identification of a broad coverage hla-dr degenerate epitope pool derived from carcinoembryonic...

Cancer Immunol Immunother (2010) 59:161–171
DOI 10.1007/s00262-009-0738-z
ORIGINAL ARTICLE

IdentiWcation of a broad coverage HLA-DR degenerate epitope pool derived from carcinoembryonic antigen

Lavakumar Karyampudi · Christopher J. Krco · Kimberly R. Kalli · Courtney L. Erskine · Lynn C. Hartmann · Karin Goodman · James N. Ingle · Matthew J. Maurer · Aziza Nassar · Chao Yu · Mary L. Disis · Peter J. Wettstein · John D. Fikes · Melanie Beebe · Glenn Ishioka · Keith L. Knutson

Received: 14 May 2009 / Accepted: 2 July 2009 / Published online: 21 July 2009© Springer-Verlag 2009

Abstract CD4 T cells are important for anti-tumorimmune responses. Aside from their role in the activationof CD8 T cells, CD4 T cells also mediate anti-tumorimmune responses by recruiting innate immune eVectorsinto the tumor microenvironment. Thus, the search forstrategies to boost CD4 T cell immunity is an active areaof research. Our goal in this study was to identify HLA-DRepitopes of carcinoembryonic antigen (CEA), a com-monly over-expressed tumor antigen. HLA-DR epitopesof CEA were identiWed using the epitope predictionprogram, PIC (predicted IC50) and tested using in vitroHLA-DR binding assays. Following CEA epitope conWr-mation, IFN-� ELIspot assays were used to detect existingimmunity against the HLA-DR epitope panel of CEAin breast and ovarian cancer patients. In vitro generatedpeptide-speciWc CD4 T cells were used to determinewhether the epitopes are naturally processed from CEA

protein. Forty-three epitopes of CEA were predicted, 15of which had high binding aYnity for 8 or more commonHLA-DR molecules. A degenerate pool of four, HLA-DRrestricted 15 amino acid epitopes (CEA.24, CEA.176/354,CEA.488, and CEA.653) consisting of two novel epitopes(CEA.24 and CEA.488) was identiWed against which 40%of breast and ovarian cancer patients had pre-existentT cell immunity. All four epitopes are naturally processedby antigen-presenting cells. Hardy–Weinberg analysisshowed that the pool is useful in »94% of patients.Patients with breast or ovarian cancer demonstrate pre-existent immune responses to the tumor antigen CEA. Thedegenerate pool of CEA peptides may be useful foraugmenting CD4 T cell immunity.

Keywords MHC class II · HLA-DR · Helper T cells · Vaccines · Peptides

L. Karyampudi and C. J. Krco are co-Wrst authors. G. Ishioka and K. L. Knutson are co-senior authors.

L. Karyampudi · C. J. Krco · C. L. Erskine · C. Yu · P. J. Wettstein · K. L. Knutson (&)Department of Immunology, College of Medicine, Mayo Clinic, 342C Guggenheim, 200 First St. SW, Rochester, MN 55905, USAe-mail: [email protected]

K. R. Kalli · L. C. Hartmann · K. Goodman · J. N. IngleDepartment of Oncology, Mayo Clinic, Rochester, MN 55905, USA

M. J. MaurerDepartment of Health Sciences Research, Mayo Clinic, Rochester, MN 55905, USA

A. NassarDepartment of Anatomic Pathology, Mayo Clinic, Rochester, MN 55905, USA

M. L. DisisTumor Vaccine Group, Center for Translational Medicine in Women’s Health, Seattle, WA 98195, USA

J. D. Fikes · M. Beebe · G. IshiokaPharmexa-Epimmune, Inc., San Diego, CA 92121, USA

Present Address:J. D. FikesKalypsys, Inc., 10420 Waterridge Circle, San Diego, CA 92121, USA

Present Address:G. Ishioka11516 Hadar Dr., San Diego, CA 92126, USA

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162 Cancer Immunol Immunother (2010) 59:161–171

Introduction

In recent years, T cell (CD8 and CD4) targeted immuno-therapy against cancer has generated increasing interest.Results associated with several ongoing clinical trials, par-ticularly adoptive T cell therapy, have been quite dramatic[1]. Cytotoxic CD8 T cells lyse tumor cells directly andbecause of this property, these cells have frequently beentargeted when designing vaccines for the treatment ofcancer. Helper CD4 T cells play a central role in adaptiveanti-tumor immunity in several pathways, including (1)production of cytokines that have important roles in thelongevity and eVector functions of antigen-activated CD8T cells, (2) activation of antigen-presenting cells (APCs) byCD40 and nCD40L interactions which enables improvedpriming of T cell immunity, and (3) activation of CD8T cell-independent mechanisms of tumor eradication [2].This latter eVector function is multi-dimensional andincludes potential anti-angiogenic properties of IFN-� andthe intratumoral recruitment of innate immune eVector cellssuch as macrophages and eosinophils [2].

