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Isolation of a New Melanoma Antigen, MART-2, Containing a Mutated Epitope Recognized by Autologous Tumor-Infiltrating T Lymphocytes¹

Yutaka Kawakami^2,*, Xiang Wang, Tomoko Shofuda^*, Hidetoshi Sumimoto^*, Janis P. Tupesis, Ellen Fitzgerald and Steven A. Rosenberg

^* Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo, Japan; and Surgery Branch, Division of Clinical Sciences, National Cancer Institutes, National Institutes of Health, Bethesda, MD 20892

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Using cDNA expression cloning, a cDNA encoding a novel human melanoma Ag, MART-2 (melanoma Ag recognized by T cells-2), recognized by HLA-A1-restricted CD8⁺ T cells from tumor-infiltrating lymphocytes (TIL1362) was isolated from an autologous melanoma cell line, 1362 mel. Homologous sequences to the cDNA had been registered in the EST database. This gene encoded an uncharacterized protein expressed ubiquitously in most normal and cancer cells. A mutation (A to G transition) was found in the cDNA obtained from the1362 mel melanoma cell line in the sequences encoding the phosphate binding loop (P-loop) that resulted in loss of the ability to bind GTP. Transfection of NIH-3T3 with the mutated MART-2 did not result in the development of significant foci. By screening 36 various cancer cell lines using single-strand conformation polymorphism, a possible mutation in the P-loop of MART-2 was found in one squamous cell lung cancer cell line, EBC1. The T cell epitope for TIL1362, FLEGNEVGKTY, was identified to be encoded by the mutated sequence of the MART-2 Ag. The mutation substituted glycine in the normal peptide with glutamic acid at the third amino acid of the epitope, which is an important primary anchor amino acid for HLA-A1 peptide binding. The normal peptide, FLGGNEVGKTY, was not recognized by TIL1362, suggesting that this T cell response was specific for the autologous tumor. Although transforming activity was not detected in the NIH-3T3 assay, MART-2 with the mutation in the P-loop may be involved in the generation of melanoma through a loss of GTP binding activity.

	Introduction

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Melanoma is known to be a relatively immunogenic cancer that can respond to IL-2 administration (1). The antitumor reactivity of T cells plays an important role in the in vivo regression of melanoma after immunotherapy (2). In the past several years, many human melanoma Ags recognized by T cells have been identified using various methods, including DNA cloning, peptide isolation, and T cell induction against candidate proteins. These Ags can be classified as melanocyte-specific proteins, including tyrosinase, melanoma Ag recognized by T cells (MART-1)³/Melan A, gp100, tyrosinase-related protein (TRP-1), and TRP2; cancer-testis Ags that are expressed in various cancers and normal testis, including MAGE, BAGE, GAGE, and NY-ESO-I; mutated peptides encoded by genetic alterations in tumor cells including -catenin and cyclin-dependent kinase 4 (CDK4); and others, such as p15, GnT-V, and PRAME (3). Because T cells possibly recognizing unknown Ags have been found, isolation of additional melanoma Ags is important for further analysis of anti-melanoma immune responses as well as for the isolation of Ags with better tumor rejection ability.

We have previously isolated a mutated -catenin peptide recognized by CD8⁺ T cells (4) and found that the mutated -catenin was probably involved in the tumorigenesis of melanoma through abnormalities in the Wnt signaling pathway (5). Similarly, association of the mutated CDK4 Ag with the generation of melanoma was suggested through its inability to bind to the CDK4 inhibitor, p16^INK4a (6). Therefore, many mutated Ags recognized by tumor-specific CD8⁺ T cells appear to be important molecules in the generation of tumors, and the analysis of such mutated Ags recognized by CD8⁺ T cells is important not only for the understanding of immune responses to human tumor cells and the development of immunotherapy, but also for the understanding of tumorigenesis.

In the present study we isolated a cDNA encoding a novel melanoma Ag we have called MART-2, which is recognized by CD8⁺ T cells derived from tumor-infiltrating lymphocytes (TIL). The T cell epitope was identified to be a mutated peptide capable of binding to HLA-A1 Ag and specifically recognized by autologous TIL. The mutation resided in the phosphate binding loop (P-loop) of the MART-2 Ag, and the mutated MART-2 lost GTP binding activity, suggesting possible involvement in 1362 melanoma development.

