Camelid single-domain antibody directed against amyloid beta and methods for producing conjugates thereof

Patent metadata

Field	Value
Publication number	US-9738712-B2
Application number	US-201415035582-A
Country	US
Kind code	B2
Filing date	Nov 13, 2014
Priority date	Nov 13, 2013
Publication date	Aug 22, 2017
Grant date	Aug 22, 2017

Abstract

The present invention relates to variable domain of a camelid heavy-chain antibodies directed to amyloid β and conjugates thereof. The present invention also relates to the use of these antibody conjugates for treating or diagnosing disorders mediated by amyloid β deposits.

First claim

The invention claimed is:

1. An isolated variable domain of a camelid heavy-chain antibody (VHH) directed against the fibrillar form of amyloid β, characterized in that its amino acid sequence comprises, from the N-terminus to the C-terminus, the amino acid sequence SEQ ID NO:1 (corresponding to the CDR1), the amino acid sequence SEQ ID NO:2 (corresponding to the CDR2) and the amino acid sequence SEQ ID NO:3 (corresponding to the CDR3).

2. The VHH according to claim 1 , characterized in that it comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7.

3. A VHH derivative consisting of a polypeptide comprising the VHH of claim 1 , provided that said VHH comprised in said polypeptide is able to bind the fibrillar form of amyloid β.

4. A VHH derivative characterized in that it has the amino acid sequence SEQ ID NO:8.

5. An isolated polynucleotide encoding the VHH of claim 1 .

6. A recombinant expression cassette comprising the polynucleotide of claim 5 under the control of a transcriptional promoter allowing the regulation of the transcription of said polynucleotide in a host cell.

7. A recombinant vector comprising the polynucleotide of claim 5 .

8. A host cell containing the recombinant expression cassette of claim 6 .

9. A host cell containing the recombinant vector of claim 7 .

10. A composition comprising the VHH of claim 1 linked to a substance of interest.

11. The composition of claim 10 , wherein the substance of interest is selected from a peptide, an enzyme, a nucleic acid, a virus and a chemical entity.

12. The composition of claim 11 , wherein the substance of interest is a NMR or MRI contrast agent selected from paramagnetic agents gadolinium (Gd), dysprosium (Dy) and manganese (Mn), and the superparamagnetic agents based on iron oxide or iron platinium, and the X-nuclei 18 F, 13 C, 23 Na, 17 O, and 15 N.

13. The composition of claim 10 , wherein the substance of interest is selected from the group consisting of an enzyme, a fluorophore, a NMR or MRI contrast agent, a radioisotope, or a nanoparticle.

14. The composition of claim 10 , wherein the substance of interest is selected from the group consisting of an analgesic compound, an anti-inflammatory compound, an antidepressant compound, a cytotoxic compound, an anticonvulsant compound or an anti-neurodegenerative compound.

15. The composition of claim 10 , wherein the substance of interest is a liposome or a polymeric entity.

16. A pharmaceutical composition comprising the composition of claim 10 and a pharmaceutically acceptable carrier.

17. A kit for brain imaging, or for diagnosing or monitoring a disorder mediated by amyloid β deposits comprising a VHH of claim 1 and a diagnostic agent.

18. An in vitro or ex vivo method for diagnosing a disorder mediated by amyloid β deposits in a subject, comprising the steps of:
a) contacting in vitro a biological sample from said subject with the composition of claim 13 , and
b) determining the presence or the absence of amyloid β deposits in said biological sample,
the presence of said amyloid β deposits indicating that said subject has a disorder mediated by amyloid β deposits.

19. An in vitro or ex vivo method for monitoring the progression or regression of a disorder mediated by amyloid β deposits in a subject, comprising the steps of:
a) contacting in vitro a biological sample from said subject with the composition of claim 13 ,
b) determining the amount of fibrillar form of amyloid β in said biological sample, and
c) comparing the amount determined in step (b) with the amount of fibrillar form of amyloid β previously obtained for said subject,
wherein a significant increase in amount of fibrillar form of amyloid β constitutes a marker of the progression of said disorder mediated by amyloid β deposits and a significant decrease of fibrillar form of amyloid β constitutes a marker of the regression of said disorder mediated by amyloid β deposits.

