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WO1992002553A1 - Molecule de liaison a des cellules tumorales et ayant une region d'inhibition de protease - Google Patents

Molecule de liaison a des cellules tumorales et ayant une region d'inhibition de protease Download PDF

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Publication number
WO1992002553A1
WO1992002553A1 PCT/GB1991/001322 GB9101322W WO9202553A1 WO 1992002553 A1 WO1992002553 A1 WO 1992002553A1 GB 9101322 W GB9101322 W GB 9101322W WO 9202553 A1 WO9202553 A1 WO 9202553A1
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Prior art keywords
upa
protease
molecule according
region
receptor
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PCT/GB1991/001322
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English (en)
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David James Ballance
Michael George Courtney
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Delta Biotechnology Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6462—Plasminogen activators u-Plasminogen activator (3.4.21.73), i.e. urokinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8132—Plasminogen activator inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21073—Serine endopeptidases (3.4.21) u-Plasminogen activator (3.4.21.73), i.e. urokinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)

Definitions

  • tissue-type plasminogen activator tPA
  • uPA urokinase-type plasminogen activator
  • Such processes are involved in growth and spread of tumour cells, mammary gland involution, ovulation, embryo development, development of the nervous system, in the normal inflammatory response and in a number of inflammatory diseases (Dan ⁇ et al , 1985) . Elevated levels of uPA have been found in malignant tissue and there is believed to be a general correlation between the amount of uPA and the invasiveness of the tumour. Studies of experimental Lewis lung tumours revealed concentration of uPA at the invasive edge of the tumours. In addition, anti-catalytic antibodies to uPA inhibited the establishment of tumour cells after i.v. injection into mice and inhibited metastasis in chick embryos.
  • the proteolytic activity released by uPA is localized by the binding of plasminogen, plasmin and uPA to specific cell surface receptors (Vassalli et al, 1985; Miles and Plow, 1988) and the importance of receptor-bound uPA in tissue invasion has been demonstrated (Ossowski, 1988).
  • the receptor-binding region has been localized to the growth factor domain of the uPA molecule in the region of amino acids 12-32 of mature uPA since peptides corresponding to this sequence are able to block the uPA receptor interaction by competition (Appella et al , 1987).
  • PAI-1 plasminogen activator inhibitor 1
  • PAI-2 plasminogen activator inhibitor 2
  • PAI-2 is a slower inhibitor of tPA and is reported to be inactive against the physiologically important fibrin- stimulated tPA.
  • PAI-2 has been shown to inhibit receptor-bound uPA (Kirchheimer and Remold, 1989a; P ⁇ llanen et al, 1990) and uPA-dependent tissue invasion (Kirchheimer and Remold, 1989b) and thus represents a potentially useful anti-tumour or anti- metastatic agent.
  • Natural sources of PAI-2 yield very small quantities of the protein but EP-A-238 275 discloses the production of recombinant PAI-2 in E. coli .
  • Our co-pending application GB8918191.1 discloses the advantageous production of recombinant PAI-2 in Saccharomyces cerevisiae .
  • WO 88/08451 discloses uPA-tPA fusions, but the portion of uPA which is used is the B chain and the intention is to retain the activities of both tPA and uPA.
  • the uPA-derived portion (if present) of the molecules of the present invention preferably has substantially no uPA-like a idolytic or proteolytic activity.
  • One aspect of the invention provides a molecule comprising a first region which binds to a tumour cell and a second region which inhibits a first protease.
  • tumour-binding may be enabled by the existence on the tumour of structures unique to the tumour, for example tumour-specific antigens such as CEA (carcino-embryonic antigen) , pan carcinoma antigen, placental alkaline antigen or polymorphic epithelial mucin antigen.
  • tumour-specific antigens such as CEA (carcino-embryonic antigen)
  • pan carcinoma antigen pan carcinoma antigen
  • placental alkaline antigen or polymorphic epithelial mucin antigen.
  • uPAR receptor for uPA
  • the tumour-binding region is preferably an entity which will bind to the receptor for uPA (uPAR) , as is described in more detail below.
  • uPAR uPA
  • it may be any other entity which binds preferentially to a tumour cell, for example the epidermal growth factor receptor present in some breast carcinomas.
  • the first protease is preferably tumour-associated and is preferably urokinase-type plasminogen activator (uPA) as is described in more detail below.
