EP3532495A1

EP3532495A1 - Anti-tau nanobodies

Info

Publication number: EP3532495A1
Application number: EP17798139.6A
Authority: EP
Inventors: Daniel Fagret; Marcelle MOULIN; Catherine Ghezzi; Pascale Perret; Sabine CHIERICI
Original assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Universite Grenoble Alpes; Centre Hospitalier Universitaire Grenoble Alpes
Current assignee: Institut National de la Sante et de la Recherche Medicale INSERM; Universite Grenoble Alpes; Centre Hospitalier Universitaire Grenoble Alpes
Priority date: 2016-10-27
Filing date: 2017-10-27
Publication date: 2019-09-04
Also published as: JP7197494B2; WO2018078140A1; US10590191B2; CN110225926A; JP2020505056A; FR3058143B1; CA3041066A1; FR3058143A1; US20190263898A1

Abstract

The invention relates to anti-Tau nanobodies.

Description

Anti-Tau nanobody

The present invention relates to anti-Tau nanobodies. There is currently a consensus that the sporadic form of Alzheimer's disease (AD) is a continuum, from a preclinical, asymptomatic form to a clinically-characterized disease using cognitive and memory tests, as well as different biomarkers (Dubois et al Lancet Neurology 13, 614-629 (2014)). However, the development of new therapeutic strategies requires diagnostic methods to early detect and track neurodegenerative signs in the at-risk population in order to select the patients to be treated and to control the effects of treatments.

AD is defined by the association of progressive dementia syndrome and two characteristic brain lesions: extracellular senile plaques (PA) composed of amyloid peptide (Ab) and neurofibrillary tangles (DNFs) composed of protein aggregates Hyperphosphorylated Tau. DNFs appear in enthorino-cortical structures before PAs are observed in the cortex (Braak et al., Journal of Neuropathological Experimental Neurology, 70 (11): 960-969 (201 1)). In addition, the cortical distribution and the number of DNFs are correlated with memory disorders in patients with AD (Nelson et al., Journal of Neuropathological Experimental Neurology, 71 (5): 362-381 (2012).). Recently, the oligomeric forms of Tau have been shown to be toxic forms (Usenovic et al., Journal of Neuroscience, 35 (42): 14234-14250 (2015)) involved in propagation (Goedert et al., Current Neurology and Neuroscience 14: 495 (2014) tauopathies, such as AD, by gradually contaminating neurons.

Nuclear cerebral imaging (single-photon emission computed tomography (SPECT) and positron emission tomography (PET)) is an appropriate tool for determining the presence and progression of AD-related molecular lesions (Dubois et al., Alzheimer's & Dementia). 12 (3): 292-323 (2016)). Radioligand of PAs are already available (and Catafau Bullich // C ^'n / ^AC / 3 and Translational Imaging (1)... 39-55 (2015)) and some radiotracers. Tracers targeting beta sheets within DNFs are currently under development. Two of them, 18F-AV-1451 (Ossenkoppele et al., Brain 139 (5): 1551-1567 (2016).) And 18F-THK51 (Jonasson et al., Journal of Nuclear Medicine, 57 ( 4): 574-581 (2016).) Could facilitate the stratification of patients with DNF lesions. On the other hand, none allows the detection and monitoring of soluble oligomeric forms of Tau. Under physiological conditions, the highly soluble Tau protein interacts with microtubules to stabilize them, its partial phosphorylation allowing its alternative detachment of microtubules. Tau protein is normally very soluble and is in unstructured form Under certain pathological conditions, post-translational modifications of Tau, in particular hyper-phosphorylation lead the protein to structure and form aggregates: dimers, oligomers, fibers : helical pairs of filaments leading to the formation of DNFs. A prominent importance is currently attributed to Tau oligomers, both from the point of view of toxicity and the spread of AD lesions and other tauopathies.

ScFvs directed against the Tau protein in the form of oligomers, in particular in the form of dimers and trimers, have been described in application US 2015/0266947. These scFvs, about 30 kDa in size, are not directed against Tau as fibers.

In order to develop new markers of pathological forms of Tau for nuclear imaging targeting the various aggregated forms of this molecule, the present inventors have developed nanobodies or nanobodies (Nbs), in particular VHHs, targeting the pathological forms, in particular the early pathological forms of Tau, more particularly Tau in the form of oligomers and optionally in the form of fibers. These nanobodies are thus likely to improve the diagnosis and monitoring of AD, as well as the evaluation of the effects of potential treatments.

These nanobodies also make it possible to target and thus identify the presence of pathological forms of Tau in other tauopathies than AD. The nanobodies according to the invention, which are in VHH format, are small antibody fragments corresponding to the variable domains of the heavy chains of certain camelid antibodies. These are the smallest functional elements (10-15 kDa) derived from immunoglobulins possessing an antigen-binding domain and have antigen affinities of the nanomolar order. Because of their simple Fc-like domain structure and low molecular weight they are suitable tools for crossing the blood-brain barrier (Caljon et al., British Journal of Pharmacology, 165 (7): 2341-2353 (2012)). .).

Summary of the invention

The present invention thus relates to a nanobody which binds to the pathological forms of the Tau protein, in particular the early pathological forms of Tau, such as Tau in the form of oligomers and optionally Tau in the form of fibers.

The subject of the present invention is therefore a nanobodies directed against the Tau protein in the form of oligomers and directed against the Tau protein in the form of fibers. The present invention also relates to a nanobodies directed against the Tau protein, said Tau protein being in the form of oligomers and said nanobodies being devoid of light chain.

The subject of the present invention is therefore a nanobodies directed against the Tau protein, said Tau protein being in pathological form, in particular in the form of oligomers, and said nanobodies competing for binding to the Tau protein in the form of oligomers with a protein. nanobodies comprising the amino acid sequences (i) GRTFSXiX ₂ X ₃ (SEQ ID NO: 1) as CDR1 in which the amino acid Χ _Ί is S or R, X ₂ is D or Y and X ₃ is T or A ,

(ii) ISX1SGGX ₂ T (SEQ ID NO: 2) as CDR2 in which the amino acid Χ _Ί is P or R and X ₂ is S or V, and

(iii) NRDPKYGNTRY (SEQ ID NO: 5) or TARRRISGTPQWHY (SEQ ID NO: 8) as CDR3.

The present invention also relates to a nanobody against the Tau protein, said Tau protein being in pathological form, in particular in the form of oligomers and said nanobodies competing for binding to the Tau protein in the form of oligomers with a nanobody comprising:

a) the amino acid sequences (i) GRTFSSDT (SEQ ID NO: 3) as CDR1, (ii) ISPSGGVT (SEQ ID NO: 4) as CDR2 and (iii) NRDPKYGNTRY (SEQ ID NO: 5) as CDR3; or

b) the amino acid sequences (i) GRTFSRYA (SEQ ID NO: 6) as CDR1, (ii) ISRSGGST (SEQ ID NO: 7) as CDR2 and (iii) TARRRISGTPQWHY (SEQ ID NO:

8) as CDR3.

The present invention also relates to nanobodies directed against the Tau protein, said nanobodies comprising the amino acid sequences:

(i) GRTFSX1X ₂ X ₃ (SEQ ID NO: 1) as CDR1 in which the amino acid Χ _Ί is S or R, X ₂ is D or Y and X ₃ is T or A,

(iii) NRDPKYGNTRY (SEQ ID NO: 5) or TARRRISGTPQWHY (SEQ ID NO: 8) as The invention also relates to nanobodies, directed against the Tau protein, comprising:

b) the amino acid sequences (i) GRTFSRYA (SEQ ID NO: 6) such as CDR1, (ii ISRSGGST (SEQ ID NO: 7) as CDR2 and (iii) TARRRISGTPQWHY (SEQ ID NO: 8) as CDR3, or

a functionally conservative variant of the nanobodies defined in a) or b) comprising a conservative substitution of one or two amino acids in one, two or three of the sequences SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5 respectively; , or SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

The present invention also relates to the nanobody as defined above for its use as a contrast agent in in vivo, non-invasive medical imaging for its use in diagnostic or prognostic methods, preferably a tauopathy or for its use as a medicine.

The subject of the invention is also the use of this nanobodies for the in vitro detection of Tau protein, in particular in pathological form, preferably an early pathological form, more particularly in the form of oligomers and optionally in the form of fibers. in a sample.

Finally, it relates to a pharmaceutical composition comprising this nanobody in association with a pharmaceutically acceptable vehicle.

Detailed description of the invention

tauopathy

The term "tauopathy" includes about twenty pathologies that share the existence of intracerebral Tau protein deposits, particularly in pathological form, and share clinical, pathological, biochemical and genetic similarities. The term "intracerebral deposits of tau protein" means the presence of Tau aggregates and thus the presence of Tau in pathological form.

Some tauopathies, such as progressive supranuclear palsy, corticobasal degeneration and Gerstmann-Straussier-Scheinker's disease, are characterized by the presence of neurofibrillary tangles (DNFs) that are indistinguishable from those of Alzheimer's disease. In this case the term "deposits Intracerebral tau protein "or" presence of pathological forms of Tau "denotes for example the presence of neurofibrillary tangles (DNFs).

Pick's disease, for example, is characterized by spherical neural inclusions called Pick's body. These Pick bodies consist of disordered straight filaments and thus also of a pathological Tau form.

"Neurofibrillary degeneration" (also referred to as neurofibrillary tangles or tangles) refers to areas, for example in the cerebral cortex, within which the neuronal population has fibrillar tangles made up of paired filaments around the nucleus and in cellular processes. helically arranged (PHF) and / or straight filaments (SF).

It should be noted that the formation of DNFs includes the aggregation of Tau, also called "fibrillar aggregation" or "fibrillogenesis". During this process, the Tau protein is organized in the form of oligomers and then in the form of fibers which is organized in the form of helicoidal pairs of filaments and in the form of straight filaments. The shapes of the helical pairs of filaments and the forms of straight filaments constitute the DNFs.

The term "pathological form of Tau" encompasses Tau protein in the form of oligomers, in the form of fibers, in the form of helicoidal pairs of filaments and in the form of straight filaments. However, it has been discovered that the intermediate aggregation forms, i.e., Tau as oligomers, and Tau as fibers, are toxic, with oligomers being the most toxic. These are referred to by the term "early pathological forms of Tau" which therefore means in particular Tau in the form of oligomers and in the form of fibers, more particularly tau in oligomeric form.

