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[Cancer Research 63, 6506-6515, October 1, 2003]
© 2003 American Association for Cancer Research

Immunology

The Alternative Open Reading Frame of LAGE-1 Gives Rise to Multiple Promiscuous HLA-DR-restricted Epitopes Recognized by T-helper 1-type Tumor-reactive CD4+ T Cells1

Maja Mandic, Christine Almunia, Stephanie Vicel, Daniel Gillet, Bratislav Janjic, Kara Coval, Bernard Maillere, John M. Kirkwood and Hassane M. Zarour2

Departments of Medicine and Melanoma Center [M. M., B. J., K. C., J. M. K., H. M. Z.] and Immunology [H. M. Z.], University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, and Protein Engineering and Research Department, CEA-Saclay, 91191 Gif-sur-Yvette, France [C. A., S. V., D. G., B. M.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The NY-ESO-1 and LAGE-1 genes are expressed by many human cancers, but not by normal tissues, with the exception of testis and placenta. The NY-ESO-1 and LAGE-1 genes give rise to multiple MHC class I and class II-presented epitopes derived from the open reading frames (ORF) 1 and 2. Here, we have investigated whether NY-ESO-1/LAGE-1 ORF2 encodes promiscuous MHC class II-restricted epitopes. Using a set of overlapping peptides from the ORF2 protein sequence and autologous dendritic cells (DCs) from normal donors and melanoma patients, we have identified three HLA-DRB1*0401-restricted peptide sequences from the LAGE-1 ORF2 that are capable of stimulating T-helper 1-type melanoma-reactive CD4+ T cells. From these bulk CD4+ T cells, we have generated CD4+ T-cell clones able to recognize not only peptide-pulsed DCs but also autologous DCs loaded with the LAGE-1 ORF2 protein. We have demonstrated that these peptides not only bind to multiple HLA-DR molecules apart from HLA-DRB1*0401 but also stimulate CD4+ T cells when presented in the context of these HLA-DR molecules. Furthermore, our binding data have delineated two additional sequences capable of broadly binding to multiple HLA-DR molecules. Altogether, these data support the immunogenicity of NY-ESO-1/LAGE-1 ORF2 gene products and clearly demonstrate their capability to stimulate T-helper 1 type CD4+ T cells. Because of the role of these cells in promoting long-lasting antitumor CTL responses, our data provide a rationale for cancer vaccine trials with peptides derived from the NY-ESO-1/LAGE-1 ORF2 for a large fraction of patients with NY-ESO-1/LAGE-1+ tumors.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The NY-ESO-1LAGE-2 and LAGE-1NY-ESO-2 genes are expressed by many human tumors but not by normal tissues, except testis and placenta (1) . These antigens belong to the category of tumor-associated antigen alternatively called CT3 (2) , tumor-specific shared (3) , or cancer-germ line antigens (4) . Because of their pattern of expression and because the germ cells in testis do not express MHC molecules (5) , the CT-derived epitopes are specifically expressed by tumor cells in the context of MHC molecules. As a consequence they represent a very interesting category of tumor antigens for use as cancer vaccines, and may be less likely to induce tolerance or autoimmunity.

The LAGE-1NY-ESO-2 gene yields two mRNA transcripts, respectively named LAGE-1 S (or LAGE-1a) and LAGE-1 L (or LAGE-1b). The primary ORF (ORF1) of the genes NY-ESO-1 and LAGE-1 S encode two homologous 180 aa-long proteins, whereas the LAGE-1 L ORF1 encodes a putative 210 aa-long protein. The alternative or nonprimary ORFs (ORF2) of the genes NY-ESO-1 and LAGE-1 encode two putative proteins that are 58 and 109 aa-long, respectively.

The NY-ESO-1/LAGE-1 gene products derive from the ORF1 appear to be very immunogenic, inducing both natural cellular and humoral responses in ~50% of patients with NY-ESO-1+ tumors (6 , 7) . The induction of primary NY-ESO-1-specific CD8+ T-cell responses has been reported after intradermal peptide vaccination in patients with NY-ESO-1+ tumors (8) . The NY-ESO-1 and LAGE-1 genes also give rise to multiple epitopes from the ORF1 presented in the context of MHC class I (2 , 9 , 10) or class II molecules (11, 12, 13, 14) and recognized by T cells. Two MHC class I-derived tumor epitopes encoded by the ORF2 of NY-ESO-1 and LAGE-1 have also been identified (9 , 15) . These two epitopes are localized in the NH2-terminal portion of the putative NY-ESO-1 and LAGE-1 ORF2 proteins, and may possibly be defective ribosomal products as suggested previously (9 , 16) .

Here we report five promiscuous HLA-DR-binding sequences that are encoded by the LAGE-1 ORF2. One of these sequences corresponds to the previously identified HLA-DR11-presented sequence reported recently by Slager et al. (17) . These data add to the multiplicity of the epitopes encoded by the genes NY-ESO-1/LAGE-1that support the strong immunogenicity of NY-ESO-1/LAGE-1 gene products in patients with NY-ESO-1/LAGE-1-expressing tumors.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines, Media, and Antibodies.
Tissues and blood samples used for all of the studies reported in this article were obtained under the UPCI Institutional Review Board-approved protocol 96–99. Patients UPCI-MEL 285 and UPCI-MEL 527 are long-lived patients who remain disease-free several years after successful therapy for disseminated NY-ESO-1-expressing metastatic melanoma. UPCI-MEL 285.1 and UPCI-MEL 527.1 cell lines were derived from one metastatic lesion of each patient. Patients UPCI-MEL 285 and UPCI-MEL 527 have been genotyped as HLA-DRB1*0401+/DRB1*0101+/DRB4*0101+ and HLA-DRB1*0401+/DRB1*1701+/DRB4*0101+, respectively. HLA-DR genotyping of melanoma patients and NDs was performed using a commercial HLA-DR typing panel of PCR primers according to the manufacturer’s instructions (Dynal, Oslo, Norway). ND1 and ND2 have been genotyped as HLA-DRB1*0401+/DRB1*1401+/DRB4*0101+ and HLA-DRB1*0701+/DRB1*1101+/DRB4*0101+, respectively. The T2.DR4 cell line was generated by transfection of HLA-DRB1*0401 cDNA into T2 cells (18) . HLA-DR-transfected mouse cells, i.e. L.DR1, L.DR3, L.DR7, L.DR11, and L.DR53 were a gift of Dr. Robert Karr (Searle Inc., St. Louis, MO). All of the cell lines were cultured in RPMI 1640 (Life Technologies, Inc., Grand Island, NY) supplemented with 10% FCS, L-arginine (116 mg/liter), L-asparagine (36 mg/liter), and L-glutamine (216 mg/liter). The HB55 and HB95 hybridoma, secreting the L243 anti-HLA-DR (class II) mAb and the W6/32 anti-HLA-A, B, and C (class I) mAb, respectively, were purchased from the American Type Culture Collection (Rockville, MD). Flow cytometry of T cells was performed using the following mAbs: CD4-PyC5 (BD PharMingen, San Diego, CA) and CD3-ECD (Beckman Coulter-Immunotech, Miami, FL).

