of February 12, 2018.This information is current as

That Generates Cross-Reactive T CellsEnzymatic Discovery of a HER-2/neu Epitope

Raphael Clynes and Keith L. KnutsonAndrea M. Henle, Courtney L. Erskine, Linda M. Benson,

http://www.jimmunol.org/content/190/1/479doi: 10.4049/jimmunol.1201264November 2012;

2013; 190:479-488; Prepublished online 23J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2012 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Enzymatic Discovery of a HER-2/neu Epitope That GeneratesCross-Reactive T Cells

Andrea M. Henle,* Courtney L. Erskine,* Linda M. Benson,† Raphael Clynes,‡ and

Keith L. Knutson*

Patients with HER-2/neu–expressing breast cancer remain at risk for relapse following standard therapy. Vaccines targeting HER-

2/neu to prevent relapse are in various phases of clinical testing. Many vaccines incorporate the HER-2/neu HLA-A2–binding

peptide p369–377 (KIFGSLAFL), because it has been shown that CTLs specific for this epitope can directly kill HER-2/neu–

overexpressing breast cancer cells. Thus, understanding how tumors process this epitope may be important for identifying those

patients who would benefit from immunization. Proteasome preparations were used to determine if p369–377 was processed from

larger HER-2/neu–derived fragments. HPLC, mass spectrometry, cytotoxicity assays, IFN-g ELISPOT, and human breast cancer

cell lines were used to assess the proteolytic fragments. Processing of p369–377 was not detected by purified 20S proteasome and

immunoproteasome, indicating that tumor cells may not be capable of processing this Ag from the HER-2/neu protein and

presenting it in the context of HLA class I. Instead, we show that other extracellular domain HER-2/neu peptide sequences

are consistently processed by the proteasomes. One of these sequences, p373–382 (SLAFLPESFD), bound HLA-A2 stronger than

did p369–377. CTLs specific for p373–382 recognized both p373–382 and p369–377 complexed with HLA-A2. CTLs specific for

p373–382 also killed human breast cancer cell lines at higher levels than did CTLs specific for p369–377. Conversely, CTLs specific

for p369–377 recognized p373–382. Peptide p373–382 is a candidate epitope for breast cancer vaccines, as it is processed by

proteasomes and binds HLA-A2. The Journal of Immunology, 2013, 190: 479–488.

Breast cancer is one of the leading causes of cancer deathfor women worldwide and is the leading cancer by site forfemales in the United States (1, 2). This year alone it is

estimated that the United States will have ∼226,870 new cases ofinvasive breast cancer and 39,510 deaths from breast cancer infemales (1). Breast cancer is generally divided into three subtypes:estrogen receptor/progesterone receptor–positive, HER-2/neu re-ceptor–positive, and triple-negative breast cancers. Despite recentadvances in therapy for primary breast tumors, relapse remainsa significant concern, particularly for the HER-2/neu and triple-negative subsets. Tumors that recur in these patients are oftenmore aggressive, metastatic, and chemoresistant and are theleading cause of death among women with breast cancer. Survivalrates for patients with recurrent breast cancer are ∼50%, whereasthe 5-y survival rate for patients with primary breast cancer isalmost 90% (3).Vaccines are being developed to prevent the recurrence of breast

cancer. The majority of vaccines being tested in clinical trials aredesigned to augment the immune response against tumor Ags,

which are often overexpressed in breast tumors (4, 5). HER-2/neu,which is expressed in 20–40% of invasive breast tumors, is one Agcommonly targeted with vaccines (6). HER-2/neu is a transmem-brane domain–spanning receptor with an intracellular tyrosinekinase–signaling domain. The HER2-signaling domain is acti-vated upon heterodimerization with other members of the HERfamily (HER1, HER3, or HER4) or upon homodimerization (7, 8).Overexpression of this oncogenic protein leads to cellular trans-formation through increased proliferation and survival (7).Although many different HER-2/neu vaccination strategies have

been or are being investigated, vaccines formulated with syntheticcytotoxic T cell–activating peptide epitopes are among the mostadvanced (9–13). For example, one vaccine that has generatedsignificant interest is composed of the adjuvant, GM-CSF, mixedwith the HLA-A2–binding HER-2/neu extracellular domain(ECD)–derived peptide p369–377 (E75) (4, 5). Fisk and colleagues(14) identified p369–377 in 1995 on the basis of the presence ofHLA-A2 anchoring motifs. Experiments using peptide-elicitedCD8+ T cell clones or HLA-A2+ transgenic mice provided strongevidence of natural processing of p369–377 and HLA-A2 presen-tation (14, 15). Some controversy, however, emerged in 1998,when Zaks and Rosenberg (16) failed to demonstrate that vaccine-elicited p369–377–specific T cells could readily recognize HLA-A2+ HER-2/neu+ tumors, casting some doubt on the utility of thisepitope in eliciting therapeutic T cells. However, human studies byKnutson and colleagues (17, 18) further supported the prior studiessuggesting natural presentation in HLA-A2.Subsequent to this early work, Mittendorf and colleagues (5)

conducted exploratory Phase I and II clinical trials in early-stagenode-negative and node-positive breast cancer patients. In themost recent 24-mo landmark analysis, Mittendorf (4) reportedsummary results of those trials. Of 182 evaluable patients, 24-modisease-free survival was 94.3% in the vaccine group and 86.8% inthe control. Although not reaching statistical significance (p =0.08) when considering all evaluable patients, further analysis re-

*Department of Immunology, College of Medicine, Mayo Clinic, Rochester, MN55905; †Department of Biochemistry and Molecular Biology, College of Medicine,Mayo Clinic, Rochester, MN 55905; and ‡Department of Medicine and Microbiol-ogy, Columbia University Medical Center, New York, NY 10032

Received for publication May 2, 2012. Accepted for publication October 22, 2012.