Tau protein is an intracellular protein, however the pathological forms of Tau can, in some cases, be extracellular.

In a particular embodiment, the pathological forms of Tau, preferably Tau in the form of oligomers, are extracellular.

Accordingly, in one embodiment "a pathological form of Tau" refers to Tau in the form of oligomers, in the form of fibers, in the form of helical pairs of filaments and / or in the form of straight filaments. Preferably, a pathological form of Tau is an early pathological form of Tau, more particularly Tau in the form of oligomers and optionally Tau in the form of fibers, for example Tau in the form of oligomers or Tau in the form of fibers.

In one embodiment, tauopathy means a disease that is associated with the existence of pathological forms of Tau, as defined above. Tauopathy is selected from Alzheimer's disease, progressive supranuclear palsy (or Steele-Richardson-OIzewski's disease), corticobasal degeneration, Pick's disease, Niemann-Pick type C disease, Gerstmann-Straussler's disease - Scheinker and fronto-temporal degeneration related to a chromosome 17 mutation, preferably Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration and Gerstmann-Straussler-Scheinker's disease, especially Alzheimer's disease.

Tau

The term "Tau protein" or "Tau" refers to the Tau protein (in English: tubule-associated unit) which is a mammalian protein. Tau protein is a member of the family of microtubule-associated proteins (microtubule associating protein). It is encoded by the MAPT gene located on chromosome 17 at position 17q21. Tau protein is also called MSTD; PPND; DDPAC; MAPTL; MTBT1; MTBT2; FTDP-17; PPP1 R103. In humans, Tau protein is synthesized primarily at the level of neurons.

Tau's primary transcript contains 16 exons. In the brain, some exons are not translated. Exons 2, 3 and 10 are alternately spliced and are specific for adult brain tissue. The alternative splicing of these 3 exons produces 6 possible combinations (2-3-10- to 2 + 3 + 10 +). At the protein level, there are therefore six isoforms of Tau proteins in the adult brain. It should be noted that the expression of Tau proteins is regulated during development. Thus, a single isoform, called fetal, is present at birth and does not include inserts coded by exons 2, 3 or 10. The other isoforms appear during the subsequent development. The length of their sequences varies from 352 to 441 amino acids.

The amino-terminal part of the Tau proteins, also called the projection domain, has a role that is still poorly understood. This projection domain may interact with the plasma membrane and some organelles such as mitochondria. As for the carboxy-terminal domain, it comprises 3 (without exon 10) or 4 (with exon 10) repetitive segments, specific binding domains (called 3R or 4R) comprising the conserved tubulin binding domain motif, which controls the stability of microtubules. These R motifs are the anchor point of the tau protein on microtubules. The three isoforms without the sequence encoded by exon 10 (10-) have three microtubule binding domains (3R), are named 2N3R, 1 N3R and 0N3R, and are distinguished by the amino-terminal portion. The three isoforms with the sequence encoded by exon 10 have four microtubule binding domains (4R), are named 2N4R, 1 N4R and 0N4R, and are also distinguished by the amino-terminal portion. The interaction with Tubulin dimers are stronger with this fourth domain, which further stabilizes microtubules and can modulate the length of neuritic extensions, as well as neuronal plasticity.

In one embodiment of the invention, said Tau protein is selected from the group consisting of 2N4R, 1N4R, 0N4R, 2N3R, 1N3R and 0N3R isoforms. Preferably the Tau protein is under the 2N4R isoform.

The amino acid sequence of the 2N4R isoform of the Tau protein, also called Tau-F or Tau441 or htau40, with 441 amino acids can be found on the UniprotKb database in its version of November 16, 2016 under the number d accession P10636-8 (SEQ ID NO: 21).

The amino acid sequence of the 1 N4R isoform of the Tau protein, also called Tau-E or Tau412, with 412 amino acids can be found on the UniprotKb database in its version of November 16, 2016 under the number of accession P10636-7 (SEQ ID NO: 22).

The amino acid sequence of the 0N4R isoform of the Tau protein, also called Tau-D or Tau383, with 383 amino acids can be found on the UniprotKb database in its version of November 16, 2016 under accession number P10636-6 (SEQ ID NO: 23).

The amino acid sequence of the 2N3R isoform of the Tau protein, also called Tau-C or Tau410, with 410 amino acids can be found on the UniprotKb database as of November 16, 2016 under accession number P10636-5 (SEQ ID NO: 24).

The amino acid sequence of the 1 N3R isoform of the Tau protein, also called Tau-B or Tau 381, with 381 amino acids can be found on the UniprotKb database in its version of November 16, 2016 under the number d accession P10636-5 (SEQ ID NO: 25).

The amino acid sequence of the 0N3R isoform of the Tau protein, also called Fetal-Tau or Tau 352, with 352 amino acids can be found on the UniprotKb database in its version of November 16, 2016 under the number of accession P10636-2 (SEQ ID NO: 26).

Tau protein is a neuronal protein that is often located in the axon, more rarely in dendrites and exceptionally in the cell body. In native form, Tau is an unstructured protein and in the form of a monomer. Therefore, in the context of the present invention, the term "native Tau" means Tau protein in monomeric form and vice versa. The function of tau proteins Native form is to interact with microtubules via specific binding domains (3R and 4R) and to promote assembly and stability of microtubules. The interaction of Tau protein with microtubules is regulated mainly by phosphorylation. Tau is a phosphoprotein that contains about 80 potential sites of phosphorylation. The regulation of the phosphorylation state of the Tau protein results from the joint activities of protein kinases and protein phosphatases.

In general, hyperphosphorylation of the Tau protein, particularly in the specific binding domains (3R and 4R), decreases its affinity for microtubules, which can lead to their destabilization and hence cytoskeletal disruption. In addition, this hyperphosphorylation can lead to the aggregation of Tau and thus the formation of DNFs, as described above in the "tauopathy" part.

One skilled in the art distinguishes the abnormal phosphorylation and hyperphosphorylation of Tau protein.

"Abnormal phosphorylation" consists of phosphorylation at sites that under physiological conditions are not affected by phosphorylation. We speak of a non-physiological epitope; these are, for example, the epitopes recognized by the AT100 and TG-3 antibodies.

In contrast, the term "hyperphosphorylated" means phosphorylation at physiological epitopes in greater numbers than in a normal adult brain or when for a given site a high percentage of Tau protein is phosphorylated.

Hyperphosphorylation of the tau protein can cause tau detachment from microtubules and an increase in intracellular tau concentration, causing the formation of oligomers and then fibers, until the helicoidal pairs of filaments and straight filaments are formed whose entanglement will constitute the fibrillar degenerations. The result is an accumulation of tau protein in neurons and involves more than 20 different neurodegenerative diseases called tauopathy. The accumulation of Tau protein is sometimes even the only cause of such tauopathy.

It is known to those skilled in the art that Tau aggregation can be studied using non-phosphorylated Tau protein (Xu, S et al., Alzheimers Dément, 2010 Mar; 6 (2): 1-10-7, Kumar S. et al., J Biol Chem., 2014 Jul 18; 289 (29): 20318-32; Flach, K et al., J Biol Chem., 2012 Dec 21; 287 (52): 43223-33).

The inventors of the present invention have developed the nanobodies of the invention directed against non-phosphorylated Tau protein, in particular in the form of oligomers. However, as demonstrated by immunohistochemistry, the nanobodies of The invention also binds specifically to Tau protein in neurofibrillary degenerations of human brain slices in which the Tau protein is present in phosphorylated form.

Accordingly, in one embodiment of the invention the Tau protein is non-phosphorylated and / or phosphorylated.

In some intracerebral deposits of Tau protein, it may also be degraded and lose the N-terminal portion while retaining the microtobule binding domains. This truncated Tau protein comprises the carboxy-terminal portion of the Tau proteins, and therefore, depending on the starting isoform, is the 3R region (R1, R2, R3, R4) or the 4R region (R1, R3 and R4). This proteolysis promotes the formation of fibers and then the aggregation of pairs of helical filaments that become insoluble and are also indicative of certain Tauopathies.

Accordingly, in one embodiment the Tau protein is a truncated Tau protein comprising the carboxy-terminal portion of the Tau protein, particularly the R3 or R4 region of the Tau repeats, preferably the R3 region. Preferably, the truncated Tau protein lacks N-terminal domains. In a particular embodiment, the Tau protein comprises or consists of a peptide having the amino acid sequence VQIVYKPVDLSKVTSKCG (SEQ ID NO: 27) which corresponds to amino acids 306 to 323 of tau441, as defined above. Anti-Tau nanobody

In the context of the present invention, the terms "antibodies" and

"immunoglobulin" have the same meaning and are used interchangeably. In conventional antibodies, the two heavy chains are linked to one another by disulfide bridges and each heavy chain is linked to a light chain by a disulfide bridge. There are two types of light chain: lambda (λ) and kappa (κ) light chains. There are five major classes of heavy chains (or isotypes) that determine the functional activity of an antibody: IgM, IgD, IgG, IgA and IgE. Each string contains separate sequence domains. The light chain has two domains: a variable domain (VL) and a constant domain (CL). The heavy chain comprises four domains: a variable domain (VH) and three constant domains (CH1, CH2 and CH3, collectively called CH). Heavy (VH) and light (VL) variable regions determine binding recognition and antigen specificity. The domains of the light chain (CL) and heavy (CH) constant regions confer important biological properties such as the association of antibody chains, secretion, transplacental mobility, complement binding and Fc receptor binding. The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin consisting of the variable portions of a light chain and a heavy chain. The specificity of the antibody lies in the structural complementarity between the site of combination of the antibody and the antigenic determinant. The antibody combining sites are made of residues that originate primarily from hypervariable regions or complementarity determining regions (CDRs). Occasionally, residues from non-hypervariable regions or "framework" (FR) regions may influence the overall structure of the domain and hence the combining site.

In the context of the invention, the term "CDR" refers to amino acid sequences, which together define the binding affinity and specificity of the natural Fv region of a native binding site of a immunoglobulin. The heavy and light chains of an immunoglobulin each have three CDRs, designated H-CDR1, H-CDR2, H-CDR3 and L-CDR1, L-CDR2, L-CDR3 respectively. An antigen binding site therefore includes 6 CDRs, including all the CDRs of a variable region of a heavy chain and a variable region of a light chain.

