Identification using phage display of peptides promoting targeting and internalization into...

JOURNAL OF MOLECULAR RECOGNITIONJ. Mol. Recognit. 2005; 18: 175–182Published online 22 September 2004 in Wiley InterScience (www.interscience.wiley.com). DOI:10.1002/jmr.723

Identification using phage display ofpeptides promoting targeting andinternalization into HPV-transformed cell lines

Philip Robinson, Denise Stuber, Francois Deryckere, Philip Tedbury, Magali Lagrangeand Georges Orfanoudakis*UMR7100-CNRS Ecole Superieure de Biotechnologie de Strasbourg, Universite Louis Pasteur, boulevard Sebastien Brandt, 67400-Illkirch, France

‘High-risk’ human papilloma viruses (HPVs) cause cervical tumours. In order to treat these tumourstherapeutic approaches must be developed that efficiently target the tumour cells. Using phage display, weselected tumour-targeting peptides from a library of constrained nonamer peptides presented multivalentlyon pVIII of M13. Three different consensus peptide sequences were isolated by biopanning on HPV16-transformed SiHa cells. The corresponding phage-peptides targeted and were internalized in HPV16transformed SiHa and CaSki cells as well as in HPV18-transformed HeLa cells, but failed to bind a panel ofnormal or transformed cell lines. Two of the three selected peptides targeted cells only when presented onphage particles in a constrained conformation. However, all three peptides retained their targeting capacitywhen presented on the reporter protein enhanced green fluorescent protein (EGFP) in a monovalent form.These peptides may be useful for the design of drug or gene delivery vectors for the treatment of cervicalcancer. Copyright # 2004 John Wiley & Sons, Ltd.

Keywords: phage display; HPV; cervical cancer; cell internalization; targeting

Received 27 May 2004; revised 7 July 2004; accepted 7 July 2004

INTRODUCTION

Cervical cancer is the third most common cancer worldwideand, for women, the second one after breast cancer. Abouthalf a million new cases occur globally each year, half ofwhich prove fatal (Garland, 2003). Human papillomavirus(HPV) infection has been shown to be the major cause ofcervical carcinogenesis (Wolf et al., 2003). HPVs are smallDNA viruses of which over 100 distinct types have beencharacterized (Chan et al., 1995). They are classified aseither ‘low risk’ or ‘high risk’, depending on their clinicalassociations (zur Hausen, 1996). ‘High risk’ HPVs (HPV16,18 and 31 for instance) have been identified as the etiolo-gical agent in over 99% of cervical cancers. Present in 50–60% of all HPV-positive biopsies (Bosch et al., 1995),HPV16 is the predominant type and the principal targetfor the development of therapeutics.

The treatment of cervical cancer is currently based onsurgery, radiotherapy and the use of chemotherapeuticdrugs. Radiotherapy fails to control the progression ofcervical cancer in 35–90% of women with locally advanceddisease (Rose et al., 1999), while chemotherapy has manyside effects because it discriminates poorly between targetand normal cells or tissues. Novel therapeutical strategieshave targeted the HPV oncoproteins E6 and/or E7 (Butzet al., 2000; Giovane et al., 1999; Wang-Johanning et al.,1998) or the mRNA coding for these (Alvarez-Salas et al.,2003; Butz et al., 2003); however, targeting of the affectedcells has not been addressed. Type-specific overexpressionof cell surface proteins is one approach that has been used toobtain organ- or tumour-targeting peptides or antibodieswith no prior knowledge of the target proteins. Phagedisplay technology has been used to isolate, from largepeptide or antibody fragment libraries, phage particles thatare all able to become internalized in living cells (Ivanenkovet al., 1999a; Poul et al., 2000). These targeting peptides orantibody fragments have been successfully used in a widerange of therapeutic approaches using liposomes (Jaafariand Foldvari, 2002; Park et al., 2001), recombinant viruses(Grifman et al., 2001; Nicklin and Baker, 2002), chemicals(Aina et al., 2002) and pro-apoptotic peptides (Ellerby et al.,1999).

In this study, phage display peptides were identifiedwhich efficiently target HPV-transformed cells. Using aconstrained nonamer library presented on the protein pVIIIof M13 phage, we identified three peptides with consensusmotifs C (R/Q) L/R T (G/N) X X G (A/V) G C, C A D P N S

Copyright # 2004 John Wiley & Sons, Ltd.

*Correspondence to: G. Orfanoudakis, UMR7100-CNRS, Ecole Superieure de

Biotechnologie de Strasbourg, Universite Louis Pasteur, boulevard Sebastien

Brandt, 67400-Illkirch, France.

E-mail: [email protected]

Contract/grant sponsors: Association pour la Recherche sur le Cancer; La Ligue

Nationale Contre le Cancer.

Abbreviations used: cfu, colony forming units; DMEM, Dulbecco’s modified

Eagle’s medium; EGFP, enhanced green fluorescent protein; EDTA, ethylene

diamine tetra-acetic-acid; FITC, fluoresceine isothiocyanate; IPTG, isopropyl-

D-thiogalactopyranoside; LB, Lauria Bertani; PBS, phosphate buffered saline;

PEG, polyethylene glycol; TBS, Tris-buffered saline; TRITC, tetramethyl

rhodamine isothiocyanate; TEV, tobacco etch virus.

V R A (M/H) C and C A A H Y R V G P W C. Thesepeptides, which were labeled �, � and � respectively, wereable to bind and translocate phage particles only in SiHa,CaSki and HeLa cells but not in several normal cells ortumour cell lines. Furthermore, the � and � phage-peptideswere functional only in their constrained conformationwhile the � phage-peptide possessed binding and translo-cating capacity when presented both in a linear and aconstrained form. When presented monovalently on a dif-ferent protein scaffold (EGFP), the selected peptides wereable to direct cellular internalisation, which demonstratestheir potential for the delivery of cargo molecules to targetcells.

