Identification of Breast Cancer Peptide Epitopes Presented by HLA-A*0201

Identification of Breast Cancer Peptide Epitopes Presented by

HLA-A*0201

Oriana E. Hawkins,† Rodney S. VanGundy,‡ Annette M. Eckerd,† Wilfried Bardet,† Rico Buchli,‡

Jon A. Weidanz,§,| and William H. Hildebrand*,†,‡

University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, Pure Protein, LLC,Oklahoma City, Oklahoma 73104, Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech

University Health Sciences Center, Abilene, Texas 79601, and Receptor Logic, Ltd., Abilene, Texas 79601

Received November 15, 2007

Cellular immune mechanisms detect and destroy cancerous and infected cells via the human leukocyteantigen (HLA) class I molecules that present peptides of intracellular origin on the surface of all nucleatedcells. The identification of novel, tumor-specific epitopes is a critical step in the development ofimmunotherapeutics for breast cancer. To directly identify peptide epitopes unique to cancerous cells,secreted human class I HLA molecules (sHLA) were constructed by deletion of the transmembraneand cytoplasmic domain of HLA A*0201. The resulting sHLA-A*0201 was transferred and expressed inbreast cancer cell lines MCF-7, MDA-MB-231, and BT-20 as well as in the immortal, nontumorigeniccell line MCF10A. Stable transfectants were seeded into bioreactors for production of >25 mg of sHLA-A*0201. Peptides eluted from affinity purified sHLA were analyzed by mass spectroscopy. Comparativeanalysis of HLA-A*0201 peptides revealed 5 previously uncharacterized epitopes uniquely presentedon breast cancer cells. These peptides were derived from intracellular proteins with either well-definedor putative roles in breast cancer development and progression: Cyclin Dependent Kinase 2 (Cdk2),Ornithine Decarboxylase (ODC1), Kinetochore Associated 2 (KNTC2 or HEC1), Macrophage MigrationInhibitory Factor (MIF), and Exosome Component 6 (EXOSC6). Cellular recognition of the MIF, KNTC2,EXOSC6, and Cdk2 peptides by circulating CD8+ cells was demonstrated by tetramer staining andIFN-γ ELISPOT. The identification and characterization of peptides unique to the class I of breast cancercells provide putative targets for the development of immune diagnostic tools and therapeutics.

Keywords: Human Leukocyte Antigen • Major Histocompatibility Complex • class I molecule • breastcancer • epitope • mass spectrometry

Introduction

Cells undergo a vast number of cellular changes duringtumorigenesis, including genetic mutation, alterations in geneexpression, and changes in protein processing. Some of thesechanges result in the secretion of biomarkers, such as theprostate specific antigen (PSA),1 which serve as indicators ofdisease. Other cancer-related markers can be recognized onthe outer surface of the cell by antibodies such as trastuzumabwhich binds the erbb2 growth factor receptor, specificallytargeting HER-2/neu overexpressing tumors.2 Unfortunately,the vast majority of cellular changes associated with tumori-genesis are not secreted or found at the surface of cancerouscells; most cancer markers are intracellular in nature.

To convey intracellular health to the immune system,mammals utilize the major histocompatibility complex (MHC)

class I molecule. Class I MHC molecules are nature’s proteomescanning chip. The MHC I molecules gather many smallpeptides of intracellular origin, including the products ofproteasomal processing and of defective translation, and carrythese intracellular peptides to the cell surface. Intracellularpeptides derived from proteins found in multiple compart-ments within the cell, and derived from proteins of manycellular functions, are sampled and presented at the cell surfaceby class I MHC.3,4 Immune cells including CD8+ cytotoxicT-lymphocytes (CTL) survey the peptides presented by class IMHC and target cells displaying cancer-specific peptides.5–7

Therefore, class I MHC presented peptides distinguish andpromote the recognition of cancerous cells by the adaptiveimmune system.

Given that MHC class I distinguish cancerous cells fromhealthy cells, a number of studies have aimed to identify classI MHC presented cancer antigens. Because class I MHCmolecules can be difficult to produce and purify, immune-based studies using CTL raised to autologous tumors have beenutilized to identify cancer immune targets.8 Other immune-based methods have relied upon predictive algorithms and invitro class I MHC peptide binding assays.9–11 Although these

* Corresponding author. University of Oklahoma Health Sciences Center,Department of Microbiology and Immunology, 975 NE 10th St., BRC 317,Oklahoma City, OK 73104. E-mail: [email protected].

† University of Oklahoma Health Sciences Center.‡ Pure Protein, LLC.§ Texas Tech University Health Sciences Center.| Receptor Logic, Ltd.

10.1021/pr700761w CCC: $40.75 2008 American Chemical Society Journal of Proteome Research 2008, 7, 1445–1457 1445Published on Web 03/18/2008

indirect approaches have identified putative tumor antigens,a direct proteomics based approach for identifying class I MHCtumor antigens is desirable. Proteomics-based methods arepositioned to directly indicate the number of epitopes thatuniquely decorate a cancer cell, serving to complement indirectimmune-based methods for cancer epitope discovery.

