Diagnostic methods using neurite growth regulatory factors

• US 5,250,414 A
• Filed: 06/24/1991
• Issued: 10/05/1993
• Est. Priority Date: 11/04/1988
• Status: Expired due to Fees

• Current Assignee: Universite Zurich
• Original Assignee: Erziehungsdirektion Of The Canton Zurich
• Inventors: Schwab, Martin E., Caroni, Pierenrico W., Paganetti, Paolo A.
• Primary Examiner(s): Hill, Jr., Robert J.
• Assistant Examiner(s): GUEST CERMAK, SHELLY
• Application Number:
  US07/719,692
• Time in Patent Office: 834 Days
• Field of Search: 530/350, 514/2, 514/21, 435/7.72, 435/7.23, 436/64, 436/813
• US Class Current: 435/7.72
• 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: C07K 14/475 Growth factors; Growth regulators
• CPC Class Codes: C07K 14/71 for growth factors; for growth regulators
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C07K 5/1016 and aromatic or cycloaliphatic
• CPC Class Codes: C12N 9/6489 Metalloendopeptidases (3.4.24)
• CPC Class Codes: G01N 33/57484 involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
• CPC Class Codes: G01N 33/74 involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
• CPC Class Codes: Y10S 436/813 Cancer

Abstract

The proteins of the present invention include central nervous system myelin associated proteins and metalloproteases associated with glioblastoma cells and other malignant tumors which can metastasize to the brain. The CNS myelin associated proteins inhibit neurite outgrowth in nerve cells and neuroblastoma cells, and can also inhibit fibroblast spreading. Such inhibitory proteins include a 35,000 dalton and a 250,000 dalton molecular weight protein. The CNS myelin associated inhibitory proteins may be used in the treatment of malignant tumors. Antibodies to the CNS myelin associated proteins can be used in the diagnosis and therapies of nerve damage. Monoclonal antibody IN-1 may be used to promote regeneration of nerve fibers over long distances in spinal cord lesions. The metalloproteases of the invention have value in diagnosis of malignancies and the treatment of nerve damage and degenerative disorders of the nervous system. Inhibitors of the metalloproteases in combination with the CNS myelin associated inhibitory proteins can be used in the treatment of malignant tumors. Methods of determining malignant potential of a cell by measuring metalloprotease activity are provided.

Claims

1. A method for diagnosing a malignancy invasive in central nervous system white matter in a patient in vitro comprising detecting the presence in a sample of a plasma membrane-associated metalloprotease characterized by the following properties:

(a) a metalloendoprotease activity which cleaves under suitable conditions the peptide benzyloxycarbonyl-Phe-Ala-Phe-Tyr, which activity is maximally active at a pH in the range of 5.5 to 7.0 and is substantially lost in 1% (w/v) CHAPS; and

(b) the ability to neutralize the inhibitory substrate property of central nervous system myelin of a higher vertebrate, in which the neutralization is detected by observing the ability of the central nervous system myelin in the presence of the metalloprotease to support neurite outgrowth or fibroblast spreading in vitro;

said sample containing cells of the patient suspected of being malignant;

in which the presence of the metalloprotease indicates the presence of the malignancy.

2. The method according to claim 1 in which the malignancy is a glioblastoma.

3. A method of determining the malignant potential of a cell suspected of being invasive in the central nervous system comprising measuring in vitro a plasma membrane-associated metallodependent degradative activity of the cell;

in which the metallodependent degradative activity neutralizes under suitable conditions the inhibitory substrate property of central nervous system myelin of a higher vertebrate, in which the neutralization is detected by observing the ability of the central nervous system myelin in the presence of the metallodependent degradative activity to support neurite outgrowth or fibroblast spreading in vitro; and

in which the amount of metallodependent degradative activity of the cell indicates the malignant potential of the cell.

4. The method according to claim 3 in which the metallodependent degradative activity is measured by an in vitro peptide substrate assay.

5. The method according to claim 4 in which the assay is carried out by determining the concentration of peptide substrate necessary to achieve a defined level of inhibition of spreading by the cell on central nervous system myelin.

6. The method according to claim 5 in which the peptide substrate is benzyloxycarbonyl-Phe-Ala-Phe-Tyr.

14. The method according to claim 3, 7 or 4 in which the cell is a glial cell.

15. The method according to claim 8, 5 or 9 in which the cell is a glial cell.

7. A method of determining the malignant potential of a cell suspected of being invasive in the central nervous system comprising measuring in vitro a plasma membrane-associated metalloendoproteolytic activity of the cell, which activity:

(a) cleaves under suitable conditions the peptide benzyloxycarbonyl-Phe-Ala-Phe-Tyr;

(b) is maximally active at a pH in the range of 5.5 to 7.0; and

(c) is substantially lost in 1% (w/v) CHAPS;

in which the amount of metalloendoproteolytic activity of the cell indicates the malignant potential of the cell.

8. The method according to claim 7 in which the metalloendoproteolytic activity is measured by an in vitro peptide substrate assay.

9. The method according to claim 8 in which the assay is carried out by determining the concentration of peptide substrate necessary to achieve a defined level of inhibition of spreading by the cell on central nervous system myelin.

10. The method according to claim 9 in which the peptide substrate is benzyloxycarbonyl-Phe-Ala-Phe-Tyr.

16. The method according to claim 6, 10 or 11 in which the cell is a glial cell.

11. A method of determining the malignant potential of a cell suspected of being invasive in the central nervous system comprising measuring in vitro the amount of a metalloprotease inhibitor necessary to achieve a defined level of inhibition of spreading by the cell on central nervous system myelin, in which the amount of metalloprotease inhibitor indicates the malignant potential of the cell.

12. The method according to claim 11 in which the metalloprotease inhibitor is a peptide substrate of the metalloprotease.

17. The method according to claim 12 or 13 in which the cell is a glial cell.

13. A method of determining the malignant potential of a cell suspected of being invasive in the central nervous system comprising measuring in vitro the amount of benzyloxycarbonyl-Phe-Ala-Phe-Tyr necessary to achieve a defined level of inhibition of spreading by the cell on central nervous system myelin, in which the amount of benzyloxycarbonyl-Phe-Ala-Phe-Tyr indicates the malignant potential of the cell.