  • uPA urokinase-type plasminogen activator
  • the protease is one that can be activated or converted from a pro-enzyme -or zymogen form by uPA or receptor-bound uPA or by plasmin to generate localised proteolytic activity.
  • the inhibitor region in the molecule of the invention may be an inhibitor of plasmin such as the Pittsburgh variant of ⁇ -antitrypsin, an ⁇ ⁇ AT variant having lysine at the P position, ⁇ 2 -antiplasmin, ⁇ 2 - macroglobulin, aprotinin or any other inhibitor with plasmin inhibitory activity, or it may be an inhibitor of a collagenase such as tissue inhibitor of metallo-proteinases (TIMP) or the related TIMP2.
  • TIMP tissue inhibitor of metallo-proteinases
  • Other proteases believed to be involved in tissue remodelling in neoplasia are proteases of the cathepsin and stromelysin families and it may be beneficial to provide an inhibitor of these activities.
  • thrombin inhibitor such as ⁇ 1 -antitrypsin Pittsburgh.
  • An application of the concept of the current invention to this system is to enhance the anti-thrombin-receptor-activating activity of thrombin by directing the ⁇ ⁇ -AT Pittsburgh to the thrombin receptor or an adjacent cell surface molecule.
  • the receptor- binding moiety of the hybrid may be a receptor-binding peptide or an antibody or antibody fragment directed against the thrombin receptor.
  • an inhibitory portion of the protease inhibitor may be used instead of the whole molecule.
  • the molecule thus provides for the targeting of the protease inhibiting region to the tumour cell.
  • protease the activity of which is inhibited may be free, bound to its receptor, associated with the cell surface via non ⁇ specific interactions or bound to adjacent cells or to extracellular matrix.
  • the first region is preferably a peptide sequence corresponding to a receptor-binding a region of the tumour- associated protease, which may or may not be the same protease as the protease which is inhibited.
  • the first region may comprise amino acids 1-32 of uPA or a variant or fragment thereof.
  • variant we mean a region with one or more minor alterations to the amino acid sequence thereof which region is nevertheless recognisably similar to the said 1-32 region and which still binds to the uPA-binding site on the uPAR.
  • the region is generally "EGF-like", epidermal growth factor (EGF) itself does not bind to the said site.
  • fragment we mean smaller sequences (preferably at least 10, 15, 19 or 20 amino acids long) which still bind to the binding site of the receptor, for example the 20-30 region of uPA. "Variants” of “fragments” may also be used.
  • the receptor- binding region of the protease may, in the molecule of the invention, be accompanied by other parts of the protease or a pro-form of the protease or a mutant thereof. Such parts or mutants should have at least 50% homology, preferably 60%, 70%, 80%, 90% or 95% homology with the protease (or, in the case of parts, with the corresponding region of the protease) in order to be regarded as parts or mutants thereof.
  • the protease-derived part of the molecule of the invention at least when present in the said molecule, has substantially no protease-like proteolytic activity.
  • uPA it may be possible to achieve this simply by using a pro- uPA mutant in which Lys 158 has been altered, for example to Gin or Gly, to prevent cleavage by plasmin.
  • the 12-19 and 31-32 residue regions adjacent the 20-30 bindir.g region of uPA seem to help the binding region to bind optimally and are preferably present, or functionally equivalent flanking regions are present.
  • the first region may alternatively be a monoclonal antibody or part thereof or a genetically engineered counterpart thereof which binds to the protease receptor, preferably uPAR. Preferably, it binds to the protease-binding site of the receptor or it binds to an area of the receptor which is sufficiently close to the said binding site for the bulk of the molecule as a whole to block binding of the protease to the receptor.
  • Monoclonal antibodies to the protease receptor or other target entity may be prepared by immunising an animal with the target entity or an antigenic part thereof and preparing and selecting an appropriate hybridoma in the conventional way (see Monoclonal Hybridoma Antibodies Ed. J.G.R. Hurrell, CRC Press 1982, incorporated herein by reference). Alternatively, a polyclonal collection of antibodies of restricted specificity may be used.
  • the antibody is a monoclonal antibody, an F ab ', (F ab ) 2 , F v , scF v or dAb fragment or a synthetic molecule including the minimum recognition unit(s) of a target-entity-specific immunoglobulin.
