Direct discovery and validation of a peptide/MHC epitope expressed in primary human breast cancer...

Cancer Immunol Immunother (2010) 59:563–573
DOI 10.1007/s00262-009-0774-8
ORIGINAL ARTICLE

Direct discovery and validation of a peptide/MHC epitope expressed in primary human breast cancer cells using a TCRm monoclonal antibody with profound antitumor properties

Bhavna Verma · Oriana E. Hawkins · Francisca A. Neethling · Shannon L. Caseltine · Sherly R. Largo · William H. Hildebrand · Jon A. Weidanz

Received: 7 August 2009 / Accepted: 14 September 2009 / Published online: 25 September 2009© Springer-Verlag 2009

Abstract The identiWcation and validation of new cancer-speciWc T cell epitopes continues to be a major area ofresearch interest. Nevertheless, challenges remain todevelop strategies that can easily discover and validate epi-topes expressed in primary cancer cells. Regarded as targetsfor T cells, peptides presented in the context of the majorhistocompatibility complex (MHC) are recognized bymonoclonal antibodies (mAbs). These mAbs are of specialimportance as they lend themselves to the detection of epi-topes expressed in primary tumor cells. Here, we use anapproach that has been successfully utilized in two diVerentinfectious disease applications (WNV and inXuenza). Adirect peptide-epitope discovery strategy involving massspectrometric analysis led to the identiWcation of peptideYLLPAIVHI in the context of MHC A*02 allele (YLL/A2)from human breast carcinoma cell lines. We then generatedand characterized an anti-YLL/A2 mAb designated asRL6A TCRm. Subsequently, the TCRm mAb was used todirectly validate YLL/A2 epitope expression in humanbreast cancer tissue, but not in normal control breast tissue.

Moreover, mice implanted with human breast cancer cellsgrew tumors, yet when treated with RL6A TCRm showed amarked reduction in tumor size. These data demonstrate forthe Wrst time a coordinated direct discovery and validationstrategy that identiWed a peptide/MHC complex on primarytumor cells for antibody targeting and provide a novelapproach to cancer immunotherapy.

Keywords T cell epitope discovery · Human leukocyte antigens · Major histocompatibility complex · Peptide · T cell receptor mimics · Monoclonal antibody · P68 RNA helicase/dead box protein · Mass spectrometry · Orthotopic model · Therapeutic eVect

Introduction

Monoclonal antibodies (mAb) are able to recognize anti-genic determinants of diverse chemical composition withhigh aYnity and speciWcity making them good candidatesfor therapeutic development and active targeting. As such,mAb made against tumor markers have been successfullyexploited as cancer therapeutics [1–7]. Yet, for all of thesesuccesses only a limited number of relevant targets havebeen identiWed that are accessible for antibody targetingand induction of antitumor responses. EVorts continue to bemade to develop new technologies for the identiWcation ofnovel targets for antibody generation.

A potential class of surface markers not yet explored forantibody targeting is the major histocompatibility complex(MHC) or human leukocyte antigen (HLA) system. MHC(HLA) class I molecules are an attractive target as they areconstitutively expressed on the surface of all nucleated cellsin the body. The MHC class I molecules load peptide frag-ments derived from degraded cellular proteins, many of

B. Verma · J. A. Weidanz (&)Department of Pharmaceutical Sciences, School of Pharmacy, Center for Immunotherapeutic Research, Texas Tech University Health Sciences Center, 1718 Pine Street, Abilene, TX 79601, USAe-mail: [email protected]

O. E. Hawkins · W. H. HildebrandDepartment of Microbiology and Immunology, School of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA

F. A. Neethling · S. L. Caseltine · S. R. Largo · J. A. WeidanzReceptor Logic, Inc, Abilene, TX 79601, USA

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564 Cancer Immunol Immunother (2010) 59:563–573

which are localized in intracellular compartments, for trans-port and eventual presentation on the cell surface [8, 9].CD8+ cytotoxic T lymphocytes (CTL) survey the peptidespresented by class I MHC and are thought to target the cellsdisplaying disease-speciWc peptides [10, 11]. Thus, MHCclass I molecules loaded with speciWc peptides can be usedto distinguish diseased from healthy cells [12].

Several diVerent strategies are currently in use to dis-cover new peptide/MHC class I epitopes. These include (1)an indirect classical strategy that relies on the expressionproWling of uniquely expressed tumor-associated antigens(TAA) by tumor cells and (2) a direct strategy involvingbiochemical processes that relies on directly eluting pep-tides from MHC molecules expressed on the surface oftumor cells and then performing mass spectrometric analy-sis [13]. In expression proWling the TAA is examined forpeptides that might be processed and bind to a particularHLA allele [14–17]. The limitation of this strategy is theimperfection of the predictive algorithms used to identifythe peptides. The direct discovery strategy provides speciWcinformation regarding the number of epitopes that uniquelydecorate a cancer cell [18]. However, this approach is lim-ited by the minute amounts of peptide available for analysisand is further complicated by the impurities of the peptidesample that includes peptides simultaneously eluted fromup to six diVerent HLA class I molecules. We have recentlyreported on a more robust system that allows for the directidentiWcation of peptides [19–21]. However, this systemuses tumor cell lines for peptide identiWcation and itremains to be determined whether the peptides expressedby cell lines are truly representative of those found in pri-mary tumor cells. Further, it is not possible for T cells andmass spectrometric analysis to directly validate the expres-sion of discovered epitopes in primary tumor tissue. How-ever, antibodies speciWc for peptide/MHC molecules couldbe used to directly validate epitope expression in primarytumor cells and might also have therapeutic applications.

