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Keywords:

  • ovarian carcinoma;
  • sperm protein 17 (Sp17);
  • tumor vaccines;
  • cytotoxic T-lymphocytes (CTLs)

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

BACKGROUND

The authors previously identified sperm protein 17 (Sp17) as being expressed in patients with multiple myeloma. The restricted expression of Sp17 in normal tissue makes it an ideal target for tumor vaccine. In the current study, the authors extended their research to include ovarian carcinoma.

METHODS

A pair of sequence specific primers was used in reverse transcriptase-polymerase chain reaction to determine the gene expression of Sp17. A recombinant Sp17 protein was used with monocyte-derived dendritic cells and autologous peripheral blood mononuclear cells to generate Sp17 specific cytotoxic T-lymphocytes (CTLs). The successful generation of Sp17 specific CTLs was confirmed using standard 51chromium-release assays.

RESULTS

Sp17 was found to be expressed in the primary tumor cells from 70% of the patients with ovarian carcinoma. Human leukocyte antigen (HLA) class I- restricted Sp17 specific CTLs were generated successfully from the peripheral blood of three patients with ovarian carcinoma at the time of disease presentation. These CTLs were able to lyse autologous Epstein-Barr virus-transformed lymphoblastoid cells in a Sp17-dependent manner. Target lysis was HLA class I-dependent and could be blocked by antibodies against monomorphic HLA class I but not HLA class II molecules. The CTLs also lysed Sp17-positive autologous tumor cells, suggesting that Sp17 is processed and presented in association with the HLA class I molecules in Sp17- positive tumor cells in a concentration and configuration that could be recognized by recombinant protein-primed CTLs. Tumor cell killing by the CTLs appeared to be mediated through the perforin pathway. Flow cytometric analysis of the CTLs indicated that they predominantly were CD8 in phenotype and produced interferon-γ and scant amounts of interleukin-4.

CONCLUSIONS

The results of the current study suggest the potential of Sp17 as a target for immunotherapy in patients with ovarian carcinoma. Cancer 2002;94:2447–53. © 2002 American Cancer Society.

DOI 10.1002/cncr.10506

Among gynecologic malignancies, ovarian carcinoma remains the cancer with the highest mortality rate. Each year in the U.S. alone, approximately 18,500–22,000 new cases of ovarian carcinoma are diagnosed and between 11,500–15,000 women die of the disease.1 The lifetime risk among women with no history of familial ovarian carcinoma is estimated to be 1.4%.1 The overall 5-year survival rate does not appear to have improved in over a decade and continues to remain low in spite of significant improvements in conventional therapeutic modalities such as surgery, chemotherapy, and radiotherapy. Even in those women who achieve a complete histologic disease remission, the risk of recurrence is approximately 50%. Therefore, there is an urgent need for other therapeutic approaches in the treatment of patients with ovarian carcinoma. Targeted specific immunotherapy is an attractive option. This treatment is more specific and less toxic and may be an important approach in maintaining long-term disease remission. However, in practice, immunotherapy has been limited by the paucity of cloned ovarian tumor antigens with a favorable expression profile.

Cancer-testis (CT) antigens are a group of normal testicular specific proteins that are expressed aberrantly by tumor cells.2 CT antigens include members of the MAGE family, BAGE, GAGE, and NY-ESO-1. Their restricted expression in normal tissue makes them ideal molecules for immunotherapy. To our knowledge the reason for the aberrant expression of these antigens by tumor cells is unclear but most likely is related to the genetic hypomethylation that often occurs during malignant transformation of normal cells. These antigens have been found by various researchers to be immunogenic in vivo in patients with malignant diseases.

We recently investigated and identified sperm protein 17 (Sp17) to be a new member of the CT antigens.3 Sp17 is a 22–24- kilodalton protein initially isolated from a rabbit epididymal sperm membrane and testis membrane pellet as a member of the rabbit sperm autoantigen family.4–6 It was shown to be available on the surface of rabbit and mouse spermatozoa after the acrosome reaction (in the mouse)7 and after sperm-zona pellucida contact was initiated (in the rabbit).6 Subsequently, human Sp17 was discovered.8 Vasectomy in males often leads to the spontaneous formation of Sp17 antibodies. These antibodies are detectable in the serum and reproductive tract of affected individuals and have been implicated in secondary infertility.9, 10 Sp17 therefore is likely to be a highly immunogenic autoantigen in humans. B-cell epitope mapping using sera from vasectomized males suggests that Sp17 contains numerous B-cell epitopes.11 In addition, we have demonstrated that Sp17 also contains functional cytotoxic T-lymphocyte (CTL) epitopes,12 suggesting that Sp17 could be a target for tumor vaccine. Provided that tumor cells in patients with ovarian carcinoma express the protein, Sp17 is an ideal target for the immunotherapy for ovarian carcinoma for the following reasons. First, Sp17 has a very restricted distribution in normal tissue. Therefore, minimal nonspecific toxicities can be expected from a Sp17-based tumor vaccine. The in vivo clinical safety of a Sp17-based vaccine also could be deduced from the apparent lack of ill health in the large number of men who develop anti-Sp17 immunity after undergoing vasectomy. Second, unlike other autoantigens, Sp17 is highly immunogenic in vivo, based on the high proportion of men who spontaneously develop Sp17 immunity after undergoing vasectomy.

