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Prepublished online as a Blood First Edition Paper on September 12, 2002; DOI 10.1182/blood-2002-02-0391.
NEOPLASIA
From the Department of Immunology, The Scripps Research
Institute, La Jolla, CA; Department of Pediatrics, Charité
Children's Hospital, Berlin, Germany; and EMD-Lexigen
Research Center, Bedford, MA.
The successful induction of a T-cell-mediated tumor-protective
immunity against poorly immunogenic malignancies remains a major
challenge for cancer immunotherapy. We achieved this by immunization
with a tyrosine hydroxylase (mTH)-based DNA vaccine, enhanced with the
posttranscriptional regulatory acting RNA element (WPRE), derived from
woodchuck hepatitis virus in combination with an antibody-cytokine
fusion protein (ch14.18-IL-2) that targets interleukin-2 (IL-2) to the
tumor microenvironment. This DNA vaccine mTH-WPRE was carried by
attenuated Salmonella typhimurium and applied by oral
gavage in a mouse model of neuroblastoma. Mice immunized with the
mTH-WPRE vaccine, and which additionally received a boost with
suboptimal doses of ch14.18-IL-2, were completely protected against
hepatic neuroblastoma metastases. In contrast, all controls presented
with disseminated metastases. Both T-cell and natural killer (NK)
cell-dependent mechanisms were involved in the induction of a systemic
tumor-protective immunity. Thus, up-regulation of interferon- The induction of an effective systemic protective
immunity against malignancies is a major objective for cancer
immunotherapy. The goal is to break peripheral tolerance of the immune
system toward poorly immunogenic tumors by activating cytotoxic T cells (CTLs) and/or natural killer (NK) cells to effectively eradicate disseminated tumor metastases. In the field of DNA vaccination, it has
been shown by others and ourselves1-3 that this can be achieved in melanoma with vaccines encoding TRP1, TRP2, and gp100 antigens4 and in colon carcinoma with the CEA
antigen.5-7 In this regard, it has been reported that
autoreactive T cells that escape negative selection in the thymus can
be activated in the periphery8 to be directed against the
tumor, resulting in an efficient tumor-protective immune response.
Importantly, there is also evidence that, aside from T cells, the
innate immune system plays a role in the host's defense against
neuroblastoma via NK cells, in particular if interleukin-2 (IL-2) is
included in the therapy protocol. In fact, eradication of murine
neuroblastoma metastases by a recombinant human/mouse chimeric
antiganglioside GD2-IL-2 fusion protein (ch14.18-IL-2) was entirely
NK-cell dependent when administered in therapeutic doses.9
Moreover, once neuroblastoma patients were treated with
IL-2-transduced autologous neuroblastoma tumor cells, they presented
with an increase in activated T cells and NK cells, indicating a role
for both effector populations in IL-2 immunotherapy of this
malignancy.10 We also recently reported that a mouse
tyrosine hydroxylase (mTH)-based DNA vaccine elicits an
antitumor effect in a poorly immunogenic murine model of
neuroblastoma.11 However, therapy with this DNA vaccine
containing solely the tumor-associated antigen mTH was of limited
efficacy as indicated by only a partial reduction in subcutaneous tumor growth. Therefore, we tested the hypothesis that the PRE sequence of
the woodchuck hepatitis virus (WPRE), in conjunction with mTH as a
target antigen, could improve the systemic tumor-protective immune
response against neuroblastoma metastases and provide complete tumor
protection. The rationale for this approach is based on the observation
that human hepatitis B virus (HBV) and woodchuck hepatitis B virus
(W-HBV) contain posttranscriptional-acting RNA elements (PRE) that
significantly enhance viral gene expression; however, woodchuck PRE
sequences enhance gene expression to a greater extent than their
equivalents of the human hepatitis B virus.12 In fact, its
WPRE sequence acts independently of transcription and splicing and thus
may improve gene expression by modification of polyadenylation, RNA
export, or translation.13,14 Moreover, recent in vitro
studies demonstrated gene expression of human multidrug resistance 1 (MDR1) and enhanced green fluorescent protein (EGFP) genes to be
enhanced by posttranscriptional modification with the WPRE
sequence.15 In addition, Popa et al suggested that
WPRE is directing messages into a CRM1-dependent mRNA export pathway in
somatic mammalian cells.16 These observations raised the
question as to whether this event could also occur in vivo and be used
to optimize the mTH-based DNA vaccine.
