EP0906419A2 - Peptid-liganden für den urokinase-rezeptor - Google Patents
Peptid-liganden für den urokinase-rezeptorInfo
- Publication number
- EP0906419A2 EP0906419A2 EP97917723A EP97917723A EP0906419A2 EP 0906419 A2 EP0906419 A2 EP 0906419A2 EP 97917723 A EP97917723 A EP 97917723A EP 97917723 A EP97917723 A EP 97917723A EP 0906419 A2 EP0906419 A2 EP 0906419A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- upar
- peptide
- seq
- binding
- amino acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70546—Integrin superfamily
- C07K14/7055—Integrin beta1-subunit-containing molecules, e.g. CD29, CD49
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to the identification of novel functional sites on the urokinase receptor in the presence of the receptor binding region of urokinase. Described herein are peptides derived from bacteriophage display that identify the sites, and a general method for identifying functional sites on proteins using bacteriophage display. Also, methods of using urokinase receptor functional sites for studies of vitronectin and integrin interaction with urokinase: urokinase receptor complex interaction are described. Also described are uses of the instant peptides for developing therapeutic molecules capable of antagonisting interactions of the vitronectin and integrin peptides with the urokinase:urokinase receptor complex.
- uPA urokinase plasminogen activator
- uPAR cell surface receptor
- the uPA:uPAR complex converts plasminogen into plas in which is known to degrade various matrix glycoproteins as described in Ellis et al, J. Biol. Chem. 264: 2185-2188 (1989), Vassili et al, J. Clin. Invest. 88: 1067-1072 (1991), and Mignatti and Rifkin, Physiol. Rev. 73: 161-195 (1993).
- the simaltaneous expression of uPA and its receptor has been associated with localized plasminogen activation and pericellular matrix degradation during directed cell migration of normal and tumor cells.
- the urokinase receptor is a 283 amino acid glycosylphosphatidyl-inositol (GPI)- anchored receptor protein of urokinase and vitronectin which appears to be a triplication of a 90 amino acid domain as described in Plough, and Ellis, FEiSS Lett. 349:163-168 (1994) and Roldan et al, EMBO J. 9: 461-414 (1990). Proteolysis of uPAR can yield fragments composed of domain 1 and domains 2-3. and subsequent analysis has shown that disulfide bonding pattern of domain 1 is completely internal to the domain, as described in Plough et al, J.Biol. Chem.
- uPAR can function as a cell adhesion receptor for vitronectin in a uPA dependent manner as desc ⁇ bed in Wei et al, 3. iol Chem. 269. 32380-32388 (1994)
- a first embodiment of the invention is a method of identifying an orphan binding site on a target polypeptide sequence by (a) providing (1) a library of potential ligands,
- Another embodiment of the invention is an isolated peptide that binds a urokinase plasminogen activator receptor (uPAR) and inhibits uPAR binding to an integrin.
- the isolated peptide can be YHXLXXGYMYT (SEQU ID NO:5) or
- Another embodiment of the invention is an isolated peptide that binds a urokinase plasminogen activator (uPAR) and inhibits uPAR binding to vitronectin.
- the isolated peptide can be AEPVYQYELDSYLRSYY (SEQ ID NO: 1), AEFFKLGPNGYVYLHSA
- Yet another embodiment of the invention is an isolated nucleic acid sequence that encodes a peptide that binds a urokinase plasminogen activator receptor (uPAR) and inhibits uPAR binding to an integrin.
- the isolated nucleic acid sequence can encode the amino acid sequence of YHXLXXGYMYT (SEQ ID NO:5) or STYHHLSLGYMYTLN
- Still another embodiment of the invention is an isolated nucleic acid sequence that encodes a peptide that binds a urokinase plasminogen activator receptor (uPAR) and inhibits uPAR binding to vitronectin.
- the isolated nucleic acid sequence can encode the amino acid sequence of AEPVYQYELDSYLRSYY (SEQ ID NO: 1 ), or
- FFKLGPNGYVYLHSA SEQ ID NO:2
- AELDLSTFYDIQYLLRT SEQ ID NO:3
- FKLXXXGYVYL SEQ ID NO:6
- Yet another embodiment of the invention is a method of treating a patient with a disorder characterized by upregulation of uPA and uPAR by providing an effective amount of an antagonist of a uPAR. integrin binding pair, and administering the antagonist to the patient.
- An additional embodiment of the invention is a method of screening tor an antagonist of uPAR.integ ⁇ n interaction comprising the steps of providing a peptide antagonist ol a uPAR integrin interaction, competing the peptide antagonist with a candidate antagonist for binding to uPAR, and identifying a candidate antagonist by the ability to compete with the peptide antagonist for uPAR binding
- Still a further embodiment of the invention is a small molecule antagonist of a uPAR.integ ⁇ n interaction identified by the just desc ⁇ bed method; a peptide antagonist of a uPAR.integ ⁇ n interaction identified by that method, and a peptoid antagonist of a uPAR.integ ⁇ n interaction identified by the same method
- Another embodiment of the invention is a pharmaceutical composition for treating a disorder characterized by upregulation of uPA and uPAR comprising an effective amount of an antagonist of a uPAR: integrin binding pair and a pharmaceutically acceptable carrier.
- Yet another embodiment of the invention is a pharmaceutical composition for treating a patient with a disorder characte ⁇ zed by upregulation of uPA and uPAR comprising an effective amount of a nucleic acid encoding a peptide antagonist of a uPAR integrin binding pair and a pharmaceutically acceptable carrier suitable for expressing the peptide in the patient
- FIG 1 UPA 1-48 is required for sUPAR binding to vitronectin
- Various concentrations of uPAl-48 were incubated with biotinylated sUPAR in vitronectm- coated wells and vitronectin-bound sUPAR detected as described m the examples. Each determination was in duplicate and the results are reported as the mean absorbance at 450 nm of the sUPAR plus uPAl-48 samples minus the mean absorbance of the sUPAR alone sample (approximately 0.03)
- FIG 2 Effects of various peptide ligands on sUPAR binding to vitronectin
- the effects of the indicated peptides on sUPAR vitronectin interaction were determined by incubating the peptides with biotinylated sUPAR in vitronectin-coated wells in the presence of uPAl-48 as in FIG 1 All peptides were solubi zed in 100% DMSO before diluting to the indicated concentrations with PBS/2% BSA for the assay. Control samples included suPAR plus 20 nM UPAl-48 and sUPAR alone.
- Peptides 7 and 18 are Homologous to the Somatomedin B Domain of Vitronectin.
- the sequence of vitronectin from residues 1 - 47 including the somatomedin B domain and RGD motif is compared with the sequences of clones 7 and 18.
- Homologous residues at positions 22 -28 in vitronectin and in the bacteriophage derived peptides are in bold as is the RGD sequence in vitronectin.
- FIG 4. Alanine Replacement of Peptide 7 Affects Both Bacteriophage and Vitronectin Binding to UPAR. Synthetic peptides at 40 ⁇ M were tested as competitors for binding of bacteriophage 7 to biotinylated suPAR as described in Materials and Methods and shown in panel A. Bacteriophage were detected with a rabbit anti-M13 antibody as described. The indicated values are the mean of triplicate determinations. The same peptides were tested in triplicate at 50 ⁇ M in the uPAl-48:uPAR:vitronectin binding ELISA.
- FIG. Bacteriophage binding to sUPAR domain 2/3. Phage were added to wells containing sUPAR domain 2/3 immobilized by its epitope tag via protein G and monoclonal antibody to the epitope tag. Wells containing protein G and antibody but no domain 2/3 were included to determine nonspecific phage binding. Urea-eluted phage and the input stocks were titered by plaque formation assay. Results were single point determinations calculated as a percent of the input titer and were repeated in three separate experiments.
- FIG 6 Table. The table of FIG 6 depicts the sequences, phage yields, and IC50s in uPAR binding assays for selected phage peptides.
- orphan binding site refers to a previously unidentified site on a polypeptide sequence that is capable of binding to another peptide or polypeptide sequence.
- the orphan binding site is distinguishable from a binding site for which the native ligand is known.
- the orphan binding sites of the invention are discovered by phage display of a peptide sequence that is capable of binding a site on a target polypeptide.
- the binding site may involve binding of a third or fourth additional polypeptide, for example, where the urokinase plasminogen activator receptor (uPAR) binds urokinase plasminogen activator (uPA) in addition to binding other ligands or polypeptides, such as, for example vitronectin and integrin.
- uPAR urokinase plasminogen activator receptor
- uPA urokinase plasminogen activator
- orphan polypeptide refers to a polypeptide sequence capable of binding at an orphan binding site.
- the orphan polypeptide may be, for example, a peptide used in a phage display screening to determine orphan binding sites, or may be the polypeptide sequence of a native or synthetic molecule that binds the orphan binding site, and is homologous in sequence to the peptide used to determine the location of the orphan binding site.
- potential ligand refers to any peptide, polynucleotide, polysaccharide, or other molecule that could potentially bind to the target polypeptide.
