NZ500656A - Vector containing a nucleic acid insertion expressing a hybrid polypeptide with a protease inhibitor domain and a receptor binding domain - Google Patents

Abstract

A recombinant nucleic acid molecule is provided comprising a vector useful for transfection or transduction of mammalian cells, wherein the vector contains a nucleic acid insertion encoding an expressible hybrid polypeptide or protein which comprises a domain with a receptor binding function and a domain with enzymatic activity, in particular protease inhibitor activity. The vector may be a viral (e.g. adenovirus or retrovirus) or non-viral vector, and the nucleic acid insertion encoding the expressible hybrid polypeptide or protein may be under the control of a cell- or tissue-specific promoter. The vector may be used to express the hybrid polypeptide in a patient in a method to prevent local proteolytic activity, extracellular matrix degradation, cell migration, cell invasion or tissue remodelling.

Description

Title: Method and Construct for inhibition of cell migration FIELD OF THE INVENTION The invention is m the field of therapeutic means and therapeutic methods for treatment of diseases in which cell migration and/or tissue remodeling occurs. Furthermore, the invention is m the field of biotechnology, in particular recombinant DNA technology and gene therapy.

BACKGROUND OF THE INVENTION -Migration of cells is an essential step in many physiological and pathological processes m which tissue remodeling occurs, such as tumor metastasis, wound healing, restenosis, angiogenesis or rheumatic arthritis. Migrating cells have to pass through the surrounding extracellular matrix. Limited proteolytic degradation of the components of the extracellular matrix is often seen during cell migration. To mediate this cell migration migrating cells produce, or recruit from their direct environment, proteolytic enzymes, such as plasminogen activators, metalloproteinases or elastases. Induction of cell migration e.g. during tumor metastasis or wound healing often correlates with the induction of the production of these enzymes.

Although the involvement of proteolytic enzymes in cell migration under pathophysiological conditions is well accepted, little attempts have been made to inhibit cell migration by inhibiting these proteolytic enzymes. A possible explanation for the limited use of protease inhibitors is the fact that these proteolytic enzymes are involved in many processes both pathological and physiological (including fibrinolysis, wound healing, growth factor activation etc.) and that inhibition of these protease systems by systemically applied protease inhibitors might have either strong side effects or may lead to a diffusion or clearance of the inhibitory compounds without having a strong effect on the local cell migration processes.

Printed from Mimosa 10/29/1999 17:23:46 page -3- Another problem in the use of protease inhibitors to interfere in cell migration and tissue remodeling is that proteases mediating these processes can bind to receptors at the cell surface. In this way the proteolytic enzymes might 5 be active locally in a pericellular microenvironment where they are protected against the action of the present inhibitors.

It has been disclosed that conjugates between the receptor binding part of u-PA (the ammotermmal fragment or ATF) and urinary trypsin inhibitor produced in vitro, inhibit migration of tumor cells in vitro (Kobayashi, Gotoh, Hirashima, Fujie, Sugmo and Terao, Inhibitory effect of a conjugate between human urokinase and urinary trypsin inhibitor on tumor cell invasion m vitro. J. Biol. Chem. (1995) 270, 8361-8366). The conjugate these authors have used is made synthetically by mixing the isolated ATF fragments with the trypsin inhibitor.

Recently it has been disclosed that these conjugates also can be produced recombinantly (WO 97/25422).

A comparable construct consisting of a receptor binding u-PA fragment and its inhibitor PAI-2, to be produced recombinantly in yeast, has been described to inhibit tumor cell migration m WO 92/02553 (PCT/GB91/01322). In this way they have made a protease inhibitor that can bind to a specific receptor at the cell surface, the urokinase receptor, and this inhibitor can inhibit cell migration (in vitro). As to the use of these constructs in vivo, a problem is the application to and the prolonged presence at the site of desired action in vivo. It is an object of the present ^ invention to go some way towards overcoming the prior art problems and/or to provide the public with a useful choice.

SUMMARY OF THE INVENTION This invention provides a recombinant nucleic acid molecule comprising a vector useful for transfection or transduction of mammalian, e.g. human, cells, wherein said 3 5 vector contains a nucleic acid insertion encoding_an- ^ v INTf LLECT'JAL PROPERTY expressible hybrid polypeptide or protein wiucr^^omgfi^es a I 1 o kov ra p " o r," v R n 50065 intellectual property office of n.z. 3 - 1 OCT 2001 received domain with a binding function and a domain with an effector function, wherein the domain with a binding function is a cell surface receptor binding domain. Preferably the domain with an effector function has enzymatic 5 activity, most preferably protease inhibitor activity. from the group consisting of urokinase receptor binding domain of urokinase, receptor binding domain of epidermal growth factor, receptor associated protein that binds to LDL 10 Receptor related protein (a2-macroglobulin receptor) and VLDL Receptor. has protease inhibitor activity and comprises a protease inhibitor or active part thereof, said protease inhibitor 15 being selected from the group consisting of (bovine) pancreatic trypsin inhibitor, (bovine) splenic trypsin inhibitor, urinary trypsin inhibitor, tissue inhibitor of matrix metalloproteinase 1, tissue inhibitor of matrix metalloprotemase 2, tissue inhibitor of matrix metallo-2 0 proteinase 3, and elastase inhibitor. The domain with an effector function may comprise (an active part of) two or more different protease inhibitors, or two or more copies of (an active part of) a protease inhibitor, or both. consisting of viral and non-viral vectors useful for trans- fection or transduction of mammalian cells. The vector may be an adenovirus vector or a retrovirus vector useful for trans-fection or transduction of human cells. 3 0 hybrid polypeptide or protein may be under the control of a cell- or tissue-specific promoter, such as an endothelial cell-specific promoter, or a vascular smooth muscle cell-specific promoter, or a liver-specific promoter. preventing local proteolytic activity, extracellular matrix degradation, cell migration, cell invasion, or tissue Preferably, the receptor binding domain is selected Preferably, the domain with an effector function Preferably, the vector is selected from the group The nucleic acid insertion encoding an expressible Disclosed but not claimed is a process for remodeling, comprising transfecting or transducing the cells involved or cells in their environment with a recombinant nucleic acid molecule as defined herein to obtain local expression of the hybrid polypeptide or protein encoded by 5 said nucleic acid molecule.

Also, this invention provides a process for producing a hybrid polypeptide or protein which comprises a domain with a binding function and a domain with an effector function, comprising transfecting or transducing mammalian 10 cells with a recombinant nucleic acid molecule as defined herein to obtain expression of the hybrid polypeptide or protein encoded by said nucleic acid molecule, and optionally recovering the hybrid polypeptide or protein produced.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 schematically depicts the plasmids pCRII-uPA (left) and pCRII-ATF (right).

Figure 2 schematically depicts the plasmid pCRII- ATF-BPTI.

Figure 3 schematically depicts the plasmid pMAD5~ ATF-BPTI.

Figure 4 shows the results of proteolytic matrix degradation experiments.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the use of hybrid proteins in which a receptor binding domain is linked to a functional protein m order to induce a local action of this protein and to prevent systemic effects and/or diffusion. In 3 0 particular this invention relates to such hybrid proteins that might be produced by a subset of cells as target cells after transfection or transduction with expression vectors. More specifically the invention relates to the use of such expression vectors, coding for hybrid proteins consisting of 35 a receptor binding domain and a protease inhibitor domain, for the prevention of cell migration and tissue remodeling by Printed from Mimosa 10/29/1999 17:23:46 page -6- WO 98/51788 PCT/NL98/00259 inhibition of proteases at the surface of migrating or invading cells.

The method and construct described in the present invention can be applied as therapy in diseases in which cell 5 migration and/or tissue remodeling occurs.

The present invention addresses the solution of several negative aspects involved in the above described use of inhibitors according to the prior art: High local concentrations of hybrid proteins in the 10 direct environment of the target cells can be obtained by production of the protein by the migrating cells themselves or cells m their immediate environment. This production can be induced by transfection or transduction of a certain subset of the cell population with a suitable vector encoding 15 the hybrid protein. For this purpose, one may use recombinant adenoviral vectors, retroviral vectors, plasmid vectors, etc.

