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A ras-Mutated Peptide Targeted by CTL Infiltrating a Human Melanoma Lesion¹

Institut de Biologie, Institut National de la Santé et de la Recherche Médicale, Unité 463, and Faculté des Sciences et Techniques de Nantes, Nantes, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ags derived from commonly mutated oncogenic proteins seem ideally suited as targets for tumor immunotherapy. Nonetheless, only a few mutated epitopes efficiently presented by human tumors have thus far been identified. We describe here an approach to identify such epitopes. This approach involves: 1) identifying tumors expressing a ras mutation and isolating the tumor-infiltrating lymphocytes (TIL); 2) transfecting COS cells to induce expression of unknown mutated peptides in the context of a patient’s HLA class I molecules; and 3) screening epitope recognition by using TIL from the tumors expressing a ras mutation. By using this approach, there appeared to be a N-ras mutation (a glutamine-to-arginine exchange at residue 61 (Q61R)), detected in a melanoma lesion, which was recognized specifically by the autologous TIL in the HLA-A*0101 context. The ras peptide 55–64^Q61R was the epitope of these TIL and was regularly presented by Q61R-mutated HLA-A*0101⁺ melanoma cell lines. This peptide and its wild-type homolog (55–64^wt) bound to HLA-A*0101 with similar affinities. However, only the mutated peptide could induce specific CTL expansion from PBL. All the CTL clones specific to the mutated peptide, failed to recognize the wild-type sequence on both COS and melanoma cells. These data thus show that oncogenic protein mutations can create shared tumor-specific CTL epitopes, efficiently presented by tumor cells, and that screening for oncogene-transfected COS cell recognition by TIL (from tumors containing mutations) is a powerful approach for the identification of these epitopes.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Tumor rejection Ags have been identified in animal cancer models, and many of these are mutated proteins (1, 2, 3). In humans, although many tumor-associated Ags have recently been identified and although many epitopes from these are currently being used in immunotherapy trials, none of these has yet reached the status of tumor rejection Ags. CTL recognition of unique mutations of tumor cell proteins have been reported to be associated with unusually favorable clinical evolution in melanoma lesions, suggesting that mutated Ags could play such a role (4, 5).

CD8⁺ CTL have been implicated as important cellular components involved in the recognition and eradication of tumor cells in both murine and human systems (6, 7). These T cells recognize epitopes, classically 8–11 aa long and bound to MHC class I molecules on APC or tumor cells. To check whether oncogenic mutated proteins represent appropriate Ag to target for tumor-specific responses, it is necessary to know the MHC class I-restricted peptides derived from these proteins shared by a significant fraction of human tumors.

ras protooncogenes are activated by point mutations in a high fraction of human malignancies. The mutation primarily occurs at codon 12 or 61 and results in the expression of p21^ras oncoproteins with a single substituted amino acid (8, 9). Because a limited number of oncogenic amino acid substitutions occur, identical ras mutations are shared by many tumors, and this may therefore generate shared tumor epitopes (9). Nonetheless, only a few MHC class-I-restricted ras-mutated epitopes have been identified thus far (10, 11, 12, 13). Many of these did not allow efficient tumor cell killing by specific CTL (10, 12, 13). This clearly limits their use in immunotherapy. This shortage of potentially antigenic ras CTL epitopes may be due in part to the method commonly used for their identification, i.e., reverse immunology. Briefly, this method consists of checking for a peptide aggretope resulting from common oncogenic mutations, using these peptides to induce specific CTL in vitro, and finally determining whether specific CTL can kill tumor cells. The poor lytic capability of CTL generated by this approach (10, 12, 13) suggests that HLA binding sequences, identified by predictive anchoring, might not be processed efficiently. We tested here a different approach to identify HLA class I-restricted mutated ras peptides, which are efficiently expressed by tumor cells. This approach relied on: 1) identifying ras-mutated tumors by sensitive allele-specific PCR and isolating the tumor-infiltrating lymphocytes (TIL)³); 2) inducing COS cells to express unknown ras-mutated peptides in the context of a patient’s HLA class I molecules by transfection; and 3) screening for epitope recognition using TIL from the mutated tumors.

