Induction of human tumor-associated differentially expressed gene-12 (TADG-12/TMPRSS3)-specific cytotoxic T lymphocytes in human lymphocyte antigen-A2.1–positive healthy donors and patients with advanced ovarian cancer

Authors


Abstract

BACKGROUND:

Tumor-associated differentially expressed gene-12 (TADG-12) is a serine protease recently found highly differentially expressed in epithelial ovarian cancer. The goal of this study was to identify potential immunogenic peptides derived from TADG-12 for immunotherapy of ovarian carcinoma.

METHODS:

A bioinformatics approach (ie, the BIMAS algorithm, National Institutes of Health, http://bimas.dcrt.nih.gov/molbio/hla_bind) was used to identify multiple immunogenic peptides derived from TADG-12 that bind to human leukocyte antigen-A2.1 and elicit peptide-specific human cytotoxic T lymphocyte (CTL) responses in healthy individuals and in patients with advanced stage ovarian cancer.

RESULTS:

CD8+ CTL populations generated against 5 TADG-12–derived peptides were consistently able to induce lysis of autologous peptide-loaded target cells above background. Importantly, TADG-12 YLPKSWTIQV peptide-specific CTLs from healthy donors and ovarian cancer patients were found to effectively kill ovarian cancer cells naturally expressing TADG-12. Cytotoxicity was significantly inhibited by anti–human lymphocyte antigen (HLA)-A2.1 (BB7-2) and anti–HLA class I (W6 of 32) monoclonal antibodies, whereas natural killer–sensitive K562 cells were not lysed. TADG-12 YLPKSWTIQV peptide-specific CTL precursor frequency was low in peripheral blood leukocytes of normal donors and ovarian cancer patients, as determined by interferon-γ production in enzyme-linked immunosorbent spot-forming cell assays. Intracellular cytokine expression measured by flow cytometry after OKT-3 monoclonal antibody stimulation showed a type 1 cytokine profile in YLPKSWTIQV peptide-specific CTLs.

CONCLUSIONS:

The TADG-12 YLPKSWTIQV peptide is an immunogenic epitope in ovarian tumors and may represent an attractive target for immunotherapy of ovarian cancer. These data may pave the way for TADG-12 peptide-derived cell-based therapy, including dendritic cell immunotherapy, for the vaccination of ovarian cancer patients harboring chemotherapy-resistant or residual disease. Cancer 2009. © 2008 American Cancer Society.

Ovarian cancer remains the most lethal gynecologic tumor in the United States and in the Western world.1 Although aggressive cytoreductive surgery followed by adjuvant platinum-based chemotherapy is able to induce clinical responses in the majority of ovarian cancer patients, most of these women eventually relapse and die from the development of chemotherapy-resistant disease.2 Because T cell responses are able to eliminate residual tumor cells independently of their proliferative state as well as their resistance to chemotherapy, ovarian tumor-specific immunotherapy may offer the prospect of an effective treatment for patients with refractory or residual disease after completion of primary standard treatment (ie, cytoreductive surgery plus adjuvant chemotherapy).

Serine proteases are enzymes involved in many biological functions of cancer cells, including activation of growth and angiogenic factors and other proteases for invasion and metastasis, and thus represent excellent candidates as tumor markers.3 Recently, with the goal of identifying genes highly and differentially expressed in epithelial ovarian cancer and to use this knowledge for the development of novel diagnostic and therapeutic markers against this disease, our group has used high-throughput technologies, such as high-density oligonucleotide microarrays to analyze ovarian serous papillary carcinoma genetic fingerprints.4 Among the several candidate target genes identified, the gene encoding for the serine protease human tumor-associated differentially expressed gene-12 (TADG-12/TMPRSS3) was found to be 1 of the top differentially expressed genes in ovarian serous carcinomas when compared with normal cell controls.4 These results were confirmatory of our previous work using redundant polymerase chain reaction (PCR) primers to detect protease genes highly differentially expressed in ovarian carcinomas,5 which also found TADG-12 to be overexpressed in 41 of 55 (79%) of the serous papillary ovarian carcinomas tested, 58% of the mucinous, and 100% and 50% of the endometrioid and clear cell carcinomas, respectively.5 Normal ovarian tissues controls were consistently negative for TADG-12 expression; minimal expression was detected in other human adult tissues.5 Of interest, a truncated TADG-12 protein was also identified in these studies, suggesting that TADG-12 variants may be present and useful both as further molecular targets for therapy and/or novel diagnostic markers.5, 6 Importantly, Wallrapp et al7 and Iacobuzio-Donahue et al8 reported that TADG-12 is also highly overexpressed in pancreatic cancers and possibly colorectal and gastric cancer, suggesting TADG-12 as a potential tumor target for multiple highly aggressive human malignancies. This information, combined with the current knowledge of the primary sequence and structure of TADG-12, which may allow identification of epitopes recognized by CD8+ cytotoxic T lymphocytes (CTLs) and potentially present on ovarian cancer cells, may now allow exploration of the utility of TADG-12 for cell-based therapy, including dendritic cell (DC) immunotherapy for the vaccination of ovarian cancer patients harboring residual disease after standard therapy.

