TWI592423B - Antibodies against pathological forms of tdp-43 and uses thereof - Google Patents

Antibodies against pathological forms of tdp-43 and uses thereof Download PDF

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TWI592423B
TWI592423B TW104107500A TW104107500A TWI592423B TW I592423 B TWI592423 B TW I592423B TW 104107500 A TW104107500 A TW 104107500A TW 104107500 A TW104107500 A TW 104107500A TW I592423 B TWI592423 B TW I592423B
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tdp
oligomer
antibody
disease
dementia
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TW201632549A (en
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陳韻如
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中央研究院
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Description

可辨識致病性TDP-43之抗體及其用途 Antibodies recognizing pathogenic TDP-43 and uses thereof

本揭示內容是有關於新穎之單體分子量為43道耳吞的致病性轉活化反應-去氧核糖核酸-結合蛋白(transactivation responsive-DNA-binding protein,43kDa,TDP-43)、可結合至該致病性TDP-43的抗體及該抗體的用途。 The present disclosure relates to a novel transactivation responsive-DNA-binding protein (43 kDa, TDP-43) having a molecular weight of 43 amps. The antibody to the pathogenic TDP-43 and the use of the antibody.

神經退化性疾病已成為現代社會不可忽視的健康議題。依據世界衛生組織報告,截至2025年,全球將會有超過75%的老年人口患有各式神經退化性疾病。額顳葉退化症(Frontotemporal lobar degeneration,FTLD)係美國年齡小於65歲的人口中第二常見的失智症;已知TDP-43是造成FTLD及肌肉萎縮性脊髓側索硬化症(amyotrophic lateral sclerosis,ALS)的主因之一。然而,針對該些疾病,目前尚無有效的治療方法,故各式研究仍致力於探討參與調控致病性TDP-43形成的分子及相關機制。 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.

有鑑於此,相關領域亟需確認參與調控致病 性TDP-43的分子,藉此製備可用以預防或治療由致病性TDP-43所造成之神經退化性疾病的藥物。 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.

本發明是基於發現單體分子量為43道耳吞的轉活化反應-去氧核糖核酸-結合蛋白(transactivation responsive-DNA-binding protein,43kDa,TDP-43)寡聚體存在於病人腦中,此寡聚體於活體內外皆具有神經毒性,並會交聯至阿滋海默症之類澱粉蛋白-β(Alzheimer’s amyloid-β,Aβ)而使Aβ形成類澱粉蛋白寡聚體。因此,一種能結合至TDP-43寡聚體的分子(例如,抗體)將可用以抑制TDP-43蛋白病變(proteinopathies),並藉以製備出可診斷、預防或治療由致病性TDP-43所造成之神經退化性疾病的藥物(例如,疫苗及被動式免疫法)。 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 ampoules is present in the patient's brain. The oligomers are neurotoxic in vivo and outside, and cross-link to Alzheimer's amyloid-β (Aβ) such that Alzheimer's amyloid-β (Aβ) forms Aβ-like 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).

據此,本揭示內容旨在提供一種可專一結合至TDP-43寡聚體之人類抗體或其之片段。該人類抗體或其之片段包含一重鏈變異區及一輕鏈變異區,其中該重鏈變異區包含序列編號:1、2及3的胺基酸序列,而該輕鏈變異區則包含序列編號:5、6及7的胺基酸序列。 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.

TDP-43寡聚體為一直徑約2至400奈米(nanometer,nm)的顆粒。較佳的情況是,TDP-43寡聚體為一直徑約40至60奈米的球型或環狀顆粒。 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.

依據某些較佳的實施方式,該人類抗體或其之片段的重鏈變異區具有序列編號:4的胺基酸序列,而其輕鏈變異區則具有序列編號:8的胺基酸序列。 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.

在一實施方式中,該人類抗體或其之片段是由一融合瘤細胞株所製備,而該融合瘤細胞株已寄存於生物資源保存及研究中心(Bioresource Collection and Research Center,BCRC),寄存編號為BCRC 960494。 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). For BCRC 960494.

在另一實施方式中,該人類抗體或其之片段是由一融合瘤細胞株所製備,而該融合瘤細胞株已寄存於生物資源保存及研究中心,寄存編號為BCRC 960495。 In another embodiment, the human antibody or fragment thereof is produced by a fusion cell line that has been deposited with the Center for Conservation and Research of Biological Resources under the accession number BCRC 960495.

在再另一實施方式中,該人類抗體或其之片段是由一融合瘤細胞株所製備,而該融合瘤細胞株已寄存於生物資源保存及研究中心,寄存編號為BCRC 960496。 In still another embodiment, the human antibody or fragment thereof is produced by a fusion cell line that has been deposited with the Center for Conservation and Research of Biological Resources under the accession number BCRC 960496.

在另一實施方式中,該人類抗體或其之片段是由一融合瘤細胞株所製備,而該融合瘤細胞株已寄存於生物資源保存及研究中心,寄存編號為BCRC 960497。 In another embodiment, the human antibody or fragment thereof is produced by a fusion cell line that has been deposited with the Center for Conservation and Research of Biological Resources under the accession number BCRC 960497.

在另一實施方式中,該人類抗體或其之片段是由一融合瘤細胞株所製備,而該融合瘤細胞株已寄存於生物資源保存及研究中心,寄存編號為BCRC 960498。 In another embodiment, the human antibody or fragment thereof is produced by a fusion cell line that has been deposited with the Center for Conservation and Research of Biological Resources under the accession number BCRC 960498.

因此,本揭示內容的第二態樣提供了該抗體的用途,即,用以製備一種用以預防或治療與TDP-43寡聚體相關疾病的藥物或醫藥組合物。該藥物或醫藥組合物包含一有效量之上述人類抗體或其之片段及一藥學上可接受之賦形劑。 Accordingly, a second aspect of the present disclosure provides the use of the antibody, i.e., 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.

依據本揭示內容較佳的實施方式,該人類抗體或其之片段包含一重鏈變異區及一輕鏈變異區,其中該重鏈變異區包含序列編號:1、2及3的胺基酸序列, 而該輕鏈變異區則包含序列編號:5、6及7的胺基酸序列。 According to a preferred embodiment of the present disclosure, the human antibody or 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.

依據本揭示內容其餘較佳的實施方式,該抗體是由一融合瘤細胞株所製備,而該融合瘤細胞株已寄存於生物資源保存及研究中心,寄存編號為BCRC 960494、BCRC 960495、BCRC 960496、BCRC 960497或BCRC 960498。 According to other preferred embodiments of the present disclosure, the antibody is prepared from a fusion cell line that has been deposited with a biological resource conservation and research center under the accession numbers BCRC 960494, BCRC 960495, BCRC 960496. , BCRC 960497 or BCRC 960498.

本發明抗體的重量約佔醫藥組合物總重量之0.1%至99%。在一實施方式中,本發明抗體的重量至少佔醫藥組合物總重量之1%。在另一實施方式中,本發明抗體的重量至少佔醫藥組合物總重量之5%。在再另一實施方式中,本發明抗體的重量至少佔醫藥組合物總重量之10%。在另一實施方式中,本發明抗體的重量至少佔醫藥組合物總重量之25%。 The weight of the antibody of the invention is from about 0.1% to about 99% by weight based on the total weight of the pharmaceutical composition. 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.

本揭示內容之藥物或醫藥組合物可用以治療與TDP-43寡聚體相關的疾病,該疾病可以是阿滋海默症(Alzheimer’s disease)、嗜銀顆粒性認知症(argyrophilic grain disease)、肌肉萎縮性脊髓側索硬化症(amyotrophic lateral sclerosis,ALS)、關島肌肉萎縮性脊髓側索硬化-巴金森氏失智症(ALS-parkinsonism dementia complex of Guam)、血管性失智症(vascular dementia)、額顳葉失智症(frontotemporal dementia)、語意失智症(semantic dementia)、雷維體失智(dementia with Lewy bodies)、亨汀頓氏舞蹈症(Huntington’s disease)、小腦萎縮症(Spinocerebellar ataxia)、包涵體肌病(inclusion body myopathy)、包涵體肌炎(inclusion body myositis)或巴金森氏症(Parkinson’s disease)。 The medicament or pharmaceutical composition of the present disclosure may be used to treat a disease associated with a TDP-43 oligomer, which may be Alzheimer's disease, argyrophilic grain disease, muscle Amytrophic lateral sclerosis (ALS), Guam muscle atrophic lateral sclerosis - ALS-parkinsonism dementia complex of Guam, vascular dementia, Frontotemporal dementia, semantic dementia, dementia with Lewy bodies, Huntington's disease, Spinocere bellar ataxia Inclusion body Myopathy), inclusion body myositis or Parkinson's disease.

因此,本揭示內容之第三態樣提供了一種用以預防或治療一個體之與TDP-43寡聚體相關疾病的方法。該方法包含投予該個體一治療有效量之本發明抗體或其之片段,藉以抑制TDP-43蛋白病變。 Accordingly, a third aspect of the present disclosure provides 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.

依據某些較佳的實施方式,本發明抗體或其之片段包含一重鏈變異區及一輕鏈變異區,其中該重鏈變異區包含序列編號:1、2及3的胺基酸序列,而該輕鏈變異區則包含序列編號:5、6及7的胺基酸序列。 According to certain preferred embodiments, the antibody of the present invention or a fragment thereof comprises a heavy chain variant region and a light chain variant region, wherein the heavy chain variant region comprises the amino acid sequence of SEQ ID NO: 1, 2 and 3, and The light chain variant region comprises the amino acid sequence of SEQ ID NOs: 5, 6, and 7.

依據本揭示內容其餘較佳的實施方式,該抗體或其之片段是由一融合瘤細胞株所製備,而該融合瘤細胞株已寄存於生物資源保存及研究中心,寄存編號為BCRC 960494、BCRC 960495、BCRC 960496、BCRC 960497或BCRC 960498。 According to other preferred embodiments of the present disclosure, the antibody or fragment thereof is prepared from a fusion tumor cell line which has been deposited with the Center for Conservation and Research of Biological Resources, and the accession number is BCRC 960494, BCRC. 960495, BCRC 960496, BCRC 960497 or BCRC 960498.

本揭示內容之方法可用以治療與TDP-43寡聚體相關的疾病,該疾病可以是阿滋海默症、嗜銀顆粒性認知症、肌肉萎縮性脊髓側索硬化症、關島肌肉萎縮性脊髓側索硬化-巴金森氏失智症、血管性失智症、額顳葉失智症、語意失智症、雷維體失智、亨汀頓氏舞蹈症、小腦萎縮症、包涵體肌病、包涵體肌炎或巴金森氏症。 The methods of the present disclosure can be used to treat diseases associated with TDP-43 oligomers, which can be Alzheimer's disease, argyrophilic granulosuscitation, amyotrophic lateral sclerosis, Guam muscle atrophic spinal cord Lateral cord sclerosis - Bajinsen's dementia, vascular dementia, frontotemporal dementia, semantic dementia, Levi's dementia, Huntington's disease, cerebellar atrophy, inclusion body myopathy , inclusion body myositis or Parkinson's disease.

本揭示內容之第四態樣提供了一種用以診斷一個體是否罹患與TDP-43寡聚體相關之疾病的方法。該方法包含由該個體取得一生物檢體;將該生物檢體與本 發明人類抗體或其之片段接觸,以決定該生物檢體中TDP-43寡聚體的含量;以及將該生物檢體中TDP-43寡聚體的含量與取自一健康個體之對照檢體的TDP-43寡聚體含量進行比對;其中,若該生物檢體中TDP-43寡聚體的含量與該對照檢體的TDP-43寡聚體含量有顯著差異,即代表該個體患有與神經退化性疾病。 A fourth aspect of the present disclosure provides a method for diagnosing whether a subject 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 contact to determine the content of 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.

依據本揭示內容較佳的實施方式,該生物檢體可以是腦部切片檢體、腦脊液檢體、全血檢體、血清檢體、血漿檢體、尿液檢體或黏液檢體。 According to a preferred embodiment of the present disclosure, the biological specimen may 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.

依據某些較佳的實施方式,該人類抗體或其之片段包含一重鏈變異區及一輕鏈變異區,其中該重鏈變異區包含序列編號:1、2及3的胺基酸序列,而該輕鏈變異區則包含序列編號:5、6及7的胺基酸序列。 According to some preferred embodiments, the human antibody or fragment thereof comprises a heavy chain variant region and a light chain variant region, wherein the heavy chain variant region comprises amino acid sequences of SEQ ID NO: 1, 2 and 3, and The light chain variant region comprises the amino acid sequence of SEQ ID NOs: 5, 6, and 7.

依據其餘較佳的實施方式,該抗體或其之片段是由一融合瘤細胞株所製備,而該融合瘤細胞株已寄存於生物資源保存及研究中心,寄存編號為BCRC 960494、BCRC 960495、BCRC 960496、BCRC 960497或BCRC 960498。 According to other preferred embodiments, the antibody 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.

因此,本揭示內容的第五態樣提供了一種用以偵測一生物檢體之致病性TDP-43表現的套組。偵測的結果可用以評估該個體是否患有與TDP-43寡聚體相關之疾病。該套組至少包含一容器及用以偵測TDP-43寡聚體的試劑,其中該試劑包含本發明之抗-TDP-43寡聚體抗體及一附於容器內的說明書,該說明書係用以告知利用本發明之抗-TDP-43寡聚體抗體來偵測致病性TDP-43 (即上述之TDP-43寡聚體)的方法。該說明書可以是書冊、磁帶、CD、VCD或DVD。該套組可另包含一用以對照指出TDP-43寡聚體於健康個體之正常含量的對照組。 Accordingly, a fifth aspect of the present disclosure provides a kit for detecting the pathogenic TDP-43 performance 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.

在參閱下文實施方式後,本發明所屬技術領域中具有通常知識者當可輕易瞭解本發明之基本精神及其他發明目的,以及本發明所採用之技術手段與實施態樣。 The basic spirit and other objects of the present invention, as well as the technical means and implementations of the present invention, will be readily apparent to those skilled in the art of the invention.