Peptide Synthesis.
The NY-ESO-1LAGE-2 ORF1 and LAGE-1NY-ESO-2 ORF2-derived peptides were synthesized using standard Fmoc chemistry by the University of Pittsburgh Peptide Synthesis Facility (Shared Resource), were >90% pure as indicated by analytical HPLC, and were validated for identity by mass spectrometry. Lyophilized peptides were dissolved in 100% DMSO at a concentration of 2 mg/ml and stored at -20°C until use. Synthesis of LAGE-1 peptides was based on the sequence of the putative ORF2 of the LAGE-1 L gene published by Aarnoudse et al. (Ref. 15 ; GenBank accession no. AJ 012835). The peptides used in the binding assays were synthesized using Fmoc chemistry as described previously (19) . Biotinylated peptides were obtained by reaction with biotinyl-6-aminocaproic acid (Fluka Chimie, St. Quentin Fallavier, France) at the NH2 terminus of the molecule. All of the peptides were purified by reverse-phase HPLC on a C18 Vydac column, and their quality was assessed by electrospray mass spectroscopy and analytical HPLC.

Recombinant Proteins.
The full-length NY-ESO-1 ORF1 recombinant protein was produced in baculovirus and was kindly provided by Drs. Lloyd J. Old and Gerd Ritter (Ludwig Institute for Cancer Research, New York, NY; Ref. 7 ). The cDNA for NY-ESO-1 cloned into the vector pcDNA3.1(-) was kindly provided by Dr. Yao-Tseng Chen and Dr. Alexander Knuth (Ludwig Institute for Cancer Research; Ref. 1 ). The coding sequence for LAGE-1 ORF2 (Ref. 15 ; Protein Data Bank accession no. CAA10197) was constructed in two steps. Its 5' half, which corresponds to a frameshift of the cDNA for NY-ESO-1, was isolated by restriction enzyme digestion of this cDNA. The 3' end of the LAGE-1 ORF2 coding sequence was constructed by hybridization of complementary synthetic oligonucleotides with codons optimized for expression in Escheria coli. Codon optimization fully respected the native coding sequence of LAGE-1 ORF2. The 5' and 3' halves of the LAGE-1 ORF2 coding sequence were introduced into the pQE80 vector (Qiagen) at the BamHI and HindIII cloning sites, leading to plasmid pLAGE-1 ORF2. The expressed LAGE-1 ORF2 protein is preceded by the sequence MRGSHHHHHHGSG. The LAGE-1 ORF2 protein was expressed in the E. coli strain Rosetta (DE3; Novagen) after induction by isopropyl-1-thio-ß-D-galactopyranoside. The inclusion bodies were solubilized in 6 M guanidinium chloride, and the Cys residues were sulfonated by incubation for 1 h at 20°C by adding 0.3 M anhydrous Na2SO3 and 0.5 ml of Thannhauser reagent (20) . The LAGE-1 ORF2 protein was purified by immobilized metal ion (Ni+) affinity chromatography. The eluted protein was refolded by two dialysis against 25 volumes of renaturation buffer [4 mM EDTA, 100 mM Tris-HCl, 10% glycerol (v/v), 8 mM cysteine, and 1 mM cystine (pH 8.3)] at a concentration of 0.5 mg/ml for 48 h at 20°C. Aggregated proteins were removed by centrifugation at 11,000 rpm for 30 min at 4°C. The protein sample was dialyzed against ammonium carbonate 10 mM (pH 8.3), freeze-dried, and stored at -20°C.

Purification of HLA-DR Molecules.
HLA-DR molecules were purified from HLA-homozygous EBV cell lines by affinity chromatography using the monomorphic mAb L 243 coupled to protein A-Sepharose CL 4B gel (Amersham Pharmacia Biotech, Orsay, France) as described previously (19 , 21) .

HLA-DR Peptide Binding Assays.
The binding to the multiple HLA-DR molecules was performed as reported previously (14 , 19 , 21) . Maximal binding was determined by incubating the biotinylated peptide with the MHC class II molecule in the absence of competitor. Binding specificity for each HLA-DR was ensured by the choice of the biotinylated peptides as described previously (21) . The biotinylated peptides were the following: HA 306–318 (PKYVKQNTLKLAT) for HLA-DRB1*0101 (1 nM; pH 6), HLA-DRB1*0401 (30 nM; pH 6), HLA-DRB1*1101 (20 nM; pH 5) and HLA-DRB5*0101 (10 nM; pH 5.5), YKL (AAYAAAKAAALAA) for HLA-DRB1*0701 (10 nM; pH 5), A3 152–166 (EAEQLRAYLDGTGVE) for HLA-DRB1*1501 (10 nM; pH 4.5), MT 2–16 (AKTIAYDEEARRGLE) for HLA-DRB1*0301 (200 nM; pH 4.5), B1 21–36 (TERVRLVTRHIYNREE) for HLA-DRB1*1301 (200 nM; pH 4.5), LOL 191–210 (ESWGAVWRIDTPDKLTGPFT) for HLA-DRB3*0101 (10 nM; pH 5.5) and E2/E168 (AGDLLAIETDKATI) for HLA-DRB4*0101 (10 nM; pH 5). Data were expressed as the concentration of peptide that prevented binding of 50% of the labeled peptide (IC50).

Induction of CD4+ T Cells with Peptides.
The induction of CD4+ T cells in vitro with DCs and the LAGE-1 ORF2-derived peptides was performed as reported previously (13 , 22) . The CD4+ T cells were cloned by limiting dilution using allogeneic PBL and EBV-B cells as feeders in the presence of IL-2 and phytohemagglutinin, and subsequently tested for specificity in IFN-{gamma} ELISPOT assays. The CD4+ T-cell clones were maintained by restimulation every 2 weeks, by alternating irradiated allogeneic PBL and EBV-B cells or autologous peptide-pulsed DCs as stimulators.