A ras-mutated peptide restricted by HLA-A*0101 was identified. This peptide was expressed by the autologous melanoma cell line but also by all other HLA-A*0101 melanoma cell lines that expressed the same mutation. This approach should permit the identification of well-expressed mutated tumor epitopes and could thus be instrumental in the development of successful immunotherapies.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
RNA extraction and cDNA check

Total RNA was extracted from tumor fragments or from tumor cell lines by the guanidinium-cesium chloride procedure and used for cDNA synthesis using Moloney murine leukemia virus reverse transcriptase (Life Technologies, Cergy-Pontoise, France). To test the quality of RNA extraction and of reverse transcription, PCR amplification of human -actin cDNA was performed on a cDNA solution aliquot using 5'-GGCATCGTGATGGACTCCG-3' and 5'-GCTGGAAGGTGGACAGCGA-3' primers. PCR was run for 21 cycles of 1 min at 94°C, 1 min at 65°C, and 1 min at 72°C.

Allele-specific PCR amplification

Exon 2 mutations of the N-ras gene were checked using a new sensitive allele-specific PCR method derived from mutant-allele-specific amplification (MASA) PCR (14). The reliability of this technique was improved by using double mismatching at the 3' end of the primer, as described in the amplification-refractory mutation system method (15, 16). 5' wild-type (wt) PCR primer N61F and mutated PCR primers are described in Fig. 1 (Sigma-Genosys, Cambridge, U.K.). The reverse primer N61R2 (5'-TGACTTGCTATTATTGATGG-3') was used for all the PCR. The PCR mixture contained 10 mM Tris-HCl, 1.5 mM MgCl₂, 0.01% (w/v) gelatin, 50 mM KCl, 200 mM concentrations of each dNTP (Pharmacia, Uppsala, Sweden), 50 pM concentrations of each primer, 1.25 U of AmpliTaq Gold (PerkinElmer, Norwalk, CT), and 100 ng of cDNA. PCR was performed for 38 cycles of 0.5 min at 94°C, 1.5 min at 54°C, and 1.5 min at 70°C. A 10-min step at 94°C was performed before the cycles.

View larger version (42K): [in this window] [in a new window]   FIGURE 1. Primers used to check N-ras mutations at residue 61 in melanoma tumors. Each primer has mismatching bases at its 3' end, except primer N61F used for the amplification of the normal allele. The amino acid modifications induced by mutations are shown in parentheses.

A 334-bp cDNA coding for a fragment of the wt N-ras protein was obtained by PCR amplification of a nonmutated N-ras cDNA. cDNA coding for mutated ras proteins were obtained by directional mutagenesis. Site-directed mutagenesis was performed using primers with nucleotide substitutions at codon 61, thus generating oncogenic mutations. Mutated DNA were inserted into pcDNA3 vector and amplified in a bacterial strain (Escherichia coli TOP 10 F'). Each construction was sequenced on both strands by the dideoxy method (17).

Cell line and TIL cultures

After the patient’s consent was given, tumors were taken from melanoma lesions and used for in vitro experimentation. This study has been approved by a committee involved with ethical issues in Nantes. All cells were cultured at 37°C under a CO₂ atmosphere. Melanoma cells lines were established from fragments of s.c. metastatic tumors or tumor-invaded lymph nodes, cultured in RPMI 1640 (BioWhittaker, Walkersville, MD) containing 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin (Sigma-Aldrich, St. Louis, MO), and 2 mM L-glutamine (Sigma-Aldrich). Mouse fibrosarcoma WEHI 164 clone 13 (used for TNF production assay) and COS-7 cells were obtained from T. Boon (Ludwig Institute for Cancer Research, Brussels, Belgium). COS-7 cells were cultured in DMEM (BioWhittaker) containing 10% FCS, streptomycin, penicillin, and L-glutamine, as mentioned above. The EBV-B-transformed cell line LAZ 338 was a gift from T. Hercend (Vertex Pharmaceutical, Abingdon, U.K.). The BM36-1 cell line (18) was a gift from A. Ziegler (Universitatsklinikum Charite, Berlin, Germany). Polyclonal TIL were obtained by a 12-day culture of tumor or tumor-invaded lymph node fragments in RPMI 1640 (Sigma-Aldrich) containing 8% AB human serum (local production), antibiotics, and glutamine (as mentioned above) and 150 U/ml rIL-2 (Chiron, Amsterdam, The Netherlands). TIL amplification was induced by PE-PHA-P (Difco, Detroit, MI). In brief, TIL were seeded in 96-well multiplates at 1000 TIL per well, in the presence of irradiated feeder cells (2 x 10⁴ LAZ cells and 10⁵ PBL per well), PHA-P, and rIL-2. TIL clones were produced in the same way, but limiting dilution culture of TIL was used instead.