為讓本發明的上述與其他目的、特徵、優點與實施例能更明顯易懂,所附圖式之說明如下:第1圖 全長TDP-43會形成與類澱粉蛋白相似的寡聚體 (a)全長TDP-43之分析式粒徑篩析層析(analytical size-exclusion chromatography,SEC)結果,其中由大腸桿菌純化的TDP-43是以光波波長為280奈米時的吸光值進行偵測(實線),而由人類HEK293細胞純化的TDP-43則是由隙縫點墨法(slot blotting)的強度進行定量(虛線);圖中所示之停留時間係為分子量標準組的停留時間(b)TDP-43的點漬法(Dot blotting)結果,其係利用抗-類澱粉蛋白寡聚體之抗體A11來進行偵測;以A11分別對新鮮純化且未經粒徑篩析層析處理之TDP-43(預載TDP-43)、經粒徑篩析層析處理之TDP-43(沖提體積,TDP-43寡聚體)及緩衝液進行免疫染色;(c)將新鮮純化之TDP-43分別沾點於緩衝液、包含9M尿素之緩衝液、包含7.2M脈鹽酸鹽(guanidine hydrochloride,GdnHCl) 之緩衝液或包含2%十二烷基硫酸鈉(sodium dodecyl sulfate,SDS)之緩衝液中,並以90℃加熱或不加熱1小時;該試驗重覆三次,並分別以A11抗體、抗-TDP-43之N端(殘基1-260)的抗體及抗-TDP-43之C端(殘基250-414)的抗體來進行偵測;以(d)穿透式電子顯微鏡(transmission electron microscope,TEM)及(e)原子力顯微鏡(atomic force microscope,AFM)來檢測TDP-43寡聚體(比例尺為500奈米),左上方為單一寡聚體的放大圖(比例尺為50奈米);(f)及(g)為經粒徑篩析層析處理後,TDP-43寡聚體的動態光散射(dynamic light scattering,DLS)分析結果;其中粒徑可以散射光強度(f)及顆粒數量(g)來表示;第2圖 TDP-43的結構、硫代黃素T(Thioflavin T,ThT)螢光及DNA結合特性 (a)全長TDP-43(實線)及短式TDP-43(虛線)的遠紫外光旋光儀光譜(Far-UV CD spectra);波長範圍為250至190奈米;(b)全長TDP-43(實線)及短式TDP-43(虛線)的Bis-ANS螢光光譜;緩衝液訊號標示為點線;(c)TDP-43及Aβ纖維與硫代黃素T的結合光譜;全長TDP-43的硫代黃素T螢光發散波長係以實線連接之■表示,短式TDP-43的硫代黃素T螢光發散波長係以虛線連接之●表示,而Aβ纖維的硫代黃素T螢光發散波長則係以點線連接之▲表示;僅Aβ纖維具有與硫代黃素T結合的訊號;(d)利用螢光滴定來檢測TDP-43與TAR DNA的結合;以單股TAR DNA-A位(實心)及-B位(空心)分別滴定全長TDP-43(方形)及短 式TDP-43(圓形);利波長為280奈米的光波進行激發時,於350奈米波長可偵測到TDP-43或TDP-43s的最大發散量;該些結果是以起始點為基準進行標準化,且連接線為最適線;第3圖 TDP-43與Aβ的交聯 (a)在不含或含有0.4至4%之TDP-43時,Aβ纖維化的硫代黃素T試驗結果;該濃度是以TDP-43的莫耳百分比來表示;(b)時間點為0,不含或含有TDP-43之光致交聯(Photo-induced cross-linking,PICUP)試驗結果,其中TDP-43的濃度是以莫耳百分比來表示;(c)不含或含有4%TDP-43之Aβ終點產物的穿透式電子顯微鏡結果(比例尺為100奈米);第4圖 TDP-43寡聚體會於活體外及活體內引發神經軸突退化及毒性 TDP-43對人類BE(2)-C細胞的細胞毒性可藉由(a)溴化噻唑藍四氮唑(MTT,3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide)及(b)乳酸去氫酶(lactate dehydrogenase,LDH)LDH試驗來分析(數量為3,平均值±平均標準差);(c)利用MTT試驗來分析TDP-43對初代神經元培養的細胞毒性;在該試驗中,細胞存活率是以緩衝液對照組做為基準進行標準化(數量為3,平均值±標準差);圖(a)及圖(c)是以單因子變異數分析(one way-ANOVA)及Tukey事後測定(Tukey post test)進行統計,圖(b)則是以單因子變異數分析及雙尾、不成對之Student’s t-test(two tailed,unpaired Student’s t-test)進行分析(*p<0.05、**p< 0.01、***p<0.001);(d)以對照組或0.44μM之TDP-43處理之初代神經元的免疫細胞化學染色結果;檢體分別以MAP-2(紅色)、GFAP(綠色)及DAPI(藍色)進行螢光染色;(e)將TDP-43注射至小鼠海馬迴,會造成海馬迴之CA區的神經元減少;分別將緩衝液對照組及TDP-43注入海馬迴中(每組隻數為3隻)後,以神經元特定標誌NeuN及核特定染劑DAPI進行免疫組織化學染色;箭頭所指處為神經元減少的位置(比例尺為50微米);第5圖 TDP-O抗體可專一地辨識TDP-43寡聚體 (a)將新鮮純化之TDP-43分別溶於原緩衝液、包含9M尿素之緩衝液、包含7.2M脈鹽酸鹽之緩衝液或包含2%十二烷基硫酸鈉之緩衝液中,並於90℃加熱或不加熱1小時後,以多株抗體TDP-O來進行點漬法分析,其中該多株抗體TDP-O是以TDP-43寡聚體為免疫原所製備的抗體;TDP-O與TDP-43所產生的免疫反應相似於第1c圖所示之A11結果;(b)分別以A11及TDP-O抗體對Aβ寡聚體進行點漬法分析;結果顯示A11可辨識Aβ寡聚體,TDP-O則不會;(c)原倍TDP-43(點線)及3倍濃縮之TDP-43(實線)的粒徑篩析層析結果,其中TDP-43寡聚體的沖提體積為★,TDP-43單體的沖提體積為☆,數字則為分子量標準組;(d)收集1ml的粒徑篩析層析分液,以TDP-O(上圖)及N1-260抗體(下圖)進行點漬法分析;(e)以酵素免疫吸附法(enzyme-linked immunosorbent assay,ELISA)分析由粒徑篩析層析純化的TDP-43寡聚體及單體;將TDP-43檢體依濃度塗佈於ELISA盤後, 分別以TDP-O(■)及N1-260(□)抗體偵測TDP-43寡聚體,而分別以TDP-O(●)及N1-260(○)抗體偵測TDP-43單體;TDP-O抗體對TDP-43寡聚體具有顯著較高的專一性;第6圖 FTLD-TDP基因轉殖鼠之TDP-43寡聚體表現量會隨著年齡增加而增加 (a)將取自於野生型、6個月或12個月大之TDP-43基因轉殖鼠(TDP-43 Tg+/+ transgenic mice)腦部的切片進行免疫螢光染色,藉以偵測人類TDP-43及TDP-43寡聚體的表現;以抗-TDP寡聚體染色的細胞會呈現綠色,以抗-TDP-43染色的細胞會呈現紅色,而以DAPI染色的細胞則會呈現藍色(比例尺為100微米);框選區域進一步以更高倍率顯示於最右欄(比例尺為25微米);(b)定量結果指出具有TDP-O及TDP-43沉澱的細胞比例係與年齡相關(每組3隻,其中各小鼠隨機取5視野進行定量,資料以平均值±平均標準差表示;結果是以單因子變異數分析及Tukey事後測定進行統計分析;*p<0.05、**p<0.01、***p<0.001);第7圖 FTLD-TDP病患會表現TDP-43寡聚體 共有三名FTLD-TDP病患、三名神經及病理分析上皆為正常的同齡健康個體,以及三名罹患阿滋海默症而無TDP-43包涵體之病患(做為神經退化疾病對照)接受本研究分析;該些個體之腦部切片代表圖分列於第7圖中,其中(a)、(c)及(e)為利用TDP-O抗體對FTLD-TDP病患之海馬迴(c)及前額葉皮質部位(a,e)進行TDP-43免疫組織化學染色圖;TDP-O可辨識深染、卵圓形或不規則形之位於細胞質內的包涵體(紅色箭頭)及神經纖維網 (neuropil,為營養不良的軸突)中逗號形式之包涵體(藍色箭頭);在某些如(e)圖所示之皮質區域中,神經元細胞質會呈現粗顆粒狀之免疫反應;如(b)、(d)及(f)所示,對照個體的腦部切片並不會產生任何TDP-O的免疫染色反應;相較於針對TDP-43單體的抗體,TDP-O抗體不會染到細胞核,證實該抗體可專一地結合至不正常折疊的TDP-43;各圖中的比例尺皆為20微米;第8圖 由病人海馬迴免疫沉澱萃取之TDP-43寡聚體會被TDP-O抗體以免疫金標定法標定 萃取FTLD-TDP病患的海馬迴組織,並以TDP-O抗體進行免疫沉澱;將萃取物以N端TDP-43抗體及免疫金標記後,利用電子顯微鏡(electron microscopy,EM)分析觀察(比例尺為50奈米);(a)FTLD-TDP病患之TDP-43寡聚體(比例尺為100奈米);(b)TDP-43寡聚體的放大圖(比例尺為50奈米);第9圖 TDP-O單株抗體對TDP-43寡聚體具有高度專一性 分別將TDP-O-3、-5、-8、-9及-10融合瘤細胞株所產生的TDP-O單株抗體,以不同濃度(每毫升1-2 x 10-5微克)進行ELISA試驗分析,藉以偵測以十二烷基硫酸鈉解構變性或未解構變性之TDP-43;第10圖 TDP-O單株抗體對純化的TDP-43寡聚體具有高度專一性 利用TDP-O融合瘤細胞株之條件培養液對以粒徑篩析層析純化過的TDP-43寡聚體及單體進行ELISA試驗分析;(A)以Superdex 200 10/300 GL管柱分離並收集TDP-43寡聚體及單體;(B)在ELISA試驗中, 利用TDP-O-3、-5、-8、-9及-10融合瘤細胞株的條件培養液來偵測TDP-43寡聚體及單體;以及第11圖 5株TDP-O單株抗體之重鏈變異區及輕鏈變異區具有一致性的胺基酸序列 X表任何的胺基酸殘基,而CDR則是互補性決定區(complementarity determining region)的縮寫。 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 oligomers similar to amyloid-like proteins (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) The results of Dot blotting of TDP-43 were detected by anti-amyloid oligomer-like antibody A11; freshly purified by A11 and subjected to particle size exclusion chromatography. TDP-43 (preloaded with TDP-43), TDP-43 (extracted volume, TDP-43 oligomer) treated with particle size chromatography and immunostaining with buffer; (c) freshly purified TDP -43 separately contaminated with buffer, containing 9M urea buffer, containing 7.2M vein hydrochloride (GondaHC) l) buffer or buffer containing 2% sodium dodecyl sulfate (SDS), and heated at 90 ° C or not heated for 1 hour; the test is 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 signal Marked as dotted line; (c) Binding spectrum of TDP-43 and Aβ fiber and thioflavin T; full-length TDP-43 thioflavin T fluorescence divergence wavelength is indicated by solid line connection, short TDP -43 thioflavin T fluorescence divergence wavelength is indicated by a dotted line connection, while Aβ fiber thioflavin T fluorescence divergence wavelength is represented by ▲ dotted line; only Aβ fiber has thio (1) Fluorescence titration was used to detect the binding of TDP-43 to TAR DNA; full-length TDP-43 was titrated with single-strand TAR DNA-A (solid) and -B (open), respectively. 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; Standardized on the basis of the starting point, and the connecting line is the optimum line; Figure 3 is the cross-linking of TDP-43 with Aβ (a) Aβ-fibrillated sulfur in the absence or inclusion of 0.4 to 4% of TDP-43 Results of the flavin T test; the concentration is expressed as the percentage of moles of TDP-43; (b) is 0 at the time point, and contains or does not contain photo-induced cross-linking (PICUP). Test result, its TDP-43 is the concentration 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 induce axonal degeneration and toxicity in vitro and in vivo TDP-43 is cytotoxic to human BE(2)-C cells by (a) thiazolyl tetrazolium bromide (MTT, 3-[ 4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) and (b) lactate dehydrogenase (LDH) LDH assay (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 deviation) Fig. (a) and (c) are statistically analyzed by one-way-variance analysis (one way-ANOVA) and Tukey post-test (Tukey post test), and (b) are analyzed by single-factor variation and double Tail, unpaired Student's t-test (two tailed, unpaired Student's t-test) (* p <0.05, ** p < 0.01, *** p <0.001); (d) with control or 0.44 μM T Immunocytochemical staining results of primary neurons treated with DP-43; specimens were fluorescently stained with MAP-2 (red), GFAP (green), and DAPI (blue); (e) TDP-43 was injected In the hippocampus of mice, 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 markers NeuN and nucleus were used. 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-O antibody can specifically identify TDP-43 oligomer (a) will be freshly purified The 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 not After heating for 1 hour, the spotting 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-43 The resulting immune response was similar to the A11 results shown in Figure 1c; (b) Aβ oligomers were analyzed by spot blotting with A11 and TDP-O antibodies, respectively; 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 + / + slices transgenic mice) were immunofluorescence staining brain, thereby detecting human TDP-43 and TDP-43 oligomer Now, 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 is 100 microns); Further displayed in the rightmost column at a higher magnification (25 μm scale); (b) Quantitative results indicate that the proportion of cells with TDP-O and TDP-43 precipitation is age-related (3 in each group, with each mouse randomized) Quantification was performed with 5 fields of view, and the data were expressed as mean ± mean standard deviation; the results were statistically analyzed by single factor analysis and Tukey's post hoc measurement; * p <0.05, ** p <0.01, *** p <0.001) Figure 7 shows that FTLD-TDP patients will have three FTLD-TDP patients with TDP-43 oligomers , three healthy individuals of the same age with neurological and pathological analysis, and three patients with Alzheimer's disease. Patients 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 immunohistochemistry for hippocampal gyrus (c) and prefrontal cortex (a, e) in patients with FTLD-TDP using TDP-O antibodies Chemical staining; TDP-O identifies dark-stained, oval or irregular corpus-like inclusion bodies (red arrows) and neurofibrils (neuropils, malnourished axons) in comma-like inclusions (blue arrow); in some cortical areas as shown in (e), the neuronal cytoplasm exhibits a coarse granular immune response; as shown in (b), (d), and (f), the control individual The brain slice does not produce any immunostaining reaction of TDP-O; compared to the antibody against TDP-43 monomer, TDP-O antibody does not stain the nucleus, confirming that the antibody can specifically bind to abnormal folding 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) placement of TDP-43 oligomers FIG. (Scale 50 nm); Fig. 9 TDP-O monoclonal antibody highly specific for the TDP-43 oligomer respectively TDP-O-3, -5, -8, -9 , and -10 hybridoma The TDP-O monoclonal antibody produced by the cell strain was analyzed by ELISA test at different concentrations (1-2 x 10 -5 μg per ml) to detect TDP destructive or undestructively denaturing with sodium lauryl sulfate. -43; Figure 10 TDP-O monoclonal antibody is highly specific for purified TDP-43 oligomers. TDP-purified by particle size screening chromatography using conditioned medium of TDP-O fusion tumor cell line. 43 oligo and monomer were analyzed by ELISA test; (A) TDP-43 oligomer and monomer were separated and collected by Superdex 200 10/300 GL column; (B) TDP-O- was used in ELISA test Conditioned medium of 3, -5, -8, -9 and -10 fusion tumor cell lines to detect TDP-43 oligomers and monomers; and the heavy chain variation of TDP-O monoclonal antibodies in Fig. 11 The 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.

為了使本揭示內容的敘述更加詳盡與完備,下文針對了本發明的實施態樣與具體實施例提出了說明性的描述;但這並非實施或運用本發明具體實施例的唯一形式。實施方式中涵蓋了多個具體實施例的特徵以及用以建構與操作這些具體實施例的方法步驟與其順序。然而,亦可利用其他具體實施例來達成相同或均等的功能與步驟順序。 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. 定義I. Definition

「TDP-43蛋白病變」(TDP-43 proteinopathy)一詞在本文中是指與單體分子量為43道耳吞之轉活化反應-去氧核糖核酸-結合蛋白(transactivation responsive-DNA-binding protein,43kDa,TDP-43)相關的疾病。TDP-43為一種疾病蛋白,其係與具有泛素包涵體(ubiquitin-positive inclusion,FTLD-U)表現之額顳葉退化症及肌肉萎縮性脊髓側索硬化症相關。TDP-43蛋白病變包含,但不限於,阿滋海默症、嗜銀顆粒性認知症、肌肉萎縮性脊髓側索硬化症、關島肌肉萎縮性脊髓側索硬化-巴金森氏失智症、血管性失智症、額顳葉失智症、 語意失智症、雷維體失智、亨汀頓氏舞蹈症、小腦萎縮症、包涵體肌病、包涵體肌炎或巴金森氏症。 The term "TDP-43 proteinopathy" (TDP-43 proteinopathy) refers to a transactivation responsive-DNA-binding protein (transactivation responsive-DNA-binding protein, which has a molecular weight of 43 amps). 43kDa, 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.

「有效量」(effective amount)一詞於本說明書中是指對與TDP-43寡聚體相關之疾病達到理想治療結果的有效劑量及治療時間週期。 The term "effective amount" as used in this specification refers to an effective dose and treatment time period for achieving a desired therapeutic result for a disease associated with a TDP-43 oligomer.

「藥學上可接受的」(pharmaceutically acceptable)一詞是指當投予至人體後,通常被視為安全的分子或組合物,例如該些具有生理相容性及不會產生過敏或類似不當反應的分子或組合物。較佳的情況是,「藥學上可接受的」(pharmaceutically acceptable)一詞在本說明書中是指經由美國聯邦或州政府之管理機構認可且可應用於動物或人類的分子或組合物。 " ""pharmaceutically acceptable"" (药物可可可) means a molecule or composition which is normally considered safe when administered to a human, such as those which are physiologically compatible and which do not cause allergies or similar inappropriate reactions. Molecule or composition. 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.

「投予」(administered、administering或administration)在本說明書中係為可互換的詞彙,且係指直接給予本發明之專一性抗體或組合物的方法。 &quot;administered,administering or administration&quot; is used interchangeably herein and refers to a method of directly administering a specific antibody or composition of the invention.

