Naturally occurring peptides associated with HLA-A2 in ovarian cancer cell lines identified by mass spectrometry are targets of HLA-A2-restricted cytotoxic T cells

Venkatesh Ramakrishna1,6, Mark M. Ross1,7, Max Petersson4, Christine C. Gatlin2, Charles E. Lyons3, Cara L. Miller1,8, Helen E. Myers1, Melanie McDaniel1, Larry R. Karns1, Rolf Kiessling4, Giorgio Parmiani5 and David C. Flyer1,9

1 Upstate Inc., Charlottesville, VA 22903, USA 2 Large Scale Proteomics Corp., Rockville, MD 20876, USA 3 Department of Chemistry, University of Virginia, Charlottesville, VA 22903, USA 4 Department of Oncology and Pathology, Radiumhemmet, Karolinska Hospital, 17176 Stockholm, Sweden 5 Unit of Immunotherapy of Human Tumors, Istituto Nazionale Tumori, 20133 Milan, Italy 6 Present address: Medarex Inc., 519 Rt. 173 West, Bloomsbury, NJ 08804, USA 7 Present address: MDS Proteomics, Charlottesville, VA 22904, USA 8 Present address: Wyeth-Lederle Viral Vaccine Immunology, Pearl River, NY 10956, USA 9 Present address: IOMAI Corporation, Gaithersburg, MD 20878, USA

Correspondence to: V. Ramakrishna; E-mail: vramakrishna{at}medarex.com
Transmitting editor: H. L. Ploegh


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Identifying naturally occurring peptides bound to HLA class I molecules recognized by HLA-restricted cytotoxic T lymphocytes (CTL) is both relevant and central to the development of effective immunotherapeutic strategies against cancer. Several cancer-related genes have been reported for ovarian cancer, but very few are known to be naturally processed T cell epitopes. In the present study we used mass spectrometry to identify 16 novel HLA-A2-bound peptides from HLA-A2+ ovarian cancer cell lines. All 16 peptides are derived from source proteins with diverse functions and marked homology to known proteins found in public databases. Synthetic peptide analogues of identified sequences were found to stabilize HLA-A2.1, albeit with varying affinities. The peptides were found to be antigenic in that a primary CD8+ CTL response could be elicited from normal donor blood. The CTL generated were not only peptide specific, but failed to recognize targets pulsed with control peptides. In addition, recognition of shared HLA-A2-restricted epitopes by these CTL is suggested by their reactivity with a subset of HLA-A2+ tumor lines and freshly isolated cancer cells or cell lines established from peritoneal ascites. These results were further corroborated by competitive inhibition of lysis of an otherwise susceptible cell line in the presence of cold peptide-pulsed targets. Furthermore, lack of recognition of several HLA-A2+ control cell lines or cells isolated from normal ovaries suggests that these peptides are cancer related. These findings broaden the list of CTL-defined antigens that could lead to the development of multi-epitope vaccines for the treatment of ovarian cancer.

Keywords: antigen, cytotoxic T lymphocyte, HLA-A2, mass spectrometry, ovarian carcinoma


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Peptides that derive from proteasomal-mediated degradation of cytosolic proteins that become associated with HLA class I molecules are 9–11 amino acids in length and are available for recognition by HLA class I-restricted CD8+ T lymphocytes (1). In recent years, several peptide antigens have been isolated from HLA-A2 molecules and identified by bioanalytical techniques (2). These studies have contributed to a growing list of peptide antigens that are currently undergoing clinical evaluation for a possible anti-cancer vaccine formulation. While the majority of antigens reported in the literature have been identified in melanoma (3) and recognized by HLA class I-restricted cytotoxic T lymphocytes (CTL), only two antigens are known to be defined by CTL in ovarian cancer, i.e. HER-2/neu and MUC-1 (46).

Due to the technically challenging nature of the methods used to identify antigens, an alternative approach, based on the prediction of HLA class I-binding peptides, has been described (7,8). Synthetic peptides from the predicted high-binding category are loaded on HLA-matched dendritic cells (DC) or other antigen-presenting cells (APC) and used as stimulators in CTL induction. The CTL are then utilized in the characterization of similar epitopes in multiple tumor lines sharing a common HLA class I allele. This strategy has worked for several new epitopes recently described from the Her-2/neu protein sequence (7,8). Similarly, T cell epitopes from folate-binding protein were first predicted and tested for antigenicity with CTL derived from both patients and normal donor blood (9). However, for this method to be effective, a prior knowledge of the source protein as well as computer algorithms to run predictions against a useful and experimentally verified database of MHC-binding peptides (1014) are needed. It should be noted that epitope predictions, in general, are cost-effective as they can reduce the number experiments to be performed. This approach, however, needs additional experiments to identify epitopes which are naturally processed. In an analogous CTL-independent approach developed by Rammensee et al. (15) and Muller et al. (16), novel peptides binding to HLA class I molecules were followed by mass spectrometry (MS)/MS for identification of the peptide sequences. Yet another approach for identifying relevant antigens utilizes MHC-eluted peptides fractionated by HPLC for direct stimulation of T cells (17).