HLA DNAs

cDNAs coding for HLA class I alleles, were obtained from T. Boon (Ludwig Institute for Cancer Research, Brussels, Belgium), F. Lemonier (Institut Pasteur, Paris, France), or E. Houssaint (Unité 463, Institut National de la Santé et de la Recherche Médicale, Nantes, France).

Transient transfection of melanoma cell lines and of COS-7 cells

Melanoma cells were seeded in 96-well plates and incubated until they were 80% confluent. Cells were then incubated at 37°C with a mix of a plasmid (100 ng) and 0.5 µl of LipofectAMINE reagent (Life Technologies) in serum-free medium. After 15 h, the transfection mixture was replaced by complete medium. COS-7 cell transfection was performed by the DEAE-dextran-chloroquine method (19, 20). Details of the procedure are described by De Plaen et al. (21). COS-7 cells (16.5 x 10³) were transfected with 100 ng of one or two plasmids.

Synthetic peptides

The wt ras peptides ILDTAGQEEY (55–64^wt); the mutated decamers ILDTAGREEY (55–64^Q61R), ILDTAGKEEY (55–64^Q61K), and LLDILDTAGR (52–61^Q61R); and the MAGE-3 peptide (EVDPIGHLY) were purchased from Synt:em (Nîmes, France). Purity (>85%) was controlled by reversed phase HPLC. Peptides were lyophilized, dissolved in DMSO at 10 mg/ml, and stored at -80°C.

HLA-A*0101 binding of ras peptides

The TAP-deficient cell line (BM36-1) expressing the HLA-A*0101 was incubated for 12 h with the peptides (2 x 10⁶ cells in RPMI 1640 with 100 µM peptide and 1 µM human ₂-microglobulin at 37°C). Brefeldin A (BFA; Sigma-Aldrich) was then added to a final concentration of 10 µg/ml. One hour after BFA was added, cells were washed with PBS and incubated at 37°C in RPMI 1640 (5% FCS, 0.5 µg/ml BFA). Then, at 0 or 30 min after removal of the peptides, cells were stained with the anti-HLA class I mAb, W6/32 (American Type Culture Collection, Manassas, VA) in PBS, 0.1% BSA, 0.5 µg/ml BFA. HLA class I expression was analyzed by flow cytometry.

T cell stimulation assays

TNF secretion and cytotoxicity assays were used to measure T cell stimulation, as described in detail elsewhere (21, 22). In brief, to obtain TNF determination, TIL (5 x 10³–5 x 10⁴) or T cell clones (2 x 10³–10⁴) were added to 3 x 10⁴ stimulator cells (COS cells 48 h after transfection or melanoma cells). Culture supernatants were harvested 6 h later and tested for TNF content by measuring lysis of WEHI 164 clone 13 in a MTT colorimetric assay. T cell cytotoxicity was measured in a standard 4-h ⁵¹Cr release assay. Briefly, BM361 or melanoma cells were labeled with ⁵¹Cr (Na₂⁵¹CrO₄; Oris, Gif-sur-Yvette, France). BM36.1 cells were then pulsed for 1 h at 37°C with 10 µM concentrarions of the peptides and washed. T cells (5 x 10³) were mixed with 10³ target cells for 4 h at 37°C.