Although our previous findings in a normal healthy donor suggested the potential of Sp17 as a tumor vaccine, it remained to be determined whether CTL generation was possible in the autologous hosts in patients with ovarian carcinoma. Patients with ovarian carcinoma can be suppressed immunologically, because of a combination of the disease process and treatment. Therefore, it is possible that CTL generation may not be possible in these patients. In addition, it also remained to be determined whether Sp17 specific CTL precursors were present in the immune repertoire of these patients, especially those with Sp17-positive tumor cells. Theoretically, these T-cells may be deleted from the immune repertoire in patients with Sp17-positive tumor cells as a form of escape from tumor immunosurveillance. In the current study, we demonstrated that Sp17 is expressed in the tumor cells from a high proportion of patients with ovarian carcinoma. We also demonstrated that it was feasible to target Sp17 as a tumor vaccine in three patients with Sp17-positive ovarian carcinoma.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Patients and Materials

Three patients with ovarian carcinoma were studied at the time of disease presentation. The human leukocyte antigen (HLA) phenotypes in these patients were: Patient 1: A24 and A66 and B7 and B44; Patient 2: HLA-A24 and A29 and B7 and B58; and Patient 3: HLA-A24 and A30 and B7 and B8. All clinical materials were obtained with the consent of the patients and approval from the local ethics committee.

Reverse Transcriptase-Polymerase Chain Reaction Detection of Sp17 Transcripts

Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed as described previously.3 Briefly, total RNA was extracted from ovarian tumor cells using the Tri-reagent (Sigma Chemical Company, St. Louis, MO). All RNA specimens first were treated with DNAse I (Ambion, Inc., Austin, TX) to remove genomic DNA contamination. First strand cDNA was synthesized from 1 μg of total RNA using a random hexamer primer. The PCR primers were 5′SP17PCR 5-GGC AGT TCT TAC CAA GAA GAT-3′ and 3′SP17PCR 5-GGA GGT AAA ACC AGT GTC CTC- 3′ and they amplify a cDNA of approximately 500 base pairs. PCR was performed using 35 amplification cycles at an annealing temperature of 55 °C. Positive control amplification contained a normal testicular cDNA and a negative control of all the PCR reaction mixture except for cDNA, which was substituted with water. RNA integrity in each sample was checked by amplification of a c-abl gene segment. Successful removal of genomic DNA contamination was confirmed in each sample by amplification of the RNA without prior RT reaction. PCR products were visualized on an ethidium bromide agarose gel for a DNA band of the expected size. All results were confirmed on two independent RT-PCRs.

Isolation of Peripheral Blood Mononucelar Cells and Generation of Dendritic Cells

Peripheral blood mononuclear cells (PBMCs) were separated from heparinized venous blood by Ficoll-Hypaque (Sigma Chemical Company) density gradient centrifugation. Dendritic cells (DCs) were generated from peripheral blood monocytes. Briefly, PBMCs were seeded into 6-well culture plates containing 3 mL of RPMI 1640 and 10% fetal calf serum (FCS) at 5–10 × 106 per well. After 2 hours at 37 °C, nonadherent cells were removed and the adherent cells were cultured at 37 °C in RPMI 1640 supplemented with 10% FCS, 800 IU/mL of granulocyte-macrophage-colony-stimulating factor (Immunex, Seattle, WA), and 1000 IU/mL of interleukin-4 (IL-4) (Genzyme, Cambridge, MA). After 7 days of culture, the DCs were harvested for pulsing with a Sp17 recombinant protein that we generated from Escherichia coli.12

DC Pulsing

After culture, the DCs were washed twice and added to 50-mL polypropylene tubes. The cationic lipid DOTAP (Boehringer-Mannheim Biochemicals, Indianapolis, IN) was used to deliver the Sp17 recombinant protein because we previously found that DCs with DOTAP-delivered antigens efficiently promoted CD8 responses.13 Briefly, the recombinant protein was mixed with the lipid at room temperature for 20 minutes and added to the DCs at 37 °C in an incubator with occasional agitation for 3 hours. The cells were washed twice before being used as antigen presenting cells.