Unlike our previous studies of melanoma and lung cancer where antigen identification was largely guided by tumor-specific CTL activity (18,19), we have taken a high-throughput peptide sequencing approach using sensitive MS techniques. In the present study, we have identified 16 different HLA-A2-associated peptides from established IFN-{gamma}-treated ovarian cancer cell lines, i.e. SKOV3.A2 and OVCAR3. Using currently available cancer protein and gene databases, the peptides could be traced to proteins with functions as diverse as cell cycle, apoptosis, cell signaling and proliferation. CTL were generated against all 16 peptides regardless of their HLA-A2.1-binding capability. These peptide-stimulated CTL are not only peptide specific, they also recognize HLA-A2+ cancer cells. Taken together, our results reinforce the notion that vaccination with these T cell epitopes will most likely induce an appropriate cell-mediated immune response to cancer while largely sparing normal tissue.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Tumor cell lines
The cell line SKOV3.A2 is an HLA-A2.1 transfectant of the original ATCC (Manassas, VA) ovarian adenocarcinoma line SKOV3 (HLA-A3, 68, B18, 35, Cw5, –) and was obtained from Dr Constantin Ioannides (M. D. Anderson Cancer Center, Houston, TX). A second ovarian cancer cell line OVCAR3 (HLA-A2, 29 B7, 58) was procured from ATCC. Both tumor lines were maintained in RPMI 1640 medium containing 10% heat-inactivated FBS, 2 mM L-glutamine, 10 mM HEPES, penicillin (100 U/ml)–streptomycin (50 µg/ml) solution and 1% sodium pyruvate solution (all from Sigma, St Louis, MO). The SKOV3.A2 cell line was continuously maintained in 250 µg/ml G418 (Invitrogen). The following HLA-A2+ ovarian cancer cell lines used in the target panel were obtained from different laboratories: INT.Ov2 (Dr G. Parmiani, Milan, Italy); Cov318, Cov413 and Cov504 (Dr P. I. Schrier, Leiden, Netherlands); Ov3507 (Dr R. Kiessling, Stockholm, Sweden); ALST (Dr T. J. Eberlein, University of Washington, St Louis, MO); MDROv (Dr R. S. Freedman, M. D. Anderson Cancer Center, Houston, TX); and ES-2.A1.A2.1 is a double transfectant selected with 300 µg/ml G418 and 100 µg/ml puromycin generated in-house from the ES-2 parent line (HLA-A3, 68, B14, 41), a kind gift of Dr R. Edwards (Magee Women’s Hospital, Pittsburgh, PA).

The following cell lines served as HLA-matched controls: HLA-A2.1+ target cells, T2 and C1R-A2.1 (an HLA-A2.1 transfectant) were kindly provided by Dr Victor Engelhard (University of Virginia, Charlottesville, VA). Additional targets comprised the HLA-A2+ normal fibroblast cell line, Hs-507-sk (ATCC), and several of the cell lines generated in-house, i.e. HLA-A2+ Epstein–Barr virus-transformed B lymphoblastoid cell lines (B-LCL), freshly isolated HLA-A2+ ovarian tumor cells (OV9813, OV9846, OV9848, OV9858 and OV9865) and cells from normal or uninvolved ovaries (N.OV1-3 series). A short panel of non-ovarian targets which included HLA-A2+ breast cancer (SKBr3-A2.1; C. G. Ioannides), colon cancer (COLO-205; ATCC) and lung cancer (H-322; ATCC) was also tested.

All cell lines described above were determined to be mycoplasma-free prior to use and maintained in RPMI medium described above. All surgical specimens collected were approved by the Human Investigation Committee and the Institutional Review Boards of the respective Cancer Centers: Dr Laurel Rice (University of Virginia Health Sciences Center; Charlottesville, VA), Dr Robert Edwards (Magee Women’s Hospital, Pittsburgh, PA), Dr C. G. Ioannides (M. D. Anderson Cancer Center, Houston, TX) and Dr D. Mishev (Sheinovo Hospital, Sofia, Bulgaria).

HLA typing
HLA typing of tumor cell lines by high-resolution DNA-PCR was carried out in the Tissue Typing Laboratories of Dr Michael Cecka (University of California, Los Angeles, CA) and Dr Pete Krausa (Forensic Analytical, Los Angeles, CA). Cell-surface expression of HLA-A, -B and -C molecules was assessed by flow cytometry using the following mAb (all from ATCC): W6/32, specific for pan-class I (anti-HLA-A, -B, -C); BB7.2, anti-HLA-A2; CR11.351, anti-HLA-A2/-68; GAP A3, anti-HLA-A3; ME.1.2, anti-HLA-B7/27; Sfr6B8, anti-HLA-B8; B1.23.2, anti-HLA-B+C; and L243, specific for pan-class II (anti-HLA-DR, -DP, -DQ). HLA-A1 expression was assessed by staining with anti-HLA-A1/-A36 specific antibody (One Lambda, Canoga Park, CA) or stained with an -A1/-A9-specific antibody clone (GV5D1) from Dr Arend Mulder (University of Leiden, Netherlands). HLA-A and -B locus typing of peripheral blood mononuclear cells (PBMC) was carried out using HLA typing trays (One Lambda).

Isolation of HLA-A2-associated peptides
The two cell lines used for antigen identification, SKOV3.A2.1 and OVCAR3, were expanded in cell factories (Nunc, Rochester, NY) in supplemented RPMI medium without antibiotics with the addition of 100 U/ml recombinant human IFN-{gamma} (Peprotech, Rocky Hill, NJ) 72 h prior to harvest. Cells were harvested in 1.5 x trypsin–EDTA (Life Technologies, Long Island, NY) on a weekly basis, washed and counted. Cell pellets were snap-frozen and stored at –80°C until use. Prior to peptide extraction, cells pellets from frozen aliquots corresponding to ~5–6 x 1010 cells were pooled and processed as previously described (19,20).