MATERIALS AND METHODS

Cell lines

The following cell lines were used in this study: SiHa, CaSki(both derived from a HPV16 integrated human cervicalcarcinoma), HeLa (derived from a HPV18 integrated humancervical adenocarcinoma), MCF7 (derived from a humanmammary gland pleural adenocarcinoma), C-33A (derivedfrom a HPV negative human cervical carcinoma), NIH/3T3(non-transformed murine embryo fibroblasts), A549 (de-rived from a human epithelial lung carcinoma), 293 (derivedfrom a human kidney epithelium transformed by adenovirus5) and Saos2 (derived from a human osteosarcoma). Allthe above cells were cultured in monolayer cultures inDulbecco’s modified Eagle’s medium (DMEM) supple-mented with 2 mM L-glutamine, 10% (v/v) foetal calfserum (FCS), 50 mg/ml penicillin and 50 mg/ml streptomy-cine (Invitrogen).

The human keratinocytes immortalized by HPV16 E6and E7 (KERTr E6/E7; ATCC number CRL-2309) and theprimary human keratinocytes (Invitrogen) were cultured indefined keratinocyte serum-free medium supplemented withepidermal growth factor and bovine pituitary extracts (In-vitrogen) in the absence of antibiotics. All cells weremaintained in 5% CO2 at 37 �C.

Biopanning

The CX9C phage display peptide library (Felici et al., 1991)was kindly provided by Dr A. Luzzago, IRBM P. AngelettiSpa, Pomezia, Italy. This phagemid library presents, on theprotein VIII of M13 phage particles, peptides of nine aminoacids constrained in a loop by a disulphide bond betweenflanking cysteine residues. To eliminate clones that bindplastic, the library was preincubated on 10 cm tissue culturedishes for 1 h at room temperature (RT) in DMEM 10% v/vFCS. SiHa cells were grown on 10 cm tissue culture dishesuntil they reached an 80% confluent monolayer in theconditions mentioned above. The cells were first incubatedin DMEM 10% v/v FCS supplemented with 100 mM chlor-oquine (Sigma-Aldrich) for 30 min at 37 �C. Subsequently,1� 1011 cfu of the phagemid library diluted in 5 ml DMEM10% v/v FCS supplemented with 100 mM chloroquine wereincubated with the cells for 2 h at 37 �C. To prevent furtherinternalizing events, the dishes were placed on ice for 5 min

and all the subsequent manipulations were performed at4 �C. The cells were washed twice with 10 ml PBS Mg2þ/Ca2þ (Invitrogen), six times with 10 ml PBS (130 mM NaCl,7 mM Na2HPO4�12H2O, 3 mM NaH2PO4, pH 7.5) and, de-pending on the cycle of biopanning, one to four times with10 ml TBS (50 mM Tris, 150 mM NaCl, pH 7.5), 0.05% v/vTween-20. Extracellular phage were inactivated by incuba-tion for 45 min with TBS containing 3 mg/ml subtilisin(Sigma-Aldrich). The cells were resuspended by pipettingto obtain a uni-cellular suspension and collected by centri-fugation (200 g, 5 min) at the bottom of a 15 ml conical tube.The supernatant from the biopanning was collected and usedto determine the persistence of infectious phage by infectionof bacteria as previously described (Kempf et al., 2001). Thesubtilisin was then inactivated by resuspending the cells in5 ml of PBS supplemented with an anti-protease cocktail(Complete1, Roche Diagnostics GmbH) followed by a15 min incubation. After pelleting, the cells were washedwith PBS and finally resuspended in 500 ml of lysis buffer(2% w/v sodium deoxycholate, 10 mM Tris–HCl pH 8.0,2 mM EDTA,) and 500 ml TBS. The tubes were vortexed andincubated for 30 min at room temperature to ensure libera-tion of intracellular phage. The recovered pool of phage wasincubated for 30 min at 37 �C without shaking in the pre-sence of 1 ml Top10 F0 bacteria (Invitrogen) at an OD600 nm

of 1. In order to neutralise the EDTA in the lysis buffer andto aid infection, 50 mM MgCl2 was added to the incubationmix. The number of infectious phage recovered was deter-mined by plating out an aliquot onto LB-agar plates(5 g/l NaCl, 5 g/l yeast extract, 10 g/l bactotryptone, 13 g/lagar) supplemented with 100 mg/ml ampicillin (Bristol-Myers Squibb) and 20 mg/ml tetracycline (Sigma-Aldrich).The rest of the phage were amplified in 200 ml LB (supple-mented with 100 mg/ml ampicillin, 20 mg/ml tetracycline) byincubation at 37 �C with shaking (250 rpm). At OD600 nm of0.25, 1 mM IPTG and 10 mM MgCl2 were added (finalconcentrations). The bacteria were then superinfected withhelper phage (New England Biolabs Inc.) at a multiplicity ofinfection of 30. The culture was incubated without shakingfor 30 min then for 5 h with shaking (250 rpm). The bacteriawere eliminated by centrifugation (13 000 g, 20 min, 4 �C).The phage particles contained within the supernatant wereprecipitated during 16 h at 4 �C by the addition of 1/6 vol. ofPEG/NaCl [20% w/v poly(ethylene glycol)-8000, 2.5 M

NaCl]. The phage were then recovered by centrifugation(15 000 g, 20 min, 4 �C) and resuspended in 1 ml TBS. Theywere then precipitated a second time by the addition of 1/6vol. PEG/NaCl followed by an incubation of 1 h on ice.After centrifugation (15 000 g, 30 min, 4 �C), the pelletcontaining the phage particles was resuspended in 1 mlTBS 0.02% NaN3 and phage titre (cfu) was determined bybacterial infection. In subsequent rounds of biopanning thesame number of phage (1� 1011 cfu) were added to theSiHa cells.

