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A Novel Strategy for the Discovery of MHC Class II–Restricted

Tumor Antigens: Identification of a Melanotransferrin

Helper T-Cell Epitope

Till A. Rohn,1Annette Reitz,

3Annette Paschen,

3Xuan D. Nguyen,

4Dirk Schadendorf,

3

Anne B. Vogt,2and Harald Kropshofer

1

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

Abstract

CD4+ helper T cells play a critical role in orchestrating hostimmune responses, including antitumor immunity. Thelimited availability of MHC class II–associated tumor antigensis still viewed as a major obstacle in the use of CD4+ T cells incancer vaccines. Here, we describe a novel approach for theidentification of MHC class II tumor-associated antigens(TAAs). By combining two-dimensional liquid chromatogra-phy and nanoelectrospray ionization tandem mass spectrom-etry, we developed a highly sensitive method for the detectionof human leukocyte antigen (HLA)-DR–associated peptidesof dendritic cells upon exposure to necrotic tumor cells.This approach led to the identification of a novel MHC classII–restricted TAA epitope derived from melanotransferrin.The epitope stimulated T cells derived from melanomapatients and healthy individuals and displayed promiscuityin HLA-DR restriction. Moreover, the same peptide was alsopresented by MHC class II–positive melanoma cells. Thisstrategy may contribute to increase the number of tumorepitopes presented by MHC class II molecules and maysupport the development of more efficacious vaccines againstcancer. (Cancer Res 2005; 65(21): 10068-78)

Introduction

T cells play a crucial role in the induction and maintenance ofantitumor immunity. This could be shown in animal models andhuman cancer therapy (1–3). The activation of antitumor T-cellimmunity relies on the recognition of tumor-associated antigens(TAAs) that bear immunogenic T-cell epitopes expressed on tumorcells. The identification of the first T-cell epitope of the tumorantigen MAGE (4) paved the way for the development of cancervaccines, which trigger cellular immune responses executed bytumor-specific T cells. Most attempts focused on the activation oftumor-specific CTLs as they can directly lyse tumor cells. Humanclinical trials applying defined MHC class I–restricted tumorantigenic peptides indicated that T-cell responses are readilydetectable in vaccinated tumor patients (5, 6). Accordingly, in-hibition of tumor growth or tumor regression could be shown inclinical studies (7–9). However, the overall immune responses wereoften weak and transient (10, 11), and a clear association betweenimmunologic and clinical responses has rarely been observed.

Possible reasons for the limited success of antitumor vaccina-tions are loss of TAAs on tumor tissue, leading to tumor escapevariants (12), down-regulation of components of the antigenprocessing and presentation pathway (13) or mechanisms ofimmunosuppression exerted by the tumor (14–16). Anotherrationale may be the use of vaccines that rely exclusively onCD8+ T-cell immunity to eradicate cancer cells. An optimal vaccine,however, might require CD8+ and CD4+ T-cell antigens to generatea strong and long-lasting antitumor response (17, 18).Several studies have shown the essential role of CD4+ T cells in

the elimination of tumors, even if MHC class II molecules areabsent on the tumor tissue (19, 20). Animal models have shown theimportance of TAA-specific CD4+ T cells for recruiting, priming,and maintaining of CTLs and for their ability to infiltrate tumors(21, 22). Even in the absence of CTLs, tumor regression can bemediated by CD4+ T cells through direct and indirect killingmechanisms (23–25) as well as via recruitment and activation ofeosinophils, macrophages, and B cells (26).Although many tumor-specific CTL epitopes are known, only a

very small number of tumor-specific helper T-cell epitopes havebeen characterized. Accordingly, thus far, only very few vaccinationstudies have included epitopes that stimulate helper T cells(27–29). This is mainly due to the lack of effective methods todiscover MHC class II–restricted tumor antigenic peptides althoughefforts are being made. Still, for most HLA haplotypes and mosttypes of tumors, appropriate helper T-cell epitopes are elusive.In the present study, we describe a novel strategy for the

identification of MHC class II–restricted tumor antigens. Humanmonocyte–derived dendritic cells were pulsed with necrotictumor cells and peptides eluted from HLA-DR molecules weresequenced by a combination of two-dimensional capillary liquidchromatography and electrospray ionization tandem massspectrometry (LC ESI-MS/MS). Sequencing revealed several novelepitopes originating from candidate tumor antigens. One of theseepitopes was derived from the melanoma-associated proteinmelanotransferrin (668-683). Melanotransferrin (668-683) was verypotent in stimulating CD4+ T cells from healthy individuals andmelanoma patients and seems a candidate epitope to be includedin peptide-based immunotherapy of malignant melanoma. Thedescribed approach might offer a powerful strategy in thediscovery of novel tumor-specific helper T-cell epitopes fromdifferent types of tumor.

Materials and Methods

Antibodies. The hybridomas producing HLA-DR–specific monoclonalantibody (mAb) L243 (recognizing HLA-DRah dimers) and mAb L235

(recognizing human melanotransferrin) were purchased from the American

Type Culture Collection (Manassas, VA). Antibodies were purified from

Requests for reprints: Harald Kropshofer, Pharmaceutical Research, F. HoffmannLa Roche Ltd., CH-4070 Basel, Switzerland. Phone: 41-61-688-3569; Fax: 41-61-688-3678;E-mail: [email protected].

I2005 American Association for Cancer Research.doi:10.1158/0008-5472.CAN-05-1973

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

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hybridoma supernatants via protein A-Sepharose. Secondary antibodiesused in flow cytometry: goat anti-mouse coupled to FITC (Dianova, New

York, NY). Capture and detection antibody pairs used in ELISA were

purchased from BD PharMingen (San Diego, CA): IFN-g capture antibody

NIB42 and IFN-g detection antibody 4S.B3.Cell lines. The human melanoma cell lines UKRV-Mel-15a, Ma-Mel-18a,

UKRV-Mel-17, MZ-2 (30), SK-Mel-28 (31), and Mel-Juso (32) as well as the

T-cell/B-cell hybrid cell line T2 (33) stably transfected with DR4

(DRB1*0401; T2.DR4) or DR1 (DRB1*0101; T2.DR1) were maintained incomplete RPMI 1640 (Life Technologies/Bethesda Research Laboratories,

Rockville, MD).