In Vitro Generation of Sp17 Specific CTL

Fresh PBMCs were cocultured with antigen-pulsed DCs at a ratio of 10:1 in RPMI 1640 supplemented with 10% autologous serum, IL-2 (10 IU/mL), and IL-7 (5 ng/mL) and incubated at 37 °C. IL-2 was added to the culture every 3–4 days thereafter. Irradiated autologous PBMC feeder cells and Sp17 recombinant protein (50 μg/mL), generated as described previously,12 were added to the culture every week. The cells were harvested after four rounds of stimulation and used for cytotoxicity assays.

Cytotoxicity Assays

Standard 4-hour 51chromium (51Cr)-release assays were performed to determine the cytotoxic activity of the Sp17-stimulated T-cells. Target cells (at an effector:target [E:T] ratio of 20:1) used included autologous Epstein-Barr virus-transformed lymphoblastoid cell line (LCL), with or without Sp17, and autologous ovarian tumor cells. To determine the HLA dependency of the cytotoxicity, HLA class I (W6/32) and HLA class II (L243) antibodies were added to the cocultures at a concentration of 25 μg/mL. All experiments were performed in quadruplicate. For all targets (including autologous ovarian carcinoma cells), the maximum releases were in excess of 200 counts per minute (cpm) and the spontaneous release was < 30% of the maximum release.

Flow Cytometric Analysis of Intracellular Cytokines

Flow cytometric analysis was performed as previously described.13 Briefly, the T-cell population was tested 4 weeks after priming, after resting for 6 days after the last antigen stimulation. The T-cells were incubated at 37 °C for 6 hours in RPMI 1640 plus 10% FCS, 50 μg/mL of Sp17 recombinant protein, and 500 ng/mL of ionomycin. Brefeldin A (10 μ g/mL) was added for the final 3 hours of incubation. Controls (nonactivated cultures) were incubated in the presence of Brefeldin A only. The cells were harvested, washed, and fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 20 minutes at room temperature, after which time they were washed and stored overnight in PBS at 4 °C. For intracellular staining, the cells were washed and permeabilized by incubation in PBS plus 1% bovine serum albumin and 0.5% saponin (Sigma Chemical Company) for 10 minutes at room temperature. Activated and control cells were stained with fluorescein isothiocyanate-labeled antiinterferon-α (IFN-α), phyco- erythin-labeled anti-IL-4, and isotype-matched control antibodies (Becton Dickinson, San Jose, CA) and analyzed by flow cytometry.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

High Frequency of Sp17 Expression by Ovarian Tumor Cells

Using a pair of sequence specific primers in PCR, we first determined the frequency of Sp17 gene expression in 10 fresh ovarian tumor samples. Sp17 transcripts were detected in 7 of 10 samples (70%) (Fig. 1), indicating the high prevalence of Sp17 gene expression by ovarian tumor cells. Therefore, this result suggests that Sp17 may be a target for a tumor vaccine that is widely applicable in patients with ovarian carcinoma. Although the PCR was not designed primarily to provide an accurate mRNA quantitation, the reproducible equi-intensity signals from Sp17-positive ovarian carcinoma samples when compared with those obtained with normal testicular PCR products suggest that Sp17 is likely to be expressed as abundantly by the tumor cells as it is by normal testis.

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Figure 1. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis using a pair of sequence specific primers for sperm protein 17 (Sp17) produced a positive signal of approximately 500 base pairs (bp). Lanes 1 and 3: Sp17-positive ovarian tumor sample; Lane 2: Sp17-negative ovarian tumor sample; Lane 4: normal testicular RNA; M: molecular marker; Lane a: PCR of DNAse I-treated RNA; Lane b: PCR of RNA that had undergone RT; Lane c: control amplification for c-abl gene segment.