Deduction of the amino acid sequenceof presented peptides

Individual clones from the third round of selection wereamplified using the protocol described above. The aminoacid sequences of the peptide presented on individual clones

176 P. ROBINSON ET AL.

Copyright # 2004 John Wiley & Sons, Ltd. J. Mol. Recognit. 2005; 18: 175–182

were deduced after sequencing the corresponding DNAinsert using the Seq4� 4 Automated DNA Sequencer, theThermo Sequenase Cy 5.5 Terminator Cycle SequencingKit and the M13 (�40) primer according to the manufac-turer’s instructions (Amersham Biosciences).

Linear presentation of selected peptides on pVIII of M13

In order to obtain linear peptides, the phagemid vectorscoding for the selected peptides were modified by replacingsequence coding for the flanking cysteines by that forserines. The coding sequence of each peptide was replacedby synthetic oligonucleotide cassettes coding for the mod-ified peptides between the restriction sites EcoRI andBamHI of the phagemid vector pc89. The pc89 vectorDNA was amplified and purified by QIAprep Spin kit(Qiagen), digested by EcoRI and BamHI restriction en-zymes to remove the sequence coding for the constrainedpeptides and purified once more. All restriction enzymeswere from New England Biolabs. The cassettes coding forthe modified peptides were prepared by annealing twosynthetic oligonucleotides. The following oligonucleotideswere used for: � 50-AATTCTCGCGTCTTACCGGC-GGAAAAGGTGTCGGCTCTGGG-30 and 50-GATCC-CCAGAGCCGACACCTTTTCCGGTAAGACGCGAG-30,� 50-AATTCTCGGCGGATCCGAACAGCGTGCGTGCG-CATTCTGGG-30 and 50-GATCCCCAGAATGCGCACG-CACG-CTGTTCGGATCCGCCGAG-30, � 50-AATTCTC-GGCGGCCCATTATCGTGTGGGCCCGTGGTCTGGG-30

and 50-GATCCCCAGACCACGGGCCCACACGATAATG-GGCCGCCGAG-30. The cassettes were ligated into theopen vector using the DNA Ligation System (AmershamBiosciences).

Cloning and production of EGFP–peptide a1, b4 and c6

EGFP was generated by PCR using pEGFP (BD Bios-ciences Clontech) vector as template DNA and the oligo-nucleotides: upstream 50-ACCGGTCGCCACCATGGTGAGCAAGGGCGAG-30, downstream 50-GACGAGCTG-TACGAGCTCGGTTAACTC GAGGGCCTA-30 EGFPwas inserted between NcoI and XhoI sites of the pET19HTvector. This vector is a modification of pET19 (Novagen)with a polylinker site containing NcoI, SacI, XhoI restrictionsites, a 6His coding sequence followed by a TEV proteasedigestion site coding sequence (Orfanoudakis, unpublishedresults). �, � and � peptide coding sequences were insertedto the 30 end of the EGFP coding fragment between uniquesites SacI and XhoI and these were separated by the DNAsequence coding for the linker (Gly Gly Gly Ser). Thefollowing oligonucleotides were used for the �-taggedGFP sequence: 50 CGGTGGAGGTTCGTGCCGTCT-TACCGGCGGAA AAGGTGTCGGCTGCTAAC-30, 50-TCGAGTTAGCAGCCGACACCTTTTCCGCCG GTAA-GACGGCACGAACCTCCACCGAGCT-30, the �-taggedEGFP sequence: 50-CGGTGGAGGTTCGTGCTCCGAA-CAGCGTGCGTGCGCATTGCTAAC-30, 50-TC GAGT-TAGCAATGCGCACGCACGCTGTTCGGATCCGCGAC-GAACCTCCACCG AGCT-30; and the �-tagged GFPsequence: 50-CGGTGGAGGTTCGTGCGCGGCCCATCG-

TGTGGGCCCGTGGTGTTAAC-30, 50-TCGAGCCACCT-CCAAGCACGCGCCG GGTAATAGCACACCCGGGC-ACCACAATTGAGCT-30. Purification of DNA fragmentswas performed using the GFX kit (Amersham Biosciences)and the ligation was performed using the DNA LigationSystem (Amersham Biosciences).

For the production of the recombinant proteins BL21/DE3 E. coli bacteria (Novagen) transformed by the plasmidpET19HTEGFP, EGFP-�, EGFP-� or EGFP-� were incu-bated in LB broth with 50 mg/ml ampicilin and 34 mg/mlchloramphenicol at 37 �C until OD600 nm reached 0.5. Theexpression was induced by addition of 1 mM IPTG (finalconcentration). After a 3 h incubation at 37 �C, the bacteriawere collected by centrifugation (2500 g, 30 min) resus-pended in phosphate buffer 20 mM pH 7.4, 30 mM imidazolein presence of EDTA-free antiprotease complete1 (Roche)and disrupted using a Cell Disintegrator Basic Z (ConstantCell Disruption Systems). The filtered clarified lysatewas passed onto a 5 ml Hi-Trap Ni2þ chelate column(Amersham Biosciences). The column was washed withphosphate buffer 30 mM imidazole and then eluted with300 mM imidazole in the same buffer. Pooled fractionscontaining the protein (EGFP, EGFP-�, EGFP-�, EGFP-�)were then desalted using a PD-10 column (AmershamBiosciences), digested by recombinant 6His-TEV proteasefor 12 h at 16 �C and loaded again on a 5 ml Hi-Trap Ni2þ

chelate column. The flow through containing the proteinwas diafiltrated against PBS and concentrated using Cen-triprep Concentrator (Amicon, Millipore Corporation). Theprotein concentration in each sample was calculated fromabsorption measurements at 280 and 488 nm.