This work was supported by grants from the Susan G. Komen for the Cure Founda-tion and National Institutes of Health, National Cancer Institute Grant R01-CA113861 (to K.L.K.) and by National Institutes of Health, National Cancer InstituteGrant R01 CA152045 (to K.L.K. and R.C.).

Address correspondence and reprint requests to Dr. Keith L. Knutson, Mayo ClinicCollege of Medicine, 342C Guggenheim, 200 First Street SW, Mayo Clinic, Ro-chester, MN 55905. E-mail address: knutson.keith@mayo.edu

The online version of this article contains supplemental material.

Abbreviation used in this article: ECD, extracellular domain.

Copyright� 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1201264

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vealed statistically significant benefit in subsets of patients. PhaseII and III clinical trials investigating p369–377 in combinationwith GM-CSF, with or without trastuzumab, are ongoing (http://www.clinicaltrials.gov) to further determine clinical benefit.Considering the potential for success of the p369–377 vaccine in

preventing recurrence, our group became interested in developingbiomarkers that may further enhance selection of patients whowould benefit from the vaccine. Because p369–377 is displayed inthe context of HLA class I, our interest in the current study fo-cused on the role of the proteasome or immunoproteasome inprocessing p369–377. These proteasomes are two large multi-subunit complexes that are important for the processing of the vastmajority of HLA class I presented epitopes (19). Once processed,the peptides released from the proteasomes are translocated intothe endoplasmic reticulum via TAP1 and TAP2 and loaded ontoclass I molecules. Although p369–377 is an epitope that is em-pirically predicted to be processed by the proteasomes (A.M.Henle and K.L Knutson, unpublished observations), to the best ofour knowledge, no studies have specifically shown this bio-chemically, which was the focus of the current study. Thus, usingpurified human 20S proteasome and immunoproteasome, weasked whether p369–377 could be processed from longer HER-2/neu peptides, allowing it to be available for binding HLA classI molecules. Our data suggest that p369–377 is not processed byeither the 20S proteasome or the immunoproteasome. However,other peptides derived from the ECD of HER-2/neu were con-sistently processed by both enzymes. One newly identifiedpeptide that is processed by both the immunoproteasome and theproteasome is p373–382, a strong HLA-A2 binder that is able toprime CD8+ T cells for HER-2/neu+ breast tumor cell recogni-tion and lysis.

Materials and MethodsCancer cell lines

The breast cancer cell lines SK-BR3, HCC1419, MCF7, and BT20 werepurchased from the American Type Culture Collection, immediately re-suscitated and expanded in RPMI 1640 medium containing 10% FBS, andsupplemented with 25 mM HEPES, 1.5 g/l sodium bicarbonate, 0.1 mMMEM nonessential amino acids, 1 mM sodium pyruvate, 2 mM l-glutamine,and 50 mM 2-ME (Invitrogen), at 37˚C and 5% CO2. Prior to assays, thecell lines were incubated overnight with 500 U/ml IFN-g to ensure max-imum HLA class I expression. The esophageal adenocarcinoma cell lineFLO-1 was a gift from Dr. Harry H. Yoon (Mayo Clinic, Rochester, MN).For verification, all tumor cell lines were tested for HLA-A status, usinga Biotest HLA-A SSP kit (Dreieich, Germany), and were tested for HER-2/neu status, using RT-PCR [primer sequences: F: (59-GCTCTTTGAGGA-CAACTATGCCC-39) R: (59-GCCCTTACACATCGGAGAACAG-39)].Tumor cells were frozen as seeding stocks. For the assays, the cells werethawed rapidly in a 37˚C water bath and expanded in RPMI 1640 medium,as described above. Because HER-2/neu expression has been associatedwith loss of class I expression in some models (20, 21), the HLA-A2 andHER-2/neu status of each cell line was verified by flow cytometry fol-lowing exposure to IFN-g, as we have previously described (22), usingmouse anti-human HLA-A2 FITC and anti HER-2/neu FITC Abs. Cognateisotypes were, respectively, mouse IgG2b,k FITC isotype and mouseIgG1,k FITC isotype (BD Pharmingen).

Peptide and protein synthesis

The peptides used in this study were the OVA peptide, SIINFEKL; theHLA-A2–binding influenza matrix protein M1 peptide p58–66 (pFLU),GILGFVFTL; and the HER-2/neu peptides p369–377, KIFGSLAFL; p368–376, KKIFGSLAF; p372–380, GSLAFLPES; p364–373, FAGCKKIFGS;p373–382, SLAFLPESFD; p364–382, FAGCKKIFGSLAFLPESFD; andp362–384, QEFAGCKKIFGSLAFLPESFDGD. All peptides were manu-factured by the Mayo Clinic Proteomics Core or Elim Biopharmaceuticalsand were.80% pure, as assessed by HPLC and mass spectrometric analysis.The HER-2/neu ECD was purified by trastuzumab affinity chromatographyfrom culture supernatants of baby hamster kidney cell–produced ECD, aspreviously described by us and others (23, 24).

Prediction algorithms

Potential HLA-A2–binding peptides from large HER-2/neu peptidesequences were predicted using a matrix pattern that calculates howpeptides bind to the HLA-A2 motif based on previously publishedpeptide motifs. The prediction algorithm is “SYFPEITHI: database forMHC ligands and peptide motifs” (25). It can be accessed via www.syfpeithi.de.

The C-Term 3.0 and 20S prediction networks on the Netchop 3.1 Server(www.cbs.dtu.dk/services/NetChop/), as well as proteasome and immu-noproteasome models 1, 2, and 3 on the Proteasome Cleavage PredictionServer (http://imed.med.ucm.es/Tools/pcps/index.html), were used toidentify the cleavage of potential HLA-A2–binding peptides from largeHER-2/neu peptide sequences. Results from all possible cleavage predic-tion methods were compiled for the study.

