WO2001038346A2

WO2001038346A2 - Short peptides to target epithelial cells

Info

Publication number: WO2001038346A2
Application number: PCT/GB2000/004409
Authority: WO
Inventors: Charles Coutelle; Philipp Jakob Jost; Holm Schneider
Original assignee: Imperial College Innovations Limited
Priority date: 1999-11-24
Filing date: 2000-11-20
Publication date: 2001-05-31
Also published as: GB9927769D0; WO2001038346A3

Abstract

Targeted gene delivery into human airway epithelial cells may help to overcome the current inefficiency of gene transfer as the major problem confronting cystic fibrosis gene therapy. To elucidate novel ligands targeting abundant, apically located receptors on differentiated airway epithelial cells, a random peptide phage display library was screened for peptides binding with high affinity to such cells. This screening yielded a selectively enriched amino acid sequence, Thr-His-Ala-Leu-Trp-His-Thr (THALWHT). Subsequent binding studies revealed that THALWHT-displaying phages bound much stronger than phages displaying control peptides to the two human airway epithelial cell lines tested, whereas on a variety of non-airway-derived human cell lines no significant binding differences were observed, suggesting selective binding of the THALWHT-motif to airway epithelia. A synthetic peptide comprising a cyclic CTHALWHTC domain and a DNA-binding moiety of 16 lysine residue was shown to enable efficient, targeted gene delivery into human airway epithelial cell lines.

Description

BIOLOGICAL MATERIAL AND USES THEREOF

The present invention relates to biological material, especially compounds such as peptides which are capable of targeting selected cell types. The targeting compounds can be used to deliver to cells agents which may be useful in the prevention, treatment and/or diagnosis of diseases, such as cystic fibrosis (CF).

As progress in the field of cystic fibrosis gene therapy has been hampered by low gene transfer efficacy (Coutelle, 1997; Boucher, 1999), the development of vectors allowing more efficient delivery of the cystic fibrosis transmembrane conductance regulator (CTFR) gene has become one of its main research strategies. The lack of cellular receptors that permit high-affinity binding of present vectors to the apical surface of human airway epithelial cells appears to be one of the main reasons for the inefficiency of both adenoviral and retroviral vectors (Pickles et al., 1998; Wang et al., 1998; Walters et al. , 1999). Non-viral systems represent an interesting alternative to viruses since they avoid some crucial problems inherent to viral gene therapy vectors, such as limited capacity for foreign DNA, contamination of virus preparations with wild type/helper virus, and induction of immune responses (Gao and Huang, 1995; Paillard, 1998). They can be used in combination with any given plasmid DNA and such vectors can be adapted to a particular gene transfer required in a given disease. However, targeting of non-viral vectors to airway epithelial cells also requires a ligand capable of binding with high-affinity to abundant, apically located receptors on these cells. Several candidates including the surfactant protein A receptor (Ross et al., 1995) and monoclonal antibodies (Stray er et al., 1997) have been investigated.

Recently we identified a peptide which binds to α₉β_rintegrin, a receptor abundant throughout the human airway epithelia (Weinacker et al., 1995), and developed non-viral vectors targeting this integrin (Schneider et al.,

1998 and 1999). However, considering the efficacy and selectivity of gene transfer achieved with appropriate vectors so far (Schneider et al., 1999), the need for novel high-affinity ligands of abundant, apically located receptors on airway epithelial cells still remains.

A first aspect of the present invention provides a compound which comprises or consists of the peptide sequence Threonine - Histidine - Alanine - Leucine - Tryptophan - Histidine - Threonine (THALWHT).

A second aspect of the present invention provides a method of identifying a compound which is capable of binding to an epithelial cell comprising testing a test compound to see if it competes for binding to an epithelial cell or a component thereof with a compound comprising or consisting of the peptide sequence THALWHT.

A third aspect of the present invention provides a compound obtainable by the method of the second aspect of the invention, which compound is capable of binding to an epithelial cell.

It will be appreciated that the compounds of the invention are claimed in isolation from at least some of the material with which they may be associated in nature. In other words, the compounds of the invention are not intended to include the compounds in a form which exists in nature. However, the compounds may of course be provided in combination with a variety of other materials and such combinations are intended to fall within the scope of the invention.

In a preferred embodiment of the first and third aspects of the invention, the compound further comprises an effector portion.

By 'effector portion' we include a portion or moiety that is able to mediate an effect on the cells being targeted upon interaction with said cells. Examples of effector portions include gene transfer agents, liposomes, cytotoxic moieties and chelating moieties.

Advantageously, the effector portion is capable of delivering a biologically active agent to the cell.

By 'biologically active agent' we mean an agent which modulates, either positively or negatively, the function and/or activity of a cell or group of cells. For example, suitable biologically active agents of the present invention include nucleic acids (e.g. transgenes, oligonucleotides, mRNA, etc.), proteins, peptides, peptide-nucleic acid complexes, drugs, radioisotopes, etc.

In a preferred embodiment of the present invention the biologically active agent comprises nucleic acid.

Advantageously, invention the biologically active agent comprises the CFTR gene. Conveniently, the biologically active agent is useful in the prevention, treatment and/or diagnosis of a disease. Preferably, the disease is associated with dysfunction of epithelial cells. Suitable diseases include genetically inherited diseases (e.g. cystic fibrosis, αl-antitrypsin deficiency), cancers (e.g. lung cancer), asthma, opportunistic (especially

AIDS-related) lung infections and inflammatory disorders such as asbestosis, etc.

Advantageously, the compounds of the present invention are used in gene therapy, for example in the treatment of cystic fibrosis.

In a further preferred embodiment of the present invention, the compound comprises an effector portion that is a gene transfer agent. Suitable gene transfer agents include virus and virus-like vectors (e.g. adenovirus, retrovinis, pseudocapsids) and DNA binding moieties (e.g. polylysine peptides).