Preparation of necrotic cells. Tumor and control cells were lysed by

four cycles of freezing (in liquid nitrogen) and subsequent thawing (at roomtemperature). Lysis was monitored by light microscopy and attained an

efficacy of 80% to 90%. The lysate was used for pulsing dendritic cells in a

ratio of 3:1.Generation of dendritic cells. Dendritic cells were differentiated from

peripheral blood monocytes, as described (32). Briefly, we isolated

monocytes from peripheral blood mononuclear cells (PBMC) of HLA-typed

donors by positive selection with anti-CD14 magnetic beads (MiltenyiBiotech, Auburn, CA) and cultured them in complete RPMI supplemented

with granulocyte macrophage-colony stimulating factor (50 ng/mL,

Leukomax; Novartis, east Hanover, NJ) and interleukin-4 (IL-4, 3 ng/mL,

R&D Systems, Minneapolis, MN). Maturation was induced on day 5 byadding tumor necrosis factor-a (TNF-a, 1 ng/mL, R&D Systems) or

lipopolysaccharide (LPS, 1 Ag/mL, Sigma, St. Louis, MO).

Isolation of CD4+ T cells. CD4+ T cells were isolated from PBMCs bynegative selection using the CD4+ T-cell isolation kit (Milteny Biotech)

consisting of a hapten antibody cocktail and anti-hapten antibodies coupled

to magnetic beads. T cells were cultured in RPMI supplemented with 1%

autologous human serum.Generation of tumor antigen-specific T-cell lines. CD4+ T cells (1 �

106) were initially stimulated with autologous dendritic cells (2 � 105) that

were pulsed with LPS (1 Ag/mL) and antigenic peptide (20 Amol/L). After

5 days, IL-2 (1,250 units/mL, R&D Systems) was added. Responding T cells

were restimulated in 10- to 14-day intervals with autologous dendritic cells

pulsed with peptide (20 Amol/L) and grown in medium containing IL-2

(1,000 units/mL). After every round of restimulation, the specificity of the

growing T cells was assessed by sandwich immunoassays for IFN-g and IL-4.Peptides. Peptides were synthesized by F-moc chemistry and purified by

reversed-phase high-performance liquid chromatography (HPLC). Some

peptides were biotinylated by coupling biotinyl-amino-hexanoic acid at the

NH2 terminus during F-moc synthesis. HA (307-319), PKYVKQNTLKLAT, isan immunodominant epitope from influenza virus hemagglutinin that

binds well to HLA-DR1, HLA-DR2, HLA-DR4, and HLA-DR5. CLIP (81-105),

LPKPPKPVSKMRMATPLLMQALPMG, is derived from the MHC class II–

associated invariant chain (Ii). Melanotransferrin (668-683), GQDLL-FKDATVRAVPV, is derived from the long glycosyl phosphatidyl inositol

(GPI)–anchored variant of melanotransferrin (splicing variant 1). CDC27

(768-782), MNFSWAMDLDFKGAN, is a known tumor antigen derived from

the cell cycle protein CDC27. NY-ESO (115-132), PLPVPGVLLKEFTVSGNI,is a known tumor antigen derived from the tumor-specific protein NY-ESO.

Vim (202-217), TLQSFRQDVDNASLAR, is derived from the intermediate

filament protein Vimentin.In vitro peptide binding assay. Purified detergent-solubilized HLA-

DR1 (DRB1*0101 purified from T2.DR1), HLA-DR2 (DRB5*0101 from

T2.DR2a), HLA-DR4 (DRB4*0401 from T2.DR4), or HLA-DR5 (DRB1*1101

from T2.DR5) molecules (20 nmol/L) were coincubated with biotinylatedHA(307-319) peptide (200 nmol/L) and different concentrations of the

corresponding competitor peptide for 24 hours at 37jC in binding buffer

[50 mmol/L sodium phosphate, 50 mmol/L sodium citrate (pH 5.0), 0.1%

Zwittergent 3-12]. Hence, samples were diluted 10-fold in PBS containing0.05% Tween 20 and 1% bovine serum albumin (BSA) and incubated in a

microtiterplate (Nunc, Naperville, IL), coated with the anti-HLA-DR mAb

L243 (3 Ag/mL) for 3 hours. Plates were developed by incubation for 45minutes with 0.1 Ag/mL streptavidin-Europium (Wallac Oy, Turku, Finland)

according to the manufacturer’s protocol. Quantification of binding of

biotinylated HA(307-319) peptide to HLA-DR molecules was done usingtime-resolved Europium fluorescence and the VICTOR multilabel counter

(Wallac Oy).

Flow cytometry. Dendritic cells were stained with the indicated

antibodies (5 Ag/mL) followed by goat anti-mouse-FITC. Analysis wasdone on a Becton Dickinson FACScalibur flow cytometer with the CellQuest

software package (Becton Dickinson, Mountain View, CA). Background

fluorescence was evaluated using irrelevant isotype-matched mAbs.

Sandwich ELISA. Supernatants were diluted 1:5 in PBS/Tween (0.05%) +BSA (1%) and incubated for 2 hours in microtiter plates (Wallac Oy) that

were previously coated with the corresponding capture antibody. After

intensive washing with PBS/Tween (0.05%), samples were incubated for

1 hour with biotinylated detection antibody. Quantitation was done asdescribed for the in vitro peptide binding assay.

Analysis of T-cell responses in melanoma patients. Blood donations

from melanoma patients were approved by the Institutional Review Boardand an informed consent was given by all donors. Frozen PBMC from HLA-

DRB1*0401-typed melanoma patients were thawed and seeded at 6 � 106

cells per well of a six-well plate in 3mL Iscove’smodified Dulbecco’smedium/

HEPES/glutamine (PAA Laboratories, Colbe, Germany) supplemented with10% human AB serum (PAA Laboratories). Peptide melanotransferrin (668-

683), dissolved in DMSO, was added at a concentration of 10 Ag/mL; control

cells were incubated with DMSO only (day 0). One day later, cytokines IL-2

(20 units/mL) and IL-7 (10 ng/mL) were added to the cultures. After 7 to 14days, cells were harvested and screened for their peptide reactivity by IFN-g

enzyme-linked immunospot (ELISPOT) assay using 105 PBMCs and 2 � 104

T2.DR4 target cells.Isolation of HLA-DR–restricted peptides. Dendritic cells (4-6 � 106)

were lysed in hypotonic lysis buffer containing 1% Triton X-100 and

precipitated with mAb L243 conjugated to Sepharose beads. After several

washing steps with double-distilled water (Merck, Darmstadt, Germany)peptides were eluted from the HLA-DR binding groove with 0.1%

trifluoracetic acid at 37jC for 35 minutes and immediately lyophilized.