- potential ligand library refers to a collection or mixture of at least 50 compounds that are potential ligands as defined above, and more preferably a potential ligand library is at least 200 potential ligand compounds, and still more preferably more than 500 compounds.
- unknown ligand refers to ligands of a target polypeptide that have not yet been discovered, but that may be discovered by the method of the invention.
- a potential ligand can bind a target polypeptide, and antagonize binding of a previously unknown ligand
- the identity and existence of the unknown ligand can be determined either by structural analysis of the potential ligand that binds a target polypeptide, or by functional changes that indicate that binding has been disrupted by an antagonist
- the unknown ligand can also be determined by screening a library of polypeptides comprising sequences that occur naturally in a competition assay with the potential ligand bound to the target polypeptide at the orphan binding site
- bacteriophage library refers to the technique in molecular biology of creating a library of peptides expressed on the surface of a bacteriophage for presentation and contacting potential target polypeptides
- the library is the polynucleotides that are expressable as peptides and presented by the bacteriophage, and may be the DNA or the amino acid moieties used or generated by this technique
- Bacteriophage panning or display has applications as described herein for screening for ligands of target polypeptides, which when identified, also identifies orphan binding sites on the target polypeptides
- peptide and the term “polypeptide” as used herein refers to a peptide or a polypeptide produced in vivo or in vitro in an environment manipulated by humans using techniques of molecular biology, biochemistry or gene therapy.
- an isolated peptide or polypeptide can be produced in a cell free system by automated peptide or polypeptide synthesis, in heterologous host cells transformed with the nucleic acid sequence encoding the peptide or polypeptide and regulatory sequences for expression in the host cells, and in an animal into which the coding sequence of the peptide or polypeptide has been introduced for expression in the animal.
- isolated polypeptides or polynucleotides can be 10% pure, 20% pure, or a higher degree of pu ⁇ ty
- derivative as used herein in reference to a peptide, polypeptide or a polynucleo de means a peptide, polypepude or polynucleotide that retains the functionality of the peptide, polypeptide or polynucleotide to which it is a derivative They may be variously modified by amino acid deletions, substitutions, insertions or inversions by, for example, site directed mutagenesis of the underlying nucleic acid molecules. Derivatives of a peptide, polypeptide or polynucleotide may also be fragments thereof.
- a derivative, or a fragment retains at least some, and preferably all of the function of the peptide or polypeptide from which it is derived
- pharmaceutical composition refers to a composition for administration of a therapeutic agent
- the therapeutic agent can be. for example, a peptide, a polypeptide. a polynueleotide. a small molecule, a peptoid. or a derivative of any of these, and refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
- Administration of a therapeutic agent of the invention includes administration of a therapeutically effective amount of the agent of the invention.
- terapéuticaally effective amount refers to an amount of a therapeutic agent sufficient to treat or prevent a condition treatable by administration of a composition of the invention. That amount is the amount sufficient to exhibit a detectable therapeutic, preventitive or ameliorative effect. The effect may include, for example, treatment or prevention of the conditions listed herein.
- the precise effective amount for a subject will depend upon the subject's size and health, the nature and extent of the condition being treated, recommendations of the treating physician, and the therapeutics or combination of therapeutics selected for administration. Thus, it is not useful to specify an exact effective amount in advance. However, the effective amount for a given situation can be determined by routine experimentation.
- Administration can include admininistration of a polypeptide, and causing the polypeptide to be expressed in an animal by administration of a polynucleotide encoding the polypeptide.
- a "recombinant vector” herein refers to any vector for transfer or expression of the polynucleotides herein in a cell, including, for example, viral vectors, non-viral vectors, plasmid vectors and vectors derived from the regulatory sequences of heterologous hosts and expression systems.
- a “regulatory sequence” herein refers to a nucleic acid sequence encoding one or more elements that are capable of affecting or effecting expression of a gene sequence, including transcription or translation thereof, when the gene sequence is placed in such a position as to subject it to the control thereof.
- a regulatory sequence can be, for example, a minimal promoter sequence, a complete promoter sequence, an induced active promoter, an enhancer sequence, an upstream activation sequence ("UAS”), an operator sequence, a downstream termination sequence, a polyadenylation sequence, an optimal 5' leader sequence to optimize initiation of translation, or a Shine-Dalgarno sequence.
- the regulatory sequence can contain a hybrid of promoters of any of the above, such as a hybrid enhancer/promoter element.
- the regulatory sequence that is appropriate for expression -of the gene of interest differs depending upon the host system in which the construct is to be expressed. Selection of the appropriate regulatory sequences for use herein is within the capability of one skilled in the art. In eukaryotes, for example, such a sequence can include one or more of a promoter sequence and/or a transcription termination sequence.
- Regulatory sequences suitable for use herein may be derived from any source including a prokaryotic source, an eukaryotic source, a virus, a viral vector, a bacteriophage or a linear or circular plasmid.
- the regulatory sequence herein can also be a synthetic sequence, for example, one made by combining the UAS of one gene with the remainder of a requisite promoter from another gene, such as the GADP/ADH2 hybrid promoter.
- a regulatory sequence can also be a repressor sequence.
- "Mammalian cell” as used herein refers to a subset of eukaryotic cells useful in the invention as host cells, and includes human cells, and animal cells such as those from dogs, cats, cattle, horses, rabbits, mice, goats, pigs, etc. The cells used can be genetically unaltered or can be genetically altered, for example, by transformation with appropriate expression vectors, marker genes, and the like.
- Mammalian cells suitable for the method of the invention are any mammalian cell capable of expressing the genes of interest, or any mammalian cells that can express a cDNA library, cRNA library, genomic DNA library or any protein or polypeptide useful in the method of the invention.
- Mammalian cells also include cells from cell lines such as those immortalized cell lines available from the American Type Culture Collection (ATCC).
- ATCC American Type Culture Collection
- Such cell lines include, for example, rat pheochromocytoma cells (PC 12 cells), embryonal carcinoma cells (PI 9 cells), Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human embryonic kidney cells, mouse sertoli cells, canine kidney cells, buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumor cells, as well as others. Also included are hematopoetic stem cells, neuronal stem cells such as neuronal sphere cells, and embryonic stem cells (ES cells).
- PC 12 cells rat pheochromocytoma cells
- PI 9 cells Chinese hamster ovary
- HeLa cells HeLa cells
- baby hamster kidney (BHK) cells baby hamster kidney (BHK) cells
- COS monkey kidney cells
- human hepatocellular carcinoma cells e.g., Hep G2
- a "polynucleotide sequence,” a "nucleic acid molecule,” a “nucleic acid sequence,” or a “coding sequence,” as used herein, refers to either RNA or DNA that encodes a specific amino acid sequence or its complementary strand.
- a nucleic acid molecule may also be an oligonucleotide probe that may or may not encode a functional peptide, for example, an antisense oligonucleotide sequence, or a ribozyme.
- analog refers to splice variants, truncations, variants, alleles and derivatives and the like, of a mature protein Unless specifically mentioned otherwise, the “analogs” possess one or more of the bioactivities of the "mature protein,” or possess the bioactivity of the peptide
- peptides or polypeptides that are identical or contain at least 60%, preferably 70%, more preferably 80%, and most preferably 90% amino acid sequence homology to the amino acid sequence of the mature protein or the peptide wherever derived, from human or nonhuman sources, are included within this definition
- amino acid substitutions can be conservative ammo acid substitutions or substitutions to eliminate non-essential ammo acid residues such as to alter a glycosylation site, a phosphorylation site, an acetylation site, or to minimize misfolding by substitution or deletion of one or more cysteine residues that are not necessary for function.
- Conservative amino acid substitutions are those that preserve the general charge, hydrophobic ⁇ ty/ hydrophihcity and or ste ⁇ c bulk of the am o acid substituted, for example, substitutions between the members of the following groups are conservative substitutions: Gly/Ala, Val/Ile/Leu, Asp/Glu, Lys/Arg, Asn/Gln, Ser/Thr/Cys and Phe/Trp/Tyr.
- the analogs herein further include peptides having one or more peptide mimics, also known as peptoids, that possess the bioactivity of the protein.
- polypeptides containing one or more analog ammo acid including, for example, unnatural ammo acids, etc.
- polypeptides with substituted linkages as well as other modifications known in the art, both naturally occur ⁇ ng and non-naturally occurring.
- polypeptide also does not exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like
- binding pair refers to a pair of molecules, usually referring to a protein/protein pair, but does not exclude a protein/DNA pair, or a protein/RNA pair, or DNA/DNA pair, DNA/RNA pair or RNA RNA pair, and can include small molecules that bind protein or DNA or RNA
- the components of such pair bind specifically to each other with a higher affinity than to a random molecule, such that upon binding, for example, in case of a ligand receptor interaction, the binding pair triggers a cellular or an intracellular response, or forms a complex.
- An example of a ligand/receptor binding pair -I l ⁇ l s a pair formed between PDGF (platelet derived growth factor) and a PDGF receptor.
- An example of a different binding pair is an antigen/antibody pair in which the antibody is generated by immunization of a host with the antigen.