  • the first region consists of any peptide which will bind to the protease-binding site, or sufficiently close to it to block it, and need not be homoxogous with any part of the protease.
  • the second region may be any compound which inhibits the action of the protease (preferably uPA) .
  • a compound may be an antibody (or antibody fragment etc, as discussed above) directed against the enzymatically active site of the protease, or against such a part of the protease molecule as to block access of the substrate to the active site or against any other part of the protease such that binding of the antibody to the said part prevents or at least reduces the activity of the protease towards its substrate.
  • the second region is PAI-2 or part thereof or a mutant PAI-2 or part thereof.
  • the mutant or part of PAI-2 retains a useful level of the uPA-inhibiting activity of PAI-2. Fusions of the uPAR-binding domain of uPA and PAI-2 or a part or mutant thereof (hereinafter called "uPA-PAI-2 fusions") may exhibit the following properties:
  • the molecule should be stable In vivo.
  • stable in vivo we mean that the molecule is sufficiently stable when administered parenterally, preferably intravenously, to allow useful quantities of it to reach a tumour and bind to the receptors on the tumour cells.
  • a molecule with a half-life in the human circulation of 30 minutes or longer is considered to be stable.
  • the half-life is at least 1 hour, 24 hours, 3 days or more.
  • the molecules of the invention may be dissolved in suitable delivery vehicles and administered to patients to inhibit or prevent the growth or spread of an actual or suspected tumour.
  • the patient is preferably a human but may be another animal, preferably a mammal, such as a pet (dogs, cats, etc) or an economically important animal (sheep, cattle, pigs, fowl, horses, etc) .
  • the tumour- binding domain is modified for optimal binding to the animal receptor and ideally is the sequence of the said domain of the protease of that animal.
  • the inhibitor moiety may be modified to effect efficient inhibition of the protease of that animal.
  • the molecules are preferably administered parenterally, for example intravenously, intramuscularly or subcutaneously, by injection or infusion. Clinically qualified people will be able to determine suitable dosages, delivery vehicles and administrative routes.
  • the molecules of the invention may be produced by chemically linking the said first and second regions by methods known to those in the art of protein chemistry, for example using the methods of O 1 Sullivan et al (1979) and bifunctional linking reagents suchasm-maleimido-benzoyl-N-hydroxy-succinimideester.
  • hosts are legion and include E . coli , B . subtil is, Aspergillus and other filamentous fungi, yeasts, animal cells, plant cells, insect cells and transgenic whole animals and plants.
  • Exemplary genera of yeast contemplated to be useful in the practice of the present invention are Pichia, Saccharomyces , Kl uyveromyces , Candi da , Torulopsis , Hansenul a ,
  • Schizosaccharomyces Citeromyces, Pachysolen, Debaromyces, Metschunikowia, Rhodosporidium, Leucosporidium, Botryoascus , Sporidiobolus , Endomycopsis , and the like.
  • Preferred genera are those selected from the group consisting of Pichia, Saccharomyces, Kluyveromyces, Yarrowia and Hansenula , because the ability to manipulate the DNA of these yeasts has, at present, been more highly developed than for the other genera mentioned above.
  • Saccharomyces are Saccharomyces cerevisiae, Saccharomyces italicus and Saccharomyces rouxii .
  • Kluyveromyces are Kluyveromyces fragilis and Kluyveromyces lactis .
  • Hansenula examples include Hansenula polymorpha, Hansenula anomala and Hansenula capsulata .
  • Yarrowia lipolytica is an example of a suitable Yarrowia species.
  • Yeast cells can be transformed by: (a) digestion of the cell walls to produce spheroplasts; (b) mixing the spheroplasts with transforming DNA (derived from a variety of sources and containing both native and non-native DNA sequences) ; and (c) regenerating the transformed cells. The regenerated cells are then screened for the incorporation of the transforming DNA.
  • yeast cells of the genera Pichia, Saccharomyces , Kluyveromyces , Yarrowia and Hansenula can be transformed by enzymatic digestion of the cell walls to give spheroplasts; the spheroplasts are then mixed with the transforming DNA and incubated in the presence of calcium ions and polyethylene glycol, then transformed spheroplasts are regenerated in regeneration medium.
  • the protein is preferably produced as a soluble intracellular protein.
