Oligopeptide and methods for producing conjugates thereof

Patent metadata

Field	Value
Publication number	US-2016355575-A1
Application number	US-201415035602-A
Country	US
Kind code	A1
Filing date	Nov 13, 2014
Priority date	Nov 13, 2013
Publication date	Dec 8, 2016
Grant date	—

Abstract

The present invention relates to an oligopeptide and conjugates thereof. The present invention also relates to the use of these oligopeptide conjugates for treating or diagnosing disorders mediated by amyloid β deposits. Finally, the present invention also relates to a coupling method for obtaining oligopeptide coupled with a substance of interest (functional conjugates).

First claim

1 . An oligopeptide of formula P-C-Z or Z-C-P, wherein:
P is a 8 to 800 amino acid peptide having no reduced cystein residue, C is a cystein residue, Z represents a 1-10 amino acid spacer, wherein the amino acid residues of Z are identical or different and wherein Z does not contain a cystein residue, characterized in that said cystein residue C is linked to a substance of interest through a maleimido compound of formula (I) bearing said substance of interest:

wherein:
B, B′ 1 , B′ 2 , and B″, identical or different, are independently single bonds or spacers selected from polyols, polyolefins, polyalkyls, vinyl polymers, polyaldehydes polyacid esters,

D, D′ and D″, identical or different, are independently selected from amine, amide, amino-alcohol, urea, thiourea, carbamate, carbonate, ester, ether, thioether, aryl, heteroaryl, oxime groups,
A is a single bond or a chelating agent,
SI is the substance of interest,
X′ is an acid, amine, amide, ester, ether, alkyl, alkenyl, alkynyl, aryl or heteroaryl function, and
n=1 to 100.

2 . The oligopeptide according to claim 1 , characterized in that Z consists of a 2 amino acid sequence.

3 . The oligopeptide according to claim 1 , characterized in that P comprises a peptide P′ selected from the group consisting of a variable domain of a camelid heavy-chain antibody (VHH), a Fab, F(ab)′ 2 Fv or scFv fragment of a conventional antibody, an immunoglobulin new antigen receptor (IgNAR), a nanofitin, a DARPin, an anticalin, an affibody an affilin, an avimer, a monobody and a kunitz domain.

4 . The oligopeptide according to claim 1 , characterized in that the amino acid peptide P of the oligopeptide of formula P-C-Z has at its C-terminus a 1-10 amino acid spacer Y, or the amino acid peptide P of the oligopeptide of formula Z-C-P has at its N-terminus a 1-10 amino acid spacer Y, wherein the amino acid residues of said amino acid spacer Y are identical or different, and wherein said amino acid spacer Y does not contain a cystein residue.

5 . The oligopeptide according to claim 4 , characterized in that Y represents a 4 neutral amino acid spacer.

6 . The oligopeptide according to claim 1 , characterized in that the amino acid peptide P of the oligopeptide of formula P-C-Z has at its N-terminus a 1-50 amino acid sequence X or the amino acid peptide P of the oligopeptide of formula Z-C-P has at its C-terminus a 1-50 amino acid sequence X, wherein the amino acid residues of said amino acid sequence X are identical or different, and wherein said amino acid sequence X does not contain a cystein residue.

7 . The oligopeptide according to claim 6 , characterized in that X comprises a tag and an enzyme cleavage site.

8 . The oligopeptide according to claim 3 , characterized in that the peptide P′ is a VHH and the oligopeptide formula is VHH-C-Z, VHH-Y-C-Z, X-VHH-C-Z, X-VHH-Y-C-Z, Z-C-VHH, Z-C-Y-VHH, Z-C-VHH-X, or Z-C-Y-VHH-X.

9 . The oligopeptide according to claim 1 , characterized in that the substance of interest is a diagnostic compound, selected from the group consisting of an enzyme, a fluorophore, a NMR or MRI contrast agent, a radioisotope and a nanoparticle.

10 . The oligopeptide according to claim 9 , characterized in that the diagnostic compound 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.

11 . The oligopeptide according to claim 1 , characterized in that the substance of interest is a therapeutic compound selected from a peptide, an enzyme, a nucleic acid, a virus and a chemical entity.

12 . The oligopeptide according to claim 1 , characterized in that A in the maleimido compound of formula (I) is a chelating agent and the substance of interest SI is a NMR or MRI contrast agent.

13 . The oligopeptide according to claim 1 , characterized in that the chelating agent A of the maleimido compound of formula (I) is selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA), diethylene triamine pentaacetic 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).

14 . The oligopeptide according to claim 1 , characterized in that the substance of interest SI is gadolinium (Gd), and the chelating agent A is DOTA.

15 . The oligopeptide according to claim 1 , characterized in that the maleimido compound is of formula (I′):

.

16 . A maleimido compound characterized in that it is of formula (I′):

wherein B, B′ 1 , B′ 2 , B″, A, SI and n are as defined in claim 1 .

17 . (canceled)

18 . (canceled)

19 . Site specific method for coupling a substance of interest with an oligopeptide of formula P-C-Z or Z-C-P, wherein:
P is a 8 to 800 amino acid peptide having no reduced cystein residue, C is a cystein residue, Z represents a 1-10 amino acid spacer, wherein the amino acid residues of Z are identical or different and wherein Z does not contain a cystein residue, characterized in that said method comprises a conjugation step between said oligopeptide and a maleimido compound of formula (I) as defined in claim 1 .

20 . The site specific method according to claim 19 , characterized in that the conjugation step is implemented at a pH ranging from 4 to 7.5.

21 . The site specific method according to claim 19 , characterized in that Z consists of a 2 amino acid sequence.

22 . The site specific method according to claims 19 , characterized in that P comprises a peptide P′ selected from the group consisting of a variable domain of a camelid heavy-chain antibody (VHH), a Fab, F(ab)′ 2 Fv or scFv fragment of a conventional antibody, an immunoglobulin new antigen receptor (IgNAR), a nanofitin, a DARPin, an anticalin, an affibody an affilin, an avimer, a monobody and a kunitz domain.

23 . The site specific method according to claim 19 , characterized in that the amino acid peptide P of the oligopeptide of formula P-C-Z has at its C-terminus a 1-10 amino acid spacer Y, or the amino acid peptide P of the oligopeptide of formula Z-C-P has at its N-terminus a 1-10 amino acid spacer Y, wherein the amino acid residues of said amino acid spacer Y are identical or different, and wherein said amino acid spacer Y does not contain a cystein residue.

24 . The site specific method according to claim 23 , characterized in that Y represents a 4 neutral amino acid spacer.

25 . The site specific method according to claim 19 , characterized in that the amino acid peptide P of the oligopeptide of formula P-C-Z has at its N-terminus a 1-50 amino acid sequence X or the amino acid peptide P of the oligopep

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

• 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

• C07K16/18 Primary

  against material from animals or humans · CPC title

• C07K2317/569

  Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody® · CPC title

• G01N33/6854

  Immunoglobulins · CPC title

• C07K2317/34

  Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues · CPC title