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WO1996019501A1 - Modulateurs des fonctions des cellules osseuses et leurs utilisations - Google Patents

Modulateurs des fonctions des cellules osseuses et leurs utilisations Download PDF

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Publication number
WO1996019501A1
WO1996019501A1 PCT/US1995/016826 US9516826W WO9619501A1 WO 1996019501 A1 WO1996019501 A1 WO 1996019501A1 US 9516826 W US9516826 W US 9516826W WO 9619501 A1 WO9619501 A1 WO 9619501A1
Authority
WO
WIPO (PCT)
Prior art keywords
factor
bone
pancreatic
cells
antibody
Prior art date
Application number
PCT/US1995/016826
Other languages
English (en)
Inventor
Toshiyuki Yoneda
Elzbieta Izbicka
Gregory R. Mundy
Original Assignee
Osteosa Inc.
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 Osteosa Inc. filed Critical Osteosa Inc.
Priority to AU46075/96A priority Critical patent/AU4607596A/en
Publication of WO1996019501A1 publication Critical patent/WO1996019501A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6448—Elastases, e.g. pancreatic elastase (3.4.21.36); leukocyte elastase (3.4.31.37)
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/51—Bone morphogenetic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • FIGURE 21 shows the effect of human recombinant elastase III B on rat osteoclast pit formation.
  • the invention concerns monoclonal and polyclonal antibodies to these factors as well as uses of these antibodies therapeutically and diagnostically.
  • the invention also contains an assay for this peptide.
  • the invention also concerns synthetic antagonists which neutralize the biological activity of these factors and can be used therapeutically. These antagonists were synthesized based on our knowledge of amino acid sequence of these factors.
  • EXAMPLE 6 PREPARATION OF MONOCLONAL ANTIBODIES TO PX Monoclonal antibodies (MAb) to PX were made using an in vitro immunization technique (Van Ness et al. (1983) Nature 301: 425-427).
  • Spleen cells of 8 to 12 week-old female Balb/C mice were immunized with purified PX in the presence of 100 ⁇ g N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP, Sigma), 125 ⁇ g lipopolysaccharide (LPS, Difco) and 500 ml culture supernatants of concanavalin A (50 ⁇ g/ml)-stimulated spleen cells (5 x 10 6 /ml) in 5 ml IMDM supplemented with 20% FBS in 6-well plates for 4 days. The immunized spleen cells were then hybridized with mouse myeloma FO cells (kindly provided by Dr.
  • the receptor is isolated and purified, and traditional techniques for isolation and purification may be employed. Two approaches to receptor identification may be employed. Accordingly, the receptor may be identified by affinity labeling. A chemical cross-linking agent which covalently crosslinks labeled factor to the receptor will be used. The factor covalently bound to an inert matrix will be utilized to construct an affinity column. The receptor will be solubilized by non-ionic detergents. The receptor will then be eluted from the affinity column in an acidic urea solution, dialyzed and then pH increased to lead to renaturation of the receptor.
  • affinity labeling A chemical cross-linking agent which covalently crosslinks labeled factor to the receptor will be used. The factor covalently bound to an inert matrix will be utilized to construct an affinity column. The receptor will be solubilized by non-ionic detergents. The receptor will then be eluted from the affinity column in an acidic urea solution, dialyzed and then pH increased to lead to renaturation of the receptor.
  • the cDNA library from MG-63 cells may be used, which cells express the receptor, and for which the receptor has been characterized.
  • the cDNA library is subfractionated into 500 pools of 2x10 4 clones each and then DNA from each pool is transfected into 1.5 x 10 6 cells by electroporation. This technique detects single clones from pools of 2x10 4 clones (compared with 1x10 3 clones using previous methods).
  • the cells are cultured for 48 hours on glass slides and incubated with a labeled peptide. Following the 48 hour incubation period, the slides are fixed and dipped in photographic emulsion and the positive pools identified. Individual clones expressing the PX receptor are identified.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

