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*
Department of Oncology and Pathology, Radiumhemmet, Karolinska Hospital, Stockholm, Sweden;
Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden;
First Department of Surgery, Yamanashi Medical University,Yamanashi, Japan;
§
Department of Health and Human Services, National Cancer Institute, National Institute of Health, Bethesda, MD 20892; and
¶
Epimmune, San Diego, CA 92121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As an alternative to the genetic and biochemical approach for identifying tumor- associated CTL epitopes, a reverse immunology method has been developed, which is not constrained by the availability of tumor-specific CTL, and thereby not limited to the analysis of only immunodominant epitopes already recognized by cancer patients during the course of their disease. In this method, MHC class I-binding epitopes are identified and corresponding synthetic peptides are tested for their capacity to induce peptide- and tumor-specific CTL derived from healthy individuals or cancer patients. This approach has recently been used for the definition of several new CTL epitopes in different melanoma Ags (7, 9, 10, 11).
The identification of tumor-associated Ags in melanomas has been paralleled by the demonstration that CTL derived from solid tumors or ascites of patients with ovarian cancer are capable of recognizing autologous and HLA class I-matched allogeneic tumors (12, 13, 14, 15, 16). The HER2/neu gene, which encodes a 185-kDa transmembrane glycoprotein with tyrosine-specific kinase activity and has a similarity in structure and sequence to the epidermal growth-factor receptor (17), is frequently recognized by tumor-specific CTL lines and clones derived from patients with carcinomas. HLA-A2-restricted CTL epitopes in the HER2/neu protein recognized by ovarian (12, 13) and breast cancer-specific (18, 19) CTL have previously been defined. Our previous work (20) has recently resulted in the identification of two new HLA-A2.1-restricted CTL epitopes from HER2/neu.
In the present study, we have tested a set of new HLA-A2-binding HER2/neu-derived peptides, using the reverse immunology approach, for their ability to elicit peptide- and tumor-specific CTL responses in patients with ovarian carcinomas. A method was developed in which peptide-loaded DC derived from the ascitic fluid of patients were used to induce peptide-specific CTL. We found that HER2/neu-specific CTL, elicited by several HER2/neu-derived peptides or isolated from the ascitic fluid of patients with ovarian carcinoma, were able to kill autologous and allogeneic HLA-A2+ ovarian and colorectal carcinoma cells in a tumor-specific manner. Furthermore, as a new finding, we report that melanomas, previously reported to overexpress HER2/neu (21), are also efficiently lysed by HER2/neu-specific CTL. The findings that several new HER2/neu-derived epitopes can efficiently elicit tumor-specific CTL against carcinomas and melanomas by using T cells and dendritic cells (DC)5 from cancer patients broaden the potential application for epitope-based immunotherapy based on this protooncogene.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tumor cells were isolated as described (22) from the ascitic fluid of HLA-A2.1+ patients with advanced epithelial ovarian cancer. All patients had undergone various regiments of chemotherapy due to recurrent disease. The ascitic fluid was collected in sterile heparinized containers. The samples were centrifuged at 800 x g for 10 min, and the cell pellet was resuspended in PBS containing 5% FBS, 100 U/ml penicillin, and streptomycin. Ascitic cells were then isolated by centrifugation on Ficoll-Hypaque and cultured at 106 cell/ml. Lymphocyte growth was inhibited by adding 1 µg/ml cyclosporin A to the medium. Nonadherent cells and debris were removed and the tumor cells were cultured in RPMI 1640 medium (Life Technologies, Auckland, NZ), supplemented with 10% FBS (Life Technologies), 2 mM glutamine (Sigma-Aldrich, Tyresö Sweden), 20% autologous ascitic fluid, 2.5 µg/ml insulin, and 1% penicillin-streptomycin (Sigma-Aldrich). Every 5 to 6 days, the medium was replaced with fresh supplemented medium.
Preparation of DC
DC were produced from monocytes according to a previously described protocol (23, 24, 25, 26). Briefly, after Ficoll-Hypaque separation, cells from ascitic fluid were resuspended in RPMI 1640 medium, supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin-streptomycin. The cells (106 cell/ml) were incubated overnight at 37°C, and the nonadherent cells were removed by gentle pipetting. The adherent cells were cultured in RPMI 1640 and supplemented with 10% FBS medium containing 1000 U/ml GM-CSF (Schering-Plough, Stockholm, Sweden) and 1000 U/ml IL-4 (Schering-Plough, Brinny, Cork, Ireland). After 9–12 days, floating DC-like cells were harvested in PBS and used as stimulators after peptide loading. Cell morphology was determined by standard microscopic techniques.
Peptide-specific CTL lines and antitumor CTL clones
DC were incubated with 25 µg/ml peptide for 12 h at room temperature, then irradiated and washed twice to remove excess of peptide, before being used as stimulator cells. Lymphocytes derived from tumor-associated lymphocytes (TAL) were stimulated with irradiated autologous peptide-loaded DC cells in AIM-V medium (Life Technologies) containing 12.5 U/ml IL-2 (kindly supplied by Dr. P. Simon, DuPont Merck Pharmaceutical, Glenolden, PA) and 10 ng/ml IL-7 (Becton Dickinson, Stockholm, Sweden). Stimulator to T cell ratio was 1:20. After at least four rounds of stimulation at weekly intervals, the cytotoxicity of CTL was measured by a standard 51Cr release assay. Antitumor CTL clones were then obtained from reactive CTL by limiting dilution after three weekly stimulations with irradiated autologous tumor cells.
