A Novel Strategy for the Discovery of MHC Class II–Restricted Tumor Antigens: Identification of a Melanotransferrin Helper T-Cell Epitope

Research Article

Till A. Ro¨hn,1 Annette Reitz,3 Annette Paschen,3 Xuan D. Nguyen,4 Dirk Schadendorf,3 Anne B. Vogt,2 and Harald Kropshofer1

1Pharmaceutical Research and 2Roche Center for Medical Genomics, F. Hoffmann La Roche Ltd., Basel, Switzerland; 3German Cancer Research Center, Skin Cancer Unit; and 4Institute of Transfusion Medicine and Immunology, Mannheim, Germany

Abstract Possible reasons for the limited success of antitumor vaccina- CD4+ helper T cells play a critical role in orchestrating host tions are loss of TAAs on tumor tissue, leading to tumor escape immune responses, including antitumor immunity. The variants (12), down-regulation of components of the antigen limited availability of MHC class II–associated tumor antigens processing and presentation pathway (13) or mechanisms of is still viewed as a major obstacle in the use of CD4+ T cells in immunosuppression exerted by the tumor (14–16). Another cancer vaccines. Here, we describe a novel approach for the rationale may be the use of vaccines that rely exclusively on CD8+ T-cell immunity to eradicate cancer cells. An optimal vaccine, identification of MHC class II tumor-associated antigens + + (TAAs). By combining two-dimensional liquid chromatogra- however, might require CD8 and CD4 T-cell antigens to generate a strong and long-lasting antitumor response (17, 18). phy and nanoelectrospray ionization tandem mass spectrom- + etry, we developed a highly sensitive method for the detection Several studies have shown the essential role of CD4 T cells in the elimination of tumors, even if MHC class II molecules are of human leukocyte antigen (HLA)-DR–associated peptides absent on the tumor tissue (19, 20). Animal models have shown the of dendritic cells upon exposure to necrotic tumor cells. + This approach led to the identification of a novel MHC class importance of TAA-specific CD4 T cells for recruiting, priming, II–restricted TAA epitope derived from melanotransferrin. and maintaining of CTLs and for their ability to infiltrate tumors (21, 22). Even in the absence of CTLs, tumor regression can be The epitope stimulated T cells derived from melanoma + patients and healthy individuals and displayed promiscuity mediated by CD4 T cells through direct and indirect killing in HLA-DR restriction. Moreover, the same peptide was also mechanisms (23–25) as well as via recruitment and activation of presented by MHC class II–positive melanoma cells. This eosinophils, macrophages, and B cells (26). strategy may contribute to increase the number of tumor Although many tumor-specific CTL epitopes are known, only a epitopes presented by MHC class II molecules and may very small number of tumor-specific helper T-cell epitopes have support the development of more efficacious vaccines against been characterized. Accordingly, thus far, only very few vaccination cancer. (Cancer Res 2005; 65(21): 10068-78) studies have included epitopes that stimulate helper T cells (27–29). This is mainly due to the lack of effective methods to discover MHC class II–restricted tumor antigenic peptides although Introduction efforts are being made. Still, for most HLA haplotypes and most T cells play a crucial role in the induction and maintenance of types of tumors, appropriate helper T-cell epitopes are elusive. antitumor immunity. This could be shown in animal models and In the present study, we describe a novel strategy for the human cancer therapy (1–3). The activation of antitumor T-cell identification of MHC class II–restricted tumor antigens. Human immunity relies on the recognition of tumor-associated antigens monocyte–derived dendritic cells were pulsed with necrotic (TAAs) that bear immunogenic T-cell epitopes expressed on tumor tumor cells and peptides eluted from HLA-DR molecules were cells. The identification of the first T-cell epitope of the tumor sequenced by a combination of two-dimensional capillary liquid antigen MAGE (4) paved the way for the development of cancer chromatography and electrospray ionization tandem mass vaccines, which trigger cellular immune responses executed by spectrometry (LC ESI-MS/MS). Sequencing revealed several novel tumor-specific T cells. Most attempts focused on the activation of epitopes originating from candidate tumor antigens. One of these tumor-specific CTLs as they can directly lyse tumor cells. Human epitopes was derived from the melanoma-associated protein clinical trials applying defined MHC class I–restricted tumor melanotransferrin (668-683). Melanotransferrin (668-683) was very antigenic peptides indicated that T-cell responses are readily potent in stimulating CD4+ T cells from healthy individuals and detectable in vaccinated tumor patients (5, 6). Accordingly, in- melanoma patients and seems a candidate epitope to be included hibition of tumor growth or tumor regression could be shown in in peptide-based immunotherapy of malignant melanoma. The clinical studies (7–9). However, the overall immune responses were described approach might offer a powerful strategy in the often weak and transient (10, 11), and a clear association between discovery of novel tumor-specific helper T-cell epitopes from immunologic and clinical responses has rarely been observed. different types of tumor.

Materials and Methods Requests for reprints: Harald Kropshofer, Pharmaceutical Research, F. Hoffmann Antibodies. The hybridomas producing HLA-DR–specific monoclonal La Roche Ltd., CH-4070 Basel, Switzerland. Phone: 41-61-688-3569; Fax: 41-61-688-3678; ah E-mail: [email protected]. antibody (mAb) L243 (recognizing HLA-DR dimers) and mAb L235 I2005 American Association for Cancer Research. (recognizing human melanotransferrin) were purchased from the American doi:10.1158/0008-5472.CAN-05-1973 Type Culture Collection (Manassas, VA). Antibodies were purified from

