EP0642354A1 - PROCEDES PERMETTANT DE DETECTER ET D'ISOLER L'uPA-R ET D'INHIBER LA LIAISON DE L'uPA A L'uPA-R - Google Patents

PROCEDES PERMETTANT DE DETECTER ET D'ISOLER L'uPA-R ET D'INHIBER LA LIAISON DE L'uPA A L'uPA-R

Info

Publication number
EP0642354A1
EP0642354A1 EP92922134A EP92922134A EP0642354A1 EP 0642354 A1 EP0642354 A1 EP 0642354A1 EP 92922134 A EP92922134 A EP 92922134A EP 92922134 A EP92922134 A EP 92922134A EP 0642354 A1 EP0642354 A1 EP 0642354A1
Authority
EP
European Patent Office
Prior art keywords
upa
antibody
vol
cells
binding
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP92922134A
Other languages
German (de)
English (en)
Other versions
EP0642354A4 (fr
Inventor
Ikuko F. Mizukami
Robert F. Todd, Iii
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Michigan System
University of Michigan Ann Arbor
Original Assignee
University of Michigan System
University of Michigan Ann Arbor
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 University of Michigan System, University of Michigan Ann Arbor filed Critical University of Michigan System
Publication of EP0642354A1 publication Critical patent/EP0642354A1/fr
Publication of EP0642354A4 publication Critical patent/EP0642354A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to methods for detecting and isolating uPA-R utilizing monoclonal antibodies.
  • the present invention also relates to methods for inhibiting the binding of uPA to uPA-R.
  • Uro inase-type plasminogen activator is an enzyme responsible for the generation of the protease, plasmin.
  • uPA is thought to be involved in the generation of extracellular proteolytic activity in physiological and pathological processes such as cell migration, tissue remodeling, and tumor invasion and metastasis (Adv. Cancer Res.. vol. 44, pp. 139266 (1985).
  • the evidence that uPA plays a specific role in these processes includes the observation that uPA binds to a specific cellular receptor (uPA-R) on a wide variety of cell types of normal and malignant origin (J. Cell Biol.. vol. 100, pp. 86-96 (1985) ; Pro. Natl. Acad. Sci. U.S.A.. vol.
  • Plasminogen is also able to bind to many cell types (J. Biol. Chem. , vol. 260, pp. 4303-4311 (1985)) and it has been demonstrated that the concomitant binding of uPA and plasminogen results in the generation of plasmin activity on the cell surface (J. Biol. Chem.. vol. 264, pp. 2185-2188 (1989); and J. Cell Biol.. vol. 108, pp. 1987-1995 (1989)) and also that this system constitutes a mechanism for accelerating the activation of the pro-enzyme form of uPA (J. Biol. Chem. , vol. 264, pp. 2185-2188 (1989)) .
  • Mo3 is barely detectable on freshly isolated monocytes, but is prominent on the surface of monocytes activated by culture in media containing soluble inflammatory factors such as LCP, MDP, and cytokines including TNF, M-CSF, GM-CSF, and IL-3.
  • soluble inflammatory factors such as LCP, MDP, and cytokines including TNF, M-CSF, GM-CSF, and IL-3.
  • Mo3 expression appears to be more constitutive: normal tonsillar epithelium, hepatocytes, and dermal collagen were all positive for Mo3 staining.
  • uPA receptors have been described to be associated with adhesion (J. Cell Biol.. vol. 104, p. 1085 (1987)) and cytoskeletal proteins such as vinculin (J. Cell Biol. , vol. 106,, vol. 1241 (1988)), alpha-actinin and actin in contact sites on metastatic tumor cells (Thromb. Res. Suppl. , vol. 10, p. 55 (1990)) .
  • one object of the present invention is to provide a method for isolating uPA-R.
  • Figure 1 illustrates the competitive blocking of uPA-FITC by murine monoclonal and rabbit polyclonal antibodies specific for Mo3. Acid washed, PMA-stimulated U-937 cells were preincubated in buffer containing the indicated dilutions of murine monoclonal antibodies: IgG2a anti-Mo3f (—•—) , IgM anti-Mo3e (— ⁇ —) , or isotype-identical negative control antibodies, IgG2a 5B7 (—o—) , or IgM anti-CD14 (—D—) ; or polyclonal rabbit anti-Mo3 (— ⁇ —) or normal rabbit serum (— ⁇ —) , or no antibody (X) .
  • murine monoclonal antibodies IgG2a anti-Mo3f (—•—) , IgM anti-Mo3e (— ⁇ —) , or isotype-identical negative control antibodies, IgG2a 5B7 (—o—) , or Ig
  • uPA-FITC was added, and the cell suspension was incubated for an additional 60 in at 4°C. After washing and fixation, the cells were subjected to flow cyto etric analysis in which 5000 cells per determination were assessed for uPA-FITC binding. The mean channel fluorescence of each stained population is indicated on the y-axis.
  • Mo3 is a cell surface protein that is anchored to the plasma membrane by a GPI linkage (J. Immunol., vol. 144, p. 1841 (1990)). Supporting this conclusion is the fact that in addition to being susceptible to cleavage from the cell surface by PI-PLC, Mo3 surface expression is deficient in a patient with paroxysmal nocturnal hemoglobinuria (J. Immunol.. vol. 144, p. 1841 (1990)), a disease that is characterized by the absence of GPI-linked determinants on leukocytes (J. Exp. Med.. vol. 166, p. 1011 (1987)).
  • Mo3 is the same as uPA-R.
  • a computer search of the NBRF database suggested that Mo3 is identical to the human receptor for urokinase plasminogen activator (uPA-R) , and this was confirmed by comparison of the complete sequence for uPA-R (EMBO J.. vol. 9, pp. 467-474 (1990)) with that of Mo3.