Peptide synthesis and HLA-A2.1-binding assay
HER2/neu-derived peptides were identified on the basis of the HLA-A2.1-binding motif using a computer program (27). Nonamer and decamer peptides were synthesized by a solid phase method, using a multiple peptide synthesizer, and purified by HPLC, as previously described (10, 11). Peptide binding to HLA-A2 was measured as described (27, 28). Briefly, various doses of the test peptides (ranging from 100 µM-1 nM) were coincubated with 0.5 nM radiolabeled HBVc18–27 (FLPSDYFPSV) peptide and HLA-A2.1 heavy chain and ß2-microglobulin for 2 days at room temperature in the presence of a mixture of protease inhibitors. Percentage of MHC-bound radioactivity was determined by gel filtration, and the IC50, the concentration required to inhibit 50% of the radiolabeled peptide binding, was calculated for each peptide.
Cell lines
CAOV-4, SW-626 ovarian tumor cell lines and colon carcinoma cell line SW-620 were furnished by the American Type Culture Collection (ATCC, Manassas, VA). OVA-9301, OVA-0826, OVA-6906, OVA-320929, OVA-360622, OVA-1226 ovarian tumor lines, and BL, BE, DFB melanoma lines were established at Microbiology and Tumor Biology Center in Karoliska Institute. FM-55 and FM-3D melanoma lines were kindly provided by Dr. J. Zeuthen (Danish Cancer Society Research Center, Copenhagen, Denmark). FMS melanoma line was from Dr. Gaudernack (Norwegian Radium Hospital, Oslo, Norway). C1R/A2, a MHC class I-defective LCL cell line transfected with HLA-A2 (29) was provided by Dr. M. Mosac, Karolinska Institute. The TAP-defect HLA-2.1 T2 cell line derived from the human T cell leukemia/B cell LCL hybrid 174 (30) was a generous gift of Dr. P. Cresswell (Yale University School of Medicine, New Haven, CT).
Transfections
Transfections were performed using Lipofectin (Life Technologies, Grand Island, NY), according to the manufacturer’s protocols. The C1R/A2 cell line was transfected in our laboratory with the gene encoding for the HER2/neu protooncogene (LTR-2/erbB-2 encoding a full-length HER2/neu cDNA) kindly provided by Dr. J. Pierce (Laboratory of Cellular and Molecular Biology, National Cancer Institute). The HER2/neu-transfected C1R/A2 line (C1R/A2. HER2) was selected in medium containing 1 µg/ml mycofenolic acid, 250 µg/ml xantine, 15 µg/ml hypoxantine, 10 µg/ml thymidine, and 2 µg/ml aminopterine (Sigma-Aldrich). The ovarian tumor cell line SW-626 was transfected with the HLA-A2 expression vector containing the full-length HLA-A2.1 cDNA. The transfected cells were selected with 200 µg/ml hygromycin B (Boehringer Mannheim, Mannheim, Germany).
mAbs and FACS analysis
The anti-HER2/neu (mAb TA-1) recognizing the extracellular domain of HER2/neu was purchased from Oncogene Science (New York, NY). The mAb BB7.2 (HB82, ATCC) was used to detect HLA-A2.1. FITC-conjugated goat anti-mouse IgG was purchased from Becton Dickinson (Mountain View, CA). For cytometric analysis of tumor cells, 1 x 105 cells per staining were washed in PBS, incubated with a primary murine anti-human mAb for 20 min on ice. Cells were washed twice before incubation with a secondary FITC-labeled rabbit anti-mouse IgG Ab for 20 min. Cells were then washed twice before being resuspended in PBS containing 1% paraformaldehyde and 1% FBS.
FITC-labeled anti-CD3 (mAb UCHT1), CD4 (mAb MT310), and CD8 (mAb DK25), plus PE-labeled anti-CD4 (mAb MT310), CD8 (mAb DK25), and CD56 (mAb MOC-1) were purchased from Dako (Dakopatts, Älvsjö, Sweden). After staining, the cells were fixed with 1% paraformaldehyde and kept at 4°C until analysis by FACS (Becton Dickinson). For every staining, isotype-matched control mAb was used as a negative control.
Cytotoxic assay
To analyze tumor recognition, tumor cells were labeled for 1 h at 37°C with 125 µCi/106 cells 51Cr (Amersham, Amersham Sweden, Solna). For peptide recognition, T2 cells or C1R-A2 cells were incubated overnight at 26°C together with 25 µg/ml peptide, washed, and then labeled. Cytotoxic assays were performed by incubating 51Cr-labeled target cells with effector cells at various E:T ratios at 37°C for 4 h. In some experiments, blocking with an anti-HLA-A2 Ab was performed by incubating the target cells for 20 min with 1/50 dilution of the supernatant of the BB-7.2 hybridoma. Cold target inhibition was also done using nonradiolabeled T2 loaded with HER2/neu peptide or with the HLA-A2-binding irrelevant peptide MP58–66 (GILGFVFTL) used as negative control. The cold target to hot target (51Cr labeled) ratio was 10:1, unless otherwise indicated. Supernatants were harvested and radioactivity was determined using a gamma counter. The percentage of 51Cr release was calculated according to the following formula: percent lysis = 100 x (experimental release - spontaneous release)/(maximum release - spontaneous release). All determinations were made in triplicates.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously screened the amino acid sequence of the
HER2/neu protooncogene for the most probable HLA-A2 nona-
and deca-mer peptide epitopes by the use of a computer program that
takes into account the presence of main HLA-A2.1-specific anchor
residues, and specific secondary anchor residues (20, 27).