Diffusion of the inhibitor and systemic side effects are prevented by binding the hybrid protein (by its receptor binding domain) to the cell surface of the target 20 cell. Local expression of this hybrid protein also contributes to the reduction of systemic side effects, while the negative effect of diffusion of the protein is reduced by the production at the site where action is required. The local expression of the hybrid protein m specific sub-25 populations of cells, e.g. endothelial cells prone to migrate during angiogenesis, can be enhanced using cell type-specific or tissue-specific expression vectors, in which the expression of the protein is under control of a promoter with cell type-specific or tissue-specific regulatory elements. 30 - Binding of a protease inhibitor to a cell surface receptor can locate the inhibitor close to its molecular target, the cell surface bound proteolytic enzyme. Local inhibition of the proteolytic activity in the pericellular microenvironment may be achieved in this way. 35 - Binding of a protease inhibitor to a cell surface receptor for a proteolytic enzyme, such as the urokinase Printed from Mimosa 10/29/1999 17:23:46 page -7- WO 98/51788 PCT/NL98/00259 6 receptor, may have an additional inhibitory effect. It prevents the binding of the proteolytic enzyme to its receptor, and thus strongly reduces the action of this enzyme as has been shown for blocking the binding of u-PA to its 5 receptor which can strongly inhibit cell migration.

Hybrid proteins, for which the expression vectors (e.g. adenoviral or retroviral expression vectors) contain the encoding DNA sequences, might contain a region that binds to a cell surface receptor and that is not subsequently 10 internalized. Receptor binding domains that can be used for this purpose are e.g. the u-PAR binding domain of urokinase plasminogen activator, the receptor binding domain of epidermal growth factor, the receptor associated protein (RAP) that binds to the LDL-R related protein (LRP), also 15 called a2-macroglobulin receptor, and the VLDL-receptor.

The inhibitor part of the encoded hybrid protein might consist of various protease inhibitors such as bovine pancreatic trypsin inhibitor, also called aprotmin or Trasylols, other trypsin inhibitors such as urinary trypsin 20 inhibitor, inhibitors for matrix-degrading metalloproteinases such the tissue inhibitors of metalloproteinases TIMP-1, TIMP-2 and TIMP-3, or variants thereof. Also inhibitors for other proteases like elastase are very suitable for being incorporated into the expression vector containing the DNA 25 sequences encoding the hybrid proteins. Multiple copies of the DNA sequences encoding the functional protein part of the hybrid protein e.g. the inhibitor part, or combinations of different inhibitors or derivatives thereof might be incorporated into the DNA construct in the expression vector. 30 Another very attractive possibility would be to use such an expression vector encoding hybrid receptor binding protein to apply any functional protein that should exert its action m the local environment of the target cell, e.g. a protease involved in the activation of a growth factor or an 35 other e.g. vasoregulatory component.

Printed from Mimosa 10/29/1999 17:23:46 page -8- The action of the functional protein or protein domains of the hybrid protein is localized to the direct microenvironment of the target cells by binding of the receptor binding domain to a receptor at the surface of the 5 target cells. Production of the hybrid protein in the direct environment of the target cells or even by the target cells themselves can be obtained by transfection or transduction of these cells by the use of expression vectors that might be based on a non-viral or an adeno- or retroviral vector 10 system. Expression m specific cell or tissue types might be achieved by the use of specific promoter elements in the expression vectors. For example, for endothelial cell-specific expression (elements of) the promoter region of the human or murine pre-pro-endothelm gene (HUMEDN1B and 15 MMU07982, respectively, GENBANK) can be used, for vascular smooth muscle cell-specific expression (elements of) the promoter region of the human vascular smooth muscle a-actin gene (HUMACTSA, GENBANK) can be used, and for liver-specific expression the promoter of the human albumin gene (HUMALBGC, 20 GENBANK) can be used.

Local delivery of these vectors might be obtained using various commonly used methods, including catheters, topically applied gels containing the vectors or targeted delivery systems. For site-specific delivery to the vessel 25 wall, e.g. to prevent restenosis and vessel wall remodeling after angioplasty, special catheters can be used. At the moment double balloon catheters, channeled balloon catheters, multiple needle catheters and balloon catheters coated with a vector containing a hydrogel are being used for vessel wall-30 specific delivery. Other ways to deliver the vectors directly into the vessel wall are the use of stents coated with vector containing coatings, topical application of vector containing hydrogels to the outside of the blood vessel or ex vivo delivery directly into the blood vessel during trans-35 plantation surgery. Ex vivo transduction of proliferating cells using retroviral vectors followed by a reinjection may Printed from Mimosa 10/29/1999 17:23:46 page -9- WO 98/51788 PCT/NL98/00259 also be used to deliver the vector constructs at the site where their action is required.

The present application will be described herein-5 after in further detail, while referring to the following examples. It is to be noted that these examples merely serve to illustrate the invention, not to restrict it.

EXAMPLE 1 An expression plasmid encoding the aminoterminal fragment of urokinase plasminogen activator (u-PA), amino acids 1-138, hereafter referred to as ATF, can be constructed by deleting the DNA sequences encoding amino acids 13 9 till 401 in an expression plasmid for the full length u-PA using a 15 polymerase chain reaction (PCR) with the following oligonucleotides: 5'-cccgggcttttttccatctgcgcagtc-31 and 5'-agggtcaccaaggaagagaatggc-31. After amplification by PCR the newly formed DNA fragment can be circularized by ligation to restore the circular character of the expression plasmid. 20 In this way an expression plasmid encoding the ATF and the C terminal last 11 amino acid residues including the stop codon can be constructed.

The sequence of the thus formed DNA construct encoding the u-PA ATF fragment then is determined and compared 25 with the predicted sequence as a control for possible mutations introduced during the construction procedure.

The construction pCRII-ATF from pCRII-uPA using PCR is shown in Figure 1. In figure 1, the area indicated between the lines was removed during the PCR amplification, resulting 30 in the ATF plasmid. The plasmid pCRII-uPA is shown to the left, plasmid pCRII-ATF to the right.

EXAMPLE 2 DNA fragments encoding amino acid residues 36-93 of 35 bovine pancreatic trypsin inhibitor (BPTI) and the homologous amino acid residues of bovine spleen trypsin inhibitor (BSTI) Printed from Mimosa 10/29/1999 17:23:46 page -10- 9 can be isolated by performing a PCR reaction on genomic DNA isolated for bovine aortic endothelial cells using the following oligonucleotides: 5'-_tcgcgacctgacttctgcctagagc-3' covering nucleotides 2509 to 2533 (with modifications, 5 indicated in italics, in the 5' region of the oligonucleotide to introduce a Nrul site (underlined) for cloning purposes) of the BPTI gene according to the published sequence (GENBANK, BTBPTIG), and nucleotide 2442 to 2462 of the BSTI gene according to the published sequence (GENBANK, BTBSTIG) 10 and 5 ' -cr<7tcacccaqgqcccaatattaccacc-3 ' covering nucleotides 2677 to 2704 of the BPTI gene and 2610 to 2636 of the BSTI gene (modified in the indicated nucleotides (italics) to introduce a BstEII and a Sspl site respectively (underlined)). The amplified DNA fragments then were cloned 15 into an appropriate plasmid vector, pCRII or pUC13, and then the exact sequence of the amplified DNA fragments in the isolated clones was analyzed to differentiate between BPTI and BSTI which have a very high degree of homology.

EXAMPLE 3 The DNA fragment encoding amino acids 1 to 207 of the human tissue inhibitor of metalloproteinase type 1 is isolated by performing a reverse transcriptase polymerase chain reaction on total RNA isolated from human foreskin 25 fibroblasts by using the following oligonucleotides 51-agagagacaccagagaacccaccat-31 covering nucleotides 41 to 65 of the human TIMP-1 cDNA (according to the sequence in GENBANK HSTIMPR) and 5'-tcattgtccggaagaaagatgggag-3' covering nucleotides 740 till 755. The amplified DNA fragment was 3 0 cloned into an appropriate host vector, pUC13, and then the exact sequence of the amplified DNA fragment in the isolated clones was analyzed.