HLA-A2 stabilization assays

The lymphoblastic cell line T2 (American Type Culture Collection) wasmaintained at 37˚C and 5% CO2 in IMDM supplemented with 20% FBS.For stabilization assessment, T2 cells were washed, counted, and resus-pended at 1 3 106 cellsper milliliter in IMDM in a 24-well plate andpulsed with a serial dilution of 0.2 2 100 mM peptide for 16 h at roomtemperature. Subsequently, cells were washed 33 in PBS/0.5% BSA andstained with anti–HLA-A2 FITC Ab (clone BB7.2; BD Pharmingen) orisotype control mouse IgG2b,k FITC Ab (BD Pharmingen) and analyzedby flow cytometry on a FACScan with CellQuest software (BD Bio-sciences). Influenza peptide (pFLU, GILGFVFTL; see Ref. 26) served asa positive control. SIINFEKL served as the negative control peptide.

Human T cell isolation and cultures

Whole-blood samples obtained from Leuko Reduced Separator Chambers(Trima) from the Department of Transfusion Medicine at Mayo Clinic werelysed in ammonium-chloride-potassium buffer and separated over Ficoll-Paque (GE Healthcare). A total of 1 3 106 cells from the PBMC frac-tion were stained with mouse anti-human HLA-A2 FITC BB7.2 Ab (BDPharmingen) or mouse IgG2b,k FITC Ab (BD Pharmingen) and analyzedwith a FACScan flow cytometer (BD Biosciences). PBMC samples thatstained positive for HLA-A2 were further enriched with the CD8+ T CellIsolation Kit II (Miltenyi Biotec) and separated by AutoMACS (MiltenyiBiotec) per the manufacturer’s protocol.

Enriched CD8+ T cells were plated in 2 ml media in six-well platesat 2 3 106 cells per milliliter. Peptide was added at a concentration of10 mg/ml. Remaining CD82 PBMCs were frozen for future restimulations.On days 3 and 5, human rIL-2 was added to the wells at 50 U/ml, andhuman rIFN-g was added at 100 U/ml. On day 7, cryopreserved PBMCswere thawed, washed three times, pulsed with 10 mg/ml peptide for 2 h,and irradiated to 4000 rads. The PBMCs were washed and suspended infresh media and then added to the CD8+ T cells. One additional restim-ulation was done on day 14. The cells were used for assays on days 21–28.

Human ELISPOT

An IFN-g ELISPOT assay was performed as described previously (18).Briefly, cultured CD8+ T cells were incubated at 37˚C for 48 h with breastcancer cell lines or PBMCs that had been pulsed with 100 mM peptide at37˚C and 5% CO2 for 16 h, media alone, or PMA–ionomycin. Respectivewells containing target cells were, in some assays, incubated with blockingAbs for 1 h at a concentration of 1 mg/100 ml RPMI + 10% FBS mediaprior to CD8+ T cell addition. All Abs were from BD Pharmingen (purifiedmouse anti-human HLA-A2 BB7.2; purified mouse anti-human HLA-ABCW6/32; purified mouse IgG2b,k isotype control; and purified IgG2a,kisotype control).

Cytolytic T cell assays

Tumor cells were lysed using an impedance-based approach as previouslydescribed (22, 27). Then 100 ml RPMI 1640 (Mediatech) with 10% FBSwas added to each well in an E-Plate 16 (Roche). Background impedanceon the plate was measured on the xCELLigence RTCA SP instrument(Roche) at 37˚C and 5% CO2. Cells from human tumor cell lines (SK-BR3,MCF7, BT20, FLO, and HCC1419) were harvested with trypsin, counted,washed, and resuspended in RPMI 1640 with 10% FBS and 500 U/mlhuman IFN-g (PeproTech) at 5 3 104 cells per milliliter. IFN-g wasused to ensure maximal upregulation of HLA class I molecules. A total of100 ml tumor cells was added to each well of the E-Plate 16, which wasthen placed in the xCELLigence RTCA DP. Impedance was measuredevery 5 min for ∼20 h at 37˚C and 5% CO2 (until the cells adhered to thegold electrodes on the bottom of each well and proliferated to ∼1 3 104

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cells per well). T cells were counted and resuspended at a concentration of5 3 105 cells per milliliter in RPMI 1640 + 10% FBS media. Then 100 mlT cells or media alone was added to the respective wells. The E-plate 16 wasplaced in the xCELLigence RTCA SP, and impedance measurements wererecorded every 5 min for.10 additional hours at 37˚C and 5% CO2. T cell–mediated death of tumor cells was monitored in real time and was indicatedby a decrease in cell index. Data were analyzed with RTCA Software 1.2(Acea Biosciences). Results were normalized 1–2 h after T cell addition.Cytolytic activity was calculated as the percentage of cytolysis 10 h afterthe normalization time (= [CIno effector 2 CIeffector]/CIno effector 3 100). ForHLA class I blocking, Abs were added at a concentration of 1 mg/100 mlRPMI 1640 + 10% FBS media.

Proteasome and immunoproteasome cleavage assay, HPLC,and mass spectrometry

Purified human 20S proteasome, immunoproteasome, PA28 activator asubunit, and synthetic lactacystin were obtained from Boston Biochem.Proteasome and immunoproteasome enzymes were assayed for activitywith the fluorescent Suc-LLVY-AMC substrate (Boston Biochem) per themanufacturer’s protocol. Cleavage assays were performed in 400 ml re-action buffer (25 mM HEPES, 0.5 mM EDTA, pH 7.6; Boston Biochem).PA28 activator (stock 20 mM) was added to the reaction buffer, to a finalconcentration of 14.5 nM. Human proteasome or immunoproteasomeenzymes (stock 2 mM) were added to the reaction buffer, to a final con-centration of 1.45 nM. A 10 mM peptide substrate (HER-2/neu, 19 mer or23 mer) was diluted in the reaction mix 1:1000. Some samples had lac-tacystin added to the reaction, to a final concentration of 10 mM, to inhibitthe proteasome activity. Blanks without proteasome or immunoproteasomewere run in parallel. Samples were incubated in a 37˚C water bath for 30–60 min. Preliminary studies suggested no difference in the cleavageproducts detected between 30 min and 1 h (data not shown). Samples werethen filtered using prewashed YM-30 30,000-kDa MWCO microcon filters

(Millipore) at 14,000 3 g for 15 min. Filter retentates were collected byinverted centrifugation at 3000 3 g for 3 min.