Recognizing the protein production, isolation, and charac-terization challenges associated with the direct analysis of classI MHC proteome scanning chips,12 we set out to obtainplentiful quantities of individual human class I MHC (HLA)from well-characterized cancer cell lines. Through expressionof a secreted human class I MHC (sHLA), the cell’s own classI remain on the cell surface and only the transfected sHLA isharvested. Moreover, secretion of the human class I MHCmolecule allows purification of the desired protein from tissueculture supernatants rather than isolating class I MHC frommore complex detergent lysates. We have shown that sHLApresent intracellular peptides as do cell surface class I.13 Thus,we have developed a method for producing and purifyingplentiful class I from cancerous cell lines. Once the class I isharvested from cancerous cells, we strip the sHLA of its peptidecargo and use comparative mass spectrometry to perusecancer-specific class I peptide epitopes.

Here, we directly compare class I HLA A*0201 presentedpeptide epitopes of breast cancer cell lines to those presentedby a nontumorigenic line. The class I HLA A*0201 allele wasselected for its high frequency in the population. Tumorigeniccell lines, MDA-MB-231, MCF-7, BT-20, and the nontumori-genic cell line MCF10A were transfected with the sHLA-A*0201construct. Peptides were purified from 25 mg of harvestedsHLA-A*0201 produced by each cell line. Comparative mappingof thousands of sHLA-A*0201 derived peptides by mass spec-trometry identified 5 previously uncharacterized epitopesunique to the tumorigenic cell lines. Through characterizationof protein expression, and by testing immune recognition ofthe epitopes, we provide preliminary validation for these 5putative breast cancer epitopes. The nature of these peptidesand their potential for immune targeting is discussed.

Materials and Methods

Tissue Culture. Tumorigenic breast cancer cell lines, MDA-MB-231, MCF-7, and BT-20 (ATCC), and a nontumorigenic,immortalized cell line, MCF10A (ATCC), were cultured inDMEM/F12K (Caisson Laboratories, North Logan, UT), 10%heat-inactivated FCS, and 100 units/mL Penn-Strep (Invitrogen,Carlsbad, CA). MCF10A culture medium was further supple-mented with 20 ng/mL cholera toxin (Calbiochem, San Diego,CA), 0.5 µg/mL hydrocortisone (Sigma, St. Louis, MO), 10 µg/mL recombinant human insulin (Wisent, Saint-Jean-Baptistede Rouville, Quebec, Canada), and 20 ng/mL recombinanthuman epidermal growth factor (Wisent).14

Secreted HLA Production. To produce secreted HLA mol-ecules, R-chain cDNAs of the most common HLA allele, A*0201,were modified at the 3′ end by PCR mutagenesis to deletecodons 5–7 encoding the transmembrane and cytoplasmicdomains and to add a 30 base-pair tail encoding the 10 aminoacid rat very low density lipoprotein receptor (VLDLr), SVVST-DDDLA, for purification purposes.15 sHLA-VLDLr was clonedinto the mammalian expression vector pcDNA3.1(-) Geneticin(Invitrogen) and then sequenced to ensure fidelity of eachclone.

Cell Transfection. Breast cancer cell lines (MCF-7, MDA-MB-231, and BT-20) and an immortal, nontumorigenic breast

epithelial cell line (MCF10A) were transfected with sHLA-A*0201using the FuGENE 6 Transfection Reagent kit (Roche Diagnos-tics Corp., Indianapolis, IN). Briefly, cells were grown incomplete media to 80–85% confluency after which they weretrypsinized and plated at 2 × 105 cells/well in a 6-well tissueculture plate (Falcon, Becton Dickinson Labware, FranklinLakes, NJ) in 1 mL of serum-free media and grown overnightto reach 50–80% confluency before transfection. Cells weretransfected by adding 100 µL of serum-free media containing1 µg of DNA at 1:3 ratio DNA/FuGENE. Plates were incubatedafter transfection for 24 h, and then received 1 mL of completeselective media containing the appropriate concentration ofantibiotic.

VLDLr Capture ELISA. Ninety-six well StarWell Maxisorpplates (Nalge Nunc International) were coated with 200 µL ofmouse monoclonal anti-VLDLr (ATCC clone CRL-2197) anti-body at 10.0 µg/mL in carbonate buffer, pH 9.0. Plates wereincubated at 4 °C overnight and then blocked with 3% BSA inPBS for 2 h at room temperature. Standards were set intriplicates at 100, 80, 60, 40, 20, 10, 5, and 0 ng/mL (blank),using a known VLDLr-tagged sHLA molecule as a standardprotein. Samples were incubated for 1 h at 37 °C. Detection ofsHLA molecules was performed using rabbit anti-�2microglo-bulin (DAKO, Denmark) and HRP-donkey anti-rabbit (JacksonImmunoResearch Laboratories) incubated 30 min each at roomtemperature. Following a 30 min development with OPD(Sigma), the reaction was stopped with 3N H2SO4 and plateswere read at 490 nm. Samples were quantified by comparingthem to sHLA standards.

