EP0451211A1 - Clonage et production d'analogues polypeptidiques de fibronectine humaine et procedes d'utilisation desdits analogues polypeptidiques - Google Patents

Clonage et production d'analogues polypeptidiques de fibronectine humaine et procedes d'utilisation desdits analogues polypeptidiques

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Publication number
EP0451211A1
EP0451211A1 EP90902086A EP90902086A EP0451211A1 EP 0451211 A1 EP0451211 A1 EP 0451211A1 EP 90902086 A EP90902086 A EP 90902086A EP 90902086 A EP90902086 A EP 90902086A EP 0451211 A1 EP0451211 A1 EP 0451211A1
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EP
European Patent Office
Prior art keywords
polypeptide
naturally
human fibronectin
binding domain
plasmid
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Withdrawn
Application number
EP90902086A
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German (de)
English (en)
Other versions
EP0451211A4 (en
Inventor
Tikva Vogel
Avigdor Levanon
Moshe Werber
Rachel Guy
Amos Panet
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Savient Pharmaceuticals Inc
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Savient Pharmaceuticals Inc
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Application filed by Savient Pharmaceuticals Inc filed Critical Savient Pharmaceuticals Inc
Publication of EP0451211A1 publication Critical patent/EP0451211A1/fr
Publication of EP0451211A4 publication Critical patent/EP0451211A4/en
Withdrawn legal-status Critical Current

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    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M25/00—Catheters; Hollow probes
    • A61M25/0043—Catheters; Hollow probes characterised by structural features
    • A61M25/0045—Catheters; Hollow probes characterised by structural features multi-layered, e.g. coated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/08—Vasodilators for multiple indications
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo
    • 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 cloning and production of analogs of human fibronectin and methods of using such polypeptide analogs.
  • Fibronectin is a glycoprotein composed of two identical subunits each of approximately 220,000 molecular weight. Two major forma of fibronectin are produced and secreted by human cells in culture and in vivo (1). The cell-associated fibronectin is relatively insoluble and participates in cell adhesion, wound healing, cell differentiation and phagocytosis. The plasma fibronectin, produced primarily in the liver, is a soluble serum protein with biological properties similar to those of cell fibronectin. Fibronectin is considered a multifunctional modular protein since limited proteolytic cleavage produces polypeptides with distinct activities. The different functional domains of the fibronectin molecule have been defined by partial proteolysis digests, and include heparin-, DNA-, fibrin-, gelatin-, and cell- binding domains (2-6).
  • Obara, et al. (1987) disclose the expression of a portion of the cell binding domain of human fibronectin fused to Eacherichia coli ß-galactosidasa (8). Additionally, Obara, et al. (1988) disclose the expression of portions of the cell binding domain fused to ß-galactosidase which have been mutagenized, i.e., site specific deletions of portions of the cell binding domain were obtained as fused proteins (9).
  • Ruoslahti and Pierschbacher disclose a tetrapeptide exhibiting the cell-attachment activity of fibronectin with the formula X-Arg-Gly-Asp-R-Y (RGD).
  • the preferred R is Ser, but R may also be other amino acids such as Cys or Thr.
  • the tetrapeptide may be used to promote cell attachment to a substrate (such as a prosthetic device) by immobilizing the peptide on the substrate, to inhibit attachment to a substrate by using the peptide in solubilized or suspended form, and to enhance the phagocytic activity of cells.
  • peptides disclosed in the above patent applications although having the activity of the cell binding domain of fibronectin, are not as active in cell binding as larger fibronectin polypeptides. It is thought that the smaller peptides are not able to obtain the correct spatial conformation to fully mimic the complete fibronectin molecule. It is also believed that these polypep tides are missing additional sites or regions in the cell binding domain which are important in enhancing the cell attachment activity of the fibronectin molecule.
  • the use of the entire cell binding domain or large fragments of the cell binding domain instead of short synthetic peptides (RGD) for in vivo experimentation has several advantages including: (i) the affinity of the plasma-derived cell binding domain (75,000 daltons) to the fibronectin receptor is about 200-fold higher compared with that of the short RGD peptides; (ii) the stability in vivo of the entire cell binding domain to proteases, especially exopeptidases, is higher than that of the short synthetic peptides; and (iii) the rate of clearance by the kidneys is much slower for the larger proteins than the synthetic short peptides, resulting in an extended half-life of the larger fragments of the cell binding domain (CBD).
  • Applicants' invention provides plasmids which are used to produce polypeptides.
  • the polypeptides may be used to inhibit platelet aggregation. Drugs developed in the past to inhibit aggregation have interfered with the physiological functions of the platelet without achieving total inhibition.
  • the polypeptides of applicants' invention overcome these difficulties by obstructing the mechanism of platelet aggregation via competition with the natural molecules which are responsible for inter-platelet interactions as well as interactions between platelets and the substratum.
  • Applicants' polypeptides may be used as therapeutic agents and may have a considerably longer half-life than the prostacyclins. simultaneously, they may have significantly shorter half-lives than aspirin or other commonly used antiplatelet aggregation drugs. Hence, they may not have the complications associated with these drugs. Even though they act during the critical stage of injured lumen blood vessel repair, applicants' polypeptides may not interfere with the indispensable coagulation-induced events necessary for wound repair. Hence, uncontrolled bleeding may not occur. This is because applicants' polypeptides may act by interfering specifically with the mechanism of adhesion and spreading of various moieties involved in aggregation, unlike existing therapies which interfere with the physiological functions of platelet aggregation. The pharmacokinetics of the therapeutics may be modified by design, thereby assuring reversibility of the process and ideal management of the coagulation and platelet aggregation process.
  • Wound healing therapeutics currently under development use the entire fibronectin molecule derived from pooled donor blood or autologous blood.
  • Applicants' invention uses the individual functional domains for wound healing.
  • the recombinant cell binding domain may be used instead of the whole fibronectin molecule to promote wound healing.
  • the recombinant polypeptide is more stable and may be produced in virtually unlimited amounts, free of any blood contaminants.
  • the fibrin binding domain of fibronectin may be used as an anti-infective agent to prevent sepeis in wounds.
  • Humphries, et al. (12) have demonstrated that co-injection of the RGD peptide with mouse melanoma cells dramatically inhibited formation of lung metastatic colonies in mice.
  • Applicants' fibronectin polypeptide analogs may also be useful in detecting clotting of fibrin in leg veins, a common clinical manifestation in the elderly. Localization of a fibrin thrombus is a very complicated procedure performed in nuclear medicine departments. The assay is based on the low affinity of injected radiolabeled fibrinogen for the thrombus (21). However, the labeled fibrinogen is diluted by the large amounts of endogenous fibrinogen in the blood circulation (about 10 gr.). Thus, the method is not sensitive.
  • Applicants' invention provides for the use of the radiolabeled amino-terminal domain of fibronectin (31 kD), which has high binding affinity to fibrin, as a tracer for detection of fibrin thrombi. Since the amount of fibronectin in the blood is considerably lower than that of fibrinogen, applicants' invention provides a more sensitive tracer for thrombus detection. Moreover, as this domain of fibronectin is covalently cross-linked to the fibrin clot by factor XIII, considerable accumulation of the radiolabeled domain at the clot site will occur.
  • this invention providse novel polypeptide analogs of fibronectin which are similar in sequence to domains of the human fibronectin molecule.
  • the invention further provides uses of individual domains of fibronectin in the treatment of subjects with conditions such as cerebrovascular disorders, cardiovascular disorders, wounds, and cancer.
  • This invention provides a plasmid for expression of a polypeptide which comprises a substantial portion of the amino acid sequence of one of the domains of naturally-occurring human fibronectin and which does not correspond to a product of proteolytic digestion of naturally-occurring human fibronectin comprising DNA encoding the polypeptide and DNA encoding suitable regulatory elements positioned relative to the DNA encoding the polypeptide so as to effect expression of the polypeptide in a suitable host cell.
  • a plasmid for expression of a polypeptide which comprises a substantial portion of the amino acid sequence of the cell binding domain of naturally-occurring human fibronectin and which does not correspond to a product of proteolytic digestion of naturally-occurring human fibronectin comprising DNA encoding the polypeptide and DNA encoding suitable regulatory elements positioned relative to the DNA encoding the polypeptide so as to effect expression of the polypeptide in a suitable host cell.
  • the invention provides a plasmid for expression of a polypeptide which comprises a substantial portion of the amino acid sequence of the fibrin binding domain of naturally-occurring human fibronectin and which does not correspond to a product of proteolytic digestion of naturally-occurring human fibronectin comprising DNA encoding the polypeptide and DNA encoding suitable regulatory elements positioned relative to the DNA encoding the polypeptide so as to effect expression of the polypeptide in a suitable host cell.
  • the polypeptide is about a 75 kD polypeptide fragment of the cell binding domain of human fibronectin; about a 40 kD polypeptide fragment of the cell binding domain of human fibronectin; about a 33 kD polypeptide fragment of the cell binding domain of human fibronectin; about a 31 kD polypeptide fragment of the fibrin binding domain of human fibronectin; or about a 20 kD polypeptide fragment of the fibrin binding domain of human fibronectin.
  • the polypeptide is a 75 kD polypeptide fragment of the cell binding domain of human fibronectin comprising amino acids 1102-1851, but deleted of amino acids 1600-1689; a 40 kD polypeptide fragment of the cell binding domain of human fibronectin comprising amino acids 1380-1851, but deleted of amino acids 1600-1689; a 33 kD polypeptide fragment of the cell binding domain of human fibronectin comprising amino acids 1329-1722, but deleted of amino acids 1600-1689; a 31 kD polypeptide fragment of the fibrin binding domain of human fibronectin comprising amino acids 1-262; or a 20 kD polypeptide fragment of the fibrin binding domain of human fibronectin comprising amino acids 1-153 and 13 additional amino acids.
  • the plasmids are preferably exprsesed in suitable strains of Escherichia coli.
  • An example of a suitable Escheriehia coli strain is A4255 (F + ) [ATCC Accession No. 67830], Escherlchia coll strain A1645 [ATCC Accession No. 67829], or Escherlchia coli strain A4255 [ATCC Accession No. 67910].
  • This invention also provides a method of producing a polypeptide which comprises a substantial portion of the amino acid sequence of one of the domains of naturally-occurring human fibronectin which comprises treating an Escherlchia coli cell containing a plasmid comprising DNA encoding the polypeptide so that the DNA directs expression of the polypeptide and recovering from the cell the polypeptide so expressed.
  • a method of producing a polypeptide which comprises a substantial portion of the amino acid sequence of the cell binding domain or the fibrin binding domain of naturally-occurring human fibronectin which comprises treating an Escherichia coll cell containing a plasmid comprising DNA encoding the polypeptide so that the DNA directs expression of the polypeptide and recovering from the cell the polypeptide so exprsesed.
  • the polypeptide so produced is a 75 kD, 40 kD, or 33 kD polypeptide fragment of the cell binding domain of naturally-occurring human fibronectin, or a 31 kD or 20 kD polypeptide fragment of the fibrin binding domain of naturally-occurring human fibronectin.
  • the invention providse a purified polypeptide substantially free of other substances of human origin which comprises a substantial portion of the amino acid sequence of one of the domaina of naturally-occurring human fibronectin and which does not correspond to a product of proteolytic digestion of naturally-occurring human fibronectin.
  • Preferred domains are the cell binding domain and the fibrin binding domain.
  • Preferred polypeptides are the 75 kD, 40 kD, and 33 kD polypeptides of the cell binding domain and the 31 kD and 20 kD polypeptidse of the fibrin binding domain.
  • the invention provides a bacterially-produced polypeptide which comprises a substantial portion of the amino acid sequence of one of the domains of naturally-occurring human fibronectin and which does not correspond to a product of proteolytic digestion of naturally-occurring human fibronectin.
  • Preferred domains are the cell binding domain and the fibrin binding domain.
  • Preferred polypeptides are the 75 kD, 40 kD, and 33 kD polypeptides of the cell binding domain and the 31 kD and 20 kD polypeptides of ths fibrin binding domain.
  • This invention further provides a polypeptide which comprises a substantial portion of the amino acid sequence of one of the domains of naturally-occurring human fibronectin which does not correspond to a product of proteolytic digestion of human fibronectin. Also provided is a polypeptide which comprises a substantial portion of the amino acid sequence of the cell binding domain or the fibrin binding domain of naturally-occurring human fibronectin which does not correspond to a product of proteolytic digestion of human fibronectin.
  • Preferred polypeptides are the 75 kD, 40 kD, and 33 kD polypeptides of the cell binding domain and the 31 kD and 20 kD polypeptides of the fibrin binding domain.
  • the invention provides a composition comprising at least two of the polypeptides described above and a suitable carrier, and a hybrid polypeptide consisting essentially of at least two such polypeptides.
  • the invention further provides pharmaceutical compositions comprising an amount of the polypeptides of the subject invention effective to inhibit platelet aggregation and a pharmaceutically acceptable carrier.
  • pharmaceutical compositions comprising an amount of the polypeptides of the subject invention effective to inhibit thromboxane release from platelets and a pharmaceutically acceptable carrier.
  • the polypeptides of this invention may be used to inhibit platelet aggregation or to inhibit thromboxane release from platelets. They may also be used to treat a subject with a cerebrovascular disorder, a cardiovascular disorder, a wound, a bacterial infection, a cancer, or to detect a tumor or thrombi (by imaging of the thrombi).
  • polypeptides of this invention may be bound to thrombolytic agents, growth factors, serum albumin, blood factors, polyethyleneglycol, or superoxide dismutase.
  • polypeptides of the subject invention may be used to coat a medical device, such as a catheter, so as to minimize risk of bacterial infection associated with use of such medical devices.
  • the cDNA sequence applicants have cloned and expressed is missing the 270 bp extra domain (ED) segment which extends from nucleotides 4811 to 5080, inclusive, on the Baralle map (aee Figure 41).
