cell binding and internalization by filamentous phage displaying a

THE JOURNAL OF BIOLOCIC~~ CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 17, Issue of April 29, pp. 12468-12474, 1994 Printed in U.S.A.

Cell Binding and Internalization by Filamentous Phage Displaying a Cyclic Arg-Gly-Asp-containing Peptide*

(Received for publication, December 9, 1993, and in revised form, February 28, 1994)

Stephen L. Hart*, Andrew M. Knight, Richard P. Harbottle, Ajaykumar Mistry, Hans-Dieter Hungere, Daniel F. Cutlern, Robert Williamson, and Charles Coutelle From the Department of Biochemistry and Molecular Genetics, St. Mary’s Hospital Medical School, Norfolk Place, London, W2 lPG, United Kingdom, §Max Delbriick Centre for Molecular Medicine, Robert Rossle Strasse 10, 13122 Berlin, Federal Republic of Germany, and IMRC Laboratory of Molecular Cell Biology, University College London, London, WClE 6BT United Kingdom

Ligands that bind mammalian cell surface integrins with high affinity can mediate cellular internalization. We show that particles of the bacteriophage fd that display the cyclic integrin-binding peptide sequence GGCRGDMFGC in a proportion of their major coat protein subunits bind to cells and are efficiently internalized. In the displayed peptide the conformation of the RGD motif is restricted within a hairpin loop formed by a disulfide bridge between the 2 cysteine residues. Cel- lular internalization of phage was demonstrated by confocal and non-confocal immunofluorescence microscopy of tissue-cultured cells incubated with phage particles. The phage were contained in juxtanuclear vesicles in the same serial sections as transferrin receptor but were not colocalized with the cell surface marker alkaline phosphatase. Cell binding and internalization was inhibited by preincubation of cells with the integrin-binding peptide GRGDSP, whereas the control peptide GRGESP had no inhibitory effect. These results indicate that cyclic integrin-binding peptides can be used to target and enter cells and that it should be possible to exploit such peptides for the introduction of DNA, drugs, or other macromolecules.

We are investigating the targeting of cell-surface integrin receptors as a means of facilitating cell entry of artificial constructs which may be applicable in the design of new vectors for gene therapy or drug delivery. Integrins are a superfamily of heterodimeric cell adhesion molecules that consist of several different a and p subunits. Their cellular function is to mediate the movement, shape, and polarity of cells through binding with proteins of the extracellular matrix. In addition, integrins are exploited as receptors for cell entry by pathogenic bacteria, such as Yersinia pseudotuberculosis (Isberg, 1991) and Borda- tella pertussis (Relman et al., 1990), eukaryotic viruses, includ- ing adenovirus (Wickham et al . , 19931, echovirus (Bergelson et al . , 1992), and foot-and-mouth disease virus (Logan et al., 1993), and probably mediate sperm-egg binding (Blobel et al., 1992). The widespread biological exploitation of integrin-mediated cell binding and internalization suggests its use for the development of receptor-mediated gene delivery constructs.

Internalization of E: pseudotuberculosis is facilitated by the bacterial membrane protein invasin binding to several chain

* This work was supported in part by the Leopold Muller Bequest and the Medical Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom correspondence should be addressed. Tel.: 44-71-723-1252 (ext. 5478); Fax: 44-71-706-3272.

integrins on the cell surface (Isberg and Leong, 1990). Invasin mediates both the initial binding and internalization of the bacterial cell (Isberg and Leong, 1990). Studies on the binding of invasin with its particular integrin receptors and the subse- quent process of bacterial internalization suggest that integrin- mediated internalization of Z pseudotuberculosis proceeds by a phagocytic-like process that requires evenly distributed ligands with a high receptor-binding affinity (Isberg, 1991; “ran Van Nhieu and Isberg, 1993).

Many other integrin-binding proteins contain the conserved amino acid sequence arginine-glycine-aspartic acid (RGD) (Pierschbacher and Ruoslahti, 1984). The specificity and affinity of the interaction of this tripeptide with integrin molecules is dependent upon the amino acids flanking the RGD motif and the secondary structure of the protein in that region. Cyclic peptides, for example, in which the conformational freedom of the RGD sequence is restricted, have a higher affinity for integrins than their linear relations (Pierschbacher and Ruo- slahti, 1987). Some disintegrin snake venoms that bind certain integrins with very high affinities contain an RGD sequence within a sterically restrained conformation (Gould et al., 1990; Scarborough et al., 1993a). It has also been demonstrated that small cyclic peptides containing RGD motifs can mimic the specificity and potency of various disintegrin sequences (Scar- borough et al., 1993b). These observations suggested that cyclic peptides containing an RGD sequence may bind integrin molecules with a high affinity and, thus, allow internalization by a similar mechanism to invasin-mediated internalization of E: pseudotuberculosis.

