WO2016053610A1 - Anticorps contre les formes pathologiques de tdp-43 et utilisations de ces derniers - Google Patents

Anticorps contre les formes pathologiques de tdp-43 et utilisations de ces derniers Download PDF

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Publication number
WO2016053610A1
WO2016053610A1 PCT/US2015/050114 US2015050114W WO2016053610A1 WO 2016053610 A1 WO2016053610 A1 WO 2016053610A1 US 2015050114 W US2015050114 W US 2015050114W WO 2016053610 A1 WO2016053610 A1 WO 2016053610A1
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Prior art keywords
tdp
antibody
oligomer
seq
disease
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PCT/US2015/050114
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English (en)
Inventor
Yun-Ru Chen
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Academia Sinica
Liang, Chi-Ming
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Priority claimed from TW104107500A external-priority patent/TWI592423B/zh
Application filed by Academia Sinica, Liang, Chi-Ming filed Critical Academia Sinica
Priority to JP2017517321A priority Critical patent/JP2018503596A/ja
Priority to CN201580053162.4A priority patent/CN106999579A/zh
Priority to US15/516,015 priority patent/US9796778B1/en
Priority to EP15847220.9A priority patent/EP3200825A4/fr
Publication of WO2016053610A1 publication Critical patent/WO2016053610A1/fr

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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

Definitions

  • the present disclosure relates to novel pathological form of transactivation responsive (TAR)-DNA-binding protein 43 kDa (TDP-43), antibodies against such pathological form of TDP-43, and uses thereof.
  • TAR transactivation responsive
  • TDP-43 transactivation responsive protein 43 kDa
  • TDP-43 transactivation responsive lateral sclerosis
  • TDP-43 transactivation responsive (TAR)-DNA-binding protein 43 kDa
  • a molecule capable of binding e.g., an antibody
  • TDP-43 oligomer may suppresses the TDP-43 proteinopathy and thus are useful for the manufacture of a medicament (i.e., a vaccine and passive immunization) suitable for diagnosing, preventing or treating a neurodegenerative disease resulted from deposit of pathological form of TDP-43.
  • the present disclosure aims to provide an antibody or a fragment thereof that specifically binds to TDP-43 oligomer.
  • the antibody comprises a heavy chain variable region comprising amino acid sequences of SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID NO: 3; and a light chain variable region comprising amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.
  • the TDP-43 is characterized with a particle size of about 2 to 400 nm in diameter.
  • TDP-43 oligomer has a spherical particle size of about 40 to 60 nm in diameter.
  • the heavy chain variable region of the present antibody has the amino acid sequence of SEQ ID NO: 4
  • the light chain variable region of the present antibody has the amino acid sequence of SEQ ID NO: 8.
  • the present antibody is produced by the hybridoma cell line deposited in the Bioresource and Collection Center (BCRC) of the Food Industry Development and Research Institute (FIDRI) in Taiwan (HsinChu, Taiwan, R.O.C.) as accession number of BCRC960494.
  • BCRC Bioresource and Collection Center
  • FIDRI Food Industry Development and Research Institute
  • the present antibody is produced by the hybridoma cell line deposited with the BCRC as accession number of BCRC960495.
  • the present antibody is produced by the hybridoma cell line deposited with the BCRC as accession number of BCRC960496.
  • the present antibody is produced by the hybridoma cell line deposited with the BCRC as accession number of BCRC960497.
  • the present antibody is produced by the hybridoma cell line deposited with the BCRC as accession number of BCRC960498.
  • the medicament or the pharmaceutical composition comprises an effective amount of the antibody described above; and a therapeutically acceptable excipient.
  • the present antibody comprises a heavy chain variable region comprising amino acid sequences of SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID NO: 3; and a light chain variable region comprising amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.
  • the heavy chain variable region has the amino acid sequence of SEQ ID NO: 4
  • the light chain variable region has the amino acid sequence of SEQ ID NO: 8.
  • the antibody may be produced by any of the hybridoma cell lines deposited with the BCRC as accession number of BCRC960494, BCRC960495, BCRC960496, BCRC960497, or BCRC960498.
  • the antibody of this invention is present at a level of about 0.1 % to 99% by weight, based on the total weight of the pharmaceutical composition. In some embodiments, the antibody of this invention is present at a level of at least 1 % by weight, based on the total weight of the pharmaceutical composition. In certain embodiments, the antibody of this invention is present at a level of at least 5% by weight, based on the total weight of the pharmaceutical composition. In still other embodiments, the antibody of this invention is present at a level of at least 10% by weight, based on the total weight of the pharmaceutical composition. In still yet other embodiments, the antibody of this invention is present at a level of at least 25% by weight, based on the total weight of the pharmaceutical composition.
  • the TDP-43 oligomer associated disease treatable by the medicament or the pharmaceutical composition of the present disclosure may be any of, Alzheimer's disease, argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), ALS-parkinsonism dementia complex of Guam, vascular dementia, frontotemporal dementia, semantic dementia, dementia with Lewy bodies, Huntington's disease, Spinocerebellar ataxia, inclusion body myopathy, inclusion body myositis, hippocampal sclerosis, or Parkinson's disease.
  • ALS amyotrophic lateral sclerosis
  • ALS-parkinsonism dementia complex of Guam vascular dementia, frontotemporal dementia, semantic dementia, dementia with Lewy bodies, Huntington's disease, Spinocerebellar ataxia, inclusion body myopathy, inclusion body myositis, hippocampal sclerosis, or Parkinson's disease.
  • the method includes the step of, administering to the subject a therapeutically effective amount of the antibody of this invention, so as to inhibit or suppress the TDP-43 proteinopathies.
  • the present antibody comprises a heavy chain variable region comprising amino acid sequences of SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID NO: 3; and a light chain variable region comprising amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.
  • the heavy chain variable region has the amino acid sequence of SEQ ID NO: 4
  • the light chain variable region has the amino acid sequence of SEQ ID NO: 8.
  • the antibody may be produced by the hybridoma cell line deposited with the BCRC as accession number of BCRC960494, BCRC960495, BCRC960496, BCRC960497, or BCRC960498.
  • the TDP-43 oligomer associated disease that may be treated by the method of the present disclosure is Alzheimer's disease (AD), argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), ALS-parkinsonism dementia complex of Guam, vascular dementia, frontotemporal dementia, semantic dementia, dementia with Lewy bodies, Huntington's disease, Spinocerebellar ataxia, inclusion body myopathy, inclusion body myositis, hippocampal sclerosis, or Parkinson's disease (PD).
  • AD Alzheimer's disease
  • ALS amyotrophic lateral sclerosis
  • ALS-parkinsonism dementia complex of Guam vascular dementia, frontotemporal dementia, semantic dementia, dementia with Lewy bodies, Huntington's disease, Spinocerebellar ataxia, inclusion body myopathy, inclusion body myositis, hippocampal sclerosis, or Parkinson's disease (PD).
  • AD Alzheimer's disease
  • ALS amyotrophic lateral sclerosis
  • the method includes steps of, obtaining a biological sample from the subject; determining the amount of the TDP-43 oligomer in the biological sample by contacting the biological sample with an effective amount of the antibody of this invention; and comparing the detected amount of the TDP-43 oligomer in the biological sample with that of a control sample obtained from a healthy subject; wherein a significantly higher or lower amount of the detected TDP-43 oligomer in the biological sample than that of the control sample indicates that the subject suffers from the neurodegenerative disease.
  • the biological sample is brain biopsy sample, a cerebrospinal fluid sample, a whole blood sample, a serum sample, a plasma sample, a urine sample, or a mucus sample.
  • the present antibody comprises a heavy chain variable region comprising amino acid sequences of SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID NO: 3; and a light chain variable region comprising amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.
  • the heavy chain variable region has the amino acid sequence of SEQ ID NO: 4
  • the light chain variable region has the amino acid sequence of SEQ ID NO: 8.
  • the antibody may be produced by any of the hybridoma cell lines deposited with the BCRC as accession number of BCRC960494, BCRC960495, BCRC960496, BCRC960497, or BCRC960498.
  • the detecting result may be used as a reference for determining whether a subject of the biological sample suffers from a TDP-43 oligomer associated disease.
  • the kit includes, at least, a container, and reagents for detecting TDP-43 oligomer in the biological sample, wherein the reagents comprise the anti-TDP-43 oligomer antibody of this invention and a legend associated with the container and indicating how to use the anti-TDP-43 oligomer antibody of this invention for detecting the pathological form of TDP-43 (i.e., TDP-43 oligomer as described herein) in the biological sample.
  • the legend may be in a form of pamphlet, tape, CD, VCD or DVD.
  • the kit may further include a negative control that indicates either absence of TDP-43 oligomer or the normal level of the TDP-43 oligomer in a healthy subject.
  • FIG 1 depicts that full-length TDP-43 forms amyloid-like oligomers.
  • FIG 1A Analytical size-exclusion chromatography (SEC) of full-length TDP-43 purified from E. coli monitored by absorption at 280 nm (solid line) or human HEK293 cells quantified by slot blotting intensity (dashed line). The retention times of the molecular weight standards are indicated.
  • FIG 1 B Dot blotting of TDP-43 using anti-amyloid oligomer antibody, A1 1 . Freshly purified TDP-43 before (pre-load TDP-43) and after SEC (the void volume, TDP-43 oligomers) and the buffers were immunostained with A1 1 .
  • FIG 1 C Freshly purified TDP-43 was dotted in buffer or buffers containing 9 M urea, 7.2 M GdnHCI, or 2% SDS with or without heating at 90°C for 1 h. Three replicates were generated and probed separately by A1 1 antibody, anti-N-terminal TDP-43 antibody (residues 1 -260), and anti-C-terminal TDP-43 antibody (residues 250-414).
  • FIG 1 D TEM image of TDP-43 oligomers (scale bar, 500 nm) and zoomed-in images of a single oligomer are shown in the upper left (scale bar, 50 nm).
  • FIG 1 E AFM image of TDP-43 oligomers (scale bar, 500 nm) and zoomed-in images of a single oligomer are shown in the upper left (scale bar, 50 nm).
  • FIGs 1 F and 1 G DLS analysis of the oligomer fraction of TDP-43 from SEC. Particle diameters are plotted against scattered light intensity and particle number.
  • FIG 2 depicts the conformation, ThT fluorescence, and DNA binding of TDP-43.