	Materials and Methods

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Generation of melanoma reactive CTL from TIL of a patient with metastatic melanoma

TIL1362 was expanded from TIL of a metastasis on the right flank of a 43-year-old woman. TIL were grown in RPMI 1640 medium containing 6000 IU/ml of IL-2 (Cetus-Oncology Division, Chiron, Emeryville, CA) for 30 days as previously described (7). The patient received treatment with chemotherapy (dacarbazine, cisplatinum, and tamoxifen) plus TIL, IL-2, and IFN- for multiple metastases in the breast and lymph nodes. After the treatment, partial regression of six metastases occurred, but a breast metastasis continued to grow.

Construction of a cDNA library from 1362 mel melanoma cell line and screening using TIL1362

A cDNA library was constructed from the 1362 mel melanoma cell line in the VR1012 vector that was provided by Chiron/Viagene (San Diego, CA) as previously described (8). Briefly, the multiple cloning site was modified in this vector to contain EcoRI and HindIII restriction sites following destruction of a HindIII site in the vector backbone conducted using site-directed mutagenesis. Poly(A)⁺ mRNA was isolated from the 1363 mel melanoma cell line using an oligo(dT) column, and a cDNA library was prepared using directional random primers (Novagen, Madison, WI). Briefly, random primers containing two T residues at the 5' end were used for RT mRNA. Following ligation of the oligomer 5'-GCTTGAATTCAAGC-3' to the cDNA, DNA was digested with HindIII, resulting in clones containing an EcoRI site at the 5' end and a HindIII site at the 3' end. These cDNAs were ligated in the modified VR1012 plasmid that was digested with EcoRI and HindIII.

cDNA pools containing 100 bacteria/pool were grown overnight, and plasmid DNAs were isolated using the Wizard 9600 DNA purification system (Promega, Madison, WI). Pools of cDNAs (100–200 ng) were transfected into COS cells with HLA cDNA (100 ng) using Lipofectamine (Life Technologies, Gaithersburg, MD). Fifty thousand TIL1362 were mixed with 5 x 10⁴ COS cells transfected with a pool of the cDNA library for about 20 h, supernatants were harvested, and IFN- was measured using ELISA as previously reported (9). Bacteria were transformed with cDNA pools from positive wells in the first screening, and plasmids containing a single cDNA were isolated and used for the second screening with the CTLs. After the second or third screening, cDNAs encoding melanoma Ags were sequenced using an ABI Prism 310 genetic analyzer with dye terminator cycle sequencing kits (Perkin-Elmer, Foster City, CA). Sequences were compared with the GenBank database using the BLAST algorithm.

5'- and 3'-rapid amplification of cDNA ends (RACE) and cloning of the full-length cDNA

The RACE protocol was performed using the Marathon cDNA Amplification Kit (Clontech, Palo Alto, CA), according to the manufacturer’s instructions. One microgram of poly(A)⁺ RNA from the SKmel23 melanoma cell line was reverse transcribed using a modified locking oligo(dT) primer containing two degenerate nucleotide sequences at its 3' end and avian myeloblastosis virus reverse. Second-strand synthesis was accomplished with a mixture of Escherichia coli DNA polymerase I, RNase H, and DNA ligase. Double-stranded cDNA was blunt-ended with T4 DNA polymerase and ligated to the Marathon cDNA adaptor 1 (5'-CTA ATA CGA CTC ACT ATA GGG CTC GAG CGG CCG CCC GGG CAG GT-3') using T4 DNA ligase. The anchor-ligated cDNAs were then subjected to PCR with the adaptor primer 1 (5'-CCA TCC TAA TAC GAC TCA CTA TAG GGC-3') and the clone 8B6-specific primer (5'-GTC GTA GCC GCC ATG CCA GTA GCT CAC A-3') complementary to nucleotides +1137 to +1164 for 5'-RACE or with the adaptor primer 1 and the 8B6-specific primer (5'-CGC CTT CTT TGC TGG TGG TGG CTG GC-3') complementary to nucleotides +763 to +788 for 3'-RACE. The PCR condition was as follows: 94°C for 30 min, followed by 94°C for 5 s, 68°C for 4 min for five cycles, 94°C for 5 s, 70°C for 4 min for five cycles, 94°C for 5 s, 72°C for 4 min for 25 cycles, and a final step of 72°C for 4 min for one cycle. Five microliters of the amplified products were electrophoresed in a 1% agarose gel. The remaining products were diluted 1:50 in Tricine-EDTA buffer and subjected to the second round of PCR described above using 20 cycles (instead of 25 cycles) with the nested adaptor primer 2 (5'-ACT CAC TAT AGG GCT CGA GCG GC-3') and the nested 8B6-specific primer (5'-GTA AGA AAT GCA GAA ACA CAG GAA GGC C-3') complementary to nucleotides +1459 to +1486 for 5'-RACE or with the nested adaptor primer and the nested 8B6-specific primer (5'-TGG CCC GAT ACT TCT CCC CAC AA-3) complementary to nucleotides +1259 to +1281 for 3' RACE. The final amplified products were subcloned into pGEM-T (Promega).