The utilization of antibodies to recognize MHC-restricted peptide targets on the surface of cancer cells haveseveral signiWcant beneWts. First, anti-peptide/MHC mAbscould be used to directly validate epitope expression infresh tissue. Second, these mAbs might lead to the detec-tion of novel biomarkers for cancer diagnosis. Finally, itmight be possible to markedly expand the repertoire oftherapeutic mAbs if it were shown that these molecules hadantitumor properties. In recent years, antibodies have beendescribed for the direct detection and visualization ofspeciWc peptide–MHC complexes (T cell epitopes) on thesurface of cells [22, 23]. Several groups including our labo-ratory have consistently been able to generate anti-peptide/MHC mAbs that we designate as TCRm [24, 25]. TCRmreagents would presumably be well suited for directlydeWning tumor peptide-epitopes expressed in primary

tumor cells. This could lead to new targets for cancer diag-nosis and therapeutic intervention.

Here, we demonstrate the successful integration of twotechnologies that were used to directly identify theYLLPAIHVI peptide from RNA helicase p68 protein pres-ent in the context of HLA-A2 from human breast carci-noma cell lines and then use the RL6A TCRm to directlyvalidate expression in primary human breast tissue. Finally,we show the potential therapeutic application of the RL6AmAb to inhibit tumor growth in an orthotopic breast cancermodel.

Materials and methods

IdentiWcation of YLLPAIHVI p68 peptide

We have previously reported on the strategy for peptide-epitope discovery [19–21, 25, 26]. In brief, in the presentexperiments human breast cancer cell lines MDA-MB-231,MCF-7 and BT-20 were obtained from the AmericanTissue Culture Collection (Manassas, VA) and weretransfected with plasmid constructs expressing solubleHLA-A*0201 (sHLA) in the expression vector pcDNA3.1.Stable transfectants were established by selection in G418Sulfate (CELLGRO-Mediatech, Lawrence, KS). Resistantcolonies were subcloned by single cell sorting (InXuxSorter, Cytopeia, Seattle, WA), grown to near conXuencyand sHLA production screened by ELISA with the highestsHLA-secreting subclones used for epitope-identiWcationexperiments.

Successfully transfected cancer cell lines that produce>40 ng/ml of sHLA-A*0201 protein in Xasks were grownin bioreactors (Toray, Tokyo, Japan) in a CP2500 CellPharm (Biovest International, Minneapolis, MN). Cellgrowth was maintained for 8–12 weeks in DMEM/F12K(Caisson Laboratories, North Logan, UT) under optimalpH, oxygen and glucose. sHLA-A*0201 molecules werepuriWed from harvested pools by aYnity chromatographyusing anti-HLA W6/32 monoclonal antibody (HB95,ATCC) that was linked to CnBr-activated Sepharose 4Bresin (GE Healthcare, Piscataway, NJ). sHLA–peptidecomplexes were eluted with 0.2 N acetic acid, after whichfractions containing sHLA were brought up to 10% aceticacid and heated at 76°C for 10 min to dissociate endoge-nous peptides from sHLA heavy chains and �2-microglobu-lin. Eluted peptides were then separated from MHC heavyand light chains by ultraWltration in a stirred cell with a3 kDa molecular weight cutoV cellulose membrane (Millipore,Bedford, MA) and Xash frozen prior to lyophilization. Aharvest of 20–30 mg of sHLA complex provided 250–500 �gof free isolated peptides representing the endogenouslybound class I peptides. The eluted peptides were desalted

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and fractionated by reverse-phase HPLC using a standardgradient of acetonitrile.

Mass spectrometric analysis

Each peptide fraction obtained from the HPLC was mappedby mass spectrometry. Initial ion scans were performed ona QStar Elite mass spectrometer (Applied Biosystems,Carlsbad, CA) equipped with a NanoSpray nano-ESI ioni-zation source, with windows of 300–1,200 atomic massunits (amu), and later, locked into 15 amu increments andvisually compared between three diVerent cancer cell lines.IdentiWed ions corresponding to the presence of speciWcpeptides were then subjected to tandem mass spectroscopy(MS/MS) to obtain peptide sequences. Comparison ofpeptides in the mass spectrometric maps indicated peptideepitopes, including their number and relative abundance,consistently found in cancer cell lines.

An ion with an m/z of 519.82 was identiWed as sharedin the MS ion maps and was selected for MS/MS analysis.A sequence of YLLPAIVHI was assigned to this ion. TheMS/MS spectra obtained were compared with the spectra ofsynthetic p68 peptide (YLLPAIVHIaa128–136). The syntheticpeptide contained the identical amino acid sequence corre-sponding to the putative p68 peptide found, when subjectedto MS/MS under identical conditions as the naturallyoccurring peptide and overlaid to conWrm sequence iden-tity, conWrming the endogenous p68 peptide sequence.Fragmentation patterns generated from MS/MS were inter-preted with the aid of web-based applications BioExplorer,MASCOT, Protein Prospector and BLAST, to identify thep68 protein origin [20].

