WO1999053955A1 - Creation of bioactive surfaces through selective adsorption - Google Patents

Abstract

Implantable articles contacted with antibodies to endogenous biomolecules which promote or inhibit the activity of these biomolecules, and methods of using these articles. These articles include medical devices contacted with antibodies which inhibit the activity of proteins which promote blood clot formation and antibodies which promote epithelial cell adhesion.

Description

CREATION OF BIOACTIVE SURFACES THROUGH SELECTIVE ADSORPTION Field of the Invention The present invention relates to capturing and promoting or inhibiting the activity of bioactive molecules by antibodies or other agents applied to a medical device, including an implantable or transcutaneous article.

Background of the Invention Medical devices implanted into a patient either transiently or for extended periods of time have inherent risks associated therewith. For example, artificial heart valves, vascular stents and vascular shunts can promote blood clot formation and trans- epithelial medical appliances, including indwelling catheters and colostomy tubes, which breach the skin for extended periods of time, may result in inflammation and/or infection. Coating the surface of medical devices used in the heart and vasculature with anti-thrombotic agents such as heparin is one approach to inhibiting blood clot formation. However, many of these agents are not particularly stable in vivo.

As described in U.S. Patent No. 5,585,267, coating trans-epithelial appliances with epithelial cells prior to or after insertion into the skin helps prevent these undesirable processes. Coating surgical meshes with epithelial cells for use in skin allografts is also desirable to recruit more epithelial cells to the wound site which facilitate healing of the wound. In addition, periodontitis, a severe form of gum disease resulting in destruction of gum tissue epithelium and bone erosion, is amenable to treatment with dental abutment pieces coated with epithelial cells which promotes reattachment of detached gum tissue to the tooth surface as disclosed in U.S. Patent No. 5,585,267. U.S. Patent No. 5,585,267 describes the use of an extracellular matrix protein secreted by rat bladder carcinoma cells in coating trans-epithelial appliances for promoting epithelial cell attachment thereto. International Application No. WO97/36621 discloses trans-epithelial appliances coated with laminin 5, a form of laminin found in rat and human cells, for promoting epithelial cell attachment thereto and in vivo placement of a laminin 5 -coated surface for inducing epithelial cell adhesion thereto. In other biological processes, such as wound healing, it is desirable to promote adhesion of epithelial cells at the wound site. The present invention provides a method for delivering bioactive molecules to a particular in vivo location, and for promoting or inhibiting the activity of bioactive molecules near a medical device. These bioactive molecules play important roles in many physiological processes.

Summary of the Invention One embodiment of the present invention is a method of capturing and promoting or inhibiting the activity of one or more endogenous bioactive molecules in a vertebrate, preferably a mammal, more preferably a human, comprising the steps of applying to the article one or more agents having affinity for the one or more endogenous bioactive molecules; and placing the article in contact with the vertebrate. Preferably, the endogenous biological molecule is a cell adhesion molecule, growth factor, growth factor receptor, adhesion receptor, enzyme, cytokine, blood clotting protein, and the like. In one aspect of this preferred embodiment, the vertebrate is a human. Advantageously, the article is a transepithelial appliance. Alternatively, the article is an implantable medical device. In another aspect of this preferred embodiment, the agent is an antibody. Preferably, the antibody is monoclonal. Alternatively, the antibody is polyclonal. Preferably, the applying step comprises immersion of the article in a solution of the agent or spraying the article with a solution of the agent. More preferably, the applying step comprises covalent attachment of the agent to the article. In one aspect of this preferred embodiment, the placing step comprises inserting the article in the vertebrate. In another aspect of this preferred embodiment, the placing step comprises implanting the article in the vertebrate.

Another embodiment of the present invention is a method of capturing and promoting or inhibiting the activity of one or more bioactive molecules in a vertebrate, preferably a mammal, more preferably a human, comprising the steps of applying to an article one or more agents having affinity for the one or more bioactive molecules; contacting the article with the one or more bioactive molecules; and placing the article in contact with the vertebrate. The present invention also provides a method for providing a bioactive molecule on an article, comprising the steps of: providing an article having one or more agents having affinity for one or more bioactive molecules attached thereto; and placing the article into an environment that includes the bioactive molecule and allowing the bioactive molecule to bind to the one or more agents on the article. In a preferred embodiment, this results in formation of a coating of the bioactive molecules on the article. Preferably, the placing step comprises introducing the article in vivo. More preferably, the placing step comprises contacting the article with a solution of the bioactive molecule in vitro. The method may further comprise the step of introducing the article into an environment including living cells in which the bioactive coating has an effect on the living cells. Advantageously, the article is a prosthesis, tissue growth scaffold or implantable medical device. In one aspect of this preferred embodiment, the article is a vascular device. Preferably, the coating has antithrombotic properties. In another aspect of this preferred embodiment, the one or more agents is an antibody. The method may further comprise a second agent having affinity for a second bioactive molecule. Another embodiment of the present invention is a method for targeting a bioactive molecule to an implanted article, comprising the steps of: implanting an article in a vertebrate in vivo, wherein the article has one or more agents having affinity for one or more bioactive molecules attached thereto; and administering a bioactive molecule to the vertebrate, whereby the one or more agents binds the bioactive molecule to the article.