Fluorescence microscopy

Cells (SiHa, HeLa, CaSki, MCF7, C-33 A, NIH/3T3, A549,293, Saos2, KERTrE6/E7 and the primary human keratino-cytes) were grown to subconfluency on eight chamber Lab-Tek permanox slides (Nunc Inc.). They were preincubatedwith 200 ml of DMEM 10% FCS supplemented with 100 mM

chloroquine for 30 min at 37 �C before the addition of2� 109 phage in 200 ml of the same medium. The cellswere incubated for 1 h at 4 �C, washed four times with PBSMg2þ/Ca2þ in order to eliminate phage that bind non-specifically and were then incubated at 37 �C for 2 h inculture medium supplemented with chloroquine. Half theslides were incubated at 37 �C and the other half weredirectly treated after the incubation at 4 �C. Thereafter, thesame protocol was applied to all the slides. At 4 �C, the cellswere washed three times with: PBS Mg2þ/Ca2þ for 5 min,once with 0.1 mM glycine–HCl pH 2.2, 1% BSA for 5 minand finally three times with PBS Mg2þ/Ca2þ for 5 min. Thefollowing treatments were performed at room temperature:the cells were fixed by incubation with PBS containing 4%w/v paraformaldehyde for 15 min, washed three times withPBS, permeabilized by incubation with PBS containing0.1% w/v saponin (PBS-S, Sigma-Aldrich) for 15 minand blocked by incubation with PBS-S containing 1% w/vBSA (PBS-SB). They were then incubated for 1 h withpolyclonal rabbit anti-M13 bacteriophage IgG diluted1:1400 in PBS-SB. After four washes with PBS-S, thecells were incubated for 1 h with TRITC-conjugated goat

INTERNALIZING PEPTIDES FOR HPV-TRANSFORMED CELLS 177


anti-rabbit immunoglobulins IgG (Sigma-Aldrich) diluted1:1000 in PBS-SB. The cells were then washed four timeswith PBS-S. After removing the chambers and gaskets, thecells were mounted in Fluoromount-G (Southern Biotech-nology Associates). They were examined using a ZeissAxioplan through a 40� objective and a TRITC filter.

For the EGFP–peptide internalization experiments, theprotocol described above was applied with the followingmodifications. SiHa cells were incubated with purifiedEGFP, EGFP-�, EGFP-� and EGFP-� at 5 mM in the pre-sence of 100 mM chloroquine and 10 mg/ml Alexa Fluor1

546-conjugated human transferrin (Invitrogen) for 2 h at37 �C, then washed with PBS Mg2þ/Ca2þ three times beforefixation. Mouse monoclonal Anti-GFP antibodies (Roche)and Alexa Fluor1 488 goat-anti mouse IgG antibodies(Invitrogen) were used, both at a dilution of 1:1000.

RESULTS

Identification of SiHa internalizing phage-peptides

The pC89 phagemid library of type CX9C used here haspreviously been screened successfully for peptides whichtranslocate into mammalian cells (Ivanenkov et al.,1999a,b). This library has an estimated diversity of 108

individual clones, 25% of which express a fused peptide(Felici et al., 1991) and presents, on the protein VIII of M13phagemid particles, a random nine-amino acid motif con-strained into a loop by a disulfide bond between flankingcysteine residues.

Phage clones that become internalized in SiHa cells wereisolated by three rounds of selection. Chloroquine, a lyso-somotropic agent known to increase the survival of inter-nalised phage (Ivanenkov et al., 1999b), was used in bothpreincubation and incubation of the SiHa cells with thephage library. In order to select only internalized phage,cells were incubated in the presence of subtilisin whichpartially digests the protein III of the cell surface boundphage, rendering them non-infectious. Internalized phagewere released by cell lysis, titrated and propagated inbacteria for the following round of selection. After threerounds of selection, an enrichment of 105 was obtained. Therandom insert region of the single-stranded DNA from 50individual clones in the third round of selection wassequenced and the amino acid coding sequence deduced(Table 1). Clone 1 represented 64% (32/50) of the phagemidsequenced. Clones 2 (6/50) and 3 (2/50) are very similar toclone 1. These three clones were grouped together as the �class. A second class of sequences, �, was constituted byclone 4 (4/50) and 5 (4/50) which differ by only one aminoacid. Clone 6 was found twice among the sequencedphagemids and was labelled � peptide.

Cell specificity of the selected peptides

We wanted to determine first whether the �, � and �peptides were able to distinguish between SiHa cells, otherHPV 16- or 18-transformed cervical cell lines and/or non-HPV-transformed cell types. For this purpose, we usedimmunofluorescence microscopy to visualize the binding