Cancer Res 2005; 65: (21). November 1, 2005 10068 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Discovery of MHC Class II–Restricted Tumor Antigens hybridoma supernatants via protein A-Sepharose. Secondary antibodies biotinylated HA(307-319) peptide to HLA-DR molecules was done using used in flow cytometry: goat anti-mouse coupled to FITC (Dianova, New time-resolved Europium fluorescence and the VICTOR multilabel counter York, NY). Capture and detection antibody pairs used in ELISA were (Wallac Oy). purchased from BD PharMingen (San Diego, CA): IFN-g capture antibody Flow cytometry. Dendritic cells were stained with the indicated NIB42 and IFN-g detection antibody 4S.B3. antibodies (5 Ag/mL) followed by goat anti-mouse-FITC. Analysis was Cell lines. The human melanoma cell lines UKRV-Mel-15a, Ma-Mel-18a, done on a Becton Dickinson FACScalibur flow cytometer with the CellQuest UKRV-Mel-17, MZ-2 (30), SK-Mel-28 (31), and Mel-Juso (32) as well as the software package (Becton Dickinson, Mountain View, CA). Background T-cell/B-cell hybrid cell line T2 (33) stably transfected with DR4 fluorescence was evaluated using irrelevant isotype-matched mAbs. (DRB1*0401; T2.DR4) or DR1 (DRB1*0101; T2.DR1) were maintained in Sandwich ELISA. Supernatants were diluted 1:5 in PBS/Tween (0.05%) + complete RPMI 1640 (Life Technologies/Bethesda Research Laboratories, BSA (1%) and incubated for 2 hours in microtiter plates (Wallac Oy) that Rockville, MD). were previously coated with the corresponding capture antibody. After Preparation of necrotic cells. Tumor and control cells were lysed by intensive washing with PBS/Tween (0.05%), samples were incubated for four cycles of freezing (in liquid nitrogen) and subsequent thawing (at room 1 hour with biotinylated detection antibody. Quantitation was done as temperature). Lysis was monitored by light microscopy and attained an described for the in vitro peptide binding assay. efficacy of 80% to 90%. The lysate was used for pulsing dendritic cells in a Analysis of T-cell responses in melanoma patients. Blood donations ratio of 3:1. from melanoma patients were approved by the Institutional Review Board Generation of dendritic cells. Dendritic cells were differentiated from and an informed consent was given by all donors. Frozen PBMC from HLA- peripheral blood monocytes, as described (32). Briefly, we isolated DRB1*0401-typed melanoma patients were thawed and seeded at 6 Â 106 monocytes from peripheral blood mononuclear cells (PBMC) of HLA-typed cells per well of a six-well plate in 3 mL Iscove’s modified Dulbecco’s medium/ donors by positive selection with anti-CD14 magnetic beads (Miltenyi HEPES/glutamine (PAA Laboratories, Co¨lbe, Germany) supplemented with Biotech, Auburn, CA) and cultured them in complete RPMI supplemented 10% human AB serum (PAA Laboratories). Peptide melanotransferrin (668- with granulocyte macrophage-colony stimulating factor (50 ng/mL, 683), dissolved in DMSO, was added at a concentration of 10 Ag/mL; control Leukomax; Novartis, east Hanover, NJ) and interleukin-4 (IL-4, 3 ng/mL, cells were incubated with DMSO only (day 0). One day later, cytokines IL-2 R&D Systems, Minneapolis, MN). Maturation was induced on day 5 by (20 units/mL) and IL-7 (10 ng/mL) were added to the cultures. After 7 to 14 adding tumor necrosis factor-a (TNF-a, 1 ng/mL, R&D Systems) or days, cells were harvested and screened for their peptide reactivity by IFN-g lipopolysaccharide (LPS, 1 Ag/mL, Sigma, St. Louis, MO). enzyme-linked immunospot (ELISPOT) assay using 105 PBMCs and 2 Â 104 Isolation of CD4+ T cells. CD4+ T cells were isolated from PBMCs by T2.DR4 target cells. negative selection using the CD4+ T-cell isolation kit (Milteny Biotech) Isolation of HLA-DR–restricted peptides. Dendritic cells (4-6 Â 106) consisting of a hapten antibody cocktail and anti-hapten antibodies coupled were lysed in hypotonic lysis buffer containing 1% Triton X-100 and to magnetic beads. T cells were cultured in RPMI supplemented with 1% precipitated with mAb L243 conjugated to Sepharose beads. After several autologous human serum. washing steps with double-distilled water (Merck, Darmstadt, Germany) Generation of tumor antigen-specific T-cell lines. CD4+ T cells (1 Â peptides were eluted from the HLA-DR binding groove with 0.1% 106) were initially stimulated with autologous dendritic cells (2 Â 105) that trifluoracetic acid at 37jC for 35 minutes and immediately lyophilized. were pulsed with LPS (1 Ag/mL) and antigenic peptide (20 Amol/L). After Mass spectrometry. Peptide identification was achieved by the 5 days, IL-2 (1,250 units/mL, R&D Systems) was added. Responding T cells multidimensional protein identification technology, which is based on a were restimulated in 10- to 14-day intervals with autologous dendritic cells two-dimensional liquid chromatographic fractionation followed by mass pulsed with peptide (20 Amol/L) and grown in medium containing IL-2 spectrometric sequencing (LC-ESI-MS/MS). Briefly, lyophilized peptides (1,000 units/mL). After every round of restimulation, the specificity of the were resuspended in 5% acetonitrile, 0.5% acetic acid, 0.012% heptafluoro growing T cells was assessed by sandwich immunoassays for IFN-g and IL-4. butyric acid, and 1% formic acid. Peptide fractionation was achieved on a Peptides. Peptides were synthesized by F-moc chemistry and purified by fused-silica microcapillary column (100-Am inner diameter) packed with reversed-phase high-performance liquid chromatography (HPLC). Some C18 reverse-phase material C18-ACE 3 Am (ProntoSIL 120-3-C18 ACE-EPS; peptides were biotinylated by coupling biotinyl-amino-hexanoic acid at the Bischoff Chromatography, Atlanta, GA) followed by cation exchange

NH2 terminus during F-moc synthesis. HA (307-319), PKYVKQNTLKLAT, is material (Partishere SCX; 5-Am particle size, Whatman, Hillsboro, OR). an immunodominant epitope from influenza virus hemagglutinin that A fully automated 10-step gradient separation was carried out on an binds well to HLA-DR1, HLA-DR2, HLA-DR4, and HLA-DR5. CLIP (81-105), ULTIMATE nanoflow HPLC (Dionex, Sunnyvale, CA). The first six steps LPKPPKPVSKMRMATPLLMQALPMG, is derived from the MHC class II– consisted of a short 5-minute salt elution step with increasing concen- associated invariant chain (Ii). Melanotransferrin (668-683), GQDLL- trations of ammonium acetate (0-225 mmol/L) followed by a nonlinear FKDATVRAVPV, is derived from the long glycosyl phosphatidyl inositol acetonitrile gradient (5-64%); the last four steps consisted each of a (GPI)–anchored variant of melanotransferrin (splicing variant 1). CDC27 20-minute salt elution step (250-1,500 mmol/L) followed by a nonlinear (768-782), MNFSWAMDLDFKGAN, is a known tumor antigen derived from acetonitrile gradient. The HPLC column was directly coupled to a Finnigan the cell cycle protein CDC27. NY-ESO (115-132), PLPVPGVLLKEFTVSGNI, LCQ ion trap mass spectrometer (Finnigan, San Jose, CA) equipped with a is a known tumor antigen derived from the tumor-specific protein NY-ESO. nano-LC ESI source. MS in the MS/MS mode was done according to the Vim (202-217), TLQSFRQDVDNASLAR, is derived from the intermediate manufacturer’s protocol. The identification of peptides was done by the filament protein Vimentin. SEQUEST algorithm against the Swiss-Prot database (http://www.expasy. In vitro peptide binding assay. Purified detergent-solubilized HLA- org/sprot/sprot-top.html). Peptide evaluation was done according to strict DR1 (DRB1*0101 purified from T2.DR1), HLA-DR2 (DRB5*0101 from quality requirements based on the sequence variables cross-correlation

T2.DR2a), HLA-DR4 (DRB4*0401 from T2.DR4), or HLA-DR5 (DRB1*1101 (Xcorr), delta cross-correlation (dCn), preliminary score (Sp), and ranking of from T2.DR5) molecules (20 nmol/L) were coincubated with biotinylated the peptide (Rsp). That is, only those peptides were considered that showed HA(307-319) peptide (200 nmol/L) and different concentrations of the a Xcorr value of 1.8 for singly charged ions, 2.0 for doubly charged ions, and corresponding competitor peptide for 24 hours at 37jC in binding buffer 2.8 for triply charged ions. Furthermore, peptides had to exhibit a dCn value

[50 mmol/L sodium phosphate, 50 mmol/L sodium citrate (pH 5.0), 0.1% of >0.1, a Sp of >500, and an ion coverage of >50% to be considered. In Zwittergent 3-12]. Hence, samples were diluted 10-fold in PBS containing addition, each spectrum was evaluated manually with respect to its 0.05% Tween 20 and 1% bovine serum albumin (BSA) and incubated in a plausibility. microtiterplate (Nunc, Naperville, IL), coated with the anti-HLA-DR mAb Single target expression profiling. Single target expression profiling L243 (3 Ag/mL) for 3 hours. Plates were developed by incubation for 45 (STEP) analysis was done on a plate containing cDNA of cancer and normal minutes with 0.1 Ag/mL streptavidin-Europium (Wallac Oy, Turku, Finland) tissues from a variety of sources. Data was generated by quantitative real- according to the manufacturer’s protocol. Quantification of binding of time PCR to the gene of interest and glycerine aldehyde-3-phosphate www.aacrjournals.org 10069 Cancer Res 2005; 65: (21). November 1, 2005