Cell lines and human PBLs

The human tumor cell lines SW620 (colorectal), SKOV-3-A2 (ovarian), the human monocytic cell line THP-1 and T2cells, a human B lymphoblast cell line deWcient in TAP 1and 2 proteins [27] were obtained from the ATCC. Thebreast cancer cell line 1520 was a kind gift from Dr. Sold-ano Ferrone, University of Pittsburgh, Pittsburgh, PA.Human PBMCs from anonymous donors were obtainedfrom separation cones of discarded apheresis units from theCoVee Memorial Blood Center (Amarillo, TX) after plate-let harvest. Monocytes were isolated and used to generateimmature dendritic cells (iDC).

Antibodies and synthetic peptide

Polyclonal antibody goat anti-mouse IgG heavy chain–phycoerythrin (PE) was purchased from Caltag Laborato-ries (Burlingame, CA). Isotype control antibodies, mouseIgG1, IgG2a and IgG2b, were purchased from Southern

Biotech (Birmingham, AL). The BB7.2 (anti-HLA-A2.1mAb) expressing mouse hybridoma cell line was purchasedfrom the ATCC. Peptide p68 RNA helicase YLLPAIVHI(residues 128–136, designated as YLL 128); peptides TLAYLIFLL and KLMSPKLYV from CD19; peptide LLGRNSFEV from p53 tumor suppressor protein; peptide VLMTEDIKL from eIF4G protein; peptide ILDQKINEV fromornithine decarboxylase protein; and peptide KIFGSLAFLfrom Her2/neu protein were synthesized at the Universityof Oklahoma Health Science Center, Oklahoma City, OK,using a solid-phase method and puriWed by HPLC togreater than 90%.

Generation of HLA-A2 monomer and tetramers

HLA-A2 extracellular domain containing a C-terminusBirA sequence and �2m were produced as inclusion bodiesin Escherichia coli and refolded essentially as describedpreviously [28]. After refolding, the peptide–HLA-A2 mix-ture was concentrated and properly folded monomer com-plexes were isolated from contaminants on a Superdex 75sizing column (GE Healthcare Bio-Sciences AB, Piscata-way, NJ). Monomer concentration was determined by BCAprotein assay (Pierce, Rockford, IL) and then biotinylatedusing biotin ligase (Avidity, Boulder, CO). After a secondpuriWcation on the Superdex 75 sizing column, biotin-labeled monomer was then used to generate tetramercomplexes by addition of streptavidin that were used intetramer blocking assays or used in plasmon surfaceresonance studies.

Generation of RL6A TCRm

TCRm antibodies were generated as described previously[24, 25]. BrieXy, female BALB/c mice were immunizedwith a solution containing 50 �g of puriWed YLL peptide/HLA-A2 complex and Quil-A adjuvant (Sigma, St. Louis,MO) at 14-day intervals. Hybridoma supernatants werescreened by ELISA followed by conWrmation analysisusing peptide-pulsed T2 cells.

ELISA

ELISA assays were performed using Maxisorb 96-wellplates (Nunc, Rochester, NY). Assays to evaluate bindingspeciWcity of the TCRm antibodies were done on platescoated with 100 ng/well HLA/peptide tetramer. Boundantibodies were detected with peroxidase-labeled goat anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA)followed by the addition of ABTS (Pierce). Reactions werequenched with 1% SDS. Absorbance was measured at405 nm on a Victor II plate reader (PerkinElmer, Wellesley,MA). The SBA Clonotyping System/HRP and mouse

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immunoglobulin panel from Southern Biotech were used toestimate the concentration of RL6A (isotype IgG2a) in thesupernatant of FBS-containing medium. The assay was runaccording to manufacturer’s directions and RL6A signal wascompared with that of an IgG2a standard supplied by themanufacturer. Development, quenching and analysis of theplates were performed as described above for the TCRms.

Cell staining

T2 is a mutant cell line that lacks transporter-associatedprotein (TAP) 1 and 2 which allows for eYcient loading ofexogenous peptides. The T2 cells were pulsed with the pep-tides at 20 �g/ml for 4 h in AIM-V with the exception ofthe peptide-titration experiments in which the peptide con-centration was varied as indicated. Cells were washed andresuspended in FACS buVer (PBS + 0.5% FBS + 2 mMEDTA) and then stained with 25 and 50 ng/ml of RL6ATCRm, BB7.2 or isotype control antibody for 15–30 min in100 �l FACS buVer. Cells were then washed with 2 mlFACS buVer and the pellet was resuspended in 100 �l ofFACS buVer containing 2 �l of either of two goat anti-mouse secondary antibodies (FITC or PE labeled). Afterincubating for 15–30 min at room temperature, the washwas repeated and cells were resuspended in 0.25 ml FACSbuVer, analyzed on a FACS Canto II instrument and evalu-ated using FlowJo analysis software (Tree Star Inc., Ash-land, OR). Tumor cell lines were stained and evaluated inthe same manner as the T2 cells, after being released fromplates and washed in FACS buVer.