Interleukin-2 (IL-2), a well-known, strong promoter of T-cell
proliferation, which at low levels induces T-cell
expansion,17,18 provides the rationale for boosting an
initial immune response provoked by the mTH-WPRE vaccine with the
recombinant antibody-IL-2 fusion protein ch14.18-IL-2. This fusion
protein uses disialoganglioside GD2 (GD2) as a docking site on
neuroblastoma cells, where it is overexpressed and was already
successfully used as a target for immunotherapy in neuroblastoma
patients19-21 and mouse models of this malignancy as well
as other GD2-expressing tumors.22,23 In fact, IL-2
directed to the tumor microenvironment could locally amplify and expand
an initial immune response provoked by the mTH-WPRE vaccine.
Here, we demonstrate for the first time that posttranscriptional
modification of an antitumor DNA vaccine with the WPRE sequence results
in an enhanced immune response that in combination with the
ch14.18-IL-2 fusion protein leads to complete T-cell- and NK-cell-mediated protection from neuroblastoma metastases.
Construction and characterization of DNA vaccines encoding mTH
and WPRE
Generation and characterization of tumor-specific antibody-IL-2
fusion protein
Neuroblastoma tumor model and treatment protocol
Transformation of attenuated S typhimurium (SL 7207) with plasmids encoding a DNA vaccine Cultured bacteria were washed with deionized water and then admixed with either mTH-WPRE, mTH, or the empty vector DNA (3 × 1010 bacteria, 1 µg plasmid DNA) and electroporated with a Bio-Rad Gene Pulser (Hercules, CA) at 2.5 kV, 25 microfarads (µF), and 200 in a 0.1-cm
electroporation cuvette. The transformed bacteria were cultured in
Luria-Bertani medium (LB) supplemented with 20 mM glucose (SOC)
medium for 30 minutes at 37°C under vigorous shaking and then plated
onto LB plates under the selection of 50 µg/mL ampicillin.
Immunofluorescence staining and confocal microscopy COS-7 cells were maintained in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum (FCS), 1% glutamine, and 1% penicillin/streptomycin. Transfection of COS-7 cells was performed with Superfect (Qiagen, Valencia, CA) according to the manufacturer's guidelines, and 24 hours later cells were analyzed by fluorescence microscopy. Cells grown on coverslips were fixed for 10 minutes with 4% paraformaldehyde; mTH was detected with rabbit-anti-mTH antibody (Chemicon, Temecula, CA) diluted 1:100 in phosphate-buffered saline (PBS) containing 10% FCS and 0.1% saponin and stained with Texas red-labeled donkey antirabbit antibody immunoglobulin G (IgG) (Jackson Immuno Research, West Grove, PA). Cells were analyzed with a MRC1024 confocal microscope (Bio-Rad).Cytotoxicity assay Mouse splenocytes were cultured for 2 days with irradiated NXS2 tumor cells at a tumor cell-effector cell ratio of 1:100. Subsequently a 4-hour 51chromium (51Cr) release assay was performed as described previously.25 The percentage of target cell lysis was calculated as follows: experimental release (cpm) spontaneous release (cpm)/total release
(cpm) spontaneous release (cpm) × 100 = percent cytotoxicity.
Flow cytometry Staining for expression of cell surface antigens and intracellular antigens was performed as described previously.25 Splenocytes of control mice and immunized mice were isolated as described for the cytotoxicity assay and stimulated for 5 hours with 5 ng/mL phorbol myristate acetate (PMA) (Sigma, St Louis, MO) and 500 ng/mL ionomycin (Sigma). All stimulated cultures contained 1 µg/mL Brefeldin A (Sigma) to block protein transport into post-Golgi compartments and to allow cytokines to accumulate within cells. Cells were analyzed on a FACScan flow cytometer using Cell Quest software (Becton Dickinson, San Diego, CA).Statistics The statistical significance of differential findings between experimental groups of animals was determined by the 2-tailed Student t test. Findings were regarded as significant if 2-tailed P values were .05.