Strategies using deWned peptide epitopes have been usedto generate anti-tumor T cell responses, either in vivo (e.g.vaccination) or in vitro (e.g. for adoptive T cell therapy),due to advantages such as chemical deWnition, ease of syn-thesis, long-term stability, and targeting speciWcity (e.g.MHC class I). Recently, increasing attention has focused onidentifying CD4 T cell-activating MHC class II epitopesfrom a variety of diVerent tumor antigens, including carci-noembryonic antigen (CEA), folate receptor alpha, tyrosi-nase, gp100, MART1/Melan-A, NY-ESO-1, p53, HER-2/neu, and insulin-like growth factor binding protein 2(IGFBP-2) [3–12]. However, the human MHC class IIlocus is very polymorphic making it diYcult to developeVective strategies that can be tested in the majority ofpatients. Despite the polymorphism, however, the peptidebinding characteristics of each variant do not diVer signiW-cantly making it possible to identify degenerate peptidescapable of binding multiple allelic variants of HLA-DR.IdentiWcation of degenerate peptides of TAAs may lead toeVective vaccination strategies against cancer.

CEA is a membrane glycoprotein and is over-expressed inseveral human malignancies, such as colorectal, gastric, pan-creatic, non-small-cell lung, breast, cervical, ovarian, pros-trate, and head and neck cancers [13–15]. CEA is shown tohave an important role in the development of metastatic dis-ease by inhibiting cell death [16] and cooperating in cellulartransformation with several proto-oncogenes such as BCL2and c-myc [17]. The utility of this protein as a target antigenfor immunotherapies is well documented. Several CD8 T cellepitopes of CEA have been identiWed so far [18–20] andDNA encoding the whole protein has been used in advancedvaccine clinical trials [21]. Lastly, it has been reported by

Bos and colleagues [22] that CD4 T cells are important ineliciting protective immunity against CEA in murine models.Thus, the identiWcation of a degenerate MHC class II-bindingpool from CEA is important for use as a vaccine, alone or incombination with MHC class I epitopes, and for monitoringimmune responses in whole antigen approaches.

We used the PIC (predicted IC50) epitope prediction pro-gram, as previously described [5], to identify a pool of 43potential HLA-DR binding epitopes of CEA. Four of theseCEA peptides were naturally targeted in patients previouslydiagnosed with either breast or ovarian cancer. This pool offour epitopes is calculated to bind to HLA-DR in »94% ofpeople suggesting its potential utility as a broad coverageCD4 T cell-activating vaccine component.

Materials and methods

Reagents

PuriWed CEA protein was obtained from Abcam (Cambridge,MA). CEA peptides were synthesized either at Mayo Proteo-mics Research Center or by Pharmexa-Epimmune, Inc.(San Diego, CA). The purity (>95%) and identity of peptideswere determined by reverse phase HPLC and mass spectrom-etry analysis, respectively. The synthetic peptides werelyophilized, resuspended in DMSO and then diluted in PBS.

Epitope prediction

A modiWed linear coeYcient or matrix-based method calledPIC (predicted IC50) was used for predicting peptides withHLA-DR binding capacity [23, 24]. PIC is proprietaryto Pharmexa-Epimmune, Inc., but another predictionalgorithm (developed by Dr. Alessandro Sette) similar toPIC is available at NIH-supported public website, http://www.iedb.org. PIC is predicted on the assumption that each resi-due along a peptide molecule can independently contributeto binding aYnity. PIC generates a score for individual pep-tides that is derived from polynomial coeYcients describingthe relative binding associated with each of the 20 naturallyoccurring amino acid residues for each peptide position.Next, mathematical transformations are performed, includ-ing linear polynomial scaling, an experimental power trans-formation, and a further linear correction based on minimizingthe deviation of predicted values from experimental values.Based on these operations, the algorithm yields a predictedIC50 value (designated as PIC) for the corresponding inputsequence. PIC converts coeYcient-based scores into anIC50 prediction and enables prioritization of peptides forfurther screening based on the predicted strength of HLA-DR binding. Lower PIC values indicate higher bindingaYnity to HLA. The program analyzes 15 amino acid long

123

http://www.iedb.org

http://www.iedb.org

Cancer Immunol Immunother (2010) 59:161–171 163

sequences oVset by 3 residues encompassing the entire pro-tein.

