TWI676635B

TWI676635B - Peptide immunogens from the c-terminal end of alpha-synuclein protein and formulations thereof for treatment of synucleinopathies

Info

Publication number: TWI676635B
Application number: TW107120762A
Authority: TW
Inventors: 長怡王; Chang-Yi Wang
Original assignee: 英屬開曼群島商聯腦科學公司; United Neuroscience
Priority date: 2018-06-15
Filing date: 2018-06-15
Publication date: 2019-11-11
Also published as: TW202000689A

Abstract

本發明有關於α-突觸核蛋白(α-Syn)胜肽免疫原結構、含此結構的組成物、此結構所誘發的抗體、以及製造及使用此結構及組成物的方法。α-Syn胜肽免疫原結構含有α-Syn的B細胞抗原決定位，其直接或透過選擇性異源連接子連接異源性T輔助細胞(Th)抗原決定位。胜肽免疫原結構的B細胞抗原決定位含有α-Syn的約10至約25個胺基酸，對應於全長α-Syn之自約第111個位置的甘胺酸(G111)至約第135個位置的天門冬胺酸(D135)的序列。α-Syn胜肽免疫原結構刺激高特異性抗體的產生，此高特異性抗體與為單體、寡聚物及原纖維之α-Syn的β摺疊，而非天然之α-螺旋的α-Syn交叉反應，對具罹患突觸核蛋白病風險的個體提供治療性免疫反應。 The invention relates to an α-synuclein (α-Syn) peptide immunogen structure, a composition containing the structure, an antibody induced by the structure, and a method for manufacturing and using the structure and the composition. The α-Syn peptide immunogen structure contains the B-cell epitope of α-Syn, which is connected to the heterologous T helper cell (Th) epitope directly or through a selective heterolinker. The B-cell epitope of the peptide immunogen structure contains about 10 to about 25 amino acids of α-Syn, corresponding to the glycine (G111) to about 135 of the full-length α-Syn Sequence of aspartic acid (D135) at three positions. The α-Syn peptide immunogen structure stimulates the production of highly specific antibodies that are β-sheets with α-Syn, which are monomers, oligomers, and fibrils, rather than the natural α-helix α- Syn cross-reactivity provides a therapeutic immune response to individuals at risk for synucleinopathy.

Description

α -突觸核蛋白C端胜肽免疫原及其用於治療突觸核蛋白疾病的配方Alpha-synuclein C-terminal peptide immunogen and its formula for treating synuclein diseases

本發明係有關於α-突觸核蛋白(α-Syn)C端的胜肽免疫原及其用於治療突觸核蛋白疾病的配方。 The invention relates to a peptide immunogen at the C-terminus of α-synuclein (α-Syn) and its formula for treating synuclein diseases.

突觸核蛋白(參照網站：en.wikipedia.org/wiki/Synuclein)為脊椎動物常見的可溶性蛋白家族，主要表現於神經組織及某些腫瘤中。突觸核蛋白家族包括三種已知蛋白：α-突觸核蛋白(參照網站：en.wikipedia.org/wiki/Alpha-synuclein)、β-突觸核蛋白(參照網站：en.wikipedia.org/wiki/Beta-synuclein)、以及γ-突觸核蛋白。所有突觸核蛋白通常具有一高度保守的α-螺旋脂質結合區，其類似於可交換載脂蛋白的A2類脂質結合結構域。僅管已有數據推測突觸核蛋白可能參與膜的穩定性及/或翻轉，但目前仍未顯示與正常細胞功能有關。 Synuclein (see website: en.wikipedia.org/wiki/Synuclein) is a common soluble protein family in vertebrates, which is mainly expressed in neural tissues and certain tumors. The synuclein family includes three known proteins: α-synuclein (see website: en.wikipedia.org/wiki/Alpha-synuclein) and β-synuclein (see website: en.wikipedia.org/ wiki / Beta-synuclein), and gamma-synuclein. All synucleins typically have a highly conserved α-helical lipid-binding region, which is similar to the A2 lipid-binding domain of exchangeable apolipoproteins. Although the existing data speculate that synuclein may be involved in membrane stability and / or turnover, it has not been shown to be related to normal cell function.

全長α-突觸核蛋白(α-Syn)為具有140個胺基酸的蛋白質(Accession No.NP_000336)，由SNCA基因所編碼。經由選擇性剪接可產生至少3種同型的α-Syn。主要的形式為全長蛋白。其它同型蛋白為α-Syn-126，其缺少第41-54個殘基，沒有外顯子3(exon 3)；α-Syn-112缺少第103-130個殘基，沒有外顯子5(exon 5)。 Full-length α-synuclein (α-Syn) has 140 amine groups Acidic protein (Accession No. NP_000336), encoded by the SNCA gene. At least 3 isoforms of α-Syn can be generated by alternative splicing. The main form is full-length protein. The other homologous protein is α-Syn-126, which lacks residues 41-54 and no exon 3; α-Syn-112 lacks residues 103-130 and no exon 5 ( exon 5).

α-Syn的一級結構通常分成3種不同的區域：(1)第1-60個胺基酸：由4個重複的11-胺基酸組成的雙極性N端區，其包括KTKEGV共有序列，具有α-螺旋結構，與載脂蛋白結合域類似；(2)第61-95個胺基酸：中央疏水性區，其包括非類澱粉蛋白-β(NAC)區，與蛋白聚集有關；以及(3)第96-140胺基酸：高度酸性及富含脯氨酸區，其無明顯的結構傾向。NAC區的35個胺基酸的α-Syn片段在富含非類澱粉蛋白部分中與Aβ一同存在。NAC為其前驅蛋白NACP的片段，於太平洋電鰩(Torpedo californica)中發現全長人類突觸核蛋白的同源物，在此稱為人類α-Syn。 The primary structure of α-Syn is usually divided into 3 different regions: (1) amino acids 1-60: bipolar N-terminal region consisting of 4 repeating 11-amino acids, which includes the KTKEGV consensus sequence, Has an alpha-helical structure, similar to apolipoprotein binding domains; (2) 61-95 amino acids: a central hydrophobic region, which includes non-amyloid-beta (NAC) regions, and is associated with protein aggregation; and (3) Amino acids 96-140: highly acidic and proline-rich regions, which have no obvious structural tendency. The α-Syn fragments of the 35 amino acids in the NAC region are present with Aβ in the non-amyloid-rich portion. NAC is a fragment of its precursor protein NACP, and a full-length human synuclein homologue was found in Torpedo californica , and is referred to herein as human α-Syn.

使用高解析度離子遷移質譜儀(IMS-MS)與HPLC純化的α-Syn顯示α-Syn具自身蛋白水解(self-proteolytic)，培養後會產生各種的小分子量片段。全長14.46kDa蛋白可產生許多較小片段，包括一個12.16kDa片段(第14-133胺基酸)及一個10.44kDa片段(第40-140胺基酸)的C-及N-端截斷，以及一個7.27kDa片段(第72-140胺基酸)。7.27kDa片段含有複數個NAC區，聚集速度明顯比全長α-Syn快。這些自身蛋白水解產物在 α-Syn聚集中扮演中間體或輔助因子的角色。 Α-Syn purified using high-resolution ion mobility mass spectrometry (IMS-MS) and HPLC showed that α-Syn had self-proteolytic, and various small molecular weight fragments were produced after cultivation. The full-length 14.46 kDa protein produces many smaller fragments, including a 12.16 kDa fragment (14-133 amino acids) and a 10.44 kDa fragment (40-140 amino acids) with C- and N-terminal truncations, and a 7.27 kDa fragment (amino acids 72-140). The 7.27kDa fragment contains multiple NAC regions, and the aggregation rate is significantly faster than the full-length α-Syn. These self-proteolysates α-Syn aggregates play an intermediate or cofactor role.

α-Syn為腦部多餘的組成，其佔腦及膠質細胞之細胞質中所有蛋白的1%。α-Syn廣泛地在新皮質、海馬、齒狀回、嗅球、紋狀體、丘腦及小腦中表現。α-Syn也在造血細胞中高度表現，包括B細胞、T細胞與NK細胞以及單核細胞和血小板。在腦中，α-Syn主要存在於突觸前端的神細胞(神經元)。在這些結構中，α-Syn與磷脂質和蛋白質相互作用。突觸前端釋放化學訊息，稱為神經傳導物質，例如來自於突觸小泡的多巴胺。神經傳導物質的釋放可在神經元間傳遞訊息，且對於腦部正常功能，包括認知是非常重要的。 α-Syn is an extra component of the brain, which accounts for 1% of all proteins in the cytoplasm of the brain and glial cells. α-Syn is widely expressed in neocortex, hippocampus, dentate gyrus, olfactory bulb, striatum, thalamus and cerebellum. α-Syn is also highly expressed in hematopoietic cells, including B cells, T cells and NK cells, as well as monocytes and platelets. In the brain, α-Syn is mainly found in neurons (neurons) at the front of the synapse. In these structures, α-Syn interacts with phospholipids and proteins. The front of the synapse releases chemical information called neurotransmitters, such as dopamine from synaptic vesicles. The release of neurotransmitters can transmit messages between neurons and is important for normal brain function, including cognition.

溶液中的α-Syn為一無穩定構型的蛋白質，因此其不具有單一穩定的3D結構。已顯示α-Syn與微管蛋白顯著地相互反應，且α-Syn可能具有微管相關蛋白(如tau)的活性。α-Syn一般被認為是非結構可溶性蛋白，非突變α-Syn可形成一種對抗聚集的穩定折疊四聚體。然而，α-Syn可在路易氏體的病理狀態下聚集形成不溶性纖維。此病狀即為已知的突觸核蛋白疾病(請參照網頁：en.wikipedia.org/wiki/Synucleinopathies)。 Α-Syn in solution is a protein with no stable configuration, so it does not have a single stable 3D structure. It has been shown that α-Syn and tubulin react significantly with each other, and α-Syn may have the activity of a tubulin-related protein such as tau. α-Syn is generally considered to be a non-structurally soluble protein. Non-mutated α-Syn forms a stable folded tetramer that resists aggregation. However, α-Syn aggregates to form insoluble fibers in the pathological state of Lewy bodies. This condition is known as synuclein disease (see webpage: en.wikipedia.org/wiki/Synucleinopathies).

突觸核蛋白疾病是一組不同的神經變性疾病，其具有共同的病理特徵：在神經病理學檢查中，不溶性α-Syn異常聚集體特徵的病變存在於神經元和神經膠質細胞損傷族群中。最常見的突觸核蛋白疾病包括路易氏體症(LBDs)，如帕金森氏症(PD)、帕金森病性癡呆(PDD)及路易氏體性癡呆(DLB)，以及多發性系統退化症(MSA)或第一型腦內鐵沉積性神經系統退化症。目前這些疾病的治療方式包括症狀療法，如，L-dopa、抗膽鹼能藥物以及單胺氧化醇抑製劑。然而，目前所有治療機會僅可症狀緩解，無法對患者達到持續的疾病改善效果。 Synuclein diseases are a different group of neurodegenerative diseases that share common pathological characteristics: In neuropathological examination, lesions characterized by insoluble α-Syn abnormal aggregates are present in the neuron and glial cell injury population. The most common synuclein diseases include Lewy body disease (LBDs) such as Parkinson's disease (PD), Parkinson's disease dementia (PDD), and road disease. Degenerative Dementia (DLB), as well as multiple systemic degeneration (MSA) or type 1 degenerative neurological degeneration of the brain. Current treatments for these diseases include symptomatic treatments, such as L-dopa, anticholinergic drugs, and monoamine oxide alcohol inhibitors. However, at present, all treatment opportunities can only relieve symptoms and cannot achieve sustained disease improvement for patients.

LBD為進行性神經變性疾病，其特徵為震顫、僵硬、運動遲緩和腦中多巴胺能神經元損失。DLB及PDD的症狀包括認知障礙。在西方國家中，60歲以上人口中超過2%會發展為PD/LBD的典型症狀。其顯示遺傳感受性和環境因素似乎與疾病的發展有關。此疾病患者在腦的皮質和皮質區域，特別是高含量的多巴胺神經元或神經元突出的區域發展出細胞內包涵體，被稱為路易氏體(LBs)。在LBD，路易氏體中累積的α-Syn會影響腦部。此外，已證明點突變及α-Syn基因重組或增殖與罕見的家族性帕金森氏綜合症有關。 LBD is a progressive neurodegenerative disease characterized by tremor, stiffness, bradykinesia, and loss of dopaminergic neurons in the brain. Symptoms of DLB and PDD include cognitive impairment. In Western countries, more than 2% of the population over the age of 60 will develop the typical symptoms of PD / LBD. It shows that genetic susceptibility and environmental factors seem to be related to the development of the disease. Patients with this disease develop intracellular inclusions in the cortex and cortical areas of the brain, especially in areas with high levels of dopamine neurons or neuronal protrusions, known as Lewy bodies (LBs). In LBD, α-Syn accumulated in Lewy bodies affects the brain. In addition, point mutations and α-Syn gene recombination or proliferation have been shown to be associated with rare familial Parkinson's syndrome.

多發性系統退化症(MSA)為一偶發性老化神經退化性疾病，其特徵為L-DOPA抗性的帕金森綜合症、小腦性運動失調症及自主神經障礙。患有多系統神經元損傷患者的各種腦區，包括紋狀體、黑質、小腦、橋腦及下橄欖核與脊髓受到影響。MSA的特徵為在整個中樞神經系統中具有α-Syn陽性膠質細胞質(GCI)和罕見的神經元雜物。 Multiple system degeneration (MSA) is a sporadic aging neurodegenerative disease characterized by L-DOPA-resistant Parkinson's syndrome, cerebellar dyskinesia, and autonomic disorders. Various brain regions in patients with multisystem neuron damage, including striatum, substantia nigra, cerebellum, pontine, and inferior olive nucleus and spinal cord are affected. MSA is characterized by alpha-Syn positive glial cytoplasm (GCI) and rare neuronal debris throughout the central nervous system.

其它罕見疾病，如各種神經突起失調症，也具有α-Syn病變，且α-Syn是路易氏體纖維的主要結構。通常，路易氏體含有tau蛋白；然而α-Syn及tau在相同的包涵體中構成2個不同的中間絲次單元(subsets)。在偶發及家族性的阿茲海默症中也發現α-Syn病變。 Other rare diseases, such as various neurite disorders, also have α-Syn lesions, and α-Syn is the main structure of Lewy body fibers. Usually, Leu body contains tau protein; however, α-Syn and tau constitute two different intermediate filament subunits in the same inclusion body. Alpha-Syn lesions are also found in sporadic and familial Alzheimer's disease.

α-Syn累積的機制並不清楚。α-Syn單體在溶液中並不折疊，但可以α-螺旋形式與膜結合。未折疊的單體可先聚集成小的寡聚物，其可以藉由類β-摺疊的相互作用而穩定，以形成更高分子量的不溶性纖維。α-Syn的存在與非結構、α-螺旋和富含β-摺疊結構的混合物維持一平衡。已知突變或緩衝液條件可增強聚集，大幅地增加β構象異構體的群體，因此其可能與致病性聚集有關。已有證據證明結構化中間體富含β結構，其為聚集體前驅物，最終形成路易4體。 The mechanism of α-Syn accumulation is unclear. The α-Syn monomer does not fold in solution, but can bind to the membrane in an α-helical form. Unfolded monomers can first aggregate into small oligomers, which can be stabilized by β-sheet-like interactions to form higher molecular weight insoluble fibers. The presence of α-Syn maintains a balance with unstructured, α-helix and β-sheet-rich mixtures. Mutations or buffer conditions are known to enhance aggregation and greatly increase the population of beta conformers, so it may be related to pathogenic aggregation. There is evidence that structured intermediates are rich in beta structures, which are aggregate precursors that eventually form the Louis 4 body.

許多生理因素可改變α-Syn導致聚集的形成，包括(1)藉由一或多種酵素進行磷酸化，(2)藉由蛋白酶，如鈣蛋白酶進行切割；(3)發炎期間，以一氧化氮(NO)或其他活性氮物質進行硝化。ER-高基氏體運輸、突觸小泡、線粒體、溶酶體等其它蛋白水解物質為因聚集所造成之α-Syn毒性的細胞標的。 Many physiological factors can alter the formation of α-Syn leading to the formation of aggregates, including (1) phosphorylation by one or more enzymes, (2) cleavage by proteases such as calpain, and (3) nitric oxide during inflammation (NO) or other active nitrogen substances for nitration. Other proteolytic substances such as ER-high martensite transport, synaptic vesicles, mitochondria, and lysosomes are the target cells of α-Syn toxicity caused by aggregation.

治療突觸核蛋白疾病的策略為使用抑制α-Syn聚集的化合物。已顯示小分子枯茗醛(cuminaldehyde)可抑制α-Syn的纖維化。除了小分子治療外，已有文獻揭示α-Syn聚集與免疫治療有關(Lee JS and Lee S-J,2016)。然而，文獻指出在開發α-Syn免疫治療仍有許多潛在的問題，包括(1)對α-Syn正常生理功能的潛在干擾；(2)將抗體藥物傳送至腦實質(brain parenchyma)的困難；以及(3)免疫治療的功效。 The strategy for treating synuclein diseases is to use compounds that inhibit α-Syn aggregation. The small molecule cuminaldehyde has been shown to inhibit fibrosis of α-Syn. In addition to small molecule therapy, literature has revealed that α-Syn aggregation is associated with immunotherapy (Lee JS and Lee S-J, 2016). However, the literature indicates that there are still many potential problems in developing α-Syn immunotherapy, including (1) potential interference with the normal physiological functions of α-Syn; (2) anti- Difficulties in delivering body drugs to the brain parenchyma; and (3) efficacy of immunotherapy.

至目前為止，對於開發定點胜肽免疫原及其製劑以治療突觸核蛋白疾病患者尚未滿足。 So far, the development of site-specific peptide immunogens and their preparations to treat patients with synuclein diseases has not been satisfied.

參考文獻 references

"Alpha-synuclein," Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org/w/index.php？title=Alpha-synuclein&oldid=781366541 (accessed May 30, 2017). "Alpha-synuclein," Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org/w/index.php? title = Alpha-synuclein & oldid = 781366541 (accessed May 30, 2017).

"Synucleinopathies," Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org/w/index.php？title=Synucleinopathies&oldid=686287116 (accessed May 30, 2017). "Synucleinopathies," Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org/w/index.php? title = Synucleinopathies & oldid = 686287116 (accessed May 30, 2017).

"Beta-synuclein," Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org/w/index.php？title=Beta-synuclein&oldid=763171134 (accessed May 30, 2017). "Beta-synuclein," Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org/w/index.php? title = Beta-synuclein & oldid = 763171134 (accessed May 30, 2017).

LEE, J.S., et al., “Mechanism of Anti-α-Synuclein Immunotherapy”, J Mov Disord.; 9(1):14-19 (2016) LEE, J.S., et al., "Mechanism of Anti-α-Synuclein Immunotherapy", J Mov Disord .; 9 (1): 14-19 (2016)

TRAGGIAI, E., et al. “An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus”, Nat Med.; 10(8):871-875 (2004) TRAGGIAI, E., et al. "An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus ", Nat Med .; 10 (8): 871-875 (2004)

WANG, C., et al. “Versatile Structures of α-Synuclein”, Front Mol Neurosci. 9:48 (2016) WANG, C., et al. “Versatile Structures of α-Synuclein”, Front Mol Neurosci. 9:48 (2016)

本發明係有關於α-突觸核蛋白(α-Syn)胜肽免疫原結構。本發明也有關於含此胜肽免疫原結構的組成物，製造及使用此胜肽免疫原的方法，以及因胜肽免疫原所產生的抗體。 The invention relates to the structure of an α-synuclein (α-Syn) peptide immunogen. The present invention also relates to a composition containing the peptide immunogen structure, a method for manufacturing and using the peptide immunogen, and antibodies produced by the peptide immunogen.