Briefly, the cell pellets were solubilized at 1 x 106 cells/ml in 20 mM Tris (pH 8.0), 150 mM NaCl, 1% CHAPS, 5 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 5 nM EDTA, 0.2% sodium azide and 17.4 µg/ml PMSF (Calbiochem-Novabiochem) for 1 h at 4°C. The lysates were centrifuged at 100,000 g, pellets discarded and the supernatant passed through a 0.22-µm filter. The supernatant was then passed over a series of recombinant Protein A–Sepharose columns (Roche, Nutley, NJ): first, a pre-column of CNBr-activated Sepharose 4B column saturated with Tris–NaCl followed by an immunoaffinity column conjugated to the HLA-A2-specific mAb BB7.2. After a series of wash steps, peptides were eluted from the antibody column with 10% acetic acid and boiled for 5 min to further dissociate any bound peptide from the class I heavy chains. The peptides were then separated from the co-purifying heavy chains and ß2-microglobulin by centrifugation on an Ultrafree-CL membrane with a nominal cut-off of Mr 5000 (Millipore, Bedford, MA.). The resulting peptide solution was concentrated in a speedvac to 50 µl. Aliquots of peptide-free fractions containing class I HLA molecules were analyzed by SDS–PAGE (Laemmli), and were found to be 95% pure with expected bands of 44 and 12 kDa for class I heavy chains and ß2-microglobulin respectively.

Peptide fractionation
The peptide extracts were fractionated by RP-HPLC5 using an Applied Biosystems model 140B system. The extracts were first concentrated by vacuum centrifugation from ~20 ml to 250 µl and injected into a Higgins (Mountain View, CA) C18 HAISIL column (2.1 mm x 4 cm, 300 A, 5 µm). The peptides were eluted by first using a gradient of acetonitrile:0.085% trifluoroacetic acid (TFA) in 0.1% TFA:water with the concentration of acetonitrile increasing from 0 to 9% (0–5 min), 9 to 36% (5–55 min) and 36 to 60% (55–62 min). The flow rate was 200 µl/min and fractions were collected at 40- or 60-s intervals. Peptides were detected by monitoring UV absorbance at 214 nm.

Mass spectrometric analysis
Mass spectrometric analysis of HPLC fractions was performed by online electrospray ionization tandem MS (LC-ESI-MS-MS) and MS/MS using a Finnigan LCQ ion trap mass spectrometer and LC-ESI materials, with configuration and conditions as previously described (21). An aliquot corresponding to ~1/10th of a HPLC fraction was loaded onto the reverse-phase C18 microcapillary HPLC column and gradient (A = 0.1 M acetic acid in H2O, B = 70% acetonitrile, 0.1 M acetic acid in H2O, 0 to 60% B in 60 min) eluted directly into the mass spectrometer. The instrument was operated in a data-dependent fashion, in which acquisition of full-scan mass spectra (300 < m/z < 1500) was followed by collision-activated dissociation analysis of the five most abundant ions. Each cycle (MS scan followed by five MS/MS scans) was performed in ~14 s and cycles were repeated continuously for the duration of peptide elution. MS/MS spectra were searched using the SEQUEST algorithm against the non-redundant protein database (http://www.ncbi.nlm.nih.gov/Entrez/) maintained at the National Center for Biotechnology Information.

Peptide synthesis
Synthetic peptides were produced using a Gilson AMS 422 multiple peptide synthesizer (Madison, WI) and standard Fmoc chemistry to yield small quantities of peptides (~10 µmol). Peptides were purified by reverse-phase HPLC using a 4.6 mm i.d. x 100 mm long POROS column (PerSeptive Biosystems, Cambridge MA) and a 10 min 0–60% acetonitrile in 0.1% TFA gradient. Peptides thus made were found to be >85% pure. Custom-scaled peptides of higher purity (~98%) were purchased from Research Genetics (Huntsville, AL) or Peptidogenic Research (Livermore, CA). Synthetic peptides were aliquoted in DMSO at a concentration of 5 mg/ml and stored at room temperature under nitrogen for immunological experiments.

Homology searches of identified peptide sequences
Proteins containing peptides corresponding to the masses identified by MS were analyzed with the search algorithm, SEQUEST. Searches were also carried out on the GenBank non-redundant sequence database (http://www.ncbi.nlm.nih.gov/Entrez/) as well as on our own unique database of 2943 specific sequences compiled from GenBank and EST database entries. Upon experimental confirmation of the peptide sequence, a tBLASTn search of the GenBank non-redundant database was performed to identify any genes containing the DNA sequence encoding the peptide.

HLA-A2.1 peptide-stabilization assay
Each peptide was tested for concentration-dependent binding to T2 cells in a HLA-A2.1 stabilization assay (22). TAP-defective T2 cells were incubated at room temperature for 16 h with peptide concentrations ranging from 0.1 to 100 µM in AIM-V medium containing 1.5 µg/ml ß2-microglobulin. Stability of HLA-A2.1 was assessed by flow cytometry following staining with MA2.1 mAb (ATCC) and rabbit F(ab)2 anti-mouse-FITC secondary antibody (Dako, Glostrup, Denmark). The HLA-A2.1 standard binding peptides, flu M1 (GILGFVFTL) and hepatitis B virus core antigen HBVc18–27 P6Y~C (FLPSDCFPSV), were used as positive controls. The t1/2 of dissociation of HLA-A2.1-binding peptides was predicted from Parker’s HLA-binding peptide algorithm (http://www-bimas.dcrt.nih.gov/molbio/hla_bind/) (10,12).