Subjects

Blood specimens were obtained from 18 healthy donors and38 (9 breast and 29 ovarian) disease-free cancer patientsfrom Mayo Clinic. Ten breast cancer patient samples wereobtained from University of Washington (Seattle, WA) andwere processed and stored using the same procedures andprotocols as Mayo Clinic samples. FFPE tissue sampleswere obtained from patients at the time of initial surgicalprocedure. This study was approved by Institutional ReviewBoards at both the Mayo Clinic and the University of Wash-ington. Patients were free from active treatment for at least30 days when blood was collected. For T cell studies, themean (§SEM) ages of healthy donors and patients were42 § 11 and 55 § 2 years, respectively (p < 0.0001).Tumor grade information was available for 34 patients(1 grade 2, 2 grade 7, 3 grade 5 and 4 grade 20) and stageinformation was available for 35 patients (12 stage I, 7 stageII, 15 stage III and 1 stage IV). Tumor tissues for CEAstaining were available for 26 patients (breast tumor 6,ovarian tumor 20). The patient and healthy donor popula-tions, as described in our previously published study, hadno diVerences of T cell reactivity to non-speciWc mitogen(PMA/ionomycin) or viral antigens between the groups [5].

Preparation of peripheral blood mononuclear cells (PBMCs)

Peripheral blood mononuclear cells were isolated fromblood by density gradient centrifugation as described previ-ously [25]. Cells were cryopreserved in liquid nitrogen infreezing medium (RPMI with 45% FBS and 10% dimethyl-sulfoxide) at a cell density of 25–50 £ 106 cells/ml.

AYnity puriWcation of HLA-DR molecules

HLA-DR molecules used in this study (list in Table 1) werechosen to allow balanced population coverage [26]. HLA-DR molecules were puriWed from EBV-transformed homo-zygous cell lines or from transfected Wbroblasts by aYnitychromatography as previously described [24] using mAbLB3.1 coupled to Sepharose CL-4B beads. Eluates contain-ing HLA-DR molecules were obtained after passing celllysates through an anti-DR column. The eluate was thenconcentrated by centrifugation.

HLA-DR binding assay

The binding aYnity of peptides to diVerent HLA-DR mole-cules was determined by their ability to inhibit the binding

of high-aYnity radiolabeled probe peptides to speciWcHLA-DR molecules using gel-Wltration radioimmunoassay[27]. BrieXy, puriWed HLA-DR molecules and radiolabeledpeptides were incubated in the presence of the inhibitorpeptide in a reaction vessel for 2 days either at room tem-perature or at 37°C in the presence of protease inhibitors.After incubation, the percentage of HLA-DR bound radio-activity was determined by capturing HLA-DR/peptidecomplexes on Optiplates (Packard Instruments) coated withthe LB3.1 antibody and determining bound counts per min-ute followed by aYnity calculations. As in previous studies,peptides with aYnities for speciWc HLA-DR molecules of1,000 nmol/l or better were deWned as high-aYnity binders.

Enzyme-linked immunosorbent spot assay (ELIspot assay)

A 10-day ELIspot for detecting low-frequency T cells inPBMCs of healthy donors and breast/ovarian cancerpatients was used to determine reactivity to the CEA-derived peptides (Table 1) and was done in groups of two(two healthy donors, one healthy/one cancer patient, or twocancer patients) essentially as previously described [25].A patient was identiWed as having an immune response to aspeciWc peptide if the calculated T cell frequency to thatpeptide exceeded the mean frequency of the control popula-tion plus two standard deviations [5]. A peptide was con-sidered naturally immunogenic if greater than 10% of thepatients demonstrated signiWcantly elevated immunity tothat peptide relative to the controls.

Generation of antigen-speciWc CD4+ T cells

Dendritic cells were generated from PBMCs as describedpreviously [5]. BrieXy, PBMCs were seeded into six-wellplates (6 £ 106 cells/well) in culture medium (completeRPMI medium with human AB serum) containing granulo-cyte macrophage colony-stimulating factor and interleukin-4. On day 5, bacterial CpG was added to the cultures at theconcentration of 1 �g/ml. On day 6, peptide (10 �g/ml) andB7-DC crosslinking antibody (10 �g/ml) (a gift fromDr. Larry Pease, Mayo Clinic) were added to the DCcultures. After 4 h incubation, pure CD4 T cells isolatedfrom PBMCs by magnetic separation (purity »99%) wereadded and the cultures were incubated at 37°C with peri-odic interleukin-2 and interleukin-12 addition.