  • ED extra domain
  • the cDNA sequence which is said to extend from nucleotide 3317 to 5566 on the Baralle map contains only 1980 nucleotides, because it is missing the 270 nucleotides of the ED segment, namely from nucleotides 4811 to 5080 inclusive; this region is also known in the art as the ED-A region.
  • the protein expressed by that DNA fragment would encode from amino acid 1102 to amino acid 1851 on the Baralle map but would be missing the 90 amino acids encoded by the ED region, namely amino acids 1600-1689 inclusive, and thus it would contain only 660 amino acids. This is true for all fragments described in this application which span the ED region. (The region known in the art as the ED-B region is missing both in Baralle's sequence and in applicants' cDNA.)
  • the definition of the proteins expressed as 40 kD, 31 kD, 75 kD, 33 kD and 20 kD is an operational definition, based on their mobility on SDS polyacrylamide gels compared to that of markers of known molecular weight.
  • Figure 1 The upper portion of Figure 1 shows various fibronectin cDNA clones which have been isolated and the alignment of such cDNA clones relative to one another and to the full length sequence of fibronectin cDNA.
  • the lower portion of Figure 1 is a schematic representation of the various domains present within the human fibronectin polypeptide.
  • FIG. 2 This figure shows the construction of plasmid pFN100 from plasmids pBR322 and pFN919 using a synthetic linker. Plasmid pFN100 encodes the 5' end of the cDNA corresponding to the fibronectin cell binding domain, which corresponds to nucleotides 3317-4090.
  • FIG. 3 This figure shows the construction of plasmid pFN101 from plasmids pBR322 and pFN919 using a synthetic linker.
  • the plasmid pFN101 contains cDNA corresponding to nucleotides 3611-4090 of fibronectin.
  • FIG. 4 This figure shows the construction of plasmid pFN102 from plasmids pBR322 and pFN916 using a synthetic linker.
  • An EcoRI-BglII fragment corresponding to nucleotides 4151-5566 was ligated to the synthetic linker (as shown in the figure) and the large pBR322 fragment derived from an EcoRI-Sal 1 digestion of pBR322.
  • Plasmid pFN102 encodes the 3' end of the cDNA corresponding to the cell binding domain.
  • FIG. 5 This figure shows the construction of plasmid pFN103 from plasmids pFN100 ( Figure 2) and pFN102 ( Figure 4) using a synthetic linker. Plasmid pFN103 encodes the cDNA region corrseponding to the cell binding domain of fibronectin from nucleotides 3317 to 5566.
  • Figure 6. This figure shows the construction of plasmid pFN105 from plasmid pFN103 ( Figure 5) and plasmid pTV301 ( Figure 7) .
  • the cDNA corresponding to the cell binding domain was removed from plasmid pFN103 and inserted into a fragment of plasmid pTV301 under the control of the ⁇ P L promoter and the cII ribosomal binding site. It was expected that plasmid pFN105 would direct the expression of the entire cell binding domain
  • FIG. 7 This figure shows the construction of plasmid pTV301 from plasmids pTV104(2) and p579.
  • Plasmid pTV301 directs the exprsesion of a human growth hormone analog under the control of the ⁇ P L promoter and the ell riboeomal binding site.
  • the plasmid also contains a T 1 T 2 transcription terminator downstream of the cDNA encoding human growth hormone.
  • FIG. 8 This figure shows the construction of plasmid pFN104 from plasmids pFN101 and pFN102 using a synthetic linker.
  • Pleemid pFN104 containa cDNA encoding the cell binding domain from nucleotides 3611 to 5566.
  • pjgure 9. This figure shows the construction of plasmid pFN106 from plasmids pTV301 and pFN104.
  • Plasmid pFN106 was expected to direct the expression of a 65 kD polypeptide under the control of the ⁇ P L promoter and the cII ribosomal binding site. The 65kD polypeptide was expected to contain the RGD sequence. However, due to a single thymidine deletion at position 4030, a termina tion codon within the cDNA sequence was generated and plasmid pFN106 directed the expression of a 35kD protein.
  • FIG. 10 This figure shows the construction of plasmid pFN126-3 from plasmids pFN105 and pFN114-4.
  • Plasmid pFN114-4 is an additional plasmid harboring FN cDNA isolated independently from the cDNA library; PFN114-4 haa no deletion of the thymidine in position 4030.
  • An SstI-BglII fragment from plasmid pFN114-4 was used to replace the SstI-BglII fragment from plasmid pFN105.
  • the rseulting plasmid pFN 117-4 directed the expression of the full length 75 kD cell binding domain protein but the Alfaid does not contain the translation termination codon and terminates within the pBR322 sequence. To correct this fault, the following linker was constructed:
  • This linker contains BglII and HindIII sites at the 5' and 3' terminals respectively, and codes for the 3' end of the 75 kD CBD including the translation termination codon. It was ligated to plasmid pFNH7-4 which had been cleaved with BglII and HindIII. The resulting plasmid pFN 126-3 directed the expression of the full length 75 kD cell binding domain protein, with the authentic C-terminus.
  • FIG 11. This figure shows the construction of plasmid pFN118 (alternatively designated pFN 118-2) from plasmids pTV301 and pFN102 using a synthetic linker.
  • Plasmid pFN118 was designed to express a 40 kD cell binding domain protein. However, due to a mistake in the synthesis of the linker the protein was not terminated within the linker, but rather was only terminated in the sequences derived from the vector pBR322. Thus, the protsin obtained was a fused protein.
  • FIG. 12 This figure shows the construction of plasmid pFN132-5 from plasmid pFN118 and plasmid pTV301 using a synthetic linker.
  • the synthetic linker was used to replace a portion of the defective synthetic linker in plasmid pFN118.
  • the rseulting plasmid pFN132-5 directed the expression of a 40kD cell binding domain protein.
  • FIG. 13 This figure shows the collagen and DNA synthesis in applicants' wound healing model. The experiments were carried out as described in Examples 10 and 22, with 50 ⁇ g of the 40 kD protein per sponge. Bovine serum albumin (BSA) at 100 ⁇ g per sponge and saline were used as negative controls.
  • BSA bovine serum albumin
  • FIG. 14 This figure shows the pharmacokinetics of the 40kD recombinant CBD protein.
  • Figure 15 This figure shows the nucleotide sequence of a BamHI-EcoRI synthetic fragment used in the subcloning of the cell binding domain.
  • FIG 16. This figure shows the construction of plasmid p579.
  • the rRNA operon T 1 T 2 transcription termination fragment was isolated from plasmid pPS1 (deposited with the ATCC under Accsesion No. 39807) which had been digested with HindIII.
  • the T 1 T 2 fragment was inserted into the unique HindIII site of pRO211 ( Figure 17) which had been digested with HindIII.
  • the rseulting expression vector, p579 contains the ⁇ P L promoter and the C II ribosomal binding site, followed by the T 1 T 2 transcription termination sequences.
  • FIG. 17 This figure shows the construction of plasmids pRO211 and pRO12.
  • the plasmid pJH200 (deposited with the ATCC under Accession No. 39783) was partially digested with NdeI, treated with DNA polymerase I
  • the expression vector pR0211 was digested with NdeI and HindIII, the large fragment isolated and ligated to an
  • FIG. 18 This figure shows various fibronectin plasmids which contain the cell binding domain, and the alignment of the plasmids to one another and to the full length sequence of the cell binding domain. (The alignment of the cell binding domain relative to the fibronectin cDNA is shown in Figure 1.)
  • CBD I Cell Binding Domain I (includes the RGD sequence)
  • CBD II Cell Binding Domain II [as disclosed by
  • Figure 19 This figure summarizes information about the plasmids encoding various fragments of the cell binding domain and the proteins exprsesed by them.
  • Figure 20 This figure shows the construction of plaraid pFN128-4 by ligation of synthetic linker A ( Figure 26) to the large fragment produced by EcoRV and BglII digestion of plasmid pFNH7-4 ( Figure 10).
  • Plasmid PFN128-4 contains cDNA encoding the cell binding domain from nucleotidse 3317 to 5179; the 3' terminus is formed by linker A.
  • the plasmid exprseses a 65 kD cell binding domain protein under the control of the ⁇ P L promoter and the cII ribosomal binding site.
  • this plasmid could have been identically constructed from plasmid PFN126-3 (ATCC Accession No. 67829), which differs from plasmid pFN117-4 only at the 3' terminus of the cDNA encoding the cell binding domain.
  • FIG. 21 Thia figure shows the construction of plasmid pFN130-11 by ligation of synthetic linker B ( Figure 26) to the large fragment obtained by digestion of plasmid pFN117-4 by BamHI and HindIII.
  • Plasmid PFN130-11 contains cDNA encoding the cell binding domain from nucleotidse 3317 to 4090; the 3' terminus was formed by linker B.
  • This plasmid exprseses a 28 kD cell binding domain protein under the control of the ⁇ P L promoter and the cII ribosomal binding site. Note that this plasmid could have been identically constructed from plasmid pFN126-3 (ATCC Accession No. 67829), which differs from plasmid pFNH7-4 only at the 3' terminus of the cDNA encoding the cell binding domain.
  • FIG 22 This figure shows the construction of plasmid pFN135-12 by tri-partite ligation of synthetic linker C ( Figure 26), synthetic linker D ( Figure 26) and the large fragment obtained by digestion of plasmid
  • Plasmid pFN135-12 contains cDNA encoding the cell binding domain from nucleotides 3991 to approximately 5566; the 5' terminus was formed by linkers C and D.
  • This plasmid expresses a 45 kD cell binding domain protein under the control of the 7.
  • P L promoter and the cII riboeomal binding site Note that there is the seme error in the C-tsrminus of this protein as in the 75 kD protein expressed by the parent plasmid
  • PFN117-4 (see Description to Figure 10); i.e., there is no translation termination codon at the end of the fibro nectin cDNA, and thus translation terminates in the pBR322 coding sequence.
  • FIG 23 This figure shows the construction of plasmid pFN137-2 by ligation of synthetic linker A ( Figure 26) to the large fragment produced by EcoRV and BglII digestion of plasmid pFNl35-12 ( Figure 22).
  • Plasmid pFN137-2 contains cDNA encoding the cell binding domain from nucleotides 3998 to 5179; the 3' terminus was formed by linker A.
  • This plasmid expressse a 33 kD cell binding domain protein under the control of the 7 ⁇ P L promoter and the cII ribosomal binding site.
  • Plasmid PFN137-2 has been deposited with the ATCC under Accession No. 67910.
  • FIG. 24 This figure shows the construction of plasmid pFN143-1 by tri-partite ligation of synthetic linker E ( Figure 26), the large fragment obtained by digestion of plasmid pFN137-2 by NdeI and BamHI, and the small fragment obtained by digestion of plasmid pFN126-3 by SstI and BamHI.
  • Plasmid pFN143-l contains cDNA encoding the cell binding domain from nucleotides 3602 to 5179; the 5' terminus was formed by linker E.
  • This plasmid expresses a 55 kD cell binding domain protein under the control of the ⁇ P L promoter and the cII ribosomal binding site.
  • FIG. 25 This figure shows the construction of plasmid pFN134-9 by triple ligation of aynthetic linker A ( Figure 26), the large fragment derived by digestion of plasmid p578 (see below) by NdeI and BglII, and the small fragment derived by digestion of plasmid pFN118 ( Figure 11) by NdeI and EcoRV.
  • Plasmid pFN134-9 contains cDNA encoding the cell binding domain from nucleotides 4151 to 5179; the 3' terminus was formed by linker A. Downstream of the 3' terminus are the T 1 T 2 transcription termination sequences.
  • the plasmid expresses a 28 kD cell binding domain protein under the control of the ⁇ P L promoter and the ell ribosomal binding site.
  • Plasmid p578 is another isolate from the same ligation that produced p579 (see Figure 16). Plasmids p578 and p579 are understood to be identical.
  • Plasmid pFN134-9 could have been constructed in a similar manner from plasmid pFN132-5 (ATCC Accession
  • FIG. 26 Synthetic linkers were used in the construction of the subject plasmids. This figure shows the base sequence of synthetic oligonucleotides A, B, C, D and E used as linkers in the construction of plasmids described in the preceding figures.
  • FIG. 27 This figure shows the pharmacokinetics of the radioactivsly-labeled 33 kD protein when injected intravenously into rats.
  • FIG 28 This figure shows the dose-response effect of the 33 kD and 40 kD proteins and also of the plasmatic 75 kD protein (P-75 kD) and the synthetic pentapeptide GRGDS (Sigma) on the in vitro human platelet aggregation assay system described in Example 7.
  • the plasmatic 75 kD protein was purified from a tryptic digest of fibronectin by an adaptation of the method described by Hayashi, M. and Yamada, K.M. (28).
  • the proteins (and GRGDS) were added in the amounts shown in PBS solution to separate reaction tubes containing 125 ⁇ l of platelet-rich plasma (PRP) (from donor T).
  • PRP platelet-rich plasma
  • the final reaction volume was made to 250 ⁇ l by the addition of PBS after equilibration.
  • Platelet aggregation was induced by addition of ADP solution to a final concentration of 10 ⁇ M ADP.
  • the transmittance of each reaction tube was measured (during 3-minute reaction time) and inhibition of platelet aggregation, calculated as a percent maximum tranemittancs, was plotted against protein concentration ( ⁇ M).
  • ⁇ M protein concentration
  • Figure 29 This figure shows the doee-response effect of the 33 kD and the 40 kD proteins on platelet aggregation in whole blood, using the in vitro platelet aggregation assay system described in Example 7, modified as follows: the reaction tube mixture contained 500 ⁇ l whole blood (from R) plus 500 ⁇ l PBS or protein solution in PBS. The experiments were performed in the presence of 0 (control), 0.65 ⁇ M and 2.6 ⁇ M 33 kD protein and in the presence of 0 (control), 1.5 ⁇ M and 7.5 ⁇ M 40 kD protein. The solutions were equilibrated to 37oC for 3 minutes, and then platelet aggregation was induced by addition of ADP solution to a final concentration of 10 ⁇ M ADP.