The present invention also provides a medical device having a biocoating thereon, wherein the biocoating comprises one or more agents having affinity for one or more bioactive molecules attached to the device; and the bioactive molecule, which is bound by the one or more agents. The present invention also provides a method for identifying a novel agent having a particular biological activity, or a known agent having a new biological activity, comprising the steps of: generating monoclonal antibodies against a sample containing the agent, contacting the antibodies with the sample, wherein the antibody binds the agent, assaying the bound agent for a desired biological activity, and determining the identity of the agent. Preferably, the agent is a protein, carbohydrate, lipid, glycoprotein, glycolipid, lectin, receptor, ligand, enzyme substrate analog, or other organic molecule. In another aspect of this preferred embodiment, the sample is a tissue extract, cell extract, body fluid or cell culture supernatant. Advantageously, the biological activity is cell adhesion, cell migration, cell activation, cell survival, cell proliferation, apoptosis, fibrosis, inflammation, angiogenesis, transport or ligand bonding.

Brief Description of the Drawings Figure 1 is a graph which shows human keratinocyte adhesion to a plastic substrate coated with a non-blocking antibody to laminin 5 (KS12), a blocking antibody to laminin 5 (RG13), laminin 5, bovine serum albumin and an irrelevant antibody (MOPc). Higher values of OD 595 indicate greater cell attachment.

Detailed Description of the Preferred Embodiments It is well known that bioactive molecules can be captured in vitro by antibodies or other agents having specific affinity therefor. The present invention includes the discovery that antibodies or other agents can be bound to an article, particularly a medical device, and can capture bioactive molecules either in vitro or in vivo.

Depending on the nature of the binding between the bioactive molecule and the antibody or other agent, the bioactive molecule is presented in either an active or inactive orientation.

If the antibody (or other agent) is a "non-blocking" antibody (or other agent), the bioactive molecule is captured and presented in an active orientation. Conversely, if the antibody (or other agent) is a "blocking" antibody (or other agent), the bioactive molecule is captured and presented in an inactive orientation. Whether a particular agent is "blocking" or "non-blocking" can be determined using assays well known in the art. For example, the determination of whether an agent having affinity for a particular cell adhesion protein is "blocking" or "non-blocking" can be made by standard cell adhesion assays using microtiter plates coated with an adhesion protein and then incubated with the agent. If cells bind to the wells, the agent is a non- blocking agent. If cells do not bind, the agent is a blocking agent.

As used herein, the term "agent" denotes a protein, carbohydrate, lipid, glycoprotein, glycolipid, lectin, receptor, ligand, enzyme substrate analog, or other organic molecule which specifically binds to a bioactive molecule and either promotes or inhibits its activity. Specific examples of agents include streptavidin which binds biotin, concanavalin A which binds to high-mannose glycoproteins, interleukin-1 which binds to the interleukin 1 receptor, epidermal growth factor which binds to the epidermal growth factor receptor, and the like. In a preferred embodiment, the agent is an antibody which specifically binds to the antigen to which it was generated.

As used herein, the term "bioactive molecule" denotes any molecule having biological activity. These molecules include cytokines, cell adhesion molecules (e.g., integrins, neural cell adhesion molecules, selectins, cadherins), cell surface antigens, blood clot formation-inducing proteins, fibrinolytic proteins, extracellular matrix proteins, growth factors, neurotransmitters, neuropeptides, neuronal enzymes, hormones, enzymes, antibiotics and the like.

Rather than applying a bioactive molecule, which is intended to evoke a desired biological response, to an article, such as an implantable medical device, one or more agents which captures and presents the bioactive molecule in an active or inactive orientation is applied to the device. In another preferred embodiment, the agent targets a cell surface receptor and either promotes or inhibits signalling events normally mediated by the receptor ligand. The agent may also capture and present a bioactive molecule for immune recognition and response, or for destruction by cells, proteases or other molecules. The present invention can be used for a wide variety of applications, e.g., to promote or inhibit angiogenesis, cell adhesion, inflammation, cell migration, cell proliferation, blood coagulation, cell survival, guided nerve regeneration, fϊbrosis, and the like. For example, rather than coating a trans-epithelial appliance with 804G soluble matrix protein to stimulate attachment of epithelial cells thereto as described in U.S. Patent No. 5,585,267, the appliance is coated with an antibody to 804G matrix protein (laminin 5), such as TR1 antibody. The device is then implanted at a wound site and the anti-laminin 5 antibodies bind endogenous laminin 5 and thus promote the in vivo attachment of epithelial cells at the site to promote healing of the wound.

Conversely, in situations where it is desirable to inhibit the activity of an endogenous biomolecule, a blocking agent can be used. Such an agent binds to a region of a molecule necessary for activity, thus acting as an inhibitor of molecular activity. Alternatively, the agent may bind to a site in such a way as to expose or protect the remainder of the endogenous biomolecule to proteases or other molecules. The ability of the extracellular matrix protein laminin 5 to promote epithelial cell adhesion can be inhibited by the blocking antibody EM- 11 (Desmos, Inc., San Diego, CA). This may be desirable for enabling fibroblast attachment to the subcutaneous region of a prosthetic tooth.