Figure 1. MAPPs strategy for the differential analysis of MHC class II–restricted peptides of dendritic cells (DC). A, dendritic cells were stimulated with TNF-a in the presence or absence of necrotic tumor cells. HLA-DR molecules were purified and peptides eluted. Peptide mixtures were fractionated by two-dimensional nano-HPLC and peptides concomitantly sequenced online by ESI-MS/MS. Identified peptides of pulsed and unpulsed dendritic cells were comparatively analyzed and those peptides presented only after encounter of necrotic tumor cells were further evaluated. B, fragment spectrum of [M+H+] of peptide GQDLLFKDATVRAVPV (m/z observed = 1,728.7) derived from melanotransferrin (668-683) identified only in the HLA-DR-associated peptide mixture of dendritic cells after exposure to necrotic Ma-Mel-18a cells. List of theoretical masses of the b- and y-ion series of the corresponding peptide. The fragments actually detected by MS/MS are in bold letters and are annotated in the fragment spectrum. dehydrogenase (GAPDH; for standardization) in the same tube at the same Results time on a Taqman (Applied Biosystems, Foster City, CA), running 40 cycles. A primer pair was generated, specifically picking up a sequence from exon Identification of HLA-DR–restricted tumor-associated pep- 9 of the melanotransferrin gene therefore being restricted to the long tides on dendritic cells. In the necrotic zones of tumors, dead transcript of melanotransferrin from which the antigenic epitope was tumor cells or tumor cell debris may be ingested by immature derived: 5V-Mtf1-I (5V-CAGTGCGTGTCAGCCAAGTC) and 3V-Mtf1-I (5V- dendritic cells and delivered to the draining lymph node. In the TTCCCCGCCGTGTAAATGT). A site-specific probe sequence labeled with T cell–rich areas, dendritic cells might initiate a T-cell response a fluorescent reporter dye and a fluorescent quencher dye was used for against tumor-specific epitopes presented by MHC molecules and detection, P-Mtf1-I (5V-AGCGTCGACCTGCTCAGCCTGG). The relative DCT induce tumor-infiltrating lymphocytes (34, 35). To mimic in vitro expression of the gene of interested was E =2 . DCT is the difference in the thermocycles of the GAPDH gene versus gene of interest after which tumor cell uptake, processing and tumor antigen presentation by the fluorescent signal pierces the threshold. The expression of GAPDH in dendritic cells and to exploit this scenario for the identification of each tissue was adjusted to the expression level of a panel of eight MHC class II–restricted tumor antigens, we established a strategy, housekeeping genes. denoted as MHC class II–associated peptide proteomics (MAPPs),

Cancer Res 2005; 65: (21). November 1, 2005 10070 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Discovery of MHC Class II–Restricted Tumor Antigens which allows the identification of self and foreign HLA-restricted even exceeding peptide HA (307-319), which is a strong binder in the peptides on as little as 1 to 5 Â 106 dendritic cells (ref. 36; Fig. 1A). context of HLA-DR4, HLA-DR1, HLA-DR2, and HLA-DR5. Melano- That is, we stimulated dendritic cells with TNF-a and concomi- transferrin (668-683) was also superior to CDC27 (768-782) in tantly exposed them to necrotic MHC class II–negative tumor cells. binding to HLA-DR4. In contrast to CDC27 (768-782), which was Dendritic cells were allowed to ingest necrotic tumor cells for 24 hours, whereupon dendritic cells were lysed, HLA-DR-associated Table 1. Neoantigens on dendritic cells (DRB1*0401/1301) peptides isolated via affinity beads and analyzed by two- after pulse with necrotic Ma-Mel-18a melanoma cells dimensional LC ESI-MS/MS. Through analysis of dendritic cells that were exposed to necrotic tumor cells, compared with dendritic Protein Peptide sequence Epitope cells that were not, peptides that relied on the presence of tumor cells could be identified. Table 1 shows a list of peptides that were Melanotransferrin GQDLLFKDATVRAVPVG 668-684 (melanoma-associated GQDLLFKDATVRAVPV 668-683 presented on HLA-DR molecules of dendritic cells, haplotype HLA- antigen p97)* DRB1* 0401/1301, after encounter of necrotic Ma-Mel-18a mela- Regulator of G-protein PALLPTPVEPTAACGPGGGD 445-464 c noma cells and absent on unpulsed dendritic cells. Only those signaling 11 (RGS11) c among f600 identified peptides are enlisted that were present in Cathepsin D IHHKYNSDKSSTYVK 119-133 two independent measurements of tumor-pulsed dendritic cells VDQNIFSFYLSRDPDAQPGGE 224-244 and absent in both corresponding measurements of the autologous NIFSFYLSRDPDAQPGGE 227-244 unpulsed dendritic cells. Peptides were further evaluated and NIFSFYLSRDPDAQPGGEL 227-245 selected according to several quality criteria (see Materials and IFSFYLSRDPDAQPG 228-242 NIFSFYLSRDPDAQPGG 227-243 Methods). By this means, 40 peptides derived from 13 different NIFSFYLSRDPDAQPG 227-242 proteins were identified. Many of these peptides were derived from Signal regulatory EPNNHTEYASIQTSPQPA 445-462 c proteins that are ubiquitously expressed (e.g., from cytoskeletal or protein a 1 (Sirp a 1) NHTEYASIQTSPQPA 448-462 c matrix proteins like tubulin or collagen, or constitutively expressed Vimentin LTNDKARVEVERDNLAEDIM 163-182 enzymes, like Lysyl hydroxylase or Ribophorin I). Others were derived TNDKARVEVERDNLAEDIM 164-182 from proteins that have been described to be overexpressed in DKARVEVERDNLAEDIM 166-182 certain cancers, like the regulator of G-protein signaling proteins NDKARVEVERDNLAEDI 165-181 (37), Cathepsin D (38), Sirp a1 (39), or Vimentin (40). These proteins, NDKARVEVERDNLAEDIM 165-182 LQEEIAFLKKLHEEEIQ 226-242 however, are also ubiquitously expressed on healthy tissues. One of EEIAFLKKLHEEEIQ 228-242 the peptides that were selectively found only in the HLA-DR- LQEEIAFLKKLHE 226-238 associated peptide repertoire of tumor-pulsed dendritic cells was LQEEIAFLKKLHEEE 226-240 b derived from the protein melanotransferrin, shown to be strongly Ribophorin I GAKNIEIDSPYEISRAPD 377-394 LPEGAKNIEIDSPYEISRAPD 374-394 expressed on melanoma cells (41). The melanotransferrin peptide b Lysyl hydroxylase 3 NVPTVDIHM*KQVGYEDQ 606-622 melanotransferrin (668-683) as well as its length variant melano- b transferrin (668-684) could be identified. The MS/MS spectrum of the Lysyl hydroxylase 1 NVPTIDIHMNQIGFERE 595-611 identified epitope melanotransferrin (668-683) is depicted in Fig. 1B. NVPTIDIHM*NQIGFER 595-610 VPTIDIHM*NQIGFER 596-610 The very same peptide could also be detected in an independent NVPTIDIHM*NQIGFERE 595-611 experiment in which dendritic cells of a different donor of the Putative KRTTDVMFGGKQVVVCG 272-288 b haplotype DRB1*0101/04011 were pulsed with necrotic Ma-Mel-18a adenosylhomocysteinase 2 b cells (data not shown), suggesting that the epitope is HLA-DR4 Protein pM5 precursor NAM*TFTFDNVLPGKYK 541-556 b restricted and very abundant on MHC class II molecules of dendritic Collagen a 1(II) chain KSGDYWIDPNQGCTL 1225-1239 cells after uptake of necrotic Ma-Mel-18a melanoma cells. KSGDYWIDPNQGCT 1225-1238 h b To verify the identity of the discovered peptide, the synthetic Tubulin -5 chain GAKFWEVISDEHGIDPT 17-33 analogue of melanotransferrin (668-683) was applied to MS/MS IGAKFWEVISDEHGIDPT 16-33 KFWEVISDEHGIDPT 19-33 fragmentation on the same Finnigan LCQ Ion Trap MS instrument. AKFWEVISDEHGIDPT 18-33 The fragmentation pattern of the synthetic peptide was almost HUMAN 40S ribosomal DSHGVAQVRFVTGNKIL 55-71 b identical to the naturally processed peptide thereby confirming the protein S13 SDDVKEQIYKLAKKGLTPSQ 29-48 identity of melanotransferrin (668-683) (Fig. 2A). To our knowledge, DDVKEQIYKLAKKGLTPS 30-47 neither CTL nor helper T-cell epitopes of melanotransferrin have VKEQIYKLAKKGLTPS 32-47 been identified thus far. HLA-DR binding of melanotransferrin (668-683). The identified melanotransferrin peptide possessed two overlapping NOTE: HLA-DR-associated peptides of dendritic cells pulsed with Ma- HLA-DR binding motives, which may confer binding to a diverse Mel-18a and unpulsed autologous control dendritic cells were analyzed twice by two-dimensional LC ESI-MS/MS. Only peptides that were set of HLA-DR molecules. The first motif comprises anchor present in the peptide repertoire of both pulsed dendritic cell samples residues L-672, D-675, T-677, and A-680, whereas residues F-673, and absent in both unpulsed dendritic cell samples are listed. A-676, V-678, and V-681 constitute the second motif. As *Protein for which melanoma-associated expression has been melanotransferrin (668-683) was identified in the peptide described. No ubiquitous expression. repertoire of two donors sharing the HLA-DRB1*0401 molecules, cUbiquitously expressed proteins that have been shown overexpressed binding of the synthetic peptide to HLA-DR4 was assessed by an in cancer tissues. b in vitro peptide binding assay (Fig. 2B). As expected, melano- Ubiquitously expressed proteins without apparent association to cancer. transferrin (668-683) showed high binding affinity to HLA-DR4, www.aacrjournals.org 10071 Cancer Res 2005; 65: (21). November 1, 2005