Detection of the mutation by PCR-restriction fragment length polymorphism (PCR-RFLP)

To detect the mutation of clone 8B6 by RFLP, we designed a forward primer (5'-TGG CCC GAT ACT TCT CCC CAC AA-3') complementary to nucleotides +1259 to +1281 and a reverse primer (5'-CAG TAG GTT TTC CCA ACC TCA TGG GCC-3') complementary to nucleotides +1359 to +1385, which amplify a 127-bp PCR product. The reverse primer was designed to mismatch to the wild-type 8B6 sequence by changing G to C at position 1361 and A to C at position 1363 to create an ApaI site, GGGCCC, in the PCR product from the wild-type 8B6 sequence (G allele). The PCR product from the wild-type sequence was digested by ApaI to two 104- and 23-bp fragments. We also designed a forward primer (5'-TCC TGT CCA ACC TGG TAT TTC TAG-3') complementary to nucleotides +1334 to +1357 and a reverse primer (5'-CCT TGT ATG AAG ATC CTA TTC CAG-3') complementary to nucleotides +1383 to +1406. The forward primer was designed to create a XbaI site, TCTAGA, by changing T to A at position 1356. A 73-bp PCR product from the mutated 8B6 sequence (A allele) was cleaved by XbaI into two 20- and 53-bp fragments. PCR products from 50 ng of genomic DNA were digested by ApaI or XbaI (New England Biolabs, Beverly, MA) for 1 h at 37°C. The mutation was detected by electrophoresis of the digested PCR products on a 3% agarose gel (AmpliSize Agarose; Bio-Rad, Hercules, CA).

Fluorescence-based single-strand conformation polymorphism (SSCP) analysis

PCR was conducted using fluorescent dye 5'-end-labeled primers customized by PE Biosystems (10). The forward strand was labeled using blue fluorescent dye FAM-labeled forward primer (5'-TGG CCC GAT ACT TCT CCC CAC AA-3') complementary to nucleotides +1259 to +1281, and the reverse strand was labeled using green fluorescent dye HEX-labeled reverse primer (5'-CCT TGT ATG AAG ATC CTA TTC CAG TAG G-3') complementary to nucleotides +1379 to +1406. PCR amplification and SSCP analysis were performed on various cell lines. The 148-bp PCR products were purified using Microcon YM-100 columns (Millipore, Bedford, MA) to remove excess primer. One microliter of the PCR products diluted to an appropriate concentration was added to loading buffer (10.5 ml deionized formamide/0.5 ml GeneScan internal Lane Size Standard (GeneScan-500; PE Applied Biosystems)/0.5 ml of 0.3 N NaOH). The mixture was heated to 95°C for 5 min followed by snap-cooling on ice. Fluorescence-based SSCP was performed by the ABI PRISM 310 Genetic Analyzer. Electrophoresis was carried in a 41-cm, 50-mm internal diameter capillary filled with 3% GeneScan Polymer in 1x Tris-borate-EDTA electrophoresis buffer containing 10% glycerol at 317 V/cm at 30°C and analyzed using GeneScan software.