In another preferred embodiment, one or more antibodies which binds to and blocks the activity of one or more pro-inflammatory molecules such as interleukin- 1, interleukin-2, interleukin-6, TNF-α and prostaglandins, is applied to an artificial joint or ligament. Similarly, an indwelling catheter contacted with such antibodies will bind to and inhibit the activity of these molecules, thus reducing the amount of inflammation around the catheter.

Different agents exhibiting different activities can be applied to the same device.

For example, a surface can be contacted with one antibody which captures antithrombin III (A5816, Sigma, St. Louis, MO), an antibody which captures tissue plasminogen activator (tPA) (DC-423, Biodesign, Kennebunk, ME), and a third antibody which captures laminin 5 (GB3, Harlan, Madison, WI). Such a surface would promote epithelial cell adhesion and prevent clot formation.

In another preferred embodiment, a bioactive molecule can be captured and either activated or inactivated indirectly via a secondary molecule. For example, an agent having affinity for an inhibitor of an enzyme is applied to a medical device. The agent binds the endogenous inhibitor which is then bound by the enzyme, resulting in enzyme inhibition in the vicinity of the device. Conversely, an agent having affinity for an activator of an enzyme is applied to a medical device which binds endogenous activator which is then bound by the enzyme, resulting in localized enzyme activation.

In another preferred embodiment, the device contacted with the agent is contacted with the bioactive molecule in vitro prior to implantation of the medical device.

Implantable or insertable devices contacted with one or more agents may also be contacted with a desired biological molecule in vitro, then implanted or inserted into a vertebrate, preferably a mammal, more preferably a human. For example, an implantable or insertable medical device is contacted with an antibody to laminin, then contacted with laminin which becomes bound to the antibody. The device is then implanted or inserted at a site where epithelial cell recruitment and adhesion is desired, such as a wound or burn site. An antibody to one or more antibiotics or other drugs can also be applied to an implantable bead, particulate, object, or medical device, followed by implantation of the device and oral or parenteral administration of the corresponding antibiotic or other drug. Bacterial colonization on the device, or in the vicinity of the implanted object, is inhibited by virtue of the antibiotic bound to the treated device or object. Alternatively, one can direct the drug to the surface after implantation.

Polyclonal antibodies to a desired endogenous or exogenous bioactive molecule are either obtained from a commercial supplier, or are produced by inoculating animals, preferably rabbits, with the molecule, then obtaining blood serum after several booster injections as is well known in the art. Monoclonal antibodies to any desired endogenous or exogenous bioactive molecule are obtained from a commercial supplier, or are prepared using hybridoma technology which is well known in the art (Galfre et al., Meth. Enzymol. 73:3-46, 1981). These mouse monoclonal antibodies can be humanized, if desired, using well known methods to reduce their immunogenicity for human use. The entire antibody, Fab' fragment, (Fab)'₂ fragment, genetically engineered single chain antibodies (SCABs), or bivalent antibodies which recognize two different antigens can be used to coat an article. However, monovalent antibodies could afford some control with respect to affinity and clustering.

Although any biocompatible article capable of being implanted in vivo can be used in the present invention, manufactured or shaped articles, particularly medical devices, are preferred. The shaped articles contemplated for use in the invention include such forms as sheets, fibers, sponges, porous matrices, fabrics, prostheses, metal articles, ceramic articles, bioerodible articles, implantable articles and the like. Implantable shaped articles include heart valves, pacemakers, artificial bones and joints, powders, particulates, beads, vascular grafts, vascular shunts, vascular stents, artificial tear ducts, eye lenses, dental implants, artificial tendons and ligaments, rods, plates, pins, screws, surgical textiles (meshes), sutures, defϊbrillators, catheters, implanted ports for injection into a specific location, breast implants, cochlear implants and any other device intended for insertion, injection, or implantation into a vertebrate, preferably a mammal, more preferably a human. One or more desired antibody(ies) or other protein(s) having affinity for a particular bioactive molecule is applied to the article, which is then implanted or inserted at the appropriate site using surgical and medical procedures well known in the art (e.g., Sabiston, D. C, Jr., M.D., Textbook of Surgery: The Biological Basis of Modern Surgical Practice, 15th Edition, W. B. Saunders Co., Philadephia, 1997).

The implanted article typically contacts at least one of a body surface and a body fluid, including bones, teeth, gums, skin, connective tissue, peritoneum, neural tissue, muscle, internal organs, blood, cerebrospinal fluid and the like.

Suitable substrate materials include articles made of organic materials such as collagen, regenerated collagen, hyaluronic acid, polyglycolic acid, polygalactose, polylactic acid or derivatives thereof and the like; biocompatible metals such as stainless steel and titanium; ceramic materials including prosthetic material such as hydroxylapatite; synthetic polymers including nylons; polyesters; polystyrenes; polypropylenes; polycarbonates; polyethylenes; polysulfones; polyacrylates; polytetrafluoroethylenes; silicones; and virtually any other material to which biological molecules can readily adhere or be bound. These materials are typically non- immunogenic, stable and retain their structural integrity after implantation in vivo.