  • Suitable promoters for S. cerevisiae include those associated with the PGK1 gene, GAL1 or GAL10 genes, CYC1, PH05, TRP1 , ADH1, ADH2, the genes for glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, triose phosphate isomerase, phosphoglucose isomerase, glucokinase, ⁇ -mating factor pheromone, a-mating factor pheromone, the PRB1 promoter, the GOT2 promoter, and hybrid promoters involving hybrids of parts of 5' regulatory regions with parts of 5• regulatory regions of other promoters or with upstream activation sites (eg the promoter of EPA-258067) .
  • the preferred promoter is the PRB1 promoter.
  • the transcription termination signal is preferably the 3 ' flanking sequence of a eukaryotic gene which contains proper signals for transcription termination and polyadenylation.
  • Suitable 3 1 flanking sequences may, for example, be those of the gene naturally linked to the expression control sequence used, ie may correspond to the promoter. Alternatively, they may be different in which case the termination signal of the S. cerevisiae ADH1 gene is preferred.
  • Suitable secretion leader sequences include mammalian leader sequences, such as the HSA and pro-uPA leader sequences, S . cerevisiae leader sequences such as the ⁇ -mating factor pheromone pre- and prepro- sequence, the invertase (SUC2 ) leader sequence, the PH05 leader sequence, or hybrid leader sequences such as the leader sequence of WO 90/01063.
  • mammalian leader sequences such as the HSA and pro-uPA leader sequences
  • S . cerevisiae leader sequences such as the ⁇ -mating factor pheromone pre- and prepro- sequence
  • the invertase (SUC2 ) leader sequence the PH05 leader sequence
  • hybrid leader sequences such as the leader sequence of WO 90/01063.
  • amino acid sequence of the N-terminal portion of uPA encompassing the growth factor domain is as follows: 10 20 30
  • the sequence of residues 20-30 is believed to be responsible for the specificity of binding to the receptor and this sequence may represent the minimum requirement for binding to the receptor. Sequences adjacent to this region provide the proper conformation for optimum binding but may be substituted by a functionally equivalent structure.
  • the uPA sequence incorporated into the fusion molecules preferably includes residues 12-32 and in the preferred embodiment includes residues 1-47 to ensure optimum binding to uPAR. It is not necessary for the first region of the molecule of the invention to be at or adjacent the N-terminal of the molecule. The second region can be N-terminal to the first region.
  • DNA sequences for expression of the molecules of the invention may be prepared by known techniques, for example by fusing cDNA fragments prepared from mRNAs corresponding respectively to the receptor-binding region of the protease and to a protease inhibitor or a part thereof.
  • Monoclonal antibodies may be prepared generally by the techniques of Zola (1988) which is incorporated herein by reference.
  • Useful antigens for preparing either monoclonal or polyclonal antibodies are (for the second region) uPA, low molecular weight uPA or the heavy (B) chain of uPA, and (for the first region) uPAR or peptides based on the uPA-binding domain thereof.
  • Antibody fragments such as F ab fragments, may be prepared therefrom in known ways.
  • the antibodies may be humanized in known ways.
  • Antibody-like molecules may be prepared using the recombinant DNA techniques of WO 84/03712.
  • bispecific antibodies specific for an appropriate receptor and an appropriate site on the protease, may be made by any of the methods described by Williams (1988) .
  • Such molecules will block the protease-binding site of the receptor and also inhibit the proteas ' e.
  • the -antibody may alternatively be bispecific for the receptor and the protease inhibitor (or a mutant or fragment thereof) so that the antibody acts to bind the inhibitor to the receptor.
  • the combination of the antibody bound to the inhibitor thus constitutes a molecule of the invention, whereas the antibody itself constitutes a precursor thereof, forming a separate aspect of the invention.
  • the antibody specific for the receptor and the inhibitor may be combined with the inhibitor before being administered or may be co-administered therewith.
  • co- administered we do not necessarily mean that the antibody and the inhibitor must be administered simultaneously; it is sufficient for them to combine in a therapeutically useful way in the body.
  • Chimaeric antibodies where the F c region is a human immunoglobin or part thereof, may be desirable for long term treatment, to reduce adverse im unological responses.
  • Single chain antibodies may be used for either or both of the two regions of the molecules of the invention.