- An example of an organic molecule - protein binding pair is the binding of retmoic acid with its protein receptor, the retmoic acid receptor.
- Specific binding indicates a binding interaction having a low dissociation constant, which distinguishes specific binding from non-specific, background, binding.
- a low dissociation constant would be, for example, 1.0 ⁇ M, more preferably 10 nM, still more preferably 1.0 nM or less.
- antagonist refers to a molecule that blocks signalling to a detectable degree, as for example, a molecule that can bind a receptor, but which does not cause a signal to be transduced by the receptor to the cell.
- the peptide antagonist can bind, for example, the uPAR receptor at or near the integrin binding site, and prevent integrin from forming a binding pair with uPAR.
- agonist refers to a molecule that mimics the signalling in the pathway under study, for example, by binding a receptor and promoting a signal transduction to the cell through the receptor.
- an agonist of a peptide antagonist of uPAR would mimic or be able to compete with the peptide antagonist for blocking the formation of a uPAR:integrin binding pair.
- Small molecules or peptoids can be screened for the ability to perform the same or similar function of a peptide antagonist of the uPAR: ⁇ nteg ⁇ n binding pair interaction.
- uPAR The urokinase plasminogen activator receptor "uPAR” as used herein refers to the urokinase plasminogen activator receptor.
- uPAR is a glycosylphosphatidyl -mositol- linked urokinase and vitronectin receptor. uPAR is expressed on many cells as a consequence of cytokine stimulation or malignant transformation as desc ⁇ bed in Blasi et al, J. Cell. Biol. 104: 801 (1987).
- Urokinase plasminogen activator refers to a se ⁇ ne protease capable of activating urokinase plasminogen. When bound to its cell surface receptor, uPAR, uPA converts plasminogen to plasmin.
- uPA a se ⁇ ne protease capable of activating urokinase plasminogen. When bound to its cell surface receptor, uPAR, uPA converts plasminogen to plasmin.
- “Integ ⁇ n” as used herein refers to the integrin family of cell adhesion receptors known to mediate cell attachment to extracellular matrix proteins and also known to play a critical role in cell moti ty
- cytoskeletal disorder refers to a disorder in a patient that can be characterized at least in part by the formation of an abnormal condition in the cytoskeleton of at least one tissue of the patient Cytoskeletal abnormalities can be associated with a variety of conditions, including, for example, tumor growth, metastatic cancer, angiogenesis, wounds, and other disorders
- EGF epidermal growth factor
- uPA urokinase plasminogen activator
- uPAl-48 am o acids 1 to 48 of urokinase
- uPAR urokinase plasminogen activator receptor
- sUPAR soluble truncated form of the urokinase receptor
- uPA 13-32 amino acids 13-32 of human urokinase with Cysl9 converted to Ala
- PAI-1 plasminogen activator inhibitor type-1, ATF, amino terminal fragment of uPA
- HRP horse radish peroxidase, PBS, phosphate buffered saline, BSA, bovine serum albumin
- the invention is the use of bacteriophage display to identify novel functional sites on proteins Using this novel application of bacte ⁇ phage display techniques, the inventors have identified novel peptide sequences that bind to the human urokinase receptor in the presence of the receptor binding region of human urokinase, and so identified novel functional sites
- the identified peptides define two new functional sites on the urokinase receptor.
- the first is a site that corresponds to the interaction site of urokinase:urok ase receptor complexes with vitronectin and show homology to the somatomedin B domain of vitronectin.
- the second functional site is involved m a previously unexpected interaction of the urokinase receptor with mteg ⁇ ns and likely defines the ⁇ nteg ⁇ n:urok ⁇ nase receptor interface Modulation of this second site can lead to alterations in integrin activity/specificity and affect cell adhesion and other integrin mediated events.
- the invention includes three peptides that inhibit the uPAR vitronectin binding interaction, peptide 7 (SEQ ID NO 1 ), peptide 9 (SEQ ID NO 2), and peptide 18 (SEQ ID NO 3), and use of these peptides to inhibit the uPAR vitronectin interaction
- Vitronectin has been implicated in binding to uPAR as desc ⁇ bed in Waltz et al J.Bwl Chem 269 14746-14750-f 1994) It has been shown that the urokinase receptor can be a uPA dependent adhesion receptor for vitronectin as desc ⁇ bed in Wei et al, J Biol Chem 269 32380-32388 ( 1994)
- Vitronectin is a complex giycoprotein with a modular domain structure which exists in both circulating and extracellular matrix forms as desc ⁇ bed in Preissner et al, Annu.Rev Cell Biol 7 275-310 ( 1991 ) It interacts with
- integrin site such as peptide 25 (SEQ ID NO:4) Clone 25 represents a distinct sequence motif, and based on the equivalent binding to D23, identify a unique binding site on suPAR.
- sequence motif we have determined to be necessary but not sufficient for inhibiting the binding pair interaction between uPAR and integrin is
- GYZY where Z is M or V.
- Peptide 25 has been shown to bind to the urokinase receptor and modulates integrin function.
- the sequence of peptide 25 is AESTYHHLSLGYMYTLN, where, by alanine replacement the am o acids YHXLXXGYMYT, where X is any amino acid were determined to be important for inhibiting uPAR binding to integrin.
- a further aspect of the invention is the use of peptide 25 as a lead compound and a tool for assay development of other molecules with the same activity, for example, small molecules and peptoids.
- the EGF-like domain is the receptor binding motif and binds to uPAR with similar affinity (0.1 - 5 nM) as uPA.
- the 15mer random peptide bacteriophage library as described in Devlin et al, Science 249' 404-406 (1990) was affinity selected on suPAR:uPAl-48 complexes immobilized on magnetic beads.
- an in vitro ELISA based assay for this interaction. Under the conditions of the assay binding of biotinylated uPAR to vitronectin is strictly dependent on uPAl-48.
- clones 7 and 18, which show greatly reduced competition for uPAl-48 binding also inhibit complex binding, while a scrambled version of clone 7 (having the same amino acids as clone 7, but in a different order) does not. None of the peptides when tested alone increased the binding of biotinylated sUPAR to vitronectin.
- vitronectin binding by uPAR correlated with cell adhesion of stimulated U937 cells as described in Wei et al, J. Biol.Chem.
- bacteriophage peptides that are homologous to the somatomedin B domain of vitronectin, the binding site of PAI- 1.
- clones 7 and 18 share the sequence E-L-D just N-terminal to the conserved leucine, whereas the related sequence D-E-L is found in the somatomedin B domain of vitronectin at residues 22-24, adjacent to the conserved sequence LCSYY.
- residues in peptide 7 are important for uPAR binding and inhibition of vitronectin binding
- the results shown in Fig 5, indicate that the residues conserved between the peptides and vitronectin are important for activity in these assays.
- uPAR domain2-3 fragment binds bacteriophage but not uPAl-48.
- uPAR is the only member of the Ly6/CD59 family to contain three repeats of the homologous cysteine containing domain Plough et al, FEBS Lett. 349: 163-168 (1994).
- the binding site for vitronectin on uPAR is in domains 2 and 3 (D23) as described in Wei et al. J. Biol.Chem. 269: 32380-32388 (1994).
- the secreted protein was purified on an anti-epitope affinity column, and was tested first for its ability to compete in the suPAR binding assay. There was no competition in this assay at 100 nM D23, in contrast to intact suPAR which shows an IC50 of 0.1 nM under the same conditions.
- the inventors then tested the ability of various uPAR bacteriophage displayed ligands to bind to immobilized D23.
- the results shown in FIG 6, indicate that the ligands fall into three different classes with respect to binding to D23 and sUPAR.
- Clone 20 and 13-32 bind signficantly only to intact sUPAR, whereas clones 9 and 25 bind equivalently to the D23 fragment and full-length receptor.
- Bacteriophage bearing clones 7 and 18 peptides show an intermediate degree of binding to D23, and substantially better binding to an intact receptor.
- Integrins are a class of heterodimeric receptors implicated in adhesive interactions that regulate cell trafficking and intracellular signalling events important to cellular differentiation, migration and survival as described in Dustin et al, Nature 329: 846 (1987) and Shattil et al, Curr. Opin. Cell Biol. 6: 695 (1994). Adhesion of cells via integrins requires, in addition to ligand binding, a reorganization of intregrin distribution and assembly of connecting elements that link integrins to the cytoskeleton as described in Miyamoto et al, Science 267 :883 (1995) and Burridge et al, Annu. Rev. Cell Biol. 4: 487 (1988).
- ⁇ l integrins have been extensively studied in this regard.
- the cytoplasmic tail of ⁇ 1 chains binds talin and alpha-actinin, which themselves interact directly with actin as described in Otey et al, J. Cell. Biol. 111: 121 (1990), and Schaller et al, J. Cell. Biol. 130: 1181 (1988).
- the assembly of such cytoskeletal connections is not strictly a consequence of cell surface expression, but frequently requires secondary cell signaling as described in Faull et al, J. Cell. Biol. 121 : 155 (1993), Masumoto et al, J. Biol. Chem. 268: 228 (1993), and Burn et al, Proc. Nat'l.