Subcloning and Large-Scale Production. Transfected cellsgrown in selective antibiotic media were tested for productionof sHLA molecules by ELISA. Positive wells were trypsinizedand subcloned into 96-well plates (Falcon) by single cell sortingusing the Influx Cell Sorter (Cytopeia, Seattle, WA). Individualwells with subcloned cells were tested for the production ofsHLA, and positive wells were expanded for inoculation intobioreactors (Toray, Tokyo, Japan) in a CP2500 Cell Pharm(Biovest International, Minneapolis, MN) (Scheme 1).

Peptide Purification. Cell supernatants were passed over asepharose 4B precolumn to remove excess milk fat. Ap-proximately 25 mg of A*0201VLDLr molecules from each cellline was purified over an affinity column composed of anti-VLDLr antibody coupled with CNBr activated Sepharose 4B (GEHealthcare, Piscataway, NJ). sHLA molecules were then elutedin 0.2 N acetic acid, brought up to 10% acetic acid, and heatedto 78 °C for 10 min. Peptides were separated from heavy andlight chains by ultrafiltration in a stirred cell with a 3 kDamolecular weight cutoff cellulose membrane (Millipore, Bed-ford, MA). Each peptide batch was flash-frozen and lyophilized.The peptides were then reconstituted in 10% acetic acid. Tobe certain the peptides were derived from the HLA moleculeof interest, 10% of the pooled peptides from each cell line wassubjected to 14 rounds of Edman sequencing to confirm anA*0201 binding motif.16

Reversed-Phase HPLC. Peptides were reversed-phase HPLCfractionated with a 4 µm, 90 Å, 2 × 150 mm Jupiter Proteo C12column (Phenomenex, Torrance, CA) on a Paradigm MG4system (Michrom Bioresources, Auburn, CA) with a 1 mLstainless loop using an CH3CN gradient as follows: 2% B for 11min. (80 µL/min), 2–5% B in 0.02 min (80–160 µL/min), 5–40%B in 40 min (160 µL/min), and 40–80% B in 20 min (160 µL/min). Composition of solvents was as follows: solvent A, 98%H20, 2% CH3CN, and 0.1% TFA (trifluoroacetic acid); and

research articles Hawkins et al.

1446 Journal of Proteome Research • Vol. 7, No. 4, 2008

solvent B, 95% CH3CN, 5% H20, and 0.08% TFA. Approximately250 µg of total peptide was separated into 40 0.7-min fractions.UV absorption was monitored at 215 nm. Consecutive andidentical peptide separations were performed for each peptide-batch.

Mass Spectrometric Analysis. Peptide fractions were con-centrated to dryness by Speed-Vac and reconstituted in 20 µLof nanospray buffer composed of 50% methanol, 50% H20, and0.5% acetic acid. Nanoelectrospray capillaries (Proxeon, Den-mark) were loaded with 1 µL of each peptide fraction andinfused at 1100 V on a Q-Star Elite quadrupole mass spectrom-eter with a TOF (time-of-flight) detector (Applied Biosystems,Foster City, CA) for 5 min. Triplicate ion maps were generatedfor each fraction in a mass range of 300–1200 amu. MS peaklists were generated with a threshold of 500 counts in AnalystQS 2.0 (ABI/MDS Sciex) and aligned for each fraction and eachcell line using an internally generated Excel (Microsoft, 2003)script. Ions excluded from the alignment of tumorigenic versusnontumorigenic peak lists were selected as potentially unique.Spectra from corresponding fractions of each cell line were,also, aligned and visually assessed with a 20 amu window forthe presence of unique ion peaks.

Unique peaks selected for further analysis were subjectedto tandem mass spectrometry (MS/MS) and an amino acidsequence assigned to centroided, deisotoped data using thepublicly available, Web-based MASCOT (Matrix Science Ltd.,London, U.K.) and/or de novo sequencing. Search engine

parameters were as follows: database NCBinr, human species,no enzyme, phosphorylation or sulfation allowed, and masstolerance of 0.5 Da for both precursor and MS/MS data.Synthetic peptides, corresponding to each putative sequence,were produced and subjected to MS/MS under identicalcollision conditions as the naturally occurring peptide. Thespectra produced were compared to confirm peptide sequenceidentity.

Synthetic Peptides. Unmodified peptides were synthesizedand purified by the Molecular Biology Resource Facility (TheUniversity of Oklahoma HSC, Oklahoma City, OK). Purity wasdetermined to be greater than 95%. The composition wasascertained by mass spectrometric analysis.

IC50. The binding affinity of the individual peptides for theHLA A*0201 was determined using a competitive binding,fluorescence polarization based assay, PolyTest (Pure Protein,LLC, Oklahoma City, OK).17 In brief, the peptide of interest isincubated with a FITC (fluorescein isothiocyanate) labeledreference peptide and soluble HLA class I heavy and lightchains. Displacement of the reference peptide by the competi-tor results in increased rotational mobility of the labeledpeptide and decreased polarization. The IC50 is determined bythe concentration of the competing peptide required to inhibit50% binding of the reference peptide to the HLA molecule. Forthis assay, an IC50 < 5000 nM is considered high affinity.