An agent having affinity for an endogenous bioactive molecule is applied to an article by either passive adsorption or covalent linkage. Passive adsorption can be accomplished in a variety of ways, e.g., by immersion of an article in an antibody- or other agent-containing solution, spraying the article with the solution and the like. Alternatively, agents may be covalently linked to a surface using any of a variety of well known cross-linking reagents 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.2HCl (DTBP) and disuccinimidyl suberate (DSS). Sulfhydryl-specifϊc reagents include bismaleimidohexane (BMH) and l,4-di-[3'-2'-pyridyldithio(propionamido)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 succimidyl 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 hydrazides, resulting in formation of amide or hydrazone 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 particular agent and the surface chemistry of the article to which the agent is to be attached. A surface can be chemically modified or manufactured to contain particular reactive groups using methods well known in the art. Agents can also be covalently coupled to a substrate. For example, substrates can be manufactured which contain epoxide groups on their surface. Agents can be covalently linked to these 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, radiofrequency (RF) energy, laser or strong acid or base treatment may facilitate attachment of cross-linking agents.

In a preferred embodiment, the amount of antibody or agent applied to the surface of an article is between about 0.01 μg/cm² and 100 μg/cm². In a particularly preferred embodiment, the amount of antibody or agent applied to the surface of the article is between about 0.1 μg/cm² and 10 μg/cm². Particular types of antibodies contemplated for coating the articles include those which bind to and inhibit the activity of blood clot formation-promoting proteins including thrombin to prevent blood clot formation, particularly for articles implanted within the vascular system such as vascular shunts, grafts and heart valves. Other types of antibodies contemplated for use in the present invention are those having affinity for extracellular matrix and other cell adhesion-promoting proteins; those having affinity for growth factors, growth factor receptors, adhesion receptors and cytokines; and those having affinity for hormones.

There are thousands of known antibodies, both monoclonal and polyclonal, that are suitable for use in the present invention. Many of the antibodies listed below are available from Sigma Chemical Co, St. Louis, MO. Antibodies can be used in the present invention to either inhibit or promote many physiological processes as described below.

Blocking antibodies to proteins involved in blood clotting are applied to medical devices used in cardiovascular applications, such as vascular stents and shunts, to promote an anti-thrombotic state. Blocking antibodies useful for inducing an anti- thrombotic state include those generated against blood clotting proteins including thrombin, Factor V, Factor VII, Factor IX, Factor X, fibrinogen, Protein C, Protein S, von Willebrand Factor, plasminogen activator inhibitor I (PAI-I) and plasminogen activator inhibitor II (PAI-II).

In another preferred embodiment, non-blocking antibodies to proteins involved in fibrinolysis are used to maintain an anti-thrombotic state. Such antibodies include those generated against tPA, urokinase (uPA) and plasmin. In yet another preferred embodiment, both blocking antibodies to proteins involved in blood clotting and non- blocking antibodies to proteins involved in fibrinolysis are applied to an implantable medical device to promote and anti-thrombotic state. Non-blocking or blocking antibodies to extracellular matrix proteins useful in promoting or inhibiting cell adhesion, respectively, for use in the present invention include, but are not limited to, antibodies generated against aggrecan, biglycan, bone sialoprotein, cartilage matrix protein, cartilage oligomeric matrix protein, collagen types I-XVI, decorin, elastin, entactin, fibrilin, fibrinogen, fibromodulin, fibulin, laminins (all isoforms), link protein, lumican, matrix gla protein, microfibril-associated glycoprotein, osteocalcin, osteonectin, osteopontin, perlecan, phosphoryn, tenascin, thrombospondin,

10 versican, vitronectin and von Willebrand factor.

Antibodies to growth factors and growth factor receptors can be used as biocoating to inhibit cell growth. For cells requiring these growth factors or having growth factor receptors, antibodies which capture and sequester these factors will inhibit cell growth. Similarly, antibodies to cytokines which capture and sequester cytokines will inhibit the growth of cells which require such cytokines. Conversely, if the antibodies capture and present the molecules to cells in a useful way, cell growth will be stimulated. Examples of such antibodies include those generated against epidermal growth factor (EGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), fibroblast growth factor (FGF), granulocyte macrophage colony stimulating factor (GM- CSF), receptors for all of the above growth factors, tumor necrosis factor-α (TNF-α), interferon-α, β, γ (IFN-α, β, γ), interleukins, erythropoietin, and any other desired growth factor, growth factor receptor or cytokine. Antibodies to neurotransmitters, neuropeptides and neuronal enzymes are used as biocoatings for modulating the function of the peripheral and central nervous system. Such antibodies include those generated against aspartate, DOPA decarboxylase (DDC), β-endorphin, leu-enkephalin, met-enkephalin, γ-aminobutyric acid (GABA), glutamate, neuropeptide Y (NPY), nitric oxide synthase-brain (bNOS), serotonin, substance P (SP), vasoactive intestinal peptide (VIP), and the like.

Antibodies to hormones and enzymes can be used to coat implantable devices to modulate the function of the hormone. Such hormones include adrenocorticotrophic hormone (ACTH), angiotensin I, calcitonin, calcitonin gene related polypeptide (CGRP), cholecystokinin, endothelin, gastrin, luteinizing hormone releasing hormone (LHRH), estrogen, oxytocin, tryptophan hydroxylase and tyrosine hydroxylase, and the like.