PBL stimulation by the wt and 55–64^Q61R ras peptides

CD8⁺ HLA-A*0101 PBL were obtained by negative sorting of CD4⁺ T cells using immunomagnetic beads (Miltenyi Biotec, Paris, France). CD8⁺ T cells were stimulated three times at 1-wk intervals using autologous mature dendritic cells (DC) pulsed with either the wt or the mutated peptides. DC were prepared within six-well culture plates, from adherent PBMC, by a 7-day culture with RPMI 1640 containing 10% FCS, 50 ng/ml GM-CSF (Sigma-Aldrich), and 50 ng/ml IL-4 (Sigma-Aldrich). At day 7, DC maturation was induced by a 2-day culture at 37°C in RPMI 1640 containing 10% FCS, 10 ng/ml TNF- (Sigma-Aldrich), and 100 µg/ml poly(IC) (Sigma-Aldrich). At day 9, mature DC were loaded with one of the ras peptides by incubation for 2 h at 37°C with 5 µM peptide. After a washing, DC were used to stimulate CD8⁺ PBL at the stimulatory-responder cell ratio of 10:1. Each culture well was tested for the presence of peptide specific CTL. To this aim, 7 days after the last stimulation, PBL were stimulated for 6 h by BM36-1 cells pulsed with the appropriate peptide. The specific response was measured by intracellular cytoplasmic IFN- labeling as described (22). Lymphocytes from one culture well, containing 0.5% responding T cells, were cloned by limiting dilution culture.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of melanoma tumors expressing codon 61 N-ras mutations

Melanoma tumors or melanoma-invaded lymph nodes from 42 melanoma patients were screened for the presence of N-ras mutations at codon 61, using the MASA PCR. Nine of 42 expressed such a mutation. PCR results obtained with melanoma tumor M6 are shown in Fig. 2. Table I summarizes the mutations that were detected. The arginine to glutamine exchange, Q61R, was detected in four tumors. Mutations inducing lysine and histidine substitutions were found, respectively, in three and two tumors.

View larger version (98K): [in this window] [in a new window]   FIGURE 2. PCR detection of the Q61R ras mutation in melanoma lesion M6. A first step including multiplex PCR were done for each patient, to localize ras mutations at the first, second, or third base of codon 61, using, respectively, the following mixes of primers: N6111/12; N6121/22/23; and N6131/32. A simplex PCR using the wt ras primers N61F was done in parallel, as a positive control. According to the results obtained in the multiplex PCR, three to four simplex PCR (including the positive control) were then done. At this time, a negative control was also done, using cDNA from a nonmutated cell line. For patient M6, the multiplex PCR (N6121/22/23) and the simplex PCR (N6122) gave a 106-bp (pb) signal. This indicates the substitution of the adenine with a guanine.

TIL from the seven tumors bearing N-ras mutations were stimulated by COS-7 cells cotransfected with a cDNA coding for a patient HLA class I molecule (between 3 and 5 HLA cDNA were available per patient) and with a cDNA coding for the appropriate N-ras mutation (see Table I). As shown in Fig. 3, M6 TIL responded to COS-7 cells expressing HLA-A*0101 and the arginine mutated N-ras DNA. No recognition was found in the other two HLA contexts tested. Other TIL did not recognize the transfected COS cells (data not shown).

View larger version (23K): [in this window] [in a new window]   FIGURE 3. M6 TIL response to COS-7 cells cotransfected by the HLA-A*0101 and Q61R ras DNA. COS cells were transfected with pcDNA3 vectors coding for one HLA class I allospecificity from M6 patient and for a ras fragment bearing the Q61R mutation detected in the M6 tumor. 48 h after transfection, COS-7 cells were incubated for 6 h with M6 TIL. The amount of TNF secreted was determined using a biological assay with Wehi cells. Results are expressed as mean ± SD of six replicates. *, p < 0.01.

From M6 TIL, 112 CD8⁺ clonal colonies were derived by a limiting dilution culture. About 80 of these recognized the autologous melanoma cells, among which 3 also recognized COS-7 cells cotransfected with the HLA-A*0101 and the Q61R ras DNA. One of these colonies expressed the TCR V8 chain, whereas the other two expressed undetermined V. These clones failed to recognize COS-7 cells transfected with the wt ras and with the other ras mutations. Therefore, at least two distinct TIL clones from M6 tumor specifically recognized a Q61R ras epitope in the HLA-A*0101 context (Fig. 4).