- the invention includes the development of reagents such as that prototyped by peptide 25 demonstrated to disupt uPAR integrin associations and restore integrin function, or reagents comparable o soluble uPAR which impair integrin function, such as for example, antibodies to the site on integrin of uPAR: ⁇ ntegrin binding, for use in modifying inflammation and tumor progression.
- reagents such as that prototyped by peptide 25 demonstrated to disupt uPAR integrin associations and restore integrin function
- reagents comparable o soluble uPAR which impair integrin function such as for example, antibodies to the site on integrin of uPAR: ⁇ ntegrin binding
- peptide 18 blocks binding of complexes to vitronectin, as does peptide 7.
- the invention also includes methods for screening for molecular mimics of the inhibitory activity of the peptides of the invention, for example peptide 7 and peptide 25, for the purpose of identifying, for example, small molecule or peptoid inhibitors of uPAR:vitronectin or uPAR:integrin binding interactions.
- Such antagonists of uPAR interactions can be, for example, peptide derivatives such as peptoids, small molecules, or polynucleotides.
- antagonists are useful for development of therapeutics for treatment of conditions characterized by uPAR: vitronectin binding or by uPAR:integrin binding, or more generally, by upregulation of uPA and uPAR, where cell adhesion is compromised.
- the instant peptides and antagonist can be useful in treating a disease state or malady which is caused or exacerbated by the biological activity of uPA or uPAR.
- the conditions may also be characterized, for example, by cell migration and invasion, as seen in such disorders as, for example, tumor cell invasion, metastatic disease, and the condition may also be chronic inflammation.
- the molecular mimics, peptoids or small molecules; or analogs, variants, or derivatives of the instant peptides exhibit a I of less than lO ⁇ M: more preferably, less than 5 ⁇ M, even more preferably less than l ⁇ M: even more preferably less than lOOnM; even more preferably less than 10 nM.
- huPAR or the complex of huPAR:integrin or vitronectin even more preferably less than 10 nM.
- any of the full-length, derivatives, or polypeptide or peptide inhibitors or antagonists of the invention can be cloned, expressed, or synthesized by standard recombinant DNA or chemical techniques. Some exemplary expression systems that can be applied for these purposes follow.
- Administration of the peptide, polypeptide. and polynucleotide therapeutics of the invention can conducted by administration of the synthesizied peptide or polypeptide, or by administration of a polynucleotide for expression in an animal, or by administration of a non-coding polynucleotide inhibitor. Further below are also provided methods of making small molecule and peptoid library pools for screening for the desired activity.
- gene therapy techniques for administering a polynucleotide of the invention to a patient for the purpose of expressing the polypeptide or peptide encoded by the polynucleotide or nucleic acid molecule in the animal.
- non-coding nucleic acid molecules such as for example, ribozymes and antisense molecules can be administered with an appropriate pharmaceutically acceptable carrier.
- Control elements for use in bacteria include promoters, optionally containing operator sequences, and ribosome binding sites.
- Useful promoters include sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp), the ⁇ -lactamase (bla) promoter system, bacteriophage ⁇ PL, and T7.
- synthetic promoters can be used, such as the tac promoter.
- ⁇ - lactamase and lactose promoter systems are described in Chang et al, Nature (1978) 275: 615, and Goeddel et al., Nature (1979) 281: 544; the alkaline phosphatase, tryptophan (tip) promoter system are described in Goeddel et al., Nucleic Acids Res. (1980) 8: 4057 and EP 36,776 and hybrid promoters such as the tac promoter is described in U.S. Patent No. 4,551,433 and de Boer et al, Proc. Natl. Acad. Sci. USA (1983) 80: 21-25. However, other known bacterial promoters useful for expression of eukaryotic proteins are also suitable.
- Promoters for use in bacterial systems also generally will contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the target polypeptide.
- SD Shine-Dalgarno
- the signal sequence can be substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, Ipp, or heat stable enterotoxin II leaders.
- the origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria.
- the foregoing systems are particularly compatible with Escherichia coli.
- numerous other systems for use in bacterial hosts including Gram-negative or Gram-positive organisms such as Bacillus spp., Streptococcus spp., Streptomyces spp., Pseudomonas species such as P. aeruginosa. Salmonella typhimurium, or Serratia marcescans, among others.
- Methods for introducing exogenous DNA into these hosts typically include the use of CaCb or other agents, such as divalent cations and DMSO.
- DNA can also be introduced mtcrbacte ⁇ al cells by electroporation, nuclear injection, or protoplast fusion as described generally in Sambrook et al ( 1989), MOLECULAR CLONING. A LABORATORY MANUAL. 2d edition (Cold Spring Harbor Press, Cold Spring Harbor, N Y ) These examples are illustrative rather than limiting
- the host cell should secrete minimal amounts of proteolytic enzymes
- in vitro methods of cloning e.g., PCR or other nucleic acid polymerase reactions, are suitable
- Prokaryotic cells used to produce the target polypeptide of this invention are cultured suitable media, as described generally in Sambrook et al. ( 1989), MOLECULAR CLONING A LABORATORY MANUAL, 2d edition (Cold Sp ⁇ ng Harbor Press, Cold Sp ⁇ ng Harbor, N Y ),
- Expression and transformation vectors have been developed for transformation into many yeasts
- expression vectors have been developed for, among others, the following yeasts: Saccharomyces cerevisiae ,as desc ⁇ bed in Hmnen et al, Proc. Natl Acad. Sci. USA (1978) 75. 1929; Ito et al, J. Bactenol. (1983) 153. 163, Candida albicans as desc ⁇ bed in Kurtz et al, Mol Cell. Biol (1986) 6. 142, Candida maltosa, as desc ⁇ bed in Kunze et al, J.
- Control sequences for yeast vectors are known and include promoters regions from genes such as alcohol dehydrogenase (ADH), as described in EP 284,044, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglycerate mutase. and pyruvate kinase (PyK), as described in EP 329,203.
- the yeast PH05 gene, encoding acid phosphatase also provides useful promoter sequences, as described in Myanohara et al, Proc. Natl. Acad.
- promoter sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase, as described in Hitzeman et al, J. Biol. Chem. (1980) 255: 2073, or other glycolytic enzymes, such as pyruvate decarboxylase, triosephosphate isomerase, and phosphoglucose isomerase, as described in Hess et al, J. Adv. Enzyme Reg. (1968) 7: 149 and Holland et al, Biochemistry (1978) 77:4900.
- Inducible yeast promoters having the additional advantage of transcription controlled by growth conditions, include from the list above and others the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
- suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EP 073,657.
- Yeast enhancers also are advantageously used with yeast promoters.
- synthetic promoters which do not occur in nature also function as yeast promoters.
- upstream activating sequences (UAS) of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter.
- hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region, as described in U.S. Patent Nos. 4,876,197 and 4,880,734.
- Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2, GALA, GAL10, or PH05 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK, as described in EP 164,556.
- a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription
- control elements which may be included in the yeast expression vectors are terminators, for example, from GAPDH and from the enolase gene, as described in Holland et al , J Biol Chem (1981) 256 1385, and leader sequences which encode signal sequences for secretion DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast mvertase gene as described in EP 012,873 and JP 62,096,086 and the a-factor gene, as described in U.S.
- Methods of introducing exogenous DNA into yeast hosts are well known in the art, and typically include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations Transformations into yeast can be earned out according to the method desc ⁇ bed in Van Sohngen et al, J. Bact. (1977) 130:946 and Hsiao et al , Proc Natl. Acad.
- yeast secretion the native target polypeptide signal sequence may be substituted by the yeast mvertase, ⁇ -factor, or acid 5-phosphatase leaders
- yeast A suitable selection gene for use in yeast is the trp ⁇ gene present in the yeast plasmid described in Kingsman et al. Gene (1979) 7 141 or Tschemper et al, Gene (1980) 10.
- the trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan Similarly, Leu2-defic ⁇ ent yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 Gene
- a sequence encoding a yeast protein can be linked to a coding sequence of the polypeptide to produce a fusion protein that can be cleaved intracellularly by the yeast cells upon expression
- a yeast leader sequence is the yeast ubiquitin gene Expression in Insect Cells
- Baculovirus expression vectors are recombinant insect viruses in which the coding sequence for a foreign gene to be expressed is inserted behind a baculovirus promoter in place of a viral gene, e.g., polyhedrin, as described in Smith and Summers. U.S. Pat. No., 4,745,051.
- An expression construct herein includes a DNA vector useful as an intermediate for the infection or transformation of an insect cell system, the vector generally containing DNA coding for a baculovirus transcriptional promoter, optionally but preferably, followed downstream by an insect signal DNA sequence capable of directing secretion of a desired protein, and a site for insertion of the foreign gene encoding the foreign protein, the signal DNA sequence and the foreign gene being placed under the transcriptional control of a baculovirus promoter, the foreign gene herein being the coding sequence of the polypeptide.
