WO2021035210A1 - Anticorps à chaîne unique se liant à des oligomères de protéines tau et inhibant l'ensemencement par des extraits pathologiques de la maladie d'alzheimer - Google Patents

Anticorps à chaîne unique se liant à des oligomères de protéines tau et inhibant l'ensemencement par des extraits pathologiques de la maladie d'alzheimer Download PDF

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WO2021035210A1
WO2021035210A1 PCT/US2020/047622 US2020047622W WO2021035210A1 WO 2021035210 A1 WO2021035210 A1 WO 2021035210A1 US 2020047622 W US2020047622 W US 2020047622W WO 2021035210 A1 WO2021035210 A1 WO 2021035210A1
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tau
antibody
scfv
monoclonal antibody
oligomers
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PCT/US2020/047622
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David S. Eisenberg
Romany ABSKHARON
Paul M. SEIDLER
Charles Glabe
Stephan Philipp
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The Regents Of The University Of California
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    • 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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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

  • AD Alzheimer's disease
  • AD is associated with the accumulation of extracellular plaques composed of Ab peptides, and intracellular neurofibrillary tangles of hyperphosphorylated tau protein (2).
  • the cascade of molecular events that leads to AD is not fully understood, the spreading of tau pathology through the brain tracks with cognitive decline (3), and small oligomers and fibrillar inclusions are thought to seed the spread of tau pathology in the brains of transgenic mice expressing human P301S mutant, and possibly in humans (4).
  • numerous other neurodegenerative disorders are associated with the deposition of aggregated tau in the brain.
  • tauopathies include chronic traumatic encephalopathy (CTE), frontotemporal dementia with parkinsonism-17 (FTDP-17), progressive supranuclear palsy (PSP), and Pick’s disease among others (5, 6).
  • CTE chronic traumatic encephalopathy
  • FTDP-17 frontotemporal dementia with parkinsonism-17
  • PSP progressive supranuclear palsy
  • Pick s disease among others
  • tauopathies occur in the absence of fibrillar inclusions of amyloid beta, although AD and tauopathies are both thought to progress by the seeded spreading of aggregated tau from cell to cell in a prion-like manner (7).
  • Oligomeric inclusions of soluble tau could form prior to the deposition of larger neurofibrillary tangles in the brain, and soluble oligomers have been shown to provoke neuronal toxicity and the accumulation of fibrillar tau inclusions (8-11).
  • IL15-induced recombinant tau oligomers but not tau monomers or fibrils, react with a newly designed/discovered monoclonal antibody (M204), and further that an engineered M204 single-chain variable-fragment (M204-scFv) embodiments of this antibody inhibit seeding by IL15-induced tau oligomers, as well as inhibiting seeding in pathological tau extracts from donors with AD and Chronic Traumatic Encephalopathy (CTE).
  • M204-scFv engineered M204 single-chain variable-fragment
  • Biochemical characterizations of the M204-scFv antibody further show that it partitions into oligomeric forms that inhibit seeding differently, and crystal structures of the M204-scFv monomer, dimer and trimer reveal conformational differences that explain increased binding and inhibition by oligomeric forms the antibody.
  • monoclonal M204 and/or M204-scFv has the potential as an early- stage diagnostic for AD and tauopathies, and is a promising therapeutic scaffold because antibodies have ability, albeit low, to cross the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • monoclonal M204 or M204-scFv has the potential to inhibit one or more of dozens of different tauopathies. Although aggregated tau is thought to be a common etiology shared by all tauopathies, evidence suggests that tau aggregation can ensue in different ways to result in different molecular structures that are referred polymorphs. Monoclonal M204 or M204-scFv appear to target a subset of possible polymorphs, and therefore could be used as clinical imaging agents and/or as therapeutic agents for these polymorphs.
  • the invention disclosed herein has a number of embodiments.
  • One embodiment of the invention is a monoclonal antibody that binds oligomeric tau, does not bind tau monomers, and does not bind tau fibrils.
  • the monoclonal antibody is disposed in a composition that further contain pharmaceutically acceptable excipients such as preservatives and/or antimicrobial agents and/or pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents and the like.
  • the monoclonal antibody recognizes the epitope bound by M204 monoclonal antibody (e.g. a monoclonal antibody having amino acid sequences encoded by SEQ ID NO: 1 and/or 3).
  • the monoclonal antibody is a M204 single-chain variable fragment (ScFv) antibody comprising the amino acid sequence shown in SEQ ID NO: 7.