  • Figure 1 illustrates the construction of plasmids pDBPl, pDBP2 and pDBP3.
  • E EcoRI
  • P PstI
  • Bg Bglll
  • H tfindlll
  • B BajjiHI
  • A Aflll. Only the Alll site present in the PAI-2 encoding sequence is shown;
  • Figure 2 shows a DNA sequence encoding, and corresponding amino acid sequence of, PAI-2 in pDBPl;
  • Figure 3 shows the DNA sequence of the PRB1 promoter
  • Figures 4 to 8 respectively illustrate plasmids pAYE333, PAYE334, pAYE335, pDBP5 and pDBP6;
  • FIG 9 illustrates the construction of plasmids pHJB117 and pDJBll ⁇
  • Figure 10 illustrates the construction of plas id pDBUPl
  • Figure 11 shows flow cytometer histograms of U937 cells to detect binding of the uPA-PAI-2 hybrid protein to cells. Control cells are shown by the left hand peak and cells incubated with the hybrid protein are shown by the right hand peak. Detection of bound hybrid protein was with anti-PAI-2 antibody and FITC- conjugated secondary antibody;
  • Figure 12 illustrates the construction of plasmids pDBA2, pDBUAl, pDBUA2 and pDBUA3.
  • Figure 13 shows the DNA sequences and amino acid sequence encoded thereby, for o ⁇ antitrypsin in pDBAl.
  • a plasminogen activator inhibitor 2 coding sequence ( Figure 1) may be derived from EP-A-238 275.
  • the DNA may be obtained from E. coli deposited as ATCC 53585 in connection with EP 238 275. This contains the plasmid pBTA438, comprising the PAI-2 sequence.
  • the sequences encoding the uPA growth factor domain may be assembled from synthetic oligonucleotides by standard procedures.
  • the library was screened using radioactively labelled oligonucleotide probes corresponding to the DNA sequences encoding the N-terminus (oligo 1) and C-terminal end (amino acids 400-410) (oligo 2) of the PAI-2 protein, respectively by standard procedures.
  • restriction enzyme recognition sites were created at the 5' and 3 1 ends of the PAI-2 gene.
  • a Bglll site was created at the 5 1 end of the gene using the anti-sense oligonucleotide primer 1:
  • oligonucleotide-directed in vitro mutagenesis system-version 2, Amersham pic A clone derived from this procedure and with the correct changes was designated pDBP2 ( Figure 1) .
  • Oligonucleotide linkers were then used to position restriction sites at either end of the gene which are suitable for insertion of the gene into an expression vector.
  • the linker positioned at the 5' end of the gene was Linker 1
  • the structural gene, PRB1 for the Saccharomyces cerevisiae vacuolar endoprotease B has been isolated- (Moehle et al, 1987a) on two prJbl complementing plasmids called MK4 and FP8.
  • MK4 and FP8 The structural gene, PRB1 for the Saccharomyces cerevisiae vacuolar endoprotease B has been isolated- (Moehle et al, 1987a) on two prJbl complementing plasmids called MK4 and FP8.
  • the DNA sequence of the PRB1 gene has been reported, as has 150bp of the PRB1 promoter (Moehle et al, 1987b). A more extensive DNA sequence of the PRB1 promoter is also available as an entry in the Genbank database, release 60, accession number M18097, locus YSCPRB1, Figure 3.
  • the whole of the PRB1 promoter may be used, or a smaller portion thereof, as may readily be determined.
  • the roughly lkbp sequence extending upstream from the start codon to the SnaBl site is effective.
  • the promoter element was further modified by site directed mutagenesis (oligonucleotide direct in vitro mutagenesis system-Version 2, Amersham) according to the manufacturer's instructions. Mutagenesis with the oligonucleotide
  • HindiII Plasmid pAAH5 (Goodey et al, 1987) was linearised by partially digesting with BamHI. The 5' protruding ends were blunt-ended with T4 DNA polymerase and ligated with the double- stranded oligonucleotide Linker 3.
  • a recombinant plasmid pAYE334 ( Figure 5) was selected in which a WotI restriction site had replaced the BamHI site at the 3' end of the ADH1 terminator.
  • the 0.8kbp Wotl-Hindlll modified protease B promoter sequence was placed upstream of the 0.45kbp tfindlll-Wotl ADH1 transcription terminator on a pAT153-based plasmid (Twigg and Sherratt, 1980) to generate pAYE335 ( Figure 6) .