「個體」(subject)或「病患」(patient)是指包含人類的動物,其係可接受本發明之組合物及/或方法的治療。除非特定指出單一性別,否則「個體」(subject)或「病患」(patient)同時意指雄性及雌性。因此,「個體」(subject)或「病患」(patient)包含任何可接受治療的哺乳類動物。「個體」(subject)或「病患」(patient)包含,但不限於,人類、大鼠、小鼠、天竺鼠、猴子、豬、羊、牛、馬、狗、貓、鳥及家禽。在本發明實施例中,病患為人類。 &quot;Subject&quot; or &quot;patient&quot; (patient) refers to a human-containing animal that is capable of receiving treatment of the compositions and/or methods of the present 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.

「治療」(treat或treatment)在本說明書是 指產生一藥學及/或生理的效果,例如偵測致病性TDP-43的表現、預防或治療器官萎縮,或是抑制失智症、肌肉無力及僵直。該效果可以是預防性的,即完全或部分預防一疾病或其病徵;及/或是治療性的,即部分或完全治癒一疾病及/或其所造成的不適。「治療」(Treatment)在本說明書包含預防、治療或減緩一哺乳類動物(特別是人類)的疾病;該治療包含:(1)預防、治療或減緩一個體罹患一疾病(例如神經退化性疾病),其中該個體為罹患該疾病之高風險族群,或是已罹患該疾病而尚未確診斷定;(2)抑制一疾病(例如抑制其發生);或(3)減輕一疾病(例如減輕與該疾病相關之徵狀)。 "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).

「抗體」(antibody或antibodies)在本說明書中採廣義定義,包含具有生物活性的單株抗體、多株抗體、多專一性抗體(例如雙專一性抗體)及上述任一抗體片段;該生物活性是指在包含他種蛋白及/或分子的混合物中,能辨識標的蛋白並專一結合至抗原的特性。依據本揭示內容的一實施方式,本發明抗體為一多株抗體,其係可專一地辨識TDP-43寡聚體。 "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.

「單株抗體」(monoclonal antibody)一詞在本說明書是指一抗體,其係由一群具有相同質性的抗體分離出來,且不需經過特定方法加以建構。相較於多株抗體,其係包含可分別辨識不同抗原決定位置(epitope)的多種抗體,單株抗體僅能專一辨識一抗原的單一抗原決定位置。本揭示內容之單株抗體可以藉由融合方法或重組DNA方法來製備。本說明書之單株抗體可包含「嵌 合型」(chimeric)或「重組型」(recombinant)抗體,其中該抗體重鏈及/或輕鏈的部分區域是與一源自一特定物種之抗體或一分屬一特定抗體型或亞型之抗體,具有相同或同源的對應序列;而重鏈及輕鏈的其他區域則是與源自其他物種或分屬其他抗體型或亞型的另一抗體,或是抗體片段(只要該片段具有相關的生物活性),具有相同或同源的對應序列。依據本揭示內容的一特定實施方式,本發明抗體為一單株抗體,其係可專一地辨識TDP-43寡聚體。 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.

非人類(例如小鼠)抗體的「人類化」(humanized)抗體是指一種包含源自非人類免疫球蛋白之最小序列的嵌合型抗體。人類化抗體為人類的免疫球蛋白,其變異區的殘基由源自非人類物種(例如小鼠、大鼠、兔子或其他具有所需專一性或親合性的非人類靈長類)之變異區殘基所取代。在某些情況下,人類免疫球蛋白的Fv骨架區域(Fv framework region,FR)殘基是由相對應之非人類殘基所取代。一般來說,人類化抗體大致包含至少一個(通常為二個)可變異區,其中全部或多數FR區域為人類免疫球蛋白的序列。人類化抗體可以選擇性地包含一免疫球蛋白(通常為一人類免疫球蛋白)之恒定區的部分序列。 A "humanized" antibody to a non-human (eg, mouse) 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.

本說明書所用的單數名詞「一」(a或an)及「該」(the)涵蓋該名詞的複數型;而所用的複數名詞時亦涵蓋該名詞的單數型。 The singular nouns "a" and "the" and "the" are used in the singular terms of the noun.

II. 發明說明II. Description of the invention

已知單體分子量為43道耳吞之轉活化反應-去氧核糖核酸-結合蛋白(transactivation responsive-DNA-binding protein,43kDa,TDP-43)為一種致病性蛋白,其係與具有泛素包涵體(ubiquitin-positive inclusion,FTLD-U)表現的額顳葉退化症及肌肉萎縮性脊髓側索硬化症相關。本申請案發明人發現全長的TDP-43會形成結構穩定且具神經毒性的球形寡聚體,該寡聚體會交聯至阿滋海默症之類澱粉蛋白-β(Alzheimer’s amyloid-β,Aβ)並導致Aβ形成類澱粉蛋白寡聚體;因此,一能與TDP-43寡聚體結合的藥劑(例如抗體)將可用以抑制TDP-43蛋白病變;據此,該藥劑可用以製備一種藥物(例如疫苗或被動式免疫法),藉以預防或治療由致病性TDP-43所造成的神經退化性疾病。 It is known that the molecular weight of the monomer is 43 transaminating transactivation-DNA-binding protein (43kDa, TDP-43) is a pathogenic protein with ubiquitin The frontotemporal lobe degeneration and musculoskeletal lateral sclerosis associated with ubiquitin-positive inclusion (FTLD-U) 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.

因此,本揭示內容的第一態樣提供了一種抗體,更具體來說,該抗體為一種能辨識TDP-43寡聚體的抗體,並藉此抑制一蛋白(特別是TDP-43)的聚集,其中該聚集現象係與一疾病相關。一般來說,蛋白聚集是一種自傳播反應(self-propagating manner),一旦開始,聚集反應將會接連自動地發生,並引發構型改變及/或更多蛋白分子的聚合,最終形成無法被蛋白酶分解的有毒產物。經此形成的蛋白寡聚體被認為是造成神經退化性疾病的近因之一,該些神經退化性疾病包含阿滋海默症、嗜銀顆粒性認知症、肌肉萎縮性脊髓側索硬化症、關島肌肉萎縮性脊髓側索硬化-巴金森氏失智症、血管 性失智症、額顳葉失智症、語意失智症、雷維體失智、亨汀頓氏舞蹈症、小腦萎縮症、包涵體肌病、包涵體肌炎及巴金森氏症。 Accordingly, a first aspect of the present disclosure provides an antibody, and more particularly, 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.

依據本揭示內容較佳的實施方式,TDP-43寡聚體為粒徑約2至400奈米的顆粒。較佳的情況是,TDP-43寡聚體為粒徑約2至400奈米的顆粒,例如20至30奈米、30至40奈米、40至50奈米、50至60奈米、60至70奈米、70至80奈米、80至90奈米、90至100奈米、100至120奈米、120至140奈米、140至160奈米、160至180奈米、180至200奈米、200至220奈米、220至240奈米、240至260奈米、260至280奈米、280至300奈米、300至320奈米、320至340奈米、340至360奈米、360至380奈米及380至400奈米。更佳的情況是,TDP-43寡聚體為粒徑約40至60奈米的球形或環狀顆粒。 In accordance with a preferred embodiment of the present disclosure, the TDP-43 oligomer is a particle having a particle size of from about 2 to about 400 nanometers. 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.

本揭示內容之抗體可專一地結合至上述TDP-43寡聚體、其抗原決定位置、其不同構型及各構型之抗原決定位置。在較佳實施方式中,本抗體可優先結合至致病性TDP-43,更具體來說,該致病性TDP-43為一全長的TDP-43寡聚體顆粒,其粒徑大小約為2至400奈米。 The antibodies of the present disclosure can specifically bind to the above-described TDP-43 oligomers, their epitopes, their different configurations, 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.

為製備所述之單株抗體,先將適量之TDP-43寡聚體注入諸如小鼠、大鼠或兔子等動物體內,使其產生免疫反應。一般來說,佐劑會先與TDP-43寡聚體溶液混合,再將其注入動物體內。適用於本發明之佐劑包含 弗氏完全佐劑(Freund’s complete adjuvant,FCA)、弗氏不完全佐劑(Freund’s incomplete adjuvant,FIA)及氫氧化鋁佐劑。抗體的注射可以血管、皮下、腹腔或肌肉等方式給予。抗原注射的時間間隔並無特定限定。抗原注射的間隔時間可為數天至數週,較佳的方法是,2至3週,共1至10次(較佳為2至5次)。注射後,一旦抗體的效價到達2或更高的吸光值,則停止後續注射,並繼續飼養該動物1個月。接著,至少再注射一次。於最後一劑注射數天後(較佳約3至5天),取出該動物之脾臟細胞及區域淋巴結。採取血液檢體並將之離心處理以分離血清。可利用任何適合的方法來檢測所得血清中的抗體效價,例如酵素免疫吸附法(enzyme-linked immunosorbent assay,ELISA)、酵素免疫分析(enzyme immunoassay,EIA)或放射免疫分析(radio immunoassay,RIA)。在一較佳的實施例中,是利用ELISA來測定抗體之效價。 To prepare the monoclonal antibodies, an appropriate amount of TDP-43 oligomer is first injected into an animal such as a mouse, rat or rabbit to produce 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.

由分離之脾臟細胞及區域淋巴結製備可產生抗體的細胞。在製備可產生抗體的細胞時,宜先盡可能移出組織殘渣及紅血球。在此步驟可使用商業購買之紅血球去除劑。或者是,可以使用包含氯化銨及三羥甲基氨基甲烷(Tris(hydroxymethyl)aminomethane(Tris))的緩衝液。立即將可產生抗體的細胞與永生細胞(例如骨髓瘤細胞)進行融合,以製得融合瘤細胞;融合瘤細胞為一種可持續生長繁殖,並產生抗體的半永生細胞。可以使用由動物(例如小鼠)取得之常用的細胞株。較適 用於本發明的細胞株為可有效融合、持續產生高量抗體、對次黃嘌呤-胺蝶呤-胸腺核苷(hypoxanthine-aminopterin-thymidine,HAT)培養液具有敏感性,且唯有在與可產生抗體之細胞融合後方可存活的細胞株。適合的骨髓瘤細胞包含,但不限於,小鼠骨髓瘤細胞株(例如骨髓瘤FO細胞)及人類骨髓瘤細胞株(例如Karpas 707H)。 An antibody-producing cell is prepared from isolated spleen cells and regional lymph nodes. 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).

細胞融合通常是將脾臟細胞或淋巴結細胞與商業取得之骨髓瘤細胞在一促進細胞融合因子的作用下,進行融合反應,例如平均分子量約為200至20,000kDa的聚乙二醇(Polyethylene glycol,PEG)或其他類似物。或者是,細胞融合可以利用商業細胞融合儀,藉由電刺激(例如電融合)來進行融合反應。融合後,將所得細胞稀釋並培養於HAT培養液中。 Cell fusion is usually carried out by spleen cells or lymph node cells and commercially available myeloma cells under the action of a cell fusion factor, such as polyethylene glycol (PEG) having an average molecular weight of about 200 to 20,000 kDa. ) or other analogues. 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.

由該些融合細胞中挑選出適合的融合瘤細胞。於HAT培養液中存活的融合細胞會形成聚集群落。收集各培養液的上清液,且以TDP-43寡聚體檢測是否具有抗體效價。如上所述,可以使用酵素免疫吸附法、酵素免疫分析或放射免疫分析來進行檢測,其係將TDP-43寡聚體或寡聚體解構後形成TDP-43的單體塗佈於盤中,並由此進行篩選。一旦確認可產生抗體的孔洞,即可將其中的細胞轉移至不含胺蝶呤的次黃嘌呤-胸腺核苷(hypoxanthine-thymidine,HT)培養液中培養。經過培養,再次確認培養上清液中的抗體效價。由最終篩選出的細胞中,分離出單一顆細胞。待單一顆細胞生長繁殖 成一聚集群落後,篩選對TDP-43寡聚體具有高度專一性的聚集群落,並使其持續生長繁殖以製成融合瘤細胞株。 Suitable fusion tumor cells are selected 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.

依據本揭示內容較佳的實施方式,共篩選出5株融合瘤細胞株:TDP-O-3、TDP-O-5、TDP-O-8、TDP-O-9及TDP-O-10,單株抗體可由該些融合瘤細胞株以任何方式進行分離或製備。舉例來說,可以將融合瘤細胞株培養於低血清濃度的培養液中,再由該培養上清液製備抗體。或者是,將融合瘤細胞株注入動物的腹腔,由腹水來製備抗體。抗體可以任何方式進行純化,該方式包含親和性管柱、膠濾層析、離子交換層析或相關技術。可以使用任何相關領域習知技藝人士所熟知的方法或其組合。 According to a preferred embodiment of the present disclosure, five fusion tumor cell lines were selected: TDP-O-3, TDP-O-5, TDP-O-8, TDP-O-9 and TDP-O-10. The monoclonal antibodies can be isolated or prepared by any of these fusion tumor cell lines in any manner. 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.

依據特定的實施方式,本揭示內容的單株抗體係分別由融合瘤細胞株TDP-O-3、TDP-O-5、TDP-O-8、TDP-O-9及TDP-O-10製備,該些融合瘤細胞株已寄存於台灣新竹的食品工業發展及研究中心(Development and Research Institute,FIDRI)的生物資源保存及研究中心(Bioresource Collection and Research Center,BCRC),其寄存編號分別為BCRC 960494、BCRC 960495、BCRC 960496、BCRC 960497及BCRC 960498。 According to a specific embodiment, the monoclonal antibody system of the present disclosure is 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 at the Bioresource Collection and Research Center (BCRC) of the Development and Research Institute (FIDRI) in Hsinchu, Taiwan, with registration numbers BCRC. 960494, BCRC 960495, BCRC 960496, BCRC 960497 and BCRC 960498.

依據較佳實施方式,本單株抗體的重鏈及輕鏈變異區分別包含一致性的胺基酸序列(consensus sequence)。據此,本單株抗體分別包含一重鏈變異區及一輕鏈變異區,其中該重鏈變異區包含序列編號:1、2 及3的胺基酸序列,而該輕鏈變異區則包含序列編號:5、6及7的胺基酸序列。較佳的實施方式為,人類化單株抗體的重鏈變異區具有序列編號:4的胺基酸序列,而其輕鏈變異區則具有序列編號:8的胺基酸序列。 According to a preferred embodiment, the heavy and light chain variant regions of the monoclonal antibodies comprise a consensus amino acid sequence, respectively. 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.

或者是,抗-TDP-43寡聚體的單株抗體可以由DNA基因選殖來製備。將用以編碼抗-TDP-43寡聚體單株抗體的DNA分離出來後,再用傳統步驟進行定序,例如可專一結合至用以編碼單株抗體之重鏈及輕鏈基因的寡核苷酸探針。較佳的DNA來源是利用該些融合瘤細胞株(例如TDP-O-3、TDP-O-5、TDP-O-8、TDP-O-9或TDP-O-10融合瘤細胞株)。一旦分離後,可將DNA建構至表現載體,再將表現載體轉染至宿主細胞(例如大腸桿菌細胞、猿猴細胞COS、中國倉鼠卵巢細胞CHO或不會產生免疫球蛋白的骨髓瘤細胞),藉此於重組宿主細胞中製備出單株抗體。 Alternatively, a monoclonal antibody against an anti-TDP-43 oligomer can be 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.

所製得的單株抗體及用以編碼該些單株抗體的DNA可用以製備嵌合型抗體(例如雙專一性抗體)、人類化抗體及/或其中的抗體片段。 The monoclonal antibodies produced and the DNA encoding the monoclonal antibodies can be used to prepare chimeric antibodies (e.g., bispecific antibodies), humanized antibodies, and/or antibody fragments thereof.

非人類來源的單株抗體因具有免疫抗原性(immunogenicity)而會造成過敏反應。單株抗體多是源自小鼠,其注射至人體後往往會引起嚴重的免疫反應。為降低本發明抗-TDP-43寡聚體單株抗體的免疫抗原性,將小鼠抗-TDP-43寡聚體單株抗體的重鏈及輕鏈變異區與人類抗體之恒定區接合,藉此製備人類化抗體。 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.

為製備人類化的抗-TDP-43寡聚體抗體,先 將該些用以編碼抗體的DNA分離並定序,再據以進行人類化建構。 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.

