EP3532495A1 - Anti-tau-nanokörper - Google Patents

Anti-tau-nanokörper

Info

Publication number
EP3532495A1
EP3532495A1 EP17798139.6A EP17798139A EP3532495A1 EP 3532495 A1 EP3532495 A1 EP 3532495A1 EP 17798139 A EP17798139 A EP 17798139A EP 3532495 A1 EP3532495 A1 EP 3532495A1
Authority
EP
European Patent Office
Prior art keywords
seq
nanobodies
tau
nanobody
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP17798139.6A
Other languages
English (en)
French (fr)
Inventor
Daniel Fagret
Marcelle MOULIN
Catherine Ghezzi
Pascale Perret
Sabine CHIERICI
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Grenoble Alpes
Centre Hospitalier Universitaire Grenoble Alpes
Original Assignee
Institut National de la Sante et de la Recherche Medicale INSERM
Universite Grenoble Alpes
Centre Hospitalier Universitaire Grenoble Alpes
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institut National de la Sante et de la Recherche Medicale INSERM, Universite Grenoble Alpes, Centre Hospitalier Universitaire Grenoble Alpes filed Critical Institut National de la Sante et de la Recherche Medicale INSERM
Publication of EP3532495A1 publication Critical patent/EP3532495A1/de
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to anti-Tau nanobodies.
  • AD Alzheimer's disease
  • cognitive and memory tests as well as different biomarkers.
  • biomarkers Dubois et al Lancet Neurology 13, 614-629 (2014).
  • 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)).
  • 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).).
  • Nuclear cerebral imaging 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.
  • 18F-AV-1451 (Ossenkoppele et al., Brain 139 (5): 1551-1567 (2016).)
  • 18F-THK51 (Jonasson et al., Journal of Nuclear Medicine, 57 ( 4): 574-581 (2016).)
  • 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.
  • nanobodies or nanobodies 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.
  • Nbs nanobodies or nanobodies
  • 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)). .).
  • 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 ⁇ ⁇ is S or R, X 2 is D or Y and X 3 is T or A ,
  • NRDPKYGNTRY SEQ ID NO: 5
  • TARRRISGTPQWHY SEQ ID NO: 8
  • 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:
  • the present invention also relates to nanobodies directed against the Tau protein, said nanobodies comprising the amino acid sequences:
  • NRDPKYGNTRY SEQ ID NO: 5
  • TARRRISGTPQWHY SEQ ID NO: 8
  • 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.
  • composition comprising this nanobody in association with a pharmaceutically acceptable vehicle.
  • 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.
  • 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.
  • DNFs neurofibrillary tangles
  • 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).
  • PHF helically arranged
  • SF straight filaments
  • DNFs includes the aggregation of Tau, also called “fibrillar aggregation” or “fibrillogenesis”.
  • 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.
  • 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.
  • the intermediate aggregation forms i.e., Tau as oligomers, and Tau as fibers
  • Tau is toxic, with oligomers being the most toxic.
  • 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.
  • the pathological forms of Tau preferably Tau in the form of oligomers, are extracellular.
  • 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.
  • 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.
  • 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 protein 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.
  • 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.
  • 4R microtubule binding domains
  • 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.
  • said Tau protein is selected from the group consisting of 2N4R, 1N4R, 0N4R, 2N3R, 1N3R and 0N3R isoforms.
  • 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.
  • 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.
  • 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.
  • hyperphosphorylation of the Tau protein decreases its affinity for microtubules, which can lead to their destabilization and hence cytoskeletal disruption.
  • this hyperphosphorylation can lead to the aggregation of Tau and thus the formation of DNFs, as described above in the "tauopathy" part.
  • Abnormal phosphorylation consists of phosphorylation at sites that under physiological conditions are not affected by phosphorylation.
  • a non-physiological epitope these are, for example, the epitopes recognized by the AT100 and TG-3 antibodies.
  • hypophosphorylated 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.
  • 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.
  • the Tau protein is non-phosphorylated and / or phosphorylated.
  • 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.
  • 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.
  • the truncated Tau protein lacks N-terminal domains.
  • 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.
  • immunoglobulin have the same meaning and are used interchangeably.
  • 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.