  • linker 5 to encode the C-terminal portion of the uPA growth factor domain (amino acids 24-47 of mature uPA) and the start of the PAI-2 sequence.
  • This linker was ligated with a Bglll-Bindlll fragment of pDBP3, representing the remainder of the PAI-2 sequence, into pUC18 at Sall-Hindlll to form the plasmid pDJB117 ( Figure 9) .
  • a second linker representing the PRB1 ATG environment, a methionine initiation codon and DNA encoding the N-terminal portion of the uPA growth domain was assembled by annealing the four oligonucleotides shown below:- Linker 6 oligo 3
  • Plasmid pDBUPl contains a PRBl promoter fragment to control transcription of the uPA-PAI-2 sequence and the ADH1 transcription terminator.
  • the plasmid was introduced into a suitable S. cerevisiae strain wherein it effected production of the hybrid protein as a soluble intracellular protein which was recognised by anti-PAI-2 and anti-uPA antibodies in Western blotting experiments.
  • the protein was purified from cell lysates by copper chelate and anion exchange chromatography and was found to inhibit uPA with similar kinetics to PAI-2 itself, ie second order inhibition rate constant of l. ⁇ x 10 6 M ' ⁇ -S "1 , determined according to Thorsen et al (1988) .
  • the uPA growth factor domain was shown to be functional by its ability to mediate specific binding to U937 cells.
  • the cells were first treated with 50mM glycine HCl, 0.1M NaCl, pH3.0 for 3 minutes at 4°C to remove endogenous cell-bound uPA.
  • the cell suspension was neutralised by the addition of one-fifth volume of 0.5M Hepes, 0.1M NaCl, pH7.5 and then the cells were washed twice with PBS (Dulbecco's Ca 2+ and Mg 2+ free phosphate buffered saline) containing 0.1% (w/v) bovine serum albumin (PBS + BSA).
  • a human ⁇ 1 -antitrypsin cDNA identical in sequence to that described in the Genbank database, release 60, accession number X01683 V00496, locus HUMAIATR was amplified by Polymerase Chain Reaction using a Perkin Elmer Cetus DNA thermal cycler according to the manufacturer's instructions.
  • the DNA sequence of the two primary oligonucleotides were:
  • the 1.22kbp modified cDNA was purified, digested with Hindlll and BainHI inserted and cloned into the Hindlll-BamHI sites of M13mpl9 (Yanisch-Perron et al (1985) Gene 33, 103-119) generating pDBAl ( Figure 12) .
  • the integrity of human 0 ⁇ - antitrypsin was confirmed by dideoxynucleotide sequencing.
  • a sequence encoding ⁇ ⁇ -antitrypsin Pittsburgh ( ⁇ AT-P) (Owen et al, 1983) was created using oligonucleotide-directed mutagenesis of pDBAl using the oligonucleotide shown below
  • Linker 7 Two oligonucleotides were synthesised and then annealed to form a linker (Linker 7) to encode the C-terminal portion of the uPA growth factor domain (amino acids 24-47) and the start of the ⁇ AT-P sequence.
  • a second linker representing the PRBl ATG environment, a methionine initiation codon and DNA encoding the N-terminal portion of the uPA growth domain was assembled by annealing the four oligonucleotides shown below: -
  • Double stranded pDBUAl was digested with Seal and EcoRI and then ligated with linker 8 to form pDBUA2 (Fig 12) .
  • the Hindlll fragment of pDBUA2 was then introduced into the expression plasmid pDBP6 to form pDBUA3 (Fig 12) .
  • This plasmid was then introduced into a suitable S. cerevisiae strain wherein it effected production of the hybrid protein which was recognised by anti-a x AT and anti-uPA antibodies in Western blotting experiments.

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Abstract

Molécule comprenant une première région qui se lie à une tumeur (de préférence, le récepteur de uPA) et une deuxième région qui inhibe une protéase (de préférence associée à la tumeur), par exemple uPA. La première et la deuxième régions peuvent être des anticorps respectifs ou des parties de ceux-ci, spécifiques à des parties appropriées de uPAR et uPA. De préférence, la première région est une partie de uPA de liaison de uPAR, par exemple sa région 12-32, et la deuxième région est PAI-2 ou un analogue d'inhibition de uPA ou une partie de celui-ci, et les première et deuxième régions se combinent pour former un polypeptide simple, pouvant s'exprimer à partir d'un hôte transformé.