The location of the CDRs in the sequence of an antibody or nanobody can be determined by those skilled in the art using previously described techniques. Typically, CDRs can be identified by sequencing the DNA of the antibody or nanobodies with a suitable system, such as the 3730XL DNA Analyzer and ABI PRISM BigDye Terminator cyc, and then analyzing the sequences thus obtained using bases. dedicated data such as the international ImMunoGeneTics database or IMTG (Lefranc (2003) Dev Comp., Immunol 27:55) or Kabat, et al. (National Institutes of Health, Bethesda, Md., 1991), preferably IMGT.

In the context of the invention, the term "framework region", "framework" or "FR" refers to the amino acid sequences intercalated between the CDRs.

In the context of the invention, the terms "nanobody", "nanobody", "VHH", "VHH antibody fragment" and "unic domain antibody" are used interchangeably and denote the variable domain of the unique heavy chain. antibodies of the type found in camelids, which are naturally devoid of light chains. In the absence of light chain the nanobodies each have three CDRs, designated CDR1, CDR2, and CDR3 respectively. The nanobodies according to the invention may in particular be nanobodies of camels, dromedaries, llamas or alpacas. Preferably, the nanobodies according to the invention are lamas nanobodies.

By "nanobodies directed against the Tau protein" is meant here a nanobodies capable of selectively binding to the Tau protein, as defined in the "Tau" section. Preferably, the nanobody is specific for Tau, that is to say that it binds Tau to the exclusion of any other molecule.

The inventors have prepared nanobodies directed against pathological forms of the Tau protein, such as the Tau protein in the form of oligomers and optionally in the form of fibers. The nanobodies can also be directed against the truncated Tau protein in fiber form.

In particular, the inventors have immunized llamas with a protein enriched Tau Tau protein preparation in the form of oligomers, with a protein enriched Tau protein preparation in Tau form and a Tau protein enriched truncated Tau protein preparation. truncated in the form of fibers. The inventors then identified more precisely two nanobodies directed against Tau and having additional features not expected that do not have other anti-Tau nanobodies. Indeed, these two nanobodies, 2C5 and S2T2M3_E6, in particular 2C5, recognize Tau as oligomers and optionally in the form of fibers. Tau protein in fiber form is also recognized when it is a truncated Tau protein as defined above. At the same time, these two nanobodies, 2C5 and S2T2M3_E6, in particular 2C5, do not bind to the native Tau protein in monomeric form. In addition, these two nanobodies, 2C5 and S2T2M3_E6, in particular 2C5, do not bind to beta amyloid peptide fibers.

Accordingly, in a preferred embodiment, the nanobodies are directed against the Tau protein as oligomers.

The term "oligomers" in the context of the present invention relates to a Tau preparation obtained by an in vitro fibrillation method as described in the examples of this application. Typically, the fibrillation of Tau (40 μl) is carried out for example at 37 ° C. in buffer, for example MOPS (3- (N-morpholino) propanesulfonic acid), typically 20 mM, typically at pH 7 in the presence typically of heparin (10 μl), NaN3 (4%) for a final volume of typically 1.5 ml. Fibrillation is typically stopped by freezing the samples at -80 ° C after typically 48 hours to obtain Tau as oligomers. Depending on the exact time of fibrillation this Tau protein preparation is enriched in Tau protein as oligomers.

The term "Tau protein enriched as oligomers" means a Tau protein preparation in the form of oligomers of which more than 50% of Tau proteins are in the form of oligomers. For example, more than 50%, more than 55%, more than 60%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90% of Tau proteins are in the form of oligomers . It is known to those skilled in the art that an enriched preparation Tau proteins in the form of oligomers further contains Tau protein in native form and Tau protein in the form of fibers.

Accordingly, in one embodiment, the oligomeric Tau protein is a mixture of Tau proteins in the form of monomers, in the form of oligomers and in fiber form, of which more than 50% of the Tau proteins are in the form of oligomers. Oligomers, in particular more than 55%, more than 60%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90% of the Tau proteins are in the form of oligomers.

It is also known to those skilled in the art that it is possible to purify the Tau protein in the form of oligomers from such a preparation, using several methods known to those skilled in the art, including for example chromatography. steric exclusion.

The oligomers preferably comprise between 2 and 45 units of Tau. Oligomers having 2 to 8 units of Tau are referred to as soluble oligomers and oligomers having more than 8 to 45 units of Tau are referred to as granular oligomers and may be soluble or insoluble. In one embodiment these oligomers can be characterized, using an atomic force microscope or electron microscope, as round points having a diameter of about 12 to 29 nm.

In another embodiment these oligomers can be characterized using size exclusion chromatography as being spherical molecules having a diameter of about 12 to 35 nm.

Accordingly, the term "oligomer" in the context of the invention refers to an aggregate or Tau polymer having from 2 to 50 Tau units, particularly 2 to 20, most preferably 2 to 12 tau units. , for example a dimer, a trimer, a tetramer, a pentamer, a hexamer, heptamer, octamer, nonamer, decamer, undecamer or dodecamer of Tau or having 20 to 45, preferably 30 to 45, units of Tau, for example at 45, preferably 38 to 42 units of Tau. In some embodiments, the tau protein is dimeric or trimeric. In some embodiments, the oligomeric Tau protein is soluble and / or non-soluble, more preferably soluble.

In one embodiment, the nanobodies of the invention have an affinity for Tau protein in the form of oligomers which is <20nM, for example <10nM, <8nM, <7nM or <5nM, for example an affinity of 0.1nM to 20 nM, in particular from 1 nM to 10 nM, or from 1 nM to 7 nM, for example 5 nM.

By "affinity" is meant the binding capacity between a macromolecule and the antigen it fixes, in particular the binding capacity between a nanobody and the antigen that it fixes, for example a nanobody of the invention and the Tau protein in the pathological forms as defined above.

The affinity and therefore the ability of the nanobodies of the invention to bind to the Tau protein, for example in the form of oligomers or fibers, can be measured in vitro by several methods, including surface plasmon resonance (SPR). especially using a BIAcore 2000-Pharmacia Biosensor type device, Upsala, Sweden) or for example by an ELISA test, as described in the examples.

In one embodiment, the nanobodies of the invention further bind to Tau protein in the form of fibers.

The term "fibers" in the context of the invention relates to a preparation of the Tau protein obtained by a fibrillation process as described in the examples of this application. Typically, the fibrillation of Tau (40 μl) is carried out for example at 37 ° C. in buffer, for example MOPS (3- (N-morpholino) propanesulfonic acid), typically 20 mM, typically at pH 7 in the presence typically of heparin (10 μl), NaN3 (4%) for a final volume of typically 1.5 ml. Fibrillation is typically stopped by freezing the samples at -80 ° C after typically 72 hours to obtain Tau as fibers. Such a preparation of Tau proteins in the form of fibers is therefore a preparation of Tau proteins enriched in Tau protein in the form of fibers.

The term "fiber-enriched Tau" means a Tau protein preparation in the form of fibers of which more than 50% of the Tau proteins are in the form of fibers. For example, more than 50%, more than 55%, more than 60%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90% of the Tau proteins are in the form of fibers. It is known to a person skilled in the art that a preparation enriched with Tau proteins in the form of fibers also contains Tau protein in native form and Tau protein in the form of oligomers.

Accordingly, in one embodiment, the Tau protein in the form of fibers is a mixture of Tau proteins in the form of monomers, in the form of oligomers and in the form of fibers, of which more than 50% of the Tau proteins are in the form of fibers, especially more than 55%, more than 60%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90% of the Tau proteins are in the form of fibers.

It is also known to those skilled in the art that it is possible to purify Tau protein in fiber form from such a preparation, using several methods known to those skilled in the art, including for example chromatography. steric exclusion. In the context of the invention, the term fiber describes a polymer or Tau aggregate obtained in an artificial manner mimicking the Tau fibrillation in vivo and thus mimicking the helical pairs of filaments and the straight filaments that make up the DNFs. Accordingly, Tau fibers as used in the context of the invention encompass pairs of helical filaments and straight filaments. By electron microscopy these fibers can be characterized as having a filamentous appearance. In one embodiment, the nanobodies of the invention have an affinity for the Tau protein in the form of fibers which is <20nM, for example <10nM, <8nM, <7nM or <6nM, for example an affinity of 0.1nM to 20nM. nM, in particular from 1 nM to 10 nM, or from 1 nM to 8 nM, for example 6 nM.

In another embodiment, the nanobodies of the invention further bind to the truncated Tau protein. Preferably, this truncated protein is in the form of fibers and may also be named R3 peptide fibers. The truncated Tau protein is as defined in the "Tau" section above. In particular, this truncated Tau protein comprises or consists of the amino acid sequence SEQ ID NO: 27.

In one embodiment, the nanobodies of the invention have an affinity for the truncated Tau protein in the form of fibers which is <100nM, for example <80nM, <70nM, <60nM or <50nM, for example an affinity of 1nM to 100 nM, in particular from 1 nM to 80 nM, or from 40 nM to 60 nM, for example 50 nM.

In one embodiment, the nanobodies of the invention have an affinity for the truncated Tau protein in the form of fibers which is <100 nM, for example an affinity of 1 nM to 100 nM, in particular 1 nM to 80 nM, of 1 nM at 20nM, for example an affinity of 10 to 20nM.

In the context of the invention, "the truncated Tau protein in the form of fibers" or "the peptide fibers R3" relates to an R3 peptide preparation obtained by an in vitro fibrillation method as described in the examples of this application.

Typically, the fibrillation of R3 (0.4 μl) is carried out for example at 37 ° C. in buffer, for example PBS (50 mM salt buffer phosphate, typically at pH 7 in the presence of heparin (0.4 μl), NaN 3 (4%) for a final volume of typically 1.5 ml Fibrillation is typically stopped by freezing the samples at -80 ° C after typically 72 hours to obtain truncated Tau (R3) as fibers.

