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Lung Cancer 77 (2012) 312– 318

Contents lists available at SciVerse ScienceDirect

Lung Cancer

jou rn al h om epa ge: www.elsev ier .com/ locate / lungcan

verexpression and potential targeting of the oncofoetal antigen 5T4 inalignant pleural mesothelioma

aly Al-Taeia, Josephine Salimua, Jason F. Lesterb, Seamus Linnanec,d, Madusha Goonewardenae,ichard Harrope, Malcolm D. Masona, Zsuzsanna Tabia,∗

Department of Oncology, School of Medicine, Cardiff University, Velindre Cancer Centre, Velindre Road, Whitchurch, Cardiff CF14 2TL, UKVelindre NHS Trust, Velindre Cancer Centre, Velindre Road, Whitchurch, Cardiff CF14 2TL, UKCardiff and Vale NHS Trust, Llandough Hospital, Penlan Road, Llandough, Penarth CF64 2XX, UKBlackrock Clinic, Suite 13, Rock Road, Blackrock, Co Dublin, IrelandOxford BioMedica UK Ltd., The Medawar Centre, Robert Robinson Avenue, Oxford Science Park, Oxford OX4 4GA, UK

r t i c l e i n f o

rticle history:eceived 10 January 2012eceived in revised form 2 March 2012ccepted 14 March 2012

eywords:alignant pleural mesothelioma

umour-associated antigenovel targetT4ancer vaccine-cell killing

a b s t r a c t

Malignant pleural mesothelioma (MPM) is resistant to conventional treatments. Novel, targeted treat-ments are hampered by the relative lack of MPM-associated tumour antigens. The aim of this studywas to evaluate the level of expression and the relevance of 5T4 as a tumour-associated antigen inMPM. 5T4 expression was assessed by Western blotting, flow cytometry, immuno-cytochemistry and-histochemistry in 11 mesothelioma cell lines, 21 tumour biopsies, and ex vivo tumour cells obtainedfrom the pleural fluid (PF) of 10 patients. 5T4 antibody levels were also determined in the plasma ofpatients and healthy donors. The susceptibility of MPM cells to 5T4-specific T-cell-mediated killing wasdetermined using an HLA-A2+, CD8+ T-cell line, developed against the 5T417–25 peptide. We report herethat cell surface 5T4 expression was detected in all mesothelioma cell lines and PF cell samples. Mesothe-lin and CD200, a suggested mesothelioma marker, were co-expressed with 5T4 on tumour cells in PF.Immunohistochemistry confirmed overexpression of 5T4, similar to mesothelin, on tumour cells but noton reactive stroma in all tissue sections tested. Median 5T4 antibody levels were 46% higher in patient

+

than in healthy donor plasma, indicating immune recognition. Importantly, 5T4-specific CD8 T-cellswere able to kill four out of six HLA-A2+ MPM cell lines but not an HLA-A2− cell line, demonstratingimmune recognition of MPM-associated 5T4 antigen at the effector T-cell level. We conclude that 5T4 isa potential new antigen for targeted therapies such as immunotherapy in MPM, as it is overexpressed onmesothelioma cells and recognised by 5T4-specific cytotoxic T-cells. Our findings have been translated

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into a Phase II clinical tria

. Introduction

Malignant pleural mesothelioma (MPM) is an incurable malig-ancy of the pleural membranes. In 2008, 2249 people in theK died from MPM and median survival is only 6–18 months.emetrexed–cisplatin chemotherapy is the current standard ofare, but treatment results in median survival advantage of lesshan 3 months [1], dictating an urgent need for the development ofew therapies.

The impetus to pursue immunotherapy in MPM stems from var-ous observations. Tumour-infiltrating CD8+ T-cells correlate withavourable prognosis in MPM [2,3], whilst a rare case of regression

∗ Corresponding author at: CRW Research Laboratories, Velindre Cancer Centre,elindre Road, Whitchurch, Cardiff CF14 2TL, UK. Tel.: +44 2920 196137;

ax: +44 2920 529625.E-mail address: zsuzsanna.tabi@wales.nhs.uk (Z. Tabi).