  • impedance was monitored instead of transmittance.
  • the impedance method detects aggregation by passing a very small electric current between two electrodes immersed in a sample of blood, and measuring the electrical impedance between the electrodes (22).
  • the impedance results in the above experiments were converted to percent inhibition (calculated as percentage of maximum impedance in control tubes), and plotted against the concentration ( ⁇ M) of the 33 kD and 40 kD proteins.
  • the 40 kD protein sample had been reactivated by urea treatment as described in Example 20.
  • Figure 30 This figure shows the effect of the 33 kD and 40 kD proteins in the in vitro platelet aggregation system described in Example 7.
  • Platelet-rich plasma from R was used and the 33 kD and 40 kD proteins were added to the reaction tube mixture in the concentrations indicated.
  • Two different preparations of 40 kD protein were tested: the 40 kD sample designated "TSK-1-Urea-PBS" was reactivated by urea treatment as described in Example 20, after Fractogel filtration (as described in Example 15); the other 40 kD preparation was not reactivated, calculation of inhibition of aggregation was performed by comparing the aggregation aftar 0.5 min. reaction time.
  • Figure 31 The effect of protein concentration on the binding to platelets of the 33 kD and 40 kD proteins and human plasmatic fibronectin was examined using the method described in Example 19.
  • the reaction mixture contained 5 x 10 8 washed platelets and thrombin. In each case the amount of labeled protein was as indicated in the figure.
  • Figure 32 The effect of the synthetic pentapeptide GRGDS (Sigma) on the binding to washed platelets of human plasma fibronectin (h-FN) and the 40 kD and 33 kD proteins was examined uaing the method described in Example 19 and using thrombin as stimulant. Experiments were performed using 0.1 ⁇ M 125 I-labeled 40 kD, 33 kD and plasmatic FN alone and also in the presence of a 10-fold excess of cold (unlabeled) homologous protein (40 kD and 33 kD respectively) and in the presence of 50 ⁇ M GRGDS pentapeptide. The figures were normalized by considaring the binding by each labeled protein alone as 100%.
  • Figure 33 The effect of the presence or absence of thrombin on the binding of the 40 kD and 33 kD proteins to washed platelets wee examined using the method described in Example 19.
  • the platelet concentration was 5 ⁇ 10 8 platelets/ml.
  • reaction mixture contained 100 nM 125 I-40 kD in the presence or absence of thrombin and in the presence or absence of additional 1 ⁇ M unlabeled 40 kD, as indicated.
  • reaction mixture contained 100 nM 13S I-33 kD in the presence or absence of thrombin and in the presence or absence of additional 1 ⁇ M unlabeled 33 kD, as indicated.
  • the percentage binding indicated is the percentage of input radioactive label which was bound.
  • Figure 34 The effect of the 40 kD and 33 kD proteins, plasmatic 75 kD (p-75 kD) and GRGDS on the binding of fibrinogen to weehed platelets was studied.
  • the method was as described in Example 19, except as follows.
  • the reaction mixture contained 5 ⁇ 10 8 platelets and the experiment was performed both in the presence and absence of thrombin. After pre-incubation for 10 minutes at 25oC, 4 unite per ml of hirudin (Bio-Makor, Israel) was added and pre-incubation continued for 5 additional minutes at 25oC. To the reaction mixture 100 ⁇ M 125 I-fibrinogen was added.
  • control samples contained no further additions, and the test samples contained 5 ⁇ M 40 kD, 5 ⁇ M 33 kD, 5 ⁇ M plasmatic 75 kD, or 50 ⁇ M GRGDS pentapeptide, added at the same time as the 125 I-fibrinogen, all as indicated in the figure. Incubation continued for an additional 20 minutes before termination of the reaction and measurement of radioactivity. The percentage binding indicated is the percentage of input radioactive label which was bound.
  • Figure 35 The dose response of the binding of the 40 kD and 23 kD proteins to washed platelets in the presence and absence of thrombin was determined using the method described in Example 19.
  • the reaction mixture contained 5 ⁇ 10 8 platelets.
  • Figure 38 The cell binding activity of the 40 kD protein is compared to that of plasmatic fibronectin (FN).
  • Example 16 The cell binding activity of the 40 kD protein before and after urea reactivation, 40 kD and 40 kD(U), and of plasmatic fibronectin, FN, was measured by a cell attachment assay system (23) (see Example 16), using a BALB/C 3T3 fibroblast cell line (ATCC Accession No. CCL 163). Urea reactivation was carried out as described in Example 20.
  • the cell binding activity of the 40 kD protein and of plasmatic fibronectin (FN) was measured by a cell attachment assay system using either the 3T3 fibroblast cell line described above or an NRK-52E cell line (ATCC Accsesion No. CRL 1571). The assay technique was as described in A above.
  • FIG. 40 This figure shows the effect of the 40 kD protein on collagen and DNA synthesis in a sponge wound healing model in rats.
  • the experiments were carried out as described in Example 22.
  • Plasma fibronectin (FN) and epidermal growth factor (EGF, Biomedical Technologies Inc.) were used as positive controls at the level of 100 ⁇ g per sponge and 8 ⁇ g per sponge, respectively.
  • FIG. 41 This figure shows the nucleotide sequence of human fibronectin cDNA.
  • Figure 42 Seven pairs of chemically synthesized oligomers were prepared. The synthetic oligomers code for the first 153 N-terminal amino acids of human FN. This figure shows the sequence of these 7 pairs of synthetic oligomers.
  • Figure 43 The DNA fragment coding for amino acids 1 to 153 of N-terminal domain of human FN was assembled from the 7 pairs of chemically synthesized oligomers shown in Figure 42 as follows:
  • Oligomers 3/4, 5/6, 7/8 and 9/10, each pair in a separate tube were annealed and then phosphorylated at the 5' end using T4 polynucleotide kinase enzyme.
  • pairs 3/4 and 5/6 were ligated to each other using T4 DNA ligase.
  • reaction pairs 7/8 and 9/10 were ligated to each other.
  • an aliquot of the ligation mixture was analyzed on gel to determine the size of the newly formed fragments and the efficiency of ligation.
  • the two above mentioned ligation mixtures were mixed together and pair 6, oligomers 11/12 which had been annealed and phosphorylated previously in a separate tube were added to the mixture.
  • a 326 base pair DNA fragment obtained from the above ligation mixture was isolated from an agarose gel and purified.
  • the purified synthetic 326 fragment was added to two additional pairs of synthetic linkers: Pair 1, oligomers 1/2 and Pair 7 oligomers 13/14. In Pair 1 only oligomer 2 was phosphorylated at the 5' end and in Pair 7 only oligomer 13 was phosphorylated at the 5'end.
  • the plasmid obtained, designated pFN 932-18 contained the entire synthetic EcoRI (5'end) - BamHI (3'end) restriction fragment coding for N-terminal 153 amino acids of human FN, in a pBR322 vector.
  • Figure 44 Expression of the N-terminal 153 amino acid ssousncs of FN.
  • Plasmid pFN 932-18 was digested with NdeI and BamHI endonucleases.
  • the NdeI-BamHI DNA fragment coding for FN (first 153 amino acids + additional N-terminal methionine) was isolated and ligated into the large fragment obtained by digestion of plasmid pTV301 with NdeI and Bgl II endonucleases.
  • (Plasmid pTV301 ( Figure 7) expresses human growth hormone, hGH, under the control of lambda P L promoter and the cII RBS).
  • the plasmid obtained was designated pFN949-2.
  • a synthetic oligonucleotide containing a TAA termination codon and a BglII site having the following sequence:
  • CTGTTTAAGCA GACAAATTCGTCTAG was ligated to the 3' end (PvuII site) of an EcoRI-PvuII FN fragment isolated from cDNA clone plasmid p931-5 (see Figure 1) digested with EcoRI and PvuII.
  • the ligation was carried out in the presence of DNA vector plasmid pBR322 digested with EcoRI and BamHI (large fragment).
  • the plasmid obtained was designated pFN935-12.
  • Figure 46 Subcloning of the carboxy-terminal region of FBD in ⁇ P L expression vector.
  • Plasmid pFN 935-12 was digested with EcoRI and HincII.
  • the EcoRI-HincII fragment coding for FN was isolated and ligated to DNA, the large fragment obtained by digestion of plasmid pTV194-80 with EcoRI and SmaI.
  • Plasmid pTVl94-80 expresses human ApoE under the control of the ⁇ P L promoter and ß-lactamase promoter and RBS).
  • the plasmid obtained was designated pFN 946-12. This plasmid is deleted of the PBLA sequences and therefore does not express the carboxy domain of FBD.
  • Oligomers 15/16 and 17/18 were annealed and phosphorylated at the 5' end each in a separate tube and then mixed together for ligation using T4 DNA ligase. After 3 hours of ligation, oligomers 19/20 (previously annealed and kinased at their 5' ends) were added for an additional 3 hours ligation at room temperature.
  • the synthetic DNA fragment obtained was used for further ligation with an EcoRI-DdeI FN coding sequence obtained from plasmid pFN 932-18 digested with EcoRI and DdeI.
  • the ligation was carried out in the presence of plasmid pUC19 digested with EcoRI and XbaI (large fragment).
  • the plasmid obtained was designated pFN948-4.
  • Plasmid pFN948-4 was digested with EcoRI and XbaI.
  • the EcoRI-XbaI fragment coding for the N-terminal region of FBD was isolated and ligated to the carboxy terminal region of FBD by digestion of plasmid pFN946-12 with EcoRI and XbaI (using the large fragment).
  • the plasmid obtained was designated pFN 957.
  • Plasmid pFN 957 was digested with NdeI and HindIII. The
  • NdeI-HindIII fragment coding for the FBD was isolated and ligated into the isolated vector fragment of plasmid pTV301 digested with NdeI and HindIII in the presence of isolated purified HindIII-HindIII T 1 T 2 coding DNA fragment.
  • the plasmid obtained was designated pFN962-3.
  • Figure 50 Expression of the entire FBD protein under ⁇ P L promoter and PBLA ribosomal binding site
  • Plasmid pBLA11 (ATCC No. 39788) was digested with EcoRI and AluI.
  • NdeI then treated with Klenow enzyme in the presence of all four dNTPs to fill in the NdeI site and digested with EcoRI (using the large fragment).
  • the plasmid obtained was designated pFN 975-25.
  • Figure 51 Refolding/reoxidation and purification of r31 kD as followed by SDS-PAGE under reducing and non-reducing conditions
  • the gel (12% acrylamide) under reducing conditions (with ß-mercaptoethanol (ME) ) monitors the process of purification, whereas the non-reducing conditions (without ME) are indicative of the refolding/reoxidation, leading to faster moving and less diffuse bands. Note (in the absence of ME) that the band of 'After Phenyl-S' is much sharper than that of 'Refolded', indicating that reoxidation continues even during the purification.
  • ME ß-mercaptoethanol
  • Refolded (GSH/GSSG): r31kD which has been refolded/reoxidized - after having been extracted from the crude pellet in the presence of GSH/GSSG 3 mM/0.3 mM at pH 8.0; Phenyl-S; Phenyl-Sepharose; Q-S; Q-Sepharose; p31 kD; plasma-derived 31 kD (obtained by tryptic digestion); molecular weight markers: Low Molecular Weight protein calibration kit (Pharmacia Fine Chemicals), containing markers whose molecular weights are 94 kD, 67 kD, 43 kD, 30 kD, 20.1 kD and 14.4 kD.
  • Figure 52 Purification of GSH/GSSG-refolded r31 kD by Phenyl-Sepharose chromatography
  • the supernatant (1,280 ml) was brought to 0.2 M in ammonium sulfate (AS) and loaded onto a 45 ml column of phenyl-Sepharose previously equilibrated with Buffer A at pH 8.5, containing also 0.2 M AS.
  • the column was washed with 150 ml of the same solution, followed by 150 ml of Buffer A, 50 ml of water and 50 ml of 6 M GuCl.
  • the purified r31kD appeared in the Buffer A fraction and at this stage it was more than 85% pure. Absorbance was measured at 280 nm.
  • the figure shows the pharmacokinetics of plasma derived 31 kD in rat serum as a function of time.
  • the rats were injected intravenously with 0.5 mg p31 kD per kg body weight.
  • Reaction I was carried out as described in Example 26 using 20 ⁇ l citrated whole blood, 0.15 ⁇ M 125 I-r-FBD (r31 kD) (5.6 ⁇ 10 5 cpm/ ⁇ g).
  • Reaction II was initiated with the formation of unlabeled Fibrin clot using 20 ⁇ l citrated whole blood as described in Example 26. After the first incubation, 0.15 ⁇ M 125 I- r-FBD was added to the existing reaction tube ("Serum”) or to the Fibrin pellet following centrifugation and resuspension in PBS ("PBS"). When CaCl 2 and Hirudin were added the concentrations were 5 mM and 3 U/ml, respectively. Reaction II was continued thereafter as described in Example 26.
  • Reaction I was carried out using several tubes containing 100 ⁇ l citrated whole blood and 0.3 ⁇ M 125 I-P-FBD (5.0 ⁇ 10 4 cpm/ ⁇ g). At the end of the incubation, the pellet was collected by centrifugation and then resuspended in PBS solution containing 1 ⁇ /ml plasmin (from porcine blood, Sigma) and was further incubated at 33°C for the indicated time intervals. The reaction was terminated by cooling and immediate centrifugation. The radioactivity in the supernatant and the pellet was measured by a gamma counter.