Western Blot. Cell lysates were generated from cell linesusing the RIPA buffer and HALT Protease Inhibitor Cocktail

Scheme 1. View of the Overall Strategy for Identifying Peptide Epitopes Uniquely Presented by the HLA A*0201 of Breast CancerCells

Breast Cancer Peptide Epitopes Presented by HLA-A*0201 research articles

Journal of Proteome Research • Vol. 7, No. 4, 2008 1447

Kits (Pierce) according to manufacturer’s instructions. SDS-PAGE was performed with 10 µg of total protein loaded onto4–12% NuPAGE Bis-Tris precast gels in MOPS buffer (Invitro-gen). Protein was blotted on PVDF membrane and probed withmouse monoclonal anti-ODC1, anti-Cdk2, anti-KNTC2 (NovusBiologicals), or rabbit polyclonal anti-EXOSC6 (Abcam). Pro-teins were detected using HRP-conjugated Donkey anti-mouseIgG or Donkey anti-rabbit IgG (Jackson Immunoresearch) andSuperSignal Chemiluminescent Substrate (Pierce). All blotswere stripped with Restore Western Blot Stripping Buffer(Pierce) and reprobed with mouse monoclonal anti-�actin(Sigma).

MIF protein was immunoprecipitated from 1 mL of 3 dayconfluent cell culture supernatants using mouse monoclonalanti-MIF (Novus Biologicals) coated Protein G sepharose beads(GE Healthcare). Electrophoresis and detection were performedas above.

Research Participants. Subjects, with or without a priorhistory of breast cancer (Ductal Carcinoma In Situ or InfiltratingDuctal Carcinoma), were recruited according to OUHSC In-stitutional Review Board approved protocol number 13571.Participant HLA type was determined by Sequence BasedTyping and confirmed by flow cytometry of PBMC stained withFITC labeled BB7.2 anti-HLA-A*0201 antibody. Nine HLA-A*0201 positive subjects were identified from each group. Fortymilliliters of whole blood was collected from each participantand processed for Peripheral Blood Mononuclear Cells (PBMC).

Tetramer Staining. HLA-A*0201 positive PBMC were sepa-rated by Lymphoprep gradient (Axis-Shield). PBMC wereresuspended in Cell Staining Buffer (Biolegend), and 1 × 106

cells were stained for 30 min at 4 °C in a 1:100 dilution ofAllophycocyanin (APC) labeled MHCI tetramer (NIH TetramerFacility or Protein Chemistry Core, Baylor College of Medicine),FITC anti-CD8R (Biolegend), and PerCP-Cy5.5 anti-CD3 (Bec-ton Dickenson). PBMC were washed, fixed in 1% paraformal-dehyde (PFA) in phosphate buffered saline (PBS), and analyzedon a FACScaliber (Becton Dickenson).

ELISPOT. Fresh PBMC were stimulated for 1 week with 2µg of peptide in RPMI 1640 with 10% fetal bovine serum.Recombinant human IL-2 (Invitrogen) was added at 0.3 ng/mL on days 3, 5, and 7. PBMC were rested 48 h prior toELISPOT. A total of 1 × 105 cells/well were plated on anti-human IFN-γ coated plates (SeraCare) with 2 µg of peptide orPhytohemagglutinin-H (Sigma) as a positive control. Plateswere developed according to kit instructions. Plates were readon an ImmunoSpot plate reader and analyzed using Immu-noSpot v. Four (Cellular Technology, Ltd.).

Results

MS Comparative Analysis. Peptides were eluted from 25 mgof purified sHLA A*0201 harvested from three tumorigenicbreast epithelial cell lines (MCF-7, MDA-MB-231, and BT-20)and the nontumorigenic breast epithelial line (MCF10A). TheA*0201 peptide binding motif was confirmed by Edmansequencing 10% of pooled peptides from each cell line (datanot shown). The peptide batches were consecutively fraction-ated by RP-HPLC (Supplementary Figure 1 in SupportingInformation). During mass spectrometric analysis, alignmentof corresponding fractions was confirmed by the presence ofidentical peptides across the panel.

Triplicate MS ion maps were generated from each of 40peptide containing fractions for each cell line. Peak lists fromtumorigenic and nontumorigenic peptide batches were aligned

from corresponding fractions and excluded peaks were treatedas potentially unique to the tumorigenic lines. Additionally,spectra from corresponding fractions were examined visuallyto identify or confirm the presence of ion peaks that couldrepresent peptides unique to the tumorigenic lines. Five ionpeaks were identified as being shared among tumorigenic celllines and absent from the MCF10A. These peaks were +2 ionsat 536.32 m/z (Figure 1), 539.8 m/z (Figure 3), 492.77 m/z,462.24 m/z, and 500.77 m/z (Supplementary Figures 2, 4, and6 in Supporting Information).

