WO2000034441A1 - CONVERSION OF LAMININ 5 TO A MIGRATORY SUBSTRATE BY PROTEOLYSIS OF THE η2 CHAIN THEREOF - Google Patents

CONVERSION OF LAMININ 5 TO A MIGRATORY SUBSTRATE BY PROTEOLYSIS OF THE η2 CHAIN THEREOF Download PDF

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WO2000034441A1
WO2000034441A1 PCT/US1999/029433 US9929433W WO0034441A1 WO 2000034441 A1 WO2000034441 A1 WO 2000034441A1 US 9929433 W US9929433 W US 9929433W WO 0034441 A1 WO0034441 A1 WO 0034441A1
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protease
laminin
medical device
chain
cathepsin
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PCT/US1999/029433
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French (fr)
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Mark Fitchmun
Jutta Falk-Marzillier
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Desmos, Inc.
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Priority to AU21752/00A priority Critical patent/AU2175200A/en
Publication of WO2000034441A1 publication Critical patent/WO2000034441A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28Materials for coating prostheses
    • A61L27/34Macromolecular materials
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L29/00Materials for catheters, medical tubing, cannulae, or endoscopes or for coating catheters
    • A61L29/08Materials for coatings
    • A61L29/085Macromolecular materials
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/08Materials for coatings
    • A61L31/10Macromolecular materials
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61FFILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/0077Special surfaces of prostheses, e.g. for improving ingrowth
    • A61F2002/0086Special surfaces of prostheses, e.g. for improving ingrowth for preferentially controlling or promoting the growth of specific types of cells or tissues
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61FFILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F13/00Bandages or dressings; Absorbent pads
    • A61F2013/00089Wound bandages
    • A61F2013/00157Wound bandages for burns or skin transplants

Definitions

  • the present invention relates to conversion of laminin 5 (LN5) to a cell migration-promoting substrate by proteases. More specifically, the invention relates to cleavage of the ⁇ 2 chain of laminin 5.
  • Cell migration is a process which occurs during vital naturally occurring physiological processes such as wound healing, immune responses, bone repair, inflammation and the like.
  • stimulation of cell migration is desirable in promoting recruitment of epithelial cells to a wound site to facilitate healing of the wound, and in promoting migration of immune cells to sites of infection.
  • Laminins are heterotrimeric extracellular matrix proteins consisting of three subunits: ⁇ , ⁇ and ⁇ . There are at least five known ⁇ subunits ( ⁇ 1 , ⁇ 2 , ⁇ 3 , ⁇ 4 , ⁇ 5 ), three known ⁇ subunits ( ⁇ instruct ⁇ 2 , ⁇ 3 ) and two known ⁇ ( ⁇ instruct ⁇ 2 ) subunits (Miner et al., J. Cell. Biol. 137:685-701, 1997).
  • Laminin 5 (LN5) is an ⁇ 3 ⁇ 3 ⁇ 2 heterotrimer which is typically associated with epithelial cell adhesion and sometimes with hemidesmosome formation. The designation "laminin 5" was coined by Burgeson et al.
  • LN5 is also produced by 804G and NBT-II rat bladder carcinoma cells (U.S. Patent Nos. 5,541,106 and 5,422,264, hereby incorporated by reference).
  • a human epithelial cell line, MCF-10A produces a LN5 extracellular matrix which also induces hemidesmosome formation.
  • This extracellular matrix protein is described in U.S. Patent No. 5,770,448, the entire contents of which are hereby incorporated by reference.
  • U.S. Patent Numbers 5,422,264 and 5,541,106 describe the isolation of rat LN5 and its ability to induce adhesion and hemidesmosome formation in epithelial cells. The purification of soluble LN5 is described in U.S. Patent No.
  • U.S. Patent No. 5,672,361 the entire contents of which are hereby incorporated by reference, discloses the growth of pancreatic islet cells on human LN5-coated substrates.
  • U.S. Patent No. 5,585,267 discloses the growth of epithelial cells on trans-epithelial appliances coated with rat LN5.
  • LN5 is a matrix component of epithelial tissue basement membranes and plays an important role in the initiation and maintenance of epidermal cell anchorage to the underlying connective tissue. Cell interaction with elements of the extracellular matrix impacts their adherence, motiiity as well as protein and gene expression (Adams et al., Develop. 117:1183-1198, 1993). In intact, normal tissue, epithelial cells bind to extracellular matrix molecules which are organized into a complex multiprotein structure called the basement membrane. The major components of the basement membrane include type IV collagen, proteogl ⁇ ca ⁇ s and laminins.
  • LN5 plays an important role in establishing firm adherence of epithelial cells to the basement membrane since it is necessary for the assembly and maintenance of stable anchorage devices between epithelial cells and hemidesmosomes (Green et al., FASEB J. 10:871-880, 1996; Baker et al., J. Cell Sci. 109:2509-2520, 1996). LN5 is also expressed at the budding tips of invading tumor cell populations, i.e. at sites where cancer cells are undergoing cell division but where there are most likely no hemidesmosomes (Pyke et al., Am. J. Pathol. 145:782-791, 1994; Pyke et al., Cancer Res. 55:4132-4139, 1995).
  • Proteases play an important role in the remodeling of basement membranes during wound healing, metastasis and tumor invasion. These proteases may induce specific cleavage events that result in alteration of function. For example, specific cleavage of LN5 by matrix metalloprotease-2 (MMP2) was shown to induce migration of breast epithelial cells by cleaving the LN5 ⁇ 2 subunit at residue 587, exposing a putative cryptic promigratory site on LN5 that triggers cell motiiity (Giannelli et al., Science 277:225-228, 1997). This cleavage converted LN5 to a molecule that mediates cell migration in addition to cell adhesion.
  • MMP2 matrix metalloprotease-2
  • One embodiment of the present invention is a method for converting laminin 5 to a migration-promoting substrate, comprising the step of contacting said laminin 5 with a protease which releases all or part of domains III, IV and V of the ⁇ 2 chain thereof, with the proviso that said protease is not a matrix metalloprotease.
  • the protease is elastase or ch ⁇ motrypsin.
  • the protease is a serine protease.
  • the serine protease is cathepsin G, coagulation factor Xa, cucumisin, endoproteinase Arg-C, endoproteinas ⁇ Glu-C, kallikrein, subtilisin A, thrombocytin or tr ⁇ psin.