Here, we use a bioinformatics approach based on the BIMAS algorithm of the Center for Information Technology, National Institutes of Health (http://bimas.dcrt.nih.gov/molbio/hla_bind) to predict human lymphocyte antigen (HLA)-A2–binding peptides from TADG-12. We demonstrate for the first time that human CTLs can be generated against several TADG-12–derived peptides in healthy women as well as women harboring advanced stage ovarian cancer. More importantly, we show that at least 1 of these TADG-12 peptide-specific CTL populations is capable of killing autologous TADG-12–positive ovarian tumor cells in an HLA class I–restricted fashion, whereas TADG-12–negative HLA-identical normal cells are not killed. Thus, TADG-12 overexpression in ovarian carcinoma may potentially be used as a target for therapeutic vaccination in ovarian cancer patients refractory to standard treatment modalities.

MATERIALS AND METHODS

Peptide Prediction

The entire cloned sequence of TADG-125, 6 was reviewed for 9- and 10-mer peptides that could potentially bind to major histocompatibility complex (MHC) class I molecules using a peptide-motif scoring system (ie, http://bimas.dcrt.nih.gov/molbio/hla_bind). This software uses a quantitative scoring matrix derived from coefficients representing the contribution of any given amino acid at a specified position in the peptide to binding to each HLA molecule. Analysis was performed for HLA-A2.1 (a MHC class I allele expressed by nearly 50% of cancer patients).

Peptides

All TADG-12 peptides were purchased from Alpha Diagnostic International (San Antonio, Tex) and provided at >90% purity, as verified by high-performance liquid chromatography and mass spectrometry analysis. Peptides used for DC priming are listed in Table 1. As a positive control for the HLA-A2–binding assay, the influenza virus matrix peptide (GILGFVFTL) was used. Peptides were dissolved in dimethylsulfoxide and frozen at −80°C until use.

Table 1. Peptides Used for Dendritic Cell Priming
Peptide IDTADG-12 PeptidesNo. of AABIMAS ScoreFold Increase±SD
  1. AA, amino acid; BIMAS, Bioinformatics Molecular Analysis Section; SD, standard deviation.

P1 TADG-12/9AQLGFPSYV9545.3160.79 ± 0.28
P2 TADG-12/9LLPLKFFPI9195.4481.03 ± .27
P3 TADG-12/10SLLPlKFFPI10425.3871.43 ± 0.38
P4 TADG-12/10LLPDdKVTAL10342.4612.26 ± 0.47
P5 TADG-12/10YLPKsWTIQV10319.9391.52 ± 0.23
P6 TADG-12/10GLDDlKISPV10262.351.45 ± 0.38
P7 TADG-12/10KLVGaTSFGI10211.7862.19 ± 0.43
P8 TADG-12/10SLLSqWPWQA10137.8621.11 ± 0.11
M1 FLUGILGFVFTL9550.9271.38 ± 0.29

Peptide-binding Assay

Peptide-induced stabilization of HLA-A2.1 molecules on T2 cells was done as described previously.9 Two × 105 T2 cells were incubated with 50 μg/mL of the indicated synthetic peptides and 3 μg/mL human B2μ (Calbiochem, Gibbstown, NJ) for 18 to 20 hours at 37°C. The cells were then stained with the HLA-A2.1–specific antibody, BB7.2, for 45 minutes, washed with fluorescent-activated cell sorter analysis buffer (phosphate-buffered saline, 5% fetal bovine serum [FBS], and 0.01% sodium azide), and stained with a secondary fluorescein isothiocyanate–conjugated anti–immunoglobulin G antibody (Biosource International Inc., Camarillo, Calif). The cells were fixed in 4% formaldehyde before flow cytometry analysis. The negative control consisted of T2 cells without peptide. The positive control consisted of T2 cells loaded with the Flu matrix peptide, GILGFVFTL. Flow cytometric analysis was done using a FACScanTM flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Experimental results are depicted as X-fold increase = mean fluorescent intensity of T2 cells loaded with peptide/mean fluorescent intensity of T2 cells with no peptide. An X-fold increase of >1 indicates that the peptide binds to HLA-A2.1.

