ARTICLE

5-Fluorouracil–Based Chemotherapy Enhances the Antitumor Activity of a Thymidylate Synthase–Directed Polyepitopic Peptide Vaccine

Pierpaolo Correale, Maria Teresa Del Vecchio, Giuseppa Di Genova, Gianni Gori Savellini, Marco La Placa, Chiara Terrosi, Marzio Vestri, Renato Urso, Francois Lemonnier, Angelo Aquino, Enzo Bonmassar, Giorgio Giorgi, Guido Francini, Maria Grazia Cusi

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).


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Background: Thymidylate synthase (TS), a key enzyme in DNA synthesis, is often overexpressed in cancer cells. Some chemotherapeutic agents, such as 5-fluorouracil (5-FU), act by inhibiting TS expression. We evaluated whether a novel 28-amino acid multiepitope peptide, TS/PP, that contains the sequences of three TS-derived epitopes with binding motifs for HLA-A(*)02.01 could induce a TS-directed cytotoxic T-lymphocyte (CTL) response with antitumor activity. Methods: TS/PP peptide immunologic activity in CTL lines derived from human leukocyte antigen (HLA)-A(*)02.01+ peripheral blood mononuclear cells (PBMCs) was tested in the presence of interleukin-2 and autologous TS/PP peptide-loaded dendritic cells. Immunologic and antitumor activities of TS/PP and its toxicity were also evaluated in vivo in HLA-A(*)02.01 transgenic (HHD) mice that were vaccinated with TS/PP, control, or TS-peptide cocktail and treated with or without 5-FU chemotherapy. The mice were also inoculated subcutaneously with TS-expressing EL-4/HHD lymphoma cells to assess immune response against these tumor cells. Results: TS/PP-specific CTL lines showed a TS-multiepitopic specificity and were able to kill TS+/HLA-A(*)02.01+ breast and colon carcinoma cells. The killing ability against target cells previously exposed to sublethal doses of 5-FU was statistically significantly greater than against untreated target cells (43.5% versus 26.5% at 25/1 effector to target ratio [Difference {diff} = 17.0]; 95% confidence interval [CI] = 12.6 to 20.4) for MDA-MB-231 breast carcinoma cells and 73.5 versus 48.5 (diff = 25.0; 95% CI = 16.2 to 33.8) for the SW-1463 colon carcinoma cells. HHD mice vaccinated with TS/PP manifested a TS-peptide-specific CTL response with no sign of autoimmunity or toxicity. Furthermore, treatment of these mice with 5-FU delayed or prevented the occurrence of tumors formed by inoculation with autologous (TS+)EL-4/HHD lymphoma cells. Conclusions: The multiepitopic TS/PP vaccine induces a tumor-specific immune response in mice and is especially potent when used in combination with 5-FU-based chemotherapy.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Thymidylate synthase (TS) is an intracellular enzyme that catalyzes the methylation of deoxyuracil in the presence of reduced folates (1,2) and thereby plays an essential role in one of the rate-limiting steps in DNA synthesis. This reaction provides the main source of thymidylate in eukaryotic cells. TS expression is tightly regulated by the genes involved in cell cycle control in normal cells. By contrast, TS is constitutively overexpressed in tumor cells which show high proliferative activity. Consequently, chemotherapeutic agents, such as fluoropyrimidine-based analogs, have been developed to target TS. 5-Fluorouracil (5-FU) is fluoropyrimidine pro-drug, one of whose cytotoxic metabolites, 5-fluorouridin-triphosphate or 5-fluoro-deoxy-uridin-monophosphate, inhibits TS by forming a tertiary and stable complex composed of TS, 5-fluoro-deoxy-uridin-monophosphate, and tetrahydrofolate, which is rapidly degraded in the cell cytoplasm (1,3). Several studies have suggested that increased expression of TS enzyme or mutations in the TS gene can make tumor cells resistant to 5-FU (1,4,5). In addition, these traits have been reported to be negative prognostic factors in patients with gastric and colon cancer (4,5).

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.