Of 165 peptides (9 mers and 10 mers) containing HLA-A2.1-binding
motifs, 23 were found to bind with an IC50 < 500
nM to purified HLA-A2.1 molecules, and 22 of these were included in the
present analysis (Table I
). The HER2
(9369) peptide previously reported as a CTL
epitope in cancer patients (14, 31) and also the HER2
(9689) epitope previously found by us to be
immunodominant in tumor-specific tumor-infiltrating lymphocytes from
gastric cancer (32) were among the highest affinity
HLA-A2.1-binding peptides.
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Three of the five high affinity binding (IC50 <
50) peptides, HER2(9665),
HER2(9689), and the previously defined
HER2(9369) (14, 31), were able to
induce peptide-specific lysis of T2 by CTL derived from at least four
of eight different patients with ovarian carcinomas, as shown with CTL
from patients OVA-320929 and OVA-360622 (Fig. 1, data not shown for
HER2(9665)). Six among the seventeen intermediate
binders (IC50 = 51–500 nM), including
HER2(948), HER2(9435),
HER2(9767), HER2(10785),
HER2(9789), and
HER2(10952), were able to elicit peptide-specific
CTL in at least three of eight different patients analyzed (Fig. 1, data only shown for HER2(9435)).
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). The peptide specificity of these CTL
was observed even in the presence of a 20-fold excess of K562 cells
(data not shown). In our initial protocol, stimulations were conducted
in the presence of IL-2 (12.5 U/ml) and with a ratio of DC to T cells
of 1:20 (Fig. 1, A–C). By adapting this to a protocol in
which stimulations were conducted without IL-2 and with a higher ratio
of DC to T cells (1:2.5–5) in the presence of 10 ng/ml IL-7, less
nonspecific killing of nonpulsed T2 was observed (Fig. 1, D
and E). Thus, our CTL induction protocol made it possible to produce HER2/neu-derived peptide-specific CTL from TAL of four of eight tested HLA-A2+ ovarian carcinoma patients, using nine different HLA-A2-restricted epitopes from HER2/neu. Notably, all four of the eight patients, OVA-9301, OVA-0826, OVA-320929, and OVA-360622, from which HER2/neu-specific CTL were generated, had tumors that expressed HER2/neu (data not shown).
HER2/neu-specific CTL can specifically recognize cells expressing HLA-A2 and HER2/neu
To establish whether peptide-induced CTL could recognize
HLA-A2-associated naturally processed epitopes, we next transfected the
HLA-A2+ gene in the
HLA-A2-, HER2/neu-expressing ovarian
carcinoma line SW-626. Six peptide-specific CTL lines from patient
OVA-9301, selected on the basis of availability of cells, were tested
against this HLA-A2+ transfectant and its
control. The CTL raised against peptide
HER2(9369), HER2(9435),
HER2(9665), HER2(9689), and
HER2(10952) all showed cytotoxic activity against
the HLA-A2+ transfectant of SW-626, although the
cytotoxic activity of the CTL raised against
HER2(9435) was low in this experiment. The
HLA-A2- control line was relatively resistant to
cytotoxicity (Fig. 2A). As
control, the influenza virus peptide (MP58–66)-specific CTL were not
able to lyse these targets.
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B). Again, the cytotoxic activity of
the CTL raised against HER2(9435) was low in this
experiment. Similarly, MP58–66 peptide-specific CTL did not lyse the
transfectant. Our data therefore show that the CTL lines induced by at least five of the high or intermediate HER2/neu HLA-A2 binders can induce specific CTL that also recognize naturally processed MHC class I-associated peptides.
HER2/neu-specific CTL can specifically recognize autologous and allogeneic carcinomas overexpressing HER2/neu
We succeeded in establishing tumor lines from patients 9301 and
0826. Both of these lines were found to overexpress HER2/neu
(data not shown). HER2/neu-specific CTL were tested for
their capacity to kill autologous tumor cells, while the
HLA-A2-, HER2/neu+ ovarian
tumor line SW-626 was used as control. CTL from patient 9301 induced by
peptides HER2(9369),
HER2(9435), HER2(9665),
HER2(9689), and HER2(10952)
were able to kill the autologous tumor, while the
HLA-A2- SW-626 tumor or the NK-sensitive K562
line was relatively resistant to lysis by the same CTL (Fig. 3). HER2(10785)
peptide-specific CTL did not specifi- cally lyse autologous
tumor cells (data not shown). Similarly, the CTL lines from patient
OVA-0826 induced by the same four peptides showed cytotoxic activity
against the autologous tumor, although this tumor was in general more
resistant to cytotoxicity (data not shown).