EXAMPLE 4 For construction of a recombinant adenovirus containing sequences encoding the ATF.BPTI hybrid protein, Printed from Mimosa 10/29/1999 17:23:46 page -11- this sequence is cloned in the adenoviral vector construction adapter and expression plasmid pMAD5. This plasmid contains part of the wildtype adenovirus type 5 DNA sequences, a Major Late Promoter (MLP), and a poly-adenylation (polyA) signal 5 and can be used as either an expression vector or a shuttle vector to construct a recombinant adenovirus. This plasmid was derived from plasmid pMLPIO as follows. First pMLPlO-lin was constructed by insertion of a synthetic DNA fragment with unique sites for the restriction endonucleases Mlul, SplI, 10 SnaBl, Spel, AsuII and Muni into the Hindlll site of pMLPIO. Subsequently, the adenovirus Bglll fragment spanning nt 3328 to 8914 of the Ad5 genome was inserted into the Muni site of pMLPIO - lm. Finally, the Sall-BamHI fragment was deleted to inactivate the tetracycline resistance gene, resulting in 15 plasmid pMAD5. To clone the ATF.BPTI sequence into the pMAD5" plasmid between the MLP promoter and the polyA signal the following strategy has been followed.

Starting from a pCRII plasmid m which a 1373 base pair fragment of the uPA cDNA was cloned, a PCR reaction with 20 the oligonucleotides 5'-cccgggcttttttccatctgcgcagtc-31 (Smal site underlined and nucleotides changed in italics) and 5'-aqcfgtcaccaaaqaaaaaaataac-3' (BstEII site underlined and nucleotides changed m italics) was performed as described in example 1 to make a pCRII-ATF plasmid (see figure 1). 25 Subsequently this pCRII-ATF plasmid was digested with the restriction enzymes Smal and Bstell. In parallel the pCRII-BPTI plasmid was digested with the restriction enzymes Nrul and Bstell and the BPTI containing fragment was cloned into the pCRII-ATF plasmid (see figure 2). The construction pCRII-30 ATF-BPTI is shown in Fig. 2.

In a next step the ATF-BPTI sequence was cloned into pMAD5. This was done by digestion of the pCRII-ATF-BPTI plasmid with the restriction enzymes EcoRV and Spel, isolation of the ATF-BPTI encoding DNA fragment and cloning 35 of this fragment into the SnaBI and Spel digested pMAD5 Printed from Mimosa 10/29/1999 17:23:46 page -12- 11 plasmid. The cloning was tested by restriction analysis and sequence analysis.

The pMAD5-ATF-BPTI shuttle vector for the construction of ATF-BPTI adenoviral vector is shown in Figure 3.

EXAMPLE 5 In a similar way as described m example 4 for pMAD5-ATF-BPTI a plasmid containing the BSTI-gene (pMAD5-ATF-BSTI) was constructed using the pCRII-BSTI plasmid instead of 10 the pCRII-BPTI plasmid.

EXAMPLE 6 For construction of a recombinant adenovirus containing sequences encoding the ATF-TIMP1 hybrid protein, 15 this sequence is cloned in the pMAD5 expression plasmid. This plasmid contains part of the wildtype adenovirus type 5 DNA sequences, a Major Late Promoter (MLP), and a polyadenylation (polyA) signal and can be used as either an expression vector or a shuttle vector to construct a recombinant adenovirus. To 2 0 clone the ATF-TIMP1 sequence into the pMAD5 plasmid between the MLP promoter and the polyA signal, the following strategy has been followed.

Starting from a pCRII plasmid m which a 1373 base pair fragment of the uPA cDNA was cloned, a PCR reaction with 25 the oligonucleotides 51-cccgggcttttttccatctgcgcagtc-31 and 51-agggtcaccaaggaagagaatggc-3' was performed as described in example 1 to make a pCRII-ATF plasmid (see figure 1). Subsequently this pCRII-ATF plasmid was digested with the restriction enzymes Smal and Bstell.

In parallel a fragment of the cDNA of TIMP1 in pUC13-TIMP1 encoding amino acid residues 1 to 184 of the mature protein, but lacking the signal peptide and the stop codon, was amplified using the following oligonucleotides 51-tcgcgatgcacctgtgtcccacc-3' and 35 5'-qqtcacccaaatattqactatataaaaccqcaaqg-3'. These oligonucleotides contain recognition sites for the restriction Printed from Mimosa 10/29/1999 17:23:46 page -13- WO 98/51788 PCT/NL98/00259 12 enzymes Nrul (first oligonucleotide, underlined) and BstEII and Sspl respectively (second oligonucleotide, underlined); these sites are needed for the cloning procedure.

The amplified DNA fragment was cloned into a pCRII 5 vector and called pCRII-TIMPl. This vector was subsequently digested with the restriction enzymes Nrul and Bstell and the TIMP1 containing DNA fragment was cloned into the pCRII-ATF plasmid (see figure 1).

In a next step the ATF-TIMP sequence was cloned 10 into pMAD5. This was done by digestion of the pCRII-ATF-TIMP plasmid with the restriction enzymes EcoRV and Spel, isolation of the ATF-TIMP encoding DNA fragment and cloning of this fragment into the SnabI and Spel digested pMAD5 plasmid. The cloning was tested by restriction analysis and 15 sequence analysis.

For construction of a recombinant adenovirus containing sequences encoding the ATF.TIMPl hybrid protein, this sequence is cloned in the pMAD5 expression plasmid. This plasmid contains part of the wildtype adenovirus type 5 DNA 20 sequences, a Major Late Promoter (MLP), and a poly- adenylation (polyA) signal and can be used as either an expression vector or a shuttle vector to construct a recombinant adenovirus. To clone the ATF.TIMPl sequence into the pMAD5 plasmid between the MLP promoter and the polyA 25 signal the following strategy has been followed.

Starting from a pCRII plasmid in which a 1373 base pair fragment of the uPA cDNA was cloned, a PCR reaction with the oligonucleotides 5'-cccgggcttttttccatctgcgcagtc-31 and 5'-agggtcaccaaggaagagaatggc-31 was performed as described in 30 example 1 to make a pCRII-ATF plasmid (see figure 1).

Subsequently on this pCRII-ATF plasmid a PCR reaction was performed using the oligonucleotides 51-aatattattgaacttcatcaagttcc-31 and 51-gactctagagcaaaaatgacaaccag-3' and the resulting DNA 35 fragment was cloned into the pCRII cloning vector. In this way the signal peptide of u-PA is removed and a Sspl Printed from Mimosa 10/29/1999 17:23:46 page -14- / 13 restriction enzyme recognition site is introduced (underlined) . The resulting plasmid DNA is designated pCRIIATF*.

In parallel a fragment of the cDNA of TIMPl m pUC13-TIMPl encoding amino acid residues -23 to 184 of the 5 TIMP-1 protein, including the signal peptide but lacking the stop codon, was amplified using the oligonucleotides 5'-agagagacaccagagaacccaccat-31 and 51-aatattggctatctgggaccgcagg-3' containing a recognition site for the restriction enzyme Sspl (underlined) and cloned into 10 a pCRII cloning vector. The resulting plasmid DNA is designated pCRII-TIMPl*.

This vector was subsequently digested with the restriction enzymes Sspl and EcoRV and the TIMPl containing DNA fragment was cloned into a EcoRV-SspI digested pCRII-ATF* 15 plasmid. The resulting plasmid containing the TIMP-ATF DNA fragment was called pCRII-TIMP-ATF. In a next step, the TIMP-ATF sequence was cloned into pMAD5. This was done by digestion of the pCRII-TIMP-ATF plasmid with the restriction enzymes EcoRV and Spel, isolation of the TIMP-ATF encoding 20 DNA fragment and cloning of this fragment into the SnabI and Spel digested pMAD5 plasmid. The cloning was tested by restriction analysis and sequence analysis.

