# US 5,703,206 A · Macrophage inflammatory protein 2 (MIP-2)

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- Number: US 5,703,206 A
- Document ID: US5703206A
- Status: Granted
- Kind: A
- Application number: 08/285,498
- Filed: 1994-08-03
- Granted: 1997-12-30
- Earliest claimed: 1981-09-08
- Inventors: Stephen D. Wolpe; Anthony Cerami; Barbara Sherry
- Web page: https://patentgrep.com/patent/US5703206A

## CPC

- G01N 33/6863: {Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors}
- C07K 14/522: {Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4, KC}
- C07K 14/523: {Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2}
- C07K 16/241: {Tumor Necrosis Factors}
- 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)
- C12N 2501/21: Chemokines, e.g. MIP-1, MIP-2, RANTES, MCP, PF-4
- C12N 2501/22: Colony stimulating factors (G-CSF, GM-CSF)
- G01N 2333/52: Assays involving cytokines
- G01N 2400/40: Glycosaminoglycans, i.e. GAG or mucopolysaccharides, e.g. chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate, and related sulfated polysaccharides
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- 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)
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

An inflammatory cytokine is disclosed which has been isolated from cells that have been incubated with a stimulator material. The inflammatory cytokine comprises a protein that is capable of binding to heparin, inducing localized inflammation characterized by polymorphonuclear cell infiltration when administered subcutaneously and having potent in vitro chemotactic activity while inducing little or no in vitro chemokinesis in polymorphonuclear cells, while lacking the ability to suppress the activity of the anabolic enzyme lipoprotein lipase, cause the cytotoxicity of cachectin/TNF-sensitive cells, stimulate the blastogenesis of endotoxin-resistant C3H/HeJ thymocytes, or induce the production of cachectin/TNF by primary thioglycollate-elicited mouse macrophage cells. A particular inflammatory cytokine has been isolated and its cDNA has been sequenced. The sequence predicts a protein cDNA of 73 amino acids in length and a molecular weight of 7,851. Diagnostic and therapeutic utilities are proposed, and testing procedures, materials in kit form, recombinant materials and procedures, and pharmaceutical compositions are likewise set forth.

## Claims

### Claim 1

An inflammatory cytokine in purified form which elutes from a heparin affinity column in a 0-2M NaCl gradient at 0.75M NaCl, is cationic under physiological conditions, and is characterized by inducing localized inflammation characterized by polymorphonuclear cell infiltration when administered subcutaneously and having potent in vitro chemotactic cell activity while inducing little or no in vitro chemokinesis in polymorphonuclear cells, while lacking the ability to suppress the activity of the anabolic enzyme lipoprotein lipase, cause the cytotoxicity of cachectin/TNF-sensitive cells, stimulate the blastogenesis of endotoxin-resistant C3H/HeJ thymocytes, or induce the production of cachectin/TNF by primary thioglycollate-elicited mouse macrophage cells, which cytokine has as apparent molecular mass of approximately 6000 Daltons by SDS-PAGE, and fractionates from a gel filtration column with an apparent molecular mass of approximately 10,000 Daltons.

### Claim 2 (depends on 1)

The inflammatory cytokine of claim 1 wherein said cytokine is incapable of inducing fever in rabbits or inducing superoxide formation or respiratory burst in human neutrophils in vitro.

### Claim 3 (depends on 1)

The inflammatory cytokine of claim 1 wherein said cytokine is derivable from animal macrophage cells that may have been incubated with a stimulator material such as might accompany an invasive stimulus.

### Claim 4 (depends on 1)

The inflammatory cytokine of claim 1 comprising an amino acid sequence set forth in FIG. 7.

### Claim 5 (depends on 1, 4)

The inflammatory cytokine of either of claims 1 or 4 labeled with a detectable label.

### Claim 6 (depends on 5)

The inflammatory cytokine of claim 5 wherein the label is selected from enzymes, chemicals which fluoresce and radioactive elements.

### Claim 7

A composition comprising mature macrophage inflammatory protein 2 (mMIP-2) substantially free of other polypeptides.

### Claim 8

A polypeptide comprising an amino acid sequence of mMIP-2 set forth in FIG. 7.

### Claim 9 (depends on 1)

The inflammatory cytokine of claim 1 which is murine MIP-2 having the amino acid sequence set forth in FIG. 7.

### Claim 10 (depends on 1)

The inflammatory cytokine of claim 1 which is human.

### Claim 11

A method for preparing an inflammatory cytokine comprising:
A. gathering a sample of cells from a mammal;
B. incubating a portion of said cells with a stimulator material associated with an invasive event for a mammal;
C. inducing said cells to produce said inflammatory cytokine; and
D. isolating said inflammatory cytokine from a supernatant harvested form the mass of said cells, which inflammatory cytokine is isolated on the basis of eluting from a heparin affinity column in a 0-2M NaCl gradient at 0.7M NaCl having an apparent molecular mass of approximately 6000 Daltons by SDS-PAGE, and fractionates from a gel filtration column with an apparent molecular mass of approximately 10,000 Daltons being cationic under physiological conditions, inducing localized inflammation characterized by polymorphonuclear cell infiltration when administered subcutaneously and having potent in vitro chemotactic activity while inducing little or no in vitro chemokinesis in polymorphonuclear cells, while lacking the ability to suppress the activity of the anabolic enzyme lipoprotein lipase, cause the cytotoxicity of cachectin/TNF-sensitive cells, stimulate the blastogenesis of endotoxin-resistant C3H/HeJ thymocytes, or induce the production of cachectin/TNF by primary thioglycollate-elicited mouse macrophage cells.

### Claim 12 (depends on 11)

The method of claim 11 wherein said stimulator material comprises endotoxin.