  • light chain There are two types of light chain: lambda ( ⁇ ) and kappa ( ⁇ ) light chains.
  • ⁇ lambda
  • ⁇ kappa
  • 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.
  • CDRs complementarity determining regions
  • FR framework
  • 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.
  • CDRs in the sequence of an antibody or nanobody can be determined by those skilled in the art using previously described techniques.
  • 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.
  • framework region refers to the amino acid sequences intercalated between the CDRs.
  • nanobody 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.
  • 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.
  • 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.
  • 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.
  • these two nanobodies, 2C5 and S2T2M3_E6, in particular 2C5 do not bind to the native Tau protein in monomeric form.
  • these two nanobodies, 2C5 and S2T2M3_E6, in particular 2C5 do not bind to beta amyloid peptide fibers.
  • the nanobodies are directed against the Tau protein as oligomers.
  • 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.
  • the fibrillation of Tau (40 ⁇ l) 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 ⁇ l), 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.
  • this Tau protein preparation is enriched in Tau protein as oligomers.
  • 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.
  • 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.
  • 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.
  • these oligomers can be characterized using size exclusion chromatography as being spherical molecules having a diameter of about 12 to 35 nm.
  • 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.
  • the tau protein is dimeric or trimeric.
  • the oligomeric Tau protein is soluble and / or non-soluble, more preferably soluble.
  • 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.
  • 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.
  • SPR surface plasmon resonance
  • the nanobodies of the invention further bind to Tau protein in the form of fibers.
  • 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.
  • the fibrillation of Tau (40 ⁇ l) 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 ⁇ l), 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.
  • 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.
  • 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.
  • Tau fibers 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.
  • 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.
  • the nanobodies of the invention further bind to the truncated Tau protein.
  • 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.
  • this truncated Tau protein comprises or consists of the amino acid sequence SEQ ID NO: 27.
  • 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.
  • 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.
  • 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.
  • the fibrillation of R3 (0.4 ⁇ l) 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 ⁇ l), 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.
  • buffer for example PBS (50 mM salt buffer phosphate, typically at pH 7 in the presence of heparin (0.4 ⁇ l), 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.
  • 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
  • BSA buffer phosphate buffer
  • 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.
  • the nanobodies of the invention additionally bind to helical pairs of filaments and / or straight filaments.
  • 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.
  • 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.
  • the nanobodies of the invention do not interact substantially with the polymerized amyloid beta 1 -42 protein.
  • 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.
  • the affinity for monomeric Tau protein can not be measured because the binding response is too weak.
  • 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.
  • the The concentration of the nanobodies used may be the EC 50 concentration or the concentration necessary to reach the saturation plateau.
  • 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.
  • 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 2C5 nanobodies have the amino acid sequence QVQLVQSGG
  • the nanobody S2T2M3_E6 has the amino acid sequence EVQLVESGGGLVQAGGSLRLSCAASGRTFSRYAMGWFRQAPGKEREF
  • VASISRSGGSTRYADAVKGRFTISRDNTNNTVYLLMNNLKPEDTAVYYC TARRRISGTPQWHYWGQGTQVTVSS (SEQ ID NO: 10).
  • o CDR2 ISPSGGVT (SEQ ID NO: 4)
  • CDR1, CDR2 and CDR3 are 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.
  • 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 ⁇ ⁇ is S or R, X 2 is D or Y and X 3 is T or A,
  • NRDPKYGNTRY SEQ ID NO: 5
  • TARRRISGTPQWHY SEQ ID NO: 8
  • a candidate nanobicide to compete for Tau protein binding for example in the form of oligomers
  • a nanobodies comprising the CDRs of a 2C5 and / or S2T2M3_E6 nanobodies as defined above, ( hereinafter a "reference" nanobodies)
  • the antigen ie Tau protein in the form of oligomers