Cell Line Cultures

The human T2 cells and the natural killer cell (NK)-sensitive target K562 cell line were obtained from American Type Culture Collection (Manassas, Va) and cultured in RPMI 1640 containing 10% fetal bovine serum (FBS, Gemini Bio-Products, West Sacramento, Calif). Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) were established from HLA-A2.1-positive donors and HLA-A2.1-positive ovarian cancer patients by coculture of peripheral blood leukocytes (PBLs) with EBV-containing supernatant from the B95.8 cell line in the presence of 1 μg/mL cyclosporine A (Novartis Pharmaceutical UK Ltd, Camberley, UK) as previously described.10, 11 Primary autologous serous papillary ovarian cancer cell lines were established after sterile processing of the samples from surgical biopsies as previously described for ovarian carcinoma specimens.10 Specimens were obtained from 3 patients harboring advanced stage disease (ie, 1 with stage III C and 2 with stage IV ovarian cancer) at the time of surgery through the Division of Gynecologic Oncology and the Pathology Department at the University of Arkansas for Medical Sciences, Little Rock, Arkansas, under approval of the institutional review board. All patients had ovarian cancer with serous papillary histology. Patient 1 was aged 65 years (OVA-1), Patient 2 was aged 72 years (OVA-2), and Patient 3 was aged 52 years (OVA-3). OVA-1 and OVA-3 primary cell lines were collected from patients at the time of primary debulking surgery and before the administration of adjuvant chemotherapy, whereas the OVA-2 primary cell line was derived from a patient harboring platinum-resistant disease at the time of tumor recurrence. OVA-2 tumor resistance to multiple chemotherapeutic agents in vivo was confirmed in vitro by measuring chemotherapy resistance as percentage cell inhibition by in vitro extreme drug resistance (EDR) assay (Oncotech Inc. Irvine, Calif).12 All of the tumor lines were cultured in Roswell Park Memorial Institute 1640 supplemented with N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid buffer, L-glutamine, penicillin, and 10% heat inactivated FBS. The epithelial nature and the purity of primary tumor cultures was verified by immunohistochemical staining and flow cytometric analysis with antibodies against cytokeratin and vimentin as previously described.10, 11 Only primary cultures that had at least 90% viability and contained >99% epithelial cells were used for cytotoxicity assays.

Screening for Peptide-specific HLA-restricted T Cells

Peptide-specific CTLs were generated in vitro using fully mature autologous dendritic cells separately pulsed with all 8 TADG-12–derived peptides and tested for their ability to kill peptide-pulsed targets, normal unpulsed LCLs, or tumor cells in a TADG-12–dependent fashion. Briefly, fresh and/or cryopreserved PBLs from 2 healthy HLA-A2+ female donors were used for generation of DCs. Similarly, PBLs from 3 consecutive HLA-A2–positive patients harboring advanced ovarian carcinoma overexpressing TADG-12 and from whom primary autologous cell cultures were available were also used for DC generation. The HLA types of the ovarian cancer patients studied were the following: Patient 1: A2, B35, B51, Cw4, Cw14; Patient 2: A2, A24, B35, B44, Cw2, Cw6; and Patient 3: A2, A24 B7, B49, Cw7. Monocyte-derived DCs were cultured in AIM-V (InVitrogen, Inc., Carlsbad, Calif) supplemented with granulocyte-macrophage colony-stimulating factor (Immunex, Seattle, Wash) and IL-4 (R & D Systems, Minneapolis, Minn).11 After culture for 5 days, DC maturation was induced by addition of tumor necrosis factor-α, interleukin (IL)-1β (both from R & D Systems), and prostaglandin E2 (Sigma, St. Louis, Mo).11 Mature DCs were pulsed for 1 to 2 hours at 37°C with 50 μg/mL of the 8 selected TADG-12–derived peptides described in Table 1, and washed twice before culture with PBLs at a responder:stimulator ratio of 30:1. The culture medium was AIM-V plus 5% human AB serum (Gemini Bio-Products). No IL-2 was added. After 7 days, responder T cells were collected and restimulated with peptide-pulsed DCs. For the second and third DC stimulations, the medium was supplemented with 50 U/mL IL-2, and the culture period was extended to 14 days. After 3 restimulations, CD8+ T cells were recovered by positive selection with anti-CD8 magnetic beads (Dynal Biotech, Brown Deer, Wis). Subsequent restimulations of CD8+ T cells used peptide-loaded irradiated autologous PBLs as antigen-presenting cells. The cytotoxicity of generated CTL lines was assessed using a standard 5-hour chromium release assay (51Cr-release). MHC-restriction of lytic activity was tested by a blockade of the killing of HLA-A2–positive tumors using the anti–pan-HLA monoclonal antibody (MoAb) W6 of 32 and the anti–HLA-A2 MoAb BB7.2 compared with the isotype-matched controls. Specific lysis of target cells is presented as percentage of specific 51Cr release, calculated from the formula: (E − S/T − S) × 100, where E is experimental 51Cr release, S is the spontaneous 51Cr release, and T is the total 51Cr release by 1% Triton X-100.