Generation of monocyte-derived DC and peptide loading
PBMC were purified from HLA-A2+ normal donor blood using lymphocyte separation media (Cappel ICN Biomedical, Aurora, OH). PBMC (5 x 106) were added to individual wells of a 24-well cluster plate (Costar, Corning, NY) in 1.0 ml of serum-free AIM-V medium (Life Technologies) and allowed to adhere for 60 min at 37°C. Non-adherent cells were removed and saved as a source of effector T cells. Adherent PBMC (~8 x 105/well) were then pulsed with 50 µg/ml synthetic peptides in serum-free AIM-V medium containing 1.5 µg/ml ß2-microglobulin (Calbiochem-Novabiochem, San Diego, CA) and incubated for 2 h at 37°C. Unbound peptides were aspirated and the wells washed with media.

Monocyte-derived DC were generated as follows. PBMC (5 x 107) were allowed to adhere in T-75 flasks (Corning) in 10 ml of serum-free AIM-V medium for 60 min at 37°C. Non-adherent cells were collected as a source of effector T cells and pooled with the previous collection above. Adherent monocytes in flasks were then exposed to recombinant human granulocyte macrophage colony stimulating factor (GM-CSF, 25 ng/ml; Peprotech) and recombinant human IL-4 (100 ng/ml; Peprotech) in 10 ml of AIM-V medium containing 10% heat-inactivated FBS. DC obtained by this method [immature DC (iDC)] are characterized by expression of low levels of CD83, CD80, CD86, and HLA class I and class II molecules (data not shown).

Mature DC (mDC) were obtained by exposing day 5 DC cultures to recombinant soluble CD40 ligand (sCD40L; Peprotech) at 1.5 µg/ml for 24 h in the presence of 25 ng/ml GM-CSF and are characterized by expression of high levels of CD80, CD86, and HLA class I and class II molecules. mDC were harvested, washed, pulsed with 5 µg/ml peptide in serum-free AIM-V medium and irradiated (5000 rad) prior to use as stimulators.

Generation of peptide-specific CTL
The protocol used here is a modification of the method described by Plebanski et al. (23). CTL to peptide were generated by 3–4 cycles of stimulation with peptide-loaded APC. For the first round of stimulation (day 0), T cells or non-adherent PBMC from above (2 x 106/ml or 4 x 106 per well) were added in bulk (CD4+, CD8+, NK, etc.) to adherent PBMC-loaded peptides in serum-free medium (50 µg/ml), ß2-microglobulin (1.5 µg/ml) (Calbiochem-Novabiochem), recombinant human IL-7 (5 ng/ml) (Peprotech) and keyhole limpet hemocyanin (5 µg/ml) (Sigma, St Louis, MO). Cultures were re-stimulated with iDC every 7 days, pulsed with varying amounts of peptide (second round 25 µg/ml, third round 10 µg/ml) and irradiated (5000 rad) on day 8. At each re-stimulation, the T cells were transferred to new plates by first aspirating 70% of spent media in wells and then transferring the pooled contents to a new plate. Fresh IL-7 was added at each re-stimulation. The responder:stimulator (T cell:DC) ratio was set at 20 for each stimulation. Recombinant human IL-2 (10 U/ml) was added on day 5 after each re-stimulation.

Prior to 51Cr-release assay, the T cells were harvested and CD8+ T cells were purified by positive selection using CD8+ microbeads immunomagnetic cell separation with MACS kit (Miltenyi Biotec, Auburn, CA). If a fourth round of stimulation was necessary following CTL analysis, the CTL were pulsed as before, except with 5–10 µg/ml of peptide.

Generation of allospecific CTL
HLA-A2-allospecific CTL were obtained in a mixed lymphocyte reaction by repeated stimulation of HLA-A3+ PBMC (responders) with irradiated HLA-A2+ stimulator PBMC at a ratio of 10:1 in the presence of 10 U/ml IL-2. Stimulation was repeated weekly with PBMC from different HLA-A2+ donors so as to minimize alloresponse to non-HLA-A2 antigens. T cells were assessed for lysis on several HLA-A2+ targets including tumor cells, EBV-B cells and HLA-A3+ targets every week after the third round of stimulation.

CTL expansion
Expansion of large numbers of peptide-specific or HLA-A2-allospecific CTL was achieved by culturing 5 x 104–1 x 105 T cells around day 6 or 7 post peptide- or allostimulation in the presence of 2.5 x 107 irradiated (5000 rad) allogeneic normal donor PBMC coated with anti-CD3 antibody at 10 ng/ml (BD PharMingen, San Diego, CA) and 25 U/ml of recombinant human IL-2 (Peprotech) in a final volume of 30 ml RPMI medium. Media changes with IL-2 addition (50 U/ml) were effected on days 5 and 8. Cells were harvested for cytotoxicity assays on days 10–12 and re-stimulated or frozen for later use.

51Cr-release cytotoxicity assay
The standard 4-h Cr-release assay was performed in 96-well V-bottomed microplates. Target cells in suspension (T2, C1R.A2, B-LCL and K562) were labeled with 100 µCi Na251CrO4 (NEN Life Science, Boston, MA) per 1 x 106 cells either overnight (~ 6–18 h) in 5 ml RPMI 1640 media containing 2–5% FBS or for 60–90 min at 37°C directly with the cell pellet in the case of adherent cells (tumor cell lines and control lines). Labeling was terminated by washing the targets with cold media containing 5% FBS for a total of three washes. Target cells were resuspended at a concentration of 2 x 104/ml. About 2 x 103 targets in 100 µl were delivered to each well containing CTL (effectors) seeded at different E:T ratios. Spontaneous release wells contained targets in media alone, while maximal release wells contained targets in 2% NP-40 detergent (Igepal CA-630; Sigma). HLA restriction of CTL-mediated killing was achieved by preincubation of targets with HLA-specific antibodies prior to incubation with CTL.