IFN-{gamma} and IL-5 ELISPOT Assays.
The recognition of APC pulsed with peptides, proteins, and tumor cells was assessed by ELISPOT assays specific for hu-IFN-{gamma} and IL-5 as reported previously (13 , 14) . Alternatively, 2000 protein-loaded DCs were added to 103 CD4+ T-cell clones/well. The protein-loaded DCs were prepared as reported previously (13) in the presence of the recombinant NY-ESO-1 ORF1 or LAGE-1 ORF2 proteins (30 µg/ml). Spot numbers and spot sizes were determined with the use of computer-assisted video image analysis as described previously (23) . For statistical evaluation, a t test for unpaired samples was used. Values of P < 0.05 were considered significant.

IFN-{gamma} and IL-4 Cytokine Secretion Assays.
The recognition of autologous DCs pulsed with peptides (10 µg/ml) or proteins (30 µg/ml) was also assessed by MACS secretion assays for IFN-{gamma} and IL-4 (Miltenyi Biotech, Auburn, CA). Briefly, 1 x 106 CD4+ T-cell clones were incubated for 6 h at 37°C in 24-well plate, in the presence of 1 x 105 autologous DCs pulsed with peptides (10 µg/ml) or protein (30 µg/ml) as reported previously (13) . The cells were then labeled for 5 min at 4°C with either IFN-{gamma}-specific or IL-4-specific high-affinity capture matrix, also called catch reagents (Miltenyi Biotech). After 45 min of incubation, the secreted cytokine was stained with either IFN-{gamma} detection antibody-FITC (Miltenyi Biotech)/anti-CD4-PyC5/anti-CD3-ECD or IL-4 detection antibody-PE (Miltenyi Biotech)/anti-CD4-PyC5/anti-CD3-ECD. Cells were then washed and fixed with 1% paraformaldehyde before flow cytometry analysis (Beckman-Coulter Epics XL and Expo 32 software).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Three HLA-DRB1*0401-restricted Peptides Encoded by LAGE-1 ORF2 Stimulate Th-1-type CD4+ T Cells from PBL of Melanoma Patients and NDs.
We synthesized short overlapping peptide sequences that span the whole LAGE-1 ORF2 putative protein. The sequence of the LAGE-1 L ORF2 protein was obtained from the GENEBANK (accession no. AJ012835). Overall, 17 peptides each of 18 amino acids in length were chosen for subsequent analyses. In a series of in vitro experiments, we "primed" CD4+ T cells from 2 HLA-DRB1*0401+ melanoma patients with NY-ESO-1-expressing tumors and two NDs. "Mature" DC were incubated with each of the 17 peptides (10 µg/ml), irradiated, and used to stimulate autologous CD4+ T cells (isolated previously from the peripheral blood, as described in "Materials and Methods"). The individual responder cell cultures were restimulated on a weekly basis with irradiated autologous mature DC loaded with the corresponding peptide used in the primary stimulation. After at least three restimulations, the immunoreactivity of the CD4+ T-cell cultures was analyzed in IFN-{gamma} ELISPOT assays. As shown in Fig. 1, A and BCitation , bulk CD4+ T cells from patient UPCI-MEL 285 that were stimulated either with LAGE-1 ORF2 53–67 peptide or with LAGE-1 ORF2 85–102 specifically recognized T2.DR4 cells pulsed with the relevant immunogenic peptide. These CD4+ T cells also displayed reactivity against the autologous NY-ESO-1/LAGE-1+ melanoma cell line UPCI-MEL 285.1, which was partially inhibited by addition of anti-HLA-DR mAb (L243) but not anti-HLA-A,B,C mAb (W6/32) to ELISPOT wells. No IFN-{gamma} spots were produced by CD4+ T cells cultured with T2.DR4 cells alone, or pulsed with an irrelevant HLA-DR4-restricted peptide (NY-ESO-1 ORF1 119–143; Ref. 13 ) or with the NY-ESO-1/LAGE-1- cell line, UPCI-MEL 136.1 (data not shown).



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  		Fig. 1. Recognition of peptides LAGE-1 ORF2 53–67, 85–102 and 7–24; and NY-ESO-1/LAGE-1+, HLA-DRB1*0401+ melanoma cell line, UPCI-MEL 285.1 by Th1-type CD4+T cells of HLA-DRB1*0401+ melanoma patient and ND. CD4+ T cells from HLA-DRB1*0101+/DRB1*0401+/DRB4*0101+ melanoma patient (UPCI-MEL 285) and from HLA-DRB1*0701+/DRB1*1101+/DRB4*0101+ ND 2 (ND1), underwent three rounds of in vitro stimulation with autologous DC pulsed with peptide LAGE-1 53–67, LAGE-1 85–102, or LAGE-1 7–24 as described in "Materials and Methods." Ten-thousand of the resulting responder CD4+ T cells were incubated in a 48-h IFN-{gamma} assay in the presence of T2.DR4 cells pulsed with peptide LAGE-1 ORF2 53–67 (A), LAGE-1 ORF2 85–102 (B), or LAGE-1 ORF2 7–24 (C; 10 µg/ml). The CD4+ T cells were also incubated in the presence of the autologous or HLA-matched HLA-DRB1*0401+/DRB4*0101+ melanoma cell line, UPCI-MEL 285.1 ± anti-HLA-DR antibodies (L243), or UPCI-MEL 285.1 cells ± anti-HLA-A,B,C antibodies (W6/32). IFN-{gamma} spots were developed and counted by computer-assisted video image analysis. Each bar represents the mean spot number of triplicates ± SD with 104 CD4+ T cells initially seeded per well (Ps <0.05 were considered significant and are indicated with *). Data from one representative experiment out of three performed is depicted.

 
Additionally, peptide LAGE-1 ORF2 7–24 but not LAGE-1 ORF2 1–18 stimulated bulk CD4+ T cells from one HLA-DRB1*0401+/DRB1*1401+/DRB4*0101+ ND (ND1) that specifically recognized T2.DR4 cells pulsed with the relevant immunogenic peptide (Fig. 1C)Citation . These CD4+ T cells also displayed reactivity against the HLA-DRB1*0401-matched, NY-ESO-1/LAGE-1+ melanoma cell line UPCI-MEL 285.1, which was partially inhibited by addition of anti-HLA-DR mAb (L243) but not anti-HLA-A,B,C mAb (W6/32) to ELISPOT wells. Similar data were also observed with PBL isolated from an additional HLA-DRB1*0401+ melanoma patient (UPCI-MEL 527; data not shown).

These data support the existence of three HLA-DRB1*0401-restricted epitopes encoded by LAGE-1 ORF2 that are capable of stimulating autologous Th1-type melanoma-reactive CD4+ T cells.