PCT/GB1991/001322 1990-08-03 1991-08-02 Molecule de liaison a des cellules tumorales et ayant une region d'inhibition de protease WO1992002553A1 (fr)

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GB9017083A GB2246779B (en) 1990-08-03 1990-08-03 Tumour-associated protease inhibitors targeted to tumour cells
GB9017083.8 1990-08-03

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WO1992002553A1 true WO1992002553A1 (fr) 1992-02-20

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AU (1) AU8318591A (fr)
GB (1) GB2246779B (fr)
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WO1994028014A3 (fr) * 1993-05-28 1995-01-26 Chiron Corp Peptides inhibiteurs de l'activite des recepteurs d'urokinase
US5712136A (en) * 1994-09-08 1998-01-27 Genvec, Inc. Adenoviral-mediated cell targeting commanded by the adenovirus penton base protein
EP0692968A4 (fr) * 1993-04-02 1998-05-20 Gen Hospital Corp Fragments de l'activateur du plasminogene urokinase
US5770442A (en) * 1995-02-21 1998-06-23 Cornell Research Foundation, Inc. Chimeric adenoviral fiber protein and methods of using same
US5846782A (en) * 1995-11-28 1998-12-08 Genvec, Inc. Targeting adenovirus with use of constrained peptide motifs
WO1998051788A3 (fr) * 1997-05-12 1999-05-20 Tno Procede et construction pouvant inhiber une migration cellulaire
US5965541A (en) * 1995-11-28 1999-10-12 Genvec, Inc. Vectors and methods for gene transfer to cells
US6077508A (en) * 1998-03-23 2000-06-20 American Diagnostica Inc. Urokinase plasminogen activator receptor as a target for diagnosis of metastases
US6127525A (en) * 1995-02-21 2000-10-03 Cornell Research Foundation, Inc. Chimeric adenoviral coat protein and methods of using same
US6509445B1 (en) 1996-01-08 2003-01-21 Nissin Food Products Co., Ltd. Cancerous metastasis inhibitor
US6913922B1 (en) 1999-05-18 2005-07-05 Crucell Holland B.V. Serotype of adenovirus and uses thereof
US6929946B1 (en) 1998-11-20 2005-08-16 Crucell Holland B.V. Gene delivery vectors provided with a tissue tropism for smooth muscle cells, and/or endothelial cells
US6951755B2 (en) 1994-09-08 2005-10-04 Genvec, Inc. Vectors and methods for gene transfer
US6974695B2 (en) 2000-11-15 2005-12-13 Crucell Holland B.V. Complementing cell lines
US7052881B2 (en) 1995-06-15 2006-05-30 Crucell Holland B.V. Packaging systems for human recombinant adenovirus to be used in gene therapy
US7235233B2 (en) 2000-09-26 2007-06-26 Crucell Holland B.V. Serotype 5 adenoviral vectors with chimeric fibers for gene delivery in skeletal muscle cells or myoblasts
US7468181B2 (en) 2002-04-25 2008-12-23 Crucell Holland B.V. Means and methods for the production of adenovirus vectors
US7749493B2 (en) 1998-07-08 2010-07-06 Crucell Holland B.V. Chimeric adenoviruses
US7807621B2 (en) 1997-07-25 2010-10-05 Angstrom Pharmaceuticals, Inc. Anti-invasive and anti-angiogenic compositions

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EP0692968A4 (fr) * 1993-04-02 1998-05-20 Gen Hospital Corp Fragments de l'activateur du plasminogene urokinase
US5679782A (en) * 1993-05-28 1997-10-21 Chiron Corporation Oligonucleotides encloding peptide inhibitors of urokinase receptor activity
WO1994028014A3 (fr) * 1993-05-28 1995-01-26 Chiron Corp Peptides inhibiteurs de l'activite des recepteurs d'urokinase
US5712136A (en) * 1994-09-08 1998-01-27 Genvec, Inc. Adenoviral-mediated cell targeting commanded by the adenovirus penton base protein
US6951755B2 (en) 1994-09-08 2005-10-04 Genvec, Inc. Vectors and methods for gene transfer