  • 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).
  • 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.
  • 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.
  • the invention in another subject 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 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
  • 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 ⁇ ⁇ is S or R, X 2 is D or Y and X 3 is T or A,
  • NRDPKYGNTRY SEQ ID NO: 5
  • TARRRISGTPQWHY SEQ ID NO: 8
  • 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
  • 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.
  • 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:
  • Region FR1 QVQLVQSGGGLVQAGGSLRLSCAAS (SEQ ID NO: 1 1)
  • Region FR2 LAWFRQAPGKEREFVAS (SEQ ID NO: 12)
  • o Region FR3 YYEDSVKGRFTISRDNSKNTVLLQMNSLTPEDTAVYYC (SEQ ID NO: 13)
  • 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)
  • 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.
  • 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.
  • 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.
  • 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.
  • the nanobody according to the invention is a nanobody comprising or consisting of the amino acid sequence SEQ ID NO: 9.
  • 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.
  • isoleucine, a hydrophobic amino acid may be replaced by leucine, methionine or valine.
  • 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.
  • 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.
  • 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.
  • 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.
  • nanobodies of interest 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.
  • 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.
  • 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.
  • the nanobodies according to the invention comprise a myc and / or hexahistidine label.
  • 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.
  • a protein when 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.
  • 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.
  • 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.
  • 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 .
  • vector means 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.
  • 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.
  • regulatory elements such as a promoter, activator, terminator, etc.
  • 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.
  • 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.
  • Plasmids include replicative plasmids comprising an origin of replication, or integrative plasmids, such as for example pUC, pcDNA, pBR, etc.
  • viral vectors include adenoviral, retroviral, herpesvirus and AAV vectors.
  • 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.
  • 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.
  • host cells include prokaryotic cells (such as bacteria), and eukaryotic cells (such as yeast cells, mammalian cells, insect cells, plant cells, etc.).
  • 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:
  • 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.
  • the invention furthermore relates to a method for producing a nanobodies according to the invention, said method comprising the steps of:
  • 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.
  • immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxilapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.
  • the nanobodies according to the invention are particularly useful for medical imaging.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • the detectable marker is preferably a radioelement such as gallium ( 68 Ga) for PET imaging, or technetium 99m ( 99m Tc) for SPECT imaging.
  • the detectable marker is a radioelement.
  • 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.
  • 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 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.
  • nanobody as defined above for the manufacture of a contrast agent useful for medical imaging, in particular in vivo, non-invasive medical imaging.
  • 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.
  • contrast agent is used to designate a tracer associated with a marker as defined above.
  • imaging methods refer to methods that allow the visualization of the interior of an organism or organs of an organism.
  • 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).
  • SPECT single photon emission computed tomography
  • PET positron emission tomography
  • 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.
  • a contrast agent also called radiopharmaceutical
  • 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.
  • 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 ).
  • the individual is human.
  • 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.
  • the nanobody can be administered, for example, orally, by inhalation or parenterally (in particular by intravenous injection).
  • parenteral route 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.