  • the protease is a cysteine protease.
  • the cysteine protease is actinidin, calpain I, calpain II, cathepsin B or clostripain.
  • the protease is papain, bromelain, ficin, S. aureus V8 protease, cathepsin D or pepsin.
  • the laminin 5 is rat laminin 5.
  • the laminin 5 is human laminin 5.
  • the present invention also provides an isolated laminin 5 protein resulting from proteoi ⁇ sis of the ⁇ 2 chain and release of all or part of domains III, IV and V therefrom, with the proviso that the protease is not a matrix metalloprotease.
  • the protease is elastase or chymotrypsin.
  • the protease is a serine protease.
  • the serine protease is cathepsin G, coagulation factor Xa, cucumisin, endoproteinase Arg-C, endoproteinase Glu-C, kallikrein, subtilisin A, thrombocytin or trypsin.
  • the protease is a cysteine protease.
  • the cysteine protease is actinidin, calpain I, calpain II, cathepsin B or clostripain.
  • the protease is papain, bromeiain, ficin, S. aureus V8 protease, cathepsin D or pepsin.
  • the laminin 5 is rat laminin 5.
  • the laminin 5 is human laminin 5.
  • Another embodiment of the present invention is a method for promoting migration of cells onto a medical device, comprising the steps of: contacting a medical device with an isolated laminin 5 protein resulting from proteolysis of the ⁇ 2 chain and release of all or part of domains III, IV and V therefrom, with the proviso that said protease is not a matrix metalloprotease; and placing the medical device in the vicinity of the cells.
  • the medical device is contacted in vitro.
  • the medical device is contacted in vivo.
  • the cells are epithelial cells.
  • the present invention also provides a medical device or labware item to which an isolated laminin 5 large protein fragment resulting from proteolysis of laminin 5 in the hinge region of the ⁇ 2 chain with a protease, with the proviso that the protease is not a matrix metalloprotease, has been applied.
  • Figure 1 is a graph showing that cleavage of rat laminin 5 (rtLN5) with chymotrypsin or elastase results in conversion of reLN5 to a migratory substrate.
  • Elastase or chymotrypsin-digested rtLN5 or intact control rtLN5 was coated onto pre-treated glass coversiips and used as a substrate for human gingivai epithelial (GEH25) cells in a gold migration assay.
  • GEH25 ceils did not migrate on control intact rtLN5, but migrated on rtLN5 digested for 1, 2 or 3 hours with either chymotrypsin or elastase.
  • Figure 2 is a graph showing that chymotrypsin and elastase digested rat laminin 5 bind with identical coating efficiencies to glass coversiips compared to intact control rtLN5.
  • the coating efficiency of digested or intact rtLN5 was determined by incubating coated and control glass coversiips with the LN5 3 chain-specific monoclonal antibody CM6. After incubation with a horseradish peroxidase (HRP)-coupled secondary antibody, the immune complex was developed with OPD substrate in citrate buffer and samples were read at 490 nm in a plate reader. There was no difference in coating efficiencies between protease-treated rtLN5 and control rtLN5.
  • HRP horseradish peroxidase
  • Figure 3 is a graph showing the adhesion of human FGmet2 pancreatic carcinoma cells to tissue culture dishes coated with intact rtLN5, rtLN5 cleaved with chymotrypsin for 1 hour, or rtLN5 cleaved with elastase for 1 hour.
  • Chymotrypsin or eiastase-treated rtLN5 or intact rtLN5 were used to coat a 96-well plate in serial dilutions starting with 10 ⁇ g/ml.
  • a suspension of FGmet2 cells (80,000/well) was incubated for 30 min at 37°C, washed, fixed, stained with crystal violet and solubilized in 1 % SDS.
  • the present invention includes the observation that cleavage of the ⁇ 2 chain of LN5 with elastase, chymotrypsin or endoproteinase L ⁇ s-c in the hinge region thereof, results in the release of all or part of domains III, IV and V and converts LN5 to a substrate that promotes cell migration as shown by migration of an epithelial cell line on immobilized elastase- or chymotrypsin-treated LN5 (Example 3, Fig. 1).
  • the "hinge region" of the ⁇ 2 chain of LN5 is the region between domains II and III thereof.
  • the protease-cleaved LN5 also retains its adhesion-promoting properties (Fig. 3). Cleavage of rtLN5 or human LN5 (hLN5)with elastase or chymotrypsin results in release of a protein fragment having a molecular weight of about 70 kDa. The remainder of the LN5 molecule, having a molecular weight of about 380 kDa, promotes cell migration in addition to cell adhesion. Because MMP2 also cleaves the LN5 ⁇ 2 chain in the hinge region, this indicates that this region is uniquely labile to proteolytic degradation.
  • MMP2 and elastase have been implicated in the modification of basement membranes in processes of tissue invasion.
  • the proteol ⁇ ticall ⁇ sensitive area in the ⁇ 2 chain may be important in modulating LN5 activity, and may be affected by different enzymes in distinct situations.
  • MMP2 only cleaves rtlN5, but not hLN5 (Example 5), indicating that another protease which does cleave LN5, such as elastase, is physiologically relevant in humans.
  • elastase may be the most physiologically relevant because it is present in wound fluid, breast cancer tumor extracts and in the metastatic human lung carcinoma cell line EBC-1.
  • LN5 conversion of LN5 to a migration-promoting substrate by cleavage of LN5 with elastase, chymotrypsin and endoproteinase Lys-c is disclosed herein, the use of any non-matrix metalloprotease is also contemplated for this cleavage and conversion.
  • Types of proteases contemplated for use in the present invention include serine proteases, cysteine proteases and other non-matrix metalloproteases.
  • serine proteases examples include cathepsin G, coagulation factor Xa, cucumisin, endoproteinase Arg-C, endoproteinase Glu-C, kallikrein, subtilisin A, thrombocytin and trypsin.
  • cysteine proteases examples include actinidin, calpain I, calpain II, cathepsin B and clostripain.
  • Other proteases suitable for use in the present invention include papain, bromeiain, ficin, S. aureus V8 protease, cathepsin D and pepsin.
  • the peptides listed above are commercially available from many sources, for example Calbiochem, San Diego, CA and SIGMA, St. Louis, MO.
  • the ability of any particular protease to cleave laminin 5, resulting in release of all or part of domains III, IV and V of the ⁇ 2 chain thereof, and to convert LN5 from an adhesion-promoting to a migration-promoting substrate can be determined using the migration assay described in Example 3.