We decided to test an integrin-binding, RGD-containing cyclic peptide in a filamentous phage display system by fusing exogenous DNA sequences with the major coat protein gene of fd phage, gene VI11 (Greenwood et al., 1991). The aim of this approach was to display multiple copies of the integrin-binding peptide in order to maximize the opportunity for interactions between phage and cells. Approximately 3,000 subunits of the major coat protein, pVIII, make up the tubular capsid coating the single-stranded DNA genome of the phage and peptides of up to 12 amino acid residues have been displayed as pVIII fusions when the capsid contains a hybrid mixture of wild-type pVIII and fusion pVIII subunits (Greenwood et al., 1991).

We designed DNA oligonucleotides encoding the cyclic peptide sequence GGCRGDMFGC to display on the phage in multiple copies. This peptide was originally isolated and described in a phage display library in the gene III-encoded minor coat protein of filamentous phage and demonstrated to have a high integrin-binding affinity (O’Neil et al., 1992). Bacteriophage fd particles displaying this peptide in their major coat protein subunits also have a high binding affinity for integrins. We have shown that this interaction is responsible for phage bind-

12468

Cell Targeting and Entry with Filamentous Phage 12469

ing and entering cells in culture, whereas control phage show no specific interactions.

EXPERIMENTAL PROCEDURES Cell Lines, Bacterial Strains, Plasmids, and Phage-HEp-2 cells, a

human laryngeal epithelial cell line, were grown in Dulbecco's modified Eagle's medium (DMEM)' supplemented with 2 m L-glutamine; GIBCOISRL) containing 10% fetal calf serum.

The bacterial strain Escherichia coli TG1 (F', supE, LacP, rec0) and the plasmid pKfdH (Ap') were provided by Dr. Richard Perham (De- partment of Biochemistry, University of Cambridge). E. coli TG1 was maintained on M9 minimal medium and LB media (Sambrook et al., 1989). The bacteriophage vector fd-tet (Tet'), originally called fdDOGl (Clackson et al., 1991), was a gift from Dr. John McCafferty (Cambridge Antibody Technology).

RGDfor (5'-GGTGGCTGCCGTGGCGATATG"CGG'ITGC-3') and RG- Oligonucleotides and Peptides-The complementary oligonucleotides

D"' (5'-GCAACCGAACATATCGCCACGGCAGCCACC-3') were used in the construction of gene VI11 fusion proteins. The primer 5"GGATAA- CAATTTCACACACAGG-3' was used for sequencing pKfdH gene VI11 recombinants. All oligonucleotides were purchased from Oswel (Univer- sity of Edinburgh). The synthetic peptides GRGDSP and GRGESP were obtained from Genosys Biotechnologies Inc. (Cambridge) or GIBCOI BRL.

The complementary oligonucleotides RGD'"' and RGD"' were phos- phorylated at their 5' ends with T4 DNAphosphorylase and transferred to an annealing buffer (0.15 M KC1, 0.01 M "ris-cl, pH 7.5). After incu- bating at 65 "C for 2 min the complementary oligonucleotides were annealed by cooling the mixture slowly to room temperature.

Preparation of Plasmid DNA and Replicating Form Bacteriophage DNA-Plasmid and replicating form phage DNA were prepared from E. coli by the alkaline lysis method (Ish-Horowitz and Burke, 1981) and purified on Qiagen columns (Diagen, Germany).

Plasmids, replicating form phage DNA, and the DNA products of ligation reactions were transformed into E. coli TG1 competent cells by the method of Chung and Miller (1988). Transformants were selected on LB agar plates containing the appropriate antibiotic.

DNA Subcloning and Sequencing-Restriction endonuclease diges- tions and incubations with calf intestinal alkaline phosphatase, T4 DNA phosphorylase, and T4 DNA ligase were performed using standard protocols (Sambrook et al., 1989). Electrophoresis of DNA was carried out in 0.8% agarose gels containing 0.5 pg ml-' ethidium bromide in TAE buffer (0.04 M Tris-acetate, 1 m EDTA) (Sambrook et al., 1989). Dideoxy sequencing (Sanger et al., 1977) was done with a Sequenase kit (U. S. Biochemical Corp.) following the manufacturer's instructions. DNA in the sequencing reactions was radiolabeled with [36SldATPaS (-600 Ci (-22 TBq) mmol") (Amersham Corp.).

Phage Growth Conditions-Phage were prepared from transduced TG1 cultures in LB broth containing 15 pg ml-' tetracycline as described previously (McCafYerty et al., 1990). Bacterial cells were pelleted by centrifugation, and the clarified supernatant was decanted. Phage were precipitated from the supernatant by adding 0.2 volume of 20% polyethylene glycol (molecular mass 8,000 Da) in 2.5 M NaCl and standing at 4 "C for 1 h. Phage were pelleted by centrifugation (12,000 x g, 20 minl then resuspended in 0.01 supernatant volume of water or TE buffer (10 ~ l l ~ Tris-C1, pH 8.0, 1 m EDTA). Insoluble material was removed by two further rounds of centrifugation a t 12,000 x g for 10 min.