Figure 2. Fragmentation and HLA-DR binding of synthetic melanotransferrin (668-683). A, fragment spectrum [M+H+] of synthetic GQDLLFKDATVRAVPV. Observed peptide fragments are indicated in bold. B, peptide binding assay. The concentration of each peptide required to reduce binding of biotinylated HA (307-319) by 50% (IC50) through competition was determined by capture ELISA. The reciprocal 1/IC50 is given which directly correlates with peptide binding affinity. The IC50 values were determined in the context of HLA-DR4, HLA-DR1, HLA-DR2, and HLA-DR5 (left to right). Columns, means of three independent experiments for each haplotype; bars, FSD. identified as a tumor antigen in the context of HLA-DRB1*0401 a panel of other melanoma cell lines and gave rise to the (35), melanotransferrin (668-683) displayed mediocre binding presentation of melanotransferrin (668-683) by dendritic cells. affinities to additional HLA-DR alleles, such as HLA-DR1, HLA- Interestingly, when necrotic UKRV-Mel-15a cells that express DR2, and HLA-DR5, accounting for almost 50% of the HLA-DR substantially less melanotransferrin than Ma-Mel-18a cells were genotypes among Caucasians. Haplotype promiscuity of melano- subjected to the same dendritic cells, no presentation of any transferrin (668-683) was also higher than that of the tumor melanotransferrin peptide could be observed (data not shown). antigen NY-ESO (115-132), recognized by CD4+ T cells of patients Both UKRV-Mel-15a and Ma-Mel-18a melanoma cell lines were with NY-ESO-expressing tumors (42). Thus, melanotransferrin negative for MHC class II (Fig. 3B). (668-683) apparently exhibits a reasonable degree of promiscuity MHC class II–positive UKRV-Mel-17 cells present melano- with regard to binding to allelic variants of HLA-DR. transferrin (668-683). We asked whether melanoma cells that Expression of melanotransferrin in melanoma cell lines. coexpress both melanotransferrin and HLA-DR molecules would Melanotransferrin is a cell membrane protein that is attached themselves present the melanotransferrin peptide. We therefore to the cell surface through a GPI anchor. We compared the analyzed the HLA-DR-restricted self-peptide repertoire of the expression of melanotransferrin on melanoma cells and dendritic melanoma cell line UKRV-Mel-17, which strongly expresses both cells by flow cytometry using anti–melanotransferrin-specific mAb melanotransferrin and HLA-DRB1* 0401/03011 (Fig. 3A and B). As L235 (Fig. 3A). As expected from our experiments described above, melanotransferrin (668-683) was identified in the context of HLA- dendritic cells apparently do not express melanotransferrin neither DRB1*0401 (Table 1) and showed strong binding affinity for HLA- in the immature nor in the mature state. The melanoma cell line DR4 (Fig. 2B), we expected melanotransferrin (668-683) to be also Ma-Mel-18a, however, which was used as antigen source, exhibited present in the HLA-DR-restricted peptide repertoire of UKRV-Mel- a relatively strong expression of melanotransferrin compared with 17 cells.

Cancer Res 2005; 65: (21). November 1, 2005 10072 www.aacrjournals.org

7 HLA-DR-molecules of ca. 5 Â 10 UKRV-Mel-17 cells were Table 2. HLA-DR-restricted peptides of tumor-associated isolated, peptides eluted and subjected to two-dimensional LC ESI- proteins eluted from UKRV-Mel-17 melanoma cells MS/MS. 971 HLA-DR-associated peptides could be sequenced, by (DRB1*0401/03011) this approach. Table 2 displays a subset of these peptides. We listed only those peptides that were derived from proteins described to Protein Peptide sequence Epitope be associated with cancer. Indeed, melanotransferrin (668-683) and its elongation variant melanotransferrin (668-684) were among the Melanotransferrin GQDLLFKDATVRAVPV 668-683 set of HLA-DR-associated self-peptides presented by this melano- (melanoma-associated GQDLLFKDATVRAVPVG 668-684 ma cell line. antigen p97) Other epitopes present in the self-peptide repertoire of UKRV-Mel- Gp100 (melanoma- NRQLYPEWTEAQR 45-57 17 cells were derived from gp100, MART-1, melanoma-associated associated ME20 NRQLYPEWTEAQRLD 45-59 chrondoitinsulfate-proteoglycan, matrix metalloproteinase-14 antigen) RQLYPEWTEAQR 46-57 (MMP-14), and melanoma differentiation–associated protein-9 WNRQLYPEWTEAQR 44-57 WNRQLYPEWTEAQRLD 44-59 (syntenin). From each parent protein, one epitope and several of MART-1 (Melan-A) APPAYEKLSAEQSPPPY 100-116 the respective length variants could be identified (Table 2). The APPAYEKLSAEQSPPP 100-115 peptide derived from gp100 sequenced here has been described as Melanoma-associated GPWPQGATLRLDPTVLDAGEL 2053-2073 helper T-cell epitope before (43). A helper T-cell epitope of MART-1 chondroitinsulfate- GPWPQGATLRLDPTVLDAGE 2053-2072 has already been discovered in the context of HLA-DRB1*0401 proteoglycan NG2 GATLRLDPTVLDAGEL 2058-2073 (44), The peptide identified here, however, has not been described (HSN tumor-specific GPWPQGATLRLDPTVLDAGELA 2053-2074 before. The other tumor-associated MHC class II peptides listed antigen) here, to our knowledge, have not been identified, as yet. MMP-14 (MT-MMP 1, GDKHWVFDEASLEPG 384-398 Melanotransferrin (668-683) is a helper T-cell epitope. To membrane-type-1 GDKHWVFDEASLEPGYPK assess whether melanotransferrin (668-683) may sensitize helper MMP) T cells, we isolated CD4+ TcellsfromPBMCsofanHLA- Syntenin 1 (melanoma ITSIVKDSSAARNGLL 218-234 differentiation– ITSIVKDSSAARNGL 218-233 associated protein-9) ITSIVKDSSAARN 218-231 ITSIVKDSSAARNGLLT 218-235

NOTE: HLA-DR molecules were isolated from 5 Â 107 cells, peptides eluted and subjected to two-dimensional LC ESI-MS/MS. Enlisted peptides are derived from proteins known to be tumor associated.