  • the incubation conditions for any of these proteases are well known, and may be found, for example, in the manufacturer's instructions provided with the proteases.
  • the large fragment of LN5 resulting from protease cleavage of the ⁇ 2 chain in the hinge region is used to coat the surface of a medical device to which migration and colonization of a particular cell type is desired.
  • the device is then placed into contact with a patient, for example implanted into a patient, using conventional surgical techniques as described in Sabiston, D. C, Jr., M.D., Textbook of Surgery: the Biological Basis of Modern Surgical Practice, 15th ed., W.B. Saunders Co., Philadelphia, 1997).
  • Such medical devices include, but are not limited to, surgical meshes, catheters, metal rods, metal pins, artificial joints, dental implants, wires and pacemakers.
  • protease- digested LN5 is applied to a surgical mesh which is placed onto a burn site of a patient.
  • the protease-digested LN5 stimulates migration of epithelial cells to the mesh which accelerates the healing process in the wounded area.
  • protease-digested LN5 is applied to a surgical mesh and placed into a culture dish containing epithelial cells. The LN5 promotes migration of epithelial cells onto the mesh. The mesh is then placed onto the burn site.
  • the cells can be contacted with the LN5-coated device either in vivo or in vitro.
  • Protease-treated LN5 is purified using any conventional chromatographic method in which the small (cleaved) fragment of LN5 and the protease elute well separated from the large fragment (e.g., gel filtration chromatography). LN5 may also be purified as described in U.S. Patent No. 5,760,179.
  • LN5 is is provided in a pharmaceutically acceptable excipient or diluent which are no ⁇ toxic to recipients at the dosages and concentrations employed.
  • These pharmaceutical formulations can be prepared by conventional techniques, e.g. as described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 1985.
  • Suitable carriers, excipients or diluents for the preparation of solutions and syrups include water, pol ⁇ ols, sucrose and glucose. These formulations can additionally contain preservatives, solubilizers, stabilizers, viscosity agents, wetting agents, emuisifiers, buffers, atioxidants and diluents.
  • Protease-digested LN5 is applied to medical device, including transepithelial appliances and other shaped articles as disclosed in U.S. Patent No. 5,585,267, by either passive absorption or covalent linkage. Passive adsorption can be accomplished in a variety of ways, e.g. by immersion of an article in a LN5-contai ⁇ ing solution, spraying the article with the solution, and the like.
  • LN5 may be covalently linked to the medical device using any of a variety of well known cross-linking agents which target different chemical groups on proteins, including amino, carboxyl, sulfhydryl, aryl, hydroxyl and carbohydrate groups.
  • cross-linking reagents described below are available from Pierce Chemical Co. (Rockford, IL) and described in the Pierce catalog. Other cross-linking reagents are well known in the art, and may be available from other suppliers. There are two main types of cross-linking agents: homobifunctional and heterobifunctional. Homobifunctional cross-linkers have at least two identical reactive groups and can target primary amine or sulfhydryl groups. Examples of homobifunctional cross-linking reagents which target amine groups include dimethyl 3,3,-dithiobispropionimidate 2HCI (DTBP) and disuccimidyl suberate (DSS).
  • DTBP dimethyl 3,3,-dithiobispropionimidate 2HCI
  • DSS disuccimidyl suberate
  • Sulfhydryl-specific reagents include bismaleimidohexane (BMH) and 1,4-di-[3'-2'-pyridyldithio(propinamido)butane] (DPDPB).
  • BMH bismaleimidohexane
  • DPDPB 1,4-di-[3'-2'-pyridyldithio(propinamido)butane]
  • the main disadvantages of such homobifunctional cross-linkers are self-conjugation, intramolecular cross- linking and/or polymerization.
  • Heterodifunctional cross-linkers contain two or more different reactive groups that allow for sequential conjugations with specific groups of proteins, minimizing undesirable polymerization or self-conjugation.
  • Heterodifunctional cross-linkers which react with primary or secondary amines include imidoesters and N- hydroxysuccinimide (NHS)-esters such as sicci idyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and succimidyl-4-(p-maleimidophenyl)-butyrate (SMPB).
  • Cross-linkers which react with sulfhydryl groups include maleimides, haloacetyls and pyridyl disulfides.
  • Carbodiimide cross-linkers couple carboxyls to primary amines or h ⁇ drazides, resulting in formation of amide or h ⁇ drazone bonds.
  • One widely used carbodiimide cross-linker is l-ethyl-3- (3-dimethylaminopropyl)-carbodiimide hydrochloride).
  • cross-linking agent is dependent upon the surface chemistry of the article to which the LN5 is to be attached.
  • a surface can be chemically modified or manufactured to contain particular reactive groups using methods well known in the art.
  • substrates can be manufactured which contain epoxide groups using methods well known in the art.
  • surface activation or modification e.g., by plasma discharge, corona discharge, electron beam, RF energy, laser or strong acid or base treatment
  • the amount of LN5 applied to a medical device is between about 0.01 ⁇ g/cm 2 and
  • the amount of LN5 to the medical device is between about 0.1 ⁇ g/cm 2 and 10 ⁇ g/cm 2 .
  • the device is immersed in or sprayed with LN5 solutions having concentrations of between about 0.001 mg/ml and 100 mg/ml, more preferably between about 1 mg/ml and about 10 mg/ml.
  • Another embodiment of the present invention is a medical device, including transepithelial appliances and other shaped articles as disclosed in U.S. Patent No. 5,585,267, the entire contents of which are hereby incorporated by reference, to which the protease-cleaved LN5 of the invention has been applied.
  • Protease-cleaved LN5 may also be used in research and diagnostics.
  • the protease-cleaved LN5 of the invention may be applied to any conventional labware item such as a tissue culture dish, microtiter plate, flask, bioreactor, slide, and the like, to which it induces migration of cells applied to or contained within the labware item.
  • Another embodiment of the invention is a labware item to which protease-cleaved LN5 has been applied.
  • Another embodiment of the present invention is non-metalloprotease-digested LN5 which is cleaved in the hinge region of the ⁇ 2 chain. Cleavage of LN5 results in release of a small protein fragment containing all or part of domains ill, IV and V of the ⁇ 2 chain of the LN5 heterotrimer.