依據本揭示內容較佳的實施方式,對互補性決定區(complementary determining region,CDR)進行轉接(grafting),其係將人類抗體之VH及VL基因中的CDR置換為特定的CDR編碼片段(例如該些抗-TDP-43寡聚體抗體中,用以編碼與TDP-43寡聚體結合之胺基酸片段的DNA片段)。由此製備出的抗體僅有CDRs係源自於小鼠的抗體,而VH及VL基因中其他的骨架區,以及恒定區的基因(即CK或CH1-H-CH2-CH3)則是人類的IgG。 According to a preferred embodiment of the present disclosure, a complementation determining region (CDR) is grafted by replacing a CDR in a VH and VL gene of a human antibody with a specific CDR coding fragment ( For example, among these anti-TDP-43 oligomer antibodies, a DNA fragment encoding an amino acid fragment bound 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.

在較佳的實施方式中,人類化抗-TDP-43寡聚體單株抗體包含一重鏈變異區及一輕鏈變異區。所製成的人類化抗-TDP-43寡聚體單株抗體可依據相關領域中的標準步驟來進行純化,包含掃流過濾(cross-flow filtration)、親和性管柱層析(affinity column chromatography)或膠體過慮(gel filtration)等方法。須知,人類化抗體的作用應與小鼠抗-TDP-43寡聚體抗體的作用相同或大致相似。較佳的情況是,相較於老鼠抗體,人類化抗-TDP-43寡聚體抗體(不論是Fab或全長IgG的形式)在施用至人類個體時,應會產生較佳的作用。在某些實施方式中,人類化抗-TDP-43寡聚體抗體可用以製備本揭示內容之雙專一性抗體。 In a preferred embodiment, the humanized anti-TDP-43 oligomer 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.

可利用任何活體外或活體內的TDP-43蛋白病變模式來確認本發明之抗-TDP-43寡聚體抗體。習知技 藝人士應知,可利用動物模式(例如實施例7所述之動物模式)來確認本發明之抗-TDP-43寡聚體抗體。或者是,可利用實施例8所述之相關病人檢體來確認本發明之抗-TDP-43寡聚體抗體。 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.

習知技藝人士應知,TDP-43蛋白病變的實驗模式可做為預防性測試或治療性測試。在預防性測試中,在動物產生TDP-43蛋白病變或其徵症前,即先投予藥物,藉以測試本發明之抗-TDP-43寡聚體抗體對TDP-43蛋白病變或其徵症之預防、減緩或延緩的功效。在治療性測試中,藥物是在動物產生TDP-43蛋白病變或其徵症後方進行投予,藉以測試本發明之抗-TDP-43寡聚體抗體對TDP-43蛋白病變或其徵症之治療、減輕或舒緩的功效。TDP-43蛋白病變的徵狀包含,但不限於,致病性TDP-43於測試個體之腦部、脊髓、腦脊液或血清的數量變化。 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.

據此,本揭示內容提供一種醫藥組合物或一藥物,其係可用以治療一種與Aβ堆積相關的神經退化性疾病。該醫藥組合物包含一有效量之本發明抗-TDP-43寡聚體抗體及一藥學上可接受之賦形劑。該種可利用本揭示內容之醫藥組合物或藥物進行治療的TDP-43寡聚體相關疾病包含,但不限於,阿滋海默症、嗜銀顆粒性認知症、肌肉萎縮性脊髓側索硬化症、關島肌肉萎縮性脊髓側索硬化-巴金森氏失智症、血管性失智症、額顳葉失智症、語意失智症、雷維體失智、亨汀頓氏舞蹈症、小腦萎縮症、包涵體肌病、包涵體肌炎及巴金森氏症。 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.

一般來說,本發明之抗-TDP-43寡聚體抗體 的重量約佔醫藥組合物總重量之0.1%至99%。在一實施方式中,本發明抗-TDP-43寡聚體抗體的重量至少佔醫藥組合物總重量之1%。在另一實施方式中,本發明抗-TDP-43寡聚體抗體的重量至少佔醫藥組合物總重量之5%。在再另一實施方式中,本發明抗-TDP-43寡聚體抗體的重量至少佔醫藥組合物總重量之10%。在另一實施方式中,本發明抗-TDP-43寡聚體抗體的重量至少佔醫藥組合物總重量之25%。 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.

在某些實施方式中,本發明醫藥組合物或藥物更可包含一藥劑,該藥劑為一已知可用以改善一神經退化性疾病之徵狀的藥劑;例如乙醯膽鹼酵素抑制劑(acetylcholinesterase inhibitor,AChEI)、Aβ抑制劑或毒蕈鹼受器(muscarinic receptor)拮抗劑及其類似物。 In certain embodiments, the pharmaceutical composition or medicament of the present invention may further comprise a medicament which is an agent known to be useful for ameliorating a neurodegenerative disease; for example, an acetylcholinesterase inhibitor Inhibitor, AChEI), Aβ inhibitor or muscarinic receptor antagonist and analogs thereof.

本發明醫藥組合物或藥物係依照可接受的藥物製作方法來進行製備,例如於Remington’s Pharmaceutical Sciences,17th edition,ed.Alfonoso R.Gennaro,Mack Publishing Company,Easton,Pa(1985)所述的方法。藥學上可接受之賦形劑為該些與劑型中其他成分相容且可為生物體接受的物質。 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.

本發明之抗-TDP-43寡聚體抗體可單獨或與藥學上可接受之賦形劑一起以口服、腹腔注射、顱內注射、脊內注射、肌肉注射、靜脈注射、皮膚吸收、腸內注射或吸入投予等方式投予至個體。在一較佳的實施方式中,本發明之抗-TDP-43寡聚體抗體係以靜脈注射的方式投予至個體。在另一較佳實施方式中,本發明之抗 -TDP-43寡聚體抗體是以脊內注射的方式投予至個體。 The anti-TDP-43 oligomer antibody of the present invention may be administered orally, intraperitoneally, intracranially, intrathecally, intramuscularly, intravenously, dermally, enterally, alone or together with a pharmaceutically acceptable excipient. It is administered to an individual by injection 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.

可使用之固體賦形劑可以包含一或多種物質,該物質可以是調味劑、潤滑劑、增溶劑、懸浮劑、填充劑、助流劑、壓縮助劑、粘合劑、片劑崩解劑或包覆性材料。若以粉末形式存在,該賦形劑係為一可與細碎活性成分混合的細碎的固體。若以片劑方式存在,活性成分是與一具有壓縮特性的賦形劑,以適當比例壓縮為所需的形狀及大小。粉末及片劑最好包含高達99%的活性成分。適用於本發明之固體賦形劑可以是磷酸鈣、硬脂酸鎂、滑石、糖、乳糖、糊精、澱粉、明膠、纖維素、甲基纖維素、羧甲基纖維素鈉、聚乙烯吡咯烷酮或其類似物。 A solid excipient that can be used may contain one or more substances, which may be flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders, tablet disintegrating agents. Or coated materials. 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.

本發明之抗-TDP-43寡聚體抗體亦可配製為無菌溶液或懸浮液形式之液體醫藥組合物,其係可以靜脈、肌肉、皮下、脊內、腹腔或顱內等方式注射至個體。口服投予可以是液體或固體形式。 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.

為因應以黏膜方式投予,本發明之藥物或醫藥組合物可以配製為各式劑量形式,例如若要經由口腔黏膜吸收,本發明之藥物或醫藥組合物可以頰及/或舌下藥物劑量單位進行配製。各式藥學上可接受之具有生物降解性的聚合賦形劑亦可使用於本發明,該類賦形劑不但提供適度的生物附著及藥物釋放特性,更可與頰及/或舌下藥物劑量單位中的活性試劑及其他成分相容。一般來說,聚合賦形劑包含可附著於口腔黏膜之溼潤表面的親水性聚合物。適用於本發明之聚合賦形劑包含, 但不限於,丙烯酸聚合物及共聚物、水解聚乙烯醇、聚乙烯氧化物、聚丙烯酸酯、乙烯聚合物及共聚物、聚乙烯吡咯烷酮、聚葡萄糖、瓜爾膠、果膠、澱粉及纖維素聚合物。 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.

因此,本發明亦提供一種治療哺乳類動物(特別是人類)之與TDP-43寡聚體相關之疾病的方法,該方法包含投予本發明包含抗-TDP-43寡聚體抗體之藥物及醫藥組合物至該個體。該藥物或醫藥組合物可以任何能將組合物活性成分有效傳遞至哺乳類動物(特別是人類)之特定位置的方式投予,例如透過口、鼻、肺、皮膚(例如被動或離子電滲傳遞)或非口服(例如直腸、脂肪、皮下、靜脈、脊內、肌肉、鼻腔、小腦內、眼液或軟膏)來投予。此外,本發明組合物的投予可以與其他活性成分同時投予。 Accordingly, the present invention also provides a method for treating a disease associated with a TDP-43 oligomer in a mammal, particularly a human, comprising administering a medicament and a medicament comprising the anti-TDP-43 oligomer antibody of the present invention. The composition is to the individual. 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.

在某些實施方式中,投予至個體的有效劑量約為每公斤個體體重之1至100毫克,例如每公斤個體體重之10、20、30、40、50、60、70、80、90或100毫克;較佳的是每公斤個體體重之50至70毫克,例如每公斤個體體重之50、60或70毫克;最佳的是每公斤個體體重之50毫克。該些劑量可以是單一次投予劑量,或是分成多次投予。 In certain embodiments, the effective dose administered to the individual is from about 1 to 100 mg per kg of body weight, such as 10, 20, 30, 40, 50, 60, 70, 80, 90 per kg of body weight. 100 mg; preferably 50 to 70 mg per kg of body weight, 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.

依據本揭示內容可選擇的實施方式,該方法更可包含投予該個體一乙醯膽鹼酵素抑制劑、Aβ抑制劑或毒蕈鹼受器拮抗劑,其中該抑制劑/拮抗劑可與本發明之抗-TDP-43寡聚體抗體同時或先後投予。 According to an alternative embodiment of the present disclosure, the method may further comprise administering to the individual an acetylcholine inhibitor, an Aβ inhibitor or a muscarinic receptor antagonist, wherein the inhibitor/antagonist is compatible with the present The anti-TDP-43 oligomer antibody of the invention is administered simultaneously or sequentially.

在某些實施方式中,該乙醯膽鹼酵素抑制劑可以是阿蘭他敏(alantamine)、星斯林(cymserine)、多奈派劑(donepezil)、ER 127528、加蘭他敏(galantamine)、甘斯替敏(ganstigmine)、石杉鹼甲(huperzineA)、苯羥基丙氨酸(phenserine)、苯乙基正星斯林(phenethylnorcymserine)、卡巴拉汀(rivastigmine)、RS 1259、SPH 1371、他克林(tacrine)、噻星斯林(thiacymserine)或蘭奈派齊(zanapezil)。在其他實施方式中,該Aβ抑制物可以是巴比留主(bapineuzumab)、PTB2、鯊肌醇(scyllo-inositol)、PPI 1019、RS 0406、SP 233、EGCG、Exberyl-1或SEN 606。而該毒蕈鹼受器拮抗劑可以是氧化震顫素(oxotremorine)或占諾美林(xanomeline)。 In certain embodiments, the acetylcholine enzyme inhibitor can be alanamine, cymserine, donepezil, ER 127528, galantamine, gan Ganstigmine, huperzine A, phenserine, phenethylnorcymserine, rivastigmine, RS 1259, SPH 1371, tacrine (tacrine), thiacymserine or 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.

本發明之抗-TDP-43寡聚體抗體可做為一種用以偵測或診斷一個體是否罹患與TDP-43寡聚體相關之疾病的工具。因此,本發明提供一種用以偵測或診斷一個體罹患或疑似患有TDP-43寡聚體相關疾病的方法。該方法包含下述步驟:由該個體取得一生物檢體;將該生物檢體與本發明之人類抗體接觸,藉以決定該生物檢體中TDP-43寡聚體的含量;以及將該生物檢體中TDP-43寡聚體的含量與取自一健康個體之對照檢體的TDP-43寡聚體含量進行比對;其中,若該生物檢體中TDP-43寡聚體的含量與該對照檢體的TDP-43寡聚體含量有顯著差異時,即代表該個體患有與神經退化性疾病。在本文中所謂的「顯著差異」係指由待測生物檢體所測得TDP-43 寡聚體含量,在統計學上明顯高於或低於由健康個體之對照檢體所測得的TDP-43寡聚體含量。 The anti-TDP-43 oligomer antibody of the present invention can be used as a tool for detecting or diagnosing whether a body is suffering from a disease associated with TDP-43 oligomer. 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.

上述生物檢體包含,但不限於,腦部切片檢體、腦脊液檢體、全血檢體、血清檢體、血漿檢體、尿液檢體、黏液檢體及該些檢體的純化或過濾產物。 The biological sample includes, but is not limited to, a brain slice, a cerebrospinal fluid sample, a whole blood sample, a serum sample, a plasma sample, a urine sample, a mucus sample, and purification or filtration of the sample. product.

可利用任何相關領域習知技藝人士所熟知的方法來檢測抗體之結合,例如酵素免疫吸附法(enzyme-linked immunosorbent assay,ELISA)、三明治免疫分析法(sandwich immunoassay)、原位免分析法(in situ immunoassays,例如利用膠態金、酵素或同位素標的)、西方墨點法、凝集分析法(agglutination assay,例如膠體凝集分析法、血球凝集分析法等)、補體結合分析法(complement fixation assay)、免疫螢光染色法(immunofluorescence assay)及免疫電泳法(immunoelectrophoresis assay)等。在一實施方式中,是利用ELISA來測定抗體的結合。在某些實施方式中,可利用體液(例如腦脊液、全血、血清、血漿、黏液及該些體液的純化或過濾產物)來檢測自體抗體。在一較佳的實施方式中,是利用腦脊液檢體來偵測抗體。在其他實施方式中,是利用腦部切片檢體來偵測抗體。 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.

為確保相關領域習知技藝人士可有效利用本發明,本發明之抗-TDP-43寡聚體抗體可組裝為套組形式,便於診斷、偵測或確認一神經退化性疾病。在較佳的實施方式中,是利用能與本發明之抗-TDP-43寡聚體抗體反應的致病性TDP-43來診斷一個體是否罹患神經退化性 疾病。舉例來說,相較於對照組(取自一健康個體的檢體),若在一生物檢體中發現較高量或較低量之可與本發明之抗-TDP-43寡聚體抗體反應的致病性TDP-43,表示該個體罹患與TDP-43寡聚體相關的疾病。該資料可用以進行後續相關治療。舉例來說,若一個體發現具有致病性TDP-43,即可開始進行TDP-43寡聚體相關疾病(例如AD、ALS及PD)之治療,早期的治療往往具有較佳的療效。 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.

在一實施方式中,本發明利用抗-TDP-43寡聚體抗體提供一種用以診斷TDP-43寡聚體相關疾病的套組。套組的成分包含:一容器、用以偵測生物檢體中TDP-43寡聚體的試劑及一附於容器內的說明書,其中該試劑包含利用本揭示內容任一實施例揭示方法所製備的抗-TDP-43寡聚體抗體,而該說明書是用以告知利用本發明之抗-TDP-43寡聚體抗體來偵測致病性TDP-43(即上述之TDP-43寡聚體)的方法。該說明書可以是書冊、磁帶、CD、VCD或DVD。該套組可另包含一用以對照指出TDP-43寡聚體於健康個體之正常含量的負對照。 In one embodiment, the invention utilizes an anti-TDP-43 oligomer antibody to provide a kit for diagnosing a TDP-43 oligomer-associated disease. 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.

下文提出多個實驗例來說明本發明的某些態樣,以利本發明所屬技術領域中具有通常知識者實作本發明。不應將這些實施例視為對本發明範圍的限制。據信習知技藝者在閱讀了此處提出的說明後,可在不需過度解讀的情形下,完整利用並實踐本發明。此處所引用的所有公開文獻,其全文皆視為本說明書的一部分。 A number of experimental examples are set forth below to illustrate certain aspects of the present invention, and the present invention may be practiced by those of ordinary skill in the art. 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.

