Leukaemia inhibitory factor

• US 6,261,548 B1
• Filed: 04/21/1997
• Issued: 07/17/2001
• Est. Priority Date: 04/02/1987
• Status: Expired due to Fees

• Current Assignee: EMD Millipore Corporation (Merck & Co., Inc.)
• Original Assignee: Zenyth Therapeutics Limited (CSL Limited)
• Inventors: Gearing, David Paul, King, Julie Ann, Nice, Edouard Collins, Metcalf, Donald, Nicola, Nicos Anthony, Gough, Nicholas Martin, Hilton, Douglas James, Willson, Tracy Ann, Simpson, Richard John
• Primary Examiner(s): MERTZ, PREMA MARIA
• Application Number:
  US08/839,909
• Time in Patent Office: 1,548 Days
• Field of Search: 530/351, 530/412, 530/413, 530/416, 530/417, 514/2, 514/8, 514/12, 435/69.5, 435/71.1, 435/71.2, 435/172.3, 435/325, 435/252.3, 435/254.11, 435/254.2, 435/320.1, 435/471, 424/85.1
• US Class Current: 424/85.1
• CPC Class Codes: 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
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: C07K 14/5415 Leukaemia inhibitory factor [LIF]
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/23 containing a GST-tag
• CPC Class Codes: C07K 2319/50 containing protease site
• CPC Class Codes: C07K 2319/74 containing a fusion for binding to a cell surface receptor
• CPC Class Codes: C07K 2319/95 containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2740/13043 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00

Abstract

The present invention provides isolated DNA molecules encoding human and murine LIF and methods of producing LIF by the expression of the isolated DNA and suitable host cells. The invention further provides human and murine LIF polypeptides, pharmaceutical compositions containing LIF, and methods of use thereof.

Claims

1. A composition comprising a polypeptide in essentially pure form having leukemia inhibitory factor (LIF) activity, said polypeptide comprising an amino acid sequence set forth in FIGS. 15A or 15B and a pharmaceutically acceptable carrier or diluent.

2. A purified and isolated polypeptide having murine leukemia inhibitory factor (LIF) activity, said polypeptide comprising an amino acid sequence set forth in FIG. 15A or 15B which polypeptide is substantially free of other murine proteins.

3. A transformed host cell comprising a DNA molecule encoding the polypeptide of claim 2 having murine LIF activity.

4. The host cell of claim 3 wherein said host cell is a bacterial or yeast cell.

5. A method for purifying a glycosylated polypeptide having murine leukemia inhibitory factor (LIF) activity, said polypeptide comprising an amino acid sequence set forth in FIGS. 15A or 15B, which comprises:

(a) chromatographing said polypeptide in crude form on an anion exchange column, and eluting said polypeptide by an increasing salt gradient,

(b) chromatographing the product of step (a) on a lectin affinity column, said lectin having an affinity for mannose and eluting said polypeptide with a mannose derivative,

(c) chromatographing the product of step (b) on a cation exchange column and eluting said polypeptide with an increasing salt gradient, and

(d) chromatographing the product of step (c) under HPLC conditions on a reverse phase column and eluting said polypeptide with an increasing gradient of acetonitrile.

6. The method of claim 5 wherein said polypeptide in crude form comprises human bladder carcinoma cell line 5637 (ATCC Accession No. HTB9) conditioned medium.