The plate was gently spun for 1–2 min and incubated at 37°C for 4 h. For harvesting assay plates, 100 µl of supernatants from the wells was transferred to counting tubes (USA Scientific) and {gamma} counts were determined in a {gamma} counter (ICN Micromedic Systems, Huntsville, AL). Cytolytic activity of T cells was expressed in percent specific lysis as follows: specific lysis = {[experimental release (c.p.m.) – spontaneous release (c.p.m.)]/[maximal release (c.p.m.) – spontaneous release (c.p.m.)]} x 100.

Competitive inhibition assay
Peptide-stimulated CTL were reacted with 51Cr-labeled Ov2 tumor cells (E:T ratio of 40) in the presence of excess of cold targets in a 4-h Cr-release assay. Cold targets were either empty T2 cells, T2 cells pulsed with 1 µg/ml relevant peptide (used to stimulate CTL) or irrelevant (control) peptides (HER-2/neu369–377 or MART-127–35), or IFN-{gamma} pre-treated tumor cells (SKOV3.A2 and OVCAR 3) with the cold target in 5-fold excess of the hot target.

Statistical analysis
Two-tailed Student’s t-test was used for statistical analysis of 51Cr release and peptide-binding assays.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Analysis of HPLC-fractionated HLA-A2-eluted peptides
Peptides were purified from HLA-A2 molecules of IFN-{gamma}-treated SKOV3.A2 and OVCAR3 ovarian cancer cell lines, and subjected to reverse-phase HPLC analysis. Forty HPLC fractions per MHC cell preparation were analyzed by MS. Approximately 500 MS/MS spectra per HPLC fraction were collected and a total of 20,000 MS/MS spectra per cell line extract, corresponding to 1 x 108 cell equivalents per LC-MS/MS run, were put through analysis. A fraction of these spectra (10%) yielded database hits with SEQUEST cross-correlation scores (XCORR) >2.0. Of these, <1% of the database protein/genes matched appear to be cancer related. An XCORR threshold of interesting candidate peptides was set at 2.30. Where ambiguous results were encountered, spectra were interpreted manually. All identified peptides were confirmed by LC-MS co-elution experiments using the HPLC fraction and the corresponding candidate synthetic peptide (data not shown). The peptides possess the canonical motif for binding to HLA-A2.1 molecule, i.e. N-terminal anchor residues (L > M > I > V) at P2 and C-terminal residues (V > L > I > M) at P9 or P10 positions of the peptide sequence.

The results summarized in Table 1 show 16 HLA-A2-associated peptides and their corresponding source proteins. The majority of the peptides identified in both cell lines are 9mers, although two 10mer peptides (P2 and P15) were found in OVCAR3, whereas a 10mer and an 11mer (P9 and P10) were found in SKOV3.A2. Interestingly, three peptides were found from the preparations of both cell lines (P3, P7 and P16).


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Table 1. Analysis of HLA-A2.1-eluted peptides by MS
 
Homology searches reveal proteins with functional diversity
The analysis of peptide sequences searched in public databases shows that the proteins identified are expressed in a wide variety of tissues including neoplasia. Homology searches returned 100% matches for all 16 peptides but one (P7) derived from Abl-binding protein 3 (AblBP3, U31089) since two additional entries against this sequence were found containing this epitope, i.e. human Abl-interactor 2 (Abi-2, U23435) and human Arg protein tyrosine kinase-binding protein (ArgBP, X95632).

Comparing the coding region of each of the entries, it appears that both AblBP3 are wholly contained in ArgBP, possibly representing an alternatively spliced mRNA isoform or a partial cDNA clone with a truncated 5' end. Abi-2 and AblBP3 are homologous over the majority of the coding region, including the region encoding the identified peptide sequence. These two differ in the first 10 amino acids of AblBP3, which are replaced with an alternate 50 amino acids in Abi-2, possibly due to alternative splicing of the first coding exon. In addition, it was not possible to resolve the origin of peptide P4, since the peptide exists in both {alpha} and ß forms of topoisomerase II. The remainder of the peptide sequences listed in Table 1 could be traced to well-known proteins catalogued in the NCBI nucleotide and SwissProt database. Searching against our own database of overexpressed genes in cancer, we find that the APC gene product, the ADAM17 protein, {gamma}-catenin and the RTK6 appear to be particularly interesting, not only in view of their potential in cell transformation and the important role in cell signaling and apoptosis, but also from the standpoint of targeting specific therapy.

HLA-A2.1-stabilizing peptides
In order to determine whether the 16 peptides identified indeed bind to HLA-A2.1, synthetic peptides analogues were tested for their ability to stabilize the peptide–HLA-A2.1 complex in the presence of ß2-microglobulin. All 16 peptides were pulsed on TAP-defective T2 cells overnight (~16 h) and cells were analyzed for HLA-A2.1 expression by flow cytometry. Binding data shown in Table 2 indicate all 16 peptides tested were found to stabilize HLA-A2.1, albeit to different extents. Based on the shift in MFI values, the peptides were arbitrarily ranked as low (90–110), intermediate (110–140) or high (140–200) affinity binding peptides. For a relative comparison, standard peptides used were flu M1 and HBVc18–27 peptides.


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Table 2. Stabilization of HLA-A2.1 using synthetic peptides
 
Interestingly, only three peptides appear to be weakly stabilizing (P2, P3 and P10), while three other peptides displayed intermediate affinity and were, therefore, moderately stabilizing HLA-A2.1 molecules (P1, P6, P8 and P11). The rest of the peptides bound HLA-A2.1 with high affinity (P4, P5, P7, P9 and P12–P16). Some peptides were able to stabilize HLA-A2.1 at peptide concentrations <10 µM (P5, P12 and P16), while peptides P1 and P2 needed higher peptide concentrations (>10 µM). The affinity estimates here are not absolute as would be expected in binding of peptides to purified HLA-A2.1 folded chains in solution. Rather, they give an estimate of the ‘off-rate’ and are useful in relative rank ordering. In other words, the peptides that stabilize the HLA-A2.1 molecule display a slower off-rate and vice versa.

