Steroidogenic Acute Regulatory (StAR)-Directed Immunotherapy Protects against Tumor Growth of StAR-Expressing Sp2-0 Cells in a Rodent Adrenocortical Carcinoma Model
DÖRTE ORTMANN, JÜRGEN HAUSMANN, FELIX BEUSCHLEIN, KAI SCHMENGER, MAIK STAHL, MICHAEL GEISSLER, AND MARTIN REINCKE
Department of Internal Medicine 2, Division of Endocrinology (D.O., F.B., K.S., M.S., M.R.) and Division of Gastroenterology (M.G.), University Hospital of Freiburg, D-79106 Freiburg, Germany; and Institute of Medical Microbiology and Hygiene, Department of Virology (J.H.), University of Freiburg, D-79104 Freiburg, Germany
Adrenocortical carcinoma (ACC) is a highly malignant tumor with poor response to classical antitumor therapy. Steroido- genic acute regulatory (StAR) protein is expressed in most human ACCs. The aim of this study was to induce antitumoral T cells directed against StAR in a murine tumor model. Be- cause a suitable syngenic adrenocortical mouse tumor model is lacking, we established a clone of the mouse myeloma Sp2-0 tumor cell line stably expressing murine StAR (Sp2-mStAR). Using repeated im injections of plasmid DNA encoding mStAR followed by infection with a recombinant vaccinia virus (rVV) expressing mStAR, we induced a cytotoxic T-cell response as measured by enzyme-linked immunospot assay. To demon- strate antitumor activity of the vaccination procedure, mice were treated as follows: group A, mice immunized with plas- mids and rVV encoding mStAR receiving Sp2-mStAR cells;
control group B, mice immunized with the empty plasmid and the empty rVV receiving Sp2-mStAR cells; control group C, mice immunized with the empty plasmid and rVV encoding P450 side-chain cleavage enzyme receiving Sp2-mStAR cells; and control group D, mice immunized with plasmid and rVV encoding mStAR receiving parental Sp2-0 cells. A high pro- portion (89-100%) of the control groups B, C, and D developed subcutaneous tumors. In contrast, immunization specific for mStAR (group A) was highly protective against tumor growth (percentage of tumor-free animals, 67%; P < 0.001 vs. controls). In summary, these results show that T-cell tolerance toward mStAR can be broken, resulting in antitumoral immunity. Thus, StAR represents a candidate target antigen for immu- notherapeutic strategies against ACC. (Endocrinology 145: 1760-1766, 2004)
A DRENOCORTICAL CARCINOMA (ACC) is a highly malignant but rare endocrine neoplasm, with a yearly incidence of approximately two per million people (1-3). Tumors may be functional or nonfunctional, depending on whether they produce cortisol, aldosterone, androgens, or estrogens. Accordingly, more than 50% of patients with ACC have clinical evidence of excess hormone production. The etiology of ACC is largely unknown, although recent studies documenting chromosomal abnormalities and alterations in growth factor production have provided insight into possible mechanisms of molecular pathogenesis (4, 5). Seventy per- cent of the patients suffer from an advanced tumor stage, with local invasion or distant metastasis at the time of di- agnosis. The 5-yr survival rate for stage III and IV disease of the MacFarlane classification is around 20% (6, 7). Complete surgical resection is currently the only potentially curative therapy for localized ACC. Locally recurrent or isolated dis- tant metastatic disease may also be amenable to surgical
resection in carefully selected patients. Response rates to cytotoxic systemic chemotherapy and the adrenolytic agent mitotane have been modest (8). Therefore, improved treat- ment strategies are clearly required for this tumor entity and might include tumor antigen-specific immunotherapeutic strategies.
ACC expresses a broad spectrum of tumor-associated an- tigens relatively specific to the adrenal cortex, such as ACTH receptor (9), angiotensin II type 1 receptor (10), steroidogenic enzymes like P450 side-chain cleavage enzyme (P450scc) (11), P450 21-hydroxylase (12), and steroidogenic acute reg- ulatory protein (StAR) (13), which could serve as potential immunotherapeutic targets. However, in undifferentiated tumors, the expression of many of these antigens is lost (11). By contrast, StAR expression was present in all ACCs studied in a recent series in our laboratory (13). Based on this evi- dence, we attempted to evoke a specific immune response against murine StAR (mStAR) protein using a DNA-based immunization approach. Immune recognition of self anti- gens in humans has been best characterized in patients with malignant melanoma (14). Melanoma-associated antigens can be recognized by antibodies and CD4+ and CD8+ T cells, suggesting that broad-based immune responses against these antigens may be induced. Immunity to self-differen- tiation antigens is difficult to elicit because of immune tol- erance and ignorance. Here, we show that immunological
Abbreviations: ACC, Adrenocortical carcinoma; CTL, cytotoxic T- lymphocyte; ELISPOT, enzyme-linked immunospot; FBS, fetal bovine serum; IFN, interferon; MHC, major histocompatibility complex; P450scc, P450 side chain cleavage enzyme; rVV, recombinant vaccinia virus; StAR, steroidogenic acute regulatory.
Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.
tolerance toward the antigen mStAR can be broken by a prime-boost immunization protocol using DNA plasmids encoding mStAR and a recombinant vaccinia virus (rVV) vector directing mStAR expression. Moreover, we present evidence that the mStAR-specific immune response results in prophylactic antitumoral effects against a mStAR-expressing murine tumor cell line in vivo.
Materials and Methods
Animals and cell lines
All experiments involving animals were performed in accordance with institutionally approved animal care guidelines. The protocols used were approved by the institutional animal review committee. Fe- male BALB/c mice (H-2ª), 6-8 wk of age, were obtained from Charles River Laboratories (Sulzfeld, Germany). The mouse myeloma cell line Sp2-0 (H-2ª, ATCC CRL1581) was grown in DMEM supplemented with 20% heat-inactivated fetal bovine serum (FBS). Sp2-0 cells were stably transfected using expression constructs encoding mStAR (pSecTagA- mStAR) and selected for stable expression of the transgene by addition of Zeocin (Cayla, Toulouse, France) into the culture medium at a final concentration of 100 µg/ml. Individual cell clones were picked and subsequently screened by Western blotting, radioimmunoprecipitation, and immunofluorescence for expression of the transgene. The Sp2- mStAR cells were used as an adrenocortical tumor model in BALB/c mice. Subcutaneous injection of BALB/c mice with Sp2-mStAR cells or (as a control) parental Sp2-0 cells resulted in palpable tumors after 8-10 d with a penetrance of more than 95%.
DNA constructs
The complete cDNAs of mStAR and murine P450scc (mP450scc) were kind gifts from D. Stocco (Southwestern Medical Center, Dallas, TX) (15) and K. Parker (Tech University Health Science Center, Lubbock, TX) (16), respectively. Both inserts were cloned into the eukaryotic expres- sion vector pcDNA3.1 (Invitrogen, San Diego, CA). To obtain a more immunogenic expression plasmid, the plasmid pSecTagA-mStAR con- taining a secretory IgG leader sequence was created by subcloning the full-length mStAR cDNA in frame into the eukaryotic secretion vector pSecTagA (Invitrogen). The correct sequence of the inserted cDNA was verified by automated sequence analysis (Toplab, Munich, Germany). To further enhance the immune response, plasmids expressing inter- leukin 12p70 (pApIL-12p70) and granulocyte macrophage-colony stim- ulating factor (pRJB-GM-CSF) were used (17, 18).
rVV
The cDNAs encoding mStAR or mP450scc, respectively, were sub- cloned into the vaccinia virus transfer vector pSC11 (19). Recombinant viruses were generated as described previously (20). Briefly, rVV (rVV- mStAR and rVV-mP450scc) was isolated after homologous recombina- tion by three rounds of plaque purification and grown to high titers on CV-1 cells. To verify correct protein expression, cell lysates of vaccinia virus-infected CV-1 cells were examined by radioimmunoprecipitation as described in Radioimmunoprecipitation.
Immunofluorescence
Sp2-0 and stably transfected cell lines (Sp2-mStAR) were fixed with 3.7% paraformaldehyde and permeabilized by PBS/0.5% Triton X-100. Transgene expression was detected by incubating with antihistidine antibody (Qiagen, Hilden, Germany) and detected with fluorescein iso- thiocyanate or Cy3-labeled antirabbit IgG antibody.
Western blotting
Immunoblots were performed using whole-cell extracts from Sp2- mStAR cells employing an antibody directed against the 6-histidine tag (Qiagen) or a polyclonal mouse antiserum directed against mStAR and visualized by enhanced chemiluminescence (Amersham Corp., Arling- ton Heights, IL) according to manufacturer’s specifications. For analysis
of mStAR expression by Sp2 tumors, the tumor tissue was homogenized in radioimmunoprecipitation assay buffer (10% Triton X-100; 10% so- dium deoxycholate; 10% sodium dodecyl sulfate; 5% aprotinin; 150 mm NaCl; 50 mM Tris-Cl, pH 7.5), and 1.6 µg of total tumor protein per lane was separated by SDS-PAGE.
