TW201632549A - Antibodies against pathological forms of TDP-43 and uses thereof - Google Patents

Abstract

Disclosed herein are novel pathological form of TDP-43, monoclonal antibodies against such pathological form of TDP-43, and uses thereof. The novel pathological form of TDP-43 is characterized in having a particle size of about 2 to 400 nm in diameter.

Description

Antibodies recognizing pathogenic TDP-43 and uses thereof

The present disclosure relates to a novel transactivation responsive-DNA-binding protein (43 kDa, TDP-43) having a molecular weight of 43 kD. An antibody to disease TDP-43 and the use of the antibody.

Neurodegenerative diseases have become a health issue that cannot be ignored in modern society. According to the World Health Organization, as of 2025, more than 75% of the elderly population worldwide will suffer from a variety of neurodegenerative diseases. Frontotemporal lobar degeneration (FTLD) is the second most common dementia in the US population younger than 65 years old; TDP-43 is known to cause FTLD and amyotrophic lateral sclerosis One of the main reasons for ALS). However, there is currently no effective treatment for these diseases, so various studies are still devoted to exploring the molecules involved in the regulation of pathogenic TDP-43 formation and related mechanisms.

In view of this, it is urgent to confirm the involvement in the regulation of disease-related diseases. The molecule of sex TDP-43, thereby preparing a medicament for preventing or treating a neurodegenerative disease caused by pathogenic TDP-43.

The present invention is based on the discovery that a transactivation responsive-DNA-binding protein (43 kDa, TDP-43) oligomer having a molecular weight of 43 thousand tracts is present in the brain of a patient. The body is neurotoxic in vivo and outside, and cross-links to Alzheimer's amyloid-β (Aβ) such as Alzheimer's disease to form amyloid oligomers. Thus, a molecule (eg, an antibody) that binds to a TDP-43 oligomer will be used to inhibit TDP-43 proteinopathies and thereby produce a diagnostic, prophylactic or therapeutic treatment by pathogenic TDP-43. Drugs that cause neurodegenerative diseases (eg, vaccines and passive immunization).

Accordingly, the present disclosure is directed to providing a human antibody or fragment thereof that specifically binds to a TDP-43 oligomer. The human antibody or fragment thereof comprises a heavy chain variant region comprising an amino acid sequence of SEQ ID NO: 1, 2 and 3, and a fragment of the light chain comprising the sequence number : Amino acid sequences of 5, 6 and 7.

The TDP-43 oligomer is a particle having a diameter of about 2 to 400 nanometers (nm). Preferably, the TDP-43 oligomer is a spherical or cyclic particle having a diameter of from about 40 to about 60 nanometers.

According to certain preferred embodiments, the heavy chain variant region of the human antibody or fragment thereof has the amino acid sequence of SEQ ID NO: 4, and the light chain variant region thereof has the amino acid sequence of SEQ ID NO: 8.

In one embodiment, the human antibody or fragment thereof is Prepared from a fusion tumor cell line that has been deposited with the Bioresource Collection and Research Center (BCRC) under the accession number BCRC 960494.

In another embodiment, the human antibody or fragment thereof It is prepared by a fusion tumor cell line which has been deposited in the Center for Biological Resource Conservation and Research under the accession number BCRC 960495.

In still another embodiment, the human antibody or fragment thereof The segment was prepared from a fusion tumor cell line which has been deposited with the Bioresource Conservation and Research Center under the accession number BCRC 960496.

In another embodiment, the human antibody or fragment thereof It is prepared by a fusion tumor cell line which has been deposited in the Center for Conservation and Research of Biological Resources, and the accession number is BCRC 960497.

In another embodiment, the human antibody or fragment thereof It is prepared by a fusion tumor cell line which has been deposited in the Center for Conservation and Research of Biological Resources, and the accession number is BCRC 960498.

Thus, a second aspect of the disclosure provides the antibody Use, that is, to prepare a pharmaceutical or pharmaceutical composition for preventing or treating a disease associated with a TDP-43 oligomer. The pharmaceutical or pharmaceutical composition comprises an effective amount of the above human antibody or fragment thereof and a pharmaceutically acceptable excipient.

According to a preferred embodiment of the present disclosure, the human anti- The fragment or a fragment thereof comprises a heavy chain variant region and a light chain variant region, wherein the heavy chain variant region comprises an amino acid sequence of sequence numbers: 1, 2 and 3, The light chain variant region comprises the amino acid sequence of SEQ ID NOs: 5, 6, and 7.

According to the remaining preferred embodiments of the present disclosure, the anti- The body is prepared from a fusion cell line that has been deposited with the Center for Conservation and Research of Biological Resources, under the accession numbers BCRC 960494, BCRC 960495, BCRC 960496, BCRC 960497 or BCRC 960498.

The weight of the antibody of the present invention is about the total weight of the pharmaceutical composition. 0.1% to 99%. In one embodiment, the antibody of the invention has a weight of at least 1% by weight of the total weight of the pharmaceutical composition. In another embodiment, the antibody of the invention has a weight of at least 5% of the total weight of the pharmaceutical composition. In still another embodiment, the antibody of the invention has a weight of at least 10% of the total weight of the pharmaceutical composition. In another embodiment, the antibody of the invention has a weight of at least 25% of the total weight of the pharmaceutical composition.

The medicament or pharmaceutical composition of the present disclosure can be used for treatment A disease associated with TDP-43 oligomers, which may be Alzheimer's disease, argyrophilic grain disease, amyotrophic lateral sclerosis (amyotrophic lateral sclerosis). ALS), Guam muscle atrophic lateral sclerosis - ALS-parkinsonism dementia complex of Guam, vascular dementia, frontotemporal dementia, semantics Dementia (dementia with Lewy bodies), Huntington's disease, Spinocere bellar ataxia, inclusion body Myopathy), inclusion body myositis or Parkinson's disease.

Therefore, the third aspect of the present disclosure provides a use A method for preventing or treating a disease associated with a TDP-43 oligomer. The method comprises administering to the individual a therapeutically effective amount of an antibody of the invention or a fragment thereof, thereby inhibiting TDP-43 proteinopathy.

According to certain preferred embodiments, the antibody of the present invention or The fragment comprises a heavy chain variant region comprising a nucleotide sequence of sequence numbers 1, 2 and 3, and a light chain variant region comprising the sequence number: 5, 6 and The amino acid sequence of 7.

According to the remaining preferred embodiments of the present disclosure, the anti- The fragment or fragment thereof is prepared from a fusion cell line that has been deposited with the Center for Conservation and Research of Biological Resources under the accession numbers BCRC 960494, BCRC 960495, BCRC 960496, BCRC 960497 or BCRC 960498.

The method of the present disclosure can be used to treat with TDP-43 A polymer-related disease, which may be Alzheimer's disease, argyrophilic granulosuscitation, amyotrophic lateral sclerosis, Guam muscle atrophic lateral sclerosis - Parkinson's dementia, vascular Dementia, frontotemporal dementia, semantic dementia, Levi dementia, Huntington's disease, cerebellar atrophy, inclusion body myopathy, inclusion body myositis or Parkinson's disease.

The fourth aspect of the present disclosure provides a method for diagnosis Whether a body is suffering from a disease associated with TDP-43 oligomers. The method comprises obtaining a biological sample from the individual; the biological sample and the present Inventing a human antibody or a fragment thereof to determine the content of the TDP-43 oligomer in the biological sample; and the content of the TDP-43 oligomer in the biological sample and the control sample taken from a healthy individual The content of TDP-43 oligomer is compared; wherein if the content of TDP-43 oligomer in the biological sample is significantly different from the TDP-43 oligomer content of the control sample, it means that the individual suffers from There are diseases with neurodegenerative diseases.

According to a preferred embodiment of the present disclosure, the biopsy The body may be a brain slice sample, a cerebrospinal fluid sample, a whole blood sample, a serum sample, a plasma sample, a urine sample or a mucus sample.

According to certain preferred embodiments, the human antibody or The fragment comprises a heavy chain variant region comprising a nucleotide sequence of sequence numbers 1, 2 and 3, and a light chain variant region comprising the sequence number: 5, 6 and The amino acid sequence of 7.

According to other preferred embodiments, the antibody or a fragment thereof The segment is prepared from a fusion tumor cell line that has been deposited with the Center for Conservation and Research of Biological Resources with accession numbers BCRC 960494, BCRC 960495, BCRC 960496, BCRC 960497 or BCRC 960498.

Therefore, the fifth aspect of the present disclosure provides a use A kit for detecting the presence of a pathogenic TDP-43 of a biological specimen. The results of the assay can be used to assess whether the individual has a disease associated with TDP-43 oligomers. The kit comprises at least one container and an agent for detecting TDP-43 oligomer, wherein the reagent comprises the anti-TDP-43 oligomer antibody of the present invention and a specification attached to the container, the instruction is used To inform the use of the anti-TDP-43 oligomer antibody of the present invention to detect pathogenic TDP-43 (i.e., the above TDP-43 oligomer) method. The instructions can be a book, a tape, a CD, a VCD or a DVD. The kit may additionally comprise a control group for controlling the normal levels of TDP-43 oligomers in healthy individuals.

After referring to the following embodiments, the technical field of the present invention The basic spirit and other objects of the invention, as well as the technical means and implementations of the invention, are readily apparent to those of ordinary skill in the art.