In one example, the affinity for the truncated Tau protein in fiber form is measured, for example, by ELISA, in, for example, a buffer phosphate buffer. Saline (NaCl, 137mM, KCl, 2.7mM, Na ₂ HPO ₄ , 10mM KH ₂ PO ₄ , 1.8mM) comprising, for example, 1% BSA and having, for example, a pH of 7.5.

The inventors have also demonstrated for the nanobodies of the invention a specific labeling of the hippocampal cell bodies, the entorhinal cortex and the temporal cortex in patients with Alzheimer's disease.

Accordingly, in one embodiment, the nanobodies of the invention additionally bind to helical pairs of filaments and / or straight filaments.

The term "helical pair of filaments" refers to helical paired filaments comprising at least 10, at least 12, at least 20, preferably at least 40, and in particular at least 50 or at least 60 units of Tau. Normally these filaments can be observed by electron microscopy and have a diameter of 8 to 20 nanometers, for example 10 to 20 nm and a helical pitch of about 80 nm, for example 70 to 90 nm.

The term "straight filaments" means filaments which are not helically matched and comprising at least 10, at least 12, at least 20, preferably at least 40 units of Tau, in particular at least 50 or at least 60 units of Tau. Tau. Normally these filaments can be observed by electron microscopy and have a diameter of about 10 nanometers, for example from 8 to 17 nm, in particular from 9 to 12 nm, for example 10 nm.

The helical pairs of filaments and straight filaments that make up the DNFs are not soluble.

In another embodiment, the nanobodies of the invention do not interact substantially with the polymerized amyloid beta 1 -42 protein.

In one embodiment, the nanobodies do not substantially interact with the Tau protein in monomeric form.

A nanobodies "do not substantially intercalate" with a protein, for example, polymerized amyloid beta 1 -42 protein or Tau in monomeric form, when the affinities for Tau protein as an oligomer and the affinity for Tau protein under The monomer form or the polymerized amyloid beta-42 protein are very different. In one example, the affinity for monomeric Tau protein can not be measured because the binding response is too weak. In another example, a nanobodies do not substantially interact with the Tau protein in monomeric form or the polymerized amyloid beta 1 -42 protein, when the nanobody-binding reaction with Tau as a monomer is less than 5% of the response. binding of the same nanobodies with Tau as an oligomer in the same experimental condition and at the same concentration of the nanobody. In practice, the The concentration of the nanobodies used may be the EC 50 concentration or the concentration necessary to reach the saturation plateau.

For example, the affinity of the nanobodies of the invention for Tau protein in monomeric form is> 1200 nM, for example> 1400 nM,> 1600 nM,> 1800 nM, in particular> 1800 nM.

For example, the affinity of the nanobodies of the invention for the polymerized amyloid beta 1 -42 protein is> 5000 nM, for example> 8000 nM,> 9000 nM,> 10000 nM, in particular> 10000 nM.

The nanobodies of the invention have been sequenced:

the 2C5 nanobodies have the amino acid sequence QVQLVQSGG

GLVQAGGSLRLSCAASGRTFSSDTLAWFRQAPGKEREFVASISPSGGV TYYEDSVK.GRFTISRDNSKNTVLLQMNSLTPEDTAVYYCNRDPKYGNT RYWGQGTQVTVSS (SEQ ID NO: 9),

the nanobody S2T2M3_E6 has the amino acid sequence EVQLVESGGGLVQAGGSLRLSCAASGRTFSRYAMGWFRQAPGKEREF

VASISRSGGSTRYADAVKGRFTISRDNTNNTVYLLMNNLKPEDTAVYYC TARRRISGTPQWHYWGQGTQVTVSS (SEQ ID NO: 10).

The CDRs of these two nanobodies have been more specifically sequenced and are as follows:

· 2C5

o CDR1: GRTFSSDT (SEQ ID NO: 3)

o CDR2: ISPSGGVT (SEQ ID NO: 4)

o CDR3: NRDPKYGNTRY (SEQ ID NO: 5)

• S2T2M2_E6

o CDR1: GRTFSRYA (SEQ ID NO: 6)

o CDR2: ISRSGGST (SEQ ID NO: 7)

o CDR3: TARRRISGTPQWHY (SEQ ID NO: 8)

As is well known to those skilled in the art, the combination of CDR1, CDR2 and CDR3 is sufficient to define an antigen binding site. It is also known to those skilled in the art that two nanobodies that recognize the same antigen compete for binding to this antigen.

Accordingly, an object of the invention relates to a nanobodies directed against the Tau protein, said Tau protein being in pathological form, in particular in the form of oligomers and said nanobodies competing for binding to the Tau protein in the form of oligomers with a nanobody including amino acid sequences (i) GRTFSX1X ₂ X ₃ (SEQ ID NO: 1) as CDR1 in which the amino acid Χ _Ί is S or R, X ₂ is D or Y and X ₃ is T or A,

(iii) NRDPKYGNTRY (SEQ ID NO: 5) or TARRRISGTPQWHY (SEQ ID NO: 8) as CDR3.

The ability of a candidate nanobicide to compete for Tau protein binding, for example in the form of oligomers, with a nanobodies comprising the CDRs of a 2C5 and / or S2T2M3_E6 nanobodies as defined above, ( hereinafter a "reference" nanobodies) can easily be verified, for example, by competitive ELISA in which the antigen (ie Tau protein in the form of oligomers) is bound to a solid support and two solutions containing the nanobody The candidate and the reference nanobodies, respectively, are added and the nanobodies will compete to bind to the antigen. The amount of reference nanobodies bound to the antigen can then be measured and compared to the amount of reference nanobodies bound to the antigen when measured against a negative control (eg solution free of candidate nanobodies). An amount of bound reference nanobodies in the presence of candidate nanobodies that is decreased relative to the amount of bound reference nanobodies in the presence of the negative control indicates that the candidate nanobodies compete for binding to Tau protein as oligomers. . Ideally, the reference nanobodies can be labeled (e.g. by fluorescence) to facilitate the detection of bound reference nanobodies. Repeated measurements can be performed with successive dilutions of the candidate and / or reference nanobodies.

In another subject of the invention, the invention relates to a nanobodies directed against the Tau protein, said Tau protein being in pathological form, in particular in the form of oligomers and said nanobodies competing for binding to the Tau protein under oligomer form with a nanobodies selected from nanobodies comprising: a) amino acid sequences (i) GRTFSSDT (SEQ ID NO: 3) such as CDR1, (ii) ISPSGGVT (SEQ ID NO: 4) such as CDR2 and ( iii) NRDPKYGNTRY (SEQ ID NO: 5) as CDR3; or

b) the amino acid sequences (i) GRTFSRYA (SEQ ID NO: 6) as CDR1, (ii) ISRSGGST (SEQ ID NO: 7) as CDR2 and (iii) TARRRISGTPQWHY (SEQ ID NO: 8) as CDR3. An object of the present invention relates to a nanobody against protein

Tau comprising the amino acid sequences (i) GRTFSX1X ₂ X ₃ (SEQ ID NO: 1) as CDR1 in which the amino acid Χ _Ί is S or R, X ₂ is D or Y and X ₃ is T or A,

(iii) NRDPKYGNTRY (SEQ ID NO: 5) or TARRRISGTPQWHY (SEQ ID NO: 8) as CDR3.

The invention also relates to a nanobodies directed against the Tau protein comprising: a) the amino acid sequences (i) GRTFSSDT (SEQ ID NO: 3) as CDR1, (ii) ISPSGGVT (SEQ ID NO: 4) as CDR2 and (iii) NRDPKYGNTRY (SEQ ID NO: 5) as CDR3; or

In a preferred embodiment, this Tau protein is in the form of oligomers and optionally in the form of fibers.

The inventors have also sequenced the "framework" (FR) regions of the 2C5 and S2T2M2 E6 nanobodies. The corresponding sequences are as follows:

• 2C5

o Region FR1: QVQLVQSGGGLVQAGGSLRLSCAAS (SEQ ID NO: 1 1) o Region FR2: LAWFRQAPGKEREFVAS (SEQ ID NO: 12)

o Region FR3: YYEDSVKGRFTISRDNSKNTVLLQMNSLTPEDTAVYYC (SEQ ID NO: 13)

o Region FR4: WGQGTQVTVSS (SEQ ID NO: 14)

• S2T2M2_E6

Region FR1: EVQLVESGGGLVQAGGSLRLSCAAS (SEQ ID NO: 15) Region FR2: MGWFRQAPGKEREFVAS (SEQ ID NO: 16)

Region FR3: RYADAVKGRFTISRDNTNNTVYLLMNNLKPEDTAVYYC (SEQ ID NO: 17)

Region FR4: WGQGTQVTVSS (SEQ ID NO: 18)

In a particular embodiment, the invention relates to a nanobody against the Tau protein, said Tau protein being in pathological form, in particular in the form of oligomers and said nanobodies competing for binding to the Tau protein as oligomers with a nanobodies selected from a nanobodies comprising an amino acid sequence selected from the group consisting of SEQ amino acid sequences ID NO: 9 and SEQ ID NO: 10.

In a particular embodiment, the invention relates to a nanobody comprising or consisting of the sequence of FR1 -CDR1 -FR2-CDR2-FR3-CDR3-FR4 sequences as defined above of one of the nanobodies identified by the inventors.

Preferably, the nanobody according to the invention is therefore a nanobodies comprising or consisting of an amino acid sequence selected from the group consisting of the amino acid sequences SEQ ID NO: 9 and SEQ ID NO: 10 or a functionally variant conservative thereof comprising a conservative substitution of one or two amino acids in one, two or three of the CDRs included respectively in the amino acid sequence SEQ ID NO: 9 or SEQ ID NO: 10. The functionally conservative variant as as defined above may further comprise one or more substitutions, in particular one or more conservative substitutions in the regions respectively of the amino acid sequences SEQ ID NO: 9 or SEQ ID NO: 10 which are not CDRs, such as that the regions "frame", especially the "framework" regions defined above. More preferably, the nanobody according to the invention is a nanobodies comprising or consisting of an amino acid sequence selected from the group consisting of amino acid sequences SEQ ID NO: 9 and SEQ ID NO: 10. most preferred, the nanobody according to the invention is a nanobody comprising or consisting of the amino acid sequence SEQ ID NO: 9.