DRB1*0401-positive healthy donor and repeatedly stimulated them with autologous dendritic cells that had been pulsed with melanotransferrin (668-683). Melanotransferrin (668-683)–pulsed dendritic cells induced a helper T-cell type 1 (TH1) response, as indicated by the specific release of IFN-g, whereas dendritic cells pulsed with control peptide did not (Fig. 4A). Release of TH2 cytokines, such as IL-4, could not be observed in response to melanotransferrin (668-683)–pulsed dendritic cells (data not shown). To verify that the melanotransferrin (668-683)–specific T-cell line recognized melanotransferrin (668-683) in the context of HLA-DR4, we did an MHC restriction analysis. The T-cell line was stimulated with peptide-pulsed T2.DR4 or T2.DR1 transfectants. T2.DR4 cells were capable of inducing a substantial T-cell response, whereas peptide-pulsed T2.DR1 cells provoked only a weak release of IFN-g (Fig. 4B). T-cell recognition of T2.DR4 cells could be substantially inhibited by 10 Ag/mL anti-HLA-DR mAb L243, whereas no effect was observed at the same concentration of MHC class I–specific control mAb W6/32. When T-cell lines were stimulated by melanoma cell line UKRV- Mel-17, which is presenting the melanotransferrin (668-683) Figure 3. Expression of melanotransferrin (MTf ) and HLA-DR on melanoma cells. A, flow cytometric analysis of a panel of melanoma cells, immature (IM) epitope in the context of HLA-DRB1*0401, as identified by MS and mature dendritic cells (DC; Mat, stimulated with 1 Ag/mL LPS for 24 hours) (Table 2), only a very weak T-cell response could be observed stained with the melanotransferrin-specific mAb L235 (5 Ag/mL). Columns, mean fluorescence intensity of the specific staining for each cell type from three (Fig. 4C). This weak T-cell stimulation could be inhibited by experiments; bars, FSD. Isotype controls for each cell type were set to a mean coincubation with the anti-HLA-DR mAb L243. When UKRV-Mel- fluorescence intensity of 1. B, expression of HLA-DR on melanoma cell lines. 17 cells were pretreated with IFN-g for 48 hours, carefully washed, Flow cytometry of melanoma cell lines Ma-Mel-18a, UKRV-Mel-15a, and UKRV-Mel-17 with mAb L243 (5 Ag/mL) specific for HLA-DR. Solid lines, isotype and then cocultured with T cells, a stronger T-cell stimulation control. One of three experiments with similar results. occurred which was abolished by mAb L243. IFN-g pretreatment www.aacrjournals.org 10073 Cancer Res 2005; 65: (21). November 1, 2005

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Cancer Research of UKRV-Mel-17 cells led to elevated expression of HLA-DR, specific T cells. Pulsing of UKRV-Mel17 cells with exogenous melanotransferrin, and intercellular adhesion molecule-1 (data melanotransferrin (668-683) peptide to further increase the not shown), aspects that may all contribute to the stronger number of melanotransferrin (668-683)/HLA-DR complexes led recognition of UKRV-Mel-17 cells by melanotransferrin (668-683)– to a roughly 25-fold elevation of IFN-g release. When the same set

Cancer Res 2005; 65: (21). November 1, 2005 10074 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Discovery of MHC Class II–Restricted Tumor Antigens of experiments was done with the MHC class II–negative control cancer and colon cancer primary tissues exhibited elevated melanoma cell line UKRV-Mel-15a, no T-cell stimulation could be expression of melanotransferrin. In the case of lung cancer observed (Fig. 4C). tissues, 4 of 14 assessed samples (f30%) showed an expression As the response of the T-cell line to naturally processed that was 12- to 45-fold increased over the average expression of melanotransferrin (668-683) epitope presented by UKRV-Mel-17 normal lung tissues. The average expression of all lung cancer was rather weak but could be increased by IFN-g pretreatment samples was about eight times higher than the average or exogenous peptide administration, we supposed that the expression of all normal lung tissues. With respect to colon T-cell line that we generated from a healthy donor was a low- cancer and colon cancer metastasis, elevation of melanotrans- avidity melanotransferrin-specific T-cell line that required rather ferrin expression was not that distinct, however, in 9 of 40 high numbers of surface peptide/MHC class II complexes. samples (f25%) expression levels were increased by >5-fold Peptide titration experiments using mature autologous dendritic compared with normal tissues. No increased expression could be cells as antigen-presenting cells revealed that T-cell responses observed in prostate cancer (Fig. 5A), bladder, or kidney cancer could only be observed with peptide concentrations higher than (data not shown). Importantly, melanotransferrin was hardly 0.1 Amol/L (Fig. 4D). Our results show that the mass spectro- expressed in normal tissues tested (Fig. 5A, dotted line). Slightly metrically identified HLA-DR-restricted melanotransferrin pep- elevated expression could only be observed in normal breast and tide presented by dendritic cells after uptake of necrotic tumor small intestine tissues (data not shown). In depth analysis of cells is a true helper T-cell epitope as it proved to be immu- mRNA expression in different types of lung cancer further nogenic. revealed that melanotransferrin is overexpressed in different Recognition of melanotransferrin (668-683) by T cells of neoplastic lung cell types but most pronounced in large cell lung melanoma patients. We further wanted to explore whether cancer cells (Fig. 5B). Based on these results, melanotransferrin melanotransferrin (668-683) would also be suitable to induce a seems specifically overexpressed in certain cancers, whereas it is T-cell response in late-stage melanoma patients, as in these absent on most normal tissues. patients potentially reactive T cells might have been anergized or deleted due to the induction of antigen-specific tolerance exerted by the tumor (45). PBMCs of three HLA-DRB1*0401-positive Discussion melanoma patients were isolated and incubated with or without The importance of tumor-reactive CD4+ helper T cells in the melanotransferrin (668-683). After 7 to 14 days, these PBMCs development of antitumor immunity has become increasingly clear were analyzed for their melanotransferrin (668-683)–specific over the past decade (17, 18, 46). The development of broadly reactivity in the presence of DRB1*0401-expressing T2 trans- applicable methods to expand the number of MHC class II– fectants. Reactive T cells were enumerated by ELISPOT analysis. restricted tumor antigens, therefore, remains an important task in Melanotransferrin (668-683)–specific T-cell responses could be tumor immunotherapy. In tumor vaccination, the use of synthetic detected in all three patients when PBMCs were presensitized peptides compared with whole cell preparations or adoptive with melanotransferrin (668-683) as indicated by the significantly transfer approaches has the advantage that peptides can easily increased number of IFN-g-producing cells (Fig. 4E). We be manufactured and pharmaceutically formulated. conclude that late-stage melanoma patients do bear helper We developed a procedure that allows us to identify HLA-DR T cells that respond to the melanotransferrin (668-683) peptide ligands of as little as 106 dendritic cells (36). Dendritic cells are antigen. likely to be most critical in mounting antitumor immunity, as Expression of melanotransferrin in normal and cancer they take up antigens from damaged tumor cells in tumor tissues. Melanotransferrin has been described to be melanoma lesions, traffic to draining lymph nodes, and prime antigen- associated and absent on most other normal tissues (41). To specific naive T cells (47, 48). To mimic this process in vitro,we extend this knowledge and to verify melanotransferrin (668-683) incubated dendritic cells with necrotic tumor cells and identified as a tumor antigen that could be employed in immunotherapy HLA-DR-restricted antigenic peptides derived from tumor cells against cancer, we assessed the expression of melanotransferrin through a highly sensitive two-dimensional LC ESI-MS/MS (transcription variant 1, of which the epitope was derived) by method. We believe that this approach has several advantages real-time PCR in a panel of 189 normal and 119 cancer tissues. over other current strategies: A selection of several normal versus cancer tissues is shown in (a) Approaches using T cells as screening tools have the Fig. 5A. Interestingly, not only melanoma cells but also lung limitation that T-cell clones from individual cancer patients need