To make the above and other objects, features, advantages and embodiments of the present invention more apparent, the description of the drawings is as follows: Figure 1 shows that the full length TDP-43 forms an oligomer similar to the amyloid-like protein (a The results of analytical size-exclusion chromatography (SEC) of full-length TDP-43, wherein TDP-43 purified by E. coli is detected by the absorbance at a wavelength of 280 nm. The solid line), while TDP-43 purified from human HEK293 cells was quantified by the intensity of slot blotting (dashed line); the residence time shown in the figure is the residence time of the molecular weight standard group; b) Dot blotting of TDP-43, which was detected by anti-amyloid oligomer-like antibody A11; freshly purified and not subjected to particle size chromatography by A11 TDP-43 (preloaded with TDP-43), TDP-43 (extracted volume, TDP-43 oligomer) treated with particle size exclusion chromatography and buffer for immunostaining; (c) freshly purified TDP-43 is spotted in buffer, containing 9M urea buffer, containing 7.2M guanidine hydrochloride (Gd) nHCl) buffer or buffer containing 2% sodium dodecyl sulfate (SDS), and heated at 90 ° C or not heated for 1 hour; the test was repeated three times, and respectively with A11 antibody, Anti-TDP-43 N-terminal (residue 1-260) antibody and anti-TDP-43 C-terminal (residue 250-414) antibody for detection; (d) transmission electron microscope ( Transmission electron microscope, TEM) and (e) atomic force microscope (AFM) to detect TDP-43 oligomer (scale bar is 500 nm), and the upper left is a magnified view of a single oligomer (scale bar is 50 nm) (f) and (g) are the results of dynamic light scattering (DLS) analysis of TDP-43 oligomers after particle size chromatography; wherein the particle size can scatter light intensity (f And the number of particles (g); Figure 2 TDP-43 structure, thioflavin T (ThT Flavin T, ThT) fluorescence and DNA binding properties (a) full-length TDP-43 (solid line) and short Far-UV CD spectra of TDP-43 (dashed line); wavelength range from 250 to 190 nm; (b) full-length TDP-43 (solid line) and short TDP-43 (dashed line) Bis-ANS fluorescence spectrum; buffer The number is marked as a dotted line; (c) the binding spectrum of TDP-43 and Aβ fiber to thioflavin T; the full-length TDP-43 thioflavin T fluorescence divergence wavelength is indicated by the solid line connection, short The thioflavin T fluorescence divergence wavelength of TDP-43 is indicated by the dotted line connection, while the thioflavin T fluorescence emission wavelength of Aβ fiber is represented by the ▲ dotted line; only Aβ fiber has sulfur (1) Fluorescence titration was used to detect the binding of TDP-43 to TAR DNA; single-strand TAR DNA-A (solid) and -B (open) were titrated to full length TDP-43 (square) and short TDP-43 (circular); when excited by a light wave with a wavelength of 280 nm, the maximum divergence of TDP-43 or TDP-43s can be detected at a wavelength of 350 nm; Standardization is based on the starting point, and the connecting line is the optimum line; Figure 3 is the cross-linking of TDP-43 with Aβ (a) Aβ fibrillation in the absence or inclusion of 0.4 to 4% TDP-43 The result of the thioflavin T test; the concentration is expressed as the percentage of moles of TDP-43; (b) the time point is 0, and there is no photo-induced cross-linking (T---). PICUP) test results, The concentration of the TDP-43 are expressed as a percentage mole; a transmission electron microscope the results of (c) contains no or Aβ 4% TDP-43 of the end product (100 nm scale bar); FIG. 4 TDP- 43 oligomers can induce axonal degeneration and toxicity in vitro and in vivo. The cytotoxicity of TDP-43 on human BE(2)-C cells can be achieved by (a) thiazolyl tetrazolium bromide (MTT, 3- [4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) and (b) lactate dehydrogenase (LDH) LDH assay for analysis (quantity 3, mean ± mean standard deviation) (c) The MTT assay was used to analyze the cytotoxicity of TDP-43 on primary neuronal culture; in this assay, cell viability was normalized to the buffer control group (quantity 3, mean ± standard) (a) and (c) are statistically analyzed by one-way-variance analysis (one way-ANOVA) and Tukey post-test (Tukey post test), and (b) is analyzed by single factor variance. Two-tailed, unpaired Student's t-test (two tailed, unpaired Student's t-test) for analysis (* p <0.05, ** p < 0.01, *** p <0.001); (d) with control group or 0.44 μM Results of immunocytochemical staining of primary neurons treated with TDP-43; specimens were fluorescently stained with MAP-2 (red), GFAP (green), and DAPI (blue); (e) injection of TDP-43 When the hippocampus returned to the mouse, the neurons in the CA area of the hippocampus were reduced. The buffer control group and TDP-43 were injected into the hippocampus (only 3 in each group), and the neuron-specific marker NeuN and Nuclear specific stain DAPI was used for immunohistochemical staining; the arrow pointed to the location of neuron reduction (scale bar is 50 microns); Figure 5 TDP-0 antibody can specifically identify TDP-43 oligomer (a) will be fresh The purified TDP-43 is dissolved in the original buffer, the buffer containing 9M urea, the buffer containing 7.2M pulse hydrochloride or the buffer containing 2% sodium lauryl sulfate, and heated at 90 ° C or After heating for 1 hour, the spotted method was performed using a multi-drug antibody TDP-O, which was prepared by using TDP-43 oligomer as an immunogen; TDP-O and TDP- The immune response produced by 43 is similar to the A11 results shown in Figure 1c; (b) the Aβ and TDP-O antibodies were used to analyze the Aβ oligomers by spot blotting; It is shown that A11 can recognize Aβ oligomers, but TDP-O does not; (c) particle size screening and chromatography results of original TDP-43 (dotted line) and 3-fold concentrated TDP-43 (solid line), among which The elution volume of TDP-43 oligomer is ★, the elution volume of TDP-43 monomer is ☆, the number is the molecular weight standard group; (d) The 1ml particle size fractionation chromatography liquid is collected to TDP- O (top) and N _1-260 antibodies (bottom) were subjected to spot blotting analysis; (e) analysis of TDP-purified by particle size exclusion chromatography by enzyme-linked immunosorbent assay (ELISA) 43 oligomers and monomers; TDP-43 samples were applied to the ELISA plate according to the concentration, and TDP-O (■) and N _1-260 (□) antibodies were used to detect TDP-43 oligomers, respectively. TDP-43 monomer was detected by TDP-O(•) and N _1-260 (○) antibodies respectively; TDP-O antibody has significantly higher specificity for TDP-43 oligomer; Figure 6 FTLD-TDP The expression of TDP-43 oligomers in genetically transformed mice increases with age (a) will be obtained from wild-type, 6-month or 12-month-old TDP-43 gene-transforming mice (TDP-43 Tg ^+/+ transgenic mice) sections of the brain for immunofluorescence staining to detect human TDP-43 and TDP-43 oligomers Performance; cells stained with anti-TDP oligomers will appear green, cells stained with anti-TDP-43 will appear red, while cells stained with DAPI will appear blue (scale bar 100 microns); The region is further displayed in the rightmost column at a higher magnification (25 micrometer scale); (b) Quantitative results indicate that the proportion of cells with TDP-O and TDP-43 precipitation is age-related (3 in each group, of which each mouse Five fields were randomly selected for quantification, and the data were expressed as mean ± mean standard deviation; the results were statistically analyzed by single factor analysis and Tukey post hoc determination; * p <0.05, ** p <0.01, *** p <0.001 Figure 7: FTLD-TDP patients will have three FTLD-TDP patients with TDP-43 oligomers , three healthy and healthy individuals of the same age with neurological and pathological analysis, and three patients with Azheimer's disease. Patients with symptoms without TDP-43 inclusion bodies (as a neurodegenerative disease control) were analyzed in this study; the representative brain slices of these individuals are listed in Figure 7, where (a), (c) and (e) TDP-43 immunization of hippocampal gyrus (c) and prefrontal cortex (a, e) in FTLD-TDP patients using TDP-O antibodies Weaving chemical staining; TDP-O can identify dark-stained, oval or irregular-shaped inclusion bodies (red arrows) in the cytoplasm and neuropiles (neuropils, malnourished axons) in the form of commas Body (blue arrow); in some cortical areas as shown in (e), the neuronal cytoplasm will exhibit a coarse granular immune response; as shown in (b), (d) and (f), Individual brain sections do not produce any TDP-O immunostaining reaction; compared to antibodies against TDP-43 monomer, TDP-O antibody does not stain the nucleus, confirming that the antibody can specifically bind to abnormal Folded TDP-43; the scales in each figure are 20 microns; Figure 8: TDP-43 oligomers extracted from the patient's hippocampus by immunoprecipitation will be labeled with TDP-O antibody by immunogold calibration for FTLD-TDP patients. The hippocampus was returned to the tissue and immunoprecipitated with TDP-O antibody; the extract was labeled with N-terminal TDP-43 antibody and immunogold, and analyzed by electron microscopy (EM) (scale bar is 50 nm); (a) TDP-43 oligomers in patients with FTLD-TDP (scale bar is 100 nm); (b) TDP-43 oligomers Enlarged view (scale bar is 50 nm); Figure 9 TDP-O monoclonal antibody is highly specific for TDP-43 oligomers. TDP-O-3, -5, -8, -9 and -10 are fused, respectively. The TDP-O monoclonal antibody produced by the tumor cell strain was analyzed by ELISA test at different concentrations (1-2 x 10 ^-5 μg per ml) to detect destructive or undestructive denaturing with sodium lauryl sulfate. TDP-43; Figure 10 TDP-O monoclonal antibody is highly specific for purified TDP-43 oligomers. TDP purified by size screening chromatography using conditioned medium of TDP-O fusion tumor cell line. -43 oligomers and monomers were analyzed by ELISA assay; (A) TDP-43 oligomers and monomers were isolated and collected on a Superdex 200 10/300 GL column; (B) TDP-O was used in an ELISA assay Conditioned medium of -3, -5, -8, -9, and -10 fusion tumor cell lines to detect TDP-43 oligomers and monomers; and the heavy chain of TDP-O monoclonal antibodies of Figure 11 The variant region and the light chain variant region have a uniform amino acid residue of the amino acid sequence X, and the CDR is an abbreviation for the complementarity determining region.

The description of the embodiments of the present invention is intended to be illustrative and not restrictive. The features of various specific embodiments, as well as the method steps and sequences thereof, are constructed and manipulated in the embodiments. However, other specific embodiments may be utilized to achieve the same or equivalent function and sequence of steps.

I. Definition

The term "TDP-43 proteinopathy" (TDP-43 proteinopathy) refers to a transactivation responsive-DNA-binding protein (43 kDa) with a molecular weight of 43 kD. TDP-43) related diseases. TDP-43 is a disease protein associated with frontal lobe degeneration and musculoskeletal lateral sclerosis with ubiquitin-positive inclusion (FTLD-U). TDP-43 protein lesions include, but are not limited to, Alzheimer's disease, argyrophilic granulosuscitation, amyotrophic lateral sclerosis, Guam muscle atrophic lateral sclerosis - Parkinson's dementia, blood vessels Sexual dementia, frontotemporal dementia, Semantic dementia, Levi dementia, Huntington's disease, cerebellar atrophy, inclusion body myopathy, inclusion body myositis or Parkinson's disease.

The word "effective amount" is used in this description. The book refers to the effective dose and treatment time period for achieving the desired therapeutic outcome for a disease associated with TDP-43 oligomers.

"pharmaceutically acceptable" The term acceptable refers to a molecule or composition that is generally considered safe when administered to a human, such as such molecules or compositions that are physiologically compatible and that do not produce an allergic or similar untoward reaction. Preferably, the term "pharmaceutically acceptable" as used in this specification refers to a molecule or composition that is approved by a regulatory agency of the United States federal or state government and that is applicable to animals or humans.

"administered" (administered, administering or Administration, in this specification, is an interchangeable term and refers to a method of directly administering a specific antibody or composition of the invention.

"subject" or "patient" is Refers to an animal comprising a human that is capable of receiving treatment of the compositions and/or methods of the invention. "Subject" or "patient" means both male and female, unless a specific gender is specified. Therefore, a "subject" or "patient" contains any mammal that is acceptable for treatment. A "subject" or "patient" includes, but is not limited to, humans, rats, mice, guinea pigs, monkeys, pigs, sheep, cattle, horses, dogs, cats, birds, and poultry. In an embodiment of the invention, the patient is a human.

"treat or treatment" in this manual is Refers to the production of a pharmaceutically and/or physiological effect, such as detecting the manifestations of pathogenic TDP-43, preventing or treating organ atrophy, or inhibiting dementia, muscle weakness and stiffness. The effect may be prophylactic, i.e., complete or partial prevention of a disease or a condition thereof; and/or therapeutic, i.e., partial or complete cure of a disease and/or discomfort caused thereby. "Treatment" in this specification includes the prevention, treatment or alleviation of a disease in a mammal (especially human); the treatment comprises: (1) preventing, treating or slowing down a disease (eg neurodegenerative disease) , wherein the individual is at a high risk group suffering from the disease, or has not been diagnosed with the disease; (2) inhibiting a disease (eg, inhibiting its occurrence); or (3) reducing a disease (eg, alleviating the disease) Related symptoms).

"Antibody" or "antibodies" are defined broadly in this specification and include biologically active monoclonal antibodies, polyclonal antibodies, polyspecific antibodies (eg, bispecific antibodies), and any of the above antibody fragments; It refers to the property of recognizing a target protein and specifically binding to an antigen in a mixture containing other proteins and/or molecules. In accordance with an embodiment of the present disclosure, the antibody of the present invention is a multi-strain antibody that specifically recognizes TDP-43 oligomers.

The term "monoclonal antibody" as used in this specification refers to an antibody which is isolated from a population of antibodies of the same nature and which need not be constructed by a specific method. Compared with a plurality of antibodies, the system comprises a plurality of antibodies which can respectively identify different epitopes, and the monoclonal antibodies can only uniquely identify a single antigen-determining position of an antigen. The monoclonal antibodies of the present disclosure can be produced by a fusion method or a recombinant DNA method. The monoclonal antibodies of this specification may contain "embedded "chimeric" or "recombinant" antibody, wherein a portion of the heavy and/or light chain of the antibody is associated with an antibody or a subspecies of a particular antibody or subtype Antibodies having the same or homologous corresponding sequences; and other regions of the heavy and light chains are another antibody or antibody fragment derived from other species or subtypes of other antibody types or subtypes (as long as the fragment Corresponding biological activity), having the same or homologous corresponding sequences. According to a particular embodiment of the present disclosure, the antibody of the invention is a monoclonal antibody that specifically recognizes TDP-43 oligomers.

"Humanization" of non-human (eg mouse) antibodies A (humanized) antibody refers to a chimeric antibody comprising a minimal sequence derived from a non-human immunoglobulin. Humanized antibodies are human immunoglobulins whose residues are derived from non-human species (eg, mice, rats, rabbits, or other non-human primates with the desired specificity or affinity). Replacement of residues in the variant region. In some cases, the Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Generally, a humanized antibody will generally comprise at least one (usually two) variable regions, wherein all or a majority of the FR regions are sequences of human immunoglobulins. A humanized antibody can optionally comprise a partial sequence of a constant region of an immunoglobulin, typically a human immunoglobulin.

The singular nouns "a" and "the" and "the" are used in the singular terms of the noun.

II. Description of the invention

It is known that the molecular weight is 43 thousand guanines and the activation reaction - deoxygenation Transactivation responsive-DNA-binding protein (43kDa, TDP-43) is a pathogenic protein linked to the frontotemporal lobe with ubiquitin-positive inclusion (FTLD-U) expression. Degenerative and muscular atrophic lateral sclerosis are associated. The inventors of the present application found that full-length TDP-43 forms a structurally stable and neurotoxic spherical oligomer which crosslinks to amyloid-β (Alzheimer's amyloid-β, Aβ). And causing Aβ to form amyloid-like oligomers; therefore, an agent (eg, an antibody) capable of binding to a TDP-43 oligomer will be used to inhibit TDP-43 proteinopathy; accordingly, the agent can be used to prepare a drug (eg vaccine or passive immunization) to prevent or treat neurodegenerative diseases caused by pathogenic TDP-43.

Thus, the first aspect of the present disclosure provides an anti- More specifically, the antibody is an antibody that recognizes a TDP-43 oligomer and thereby inhibits aggregation of a protein, particularly TDP-43, wherein the aggregation phenomenon is associated with a disease. In general, protein aggregation is a self-propagating manner. Once initiated, aggregation reactions will occur automatically and initiate structural changes and/or polymerization of more protein molecules, ultimately resulting in the inability to be proteases. Decomposed toxic products. The resulting protein oligomers are considered to be one of the proximate causes of neurodegenerative diseases including Alzheimer's disease, argyrophilic granuloceptive disorder, and amyotrophic lateral sclerosis. , Guam muscle atrophic lateral sclerosis - Parkinson's dementia, blood vessels Dementia, frontotemporal dementia, semantic dementia, Levi dementia, Huntington's disease, cerebellar atrophy, inclusion body myopathy, inclusion body myositis and Parkinson's disease.

According to a preferred embodiment of the present disclosure, the TDP-43 is The polymer is a particle having a particle size of from about 2 to 400 nm. Preferably, the TDP-43 oligomer is a particle having a particle size of from about 2 to 400 nm, such as from 20 to 30 nm, from 30 to 40 nm, from 40 to 50 nm, from 50 to 60 nm, 60. Up 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 Nano, 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. More preferably, the TDP-43 oligomer is a spherical or cyclic particle having a particle size of from about 40 to about 60 nanometers.

The antibodies of the present disclosure can be specifically bound to the above The TDP-43 oligomer, its antigen-determining position, its different configuration, and the epitope of each configuration. In a preferred embodiment, the antibody preferentially binds to pathogenic TDP-43, and more specifically, the pathogenic TDP-43 is a full-length TDP-43 oligomer particle having a particle size of about 2 to 400 nm.