Cleavage products (5 ml reaction mixture) from the HER-2/neu peptideincubations were separated and identified using an Agilent 1100 HPLCsystem interfaced to an Agilent MSD/TOF mass spectrometer. The prod-ucts were resolved on an Agilent Zorbax 300SB-C18 column (1 3 150mm; 3.5 m; 45˚C), using mobile phases containing H2O, acetonitrile,isopropanol, and formic acid [Pump A: 98:1:1:0.1 and Pump B:10:80:10:0.1 (v/v/v/v)]. The separation gradient was 5–60% B over 23min; 80% B for 5 min; with a 7-min equilibration at 5% B, using a flowrate of 60 mL/min. Mass spectra were collected in positive mode, usingan electrospray ionization interface over an m/z range of 300–2800.Other instrument parameters used were spray voltage, 3500 V; fragmentor,175 V; skimmer, 65 V; radio frequency octopole, 250 V; gas temperature,325˚C; gas flow, 5 L/min; and nebulizer gas, 30 c. Raw spectra were ob-tained and peaks transformed to molecular masses using Agilent Mass-Hunter Qualitative Analysis with BioConfirm software (version B.02.00).The observed masses were then compared with the theoretical massesof the known HER-2/neu peptide sequences and assigned a sequence.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 5. Data wereanalyzed using one-way ANOVA, Tukey, Mann–Whitney, or Student t testsas stated in the figure legends, and the results were considered statisticallysignificant if p , 0.05.

ResultsThe proteasome and immunoproteasome fragment syntheticHER-2/neu–derived peptides into multiple shorter peptides

A 19 aa sequence (p364–382, FAGCKKIFGSLAFLPESF) derivedfrom the ECD of HER-2/neu was synthesized to study whether the

FIGURE 1. The proteasome and immunoproteasome

fragment synthetic HER-2/neu–derived peptides into

multiple shorter peptides, not including p369–377. (A)

Total ion chromatograms for the 19 mer in reaction

buffer, 19 mer + PA28 activator + immunoproteasome,

19 mer + PA28 activator + proteasome, and 19 mer +

PA28 activator + proteasome + lactacystin. *Starting

19-mer FAGCKKIFGSLAFLPESFD; ** and *** indi-

cate deletion products from the peptide synthesis. The

numbered peaks are notable peptides identified by ac-

curate monoisotopic mass as fragment peptides of the

19 mer and correspond to the sequences in Table I.

Synthesized 9-mer p369–377 is also shown. Other

peaks observed on the baseline are background prod-

ucts from the assay. (B) Extracted ion chromatograms

for HER-2/neu p369–377 KIFGSLAFL-OH fragment

for 19 mer + PA28 activator + immunoproteasome and

19 mer + PA28 activator + proteasome. HER-2/neu

p369–377 was not detected.

The Journal of Immunology 481

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proteasome and immunoproteasome could cleave the HLA-A2HER-2/neu epitope, p369–377. Processing studies using longerpeptides are common in the cancer epitope discovery field andprovide greater detection in vitro compared with cleavage assaysusing full-length rHER-2/neu protein (28–30). HER-2/neu p369–377 is embedded in the synthesized 19 mer, with an extra 5 aa onboth the N- and the C-termini.Purified proteasome and immunoproteasome enzymes were

individually incubated with the 19-mer substrate and PA28 acti-vator. Cleaved products were analyzed via liquid chromatographyand mass spectrometry (Fig. 1A). Several peptides between 8 and10 aa in length, the appropriate length for binding to HLA class Imolecules, were detected in the cleaved samples. However, p369–377 was not detected in any of the samples (Fig. 1B). These re-sults suggest that p369–377 is not a reaction product of the pro-teasome or immunoproteasome.

Peptides generated in vitro by proteasomes are predicted tobind HLA-A2

Because peptides other than p369–377were detected as degradationproducts, we questioned whether any of these peptides may serveas potential candidates for HER-2/neu cancer immunotherapies.Thus, an HLA binding prediction server, SYFPEITHI, was used topredict the ability of these other peptides to bind HLA-A2 mole-cules (Table I). Several peptides scored higher than 10, suggestingthat these epitopes may bind HLA-A2. For comparison, p369–377had a score of 28, indicating that it is predicted to bind strongly toHLA-A2, a finding consistent with prior studies (5). Proteasome andimmunoproteasome cleavage servers were also used to compare al-gorithm predictions with the in vitro results. The cleavage predictionsfrom the algorithms did not always correspond to the in vitro bio-chemical data (Table I). Specifically, the algorithm predictions alignedwith the in vitro cleavage data in 6 of 12 (50%) peptides (Table I).