Figure 4. Stimulation of melanotransferrin [MTf (668-683)]–specific T cells. A, CD4+ T cells of a HLA-DRB1* 0401/0701 donor were isolated and probed for its specific recognition of melanotransferrin (668-683) after five rounds of restimulation with autologous dendritic cells pulsed with peptide. Dendritic cells were activated with LPS (1 Ag/mL) and pulsed with 20 Amol/L of melanotransferrin (668-683) or control peptide CLIP (81-104) for 24 hours before T-cell stimulation. T cells were cocultured in a ratio of 10:1 with pulsed autologous dendritic cells for 16 hours. Supernatants were probed for secreted IFN-g by sandwich ELISA. B, 5 Â 104 T2.DR4 or T2.DR1 cells were used as antigen-presenting cells (APC) and incubated with 1 Â 105 T cells for 24 hours in the presence of 20 Amol/L peptide or peptide + 10 Ag/mL of the blocking antibodies W6/32 (anti MHC class I) or L243 (anti-HLA-DR). Secreted IFN-g was determined as in (A). C, melanoma cells were either pretreated with 100 units/mL IFN-g for 48 hours or left untreated. UKRV-Mel-17 or UKRV-Mel-15a melanoma cells (5 Â 104) were then incubated with (+TCL) or without 1 Â 105 melanotransferrin-specific T-cell lines for 24 hours in the presence of 10 Ag/mL mAb W6/32 or L243 or peptide melanotransferrin (668-683). D, IFN-g release of melanotransferrin (668-683)–reactive T-cell line in response to dendritic cells pulsed for 24 hours with LPS and various concentrations of melanotransferrin (668-683) or control CLIP (81-104). Columns (points), mean concentration in ng/mL (n = 3); bars, FSD. *, P < 0.05 (significant). E, detection of melanotransferrin (668-683)–specific T cells in the peripheral blood of HLA-DRB1*0401+ melanoma patients. PBMCs (6 Â 106) from melanoma patient EM (HLA-DRB1*0301 and HLA-DRB1*0401), MD (HLA-DRB1*0401 and HLA-DRB1*1101), and ASn (HLA-DRB1*0401 and HLA-DRB1*1302) were seeded in the absence or presence of peptide melanotransferrin (668-683). After an incubation period of 7 days (for EM and MD) and 14 days (for ASn), T cells were analyzed for their specificity in an IFN-g ELISPOT assay by incubation with T2.DR4 cells as antigen presenters in the presence of melanotransferrin (668-683), Vim (202-217), or without (w/o) peptide. Columns, means of three-well (EM, MD) and two-well (Asn) determinations; bars, FSD. *, P < 0.05, (significant). www.aacrjournals.org 10075 Cancer Res 2005; 65: (21). November 1, 2005

Figure 5. Single target expression profiling for melanotransferrin. cDNA was generated from a panel of normal versus cancer tissues. Quantitative real time PCR was done with primers specific for melanotransferrin (MTf ) and the reference gene GAPDH. Expression levels are given as arbitrary units based on the relative expression of melanotransferrin mRNA versus normalized GAPDH in the corresponding tissue. A, each filled box is indicative of the expression of melanotransferrin mRNA per sample. The numbers above the panels for each tissue show the average expression of all samples of the corresponding tissue (horizontal line). The following numbers of samples per tissue were analyzed: testis, n = 8; brain, n = 6; spleen, n = 5; muscle, n = 18; lymph node, n = 3; adipose, n = 19; lung, n = 21; lung cancer, n = 14; melanoma cell lines, n = 3; colon, n = 16; colon cancer, n = 25; colon cancer metastases, n = 15; prostate, n =8; prostate cancer, n = 11. Dotted line, average expression of melanotransferrin in all normal tissues that were assessed. B, expression of melanotransferrin in different cell types of normal lung and lung cancer tissues. Expression is given in arbitrary units as in (A). Abbreviation: NSCLC, non–small cell lung cancer.

to be generated, which can be particularly difficult for to approach, it is independent of purification or cloning steps of nonmelanoma tumors. (b) In case only low-affinity T-cell clones preselected tumor antigens pulsed onto antigen presenting cells are available, T-cell screenings might give negative results, or cloned as invariant chain fusion proteins. This approach, although the antigenic peptide is presented by the tumor. (c) however, bears the imminent danger that dendritic cells not only Screening by T cells is laborious and time consuming as it take up tumor-specific proteins but also housekeeping proteins depends on testing of overlapping peptides or peptides that have expressed by any type of cell. To solve this issue, we subtract the been predicted in silico by algorithms that are still far less broad panel of HLA-DR–associated self-peptides derived from reliable in the context of MHC class II than MHC class I dendritic cell–resident proteins (36) from those presented after molecules. (d) Epitopes identified by T-cell screening need to be ingestion of necrotic tumor material. Hence, we focus on confirmed by testing of the respective recombinant protein in a epitopes that are either exclusively presented after ingestion of T-cell assay, because cryptic epitopes might have been identified. tumor cells and absent among the naturally occurring self- (e) Our approach circumvents the limitation that most tumors peptides on dendritic cells or peptides that are strongly are MHC class II negative, as it identifies tumor antigens that up-regulated in the presence of tumor cells. Following this are presented by MHC class II molecules of dendritic cells. (f) strategy, several epitopes could be identified, which were As whole tumor cell preparations are used in the present exclusively present on tumor-pulsed dendritic cells. However,