Surgical meshes for promoting wound healing may be contacted with antibodies which bind to wound healing-promoting proteins such as H3, a protein described in U.S. Patent No. 5,599,788, and TGF-β.

Antibodies to proinflammatory cytokines such as interleukin- 1, interferon-γ and TNF-α can also be used to coat articles to prevent inflammatory reactions at the implantation site. For example, a biocompatible article is placed at an incision site after

11 surgery to minimize inflammation during the healing process.

Antibodies to various antibiotics are used to coat implanted devices which then capture and concentrate an antibiotic administered after surgery at the implantation site to prevent infection. For example, antibodies to gentamicin (catalog nos. 101, 102) and penicillin (catalog nos. 701, 704) are available from Biodesign (Kennebunk, ME). One significant advantage of this embodiment is that smaller, less frequent dosages of antibiotic are required due to the ability of the antibodies to capture the circulating antibiotic.

The endogenous biomolecules recruited by agents bound to articles function better than those produced in a laboratory. Antibodies are more resistant to degradation than are most biological molecules. Thus, the antibodies bound to the implanted or inserted medical devices will persist in vivo. In addition, bioactive molecule turnover can be modulated by antibody affinity.

In another preferred embodiment one or more agents having affinity for one or more cell surface antigens is applied to a medical device. Cells possessing these cell surface antigens bind to the agent which is, in turn, bound to the medical device. Cell surface antigens include, for example, growth factor receptors, cytokine receptors, F_c receptors, T cell receptors, cell adhesion receptors, neural cell adhesion molecules and the like. Heterobivalent or homobivalent antibodies can be used to crosslink cell surface receptors, thereby inducing a signaling event. Antibodies to cell adhesion receptors are used to induce cell attachment to a medical device. For example, antibodies to the cell adhesion receptor α5βl, expressed predominantly on fibroblasts, are used to induce fibroblast attachment.

Medical devices contacted with one or more agents can also serve as "reservoirs" for the bioactive molecule. For example an agent having specific affinity for a particular cytokine is applied to a medical device which is then implanted into a vertebrate. The circulating cytokine binds to the agent and is thus, in effect, concentrated at the surface of the medical device. Cells which have the corresponding cytokine receptor can bind to the bound cytokine, thus removing it from its specific binding agent. The medical device is then "recharged" with circulating cytokine to replace the cytokine removed by the cell containing its receptor.

12 In another preferred embodiment of the invention, articles contacted with antibodies are also used to discover new biological molecules possessing interesting or desirable activities, or to discover new uses for known biological molecules. For example, monoclonal antibodies are created against impure preparations from tissues, cells, body fluids or cell culture products. These antibodies are applied to a surface, the surface is exposed to the original inoculum and then assayed for the activity of interest using standard assays. Activities of interest include cell adhesion, cell migration, cell activation, cell survival, cell proliferation, cell differentiation, apoptosis, fibrosis, inflammation, angiogenesis, transport, binding to other ligands, drug binding, and the like. For example, cell activation is assayed by cytokine release using a standard enzyme-linked immunosorbent assay (ELISA); cell proliferation is assayed by measuring incorporation of tritiated thymidine; cell adhesion is monitored by measuring the optical density at 595 nm of cells stained with crystal violet; and cell differentiation is assessed visually based on changes in morphology. The protein to which the antibody binds is then identified. If the protein is known, but not known to have the particular activity, then a new activity for a known protein has been discovered. If the protein is new, then a new protein having a particular activity has been discovered. For example, agents which promote guided nerve regeneration can be discovered as described in Example 20. In addition to treating man-made or exogenous articles, it will be appreciated that naturally occurring and endogenous articles can also be coated or treated. Thus, the present invention contemplates application to any suitable body part, such as bone, tooth, connective tissue, membranes, vessels, and the like. Thus, for example, antithrombogenic molecules can thereby be attracted to blood vessels, cell attachment factors can be attracted to teeth, osteogenic factors can be attracted to bones or fractures, and the like. Many other examples of use will be readily apparent to one of ordinary skill in the art.

The ability of an antibody to laminin 5 to promote human keratinocyte adhesion in vitro was determined as described in the following example. Example 1

Promotion of keratinocyte cell adhesion by antibody to laminin 5

13 Normal human keratinocytes (NHKs) are epidermal cells which secrete small amounts of laminin 5. These cells will gradually adhere to plastic over a time frame of approximately 5 hours. These cells were plated on a plastic surface coated with a control antibody or BSA (MOPc; Figure 1) which was not generated against an extracellular matrix protein and took approximately 5 hours. The same result was observed with BSA. When NHKs were plated onto a plastic surface coated with an antibody to laminin 5 which captures and presents laminin 5 in a manner which allows keratinocyte attachment thereto (KS12), the cells adhered to the surface in about 90 minutes. However, when a blocking antibody (RG13) was used which inhibits the ability of laminin 5 to promote attachment of NHKs to the substrate, very little cell attachment was observed at 5 hours. Thus, the non-blocking antibody to laminin 5 amplified the ability of the cells to bind to the surface by capturing and presenting the small amount of laminin 5 produced by the cells which induced cell attachment to the surface. Conversely, a blocking antibody attenuated the ability of the cells to bind to the surface by capturing and blocking the small amount of laminin 5.