本發明之胜肽免疫原結構含有一B細胞抗原決定位，其直接或經由選擇性異源性連接子連接異源性T輔助細胞(Th)。胜肽免疫原結構中的B細胞抗原決定位部分含有α-Syn C端的10至25個胺基酸殘基，其為全長α-Syn(SEQ ID NO：1)中約第111個胺基酸的甘氨酸(G111)至約第135個胺基酸的天門冬胺酸(D135)。胜肽免疫原結構的異源性Th抗原決定位部分源自於病原體蛋白質的胺基酸序列。胜肽免疫原結構的B細胞抗原決定位及Th抗原決定位部分在給予至宿主時，可共同作用刺激產生抗體，此抗體可專一性辨識及結合至α-Syn B細胞抗原決定位。 The peptide immunogen structure of the present invention contains a B-cell epitope, which is directly or via a selective heterologous linker connected to a heterologous T helper cell (Th). The B-cell epitope in the peptide immunogen structure contains 10 to 25 amino acid residues at the C-terminus of α-Syn, which is about the 111th amino acid in the full-length α-Syn (SEQ ID NO: 1). From glycine (G111) to about 135th aspartic acid (D135). The heterologous Th epitope of the peptide immunogen structure is derived in part from the amino acid sequence of the pathogen protein. When the B-cell epitope and Th epitope portion of the peptide immunogen structure are administered to the host, they can work together to stimulate the production of antibodies. This antibody can specifically recognize and bind to the α-Syn B-cell epitope.

在某些實施例中，α-Syn胜肽免疫原結構包括：(a)一B細胞抗原決定位，包含10至25個胺基酸，其為SEQ ID NO：1中約G111至約D135；(b)T輔助抗原決定位，其擇自於SEQ ID NO：70-98所組成的群組；以及(c)選擇性異源性連接子，其擇自於Lys-、Gly-、Lys-Lys-Lys-、(α,ε-N)Lys及ε-N-Lys-Lys-Lys-Lys(SEQ ID NO：148)所組成的群組，其中B細胞抗原決定位直接或透過選擇性異源性連接子共價連接T輔助細胞抗原決定位。在一特定實施例中，α-Syn胜肽免疫原結構包括擇自於SEQ ID NOs：107、108、111-113及115-147所組成之群組。 In certain embodiments, the α-Syn peptide immunogen structure includes: (a) a B-cell epitope comprising 10 to 25 amino acids, which are about G111 to about D135 in SEQ ID NO: 1; (b) T-assisted antigen determination Mapping, selected from the group consisting of SEQ ID NOs: 70-98; and (c) selective heterologous linkers, selected from Lys-, Gly-, Lys-Lys-Lys-, (α , ε-N) Lys and ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148), in which the B cell epitope is covalently linked directly or through a selective heterologous linker T helper epitope. In a specific embodiment, the α-Syn peptide immunogen structure includes a group selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147.

本發明更有關於含有本發明胜肽免疫結構的組成物，包括醫藥組成物。本發明之醫藥組成物為可誘發免疫反應及刺激宿主生成針對本發明醫藥組成物的抗體。本發明組成物可含有一或多個胜肽免疫結構的混合物。在某些實施例中，此組成物含有本發明胜肽免疫結構及其它額外成分，包括載體、佐劑、緩衝液及其它適當的試劑。在特定實施例中，組成物含有胜肽免疫結構，其以穩定的免疫刺激複合物形式存在，並選擇性補充作為佐劑的CpG寡聚物。 The invention further relates to a composition containing a peptide immune structure of the present invention, including a pharmaceutical composition. The pharmaceutical composition of the present invention is capable of inducing an immune response and stimulating the host to produce antibodies against the pharmaceutical composition of the present invention. The composition of the invention may contain a mixture of one or more peptide immune structures. In certain embodiments, this composition contains the peptide immune structure of the present invention and other additional ingredients, including carriers, adjuvants, buffers, and other appropriate agents. In a specific embodiment, the composition contains a peptide immune structure, which is in the form of a stable immune stimulating complex, and optionally supplements the CpG oligomer as an adjuvant.

在某些實施例中，組成物包括一α-Syn胜肽免疫原結構，其含有一擇自於SEQ ID NOs：107、108、111-113、115-147所組成群組的胺基酸序列。在特定實施例中，組成物為一醫藥組成物，包括一α-Syn胜肽免疫原結構，其含有一擇自於SEQ ID NOs：107、108、111-113、115-147所組成群組的胺基酸序列及一藥學上可接受之載體或佐劑。 In some embodiments, the composition includes an α-Syn peptide immunogen structure containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, 115-147 . In a specific embodiment, the composition is a pharmaceutical composition, including an α-Syn peptide immunogen structure, which contains a group selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147. Amino acid sequence and a pharmaceutically acceptable carrier or adjuvant.

本發明更有關於以胜肽免疫原結構免疫宿主所生成的抗體。本發明之抗體專一性地辨識及結合胜肽免疫原結構的α-Syn B細胞抗原決定位部分。本發明α-Syn抗體具有不可預期之與單體、寡聚物及纖維狀α-Syn之β-摺疊的高度交互反應。基於其獨特的特徵及特性，本發明之抗體可提供一免疫治療方式以針對、辨識及治療突觸核蛋白疾病。 The invention further relates to antibodies produced by immunizing a host with a peptide immunogen structure. The antibody of the present invention specifically recognizes and binds to the α-Syn B cell epitope portion of the peptide immunogen structure. The α-Syn antibodies of the present invention have an unexpectedly high degree of interaction with β-sheets of monomers, oligomers, and fibrous α-Syn. Based on its unique characteristics and properties, the antibodies of the present invention can provide an immunotherapy approach to target, identify and treat synuclein diseases.

在特定實施例中，抗體或其抗原決定位結合片段可專一性地結合至α-Syn胜肽免疫原結構的B細胞抗原決定位，其擇自於SEQ ID NOs：107、108、111-113、115-147所組成的群組。 In a specific embodiment, the antibody or epitope-binding fragment thereof can specifically bind to the B-cell epitope of the α-Syn peptide immunogen structure, which is selected from SEQ ID NOs: 107, 108, 111-113 , 115-147.

本發明更有關於製造及使用本發明胜肽免疫結構、抗體及組成物的方法。本發明之方法可提供低製造成本及高品質管制的胜肽免疫結構及含此結構的組成物，以抑制及治療突觸核蛋白疾病。 The invention further relates to methods for making and using the peptide immune structures, antibodies and compositions of the invention. The method of the present invention can provide a peptide immune structure with low manufacturing cost and high quality control, and a composition containing the same to inhibit and treat synuclein diseases.

本發明更包括治療及/或抑制突觸核蛋白疾病的方法，包括使用本發明胜肽免疫結構及/或針對胜肽免疫結構的抗體。在某些實施例中，治療及/或抑制突觸核蛋白疾病的方法，包括給予一宿主一含有本發明胜肽抗原結構的組成物。在特定實施例中，此方法中所使用的組成物包含一本發明胜肽免疫原結構以及帶負電荷的寡核苷酸，如CpG寡聚物，透過靜電結合形成穩定的免疫刺激複合物形式，此複合物更包括選擇性作為佐劑的礦物鹽或礦物油，給予至罹患突觸核蛋白疾病的患者。本發明之方法包括將胜肽免疫原結構給予至罹患突觸核蛋白疾病或具罹患風險宿主的給藥方式、劑型及途徑。 The invention further includes a method for treating and / or inhibiting a synuclein disease, including using the peptide immune structure of the invention and / or an antibody against the peptide immune structure. In certain embodiments, a method of treating and / or inhibiting a synuclein disease comprises administering to a host a composition comprising a peptide antigen structure of the invention. In a specific embodiment, the composition used in this method comprises a peptide immunogen structure of the present invention and a negatively charged oligonucleotide, such as a CpG oligomer, which forms a stable immunostimulatory complex form through electrostatic binding. This complex further includes mineral salts or mineral oils that are selectively used as adjuvants for administration to patients suffering from synuclein diseases. The invention The method includes the administration mode, dosage form and route of administering the peptide immunogen structure to a host suffering from a synuclein disease or at risk.

在各種實施例中，本發明包括使用α-Syn胜肽免疫原結構及/或α-Syn胜肽免疫原結構所誘發之抗體的方法。在特定實施例中，本發明包括產生抗體、抑制α-Syn聚集、減少α-Syn聚集及辨識不同大小α-Syn聚集的方法。各種方法包括給予一所欲宿主一藥學上有效量之α-Syn胜肽免疫原。 In various embodiments, the invention includes methods of using antibodies elicited by the α-Syn peptide immunogen structure and / or the α-Syn peptide immunogen structure. In particular embodiments, the invention includes methods for generating antibodies, inhibiting α-Syn aggregation, reducing α-Syn aggregation, and identifying α-Syn aggregation of different sizes. Various methods include administering a pharmaceutically effective amount of an alpha-Syn peptide immunogen to a desired host.

第1圖顯示在α-Syn C端抗體(樣本1-4)或載體控制組(樣本5)存在下的6天，in vitro α-Syn胜肽免疫原聚集的程度。特別是，在以α-Syn_111-132(樣本1)；α-Syn_121-135(樣本2)；α-Syn_123-135(樣本3)；α-Syn_126-135(樣本4)誘發之抗-α-Syn抗體存在下，或載體控制組(樣本5)的α-Syn聚集。α-Syn聚集的程度以硫黃素-T(ThT)染色測量。樣本1-4以樣本5載體控制組進行標準化。誤差線代表各重複試驗的SEM(平均值標準誤差)。 Figure 1 shows the degree of in vitro α-Syn peptide immunogen accumulation in the presence of an α-Syn C-terminal antibody (Sample 1-4) or a vector control group (Sample 5). In particular, in order to α-Syn _111-132 (Sample 1); α-Syn _121-135 (Sample 2); α-Syn _123-135 (Sample 3); α-Syn _126-135 (Sample 4) of the induced Aggregation of α-Syn in the presence of an anti-α-Syn antibody or the vector control group (Sample 5). The degree of α-Syn aggregation was measured by thioflavin-T (ThT) staining. Samples 1-4 were normalized with the sample 5 vector control group. Error bars represent the SEM (mean standard error) of each replicate.

第2圖顯示在針對α-Syn C端抗體(樣本1-3)或免疫前血清控制組(樣本4)的存在下的培養3天，預形成in vitro α-Syn聚集體的解聚集程度。特別是，將預形成的α-Syn聚集體以α-Syn_111-132(樣本1)、α-Syn_126-135(樣本2)誘發之抗-α-Syn抗體、以α-Syn_111-132及α-Syn_126-135誘發之抗體組合；或免疫前血清控制組(樣本4)進行培養。α-Syn聚集的程度以硫黃素-T(ThT)染色測量。樣本1-3以樣本4載體控制組進行標準化。誤差線代表各重複試驗的SEM(平均值標準誤差)。 Figure 2 shows the degree of deaggregation of pre-formed in vitro α-Syn aggregates after 3 days of culture in the presence of α-Syn C-terminal antibody (Sample 1-3) or pre-immune serum control group (Sample 4). In particular, α-Syn preformed aggregates in α-Syn _111-132 (sample 1), α-Syn _126-135 (Sample 2) induce the anti -α-Syn antibody to α-Syn _111-132 And α-Syn _126-135- induced antibody combinations; or the pre-immune serum control group (sample 4) was cultured. The degree of α-Syn aggregation was measured by thioflavin-T (ThT) staining. Samples 1-3 were normalized with the sample 4 vector control group. Error bars represent the SEM (mean standard error) of each replicate.

第3圖顯示過度表現α-Syn之PC12細胞在針對α-Syn C-端抗體(樣本 1-4)或載體控制組(樣本5)存在下與神經生長因子(NGF)進行培養，α-Syn聚集及α-Syn解聚集的程度。特別是，將PC12細胞以α-Syn_111-132(樣本1)、α-Syn_121-135(樣本2)、α-Syn_123-135(樣本3)、α-Syn_126-135(樣本4)誘發之抗-α-Syn抗體；或載體控制組(樣本5)進行培養。樣本1-4以樣本5載體控制組進行標準化。誤差線代表各重複試驗的SD(標準差)。 Figure 3 shows that PC12 cells overexpressing α-Syn were cultured with nerve growth factor (NGF) in the presence of α-Syn C-terminal antibodies (Sample 1-4) or the vector control group (Sample 5). Α-Syn Degree of aggregation and α-Syn deaggregation. In particular, PC12 cells were treated with α-Syn _111-132 (sample 1), α-Syn _121-135 (sample 2), α-Syn _123-135 (sample 3), and α-Syn _126-135 (sample 4). The induced anti-α-Syn antibody; or the vector control group (sample 5) was cultured. Samples 1-4 were normalized with the sample 5 vector control group. Error bars represent SD (standard deviation) of each replicate.

第4圖顯示細胞培養在針對α-Syn C-端抗體(樣本1-4)或載體控制組(樣本5)下，α-Syn誘導之TNF-α及IL-6的釋放程度。特別是，將微膠細胞(microglia)以α-Syn_111-132(樣本1)、α-Syn_121-135(樣本2)、α-Syn_123-135(樣本3)、α-Syn_126-135(樣本4)誘發之抗-α-Syn抗體；或載體控制組(樣本5)進行培養。樣本1-4以樣本5載體控制組進行標準化。誤差線代表各重複試驗的SD(標準差)。 Figure 4 shows the degree of α-Syn-induced release of TNF-α and IL-6 by cell culture under α-Syn C-terminal antibody (samples 1-4) or vector control group (sample 5). In particular, microglia cells were treated with α-Syn _111-132 (Sample 1), α-Syn _121-135 (Sample 2), α-Syn _123-135 (Sample 3), and α-Syn _126-135. (Sample 4) The induced anti-α-Syn antibody; or the vector control group (Sample 5) was cultured. Samples 1-4 were normalized with the sample 5 vector control group. Error bars represent SD (standard deviation) of each replicate.

第5A-5C圖顯示抗α-Syn抗體在in vitro神經變性模型中對預形成α-Syn聚集NGF誘導之神經元分化PC12細胞的影響。第5A圖為評估經NGF處理(黑色實線)；經NGF處理且具有外源性預形成α-Syn聚集(短虛線)；經NGF處理且具有免疫前血清(淺色實線)；以及經NGF處理且具有外源性預形成α-Syn聚集及免疫前血清(長虛線)之PC12細胞的神經元突長度。第5B圖為評估經NGF處理且具有載體(黑色實線)；經NGF處理且具有外源性預形成α-Syn聚集(短虛線)；經NGF處理且具有受α-Syn_111-132(SEQ ID NO：113)誘導之抗-α-Syn抗體(淺色實線)；以及經NGF處理且具有外源性預形成α-Syn聚集及受α-Syn_111-132(SEQ ID NO：113)誘導之抗-α-Syn抗體(dashed line)之PC12細胞的神經元突(neurite)長度。第5C圖為評估經NGF處理且具有載體(黑色實線)；經NGF處理且具有外源性預形成α-Syn聚集(長虛線)；經NGF處理且具有受α-Syn_126-135(SEQ ID NO：112)誘導之抗-α-Syn抗體(淺色實線)；以及經NGF處理且具有外源性預形成α-Syn聚集及受α-Syn_126-135(SEQ ID NO：112)誘導之抗-α-Syn抗體(長虛線)之PC12細胞的神經元突長度。 Figures 5A-5C show the effect of anti-α-Syn antibodies on NGF-induced PC12 cells induced by preformed α-Syn aggregates in an in vitro neurodegeneration model. Figure 5A is an evaluation of NGF treatment (solid black line); NGF treatment with exogenous preformed α-Syn aggregates (short dashed lines); NGF treatment with pre-immune serum (light solid line); and Nerve process length of PC12 cells treated with NGF and exogenous pre-formed α-Syn aggregation and pre-immune serum (long dashed line). Figure 5B is an evaluation of NGF-treated and with a carrier (solid black line); NGF-treated and with exogenous preformed α-Syn aggregates (short dashed lines); NGF-treated and with α-Syn _111-132 (SEQ ID NO: 113) induced anti-α-Syn antibody (light solid line); and NGF-treated and exogenous preformed α-Syn aggregates and affected by α-Syn _111-132 (SEQ ID NO: 113) Induced anti-α-Syn antibody (dashed line) PC12 cell neuron neurite length. Figure 5C is an evaluation of NGF-treated and with a carrier (black solid line); NGF-treated and with exogenous pre-formed α-Syn aggregates (long dashed lines); NGF-treated and with α-Syn _126-135 (SEQ ID NO: 112) induced anti-α-Syn antibody (light solid line); and NGF-treated and exogenous preformed α-Syn aggregates and affected by α-Syn _126-135 (SEQ ID NO: 112) Neuronal process length of PC12 cells induced by anti-α-Syn antibody (long dashed line).

第6A-6B顯示在使用NGF誘導之神經元分化野生型過度表現α-Syn之PC12細胞in vitro神經變性模型中抗-α-Syn抗體對細胞數及神經元突長度的影響。細胞經載體控制組(樣本1)；受α-Syn_101-132(樣本2)、α-Syn_111-132(樣本3)、α-Syn_121-135(樣本4)、α-Syn_123-135(樣本5)、α-Syn_126-135(樣本6)誘導之抗-α-Syn抗體、或受α-Syn_111-132及α-Syn_126-135誘導之抗-α-Syn抗體組合(樣本7)；或免疫前血清控制組(樣本8)。第6A圖為評估各樣本對恢復PC12細胞數的保護能力。第6B圖為評估經各樣本處理之細胞的神經元突長度。樣本1-8以NGF誘導之神經元分化野生型過度表現α-Syn之PC12細胞進行標準化。使用t檢測進行顯著性檢定(p值少於0.05代表具有統計上顯著並以*號表示)。 Figures 6A-6B show the effect of anti-α-Syn antibodies on cell number and neuronal process length in an in vitro neurodegeneration model of PC12 cells in vitro with overexpression of α-Syn using NGF-induced neuronal differentiation. Cell control group (Sample 1); α-Syn _101-132 (Sample 2), α-Syn _111-132 (Sample 3), α-Syn _121-135 (Sample 4), α-Syn _123-135 (sample 5), α-Syn _126-135 (sample 6) -α-Syn-induced anti-antibody, or by α-Syn _111-132 _126-135 -α-Syn-induced anti-antibody combinations and α-Syn (sample 7); or pre-immune serum control group (sample 8). Figure 6A is an assessment of the protective ability of each sample to restore the number of PC12 cells. Fig. 6B is an assessment of the neuronal process length of cells treated with each sample. Samples 1-8 were normalized with NGF-induced neuronal differentiation of wild-type overexpressing α-Syn PC12 cells. A t-test was used to test for significance (p-values less than 0.05 represent statistically significant and are indicated by *).