    MATERIAL AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Cell Cultures

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 {beta}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 = 245–253), TS/2 (YMIAHITGL, amino acid TS position = 229–237), TS/3 (FLDSLGFST, amino acid TS position = 111–119), 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 10–4 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 24–48 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:

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 Peptide–Specific 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.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Polyepitope Peptide Characterization

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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Fig. 1. Cytotoxic activity of cytotoxic T-lymphocyte (CTL) lines directed against thymidylate synthase (TS)-derived epitope peptides. CIR-A2 target cells were pulsed for 4 hours with 25 µg /mL of TS/PP peptide, with 25 µg/mL of one of the specific peptides (TS/1, TS/2, or TS/3), or CIR-A2 target cells transfected with the plasmid containing the TS gene (pTS). CIR-A2 target cells transfected with plasmid backbone (pcDNA3) (data not shown), either unpulsed or pulsed with nonreacting peptides with human leukocyte antigen (HLA)-A(*)02.01 binding motifs derived from the parathyroid hormone related protein (PTR-4) and/or influenza matrix peptide, were used as negative controls. The results are expressed as the percentage of specific lysis (i.e., chromium release) at different effector/target (E/T) cell ratios from three independent experiments. A) CTL lines generated with each of the single TS peptides (TS/1, TS/2, and TS/3). B) CTL lines generated with the multiepitopic TS/PP peptide. Solid circles = unpulsed CIR-A2 target cells; open circles = CIR-A2 transfected with pTS; solid squares= CIR-A2 loaded with TS/PP peptide; open squares = CIR-A2 pulsed with the nonreacting PTR-4 peptide; open diamonds = CIR-A2 pulsed with TS/1; solid diamonds = CIR-A2 pulsed with TS/2; solid triangles = CIR-A2 pulsed with TS/3; open triangles = CIR-A2 pulsed with the nonreacting influenza matrix peptide.

 
Processing and Immunogenicity of the TS/PP Peptide

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 HLA–Matched Breast and Colon Carcinoma Cells and 5-FU–Induced 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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Fig. 2. Lytic activity of cytotoxic T-lymphocyte (CTL) lines against MDA-MB-231 breast carcinoma cells. CTL lines were generated with the TS/PP peptide and with each of the three thymidylate synthase (TS)-epitope peptides alone. The results are expressed as the percentage of specific lysis at different effector/target (E/T) ratios. In some experiments, blocking antibodies against human leukocyte antigen (HLA)-A(*)02.01 (A2.69) were used at 10 mg/mL. Open circles = MDA-MB-231 target cells; open triangles = 5-fluorouracil (5-FU)-treated MDA-MB-231 target cells in the presence of A2.69 monoclonal antibodies; solid squares = MDA-MB-231 target cells exposed to 5-FU; open squares = MDA-MB-231 exposed to 5-FU and to A2.69 monoclonal antibodies.

 


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Fig. 3. Lytic activity of cytotoxic T-lymphocyte (CTL) lines against colon carcinoma cells with or without treatment with 5-fluorouracil (5-FU). Killing ability against tranfected or untransfected HT-29 and SW-1463 colon carcinoma cell lines by CTL lines generated with TS/PP peptide or with each of the thymidylate synthase (TS) peptides (TS/1, TS/2, TS/3) was measured by cytotoxic assays. The results are expressed as the percentage of specific lysis at different effector/target (E/T) ratios (means and standard deviations). 1) Human leukocyte antigen (HLA)-A(*)02.01-transfected-HT-29 target cells; 2) CTL lines against SW-1463 versus 5-FU treated SW-1463 target cells. Open circles = HT-29 target cells; solid circles = HT-29 transfected with HLA-A(*)02.01 gene; open triangles = HT-29 transfected with HLA-A(*)02.01 gene and exposed to 5-FU; open squares = SW-1463 target cells; solid squares = SW-1463 exposed to 5-FU.

 
To evaluate the peptide specificity of these CTL lines, a cold competition assay was performed. CIR-A2 cells were loaded with TS/PP as 51Cr-labeled CTL targets, and HLA-A(*)02.01-gene-transfected HT-29 or 5-FU-treated/HLA-A(*)02.01-gene-transfected HT-29 colon carcinoma cells were used as cold competitors at different ratios of labeled targets to cold competitor ratios (L/C ratio). Lysis of CIR-A2 targets loaded with TS/PP was reduced by the cold competitors and completely abrogated at low L/C ratios (1/5) (Table 2). We also found that HLA-A(*)02.01-gene-transfected HT-29 (A2-HT-29) cells treated with 5-FU were better competitors than the untreated A2-HT-29 cells because complete abrogation of target cell lysis occurred at a much lower L/C ratio (1/1) (Table 2), suggesting that the immune-sensitizing effect of 5-FU is related to an increase in TS-epitope peptides. Cytofluorimetric analysis and immunoblotting performed on 5-FU-treated cells showed that this treatment did not induce changes in class I HLA expression, although it did induce a greater than 50% increase in TS expression in the MDA-MB-231, HT-29, and SW-1463 target cells (data not shown) that was statistically significant.