Kinetic of Expression of TADG-12 in Primary Ovarian Cancer Cell Lines

Because tissue digestion and prolonged in vitro cell culture may potentially alter TADG-12 antigen expression on freshly established primary tumors, primary ovarian cancer cell lines were evaluated before and after several in vitro passages for TADG-12 expression by real time PCR. Briefly, RNA extraction was performed on the autologous primary ovarian cancer cell line when tumor cells were 50% to 80% confluent after no passages (ie, P0) and up to 20 passages in vitro. RNA isolation was performed using TRIzol Reagent (Invitrogen, Carlsbad, Calif) according to the manufacturer's instructions. Quantitative real time PCR was performed with an ABI Prism 7000 Sequence Analyzer (Applied Biosystems, Foster City, Calif) to evaluate expression of TADG-12 in the primary tumors. The comparative threshold cycle method was used for the calculation of amplification fold as specified by the manufacturer. TADG-12 primers were obtained from Applied Biosystems as assay on demand products (Assay ID: HS00225161_m1).

Cytokine assays

Intracellular cytokine expression was measured by flow cytometry after overnight coculture of T cells with peptide-pulsed LCLs or DCs or anti-CD3 MoAb (solid phase OKT-3, 10 μg/mL, Ortho Pharmaceutical Corp., Raritan, NJ) as previously described.10, 11 Negative controls included T cells cultured alone, or with unpulsed LCLs or DCs. Fluorescence was measured with a FACSCalibur (BD Biosciences, San Jose. Calif), and data were analyzed with CellQuest software (BD Biosciences).

Enzyme-linked immunosorbent spot-forming cell assay

Interferon (IFN)-γ enzyme-linked immunosorbent spot-forming cell (ELISPOT) kits (R&D Systems, Minneapolis, Minn) was used to determine the frequency of cytokine-expressing CD8+ T cells after overnight activation with peptides, tumor cells, or OKT-3 MoAb. T cells (5 × 104/well) and targets (5 × 103/well) were added to duplicate wells and left overnight for T cell activation in 96-well plates precoated with diluted capture antibody (Multiscreen-IP opaque plate; Millipore, Billerica, Mass) (1:60 dilution in phosphate-buffered saline [PBS]) following manufacturer instructions. Streptavidin-AP (1:60 dilution in 1% bovine serum albumin in PBS) was added and incubated at room temperature for 2 hours, 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium chromogen solution was added to each well, and the plates were left in the dark for about 10 minutes at room temperature before deionized water was added to stop development. Plates were scanned by Cellular Technology Ltd. (Shaker Heights, Ohio), and results were analyzed with Immunospot software (Cellular Technology).

Statistical Analysis

Data were analyzed using Student t test. All data were expressed as mean percentages of positive cells ± standard deviation. In all tests, the difference was considered significant when P values were less than .05.

RESULTS

Binding Affinity of TADG-12 Peptides

Eight peptides (2 nonamer and 6 decamer) derived from TADG-12, which were predicted for binding according to the computer prediction programs, were synthesized and included in the peptide-binding study (Table 1). With the exception of TADG-12-p1 peptide, which failed to elevate HLA-A2.1 expression above background levels in multiple experiments, the remaining predicted HLA-A2.1–binding peptides tested were able to induce elevations in human T2 cell surface HLA-A2.1 expression (Table 1). Five of the TADG-12–derived peptides (TADG-12-p3, -p4, -p5, -p6, and -p7) were found to bind to HLA-A2.1 molecules with a mean fluorescence intensity (MFI) equal or superior to the Flu-matrix peptide used as positive control (Table 1).