- the promoter for use herein can be a baculovirus transcriptional promoter region derived from any of the over 500 baculoviruses generally infecting insects, such as, for example, the Orders Lepidoptera, Diptera, Orthoptera, Coleoptera and Hymenoptera including, for example, but not limited to the viral DNAs of Autographo californica MNPV, Bombyx mori NPV, rrichoplusia ni MNPV, Rachlplusia ou MNPV or Galleria mellonella MNPV.
- the baculovirus transcriptional promoter can be, for example, a baculovirus immediate-early gene EEI or DEN promoter; an immediate-early gene in combination with a baculovirus delayed-early gene promoter region selected from the group consisting of a 39K and a Hr ' ndlll fragment containing a delayed-early gene; or a baculovirus late gene promoter.
- the immediate-early or delayed-early promoters can be enhanced with transcriptional enhancer elements.
- Particularly suitable for use herein is the strong polyhedrin promoter of the baculovirus, which directs a high level of expression of a DNA insert, as described in Friesen et al. (1986) "The Regulation of Baculovirus Gene Expression” in: THE MOLECULAR BIOLOGY OF BACULOVIRUSES (W.Doerfler, ed.); EP 127.839 and EP 155,476; and the promoter from the gene encoding the plO protein, as described in Vlak et al, J. Gen. Virol. (1988) 69:165-116.
- the plasmid for use herein usually also contains the polyhedrin polyadenylation signal, as described in Miller et al , Ann Rev Microbiol ( 1988) 42 177 and a procaryotic ampicil n-resistance (amp) gene and an origin of replication for selection and propagation in E coli
- DNA encoding suitable signal sequences can also be included and is generally derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene, as described in Carbonell et al, Gene ( 1988) 73 409, as well as mammalian signal sequences such as those derived from genes encoding human a-mterferon as described in Maeda et al , Nature ( 1985) J75:592-594, human gast ⁇ n- releasing peptide, as described in Lebacq-Verheyden et al, Mol Cell Biol (1988) 8 3129; human IL-2, as described in Smith et al, Proc Natl.
- baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frug ⁇ erda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosoph ⁇ a melanogaster (fruitfly), and
- Bombyx mon host cells have been identified and can be used herein See, for example, the descnption m Luckow et al., B ⁇ o/Technology(198&) 6: 47-55, Miller et al , in GENETIC ENGINEERING (Setlow, J.K. et al. eds.), Vol 8 (Plenum Publishing, 1986), pp. 277-279, and Maeda et al, Nature, (1985) 315 592-594.
- viruses are publicly available, e g., the L-l variant of Autographa californica NPV and the Bm-5 strain oi Bombyx mon NPV Such viruses may be used as the virus for transfection of host cells such as Spodoptera frug ⁇ erda cells
- baculovirus genes in addition to the polyhedrin promoter may be employed to advantage in a baculovirus expression system. These include immediate-early (alpha), delayed-early (beta), late (gamma), or very late (delta), according to the phase of the viral infection during which they are expressed The expression of these genes occurs sequentially, probably as the result of a ' cascade" mechanism of transcriptional regulation.
- the immediate-early genes are expressed immediately after infection, in the absence of other viral functions, and one or more of the resulting gene products induces transcription of the delayed-early genes
- Some delayed-early gene products induce transcription of late genes, and finally, the very late genes are expressed under the control of previously expressed gene products from one or more of the earlier classes.
- IEI a preferred immediate-early gene of Autographo calif ornica nuclear polyhedrosis virus (AcMNPV).
- AcMNPV Autographo calif ornica nuclear polyhedrosis virus
- IEI is pressed in the absence of other viral functions and encodes a product that stimulates the transcription of several genes of the delayed-early class, including the preferred 39K gene, as described in Guarino and Summers, J. Virol. ( 1986) 57:563-571 and J. Virol. (1987) 67:2091-2099 as well as late genes, as described in Guanno and Summers, Virol (1988) 762:444-451.
- Immediate-early genes as described above can be used in combination with a baculovirus gene promoter region of the delayed-early category. Unlike the immediate-early genes, such delayed-early genes require the presence of other viral genes or gene products such as those of the immediate-early genes.
- the combination of immediate-early genes can be made with any of several delayed-early gene promoter regions such as 39K or one of the delayed-early gene promoters found on the Hi/idi ⁇ fragment of the baculovirus genome. In the present instance, the 39 K promoter region can be linked to the foreign gene to be expressed such that expression can be further controlled by the presence of IEI, as described in L. A.
- enhancement of the expression of heterologous genes can be realized by the presence of an enhancer sequence in direct cis linkage with the delayed-early gene promoter region.
- enhancer sequences are characterized by their enhancement of delayed-early gene expression in situations where the immediate-early gene or its product is limited.
- the hr5 enhancer sequence can be linked directly, in cis, to the delayed-early gene promoter region, 39K, thereby enhancing the expression of the cloned heterologous DNA as described in Guarino and Summers (1986a), (1986b), and Guarino et al. (1986).
- the polyhedrin gene is classified as a very late gene. Therefore, transcription from the polyhedrin promoter requires the previous expression of an unknown, but probably large number of other viral and cellular gene products. Because of this delayed expression of the polyhedrin promoter, state-of-the-art BEVs, such as the exemplary BEV system described by Smith and Summers in, for example. U.S. Pat. No.. 4.745.051 will express foreign genes only as * a result of gene expression from the rest of the viral genome, and only after the viral infection is well underway. This represents a limitation to the use of existing BEVs. The ability of the host cell to process newly synthesized proteins decreases as the baculovirus infection progresses.
- gene expression from the polyhedrin promoter occurs at a time when the host cell's ability to process newly synthesized proteins is potentially diminished for certain proteins such as human tissue plasminogen activator.
- the expression of secretory glycoproteins in BEV systems is complicated due to incomplete secretion of the cloned gene product, thereby trapping the cloned gene product within the cell in an incompletely processed form.
- an insect signal sequence can be used to express a foreign protein that can be cleaved to produce a mature protein
- the present invention is preferably practiced with a mammalian signal sequence appropriate for the gene expressed.
- An exemplary insect signal sequence suitable herein is the sequence encoding for a Lepidopteran adipokinetic hormone (AKH) peptide.
- the AKH family consists of short blocked neuropeptides that regulate energy substrate mobilization and metabolism in insects.
- a DNA sequence coding for a Lepidopteran Manduca sexta AKH signal peptide can be used.
- Other insect AKH signal peptides, such as those from the Orthoptera Schistocerca gregaria locus can also be employed to advantage.
- Another exemplary insect signal sequence is the sequence coding for Drosophila cuticle proteins such as CPI, CP2, CP3 or CP4.
- the desired DNA sequence can be inserted into the transfer vector, using known techniques.
- An insect cell host can be cotransformed with the transfer vector containing the inserted desired DNA together with the genomic DNA of wild type baculovirus, usually by cotransfection.
- the vector and viral genome are allowed to recombine resulting in a recombinant virus that can be easily identified and purified.
- the packaged recombinant virus can be used to infect insect host cells to express a desired polypeptide.
- Typical promoters for mammalian cell expression of the polypeptides of the invention include the SV40 early promoter, the CMV promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), and the herpes simplex virus promoter, among others.
- Other non-viral promoters such as a promoter derived from the murine metallothionein gene, will also find use in mammalian constructs.
- Mammalian expression may be either constitutive or regulated (inducible), depending on the promoter. Typically, transcription termination and polyadenylation sequences will also be present, located 3' to the translation stop codon.
- a sequence for optimization of initiation of translation located 5' to the polypeptide coding sequence, is also present.
- transcription terminator/polyadenylation signals include those derived from SV40, as described in Sambrook et al. ( 1989). cited previously. Introns, containing splice donor and acceptor sites, may also be designed into the constructs of the present invention.
- Enhancer elements can also be used herein to increase expression levels of the mammalian constructs. Examples include the SV40 early gene enhancer, as desc ⁇ bed in Dijkema et al, EMBO J. ( 1985) 4:761 and the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al, Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and human cytomegalovirus, as described in Boshart et al, Cell ( 1985) 41:521.
- a leader sequence can also be present which includes a sequence encoding a signal peptide, to provide for the secretion of the foreign protein in mammalian cells.
- adenovirus t ⁇ partite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.
- the mammalian expression vectors can be used to transform any of several mammalian cells.
- Methods for introduction of heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
- dextran-mediated transfection calcium phosphate precipitation
- polybrene mediated transfection protoplast fusion
- electroporation electroporation
- encapsulation of the polynucleotide(s) in liposomes and direct microinjection of the DNA into nuclei.
- General aspects of mammalian cell host system transformations have been described by Axel in U.S. Patent No. 4,399,216.
- the mammalian host cells used as responsive cells or producing cells in the invention may be cultured in a variety of media.
- Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM1, Sigma) are suitable for cultu ⁇ ng the host cells.