Identification of the epitope for TIL1362

The pcDNA3 plasmid containing truncated variants of the cDNA encoding the 8B6 Ag was generated by PCR as previously described (11). The region containing the epitope was determined by testing recognition by TIL1362 of COS cells transfected with the various cDNA fragments and HLA-A1 cDNAs. Candidate peptides in the regions containing the epitope were synthesized on the AMS 222 multiple peptide synthesizer (Gilson, Worthington, OH) using standard F-moc chemistry and were further purified on a R2 reverse HPLC column (PerSeptive Biosystems, Framingham, MA) as well as a C₈ column (Vydac, Hesperia, CA; >98% purity) using an acetonitrile gradient in water with 0.05% trifluoroacetic acid. The m.w. of the peptides were verified by mass spectrometry. To assess peptide recognition by T cells, an IFN- release assay was performed using peptide-pulsed indicator cells, HLA-A1-transfected COS cells, as previously described (8).

[³⁵S]GTPS binding assay

[³⁵S]GTPS-binding was performed as previously described (12). Briefly, COS-7 cells plated at a density of 1 x 10⁶ in 10-cm dishes were transfected with 10 µg of vector, pcDNA3.1(-), and wild-type or mutant 8B6 in the pcDNA3.1(-). Transfected COS cells were resuspended in 1 ml of whole cell extract buffer (50 mM HEPES-KOH (pH 7.8), 420 mM KCl, 0.1 mM EDTA (pH 8.00), and 5 mM MgCl₂) containing 1 mM DTT and the protease inhibitor mixture Complete (Roche Molecular Biochemicals, Indianapolis, IN) and lysed by 10 times passage through a 25-gauge needle. Cellular debris was removed by centrifugation. Thirty micrograms of whole cell extracts were incubated for 2 h at 30°C in 40 µl of the reaction mixture containing 20 mM HEPES-KOH (pH 7.8), 1 mM EDTA, 5 mM MgCl₂, 2.5 mM L--dimyristoyl phosphatidylcholine, 0.3% 3-[(3-cholamidopropyl)dimethylammonio]propane sulfonic acid, and 1 mM [³⁵S]GTPS. The reaction was stopped by addition of 2 ml of iced-cold stop buffer (20 mM Tris-HCl, pH 8.0) containing 25 mM MgCl₂ and 100 mM NaCl), followed by rapid filtration on nitrocellulose filters. Filters were washed five times with stop buffer. After the filters dissolved in 10 ml of toluene scintillator, the radioactivity was counted.

NIH-3T3 transforming assay

Ten micrograms of plasmid DNA containing MART-2 cDNA was mixed with 65 µg of salmon sperm DNA and used to transfect 5 x 10⁵ NIH-3T3 cells in 10-cm tissue culture dishes using the calcium phosphate precipitation method as previously described (13). The cells were detached by trypsinization 24 h after transfection, split 1:3, and maintained in 5% FCS/DMEM medium. Focus formation was scored 3 wk after the transfection.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Recognition of novel shared melanoma Ags by TIL1362 in the context of HLA-A1

TIL1362 recognized most HLA-A1⁺ allogeneic melanoma cell lines tested, but did not recognize HLA-A1⁺ EBV-B cells, including 888EBV-B cells, 1088EBV-B cells, and 1359EBV-B cells; the HLA-A1-transfected 293 human embryonic kidney epithelial cell line; or the HLA-A1-transfected COS monkey kidney cell line, suggesting that TIL1362 recognized shared melanoma Ags presented by HLA-A1 Ag (Tables I and II) (14). This response was blocked by anti-HLA class I Ab, W6/32, suggesting MHC class I-restricted recognition of melanoma cells (data not shown). Further analysis was conducted by analyzing the ability of TIL1362 to recognize a melanoma cell line that had been stably transfected with HLA-A1 cDNA. TIL1362 recognized HLA-A1-transfected 526 mel, but did not recognize the parental HLA-A1-negative melanoma cell lines, confirming that the TIL recognized shared melanoma Ags in an HLA-A1-restricted manner (Table I).