To prepare the monoclonal antibody, firstly, the appropriate amount of TDP-43 The oligomer is injected into an animal such as a mouse, a rat or a rabbit to cause an immune response. Generally, the adjuvant will first be mixed with the TDP-43 oligomer solution and injected into the animal. Adjuvants suitable for use in the present invention comprise Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA) and aluminum hydroxide adjuvant. Injection of the antibody can be administered by blood vessel, subcutaneous, abdominal cavity or muscle. The time interval for antigen injection is not specifically limited. The interval between antigen injections may be from several days to several weeks, and preferably from 2 to 3 weeks, from 1 to 10 times (preferably from 2 to 5 times). After the injection, once the titer of the antibody reached an absorbance of 2 or higher, the subsequent injection was stopped and the animal was kept for 1 month. Then, at least one more injection. The spleen cells and regional lymph nodes of the animal are removed several days after the last dose (preferably about 3 to 5 days). Blood samples were taken and centrifuged to separate the serum. Any suitable method can be used to detect antibody titers in the obtained serum, such as enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA) or radio immunoassay (RIA). . In a preferred embodiment, the titer of the antibody is determined by ELISA.

Preparation from isolated spleen cells and regional lymph nodes can be produced The cells of the antibody. In the preparation of cells that produce antibodies, it is desirable to remove tissue debris and red blood cells as much as possible. A commercially available red blood cell remover can be used in this step. Alternatively, a buffer containing ammonium chloride and Tris (hydroxymethyl) aminomethane (Tris) may be used. The antibody-producing cells are immediately fused with immortalized cells (such as myeloma cells) to produce a fusion tumor cell; the fusion tumor cell is a semi-immanent cell that sustainably grows and produces antibodies. A commonly used cell strain obtained from an animal such as a mouse can be used. More suitable The cell strain used in the present invention is effective for fusion, and continuously produces high amounts of antibodies, and is sensitive to hypoxanthine-aminopterin-thymidine (HAT) culture solution, and only A cell strain that can survive after cell fusion of the antibody. Suitable myeloma cells include, but are not limited to, mouse myeloma cell lines (eg, myeloma FO cells) and human myeloma cell lines (eg, Karpas 707H).

Cell fusion is usually the process of spleen cells or lymph node cells Commercially obtained myeloma cells undergo a fusion reaction under the action of a cell fusion factor, such as polyethylene glycol (PEG) or the like having an average molecular weight of about 200 to 20,000 kDa. Alternatively, cell fusion can be performed by electrical stimulation (e.g., electrofusion) using a commercial cell fusion instrument. After fusion, the resulting cells were diluted and cultured in HAT medium.

Selecting suitable fusion tumor cells from the fused cells The fused cells that survived the HAT medium formed aggregate communities. The supernatant of each culture solution was collected, and whether the antibody titer was detected by TDP-43 oligomer. As described above, the enzyme immunosorbent assay, the enzyme immunoassay, or the radioimmunoassay can be used to detect the TDP-43 oligomer or the oligomer to form a TDP-43 monomer, which is coated on the tray. And thus screening. Once the pores of the antibody are confirmed, the cells can be transferred to a hypoxanthine-thymidine (HT) culture medium containing no pterin. After the culture, the antibody titer in the culture supernatant was confirmed again. From the finally selected cells, a single cell was isolated. Waiting for a single cell to grow and multiply After forming a cluster of colonies, a highly specific aggregated community of TDP-43 oligomers was screened and allowed to continue to grow and grow to form a fusion tumor cell line.

According to a preferred embodiment of the present disclosure, a total of 5 fusion tumor cell lines: TDP-O-3, TDP-O-5, TDP-O-8, TDP-O-9 and TDP-O-10, and the monoclonal antibodies can be obtained from any of these fusion tumor cell lines in any way. Separation or preparation. For example, the fusion tumor cell strain can be cultured in a culture medium having a low serum concentration, and an antibody can be prepared from the culture supernatant. Alternatively, the fusion tumor cell strain is injected into the abdominal cavity of the animal, and the antibody is prepared from ascites. The antibody can be purified in any manner, including affinity columns, gel filtration chromatography, ion exchange chromatography or related techniques. Any method known to those skilled in the relevant art or a combination thereof may be used.

Individual resistance of the disclosure according to a particular embodiment The system was prepared from the fusion tumor cell lines TDP-O-3, TDP-O-5, TDP-O-8, TDP-O-9 and TDP-O-10, respectively. These fusion tumor cell lines have been deposited in Hsinchu, Taiwan. The Bioresource Collection and Research Center (BCRC) of the Development and Research Institute (FIDRI) has registration numbers BCRC 960494, BCRC 960495, BCRC 960496, BCRC 960497 and BCRC 960498. .

According to a preferred embodiment, the heavy chain and light weight of the monoclonal antibody The strand variant regions each comprise a consensus amino acid sequence. Accordingly, the monoclonal antibodies comprise a heavy chain variant region and a light chain variant region, wherein the heavy chain variant region comprises the sequence number: 1, 2 And the amino acid sequence of 3, and the light chain variant region comprises the amino acid sequence of SEQ ID NOs: 5, 6, and 7. In a preferred embodiment, the heavy chain variant region of the humanized monoclonal antibody has the amino acid sequence of SEQ ID NO: 4, and the light chain variant region thereof has the amino acid sequence of SEQ ID NO: 8.

Alternatively, the monoclonal antibody against the anti-TDP-43 oligomer can Prepared by DNA gene selection. The DNA encoding the anti-TDP-43 oligomer monoclonal antibody is isolated and sequenced using conventional procedures, such as oligonucleotides that are specifically conjugated to the heavy and light chain genes encoding the monoclonal antibodies. Glycosidic probe. A preferred source of DNA is the use of such fusion tumor cell lines (e.g., TDP-O-3, TDP-O-5, TDP-O-8, TDP-O-9 or TDP-O-10 fusion tumor cell lines). Once isolated, the DNA can be constructed into a performance vector, and the expression vector can be transfected into a host cell (eg, E. coli cells, simian cell COS, Chinese hamster ovary cell CHO, or myeloma cells that do not produce immunoglobulin), This produces a monoclonal antibody in a recombinant host cell.

Monoclonal antibodies produced and used to encode the monoclonal antibodies The DNA can be used to prepare chimeric antibodies (eg, bispecific antibodies), humanized antibodies, and/or antibody fragments thereof.

Individual antibodies of non-human origin cause an allergic reaction due to immunogenicity. Most of the monoclonal antibodies are derived from mice, which often cause a serious immune response after injection into the human body. In order to reduce the immunogenicity of the anti-TDP-43 oligomer monoclonal antibody of the present invention, the heavy chain and light chain variant regions of the mouse anti-TDP-43 oligomer monoclonal antibody are ligated to the constant region of the human antibody, Thereby a humanized antibody is prepared.

For the preparation of humanized anti-TDP-43 oligomer antibodies, The DNAs used to encode the antibodies are isolated and sequenced, and then humanized for construction.

Complementary in accordance with a preferred embodiment of the present disclosure The decision determining region (CDR) is grafted by replacing the CDRs in the VH and VL genes of the human antibody with specific CDR coding fragments (eg, among the anti-TDP-43 oligomer antibodies) a DNA fragment encoding an amino acid fragment that binds to a TDP-43 oligomer). The antibodies thus prepared are only CDRs derived from mouse antibodies, while the other framework regions of the VH and VL genes, as well as the constant region genes (ie CK or CH1-H-CH2-CH3) are human IgG.

In a preferred embodiment, the humanized anti-TDP-43 oligo The monoclonal antibody comprises a heavy chain variant region and a light chain variant region. The prepared humanized anti-TDP-43 oligomer monoclonal antibody can be purified according to standard procedures in the related art, including cross-flow filtration, affinity column chromatography (affinity column chromatography). Or methods such as gel filtration. It is to be understood that the effect of the humanized antibody should be identical or substantially similar to that of the mouse anti-TDP-43 oligomer antibody. Preferably, the humanized anti-TDP-43 oligomer antibody (whether in the form of Fab or full-length IgG) will produce a better effect when administered to a human subject than a mouse antibody. In certain embodiments, a humanized anti-TDP-43 oligomer antibody can be used to prepare a bispecific antibody of the present disclosure.

Any of the in vitro or in vivo TDP-43 protein lesion patterns can be used to confirm the anti-TDP-43 oligomer antibody of the present invention. Traditional technology The artisan will recognize that the anti-TDP-43 oligomer antibody of the present invention can be confirmed using an animal model (e.g., the animal model described in Example 7). Alternatively, the anti-TDP-43 oligomer antibody of the present invention can be confirmed using the relevant patient sample described in Example 8.

It should be understood by those skilled in the art that the experimental mode of TDP-43 protein lesions can be used as a preventive test or a therapeutic test. In a prophylactic test, an anti-TDP-43 oligomer antibody of the present invention is tested for TDP-43 protein lesion or its syndrome before the animal produces TDP-43 protein lesion or its symptom. The effectiveness of prevention, slowing or delaying. In a therapeutic test, the drug is administered after the animal produces TDP-43 protein lesion or its symptom, thereby testing the anti-TDP-43 oligomer antibody of the present invention for TDP-43 protein lesion or its symptom Therapeutic, palliative or soothing effects. Symptoms of TDP-43 proteinopathy include, but are not limited to, changes in the number of pathogenic TDP-43 in the brain, spinal cord, cerebrospinal fluid, or serum of the test subject.

Accordingly, the present disclosure provides a pharmaceutical composition or a medicament that can be used to treat a neurodegenerative disease associated with A[beta] accumulation. The pharmaceutical composition comprises an effective amount of an anti-TDP-43 oligomer antibody of the invention and a pharmaceutically acceptable excipient. Such TDP-43 oligomer-related diseases which can be treated with the pharmaceutical composition or medicament of the present disclosure include, but are not limited to, Alzheimer's disease, argyrophilic granulosuscitation, amyotrophic lateral sclerosis Disease, Guam muscle atrophic lateral sclerosis - Parkinson's dementia, vascular dementia, frontotemporal dementia, semantic dementia, Levi's dementia, Huntington's disease, cerebellum Atrophy, inclusion body myopathy, inclusion body myositis and Parkinson's disease.

In general, the anti-TDP-43 oligomer antibody of the present invention The weight is from about 0.1% to about 99% by weight based on the total weight of the pharmaceutical composition. In one embodiment, the anti-TDP-43 oligomer antibody of the invention comprises at least 1% by weight of the total weight of the pharmaceutical composition. In another embodiment, the anti-TDP-43 oligomer antibody of the invention has a weight of at least 5% of the total weight of the pharmaceutical composition. In still another embodiment, the anti-TDP-43 oligomer antibody of the invention has a weight of at least 10% of the total weight of the pharmaceutical composition. In another embodiment, the anti-TDP-43 oligomer antibody of the invention comprises at least 25% by weight of the total weight of the pharmaceutical composition.

In certain embodiments, the pharmaceutical compositions or medicaments of the invention The agent may further comprise an agent which is an agent known to be useful for ameliorating a neurodegenerative disease; for example, an acetylcholinesterase inhibitor (AChEI), an Aβ inhibitor or a muscarinic agent; Muscarinic receptor antagonists and analogs thereof.

The pharmaceutical composition or medicament of the present invention is based in accordance with acceptable pharmaceutical manufacturing method be prepared, for example, in Remington's Pharmaceutical Sciences, 17 ^th method edition, ed.Alfonoso R.Gennaro, Mack Publishing Company , Easton, Pa (1985) the . Pharmaceutically acceptable excipients are those which are compatible with the other ingredients of the dosage form and which are acceptable to the organism.

The anti-TDP-43 oligomer antibody of the present invention may be used alone or in combination with The pharmaceutically acceptable excipients are administered to the individual by oral, intraperitoneal, intracranial, intrathecal, intramuscular, intravenous, dermal, enteral or inhalation administration. In a preferred embodiment, the anti-TDP-43 oligomer anti-system of the invention is administered to an individual by intravenous injection. In another preferred embodiment, the antibody of the present invention The -TDP-43 oligomer antibody is administered to an individual by intrathecal injection.

Solid excipients that can be used can contain one or more substances, The substance may be a flavoring agent, a lubricant, a solubilizer, a suspending agent, a filler, a glidant, a compression aid, a binder, a tablet disintegrating agent or a coating material. If present in powder form, the excipient is a finely divided solid which can be combined with the finely divided active ingredient. If present in the form of a tablet, the active ingredient is compressed to the desired shape and size in an appropriate ratio with an excipient having compressible properties. The powders and tablets preferably contain up to 99% of the active ingredient. Solid excipients suitable for use in the present invention may be calcium phosphate, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone Or an analogue thereof.

The anti-TDP-43 oligomer antibody of the present invention may also be formulated as a liquid pharmaceutical composition in the form of a sterile solution or suspension which can be administered to an individual intravenously, intramuscularly, subcutaneously, intrathecally, intraperitoneally or intracranically. Oral administration can be in liquid or solid form.

In order to be administered in a mucosal manner, the medicament or pharmaceutical composition of the present invention can be formulated into various dosage forms, for example, if absorbed through the oral mucosa, the pharmaceutical or pharmaceutical composition of the present invention can be administered in buccal and/or sublingual dosage units. Formulation. Various pharmaceutically acceptable biodegradable polymeric excipients can also be used in the present invention which provide not only modest bioadhesion and drug release properties, but also buccal and/or sublingual drug dosages. The active agent in the unit is compatible with the other ingredients. Generally, polymeric excipients comprise a hydrophilic polymer that can adhere to the wet surface of the oral mucosa. Polymeric excipients suitable for use in the present invention comprise, But not limited to, acrylic polymers and copolymers, hydrolyzed polyvinyl alcohol, polyethylene oxides, polyacrylates, ethylene polymers and copolymers, polyvinylpyrrolidone, polydextrose, guar gum, pectin, starch and cellulose polymer.