169-5002/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.oi:10.1016/j.lungcan.2012.03.008

lying 5T4-targeted therapies in MPM patients.© 2012 Elsevier Ireland Ltd. All rights reserved.

has correlated with positive immune parameters [4]. Furthermore,mesothelioma models respond to immunotherapeutic strategies[5]. For example, intrapleural interferon-� triggered local immuneresponses, detectable even at distant sites with clinical benefit [6].In patients, a trial utilised autologous tumour cell lysate loadedon patients’ dendritic cells (DC) in order to trigger anti-tumourimmune responses with positive results [7].

Targeted immunotherapy relies on the overexpression oftumour-associated antigens (TAA). Mesothelin, a differentiationantigen normally present on mesothelial cells but overexpressedin malignancy, is currently the main target antigen [8], but itsoverexpression is restricted to epithelioid mesothelioma [9]. Inthis study, we aimed to determine the expression profile of 5T4and its relevance for immunological targeting in MPM. 5T4 is a

72 kDa oncofoetal glycoprotein with cell surface expression [10].It has restricted expression in normal tissues but is overexpressedin numerous malignancies, including testicular, breast and coloncancer [11]. It alters cellular dynamics facilitating metastatic spread

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12,13] and is a negative prognostic factor [14]. Consequently, 5T4argeted therapies are under development such as 5T4 antibodyonjugated to calicheamicin [15] or Staphylococcal enterotoxin EABR-214936) which proved effective in Phase II trials in advancedenal cell carcinoma patients [16]. The cancer vaccine Trovax® (aodified vaccinia Ankara virus encoding 5T4) is also undergoing

linical trials. It has been tested in 500 patients in Phase I, II andII clinical trials in advanced colorectal, renal and prostate can-er. The vaccine was well tolerated and a positive association wasbserved between the level of vaccine-induced antibody responsesnd clinical outcome [17].

In this study, 5T4 expression was assessed in mesothelioma cellines, tumour tissue and pleural fluid (PF) cells, representing thehree disease subtypes; epithelioid, sarcomatoid and biphasic. 5T4-pecific antibody responses were studied in patient plasma, and theusceptibility of mesothelioma cells to 5T4-specific T-cell killingas established in cytotoxicity assays.

. Materials and methods

.1. Tumour tissue

Ethical approval for obtaining human-derived material wasranted by the South East Wales Research Ethics Committee andnformed consent was obtained from all subjects. Biopsy specimens

ere snap-frozen and stored in liquid-nitrogen vapour. Formalin-xed paraffin-embedded (FFPE) tissue sections were obtained fromhe Histopathology Department at Llandough Hospital (Cardiff,K).

.2. Peripheral blood mononuclear cells (PBMC), PF cells andlasma

PF cells were isolated by centrifugation from PF collected viandwelling catheters. Venous blood of healthy donors or the pel-eted PF cells were subjected to density gradient centrifugationn Histopaque® (Sigma, UK) then the cells (PBMC or PF cellsespectively) were cryopreserved in RPMI-1640 media (Lonza, UK)upplemented with 100 U/ml penicillin, 100 �g/ml streptomycin,

mM l-glutamine, 25 mM HEPES, 1 mM Na-pyruvate (sRPMI) plus0% DMSO and 20% foetal bovine serum (FBS; PAA, UK) were added.lasma collected from the uppermost fraction of separated bloodas frozen at −20 ◦C.