Advantageously, the gene transfer agent is an adenovirus. Compounds of the present invention may be linked to adenovirus using methods known in the art to target delivery of the adenovirus to epithelial cells. For example, the penton base coat protein may be replaced with a compound of the present invention to target the adenovirus to epithelial cells, using the methodology of Wickham et al. (1995) Gene Therapy 2, 750-756. In an alternative strategy, anti-fiber knob antibodies conjugated to a compound of the present invention may be used to 'redirect' delivery of adenovirus (see Douglas et al. (1996) Nature Biotechnology 14, 1574- 1578). A further methodology is described by Romanczuk et al. (1999) Human Gene Therapy 10:2615-2626, involving the covalent attachment to the adenovirus of biftmctional PEG displaying the targeting peptide ligand.

Suitably, the effector portion comprises a pseudocapsid. The use of pseudocapsids for the delivery of genetic material is known in the art, for example see WO 98/48841.

Conveniently, the gene transfer vector is a retrovinis. For example, vectors based on replication defective murine leukaemia viruses may be used to mediate mtegrative gene transfer. Compounds of the present invention may be linked to retrovinis vectors using methods known in the art, for example see Valsesia-Wittmann et al. (1994) and Neda et al. (1991).

Preferably, the effector portion comprises a liposome, such as a cationic liposome. Methods of liposome-mediated delivery of genes using targeting peptides are known in the art. For example, Colin et al. (1998) Gene Therapy 5, 1488-1498 and Gyongyossy-Issa et al. (1998) Arch. Biochem. Biophys. 353, 101-108 disclose the integrin-mediated targeting of epithelial cells using an RGD tripeptide linked to cationic liposomes.

In a preferred embodiment of the present invention, the compound comprises a cyclic CTHALWHTC domain and a DNA binding moiety. Such a compound may be delivered alone or complexed to a liposome.

Conveniently, the DNA binding moiety comprises poly ly sine, preferably 16 lysine residues. In a further embodiment of the present invention the compound comprises an effector portion which is directly or indirectly cytotoxic.

By "directly or indirectly cytotoxic", we include the meaning that the moiety may itself be toxic (for example radionuclides; ricin; tumour necrosis factor; interleukin-2; interferon-gamma; ribonuclease; deoxyribonuclease; Pseudomonas exotoxin A) or it may be metabolised to form a toxic product, or it may act on something else to form a toxic product.

In relation to the indirectly cytotoxic moiety, the moiety may be an enzyme capable of converting a substantially non-toxic prodrug to a toxic drug. Examples of such enzymes include, but are not limited to, cytosine deaminase, which converts 5-fluorocytosine (5FC) to 5-fluorouracil (5FU) (Mullen et al (1922) PNAS 89, 33); the herpes simplex enzyme thymidine kinase, which sensitises cells to treatment with the antiviral agent ganciclovir (GCV) or aciclovir (Moolten (1986) Cancer Res. 46, 5276; Ezzedine et al (1991) New Biol 3, 608). Additionally, the cytosine deaminase of any organism, for example E. coli or Saccharomyces cerevisiae, may be used.

In an additional embodiment of the present invention the compound comprises an effector portion which is directly or indirectly detectable. For example, such detectable effector portions may comprise chelating moieties complexed with radioisotopes, chemiluminescent tags or, alternatively, may comprise gene transfer agents capable of delivering reporter genes (e.g. the firefly luciferase gene). In a fourth aspect of the present invention there is provided a composition comprising a compound of the invention and a lysosomolytic agent.

Suitable lysosomolytic agents include chloroquine, poly-ethyleneimine and lysosomolytic peptides (see Harbottle et al. , 1998, Human Gene Therapy 9, 1037-1047; Cooper et al, 1999, Angew. Chem. Int. Ed. 38, 1949-

1952).

A fifth aspect of the present invention provides a pharmaceutical formulation comprising a compound according to the first or third aspect of the invention or a composition according to the fourth aspect of the invention and a pharmaceutically acceptable excipient or carrier. This could for instance be applied in an aerosolised form orally or directly into the bronchi using bronchoscopy.

A sixth aspect of the present invention provides the use of a compound, composition or formulation of the invention to target epithelial cells.

In a preferred embodiment of the sixth aspect of the invention, the compound is capable of targeting the apical surface of the cells. Advantageously, the epithelial cells are airway epithelial cells. More preferably, the epithelial cells are cells of the bronchus or trachea.

In a seventh aspect of the invention there is provided the use of the peptide sequence THALWHT in the identification of an apical receptor of an epithelial cell or component thereof.

A eighth aspect of the invention provides a receptor or component thereof identifiable by the use according to the fifth aspect of the invention. Preferred, non-limiting examples which embody certain aspects of the invention will now be described with reference to the accompanying figures:

Figure legends

Fig 1: Peptides isolated by biopanning of a random heptamer phage display library on the human airway epithelial cell line 16HBE14o^"

Shown are sequences of heptamer peptides displayed on M13 bacteriophages from a library of phage-displayed peptides after three rounds of selection on the human bronchial epithelial cell line 16HBE14o. Phages were left to bind to the cells and unspecifically bound phages were washed away. Specifically bound phages were eluted, amplified in E. coli and taken through additional binding/amplification cycles to enrich the pool of specifically binding phages. Two fractions of specifically bound phages were eluted in each round. An acidic fraction was recovered by addition of Glycin-HCl (pH 2.2) to the cells and a cell-lysis fraction was collected after lysing the cells. Stringency of selection was increased during the procedure by decreasing binding times from 60 min in 1^st round to 10 min in 3^rd round of biophanning. In addition washing buffer containing increasing amounts of the detergent Tween-20 (0% - 0.2%) was used to wash away unspecifically bound phages. Twenty phages per fraction were characterised by DNA sequencing and the first ten sequenced phages are shown. Phages displaying the peptide THALWHT occurred in 3 out of 20 sequenced phages. Fig. 2: Binding of peptide-displaying bacteriophages to human bronchial epithelial cells (16HBE14o)

Binding of bacteriophages displaying the heptamer peptide THALWHT was compared with the binding of several control phages. Two-fold serial dilutions ranging from 6.3xl0³ to 4.87xl0⁸ phages/ 100 μl were prepared in washing buffer containing 0.2% Tween-20 and added to 16HBE14o^" cells followed by incubation for 1 hr. Detection was performed using anti-M13 antibody conjugated to horseradish peroxidase and absorbances were measured at 405 nm. Each data point is the average from six individual experiments. Error bars represent the standard deviation.