EXAMPLE 7 Vectors encoding hybrid proteins containing multiple copies of the BPTI unit coupled to the ATF domain have been constructed. To construct these multiple BPTI vectors, the following strategy is followed.

The pMAD5-ATF-BPTI described in EXAMPLE 4 is 30 digested with the restriction enzymes Sspl and BstEII. In this way the vector is opened exactly in the open reading frame at the end of the BPTI sequence. The pCRII-BPTI plasmid described in EXAMPLE 2 is digested with Nrul and BstEII resulting in a BPTI encoding DNA fragment with one blunt end 35 (Nrul). The fragment was then monodirectionally cloned into the Sspl BstEII pMAD5-ATF-BPTI vector. The thus constructed Printed from Mimosa 10/29/1999 17:23:46 page -15- 14 plasmid named pMAD5-ATF-BPTI-BPTI was used as a shuttle vector for the construction of recombinant adenoviruses.

This approach can be repeated multiple times to construct vectors containing multiple BPTI-domams.

EXAMPLE 8 A vector encoding a hybrid protein containing both a BPTI unit and a TIMPl unit coupled to the ATF domain has been constructed. To construct this BPTI-TIMP vector, the 10 following strategy is followed.

The pMAD5-ATF-BPTI described in EXAMPLE 4 is digested with the restriction enzymes Sspl and BstEII. In this way the vector is opened right behind the BPTI sequence. The pCRII-TIMP plasmid described m EXAMPLE 6 is digested 15 with Nrul and BstEII resulting in a TIMPl encoding DNA fragment with one blunt end. The fragment was then cloned into the Sspl BstEII pMAD5-ATF-BPTI vector. The thus constructed plasmid named pMAD5-ATF-BPTI-TIMP was used as a shuttle vector for the construction of recombinant adenoviruses.

EXAMPLE 9 To monitor the production of a functional ATF-BPTI hybrid protein after transfection of cells with pMAD5 or transduction with a recombinant replication-deficient ATF-25 BPTI encoding adenovirus, the following tests have been performed.

The production of the hybrid ATF-BPTI protein by CHO cells transfected with the pMAD5-ATF-BPTI was analyzed using a uPA ELISA that recognizes the ATF, the aminoterminal 3 0 fragment of u-PA. Production of ATF-BPTI was clearly detectable both after transient transfection of CHO cells with the pMAD5-ATF-BPTI plasmid (50-100 ng/ml/24hrs) and after transduction with an ATF-BPTI encoding adenoviral vector (up to 1.5 ng/ml/24hrs).

- The cell culture media of CHO cells transduced with the ATF-BPTI adenovirus were analyzed using western blotting Printed from Mimosa 10/29/1999 17:23:46 page -16- techniques. After electrophoresis and blotting, parallel filters were analyzed with polyclonal antibodies against either u-PA or BPTI (raised against Trasylol®) . In both filters a signal was detected at the same expected position 5 at approximately 20kDa. This indicates that the protein produced indeed contains fragments of u-PA and BPTI, thus that the hybrid protein is produced.

The function as an inhibitor of plasmin activity of the ATF-BPTI protein was first analyzed in solution using 10 dilutions of the culture medium of ATF-BPTI virus infected CHO cells (approximately 1.8 ^g/ml). They were incubated with plasmin (1 nM) and the activity of plasmin was measured using a chromogenic substrate. Trasylol" dilutions were used as control references. Plasmin inhibition by ATF-BPTI medium was 15 very effective, diluting the medium lOOOx (i.e. 100 nM ATF-BPTI) resulted in a 50% inhibition of the activity of 1 nM plasmin, a similar inhibition as was observed with 100 nM Trasylol®. Thus the activity of ATF-BPTI is comparable to that of commercially available Trasylol" (Bayer, Germany). 20 - The function of ATF-BPTI as an inhibitor for plasmin bound to the cell surface via the interaction of the ATF domain with the u-PA receptor (uPAR) was tested using mouse cell lines that are either or not transfected with the human uPA receptor gene. These cells were incubated for 6 hrs 25 with diluted medium of the ATF-BPTI virus-infected CHO cells. Cell extracts were made of the uPAR-transfected cells and the parental mouse cells lacking the human uPAR. Parallel cultures underwent a short acid treatment (pH 3, 3 min) before the cell extracts were made. This treatment will 30 remove any u-PA or ATF bound to the u-PA receptor. The cell extracts were incubated with InM plasmin and the plasmin activity was determined. Plasmin activity could only be inhibited by the cell extract of the u-PAR containing cell line. No inhibition of plasmin activity was observed in the 35 cell extracts of parental cell line, lacking the u-PA receptor, and in the acid-treated u-PAR containing cell line.

Printed from Mimosa 10/29/1999 17:23:46 page -17- 16 This clearly indicates that ATF-BPTI can function as a u-PAR bound plasmin inhibitor.

TABLE 1 % INHIBITION OF PLASMIN ACTIVITY cell line uPAR transfected cell line parental cell line acid treatment - + - + % inhibition 93% 0% 0% 0% EXAMPLE 10 Cell-specific expression of ATF-BPTI in endothelial cells e.g. to specifically inhibit the migration of endothelial cells during angiogenesis, is achieved by cloning sequences of the promoter of the human pre-pro-endothelin 1 gene (nucleotide 2180-3680 of HUMEDN1B (GENBANK)) in front of the ATF-BPTI encoding DNA in an adenoviral vector. In this way, highly endothelial cell-specific expression of the ATF-BPTI hybrid protein can be obtained.

EXAMPLE 11 Proteolytic degradation of the extracellular matrix is a key event in many cell migration and tissue remodeling processes. This proteolytic matrix degradation is often found to be mediated by urokinase-type plasminogen activation. In order to test whether infection with an ATF-BPTI encoding adenovirus can inhibit plasmin mediated extracellular matrix degradation, an experiment was performed using human synoviocytes. These cells were infected with an ATF-BPTI adenovirus while they were seeded on an 3H-labeled extracellular matrix existing of bovine cartilage material. Profound inhibition of matrix degradation could be observed in the virus treated cells (figure 4) indicating that matrix Printed from Mimosa 10/29/1999 17:23:46 page -18- degradation can be inhibited by infecting cells with the ATF-BPTI encoding virus.

Figure 4 shows the degradation of cartilage matrix by human synoviocytes in the presence of plasminogen. Matrix 5 is incubated with control medium (lane 1), synoviocytes (lane 2), synoviocytes infected with ATF-BPTI adenovirus (lane 3), and synoviocytes incubated with TrasylolB (lOOKIU/ml) (lane 4).

EXAMPLE 12 In the process of restenosis smooth muscle cell migration and vessel wall remodeling are key events in which plasmin mediated proteolysis of extracellular matrix components is involved. In vivo application of general plasmin inhibitors to interfere in this process may have 15 systemic side effects. Application of a plasmin inhibitor to the surface of the migrating cells might prevent these side effects. Infection of the blood vessel wall with an ATF-BPTI adenovirus at a site where neointima formation can be expected, e.g. in a transplanted "coronary by-pass" graft, 20 might be a ideal way to produce the ATF-BPTI locally, and thus inhibit plasmin activity in the direct surroundings of the migrating (smooth muscle) cells, resulting in a reduced neointima formation.

This principle was tested using human saphenous 25 vein organ cultures, a model system in which neointima formation can be mimicked very realistically. In parallel cultures, either or not infected with an ATF-BPTI adenovirus, the neointima formation was analyzed after three and four weeks. In the untreated tissues a clear neointima formation 3 0 could be observed. Profound inhibition of the neointima formation could be observed in the tissues treated with 1010 pfu/ml ATF-BPTI adenovirus.

Printed from Mimosa 10/29/1999 17:23:46 page -19- 18 Appendix Description and Nucleotide sequence of the pMAD5-ATF-BPTI plasmid.