  • FIG 2A Far-UV CD spectra of full-length TDP-43 (solid line) and short-form TDP-43 (dashed line). The spectra from 250 to 190 nm are shown.
  • FIG 2B Bis-ANS fluorescence spectra of full-length TDP-43 (solid line) and short-form TDP-43 (dashed line). The buffer signal is shown (dotted line).
  • FIG 2C ThT binding of TDP-43 in comparison to ⁇ fibrils.
  • FIG 2D TAR DNA binding of TDP-43 monitored by fluorescence titration.
  • Full-length (squares) and short-form (circles) of TDP-43 were titrated with single strand TAR DNA-A (solid symbols) and -B sites (empty symbols).
  • the emission maxima of TDP-43 or TDP-43s at 350 nm were monitored while exciting at 280 nm. The data shown were normalized to the starting point and the lines shown are fit lines.
  • FIG 3 depicts the cross-seeding of TDP-43 to ⁇ .
  • FIG 3A ThT assay of ⁇ fibrillization without and with TDP-43 in concentrations ranging from 0.4 to 4%. Percentages of TDP-43 seeded in molar ratio are indicated.
  • FIG 3B Photo-induced cross-linking (PICUP) assay of ⁇ without and with TDP-43 at time 0. The percentages of TDP-43 seeded in molar ratio are indicated.
  • FIG 3C TEM images of end-point products of ⁇ without and with 4% TDP-43 (scale bar, 100 nm).
  • FIG 4D The immunocytochemistry of primary neurons treated with control or TDP-43, 0.44 ⁇ . The samples were subjected to MAP-2 immunostaining, GFAP immunostaining, and DAPI staining.
  • FIG 5 depicts that TDP-0 antibody recognizes TDP-43 oligomers specifically.
  • FIG 5A Freshly purified TDP-43 in native buffer or buffers containing 9 M urea, 7.2 M GdnHCI, or 2% SDS with or without heating at 90°C for 1 h was subjected to dot blotting probed by the newly generated polyclonal antibody, TDP-O, using TDP-43 oligomers as immunogen. The immunoreactivity of TDP-43 conformation by TDP-0 was similar to that by A1 1 as shown in FIG 1 C.
  • FIG 5B ⁇ oligomers were subjected to dot blotting probed by A1 1 and TDP-0 antibodies.
  • FIG 5C TDP-43 with (solid line) and without (dotted line) 3 fold concentration were subjected to SEC. The elution volumes of TDP-43 oligomer (*), TDP-43 monomer (it), and the molecular weight standards are indicated.
  • FIG 5D The 1 ml SEC fractions were collected and subjected to dot blotting by TDP-0 (upper blot) and N -2 6o antibodies (lower blot).
  • FIG 5E The purified TDP-43 oligomers and monomers from SEC were characterized by ELISA. The TDP-43 samples were coated dose-dependently onto the ELISA plates.
  • TDP-43 oligomers were probed by TDP-0 ( ⁇ ) and N-i-260 ( ⁇ ) antibodies and TDP-43 monomers were probed by TDP-0 ( ⁇ ) and N-i-260 (o) antibodies.
  • TDP-0 antibody possesses significant higher specificity against TDP-43 oligomers.
  • FIG 6 depicts that TDP-43 oligomers are present and increase with age in transgenic mouse model of FTLD-TDP.
  • FIG 6A Immunofluorescent staining of human TDP-43 and TDP-43 oligomers in the brain slices of wild type and 6- and 12-month-old TDP-43 Tg +/+ transgenic mice. Cells with anti-TDP oligomer staining, TDP-43 staining, and DAPI staining are shown (scale bar, 100 ⁇ ). Blocked area was presented at higher magnification in the last column (scale bar, 25 ⁇ ).
  • FIG 6B Quantified results showed that the ratio of cells with deposition of TDP-0 and TDP-43 in cytosol were age dependent.
  • FIG 7 depicts that TDP-43 oligomers are present in FTLD-TDP patients.
  • TDP-0 Immunohistochemical staining of TDP-43 in the hippocampal (C) and frontal cortical sections (A, E) of FTLD-TDP patients by the TDP-0 antibody.
  • TDP-0 identified densely stained, ovoid or irregularly shaped but discreet cytoplasmic inclusions (arrow) as well as comma-shaped profiles in the neuropil representing dystrophic neurites (arrowhead). In some cortical areas such as shown in (E), neuronal cytoplasm showed coarse granular immunoreactivities.
  • B), (D), and (F) TDP-0 did not stain the brains of control subjects.
  • TDP-0 antibody in contrast to antibodies for monomeric TDP-43, did not stain nuclei demonstrating specificity toward misfolded TDP-43. Scale bars in all panels are 20 ⁇ .
  • FIG 8 depicts the immunogold labeling of TDP-43 oligomers immunoprecipitated from the diseased hippocampus.
  • the hippocampus of a FTLD-TDP patient was extracted and immunoprecipated by TDP-0 antibody.
  • the eluent was subjected to EM with immunogold labeling with the N-term TDP-43 antibody (scale bar, 50 nm).
  • FIG 8A TDP-43 oligomers in FTLD-TDP (scale bar, 100 nm).
  • FIG 8B zoom-in images of TDP-43 oligomers (scale bar, 50 nm).
  • FIG 9 depicts that TDP-0 mAbs exhibit higher specificities toward TDP-43 oligomer.
  • concentrations of TDP-0 mAbs (1 -2 x 10 " ⁇ g/mL) respectively produced by TDP-O-3, -5, -8, -9, and -10 hybridoma cells were used in ELISA assay to detect the SDS denatured or non-denatured TDP-43.
  • FIG 10 depicts that TDP-0 mAbs exhibit higher specificities toward purified TDP-43 oligomer.
  • the conditional medium of TDP-0 hyb doma cells were used to detect purified TDP-43 oligomers and monomers from size-exclusion chromatography by ELISA assay.
  • FIG 10A TDP-43 oligomers and monomers were separated and collected by Superdex 200 10/300 GL column.
  • FIG 10B Conditional medium of TDP-O-3, -5, -8, -9, and -10 hybridoma cell lines were used to detect the TDP-43 oligomers and monomers by ELISA assay.
  • FIG 11 depicts the consensus amino acid sequences of heavy chain variable region (V H ) and light chain variable region (V L ) of the 5 isolated TDP-0 monoclonal antibodies.
  • V H heavy chain variable region
  • V L light chain variable region
  • X represents any amino acid residues
  • CDR is the abbreviation of the complementarity determining region.
  • FIG 12 depicts that TDP-0 monoclonal antibody rescues TDP-43 oligomers-induced cytotoxicity.
  • MTT assay was performed to examine cell viability of BE(2)-C cells. Data are presented as mean ⁇ standard deviation. Statistical analysis was performed by one-way ANOVAs, *p ⁇ 0.05, **p ⁇ 0.01 , ***p ⁇ 0.001 . The result showed the toxicity induced by TDP-43 oligomers was significantly rescued by the treatment of TDP-0 antibody.
  • TDP-43 proteinopathy refers to diseases particularly linked to transactivation responsive (TAR)-DNA-binding protein of 43 kDa (TDP-43).
  • TDP-43 is a disease protein known to link with frontotemporal lobar degeneration with ubiquitin-positive inclusion (FTLD-U), and amyotrophic lateral sclerosis (ALS).
  • TDP-43 proteinopathy thus includes, but is not limited to, Alzheimer's disease (AD), argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), ALS-parkinsonism dementia complex of Guam, vascular dementia, frontotemporal dementia, semantic dementia, dementia with Lewy bodies, Huntington's disease, Spinocerebellar ataxia, inclusion body myopathy, inclusion body myositis, hippocampal sclerosis, or Parkinson's disease (PD).
  • AD Alzheimer's disease
  • ALS amyotrophic lateral sclerosis
  • ALS-parkinsonism dementia complex of Guam vascular dementia, frontotemporal dementia, semantic dementia, dementia with Lewy bodies, Huntington's disease, Spinocerebellar ataxia, inclusion body myopathy, inclusion body myositis, hippocampal sclerosis, or Parkinson's disease (PD).
  • AD Alzheimer's disease
  • ALS amyotrophic lateral sclerosis
  • an effective amount refers to an amount effective, at dosages, and for periods of time necessary, to achieve the desired therapeutically result with respect to the treatment of a TDP-43 oligomer associated disease.
  • phrases "pharmaceutically acceptable” refers to molecular entities and compositions that are "generally regarded as safe", e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • administered means either directly administering a bi-specific antibody or a composition of the present disclosure.
  • the term “subject” or “patient” refers to an animal including the human species that is treatable with the compositions and/or methods of the present disclosure.
  • treat or “treatment” as used herein are intended to mean obtaining a desired pharmacological and/or physiologic effect, e.g., detecting the presence of pathologic form of TDP-43, preventing or rescuing organ atrophy, or inhibiting development of dementia or muscular weakness and stiffness.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment includes preventative (e.g., prophylactic), curative or palliative treatment of a disease in a mammal, particularly human; and includes: (1 ) preventative (e.g., prophylactic), curative or palliative treatment of a disease or condition (e.g., a cancer or heart failure) from occurring in an individual who may be pre-disposed to the disease but has not yet been diagnosed as having it; (2) inhibiting a disease (e.g., by arresting its development); or (3) relieving a disease (e.g., reducing symptoms associated with the disease).
  • preventative e.g., prophylactic
  • a disease or condition e.g., a cancer or heart failure
  • antibody or “antibodies” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bi-specific antibodies), and antibody fragments so long as they exhibit the desired biological activity, that is, to specifically bind to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or other molecules.
  • the antibody of this invention is a polyclonal antibody that specifically recognizes TDP-43 oligomer.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, and is not to be constructed as requiring production of the antibody by any particular method. In contrast to polyclonal antibodies which typically include different antibodies directed to different epitopes, each monoclonal antibody is directed against a single determinant (i.e., epitope) on the antigen.
  • the monoclonal antibodies of the present disclosure may be made by hybridoma method or by recombinant DNA methods.
  • the monoclonal antibodies herein specifically include “chimeric” or “recombinant” antibodies, in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a antibody class or subclass, while the remainder of the chain identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired biological activity.
  • "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin.