Figure 3: Comparison of phage-binding to different human cell lines

Binding of 6.3xl0¹⁰ phage particles/ 100 μl was tested on the following human cell lines 16HBE14o^" (bronchial epithelium-derived), HTEo^" (tracheal epithelium-derived), CaCo2 (colon carcinoma), HeLa (cervix epitheloid carcinoma), HUH-7 (hepatocyte-derived), Mz-ChAl (gall bladder epithelium-derived) and SW480 (colon epithelium-derived). The attached phages were quantified using anti-M13 antibody conjugated to horseradish peroxidase and measuring the absorbances at 405 nm.

Black bars: phages displaying the heptamer THALWHT; white bars: control phage displaying the heptamer IPACPSC. The average of three independent experiments is shown (16HBE14o^": n=8). Fig. 4: Reporter gene delivery using a cyclic K[16]-GGCTHALWHTC peptide

Peptide-pGL3 complexes containing one, two or four retardation units of the peptide (retardation unit = the amount of peptide required for complete retardation of the electrophoretic mobility of 1 μg of plasmid DNA) formed in HEPES-buffered saline were incubated with confluent 16HBE14o^" cells on 24- well dishes for 4 h at 37 °C. Following further incubation for additional 44 h, luciferase activity in the cell lysates was determined. Each column represents the average results of 4 experiments each performed in sextuplets. Error bars indicate the SEM.

Fig. 5: Effect of chloroquine on gene transfer mediated by a cyclic K[16]-GGCTHALWHTC peptide

Four retardation units of [K] 16-GGCTHALWHTC complexed with pGL3 DNA were added together with increasing amounts of the lysosomotropic agent chloroquine (0 μM - 300 μM) to semi-confluent 16BHE14o^" or CaCo2 cells followed by incubation for 4 h at 37°C. Forty-eight hours after gene delivery the luciferase activity in the cell lysates was determined.

Each column shows the average result of sextuplet wells. Gene expression is represented as percentage of that obtained with the cationic lipid Lipofect AMINE in the same experiment (black bars: 16HBE14o^" cells; white bards: CaCo2 cells). Error bars indicate the standard deviation. EXAMPLE 1

Materials and methods

Cell lines and synthetic peptides

The human cell lines 16HBE14o^" (bronchial epithelium-derived) and HTEo^" (tracheal epithelium-derived) were maintained in Eagle's minimal essential medium (MEM) with L-glutamine (Sigma, Poole), supplemented with 10% foetal calf serum (FCS) and penicillin/streptomycin (100 U/ml of each). The human cell lines CaCo2 (colon carcinoma), HeLa (cervix epitheloid carcinoma), HUH-7 (differentiated hepatoma) and SW480 (colon carcinoma) were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% FCS (HeLa, SW480, HUH-7) or 20% FCS (CaCo2) and penicillin/treptomycin (100 U/ml of each). Mz-ChAl cells (gall bladder adenocarcinoma) were maintained in CMRL-1066 medium (Life Technologies, Paisley) containing 10% FCS and penicillin/streptomycin (100 U/ml of each). Standard tissue culture reagents were purchased from Life Technologies, Paisley.

The peptides K[16]-GGCRGDMFGCA and K[16]-GGCRGEMFGCA were synthesised on a solid-phase batch peptide synthesiser (Applied Biosystems, Foster City, CA) and disulphide-cyclised as described previously (Harbottle et al., 1998). Peptides K[16]-GGCTHALWHTC (disulphide-cyclised) and K[16] were produced by Affiniti Research Products, Exeter. The ability of each peptide to bind DNA was analysed by addition of increasing amounts of peptide to a constant amount of plasmid DNA followed by electrophoresis on a 1 % agarose gel, which indicated the amount of peptide required for complete retardation of the electrophoretic mobility of 1 μg of plasmid DNA (= 1 retardation unit).

Biopanning

The random peptide phage library Ph.D. -7 (New England Biolabs) was applied to 16HBE14o^" cells grown in 6- well plates (4 x 10⁶ cells/ well) to confluency. In this library 7 amino acid-polymers are displayed on the surface of M13-derived phages as fusions with the phage coat protein pill. Three hours prior to the biopanning procedure the cell culture medium was replaced by serum-free MEM, followed after 2 hours by application of 1 % bovine serum albumin (BSA) in Dulbecco's phosphate-buffered saline (PCS), further incubation for 1 hour and washing with Tris-buffered saline (TBS; 50 mM Tris-HCL, pH 7.5, and 150 mM NaCl). 2 x 10¹¹ phages in 1 ml of TBS were applied to a single well and allowed to bind to the cells at 40 °C for 60 mins, followed by washing away unspecifically bound phages with TBS (10 washes) and elution of bound phages with 0.2 M glycine-HCl buffer, pH 2.2. This eluate was neutralised with 1 M Tris-HCl, pH 9.1, (150 μl/ml eluate) and stored as acid-eluted fraction. Subsequent lysis of the cells using 30 mM Tris-CHl, 1 mM EDTA (Barry et al., 1996) yielded the cell-lysis fraction of the phage eluate.

