Nanobodies suitable for neuron regeneration therapy

• US 10,640,552 B2
• Filed: 09/09/2015
• Issued: 05/05/2020
• Est. Priority Date: 09/09/2014
• Status: Active Grant

• Current Assignee: Centre National De La Recherche Scientifique, Institut Pasteur
• Original Assignee: Institut Pasteur
• Inventors: Prehaud, Christophe, Lafon, Monique, Lafaye, Pierre
• Primary Examiner(s): Hayes, Robert C
• Application Number:
  US15/508,486
• Publication Number:
  US 20170283490A1
• Time in Patent Office: 1,700 Days
• CPC Class Codes: A61K 39/0007 Nervous system antigens; Prions
• CPC Class Codes: A61K 47/6843 the antibody targeting a material from animals or humans
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C07K 16/18 against material from animals or humans
• CPC Class Codes: C07K 2317/22 from camelids, e.g. camel, llama or dromedary
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/569 Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
• CPC Class Codes: C07K 2319/22 containing a Strep-tag
• CPC Class Codes: C07K 2319/33 fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• CPC Class Codes: C12N 2760/20122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Abstract

The invention is in the domain of delivery of molecules to brain cells across the blood-brain barrier. The invention relates to a novel polypeptide-based carrier that allows the efficient delivery of an effector peptide, to neuron cells across the blood-brain barrier, and to methods for the production and testing of such carrier, including a model for testing the capacity of such molecule to cross the blood-brain barrier and/or the toxicity of molecules on the blood brain barrier and/or the capacity of molecules that have crossed to target human brain cells (e.g. neurons, astrocytes and microglial cells).

Claims

1. A polypeptide comprising a variable heavy chain domain (VHH) of a camelid heavy-chain antibody,

wherein said VHH comprises:

i) a CDR1 region with the sequence IDVINNMA (SEQ ID NO;

47),

ii) a CDR2 region with the sequence TITSGFSTNY (SEQ ID NO;

48), and

iii) a CDR3 region with the sequence KVHLIRLGAARAYDY (SEQ ID NO;

49);

wherein the amino acid sequence of said VHH is at least 90% identical to SEQ ID NO;

3; and

wherein said polypeptide is permeable across the blood-brain barrier and has an isoelectric point of at least 8.5.

2. The polypeptide of claim 1, further comprising a neuron cell-targeting peptide fused in-frame with the VHH, wherein the neuron cell-targeting peptide comprises a sequence selected from the group consisting of SEQ ID NO:

1, SEQ ID NO;

32, SEQ ID NO;

33, and SEQ ID NO;

34.

3. The polypeptide of claim 2, wherein the neuron cell-targeting peptide is at the NH2-terminal extremity of said polypeptide.

4. The polypeptide of claim 1, wherein the polypeptide has dimerization capacity.

5. The polypeptide of claim 2, further comprising a tagging peptide or an affinity peptide.

6. The polypeptide of claim 2, further comprising an effector polypeptide fused in-frame, wherein the effector polypeptide comprises the amino acid sequence of SEQ ID NO:

7.

7. The polypeptide of claim 6 further comprising a peptide spacer between the VHH and the effector polypeptide.

9. A composition comprising a polypeptide of claim 1 in association with a physiologically acceptable vehicle suitable for in vivo administration.

10. The polypeptide of claim 1, wherein said polypeptide has an isoelectric point of at least 9.0.

8. A polypeptide comprising the amino acid sequence of SEQ ID NO:

11.

11. A polypeptide comprising the amino acid sequence of SEQ ID NO:

45.