  • buffers based on phosphate salts may for example use buffers based on phosphate salts as buffers.
  • suspending agents include methylcellulose, acacia, and sodium carbocymethylcellulose.
  • 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.
  • 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.
  • 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.
  • the nanobody according to the invention is used as a contrast agent in medical imaging to visualize neurofibrillary tangles (DNFs) in a patient.
  • DNFs neurofibrillary tangles
  • cortical distribution and the number of, for example, DNFs are correlated with memory disorders in patients with, for example, AD.
  • pathological forms of Tau protein is a marker of tauopathy.
  • 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 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.
  • diagnosis method or “diagnosis” is meant here a method for determining whether an individual suffers from a pathology.
  • prognostic method or “prognosis” is meant here a method for determining whether an individual is at risk of developing a pathology.
  • the nanobody as defined above is used for the diagnosis or prognosis of a tauopathy.
  • 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.
  • 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.
  • 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.
  • 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:
  • these methods of diagnosing tauopathy are in vivo methods.
  • 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.
  • 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).
  • 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.
  • progressive supranuclear paralyzation is characterized by the presence of DNF in the brain stem and frontal neocortex.
  • corticobasal degeneration is characterized by the presence of DNF in the parietal cortex.
  • 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.
  • 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.
  • 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.
  • 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.
  • the use according to the invention may comprise the steps of:
  • the "pathological form of Tau” is as defined in the "tauopathy” section above.
  • the treatment is a treatment of a taupopathy and comprises for example the use of inhibitors of Tau aggregation.
  • the treatment of a taupopathy includes the use of the nanobodies of the invention.
  • tauopathies 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.
  • PHFs helical paired filament pairs
  • SFs straight filaments
  • 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.
  • 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.
  • 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.
  • 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.
  • parenterally in particular by intravenous injection
  • 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.
  • buffers those skilled in the art may use buffers based on organic phosphate salts.
  • suspending agents examples include methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, acacia and sodium carboxymethylcellulose.
  • 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
  • a pharmaceutical composition comprising a nanobody as defined above in association with a pharmaceutically acceptable vehicle.
  • 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.
  • 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.
  • 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.
  • sample is meant here a part of a larger element.
  • the sample is a substance of biological origin.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • Fibrillation of Tau (40 ⁇ l) 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 ⁇ l) 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.
  • the fibrillation of R3 (0.4 ⁇ l) is carried out at 37 ° C. in PBS buffer (50 mM saline phosphate buffer, at pH 7 in the presence of heparin (0.4 ⁇ l), 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.
  • 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.
  • Nanobodies have been characterized for their affinity and specificity through two stages.
  • 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).
  • ELISA II Tests The nanobody 2C5 was radiolabeled with iodine 125.
  • 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:
  • Nbs single variable domain camelide antibodies
  • 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).
  • Nb S2T2M3 E6 was selected for its affinity for low molecular weight tau polymers.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Psychiatry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Hospice & Palliative Care (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Peptides Or Proteins (AREA)
  • Lubricants (AREA)
  • Carbon And Carbon Compounds (AREA)
  • Catalysts (AREA)
EP17798139.6A 2016-10-27 2017-10-27 Anti-tau-nanokörper Pending EP3532495A1 (de)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR1660450A FR3058143B1 (fr) 2016-10-27 2016-10-27 Nanocorps anti-tau
PCT/EP2017/077691 WO2018078140A1 (fr) 2016-10-27 2017-10-27 Nanocorps anti-tau