Both phage eluates were amplified separately in E. coli ER2567 cells according to the protocol provided by the manufacturer of the library and then re-applied to the cells. In further panning rounds the phage binding time was reduced to 15 mins (2^nd round) and 10 mins (3^rd round) to increase the stringency of the procedure. Stringency was also increased by using washing buffers containing Tween-20 detergent (0.1 % in the 2^nd round, 0.2% in the 3^rd round). The phages were quantified both after elution and amplification to enable the use of equal amounts 2 x 10¹¹ phages) in each round of biopanning. After three rounds of phage- selection, 20 phages per fraction were characterised by DNA sequencing on an ABI PRISM 377 DNA sequencer using a BigDye Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA).

Phage Binding Assays

Binding studies were performed using enzyme-linked immunosorbant assays on different human cells grown in 96-well plates to confluency. After blocking the plates with 1 % BSA, two-fold serial dilutions of the phages (6.3xl0¹⁰ to 4.87xl0⁸) were applied to the wells. Phages were allowed to bind to the cells for 60 mins at 4°C. Unbound phages were removed by washmg the plates six times with a wash buffer containing 0.2% Tween-20. An anti-M13 antibody conjugated with horseradish peroxidase was then added to each well, followed by addition of 2,2'- Asino-bis(3-ethylbenzthiazoline-6-sulfonic acid)/H₂0₂ solution after 1 hour and measurement of the absorbance at 405 nm.

Gene transfer experiments

The American firefly luciferase gene under the control of a simian virus 40 promoter and enhancer in the plasmid vector pGL3 (Promega, Madison) was used as reported gene. Plasmid DNA was purified from overnight cultures of transformed Escherichia coli JM109 using an endotoxin extraction kit (Qiagen, Hilden). Cells were seeded in tissue culture-treated 24- well plates at a density of 5xl0⁴ cells/ well and incubated until confluent. Peptide-DNA and DNA-LipofectAMINE Gibco complexes (24 μg of LipofectAMINE/ 1 μg of DNA) were prepared in

50 μl of serum-free OptiMem medium (Life Technologies, Paisley). The cells were washed and incubated in OptiMem for 30 min at 37 °C prior to addition of the complexes and further incubation for 4 h. The medium was then replaced by DMEM containing 10% FCS supplemented with penicillin (100 U/ml) and streptomycin (100 U/ml) and the incubation was continued for an additional 44 h. If chloroquine was used, this was added to the medium 20 min before the peptide-DNA complexes were applied. Luciferse activity was determined after harvesting the cells in 200 μl of reporter lysis buffer (Promega, Madison) using the luciferase assay kit from Promega and a Berthold luminometer.

Results

Isolation of a peptide binding with high affinity to human airway epithelial cells from a random-peptide phage display library

Several groups including our own have tested non-viral gene therapy vectors targeting apically located receptors on human airway epithelial cells (Ross et al., 1995; Schneider et al., 1999), but none of these systems appears to bind with an affinity sufficient for most gene therapy applications. We screened a linear heptamer phage display library on the human bronchial epithelium-derived cell line 16HBE14o^". This cell line has been described to develop in vitro the morphologic features of differentiated airway epithelial cells (Cozens et al., 1994). On cells forming a confluent layer the presence of ciliae on the apical surface and tight junctions separating apical and basolateral cell membranes were confirmed by electron microscopy.

During the biopanning steps the stringency of the procedure was increased by reducing the phage binding time to 10 min and by using washing buffers containing Tween-20 detergent. Specifically bound phages were eluted using a glycine buffer and collected as acidic fraction, followed by lysis of the cells to obtain a cell-lysis fraction of the remaining phages. After three rounds of phage-selection, 20 phage clones per fraction were characterised by DNA sequencing. Amongst those only the amino acid sequence Thr-His-Ala-Leu-Trp-His-Thr (THALWHT) from the linear heptamer library eluted by acid-stripping was found repeatedly, in 15% of the phage clones of the linear heptamer library (fig. 1).

Phages displaying a THALWHT peptide bind with high affinity to human airway epithelial cells but not to non-airway derived cells

Using ELISA the binding of the selectively enriched phage to 16HBE14o^" cells was compared with that of several control phages across a range of concentrations between 4.87xl0⁸ and 6.3xl0¹⁰ particles/ well. In this assay the THALWHT-displaying phage bound markedly better than the control phages to 16HBE14o^" cells, at all concentrations tested (fig. 2). When the same assay was performed on 5 other human cell lines derived from various other tissues the binding of the THALWHT-displaying phage and control phages did not differ significantly. This suggested a strong binding of the peptide THALWHT specifically to 16HBE14o^" cells via a receptor abundant on these cells but not on non-airway derived cells. To investigate whether the THALWHT motif would also bind to airway epithelial cells different from 16HBE14o^" we performed the same ELISA also on the human tracheal epithelial cell line HTEo^' and observed significantly better binding of the THALWHT-displaying phage compared with various control phages. The ELISA results are summarised in Fig. 3.

A cyclic K[16]-GGCTHALWHTC peptide enables highly efficient, targeted gene delivery into human airway epithelial cell lines

As the peptide THALWHT appeared to be an high-affinity ligand of receptors on human airway epithelia, we synthesised a peptide comprising a receptor-targeting domain (disulphide-cyclised CTHALWHTC) and a

DNA-binding moiety consisting of 16 lysine residues. With this bifunctional synthetic peptide specific gene delivery into 16HBE14o^" cells could be achieved and the relative gene transfer efficacy (standardised as percentage of lipofectAMINE-mediated transfection) was higher than with the systems for integrin-mediated gene transfer developed in our lab

(Harbottle et al., 1998; Schneider et al., 1999). Complexes formed with two or four retardation units of this peptide were found to deliver a luciferase reporter gene one to two orders of magnitude more efficiently into 16HBE14o cells than complexes formed with two retardation units of

K[16] or K[16]-GGCRGEMFGCA (fig. 4).

