Affiliations of authors: Medical Oncology Section (PC, MLP, GF), Pathology Section, Department of Human Pathology and Oncology (MTDV, MV), Virology Section, Department of Molecular Biology (GDG, GGS, CT, MGC), "Giorgio Segre" Department of Pharmacology (RU, GG), Siena University School of Medicine, Siena, Italy; Immunité Cellulaire Antivirale, Institut Pasteur, Paris Cedex, France (FL); Medical Oncology and Pharmacology Section, Department of Neuroscience, University of Roma "Tor Vergata," Rome, Italy (AA, EB)
Correspondence to: Maria Grazia Cusi, PhD, Department of Molecular Biology, Virology Section, Siena University School of Medicine, Viale Bracci 1, 53100 Siena, Italy (e-mail: cusi{at}unisi.it) or Pierpaolo Correale, PhD, Department of Human Pathology and Oncology, Medical Oncology Section, Siena University School of Medicine, Viale Bracci 1, 53100 Siena, Italy (e-mail: correale{at}unisi.it).
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ABSTRACT |
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INTRODUCTION |
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To overcome such treatment limitations, recombinant cancer vaccines have been studied for their efficacy. One approach is vaccination with synthetic peptide epitopes that correspond to peptides presented by class I major histocompatibility complex (MHC) molecules on tumor cells. Recognition of these peptide-MHC complexes by cytotoxic T lymphocytes (CTL) induces an immune response against the tumor cells.
Previously, we investigated whether TS could be used as a target antigen for active immunotherapy (6). We identified three different TS-derived epitopes (TS/1, TS/2, and TS/3) with human leukocyte antigen (HLA)-A(*)02.01 binding ability and tested their immunologic activity by generating and characterizing CTL lines derived from HLA-A(*)02.01+ peripheral blood mononuclear cells (PBMCs) in the presence of interleukin-2 and autologous TS-peptide-pulsed dendritic cells. These CTL lines were able to recognize CIR-A2 lymphoma target cells pulsed with the same peptide used for CTL stimulation, but they showed only a weak ability to kill breast and colon carcinoma cells if these target cells had not previously been exposed to sublethal doses of 5-FU (6).
In this study, we generated a 28-amino acid peptide (TS/PP) that contains the TS/1, TS/2, and TS/3 epitope sequences, all of which are presented by the MHC class I molecule HLA-A(*)02.01. We investigated whether this peptide could induce a TS-specific CTL response with antitumor activity by performing multiple in vitro stimulations of human PBMCs. We also investigated the toxicity and the antitumor activity of the TS/PP peptide vaccination alone and in combination with 5-FU-based chemotherapy in HLA-A(*)02.01 (HHD) transgenic mice challenged with syngeneic tumor cells (EL-4 HHD lymphoma cells) whose TS expression can be increased by 5-FU treatment.
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MATERIAL AND METHODS |
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MDA-MB-231 breast carcinoma, HT29, and SW1463 colon carcinoma cell lines were purchased from the American Type Culture Collection (Manassas, VA) and cultured as suggested by the supplier. The CIR-A2 B-cell lymphoma (7) and the EL-4/HHD cell lines (mouse 2 microglobulin-deficient thymoma cells transfected with the HHD HLA-A2.1 monochain construct) were provided by Jeffrey Schlom (Experimental Oncology Section, Laboratory of Tumor Immunology and Oncology, National Cancer Institute, National Institutes of Health, Bethesda, MD), and Antonio Scardino (INSERM, Goustav Roussy Institute, Vilejuif, France), respectively. Both of these lines were maintained in complete RPMI-1640 (Hyclone Europe, Cramligton, United Kingdom) medium with the addition of 10% heat-inactivated fetal calf serum, 2mM L-glutamine, and 100 U/mL penicillin/streptomycin (Hyclone Europe). Adherent cells were removed using trypsin-EDTA solution (0.05% trypsin and 0.02% EDTA in phosphate-buffered saline without calcium and magnesium).