Identification of Peptides Uniquely Presented by HLA-A*0201. Potentially unique ions were selected for MS/MSfragmentation and a sequence assigned using MASCOT. Theproduct ion spectra are shown in Figures 2 and 4 and inSupplementary Figures 3, 5, and 7 in Supporting Information.The peptide sequence of ion peak 536.32 m/z was ILDQKINEVwhich corresponds to postions 23–31 of Ornithine Decarboxy-lase (ODC1). The sequence of ion peak 539.8 m/z wasFLSELTQQL which corresponds to positions 19–27 of Mac-rophage Migration Inhibitory Factor (MIF). The sequence ofion peak 492.77 m/z was ALMPVLNQV which corresponds topositions 214–222 of Exosome Component 6 (EXOSC6). Thesequence of ion peak 462.24 m/z was KIGEGTYGV whichcorresponds to positions 9–17 of Cyclin Dependent Kinase 2(Cdk2). The sequence of ion peak 500.77 m/z was GLNEEIARVwhich corresponds to positions 330–338 of Kinetochore As-sociated 2 (KNTC2 or HEC1). Table 1 provides a more detaileddescription of the peptides. Most of the source proteins of thesepeptides, such as ODC, MIF, KNTC2, and Cdk2, have well-defined roles in the development and progression of manycancers which are addressed in the Discussion. The role ofEXOSC6 in tumor development is unclear, but putative as-sociations are possible.

We sequenced over 150 peptides presented by the HLAA*0201 from the 3 tumorigenic cell lines and the nontumori-genic line. Most of these peptides were shared by all 4 cell linesand were used to ensure proper alignment of the fractions,including the bona fide CTL epitope, GLIEKNIEL from DNAmethyl transferase 1.18 A few peptides were unique to anindividual cell line, such as LLQEVEHQL from the E3 ubiquitinligase TRIM37 found only in the MCF-7 peptide pool. Thepeptides corresponding to ODC1, Cdk2, EXOSC6, and KNTC2were presented by the HLA A*0201 of all three tumorigenic linesand missing from the MCF10A pool. The MIF peptide was onlyidentified in the MCF-7 and MDA-MB-231 batches. Althoughseemingly absent from the BT-20 batch, the MIF peptide mayin fact be present at low concentration and therefore maskedby the isotope of the overlapping peptide at 539.26 m/z. Furtherseparation would be required to confirm or deny the possibility.However, the relevance of MIF to tumor development, progres-sion, and metastasis makes it an attractive target even if itspresentation is limited to a subset of tumors.

Validation of Unique Peptide Ligands. Three fractionspreceding and following the fraction of interest were examinedto confirm the unique nature of these peptides. Syntheticpeptides were produced and subjected to MS/MS underidentical collision conditions, and spectra were compared withnative peptide to confirm peptide sequences (Figures 2 and 4,Supplementary Figures 3, 5, and 7 in Supporting Information).The 5 peptides identified were determined to have high affinityfor the HLA A*0201 using a competitive binding, fluorescencepolarization based assay (Table 1).



Confirmation of Protein Expression. Western blotting wasperformed to confirm expression of the peptide source proteinsby the different cell lines (Figure 5). A 75 kDa band was detectedby the anti-KNTC2 antibody at various levels in lysates fromall four cell lines. A 32 kDa band was detected by the anti-Cdk2 antibody at almost identical levels in all four cell lines. A28 kDa band was present in all four lysates, corresponding to

EXOSC6. The secreted protein MIF was not detected in anylysates but could be immunoprecipitated from tissue culturesupernatants. In Figure 5, immunoprecipitated MIF is visibleas a band at 12 kDa. Interestingly, the BT20 cell line producedthe highest level of MIF but did not present MIF peptide onthe HLA molecule. ODC1 was faintly detected as a 53 kDa bandin lysates from MDA-MB-231, BT-20, MCF-7, and MCF10A cell

Figure 1. MS ion map showing a unique +2 peak at 536.32 m/z in corresponding peptide fractions from (A) MCF-7, (B) MDA-MB-231,(C) BT-20, and (D) the nontumorigenic MCF10A. Note: The ion peak at 532.79 m/z is shared by all four cell lines and corresponds to apeptide derived from RPL5.



lysates (Figure 5). The source proteins were expressed by allcell lines, suggesting a disconnection between expression andclass I presentation.

Immune Recognition of Breast Cancer Associated Pep-tides. Fresh PBMC from 11 HLA-A*0201 subjects with orwithout a history of breast cancer were stained with HLA-A*0201 tetramers comprised of ODC1, Cdk2, KNTC2, EXOSC6,MIF, and the Epstein–Barr Virus BMLF1 peptides. EBV BMLF1represents a positive control. Subject 6, with a positive history

of breast cancer, displayed CD8+ recognition of the Cdk2,EXOSC6, EBV control tetramers (Figure 6).

