NANOBODIES SUITABLE FOR NEURON REGENERATION THERAPY

• US 20170283490A1
• Filed: 09/09/2015
• Published: 10/05/2017
• 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
• Granted Patent: US 10,640,552 B2
• Time in Patent Office: 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 or consisting of a VHH of a camelid heavy-chain antibody, wherein said VHH:

i) has the sequence of SEQ ID NO;

3;

ii) is a sequence variant of the VHH of i), which at least 90% identity with the sequence of SEQ ID NO;

3;

or

iii) is a portion of the VHH of i) or ii);

and wherein such polypeptide is permeable across the blood-brain barrier and has an isoelectric point equal to or higher than 8.5, preferably equal to or higher than 9.

2. The polypeptide of claim 1 wherein the sequence length of the portion is at least 90% of the sequence length of the VHH fragment.

3. The polypeptide of claim 1, wherein said polypeptide comprises the CDR regions of the VHH fragment with the sequence of SEQ ID NO:

3.

4. The polypeptide of claim 1, which is a VHH-comprising polypeptide, wherein said VHH or portion thereof is fused in-frame with additional peptide(s).

5. The polypeptide of claim 4 which comprises a neuron cell-targeting peptide, preferably at the NH2-terminal extremity of said polypeptide.

6. The polypeptide of claim 5 wherein the neuron cell-targeting peptide consists of or comprises the sequence selected among the group consisting of SEQ ID NO:

1 and SEQ ID NO;

32 to SEQ ID NO;

34.

7. The polypeptide of claim 1 which has dimerization capacity, in particular homodimerization capacity and preferably wherein homodimerization is achieved by disulfide bridge(s).

8. The polypeptide of claim 4 comprising a tagging peptide or an affinity peptide.

9. The polypeptide of any of claim 4 comprising an effector polypeptide fused in-frame, in particular an effector polypeptide which has an effect on :

cell viability, cell-cycle state, apoptosis, cell survival, cell death, mitosis, cellular differentiation, size or morphology of cells, cell growth, neurite outgrowth, connections between cells, activation state of cell signalling pathways, signal transduction, cell mobility, cell motility, disease-related conditions, post-injury or post-lesion related conditions, diagnostic-related condition.

10. The polypeptide of claim 9 wherein the effector polypeptide consists of or comprises a peptide derived from the G protein of a Rabies virus which has an effect on the survival and/or protection and/or motility of neurons and/or which promotes neurite outgrowth, in particular a peptide which has the sequence of SEQ ID NO:

7.

11. The polypeptide of any of claim 10 wherein a peptide spacer is inserted between the VHH and the effector polypeptide.

12. The polypeptide of any of claim 1 which comprises a VHH sequence, such as SEQ ID NO:

3, a neuron cell-targeting peptide such a SEQ ID NO;

1, a Neurovita sequence, such as SEQ ID NO;

7 and optionally a tag sequence, such as SEQ ID NO;

5 and which preferably has the sequence selected from the group consisting of SEQ ID NO;

11 and SEQ ID NO;

45.

13. A polynucleotide encoding the polypeptide of any of claim 1.

14. A vector containing the polynucleotide of claim 13.

15. A vector of claim 14 suitable for expression of the polypeptide encoded by said polynucleotide in bacteria.

16. A cell or cell line polynucleotide of claim 13.

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

18. A method of treating neurodegenerative diseases, brain stroke, trauma, lesion or injury, comprising administering the composition of claim 17 to a subject in need thereof.