Peptides
TS/1 (TLGDAHIYL, amino acid TS position = 245253), TS/2 (YMIAHITGL, amino acid TS position = 229237), TS/3 (FLDSLGFST, amino acid TS position = 111119), and TS/PP (YMIAHITGLFLDSLGFSTTLGDAHIYL) peptides were synthesized as previously described (6). The previously characterized TS/1, TS/2, and TS/3 peptides were selected because of their high HLA-A(*)02.01+ binding score predicted according to Parker's algorithm (8) and their ability to bind HLA-A(*)02.01 molecules in the immunocytofluorimetric T2 class-I-HLA up-regulation test (10), which indirectly measures peptide binding to different HLA class I molecules on T2-A2 cells. These lymphoblastoid cells are defective in transporter associated with antigen processing and present unstable empty class-I HLA molecules on their surface. This test takes advantage of the fact that effective peptide binding stabilizes HLA complexes, prolonging their half-life on the membrane, allowing for cytofluorimetric measurement of the binding as an increase in main fluorescence intensity per cell (9). TS/PP contains TS/1, TS/2, and TS/3 epitope amino acid sequences and consensus motifs predicted to bind not only HLA-A(*)02.01 (five sequences) but also -A3, -A1, -A24 (five sequences), B44, and HLA-Dr (eight sequences), respectively.
Generation of Dendritic Cells and CTL Cultures
PBMCs were obtained by Ficoll-Hypaque gradient separation of buffy coats (12) of blood samples collected from four different HLA-A(*)02.01-typed healthy human donors and two colon cancer patients who gave written informed consent. The dendritic cells used for in vitro CTL stimulation were generated from autologous PBMCs as previously described (13).
Generation of TS/PP-Specific CTL Lines
CTL lines were generated from PBMCs as previously described (6,13,14). In brief, PBMCs from two different healthy HLA-A(*)02.01+ donors were performed with autologous irradiated dendritic cells loaded with TS/1, TS/2, or TS/3 (25 µg/mL per 106 cells) for 1 hour or with 25 µg/mL per 106 cells of TS/PP for 4 hours at a PBMC/dendritic cell ratio of five to one. After a 5-day coculture of PBMCs and dendritic cells in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4, the cells were maintained in complete AIM-V medium containing 5% human AB (AB blood group) serum and low-dose [(25 U/mL)] interleukin-2 for another 10 days and were then restimulated as described above. After at least four in vitro stimulations, the CTL cultures were evaluated for immunophenotype and cytotoxic activity. All CTL lines were examined monthly by flow cytometry and showed the following immunophenotype: CD3+ = 90%95%; CD56+ = 10%22%; CD4+ = 37%40%; and CD8+ = 40%50%.
5-FU Treatment and Transfection of Tumor Cells Before CTL Assay
Target cells were treated with a sublethal dose of 5-FU as previously described (13). Tumor cells (HT-29, MDA-MB-231, EL-4/HHD) were seeded at a concentration of 2 x 105 cells/mL in 25-cm2 flasks (Falcon, Lincoln Park, NJ). On the third day after seeding, 5-FU was added to a concentration of 104 M for 1 hour. The drug was then removed by multiple washings with phosphate-buffered saline, and fresh medium was then added to the cells. TS expression was evaluated by immunoblotting and by immunocytofluorimetric analysis 6, 24, and 48 hours later by using TS monoclonal antibody 4130 as described previously (15). All cell lines constitutively expressed TS in the range of 25% to 35% of the cell population, which increased to 60% to 75% after 2448 hours of treatment with 5-FU. With this dose of 5-FU, which was chosen on the basis of previous experiments (data not shown), the highest rate of TS increase could be obtained without substantial levels of cell death. Target cells showed 95% cell survival in a trypan blue viability assay before being used in the cytotoxic assays.