To test for functional immune recognition of the newlydiscovered breast cancer epitopes, PBMC from 6 subjectswere stimulated in vitro for 1 week prior to IFN-γ ELISPOTtesting. Subjects 1, 3, 4, 5, and 6 had a positive history ofbreast cancer, while subject 2 had no history of breast cancer.Subject 1 produced a relatively robust IFN-γ response toKNTC2 and MIF (Figure 7). Subject 6 produced a robust

Figure 2. Product-ion spectra of an ESI produced +2 ion, 536.32 m/z, in corresponding peptide fractions from (A) MCF-7, (B)MDA-MB-231, (C) BT-20, (D) the nontumorigenic MCF10A, and (E) Synthetic. Sequence of A-C and E is peptide 23–31 (ILDQKINEV)of ODC1.



response to EXOSC6, which recapitulates the tetramer stain-ing (Figure 6). Interestingly, subject 6 PBMC did not produceIFN-γ in response to Cdk2 (Figure 7) despite staining ofCD8+ cells with Cdk2 tetramers (Figure 6). Further pheno-typic characterization of these Cdk2 tetramer +/IFN-γELISPOT– cells is warranted.

Discussion

Given that class I HLA molecules decorate the cell surfacewith intracellular peptide epitopes, a number of indirectmethods have been used to identify HLA associated tumorrejection antigens. Purification of HLA associated peptides from

Figure 3. MS ion map showing a unique +2 peak at 539.8 m/z in corresponding peptide fractions from (A) MCF-7, (B) MDA-MB-231, (C)BT-20, and (D) the nontumorigenic MCF10A. Note: Peak 539.76 m/z in panel C is an isotope of 539.26 m/z.



cell lysates has also revealed a small number to tumor antigens.However, innate difficulties associated with protein production,purification, and peptide yield make direct analysis with theHLA molecule and its peptide ligands problematic whenworking from detergent cell lysates. Recognizing the power ofHLA class I to distinguish cancerous cells, we developed amethod for producing plentiful class I without detergent lysis.We then isolated the class I peptide cargo and comparedcancerous and noncancerous peptide epitopes by mass spec-troscopy. This approach provides a direct proteomics view of

the peptide epitopes that decorate well-characterized breastcancer cell lines. In this study, we identified five epitopes thatrepresent intuitive targets for breast cancer therapies as wellas therapies directed to a variety of other tumor types. Below,we discuss the characteristics of the parental proteins and theirrelevance to cancer.

ODC. Ornithine decarboxylase is an enzyme required forpolyamine synthesis. It catalyzes the initial conversion ofL-ornithine to putrescine, which is subsequently converted tospermidine and then spermine by S-adenosylmethionine de-

Figure 4. Product-ion spectra of an ESI produced +2 ion, 539.8 m/z, in corresponding peptide fractions from (A) MCF-7, (B) MDA-MB-231, (C) BT-20, (D) the nontumorigenic MCF10A, and (E) Synthetic. Sequence of A, B, and E is peptide 19–27 (FLSELTQQL) of MIF.



carboxylase. These polyamines act as organic cations and arerequired for cellular proliferation, differentiation, and trans-formation. The Odc gene promoter is a target of the oncogenemyc, Ras activation pathways19 and estrogen mediated activa-tion through cAMP/PKA.20 Through mRNA microarray, Westernblot, enzymatic activity, and immunohistochemistry, a greatdeal is known about the expression patterns of ODC in primarytissues and numerous cell lines, including those examined inthis study.21–23 Expression of ODC tends to be very low interminally differentiated tissues but very high and even prog-nostic in numerous tumors, including but not limited to breast,lung, and prostate cancer.24,25 Polyamine analogues and ODC-targeted siRNA have been shown to induce cell cycle arrest andinhibit proliferation and tumor invasiveness.26 ODC protein hasa high turnover rate mediated by ubiquitin-dependent and-independent mechanisms that target the protein to theproteasome,19 the source of MHC class I peptides. The IL-DQKINEV peptide was previously eluted from the TAP defi-cient, HLA- A*0201 positive T2 cell line, a T × B cell hybridoma,transfected with TAP1 and either the TAP2*B or TAP2*Bky2alleles.27 Overexpression coupled with a high rate of protea-somal degradation make ODC a prime target for HLA presenta-tion on cancer cells. Although we detected no immune recog-nition by our cohort of study participants, we recognize thepossibility of cellular recognition with immunization or target-ing of the peptide/HLA complex by T cell Receptor mimicantibodies (TCRm).28

MIF. Macrophage Migration Inhibitory Factor is a multi-functional cytokine produced by a variety of normal and tumorcell types. MIF protein suppresses T29 and NK30 cell activity

and may play at least a contributory role in maintainingimmune privilege in the eye and the maternal/fetal interface.31