Quantification of Bacteriophage fd-The total number of phage particles was determined from the amount of single-stranded DNA derived from a known volume of phage suspension. Single-stranded phage DNA was prepared by phenol extraction of a phage suspension followed by ethanol precipitation (Sambrook et al., 1989). After washing with 70% ethanol, the DNA pellet was resuspended in 610 pl of water. A 10-pl sample of the single-stranded DNA preparation was removed for agarose gel analysis. The uv absorption spectrum of the remaining DNA sample was determined with a Beckman DU-64 spectrophotometer, and the amount of single-stranded DNA was calculated from A260nm.

The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium; Ap', ampicillin resistance; FITC, fluorescein isothiocyanate; I m G , isopropyl-1-thio-P-D-galactopyranoside; LB, Luna-Bertani; pVIII, fd gene VIII-encoded major coat protein; T e r , tetracycline resistance; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1- bis(hydroxymethyl)ethyl]glycine; for, forward; rev, reverse; dATP, deoxy adenosine triphosphate (as, 35S atom attached to P in a position).

The number of infectious phage particles was determined by the addition of 10 pl of phage to 1 ml of LB broth containing E. coli TG1 in early log phase of growth and incubation for 30 min a t 37 "C with gentle shaking. A 10-fold dilution series of the culture was prepared in LB broth, and 100 pl of each diluted sample were spread on LB-tetracycline plates. Tetracycline-resistant colonies were counted after overnight incubation at 37 "C.

Production and Purification of Hybrid Phage Particles-Phage particles containing a hybrid mixture of gene VI11 major coat protein subunits and gene VI11 oligo-encoded fusion proteins were prepared in a similar way to that of Rowitch et al. (1988). Single colonies ofE. coli TGl cells containing the plasmids pKREV or pKRGD (both Ap') were inoculated in 5 ml of LB broth containing 100 pg ml-' ampicillin and incubated a t 37 "C with vigorous shaking. Bacteriophage fd-tet was added to the bacteria at a multiplicity of infection of 25 when the culture reached early exponential growth phase (A2a = 0.2-0.3) and incubated for a further 30 min. A 10-fold serial dilution of the infected culture was prepared in LB broth, and 100 pl of the dilutions were spread on LB agar plates containing ampicillin (100 pg m1-I) and tetracycline (15 pg ml-I). After overnight incubation at 37 "C, single colonies resistant to both ampicillin and tetracycline were inoculated in 5 ml of LB broth containing both antibiotics and incubated overnight at 37 "C with vigorous shaking. The simultaneous presence of fd-tet, and pKREV or pKRGD, in the same bacterial cell was confirmed by DNA analysis. Plasmid DNA was prepared from ApTet' TG1 cells and analyzed by agarose gel electrophoresis after digestion with EcoRI restriction endonuclease.

Bacteriophage were prepared from E. coli TG1 cells harboring both bacteriophage fd-tet and pKREV or pKRGD by growing TGl (ApTet') in 200 ml of LB broth containing ampicillin ( 100 pg ml-I), tetracycline ( 15 pg ml-'1, and 1 m IPTG for 16-18 h. Phage samples were prepared and quantified as described above. In Vitro Danscription-Danslation-A prokaryotic DNA-directed

translation kit (Amersham Corp.) was used according to the manufacturer's instructions for the in vitro transcription and translation of plasmid DNA. ~-[~'S]Methionine (>1,000 Ci (37 TBq) mmol") (Amer- sham) was used as the radiolabeled amino acid. The labeled proteins were separated by electrophoresis in a 15% (wfv) polyacrylamide gel containing 0.1% SDS (Laemmli, 1970) and then transferred to Immo- bilon-P membrane (Millipore) by electroblotting. The membrane was dried and then exposed to x-ray film for 6 h. Rainbow-colored molecular mass markers (2.35-46.0 kDa; Amersham) were used to estimate the size of labeled proteins.

SDS-PAGE Phage Analysis-Five-microliter samples of phage suspension were mixed with an equal volume of loading buffer and were denatured by boiling for 5 min. The proteins were then separated by electrophoresis on an SDS-PAGE gel (16.5% polyacrylamide) in a Tricine buffer (Schagger and von Jagow, 1987) then electroblotted to an Immobilon-P (poly(viny1idene fluoride)) membrane (Millipore). The membrane was blocked and probed with antibodies raised in sheep against fd phage and subsequently anti-sheep antibodies (Sigma). The immunocomplexes formed were then detected by the "contact copy" method using a protein A-neomycin phosphotransferase I1 fusion protein (Hunger et al., 1990).