第7A-7B顯示利用西方墨點法分析抗-α-Syn抗體對辨識及結合不同大小之α-Syn聚集的能力。第7A圖為一西方墨點法分析結果，其與市售抗-α-Syn抗體、Syn211(泳道1)、免疫前血清控制組(泳道2)、以Syn_111-132誘導之抗α-Syn抗體(泳道3)、以Syn_111-135誘導之抗α-Syn抗體(泳道4)、以Syn_121-135誘導之抗α-Syn抗體(泳道5)、以Syn_123-135誘導之抗α-Syn抗體(泳道6)、以及以Syn_126-135誘導之抗α-Syn抗體(泳道7)比較。第7B圖為一柱狀圖，其顯示各抗體結合至各種大小之α-Syn分子複合物(包括單體、二聚體、三聚體、四聚體及寡聚物)的相對能力。西方墨點法的化學冷光訊號如第7A圖所示，經定量後以第7B圖的柱狀圖表示。 Figures 7A-7B show the ability of anti-α-Syn antibodies to recognize and bind α-Syn aggregates of different sizes by Western blot analysis. Figure 7A shows the results of a western blot analysis with commercially available anti-α-Syn antibodies, Syn211 (lane 1), pre-immune serum control group (lane 2), and anti-α-Syn induced by Syn _111-132 . Antibody (lane 3), anti-α-Syn antibody induced by Syn _111-135 (lane 4), anti-α-Syn antibody induced by Syn _121-135 (lane 5), anti-α- induced by Syn _123-135 _{Comparison of} Syn antibody (lane 6) and anti-α-Syn antibody (lane 7) induced with Syn _126-135 . Figure 7B is a bar graph showing the relative ability of each antibody to bind to various sizes of α-Syn molecular complexes, including monomers, dimers, trimers, tetramers, and oligomers. The chemical cold light signal of western ink dot method is shown in Fig. 7A, and after quantification, it is represented by the histogram of Fig. 7B.

第8A-8C為圓漬點墨法(dot blot)分析結果，其顯示針對α-Syn C-端的抗體僅會辨識與結合至不同種類的α-Syn(即α-螺旋單體、β-折疊單體、β-折疊寡聚物及β-折疊纖維)，而不會結合至相同種類的其它澱粉樣蛋白(即 Aβ1-42和Tau441)。第8A圖為一控制組樣本，由天竺鼠免疫前血清純化之抗體不會與任何蛋白結合。第8B圖顯示以Syn_111-132(SEQ ID NO：113)誘導之抗α-Syn抗體會辨識及結合不同種類的α-Syn、Aβ1-42與Tau441蛋白。第8C圖顯示以Syn_126-135(SEQ ID NO：112)誘導之抗α-Syn抗體會辨識及結合至不同種類的α-Syn、Aβ1-42與Tau441蛋白。 Sections 8A-8C are the results of dot blot analysis, which show that antibodies against the C-terminus of α-Syn will only recognize and bind to different types of α-Syn (i.e., α-helical monomers, β-sheets). Monomers, β-sheet oligomers and β-sheet fibers) without binding to other amyloids of the same species (ie Aβ1-42 and Tau441). Figure 8A is a control group sample. Antibodies purified from guinea pig pre-immune sera will not bind to any protein. FIG. 8B shows that the anti-α-Syn antibody induced by Syn _111-132 (SEQ ID NO: 113) recognizes and binds different types of α-Syn, Aβ1-42 and Tau441 proteins. Figure 8C shows that anti-α-Syn antibodies induced by Syn _126-135 (SEQ ID NO: 112) recognize and bind to different types of α-Syn, Aβ1-42 and Tau441 proteins.

第9圖為一表格，其整理針對α-Syn C-端之抗體對各種PC12細胞之胞外α-Syn的結合親和力，測量免疫細胞化學(ICC)分析中的陽性訊號。特別是，受α-Syn_111-132、α-Syn_121-135、α-Syn_126-135或免疫前血清控制組誘發之抗α-Syn抗體在以NGF處理之PC12母細胞、陰性控制PC12細胞、野生型α-Syn過度表現PC12細胞、A53T突變α-Syn過度表現PC12細胞中的相對結合親和力。 Figure 9 is a table summarizing the binding affinities of antibodies against the α-Syn C-terminus to the extracellular α-Syn of various PC12 cells, and measuring the positive signals in immunocytochemical (ICC) analysis. In particular, by _{α-Syn 111-132, α-Syn} 121-135, α-Syn 126-135 or preimmune serum control group induced the anti-α-Syn antibody PC12 cells to parent NGF treatment, the negative control PC12 cells Wild-type α-Syn overexpresses PC12 cells, and A53T mutant α-Syn overexpresses relative binding affinity in PC12 cells.

第10A-10C圖顯示針對α-Syn C-端之抗體僅與PD腦切片中的α-Syn結合，而不與健康腦切片結合。第10A圖顯示α-Syn胜肽免疫原結構誘發之α-Syn抗體及免疫前抗體在正常人腦組織中未偵測到免疫反應。第10B圖顯示針對α-Syn聚集之抗體在PD丘腦切片中的免疫反應，如箭頭所示。第10C圖為一表格，顯示針對α-Syn C-端與免疫前控制組對α-Syn之抗體在PD及健康腦切片中的免疫反應，以顯微觀察計數陽性染色的數目。 Figures 10A-10C show that antibodies directed against the C-terminus of α-Syn only bind to α-Syn in PD brain sections and not to healthy brain sections. FIG. 10A shows that the α-Syn antibody and pre-immune antibody induced by the α-Syn peptide immunogen structure did not detect an immune response in normal human brain tissue. FIG. 10B shows the immune response of antibodies against α-Syn aggregation in PD thalamus sections, as indicated by arrows. Figure 10C is a table showing the immune response to the α-Syn C-terminus and the pre-immune control group antibodies to α-Syn in PD and healthy brain sections. The number of positive staining was counted by microscopic observation.

第11A-11B圖顯示在免疫3次佐劑(空心圓圈)或含有α-Syn_111-132(空心方塊)、α-Syn_126-135(實心圓圈)、或α-Syn_111-132及α-Syn_126-135之組合(實心方塊)的胜肽免疫原後，PD小鼠模型中抗-α-Syn IgG的量。第11A圖顯示MPP+小鼠模型中IgG的程度。第11B圖顯示接種α-Syn纖維小鼠模型中IgG的量。 Figures 11A-11B show 3 adjuvants (open circles) or alpha-Syn _111-132 (open squares), α-Syn _126-135 (closed circles), or α-Syn _111-132 and α- The amount of anti-α-Syn IgG in the PD mouse model following the peptide immunogen of the combination (solid square) of Syn _126-135 . Figure 11A shows the extent of IgG in the MPP + mouse model. Figure 11B shows the amount of IgG in a mouse model inoculated with alpha-Syn fibers.

第12A-12B圖顯示PD小鼠模型在接受單純佐劑(空心圓圈)、含有α-Syn_111-132(空心方塊)、α-Syn_126-135(實心圓圈)的胜肽免疫原、或α-Syn_111-132 及α-Syn_126-135(實心方塊)組合3次免疫後，末梢循環的α-Syn量。第12A圖顯示MPP+誘發小鼠模型的α-Syn量。第12B圖顯示接種α-Syn纖維小鼠模型中α-Syn的量。 Figures 12A-12B show PD mouse models receiving peptide immunogens containing pure adjuvant (open circles), α-Syn _111-132 (open squares), α-Syn _126-135 (closed circles), or α after -Syn _111-132 and α-Syn _126-135 (solid squares) immunizations composition 3, α-Syn amount of peripheral circulation. Figure 12A shows the amount of α-Syn in the MPP + induced mouse model. Figure 12B shows the amount of α-Syn in a mouse model inoculated with α-Syn fibers.

第13A-13B圖顯示未處理健康小鼠模型(lane 1)或接受3次單純佐劑(lane 2)或含有α-Syn_111-132胜肽免疫原之PD小鼠模型(lane 2-3)腦樣本的α-Syn寡聚物量。未處理Balb/c小鼠為健康小鼠模型，MPP+誘發小鼠模型為PD小鼠模型。第13A圖為西方墨點法結果，其顯示樣本中α-Syn寡聚物以及作為蛋白對照組(protein loading control)的GAPDH量。第13B圖比較第13A西方墨點法中所顯示的α-Syn寡聚物量，以GAPDH標準化蛋白的量，且未處理健康小鼠模型的溶解物的量經標準化為1.00。 Figures 13A-13B show an untreated healthy mouse model (lane 1) or a PD mouse model (lane 2-3) receiving 3 times of simple adjuvant (lane 2) or containing an α-Syn _111-132 peptide immunogen Amount of α-Syn oligomers from brain samples. Untreated Balb / c mice were healthy mouse models, and MPP + induced mouse models were PD mouse models. Figure 13A is the result of Western blot method, which shows the amount of α-Syn oligomer and GAPDH as a protein loading control in the sample. FIG. 13B compares the amount of α-Syn oligomers shown in 13A Western blot method, normalizes the amount of protein with GAPDH, and normalizes the amount of lysate in untreated healthy mouse models to 1.00.

第14A-14G圖顯示未處理之健康小鼠模型(lane 1)或PD小鼠模型(lane 2-4)接受單純佐劑(lane 2)或含有α-Syn_111-132(lane 3)或α-Syn_126-135(lane 4)的胜肽免疫原後，腦部組織中α-Syn寡聚物量及酪氨酸羥化酶的量。未處理FVB小鼠為健康小鼠模型，α-Syn纖維小鼠模型為PD小鼠模型。第14A圖為西方墨點法分析結果，其顯示同側黑質溶解物中，α-Syn寡聚物、酪氨酸羥化酶以及作為蛋白對照組(protein loading control)的GAPDH量。第14B圖顯示比較第14A圖西方墨點法中所顯示的α-Syn寡聚物量，以GAPDH標準化蛋白的量。將蛋白質的量以GAPDH標準化後，第14B圖顯示比較第14A圖之西方墨點法中相對的α-Syn寡聚物量。將蛋白的量以GAPDH標準化後，第14C圖顯示比較第14A圖之西方墨點法中相對的酪氨酸羥化酶蛋白量。第14D圖為一西方墨點法，其顯示同側紋狀體之α-Syn寡聚物的量，並以GAPDH作為加載控制。將蛋白質的量以GAPDH標準化後，第14E圖顯示比較第14C圖之西方墨點法中相對的α-Syn寡聚物的量。第14F圖為一西方墨點法，其顯示同側紋狀體之α-Syn寡聚物的量，並以GAPDH作為加載控制。將蛋白的量以GAPDH標準化後，第14G圖顯示比較第14E圖之西方墨點法中相對的α-Syn寡聚物量。 Figures 14A-14G show an untreated healthy mouse model (lane 1) or PD mouse model (lane 2-4) that received a simple adjuvant (lane 2) or contained α-Syn _111-132 (lane 3) or α -Syn _126-135 (lane 4) after peptide immunogen, the amount of α-Syn oligomers and the amount of tyrosine hydroxylase in brain tissue. Untreated FVB mice were healthy mouse models, and α-Syn fiber mouse models were PD mouse models. Figure 14A is the result of Western blot analysis, which shows the amount of α-Syn oligomer, tyrosine hydroxylase, and GAPDH as a protein loading control in the ipsilateral nigra solute. Figure 14B shows the amount of α-Syn oligomers compared to the Western blot method shown in Figure 14A to normalize the amount of protein by GAPDH. After normalizing the amount of protein with GAPDH, FIG. 14B shows the relative amount of α-Syn oligomers in the Western blot method compared to FIG. 14A. After normalizing the amount of protein with GAPDH, Fig. 14C shows the relative amount of tyrosine hydroxylase protein in the Western blot method compared to Fig. 14A. Figure 14D is a Western blot method, which shows the amount of α-Syn oligomers in the ipsilateral striatum, with GAPDH as the loading control. After normalizing the amount of protein with GAPDH, Fig. 14E shows the relative amount of α-Syn oligomers in the Western blot method comparing Fig. 14C. Fig. 14F is a Western blot method, which shows the amount of α-Syn oligomers in the ipsilateral striatum, with GAPDH as the loading control. After normalizing the amount of protein with GAPDH, Figure 14G shows the relative amount of α-Syn oligomers in the Western blot method compared to Figure 14E.

第15A-15C圖顯示以CatWalk^TM XT分別分析注射食鹽水(lane 1)或佐劑(lane 2)健康小鼠(lane 1-2)，或以佐劑(lane 3)、含有α-Syn_126-135(lane 4)或α-Syn_111-132(lane 5)之胜肽免疫原免疫PD小鼠模型(lanes 3-5)的運動功能。以t檢定進行顯著性分析(小於0.05的p值被定義為具統計上顯著差異，並以星號“*”表示)。第15A圖為分析經治療之小鼠的左後肢，其中未治療之FVB小鼠作為健康小鼠模型，α-Syn纖維免疫之小鼠作為PD小鼠模型。第15B圖為分析治療小鼠的跑步時期，其中未治療之FVB小鼠作為健康小鼠模型，且α-Syn纖維免疫之小鼠作為PD小鼠模型。第15C圖為分析治療小鼠的跑步時間，其中以未治療之Balb/c小鼠作為健康小鼠模型，MPP+誘導小鼠作為PD小鼠模型。 Figures 15A-15C show CatWalk ^TM XT analysis of healthy mice (lane 1-2) injected with saline (lane 1) or adjuvant (lane 2), or α-Syn ₁₂₆ containing adjuvant (lane 3), respectively. _-135 (lane 4) or α-Syn _111-132 (lane 5) peptide immunogen immunized PD mouse model (lanes 3-5) for motor function. Significance analysis was performed by t test (p-values less than 0.05 were defined as statistically significant differences and indicated by an asterisk "*"). Fig. 15A is an analysis of the left hind limb of treated mice, in which untreated FVB mice were used as healthy mouse models and α-Syn fiber immunized mice were used as PD mouse models. FIG. 15B is an analysis of the running period of the treated mice, in which untreated FVB mice were used as healthy mouse models, and α-Syn fiber-immunized mice were used as PD mouse models. Figure 15C is the analysis of the running time of treated mice, in which untreated Balb / c mice were used as healthy mouse models, and MPP + induced mice were used as PD mouse models.

第16A圖顯示PD-021514抗體(α-Syn_85-140，wpi 08)可識別具最高親和力的α-Syn纖維。觀察到與帶狀(ribbons)和纖維-91有良好的結合。與寡聚物和纖維-65的結合力差。與α-Syn單體和缺少C-端30個胺基酸殘基(Fib-110)之纖維的結合力差。第16B圖顯示PD-021522(α-Syn_85-140，wpi 13)可與所有的種類/寡聚物結合，而不與單體結合。偵測訊號不具有明顯的濃度依賴性增加。抗體可結合至缺少C-端30個胺基酸殘基(Fib-110)的纖維。因此，抗原決定位不在此區域內。第16C圖顯示PD-100806(α-Syn_126-135，wpi 09)可與所有的種類結合，與帶狀α-Syn具有最高的親和力。與天然α-Syn寡聚物結合效率較低。幾乎不與戊二醛、多巴胺交聯低聚物及單體α-syn結合。由於抗體不與缺少C-端30個胺基酸殘基(Fib-110)之纖維結合，因此抗體可能是針對α-syn的30個C-端氨基酸殘基。第16D圖顯示市售抗體Syn-1(clone 42,BD bioscience)除了戊二醛交聯之外，可與所有α-Syn 種類和寡聚體結合。其也可與單體α-Syn結合。其抗原決定位橫跨91至96/99殘基。同樣地，其可與缺少C-端30個胺基酸殘基(Fib-110)之纖維結合。第16E圖顯示與α-Syn單體相比，PRX002抗體對纖維狀α-Syn具有稍微較佳的辦識親和力。第16F圖顯示控制天竺鼠產生的抗體背景。第16G圖顯示控制抗體Syn-1的背景。第16H圖顯示PRX002抗體的背景控制組。 Figure 16A shows that PD-021514 antibody (α-Syn _85-140 , wpi 08) can recognize α-Syn fibers with the highest affinity. Good binding with ribbons and fiber-91 was observed. Poor binding to oligomers and fiber-65. Poor binding with α-Syn monomers and fibers lacking 30 C-terminal amino acid residues (Fib-110). Figure 16B shows that PD-021522 (α-Syn _85-140 , wpi 13) can bind to all species / oligomers, but not to monomers. The detection signal does not have a significant concentration-dependent increase. The antibody can bind to fibers lacking the C-terminal 30 amino acid residues (Fib-110). Therefore, the epitope is not in this region. Figure 16C shows that PD-100806 (α-Syn _126-135 , wpi 09) can be combined with all species, and has the highest affinity with ribbon α-Syn. Low binding efficiency to natural α-Syn oligomers. It hardly binds to glutaraldehyde, dopamine crosslinked oligomer, and monomer α-syn. Since the antibody does not bind to a fiber lacking 30 C-terminal amino acid residues (Fib-110), the antibody may be directed to the 30 C-terminal amino acid residues of α-syn. Figure 16D shows that the commercially available antibody Syn-1 (clone 42, BD bioscience) can bind to all α-Syn species and oligomers except for glutaraldehyde cross-linking. It can also be combined with the monomer α-Syn. Its epitope spans between 91 and 96/99 residues. Similarly, it can bind to fibers lacking 30 C-terminal amino acid residues (Fib-110). Figure 16E shows that PRX002 antibody has slightly better affinity for fibrous α-Syn compared to α-Syn monomer. Figure 16F shows the background of the antibodies produced by guinea pigs. Figure 16G shows the background of the control antibody Syn-1. Figure 16H shows the background control group of PRX002 antibody.

第17A-17D圖為IHC分析UNS抗體對路易氏體(DLB)癡呆患者之基底核中α-Syn的特異性分析。以各抗體(PD062220、PD062205、PD100806及NCL-L-ASYN)染色α-Syn聚集體的平均面積百分比，來測定總面積7.5mm²的殼核(第17A圖)、內囊(第17B圖)及島葉(第17C圖)。第17D圖顯示以各抗體免疫染色殼核中所獲得的代表性顯微影像。UNS抗體可檢測到殼核(F(3,7)=1.550，p=0.284，ANOVA)、內囊(F(3,7)=1.356，p=0.332，ANOVA)和島葉皮質(F(3,8)=2.050，p=0.195，ANOVA)中較高面積百分比的α-Syn聚集體。P<0.05(*)；P<0.01(**)；P<0.001(***)。數值以平均值+SD(誤差線)表示。 Figures 17A-17D show the specificity analysis of α-Syn in the basal nucleus of patients with Lewy body (DLB) dementia by IHC analysis of UNS antibodies. The average area percentage of α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was used to determine the putamen (Figure 17A) and inner capsule (Figure 17B) with a total area of 7.5 mm ² And island leaves (Figure 17C). Figure 17D shows representative microscopic images obtained from immunostaining of the putamen with each antibody. UNS antibodies can detect the putamen (F (3,7) = 1.550, p = 0.284, ANOVA), the inner capsule (F (3,7) = 1.356, p = 0.332, ANOVA), and the insular cortex (F (3 8) = 2.050, p = 0.195, ANOVA) a higher area percentage of α-Syn aggregates. P <0.05 (*); P <0.01 (**); P <0.001 (***). Values are expressed as mean + SD (error bar).

