WO1989010133A1 - Inhibiteurs de cellules a tiges - Google Patents

Inhibiteurs de cellules a tiges Download PDF

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Publication number
WO1989010133A1
WO1989010133A1 PCT/GB1989/000396 GB8900396W WO8910133A1 WO 1989010133 A1 WO1989010133 A1 WO 1989010133A1 GB 8900396 W GB8900396 W GB 8900396W WO 8910133 A1 WO8910133 A1 WO 8910133A1
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WO
WIPO (PCT)
Prior art keywords
inhibitor
cells
sepharose
stem cells
resin
Prior art date
Application number
PCT/GB1989/000396
Other languages
English (en)
Inventor
Ian Bernard Pragnell
Original Assignee
Cancer Research Campaign Technology Limited
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.)
Filing date
Publication date
Application filed by Cancer Research Campaign Technology Limited filed Critical Cancer Research Campaign Technology Limited
Publication of WO1989010133A1 publication Critical patent/WO1989010133A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • THIS INVENTION relates to stem cell inhibitors and is particularly concerned with improvements in the management of cancer chemotherapy.
  • medullary aplasia represents a limiting factor for the clinical use of cytotoxic drugs which are active in cycling cells during chemotherapy of cancer. It has been recognised for many years that existing methods of chemotherapy could be improved if it were possible to protect the haemopoietic stem cells during treatment with the cytotoxic drug but, in spite of extensive research in this area, no suitably specific inhibitory agent from a readily amenable source has previously been discovered. One of the reasons for this lack of progress is believed to be the difficulty in developing a suitable ii vitro assay to monitor stem cell regulation.
  • the present invention provides a haemopoietic stem cell inhibitor characterised by the following properties: 1.
  • the inhibitor activity is sensitive to degradation with trypsin and the inhibitor is therefore proteinaceous.
  • the inhibitor when partially purified by treatment on an anion-exchanger shows a molecular weight range 45-60 Kd on a molecular weight analysis resin.
  • the inhibitor when purified to a single band on polyacrylamide gel electrophoresis, under reducing conditions, shows a molecular weight range of 8-10 Kd and a slightly higher molecular weight range under non-reducing conditions.
  • the inhibitor is heat-stable as follows:
  • the inhibitor binds to anion-exchangers and can be eluted with a salt gradient at between 0.26 and 0.28 molar NaCl. 6.
  • the inhibitor binds to a Heparin Sepharose affinity chromatography resin and can be eluted from the resin with one molar sodium chloride buffer.
  • the inhibitor binds avidly to Blue Sepharose affinity chromatography resin and can be eluted from the resin with 5M magnesium chloride.
  • L929 cell conditioned medium L929 CM, a source of the growth factor CSF-1
  • AF1-19T CM 10% AF1-19T cell conditioned medium
  • the inhibitor of the present invention is obtainable from several macrophage cell lines. Interest centres initially upon the various known macrophage cell lines of bone marrow origin, typically murine bone marrow as this represents a major source of such macrophage cell lines. Our screening of a population of such cell lines has identified at least two known cell lines capable of producing our inhibitor. In the Examples illustrating this invention, we describe the isolation from one such known macrophage cell line.
  • the inhibitor of the invention can be produced quite simply from the inhibitor-producing macrophage cell line by cultivating the cell line in an appropriate growth medium under conventional conditions, e.g. 37 C, the use of aerobic conditions with 5% v/v carbon dioxide in air provides an ideal growth environment, until the cell concentration is approximately 10 per ml. At this stage, the still growing cells are separated from the growth medium, e.g. by centrifugation or preferably membrane filtration and the inhibitor can then be recovered from the supernatant. In order to demonstrate inhibitor activity, it is desirable that the supernatant first be concentrated, e.g. using membrane dialysis to give a concentration of about 20-fold. The inhibitor can then be isolated from the concentrated supernatant by chromatographic procedures.
  • a first step of purification may include passing the concentrated supernatant over an anion-exchange resin and eluting a fraction using a 0.25-0.30M NaCl solution.
  • a second step of purification may include passing the product from the first step over a
  • Sepharose-Heparin column and eluting a fraction using NaCl solution of molarity at least 1M.
  • a third step of purification may include passing the product from the second step over Sepharose-Blue and eluting a fragment using MgCl 2 solution of molarity at least 3M. This elution gives a product that shows a single band on polyacrylamide gel electrophoresis under reducing conditions.
  • the inhibitor or i munogenically active fragment thereof can also be used as an immunogen to raise antibodies that will recognise part or all of the inhibitor, by immunising a host animal and recovering from the host animal antibodies or antibody producing cells.