Cell Binding and Inhibition Assays-Filamentous phage and the proteins fibronectin and BSA were attached to wells in 96-well plates and incubated with a cell suspension. The number of attached cells remaining in wells was estimated after washing. Fifty microliters of fibronectin (1 mg ml-') or 50 pl of serial dilutions of phage in the range 1 x 10" to 1 x 10" phage particledml were added to individual wells in a 96-well plate (Costar EIA/RIA 3590). The plates were left overnight at room temperature then washed with PBS the following day and blocked for 1 h at 37 "C with 3% (w/v) BSA (fraction V bovine serum albumin; Sigma) in PBS. Finally, wells were again washed with PBS. HEp-2 cells were harvested from 500-ml tissue culture flasks (Nunc) when subconfluent by adding 2 m EDTA in PBS for 20 min a t 37 "C. Cells were washed with DMEM containing 10% fetal calf serum, and the final cell pellet was resuspended in medium a t a concentration of - 1 x lo6 cells/ ml. 5 x lo4 cells were added to each well and incubated for 1.5 h at 37 "C.

For competitive binding inhibition assays HEp-2 cells were pre- treated before adding to the phage-containing wells by adding 500 pl of peptide solution GRGDSP or GRGESP (1 mg ml-I) to 1.7-ml samples of the cell suspension and mixing gently by rotation for 1 h at 4 "C. Wells were washed gently with PBS, and attached cells were fxed with methanol. Remaining cells were stained with 0.1% (wIv) crystal violet in distilled water (Brasaemle and Attie, 1988). Excess stain was washed from the wells with PBS and air-dried. Cells were lysed in the wells by

12470 Cell Targeting and Entry with Filamentous Phage pWdH

taC gene WI a

pKRCD

b w d- REV oligo P W V

Lranslaaon and manuadon J

sire

FIG. 1. The construction of pKRW and pKRGD. The annealed oligonucleotides RGD'"' and RGD"' were blunt end-ligated into the HpaI site of pKfdH ( a ) . The insert (open box) can ligate in both orien- tations, generating either pKRGD or pKREV ( b ) . The alternative amino acid sequences are the N-terminal ends of the mature fusion proteins generated by pKREV and pKRGD, after cleavage of the leader sequence ( c ) . The arrow points to the cleavage site of the pVIII leader sequence which is removed during the phage assembly process.

adding 1% SDS. was determined in a multiwell plate spectrophotometer.

Internalization Assays-HEp-2 cells were grown on 13-mm coverslips in 24-well tissue culture grade plates (Falcon) in DMEM supplemented with 10% fetal calf serum for up to 3 days or until a subconfluent monolayer was obtained. The coverslips were then incubated in the same medium containing 1 x 10'' phage particles and, in competition assays, the peptides GRGDSP (0.5 m) or GRGESP (0.5 mM), for 6 h. The cells were then washed in PBS five times, futed in 3% paraformal- dehyde in PBS, blocked with 50 mM NH,CI in PBS, and incubated in 1% BSA and 0.05% saponin in PBS. This buffer was used subsequently for both incubation and washing. The cells on coverslips were then inverted onto Parafilm with 50 pl of sheep anti-fd antibody diluted 1/1000 in the presence of either the anti-transferrin receptor monoclonal antibody B3/25 (a gift from Dr. I. Trowbridge) or anti-alkaline phosphatase antiserum (Dako) in 1% BSA in PBS. The cells were washed with PBS before incubation with anti-goat Ig antibody conjugated to fluorescein isothiocyanate (Dako) and anti-mouse Ig antibody conjugated to rhodamine (Dako). The cells were washed in 5% PBS, mounted in 3% propyl gallate and 90% glycerol in PBS, and sealed in parafiddental wax or nail polish.

Microscopy-Optical confocal sections were taken using a Bio-Rad MRC 600 scanning laser confocal unit in conjunction with a Nikon Optiphot microscope with the apertures set no greater than 30%. Im- munofluorescent microscopy was performed with a Leica Aristoplan uv fluorescent microscope.

RESULTS Construction ofpKREV and pKRGD-The annealed comple-

mentary oligonucleotides RGD'"' and RGD"' were phospho- rylated, then ligated into the HpaI site of the vector pKfdH (Greenwood et al., 1991) to produce an in-frame fusion of the oligonucleotide with gene VlII, the fd major coat protein gene (Fig. 1). The ligation mixture was used to transform E. coli TG1 cells. DNA was prepared from ampicillin-resistant (Ap') transformants and digested with HpaI restriction endonuclease to identify recombinant plasmids. The orientation of the insert and the reading frame was established by DNA sequencing. The recombinant plasmid pKRGD consists of pKfdH with the annealed oligonucleotides in the orientation encoding an in- frame gene VI11 fusion with the predicted amino acid sequence GGCRGDMFGC, whereas pKREV contains the insert in-frame with gene VI11 but in the reverse orientation, encoding the amino acid sequence ATEHIATAAT (Fig. 1).

pKREVandpKRGD Produce Fusion Proteins-The 73 amino acid residues of pVIII and its leader sequence encoded by pKfdH (Greenwood et al., 1991) have a deduced molecular mass