Determination of HLA class II restriction and reactivity of peptide-speciWc CD4+ T cells against CEA protein

On day 15 of in vitro stimulation, peptide-speciWc CD4 Tcells were assayed for reactivity with the CEA antigen(CEA peptides and CEA protein) and irrelevant antigens byELIspot and T cell proliferation assays. For these assays, in

123


Tab

le1

Bin

ding

aY

niti

es o

f C

EA

pep

tides

to p

uriW

ed H

LA

-DR

Pept

ides

that

con

stit

ute

dege

nera

te p

ool a

re in

bol

d

ND

not

det

erm

ined

aPo

sitio

n of

N-t

erm

inal

am

ino

acid

Sequ

ence

Pep

tide

nam

ePo

sitio

naIC

50 n

M to

pur

iWed

HL

A

DR

B1

*010

1D

RB

1*0

301

DR

B1

*040

1D

RB

1*0

404

DR

B1

*040

5D

RB

1*0

701

DR

B1

*080

2D

RB

1*0

901

DR

B1

*110

1D

RB

1*1

201

DR

B1

*130

2D

RB

1*1

501

DR

B3

*010

1D

RB

4*0

101

DR

B5

*010

1

LL

TF

WN

PP

TT

AK

LT

IC

EA

.24

246.

916

,313

273

5225

83.

717

45,

779

524,

995

245

46N

D2,

171

31

TA

KL

TIE

STPF

NV

AE

CE

A.3

333

7261

310

641

383

701,

736

4,01

95,

977

2,90

735

140

ND

533,

350

EV

LL

LV

HN

LP

QH

LF

GC

EA

.50

502.

783

03.

41.

730

5.4

5998

940

5.4

0.36

4.7

334

501,

088

YS

WY

KG

ER

VD

GN

RQ

IC

EA

.65

6551

1N

D34

585

360

866

8,43

2N

D1,

840

ND

533

306

3,00

245

31,

043

NR

QII

GY

VIG

TQ

QA

TC

EA

.76

7621

6N

D10

81.

512

946

4634

536

4,35

199

02.

6N

D5.

22,

230

GR

EII

YPN

ASL

LIQ

NC

EA

.97

9762

433

251

8855

029

1,95

91,

355

2,20

921

224

494,

035

4310

,612

DT

GFY

TL

HV

IKSD

LV

CE

A.1

1611

664

984

8426

095

2390

8317

417

41,

072

6514

,943

1856

4

FYT

LH

VIK

SD

LV

NE

EC

EA

.119

119

101

8018

416

941

5651

471

86,

385

616

1,34

014

14,3

4322

4,50

1

YL

WW

VN

NQ

SLP

VSP

RC

EA

.176

/354

176/

354

563

167

422

226

8418

181

1,10

528

1,31

697

117

266

870

81,

606

QE

LFI

PNIT

VN

NSG

SC

EA

.282

282

147

644

2522

737

91,

658

293

1,08

635

81,

299

2674

0N

D3,

880

3,86

9

SYT

YY

RP

GV

NL

SLS

CC

EA

.423

423

1.6

4,42

56.

84,

036

300

5.4

211,

372

776

371

7.2

462,

626

1,78

413

5

RT

TV

KT

ITV

SAE

LP

KC

EA

.488

488

891,

267

5854

114.

23,

763

367

6,98

82,

758

9629

ND

1,26

31,

917

NG

TY

AC

FV

SNL

AT

GR

CE

A.6

5065

083

981

811

558

3020

7037

72,

174

2,05

230

2,35

16,

963

3,24

737

YA

CF

VSN

LA

TG

RN

NS

CE

A.6

5365

318

377

422

541

327

531

1,77

44,

520

217

2,39

910

71,

237

ND

1,56

917

NN

SIV

KSI

TV

SASG

TC

EA

.665

665

3410

343

1.8

128

3418

446

920

91,

328

4.4

274

15,8

0826

2,28

0

123


vitro stimulated CD4 T cells (1 £ 105 cells/well) and autol-ogous irradiated PBMCs (1 £ 105 cells/well) were added at1:1 ratio in each well (in 96-well plates for proliferationassay or in 96-well NC plates coated with anti-humanIFN-� Ab for ELIspot assay) and incubated at 37°C at 5% CO2

for 20–24 h in the presence of diVerent stimulants. Stimu-lants were each CEA peptide (10 �g/ml) and CEA protein(1 �g/ml). For the irrelevant peptide, C140, cyclin D1 pep-tide (MELLLVNKLKWNLAA) (10 �g/ml) was used andfor irrelevant protein, PKC nu (1 �g/ml), a protein of simi-lar preparation and size to CEA was used. To determineHLA class II restriction of peptide-speciWc CD4 T cells,anti-human HLA-DR and HLA-DP, DQ, DR antibodies(BD Biosciences, San Jose, CA) (10 �g/ml) were used inELIspot and proliferation assays. Wells with CD4 T cellsand irradiated PBMCs alone were considered as back-ground. ELIspot and proliferation assay results (back-ground subtracted) are expressed as antigen-speciWc CD4 Tcells/million PBMCs and CPM, respectively. These experi-ments were repeated three times using CD4 T cells isolatedfrom diVerent healthy donors.