  • Unlabeled fibrin clot was formed using 20 ⁇ l citrated whole blood and using the conditions described for reaction II in Example 26.
  • Figure 61 Binding of 125 I-r31 kD to preformed fibrin clot (Reaction II): effect of fibrin clot age The unlabeled fibrin clot was formed using 20 ⁇ l citrated whole blood and using the conditions described for reaction II in Example 26.
  • reaction II I-r31 kD (2.9x10 4 cpm/ ⁇ g) was added, and incubation terminated after an additional 2 hours (reaction II).
  • control + T.G. 0.2 units/ml; Sigma
  • the biological activity of the FBD was studied in a model of vascular injury as indicated in Example 18, using the Extra Cellular Matrix, "ECM", of cultured endothelial cells.
  • ECM Extra Cellular Matrix
  • the binding reaction was carried out in a solution using 5 ⁇ 10 8 PFU/ml of S. aureus SA113 (ATCC Accession No. 35556) and 12S I-FN (4 ⁇ 10 4 cpm/ ⁇ g) or 125 I-FBD (1.3 ⁇ 10 5 cpm/ ⁇ g; r31 kD FBD, "reoxidized-refolded") at concentrations indicated in the figure and as described in methods.
  • concentration of the labeled molecules described is calculated using molecular weights of 220,000 and 31,000 daltons for FN and r31 kD, respectively.
  • Figure 66 Binding of FBD to S. aureus: competition with folded, and reduced, forms
  • Binding in solution of 1.25 ⁇ g 125 I-p31 kD (2.3 ⁇ 10 5 cpm/ ⁇ g) to 5x10 8 PFU/ml of S. aureus SA113 was carried out in the presence of the indicated concentrations of the following unlabeled proteins: P-FBD (p31 kD), r-FBD (r31 kD FBD “reoxidized-refolded"), r-FBD-R (r31kD FBD “Reduced Carboxyamidated), and r-CBD (r-33 kD cell binding domain of FN).
  • the binding reaction was carried out as indicated in the methods section.
  • Binding of 7.5 ⁇ 10 8 PFU/ml of 3H-leucine-S. aureus (5.8 cpm/10 5 PFU) to FN immobilized onto plastic vials was carried out as described in methods and in the presence of human plasma FN, r-FBD (r31 kD FBD “reoxidized-refolded"), P-FBD (p31 kD), or BSA (Bovine Serum Albumin, Sigma). Binding of "control" reaction in the absence of competitors (9.3% of input bacteria) was normalized to 100%.
  • Binding of 3.0 ⁇ 10 6 PFU/ml of 125 I-S. aureus (1 CPM/3 PFU) to "Uno" bronchial plastic catheters (3 cm for each reaction, in duplicate) coated with FN was carried out as described in methods.
  • competition reaction was performed, the bacteria and the added protein were preincubated at room temperature for 30 minutes and then added to the catheters for further incubation as described in the methods section.
  • the proteins used in the competition reactions were: P- 31 (p31 kD), r-20 (recombinant derived 20 kD FBD) and r- 31 (reoxidated and refolded r31kD). Some of the reactions (see figure) were measured in the presence of 5 ⁇ m Heparin (from porcine intestinal mucosa, molecular weight of 10,000; Sigma).
  • the figure shows the distribution of radioactivity in serially sectioned aorta segments from balloon catheterized rabbits. The measurements were taken 72 hours after injection of 125 I-labeled Fibronectin (FN) or plasma derived 31kD FBD (31kD).
  • FN 125 I-labeled Fibronectin
  • 31kD plasma derived 31kD FBD
  • FIG. 70 Construction of pTV-170
  • the NdeI-NdeI ApoE fragment was isolated from plasmid pApoE-EX2 (ATCC Accession No. 39787) and inserted into the unique NdeI site of the expression vector p579 ( Figure 16) which had been digested with NdeI.
  • the resulting plasmid pTV-170 expresses an analog of natural human ApoE protein having a methionine residue added at the N-terminus.
  • the ß-lactamase promoter and ribosomal binding site fragment was isolated from plasmid pBLA11 (ATCC Accession No. 39788) after digestion with EcoRI and AluI. This fragment was ligated to the large fragment of pTV-170 ( Figure 70) plasmid which had been digested with NdeI, filled in with DNA polymerase I (Klenow) and then digested with EcoRI. DETAILED DESCRIPTION OF THE INVENTION
  • the plasmids pFN 126-3, pFN 132-5, pFN 975-25, pFN 949-2, and pFN 137-2 were deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the
  • the subject invention provides a plasmid for expression of a polypeptide which comprises a substantial portion of the amino acid sequence of one of the domains of naturally-occurring human fibronectin and which does not correspond to a product of proteolytic digestion of naturally-occurring human fibronectin comprising DNA encoding the polypeptide and DNA encoding suitable regulatory elements positioned relative to the DNA encoding the polypeptide so as to effect expression of the polypeptide in a suitable host cell.
  • a plasmid for expression of a polypeptide which comprises a substantial portion of the amino acid sequence of the cell binding domain of naturally-occurring human fibronectin and which does not correspond to a product of proteolytic digestion of naturally-occurring human fibronectin comprising DNA encoding the polypeptide and DNA encoding suitable regulatory elements positioned relative to the DNA encoding the polypeptide so as to effect expression of the polypeptide in a suitable host cell.
  • a plasmid for expression of a polypeptide which comprises a substantial portion of the amino acid sequence of the fibrin binding domain of naturally-occurring human fibronectin and which does not correspond to a product of proteolytic digestion of naturally-occurring human fibronectin comprising DNA encoding the polypeptide and DNA encoding suitable regulatory elements positioned relative to the DNA encoding the polypeptide so as to effect expression of the polypeptide in a suitable host cell.
  • fibrin binding domain polypeptide analogs include the p31 kD, r31 kD, and r20 kD polypeptides. These polypeptides exhibit the binding and adhesive properties of portions of naturally-occurring human fibronectin. The scope of the claims of the subject application are not intended to be limited to these three FBD analogs, which are examples of preferred embodiments only.
  • r31 kP fibrin binding domain polypeptide encompasses the three forms of such protein defined in Example 24 as:
  • the "scrambled" r31 kP polypeptide is r31 kP protein which is apparently improperly folded due to the formation of one or more incorrect disulfide bonds.
  • the polypeptide is about a 75 kD polypeptide fragment of the cell binding domain of human fibronectin, about a 40 kD polypeptide fragment of the cell binding domain of human fibronectin, about a 33 kD polypeptide fragment of the cell binding domain of human fibronectin, about a 31 kD polypeptide fragment of the fibrin binding domain of human fibronectin, or about a 20 kD polypeptide fragment of the fibrin binding domain of human fibronectin.
  • the polypeptide is a 75 kD polypeptide fragment of the cell binding domain of human fibronectin comprising amino acids 1102-1851, but deleted of amino acids 1600-1689; a 40 kD polypeptide fragment of the cell binding domain of human fibronectin comprising amino acids 1380-1851, but deleted of amino acids 1600-1689; a 33 kD polypeptide fragment of the cell binding domain of human fibronectin comprising amino acids 1329-1722, but deleted of amino acids 1600-1689; a 31 kD polypeptide fragment of the fibrin binding domain of human fibronectin comprising amino acids 1-262; or a 20 kD polypeptide fragment of the fibrin binding domain of human fibronectin comprising amino acids 1-153 and 13 additional amino acids.
  • Naturally-occurring human fibronectin is as it occurs in the human body (in plasma).
  • a polypeptide which has the biological activity of naturally-occurring human fibronectin exhibits binding or adhesive properties similar to naturally-occurring human fibronectin when the level of such activity is assayed or determined.
  • a polypeptide which has the biological activity of one of the domains of naturally-occurring human fibronectin, such as the cell binding domain exhibits binding or adhesive properties similar to the domain of naturally-occurring human fibronectin when the level of such activity is assayed or determined.
  • nucleotide sequences of the various functional domains are determined by cleavage with restriction enzymes, and do not correspond to the nucleotide sequences of the domains as defined by proteolytic digestion of fibronectin.
  • the plasmid of this invention further comprises suitable regulatory elements positioned relative to the DNA encoding the polypeptide so as to effect expression of the polypeptide in a suitable host cell, such as promoters and operators, e.g. ⁇ P L O L , ribosomal binding sites, e.g. C II , and repressers.
  • suitable regulatory elements include, for example, the lac, trp, tac, lpp and deo promoters (European Patent Application Publication No. 0303972, published February 22, 1989).
  • the suitable regulatory elements are positioned relative to the DNA encoding the polypeptide so as to effect expression of the polypeptide in a suitable bacterial host cell.
  • the regulatory elements are positioned close to and upstream of the DNA encoding the polypeptide.
  • the invention provides a plasmid designated pFN 126-3 having the restriction map shown in Fig. 10 and deposited in Escherichia coli strain A1645 under ATCC Accession No. 67829. Plasmid pFN 126-3 encodes a 75 kD polypeptide fragment of the cell binding domain of human fibronectin comprising amino acids 1102-1851, but deleted of amino acids 1600-1689. Also provided is a plasmid designated pFN 132-5 having the restriction map shown in Figure 12 and deposited in Escherichia coli strain A4255 (F + ) under ATCC Accession No. 67830. Plasmid pFN 132-5 encodes a 40 kD polypeptide fragment of the cell binding domain of human fibronectin comprising amino acids 1380-1851, but deleted of amino acids 1600-1689.
  • Plasmid pFN 137-2 encodes a 33 kD polypeptide fragment of the cell binding domain of human fibronectin comprising amino acids 1329-1722, but deleted of amino acids 1600-1689.
  • the invention also provides a plasmid designated pFN 975-25 and deposited in Escherichia coli strain A4255 (F + ) under ATCC Accession No. 67832. Plasmid pFN 975-25 encodes a 31 kD polypeptide fragment of the fibrin binding domain of human fibronectin comprising amino acids 1-262.
  • Plasmid pFN 949-2 encodes a 20 kD polypeptide fragment of the fibrin binding domain of human fibronectin comprising amino acids 1-153 and 13 additional amino acids.
  • the plasmids of this invention may be introduced into suitable bacterial host cells, preferably Escherichia coli.
  • suitable Escherichia coli is strain A4255 (F + ) [ATCC Accession No. 67830], strain A1645 [ATCC Accession No. 67829], and strain A4255 [ATCC Accsssion No. 67910], but other Escherichia coli strains and other bacteria can also be used as host cells for the plasmids.
  • Such bacteria include Pseudomonas aeruoinosa and Bacillus subtilis.
  • the bacteria used as hosts may be any strain including auxotrophic (such as A1645), prototrophic (such as A4255), and lytic strains; F + and F- strains; strains harboring the cI 857 repressor sequence of the prophage (such as A1645 and A4255); and strains deleted for the deo repressers and the deo gene (see European Patent Application Publication No. 0303972, published February 22, 1989).
  • Escherichia coli strain A4255 (F + ) has been deposited harboring plasmids pFN 132-5 and pFN 137-2 under ATCC Accession Nos. 67830 and 67910, respectively.
  • Escherichia coli strain A1645 has been deposited harboring plasmid pFN 126-3 under ATCC Accession No. 67829.
  • the invention provides an Escherichia coli cell containing the plasmid designated pFN 126-3 and wherein the cell and wherein the cell is deposited under ATCC Accession No. 67829.
  • Escherichia coli cell containing the plasmid designated pFN 132-5 and wherein the cell is deposited under ATCC Accession No. 67830.
  • the invention provides a Escherichia coli cell containing the plasmid designated pFN 137-2 and wherein the cell is deposited under ATCC Accession No. 67910.
  • Escherichia coli cell containing the plasmid designated pFN 975-25 and wherein the cell is deposited under ATCC Accession No. 67832.
  • the invention provides a Escherichia coli cell containing the plasmid designated pFN 949-2 and wherein the cell is deposited under ATCC Accession No. 67831.
  • the invention further provides a method of producing a polypeptide which comprises a substantial portion of the amino acid sequence of one of the domains of naturally-occurring human fibronectin which comprises treating an Escherichia coli cell containing a plasmid comprising DNA encoding the polypeptide so that the DNA directs expression of the polypeptide and recovering from the cell the polypeptide so expressed.
  • Also provided is a method of producing a polypeptide which comprises a substantial portion of the amino acid sequence of the cell binding domain of naturally-occurring human fibronectin which comprises treating an Escherichia coli cell containing a plasmid comprising DNA encoding the polypeptide so that the DNA directs expression of the polypeptide and recovering from the cell the polypeptide so expressed.
  • the invention provides a method of producing a polypeptide which comprises a substantial portion of the amino acid sequence of the fibrin binding domain of naturally-occurring human fibronectin which comprises treating an Escherichia coli cell containing a plasmid comprising DNA encoding the polypeptide so that the DNA directs expression of the polypeptide and recovering from the cell the polypeptide so expressed.
  • the polypeptide so produced is a 75 kD, 40 kD, or 33 kD polypeptide of the cell binding domain, or a 31 kD or 20 kD polypeptide of the fibrin binding domain.
  • Ths invention provides a purified polypeptide substantially free of other substances of human origin which comprises a substantial portion of the amino acid sequence of one of the domains of naturally-occurring human fibronectin and which does not correspond to a product of proteolytic digestion of naturally-occurring human fibronectin.
  • a purified polypeptide substantially free of other substances of human origin which comprises a substantial portion of the amino acid sequence of the cell binding domain of naturally-occurring human fibronectin and which does not correspond to a product of proteolytic digestion of naturally-occurring human fibronectin.
  • a purified polypeptide substantially free of other substances of human origin which comprises a substantial portion of the amino acid sequence of the fibrin binding domain of naturally-occurring human fibronectin and which does not correspond to a product of proteolytic digestion of naturally-occurring human fibronectin.