  • Such antibodies can be prepared e.g. in rabbits where polyclonal antibodies can be recovered from the serum of the rabbit or can be used as immunogens in mice for the production of monoclonal antibodies by conventional techniques.
  • the present invention also extends to DNA sequences encoding the inhibitor of the invention.
  • DNA is of interest in the production of the inhibitor by recombinant DNA techniques.
  • Such techniques can involve two different approaches. Both approaches require, as a first step, the production of a gene library from the messenger RNA of the macrophage cell line that naturally produces the inhibitor. This involves the production of a complementary DNA expressible in a bacterial or other host cells.
  • a first approach involves the production and use of DNA probes. Limited sequence analysis of the inhibitor will permit the synthesis of oligonucleotides that can be used to probe the DNA library to identify those cDN 's in the library that hybridise with the probe and so permits the identification of the messenger RNA encoding the whole inhibitor.
  • a gene library of human origin can be probed to identify and isolate a DNA encoding an inhibitor of human origin which is expressable, using techniques now well-known, in a host cell system.
  • the inhibitor of the invention In clinical application, it is desirable to target the inhibitor of the invention to the blood-forming tissue. This targetting can be achieved by injecting the inhibitor, normally by infusion or bolus intravenous administration and the present invention extends to pharmaceutical compositions containing the inhibitor and appropriate diluents or carriers suitable for such parenteral administration.
  • the interest in the inhibitor of this invention is not restricted to stem cells specific to the haemopoietic system but extends to other stem cells e.g. epithelial stem cells, making the inhibitor of interest not only in relation to alleviating the side effects of cytotoxic drug therapy on bone marrow cells but also in the treatment of solid tumours.
  • the inhibitor is also of interest in the treatment of leukaemia where leukaemic bone marrow cells are treated vitro or vivo, with inhibitor so that proliferation of normal stem cells is prevented and the proliferating leukaemic cells can then be treated with a cytotoxic agent.
  • J774.2 cells were originally growing in a modified Eagles medium supplemented with 10% foetal calf serum. The cells were subcultured into the medium: Dulbeccos x 10 50 ml
  • the 774.2 cells were subcultured into the above medium with added foetal calf serum (5%) for one week, with a further subculture at three days in the same medium. Cells were further subcultured into medium plus 2.5% foetal calf serum for a week, then into 1% foetal calf serum for a further week until finally all serum was removed. At this stage the cells were subcultured every two days, allowed to grow to a concentration of 8 x 10 /ml and diluted to 2 x 10 /ml at subculture. Cells were then transferred to spinner culture and acclimatised to growth in suspension, subcultering every two days as described above. This cell line designated J774.2(S) and capable of growth in serum free suspension culture was then used for inhibitor production.
  • Undiluted medium from 8 x 10 cells is separated from the cells using a Millipore Pellicon-Casette system with a 0.45u microporous membrane.
  • the medium minus cells is then concentrated inthe same apparatus using a 10K cut-off membrane to a final volume of about 400 ml. This concentrate is then processed as described below for biochemical purification.
  • Stage 1 The 400 ml concentrate is desalted on a G-25 HR16/50 (Pharmacia) in 0.02M Tris-HCl pH 7.6, applied to the anion-exchange Mono Q HRlO/10 column (Pharmacia, FPLC system) and finally eluted with a salt gradient (0 to 1M NaCl) in the same buffer.
  • the active fractions of inhibitor elute between 0.26 and 0.28M NaCl.
  • Stage 2 The impure inhibitor from Stage 1 is applied directly to a Sepharose-Heparin (Pharmacia) column, HRlO/10.
  • the inhibitor binds to the resin and the majority of proteins (more than 90%) can be washed off the resin using 0.1M NaCl-0.02M Tris HC1, pH 7.6.
  • the inhibitor is thn eluted from the resin in 1M NaCl-0.02M Tris HC1, pH 7.6.
  • Stage 3 The partially purified inhibitor from Stage 2 is applied directly to a Blue Sepharose (Pharmacia) column HR5/5 without desalting. The column is then washed with the buffer used to elute the inhibitor in Stage 2, to remove other protein. The purified inhibitor, which binds strongly, is eluted with 5M MgCl 2 -0.02M Tris-HCl pH 7.6
  • the purified inhibitor can be seen as a single band of molecular weight 8-10Kd on polyacrylamide gels under reducing conditions. When the inhibitor is applied to polyacrylamide gel electrophoresis under non-reducing conditions, it can be seen to be of slightly higher molecular weight.
  • CFU-A ultipotential stem cells
  • Bone marrow cells were incubated in paired tubes containing 5 x 10 cells in 1 ml Fischer's medium supplemented with 20% horse serum. The inhibitor or alpha-MEM was added to each tube and Fischer's medium was added to control tubes. The mixtures were incubated at