From To Description 1 184 adenovirus sequence 51 184 447 adenovirus Major Late Promoter (MLP) 447 644 tripartite leader sequence (TPL) 685 1167 urokinase ATF sequence 1168 1353 bovine prancreas trypsin inhibitor sequence 1360 1443 urokinase 3' sequence (including stop codon) 1514 1615 sequence derived form pSP65 and LacZ 1616 1751 SV4 0 poly A signal 1752 7334 adenovirus sequence 3' 9831 8971 p-lactamase 2 0 Nucleotide sequence: l CATTTTCGCG GGAAAACTGA ATAAGAGGAA GTGAAATCTG AATAATTTTG TGTTACTCAT 61 AGCGCGTAAT ATTTGTCTAG GGCCGCGGGG ACTTTGACCG TTTACGTGGA GACTCGCCCA 121 GGTGTTTTTC TCAGGTGTTT TCCGCGTTCC GGGTCAAAGT TGGCGTTTTA TTATTATAGT 181 CAGCTGATCG AGCGGTGTTC CGCGGTCCTC CTCGTATAGA AACTCGGACC ACTCTGAGAC 241 GAAGGCTCGC GTCCAGGCCA GCACGAAGGA GGCTAAGTGG GAGGGGTAGC GGTCGTTGTC 301 CACTAGGGGG TCCACTCGCT CCAGGGTGTG AAGACACATG TCGCCCTCTT CGGCATCAAG 361 GAAGGTGATT GGTTTATAGG TGTAGGCCAC GTGACCGGGT GTTCCTGAAG GGGGGCTATA 421 AAAGGGGGTG GGGGCGCGTT CGTCCTCACT CTCTTCCGCA TCGCTGTCTG CGAGGGCCAG 481 CTGTTGGGGC TCGCGGTTGA GGACAAACTC TTCGCGGTCT TTCCAGTACT CTTGGATCGG 541 AAACCCGTCG GCCTCCGAAC GGTACTCCGC CACCGAGGGA CCTGAGCGAG TCCGCATCGA 601 CCGGATCGGA AAACCTCTCG AGAAAGGCGT CTAACCAGTC GCTGATCGAT AAGCTAGCTT 661 ACGCGTACAT CTGCAGAATT CGGCTTAACT CTAGACCATG AGAGCCCTGC TGGCGCGCCT 721 GCTTCTCTGC GTCCTGGTCG TGAGCGACTC CAAAGGCAGC AATGAACTTC ATCAAGTTCC 781 ATCGAACTGT GACTGTCTAA ATGGAGGAAC ATGTGTGTCC AACAAGTACT TCTCCAACAT 841 TCACTGGTGC AACTGCCCAA AGAAATTCGG AGGGCAGCAC TGTGAAATAG ATAAGTCAAA Printed from Mimosa 10/29/1999 17:23:46 page -20- 19 901 AACCTGCTAT GAGGGGAATG GTCACTTTTA CCGAGGAAAG GCCAGCACTG ACACCATGGG 961 CCGGCCCTGC CTGCCCTGGA ACTCTGCCAC TGTCCTTCAG CAAACGTACC ATGCCCACAG 1021 ATCTGATGCT CTTCAGCTGG GCCTGGGGAA ACATAATTAC TGCAGGAACC CAGACAACCG 1081 GAGGCGACCC TGGTGCTATG TGCAGGTGGG CCTAAAGCCG CTTGTCCAAG AGTGCATGGT 1141 GCATGACTGC GCAGATGGAA AAAAGCCCCG ACCTGACTTC TGCCTAGAGC CTCCATATAC 1201 GGGTCCCTGC AAGGCCAGAA TTATCAGATA CTTCTACAAC GCCAAGGCTG GGCTCTGCCA 1261 GACCTTTGTA TATGGCGGCT GCAGAGCTAA AAGAAACAAT TTCAAGAGCG CAGAGGACTG 1321 CATGAGGACC TGTGGTGGTA ATATTGGGCC CTGGGTCACC AAGGAAGAGA ATGGCCTGGC 1381 CCTCTGAGGG TCCCCAGGGA GGAAACGGGC ACCACCCGCT TTCTTGCTGG TTGTCATTTT 1441 TGCTCTAGAG TCAAGCCGAA TTCTGCAGAT ATCGTCCATT CCGACAGCAT CGCCAGTCAC 1501 TATGGCGTGC TGCTAGAGGA TCCCCGGGCG AGCTCGAATT CCAGCTGAGC GCCGGTCGCT 1561 ACCATTACCA GTTGGTCTGG TGTCAAAAAT AATAATAACC GGGCAGGGGG GATTCTGAAC 1621 TTGTTTATTG CAGCTTATAA TGGTTACAAA TAAAGCAATA GCATCACAAA TTTCACAAAT 1681 AAAGCATTTT TTTCACTGCA TTCTAGTTGT GGTTTGTCCA AACTCATCAA TGTATCTTAT 1741 CATGTCTGGA TCTGGAAGGT GCTGAGGTAC GATGAGACCC GCACCAGGTG CAGACCCTGC 1801 GAGTGTGGCG GTAAACATAT TAGGAACCAG CCTGTGATGC TGGATGTGAC CGAGGAGCTG 1861 AGGCCCGATC ACTTGGTGCT GGCCTGCACC CGCGCTGAGT TTGGCTCTAG CGATGAAGAT 1921 ACAGATTGAG GTACTGAAAT GTGTGGGCGT GGCTTAAGGG TGGGAAAGAA TATATAAGGT 1981 GGGGGTCTTA TGTAGTTTTG TATCTGTTTT GCAGCAGCCG CCGCCGCCAT GAGCACCAAC 2041 TCGTTTGATG GAAGCATTGT GAGCTCATAT TTGACAACGC GCATGCCCCC ATGGGCCGGG 2101 GTGCGTCAGA ATGTGATGGG CTCCAGCATT GATGGTCGCC CCGTCCTGCC CGCAAACTCT 2161 ACTACCTTGA CCTACGAGAC CGTGTCTGGA ACGCCGTTGG AGACTGCAGC CTCCGCCGCC 2221 GCTTCAGCCG CTGCAGCCAC CGCCCGCGGG ATTGTGACTG ACTTTGCTTT CCTGAGCCCG 2281 CTTGCAAGCA GTGCAGCTTC CCGTTCATCC GCCCGCGATG ACAAGTTGAC GGCTCTTTTG 2341 GCACAATTGG ATTCTTTGAC CCGGGAACTT AATGTCGTTT CTCAGCAGCT GTTGGATCTG 2401 CGCCAGCAGG TTTCTGCCCT GAAGGCTTCC TCCCCTCCCA ATGCGGTTTA AAACATAAAT 2461 AAAAAACCAG ACTCTGTTTG GATTTGGATC AAGCAAGTGT CTTGCTGTCT TTATTTAGGG 2521 GTTTTGCGCG CGCGGTAGGC CCGGGACCAG CGGTCTCGGT CGTTGAGGGT CCTGTGTATT 2581 TTTTCCAGGA CGTGGTAAAG GTGACTCTGG ATGTTCAGAT ACATGGGCAT AAGCCCGTCT 2641 CTGGGGTGGA GGTAGCACCA CTGCAGAGCT TCATGCTGCG GGGTGGTGTT GTAGATGATC 2701 CAGTCGTAGC AGGAGCGCTG GGCGTGGTGC CTAAAAATGT CTTTCAGTAG CAAGCTGATT 2761 GCCAGGGGCA GGCCCTTGGT GTAAGTGTTT ACAAAGCGGT TAAGCTGGGA TGGGTGCATA 2821 CGTGGGGATA TGAGATGCAT CTTGGACTGT ATTTTTAGGT TGGCTATGTT CCCAGCCATA 2881 TCCCTCCGGG GATTCATGTT GTGCAGAACC ACCAGCACAG TGTATCCGGT GCACTTGGGA 2941 AATTTGTCAT GTAG CTTAGA AGGAAATGCG TGGAAGAACT TGGAGACGCC CTTGTGACCT 3001 CCAAGATTTT CCATGCATTC GTCCATAATG ATGGCAATGG GCCCACGGGC GGCGGCCTGG Printed from Mimosa 10/29/1999 17:23:46 page -21- 3061 GCGAAGATAT TTCTGGGATC ACTAACGTCA TAGTTGTGTT CCAGGATGAG ATCGTCATAG 3121 GCCATTTTTA CAAAGCGCGG GCGGAGGGTG CCAGACTGCG GTATAATGGT TCCATCCGGC 3181 CCAGGGGCGT AGTTACCCTC ACAGATTTGC ATTTCCCACG CTTTGAGTTC AGATGGGGGG 3241 ATCATGTCTA CCTGCGGGGC GATGAAGAAA ACGGTTTCCG GGGTAGGGGA GATCAGCTGG 3301 GAAGAAAGCA GGTTCCTGAG CAGCTGCGAC TTACCGCAGC CGGTGGGCCC GTAAATCACA 3361 CCTATTACCG GGTGCAACTG GTAGTTAAGA