  • Humanized antibodies are human immunoglobulins in which hypervarible region residues are replaced by hypervarible region residues from a non-human species such as mouse, rat, rabbit, or non-human primate having the desired specificity or affinity.
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the FR regions are those of a human immunoglobulin sequence.
  • the humanized antibody may optionally comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • Transactivation responsive (TAR)-DNA-binding protein of 43 kDa has been identified as the disease protein in ubiquitinated misfolded aggregates of all subtypes of sporadic frontotemporal lobar degeneration with ubiquitin-positive inclusion (FTLD-U), as well as amyotrophic lateral sclerosis (ALS).
  • TDP-43 forms structurally stable spherical oligomers that cross-seed Alzheimer's amyloid- ⁇ ( ⁇ ) to form amyloid oligomers, thus if an agent (e.g., an antibody) could bind such TDP-43 oligomer, such agent may suppress or inhibit TDP-43 proteinopathy and/or TDP-43 effect to a subsequent event such as, but not limited to, ⁇ oligomerization, and accordingly is useful for the manufacture of a medicament (e.g., a vaccine or passive immunization) suitable for preventing or treating a neurodegenerative disease resulted from the deposit and/or aggregation of pathological form of TDP-43.
  • a medicament e.g., a vaccine or passive immunization
  • an antibody particularly, an antibody that recognizes the TDP-43 oligomer identified in the examples of the present invention, thereby inhibits the aggregation of a protein, particularly, TDP-43, which aggregation is associated with a disease.
  • the protein aggregation process proceeds in a self-propagating manner, once initiated, an aggregation cascade ensues that involves induced conformation change and/or polymerization of further protein molecules, leading to the formation of toxic product that is resistant to proteolysis.
  • the thus formed protein aggregation is thought to be the proximal cause of neurodegeneration diseases, such as Alzheimer's disease (AD), argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), ALS-parkinsonism dementia complex of Guam, vascular dementia, frontotemporal dementia, semantic dementia, dementia with Lewy bodies, Huntington's disease, Spinocerebellar ataxia, inclusion body myopathy, inclusion body myositis, hippocampal sclerosis, or Parkinson's disease (PD).
  • AD Alzheimer's disease
  • ALS amyotrophic lateral sclerosis
  • ALS-parkinsonism dementia complex of Guam vascular dementia, frontotemporal dementia, semantic dementia, dementia with Lewy bodies, Huntington's disease, Spinocerebellar ataxia, inclusion body myopathy, inclusion body myositis, hippocampal sclerosis, or Parkinson's disease (PD).
  • AD Alzheimer's disease
  • ALS amyotrophic lateral sclerosis
  • the TDP-43 oligomer is characterized in having a particle size of about 2 to 400 nm in diameter.
  • the TDP-43 oligomer has a particle size of about 20 to 400 nm in diameter, such as 20 to 30 nm, 30 to 40 nm, 40 to 50 nm, 50 to 60 nm, 60 to 70 nm, 70 to 80 nm, 80 to 90 nm, 90 to 100 nm, 100 to 120 nm, 120 to 140 nm, 140 to 160 nm, 160 to 180 nm, 180 to 200 nm, 200 to 220 nm, 220 to 240 nm, 240 to 260 nm, 260 to 280 nm, 280 to 300 nm, 300 to 320 nm, 320 to 340 nm, 340 to 360 nm, 360 to 380 nm, and 380 to 400 nm in diameter. More preferably,
  • the antibodies of the present invention specifically bind the TDP-43 oligomer described above, its epitopes, as well as various conformations and epitopes thereof.
  • antibodies disclosed herein preferentially bind pathologic TDP-43, specifically, full-length TDP-43 oligomer characterized in having a spherical particle size of about 2 to 400 nm in diameter.
  • mice, rats or rabbits are first immunized with TDP-43 oligomer at a suitable dose.
  • adjuvant and the TDP-43 oligomer solution are mixed together when immunizing the animals with TDP-43 oligomer.
  • adjuvants useful for this invention include Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), and aluminum hydroxide adjuvant.
  • FCA Freund's complete adjuvant
  • FIA Freund's incomplete adjuvant
  • Immunization is generally carried out mainly by intravenous, subcutaneous, intraperitoneal or intramuscular injection of the antigen. The immunization interval is not particularly limited.
  • Immunization may be carried out at intervals of several days to several weeks, preferably 2 to 3 weeks, for 1 to 10 times, preferably 2 to 5 times. Once antibody titers reaches 2 or more in the absorbance level as the result of immunization, the animals are left for about 1 month. Then, re-immunization is carried out for at least once. Several days, preferably 3 to 5 days, after the final immunization, splenic cells and regional lymph nodes are removed. Blood samples are taken regularly after immunization and subject to centrifugation to separate sera.
  • the resultant sera are then subject to measurement of antibody titers by any suitable method, which includes, and is not limited to, enzyme linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), or radio immunoassay (RIA).
  • ELISA enzyme linked immunosorbent assay
  • EIA enzyme immunoassay
  • RIA radio immunoassay
  • antibody titers are measured by ELISA.
  • final immunization is given to those animals showing high antibody titers to TDP-43 oligomer.
  • Antibody-producing cells are prepared from splenic cells and regional lymph nodes or the like of the immunized animals. In the preparation of antibody-producing cells, it is preferably to remove tissue debris and erythrocytes as much as possible. Commercial erythrocyte remover may be used to this purpose. Alternatively, a buffer ammonium chloride and Tris may be prepared and used. The thus prepared antibody-producing cells are immediately fused with immortal cells such as myeloma cells to produce hybridoma cells, which semi-eternally continue to proliferate while producing antibodies. Commonly available cell strain derived from an animal such as mouse may be used.
  • a preferable cell strain to be used in this invention should be those that fuse efficiently, support stable high level production of antibody and are sensitive to HAT selection medium, which contains hypoxanthine, thymidine and aminopterin, and should survive there only when fused with antibody-producing cells.
  • myeloma cells include, but are not limited to, mouse myeloma cell line (such as myeloma FO cells) and human myeloma cell line (such as Karpas 707H).
  • Cell fusion is usually carried out by mixing splenic cells or lymph node cells with a commercial available myeloma cells in the presence of a cell-fusion promoter, such as PEG having an average molecular weight from about 200 to 20,000 daltons or the like.
  • a cell-fusion promoter such as PEG having an average molecular weight from about 200 to 20,000 daltons or the like.
  • cell fusion may be carried out in a commercial cell fusion device utilizing electric stimulation such as electro-fusion. After the fusion, the resultant cells are then diluted and cultured in HAT medium.
  • Hybridomas of interest are then selected from the fused cells.
  • the fused cells surviving cultured in HAT medium would form colonies.
  • the supernatant of each culture well is then collected and examine for the presence or absence of antibody titers to TDP-43 oligomer.
  • ELISA, EIA or RIA may be used, in which TDP-43 oligomer or TDP-43 monomer is coated onto the plates and used as a screening criteria.
  • a HT medium which does not contain aminopterin. After culturing for a while, antibody titers in the culture supernatant are confirmed again.
  • Cells that are finally selected are then subject to cloning to obtain single cells. Clones that exhibit high specificity to TDP-43 oligomer are selected, and are proliferated to some extent to establish hybridomas.
  • 5 hybridomas, TDP-O-3, TDP-O-5, TDP-O-8, TDP-O-9 and TDP-O-10 were selected, and monoclonal antibodies may be isolated or prepared by any known method.
  • antibodies may be prepared from cultured supernatant obtained by culturing hybridomas in a medium with low serum concentration.
  • hybridomas may be injected into abdominal cavities of animals and the resultant abdominal dropsies are collected to prepare antibodies.
  • Antibodies may be purified or isolated by methods that employ affinity column, gel filtration chromatography, ion exchange chromatography or the like. Any of these known methods may be appropriately selected or used in combination.
  • the monoclonal antibodies of the present disclosure are respectively produced by the hybridoma cell line TDP-O-3, TDP-O-5, TDP-O-8, TDP-O-9 and TDP-O-10, which are deposited in the Bioresource Collection and Research Center (BCRC) of the Food Industry Development and Research Institute (FIDRI) (HsinChu, Taiwan, R.O.C.) as accession number of BCRC960494, BCRC960495, BCRC960496, BCRC960497, or BCRC960498.
  • BCRC Bioresource Collection and Research Center
  • FIDRI Food Industry Development and Research Institute
  • the monoclonal antibodies disclosed herein comprise consensus sequences respectively located at the heavy and light chain variable regions.
  • the monoclonal antibodies described herein respectively comprise a heavy chain variable region comprising amino acid sequences of SEQ ID NO: 1 , SEQ ID NO: 2 and SEQ ID NO: 3; and a light chain variable region comprising amino acid sequences of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.
  • the humanized monoclonal antibody comprises a heavy chain variable region of SEQ ID NO: 4, and a light chain variable region of SEQ ID NO: 8.
  • Xaa represents any L- or D-form amino acid residues known in the art.
  • Xaa is an acidic amino acid residue (e.g., aspartate or glutamate).
  • Xaa is a basic amino acid residue (e.g., lysine, arginine, or histidine).
  • Xaa is a nonpolar amino acid residue (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan).
  • Xaa is an uncharged polar amino acid residue (e.g., glycine, asparagine, glutamine, cysteine, serine, threonine, or tyrosine).
  • anti-TDP-43 oligomer monoclonal antibodies may be produced by DNA cloning or DNA synthesis.
  • DNA encoding anti-TDP-43 oligomer mAbs may be easily isolated and sequenced by use of conventional procedures, such as using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies.
  • the hybridoma cells serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as E. Coli cells, simian COS cells or Chinese hamster ovary (CHO) cells or myeloma cells that do not produce immunoglobulin proteins, to synthesize the desired monoclonal antibodies in the recombinant host cells.
  • host cells such as E. Coli cells, simian COS cells or Chinese hamster ovary (CHO) cells or myeloma cells that do not produce immunoglobulin proteins, to synthesize the desired monoclonal antibodies in the recombinant host cells.
  • the monoclonal antibodies thus produced and the DNA encoding such antibodies can then be used to produce chimeric antibodies (e.g., bi-specific antibodies), humanized antibodies and/or antibody fragments derived thereof.
  • the major concern of a non-human origin monoclonal antibody is its immunogenicity to the recipient, in some cases, caused dangerous allergic reactions.
  • Most monoclonal antibodies are of murine origin, and have been found to be immunogenic when injected to human.