Gene delivery mediated by K[16I-GGCTHALWHTC can be markedly enhanced by a lysomolytic agent or a cationic liposome

However, as the cationic liposome LipofectAMINE that served as assay standard gave on average 6- to 8-fold higher reporter gene expression values, we tried to enhance gene delivery by addition of the lysosomolytic agent chloroquine. This approach allowed us to increase the gene transfer efficacy by more than one order of magnitude (fig. 5), demonstrating that the peptide-DNA complexes are taken up via an endocytic route after binding to the cells and can be released efficiently from the endosomes.

We also tried to improve the efficiency of the gene transfer by including a cationic liposome in the peptide/DNA complexes, which has been shown to enhance the transfection mediated by our system for integrin-mediated gene transfer (Colin et al., 1998; Schneider et al, 1999). With this approach we were able to increase the gene transfer efficacy mediated by the liposome alone by more than one order of magnitude. However, as with other systems (Colin et al., 1998; Schneider et al , 1999) this increase in efficiency was associated with reduced, but significant targeting specificity.

Discussion

Random peptide libraries in bacteriophage have proved useful for the identification of novel targeting ligands (Barry et al, 1996; Pasqualini & Ruoslahti, 1996). By biopanning of such libraries on the human bronchial epithelial cell line 16HBE14o^" we found a phage displaying the peptide THALWHT, which binds significantly better to 16HBE14o^" cells than several control phages. However, the binding of this phage to several non-airway derived human cell lines did not differ from that of the control phages, suggesting involvement of a receptor abundant on 16HBE14o^" but not on the other cells. Our group has been investigating the use of small synthetic peptides comprising a cell surface receptor-targeting domain and a cationic DNA binding moiety as novel non- viral vectors for gene therapy (Hart et al.,

1995; Harbottle et al., 1998; Colin et al, 1998; Schneider et al, 1999). These vectors avoid some crucial problems inherent to other gene therapy vectors, such as induction of an immune response or limited capacity for foreign DNA. They can be synthesised in large quantities and high purity and allow the combination with any plasmid DNA. Although systems for receptor-mediated gene transfer have not yet reached the gene transfer efficiency of the best viral vectors, combinations with lysosomolytic agents and liposomes may approach it and appear to be promising with respect to in vivo application of this peptide. The possibility to synthesise and modify well-defined gene therapy vectors may therefore be a major advantage of our synthetic peptides.

EXAMPLE 2

Competition binding assay

The THALWHT peptide or derivative thereof (e.g. pep tide-protein conjugates) is added to individual wells of a non-tissue culture treated 96- well plate (Costar) and attached overnight at 37 °C. The wells are then blocked with 3 % BS A/PBS for 1 hour at 37 °C.

Cultured cells are harvested from 250 ml tissue culture flasks by incubation with EDTA and/or trypsin for 5 minutes at 37 °C. The cells are washed in medium and diluted to a concentration of approximately 1 x 10⁶ cells/ml. A fraction of the cells are then mixed gently by rotation for 1 hour at 4°C with the competing peptide. A 50 μl aliquot of the cell suspension treated with the peptide (5 x 10⁴ cells) and a 50 μl aliquot of untreated cell suspension (same number of cells) are then added to each well and incubated at 37° C for 1.5 hours.

Following incubation, the wells are washed gently with PBS and attached cells are fixed by addition of 50 μl of methanol for 15 minutes. The methanol is then removed and the plates are air-dried before the remaining cells are stained with 0.1 % (w/v) crystal violet in distilled water. The excess crystal violet is removed by washing with PBS and the stained cells are solubilised by addition of 50 μl of 1 % SDS to each well.

The absorbances of individual wells at 595nm is then measured using a spectrophotometer. The absorbances indicate the numbers of cells binding to THALWHT. If the other peptide with which a fraction of the cells was pre-incubated inhibits this binding competitively, the absorbance in the appropriate wells should be lower than that of control wells.

A peptide is positively selected (i.e. is considered to compete with binding to THALWHT) if absorbance is decreased by at least 50% compared to the absorbance of the well containing untreated cells. Preferably, absorbance is decreased to 30% , 20% , 10%, 5% or 1 % or less than 5% of untreated values. Ideally, the absorbance corresponding to cells treated with the competing peptide is substantially zero.

Skilled persons will appreciate that the above assay can be modified in a variety of ways. For example, a different component, e.g. the cells, may be fixed to the solid support and/or different binding detection systems employed.

EXAMPLE 3

Methods of linking peptides with an effector portion

Linking peptides to adenovirus

Methods of directing the delivery of adenoviral vectors to specific cell populations using targeting peptides are known in the art. For example, see the methods described by :

(i) Wickham et al. (1995) Gene Therapy 2, 750-756, in which the gene sequence for the new ligand is inserted into the virus genome to replace the region coding for the RGD peptide motif in the penton base;

(ii) Douglas et al. (1996) Nature Biotechnology 14: 1574-1578, in which an anti-fibre antibody, chemically conjugated to a cell specific ligand, attaches to the fibre and retargets cell binding of adenovirus via its fibre- protein; and

(iii) Romanczuk et al. (1999) Human Gene Therapy 10:2615-2626 in which bifunctional PEG displaying the targeting peptide ligand is covalently attached to the adenovirus. Linking peptides to retrovinis

From the envelope gene of an avian leukosis virus of subgroup A, mutants are constructed in which the small regions I and II of the receptor-binding domains are replaced by the THALWHT peptide or its derivatives. Substitutions in these two regions do not affect the processing and the viral incorporation of the mutants. Deglycosylated helper-free retroviral particles obtained by mild treatment with N-glycosidase F are produced with the mutant envelopes. Plastic dishes coated with re-targeted viral particles are used to investigate whether the mutants promote binding of mammalian cells resistant to infection by native subgroup A avian leukosis viruses. The infections can be monitored by neomycin selection. No neomycin-resistant clones should be obtained after infection by viruses with wild-type envelopes. Conversely, colonies should be obtained after infection by viruses with envelopes bearing the new targeting domain in region II, and the genome of the retroviral vector should be found correctly integrated in cell DNA of these colonies. By using blocking peptide containing the THALWHT sequence it should be possible to show that preincubation of target cells specifically inhibits infection by retargeted viruses (Valsesia-Wittmann et al., 1994).