Mass spectrometry. Peptide identification was achieved by the

multidimensional protein identification technology, which is based on a

two-dimensional liquid chromatographic fractionation followed by mass

spectrometric sequencing (LC-ESI-MS/MS). Briefly, lyophilized peptides

were resuspended in 5% acetonitrile, 0.5% acetic acid, 0.012% heptafluoro

butyric acid, and 1% formic acid. Peptide fractionation was achieved on a

fused-silica microcapillary column (100-Am inner diameter) packed with

C18 reverse-phase material C18-ACE 3 Am (ProntoSIL 120-3-C18 ACE-EPS;

Bischoff Chromatography, Atlanta, GA) followed by cation exchange

material (Partishere SCX; 5-Am particle size, Whatman, Hillsboro, OR).

A fully automated 10-step gradient separation was carried out on an

ULTIMATE nanoflow HPLC (Dionex, Sunnyvale, CA). The first six steps

consisted of a short 5-minute salt elution step with increasing concen-

trations of ammonium acetate (0-225 mmol/L) followed by a nonlinear

acetonitrile gradient (5-64%); the last four steps consisted each of a

20-minute salt elution step (250-1,500 mmol/L) followed by a nonlinear

acetonitrile gradient. The HPLC column was directly coupled to a Finnigan

LCQ ion trap mass spectrometer (Finnigan, San Jose, CA) equipped with a

nano-LC ESI source. MS in the MS/MS mode was done according to the

manufacturer’s protocol. The identification of peptides was done by the

SEQUEST algorithm against the Swiss-Prot database (http://www.expasy.

org/sprot/sprot-top.html). Peptide evaluation was done according to strict

quality requirements based on the sequence variables cross-correlation

(Xcorr), delta cross-correlation (dCn), preliminary score (Sp), and ranking of

the peptide (Rsp). That is, only those peptides were considered that showed

a Xcorr value of 1.8 for singly charged ions, 2.0 for doubly charged ions, and

2.8 for triply charged ions. Furthermore, peptides had to exhibit a dCn value

of >0.1, a Sp of >500, and an ion coverage of >50% to be considered. In

addition, each spectrum was evaluated manually with respect to its

plausibility.

Single target expression profiling. Single target expression profiling

(STEP) analysis was done on a plate containing cDNA of cancer and normaltissues from a variety of sources. Data was generated by quantitative real-

time PCR to the gene of interest and glycerine aldehyde-3-phosphate

Discovery of MHC Class II–Restricted Tumor Antigens

www.aacrjournals.org 10069 Cancer Res 2005; 65: (21). November 1, 2005



dehydrogenase (GAPDH; for standardization) in the same tube at the sametime on a Taqman (Applied Biosystems, Foster City, CA), running 40 cycles.A primer pair was generated, specifically picking up a sequence from exon9 of the melanotransferrin gene therefore being restricted to the longtranscript of melanotransferrin from which the antigenic epitope wasderived: 5V-Mtf1-I (5V-CAGTGCGTGTCAGCCAAGTC) and 3V-Mtf1-I (5V-TTCCCCGCCGTGTAAATGT). A site-specific probe sequence labeled witha fluorescent reporter dye and a fluorescent quencher dye was used fordetection, P-Mtf1-I (5V-AGCGTCGACCTGCTCAGCCTGG). The relativeexpression of the gene of interested was E = 2DCT. DCT is the differencein the thermocycles of the GAPDH gene versus gene of interest after whichthe fluorescent signal pierces the threshold. The expression of GAPDH ineach tissue was adjusted to the expression level of a panel of eighthousekeeping genes.

Results

Identification of HLA-DR–restricted tumor-associated pep-tides on dendritic cells. In the necrotic zones of tumors, dead

tumor cells or tumor cell debris may be ingested by immature

dendritic cells and delivered to the draining lymph node. In the

T cell–rich areas, dendritic cells might initiate a T-cell responseagainst tumor-specific epitopes presented by MHC molecules and

induce tumor-infiltrating lymphocytes (34, 35). To mimic in vitro

tumor cell uptake, processing and tumor antigen presentation bydendritic cells and to exploit this scenario for the identification of

MHC class II–restricted tumor antigens, we established a strategy,

denoted as MHC class II–associated peptide proteomics (MAPPs),

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 thepresence or absence of necrotic tumor cells. HLA-DR molecules were purified and peptides eluted. Peptide mixtures were fractionated by two-dimensional nano-HPLCand peptides concomitantly sequenced online by ESI-MS/MS. Identified peptides of pulsed and unpulsed dendritic cells were comparatively analyzed and thosepeptides presented only after encounter of necrotic tumor cells were further evaluated. B, fragment spectrum of [M+H+] of peptide GQDLLFKDATVRAVPV(m/zobserved = 1,728.7) derived from melanotransferrin (668-683) identified only in the HLA-DR-associated peptide mixture of dendritic cells after exposure to necroticMa-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 lettersand are annotated in the fragment spectrum.

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which allows the identification of self and foreign HLA-restrictedpeptides on as little as 1 to 5 � 106 dendritic cells (ref. 36; Fig. 1A).