View larger version (39K): [in this window] [in a new window]   FIGURE 4. TIL clones M6ras5 and M6ras24 specifically recognize a Q61R ras epitope. COS-7 cells, transfected or not transfected, by either the ras wt cDNA or a ras-mutated cDNA and/or by the HLA-A*0101 were incubated with M6 TIL clones for 6 h. The amount of TNF secreted was determined using a biological assay with Wehi cells.

A TAP-deficient cell line (BM36-1) expressing the HLA-A*0101 was used to present ras peptides encompassing the mutated position 61 and exhibiting consensus binding sequences for HLA-A*0101. 55–64^Q61R and 52–61^Q61R ras peptides were the peptides tested. The peptide controls that were used were the 55–64^Q61K ras peptide and the 55–64^wt ras peptide. As shown in Fig. 5, only the 55–64^wt and the 55–64^Q61R ras peptides sensitized the BM36-1 cell line to clone lysis. However, the sensitizing activity of the wt peptide was very low.

View larger version (28K): [in this window] [in a new window]   FIGURE 5. Peptide specificity of M6 TIL clones specific for the Q61R ras mutation. The TAP-deficient cell line BM36-1 expressing the HLA-A*0101 was pulsed with different ras peptides: the 55–64wt (ILDTAGQEEY); the 55–64Q61R (ILDTAGREEY); the 55–64Q61K (ILDTAGKEEY); and the 52–61Q61R (LLDILDTAGR). Lytic activity of TIL clones M6ras5 and 24 was measured, at an E:T ratio of 3:1, by the classical 4-h 51Cr release assay.

To ascertain whether the 55–64^Q61R ras peptide was recognized because the mutation induced peptide anchoring to the HLA-A*0101, we compared the binding of the 55–64^wt and the 55–64^Q61R ras peptides to HLA-A*0101. Fig. 6 shows that both peptides induced a similar stabilization of HLA-A*0101 molecules on the BM36-1 cells. This was at a lower level and for a shorter period of time than the MAGE-3 peptide used as the positive control. Therefore, the 55–64^wt ras peptide is naturally an aggretope.

View larger version (47K): [in this window] [in a new window]   FIGURE 6. HLA-A*0101 binding of ras peptides. The TAP-deficient cell line BM36-1 was pulsed overnight with ras peptides (55–64wt (ILDTAGQEEY), 55–64Q61R (ILDTAGREEY), the 52–61Q61R (LLDILDTAGR)) and with the MAGE-3 peptide 168–176 (EVDPIGHLY), as a positive control. One hour after adding BFA, cells were washed and stained with the anti-MHC I mAb W632 (ATCC) at 0 or 30 min after removal of the peptides. MHC I expression was analyzed by flow cytometry. The gray histograms show MHC I expression by BM36.1 cells in the absence of added peptide. *, Fluorescence geometrical mean.

Three HLA-A*0101 melanoma cell lines expressing the Q61R ras mutation and three HLA-A*0101 lines that did not were compared for their capacity to stimulate the specific TIL clones. Both clones recognized the mutated cell lines but not the nonmutated ones (Fig. 7A). Furthermore, transfection of HLA-A*0101 into three non-HLA-A*0101 melanoma cell lines that expressed the Q61R ras mutation induced the recognition of two of these cells (Fig. 7B).

View larger version (35K): [in this window] [in a new window]   FIGURE 7. The Q61R ras epitope is efficiently presented on HLA-A*0101 melanoma cells that express the Q61R ras mutation. TIL clones M6ras5 and 24 were incubated for 6 h at 37°C with HLA-A*0101 melanoma cell lines expressing (M6, M90, and Mel4) or not expressing (M171 and FM28Z) the Q61R ras mutation (A) or with non-HLA-A*0101 melanoma cell lines expressing the Q61R ras mutation, transfected or not with the HLA-A*0101 DNA (B). Transfection was done using LipofectAMINE reagent. The amount of TNF secreted was determined using a biological assay with Wehi cells.