.3. Cell lines and culture

Mesothelioma cell lines were generated by long term culturef explanted tumour biopsies and maintained by regular passag-ng in sRPMI 5% FBS. PF-derived tumour cell lines were obtainedy long-term culture of adherent PF-cells. Tumour subtype wasetermined at the time of surgery. Cell lines were used betweenassages 6–12. B lymphocytic cell lines (BLCL) were generated fromBMC by standard EBV-immortalisation. LNCaP human prostatedenocarcinoma cell line (ECACC, UK) and T2 cells (HLA-A2+ humanAP-deficient hybridoma; Cardiff University, MRC Cooperative)ere grown in sRPMI 10% FBS. Cell lines were HLA-typed by theelsh Blood Service and underwent regular mycoplasma testing

sing Vector® GeM mycoplasma detection kit (Cambio Ltd., UK).

.4. Western blotting

Lysates were extracted from cell lines using lysis buffer (2%P-40, Sigma). Snap frozen tumour tissue was thawed in ice-old RIPA buffer (50 mM Tris–HCl pH 7.4, 1% NP-40, 0.25%a-deoxycholate, 1 mM EDTA and 150 mM NaCl) then crushed.

r 77 (2012) 312– 318 313

Protein concentration was determined using micro BCATM pro-tein assay kit (Thermo Fisher, UK). 5T4 protein was included(Oxford BioMedica, UK). Broad-spectrum protein markers (Biorad,UK) were used. Lysates were separated on a 12% polyacrylamide gel(30% acrylamide/bis-acrylamide; Sigma) by SDS-PAGE under non-reducing conditions, transferred to HybondTM-P PVDF membrane(GE Healthcare, UK), blocked in PBS/Tween 20 (Sigma) with 5% non-fat dry milk and probed with anti-5T4 (Oxford Biomedica) [10].Horseradish-peroxidase-conjugated secondary antibody was used(Insight Biotechnology Ltd., UK). Bands were developed by ECL+ onfilm (GE Healthcare, UK) and visualised using the Xograph Compactx4 X-ray film processor (Xograph Healthcare Ltd., UK). For calre-tinin, the transfer was carried out using the iBlotTM Dry BlottingSystem with PVDF gel-transfer stacks (Invitrogen, UK). After prob-ing for calretinin (BD, UK), the Western DotTM 625 Western blotsecondary detection kit was used (Invitrogen). Membranes werevisualised using MiniBIS-Pro with GelCapture software (BertholdTechnologies UK Ltd., UK).

2.5. Flow cytometry

The 5T4 antibody (Oxford Biomedica) was conjugated toAlexa-488 (Invitrogen). Cells were blocked with FcR block-ing reagent (Miltenyi Biotec, UK), labelled with 5T4-Alexa-488,mesothelin (Rockland Immunochemicals, USA) followed by Alexa-488-conjugated secondary antibody, or isotype controls. PF cellswere labelled with 7-AAD (eBioscience), CD14-APC-Cy7, CD200-PE, CD3-APC (BD), 5T4-Alexa-488 and mesothelin-APC (R&D, UK).Flow cytometry was performed on a FACSCanto flow cytometer(BD) using FACSDiva software.

2.6. Immunocytochemistry

Mesothelioma cells (104) were seeded onto coverslips. Once60% confluent, cells were fixed in 4% paraformaldehyde (Sigma),permeabilised in 0.05% Triton-X100 (Sigma) and blocked in 0.1%mouse serum (Gibco) before labelling with 5T4 (Oxford Biomedica)or isotype antibody (Dako, UK), followed by Alexa-488-conjugatedsecondary antibody (Invitrogen). Coverslips were mounted usingVectashield mounting media with DAPI (Vector Laboratories, UK).Imaging was performed on Zeiss Axiovert 40 CFL epifluorescencemicroscope, connected to Cannon Powershot S45 digital cameraand acquired with RemoteCapture software.