That is, we stimulated dendritic cells with TNF-a and concomi-

tantly exposed them to necrotic MHC class II–negative tumor cells.Dendritic cells were allowed to ingest necrotic tumor cells for 24

hours, whereupon dendritic cells were lysed, HLA-DR-associated

peptides isolated via affinity beads and analyzed by two-

dimensional LC ESI-MS/MS. Through analysis of dendritic cellsthat were exposed to necrotic tumor cells, compared with dendritic

cells that were not, peptides that relied on the presence of tumor

cells could be identified. Table 1 shows a list of peptides that werepresented on HLA-DR molecules of dendritic cells, haplotype HLA-

DRB1* 0401/1301, after encounter of necrotic Ma-Mel-18a mela-

noma cells and absent on unpulsed dendritic cells. Only thoseamong f600 identified peptides are enlisted that were present in

two independent measurements of tumor-pulsed dendritic cells

and absent in both corresponding measurements of the autologous

unpulsed dendritic cells. Peptides were further evaluated andselected according to several quality criteria (see Materials and

Methods). By this means, 40 peptides derived from 13 different

proteins were identified. Many of these peptides were derived fromproteins that are ubiquitously expressed (e.g., from cytoskeletal or

matrix proteins like tubulin or collagen, or constitutively expressed

enzymes, like Lysyl hydroxylase or Ribophorin I). Others were derivedfrom proteins that have been described to be overexpressed in

certain cancers, like the regulator of G-protein signaling proteins

(37), Cathepsin D (38), Sirp a1 (39), or Vimentin (40). These proteins,however, are also ubiquitously expressed on healthy tissues. One of

the peptides that were selectively found only in the HLA-DR-

associated peptide repertoire of tumor-pulsed dendritic cells was

derived from the protein melanotransferrin, shown to be stronglyexpressed on melanoma cells (41). The melanotransferrin peptide

melanotransferrin (668-683) as well as its length variant melano-

transferrin (668-684) could be identified. The MS/MS spectrum of theidentified epitope melanotransferrin (668-683) is depicted in Fig. 1B .

The very same peptide could also be detected in an independent

experiment in which dendritic cells of a different donor of thehaplotype DRB1*0101/04011 were pulsed with necrotic Ma-Mel-18a

cells (data not shown), suggesting that the epitope is HLA-DR4

restricted and very abundant on MHC class II molecules of dendritic

cells after uptake of necrotic Ma-Mel-18a melanoma cells.To verify the identity of the discovered peptide, the synthetic

analogue of melanotransferrin (668-683) was applied to MS/MSfragmentation on the same Finnigan LCQ Ion Trap MS instrument.The fragmentation pattern of the synthetic peptide was almostidentical to the naturally processed peptide thereby confirming theidentity of melanotransferrin (668-683) (Fig. 2A). To our knowledge,neither CTL nor helper T-cell epitopes of melanotransferrin havebeen identified thus far.HLA-DR binding of melanotransferrin (668-683). The

identified melanotransferrin peptide possessed two overlappingHLA-DR binding motives, which may confer binding to a diverseset of HLA-DR molecules. The first motif comprises anchorresidues L-672, D-675, T-677, and A-680, whereas residues F-673,A-676, V-678, and V-681 constitute the second motif. Asmelanotransferrin (668-683) was identified in the peptiderepertoire of two donors sharing the HLA-DRB1*0401 molecules,binding of the synthetic peptide to HLA-DR4 was assessed by anin vitro peptide binding assay (Fig. 2B). As expected, melano-transferrin (668-683) showed high binding affinity to HLA-DR4,

even exceeding peptide HA (307-319), which is a strong binder in thecontext of HLA-DR4, HLA-DR1, HLA-DR2, and HLA-DR5. Melano-transferrin (668-683) was also superior to CDC27 (768-782) inbinding to HLA-DR4. In contrast to CDC27 (768-782), which was

Table 1. Neoantigens on dendritic cells (DRB1*0401/1301)after pulse with necrotic Ma-Mel-18a melanoma cells

Protein Peptide sequence Epitope

Melanotransferrin GQDLLFKDATVRAVPVG 668-684(melanoma-associated

antigen p97)*

GQDLLFKDATVRAVPV 668-683

Regulator of G-proteinsignaling 11 (RGS11)

cPALLPTPVEPTAACGPGGGD 445-464

Cathepsin Dc

IHHKYNSDKSSTYVK 119-133

VDQNIFSFYLSRDPDAQPGGE 224-244

NIFSFYLSRDPDAQPGGE 227-244NIFSFYLSRDPDAQPGGEL 227-245

IFSFYLSRDPDAQPG 228-242

NIFSFYLSRDPDAQPGG 227-243

NIFSFYLSRDPDAQPG 227-242Signal regulatory EPNNHTEYASIQTSPQPA 445-462

protein a 1 (Sirp a 1)c

NHTEYASIQTSPQPA 448-462

Vimentinc

LTNDKARVEVERDNLAEDIM 163-182

TNDKARVEVERDNLAEDIM 164-182DKARVEVERDNLAEDIM 166-182

NDKARVEVERDNLAEDI 165-181

NDKARVEVERDNLAEDIM 165-182LQEEIAFLKKLHEEEIQ 226-242

EEIAFLKKLHEEEIQ 228-242

LQEEIAFLKKLHE 226-238

LQEEIAFLKKLHEEE 226-240Ribophorin I

bGAKNIEIDSPYEISRAPD 377-394

LPEGAKNIEIDSPYEISRAPD 374-394

Lysyl hydroxylase 3b

NVPTVDIHM*KQVGYEDQ 606-622

Lysyl hydroxylase 1b

NVPTIDIHMNQIGFERE 595-611NVPTIDIHM*NQIGFER 595-610

VPTIDIHM*NQIGFER 596-610

NVPTIDIHM*NQIGFERE 595-611Putative

adenosylhomocysteinase 2bKRTTDVMFGGKQVVVCG 272-288

Protein pM5 precursorb

NAM*TFTFDNVLPGKYK 541-556

Collagen a 1(II) chainb

KSGDYWIDPNQGCTL 1225-1239KSGDYWIDPNQGCT 1225-1238

Tubulin h-5 chainb

GAKFWEVISDEHGIDPT 17-33

IGAKFWEVISDEHGIDPT 16-33

KFWEVISDEHGIDPT 19-33AKFWEVISDEHGIDPT 18-33

HUMAN 40S ribosomal DSHGVAQVRFVTGNKIL 55-71

protein S13b

SDDVKEQIYKLAKKGLTPSQ 29-48

DDVKEQIYKLAKKGLTPS 30-47VKEQIYKLAKKGLTPS 32-47

NOTE: HLA-DR-associated peptides of dendritic cells pulsed with Ma-Mel-18a and unpulsed autologous control dendritic cells were analyzedtwice by two-dimensional LC ESI-MS/MS. Only peptides that werepresent in the peptide repertoire of both pulsed dendritic cell samplesand absent in both unpulsed dendritic cell samples are listed.*Protein for which melanoma-associated expression has beendescribed. No ubiquitous expression.cUbiquitously expressed proteins that have been shown overexpressedin cancer tissues.bUbiquitously expressed proteinswithout apparent association to cancer.