  • the resulting migration-inducing iarge fragment of LN5 is itself a novel molecule because the cleavage which occurs by using a non-matrix metalloprotease is not at the same site as that induced by the MMP2 of Giannelli et al., supra.
  • MMP2 cleaves rtLN5 rtLN5 was purified from 804G rat bladder carcinoma cell conditioned medium as described in U.S. Patent No. 5,760,179. Purified rtLN5 was incubated with 2 units or 0.2 units MMP2 (gelatinase 72 kD; Boehringer Mannheim,
  • LN5 heterotrimers resolve as a doublet. Under these conditions, exposure of rtLN5 to MMP2 results in release of an approximately 65 kD fragment. Under reducing conditions, the ⁇ 2 150 kD and ⁇ 100 kD polypeptide chains are converted into an 80 kDa fragment. The ⁇ 3 and ⁇ 3 polypeptides are not affected by MMP2 treatment.
  • cleavage of rtLN5 occurs in the same region using elastase and chymotrypsin as with MMP2.
  • Western blot analysis using the ⁇ 2 chain-specific antiserum J20 showed that the 80kD polypeptide was a ⁇ 2 chain digestion product.
  • Example 3 Conversion of rtLN5 to a migratory substrate Elastase- and ch ⁇ motr ⁇ psin-digested rtLN5 or intact control rtLN5 was coated onto pre-treated glass coversiips and used as a substrate for primary human gingival epithelial (GEH25) cells in a gold migration assay. Briefly, glass coversiips were washed with 100% ethanol, air dried and briefly dipped into 1 % bovine serum albumin
  • BSA phosphate buffered saline
  • GEH25 cells did not migrate on control rtLN5, but migrated on rtLN5 digested for 1, 2 or 3 hours with either chymotrypsin or elastase ( Figure 1).
  • Example 4 Binding efficiencies of chymotrypsin- and elastase-digested rtLN5
  • the coating efficiency of chymotrypsin- or elastase-digested or intact rtLN5 was determined by incubating coated and control glass coversiips with the LN5 3 -specific monoclonal antibody CM6. After incubation with a horseradish peroxidase-coupled secondary antibody, the immune complex was developed with OPD substrate in citrate buffer. The results show that chymotrypsin- and elastase-digested rtLN5 bind with identical coating efficiencies to the glass coversiips compared to control rtLN5.
  • Example 5 MMP2 cleaves rtLN5, but not human LN5 rtLN5 and hLN5, purified from the human epithelial cell line MCF 10A using the method described in U.S. Patent No. 5,760,179, were digested under identical conditions using either 2 units or 0.2 units of MMP2 (gelatinase 72 kD, Boehringer Mannheim) for 18 hours at 37°C. The digested samples were analyzed by 6% SDS-PAGE and stained with Coomassie Brilliant Blue. While the rtLN5 was cleaved as described in Example 1, cleavage of hLN5 did not occur.
  • Coomassie Brilliant Blue Elastase and chymotrypsin induce cleavage of the ⁇ 2 100 kD polypeptide chain and generate fragments of approximately 75 kD and 65 kD, respectively.
  • Chymotrypsin- or elastase-treated rtLN5 or intact LN5 were used to coat a 96-well plate in serial dilutions starting with 10 ⁇ g/ml. for attachment, a suspension of FGmet 2 ceils (80,000/well) was incubated for 30 min at
  • Example 8 Promotion of epithelial cell migration in vivo
  • a standard surgical mesh obtained from, for example, Johnson & Johnson Medical, Inc. is immersed in a pharmaceutically acceptable solution of hLN5 (5 mg/ml) purified from human MCF-10A epithelial cells as described in U.S. Patent No. 5,760,179, which has been cleaved with neutrophil or pancreatic elastase.
  • the mesh is allowed to dry, and placed onto a laceration of a patient under sterile conditions.
  • the elastase-cleaved hLN5 facilitates healing of the laceration by promoting migration and adhesion of epithelial cells at the wound site.

Abstract

Conversion of laminin 5 to a migration-promoting molecule by treatment with a protease other than a matrix metalloprotease. The protease-treated laminin 5 is applied to a medical device to promote colonization by a desired cell type.

Description

CONVERSION OF LAMININ 5 TO A MIGRATORY SUBSTRATE BY PROTEOLYSIS OF THE γ, CHAIN THEREOF
Field of the Invention The present invention relates to conversion of laminin 5 (LN5) to a cell migration-promoting substrate by proteases. More specifically, the invention relates to cleavage of the γ2 chain of laminin 5.
Background of the Invention Cell migration is a process which occurs during vital naturally occurring physiological processes such as wound healing, immune responses, bone repair, inflammation and the like. For example, stimulation of cell migration is desirable in promoting recruitment of epithelial cells to a wound site to facilitate healing of the wound, and in promoting migration of immune cells to sites of infection.
Laminins are heterotrimeric extracellular matrix proteins consisting of three subunits: α, β and γ. There are at least five known α subunits (α12345), three known β subunits (β„β23) and two known γ (γ„γ2) subunits (Miner et al., J. Cell. Biol. 137:685-701, 1997). Laminin 5 (LN5) is an α3β3γ2 heterotrimer which is typically associated with epithelial cell adhesion and sometimes with hemidesmosome formation. The designation "laminin 5" was coined by Burgeson et al. (Matrix Biol. 14:209-211, 1994) to refer to a protein which is secreted into the culture medium by human keratinocytes and enhances keratinocγte attachment (Rousselle et al., J. Cell Biol. 114:567-576, 1991; International Publication Nos. W092/17498 and W094/0531). A similar protein was also identified by Carter et al. (Cell 65:599-619, 1991; International Publication No. W095/06660) and called epiligrin. This protein is similar to the basement membrane glycoprotein recognized by the GB3 antibody in human keratinocγte culture medium called nicein (Hsi et al., Placenta 8:209-217, 1987).