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Table 2.  A TS/PP peptide–specific cytotoxic T-cell cold competition assay*

 
Immunologic, Toxicologic, and Antitumor Activity of TS/PP Peptide in HLA-A(*)02.01 Transgenic (HHD) Mice

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 30–40 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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Fig. 4. Tumor diameter in human leukocyte antigen (HLA)-A(*)02.01 transgenic HHD mice vaccinated with thymidylate synthase (TS)/PP, TS-peptide cocktail, or with control peptide. Some mice were treated with 5-fluorouracil (5-FU). Tumor growth between mice vaccinated with TS/PP with 5-FU treatment versus those vaccinated with control peptide or TS-peptide cocktail with or without 5-FU treatment. The results are reported as average major diameter ± standard deviation. Open squares = mice vaccinated with mumps control peptide; open triangles = with TS-epitope peptide cocktail; open circles = with TS/PP peptide; solid squares = mumps control peptide and 5-FU chemotherapy; solid triangles = TS-epitope peptide cocktail and 5-FU chemotherapy; solid circles = TS/PP peptide and 5-FU chemotherapy.

 


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Fig. 5. Tumor growth in vaccinated mice sacrificed thirty days after inoculation with 2 x 106 EL4/HHD lymphoma cells subcutaneously. A) Mice vaccinated with mumps control peptide; B) mice vaccinated with mumps control peptide and treated with 5-fluorouracil (5-FU); C) mice vaccinated with the thymidylate synthase (TS)-peptide cocktail; D) mice vaccinated with the TS-peptide cocktail and treated with 5-FU; E) mice vaccinated with TS/PP peptide; and F) mice vaccinated with TS/PP peptide and treated with 5-FU.

 
All mice vaccinated with TS/PP were sacrificed for pathologic and immunologic study 30 days after the sacrifice of the other groups of mice. The pathology study performed on tissue samples of liver, brain, spleen, lung, gastroenteric mucosa, and hair did not reveal any sign of autoimmunity or toxicity (data not shown). Histologic evaluation of the primary tumors showed extensive degenerative processes (apoptotic bodies, intercellular and pseudocystic spaces, desmoplastic reaction) in tumors in mice vaccinated with the TS/PP peptide or with the cocktail of TS-peptide epitopes in mice that had also received 5-FU. In particular, the tumors of mice vaccinated with TS/PP peptide had more small lymphocytes clusters intermingled with dying tumor cells than mice vaccinated with TS-peptide cocktail (Fig. 6). Similarly, we observed immunohistochemical differences between tumors of the control group and the groups vaccinated with the TS-peptide cocktail or TS/PP peptide as shown in Fig. 6. In the control group, immunohistochemical analysis showed disseminated TS-positive tumor cells. The number of disseminated TS-positive tumor cells was substantially increased among control mice that were also treated with 5-FU compared with mice vaccinated with the cocktail of TS-peptides and compared, in particular, with mice vaccinated with the TS/PP peptide, with or without 5-FU treatment (Fig. 6).



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Fig. 6. Pathologic analysis of tumor tissue removed from sacrificed mice. Thymidylate synthase (TS) protein was detected by immunostaining (larger photos); insets show the hematoxylin-eosin staining of the same samples. Each photo was taken from a single mouse and was representative of the group of mice receiving the same treatment. A) Control mice vaccinated with mumps control peptide. The arrow points to a TS-positive cell; inset, tumor cells with scattered apoptotic bodies. B) Mice vaccinated with mumps peptide and treated with 5-fluorouracil (5-FU); inset, degenerative changes in tumor cells and the presence of intercellular spaces. C) Mice vaccinated with the TS-peptide cocktail (rare TS-positive tumor cells); inset, many apoptotic bodies, intercellular spaces, and desmoplastic reactions. D) Mice vaccinated with the TS-peptide cocktail and treated with 5-FU (rare TS-positive cells); inset, pseudocystic spaces in areas with conspicuous degenerative changes. E) Mice vaccinated with TS/PP peptide (some TS-positive tumor cells and clusters of small lymphocytes among tumor cells); inset, clusters of small lymphocytes in tumor areas. F) Mice vaccinated with TS/PP peptide and treated with 5-FU (almost no TS-positive tumor cells and clusters of small lymphocytes surrounding TS-negative areas); inset, clusters of small lymphocytes among tumor cells; inset, pseudocystic spaces. Magnification = 250x.