第18A-18D圖為IHC分析UNS抗體對帕金森病(PD)患者之基底核α-Syn的特異性分析。以各抗體(PD062220、PD062205、PD100806及NCL-L-ASYN)染色α-Syn聚集體的平均面積百分比，來測定總面積7.5mm²的殼核(第18A圖)、內囊(第18B圖)及島葉(第18C圖)。第18D圖顯示免疫染色殼核所獲得的代表性顯微影像。UNS抗體可檢測到殼核(F(3,18)=4.152，p=0.047，ANOVA)、內囊(F(3,8)=1.995，p=0.1934，ANOVA)與島葉皮質(F(3,8)=0.4044，p=0.754，ANOVA)中較高面積百分比的α-Syn聚集體。與NCL-L-ASYN相比，PD100806可檢測到顯著更高比例的α-Syn面積。(p=0.023，PD100806 vs NCL-L-ASYN；n=3)。P<0.05(*)；P<0.01(**)； P<0.001(***)。單因子變異數分析ANOVA(One-way ANOVA)後進行Dunnett檢驗。數值以平均值+SD(誤差線)表示。 Figures 18A-18D are specific analysis of IHC analysis of UNS antibodies on basal nucleus α-Syn of patients with Parkinson's disease (PD). The average area percentage of α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was used to determine the putamen (Figure 18A) and inner capsule (Figure 18B) with a total area of 7.5 mm ² And island leaves (Figure 18C). Figure 18D shows a representative microscopic image obtained by immunostaining the putamen. UNS antibodies can detect the putamen (F (3,18) = 4.152, p = 0.047, ANOVA), the inner capsule (F (3,8) = 1.995, p = 0.1934, ANOVA) and the insular cortex (F (3 8) = 0.4044, p = 0.754, ANOVA) a higher area percentage of α-Syn aggregates. Compared with NCL-L-ASYN, PD100806 can detect a significantly higher proportion of α-Syn area. (p = 0.023, PD100806 vs NCL-L-ASYN; n = 3). P <0.05 (*); P <0.01 (**); P <0.001 (***). Dunnett's test was performed after One-way ANOVA (One-way ANOVA). Values are expressed as mean + SD (error bar).

第19A-19B圖為IHC分析UNS抗體對多重系統退化症(MSA)患者之基底核α-Syn的特異性分析。以各抗體(PD062220、PD062205、PD100806及NCL-L-ASYN)染色α-Syn聚集體的平均面積百分比，來測定3名MSA患者總面積7.5mm²的殼核(第19A圖)及內囊(第19B圖)。在MSA患者的島葉皮質中未檢測到病理特徵，因此未進行定量。UNS抗體可檢測到殼核(F(3,8)=1.56，p=0.273，ANOVA)與內囊(F(3,8)=1.126，p=0.395，ANOVA)中較高面積百分比的α-Syn聚集體。P<0.05(*)；P<0.01(**)；P<0.001(***)。數值以平均值+SD(誤差線)表示。 Figures 19A-19B are specific analysis of the basal nucleus α-Syn of patients with multiple system degenerative disease (MSA) by IHC analysis of UNS antibodies. Average area percentages of each antibody (PD062220, PD062205, PD100806 and NCL-L-ASYN) α-Syn stained aggregates, MSA patients to determine the total area of 7.5mm ² 3 putamen (second FIG. 19A) and internal capsule ( (Figure 19B). No pathological features were detected in the insular cortex of MSA patients, and therefore were not quantified. UNS antibody can detect α- in the higher area percentage in the putamen (F (3,8) = 1.56, p = 0.273, ANOVA) and inner capsule (F (3,8) = 1.126, p = 0.395, ANOVA). Syn aggregates. P <0.05 (*); P <0.01 (**); P <0.001 (***). Values are expressed as mean + SD (error bar).

第20A-20E圖為IHC分析UNS抗體對不同突觸核蛋白疾病患者之基底核α-Syn的特異性分析。以各抗體(PD062220、PD062205、PD100806及NCL-L-ASYN)染色α-Syn聚集體的平均面積百分比，來測定PD(第20A圖)、DLB(第20B圖)與MSA(第20C圖)患者總面積7.5mm²的黑質。比較各種抗體與診斷抗體NCL-L-ASYN的染色面積百分比。UNS抗體可檢測到MSA(F(3,8)=0.830，p=0.51，ANOVA)、DLB(F(3,7)=2.493，p=0.144，ANOVA)與PD(F(3,7)=0.189，p=0.900，ANOVA)患者黑質中α-Syn聚集體的面積百分比較高。第20D圖(MSA)和第20E圖中顯示各抗體免疫染色的代表性顯微鏡圖像。P<0.05(*)；P<0.01(**)；P<0.001(***)。數值以平均值+SD(誤差線)表示。 Figures 20A-20E are specific analysis of the basal nucleus α-Syn of patients with different synuclein diseases by IHC analysis of UNS antibodies. The average area percentage of α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was used to determine PD (Figure 20A), DLB (Figure 20B), and MSA (Figure 20C) patients. Substantia nigra with a total area of 7.5 mm ² . Compare the percentage of stained area of various antibodies with the diagnostic antibody NCL-L-ASYN. UNS antibodies can detect MSA (F (3,8) = 0.830, p = 0.51, ANOVA), DLB (F (3,7) = 2.493, p = 0.144, ANOVA) and PD (F (3,7) = 0.189, p = 0.900, ANOVA). The percentage of α-Syn aggregates in the substantia nigra was higher. Figures 20D (MSA) and 20E show representative microscope images of each antibody immunostaining. P <0.05 (*); P <0.01 (**); P <0.001 (***). Values are expressed as mean + SD (error bar).

第21A-21F圖為IHC分析UNS抗體對具不同突觸核蛋白病患之顳葉皮質的白質與灰質中α-Syn的特異性分析。以各抗體(PD062220、PD062205、PD100806及NCL-L-ASYN)染色α-Syn聚集體的平均面積百分比，來測定PD(第21A與21D圖)、DLB(第21B與21E圖)與MSA(第21C與21F圖) 患者總面積7.5mm²的皮質灰質及皮質白質。比較各抗體與診斷抗體NCL-L-ASYN的染色面積百分比。P<0.05(*)；P<0.01(**)；P<0.001(***)。單因子變異數分析ANOVA(One-way ANOVA)後進行Dunnett檢驗。數值以平均值+SD(誤差線)表示。 Figures 21A-21F show IHC analysis of the specificity of the UNS antibody for α-Syn in the white and gray matter of the temporal lobe cortex of patients with different synuclein diseases. The average area percentage of α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was used to determine PD (Figure 21A and 21D), DLB (Figure 21B and 21E), and MSA (Figure 21C and 21F) Cortical gray matter and cortical white matter with a total area of 7.5 mm ² . The percentage of stained area between each antibody and the diagnostic antibody NCL-L-ASYN was compared. P <0.05 (*); P <0.01 (**); P <0.001 (***). Dunnett's test was performed after One-way ANOVA (One-way ANOVA). Values are expressed as mean + SD (error bar).

第22A-22C圖為IHC分析UNS抗體對具不同突觸核蛋白病患之小腦中α-Syn的特異性分析。以各抗體(PD062220、PD062205、PD100806及NCL-L-ASYN)染色α-Syn聚集體的平均面積百分比，來測定PD(第22A圖)、DLB(第22B圖)與MSA(第22C圖)患者總面積7.5mm²的小腦。UNS抗體可檢測到MSA(F(3,8)=0.929，p=0.469，ANOVA)；DLB(F(3,6)=1.426，p=0.325，ANOVA)與PD(F(3,6)=2.509，p=0.157，ANOVA)患者中α-Syn聚集體的面積百分比較高。比較各抗體與診斷抗體NCL-L-ASYN的染色面積百分比。P<0.05(*)；P<0.01(**)；P<0.001(***)。數值以平均值+SD(誤差線)表示。 Figures 22A-22C are specific analysis of α-Syn in cerebellum of patients with different synuclein by IHC analysis of UNS antibodies. The average area percentage of α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was used to determine patients with PD (Figure 22A), DLB (Figure 22B), and MSA (Figure 22C). Cerebellum with a total area of 7.5 mm ² . UNS antibody can detect MSA (F (3,8) = 0.929, p = 0.469, ANOVA); DLB (F (3,6) = 1.426, p = 0.325, ANOVA) and PD (F (3,6) = 2.509, p = 0.157, ANOVA) The area percentage of α-Syn aggregates was higher in patients. The percentage of stained area between each antibody and the diagnostic antibody NCL-L-ASYN was compared. P <0.05 (*); P <0.01 (**); P <0.001 (***). Values are expressed as mean + SD (error bar).

第23A-23B圖為非病患對照組大腦黑質(第23A圖)和殼核(第23B圖)以各抗體免疫染色的代表性圖像。UNS抗體未檢測到任何α-Syn病理，與診斷抗體NCL-L-ASYN結果一致。 Figures 23A-23B are representative images of brain substantia nigra (Figure 23A) and putamen (Figure 23B) of non-patient control groups with each antibody immunostaining. UNS antibody did not detect any α-Syn pathology, which was consistent with the results of the diagnostic antibody NCL-L-ASYN.

第24A-24D圖為IHC分析UNS抗體對DLB或PD患者基底核島葉中的LBs的α-Syn特異性分析。以各抗體(PD062220、PD062205、PD100806及NCL-L-ASYN)陽性免疫LBs的平均面積百分比，來測定PD(第24A圖)與DLB(第24B圖)患者總面積7.5mm²的島葉。LB的面積百分比為各抗體檢測到的總α-Syn比例。以各抗體(PD062220、PD062205、PD100806及NCL-L-ASYN)染色α-Syn聚集體的平均面積百分比，來測定PD(第24A圖)與DLB(第24B圖)患者總面積7.5mm²的島葉。LBs的百分比面積為各抗體檢測到的總α-Syn比例。UNS抗體可檢測到DLB(F(3,7)=0.836， p=0.516，ANOVA)與PD(F(3,4)=0.913，p=0.510，ANOVA)患者島葉中LBs(或較高比例LNs)的面積百分比較低。比較各抗體與診斷抗體NCL-L-ASYN的染色面積百分比。第24C(PD)圖與第24D(DLB)圖顯示各抗體免疫染色的代表性顯微鏡圖像。P<0.05(*)；P<0.01(**)；P<0.001(***)。數值以平均值+SD(誤差線)表示。 Figures 24A-24D are IHC analysis of α-Syn specific analysis of LBs in basal nucleus leaves of DLB or PD patients by UNS antibodies. The average area percentage of each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) -positive LBs was used to determine the total leaf area of 7.5 mm ² in patients with PD (Figure 24A ) and DLB (Figure 24B ). The area percentage of LB is the total α-Syn ratio detected by each antibody. The average area percentage of α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was used to determine an island with a total area of 7.5 mm ² in patients with PD (Figure 24A) and DLB (Figure 24B). leaf. The percentage area of LBs is the ratio of total α-Syn detected by each antibody. UNS antibody can detect LBs (or higher proportion) in island leaves of patients with DLB (F (3,7) = 0.836, p = 0.516, ANOVA) and PD (F (3,4) = 0.913, p = 0.510, ANOVA) LNs) has a lower area percentage. The percentage of stained area between each antibody and the diagnostic antibody NCL-L-ASYN was compared. Figures 24C (PD) and 24D (DLB) show representative microscope images of each antibody immunostaining. P <0.05 (*); P <0.01 (**); P <0.001 (***). Values are expressed as mean + SD (error bar).

第25A-25D圖為IHC分析UNS抗體對DLB或PD病患之顳葉皮質中灰質的特異性分析。以各抗體(PD062220、PD062205、PD100806及NCL-L-ASYN)染色α-Syn聚集體的平均面積百分比，來測定PD(第25A圖)與DLB(第25B圖)患者總面積7.5mm²的灰質。LBs的面積百分比為各抗體檢測到之總α-突觸核蛋白的比例。UNS抗體可檢測到PD(F(2,3)=1.983，p=0.282，ANOVA)與DLB(F(3,7)=1.906，p=0.217，ANOVA)患者灰質中LBs(或較高比例LNs)的面積百分比較低。比較各抗體與診斷抗體NCL-L-ASYN的染色面積百分比。第25C圖(PD)與第25D圖(DLB)顯示各抗體免疫染色的代表性顯微鏡圖像。P<0.05(*)；P<0.01(**)；P<0.001(***)。數值以平均值+SD(誤差線)表示。 Figures 25A-25D are specific analysis of gray matter in temporal lobe cortex of patients with DLB or PD by UNS antibody analysis by IHC. The average area percentage of α-Syn aggregates stained with each antibody (PD062220, PD062205, PD100806, and NCL-L-ASYN) was used to determine the gray area of 7.5 mm ² in the total area of patients with PD (Figure 25A) and DLB (Figure 25B). . The area percentage of LBs is the proportion of total α-synuclein detected by each antibody. UNS antibody can detect LBs (or higher proportion of LNs) in gray matter of patients with PD (F (2,3) = 1.983, p = 0.282, ANOVA) and DLB (F (3,7) = 1.906, p = 0.217, ANOVA) ) Has a lower area percentage. The percentage of stained area between each antibody and the diagnostic antibody NCL-L-ASYN was compared. Figures 25C (PD) and 25D (DLB) show representative microscope images of each antibody immunostaining. P <0.05 (*); P <0.01 (**); P <0.001 (***). Values are expressed as mean + SD (error bar).

第26A-26B圖為以UNS抗體及NCL-L-ASYN免疫染色DLB(第26A圖)與PD(第26B圖)患者中腦黑質的代表性圖像。與NCL-L-ASYN相比，UNS抗體對LN具有較高程度的檢測能力。 Figures 26A-26B are representative images of brain substantia nigra in patients with DLB (Figure 26A) and PD (Figure 26B) immunostained with UNS antibodies and NCL-L-ASYN. Compared with NCL-L-ASYN, UNS antibodies have a higher degree of detection of LN.

第27A-27C圖為α-Syn的細胞特異性聚集。PD(第27A圖)、DLB(第27B圖)與MSA(第27C圖)患者基底核和人類中腦α-Syn聚集體的最大投影覆蓋共軛焦影像。神經元(HuD，綠色)內的α-Syn(PD062205，紅色)聚集存在於PD與DLB患者，而非MSA。於黑白圖像中標記α-Syn(PD062205)和HuD，可依要求提供彩色圖像。比例尺：10μM。 Figures 27A-27C show cell-specific aggregation of α-Syn. The largest projections of basal nucleus and human midbrain α-Syn aggregates in patients with PD (figure 27A), DLB (figure 27B) and MSA (figure 27C) covered conjugate focus images. Α-Syn (PD062205, red) in neurons (HuD, green) aggregated in patients with PD and DLB, but not MSA. Mark α-Syn (PD062205) and HuD in black and white images, and provide color images on request. Scale bar: 10 μM.

第28A-28C圖為α-Syn的細胞特異性聚集。PD(第28A圖)、DLB(第 28B圖)與MSA(第28C圖)患者中α-Syn聚集體的最大投影覆蓋共軛焦影像。少突膠質細胞內(Olig2，綠色)的α-Syn(PD062205，紅色)聚集存在於MSA患者，而非PD與DLB患者。於黑白圖像中標記α-Syn(PD062205)與Olig2，可依要求提供彩色圖像。比例尺：10μM。 Figures 28A-28C show cell-specific aggregation of α-Syn. The largest projections of α-Syn aggregates in patients with PD (Figure 28A), DLB (Figure 28B), and MSA (Figure 28C) covered conjugate focus images. Oligodendrocyte (Olig2, green) α-Syn (PD062205, red) aggregates exist in patients with MSA, not patients with PD and DLB. Mark α-Syn (PD062205) and Olig2 in black and white images, and provide color images on request. Scale bar: 10 μM.

本發明係有關於α-突觸核蛋白(α-Syn)胜肽免疫原結構。本發明更有關於含有胜肽免疫原結構的組成物、製造及使用此胜肽免疫原結構的方法，以及此胜肽免疫原結構所產生的抗體。 The invention relates to the structure of an α-synuclein (α-Syn) peptide immunogen. The invention further relates to a composition containing a peptide immunogen structure, a method for manufacturing and using the peptide immunogen structure, and an antibody generated by the peptide immunogen structure.

本發明之胜肽免疫原結構含有一源自α-Syn的B細胞抗原決定位，其直接或透過一選擇性異源性連接子連接一異源性T輔助細胞(Th)抗原決定位。胜肽免疫原結構的B細胞抗原決定位含有源自α-Syn C-端區域約10至約25個胺基酸，其對應於全長α-Syn(SEQ ID NO：1)的約第111個甘氨酸(G111)至約第135個天門冬胺酸(D135)的序列。胜肽免疫原結構的異源性Th抗原決定位部分源自於致病蛋白的胺基酸序列。胜肽免疫原結構的B細胞抗原決定位及Th抗原決定位在給予至一個體時，可共同作用刺激專一性辨識及結合α-Syn B細胞抗原決定位的抗體。 The peptide immunogen structure of the present invention contains a B-cell epitope derived from α-Syn, which is connected to a heterologous T helper cell (Th) epitope directly or through a selective heterologous linker. The B cell epitope of the peptide immunogen structure contains about 10 to about 25 amino acids derived from the C-terminal region of α-Syn, which corresponds to about 111th of the full-length α-Syn (SEQ ID NO: 1) Sequence of glycine (G111) to about 135th aspartic acid (D135). The heterologous Th epitope of the peptide immunogen structure is derived in part from the amino acid sequence of the pathogenic protein. When the B-cell epitope and Th epitope of the peptide immunogen structure are administered to a single body, they can work together to stimulate antibodies that specifically recognize and bind to the α-Syn B-cell epitope.

本發明更有關於含有胜肽免疫原結構的組成物，包括醫藥組成物。此醫藥組成物可在一個體中刺激免疫反應及產生針對胜肽免疫原結構的抗體。此組成物可含有一或一個以上之胜肽免疫原結構的混合物。在某些實施例中，此組成物含有本發明胜肽免疫原結構及額外的成分，包括載體、佐劑、緩衝液及其它適合的試劑。在特定實施例中，此組成物含有本發明胜肽免疫原結構與一CpG寡聚物形成穩定的免疫刺激複合物，其選擇性地補充一佐劑。 The invention further relates to a composition containing a peptide immunogen structure, including a pharmaceutical composition. This pharmaceutical composition can stimulate the immune response and produce antibodies against the peptide immunogen structure in one body. This composition may contain A mixture of one or more peptide immunogen structures. In certain embodiments, this composition contains the peptide immunogen structure of the present invention and additional ingredients, including carriers, adjuvants, buffers, and other suitable reagents. In a specific embodiment, the composition contains the peptide immunogen structure of the present invention and a CpG oligomer to form a stable immunostimulatory complex, which selectively supplements an adjuvant.

本發明更有關於免疫一個體本發明胜肽免疫原結構以產生的抗體。本發明抗體專一性辨識及結合胜肽免疫原結構的α-Syn B細胞抗原決定位部分。本發明α-Syn抗體對於單體、寡聚物或α-Syn纖維之β-sheet具有一不可預期之高交叉反應性。基於其獨特的特徵和性質，本發明抗體可提供一免疫治療方式以標定、辨別及治療突觸核蛋白疾病。 The invention is even more concerned with antibodies produced by immunizing a peptide immunogen structure of the invention. The antibody of the present invention specifically recognizes and binds an epitope portion of an α-Syn B cell of a peptide immunogen structure. The α-Syn antibodies of the present invention have an unexpectedly high cross-reactivity to β-sheets of monomers, oligomers or α-Syn fibers. Based on its unique characteristics and properties, the antibodies of the present invention can provide an immunotherapeutic method for calibrating, identifying, and treating synuclein diseases.

本發明更有關於製造及使用本發明胜肽免疫原結構、抗體及組成物的方法。本發明方法提供低製造成本及胜肽免疫原結構的品質控管及含有胜肽免疫原結構的組成物，其可用於抑制及治療病症。 The invention further relates to methods for making and using the peptide immunogen structure, antibodies, and compositions of the invention. The method of the present invention provides low-cost manufacturing quality control of the peptide immunogen structure and a composition containing the peptide immunogen structure, which can be used for inhibiting and treating disorders.