2.7. Immunohistochemistry

FFPE sections were de-waxed in xylene (Genta Medical Ltd.,UK) then rehydrated in 100%–92%–70% ethanol-gradient. Antigenretrieval was performed by heating slides in retrieval solution(Dako) at 95 ◦C then at room temperature and washing in distilledwater. Sections were stained for 5T4 (R&D), isotype (eBioscience)or mesothelin (R&D) using a signal amplification kit (Dako). Sec-tions were counterstained with haematoxylin (Thermo-Fisher),dehydrated in 70%–92%–100% ethanol-gradient then in xylene andmounted with DPX (Thermo-Fisher). Slides were imaged on MiraxScanner (Zeiss, UK) and analysed using Pannoramic Viewer soft-ware (3DHistech, Hungary).

2.8. 5T4 antibody levels

Plasma 5T4-specific antibody levels were determined using avalidated semi-quantitative ELISA as described previously [18]. ABserum (Sigma), pooled from 200 individuals, was used as a control.

3 Cancer 77 (2012) 312– 318

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Fig. 1. Characterisation of mesothelioma cell lines for 5T4 expression. (A) West-ern blot of total 5T4 expression. Equal amounts of cell-lysate protein (30 �g) fromLNCaP or mesothelioma cells or purified 5T4 protein (100 ng) were probed for 5T4(10 �g/ml; top) and cell-lysates also for calretinin (1 �g/ml; bottom). The numberson top of the gels represent the sample IDs. (B) Flow cytometry analysis of 5T4cell surface expression on LNCaP and mesothelioma cell lines. Cells were labelledwith either IgG1-Alexa488 isotype (black bar) or 5T4-Alexa488 (10 �g/ml; greybar) conjugated antibodies. Means and SD of mfi (mean fluorescence intensity) areshown, calculated from two independent experiments. (C) Flow cytometry analysisof mesothelin surface expression. Cells were labelled with either rat IgG2A isotype(black bar) or mesothelin antibody (2 �g/ml; grey bar), followed by a goat anti-

14 S. Al-Taei et al. / Lung

.9. Generation of a 5T4 peptide-specific T-cell line

A CD8+ T-cell line was developed from a HLA-A2+ healthy donory stimulation of non-adherent PBMC with autologous monocyte-erived day-six DC, generated from adherent PBMC with 500 ng/mlM-CSF (Prospec-Tany Technogene Ltd., Israel) and 500 U/ml IL-

(Peprotech, UK). DC were loaded with 2 �g/ml 5T417–25 peptideRLARLALVL; 90.4% purity; ProImmune, UK) [19] and treated simul-aneously with 5 ng/ml LPS for 1 h. DC were washed and platedut in 24-well trays at 2 × 105 cells/well with 4 × 106 non-adherentBMC in 2 ml. Cultures were supplemented with 1000 U/ml IL-6nd 5 ng/ml IL-12 [20] and grown for eight days. Peptide-specifictimulation was repeated weekly with peptide-loaded autologousC as above, supplemented with 5 ng/ml IL-7 and 10 U/ml IL-2 [20].fter 24 days, peptide-specific cells were separated using IFN-� cellnrichment (Miltenyi Biotech, UK). Separated T-cells (1–5 × 105)ere expanded weekly with a mixture of 5 × 106 peptide-pulsed

utologous BLCL irradiated with 4000 rad; 5 × 107 allogeneic PBMCixed from 2 to 3 donors and irradiated with 3000 rad; 50 U/ml

L-2 and 1 �l/ml OKT3 hybridoma supernatant (MRC Coopera-ive, Cardiff University) in 50 ml sRPMI 10% FBS and 1% AB-serumSigma) in a T75 flask. Half the media was replaced at day four withresh media containing cytokines. CD8+ T-cell separation was car-ied out after seven days using the EasySep CD8+ T-cell enrichmentit (StemCell Technologies, UK). Cytotoxicity assay was carried outfter 7–9 days in expansion.

