# US 5,352,668 A · Product and method for improving keratinocyte adhesion to the dermis

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- Number: US 5,352,668 A
- Document ID: US5352668A
- Status: Granted
- Kind: A
- Application number: 07/936,850
- Filed: 1992-08-28
- Granted: 1994-10-04
- Earliest claimed: 1991-03-26
- Assignees: The State of Oregon Acting by and through the State Board of Higher Education on Behalf of Oregon Health Sciences University
- Inventors: Robnert E. Burgeson; Gregory P. Lunstrum; Patricia Rousselle; Douglas R. Keene; M. Peter Marinkovich
- Web page: https://patentgrep.com/patent/US5352668A

## CPC

- C12N 5/0629: {Keratinocytes; Whole skin}
- A61K 35/36: Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells (islets of Langerhans A61K35/39)
- A61K 38/39: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
- A61L 27/227: {Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24}
- A61L 27/60: Materials for use in artificial skin
- A61P 17/00: Drugs for dermatological disorders
- C07K 14/47: from mammals
- C07K 14/78: Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
- C07K 16/18: against material from animals or humans
- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- A61K 35/12: Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells (vaccines or medicinal preparations containing antigens or antibodies A61K39/00)
- C12N 2501/58: Adhesion molecules, e.g. ICAM, VCAM, CD18 (ligand), CD11 (ligand), CD49 (ligand)
- C12N 2533/50: Proteins
- C12N 2533/52: Fibronectin; Laminin
- Y10S 530/842: Skin; hair; nails; sebaceous glands; cerumen
- Y10S 530/851: Placenta; amniotic fluid
- Y10S 623/915: Method or apparatus for preparing biological material
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61L: METHODS 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 (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

A purified protein kalinin is disclosed that provides adhesion between epidermal keratinocytes and the underlying dermis. Purified kalinin localizes to the anchoring filaments of basement membranes or human subepithelial skin, trachea, esophagus, cornea and amnion when such areas are probed with BM165 monoclonal antibody after localization. The protein has a molecular weight of approximately 400-460 kDa and exists in a cell-associated form (about 460 kDa) and two medium-associated forms (about 440 and 400 kDa, respectively). The cell-associated form comprises a 200-, a 155- and a 140-kDa subunit, all normally held together by disulflde bonds. The cell-associated form is subjected to extracellular processing to produce the two medium-associated forms, wherein, in the 440-kDa form, the 200-kDa subunit has been processed to a 165-kDa subunit and, in the 400-kDa form, the 155-kDa subunit has been processed to a 105-KDa subunit. The BM165 epitope is located on the 165-kDa subunit. Kalinin has a rotary-shadow image revealing an asymmetric rod 107-nm long having two globules at a first end and a single globule at an opposing end. A method is also disclosed for improving adhesion of transplanted keratinocytes to an underlying substrate, such as the human dermis, by optimizing production of kalinin from cultured keratinocytes, or by providing an exogenous source of kalinin between the keratinocytes and substrate.

## Claims

### Claim 1

A method of improving adhesion of transplanted keratinocytes to a substrate, comprising the step of providing an amount of purified kalinin between the keratinocytes and the substrate wherein the kalinin is of molecular weight about 400 to about 460 kDa and wherein the kalinin provides adhesion between human dermis and epidermis.

### Claim 2

A method of improving adhesion of transplanted keratinocytes to a substrate, comprising:
monitoring the production of a protein kalinin by cell cultures of keratinocytes, wherein said kalinin is of molecular weight about 400 to about 460 kDa and provides adhesion between human dermis and epidermis; and
transplanting keratinocytes while they are actively producing kalinin.

### Claim 3 (depends on 1)

The method of claim 1 wherein the substrate is the surface of a burn wound.

### Claim 4 (depends on 2)

The method of claim 2 wherein the substrate is the surface of a burn wound.

### Claim 5 (depends on 4)

The method of claim 4 wherein the substrate is human dermis or subcutaneous tissue.

### Claim 6 (depends on 3)

The method of claim 3 wherein the substrate is human dermis or subcutaneous tissue.

### Claim 7

A method of improving adhesion of transplanted keratinocytes to a substrate, comprising the step of providing a sufficient amount of kalinin between the keratinocytes and the substrate, wherein the kalinin is a protein of molecular weight about 400 to about 460 kDa having the characteristic of providing adhesion between human dermis and epidermis and wherein the provided kalinin is exogenous to the transplanted keratinocytes and the substrate.

### Claim 8 (depends on 7)

The method of claim 7 wherein the substrate is the surface of a burn wound.

### Claim 9 (depends on 7)

The method of claim 7 wherein the substrate is human dermis or subcutaneous tissue.