# US 5,707,805 A · Assay for detecting keratinocyte growth factor (KGF) and its activity

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- Number: US 5,707,805 A
- Document ID: US5707805A
- Status: Granted
- Kind: A
- Application number: 08/455,641
- Filed: 1995-05-31
- Granted: 1998-01-13
- Earliest claimed: 1989-01-31
- Assignees: The United States of America as represented by the Department of Health and Human Services
- Inventors: Jeffrey S. Rubin; Paul W. Finch; Stuart A. Aaronson
- Web page: https://patentgrep.com/patent/US5707805A

## CPC

- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- A61P 17/00: Drugs for dermatological disorders
- A61P 17/02: for treating wounds, ulcers, burns, scars, keloids, or the like
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 14/50: Fibroblast growth factor [FGF]
- C07K 14/501: {acidic FGF [aFGF]}
- C07K 16/22: against growth factors {; against growth regulators}
- C12N 15/70: Vectors or expression systems specially adapted for E. coli
- C12N 15/79: Vectors or expression systems specially adapted for eukaryotic hosts
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2319/00: Fusion polypeptide
- C07K 2319/02: containing a signal sequence
- C07K 2319/75: containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
- C12Q 2600/158: Expression markers
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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)
- 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)

## Abstract

Discoveries are disclosed that show particular aspects of recombinant DNA technology can be used successfully to produce hitherto unknown human keratinocyte growth factor (KGF) protein free of other polypeptides. These proteins can be produced in various functional forms from spontaneously secreting cells or from DNA segments introduced into cells. These forms variously enable biochemical and functional studies of this novel protein as well as production of antibodies. Means are described for determining the level of expression of genes for the KGF protein, for example, by measuring mRNA levels in cells or by measuring antigen secreted in extracellular or body fluids.

## Claims

### Claim 1

A method for assaying Keratinocyte Growth Factor (KGF) activity comprising:
(a) in a first reaction, determining the level of .sup.3 H thymidine incorporation into DNA after adding an aliquot of a test sample comprising isolated KGF to a culture of keratinocytes grown to confluence and maintained in a serum-free medium;
(b) in a second reaction, determining the level of .sup.3 H thymidine incorporation into DNA after adding another aliquot of the test sample of step (a) to a culture of fibroblasts grown to confluence and maintained in a serum free medium; and
(c) assessing KGF activity by comparing the results of the first reaction with the results of the second reaction, wherein a fold stimulation of .sup.3 H thymidine incorporation into the DNA in the first reaction that is at least about 50 times greater than in the second reaction indicates KGF activity.

### Claim 2

An assay for detecting human Keratinocyte Growth Factor (KGF) comprising contacting a test sample suspected of containing KGF with an antibody which specifically binds KGF and determining the amount of polypeptide antibody complex formed.

### Claim 3

A method for detecting Keratinocyte Growth Factor (KGF) in a test sample comprising:
(a) determining the level of .sup.3 H thymidine incorporation into DNA of keratinocytes after adding, in the presence of a KGF-neutralizing antibody, a test sample suspected of containing KGF to a culture of keratinocytes grown to confluence and maintained in a serum-free medium;
(b) performing step (a) in the absence of a KGF-neutralizing antibody;
wherein a decrease in .sup.3 H thymidine incorporation in (a) relative to (b) indicates the presence of KGF in the test sample.