- U.S. RE 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary to create optimal conditions for the function of the cells according to the method of the invention, including supplementation as necessary with hormones and/or other growth factors such as insulin, transfer ⁇ n, or epidermal growth factor, salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (sueh as adenosine and thymidine), antibiotics (such as GentamycmTM M drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source range) Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art
- the culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan
- Gene therapy strategies for delivery of constructs of the invention can utilize viral or non- viral vector approaches in in vivo or ex vivo modality Expression of such coding sequence can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence in vivo can be either constitutive or regulated
- any of a number of viral vectors can be used, as descnbed in Jolly, Cancer Gene Therapy 1: 51-64 (1994).
- the coding sequence can be inserted into plasmids designed for expression retroviral vectors, as described in Kimura et al, Human Gene Therapy (1994) 5: 845-852, adenoviral vectors, as desc ⁇ bed in Connelly et al, Human Gene Therapy (1995) 6. 185-193, adeno- associated viral vectors, as described in Kap tt et al, Nature Genetics (1994) 6: 148-153 and smdbis vectors.
- Promoters that are suitable for use with these vectors include the Moloney retroviral LTR, CMV promoter and the mouse albumin promoter. Replication incompetent free virus can be produced and injected directly into the animal or humans or by transduction of an autologous cell ex vivo, followed by injection in vivo as described in Zatloukal et al., Proc Natl. Acad. Sci USA (1994) 91 5148-5152.
- the altered coding sequence can also be inserted into plasmid for expression of the uPAR polypeptide in vivo or ex vivo
- the coding sequence can be delivered by direct injection into tissue or by intravenous infusion
- Promoters suitable for use in this manner include endogenous and heterologous promoters such as CMV
- a synthetic T7T7 T70B promoter can be constructed in accordance with Chen et al (1994), Nucleic Acids Res. 22.
- the T7 polymerase is under the regulatory control of its own promoter and drives the transc ⁇ ption of the uPAR coding sequence, which is also placed under the control of a T7 promoter
- the coding sequence can be injected in a formulation comprising a buffer that can stablize the coding sequence and facilitate transduction thereof * into cells and or provide targeting, as described in Zhu et al. Science ( 1993) 267: 209-21 1
- Expression of the coding sequence in vivo upon delivery for gene therapy purposes by either viral or non-viral vectors can be regulated for maximal efficacy and safety by use of regulated gene expression promoters as described in Gossen et al, Proc. Natl. Acad. Sci. USA (1992) 89:5547-5551
- the uPAR coding sequence can be regulated by tetracycline responsive promoters. These promoters can be regulated in a positive or negative fashion by treatment with the regulator molecule.
- the sequence can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polyme ⁇ c DNA-binding cations like polylysine, protarmne, and albumin, linked to cell targeting ligands such as asialoorosomucoid, as described in Wu and Wu, J. Biol. Chem. (1987) 262: 4429-4432; insulin, as described in Hucked et al, Biochem. Pharmacol. 40: 253-263 (1990); galactose, as described in Plank et al, Bioconjugate Chem.
- synthetic gene transfer molecules such as polyme ⁇ c DNA-binding cations like polylysine, protarmne, and albumin, linked to cell targeting ligands such as asialoorosomucoid, as described in Wu and Wu, J. Biol. Chem. (1987) 262: 4429-4432; insulin, as described in Hucked et al, Bio
- non-viral delivery suitable for use includes mechanical delivery systems such as the biolistic approach, as desc ⁇ bed in Woffendin et al, Proc. Natl. Acad. Sci. USA (1994) 91(24): 11581-1 1585.
- the uPAR coding sequence and the product of expression of such can be delivered through deposition of photopolyme ⁇ zed hydrogel materials.
- Other conventional methods for gene delivery that can be used for delivery of the uPAR coding sequence include, for example, use of hand held gene transfer particle gun. as described in U.S. 5,149,655; use of ionizing radiation for activating transferred gene, as described in U.S. 5,206,152 and PCT application WO92/11033.
- gene therapy technology with regard to the peptides and polypeptides of the invention and their analogues or variants can be made in disease states where, for example, activity of any of uPAR is detrimental to the patien
- gene therapy using the polypeptides and peptides of the invention and their analogues or variants is appropriate when treating conditions of cytoskeletal disruption, for example, in vivo expression of antagonists or dominant negatives to interupt, for example, the uPAR:mteg ⁇ n binding pair formation and the cellular responses, such as cellular migration, that result from the binding pair formation of uPAR and integrin.
- gene therapy can be applied according to the invention m all situations where uPAR forms a binding pair interaction with vitronectin or integrin and acts to modulate cytoskeletal integrity and affect cellular migration, by administering according to a gene therapy protocol, of a sufficient amount of a peptide of the invention or its analogue, variant, or dominant negative, for example, for modulating the normal activity of uPAR binding pair interactions.
- peptides of the invention can be made in the context of treatment of a patient afflicted by a condition characterized by cytoskeletal disruption and/or also including cellular migration.
- Conditions of cancer and/or inflammatory conditions are examples of such conditions.
- assays can be developed for screening small molecule library pools for funcuonal uPAR.vitronect and uPAR:mtegnn inhibitors, antagonists, and agonists for use in controlling, for example, cytoskeletal disruption and cellular migration.
- inhibitors, antagonists, or agonists can be administered to the animal, and can be administered with a pharmaceutically acceptable carrier, including, for example, liposomes compositions such as DepofoamTM, and other carriers such as, for example, FocalgelTM.
- Small molecule libraries may be used to screen for the ability of the small molecule to mimic, synergize or attenuate any action of SIP, and can be made as follows
- a "library" of peptides may be synthesized and used following the methods disclosed in U S Patent No. 5,010,175, (the ' 175 patent) and in PCT W091/17823
- a suitable peptide synthesis support for example, a resin, is coupled to a mixture of appropriately protected, activated amino acids
- the method desc ⁇ bed in W091/17823 is similar However, instead of reacting the synthesis resin with a mixture of activated am o acids, the resin is divided into twenty equal portions, or into a number of portions corresponding to the number of different ammo acids to be added in that step, and each amino acid is coupled individually to its portion of resin The resin portions are then combined, mixed, and again divided into a number of equal portions for reaction with the second amino acid. Additionally, one may maintain separate "subpools" by treating portions in parallel, rather than combining all resins at each step. This simplifies the process of determining which peptides are responsible for any observed alteration of gene expression in a responsive cell
- a further alternative agents include small molecules, including peptide analogs and derivatives, that can act as stimulators or inhibitors of gene expression, or as ligands or antagonists.
- Some general means contemplated for the production of peptides, analogs or de ⁇ vatives are outlined in CHEMISTRY AND BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES, AND PROTEINS - A SURVEY OF RECENT DEVELOPMENTS, Wemstein, B. ed , Marcell Dekker, Inc , publ New York ( 1983)
- substitution of D-amino acids for the normal L-stereoisomer can be carried out to increase the half-life of the molecule.
- Peptoids polymers comprised of monomer units of at least some substituted amino acids, can act as small molecule stimulators or inhibitors herein and can be synthesized as described in PCT 91/19735
- Presently preferred amino acid substitutes are N-alkylated derivatives of glycine, which are easily synthesized and incorporated into polypeptide chains
- any monomer units which allow for the sequence specific synthesis of pools of diverse molecules are appropriate for use in producing peptoid molecules.
- the benefits of these molecules for the purpose of the invention is that they occupy different conformational space than a peptide and as such are more resistant to the action of proteases.
- Peptoids are easily synthesized by standard chemical methods. The preferred method of synthesis is the "submonomer" technique described by R.
- Combinatorial libraries of mixtures of such heterocyclic organic compounds can then be assayed for the ability to alter gene expression.
- Suitable carriers for the therapeutics of the invention for administration in a patient including but not limited to molecules capable of antagonizing the inhibitory effects of the peptides of the invention (for example peptides 7, 9, 18, and 25 and analogs or variants of these), including, for example small molecules, peptides, peptoids, polynucleotides and polypeptides, may be large, slowly metabolized macromolecules such as proteins, poly sacchar ides, poly lactic acids, poly gly colic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles.
- Such carriers are well known to those of ordinary skill in the art.
- Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
- mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like
- organic acids such as acetates, propionates, malonates, benzoates, and the like.
- Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
- the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
- Liposomes are included within the definition of a pharmaceutically acceptable carrier.
- liposomes refers to, for example, the liposome compositions described in U.S.
- Liposomes may be pharmaceutical carriers for the peptides, polypeptides or polynucleotides of the invention, or for combination of these therapeutics.
- Example 1 Affinity Selection of 15mer random peptide library on UPAR:uPAl-48 complexes
- Soluble recombinant human urokinase receptor (suPAR) was expressed and secreted from baculovirus-infected Sf9 insect cells, as described in Goodson et al, Proc.Natl.Acad.Sci.USA 91: 7129-7133 (1994).
- the EGF-like domain of human urokinase (uPA residues 1-48) was expressed from recombinant yeast as described Stratton-Thomas et al, Prot.Eng. 8: 463-470 (1995).
- UPAl-48 was purified by a revision of the published procedure, involving ion exchange chromatography and reverse phase HPLC under reducing conditions, followed by a refolding step and rechromatography on reversed phase HPLC of the oxidized material. Soluble uPAR was purified on a column of immobilized uPAl-48, eluted at low pH. biotinylated according to Kaufman et al. Anal. Biochem. 211: 261-266 ( 1993) and purified on a Soft-Avidin column (Promega Corporation. Madison, WI).