and internalization of the selected phage-peptides in SiHa,CaSki and HeLa cell lines, primary human keratinocytes(Fig. 1) and a battery of other cell lines (MCF7, HT3, C-33A, A549, 293, Saos2, NIH/3T3 and KerE6/E7; data notshown). The cells were incubated, in the presence ofchloroquine, with 2� 109 cfu (1� 1010 cfu/ml) of phage-peptide control, �1, �5 or �6. In parallel experiments, oneset of cells were incubated with the phage-peptides for 1 h at4 �C, the other set for 1 h at 4 �C, washed and then incubateda further 2 h at 37 �C in the absence of phage. Figure 1 showscells following this 37 �C incubation. The focal planethrough the middle of the cells, used throughout Fig. 1,allows internalized and extracellularly bound phage-peptides to be visualized as distinct white dots. When thecells were incubated only at 4 �C, the phage-peptides boundto the cell surface but internalization did not occur. Repre-sentative results obtained with SiHa cells are shown inFig. 2. With a medial focal view, phage-peptides werevisualized as white dots localized at the cell surface accom-panied by a cloudy white staining in the middle of the cell[Fig. 2(A)] which corresponds to phage bound to the apicalface of the cell [Fig. 2(B)]. �1, �5 and �6 peptides were ableto bind and facilitate internalization of phage to SiHa cells[Fig. 1(B–D)]. �1 and �5 phage peptides were not able todiscriminate between SiHa, HeLa and CaSki cells [Fig. 1(B,C, F, G, J and K)]. However, the �6 peptide becameinternalized only in SiHa cells [Fig. 1(D)]. Furthermore,the selected peptides were not able to bind the primaryhuman keratinocytes [Fig. 1(N–P)] or the other cell linestested (data not shown). The data obtained with the primaryhuman keratinocytes are representative of the other cellstested.

Importance of the disulphide bond in thebinding of the phage peptides

In order to determine whether the disulphide bond isnecessary for the binding and internalization of the �, �and � peptides, both the N- and C-terminal cysteineswere replaced by serine. The capacity of linear �, � and �

Table 1. Amino acid sequence of the selectedpeptides

Family Clone Amino acid sequence Frequencyof the presented peptide

� 1 CRLTGGKGVGC 322 CRRTNWQGAGC 63 CQLTGTHGAGC 2

� 4 CADPNSVRAMC 45 CADPNSVRAHC 4

� 6 CAAHYRVGPWC 2Total 50

Control 7 CSSRFRPALSC

After three rounds of biopanning on SiHa cells, the DNA of 50individual clones was purified, sequenced and the amino acid sequenceof the presented peptides deduced. The six individual sequences weregrouped into three distinct classes. Identical amino acids are in boldand those which are found at least twice in the same position within thesame family are underlined. Phage-peptide 7 was randomly selectedfrom the CX9C library and used as the negative control in laterexperiments.



phage-peptides to target SiHa cells was tested by immuno-fluorescence. Only the linear � phage-peptide retained itscapacity to bind [Fig. 3(B)] and to become internalized [Fig.3(H)] in SiHa cells.

a1, b5 and �6 peptides mediate internalisationof EGFP fusion

To determine whether the selected peptides are functionaloutside the context of the multivalent phage presentation atthe N-terminus of M13 pVIII, an EGFP fusion protein wasprepared. The peptides were genetically fused to the C-terminus of the reporter protein EGFP in a pET expressionvector. The constructs were expressed in E. coli cells andpurified by Ni2þ-chelate chromatography. The 6his tag was

removed by digestion with the recombinant 6his-TEV pro-tease and a second Ni2þ-chelate purification step removedboth free 6his tag and the 6his-TEV protease. EGFP andEGFP-peptides were applied at 5 mM to SiHa cells for 2 h at37 �C. Immunofluorescence microscopy was used to followEGFP alone, EGFP-�1, -�5 and -�6 (Fig. 4). Owing tothe pH quenching of the natural fluorescence of EGFP in theendosomal system, we used anti-GFP antibodies to improvethe fluorescence signal. In the presence of chloroquine andfluorescently labelled human transferrin, SiHa cells wereincubated with EGFP, EGFP-�, -� and -� (Fig. 4). Partial co-localization of EGFP-�, -� and -� [Fig. 4(A)] with humantransferrin [Fig. 4(B)] was seen [Fig. 4(C)], whereas nosignal was produced by EGFP alone [Fig. 4(A)]. Thisindicates that the selected peptides promote cellular inter-nalization of EGFP in a C-terminal configuration and follow,at least in part, the same intracellular route as transferrin, amarker of early endosomes.

DISCUSSION

Generation of homing ligands specific for particular tumourcells could greatly improve the diagnosis and treatment ofcancer. Tumour cells are known to up-regulate membranereceptors (Oyama et al., 2003), a phenomenon which can beexploited for isolating tumour-homing ligands. The phagedisplay technology makes it possible to identify peptideligands without prior knowledge of the molecular topologyof tumour cell surfaces. This in vitro approach has been usedsuccessfully by several groups to isolate cell type-specificpeptides (reviewed in [Landon and Deutscher, 2003), e.g.

Figure 1. Visualization of internalized phage-peptides and their cellularspecificity by immunofluorescence microscopy. SiHa (A–D), CaSki (E–H)HeLa (I–L) and primary human keratinocytes (M–P) were incubated, in thepresence of chloroquine (100mM), with the selected phage-peptides con-trol, �1, �5 and �6 (1�1010 cfu/ml) for 1 h at 4 �C, extensively washed andincubated for a further 2h at 37 �C. Phage particles were stained usingrabbit anti-M13 and TRITC-conjugated anti-rabbit antibodies. The magni-fication was 400 times and the focal plane medial with respect to the cells.

Figure 2. Medial and apical focal planes. SiHa cells were incu-bated with phage-peptide �1 (1�1010 cfu/ml) at 4�C for 1h andwashed extensively. Phage particles were stained using rabbitanti-M13 and TRITC-conjugated anti-rabbit antibodies. Themag-nification was 400 times and the focal plane medial (A) or apical(B) with respect to the cells.



phage-peptides that bind neutrophils (Mazzucchelli et al.,1999), neuroblastoma binding peptides (Zhang et al., 2001),colon tumour RPMrel-specific binding peptide (Kelly andJones, 2003) and peptides specific for three lung tumour celllines (Oyama et al., 2003). In this study, we employed thephage display technology to isolate cell-internalizingphage-peptides for the HPV16-transformed cell line, SiHa.This is a novel way to address the problem, since all othertargeted strategies for the treatment of cervical cancer haveused the HPV oncoproteins E6 and/or E7 or the mRNAencoding these as their target (Alvarez-Salas et al., 2003;

Butz et al., 2000, 2003; Giovane et al., 1999; Wang-Johanning et al., 1998).