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). Among the
ovarian carcinomas, CAOV-4 and OVA-6906, both of which are
HLA-A2+ and
HER2/neu+, were lysed, while the SW-626
(HLA-A2-, HER2/neu+)
was very poorly recognized and OVA-1226
(HLA-A2-neu-) were not
lysed at all. The colorectal cancer line SW-620
(HLA-A2+, HER2/neu+)
was also highly sensitive to these HER2/neu-specific CTL.
These data confirm the widespread expression of the HER2/neu
Ag in various types of carcinomas, and show that it is processed and
presented to MHC class I-restricted CTL in all of the recognized
carcinomas. The carcinoma-specific cytotoxicity of this
HER2(9369)-specific CTL line was further
established by blocking with HLA-A2-specific mAb (data not shown).
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In the next series of experiments, five of eight melanoma lines,
BL, BE, FMS, FM-3D, and FM-55, were found to express
HER2/neu, further confirming (21) that this
molecule can also be expressed in tumors of neuroectodermal origin
(Table II). Two of these melanoma lines (FMS and BL) that were
HLA-A2+ and HER2/neu+were also highly sensitive to the
HER2(9369)-specific CTL line from donor OVA-0826
(Table II), from OVA-320929, and to the
HER2(9689)- and
HER2(9435)-specific CTL lines from donor
OVA-320929, and from donor OVA-360622 (data not shown). However, two of
the HLA-A2+ and
HER2/neu+ melanoma lines (BE and FM-3D)
showed weak sensitivity to the
HER2(9369)-specific CTL (Table II). The
HLA-A2+, HER2/neu-melanoma line DFB was resistant (Table II) or only weakly sensitive
(data not shown) to HER2/neu-specific CTL.
The specificity of the CTL lines against HER2/neu+ melanomas was further tested by adding an excess of nonlabeled cold T2 cells pulsed with the specific peptide to the mixture of CTL and51Cr-labeled melanoma targets. Recognition of the melanoma targets by the CTL lines specific for HER2(9689), HER2(9369), or HER2(9435) was significantly inhibited (20–40% inhibition) by unlabeled T2 cells only when pulsed with the cognate peptide, but not by unlabeled T2 cells pulsed with an irrelevant HLA-A2.1-binding peptide or by unlabeled control T2 cells. These results further confirm that a substantial part of the cytotoxicity of the HER2/neu-specific CTL is directed against naturally processed HER2-derived epitopes presented at the surface of the HLA-A2+ HER2/neu+melanomas.
CTL clones specific for new HER2/neu-derived epitopes expressed on ovarian carcinomas can be isolated from ovarian-specific CTL lines
We next asked whether CTL clones specific for HER2/neu
epitopes on ovarian carcinomas and melanomas can be isolated from
ovarian-specific CTL lines that were derived from expanding ascitic
derived T cells in IL-2 without stimulation with synthetic peptides. A
tumor-specific CTL line (OVA-3507) produced by repeated stimulation
with the autologous tumor and previously shown to be cytotoxic against
the autologous tumor and against other HLA-A2.1-expressing allogeneic
tumors (31) was cloned by limiting dilution. Seven of
fifty-eight clones derived from the OVA-3507 line were shown to be
cytotoxic for HLA-A2-expressing ovarian carcinoma lines (data not
shown). Three of them were further analyzed for their cytotoxicity
against the panel of HER-2/neu-derived peptides loaded onto
T2 cells. Clone 9 recognized HER2(9435), and
clone 29 recognized the HER2(9689) epitopes (Fig. 4) when loaded at concentrations of
100–0.1 µg/ml to T2 cells, thus demonstrating that these two
epitopes can also be immunodominant, as we already have shown for the
HER2(9689) epitope (32). None of
these two clones recognized other HLA-A2-binding peptides (Fig. 4). One
of the three clones (43) was found to recognize the
HER2(9369) peptide (data not shown), thus
confirming previous results (31).