  • the monoclonal antibody is coupled to a heterologous amino acid sequence (e.g. a cell penetrating peptide such one disclosed in PCT/US2018/022742) or another element such as an imaging agent (e.g. a fluorophore).
  • a heterologous amino acid sequence e.g. a cell penetrating peptide such one disclosed in PCT/US2018/022742
  • an imaging agent e.g. a fluorophore
  • Another embodiment of the invention is a polynucleotide encoding a M204 monoclonal antibody (e.g. SEQ ID NO: 1 and/or 3); or a M204 single-chain variable fragment antibody having an amino acid sequence shown in SEQ ID NO: 7.
  • the antibody amino acid sequence encoded by the polynucleotide is fused in frame to heterologous amino acid sequence (e.g. a cell penetrating peptide, a PelB leader sequence or the like) or coupled to an imaging agent (e.g. a fluorescent protein or the like).
  • a related embodiment of the invention is a vector comprising a polynucleotide encoding a M204 monoclonal antibody in combination with one or more regulatory nucleotide sequences that facilitate expression of the antibody in mammalian cells (e.g. a promoter, an enhancer a polyA signal or the like).
  • embodiments of the invention also include mammalian cells comprising these vectors.
  • inventions include methods of binding a tau protein with an monoclonal antibody disclosed herein, the methods comprising combining a monoclonal antibody disclosed herein with tau protein; and then allowing the antibody to bind tau protein, so that tau protein is bound by the monoclonal antibody.
  • these methods can be practiced in a number of protocols in vitro (e.g. in diagnostic methods) and in vivo (e.g. in therapeutic methods).
  • the tau protein is derived from or present in brain tissue.
  • the brain tissue is derived from an individual having or suspected of having Alzheimer’s disease or chronic traumatic encephalopathy.
  • the monoclonal antibody is combined with tau protein so as to inhibit seeding of tau oligomers.
  • the methodology is used to inhibit tau aggregation occurring in a tauopathy.
  • the monoclonal antibody is coupled to an imaging agent; and the method is used to diagnose a tauopathy.
  • Embodiments of the invention include adding monoclonal M204 or M204- scFv in trans to a solution (or cell) containing Tau protein to prevent its seeded aggregation, or to solutions of aggregated tau to prevent seeding.
  • nucleic acid encoding monoclonal M204 or M204-scFv can be functionally expressed to synthesize monoclonal M204 or M204-scFv in cells, which then go on to inhibit intracellular and/or extracellular tau aggregates in proximity.
  • cell- penetrating sequences may allow for improved action of monoclonal M204 or M204-scFv by mediating delivery across the cell membrane, and modification of the sequence with chemicals such as polyethylene glycols, amino acids (natural and non-natural), etc. may improve metabolic stability and efficacy.
  • Monoclonal M204 or M204-scFv could be delivered peripherally into the bloodstream, potentially accompanied by transient BBB disruption, for therapeutic use, or by other means such as intranasal delivery.
  • imaging agents e.g. fluorophores, iron-oxide nanoparticles and the like
  • imaging agents e.g. fluorophores, iron-oxide nanoparticles and the like
  • Monoclonal M204 or M204-scFv addresses an unmet need by providing a potential therapeutic treatment and diagnostic imaging option for Alzheimer’s disease, and other tau pathologies.
  • Monoclonal M204 or M204-scFv differs from current antibodies that are being tested in clinical trials as m204 is a conformational antibody that targets aggregated tau.
  • engineered single- chain M204-scFv is much smaller than conventional antibodies, allowing it a better opportunity to diffuse in tissue.
  • the smaller size of M204-scFv compared to the monoclonal antibody format will allow cheaper and faster production in microbial systems.
  • Figure 1(A) shows the kinetics of the amyloid fibrils formation monitored by ThT fluorescence dye at 480 nm; tau-K18 fibrils induced by heparin (blue plot), tau-K18 fibrils induced by IL15 (green plot) and tau-K18 fibrils induced by IL23 (magenta plot).
  • Figure 1(B) shows the separation of tau-K18 oligomers induced by IL15 using size exclusion chromatography (HiLoad 16/600 Superdex 75 pg column).
  • Figure 1(C) shows immunoblot analysis of tau-K18 oligomer and monomer fractions from the SEC peaks using Anti-oligomer A11 polyclonal antibody.
  • Figure 1(D) shows TEM image of tau-K18 monomer.
  • Figure 1(E) shows TEM image of tau-K18 oligomer.
  • Figure 1(F) shows TEM image of tau-K18 fiber at the ThT end point.