  • the present invention relates to the detection of uPA-R by observing the binding of monoclonal anti-Mo3 antibodies to uPA-R.
  • Suitable monoclonal anti-Mo3 antibodies include anti-Mo3a-f.
  • the production of anti-Mo3f is described in J. Immunol. , vol. 144, pp. 1841-1848 (1990)
  • the generation of anti-Mo3a-e is described in J. Immunol.. vol 137, pp. 448-455 (1986) and Blood, vol. 59, p. 775 -1-
  • the present detection method may be carried out on free or membrane bound uPA-R.
  • the uPA-R may be detected by the use of an ELISA.
  • a cell suspected of expressing uPA-R may be incubated with one of the present antibodies. After washing, the cells may be incubated with another antibody, which binds to the present antibody, and which is conjugated with an enzyme suitable for ELISA, such as horse radish peroxidase, HRP. After another washing, the cell may be incubated with a substrate for the enzyme and the activity of the enzyme may be determined. The activity of the enzyme will be related to the amount of uPA-R on the surface of the cell.
  • an ELISA may also be used to detect soluble uPA-R in body fluids, (e.g., plasma, urine, and inflammatory exudates) as a marker of inflammation and/or cancer.
  • the cells may be incubated with one of the present antibodies which has been modified to carry a label.
  • the antibody may be treated with periodate to generate carbonyl groups on the sugar groups of the antibody.
  • a suitable label may be covalently linked to the antibody via a difunctional linking group. Suitable labels and linking groups are disclosed in Hau ⁇ land. Handbook of Flourescent Probes and Research Chemicals. Molecular Probes, Inc., Eugene, Oregan (1989) and Pierce Immunotechnology Catalog and Handbook. Pierce, Rockford, IL (1990) .
  • the present invention also relates to the isolation of uPA-R, by use of an antibody specific for Mo3.
  • uPA-R may be isolated by immunoprecipitation with an antibody specific for Mo3. It is preferred to use anti-Mo3f for the immunoprecipitation.
  • the antibody may be bound on a suitable support, e.g., proteinA-Sephorose® (Pharmacia-LKB Biotechnology, Inc., Piscataway, NJ) as described in J. Immunol. , vol. 147, pp.
  • the bound antibody may be used to isolate uPA-R obtained from lysed cells or from the supernatants of uPA-R bearing cells exposed to phosphatidylinositol-specific phospholipase.
  • the uPA-R may be labelled before lysing of the cells by either treatment with the N-hydroxysuccinimide ester of biotin or incubation of the cell with a nutrient containing a radio label, e.g. 35 s-methionine or 3 H-mannose.
  • the present invention also relates to a method for inhibiting the binding of uPA to uPA-R by treating uPA-R with an antibody specific for Mo3» It is preferred that the antibody be anti-Mo3e or anti-Mo3f. It is especially preferred that the antibody be anti-Mo3f.
  • the inhibition of the binding of uPA to uPA-R may be carried out in vitro (extracorporeal) or in vivo. Thus, the inhibition of the binding uPA to uPA-R may comprise one aspect of an in vitro assay for the presence of uPA-R.
  • the present method of inhibiting the binding of uPA to uPA-R may also be carried out in the body.
  • administration of an antibody the in vivo binding of uPA to uPA-R may be inhibited.
  • the present method encompasses preventing or reducing tissue damage by inflammatory phagocytic cells (macrophages and neutrophils) and metastatic invasion by tumor cells.
  • the antibody is administered systematically (e.g. by intravenous injection) .
  • inflammatory processes in which the method may be applied include the following: rheumatoid arthritis, immune vasculitis, glomerulonephritis, inflammatory bowel disease, and adult respiratory distress syndrome.
  • the method may also be applied to inhibit tumor cell invasion (metastases invasion) by various human tumors expressing uPA-R, e.g., malignant melanoma, breast cancer, and sarcoma.
  • the actual dosage and regimen of antibody administration will of course depend on the health of the patient and the condition being treated. However, good results may be achieved with dosages of 0.01 to 5 mg/kg of body weight, preferably 0.1 to 1 mg/kg of body weight.
  • This schedule of administration may be carried out for a few (1 to 10) days.
  • the antibody may be administered early in the course of inflammation, and in the case of cancer, at the time of surgical resection to prevent metastasis formation from surgically-dislodged tumor cells.
  • the antibody may be administered in various pharmaceutical compositions in the form of an injectable solution or suspension.
  • the present invention provides a method for inhibiting the binding of uPA to uPA-R.
  • antibodies specific for Mo3 are capable of inhibiting the binding of uPA to uPA-R.
  • Figure 1 shows the results of the competitive blocking of uPA-FITC (fluorescein isothiocyanate) with a variety of antibodies specific for Mo3.
  • the results in Figure 1 clearly demonstrate that the antibodies specific for Mo3 (anti-Mo3f, (—•—) ; anti-Mo3e (— ⁇ —) ; and polyclonal rabbit anti-Mo3 (—A—) are capable of blocking the binding of uPA-FITC to uPA-R.
  • uPA High molecular weight urokinase
  • uPA-FITC FITC-conjugated uPA