  • humanized antibodies are produced by attaching variable domains in the heavy and light chains of murine anti-TDP-43 oligomer Abs onto the constant regions of human antibodies.
  • the DNA encoding such antibodies was isolated and sequenced, and then used to create humanized constructs.
  • CDR (complementary determining region) grafting is employed, in which the CDR regions in the VH and VL genes of a human antibody are replaced with the appropriate CDR coding segments (such as those DNA segments in anti- TDP-43 oligomer Abs that code amino acid segments responsible for bindingTDP-43 oligomer).
  • the resulting antibodies therefore have variable regions in which only the CDRs are from the original mouse antibodies, while the framework regions in the VH and VL genes as well as the constant region genes (i.e., CK or CH1 -H-CH2-CH3) are those of human IgG.
  • the humanized anti-TDP-43 oligomer mAb comprises a heavy chain variable domain and a light chain variable domain.
  • the humanized anti-TDP-43 oligomer mAbs may be purified according to standard procedures in the art, including cross-flow filtration, affinity column chromatography, gel filtration and the like. It should be understood that the humanized antibodies shall perform in a manner identical or substantially similar to that of murine anti-TDP-43 oligomer Abs.
  • the humanized anti-TDP-43 oligomer Abs (either in the form of Fab or full length IgG) shall be more advantages to use in a human subject, as compared to the murine version.
  • the humanized anti-TDP-43 oligomer Abs are used in the production of bi-specific antibodies of the present disclosure.
  • Anti-TDP-43 oligomer antibodies of the present invention can be characterized by use of any in vivo or in vitro models of TDP-43 proteinopathies.
  • a skilled artisan readily understands that an anti-TDP-43 oligomer antibody of the present invention can be characterized in a mouse model, such as the animal model described in Example 7.
  • the anti-TDP-43 oligomer antibody of the present invention can be characterized by human AD samples as described in Example 8.
  • the anti-TDP-43 oligomer antibody of the present invention possesses an inhibitory effect on the cell cytotoxicity induced by TDP-43 oligomers.
  • the concentration of the present anti-TDP-43 oligomer antibody sufficient to inhibit the TDP-43 oligomer-induced cytotoxicity is about 0.001 -5.0 mg/ml; for example, 0.001 , 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01 , 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 mg/ml.
  • the concentration is about 0.01 -1 .0 mg/ml. More preferably, the
  • an experimental model of TDP-43 proteinopathy can be used in a preventive setting or in a therapeutic setting.
  • a preventive setting the dose of animals starts prior to the onset of the TDP-43 proteinopathy or symptoms thereof, and the Anti-TDP-43 oligomer antibody of the present invention is evaluated for its ability to prevent, reduce or delay the onset of TDP-43 proteinopathy or symptoms thereof.
  • the dose of animals starts after the onset of the TDP-43 proteinopathy or symptoms thereof, and the Anti-TDP-43 oligomer antibody of the present invention is evaluated for its ability to treat, reduce or alleviate the onset of TDP-43 proteinopathy or symptoms thereof.
  • Symptoms of TDP-43 proteinopathy include, but are not limited to, accumulation of pathological TDP-43 deposits in the brain, spinal cord, cerebrospinal fluid or serum of a test subject.
  • the disclosure provides a pharmaceutical composition or a medicament for treating a neurodegenerative disease associated with the aggregation of ⁇ .
  • the composition comprises an effective amount of the anti-TDP-43 oligomer antibody of the present invention as described herein; and a pharmaceutically acceptable excipient.
  • the TDP-43 oligomer associated disease treatable by the pharmaceutical composition or the medicament of the present disclosure includes, but is not limited to, Alzheimer's disease, argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), ALS-parkinsonism dementia complex of Guam, vascular dementia, frontotemporal dementia, semantic dementia, dementia with Lewy bodies, Huntington's disease, Spinocerebellar ataxia, inclusion body myopathy, inclusion body myositis, hippocampal sclerosis, or Parkinson's disease.
  • ALS amyotrophic lateral sclerosis
  • ALS-parkinsonism dementia complex of Guam vascular dementia, frontotemporal dementia, semantic dementia, dementia with Lewy bodies, Huntington's disease, Spinocerebellar ataxia, inclusion body myopathy, inclusion body myositis, hippocampal sclerosis, or Parkinson's disease.
  • the anti-TDP-43 oligomer antibody of this invention is present at a level of about 0.1 % to 99% by weight, based on the total weight of the pharmaceutical composition. In some embodiments, the anti-TDP-43 oligomer antibody of this invention is present at a level of at least 1 % by weight, based on the total weight of the pharmaceutical composition. In certain embodiments, the anti-TDP-43 oligomer antibody is present at a level of at least 5% by weight, based on the total weight of the pharmaceutical composition. In still other embodiments, the anti-TDP-43 oligomer antibody is present at a level of at least 10% by weight, based on the total weight of the pharmaceutical composition. In still yet other embodiments, the anti-TDP-43 oligomer antibody) is present at a level of at least 25% by weight, based on the total weight of the pharmaceutical composition.
  • the medicament of said pharmaceutical composition of this invention further includes an agent that is known to improve the symptoms of a neurodegenerative disease.
  • agent include, and are not limited to, AChEI, an ⁇ inhibitor, or a muscarinic receptor agonist, and the like.
  • the medicament or said pharmaceutical composition is prepared in accordance with acceptable pharmaceutical procedures, such as described in Remington's Pharmaceutical Sciences, 17 th edition, ed. Alfonoso R. Gennaro, Mack Publishing Company, Easton, Pa (1985).
  • Pharmaceutically acceptable excipients are those that are compatible with other ingredients in the formulation and biologically acceptable.
  • the anti-TDP-43 oligomer antibody of this invention may be administered by any means known in the art, such as orally, intraperitoneally, intracranially, intrathecally, intramuscularly, intraveneously, transdermally, rectally or by inhalation, alone or in combination with conventional pharmaceutically acceptable excipients.
  • the anti-TDP-43 oligomer antibody of this invention is administered intraveneously to the subject.
  • the anti-TDP-43 oligomer antibody of this invention is administered intrathecally to the subject.
  • Applicable solid excipients may include one or more substances that may also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents or an encapsulating material.
  • the excipient is a finely divided solid that is in admixture with the finely divided active ingredient.
  • the active ingredient is mixed with an excipient having the necessary compression properties in suitable proportions and compacted in the shape and size desired.
  • the powders and tablets preferably contain up to 99% of the active ingredient.
  • Suitable solid excipient includes, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine and the like.
  • the anti-TDP-43 oligomer antibody of the present invention may also be formulated into liquid pharmaceutical compositions, which are sterile solutions or suspensions that can be administered by, for example, intravenous, intramuscular, subcutaneous, intrathecal, intraperitoneal, or intra-cerebella injection. Oral administration may be either liquid or solid composition form.
  • the medicament or said pharmaceutical compositions of this invention may also be formulated in a variety of dosage forms for mucosal application, such as buccal and/or sublingual drug dosage units for drug delivery through oral mucosal membranes.
  • a wide variety of biodegradable polymeric excipients may be used that are pharmaceutically acceptable, provide both a suitable degree of adhesion and the desired drug release profile, and are compatible with the active agents to be administered and any other components that may be present in the buccal and/or sublingual drug dosage units.
  • the polymeric excipient comprises hydrophilic polymers that adhere to the wet surface of the oral mucosa.
  • polymeric excipients include, but are not limited to, acrylic acid polymers and copolymers; hydrolyzed polyvinylalcohol; polyethylene oxides; polyacrylates; vinyl polymers and copolymers; polyvinylpyrrolidone; dextran; guar gum; pectins; starches; and cellulosic polymers.
  • this invention also provides methods of treating mammals, preferably humans, of a TDP-43 oligomer associated disease, which comprises the administration of the medicament or said pharmaceutical composition of this invention that contains anti-TDP-43 oligomer antibody as described herein.
  • a mammal preferably human
  • Such medicament or composition is administered to a mammal, preferably human, by any route that may effectively transports the active ingredient(s) of the composition to the appropriate or desired site of action, such as oral, nasal, pulmonary, transdermal, such as passive or iontophoretic delivery, or parenteral, e.g., rectal, depot, subcutaneous, intravenous, intrathecal, intramuscular, intranasal, intra-cerebella, ophthalmic solution or an ointment.
  • the administration of the compound of this invention with other active ingredients may be concurrent or simultaneous.
  • the effective dose administered to the subject is from about 1 to 100 mg/Kg body weight of the subject, such as about 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg/Kg body weight of the subject, preferably about 50 to 70 mg/Kg body weight of the subject, such as 50, 60 or 70 mg/Kg body weight of the subject; most preferably about 50 mg/Kg body weight of the subject.
  • the dose can be administered in a single aliquot, or alternatively in more than one aliquot.
  • the method may further include the step of, administering to the subject an acetylcholinesterase inhibitor (AChEI), an ⁇ inhibitor, or a muscarinic receptor agonist, either simultaneously or sequentially with the anti-TDP-43 oligomer antibody as described above.
  • an acetylcholinesterase inhibitor AChEI
  • an ⁇ inhibitor a muscarinic receptor agonist
  • the AChEI is any of alantamine, cymserine, donepezil, ER 127528, galantamine, ganstigmine, huperzine A, phenserine, phenethylnorcymserine, rivastigmine, RS 1259, SPH 1371 , tacrine, thiacymserine, or zanapezil.
  • the ⁇ inhibitor is any of bapineuzumab, PTB2, scyllo-inositol, PPI 1019, RS 0406, SP 233, EGCG, Exberyl-1 , or SEN 606.
  • the muscarinic receptor agonist is oxotremorine or xanomeline.
  • the anti-TDP-43 oligomer antibody of the present invention may also be used as a tool for the detection or diagnosis of a TDP-43 oligomer associated disease in a subject. Accordingly, this invention provides a method for detecting or diagnosing a subject having or suspected of having a TDP-43 oligomer associated disease.
  • the method includes steps of, obtaining a biological sample from the subject; determining the amount of the TDP-43 oligomer in the biological sample by contacting the biological sample with an effective amount of the present antibody; and comparing the detected amount of the TDP-43 oligomer in the biological sample with that of a control sample obtained from a healthy subject; wherein a significantly higher or lower amount of the detected TDP-43 oligomer in the biological sample than that of the control sample indicates that the subject suffers from the TDP-43 oligomer associated disease.