  • the purified polypeptide is a 75 kD polypeptide substantially free of other substances of human origin which comprises amino acids 1102-1851 of the cell binding domain of naturally-occurring human fibronectin, but deleted of amino acids 1600-1689, which does not correspond to a product of proteolytic digestion of naturally-occurring human fibronectin.
  • Another preferred purified polypeptide is a 40 kD polypeptide substantially free of other substances of human origin which comprises amino acids 1380-1851 of the cell binding domain of naturally-occurring human fibro nectin, but deleted of amino acids 1600-1689, which does not correspond to a product of proteolytic digestion of naturally-occurring human fibronectin.
  • the invention also provides a purified polypeptide of 33 kD substantially free of other substances of human origin which comprises amino acids 1329-1722 of the cell binding domain of naturally-occurring human fibronectin, but deleted of amino acids 1600-1689, which does not correspond to a product of proteolytic digestion of naturally-occurring human fibronectin.
  • the invention also provides a bacterially-produced polypeptide which comprises a substantial portion of the amino acid sequence of one of the domains of naturally-occurring human fibronectin which does not correspond to a product of proteolytic digestion of naturally-occurring human fibronectin.
  • the domain is the cell binding domain or the fibrin binding domain of naturally-occurring human fibronectin.
  • Preferred polypeptides include: a bacterially-produced 75 kD polypeptide which comprises amino acids 1102-1851 of the cell binding domain of naturally-occurring human fibronectin, but deleted of amino acids 1600-1689 which does not correspond to a product of proteolytic digestion of naturally-occurring human fibronectin; a bacterially- produced 40 kD polypeptide which comprises amino acids 1380-1851 of the cell binding domain of naturally- occurring human fibronectin, but deleted of amino acids 1600-1689 which does not correspond to a product of proteolytic digestion of naturally-occurring human fibronectin; a bacterially-produced 33 kD polypeptide which comprises amino acids 1329-1722 of the cell binding domain of naturally-occurring human fibronectin, but deleted of amino acids 1600-1689 which does not correspond to a product of proteolytic digestion of naturally-occurring human fibronectin; a bacterially-produced 31 kD polypeptide of the fibrin binding domain of naturally-occurring human fibronectin which
  • the invention provides a polypeptide which comprises a substantial portion of the amino acid sequence of one of the domains of naturally-occurring human fibronectin which does not correspond to a product of proteolytic digestion of human fibronectin.
  • the domain preferably is the cell binding domain or the fibrin binding domain.
  • the polypeptide is a 75 kD polypeptide comprising amino acids 1102-1851, but deleted of amino acids 1600-1689, a 40 kD polypeptide comprising amino acids 1380-1851, but deleted of amino acids 1600-1689, a 33 kD polypeptide comprising amino acids 1329-1722, but deleted of amino acids 1600-1689, a 31 kD polypeptide comprising amino acids 1-262, or a 20 kD polypeptide comprising amino acids 1-153 and 13 additional amino acids.
  • the invention provides a composition comprising at least two of the polypeptides disclosed above and a suitable carrier.
  • the polypeptides in such a composition may be bound to one another.
  • bound encompasses polypeptides bound covalently, non-covalently, or conjugated.
  • the polypeptides may be conjugated through other chemical moities including amino acid or polypeptide linkers, which are standardly used in the art and are well-known to those skilled in the art to which the subject invention pertains.
  • hybrid polypeptide consisting essentially of at least two of the polypeptides disclosed above.
  • the invention also provides a pharmaceutical composition comprising an amount of the composition comprising at least two of the polypeptides effective to inhibit platelet aggregation and a pharmaceutically acceptable carrier and a pharmaceutical composition comprising an amount of the hybrid polypeptide effective to inhibit platelet aggregation and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising an amount of any one of the disclosed polypeptides effective to inhibit platelet aggregation and a pharmaceutically acceptable carrier, and a pharmaceutical composition comprising an amount of any of the disclosed polypeptides effective to inhibit thromboxane release from platelets and a pharmaceutically acceptable carrier.
  • the invention also provides a method of inhibiting platelet aggregation which comprises contacting platelets under suitable conditions with an amount of any of the disclosed polypeptides effective to inhibit platelet aggregation, and a method of inhibiting thromboxane release from platelets which comprises contacting platelets under suitable conditions with an amount of any of the disclosed polypeptides effective to inhibit thromboxane release from the platelets.
  • the invention further provides a method of treating a subject with a cerebrovascular or cardiovascular disorder which comprises administering to the subject an amount of any of the disclosed polypeptides effective to inhibit platelet aggregation.
  • cardiovascular disorders susceptible to treating include acute myocardial infarction, or angina.
  • Subjects who have undergone angioplasty or coronary bypass surgery or who have received thrombolytic treatment may also be treated with the polypeptides of the subject invention so as to inhibit platelet aggregation.
  • the invention provides a method of treating a subject with a wound which comprises administering to the subject an amount of any of the disclosed polypeptides effective to promote healing of the wound.
  • the wound may be a cutaneous wound, such as an incisional, a skin deficit, a skin graft or a burn wound.
  • the wound may also be an eye wound, such as a corneal epithelial wound or a corneal stromal wound, or a tendon injury.
  • This invention further provides a method of treating a subject susceptible to, or afflicted with, a bacterial infection which comprises administering to the subject an amount of any of the disclosed polypeptides effective to prevent or treat the bacterial infection.
  • a bacterial infection may be due to the presence of a catheter or an implant in the subject.
  • the invention also provides a method of treating a subject with cancer which comprises administering to the subject an amount of any of the disclosed polypeptides effective to retard tumor metastasis, and a method of detecting a tumor in a subject which comprises administering to the subject an amount of any of the disclosed polypeptides effective to detect the tumor.
  • polypeptides of the subject invention can also be used in a method of detecting a thrombus in a subject which comprises administering to the subject an amount of the polypeptide effective to detect the thrombus.
  • the polypeptide is used as a diagnostic agent for detecting the tumor or thrombus.
  • Numerous methods are known in the art for such detection, such as radioactive labeling (nuclear medicine use of isotopes), radio- opaque labeling (such as CAT scan), and Magnetic Resonance Imaging (MRI). Any of these labeling methods can be used in the method of the subject invention for detecting the tumor or thrombus.
  • the invention also provides any of the disclosed polypeptides bound to a thrombolytic agent.
  • the thrombolytic agent is selected from the group consisting, of: tissue plasminogen activator (TPA), urokinase, strepotokinase, prourokinase, Anisoylated Plasminogen-Streptokinase Activator Complex (EminaseTM), or TPA analogs.
  • the invention provides any of the disclosed polypeptides bound to a growth factor, such as: EGF, PDGF; ⁇ -TGF, ß-TGF, FDGF, TNF, interleukins, interferons, erythropoietin, colony-stimulating factor (CSF), GM-CSF, G-CSF, or CSF-I.
  • a growth factor such as: EGF, PDGF; ⁇ -TGF, ß-TGF, FDGF, TNF, interleukins, interferons, erythropoietin, colony-stimulating factor (CSF), GM-CSF, G-CSF, or CSF-I.
  • the polypeptides may also be bound to serum albumin, a blood factor such as Factor VIII or Factor XIII, polyethyleneglycol, or to superoxide dismutase.
  • the invention provides a coated medical device comprising a medical device and the polypeptides of the fibrin binding domain of naturally-occurring human fibronectin applied as a coating to the surface of the medical device.
  • medical devices which may be coated include catheters, medical implants (such a hip replacement and prostheses), tubings and syringes.
  • the invention provides a method of minimizing risk of bacterial infection associated with use of medical devices which comprises:
  • Applicants' invention also provides uses of tryptic fragments of the fibrin binding domain.
  • tryptic fragments are obtained by proteolytic digestion of plasma derived fibronectin.
  • These tryptic fragments may be used in a method of inhibiting platelet aggregation which comprises contacting platelets under suitable conditions with an amount of the polypeptide effective to inhibit platelet aggregation, such polypeptide comprising a substantial portion of the amino acid sequence of the fibrin binding domain of naturally-occurring human fibronectin.
  • a method of inhibiting thromboxane release from platelets which comprises contacting platelets under suitable conditions with an amount of a polypeptide effective to inhibit thromboxane release from the platelets, such polypeptide comprising a substantial portion of the amino acid sequence of the fibrin binding domain of naturally-occurring human fibronectin.
  • Also provided is a method of treating a subject with a cerebrovascular disorder which comprises administering to the subject an amount of a polypeptide effective to inhibit platelet aggregation, such polypeptide comprising a substantial portion of the amino acid sequence of the fibrin binding domain of naturally-occurring human fibronectin.
  • the polypeptide of the fibrin binding domain may be used to treat a subject with a cardiovascular disorder which comprises administering to the subject an amount of a polypeptide effective to inhibit platelet aggregation, such polypeptide comprising a substantial portion of the amino acid sequence of the fibrin binding domain of naturally-occurring human fibronectin.
  • the cardiovascular disorder may include acute myocardial infarction or angina.
  • Subjects who have undergone angioplasty or coronary bypass surgery or who have received thrombolytic treatment may also be treated with the plasma-derived fibrin binding domain polypeptides.
  • the invention also provides a method of treating a subject with a wound which comprises administering to the subject an amount of a fibrin binding domain polypeptide effective to promote healing of the wound, such polypeptide comprising a substantial portion of the amino acid sequence of the fibrin binding domain of naturallyoccurring human fibronectin.
  • the wound may be a cutaneous wound, such as an incisional wound, a skin deficit wound, a skin graft or a burn wound.
  • the wound may also be an eye wound, such as a corneal epithelial wound or a corneal stromal wound, or a tendon injury.
  • the invention also provides a method of treating a subject susceptible to, or afflicted with, a bacterial infection which comprises administering to the subject an amount of a polypeptide effective to prevent or treat the bacterial infection, such polypeptide comprising a substantial portion of the amino acid sequence of the fibrin binding domain of naturally-occurring human fibronectin.
  • the bacterial infection may be due to the presence of a catheter or an implant in the subject.
  • a method of treating a subject with cancer which comprises administering to the subject an amount of the fibrin binding domain polypeptide effective to retard tumor metastasis, such polypeptide comprising a substantial portion of the amino acid sequence of the fibrin binding domain of naturally-occurring human fibronectin.
  • the invention provides a method of detecting a tumor in a subject which comprises administering to the subject an amount of a FBD polypeptide effective to detect the tumor, such polypeptide comprising a substantial portion of the amino acid sequence of the fibrin binding domain of naturally-occurring human fibronectin, and a method of detecting a thrombus in a subject which comprises administering to the subject an amount of such a polypeptide effective to detect the thrombus, such polypeptide comprising a substantial portion of the amino acid sequence of the fibrin binding domain of naturally-occurring human fibronectin.
  • the cDNA sequence applicants have cloned and expressed is missing the 270 bp extra domain (ED) segment which extends from nucleotides 4811 to 5080, inclusive, on the Baralle map (see Figure 41).
  • ED extra domain
  • the cDNA sequence which is said to extend from nucleotide 3317 to 5566 on the Baralle map contains only 1980 nucleotides, because it is missing the 270 nucleotides of the ED segment, namely from nucleotides 4811 to 5080 inclusive; this region is also known in the art as the ED-A region.
  • the protein expressed by that DNA fragment would encode from amino acid 1102 to amino acid 1851 on the Baralle map but would be missing the 90 amino acids encoded by the ED region, namely amino acids 1600-1689 inclusive, and thus it would contain only 660 amino acids. This is true for all fragments described in this application which span the ED region. (The region known in the art as the ED-B region is missing both in Baralle's sequence and in applicants' cDNA.)
  • the EcoRI cleavage site shown at position 3317 was constructed by applicants during the cloning procedure by use of EcoRI linkers.
  • This GAATTC sequence at positions 3313 to 3318 differs in 1 nucleotide from the corresponding Baralle sequence GATTC. This introduces a single nucleotide change C to A at nucleotide 3315. This changes the corresponding amino acid from Thr to Asn.
  • EXAMPLE 1 Preparation of a Fibronectin cDNA Library
  • a cDNA library was prepared in gt11 from poly A+ mRNA isolated from human liver according to the published procedures (13,14). The cDNA fragments were cloned using EcoRI linkers and the cDNA library was screened for fibronectin (FN) positive plasmids using the following synthetic DNA probes.
  • cDNA has a 270 base pair deletion from base 4811 to base 5080 on the FN physical map (the complete non spliced cDNA).
  • CBD Cell Binding domain
  • ACCTAGTTAAGTTAA was ligated to an EcoRI-BamHI fragment derived from cDNA clone pFN919 (bases 3317 to 4151) to create an NdeI site.
  • the ligated fragment was inserted into NdeI-BamHI digested pBR322 ( Figure 2).
  • the plasmid obtained was designated pFN100.
  • the plasmid cloning was carried out in Escherichia coli strain MC 1061.
  • TATGGATCAGAGCT ACCTAGTC was ligated to a SstI-BamHI fragment derived from cDNA clone pFN919 (bases 3611 to 4090) to create an NdeI site.
  • the ligated fragment was inserted into NdeI-BamHI digested pBR322 ( Figure 3).
  • the plasmid obtained was designated pFN101 and was maintained in Escherichia coli strain MC 1061.
  • a synthetic oligomer containing a termination codon and HindIII and Sail restriction sites was ligated to the 3' end of an EcoRI-BglII fragment derived from cDNA clone pFN916 (bases 4151 to 5566). The ligated fragment was inserted into an EcoRI-Sall digested pBR322 plasmid ( Figure 4). The plasmid obtained was designated pFN102 and was maintained in Escherichia coli strain MC 1061.
  • the synthetic oligomer had the following sequence:
  • plasmid pFN102 was digested with EcoRI and HindIII enzymes, and the 3' end EcoRI-HindIII of the CBD was isolated and ligated to plasmid pFN100 which had been digested with BamHI and HindIII ( Figure 5). The ligation was performed in the presence of a BamHI-EcoRI synthetic fragment with the sequence shown in Figure 15. The plasmid obtained was designated pFN103 and was maintained in Escherichia coli strain MC 1061.