identified as a tumor antigen in the context of HLA-DRB1*0401(35), melanotransferrin (668-683) displayed mediocre bindingaffinities to additional HLA-DR alleles, such as HLA-DR1, HLA-DR2 , and HLA-DR5 , accounting for almost 50% of the HLA-DRgenotypes among Caucasians. Haplotype promiscuity of melano-transferrin (668-683) was also higher than that of the tumorantigen NY-ESO (115-132), recognized by CD4+ T cells of patientswith NY-ESO-expressing tumors (42). Thus, melanotransferrin(668-683) apparently exhibits a reasonable degree of promiscuitywith regard to binding to allelic variants of HLA-DR.Expression of melanotransferrin in melanoma cell lines.

Melanotransferrin is a cell membrane protein that is attachedto the cell surface through a GPI anchor. We compared theexpression of melanotransferrin on melanoma cells and dendriticcells by flow cytometry using anti–melanotransferrin-specific mAbL235 (Fig. 3A). As expected from our experiments described above,dendritic cells apparently do not express melanotransferrin neitherin the immature nor in the mature state. The melanoma cell lineMa-Mel-18a, however, which was used as antigen source, exhibiteda relatively strong expression of melanotransferrin compared with

a panel of other melanoma cell lines and gave rise to thepresentation of melanotransferrin (668-683) by dendritic cells.Interestingly, when necrotic UKRV-Mel-15a cells that expresssubstantially less melanotransferrin than Ma-Mel-18a cells weresubjected to the same dendritic cells, no presentation of anymelanotransferrin peptide could be observed (data not shown).Both UKRV-Mel-15a and Ma-Mel-18a melanoma cell lines werenegative for MHC class II (Fig. 3B).MHC class II–positive UKRV-Mel-17 cells present melano-

transferrin (668-683). We asked whether melanoma cells thatcoexpress both melanotransferrin and HLA-DR molecules wouldthemselves present the melanotransferrin peptide. We thereforeanalyzed the HLA-DR-restricted self-peptide repertoire of themelanoma cell line UKRV-Mel-17, which strongly expresses bothmelanotransferrin and HLA-DRB1* 0401/03011 (Fig. 3A and B). Asmelanotransferrin (668-683) was identified in the context of HLA-DRB1*0401 (Table 1) and showed strong binding affinity for HLA-DR4 (Fig. 2B), we expected melanotransferrin (668-683) to be alsopresent in the HLA-DR-restricted peptide repertoire of UKRV-Mel-17 cells.

Figure 2. Fragmentation and HLA-DR binding of synthetic melanotransferrin (668-683). A, fragment spectrum [M+H+] of synthetic GQDLLFKDATVRAVPV. Observedpeptide fragments are indicated in bold. B, peptide binding assay. The concentration of each peptide required to reduce binding of biotinylated HA (307-319) by50% (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 foreach haplotype; bars, FSD.

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HLA-DR-molecules of ca. 5 � 107 UKRV-Mel-17 cells wereisolated, peptides eluted and subjected to two-dimensional LC ESI-MS/MS. 971 HLA-DR-associated peptides could be sequenced, bythis approach. Table 2 displays a subset of these peptides. We listedonly those peptides that were derived from proteins described tobe associated with cancer. Indeed, melanotransferrin (668-683) andits elongation variant melanotransferrin (668-684) were among theset of HLA-DR-associated self-peptides presented by this melano-ma cell line.Other epitopes present in the self-peptide repertoire of UKRV-Mel-

17 cells were derived from gp100, MART-1, melanoma-associatedchrondoitinsulfate-proteoglycan, matrix metalloproteinase-14(MMP-14), and melanoma differentiation–associated protein-9(syntenin). From each parent protein, one epitope and several ofthe respective length variants could be identified (Table 2). Thepeptide derived from gp100 sequenced here has been described ashelper T-cell epitope before (43). A helper T-cell epitope of MART-1has already been discovered in the context of HLA-DRB1*0401(44), The peptide identified here, however, has not been describedbefore. The other tumor-associated MHC class II peptides listedhere, to our knowledge, have not been identified, as yet.Melanotransferrin (668-683) is a helper T-cell epitope. To

assess whether melanotransferrin (668-683) may sensitize helperT cells, we isolated CD4+ T cells from PBMCs of an HLA-

DRB1*0401-positive healthy donor and repeatedly stimulatedthem with autologous dendritic cells that had been pulsed withmelanotransferrin (668-683). Melanotransferrin (668-683)–pulseddendritic cells induced a helper T-cell type 1 (TH1) response, asindicated by the specific release of IFN-g, whereas dendriticcells pulsed with control peptide did not (Fig. 4A). Release ofTH2 cytokines, such as IL-4, could not be observed in responseto melanotransferrin (668-683)–pulsed dendritic cells (data notshown). To verify that the melanotransferrin (668-683)–specificT-cell line recognized melanotransferrin (668-683) in thecontext of HLA-DR4, we did an MHC restriction analysis. TheT-cell line was stimulated with peptide-pulsed T2.DR4 orT2.DR1 transfectants. T2.DR4 cells were capable of inducing asubstantial T-cell response, whereas peptide-pulsed T2.DR1 cellsprovoked only a weak release of IFN-g (Fig. 4B). T-cellrecognition of T2.DR4 cells could be substantially inhibited by10 Ag/mL anti-HLA-DR mAb L243, whereas no effect wasobserved at the same concentration of MHC class I–specificcontrol mAb W6/32.When T-cell lines were stimulated by melanoma cell line UKRV-

Mel-17, which is presenting the melanotransferrin (668-683)epitope in the context of HLA-DRB1*0401, as identified by MS(Table 2), only a very weak T-cell response could be observed(Fig. 4C). This weak T-cell stimulation could be inhibited bycoincubation with the anti-HLA-DR mAb L243. When UKRV-Mel-17 cells were pretreated with IFN-g for 48 hours, carefully washed,and then cocultured with T cells, a stronger T-cell stimulationoccurred which was abolished by mAb L243. IFN-g pretreatment