  • the biological sample described herein includes, but is not limited to, a brain biopsy sample, a cerebrospinal fluid sample, a whole blood sample, a serum sample, a plasma sample, a urine sample, a mucus sample and purified or filtered forms thereof.
  • Antibody binding may be detected by techniques known in the art, such as radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), "sandwich” immunoassay, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels), western blot, agglutination assay (e.g., gel agglutination assay, hemagglutination assay and etc), complement fixation assay, immunofluorescence assay, and Immunoelectrophoresis assay and etc.
  • ELISA enzyme-linked immunosorbent assay
  • sandwich immunoassays e.g., using colloidal gold, enzyme or radioisotope labels
  • western blot e.g., using colloidal gold, enzyme or radioisotope labels
  • agglutination assay e.g., gel agglutination assay, hemagglutination assay and etc
  • complement fixation assay e.g
  • autoantibodies are detected in bodily fluids, including but are not limited to cerebrospinal fluid, whole blood, serum, plasma, mucus, and purified or filtered forms thereof.
  • bodily fluids including but are not limited to cerebrospinal fluid, whole blood, serum, plasma, mucus, and purified or filtered forms thereof.
  • antibodies were detected from a cerebrospinal fluid sample.
  • antibodies are detected from a brain biopsy sample.
  • the anti-TDP-43 oligomer antibody of the invention is assembled into kits for the diagnosis, detection or confirmation of a neurodegenerative disease.
  • the presence of pathologic forms of TDP-43 reactive to the anti-TDP-43 oligomer antibody of this invention is used to provide prognosis to a subject.
  • the detection of a significant different level of pathologic forms of TDP-43 reactive to the anti-TDP-43 oligomer antibody of this invention, as compared to controls (derived from a healthy subject), in a biological sample is indicative of occurrence of the TDP-43 oligomer associated disease.
  • the information provided is also used to direct the course of treatment.
  • therapies for the treatment of the TDP-43 oligomer associated disease such as AD, ALS and PD, may be started at an earlier time when they are more likely to be effective.
  • the present invention provides a kit for the diagnosis of a TDP-43 oligomer associated disease by use of the anti-TDP-43 oligomer antibody of this invention.
  • the components included in the kits are: a container, reagents for detecting TDP-43 oligomer in a biological sample, wherein the reagents comprise the anti-TDP-43 oligomer antibody of this invention prepared in accordance with the procedure described in one example of this invention; and a legend associated with the container and indicating how to use the anti-TDP-43 oligomer antibody of this invention for detecting the pathological form of TDP-43 (or TDP-43 oligomer as described herein) in the biological sample.
  • the legend may be in a form of pamphlet, tape, CD, VCD or DVD.
  • the kit may further comprise a negative control that indicates the normal level of the TDP-43 oligomer that forms a complex with the anti-TDP-43 oligomer antibody in a healthy subject.
  • TDP-43 The isolated human TDP-43 from HEK cells was a gift from OriGene Technologies, Inc. (Rockville, MD, USA). Based on the product description, TDP-43 was obtained from transiently transfected human HEK 293 cells with a TrueORF clone, RC210639. The recombinant TDP-43 possessed a C-terminal Myc-DDK tag. The overexpressed recombinant TDP-43 protein was purified using an anti-DDK affinity column. The short-form of TDP-43 (residues 101 -285) is prepared in accordance with procedures described by Kuo et al (Nucleic Acids Res (2009) 37, 1799-1808).
  • Anti-N-terminal residues 1 -260 TDP-43 antibody (denoted as N -2 6o) for dot blotting and anti-TDP-43 antibody for immunohistochemistry were both purchased from Abeam (Cambridge, UK).
  • Anti-C-terminal residues 350-414 TDP-43 antibody (denoted as C35o- 4 i 4 ) was from Novus (Littleton, CO, USA) and anti-amyloid oligomer antibody A1 1 was from BioSource (Invitrogen, Carlsbad, CA, USA).
  • Anti-DDK monoclonal antibody was from Origene Technologies, Inc.
  • ⁇ peptide was synthesized by peptide synthesis facility in the Genomics Research Center, Academia Sinica. Other chemicals were purchased either from Sigma Aldrich (St. Louis, MO, USA) or Amresco (Solon, OH, USA). All cell culturing reagents were purchased from Gibco (Invitrogen) except for further indication.
  • TDP-43 Purification of TDP-43.
  • the N-terminal His-Tag TDP-43 was cloned from the plasmid pCMV-Taq2B containing the cDNA encoding full-length TDP-43.
  • the amplified product was double digested with Xhol/BamHI and subcloned into pET14b vector (Novagen, Merck KGaA, Darmstadt, Germany) to generate an N-terminal His-tag.
  • the N-terminal His-tagged TDP-43 was transformed and overexpressed in E. coli strain Rosetta 2. (Novagen, Merck KGaA, Darmstadt, Germany).
  • the cells were harvested and lysed by a microfluidizer on ice in 30 mM Tris-HCI buffer, pH 8, containing 500 mM NaCI, 10% glycerol, 1 mM DTT, 2% RNase A, 2% DNase I, and protease inhibitor cocktail (Complete, EDTA-free, Roche Applied Science, Mannheim, Germany). The lysate was centrifuged at 27,000 ⁇ g, 4 °C.
  • TDP-43 was eluted at approximately 200 mM imidazole. Purified his-tagged TDP-43 protein was run on SDS-PAGE and identified by Coomassie blue staining. The recombinant TDP-43 contained extra N-terminal residues MGSSHHHHHHSSGLVPRGSHMLE. The calculated molecular mass is 47,147 Da.
  • the protein was further dialyzed as indicated. Protein concentration was quantified after background subtraction by absorption at 280 nm with the extinction coefficient of 44,920 cm “1 M “1 according to the equation described by Nick Pace (Pace et al., (1995) Protein Sci 4, 241 1 -2423). For the short-form of TDP-43, the extinction coefficient of 15,470 cm “1 M "1 was used.
  • One ml fractions were collected automatically by a fraction collector.
  • the preload sample and the oligomer fraction were collected and subjected to dot blotting probed by A1 1 (1 :1000) with different exposure times due to much diluted signal from the oligomer fraction.
  • the oligomer fraction was characterized with dynamic light scattering.
  • a volume of 100 ⁇ of TDP-43 obtained from HEK cells at 2 ⁇ were also examined with the same procedure. The fractions were analyzed by slot blotting.
  • TDP-43 Dot blotting.
  • Purified TDP-43 was diluted 10 times into 10 mM Tris-HCI buffer, pH 8, with or without denaturants. Each sample in different conditions contains either no denaturant, 9 M urea, 7.2 M GdnHCI, or 2% SDS. The final TDP-43 concentration was 0.4 ⁇ . The samples were either incubated for 1 hr at the room temperature or 90 °C. The TDP-43 samples, 2 ⁇ , were dotted onto nitrocellulose membranes and were subjected to dot blotting.
  • the membranes were subjected to anti-N-terminal residues 1 -260 TDP-43 antibody (1 :2,000), Anti-C-terminal residues 350-414 TDP-43 antibody (1 :2,000), and anti-amyloid oligomer antibody, A1 1 , (1 :1 ,000) in 5% milk with TBST followed by the corresponding horseradish peroxidase-conjugated secondary antibodies anti-rabbit or anti-mouse IgG (1 :5,000; Millipore, Billerica, Massachusetts, USA).
  • the membranes were developed with ECL chemiluminescence reagent (Millipore).
  • TDP-43 Transmission electron microscopy. Freshly purified TDP-43 was dialyzed into a buffer containing 10 mM Tris, pH 8, at 4 °C overnight. The sample was centrifuged at 1 7,000xg, 4°C, for 30 min to remove precipitates and the supernatant was quantified and subjected to TEM imaging. The TDP-43 samples were placed on glow-discharged, 400-mesh Formvar carbon-coated copper grids (EMS Inc., Hatfield, PA, USA) for 5 min, rinsed, and negatively stained with 2% uranyl acetate. The samples were examined with Tecnai G2 Spirit TWIN TEM (FEI, Hillsboro, OR, USA) or Hitachi H-7000 TEM (Hitachi Inc., Japan) with an accelerating voltage of 75 kV.
  • EMS Inc. Hatfield, PA, USA
  • the sample was left to dry in the room temperature and subjected to AFM imaging (Nanonics, Jerusalem, Israel) using tapping mode.
  • AFM tips, PPP-ZEILR (Nanosensors, Neuchatel, Switzerland) with a spring constant of 1 .6 N/m and the tip radius ⁇ 10 nm were employed for the experiment.
  • Circular dichroism Freshly purified TDP-43 was dialyzed into a buffer containing 10 mM Tris, pH 8, at 4°C overnight. The sample was centrifuged at 17,000xg, 4°C, for 30 min to remove precipitates and the supernatant was quantified and subjected to CD measurement.
  • Far-UV CD spectra were measured in a circular quartz cell (Hellma, Forest Hills, NY, USA) by Jasco J-815 spectropolarimeter (Jasco Inc., Easton, MD, USA) with 1 mm path length at room temperature. The spectra were collected from 250 to 190 nm and corrected with buffer background.
  • TDP-43 Intrinsic and Bis-ANS fluorescence spectroscopy. Freshly purified TDP-43 was dialyzed into a buffer containing 10 mM Tris, pH 8, at 4°C overnight. The sample was centrifuged at 17,000xg, 4°C, for 30 min to remove precipitates and the supernatant was quantified. The intrinsic fluorescence of TDP-43, at 1 .5 ⁇ , was collected from 305 to 400 nm while excitation at 280 or 295 nm. The Bis-ANS spectra of TDP-43, TDP-43s, and buffer control were collected from 450 to 600 nm with excitation at 400 nm. The samples contain 0.8 ⁇ TDP-43 and 5 ⁇ Bis-ANS. All experiments were done at 25°C with a circulating water bath using FluoroMax-3 spectrofluorometer (Horiba Jobin Yvon, Kyoto, Japan).