Effect of WPRE-enhanced DNA vaccine on in vitro expression of mTH antigen The posttranscriptional regulatory PRE element of the woodchuck hepatitis virus was cloned and inserted as described in "Materials and methods," directly downstream of the tumor-associated mTH antigen and following the stop codon. The correct sizes of the mTH and the WPRE fragments were analyzed by restriction enzyme digestion with a 1497-bp fragment encoding for mTH and a 641-bp fragment for the WPRE sequence (Figure 1). Integrity of the genes was verified by nucleotide sequencing. Gene expression of the DNA constructs was determined by confocal microscopic analysis. For this purpose, COS-7 cells were transduced with plasmids encoding mTH in the presence or absence of the WPRE sequence. Figure 2 illustrates protein expression of the mTH antigen in the cytoplasm (original magnification × 20), with protein accumulation appearing predominantly perinuclear as demonstrated at higher magnification (× 40).
Induction of complete systemic tumor protection by posttranscriptionally enhanced WPRE DNA vaccine in combination with a targeted IL-2 boost To assess whether the WPRE-enhanced DNA vaccine could induce effective systemic tumor-protective immunity, A/J mice were immunized twice by oral gavage at 2-week intervals with an attenuated strain of S typhimurium (SL7207) as vaccine carrier as described previously.11 This attenuated strain of salmonella is unable to produce aromatic amino acids necessary for its replication in vivo and consequently dies once it reaches the secondary lymphoid tissue after passing through the M cells of intestinal mucosa into the Peyer patches, where it liberates copies of the DNA vaccine. The animals received either mTH, the mTH-WPRE DNA vaccine with or without an additional boost of subcurative doses of ch14.18-IL-2 fusion protein, or the empty vector as a control. One week after immunization, experimental metastases were induced intravenously by a lethal challenge with wild-type NXS2 neuroblastoma cells. Mice immunized with the mTH-WPRE vaccine revealed significantly less metastases as assessed by liver weight and compared with mice immunized with mTH alone or empty vector controls (P .04), indicating that addition
of the WPRE sequence significantly enhanced the antitumor immune
response. Moreover, boosts with the ch14.18-IL-2 fusion protein
following immunization with the mTH vaccine also significantly enhanced antitumor immune response, with 4 of 6 mice remaining tumor free compared with animals immunized with mTH alone or empty vector controls, where all mice developed fulminant metastases
(P .03). However, only if the mTH-WPRE vaccine was
combined with ch14.18-IL-2 boosts, 100% of the mice were completely
protected against hepatic neuroblastoma metastases when evaluated after
28 days (Figure 3; Table
1). These findings indicate
that the WPRE-enhanced DNA vaccine plus the ch14.18-IL-2
fusion protein boost is most effective in inducing complete
tumor-protective immunity against neuroblastoma metastases in all
experimental animals. Thus, the WPRE sequence can effectively enhance
the in vivo antitumor immune response in conjunction with a
tumor-associated antigen. However, a combination of both of
these components is required to markedly enhance tumor-protective immunity, because neither one of the components alone is capable of
protecting 100% of the mice.
Effector mechanisms involved in systemic tumor-protective immune response induced by the WPRE-enhanced DNA vaccine boosted with the ch14.18-IL-2 fusion protein To identify immune effector cells responsible for therapy-induced tumor protection, splenocytes were harvested 2 days after the final boost with ch14.18-IL-2 from mice of all experimental groups. These cells were stained for CD8 and intracellular interferon- (IFN-) and analyzed by flow cytometry. Both treatment
groups receiving the antibody-IL-2 fusion protein revealed
significant up-regulation of IFN-+ CD8+ T
cells. This effect was independent of whether previous DNA vaccination
included the WPRE-sequence or not, indicating that the boost with
targeted IL-2 leads to the activation of tumor-specific T cells.
Interestingly, enhancement of the mTH vaccine with the WPRE sequence
alone did not affect the CD8+ T-cell compartment, and
administration of only the ch14.18-IL-2 fusion protein in subcurative
dosages resulted neither in antitumor effects nor in up-regulated
IFN-+ CD8+ T cells (Figure
4). In contrast, only the ch14.18-IL-2
boosts administered after DNA vaccination with or without WPRE produced a doubling of IFN-+ CD8+ T cells. This
finding points to the importance of combination treatments with DNA
vaccines followed by cytokine boosts for the induction of optimal
CD8+ T-cell-mediated immune responses.