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Rheumatology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Pain & Pain Management (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

Des anticorps dirigés contre la protéine Mo3 sont susceptibles de se lier au récepteur de l'activateur de plasminogène du type urokinase (uPA-R), ou d'inhiber la liaison de l'activateur de plasminogène du type urokinase (uPA) à l'uPA-R.
EP92922134A 1991-11-18 1992-10-27 PROCEDES PERMETTANT DE DETECTER ET D'ISOLER L'uPA-R ET D'INHIBER LA LIAISON DE L'uPA A L'uPA-R. Withdrawn EP0642354A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US79320891A 1991-11-18 1991-11-18
US793208 1991-11-18
PCT/US1992/008977 WO1993009808A1 (fr) 1991-11-18 1992-10-27 PROCEDES PERMETTANT DE DETECTER ET D'ISOLER L'uPA-R ET D'INHIBER LA LIAISON DE L'uPA A L'uPA-R

Publications (2)

Publication Number Publication Date
EP0642354A1 true EP0642354A1 (fr) 1995-03-15
EP0642354A4 EP0642354A4 (fr) 1995-07-12

Family

ID=25159386

Family Applications (1)

Application Number Title Priority Date Filing Date
EP92922134A Withdrawn EP0642354A4 (fr) 1991-11-18 1992-10-27 PROCEDES PERMETTANT DE DETECTER ET D'ISOLER L'uPA-R ET D'INHIBER LA LIAISON DE L'uPA A L'uPA-R.

Country Status (4)

Country Link
EP (1) EP0642354A4 (fr)
JP (1) JPH07501524A (fr)
CA (1) CA2123874A1 (fr)
WO (1) WO1993009808A1 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994028014A2 (fr) * 1993-05-28 1994-12-08 Chiron Corporation Peptides inhibiteurs de l'activite des recepteurs d'urokinase
JP2852192B2 (ja) * 1994-07-08 1999-01-27 カンセアフォースクニングスフォンデン・アフ・１９８９（フォンデン・チル・フレメ・アフ・エクスペリメンテル・カンセアフォースクニング） ｕＰＡＲのドメイン２＋３のｕＰＡ結合部位および抗体
US6077508A (en) * 1998-03-23 2000-06-20 American Diagnostica Inc. Urokinase plasminogen activator receptor as a target for diagnosis of metastases
EP0982036A1 (fr) * 1998-08-28 2000-03-01 Wilex Biotechnology GmbH Modulation de l'adhesion cellulaire a médiation de la Beta-2-integrin
TW200813091A (en) * 2006-04-10 2008-03-16 Amgen Fremont Inc Targeted binding agents directed to uPAR and uses thereof
PL2117571T3 (pl) 2006-12-08 2017-08-31 Monopar Therapeutics Inc. Epitop receptora aktywatora plazminogenu typu urokinazy

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF IMMUNOLOGY, vol.148, no.11, 1992, USA pages 3636 - 3642 H.Y. MIN ET AL. 'cDNA for Mo3, amonocyte activation antigen, encodes the human receptor for urokinase plasminogen activator.' *
See also references of WO9309808A1 *

Also Published As

Publication number Publication date
CA2123874A1 (fr) 1993-05-27
JPH07501524A (ja) 1995-02-16
WO1993009808A1 (fr) 1993-05-27
EP0642354A4 (fr) 1995-07-12

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