LN5 is also produced by 804G and NBT-II rat bladder carcinoma cells (U.S. Patent Nos. 5,541,106 and 5,422,264, hereby incorporated by reference). A human epithelial cell line, MCF-10A, produces a LN5 extracellular matrix which also induces hemidesmosome formation. This extracellular matrix protein is described in U.S. Patent No. 5,770,448, the entire contents of which are hereby incorporated by reference. U.S. Patent Numbers 5,422,264 and 5,541,106 describe the isolation of rat LN5 and its ability to induce adhesion and hemidesmosome formation in epithelial cells. The purification of soluble LN5 is described in U.S. Patent No. 5,760,179, the entire contents of which are hereby incorporated by reference. U.S. Patent Nos. 5,510,263 and 5,681,587, the entire contents of which are hereby incorporated by reference, disclose the successful passaging of fetal and adult islet cells when plated on a rat LN5-coated substrate. U.S. Patent No. 5,672,361, the entire contents of which are hereby incorporated by reference, discloses the growth of pancreatic islet cells on human LN5-coated substrates. U.S. Patent No. 5,585,267, the entire contents of which are hereby incorporated by reference, discloses the growth of epithelial cells on trans-epithelial appliances coated with rat LN5. U.S. Patent Application Serial No. 09/145,387, the entire contents of which are hereby incorporated by reference, discloses the use of LN5 for treatment of bone defects. LN5 is a matrix component of epithelial tissue basement membranes and plays an important role in the initiation and maintenance of epidermal cell anchorage to the underlying connective tissue. Cell interaction with elements of the extracellular matrix impacts their adherence, motiiity as well as protein and gene expression (Adams et al., Develop. 117:1183-1198, 1993). In intact, normal tissue, epithelial cells bind to extracellular matrix molecules which are organized into a complex multiprotein structure called the basement membrane. The major components of the basement membrane include type IV collagen, proteoglγcaπs and laminins. LN5 plays an important role in establishing firm adherence of epithelial cells to the basement membrane since it is necessary for the assembly and maintenance of stable anchorage devices between epithelial cells and hemidesmosomes (Green et al., FASEB J. 10:871-880, 1996; Baker et al., J. Cell Sci. 109:2509-2520, 1996). LN5 is also expressed at the budding tips of invading tumor cell populations, i.e. at sites where cancer cells are undergoing cell division but where there are most likely no hemidesmosomes (Pyke et al., Am. J. Pathol. 145:782-791, 1994; Pyke et al., Cancer Res. 55:4132-4139, 1995).
Proteases play an important role in the remodeling of basement membranes during wound healing, metastasis and tumor invasion. These proteases may induce specific cleavage events that result in alteration of function. For example, specific cleavage of LN5 by matrix metalloprotease-2 (MMP2) was shown to induce migration of breast epithelial cells by cleaving the LN5 γ2 subunit at residue 587, exposing a putative cryptic promigratory site on LN5 that triggers cell motiiity (Giannelli et al., Science 277:225-228, 1997). This cleavage converted LN5 to a molecule that mediates cell migration in addition to cell adhesion. However, the serine protease plasmin and another matrix metalloprotease, MMP9, were unable to cleave LN5 (Giannelli et al., supra.). There is an ongoing need for promoting cell migration both in vitro and in vivo. The present invention addresses this need.
Summary of the Invention One embodiment of the present invention is a method for converting laminin 5 to a migration-promoting substrate, comprising the step of contacting said laminin 5 with a protease which releases all or part of domains III, IV and V of the γ2 chain thereof, with the proviso that said protease is not a matrix metalloprotease. Preferably, the protease is elastase or chγmotrypsin. In one aspect of this preferred embodiment, the protease is a serine protease. Preferably, the serine protease is cathepsin G, coagulation factor Xa, cucumisin, endoproteinase Arg-C, endoproteinasε Glu-C, kallikrein, subtilisin A, thrombocytin or trγpsin. In another aspect of this preferred embodiment, the protease is a cysteine protease. Preferably, the cysteine protease is actinidin, calpain I, calpain II, cathepsin B or clostripain. In yet another aspect of this preferred embodiment, the protease is papain, bromelain, ficin, S. aureus V8 protease, cathepsin D or pepsin. Preferably, the laminin 5 is rat laminin 5. Alternatively, the laminin 5 is human laminin 5.
The present invention also provides an isolated laminin 5 protein resulting from proteoiγsis of the γ2 chain and release of all or part of domains III, IV and V therefrom, with the proviso that the protease is not a matrix metalloprotease. Preferably, the protease is elastase or chymotrypsin. In one aspect of this preferred embodiment, the protease is a serine protease. Preferably, the serine protease is cathepsin G, coagulation factor Xa, cucumisin, endoproteinase Arg-C, endoproteinase Glu-C, kallikrein, subtilisin A, thrombocytin or trypsin. In another aspect of this preferred embodiment, the protease is a cysteine protease. Preferably, the cysteine protease is actinidin, calpain I, calpain II, cathepsin B or clostripain. In yet another aspect of this preferred embodiment, the protease is papain, bromeiain, ficin, S. aureus V8 protease, cathepsin D or pepsin. Preferably, the laminin 5 is rat laminin 5. Alternatively, the laminin 5 is human laminin 5.
Another embodiment of the present invention is a method for promoting migration of cells onto a medical device, comprising the steps of: contacting a medical device with an isolated laminin 5 protein resulting from proteolysis of the γ2 chain and release of all or part of domains III, IV and V therefrom, with the proviso that said protease is not a matrix metalloprotease; and placing the medical device in the vicinity of the cells. Preferably, the medical device is contacted in vitro. Alternatively, the medical device is contacted in vivo. Advantageously, the cells are epithelial cells.
The present invention also provides a medical device or labware item to which an isolated laminin 5 large protein fragment resulting from proteolysis of laminin 5 in the hinge region of the γ2 chain with a protease, with the proviso that the protease is not a matrix metalloprotease, has been applied. Brief Description of the Figures
Figure 1 is a graph showing that cleavage of rat laminin 5 (rtLN5) with chymotrypsin or elastase results in conversion of reLN5 to a migratory substrate. Elastase or chymotrypsin-digested rtLN5 or intact control rtLN5 was coated onto pre-treated glass coversiips and used as a substrate for human gingivai epithelial (GEH25) cells in a gold migration assay. GEH25 ceils did not migrate on control intact rtLN5, but migrated on rtLN5 digested for 1, 2 or 3 hours with either chymotrypsin or elastase.
Figure 2 is a graph showing that chymotrypsin and elastase digested rat laminin 5 bind with identical coating efficiencies to glass coversiips compared to intact control rtLN5. The coating efficiency of digested or intact rtLN5 was determined by incubating coated and control glass coversiips with the LN5 3 chain-specific monoclonal antibody CM6. After incubation with a horseradish peroxidase (HRP)-coupled secondary antibody, the immune complex was developed with OPD substrate in citrate buffer and samples were read at 490 nm in a plate reader. There was no difference in coating efficiencies between protease-treated rtLN5 and control rtLN5.