Induction of peptide-specific CTL
We next tested whether peptides that bound to HLA-A2.1 could induce a CTL response to the peptide. Several in vitro stimulation protocols that were attempted, from multiple HLA-A2+ donor blood, largely resulted in CTL with short-lived peptide-specific reactivity. Using a modification of the method described by Plebanski et al. (23), CTL with sustained activity could be generated using peptide-pulsed autologous APC. Peptide-reactive T cells were first assayed for activity using T2 targets which were either not pulsed or pulsed with peptide to which CTL was raised (Fig. 1 A and B).



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Fig. 1. Generation of peptide-specific CTL. T cells were stimulated with autologous DC or PBMC pulsed with synthetic peptides on a weekly basis for 4–5 weeks, enriched for CD8+ T cells and assayed for cytotoxicity as described in Methods. Targets (T2 cells) were either left empty (open circles) or pulsed with 1.0 µg/ml peptide (filled circles) in serum-free AIM-V medium at 37°C for 1 h and washed before use as targets. (A) P11, KLQELNYNL; P12, ILIEHLYGL; P13, YLIELIDRV; P14, NLMEQPIKV; P15, FLAEDALNTV; P16, TLLNVIKSV; P4, FLYDDNQRV; and P7, ILDDIGHGV; and (B) P1, KIMDQVQQA; P3, KLDVGNAEV; P9, LLIDDKGTIKL; P6, YLMDTSGKV; P2, RLQEDPPAGV; P5, ALMEQQHYV; P8, LLDRFLATV; and P10, RLYPWGVVEV, all show reactivity of peptide-stimulated CTL to their cognate peptide-pulsed T2 cells. Results are representative of five independent experiments performed in triplicate, P < 0.01.

 
The peptide-stimulated CTL recognize only peptide-pulsed targets (T2), but not empty (non-pulsed) T2 cells or T2 cells loaded with irrelevant control peptides: HER-2/neu369–377 (KIFGSLAFL) or MART-127–35, (AAGIGILTV), indicating the peptide-specific reaction of all CTL cultures (Fig. 2). In addition, peptide-specific T cells were HLA class I- and HLA-A2-restricted since their activity was effectively blocked only in the presence of W6/32 (20 µg/ml) and BB7.2 (10 µg/ml) antibody, but not when isotype-matched control antibodies were used at the same concentration (Table 3). These results show that peptides identified by MS are antigenic in normal donors in whom a primary CTL response could be induced under conditions of appropriate stimulation (24).



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Fig. 2. Peptide-specific T cells recognize HLA-A2+ tumor cells. T cells were generated as described in Methods and assayed for cytotoxicity on IFN-{gamma}-treated HLA-A2+ tumor cells and control target cells (including irrelevant peptide-pulsed on T2 cells at 1.0 µg/ml and incubated as described in the legend to Fig. 1). CTL raised to synthetic peptides (A) P1, KIMDQVQQA; P3, KLDVGNAEV; P9, LLIDDKGTIKL; P6, YLMDTSGKV; P2, RLQEDPPAGV; P5, ALMEQQHYV; P8, LLDRFLATV; and P10, RLYPWGVVEV; and (B) P11, KLQELNYNL; P12, ILIEHLYGL; P13, YLIELIDRV; P14, NLMEQPIKV; P15, FLAEDALNTV; P16, TLLNVIKSV; P4, FLYDDNQRV; and P16, ILDDIGHGV, show reactivity of peptide-specific CTL with tumor cells, but not with targets pulsed with irrelevant peptides. T2 and K562 cells were left untreated. Results shown are representative of three independent experiments performed in triplicate, P < 0.05.

 

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Table 3. HLA restriction of peptide-specific CTL
 
Peptide-specific CTL recognize HLA-A2+ tumor cells
In order to determine whether peptides found on SKOV3.A2 are also shared among other ovarian tumor lines or cells isolated from fresh cancer material, we tested a selected group of peptide-specific CTL against a broad panel of 22 different HLA-A2+ targets, which included ovarian, lung and colorectal tumor cells. Tumor lines used in the target panel were previously tested for HLA-A2 expression by flow cytometry and determined to be suitable targets using HLA-A2-allospecific CTL (data not shown), indicating appreciable levels of HLA-A2 expression on the cell surface. Control targets in the panel included HLA-A2+ T2, B-LCL, Hs-507-sk fibroblast line and normal ovarian cells. The average specific lysis of the control targets was low (~5%). CTL response was considered positive if lysis on tumor targets was >=2.5-fold than that of control targets. The results show that different tumor targets are lysed to different extents by peptide-specific CTL and in some cases background to no reactivity is seen (Fig. 2A and B).

Reactivity of a select group of peptide-stimulated CTL cultures with a broad panel of targets revealed that CTL reacted with a subset of HLA-A2+ tumor cells in the panel indicating the presence of similar epitopes shared among several ovarian tumors restricted by the HLA-A2.1 allele (Fig. 3A–H). Thus, peptides P3–P6, P8, P10 and P11 represent T cell epitopes shared among five of 15 HLA-A2+ ovarian carcinomas, while epitope P7 appears to be shared between just two tumors, SKOV3.A2 and Ov2 cell lines. Interestingly, peptides P3 and P6 appear to be shared antigens outside of the ovarian panel, i.e. on a breast cancer cell line, SKBr3.A2. Peptides P3, P4 and P6, on the other hand, appear to be shared T cell antigens on colorectal cancer (OV9865). Since the antigen density on target cells is expected to be in the nanomolar to picomolar range, the observed recognition of tumor cells indicates that T cells display sufficient avidity to exert effector function in that range.