Plasmon surface resonance

Experiments were performed using SensiQ (ICx Nomadics,Oklahoma City, OK) biomolecular interaction analysisoptimized for doing surface plasmon resonance. BrieXy,Protein A was coupled to SensiQ sensors using EDC/NHSchemistry and then soluble RL6A TCRm was captured bypassing a solution of the TCRm (3.125 nM) over sensor at arate of 1 ml/min to achieve 1.578 response units (RUs). Thesensor was washed with HBS-TE buVer and then six con-centrations (10–311 nM) of soluble YLL peptide/HLA-A2monomer was sequentially run over the sensor and theamount of captured monomer was used to calculate thebinding aYnity constant and half-life of dissociation usingQ-DAT analysis software (ICx Nomadics).

Staining of human cells and lines

Tumor cells (3 £ 105) were washed and re-suspended in(0.1 ml) FACS buVer followed by primary incubation withRL6A TCRm at concentrations of 25 and 50 ng/ml. Isotypecontrol antibodies (IgG1, IgG2a, IgG2b) were used at either

25 or 50 ng/ml to stain cells and the pan-HLA-A2 antibody,BB7.2 was used at 500 ng/ml. Cells were stained for40 min protected from the light followed by addition of2 ml FACS buVer to each tube and then centrifuged at1,000 RPM for 10 min. The cell pellets were re-suspendedin 0.1 ml of FACS buVer and then incubated with goat anti-mouse Ig 1:200 dilution of concentrated antibody solution(FITC or PE labeled) for 15 min protected from the light.After incubation, the cells were washed and re-suspendedin 0.25 ml FACS buVer. Cells were run on FACS Canto IIand analyzed using FlowJo analysis software.

Western blot analysis

Denaturing gel electrophoresis was performed with 20–50 �g of total cellular protein from cell lysates per lane andthen transferred to nitrocellulose membranes for the detec-tion of proteins from which peptides were identiWed. Com-mercially available anti-p68 antibodies were purchasedfrom Santa Cruz Biotechnology (Santa Cruz, CA) for pro-tein detection.

Human tissue procurement

The institutional review board at Hendrick Medical Center(Abilene, TX) gave approval for patient consent collectionof normal and breast cancer tissue.

Immunohistochemistry

Tumor samples from each patient were placed on Cryo-mould, covered in OCT media, Xash frozen using isopen-tane and dry ice and stored at ¡80°C until used. Tissuesections were made at 5-�m size, Wxed using 5% methanoland stained with RL6A, IgG2a, BB7.2, IgG2b at 0.5 �g/mlfor 1 h in diluent containing 2.5% BSA to prevent non-spe-ciWc staining of tissue. Detection of primary antibody bind-ing was determined using goat anti-mouse Ig–horseradishperoxidase (Vector Labs) that in the presence of substrateDAB provides an indicator system (formation of brownprecipitate) to visualize the location of antigen/antibodybinding using light microscopy. Hematoxylin QS was usedas a nuclear counterstain (Vector Labs). Hematoxylin andeosin stains (Sigma) were used to assess cell morphologyand tumor cell presence in tissue. Tissue sections were ana-lyzed using light microscopy (Nikon Eclipse TE 2000,inverted, deconvolution microscope with Simple PCI Suitesoftware, Nikon, Melville, NY).

Tetramer competition assay

In several studies, tissue staining was performed in thepresence of soluble peptide/HLA-A2 tetramer to conWrm

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speciWc binding of the RL6A TCRm to YLL peptide/HLA-A2 target expressed on tumor cells in tissue. Prior to thestaining, RL6A TCRm and the BB7.2 mAbs at 1 �g/mlwere each mixed with soluble YLL peptide/A2, KIFS pep-tide/A2 or ILD peptide/A2 tetramers (10 �g/ml). After 1 h,tissue was rinsed with PBS and stained with secondary anti-body-peroxidase conjugate for 30 min (described above).Hematoxylin was used as a nuclear counterstain.

In vivo models

The IACUC committee at TTUHSC approved all animalstudies. Athymic nude mice (Crl:NU-Foxn1nu) wereobtained from Charles River (Wilmington, MA) andhoused under sterile conditions in barrier cages. Mice weresubcutaneously injected with 5 £ 106 freshly harvestedMDA-MB-231 cells at 97% viability in Matrigel in theright mammary pad of mice. Matrigel provides essentialnutrients for tumor cells to implant more eYciently andthus promote more rapid tumor growth. After 5 weeks, pal-pable tumors were detected (¸50 mm3) and mice received5 weekly i.p. injections with either 100 �g of an isotypeIgG2a control antibody (n = 5) or 100 �g of RL6A TCRm(n = 5). The RL6A TCRm and isotype control antibodieswere injected into mice using a diluent of Wltered sterile

phosphate buVered solution. This dosage regimen followedthe prophylactic and therapeutic settings for RL4B [29].

Tumor volume was measured twice weekly. Tumorvolumes were calculated by assuming a spherical shapeand using the following formula: volume = L £ (b2)/2(L = longest diameter, b = shortest diameter) where themean tumor diameter was measured in two dimensions.