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A). A similar pattern
of recognition was observed for both of these CTL clones, in which the
carcinomas CAOV-4 and SW-620 (HLA-A2.1+,
HER2/neu+) and the melanoma lines FMS and
BL (HLA-A2.1+,
HER2/neu+) were lysed, while the
HLA-A2.1+, HER2/neu-melanoma line DFW was not. To confirm that the antitumor cytotoxic
activity of the OVA-3507-derived clones 9 and 29 (Fig. 5) was specific
for the cognate peptide, an excess of nonlabeled cold T2 cells pulsed
with the corresponding specific HER2 peptide was added to the mixture
of CTL and 51Cr-labeled ovarian carcinoma CAOV-4
(Fig. 5B), colon carcinoma SW-620 (Fig. 5C), and
melanoma BL (Fig. 5D) targets. Recognition of the ovarian
carcinoma and melanoma targets was significantly inhibited by unlabeled
T2 cells pulsed with the cognate HER2-derived peptide, but not or much
less by unlabeled T2 cells or T2 cells pulsed with an irrelevant
HLA-A2.1-binding peptide (MP58–66).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Epitope identification was performed by the reverse immunology approach, previously used to define other CTL epitopes from human tumor-associated Ags. Herein, besides the known HER2(9369) epitope (12, 31), we report the identification of at least four more HER2/neu-derived epitopes, including HER2(9435), HER2(9665), HER2(9689), and HER2(10952), that were able to elicit CTL in patients with ovarian cancer. These epitopes elicited CTL that specifically killed peptide-sensitized target cells and, most importantly, autologous tumor cells. The HER2(9689) epitope was recently found to be immunodominant in gastric cancer-specific CTL (32). The HER2(95), HER2(9369), and HER2(9435) peptides were shown to be able to elicit peptide- and tumor-specific CTL from PBL of HLA-A2.1+ healthy donors (20), although a different T cell sensitization protocol was used. Two of these peptides, HER2(9369) and HER2(9435), were also found to elicit CTL in cancer patients. Our CTL induction protocol, with repeated stimulations over an extended period of time, does however not allow us to draw conclusions regarding the immunodominance of these epitopes in the tumor-specific CTL repertoire of the patients. The response in healthy donors may represent an example of an in vitro induction of a primary response, or alternatively, a weak secondary response following sensitization in vivo by peptides expressed on HER2/neu+ normal tissues. A comparison of epitope-specific CTL precursor frequencies between cancer patients and healthy donors would seem necessary to establish whether the ascitic fluid does indeed contain enhanced levels of HER2/neu-specific CTL precursors.
Three additional epitopes that were able to elicit peptide-specific CTL were defined, including HER2(948), HER2(9767), and HER2(9785) (data not shown).
T cells induced in vitro with APC pulsed with peptides selected for
their ability to bind to MHC class I alleles frequently have been shown
to recognize peptide-pulsed target cells, but not target cells
presenting naturally processed epitopes (33, 34).
Presumably, this is due to the low levels of epitope presented by the
latter. It is therefore of particular interest that CTL generated
against several of the new HER2/neu-derived epitopes
characterized in this study were able to recognize the autologous
HER2/neu+ HLA-A2+ovarian carcinomas, but not HLA-A2- ones (Fig. 3). The specificity of these peptide-induced lines was further
established by their lysis of C1R cells transfected with
HER2/neu and HLA-A2 genes (Fig. 2B) and by
analyzing a HLA-A2 transfectant of a HLA-A2-,
HER2/neu-expressing ovarian carcinoma (Fig. 2A).
CTL specific for the HER2(9369) (31, 35), HER2(9971) (12), and
HER2(10654) (35, 36) epitopes were
previously shown to recognize HLA-A2+,
HER2/neu+ tumor targets, demonstrating that
several epitopes from this molecule appear to be naturally processed
and presented on the surface of carcinomas. Our method for CTL
generation may favor the outgrowth of high affinity CTL precursors
bearing the capacity to recognize cell surface epitopes expressed at
low density.
Immunodominance was addressed by testing CTL clones developed from patient-derived CTL lines stimulated solely with autologous tumor cells. In agreement with previous results (12, 31), one CTL clone was found to be specific for the HER2(9369) epitope, while two additional CTL clones from the same donor also recognized the HER2(9435) and HER2(9689) epitopes, further confirming that HER2/neu-derived epitopes constitute a dominant part of the tumor-specific CTL response against ovarian carcinomas. Cold target competition assays confirmed that the cytotoxicity of these clones against HLA-A2+ carcinomas and melanomas was specific for the cognate naturally processed peptide. The immunodominance of HER2/neu could be explained by the fact that this molecule is a surface receptor (37) that may recycle into the cytoplasm and therefore be easily accessible to the MHC class I Ag presentation pathway. We have recently defined immunodominant CTL epitopes from another cell surface receptor (38), the MC1R receptor expressed on the majority of human melanomas (39). Thus, it is possible that cell surface receptors in general may frequently generate CTL epitopes.
Herein we also present the novel finding that HER2/neu-specific CTL lines and clones, recognizing epitopes HER2(9369), HER2(9435), or HER2(9689), also can kill some HLA-A2+, HER2/neu+ melanoma lines. Immunohistochemical studies have previously shown that human melanomas could express significant levels of HER2/neu (21). We find that five of the eight melanoma lines express significant levels of HER2/neu, as analyzed by flow cytometry, although their levels of expression were somewhat lower than that seen in the ovarian carcinoma lines. Two of the freshly isolated melanoma samples from which these tumor lines were established (BL and BE) were also analyzed for HER2/neu expression by immunohistochemistry and were found to express HER2/neu (data not shown), demonstrating that their HER2/neu expression was not induced by in vitro culture. Four of these melanoma lines were killed by our HER2/neu-specific CTL, although two (BE and FM-3D) only to a relatively low degree. The specificity of the killing of the melanoma lines by HER2/neu-specific CTL was further confirmed by cold target competition assays. Our finding of HER2/neu-specific CTL killing of melanomas may broaden the potential use of immunotherapy based on HER2/neu.
It should be noted that as we have not tested any low affinity binding HER2/neu epitopes, we cannot exclude that some of these may also be able to induce CTL with our CTL sensitization protocol. Our results demonstrate that the reverse immunology approach can be utilized to greatly broaden the repertoire of epitopes available to specific immunotherapy, allowing to tap into a rich collection of subdominant epitopes. Several of these epitopes would remain unavailable for exploitation if one were limited to only the most dominant epitopes.