In the context of the invention, the term "functionally conservative variant" refers to variants in which a given amino acid in a nanobody according to the invention is substituted without altering the overall conformation and function of the nanobody, including replacement. from one amino acid to another having similar properties (eg polarity, hydrogen bonding potential, acidity, basicity, hydrophobicity, presence of an aromatic group, etc.). Amino acids with similar properties are well known to those skilled in the art. For example, arginine, histidine and lysine of hydrophilic-basic amino acids and may be interchangeable. Similarly, isoleucine, a hydrophobic amino acid, may be replaced by leucine, methionine or valine. Such changes should have little or no effect on the apparent molecular weight or isoelectric point of the nanobodies. A natural amino acid can be replaced by an unnatural amino acid, such as an amino acid in D-configuration, beta-amino acid or gamma. Examples of conservative substitutions are shown in Table 1 below.

Table 1: Conservative Substitutions I

Alternatively, the conservative amino acids can be grouped as described in Lehninger (1975, Biochemistry, 2nd Edition, Worth Publishers, Inc. New York: NY, pp. 71-77), as shown in Table 2 below.

Table 2: Conservative Substitutions II

According to another alternative, examples of conservative substitutions are shown in Table 3 below.

Table 3: Conservative Substitutions III

Residue of origin Example of substitution

A V L I

R K Q N

N Q H K R

D E

C S

G N

E D

H N Q K R

I L V M A F

L I V M A F

K R Q N

M L F I

F L V I A

P G

ST τ s

w Y

Y W F T S

V I L M F A

These functionally conservative variants retain the ability to bind the Tau protein, in particular the Tau protein in oligomeric form and optionally in the form of fibers. Preferably, these functionally conservative variants have a binding affinity with Tau, in particular with Tau in the form of an oligomer and optionally in the form of fibers, which is equal to or greater than that of the corresponding nanobody.

Knowing the amino acid sequence of the nanobodies of interest, those skilled in the art are capable of producing the nanobodies according to the invention, in particular the 2C5 and S2T2M2 E6 nanobodies defined above, by conventional techniques for the production of polypeptides. . For example, they can be synthesized using the well known method of solid phase synthesis (Merrifield (1996) Proc Soc Ex Boil 21: 412 Merrifield (1963) J. Am Chem Soc 85: 2149 Tarn et al (1983) J Am Chem Soc 105: 6442), preferably using a commercially available peptide synthesizer (such as that made by Applied Biosystems, Foster City, Calif.) And following the manufacturer's instructions.

Alternatively, the nanobodies according to the invention can be synthesized by recombinant DNA techniques well known to those skilled in the art (Maniatis et al., (1982) Molecular Cloning: a laboratory manual, Cold Spring Harbor Laboratories, NY, 51- 54 and 412-430). For example, they can be obtained as DNA expression products after incorporation of DNA sequences encoding the polypeptide of interest into expression vectors and introduction of these vectors into appropriate prokaryotic or eukaryotic hosts which will express the polypeptide. of interest, from which they can then be isolated using techniques well known to those skilled in the art. As is well known to those skilled in the art, when a protein is synthesized by recombinant DNA techniques, it is generally synthesized associated with a label facilitating its purification. Such labels are well known to those skilled in the art and include, for example, hexahistidine (6His), glutathione-S-transferase (GST), myc tag or hemagglutinin of influenza virus (HA). Preferably, the nanobodies according to the invention comprise a myc and / or hexahistidine label. Accordingly, an object of the invention is also constituted by a nanobody comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NO: 9, and SEQ ID NO: 10 further comprising at their C-terminus or N-terminus, preferably at their C-terminus, a myc tag and / or six histidine residues, more preferably a myc tag and six histidine residues. As is well known to those skilled in the art, when a protein is associated with a label facilitating its purification, such a protein comprises between the native sequence and this label a sequence allowing an enzymatic cleavage between the protein and this label. A nanobody comprising or consisting of the amino acid sequence selected from the group consisting of SEQ ID NO: 19 and SEQ ID NO: 20 is also part of the invention.

Another subject of the invention relates to a nucleic acid comprising a nucleic sequence coding for the nanobody according to the present invention.

In a particular embodiment, the nucleic acid according to the invention comprises or consists of a nucleic sequence encoding a nanobody defined by one of the amino acid sequences SEQ ID NO: 9 or SEQ ID NO: 10. preferred, the nucleic acid according to the invention comprises or consists of a nucleic acid sequence encoding the nanobodies defined by the amino acid sequence SEQ ID NO: 9.

Typically, said nucleic acid is a DNA or RNA molecule, which can be included in any suitable vector, such as a plasmid, a cosmid, an episome, an artificial chromosome, a phage or a viral vector .

The terms "vector", "cloning vector" and "expression vector" mean the vehicle by which the DNA or RNA sequence can be introduced into the host cell, so as to transform the host and promote the expression (eg transcription and translation) of the introduced sequence.

Therefore, another object of the invention relates to a vector comprising a nucleic acid according to the invention.

Such vectors may include regulatory elements, such as a promoter, activator, terminator, etc., to cause or direct the expression of the polypeptide. Examples of promoters and enhancers used in animal cell expression vectors include the SV40 early promoter and activator (Mizukami et al (1987) J. Biochem 101: 1307-1310), the LTR promoter. and Moloney mouse leukemia virus activator, the promoter (Mason et al (1985) Ce / 41: 479-487) and the enhancer (Gillies et al (1983) Cell 33: 717- 728) of the immunoglobulin chain, etc.

Any expression vector for animal cells can be used. Examples of suitable vectors include pAGE107 (Miyaji et al (1990) Cytotechnology 3: 133-140), pAGE103 (Mizukami et al (1987) J. Biochem 101: 1307-1310), pHSG274. (Brady et al (1984) Gene 27: 223-232), pKCR (O'Hare et al (1981) Proc Natl Acad Sci USA 78: 1527-1531), pSG1 beta d2-4 (Miyaji et al (1990) Cytotechnology 3: 133-140), etc.

Other examples of plasmids include replicative plasmids comprising an origin of replication, or integrative plasmids, such as for example pUC, pcDNA, pBR, etc.

Other examples of viral vectors include adenoviral, retroviral, herpesvirus and AAV vectors. Such recombinant viruses can be produced by techniques well known to those skilled in the art, such as by transfection of packaging cells or by transient transfection with plasmids or helper viruses. Typical examples of virus packaging cells include PA317 cells, PsiCRIP cells, GPenv + cells, 293 cells, and the like. Detailed protocols for producing such replication-deficient recombinant viruses can be found, for example, in WO 95/14785, WO 96/22378, US 5,882,887, US 6,013,516, US 4,861,719, US 5,278,056, and WO 94/19478.

Another object of the present invention relates to a cell which has been transfected, transduced or transformed with a nucleic acid and / or a vector according to the invention.

The terminating "transformation" means the introduction of a "foreign" (extrinsic or extracellular) DNA or RNA gene or sequence into a host cell, such that the host cell will express the gene. or the sequence introduced to produce the substance of interest, typically a protein encoded by the gene or sequence introduced. A host cell that receives and expresses the introduced DNA or RNA has been "transformed".

The nucleic acids according to the invention can be used to produce a nanobody according to the invention in an appropriate expression system. The term "expression system" means a host cell and a compatible vector under appropriate conditions, e.g. for the expression of a protein encoded by the foreign DNA carried by the vector and introduced into the host cell.

Conventional expression systems include the host cells Escherichia co // ^'and plasmid vectors, insect host cells and Baculovirus vectors, and mammalian host cells and vectors. Other examples of host cells include prokaryotic cells (such as bacteria), and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.). Specific examples include Escherichia coli, Kluyveromyces or Saccharomyces yeasts, mammalian cell lines (eg Vero cells, CHO cells, 3T3 cells, COS cells, etc.) as well as primary or established mammalian cell cultures. (eg produced from lymphoblasts, fibroblasts, epithelial cells, nerve cells, adipocytes, etc.). Examples also include mouse SP2 / 0-Ag14 cells (ATCC CRU 581), mouse P3X63-Ag8.653 cells (ATCC CRU 580), CHO cells in which a dihydrofolate reductase gene is defective, YB2 cells / Rat 3HL.P2.G1 1 .16Ag.20 (ATCC CRU 662), etc.

The present invention also relates to a method for producing a recombinant host cell expressing a nanobody according to the invention, said method comprising the steps of:

(i) introducing in vitro or ex vivo a nucleic acid as described above into a competent host cell,

(ii) culturing in vitro or ex vivo the recombinant host cell obtained and

(iii) optionally selecting the cells that express and / or secrete said nanobody. Such recombinant host cells can be used for the production of nanobodies according to the invention.

The nanobodies according to the invention may be produced by any technique known to those skilled in the art, such as for example any chemical, biological, genetic or enzymatic technique, alone or in combination.

In particular, the invention furthermore relates to a method for producing a nanobodies according to the invention, said method comprising the steps of:

(i) culturing a transduced or transfected or transformed cell according to the invention under conditions suitable to allow the expression of said nanobody, and

(ii) recover the expressed nanobodies.

The nanobodies according to the invention can be conveniently separated from the culture medium by conventional immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxilapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

Nanobodies marked

The nanobodies according to the invention are particularly useful for medical imaging. In this context, it is particularly advantageous to have nanobodies associated with a detectable marker. The present inventors have shown that the 2C5 and S2T2M3_E6 nanobodies, in particular 2C5, retained their properties when they were associated with a detectable marker.

The present invention therefore also relates to a nanobody as defined above linked to a detectable marker.

By "nanobodies bound to a detectable marker" is meant herein that the detectable label is directly or indirectly linked to the nanobody, for example via a cleavable or non-cleavable linker peptide, or is incorporated into the nanobody. The detectable marker may in particular be bound to the nanobodies by substitution (for example, by substituting an H for an I at the tyrosine residues), by complexing or by chelation.