GAGCTGCAGC TGCCGTCATC CCTGAGCAGG 3421 GGGGCCACTT CGTTAAGCAT GTCCCTGACT CGCATGTTTT CCCTGACCAA ATCCGCCAGA 3481 AGGCGCTCGC CGCCCAGCGA TAGCAGTTCT TGCAAGGAAG CAAAGTTTTT CAACGGTTTG 3541 AGACCGTCCG CCGTAGGCAT GCTTTTGAGC GTTTGACCAA GCAGTTCCAG GCGGTCCCAC 3601 AGCTCGGTCA CCTGCTCTAC GGCATCTCGA TCCAGCATAT CTCCTCGTTT CGCGGGTTGG 3661 GGCGGCTTTC GCTGTACGGC AGTAGTCGGT GCTCGTCCAG ACGGGCCAGG GTCATGTCTT 3721 TCCACGGGCG CAGGGTCCTC GTCAGCGTAG TCTGGGTCAC GGTGAAGGGG TGCGCTCCGG 3781 GCTGCGCGCT GGCCAGGGTG CGCTTGAGGC TGGTCCTGCT GGTGCTGAAG CGCTGCCGGT 3841 CTTCGCCCTG CGCGTCGGCC AGGTAGCATT TGACCATGGT GTCATAGTCC AGCCCCTCCG 3901 CGGCGTGGCC CTTGGCGCGC AGCTTGCCCT TGGAGGAGGC GCCGCACGAG GGGCAGTGCA 3961 GACTTTTGAG GGCGTAGAGC TTGGGCGCGA GAAATACCGA TTCCGGGGAG TAGGCATCCG 4021 CGCCGCAGGC CCCGCAGACG GTCTCGCATT CCACGAGCCA GGTGAGCTCT GGCCGTTCGG 4081 GGTCAAAAAC CAGGTTTCCC CCATGCTTTT TGATGCGTTT CTTACCTCTG GTTTCCATGA 4141 GCCGGTGTCC ACGCTCGGTG ACGAAAAGGC TGTCCGTGTC CCCGTATACA GACTTGAGAG 4201 GCCTGTCCTC GAGCGGTGTT CCGCGGTCCT CCTCGTATAG AAACTCGGAC CACTCTGAGA 4261 CAAAGGCTCG CGTCCAGGCC AGCACGAAGG AGGCTAAGTG GGAGGGGTAG CGGTCGTTGT 4321 CCACTAGGGG GTCCACTCGC TCCAGGGTGT GAAGACACAT GTCGCCCTCT TCGGCATCAA 4381 GGAAGGTGAT TGGTTTGTAG GTGTAGGCCA CGTGACCGGG TGTTCCTGAA GGGGGGCTAT 4441 AAAAGGGGGT GGGGGCGCGT TCGTCCTCAC TCTCTTCCGC ATCGCTGTCT GCGAGGGCCA 4501 GCTGTTGGGG TGAGTACTCC CTCTGAAAAG CGGGCATGAC TTCTGCGCTA AGATTGTCAG 4561 TTTCCAAAAA CGAGGAGGAT TTGATATTCA CCTGGCCCGC GGTGATGCCT TTGAGGGTGG 4621 CCGCATCCAT CTGGTCAGAA AAGACAATCT TTTTGTTGTC AAGCTTGGTG GCAAACGACC 4681 CGTAGAGGGC GTTGGACAGC AACTTGGCGA TGGAGCGCAG GGTTTGGTTT TTGTCGCGAT 4741 CGGCGCGCTC CTTGGCCGCG ATGTTTAGCT GCACGTATTC GCGCGCAACG CACCGCCATT 4801 CGGGAAAGAC GGTGGTGCGC TCGTCGGGCA CCAGGTGCAC GCGCCAACCG CGGTTGTGCA 4861 GGGTGACAAG GTCAACGCTG GTGGCTACCT CTCCGCGTAG GCGCTCGTTG GTCCAGCAGA 4921 GGCGGCCGCC CTTGCGCGAG CAGAATGGCG GTAGGGGGTC TAGCTGCGTC TCGTCCGGGG 4981 GGTCTGCGTC CACGGTAAAG ACCCCGGGCA GCAGGCGCGC GTCGAAGTAG TCTATCTTGC 5041 ATCCTTGCAA GTCTAGCGCC TGCTGCCATG CGCGGGCGGC AAGCGCGCGC TCGTATGGGT 5101 TGAGTGGGGG ACCCCATGGC ATGGGGTGGG TGAGCGCGGA GGCGTACATG CCGCAAATGT 5161 CGTAAACGTA GAGGGGCTCT CTGAGTATTC CAAGATATGT AGGGTAGCAT CTTCCACCGC Printed from Mimosa 10/29/1999 17:23:46 page -22- 21 5221 GGATGCTGGC GCGCACGTAA TCGTATAGTT CGTGCGAGGG AGCGAGGAGG TCGGGACCGA 5281 GGTTGCTACG GGCGGGCTGC TCTGCTCGGA AGACTATCTG CCTGAAGATG GCATGTGAGT 5341 TGGATGATAT GGTTGGACGC TGGAAGACGT TGAAGCTGGC GTCTGTGAGA CCTACCGCGT 5401 CACGCACGAA GGAGGCGTAG GAGTCGCGCA GCTTGTTGAC CAGCTCGGCG GTGACCTGCA 5461 CGTCTAGGGC GCAGTAGTCC AGGGTTTCCT TGATGATGTC ATACTTATCC TGTCCCTTTT 5521 TTTTCCACAG CTCGCGGTTG AGGACAAACT CTTCGCGGTC TTTCCAGTAC TCTTGGATCG 5581 GAAACCCGTC GGCCTCCGAA CGGTAAGAGC CTAGCATGTA GAACTGGTTG ACGGCCTGGT 5641 AGG CGCAGCA TCCCTTTTCT ACGGGTAGCG CGTATGCCTG CGCGGCCTTC CGGAGCGAGG 5701 TGTGGGTGAG CGCAAAGGTG TCCCTGACCA TGACTTTGAG GTACTGGTAT TTGAAGTCAG 5761 TGTCGTCGCA TCCGCCCTGC TCCCAGAGCA AAAAGTCCGT GCGCTTTTTG GAACGCGGAT 5821 TTGGCAGGGC GAAGGTGACA TCGTTGAAGA GTATCTTTCC CGCGCGAGGC ATAAAGTTGC 5881 GTGTGATGCG GAAGGGTCCC GGCACCTCGG AACGGTTGTT AATTACCTGG GCGGCGAGCA 5941 CGATCTCGTC AAAGCCGTTG ATGTTGTGGC CCACAATGTA AAGTTCCAAG AAGCGCGGGA 6001 TGCCCTTGAT GGAAGGCAAT TTTTTAAGTT CCTCGTAGGT GAGCTCTTCA GGGGAGCTGA 6061 GCCCGTGCTC TGAAAGGGCC CAGTCTGCAA GATGAGGGTT GGAAGCGACG AATGAGCTCC 6121 ACAGGTCACG GGCCATTAGC ATTTGCAGGT GGTCGCGAAA GGTCCTAAAC TGGCGACCTA 6181 TGGCCATTTT TTCTGGGGTG ATGCAGTAGA AGGTAAGCGG GTCTTGTTCC CAGCGGTCCC 6241 ATCCAAGGTT CGCGGCTAGG TCTCGCGCGG CAGTCACTAG AGGCTCATCT CCGCCGAACT 6301 TCATGACCAG CATGAAGGGC ACGAGCTGCT TCCCAAAGGC CCCCATCCAA GTATAGGTCT 6361 CTACATCGTA GGTGACAAAG AGACGCTCGG TGCGAGGATG CGAGCCGATC GGGAAGAACT 6421 GGATCTCCCG CCACCAATTG GAGGAGTGGC TATTGATGTG GTGAAAGTAG AAGTCCCTGC 6481 GACGGGCCGA ACACTCGTGC TGGCTTTTGT AAAAACGTGC GCAGTACTGG CAGCGGTGCA 6541 CGGGCTGTAC ATCCTGCACG AGGTTGACCT GACGACCGCG CACAAGGAAG CAGAGTGGGA 6601 ATTTGAGCCC CTCGCCTGGC GGGTTTGGCT GGTGGTCTTC TACTTCGGCT GCTTGTCCTT 6661 GACCGTCTGG CTGCTCGAGG GGAGTTACGG TGGATCGGAC CACCACGCCG CGCGAGCCCA 6721 AAGTCCAGAT GTCCGCGCGC GGCGGTCGGA GCTTGATGAC AACATCGCGC AGATGGGAGC 6781 TGTCCATGGT CTGGAGCTCC CGCGGCGTCA GGTCAGGCGG GAGCTCCTGC AGGTTTACCT 6841 CGCATAGACG GGTCAGGGCG CGGGCTAGAT CCAGGTGATA CCTAATTTCC AGGGGCTGGT 6901 TGGTGGCGGC GTCGATGGCT TGCAAGAGGC CGCATCCCCG CGGCGCGACT ACGGTACCGC 6961 GCGGCGGGCG GTGGGCCGCG GGGGTGTCCT TGGATGATGC ATCTAAAAGC GGTGACGCGG 7021 GCGAGCCCCC GGAGGTAGGG GGGGCTCCGG ACCCGCCGGG AGAGGGGGCA GGGGCACGTC 7081 GGCGCCGCGC GCGGGCAGGA GCTGGTGCTG CGCGCGTAGG TTGCTGGCGA ACGCGACGAC 7141 GCGGCGGTTG ATCTCCTGAA TCTGGCGCCT