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Fig. 3. Shared T cell epitopes recognized by peptide-specific CTL. Peptide-stimulated CTL cultures were selected for screening a broad target panel consisting of HLA-A2+ tumor cells pretreated with IFN-{gamma}. Control targets included C1R.A2, JY, T2 and IFN-{gamma}-treated Hs-507-sk fibroblast cell line. Peritoneal ascites-derived tumor cells (OV9813, OV9846 and OV9848) or cell lines (OV9858 and OV9865) were thawed from frozen aliquots, introduced into flasks with growth medium for 3 days and exposed to IFN-{gamma} for 48 h prior to assay. Panels show reactivity of CTL raised against peptides (A) P3, (B) P6, (C) P5, (D) P8, (E) P10, (F) P11, (G) P4 and (H) P7 tested for reactivity against an HLA-A2+ target panel. E:T = 40. Results are representative of two independent experiments performed in triplicate, P < 0.05.

 
No reactivity was noted in other HLA-A2+ tumors or control lines, including cells isolated from normal ovary (Table 4), suggesting that peptides for which T cells are specific are either not presented on the cell surface or present in low copy numbers. However, lack of recognition of normal ovarian target cells could also be due to a reduced HLA-A2 expression on the cell surface. Flow cytometric analysis of anti-HLA-A2 antibody (BB7.2)-stained cells showed modest levels (MFI 90–120 compared to 190 in C1R.A2.1 cells), while staining with anti-HLA-A, -B, -C pan-class I antibody (W6/32) revealed that these cells are all HLA class I+ (MFI 400–600 compared to 450 in C1R.A2.1). Using HLA-A2-allospecific CTL, the lysability of the various normal targets was found to be variable (Table 5). These results indicate that HLA-A2 expression is crucial for antigen presentation, but does not render the target susceptible to CTL-mediated lysis unless loaded with cognate peptide as noted in the pulsed versus non-pulsed N.OV-2 subset of targets tested.


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Table 4. Peptide-reactive T cells fail to recognize normal ovarian cells
 

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Table 5. Sensitivity of normal ovarian cells to CTL is limited by HLA-A2 expression and peptide presentation
 
CTL reactivity on tumor is antigen dependent
Since peptide-specific CTL react with some HLA-A2+ tumor lines, the possibility that CTL may non-specifically recognize targets, by antigen-independent mechanisms, was not ruled out in the above experiments. To directly address the issue of peptide specificity of CTL, a cold-target inhibition/competition assay was set up. CTL raised to 10 peptides were selected and tested for Ov2 lysis in the presence of inhibitor cold targets using the standard 4-h cytotoxicity assay. With the exception of peptide P15, where no inhibition of Ov2 lysis was seen, all peptide-pulsed targets caused reduction in Ov2 lysis, suggesting common determinants were recognized (Fig. 4). Barring background inhibition (~15%) seen with empty T2 cells or T2 loaded with irrelevant control peptides, the window of specific inhibition was 20–50%.

When competition was carried out in the presence of cold tumor cells, SKOV3.A2 and OVCAR 3, inhibition was more pronounced with some epitopes (P3, P5–P8, P10 and P14). In addition, the activity of CTL raised to peptides P2 and P5 was blocked in the presence of cold OVCAR3, but not cold SKOV3.A2 (Fig. 4). This result is consistent with the identification of peptides P2 and P5 in the OVCAR3 cell line. Peptides common to both cell lines (P3, P7, P8 and P16) are competed by their respective CTL as expected.



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Fig. 4. Competitive inhibition of Ov2 lysis by cold peptide-pulsed targets reveals peptide-specificity of CTL. CTL raised to 10 different peptides were assayed for cytotoxicity against 51Cr-labeled Ov2 tumor cells in the absence (None) or presence (T2 empty or pulsed and IFN-{gamma}-treated tumor cells) of excess cold competitor targets. E:T = 40; the competitor:hot target ratio was set at 5.0 (~10,000 cold targets). Results are representative of two independent experiments performed in triplicate, P < 0.05.

 
These results indicate that (i) CTL show specific interaction with the peptide to which they are sensitized to, (ii) CTL compete for similar epitopes presented on Ov2 as were found in SKOV3.A2 and OVCAR3 cell lines by MS, and (iii) the CTL response to Ov2 cells is not inhibited by peptide P15-pulsed T2 cells, because it nearly overlaps the response noted in the presence of control peptides (data not shown).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
There is a growing body of evidence that IFN-{gamma} can augment antigen presentation by stimulating several components of the MHC class I presentation pathway (1,25). In this study we have identified 16 novel peptide sequences associated with the HLA-A2.1 molecule expressed in two ovarian cancer cell lines previously exposed to IFN-{gamma}. All 16 peptides are homologous to known proteins in the database (Table 1). Mass spectrometric detection of HLA-A2.1-associated peptides in SKOV3.A2 and OVCAR3 indicates that the proteins from which they originate have been targeted to the class I presentation pathway. The proteins are interesting in terms of their role in several cancer cell pathways. It is important to point out that some or all of the peptide sequences identified here could be due to a shift in antigen processing and transport as a result of IFN-{gamma} exposure, as observed in the mass spectrum of A2 peptides of IFN-{gamma}-treated versus untreated SKOV3.A2 cells (data not shown).