10-3 x MJ 1 2 3 4 5 6

14.0-

- a . . o * * 6.5 -

translation SDS-PAGE electroblot. Lanes 1 and 4, pKREV; 2 and 5, FIG. 2. Autoradiogram of the cell-free coupled transcription-

pKRGD; 3 and 6 , pKfdH. The lower band with an approximate molecular mass of 7.5 kDa represent pVIII with the leader sequence while the higher bands a t about 8.6 kDa represent the fusion proteins produced by pKREV and pKRGD.

of 7.64 kDa. The deduced amino acid sequences of the pVIII fusion proteins of pKRGD and pKREV, with the leader sequences, were each 83 residues long with calculated molecular masses of 8.62 and 8.60 kDa, respectively. The sizes of the major labeled proteins produced by the in vitro transcription- translation of pKRGD and pKREV were estimated from SDS- PAGE gels and were of the predicted molecular masses (Fig. 2).

Generation of Hybrid Phage-During phage replication and assembly the pVIII leader sequence is cleaved leaving the N- terminal part of the 3,000 pVIII subunits on the outside of the phage capsid. N-terminal fusions with gene VlII are, therefore, displayed on the outer phage surface.

Phage capsids containing both wild-type and fusion pVIII subunits were generated from TG1 cells containing fd-tet DNA and plasmid pKREV or pKRGD DNA (Fig. 3). To obtain these hybrid phage E. coli TG1 cells containing either pKREV or pKRGD were inoculated into LB broth containing ampicillin and grown to early log phase when they were transduced with fd-tet. TG1 cells containing fd-tet and either pKREV or pKRGD were selected on LB agar containing ampicillin and tetracycline. Episomal DNA was prepared from Ap' Tet' TG1 Cells and analyzed by agarose gel electrophoresis after EcoRI restriction endonuclease digestion (Fig. 4). Restriction analysis revealed the presence of two species of episomal DNA molecules in these cells. Their sizes corresponded to linearized fd-tet double stranded DNA(9.2 kilobases) and linearized plasmid pKREV or pKRGD DNA (4.8 kilobases). The ratio of plasmid DNA to phage replicating form DNA was visually estimated to be approximately three to one in agarose gels stained with ethidium bromide.

Hybrid phage were prepared from cultures of those TG1 cells shown to contain both fd-tet and either of the plasmids pKREV or pKRGD. Cells were inoculated in media containing ampicillin, tetracycline, and the gratuitous tac inducer IPTG (1 mM), and phage were prepared by polyethylene glycol precipitation. The number of phage particles isolated was estimated indirectly from the amount of single-stranded DNA purified from a known volume of phage suspension. The titer of infectious particles was determined and the infectivity calculated as a percentage of the number of phage particles that are infectious (Table I). Yields of hybrid phage were similar to those of fd-tet parental phage suggesting that the assembly and structural stability of hybrid phage was unimpaired. Hybrid phage, however, infected TG1 cells less efficiently than fd-tet phage (Table I).

Fig. 5 shows the protein separation and detection of fd by Western blotting. The major coat protein, pVIII, was detected at about 5.0 kDa in all samples. The phage fdRGD, however, shows an extra band at about 6.0 kDa which corresponds with the predicted size of the pVIII fusion with the cyclic RGD- containing peptide. The relative band intensities on the West- ern blot suggest that the pVIII fusion protein subunits repre-

Cell Targeting and Entry with Filamentous Phage

FIG. 3. Cartoon of the hybrid phage. fdRGD displays the cyclic RGD-containing peptide while fdREV displays an irrelevant linear peptide.

1 2 3 4 5 6 7

I FIG. 4. Agarose gel electrophoresis of plasmid and RF DNA

samples. DNA samples were prepared from E. coli TG1 cells resistant to one or two antibiotics and digested with EcoRI restriction endonuclease. Lane 1, 1-kilobase ladder (GIBCO); 2, DNA from TG1 containing fd-tet only; 3, DNAfrom TG1 containing fd-tet and pKREV; 4, DNAfrom TG1 containing pKREV only; 5, DNA from TG1 containing fd-tet and pKRGD; 6, DNA from TG1 containing pKRGD only; 7, DNA from TG1 containing pKfdH only.

TABLE I Infectivity of hybrid phage

Phage preparations were used to infect TG1 cells to determine the infectious particles and the total number of phage particles was determined indirectly by the amount of single-stranded DNA on the basis of one DNA molecule representing one phage particle. The percentage ratio of infectious particles relative to the total number of particles is the infectivity of the phage sample.