.10. Cytotoxicity assay

T-cell cytotoxic activity was tested on peptide-pulsedr unpulsed T2 cells labelled with 51Cr. Target cells3 × 103)/100 �l/well and increasing numbers of T-cells in 100 �l,t Effector:Target (E:T) ratios ranging from 80:1 to 2.5:1, werelated in triplicates in a 96-well tray. Control wells containedarget cells only with 100 �l media or 100 �l 5% Triton-X100.upernatant (50 �l) was collected from each well 4 h later and1Cr-release was measured on a Microbeta-3 (Perkin-Elmer, UK)ounter. Tumour cell killing was tested the same way as above, onix HLA-A2+ and one HLA-A2− mesothelioma line (3 × 103 tumourells/well) in a 5 h 51Cr-release assay at 20:1 E:T ratio. Percentpecific lysis was calculated as described [21].

.11. Statistical analysis

Statistical analyses were carried out using Student’s t-testSigmaPlot 10.0.1) or Mann–Whitney U-test (GraphPad Prism 5),s indicated. Differences were considered statistically significantt p < 0.05.

. Results

.1. Characterisation of mesothelioma cell lines for 5T4 expression

In this study cell lysates from five biphasic (M2, 18, 19, 26, 36),ve epithelioid (M1, 15, 30, 38, 43) and one sarcomatoid (M34)umour cell lines and LNCaP cells were subjected to Western blot-ing to assess total 5T4 expression. All mesothelioma lines wereositive for 5T4, including the sarcomatoid line M34 (Fig. 1A),hilst LNCaP cells were negative. The observed 5T4 band co-igrated with purified 5T4 protein. Flow cytometry revealed 5T4

urface expression on all mesothelioma lines but not on LNCaP cells

Fig. 1B). To confirm that the cell lines were true mesothelioma cells,xpression of mesothelin and calretinin, both diagnostic markersor MPM [9], was assessed. All epithelioid cell lines were positiveor either mesothelin (Fig. 1C) or calretinin (Fig. 1A) or both.

mouse-Alexa-488 antibody. Means and SD of mfi are shown, calculated from twoindependent experiments. (D) 5T4 cellular distribution in M15 (i), M38 (ii), M43(iii) and M19 (iv) mesothelioma cell lines by epifluorescence microscopy. Cells werestained with 5T4 primary antibody (10 �g/ml) followed by Alexa-488-conjugatedanti-mouse secondary antibody. Cells were imaged at 100× magnification.

Cancer 77 (2012) 312– 318 315

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Fig. 2. 5T4 expression on tumour cells resident in PF. (A) Inset dot plot demon-strates the gating strategy used to identify tumour cells in PF samples. A mixtureof PBMC and M15 mesothelioma cells were labelled with CD3-APC, CD14-APC-Cy7, IgG1-Alexa488 or 5T4-Alexa488. P1 represents the tumour cell gate (5T4+)and P2 the immune cell gate (CD3+, CD14+ and 5T4−). The graph shows 5T4 cellsurface expression on ex vivo tumour cells in PF. The values represent the per-cent of 5T4+ tumour cells present within the P1 gate, as determined in the dot plotabove, following IgG1-Alexa-488 isotype (black) or 5T4-Alexa-488 (10 �g/ml; grey)antibody labelling. (B) Dot plot of live PF cells (7AAD) from patients M2.10, M2.13and M2.21, labelled with CD200-PE, and either with IgG1-Alexa488/IgG2A-APC (iso-type controls) or 5T4-Alexa488/mesothelin-APC antibodies. (i) Cells labelled withCD200-PE and IgG1 isotype-Alexa488 for 5T4, (ii) Cells labelled with CD200-PE and5T4-Alexa488 antibodies. Mesothelin expression by either the 5T4+CD200+ (iii) or5T4−CD200− cells (iv) is shown on histogram overlays of isotype-Alexa488 (grey)vs mesothelin-Alexa488 antibody binding (black).