Therefore, the present invention also provides a treatment for mammals (special A method of a disease associated with a TDP-43 oligomer, which comprises administering to a subject a pharmaceutical and pharmaceutical composition comprising an anti-TDP-43 oligomer antibody of the invention. The medicament or pharmaceutical composition can be administered in any manner effective to deliver the active ingredient of the composition to a particular location in a mammal, particularly a human, such as through the mouth, nose, lungs, skin (eg, passive or iontophoretic delivery). Or parenteral (eg rectal, fat, subcutaneous, intravenous, intrathoracic, intramuscular, nasal, cerebellum, ocular or ointment). Furthermore, the administration of the compositions of the invention may be administered simultaneously with other active ingredients.

In certain embodiments, the effective dose administered to the individual It is about 1 to 100 mg per kg of body weight, for example 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 mg per kg of body weight; preferably 50 to 100 kg per kg body weight 70 mg, for example 50, 60 or 70 mg per kg of body weight; optimally 50 mg per kg of body weight. These doses may be administered in a single dose or divided into multiple doses.

According to an alternative embodiment of the present disclosure, the method More preferably, the subject can be administered an acetylcholine inhibitor, an Aβ inhibitor or a muscarinic receptor antagonist, wherein the inhibitor/antagonist can be simultaneously with the anti-TDP-43 oligomer antibody of the present invention. Or have been given in succession.

In certain embodiments, the acetylcholine enzyme inhibitor It can be alanamine, cymserine, donepezil, ER 127528, galantamine, ganstigmine, huperzine A , phenhydroxyrine (phenenserine), phenethylnorcymserine, rivastigmine, RS 1259, SPH 1371, tacrine, thiacymserine or lanina Qi (zanapezil). In other embodiments, the A[beta] inhibitor can be bapineuzumab, PTB2, scyllo-inositol, PPI 1019, RS 0406, SP 233, EGCG, Exberyl-1 or SEN 606. The muscarinic receptor antagonist can be oxotremorine or xanomeline.

The anti-TDP-43 oligomer antibody of the invention can be used as a kind A tool used to detect or diagnose whether a body is suffering from a disease associated with TDP-43 oligomers. Accordingly, the present invention provides a method for detecting or diagnosing a disease associated with or suspected of having a TDP-43 oligomer. The method comprises the steps of: obtaining a biological sample from the individual; contacting the biological sample with the human antibody of the invention to determine the content of the TDP-43 oligomer in the biological sample; and the biopsy The content of TDP-43 oligomer in the body is compared with the content of TDP-43 oligomer obtained from a control sample of a healthy individual; wherein, if the content of TDP-43 oligomer in the biological sample is When there is a significant difference in the TDP-43 oligomer content of the control sample, it means that the individual has a neurodegenerative disease. The term "significant difference" in this context refers to the TDP-43 measured by the biological specimen to be tested. The oligomer content is statistically significantly higher or lower than the TDP-43 oligomer content measured by a control sample from a healthy individual.

The above biopsy includes, but is not limited to, brain biopsy Body, cerebrospinal fluid sample, whole blood sample, serum sample, plasma sample, urine sample, mucus sample, and purified or filtered product of the sample.

The binding of antibodies can be detected by methods well known to those skilled in the relevant art, such as enzyme-linked immunosorbent assay (ELISA), sandwich immunoassay, in situ immunoassay ( in Situ immunoassays, for example, using colloidal gold, enzymes or isotopes, Western blotting, agglutination assays (eg, colloidal agglutination assays, hemagglutination assays, etc.), complement fixation assays, Immunofluorescence assay (immunofluorescence assay) and immunoelectrophoresis assay (immunoelectrophoresis assay). In one embodiment, the binding of the antibody is determined using an ELISA. In certain embodiments, autologous antibodies can be detected using bodily fluids (eg, cerebrospinal fluid, whole blood, serum, plasma, mucus, and purified or filtered products of such bodily fluids). In a preferred embodiment, the cerebrospinal fluid sample is used to detect antibodies. In other embodiments, brain slices are used to detect antibodies.

To ensure that the present invention is effectively utilized by those skilled in the relevant art, the anti-TDP-43 oligomer antibodies of the present invention can be assembled into a kit format for facilitating the diagnosis, detection or confirmation of a neurodegenerative disease. In a preferred embodiment, the pathogenic TDP-43 reactive with the anti-TDP-43 oligomer antibody of the present invention is used to diagnose whether a body is suffering from neurodegenerative disease. For example, a higher or lower amount of an anti-TDP-43 oligomer antibody of the present invention can be found in a biological sample compared to a control group (a sample taken from a healthy individual). The pathogenic TDP-43 of the response indicates that the individual is suffering from a disease associated with TDP-43 oligomers. This information can be used for subsequent related treatments. For example, if a body is found to have pathogenic TDP-43, treatment with TDP-43 oligomer-related diseases (such as AD, ALS, and PD) can begin, and early treatment often has better efficacy.

In one embodiment, the invention utilizes an anti-TDP-43 oligo The polymer antibody provides a kit for diagnosing TDP-43 oligomer-related diseases. The components of the kit comprise: a container, a reagent for detecting TDP-43 oligomers in the biological sample, and a instructions attached to the container, wherein the reagent comprises the method disclosed by any of the methods disclosed in the present disclosure. Anti-TDP-43 oligomer antibody, and the instructions are for informing the use of the anti-TDP-43 oligomer antibody of the invention to detect pathogenic TDP-43 (ie, the above TDP-43 oligomer) )Methods. The instructions can be a book, a tape, a CD, a VCD or a DVD. The kit may additionally include a negative control for comparing the normal levels of TDP-43 oligomers to healthy individuals.

A number of experimental examples are presented below to illustrate certain aspects of the invention. The present invention has been made by those having ordinary skill in the art to which the present invention pertains. These examples should not be construed as limiting the scope of the invention. It is believed that the skilled artisan, after reading the description set forth herein, may fully utilize and practice the invention without undue interpretation. All publications cited herein are hereby incorporated by reference in their entirety.

Example

Materials and methods

Materials Human TDP-43 isolated from HEK cells was obtained from OriGene Technologies, Inc. (Rockville, MD, USA). From the product data, a recombinant TDP-43 protein can be obtained by transfecting a TrueORF strain (ie RC210639) into human HEK 293 cells; the C-terminus of the recombinant TDP-43 protein has a Myc-DDK marker (tag) Therefore, it can be purified by using an anti-DDK affinity column. Short TDP-43 (residues 101-285) was prepared according to the procedure described by Kuo et al. (Nucleic Acids Res (2009) 37, 1799-1808). TDP-43 antibody (labeled N _1-260 ) for western blot, anti-N-terminal residues 1-260 and anti-TDP-43 antibody for immunohistochemistry were purchased from Abcam (Cambridge) , UK). The TDP-43 antibody (labeled C _350-414 ) with anti-C-terminal residues 350-414 was purchased from Novus (Littleton, CO, USA), while the antibody A11 against anti-amyloid oligomers was purchased from BioSource (Invitrogen, Carlsbad, CA, USA). Anti-DDK monoclonal antibodies were purchased from Origene Technologies, Inc. The Aβ peptide is synthesized by the peptide synthesis laboratory of the Central Research Institute's genomic center. The remaining chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA) or Amresco (Solon, OH, USA). All cell culture reagents were purchased from Gibco (Invitrogen) unless otherwise indicated.

Purified TDP-43 was first cloned from the transformant pCMV-Taq2B containing a cDNA encoding full-length TDP-43. The selected product was further transferred to the vector pET14b (Novagen, Merck KGaA, Darmstadt, Germany) with restriction enzyme XhoI/BamHI to generate an N-terminal histidine marker (His-Tag). The resulting TDP-43 having a set of amino acid markers at the N-terminus was transfected into E. coli Rosetta 2 (Novagen, Merck KGaA, Darmstadt, Germany) for performance. The cells were collected, suspended in 30 mM Tris-HCl (pH 8) buffer, placed on ice, and disrupted with a microfluidizer containing 500 mM NaCl, 10% glycerol, 1 mM DTT, 2% RNase. A, 2% DNase I and protease inhibitor (completely, without EDTA, purchased from Roche Applied Science, Mannheim, Germany). The lysate was centrifuged at 27,000 x g at 4 °C. A Ni-NTA affinity column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) was first equilibrated with a buffer containing 30 mM Tris (pH 8), 500 mM NaCl, 1 mM DTT, 20 mM imidazole, and 10% glycerol. Thereafter, the resulting supernatant is injected into the column. After dissolving the imidazole in the above buffer, the protein product was eluted with different concentrations of imidazole. About 200 mM of imidazole can rush to the TDP-43 protein. The purified histidine-tagged TDP-43 protein was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue. Purified TDP-43 contains the N-terminal residue MGSSHHHHHHSSGLVPRGSHMLE. The estimated molecule is approximately 47,147 Da. The protein product was further subjected to dialysis. According to the formula described by Nick Pace (Pace et al., (1995) Protein Sci 4, 2411-2423), the absorbance background value at 280 nm was subtracted, and the protein concentration was calculated with an extinction coefficient of 44,920 cm ^-1 M ^-1 . . When calculating the concentration of the short TDP-43, the extinction coefficient is 15,470 cm ^-1 M ^-1 .

Size exclusion chromatography (SEC) first standardized Superdex-200 10/300 GL analytical gel filtration column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) with molecular weight marker set, wherein the molecular weight The marker group was dissolved in ferritin (440 kDa), β-amylase (200 kDa), bovine serum albumin (66 kDa) and cytochrome C (12.4 kDa), respectively. Buffer containing 30 mM Tris (pH 7.4) and 150 mM NaCl. The flow rate is 0.5 ml per minute. The recombinant TDP-43 prepared from E. coli was first filtered through a 0.2 micron filter membrane, and a volume of 300 microliters of the filtrate was injected into a Superdex-200 column. The liquid separation was automatically collected using a fraction collector, wherein each tube was dispensed in 1 ml. Pre-loaded samples and fractions containing oligomers were collected and analyzed by spotting method with A11 antibody (1:1000) at different exposure times. The separation of each oligomer was confirmed by dynamic light scattering. A volume of 100 microliters was prepared from HEK cells, and TDP-43 at a concentration of 2 micromoles was also tested in the same procedure. Further, the spotting method was used to analyze each liquid separation.

Point Method ink slot (slot blotting) since the concentration is too low, it is isolated from HEK293 cells were analyzed using dot blot, and the particle diameter by size exclusion chromatography of the TDP-43. From a 1 ml portion of each tube, 200 microliters of a Bio-Dot SF microfilter (Bio-Rad, Hercules, CA, USA) first equilibrated with an internal vacuum system was injected. At the time of detection, the primary antibody was an anti-DDK monoclonal antibody (OriGene Technologies, Inc., Rockville, MD, USA). Signal intensity was quantified using Image J 1.42 (National Institutes of Health, MD, USA).

The purified TDP-43 was diluted 10-fold with 10 mM Tris-HCl buffer in the presence or absence of a denaturant in dot mM. The samples of different concentrations contained no denaturing agent, buffer containing 9M urea, containing 7.2M vein hydrochloride or containing 2% sodium lauryl sulfate. The final concentration of TDP-43 was 0.4 μM. The samples were separately treated at room temperature or 90 ° C for 1 hour. Each TDP-43 sample was spotted on a nitrocellulose membrane in a volume of 2 μl to carry out a spotting method. Briefly, after blocking and washing with physiological saline (ie TBST) containing Tris as a buffer base and containing 0.002% Tween 20, the membrane was separately TDP with anti-N residues 1-260. -43 antibody (1:2,000), anti-C-terminal residue 350-414 of TDP-43 antibody (1:2,000) and anti-precipitated protein oligomer antibody A11 (1:1,000) were reacted, wherein These antibodies are all dissolved in TBST buffer containing 5% milk; then secondary antibody anti-rabbit or anti-mouse IgG (1:5,000) conjugated with horseradish peroxidase (HRP) , Millipore, Billerica, Massachusetts, USA). The performance was finally analyzed using ECL chemiluminescence reagent (Millipore).

Transmission electron microscope ( TEM) The freshly purified TDP-43 was placed in a buffer containing 10 mM Tris (pH 8) for dialysis reaction at 4 ° C until the next day. The specimen was again placed at 4 ° C, centrifuged at 17,000 × g for 30 minutes to remove the precipitate, and the supernatant was taken and quantified by transmission electron microscopy. The TDP-43 specimens were first placed in a glow-discharged, 400-mesh Formvar carbon-coated copper grids (EMS Inc). , Hatfield, PA, USA) 5 minutes, washed and then negatively stained with 2% uranyl acetate. Each sample was examined under a pressure of 75 kV under a Tecnai G2 Spirit TWIN TEM (FEI, Hillsboro, OR, USA) or Hitachi H-7000 TEM (Hitachi Inc., Japan).

Atomic force microscope ( AFM) The freshly purified TDP-43 was placed in a buffer containing 10 mM Tris (pH 8) for dialysis reaction at 4 ° C until the next day. The specimen was again placed at 4 ° C, centrifuged at 17,000 × g for 30 minutes to remove the precipitate, and the supernatant was taken and quantified by atomic force microscopy. Ten microliters of TDP-43 was dropped on mica sheets (Ted Pella, Redding, CA, USA) and allowed to stand for 5 minutes to attach the cells. The specimen was washed with 1 ml of secondary water, and the specimen was carefully removed from the mica sheet. The specimen was left at room temperature until dry, and subjected to atomic force microscopy analysis in a tapping mode (Nanonics, Jerusalem, Israel). This experiment was carried out using a PPP-ZEILR (Nanosensors, Neuchatel, Switzerland) with a spring constant of 1.6 N/m and an atomic force microtip with a tip radius of <10 nm.

Dynamic light scattering (DLS) was used to perform dynamic light scattering analysis on the oligomers eluted by particle size chromatography. First, the sample was dissolved in a buffer containing 30 mM Tris (pH 7.4) and 150 mM NaCl. After a 50 mW fiber balance Zetasizer Nano ZS dynamic light scattering instrument (Malvern Instruments, Worcestershire, UK), sample analysis was performed. The appropriate viscosity and refractive index were set according to each solution, and the temperature was fixed at 25 °C.