Isolation of cDNA encoding the melanoma Ag recognized by TIL1362

A cDNA library made from the autologous melanoma cell line 1362 mel was screened with TIL1362 by transfecting COS cells with cDNA pools (each containing 100 cDNAs) and HLA-A1 cDNA, and measuring IFN- release from TIL1362 when TIL and transduced COS cells were mixed. After screening 800 cDNA pools (8 x 10⁴ cDNAs), four strong positives (7F12, 8A12, 8B6, 8E12) and four weak positives (7A4, 7A11, 7E8, 8A6) were identified. Eighty colonies derived from each positive cDNA pool were rescreened with TIL1362, and three positives from the pool 8B6 and five positives from the pool 7F12 were identified. After the third screening of these eight positives, 8B6 and 7F12 were found to contain the same 1552-kb cDNA, and this cDNA fragment (8B6) was confirmed to encode the melanoma Ag recognized by TIL1362. TIL1362 secreted high amounts of IFN- when stimulated with COS cells transfected with both the 8B6 fragment and HLA-A1 cDNA (Table II).

Structure of the isolated melanoma Ag

To isolate the full-length cDNA for the 8B6 Ag, 5'- and 3'-RACE was performed using mRNA obtained from the 1362 mel autologous melanoma cell line and the SKmel23 allogeneic melanoma cell line. From 1362 mel, a 3587-bp cDNA was isolated and sequenced as shown in Fig. 1. The presence of a Kozack sequence and a stop codon in the 5'-flanking sequence of the indicated ATG suggested that this cDNA encoded a protein consisting of 453 aa. The protein encoded by this gene was named MART-2, because this is the second T cell-defined melanoma Ag encoded by a novel gene isolated in our group. From SKmel23, a 3728-bp cDNA with an additional 195-bp insertion was isolated (Fig. 2). This insertion added 65 aa without a frame shift. Database analysis revealed a homologous cDNA (AK001586) that was isolated from the NT2 teratocarcinoma as well as genomic DNAs (AL035414, AL034351) derived from chromosome 1q32. Comparison between the two isolated cDNAs and the cDNA and genomic DNAs in GenBank revealed that the 195-bp insertion was generated by alternative splicing (Fig. 2A). The difference in the protein structure among three identified MART-2 sequences is shown in Fig. 2B. By PCR analysis with the primers flanking the 195-bp insertion, the presence of these different cDNAs was examined, and bothcDNAs, with or without this insertion, were found in 1362 mel, SKmel23, and primary cultured melanocytes (data not shown).

View larger version (50K): [in this window] [in a new window]   FIGURE 1. Nucleotide and amino acid sequence of the MART-2 Ag isolated from the 1362 mel melanoma cell line. The T cell epitope is underlined.

View larger version (41K): [in this window] [in a new window]   FIGURE 2. Structure of genes and proteins of the MART-2 Ag. A, The MART-2 mRNA isolated from 1362 mel, SKmel23 melanoma cell lines, and NT2 teratocarcinoma were generated by alternative splicing from the MART-2 gene on chromosome 1q32. B, The MART-2 proteins isolated from SKmel23 and NT2 contain additional polypeptides, which are underlined. Structure of the C-terminal ends was also different among the isolated proteins. a, 1362 mel; b, SKmel23; c, NT2.

Expression of this gene was examined by RT-PCR and Northern blot analysis. It was expressed in normal tissues, including brain, lung, heart, spleen, liver, small intestine, colon, kidney, muscle, placenta, stomach, and testis; in noncancer cell lines, including melanocytes, T cells, and fibroblasts; as well as in cancer cell lines, including melanoma (SKmel23, 888 mel, A375, 1362 mel, 1363 mel, 928 mel, 624 mel, 586 mel, 526 mel, 501 mel, 397 mel), brain tumors (U87-MO, T98G), esophageal cancer (TE8, TE10), lung cancer (K1S, LU99, LK2, EBC1, SBC2, RERF-LCMA), bladder cancer (KU7, BC47), renal cell cancer (RCC6, RCC7, RCC8), pancreatic cancer (PK1, PK59), prostate cancer (JCA1, PC3), breast cancer (HS578, MDA231), hepatoma (HepG2), and leukemia and lymphoma (HL60, K562, Molt4, Daudi), suggesting the ubiquitous expression of the MART-2 Ag (data not shown).