  • the plasmid obtained was designated pFN105 and was maintained in Escherichia coli strain A 1645.
  • Plasmid pTV301 was constructed as shown in Figure 7 by
  • Plasmid pTV104(2) has been deposited with the ATCC under Accession No. 39384.
  • the NdeI-NdeI hGH fragment was inserted into the NdeI site of expression vector plasmid p579.
  • Plasmid p579 was constructed from plasmids pRO211 and pPS1 ( Figure 16). Plasmid pPS1 has been deposited with the ATCC under Accession No. 39807.
  • Plasmid pRO211 was constructed from plasmid pJH200 ( Figure 17). Plasmid pJH200 has been deposited with the ATCC under Accession No. 39783.
  • plasmid pFN102 was digested wwith EcoRI-HindIII, and the fragment containing the CBD was isolated and ligated to plasmid pFN101 in the presence of a BamHI-EcoRI synthetic fragment with the sequence shown in Figure 15 (See Example 2) ( Figure 8).
  • the plasmid obtained was designated pFN104 and was maintained in Escherichia coli strain MC 1061.
  • NdeI-HindIII fragment of FN was isolated from plasmid pFN104 and ligated into plasmid pTV301 digested with NdeI and HindIII ( Figure 9).
  • the plasmid obtained was designated pFN106 and was maintained in Escherichia coli strain A 1645.
  • Plasmids pFN105 and pFN106 were used to transform Escherichia coli strain A1645.
  • the clones obtained were grown in LB medium containing Ampicillin (100 ⁇ g/ml) at 30°C to O.D. of 0.7. Expression was obtained upon induction at 42oC for 1-3 hours.
  • the SstI-BglII fragment of clone pFN105 was replaced by an SstI-BglII fragment of cDNA clone pFN114-4 ( Figure 10).
  • Plasmid pFN114-4 is an additional plasmid harboring FN cDNA isolated independently from the cDNA library; pFN114-4 has no deletion of the thymidine in position 4030.
  • An SstI-BglII fragment from plasmid pFN114-4 was used to replace the SstI-BglII fragment from plasmid pFN105.
  • the resulting plasmid p117-4 directed the expression of the full length 75kD cell binding domain protein but the plasmid does not contain the translation termination codon and terminates within the pBR322 sequence.
  • the following linker was constructed: GATCTACTAAGGCCTA
  • This linker contains BglII and HindIII sites at the 5' and 3' terminals respectively, and codes for the 3' end of the 75 kD CBD including the translation termination codon. It was ligated to plasmid pFN117-4 which had been cleaved with BglII and HindIII ( Figure 10). The resulting plasmid pFN 126-3 directed the expression of the full length 75 kD cell binding domain protein, with the authentic C-terminus. Plasmid pFN 126-3 has been deposited in Escherichia coli A1645 with the American Type Culture Collection under Accession No. 67829.
  • the protein expressed has the amino acids met-asp-gln-phe-asn-ser (MDQFNS) added to the amino terminus of a protein which begins at amino acid 1102 and extends to amino acid 1851 of the fibronectin molecule (minus amino acids 1600-1689). This molecule differs from the 75 kD protein obtained by partial tryptic digests of fibronectin. The tryptic polypeptide fragment extends from amino acid no. 873 to amino acid no. 1555.
  • MDQFNS met-asp-gln-phe-asn-ser
  • Plasmid pFN102 was digested by EcoRI and HindIII.
  • the FN EcoRI-HindIII fragment was isolated and ligated to plasmid pTV301 which had been digested with NdeI and HindIII in the presence of a synthetic DNA fragment having the sequence:
  • plasmid pFN118 was digested by Bglll and NdeI, and the BglII-NdeI FN fragment was isolated and ligated to both the
  • NdeI-HindIII large fragment isolated from plasmid pTV301 and also to a synthetic linker ( Figure 12).
  • the synthetic linker had the following sequence:
  • the plasmid obtained, pFN132-5 was found to express the 40kD CBD protein which terminated at the correct site.
  • the original transformation of plasmid pFN132-5 was carried out in Escherichia coli strain A 1645. After plasmid analysis (restriction enzymes, DNA sequencing and determination of the 40 kP protein expression) the plasmid was then used to transform Escherichia coli strain 4255 (F + ). Plasmid pFN 132-5 has been deposited in Escherichia coli with the American Type Culture Collection under Accession No. 67830.
  • the protein expressed has the amino acids met-asp-gln-phe (MDQF) added to the amino terminus of a polypeptide which begins at fibronectin amino acid 1380 and extends to amino acid 1851 of the fibronectin molecule (minus amino acids 1600-1689). This molecule differs from the 75 kP protein obtained by partial tryptic digests of fibronectin. The tryptic polypeptide fragment extends from amino acid no. 873 to amino acid no. 1555. Growth conditions
  • the 40 kD CBD protein was further purified by solubilization of the pellet in 6M Urea (in the above buffer) and fractionation by DEAE cellulose ion exchange column chromatography.
  • the 40kD protein was pooled and dialyzed against PBS.
  • An improved purification method for the 40kD protein is disclosed in Example 15. EXAMPLE 6
  • the purified 40kP CBD was assayed for biological activity.
  • Authentic plasma FN was used as a positive control.
  • Plasma FN and recombinant 40 kD CBD were immobilized on a plastic surface.
  • the binding of Tritium-labeled mouse 3T3 cells to the layer was determined as a function of the immobilized proteins concentration, as described by Pierschbacher, M., et al. (15, 18). Results indicate that the extent of cell binding by the 40 kD protein is comparable to that of the authentic plasma FN. Additional results are disclosed in Example 16.
  • PRP platelet-rich plasma
  • PPP platelet-poor plasma
  • the aggregometer was calibrated on 0.1 and 0.9 transmittance units using the PRP and PPP, respectively.
  • Reaction tube mixture a. 125 ⁇ l of PRP.
  • Platelet aggregation was induced by adding an APP solution to a final concentration of 10 ⁇ M (5 ⁇ l of 0.5mM ADP solution).
  • the results are summarized in Table I and demonstrate that both the 40 kD CBD and the 31 kD FBD proteins partially inhibit the APP induced platelet aggregation, while undigested, whole plasmatic fibronectin had no effect.
  • GRGDS synthetic pentameric peptide
  • the RGP-containing synthetic pentapeptide at relatively high molar concentrations, exhibited an inhibitory effect, as reported in the literature.
  • the purified plasma FBD inhibits platelet aggregation in vitro. This finding will be further corroborated using recombinant FBD. More results showing the effect of the 40 kD protein on platelet aggregation are disclosed in Example 17.
  • a 31 kD tryptic fragment from plasmatic FN was isolated and purified by chromatography on PEAE-cellulose, CM-Sepharose and Heparin-Sepharose. The purified fragment was found to be effective in preventing platelet aggregation (40% inhibition at concentrations between 0.5 and 1.1 ⁇ M).
  • the 31 kP fragment was obtained by cleavage of plasmatic FN (purified on a Gelatin-Sepharose column from which it was eluted and stored in 1M guanidinium hydrochloride).
  • plasmatic FN purified on a Gelatin-Sepharose column from which it was eluted and stored in 1M guanidinium hydrochloride.
  • 206 mgs of FN - after dialysis against 10 mM of Tris-HCl - were digested with 0.01% of TPCK-trypsin at 37°C for 5 min.
  • the tryptic digest was loaded on a DE52 column (6ml) and 1/5 of the flow-through fraction (50 ml) was applied on a CM-Sepharose column (3 ml) and eluted with a NaCl gradient (0-0.5 M) .
  • the purified fragment was characterized by SPS-PAGE under reducing and non-reducing conditions, by gel filtration chromatography on Superose 12 (molecular weight 26 kP) and by N-terminal sequencing (the N-terminus was found to be blocked, as expected for a pyroglutamate residue). Furthermore, the material did react in immunoblots with anti-FN and anti-rec20 kP antibodies. (Rec 20 kP represents a recombinant FN molecule which contains amino acids 1-190.) Its binding characteristics to Heparin-Sepharose also constitute evidence for its nature. This 31 kP fragment also reacted with Staphylococcus aureus in a bacteria binding assay. EXAMPLE 9
  • nucleotide number 19 was changed to adenine (A), thereby eliminating a Pdel restriction site without altering the amino acid sequence.
  • A adenine
  • the various steps for the cloning of the above synthetic DNA fragment into pBR322 plasmid vector digested with EcoRI and BamHI are described in Figure 43.
  • the plasmid obtained was designated pFN 932-18.
  • the protein has a mobility of about 20 kD on reduced SDS polyacrylamide gels as determined from the mobility of the size markers.
  • the protein comprises methionine followed by the first 153 amino acids of fibronectin followed by 4 amino acids coded for by a synthetic linker and then 9 amino acids resulting from readthrough into the pBR322 vector, i.e. a total of 167 amino acids.
  • the protein is referred to as the r20 kD protein or the r20 kD FBD.
  • a synthetic oligonucleotide containing a TAA termination codon and a BglII site having the following sequence:
  • 3' GACAAATTCGTCTAG was ligated to the 3' end of an EcoRI-PvuII fragment isolated from FN cDNA clone p931-5 and to a pBR322 vector digested with EcoRI and BamHI as described in Figure 45.
  • the plasmid obtained was designated pFN935-12.
  • NdeI-HindIII fragment containing the FBD coding region and the T 1 T 2 transcription terminators was isolated from plasmid pFN-957 and inserted into plasmid pTV 301 ( Figure 7) digested with NdeI and HindIII as described in Figure 49.
  • the resulting plasmid designated as pFN 962-3, directs the expression of a FBD analog protein under the control of ⁇ P L promoter and cII ribosomal binding site.
  • Escherichia coli strains A1645 and A4255 transformed with this plasmid expressed only small amounts of the FBD protein.
  • the expression of the FBD protein was detectable only by Western blot analysis using polyclonal antibodies directed against human plasma derived FN.
  • PBLA DNA fragment coding for the s-lactamase promoter and ß-lactamase RBS
  • the DNA fragment coding for PBLA was isolated from plasmid pBLA11 (ATCC No. 39788) and inserted into plasmid pFN 962-3 digested with NdeI, filled in with Klenow enzyme and digested with EcoRI as described in Figure 50.
  • the clone expressing the r31 kD FBD protein was fermented in rich medium (yeast extract and casein hydrolysate) containing ampicillin. Growth was carried out at 30°C. Expression was obtained upon induction at 42°C for 2 hours.
  • rich medium yeast extract and casein hydrolysate
  • the process is made up of three stages:
  • the cake is disrupted first in 5 volumes of 50mM Tris-HCl/50 mM Na-EPTA, pH 8 (Buffer 1); the pellet is then treated with 1.2 volumes of Buffer 1 containing 100 mg/liter lysozyme (2 hours agitation at 37oC). Triton X 100 is added to the resulting suspension (to 1%), and after 30 min. at room temperature the suspension is centrifuged and the pellet is resuspended and washed twice with water. All these steps are performed by disruption of the pellet and centrifugation and the 31 kD stays in the pellet, as evidenced from SDS-PAGE gels.
  • the washed pellet is suspended in 14 volumes of 10 mM Tris-HCl/5 mM EDTA/2mM PMSF/2mM 6-aminocaproate, pH 7.5 (Buffer A) and then treated successively with Buffer A containing: 1% decyl sulfate, 1% decyl sulfate/5% glycerol and 5% glycerol.
  • Buffer A containing: 1% decyl sulfate, 1% decyl sulfate/5% glycerol and 5% glycerol.
  • the final treatment is with Buffer A without additives.
  • the washed pellet from step 1 above is dissolved in 150-700 volumes of 6M GuCl/3mM GSH in Buffer A.
  • the concentration of GuCl is lowered gradually, i.e., first 2 M, then 1 M and 0.5 M, while keeping the concentration of all other components constant, except for the volume, which at this stage is brought to 500-1000 fold higher than that of the pellet.
  • refolding is initiated by the addition of 0.3 mM of GSSG and incubation at room temperature for 24-48 hours.
  • the refolded 31 kP is then dialyzed against Buffer A without additives.
  • the r40 kD CBD fragment domain was tested in a rat wound healing model.
  • a polyvinyl sponge is implanted subcutaneously, and test substances are injected in situ.
  • the effect of the test materials in terms of chemotaxis and extracellular matrix formation, is reflected by increased DNA and collagen accumulation in the sponge (see Figure 13).
  • r31 kD FBD To estimate the capacity of r31 kD FBD to interfere with the adherence of bacteria to the extracellular matrix in wounds, a competition assay was developed. In this assay, adherence of Staphylococcus aureus to a plastic surface coated with fibronectin and the interference of FBD with adherence were measured. Both authentic FBD and r31 kD FBD were active in inhibiting bacterial adhesion to the fibronectin coated surface.
  • the pharmacokinetic behavior of the 40 kP recombinant protein was studied in rats.
  • the half-life of the RGD peptide is 8 minutes.
  • FIG. 18 shows diagramatically the length and location of the cDNA in each plasmid and indicates the presence or absence of cDNA encoding Cell Binding Pomains I and II (CBD I and CBD II).
  • Figure 19 summarizes information about the proteins expressed by the resulting plasmids.
  • the plasmids were originally used to transform Escherichia coli strain A1645 and, after plasmid analysis (restriction enzyme data and determination of expression), the plasmids were then used to transform Escherichia coli strain A4255 (F + ).
  • expression plasmid pFN 128-4 was constructed as shown in Figure 20.
  • This plasmid expresses a protein with a molecular weight as measured by polyacrylamide gel electrophoresis of 65 kD; this protein was therefore designated "the 65 kD protein".