Thus, NHKs which produce laminin 5 adhere more rapidly to a substrate coated with a nonblocking monoclonal antibody specific to laminin 5 than to a substrate coated with an irrelevant antibody or no antibody. These cells adhered more slowly to a substrate coated with a blocking monoclonal antibody specific to laminin 5 than to plastic coated with an irrelevant antibody or no antibody. Cell adhesion to KS12-coated plastic is prevented if the cells are treated with cycloheximide to prevent protein synthesis. These same cycloheximide-treated cells adhere well to plastic coated with laminin 5. This supports the hypothesis that the cells produce the laminin 5, but the antibody facilitates the adhesion-promoting activity thereof. Example 2

Inhibition of blood coagulation A blocking antibody to a coagulation-inducing agent such as Factor Xa (e.g., antibody 5224, American Diagnostics, Greenwich, CT) is applied to an artificial heart valve, vascular stent, or vascular shunt prior to implantation into a patient who has undergone standard cardiovascular surgical procedures. The antibody inhibits thrombus formation around the implanted vascular device.

14 Example 3

Activation of fibrinolysis

A non-blocking antibody to a thrombolytic agent such as tPA (e.g., DC-423.

Biodesign) is applied to an artificial heart valve, vascular stent, or vascular shunt prior to implantation into a patient who has undergone standard cardiovascular surgical procedures. The tPA activates plasminogen which degrades of any clots which may form during the surgical procedure.

Example 4

Concurrent inhibition of blood coagulation and activation of fibrinolysis A non-blocking antibody to a thrombolytic agent such as tPA (e.g., DC-423,

Biodesign) and a blocking antibody to a coagulation-inducing agent such as Factor Xa

(e.g., 5224, American Diagnostics) are applied to an artificial heart valve, vascular stent, or vascular shunt prior to implantation into a patient who has undergone standard cardiovascular surgical procedures. The combination of antibodies promotes an overall anti-thrombotic state by both inhibiting blood coagulation and stimulating fibrinolysis.

Example 5 Promotion of wound healing - laminin 5 attachment in vivo A non-blocking monoclonal antibody to human laminin 5 (e.g., GB3) is applied to a substrate, such as surgical mesh, which is then placed at a wound site in a patient. The antibody promotes epithelial cell adhesion and migration to the site and more rapid healing of the wound by capturing endogenous laminin 5 and promoting its activity.

Example 6 Promotion of wound healing - laminin 5 attachment in vitro A non-blocking antibody to human laminin 5 is applied to a surgical mesh, followed by contacting the mesh with human laminin 5 in vitro. The mesh is then placed at a wound site where the bound laminin 5 promotes epithelial cell recruitment and adhesion to the wound site.

Example 7 Localization of antibiotic to implant site A monoclonal antibody to gentamycin (e.g., antibody 101, Biodesign) is to a medical device, such as an artificial joint, prior to surgical placement of the joint into

15 a patient. Before, during and/or after the surgical procedure, the patient is given oral, parenteral or local gentamycin which concentrates at the site of the implant due to the antibody bound to the implant and reduces the risk of bacterial infection at the implant site. This concentration effect also reduces the required dosage of antibiotic. Example 8

Inhibition of inflammation induced by central venous catheter A blocking antibody to a pro-inflammatory cytokine, such as TNF-α, is applied to a central venous catheter (Hickman catheter) (Bard Instruments, Salt Lake City, UT), prior to insertion into a vein by standard surgical procedures. The antibody reduces inflammation around the catheter.

Example 9 Inhibition of capsule formation and promotion of angiogenesis around implanted drug delivery devices

A drug delivery device, such as an implanted port with an open-ended catheter, is implanted into patients to allow delivery of drugs when needed. However, these devices often induce formation of collagen structures which are poorly vascularized. Therefore, to inhibit encapsulation and promote angiogenesis around the implant site, a non-blocking antibody to fibronectin (e.g., antibody 3E1, GIBCO, Grand Island, NY) to inhibit encapsulation and a non-blocking antibody against vascular endothelial growth factor (VEGF) (e.g., antibody A-20, Santa Cruz Biotechnology, Santa Cruz, CA) to promote angiogenesis is applied to the device.

Example 10 Inhibition of inflammation around implanted artificial joint Prior to surgical implantation into a patient, one or more blocking antibodies to pro-inflammatory cytokines such as TNF-α and/or II- 1 is applied to an artificial knee.

The antibody(ies) reduce post-surgical inflammation by inhibiting the activity of these cytokines.

Example 11 Promotion of epithelial cell adhesion around catheter insertion site GB3, a non-blocking monoclonal antibody to laminin 5 (available from Harlan

Biotechnology), is applied to the transcutaneous region of an indwelling catheter. By

16 capturing endogenous laminin 5, the GB3 promotes epithelial cell adhesion around the catheter entry site to seal the skin around the catheter, thus inhibiting bacterial colonization of the subcutaneous portion of the catheter.

Example 12 Promotion of adhesion of gingival epithelium to dental implant

GB3 is applied to the transcutaneous region of a titanium dental implant to promote adhesion of gingival epithelial cells around the implant site. This prevents bacterial colonization of the subcutaneous portion of the implant.