TADG-12 expression by real time PCR

Because in recent gene expression profiling studies in ovarian carcinoma cell lines4 we observed major alterations in gene expression because of prolonged in vitro cell culture, a thorough analysis of TADG-12 antigen expression on the fresh ovarian tumors used in this study, before and after several in vitro passages, was performed by real time PCR. As representatively shown for 2 (ie, OVA-1 and OVA-2) of the 3 primary autologous ovarian tumors used in cytotoxicity assays, a consistent down-regulation of the expression levels of TADG-12 in the more advanced in vitro passages of the primary ovarian carcinoma cell lines was observed by real time PCR (Fig. 1). Thus, primary cell lines cultured for several passages in vitro may represent suboptimal models for evaluating the potential of TADG-12 peptide pulsed DC–stimulated CTL therapy against ovarian cancer. On the basis of these findings, early passages of primary ovarian cancer cell lines overexpressing TADG-12 were cryopreserved and used in the cytotoxicity assays shown below.

Figure 1.

Quantitative real time polymerase chain reaction analysis of TADG-12 expression in ovarian carcinoma is shown. The Y axis represents the fold induction relative to normal ovary expression. The X axis represents ovarian cancer sample from Patients 1 (Ova-1) and 2 (Ova-2) tested for TADG-12 expression at different time points of in vitro culture (ie, tumors never passed in culture to a new flask = P0, and P1, P3, P13, and P20). A clear down-regulation of the expression levels of TADG-12 in the more advanced in vitro passages is evident.

CTL response against TADG-12 peptides in healthy donors

PBLs from 2 HLA-A2–positive healthy female donors were stimulated with TADG-12 peptide–pulsed DCs as described in Materials and Methods. We included all 8 TADG-12–deduced peptides in the first line of experiments. Consistent cytotoxic responses against peptide-loaded autologous LCL targets were seen from the fourth passage onward in 5 (ie, p2, p3, p5, p7, p8) of 8 TADG-12 peptide–stimulated CTL populations from both healthy female donors (Fig. 2). Because CTL cell lines generated by in vitro primary stimulation with a high concentration of peptides often fail to lyse targets expressing endogenous antigens and/or tumor cells may not be competent to process and express specific TADG-12 peptides, HLA-A2–matched primary ovarian cancer cells expressing TADG-12 (ie, OVA-1) and HLA-A2–matched LCLs derived from the same cancer patient were also used as means to reliably demonstrate specific lysis of targets endogenously expressing the TADG-12 antigen. Unlike CTLs stimulated with peptides 2, 3, 7, and 8 (data not shown), TADG-12 peptide YLPKSWTIQV (P5)-specific CTL populations from both donors were found consistently cytotoxic against HLA-A2–matched primary ovarian cancer target cells (Fig. 3). Importantly, cytotoxicity was significantly inhibited by blocking MoAb against nonpolymorphic HLA A, B, (P < .05); NK-sensitive K562 cells and HLA-matched and autologous LCLs were lysed at a much lower level (Fig. 3).

Figure 2.

CD8+ cytotoxic T lymphocyte recognition of 8 TADG-12–derived peptides in healthy donors is shown. Cytotoxicity was tested in a 5-hour 51Cr-release assay against autologous lymphoblastoid cell lines (LCLs) and autologous LCLs pulsed with 50 μg/mL peptide. Bars represent percent cytotoxicity of TADG-12–derived peptide–specific CD8+ T cell populations against LCL pulsed with each peptide after subtraction of the cytotoxic activity against LCL controls (ie, unpulsed). Effector/target cell ratio is shown at 20:1. A representative experiment of 5 is shown.

Figure 3.

CD8+ cytotoxic T lymphocyte recognition of p5-peptide–pulsed and unpulsed autologous and human lymphocyte antigen (HLA)-A2–matched lymphoblastoid cell lines (LCLs) and an HLA-A2–matched Patient 1 (OVA-1) TADG-12–positive primary ovarian carcinoma cell line in healthy donors is shown. Cytotoxicity was tested in a 5-hour 51Cr-release assay against LCLs pulsed with 50 μg/mL peptide, OVA-1, and K-562 cells. Effector/target cell ratio is shown at 20:1. Anti-HLA class I blocking antibody (W6/32) and isotype control monoclonal antibody (mAb; data not shown) were used at 50 μg/mL. A representative experiment of 3 is shown.

CTL response against TADG-12 peptides in ovarian cancer patients

We next assessed whether PBLs from 3 HLA-A2–positive ovarian cancer patients stimulated with TADG-12 peptide–pulsed DCs may specifically lyse autologous TADG-12–expressing tumor cells. Again, all 8 TADG-12–deduced peptides were used in the first line of experiments. Similarly to the results obtained with healthy female donors, we were able to detect cytotoxic T cell responses against p2, p3, p5, p7, and p8 TADG-12 peptides in all 3 ovarian cancer patients after several weekly restimulations (data not shown). However, only p5 stimulated CTLs were able to consistently induce HLA class I–restricted T cell responses against autologous LCLs loaded with p5 peptide in all 3 patients (Fig. 4). Cytotoxicity was significantly inhibited by blocking MoAb against nonpolymorphic HLA A, B, and C determinants (P < .05) and anti-HLA-A2 MoAb BB7.2 (not shown); NK-sensitive K562 cells were not lysed (Fig. 4).