Peptide LAGE-1 ORF2 53–67 Stimulates Th1-type CD4+ T-Cell Clones from One HLA-DRB1*0401+ Melanoma Patient That Recognized Autologous Protein-loaded DCs.
Several clones were obtained by limiting dilution from the CD4+ bulk T cells of patient UPCI-MEL 285, stimulated in vitro with peptide LAGE-1 ORF2 53–67. One representative clone, 31/5, specifically recognized autologous DCs pulsed with the LAGE-1 ORF2 53–68 peptide or loaded with the LAGE-1 ORF2 protein (Fig. 2A)Citation . Unloaded DCs, DCs pulsed with an irrelevant HLA-DR4-restricted peptide (i.e., LAGE-1 ORF2 85–102), and DCs fed with the NY-ESO-1 ORF1 protein served as baseline and controls. Clone 31/5 did not produce IL-5 in IL-5 ELISPOT assays (data not shown).



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  		Fig. 2. Peptide LAGE-1 ORF2 53–67 stimulates Th1-type CD4+ T-cell clone 31/5 derived from PBL of an HLA-DRB1*0401+ melanoma patient. The CD4+ T-cell clone 31/5 was obtained by limiting dilution from the anti-LAGE-1 ORF2 53–67 bulk CD4+ T cells of patient UPCI-MEL 285 as described in "Materials and Methods." One-thousand CD4+ T cells from clone 31/5 were incubated in a 20 h IFN-{gamma} ELISPOT assay in the presence of: (A) autologous DCs pulsed with LAGE-1 ORF2-derived peptides, or loaded with LAGE-1 ORF2 protein or with NY-ESO-1 ORF1 protein; (B) T2.DR4 cells pulsed with short internal peptide sequences from peptide LAGE-1 ORF2 53–67 to delineate the minimal peptide sequence recognized by clone 31/5, i.e. LAGE-1 ORF2 56–65; (C) T2.DR4 cells pulsed with titered-doses of peptides LAGE-1 ORF2 53–67 ({bullet}) and LAGE-1 ORF2 56–65 ({circ}). IFN-{gamma} spots were developed and counted by computer-assisted video image analysis. Each bar represents the mean spot number of triplicates ± SD with 103 CD4+ T cells initially seeded per well (Ps <0.05 were considered significant and are indicated with *). Data from one representative experiment of three performed is depicted.

 
We synthesized a series of peptides from the internal sequence of the immunogenic peptide LAGE-1 ORF2 53–67 to delineate the minimal peptide sequence recognized by the CD4+ T cells. Multiple LAGE-1 ORF2 53–67-reactive CD4+ T-cell clones were capable of recognizing T2.DR4 cells pulsed with peptide LAGE-1 ORF2 56–65 as shown for clone 31/5 in Fig. 2BCitation .

The ability of CD4+ T cell clone 31/5 to produce IFN-{gamma} in the presence T2.DR4 cells, preincubated with the LAGE-1 ORF2 53–67 and 56–65 peptides at various concentrations, was evaluated to determine the peptide-dose "threshold" for effector T-cell recognition. The half-maximal stimulation of LAGE-1 ORF2 53–67 peptide-reactive CD4+ T cells required peptide "loading" concentrations of ~15 nM (Fig. 2C)Citation .

Peptide LAGE-1 ORF2 85–102 Stimulates HLA-DRB1*0401-restricted Th1-type CD4+ T-Cell Clones from a Melanoma Patient That Recognized Autologous Protein-pulsed DCs.
Several clones were also obtained by limiting dilution from the CD4+ bulk T cells of patient UPCI-MEL 285 that recognized the LAGE-1 ORF2 85–102 peptide. In particular, one representative clone 32/28 specifically produced IFN-{gamma} in the presence of autologous DCs pulsed with the LAGE-1 ORF2 85–102 peptide or loaded with the LAGE-1 ORF2 protein both in IFN-{gamma} secretion assays (Fig. 3A)Citation and IFN-{gamma} ELISPOT assays (data not shown). Unloaded DCs, DCs pulsed with an irrelevant HLA-DR4-restricted peptide (i.e., NY-ESO-1 ORF1 119–143), and DCs fed with the NY-ESO-1 ORF1 protein served as baseline and controls. Clone 32/28 did not produce IL-4 (IL-4 secretion assay) or IL-5 (IL-5 ELISPOT assay) in the presence of peptide-pulsed or protein-loaded DCs (data not shown).



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  		Fig. 3. Peptide LAGE-1 ORF2 85–102 stimulates Th1-type CD4+ T-cell clone 32/28 derived from PBL of an HLA-DRB1*0401+ melanoma patient. The CD4+ T-cell clone 32/28 was obtained by limiting dilution from the anti- LAGE-1 ORF2 85–102 bulk CD4+ T cells of patient UPCI-MEL 285 as described in "Materials and Methods." Clone 32/28 was incubated in the presence of: (A) autologous DCs pulsed with peptide LAGE-1 ORF2 85–102 (10 µg/ml), peptide NY-ESO-1 ORF1 119–143 (10 µg/ml), protein LAGE-1 ORF2 (30 µg/ml), or protein NY-ESO-1 ORF1 (30 µg/ml) in an IFN-{gamma}-secretion assay as described in "Materials and Methods;" (B) T2.DR4 cells pulsed with short internal peptide sequences from peptide LAGE-1 ORF2 85–102 to delineate the minimal peptide sequence recognized by clone 32/28, i.e. LAGE-1 ORF2 85–98 in a 20 h IFN-{gamma} ELISPOT assay; (C) T2.DR4 cells pulsed with titered-doses of peptides LAGE-1 ORF2 85–102 ({bullet}) and LAGE-1 ORF2 85–98 ({circ}) in a 20 h IFN-{gamma} ELISPOT assay. IFN-{gamma} spots were developed and counted by computer-assisted video image analysis. Each bar represents the mean spot number of triplicates ± SD with 103 CD4+ T cells initially seeded per well (Ps <0.05 were considered significant and are indicated with *). Data from one representative experiment out of three performed is depicted.

 
From the internal sequence of peptide LAGE-1 ORF2 85–102, we synthesized shorter sequences and tested their ability to be recognized by the anti-LAGE-1 ORF2 85–102 CD4+ T-cell clones in IFN-{gamma} ELISPOT assays. Peptide LAGE-1 ORF2 85–98 represents the minimal peptide sequence recognized by several LAGE-1 ORF2 85–102-specific CD4+ T-cell clones, including clone 32/28 (Fig. 3B)Citation .

The ability of clone 32/28 to produce IFN-{gamma} in the presence T2.DR4 cells, preincubated with various concentrations of peptides LAGE-1 ORF2 85–102 and 85–98, was evaluated. Half-maximal stimulation of clone 32/28 required peptide "loading" concentrations of ~2 nM (Fig. 3C)Citation .