Results

Direct discovery of a peptide derived from the p68 helicase protein that is common to breast tumor cell lines

The breast cancer cell lines, MDA-MB-231, MCF-7 andBT-20 were transfected with the sHLA-A*0201 (HLA-A2)construct. Peptides were puriWed from 25 mg of harvestedsHLA-A2 produced by each cell line. Comparative map-ping of sHLA-A2-derived peptides was performed and thep68 RNA helicase peptide YLLPAIVHI, located at aminoacid residues 128–136 of the protein and designated as theYLL peptide, was found to be expressed by the three breastcarcinoma cell lines (Fig. 1). For conWrmation purposes,the p68 peptide-containing fraction of each cell linewas compared with synthetic p68 peptide. These data

Fig. 1 Direct discovery of p68 peptide YLLPAIVHI expressed bybreast cancer cells. Ion map displaying the peptides eluted from solubleHLA-A2 complexes in three breast adenocarcinoma cell lines.Peptides were Wrst separated by HPLC (see “Materials and methods”).

In the ion map, the ion peak at 519.82 amu, highlighted with a bluebox, represents the YLLPAIVHI peptide common to all three breastcancer cell lines and was identiWed as originating from p68

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demonstrate that the YLLPAIHVI peptide is indeed loadedinto the HLA-A2 peptide-binding pocket in the threehuman breast carcinoma cell lines investigated. Further, thepeak that is prominent in the three breast cancer cell lines isminimal in the non-tumorigenic (control) mammary epithe-lial cell line; this, therefore, seemed like a good candidatefor an over expressed peptide.

RL6A TCRm is speciWc for the YLL-peptide/HLA2 complex

The RL6A TCRm mAb was generated as previouslydescribed [25, 26]. To establish that the RL6A TCRm mAbisolated in the initial screening was HLA-A2 restricted andpeptide-speciWc, a series of assays were performed to char-acterize its binding speciWcity. The Wrst assessment usedrefolded peptide/HLA-A2 molecules immobilized at 1 �g/ml in wells of 96-well plate as targets for testing the RL6ATCRm in ELISA. Figure 2a shows the results of ELISAanalysis of puriWed RL6A TCRm binding to refolded tetra-mer of HLA-A2/�2m loaded with YLL-peptide comparedwith four other HLA-A2 tetramer complexes loaded with

irrelevant peptides, TLAYLIFLL, LLGRNSFEV, KLMSP-KLYV and VLMTEDIKL. SigniWcant reactivity above thebackground was observed for all dilutions of RL6A TCRm(ranging from 25 to 0.78 ng/well) in wells containing YLL/A2 tetramer illustrating the Wne recognition speciWcity ofthe RL6A TCRm. To conWrm that each well was coated theHLA-A2 conformation-speciWc antibody BB7.2 was used(data not shown).

To further establish the binding speciWcity of RL6ATCRm for the YLL/A2 complex on the cell surface, T2cells were either used unpulsed or pulsed with the speciWcpeptide YLLPAIVHI or an irrelevant peptide (LLGRNS-FEV) at 20 �M concentration. RL6A TCRm was used tostain cells at 25 and 50 ng/ml, respectively and the resultsof the study are shown in Fig. 2b. The RL6A TCRm did notstain unpulsed T2 cells or T2 cells pulsed with irrelevantpeptide indicating that like the ELISA assay results RL6ATCRm binding was not due to the recognition of the heavychain of HLA, the �2m protein or the peptide alone.Instead, the RL6A TCRm showed a strong binding signalfor T2 cells pulsed only with the speciWc peptide (Fig. 2b)and that the signal was more intense with 50 ng/ml of

Fig. 2 Characterization of the binding speciWcity for RL6A TCRm. a PuriWed RL6A TCRm was used to probe wells coated with HLA-A2 tetramer refolded with the peptides indicated (CD19, TLAYLIFLL; p53 tumor suppressor protein, LLGRNSFEV; CD19, KLMSP-KLYV; eIF4G, VLMTEDIKL and DEAD Box RNA helicase p68; YLLPAIVHI). Bound mAb was detected with a goat anti-mouse IgG–peroxidase conju-gate and developed using ABTS substrate. b PuriWed RL6A TCRm (25 and 50 ng/ml) was used to stain 5 £ 105 T2 cells pulsed with the irrelevant pep-tide (LLGRNSFEV) or speciWc peptide (YLLPAIVHI) or no peptide. After washing, cells were probed with a goat anti-mouse IgG–PE, washed and ana-lyzed by Xow cytometry. Counts are mean Xuorescence intensity (MFI). c T2 cells were pulsed with varying levels of YLL pep-tide (0–160 ng/ml) and stained with 50 ng/ml RL6A TCRm followed by a secondary goat anti-mouse IgG. MFI values are shown for the various peptide concentrations

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TCRm. In total, RL6A TCRm did not stain T2 cells pulsedwith 30 diVerent irrelevant peptides (data not shown).These data demonstrate that RL6A TCRm is speciWc for theYLL peptide only in the context of HLA-A2. This pointwill be further expanded below.

Next, we examined the relationship between target den-sity and mean Xuorescence intensity (MFI) (Fig. 2c). Here,T2 cells were pulsed with decreasing concentrations of theYLLPAIVHI peptide ranging from 160 to 0.63 ng/ml fol-lowed by staining with 50 ng/ml of RL6A TCRm. Theresults show that RL6A TCRm staining intensity decreasedin parallel with decreasing concentrations of peptide(160 ng/ml of peptide resulted in MFI >5,000; 0.63 ng/mlof peptide resulted in MFI <1,000) indicating a dependenceof RL6A TCRm staining intensity on target density.