實施例Example

材料及方法Materials and methods

材料 由HEK細胞所分離的人類TDP-43係取自於OriGene Technologies,Inc.(Rockville,MD,USA)。由產品資料可知,將一TrueORF殖株(即RC210639)轉染至人類HEK 293細胞後,可製得一重組TDP-43蛋白;該重組TDP-43蛋白的C端具有一Myc-DDK標誌(tag),故可利用抗-DDK親和性管柱進行純化。短式TDP-43(殘基101-285)則是依照Kuo等人所述的步驟(Nucleic Acids Res(2009)37,1799-1808)來製備。用於西方墨點法之抗-N端殘基1-260的TDP-43抗體(標記為N1-260)及用於免疫組織化學染色的抗-TDP-43抗體皆是購買自Abcam(Cambridge,UK)。抗-C端殘基350-414之TDP-43抗體(標記為C350-414)是購買自Novus(Littleton,CO,USA),而抗-類澱粉蛋白寡聚體的抗體A11則是購買自BioSource(Invitrogen,Carlsbad,CA,USA)。抗DDK單株抗體是購自Origene Technologies,Inc。Aβ胜肽是由中央研究院基因體中心的胜肽合成實驗室所合成。其餘化學藥劑則皆是購自Sigma Aldrich(St.Louis,MO,USA)或Amresco(Solon,OH,USA)。除非另有所指,否則所有細胞培養試劑皆是購自Gibco(Invitrogen)。 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.

純化TDP-43 先由包含一用以編碼全長TDP-43之cDNA的轉體pCMV-Taq2B選殖出TDP-43片段。再將該選殖產物以限制酶XhoI/BamHI轉殖至載體pET14b(Novagen,Merck KGaA,Darmstadt,Germany),以產生N端組胺酸標誌(His-Tag)。將所得之N端具有 一組胺酸標誌的TDP-43轉殖至大腸桿菌株Rosetta 2(Novagen,Merck KGaA,Darmstadt,Germany)進行表現。收集細胞,將細胞懸浮於30mM Tris-HCl(pH 8)緩衝液後置於冰上,以微流儀(microfluidizer)打破細胞,其中該緩衝液包含500mM NaCl、10%甘油、1mM DTT、2%RNase A、2%DNase I及蛋白酶抑制劑(完全,不含EDTA,購買自Roche Applied Science,Mannheim,Germany)。於4℃,以27,000×g離心該溶菌產物。先以包含30mM Tris(pH 8)、500mM NaCl、1mM DTT、20mM咪唑(imidazole)及10%甘油的緩衝液平衡一Ni-NTA親合性管柱(GE healthcare Bio-Sciences AB,Uppsala,Sweden)後,將所得之上清液注入該管柱。將咪唑溶於上述緩衝液後,利用不同濃度的咪唑來沖提蛋白產物。約200mM的咪唑可沖提出TDP-43蛋白。利用十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)及古瑪西藍(Coomassie blue)來確認所純化的組胺酸標誌TDP-43蛋白。純化的TDP-43包含N端殘基MGSSHHHHHHSSGLVPRGSHMLE。估算的分子約為47,147Da。再進一步將該蛋白產物進行透析。依據Nick Pace(Pace et al.,(1995)Protein Sci 4,2411-2423)所述之公式,扣除於280奈米時的吸光背景值,以消光係數為44,920cm-1M-1計算蛋白濃度。在計算短式TDP-43之濃度時,消光係數則為15,470cm-1M-1 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 .

粒徑篩析層析(Size exclusion chromatography,SEC) 先以分子量標記組來標準化Superdex-200 10/300 GL分析式凝膠過濾管柱(GE healthcare Bio-Sciences AB,Uppsala,Sweden),其中該分子量標記組是分別將鐵蛋白(ferritin,440kDa)、β-澱粉酶(β-amylase,200kDa)、胎牛血清白蛋白(bovine serum albumin,66kDa)及細胞色素C(cytochrome C,12.4kDa)溶於包含30mM Tris(pH 7.4)及150mM NaCl的緩衝液中。流速為每分鐘0.5毫升。由大腸桿菌製備之重組TDP-43先以0.2微米的過濾膜過濾,再將體積為300微升的過濾液注入Superdex-200管柱。利用分液收集器(fraction collector)自動收集分液,其中每管分液為1毫升。收集預載檢體及包含寡聚體的分液,並利用點漬法以A11抗體(1:1000)在不同的曝光時間下進行分析。藉由動態光散射來確認各寡聚體分液。由HEK細胞製得、體積為100微升,而濃度為2微莫耳的TDP-43亦同時以相同步驟進行檢測。進一步利用點漬法來分析各分液。 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.

隙縫點墨法(slot blotting) 由於濃度過低,故利用點漬法來分析由HEK293細胞分離,且經粒徑篩析層析處理的TDP-43。自每管1毫升的分液中,取200微升注入先以內部真空系統平衡的Bio-Dot SF微過濾儀(Bio-Rad,Hercules,CA,USA)。偵測時,一級抗體為抗-DDK單株抗體(OriGene Technologies,Inc.,Rockville,MD,USA)。訊號強度是利用Image J 1.42(National Institutes of Health,MD,USA)來進行定量。 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).

點漬法(dot blotting) 以10mM Tris-HCl 緩衝液在有或無變性劑的情況下,10倍稀釋純化的TDP-43。不同濃度的檢體分別包含無變性劑、含9M尿素、含7.2M脈鹽酸鹽或含2%十二烷基硫酸鈉的緩衝液。最終TDP-43的濃度為0.4μM。分別將檢體置於室溫或90℃處理1小時。將各TDP-43檢體,以2微升的體積點漬於硝化纖維膜上進行點漬法。簡單來說,經阻斷及以Tris為緩衝基底且包含0.002%妥文20(Tween 20)的生理食鹽水(即TBST)洗滌後,將膜分別與抗-N端殘基1-260之TDP-43抗體(1:2,000)、抗-C端殘基350-414之TDP-43抗體(1:2,000)及抗-類澱法蛋白寡聚體之抗體A11(1:1,000)進行反應,其中,該些抗體皆係溶於包含5%牛奶之TBST緩衝液中;之後再以與山葵過氧化酶(horseradish peroxidase,HRP)接合之二級抗體抗-兔子或抗-小鼠IgG(1:5,000,Millipore,Billerica,Massachusetts,USA)偵測之。最終利用ECL化學冷光試劑(ECL chemiluminescence reagent,Millipore)分析表現。 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).

穿透式電子顯微鏡(transmission electron microscope,TEM) 於4℃的環境下,將新鮮純化的TDP-43置於包含10mM Tris(pH 8)之緩衝液進行透析反應至隔日。再將檢體置於4℃,以17,000×g離心30分鐘去除沉澱物後,取上清液定量並進行穿透式電子顯微鏡分析。先將TDP-43檢體置於輝光-放電(glow-discharged)、400-篩孔福爾瓦碳包覆之銅柵(Formvar carbon-coated copper grids 400-mesh Formvar carbon-coated copper grids,EMS Inc.,Hatfield,PA,USA)5分鐘,清洗後再以2%醋酸鈾醯(uranyl acetate)進行負染。以Tecnai G2 Spirit TWIN TEM(FEI,Hillsboro,OR,USA)或Hitachi H-7000 TEM(Hitachi Inc.,Japan)在75kV加壓的環境下,檢測各檢體。 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).

原子力顯微鏡(atomic force microscope,AFM) 於4℃的環境下,將新鮮純化的TDP-43置於包含10mM Tris(pH 8)之緩衝液進行透析反應至隔日。再將檢體置於4℃,以17,000×g離心30分鐘去除沉澱物後,取上清液定量並進行原子力顯微鏡分析。將10微升的TDP-43滴於雲母片(Ted Pella,Redding,CA,U.S.A.)上,靜置5分鐘以使細胞貼附。以1毫升之二次水洗滌檢體,並將檢體由雲母片上小心取下。將檢體置於室溫直至乾燥,再以輕敲模式(tapping mode)進行原子力顯微鏡分析(Nanonics,Jerusalem,Israel)。本實驗是使用彈性常數為1.6N/m的PPP-ZEILR(Nanosensors,Neuchatel,Switzerland),且尖端半徑<10奈米的原子力顯微尖端來進行。 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.

動態光散射(dynamic light scattering,DLS) 將該些由粒徑篩析層析沖提之寡聚體分液進行動態光散射分析。首先,將檢體溶於包含30mM Tris(pH 7.4)及150mM NaCl的緩衝液中。以50mW後纖維平衡Zetasizer Nano ZS動態光散射儀(Malvern Instruments,Worcestershire,UK)後,進行檢體分析。依據各溶液設定適當的黏度及折射率,溫度則固定為25℃。 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.

旋光儀(circular dichroism,CD) 於4℃的環境下,將新鮮純化的TDP-43置於包含10mM Tris(pH 8)之緩衝液進行透析反應至隔日。再將檢體置於4℃,以17,000×g離心30分鐘去除沉澱物後,取上清液定量並進行旋光儀分析。在一圓形的石英電池(Hellma,Forest Hills,NY,USA)中,以設定1毫米路徑長度及室溫條件之Jasco J-815分光光譜儀(Jasco Inc.,Easton,MD,USA)來測量遠紫外光旋光儀光譜。收集250至190毫米的光譜,並以緩衝液背景值校正之。 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.

內源性及Bis-ANS螢光顯微鏡(intrinsic and Bis-ANS fluorescence spectroscopy) 於4℃的環境下,將新鮮純化的TDP-43置於包含10mM Tris(pH 8)之緩衝液進行透析反應至隔日。再將檢體置於4℃,以17,000×g離心30分鐘去除沉澱物後,取上清液定量並進行定量。以波長為280或295奈米的光源激發,收集1.5μM之TDP-43在波長為305至400奈米之間的內源性螢光。以波長為400奈米的光源激發,收集TDP-43、TDP-43s及緩衝液對照組在波長為450至600奈米之間的Bis-ANS光譜。該些檢體包含0.8μM的TDP-43及5μM的Bis-ANS。所有實驗皆是於溫度為25℃的循環式水浴槽,以FluoroMax-3光譜螢光計(Horiba Jobin Yvon,Kyoto,Japan)完成。 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).

硫代黃素T(Thioflavin T,ThT)結合 於4℃的環境下,將新鮮純化的TDP-43置於包含10mM Tris(pH 8)之緩衝液進行透析反應至隔日。再將檢體置於 4℃,以17,000×g離心30分鐘去除沉澱物後,取上清液定量並進行定量。以TDP-43檢體及Aβ40纖維進行硫代黃素T結合檢測。先將1μM的檢體與相同莫耳濃度的硫代黃素T混合,以波長為442奈米的光源激發後,收集波長為455至505奈米的發散光譜。依照前述方法來製備濃度為25μM的Aβ40纖維原液(Chen and Glabe,J.Biol.Chem.(2006)281,24414-24422)。簡單來說,將合成的Aβ溶於6M的脈鹽酸鹽中,再於10mM的磷酸鹽緩衝液(PBS,pH 7.4)重新折疊。再來,將25μM的Aβ於25℃的環境下,靜置超過10天。在進行實驗前,須先將成熟的纖維稀釋至1μM。將所得光譜扣除緩衝背景值。 The thioflavin T (ThT) was combined with a 4 ° C environment, 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.

剛果紅光譜(Congo Red spectroscopy) 加入10μM的剛果紅,並以紫光/可見光譜儀DU800(Beckman Coulter,CA)測量0.5μM之經透析的TDP-43及成熟的Aβ纖維於400至600奈米的吸光值。 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.

以OC抗體進行點漬法 將經透析的TDP-43及A β纖維(2微升,0.5μM)點漬於硝化纖維膜上,並利用標準點漬法實驗步驟進行後續分析。其中,實驗所使用的抗體分別為OC抗體(Millipore,1:10,000)及HRP接合之抗-兔子IgG(1:10,000)。 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).

以螢光滴定(Fluorescence titration)來偵測DNA結合 使用螢光滴定來偵測DNA結合所造成的蛋白構型改變。以單股TAR DNA來滴定新鮮純化且經透析、濃度為1.5μM的全長TDP-43及短式TDP-43(殘基 101-285),其中該單股TAR DNA包含TAR DNA A-位置(5'-CTTTTTGCCTGT-3')及B-位置(5'-TGGGTCTCTCTG-3')。以波長為280奈米的光源激發內源性蛋白螢光,並觀察TDP-43構型的改變。以FluoroMax-3光譜螢光計(Horiba Jobin Yvon,NJ,USA)收集發散光譜。收集全長或短式TDP-43於350奈米的發散光,扣除DNA對照組的數值後,以稀釋倍數校正之。之後,以起始強度標準化所得數值,再代入單一蛋白與配體(ligand)的結合公式(Chen et al.,Protein Sci(2004)13,2196-2206):P+L PL r=(2Pt)-1*[(Kd+Lt+Pt)-((Kd+Lt+Pt)2-4PtLt)1/2] Fluorescence titration was used to detect DNA binding . Fluorescence titration was used to detect changes in protein conformation caused 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 ]

其中,γ為所觀察的訊號改變,其係代表結合蛋白;P t 為總TDP-43濃度;L t 為總配體濃度;而Kd則為解離常數。依照DNA及TDP-43的比例來繪製結果。 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.

聚合物交聯及硫代黃素T試驗 依照先前流程來製備Aβ(Chen et al.,J.Biol.Chem.(2011)286,9646-9656;Ni et al.,FASEB(2011)25,1390-1401)。將Aβ40溶於包含8M脈鹽酸鹽的緩衝液A(10mM磷酸鈉,pH 7.4)中,進行再折疊,並以280奈米的吸光值進行定量(ε=1,280cm-1M-1)。以10mM的Tris-HCl(pH 8)及150mM的NaCl配製包含25μM之Aβ、50μM之硫代黃素T及不同濃度且經透析之TDP-43(濃度為0到1μM,即0-4%)的實驗檢體。將該些檢體靜置於ELISA之96-孔盤,並以透明薄膜密封,利用微盤分析儀 (SpectraMax M5,Molecule Devices)於25℃、不同時間點下進行測量。硫代黃素T是以442奈米激發,而以485奈米測量其發散光譜。 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.

光致交聯(Photo-induced cross-linking,PICUP) 本實驗依前述之步驟流程進行(Bitan et al.,J.Biol.Chem.(2001)276,35176-35184)。簡單來說,以包含8M尿素及10mM磷酸鈉的緩衝液來製備Aβ原液,以利後續SDS-PAGE分析。以包含10mM Tris-HCl(pH 8)及150mM NaCl的緩衝液,在包含或不包含不同濃度之TDP-43的情況下,配製濃度為25μM的Aβ檢體。立即進行光致交聯試驗分析。將90%之Aβ溶液與各5%的3mM Ru(Bpy)及20mM APS均勻混合。之後,將檢體置於一封閉的空間,以手動曝露於藍光發光二極體30秒。加入SDS-PAGE的檢體緩衝液可中止光致交聯反應,再以16%的Tris-tricine SDS-PAGE分析該檢體。所有的反應皆需立即性處理。利用可辨識Aβ殘基1-17之抗-Aβ抗體6E10(Chemicon Inc.,Billerica,MA)及抗-N端TDP-43(殘基1-260)之抗體,以西方墨點法來分析凝膠。 Photo-induced cross-linking (PICUP) This experiment was carried out according to the procedure described above (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.

TDP-43 對於人類神經母細胞瘤的細胞毒性 分別利用溴化噻唑藍四氮唑(MTT,3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide)及乳酸去氫酶(lactate dehydrogenase,LDH,Promega,Madison,WI,United States)試驗來檢測TDP-43對人類神經母細胞瘤BE(2)-C細胞株(美國菌種保存中心編號CRL-2268)的細胞毒性。將細胞培養於包含10% 胎牛血清(fetal bovine serum,FBS,Biological Industry)之RPMI生長培養液(Gibco)中,並置於37℃、5%CO2的潮溼環境中生長。將60,000細胞種植於透明的96-孔洞ELISA盤(Corning,NY,USA)中,並靜置至隔夜。洗滌細胞後,將培養液置換為40微升之無血清的RPMI培養液,接著加入10微升之以透析緩衝液連續稀釋的TDP-43檢體,該TDP-43檢體為新鮮透析及離心的檢體。收取上清液,並以透析緩衝液做為一緩衝液對照組。將細胞靜置24小時,再利用MTT試驗依照標準程進行分析。簡言之,將7微升之濃度為每毫升5微克的MTT加入各孔洞,靜置3小時。去除培養液後,加入二甲基亞碸(dimethyl sulfoxide,DMSO)溶解細胞,直到結晶紫染劑完全溶出。以ELISA盤分析儀(SpectraMax M5,Molecule Devices,USA)測量570到690奈米的吸光值,再將690奈米的讀值訊號減去570奈米的讀值訊號。平均五次實驗結果,並以不含細胞的背景值予以校正。細胞存活率是將投予緩衝液之細胞讀值設為100%後,進行標準化的計算結果。LDH試驗則是依照說明書指示步驟進行。簡單來說,將20,000細胞種植於生長培養液中,靜置24小時。細胞的處理方法如MTT試驗所述。將LDH受質與試劑緩衝液均勻混合後,放置於室溫。將細胞冷卻至室溫30分鐘,再加入受質反應。避光1小時,以560奈米激發受質螢光,並以ELISA盤分析儀讀取590奈米的發散值。取三次實驗結果平均,並以不含細胞的背景值予以校正。MTT及LDH試驗的p-值皆是以不成對之Student’s t-test計算。 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.