Transfection of HT-29 Target Cells With HLA-A(*)02.01 Molecule
To use colon cancer cells as MHC class I-HLA-restricted target cells, plasmid carrying the HLA-A(*)02.01 gene sequence [provided by Antonio Scardino, INSERM, Villejuif Cedex, France (10)] was transfected into HT-29 cells as previously described (6,13). HLA-A(*)02.01 expression was evaluated before each experiment on target cells by indirect flow cytometry using an anti-HLA-A1.2 monoclonal antibody (A2.69 One Lambda, Inc.). Statistically significant A(*)02.01 expression was found in CIR-A2 (90%95%), SW-1463 (50%65%), and HLA-A(*)02.01 gene-transfected HT-29 (55%75%) cell lines.
Transfection of Other Targets
To use CIR-A2 section cells as possible target of TS-specific/HLA-A(*) 02.01-restricted CTL subsets, these cells were transfected with a plasmid expressing the TS gene sequence (6). Briefly, the human TS gene was amplified from the SW-1463 cells by reverse transcriptase-polymerase chain reaction using the sense primer 5'AAGCTTATGCCTGTGGCCGGCTC3' and the antisense primer 5'AAGCTTCTAAACAGCCATTTCCA3'. The PCR product was then purified and cloned in the HindIII site of the pcDNA3 expression vector (Invitrogen, San Diego, CA), which was then introduced into CIR-A2 target cells. Antigen expression on target cells was evaluated before each experiment by indirect flow cytometry using the TS monoclonal antibody. Only 25%30% of the untransfected CIR-A2 cell population expressed TS, whereas 50%55% of the cells expressed TS after gene transfection.
Cytotoxic Assays
51Chromium (51Cr) release assays were performed as described in previous studies (14,15). Human CTL lines (TS/PP CTL lines and the TS-epitope peptide-specific CTL lines) were tested against several target cells including CIR-A2, peptide-pulsed (TS/1, TS/2, TS/3, PTR-4, TS/PP), and TS gene-transfected CIR-A2 target cells and untreated and 5-FU-treated MDA-MB-231, HT29, and HLA-A(*)02.01 gene transfected-HT29 and SW1463 cell lines. Spleen cells derived from HHD mice were stimulated with TS/PP peptide and were tested against EL-4/HHD and 5-FU-treated EL-4/HHD, respectively. These tests were performed at 25/1, 12.5/1, 6.25/1, and 1/1 effector (E)/ target (T) ratios.
HLA-A(*)02.01 molecule expression on HT-29 target cell membranes was obtained by gene transfection as described previously (14,15). Specific lysis was calculated as follows:
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Spontaneous release was determined from the wells to which 100 mL of medium had been added instead of effector cells. Total releasable radioactivity was determined after treating target cells with 2.5% Triton X-100. For the HLA-blocking experiments, the targets were exposed to UPC-10 (Cappel/Organon Technique Corp., West Chester, PA) control monoclonal antibody or anti-HLA-A2 (A2.69; One Lambda, Inc., Canoga Park, CA) for 1 hour before carrying out the cytotoxic assay.
Competition Assay
Competition assays were performed as described in a previous study (14). Briefly, CTL assays were performed against TS/PP peptide-loaded 51Cr-labeled CIR-A2 target (L) cells in the presence of unlabeled cold competitors (C) at different L/C ratios. Cold competitors were represented by untreated and 5-FU-treated HLA-A(*)02.01 gene-transfected HT-29 cells.