Tumor-specific cytotoxic activity, determined by a standard
51Cr release assay with splenocytes isolated from mice of
each experimental group, indicated that only animals immunized with the
mTH-WPRE DNA vaccine plus ch14.18-IL-2 boosts produced a 3-fold
increase in specific cytolysis of NXS2 tumor target cells over controls (Figure 5). This finding indicates that a
tumor-specific CTL response requires this combined treatment regimen
because neither of these components alone could induce increased
tumor-specific cytotoxic activity.
Significantly, previous experiments demonstrated that, once the
ch14.18-IL-2 fusion protein was administered in therapeutic dosages,
the eradication of neuroblastoma metastases was entirely NK-cell
dependent.9 This finding led to the assumption that besides CD8+ T cells the innate immune system may be
activated by the mTH-WPRE DNA vaccine, especially following boosts with
the ch14.18-IL-2 fusion protein. Therefore, we investigated the role
of NK cells in a second set of experiments where all A/J mice in the
experimental groups were depleted of NK cells in vivo with
anti-asialo-GM1 antiserum (Table 2). In
this case, systemic protective immunity against NXS2 wild-type tumor
cells was completely abrogated in all treatment groups in contrast to
nondepleted controls, indicating that NK cells expressing GM1 play a
crucial role in the mediation of systemic tumor-protective immunity in
our model system.
For immunotherapy to achieve long-term remission in cancer patients with minimal residual disease, it is crucial to break peripheral tolerance against tumor self-antigens. This remains the goal of immunotherapy regimens designed to achieve tumor-protective immunity. Here, we tested the hypothesis that systemic tumor-protective immunity can be induced by a DNA vaccine, which was posttranscriptionally modified to enhance gene expression of the tumor-associated antigen mTH, combined with boosts of an antibody-IL-2 fusion protein. We previously reported that it was possible to induce an antitumor immune response against this mTH self-antigen following DNA vaccination.11 However, this particular DNA vaccine encoding only murine TH induced limited antitumor protection as indicated by a reduction in subcutaneous tumor growth. Therefore, we optimized this mTH-based DNA vaccine by combining its expression unit with the WPRE sequence, leading to a more effective tumor-protective immune response as documented by the absence of tumor growth after immunization. In contrast, the mTH vector alone could not induce full protection, which was in accordance with our previous results.11 Interestingly, although we were unable to detect an enhancement of mTH expression by addition of WPRE in vitro, our in vivo data clearly indicate that immunization with the WPRE-enhanced vaccine leads to significantly improved tumor-protective immunity. However, the mechanisms involved in the enhanced induction of protective immunity by the WPRE element are not entirely understood. Recent reports revealed some mechanistic features of posttranscriptional elements that affect RNA metabolism at the level of RNA stability, polyadenylation, and nucleocytoplasmic transport.13-15,26,27 Remarkably, besides these quantitative aspects, WPRE recruits the nuclear export receptor CRM1 to the transcript and thereby changes the intracellular nucleocytoplasmic transport pathway of the mRNA, which differs from the export pathway of cellular mRNAs.16 Therefore, we propose that when the mTH vaccine is applied alone the transcript expressed is exported via the cellular mRNA export pathway; however, in the presence of WPRE the mTH transcript leaves the nucleus via a pathway involving CRM1. This difference in nucleocytoplasmic transport may result in a qualitatively different association of the resulting mTH transcript with cellular compartments involved in antigen processing and presentation, which, in turn, may lead to a more effective antitumor immune response in vivo. We indeed demonstrated this to be the case, because the WPRE sequence markedly enhanced the efficacy of our DNA vaccine. Specifically, systemic tumor protection occurred in 4 of 6 animals remaining tumor free after vaccination with mTH-WPRE. In contrast, all mice immunized with mTH alone developed extensive tumor metastases. The extent of tumor metastases was assessed by liver weight and number of metastatic foci. While metastatic foci indicate visible macroscopic changes, a change in liver weight indicates even microscopic changes and therefore appears to be a more sensitive parameter. Consequently, the tumor-protective effect achieved in our experiments clearly indicates that the WPRE sequence is an important tool to enhance the efficacy of DNA-based tumor vaccines. Boosts with subcurative dosages of ch14.18-IL-2 fusion protein
targeted to the tumor microenvironment lead to an efficient amplification of the initial antitumor immunity induced by our DNA
vaccine. Two lines of evidence support this conclusion. First, boosts
with the ch14.18-IL-2 fusion protein produced a 3-fold increase in
intracellular expression of IFN- In summary, we demonstrated that a DNA vaccine based on the mTH tumor self-antigen and enhanced with the WPRE sequence can induce tumor-protective immunity and that this immune response can be amplified by subcurative doses of IL-2 targeted to the tumor microenvironment by the ch14.18-IL-2 fusion protein. Each component of the combined treatment regimen played a role, and 100% protection was obtained only when all components were included to induce effective immune responses. The mechanisms of action involved included at least 2 effector cell-types: CD8+ T cells and NK cells. It is anticipated that this strategy may lead to an improvement in the efficacy of DNA vaccines for cancer therapy.