Figure 3 is a graph showing the adhesion of human FGmet2 pancreatic carcinoma cells to tissue culture dishes coated with intact rtLN5, rtLN5 cleaved with chymotrypsin for 1 hour, or rtLN5 cleaved with elastase for 1 hour. Chymotrypsin or eiastase-treated rtLN5 or intact rtLN5 were used to coat a 96-well plate in serial dilutions starting with 10 μg/ml. A suspension of FGmet2 cells (80,000/well) was incubated for 30 min at 37°C, washed, fixed, stained with crystal violet and solubilized in 1 % SDS. Quantitation of attached cells was performed by reading the plates at 595 nm. The experiment was performed in triplicate. Cell adhesion was the same for all three substrates. Detailed Description of the Preferred Embodiments The present invention includes the observation that cleavage of the γ2 chain of LN5 with elastase, chymotrypsin or endoproteinase Lγs-c in the hinge region thereof, results in the release of all or part of domains III, IV and V and converts LN5 to a substrate that promotes cell migration as shown by migration of an epithelial cell line on immobilized elastase- or chymotrypsin-treated LN5 (Example 3, Fig. 1). The "hinge region" of the γ2 chain of LN5 is the region between domains II and III thereof. The protease-cleaved LN5 also retains its adhesion-promoting properties (Fig. 3). Cleavage of rtLN5 or human LN5 (hLN5)with elastase or chymotrypsin results in release of a protein fragment having a molecular weight of about 70 kDa. The remainder of the LN5 molecule, having a molecular weight of about 380 kDa, promotes cell migration in addition to cell adhesion. Because MMP2 also cleaves the LN5 γ2 chain in the hinge region, this indicates that this region is uniquely labile to proteolytic degradation. MMP2 and elastase have been implicated in the modification of basement membranes in processes of tissue invasion. Thus, the proteolγticallγ sensitive area in the γ2 chain may be important in modulating LN5 activity, and may be affected by different enzymes in distinct situations.
However, it has been determined that MMP2 only cleaves rtlN5, but not hLN5 (Example 5), indicating that another protease which does cleave LN5, such as elastase, is physiologically relevant in humans. Among the proteases examined, elastase may be the most physiologically relevant because it is present in wound fluid, breast cancer tumor extracts and in the metastatic human lung carcinoma cell line EBC-1.
Although the conversion of LN5 to a migration-promoting substrate by cleavage of LN5 with elastase, chymotrypsin and endoproteinase Lys-c is disclosed herein, the use of any non-matrix metalloprotease is also contemplated for this cleavage and conversion. Types of proteases contemplated for use in the present invention include serine proteases, cysteine proteases and other non-matrix metalloproteases. Examples of serine proteases include cathepsin G, coagulation factor Xa, cucumisin, endoproteinase Arg-C, endoproteinase Glu-C, kallikrein, subtilisin A, thrombocytin and trypsin. Examples of cysteine proteases include actinidin, calpain I, calpain II, cathepsin B and clostripain. Other proteases suitable for use in the present invention include papain, bromeiain, ficin, S. aureus V8 protease, cathepsin D and pepsin. The peptides listed above are commercially available from many sources, for example Calbiochem, San Diego, CA and SIGMA, St. Louis, MO. The ability of any particular protease to cleave laminin 5, resulting in release of all or part of domains III, IV and V of the γ2 chain thereof, and to convert LN5 from an adhesion-promoting to a migration-promoting substrate can be determined using the migration assay described in Example 3. The incubation conditions for any of these proteases are well known, and may be found, for example, in the manufacturer's instructions provided with the proteases.
The large fragment of LN5 resulting from protease cleavage of the γ2 chain in the hinge region is used to coat the surface of a medical device to which migration and colonization of a particular cell type is desired. The device is then placed into contact with a patient, for example implanted into a patient, using conventional surgical techniques as described in Sabiston, D. C, Jr., M.D., Textbook of Surgery: the Biological Basis of Modern Surgical Practice, 15th ed., W.B. Saunders Co., Philadelphia, 1997). Such medical devices include, but are not limited to, surgical meshes, catheters, metal rods, metal pins, artificial joints, dental implants, wires and pacemakers. For example, protease- digested LN5 is applied to a surgical mesh which is placed onto a burn site of a patient. The protease-digested LN5 stimulates migration of epithelial cells to the mesh which accelerates the healing process in the wounded area. Alternatively, protease-digested LN5 is applied to a surgical mesh and placed into a culture dish containing epithelial cells. The LN5 promotes migration of epithelial cells onto the mesh. The mesh is then placed onto the burn site. Thus, the cells can be contacted with the LN5-coated device either in vivo or in vitro. Protease-treated LN5 is purified using any conventional chromatographic method in which the small (cleaved) fragment of LN5 and the protease elute well separated from the large fragment (e.g., gel filtration chromatography). LN5 may also be purified as described in U.S. Patent No. 5,760,179. For application to a medical device which is used in vivo, LN5 is is provided in a pharmaceutically acceptable excipient or diluent which are noπtoxic to recipients at the dosages and concentrations employed. These pharmaceutical formulations can be prepared by conventional techniques, e.g. as described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 1985. Suitable carriers, excipients or diluents for the preparation of solutions and syrups include water, polγols, sucrose and glucose. These formulations can additionally contain preservatives, solubilizers, stabilizers, viscosity agents, wetting agents, emuisifiers, buffers, atioxidants and diluents.
Protease-digested LN5 is applied to medical device, including transepithelial appliances and other shaped articles as disclosed in U.S. Patent No. 5,585,267, by either passive absorption or covalent linkage. Passive adsorption can be accomplished in a variety of ways, e.g. by immersion of an article in a LN5-contaiπing solution, spraying the article with the solution, and the like. Alternatively, LN5 may be covalently linked to the medical device using any of a variety of well known cross-linking agents which target different chemical groups on proteins, including amino, carboxyl, sulfhydryl, aryl, hydroxyl and carbohydrate groups. All of the cross-linking reagents described below are available from Pierce Chemical Co. (Rockford, IL) and described in the Pierce catalog. Other cross-linking reagents are well known in the art, and may be available from other suppliers. There are two main types of cross-linking agents: homobifunctional and heterobifunctional. Homobifunctional cross-linkers have at least two identical reactive groups and can target primary amine or sulfhydryl groups. Examples of homobifunctional cross-linking reagents which target amine groups include dimethyl 3,3,-dithiobispropionimidate 2HCI (DTBP) and disuccimidyl suberate (DSS). Sulfhydryl-specific reagents include bismaleimidohexane (BMH) and 1,4-di-[3'-2'-pyridyldithio(propinamido)butane] (DPDPB). The main disadvantages of such homobifunctional cross-linkers are self-conjugation, intramolecular cross- linking and/or polymerization.