本發明更包括使用本發明胜肽免疫原結構及/或針對胜肽免疫原結構之抗體，治療及/或抑制突觸核蛋白疾病的方法。在某些實施例中，治療及/或抑制突觸核蛋白疾病的方法包括給予一個體一含有本發明胜肽免疫原結構的組成物。在特定實施例中，此方法中所使用的組成物含有本發明胜肽免疫原結構與帶負電的寡核苷酸，如CpG寡聚物，透過靜電偶合以形成一穩定的免疫刺激複合物，此複合物更選擇性補充礦物鹽或油作為佐劑，給予至突觸核蛋白疾病的患者。本發明之方法也包括給藥設計、劑型及途徑，以給予此胜肽免疫原結構至一具罹患或具有突觸核蛋白疾病的個體。 The present invention further includes a method for treating and / or inhibiting a synuclein disease using the peptide immunogen structure of the present invention and / or an antibody against the peptide immunogen structure. In certain embodiments, a method of treating and / or inhibiting a synuclein disease comprises administering to a subject a composition comprising a peptide immunogen structure of the invention. In a specific embodiment, the composition used in this method contains the peptide immunogen structure of the present invention and a negatively charged oligonucleotide, such as a CpG oligomer, through electrostatic coupling to form a stable immunostimulatory complex This complex is more selectively supplemented with mineral salts or oils as an adjuvant to patients with synuclein diseases. The method of the present invention also includes the administration design, dosage form and route to administer the peptide immunogen structure to an individual suffering from or having a synuclein disease.

發明說明中的章節標題僅用於組織目的，不用用於限制所描述的主題。所有或部份引用的文獻皆可依據其整體對任何目的併入發明說明中。 The section headings in the description are for organizational purposes only and are not intended to limit the subject matter described. All or part of the cited documents may be incorporated into the description of the invention for any purpose in its entirety.

除特別說明，本發明所屬領域人士可通常了解本發明說明中所有使用之技術及科學用語的相同含義。若無特別說，單數用語“一”及“該”包括多個標示。同樣地，除非上下文另有明確說明，用語“或”包括“和”。因此，“包括A或B”係指包含A或B，或A及B。更應了解的是，對於多胜肽所有的胺基酸大小與所有的分子量或分子量值為近似的，且揭示於本發明說明中。儘管也可用與發明說明中所述相似或相同的方法和材料進行及測試本發明之方法，但適當的方法及材料揭示於後文。發明說明中所有的文獻、專利申請案、專利及其它參考文獻皆可併入發明說明中。若有衝突，包含用語解釋在內的發明說明會進行說明。此外，材料、方法和實施例僅用於進行說明，而非用於限制。 Unless otherwise specified, those skilled in the art can generally understand the same meanings of all technical and scientific terms used in the description of the present invention. Unless otherwise specified, the singular terms "a" and "the" include plural designations. Likewise, the term "or" includes "and" unless the context clearly dictates otherwise. Therefore, "including A or B" means including A or B, or A and B. It should be further understood that all amino acid sizes for polypeptides are similar to all molecular weights or molecular weight values and are disclosed in the description of the present invention. Although methods and materials similar or identical to those described in the description of the invention can also be used in the tests and methods of the present invention, suitable methods and materials are disclosed below. All documents, patent applications, patents, and other references in the description of the invention may be incorporated into the description of the invention. In the event of conflict, the description of the invention, including terminology, will be explained. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

α-Syn胜肽免疫原結構α-Syn peptide immunogen structure

本發明說明提供胜肽免疫原結構，含有一源自α-Syn之B細胞抗原決定位，其直接或透過一選擇性異源連接子共價連接異源性T輔助細胞(Th)抗原決定位。 The invention provides a peptide immunogen structure containing a B-cell epitope derived from α-Syn, which is covalently linked to a heterologous T helper cell (Th) epitope directly or through a selective heterolinker. .

本發明所述“α-Syn胜肽免疫原結構”係指一胜肽含有(a)源自α-Syn C-端之具有約10至25個胺基酸的B細胞抗原決定位，其對應於全長α-Syn(SEQ ID NO：1)約第111個位置的甘氨酸(G111)至第135個位置的天門冬胺酸(D135)的序列；(b)一異源性Th抗原決定位；(c)一選擇性的異源性連接子。 The “α-Syn peptide immunogen structure” in the present invention means that a peptide contains (a) a B-cell epitope with about 10 to 25 amino acids derived from the C-terminus of α-Syn, which corresponds to The sequence of glycine (G111) to aspartic acid (D135) at about the 111th position of the full-length α-Syn (SEQ ID NO: 1); (b) a heterologous Th epitope; (c) A selective heterologous linker.

在特定實施例中，胜肽免疫原結構可以下式表示：(Th)_m-(A)_n-(α-Syn C-端片段)-X或(α-Syn C-端片段)-(A)_n-(Th)_m-X其中Th為一異源性T輔助抗原決定位；A為一異源性連接子；(α-Syn C-端片段)為一源自α-Syn C-端片段之具有約10至25個胺基酸的B細胞抗原決定位；X為一胺基酸的α-COOH或α-CONH₂；M為1至約4；且N為0至約10。 In a specific embodiment, the peptide immunogen structure can be represented by the formula: (Th) _m- (A) _n- (α-Syn C-terminal fragment) -X or (α-Syn C-terminal fragment)-(A ) _n- (Th) _m -X where Th is a heterologous T helper epitope; A is a heterologous linker; (α-Syn C-terminal fragment) is an α-Syn C-terminal The fragment has B-cell epitopes of about 10 to 25 amino acids; X is α-COOH or α-CONH ₂ of a monoamino acid; M is 1 to about 4; and N is 0 to about 10.

α-Syn胜肽免疫原結構的各種組成如下所示。 The various components of the α-Syn peptide immunogen structure are shown below.

a.α-Syn及α-Syn C-端片段a. α-Syn and α-Syn C-terminal fragments

本發明所述“α-Syn”、“alpha-突觸核蛋白”、“α-突觸核蛋白”及其類似係指來自於任何表現 α-Syn之組織的(a)全長α-Syn蛋白及/或(b)其片段。α-Syn具有極端的構象多樣性，在膜結合、細胞質及澱粉樣蛋白聚集中適應不同的條件，且具有多種功能。在某些實施例中，α-Syn蛋白來自於人類。在特定實施例，全長人類α-Syn蛋白具有140胺基酸(Accession No.NP_000336)(SEQ ID NO：1)。 The “α-Syn”, “alpha-synuclein”, “α-synuclein” and the like in the present invention are derived from any manifestation (a) The full-length α-Syn protein and / or (b) a fragment of α-Syn. α-Syn has extreme conformational diversity, adapts to different conditions in membrane binding, cytoplasm, and amyloid aggregation, and has multiple functions. In certain embodiments, the alpha-Syn protein is from a human. In a specific embodiment, the full-length human α-Syn protein has a 140 amino acid (Accession No. NP_000336) (SEQ ID NO: 1).

本發明所述α-Syn的“C-端區”或“C-端”係指任何源自α-SynC-端的胺基酸序列。在特定實施例中，α-Syn的C-端區或C-端為具有96-140個殘基的胺基酸序列，或其片段。α-Syn的C-端區富含脯氨酸且帶負電，其為本質上無規蛋白質中常見的特性，可維持溶解度。由於低疏水性和高淨負電荷，α-Syn的C-端區通常以無規環狀結構存在。在胞外研究中，已顯示pH下降中和負電荷可促進α-Syn的聚集。 The "C-terminal region" or "C-terminus" of α-Syn according to the present invention refers to any amino acid sequence derived from the C-terminus of α-Syn. In a specific embodiment, the C-terminal region or C-terminus of α-Syn is an amino acid sequence having 96-140 residues, or a fragment thereof. The C-terminal region of α-Syn is rich in proline and negatively charged, which is a characteristic common in essentially random proteins that maintains solubility. Due to low hydrophobicity and high net negative charge, the C-terminal region of α-Syn usually exists as a random ring structure. In extracellular studies, it has been shown that a decrease in pH to neutralize negative charges can promote the aggregation of α-Syn.

本發明所述“α-Syn C-端片段”或“源自α-Syn C-端之B細胞抗原決定位”係指全長α-Syn序列的一部分，其包括約10至25個源自α-Syn C-端的胺基酸，其對應於全長α-Syn約第111個位置的甘氨酸(G111)至第135個位置的天門冬胺酸(D135)的序列。α-Syn C-端片段也可為α-Syn G111-D135胜肽及其片段。本發明各種之α-Syn C-端片段係以全長α-Syn(SEQ ID NO：1)中相對應的各種胺基酸位置表示。 The “α-Syn C-terminal fragment” or “B-cell epitope derived from the α-Syn C-terminal” in the present invention refers to a part of the full-length α-Syn sequence, which includes about 10 to 25 -A C-terminal amino acid of Syn, which corresponds to the sequence of glycine (G111) to aspartic acid (D135) at about the 111th position of the full-length α-Syn. The α-Syn C-terminal fragment may also be α-Syn G111-D135 peptide and fragments thereof. The various α-Syn C-terminal fragments of the present invention are represented by corresponding amino acid positions in the full-length α-Syn (SEQ ID NO: 1).

α-Syn胜肽免疫結構中所使用的α-Syn C-端片段胺基酸序列係依據一設計原理進行選擇。這些原理包括使用一α-Syn胜肽序列：(i)因β-Syn可結合至α-Syn且防止其聚集，所以其與β-突觸核蛋白(β-Syn)不具有明顯的序列同源性，以避免產生會與β-Syn交叉反應的抗體；(ii)其不具有α-Syn中的自體T輔助抗原決定位，以避免自體性T細胞活化，如先前使用針對Aβ_1-42之AN1792疫苗來治療阿茲海默症的臨床試驗，其會導致腦部發炎造成急性腦膜炎(meningococcal encephalitis)；(iii)其為α-Syn中的一個區域，其容易受天然結構之構象改變影響；(iv)因其為自身分子，其為非免疫原性；(v)其可藉由一蛋白載體或一有效的T輔助抗原決定位提供免疫原性；(vi)當具有免疫原性且給予至一個體時：(a)刺激高力價針對α-Syn胜肽序列(B細胞抗原決定位)抗體，且抗體不會針對蛋白質載體或有效的T輔助抗原決定位；(b)刺激高力價抗體，其可與單體、寡聚體或α-Syn纖維的變性β-折疊反應，使此抗體可避免α-Syn形成聚集，使任何的α-Syn聚集分解，且移除毒性α-Syn寡聚體、聚集及/或纖維，以減少或避免腦內的α-Syn聚集負荷；(c)不會刺激與天然α-Syn反應的抗體，因為天然α-Syn為一組織分佈廣泛的主要細胞蛋白，可提供高安全性。 The α-Syn C-terminal fragment amino acid sequence used in the α-Syn peptide immune structure is selected according to a design principle. These principles include the use of an α-Syn peptide sequence: (i) because β-Syn can bind to α-Syn and prevent it from aggregating, it has no obvious sequence identity with β-synuclein (β-Syn) Derived to avoid the production of antibodies that will cross-react with β-Syn; (ii) it does not have the auto-T helper epitope in α-Syn to avoid autologous T cell activation, as previously used against Aβ _{1 -42} of AN1792 vaccine in clinical trials to treat Alzheimer's disease, which can cause brain inflammation and acute meningococcal (meningococcal encephalitis); (iii) it is a region in α-Syn, which is easily affected by natural structures Conformation change effect; (iv) It is non-immunogenic because it is a self-molecule; (v) It can provide immunogenicity through a protein carrier or an effective T helper epitope; (vi) When it is immune When it is native and given to one individual: (a) stimulate high-valence antibodies against α-Syn peptide sequence (B cell epitope), and the antibody will not target protein carriers or effective T helper epitopes; (b) ) Stimulate high potency antibodies, which can react with denatured β-sheets of monomers, oligomers, or α-Syn fibers This antibody can prevent the formation of α-Syn aggregation, decompose any α-Syn aggregation, and remove toxic α-Syn oligomers, aggregations and / or fibers to reduce or avoid the α-Syn aggregation load in the brain; (c) It does not stimulate antibodies that react with natural α-Syn, because natural α-Syn is a major cellular protein widely distributed in tissues, which can provide high safety.

在考慮這些設計原理下，可選擇α-Syn的C-端區域作為胜肽免疫原設計的標的。此外，基於結構特徵，與其他α-Syn區域相比，更容易被抗體或其他物理因子所調控，因此選擇α-Syn的C-端區域。 Taking these design principles into consideration, the C-terminal region of α-Syn can be selected as the target of peptide immunogen design. In addition, based on the structural characteristics, compared to other α-Syn regions, they are more easily regulated by antibodies or other physical factors, so the C-terminal region of α-Syn was selected.

評估多種源自於α-Syn的胜肽序列，以確定及選擇多種符合設計原理的α-Syn胜肽，在實施例中進一步描述。特別是，符合設計原理的序列具有約10至25個胺基酸，其相對應於全長α-Syn約第111個位置的甘氨酸(G111)至第135個位置的天門冬胺酸(D135)。 Various peptide sequences derived from α-Syn are evaluated to determine and select multiple α-Syn peptides that meet design principles, as further described in the Examples. In particular, a sequence that conforms to the design principle has about 10 to 25 amino acids, which correspond to glycine (G111) to aspartic acid (D135) at about the 111th position of the full-length α-Syn.

在某些實施例中，α-Syn的C-端片段為25個胺基酸的α-Syn G111-D135胜肽(SEQ ID NO：12)。在其它實施例中，α-Syn的C-端片段為含有α-Syn G111-D135胜肽(SEQ ID NO：12)的約10個連續胺基酸。在特定實施例中，α-Syn的C-端片段含有α-Syn G111-D135胜肽(SEQ ID NO：12)的10、11、12、13、14、15、16、17、18、19、20、21、22、23、24或25個連續胺基酸。在特定實施例中，α-Syn的C-端片段具有一胺基酸序列，如表1所述SEQ ID NOs：12-15、17或49-64。 In certain embodiments, the C-terminal fragment of α-Syn is an α-Syn G111-D135 peptide (SEQ ID NO: 12) of 25 amino acids. In other embodiments, the C-terminal fragment of α-Syn is about 10 consecutive amino acids containing α-Syn G111-D135 peptide (SEQ ID NO: 12). In a specific embodiment, the C-terminal fragment of α-Syn contains 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 of the α-Syn G111-D135 peptide (SEQ ID NO: 12). , 20, 21, 22, 23, 24, or 25 consecutive amino acids. In a specific embodiment, the C-terminal fragment of α-Syn has an amino acid sequence, such as SEQ ID NOs: 12-15, 17 or 49-64 as described in Table 1.

本發明的α-Syn C-端片段也包括α-Syn G111-D135胜肽及其片段的免疫原性功能類似物或同源物。G111-D135胜肽及其片段的功能免疫類似物或同源物包括各種實質上保有與原始胜肽相同免疫原性的變異物。免疫原性功能類似物可在一胺基酸位置上有一保守取代；在整體電荷有所改變；共價連接其它單元；或胺基酸的增加、***或刪除及/或其任何組合。 The α-Syn C-terminal fragment of the present invention also includes immunogenic functional analogs or homologues of α-Syn G111-D135 peptides and fragments thereof. The functional immune analogs or homologues of G111-D135 peptides and fragments thereof include various variants that retain substantially the same immunogenicity as the original peptide. Immunogenic functional analogues may have a conservative substitution at an amino acid position; Changes in overall charge; covalent attachment of other units; or addition, insertion or deletion of amino acids and / or any combination thereof.

保守取代為一胺基酸被置換為其它具有類似化學特性的胺基酸。例如，非極性(疏水性)胺基酸包括丙胺酸、亮胺酸、異亮胺酸、纈胺酸、脯胺酸、***酸、色胺酸與甲硫胺酸；極性中性胺基酸包括甘胺酸、絲胺酸、蘇胺酸、半胱胺酸、酪胺酸、天門冬醯胺與麩醯胺酸；帶正電(鹼性)胺基酸包括精胺酸、賴胺酸和組胺酸；以及帶負電(酸性)胺基酸包括天門冬胺酸與谷胺酸。 Conservative substitutions are replaced by other amino acids with similar chemical properties. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids Includes glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine And histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

免疫原性功能類似物包括具有1至約4個胺基酸保守性取代、增加、刪除或***的胺基酸序列，其可刺激與α-Syn G111-D135胜肽交叉反應的免疫反應。保守性取代、增加、刪除或***可以天然或非天然胺基酸完成。非天然胺基酸包括，但不限於，ε-N賴胺酸、ß-丙胺酸、鳥氨酸、正白胺酸、正纈胺酸、羥脯氨酸、甲狀腺素、γ-胺基丁酸、高絲胺酸、瓜胺酸、胺基苯甲酸、6-氨基己酸(Aca；6-Aminohexanoic acid)、羥脯胺酸、硫醇丙酸(MPA)、3-硝基-酪氨酸、焦谷胺酸及其類似。天然胺基酸包括丙胺酸、精胺酸、天門冬醯胺、天門冬胺酸、半胱胺酸、谷胺酸、麩醯胺酸、甘氨酸、組氨酸、異亮胺酸、亮胺酸、賴胺酸、甲硫胺酸、***酸、脯胺酸、絲胺酸、蘇胺酸、色胺酸、酪胺酸及纈胺酸。 Immunogenic functional analogs include amino acid sequences with conservative substitutions, additions, deletions, or insertions of 1 to about 4 amino acids, which can stimulate an immune response that cross-reacts with the α-Syn G111-D135 peptide. Conservative substitutions, additions, deletions or insertions can be done with natural or unnatural amino acids. Unnatural amino acids include, but are not limited to, ε-N lysine, ß-alanine, ornithine, n-leucine, n-valine, hydroxyproline, thyroxine, γ-amine Acid, homoseric acid, citrulamic acid, aminobenzoic acid, 6-aminocaproic acid (Aca; 6-Aminohexanoic acid), hydroxyproline, thiopropionic acid (MPA), 3-nitro-tyrosine , Pyroglutamic acid and similar. Natural amino acids include alanine, arginine, aspartic acid, aspartic acid, cysteine, glutamic acid, glutamic acid, glycine, histidine, isoleucine, leucine , Lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

在一實施例中，一特定胜肽的功能性免疫類似物包含與原胜胺相同的胺基酸序列，且更包括在α-Syn G111-D135胜肽及其片段的胺基端增加3個賴胺酸(Lys-Lys-Lys)。在此實施例中，此3個賴胺酸融合至原胜肽會改變原胜肽整體的電荷，但不會改變原胜肽的功能。 In one embodiment, the functional immune analogue of a specific peptide contains the same amino acid sequence as the original peptide, and is further included in α-Syn G111-D135 peptide and its fragment have 3 lysine (Lys-Lys-Lys) added to the amine end. In this embodiment, the fusion of the three lysines to the original peptide will change the overall charge of the original peptide, but will not change the function of the original peptide.