Others have used the ascites as a source of T cells to generate tumor-specific CTL (22, 40). As a new approach, we have in this study used the ascites as a source for both T cells and DC for the generation of peptide-specific CTL. The ascitic fluid from patients with ovarian and other types of tumors (colon cancer, liver cancer, and pancreatic cancer) may therefore be a useful starting source of T cells and DC cells for the generation of large numbers of CTL to be used in protocols of adoptive immunotherapy.
Our data clearly establish that HER2/neu is an immunodominant molecule. DC cultures can be isolated from ascitic material, loaded with peptides corresponding to the dominant and subdominant epitopes, and used to develop CTL lines from T cell cultures originating from autologous ascitic material. We hope that this approach will be useful in developing protocols for epitope-based immunotherapy against various carcinomas.
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Footnotes |
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2 F.S.-O. and J.C. made equal contribution to this article.
3 Current address: Department of Immunology, Mayo Clinic, Rochester, MN 55905.
4 Address correspondence and reprint requests to Dr. Rolf Kiessling, Department of Oncology and Pathology, Radiumhemmet, Karolinska Hospital, S-171 76 Stockholm, Sweden. E-mail address:
5 Abbreviations used in this paper: DC, dendritic cell; LCL, lymphoblastoid cell line; TAL, tumor-associated lymphocyte.
Received for publication March 8, 1999. Accepted for publication May 3, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J. Exp. Med. 179:1109.
impairs the ability of monocyte-derived dendritic cells to present tumor-specific and allo-specific antigens and reduces their expression of CD1A, CD80 and CD4. Cytokine 10:747.[Medline]
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 Q.-S. Du, Y.-T. Wei, Z.-W. Pang, K.-C. Chou, and R.-B. Huang Predicting the affinity of epitope-peptides with class I MHC molecule HLA-A*0201: an application of amino acid-based peptide prediction Protein Eng. Des. Sel., September 1, 2007; 20(9): 417 - 423. [Abstract] [Full Text] [PDF]
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C. Taylor, D. Hershman, N. Shah, N. Suciu-Foca, D. P. Petrylak, R. Taub, L. Vahdat, B. Cheng, M. Pegram, K. L. Knutson, et al. Augmented HER-2 Specific Immunity during Treatment with Trastuzumab and Chemotherapy Clin. Cancer Res., September 1, 2007; 13(17): 5133 - 5143. [Abstract] [Full Text] [PDF]
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 A. Scardino, M. Alimandi, P. Correale, S. G. Smith, R. Bei, H. Firat, M. G. Cusi, O. Faure, S. Graf-Dubois, G. Cencioni, et al. A Polyepitope DNA Vaccine Targeted to Her-2/ErbB-2 Elicits a Broad Range of Human and Murine CTL Effectors to Protect against Tumor Challenge Cancer Res., July 15, 2007; 67(14): 7028 - 7036. [Abstract] [Full Text] [PDF]
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B. J. Czerniecki, G. K. Koski, U. Koldovsky, S. Xu, P. A. Cohen, R. Mick, H. Nisenbaum, T. Pasha, M. Xu, K. R. Fox, et al. Targeting HER-2/neu in Early Breast Cancer Development Using Dendritic Cells with Staged Interleukin-12 Burst Secretion Cancer Res., February 15, 2007; 67(4): 1842 - 1852. [Abstract] [Full Text] [PDF]
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R. Singh and Y. Paterson Vaccination Strategy Determines the Emergence and Dominance of CD8+ T-Cell Epitopes in a FVB/N Rat HER-2/neu Mouse Model of Breast Cancer. Cancer Res., August 1, 2006; 66(15): 7748 - 7757. [Abstract] [Full Text] [PDF]
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H. Norell, M. Carlsten, T. Ohlum, K.-J. Malmberg, G. Masucci, K. Schedvins, W. Altermann, D. Handke, D. Atkins, B. Seliger, et al. Frequent Loss of HLA-A2 Expression in Metastasizing Ovarian Carcinomas Associated with Genomic Haplotype Loss and HLA-A2-Restricted HER-2/neu-Specific Immunity. Cancer Res., June 15, 2006; 66(12): 6387 - 6394. [Abstract] [Full Text] [PDF]
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A. D. Gritzapis, L. G. Mahaira, S. A. Perez, N. T. Cacoullos, M. Papamichail, and C. N. Baxevanis Vaccination with Human HER-2/neu (435-443) CTL Peptide Induces Effective Antitumor Immunity against HER-2/neu-Expressing Tumor Cells In vivo. Cancer Res., May 15, 2006; 66(10): 5452 - 5460. [Abstract] [Full Text] [PDF]
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F. Rohrbach, R. Weth, M. Kursar, A. Sloots, H.-W. Mittrucker, and W. S. Wels Targeted Delivery of the ErbB2/HER2 Tumor Antigen to Professional APCs Results in Effective Antitumor Immunity J. Immunol., May 1, 2005; 174(9): 5481 - 5489. [Abstract] [Full Text] [PDF]