  • TDP-43 Thioflavin T binding. Freshly purified TDP-43 was dialyzed into a buffer containing 10 mM Tris, pH 8, at 4°C overnight. The sample was centrifuged at 17,000xg, at 4°C, for 30 min to remove precipitates and the supernatant was quantified. The TDP-43 sample and ⁇ 40 fibrils were used to detect thioflavin T binding. The samples, 1 ⁇ , were mixed with equimolar thioflavin T and excited at 442 nm, and the emission spectra from 455 to 505 nm were collected. ⁇ 40 fibril stock, 25 ⁇ , was prepared as previously described (Chen and Glabe, J. Biol. Chem.
  • Fluorescence titration for DNA binding Fluorescence titration was employed to monitor protein conformational changes upon DNA binding. Freshly purified and dialyzed full-length TDP-43 and short-form of TDP-43 (residues 101 -285), at 1 .5 ⁇ , in 30 mM Tris-HCI, pH 8, were titrated with single strand TAR DNAs including TAR DNA A-site (SEQ ID NO: 9) and B-site (SEQ ID NO: 10). The intrinsic protein fluorescence was excited at 280 nm to monitor the conformational change of TDP-43.
  • the emission spectra were collected by FluoroMax-3 spectrofluorometer (Horiba Jobin Yvon, NJ, USA). The emission maxima at 350 nm for full-length or short-form TDP-43 were collected, subtracted with the DNA controls, and corrected with the dilution factor. Then, the data were normalized to the starting intensity and further fitted to a single protein and ligand binding equation (Chen et al., Protein Sci (2004) 13, 2196-2206):
  • the experimental samples containing 25 ⁇ ⁇ , 50 ⁇ ThT, and different concentrations of freshly dialyzed TDP-43 ranging from 0 to 1 ⁇ (0-4%) were prepared in 10 mM Tris-HCI, pH 8, and 150 mM NaCI.
  • the samples were then incubated in a 96-well ELISA plate in quiescence, sealed with a transparent film, and monitored by a microplate reader (SpectraMax M5, Molecule Devices) at 25°C at various times. ThT emission was measured at 485 nm while excitation was at 442 nm.
  • the cells were then washed and replaced by 40 ⁇ of serum-free RPMI media followed by addition of 10 ⁇ serially diluted TDP-43 samples with the dialysis buffer.
  • the TDP-43 samples were freshly dialyzed and centrifuged. The supernatant was employed and the dialysis buffer served as a buffer control.
  • the cells were further incubated for 24 hr, and MTT assay was performed following the standard protocol. In brief, a volume of 7 ⁇ of 5 mg/ml MTT was added in each well and cells were incubated for 3 hr. Then, the media were discarded and DMSO was added to lyze the cells until the purple formazan crystals were fully dissolved.
  • the substrate fluorescence was excited at 560 nm and emission at 590 nm was measured by the ELISA plate reader.
  • the data were obtained from three replicas, averaged, and corrected by the sample background without cells.
  • the p-values of MTT and LDH assays were calculated using unpaired Student's t-test.
  • the dialysis buffer was served as buffer control. MTT formazan crystals were then dissolved by equal volume of lysis buffer containing 10% SDS and 20 mM HCI overnight. The absorbance at wavelength 570 nm in each well was measured on an ELISA plate reader (TECAN, Switzerland). The data were obtained from three replicas, averaged, and corrected by the sample background without cells. The p-values for MTT assay were calculated using unpaired Student's t-test.
  • MAP-2 microtubule associated protein-2
  • GFAP polyclonal rabbit anti-glial fibrillary acidic protein
  • Each animal received bilateral intrahippocamal injection of recombinant full-length TDP-43.
  • the stereotaxic coordinates were in relation to bregma as follows: anteroposterior (AP), -2 mm; mediolateral (ML), ⁇ 1 mm; dorsoventral (DV), -2 mm.
  • the injection needle slowly approached to the desired depth and 2 ⁇ TDP-43, 2.2 ⁇ , was injected using a stainless-steel syringe needle (33-gauge) connected to a microsyringe (Hamilton Company, NV, USA) at an injection rate of 1 ⁇ /min. The needle was left in place for an additional 5 min to limit the diffusion of the injected TDP-43.
  • PFA paraformaldehyde
  • PBS phosphate buffered saline
  • ⁇ ⁇ - ⁇ -thick sections were treated with a blocking solution containing 0.2% Triton X-100 and 5% normal donkey serum in 0.01 M PBS at room temperature for 1 h.
  • the sections were incubated in mouse monoclonal anti-NeuN (1 :300, Millipore, Temecula, CA) at 4°C overnight and Alexa Fluor 555-conjugated donkey anti-mouse antibody (1 :300, Chemicon, Temecula, CA) at room temperature for 1 h.
  • the sections were then counterstained with DAPI and mounted with fluorescent mounting medium (DAKO, Glostrup, Denmark). All sections were examined under an upright fluorescent microscope (BX51 , Olympus, Tokyo, Japan).
  • the sections were incubated with anti-TDP-43 antibody (1 :1000, Abeam, ab104223), TDPO (1 : 1000, LTK BioLaboratories, Taiwan), Alexa Fluor 488-conjugated goat anti-rabbit antibodies and the Alex Fluor 555-conjugated donkey anti-mouse antibodies (1 :300, Invitrogen).
  • the sections were then incubated with DAPI and coverslipped with fluorescent mounting medium (fluorescent mounting medium; Dako). All sections were examined in a laser scanning confocal microscope (Nikon TE2000EPFS-C1 -Si).
  • TDP-43 oligomer-specific polyclonal antibody production and characterization.
  • the purified full-length TDP-43 was dialyzed into 10 mM Tris-HCI, pH 8.0, at 4°C, and concentrated to approximately 0.2 mg/ml to serve as immunogen.
  • New Zealand white rabbits were immunized with the immunogen following the standard protocol for polyclonal antibody production (LTK BioLaboratories, Taiwan). Briefly, the immunogen was injected 0.5 ml into the rabbit at 2-week intervals. After 6 injections, the rabbit was sacrificed, and the blood serum was obtained for usage.
  • TDP-43 For characterization of TDP-0 by dot blotting and ELISA, full-length TDP-43 was subjected to SEC (Superdex-200 10/300 GL, GE healthcare Bio-Sciences AB, Uppsala, Sweden) in a buffer containing 30 mM Tris-HCI, pH 8.0, and 150 mM NaCI with a flow rate of 0.3 ml/min. One ml fractions were collected and subjected to dot blotting probed by TDP-0 and Ni -2 6o antibodies. The TDP-43 oligomer and monomer fractions were further quantified by Micro BCATM Protein Assay Kit (Thermo Scientific, Rockford, IL) and coated onto ELISA plate with serial dilution.
  • ELISA following the standard protocol was performed. Briefly, the coated samples were incubated overnight at 4°C, then blocked for 2 hr at room temperature with 10% skim milk in TBST. The plates were washed and probed by TDP-0 (1 : 12,500 in 3% skim milk in TBST) or anti-N-terminal residues 1 -260 TDP-43 antibody (1 : 1 ,000 dilution in 3% skim milk in TBST) for 2 hr at room temperature.
  • the plates were subjected to anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1 :1 ,000; Merck Millipore, Billerica, Massachusetts, USA) for 2 hr at room temperature, washed, then developed by 100 ⁇ 3,3,5,5-tetramethylbenzidine (TMB, Merck Millipore, Billerica, Massachusetts, USA).
  • TMB 3,3,5,5-tetramethylbenzidine
  • the reaction was stopped with addition of 100 ⁇ of 250 mM HCI and the absorbance was read at 450 nm by SpectraMax M5 (Molecular Device, Sunnyvale, CA).
  • ⁇ 5 peptide within RRM2 domain of TDP-43 fragment was chemically synthesized (MDBio, Inc., USA). The peptide was solubilized by acetonitrile and diluted 10 times with PBS buffer (pH 7.4) at 5 mg/ml. The supernatant was collected after centrifugation at 17,000 * g for 30 min and filtered through a 0.2 ⁇ filter membrane (Pall, USA). Then, the sample was incubated in quiescence at room temperature for 7 days. ⁇ 5 fibrils were formed and the morphology was observed by TEM. The sample was subjected to dot blotting probed by TDP-0 antibody following the aforementioned procedure.
  • TDP-0 staining of the brain tissues of FTLD-TDP patients Paraffin-embedded tissue sections were de-waxed with three washes of Xylene. Tissues were then hydrated through an alcohol gradient, followed by a five-min wash in 1 x TBS. Antigen recovery was performed using 10 mM sodium citrate (pH 6) by microwaving for 15 min at full power (1 ,500 watts). Endogenous peroxidase activity was blocked by incubating the tissue in 3% hydrogen peroxide in 1 x TBS for 20 min, followed by a five-min wash in 1 x TBS.
  • Non-specific binding was blocked by incubating the tissues in 10% normal goat serum (Invitrogen) for 1 hr at ambient temperature.
  • Primary antibody was diluted at 1 :1000 in antibody buffer (1 x TBS supplemented with 5% bovine serum albumin and 0.5% Tween-20) and applied to the tissues overnight at 4°C. Slides were washed three times in wash buffer (1 x TBS/0.5% Tween-20). Biotinylated secondary antibodies were applied at 1 :500 dilution in antibody buffer for 1 hr at ambient temperature.
  • Tissue was washed three times in wash buffer, then incubated with Vectastain ABC peroxidase reagent (Vector Labs, PK-6100) for 30 minutes, followed by one wash in 1 x TBS. The tissues were then exposed to DAB peroxidase substrate reagent (Vector Labs, SK-4100) for 2-10 min to achieve optimal staining. Tissues were counterstained with Mayers Hematoxylin Solution (Sigma Aldrich), and then dehydrated through an alcohol gradient followed by a xylene wash. Tissue slides were mounted using Permount (Sigma Aldrich). Post-mortem brain samples from three control, three FTLD-TDP, and three AD patients were provided by the Alzheimer's Disease Center at University of California Davis. The study was approved by the Institutional Review Board. Informed consent to share research tissue after death was obtained from all patients.
  • TDP-0 antibodies on the IP beads were prepared following the manufacturer's procedure. Briefly, a total of 40 ⁇ g of TDP-0 antibody was incubated with 10 ⁇ each of protein A and G Mag SepharoseTM Xtra (GE healthcare) in the binding buffer (50 mM Tris pH 7.5, 150 mM NaCI) at room temperature for 1 hr. After antibody binding on the beads, crosslinking was performed by crosslink reagent (50 mM dimethyl pimelimidate dihydrochloride and 200 mM triethanlamine, pH 8.9) at RT for 1 hr and the reaction was stopped by the addition of 100 mM ethanolamine, pH 8.9, at RT for 30 min.