Immunohistochemistry

Formalin-Wxed paraYn-embedded tissue sections weredeparaYnized and antigen retrieval was carried out usingEDTA. Slides were treated with peroxidase blockingreagent followed by incubation with protein block for5 min. Mouse monoclonal anti-CEA antibody (Cell Signal-ing Technology Inc., Danvers, MA) was applied at a 1/500dilution for 60 min. Visualization was carried out usingDAKO’s Dual+ Envision link followed by incubation withdiaminobenzidine. Sections were counterstained withhematoxylin. Staining intensity was graded on a 0–3 scale,0 and 1 grades were considered as low expression, 2 and 3grades were considered as high expression.

Statistical analysis

Statistical analyses were performed using GraphPad InstatSoftware or GraphPad prism software. Two-tailed Mann–Whitney tests or Student’s t tests were used to analyze thedata unless otherwise stated. p < 0.05 was considered assigniWcant.

Results

IdentiWcation of CEA peptides with high binding aYnity for HLA-DR molecules

Using the epitope prediction program PIC, 43 candidateCEA HLA-DR binding peptides were identiWed (data not

shown). These peptides were tested for their binding to 15diVerent HLA-DR molecules as described in “Materialsand methods”. Fifteen (35%) out of 43 peptides (Table 1),which bound to at least 8 diVerent HLA-DR molecules withIC50 binding aYnity of ·1,000 nM, were selected for fur-ther analysis.

Detection of elevated levels of peptide-speciWc T cell immunity in cancer patients

A 10-day IFN-� ELIspot assay as described in “Materialsand methods” was used to determine whether the individu-als previously diagnosed with breast or ovarian cancer hadgenerated natural immunity to any of the peptides. Immu-nity to 4 of the 15 peptides (CEA.24, CEA.176/354,CEA.488, CEA.653) was detected in patients with eitherbreast or ovarian cancer as shown in the scattergrams inFig. 1a–d. T cell frequencies to each of the peptides rangedfrom 593 § 124 (§SE) to 989 § 150 peptide-speciWc Tcells/million PBMCs. There were no discernable diVer-ences (p > 0.05) in epitope-speciWc T cell frequenciesbetween breast and ovarian cancer patients (data notshown). As shown in Fig. 2a, the cumulative T cell fre-quency of the three peptides CEA.24, CEA.176, andCEA.653 was increased in patients, whereas for one pep-tide, CEA.488, a smaller increase was observed. The diVer-ence in cumulative T cell frequency against the poolobserved in patients and controls was statistically signiW-cant (p = 0.01). Based on the available data describing theallelic frequencies, Hardy–Weinberg calculations estimatedthat the CEA pool of four peptides covers »94% of individ-uals (Table 2) [28, 29]. As shown in Fig. 2b, 40% ofpatients demonstrated immunity to the pool and this is sig-niWcantly (p = 0.006) higher than the proportion of healthydonors responding to the pool.

T cell responses do not correlate with patient clinical features

Blood was drawn from female volunteers without majorexclusion criteria other than that they had not been previ-ously diagnosed with cancer. Patients who had been diag-nosed and treated for breast or ovarian cancers or both butwho were currently disease free were selected for immuneassessment in this study. Although this unimpeded enroll-ment resulted in an age diVerence between the healthy con-trol donors and the patients, statistical analysis showed thatage did not explain the elevated immunity to CEA(p > 0.05). Neither stage nor grade of tumor correlated withthe levels of immunity (p > 0.05). Immunohistochemicalstaining results showed that 38% of patients had high levelsof CEA expression while 62% had little or no expression(data not shown). These staining results are consistent with

123


previous reports demonstrating that 10–50% of breast andovarian cancer patients have CEA-positive tumors [30, 31].Sixty percent of patients with CEAhi tumors and 38% ofpatients with CEAlo tumors had detectable immuneresponses (p > 0.05).