Alternatively, retrovinis could be retargeted by chemical modification as demonstrated by linking lactose to the virus envelope, as described by Neda et al. (1991) J. Biol. Chem. 266: 14143-6. Use of targeting peptides in conjunction with liposomes and pseudocapsids

Synthetic gene transfer systems such as liposomes and pseudocapsids can be targeted by use of the peptides of the invention. This can be performed for lipid targeting by various methods representing the state of the art, such as those described by Zalipinsky et al. 1994; Garcia et al. 1999, Ogawa et al. 1999 and de Lima et al. 1999, WO 99/54344, or by modifications of such methods (see also Example 1).

For targeting of pseudocapsids, chemical or biological methods as described above for retargeting the adenovirus capsid and the retroviral envelop, respectively, may be applied.

Preparation of compounds of the invention comprising a cyclised peptide and a DNA binding moiety

(see Example 1)

Preparation of compounds of the invention comprising a detectable moiety

The compounds of the present invention may be linked to a directly or indirectly detectable moiety using methods known in the art. For example, said compounds may be fused to chelating agents which can then be complexed to radioisotopes such as yttrium-90, tritium, etc. Alternatively, the detectable moiety may comprise a marker gene (such as firefly luciferase; see Example 1) which is carried by a gene transfer vector coupled to a compound of the invention.

EXAMPLE 4

Medical use of the compounds of the invention

The cystic fibrosis transmembrane conductance regulator (CTFR) gene, as disclosed by Riordan et al. (1989) Science 245, 1066-1073, encodes a 170 kD cAMP-activated membrane channel, expressed by epithelial cells, which regulates chloride conductance across the epithelial cell layer of the airways. Gene therapy for CF has focussed on the delivery of the CFTR gene to airway epithelial cells, for example see review by Boucher (1999) J. Clin. Investigation 103, 441-445.

Compounds including the peptide THALWHT or any derivatives of it may be used to deliver a gene, drug, protein etc. to the lungs of CF patients. Such compounds could be applied for instance by instillation or inhalation.

Methods for aerosol delivery of gene therapy agents of the invention are described in Alton et al. (1999) Lancet 353:947-54, the disclosure of which is incorporated herein by reference. In particular, we refer to methods of admmistration and (CFTR) gene dosages together with methods of measuring successful gene transfer in humans. EXAMPLE 5

Use of the compounds of the invention for the identification of putative membrane receptors

The THALWHT-containing peptides of the present invention can be used to isolate (thus far unidentified) endogenous receptor protein(s).

For example, putative receptors for the peptides of the invention may be isolated using the following strategies:

(i) Affinity binding methods may be used to identify membrane receptors to which the peptides of the invention bind. Such an approach has been used successfully by Shahan et al. (1999) to identify putative receptors on tumour cells to which an integrin alpha 3 (IN) 179-208 peptide binds.

(ii) Alternatively, isolation of the coding sequence for the receptor proteins may be achieved by subtraction hybridisation techniques against cDΝA from epithelial cells that do not bind the THALWHT-containing peptides. Such as approach has been used by Kobayashi et al. (1997) for the isolation of a sulfate transporter. A related method applying microarrays for this purpose is described by Kononen et al. (1998).

Acknowledgements

This work was supported in part by grants from the March of Dimes Birth

Defects Foundation (19-FY97-0632 to C. C), the British Medical

Research Council (G9227430 to C. C), the Deutsche Forschungsgememschaft (Schn 569/1-1 to H. S.) and the Association

Francaise de Lutte contre la Mucovisidose. P.J.J. is recipient of a scholarship from the Studienstiftung des deutschen Volkes.

References

Barry, M.A., Dower, W.J., Johnston, S.A. : Toward cell-targeting gene therapy vectors: selection of cell-binding peptides from random peptide- presenting phage libraries. Nat. Med. 2, 299-305 (1996)

Boucher, R.C.: Status of gene therapy for cystic fibrosis lung disease. J. Clin. Invest. 103, 441-445 (1999)

Colin, M. , Harbottle, R.P., Knight, A.M., Kornprobst, M. , Cooper, R.G., Miller, A.D. , Trugnan, G., Capeau, J. , Coutelle, C, Brahimi- Horn, M.C. : Liposomes enhance delivery and expression of an RGD- oligolysine gene transfer vector in human tracheal cells. Gene Ther. 5, 1488-1498 (1998)

Coutelle C : Gene therapy for cystic fibrosis - strategies, problems and perspectives. In: Strauss M., Baranger J.A. (eds.) Concepts in Gene Therapy, Walter de Gruyter, Berlin, pp313-343 (1997)

Cozens, A.L. , Yezzi, M.J. , Kunzelmann, K. , Ohrui, T. , Chin, L., Eng, K., Finkbeiner, W.E., Widdicombe, J.H., Gruenert, D.C.: CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells. Am. J. Respir. Cell. Mol. Biol. 10, 38-47 (1994)

Cwirla, S.E. , Balasubramaman, P. , Duffin, D.J. , Wagstrom, C.R., Gates, CM. , Singer, S.C., Davis, A.M. , Tansik, R.L. , Mattheakis, L.C., Boytos, CM. , Schatz, P.J. , Baccanari, D.P., Wrighton, N.C , Barrett, R.W., Dower, W.J.: Peptide agonist of the thrombopoietin receptor as potent as the natural cytokine. Science 276, 1696-1699 (1997)

Grubb, B.R., Pickles, R.J., Ye, H., Yankaskas, J.R., Vick, R.N., Engelhardt, J.F., Wilson, J.M., Johnson, L.G., Boucher, R.C: Inefficient gene transfer by adenovirus vector to cystic fibrosis airway epithelia of mice and humans. Nature 371, 802-806 (1994)