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Successful Generation of Sp17 Specific CTLs from PBMCs from Patients with Multiple Myeloma

To determine the feasibility of generating Sp17 specific CTLs from patients with Sp17-positive ovarian carcinoma, we primed PBMCs from three consecutive Sp17-positive ovarian tumor patients with recombinant Sp17-pulsed DCs. We successfully generated Sp17 specific CTLs after three rounds of T-cell stimulation with the recombinant protein. When used as effector cells in cytotoxicity assays, we reproducibly demonstrated that these T-cells were able to kill autologous LCL pulsed with the Sp17 recombinant protein efficiently (Fig. 2). Target lysis for the autologous LCL was Sp17-dependent because no significant target lysis was observed when autologous LCLs without prior pulsing with Sp17 protein were used as targets in the cytotoxicity assays (P < 0.00001) (Fig. 2). This finding indicates that recombinant Sp17 protein, when used with autologous DCs, can prime T-cells from patients with ovarian carcinoma, and supports the successful generation of Sp17 specific CTL using autologous DCs and recombinant Sp17 protein. Target lysis was HLA class I-restricted, and could be blocked by antibodies directed against monomorphic HLA class I molecules. In contrast, HLA class II antibodies did not appear to affect the cytotoxic activity of the T-cells (P < 0.000001) (Fig. 2). These results therefore point to the involvement of CD8 T-cells in the CTL activity against Sp17-pulsed autologous target cells. They also suggest the presence of Sp17 specific T-cells in the immune repertoire of these patients.

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Figure 2. Successful generation of sperm protein 17 (Sp17) specific cytotoxic T-lymphocytes (CTLs) from peripheral blood mononuclear cells (PBMCs) of patients with ovarian carcinoma. Sp17 specific CTL-lysed autologous LCL pulsed with Sp17 recombinant protein but not the control target cells. Target cell lysis was human leukocyte antigen (HLA) Class I-dependent and could be blocked by a monoclonal antibody directed at monomorphic HLA Class I molecules but not HLA Class II molecules (E:T ratio of 20:1). Results were expressed as the mean ± the standard deviation. Figure represents one of two similar results obtained in two separate experiments. Pt: Patient.

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Recombinant Sp17 Protein-Primed CTLs Lyse Sp17-Positive Autologous Tumor Cells

To investigate the feasibility of using a Sp17-based tumor vaccine to treat ovarian carcinoma, we then investigated the relevance of the Sp17-primed CTLs in the context of autologous tumor cell lysis. Autologous tumor cells therefore were used as targets in the standard 51Cr-release assays with the Sp17 specific CTLs. We observed autologous target killing in all three Sp17-positive tumors (Fig. 3). Autologous tumor cell lysis also could be blocked by a monoclonal antibody directed at monomorphic HLA class I molecules but not HLA class II molecules (Fig. 3). Therefore, these results suggest the in vivo processing and presentation of Sp17 CTL peptides in association with the HLA class I molecules by Sp17-positive tumor cells. Furthermore, they also demonstrate that Sp17 recombinant protein could be used to prime and propagate CTLs that recognize Sp17 peptides with a concentration and configuration similar to those presented on the surface of Sp17-positive autologous tumor cells.

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Figure 3. Sperm protein 17 (Sp17)-recombinant protein-primed cytotoxic T-lymphocyte (CTL) could lyse autologous tumor cells expressing Sp17. Tumor cell cytotoxicity by CTLs could be blocked by a monoclonal antibody directed at monomorphic human leukocyte antigen (HLA) Class I molecules but not HLA Class II molecules (Patients [Pt] 1–3 were Sp17-positive) (E:T ratio 20:1). The results were expressed as the mean ± the standard deviation.

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Sp17 Specific CTLs Lysed Target Cells through a Perforin-Mediated Pathway

To determine the mode of target cytotoxicity mediated by the Sp17 specific CTLs, we treated the CTLs with either concanamycin A (CMA) or Brefeldin A. CMA selectively inhibits the perforin-mediated target lysis pathway whereas Brefeldin A Fas-mediated target apoptosis. We observed no significant effect of Brefeldin A on the ability of the effector cells to kill autologous tumor cells (Fig. 4). Conversely, CMA treatment of the effector cells nearly completely abrogated the cytotoxic ability of the CTLs to lyse autologous tumor cells (Fig. 4), suggesting that autologous tumor cell lysis by the Sp17 specific CTLs was mediated primarily through perforin.

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Figure 4. Autologous tumor cell lysis by sperm protein 17 (Sp17) specific cytotoxic T-lymphocytes (CTLs) could be blocked by cancanamycin A (CMA) but not Brefeldin A, suggesting that target cell cytotoxicity was mediated via the perforin pathway. Pt: Patient.