Figure 3. Expression of melanotransferrin (MTf ) and HLA-DR on melanomacells. A, flow cytometric analysis of a panel of melanoma cells, immature (IM)and mature dendritic cells (DC ; Mat , stimulated with 1 Ag/mL LPS for 24 hours)stained with the melanotransferrin-specific mAb L235 (5 Ag/mL). Columns, meanfluorescence intensity of the specific staining for each cell type from threeexperiments; bars, FSD. Isotype controls for each cell type were set to a meanfluorescence intensity of 1. B, expression of HLA-DR on melanoma cell lines.Flow cytometry of melanoma cell lines Ma-Mel-18a, UKRV-Mel-15a, andUKRV-Mel-17 with mAb L243 (5 Ag/mL) specific for HLA-DR. Solid lines, isotypecontrol. One of three experiments with similar results.

Table 2. HLA-DR-restricted peptides of tumor-associatedproteins eluted from UKRV-Mel-17 melanoma cells(DRB1*0401/03011)

Protein Peptide sequence Epitope

Melanotransferrin(melanoma-associated

antigen p97)

GQDLLFKDATVRAVPVGQDLLFKDATVRAVPVG

668-683668-684

Gp100 (melanoma-associated ME20

NRQLYPEWTEAQRNRQLYPEWTEAQRLD

45-5745-59

antigen) RQLYPEWTEAQR 46-57

WNRQLYPEWTEAQR 44-57

WNRQLYPEWTEAQRLD 44-59MART-1 (Melan-A) APPAYEKLSAEQSPPPY 100-116

APPAYEKLSAEQSPPP 100-115

Melanoma-associated

chondroitinsulfate-proteoglycan NG2

(HSN tumor-specific

antigen)

GPWPQGATLRLDPTVLDAGEL

GPWPQGATLRLDPTVLDAGEGATLRLDPTVLDAGEL

GPWPQGATLRLDPTVLDAGELA

2053-2073

2053-20722058-2073

2053-2074

MMP-14 (MT-MMP 1,membrane-type-1

MMP)

GDKHWVFDEASLEPGGDKHWVFDEASLEPGYPK

384-398

Syntenin 1 (melanomadifferentiation–

associated protein-9)

ITSIVKDSSAARNGLLITSIVKDSSAARNGL

ITSIVKDSSAARN

ITSIVKDSSAARNGLLT

218-234218-233

218-231

218-235

NOTE: HLA-DR molecules were isolated from 5 � 107 cells, peptides

eluted and subjected to two-dimensional LC ESI-MS/MS. Enlisted

peptides are derived from proteins known to be tumor associated.





of UKRV-Mel-17 cells led to elevated expression of HLA-DR,melanotransferrin, and intercellular adhesion molecule-1 (datanot shown), aspects that may all contribute to the strongerrecognition of UKRV-Mel-17 cells by melanotransferrin (668-683)–

specific T cells. Pulsing of UKRV-Mel17 cells with exogenousmelanotransferrin (668-683) peptide to further increase thenumber of melanotransferrin (668-683)/HLA-DR complexes ledto a roughly 25-fold elevation of IFN-g release. When the same set

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of experiments was done with the MHC class II–negative controlmelanoma cell line UKRV-Mel-15a, no T-cell stimulation could beobserved (Fig. 4C).As the response of the T-cell line to naturally processed

melanotransferrin (668-683) epitope presented by UKRV-Mel-17was rather weak but could be increased by IFN-g pretreatmentor exogenous peptide administration, we supposed that theT-cell line that we generated from a healthy donor was a low-avidity melanotransferrin-specific T-cell line that required ratherhigh numbers of surface peptide/MHC class II complexes.Peptide titration experiments using mature autologous dendriticcells as antigen-presenting cells revealed that T-cell responsescould only be observed with peptide concentrations higher than0.1 Amol/L (Fig. 4D). Our results show that the mass spectro-metrically identified HLA-DR-restricted melanotransferrin pep-tide presented by dendritic cells after uptake of necrotic tumorcells is a true helper T-cell epitope as it proved to be immu-nogenic.Recognition of melanotransferrin (668-683) by T cells of

melanoma patients. We further wanted to explore whethermelanotransferrin (668-683) would also be suitable to induce aT-cell response in late-stage melanoma patients, as in thesepatients potentially reactive T cells might have been anergized ordeleted due to the induction of antigen-specific tolerance exertedby the tumor (45). PBMCs of three HLA-DRB1*0401-positivemelanoma patients were isolated and incubated with or withoutmelanotransferrin (668-683). After 7 to 14 days, these PBMCswere analyzed for their melanotransferrin (668-683)–specificreactivity in the presence of DRB1*0401-expressing T2 trans-fectants. Reactive T cells were enumerated by ELISPOT analysis.Melanotransferrin (668-683)–specific T-cell responses could bedetected in all three patients when PBMCs were presensitizedwith melanotransferrin (668-683) as indicated by the significantlyincreased number of IFN-g-producing cells (Fig. 4E ). Weconclude that late-stage melanoma patients do bear helperT cells that respond to the melanotransferrin (668-683) peptideantigen.Expression of melanotransferrin in normal and cancer

tissues. Melanotransferrin has been described to be melanomaassociated and absent on most other normal tissues (41). Toextend this knowledge and to verify melanotransferrin (668-683)as a tumor antigen that could be employed in immunotherapyagainst cancer, we assessed the expression of melanotransferrin(transcription variant 1, of which the epitope was derived) byreal-time PCR in a panel of 189 normal and 119 cancer tissues.A selection of several normal versus cancer tissues is shown inFig. 5A . Interestingly, not only melanoma cells but also lung

cancer and colon cancer primary tissues exhibited elevatedexpression of melanotransferrin. In the case of lung cancertissues, 4 of 14 assessed samples (f30%) showed an expressionthat was 12- to 45-fold increased over the average expression ofnormal lung tissues. The average expression of all lung cancersamples was about eight times higher than the averageexpression of all normal lung tissues. With respect to coloncancer and colon cancer metastasis, elevation of melanotrans-ferrin expression was not that distinct, however, in 9 of 40samples (f25%) expression levels were increased by >5-foldcompared with normal tissues. No increased expression could beobserved in prostate cancer (Fig. 5A), bladder, or kidney cancer(data not shown). Importantly, melanotransferrin was hardlyexpressed in normal tissues tested (Fig. 5A, dotted line). Slightlyelevated expression could only be observed in normal breast andsmall intestine tissues (data not shown). In depth analysis ofmRNA expression in different types of lung cancer furtherrevealed that melanotransferrin is overexpressed in differentneoplastic lung cell types but most pronounced in large cell lungcancer cells (Fig. 5B). Based on these results, melanotransferrinseems specifically overexpressed in certain cancers, whereas it isabsent on most normal tissues.