Heterodifunctional cross-linkers contain two or more different reactive groups that allow for sequential conjugations with specific groups of proteins, minimizing undesirable polymerization or self-conjugation. Heterodifunctional cross-linkers which react with primary or secondary amines include imidoesters and N- hydroxysuccinimide (NHS)-esters such as sicci idyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and succimidyl-4-(p-maleimidophenyl)-butyrate (SMPB). Cross-linkers which react with sulfhydryl groups include maleimides, haloacetyls and pyridyl disulfides. Carbodiimide cross-linkers couple carboxyls to primary amines or hγdrazides, resulting in formation of amide or hγdrazone bonds. One widely used carbodiimide cross-linker is l-ethyl-3- (3-dimethylaminopropyl)-carbodiimide hydrochloride).
The choice of cross-linking agent is dependent upon the surface chemistry of the article to which the LN5 is to be attached. A surface can be chemically modified or manufactured to contain particular reactive groups using methods well known in the art. For example, substrates can be manufactured which contain epoxide groups using methods well known in the art. In some instances, surface activation or modification (e.g., by plasma discharge, corona discharge, electron beam, RF energy, laser or strong acid or base treatment) may facilitate attachment of cross- linking agent. In a preferred embodiment, the amount of LN5 applied to a medical device is between about 0.01 μg/cm2 and
100 μg/cm2. In a more preferred embodiment, the amount of LN5 to the medical device is between about 0.1 μg/cm2 and 10 μg/cm2. To attain these amounts, the device is immersed in or sprayed with LN5 solutions having concentrations of between about 0.001 mg/ml and 100 mg/ml, more preferably between about 1 mg/ml and about 10 mg/ml. Another embodiment of the present invention is a medical device, including transepithelial appliances and other shaped articles as disclosed in U.S. Patent No. 5,585,267, the entire contents of which are hereby incorporated by reference, to which the protease-cleaved LN5 of the invention has been applied.
Protease-cleaved LN5 may also be used in research and diagnostics. For example, the protease-cleaved LN5 of the invention may be applied to any conventional labware item such as a tissue culture dish, microtiter plate, flask, bioreactor, slide, and the like, to which it induces migration of cells applied to or contained within the labware item.
Another embodiment of the invention is a labware item to which protease-cleaved LN5 has been applied.
Another embodiment of the present invention is non-metalloprotease-digested LN5 which is cleaved in the hinge region of the γ2 chain. Cleavage of LN5 results in release of a small protein fragment containing all or part of domains ill, IV and V of the γ2 chain of the LN5 heterotrimer. The resulting migration-inducing iarge fragment of LN5 is itself a novel molecule because the cleavage which occurs by using a non-matrix metalloprotease is not at the same site as that induced by the MMP2 of Giannelli et al., supra.
Example 1
MMP2 cleaves rtLN5 rtLN5 was purified from 804G rat bladder carcinoma cell conditioned medium as described in U.S. Patent No. 5,760,179. Purified rtLN5 was incubated with 2 units or 0.2 units MMP2 (gelatinase 72 kD; Boehringer Mannheim,
Indianapolis, IN) for 18 hours at 37°C. The digested samples were analyzed by 6% sodium dodecyl suifate- polγacrylamide gel electrophoresis (SDS-PAGE) under both non-reducing and reducing conditions, and stained with
Coomassie Brilliant Blue. Under non-reducing conditions, association of the three rtLN5 subunits is preserved and the
LN5 heterotrimers resolve as a doublet. Under these conditions, exposure of rtLN5 to MMP2 results in release of an approximately 65 kD fragment. Under reducing conditions, the γ2 150 kD and γ100 kD polypeptide chains are converted into an 80 kDa fragment. The α3 and β3 polypeptides are not affected by MMP2 treatment.
Example 2
Cleavage of rtLN5 with chymotrypsin and elastase rtLN5 was incubated with elastase (1:10 w/w) or chymotrypsin (1:100 w/w) for 1, 2 or 3 hours at 37°C and analyzed by 6% SDS-PAGE. The gel was transferred to nitrocellulose and analyzed by Western blotting using a rtLN5 polγclonal antiserum. The results showed the disappearance of the γ2 150 kD and γ2 100 kD polypeptides, and appearance of a polypeptide of approximately 80 kD. Thus, cleavage of rtLN5 occurs in the same region using elastase and chymotrypsin as with MMP2. Western blot analysis using the γ2 chain-specific antiserum J20 showed that the 80kD polypeptide was a γ2 chain digestion product.
Example 3 Conversion of rtLN5 to a migratory substrate Elastase- and chγmotrγpsin-digested rtLN5 or intact control rtLN5 was coated onto pre-treated glass coversiips and used as a substrate for primary human gingival epithelial (GEH25) cells in a gold migration assay. Briefly, glass coversiips were washed with 100% ethanol, air dried and briefly dipped into 1 % bovine serum albumin
(BSA). After drying, coversiips were transferred into a 24-well plate and coated with intact, proteolytically processed LN5 or 3% BSA at 4°C overnight. A 1.4 mM colloidal gold/12 mM sodium carbonate solution was prepared by boiling, followed by addition of formaldehyde to a final concentration of 0.01 %. Each coverslip was incubated with 500 μl colloidal gold solution for several hours and subsequently transferred to a new plate. GEH25 cells were seeded into each well (3,000/well) and allowed to migrate for 18 hours at 37°C in 5% C02. The cells were fixed in 3% paraformaldehyde in phosphate buffered saline (PBS) and pictures were taken. Photographs were scanned and a pixel analysis was done evaluating cleared areas generated by cell migration tracks versus the total area. Migration was evaluated by pixel analysis expressed as percentage of cleared areas generated by cell migration tracks versus the total area. GEH25 cells did not migrate on control rtLN5, but migrated on rtLN5 digested for 1, 2 or 3 hours with either chymotrypsin or elastase (Figure 1).