The antigenicity of the 55–64^Q61R peptide and of its wt analog was uncertain and was examined by testing the capacity of these peptides to induce the growth of specific CTL through in vitro stimulation of PBMC by peptide-loaded DC. Thirty million CD8⁺ PBMC from a HLA-A*0101 donor were stimulated three times a week by mature DC pulsed with 5 µg/ml amounts of the 55–64^Q61R ras peptide or of its wt analog. Two of five culture wells that were stimulated by the mutated peptide but none of those stimulated by the wt peptide developed peptide-specific CTL, as shown by intracellular cytokine labeling (0.3 and 0.5% reactive cells; data not shown). Cloning of one positive culture yielded several T cell clones (at least one CTL clone) that recognized BM36-1 cells pulsed with the mutated peptide. These clones poorly killed the same cell line pulsed with the wt peptide analog (data not shown). Using the COS transfection system, we showed that this clone had a strong preference for the Q61R ras mutation, although it did recognize the wt and the Q61K mutated ras at low levels (Fig. 8A). Among a large panel of HLA-A*0101 melanoma cell lines, only those that expressed the Q61R mutation stimulated a clone response (Fig. 8B).

View larger version (30K): [in this window] [in a new window]   FIGURE 8. CTL clones derived from HLA-A*0101 PBL stimulated in vitro by autologous DC pulsed with the 55–64Q61R peptide are specific for this epitope. TNF response of one of these clones to COS cells cotransfected by the HLA-A*0101 and various ras DNA (A) HLA-A*0101 melanoma cell lines that express (M6, M90, Mel4) or do not express (others) the Q61R ras mutation (B).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The mutated arginine, being at position 7 of this peptide, was not expected to provide an anchoring capacity to this peptide sequence. This was confirmed by showing that the homologous wt peptide had a binding affinity to HLA-A*0101 similar to that of the arginine-mutated peptide. However, even high concentrations of the wt peptide loaded on HLA-A*0101 TAP-deficient cells failed to induce a significant response of TIL clones. Therefore, the arginine mutation creates, on a wt aggretope, a neoepitope that was recognized specifically by TIL. This suggests that other mutations occurring at the same position might create additional HLA-A*0101-restricted T cell epitopes.

Despite a capacity of binding to HLA-A*0101 similar to that of the 55–64^Q61R peptide, the 55–64^wt peptide loaded on DC did not induce the growth of specific CTL among PBL. This suggests that the repertoire specific for endogenously processed wt ras peptides has been deleted as expected for a potentially autoreactive repertoire. Nonetheless, the deletion of this repertoire seems to be incomplete, as far as in vivo immunization of cancer patients with long ras peptides (14 aa) mutated at residue 12 induced the development of CTL specific for wt ras in some patients (23).

This study is the first demonstration of a spontaneous in vivo expansion of CTL specific for a mutated ras epitope inside a tumor that efficiently expressed this epitope. Another study showed mutated ras-specific CTL in the blood of a colon carcinoma patient (24). However, the autologous tumor lacked the mutation, and the target epitope failed to be identified although it was shown to be endogenously processed by a colon carcinoma line upon IFN- treatment (25). Several ras epitopes mutated at position 12, 13, or 61 had been previously designed on the basis of their anchoring capacity to given HLA class I alleles (10, 13, 26). Peptides selected in this way could possibly stimulate the growth of specific CTL from PBL in vitro. However, those CTL poorly recognized mutated tumor cells, thus suggesting that endogenous expression of these peptides was limited (10, 12, 13). In contrast, the arginine-mutated peptide identified here as a TIL target was efficiently presented by melanoma cell lines. Furthermore, DC loaded with this peptide efficiently stimulated specific CTL from a HLA-A*0101 healthy donor’s PBL. These CTL recognized all the HLA-A*0101 melanoma cell lines expressing the Q61R ras, and exclusively those cell lines, as observed before for the TIL clones. Consequently, the 55–64^Q61R ras peptide is efficiently processed and presented by mutated HLA-A*0101 tumor cells and is sufficiently immunogenic to stimulate specific TIL expansion in vivo and PBL-derived CTL expansion in vitro. This peptide is therefore a good candidate to immunize HLA-A*0101 melanoma patients bearing an arginine mutation at residue 61 of a ras gene in their tumor. Patients who may benefit from a vaccine based on this precise mutation are those sharing both the HLA-A*0101 allotype and the arginine to glutamine substitution. This represents only a small fraction of melanoma patients (i.e., 1.2%), however, because the same mutated peptide also binds to HLA-B*1501 patients expressing this allotype and the Q61R mutation might also be candidates for this vaccine. Furthermore, as stated above, because the ras mutation described creates an epitope on a preexisting aggretope, it is likely that other ras mutations should also give rise to additional epitopes efficiently expressed by melanoma lesions. In this case, a vaccine encompassing these different ras mutations should be useful to treat 20% of patients expressing HLA-A*0101 and HLA-B*1501 allotypes. Moreover, it could also be used as a target for immunotherapeutic treatment of other tumors that express the Q61R mutation at residue 61 of K-, H-, or N-ras because the three ras proteins share a 100% identical 86-aa N-terminal region (27). ras mutations affecting codon 61 have been detected with a high frequency in congenital melanocytic nevi (28), multiple myelomas (16), and thyroid tumors (29).