Binding of HER-2/neu p373-382 to HLA-A2 molecules

Because the algorithms predicted that many degradation peptidesmay serve as targets that could be displayed in HLA class I on the

surface of breast cancer cells, T2 HLA-A2 stabilization assays wereperformed. Influenza matrix protein M1 peptide was used as thepositive control because it is known to bind HLA-A2 stronglyand result in HLA-A2 stabilization on the surface of T2 cells.SIINFEKL was used as the negative control, as it does not bindHLA-A2. Four peptides (p368–376, KKIFGSLAF; p372–380,GSLAFLPES; p364–373, FAGCKKIFGS; and p373–382,SLAFLPESFD) identified via the in vitro proteasome and immu-noproteasome assays were synthesized and tested for their bindingto HLA-A2. Only the p369–377 epitope and another epitope,p373–382, resulted in increased surface levels of HLA-A2, indi-cating that these peptides are able to bind A2 and stabilize thecomplex (Fig. 2A and data not shown [nonbinding HER-2/neupeptides]). Titration studies with each peptide were conducted tocompare the peptide affinity for HLA-A2. The p373–382 epitopebound HLA-A2 strongly, resulting in HLA-A2 stabilization atconcentrations as low as 200 nM. In contrast, p369–377 did notbegin to stabilize HLA-A2 until the peptide was at a concentrationof ∼6–7 mM (Fig. 2B). Pulsing with 50 mM of the p373–382peptide consistently resulted in increased surface expression of theHLA-A2 molecule on T2 cells, at levels similar to those of thepositive control pFLU peptide (Fig. 2C). The p369–377 epitopestabilized HLA-A2 at ∼50% of the maximal stabilization seenwith pFLU HLA-A2 upregulation. The concentration of peptidethat induced half-maximal stabilization of HLA-A2 was ∼0.94 mMfor pFLU, ∼1.8 mM for p373–382, and ∼9.6 mM for p369–377(Fig. 2D).

Processing of p373–382 and other peptides from a 23-mersequence of HER-2/neu

Although the p373–382 peptide was released from the 19-merpeptide by both the proteasome and the immunoproteasome andcan bind HLA-A2, the epitope was located on the C-terminal endof the 19 mer, and therefore cleavage between two adjacent aminoacids was not possible to measure. However, in both the protea-some and the immunoproteasome samples in Fig. 1, the potentialfor processing of the 19 mer into the p373–382 decamer was

Table I. Peptide fragments processed from HER-2/neu p364–382, FAGCKKIFGSLAFLPESFD

Label onFigure

HER-2/neuAmino

Acid NumberRetentionTime (min) Peptide

PeptideGenerated by

Immunoproteasome

PeptideGenerated byProteasome

HLA-A*0201Binding Scorea

Cleavage Predictedby IP/P Serversb

1 371–381 20.4 FGSLAFLPESF + + NA Y2 364–374 15.45 FAGCKKIFGSL + + NA Y2 366–376 15.45 GCKKIFGSLAF + + NA Y3 372–382 18.4 GSLAFLPESFD + + NA Y4 373–382 18.5 SLAFLPESFD + + 13 N5 371–380 19.1 FGSLAFLPES + + 8 N5 372–381 19.1 GSLAFLPESF + + 5 Y6 364–373 12.8 FAGCKKIFGS + + 10 N7 374–382 18.2 LAFLPESFD + + 8 N8 373–381 19.2 SLAFLPESF + + 16 N2 369–377 ND KIFGSLAFL 2 2 28 Y9 375–382 16.06 AFLPESFD + + NA Y* 364–382 19.14 FAGCKKIFGSLAFLPESFD-NH4 NA NA NA NA* 364–382 19.14 FAGCKKIFGSLAFLPESFD-COOH NA NA NA NA** NA 19.54 FAGKKIFGSLAFLPESFD-NH4 NA NA NA NA** NA 19.54 FAGKKIFGSLAFLPESFD-COOH NA NA NA NA*** NA 14.78 FAGKKIFGSL NA NA NA NA*** NA 14.78 GKKIFGSLAF NA NA NA NA

Numbers and asterisks indicate peptide labels in Fig. 1A.aThe SYFPEITHI server was used to predict nonamer and decamer peptide binding to HLA-A*0201.bThe 20S and C-term 3.0 prediction methods on the Netchop 3.1 server and the Proteasome Cleavage Prediction Server with models 1, 2, and 3 for the proteasome and

immunoproteasome enzymes were used to predict whether the smaller peptides could be processed by the enzymes from the larger 23-mer sequence, irrespective of our in vitrodata.

+, The peptide was produced by the respective enzyme; –, lack of peptide detection in processed samples; NA, not applicable: Peptide is either a deletion product, is startingmaterial, or is too large for binding predictions to HLA-A*0201; ND, not detected.

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detected by monitoring C-terminal end conversion from an amideto a free acid (not shown).To determine if p373–382 could be internally processed, a

23-mer peptide (p362–384, QEFAGCKKIFGSLAFLPESFDGD)from HER-2/neu was synthesized. The in vitro proteasome andimmunoproteasome assays were repeated with the 23 mer. Al-though proteolysis was generally less efficient with the longerpeptide, liquid chromatography and mass spectrometry showedthat several peptide epitopes were detected in these samples(Supplemental Fig. 1A, Supplemental Table I). Again, the p369–377 epitope was not detected in the proteasome or the immuno-proteasome samples (Supplemental Fig. 1B). Extracted ion chro-matograms showed that the p373–382 decamer was processed inboth the proteasome and, to a lesser extent, the immunoprotea-some samples (Fig. 3). Although lower levels of the decamer weredetected in the lactacystin-inhibited reactions compared with theuninhibited proteasome reaction, generation of p373–382 was stillpresent, suggesting the proteasome uses both lactacystin-resistantand -sensitive catalysis to liberate the peptide. Overall comparisonshows that the peptide products generated by proteolysis from the23-mer peptide were similar to the products obtained with the 19-mer peptide (Figs. 3B, 3C).

Generation of HER-2/neu peptide–specific CD8+ T cells

Thus far, the results suggest that p373–382 can be processed by theproteasome and immunoproteasome and that it binds HLA-A2strongly, suggesting potential clinical relevance. To address this,blood was obtained from human donors, and short-term CD8+

T cell lines were generated against pFLU, p369–377, and p373–382. IFN-g ELISPOT analysis using autologous peptide pulsedPBMCs as targets demonstrated specificity of the lines (Fig. 4A).The pFLU T cell line recognized only pFLU and none of the otherpeptide pulsed PBMC targets. The p369–377 pulsed PBMCs wererecognized by the p369–377 CD8+ T cells and, unexpectedly, alsoby the p373–382 T cells. Similarly, the p373–382 pulsed PBMCswere recognized by the p373–382–specific CD8+ T cells but alsoby the p369–377–specific T cells. Thus, the results show thatT cells responsive to p373–382 exist in the human T cell repertoire.