CTGCGTGAAG ACGACGGGCC CGGTGAGCTT 7201 GAGCCTGAAA GAGAGTTCGA CAGAATCAAT TTCGGTGTCG TTGACGGCGG CCTGGCGCAA 7261 AATCTCCTGC ACGTCTCCTG AGTTGTCTTG ATAGGCGATC TCGGCCATGA ACTGCTCGAT 7321 CTCTTCCTCC TGGAGATCAA TTGAAGCTAG CTTTAATGCG GTAGTTTATC ACAGTTAAAT Printed from Mimosa 10/29/1999 17:23:46 page -23- 22 7381 TGCTAACGCA GTCAGGCACC GTGTATGAAA TCTAACAATG CGCTCATCGT CATCCTCGGC 7441 ACCGTCACCC TGGATGCTGT AGGCATAGGC TTGGTTATGC CGGTACTGCC GGGCCTCTTG 7501 CGGGATATCG TCCATTCCGA CAGCATCGCC AGTCACTATG GCGTGCTGCT AGCGCTATAT 7561 GCGTTGATGC AATTTCTATG CGCACCCGTT CTCGGAGCAC TGTCCGACCG CTTTGGCCGC 7621 CGCCCAGTCC TGCTCGCTTC GCTACTTGGA GCCACTATCG ACTACGCGAT CATGGCGACC 7681 ACACCCGTCC TGTGGATCTC GACCGATGCC CTTGAGAGCC TTCAACCCAG TCAGCTCCTT 7741 CCGGTGGGCG CGGGGCATGA CTATCGTCGC CGCACTTATG ACTGTCTTCT TTATCATGCA 7801 ACTCGTAGGA CAGGTGCCGG CAGCGCTCTG GGTCATTTTC GGCGAGGACC GCTTTCGCTG 7861 GAGCGCGACG ATGATCGGCC TGTCGCTTGC GGTATTCGGA ATCTTGCACG CCCTCGCTCA 7921 AGCCTTCGTC ACTGGTCCCG CCACCAAACG TTTCGGCGAG AAGCAGGCCA TTATCGCCGG 7981 CATGGCGGCC GACGCGCTGG GCTACGTCTT GCTGGCGTTC GCGACGCGAG GCTGGATGGC 8041 CTTCCCCATT ATGATTCTTC TCGCTTCCGG CGGCATCGGG ATGCCCGCGT TGCAGGCCAT 8101 GCTGTCCAGG CAGGTAGATG ACGACCATCA GGGACAGCTT CAAGGATCGC TCGCGGCTCT 8161 TACCAGCCCA GCAAAAGGCC AGGAACCGTA AAAAGGCCGC GTTGCTGGCG TTTTTCCATA 8221 GGCTCCGCCC CCCTGACGAG CATCACAAAA ATCGACGCTC AAGTCAGAGG TGGCGAAACC 8281 CGACAGGACT ATAAAGATAC CAGGCGTTTC CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG 8341 TTCCGACCCT GCCGCTTACC GGATACCTGT CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC 8401 TTTCTCATAG CTCACGCTGT AGGTATCTCA GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG 8461 GCTGTGTGCA CGAACCCCCC GTTCAGCCCG ACCGCTGCGC CTTATCCGGT AACTATCGTC 8521 TTGAGTCCAA CCCGGTAAGA CACGACTTAT CGCCACTGGC AGCAGCCACT GGTAACAGGA 8581 TTAGCAGAGC GAGGTATGTA GGCGGTGCTA CAGAGTTCTT GAAGTGGTGG CCTAACTACG 8641 GCTACACTAG AAGGACAGTA TTTGGTATCT GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA 8701 AAAGAGTTGG TAGCTCTTGA TCCGGCAAAC AAACCACCGC TGGTAGCGGT GGTTTTTTTG 8761 TTTGCAAGCA GCAGATTACG CGCAGAAAAA AAGGATCTCA AGAAGATCCT TTGATCTTTT 8821 CTACGGGGTC TGACGCTCAG TGGAACGAAA ACTCACGTTA AGGGATTTTG GTCATGAGAT 8881 TATCAAAAAG GATCTTCACC TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCT 8941 AAAGTATATA TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA 9001 TCTCAGCGAT CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC GTGTAGATAA 9061 CTACGATACG GGAGGGCTTA CCATCTGGCC CCAGTGCTGC AATGATACCG CGAGACCCAC 9121 GCTCACCGGC TCCAGATTTA TCAGCAATAA ACCAGCCAGC CGGAAGGGCC GAGCGCAGAA 9181 GTGGTCCTGC AACTTTATCC GCCTCCATCC AGTCTATTAA TTGTTGCCGG GAAGCTAGAG 9241 TAAGTAGTTC GCCAGTTAAT AGTTTGCGCA ACGTTGTTGC CATTGCTGCA GGCATCGTGG 9301 TGTCACGCTC GTCGTTTGGT ATGGCTTCAT TCAGCTCCGG TTCCCAACGA TCAAGGCGAG 9361 TTACATGATC CCCCATGTTG TGCAAAAAAG CGGTTAGCTC CTTCGGTCCT CCGATCGTTG 9421 TCAGAAGTAA GTTGGCCGCA GTGTTATCAC TCATGGTTAT GGCAGCACTG CATAATTCTC 9481 TTACTGTCAT GCCATCCGTA AGATGCTTTT CTGTGACTGG TGAGTACTCA ACCAAGTCAT Printed from Mimosa 10/29/1999 17:23:46 page -24- 23 9541 TCTGAGAATA GTGTATGCGG CGACCGAGTT GCTCTTGCCC GGCGTCAACA CGGGATAATA 9601 CCGCGCCACA TAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCT TCGGGGCGAA 9661 AACTCTCAAG GATCTTACCG CTGTTGAGAT CCAGTTCGAT GTAACCCACT CGTGCACCCA 9721 ACTGATCTTC AGCATCTTTT ACTTTCACCA GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC 9781 AAAATGCCGC AAAAAAGGGA ATAAGGGCGA CACGGAAATG TTGAATACTC ATACTCTTCC 9841 TTTTTCAATA TTATTGAAGC ATTTATCAGG GTTATTGTCT CATGAGCGGA TACATATTTG 9901 AATGTATTTA GAAAAATAAA CAAATAGGGG TTCCGCGCAC ATTTCCCCGA AAAGTGCCAC 9961 CTGACGTCTA AGAAACCATT ATTATCATGA CATTAACCTA TAAAAATAGG CGTATCACGA 10021 GGCCCTTTCG TCTTCAAGAA TTCTCATGTT TGACAGCTTA TCATCATCAA TAATATACCT 10081 TATTTTGGAT TGAAGCCAAT ATGATAATGA GGGGGTGGAG TTTGTGACGT GGCGCGGGGC 10141 GTGGGAACGG GGCGGGTGAC GTAGTAGTGT GGCGGAAGTG TGATGTTGCA AGTGTGGCGG 10201 AACACATGTA AGCGACGGAT GTGGCAAAAG TGACGTTTTT GGTGTGCGCC GGTGTACACA 10261 GGAAGTGACA ATTTTCGCGC GGTTTTAGGC GGATGTTGTA GTAAATTTGG GCGTAACCGA 10321 GTAAGATTTG GC Printed from Mimosa 10/29/1999 17:23:46 page -25- 1. A recombinant nucleic acid molecule comprising a vector useful for transfection or transduction of mammalian, e.g. human, cells, wherein said vector contains a nucleic acid insertion encoding an expressible hybrid polypeptide or protein which comprises a domain with a binding function and a domain with an effector function, wherein the domain with a binding function is a cell surface receptor binding domain. 2. A recombinant nucleic acid molecule according to