Figure 4.

CD8+ cytotoxic T lymphocyte recognition of p5-peptide–pulsed and unpulsed autologous lymphoblastoid cell lines (LCLs) and K-562 cells in ovarian carcinoma patients is shown. Cytotoxicity was tested in a 5-hour 51Cr-release assay against autologous LCLs and autologous LCLs pulsed with 50 μg/mL peptide. Anti–human lymphocyte antigen (HLA) class I blocking antibody (W6/32) and isotype control monoclonal antibody (data not shown) were used at 50 μg/mL. Effector/target (E:T) cell ratio is shown at 20:1. A representative experiment of 3 is shown. OVA-1 indicates Patient 1; OVA-2, Patient 2; OVA-3, Patient 3.

Next, we assessed whether TADG-12-p5–specific CTLs from ovarian cancer patients were able to kill autologous ovarian tumors endogenously expressing TADG-12. As shown in Figure 5, YLPKSWTIQV peptide–specific CTLs were able to efficiently recognize autologous malignant cells expressing TADG-12 in an HLA-A2.1–specific manner, whereas autologous LCLs and K562 cells were only minimally killed. In some experiments, autologous ovarian tumors were exposed to IFN-γ for 48 hours before being tested in cytotoxicity assays against P5-stimulated CTL populations. Although IFN-γ was able to significantly increase the levels of HLA-A2.1 and HLA-ABC in all 3 ovarian carcinomas (data not shown), cytotoxic activity was not significantly increased (Fig. 5). Blocking with anti–HLA-A2 MoAb BB7.2 and MoAb W6/32 was also able to reduce cytolytic capacity against each autologous cell line, which further confirmed HLA restriction of CTL activity (Fig. 5).

Figure 5.

CD8+ cytotoxic T lymphocyte recognition of autologous (interferon-γ [IFN-g] treated or untreated) TADG-12–positive ovarian tumor cells and human lymphocyte antigen (HLA)-identical TADG-12–negative autologous lymphoblastoid cell lines (LCLs) is shown. Cytotoxicity was tested in a 5-hour 51Cr-release assay against unpulsed LCLs and Patient 1 (OVA-1), Patient 2 (OVA-2), and Patient 3 (OVA-3). Percentage lysis at 5:1 (empty columns), 10:1 (gray columns), and 20:1 (black columns) effector/target (E:T) cell ratios is shown. Anti-HLA class I blocking antibody (W6 of 32), anti–HLA-A2.1 (BB7.2), and isotype control monoclonal antibody (mAb; data not shown) were used at 50 μg/mL. A representative experiment of 3 is shown.

Intracellular cytokine expression by flow cytometry

We used flow cytometric techniques to detect intracellular cytokine expression at the single-cell level. In our analysis, we tested intracellular IFN-γ and IL-4 cytokine expression of the peptide-specific CTL populations after antigen stimulation with antibody-producing cells (APCs) (ie, LCLs or DCs) loaded or unloaded with TADG-12–derived peptides or solid phase OKT-3 (ie, positive control). Only TADG-12-p3–peptide specific CTL populations were found to express significant amounts of IFN-γ after peptide antigen stimulation by flow cytometry (Fig. 6). In contrast, p5 CTLs were unable to produce significant intracellular IFN-γ expression after p5 peptide antigen stimulation, although they we able to secrete large amounts of IFN-γ when stimulated by OKT-3 (Fig. 7). Finally, no significant IL-4 secretion was found in p3 and p5 CTL stimulated with autologous peptide-loaded LCLs or OKT-3 (Figs. 6 and 7), suggesting no bias in favor of a type 2 cytokine profile.

Figure 6.

Representative dot plot analysis depicts intracellular interferon-γ (IFN-γ) (Left Panel) and interleukin-4 (IL4) (Right Panel) expression by p3-specific cytotoxic T lymphocytes against autologous lymphoblastoid cell lines (LCLs), autologous LCLs pulsed with 50 μg/mL of P3, and OKT-3 as described in Materials and Methods. Numbers in the quadrants represent the percentage of CD8+ cytokine positive T lymphocytes. FL1-H indicates anti–anti-CD8 fluorescein isothiocyanate; FL2-H, IFN-γ phycoerythrin (PE), or anti-IL4 PE.

Figure 7.