HER-2/neu p373–382–specific CTLs recognize breast cancercells and naturally processed Ag

To address whether T cells generated by p373–382 recognize HER-2/neu–expressing breast cancer cells, a panel of HER-2/neu andHLA-A2–expressing breast cancer cell lines was used in anELISPOT assay with the three different T cell lines (Fig. 4B).With IFN-g ELISPOT, the activities of the lines were compared.Despite equivalency of the cell lines, in terms of numbers ofspecific cells, as assessed in the ELISPOT using autologous tar-gets, T cells generated by p373–382 generally recognized breastcancer cells at higher levels than did p369–377–specific T cells.BT20 cells served as the negative control cell line, as they areHLA-A2 negative. These results indicate that cancer cells may beprocessing and presenting the p373–382 peptide at their surface inthe context of HLA-A2. The CD8+ T cell lines were also tested forcytotoxicity. Similar to the IFN-g ELISPOT results, the p373–382T cells lysed the cancer cell lines at higher levels than did thep369–377 T cells. The BT20 (HLA-A2 negative) and FLO (HER-2/neu negative and HLA-A2 positive) cell lines served as negativecontrols (Fig. 4C).To further demonstrate that p373–382 is a naturally processed

epitope from HER-2/neu, we pulsed autologous PBMCs withHER-2/neu p373–382, pFLU, HER-2/neu ECD, and OVA proteinand assayed for IFN-g+ ELISPOT with HER-2/neu p373–382–specific CD8+ T cells and pFLU-specific CD8+ T cells. CD8+

FIGURE 2. HER-2/neu p373–382 stabilizes HLA-A2 on T2 cells. (A)

Shown are composite histograms of HLA-A2 levels on T2 cells pulsed

with 50 mM various peptides [Influenza peptide, pFLU, served as a

positive control, cells pulsed with OVA (SIINFEKL) peptide as a nega-

tive control (nonbinding peptide)]. Unpulsed T2 cells were stained with

isotype Ab. One representative experiment of 10 is shown. (B) Shown is

the fold change in HLA-A2 staining intensity on T2 cells pulsed with

a serial dilution of 0.1952 100 mM peptide. Results are calculated as the

ratio of sample fluorescence relative to fluorescence obtained in the

absence of peptide, and presented as the mean and SEM of 10 experi-

ments. (C) Shown are the mean (6 SEM, n = 10) fluorescence intensities

of T2 cells pulsed with 50 mM peptide relative to the maximal response

seen with pFLU. ***p , 0.001, calculated using one-way ANOVA with

the Tukey multiple comparison test. (D) Shown are EC50 (i.e., half-

maximal effective concentration) curves depicting concentration-de-

pendent HLA-A2 stabilization over various concentrations of peptide

(n = 10). Inset data show extrapolated EC50 values calculated using a

one-site saturation model.

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T cells specific for p373–382 had a strong response against p373–382 pulsed PBMCs and against HER-2/neu ECD pulsed PBMCs(Fig. 4D). The amount of nonspecific activity displayed by theT cells was minimal, as control pFLU-specific T cells had littlerecognition of targets pulsed with HER-2/neu peptide and OVA,and similarly, p373–382–specific T cells recognized control OVAand pFLU pulsed targets at low levels.

IFN-g and lytic responses of p373–382–generated T cells areHLA class I restricted

To address whether the IFN-g response of p373–382–generatedT cells was HLA class I restricted, T cell lines were generatedagainst all three peptides, pFLU, p369–377, and p373–382, fol-lowed by IFN-g ELISPOT testing in the presence or absence ofa blocking anti–HLA class I H chain Ab. The pFLU T cells did notrecognize p373–382 pulsed PBMC targets. As expected, bothp369–377 and p373–382–generated T cells recognized p373–382pulsed PBMCs with IFN-g release (Fig. 5A). The addition of anti-HLA class I H chain (anti–HLA-ABC) Ab resulted in a significant90% reduction in the numbers of cells responding with IFN-g. Thesame cell line was tested several days later, replacing anti–HLA-

ABC with specific anti–HLA-A2 blocking Ab. As shown in Fig.5B, anti–HLA-A2 blocked IFN-g release to the same extent asanti–HLA-ABC, suggesting that HLA-A2 is the primary mediatorof reactivity. Also shown in Fig. 5A and 5B, the number of p369–377 T cells releasing IFN-g in response to p373–382 pulsed tar-gets decreased completely with the addition of anti–HLA-ABCand anti–HLA-A2 Abs, further supporting cross-reactivity of thep369–377 and p373–382 T cell lines. Consistent with the ELI-SPOT analysis, blocking of HLA class I on the surface of SKBR3breast cancer cells nearly abolished lysis of the tumor cells byboth p369–377 T cells and p373–382 T cells (Fig. 5C).

DiscussionHER-2/neu peptide p369–377 was one of the earliest tumor Ag–derived cytotoxic T cell epitopes to have been identified (14). Onthe basis of successfully moving this epitope into human clinicaluse, we chose to determine whether this epitope is cleaved forHLA class I presentation by the immunoproteasome or protea-some. Despite a positive prediction result with the proteasome andimmunoproteasome cleavage servers, cleavage of the peptide wasnot detected in the in vitro proteasome and immunoproteasome

FIGURE 3. HER-2/neu p373–382 is processed from

HER-2/neu 23 mer by the immunoproteasome and

proteasome. (A) Extracted ion chromatograms exam-

ining for the HER-2/neu p373–382 fragment from the

23 mer in reactions containing (top to bottom) reaction

buffer, immunoproteasome, proteasome, and protea-

some + lactacystin. Also shown is purified HER-2/neu

10 mer p373–382. (B) Predominant cleavage products

from digestion of human HER-2/neu 23-mer peptide

362–384 with 20S proteasome and immunoproteasome.