We express our appreciation to Collette Beaton for the preparation of this manuscript.
Submitted August 13, 2002; accepted August 20, 2002.
Prepublished online as Blood First Edition Paper, September 12, 2002; DOI 10.1182/blood-2002-02-0391.
Supported by National Institutes of Health grant 1R21CA83140-01 (H.N.L., R.A.R.) and Emmy Noether Programm of the Deutsche Forschungsgemeinschaft (H.N.L.) (Lo 635/2-1). J.M.R. is a fellow of the Deutsche Forschungsgemeinschaft.
U.P. and H.W. contributed equally to this study, and H.N.L. and R.A.R. contributed equally to this study.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Ralph A. Reisfeld, The Scripps Research Institute, Department of Immunology, R218, IMM13, 10550 N Torrey Pines Rd, La Jolla, CA 92037; e-mail: reisfeld{at}scripps.edu.
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© 2003 by The American Society of Hematology.
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  R. J. Petrovan, C. D. Kaplan, R. A. Reisfeld, and L. K. Curtiss DNA Vaccination Against VEGF Receptor 2 Reduces Atherosclerosis in LDL Receptor-Deficient Mice Arterioscler Thromb Vasc Biol, May 1, 2007; 27(5): 1095 - 1100. [Abstract] [Full Text] [PDF]
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 S. Fest, N. Huebener, S. Weixler, M. Bleeke, Y. Zeng, A. Strandsby, R. Volkmer-Engert, C. Landgraf, G. Gaedicke, A. B. Riemer, et al. Characterization of GD2 Peptide Mimotope DNA Vaccines Effective against Spontaneous Neuroblastoma Metastases Cancer Res., November 1, 2006; 66(21): 10567 - 10575. [Abstract] [Full Text] [PDF]
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 Z. C. Neal, J. C. Yang, A. L. Rakhmilevich, I. N. Buhtoiarov, H. E. Lum, M. Imboden, J. A. Hank, H. N. Lode, R. A. Reisfeld, S. D. Gillies, et al. Enhanced Activity of Hu14.18-IL2 Immunocytokine against Murine NXS2 Neuroblastoma when Combined with Interleukin 2 Therapy Clin. Cancer Res., July 15, 2004; 10(14): 4839 - 4847. [Abstract] [Full Text] [PDF]
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 S. Garg, A. E. Oran, H. Hon, and J. Jacob The Hybrid Cytomegalovirus Enhancer/Chicken {beta}-Actin Promoter along with Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances the Protective Efficacy of DNA Vaccines J. Immunol., July 1, 2004; 173(1): 550 - 558. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
3FUB
plasmid encoding for mTH as described earlier
); boosted with the ch14.18-IL-2 fusion protein in addition to
mTH-vaccination (
); WPRE-enhanced mTH vaccine, pcDNA3.1mTH-WPRE
(
); and a combination therapy of mTH-WPRE vaccination plus
ch14.18-IL-2 fusion protein boost (
). Results were compared with
controls either immunized with the empty vector (
) or treated with
the ch14.18-IL-2 fusion protein alone (
).
.
J Immunol.
1996;157:126-137