TDP-43 對小鼠初代皮質神經元的毒性 由國家實驗動物中心(NLAC,台灣)購買懷孕的C57BL/6JNarl小鼠,相關動物實驗皆是經過台灣台北國立陽明大學之實驗動物照護及使用委員會(Institutional Animal Care and Use Committee,IACUC)許可後進行。自第19天的小鼠胚胎取出初代皮質神經元,以每孔洞2 x 105細胞的細胞量種植於96-孔盤,並培養於包含2μM之FdU的神經基底培養液(21103049,GIBCO,USA)中。於活體外生長8天後,投予初代皮質神經元新鮮透析的TDP-43,靜置24小時,再加入每毫升0.5毫克的MTT,靜置3小時。透析緩衝液係為本實驗的緩衝液對照組。加入與MTT試劑等量之包含10%十二烷基硫酸鈉及20mM HCl的溶解緩衝液,靜置至隔日,使MTT的結晶紫染劑可以完全溶解。以ELISA盤分析儀(TECAN,Switzerland)測量各孔洞570奈米的吸光值。取三次實驗結果平均,並以不含細胞的背景值予以校正。MTT試驗的p-值是以不成對之Student’s t-test計算。 Toxicity of TDP-43 to primary cortical neurons in mice The pregnant C57BL/6JNarl mice were purchased from 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.

免疫細胞化學染色(Immunocytochemistry) 將初代神經元細胞培養於一置於24-孔盤且以聚-D-離胺酸(poly-D-lysine)塗佈之12毫米蓋玻片上(每孔洞含3×105細胞)。於37℃、5%CO2的潮溼環境,以TDP-43(0.6μM)或緩衝液對照處理細胞24小時後,以4%之三聚甲醛(paraformaldehyde)於室溫固定細胞20分鐘。之後,以PBS洗滌細胞3次,每次10分鐘,再以包含0.3%采酮x-100(triton x-100)及10%胎牛血清之阻斷緩 衝液於室溫處理1小時。接著,以抗-微管相關蛋白-2(microtubule associated protein-2,MAP-2,MAB378,Millipore,USA)抗體及兔子抗-膠質纖維酸性蛋白(glial fibrillary acidic protein,GFAP,Z0334,DAKO,Glostrup,Denmark)抗體於室溫處理2小時,再以接有德克薩斯紅(Texas-Red)之山羊抗-小鼠IgG二級抗體(AbD Serotec,UK)或接有螢光異硫氰酸鹽(Fluorescein isothiocyanate,FITC)之山羊抗-小鼠IgG二級抗體(Millipore,USA)於室溫偵測1小時。最後,以包含DAPI的包埋膠(VECTASHIELD®+DAPI,H-1200,Vector Lab.)包埋細胞,再利用Olympus螢光顯(BX-61,Olympus)微鏡觀察染色結果。 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.

將TDP-43注入至海馬迴組織 由台灣台南國立成功大學購入並於該校飼養C57/BL6J小鼠,在該校之實驗動物照護及使用委員會規範下,將不同藥劑投予至2個月大之C57/BL6J小鼠的海馬迴中。首先由鼻管投予小鼠異氟烷吸入性麻醉劑(氧氣之1.2%)。將重組全長之TDP-43投予至各小鼠雙側的海馬迴組織中。相對於前囟(bregma)的立體定向座標為前後側(anteroposterior,AP)-2mm、中側(mediolateral,ML)±1mm及背側(dorsoventral,DV)-2mm。將注射針頭緩慢地刺入特定深度後,以帶有一微量注射器(Hamilton Company,NV,USA)的不鏽鋼注射針將2微升的TDP-43(2.2μM)以每分鐘1微升的速度注入。注射完後將針頭放置5分鐘後再抽出,以防包含TDP-43的注射液滲出。 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.

小鼠腦部的免疫螢光染色 將20週大的雄性野生型小鼠,以及6及12個月大的TDP-43基因轉殖小鼠(TDP-43 Tg+/+)進行免疫螢光染色。所有的小鼠皆飼養於國立成功大學,且實驗流程係依照該校實驗動物照護及使用委員會的規範來進行。在投予TDP-43兩週後,將小鼠深層麻醉,再以溶於0.01M之PBS(pH 7.4)的4%三聚甲醛由心臟進行灌流。取出腦組織並將其置於30%蔗糖/4%PFA溶液至隔日。將腦組織細切成厚度為10微米的薄片,再以阻斷緩衝液於室溫作用1小時,該阻斷緩衝液是將0.2%采酮x-10及5%正常驢血清溶於0.01M PBS調配而成。在進行海馬迴組織注射實驗時,將腦組織置於小鼠單株抗-NeuN之抗體(1:300,Millipore,Temecula,CA)中,於4℃放置至隔日;再以與Alexa Fluor 555螢光染劑接合之驢抗-小鼠抗體(1:300,Chemicon,Temecula,CA)於室溫作用1小時。最後以DAPI複染該腦組織,並以包含螢光包埋介質(DAKO,Glostrup,Denmark)進行包埋。利用正立螢光顯微鏡(BX51,Olympus,Tokyo,Japan)來檢測各腦組織的染色結果。在進行基因轉殖鼠之共存研究時,將腦組織切片分別置於抗-TDP-43抗體(1:1000,Abcam,ab104223)、TDPO(1:1000,LTK BioLaboratories,Taiwan)、與Alexa Fluor 488螢光染劑接合之山羊抗-兔子抗體及與Alex Fluor 555螢光染劑接合之驢抗-小鼠抗體(1:300,Invitrogen)中。將該腦組織置於DAPI染劑中,最後以包含螢光包埋介質(DAKO,Glostrup,Denmark)封片。利用雷射掃描共焦顯微鏡來(Nikon TE2000EPFS-C1-Si)檢測各腦組織的 染色結果。 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).

製備並確認對TDP-43寡聚體具有專一性之多株抗體 在4℃的環境下,以10mM Tris-HCl(pH 8.0)透析經純化之全長TDP-43,並將所得產物進行離心,以製備最終濃度約為每毫升0.2毫克的免疫原蛋白。以該免疫原蛋白免疫注射紐西蘭白兔,並以標準製備流程(台灣LTK BioLaboratories)來製備多株抗體。簡單來說,免疫原蛋白是以2週的間隔時間,每次以0.5毫升的體積注入至白兔體內。經6次注射後,犠牲白兔,取出其血清。為以點漬法及ELISA分析確認TDP-O,將全長的TDP-43注入粒徑篩析層析(Superdex-200 10/300 GL,GE healthcare Bio-Sciences AB,Uppsala,Sweden)處理,其中該緩衝液包含30mM之Tris-HCl(pH 8.0)及150mM之NaCl,流速則為每分鐘0.3毫升。以1毫升為單位體積收集沖提液,將利用TDP-O及N1-260抗體進行點漬法分析。另外以微BCATM蛋白分析套組(Micro BCATM Protein Assay Kit,Thermo Scientific,Rockford,IL)定量TDP-43寡聚體及單體後,將其以連續稀釋法塗佈於ELISA盤。利用標準流程來進行ELISA分析。簡言之,將塗佈的檢體置於4℃至隔日,再於室溫以包含10%脫脂牛奶的TBST處理2小時。洗滌ELISA盤後,利用TDP-O(以1:12,500的比例溶於包含3%脫脂牛奶的TBST中)或抗-N端殘基1-260的TDP-43抗體(以1:1,000的比例溶於包含3%脫脂牛奶的TBST中)於室溫偵測2小時。洗滌後,再以與HRP接合之抗-兔子抗體(1:1,000,Merck Millipore,Billerica,Massachusetts,USA)於室溫偵測2小時,經再次洗滌後,以100毫升之3,3,5,5-四甲基聯苯苯胺(3,3,5,5-tetramethylbenzidine,TMB,Merck Millipore,Billerica,Massachusetts,USA)呈色反應。以100毫升之250mM HCl中止反應,再利用SpectraMax M5(Molecular Device,Sunnyvale,CA)於450奈米的波長下讀取檢體的吸光值。 Preparation and confirmation of multiple strains of antibodies specific for TDP-43 oligomers Purified full-length TDP-43 was dialyzed against 10 mM Tris-HCl (pH 8.0) at 4 ° C, and the resulting 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.

製備β5纖維 利用化學方法(MDBio,Inc.,USA)來合成位於TDP-43片段之RRM2區的β5胜肽。將該胜肽溶於乙腈後,以PBS緩衝液(pH 7.4)將起始濃度為每毫升5毫克的胜肽進行十倍稀釋。以17,000×g的轉速離心30分鐘,收取上清液並利用0.2微米的過濾膜(Pall,USA)過濾。之後,將檢體靜置於室溫7天。利用穿透式電子顯微鏡觀察形成的β5纖維。依前述方法以TDP-O抗體進行點漬法來分析各檢體。 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.

FTLD-TDP病患之腦組織的TDP-O染色 以二甲苯洗滌以石臘包埋的腦組織切片三次進行脫臘。利用不同濃度的酒精水合該腦組織切片後,以1倍TBS洗滌5分鐘。將切片組織置於檸檬酸鈉(pH 6)以全火力(1,500瓦)微波15分鐘,以恢復抗原表現。將組織置於包含3%過氧化氫之1倍TBS,作用20分鐘,藉以阻斷內源性過氧化酶的活性;接著再以1倍TBS洗滌5分鐘。將組織置於10%正常山羊血清(Invitrogen),於室溫下作用1小時以阻斷非專一性結合。加入以1:1000比例稀釋於抗體緩衝液(包含5%胎牛血清白蛋白及0.5%妥文 -20之1倍TBS)的一級抗體,並於4℃作用至隔日。以洗滌緩衝液(包含0.5%妥文-20之1倍TBS)洗滌檢體3次。將帶有生物素標記之二級抗體以1:500的比例稀釋於抗體緩衝液後,加入檢體,於室溫作用1小時。以洗滌液洗滌檢體三次,再將檢體置於Vectastain ABC過氧化酶試劑(Vectastain ABC peroxidase reagent,Vector Labs,PK-6100)30分鐘後,以1倍TBS洗滌一次。加入DAB過氧化酶受質試劑(DAB peroxidase substrate reagent,Vector Labs,SK-4100)2-10分鐘,以達到最佳的染色效果。以梅爾氏蘇木精溶液(Mayers Hematoxylin Solution,Sigma Aldrich)進行複染,再以不同濃度的酒精及二甲苯進行脫水反應。以Permount(Sigma Aldrich)包埋脫水後的組織切片。由加州戴維斯分校之阿滋海默中心所提供之取自3組對照、3位罹患FTLD-TDP病患及3位罹患阿滋海默症病患的腦部檢體亦同步進行分析。本實驗是經機構審查委員會之認證許可,且取得各病患之知情同意書。 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.

FTLD-TDP腦部之TDP-43寡聚體的免疫沉澱及免疫標記 以每毫升約0.05克的溶解緩衝液溶解取自2對照及1罹患FTLD-TDP病患的冷凍腦組織,該溶解緩衝液包含50mM Tris(pH 7.4)、150mM NaCl、0.5%妥文X-100及蛋白酶抑制劑(Calbiochem,Merck Millipore)。以組織研磨儀(Wheaton,NJ,USA)於冰上均質化溶解的檢體。之後,以17,000 xg的轉速於4℃離心10分鐘,收集可溶部分進行免疫沉澱分析。在進行組 織分層前,依照使用說明書將TDP-O抗體交聯至免疫沉澱微珠(bead)。簡單來說,將40微克的TDP-O抗體加入包含10微升蛋白A-及10微升蛋白G-Mag SepharoseTM Xtra(GE healthcare)的結合緩衝液(50mM Tris,pH 7.5,150mM NaCl),於室溫靜置1小時。在抗體結合至微珠後,加入包含50mM之庚二亞氨酸二甲酯二鹽酸鹽(dimethyl pimelimidate dihydrochloride)及200mM之三乙醇胺(triethanlamine,pH 8.9)的交聯試劑,於室溫作用1小時,以進行交聯反應;加入100mM的乙醇胺(pH 8.9)於室溫作用30分鐘以中止反應。接著,將海馬迴組織之可溶部分及與TDP-O抗體交聯之免疫沉澱微珠於室溫反應1小時。以結合緩衝液至少洗滌6次反應物,藉以移除未結合的蛋白成份。以緩和抗原/抗體沖提緩衝液(Gentle Ag/Ab Elution Buffer,Thermo,pH 6.6)沖提標的蛋白,再利用電子顯微鏡及免疫金標記來分析沖提產物。在進行免疫金標記時,將10微升的沖提產物置於400-篩孔福爾瓦碳包覆之銅柵(400-mesh Formvar carbon-coated copper grids,EMS Inc.,Hatfield,PA,USA)5分鐘,以PBS洗滌後,加入包含1%BSA的PBS,於室溫作用1小時。之後,以包含0.1%BSA及N1-260抗體(1:5,000,ab57105,Abcam)的PBS於室溫反應1小時,以標記該銅柵;接著以包含50mM之Tris(pH 7.5)、500mM之NaCl及0.1%之Tween-20的高鹽妥文緩衝液(high salt tween buffer,HST buffer)及PBS洗滌該銅柵。而後將該銅柵置於6奈米、以金接合之二級抗-小鼠IgG抗體(1:40,Jackson ImmunoResearch),並於室溫反應1小時。以HST 及PBS洗滌銅柵以移除未結合之抗體。利用包含1%戊二醛之PBS於室溫作用10分鐘以固定該銅柵後,以二次水洗滌6次。最後,加入2%醋酸鈾醯(uranyl acetate)進行負染色(negatively stained)後,利用穿透式電子顯微鏡(FEI Tecnai G2 F20 S-TWIN TEM)觀察染色結果。 Immunoprecipitation and immunolabeling of TDP-43 oligomers in the FTLD-TDP brain were lysed from approximately 2 g of lysing buffer per ml of frozen brain tissue from 2 controls and 1 patient with FTLD-TDP, the lysis buffer Contains 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% toxic X-100, and 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.

純化單株TDP-O抗體 單株TDP-O抗體係利用蛋白G瓊脂糖套組(protein G agarose kit,KPL)依照使用說明書進行純化。簡單來說,將混合於20%乙醇之蛋白G瓊脂糖(1.5毫升)注入一可拋棄式管柱。以10毫升之包含0.1M磷酸鈉(pH 7.4)及0.15M氯化鈉的洗滌緩衝液洗滌管柱中的樹脂。利用結合緩衝液(5毫升)稀釋取自TDP-O融合瘤細胞之條件培養液(5毫升)後,將該混合液注入管柱,並倒置管柱使混合液均勻混合。之後,以5毫升洗滌緩衝液洗滌管柱4次,直到OD280區近於0。加入1毫升之包含0.2M甘胺酸(pH 2.85)的沖提緩衝液,緩慢地沖提抗體,並收集於含有240微升之5倍洗滌液的收集管中。濃縮收集之抗體,利用Amicon Ultra-4 30kDa cutoff(Millipore)將緩衝液置換為洗滌緩衝液。利用抗體於280奈米之吸光值來定量抗體濃度(1毫克抗體的吸光值為1.34)。 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).