Experiments were then performed that were aimed atcharacterizing the binding kinetics for the RL6A TCRm. Tothis end, surface plasmon resonance was employed to exam-ine the binding aYnity (KD), the rates of association and dis-sociation and the half-life of dissociation for RL6A TCRmbinding to the YLL peptide/HLA-A2 complex. The TCRmexhibited strong binding aYnity (KD = 4.2 £ 10¡10 M) forcognate peptide/HLA-A2 complex displaying a rapid on rate(1.438 £ 105) and a noticeably slow oV rate (6.0 £ 10¡5).Further, TCRm binding to cognate peptide/HLA-A2 wascharacterized by a half-life of dissociation (t1/2) of greaterthan 190 min. These data indicate the RL6A TCRm isendowed with favorable binding characteristics for cognatepeptide/HLA-A2 complex supporting its application as aunique tumor-targeting agent.

RL6A TCRm detects endogenous YLL peptide-HLA-A2 presented on human tumor cell lines

The ability of RL6A TCRm antibody to detect endoge-nously processed peptide in the context of the HLA-A2molecule was evaluated by immunoXuorescence stainingand Xow cytometric analysis using a panel of HLA-A2-pos-itive human tumor cell lines that included MDA-MB-231(breast), 1520 (breast), SW620 (colorectal) and SKOV-3-A2 (ovarian). In addition, THP-1 cells (a human monocyticcell line) and human iDCs (normal iDCs) were used and areHLA-A2 positive. Western blot analysis was performedto determine the cellular expression status for p68 protein.All cancer cell lines (MDA-MB-231, 1520, SW620 andSKOV-3-A2) were shown to express p68 protein, however;expression was not detected in THP-1 cells and humaniDCs (Fig. 3a). The anti-HLA-A2-speciWc antibody, BB7.2conWrmed that the four tumor cell lines and THP-1 cellsand iDCs indeed expressed HLA-A2 (Fig. 3b, pink line).Next, speciWc YLL/A2 epitope expression was evaluatedby staining cells with the RL6A TCRm at 50 ng/ml(Fig. 3b). Only MDA-MB-231, 1520, SW620 and SKOV-3-A2 cells, which expressed both the p68 protein and HLA-A2 antigen, were positive when stained with the RL6ATCRm. In contrast, p68-negative cells, THP-1 cells andiDCs were not stained with RL6A TCRm. In addition,BT20 cells (human breast cancer cell line), used for the ini-tial discovery of YLLPAIVHI peptide, but lacking surfaceexpression of HLA-A2 were not stained with RL6A TCRm(data not shown). Collectively, these studies further support

Fig. 3 Detection of endogenous YLLpeptide/HLA-A2 complexes byXow cytometry and immunoXuorescent staining. a Western blot anal-ysis of protein samples from cell lysates. After protein transfer, nylonmembranes containing proteins were probed with an antibody speciWcfor p68 protein. b Cell staining was performed using 50 ng/ml of iso-type control, RL6A TCRm or BB7.2 antibodies on human immatureDCs (iDCs), THP-1 (human monocyte cell line) and four human tumor

cell lines, SW620, SKOV-3-A2, MDA-MB-231 and 1520. Antibodybinding was detected using goat-anti-mouse IgG–PE conjugate. Iso-type control antibody (IgG2a and IgG2b) staining is represented by thepurple Wlled peak and represents background staining level. RL6ATCRm cell staining is indicated with green line and staining withBB7.2 mAb is shown in pink line

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the conclusion that RL6A TCRm is highly speciWc for theYLL peptide/HLA-A2 complex.

RL6A TCRm stains primary human breast tumor tissue validating the peptide-epitope discovery technology

To further validate our peptide-epitope discovery technol-ogy, it was important to demonstrate RL6A TCRm bindingto epitopes on tumor cells in fresh human breast cancer tis-sue. A potential limitation of using human breast cancercell lines grown in long-term culture to discover peptideepitopes is the relevance of these targets to primary breasttumor cells. To begin to address the clinical relevance ofYLL/A2 epitope expression in human primary breast tumorcells, tissue was collected under an IRB protocol, Xash fro-zen, cut into sections (5 �m thick) and stained with eitherisotype control antibodies, anti-HLA-A2 antibody (BB7.2mAb) or RL6A TCRm antibody. The results are shown inFig. 4 and demonstrate that RL6A TCRm does not staincells in normal breast tissue (Fig. 4a), but does stain cells(shown by brown precipitate around cells) present in pri-mary breast tumor tissue (Fig. 4b) indicating that the YLL/A2 epitope is uniquely expressed in primary tumor cells.Further, we show by H&E staining that tumor cells arepresent in tumor tissue and are stained using the anti-p68mAb indicating the expression of p68 antigen (Fig. 4b). Incontrast, cells in normal tissue were not stained with theanti-p68 mAb (Fig. 4a). We have examined tissue fromHLA-A2-positive patients (n = 15) and all showed strongreactivity with RL6A TCRm (data not shown).