After three cycles of selection, the sub-library showed a105-fold higher internalization efficiency for SiHa cellscompared with the original library. This was obtained bythe stringent conditions used for biopanning which elimi-nated non-specific and low-affinity binders and by thespecific selection of internalised phage particles by usingthe protease subtilisin to inactivate extracellular-boundphage-peptides. As observed by others (Ivanenkov et al.,1999a), the use of phagemid instead of M13 phage and the

Figure 3. Targeting capacity of linear phage-peptides investigated usingimmunofluorescence microscopy. SiHa cells were incubated, in the pre-sence of chloroquine (100 mM), with linear and constrained �1, �5 and �6phage-peptides (1�1010 cfu/ml) for 1 h at 4�C (A–F) and for a further 2h at37�C (G–L). Phage particles were stained using rabbit anti-M13 and TRITC-conjugated anti-rabbit antibodies. The magnification was 400 times and thefocal plane medial with respect to the cells.

Figure 4. Visualization of EGFP–peptides and human transferrin co-localization inSiHa cells by immunofluorescent microscopy. In the presence of chloroquine(100mM) and Alexa Fluor1 546-conjugated human transferrin (10mg/ml), SiHa cellswere incubated with EGFP, -�, -� and -� (5 mM) for 1h at 37�C. EGFP fluorescencewas augmented using mouse anti-GFP and anti-mouse Alexa Fluor1 488 anti-bodies. Magnification was 400 times.



multivalent (pVIII) presentation of peptides appears toimprove the cellular internalization process. One explana-tion may be that the internalization of phage particles canonly be achieved by an active cellular import system, whichrequires the presence of a high number of receptors.

The biopanning on SiHa cells led to the selection of sixindividual peptide sequences presented on M13 phageparticles which were grouped into three classes �, � and �(Table 1). Composed of three individual sequences, a con-sensus sequence could be resolved for the � class, which is:C (R/Q) (L/R) T (G/N) X X G (A/V) G C. The twosequences of the � class differ only by a methionine orhistidine at position 9. Finally, for the � class, only onesequence was found. The � class represents 64% of theclones sequenced. This dominant representation of the �class could indicate a greater capacity of the � peptide tointernalize the target cells. However, it is also possible thatthis peptide sequence is more readily expressed in E. colicells, which could increase the proportion of �1 cloneswithin the phage population following amplification duringthe cycles of biopanning. Interestingly, since the � and �phage-peptides bound and became internalised only in theirconstrained form, the use of the CX9C library was determi-nant for their selection. On the other hand, the binding andinternalization of the � phage-peptide was not dependent onthe presence of the cysteine residues. This observationsuggests that the � phage-peptide could have been selectedfrom a linear library. The cell-specificity of the selectedphage-peptides was characterized in detail by immunofluor-escence microscopy. We confirmed the uptake of the threephage-peptides into the SiHa cell line. After binding at 4 �C,the three phage-peptides were internalized at 37 �C by SiHacells via receptor-mediated endocytosis. Among the 11 celllines tested, only the HPV-transformed SiHa, CaSki andHeLa cells were found to take up � and � phage-peptides.No binding was observed in the other cell lines tested,suggesting a specific targeting to HPV-transformed cervicalcell lines. This may reflect a cell membrane receptorregulation in the HPV-transformed cervical cancer-derivedcell lines different from that of other cell lines. For example,endothelin receptors are differentially expressed in twocervical cancer cell lines. CaSki cells posses endothelin Aand B receptors whereas C-33 A, an HPV-negative cervicalcancer cell line, does not possess the endothelin A receptor(Venuti et al., 2002). Interestingly, human keratinocytesimmortalized by expressed HPV16 E6 and E7 oncoproteinswere not targeted. This finding could be explained by theobservation that the co-expression of E6 and E7 oncopro-teins can induce immortalization of keratinocytes, but theseimmortalized cells are not tumorigenic, implying that addi-tional mutations are required for malignant progression(Horner et al., 2004).

The ability of the �1 phage-peptide to become interna-lized in HeLa cells has been confirmed by an independantstudy (Ivanenkov et al., 1999a). The cell line used was theHEp-2 cell line (ATCC number CCL-23). Originally thoughtto be derived from an epidermoid carcinoma of the larynx, itis now known that this cell line was established from a HeLacontamination (information obtained from ATCC).

Although the �6 phage-peptide was able to bind and beinternalized in SiHa cells, no such uptake by HeLa andCaSki cells could be demonstrated. One can speculate that

this is due to a different presentation or level of expressionbetween the receptors presented at the surface of the threeHPV-transformed cell lines. This issue will only be settledwhen the cellular receptors targeted by the peptides havebeen identified. Although the expression of molecular mar-kers correlating to HPV16-induced tumours has been in-vestigated (Cheng et al., 2002; Nees et al., 1998; Rey et al.,1999), no comparison has yet been made between cell linestransformed by HPV16 or HPV18. Such studies would bevaluable for understanding tumorigenic mechanisms.