Cancer Res 2005; 65: (21). November 1, 2005 10076 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. Discovery of MHC Class II–Restricted Tumor Antigens we also found peptides of ubiquitously expressed proteins (e.g., suggests that either the precursor frequency of melanotransferrin vimentin, collagen, tubulin, and ribosomal protein S13), which (668-683)–reactive T cells is high or that melanotransferrin (668- were not present in the self-peptide repertoire of corresponding 683)–specific T cells proliferate well. This finding supports our unpulsed dendritic cells. These proteins might particularly be set contention that under favorable conditions epitope melano- free in large quantities after tumor cell necrosis, thereby giving transferrin (668–683) may be capable of inducing an anti– rise to neoepitopes. The LC-MS method applied here does not melanotransferrin-specific immune response also in melanoma identify peptides by de novo sequencing but through comparison patients. of real mass spectra with theoretical spectra of a protein Ideally, tumor peptides to be included in vaccination trials database. To date, our approach therefore has the limitation that should be applicable to a broad range of tumor patients bearing epitopes containing sequence mutations that are not covered by a variety of HLA haplotypes. A certain degree of promiscuity in the protein database can not be detected. However, as such haplotype restriction is therefore desirable. Our peptide binding mutations often only occur in distinct individuals, mutated assays showed that melanotransferrin (668-683) exhibited a very epitopes may be less broadly applicable for immunotherapy pronounced binding affinity for HLA-DR4 but also moderate compared with those derived from nonmutated antigens, such as affinities for HLA-DR1, HLA-DR2, and HLA-DR5. These four HLA- cancer testis and tissue-specific antigens expressed in a majority DR alleles together cover f50% of the HLA-DR genotypes of the of cancer patients. Caucasian population, implying that melanotransferrin (668-683) We concentrated our efforts on the peptide derived from may be a broadly applicable tumor peptide vaccine. melanotransferrin, because melanotransferrin has already been More importantly, tumor antigens to be included in cancer described to be a melanoma-associated protein (41). Melanotrans- vaccination trials have to exhibit tumor-specific expression or at ferrin was not expressed in dendritic cells, but it was strongly least substantial overexpression in tumors compared with healthy expressed by the MHC class II–negative tumor cell line Ma-Mel- tissues, to prevent the induction of potentially life-threatening 18a, which was used as an antigen source in the presence of autoreactive immune responses against tumor-unrelated tissues. dendritic cells. This provided evidence that melanotransferrin was Our expression profiling extended already existing melanotrans- transferred from necrotic Ma-Mel-18a melanoma cells to dendritic ferrin expression data (41, 51, 52) and showed that melano- cells, where it was processed and presented. Interestingly, the very transferrin mRNA is present only in trace amounts in the same melanotransferrin epitope could also be detected as a natural assessed normal tissues. However, it was strongly overexpressed self-peptide on the melanoma cell line UKRV-Mel-17, indicating in tumor tissues (e.g., melanoma) but also certain other cancer that the peptide can indeed be generated by natural processing and types such as lung and colon cancer, supporting our contention was unlikely to be created exogenously by proteases liberated that melanotransferrin may qualify as a shared tumor antigen in during necrosis. immunotherapy. Among the naturally presented self-peptides of the tumor cell Early studies using recombinant vaccinia virus vaccines against line UKRV-Mel-17, further epitopes from tumor-specific proteins melanotransferrin in mice and Macaca fascicularis monkeys have could be identified, one of which, gp100 (44–59), has already shown that cell-mediated and humoral immune responses could been described by others and proved to be immunogenic (43). be induced against melanotransferrin-expressing xenografts and We are therefore confident that the other novel HLA-DR- transfected syngeneic tumor cells resulting in in vivo rejection of restricted peptides derived from MART-1, melanoma-associated tumor cells (53, 54). Despite the expression of trace amounts of chondroitinsulfate-proteoglycan, MMP-14, and Syntenin 1 may cross-reactive melanotransferrin in normal tissues of primates, no also qualify as MHC class II–restricted tumor antigens, although adverse effects or normal tissue damage could be observed after T-cell assays will have to confirm the immunogenicity of these eliciting the immune response against melanotransferrin (53). peptides. This suggests that vaccination with melanotransferrin (668-683) For melanotransferrin (668-683), the capacity to activate T cells against melanotransferrin-expressing tumors may also be safely could be shown by in vitro T-cell sensitization experiments. applicable in man. Reactive T cells showed a high level of melanotransferrin (668- In conclusion, we have shown that our MAPPs strategy based 683)–specific IFN-g release, indicative of a TH1 response. This on human dendritic cells and LC/MS-MS is suitable for the result proves that nontolerant melanotransferrin (668-683)–specific identification of tumor-associated MHC class II peptide antigens, naive T cells exist in the naturally occurring T-cell repertoire, which which may be of value for vaccination against cancer. We believe could be stimulated by peptide vaccination. that MAPPs provides a powerful complementation to already The induction of antigen-specific unresponsiveness by cancer existing methods that aim at expanding the number of tumor- cells seems one of the predominant means by which tumors specific helper T-cell epitopes. Future vaccination with appropri- evade the attack of the immune system. It is thus likely that ate combinations of cytotoxic CD8+ and CD4+ helper T-cell antigen-specific tolerance among T cells is of paramount epitopes may contribute to increase the success rate of cancer importance for tumor survival. Tolerance induction by tumors immunotherapy. has been shown for both CD4+ and CD8+ T cells (49, 50), and it is conceivable that in tumor patients, T-cell stimulation against certain antigens may fail due to such tolerization mechanisms. Acknowledgments Hence, it was interesting to observe that peptide sensitization Received 6/7/2005; revised 7/26/2005; accepted 8/23/2005. experiments carried out with PBMCs of HLA-DRB1*0401-positive Grant support: Wilhelm-Sander-Stiftung. The costs of publication of this article were defrayed in part by the payment of page late-stage melanoma patients revealed that after a single round charges. This article must therefore be hereby marked advertisement in accordance of stimulation with melanotransferrin (668-683), substantial with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Nadine Daniel (Roche Pharmaceuticals, Basel, Switzerland) for expert T-cell responses could be detected. The fact that a single round technical assistance, Bernd Mu¨ller (Roche Pharmaceuticals) for help with MS, and of restimulation already induced a significant T-cell response Silke Schnell for helpful discussions during her diploma thesis. www.aacrjournals.org 10077 Cancer Res 2005; 65: (21). November 1, 2005