A mutation in the P-loop of MART-2

The MART-2 cDNA from SKmel23 did not contain a leader sequence or transmembrane domain, but contained a motif for a P-loop (GXXXXGKT) that may bind ATP or GTP (15). However, the cDNA isolated from 1362 mel using TIL1362 did not contain the P-loop, because glycine was substituted with glutamic acid by a single base difference. To distinguish whether this difference was due to polymorphism or mutation in tumor cells, the presence of this sequence in T cells from the same patient as well as melanoma cell lines from other patients was evaluated by RFLP analysis of the PCR product from genomic DNAs using the primers designed to distinguish between these sequence differences. XbaI, which digests the 1362 mel sequence, digested half the PCR product from 1362 mel, but did not digest the PCR products from 1363 TIL as well as other melanoma cell lines (Fig. 3). ApaI, which digests the SKmel23 sequence, digested the PCR products from all cell lines with the exception of 1362 mel. These results indicated that the sequence identified in the 1362 mel contained the mutation that occurred in one allele of the genomic DNA of the 1362 mel melanoma cells.

View larger version (34K): [in this window] [in a new window]   FIGURE 3. Detection of the mutation in MART-2 by RFLP analysis. XbaI that digests the 1362 mel sequence digested half the PCR product from the genomic DNA of 1362 mel, but did not digest the PCR products from TIL1362 or other melanoma cell lines. ApaI that digests the SKmel23 sequence digested the PCR products from all cell lines with the exception of 1362 mel, suggesting that one allele of the genomic DNA of the 1362 mel has a mutation.

The GTP binding ability of the MART-2 proteins encoded by these cDNAs with or without the mutation was examined. Using an [³⁵S]GTPS binding assay, isotope-labeled GTP bound to cell lysates obtained from COS cells transfected with the mutant MART-2 cDNA similarly to that from COS cells transfected with the control pcDNA3.1. However, GTP bound much higher to the lysates from COS cells transfected with the wild-type MART-2 cDNA obtained from SKmel23, suggesting that loss of the P-loop motif led to loss of GTP binding activity of the MART-2 protein (Fig. 4).

View larger version (12K): [in this window] [in a new window]   FIGURE 4. Loss of GTP binding activity of the mutated MART-2 Ag. An [35S]GTPS binding assay was performed as described in Materials and Methods. [35S]GTPS was bound in lesser amounts to the lysates from the COS cells transfected with the mutant MART-2 (8B6) cDNA than to the lysates from COS cells transfected with the wild-type MART-2 cDNA, suggesting loss of GTP binding activity of the mutated MART-2 protein.

Identification of the epitope recognized by TIL1362

To identify the T cell epitope in the MART-2 Ag, the region containing the epitope was first located by testing TIL1362 recognition of COS cells transfected with various sizes of truncated cDNA fragments generated by PCR. TIL1362 recognized COS cells transfected with T7 1–1407(1–1407) and T8 1–2226(1–2226) fragments as well as the originally isolated H10 fragment 1–1552(1–1552), but recognized none of T1 1–307(1–307) to T6 1–1341(1–1341) fragments. Therefore, TIL1362 appeared to recognize the epitope around the 3' end of the T6 and T7 fragments (Table IV).

View larger version (29K): [in this window] [in a new window]   FIGURE 5. Recognition of the mutated peptide by TIL1362. A, Various sizes of the candidate peptides for the TIL1362 epitope were synthesized around the peptide FLEGNEVGKY that contained the typical HLA-A1 binding motif. TIL recognition of COS cells transfected with HLA-A1 cDNA and pulsed with a series of diluted peptides was evaluated by an IFN- release assay. The 11-mer peptide was best recognized at all concentrations. B, TIL1362 recognized COS cells transfected with HLA-A1 and pulsed with the mutated MART-2 peptide, but not the wild-type peptide, suggesting an autologous tumor cell-specific T cell response of TIL1362.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In the present study we have isolated cDNA encoding a novel melanoma Ag, MART-2, recognized by autologous tumor-specific CD8⁺ T cells derived from TIL. This cDNA contained a mutation, A to G, that occurred in 1362 mel melanoma cells. The 11-mer T cell epitope FLEGNEVGKTY was found to contain a glutamic acid encoded by this mutated sequence at the third position. Because negatively charged amino acid at the third position and tyrosine at the C terminus is a typical peptide binding motif for HLA-A1 Ag (18), this mutation created the HLA-A1 binding epitope capable of inducing tumor-specific T cells. TIL1362 did not recognize the corresponding normal peptide with a glycine at the third position, suggesting that this T cell response is autologous tumor specific. Because TIL1362 contained HLA-A1-restricted, shared melanoma Ag-specific T cell, the isolation of the common Ag is in progress. We have previously found TIL containing T cell clones specific for various tumor Ags, including mutated unique and common Ags. For example, TIL888 contained T cells specific for tyrosinase, gp100, p15, and the mutated -catenin (4, 20, 21, 22). TIL586 recognized TRP1, TRP2, and NY-ESO-I (23, 24, 25).