Radioimmunoprecipitation
CV-1 cells were infected with rVV-mStAR and rVV-mP450scc, starved in methionine-free medium for 30-60 min, and then labeled with 250 µCi of [35S]methionine/cysteine. Cells were lysed in radioim- munoprecipitation assay buffer and immunoprecipitated using poly- clonal anti-mStAR and anti-P450scc antibodies. Immune complexes were recovered using staphylococcal protein A-Sepharose and subse- quently separated by a 12% SDS-PAGE.
Prime-boost immunization
Mice were immunized three times in weekly intervals by coinjecting four plasmid constructs (pcDNA-mStAR, pSecTagA-mStAR, pRJB-GM- CSF, and pApIL-12p70) into the tibialis anterior muscle. One hundred micrograms each of plasmid pcDNA-mStAR and pSecTagA-mStAR and 25 µg of plasmid pRJB-GM-CSF and 25 µg of pApIL-12p70 were applied in a total volume of 100 pl. The rVV-mStAR, the rVV-mP450scc, and the empty rVV-sc11 were delivered iv at 5 × 106 pfu/mouse 10 d after the last DNA immunization.
Enzyme-linked immunospot (ELISPOT)
MultiScreen HA sterile plates (96-well plates) (MAHAS 4510; Milli- pore, Eschborn, Germany) were coated with 1 µg of antimouse inter- feron (IFN)-y capture antibody (clone R4-6A2; PharMingen, San Diego, CA) in 100 ul of PBS overnight at 4 C. After blocking for 2 h with 200 ul of medium (DMEM supplemented with 5% FBS, penicillin, and strep- tomycin) at room temperature, responder splenocytes were seeded in duplicate serial 2-fold dilutions from 106 to 1.25 × 105 cells per well in 100 ul of medium. Each well received 105 Sp2-0 (negative control) or Sp2-mStAR cells as stimulators. One well had no spleen cells (back- ground), and one well received 7.5 µg/ml Concanavalin A (Sigma, Taufkirchen, Germany) plus 106 cells as positive control. The plates were incubated for 24 h at 37 C. Cells were removed by washing the plates three times with PBS and three times with PBS containing 0.05% Tween 20. Bound IFN-y was detected by incubation with 100 ul of 5 ng/ul biotinylated antimouse IFN-y antibody (clone XMG1.2; PharMingen) per well at 4 C overnight. Wells were washed six times with PBS con- taining 0.05% Tween 20 and incubated with 100 ul of streptavidin- alkaline phosphatase (PharMingen; 1.4 µg/ml) in PBS containing 0.05% Tween 20 and 1% FBS for 1 h at room temperature. Finally, wells were washed three times with PBS containing 0.05% Tween 20 and three times with PBS and developed for 30 min with 3-amino-9-ethylcarbazole (Sigma). The substrate solution was prepared according to the instruc- tions of the manufacturer. The reaction was stopped by rinsing the wells with water. Spots were counted, and results were presented as number of spot-forming cells per 106 responder cells.
Tumor protection studies
After immunization, BALB/c mice were inoculated with 106 Sp2-0 or Sp2-mStAR cells in 100 ul PBS into the right flank/shoulder. Tumor-free survival was determined by careful daily examination under standard- ized conditions. After tumors were detectable, tumor size was measured in vivo by volumetry using a sliding caliper. Animals were euthanized when the tumor size exceeded 1800 mm3. Tumor, spleen, and all other organs, as well as blood, were harvested for analysis of cellular immune responses, histology, hormone determination, and expression of trans- gene in the tumor.
Hormone determination
Serum corticosterone concentrations were determined in seven im- munized animals and seven control animals. After decapitation, blood was collected by puncture of the left ventricle. The blood was imme- diately mixed with EDTA, centrifuged, and stored at -20 C until mea-
surement. Corticosterone was measured by a commercial RIA (ICN, Eschwege, Germany).
Statistical analysis
The data are expressed as mean ± SEM. Differences between groups were assessed using ANOVA and Fisher’s protective least significant difference test. Tumor-free survival time was defined as the primary end point, and survival curves were calculated using the Kaplan-Meier method. P < 0.05 was considered statistically significant.