CEA.24, CEA.176/354, and CEA.488 peptides are derived from naturally processed whole CEA protein

Even though elevated T cell responses were observedagainst the CEA pool in breast and ovarian cancer patients,only those that are naturally processed are relevant toimmunotherapy. CEA.653 has already been identiWed inprevious studies as naturally processed and presented [3].To determine whether the remaining three peptides in CEApool are naturally processed from whole proteins, we gen-erated peptide-speciWc CD4 T cells by incubating themwith CEA.24, CEA176/354, or CEA.488 and then testingfor reactivity against whole CEA protein. As shown inFig. 3, we found that CD4 T cells speciWc to CEA.24,CEA.176/354, and CEA.488 peptides responded to bothwhole protein and respective peptide but did not respond toirrelevant protein (PKC nu protein) of similar size and irrel-evant peptide (cyclin D1 peptide C140). There were no dis-cernable diVerences between the reactivity of CD4 T cellsagainst peptides and CEA protein (p > 0.05). Lastly, to con-Wrm the HLA class II restriction of peptide-speciWc CD4 Tcells, inhibition experiments were performed as described

Fig. 1 Detection of CEA pep-tide-speciWc immunity in breast and ovarian cancer patients. Scattergrams showing IFN-� production by T cells derived from patients and healthy do-nors. Results are represented as peptide-speciWc T cells/million PBMCs. Percentages represent proportion of patients responded to peptides above the gray area, which represents mean + 2SD of the healthy control responses. Each dot represents an individ-ual (patient or healthy donor)

Healthy Patients0

500

1000

1500

2000

2500

3000

17%

CEA.24

Epi

tope

-spe

cific

T c

ells

(per

106 P

BM

Cs)

Healthy Patients0

500

1000

1500

2000

2500

3000

13%

CEA.176/354

Epi

tope

-spe

cific

T c

ells

(per

106 P

BM

Cs)

Healthy Patients0

500

1000

1500

2000

2500

3000

13%

CEA.488

Epi

tope

-spe

cific

T c

ells

(per

106 P

BM

Cs)

Healthy Patients0

500

1000

1500

2000

2500

3000

25%

CEA.653

Epi

tope

-spe

cific

T c

ells

(per

106 P

BM

Cs)

A B

C D

Fig. 2 Breast and ovarian cancer patients demonstrate elevated CEA-speciWc T cell immunity compared to normal healthy individuals.a Relational diagram comparing cumulative T cell frequencies of eachpeptide between control and patients. b Column graph showing pro-portion of patients and healthy donors (control) responded to pool ofCEA peptides. p values were calculated using Mann–Whitney test

Healthy Patients0

10

20

30

40

50 p=0.006

Pro

port

ion

of d

onor

sre

spon

ding

to p

ool

Healthy Patients0

50

100

150

200p=0.01

CEA.24

CEA.176

CEA.488

CEA.653

Mea

n C

EA

pep

tide

T c

ell f

requ

ency

(per

106

PB

MC

)

A

B

123


in “Materials and methods”. As shown in Fig. 4, the reac-tivity of peptide-speciWc CD4 T cells was blocked usinganti-human HLA class II antibodies, whereas use of isotypecontrol did not eVect the reactivity of these CD4 T cells.These results conWrm that the pool of peptides (CEA.24,CEA.176/354, CEA.488, and CEA.653) against whichbreast and ovarian cancer patients had elevated immunitywere naturally processed, presented and peptide-speciWcCD4 T cells are HLA class II restricted.

Discussion

The importance of CD4 T cells in anti-tumor immunity hasled to development of strategies to identify HLA class IIepitopes contained within diVerent tumor-associated anti-gens [2, 32]. Recently, there has been interest in deWningCD4 T cell epitopes in CEA because it is a tumor antigenthat is highly expressed in diVerent types of cancers and hasa role in tumorigenesis of cancers. IdentiWcation of severalCD4 T cell epitopes from tumor antigens such as HER-2/neu, IGFBP-2, and folate receptor alpha provides evidenceof pre-existing immunity against tumor antigens [5, 33].Based on these prior works demonstrating elevated tumor-speciWc immunity, we took a comprehensive approachevaluating not only many potential CEA epitopes but alsomany diVerent HLA-DR variants in order to capture a poolof epitopes that would be useful in the majority of patients.In summary, the novel Wndings of this study are (1) patientshave elevated Th1 CD4 T cell immunity to pool of CEAepitopes CEA.24, CEA.488, CEA.176, and CEA.653 and(2) a pool of four degenerate CEA epitopes consisting oftwo novel epitopes (CEA.24 and CEA.488) was establishedthat is potentially useful in 94% of patients.