  • Figure 1(G) shows X-ray fibril diffraction from tau-K18 fibrils prepared by IL15.
  • Figure 1(H) shows Tau seeding activity by tau-K18 oligomers.
  • Figure 1(I) shows Cells seeded with 1.5 nM of tau-K18 monomers.
  • Figure 1(J) shows Unseeded cells.
  • Figure 1(K) shows Quantification of seeding from representative images shown in Figures 1H, 1I and 1J. Statistical analysis was performed using one-way ANOVA (**P ⁇ 0.005, n.s., no significance) followed by Tukey's multiple comparisons test in GraphPad Prism. Error bars show the S.D. of three replicates of the ThT measurements. The white bar represents 25 ⁇ M.
  • Figures 2A-2F Tau oligomer purification and antibody binding.
  • Figure 2(A) shows a schematic representation of full-length tau (tau40) and the four repeats of microtubule binding domain of R1–R4 (K18).
  • Figure 2(B) shows S7516/600 size exclusion chromatography (SEC) purification of tau-K18 oligomer.
  • Figure 2(C) shows Dot blot of chromatographed fractions. Only the oligomer peak shows cross- reactivity with M204, whereas neither purified monomer nor fibrillar tau cross-react with monoclonal M204.
  • Figure 2(D) is an ELISA showing cross-reactivity of monoclonal M204 antibody with chromatographed S75 fractions containing tau-K18 oligomer (colored red) but not tau monomer (colored black).
  • Figure 2(E) shows S200 10/300 size exclusion chromatography (SEC) purification of oligomeric tau40.
  • Figure 2(F) is an ELISA showing cross-reactivity of monoclonal M204 with chromatographed S200 fractions containing tau40 oligomer (colored red) but not tau monomer (colored black). Error bars show the standard deviation of triplicate measurements.
  • Figures 3A-3E Epitope mapping of antibody M204 and inhibition by M204 of seeding by tau oligomers and of binding to AD human brain fractions.
  • Figure 3(A) shows Epitope mapping of M204 binding to tau using a peptide array of overlapping synthetic tau peptides. Immunoblotting shows that M204 binds most strongly to KVQIINK (SEQ ID NO: 9) and SVQIVY (SEQ ID NO: 10) sequences (colored in red).
  • Figure 3(B) shows seeding of the purified tau-K18 oligomers in HEK293 biosensor cells expressing YFP-tagged tau-K18.
  • Figure 3(C) shows seeding following treatment of tau-K18 oligomer with 10 ⁇ M M204 antibody, showing fewer puncta than in B. Representative cells that contain aggregates are marked by red arrows, and cells without by white arrows.
  • Figure 3(D) shows Quantification of seeding from representative images shown in Figures 3B and 3C. Tau oligomer seeding and inhibition by M204 antibody were determined by calculating the number of normalized puncta per well of a 96-well plate.
  • Figure 3(E) shows a Western blot of tissue from the brain of an AD patient fractionated into: crude brain homogenate, sarkosyl insoluble, and sarkosyl soluble fractions, each probed with M204 or A11 anti-oligomer antibodies.
  • Figures 4A-4D Engineering of a single chain antibody (scFv) from monoclonal M204 that binds tau oligomers.
  • Figure 4(A) shows a schematic representation of full-length monoclonal antibody and single chain variable domain (scFv) fragment.
  • Figure 4(B) shows the amino acids sequence of M204-scFv antibody.
  • the VH and VL variable domains are colored blue and green, respectively, and the sequences of the CDRs loops are labelled.
  • the VH and VL fragments are connected by a flexible linker with sequence (G4S)3.
  • Figure 4(C) shows S75 size exclusion chromatography (SEC) of M204-scFv.
  • SEC size exclusion chromatography
  • Figure 4(D) shows SDS-PAGE analysis of monomeric, dimeric and trimeric M204-scFv fractions run under reducing and non-reducing conditions shows high molecular weight bands for both dimer and trimer fractions (marked by black arrows).
  • Figures 5A-5G M204-scFv antibody fragments bind to tau oligomers and delay amyloid fibril formation in vitro.
  • Figure 5(A) shows S200 size exclusion chromatography (SEC) purification of tau-K18 oligomer.
  • Figure 5(B) is an ELISA showing cross-reactivity of M204-scFv monomer antibody with chromatographed S200 fractions containing tau-K18 oligomer, but not tau monomer.
  • Figure 5(C) is an ELISA signal showing binding of M204-scFv dimer towards tau-K18 oligomer.