Peptide fragments were detected by HPLC interfaced

with electrospray ionization time-of-flight mass spec-

trometry. Numbers above amino acid residues indicate

the number of cleavages occurring at that site. Numbers

below designate amino acid residue positions in the

HER-2/neu protein. (C) Predominant cleavage products

from digestion of human HER-2/neu 19-mer peptide

364–382 with 20S proteasome and immunoproteasome.

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assays. In contrast, an HPLC comigrating peptide, HER-2/neup373–382, was consistently observed. This peptide was able togenerate Ag-specific CD8+ CTLs from the peripheral blood ofHLA-A2+ normal donors that effectively killed HER-2/neu–expressing tumor cells. HER-2/neu p369–377–specific CD8+

CTLs were also generated from the peripheral blood of HLA-A2+

normal donors, using the p369–377 peptide, but these did notrecognize HLA-A2+ breast cancer cells as strongly as did p373–382–specific CTLs. These results suggest that HER-2/neu–positivecancer cells produce p373–382 via the proteasome and immuno-proteasome. HER-2/neu p373–382 may be an effective candidateto induce HER-2/neu–specific effector CD8+ T cell responsesin vivo in patients.As noted, HER-2/neu p369–377 was not detected in the in vitro

proteasome and immunoproteasome assays. Despite an abundanceof prior work demonstrating its potential therapeutic utility, thereason that p369–377 was not detected is unclear, but it could bebecause the proteasome and immunoproteasome, which bothfrequently cleave at residues in the middle of this sequence, rap-idly destroyed the epitope upon production. The observation,however, that products spanning the K–K bond in the substratepeptides accumulated among the reaction products, suggests thatC-terminal processing does not occur. Our data do not rule out thepossibility that p369–377 is generated in the cell by other mech-anisms; such mechanisms involve serine and cysteine proteases, aswell as aminopeptidases, which trim the N-terminal amino acidsof peptides in the cytosol and ER (31), all of which may act uponlonger HER-2/neu sequences to yield the p369–377 epitope.Unlike the casewith other epitopes, the processing of HER-2/neu

p373–382 was not completely blocked in the in vitro assays inthe presence of the proteasome inhibitor lactacystin. However,lactacystin inhibits only the trypsin, chymotrypsin, and caspase-like activities of the proteasome. The proteasome also has otherlactacystin-resistant catalytic activities, including peptidyl-glutamylpeptide hydrolyzing and branched-chain amino acid–preferringactivities, which could aid in processing of HER-2/neu p373–382and account for some of the product detected in the presence ofthe inhibitor (29, 31–33).When tested in stabilization assays, the affinity of p373–382 for

binding to HLA-A2 molecules was comparable to that of thepositive control peptide from influenza matrix protein, pFLU. Theobservation that p369–377 stabilized HLA-A2 is consistent withprevious reports (34, 35). However, the EC50 for binding of p369–377 to HLA-A2 is significantly higher than that for both pFLU andp373–382, indicating that p373–382 has an increased bindingaffinity for HLA-A2 molecules. This stronger affinity for HLA-A2may provide an explanation for the enhanced recognition of breastcancer cell lines by HER-2/neu p373–382–specific CTLs; thep373–382 peptide may remain bound to the HLA-A2 moleculeson cancer cells for a longer time, sustaining presentation to CTLs.This suggestion is consistent with prior studies showing a strong

FIGURE 4. HER-2/neu p373–382–specific CD8+ T cells have increased

recognition of human breast cancer cell lines. (A and B) Shown are the

mean (6 SEM, n = 3) numbers of ELISPOTs obtained in response to

pFLU, p369–377, or p373–382 peptide-specific CD8+ T cells derived from

short-term culture to (A) autologous PBMCs pulsed with pFLU, p369–377,

or p373–382 or (B) irradiated tumor cells. Results are from a single donor

and are representative of similar results obtained with three other donors.

(C) Shown are the mean (6 SEM, n = 4) % lysis values calculated from the

impedance-based lysis assay at 10 h following the addition of T cells. Also

shown are the expression levels of HER-2/neu and HLA-A2 genotyping

results for each of the cell lines. Results are pertinent to (B) and (C). HER-2/

neu overexpression levels in each cell line and HLA-A2 expression are

indicated below the x-axis. Expression levels were determined by flow

cytometry and RT-PCR. The p values in (B) and (C) were calculated using

an unpaired Student t test. (D) Shown are the mean (6 SEM, n = 3)

numbers of IFN-g ELISPOTs obtained in response to pFLU or p373–382

peptide-specific CD8+ T cells derived from short-term culture to autolo-

gous PBMCs pulsed with pFLU, p373–382, HER-2/neu ECD, or OVA

protein. The experiment was repeated twice in triplicate, with both

experiments yielding similar results.

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correlation between peptide:HLA complex stability and immu-nogenicity (36–39).Evidence is provided for cross-reactivity of CTL lines generated

by each of the two peptides that overlap in their sequences by 5 aa.HER-2/neu p373–382–specific CTLs recognized PBMCs pulsedwith p373–382 and, to a slightly lesser extent, PBMCs pulsed withp369–377. Vice versa, HER-2/neu p369–377–specific CTLs rec-ognized PBMCs pulsed with p369–377 and PBMCs pulsed withp373–382. A significant number of data show that T cells can becross reactive (40–45). One recent study elegantly showed thata single autoimmune TCR is able to recognize more than a milliondifferent decamer peptides in the context of a single MHC class Imolecule (45). In that study, several epitopes identified were muchbetter agonists than the wild-type index peptide, despite having