Representative dot plot analysis depicts intracellular interferon-γ (IFN-γ) (Left Panel) and interleukin-4 (IL4) (Right Panel) expression by p5-specific cytotoxic T lymphocytes against autologous lymphoblastoid cell lines (LCLs), autologous LCL pulsed with 50 μg/mL of P5, and OKT-3 as described in Materials and Methods. Numbers in the quadrants represent the percentage of CD8+ cytokine positive T lymphocytes. FL1-H indicates anti-anti-CD8 fluorescein isothiocyanate; FL2-H, IFN-γ phycoerythrin (PE), or anti-IL4 PE.

ELISPOT assays for analysis of tumor antigen-specific T cell function

PBLs derived from the 2 healthy donors and all 3 patients harboring advanced ovarian cancer were tested by ELISPOT assays for determination of the frequency of IFNγ-secreting CD8+ T cells specific for the TADG-12-p5 peptide. Although the data are not shown, we could not detect any consistent increase in spots over background controls stimulated with unpulsed DCs, indicating that the T cell precursor frequency for TADG-12-p5 peptide in PBLs is likely very low in normal donors and ovarian cancer patients. In contrast, significant numbers of IFNγ-secreting CD8+ T cells were detectable by ELISPOT assays in TADG-12-p5 CTLs when challenged against p5-pulsed DCs in both healthy donors (not shown) and ovarian cancer patients (Table 2) after a minimum of 3 rounds of restimulation.

Table 2. Interferon-γ Enzyme-linked Immunosorbent Spot-forming Cell Assays Using P5 Cytotoxic T Lymphocytes Obtained From Ovarian Cancer Patients After 3 In Vitro Restimulations
PatientUnloaded LCLs, Spots/ 100,000 CellsP5 Peptide- loaded LCLs, Spots/ 100,000 CellsSolid Phase OKT-3, Spots/ 100,000 Cells
  • LCLs indicates lymphoblastoid cell lines; OVA-1, Patient 1; OVA-2, Patient 2; OVA-3, Patient 3.

  • Each experiment was performed with 5 × 104 cells/well in duplicates, and the average number of spots was calculated.

  • *

    Significant increases over background (P < .05 by t test).

OVA-1557*>300
OVA-2988*>300
OVA-318144*>300

DISCUSSION

The TADG-12 antigen is a 454 amino acid protein that contains a cytoplasmic domain, a type II transmembrane domain, a low-density lipoprotein receptor class A (LDLR-A) domain, a scavenger receptor cysteine-rich domain, and a serine protease domain.5-8 TADG-12 is expressed in high amounts in ovarian carcinomas, most commonly with serous and endometrioid histology,4, 5 as well as in other biologically aggressive human malignancies such as pancreatic cancer and colorectal carcinomas.7, 8 In contrast, TADG-12 is physiologically expressed only in small amounts in normal tissues and is predominantly detected in the esophagus, stomach, small intestine, colon, and some tissues of the urogenital tract (kidney and bladder).7 Thus, because of TADG-12's differential expression in human tumors, it may represent a potential target for immunotherapeutic vaccination strategies aimed at chemotherapy resistant/refractory ovarian carcinoma. Consistent with this view, previous studies have suggested that TADG-12 expression is associated with the promotion of metastasis in human epithelial tumors,7 further supporting its potential use as target antigen in biologically aggressive cancer unresponsive to standard treatment modalities.

The study described here reports the results of our analysis on the use of HLA-A2–restricted TADG-12–derived peptides as potential targets for ovarian cancer immunotherapy. By using a bioinformatics approach, we synthesized 8 native peptides with predicted binding to HLA-A2.1. Seven of the 8 synthesized native peptides bound to HLA-A2.1, and 5 induced significant cytotoxic responses against peptide-pulsed APCs in healthy female volunteers and advanced stage ovarian cancer patients. DCs pulsed with p2 peptide were able to induce significant CD8+ CTL responses against peptide-pulsed LCLs, although p2 peptide was a relatively weak HLA-A2.1 binder, at least as measured in the T2 assay (Table 1). From this experience, we must conclude that the T2 binding assay is not a reliable indicator of the potential immunogenicity of any given peptide, and that peptides that give relatively poor results in this assay may nevertheless represent strong CD8+ T cell epitopes. More importantly, CTL populations generated against TADG-12-p5 peptide in both healthy donors and ovarian cancer patients were shown to lyse TADG-12 peptide–pulsed targets and autologous ovarian carcinoma cells endogenously expressing TADG-12 in an HLA-restricted manner. These results, collectively, indicate that TADG-12-p5 peptide (YLPKSWTIQV) may represent a naturally processed CTL epitope and suggest that such TADG-12–derived peptide might also serve as a basis for CTL-mediated therapy in ovarian carcinoma patients with TADG-12–expressing tumors.