Discussion

The importance of tumor-reactive CD4+ helper T cells in thedevelopment of antitumor immunity has become increasingly clearover the past decade (17, 18, 46). The development of broadlyapplicable methods to expand the number of MHC class II–restricted tumor antigens, therefore, remains an important task intumor immunotherapy. In tumor vaccination, the use of syntheticpeptides compared with whole cell preparations or adoptivetransfer approaches has the advantage that peptides can easilybe manufactured and pharmaceutically formulated.We developed a procedure that allows us to identify HLA-DR

ligands of as little as 106 dendritic cells (36). Dendritic cells arelikely to be most critical in mounting antitumor immunity, asthey take up antigens from damaged tumor cells in tumorlesions, traffic to draining lymph nodes, and prime antigen-specific naive T cells (47, 48). To mimic this process in vitro , weincubated dendritic cells with necrotic tumor cells and identifiedHLA-DR-restricted antigenic peptides derived from tumor cellsthrough a highly sensitive two-dimensional LC ESI-MS/MSmethod. We believe that this approach has several advantagesover other current strategies:(a) Approaches using T cells as screening tools have the

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 specificrecognition of melanotransferrin (668-683) after five rounds of restimulation with autologous dendritic cells pulsed with peptide. Dendritic cells were activated withLPS (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 coculturedin 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 � 104 T2.DR4 or T2.DR1cells were used as antigen-presenting cells (APC ) and incubated with 1 � 105 T cells for 24 hours in the presence of 20 Amol/L peptide or peptide + 10 Ag/mL ofthe 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 with100 units/mL IFN-g for 48 hours or left untreated. UKRV-Mel-17 or UKRV-Mel-15a melanoma cells (5 � 104) were then incubated with (+TCL) or without 1 � 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 ofmelanotransferrin (668-683)–reactive T-cell line in response to dendritic cells pulsed for 24 hours with LPS and various concentrations of melanotransferrin (668-683) orcontrol CLIP (81-104). Columns (points), mean concentration in ng/mL (n = 3); bars, FSD. *, P < 0.05 (significant). E, detection of melanotransferrin (668-683)–specificT cells in the peripheral blood of HLA-DRB1*0401+ melanoma patients. PBMCs (6 � 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 assayby incubation with T2.DR4 cells as antigen presenters in the presence of melanotransferrin (668-683), Vim (202-217), or without (w/o) peptide. Columns, meansof three-well (EM, MD) and two-well (Asn) determinations; bars, FSD. *, P < 0.05, (significant).





to be generated, which can be particularly difficult for tononmelanoma tumors. (b) In case only low-affinity T-cell clonesare available, T-cell screenings might give negative results,although the antigenic peptide is presented by the tumor. (c)Screening by T cells is laborious and time consuming as itdepends on testing of overlapping peptides or peptides that havebeen predicted in silico by algorithms that are still far lessreliable in the context of MHC class II than MHC class Imolecules. (d) Epitopes identified by T-cell screening need to beconfirmed by testing of the respective recombinant protein in aT-cell assay, because cryptic epitopes might have been identified.(e) Our approach circumvents the limitation that most tumorsare MHC class II negative, as it identifies tumor antigens thatare presented by MHC class II molecules of dendritic cells. ( f)As whole tumor cell preparations are used in the present

approach, it is independent of purification or cloning steps ofpreselected tumor antigens pulsed onto antigen presenting cellsor cloned as invariant chain fusion proteins. This approach,however, bears the imminent danger that dendritic cells not onlytake up tumor-specific proteins but also housekeeping proteinsexpressed by any type of cell. To solve this issue, we subtract thebroad panel of HLA-DR–associated self-peptides derived fromdendritic cell–resident proteins (36) from those presented afteringestion of necrotic tumor material. Hence, we focus onepitopes that are either exclusively presented after ingestion oftumor cells and absent among the naturally occurring self-peptides on dendritic cells or peptides that are stronglyup-regulated in the presence of tumor cells. Following thisstrategy, several epitopes could be identified, which wereexclusively present on tumor-pulsed dendritic cells. However,

Figure 5. Single target expression profilingfor melanotransferrin. cDNA was generatedfrom a panel of normal versus cancertissues. Quantitative real time PCRwas done with primers specific formelanotransferrin (MTf ) and the referencegene GAPDH . Expression levels are givenas arbitrary units based on the relativeexpression of melanotransferrin mRNAversus normalized GAPDH in thecorresponding tissue. A, each filled boxis indicative of the expression ofmelanotransferrin mRNA per sample.The numbers above the panels for eachtissue show the average expression ofall samples of the corresponding tissue(horizontal line ). The following numbers ofsamples 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 cancermetastases, n = 15; prostate, n = 8;prostate cancer, n = 11. Dotted line,average expression of melanotransferrin inall normal tissues that were assessed.B, expression of melanotransferrin indifferent cell types of normal lung and lungcancer tissues. Expression is given inarbitrary units as in (A ). Abbreviation:NSCLC, non–small cell lung cancer.