A circular dichroism (CD) was placed in a buffer containing 10 mM Tris (pH 8) for dialysis on a daily basis at 4 °C. The specimen was again placed at 4 ° C, centrifuged at 17,000 × g for 30 minutes to remove the precipitate, and the supernatant was taken and quantified by polarimetry. The Jasco J-815 spectrometer (Jasco Inc., Easton, MD, USA) was set up in a circular quartz cell (Hellma, Forest Hills, NY, USA) with a path length of 1 mm and room temperature (Jasco Inc., Easton, MD, USA). UV polarimeter spectrum. A spectrum of 250 to 190 mm was collected and corrected for buffer background values.

Endogenous and Bis-ANS fluorescence spectroscopy The freshly purified TDP-43 was placed in a buffer containing 10 mM Tris (pH 8) for dialysis every day at 4 °C. . The sample was again placed at 4 ° C, and the precipitate was removed by centrifugation at 17,000 × g for 30 minutes, and the supernatant was quantified and quantified. Excitation was performed with a light source having a wavelength of 280 or 295 nm, and endogenous fluorescence of 1.5 μM of TDP-43 at a wavelength of 305 to 400 nm was collected. The Bis-ANS spectra of TDP-43, TDP-43s and buffer control groups at wavelengths between 450 and 600 nm were collected by excitation with a light source of 400 nm. The samples contained 0.8 μM of TDP-43 and 5 μM of Bis-ANS. All experiments were performed in a circulating water bath at 25 ° C using a FluoroMax-3 spectrofluorometer (Horiba Jobin Yvon, Kyoto, Japan).

Thioflavin T (ThT) was combined at 4 ° C, and freshly purified TDP-43 was placed in a buffer containing 10 mM Tris (pH 8) for dialysis reaction until every other day. The sample was again placed at 4 ° C, and the precipitate was removed by centrifugation at 17,000 × g for 30 minutes, and the supernatant was quantified and quantified. The thioflavin T binding assay was performed using TDP-43 samples and Aβ40 fibers. A 1 μM sample was first mixed with the same molar concentration of thioflavin T, and after excitation with a light source having a wavelength of 442 nm, a divergence spectrum having a wavelength of 455 to 505 nm was collected. A stock solution of Aβ40 fiber having a concentration of 25 μM was prepared according to the aforementioned method (Chen and Glabe, J. Biol. Chem. (2006) 281, 24414-24422). Briefly, the synthesized Aβ was dissolved in 6 M vein hydrochloride and refolded in 10 mM phosphate buffer (PBS, pH 7.4). Further, 25 μM of Aβ was allowed to stand in an environment of 25 ° C for more than 10 days. The mature fibers must be diluted to 1 μM before the experiment. The resulting spectrum is subtracted from the buffer background value.

Congo Red spectroscopy was added with 10 μM Congo red, and 0.5 μM dialyzed TDP-43 and mature Aβ fibers were measured for absorbance at 400 to 600 nm using a violet/visible spectrometer DU800 (Beckman Coulter, CA). value.

OC to the antibody dot blot dialyzed TDP-43 and A β fibers (2 [mu] L, 0.5uM) spotting on nitrocellulose membranes, using standard experimental procedure subsequent dot blot analysis. Among them, the antibodies used in the experiments were OC antibody (Millipore, 1:10,000) and HRP-conjugated anti-rabbit IgG (1:10,000).

In fluorescence titration (Fluorescence titration) as measured by fluorescence titration to detect DNA-binding proteins to detect conformational changes created by DNA binding. Freshly purified and dialyzed, 1.5 μM full-length TDP-43 and short TDP-43 (residues 101-285) were titrated with single-stranded TAR DNA, wherein the single-stranded TAR DNA contained the TAR DNA A-position (5 '-CTTTTTGCCTGT-3') and B-position (5'-TGGGTCTCTCTG-3'). Endogenous protein fluorescence was excited with a light source having a wavelength of 280 nm, and changes in the configuration of TDP-43 were observed. The divergence spectra were collected on a FluoroMax-3 spectrofluorometer (Horiba Jobin Yvon, NJ, USA). The divergent light of the full-length or short TDP-43 at 350 nm was collected, and the value of the DNA control group was subtracted and corrected by the dilution factor. Thereafter, the resulting value is normalized to the initial intensity and substituted into a single protein-ligand binding formula (Chen et al., Protein Sci (2004) 13, 2196-2206): P + L PL

r =(2 Pt ) ^-1 *[( Kd + Lt + Pt )-(( Kd + Lt + Pt ) ² -4 PtLt ) ^1/2 ]

Wherein γ is the observed signal change, which represents the binding protein; P _t is the total TDP-43 concentration; L _t is the total ligand concentration; and Kd is the dissociation constant. The results were plotted according to the ratio of DNA and TDP-43.

Polymer Crosslinking and Thioflavin T Assays Aβ was prepared according to the previous protocol (Chen et al., J. Biol. Chem. (2011) 286, 9646-9656; Ni et al., FASEB (2011) 25, 1390 -1401). Aβ40 was dissolved in buffer A (10 mM sodium phosphate, pH 7.4) containing 8 M vein hydrochloride, refolded, and quantified by an absorbance of 280 nm (ε = 1,280 cm ^-1 M ^-1 ). Formulation of 25 μM Aβ, 50 μM thioflavin T and different concentrations of dialyzed TDP-43 (concentration 0 to 1 μM, ie 0-4%) were prepared with 10 mM Tris-HCl (pH 8) and 150 mM NaCl. Experimental specimen. The samples were placed in a 96-well plate of an ELISA and sealed with a transparent film, and measured at 25 ° C, at different time points using a microplate analyzer (SpectraMax M5, Molecule Devices). Thioflavin T was excited at 442 nm, and its divergence spectrum was measured at 485 nm.

Photo-induced cross-linking ( PICUP) This experiment was carried out according to the aforementioned procedure (Bitan et al., J. Biol. Chem. (2001) 276, 35176-35184). Briefly, Aβ stock solutions were prepared in buffer containing 8 M urea and 10 mM sodium phosphate for subsequent SDS-PAGE analysis. Aβ samples at a concentration of 25 μM were prepared in a buffer containing 10 mM Tris-HCl (pH 8) and 150 mM NaCl with or without different concentrations of TDP-43. Photocrosslinking test analysis was performed immediately. 90% of the Aβ solution was uniformly mixed with each of 5% of 3 mM Ru(Bpy) and 20 mM APS. Thereafter, the specimen was placed in a closed space for manual exposure to the blue light emitting diode for 30 seconds. The photo-crosslinking reaction was stopped by adding SDS-PAGE to the sample buffer, and the sample was analyzed by 16% Tris-tricine SDS-PAGE. All reactions require immediate treatment. Coagulation was determined by Western blotting using antibodies against the anti-Aβ antibody 6E10 (Chemicon Inc., Billerica, MA) and anti-N-terminal TDP-43 (residues 1-260) of Aβ residues 1-17. gum.

The cytotoxicity of TDP-43 on human neuroblastoma was determined by using thiazolium bromide (MTT, 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) and lactic acid. Hydrogenase (lactate dehydrogenase, LDH, Promega, Madison, WI, United States) assay to detect cells of TDP-43 against human neuroblastoma BE(2)-C cell line (American Type Culture Collection Center number CRL-2268) toxicity. The cells were cultured in RPMI growth medium (Gibco) containing 10% fetal bovine serum (FBS, Biological Industry), and grown in a humidified environment of 37 ° C, 5% CO ₂ . 60,000 cells were seeded in clear 96-well ELISA plates (Corning, NY, USA) and allowed to stand overnight. After washing the cells, the culture solution was replaced with 40 μl of serum-free RPMI medium, followed by 10 μl of TDP-43 specimen serially diluted with dialysis buffer, which was freshly dialyzed and centrifuged. The specimen. The supernatant was collected and used as a buffer control in dialysis buffer. The cells were allowed to stand for 24 hours and analyzed by the MTT assay according to the standard procedure. Briefly, 7 microliters of 5 micrograms of MTT per milliliter was added to each well and allowed to stand for 3 hours. After removing the culture solution, dimethyl sulfoxide (DMSO) was added to dissolve the cells until the crystal violet dye was completely dissolved. The absorbance of 570 to 690 nm was measured with an ELISA plate analyzer (SpectraMax M5, Molecule Devices, USA), and the reading signal of 690 nm was subtracted from the reading signal of 690 nm. The results were averaged five times and corrected for background values without cells. The cell survival rate is a result of standardization after the cell reading value of the administration buffer is set to 100%. The LDH test is carried out in accordance with the instructions indicated in the instructions. Briefly, 20,000 cells were planted in growth medium and allowed to stand for 24 hours. The treatment of cells is as described in the MTT assay. The LDH substrate was uniformly mixed with the reagent buffer and allowed to stand at room temperature. The cells were cooled to room temperature for 30 minutes and then subjected to a substrate reaction. In the dark for 1 hour, the dosed fluorescence was excited at 560 nm, and the 590 nm divergence value was read with an ELISA plate analyzer. The results of the three experiments were averaged and corrected for background values without cells. The p -values of the MTT and LDH tests were calculated as unpaired Student's t-test.

Toxicity of TDP-43 to primary cortical neurons in mice The pregnant C57BL/6JNarl mice were purchased by the National Laboratory Animal Center (NLAC, Taiwan). The relevant animal experiments were conducted by the Experimental Animal Care and Use Committee of the National Yangming University in Taipei, Taiwan ( The Institutional Animal Care and Use Committee (IACUC) is licensed. Primary cortical neurons were removed from mouse embryos on day 19, seeded in 96-well plates at a cell size of 2 x 10 ⁵ cells per well, and cultured in a neurobasal medium containing 2 μM of FdU (21103049, GIBCO, USA) )in. After 8 days of in vitro growth, freshly dialyzed TDP-43 was administered to the primary cortical neurons, allowed to stand for 24 hours, and then 0.5 mg of MTT per ml was added and allowed to stand for 3 hours. The dialysis buffer was the buffer control group of this experiment. An equal amount of 10% sodium dodecyl sulfate and 20 mM HCl in lysis buffer was added to the MTT reagent and allowed to stand until the next day, so that the MTT crystal violet dye was completely dissolved. The absorbance at 570 nm of each well was measured by an ELISA disk analyzer (TECAN, Switzerland). The results of the three experiments were averaged and corrected for background values without cells. The p -value of the MTT assay was calculated as the unpaired Student's t-test.

Immunocytotochemistry The primary neuronal cells were cultured on a 12-mm coverslip placed on a 24-well plate and coated with poly-D-lysine (3 holes per well). ×10 ⁵ cells). After 24 hours of treatment with TDP-43 (0.6 μM) or buffer control in a humidified environment of 37 ° C, 5% CO ₂ , the cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature. Thereafter, the cells were washed three times with PBS for 10 minutes, and treated with blocking buffer containing 0.3% ketamine x-100 (triton x-100) and 10% fetal bovine serum for 1 hour at room temperature. Next, anti-microtubule associated protein-2 (MAP-2, MAB378, Millipore, USA) antibody and rabbit anti-glial fibrillary acidic protein (GFAP, Z0334, DAKO, Glostrup) ,Denmark) antibody was treated at room temperature for 2 hours, followed by Texas-Red goat anti-mouse IgG secondary antibody (AbD Serotec, UK) or fluorescent isothiocyanate A goat anti-mouse IgG secondary antibody (Millipore, USA) of salt (Fluorescein isothiocyanate, FITC) was detected at room temperature for 1 hour. Finally, the cells were embedded in embedding gel (VECTASHIELD ^® + DAPI, H-1200, Vector Lab.) containing DAPI, and the staining results were observed using Olympus fluorescent (BX-61, Olympus) micromirrors.

Injecting TDP-43 into the hippocampus tissue was purchased by Tainan National University of Taiwan, and the C57/BL6J mice were raised in the school. Under the laboratory animal care and use committee, the different drugs were administered to 2 months. The hippocampus of C57/BL6J mice were in the middle. The mouse isoflurane inhalation anesthetic (1.2% oxygen) was first administered from the nasal cannula. Recombinant full-length TDP-43 was administered to the hippocampal gyrus of both sides of each mouse. The stereotactic coordinates relative to the front stalk (bregma) are anteroposterior (AP)-2 mm, medial lateral (ML) ±1 mm, and dorsal dorsoventral (DV)-2 mm. After the injection needle was slowly penetrated to a certain depth, 2 μl of TDP-43 (2.2 μM) was injected at a rate of 1 μL per minute with a stainless steel injection needle with a micro syringe (Hamilton Company, NV, USA). After the injection, the needle was placed for 5 minutes and then withdrawn to prevent the injection containing TDP-43 from oozing out.

Immunofluorescence staining of mouse brains 20-week-old male wild-type mice, and 6 and 12-month-old TDP-43 gene-transforming mice (TDP-43 Tg ^+/+ ) were immunofluorescently stained. . All mice were housed at the National Cheng Kung University and the experimental procedures were performed in accordance with the specifications of the School Animal Care and Use Committee. Two weeks after administration of TDP-43, the mice were deeply anesthetized and perfused with the heart with 4% paraformaldehyde dissolved in 0.01 M PBS (pH 7.4). Brain tissue was removed and placed in a 30% sucrose/4% PFA solution until every other day. The brain tissue was finely cut into 10 μm thick slices, and then treated with blocking buffer at room temperature for 1 hour. The blocking buffer was prepared by dissolving 0.2% ketol x-10 and 5% normal sputum serum in 0.01 M. PBS is prepared. In the hippocampus tissue injection experiment, the brain tissue was placed in mouse anti-NeuN antibody (1:300, Millipore, Temecula, CA) and placed at 4 ° C until every other day; and then with Alexa Fluor 555 The photoinhibitor-conjugated donkey anti-mouse antibody (1:300, Chemicon, Temecula, CA) was allowed to act for 1 hour at room temperature. The brain tissue was finally counterstained with DAPI and embedded in a fluorescent-embedded medium (DAKO, Glostrup, Denmark). The staining results of each brain tissue were examined using an erect fluorescent microscope (BX51, Olympus, Tokyo, Japan). Brain biopsy was placed in anti-TDP-43 antibody (1:1000, Abcam, ab104223), TDPO (1:1000, LTK BioLaboratories, Taiwan), and Alexa Fluor 488 in coexistence studies of gene-transplanted mice. Fluorescent dye-conjugated goat anti-rabbit antibody and sputum anti-mouse antibody (1:300, Invitrogen) conjugated to Alex Fluor 555 fluorescent stain. The brain tissue was placed in a DAPI stain and finally mounted in a fluorescent-embedded medium (DAKO, Glostrup, Denmark). The staining results of each brain tissue were examined using a laser scanning confocal microscope (Nikon TE2000 EPFS-C1-Si).