在特定實施例中，α-Syn C-端片段的功能類似物與原胜胺序列具有至少50%的相似度。在其它實施例中，功能類似與原胜胺具有至少80%的相似度。在其它實施例中，功能類似與原胜胺具有至少85%的相似度。在其它實施例中，功能類似與原胜胺具有至少90%或至少95%的相似度。 In a particular embodiment, the functional analogue of the α-Syn C-terminal fragment has at least 50% similarity to the original amine sequence. In other embodiments, the functional similarity has at least 80% similarity to the original amine. In other embodiments, the functional similarity has at least 85% similarity to the original amine. In other embodiments, the functional similarity has at least 90% or at least 95% similarity to the original amine.

b. 異源性T輔助細胞抗原決定位(Th抗原決定位)b. Heterologous T helper epitopes (Th epitopes)

本發明提供胜肽免疫原結構，含有一源自α-Syn之B細胞抗原決定位，其直接或透過一選擇性異源連接子共價連接異源性T輔助細胞(Th)抗原決定位。 The invention provides a peptide immunogen structure containing a B-cell epitope derived from α-Syn, which is covalently linked to a heterologous T helper cell (Th) epitope directly or through a selective heterolinker.

α-Syn肽免疫原結構中的異源性Th抗原決定位可促進α-Syn C-端片段的免疫原性，促進專一性針對透過合理設計之優化B細胞抗原決定位(即α-Syn C-端片段)高力價抗體的產生。 The heterologous Th epitope in the structure of the α-Syn peptide immunogen can promote the immunogenicity of the C-terminal fragment of α-Syn, and promote the specificity of optimized B-cell epitopes (that is, α-Syn C -Terminal fragment) production of high-valence antibodies.

本發明中所述“異源性”係指一源自於一非野生型α-Syn序列的一部分或其同源序列的胺基酸序列。因此，異源性Th抗原決定位為一源自於非天然α-Syn(即Th抗原決定位非自體的α-Syn)胺基酸序列的Th抗原決定位。由於Th抗原決定位與α-Syn為異源性，因此當異源性Th共價連接α-Syn C-端片段時，α-Syn的天然胺基酸不會延伸至N-端或C-端。 The term "heterologous" in the present invention refers to an amino acid sequence derived from a part of a non-wild-type α-Syn sequence or a homologous sequence thereof. Therefore, the heterologous Th epitope is a Th epitope derived from an unnatural α-Syn (ie, the Th epitope is not autologous α-Syn) amino acid sequence. Because the Th epitope and α-Syn are heterologous, when the heterologous Th is covalently linked to the C-terminal fragment of α-Syn, the natural amino acid of α-Syn does not extend to the N-terminus or C- end.

本發明異源性Th抗原決定位可為任何Th抗原決定位，其不含有天然α-Syn的胺基酸序列。Th抗原決定位可具有一源自於任何物種(如，人類、豬、牛、狗、大鼠、小鼠、天竺鼠等)α-Syn的胺基酸序列。Th抗原決定位也可對多種物種的第二類MHC分子具有混雜的結合區。在特定實施例中，Th抗原決定位包括複數個混雜的第二類MHC結合區，以獲得最大的T輔助細胞活性，進行免疫反應的啟動及調控。Th抗原決定位較佳本身為免疫靜默，即因α-Syn胜肽免疫結構所誘導產生的抗體中，很少是針對Th抗原決定位，因此可具有非常集中針對α-Syn胜肽免疫結構中目標B細胞抗原決定位的免疫反應。 The heterologous Th epitope of the present invention may be any Th epitope, which does not contain the amino acid sequence of natural α-Syn. The Th epitope may have an amino acid sequence derived from any species (e.g., human, pig, cow, dog, rat, mouse, guinea pig, etc.). Th epitopes can also have promiscuous binding regions for second-class MHC molecules of multiple species. In a specific embodiment, the Th epitope includes a plurality of mixed second-type MHC binding regions to obtain maximum T helper cell activity, and to initiate and regulate the immune response. The Th epitope is preferably immunosilent itself, that is, antibodies that are induced by the α-Syn peptide immune structure are rarely directed against the Th epitope, so it can have a very concentrated focus on the α-Syn peptide immunostructure Target B-cell epitope immune response.

本發明之抗原決定位包括，但不限於，源自於外來病原菌的胺基酸序列，如表2所示(SEQ ID NOs：70-98)。此外，Th抗原決定位包括優化的人工Th抗原決定位及人工Th抗原決定位的組合(如SEQ ID NOs：71及78-84)。異源性Th抗原決定位胜肽表現為一組合序列(如SEQ ID NOs：79-82)，包含在特定胜肽的同源性可變殘基中的胜肽區塊中特定位置的混合胺基酸殘基。在合成過程中，可藉由於一程序中添加設計保護胺基酸混合物，可在特定位置取代特定胺基酸，以合成組合胜肽集合。此組合異源性Th抗原決定位集合對於不同基因背景的物種具有寬廣的Th抗原決定位覆蓋。代表性的異源性Th抗原決定位胜肽組合序列包括SEQ ID NOs：79-82，如表2所示。本發明之抗原決定位胜肽可提供基因多樣化之動物及病患寬廣的反應及免疫原性。 The epitope of the present invention includes, but is not limited to, amino acid sequences derived from foreign pathogenic bacteria, as shown in Table 2 (SEQ ID NOs: 70-98). In addition, the Th epitope includes a combination of optimized artificial Th epitopes and artificial Th epitopes (eg, SEQ ID NOs: 71 and 78-84). Heterologous Th epitope peptides appear as a combined sequence (eg, SEQ ID NOs: 79-82), a mixed amine contained at specific positions in the peptide block in the homology variable residues of a specific peptide Acid residues. During the synthesis process, it is possible to synthesize a combination of peptides by adding a design-protected amino acid mixture in a procedure to replace a specific amino acid at a specific position. This combination of heterologous Th epitope sets has broad Th epitope coverage for species with different genetic backgrounds. Representative heterologous Th epitope peptide combination sequences include SEQ ID NOs: 79-82, as shown in Table 2. The epitope peptides of the present invention can provide broad response and immunogenicity in genetically diverse animals and patients.

包含Th抗原決定位的α-Syn胜肽免疫結構可在單一固相胜肽合成中與α-Syn C-端片段同時生成。Th抗原決定位更包括Th抗原決定位的免疫類似物。免疫Th類似物包括免疫刺激類似物、交叉反應類似物及上述Th抗原決定位的任何片段，其足以促進或刺激針對α-Syn C-端片段的免疫反應。 The α-Syn peptide immunostructure containing Th epitope can be generated simultaneously with the α-Syn C-terminal fragment in a single solid phase peptide synthesis. Th epitopes further include immune analogs of Th epitopes. Immune Th analogs include immune stimulating analogs, cross-reactive analogs, and any fragments of the above-mentioned epitopes of Th, which are sufficient to promote or stimulate the immune response against the α-Syn C-terminal fragment.

Th抗原決定位胜肽的功能性免疫類似物也有效果，且為本發明的一部分。功能性兔疫Th類似物包括在Th抗原決定位中保守性取代、增加、刪除及***1至5個胺基酸，其不會實質上改變Th抗原決定位的Th-刺激功能。利用天然或非天然胺基酸完成上述α-Syn C-端片段的保守性取代、增加及刪除。表2顯示其它Th抗原決定位的功能類似物。特別是，MvF1及MvF2 Th之SEQ ID NOs：71與78為MvF4及MvF5之SEQ ID NOs：81與83的類似物，其差異在於N-及C-端具有2個胺基酸的刪除(SEQ ID NOs：71與78)或增加(SEQ ID NOs：81與83)。這2個系列類似物序列的差異不會影響序列中Th抗原決定位的功能。因此，功能性免疫原Th類似物包括多種源自於麻疹病毒融合蛋白MvF1-4 Ths(SEQ ID NOs：71、78、79、81及83)與肝炎表面蛋白HBsAg 1-3 Ths(SEQ ID NOs：80、82及84)的Th抗原決定位。 Functional immune analogs of the Th epitope peptide are also effective and are part of the present invention. Functional Thyroid Epidemic Th analogs include conservative substitutions, additions, deletions, and insertions of 1 to 5 amino acids in the Th epitope, which do not substantially alter the Th-stimulating function of the Th epitope. Conservative substitutions, additions, and deletions of the α-Syn C-terminal fragments described above are accomplished using natural or unnatural amino acids. Table 2 shows functional analogs of other Th epitopes. In particular, SEQ ID NOs: 71 and 78 of MvF1 and MvF2 Th are analogs of SEQ ID NOs: 81 and 83 of MvF4 and MvF5, and the difference is that the N- and C-termini have two amino acid deletions (SEQ ID NOs: 71 and 78) or increased (SEQ ID NOs: 81 and 83). The differences in the sequences of these two series of analogs will not affect the function of the Th epitope in the sequence. Therefore, functional immunogen Th analogs include a variety of measles virus fusion proteins MvF1-4 Ths (SEQ ID NOs: 71, 78, 79, 81, and 83) and hepatitis surface protein HBsAg 1-3 Ths (SEQ ID NOs). : 80, 82, and 84).

α-Syn胜肽免疫原結構中的Th抗原決定位可共價連接至α-Syn C-端胜肽的N-或C-端。在某些實施例中，Th抗原決定位共價連結至α-Syn C-端胜肽的N-端。在其它實施例中，Th抗原決定位共價連結至α-Syn C-端胜肽的C-端。在特定實施例中，1個以上的Th抗原決定位共價連接至α-Syn C-端片段。當1個以上的Th抗原決定位共價連接至α-Syn C-端片段時，各Th抗原決定位具有相同的胺基酸序列或不同的胺基酸序列。此外，當1個以上的Th抗原決定位共價連接至α-Syn C-端片段時，Th抗原決定位可以任何方式排列。例如，Th抗原決定位可連續連結α-Syn C-端片段的N-端，或連續連結α-Syn C-端片段的C-端；或一Th抗原決定位可連續連結α-Syn C-端片段的N-端，而另一Th抗原決定位可連續連結α-Syn C-端片段的C-端。Th抗原決定位在α-Syn C端片段的排列並無限制。 The Th epitope in the α-Syn peptide immunogen structure is Covalently linked to the N- or C-terminus of the α-Syn C-terminal peptide. In certain embodiments, the Th epitope is covalently linked to the N-terminus of the α-Syn C-terminal peptide. In other embodiments, the Th epitope is covalently linked to the C-terminus of the α-Syn C-terminal peptide. In a specific embodiment, more than one Th epitope is covalently linked to an α-Syn C-terminal fragment. When more than one Th epitope is covalently linked to the α-Syn C-terminal fragment, each Th epitope has the same amino acid sequence or a different amino acid sequence. In addition, when more than one Th epitope is covalently linked to the α-Syn C-terminal fragment, the Th epitopes can be arranged in any manner. For example, a Th epitope may be continuously linked to the N-terminus of an α-Syn C-terminal fragment, or a C-terminus of an α-Syn C-terminal fragment may be continuously linked; or a Th epitope may be continuously linked to an α-Syn C-terminal fragment. The N-terminus of the terminal fragment, while another Th epitope can continuously link the C-terminus of the α-Syn C-terminal fragment. The arrangement of Th epitopes at the C-terminal fragment of α-Syn is not limited.

在某些實施例中，Th抗原決定位直接共價連結至α-Syn C-端片段。在其它實施例中，Th抗原決定位透過連接子共價連結α-Syn C-端片段。 In certain embodiments, the Th epitope is directly covalently linked to the α-Syn C-terminal fragment. In other embodiments, the Th epitope is covalently linked to the α-Syn C-terminal fragment via a linker.

c. 異源性連接子c. Heterologous linkers

α-Syn胜肽免疫原結構選擇性地包含一異源性連接子，其共價連接α-Syn之B細胞抗原決定位與異源性T輔助細胞(Th)抗原決定位。 The α-Syn peptide immunogen structure optionally comprises a heterologous linker, which covalently links the B-cell epitope of the α-Syn and the heterologous T helper cell (Th) epitope.

如上所述，本發明之“異源性”係指一源自於一非野生型α-Syn序列的一部分或其同源序列的胺基酸序列。因此，當異源性連接子共價連結α-Syn之B細胞抗原決定位時，因為連接子與α-Syn序列為異源性，所以α-Syn天然胺基酸序列不會延伸至N-端或C-端。 As mentioned above, the "heterologous" of the present invention refers to an amino acid sequence derived from a part of a non-wild-type α-Syn sequence or a homologous sequence thereof. Therefore, when heterologous linkers covalently bind α-Syn B cells At the epitope position, because the linker is heterologous to the α-Syn sequence, the natural amino acid sequence of the α-Syn does not extend to the N-terminus or C-terminus.

連接子為任何可連結2個胺基酸及/或胜肽的分子或化合物。連接子可根據應用改變長度或極性。連接子的連結可透過一胺基或羧基連接，但也可為其他官能基。連接子包括一化合物、天然的胺基酸或非天然的胺基酸。 A linker is any molecule or compound that can link two amino acids and / or peptides. Linkers can vary in length or polarity depending on the application. The linker may be linked through an amine group or a carboxyl group, but may be other functional groups. The linker includes a compound, a natural amino acid, or a non-natural amino acid.

連接子可提供α-Syn胜肽免疫原結構多種結構特徵。在結構上，連接子提供Th抗原決定位與α-Syn C-端片段之B細胞抗原決定位一種物理分離。此連接子的物理分離可破壞因連結Th抗原決定位至B細胞抗原決定位所產生的人為二級結構。此外，透過連接子物理分離抗原決定位可消除Th抗原決定位及/或B細胞抗原決定位之間所產生之反應的干擾。此外，連接子可產生或修飾肽免疫原結構的二級結構。例如，連接子可具有一可變的鉸鏈以促進Th抗原決定位及B抗原決定位的分離。可變的鉸鏈也可允許胜肽免疫原與適當之Th細胞及B細胞之間更多有效的反應，以促進Th抗原決定位及B細胞抗原決定位的免疫反應。編碼可變鉸鏈的序列可為免疫球蛋白重鏈鉸鏈區，其一般富含脯胺酸。一種可作為連接子之可變鉸鏈可為Pro-Pro-Xaa-Pro-Xaa-Pro(SEQ ID NO：148)序列，其中Xaa為任何胺基酸，較佳為天冬胺酸。 The linker can provide a variety of structural features of the α-Syn peptide immunogen structure. Structurally, the linker provides a physical separation of the Th epitope from the B-cell epitope of the α-Syn C-terminal fragment. The physical separation of this linker can destroy the artificial secondary structure produced by linking Th epitopes to B cell epitopes. In addition, the physical separation of epitopes through the linker can eliminate the interference of the reaction between Th epitopes and / or B cell epitopes. In addition, the linker can generate or modify the secondary structure of the peptide immunogen structure. For example, the linker may have a variable hinge to facilitate the separation of the Th epitope and the B epitope. The variable hinge may also allow more effective responses between the peptide immunogen and appropriate Th cells and B cells to promote Th epitope and B cell epitope immune responses. The sequence encoding the variable hinge may be an immunoglobulin heavy chain hinge region, which is generally rich in proline. A variable hinge that can be used as a linker may be a Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 148) sequence, where Xaa is any amino acid, preferably aspartic acid.

連接子也可提供α-Syn胜肽免疫原結構功能性特徵。例如，連接子可改變α-Syn胜肽免疫原結構的整體電荷，其可影響胜肽免疫原結構的溶解度。此外，改變 α-Syn胜肽免疫原結構的整體電荷可影響胜肽免疫原結構與其它化合物及試劑結合的能力。如後文所述，α-Syn胜肽免疫原結構可為具穩定免疫刺激複合物與帶高電荷之寡核苷酸，如CpG寡聚物透過靜電結合。α-Syn胜肽免疫原結構的整體電荷對於形成穩定的免疫刺激複合物是重要的。 The linker may also provide functional features of the α-Syn peptide immunogen structure. For example, a linker can change the overall charge of the α-Syn peptide immunogen structure, which can affect the solubility of the peptide immunogen structure. Also, change The overall charge of the α-Syn peptide immunogen structure can affect the ability of the peptide immunogen structure to bind to other compounds and reagents. As will be described later, the α-Syn peptide immunogen structure may be a stable immunostimulatory complex and a highly charged oligonucleotide, such as a CpG oligomer, through electrostatic binding. The overall charge of the α-Syn peptide immunogen structure is important for the formation of stable immunostimulatory complexes.

可作為連接子的化合物包括，但不限於，(2-氨基乙氧基)醋酸(AEA)、5-氨基戊酸(AVA)、6-氨基己酸(Ahx)、8-氨-3,6-二氧雜辛酸(AEEA,mini-PEG1)、12-氨-4,7,10-三氧雜十二烷酸(mini-PEG2)、15-氨-4,7,10,13-四氧雜五-癸酸(mini-PEG3)、三氧十三烷-琥珀醯胺酸(Ttds)、12-氧-十二烷酸、Fmoc-5-氨-3-氧雜戊酸(O1Pen)及其類似物。 Compounds that can be used as linkers include, but are not limited to, (2-aminoethoxy) acetic acid (AEA), 5-aminovaleric acid (AVA), 6-aminohexanoic acid (Ahx), 8-amino-3,6 -Dioxaoctanoic acid (AEEA, mini-PEG1), 12-amino-4,7,10-trioxadodecanoic acid (mini-PEG2), 15-amino-4,7,10,13-tetraoxo Heteropenta-decanoic acid (mini-PEG3), trioxadecane-succinic acid (Ttds), 12-oxo-dodecanoic acid, Fmoc-5-amino-3-oxovaleric acid (O1Pen) Its analog.

天然胺基酸包括丙胺酸、精胺酸、天門冬醯胺、天門冬胺酸，半胱胺酸、谷胺酸、谷氨醯胺、甘胺酸、組胺酸、異亮胺酸、亮胺酸、賴胺酸、甲硫胺酸、***酸、脯胺酸、絲胺酸、蘇胺酸、色胺酸、酪胺酸和纈胺酸。 Natural amino acids include alanine, arginine, aspartic acid, aspartic acid, cysteine, glutamine, glutamine, glycine, histamine, isoleucine, leucine Amino acids, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

非天然胺基酸包括，但不限於，ε-N賴胺酸、ß-丙氨酸、鳥氨酸、正亮氨酸、正纈氨酸、羥脯氨酸、甲狀腺素、γ-氨基丁酸、絲氨酸、瓜氨酸、氨基苯甲酸、6-氨基己酸(Aca；6-Aminohexanoic acid)、羥脯氨酸、巰基丙酸、3-硝基-酪氨酸、焦谷氨酸及其類似物。 Unnatural amino acids include, but are not limited to, ε-N lysine, ß-alanine, ornithine, norleucine, norvaline, hydroxyproline, thyroxine, gamma-aminobutyric acid Acid, serine, citrulline, aminobenzoic acid, 6-aminocaproic acid (Aca; 6-Aminohexanoic acid), hydroxyproline, mercaptopropionic acid, 3-nitro-tyrosine, pyroglutamic acid and the like analog.

α-Syn胜肽免疫原結構中的連接子可共價連接至Th抗原決定位及α-Syn C-端胜肽的N-或C-端。在某些實施例中，連接子共價連接至Th抗原決定位的C-端及α-Syn C-端胜肽的N-端。在其它實施例中，連接子共價連接至C-端胜肽的C-端及Th抗原決定位的N-端。在特定實施例中，例如，當胜肽免疫原結構中有一個以上的Th抗原決定位時，可使用一個以上的連接子。當使用一個以上的連接子時，各個連接子可相同或不同。再者，當胜肽有一個以上的Th抗原決定位時，Th抗原決定位可以連接子分離，此連接子可與用於分離Th抗原決定位及B細胞抗原決定位的連接子相同或不同。連接子與Th抗原決定位或α-Syn C-端胜肽片段的排列並無特別限制。 The linker in the structure of the α-Syn peptide immunogen can be covalently linked to the Th epitope and the N- or C-terminus of the α-Syn C-terminal peptide. In some In some embodiments, the linker is covalently linked to the C-terminus of the Th epitope and the N-terminus of the α-Syn C-terminal peptide. In other embodiments, the linker is covalently linked to the C-terminus of the C-terminal peptide and the N-terminus of the Th epitope. In particular embodiments, for example, when there is more than one Th epitope in the peptide immunogen structure, more than one linker may be used. When more than one linker is used, each linker may be the same or different. Furthermore, when the peptide has more than one Th epitope, the Th epitope can be separated by a linker, and this linker can be the same as or different from the linker used to separate the Th epitope and the B cell epitope. The arrangement of the linker with the Th epitope or the α-Syn C-terminal peptide fragment is not particularly limited.