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K. L. Bondurant, M. D. Crew, A. D. Santin, T. J. O'Brien, and M. J. Cannon Definition of an Immunogenic Region Within the Ovarian Tumor Antigen Stratum Corneum Chymotryptic Enzyme Clin. Cancer Res., May 1, 2005; 11(9): 3446 - 3454. [Abstract] [Full Text] [PDF]
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A. B. Riemer, M. Klinger, S. Wagner, A. Bernhaus, L. Mazzucchelli, H. Pehamberger, O. Scheiner, C. C. Zielinski, and E. Jensen-Jarolim Generation of Peptide Mimics of the Epitope Recognized by Trastuzumab on the Oncogenic Protein Her-2/neu J. Immunol., July 1, 2004; 173(1): 394 - 401. [Abstract] [Full Text] [PDF]
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I. A. Doytchinova, V. A. Walshe, N. A. Jones, S. E. Gloster, P. Borrow, and D. R. Flower Coupling In Silico and In Vitro Analysis of Peptide-MHC Binding: A Bioinformatic Approach Enabling Prediction of Superbinding Peptides and Anchorless Epitopes J. Immunol., June 15, 2004; 172(12): 7495 - 7502. [Abstract] [Full Text] [PDF]
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M. Ruiz, H. Kobayashi, J. J. Lasarte, J. Prieto, F. Borras-Cuesta, E. Celis, and P. Sarobe Identification and Characterization of a T-Helper Peptide from Carcinoembryonic Antigen Clin. Cancer Res., April 15, 2004; 10(8): 2860 - 2867. [Abstract] [Full Text] [PDF]
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K. Kono, E. Sato, H. Naganuma, A. Takahashi, K. Mimura, H. Nukui, and H. Fujii Trastuzumab (Herceptin) Enhances Class I-Restricted Antigen Presentation Recognized by HER-2/neu-Specific T Cytotoxic Lymphocytes Clin. Cancer Res., April 1, 2004; 10(7): 2538 - 2544. [Abstract] [Full Text] [PDF]
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S. Vertuani, A. Sette, J. Sidney, S. Southwood, J. Fikes, E. Keogh, J. A. Lindencrona, G. Ishioka, J. Levitskaya, and R. Kiessling Improved Immunogenicity of an Immunodominant Epitope of the Her-2/neu Protooncogene by Alterations of MHC Contact Residues J. Immunol., March 15, 2004; 172(6): 3501 - 3508. [Abstract] [Full Text] [PDF]
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M. E. Wolpoe, E. R. Lutz, A. M. Ercolini, S. Murata, S. E. Ivie, E. S. Garrett, L. A. Emens, E. M. Jaffee, and R. T. Reilly HER-2/neu-Specific Monoclonal Antibodies Collaborate with HER-2/neu-Targeted Granulocyte Macrophage Colony-Stimulating Factor Secreting Whole Cell Vaccination to Augment CD8+ T Cell Effector Function and Tumor-Free Survival in Her-2/neu-Transgenic Mice J. Immunol., August 15, 2003; 171(4): 2161 - 2169. [Abstract] [Full Text] [PDF]
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B. K. Ko, K. Kawano, J. L. Murray, M. L. Disis, C. L. Efferson, H. M. Kuerer, G. E. Peoples, and C. G. Ioannides Clinical Studies of Vaccines Targeting Breast Cancer Clin. Cancer Res., August 1, 2003; 9(9): 3222 - 3234. [Abstract] [Full Text] [PDF]
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 V. Ramakrishna, M. M. Ross, M. Petersson, C. C. Gatlin, C. E. Lyons, C. L. Miller, H. E. Myers, M. McDaniel, L. R. Karns, R. Kiessling, et al. 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 Int. Immunol., June 1, 2003; 15(6): 751 - 763. [Abstract] [Full Text] [PDF]
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A. M. Ercolini, J.-P. H. Machiels, Y. C. Chen, J. E. Slansky, M. Giedlen, R. T. Reilly, and E. M. Jaffee Identification and Characterization of the Immunodominant Rat HER-2/neu MHC Class I Epitope Presented by Spontaneous Mammary Tumors from HER-2/neu-Transgenic Mice J. Immunol., April 15, 2003; 170(8): 4273 - 4280. [Abstract] [Full Text] [PDF]
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K. Kono, A. Takahashi, H. Sugai, H. Fujii, A. R. Choudhury, R. Kiessling, and Y. Matsumoto Dendritic Cells Pulsed with HER-2/neu-derived Peptides Can Induce Specific T-Cell Responses in Patients with Gastric Cancer Clin. Cancer Res., November 1, 2002; 8(11): 3394 - 3400. [Abstract] [Full Text] [PDF]
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J.-Y. Sun, R. S. Krouse, S. J. Forman, D. Senitzer, I. Sniecinski, S. Chatterjee, and K. K. Wong Jr. Immunogenicity of a p210BCR-ABL Fusion Domain Candidate DNA Vaccine Targeted to Dendritic Cells by a Recombinant Adeno-associated Virus Vector in Vitro Cancer Res., June 1, 2002; 62(11): 3175 - 3183. [Abstract] [Full Text] [PDF]