  • crosslink reagent 50 mM dimethyl pimelimidate dihydrochloride and 200 mM triethanlamine, pH 8.9
  • the soluble fractions of hippocampus were incubated with the TDP-0 antibody-crosslinked IP beads at room temperature for 1 hr.
  • the unbound species were removed by at least 6 washes with the binding buffer.
  • the targeted protein was eluted by Gentle Ag/Ab Elution Buffer (Thermo), pH 6.6, and the eluent was saved for EM imaging and immunogold labeling.
  • 10 ⁇ of eluent was placed on 400-mesh Formvar carbon-coated copper grids (EMS Inc., Hatfield, PA, USA) for 5 min, washed by PBS, and then blocked by 1 % BSA in PBS solution at RT for 1 hr.
  • the grids were labeled by N -2 6o antibody (1 : 5,000, ab57105, Abeam) in 0.1 % BSA containing PBS solution at RT for 1 hr, and washed by high salt tween (HST) buffer (50 mM Tris, pH 7.5, 500 mM NaCI, and 0.1 % Tween-20) and PBS. After washing, grids were incubated with 6 nm gold-conjugated secondary anti-mouse IgG antibody (1 : 40, Jackson ImmunoResearch) at RT for 1 hr. The unbound antibody was removed by HST and PBS washes.
  • HST high salt tween
  • the grids were fixed by 1 % glutaraldehyde containing PBS at RT for 10 min and washed 6 times by ddH 2 0. Finally, the grids were rinsed, negatively stained with 2% uranyl acetate, and subjected to TEM imaging with FEI Tecnai G2 F20 S-TWIN TEM.
  • the monoclonal TDP-0 antibodies were purified by protein G agarose kit (KPL) following the manufacturer's procedure. Briefly, Protein G agarose (1 .5 ml) supplied in 20% ethanol was poured into a disposable column. The resin was washed with 10 ml wash buffer (0.1 M sodium phosphate, pH 7.4, 0.15 M NaCI). Conditional medium of TDP-0 hybridoma cells (5 ml) was diluted with 5 ml binding buffer. Then, the mixed sample solution was loaded into the column and the column was inverted for mixing. Then, the column was washed by 5 ml wash buffer for 4 times until OD 280 nm was close to 0.
  • wash buffer 0.1 M sodium phosphate, pH 7.4, 0.15 M NaCI
  • the antibody was then eluted gently by adding 1 ml elution buffer (0.2 M glycine, pH 2.85) and collected in a collection tube that contained 240 ⁇ 5X wash buffer.
  • ELISA 96-well plates (Nunc MaxiSorp) were coated with 20 ng full-length TDP-43 oligomer protein dissolved in 200 ⁇ in TBS (50 mM Tris, pH 7.4, 150 mM NaCI) at 4°C overnight. After removing the coating solution, TBS buffer with and without 0.2% SDS were added into the coated wells at room temperature for 1 hr. TDP-43 oligomers treated with SDS will denature, whereas, TDP-43 oligomers treated without SDS will maintain the oligomer conformation.
  • TBST buffer (20 mM Tris, pH 7.6, 137 mM NaCI, 0.001 % Tween 20) for 2 hr at room temperature.
  • 100 ⁇ of diluted mTDP-0 antibodies (from 0.00001 to 2 ⁇ g/ml) in TBST containing 5% skim milk was added and incubated for 1 hr at room temperature.
  • the bound antibodies were detected by using the following 100 ⁇ HRP conjugated anti-mouse antibody (1 :1 ,000) diluted in 5% skim milk in TBST.
  • TDP-43 oligomers and monomers for indirect ELISA assay.
  • TDP-43 protein 180 ⁇ g/ml was concentrated from 3 to 1 ml by using Amicon Ultra-4 30 kDa cutoff. Concentrated TDP-43 protein solution, 500 ⁇ , was loaded onto a Superdex 200 10/300 GL column and eluted with 30 mM Tris, pH 8.0, 50 mM NaCI at a flow rate of 0.3 ml/min. Fractions (500 ⁇ per fraction) were collected and the fractions containing TDP-43 oligomers or monomers were pooled separately. The protein concentration was quantified by micro BCA assay (Thermo).
  • TDP-43 monomers or oligomers 300 ng were coated on 96-well ELISA plates (Nunc MaxiSorp). Conditional medium of TDP-0 hybridoma cell (100 ⁇ ) (1 :1 ,000) were used to detect TDP-43 oligomers by indirect ELISA assay as mentioned above.
  • mAb monoclonal antibody
  • Thermo isotyping Kit
  • ELISA strip-well plates with individual wells pre-coated with anti-mouse heavy-chain capture antibody (anti-lgG-i , lgG 2a , lgG 2b , lgG 3 , IgA and IgM) or anti-mouse light-chain capture antibody (kappa or lambda) were used.
  • anti-mouse heavy-chain capture antibody anti-lgG-i , lgG 2a , lgG 2b , lgG 3 , IgA and IgM
  • anti-mouse light-chain capture antibody kappa or lambda
  • the HRP conjugated anti-mouse IgG + lgA+ IgM 50 ⁇ was added to each well of the 8-well strip and the reaction was incubated for an hr at room temperature. After 3 times washing, TMB substrate (75 ⁇ ) was added to each well and the plate was incubated at room temperature in dark for 10 min. Next, the reaction was stopped by adding 75 ⁇ of 0.18 M sulfuric acid. The absorbance of each well was read by SpectraMax M5 (Molecular Devices) at 450 nm.
  • Cytotoxicity assay MTT assay was employed to examine the inhibitory effect of monoclonal TDP-0 antibody on TDP-43 oligomers-induced cytotoxicity.
  • Human neuroblastoma BE(2)-C cells ATCC #CRL-2268 were incubated at 37°C under 5% C0 2 and cultured in RPMI media with 10% fetal bovine serum. Cells were seeded in RPMI media in a 96-well plate with 40,000 cells per well and incubated for 24 hr.
  • Monoclonal TDP-O-3 (5 mg/ml) was prepared without further dilution, or with 2- or 4-folds dilution with Dulbecco's PBS.
  • TDP-43 pathology was investigated.
  • a recombinant full-length human TDP-43 in E. coli (TDP-43 with N-terminal His-Tag, MW 47,145 Da) and a TDP-43 protein purified from HEK293 cells were respectively obtained in accordance with the procedures described in "Materials and Methods.”
  • the thus obtained TDP-43 proteins were then subject to SEC analysis, and results are depicted in FIG 1A.
  • FIG 1 A As evidenced in FIG 1 A, more than 86% of TDP-43 was eluted in the void volume, and slot blotting confirmed the majority of the recombinant full-length TDP-43 proteins from two different sources readily formed large aggregates.
  • TDP proteinopathies are characterized by inclusion body (IB) formation and high-molecular-weight aggregates were found in the recombinant full-length TDP-43, we speculated that TDP-43 may form oligomers resembling the amyloid oligomers in amyloidosis. Therefore, the conformation-dependent anti-amyloid oligomer-specific antibody, A1 1 , generated against ⁇ oligomer mimics, was used to examine the TDP-43 oligomers, and we found both TDP-43 samples, but not the corresponding buffers, were immunoreactive with A1 1 (FIG 1 B). The oligomer fraction containing diluted TDP-43 exhibited weaker intensity.
  • IB inclusion body
  • Example 2 Full-length TDP-43 forms heterogeneous spherical oligomers
  • TEM transmission electron microscopy
  • AFM atomic force microscopy
  • TDP-43 oligomers are conformational and functional distinct
  • TDP-43 oligomers were characterized by far-UV circular dichroism (CD), and results are illustrated in FIG 2A. Two double minima were respectively observed near 210 and 222 nm, which likely represent a-helical structures. The spectrum differs from that of the short-form mouse TDP-43 (residues 101 -265, denoted as TDP-43s in plots), which comprises two RRMs with mostly ⁇ -strands in its crystal structure (Kuo et al., Nucleic Acids Res (2009) 37, 1799-1808).
  • CD far-UV circular dichroism
  • TDP-43 oligomers were titrated with single-strand TAR DNA-A or -B sites and compared with the short-form TDP-43 that has been reported to bind these ssDNAs in submicromolar affinity (Kuo et al., Nucleic Acids Res (2009) 37, 1799-1808).
  • the dissociation constant, Kd, obtained from the fits are 7.05 ⁇ 0.82, 6.27 ⁇ 0.68, 0.20 ⁇ 0.09, and 0.38 ⁇ 0.10 ⁇ for full-length TDP-43 with TAR-A, full-length TDP-43 with TAR-B, short-form TDP-43 with TAR-A, and short-form TDP-43 with TAR-B, respectively.
  • TDP-43 oligomers either possess abnormal conformations with reduced DNA binding capability or are hindered from DNA binding because of masking of the binding regions.
  • the emission change that remained within the full-length TDP-43 could be attributed to the existence of a small population of TDP-43 monomers in the sample.
  • Example 4 TDP-43 oligomers convert ⁇ to amyloid oligomers
  • Photo-induced cross-linking was then performed to examine the transient ⁇ species appeared in the starting time point, and results indicated that ⁇ alone formed primarily monomers, dimers, trimers, and tetramers after cross-linking, whereas TDP-43 oligomer served to seed more higher-molecular-weight ⁇ species (FIG 3B).
  • ⁇ pentamers were observed dose-dependently upon TDP-43 addition. Also, two larger assemblies, migrating at -55 kDa and smearing from -105 to > 210 kDa, were observed.
  • TDP-43 oligomers In addition to the SDS-irresistant TDP-43 monomers, a -55 kDa species and some species causing a smear from -80 to > 210 kDa were found. Further analysis using TEM imaging showed that ⁇ did not undergo fibril formation, but rather was transformed into spherical oligomers with a diameter of ⁇ 10 nm in the presence of 4% TDP-43 oligomers, whereas ⁇ alone still formed mature amyloid fibrils as expected (FIG 3C). The TDP-43 oligomer seeds retained the diameter of >50 nm which was larger than those of ⁇ oligomers (data not shown). These results supported that TDP-43 oligomers can induce the ⁇ oligomer formation, indicating again that TDP-43 shares common properties with amyloids.