Vaccination of HHD Mice
The HLA-A(*)02.01 transgenic HHD mice have been previously described (11). They were obtained at 5 weeks of age and were housed in a temperature-controlled, light-cycled room. When the mice were 6 to 8 weeks of age, groups of 10 mice were injected subcutaneously with 100 µg of peptide (group A, mumps control peptide; group B, TS peptide cocktail consisting of TS/1, TS/2, and TS/3; group C, TS/PP) emulsified in complete Freund adjuvant. Another group of 10 mice was not treated and represented the control group (group D). The vaccinations were performed four times, at 3-week intervals; 2 weeks after the last injection, the mice were inoculated subcutaneously with 2 x 106 TS-expressing /HLA-A(*)02.01+- EL-4/HHD lymphoma cells (10). Beginning 1 week later, five mice from each group received 125 mg of 5-FU intraperitoneally each week for 1 month; the other five mice were left untreated. The vital functions and weights of the mice were monitored daily, and tumors were measured with a caliper each week. Untreated mice and all of those injected with the control peptide or TS-peptide cocktail, with or without 5-FU chemotherapy, were killed by cervical dislocation under anesthesia within 30 days after the tumor cell inoculation. Mice vaccinated with TS/PP (± 5-FU treatment) were killed 60 days after the tumor cell inoculation. Spleen cells were collected from the sacrificed mice and used for immunologic studies (dimer assay and CTL cultures). Paraffin sections (4-µm thick) were cut from the sampled tissues (lung, liver, spleen, hair, and brain), processed, and stained for histologic analysis. All of these experiments were subsequently repeated on a different generation of the same strain of HLA-A(*)02.01 transgenic mice (Charles River/IFFA CREDO, Lyon, France) to confirm the results. All animal experiments were carried out according to the UK Coordinating Committee for Cancer Research Guidelines (11).
Histologic and Immunohistochemical Analyses
Samples of the lung, liver, spleen, gastroenteric mucosa, brain, and hair of each mouse were fixed in 4% buffered formalin for 24 hours, embedded in paraffin, and sectioned. Paraffin sections (4 µm thick) were cut from tissue blocks and stained with hematoxylin and eosin. Immunohistochemical staining was performed on 3-µm-thick sections of each block employing the streptavidin-biotin method. After being dewaxed and rehydrated, the sections were washed in Tris-buffered saline (pH 7.6) and preincubated with normal rabbit serum (DAKO, Copenhagen, Denmark) to prevent nonspecific binding. To improve the detection of the antigen, we used pretreatment with a microwave oven: the sections were incubated in EDTA (0.05 mM, pH 8.0) at 750 W for 5 min, three times. During the treatments, distilled water was added to compensate for evaporation. The sections were then washed in distilled water for 10 min. The slides were first incubated with anti-human TS (clone TS 106, diluted 1:50; NeoMarkers, Fremont, CA), and then the reaction was revealed using the streptavidin-biotin complex (LAB Vision, Fremont, CA) and 3,3'-diaminobenzidine as chromogen. Sections were weakly counterstained with Harris's hematoxylin, mounted with aqueous mounting medium, and examined under a light microscope. Negative controls were obtained by replacing the specific antibody with aspecific immunoglobulins. Immunoreactivity was assessed using routine light microscopy.
Flow Cytometry
The procedure for single-color flow cytometric analysis has been previously described (16). Fluorochrome-conjugated monoclonal antibodies were purchased from Becton Dickinson (San Jose, CA), W6/32 (anti-HLA class I) monoclonal antibody from SCRA (Sussex, England), A2.69 (anti-HLA-A2.1) monoclonal antibody from One Lambda Inc., and COL-1 (anticarcinoembryonic antigen [CEA] monoclonal antibody) and MOPC-21 from Cappel/Organon Tecknica Corp (West Chester, PA). Flow cytometry was performed using a Becton Dickinson FACScan equipped with a blue laser with an excitation level of 15 nW at 488 nm. TS-106 (17), a monoclonal antibody against TS, was used in a single-color flow cytometric analysis, as described above, on target cells previously fixed with 2% paraformaldehyde by using a standard procedure (6).
Determination of TS PeptideSpecific CTL Precursor Frequency
A cytofluorimetric dimer assay (16) and related reagents were purchased from BD Pharmigen (San Diego, CA) and used as described by the manufacturer.
Statistical Analyses
Mean differences in antitumor response were analyzed for statistical significance using Stat View software (Abacus Concepts, Berkeley, CA). The results were expressed as the mean of three measurements, each made in separate experiments. Differences in antitumor response were determined using Bonferroni's (all-pairwise) multiple-comparison test or the Kruskal-Wallis multiple-comparison test. The rate of EL-4/HHD lymphoma cell growth in HHD mice was estimated by a log-linear regression between tumor size versus time (i.e., the coefficient of the straight line was used as a measure of tumor growth). The influence of the 5-FU and TS/PP treatments on tumor growth in the mice inoculated with lymphoma cells was tested by two-way analysis of variance comparisons of tumor size measurements between the four groups (control, 5-FU-, TS/PP-, and 5-FU+TS/PP-treated groups). P values less than .05 were considered statistically significant, and all statistical tests were two-sided.