In order to assess the functionality of HLA-bound peptides, we have used DC pulsed with synthetic peptides to generate a T cell response. Thus, using appropriate conditions for in vitro stimulation, we were able to generate CTL against all of the peptides identified regardless of their affinity for HLA-A2. The condition under which this might occur has been addressed in many earlier studies and appears to involve CD40–CD40 ligand interactions as well as the simultaneous engagement of peptides by CD4+T cells in the context of HLA class II molecules (24,2628). This is in contrast with other reports where affinity of a peptide to the HLA molecule is the sole determinant of its immunogenicity. With regard to the protocol described here, there are clear advantages in that (i) stimulation can commence on the day a blood sample is processed and (ii) non-adherent T cells need not be frozen while waiting for DC cultures to be ready. Although data from a single donor is presented for consistency, we have examined a large cohort of HLA-A2+ donors. Nearly 50 donor samples were tested with this protocol, with T cell responses to only some of the peptides (P1, P3, P4, P6, P8 and P14) on a consistent basis in about half of the donors (data not shown).

The evidence that peptides are immunologically active is based on the following set of observations: (i) peptides presented by DC can evoke a CD8+ T cell response that is restricted by HLA class I and HLA-A2; (ii) CTL are peptide specific since they fail to lyse targets loaded with HLA-A2-binding irrelevant peptides as demonstrated in direct lysis and competitive inhibition of lysis experiments; (iii) these peptide-reactive T cells recognize a panel of freshly isolated tumor cells and cell lines established from peritoneal ascites or tumor banks, suggesting the occurrence of shared T cell epitopes (Fig. 5); and (iv) peptide-sensitized T cells do not react with target cells isolated from normal or uninvolved ovaries with or without IFN-{gamma} treatment. This last observation would suggest that similar peptides may not be processed or presented by these cells or displayed in very low copy numbers. Furthermore, HLA class I expression on normal ovarian tissue is not nearly enough to elicit an effector response and we cannot exclude the possibility that other HLA molecules may be involved in delivery of inhibitory signals to CTL attack. Nonetheless, these data reasonably argue against the overriding concern of inducing untoward reaction against normal tissue.



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Fig. 5. Shared T cell epitopes in ovarian cancer. The model is based on peptide-specific CTL reactivity shown in Figs 2–4. Sequences appearing in the overlapped segments represent likely shared antigens between two or three cancer cell lines, while sequences depicted in the non-overlapped areas appear to be unique to the cell lines.

 
Evidence that IFN-{gamma} treatment could directly modulate the antigenic profile of cancer cells is demonstrated in the study by Morel et al.; the presence of ‘immunoproteasomes’ following exposure to IFN-{gamma} could impede the processing of some antigenic peptides that are otherwise more efficiently produced by the standard proteasome (29). In addition, the repertoire of peptides associated with MHC class I appears to be different in the different cell types and, as succinctly demonstrated by Toes et al., these differences could arise from the cell types harboring different sets of proteasomes (30).

Novel HLA-A2-associated peptide sequences identified here along with their antigenicity in humans extends the number of antigens in ovarian cancer. However, the question of whether the identified peptides constitute a ‘self’ or ‘non-self repertoire’ of MHC class I-presented peptides is an important one since the sensitization to self-antigens could generate autoimmune reactions. Nonetheless, tissue-specific self-peptides as immunotherapeutic targets have been used in the case of melanoma (MART-1, gp100-2M and tyrosinase), ovarian and breast cancer (HER-2/neu and MUC-1), and in other malignancies [reviewed in (3)]. T cells respond to self-peptides presented by MHC class II molecules and are generally characterized by low-affinity interactions which serve to maintain the survival of naive T cells (31). However, the response of mature T cells to self-peptides is generally not known to occur as they have been rendered tolerant. Studies of naturally processed self-peptides from several laboratories have shown that changes in the profile of MHC class I-bound self-peptides do occur after viral infection or c-myc transformation and differentiation (3237).

Analysis of MHC-bound peptides in these systems revealed several high copy numbers of self-peptides derived from overexpressed normal proteins associated with cell signaling or receptor functions. More recently, computer algorithms modeling T cell responses concluded that T cells tend to use molecular mimicry rather than self–non-self discrimination (38,39).

The similarity of identified peptides to self-peptides in cancer cell lines is not surprising given that all peptides were derived from housekeeping proteins. Our finding of novel HLA-A2-associated sequences both in cancer cell lines and fresh tumor specimens broadens the applicability of this methodology to identify candidate antigens in the development of peptide-based vaccines for the treatment of ovarian cancer and other malignancies.


    Acknowledgements
 
We thank Dr Kevin Hogan (University of Virginia Health Sciences Center, Charlottesville, VA) for laying the foundation for these studies, Drs Victor H. Engelhard, Donald F. Hunt and Jeff Shabanowitz (University of Virginia, Charlottesville, VA) for expert guidance, and the management team at Upstate Inc. [(Argonex Inc.), Charlottesville, VA] for their support of the Antigen Discovery Program.


    Abbreviations
 
APC—antigen-presenting cell

B-LCL—Epstein–Barr virus-transformed B lymphoblastoid cell                         line

CTL—cytotoxic T lymphocyte

DC—dendritic cell

ESI-MS-MS—electrospray ionization tandem mass spectrometry

GM-CSF—granulocyte macrophage colony stimulating factor

iDC—immature DC

mDC—mature DC

MFI—mean fluorescence intensity

MS—mass spectrometry

PBMC—peripheral blood mononuclear cell

TFA—trifluoroacetic acid


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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