To further conWrm the speciWcity of RL6A TCRm, com-petitive binding assays were performed. Breast tumor tissuesections were stained with RL6A TCRm alone or in thepresence of speciWc (YLL/A2) or irrelevant (KIFS/A2) tet-ramer. The Wndings shown in Fig. 4c demonstrate thatRL6A TCRm staining was inhibited only in the presence ofthe YLL/A2 tetramer providing validation for the expres-sion of the YLL/A2 epitope on primary breast cancer cells.As expected, BB7.2 mAb staining of tumor tissue sectionswas completely blocked with both peptide/A2 tetramers(Fig. 4c). In addition, we have examined tumor tissue fromHLA-A2-negative breast cancer patients (n = 3) and shownthat RL6A TCRm does not stain, indicating the speciWcityof the TCRm is for YLL peptide in the context of HLA-A2(data not shown). These studies further validate our pep-tide-epitope direct discovery technology and indicate YLL/A2 epitope expression on primary breast cancer cells.Moreover, these Wndings suggest overexpressed tumor pep-tides by primary breast tumor cells can be discovered usingcultured tumor cell lines. Finally, these data indicate thatTCRm are useful reagents for validation and detection ofspeciWc peptide/HLA complexes expressed on tumor tissue.

Fig. 4 RL6A TCRm detects the YLL peptide/HLA-A2 epitope in hu-man breast tumor tissue. Immunohistochemistry staining was performedusing a normal human breast tissue and b human breast tumor tissue fromthe same donor. RL6A TCRm, BB7.2 and isotype control antibodieswere used at 0.5 �g/ml concentration and detected using a secondarygoat-anti-mouse-IgG–HRP conjugate. Chromagen substrate DAB pre-cipitation indicates BB7.2 and TCRm binding to cells. c Competitiveblocking of RL6A TCRm staining of human breast tumor tissue. RL6Awas used to stain tissue alone at 0.5 �g/ml or mixed with 10 �g/ml of spe-ciWc (YLL peptide/A2) or irrelevant (KIFS peptide/A2) tetramer. Tumortissue staining with BB7.2 mAb (0.5 �g/ml) was completely blockedwith 10 �g/ml of YLL peptide/A2 and KIFS peptide/A2 tetramer

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In vivo eVectiveness of RL6A TCRm using an orthotopic breast cancer model

To address the therapeutic eVectiveness of RL6A TCRm,we used an orthotopic breast cancer model involving athy-mic nude mice implanted with MDA-MB-231 tumor cells.Tumors grew until they reached palpable sizes or tumorvolumes of approximately 50 mm3. Mice were treated withWve weekly i.p. injections of 100 �g or »3.0 mg/kg withisotype control antibody or RL6A TCRm. After a 5-weekcourse of treatment, mice (n = 5) receiving weekly injec-tions with isotype control antibody had developed tumorswith mean volumes greater than 900 mm3 with tumor sizeobserved in two mice growing to larger than 1,200 mm3. Incontrast, mice treated with RL6A TCRm (n = 5) hadtumors that had only grown to a mean tumor volume of117 mm3 by week 4 after treatment with RL6A TCRm. Infact, by week 4, tumor volume was no longer measureablein two mice suggesting tumor had been completed eradi-cated. Mice treated with RL6A TCRm were followed fortwo additional weeks and still did not show signs of tumorgrowth demonstrating RL6A’s potent anti-tumor activitysigniWcantly impedes tumor progression and may delayrelapse of tumor growth (Fig. 5). Collectively, these Wnd-ings demonstrate that TCRm targeting of speciWc peptide/MHC complexes have potent anti-tumor eVects that cansigniWcantly inhibit tumor growth and reduce tumor size ina mouse model.

Discussion

We show here the direct validation of a novel T cell epitopebeing expressed in primary human breast tumor cells usinga TCRm mAb. Furthermore, the same mAb was then usedto inhibit growth of human breast tumors in an orthotopicmodel. These Wndings are important as they (1) corroboratean innovative approach to T cell epitope discovery fortumor associated or speciWc peptide/HLA complexes, (2)provide a direct means for validating T cell epitope expres-sion in human tumor tissue using mAbs and (3) demon-strate the anti-tumor properties of TCRm mAbs throughbinding to peptide/MHC complexes.

To date, several hundred human TAAs have beendescribed [30]. The discovery of tumor associated/speciWcT cell epitopes has relied primarily on indirect strategiesthat make use of gene and protein expression proWling com-bined with predictive algorithms and in vitro peptide-bind-ing assays [31]. The experience with tumor antigens is that<50% of predicated peptide epitopes can actually be used togenerate CTL that kill tumors in vitro [31, 32]. Moreover, adirect association has not been established between expres-sion and peptide-epitope presentation by MHC molecules[24]. Alternatively, discovery of peptide epitopes usingdirect strategies has identiWed unique and speciWc peptides[18]. However, a number of factors including amounts ofpeptide that can be recovered from the elution phase, thecomplexity of the tissue samples (malignant and healthycells) used to elute and isolate peptides, the quality of pep-tide preparations and the sensitivity limits of the instrumen-tation hamper direct discovery strategies. A major advancefor direct discovery is the development of technology thatrelies on the production of large amounts of soluble HLA/peptide complexes that until recently had been limited tothe identiWcation of peptides for infectious agents [20, 33,34]. Here, stable breast tumor cell transfectants expressinghigh concentrations of sHLA were used to discover the p68peptide YLLPAIHVI presented in the context of HLA-A*0201; a strategy recently used by our group to discoverother breast cancer peptide/HLA-A2 epitopes [19]. Apotential limitation for this strategy was that we might notdiscover peptides expressed in primary tumor cells usingcultured tumor cell lines because long-term cultured tumorcells may lose attributes common to the primary tumor cells.Another concern has been whether long-term culturedcells express diVerent T cell epitopes than primary tumors.Therefore, because the discovery strategy used in thisreport makes use of tumor cell lines to directly identify pep-tide epitopes, it was necessary to validate the YLL peptide-epitope on primary breast tumor tissue using the RL6ATCRm. Thus, it was important to establish that the RL6ATCRm, which stained cultured tumor cell lines from whichit was originally discovered also stained frozen tissue