Results
Stable expression of mStAR in Sp2-0 cells and expression of mStAR and mP450scc by rVV
Flow cytometry demonstrated expression of significant amounts of major histocompatibility complex (MHC) class I molecules in 86% of Sp2-0 cells allowing the presentation of MHC class I-restricted peptides to CD8+ T cells (data not shown). Sp2-0 cells were stably transfected with the expres- sion vector pSecTagA coding for mStAR, and Zeocin- resistant cell clones permanently expressing mStAR were isolated. Expression of mStAR was confirmed by immuno- fluorescence (Fig. 1, A and B). Western blot analysis using polyclonal anti-mStAR or antihistidine-tag reactive antibod- ies of Sp2-mStAR lysates proofed expression of mStAR (Fig. 1C).
In addition, CV-1 cells were infected with rVV coding for mStAR and mP450scc. Immunoprecipitation was performed using polyclonal antibodies against mStAR and mP450scc, and the precipitated proteins were analyzed by SDS-PAGE and autoradiography, revealing effective expression of mStAR and mP450scc (Fig. 1D). The proteins showed the expected molecular mass for mP450scc (55 kDa) and mStAR (30 kDa) (21, 22).
Prime-boost immunization induces mStAR-specific T-cell responses
As demonstrated in preliminary experiments, DNA-based immunization alone using pSecTagA-mStAR induced only a weak and inconsistent mStAR-specific cytotoxic T-cell re- sponse in BALB/c mice (data not shown), suggesting a low antigenicity of mStAR in this experimental setting. There- fore, a booster infection with rVV-mStAR was included in the vaccination scheme to amplify the cytotoxic T-lymphocyte (CTL) response against mStAR. BALB/c mice were immu- nized three times in weekly intervals with a plasmid encod- ing a full-length form of mStAR, followed by an iv infection with 5 × 106 pfu of rVV-mStAR (Fig. 2A) as booster im- munogen. Two weeks later, splenocytes were analyzed for the frequency of mStAR-specific IFN-y-producing cells using the ELISPOT technique after restimulation with syngenic Sp2-mStAR myeloma cells. Using this approach, we were able to detect mStAR-specific IFN-y-secreting cells in the spleens of immunized mice in 0.002% of splenocytes (Fig. 3). These IFN-y-producing splenocytes were mStAR specific be- cause no specific IFN-y spots were detected after in vitro stimulation with parental Sp2-0 cells. Furthermore, there was only weak background activity in mice immunized with control plasmid and control rVV after restimulation in vitro with Sp2-mStAR cells.
A
C
Sp2-0
Sp2- mStAR
Sp2-0
132 -
78 -
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46 -
- mStAR
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D
rVV-mP450scc
rVV-mStAR
anti-SCC anti-StAR anti-SCC anti-StAR
66 -
45 -
- mP450scc
30 -
- mStAR
Protective immunity against mStAR tumor growth is induced by prime-boost vaccination
To assess protective efficacy of the mStAR-specific prime- boost immunization against Sp2-mStAR tumors, different vaccination groups were established. In all experimental groups, vaccination was performed before tumor inoculation (Fig. 2A). By calculating survival using the Kaplan-Meier method, a prolongation of tumor-free survival (P = 0.001) was observed in the specifically immunized animals (group A, Fig. 2B) compared with animals immunized with control plasmids and boostered by control rVV infection (empty rVV-sc11, group B; rVV-mP450scc, group C) and animals harboring parental Sp2-0 tumors immunized with mStAR (group D). Most of the animals in group A did not develop tumors until the end of the observation period (group A: tumor growth in 12 of 36 animals, 33%), whereas almost all
A
4 week old BALB/c
3x DNA vaccination (DDD)
booster infection with recombinant Vaccinia Virus (rVV)
transplantation of tumor cells
Group A
pSecTagA-mSTAR
rVV-mStAR
Sp2-mStAR
Group B
pSecTagA
rVV-sc11
Sp2-mStAR
Group C
pSecTagA
rVV-mP450SCC
Sp2-mStAR
Group D
pSecTagA-mSTAR
rVV-mStAR
Sp2-0
0
1
2
3.5
5
weeks
B
-O- Group A: DDDmStAR + rVV-mStAR / Sp2-mStAR cells (n=34)
-0- Group B: DDDpSecTagA + rVV-sc11 / Sp2-mStAR cells (n=9)
Group C: DDDpSecTagA + rVV-mP450scc / Sp2-mStAR (n=16)
Survival after Tumor Cell Injection (%)
-4- Group D: DDDmStAR + rVV-mStAR / Sp2-0 cells (n=12)
120
100
80
60
40
20
0
0
5
10
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25
30
35
Days after Inoculation of Tumor Cells
C
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2000
Group A
Tumor size (mm3)
Group
1500
1000
500
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Group A
0
5
7
9
11
13
15
Days after inoculation of tumor cells
control animals developed a tumor within 28 d (group B: eight of nine animals, 89%; group C: 15 of 16 animals, 94%). Protection was mediated by antigen-specific mechanisms be- cause animals that were prime-boost vaccinated with mStAR but received parental Sp2-0 cells (group D) showed a high tumor penetrance (12 of 12 animals, 100%). In those animals in group A that developed tumors despite immunization, the tumors were smaller in size than in control animals (Fig. 2, C and D).