  • Figure 5(D) is an ELISA binding signals of M204-scFv trimer to S200 fractions of tau-K18 oligomer.
  • Figure 5(E) shows M204-scFv monomer incubated with tau tau- K18 monomer at molar ratios of 1:1 and 1:0.5 (tau tau-K18: M204-scFv) prior to ThT measurements.
  • Figure 5(F) shows the effect of M204-scFv dimer on the tau tau-K18 amyloid fibril formation.
  • Figure 5(G) shows M204-scFv trimer as inhibitor for tau- K18 amyloid fibrils.
  • Figures 6A-6F M204-scFv antibodies inhibit the seeding of tau aggregation by autopsied brain extracts from three human AD brain patients.
  • Figure 6(A) shows a Negative stain electron micrograph of filaments purified from human AD brain tissues. Scale bar 200 nm. Zoom view shows a single fibril with a twist of two protofilaments, with the appearance of a paired helical filament (PHF).
  • PHF paired helical filament
  • Figures 6B-6E show Quantification of the inhibitory effect of M204-scFv antibodies on seeding by sarkosyl insoluble fractions from AD brain tissue. Statistical analysis was performed using one-way ANOVA (****P ⁇ 0.0001, ***P ⁇ 0.0002, **P ⁇ 0.001, *P ⁇ 0.01, n.s., no significance) followed by Tukey's multiple comparisons test in GraphPad Prism. Representative cells that contain aggregates are marked by red arrows, and cells without by white arrows. Error bars show the S.D. of three replicates.
  • Figure 6(F) shows Representative images of seeding and inhibition in HEK293 biosensor cells expressing YFP-tagged tau-K18.
  • FIGS 7A-7D Tests of the effectiveness of M204-scFv for inhibition of tau fibril seeding by diseased brain extracts in our HEK293 biosensor cell-based assay.
  • M204-scFv dimer and trimer inhibits seeding by extract from ( Figure 7A) CTE Donor 1, but not (Figure 7B) CTE Donor 2, ( Figure 7C) CBD donor or ( Figure 7D) a donor with a P301L familial mutation.
  • Figures 8A-8F Crystal structures of M204-scFv monomer, dimer and trimer antibodies.
  • Figure 8(A) shows the structure of M204-scFv monomer colored in pink and possess complementarity-determining region (CDR) loops of the heavy (H) and light (L) domains, which interact with tau-oligomers.
  • Figure 8(B) shows the structure of M204-scFv dimer colored in pink and cyan.
  • Figure 8(C) shows the structure of M204-scFv trimer colored in pink, cyan and green. For each antibody the CDR loops are solvent exposed, and oligomerization of the antibody could improve its interaction with the tau oligomer by polyvalent interactions.
  • Figure 8(D) is a ribbon diagram of M204-scFv showing the electron density and proximity of cysteine 201 from two adjacent molecules with the potential to form a disulfide bond (yellow).
  • Figure 8(E) shows a comparison of CDR loops of the heavy chains (H1, H2 and H3) of M204-scFv monomer (pink color), dimer (cyan color) and trimer (green color) conformations
  • Figure 8(F) CDR loops of the light chains (L1, L2 and L3) of M204-scFv monomer (pink color), dimer (cyan color) and trimer (green color) conformations.
  • Figures 9A-9C Tau seeding activity by tau-K18 fibrils.
  • Figure 9(A) shows HEK293 biosensor cells expressing YFP-tagged tau-K18 seeded with tau-K18 fibrils that were prepared by shaking tau monomer at 250 rpm at 37 ⁇ C with 2% w/v IL15.
  • Figure 9(B) shows Unseeded cells.
  • Figure 9(C) shows Quantification of seeding from representative images shown in A and B. Statistical analysis was performed using two-tailed unpaired multiple t test (***P ⁇ 0.0001) in GraphPad Prism. Error bars show the S.D. of three replicates of the ThT measurements. Representative cells that contain aggregates are marked by red arrows, and cells without by white arrows.
  • Figure 10 Purification of rabbit monoclonal of M204 antibody.
  • Hybridomas expressing M204 were grown at 37 °C for 7 days in a bioreactor flask in 5% CO2. M204 was purified using a protein A column, and the 4–12% Bis-Tris SDS-PAGE gel (run under non-reducing conditions) shows M204 production from fractions 1-3 after staining with InstantBlue dye.