在特定實施例中，異源性連接子為一天然胺基酸或一非天然胺基酸。在其它實施例中，連接子包括一個以上的天然胺基酸或非天然胺基酸。在特定實施例中，連接子為Lys-、Gly-、Lys-Lys-Lys-、(α,ε-N)Lys或ε-N-Lys-Lys-Lys-Lys(SEQ ID NO：148)。 In a particular embodiment, the heterologous linker is a natural amino acid or a non-natural amino acid. In other embodiments, the linker includes more than one natural or non-natural amino acid. In a particular embodiment, the linker is Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N) Lys or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148).

d.α-Syn胜肽免疫原的特定實施例d. Specific examples of α-Syn peptide immunogens

α-Syn胜肽免疫原結構可具下式結構：(Th)m-(A)n-(α-Syn C-端片段)-X或(α-Syn C-端片段)-(A)n-(Th)m-X The structure of α-Syn peptide immunogen can have the following structure: (Th) m- (A) n- (α-Syn C-terminal fragment) -X or (α-Syn C-terminal fragment)-(A) n -(Th) mX

其中Th為一異源性T輔助抗原決定位；A為一異源性連接子；(α-Syn C-端片段)為一源自α-Syn C-端片段之具有約10至25個胺基酸的B細胞抗原決定位；X為一胺基酸的α-COOH或α-CONH₂；M為1至約4；且N為0至約10。 Where Th is a heterologous T helper epitope; A is a heterologous linker; (α-Syn C-terminal fragment) is an α-Syn C-terminal fragment with about 10 to 25 amines B-cell epitopes of amino acids; X is α-COOH or α-CONH ₂ of a monoamino acid; M is 1 to about 4; and N is 0 to about 10.

在某些實施例中，α-Syn胜肽免疫原結構中的異源性Th抗原決定位具有一胺基酸序列，其擇自於SEQ ID NOs：70-98或其組合，如第2圖所示。在特定實施例中，Th抗原決定位具有一胺基酸序列，其擇自於SEQ ID NOs：78-84。在特定實施例中，α-Syn胜肽免疫原結構含有一個以上的Th抗原決定位。 In certain embodiments, the heterologous Th epitope in the α-Syn peptide immunogen structure has an amino acid sequence selected from SEQ ID NOs: 70-98 or a combination thereof, as shown in Figure 2 As shown. In a particular embodiment, the Th epitope has an amino acid sequence selected from SEQ ID NOs: 78-84. In a specific embodiment, the α-Syn peptide immunogen structure contains more than one Th epitope.

在某些實施例中，選擇性異源性連接子可擇自於Lys-、Gly-、Lys-Lys-Lys-、(α,ε-N)Lys、ε-N-Lys-Lys-Lys-Lys(SEQ ID NO：148)及其組合之任一種。在特定實施例中，異源性連接子為ε-N-Lys-Lys-Lys-Lys(SEQ ID NO：148)。 In certain embodiments, the selective heterologous linker may be selected from Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N) Lys, ε-N-Lys-Lys-Lys- Any of Lys (SEQ ID NO: 148) and combinations thereof. In a specific embodiment, the heterologous linker is ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148).

在某些實施例中，α-SynC-端片段具有α-SynC-端約10至25個胺基酸，其為全長α-Syn中約第111個胺基酸的甘氨酸(G111)至約第135個胺基酸的天門冬胺酸(D135)。在特定實施例中，α-SynC-端片段具有SEQ ID NOs：12-15、17或49-64的胺基酸序列，如表1所示。 In certain embodiments, the α-SynC-terminal fragment has about 10 to 25 amino acids at the α-SynC-terminus, which is glycine (G111) to about 111th amino acid in the full-length α-Syn 135 amino acids aspartic acid (D135). In a specific embodiment, the α-SynC-terminal fragment has an amino acid sequence of SEQ ID NOs: 12-15, 17 or 49-64, as shown in Table 1.

在某些實施例中，α-Syn胜肽免疫原結構具有一胺基酸序列，其擇自於SEQ ID NOs：107-108、111-113、及115-147之任一種，如表3所示。在特定實施例中，α-Syn 胜肽免疫原結構具有一胺基酸序列，其擇自於SEQ ID NOs：107-108及111-113之任一種。 In certain embodiments, the α-Syn peptide immunogen structure has an amino acid sequence selected from any one of SEQ ID NOs: 107-108, 111-113, and 115-147, as shown in Table 3. Show. In a specific embodiment, α-Syn The peptide immunogen structure has an amino acid sequence selected from any one of SEQ ID NOs: 107-108 and 111-113.

組成物Composition

本發明更揭露包括上述α-Syn胜肽免疫原結構的組成物。 The invention further discloses a composition including the aforementioned α-Syn peptide immunogen structure.

a.胜肽組成物a. Peptide composition

含有α-Syn胜肽免疫原結構的組成物為液態或固態。液態組成物可包括水、緩衝液、溶劑、鹽類及/或任何其他可接受之試劑，其其不會改變α-Syn胜肽免疫原結構的結構或功能特性。胜肽組成物可含有一或多個α-Syn胜肽免疫原結構。 The composition containing the α-Syn peptide immunogen structure is liquid or solid. The liquid composition may include water, buffers, solvents, salts, and / or any other acceptable reagents that do not alter the structural or functional properties of the α-Syn peptide immunogen structure. The peptide composition may contain one or more α-Syn peptide immunogen structures.

b.醫藥組成物b. Pharmaceutical composition

本發明另有關於含有α-Syn胜肽免疫原結構的醫藥組成物。 The present invention also relates to a pharmaceutical composition containing an α-Syn peptide immunogen structure.

醫藥組成物可含有藥學上可接受之載體及/或其它添加劑。因此，醫藥組成物可含有一藥學上有效量之α-Syn胜肽免疫原結構及藥學上可接受之載體、佐劑及/或賦形劑，如稀釋劑、添加劑、穩定劑、防腐劑、增溶劑、緩衝液及其類似物。 The pharmaceutical composition may contain a pharmaceutically acceptable carrier and / or other additives. Therefore, the pharmaceutical composition may contain a pharmaceutically effective amount of an α-Syn peptide immunogen structure and a pharmaceutically acceptable carrier, adjuvant and / or excipient, such as a diluent, an additive, a stabilizer, a preservative, Solubilizers, buffers and the like.

醫藥組成物可含有一或多個佐劑，其可加速、延長或促進對於α-Syn胜肽免疫原結構的免疫反應，而本身無特定免疫原影響。醫藥組成物中所使用的佐劑可包括脂質體、皂苷、角鯊烯、L121、Emulsigen®、單磷醯脂質A(MPL)、QS21、ISA 35、ISA 206、ISA50V、ISA51、ISA 720以及其它佐劑及乳化劑。 The pharmaceutical composition may contain one or more adjuvants, which can accelerate, prolong, or promote the immune response to the structure of the α-Syn peptide immunogen without the specific immunogen effect itself. Adjuvants used in pharmaceutical compositions may include liposomes, saponins, squalene, L121, Emulsigen®, monophosphoryl lipid A (MPL), QS21, ISA 35, ISA 206, ISA50V, ISA51, ISA 720 and other adjuvants and emulsifiers.

在某些實施例中，醫藥組成物包括Montanide^TM ISA 51(由植物油和甘露醇油酸酯組成之w/o乳化劑之油類佐劑組成物)、Tween® 80(聚山梨醇酯80或聚氧乙烯(20)脫水山梨醇單油酸酯)、CpG寡核酸及/或其組合。在其它實施例中，醫藥組成物為一以Emulsigen或Emulsigen D作為佐劑之水包油包水(w/o/w)乳化劑。 In certain embodiments, the pharmaceutical composition includes Montanide ^™ ISA 51 (an oil adjuvant composition of a w / o emulsifier composed of vegetable oil and mannitol oleate), Tween® 80 (polysorbate 80 or Polyoxyethylene (20) sorbitan monooleate), CpG oligo and / or combinations thereof. In other embodiments, the pharmaceutical composition is a water-in-oil-in-water (w / o / w) emulsifier with Emulsigen or Emulsigen D as an adjuvant.

醫藥組成物可為立即釋放或持續釋放製劑。此外，醫藥組成物可透過免疫原捕獲及共同給予微粒來刺激全身性或局部黏膜免疫。本技術領域人士可輕易了解此傳遞系統。 The pharmaceutical composition may be an immediate release or a sustained release formulation. In addition, pharmaceutical compositions can stimulate systemic or local mucosal immunity through capture of immunogens and co-administration of microparticles. Those skilled in the art can easily understand this delivery system.

醫藥組成物可為注射液或為液體溶液或者懸浮液。在注射前，先製備含有α-Syn肽免疫原結構的液體載體。醫藥組成物可以適當的模式給予，例如，i.d.、i.v.、i.p.、i.m.、經鼻、經口、皮下等。在某些實施例中，醫藥組成物可製備為靜脈、皮下、皮內或肌內給予。醫藥組成物也可製備成適合用於其它給藥方式，包括口服和鼻內給予。 The pharmaceutical composition may be an injection solution or a liquid solution or suspension. Before injection, a liquid carrier containing the α-Syn peptide immunogen structure is prepared. The pharmaceutical composition can be administered in an appropriate mode, for example, i.d., i.v., i.p., i.m., nasal, oral, subcutaneous, and the like. In certain embodiments, the pharmaceutical composition may be prepared for intravenous, subcutaneous, intradermal, or intramuscular administration. Pharmaceutical compositions can also be prepared to be suitable for other modes of administration, including oral and intranasal administration.

醫藥組成物也可製備成具有合適的劑量單位形式。在某些實施例中，醫藥組成物含有每公斤體重約0.5μg至約1mg的α-Syn肽免疫原結構。不論患者是人類或動物，預防或治療，醫藥組成物的有效劑量可根據許多不同的因子改變，包括施用手段、目標部位、患者的生理狀態、其它給予藥物。通常，患者為一人類，但也可為非人類動物，包括轉植動物。當給予複數劑量，醫藥組成物可方便地分成適當的每單位劑量。在治療領域中可根據個體的年紀、體重及健康狀況給予一劑量。 Pharmaceutical compositions can also be prepared in a suitable dosage unit form. In certain embodiments, the pharmaceutical composition contains from about 0.5 μg to about 1 mg of alpha-Syn peptide immunogen structure per kilogram of body weight. Regardless of whether the patient is human or animal, prevention or treatment, the effective dose of the pharmaceutical composition can be changed according to many different factors, including the means of administration, the target site, the physiological state of the patient, and other administered drugs. Usually, the patient is a human, but it can also be Non-human animals, including transgenic animals. When multiple doses are administered, the pharmaceutical composition can be conveniently divided into appropriate unit doses. In the field of treatment, a dose can be given according to the age, weight and health of the individual.

在某些實施例中，醫藥組成物包括一個以上的α-Syn肽免疫原結構。含有一個以上之α-Syn肽免疫原結構的醫藥組成物可協同增加結構的免疫效率。含有一個以上之α-Syn肽免疫原結構的醫藥組成物因具有寬廣的第II類MHC覆蓋，因此可提供α-Syn肽免疫原結構更佳的免疫反應。 In certain embodiments, the pharmaceutical composition includes more than one α-Syn peptide immunogen structure. A pharmaceutical composition containing more than one α-Syn peptide immunogen structure can synergistically increase the immune efficiency of the structure. A pharmaceutical composition containing more than one α-Syn peptide immunogen structure has a broad Class II MHC coverage, and therefore can provide a better immune response to the α-Syn peptide immunogen structure.

在某些實施例中，醫藥組成物含有α-Syn肽免疫原結構，其擇自於SEQ ID NOs：107-108、111-113、115-147及其同源物、類似物及/或其組合。在特定實施例中，醫藥組成物含有α-Syn肽免疫原結構，其擇自於SEQ ID NOs：107-108、111-113及其任何組合。 In certain embodiments, the pharmaceutical composition contains an α-Syn peptide immunogen structure selected from SEQ ID NOs: 107-108, 111-113, 115-147, and homologues, analogs, and / or combination. In a particular embodiment, the pharmaceutical composition contains an α-Syn peptide immunogen structure selected from SEQ ID NOs: 107-108, 111-113, and any combination thereof.

含有α-Syn肽免疫原結構的醫藥組成物可刺激宿主免疫反應及產生抗體。 The pharmaceutical composition containing the α-Syn peptide immunogen structure can stimulate the host's immune response and produce antibodies.

c.免疫刺激複合物c. immune stimulating complex

本發明也有關於含有α-Syn胜肽免疫原結構與CpG寡核酸免疫調控複合物的醫藥組成物。免疫調控複合物專一性作為一佐劑及胜肽免疫原穩定物。免疫調控複合物為一顆粒，其可對細胞的免疫系統有效地呈現α-Syn胜肽免疫原以產生免疫反應。免疫調控複合物可為一用於腸外給予的懸浮液。免疫調控複合物也可為w/o乳化液，作為懸浮液與無機鹽或原位膠凝聚合物的組合，在非腸道給予後將α-Syn肽免疫原有效地遞送到宿主免疫系統的細胞中。免疫調控複合物可產生針對β-折疊之α-Syn的免疫反應(如，實施例13的第8A、8B及8C圖)以達到保護/治療效果。 The present invention also relates to a pharmaceutical composition containing an α-Syn peptide immunogen structure and a CpG oligonucleotide immunoregulatory complex. The immunoregulatory complex specifically acts as an adjuvant and peptide immunogen stabilizer. The immunoregulatory complex is a particle that can effectively present an α-Syn peptide immunogen to the immune system of a cell to generate an immune response. The immunomodulatory complex may be a suspension for parenteral administration. Immune regulatory complexes can also be w / o emulsions, as a combination of suspensions with inorganic salts or in situ gelling polymers. The alpha-Syn peptide immunogen is efficiently delivered to cells of the host's immune system after parenteral administration. The immune regulatory complex can generate an immune response against β-sheet α-Syn (eg, Figures 8A, 8B, and 8C of Example 13) to achieve a protective / therapeutic effect.

穩定的免疫調控複合物可藉由靜電結合複合一α-Syn胜肽免疫原結構與陰離子分子、寡核苷酸、聚核苷酸或其組合。穩定的免疫調控複合物可作為一免疫傳遞系統與一醫藥組成物合併。 The stable immunoregulatory complex can be electrostatically combined with an α-Syn peptide immunogen structure and an anion molecule, an oligonucleotide, a polynucleotide, or a combination thereof. Stable immune regulatory complexes can be combined with a pharmaceutical composition as an immune delivery system.

在某些實施例中，α-Syn胜肽免疫原結構含有一陽離子部分，其在pH5.0至8.0帶正電。α-Syn胜肽免疫原結構或混合物之陽離子部分，每一個賴胺酸(K)、精胺酸(R)或組胺酸(H)的電荷為+1，每一個天冬胺酸(D)或谷胺酸(E)的電荷為-1，其它胺基酸的電荷為0，依此方式計算淨電荷。α-Syn胜肽免疫原結構內的電荷相加以表示為平均淨電荷。一種穩定的胜肽免疫原具有平均淨正電荷+1的陽離子。較佳胜肽免疫原具有大於+2的平均淨正電荷。在某些實施例中，α-Syn胜肽免疫原結構的陽離子為異源性連接子。在某些實施例中，當連接子序列為(α,ε-N)Lys、ε-N-Lys-Lys-Lys-Lys(SEQ ID NO：148)時，α-Syn胜肽免疫原結構的陽離子具有+4電荷。 In certain embodiments, the alpha-Syn peptide immunogen structure contains a cationic moiety that is positively charged at pH 5.0 to 8.0. The cationic portion of the α-Syn peptide immunogen structure or mixture, the charge of each lysine (K), arginine (R) or histidine (H) is +1, and each aspartate (D ) Or glutamic acid (E) has a charge of -1 and other amino acids have a charge of 0, and the net charge is calculated in this manner. The addition of the charges within the α-Syn peptide immunogen structure is expressed as the average net charge. A stable peptide immunogen has a cation with an average net positive charge +1. Preferred peptide immunogens have an average net positive charge greater than +2. In certain embodiments, the cation of the α-Syn peptide immunogen structure is a heterologous linker. In certain embodiments, when the linker sequence is (α, ε-N) Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148), the structure of the α-Syn peptide immunogen The cation has a +4 charge.

本發明所述“陰離子分子”係指任何在pH5.0至8.0帶正電的分子。在某些實施例中，陰離子分子為一寡聚物或聚合物。可利用每個磷酸二酯或硫代磷酸酯基團為-1電荷來計算寡聚物或聚合物的淨負電荷。適合的陰離子寡核苷酸是具有8至64個核苷酸鹼基的單鏈DNA分子，具有1至10個重覆的CpG單元。較佳CpG免疫刺激單鏈DNA分子含有18至48個核苷酸鹼基，具有3至8個重覆的CpG單元。 The "anionic molecule" as used herein refers to any molecule that is positively charged at a pH of 5.0 to 8.0. In certain embodiments, the anionic molecule is an oligomer or polymer. The net negative charge of an oligomer or polymer can be calculated using a charge of -1 per phosphodiester or thiophosphate group. Suitable anionic oligonucleotides are single-stranded DNA molecules with 8 to 64 nucleotide bases, with 1 to 10 repeating CpG units. Preferred CpG immunostimulatory single-stranded DNA molecules contain 18 to 48 nucleotide bases and have 3 to 8 repeating CpG units.

更佳陰離子寡核苷酸如下式所示：5' X1CGX2 3'，其中C及G為未甲基化；且X1為擇自A(腺嘌呤)、G(鳥嘌呤)及T(胸腺嘧啶)；X2為C(胞嘧啶)或T(胸腺嘧啶)。或是，陰離子寡核苷酸由下式表示：5'(X3)₂CG(X4)₂ 3'，其中C及G為未甲基化；且X3擇自於A、T或G，且X4為C或T。 A better anionic oligonucleotide is shown by the following formula: 5 'X1CGX2 3', where C and G are unmethylated; and X1 is selected from A (adenine), G (guanine), and T (thymine) X2 is C (cytosine) or T (thymine). Alternatively, the anionic oligonucleotide is represented by the formula: 5 '(X3) ₂ CG (X4) ₂ 3', where C and G are unmethylated; and X3 is selected from A, T or G, and X4 C or T.

最終免疫刺激複合物一般為1-50μm的顆粒，且具有許多因子的功能，包括反應物的電化學計量及分子量。顆粒免疫刺激複合物具有佐劑化及提升in vivo特定免疫反應的優點。因此，穩定化免疫刺激複合物可穩定藉由油包水乳化、無機鹽懸浮液及聚合物凝膠來製備醫藥組成物。 The final immune stimulating complex is generally 1-50 μm particles, and has the function of many factors, including electrochemical measurement and molecular weight of reactants. The granular immune stimulating complex has the advantages of adjuvanting and enhancing the specific immune response in vivo. Therefore, the stabilized immune stimulating complex can stably prepare a pharmaceutical composition by water-in-oil emulsification, an inorganic salt suspension, and a polymer gel.