Preparation and confirmation of the TDP-43 oligomer having the specific polyclonal antibody at 4 ℃ environment to 10mM Tris-HCl (pH 8.0) was dialyzed purified full-length TDP-43, and the resultant product was centrifuged to A final concentration of approximately 0.2 mg of immunogenic protein per ml was prepared. New Zealand rabbits were immunized with the immunogenic protein, and polyclonal antibodies were prepared by a standard preparation procedure (LTK BioLaboratories, Taiwan). Briefly, the immunogenic protein was injected into the white rabbit in a volume of 0.5 ml each at a 2-week interval. After 6 injections, the white rabbits were sacrificed and their serum was taken. To confirm TDP-O by spot blotting and ELISA analysis, the full-length TDP-43 was injected into a particle size exclusion chromatography (Superdex-200 10/300 GL, GE Healthcare Bio-Sciences AB, Uppsala, Sweden), where The buffer contained 30 mM Tris-HCl (pH 8.0) and 150 mM NaCl at a flow rate of 0.3 ml per minute. The extract was collected in a volume of 1 ml and _analyzed by spot blotting using TDP-O and N _1-260 antibodies. After an additional micro BCA ^TM protein assay kit ^{(Micro BCA TM Protein Assay Kit,} Thermo Scientific, Rockford, IL) quantitative TDP-43 monomer and oligomer, which was coated on ELISA plate in serial dilution method. The ELISA assay was performed using standard procedures. Briefly, the coated samples were placed at 4 ° C to every other day and treated with TBST containing 10% skim milk for 2 hours at room temperature. After washing the ELISA plate, TDP-O (dissolved in TBST containing 3% skim milk at a ratio of 1:12,500) or anti-N-terminal 1-260 TDP-43 antibody (dissolved in a ratio of 1:1,000) It was detected at room temperature for 2 hours in TBST containing 3% skim milk. After washing, the anti-rabbit antibody (1:1,000, Merck Millipore, Billerica, Massachusetts, USA) conjugated with HRP was detected at room temperature for 2 hours, and after washing again, at 100 ml 3, 3, 5, 5-tetramethylbenzidine (3,3,5,5-tetramethylbenzidine, TMB, Merck Millipore, Billerica, Massachusetts, USA) was a color reaction. The reaction was quenched with 100 ml of 250 mM HCl, and the absorbance of the sample was read using a SpectraMax M5 (Molecular Device, Sunnyvale, CA) at a wavelength of 450 nm.

Preparation of β5 fiber The β5 peptide located in the RRM2 region of the TDP-43 fragment was synthesized by a chemical method (MDBio, Inc., USA). After dissolving the peptide in acetonitrile, a 10-fold dilution of the peptide at a starting concentration of 5 mg per ml was carried out in PBS buffer (pH 7.4). The mixture was centrifuged at 17,000 x g for 30 minutes, and the supernatant was collected and filtered using a 0.2 μm filter membrane (Pall, USA). Thereafter, the specimen was allowed to stand at room temperature for 7 days. The formed β5 fibers were observed using a transmission electron microscope. Each sample was analyzed by the spotting method using a TDP-O antibody as described above.

TDP-O staining of brain tissue of FTLD-TDP patients Brain tissue sections embedded in paraffin were washed three times with xylene for dewaxing. The brain tissue sections were hydrated with different concentrations of alcohol and washed with 1 time TBS for 5 minutes. The sectioned tissue was placed in sodium citrate (pH 6) for 15 minutes at full firepower (1,500 watts) to restore antigenic performance. The tissue was placed in 1 TBS containing 3% hydrogen peroxide for 20 minutes to block the activity of endogenous peroxidase; then washed with 1 time TBS for 5 minutes. Tissues were placed in 10% normal goat serum (Invitrogen) and allowed to act for 1 hour at room temperature to block non-specific binding. A primary antibody diluted to a 1:1000 ratio in antibody buffer (containing 5% fetal bovine serum albumin and 0.5% towen-20 TBS) was added and allowed to act at 4 ° C until every other day. The samples were washed 3 times with wash buffer (containing 1% TBS of 0.5% Towen-20). After the biotin-labeled secondary antibody was diluted 1:50 in the antibody buffer, the sample was added and allowed to act at room temperature for 1 hour. The sample was washed three times with the washing solution, and the sample was placed in a Vectastain ABC peroxidase reagent (Vector Labs, PK-6100) for 30 minutes, and then washed once with 1 time TBS. DAB peroxidase substrate reagent (Vector Labs, SK-4100) was added for 2-10 minutes to achieve the best staining effect. It was counterstained with Mayers Hematoxylin Solution (Sigma Aldrich) and dehydrated with different concentrations of alcohol and xylene. The dehydrated tissue sections were embedded in Permount (Sigma Aldrich). Brain samples from three groups of controls, three patients with FTLD-TDP and three patients with Alzheimer's disease were also analyzed simultaneously by the Azheimer Center in Davis, CA. This experiment was approved by the Institutional Review Board and obtained informed consent from each patient.

Immune oligomer FTLD-TDP brain of precipitation and TDP-43 immunolabeling of about 0.05 grams per ml lysis buffer and frozen from 2 control brain tissue of patients suffering from FTLD-TDP 1, the lysis buffer The solution contained 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% toxic X-100, and a protease inhibitor (Calbiochem, Merck Millipore). The dissolved samples were homogenized on ice using a tissue grinder (Wheaton, NJ, USA). Thereafter, the mixture was centrifuged at 17,000 xg for 10 minutes at 4 ° C, and the soluble fraction was collected for immunoprecipitation analysis. The TDP-O antibody was cross-linked to immunoprecipitated beads according to the instructions for use prior to tissue stratification. Briefly, 40 micrograms of TDP-O antibodies were added to 10 [mu] l binding buffer containing 10 microliters of protein A- and protein ^{G-Mag Sepharose TM Xtra (GE} healthcare) in (50mM Tris, pH 7.5,150mM NaCl) , It was allowed to stand at room temperature for 1 hour. After the antibody was bound to the microbeads, a crosslinking reagent containing 50 mM dimethyl pimelimidate dihydrochloride and 200 mM triethanamine (pH 8.9) was added to effect at room temperature. The reaction was carried out for an hour; the reaction was stopped by adding 100 mM ethanolamine (pH 8.9) at room temperature for 30 minutes. Next, the soluble fraction of the hippocampus back tissue and the immunoprecipitated microbeads cross-linked with the TDP-O antibody were reacted at room temperature for 1 hour. The reaction was washed at least 6 times with binding buffer to remove unbound protein components. The labeled protein was eluted with a gentle antigen/antibody extraction buffer (Gentle Ag/Ab Elution Buffer, Thermo, pH 6.6), and the extracted product was analyzed by electron microscopy and immunogold labeling. In the immunogold labeling, 10 microliters of the stripped product was placed in a 400-mesh Formvar carbon-coated copper grids (EMS Inc., Hatfield, PA, USA). After 5 minutes, after washing with PBS, PBS containing 1% BSA was added and allowed to act at room temperature for 1 hour. Thereafter, the reaction was carried out for 1 hour at room temperature with PBS containing 0.1% BSA and N _1-260 antibody (1:5,000, ab57105, Abcam) to mark the copper grid; followed by containing 50 mM Tris (pH 7.5), 500 mM. The copper grid was washed with NaCl and 0.1% Tween-20 in high salt tween buffer (HST buffer) and PBS. The copper grid was then placed in a 6 nm, gold-conjugated secondary anti-mouse IgG antibody (1:40, Jackson ImmunoResearch) and allowed to react at room temperature for 1 hour. The copper grid was washed with HST and PBS to remove unbound antibody. The copper grid was fixed by using PBS containing 1% glutaraldehyde at room temperature for 10 minutes, and then washed 6 times with secondary water. Finally, the staining results were observed by a transmission electron microscope (FEI Tecnai G2 F20 S-TWIN TEM) after adding 2% uranyl acetate for negatively stained.

Purification of individual TDP-O antibodies The TDP-O anti-system was purified using the Protein G agarose kit (KPL) according to the manufacturer's instructions. Briefly, protein G agarose (1.5 ml) mixed with 20% ethanol was injected into a disposable column. The resin in the column was washed with 10 ml of a wash buffer containing 0.1 M sodium phosphate (pH 7.4) and 0.15 M sodium chloride. After diluting the conditioned medium (5 ml) taken from the TDP-O fusion tumor cells with the binding buffer (5 ml), the mixture was poured into a column, and the column was inverted to uniformly mix the mixture. Thereafter, the column was washed 4 times with 5 ml of wash buffer until the OD ₂₈₀ area was close to zero. One milliliter of a buffering buffer containing 0.2 M glycine (pH 2.85) was added, and the antibody was slowly extracted and collected in a collection tube containing 240 μl of a 5-fold washing solution. The collected antibodies were concentrated and the buffer was replaced with a wash buffer using an Amicon Ultra-4 30 kDa cutoff (Millipore). The antibody concentration was quantified by the absorbance of the antibody at 280 nm (the absorbance of the antibody of 1 mg was 1.34).

Indirect ELISA 20 nanograms (ng) of full-length TDP-43 oligomer protein was dissolved in 200 μl of TBS solution containing 50 mM Tris (pH 7.4) and 150 mM NaCl, and then applied to an ELISA 96-well plate ( Nunc MaxiSorp), placed at 4 ° C until every other day. The coating solution was removed and added to the TBS buffer with or without 0.2% sodium dodecyl sulfate, respectively, and allowed to stand at room temperature for 1 hour. The TDP-43 oligomer treated with sodium dodecyl sulfate will denature, while the TDP-43 oligo treated with buffer containing no sodium lauryl sulfate will maintain the configuration of the original oligomer. After the treatment, the buffer was removed, and 200 μl of TBST buffer (20 mM Tris, pH 7.6, 137 mM NaCl, 0.001% Tween 20) containing 10% skim milk was added and allowed to stand at room temperature for 2 hours. The wells were washed 3 times with PBST (300 μl per well) and 100 μl of TBST-diluted mTDP-O antibody (dilution concentration from 0.00001 to 2 μg per ml) containing 5% skim milk was added to room temperature. 1 hour. After washing twice with 300 μl of PBS, the expression of the bound antibody was detected by 100 μl of an anti-mouse antibody conjugated to HRP, wherein the antibody was diluted 1:1,000 in a 5% skim milk TBST solution. After 1 hour at room temperature, it was washed twice with 300 μl of PBS, and then 100 μl of 3,3,5,5-tetramethylbenzidine (TMB, 3,5,5-tetramethylbenzidine, TMB, KPL SureBlue), reacted at room temperature for 10 minutes. 100 μl of 250 mM HCl was added to stop the reaction, and the optical density (OD) at a wavelength of 450 nm was read with a SpectraMax M5 (Molecular Devices).

Preparation of TDP-43 oligomers and monomers for indirect ELISA assay 180 micrograms of TDP-43 protein per ml was concentrated 3 times (concentrated from 3 ml to 1 ml) using Amicon Ultra-4 30 kDa cutoff. 500 microliters of the concentrated TDP-43 protein solution was injected into a Superdex 200 10/300 GL column and eluted with a buffering buffer containing 30 mM Tris (pH 8.0) and 50 mM NaCl at a flow rate of 0.3 ml per minute. The extract containing TDP-43 oligomers and monomers was collected in units of 500 microliters. The protein concentration was quantified using a micro BCA protein assay kit (Thermo). 300 ng of TDP-43 monomer or oligomer was taken and applied to an ELISA 96-well plate (Nunc MaxiSorp). According to the above-described indirect ELISA assay, 100 microliters of the conditioned medium (1:1,000 dilution) from TDP-O fused cells was used to detect the performance of TDP-43 oligomers.

Isotypes of the antibodies were confirmed to confirm the isotype of mTDP-O by a mouse monoclonal antibody isotype kit (Thermo) for ELISA assay. In this assay, anti-mouse heavy chain capture antibodies (anti-IgG ₁ , IgG _2a , IgG _2b , IgG ₃ , IgA and IgM) or anti-mouse light chain capture antibodies ( κ or λ) was applied to an ELISA plate. Briefly, the conditioned medium from TDP-O was diluted in TBS solution at a 50-fold ratio, and the diluted sample was added to an ELISA plate. Thereafter, 50 μl of an HRP-conjugated anti-mouse IgG + IgA + IgM antibody was added and reacted at room temperature for 1 hour. After 3 washes, 75 μl of TMB substrate was added to each well and reacted at room temperature for 10 minutes in the dark. Next, 75 μl of 0.18 M sulfuric acid was added to terminate the reaction. The absorbance of each well at a wavelength of 450 nm was read by SpectraMax M5 (Molecular Devices).