MIF binds the cell surface CD74 receptor which signals throughthe MAPK pathway to activate proliferation via cyclin D1, AP-1mediated up-regulation of pro-inflammatory cytokines, andcellular adhesion molecules which play a role in tumor me-tastasis.32 In addition, MIF inhibits apoptosis by activation ofAkt33 and by suppression of p53 mediated via E2F pathwaymodulation34 and COX-2 activation.35 MIF expression is im-portant for neo-angiogenesis, proliferation, and invasivenessof neuroblastoma,29 hepatocellular,36 breast,37 prostate,38 andgastric carcinomas.39 Although the presentation of MIF-derivedpeptides by the HLA molecule has not been previously de-scribed, MIF protein expression by the four cell lines has beendemonstrated here and by others.40,41 Protein expression datatherefore suggests that MIF peptide presentation is plausible,while an IFN-γ response to MIF demonstrates that the immunesystem can respond to class I HLA presented MIF peptides.Given the significance of MIF relative to tumor metastasis, weintend to pursue further investigation of this MIF epitope as atarget for immune therapies.

EXOSC6. Exosome Component 6 is one of 11–16 exonu-cleases that make up the human exosome and is the homo-logue of the yeast mRNA Transport Regulator 3 (Mtr3p). Themammalian cell contains nuclear exosomes, responsible forprocessing of the 5.8 S rRNA, small nuclear RNAs (snRNA), andsmall nucleolar RNAs (snoRNA), and cytoplasmic exosomes,responsible for the 3′-5′ degradation of mRNAs containing AUrich elements (AREs) within the 3′ UTR. ARE containingmRNAs, generally, have short half-lives of 5–30 min includinga large number of tumor associated transcripts, such as c-myc,cyclin D1, and COX-2. Degradation is mediated by the AREbinding protein, AUF1. These transcripts are often stabilizedin cancer by the overexpression of Hu family ARE bindingproteins, which displace AUF1.42 A direct role for the exosomeand its components in tumorigenesis is unclear. However,deregulation of RNA turnover can result in cellular transforma-tion, so a putative role for the exosome in tumorigenesis isreasonable.43 Interestingly, several exosome components areautoantigenic with a high degree of association to HLA-DR3.Patients suffering from poly-myositis and scleroderma, forwhich the complex was originally named (PM/Scl), have hightiter antibodies primarily to the PM/Scl 100 component.44 InFigure 5, we demonstrate expression of the EXOSC6 protein inall 4 cell lines such that cancer-specific cellular mechanisms

Table 1. Peptides Identified as Uniquely Presented by the sHLA-A*0201 of Tumorigenic Breast Epithelial Lines

peptide sourceprotein presenting cell lines

precursorion charge

peptidesequence

sequencecoverage

NCBI ginumber IC50 (nM)

MASCOTscore

associatedcancers

Cyclin DependentKinase 2 (Cdk2)

MCF-7, BT-20, MDA-MB-231 462.2 +2 KIGEGTYGV 9–17 16936528 745 56 Breast, prostate,lung, colon,ovarian

MacrophageMigrationInhibitory Factor(MIF)

MCF-7, MDA-MB-231 539.8 +2 FLSELTQQL 19–27 148608029 173 47 Breast, ovarian,prostate, gastric,colon, lung

ExosomeComponent 6(EXOSC6)

MCF-7, BT-20, MDA-MB-231 492.8 +2 ALMPVLNQV 214–222 17402904 453 49 Unclear, RNAprocessing couldplay a role inmany

OrnithineDecarboxylase(ODC1)

MCF-7, BT-20, MDA-MB-231 536.3 +2 ILDQKINEV 23–31 4505489 907 50 Breast, pancreatic,colon, liver, lung,leukemia

KinetochoreAssociated 2(KNTC2 or HEC1)

MCF-7, BT-20, MDA-MB-231 500.8 +2 GLNEEIARV 330–338 116283259 501 54 Lung, prostate,breast, ovarian,lymphoma, glioma

Figure 5. Western blot showing 75, 53, 32, 28, and 12 kDa bandsrepresenting KNTC2, ODC1, Cdk2, EXOSC6, and MIF, respec-tively. �-Actin is a loading control. Lanes (1) MDA-MB-231, (2)BT-20, (3) MCF-7, and (4) MCF10A cell lysates.



affecting protein decay may be involved in the class I HLApresentation of this peptide. The EXOSC6 peptide, ALMPVL-NQV, identified here as uniquely expressed by the tumorigenic

breast epithelial lines, was previously eluted from an ovariancarcinoma line, UCI-107:45 This peptide may be presented ona range of tumor cells. With ELISPOT and tetramer staining

Figure 6. Tetramer vs CD8 staining of PBMC from Subject 6. (A) EBV BMLF1–A*0201 tetramer, (B) Cdk2–A*0201 tetramer, (C)ODC1–A*0201 tetramer, (D) EXOSC6–A*0201 tetramer, (E) KNTC2–A*0201 tetramer, and (F) MIF–A*0201 tetramer.