Fig. 5 RL6A TCRm inhibits tumor growth in an orthotopic breastcancer model. Athymic nude mice were implanted with 5 £ 106 MDA-MB-231 tumor cells in the right mammary fat pad. Tumor cells mixedwith Matrigel were implanted and allowed to grow until mean tumorvolume reached >50 mm3. Mice then received a 100 �g i.p. injectionwith either IgG2a isotype control antibody dashed line (n = 5), orRL6A TCRm solid line (n = 5) on day 0 followed by weekly i.p. injec-tions with 100 �g of antibody for 4 weeks. Tumor size was measuredtwice weekly using calipers and tumor volumes determined using thestandard formula volume = L £ (b2)/2 (L = longest diameter,b = shortest diameter) where the mean tumor diameter was measuredin two dimensions. Data are plotted as mean tumor volume + SEM.Study is representative of three independent studies

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sections of breast cancer and lymph nodes from patientswith metastatic breast cancer.

The target of RL6A TCRm is an RNA helicase protein.These proteins are part of a large family of highly con-served intracellular proteins, the so-called D (Asp) E (Glu)A (Ala) D (Asp) DEAD box family of putative helicases[35]. The p68 RNA helicase belongs to this family ofDEAD box proteins and potentially plays a number of criti-cal physiological roles [36–41]. In addition, p68 has beenwidely implicated in tumor cell growth regulation andtumor development [42]. Overexpression of p68 has beenobserved in many cancers and post-translational modiWca-tion by ubiquitination and phosphorylation of p68 has beenimplicated in tumor development and cell proliferation/transformation [43, 44]. Hunt et al. [18] Wrst discovered theYLLPAIVHI peptide in transformed human B cells. It isconceivable that peptides such as YLLPAIHVI from p68are readily translocated into the lumen of the ER and loadedinto the binding pocket of HLA-A*0201. Our Wndings withRL6A TCRm staining of p68 antigen-positive humanbreast tumor tissue and its speciWc blocking with solubleYLL/A2 tetramer but not control tetramer indicates thatp68 YLL peptide/HLA-A2 complexes are expressed on pri-mary tumor cells. In addition, evidence for YLL/A2 expres-sion on cancer cells is supported by the absence of RL6ATCRm staining of normal human breast tissue and tissuefrom HLA-A2-negative breast cancer patients. Detection ofthe YLL peptide/HLA-A2 on the surface of tumor cell linesand primary tumor cells with RL6A TCRm supports theview that the YLL peptide/HLA-A2 complex is a putativetumor associated/speciWc epitope. Thus, these Wndings vali-date the discovery strategy of using tumor cell lines and cannow be applied to directly identify peptide epitopesexpressed in primary and metastatic breast cancer cells toassist in the development of new immunotherapy strategiesthat include T cell eliciting vaccines, adoptive T cell thera-pies and TCRm mAbs.

Two distinctly diVerent strategies have been used to gen-erate anti-peptide/MHC antibodies. On the one hand, phagedisplay has been used to generate murine and human Fabsfrom immunized and non-immunized libraries [45, 46]. Onthe other hand, our strategy for making antibody to speciWcpeptide/MHC complexes led to the generation of RL6ATCRm, which relied on immunization and classical hybrid-oma technology [24, 25]. A signiWcant beneWt of ourapproach is that the TCRm antibodies routinely have high-binding aYnity and avidity and retain the ability to activateimmune eVector functions. Furthermore, tumors treated withthe RL6A TCRm remained small and did not return even4 weeks after stopping treatment. This raises the possibilitythat the TCRm had successfully killed the entire tumor.

In conclusion, we show the ability to directly discover andthen validate an epitope on primary human breast tumor tissue

using a TCRm mAb. This work is signiWcant because it pro-vides the Wrst evidence for an integrated discovery and valida-tion strategy that can be used for the direct visualizationof T cell epitopes in tumor tissue. This strategy has not beenemployed previously but could ultimately provide signiWcantinsight into the peculiarities of peptide-epitope expression intumor cells. Finally, these results indicate TCRm are promis-ing agents for cancer detection and treatment.

Acknowledgments We thank Dr. William P. Weidanz for criticaldiscussion of the data. We thank Dr. Stephen Wright and Dr. PiotrTabaczewski for his assistance with the mouse models and Dr. KathrynNorton for tissue acquisition.

ConXict of interest statement Jon A. Weidanz is Chief Scientistand founder of Receptor Logic, Inc., Abilene, Texas, 79601. Study wassupported in part by Receptor Logic, Inc.

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