Phage Total hage Infectious partictedml particledml Infectivity

~~~~

O/o

fd-tet 2.9 X 1013 8.2 x 10'0 2.80 fdRGD 3.7 x 1013 4.5 x 10'0 0.12 fdREV 1.3 x 1013 9.0 x 10'0 0.70

sent 1% or less of the total capsid subunits. However, this may be an underestimation since a previous study (Greenwood et al., 1991) suggests that antibodies against wild-type fd have a poor affinity for pVIII fusion proteins. In addition, protein bands were blotted from SDS-PAGE gels onto Immobilon-P membranes and the N-terminal amino acid sequence determined. The sequence of amino acid residues of both the pVIII fusion protein and wild-type subunits corresponded with the predicted amino acid sequence (data not shown). In contrast to the Western blotting data the partial amino acid sequence suggested that the pVIII fusion protein subunits were present in approximately 10% of the total capsid subunits, a copy number of about 300 pVIII fusion proteins per phage particle. These results demonstrate that the phage constructs are displaying the cyclic RGD-containing peptide. Different relative propor-

10-3 x Mr

6.0 - 12471

1 2 3

FIG. 5. Western blot of fdRGD phage particles. Lanes I , fdRGD; 2, fd-tet; 3, fd-tet. Proteins were detected after electroblotting by contact copy. The predominant band a t about 5 kDa represents pVIII while the fainter band in lane 1 a t about 6 kDa represents the fusion protein of pVIII and the cyclic RGD-containing peptide.

tions of pVIII subunits and pVIII fusion subunits in the capsid of fdRGD were inferred from the Western blot and the N-terminal sequencing data, suggesting that anti-fd antibodies did not react as strongly with the fusion protein as with native

Phage Bind Specifically to Cell-surface Integrins-We postu- lated that phage fdRGD, displaying the cyclic RGD peptide, would interact with the cell surface integrins. Therefore we assayed integrin-mediated cell binding by phage displaying the cyclic RGD-containing peptide in multiwell plates in competition with a synthetic integrin-binding peptide.

Wells were coated with the hybrid phage fdRGD displaying the cyclic RGD sequence and the control phage fdREV displaying an irrelevant linear peptide. Cells incubated in wells containing fdRGD and fibronectin were seen to flatten out and spread under an inverted microscope. The extent of cell binding was quantified by the crystal violet cell-staining assay (Fig. 6a ). The amount of cell binding to wells containing fdRGD was similar to the binding of cells to wells containing fibronectin. The same cells, however, bound poorly and nonspecifically to the hybrid phage fdREV.

To demonstrate that cell binding was integrin-mediated we selected the peptide sequence GRGDSP, the integrin-binding domain of fibronectin, as a competitive inhibitor and preincubated HEp-2 cell suspensions with solutions of this peptide (0.5 mM) or a control peptide GRGESP (0.5 mM). These cells were then transferred to wells containing the phage. Fig. 6 shows that these cells did not bind specifically to wells containing fdREV under any conditions. Cell attachment to wells containing fdRGD phage particles was unaffected by preincubation of the cells with the peptide GRGESP whereas preincubation of cells with the peptide GRGDSP inhibited binding (Fig. 6b).

Binding of HEp-2 cells to wells containing 5 x 10" fdRGD phage particles was assayed after preincubation of cells with increasing concentrations of RGD and RGE peptides (Fig. 7). Cell binding inhibition was observed at a GRGDSP peptide concentration above 100 PM, and the effect increased with higher peptide concentrations. The results of the cell binding inhibition assays indicate that cell binding by phage displaying the RGD cyclic peptide is mediated by cell surface integrin molecules.

Hybrid Phage Are Internalized by Mammalian Cells-Phage internalization was assessed by confocal microscopy and fluorescence microscopy in relation to two characterized cell markers, transferrin receptor and alkaline phosphatase. Transferrin receptor is recycled between the endosomal compartment and the cell surface, while alkaline phosphatase is a glycosylphos- phatidylinositol-linked cell surface marker.

Approximately 1 x lo5 cells were incubated with 1 x 10'' hybrid phage particles for 6 h and then fmed. Phage were detected with an antiserum and fluorescein isothiocyanate-conjugated secondary antibody. The same cells were probed with antibodies directed against either transferrin receptor or alka-

pVIII.

12472 Cell Targeting and Entry a

2.0 , E

1 2 3 4 5 6 7 8 Contents 01 wells

b 0.4 -

1 2 3 4 5 6 7 Contents 01 wells

FIG. 6. Cell binding studies of fdRGD and fdREV. a, HEp-2 cells (5 x 10' in 50 pl) were added to wells in a 96-well plate (Costar EIA/RIA) containing different amounts of fdRGD or fdREV phage particleslwell

bated with the peptides GRGDSP and GRGESP then added to wells as and blocked with BSA. b, HEp-2 cells (5 x 10' in 50 pl) were preincu-

in a). Crystal violet cell binding assays were performed. Ason" is the absorbance of the solubilized crystal violet stain remaining attached to the wells after washing and fixing. Each result is the mean of seven observations. Error bars represent the standard deviation about the mean. 1, no phage; 2, 5 x 10" fdREV particles; 3, 5 x lo9 fdREV particles; 4, 5 x IOR fdREV particles; 5 , 5 x 10'' fdRGD particles; 6 ,5 x IO9 fdRGD particles; 7, 5 x 10' fdRGD particles; 8,50 pg of fibronectin.

0.5 -]