Histology of the adrenal glands and corticosterone concentrations in immunized animals
To evaluate potential side effects of immunization, such as autoimmune adrenalitis, paraffin-embedded adrenal glands of vaccinated animals were stained with hematoxylin and eosin and analyzed by light microscopy. The architecture of the adrenal cortex remained intact in vaccinated animals, and there was no evidence of infiltration by lymphocytes (Fig. 4,
number of spot-forming cells / 106 splenocytes
25
Group A: DDDStAR+rVV-mStAR
20
D Group C: DDDpSecTagA+rVV-mP450scc
15
10
5
0
Sp2-mStAR
Sp2-0
stimulator cells
A and B). In addition, corticosterone levels were in the range of unvaccinated control animals, whereas animals bearing Sp2-0 tumors without vaccination had increased corticoste- rone concentrations, most likely reflecting activation of the hypothalamic-pituitary-adrenal axis due to the large tumor burden (Fig. 4℃).
Detection of mStAR expression in the tumor tissue
Twelve of 36 animals in group A developed tumors de- spite immunization with mStAR. These tumors were further analyzed to investigate possible mechanisms of tumor es- cape. Theoretically, the following reasons could account for the failure of tumor suppression by murine T cells: 1) im- mune escape due to the selection of a mStAR-negative cell clone, 2) insufficient activity of primed T cells, and 3) vac- cination failure. Because seven of eight investigated tumors strongly expressed mStAR, loss of tumor antigen expression was excluded in most cases as the cause for tumor growth (Fig. 4D). There was no evidence of infiltration with inflam- matory cells within the tumors (Fig. 4, E-G). This points toward an insufficient CTL response as the major cause of tumor growth in this subgroup of animals.
Discussion
The data presented suggest that immunization using DNA vaccination followed by vaccinia virus boosting is sufficient to break immune tolerance against the adrenal mStAR an- tigen. Using this approach, we were able to induce a mStAR- specific immune response strong enough to inhibit tumor growth of a mouse myeloma cell line constitutively express- ing mStAR. To our knowledge, this is the first report de- scribing a successful immunization strategy against an ad- renal-specific antigen. Thus, this approach might have the
potential to become a useful strategy for immune therapy of ACC.
ACC is characterized by rapid growth, early metastatic spread, low sensitivity to radiation therapy, and low re- sponse rates to polychemotherapy (4, 8). The low response rates to polychemotherapy are in part explained by high expression levels of the multidrug-resistance gene by this tumor (23). A molecular feature of ACC is increased genetic instability with a high number of chromosomal aberrations (24, 25). Standard treatment consists of aggressive surgery to achieve complete tumor resection, followed by adrenolytic therapy alone or in combination with chemotherapy in case of recurrence or metastases (26). Despite these efforts, 5-yr survival rates remain low, with a range of 20-30%. Therefore, alternative therapies, such as gene and immune therapy, might be a promising option in ACC. So far, however, only limited data from animal models on the treatment of adrenal disease by gene therapy have been published (27, 28). As for the treatment of ACC, a recent report describes a mouse model using the SW13 human ACC cell line injected into nude mice (29). Tumors were then targeted by transfection with adenoviral plasmids containing the thymidine kinase suicide gene. This approach was successful in inducing a significant tumor regression. However, a potential disad- vantage of this strategy is that it is restricted to the primary tumor and needs direct application. This approach is of less value in disseminated disease with multiple metastases, which could only be targeted by multiple injection of the vector or by a hypothetical bystander effect.
Through targeting of a highly specific antigen, the ap- proach used in our study has the potential advantage of both a specific and systemic effect. Antigen expression of steroi- dogenic enzymes like P450scc, P450c17, and the StAR protein are mainly restricted to the gonads and the adrenal cortex. This renders them potentially useful as targets for active immune-therapeutic strategies. However, prerequisites for targeted immune therapy are the expression of these anti- gens in sufficient quantities within the tumors and the an- tigen presentation by the human leukocyte antigen complex by tumor cells. Whereas the latter has not been studied, the former is well documented (10-13). Previous studies using Northern blot analysis, in situ hybridization, and immuno- histochemistry have demonstrated that many (as in the case of P450scc and P450c17) or the majority (as in the case of StAR) of human ACC express these potential targets (5). We, therefore, selected StAR as the antigen for an immune-based strategy. Because the immunogenic epitopes of the protein are not known, we subcloned the full-length cDNA into the DNA expression vector pSecTagA. To improve efficiency of immunization we used a prime-boost vaccination strategy that has been previously shown to induce strong antitumor immune responses (30). We used a genetically generated murine tumor cell line instead of a naturally occurring tumor model because a useful natural syngenic mouse model for ACC is lacking. The adrenocortical Y1 cell line expresses StAR and leads to tumor formation when given sc in the parental LAF1 mouse strain. However, the only other syn- genic cell line derived from LAF1 mice, the pituitary AtT-20 cell line, does not express MHC class I molecules (Ortmann,
A
C
140
C
serum corticosterone (ng/ml)
120
M
100
Group A
80
60
B
40
20
0
€
immunization: mStAR
-
M
tumor cells: Sp2-mStAR Sp2-0
Group C
E
F
D
Sp2-0
Sp2-mStAR
Group A
DCCAA
Group A
G
Group C
Group C
D., unpublished data) and is, therefore, inapplicable for the examination of the antitumoral immune response.
At the cellular level, ACC is often a heterogeneous tumor. Microheterogenicity has been shown for P450 enzyme ex- pression and for ACTH receptor expression in adenomas and carcinomas by in situ hybridization (11, 31) demonstrating tumor areas expressing low amounts of the respective gene product. Immunization against one adrenal antigen, such as StAR, harbors the risk that tumor cells not expressing the antigen may escape immune surveillance by the host. There- fore, for future approaches, it seems more reasonable to use several different antigens as targets at the same time rather than a single antigen. Such an approach would possibly include additional adrenal antigens like ACTH receptor, P450scc, and other steroidogenic enzymes often expressed in ACC (11, 12). In addition, in some patients, treatment of hormone excess induced by the tumor can become a major therapeutic goal. The elimination of tumor subclones ex- pressing steroidogenic enzymes that are responsible for the overt hormone production could be achieved by immuno- logical approaches specifically targeted against these cells.
In our model, we have demonstrated the efficiency of a prophylactic treatment approach. However, treatment of re- maining or recurrent tumor disease is the main goal in most patients with ACC. It will be necessary to show that the strategy of our protocol can be adopted for adjuvant tumor treatment, such as metastatic disease, as well. Nevertheless, because recurrence rates after surgical treatment are as high as 30-70%, prophylactic treatment in patients with com-
pletely resected tumors would also be desirable to achieve long-term remission after surgery.
In summary, we have shown, as proof of principle, that a combined DNA/rVV vaccination against a steroid cell- specific antigen like mStAR can break immune tolerance and induce a CTL response strong enough to reject tumor cells expressing mStAR. Future studies should be directed toward identification of HLA-restricted epitopes using human CD8+ T cells from ACC patients. Moreover, identification of H-2ª-restricted CTL epitopes in mStAR could be used to improve the sensitivity of the ELISPOT assay and to further study immunotherapeutic strategies against mStAR such as peptide-pulsed dendritic cells.
Acknowledgments
We are indebted to Keith L. Parker (Department of Internal Medicine, Southwestern Medical Center, Dallas, TX) and Douglas M. Stocco (De- partment of Cell Biology and Biochemistry, Texas Tech University Health Science Center, Lubbock, TX) for the kind gift of the mP450scc cDNA and mStAR cDNA and antibody, respectively.
Received August 1, 2003. Accepted December 31, 2003.
Address all correspondence and requests for reprints to: Martin Re- incke, M.D., Division of Endocrinology, University of Freiburg, Hug- stetter Strasse 55, D-79106 Freiburg, Germany. E-mail: reincke@med1. ukl.uni-freiburg.de.
This work was supported by a grant from the Dr. Mildred Scheel Stiftung (to F.B., M.G., and M.R.) and by the Deutsche Forschungsge- meinschaft (Re 752/11-1).
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