References histocompatibility complex class II negative tumors. 37. Furuya M, Nishiyama M, Kimura S, et al. Expression J Exp Med 1998;187:693–702. of regulator of G protein signalling protein 5 (RGS5) in 1. Houghton AN, Gold JS, Blachere NE. Immunity against 20. Mumberg D, Monach PA, Wanderling S, et al. CD4(+) the tumour vasculature of human renal cell carcinoma. cancer: lessons learned from melanoma. Curr Opin T cells eliminate MHC class II-negative cancer cells J Pathol 2004;203:551–8. Immunol 2001;13:134–40. in vivo by indirect effects of IFN-g. Proc Natl Acad Sci 38. Leto G, Gebbia N, Rausa L, Tumminello FM. 2. Rosenberg SA. Progress in human tumor immunology U S A 1999;96:8633–8. Cathepsin D in the malignant progression of neoplastic and immunotherapy. Nature 2001;411:380–4. 21. Casares N, Lasarte JJ, de Cerio AL, et al. Immunization diseases [review]. Anticancer Res 1992;12:235–40. 3. Stevanovic S. Identification of tumor-associated T-cell with a tumor-associated CTL epitope plus a tumor- 39. ChenTT,BrownEJ,HuangEJ,SeamanWE. epitopes for vaccine development. Nat Rev Cancer 2002; related or unrelated Th1 helper peptide elicits protective Expression and activation of signal regulatory protein 2:514–20. CTL immunity. Eur J Immunol 2001;31:1780–9. a on astrocytomas. Cancer Res 2004;64:117–27. 4. Van der Bruggen P, Traversari C, Chomez P, et al. Gene 22. Marzo AL, Kinnear BF, Lake RA, et al. Tumor-specific 40. Singh S, Sadacharan S, Su S, Belldegrun A, Persad S, encoding an antigen recognized by cytolytic T lympho- CD4+ T cells have a major ‘‘post-licensing’’ role in CTL Singh G. Overexpression of vimentin: role in the invasive cytes on a human melanoma. Science 1991;254:1643–7. mediated anti-tumor immunity. J Immunol 2000;165: phenotype in an androgen-independent model of 5. Jager E, Maeurer M, Hohn H, et al. Expansion of Melan 6047–55. prostate cancer. Cancer Res 2003;63:2306–11. A-specific cytotoxic T lymphocytes in a melanoma 23. Cohen PA, Peng L, Plautz GE, Kim JA, Weng DE, Shu 41. Brown JP, Woodbury RG, Hart CE, Hellstrom I, patient responding to continued immunization with S. CD4+ T cells in adoptive immunotherapy and the Hellstrom KE. Quantitative analysis of melanoma- melanoma-associated peptides. Int J Cancer 2000;86: indirect mechanism of tumor rejection. Crit Rev associated antigen p97 in normal and neoplastic tissues. 538–47. Immunol 2000;20:17–56. Proc Natl Acad Sci U S A 1981;78:539–43. 6. Disis ML, Gooley TA, Rinn K, et al. Generation of T-cell 24. Greenberg PD. Adoptive T cell therapy of tumors: 42. Jager E, Jager D, Karbach J, et al. Identification of NY- immunity to the HER-2/neu protein after active mechanisms operative in the recognition and elimina- ESO-1 epitopes presented by human histocompatibility immunization with HER-2/neu peptide-based vaccines. tion of tumor cells. Adv Immunol 1991;49:281–355. antigen (HLA)-DRB4*0101–0103 and recognized by J Clin Oncol 2002;20:2624–32. 25. Qin Z, Blankenstein T. CD4+ T cell-mediated tumor CD4(+) T lymphocytes of patients with NY-ESO-1- 7. Rosenberg SA, Yang JC, Schwartzentruber DJ, et al. rejection involves inhibition of angiogenesis that is expressing melanoma. J Exp Med 2000;191:625–30. Immunologic and therapeutic evaluation of a synthetic dependent on IFN g receptor expression by non- 43. Li K, Adibzadeh M, Halder T, et al. Tumour-specific peptide vaccine for the treatment of patients with hematopoietic cells. Immunity 2000;12:677–86. MHC-class-II-restricted responses after in vitro sensiti- metastatic melanoma. Nat Med 1998;4:321–7. 26. Hung K, Hayashi R, Lafond-Walker A, Lowenstein C, zation to synthetic peptides corresponding to gp100 and 8. Nestle FO, Alijagic S, Gilliet M, et al. Vaccination of Pardoll D, Levitsky H. The central role of CD4(+) T cells Annexin II eluted from melanoma cells. Cancer melanoma patients with peptide- or tumor lysate- in the antitumor immune response. J Exp Med 1998;188: Immunol Immunother 1998;47:32–8. pulsed dendritic cells. Nat Med 1998;4:328–32. 2357–68. 44. Zarour HM, Kirkwood JM, Kierstead LS, et al. 9. Marchand M, van Baren N, Weynants P, et al. Tumor 27. van Driel WJ, Ressing ME, Kenter GG, et al. Melan-A/MART-1(51–73) represents an immunogenic regressions observed in patients with metastatic mela- Vaccination with HPV16 peptides of patients with HLA-DR4-restricted epitope recognized by melanoma- noma treated with an antigenic peptide encoded by advanced cervical carcinoma: clinical evaluation of a reactive CD4(+) T cells. Proc Natl Acad Sci U S A 2000; gene MAGE-3 and presented by HLA-A1. Int J Cancer phase I-II trial. Eur J Cancer 1999;35:946–52. 97:400–5. 1999;80:219–30. 28. Brossart P, Wirths S, Stuhler G, Reichardt VL, Kanz L, 45. Pardoll D. Does the immune system see 10. Lee PP, Yee C, Savage PA, et al. Characterization of Brugger W. Induction of cytotoxic T-lymphocyte tumors as foreign or self? Annu Rev Immunol circulating T cells specific for tumor-associated antigens responses in vivo after vaccinations with peptide-pulsed 2003;21:807–39. in melanoma patients. Nat Med 1999;5:677–85. dendritic cells. Blood 2000;96:3102–8. 46. Toes RE, Ossendorp F, Offringa R, Melief CJ. CD4 T 11. Panelli MC, Wunderlich J, Jeffries J, et al. Phase 1 29. Slingluff CL, Yamshchikov G, Neese P, et al. Phase I cells and their role in antitumor immune responses. study in patients with metastatic melanoma of immu- trial of a melanoma vaccine with gp100 (280–288) J Exp Med 1999;189:753–6. nization with dendritic cells presenting epitopes derived peptide and tetanus helper peptide in adjuvant: 47. Finn OJ. Cancer vaccines: between the idea and the from the melanoma-associated antigens MART-1 and immunologic and clinical outcomes. Clin Cancer Res reality. Nat Rev Immunol 2003;3:630–41. gp100. J Immunother 2000;23:487–98. 2001;7:3012–24. 48. Knutson KL, Disis ML. Tumor antigen-specific T 12. Khong HT, Restifo NP. Natural selection of tumor 30. Eichmuller S, Usener D, Jochim A, Schadendorf D. helper cells in cancer immunity and immunotherapy. variants in the generation of ‘‘tumor escape’’ pheno- mRNA expression of tumor-associated antigens in Cancer Immunol Immunother 2005;54:721–8. types. Nat Immunol 2002;3:999–1005. melanoma tissues and cell lines. Exp Dermatol 2002; 49. Bogen B. Peripheral T cell tolerance as a tumor 13. Seliger B, Ritz U, Abele R, et al. Immune escape of 11:292–301. escape mechanism: deletion of CD4+ T cells specific for melanoma: first evidence of structural alterations in two 31. Carey TE, Takahashi T, Resnick LA, Oettgen HF, Old a monoclonal immunoglobulin idiotype secreted by a distinct components of the MHC class I antigen LJ. Cell surface antigens of human malignant melano- plasmacytoma. Eur J Immunol 1996;26:2671–9. processing pathway. Cancer Res 2001;61:8647–50. ma: mixed hemadsorption assays for humoral immunity 50. Staveley-O’Carroll K, Sotomayor E, Montgomery J, 14. Tada T, Ohzeki S, Utsumi K, et al. Transforming to cultured autologous melanoma cells. Proc Natl Acad et al. Induction of antigen-specific T cell anergy: an early h growth factor- -induced inhibition of T cell function. Sci U S A 1976;73:3278–82. event in the course of tumor progression. Proc Natl Susceptibility difference in T cells of various phenotypes 32. Johnson JP, Demmer-Dieckmann M, Meo T, Hadam Acad Sci U S A 1998;95:1178–83. and functions and its relevance to immunosuppression MR, Riethmuller G. Surface antigens of human melano- 51. Alemany R, Vila MR, Franci C, Egea G, Real FX, in the tumor-bearing state. J Immunol 1991;146:1077–82. ma cells defined by monoclonal antibodies. I. Biochem- Thomson TM. Glycosyl phosphatidylinositol membrane 15. Gabrilovich DI, Chen HL, Girgis KR, et al. Production ical characterization of two antigens found on cell lines anchoring of melanotransferrin (p97): apical compart- of vascular endothelial growth factor by human tumors and fresh tumors of diverse tissue origin. Eur J Immunol mentalization in intestinal epithelial cells. J Cell Sci inhibits the functional maturation of dendritic cells. Nat 1981;11:825–31. 1993;104:1155–62. Med 1996;2:1096–103. 33. Salter RD, Howell DN, Cresswell P. Genes regulating 52. Rothenberger S, Food MR, Gabathuler R, et al. 16. Salazar-Onfray F. Interleukin-10: a cytokine used by HLA class I antigen expression in T-B lymphoblast Coincident expression and distribution of melanotrans- tumors to escape immunosurveillance. Med Oncol 1999; hybrids. Immunogenetics 1985;21:235–46. ferrin and transferrin receptor in human brain capillary 16:86–94. 34. Wang RF, Rosenberg SA. Human tumor antigens for endothelium. Brain Res 1996;712:117–21. 17. Topalian SL. MHC class II restricted tumor antigens cancer vaccine development. Immunol Rev 1999;170:85–100. 53. Estin CD, Stevenson US, Plowman GD, et al. + and the role of CD4 T cells in cancer immunotherapy. 35. Wang RF, Wang X, Atwood AC, Topalian SL, Recombinant vaccinia virus vaccine against the human Curr Opin Immunol 1994;6:741–5. Rosenberg SA. Cloning genes encoding MHC class II- melanoma antigen p97 for use in immunotherapy. Proc 18. Wang RF. The role of MHC class II-restricted tumor restricted antigens: mutated CDC27 as a tumor antigen. Natl Acad Sci U S A 1988;85:1052–6. antigens and CD4+ T cells in antitumor immunity. Science 1999;284:1351–4. 54. Kahn M, Sugawara H, McGowan P, et al. CD4+ T cell Trends Immunol 2001;22:269–76. 36. Rohn TA, Boes M, Wolters D, et al. Upregulation of clones specific for the human p97 melanoma-associated 19. Ossendorp F, Mengede E, Camps M, Filius R, Melief the CLIP self peptide on mature dendritic cells antigen can eradicate pulmonary metastases from a CJ. Specific T helper cell requirement for optimal antagonizes T helper type 1 polarization. Nat Immunol murine tumor expressing the p97 antigen. J Immunol induction of cytotoxic T lymphocytes against major 2004;5:909–18. 1991;146:3235–41.

Cancer Res 2005; 65: (21). November 1, 2005 10078 www.aacrjournals.org

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 2005 American Association for Cancer Research. A Novel Strategy for the Discovery of MHC Class II− Restricted Tumor Antigens: Identification of a Melanotransferrin Helper T-Cell Epitope

Till A. Röhn, Annette Reitz, Annette Paschen, et al.

Cancer Res 2005;65:10068-10078.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/65/21/10068

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2005/10/31/65.21.10068.DC1

Cited articles This article cites 52 articles, 22 of which you can access for free at: http://cancerres.aacrjournals.org/content/65/21/10068.full#ref-list-1

Citing articles This article has been cited by 3 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/65/21/10068.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/65/21/10068. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.