone marrow-derived dendritic cells that
were pulsed with antigen, including Id/GM-CSF. As shown in
Fig 7, pulsing of dendritic cells with CpG
ODN increased production of IL-12, particularly when cells were also
pulsed with Id/GM-CSF. IL-6 production by dendritic cells was also
increased by the addition CpG ODN to Id/GM-CSF, although the effect was
less pronounced than for IL-12. The impact of GM-CSF alone on dendritic
cell production of cytokines was not studied, because these cells were
generated using GM-CSF.
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| Fig 7.
Production of IL-6 and IL-12 by dendritic cells pulsed
with Id-KLH or Id/GM-CSF. Bone marrow-derived dendritic cells were
pulsed with antigen with and without CpG ODN for 18 hours. Production
of IL-12 and IL-6 was determined by ELISA. () CpG ODN; () no CpG
ODN.
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| |
DISCUSSION |
GM-CSF and CpG ODN both enhance development of an active immune
response, yet may play very different roles in that response. Whereas
the complex effects of GM-CSF on APCs are still being defined, it is
clear that GM-CSF can stimulate granulocyte and monocyte production and
activate various aspects of APC function. There is some evidence that
much of the adjuvant effect is related to enhanced phagocytosis or
pinocytic uptake of antigen as opposed to enhanced presentation of
antigen.37 In broad terms, GM-CSF can be thought of as a
cytokine that attracts APCs to the site and induces antigen uptake by
those APCs.
Even less is known about the mechanisms responsible for the adjuvant
effects of CpG ODN. Some hints can be obtained from analysis of the
immune activation seen with bacterial DNA, because this is similar to
that seen with CpG ODN. Bacterial DNA plays a role in the response to
intracellular microbial infections and appears to enhance the response
to microbial antigen that is already present within the
APC.3,8 Bacterial DNA can induce a cellular, ie, a TH1,
response, which is likely beneficial when the invading organisms are
intracellular. Thus, bacterial DNA, and CpG ODN, appear to enhance the
immune response by upregulating costimulatory molecules and
MHC12 and inducing production of TH1-like cytokines that,
in turn, enhance the function of effector cells, including T cells, B
cells, and NK cells.9,15
The combination of GM-CSF and CpG ODN could therefore stimulate
different steps in the induction of the immune response. The primary
affect of GM-CSF could be in increasing antigen uptake, whereas the
primary affect of CpG ODN could be on enhancing the downstream response
to antigen, including production of cytokines involved in effector cell
activation. In addition, CpG ODN contributes by synergistically
promoting B-cell activation through the antigen receptor and so
preferentially activating antigen-specific B cells.1
The data presented above indicate immunization strategies involving the
combination of GM-CSF and CpG ODN are particularly effective. CpG ODN
and soluble GM-CSF were additive in their ability to induce anti-Id IgG
after immunization with Id-KLH. Murine GM-CSF has a short
half-life,38 which may explain the lack of a dramatic adjuvant effect. APCs may not have had adequate exposure to GM-CSF. CpG
ODN was particularly effective at increasing production of anti-Id IgG
after immunization with an Id/GM-CSF fusion protein. Prior studies by
Tao and Levy21 demonstrated that this fusion protein has
enhanced GM-CSF activity compared with native GM-CSF, most likely due
to the bivalent nature of the construct. In addition, this fusion
protein may have been more effective at delivering both the GM-CSF and
antigen to the APC. In our studies, remarkable levels of anti-Id IgG
were achieved after repeated immunization with Id/GM-CSF and CpG ODN.
Importantly, CpG ODN shifted the response to a IgG2a under all
conditions studied, including immunization with soluble GM-CSF and the
Id/GM-CSF fusion protein, suggesting an enhanced TH1 response.
Immunization using this approach translated into protection from tumor
growth only 3 days after immunization with Id/GM-CSF and CpG ODN. This
is the most effective protection reported to date in this extensively
studied model. As with any model system, we need to consider the
specifics of the model when interpreting results. Passive
administration of anti-Id monoclonal antibody has antitumor activity
against 38C13.26,28,36 Although monoclonal antibodies of
the IgG2a isotype are the most effective therapeutically, those of the
IgG1 isotype also demonstrate antitumor activity. This may explain the
significant antitumor activity of Id/GM-CSF alone, despite its
relatively limited ability to induce a TH1 response. Other
investigations suggest cellular immunity, which is enhanced by CpG
ODN,14,15 also plays a role in the antitumor response.20 For technical reasons we have been unable to
demonstrate unequivocally that a cellular response was playing a role
in the observed antitumor activity. This is currently being explored in
other systems. Nevertheless, it is likely that the enhanced antitumor
activity of the combination of Id/GM-CSF and CpG ODN documented above
was related to enhancement of both the humoral and cellular response.
In summary, GM-CSF can enhance antigen uptake by APC and is known to be
effective as an immune adjuvant. CpG ODN is an exciting new immune
adjuvant that appears to function by different mechanisms, including
activating immune effector cell function. In the studies outlined
above, we demonstrate that the combination of immunization strategies
involving GM-CSF and CpG ODN are effective at inducing an enhanced TH1
type response and protecting from tumor growth. Further investigation
into the precise mechanisms involved in the response to these two
agents, both separately and together, is required before the true
promise of this approach can be elucidated.
| |
ACKNOWLEDGMENT |
The authors thank Ronald Levy, MD (Stanford University, Stanford, CA)
for supplying the cell lines that produce the Id/GM-CSF fusion protein;
Elaine Thomas, PhD (Immunex) for murine GM-CSF; Justin Fishbaugh
(University of Iowa Flow Cytometry Facility); Charles Davis, PhD, for
statistical consultation; Anna M. Acosta and Laurie Love-Homan for
expert technical assistance; and Timothy Ratliff, PhD, and Donald
MacFarlane, MD, for helpful suggestions.
| |
FOOTNOTES |
Submitted February 19, 1998;
accepted July 2, 1998.
Supported by the Department of Veteran's Affairs Medical Research
Service and the University of Iowa Cancer Center.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to George J. Weiner, MD, Department of
Internal Medicine, University of Iowa, C32K GH, 200 Hawkins Dr, Iowa
City, IA 52242.
| |
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Abstract
Introduction