- VEYRDAYSYPQYLSYLE SEQ ID NO 8
- Recombinant PAI-1 was obtained from American Diagnostica.
- Horse-radish peroxidase (HRP) conjugated streptavidin was from Pierce Chemical, Rockford, IL.
- Anti-M13 antibody was from Pharmacia, Piscataway, NJ. Affinity selections were performed on streptavidin coated magnetic beads (Dynal,
- Biotinylated suPAR (1.5 ⁇ g) was mixed with 3.5 ⁇ g of uPAl-48 in a total volume of 100 ⁇ l for 30 minutes at room temperature. Magnetic beads were blocked with PBS/1% BSA (PSB/BSA) for 30 minutes and then suPAR:uPAl-48 complexes were added in PBS/0.1% BSA, and incubated at room temperature for 2 hours. Beads were then washed 3 times with PBS BSA and resuspended with an aliquot of the 15mer random peptide library in 500 ⁇ l.
- PSB/BSA PBS/1% BSA
- Streptavidin 100 ⁇ l (0.1 mg/ml) in 50 mM Na2C ⁇ 3, pH 9.6, was added to
- the urea eluate was brought to neutral pH by addition of 2M Tris base and the bacteriophage titers of input stocks and elutions measured by plaque formation assay. Results were expressed as the percent of input bacteriophage which bind to the wells. Alternatively, the amount of bacteriophage was determined in an ELISA where phage were preincubated with HRP-conjugated anti-M13 antibody for 30 minutes at room temperature before dispensing into wells prepared as above and incubated for one hour at room temperature. The final anti-M13 conjugate dilution was 1:4000. After washing, TMB substrate (100 ⁇ l/well) was added and color development was stopped with 0.8N H2SO4 (100 ⁇ l/well). The absorbance at 450 nm was then measured in a 96 well plate reader.
- Novel peptide sequences are obtained by panning uPAl-48:uPAR complexes.
- Selection of high affinity peptide ligands for the uPA binding site on uPAR was a relatively efficient process as described in Goodson et al, Proc.Natl.Acad.Sci.US A 91: 7129-7133 (1994), was extended by selecting for peptide-displaying bacteriophage with affinity for additional, functionally important sites on UPAR by including an excess of recombinant EGF-like domain of uPA (uPAl-48) to reduce selection of uPA binding site peptides as described in Stratton-Thomas et al, Prot.Eng. 8: 463-470 (1995).
- the EGF- like domain is the receptor binding motif as described in Appella et al, J. Biol.Chem. 262: 4437-444O (1987) and Robbiati et al Fibrinol. 4: 53-60 ( 1990), and binds to uPAR with similar affinity (0.1 - 5 nM) as uPA as described in Mazar et al, Fibrinol. 6: 49-55 (1992).
- the 15mer random peptide bacteriophage library as descrobed in Devlin et al, Science 249: 404-406 ( 1990) was affinity selected on suPAR:uPA 1-48 complexes lmmobihzed on magnetic beads. -The yield of bacteriophage increased 30 fold, from 0 008% at round 2 to 0.24% at round 3 suggesting enrichment for binding bacteriophage.
- Protein G 100 ⁇ l, 1 mg/ml in 50 mM Na2C ⁇ 3, pH 9.6, was added to MaxiSorp wells, incubated overnight at 4°C and then washed with PBS/BSA.
- Fifty ⁇ l of monoclonal antibody to the epitope tag EYMPME was added at 1 mg/ml in PBS/ BSA and incubated for 2 hours at room temperature The wells were washed, recombinant sUPAR domain 2-3 (1.7 ⁇ M in PBS/ BSA) was added and incubated for 1.5 hours at room temperature.
- Vitronectin was purified from human plasma by the method of Yatohgo et al, Cello Struct.and Fund. 13: 281-292 ( 1988). Purified vitronectin was diluted to 20 ⁇ g/ml in PBS containing 1 mM CaC12 and 0.5 mM MgCl2, dispensed at 50 ⁇ l/well into PBS containing 1 mM CaC12 and 0.5 mM MgCl2, dispensed at 50 ⁇ l/well into
- Immulon II wells (Dynatech, Chantilly, VA), incubated overnight at 4°C and washed with PBS/BSA.
- Biotinylated sUPAR was diluted to 20 nM in PBS/BSA, incubated with or without test ligand for 30 minutes at room temperature (22°C), dispensed at 100 ⁇ l/well and incubated for 90 minutes.
- Wells were then washed and horseradish peroxidase (HRP)-conjugated streptavidin was added at 0.4 ⁇ g/ml in PBS/2% BSA for 1 hour followed by washing and addition of 100 ⁇ l/well TMB substrate.
- HRP horseradish peroxidase
- bacteriophage derived peptides block complex binding and cell adhesion to vitronectin.
- the ability of the various bacteriophage derived peptides to affect binding of uPAl-48:uPAR complexes to vitronectin was assessed in the ELISA assay. Two classes of peptides were effective antagonists in this assay. First, clone 20 and uPA 13-32, which compete directly for uPAl-48 binding to sUPAR, reduced binding. An analog of clone 20 peptide, which shows greatly reduced receptor binding activity did not affect binding to vitronectin.
- Vitronectin reduced bacteriophage binding to the uPAl-48:suPAR complex by 5-10 fold, consistent with the hypothesis that these peptides mimic vitronectin as a uPAR ligand.
- vitronectin binding by uPAR correlated with cell adhesion of stimulated U937 cells as described in Wei et al, J. Biol.Chem. 269: 32380- 32388 (1994). It was found also that clone 7 peptide could block uPAR mediated adhesion of these cells, whereas the scrambled version of the same peptide had no effect. Additionally, binding of uPAl-48:uPAR complexes to vitronectin was shown to be blocked by PAI-1, vitronectin, and the somatomedin B domain of vitronectin.
- vitronectin Another function of vitronectin has been determined to be stabilization of the active conformation of PAI- 1 , which appears to occur via the somatomedin B domain of vitronectin, as described in Seiffert et al, J. Biol.Chem. 269: 2659-2666 (1994).
- PAI-1 is a very efficient competitor of uPAl-48:suPAR complexes binding to vitronectin, with an apparent IC50 of 10 nM. This suggested to the inventors that the binding site of uPAR and PAI-1 are overlapping.
- vitronectin binding to active PAI-1 is primarily via the somatomedin B domain, as described in Seiffert et al, J.Biol.Chem. 269: 2659-2666 (1994).
- the inventors tested whether vitronectin and recombinant somatomedin B domain would also inhibit uPAR binding to vitronectin. Accordingly, the inventors showed that molecules inhibit, whereas a point mutation of the domain does not.
- bacteriophage peptides are homologous to the somatomedin B domain of vitronectin, which is also the binding site of PAI- 1.
- sequences of bacteriophage derived peptides 7 and 18 were examined for homology to this domain.
- Example 4 SuPAR: l-An ⁇ l ⁇ no-8-Napthalenesulfonate (ANS) Fluorescence Measurements Determination of the effect of various peptide ligands on sUPAR/ANS fluorescence was performed following a procedure similar to that of Ploug et al, Biochem. 33: 8991-8997 (1994). Fluorescence emission spectra of sUPAR/ANS solutions with or without competitors were obtained using an Hitachi F-4500 fluorescence spectrophotometer with an excitation wavelength of 386 nm, 5-nm band- pass excitation and emission slits, and a 10 mm path length quartz cuvette. The emission spectra from 400 to 600 nm were recorded.
- ANS fluorescence enhancement distinguished the peptide sequences
- the inventors examined their effects on the fluorescence enhancement of ANS which occurs upon uPAR binding, and which has been shown to correlate with occupancy of the uPA binding site and the functional state of the uPAR molecule of Ploug et al, Biochem 33 8991 -8997 ( 1994)
- the effects of several peptide uPAR ligands on ANS fluorescence enhance ent in the presence of uPAR had the expected result that uPAl-48 and clone 20 reduce ANS fluorescence, consistent with their potent activity in the receptor binding assay.
- Clone 7 also reduced fluorescence in a dose dependent manner, although at higher concentrations, while clone 25 peptide has no effect at up to 20 ⁇ M. These results suggested that clone 7, 20, and uPAl-48 share some common binding determinants or a common binding conformation of uPAR with ANS, whereas clone 25 binds to a distinct site.
- UPAR is the only member of the Ly6/CD59 family to contain three repeats of the homologous cysteine containing domain as described in Plough et al, FEBS Lett. 349: 163-168 (1994).
- Previous work by the inventors suggested that the binding site for vitronectin on uPAR is in domains 2 and 3 (D23) as described by Wei et al, J. Biol.Chem. 269: 32380-32388 ( 1994).
- baculovirus infected Sf9 insect cells a fragment of suPAR, residues 93-313, predicted to encompass the second and third CD59 homologous domains with a C-terminal 6 amino acid epitope tag.
