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

Patent metadata

Field	Value
Publication number	US-2016347831-A1
Application number	US-201415035582-A
Country	US
Kind code	A1
Filing date	Nov 13, 2014
Priority date	Nov 13, 2013
Publication date	Dec 1, 2016
Grant date	—

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

1 - 29 . (canceled)

30 . 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).

31 . The VHH according to claim 30 , 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.

32 . A VHH derivative consisting of a polypeptide comprising the VHH of claim 30 , provided that said VHH comprised in said polypeptide is able to bind the fibrillar form of amyloid β.

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

34 . An isolated polynucleotide encoding the VHH of claim 30 .

35 . A recombinant expression cassette comprising the polynucleotide of claim 34 under the control of a transcriptional promoter allowing the regulation of the transcription of said polynucleotide in a host cell.

36 . A recombinant vector comprising the polynucleotide of claim 34 .

37 . A host cell containing the recombinant expression cassette of claim 35 .

38 . A host cell containing the recombinant vector of claim 36 .

39 . A composition comprising the VHH of claim 30 linked to a substance of interest.

40 . The composition of claim 39 , wherein the substance of interest is selected from a peptide, an enzyme, a nucleic acid, a virus and a chemical entity.

41 . The composition of claim 39 , 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.

42 . The composition of claim 40 , 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.

43 . The composition of claim 39 , 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.

44 . The composition of claim 39 , wherein the substance of interest is a liposome or a polymeric entity.

45 . A non-site specific method for coupling a VHH of claim 30 with a substance of interest, said method comprising a conjugation step of a substance of interest with the VHH.

46 . The non-site specific method of claim 45 , 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.

47 . The non-site specific method of claim 46 , wherein the substance of interest is a compound selected from NMR or MRI contrast agents and metallic radioisotopes.

48 . The non-site specific method of claim 47 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.

49 . The non-site specific method of claim 47 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, 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).

50 . The non-site specific method of claim 48 , 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.

51 . The non-site specific method of claim 49 , 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.

52 . The non-site specific method of claim 48 , 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).

53 . The non-site specific method of claim 49 , 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).

54 . The non-site specific method of claim 48 , wherein the substance of interest is gadolinium (Gd), and the chelating agent is DOTA.

55 . The non-site specific method of claim 49 , wherein the substance of interest is gadolinium (Gd), and the chelating agent is DOTA.

56 . 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.

57 . An in vitro or ex vivo method for diagnosing a disorder mediated by amyloid β deposits, such as Alzheimer's disease and Down's syndrome, in a subject, comprising the steps of:
a) contacting in vitro a biological sample from said subject with the composition of claim 41 , 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.

Assignees

• Hoffmann La Roche
• Pasteur Institut
• Centre Nat Rech Scient
• Commissariat Energie Atomique

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

• 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

• A61P25/04

  Centrally acting analgesics, e.g. opioids · CPC title

• A61P25/08

  Antiepileptics; Anticonvulsants · CPC title

• A61P29/00

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