  • TDP-43 oligomers may cause neurotoxicity and neurite degeneration was investigated.
  • TDP-O Since the anti-amyloid oligomer antibody (i.e., A1 1 ) cross-reacted with different amyloids, to validate whether the TDP-43 oligomers are present in the disease, a polyclonal antibody were generated using the recombinant TDP-43 oligomers as immunogen in rabbit in accordance with procedures described in "Materials and Methods" section, and the thus obtained polyclonal antibody is named TDP-O.
  • A1 1 anti-amyloid oligomer antibody
  • TDP-0 antibody against the full-length TDP-43 oligomeric conformation was examined by dot blotting under various denaturing conditions as described in "Materials and Methods" section, and results are illustrated in FIG 5A.
  • TDP-0 with 1 :125,000 dilution was capable of reacting with full-length TDP-43 in the native buffer regardless of heating (90°C, 1 h); however, the reactivity between the antibody and the TDP-43 oligomer disappeared when 2% SDS was present, with or without heating.
  • the reactivity did not diminish when the full-length TDP-43 was treated with either 7.2 M GdnHCI or 9 M urea at room temperature for more than one hr, which indicated that the protein was stable under high concentration of chemical denaturants; however, the reactivity signal started to decrease when additional heating was employed, indicating that TDP-0 is a conformational dependent antibody.
  • the dot blotting result from TDP-0 was qualitatively identical to that of A1 1 (FIG 5A vs FIG 1 C).
  • TDP-0 is TDP-43 specific rather than a common amyloid oligomer antibody like A1 1
  • the antibody specificity against ⁇ oligomers was examined using A1 1 and/or TDP-O.
  • TDP-0 failed to recognize ⁇ oligomers, which demonstrates that it is specific toward TDP-43 oligomers.
  • ELISA was also performed to quantitatively characterize the reactivity between TDP-0 and TDP-43 oligomers and monomers; while the TDP-43 antibody recognizing N-terminal residues 1 -260 (Ni -2 6o) was employed for comparison purpose.
  • the full-length TDP-43 at approximately 1 ⁇ or three times more concentrated TDP-43 samples was loaded into SEC, and 1 ml eluted fractions were collected (FIG 5C).
  • the SEC fractions of the concentrated TDP-43 from fractions 1 to 18 were then subjected to dot blotting with TDP-0 or N-i-260 antibodies, and results are illustrated in FIG 5D. It was found that TDP-0 reacted strongly with fraction numbers 5 to 7, which corresponded to the TDP-43 oligomers eluted in the void volume. By contrast, Ni -2 6o exhibited strong reactivity with fraction 6, and fractions 15 to 18; which indicates that this antibody may recognize both oligomeric and monomeric form of TDP-43.
  • TDP-43 oligomer specificity with TDP-0 was quantified by ELISA, where the Ni -2 6o antibody was used as control.
  • the TDP-43 oligomer and monomer fractions were respectively coated onto ELISA plate with serial dilutions.
  • TDP-0 and N 1-260 were applied and developed following standard ELISA protocol. Results depicted in FIG 5E indicated that TDP-0 antibody exhibited strong reactivity towards TDP-43 oligomers (EC50 ⁇ 0.5 g/ml) than to TDP-43 monomer, whereas Ni -2 6o antibody exhibited similar reactivity toward ether TDP-43 oligomer or monomer (FIG 5E).
  • TDP-0 recognizes the oligomeric rather than fibrillar form of TDP-43 as previously reported (Wang et al., J Biol Chem (2013) 288, 9049-9057)
  • the ⁇ 5 fibrils generated from the 5 th ⁇ -strand within RRM2 domain of TDP-43 were dotted (data not shown), and result clearly showed that TDP-0 recognizes only TDP-43 oligomers but not the ⁇ 5 fibrils.
  • the polyclonal TDP-0 antibody of this application may specifically recognize TDP-43 oligomeric conformation without cross-reacting with ⁇ species.
  • Example 7 TDP-43 oligomers exist in transgenic mice brain
  • FTLD-TDP-43 transgenic mice expressing full-length TDP-43 in the forebrain has been reported to recapitulate FTLD-TDP-like pathology (Tsai et al., J Exp Med (2010) 207, 1661 -1673), for their deficiency in the learning/memory capabilities and motor functions as they age.
  • the brain sections of FTLD-TDP-43 transgenic mice were immunofluorescently stained with anti-TDP-43 and TDP-0 and counterstained with DAPI.
  • TDP-43 was found predominantly in the nucleus.
  • TDP-43 was mainly found in the cytosol in the FTLD-TDP transgenic mice, while TDP-0 signals were also detected.
  • Significant amount of TDP-43 and TDP-0 signals were found to colocalize in the cells, and number of cells with double positive signals increased with age. The appearance and colocalization of the signals suggested that amyloid-like TDP-43 oligomers do exist in the FTLD-TDP transgenic mice brain and the amount increases with age.
  • Example 8 TDP-43 oligomers are present in brains of FTLD-TDP patients
  • TDP-43 oligomers In order to evaluate the relevance of TDP-43 oligomers in human diseases, the presence of TDP-43 oligomers in FTLD-TDP patients was investigated using the subgroup of FTLD patients with TDP-43 immunoreactive inclusions.
  • the TDP-0 polyclonal antibodies were used to immunostain hippocampal and frontal cortical sections of three cases of pathologically confirmed FTLD-TDP, three cases of age-matched non-dementic controls, and three cases of pathologically confirmed AD without TDP-43 pathology as the "disease controls".
  • TDP-0 antibody identified various neuronal cytoplasmic inclusions (FIG 7, panels A and C) and dystrophic neurites (FIG 7, panel A) that appeared to be similar to the TDP-43 inclusions in FTLD-TDP patients.
  • neuronal cytoplasm was intensively stained in a granular pattern (FIG 7, panel E).
  • TDP-0 did not exhibit significant immune-reactivity towards the control brains (FIG 7, panels B, D, and F), nor to AD brains lacking TDP-43 pathology (data not shown).
  • TDP-0 polyclonal antibody failed to stain the nuclei in normal tissue, which is consistent with our biochemical observation that it does not react with TDP-43 monomer normally present in the nuclei.
  • IP immunoprecipitation
  • TDP-43 oligomers exist in the brains of FTLD-TDP patients.
  • ELISA assay was performed to examine the specificity of each mAbs produced from TDP-O-3, -5, -8, -9, and -10 hybridoma cells on TDP-43 oligomers with or without SDS denaturation. Results are depicted in FIG 9. Monoclonal antibodies produced from any of the five hybridoma cell lines exhibited higher binding activities toward non-denatured TDP-43 oligomers than the denatured TDP-43 protein. The results indicated that these mAbs have specificity toward TDP-43 oligomers and they are conformational-dependent antibodies.
  • TDP-43 oligomers and monomers were purified by gel filtration (FIG 10A), and then respectively coated on plates for ELISA to confirm the specificity of each mAbs.
  • Results as depicted in FIG 10B confirmed that each of the mAbs produced from TDP-O-3, -5, -8, -9, and -10 hybridoma cell lines may specifically recognize TDP-43 oligomers.
  • ELISA mouse mAb isotyping Kit (Thermo) was also used to determine the isotypes of the TDP-0 mAbs. Results are summarized in Table 1 .
  • Example 10 TDP-43 oligomer-specific mAb inhibits TDP-43 oligomers-induced cytotoxicity
  • TDP-0 antibody can inhibit the cell cytotoxicity induced by TDP-43 oligomers
  • human neuroblastoma BE(2)-C cells were respectively treated by TDP-43 oligomers with or without monoclonal TDP-0 antibody (i.e., TDP-O-3) at the indicated dosages as described in "Materials and Methods”.
  • TDP-O-3 monoclonal TDP-0 antibody
  • the cell viability was then measured by MTT assay.
  • the present disclosure unexpectedly discovers a pathological form of TDP-43, which cross-seeds ⁇ to form amyloid oligomers and is associated with neurodegenerative diseases. Accordingly, the present disclosure aims at providing an antibody useful in suppressing the TDP-43 proteinopathy. Results from the foregoing working examples confirm and support that the present anti-TDP-43 antibodies specifically bind to the pathological form of TDP-43 and suppress the TDP-43-induced cytotoxicity; thus, they may act as a potential means to prevent and/or treat the TDP-43-associated neurodegenerative diseases.

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Abstract

L'invention concerne une nouvelle forme pathologique de TDP-43, des anticorps monoclonaux contre cette forme pathologique de TDP-43, et les utilisations de ces derniers. La nouvelle forme pathologique de TDP-43 est caractérisée par sa taille particulaire sphérique d'environ 2 à 400 nm de diamètre. Un second aspect de la présente invention concerne l'utilisation d'un anticorps pour la fabrication d'un médicament ou d'une composition pharmaceutique destinés à la prophylaxie ou au traitement de maladies associées à l'oligomère TDP-43.
PCT/US2015/050114 2014-10-03 2015-09-15 Anticorps contre les formes pathologiques de tdp-43 et utilisations de ces derniers WO2016053610A1 (fr)

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US11355328B2 (en) 2016-04-13 2022-06-07 Purdue Research Foundation Systems and methods for isolating a target ion in an ion trap using a dual frequency waveform
JP2020522475A (ja) * 2017-05-30 2020-07-30 ザ ユニヴァーシティ オブ ブリティッシュ コロンビア Ｔｄｐ−４３のｒｎａ認識モチーフ１（ｒｒｍ１）中のエピトープ、およびそれらに対するミスフォールディング選択的抗体
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JPWO2019177138A1 (ja) * 2018-03-16 2021-05-13 国立大学法人滋賀医科大学 異常ｔｄｐ−４３を分解除去する抗体断片
JP7320282B2 (ja) 2018-03-16 2023-08-03 国立大学法人滋賀医科大学 異常ｔｄｐ－４３を分解除去する抗体断片
WO2021222168A2 (fr) 2020-04-28 2021-11-04 Sola Biosciences Llc Compositions et méthodes pour le traitement de protéinopathies tdp-43
WO2022034228A1 (fr) * 2020-08-14 2022-02-17 Ac Immune Sa Molécules de liaison anti-tdp-43 humanisées et leurs utilisations
WO2022115705A2 (fr) 2020-11-30 2022-06-02 Enigma Biointelligence, Inc. Évaluation non invasive de la maladie d'alzheimer

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