By "detectable marker" is meant here a compound that produces a detectable signal. When it is associated with a tracer, it makes it possible to follow the fate of the tracer in the body. The detectable marker may be an MRI contrast agent, a scintigraphic contrast agent, an X-ray contrast agent, an ultrasound contrast agent, an optical imaging contrast agent. Examples of detectable markers include radioelements, fluorophores such as fluorescein, Alexa, cyanine; chemiluminescent compounds such as luminol; bioluminescent compounds such as luciferase or alkaline phosphatase; and contrast agents such as nanoparticles or gadolinium. The choice of the appropriate detectable marker, which depends on the detection system used, is within the abilities of those skilled in the art. For example, when the detection system is MRI, the detectable label is preferably an iron oxide nanoparticle or gadolinium; when the detection system is fluorescence imaging, the detectable marker is preferably Fluorescein, Alexa or cyanine; when the detection system is chemiluminescence imaging, the detectable label is preferably luminol; when the detection system is bioluminescence imaging, the detectable label is preferably luciferase or alkaline phosphatase; when the detection system is nuclear imaging, the detectable marker is preferably a radioelement such as gallium ( ⁶⁸ Ga) for PET imaging, or technetium 99m ( ^99m Tc) for SPECT imaging.

Preferably, the detectable marker is a radioelement. Examples of radioelements, which are more particularly used in nuclear imaging techniques, include Technetium 99m ( ^99m Tc), Iodine 123 ( ¹²³ I), 125 I ( ¹²⁵ I), and fluorine 18 ( ¹⁸ F), gallium 68 ( ⁶⁸ Ga), and any other radioelements usable in humans. Therefore, preferably, the radioelement is selected from the group consisting of ^99m Tc, ¹²³ l, ¹²⁵ l, ¹⁸ F, and ⁶⁸ Ga. Most preferably, the radioelement is ^99m Tc or ⁶⁸ Ga, still favorite ^99m Tc.

Use as a contrast agent

The inventors have demonstrated that the 2C5 nanobody allows specific labeling of the hippocampus, entorhinal cortex and temporal cortex cell bodies in patients suffering from Alzheimer's disease and in pure tauopathy. The inventors have therefore demonstrated that the nanobodies of the invention, in particular 2C5, constitute tracers specific for the pathological forms of the Tau protein, in particular early pathological forms of Tau, for example Tau in the form of oligomer and optionally in the form of fibers, and allow its detection by imaging.

The invention therefore proposes a nanobody as defined above for its use as a contrast agent in medical imaging, in particular in vivo, non-invasive medical imaging.

It also relates to the use of a nanobody as defined above for the manufacture of a contrast agent useful for medical imaging, in particular in vivo, non-invasive medical imaging.

By "contrast agent" is meant here a substance or a composition which, administered in the body, makes it possible to detectably mark organs or structures (tissue, cell, receptor) which, without a contrast agent, are or not visible in medical imaging. By extension, the term "contrast agent" is used to designate a tracer associated with a marker as defined above.

In the context of the invention, "imaging methods" refer to methods that allow the visualization of the interior of an organism or organs of an organism. Examples of imaging methods include invasive techniques such as endocoronary ultrasound, and non-invasive techniques such as magnetic resonance imaging, x-ray imaging, ultrasound, optical imaging, or nuclear medicine such as scintigraphy, especially single photon emission computed tomography (SPECT) and positron emission tomography (PET). Preferably, the imaging method according to the invention is scintigraphy, in particular SPECT or PET scintigraphy.

The scintigraphy is based on the administration (usually intravenously) of a contrast agent, also called radiopharmaceutical, consisting of a tracer labeled with a radioelement. The specific localization of this contrast agent in the body is then determined by detection of gamma or beta emitted radiation.

Single-photon emission computed tomography (PET) and positron emission tomography (PET) are scintigraphic tomography-based nuclear tomographic imaging techniques that provide three-dimensional images and reconstructions of organs and their metabolism by means of a set of cameras that revolve around the patient.

The present invention also relates to a medical imaging method, in particular non-invasive in vivo medical imaging, in which a nanobody as defined above is administered to a patient. The medical imaging method according to the invention may further comprise the steps of detecting the link or the absence binding nanobodies in patient's body areas and viewing patient's body areas in which nanobody binding can be detected.

In the context of the invention, a "patient" designates a human or non-human mammal, such as a rodent (rat, mouse, rabbit), a primate (chimpanzee), a feline (cat), a canine (dog ). Preferably, the individual is human.

In a particular embodiment, the term "patient" refers to a human who exhibits symptoms associated with tauopathy. Depending on the tauopathy, these symptoms may be for example Parkinson's syndrome, axial dystonia, capricious hand phenomenon, or cognitive disorders of the patient.

Any method of administration known to those skilled in the art can be used to administer the nanobody according to the invention to the patient. In particular, the nanobody can be administered, for example, orally, by inhalation or parenterally (in particular by intravenous injection). When the parenteral route is chosen, the nanobody can be in the form of injectable solutions and suspensions, packaged in ampoules or flasks. The forms of parenteral administration are conventionally obtained by mixing the nanobodies according to the invention with buffers, stabilizers, preservatives, solubilizing agents, isotonic agents and suspending agents. According to known techniques, these mixtures can be sterilized and packaged in the form of intravenous injections. Those skilled in the art may for example use buffers based on phosphate salts as buffers. Examples of suspending agents include methylcellulose, acacia, and sodium carbocymethylcellulose. Examples of stabilizers include sodium sulfite and sodium metasulfite, and examples of preservatives include sodium p-hydroxybenzoate, sorbic acid, cresol and chlorocresol.

The amount of nanobodies administered depends naturally on the route of administration, the size and / or weight of the patient, and the detection technique used.

In the context of the invention, the term "body area" refers to a specific region of the body. It may be, for example, an organ, a part of an organ or a tissue, such as the brain, in particular the hippocampus, the entorhinal cortex or the temporal cortex.

In a particular embodiment, the nanobody according to the invention is used as a contrast agent in medical imaging to visualize the Tau protein in pathological form, in particular to visualize Tau in the form chosen from oligomers, fibers, helicoidal pairs of filaments and straight filaments, of oligomers and fibers, more preferably oligomers, in a patient.

In one embodiment, the nanobody according to the invention is used as a contrast agent in medical imaging to visualize neurofibrillary tangles (DNFs) in a patient.

In one example, cortical distribution and the number of, for example, DNFs are correlated with memory disorders in patients with, for example, AD.

Diagnostic use

The appearance of pathological forms of Tau protein, as defined above, is a marker of tauopathy. In addition, the detection of pathological forms of Tau, in particular early pathological forms, such as Tau protein in the form of oligomers and possibly fibers, makes it possible to identify the presence of the toxic forms involved in the propagation or development of Tau. one of the tauopathies. The identification of pathological forms, in particular early pathological forms, such as Tau in the form of oligomers and possibly fibers, therefore constitutes the characterization of a tauopathy and / or the identification of the beginning of a tauopathy. On the other hand, the possibility of imaging the evolution, that is to say the progression or regression, of a pathological form of Tau previously identified represents a modality for evaluating the efficacy of a therapeutic treatment. in a patient diagnosed with tauopathy.

The invention therefore also relates to a nanobody as defined above for its use in diagnostic or prognostic methods.

By "diagnostic method" or "diagnosis" is meant here a method for determining whether an individual suffers from a pathology.

By "prognostic method" or "prognosis" is meant here a method for determining whether an individual is at risk of developing a pathology.

Preferably, the nanobody as defined above is used for the diagnosis or prognosis of a tauopathy.

The tauopathies are as defined above. Preferably, the nanobody according to the invention is used for the diagnosis or prognosis of Alzheimer's disease.

The presence, for example, of Tau in oligomeric form exposes the subject to a risk of developing tauopathy, especially Alzheimer's disease. The nanobody according to the invention can therefore be used to detect a risk of occurrence of tauopathy, in particular Alzheimer's disease, in a patient. By "risk of occurrence" is meant here the probability that an individual develops a pathology.

The present invention also relates to a method for diagnosing a tauopathy and / or detecting a risk of occurrence of tauopathy in a patient, said method comprising the steps of administering a nanobody as defined above to said patient and detecting said nanobodies in the body of said patient, detecting a preferential location of said nanobody in the brain indicative of tauopathy and / or risk of occurrence of tauopathy.

In one embodiment, said method further comprises the steps of administering an amyloid plaque marker to said patient and detecting said amyloid plaque marker in said patient's body, detecting a preferential location of said nanobody and said marker amyloid plaques in the brain indicative of tauopathy and / or risk of occurrence of tauopathy, especially Alzheimer's disease.

Consequently, the present invention also relates to a method for diagnosing a tauopathy, in particular Alzheimer's disease and / or to detecting a risk of occurrence of tauopathy, in particular Alzheimer's disease, in a patient. patient, said method comprising the steps of:

i) administering a nanobody according to the invention to said patient and detecting said nanobodies in the body of said patient, and

ii) administering an amyloid plaque marker to said patient and detecting said amyloid plaque marker in the body of said patient,

detecting a preferential location of said nanobody and said amyloid plaque marker in the brain indicative of tauopathy, particularly Alzheimer's disease and / or risk of occurrence of tauopathy, particularly of Alzheimer's disease.

In one embodiment, these methods of diagnosing tauopathy are in vivo methods.

By "amyloid plaque marker" is meant tracers bound to a detectable marker which binds specifically to amyloid plaques and is useful as a contrast agent in non-invasive, in vivo medical imaging. Markers of amyloid plaques are, for example tracer ^18F-18F as florbetapir, florbetaben 18F, 18F and 18F-flutemetanol-AZD4694 bookmark.

It is described in the prior art and it is therefore known to those skilled in the art, according to particular tauopathies, which brain region is affected by the presence of pathological forms of Tau. The localization in the brain of degeneration neurofibrillary or other pathological form of Tau and possibly amyloid plaques is described for several tauopathies, for example in Catafau, AM and Bullich, S (Clin Transi Imaging, 2015; 3 (1): 39-55. Epub 2015 Jan 21 .) or in Tranchant, C. (Medicine / Science A1997; 13: 989-97).

By "preferential localization" it is meant that the amount of nanobodies detected in the brain, in particular for example at the levels of the hippocampus, frontal neocortex, entorhinal cortex and temporal cortex cell bodies, is greater than the background noise which corresponds to a non-specific localization of the nanobody in the body.

For example, progressive supranuclear paralyzation is characterized by the presence of DNF in the brain stem and frontal neocortex.

In another example, corticobasal degeneration is characterized by the presence of DNF in the parietal cortex.

In another example, Alzheimer's disease is characterized by the presence of DNF at enthorino-cortical structures.