- the secreted protein was purified on an anti-epitope affinity column, and was tested first for its ability to compete in the suPAR binding assay. There was no competition in this assay at 100 nM D23, in contrast to intact suPAR which shows an IC50 of 0.1 nM under the same conditions.
- the inventors then tested the ability of various uPAR bacteriophage displayed ligands to bind to immobilized D23.
- the results shown in FIG 5, indicate that the ligands fall into three different classes with respect to binding to D23 and sUPAR.
- Clone 20 and 13-32 bind signficantly only to intact sUPAR, whereas clones 9 and 25 bind equivalently to the D23 fragment and full-length receptor.
- Bacteriophage bearing clones 7 and 18 peptides show an intermediate degree of binding to D23, and substantially better binding to intact receptor.
- the chimeric cDNA was subcloned into pBluescript, verified by nucleotide sequenceing (Sequenase, United States Biochemical Corp) then digested with Xbal and Xhol and finally subcloned into the pCEP4 expression vector. Co-transfectants were shown to bind equivalent amounts of vitronectin and urokinase at 4°C by methods as described in Wei et al, J. Biol. Chem. 269: 32380 (1994).
- Immunoblotting confirmed comparable expression of GPI-uPAR and TM-uPAR in 293 cells as well as comparable urokinase and vitronectin binding.
- triton X 100 (0.2%) insoluble fraction of GPI-uPAR 293 cells is solubilized in polar detergents, immunoprecipitation of ⁇ l clearly co-precipitates uPAR as described in Filardo et al, J. Cell Biol.
- the insoluble residues were solubolized at 4°C for 20 minutes in IX RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl, pH 7.5, 1% deoxycholate, 0.1% sodium dodecyl sulfate, 1% Triton X-100) supplemented with protease inhibitors.
- the triton soluble fraction was diluted 1 : 1 with 2X RIPA buffer. Both fractions were centrifuged for 10 minutes at 6000 rpm, and then precleared by incubation with nonimmune serum and protein A-agarose for 2 hours at 4°C. Supematants were transferred to fresh tubes and incubated with antibodies against ⁇ l or caveolin for 2 hours at 4°C.
- Immune complexes were recovered with protein A-agarose. The washed immunoprecipitates were subjected to 8% SDS-PAGE, and transferred onto a nitrocellulose membrane. The filters were blocked in 5% nonfat dried milk, and probed with anti-uPAR Mab R2 (from E. Ronne, Finsen Lab, Denmark), 1 ⁇ g/ml. The blots were washed and incubated with HRP conjugated antibodies for one hour. After washing, the membranes were developed using enhanced chemiluminescence (NEN Du Pont, .Wilmington, DE) according to the manufacturer's protocol.
- enhanced chemiluminescence NN Du Pont, .Wilmington, DE
- uPAR associates with and modifies function of certain integrins. This association both promotes adhesion to a migration toward a specific matrix protein, vitronectin, and destabilizes the normal adhesive function of integrins. In vivo, the ability of uPAR to destabilize integrin-dependent attachments is reinforced by the concurrent binding of the protease urokinase.
- analog sequences are listed with the amino terminus of the analogs printed on the left.
- the analog sequences utilized the one letter amino acid abbreviations unless otherwise noted.
- the lower case letters indicate a D-amino acids, for example "s” indicates a D-se ⁇ ne.
- Analogs 2-4 and 31-96 have a free ammo terminus and a C-terminal carboxamides.
- Analogs 5-30 comprise an acetylated terminus (Ac- oligomer-NH 2 ).
- the analogs were tested in an assay utilizing soluble uPAR, similar to the method desc ⁇ bed in Example 2. The analogs were tested for their ability to compete with phage displaying either peptide 9 or peptide 25. Analogs 3-61 were tested in competition with peptide 25. Analogs 62-96 were tested in competition with peptide 9. Approximately 10 8 plaque forming units of the phage were used in the assay.
- Analogs 31-61 were tested for their ability to compete with phage displaying peptide 25.
- the active analogs were tested further at two concentrations, 5 ⁇ M and 2.5 ⁇ M. These concentrations were calculated based on the synthesis reactions. However, the sequences * were further tested and determined to contain high amounts of amino acids and the quantity tested could have been higher than 5 ⁇ M or 2.5 ⁇ M.
- Analogs 80-96 were tested for their ability to compete with phage displaying peptide 9.
- the active analogs were tested further at two concentrations, 5 ⁇ M and 2.5 ⁇ M. These concentrations were calculated based on the synthesis reactions. However, the sequences * were further tested and determined to contain low amounts of amino acids and the quantity tested could have been lower than 5 ⁇ M or 2.5 ⁇ M.
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Application Number | Priority Date | Filing Date | Title |
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US62336196A | 1996-03-28 | 1996-03-28 | |
US623361 | 1996-03-28 | ||
PCT/US1997/005199 WO1997035969A2 (en) | 1996-03-28 | 1997-03-28 | Peptide ligands of the urokinase receptor |
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EP0906419A2 true EP0906419A2 (de) | 1999-04-07 |
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Family Applications (1)
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EP97917723A Withdrawn EP0906419A2 (de) | 1996-03-28 | 1997-03-28 | Peptid-liganden für den urokinase-rezeptor |
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EP (1) | EP0906419A2 (de) |
JP (2) | JP4088344B2 (de) |
AU (1) | AU2597897A (de) |
WO (1) | WO1997035969A2 (de) |
Families Citing this family (16)
Publication number | Priority date | Publication date | Assignee | Title |
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AR020101A1 (es) | 1998-07-01 | 2002-04-10 | Cancerforskingsfonden Af 1989 | Un antagonista peptido del receptor de uroquinasa humana, composicion farmaceutica que lo contiene, su uso para la manufactura de un medicamento y metodopara seleccionar un antagonista peptido adecuado. |
EP0982036A1 (de) * | 1998-08-28 | 2000-03-01 | Wilex Biotechnology GmbH | Beeinflussung der Beta-2-Integrin-Vermittelnde Zelladhäsion |
ES2279649T3 (es) | 1998-10-23 | 2007-08-16 | Amgen Inc. | Compuestos trombopoyeticos. |
US7488590B2 (en) | 1998-10-23 | 2009-02-10 | Amgen Inc. | Modified peptides as therapeutic agents |
US6660843B1 (en) | 1998-10-23 | 2003-12-09 | Amgen Inc. | Modified peptides as therapeutic agents |
AU2001257174A1 (en) * | 2000-04-21 | 2001-11-07 | Amgen Inc. | Integrin/adhesion antagonists |
CA2408812A1 (en) * | 2000-05-09 | 2001-11-15 | Dgi Biotechnologies, Inc. | Methods of identifying the activity of gene products |
WO2003051347A1 (en) * | 2001-12-19 | 2003-06-26 | Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw | Use of urokinase receptor antagonists to modulate ischemiareperfusion injury |
MX2007000216A (es) | 2004-07-08 | 2007-03-15 | Amgen Inc | Peptidos terapeuticos. |
US8008453B2 (en) | 2005-08-12 | 2011-08-30 | Amgen Inc. | Modified Fc molecules |
TW200813091A (en) * | 2006-04-10 | 2008-03-16 | Amgen Fremont Inc | Targeted binding agents directed to uPAR and uses thereof |
US9283260B2 (en) | 2006-04-21 | 2016-03-15 | Amgen Inc. | Lyophilized therapeutic peptibody formulations |
SG191142A1 (en) | 2010-12-22 | 2013-07-31 | Ifom Fond Istituto Firc Di Oncologia Molecolare | uPAR-ANTAGONISTS AND USES THEREOF |
CN103930128A (zh) | 2011-08-05 | 2014-07-16 | 意大利癌症研究基金会分子肿瘤学研究所(Ifom) | 组成型活性uPAR变体及其在抑制性抗体的生成与分离中的应用 |
WO2015004148A1 (en) | 2013-07-08 | 2015-01-15 | Ifom Fondazione Istituto Firc Di Oncologia Molecolare | Biomarker of plasminogen activation system and/or of upar/vn interaction and uses thereof |
WO2019165105A1 (en) * | 2018-02-26 | 2019-08-29 | The Board Of Trustees Of The Leland Stanford Junior University | Ligands of the urokinase receptor and their use in treating, detecting, and imaging cancer |
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- 1997-03-28 AU AU25978/97A patent/AU2597897A/en not_active Abandoned
- 1997-03-28 EP EP97917723A patent/EP0906419A2/de not_active Withdrawn
- 1997-03-28 WO PCT/US1997/005199 patent/WO1997035969A2/en not_active Application Discontinuation
- 1997-03-28 JP JP53466697A patent/JP4088344B2/ja not_active Expired - Fee Related
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JP2000509968A (ja) | 2000-08-08 |
WO1997035969A3 (en) | 1997-11-06 |
WO1997035969A2 (en) | 1997-10-02 |
JP4088344B2 (ja) | 2008-05-21 |
JP2008109934A (ja) | 2008-05-15 |
AU2597897A (en) | 1997-10-17 |
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