The mutation in the MART-2 Ag was found to reside in the motif of the P-loop, and the wild-type MART-2 has been shown to have GTP binding activity (Fig. 4). This motif consists of a glycine-rich sequence followed by a conserved lysine and a serine or threonine (GXXXXGK(TS)) and was observed in various ATP and GTP binding proteins, including ATP synthase, Ras proteins, myosin heavy chains, adenylate kinase, elongation factors, thymidine kinases, and phosphoglycerate kinases (15). Each protein has a particular submotif, such as G (GAD) X (AG) XGK (ST) XL for Ras proteins. The P-loop of MART-2 appeared to be different from the reported submotifs, suggesting that MART-2 is a new protein with unknown function. The Ras P21 protein binds GTP and GDP and possesses a GTPase activity that can be affected by mutations in the P-loop. Because the Ras P21 proteins with mutations in the P-loop are known to have a transforming activity when transfected in NIH-3T3 cells (16), transforming activity of the mutated MART-2 that lost GTP binding activity was examined using the NIH-3T3 assay, but, was not detected. However, this mutated MART-2 may still be involved in other aspects of melanoma development. In addition to 1362 mel and the reported NT2 teratocarcinoma cell line, a squamous cell lung cancer cell line, EBC1, was found to contain a different amino acid in the P-loop by screening 36 tumor cell lines using SSCP. However, it was difficult to distinguish whether these differences were caused by mutation or polymorphism because of the unavailability of normal cells from the same patients. Thus, mutation in the P-loop of MART-2 may not frequently occur, although SSCP analysis may have a limited sensitivity for detection of these mutations.

The previously identified mutated peptides, including -catenin and CDK4, were isolated using T cells from patients with a good prognosis after treatment, suggesting that T cell responses to these mutated peptides might be involved in the tumor regression (4, 6). The patient in this study was treated with adoptive transfer of TIL1362 combined with IL-2, IFN-, and chemotherapy using cisplatin, dacarbazine, and tamoxifen. Although some metastases regressed after the treatment, it is difficult to conclude whether the T cells were involved in this regression. Tumor-specific mutated Ags may be ideal targets for immunotherapy. Mutated peptides appear to be potent rejection Ags in murine tumor models (26). Ag loss variants may not develop if mutated molecules are important for tumor growth. However, it may be difficult to apply unique mutated peptides for immunotherapy unless mutations are common, or techniques for rapid identification of mutated tumor Ags or efficient immunotherapy that does not require Ag identification can be developed.

In summary, we have isolated a novel melanoma Ag, MART-2, recognized by autologous tumor-specific CD8⁺ T cells derived from TIL. The T cell epitope was found to be a mutated peptide derived from the mutated sequence in the P-loop of MART-2. The mutated MART-2 lost binding activity to GTP and may have been involved in the tumorigenesis of this patient’s melanoma.

	Footnotes

 
¹ This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sport, and Culture of Japan (10557083, 9255106, and 12217132), a Grant-in-Aid for Cancer Research (11-7) from the Ministry of Health and Welfare, the Ciba-Geigy Foundation (Japan) for the Promotion of Science, the Vehicle Racing Commemorative Foundation, the Naito Foundation, the Keio University Medical Science Fund, and Keio University Special Grant-in-Aid for Innovative Collaborative Research Projects, Keio Gijuku Academic Development Funds.

² Address correspondence and reprint requests to Dr. Yutaka Kawakami, Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, 160-8582 Tokyo, Japan.

³ Abbreviations used in this paper: MART, melanoma Ag recognized by T cells; TIL, tumor-infiltrating lymphocytes; SSCP, single-strand conformation polymorphism;P-loop, phosphate binding loop; RACE, rapid amplification of cDNA ends; RFLP, restriction fragment length polymorphism; CDK4, cyclin-dependent kinase 4; TRP, tyrosinase-related protein.

Received for publication June 19, 2000. Accepted for publication November 28, 2000.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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