間接ELISA 將20奈克(nanogram,ng)之全長TDP-43寡聚體蛋白溶於200微升之包含50mM Tris(pH 7.4)及150mM NaCl的TBS溶液後,塗佈於ELISA 96-孔洞盤(Nunc MaxiSorp),並置放於4℃至隔日。移除塗佈溶液,分別加入有或無包含0.2%之十二烷基硫酸 鈉的TBS緩衝液,置於室溫1小時。以十二烷基硫酸鈉處理之TDP-43寡聚體將會變性,而以不含十二烷基硫酸鈉之緩衝液處理的TDP-43寡體則會維持原寡聚體之構型。處理後,移除緩衝液,並加入200微升之包含10%脫脂牛奶的TBST緩衝液(20mM Tris,pH 7.6,137mM NaCl,0.001%Tween 20),於室溫作用2小時。利用PBST(每孔洞300微升)洗滌孔洞3次,再加入100微升之以包含5%脫脂牛奶之TBST稀釋的mTDP-O抗體(稀釋濃度由每毫升0.00001至2微克),置於室溫1小時。以300微升之PBS洗滌2次後,藉由100微升之與HRP接合的抗-小鼠抗體偵測結合抗體的表現,其中該抗體是以1:1,000的比例稀釋於包含5%脫脂牛奶的TBST溶液。室溫作用1小時後,以300微升之PBS洗滌2次,再加入100微升之3,3,5,5-四甲基聯苯苯胺(3,3,5,5-tetramethylbenzidine,TMB,KPL SureBlue),於室溫反應10分鐘。加入100微升之250mM的HCl來中止反應,並以SpectraMax M5(Molecular Devices)讀取波長450奈米時的光密度(optical density,OD)。 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).

製備TDP-43寡聚體及單體以進行間接ELISA試驗 以Amicon Ultra-4 30kDa cutoff將每毫升180微克的TDP-43蛋白進行3倍濃縮(由3毫升濃縮至1毫升)。將500微升之濃縮TDP-43蛋白液注入Superdex 200 10/300 GL管柱,並以包含30mM Tris(pH 8.0)及50mM NaCl的沖提緩衝液以每分鐘0.3毫升的流速進行沖提。以500微升為單位,分別收集包含TDP-43寡聚體 及單體的沖提液。利用微BCA蛋白分析套組(Thermo)定量蛋白濃度。取300奈克之TDP-43單體或寡聚體,將其塗佈於ELISA之96-孔洞盤(Nunc MaxiSorp)。依上述之間接ELISA試驗,以100微升之取自TDP-O融合細胞的調件培養液(稀釋比例為1:1,000)來偵測TDP-43寡聚體的表現。 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.

確認抗體的同型(Isotypes) 藉由一用於ELISA試驗之小鼠單株抗體同型套組(Thermo)來確認mTDP-O的同型。在該試驗中,分別將抗-小鼠重鏈之捕捉抗體(capture antibody,抗-IgG1、IgG2a、IgG2b、IgG3、IgA及IgM)或抗-小鼠輕鏈之補捉抗體(κ或λ)塗佈於ELISA盤。簡言之,將取自TDP-O的調件培養液以50倍比例稀釋於TBS溶液中,將稀釋好的檢體加入ELISA盤。之後,加入50微升之以HRP接合的抗-小鼠IgG+IgA+IgM抗體,於室溫反應1小時。經3次洗滌後,每孔洞加入75微升之TMB受質,於室溫避光反應10分鐘。接著,加入75微升之0.18M硫酸以中止反應。藉由SpectraMax M5(Molecular Devices)讀取各孔洞於波長450奈米時的吸光值。 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).

將Ig變異區基因進行轉殖及定序 以GeneJET RNA純化套組(GeneJET RNA purification kit,Thermo)萃取2×106融合瘤細胞的RNA。利用極大第一股cDNA合成套組(Maxima First Strand cDNA synthesis kit,Thermo)合成cDNA。之後,藉由小鼠Ig-引子組(Ig-Primer Sets,Novagen)及GoTaq® G2綠色擴增反應 混合劑(GoTaq® G2 Green Master Mix,Promega)來放大Ig重鏈及κ基因。將所得之放大產物以TOPO® TA Cloning®套組(Invitrogen)進行轉殖。最終產物的DNA序列是以Mission Biotech進行定序。 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.

實施例1 全長TDP-43易於形成寡聚體Example 1 Full-length TDP-43 is easy to form oligomers

在本實施例中,將探討TDP-43的病理機制。依照「材料及方法」所述之步驟,分別由大腸桿菌及HEK293細胞純化出重組之全長人類TDP-43(其N端帶有一組胺酸標誌,分子量為MW 47,145Da)及TDP-43蛋白。將所得之TDP-43蛋白進行粒徑篩析層析分析。如第1a圖所示,沖提體積包含至少86%的沖提TDP-43,而隙縫點墨法則證實兩種來源之重組全長TDP-43蛋白皆易於形成寡聚體。有鑑於TDP蛋白病變與包涵體(inclusion body,IB)的形成相關,而重組全長TDP-43會形成高分子量寡聚體,故推測TDP-43可能會形成與澱粉樣變性病(amyloidosis)之類澱粉寡聚體相似的寡聚體。因此,利用一種針對Aβ寡聚體擬態所製備,且與構型相關之抗-類澱粉專一抗體(即A11)來檢測TDP-43寡聚體;結果顯示兩種TDP-43蛋白檢體皆能與A11產生免疫反應,而其相對應之緩衝液則不會(第1b圖)。包含稀釋TDP-43的寡聚體分液具有較弱的訊號強度。 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.

為確認該抗體及抗原間的辨識是與構型相關,在利用A11、抗-N端之TDP-43抗體及抗-C端之TDP-43抗體進行點漬法分析前,先以不同變性方法來破壞TDP-43寡聚體的構型(第1c圖)。將蛋白培養於不同的緩衝液, 即,有或無9M尿素、7.2M脈鹽酸鹽或2%十二烷基硫酸鈉的緩衝液,再置於90℃處理1小時。發現在偵測A11的表現時,加熱與否並不會顯著地影響原緩衝液之訊號量。然而,加入高濃度之尿素或脈鹽酸鹽會減少偵測的訊號量,再加熱處理則會大幅減少訊號量。不論加熱與否,加入2%十二烷基硫酸鈉皆無法偵測到任何訊號。相對地,抗-N端之抗體不論在何處理下,皆能辨識TDP-43的表現;此結果亦印證了點漬於膜上的蛋白係為等量。加入尿素並加熱處理或加入脈鹽酸鹽(不論加熱與否)皆會部份或完全減少抗-C端抗體對於抗原的辨識。該些結果指出,抗-C端之TDP-43抗體對於抗體的辨識度會隨著不同變型條件而改變。 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.

整體來說,上述結果指出重組之全長TDP-43易於形成高分子量蛋白;該些高分子蛋白與類澱粉寡聚體具有共同的抗原決定位置。該些高分子蛋白對十二烷基硫酸鈉以外的化學變型處理皆相當穩定。 Overall, the above results indicate that recombinant full-length TDP-43 is prone 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.

實施例2 全長TDP-43會形成非均質之球形寡聚體Example 2 Full-length TDP-43 forms heterogeneous spherical oligomers

在本實施例中,利用穿透式電子顯微鏡(transmission electron microscope,TEM,第1d圖)及原子力顯微鏡(atomic force microscope,AFM,第1e圖)來觀察寡聚體的形態。穿透式電子顯微鏡顯示該些非均質之球形蛋白具有數個球形及環形的結構特徵,而原子力顯微鏡則顯示多數球形蛋白呈現球形結構特徵,僅有極少數具有環形的結構特徵(第1e圖之插圖)。利 用穿透式電子顯微鏡來計算顆粒大小分佈。多數的球形顆粒具有約40到60奈米的粒徑(結果未顯示)。經原子力顯微鏡分析,環形寡聚體的高度約為6奈米(結果未顯示)。此外,經動態光散射(dynamic light scattering,DLS)分析顯示,由粒徑篩析層析所得之寡聚體蛋白係呈現一非均質的分佈,其顆粒的粒徑大小約為40到400奈米(第1f圖)。大多數的顆粒粒徑約為50到60奈米(第1g圖),穿透式電子顯微鏡亦呈現相同的分析結果。若結合影像及粒徑分佈結果可知,全長TDP-43會形成具有球形超結構的非均質顆粒。該些結果符合之前的研究觀察,即野生型TDP-43及與肌肉萎縮性脊髓側索硬化症相關的TDP-43突變皆會形成寡聚體(Johnson et al.,J.Biol.Chem(2009)284,20329-20339)。整體而言,本研究結果指出,TDP-43蛋白不論是在結構上或是免疫分析上,皆與球形類澱粉寡聚體相似,而該球形類澱粉寡聚體係具有神經毒性,且會表現於不同的神經退化性疾病。 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.

實施例3 TDP-43寡聚體具有明確的構型及功能Example 3 TDP-43 oligomer has a clear configuration and function

為進一步檢測TDP-43寡聚體的構型,利用遠紫外光旋光儀光譜來分析TDP-43寡聚體的二級結構,結果顯示於第2a圖。分別於接近210及222奈米出現可能為α-螺旋結構的二個極小值。該光譜分析與短式小鼠TDP-43(殘基101-265,圖中簡記為TDP-43s)的光譜分析不同,其中該短式小鼠TDP-43在晶體結構上包含二個多為β-股的RRM(Kuo et al.,Nucleic Acids Res(2009)37, 1799-1808)。由內源性芳香族殘基激發所得之螢光光譜顯示,在光波約為340奈米時可偵測到發散最大值,該結果指出TDP-43之酪胺酸及色胺酸殘基為溶劑暴露殘基(結果未顯示)。此外,亦利用外源性螢光染劑Bis-ANS來偵測該些暴露之疏水性蛋白表面,其中螢光染劑Bis-ANS為一種常用以偵測蛋白折疊時部分尚未折疊之中間產物的染劑。如第2b圖所示,相較於短式TDP-43,全長TDP-43會與螢光染劑Bis-ANS反應而產生更高的螢光量,顯示全長TDP-43寡聚體具有與短式TDP-43不一樣的疏水性暴露表面。 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.

另外,亦使用傳統類澱粉染劑硫代黃素T(thioflavin T,ThT)來檢測全長TDP-43是否會與硫代黃素T結合,結果顯示於第2c圖。不論是全長或短式TDP-43,皆偵測不到硫代黃素T結合所發散之螢光發散波峰。相較之下,相同濃度的Aβ纖維(1μM)則具有顯著的螢光發散(第2c圖),此結果與以硫代黃素T分析之病理檢驗結果相符合。相似地,TDP-43寡聚體亦不會與剛果紅及抗-纖維抗體OC結合反應(結果未顯示)。該些結果指出,TDP-43寡聚體應無與β褶板(β sheet)交聯的結構。然而,關於原子能階的結構尚需進一步研究確認。 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 sheet) structure. However, the structure of the atomic energy level needs further research and confirmation.

有鑑於正常且具有功能性之TDP-43會與一特殊的核酸序列結合,接下來將探討TDP-43寡聚體是否會干擾DNA的結合能力。利用螢光滴定來觀察蛋白與DNA結合後之構型改變,結果顯示於第2d圖。簡單來說,以單股TAR DNA-A位或-B位滴定分析TDP-43寡聚體,並將 所得結果與短式TDP-43的滴定分析結果比較,已知短式TDP-43會以亞微莫耳(submicromolar)的親和度與單股DNA結合(Kuo et al.,Nucleic Acids Res(2009)37,1799-1808)。如第2d圖所示,在低單股DNA濃度時,TAR DNA-A位及-B位皆會減少短式TDP-43的螢光量,而加入全長TDP-43則會顯著地降低該反應。該結果符合「材料與方法」所述之單一抗體與配體的結合公式,並指出TDP-43及單股DNA的化學計量係為1:1。由該公式可得出全長TDP-43與TAR-A、全長TDP-43與TAR-B、短式TDP-43與TAR-A及短式TDP-43與TAR-B的解離常數(dissociation constant,Kd)分別為7.05±0.82、6.27±0.68、0.20±0.09及0.38±0.10μM。該些結果指出,單股DNA與短式TDP-43的結合強度約為與全長TDP-43結合強度的15倍,其中TAR-A及-B位對於全長TDP-43具有相似的親和力。該些發現顯示,全長TDP-43寡聚體的RRMs可能具有不正常的構型,因而造成與DNA結合能力的下降,或是與DNA結合的區域受到遮蔽而無法與DNA結合。全長TDP-43之發散值的改變有可能是因檢體中存有少量的TDP-43單體所致。總結來說,該些結果證實TDP-43寡聚體具有與短式TDP-43不同的生物物理及生物化學特性,亦即,二者的構型不相同。 Given that normal and functional TDP-43 will bind to a particular nucleic acid sequence, it will be explored 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.

實施例4 TDP-43寡聚體將Aβ轉換為類澱粉蛋白寡聚體Example 4 TDP-43 oligomer converts Aβ to amyloid-like oligomers

在本實施例中,將以交聯實驗來探討TDP-43是否會影響Aβ纖維化反應。 In this example, cross-linking experiments will be conducted to investigate whether TDP-43 affects Aβ fibrosis.

首先,利用硫代黃素T試驗來檢測在有或無0.4到4%之TDP-43寡聚體時,Aβ40的纖維化反應,結果顯示於第3a圖。分析指出,TDP-43可有效抑制Aβ的纖維化反應,且該抑制效果會隨著TDP-43濃度的增加而增加(第3a圖)。在將近180小時的實驗觀察期中,4%的TDP-43可完全抑制Aβ的纖維化反應。再利用光致交聯反應(Photo-induced cross-linking,PICUP)來檢驗在實驗初始時,短暫出現的Aβ蛋白;結果指出在交聯後,Aβ主要會形成單體、雙聚體、三聚體及四聚體,而TDP-43寡聚體會造成具有更高分子量之Aβ蛋白產生(第3b圖)。加入TDP-43可觀察到Aβ五聚體,且表現量係與TDP-43加入的量呈正常相關。另外,亦觀察到二個具有較大分子量的組合,分別位於~55kDa及分佈於~105至>210kDa之間。除了對十二烷基硫酸鈉不具抗性的TDP-43單體,亦發現一分子量約~55kDa蛋白及數個分佈於~80至>210kDa的蛋白。若進一步利用穿透式電子顯微鏡分析可發現,在加入4%的TDP-43後,Aβ並不會形成纖維,而是會變性為一具有<10奈米粒徑的球形寡聚體;該Aβ在不含TDP-43仍會形成成熟的類澱粉纖維(第3c圖)。TDP-43寡聚體交聯具有>50奈米的粒徑,該粒徑大於Aβ寡聚體的粒徑大小(資料未顯示)。該些數據顯示TDP-43寡聚體可以引發Aβ寡聚體的形成,且TDP-43與類澱粉蛋白具有共同的特性。 First, the thioflavin T test was used to detect the fibrosis reaction of Aβ40 with or without 0.4 to 4% of TDP-43 oligomers, and the results are 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.

實施例5 TDP-43寡聚體引發軸突退化及毒性Example 5 TDP-43 oligomers induce axonal degeneration and toxicity

在本實施例中,將進一步探討TDP-43寡聚體是否會造成神經毒性及軸突之退化。 In this example, it will be further explored whether TDP-43 oligomers cause neurotoxicity and axonal degeneration.

簡單來說,以連續稀釋之主要包含寡聚體的TDP-43檢體來處理人類神經母細胞瘤BE(2)-C細胞,再以MTT及LDH試驗來檢測所產生的細胞毒性;結果顯示於第4a到4c圖。MTT試驗指出,相較於緩衝液對照組,加入0.44μM的TDP-43會減少約20%的細胞存活率(第4a圖)。有鑑於重組TDP-43的低溶解度,無法以更高濃度之TDP-43進行該毒性試驗。LDH試驗亦呈現類似的結果,加入0.44μM的TDP-43會顯著地引發細胞死亡(第4b圖)。TDP-43亦會於小鼠的初代皮質神經元產生與劑量呈正相關的神經毒性,其中,投予0.6μM的TDP-43會減少約20%的細胞存活率(第4c圖)。 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).