20. A method for in vivo imaging amyloid β deposits in a subject comprising the steps of
a) administrating a detectable quantity of the composition of claim 13 to a subject, and,
b) detecting the substance of interest in said subject by an imaging method.

21. An in vitro or ex vivo method for detecting the presence or the absence of amyloid β deposits in a subject, comprising the steps of:
a) contacting in vitro a biological sample from said subject with the composition of claim 13 , and
b) determining the presence or the absence of amyloid β deposits in said biological sample.

22. A non-site specific method for coupling a VHH of claim 1 with a substance of interest, said method comprising a conjugation step of a substance of interest with the VHH.

23. The non-site specific method of claim 22 , wherein the substance of interest is a compound selected from the group consisting of a peptide, an enzyme, a nucleic acid, a virus, a fluorophore, a NMR or MRI contrast agent, a chemical entity, a radioisotope and a nanoparticle.

24. The non-site specific method of claim 23 , wherein the substance of interest is a compound selected from NMR or MRI contrast agents and metallic radioisotopes.

25. The non-site specific method of claim 24 comprising the following steps:
(i) the conjugation of a chelating agent activated in the form of an ester or an anhydride with lysine residues of a VHH directed against the fibrillar form of amyloid β, characterized in that its amino acid sequence comprises, from the N-terminus to the C-terminus, the amino acid sequence SEQ ID NO:1 (corresponding to the CDR1), the amino acid sequence SEQ ID NO:2 (corresponding to the CDR2) and the amino acid sequence SEQ ID NO:3 (corresponding to the CDR3), and
(ii) the chelation of the ligand of step (i) with the substance of interest.

26. The non-site specific method of claim 25 , wherein the substance of interest is a NMR or MRI contrast agent selected from the paramagnetic agents gadolinium (Gd), dysprosium (Dy) and manganese (Mn), and the superparamagnetic agents based on iron oxide or iron platinium, and the X-nuclei 18 F, 13 C, 23 Na, 17 O, and 15 N.

27. The non-site specific method of claim 25 , wherein the chelating agent is selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA), diethylene triamine penta-acetic acid (DTPA), 1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetylamide (DO3A), nitrilotriacetic acid (NTA), D-penicillamine (Pen), 2,3-dimercaptosuccinic acid (DMSA), 2,3-dimercapto-1-propanesulfonic acid (DMPS), 2,3-dimercaptopropanol (BAL), triethylenetetramine (Trien), the ammonium tetrathiomolybdate (TTM) anion, ethylenediaminetetraacetic acid (EDTA), 2-(p-isothiocyanatobenzyl)-6-methyl-diethylenetriaminepentaacetic acid (IB4M) or hydroxypyridinone (HOPO).

28. The non-site specific method of claim 25 , wherein the substance of interest is gadolinium (Gd), and the chelating agent is DOTA.

29. The non-site specific method of claim 24 comprising the following steps:
(i′) the chelation of a substance of interest with a chelating agent activated in the form of an ester or an anhydride and
(ii′) the conjugation of the pre-chelated substance of interest of step (i′) with lysine residues of a VHH directed against the fibrillar form of amyloid β, characterized in that its amino acid sequence comprises,

Assignees

• Hoffmann La Roche
• Pasteur Institut
• Centre Nat Rech Scient
• Commissariat Energie Atomique
• F Hoffman-La Roche Ag
• Centre Nat De La Rech Scient (Crns)

Inventors

• Lafaye Pierre
• Bay Sylvie
• Delatour Benoit
• Dhenain Marc
• Duyckaerts Charles
• Li Tengfei
• Vandesquille Matthias
• Czech Christian
• Grueninger Fiona

Classifications

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P29/00

  Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] · CPC title

• A61P25/28

  for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia · CPC title

• A61P25/24

  Antidepressants · CPC title

• A61P25/08

  Antiepileptics; Anticonvulsants · CPC title