Variable domains of camelid heavy-chain antibodies directed against glial fibrillary acidic proteins

• US 8,460,888 B2
• Filed: 07/07/2009
• Issued: 06/11/2013
• Est. Priority Date: 07/10/2008
• Status: Active Grant

• Current Assignee: Centre National De La Recherche Scientifique, Institut Pasteur
• Original Assignee: Institut Pasteur
• Inventors: Lafaye, Pierre, Rougeon, Francois, Bourgeois, Jean-Pierre
• Primary Examiner(s): Kemmerer, Elizabeth C
• Assistant Examiner(s): BALLARD, KIMBERLY
• Application Number:
  US13/002,910
• Publication Number:
  US 20110250211A1
• Time in Patent Office: 1,435 Days
• Field of Search: None
• US Class Current: 435/7.21
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 2039/54 characterised by the route of administration
• CPC Class Codes: A61K 2039/543 intranasal
• CPC Class Codes: A61K 2039/545 characterised by the dose, timing or administration schedule
• CPC Class Codes: A61K 51/1018 against material from animals or humans
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/04 Centrally acting analgesics, e.g. opioids
• CPC Class Codes: A61P 25/08 Antiepileptics; Anticonvulsants
• CPC Class Codes: A61P 25/24 Antidepressants
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 16/3053 Skin, nerves, brain
• CPC Class Codes: C07K 2317/22 from camelids, e.g. camel, llama or dromedary
• CPC Class Codes: C07K 2317/569 Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• CPC Class Codes: C07K 2317/92 Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• CPC Class Codes: C12N 5/0622 Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells

Abstract

The present invention relates to the use of variable domains of camelid heavy-chain antibodies (VHH domains) directed against an intracellular target and having an isoelectric point of at least 8.5, for targeting said intracellular target or for the preparation of a peptide vector. Particularly, it concerns VHH domains directed against a glial fibrillary acidic protein and uses thereof for preparing therapeutic or diagnostic agents.

Claims

1. An isolated variable domain of a camelid heavy-chain antibody (VHH domain) directed against a glial fibrillary acidic protein (GFAP), wherein said VHH domain comprises an amino acid sequence selected from the group consisting of the consensus amino acid sequence SEQ ID NO:

1, SEQ ID NO;

3, SEQ ID NO;

6, SEQ ID NO;

9 and SEQ ID NO;

12.

2. A VHH domain according to claim 1, comprising a homodimer of SEQ ID NO:

1.

3. A VHH domain according to claim 1, wherein it is obtainable by the method comprising the steps of:

(a) immunizing a camelid with a GFAP, (b) isolating peripheral lymphocytes of the immunized camelid, obtaining the total RNA and synthesizing the corresponding cDNAs, (c) constructing a library of cDNA fragments encoding VHH domains, (d) transcribing the VHH domain-encoding cDNAs obtained in step (c) to mRNA using PCR, converting the mRNA to ribosome display format, and selecting the VHH domain by ribosome display, and (e) expressing the VHH domain in a vector.

4. An isolated polypeptide, comprising a VHH domain according to claim 1.

5. The polypeptide according to claim 4, wherein it contains at its C-terminus the amino acid sequence LEHHHHHH (SEQ ID NO:

15).

6. An isolated antibody or fragment thereof, comprising a VHH domain according to claim 1.

7. A therapeutic or diagnostic agent, comprising a VHH domain according to claim 1, linked, directly or indirectly, covalently or non-covalently to a substance of interest.

8. A therapeutic or diagnostic agent according to claim 7, wherein said substance of interest is a therapeutic or diagnostic compound selected from the group consisting of a peptide, an enzyme, a virus, a fluorophore, a heavy metal, a chemical entity and a radioisotope.

9. A therapeutic or diagnostic agent according to claim 8, wherein said substance of interest is a liposome or a polymeric entity comprising a therapeutic or a diagnostic compound selected from the group consisting of a peptide, an enzyme, a virus, a fluorophore, a heavy metal, a chemical entity and a radioisotope.

10. A therapeutic or diagnostic agent according to claim 8, wherein said therapeutic compound is selected from the group consisting of an anticancer compound, an analgesic compound, an anti-inflammatory compound, an antidepressant compound, an anticonvulsant compound and an anti-neurodegenerative compound.

11. A therapeutic or diagnostic agent, comprising a polypeptide according to claim 4, linked, directly or indirectly, covalently or non-covalently to a substance of interest.

12. A therapeutic or diagnostic agent, comprising an antibody according to claim 6, linked, directly or indirectly, covalently or non-covalently to a substance of interest.

13. A kit comprising a VHH domain according to claim 1.

14. A kit comprising a polypeptide according to claim 4.

15. A kit comprising an antibody according to claim 6.

16. A kit comprising a diagnostic agent according to claim 7.

17. A pharmaceutical composition, comprising a therapeutic agent according to claim 7 and a pharmaceutically acceptable carrier.

18. A method of indicating the presence of a disorder mediated by glial fibrillary acidic proteins (GFAPs) in a subject comprising the steps of:

a) contacting in vitro or ex vivo an appropriate biological sample with a VHH domain according to claim 1, b) determining the amount of GFAPs in said biological sample, and c) comparing the amount determined in step (b) with a standard, a difference in amount indicating the presence of said disorder.

19. The method of claim 18, wherein said disorder is Alzheimer'"'"'s disease, Alexander disease, gliosis, or astrocytoma.

20. A method of indicating the presence of a disorder mediated by glial fibrillary acidic proteins (GFAPs) in a subject comprising the steps of:

a) contacting in vitro or ex vivo an appropriate biological sample with a polypeptide according to claim 4, b) determining the amount of GFAPs in said biological sample, and c) comparing the amount determined in step (b) with a standard, a difference in amount indicating the presence of said disorder.

21. The method of claim 20, wherein said polypeptide contains at its C-terminus the amino acid sequence LEHHHHHH (SEQ ID NO:

15).

22. The method of claim 20, wherein said disorder is Alzheimer'"'"'s disease, Alexander disease, gliosis, or astrocytoma.