To examine the possible monovalent use of the selectedpeptide sequences, we successfully tested the capacity of the�, � and � peptide sequences to target and induce uptake ofa functional macromolecule, EGFP, into SiHa cells. Ourresults demonstrate that the �1, �5 and �6 peptides, selectedas a fusion to the N-terminus of pVIII, retain their inter-nalizing capacity when fused to the C-terminus of a protein.EGFP-peptide fusion products bind at 4 �C (data not shown)and become internalized at 37 �C by receptor-mediatedendocytosis. Colocalization studies of cells treated withboth EGFP–peptide fusion products and Alexa fluor-568labelled-transferrin, an early endosome marker, demonstratethat the EGFP–peptide fusion products were delivered to theendosomes (Klausner et al., 1983). In the absence ofchloroquine, a lysosomotropic agent which inhibits transferof endocytosed material from endosomes to lysosomes(Wattiaux et al., 2000) and which preserves internalizedproteins (Lee et al., 2001) or phage particles (Ivanenkovet al., 1999a), EGFP–peptide fusion products are directed tothe lysosomes to be degraded. Although EGFP–peptides areinternalized, they could not escape from endosomes and bereleased into the cytosol. To examine the possible applica-tion of the selected peptides in the targeted delivery oftherapeutic molecules, escape from the endosomal pathwayis required. However, our results clearly indicate that thethree peptides can be removed from the context of a multi-valent presentation on a phage particle and be presentedmonovalently on a different scaffold while retaining theircell specificity.

In conclusion, we report the identification by phagedisplay on whole cells of three phage-peptides, �1, �5and �6 which are capable of becoming specifically inter-nalized in HPV16- and 18-transformed cells. Furthermore,we demonstrate that the function of � and � peptides isdependent on their cyclic conformation but that all theselected peptides retain their function when presentedmonovalently on a different protein scaffold. Further workwill try to identify the receptor(s) used by these peptides toevaluate the use of these peptides as targeting agents for thedelivery of therapeutical molecules to cervical cancer cells.

Acknowledgements

We are indebted to Dr Alessandra Luzzago (IRBM P. Angeletti Spa,Pomezia, Italy) for providing us the pVIII cys phage library. We thankProfessor Etienne Weiss for helpful discussions and Dr Marc vanRegenmortel for comments and critical reading of the manuscript. Wealso thank Dr Jean-Luc Galzi for his help with EGFP. P.R. was funded byan Entente Cordiale scholarship, by the European Doctoral College ofStrasbourg and by an Association pour la Recherche contre le Cancerfellowship. This work was supported by the Association pour la Recherchesur le Cancer and by La Ligue Nationale Contre le Cancer (comite du HautRhin).



REFERENCES

Aina OH, Sroka TC, Chen ML, Lam KS. 2002. Therapeutic cancertargeting peptides. Biopolymers 66: 184–199.

Alvarez-Salas LM, Benitez-Hess ML, DiPaolo JA. 2003. Advancesin the development of ribozymes and antisense oligodeox-ynucleotides as antiviral agents for human papilloma-viruses. Antivir. Ther. 8: 265–278.

Bosch FX, Manos MM, Munoz N, Sherman M, Jansen AM, PetoJ, Schiffman MH, Moreno V, Kurman R, Shah KV. 1995.Prevalence of human papillomavirus in cervical cancer: aworldwide perspective: International Biological Study onCervical Cancer (IBSCC) Study Group. J. Natl Cancer Inst.87: 796–802.

Butz K, Denk C, Ullmann A, Scheffner M, Hoppe-Seyler F.2000. Induction of apoptosis in human papillomavirus-positive cancer cells by peptide aptamers targeting theviral E6 oncoprotein. Proc. Natl Acad. Sci. USA 97: 6693–6697.

Butz K, Ristriani T, Hengstermann A, Denk C, Scheffner M,Hoppe-Seyler F. 2003. siRNA targeting of the viral E6 onco-gene efficiently kills human papillomavirus-positive cancercells. Oncogene 22: 5938–5945.

Chan SY, Delius H, Halpern AL, Bernard HU. 1995. Analysisof genomic sequences of 95 papillomavirus types:uniting typing, phylogeny, and taxonomy. J. Virol. 69:3074–3083.

Cheng Q, Lau WM, Tay SK, Chew SH, Ho TH, Hui KM. 2002.Identification and characterization of genes involved in thecarcinogenesis of human squamous cell cervical carcinoma.Int. J. Cancer 98: 419–426.

Ellerby HM, Arap W, Ellerby LM, Kain R, Andrusiak R, Rio GD,Krajewski S, Lombardo CR, Rao R, Ruoslahti E, Bredesen DE,Pasqualini R. 1999. Anti-cancer activity of targeted pro-apoptotic peptides. Nat. Med. 5: 1032–1038.

Felici F, Castagnoli L, Musacchio A, Jappelli R, Cesareni G. 1991.Selection of antibody ligands from a large library of oligo-peptides expressed on a multivalent exposition vector. J.Mol. Biol. 222: 301–310.

Garland SM. 2003. Can we really beat cervical cancer? Med. J.Aust. 178: 647–649.

Giovane C, Trave G, Briones A, Lutz Y, Wasylyk B, Weiss E. 1999.Targetting of the N-terminal domain of the human papillo-mavirus type 16 E6 oncoprotein with monomeric scFvsblocks the E6-mediated degradation of cellular p53. J. Mol.Recognit. 12: 141–152.

GrifmanM, TrepelM, Speece P, Gilbert LB, ArapW, Pasqualini R,Weitzman MD. 2001. Incorporation of tumor-targeting pep-tides into recombinant adeno-associated virus capsids.Mol.Ther. 3: 964–975.

Horner SM, DeFilippis RA, Manuelidis L, DiMaio D. 2004.Repression of the human papillomavirus E6 gene initiatesp53-dependent, telomerase-independent senescence andapoptosis in HeLa cervical carcinoma cells. J. Virol. 78:4063–4073.

Ivanenkov VV, Felici F, Menon AG. 1999a. Targeted delivery ofmultivalent phage display vectors into mammalian cells.Biochim. Biophys. Acta 1448: 463–472.