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A. Scardino, D.-A. Gross, P. Alves, J. L. Schultze, S. Graff-Dubois, O. Faure, S. Tourdot, S. Chouaib, L. M. Nadler, F. A. Lemonnier, et al. HER-2/neu and hTERT Cryptic Epitopes as Novel Targets for Broad Spectrum Tumor Immunotherapy J. Immunol., June 1, 2002; 168(11): 5900 - 5906. [Abstract] [Full Text] [PDF]
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M. P. Piechocki, S. A. Pilon, and W.-Z. Wei Complementary Antitumor Immunity Induced by Plasmid DNA Encoding Secreted and Cytoplasmic Human ErbB-2 J. Immunol., September 15, 2001; 167(6): 3367 - 3374. [Abstract] [Full Text] [PDF]
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C. Meyer zum Buschenfelde, J. Metzger, C. Hermann, N. Nicklisch, C. Peschel, and H. Bernhard The Generation of Both T Killer and Th Cell Clones Specific for the Tumor-Associated Antigen HER2 Using Retrovirally Transduced Dendritic Cells J. Immunol., August 1, 2001; 167(3): 1712 - 1719. [Abstract] [Full Text] [PDF]
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E. Keogh, J. Fikes, S. Southwood, E. Celis, R. Chesnut, and A. Sette Identification of New Epitopes from Four Different Tumor-Associated Antigens: Recognition of Naturally Processed Epitopes Correlates with HLA-A*0201-Binding Affinity J. Immunol., July 15, 2001; 167(2): 787 - 796. [Abstract] [Full Text] [PDF]
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P. Bonvini, W. G. An, A. Rosolen, P. Nguyen, J. Trepel, A. Garcia de Herreros, M. Dunach, and L. M. Neckers Geldanamycin Abrogates ErbB2 Association with Proteasome-resistant {beta}-Catenin in Melanoma Cells, Increases {beta}-Catenin-E-Cadherin Association, and Decreases {beta}-Catenin-sensitive Transcription Cancer Res., February 1, 2001; 61(4): 1671 - 1677. [Abstract] [Full Text]
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R. P. M. Sutmuller, L. R. H. M. Schurmans, L. M. van Duivenvoorde, J. A. Tine, E. I. H. van der Voort, R. E. M. Toes, C. J. M. Melief, M. J. Jager, and R. Offringa Adoptive T Cell Immunotherapy of Human Uveal Melanoma Targeting gp100 J. Immunol., December 15, 2000; 165(12): 7308 - 7315. [Abstract] [Full Text] [PDF]
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 F. Berard, P. Blanco, J. Davoust, E.-M. Neidhart-Berard, M. Nouri-Shirazi, N. Taquet, D. Rimoldi, J. C. Cerottini, J. Banchereau, and A. K. Palucka Cross-Priming of Naive Cd8 T Cells against Melanoma Antigens Using Dendritic Cells Loaded with Killed Allogeneic Melanoma Cells J. Exp. Med., December 4, 2000; 192(11): 1535 - 1544. [Abstract] [Full Text] [PDF]
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 P. J. Mosca, A. C. Hobeika, T. M. Clay, S. K. Nair, E. K. Thomas, M. A. Morse, and H. K. Lyerly A subset of human monocyte-derived dendritic cells expresses high levels of interleukin-12 in response to combined CD40 ligand and interferon-gamma treatment Blood, November 15, 2000; 96(10): 3499 - 3504. [Abstract] [Full Text] [PDF]
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B. W. Anderson, G. E. Peoples, J. L. Murray, M. A. Gillogly, D. M. Gershenson, and C. G. Ioannides Peptide Priming of Cytolytic Activity to HER-2 Epitope 369-377 in Healthy Individuals Clin. Cancer Res., November 1, 2000; 6(11): 4192 - 4200. [Abstract] [Full Text] [PDF]
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C. Meyer zum Buschenfelde, N. Nicklisch, S. Rose-John, C. Peschel, and H. Bernhard Generation of Tumor-Reactive CTL Against the Tumor-Associated Antigen HER2 Using Retrovirally Transduced Dendritic Cells Derived from CD34+ Hemopoietic Progenitor Cells J. Immunol., October 1, 2000; 165(7): 4133 - 4140. [Abstract] [Full Text] [PDF]
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H. Kobayashi, M. Wood, Y. Song, E. Appella, and E. Celis Defining Promiscuous MHC Class II Helper T-Cell Epitopes for the HER2/neu Tumor Antigen Cancer Res., September 1, 2000; 60(18): 5228 - 5236. [Abstract] [Full Text]
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J. Gong, N. Nikrui, D. Chen, S. Koido, Z. Wu, Y. Tanaka, S. Cannistra, D. Avigan, and D. Kufe Fusions of Human Ovarian Carcinoma Cells with Autologous or Allogeneic Dendritic Cells Induce Antitumor Immunity J. Immunol., August 1, 2000; 165(3): 1705 - 1711. [Abstract] [Full Text] [PDF]
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C. N. Baxevanis, I. F. Voutsas, O. E. Tsitsilonis, A. D. Gritzapis, R. Sotiriadou, and M. Papamichail Tumor-Specific CD4+ T Lymphocytes from Cancer Patients Are Required for Optimal Induction of Cytotoxic T Cells Against the Autologous Tumor J. Immunol., April 1, 2000; 164(7): 3902 - 3912. [Abstract] [Full Text] [PDF]
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