US6153435A (en) * 1995-02-21 2000-11-28 Cornell Research Foundation, Inc. Nucleic acid that encodes a chimeric adenoviral coat protein
US5770442A (en) * 1995-02-21 1998-06-23 Cornell Research Foundation, Inc. Chimeric adenoviral fiber protein and methods of using same
US6576456B2 (en) 1995-02-21 2003-06-10 Cornell Research Foundation, Inc. Chimeric adenovirus fiber protein
US6127525A (en) * 1995-02-21 2000-10-03 Cornell Research Foundation, Inc. Chimeric adenoviral coat protein and methods of using same
US7105346B2 (en) 1995-06-15 2006-09-12 Crucell Holland B.V. Packaging systems for human recombinant adenovirus to be used in gene therapy
US7052881B2 (en) 1995-06-15 2006-05-30 Crucell Holland B.V. Packaging systems for human recombinant adenovirus to be used in gene therapy
US5846782A (en) * 1995-11-28 1998-12-08 Genvec, Inc. Targeting adenovirus with use of constrained peptide motifs
US6329190B1 (en) 1995-11-28 2001-12-11 Genvec, Inc. Targetting adenovirus with use of constrained peptide motifs
US6649407B2 (en) 1995-11-28 2003-11-18 Genvec, Inc. Targeting adenovirus with use of constrained peptide motifs
US6057155A (en) * 1995-11-28 2000-05-02 Genvec, Inc. Targeting adenovirus with use of constrained peptide motifs
US5965541A (en) * 1995-11-28 1999-10-12 Genvec, Inc. Vectors and methods for gene transfer to cells
US6509445B1 (en) 1996-01-08 2003-01-21 Nissin Food Products Co., Ltd. Cancerous metastasis inhibitor
WO1998051788A3 (fr) * 1997-05-12 1999-05-20 Tno Procede et construction pouvant inhiber une migration cellulaire
US8110543B2 (en) 1997-07-25 2012-02-07 Angstrom Pharmaceuticals, Inc. Anti-invasive and anti-angiogenic compositions
US7807621B2 (en) 1997-07-25 2010-10-05 Angstrom Pharmaceuticals, Inc. Anti-invasive and anti-angiogenic compositions
US6077508A (en) * 1998-03-23 2000-06-20 American Diagnostica Inc. Urokinase plasminogen activator receptor as a target for diagnosis of metastases
US7749493B2 (en) 1998-07-08 2010-07-06 Crucell Holland B.V. Chimeric adenoviruses
US6929946B1 (en) 1998-11-20 2005-08-16 Crucell Holland B.V. Gene delivery vectors provided with a tissue tropism for smooth muscle cells, and/or endothelial cells
US6913922B1 (en) 1999-05-18 2005-07-05 Crucell Holland B.V. Serotype of adenovirus and uses thereof
US7270811B2 (en) 1999-05-18 2007-09-18 Crucell Holland B.V. Serotype of adenovirus and uses thereof
US7250293B2 (en) 1999-05-18 2007-07-31 Crucell Holland B.V. Complementing cell lines
US7906113B2 (en) 1999-05-18 2011-03-15 Crucell Holland B.V. Serotype of adenovirus and uses thereof
US8221971B2 (en) 1999-05-18 2012-07-17 Crucell Holland B.V. Serotype of adenovirus and uses thereof
US7235233B2 (en) 2000-09-26 2007-06-26 Crucell Holland B.V. Serotype 5 adenoviral vectors with chimeric fibers for gene delivery in skeletal muscle cells or myoblasts
US7344883B2 (en) 2000-11-15 2008-03-18 Crucell Holland B.V. Complementing cell lines
US6974695B2 (en) 2000-11-15 2005-12-13 Crucell Holland B.V. Complementing cell lines
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US7468181B2 (en) 2002-04-25 2008-12-23 Crucell Holland B.V. Means and methods for the production of adenovirus vectors
US7820440B2 (en) 2002-04-25 2010-10-26 Crucell Holland B.V. Means and methods for producing adenovirus vectors

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GB2246779A (en) 1992-02-12
GB2246779B (en) 1994-08-17
AU8318591A (en) 1992-03-02
GB9017083D0 (en) 1990-09-19

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