Ig variant region gene was transferred and sequenced RNA of 2×10 ⁶ fusion tumor cells was extracted with GeneJET RNA purification kit (Thermo). cDNA was synthesized using a Maxima First Strand cDNA synthesis kit (Thermo). Thereafter, the Ig heavy chain and kappa genes were amplified by a mouse Ig-primer set (Ig-Primer Sets, Novagen) and a GoTaq ^® G2 green amplification reaction mixture (GoTaq ^® G2 Green Master Mix, Promega). The resulting product was amplified in the TOPO ^® TA Cloning ^® kit (Invitrogen) were transfected colonization. The DNA sequence of the final product was sequenced by Mission Biotech.

Example 1 Full-length TDP-43 is easy to form oligomers

In this example, the pathological mechanism of TDP-43 will be explored. Recombinant full-length human TDP-43 (having a set of amino acid markers at the N-terminus, molecular weight MW 47, 145 Da) and TDP-43 protein was purified from E. coli and HEK293 cells according to the procedure described in "Materials and Methods". The obtained TDP-43 protein was subjected to particle size analysis chromatography analysis. As shown in Figure 1a, the elution volume contained at least 86% of the eluted TDP-43, and the slit point ink method confirmed that both recombinant recombinant full-length TDP-43 proteins were prone to oligomer formation. In view of the fact that TDP protein lesions are associated with the formation of inclusion bodies (IB), and recombinant full-length TDP-43 forms high molecular weight oligomers, it is speculated that TDP-43 may form and amyloidosis. A similar oligomer of starch oligomers. Therefore, a TDP-43 oligomer was detected using an anti-plasmid-specific antibody (ie, A11) prepared against the mimetic of Aβ oligomer and related to the configuration; the results showed that both TDP-43 protein samples were able to detect An immune response is produced with A11, and the corresponding buffer is not (Fig. 1b). The oligomer fraction containing diluted TDP-43 has a weaker signal intensity.

In order to confirm that the identification of the antibody and the antigen is related to the configuration, before using the A11, the anti-N terminal TDP-43 antibody and the anti-C terminal TDP-43 antibody for spot blotting analysis, different denaturation methods are used. To disrupt the configuration of the TDP-43 oligomer (Fig. 1c). Culture the protein in different buffers, Namely, buffer with or without 9 M urea, 7.2 M vein hydrochloride or 2% sodium lauryl sulfate was further treated at 90 ° C for 1 hour. It was found that heating or not does not significantly affect the amount of signal in the original buffer when detecting the performance of A11. However, the addition of high concentrations of urea or vein hydrochloride reduces the amount of signal detected, and reheating significantly reduces the amount of signal. No matter whether it is heated or not, no signal can be detected by adding 2% sodium lauryl sulfate. In contrast, the anti-N-end antibody recognizes the performance of TDP-43 regardless of the treatment; this result also confirms that the protein system spotted on the membrane is equal. Addition of urea and heat treatment or addition of pulse hydrochloride (whether heated or not) will partially or completely reduce the recognition of the antigen by the anti-C-terminal antibody. These results indicate that the recognition of antibodies by the anti-C terminal TDP-43 antibody varies with different modification conditions.

Overall, the above results indicate the recombination of the full-length TDP-43 It is easy to form high molecular weight proteins; these high molecular proteins share a common epitope with starch-like oligomers. These high molecular proteins are quite stable to chemical modification treatments other than sodium lauryl sulfate.

Example 2 Full-length TDP-43 forms heterogeneous spherical oligomers

In the present example, the morphology of the oligomer was observed by a transmission electron microscope (TEM, Fig. 1d) and an atomic force microscope (AFM, Fig. 1e). Transmission electron microscopy showed that the heterogeneous globular proteins have several spherical and circular structural features, while atomic force microscopy showed that most globular proteins exhibited spherical structural features, and only a few had circular structural features (Fig. 1e). illustration). Profit A particle size distribution was calculated using a transmission electron microscope. Most of the spherical particles have a particle size of about 40 to 60 nm (results not shown). The height of the ring-shaped oligomer was about 6 nm by atomic force microscopy (results not shown). In addition, dynamic light scattering (DLS) analysis showed that the oligomeric protein obtained by particle size chromatography showed a heterogeneous distribution with a particle size of about 40 to 400 nm. (Fig. 1f). Most of the particles have a particle size of about 50 to 60 nm (Fig. 1g), and the same analysis results are obtained by a transmission electron microscope. Combined with the image and particle size distribution results, the full-length TDP-43 will form heterogeneous particles with a spherical superstructure. These results are consistent with previous studies showing that wild-type TDP-43 and TDP-43 mutations associated with amyotrophic lateral sclerosis form oligomers (Johnson et al., J. Biol. Chem (2009). ) 284, 20329-20339). Overall, the results of this study indicate that TDP-43 protein is similar to spherical amyloid oligomers in both structural and immunological analysis, and the spherical starch oligomerization system is neurotoxic and will be expressed in Different neurodegenerative diseases.

Example 3 TDP-43 oligomer has a clear configuration and function

To further examine the configuration of the TDP-43 oligomer, the secondary structure of the TDP-43 oligomer was analyzed using a far-ultraviolet polarimeter spectroscopy and the results are shown in Figure 2a. Two minimum values, which may be alpha-helical structures, appear near 210 and 222 nm, respectively. This spectral analysis differs from the spectral analysis of short mouse TDP-43 (residues 101-265, abbreviated as TDP-43s in the figure), wherein the short mouse TDP-43 contains two more β in the crystal structure. -RRM of the stock (Kuo et al., Nucleic Acids Res (2009) 37, 1799-1808). The fluorescence spectrum obtained by excitation of endogenous aromatic residues shows that the maximum value of divergence can be detected when the light wave is about 340 nm, which indicates that the tyrosine and tryptophan residues of TDP-43 are solvents. Residues were exposed (results not shown). In addition, the exogenous fluorescent dye Bis-ANS is also used to detect the surface of the exposed hydrophobic protein, wherein the fluorescent dye Bis-ANS is a commonly used intermediate product to detect partial unfolded proteins when the protein is folded. Dyeing agent. As shown in Figure 2b, the full-length TDP-43 reacts with the fluorescent dye Bis-ANS to produce a higher amount of fluorescence compared to the short TDP-43, showing that the full-length TDP-43 oligomer has a short form. TDP-43 is not the same as the hydrophobic exposed surface.

In addition, a conventional starch dye thioflavin T (ThT) was also used to detect whether full-length TDP-43 binds to thioflavin T, and the results are shown in Figure 2c. No matter whether it is full-length or short TDP-43, the fluorescence divergence peaks fused by thioflavin T are not detected. In contrast, the same concentration of Aβ fiber (1 μM) had a significant fluorescence divergence (Fig. 2c), which was consistent with the pathological test results of the thioflavin T analysis. Similarly, TDP-43 oligomers did not bind to Congo red and anti-fibril antibody OC (results not shown). These results indicate that, TDP-43 should be no crosslinked oligomer with beta] pleated sheet (β sheet) structure. However, the structure of the atomic energy level needs further research and confirmation.

In view of the normal and functional TDP-43 will be combined with a special The combination of specific nucleic acid sequences will next explore whether TDP-43 oligomers interfere with DNA binding ability. Fluorescence titration was used to observe changes in the conformation of the protein after binding to DNA, and the results are shown in Figure 2d. Briefly, TDP-43 oligomers were analyzed by single-stranded TAR DNA-A or -B titration and The results obtained are compared with the titration analysis results of the short TDP-43, which is known to bind to single-stranded DNA with submicromolar affinity (Kuo et al., Nucleic Acids Res (2009) 37, 1799-1808). As shown in Figure 2d, at low single-strand DNA concentrations, both TAR DNA-A and -B positions reduced the amount of short-form TDP-43 fluorescence, whereas addition of full-length TDP-43 significantly reduced the response. This result is in accordance with the binding formula of single antibody and ligand described in "Materials and Methods", and indicates that the stoichiometry of TDP-43 and single-stranded DNA is 1:1. From this formula, the dissociation constants (dissociation constants) of full-length TDP-43 and TAR-A, full-length TDP-43 and TAR-B, short TDP-43 and TAR-A, and short TDP-43 and TAR-B can be obtained. Kd) were 7.05 ± 0.82, 6.27 ± 0.68, 0.20 ± 0.09, and 0.38 ± 0.10 μM, respectively. These results indicate that the binding strength of the single-stranded DNA to the short TDP-43 is about 15 times that of the full-length TDP-43, wherein the TAR-A and -B positions have similar affinities for the full-length TDP-43. These findings indicate that RRMs of full-length TDP-43 oligomers may have an abnormal configuration, resulting in a decrease in binding ability to DNA, or a region bound to DNA that is blocked from binding to DNA. The change in the divergence value of the full-length TDP-43 may be due to the presence of a small amount of TDP-43 monomer in the sample. In summary, these results demonstrate that TDP-43 oligomers have different biophysical and biochemical properties than short TDP-43, ie, the configurations are different.

Example 4 TDP-43 oligomer converts Aβ to amyloid-like oligomers

In this example, cross-linking experiments will be conducted to investigate whether TDP-43 affects Aβ fibrosis.

First, use the thioflavin T test to detect presence or absence. The fibrosis reaction of Aβ40 in the case of 0.4 to 4% of TDP-43 oligomers is shown in Fig. 3a. The analysis indicated that TDP-43 can effectively inhibit the fibrosis reaction of Aβ, and the inhibitory effect increases with the increase of TDP-43 concentration (Fig. 3a). In the experimental observation period of nearly 180 hours, 4% of TDP-43 completely inhibited the fibrosis reaction of Aβ. Photo-induced cross-linking (PICUP) was used to test the transient appearance of Aβ protein at the beginning of the experiment. The results indicated that after cross-linking, Aβ mainly formed monomers, dimers, and trimers. And tetramers, while TDP-43 oligomers cause Aβ protein production with higher molecular weight (Fig. 3b). Aβ pentamer was observed by the addition of TDP-43, and the amount of expression was normally correlated with the amount of TDP-43 added. In addition, two combinations with larger molecular weights were observed, located at ~55 kDa and distributed between ~105 and >210 kDa. In addition to the TDP-43 monomer which is not resistant to sodium lauryl sulfate, a protein with a molecular weight of about ~55 kDa and several proteins distributed from ~80 to >210 kDa were also found. Further analysis by transmission electron microscopy revealed that after the addition of 4% TDP-43, Aβ did not form fibers, but was denatured into a spherical oligomer having a particle size of <10 nm; Mature starch-like fibers will still form in the absence of TDP-43 (Fig. 3c). The TDP-43 oligomer crosslink has a particle size of >50 nm which is larger than the particle size of the A? oligomer (data not shown). These data show that TDP-43 oligomers can initiate the formation of Aβ oligomers, and that TDP-43 shares common properties with amyloid-like proteins.

Example 5 TDP-43 oligomers induce axonal degeneration and toxicity

In this example, it will be further explored whether TDP-43 oligomers cause neurotoxicity and axonal degeneration.

Briefly, human neuroblastoma BE(2)-C cells were treated with serially diluted TDP-43 samples containing mainly oligomers, and the resulting cytotoxicity was detected by MTT and LDH assays; Figure 4a to 4c. The MTT assay indicated that the addition of 0.44 [mu]M of TDP-43 reduced cell viability by approximately 20% compared to the buffer control group (Fig. 4a). In view of the low solubility of recombinant TDP-43, this toxicity test could not be performed with a higher concentration of TDP-43. The LDH test also showed similar results, and the addition of 0.44 μM of TDP-43 significantly induced cell death (Fig. 4b). TDP-43 also produces a dose-positive neurotoxicity in the primary cortical neurons of mice, wherein administration of 0.6 μM of TDP-43 reduced cell viability by approximately 20% (Fig. 4c).

In addition, immunohistochemical staining of these mouse skin neurons showed that 0.6 μM of TDP-43 caused axonal atrophy and reduced the density and number of neurons (Fig. 4d). To further test the neurotoxicity of TDP-43 oligomers in vivo, 2 μl of 2.2 μM TDP-43 was injected into the hippocampus of the mice, and the survival of the neurons was analyzed by immunofluorescence staining. Immunohistochemical staining with the neuron-specific marker NeuN and the nuclear specific stain DAPI revealed that the mice receiving TDP-43 had a smaller number of neurons in the CA1 layer than the mice treated with the buffer. (Fig. 4e). The results indicate that injection of full-length TDP-43 oligomers into the hippocampus can cause cytotoxicity.

Example 6 Preparation and Confirmation of Multiple Antibodies Specific to TDP-43 Oligomers

In view of the anti-amyloidogenic oligomer antibody A11 will be given Linked to different amyloid proteins, according to the method described in "Materials and Methods", after using recombinant TDP-43 oligomer as immunogen, immunostimulate rabbits to produce a multi-drug antibody TDP-O, in order to understand TDP- Whether the 43 oligomers will affect the disease.

According to the method described in "Materials and Methods", in different Under denaturing, the spotting method was used to analyze the specificity of the TDP-O antibody for the full-length TDP-43 oligomer configuration; the results are shown in Figure 5a. Regardless of heating (90 ° C, 1 hour) or not, TDP-O diluted in the original buffer at a ratio of 1:125,000 can react with the full-length TDP-43; however, 2% sodium lauryl sulfate destroys the antibody and TDP -43 Oligomer reaction, whether or not heat treatment. In addition, the addition of 7.2M vein hydrochloride or 9M urea, and treatment at room temperature for more than 1 hour, does not affect the reaction of full-length TDP-43, the results indicate that under the treatment of high concentration of chemical denaturant, the protein remains It is quite stable; however, additional heat treatment reduces the amount of response signal, that is, TDP-O is a configuration-related antibody. Summarizing the above, the TDP-O's statutory analysis results are consistent with A11 (Fig. 5a and Fig. 1c).