An important Wnding, which conWrms the validity of ourapproach to epitope discovery, is that two of the four epitopeswithin the HLA-DR degenerate pool have been previouslyidentiWed, namely, CEA.176/354 and CEA.653. The assump-tion was made that CEA.176/354 is essentially the same asthe previously discovered epitope CEA177–189/355–367 [4].CEA.176/354 fully encompasses this peptide. At the timeof discovery, Campi and colleagues found that, when usedduring ex vivo priming among a pool of CEA epitopes,CEA177–189/355–367 was immunodominant. While ourresults support the conclusion that this epitope is immuno-dominant, we observed that 17, 13, and 25% of breast andovarian cancer patients responded to the peptides CEA.24,CEA.488, and CEA.653, respectively, which are eitherequal or greater proportions than the proportion (13%)responding to CEA.176/354. Thus, we speculate that all thepeptides that constitute the CEA HLA-DR degenerate poolidentiWed in this study might be co-dominant. In thatprior work, CEA177–189/355–367 was found to be restrictedT

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by several HLA-DR1 variants (DRB1*03, DRB1*13,DRB1*07, DRB1*14, DRB1*1101, DRB1*1104,DRB1*0405, DRB1*14) demonstrating its profound degener-acy. The present study extends these prior studies by furtherdemonstrating binding to DRB1*0101, DRB1*0401,DRB1*0404, DRB1*0802, DRB1*1501, DRB3*0101, andDRB4*0101. The other peptide that was previously identi-Wed, CEA653–667 (i.e. CEA.653), was identiWed by Kobayashiand colleagues using a similar algorithm [3]. In that study,it was found that CEA.653 binds to three HLA-DR mole-cules HLA-DRB1*04, -DRB1*07, and -DRB1*09, whichwe extended in the current study binds, with high aYnity,to include at least Wve additional HLA-DR molecules, DR1(DRB1*0101), DR3 (DRB1*0301), DR11 (DRB1*1101),

DR13 (DRB1*1302), and DRB5*0101. Thus, based onthese Wndings, our estimate that the HLA-DR degeneratepool of CEA.24, CEA.176/354, CEA.488, and CEA.653 isuseful in more than 94% of patients may be an underesti-mate. The true utility may approach 100%, if consideringthe alleles examined in our studies along with the othersexamined in prior studies.

The use of HLA class II epitopes that contain, fullywithin their sequences, HLA class I epitopes to induce bothCD4 and CD8 T cell responses simultaneously is an eVec-tive vaccination strategy [25, 34, 35]. The CEA-derivedHLA-A2-restricted CAP-1 peptide had been used as a vac-cine in several studies [36, 37] but the weak results of clini-cal trials using this vaccine have dampened enthusiasm of

Fig. 3 Peptides in the pool are derived from natural processing of CEA protein. a–c ELIspot and d–f proliferation assay results of peptide-speciWc CD4 T cells derived from short-term culture. Peptide-speciWc CD4 T cells were tested for their response against relevant peptide (CEA.24, CEA.176, CEA.488), CEA protein, irrelevant peptide (C140) and irrelevant protein (PKC nu protein). ELIspot re-sults are shown as peptide-spe-ciWc CD4 T cells per 105 CD4 T cells. Proliferation assay results are shown as counts per minute (CPM). Each columns are the mean § SE of three replicates. In all panels, the CD4 T cell responses against CEA peptides and protein are signiWcantly higher (p < 0.05) than the responses against irrelevant peptide and irrelevant protein. Columns without any values indicate that the mean values of the triplicates are lower than the background. Data shown are the representative of one of three repeated experiments with similar results

0

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4000CEA.24

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2000

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6000

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123


moving CEA peptides further into clinical trails. Recently, itwas shown by Saha and colleagues [38] that incorporating acomponent that activates helper T cells in CEA HLA class Ipeptide vaccine is important for increasing the eYcacy ofvaccine, perhaps by increasing CD8 T cell longevity. Thus,identiWcation of new CEA peptides that encompasses bothHLA class I and class II epitopes might address some of thecauses of these previous failures. Of the four epitopes thatconstitute the pool used in the current study, CEA.24–38,CEA.176–190/354–370, and CEA.653–667 all encompasspreviously reported HLA-A2 motifs, CEA.24–32,CEA.176–184/354–362, and CEA.652–660 [20]. Use ofthis pool, therefore, as a vaccine might induce both CD4 andCD8 T cell responses. Alternatively, the current pool could

be used as a CD4 T cell-activating component when mixedwith other unrelated CEA HLA class I epitopes.