  • Figure 11 Long exposure of immunoblot from Figure 3E. Long exposure of immunoblot from Figure 3E (main text) of tissue from the brain of an AD patient fractionated into: crude brain homogenate, sarkosyl insoluble, and sarkosyl soluble fractions, and then probed with the M204 or A11 anti-oligomer antibodies, as indicated.
  • AD Alzheimer’s Disease
  • Ab amyloid beta
  • tauopathies Soluble oligomers of aggregated tau accompany the accumulation of insoluble fibrils, and both oligomers and fibrils seed the spread of tau pathology. By virtue of their low molecular weight and relative solubility, oligomers may be particularly pernicious seeds.
  • tau aggregation inhibitors targeting three amyloidogenic segments of tau, SVQIVY (SEQ ID NO: 10), VQIVYK (SEQ ID NO: 11), and VQIINK (SEQ ID NO: 9). These inhibitors are capable of blocking tau aggregation, and underscore the important role of these segments in driving tau aggregation and seeding (19-23). Taking a different approach to inhibitor design, others have exploited tau antibodies that bind various epitopes and inhibit seeding.
  • tau oligomer monoclonal antibody As shown below, we report that a novel inducer of tau aggregation: ionic liquid 15 (IL15), promotes the formation of tau oligomers on-pathway to fibrils.
  • M204 monoclonal rabbit antibody
  • scFv single-chain variable-fragment
  • Embodiments of the invention include a monoclonal antibody that specifically recognizes the epitope bound by a single-chain variable fragment (ScFv) antibody comprising the amino acid sequence shown in SEQ ID NO: 7.
  • the monoclonal antibody is the M204 monoclonal antibody or a single-chain variable fragment (ScFv) antibody comprising the amino acid sequence shown in SEQ ID NO: 7.
  • embodiments of the invention include a monoclonal antibody that specifically binds oligomeric tau; but does not specifically bind tau monomers; and further does not specifically bind tau fibrils.
  • the monoclonal antibody is coupled to a heterologous amino acid sequence (e.g. a cell penetrating peptide such one disclosed in PCT/US2018/022742) or another element such as an imaging agent (e.g. a fluorophore).
  • a heterologous amino acid sequence e.g. a cell penetrating peptide such one disclosed in PCT/US2018/022742
  • an imaging agent e.g. a fluorophore
  • embodiments of the invention further include polynucleotides that encode a monoclonal antibody that binds oligomeric tau; does not bind tau monomers; and does not bind tau fibrils.
  • the monoclonal antibody encoded by the polynucleotide recognizes the epitope bound by a single-chain variable fragment (ScFv) antibody comprising the amino acid sequence shown in SEQ ID NO: 7.
  • the antibody amino acid sequence encoded by the polynucleotide is coupled to a heterologous amino acid sequence such as a cell penetrating peptide.
  • embodiments of the invention include the polynucleotide disposed within a vector having regulatory nucleotide sequences that facilitate expression of the antibody in mammalian cells, as well as mammalian cell comprising such vectors.
  • Embodiments of the invention further include methods of binding a tau protein with an antibody comprising combining a monoclonal antibody disclosed herein with tau protein; and then allowing the antibody to bind tau protein such that tau protein is bound by the antibody.
  • the tau protein is disposed within brain tissue, for example brain tissue is derived from an individual having or suspected of having Alzheimer’s disease or chronic traumatic encephalopathy.
  • the antibody is coupled to an imaging agent; and/or the method is used to diagnose a tauopathy.
  • the antibody is used as a therapeutic agent to inhibit a tauopathy.
  • the antibody is combined with tau protein so as to inhibit seeding of Tau oligomers, for example so as to inhibit tau aggregation occurring in a tauopathy.
  • the monoclonal antibody recognizes the epitope bound by a single-chain variable fragment (ScFv) antibody comprising the amino acid sequence shown in SEQ ID NO: 7 (e.g. the M204 monoclonal antibody or the single-chain variable fragment (ScFv) antibody comprising the amino acid sequence shown in SEQ ID NO: 7).
  • ScFv single-chain variable fragment
  • IL15 catalyzed aggregation of tau more robustly than IL23, but with a lag time that was about 5 times slower than heparin-induced aggregation.
  • the slower kinetics of IL15-induced aggregation allowed oligomers to be isolated during a short kinetic window, referred to as the oligomer extraction window, which corresponds to the early appearance of ThT signal (Fig.1A).
  • IL15-induced oligomers of tau-K18 isolated during the oligomer extraction window were purified by gel filtration (Fig. 1B) and tested for immunoreactivity and seeding. As shown in Fig.