Use A11 and/or TDP-O antibodies to detect the specificity of antibodies to Aβ oligomers to see if TDP-O is a specific antibody to TDP-43, rather than a pair of amyloplasts such as A11. The polymer has a wide range of antibodies. As shown in Figure 5b, TDP-O was unable to recognize Aβ oligomers, demonstrating that this antibody is an antibody specific for TDP-43 oligomers. ELISA was also used to quantify the reaction between TDP-O antibody and TDP-43 oligomer and monomer; in this part, the TDP-43 antibody with N-terminal residue 1-260 (N1-260) was also identified. Carry out test analysis and use it for comparison. Approximately 1 μM of full-length TDP-43 or a 3-fold concentrated TDP-43 sample was subjected to particle size exclusion chromatography, and 1 ml of the extract was collected to prepare a full-length TDP-43 oligomer and monomer (the first 5c picture). The concentrated TDP-43 in the first to the 18th fractions was subjected to spot blotting analysis using TDP-O or N _1-260 antibody, and the results are shown in Fig. 5d. The results show that TDP-O has a strong response to concentrated TDP-43 in the 5th to 7th fractions, which correspond to TDP-43 oligomers eluted into the stripping volume. In contrast, N _1-260 has a strong reaction with concentrated TDP-43 in the 6th and 15th to 18th cents; that is, the antibody recognizes the oligomer of TDP-43 and monomer.

In addition, ELISA was used to quantify the specificity of TDP-43 oligomers and TDP-O, and the N _1-260 antibody was used here as a control group. After quantification by the micro BCA assay, serially diluted fractions containing TDP-43 oligomers and monomers were separately applied to ELISA discs. Detection was performed using TDP-O and N _1-260 antibodies according to the standard protocol of ELISA. The results in Figure 5e show that the TDP-O antibody has a higher reactivity with TDP-43 oligomers than the TDP-43 monomer (EC50 < 0.5 μg per ml); whereas the N _1-260 antibody is TDP-43 oligomers or monomers have similar reactivity (Fig. 5e). In addition, β 5 fibers prepared from the 5th β -strand of the RRM2 region of TDP-43 were also spotted on the membrane to confirm that TDP-O only recognized TDP-43 oligomers, instead of the prior art. Fiber (Wang et al., J Biol Chem (2013) 288, 9049-9057) (results not shown); the results clearly indicate that TDP-O can only recognize TDP-43 oligomers without recognizing the β 5 fibers. Therefore, the multi-drug antibody TDP-O of the present invention can specifically recognize the configuration of the TDP-43 oligomer without cross-linking to the A β protein.

Example 7 TDP-43 oligomers are present in the brain of genetically transgenic mice

Brain sections from 6 and 12 weeks old wild type mice and FTLD-TDP-43 gene transgenic mice were subjected to immunohistochemical staining to detect whether TDP-43 oligomers were present in vivo; the results are shown in 6 picture. It is known that FTLD-TDP-43 transgenic mice exhibiting full-length TDP-43 in the forebrain have similar conditions to those of FTLD-TDP patients (Tsai et al., J Exp Med (2010) 207, 1661-1673). With age, the learning/memory and exercise capacity of the TDP-43 transgenic mice will gradually decrease. Brain sections were immunofluorescently stained with anti-TDP-43 and TDP-O and counterstained with DAPI. Predictably, in wild-type mouse brain sections, TDP-43 is predominantly expressed in the nucleus. In contrast, brain sections of FTLD-TDP transgenic mice showed that TDP-43 is mainly expressed in the cytoplasm and can detect TDP-O signals. A large number of coexisting TDP-43 and TDP-O signals can be found in the cells, and the amount of cells with double staining increases with age. These coexistence signals indicate that TDP-43 oligomers similar to starch-like molecules will be present in the brain of FTLD-TDP transgenic mice, and the amount of expression will increase with age.

Example 8 TDP-43 oligomers will be expressed in the brain of FTLD-TDP patients

To assess the role of TDP-43 oligomers in human disease, FTLD patients with TDP-43 immunoreactive inclusion bodies were further used to study the performance of oligomers in FTLD-TDP patients. The TDP-O multi-strain antibody was used to immunostain the hippocampus and anterior cortex of the patient. The patient included 3 pathologically confirmed FTLD-TDP patients and 3 controls of similar age without dementia. Individuals, and 3 patients who had been confirmed by pathology to have Alzheimer's disease but did not have TDP-43 symptoms (as disease control individuals). The TDP-O antibody recognizes different neuronal cytoplasmic inclusion bodies (Figs. 7a and 7c) and dystrophic axons similar to TDP-43 inclusion bodies in the brain of FTLD-TDP patients (Fig. 7a). In some areas, neuronal cytoplasm is deeply stained into coarse granules (Fig. 7e). In contrast, in addition to the non-specific response of the neurolipid lipofuscin, TDP-O does not affect the brain (7b, 7d, and 7f) of the control individual and the Azheimer's disease. The brain of patients without TDP-43 symptoms (results not shown) produced a significant immune response. Both TDP-O polyclonal antibody and TDP-43 monoclonal antibody could not be stained into the nucleus of normal tissues. In addition, in order to understand the TDP-43 morphology that can be identified by the TDP-O multi-strain antibody, immunoprecipitation (IP) was performed on the hippocampus of the control individuals and patients by TDP-O. The THP-O multi-strain antibody of the hippocampal triton soluble fraction and photocrosslinking was subjected to immunoprecipitation analysis. The _extracted product was _{immunolabeled} with an N-terminal TDP-43 antibody (N _1-260 ), and subjected to electron microscopic analysis (Fig. 8). The results indicated that spherical TDP-43 oligomers with a particle size of about 50 nm were observed in the patient's specimen, but not in the control individuals; the oligomers were recognized by the N _1-260 antibody. That is, the oligomers are not N-terminal truncated proteins. Overall, it was confirmed by using the TDP-O multi-strain antibody of the present invention that TDP-43 oligomers were present in the brain of FTLD-TDP patients.

Example 9 Preparation and Confirmation of TDP-43 Oligomers Specific monoclonal antibody

In this example, an ELISA assay was used to detect monoclonal antibodies prepared from TDP-O-3, -5, -8, -9, and -10 fusion tumor cells, with or without sodium dodecyl sulfate treatment. At the time, the specificity of the TDP-43 oligomer. As shown in Fig. 9, the monoclonal antibody produced by the five fusion tumor cells had higher binding activity to the undenatured TDP-43 oligomer than the denatured TDP-43 protein. These data indicate that these monoclonal antibodies are specific for TDP-43 oligomers and are conformation-related antibodies.

In order to confirm the binding specificity of each monoclonal antibody, the TDP-43 oligomer and monomer were purified by colloidal filtration (Fig. 10a), and each product was separately applied to an ELISA plate to confirm the specificity of each monoclonal antibody. Sex. The results of Fig. 10b indicate that monoclonal antibodies prepared from TDP-O-3, -5, -8, -9 and -10 fusion tumor cells can specifically recognize TDP-43 oligomers.

The ELISA mouse monoclonal antibody isotype set (Thermo) was also used to determine the isotype of each TDP-O single plant. The results are summarized in Table 1.

According to the results of Table 1, monoclonal antibodies prepared from TDP-O-3, -5, -8, -9 and -10 fusion tumor cells were highly reactive to IgG2a and kappa light chains. Therefore, these monoclonal resistance systems are IgG2a and kappa light chain subtypes.

Five monoclonal antibodies were sequenced to determine the sequence of identity; the results are presented in Figure 11.

It will be understood that the above-described embodiments and examples are merely illustrative, and that those skilled in the art can align various modifications. The description, examples, and materials set forth above are intended to be illustrative of the invention and are illustrative of the invention. The present disclosure has been disclosed in the above embodiments, but it is not intended to limit the disclosure, and any person skilled in the art can make various changes and refinements without departing from the spirit and scope of the disclosure. The scope of protection of the disclosure is subject to the definition of the scope of the patent application.

【Biomaterial Storage】

Depository: Bioresource Conservation and Research Center

Hosting date: January 28, 104, Republic of China

Deposit number: BCRC 960494, BCRC 960495, BCRC 960496, BCRC 960497 and BCRC 960498

<110> Chen Yunru

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Claims (26)

A human antibody or fragment thereof, which specifically binds to a transactivation responsive-DNA-binding protein (43 kDa, TDP-43) having a molecular weight of 43 kD. The human antibody or the antibody fragment comprises: a heavy chain variant region comprising the amino acid sequence of SEQ ID NO: 1, 2 and 3; and a light chain variant region comprising the amino group of SEQ ID NO: 5, 6 and Acid sequence. The human antibody or fragment thereof according to claim 1, wherein the heavy chain variant region has the amino acid sequence of SEQ ID NO: 4, and the light chain variant region thereof has the amino acid sequence of SEQ ID NO: 8. The human antibody or fragment thereof according to claim 2, wherein the human antibody is prepared from a fusion tumor cell line deposited at the Bioresource Collection and Research Center (Bioresource Collection and Research Center) , BCRC), the registration number is BCRC 960494. The human antibody or a fragment thereof according to claim 2, wherein the human antibody is prepared from a fusion tumor cell line deposited in a biological resource conservation and research center, and the accession number is BCRC 960495. . The human antibody or a fragment thereof according to claim 2, wherein the human antibody is prepared from a fusion tumor cell line deposited in a biological resource conservation and research center, and the accession number is BCRC 960496. . The human antibody or a fragment thereof according to claim 2, wherein the human antibody is prepared from a fusion tumor cell line deposited in a biological resource conservation and research center, and the accession number is BCRC 960497. . The human antibody or a fragment thereof according to claim 2, wherein the human antibody is prepared from a fusion tumor cell line deposited in a biological resource conservation and research center, and the accession number is BCRC 960498. . The human antibody or fragment thereof according to claim 1, wherein the TDP-43 oligomer is a particle having a diameter of about 2 to 400 nanometers. The human antibody or fragment thereof according to claim 8, wherein the TDP-43 oligomer is a spherical or cyclic particle having a diameter of about 40 to 60 nm. A method for preventing or treating diseases associated with TDP-43 oligomers A pharmaceutical composition comprising an effective amount of a human antibody of claim 1 or a fragment thereof and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 10, wherein the disease associated with the TDP-43 oligomer is Alzheimer's disease, argyrophilic grain disease, muscle atrophy Amyotrophic lateral sclerosis (ALS), Guam muscle atrophic lateral sclerosis - ALS-parkinsonism dementia complex of Guam, vascular dementia, amount Frontotemporal dementia, semantic dementia, dementia with Lewy bodies, Huntington's disease, Spinocere bellar ataxia, Inclusion body myopathy, inclusion body myositis, or Parkinson's disease. The pharmaceutical composition according to claim 10, wherein the heavy chain variant region has the amino acid sequence of SEQ ID NO: 4, and the light chain variant region thereof has the amino acid sequence of SEQ ID NO: 8. The pharmaceutical composition according to claim 12, wherein the human antibody or a fragment thereof is prepared from a fusion tumor cell line deposited in a biological resource conservation and research center, and is deposited. The number is BCRC 960494. The pharmaceutical composition according to claim 12, wherein the human antibody or a fragment thereof is prepared from a fusion tumor cell line deposited in a biological resource conservation and research center, and the accession number is BCRC. 960495. The pharmaceutical composition according to claim 12, wherein the human antibody or a fragment thereof is prepared from a fusion tumor cell line deposited in a biological resource conservation and research center, and the accession number is BCRC. 960496. The pharmaceutical composition according to claim 12, wherein the human antibody or a fragment thereof is prepared from a fusion tumor cell line deposited in a biological resource conservation and research center, and the accession number is BCRC. 960497. The pharmaceutical composition according to claim 12, wherein the human antibody or a fragment thereof is prepared from a fusion tumor cell line deposited in a biological resource conservation and research center, and the accession number is BCRC. 960498. A method for diagnosing a disease associated with a TDP-43 oligomer by a biological sample comprising: the biological sample with an effective amount of the human antibody of claim 1 or a fragment thereof is contacted to determine the content of TDP-43 oligomer in the biological sample; and the content of TDP-43 oligomer in the biological sample is TDP-43 from a control sample taken from a healthy individual. The oligomer content is compared; wherein, if the content of the TDP-43 oligomer in the biological sample is significantly different from the TDP-43 oligomer content of the control sample, it means that the individual has TDP- 43 oligo-related diseases. The method of claim 18, wherein the disease associated with the TDP-43 oligomer is Alzheimer's disease, argyrophilic schizophrenia, amyotrophic lateral sclerosis, Guam muscle atrophy Lateral spinal sclerosis - Parkinson's dementia, vascular dementia, frontotemporal dementia, semantic dementia, Levi's dementia, Huntington's disease, cerebellar atrophy, inclusion body muscle Disease, inclusion body myositis or Parkinson's disease. The method of claim 18, wherein the biological sample can be a brain slice, a cerebrospinal fluid sample, a whole blood sample, a serum sample, a plasma sample, a urine sample or A mucus sample. The method of claim 18, wherein the heavy chain variant region has the amino acid sequence of SEQ ID NO: 4, and the light chain variant region thereof has the amino acid sequence of SEQ ID NO: 8. The method of claim 21, wherein the human antibody or a fragment thereof is prepared from a fusion tumor cell line, the fusion tumor is fine The cell line is deposited at the Center for Conservation and Research of Biological Resources under the registration number BCRC 960494. The method according to claim 21, wherein the human antibody or a fragment thereof is prepared from a fusion tumor cell strain deposited in a biological resource conservation and research center, and the accession number is BCRC 960495. The method of claim 21, wherein the human antibody or fragment thereof is prepared from a fusion tumor cell line deposited in a biological resource conservation and research center, the accession number is BCRC 960496. The method of claim 21, wherein the human antibody or fragment thereof is prepared from a fusion tumor cell line deposited in a biological resource conservation and research center, the accession number is BCRC 960497. The method according to claim 21, wherein the human antibody or a fragment thereof is prepared from a fusion tumor cell strain deposited in a biological resource conservation and research center, and the accession number is BCRC 960498.