Dendritic cells are the most potent antigen-presenting cells known in humans and play a crucial role during the priming and reactivation of antigen-specific immune responses.13-15 Several human phase I/II trials have been initiated using tumor antigen–pulsed DCs, and promising clinical results have been reported, in the absence of significant toxicity (reviewed in Ref.16). Importantly, in our study, we were able to demonstrate that not only chemotherapy-naive but also chemotherapy-resistant ovarian carcinoma cells may be susceptible to killing by TADG-12–specific CTLs. In this regard, OVA-2 was collected from a patient harboring recurrent disease refractory in vivo as well as in vitro (as confirmed by EDR assay against multiple cytotoxic agents including cisplatin, paclitaxel [Taxol], and doxorubicin) to salvage chemotherapy. These data further support the hypothesis that TADG-12–specific CTLs may potentially be effective against chemotherapy-resistant disease, which remains the main cause of death in ovarian cancer patients. Thus, on the basis of these data, TADG-12 peptide–pulsed DC immunotherapy may represent a further adjunct to the current vaccination strategies of ovarian cancer patients harboring chemotherapy-resistant/residual disease. It should also be emphasized that because in this work only T cell responses directed toward antigens displayed on HLA-A2 were examined, the contributions of other HLA-A allele products and members of the other HLA families (ie, HLA-B and HLA-C) in the presentation of naturally processed TADG-12 peptides will be likely defined in the future and further increase TADG-12's potential as a immunotherapeutic target.

When ELISPOT assays were used to evaluate the frequency of peptide-specific CTLs against TADG-12 in healthy donors and ovarian carcinoma patients, we found that only repeated in vitro stimulation allowed the detection of TADG-12–peptide specific CTLs, suggesting that precursor cells specific for TADG-12 are present at low frequencies in the peripheral circulation of normal donors and patients with ovarian cancer and, therefore, need to be expanded ex vivo for frequency determination. Flow cytometry data show that peptide-specific CTL populations were not able to express significant amounts of IFN-γ after peptide-antigen stimulation. Indeed, the only exception we found were CTLs specific against the TADG-12-p3 peptide, which although unable to kill autologous ovarian cancer cells naturally expressing TADG-12 express high levels of IFN-γ when stimulated with peptide-pulsed LCLs. In this regard, however, although it is not completely clear why TADG-12 P5 peptide–specific CTL populations are able to kill target cells naturally expressing TADG-12 but unable to express IFN-γ in response to stimulation with the P5 peptide, from previous studies it is known that different thresholds of T cell receptor stimulation in CTLs may induce diverse effector functions. Indeed, although low levels of T cell stimulation in primed CD8+ T cells may induce strong cytotoxic responses against the specific target antigen, a higher threshold of T cell receptor stimulation may be necessary to induce strong cytokine release by CTLs.17, 18 Consistent with this view, p5 CTLs express large amounts of IFN-γ in response to stimulation with OKT-3 MoAb, but negligible levels of IL-4, thus suggesting no bias in favor of a type 2 cytokine profile.

CD8+ CTLs have an important role in the immune defense against cancers, and recently Zhang et al19 have unequivocally shown that patients with advanced ovarian disease have significantly longer overall and progression-free survival if tumor-infiltrating lymphocytes are present, further underlining the importance of T cell–mediated immune control in ovarian cancer. In this context, the wide expression of TADG-12 in epithelial ovarian tumors and the finding that novel immunogenic peptides can elicit an effective immune response against TADG-12–expressing tumor cells suggest that TADG-12 may represent an antigen that may indeed be suitable for T cell–mediated immunotherapy. However, for such an approach to be successful, a sufficient T cell repertoire in the cancer-bearing patient will be required to elicit a CTL response against the tumor. We have been able to expand peptide-specific CTLs derived from blood lymphocytes against the immunogenic peptide TADG-12-P5 in HLA-A2+ patients with TADG-12–expressing ovarian cancer. On the basis of these findings, we believe that the use of TADG-12–derived peptide–pulsed DCs may be an attractive strategy for vaccination and adoptive T cell immunotherapy approaches against TADG-12–expressing tumors to prevent disease recurrence or progression in ovarian cancer patients.

Conflict of Interest Disclosures

Supported by grants from the US Department of Defense (OC020196), the Angelo Nocivelli, the Berlucchi, and the Camillo Golgi Foundations, Brescia, Italy, and the Istituto Superiore di Sanita, Rome, Italy.

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