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Phenotypes and Cytokine Profiles of CTL Lines

Flow cytometry was used to evaluate the phenotypes of all three CTL lines generated in the current study. Compared with unstimulated CTLs from the same patients, there was a predominance of CD8-positive T-cells, and a paucity of CD4-positive T-cells, in the CTL lines after in vitro restimulation with the recombinant Sp17 protein (Fig. 5). These T-cells mainly were CD56-negative. Taken together with the results of the cytotoxicity assays, the results of the current study support the involvement of CD8-positive T-cells in the CTL activities. Two-color flow cytometric analysis for intracellular IFN-γ and IL-4 after Sp17 recombinant protein restimulation of the T-cells was performed to determine the cytokine profile of the CTL line. As expected, the CTL line produced predominantly IFN-γ and scant amounts of IL-4, a pattern that is in keeping with a Th1 cytokine profile (Fig. 6).

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Figure 5. Flow cytometric analysis of the cytotoxic T-lymphocytes lines demonstrated that they were comprised predominantly of CD8+ T-cells. Pt: Patient.

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Figure 6. Flow cytometric analysis of intracellular cytokines demonstrated that the cytotoxic T-lymphocytes line predominantly produced interferon-γ and a scant amount of interleukin (IL)-4.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

The list of human tumor antigens recognized by the human immune system is growing rapidly. Of these tumor antigens, CT antigens have received considerable attention because of their unique expression pattern and their potential as targets for tumor vaccines. CT antigens share the following characteristics: 1) expression is noted predominantly in testis and not in other normal somatic tissues and 2) gene activation and expression occurs in a wide range of human tumor types.

After our recent identification of Sp17 as a novel CT antigen in patients with multiple myeloma3 and the demonstration that Sp17 specific CTLs could be generated from the PBMCs of a previously nonimmunized healthy donor,12 we extended our investigations to determine the potential of targeting Sp17 in patients with ovarian carcinoma. We found that Sp17 is indeed expressed by tumor cells from 70% of patients with ovarian carcinoma, suggesting the potential to target Sp17 in immunotherapy for this malignant disease. However, the integrity of the host immune system to respond to a vaccine is another prerequisite for successful tumor immunotherapy. The immune repertoire of healthy donors is diverse. Healthy donors also usually are immunologically intact. Therefore, it was not surprising that Sp17 specific CTLs were generated easily in our previous study.12 In contrast, patients with advanced tumors often are suppressed immunologically due to the disease process and also to chemotherapy treatment. It also is conceivable that Sp17 specific T-cells may be deleted from the immune repertoire of these patients, especially those in whom Sp17 is expressed in tumor cells, as a form of a tumor immune escape mechanism. The demonstration of the ability to generate Sp17 specific CTLs that could kill autologous tumor cells will provide the basis for the use of Sp17 as a tumor vaccine in patients with ovarian carcinoma.

Using autologous DCs pulsed with a Sp17 recombinant protein as targets in cytotoxicity assays, we observed that Sp17 specific T-cells were in fact present in the immune repertoire of ovarian carcinoma patients because Sp17-primed CTLs capable of lysing autologous LCL pulsed with Sp17 protein were generated successfully. We then determined the clinical relevance of these findings in the context of autologous tumor cytotoxicity. Although we previously demonstrated the expression of Sp17 transcripts in ovarian tumor cells, it remained to be determined whether the protein is processed and presented in association with the MHC molecules for target cell recognition by recombinant protein-primed CTLs. The current study findings that Sp17 specific CTLs were able to kill autologous tumor cells in a Sp17-dependent manner support the notion that Sp17 protein is produced by the ovarian tumor cells and is processed and presented on the tumor cell surface in association with the MHC molecules. Most important, Sp17 CTL epitopes for effector cell recognition were expressed by ovarian tumor cells. Therefore, Sp17 could be used as a tumor vaccine in patients with Sp17-positive ovarian carcinoma.

As predicted by the results obtained using blocking antibodies to HLA molecules in cytotoxicity assays, the Sp17 specific CTLs predominantly were CD8 in phenotype. These T-cells also produced IFN-γ and scant amounts of IL-4. Furthermore, these T-cells lysed autologous target cells via the perforin- mediated pathway and not through the induction of apoptosis.

Sp17 appears to be a suitable target for a tumor vaccine for ovarian carcinoma. Based on the results of the current study, a Phase I/II pilot study has been initiated in our institution regarding the use of Sp17-pulsed DCs in patients with residual disease after treatment for ovarian carcinoma. However, it remains to be determined whether the laboratory results reported herein can be translated into clinical successes in our pilot study.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES