USE OF ANTI-CD44 MONOCLONAL ANTIBODY TO ENHANCE WOUND HEALING
The subject matter of this application was made with support from the United States Government under National Institutes of Health Grant No. AG 101143-12.
FIELD OF THE INVENTION
The subject invention is directed to a method of enhancing wound healing, and more particularly to the use of an anti-CD44 monoclonal antibody to enhance wound healing .
BACKGROUND OF THE INVENTION Throughout this application various publications are referenced, many in parenthesis. Full citations for each of these publications are provided at the end of the Detailed Description. The disclosures of each of these publications in their entireties are hereby incorporated by reference in this application.
It is estimated that in 1992 (US), 35.2 million wounds required major therapeutic intervention (Medical Data International, Inc. 1993) . Surgical incisional wounds are performed with aseptic technique, and are closed by primary intention. Most repair and heal uneventfully. Many traumatic wounds and cancer extirpations, however, must be left open to heal by secondary intention. Furthermore, chronic wounds have significant tissue necrosis and fail to heal by secondary intention. It is estimated that 5.5 million people in the US have chronic, nonhealing wounds and that their prevalence is increasing secondary to the increase in age-related diseases, the increase in Acquired- immune Deficiency Syndrome (AIDS) , and the increase of radiation wounds secondary to cancer intervention. In the US approximately 1.5-2.5 million people have venous leg ulcers; 300,000-500,000, diabetic ulcers; and 2.5-3.5 million, pressure ulcers (Callam et al . 1987; Phillips and Dover 1991; Lees and Lambert 1992; Lindholm et al . 1992) . These acute and chronic open wounds require long- term care and procedures that include skin grafting and tissue flaps, debridement, frequent dressing changes and administration of pain medications. This care is costly and labor intensive. Furthermore, these wounds have a severe impact on the patients1 quality of life. The chronic dermal ulcerations can cost as much as $40,000 each to heal and more disappointing is that 50% reappear within 18 months of healing. Chronic dermal ulcers are also associated with mortality. As many as 21% of patients in intermediate-care facilities with pressure ulcers die (Bergstrom et al . 1994) . Although multiple millions of dollars have been spent on the development of numerous recombinant growth factors (Abraham and Klagsbrun 1996; Heldin and estermark 1996; Nanney and King 1996; Roberts and Sporn 1996) and organotypic skin replacements (Boyce et al . 1995) for use in open wounds over the past decade, the evidence of cost-effective benefit is meager thus far (Brown et al . 1989; Robson et al . 1992a; Robson et al . 1992b; Phillips et al . 1993).
Many attempts have been made to produce a composition which can be used to facilitate wound repair. Many of these compositions involve collagen as a component. U.S. Patent Nos . 4,950,483 and 5,024,841 each discuss the usefulness of collagen implants as wound healing matrices. U.S. Patent No. 4,453,939 discusses a wound healing composition of collagen with a fibrinogen component and a thrombin component, and optionally fibronectin. U.S. Patent No. 4,970,298 discusses the usefulness of a biodegradable collagen matrix (of collagen, hyaluronic acid, and fibronectin) for wound - 3 -
healing. Yamada et al . (1995) disclose an allogeneic cultured dermal substitute that is prepared by plating fibroblasts onto a spongy collagen matrix and then culturing for 7 to 10 days. Devries et al . (1995) disclose a collagen/alpha-elastin hydrolysate matrix that can be seeded with a stromal -vascular- fraction of adipose tissue. Lamme et al . (1996) disclose a dermal matrix substitute of collagen coated with elastin hydrolysate. U.S. Patent No. 5,489,304 and Ellis and Yannas (1996) each disclose a collagen-glycosaminoglycan matrix.
There are also numerous compositions which involve hyaluronic acid (HA) as a component. Ortonne (1996), Borgognoni et al . (1996), and Nakamura et al . (1997) each discuss the usefulness of HA for wound healing. In Nakamura et al . (1997), the HA was combined with chondroitin sulfate in one series of experiments. In U.S. Patent No. 5,604,200, medical grade HA and tissue culture grade plasma fibronectin were used in combination with calcium, phosphate, uric acid, urea, sodium, potassium, chloride and magnesium to create a moist healing environment that simulates the fetal in utero wound healing matrix. U.S. Patent No. 5,631,011 discloses a composition of HA and fibrin or fibrinogen. Various other compositions have also been explored for their wound healing capabilities. Kratz et al . (1997) used a gel of heparin ionically linked to chitosan. Bartold and Raben (1996) studied platelet- derived growth factor (PDGF) . Henke et al . (1996) disclosed that chondroitin sulfate proteoglycan mediated cell migration on fibrinogen and invasion into a fibrin matrix, while Nakamura et al . (1997) concluded that chondroitin sulfate did not affect wound closure in a corneal epithelial wound. Henke et al . (1996) also disclosed that an anti-CD44 antibody blocked endothelial cell migration on fibrinogen. U.S. Patent No. 5,641,483 discloses topical gel and cream formulations containing human plasma fibronectin for healing of cutaneous wounds. Schultz et al . (1992) disclose a composition of epidermal growth factor (EGF) , fibronectin, a synthetic collagenase inhibitor, and Aprotinin.
Various studies involving fibronectin (FN) and/or particular fibronectin peptides and wound healing have also been reported. Many of these studies involve the RGD sequence, part of the cell binding domain of FN (see Schor et al . 1996; Steed et al . 1995; Sponsel et al . 1994; Kartha and Toback 1992; Kishida et al . 1992). Schor et al . (1996) disclose that only the gelatin binding domain of FN (GBD) stimulates fibroblast migration into a 3-D matrix of native type I collagen fibrils at femtomolar concentrations; whereas peptides of the other FN functional domains do not stimulate fibroblast migration in this assay at femtomolar to nanomolar concentrations. Schor et al . (1996) also disclose that the RGDS-containing cell binding domain of FN does, however, stimulate fibroblast migration in the transmembrane (or "Boyden chamber") assay. Steed et al . (1995) disclose that the RGD peptide matrix (known as Argidene Gel™ or as Telio-Derm Gel™) promoted wound healing. On the contrary, Sponsel et al . (1994) disclose that an RGD peptide impaired healing of a mechanical wound made in a confluent monolayer of one epithelial cell line. Kartha and Toback (1992) also concluded that an RGDS peptide completely inhibited cell migration into a wound area. Kishida et al . (1992), however, disclose that an RGD-albumin conjugate adsorbed onto a polyurethane sponge exhibited tissue ingrowth-promoting activity. - 5 -
Other portions of FN have also been studied for wound healing activity. U.S. Patent No. 5,198,423 studied the effects of a polypeptide containing a cell binding domain and a heparin binding domain of FN on wound healing. U.S. Patent No. 4,589,881 studied the effects of a 108 aa polypeptide fragment of FN on wound healing, as well as a biologically active fragment thereof. Sponsel et al . (1994) studied the effect of the tetrapeptide REDV and the peptide LDVPS on wound healing. The severity of the problem of chronic, nonhealing wounds dictates that continual efforts be made to define new and more effective methods for facilitating wound healing .
SUMMARY OF THE INVENTION
This need is met by the method for enhancing wound healing according to the subject invention. The method includes providing a composition of monoclonal antibody A1G3 , and applying the composition to a wound.
BRIEF DESCRIPTION OF THE DRAWINGS These and other features and advantages of this invention will be evident from the following detailed description of preferred embodiments when read in conjunction with the accompanying drawings in which:
Fig. 1 illustrates the in vitro model for assaying cell transmigration from a collagen gel into a fibrin gel; and
Fig. 2 illustrates the effect on cell migration of varying concentrations of monoclonal antibodies A1G3 and
A3D8. DETAILED DESCRIPTION OF THE INVENTION
The subject invention provides a method of enhancing wound healing. As used herein, a "wound" is intended to include both acute and chronic dermal wounds including, for example, surgical incisional wounds, traumatic wounds, cancer extirpations, radiation wounds, venous leg ulcers, diabetic ulcers, and pressure ulcers.
The method comprises providing a composition of monoclonal antibody A1G3 and applying the composition to a wound. Monoclonal antibody A1G3 is a monoclonal antibody (mAb) to CD44, and is commercially available from the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland USA 20852) as the hybridoma deposited as ATCC Accession No. HB-177. This monoclonal has been described and referred to by numerous publications, including Rivadeneira et al . 1995; Patel et al . 1995; Liao et al . 1993; Denning et al . 1990; Telen et al . 1986; Picker et al . 1989; Lobach et al . 1985; Francke et al . 1983; Haynes et al . 1983; and Mokhtar et al . 1984. CD44 is a family of extracellular matrix receptors implicated in modulating the motility of normal and transformed cells (Stamenkovic and Aruffo 1994) . CD44 receptors are alternatively spliced products of one gene suggesting transcriptional control over cell type- specific expression and function of the various isoforms. Some CD44 receptors can interact with the extracellular matrix component hyaluronic acid (HA) , which is enriced in early granulation tissue and has been implicated in modulating the motility of cells into tissues (Laurent and Fraser 1992; Turley 1992). Although all CD44 receptors have a common structural motif for HA binding (Yang et al . 1994), they can be extensively N- and 0- glycosylated (Lesley et al . 1993; Sherman et al . 1994) or contain either heparin sulfate or chondroitin sulfate glycosaminoglycans (GAGs) which can mask HA binding (Jackson et al . 1995). These CD44 proteoglycans, however, can interact with the GAG binding domains of fibronectin and collagen. This phenomenon was originally described in keratinocytes, where heparin sulfate CD44 was identified as a type III collagen receptor and in lymphocytes, where chondroitin sulfate CD44 was shown to bind fibronectin (Jalkanen and Jalkanen 1992) . When expressed as chondroitin sulfate proteoglycan, CD44 has an important influence on the motility and invasion of tumor cells (Faassen et al . 1992; Faassen et al . 1993) and microvessel endothelial cells (Henke et al . 1996) . Although the mechanism (s) by which CD44 molecules modulate the motility of normal and transformed cells is not understood, there is clear evidence indicating their importance .
Numerous monoclonal antibodies to CD44 have been identified. These include monoclonal antibodies A1G3 and A3D8 (Patel et al . 1995). While most of these antibodies inhibit cell migration (see Fig. 2 for data on monoclonal antibody A3D8 and see Henke et al . 1996) , the subject invention results from the finding that the monoclonal antibody A1G3 enhances cell migration (see Fig. 2). Compositions comprising the antibody can therefore be used to enhance wound healing. Such compositions can be provided in the form of a solution of the antibody. The solution has about 5 μg to about 300 μg of monoclonal antibody per milliliter of solution, with about 10 μg to about 100 μg being preferred. A solution having about 30 μg of monoclonal antibody per milliliter of solution provides the most significant enhancement of cell movement .
The A1G3 component is necessary for the subject composition to enhance (e.g. improve, increase) wound healing, although additional components may also be included in the composition. These additional components, such as fibronectin, hyaluronic acid, platelet-derived growth factor, and galactosaminoglycans, may further enhance the beneficial effects of the composition on wound healing.
Enhancement (e.g. improvement, increasing) of wound healing refers to the traditional sense of wound healing where clean closure of the wound occurs. Since naturally occurring wound healing involves the movement of fibroblasts into the wound site, enhancement of wound healing can be assayed in vitro using the model for cell transmigration provided in copending, co-assigned U.S. Serial No. 08/723,789, filed September 30, 1996 (the contents of which are incorporated by reference herein) . Briefly, the model provides a contracted collagen gel containing fibroblasts surrounded by a fibrin gel (see Fig. 1) . When the composition of the subject invention replaces or is added to the fibrin gel, fibroblast movement from the collagen gel into the composition or modified fibrin gel is enhanced compared to movement into the "gold standard" fibrin gel.
The composition for use in the subject invention can be provided in any suitable form. A solution of monoclonal antibody, in, for example, sterile distilled water, sterile phosphate buffered saline, or a cell culture medium, can be used if the composition can be "poured" into and contained in the wound area. A paste or gel containing the monoclonal antibody, or other suitable more viscous form of the composition, can be
"spread" over and/or into a wound area. In either case, a dressing of some form will often cover the applied composition to help prevent contamination and infection of the wound. It should be readily apparent that the composition itself (and each of its components) must be sterile (free of biological and/or chemical contamination) to also prevent contamination and infection of the wound, and biocompatible to prevent adverse tissue reaction.
The composition can be provided in the form of a backbone matrix and the monoclonal antibody. As used herein, a "backbone matrix" refers to natural extracellular matrices as well as biocompatible synthetic polymers. These backbone matrices provide the scaffold of the extracellular matrix and when the monoclonal antibody is mixed with the backbone matrix, cells can move around on the scaffold. As further used herein, an "extracellular matrix" refers to a scaffold in a cell's external environment with which the cell may interact via specific cell surface receptors.
There are numerous examples of backbone matrices suitable for use in the subject invention. These examples include fibrin, hyaluronic acid, polyethylene glycol, poly-L-glycol , and poly-L-lactate . Presently preferred backbone matrices include fibrin and hyaluronic acid. Fibrin is provided, preferably, at about 300 μg to about 300 g, more preferably at 300 μg to 3 mg . The optimal fibrin: fibronectin molar ratio is 1:10. Therefore, if the fibrin is provided as 300 μg, the fibronectin is provided as 30 μg . Hyaluronic acid is another suitable backbone matrix, and is commercially available as a dry (for example, lyophilized) powder. The dry powder can be reconstituted to a hyaluronic acid gel (in accordance with manufacturer's suggestions) for use in the subject invention. Depending upon the viscosity desired, a hyaluronic acid gel having about 5 milligrams to about 50 milligrams of hyaluronic acid per milliliter of reconstituting solution can be used. At 5 - 10 -
milligrams/milliliter, the hyaluronic acid gel will be more liquid, and at 50 milligrams/milliliter the hyaluronic acid gel will become more viscous and less easy to manipulate. Preferably, the hyaluronic acid gel is provided as a gel having about 20 milligrams of dry hyaluronic acid per milliliter of reconstituting solution.'* Suitable reconstituting solutions include, for example, sterile distilled water, sterile phosphate buffered saline (PBS) , or a cell culture medium.
As further used herein, any reference to monoclonal antibody A1G3 is intended to include humanized forms of the antibody which are non-immunogenic .
MATERIALS AND METHODS
Normal human dermal fibroblasts
Primary cultures of human adult dermal fibroblasts, acquired from Marcia Simon (Living Skin Bank, SUNY at Stony Brook) , the ATCC (Bethesda, MD) , or the NIA (Bethesda, MD) , are cultured in Dulbecco ' s modified
Eagle's medium (DMEM, Life Technologies) containing 42 mM sodium bicarbonate and supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal bovine serum (FBS, HyClone, Logan, Utah) , at 37°C and 5% C02/95% air in a humidified atmosphere. The cells are used between passages 4 and 12.