此外,該些小鼠皮膚神經元的免疫組織化學染色法顯示,0.6μM的TDP-43會造成軸突的萎縮,並減少神經元的密度及數量(第4d圖)。為進一步測試TDP-43寡聚體於活體內的神經毒性,將2微升之2.2μM的TDP-43注射至小鼠的海馬迴組織,再利用免疫螢光染色分析神經元的存活。以神經元特定標誌NeuN及核特定染劑DAPI進行免疫組織化學染色可發現,相較於以緩衝液處理之小鼠,接受TDP-43注射之小鼠的CA1層中具有較少量的神經元(第4e圖)。該結果指出,注射全長的TDP-43寡聚體至海馬迴會引起細胞毒性。 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.

實施例6 製備及確認對TDP-43寡聚體具有專一性的多株抗體Example 6 Preparation and Confirmation of Multiple Antibodies Specific to TDP-43 Oligomers

有鑑於抗-類澱粉蛋白寡聚體抗體A11會交聯至不同的類澱粉蛋白,依照「材料及方法」所述之方法,利用重組TDP-43寡聚體做為免疫原後,免疫刺激兔子以產生一多株抗體TDP-O,藉以了解TDP-43寡聚體是否會影響疾病的產生。 In view of the fact that the anti-amyloid-like oligomeric antibody A11 is cross-linked to different amyloid-like proteins, the immune-stimulated rabbit is obtained by using the recombinant TDP-43 oligomer as an immunogen according to the method described in "Materials and Methods". To generate a multi-drug antibody TDP-O, to understand whether TDP-43 oligomers will affect the disease.

依照「材料及方法」所述之方法,在不同的變性調件下,利用點漬法來分析TDP-O抗體對全長TDP-43寡聚體構型的專一性;結果顯示於第5a圖。不論加熱(90℃,1小時)與否,以1:125,000比例稀釋於原緩衝液的TDP-O皆可與全長TDP-43反應;然而,2%十二烷基硫酸鈉會破壞抗體與TDP-43寡聚體間的反應,而不論是否予以加熱處理。另外,加入7.2M之脈鹽酸鹽或9M之尿素,並於室溫作用超過1小時,並不會影響全長TDP-43的反應,該結果指出在高濃度化學變性劑的處理下,蛋白依舊相當穩定;然而,額外予以加熱處理則會減少反應訊號量,亦即,TDP-O為一構型相關的抗體。總結上述,TDP-O的點漬法定性分析結果與A11一致(第5a圖與第1c圖)。 The specificity of the TDP-O antibody to the full-length TDP-43 oligomer configuration was analyzed by spotting method under different denaturation conditions according to the method described in "Materials and Methods"; 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).

利用A11及/或TDP-O抗體來檢測抗體對A β寡聚體的專一性,以了解TDP-O是否為一對TDP-43具有專一性的抗體,而非如A11為一對類澱粉寡聚體具有廣用性的抗體。如第5b圖所示,TDP-O無法辨識A β寡聚體,證明了該抗體為對TDP-43寡聚體具有專一性之抗體。亦利用ELISA來定量分析TDP-O抗體與TDP-43寡聚體及單體間的反應;在這個部份,會辨識N端殘基 1-260(N1-260)的TDP-43抗體亦同時進行試驗分析,做為比對之用。將約1μM的全長TDP-43或3倍濃縮之TDP-43檢體進行粒徑篩析層析,並收集1毫升沖提液,藉此製備全長TDP-43的寡聚體及單體(第5c圖)。取第1至18分液中的濃縮TDP-43,以TDP-O或N1-260抗體進行點漬法分析,結果繪示於第5d圖。該結果顯示,TDP-O對於第5至7分液中的濃縮TDP-43具有較強的反應,該些檢體乃係對應於沖提至沖提體積中的TDP-43寡聚體。相較之下,N1-260則是對於第6分液及第15至18分液中的濃縮TDP-43具有較強的反應;亦即,該抗體可辨識TDP-43的寡聚體及單體。 Use A11 and/or TDP-O antibodies to detect the specificity of antibodies to 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 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.

此外,利用ELISA來定量TDP-43寡聚體與TDP-O的專一性,N1-260抗體在此做為對照組。在以微BCA試驗定量後,將連續稀釋之包含TDP-43寡聚體及單體的分液分別塗佈至ELISA盤。依ELISA的標準操作流程,利用TDP-O及N1-260抗體進行偵測。第5e圖的結果顯示,相較於TDP-43單體,TDP-O抗體對TDP-43寡聚體具有較高的反應度(EC50<每毫升0.5微克);而N1-260抗體則對TDP-43寡聚體或單體具有相似的反應度(第5e圖)。另外,亦將由TDP-43之RRM2區第5 β-股所製備的β 5纖維點漬於膜上,以確認TDP-O僅會辨識TDP-43寡聚體,而非如先前技術所述之纖維(Wang et al.,J Biol Chem(2013)288,9049-9057)(結果未顯示);結果清楚地指出TDP-O僅能辨識TDP-43寡聚體,而不會辨識β 5纖維。故,本發明之多株抗體TDP-O可專一性 地辨識TDP-43寡聚體之構型,而不會交聯至A β蛋白。 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.

實施例7 TDP-43寡聚體存在於基因轉殖鼠之腦部Example 7 TDP-43 oligomers are present in the brain of genetically transgenic mice

將取自6及12週之野生型小鼠及FTLD-TDP-43基因轉殖鼠的腦部切片進行免疫組織化學染色,以檢測TDP-43寡聚體是否存在於活體體內;結果顯示於第6圖。已知會於前腦表現全長TDP-43的FTLD-TDP-43轉殖鼠具有與FTLD-TDP病患相似的病狀(Tsai et al.,J Exp Med(2010)207,1661-1673)。隨著年齡增長,該TDP-43基因轉殖鼠的學習/記憶力及運動能力會逐漸下降。腦部切片係以抗-TDP-43及TDP-O進行免疫螢光染色,並以DAPI進行複染。可預期地,在野生型小鼠腦部切片中,TDP-43主要係表現於細胞核內。相較之下,FTLD-TDP基因轉殖鼠的腦部切片則顯示,TDP-43主要係表現於細胞質中,且可偵測到TDP-O的訊號。在細胞中可發現大量共存的TDP-43及TDP-O訊號,且具有雙染的細胞量會隨著年齡的增加而增加。該些共存訊號指出,與類澱粉相似之TDP-43寡聚體會存在於FTLD-TDP基因轉殖鼠腦部,且表現量會隨著年紀的增長而增加。 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.

實施例8 TDP-43寡聚體會表現於FTLD-TDP病患之腦部Example 8 TDP-43 oligomers will be expressed in the brain of FTLD-TDP patients

為評估TDP-43寡聚體在人類疾病所扮演的角色,進一步利用帶有TDP-43免疫反應包涵體之FTLD病患來研究寡聚體於FTLD-TDP病患的表現。利用TDP-O 多株抗體來對病患之海馬迴及前皮質區進行免疫染色,其中該病患包含3名經病理確認之FTLD-TDP病患、3名年齡相仿而不具失智病徵之對照個體,以及3名經病理確認已罹患阿滋海默症卻不具TDP-43病徵之病患(做為疾病對照個體)。TDP-O抗體可辨識不同的神經元細胞質包涵體(第7a及7c圖)及在FTLD-TDP病患腦部中與TDP-43包涵體相似之營養不良的軸突(第7a圖)。在某些區域,神經元細胞質會深染為粗顆粒狀(第7e圖)。相較之下,除了神經元脂褐質(lipofuscin)所產生的非專一性反應,TDP-O並不會對對照個體之腦部(第7b、7d及7f)及罹患阿滋海默症卻不具TDP-43病徵之病患的腦部(結果未顯示)產生顯著的免疫反應。TDP-O多株抗體及TDP-43單株抗體皆無法染到正常組織的細胞核。此外,為了解可被TDP-O多株抗體辨識的TDP-43形態,利用TDP-O對對照個體及病患的海馬迴組織進行免疫沉澱分析試驗(immunoprecipitation,IP)。將海馬迴之采酮(triton)可溶分液與光交聯之TDP-O多株抗體進行免疫沉澱分析試驗。再將沖提產物以N端TDP-43抗體(N1-260)免疫標記後,進行電子顯微鏡分析(第8圖)。結果指出,於病患檢體可觀察到粒徑約為50奈米的圓球形TDP-43寡聚體,在對照個體之檢體則無;該些寡聚體可被N1-260抗體辨識,亦即,該些寡聚體並非N端截斷蛋白。整體來說,利用本發明之TDP-O多株抗體可證實TDP-43寡聚體會存在於FTLD-TDP病患的腦部。 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.

實施例9 製備及確認對TDP-43寡聚體具Example 9 Preparation and Confirmation of TDP-43 Oligomers 有專一性的單株抗體Specific monoclonal antibody

在本實施例中,利用ELISA試驗來檢測由TDP-O-3、-5、-8、-9及-10融合瘤細胞所製備之單株抗體,在有或無十二烷基硫酸鈉處理時,對TDP-43寡聚體的專一性。如第9圖所示,相較於變性的TDP-43蛋白,由5株融合瘤細胞所產生之單株抗體對未變性之TDP-43寡聚體具有較高的結合活性。該些數據指出,該些單株抗體對於TDP-43寡聚體具有專一性,是係為構型相關的抗體。 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.

為確認各單株抗體的結合專一性,先以膠體過濾來純化TDP-43寡聚體及單體(第10a圖),再分別將各產物塗佈於ELISA盤以確認各單株抗體的專一性。第10b圖的結果指出,由TDP-O-3、-5、-8、-9及-10融合瘤細胞所製備之單株抗體可具專一性地辨識TDP-43寡聚體。 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.

亦利用ELISA小鼠單株抗體同型套組(Thermo)來決定各TDP-O單株體的同型。結果總結於表1。 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.

依據表1的結果,由TDP-O-3、-5、-8、-9及-10融合瘤細胞所製備之單株抗體對於IgG2a及κ輕鏈具有較高的反應性。因此,該些單株抗體係為IgG2a及κ輕鏈亞型。 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.

將5株單株抗體進行定序分析,以決定一致性的序列;結果表述於第11圖。 Five monoclonal antibodies were sequenced to determine the sequence of identity; the results are presented in Figure 11.

當可理解上述實施方式與實施例僅為例示,且熟習此技藝者可對齊進行各種修飾。上文提出之說明書、實施例與資料的目的在於使本說明書的結構完備,並做為實作本發明之例示。雖然本揭示內容已以實施方式揭露如上,然其並非用以限定本揭示內容,任何熟習此技藝者,在不脫離本揭示內容之精神和範圍內,當可作各種之更動與潤飾,因此本揭示內容之保護範圍當視後附之申請專利範圍所界定者為準。 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.

【生物材料寄存】【Biomaterial Storage】

寄存機構:生物資源保存及研究中心 Depository: Bioresource Conservation and Research Center

寄存日期:民國104年1月28日 Hosting date: January 28, 104, Republic of China

寄存編號:BCRC 960494、BCRC 960495、BCRC 960496、BCRC 960497及BCRC 960498 Deposit number: BCRC 960494, BCRC 960495, BCRC 960496, BCRC 960497 and BCRC 960498

<110> 陳 韻如 <110> Chen Yunru

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<400> 8 <400> 8

Claims (8)

一種人類抗體或其之片段,其係可專一地結合至一單體分子量為43道耳吞的轉活化反應-去氧核糖核酸-結合蛋白(transactivation responsive-DNA-binding protein,43kDa,TDP-43)之寡聚體,其中該人類抗體是由一融合瘤細胞株所製備而成的,且該融合瘤細胞株寄存於生物資源保存及研究中心(Bioresource Collection and Research Center,BCRC),寄存編號為BCRC 960494、960495、960496、960497或960498。 A human antibody or fragment thereof that specifically binds to a transactivation responsive-DNA-binding protein (43 kDa, TDP-43) having a molecular weight of 43 auricular endocytosis The oligomer, wherein the human antibody is prepared from a fusion tumor cell line, and the fusion tumor cell line is deposited in the Bioresource Collection and Research Center (BCRC), and the accession number is BCRC 960494, 960495, 960496, 960497 or 960498. 如請求項1所述之人類抗體或其之片段,其中該TDP-43寡聚體為一直徑約為2至400奈米(nanometer,nm)的顆粒。 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 (nm). 如請求項2所述之人類抗體或其之片段,其中該TDP-43寡聚體為一直徑約為40至60奈米的球型或環狀顆粒。 The human antibody or fragment thereof according to claim 2, wherein the TDP-43 oligomer is a spherical or cyclic particle having a diameter of about 40 to 60 nm. 一種用以預防或治療與TDP-43寡聚體相關疾病的醫藥組合物,包含一有效量之請求項1的人類抗體或其之片段及一藥學上可接受的載體。 A pharmaceutical composition for preventing or treating a disease associated with a TDP-43 oligomer, comprising an effective amount of the human antibody of claim 1 or a fragment thereof, and a pharmaceutically acceptable carrier. 如請求項4所述之醫藥組合物,其中該種與TDP-43寡聚體相關的疾病可以是阿滋海默症(Alzheimer’s disease)、嗜銀顆粒性認知症(argyrophilic grain disease)、肌肉萎縮性脊髓側索硬化症(amyotrophic lateral sclerosis,ALS)、關島肌肉萎縮性脊髓側索硬化-巴金森氏失智症(ALS-parkinsonism dementia complex of Guam)、血管性失智症(vascular dementia)、額顳葉失智症(frontotemporal dementia)、語意失智症(semantic dementia)、雷維體失智(dementia with Lewy bodies)、亨汀頓氏舞蹈症(Huntington’s disease)、小腦萎縮症(Spinocerebellar ataxia)、包涵體肌病(inclusion body myopathy)、包涵體肌炎(inclusion body myositis)或巴金森氏症(Parkinson’s disease)。 The pharmaceutical composition according to claim 4, wherein the disease associated with the TDP-43 oligomer is Alzheimer's disease (Alzheimer's disease) Disease), argyrophilic grain disease, amyotrophic lateral sclerosis (ALS), Guam muscle atrophic lateral sclerosis - Parkinson's dementia (ALS-parkinsonism) Dementia complex of Guam), vascular dementia, frontotemporal dementia, semantic dementia, dementia with Lewy bodies, Hunting Huntington's disease, Spinocere bellar ataxia, inclusion body myopathy, inclusion body myositis, or Parkinson's disease. 一種由一個體之生物檢體來診斷一與TDP-43寡聚體相關疾病的方法,包含:將該生物檢體與一有效量之請求項1之人類抗體或其之片段接觸,以決定該生物檢體中TDP-43寡聚體的含量;以及將該生物檢體中TDP-43寡聚體的含量與取自一健康個體之對照檢體的TDP-43寡聚體含量進行比對;其中,若該生物檢體中TDP-43寡聚體的含量與該對照檢體的TDP-43寡聚體含量有顯著差異,即代表該個體患有與TDP-43寡聚體相關之疾病。 A method for diagnosing a disease associated with a TDP-43 oligomer by a biological sample comprising: contacting the biological sample with an effective amount of the human antibody of claim 1 or a fragment thereof to determine the biological sample The content of the TDP-43 oligomer in the biological sample; and the content of the TDP-43 oligomer in the biological sample is compared with the TDP-43 oligomer content of the control sample taken from a healthy individual; 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 a disease associated with the TDP-43 oligomer. 如請求項6所述之方法,其中該種與TDP-43寡聚體相關的疾病可以是阿滋海默症、嗜銀顆粒性認知症、肌 肉萎縮性脊髓側索硬化症、關島肌肉萎縮性脊髓側索硬化-巴金森氏失智症、血管性失智症、額顳葉失智症、語意失智症、雷維體失智、亨汀頓氏舞蹈症、小腦萎縮症、包涵體肌病、包涵體肌炎或巴金森氏症。 The method of claim 6, wherein the disease associated with the TDP-43 oligomer is Alzheimer's disease, argyrophilic granulosuscosis, muscle Atrophic spinal cord lateral sclerosis, Guam muscle atrophic lateral sclerosis - Parkinson's dementia, vascular dementia, frontotemporal dementia, semantic dementia, Levi's dementia, Henry Tington's chorea, cerebellar atrophy, inclusion body myopathy, inclusion body myositis or Parkinson's disease. 如請求項6所述之方法,其中該生物檢體可以是一腦部切片檢體、一腦脊液檢體、一全血檢體、一血清檢體、一血漿檢體、一尿液檢體或一黏液檢體。 The method of claim 6, 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.
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