Claims (1)

Claim 1, wherein said receptor binding domain is selected from the group consisting of urokinase receptor binding domain of urokinase, receptor binding domain of epidermal growth factor, receptor associated protein that binds to LDL 15 Receptor related protein (a2-macroglobulin receptor) and VLDL Receptor. 3. A recombinant nucleic acid molecule according to Claim 1, wherein said receptor binding domain comprises the aminotermmal part of urokinase which is capable of binding 2 0 to the urokinase receptor. 4. A recombinant nucleic acid molecule according to Claim 1, wherein said receptor binding domain comprises amino acid residues 1 through 135 of urokinase. 5. A recombinant nucleic acid molecule according to 25 Claim 1, wherein said domain with an effector function is an enzymatically active domain. 6. A recombinant nucleic acid molecule according to Claim 1, wherein said domain with an effector function has protease inhibitor activity. 30 7. A recombinant nucleic acid molecule_according to Claim 6, wherein said domain having protease~EainffiJbi£!^^7Y o:fiCE of n.z. activity comprises a protease inhibitor ot active part I 1 II flOV o ir ^ o -* i nn n ^ L L . 1 vyl Lj ^ l/' J WO 98/51788 PCT/NL98/00259 25 k f| f ^ /a JU wy.jp thereof, said protease inhibitor being selected from the group consisting of (bovine) pancreatic trypsin inhibitor, (bovine) splenic trypsin inhibitor, urinary trypsin inhibitor, tissue inhibitor of matrix metalloproteinase 1, 5 tissue inhibitor of matrix metalloproteinase 2, tissue inhibitor of matrix metalloproteinase 3, and elastase inhibitor. 8. a recombinant nucleic acid molecule according to Claim 6, wherein said domain having protease inhibitor 10 activity comprises (amino acid residues 53 through 94 of) mature bovine pancreatic trypsin inhibitor. 9. A recombinant nucleic acid molecule according to Claim 6, wherein said domain having protease inhibitor activity comprises bovine splenic trypsin inhibitor. 15 10. A recombinant nucleic acid molecule according to Claim 6, wherein said domain having protease inhibitor activity comprises a tissue inhibitor of matrix metalloproteinases . 11. A recombinant nucleic acid molecule according to 20 Claim 1, wherein said domain with an effector function comprises two or more different protease inhibitors or parts thereof having protease inhibiting activity, or two or more copies of a protease inhibitor or a part thereof having protease inhibiting activity or both. 12. A recombinant nucleic acid molecule according to 25 Claim 1, wherein said vector is selected from the group consisting of viral and non-viral vectors useful for transfection or transduction of mammalian cells. 13. A recombinant nucleic acid molecule according to Claim 1, wherein said vector is an adenovirus vector or a 3 0 retrovirus vector useful for transfection or transduction of human cells. 14. a recombinant nucleic acid molecule according to Claim 1, wherein said vector is an adenovirus vector based on shuttle vector pMAD5. 35 15. A recombinant nucleic acid molecule^according to
1. Claim 1, wherein said nucleic acid insert ion E'encddiihrg)Pa'nv jj OFF.CE OF N.Z. j 1 0 foOV UZ1 1 WO 98/51788 PCT/NL98/00259 expressible hybrid polypeptide or protein is under the control of a cell- or tissue-specific promoter. 16. A recombinant nucleic acid molecule according to Claim 1, wherein said nucleic acid insertion encoding an 5 expressible hybrid polypeptide or protein is under the control of an endothelial cell-specific promoter, or a vascular smooth muscle cell-specific promoter, or a liver-specific promoter. 17. A recombinant nucleic acid molecule comprising a vector 10 useful for transfection or cransduction of mammalian, e.g. human, cells, wherein said vector contains a nucleic acid insertion encoding an expressible hybrid polypepcide or protein which comprises a receptor binding domain selected from the group consisting of urokinase recepcor binding domain of urokinase, 15 receptor binding domain of epidermal growth factor, receptor associated protein that binds to LDL Receptor related protein (a2-macroglobulin receptor) and VLDL Receptor, and a domain with protease inhibitor activity which comprises a protease inhibitor or active part thereof, said protease inhibitor being selected 2 0 from the group consisting of (bovine) pancreatic trypsin inhibitor, (bovine) splenic trypsin inhibitor, urinary trypsin inhibitor, tissue inhibitor of matrix metalloproteinase 1, tissue inhibitor of matrix metalloproteinase 2, tissue inhibitor of matrix metalloproteinase 3, and elastase inhibitor. 25 If 'TELLiCl JAL PROPERTY O.TiCc OF N.Z. 1 u hoy 2C23 50.0 intellectual property office of n.z. " 1 OCT 2001 Received 18. A-process for producing a hybrid polypeptide or protein which comprises a domain with a binding function and a domain with an effector function, comprising transfecting or transducing mammalian cells with a recombinant nucleic acid molecule as claimed in any one of Claims 1 to 17 to obtain expression of the hybrid polypeptide or protein encoded by said nucleic acid molecule, and optionally recovering the hybrid polypeptide or protein produced, wherein said process excludes the therapeutic treatment of humans, 19. A recombinant nucleic acid molecule as claimed in claim 1 or 17 substantially as herein described or exemplified. 20. A process as claimed in claim 18 substantially as herein described or exemplified. 21. Use of a recombinant nucleic acid molecule as claimed in any one of the preceding claims for the preparation of a medicament for preventing local proteolytic activity, extracellular matrix degradation, cell migration, cell invasion or tissue remodeling. 22. A process for producing a hybrid polypeptide or protein which comprises a domain with a binding function and a domain with an effector function, comprising transfecting or transducing non-human mammalian cells with a recombinant nucleic acid molecule as claimed in any one of Claims 1 to 17 to obtain expression of the hybrid polypeptide or protein encoded by said nucleic acid molecule, and optionally recovering the hybrid polypeptide or protein produced. 23. A process for preventing local proteolytic activity, extracellular matrix degradation, cell migration, cell invasion or tissue remodeling in a non-human animal, comprising transfecting or transducing the cells involved or cells in their environment with a recombinant nucleic acid molecule as claimed in any one of the preceding claims to obtain local expression of the hybrid polypeptide or protein encoded by said nucleic acid molecule.