S. Al-Taei et al. / Lung

The cellular distribution of 5T4 was analysed by immunocyto-hemistry in four cell lines (Fig. 1D). The pattern of 5T4 was similarn three of four cell lines, characterised by prominent plasma mem-rane, diffuse cytoplasmic and dense perinuclear labelling, whilst19 cells (Fig. 1D(iv)) labelled predominantly in a punctate cyto-

lasmic manner.

.2. 5T4 expression on tumour cells in PF

We assessed 5T4 surface expression on tumour cells presentn PF. The tumour cell gate was pre-determined based on the for-

ard and side scatter profiles of cultured tumour cells mixed withBMC (Fig. 2A, top panel). PF cells from all 10 donors expressed 5T4Fig. 2A). To confirm that 5T4 was expressed on tumour cells, 7-AADegative (live) PF cells from three patients were labelled with 5T4,esothelin and CD200. There was a distinct population of CD200+

ells present, and 73%, 83% and 83% of these cells, respectively from2.10, M2.13 and M2.21, expressed 5T4 (Fig. 2B(ii) vs (i)). Mesothe-

in was also highly expressed on 5T4+CD200+ cells (mfi 1068, 792nd 1669) but not CD200−/5T4− cells (81, 63 and 52 respectively;ig. 2B(iii) vs (iv)), confirming that the majority of 5T4+ cells in theF are tumour cells. Cells with the triple-positive phenotype wereot detectable in PBMC (data not shown).

.3. 5T4 expression and distribution in tumour tissue

To further verify 5T4 expression in MPM and rule out the pos-ibility that 5T4 is upregulated during extended in vitro culture ofell lines, 5T4 expression was analysed in fresh-frozen tumour tis-ues by Western blotting (Fig. 3A). A 75 kDa band, consistent withhe typical 5T4 size, was observed in all samples. Bands were alsobserved at ∼50 kDa, consistent with an immature form of 5T4. A55 kDa band, not evident in cell lysates, was present in most sam-les and suggested 5T4 dimerisation, the relevance of which is notnown.

We also assessed 5T4 expression and distribution in FFPE tis-ue sections (Fig. 3B). There was strong labelling for 5T4 andesothelin, compared to isotype-control, and similar distribution

f the two antigens. The highest magnification (Fig. 3B(iii)) con-rms that 5T4 expression is associated with nucleated tumourells. 5T4 and mesothelin expression were confirmed in tissue sec-ions obtained from seven more patients (Supplementary Fig. 1).n contrast, there was little staining of normal pleura for 5T4 and

esothelin above that of isotype control (Fig. 3C). To confirm thatT4 expression is tumour-specific, images of non-tumour struc-ures are highlighted. A blood vessel wall (Fig. 3D(i)) is 5T4− and isdjacent to a 5T4+ tumour mass. An area of tumour surroundedy reactive stroma reveals a distinct pattern of 5T4− stromalells and 5T4+ tumour cells (Fig. 3D(ii)). Pseudo-capillary struc-ures characterised by reactive stroma (5T4−) encased in malignant

esothelium (5T4+) (Fig. 3D(iii)). Gland-like structures surroundedy malignant mesothelium show 5T4 positivity of the tumour cellsnly (Fig. 3D(iv)).

.4. 5T4-specific antibodies in plasma

Antibody responses to 5T4 were identified in the plasma of MPMatients (n = 17) and healthy donors (n = 7; Fig. 4). The median 5T4ntibody level was 46% higher in the patient plasma than in healthylasma but was not statistically significant (p = 0.472). AB-serumad a low level of 5T4 antibody.