Ivanenkov VV, Felici F, Menon AG. 1999b. Uptake and intracel-lular fate of phage display vectors in mammalian cells.Biochim. Biophys. Acta 1448: 450–462.

Jaafari MR, Foldvari M. 2002. Targeting of liposomes to mela-noma cells with high levels of ICAM-1 expression throughadhesive peptides from immunoglobulin domains. J.Pharm. Sci. 91: 396–404.

Kelly KA, Jones DA. 2003. Isolation of a colon tumor specificbinding peptide using phage display selection. Neoplasia5: 437–444.

Kempf E, Weiss E, Klein P, Glacet A, Spratt S, Bourel D,Orfanoudakis G. 2001. The rescue by phage display ofhuman recombinant Fabs to gp120 HIV-1 glycoproteinusing EBV transformed lymphocytes. Mol. Biotechnol. 17:97–108.

Klausner RD, Ashwell G, van Renswoude J, Harford JB, BridgesKR. 1983. Binding of apotransferrin to K562 cells: explana-tion of the transferrin cycle. Proc. Natl Acad. Sci. USA 80:2263–2266.

Landon LA, Deutscher SL. 2003. Combinatorial discovery oftumor targeting peptides using phage display. J. Cell. Bio-chem. 90: 509–517.

Lee JH, Engler JA, Collawn JF, Moore BA. 2001. Receptormediated uptake of peptides that bind the human transferrinreceptor. Eur. J. Biochem. 268: 2004–2012.

Mazzucchelli L, Burritt JB, Jesaitis AJ, Nusrat A, Liang TW,Gewirtz AT, Schnell FJ, Parkos CA. 1999. Cell-specific pep-tide binding by human neutrophils. Blood 93: 1738–1748.

Nees M, van Wijngaarden E, Bakos E, Schneider A, Durst M.1998. Identification of novel molecular markers which corre-late with HPV-induced tumor progression. Oncogene 16:2447–2458.

Nicklin SA, Baker AH. 2002. Tropism-modified adenoviral andadeno-associated viral vectors for gene therapy. Curr. GeneTher. 2: 273–293.

Oyama T, Sykes KF, Samli KN, Minna JD, Johnston SA, BrownKC. 2003. Isolation of lung tumor specific peptides from arandom peptide library: generation of diagnostic and cell-targeting reagents. Cancer Lett. 202: 219–230.

Park JW, Kirpotin DB, Hong K, Shalaby R, Shao Y, Nielsen UB,Marks JD, Papahadjopoulos D, Benz CC. 2001. Tumor target-ing using anti-her2 immunoliposomes. J. Control Release74: 95–113.

Poul MA, Becerril B, Nielsen UB, Morisson P, Marks JD. 2000.Selection of tumor-specific internalizing human antibodiesfrom phage libraries. J. Mol. Biol. 301: 1149–1161.

Rey O, BaludaMA, Park NH. 1999. Differential gene expression inneoplastic and human papillomavirus-immortalized oralkeratinocytes. Oncogene 18: 827–831.

Rose PG, Bundy BN, Watkins EB, Thigpen JT, Deppe G, MaimanMA, Clarke-Pearson DL, Insalaco S. 1999. Concurrent cispla-tin-based radiotherapy and chemotherapy for locally ad-vanced cervical cancer. New Engl. J. Med. 340: 1144–1153.

Venuti A, Salani D, Cirilli A, Simeone P, Muller A, Flamini S,Padley R, Bagnato A. 2002. Endothelin receptor blockadeinhibits the growth of human papillomavirus-associatedcervical carcinoma. Clin. Sci. (Lond.) 103: 310S–313S.

Wang-Johanning F, Gillespie GY, Grim J, Rancourt C, AlvarezRD, Siegal GP, Curiel DT. 1998. Intracellular expression of asingle-chain antibody directed against human papilloma-virus type 16 E7 oncoprotein achieves targeted antineoplas-tic effects. Cancer Res. 58: 1893–1900.

Wattiaux R, Laurent N, Wattiaux-De Coninck S, Jadot M. 2000.Endosomes, lysosomes: their implication in gene transfer.Adv. Drug Deliv. Rev. 41: 201–208.

Wolf JK, Franco EL, Arbeit JM, Shroyer KR,Wu TC, Runowicz CD,Tortolero-Luna G, Herrero R, Crum CP, Ramirez PT,Levenback C, Tanyi JL, Hasegawa Y, Lapushin R, MorrisAJ, Berchuck A, Lu K, Smith DI, Kalli K, Hartmann LC,McCune K, Fishman D, Broaddus R, Cheng KW,Atkinson EN, Yamal JM, Bast RC, Felix EA, Newman RA,Mills GB, Gordinier ME, Malpica A, Burke TW, Bodurka DC,Jhingran A, Ramondetta LM, Schmandt R, Bevers MW,Levenback CF, Gordinier M, Gershenson DM, Sciscione AC,Shvartsman HS, Lu KH, Lee J, Lillie J, DeaversMT, Clifford S,Bast RC, Jr., Huh JJ, Fightmaster DL, Lotan R, FollenM, FangX, Auersperg N, Sigal YJ, Gano J, Wharton JT, Kavanagh JJ,Jenkins AD, Tedjarati S, Baker CH, Apte S, Huang S, KillionJJ, Fidler IJ. 2003. Innovations in understanding the biologyof cervical cancer. Cancer 98: 2064–2069.

Zhang J, Spring H, Schwab M. 2001. Neuroblastoma tumor cell-binding peptides identified through random peptide phagedisplay. Cancer Lett. 171: 153–164.

zur Hausen H. 1996. Papillomavirus infections: a majorcause of human cancers. Biochim. Biophys. Acta 1288:F55–F78.



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