The invention also relates to the nanobody according to the invention for its use for the therapeutic monitoring of a tauopathy in a subject in whom a tauopathy has been diagnosed.

It also relates to the use of the nanobodies according to the invention for the manufacture of a contrast agent useful for the therapeutic monitoring of a tauopathy in a subject in whom a tauopathy has been diagnosed.

By "therapeutic monitoring" is meant here the observation of the subject's response to the treatment administered to him. The therapeutic effect of a treatment is generally associated with slowing or inhibiting the progression of a disease, a reversion of the disease, or one or more symptoms associated with this disease. Conversely, an absence of a therapeutic effect can result in a stability or an acceleration of the progression of the disease or one or more of its symptoms. For example, if a tauopathy was diagnosed because of the presence of pathological forms of Tau, the therapeutic follow-up can be realized by observing the disappearance, the regression, the maintenance, or the growth of the pathological forms of Tau. Thus the use according to the invention may comprise the steps of:

a) administering a nanobody as defined above to a subject in whom a pathological form of Tau has been detected;

b) detecting the binding of the nanobody to said pathological form of Tau; c) repeating steps a) and b) before and after administration of a treatment to said subject; an absence or a decrease in the binding of the nanobody to said Tau pathological form indicative of a treatment having a therapeutic effect.

The "pathological form of Tau" is as defined in the "tauopathy" section above.

Preferably the treatment is a treatment of a taupopathy and comprises for example the use of inhibitors of Tau aggregation. In another example, the treatment of a taupopathy includes the use of the nanobodies of the invention.

Use as medicine

Recently, pathological forms of Tau, for example extracellular toxic forms, have been shown to represent the toxic forms of Tau (Usenovic et al., 2015) involved in the propagation (Goedert et al., 2014) of such tauopathies. than AD in the brain, by contamination of the neurons. Consequently, targeting the toxic forms of Tau to inhibit their aggregation and thus the formation of helical paired filament pairs (PHFs) and straight filaments (SFs) is a promising treatment for tauopathies, particularly Alzheimer's disease.

Accordingly, the invention further relates to the use of a nanobody as defined above for the manufacture of a medicament, in particular a medicament for the treatment of tauopathy. A method of treatment comprising administering a therapeutically effective amount of the nanobody as defined above to a patient in need thereof is also part of the present invention.

The use of an anti-Tau nanobodies therefore makes it possible to inhibit the progress of Tau aggregation.

By "treatment" of a tauopathy is meant the "therapeutic treatment" (or curative) of a tauopathy, which includes slowing or inhibiting the evolution of a tauopathy. The "prophylactic treatment" of tauopathy is also understood to include the prevention of DNF formation.

"Prevention" refers to preventing or delaying the onset or decreasing the intensity of the clinical or biochemical manifestations associated with tauopathy.

The person skilled in the art knows, from his general knowledge, to determine what are the clinical or biochemical manifestations associated with a given tauopathy and which are likely to be improved (treatment) or prevented, delayed or diminished in intensity (prevention). In the context of tauopathies, a biological parameter of interest may be the presence and location of Tau in pathological form, in particular Tau in early pathological form, such as Tau protein in the form of oligomers and optionally Tau in the form of fibers. The invention more particularly relates to the nanobody as defined above for its use for the treatment of tauopathy and / or the prevention of tauopathy, preferably for the treatment and / or prevention of the disease of tauopathy. Alzheimer.

The invention also relates to the use of a nanobody as defined above for the manufacture of a medicament for the treatment of tauopathy and / or the prevention of tauopathy in a patient likely to present a tauopathy .

The invention also relates to a method of treating tauopathy and / or preventing tauopathy in a patient in need thereof, comprising administering a therapeutically effective amount of a nanobody as defined above to a patient in need.

Preferably, the nanobodies according to the invention are used to treat the pathological forms of Tau, in particular the early pathological forms, such as the Tau oligomer forms and optionally the Tau fiber forms.

The nanobody according to the invention can be administered, for example, orally, by inhalation, parenterally (in particular by intravenous injection), in a suitable form. When the parenteral route is envisaged, the nanobody can be in the form of injectable solutes and suspensions packaged in ampoules or flasks. Forms for parenteral administration are conventionally obtained by mixing the nanobody with buffers, stabilizing agents, preservatives, solubilizing agents, isotonic agents and suspending agents. In accordance with known techniques, these mixtures are then sterilized and then packaged in the form of intravenous injections. As a buffer, those skilled in the art may use buffers based on organic phosphate salts. Examples of suspending agents include methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, acacia and sodium carboxymethylcellulose. In addition, useful stabilizers according to the invention are sodium sulfite and sodium metasulfite, while sodium p-hydroxybenzoate, sorbic acid, cresol and chlorocresol can be mentioned as preservatives.

The amount of nanobodies administered depends naturally on the mode of administration, the size and / or weight of the patient, and the nature of the cytotoxic agent that may be associated with it.

The present invention also relates to a pharmaceutical composition comprising a nanobody as defined above in association with a pharmaceutically acceptable vehicle. The term "pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions which do not produce side, allergic or otherwise objectionable reactions when administered to a mammal, particularly a human.

In the context of the invention, the term "pharmaceutically acceptable carrier" includes any solvent, dispersion medium, coating, antibacterial or antifungal agent, isotonic or absorption delaying agent, and the like. The use of such media and agents for pharmaceutically active substances is well known to those skilled in the art. With the exception of the case where a medium or conventional agent is incompatible with the active ingredient, its use in pharmaceutical compositions is envisaged. Additional active ingredients may also be incorporated into the compositions.

Use to detect in vitro Tau

The present invention also relates to the use of a nanobody as defined above for the in vitro detection of Tau protein as oligomers in a sample.

By "sample" is meant here a part of a larger element. Preferably, the sample is a substance of biological origin. Examples of biological samples include, but are not limited to, parts of organs or tissues such as the brain, particularly the hippocampus, the entorhinal cortex or temporal cortex, blood, particularly blood brain, the cerebrospinal fluid. Preferably a sample in the context of the invention relates to a brain sample. The present invention will be further illustrated by the figures, sequences and the example below.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO: 1 shows the consensus amino acid sequence of CDR1 of the nanobodies of the invention wherein the amino acid Xi is S or R, X ₂ is D or Y and X ₃ is T or A.

SEQ ID NO: 2 shows the consensus amino acid sequence of CDR2 of the nanobodies of the invention wherein the amino acid Xi is P or R and X ₂ is S or V.

SEQ ID NO: 3 shows the amino acid sequence of CDR1 of 2C5 nanobody.

SEQ ID NO: 4 shows the amino acid sequence of CDR2 of the 2C5 nanobody.

SEQ ID NO: 5 shows the amino acid sequence of CDR3 of the 2C5 nanobody. SEQ ID NO: 6 shows the amino acid sequence of CDR1 of the S2T2M3_E6 nanobody. SEQ ID NO: 7 shows the amino acid sequence of CDR2 of the S2T2M3_E6 nanobody. SEQ ID NO: 8 shows the amino acid sequence of CDR3 of the S2T2M3_E6 nanobody. SEQ ID NO: 9 shows the amino acid sequence of the 2C5 nanobody variable region.

SEQ ID NO: 10 shows the amino acid sequence of the variable region of the S2T2M3_E6 nanobody.

SEQ ID NO: 11 shows the amino acid sequence of the FR1 region of the 2C5 nanobody. SEQ ID NO: 12 shows the amino acid sequence of the FR2 region of the 2C5 nanobody. SEQ ID NO: 13 shows the amino acid sequence of the FR3 region of the 2C5 nanobody. SEQ ID NO: 14 shows the amino acid sequence of the FR4 region of the 2C5 nanobody. SEQ ID NO: 15 shows the amino acid sequence of the FR1 region of the S2T2M2_E6 nanobody.

SEQ ID NO: 16 shows the amino acid sequence of the FR2 region of the S2T2M2_E6 nanobody.

SEQ ID NO: 17 shows the amino acid sequence of the FR3 region of the S2T2M2_E6 nanobody.

SEQ ID NO: 18 shows the amino acid sequence of the FR4 region of the S2T2M2_E6 nanobody

SEQ ID NO: 19 shows the amino acid sequence of the variable region of 2C5 nanobodies with a myc tag and six histidine residues.

SEQ ID NO: 20 shows the amino acid sequence of the S2T2M3_E6 nanowind variable region with a myc tag and six histidine residues.

SEQ ID NO: 21 shows the amino acid sequence of the 2N4R isoform of Tau protein, also called Tau-F, with 441 amino acids.

SEQ ID NO: 22 shows the amino acid sequence of the N4R 1 isoform of the Tau protein, also called Tau-E, with 412 amino acids.

SEQ ID NO: 23 shows the amino acid sequence of the 0N4R isoform of Tau protein, also called Tau-D, with 383 amino acids.

SEQ ID NO: 24 shows the amino acid sequence of the 2N3R isoform of Tau protein, also called Tau-C, with 410 amino acids.

SEQ ID NO: 25 shows the amino acid sequence of the 1 N3R isoform of the Tau protein, also called Tau-B, with 381 amino acids.

SEQ ID NO: 26 shows the amino acid sequence of the 0N3R isoform of Tau protein, also called Fetal-Tau, with 352 amino acids. SEQ ID NO: 27 shows the amino acid sequence of the truncated Tau protein consisting of the 3R region of the Tau protein.

DESCRIPTION OF THE FIGURES FIG. 1: Graph which shows the results of the ELISA tests and therefore of the affinity curves of the 2C5 nanobodies for Tau-0 (Tau oligomers), Tau-F (tau fibers), Mimes (the R3 region of Tau polymerized) and Tau-N (native tau).

Figure 2: Graph showing the lack of affinity for the polymerized Abeta1 -42 amyloid peptide

Figure 3: Comparative immunohistochemistry on sections of temporal cortex of a case of AD; A) AT8 is a monoclonal antibody directed against phosphorylated PHFs in Ser2O2 and Thr205; B) 2C5 is the nanobodies of the invention directed against Tau as oligomers C) T22 is a monoclonal antibody directed against tau oligomers, D) Ab4G8 is a monoclonal antibody directed against aggregated forms of amyloid beta peptide).