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Un polypeptide, capable d'entrer en interaction avec des cellules à tiges hémopoïétiques pour leur offrir une protection contre les médicaments cytotoxiques spécifiques au cycle cellulaire utilisés en chimiothérapie du cancer, est isolé à partir de cellules macrophages provenant de la moelle osseuse.
PCT/GB1989/000396 1988-04-21 1989-04-21 Inhibiteurs de cellules a tiges WO1989010133A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB8809419.8 1988-04-21
GB888809419A GB8809419D0 (en) 1988-04-21 1988-04-21 Stem cell inhibitors

Publications (1)

Publication Number Publication Date
WO1989010133A1 true WO1989010133A1 (fr) 1989-11-02

Family

ID=10635573

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1989/000396 WO1989010133A1 (fr) 1988-04-21 1989-04-21 Inhibiteurs de cellules a tiges

Country Status (4)

Country Link
EP (1) EP0412105A1 (fr)
JP (1) JPH03505575A (fr)
GB (1) GB8809419D0 (fr)
WO (1) WO1989010133A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994028917A1 (fr) * 1993-06-15 1994-12-22 British Biotech Pharmaceuticals Limited Formulations pharmaceutiques
WO1994028916A1 (fr) * 1993-06-15 1994-12-22 British Biotech Pharmaceuticals Limited Liberation et mobilisation de cellules hematopoïetiques
WO1999036441A2 (fr) * 1998-01-17 1999-07-22 Telistar International Inhibition de cellules

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOLOGICAL ABSTRACTS, REVIEWS-REPORTS-MEETINGS, Vol. 33, 1987, No. 61402, A. JANOWSKA-WIECZOREK et al., "Protection of Human Hematopoietic Stem Cells from Tubercidin Toxicity by Inhibitors of Nucleoside Transport"; & PROC. AM. ASSOC. CANCER RES. ANNU. MEET., 1987, Vol. 28, No. 0, page 409. *
BIOLOGICAL ABSTRACTS, REVIEWS-REPORTS-MEETINGS, Vol. 35, 1988, No. 106933, E.G. WRIGHT et al., "The Effect of Stem Cell Proliferation Regulators Demonstrated With an in-vitro Assay"; & EXPERIMENTAL HEMATOLOGY, 1988, Vol. 16, No. 6, page 473. *
CHEMICAL ABSTRACTS, Vol. 109, 1988, (Columbus, Ohio, US), I.B. PRAGNELL et al., "The Effect of Stem Cell Proliferation Regulators Demonstrated With an in Vitro Assay", page 344, Abstract 89048m; & BLOOD, 1988, 72(1), 196-201. *
CHEMICAL ABSTRACTS, Vol. 96, 1982, (Columbus, Ohio, US), M. GUIGON et al., "Protection of Mice Against Lethal Doses of 1beta-D-arabinofuranosylcytosine by Pluripotent Stem Cell Inhibitors", page 36, Abstract No. 115633h; & CANCER RES., 1982, 42(2), 638-41. *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994028917A1 (fr) * 1993-06-15 1994-12-22 British Biotech Pharmaceuticals Limited Formulations pharmaceutiques
WO1994028916A1 (fr) * 1993-06-15 1994-12-22 British Biotech Pharmaceuticals Limited Liberation et mobilisation de cellules hematopoïetiques
US5925568A (en) * 1993-06-15 1999-07-20 British Biotech Pharmaceuticals Limited Release and mobilization of haematopoietic cells
WO1999036441A2 (fr) * 1998-01-17 1999-07-22 Telistar International Inhibition de cellules
WO1999036441A3 (fr) * 1998-01-17 1999-10-28 Telistar International Inhibition de cellules

Also Published As

Publication number Publication date
GB8809419D0 (en) 1988-05-25
EP0412105A1 (fr) 1991-02-13
JPH03505575A (ja) 1991-12-05

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