Although our epitope identiWcation paradigm resulted inthe establishment of a promiscuous pool of four epitopes thatis potentially useful in a broad population, we cannot ruleout that the possibility that the other epitopes that were notchosen are not biologically relevant and potentially useful.For example, our binding assay results showed that peptidessuch as CEA.50 and CEA.116 bind 14 and 13 HLA-DRs,respectively, out of 15 HLA-DR molecules, but elevated Tcell immunity against these peptides was not seen in thepatients (data not shown). Furthermore, in previous studies,CEA.116 was reported as a naturally presented CD4 T cellepitope of CEA and shown to be immunogenic in HLA-DR4

Fig. 4 Peptide-speciWc CD4 T cells are HLA class II restricted. a–c ELIspot and d–f prolifera-tion assay results of peptide-speciWc CD4 T cells derived from short-term culture. Peptide-speciWc CD4 T cells were tested for their response against relevant peptide (CEA.24, CEA.176, CEA.488) and irrelevant peptide (C140) in the presence or absence of anti HLA-DR, HLA-DP, DQ, DR antibodies and isotype con-trol. ELIspot results are shown as peptide-speciWc CD4 T cells per 105 CD4 T cells. Prolifera-tion assay results are shown as counts per minute (CPM). Each columns are the mean § SE of three replicates. Columns without any values indicate that the mean values of the triplicates are lower than the background. Data shown are the representa-tive of one of three repeated experiments with similar results

0

50

100

150

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cific

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4+T

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0

500

1000

1500

2000

2500CEA.24

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50

100

150

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100

150

200

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123


transgenic mice [39]. Despite its promiscuous binding, thelack of response in the patients to this epitope is likely attrib-utable to immunodominance by the other peptides andimpairment of the T cell repertoire speciWc for CEA.116 byperipheral tolerization mechanisms. Tassi and colleagues[40] have recently reported that patients with pancreatic can-cer demonstrate impaired immunity (e.g. Th2 immunity) toCEA relative to normal healthy controls. In our study, someimportant CEA HLA-DR epitopes reported in previous stud-ies [41] may have been overlooked because the basis of ourdiscovery process was elevated IFN-� (i.e. Th1) immunity.Thus, our study may have focused on the discovery of epi-topes for which the eVects of tolerance are minimal. Thecentral problem that remains to be answered is whether it isbetter, in terms of clinical eYcacy, to boost existing anti-tumor immunity or to reverse (i.e. break tolerance) impairedimmune responses. Although we have shown that cancerpatients have endogenous T cell immunity against the poolof CEA HLA-DR epitopes, the role of humoral responses inCEA-speciWc immunity remains unanswered in this study.But previous studies demonstrating elevated levels of CEA-speciWc IgG antibodies in breast cancer patients suggests thepresence of CEA-speciWc CD4 T cells which is consistentwith our data [42]. Thus, our results support the previousWndings that tolerance against CEA is not complete and wespeculate that it is possible to boost the pre-existing immu-nity (both humoral and cellular immunity) against CEAusing the pool of peptides reported in this study.

Despite our prediction that 94% of patients couldrespond to the degenerate pool, we observed that only 40%of patients had elevated immunity against the pool. Severalfactors such as immunodominance, immunosuppression,and CEA expression in the patients selected for this studycan be attributed to the diVerences in predicted andobserved responses [5].The Wnding of anti-CEA T cellimmunity in patients without detectable CEA expressionwould suggest that the immune system may have selectedfor antigen-negative variants. Indeed, recent murine andhuman studies have shown that antigen-speciWc immuneeVectors (T cell and antibody) can select for antigen-nega-tive variants, i.e. immunoediting [1, 43]. For example,adoptive transfer of MART1/MelanA-speciWc CD8 T cellsinto patients with metastatic melanoma resulted in theappearance of antigen-negative tumor variants [1]. At pres-ent it remains unclear if CEA-negative tumors can developfrom CEA-positive tumors. Although CEA is associatedwith a more aggressive tumor, the fact that it is absent in ahigh proportion of patients suggests that it is dispensable.Thus, CEA should subject to immunoediting.

Lastly, given the fact that CEA is highly expressed inseveral carcinomas [30], we presume that identiWcation ofthis degenerate pool of CEA HLA-DR epitopes againstwhich cancer patients have elevated endogenous T cell

immunity might be useful in developing a multi-epitope-based CEA vaccine which would cover large population ofcancer patients.

Acknowledgments The authors gratefully acknowledge the MayoClinic Comprehensive Cancer Center Immune Monitoring Core forperforming the ELIspot assays, the Mayo Clinic Proteomics ResearchCenter and Tissue and Cell Molecular Analysis Center (TACMA). Theassistance of Corazon dela Rosa and Jennifer Childs is greatly appre-ciated. This work was supported by the Mayo Clinic ComprehensiveCancer Center, generous gifts from Martha and Bruce Atwater (KLK),K01-CA100764 (KLK), P50-CA116201 (JI), K12-CA090628 (KRK,LH), and R41-CA107590-01 (GI, KLK, JF).

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identification of a broad coverage hla-dr degenerate epitope pool derived from carcinoembryonic...

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