Differences in the cytotoxic activity of various effector CTLs against the same target cells were calculated taking into account the percentage of specific cytotoxicity at each E/T ratio. Accordingly, P values were calculated using ANCOVA (analysis of covariance) (18). Data relative to cell-mediated cytolysis were expressed as number of target cells lysed (or killed) by 106 effector cells.
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RESULTS |
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The functional binding of the polyepitope peptide TS/PP to HLA-A(*)02.01 was assessed by the T2 test as described previously (6,9). In contrast with the individual epitopes TS/1, TS/2, and TS/3 (7), the TS/PP peptide did not bind HLA-A(*)02.01 molecules in the T2 test in its native form (data not shown). However, after processing by antigen-presenting cells or other target cells, TS/PP was able to generate a multiepitopic CTL immune response (Fig. 1). A computer screening of TS/PP peptide by using Ken Parker's (8) and Rammensee's (19) algorithms revealed that it also contains the amino acid sequences of several other epitopes, suggesting that TS/PP may be able to bind to several different (human and mouse) class I and class II MHC haplotypes.
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To determine whether TS/PP could be processed on the cell membrane of dendritic cells and target cells, we studied CIR-A2 target cells loaded with the TS/PP peptide and examined whether they were recognized by human CTL lines that were specific for each of the three known TS epitopes. We found that these CTL lines were all able to kill the target cells pulsed with TS/PP as well as those pulsed with the same specific peptide (TS/1, TS/2, or TS/3) used in generating the CTL line or transfected with pTS plasmid (Fig. 1). Conversely, the CTL lines generated with the TS/PP peptide (cell lines T-4756 and T-3939) showed a multiepitopic-specific cytolytic activity. That is, these CTL lines were able to kill CIR-A2 target cells that had been pulsed with each of the known TS epitope peptides, pulsed with the TS/PP peptide, or transfected with the TS plasmid (Fig. 1, B). All of the CTL lines were also able to induce low levels of killing against the negative controls (Fig. 1).
Cytolytic Activity of TS/PP-Specific CTL Lines Against Class I HLAMatched Breast and Colon Carcinoma Cells and 5-FUInduced Immune Sensitization
We next tested the cytolytic activity of the CTL lines against breast cancer cells (MDA-MB-231) and colon cancer cells [SW-1463, HT-29, and HT-29 transfected with HLA-( *)02.01 gene] untreated or exposed to sublethal doses of 5-FU (Figs. 2 and 3). CTL lines generated with the TS/PP peptide showed a statistically significantly greater ability to kill than untreated cells at a 25/1 E/T ratio {Difference [diff] = 17.0; 95% CI = 12.6 to 20.4 for MDA-MB-231 breast carcinoma cells; diff = 25.0; 95% confidence interval [CI] = 16.2 to 33.8 for SW-1463 colon carcinoma cells; and diff = 11.5; 95% CI = 1.3 to 23.3 for HLA-A(*)02.01 gene transfected-HT29 cell line}. The killing mediated by these CTL lines was restricted to targets expressing HLA-(*)02.01 molecules (Figs. 2 and 3). We also observed that tumor cells that had previously been treated with 125 µg/mL of 5-FU for 48 hours were statistically significantly more sensitive to CTL activity compared with the target cells untreated with 5-FU (Figs. 2 and 3).