.5. Tumour cell killing by a 5T4-specific CTL line

HLA-A2+ CD8+ T-cells, with demonstrable specificity for theT417–25 peptide (Fig. 5A) were tested for their ability to kill

316 S. Al-Taei et al. / Lung Cancer 77 (2012) 312– 318

Fig. 3. 5T4 protein in tumour tissue (A) Western blot of 5T4 expression in tumour tissue. Protein samples (60 �g) from tissue homogenates were probed for 5T4. Numberson top of the blots indicate sample IDs, and the observed molecular weights are shown to the right of the blot. (B) A representative sample with H&E (highlighting the tissuestructure), isotype-specific, 5T4-specific (25 �g/ml) and mesothelin-specific (10 �g/ml) staining of an FFPE section as indicated above each column. White boxes indicatethe area magnified and shown in the row below. Scale bars shown are 5000 �m (i), 500 �m (ii) and 50 �m (iii). (C) A normal tissue section stained for H&E, isotype, 5T4 andmesothelin, respectively, as indicated. The black box indicates the area magnified and shown in the row below. The scale bars represent 5000 �m (i) and 500 �m (ii), apartfrom the 5T4 stained section in (ii) which is 200 �m. Arrow indicates layer of normal mesothelial cells. (D) Tissue sections from different donors highlighting 5T4-structures.(i) The asterisk indicates the wall of a blood vessel. (ii) The asterisk indicates reactive stroma. (iii) The white arrow indicates pseudo-capillary structures of stroma encasedi which

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n malignant mesothelium. (iv) The asterisk is placed next to a gland-like structure

LA-A2+ and HLA-A2− mesothelioma cell lines, expressing 5T4Fig. 5B). Four of the six A2+ MPM cell lines (M18, 24, 34, 36) wereilled at a range of 16–27% specific lysis at 20:1 E:T ratio in a 5 h CTLssay (Fig. 5B). The lysis of these cells was significantly higher than

hat of the A2− cell line, M38. Two cell lines (M24 and M34) sensi-ive to the killing were sarcomatous, indicating that 5T4-targetingf mesothelioma is not restricted to the epithelial subtype. Theysis was <10% for two of six A2+ MPM lines (M15 and M40). This

is surrounded by a layer of malignant mesothelium.

resistance to killing was not related to their expression level of 5T4at the time of the assay (Fig. 5C).

4. Discussion

In this study we aimed to establish whether 5T4 is a relevantantigen for potential targeted therapies in MPM. 5T4 expressionhas been observed on all cell lines, tumour and PF samples from

S. Al-Taei et al. / Lung Cancer 77 (2012) 312– 318 317

Fig. 4. 5T4 antibody levels in patient and healthy donor plasma. Plasma from MPMpatients (n = 17), healthy controls (n = 7) and AB-serum were tested for levels of5dp

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Fig. 5. MPM cell killing by a 5T4-specific T-cell line. (A) T2 cells pulsed with thepeptide (black circles) or unpulsed (white circles) were exposed to increasing num-bers of T-cells (E:T ratio shown on x axis). The symbols represent the means and SDof the percent specific lysis of target cells in a 4 h 51Cr-assay. (B) HLA-A2+ mesothe-lioma cell lines (M15–M40) and an HLA-A2− cell line (M38) were pulsed with 51Crand served as targets of T-cell killing by the 5T417–25 T-cell line in a 5 h 51Cr-releaseassay at 20:1 E:T ratio. The bars represent the means and SD of % specific lysis fromtriplicate samples. (*p < 0.05; **p < 0.01; Student’s t-test). (C) 5T4 surface expres-

T4-specific antibody using an ELISA test. The medians of antibody levels (healthyonor: 8.23 U/ml and MPM: 12.04 RU/ml) are indicated by black bars. Statistics wereerformed using the Mann–Whitney U-test.