  • M204 bound the aggregation-prone peptides VQIINK (SEQ ID NO: 9) and SVQIVY (SEQ ID NO: 10) more strongly than neighboring regions. These results provide evidence M204-tau oligomer recognition is mediated by the steric zipper forming segments that drive tau aggregation, and provide evidence that these segments are presented differently in the oligomer conformation compared to the conformations of the monomer and recombinant fibril. Since M204 recognizes epitopes and structural intermediates that are important for tau aggregation, we asked if antibody binding inhibits seeding by IL15- induced tau oligomers. To test this, we measured seeding and inhibition in HEK293 tau biosensor cells (28).
  • IL15-induced tau-K18 oligomers strongly seeded aggregation in tau biosensor cells, and seeding was inhibited by the addition of purified 10 ⁇ M M204.
  • M204 immunoreactive to M204.
  • M204 immunoreactivity was markedly stronger in the crude and sarkosyl-soluble fractions compared to the sarkosyl-insoluble fraction (Fig. 3E and Figure 9).
  • the scFv construct is a small antibody generated by connecting the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins using a short flexible linker (30).
  • VH variable regions of the heavy
  • VL light chains
  • scFvs are limited in expression due to misfolding and formation of inclusion bodies and loss of binding activity (31, 32).
  • M204-scFv M204 single- chain antibody
  • PelB leader sequence to direct the protein to the bacterial periplasm.
  • SEC Size exclusion chromatography
  • scFv antibodies may form dimers depending on the sequence of the antibody and/or the flexible linker (33, 34).
  • M204-scFv forms intermolecular disulfide bonds that stabilize the oligomer by comparing the mobility of purified M204-scFv by reducing and non-reducing SDS-PAGE (Fig. 4D).
  • Fig. 4D the mobility of purified M204-scFv by reducing and non-reducing SDS-PAGE
  • the M204-scFv protein that eluted from the SEC as monomers, dimers and trimers all migrated with similar mobility by SDS-PAGE.
  • non- reducing conditions revealed distinct high molecular weight bands for the dimer and trimer gel filtration fractions, providing evidence that the dimer and trimer species are disulfide linked.
  • M204-scFv Seeding inhibition by M204-scFv
  • M204-scFv To test the ability of M204-scFv to inhibit the primary aggregation of tau, we added different ratios of M204-scFv monomer, dimer and trimer to tau-K18 and measured aggregation using an in vitro Thioflavin T (ThT) assay. All of the M204-scFv antibodies delayed fibril formation at ratios of 1:1 and 1:0.5 (tau- K18:scFv) (Fig. 5 E-G). However, the M204-scFv dimer exhibited the most pronounced shift in lag time, whereas the M204-scFv monomer showed the least inhibitory effect.
  • M204-scFv monomer on the other hand is a poor inhibitor of seeding, and in some cases even stimulates seeding (Fig.6 B and C). Because we hypothesized that brain regions affected at different stages of disease can contain different species of aggregated tau, for Donor 3 we elected to test seeding by extracts from two different brain regions: the hippocampus, which exhibits tau aggregation at early-to-moderate stages of AD (35) (Fig. 6D), and the occipital lobe, which is affected at later stages of AD (Fig. 6E). Seeding inhibition by extracts from both brain regions was virtually identical.
  • the structures of all of the M204-scFv antibodies adopted the characteristic immunoglobulin (Ig) fold, having one variable domain from both the L and H chains per protomer (Fig. 8).
  • Each variable domain has three CDR loops (CDR1, CDR2 and CDR3) in both the heavy (H) and light (L) domains (Fig. 8A).
  • the dimer interface is formed by a face opposite the antigen binding surface, and the same interface comprises one of the two intermolecular interfaces that is formed by the M204-scFv trimer (Fig. 8B-C).
  • Our crystal structures reveal that all of the CDR loops from both the L and H domains are surface exposed in all of the structures: the monomer, dimer and trimer.
  • CDR3 CDR loop 3
  • M204-scFv revealed that the dimer interface has potential to be stabilized by a disulfide bond, as Cys202 from the two chains that form the dimer interface project towards each other, and are 3.4 ⁇ apart in the crystal structures of both the dimer and trimer (Fig.8D), although the disulfide appears to be reduced in both structures, potentially by X-ray generated electrons.
  • the buried surface areas formed by the dimer and trimer are relatively low compared to other protein interaction interfaces, with a buried surface area of 581 ⁇ 2 for the dimer and 379 ⁇ 2 for trimer.