A number of tumor CTL epitopes derived from cancer/testis or differentiation tumor Ags are currently being used as targets in immunotherapy trials (30). However, because mutated epitopes (efficiently expressed by a significant fraction of tumors) were not available, immunization strategies against oncoproteins could not be checked. Nonetheless, after immunization of pancreatic cancer patients with mutant ras peptides 14 aa long, both CD4 and CD8 T cell proliferations specific for mutated ras developed in some patients (11, 12). This showed that infusion of peptides encompassing CD8⁺ and CD4⁺ T cell epitopes might permit immunization against both peptides. As helper CD4⁺ T cells are likely essential for enhancing the CTL response in vivo (31), this approach, which may ensure a more adequate immune response, may be an attractive option. Nonetheless, a recent study showed that active immunization with a MHC class II-restricted ras mutated peptides might be risky. Indeed, enhancement of tumor growth instead of protection resulted from immunization with a HLA class II-restricted mutated ras peptide in mice (32). Although some immunization procedures using viral MHC class I-restricted peptides have been reported to be tolerogenic (33), to our knowledge, no tumor-promoting effect has been reported using MHC class I-restricted mutated peptides alone. Furthermore, immunizations targeting MHC class I-restricted mutated tumor peptides in animal cancer models have been reported to induce tumor rejection (34, 35, 36, 37, 38). These results from animal cancer models and recent observations in humans supporting a correlation between the presence of CTL specific for unique tumor mutations and good survival rate (4, 5) suggest that HLA class I-restricted mutated epitopes might be the best Ags to target. In this respect, oncogenic mutations may be especially good target Ags, as far as CTL killing of tumor cells expressing the oncogene would lead to a direct tumor growth disadvantage, even if Ag loss variant tumor cells are not eradicated. Furthermore, mutated peptides are not likely to cause side effects. To test this theory, it is critical now to identify shared mutated epitopes efficiently presented by human tumors. The approach described here to reach this goal appears efficient.

	Acknowledgments

 
We thank A. Ziegler (Universitatsklinikum Charite, Berlin, Germany) for the gift of the BM36-1 cell line.

	Footnotes

 
¹ This work was supported by grants from the Ligue Nationale Contre le Cancer (France), Axe Immunologie des Tumeurs, and the Association pour la Recherche Contre le Cancer (France).

² Address correspondence and reprint requests to Dr. Francine Jotereau, Institut de Biologie, Institut National de la Santé et de la Recherche Médicale, Unité 463, 9 Quai Moncousu, 44093 Nantes Cédex 1, France. E-mail address: blinard{at}nantes.inserm.fr

³ Abbreviations used in this paper: TIL, tumor-infiltrating lymphocyte; MASA, mutant-allele-specific amplification; wt, wild type; BFA, brefeldin A; DC, dendritic cell.

Received for publication August 29, 2001. Accepted for publication February 26, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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