O 0.1 4 0.0 I

.1 1 10 100 1000 Peptide p M

FIG. 7. Competitive inhibition of cell binding by fdRGD. fdRGD phage particles were attached to wells of a 96-well plate (Costar E M RIA) at a concentration of 5 x 1 O ' O phage particledwell. Hep-2 cell suspensions were preincubated with a logarithmic range of peptides GRGDSP or GRGESP then added to wells. Cell binding was determined by the crystal violet assay. Each point is the mean of three observations.

line phosphatase and were detected with rhodamine-conjugated secondary antibodies.

Fluorescent fdRGD phage particles were detected in vesicles in consecutive 3-pm optical sections throughout the cells. They were found in the same sections as transferrin receptor, but not in the same vesicles (Fig. 8, a and b) . The phage were predomi- nantly juxtanuclear, and there was no staining in the nuclear region. I t appeared from the analysis of different confocal sections that all cells took up the phage. fdREV phage were not detected within cells to any marked extent in optical confocal sections (Fig. 8, c and d ) . Cell surface localized alkaline phosphatase and fdRGD phage particles were detected in sections by indirect immunofluorescence microscopy. The distribution of these two fluorescent stains is very different; the alkaline phosphatase surface marker is confined to the perimeter of the cell, while the phage particles are largely detected within the cell, in juxtanuclear vesicles (Fig. 8, e and f ) . There was no specific binding or internalization of fdREV phage particles. Incuba- tions were also performed with fdRGD and HEp-2 cells in the

with Filamentous Phage

a b C d

e f g h FIG. 8. Immunofluorescent detection of phage particles. Phage

particles associated with tissue-cultured cells were detected by indirect immunofluorescence in optical confocal sections ( a d ) and by non-confocal, fluorescence microscopy (e-h). All cells were incubated with 1 x 10" phage particles then fixed and permeabilized with 0.05% saponin. The phage particles and transferrin receptor were detected by different fluorescently labeled secondary antibodies and the distribution of fluorescence examined in the same confocal sections. Cells on separate coverslips were incubated with (a and b ) fdRGD phage or (c and d ) fdREV phage, and then each coverslip was probed with both (Q and c) antiserum against fd bacteriophage and ( b and d ) antibodies against transferrin receptor. On another coverslip (e and f ) , cells were incubated with fdRGD phage, and then ( e ) phage particles and ( f , alkaline phosphatase were detected in the same cells by different fluorescently labeled secondary antibodies. On two further separate coverslips ( g and h ) HEp-2 cells were incubated with fdRGD and competed with either of the peptides ( g ) GRGESP or ( h ) GRGDSP in excess (0.5 mM).

presence of the integrin-binding peptide GRGDSP (0.5 mM) and the control peptide GRGESP (0.5 mM). Phage were not found within cells exposed to the integrin-binding peptide while the control peptide did not impair internalization of fdRGD phage particles (Fig. 8, g and h).

DISCUSSION We report here that a peptide displayed on the capsid surface

of bacteriophage fd facilitates efficient cell binding and entry. The high affinity, integrin-binding cyclic peptide sequence GGCRGDMFGC (O'Neil et al., 1992) was selected for construction of the phage fdRGD. The display of peptides in the filamentous capsid in a hybrid mix of fusion and wild-type pVIII subunits allowed the display of multiple copies, perhaps 300 or more, of the ligand on each phage particle as opposed to the maximum of four or five per phage particle when using the gIII adsorption protein phage display systems. Cell binding assays were performed as a functional confirmation of the display of integrin-binding RGD peptides on the phage surface. Cells added to wells coated with fdRGD were seen to flatten and spread in a similar way to the binding of cells to wells containing fibronectin but did not attach to wells coated with fdREV. Cell binding was confirmed by staining attached cells with crystal violet followed by spectrophotometric analysis of solubilized stain. Cells that were preincubated with the integrin- specific peptide GRGDSP, however, were no longer able to attach to phage, whereas the control peptide GRGESP had no effect on binding. The efficient attachment of cells to coated surfaces by the hybrid phage fdRGD and competitive inhibition of cell binding by integrin-specific peptides suggests that the phage are displaying high affinity, integrin-binding ligands.

Analysis of confocal sections by fluorescence microscopy demonstrated that the hybrid phage fdRGD is internalized by HEp-2 tissue cultured cells. The presence of fluorescently la-

Cell Targeting and Entry with Filamentous Phage 12473

beled phage in serial confocal sections at 3-pm intervals suggests that phage were localized within vesicles. Evidence that phage are internalized and not just associated with cell surface irregularities includes the different distribution patterns of fluorescence in serial confocal sections, the absence of fluorescence in the nuclear region and the juxtanuclear location of phage-associated fluorescence. In addition, vesicles containing phage were seen in close proximity to those containing transferrin receptor but not with the surface marker alkaline phosphatase. Phage internalization, like cell binding, was ablated by competition with the integrin-binding peptide GRGDSP. Therefore, the phage-displayed cyclic RGD-containing peptide determines internalization as well as cell attachment.