Figure 4: Alignment of the variable sequences of the nanobodies of the invention. Figure 5: Graph which shows the results of the ELISA tests and thus affinity curves of the 2C5 nanobodies (without radiolabeling) for polymers of the R3 sequence of the tau protein.

Figure 6: Graph which shows the results of the ELISA tests and thus the affinity curves of the 2C5 nanobodies (radiolabeled with iodine 125) for polymers of the R3 sequence of the tau protein.

EXAMPLE

1) Generation of nanobodies:

(1) Preparation of antigens necessary for the immunization of camelids:

The cDNA (htau40) encoding the longest Tau isoform (441 aa-45.8 kDa) was cloned into plasmid pRK172, downstream of the T7 RNA polymerase promoter. The recombinant plasmids were transformed into Escherichia coli BL21 bacteria (obtained from Goedert, M et al., Neuron, 3 (4): 519-526 (1989)) (bacteria provided). by the team of Prof. Baulieu, INSERM, Paris). Purification of the protein is carried out by two FPLC (Fast Protein Liquid Chromatography) steps: (1) by cation exchange column loaded with SO 4 - (HiTrap SP-XL, Thermoscientfic) followed by (2) by exclusion chromatography ( HP Sepharose Hi Trap column - Amersham Biosciences). To obtain enriched preparations with different polymerized forms of tau (oligomer and fiber) mimicking Tau fibrillation in vivo, we used the method of Haase et al. (Journal of Neurochemistry, 88 (6): 1509-1520, 2004) modified by Flach et al. (Journal of Biological Chemistry, 2012, (287) 52: 43223-43233). Fibrillation of Tau (40 μl) is carried out at 37 ° C. in MOPS buffer (20 mM 3- (N-morpholino) propane sulphonic acid), pH 7 in the presence of heparin (10 μl) of NaN 3 (4%). for a final volume of 1.5 mL [21] [14]. Fibrillation is analyzed as described above. Fibrillation is stopped by freezing the samples at -80 ° C after 48 hours and 72 hours. The duration of the polymerization process is suitable for the production of (a) oligomers and (b) fibers. In parallel, a peptide covering the R3 region of the Tau repeats was synthesized and polymerized in a similar manner. In particular, the fibrillation of R3 (0.4 μl) is carried out at 37 ° C. in PBS buffer (50 mM saline phosphate buffer, at pH 7 in the presence of heparin (0.4 μl), NaN 3 (4%) for a volume of The fibrillation is stopped by freezing the samples at -80 ° C after typically 72 hours to obtain truncated Tau (R3) in the form of fibers.

(2) Immunization of llamas:

A llama was immunized under the same conditions with a mixture of oligomers and Tau fibers as well as polymers of R3 ((a), (b) (c)). Three successive injections are performed in days 0, 9, 18. The blood samples are taken in days 28 and 42. The blood bags are centrifuged in a Ficoll gradient in order to separate and isolate the lymphocytes and the total RNAs are extracted.

(3) Production and screening of nanobodies: They comprise several stages:

- Amplification and production of mRNA of lymphocytes of the llama

- Generation of a library of phages expressing the nanobodies of the previously immunized dromedary.

- Selection, by ELISA and FACS, of phage expressing anti-tau-oligomeric nanobodies.

- Sequencing of selected clones and identification of distinct clones

- Determination of Nbs sequences, construction and production of the mRNA fragment encoding a Nb comprising a Myc tag and histidine in Cterm

- Insertion in bacteria and quantity production of Nb.

2. In vitro characterization of nanobodies:

Nanobodies have been characterized for their affinity and specificity through two stages.

a) ELISA I tests: with the starting antigens either tau-oligomer enriched preparation, tau fiber-enriched preparation, polymerized R3, as well as the native tau protein and beta amyloid peptide fibers (made from commercial peptide). The affinity constants of the nanobodies have been determined for each of these immunogens. (b) Immunohistochemistry: on sections of paraffin tissue from human pathology (collaboration with the Department of Mental Health and Psychiatry in Geneva). These cuts cover different cases of more or less severe tauopathies,

vs)

The tissue sections are deparaffinized and then treated in floating sections after permeabilization in 0.2% PBS-Triton buffer and unmasking of the antigen using a commercial unmixing solution (Vector H-3300). The primary antibody (Nb) is applied at different concentrations (≥ 20 nM) overnight at 4 ° C. After rinsing, a rabbit anti-histdine antibody is applied for 1 h at room temperature and then a third goat anti-rabbit antibody for 1 h is finally applied after rinsing. The revelation is carried out through two incubation steps in the presence of avidine biotin complex (Vector ABC kit) and then di-amino-benzidine (Vector ABC kit). Control sections are systematically performed in the absence of the first antibody (Nb). The sections are mounted on a slide and counterstained with hematoxylin. d) ELISA II Tests: The nanobody 2C5 was radiolabeled with iodine 125. The affinity tests for polymers of the R3 sequence of the tau protein are carried out in ELISA, in a phosphate buffered saline buffer (NaCl, 137 mM; KCl 2.7mM Na2HPO4, 10mM KH2PO4, 1.8mM BSA 1%; pH = 7.5. The ELISAs are performed before (FIG. 5) and after radiolabelling (FIG. 6). It is found that after radiolabelling, the ligand retains a good affinity for its target (FIG. 6). 3. Results:

We have produced several single variable domain camelide antibodies (Nbs), specifically directed against early pathological forms of Tau protein (oligomers and fibers) of low molecular weight (15 kDa): 2C5 (VHH), S2T2M3 E6 (VHH) .

Nb 2C5 was sequenced and characterized as VHH and characterized in vitro. It has respective affinities (Kd) of 5nM, 6nM and 50nM for oligomers, fibers and truncated polymerized forms of Tau. This Nb binds neither native tau protein (Kd> 1800 nM) nor beta amyloid peptide fibers (Kd> 10,000 nM). (Figs 1 and 2). This Nb has been tested in immunohistochemistry on sections of human brains from anatomopathology (Alzheimer's of different grades and tauopathy). It demonstrates specific staining of hippocampal, entorhinal and temporal cortex cell bodies in patients with Alzheimer's disease and in dementia with pure tauopathy (Table 4, Figure 3).

The technetium-99m radiolabelling of this SdAbs has been successfully performed.

Nb S2T2M3 E6 was selected for its affinity for low molecular weight tau polymers.

Table 4: Cases of human pathology tested for 2C5

Type Abbreviation Stage de

Age Sex CDR MMS pathology type Break

Aging

VS ? ? 1? ? normal

AD AD? ? 5? ?

AD AD 74 F 5 na 15/30

AD AD 81 F 5 na 17/30

Tangle only

T 73 M 4 na na dementia

PSP / CBD PSP / CBD? ? No ? ?

Claims

claims

1. Nanobodies directed against the Tau protein, said Tau protein being in the form of oligomers and said nanobodies being devoid of light chain.

The nanobody according to claim 1, said nanobodies competing for binding to the Tau protein as oligomers with a nanobodies comprising the amino acid sequences.

(i) GRTFSXiX ₂ X ₃ (SEQ ID NO: 1) as CDR1 in which the amino acid Χ _Ί is S or R, X ₂ is D or Y and X ₃ is T or A,

(iii) NRDPKYGNTRY (SEQ ID NO: 5) or TARRRISGTPQWHY (SEQ ID NO: 8) as CDR3.

The nanobody according to claim 1 or 2, said nanobodies compete for binding to the Tau protein as oligomers with a nanobodies selected from nanobodies comprising:

b) the amino acid sequences (i) GRTFSRYA (SEQ ID NO: 6) as CDR1, (ii) ISRSGGST (SEQ ID NO: 7) as CDR2 and (iii) TARRRISGTPQWHY (SEQ ID NO: 8) as CDR3.

4. Nanobodies according to any one of claims 1 to 3, said nanobodies comprising the amino acid sequences

(iii) NRDPKYGNTRY (SEQ ID NO: 5) or TARRRISGTPQWHY (SEQ ID NO: 8) as CDR3.

The nanobody according to any one of claims 1 to 4, said nanobody comprising: a) the amino acid sequences (i) GRTFSSDT (SEQ ID NO: 3) as CDR1, (ii) ISPSGGVT (SEQ ID NO: 4) as CDR2 and (iii) NRDPKYGNTRY (SEQ ID NO: 5) as CDR3; or

8) as CDR3; or

The nanobody according to any one of claims 1 to 5, said nanobodies comprising an amino acid sequence selected from the group consisting of amino acid sequences SEQ ID NO: 9 or SEQ ID NO: 10, preferably SEQ ID NO: 9.

The nanobody according to any one of claims 1 to 6, said nanobodies being bound to a detectable marker.

8. Nanobodies according to claim 7, said detectable marker being a radioelement.

9. Nanobody according to any one of claims 1 to 8 for its use as a contrast agent in non-invasive, in vivo medical imaging.

The nanobody according to any one of claims 1 to 9 for use in diagnostic or prognostic methods, preferably Tauopathies.

11. Nanobody according to any one of claims 1 to 6 for its use as a medicament.

12. Use of a nanobody as defined in any one of claims 1 to 6 for the in vitro detection of Tau protein as oligomers in a sample.

13. A pharmaceutical composition comprising a nanobody as defined in any one of claims 1 to 6 in association with a pharmaceutically acceptable carrier.

14. Nucleic acid comprising a nucleic acid sequence encoding the nanobody as defined in any one of claims 1 to 6.

A vector comprising a nucleic acid as defined in claim 14.

16. A cell transfected, transduced or transformed with a nucleic acid according to claim 14 or a vector according to claim 15.

A method of producing a recombinant host cell expressing a nanobody as defined in any one of claims 1 to 6, said method comprising the steps of:

(i) introducing in vitro or ex vivo a nucleic acid according to claim 14 or a vector according to claim 15 into a competent host cell,

(ii) culturing in vitro or ex vivo the recombinant host cell obtained and

(iii) optionally selecting the cells that express and / or secrete said nanobody.

18. A method of producing a nanobody as defined in any one of claims 1 to 6, said method comprising the steps of:

(i) culturing a transduced or transfected or transformed cell according to claim 16 under conditions suitable to allow the expression of said nanobody, and

(ii) recover the expressed nanobodies.