only a few amino acids in common. Several mechanisms of cross-reactivity have been identified that could explain the cross-reactivity between p373–382 and p369–377 observed in the cur-rent study (46). Although the traditional view would be that theseshared 5 aa would not occupy the same position in the closedbinding cleft in HLA class I molecules, recent data suggest fluidityand motion that may allow the different peptide:HLA complexesto assume (i.e., tune) similar three-dimensional structures that canbe seen by the TCRs (42).The binding of HER-2/neu p373–382 to HLA-A2 partially

coincides with the identified anchor residues at positions 2 and 9for nonamers (47). Leucine is a dominant amino acid residue thatoccupies position 2 of HLA-A2 motifs, and it is present in thesecond position of p373–382 while absent from position 2 ofp369–377. Valine and leucine are frequently found in position 9 ofrestricted nonomer epitopes. A leucine is found in position 9 of thep369–377 epitope, but neither leucine nor valine is found in po-sition 9 of p373–382. Rather, p373–382 contains a terminalaspartic acid that is very rarely observed in the terminal positionof an HLA-A2 epitope. Our search of the epitope database at theImmune Epitope Database and Analysis Resource (http://www.immuneepitope.org), at the time of manuscript preparation, revealsonly ∼6 HLA-A2 epitopes with terminal aspartic acid residues.Arguably, much of the information that has been used to populatethese and other databases has been based on prediction algorithmsderived from a limited set of data obtained from elution studies orstudies in which crystal structures of HLA class I peptide com-plexes have been solved (48). Despite that, however, it is clearfrom higher throughput studies that leucine and valine are favoredat position 9 in nonomeric epitopes (48).What has become more appreciated in recent times is that HLA-

A2 can present longer epitopes, from 10 to 15 aa. In a recent study,Scull and colleagues (49) found that a large fraction of HLA-A2peptides were longer than 9 aa. Furthermore, many of the longerpeptides with the favored position 2 leucine anchor had a varietyof different amino acids at putative anchor residue position 9,including charged amino acids such as threonine, lysine, andaspartic acid. Relevant to this study, several eluted longer peptideswith a position 2 leucine also contained aspartic acids at position10. In general, over the years, epitope identification has been aninherently biased approach focusing on characterizing nonamers.Although it is likely that concepts derived from nonamers could insome ways extend to longer peptides, emerging data suggest criticaldifferences exist. Myers and colleagues (50) recently showed thatHLA-A2 molecules on glioblastoma tumor cells express a novel12-mer peptide frameshift. Although a bona fide consensus HLA-A2 nonamer was fully contained within the 12 mer, molecularmodeling revealed that binding of the nonamer to HLA-A2 wasdifferent at most of the amino acid positions. The similaritiesappeared to be largely confined to the terminal NH-2 moiety, aswell as the position 1 aa binding to the HLA molecule. Relevant tothe current study, however, were the observations that the T cellsgenerated against the 12 mer were cross reactive with T cellsgenerated against the embedded 9 mer, suggesting the presenceof areas shared by the 9 mer and 12 mer that are conserved enoughfor T cell recognition.Predictive algorithms continue to improve and will likely shed

additional light on longer peptides that may be relevant to vaccine-based therapeutic or prevention strategies. Several algorithms havenow been developed that combine various factors that affectbinding and that go beyond identifying motifs and, rather, discovercontributions of all amino acids to binding. In more recent times,stabilized matrix method-based algorithms (e.g., SMMPMBECand SMM) that address interactions between amino acids within an

FIGURE 5. IFN-g and lytic responses of p373–382–generated T cells

are HLA class I restricted. (A and B) Shown are the mean (6 SEM, n = 3)

numbers of p373–382–specific ELISPOTs obtained in response to p369–

377 or p373–382 peptide-specific CD8+ T cells derived from short-term

culture to autologous PBMCs pulsed with p369–377 or p373–382 in the

presence or absence of (A) HLA-ABC blocking Ab or (B) HLA-A2

blocking Ab. One representative experiment of three is shown. (C) Shown

are the mean (6 SEM, n = 4) % lysis values calculated from the imped-

ance-based lysis assay at 10 h following the addition of T cells. The target

cells were pretreated with or without 10 mg/ml HLA-ABC blocking Ab or

HLA-A2 blocking Ab and appropriate isotype control Abs 1 h prior to T

cell addition. Similar results were obtained with T cells generated from

three other donors. The p values were calculated using a nonparametric

Mann–Whitney U test.

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epitope, rather than assuming independence, have been developedthat consistently outperform those algorithms that assume inde-pendence (e.g., SYFPEITHI) (25, 48, 51, 52). The stabilizedmatrix methods also outperform those based on neural networks(48, 51, 52). SMMPMBEC (51) and SMM (48) prediction meth-ods show that p373–382 is predicted to bind with an IC50 of 2.7mM and 0.482 mM, respectively. Our observation of an EC50 of1.8 mM, using the T2 stabilization assay, is in good agreementwith the predictions.In closing, our investigations show that the commonly employed

HLA-A2–binding HER-2/neu–derived peptide, p369–377, is notprocessed from HER-2/neu protein fragments by the proteasomeor immunoproteasome, as previously suggested. Rather, an over-lapping 10-mer peptide, p373–382, was processed. Althoughp373–382 does not have a typical HLA-A2–binding amino acidsequence, binding studies demonstrate HLA-A2–binding activity.Furthermore, the peptide appears to be naturally processed and isable to elicit peptide-specific T cells that cross react with p369–377–elicited T cells. The latter finding of cross-reactivity mayexplain the clinical utility of p369–377.

AcknowledgmentsWe thank the Mayo Proteomics Research Center for helpful discussions.

DisclosuresK.L.K. and A.M.H. have a patent pending describing some of the new epi-

topes found in this study, including p373–382. Furthermore, the Mayo

Clinic has licensed, with consideration, to Tapimmune (Seattle, WA), the

rights to develop p373–382 as a therapy. The other authors have no finan-

cial conflicts of interest.

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