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The immunologic, toxicologic, and antitumor (i.e., tumor prevention) activities of TS/PP peptide were tested in HLA-A(*)02.01 transgenic HHD mice (10) and compared with those of genetically identical (syngeneic) mice vaccinated with control peptide or TS-peptide cocktail. Tumor growth in mice vaccinated with control peptide or the TS-peptide cocktail, with or without 5-FU treatment (groups A and B), were similar to that in control mice (group D) in preventing tumor cell growth. All mice in groups A, B, and D were sacrificed within 30 days because they all developed a very large and ulcerated tumor within a few weeks and were unable to move. In contrast, tumor growth in mice vaccinated with the TS/PP peptide (group C) was delayed substantially, especially when combined with 5-FU chemotherapy (Fig. 4). The mice immunized with the TS/PP peptide started to develop a small tumor 3040 days after challenge with lymphoma cells (Fig. 5), and the rate of tumor growth in these mice was statistically significantly different when compared with the rate of tumor growth from mice in groups A and B. The coefficients of tumor growth were 0.136 in controls (group A), 0.116 in the 5-FU-treated group (group B), 0.088 in the TS/PP-vaccinated group (group E), and 0.023 in the group vaccinated with TS/PP and treated with 5-FU (group F), respectively. The differences in the coefficients of tumor growth between groups A and E and groups B and F were 0.048 (95% CI = 0.029 to 0.066) and 0.093 (95% CI = 0.066 to 0.119), respectively. The interaction between treatment with 5-FU and vaccination with TS/PP was statistically significant (P = .01).
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DISCUSSION |
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In contrast with the individual TS-epitope peptides, our results suggest that TS/PP is processed by dendritic cells and by antigen-presenting cells. First, TS/PP was able to generate multi-TS epitope-specific CTL lines. Second, TS/PP was able to stimulate human PBMCs in vitro. Third, it induced an efficient antitumor TS-specific response in vivo. Further evidence that TS/PP can be processed by target cells, producing a potential target peptide, comes from the finding that each single TS epitope peptide-specific CTL line was able to recognize CIR-A2 target cells that had been pulsed with TS/PP peptide, transfected with the TS gene, or pulsed with each of the three TS peptides. In addition, these cells were able to kill HLA-A(*)02.01+/TS-producing breast and colon carcinoma cell lines.
HHD mice vaccinated with TS/PP and engrafted with syngeneic EL-4/HHD tumor cells had a substantial increase in TS peptide-specific CTL precursors and an efficient TS-specific CTL response that was accompanied by a substantial delay in tumor growth over a 60-day period. The latter finding suggests that TS/PP may contain other epitopes that are able to bind multiple class I and class II MHC molecules, expanding the spectrum of immune response in mice that can oppose EL-4/HHD tumor cell growth.
In previous studies, we reported that sublethal doses of 5-FU can sensitize breast and colon cancer cells to the cytotoxic effects of CEA peptide-specific CTL lines by increasing CEA expression in target cells (13). Similarly, we tested the immune sensitizing effects of 5-FU in vivo and observed that this drug is able to enhance the antitumor activity of TS/PP vaccination. Pathologic analyses of tumors from these mice showed that lymphocytes infiltrated tumor cells and that expression of intracellular TS was reduced, which strongly suggested a vaccine-activated immune response. Because 5-FU treatment alone did not prevent or slow EL-4/HHD tumor growth in vivo, it seems likely that 5-FU may enhance the antitumor action of TS/PP vaccination by modulating TS expression in these target cells.
The TS/PP vaccine was also found to be very safe, because no evidence of autoimmunity or other side effects were detected in vaccinated mice. The lack of side effects may reflect the fact that normal cells express TS in a very small amount for a short period of time and consequently do not produce sufficient TS-epitopes to be recognized by the TS-specific CTL precursors. Although these studies provide preliminary information on the immunologic power and toxicity of our peptide vaccine used alone and in combination with cytotoxic drugs, the present study cannot foresee the antitumor efficacy of this therapeutic strategy in human cancer patients.
We conclude that the multiepitope peptide TS/PP could be used to generate a therapeutic multiepitopic TS-specific CTL immune response. This study raises the possibility that chemotherapy in combination with peptide-based vaccines, such as TS/PP, may be appropriate for testing for the treatment of human malignancies, such as breast and colorectal carcinoma, which are sensitive to 5-FU treatment.
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NOTES |
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Manuscript received September 7, 2005; revised May 4, 2005; accepted August 4, 2005.
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