  • Hybridomas producing rabbit monoclonal M204 antibody was generated as described previously (25).
  • Cells were grown in Hybridoma-SFM (Gibco, Cat No.: 1204-076) and seeded by 15 mL cells at a density of 1.5 x 10 6 viable cells/mL into CELLine CL1000 bioreactor flask (Argos, Cat No.: 90005).
  • the supernatants was collected after one week of seeding and used for protein purification.
  • the supernatants was filtered and loaded on a packed protein A column (Thermo Scientific, Cat No.: 82080), pre-equilibrated with PBS.
  • the protein After washing with 20 column volume of PBS, the protein eluted with 100 mM glycine, pH 2.0, and neutralized with 1 M Tris–HCl, pH 9.0. The purified antibody fractions were collected and analyzed by SDS-PAGE ( Figure 10). The protein was stored at -80 oC after buffer changed with 1x PBS using a 30 kDa cutoff Ultra centrifugal filter.
  • Thioflavin T fluorescence assay K18 tau protein was diluted into PBS buffer (pH 7.4) to 25 ⁇ M concentration in 1x PBS (pH 7.4), 10 mM DTT, 10 ⁇ M ThT and 2 % ionic liquid (HR2-214-15, Hampton Research) with shaking for 24 hours at 37 oC in 96 well plate (Thermo Scientific Nunc) with a plastic bead.
  • M204-scFv antibodies were with tau protein at 0.5:1 and 1:1 molar ratio.
  • the amyloid fibril formation signal was monitored by measuring ThT fluorescence using 440-nm excitation and 480-nm emission. The fibril kinetics was calculated by averaging from three replicates using GraphPad Prism (GraphPad Software, Inc.).
  • the recombinant M204-scFv was purified by IMAC after the periplasmic extraction using osmotic pressure.
  • Tau-K18 oligomers, monomers and brain homogenate fractions were detected by immunoblot analysis with rabbit polyclonal anti-oligomer A11 antibody (Invitrogen, Cat No.: AHB0052). Followinged by Goat anti-rabbit HRP (abcam, ab6721) as a secondary antibody. All membranes were developed using PierceTM ECL Plus substrate (Thermo Scientific, Cat No.: 32132). Crystallization and data collection Monomeric M204-scFv concentrated to 9.6 mg/mL and crystallization trials were performed at 16 °C by mixing equal volumes of the protein and the reservoir solution.
  • the trimeric M204-scFv was done at concentration of 4.4 mg/mL in 0.1 M sodium acetate, pH 5.0, 1.5 M ammonium sulfate (F11: ProPlex screen, molecular dimensions). All crystals were cryoprotected with reservoir solution contains 35% (W/V) glycerol, and flash-frozen in liquid nitrogen. Data collection, structure determination, refinement, and model building X-ray data were collected at 24-ID-C and 24-ID-E at the Advanced Photon Source at Argonne National Laboratories. Dimer-M204 data were indexed, integrated, scaled, and merged using XDS and XSCALE (49) and phases were obtained by molecular replacement using search model 5gs3 chain H in PHASER (50).
  • Tau biosensor cell line maintenance and seeding HEK293 cell lines stably expressing tau-K18 P301S-eYFP, referred to as “tau biosensor cells” were engineered by Marc Diamond’s lab at UTSW (28) and used without further characterization or authentication. Cells were maintained in DMEM (Life Technologies, cat. 11965092) supplemented with 10% (vol/vol) FBS (Life Technologies, cat. A3160401), 1% penicillin/streptomycin (Life Technologies, cat.
  • the number of seeded aggregates was determined by imaging the entire well of a 96-well plate in triplicate using a Celigo Image Cytometer (Nexcelom) in the YFP channel. Aggregates were counted using ImageJ (53) by subtracting the background fluorescence from unseeded cells and then counting the number of peaks with fluorescence above background using the built-in Particle Analyzer. The number of aggregates was normalized to the confluence of each well, and dose-response plots were generated by calculating the average and standard deviations from triplicate measurements. For IC50 calculations, does-response curves were fit by nonlinear regression in Graphpad Prism.
  • Tissue was received for neuropathlogically confirmed tauopathy cases from brain regions indicated in the figure legend. Tissue was cut into 0.2-0.3 g sections and then manually homogenized in a 15 ml ultra tissue grinder (Fisher 02-542-10) in 1 ml of 50 mM Tris, pH 7.4 with 150 mM NaCl containing 1X HALT protease.