  • the 65 kD protein is expected to be 531 amino acids long as defined in Figure 19.
  • a plasmid expressing a protein with deletion of even more of the C-terminal section (including deletion of the CBD I region) than plasmid pFN 128-4 was constructed as shown in Figure 21.
  • the resulting plasmid, pFN 130-11 expresses a protein with a molecular weight as measured on SDS-polyacrylamide gel electrophoresis of 28 kD.
  • This recombinant protein, designated "the 28 kD protein” is expected to contain 258 amino acids as defined in Figure 19.
  • expression plasmid pFN 143-1 was constructed as shown in Figures 22-24. This plasmid expresses a protein with a molecular weight of 55 kD as measured by SDS-polyacrylamide gel electrophoresis.
  • the protein designated "the 55 kD protein", is expected to contain 436 amino acids as defined in Figure 19.
  • expression plasmid pFN 135-12 was constructed as shown in Figure 22. Both the parent plasmid (pFN 117-4) and this daughter plasmid (pFN 135-12) contain the same 3' terminus, which lacks a translation termination codon. Thus, translation terminates within the pBR322 mRNA, and the resulting protein contains at its C-terminus additional non-fibronectin derived amino acids. The molecular weight of the protein expressed, as measured on SDS polyacrylamide gels, was 45 kD, and the protein was therefore designated "the 45 kD protein". Including the non-fibronectin derived C-terminus it is expected to contain approximately 433 amino acids as defined in Figure 19.
  • Plasmid pFN 137-2 was constructed as shown in Figure 23. Plasmid pFN 137-2, in Escherichia coli host A4255, was deposited with the ATCC under Accession No. 67910. This plasmid expresses a protein with a molecular weight of 33 kD as measured by SDS-polyacrylamide gel electrophoresis, and this protein was therefore designated "the 33 kD protein". It is expected to contain 304 amino acids as defined in Figure 19.
  • expression plasmid pFN 134-9 was constructed as shown in Figure 25.
  • This plasmid expresses a protein with a molecular weight of 28 kD as measured by SDS-polyacrylamide gel electrophoresis. This protein was designated "the 28(*) kD protein".
  • the 28(*) kD protein is expected to contain 253 amino acids as defined in Figure 19.
  • the 40 kD protein, produced as described in Example 5, can be purified as described for the 40 kD protein on the last page of Example 5. Alternatively, it was purified by the modified method described below.
  • the resulting suspension was sonicated for 8 minutes in ice, and this sonication was repeated twice.
  • the resulting sonicate was centrifuged for 30 minutes at 15,000 rpm at 4°C.
  • the resulting bacterial pellet was resuspended in 100 ml Buffer A containing 6M urea and sonicated for 4 minutes.
  • the sonicate was centrifuged for 30 minutes at 15,000 rpm and 500 OD units of the resulting supernatant solution was loaded on a DE52 cellulose column (250 ml) equilibrated with Buffer A.
  • the column was washed with Buffer A and fractions were collected.
  • the purified 40 kD protein (Pool I+II) eluted within 1/6 of the flow through volume. This preparation of recombinant protein was then dialyzed against Buffer A and purified 40 kD protein fractions were pooled and dialyzed against PBS.
  • Escherichia coli strain A4255 (F + ) harboring plasmid pFN 137-2 which expresses the 33 kD protein was grown in LB medium containing ampicillin at 30°C. Expression was obtained upon induction at 42°C for 1-3 hours. The wet bacterial cake was sonicated as described in (a) above. A washed bacterial pellet (70 g) was dissolved in 1600 ml of freshly deionized 6M urea in Buffer A (as described above).
  • Protein Total Purity conc. (mg/ml) protein (mg) (SDS-PAGE)
  • pool 1 0.93 1674 (92%) pool 2 0.55 990 (85%)
  • the 40 kD protein binds to 3T3 fibroblasts and BHK cells in a similar manner to plasmatic fibronectin (although it does not bind to NRK cells).
  • the 33 kD protein binds to BHK cells very poorly.
  • the 40 kD protein appears to bind to the integrin receptors which are present on the cell surface of the eucaryotic cells analyzed, since additional experiments show that its binding was completely inhibited by the pentapeptide GRGDS, similarly to that of the plasmatic fibronectin.
  • the 40 kD protein is involved in cell attachment in a similar degree to intact fibronec tin, since it does not contain the CBD II site believed to work in a synergistic manner with the CBD I (RGD) site (see 25).
  • the two-thirds of the heparin binding site present in the 40 kD protein may be serving, unexpectedly, a cell-attachment function, and may compensate for the missing CBD II site. This hypothesis is substantiated by additional cell-attachment experiments which demonstrate that heparin competes with the binding of the 40 kD protein, but not with the binding of fibronectin.
  • the 33 kD and the 40 kD proteins both contain the CBD I region but not the CBD II region (see Figure 18).
  • Example 7 The in vitro assay described in Example 7 was used to evaluate the ability of the 40 kD and 33 kD proteins to inhibit platelet aggregation. The ability of the 40 kD protein to partially inhibit ADP-induced platelet aggregation was shown in Example 7 (Tables I and II).
  • GRGDS (Sigma) 50-100 33-66
  • the 33 kD protein has also been tested to determine whether it can prevent aggregation of collagen-induced platelets. Experiments were performed, using the same platelet aggregation assay system as described above, but inducing with 8 ⁇ g/ml collagen (Type I, Sigma) instead of ADP. The 33 kD protein inhibited the aggregation of platelets by collagen. The effective dose of the 33 kD protein needed to prevent aggregation is about 5-10 fold higher than for ADP-stimulated cells. Similar results were obtained using PRP from rats. The effect of the 40 kP and 33 kP polypeptides and the GRGPS polypeptide on the aggregation of platelets in different species was studied. PRP was prepared from blood from rats, rabbits, guinea pigs, dogs and humans and the inhibition of aggregation was measured using the aggregation assay described above. The results are shown in Table V.
  • ECM extracellular matrix
  • TXB 2 thromboxane B 2
  • the 33 kD protein binds specifically in a time- and dose-dependent manner to resting and activated platelets; it also inhibits platelet aggregation in solution as well as on ECM surfaces and inhibits platelet adhesion to the ECM as well as thromboxane B 2 release.
  • This surprising combination of properties demonstrates the uniqueness of the 33 kD protein and its utility as an anti-thrombotic drug.
  • the binding of the recombinant proteins to platelets was assayed as described by Ginsberg et al. (27) with the following modifications.
  • the Sepharose 2B column was not used; instead, 5 ml of 24 hour concentrated platelets from the Israel Blood Bank (approximately 2 ⁇ 10 9 platelets/ml) was centrifuged for 10 min at 1,500 rpm in a Sorvall centrifuge. The resulting pellet, resuspended in a 1:7 solution of ACP:Saline Buffer (see below), was centrifuged again for 10 min. at 1,500 rpm in a Sorvall centrifuge.
  • the resulting pellet was resuspended in 2.5 ml Tyrode's buffer (see below) and the number of platelets was counted (approximately 4 x 10 9 platelets/ml); this platelet preparation was designated "washed platelets" and was used in all of the platelet binding experiments described.
  • the reaction mixture contained: a) 133.3 ⁇ l washed platelets (5 ⁇ 10 8 platelets).
  • the solution was aspirated and the radioactivity of the pellet containing the platelets was measured in a gamma counter. This indicates the amount of radioactive protein bound to the platelets.
  • ACD Buffer 8 g/L citric acid
  • Figures 31-38 The results of binding experiments using the above assay are shown in Figures 31-38, and described in the Pescription of the Figures.
  • Figures 31 and 35 show that 40,000-125,000 molecules of the 33 kP or the 40 kP proteins bind per platelet.
  • the 40 kD protein becomes inactive in the antiplatelet aggregation assay on storage due to aggregation.
  • Reactivation can be achieved by the following procedure. i) Concentrate the solution containing the 40 kD protein using 50% ammonium sulfate to an OD 280 of 2.0-3.0 at -4oC while stirring. ii) Centrifuge the resulting solution at 4°C for 30 minutes at 10,000 rpm. iii) Resuspend the resulting pellet in buffer A containing 6M urea, freshly treated with AG-501-X8, an analytical grade mixed bed resin (Biorad).
  • the 33 kD protein was purified as described in Example 15, and characterized as follows.
  • the major fraction (about 80%, according to the 280 nm absorption) of the size exclusion profile was run on a FPLC equipped with a Superose 12 column (Pharmacia) and displayed an apparent molecular weight of 47 kD.
  • the rest of the absorption is distributed as follows: up to 10% consists of a high molecular weight (approximately 10 3 kD) aggregate and about 10% consists of a low molecular weight (less than 10 kD) component.
  • the 33 kD fragment displays a U-shaped solubility curve with a minimum solubility between pH values 3.5 and 6.5.
  • the N-terminal sequence of the 33 kD protein was found to be Met-Ser-Pro-Thr-Gly; the Escherichia coli methionine aminopeptidase does not remove the additional methionine residue derived from the ATG coding sequence. b) Preliminary characterization of the 40 kD protein
  • the 40 kD protein was purified as described in Example 15 to greater than 92% purity and it was then characterized as follows.
  • the 40 kD protein preparation consists of an oligomeric form of molecular weight smaller than 300 kD, and greater than 40% of the preparation consists of a soluble-aggregate form of molecular weight approximately 1000 kD, as determined from FPLC runs on a Superose 12 column (Pharmacia). However, this aggregation was found to be reversible in 4M urea, since under these conditions the 40 kD protein elutes from the Superose 12 column (in 4M urea) as a monomer.
  • the N-terminal sequence of the 40 kD protein was found to be Met-Asp-Gln-Phe-Asn-Ser-Ile-Thr-Leu-Thr.
  • the Met-Asp-Gln-Phe sequence is added to the N-terminus of the fibronectin domain, which begins at the subsequent Asn (see Figure 19).
  • Purified 40 kD protein prepared as described in Examples 5 and 15, was combined with isotonic (285 m osmoles), sterile pyrogen-free saline at a final concentration of 0.2-0.5 mg/ml.
  • Sterilized polyvinylalcohol sponge disks IvalonTM (Unipoint Industries, N. Carolina), were surgically implanted subcutaneously, under the dorsal skin of (6-8 week old) female Sprague-Dawley rats (weighing 150-200 g).
  • a single longitudinal incision 2-3 cm long was made along the midline of the anesthetized animals, penetrating the full thickness of the shaved, disinfected skin. Through this incision four sponges (10 mm) were inserted and positioned beneath the panniculus carnosus at a distance 1-2 cm from the incision and 2-3 cm from each other.
  • Each animal was implanted with four sterile sponges and analysis of each experimental condition was performed on identically treated sponges from individual rats. Five days after implantation the animals were lightly anesthetized and the sponges were injected with 0.1 ml of one of the following test materials: 0.9% saline alone; the 40 kD protein in saline (0.5 mg/ml); native fibronectin purified from human plasma, stored in guanidine hydro chloride and dialyzed against three changes of PBS before use (1 mg/ml in saline); and bovine serum albumin (1 mg/ml in saline). Animals were sacrificed 48 hours after injection. Implanted sponges were dissected free of loosely adherent fat and muscle, and weighed (wet weight). Sponges were assayed for DNA, protein and collagen content. One sponge from each animal was fixed in buffered formalin and histological sections were prepared and stained with Masson's trichrome to reveal cellular and matrix elements.
  • the sponges were homogenized in 1N NH 4 OH and extracted overnight at 4°C.
  • DNA content was determined according to the method of Burton (29).
  • Protein content was measured according to the method of Lowry.
  • Hydroxyproline content as a measure of collagen content was determined by the method of Woessner (30), after acid hydrolysis of the sponge in vacuo (6N HCl at 110oC for 6 hours).
  • the pharmacokinetics of the radioactively labeled 40 kD protein were studied in the rat, and the results of intravenous injection are shown in Figure 14.
  • the half life of the 40 kD protein after intravenous injection is 3.2 hours and after intraperitoneal injection is 4.2 hours.
  • the process is made up of three stages: 1. Crude processing of the bacterial cake.
  • thioredoxin Attempts were also made to increase the rate of reoxidation of the r31 kD, by using thioredoxin. Based on SDS-PAGE profiles run in the absence of mercaptoethanol (ME), thioredoxin reduction and reoxidation of "scrambled" material seems to yield a more homogeneous preparation of r31 kD, but the concentration of thioredoxin which had to be used was about 100 ⁇ M. "Scrambled material” is r31 kD protein which is apparently improperly folded due to the formation of one or more incorrect disulfide bonds.
  • Phenyl-Sepharose chromatography The large volume of refolded r31 kD is first centrifuged to remove the insoluble pellet which contains either "scrambled" r31 kD or contaminants. The supernatant is brought to 0.2 M ammonium sulfate in Buffer A and loaded onto a phenyl- Sepharose column equilibrated with Buffer A containing the same ammonium sulfate concentration. The r31 kD protein is then purified by lowering the salt concentration, i.e., by elution in Buffer A.
  • AS ammonium sulfate
  • the concentrated 31 kD was dialyzed against Buffer A, pH 8.5, before being loaded on a 40 ml column of Q-Sepharose, which had previously been equilibrated with the same buffer.
  • the purified r231 kD fragments, which eluted in the flow-through and wash fractions were concentrated by lyophilization, before being characterized.
  • the column was washed free of the contaminant proteins by a step of 1 M NaCl (Figure 54).
  • the purified material is greater than 95% pure ( Figure 51).
  • the r31 kD protein has been characterized and compared to its plasma derived counterpart in terms of its purity (purity profile on reduced gels of SDS-PAGE), migration position in non-reduced gels of SDS-PAGE ( Figure 51), apparent molecular weight (approximately 37 kD) on Superose 12 ( Figure 55), immunoblot and behavior on Heparin-Sepharose (the NaCl concentration for elution of both materials from Heparin-Sepharose was found to be approximately 0.32 M). In all of these assays the r31 kD protein is similar to plasma derived fibrin binding domain.