Figure 7. IFN-γ ELISPOT. Subject PBMC were stimulated with peptide and IL-2 1 week prior to ELISPOT. A total of 1 × 105 cells/wellwere plated with 2 µg of peptide or PHA-P as a positive control.



confirming immune recognition, the EXOSC6 epitope may actto distinguish a number of cancerous cells for immune surveil-lance mechanisms.

Cdk2. Cyclin Dependent Kinase 2 is a serine/threoninekinase that complexes with cyclin E to mediate the terminalphosphorylation and inactivation of retinoblastoma protein.This in turn releases sequestered E2F transcription factors,allowing transcription of genes required for G1 to S phasetransition.46 Induction of cyclin D1, Cdk 4/6, cyclin E, and Cdk2can be accomplished in tumors via loss of INK4 Cdk inhibitors,such as p16,47 or stimulation by mitogens, such as insulin,insulin-like growth factor I, and estrogen.48 Increased expres-sion and increased activation of cyclin E and Cdk2 are reportedin numerous tumor types, including breast,49,50 prostate,51

ovarian,52 and lung carcinomas.53 The presentation of Cdk2derived peptides by the HLA molecule has not been previouslydescribed. However, we and others have demonstrated proteinexpression in all 4 cell lines characterized in this study, makingpeptide availability to the HLA molecule probable.54 Degrada-tion of cell cycle associated proteins is tightly regulated suchthat disregulation of Cdk2 processing in tumorigenic cells maywell result in presentation by an HLA molecule. In Figure 6,we show that CD8+ cells in a breast cancer patient recognizethe Cdk2 tetramer, although the lack of an IFN-γ response inthe presence of CD8+ tetramer staining suggests a populationof regulatory cells. How the immune response views CdK2needs further exploration.

KNTC2. Kinetochore Associated 2, also known as HighlyExpressed in Cancer (HEC1), is required for proper chromo-some segregation. KNTC2 and Nuf 2 form a contact point formicrotubule attachment to the kinetochore complex duringmitotic spindle assembly55 and may act as a spindle check-point.56 KNTC2 binds the C-terminus of retinoblastoma protein(Rb) and interacts with the 26S proteasome subunit MSS1 toinhibit degradation of mitotic cyclins during M phase.57 In theabsence of Rb, abnormal expression of spindle checkpointproteins can lead to uncoupling of mitosis from the cell cycleand aneuploidy.58 KNTC2 is expressed only in actively prolif-erating cells and was identified as 1 of 11 genes correspondingto a “Death from Cancer” expression signature.59 KNTC2 isoverexpressed in numerous tumor types, including prostate,breast, lung, ovarian, lymphoma, mesothelioma, medulloblas-toma, glioma, and acute myeloid leukemia.60–63 Presentationof KNTC2 derived peptides by HLA molecules has not beenpreviously described, but the confirmed expression of KNTC2by all cell lines is consistent with epitope presentation (Figure5).64 Again, loss of control over a highly regulated system mayexplain the differential presentation of KNTC2 peptide by thetumorigenic and nontumorigenic cell lines. An IFN-γ responseto the KNTC2 peptide indicates this protein is immunogenic.

Summary. A number of studies have sought class I HLArestricted cancer epitopes for vaccine design, yet no successfultargets for mediating tumor regression have yet been identified.A multitude of factors complicate the identification of cancerepitopes, including a lack of appreciation of the class I HLApeptide epitopes that distinguish the cancerous cell. From aproteomics perspective, the steps of HLA protein production,HLA protein purification, peptide isolation, and peptide sepa-ration (all from cancerous cells) combine to make cancerepitope discovery a complex process. In this study, we directlyidentified peptides presented by the HLA class I of tumorigenicbreast epithelial cells and absent from nontumorigenic breastepithelium. We identified 5 breast cancer specific epitopes

within this context. We then proceeded to validate theseepitopes, demonstrating immune recognition of 4 epitopes bytetramer staining and IFN-γ ELISPOT in a small cohort ofsubjects.

Future studies will determine whether these epitopes arepresented on the surface of primary breast cancer cells. Weaim to extrapolate the epitopes discovered here by testing forepitope presentation on a range of cancerous primary tumorcells and on panels of noncancerous tissues. We have devel-oped mAb to the breast cancer peptide epitope/HLA complexes(data not shown), and we will stain a range of tissues to assessthe prevalence of these epitopes on cancerous and healthytissue. Our goal is to identify a subset of the epitopes reportedhere that are consistently found on cancerous cells while absenton healthy tissue. Epitopes consistently specific to cancerouscells would then be developed for therapeutic purposes.

Acknowledgment. This work was supported in partby Advanced Technology Program, NIST grant no.70NANB4H3048 (J.A.W.) and a NSF Graduate ResearchFellowship #2006036207 (O.E.H.).

Supporting Information Available: RP-HPLC frac-tionation profile of 4 peptide batches. MS and MS/MS spectraof Cdk2, EXOSC6, and KNTC2 peptides. This material isavailable free of charge via the Internet at http://pubs.acs.org.

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Identification of Breast Cancer Peptide Epitopes Presented by HLA-A*0201

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