Example 13 Promotion of adhesion of gingival epithelium to tooth surface

GB3 is applied to the transcutaneous region of a tooth surface to promote adhesion of gingival epithelial cells around the tooth. This prevents bacterial colonization of the subcutaneous region of the tooth.

Example 14

Inhibition of transcutaneous pocket formation

One problem associated with the use of transcutaneous catheters and dental implants is the formation of pockets ("marsupialization") in the transcutaneous region due to epithelial cell downward migration. In addition, the adhesion of fibroblasts to the subcutaneous region is desirable because this inhibits pocket formation. A blocking antibody to an epithelial cell adhesion-promoting agent such as laminin 5 (e.g. monoclonal antibody EMU) is applied to the subcutaneous region of a catheter or dental implant to inhibit epithelial cell adhesion and migration to the subcutaneous region. In addition, a non-blocking antibody to fibronectin may be applied to the subcutaneous region of the catheter to promote fibroblast adhesion in this region.

Example 15 Promotion of epithelial cell adhesion and inhibition of transcutaneous pocket formation around catheter insertion site

GB3 and a blocking antibody to an epithelial cell adhesion-promoting protein such as laminin 5 (e.g. monoclonal antibody EMU) are applied to the transcutaneous region of an indwelling catheter to both promote epithelial cell adhesion around the

17 catheter insertion site and to inhibit transcutaneous pocket formation around the catheter.

Example 16 Prevention of brain electrode encapsulation Electrodes are often implanted into the brains of individuals with various neurological conditions to stimulate brain electrical activity. However, these electrodes often become encapsulated by surrounding tissue or cells which results in electrical insulation and inability to conduct electrical impulses. A non-blocking antibody to cell surface molecules of neurons, such as neural cell adhesion molecule (NCAM) (e.g., antibody NCAM-QBll, Sigma, St. Louis, MO) is applied to the electrodes to promote adhesion of neurons and prevent encapsulation.

Example 17 Promotion of bone growth using implant A non-blocking antibody to a bone growth formation-inducing protein such as one of the bone morphogenetic proteins (BMPs) (Wang et al., Proc. Natl. Acad. Sci.

U.S.A. 87:2220-2224, 1990) is applied to Gelfoam™ sponges (Upjohn, Kalamazoo, MI), Collagraft™ strips (mixture of hydroxyapatite/tricalcium phosphate powder with type I bovine fibrillar collagen) (Zimmer, Warsaw, IN), metal pins or rods. The sponges or strips are then implanted into an individual at a site of bone loss, bone fracture or demineralization to promote bone growth at the site. The pins or rods are used to join separated bone regions and promote the growth of bone therebetween.

Example 18 Prevention of surgical adhesion formation Adhesions often form between an organ and surrounding connective tissue and bone after a surgical procedure. Following surgical trauma, connective tissue surrounding the organ proliferates to form a fibrous mass that binds the organ to neighboring organs, viscera, muscle or bone. This undesirable process can be inhibited by implanted mechanical devices as described in U.S. Patent Nos. 4,013,078 and 5,611,354. An antibody which inhibits adhesion of fibrous tissue is applied to these devices to enhance their ability to inhibit adhesion formation.

Example 19

18 Catalytic inactivation of thrombin A moderate affinity, non-blocking antibody specific to free antithrombin IIIA is applied to a vascular device which is then implanted into a patient using standard cardiovascular surgical procedures. Thrombin encounters and binds antibody-bound antithrombin III, resulting in thrombin inactivation. The thrombin/antithrombin III complex dissociates from the antibody which has a low affinity for the newly formed complex. The process then repeats. Thus, an overall anti-thrombotic state is maintained near the vascular device.

Example 20 Discovery of agents which stimulate guided nerve regeneration

Rat neuronal cells are grown under standard cell culture conditions and allowed to deposit their own extracellular matrix. Cells are removed by treatment with ammonium hydroxide and the deposited extracellular matrix is used to immunize mice. Mice with a high blood antibody titer are selected for hybridoma generation by standard methods. Hybridoma supernatants are spotted onto glass coverslips (24 spots per coverslip) which have been precoated with protein G and blocked with BSA. Freshly harvested rat neuronal cells are then seeded at low density onto these coverslips, which may or may not have been previously incubated in neuronal cell conditioned media. Spots, which either recruit or repel extending axons, are likely to have captured agents useful in guided nerve regeneration. Corresponding hybridomas are subcloned and banked. Antibodies produced by these hybridomas are purified and used directly for clinical applications, or are utilized for identification, characterization or isolation of therapeutic agents.

The purified antibodies are combined with the initial inoculum to immunoprecipitate the unknown antigen to which the antibody binds. The immunoprecipitate is washed and subjected to polyacrylamide gel electrophoresis. The protein band is excised from the gel and sequenced, either by Edman degradation to obtain sequence at the N-terminus, or proteolytically cleaved followed by sequencing of the peptide fragments. These methods are all well known to one of ordinary skill in the art. The sequences are then compared to known protein sequences in computer databases using well known sequence alignment programs to determine the identity of

19 the protein. If no match is found, a new protein has been discovered based on its ability to participate in guided nerve regeneration.