Example 4 Binding efficiencies of chymotrypsin- and elastase-digested rtLN5 The coating efficiency of chymotrypsin- or elastase-digested or intact rtLN5 was determined by incubating coated and control glass coversiips with the LN5 3-specific monoclonal antibody CM6. After incubation with a horseradish peroxidase-coupled secondary antibody, the immune complex was developed with OPD substrate in citrate buffer. The results show that chymotrypsin- and elastase-digested rtLN5 bind with identical coating efficiencies to the glass coversiips compared to control rtLN5. Example 5 MMP2 cleaves rtLN5, but not human LN5 rtLN5 and hLN5, purified from the human epithelial cell line MCF 10A using the method described in U.S. Patent No. 5,760,179, were digested under identical conditions using either 2 units or 0.2 units of MMP2 (gelatinase 72 kD, Boehringer Mannheim) for 18 hours at 37°C. The digested samples were analyzed by 6% SDS-PAGE and stained with Coomassie Brilliant Blue. While the rtLN5 was cleaved as described in Example 1, cleavage of hLN5 did not occur.
Example 6
Cleavage of hLN5 with chymotrypsin and elastase LN5 was purified from MCF 10A cells and exposed to various concentrations of elastase or chymotrypsin, incubated for 1 hour at 37°C, analyzed by SDS-PAGE under reducing and non-reducing conditions and stained with
Coomassie Brilliant Blue. Elastase and chymotrypsin induce cleavage of the γ2 100 kD polypeptide chain and generate fragments of approximately 75 kD and 65 kD, respectively.
Example 7 Protease-cleaved LN5 retains adhesion-promoting activity
Chymotrypsin- or elastase-treated rtLN5 or intact LN5 were used to coat a 96-well plate in serial dilutions starting with 10 μg/ml. for attachment, a suspension of FGmet 2 ceils (80,000/well) was incubated for 30 min at
37°C, washed, fixed, stained with crystal violet and solubiiized in 1 % SDS. Quantitation of attached cells was performed by reading the plates at 595 nm. All experiments were performed in triplicate. The results show that all three substrates promoted promoted the same relative amount of cell attachment (Fig. 3)
Example 8 Promotion of epithelial cell migration in vivo A standard surgical mesh obtained from, for example, Johnson & Johnson Medical, Inc., is immersed in a pharmaceutically acceptable solution of hLN5 (5 mg/ml) purified from human MCF-10A epithelial cells as described in U.S. Patent No. 5,760,179, which has been cleaved with neutrophil or pancreatic elastase. The mesh is allowed to dry, and placed onto a laceration of a patient under sterile conditions. The elastase-cleaved hLN5 facilitates healing of the laceration by promoting migration and adhesion of epithelial cells at the wound site.
It should be noted that the present invention is not limited to only those embodiments described in the Detailed Description. Any embodiment which retains the spirit of the present invention should be considered to be within its scope. However, the invention is only limited by the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. A method for converting laminin 5 to a migration-promoting substrate, comprising the step of contacting said laminin 5 with a protease which cleaves said laminin 5 in the hinge region of the γ2 chain thereof, resulting in release of all or part of domains III, IV and V, with the proviso that said protease is not a matrix metalloprotease.
2. The method of Claim 1 , wherein said protease is elastase or chymotrypsin.
3. The method of Claim 1, wherein said protease is a serine protease.
4. The method of Claim 3, wherein said serine protease is selected from the group consisting of cathepsin G, coagulation factor Xa, cucumisin, endoproteinase Arg-c, endoproteinase Glu-c, kallikrein, subtilisin A, thrombocytin, trypsin.
5. The method of Claim 1, wherein said protease is a cysteine protease.
6. The method of Claim 5, wherein said cysteine protease is selected from the group consisting of actinidin, calpain I, calpain II, cathepsin B, clostripain.
7. The method of Claim 1, wherein said protease is selected from the group consisting of papain, bromeiain, ficin, S. aureus V8 protease, cathepsin D andpepsin.
8. The method of Claim 1, wherein said laminin 5 is rat laminin 5.
9. The method of Claim 1 , wherein said laminin 5 is human laminin 5.
10. An isolated laminin 5 large protein fragment resulting from proteolysis of laminin 5 in the hinge region of the γ2 chain with a protease, with the proviso that said protease is not a matrix metalloprotease.
11. The method of Claim 10, wherein said protease is chymotrypsin or elastase.
12. The method of Claim 10, wherein said protease is a serine protease.
13. The method of Claim 12, wherein said serine protease is selected from the group consisting of cathepsin G, coagulation factor Xa, cucumisin, endoproteinase Arg-c, endoproteinase Glu-c, kallikrein, subtilisin A, thrombocytin and trypsin.
14. The method of Claim 10, wherein said protease is a cysteine protease.
15. The method of Claim 14, wherein said cysteine protease is selected from the group consisting of actinidin, calpain I, calpain II, cathepsin B andclostripaiπ.
16. The method of Claim 10, wherein said protease is selected from the group consisting of papain, bromeiain, ficin, S. aureus V8 protease, cathepsin D and pepsin.
17. The method of Claim 10 wherein said laminin 5 is rat laminin 5.
18. The method of Claim 10, wherein said laminin 5 is human laminin 5.
19. A method for promoting migration of cells onto a medical device, comprising the steps of: contacting a medical device with an isolated laminin 5 protein resulting from proteolysis of laminin 5 in the hinge region of the γ2 chain thereof to release all or part of domains III, IV and V therefrom, with the proviso that said protease is not a matrix metalloprotease; and placing said medical device in the vicinity of said cells.
20. The method of Claim 19, wherein said medical device is contacted in vitro.
21. The method of Claim 19, wherein said medical device is contacted in vivo.
22. The method of Claim 19, wherein said cells are epithelial cells.
23. A medical device or labware item to which an isolated laminin 5 large protein fragment resulting from proteolysis of laminin 5 in the hinge region of the γ2 chain with a protease, with the proviso that said protease is not a matrix metalloprotease, has been applied.
PCT/US1999/029433 1998-12-11 1999-12-10 CONVERSION OF LAMININ 5 TO A MIGRATORY SUBSTRATE BY PROTEOLYSIS OF THE η2 CHAIN THEREOF WO2000034441A1 (en)

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