Functionally, therefore, the peptide on the surface of fdRGD resembles the Yersinia surface protein invasin, in that they both mediate cell binding and internalization. Structurally, however, they are very different. Invasin, for example, does not contain an RGD sequence. Instead a high affinity integrin- binding domain that mimics the RGD motif is found in the C-terminal192 amino acids of invasin (Leong et al., 1990). Both invasin and cyclic RGD-containing peptides, however, share a high afiinity for integrins.

The importance of a high binding affinity for bacterial internalization mediated by interaction between invasin and integrins is illustrated by the functional differences between invasin and fibronectin. The extracellular matrix protein fibronectin and invasin both bind to the a5p1 integrin receptor but fibronectin has a much lower affinity than invasin does. Consequently bacteria coated with fibronectin are internalized much less efficiently than bacteria coated with invasin (Isberg and Leong, 1990; "ran Van Nhieu and Isberg, 1991). The mechanism by which invasin appears to induce internalization is phagocytosis. This process has certain similarities with the integrin-mediated flattening and spreading of cells attaching to a substrate-covered surface (Humphries, 1990; Isberg, 1991). Another possible mechanism of integrin-mediated cell entry, however, is receptor-mediated endocytosis (Smythe and War- ren, 1991). For example, internalization of adenovirus is mediated by coated pit endocytosis after binding of cell surface integrins to the RGD-containing penton capsid protein (Wickham et al., 1993). Viral attachment and internalization exploit different receptors in adenoviral infection unlike Yersinia where invasin carries out both functions. Initial attachment of adenovirus is determined by the capsid fiber protein binding to an unknown cell-surface receptor (Defer et al., 1990). Cell-surface- bound viruses are subsequently internalized by receptor-mediated endocytosis mediated by penton-integrin interactions (Wickham et al., 1993; Greber et al., 1993). Coated pit endocytosis would be an unlikely route for internalization of fdRGD, however, as the length of the phage particles (about 1 pm) would probably exclude them from packaging into coated vesicles which have a diameter of approximately 100 nm (Wag- ner et al., 1991). I t is likely, therefore, that the phage fdRGD is binding and entering cells by the same phagocytic-like process exploited in invasin-mediated internalization of Yersinia.

Long term strategies for gene therapy require the development of new cell targeting and entry systems for delivery of genes into mammalian cells. Vectors for gene therapy have been developed that exploit the physiological process of receptor-mediated endocytosis in vitro (Wu and Wu, 1987; Curiel et al., 1992; Wagner et al., 1992; Gao et al., 1993). Such systems do not have the deleterious side effects that can be associated with viral vectors, and have the potential for both specificity and flexibility with regard to the targeting of different cell types and the DNA constructs delivered. These systems have not yet realized their full potential, however, especially in vivo where most receptor-mediated complexes are much less efficient than

in vitro. Only one system, which consists of liver-targeted polylysine-asialoglycoprotein-DNA complexes, has so far given useful levels of gene expression in vivo (Wu and Wu, 1988; Chowdhury et al., 1993). Ligands which target cell surface integrins may, therefore, offer an alternative approach to the construction of targeted gene delivery constructs.

This work demonstrates the application of phage display technology for the identification of ligands that can be used for cell targeting and entry. In this case an integrin-binding ligand has been shown to internalize phage particles. Targeted filamentous phage will probably not be used as gene transfer vectors themselves as they have a single-stranded, circular DNA genome, and single-stranded DNA is expressed poorly or not at all in mammalian cells. We have performed initial investiga- tions into the delivery of a reporter gene plasmid in complexes consisting of polylysine-packaged DNA attached to the outside of fdRGD by electrostatic interactions between polylysine and the negatively charged amino acids on the outside of the phage capsid. So far we have not detected significant levels of gene expression.2 The ligand itself, however, with the advantage of small size, may be useful for the development of new receptor- mediated gene delivery systems, or for intracellular drug delivery. Further work is aimed at a more detailed characteriza- tion of the internalization process and attempting in vitro gene transfer experiments using polylysine-RGD peptide complexes.

Acknowledgments-We are grateful to Dr. John McCafferty for gifts of fd-tet and ant-fd antiserum. We thank Dr. Richard Perham and Dr. Anne Willis for donating the plasmid pKfdH and the bacterial strain TG1. We thank Professor Colin Hopkins, University College London for his helpful advice. The N-terminal amino acid sequencing was done by Susanne Kostkaat and Dr. Regine Kraft, MDC, Berlin Buch, Germany.

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