Example 21

Prevention of blood clot formation by captured heparin A non-blocking antibody to heparin is applied to a medical device, such as a vascular stent or shunt, prior to implantation into a patient using standard surgical procedures. After surgery, the patient is administered intravenous heparin which binds to the antibody applied to the medical device and inhibits blood clot formation at the implant site. This allows administration of smaller amounts of heparin than would otherwise be required due to the ability of heparin to be targeted to a particular site.

Example 22 Prevention of blood clot formation by blocking antibody to GPHb/IIIa A blocking antibody to the platelet adhesion receptor GPIIb/IIIa is applied to a medical device, such as a vascular stent or shunt, prior to implantation of the device into a patient using standard surgical procedures. The blocking antibody binds to

GPIIb/IIIa and locks the platelets into an inactive state, thus inhibiting platelet aggregation normally mediated by this receptor.

Example 23 Inhibition of matrix metalloproteases Matrix metalloproteases (MMPs), including MMP-2, are implicated as a causative factor in periodontal disease by degrading or altering extracellular matrix associated with tooth and gum. A blocking antibody to MMP-2 which inhibits this extracellular matrix degradation is applied to the subcutaneous region of a dental implant or tooth surface. 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.

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Claims

WHAT IS CLAIMED IS:

1. A method for capturing and promoting or blocking the activity of one or more endogenous bioactive molecules in a mammal, comprising the steps of: applying one or more proteins having affinity for said one or more endogenous bioactive molecules to an article; and placing said article in contact with said mammal.

2. The method of Claim 1, wherein said endogenous bioactive molecule is selected from the group consisting of a cell adhesion molecule, growth factor, growth factor receptor, enzyme, cytokine and blood clotting protein.

3. The method of Claim 1, wherein said mammal is a human.

4. The method of Claim 1, wherein said article is a transepithelial appliance.

5. The method of Claim 1, wherein said article is an implantable medical device.

6. The method of Claim 1, wherein said protein is an antibody.

7. The method of Claim 6, wherein said antibody is monoclonal.

8. The method of Claim 6, wherein said antibody is polyclonal.

9. The method of Claim 1 , wherein said coating step comprises immersion of said article in a solution of said protein.

10. The method of Claim 1, wherein said coating step comprises spraying said article with a solution of said protein.

11. The method of Claim 1 , wherein said coating step comprises covalent attachment of said protein to said article.

12. The method of Claim 1, wherein said placing step comprises inserting said article in said mammal.

13. The method of Claim 1, wherein said placing step comprises implanting said article in said mammal.

14. A method of capturing and promoting or blocking the activity of one or more biological molecules in a mammal, comprising the steps of: applying one or more proteins having affinity for said one or more bioactive molecules to an article; contacting said article with said one or more bioactive molecules; and

21 placing said article in contact with said mammal.

15. A method for providing a bioactive coating on an article, comprising the steps of: providing an article having specific binding molecules attached thereto, wherein said specific binding molecules are specific to a bioactive molecule; and placing said article into an environment that includes said bioactive molecule and allowing said bioactive molecule to bind to said specific binding molecules on said article.

16. The method of Claim 15, wherein said placing step comprises introducing said article in vivo.

17. The method of Claim 15, wherein said placing step comprises contacting said article with a solution of said bioactive molecule in vitro.

18. The method of Claim 15, further comprising the step of introducing said article into an environment including living cells in which said bioactive coating has an effect on said living cells.

19. The method of Claim 18, wherein said article is selected from the group consisting of a prosthesis, tissue growth scaffold and implantable medical device.

20. The method of Claim 19, wherein said article is a vascular device.

21. The method of Claim 20, wherein said coating has antithrombotic properties.

22. The method of Claim 1, wherein said specific binding molecule is an antibody.

23. The method of Claim 1, further comprising a second specific binding molecule directed toward a second bioactive molecule.

24. A method for targeting a bioactive molecule to an implanted article, comprising the steps of: implanting an article in a vertebrate in vivo, wherein said article has a specific binding molecule attached thereto; and administering a bioactive molecule to said vertebrate, wherein said specific binding molecule is specific to said bioactive molecule, whereby said specific binding molecule binds said bioactive molecule to said article.

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25. A medical device having a biocoating thereon, wherein said biocoating comprises a specific binding molecule attached to said device, said specific binding molecule having specificity for a bioactive molecule; and said bioactive molecule, which is bound by said specific binding molecule.

26. A method for identifying a novel agent having a particular biological activity, or a known agent having a new biological activity, comprising the steps of: generating monoclonal antibodies against a sample containing said agent; contacting said antibodies with said sample, wherein said antibody binds said agent; assaying said bound agent for a desired biological activity; and determining the identity of said agent.

27. The method of Claim 26, wherein said agent is selected from the group consisting of a protein, carbohydrate, lipid, glycoprotein, glycolipid, lectin, receptor, ligand and enzyme substrate analog.

28. The method of Claim 26, wherein said sample is selected from the group consisting of a tissue extract, cell extract, body fluid and cell culture supernatant.

29. The method of Claim 26, wherein said biological activity is selected from the group consisting of cell adhesion, cell migration, cell activation, cell survival, cell proliferation, apoptosis, fibrosis, inflammation, angiogenesis, transport and ligand binding.

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