On a identifié un facteur dérivé du pancréas qui diminue le calcium sérique, qui inhibe la résorption osseuse et qui également stimule la prolifération des cellules MG-63, qui sont des cellules du type ostéoblastes. Ce facteur a été purifié jusqu'à obtenir un produit homogène, en partant de pancréas de porcs, et on a déterminé la séquence définitive de ses acides aminés (SEQ ID NO:1). Ce facteur et l'élastase BIII (qui est une élastase pancréatique humaine) sont homologues. Des expériences avec l'élastase pancréatique humaine IIIB de recombinaison, ainsi qu'avec des anticorps anti-élastase pancréatique humaine IIIB démontrent que les effets biologiques du facteur PX natif sont également obtenus par l'élastase pancréatique humaine IIIB de recombinaison, aussi bien in vitro que in vivo. De nombreuses applications diagnostiques et thérapeutiques sont envisagées, en particulier l'évaluation préliminaire de médicaments et la réalisation de coffrets de réactifs pour diagnostiquer et surveiller les changements dans les populations de cellules osseuses. L'invention concerne également des compositions et des procédés pour traiter des résorptions ou des accrétions osseuses indésirables.
PCT/US1995/016826 1994-12-20 1995-12-19 Modulateurs des fonctions des cellules osseuses et leurs utilisations WO1996019501A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU46075/96A AU4607596A (en) 1994-12-20 1995-12-19 Modulators of bone cell function and uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US36309294A 1994-12-20 1994-12-20
US08/363,092 1994-12-20

Publications (1)

Publication Number Publication Date
WO1996019501A1 true WO1996019501A1 (fr) 1996-06-27

Family

ID=23428765

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1995/016826 WO1996019501A1 (fr) 1994-12-20 1995-12-19 Modulateurs des fonctions des cellules osseuses et leurs utilisations

Country Status (2)

Country Link
AU (1) AU4607596A (fr)
WO (1) WO1996019501A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003053167A1 (fr) 2001-12-11 2003-07-03 Societe Des Produits Nestle S.A. Composition destinee a favoriser la croissance osseuse et l'entretien de la sante osseuse
US6642216B2 (en) * 1996-12-13 2003-11-04 Zymogenetics Corporation Isoprenoid pathway inhibitors for stimulating bone growth

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE MEDLINE ON DIALOG, US NATIONAL LIBRARY OF MEDICINE, (Bethesda, MD, USA), No. 91122277, T. YONEDA et al., "Porcine Pancreas Extract Decreases Blood-Ionized Calcium in Mice and Inhibits Osteoclast Formation and Bone Resorption in Culture"; & FEBS LETTERS, 278(2), 28 January 1991, pages 171-174. *
DATABASE MEDLINE ON DIALOG, US NATIONAL LIBRARY OF MEDICINE, (Bethesda, MD, USA), No. 94215195, T. YONEDA et al., "Extracts of Porcine Pancreas Prevent Progression of Hypercalcemia and Cachexia and Prolong Survival in Nude Mice Bearing a Human Squamous Carcinoma"; & CANCER RESEARCH, 54(9), 01 May 1994, pages 2509-2513. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6642216B2 (en) * 1996-12-13 2003-11-04 Zymogenetics Corporation Isoprenoid pathway inhibitors for stimulating bone growth
US7101907B2 (en) 1996-12-13 2006-09-05 Zymogenetics Corporation Topical administration of statins for treatment of bone disorders
WO2003053167A1 (fr) 2001-12-11 2003-07-03 Societe Des Produits Nestle S.A. Composition destinee a favoriser la croissance osseuse et l'entretien de la sante osseuse
EP2135517A1 (fr) 2001-12-11 2009-12-23 Société des Produits Nestlé S.A. Composition pour la promotion de la croissance osseuse et la conservation de la santé osseuse
EP2263480A1 (fr) 2001-12-11 2010-12-22 Société des Produits Nestlé S.A. Composition pour la promotion de la croissance des os et le maintien de la santé des os, comprenant de la menthe ou des extraits de Mentha
EP2272382A1 (fr) 2001-12-11 2011-01-12 Société des Produits Nestlé S.A. Composition pour la promotion de la croissance des os et le maintien de la santé des os, comprenant des fèves de soja ou des extraits de fève de soja
EP2286675A1 (fr) 2001-12-11 2011-02-23 Société des Produits Nestlé S.A. Composition pour la promotion de la croissance osseuse et la conservation de la santé osseuse

Also Published As

Publication number Publication date
AU4607596A (en) 1996-07-10

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