Fibroblast migration assays: transmigration from organotypic collagen gel constructs into fibrin/fibronectin gels or outmigration over protein coated surfaces
Preparation of floating, contracted collagen gels
Fibroblast cultures at 80% confluence are harvested by treatment with 0.05% trypsin/0.01% EDTA. Trypsin is - 11 -
inactivated by addition of soy bean trypsin inhibitor in PBS containing 0.2% BSA. The cells are washed twice with DMEM + 2% BSA and resuspended at a concentration of 1 x 106 cells/ml. The fibroblasts are mixed with neutralized collagen (Vitrogen 100, Celtrix Labs., Santa Clara, CA) , 2% BSA, 30 ng/ml PDGF-BB, 30 μg/ml fibronectin, and concentrated DMEM so that the final concentration of DMEM and sodium bicarbonate is lx. 600 μl of the cell mixture is added to the wells of a 24-well tissue culture plate, which has been precoated with 2% BSA. The collagen is allowed to polymerize at 37°C. The final concentration of collagen is 1.8 mg/ml and each gel contains 6 x 104 cells. After two hours incubation, the gels are gently detached from the plastic surface to allow contraction with the addition of 0.5 ml DMEM + 2% BSA and 30 ng/ml PDGF-BB per well. The gels are incubated overnight at 37°C in 100% humidity, 5% C02 and 95% air. Preparation of three-dimensional transmigration model For preparation of "gold standard" transmigration assays containing a dermal organotypic construct surrounded by a fibrin clot as previously described (Greiling and Clark 1997) , dried fibrin fibril-coated dishes are washed once with PBS and fibroblast-contracted collagen gels are placed on the surface. Fibrinogen, at a final concentration of 300 μg/ml, is mixed with DMEM and 1.0 U/ml thrombin, added to the wells so that the solution is level with the top of the collagen gel, and allowed to clot at room temperature for 30 min. When needed, other supplements such as 30 ng/ml PDGF-BB are added to the mixture. For 3 dimensional transmigration into the composition of A1G3 , the wells are coated overnight at 37°C with fibrin fibrils. The next day a fibroblast -contracted collagen gel is placed on the well and A1G3 in solution, with fibrinogen, fibronectin, and - 12 -
thrombin, with or without 30 ng/ml PDGF-BB, is added so that the solution is level with the top of the collagen gels. All migration assays are quantified after a 24 hour incubation at 37°C in 100% humidity, 5% C02 and 95% air.
Evaluation of cell migration
The number of migrated cells was quantified under a Nikon inverted phase microscope by visually counting identifiable cell nuclei located outside of the contracted collagen gel in the fibrin gel (transmigration assay) . Within a given experiment each condition was run in triplicate and means ± SD calculated. All experiments were repeated at least three times. Statistical differences among conditions can be determined by ANOVA.
EXAMPLE I Assay of Wound Healing
The composition used in the present invention was tested by use of the in vitro model as described in U.S. Patent Application Serial No. 08/723,789, which is hereby incorporated by reference. The basis of the in vitro model is a contracted collagen gel containing fibroblasts which acquire a tissue-like phenotype within the collagen matrix. Surrounding the collagen gel, or dermal equivalent, with a fibrin clot produces a simple inside- outside model of the early cutaneous wound (Fig. 1) . Without an added stimulus, no more than a few of the normal adult human dermal fibroblasts within the collagen gel migrate into the fibrin gel. However, the transmigration of fibroblasts from the collagen gel into the fibrin gel is enhanced by the replacement of the fibrin gel with the composition of the subject invention or by the addition of the composition to the fibrin gel, - 13 -
since the composition facilitates cell movement thereby enhancing wound healing.
EXAMPLE II Using the 3 -dimensional transmigration assay described above, the effectiveness of the monoclonal antibody A1G3 in enhancing cell migration was tested. Referring to Fig. 2, varying concentration of monoclonal antibody A1G3 were tested in the 3 -dimensional transmigration assay. Concentrations from about 5 μg/ml to about 300 μg/ml enhanced cell migration, with concentrations between about 10 μg/ml and about 100 μg/ml (with 30 μg/ml being preferred) providing the most significant enhancement.
Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.
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