Peptide-binding Studies to Multiple HLA-DR Molecules Demonstrated the Existence of Five Promiscuous LAGE-1 ORF2 HLA-DR-binding Sequences.
We evaluated the binding capacities of the 17 overlapping peptide sequences derived from LAGE-1 ORF2 to 10 HLA-DR molecules including the 7 molecules encoded by the HLA-DRB1 genes (i.e. HLA-DRB1*0101, HLA-DRB1*0301, HLA-DRB1*0401, HLA-DRB1*0701, HLA-DRB1*1101, HLA-DRB1*1301, and HLA-DRB1*1501) and 3 molecules encoded by the HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes. These HLA-DR molecules are present in a high frequency of the Caucasian population and, hence, cover the majority of European and American individuals. As shown previously, 3 of these sequences (i.e., 7–24, 53–67, and 85–102) stimulate tumor-reactive CD4+ T cells isolated from HLA-DRB1*0401+ patients that recognized not only peptide-pulsed but also protein-loaded autologous DCs.

As compared with the IC50 values obtained with reference peptides that are defined as good binders, high and moderate binding was found for multiple peptides, which are clustered in three separate regions of the protein, i.e., 1–30, 49–72, and 67–102 (Table 1)Citation . In the NH2-terminal portion of LAGE-1 ORF2, peptide LAGE-1 7–24 exhibited the lower IC50 values and bound to HLA-DRB1*0101, HLA-DRB1*0401, HLA-DRB1*0701, HLA-DRB1*1101, HLA-DRB1*1501, and HLA-DRB5*0101 (HLA-DR51). It is surrounded by the LAGE-1 ORF2 1–18 and 13–30 peptide sequences, which bind also to multiple HLA-DR molecules. This strongly suggests the existence of multiple distinct epitopes that contain or overlap with the previously identified HLA-A2-restricted epitope LAGE-1 ORF2 1–11.


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  		Table 1 Binding capacities of LAGE-1 ORF2 peptides to immunopurified HLA-DR molecules

IC50 are expressed in nM and have been evaluated from at least three independent experiments. Reference peptides are good binders to the HLA-DR molecules and exhibited the following IC50: HA 306–318 (PKYVKQNTLKLAT) for HLA-DRB1*0101 2 nM, for HLA-DRB1*0401 40 nM, for HLA-DRB1*1101 50 nM, and for HLA-DRB5 10 nM; YKL (AAYAAAKAAALAA) for HLA-DRB1*0701 7 nM; MT 2–16 (AKTIAYDEEARRGLE) for DRB1*0301 200 nM; B1 21–36 (TERVRLVTRHIYNREE) for HLA-DRB1*1301 440 nM; A3 152–166 (EAEQLRRAYLDGTGVE) for HLA-DRB1*1501 40 nM; Lol 191–120 (ESWGAVWRIDTPDKLTGPFT) for HLA-DRB3 15 nM; and E2/E168 (AGDLLAIETDKATI) for HLA-DRB4 15 nM. IC50 values of the active peptides are inferior to 1000 nM and are indicated in bold (31) .

 
In the 49–72 region, the binding capacities of 3 overlapping peptides 49–66, 53–68, and 55–72, were very similar. Peptide LAGE-1 ORF2 53–68 was able to bind very efficiently not only to the HLA-DRB1*0401 molecule but also to multiple additional HLA-DR alleles including HLA-DRB1*0101, HLA-DRB1*1101, HLA-DRB1*1501, HLA-DRB4*0101 (HLA-DR53), and HLA-DRB5*0101 (HLA-DR51). Short peptides overlapping the sequence of the LAGE-1 ORF2 53–68 peptide were tested to precisely characterize their binding capacities to various HLA-DR molecules (Table 2)Citation . Trimming of the NH2 and COOH termini of the peptide showed that removal of the Val 54 and Gly 66 lead to a peptide as active as LAGE-1 ORF2 53–68 on HLA-DRB1*0401. Therefore, the minimal epitope spans these two residues. The corresponding peptide (LAGE-1 ORF2 55–65) bound to the HLA-DRB1*0101, HLA-DRB1*1101, and HLA-DRB5*0101 molecules with activity similar to that of the native peptide. Thus, this peptide also contains the minimal interacting region for each of these HLA-DR molecules. Substitution of the Leu 57 by a Lys resulted in a substantial binding loss for all of the HLA-DR molecules, as well as in a loss of immunogenicity (Fig. 2B)Citation . The same substitution at other aliphatic positions produced a less significant (Met 60) or no (Leu 54 and Leu 56) binding loss (Table 2)Citation , and no loss of immunogenicity (Fig. 2B)Citation . Thus, the Leu 57 serves as P1 anchor residue for all of the HLA-DR molecules, which is in agreement with their binding specificities (24) .


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  		Table 2 HLA-DR-binding capacities of short peptide sequences and modified peptide sequences from LAGE-1 ORF2 53–68

IC50 are expressed in nM and have been evaluated from at least three independent experiments. Reference values were the following: HLA-DRB1*0101 3 nM; HLA-DRB1*0401 36 nM; for HLA-DRB1*1101 21 nM; for HLA-DRB1*1501 14 nM and HLA-DRB5 11 nM; DRB4 30 nM.

 
In the COOH-terminal portion of LAGE-1 ORF2 (i.e., 67–102), peptide LAGE-1 ORF2 85–102 was capable of binding to HLA-DRB1*0101, HLA-DRB1*0701, HLA-DRB1*1101, and HLA-DRB5*0101 (HLA-DR51; Table 1Citation ). The peptide LAGE-1 ORF2 85–98 was found to bind as efficiently as the LAGE-1 ORF2 85–102 peptide to HLA-DRB1*0401 and to a lesser extent, to HLA-DRB1*0701, HLA-DRB1*1101, and HLA-DRB5*0101 (Table 3)Citation . In contrast, the short peptide sequence LAGE-1 ORF2 88–101 bound less well to these HLA-DR molecules. The critical interacting peptide sequence appears to reside mainly in the 85–98 region. Accordingly, substitution of the Trp 87 by a Lys dramatically reduced the binding and the immunogenicity of the 85–102 peptide, whereas the substitution of the other potential anchor P1 residue (Trp 91) did not provoke any effect (Table 3Citation ; Fig. 3BCitation ). Therefore, we conclude that the minimal epitope is contained in the LAGE-1 ORF2 85–98 sequence and requires the Trp 87 as a P1 anchor residue for HLA-DRB1*0401, HLA-DRB1*0701, HLA-DRB1*1101, and HLA-DRB5*0101. However, the two short peptides exhibited a similar binding activity on HLA-DRB1*0101, and none of the Lys substitution was followed by a decline in the IC50. This strongly suggests that two distinct epitopes may reside in the peptide sequence LAGE1 ORF2 85–102 for HLA-DRB1*0101 that use as P1 anchor residue either Trp 87 or Trp 91 in a double anchorage manner (25) .