2 patients. Expression was seen in 100% of samples, regardless ofubtype. 5T4 expression has been described in most adenocarcino-as with ∼70% frequency [11,22,23] and is associated with poor

rognosis likely due to increased metastatic activity [13]. Its pres-nce has not been reported in MPM before and it may contributeo the aggressive nature of the disease by mechanisms other than

etastatic spread.5T4 expression was membrane-associated in all mesothelioma

ell lines and PF tumour cells studied. In one cell line (M19) webserved relatively low expression of membrane-associated 5T4.n non-small-cell lung cancer, membranous 5T4 expression wasbserved on poorly differentiated tumour cells and associated withhorter survival time and shorter time to recurrence [24]. The cel-ular fraction of PF also contained 5T4 positive cells co-expressingD200 and mesothelin, which strongly indicates that these cells areumour cells that entered the tumour-associated fluid. As our his-ochemistry results from FFPE biopsies reveal that non-malignant

esothelium, reactive stroma and non-malignant structures in theleura do not express 5T4, 5T4 detection in patient samples mayave diagnostic as well as prognostic value. Immune-engagementith 5T4 was also studied. 5T4-specific antibodies were observed

oth in MPM patients and healthy donors, with higher levels inatients and similar to levels observed in other solid cancers [17].

n our study, the difference did not reach statistical significance,robably due to the relatively small number of samples and oneigh healthy responder. Furthermore, 5T4 antibody levels (with-ut vaccination) had no prognostic value in prostate or colorectalarcinoma [17] and its significance in MPM is also unknown.

Finally, we demonstrated that 5T4 peptide-specific T-cells canecognise and kill mesothelioma cells in a HLA-A2-restricted man-er. 5T4-specific CD8+ T-cells have been generated against 5T4equences before [25]. High affinity T-cell receptor binding of the7–25 amino-acid sequence of 5T4 has been demonstrated (ED50.5 × 10−7 M), and strong ELISPOT responses were elicited againsthis peptide in a patient vaccinated with TroVax® [19]. We foundhat mesothelioma cells even with low 5T4 expression were killed

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y T-cells effectively. On the other hand, two HLA-A2 mesothe-ioma cell lines expressing medium-high level of 5T4 were notilled in a 5 h CTL assay. There are several possible explanations aso why this is the case. The first is low HLA-A2 expression, although

sion on the target cells of the CTL assay, determined by flow cytometry. The barsrepresent mfi of 5T4 expression (grey) or isotype control (black columns).

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18 S. Al-Taei et al. / Lung

2 expression was tested on M15 cells (not shown) and its expres-ion was not downregulated. Another possibility is that the 5 hssay was not sufficiently long; it has been shown that HPV-specific-cells only kill tumour targets after 20 h co-culture [26]. Otherechanisms, such as p53 inactivation or pSTAT3 induction may

lso be responsible for suppressing CTL responses [27,28]. How-ver, CD8+ T-cells can exert tumour control not only via cytotoxicut also cytostatic effects, as shown in a mouse model of melanoma29]. Further studies will be carried out to investigate the nature ofesistance to killing by CTL in these mesothelioma cell lines.

A Phase II clinical trial, starting this year, will study the immuno-ogical and clinical effects of Trovax®, administered to MPMatients in combination with pemetrexed and cisplatin (SKOPOSrial, Velindre Cancer Centre, Cardiff, UK). We have previouslyhown that the majority of MPM patients have normal haemato-ogical profiles and immune responses [30], so are likely to respondetter to the vaccine than end-stage renal cancer patients. Target-

ng 5T4 positive cells also has the advantage of eliminating not onlyumour cells but also cells with tumour-initiating potential [24].

. Conclusion

To conclude, 5T4 is widely expressed in mesothelioma tissuesnd cell lines and serves as a target for immune cell-mediatedilling. These observations have been translated into a Phase II trialpplying Trovax® in MPM patients.

onflict of interest statement

Dr. Richard Harrop is employed by and has shares in an organi-ation which have patent rights to 5T4-based therapies.

cknowledgements

We thank Mr. Mike Saunders and Dr. Thomas HockeyHistopathology Department at Llandough Hospital), for providingFPE slides and advice. Also, Lynda Churchill for technical assistancend Dr. Lisa Spary for helpful discussions.

The study was funded by the June Hancock Mesotheliomaesearch Fund (Leeds, UK) and the British Lung Foundation. Theponsors had no additional involvement in the study.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.lungcan.2012.03.008.

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