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we also found peptides of ubiquitously expressed proteins (e.g.,vimentin, collagen, tubulin, and ribosomal protein S13), whichwere not present in the self-peptide repertoire of correspondingunpulsed dendritic cells. These proteins might particularly be setfree in large quantities after tumor cell necrosis, thereby givingrise to neoepitopes. The LC-MS method applied here does notidentify peptides by de novo sequencing but through comparisonof real mass spectra with theoretical spectra of a proteindatabase. To date, our approach therefore has the limitation thatepitopes containing sequence mutations that are not covered bythe protein database can not be detected. However, as suchmutations often only occur in distinct individuals, mutatedepitopes may be less broadly applicable for immunotherapycompared with those derived from nonmutated antigens, such ascancer testis and tissue-specific antigens expressed in a majorityof cancer patients.We concentrated our efforts on the peptide derived from

melanotransferrin, because melanotransferrin has already beendescribed to be a melanoma-associated protein (41). Melanotrans-ferrin was not expressed in dendritic cells, but it was stronglyexpressed by the MHC class II–negative tumor cell line Ma-Mel-18a, which was used as an antigen source in the presence ofdendritic cells. This provided evidence that melanotransferrin wastransferred from necrotic Ma-Mel-18a melanoma cells to dendriticcells, where it was processed and presented. Interestingly, the verysame melanotransferrin epitope could also be detected as a naturalself-peptide on the melanoma cell line UKRV-Mel-17, indicatingthat the peptide can indeed be generated by natural processing andwas unlikely to be created exogenously by proteases liberatedduring necrosis.Among the naturally presented self-peptides of the tumor cell

line UKRV-Mel-17, further epitopes from tumor-specific proteinscould be identified, one of which, gp100 (44–59), has alreadybeen described by others and proved to be immunogenic (43).We are therefore confident that the other novel HLA-DR-restricted peptides derived from MART-1, melanoma-associatedchondroitinsulfate-proteoglycan, MMP-14, and Syntenin 1 mayalso qualify as MHC class II–restricted tumor antigens, althoughT-cell assays will have to confirm the immunogenicity of thesepeptides.For melanotransferrin (668-683), the capacity to activate T cells

could be shown by in vitro T-cell sensitization experiments.Reactive T cells showed a high level of melanotransferrin (668-683)–specific IFN-g release, indicative of a TH1 response. Thisresult proves that nontolerant melanotransferrin (668-683)–specificnaive T cells exist in the naturally occurring T-cell repertoire, whichcould be stimulated by peptide vaccination.The induction of antigen-specific unresponsiveness by cancer

cells seems one of the predominant means by which tumorsevade the attack of the immune system. It is thus likely thatantigen-specific tolerance among T cells is of paramountimportance for tumor survival. Tolerance induction by tumorshas been shown for both CD4+ and CD8+ T cells (49, 50), and itis conceivable that in tumor patients, T-cell stimulation againstcertain antigens may fail due to such tolerization mechanisms.Hence, it was interesting to observe that peptide sensitizationexperiments carried out with PBMCs of HLA-DRB1*0401-positivelate-stage melanoma patients revealed that after a single roundof stimulation with melanotransferrin (668-683), substantialT-cell responses could be detected. The fact that a single roundof restimulation already induced a significant T-cell response

suggests that either the precursor frequency of melanotransferrin(668-683)–reactive T cells is high or that melanotransferrin (668-683)–specific T cells proliferate well. This finding supports ourcontention that under favorable conditions epitope melano-transferrin (668–683) may be capable of inducing an anti–melanotransferrin-specific immune response also in melanomapatients.Ideally, tumor peptides to be included in vaccination trials

should be applicable to a broad range of tumor patients bearinga variety of HLA haplotypes. A certain degree of promiscuity inhaplotype restriction is therefore desirable. Our peptide bindingassays showed that melanotransferrin (668-683) exhibited a verypronounced binding affinity for HLA-DR4 but also moderateaffinities for HLA-DR1, HLA-DR2 , and HLA-DR5 . These four HLA-DR alleles together cover f50% of the HLA-DR genotypes of theCaucasian population, implying that melanotransferrin (668-683)may be a broadly applicable tumor peptide vaccine.More importantly, tumor antigens to be included in cancer

vaccination trials have to exhibit tumor-specific expression or atleast substantial overexpression in tumors compared with healthytissues, to prevent the induction of potentially life-threateningautoreactive immune responses against tumor-unrelated tissues.Our expression profiling extended already existing melanotrans-ferrin expression data (41, 51, 52) and showed that melano-transferrin mRNA is present only in trace amounts in theassessed normal tissues. However, it was strongly overexpressedin tumor tissues (e.g., melanoma) but also certain other cancertypes such as lung and colon cancer, supporting our contentionthat melanotransferrin may qualify as a shared tumor antigen inimmunotherapy.Early studies using recombinant vaccinia virus vaccines against

melanotransferrin in mice and Macaca fascicularis monkeys haveshown that cell-mediated and humoral immune responses couldbe induced against melanotransferrin-expressing xenografts andtransfected syngeneic tumor cells resulting in in vivo rejection oftumor cells (53, 54). Despite the expression of trace amounts ofcross-reactive melanotransferrin in normal tissues of primates, noadverse effects or normal tissue damage could be observed aftereliciting the immune response against melanotransferrin (53).This suggests that vaccination with melanotransferrin (668-683)against melanotransferrin-expressing tumors may also be safelyapplicable in man.In conclusion, we have shown that our MAPPs strategy based

on human dendritic cells and LC/MS-MS is suitable for theidentification of tumor-associated MHC class II peptide antigens,which may be of value for vaccination against cancer. We believethat MAPPs provides a powerful complementation to alreadyexisting methods that aim at expanding the number of tumor-specific helper T-cell epitopes. Future vaccination with appropri-ate combinations of cytotoxic CD8+ and CD4+ helper T-cellepitopes may contribute to increase the success rate of cancerimmunotherapy.

Acknowledgments

Received 6/7/2005; revised 7/26/2005; accepted 8/23/2005.Grant support: Wilhelm-Sander-Stiftung.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 accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Nadine Daniel (Roche Pharmaceuticals, Basel, Switzerland) for experttechnical assistance, Bernd Muller (Roche Pharmaceuticals) for help with MS, andSilke Schnell for helpful discussions during her diploma thesis.





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Cancer Research




2005;65:10068-10078. Cancer Res Till A. Röhn, Annette Reitz, Annette Paschen, et al. Melanotransferrin Helper T-Cell EpitopeRestricted Tumor Antigens: Identification of a

−A Novel Strategy for the Discovery of MHC Class II

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