 
Splenocytes from mice inoculated with either the TS/PP peptide or the TS-peptide cocktail were tested for TS-peptide precursor frequency by using the cytofluorimetric dimer assay. As Table 1 shows, the frequency of TS-epitope peptide-specific CTL response was higher in spleen cells derived from mice vaccinated with TS/PP or TS cocktail (± 5-FU) than in spleen cells derived from control mice vaccinated with the mumps peptide (± 5-FU).


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Table 1.  Peptide-specific CTL response in splenocytes from mice vaccinated with TS/PP peptide, TS peptide cocktail, or control mumps peptide, compared with control splenocytes as measured by cytofluorimetric dimer assay*

 
In vitro CTL activity of spleen cells harvested from each of the four mice groups (A, B, C, D) was also tested in 51Cr release assays against TS-positive lymphoma cells before and after a 48-hour treatment with sublethal doses of 5-FU. To increase the percentage of TS/PP-specific CTLs in splenocytes from mice in group C, the spleen cells derived from each mouse group were pooled and stimulated in vitro with TS/PP peptide and low-dose interleukin-2 before being tested in the cytotoxicity assays. We observed a modest class I HLA-restricted CTL activity against TS-positive lymphoma cells in the spleen cells of mice vaccinated with the TS/PP peptide (group C) or with the TS-peptide cocktail (group B) (at 25/1 E/T ratio, % specific release = 44% and 35%, respectively; ratio = 1.26; 95% CI = 1.05 to 1.51) This killing activity of T cells from mice vaccinated with TS/PP was, however, statistically significantly greater against the 5-FU-treated target cells than against the same target cells not exposed to the cytotoxic drug [at 25/1 E/T ratio, % specific release = 65% and 48%, respectively (P = .05); ratio = 1.34; 95% CI = 1.08 to 1.65]. Minimal cytotoxic activity (defined as 15%–25%) was detected in the spleen cells of mice in the control group; however, no increase in killing was observed against the 5-FU-treated target cells. No statistically significant difference was observed between the CTL activity of spleen cells of TS/PP-vaccinated mice that had been treated with 5-FU and that of spleen cells of TS/PP-vaccinated mice that had not been treated with 5-FU treatment (data not shown). These results suggest that treatment of mice with 5-FU does not affect the TS-specific antitumor activity of the effector lymphocytes in this model. All of these experiments were subsequently repeated on a different generation of the same strain of HLA-A(*)02.01 transgenic mice, and we observed the same antitumor and immunologic effects we observed in the mice of the first strain we used.


    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Peptide-based vaccination against neoplastic diseases is an active and promising area of investigation; cancer vaccines directed against proteins that are necessary for tumor cell growth, survival, or metastasis, such as TS, may be especially useful. We developed a multiepitope peptide vaccine, TS/PP, and found that it elicited a TS-specific CTL response and had antitumor activity both in vitro and in vivo. In addition, the antitumor activity of TS/PP in vaccinated mice was enhanced when combined with 5-FU treatment. In contrast, although the TS-peptide cocktail (± 5-FU treatment) was able to induce CTL precursors specific for the three known TS epitopes and regressive phenomena in the tumor tissue, these responses were not sufficiently strong enough to inhibit the rapid tumor growth and progression in the animals. There could be a number of reasons why the TS-peptide cocktail, in contrast with the TS/PP peptide, did not show a sufficient protective activity and was unable to limit tumor growth. The individual small peptides, TS/1, TS/2, and TS/3, may be rapidly degraded in vivo by tissue proteases. If so, this short half-life could limit their ability to reach enough antigen-presenting cells to give rise to an efficient and fast CTL response. Furthermore, due to their very small size, TS-epitope peptides may easily reach the bloodstream, where they can bind with any empty HLA-A(*)02.01 molecule available, thus leading to immune suppression instead of CTL stimulation, as reported in a previous study (20).

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.


    NOTES
 Top
 Notes
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
This work was supported by a grant from the Italian Ministry of Education (MIUR 2004061578_002).


    REFERENCES
 Top
 Notes
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 

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Manuscript received September 7, 2005; revised May 4, 2005; accepted August 4, 2005.


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