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  		Table 3 HLA-DR-binding capacities of short peptide sequences and modified peptide sequences from LAGE-1 ORF2 85–102

IC50 are expressed in nM and have been evaluated from at least three independent experiments. Reference values were the following: HLA-DRB1*0101 (3 nM); HLA-DRB1*0401 (36 nM), for HLA-DRB1*0701 (7 nM) and HLA-DRB1*1101 (21 nM), for HLA-DRB5 (11 nM).

 
Additionally, our binding data demonstrate the existence of two promiscuous HLA-DR-binding sequences. Peptide LAGE-1 ORF2 67–84 binds to all of the tested HLA-DR molecules but only weakly to HLA-DRB1*0401, and not to HLA-DRB3*0101. Peptide LAGE-1 ORF2 73–90 binds to HLA-DRB1*1101, HLA-DRB4*0101 (HLA-DR53), and HLA-DRB5*0101 (HLA-DR51). This latter sequence appears to overlap with the sequence LAGE-1 ORF2 71–92 that has been shown recently to stimulate CD4+ T cells from the PBL of a melanoma patient (17) .

Altogether, our binding data define multiple promiscuous HLA-DR-binding sequences derived from LAGE-1 ORF2, which are mainly covered by five distinct peptide sequences, namely LAGE-1 ORF2 7–24, 53–68, 67–84, 73–90, and 85–102. On the basis of these binding data we next performed additional experiments to investigate the immunogenicity of these peptide sequences.

Peptides LAGE-1 ORF2 7–24, 53–67, and 85–102 Are Presented by Multiple HLA-DR Molecules to Stimulate CD4+ T Cells from Melanoma Patients and NDs.
In an independent series of in vitro experiments, we primed CD4+ T cells from 1 HLA-DRB1*0701+/HLA-DRB1*1101+/HLA-DRB4*0101+ ND2 and 1 HLA-DRB1*0101+/HLA-DRB1*0401+ melanoma patient (UPCI-MEL 285) against peptides LAGE-1 ORF2 1–18, 7–24, 53–67, 55–66, 85–102, and 85–98. Mature DC were incubated with each peptide (10 µg/ml), irradiated, and used to stimulate autologous CD4+ T cells (isolated previously from the peripheral blood, as described in "Materials and Methods"). The individual responder cell cultures were restimulated on a weekly basis with irradiated autologous mature DCs loaded with the corresponding peptide used in the primary stimulation. After at least three restimulations, the immunoreactivity of the CD4+ T-cell cultures was analyzed in IFN-{gamma} ELISPOT assays. L cells that have been genetically engineered to express HLA-DR1 (L.DR1), HLA-DR7 (L.DR7), HLA-DR11 (L.DR11), or HLA-DR 53 (L.DR53) were used as APCs in IFN-{gamma} ELISPOT assays. Bulk CD4+ T cells from UPCI-MEL 285 were stimulated with peptide LAGE-1 ORF2 1–18, peptide LAGE-1 ORF2 27–24, peptide LAGE-1 ORF2 53–67, or LAGE-1 ORF2 85–102. As shown in Fig. 4Citation A, peptide LAGE-1 ORF2 7–24 stimulated bulk CD4+ T cells that specifically recognized LAGE-1 ORF2 7–24 peptide-pulsed L.DR1 cells and reacted only weakly against peptide LAGE-1 ORF2 1–18. These CD4+ T cells also displayed reactivity against the autologous NY-ESO-1+ melanoma cell line UPCI-MEL 285, which was partially inhibited by addition of anti-HLA-DR mAb (L243) but not anti-HLA-A,B,C mAb (W6/32) to ELISPOT wells. From these bulk CD4+ T cells, we generated a series of Th1-type CD4+ T-cell clones. One representative Th1-type clone, 47/13, specifically recognized autologous DCs pulsed with peptide LAGE-1 ORF2 7–24 or loaded with the LAGE-1 ORF2 protein (Fig. 4B)Citation . Additionally, peptide LAGE-1 ORF2 1–18 stimulated CD4+ T cells capable of recognizing L.DR1 cells pulsed either with LAGE-1 ORF2 1–18 or 7–24 (data not shown). Peptide LAGE-1 ORF2 53–67 and 85–102 were able to stimulate CD4+ T cells from PBL of patient UPCI-MEL 285 that recognized L.DR1 cells pulsed with each of these peptide but not when pulsed with an irrelevant peptide (i.e. NY-ESO ORF1 119–143). The data are presented in Fig. 5Citation for peptide LAGE-1 ORF2 85–102 (data not shown for peptide LAGE-1 ORF2 53–67). These CD4+ T cells also displayed reactivity against autologous DCs loaded with the LAGE-1 ORF2 protein but not against DCs loaded with an irrelevant protein (i.e. NY-ESO-1 ORF1 protein). No IFN-{gamma} ELISPOT reactivity developed in wells containing L.DR1 cells in the absence of added CD4+ T cells (data not shown).



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  		Fig. 4. HLA-DR1-restricted CD4+ T cells from a melanoma patient recognize peptide LAGE-1 ORF2 7–24-pulsed APCs and autologous DCs loaded with the LAGE-1 ORF2 protein. CD4+ T cells from HLA-DRB1*0101+/DRB1*0401+/DRB4*0101+ melanoma patient UPCI-MEL 285 underwent three rounds of in vitro stimulation with autologous DCs pulsed with peptide LAGE-1 ORF2 7–24 as described in "Materials and Methods." Ten-thousand of the resulting responder CD4+ T cells were incubated in a 20-h IFN-{gamma} assay in the presence of L.DR1 cells pulsed with peptide LAGE-1 ORF2 7–24, peptide LAGE-1 ORF2 1–18, or peptide NY-ESO-1 ORF1 119–143 (A; 10 µg/ml). The CD4+ T cells were also incubated in the presence of the autologous HLA-DRB1*0401+/DRB4*0101+ melanoma cell line, UPCI-MEL 285.1 ± anti-HLA-DR antibodies (L243) or UPCI-MEL 285.1 cells ± anti-HLA-A,B,C antibodies (W6/32). One representative clone 47/13 was obtained by limiting dilution from the bulk CD4+ T cells. One-thousand CD4+ T cells from clone 47/13 were incubated in a 20-h IFN-{gamma} assay in the presence of autologous DCs (2000 cells/well) pulsed with LAGE-1 ORF2-derived peptides (10 µg/ml) or loaded with LAGE-1 ORF2 protein or with NY-ESO-1 ORF1 protein (30 µg/ml; B). IFN-{gamma} spots were developed and counted by computer-assisted video image analysis. Each bar represents the mean spot number of triplicates ± SD (Ps <0.05 were considered significant and are indicated with *). Data from one representative experiment out of three performed is depicted.