Publications (1)

Publication Number Publication Date
EP3532495A1 true EP3532495A1 (de) 2019-09-04

Family

ID=58314353

Family Applications (1)

Application Number Title Priority Date Filing Date
EP17798139.6A Pending EP3532495A1 (de) 2016-10-27 2017-10-27 Anti-tau-nanokörper

Country Status (7)

Country Link
US (1) US10590191B2 (de)
EP (1) EP3532495A1 (de)
JP (1) JP7197494B2 (de)
CN (1) CN110225926B (de)
CA (1) CA3041066A1 (de)
FR (1) FR3058143B1 (de)
WO (1) WO2018078140A1 (de)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110520440A (zh) 2017-02-17 2019-11-29 戴纳立制药公司 抗τ抗体及其使用方法
CN112649615A (zh) * 2020-12-21 2021-04-13 大连理工大学 一种检测不同大小的可溶性Aβ寡聚体的方法及应用
FR3134390A1 (fr) 2022-04-11 2023-10-13 Universite Grenoble Alpes Peptide utile pour le transport de molecules au travers de barrieres cellulaires

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4861719A (en) 1986-04-25 1989-08-29 Fred Hutchinson Cancer Research Center DNA constructs for retrovirus packaging cell lines
US5278056A (en) 1988-02-05 1994-01-11 The Trustees Of Columbia University In The City Of New York Retroviral packaging cell lines and process of using same
WO1994019478A1 (en) 1993-02-22 1994-09-01 The Rockefeller University Production of high titer helper-free retroviruses by transient transfection
FR2712812B1 (fr) 1993-11-23 1996-02-09 Centre Nat Rech Scient Composition pour la production de produits thérapeutiques in vivo.
SI9520093A (en) 1994-09-12 1997-08-31 Schering Ag Process for manufacture of a modified collagen-induced platelet aggregation inhibitor pallidipin.
IL116816A (en) 1995-01-20 2003-05-29 Rhone Poulenc Rorer Sa Cell for the production of a defective recombinant adenovirus or an adeno-associated virus and the various uses thereof
US6013516A (en) 1995-10-06 2000-01-11 The Salk Institute For Biological Studies Vector and method of use for nucleic acid delivery to non-dividing cells
WO2011026031A1 (en) * 2009-08-28 2011-03-03 The Board Of Regents Of The University Of Texas System Antibodies that bind tau oligomers
GB201112056D0 (en) * 2011-07-14 2011-08-31 Univ Leuven Kath Antibodies
AU2012311234B2 (en) * 2011-09-19 2017-09-28 Axon Neuroscience Se Protein-based therapy and diagnosis of tau-mediated pathology in Alzheimer's disease
WO2014059442A2 (en) 2012-10-12 2014-04-17 Arizona Board Agents, A Body Corporate Of The State Of Arizona, Acting For And On Behalf Of Arizona State University Antibody based reagents that specifically recognize toxic oligomeric forms of tau
EP3760228A1 (de) * 2013-06-10 2021-01-06 Ipierian, Inc. Verfahren zur behandlung einer tauopathie
EP2899208A1 (de) * 2014-01-28 2015-07-29 F.Hoffmann-La Roche Ag Gegen Amyloid-Beta gerichtete Einzeldomänen-phosphorylierte Tau-Proteine und Verfahren zur Herstellung von Konjugaten davon

Also Published As

Publication number Publication date
WO2018078140A1 (fr) 2018-05-03
CA3041066A1 (fr) 2018-05-03
FR3058143A1 (fr) 2018-05-04
JP2020505056A (ja) 2020-02-20
US10590191B2 (en) 2020-03-17
FR3058143B1 (fr) 2021-03-12
CN110225926A (zh) 2019-09-10
US20190263898A1 (en) 2019-08-29
JP7197494B2 (ja) 2022-12-27
CN110225926B (zh) 2024-05-03

Similar Documents

Publication Publication Date Title
EP3160999B1 (de) Antikörper- und antigenbindende fragmente zur spezifischen bindung an mit mikroröhren verknüpftes tau-protein
AU2013286680B2 (en) Antibodies to tau
JP5765857B2 (ja) Ａβオリゴマーに特異的に結合する抗体およびその利用
EP2731969B1 (de) Antikörper gegen phosphorylierte tau-aggregate
US11028157B2 (en) Binding molecules that specifically bind to tau
BR112020018868A2 (pt) métodos baseados em anticorpo para detectar e tratar doença de alzheimer
GB2507207A (en) Treatment of Tauopathies
HUE027649T2 (en) Phosphonus recognition antibodies
US20200369754A1 (en) Binding molecules that specifically bind to tau
US20130184441A1 (en) Detection of Neurodegenerative Disease
JP2018510617A (ja) 抗トランスサイレチン抗体
JP2019530428A (ja) 抗トランスサイレチン抗体
WO2018078140A1 (fr) Nanocorps anti-tau
CA2846251C (fr) Nanocorps anti-vcam-1
JP2023521763A (ja) 抗ｐｈｆ－タウ抗体及びその使用
BR112020014940A2 (pt) Adrenomedulina (adm) para diagnóstico e/ou predição de demência e ligante antiadrenomedulina para uso em terapia e prevenção de demência
Jiang et al. Single-domain antibody–based noninvasive in vivo imaging of α-synuclein or tau pathology
JP2018535921A (ja) タウの｛ｐ｝Ｓｅｒ４０４エピトープに対し選択的な、抗体を基にした分子、ならびにタウ異常症の診断および治療におけるそれらの使用

Legal Events

Date Code Title Description
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: UNKNOWN

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20190506

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

DAV Request for validation of the european patent (deleted)
DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: EXAMINATION IS IN PROGRESS

17Q First examination report despatched

Effective date: 20220217

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE

Owner name: CENTRE HOSPITALIER UNIVERSITAIRE GRENOBLE ALPES

Owner name: UNIVERSITE GRENOBLE ALPES