  • Samples were then sonicated in a cuphorn bath for 120 min under 30% power at 4 °C in a recirculating ice water bath, according to reference (54), and were used for seeding without further purification.
  • sarkosyl insoluble fibril fractions 1-2 g of brain tissue were manually homogenized in 10 mM Tris–HCl, pH 7.4, 0.8 M NaCl, 10% sucrose, and 1X HALT protease inhibitor (Thermo) using a disposable ultra tissue grinder. After clarifying by centrifugation at 15,000 rpm for 10 min, sarkosyl was added to the supernatant to a final concentration of 1% and incubated at 37 ⁇ C with shaking at 200 rpm for 1 hr.
  • Biotinylated peptides bind to NeutrAvidin producing a large 65kd tetramer which binds tightly to nitrocellulose while exposing peptides above the nitrocellulose surface that can be recognized by antibodies.
  • the biotinylated amyloid peptides (12 amino acids with average m.w. 2 kDa) were pre-incubated with NeutrAvidin (ThermoFisher Scientific, MA, USA) in phosphate-buffered saline (PBS) at molar ratio of 4:1 for 1 hr at room temperature with gentle agitation. Final concentration of peptides in the complex was 0.5 mg/ml.
  • Tween 20 Tween 20 (T-PBS) was added to the peptide complex solution at final concentration of 0.001%, and then the solution was spotted onto nitrocellulose coated glass AVID slides (Grace Bio-Labs, Inc., OR, USA) using an Omni Grid 100 contact microarray printer (Genomic Solutions). One nanoliter of peptide solution was delivered onto the membrane, corresponding approximately 0.5 ng of peptide per spot. Slides were stored in desiccator until use. Statistical analysis Data were analyzed using GraphPad Prism by performing one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test and two-tailed unpaired multiple t test.
  • ANOVA analysis of variance
  • Meraz-Rios The role of tau oligomers in the onset of Alzheimer's disease neuropathology. ACS Chem Neurosci 5, 1178-1191 (2014); published online EpubDec 17 (10.1021/cn500148z). 9. M. Tabaton, T. I. Mandybur, G. Perry, M. Onorato, L. Autilio-Gambetti, P. Gambetti, The widespread alteration of neurites in Alzheimer's disease may be unrelated to amyloid deposition. Ann Neurol 26, 771-778 (1989); published online EpubDec (10.1002/ana.410260614). 10. J. Hardy, D. J.

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Abstract

Les oligomères solubles de protéines tau agrégées accompagnent l'accumulation de fibrilles insolubles, une marque histologique de la maladie d'Alzheimer (AD) et deux douzaines de maladies neurodégénératives associées. Ici, nous faisons état de la formation d'oligomères de protéines tau in vitro formés par un liquide ionique comprenant du sulfate de méthyle et de 1-butyl-3-méthylimidazolium (IL15). À l'aide d'oligomères de protéines tau recombinés induits par IL15, nous avons conçu et développé un anticorps monoclonal (M204) qui se lie à la protéine tau oligomère, mais pas aux fibrilles ou aux monomères de de protéines tau. L'anticorps monoclonal M204, ainsi qu'un mode de réalisation d'un fragment variable de chaîne unique modifié (ScFv) de cet anticorps, ont démontré une aptitude à inhiber l'ensemencement par des oligomères de protéines tau induits par IL15 ainsi que d'extraits pathologiques de donneurs atteints de la maladie d'Alzheimer et d'encéphalopathie traumatique chronique (ETC), fournissant une preuve que M204-scFv cible des structures pathologiques qui sont formées par les protéines tau dans des maladies neurodégénératives. Les modes de réalisation de l'invention tels que M204-scFv présentent un potentiel en tant que diagnostic à un stade précoce pour la maladie d'Alzheimer et les tauopathies, et ouvrent la voie à l'élaboration d'anticorps thérapeutiques prometteurs.
PCT/US2020/047622 2019-08-22 2020-08-24 Anticorps à chaîne unique se liant à des oligomères de protéines tau et inhibant l'ensemencement par des extraits pathologiques de la maladie d'alzheimer WO2021035210A1 (fr)

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US20120301473A1 (en) * 2011-04-27 2012-11-29 Northwestern University Antibodies selective for pathological tau dimers and prefibrillar pathological tau oligomers and their uses in treatment, diagnosis and monitoring of tauopathies
US20150266947A1 (en) * 2012-10-12 2015-09-24 Arizona Board Of Regents On Behalf Of Arizona State University Antibody based reagents that specifically recognize toxic oligomeric forms of tau
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