Ferkol, T., Perales, J.C, Eckman, E., Kaetzel, C.S., Hanson, R.W., Davis, P.B.: Gene transfer into the airway epithelium of animals by targeting the polymeric immunoglobulin receptor. J. Clin. Invest.95, 493- 502 (1995)

Ferkol, T., Pellicena-Palle, A., Eckman, E., Perales, J.C, Trzaska, T., Tosi, M., Redline, R., Davis, P.B.: Immunologic responses to gene transfer into mice via the polymeric immunoglobulin receptor. Gene Ther. 3, 669-678 (1996)

Forstova, J., N. Krauzewicz, V. Sandig, J. Elliott, Z. Palkova, M. Strauss, and B. E. Griffin. Polyoma virus pseudocapsids as efficient carriers of heterologous DNA into mammalian cells. Hum Gene Ther. 6:297-306 (1995).

Gao, X., Huang, L.: Cationic liposome-mediated gene transfer. Gene Ther.2, 710-722 (1995) Garcia, M. , M. A. Alsina, F. Reig, and I. Haro. Liposomes as vehicles for the presentation of a synthetic peptide containing an epitope of hepatitis A virus. Vaccine 18:276-283 (1999).

Gruenert, D.C , Basbaum, C.B., Welsh, M.J. , Li, M., Finkbeiner, W.E., Nadel, J.A. : Characterization of human tracheal epithelial cells transformed by an origin-defective simian virus 40. Proc. Natl. Acad. Sci. USA 85, 5951-5955 (1988)

Harbottle, R.P., Cooper, R.G., Hart, S.L., Ladhoff, A. , McKay, T., Knight, A.M. , Wagner, E., Miller, A.D. , Coutelle, C : An RGD- oligolysine peptide: a prototype construct for integrin-mediated gene delivery. Hum. Gene Ther. 9, 1037-1047 (1998)

Hart, S.L., Harbottle, R.P., Cooper, R.G., Miller, A.D., Williamson, R., Coutelle, C : Gene delivery and expression mediated by an integrin- binding peptide. Gene Ther. 2, 552-554 (1995)

Kobayashi, T., Sugimoto, T., Saijoh, K. , Fukase, M. , Chihara, K: Clomng of mouse diastrophic dysplasia sulfate transporter gene induced during osteoblast differentiation by bone morphogenetic protein-2 Gene 198 341-9 (1997)

Kononen, J. , Bubendorf, L., Kallioniemi, A., Barlund, M. , Schraml, P., Leighton, S., Torhorst, J. , Mihatsch, M. J., Sauter, G. , Kallioniemi, O. P. : Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat. Med. 4, 844-7(1998) de Lima, M. C, S. Simoes, P. Pires, R. Gaspar, V. Slepushkin, and N.

Duzgunes. Gene delivery mediated by cationic liposomes: from biophysical aspects to enhancement of transfection. Mol Membr Biol.

16: 103-9 (1999).

Matsui, H., Johnson, L.G. , Randell, S.H., Boucher, R.C : Loss of binding and entry of liposome-DNA complexes decreases transfection efficiency in differentiated airway epithelial cells. /. Biol. Chem. 272, 1117-1126 (1997)

Neda, H. Wu C.H. , Wu G.Y. : Chemical modification of an ecotropic murine leukemia virus results in redirection of its target cell specificity. J Biol Chem 266, 14143-6 (1991)

Ou, W. C, M. Wang, C Y. Fung, R. T. Tsai, P. C Chao, T. H. Hseu, and D. Chang. The major capsid protein, VP1, of human JC virus expressed in Escherichia coli is able to self-assemble into a capsid-like particle and deliver exogenous DNA into human kidney cells. J Gen Virol. 80:39-46 (1999).

Ogawa, Y. , H. Kawahara, N. Yagi, M. Kodaka, T. Tomohiro, T. Okada, T. Konakahara, and H. Okuno. Synthesis of a novel lipopeptide with alpha-melanocyte-stimulating hormone peptide ligand and its effect on liposome stability. Lipids 34:387-94 (1999).

Paillard, F. : Oligoplexes: nonviral vehicles for receptor-mediated delivery. Hum. Gene Ther. 9, 987-988 (1998) Pasqualini, R., Ruoslahti, E. : Organ targeting in vivo using phage display peptide libraries. Nature 380, 364-366 (1996)

Pickles, R.J., McCarty, D., Matsui, H., Hart, P.J. , Randell, S.H., Boucher, R.C : Limited entry of adenovirus vectors into well- differentiated airway epithelium is responsible for inefficient gene transfer. J. Virol. 72, 6014-6023 (1998)

Rajotte, D., Ruoslahti, E.: Membrane dipeptidase is the receptor for a lung-targeting peptide identified by in vivo phage display. J. Biol. Chem. 274, 11593-11598 (1999)

Romanczuk, H. Galer, C Zabner, J. Barsomian, G., Wadsworthe, S.C, O'Riordan C.R. : Modification of an adenoviral vector with biologically selected peptides: A novel strategy for gene delivery to cells of choice. Human Gene Therapy 10, 2615-2626 (1999)

Ross, G.F., Morris, R.E., Ciraolo, G. , Huelsman, K. , Bruno, M., Whitsett, J.A. , Baatz, J.E., Korfhagen, T.R.: Surfactant protein A- polylysine conjugates for delivery of DNA to airway cells in culture. Hum. Gene Ther. 6, 31-40 (1995)

Schneider, H., Harbottle, R.P., Yokosaki, Y., Kunde, J., Sheppard, D., Coutelle, C : A novel peptide, PLAEIDGIELTY, for the targeting of α₉β_r integrins. FEBS-Lett. 429, 269-273 (1998) Schneider, H., Harbottle, R.P., Yokosaki, Y. , Jost, P. , Coutelle, C:

Targeted gene delivery into ^βrintegrin-displaying cells by a synthetic peptide. FEBS-Lett., in press (1999)

Schneider, H. and Coutelle, C: In utero gene therapy: The case for. Nat. Med. 5, 256-257 (1999)

Shahan, T. A. , Ziaie, Z., Pasco, S. , Fawzi, A. , Bellon, G., Monboisse, J. C, Kefalides, N. A. : Identification of CD47/integrin-associated protein and alpha(v)beta3 as two receptors for the alpha3(IV) chain of type IN collagen on tumor cells. Cancer Res. 18, 4584-90 (1999)

Strayer, D.S., Schwarting, R. : Recognition of normal, neoplastic, and fetal airway epithelial cell membranes by two monoclonal antibodies. Hum. Pathol. 28, 840-846 (1997)

Szardenings, M. , Tornroth, S., Mutulis, F., Muceniece, R., Keinanen, K. , Kuusinen, A., Wikberg, J.E.: Phage display selection on whole cells yields a peptide specific for melanocortin receptor 1. /. Biol. Chem. 272, 27943-27948 (1997)

Valsesia-Wittmann, S. , Drynda, A., Deleage, G., Aumailley, M., Heard, J.M., Danos, O. , Verdier, G., Cosset, F.L. : Modifications in the binding domain of avian retrovinis envelope protein to redirect the host range of retroviral vectors. J. Virol. 68, 4609-19 (1994)

Wang, G. , Davidson, B.L., Melchert, P., Slepushkin, V.A. , van Es, H.H. , Bodner, M., Jolly, D.J., McCray, P.B. : Influence of cell polarity on retrovirus-mediated gene transfer to differentiated human airway epithelia. J. Virol. 72, 9818-9826 (1998)

Walters, R.W., Grunst, T., Bergelson, J.M. , Finberg, R.W. , Welsh, M.J., Zabner, J. : Basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia. J. Biol. Chem. 274, 10219-10226 (1999)

Weinacker, A., Ferrando, R. , Elliott, M., Hogg, J. , Balmes, J., Sheppard, D: Distribution of integrins α_vβ₆ and α₉β_ι and their known ligands, fibronectin and tenascin, in human airways. Am. J. Respir. Cell Mol. Biol. 12, 547-557 (1995)

Zabner, J. , Freimuth, P. , Puga, A. , Fabrega, A., Welsh, M.J. : Lack of high affinity fiber receptor activity explains the resistance of ciliated airway epithelia to adenovirus infection. J. Clin. Invest. 100, 1144-1149 (1997)

Zalipinsky, S., Brandeis, E. , Newman, M.S. , Woodle, M.C : Long circulating, cationic liposomes containing amino-PEG- phosphatidylethanolamine. FEBS-Letters 353, 71-74 (1994)

Claims

1. A compound which comprises or consists of peptide sequence Threonine - Histidine - Alanine - Leucine - Tryptophan - Histidine - Threonine (THALWHT).

2. A method of identifying a compound which is capable of binding to an epithelial cell comprising testing a test compound to see if it competes for binding to an epithelial cell or a component thereof with a compound comprising or consisting of the peptide sequence THALWHT.

3. A compound obtainable by the method of claim 2, which compound is capable of binding to an epithelial cell.

4. A compound as claimed in claim 1 or 3 further comprising an effector portion.

5. A compound as claimed in claim 4 wherein the effector portion is capable of delivering a biologically active agent to the cell.

6. A compound as claimed in claims 4 or 5 wherein the agent is useful in the prevention, treatment and/or diagnosis of a disease.

7. A compound as claimed in claim 5 or 6 wherein the agent comprises nucleic acid.

8. A compound as claimed in claim 7 wherein the nucleic acid comprises the CFTR gene.

9. A compound as claimed in any one of claims 6 to 8 wherein the disease is cystic fibrosis.

10. A compound as claimed in any one of claims 4 to 9 wherein the effector portion is a gene transfer agent.

11. A compound as claimed in claim 10 wherein the effector portion comprises a virus.

12. A compound as claimed in claim 11 wherein the virus is an adenovirus.

13. A compound as claimed in claim 11 wherein the virus is a retrovinis.

14. A compound as claimed in claim 11 wherein the effector portion comprises a pseudocapsid.

15. A compound as claimed in claim 4 wherein the effector portion comprises a liposome.

16. A compound as claimed in claim 4 comprising a cyclic CTHALWHTC domain and a DNA binding moiety.

17. A compound as claimed in claim 16 wherein the DNA binding moiety comprises poly ly sine, preferably 16 lysine residues.

18. A compound as claimed in any one of claims 4 to 7, or 9 to 17 wherein the effector portion is directly or indirectly cytotoxic.

19. A compound as claimed in any one of claims 4 to 7, or 9 to 17 wherein the effector portion is directly or indirectly detectable.

20. A composition comprising a compound accordmg to any one of claims 1 or 3 to 19 and a lysosomolytic agent.

21. A composition according to claim 20 wherein the lysosomolytic agent is selected from chloroquine, polyethyleneimine and lysosomolytic peptides.

22. A pharmaceutical formulation comprising a compound according to any one of claims 1 or 3 to 19 or a composition according to claim 20 or

21 and a pharmaceutically acceptable excipient.

23. A pharmaceutical formulation according to claim 22 wherein the formulation is in the form of an aerosol.

24. Use of a compound, composition or formulation as claimed in any preceding claim to target epithelial cells.

25. Use as claimed in claim 24 wherein the compound is capable of targeting the apical surface of the cells.

26. Use as claimed in claim 24 or 25 wherein the epithelial cells are airway epithelial cells.

27. Use as claimed in claim 262 wherein the cells are cells of the bronchus or trachea.

28. Use of the peptide sequence THALWHT in the identification of an apical receptor of an epithelial cell or a component thereof.

29. A receptor or a component thereof identifiable by the use as claimed in claim 28.