  • Form a The protein as it is obtained from the washed pellet, after dissolution in 6 M GuCl, i.e., in "scrambled" form.
  • Form b The fully reduced protein, present after treatment with a reducing agent such as GSH in the presence of 6 M GuCl.
  • Form c The reoxidized-refolded protein, obtained by treatment with the GSH/GSSG as described above.
  • the protein seems to be less soluble in this form, as evidenced from the large amounts remaining in the pellet after extraction.
  • the scrambled protein differs from the refolded protein in its binding characteristics to both phenyl-Sepharose and Q-Sepharose.
  • Purified plasma derived or recombinant 31 kD protein (approximately 0.6 mg/ml) were reduced in 4.3 M Guanidinium Hydrochloride (GuCl), 40 mM s-mercaptoethanol (ME) , in 10 mM Tris-HCl, pH 8.5 for 24 hours at room temperature.
  • GuCl Guanidinium Hydrochloride
  • ME s-mercaptoethanol
  • Carboxamidation was achieved by adding iodoacetamide to the protein in four-fold excess over the concentration of ME and the solution incubated for 1 hour at room temperature. Subsequently the GuCl concentration was reduced by gradual dialysis (to 3M, 2M, 1M and 0.5M GuCl), before being dialyzed against Buffer A. The precipitate formed was centrifuged and the concentration of the resulting 31 kD protein in the supernatants were 0.34 and 0.19 mg/ml for the plasma derived and recombinant 31 kD, respectively. 6. Biological activity
  • Anti-platelet aggregation activity in preliminary experiments with the plasma derived 31 kD, an inhibitory effect had been observed using the platelet aggregation assay (Example 8). Thus far attempts to repeat the experiments demonstrating anti-aggregation properties of the plasma derived 31 kD in the APA have been unsuccessful.
  • a purified preparation of the tryptic 31 kP fragment of human plasma derived FN was iodinated with 125 I by the IC1 method (Vogel et al., DNAS 69: 180-184 (1972)) and injected intravenously into 9 rats (4.5 ⁇ 10 6 cpm; 0.5 mg/Kg). Blood samples were withdrawn 10 minutes after injection and then at 1, 4, 7 and 24 hours after injection. Groups of 3 rats each were sacrificed at 1.7 and 24 hours, and a variety of organs were excised and analyzed for radioactivity.
  • the 24 hour group rats were kept in individual metabolic cages, and accumulated urine and feces were collected at 7 hours and 24 hours. In order to determine whether the radioactivity detected represents the intact protein or small degraded fragments, the samples were subjected to TCA precipitation.
  • the relatively short half-life of the 31 kD FBD is important for its possible use in diagnostic imaging of thrombi.
  • the plasma derived 31 kD FBD or the recombinant 31 kD FBD (r31 kD) may be labeled radioactively or by other means and then introduced into the blood for the purpose of imaging thrombi.
  • the biological activity of the purified recombinant 31 kD FBD protein was compared to biological activity of either human plasma derived FN or a 31 kD FBD protein derived from partial tryptic digest (Example 8) of human plasma derived FN.
  • the biological activities assayed were binding to fibrin clot in vivo and in vitro, binding to bacteria (Staphylococcus aureus) and binding to extracellular matrix.
  • Two sets of experiments were carried out. In the first set, the binding of 125 I-r31 kD to fibrin was monitored during clot formation (Reaction I), while in the second set of experiments, the binding of 125 I-r31 kD to fibrin was monitored at various time periods after clot formation (Reaction II). Thrombin or Ca ++ were added to the mixtures in order to enable clot formation in citrated blood.
  • Plasma derived 31 kD FBD was derived by partial tryptic digest of human fibronectin (see Example 8).
  • the complete reaction mixture in siliconized microfuge tubes contained in a final volume of 250 ⁇ l the following components:
  • the competing protein was added together with the 125 I-labeled protein at the concentrations indicated in the figure legends.
  • reaction II Binding of 125 I-r31 kD FBD to preformed fibrin clots: The reaction mixture contains the same components as indicated for Reaction I, except for the 125 I-r31 kD. The first incubation was carried out at 37oC for 30 minutes, and only then the 125 I-r31 kD was added and the reaction was further incubated for a second period of 30 minutes. Reaction II was terminated and measured as described above for Reaction I.
  • Hirudin a specific inhibitor of thrombin (32) reduced the binding of 125 I-r31 kP to the fibrin clot in Reaction I, indicating that thrombin is needed for the binding.
  • thrombin is inhibited there is a reduction in clot formation and less fibrin is available for binding.
  • the addition of citrate to blood significantly reduces the concentration of free Ca ++ in the serum and therefore the addition of Ca ++ for fibrin clot formation is obligatory.
  • binding of r31 kP to a preformed clot is reduced when carried out in serum which has been already depleted of free Ca ++ ions by the preformed clot.
  • Transglutaminases are a class of calcium ion-dependent enzymes that catalyze an amidation reaction in which a carboxamide group of peptide bound glutaminyl residues and primary amines, including the epsilon amino group of peptide bound lysyl residues, are crosslinked.
  • Plasma FN is a substrate for transglutaminase from plasma, factor Xllla, (thrombin activated blood coagulation factor XIII) or liver; FN can be crosslinked to itself, fibrin and collagen.
  • the glutaminyl residues which are susceptible to factor XIII crosslinking are localized in the FBD region of FN (35).
  • the binding of 125 I-r31 kD to the fibrin clot was higher after 24 hours than the binding after 1 or 4 hours, presumably due to the fact that the clot formed became larger with time.
  • the presence of free Ca ++ ions in citrated blood is important in order to obtain optimal binding and crosslinking of 125 I-r31 kP to the fibrin clot.
  • the binding of 125 I-r31 kD to the fibrin clot was low while in the PBS containing 5 mM CaCl 2 , the binding was high at all incubation times.
  • 300 ⁇ g/ml of plasma derived FN was added as competitor in the presence of Ca ++ , there was a 20% reduction in 125 I- r31 kP binding to the fibrin clot probably due to some competition on the available fibrin binding sites.
  • Figure 62 shows experiments designed to compare 125 I-r31 kD binding to "old” and “newly formed” clots.
  • 125 I-r31 kD was added at the same time as the initiation of clot formation ("newly formed") and was allowed to interact with the clot during seven days of incubation. It is evident that FBD is incorporated efficiently and the amount incorporated increased during the first two days, probably representing an increase in the clot size during that period.
  • Adhesive molecules such as von Willebrand factor, fibronectin, fibrinogen, thrombospondin, collagen and laminin bind to ECM formed by removal of endothelial cells.
  • r31 kD FBD can serve for imaging the initial steps in plague formation at the site of injury.
  • the binding of 125 I-r31 kD at various concentrations to ECM was studied in the presence or absence of thrombin.
  • the results demonstrated that the binding of 125 I-r31 kD at low concentrations to ECM was not affected by thrombin (0.3 ⁇ M).
  • thrombin binding of r31 kD was slightly higher indicating that the number of binding sites naturally present are limited and thrombin digestion might expose additional binding sites (Figure 64).
  • R FBD Reduced-carboxasidated p31kD.
  • fibronectin The involvement of fibronectin in adhesion and invasion of wounds by a wide range of gram-positive bacteria is well established (36).
  • the fibrin binding domain of authentic plasma derived FN has been shown to interact with high affinity to specific receptors on the surface of bacteria.
  • the sites at which Staphylococcus aureus typically initiates infection are rich in FN, e.g. blood clots and subendothelium.
  • exogenous FN enhances bacterial adhesion to these sites.
  • FN binds to S. aureus through saturable, specific surface protein receptors. Scatchard analysis has revealed high affinity receptors with binding constant of 5 ⁇ 10 -9 M, and a range of 100-20,000 receptors per bacterium (37).
  • FN receptors correlates with invasiveness and pathogenicity of the clinical isolates. Removal of the receptors from S. aureus by mechanical means, or by growth of the bacteria in the presence of antibiotics decreases their ability to adhere to FN.
  • FN is a divalent molecule consisting of multiple functional domains with cell binding and collagen binding activities in addition to bacterial binding, it can anchor the bacteria to the wound via the various components of the extracellular matrix as well as via the FN receptor in tissue cells. Materials and Methods Binding of Bacteria to labeled FN or FBD in solution
  • the mixture was incubated for 2 hours at 20°C.
  • the amount of binding was assayed by removing 100 ⁇ l of the incubation mixture and layering on top of 0.5 ml PBS layered on 3 ml 10% percol, 0.15 M NaCl in a 5 ml siliconized tube. This was then centrifuged at 1,350 ⁇ g (4,000 rpm in SW bucket rotor) for 15 minutes at 20°C. The supernatant was aspirated and the pellet assayed for radioactivity.
  • Plastic vials were coated with 0.3 ml of 50 ⁇ g/ml FN, or 1% BSA. The tubes were incubated with shaking at 4°C overnight. The tubes were then washed with 5 ml PBS three times. Then 0.3 ml of 1% BSA in PBS was added and the tubes were further incubated with shaking for 2-3 hours at 20°C (for blocking free sites).
  • the bacteria (4 ⁇ 10 6 pfu/ml, 3 pfu/cpm) were added to the vials at concentrations indicated in the figure legends.
  • the final volume of the assay mix was 0.3 ml PBS.
  • the .mix was slowly agitated at 4°C for 90-120 minutes.
  • the tubes were then decanted and washed with 5 ml PBS three times.
  • the catheter pieces were incubated with 50 ⁇ g/ml FN at 4oC overnight with shaking.
  • the controls were incubated with PBS under the same conditions.
  • the FN solution was then decanted and the catheters were washed three times in PBS.
  • BSA-blocking was performed by adding 1% BSA in PBS for 1-2 hours at 20°C. The catheters were again washed three times with PBS.
  • Figure 68 demonstrates that the binding of S. aureus to the FN coated catheters is quite high, approximately 10 4 PFU/cm 2 .
  • r31 kD FBD or the plasma derived 31 kD FBD may be used therapeutically in preventing bacterial colonization of wounds.
  • the various FBD proteins will be formulated in suitable pharmaceutical formulations well-known to the average man of the art, and then used to "irrigate” or “flood” or treat the wound area for a suitable period of time, thereby preventing bacterial colonization of the wound.
  • the two molecules were iodinated with 125 I using the ICL method.
  • the radio-labeled molecules were injected intravenously into rabbits (20 ⁇ Ci; 100 micrograms/kg; 3 rabbits per group).
  • the aortas were removed, the scraped (abdominal) and intact (thoracic) areas were separated and each part was cut into several segments (5-6 segments for the lesion part and 2 segments for the control part). The tissue pieces were weighed and radioactivity counted.
  • Figure 69 summarizes the specific activity values found in the sequential aorta slices in rabbits injected with 125 I-FN (rabbits No. 1-3) or 125 I-p31 kD FBD (rabbits 4-6). As can be seen in Figure 69, enhanced localization of the labeled molecules was found in the de-endothelialized segments (thrombus zone). Also more radioactivity was localized in the clots when labeled p31 kD FBD was used than when labeled FN was used.
  • FBD plasma derived or recombinant
  • markers useful in imaging such as enzymes or radio-opaque inert molecules.
  • Aggregions in platelet rich plasma (PRP) or whole blood (blood) were induced by APP (10 ⁇ M). Extent of aggregation in PRP was determined by light transmittance. Extent of aggregation in whole blood was measured by. the impedance technique. The recombinant human fibronectin cell binding domain (33 kD) or the pentapeptide GRGDS were added to the reaction mixture to the final concentrations indicated in the table.

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Abstract

L'invention concerne des plasmides d'expression bactérienne de polypeptides comprenant une partie substantielle de la séquence d'acides aminés, et présentant l'activité biologique, d'un des domaines de fibronectine humaine se produisant naturellement, tel que le domaine de liaison de cellules ou le domaine de liaison de fibrine, comprenant de l'ADN codant le polypeptide et de l'ADN codant des éléments régulateurs adaptés positionnés par rapport à lADN codant le polypeptide, de manière à procéder à l'expression du polypeptide dans une cellule hôte adéquate. Dans le mode de réalisation actuellement préféré de l'invention, le polypeptide est un polypeptide 75 kD, 40 kD ou 33 kD du domaine de liaison de cellules, ou un polypeptide 31 kD ou un 20 kD du domaine de liaison de fibrine. L'invention concerne également des procédés de production des polypeptides ainsi que des compositions pharmaceutiques les comprenant et des supports pharmaceutiquement acceptables. On peut utiliser les polypeptides de l'invention afin d'inhiber l'agrégation de plaquettes, d'inhiber la libération de thromboxane par les plaquettes, ou afin de traiter un sujet atteint de troubles cérébrovasculaires, de troubles cardiovasculaires, souffrant d'une blessure, d'une infection bactérienne, d'un cancer ou afin de détecter un thrombi de fibrine. L'invention concerne en outre les polypeptides conjugués à des agents thrombolytiques, des facteurs de croissance, de l'albumine de sérum, des facteurs sanguins ou du polyéthylène-glycol.
EP19900902086 1988-12-29 1989-12-29 Cloning and production of polypeptide analogs of human fibronectin and method of using such polypeptide analogs Withdrawn EP0451211A4 (en)

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US5270030A (en) * 1988-12-29 1993-12-14 Bio-Technology General Corp. Fibrin binding domain polypeptide and method of producing
US5792742A (en) * 1991-06-14 1998-08-11 New York University Fibrin-binding peptide fragments of fibronectin
GB9401689D0 (en) * 1994-01-28 1994-03-23 Univ Manchester Diagnosis and treatment of endocarditis
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