 


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  		Fig. 5. Peptide LAGE-1 ORF2 85–102 stimulates HLA-DR1-restricted CD4+ T cells from an HLA-DRB1*0101+ melanoma patient that recognizes the autologous NY-ESO-1/LAGE-1+ melanoma cell line and the autologous DCs loaded with the LAGE-1 ORF2 protein. CD4+ T cells from patient UPCI-MEL 285 underwent three rounds of in vitro stimulation with autologous DCs pulsed with the LAGE-1 ORF2 85–102 peptide as described in "Materials and Methods." Ten-thousand of the resulting responder CD4+ T cells were incubated in a 20-h IFN-{gamma} ELISPOT assay in the presence of L.DR1 cells pulsed with peptides LAGE-1 ORF2 85–102 or NY-ESO-1 ORF1 119–143 (10 µg/ml) and the autologous melanoma cell line, MEL 285.1, ± anti-HLA-DR antibodies (L243), ± anti-HLA-A,B,C antibodies (W6/32). The CD4+ T cells were also incubated with autologous DCs (2000 cells/well) loaded with LAGE-1 ORF2 protein or with NY-ESO-1 ORF1 protein (30 µg/ml) as reported in "Materials and Methods." IFN-{gamma} spots were developed and counted by computer-assisted video image analysis. Each bar represents the mean spot number of triplicates ± SD with 104 CD4+ T cells initially seeded per well (Ps <0.05 were considered significant and are indicated with *). Data from one representative experiment out of two performed is depicted.

 
Bulk CD4+ T cells from donor 2 stimulated with the LAGE-1 ORF2 53–67 recognized L.DR11 and L.DR53 cells pulsed with the relevant peptide (Fig. 6A)Citation . Bulk CD4+ T cells from ND2 stimulated with LAGE-1 ORF2 85–102 peptide recognized L.DR7 and L.DR11 cells pulsed with the relevant peptide (Fig. 6B)Citation . These CD4+ T cells also recognized autologous DCs loaded with the LAGE-1 ORF2 protein but not the DCs loaded with an irrelevant protein (i.e. NY-ESO-1 ORF1).



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  		Fig. 6. Peptides LAGE-1 ORF2 53–67 and 85–102 stimulate HLA-DR7 and HLA-DR11-restricted CD4+ T cells from an HLA-DRB1*0701+/DRB1*1101+/DRB4*0101+ ND. CD4+ T cells from an HLA-DRB1*0701+/DRB1*1101+/DRB4*0101+ ND underwent three rounds of in vitro stimulation with autologous DC pulsed with either peptide LAGE-1 ORF2 53–67 or peptide LAGE-1 ORF2 85–102 as described in "Materials and Methods." Ten-thousand of the resulting responder CD4+ T cells stimulated previously with either peptide LAGE-1 ORF2 53–68 (A) or with peptide LAGE-1 ORF2 85–102 (B) were incubated in a 20-h IFN-{gamma} ELISPOT assay in the presence of L.DR7, L.DR11, or L.DR53 cells pulsed with peptides (10 µg/ml). The CD4+ T cells were also incubated in the presence of autologous DCs loaded with either protein LAGE-1 ORF2 or protein NY-ESO-1 ORF1. IFN-{gamma} spots were developed and counted by computer-assisted video image analysis. Each bar represents the mean spot number of triplicates ± SD with 104 CD4+ T cells initially seeded per well (Ps <0.05 were considered significant and are indicated with *). Data from one representative experiment of three performed is depicted.

 
Altogether, these data corroborate our binding data and demonstrate the ability of peptides LAGE-1 ORF2 53–67 and 85–102 to stimulate CD4+ T cells restricted by HLA-DR1, HLA-DR11, and HLA-DR53, or HLA-DR1, HLA-DR7, and HLA-DR11, respectively. They also confirm the existence of two distinct epitopes in the NH2-terminal portion of LAGE-1 ORF2 that are capable of stimulating either HLA-DR1-restricted CD4+ T cells or HLA-DR1 and HLA-DR4-restricted CD4+ T cells.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
NY-ESO-1/LAGE-1, unlike the majority of identified tumor-associated antigens appears to be particularly immunogenic in vivo as evidenced by spontaneous immune responses repetitively observed by different groups evaluating patients with NY-ESO-1+ tumors (6 , 26) . These observations prompted us to focus our studies on the NY-ESO-1/LAGE-1-derived products to better understand the reasons promoting this unusually strong natural immunogenicity.

Four MHC class I-restricted tumor epitopes derived from ORF2 have been reported to date: one from TRP1 (27) , one from BING-4 (28) , and two from NY-ESO-1/LAGE-1 (9 , 15) . In addition, Slager et al. (17) have reported recently two LAGE-1 ORF2-derived epitopes capable of stimulating CD4+ T cells from peripheral blood mononuclear cells of patients with melanoma. Whether or not other tumor antigens may give rise to MHC-class II ORF2-encoded tumor epitopes remained to be studied. As suggested previously by Rosenberg et al. (28) for MHC class I epitopes, and as underlined by others and by this study for MHC class II epitopes (17) , it appears worthwhile to evaluate the ability of the ORF2 of tumor antigens to encode tumor epitopes. Such analysis may support a possible correlation between ORF2-derived epitopes and the immunogenicity of a given tumor antigen. In particular, the existence of multiple epitopes encoded not only by the ORF1 but also by the ORF2 of the NY-ESO-1/LAGE-1 genes may play a critical role in the induction of spontaneous immune responses among patients with NY-ESO-1/LAGE-1-expressing tumors.

