WO1999042132A1 - Utilisation d'un anticorps monoclonal anti-cd44 pour acclerer la cicatrisation des blessures - Google Patents

Utilisation d'un anticorps monoclonal anti-cd44 pour acclerer la cicatrisation des blessures Download PDF

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Publication number
WO1999042132A1
WO1999042132A1 PCT/US1999/002775 US9902775W WO9942132A1 WO 1999042132 A1 WO1999042132 A1 WO 1999042132A1 US 9902775 W US9902775 W US 9902775W WO 9942132 A1 WO9942132 A1 WO 9942132A1
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WO
WIPO (PCT)
Prior art keywords
monoclonal antibody
wound healing
composition
wound
gel
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PCT/US1999/002775
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English (en)
Inventor
Richard A. Clark
Doris Greiling
Original Assignee
The Research Foundation Of State University Of New York
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by The Research Foundation Of State University Of New York filed Critical The Research Foundation Of State University Of New York
Priority to AU26658/99A priority Critical patent/AU2665899A/en
Publication of WO1999042132A1 publication Critical patent/WO1999042132A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2884—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD44
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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 .
  • 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 -
  • 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.
  • HA hyaluronic acid
  • Ortonne 1996, Borgognoni et al . (1996), and Nakamura et al . (1997) each discuss the usefulness of HA for wound healing.
  • the HA was combined with chondroitin sulfate in one series of experiments.
  • 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.
  • 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.
  • 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.
  • the method includes providing a composition of monoclonal antibody A1G3 , and applying the composition to a wound.
  • Fig. 1 illustrates the in vitro model for assaying cell transmigration from a collagen gel into a fibrin gel
  • Fig. 2 illustrates the effect on cell migration of varying concentrations of monoclonal antibodies A1G3 and
  • 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 .
  • 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).
  • HA extracellular matrix component
  • 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).
  • GAGs chondroitin sulfate glycosaminoglycans
  • 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 ⁇ g to about 300 ⁇ g of monoclonal antibody per milliliter of solution, with about 10 ⁇ g to about 100 ⁇ g being preferred.
  • a solution having about 30 ⁇ g 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
  • Enhancement 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.
  • 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
  • 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.
  • 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.
  • an "extracellular matrix” refers to a scaffold in a cell's external environment with which the cell may interact via specific cell surface receptors.
  • 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 ⁇ g to about 300 g, more preferably at 300 ⁇ g to 3 mg .
  • the optimal fibrin: fibronectin molar ratio is 1:10. Therefore, if the fibrin is provided as 300 ⁇ g, the fibronectin is provided as 30 ⁇ g .
  • 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.
  • a hyaluronic acid gel having about 5 milligrams to about 50 milligrams of hyaluronic acid per milliliter of reconstituting solution can be used.
  • 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.
  • 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.
  • any reference to monoclonal antibody A1G3 is intended to include humanized forms of the antibody which are non-immunogenic .
  • DMEM Eagle's medium
  • FBS HyClone, Logan, Utah
  • Fibroblast migration assays transmigration from organotypic collagen gel constructs into fibrin/fibronectin gels or outmigration over protein coated surfaces
  • Fibroblast cultures at 80% confluence are harvested by treatment with 0.05% trypsin/0.01% EDTA. Trypsin is - 11 -
  • fibroblasts are mixed with neutralized collagen (Vitrogen 100, Celtrix Labs., Santa Clara, CA) , 2% BSA, 30 ng/ml PDGF-BB, 30 ⁇ g/ml fibronectin, and concentrated DMEM so that the final concentration of DMEM and sodium bicarbonate is lx. 600 ⁇ l 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 10 4 cells.
  • 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% C0 2 and 95% air.
  • 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% C0 2 and 95% air.
  • 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.
  • 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.
  • 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 -
  • 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 ⁇ g/ml to about 300 ⁇ g/ml enhanced cell migration, with concentrations between about 10 ⁇ g/ml and about 100 ⁇ g/ml (with 30 ⁇ g/ml being preferred) providing the most significant enhancement.
  • Lamme E.N., et al . , J Histochemistry and Cytochemistry 44 (11) : 1311-1322 (1996) .

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Dermatology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention porte sur un procédé visant à accélérer la cicatrisation des blessures. Ce procédé consiste à produite une composition d'anticorps monoclonal A1G3 et à appliquer cette composition sur une blessure.
PCT/US1999/002775 1998-02-18 1999-02-10 Utilisation d'un anticorps monoclonal anti-cd44 pour acclerer la cicatrisation des blessures WO1999042132A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU26658/99A AU2665899A (en) 1998-02-18 1999-02-10 Use of anti-cd44 monoclonal antibody to enhance wound healing

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US2565698A 1998-02-18 1998-02-18
US09/025,656 1998-02-18

Publications (1)

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WO1999042132A1 true WO1999042132A1 (fr) 1999-08-26

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996028549A2 (fr) * 1995-03-10 1996-09-19 Incyte Pharmaceuticals, Inc. Recepteur d'hyaluronan exprimes dans les cellules endotheliales de la veine ombilicale de l'humain

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996028549A2 (fr) * 1995-03-10 1996-09-19 Incyte Pharmaceuticals, Inc. Recepteur d'hyaluronan exprimes dans les cellules endotheliales de la veine ombilicale de l'humain

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ALAISH S M, ET AL.: "BIOLOGY OF FETAL WOUND HEALING: HYALURONATE RECEPTOR EXPRESSION IN FETAL FIBROBLASTS", JOURNAL OF PEDIATRIC SURGERY., W. B. SAUNDERS COMPANY., US, vol. 29, no. 08, 1 August 1994 (1994-08-01), US, pages 1040 - 1043, XP002917579, ISSN: 0022-3468, DOI: 10.1016/0022-3468(94)90275-5 *
LIAO H-X, ET AL.: "REGULATION OF HUMAN CD44H AND CD44E ISOFORM BINDING TO HYALURONAN BY PHORBOL MYRISTATE ACETATE AND ANTI-CD44 MONOCLONAL AND POLYCLONAL ANTIBODIES", THE JOURNAL OF IMMUNOLOGY, THE AMERICAN ASSOCIATION OF IMMUNOLOGISTS, US, vol. 151, no. 11, 1 December 1993 (1993-12-01), US, pages 6490 - 6499, XP002917580, ISSN: 0022-1767 *

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