Several peptides capable of binding to multiple MHC class II alleles have been reported in the field of cancer immunology. In particular, HER-2/neu, MAGE-3, and NY-ESO-1 ORF1 have been shown to give rise to promiscuous HLA-DR epitopes (14 , 29 , 30) . Here we have demonstrated the good binding capabilities of five peptide sequences, including LAGE-1 ORF2 7–24, 53–68, 67–84, 73–90, and 85–102, to multiple HLA-DR molecules. Of note, NY-ESO-1 may give rise to the epitopes located in the NH2-terminal portion of the ORF2 protein, i.e., NY-ESO-1/LAGE-1 ORF2 1–18 and 7–24. In a series of in vitro experiments using DCs and PBL of melanoma patients and NDs, we have confirmed the implications of the binding data and demonstrated the immunogenicity of peptides LAGE-1 ORF2 1–18, 7–24, 53–67, and 85–102 in the context of several HLA-DR molecules. In particular, we have demonstrated the capability of peptides LAGE-1 ORF2 53–67 and 85–102 to stimulate CD4+ T cells that recognized the relevant peptide in the context of HLA-DRB1*0101, -DRB1*1101, and -DRB4*0101, or HLA-DRB1*0101, -DRB1*0701, and -DRB1*1101, respectively. Our data confirm the ability of peptide LAGE-1 ORF2 85–102 to bind to HLA-DR11 as reported previously (17) , and demonstrate the ability of this peptide to bind to a broader array of HLA-DR molecules. Our data clearly demonstrate that the 9 aa core peptide sequence capable of binding to HLA-DRB1*0401, HLA-DRB1*0701, HLA-DRB1*1101, and HLA-DRB5*0101 is 87–95 with Trp 88 as a P1 anchor residue. Interestingly, the two peptide-binding sequences from the sequence of LAGE-1 85–102 that we have identified, i.e. 87–96 (for HLA-DRB1*0401, HLA-DRB1*0701, HLA-DRB1*1101, and HLA-DRB5*0101) and 91–99 (for HLA-DRB1*0101), fits perfectly in the peptide binding motifs described previously by Southwood et al. (31) . In addition, we have demonstrated that the anti-LAGE-1 ORF2 53–68 and 85–102 CD4+ T cells recognized autologous DCs from HLA-DRB1*0701+/DRB1*1101+/DRB4*0101+ and HLA-DRB1*0101+/DRB4*0101+ patients fed with the LAGE-1 ORF2 but not the NY-ESO-1 ORF1 protein. This indicates that peptides LAGE-1 ORF2 53–68 and 85–102 contain epitopes that are naturally processed and presented in the context of multiple HLA-DR molecules.

Our studies have demonstrated the ability of the two sequences in the NH2 terminal of LAGE-1 ORF2, i.e., 1–18 and 7–24, to stimulate either HLA-DRB1*0101 or HLA-DRB1*0101 and HLA-DRB1*0401-restricted CD4+ T cells from melanoma patients and NDs, respectively. Interestingly, peptide LAGE-1 ORF2 1–18 encompasses the HLA-A2-restricted LAGE-1 ORF2 1–11 (15) and could possibly be used to stimulate both anti-LAGE-1 ORF2 CD8+ and CD4+ T cells. The clustering of MHC class I and MHC class II epitopes from LAGE-1 ORF2 may be of great value for peptide-based vaccines as shown previously for peptide NY-ESO-1 ORF1 157–170 (32) . We have also identified one peptide sequence, LAGE-1 ORF2 67–84, which binds well to nine distinct HLA-DR molecules, and we are currently performing additional experiments to confirm the immunogenicity of this sequence in the context of these multiple HLA-DR molecules.

Whereas Slager et al. (17) have reported Th-2 type CD4+ T-cell responses against one LAGE-1 ORF2-derived epitope, our data clearly demonstrate the ability of the LAGE-1 ORF2-derived peptides to stimulate Th1-type responses under appropriate conditions that promote the Th1 polarization (mature DCs and Th1-type cytokines). These data strongly support the use of these epitopes as immunogens in cancer vaccines designed to either enhance Th1-type responses or promote the shift of Th2-type responses toward a Th1 phenotype. This may be best accomplished using adjuvants to promote DC maturation and migration, and also with the activation of memory T cells by CD4+ T-cell epitopes that can stimulate IL-12 production from DCs through CD40L interactions (33 , 34) .

The immunogenicity and the promiscuity of the LAGE-1 ORF2 7–24, 53–67, and 85–102 ORF2 peptides support their relevance as potential immunogens for cancer vaccines designed to treat patients with LAGE-1+ tumors. NY-ESO-1/LAGE-1 is expressed by 50% of breast and prostate carcinomas and by 30% of metastatic melanoma, non-small cell lung cancer, bladder, and head and neck tumors (35) . Here, we have shown that peptides LAGE-1 ORF2 7–24, 53–67, and 85–102 can be presented in the context of multiple HLA-DR molecules expressed, respectively, in 70.9%, 91%, and 63% of the American-Caucasian population. Therefore, LAGE-1 ORF2 peptides 7–24, 53–67, and 85–102 will potentially be clinically relevant in 21–35%, 27–45%, and 19–32% of Caucasian patients with LAGE-1+ tumors, respectively.


   ACKNOWLEDGMENTS
 
We thank the patients and their physicians for giving time and the blood samples for the performance of these experiments. We thank Drs. L. J. Old and G. Ritter (Ludwig Institute for Cancer Research, New York, NY) for providing the NY-ESO-1 protein. We thank Drs. Stephanie Land (Department of Biostatistics, University of Pittsburgh), Walter Storkus (Department of Surgery, University of Pittsburgh), and Fred Moolten and Vijay Gandhi (UPCI) for their critical review of the manuscript. We also thank Sandra Pouvelle-Moratille (CEA-Saclay, France) and Sylvain Pichard (CEA-Saclay, France) for technical assistance, and Bonnie Mislanovitch for help in the preparation of this manuscript.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by NIH Grants 90360 (to H. Z.) and CA 56774 (to J. M. K.), a Clinical Trial Grant/Melanoma Initiative from the Cancer Research Institute (to J. M. K.) and a fellowship from the Robert Johnson Foundation (to M. M.). Back

2 To whom requests for reprints should be addressed, at the University of Pittsburgh Cancer Institute, Hillman Cancer Center, Research Pavilion, Suite 1.32, 5117 Centre Avenue, Pittsburgh, PA 15213-2582. Phone: (412) 623-3272; Fax: (412) 623-7707; E-mail: zarourhm{at}msx.upmc.edu Back

3 The abbreviations used are: CT, cancer-testis; Th, T-helper; PBL, peripheral blood lymphocyte; UPCI, University of Pittsburgh Cancer Institute; APC, antigen-presenting cell; DC, dendritic cell; mAb, monoclonal antibody; IL, interleukin; ELISPOT, enzyme-linked immunospot; ORF, open reading frame; aa, amino acid; ND, normal donor; HPLC, high-performance liquid chromatography. Back

Received 3/17/03. Revised 6/30/03. Accepted 7/ 9/03.


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Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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