抗體antibody

本發明更提供以α-Syn胜肽免疫原結構所刺激產生的抗體。 The present invention further provides antibodies that are stimulated with the structure of the α-Syn peptide immunogen.

α-Syn C端片段含有α-Syn的約10至約25個胺基酸，為全長α-Syn中約第111個位置的甘胺酸(G111)至約第135個位置的天門冬胺酸(D135)的序列，其為非免疫原性或弱免疫原性。然而，包括α-Syn C端片段、異源性Th抗原決定位與選擇性的異源性連接子的α-Syn胜肽免疫原結構在給予至宿主時，可刺激免疫反應及產生抗體。α-Syn胜肽免疫原結構可超越α-Syn本身，且促進可辨識非線性、構象抗原決定位之特定抗體的生成。 The C-terminal fragment of α-Syn contains about 10 to about 25 amino acids of α-Syn, which is the glycine (G111) to about 135 positions of the full-length α-Syn (D135) The sequence is non-immunogenic or weakly immunogenic. However, α-Syn peptides including an α-Syn C-terminal fragment, a heterologous Th epitope, and a selective heterologous linker When given to the host, the immunogen structure can stimulate the immune response and produce antibodies. α-Syn peptide immunogen structure can surpass α-Syn itself, and promote the generation of specific antibodies that can recognize non-linear, conformational epitopes.

令人驚訝的是，由α-Syn胜肽免疫原結構不會產生與以天然形式之α-Syn單體的天然α-螺旋結合。然而，α-Syn胜肽免疫原結構所產生之抗體可辨識及結合單體、寡聚體、纖維形式的α-Syn變性β-折疊。此外，α-Syn胜肽免疫原結構所產生之抗體不會與其它類澱粉蛋白類似結構(i.e.,Aβ1-42與Tau441)結合。因此，α-Syn胜肽免疫原結構的特殊設計(包括α-Syn C端片段、異源性Th抗原決定位及選擇性異源連接子)已經改變多功能α-Syn C端片段的構象，以具有類β-折疊構象。 Surprisingly, the α-Syn peptide immunogen structure does not produce natural α-helical binding to the α-Syn monomer in its native form. However, antibodies generated by the α-Syn peptide immunogen structure recognize and bind α-Syn denatured β-sheets in the form of monomers, oligomers, and fibers. In addition, the antibodies produced by the α-Syn peptide immunogen structure will not bind to other amyloid-like structures (i.e., Aβ1-42 and Tau441). Therefore, the special design of the α-Syn peptide immunogen structure (including the α-Syn C-terminal fragment, the heterologous Th epitope, and the selective heterolinker) has changed the conformation of the multifunctional α-Syn C-terminal fragment, To have a β-sheet-like conformation.

在許多功能性測試中，對源自以α-Syn胜肽免疫原結構免疫動物所獲得之血清進行廣泛的比較。這些比較證明抗體可高特異性結合經NGF處理之PC12細胞中單體及寡聚體之β-折疊的α-Syn，且不會與其它類澱粉蛋白結合(參照實施例9)。 In many functional tests, extensive comparisons have been made of sera obtained from animals immunized with an α-Syn peptide immunogen structure. These comparisons demonstrate that antibodies can specifically bind β-sheet α-Syn of monomers and oligomers in NGF-treated PC12 cells without binding to other amyloids (see Example 9).

α-Syn胜肽免疫原結構所誘發的抗體可抑制α-Syn的聚集(抗聚集活性)，且解聚集預先形成的α-Syn聚集體(裂解活性)。此外，令人驚訝抗體可以減少小神經膠質細胞(microglial cell)誘導之TNF-α和IL6的產生，這些抗體可有效地減少α-Syn聚集體或纖維介導的小膠質細胞活性。在α-Syn過度表現的細胞中，此抗體也可降低由外源α-Syn聚集體及內源α-Syn聚集體所造成的神經退化。此外，此抗體可辨識及專一性結合病理性α-Syn寡聚體聚集體或纖維，但不會與非病理性的α-Syn反應。特別是，抗體可與來自患有α突觸核蛋白相關疾病之帕金森病患者中腦部切片中的路易氏體(Lewy bodies)反應，但不會與正常人類組織反應。 Antibodies induced by the α-Syn peptide immunogen structure can inhibit the aggregation of α-Syn (anti-aggregation activity) and de-aggregate pre-formed α-Syn aggregates (cleavage activity). In addition, surprisingly, antibodies can reduce the production of TNF-α and IL6 induced by microglial cells, and these antibodies can effectively reduce α-Syn aggregates or fiber-mediated microglia activity. In α-Syn overexpressing cells, this antibody can also reduce neurodegeneration caused by exogenous α-Syn aggregates and endogenous α-Syn aggregates. Into. In addition, this antibody recognizes and specifically binds pathological α-Syn oligomer aggregates or fibers, but does not react with non-pathological α-Syn. In particular, the antibodies can react with Lewy bodies in brain slices from patients with Parkinson's disease who have alpha synuclein-related diseases, but do not react with normal human tissues.

同樣另人驚訝的是，2種帕金森氏症老鼠模型(MPP+誘導之老鼠模型及接種α-Syn纖維之老鼠模型)在給予含有α-Syn胜肽免疫原結構的組成物可(a)產生與β-折疊α-Syn高度交叉反應的抗體，(b)降低α-Syn血清，(c)降低腦部α-Syn寡聚物，以及(d)降低神經病理以恢復運動功能。 It is also surprising that two mouse models of Parkinson's disease (MPP + induced mouse model and mouse model inoculated with α-Syn fiber) can (a) produce a composition containing the α-Syn peptide immunogen structure Antibodies that are highly cross-reactive with β-sheet α-Syn, (b) decrease α-Syn serum, (c) decrease brain α-Syn oligomers, and (d) decrease neuropathology to restore motor function.

免疫本發明α-Syn胜肽免疫原結構之動物所產生的免疫反應證實此結構可產生有效地針對特定位置的抗體，其可與單體、寡聚物及纖維形式之突變的β-折疊α-Syn反應，且不會與天然形式之α-Syn C端的隨機捲曲結構反應。 Immune response produced by animals immunized with the α-Syn peptide peptide immunogen structure of the present invention confirms that this structure can produce antibodies specific to specific positions, which can interact with mutant β-sheet α in the form of monomers, oligomers and fibers -Syn reaction and does not react with the random coil structure of the C-terminus of α-Syn in its natural form.

In vitroIn vitro 功能試驗Functional test

利用α-Syn胜肽免疫原結構所產生之抗體進行in vitro功能分析。此功能分析包括，但不限於：(a)抑制in vitro重組α-Syn聚集體；且分解重組α-Syn聚集體(參照實施例8)；(b)抑制in vitro細胞性α-Syn聚集體；且分解細胞內α-Syn聚集體(參照實施例9)；(c)減少小膠質細胞TNF-α和IL6的分泌(參照實施例 10)；(d)減少由外源α-Syn聚集體誘發的神經變性(參照實施例11)；(e)減少α-Syn過度表現細胞中的神經變性(參照實施例12)；(f)證明接種α-Sy纖維和MPP+誘導之帕金森病之小鼠模型的血清中α-Syn含量降低，腦中α-Syn寡聚物含量的降低、神經變性的降低以及運動活性的恢復(參照實施例15)。 In vitro functional analysis was performed using antibodies produced by the α-Syn peptide immunogen structure. This functional analysis includes, but is not limited to: (a) inhibition of in vitro recombinant α-Syn aggregates; and decomposition of recombinant α-Syn aggregates (see Example 8); (b) inhibition of in vitro cellular α-Syn aggregates And break down intracellular α-Syn aggregates (see Example 9); (c) reduce the secretion of microglia TNF-α and IL6 (see Example 10); (d) reduce the amount of exogenous α-Syn aggregates Induced neurodegeneration (see Example 11); (e) Reduced neurodegeneration in α-Syn overexpressing cells (see Example 12); (f) Demonstrated the smallness of Parkinson's disease induced by inoculation of α-Sy fibers and MPP + The murine model had a decrease in the α-Syn content in the serum, a decrease in the α-Syn oligomer content in the brain, a decrease in neurodegeneration, and a recovery in motor activity (see Example 15).

方法method

a.製備α-Syn胜肽免疫原的方法a. Method for preparing α-Syn peptide immunogen

本發明α-Syn胜肽免疫原結構可以本技術領域人士所習知的化學合成法製備(參照Fields et al.,Chapter 3 in Synthetic Peptides：A User’s Guide,ed.Grant,W.H.Freeman & Co.,New York,NY,1992,p.77)。α-Syn胜肽免疫原結構可利用自動Merrifield固相合成技術，以t-Boc或F-moc保護之α-NH₂的支鏈胺基酸進行合成，例如，Applied Biosystems肽合成儀型430A或431。利用可替代之胺基酸在特定的可變位置進行耦合，以製備包含Th組合胜肽庫之α-Syn胜肽免疫原結構。 The α-Syn peptide immunogen structure of the present invention can be prepared by chemical synthesis methods known to those skilled in the art (see Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, WHFreeman & Co., New York, NY, 1992, p. 77). The α-Syn peptide immunogen structure can be synthesized using t-Boc or F-moc-protected α-NH ₂ branched-chain amino acids using automated Merrifield solid-phase synthesis technology, for example, Applied Biosystems Peptide Synthesizer Model 430A or 431. Alternative amino acids are used for coupling at specific variable positions to prepare an α-Syn peptide immunogen structure comprising a Th peptide library.

在α-Syn胜肽免疫原結構完成組裝後，處理樹脂將胜肽由樹脂上切割下來，並解除胺基酸支鏈的封阻。游離的胜肽可利用HPLC純化，並利用胺基酸分析或定序進行生化特性分析。胜肽的純化及生化特性分析方法為本技術領域人士所習知。 After the assembly of the α-Syn peptide immunogen structure is completed, the processing resin cuts the peptide from the resin and unblocks the amino acid branch chain. Free peptides can be purified by HPLC using amino acids Analysis or sequencing for biochemical analysis. Methods for the purification and analysis of peptides are well known to those skilled in the art.

此化學程序所產生的胜肽質量可被控制和確定，以確保α-Syn胜肽免疫原結構、免疫原性及產量的再現性。以固相胜肽合成法製造α-Syn胜肽免疫原結構的詳細描述如實施例1所示。 The quality of the peptides produced by this chemical procedure can be controlled and determined to ensure the reproducibility of the structure, immunogenicity and yield of the α-Syn peptide. A detailed description of the structure of the α-Syn peptide immunogen produced by the solid-phase peptide synthesis method is shown in Example 1.

保留預期的免疫活性的結構可變性範圍遠大於小分子藥物或與生物衍生藥物的結構可變性。因此，胜肽類似物，有意設計或由於合成過程的錯誤不可避免所產生缺失序列副產物的混合物(具有類似的色譜和免疫學性質)，通常與所欲胜肽之純化製劑相同有效。特定設計的類似物和非所欲類似物混合物只要有可辨別的QC程序監測製程及產品評估，以確保這些胜肽最終產品的再現性和功效。 The range of structural variability that retains the expected immune activity is much greater than the structural variability of small molecule drugs or biologically derived drugs. Therefore, peptide analogs, intentionally designed or a mixture of deleted sequence by-products due to errors in the synthetic process (having similar chromatographic and immunological properties), are usually as effective as the purified peptide preparations. As long as there are identifiable QC procedures to monitor the process and product evaluation of specific designed analog and undesired analog mixtures, to ensure the reproducibility and efficacy of these peptides.

α-Syn胜肽免疫原結構也可利用重組DNA技術，包括核苷酸分子、載體及/或宿主細胞製備。因此，編碼核α-Syn胜肽免疫原結構及其免疫原功能性類似物的苷酸分子也屬於本發明的一部分。同樣地，含有核苷酸分子之載體(包括表現載體)及含有載體的宿主細胞也屬於本發明的一部分。 The α-Syn peptide immunogen structure can also be prepared using recombinant DNA technology, including nucleotide molecules, vectors, and / or host cells. Therefore, a nucleotide molecule encoding a nuclear α-Syn peptide immunogen structure and a functional analogue of the immunogen also belongs to the present invention. Likewise, vectors (including expression vectors) containing nucleotide molecules and host cells containing vectors also form part of the present invention.

其它實施例也包括製備α-Syn胜肽免疫原結構及源自於胜肽免疫原結構之α-Syn G111-D135片段之免疫原功能性類似物的方法。例如，此方法可包括在可表現胜肽的條件下培養一含有編碼α-Syn胜肽免疫原結構及/或其免疫原功能性類似物核苷酸的表現載體的細胞。較長的合成胜肽免疫原可以習知的重組DNA技術合成。此技術已記載於習知的標準操作手冊中。為構築編碼本發明胜肽的基因，此胺基酸序列被反向轉譯以獲得編碼編碼此胺基酸序列的核苷酸序列，較佳具有此宿主最佳的密碼子。接著，若有需要，通常可通過合成編碼胜肽及任何調控元件的寡核苷酸來合成基因。將此合成基因***一適合的載體並轉染至一宿主細胞。接著，在一適合此表現系統及宿主的條件下表現胜肽。以標準方法純化及分析胜肽。 Other embodiments also include methods for preparing the α-Syn peptide immunogen structure and immunogenic functional analogs of the α-Syn G111-D135 fragment derived from the peptide immunogen structure. For example, the method can include culturing a peptide containing an alpha-Syn peptide Cells expressing vectors of epidemic structure and / or their immunogenic functional analogue nucleotides. Longer synthetic peptide immunogens can be synthesized using conventional recombinant DNA techniques. This technique is documented in the well-known standard operating manual. In order to construct a gene encoding the peptide of the present invention, the amino acid sequence is reverse-translated to obtain a nucleotide sequence encoding the amino acid sequence, preferably having the best codon for the host. Then, if necessary, genes can usually be synthesized by synthesizing oligonucleotides encoding peptides and any regulatory elements. This synthetic gene is inserted into a suitable vector and transfected into a host cell. The peptide is then expressed under conditions suitable for the performance system and host. The peptide was purified and analyzed by standard methods.

b.製備免疫複合物的方法b. Method for preparing immune complex

本發明各種實施例也包括製備包含α-Syn胜肽免疫原結構及CpG寡核苷酸(ODN)分子之免疫刺激複合物的方法。穩定的免疫刺激複合物(ISC)來自於α-Syn肽免疫原結構的陽離子部分及聚陰離子CpG ODN分子。可利用電荷的靜電中和完成自我裝配系統。α-Syn胜肽免疫原結構的陽離子部分與陰離子寡聚體的摩爾電荷比可確定結合程度。α-Syn胜肽免疫原結構和CpG ODN的非共價靜電結合是一可完全重複的過程。胜肽/CpG ODN免疫刺激複合物複合體聚集，其可促進呈現給免疫系統之“專業”抗原呈現細胞(APC)，進而增強複合物的免疫原性。此複合物可輕易地在製造過程中控制品質。胜肽/CpG ISC在體內(in vivo)具有良好的耐受性。此包括CpG ODN及α-Syn G111-D135片段胜肽免疫原結構之新穎的顆粒系統具有與CpG ODN相關之廣義B細胞有絲***的優點，但促進Th-1/Th-2反應的平衡。 Various embodiments of the present invention also include methods of preparing an immunostimulatory complex comprising an α-Syn peptide immunogen structure and a CpG oligonucleotide (ODN) molecule. The stable immune stimulating complex (ISC) is derived from the cationic portion of the α-Syn peptide immunogen structure and the polyanionic CpG ODN molecule. Self-assembly systems can be completed using electrostatic neutralization of charge. The molar charge ratio of the cationic portion of the α-Syn peptide immunogen structure to the anionic oligomer determines the degree of binding. The non-covalent electrostatic binding of α-Syn peptide immunogen structure to CpG ODN is a completely repeatable process. The peptide / CpG ODN immune stimulating complex complex aggregates, which can promote "professional" antigen-presenting cells (APC) presented to the immune system, thereby enhancing the immunogenicity of the complex. This composite can easily control quality during the manufacturing process. Peptide / CpG ISC is well tolerated in vivo . This novel particle system including CpG ODN and α-Syn G111-D135 fragment peptide immunogen structure has the advantage of mitosis in generalized B cells associated with CpG ODN, but promotes the balance of Th-1 / Th-2 response.

CpG ODN在本發明醫藥組成物中在相反電荷的靜電中和過程中100%與免疫原結合，形成奈米大小的顆粒。此顆粒形式可顯著地降低傳統CpG佐劑使用的劑量，減少可能的不良先天免疫反應，並有助於包括抗原呈現細胞(APC)的選擇性免疫程序。因此，此製劑為創新的概念並且通過替代機制促進免疫反應的刺激而提供可能的優點。 In the pharmaceutical composition of the present invention, CpG ODN is 100% bound to the immunogen during the electrostatic neutralization of opposite charges, and forms nano-sized particles. This granular form can significantly reduce the doses used with traditional CpG adjuvants, reduce possible adverse innate immune responses, and facilitate selective immune procedures including antigen-presenting cells (APC). Therefore, this formulation offers possible advantages for an innovative concept and the stimulation of an immune response through alternative mechanisms.

c.製備醫藥組成物的方法c. Method for preparing pharmaceutical composition

各種實施例還包括含有α-Syn胜肽免疫原結構建的藥物組合物。在某些實施方案中，醫藥組成物使用油包水乳化及礦物鹽的懸浮劑。 Various embodiments also include pharmaceutical compositions containing an alpha-Syn peptide immunogenic construct. In certain embodiments, pharmaceutical compositions use water-in-oil emulsification and suspensions of mineral salts.

為了使醫藥組成物可使用於大部分族群，且抑制α-Syn聚集也是管理目的之一，安全性成為另一考量的重要因子。儘管在臨床試驗中許多製劑在人體內使用油包水乳劑，但基於安全性，明礬仍是製劑中主要使用的佐劑。因此，明礬或其礦物鹽磷酸鋁(ADJUPHOS)經常作為臨床使用的佐劑。 In order to make the pharmaceutical composition usable in most ethnic groups, and to suppress the aggregation of α-Syn is also one of the management purposes, safety has become another important factor for consideration. Although many formulations use water-in-oil emulsions in humans in clinical trials, alum is still the main adjuvant used in formulations based on safety. Therefore, alum or its mineral salt aluminum phosphate (ADJUPHOS) is often used as an adjuvant for clinical use.

d.使用醫藥組成物的方法d. Method of using pharmaceutical composition

本發明更包括使用含有α-Syn胜肽免疫原結構之醫藥組成物的方法。 The present invention further includes a method for using a pharmaceutical composition containing an α-Syn peptide immunogen structure.

在某些實施例中，含有α-Syn胜肽免疫原結構之醫藥組成物可用於： In certain embodiments, a pharmaceutical composition containing an α-Syn peptide immunogen structure can be used for:

(a)抑制宿主中α-Syn的聚集； (a) inhibiting the aggregation of α-Syn in the host;

(b)促進宿主中預形成之α-Syn聚集體的分解； (b) promote the breakdown of pre-formed α-Syn aggregates in the host;

(c)減少宿主中小膠質細胞TNF-α和IL6的分泌； (c) reduce the secretion of TNF-α and IL6 by microglia in the host;

(d)減少宿主中由外部α-Syn聚集體所誘發的神經變性 (d) Reduce neurodegeneration in the host induced by external α-Syn aggregates

(e)減少α-Syn過度表現細胞中的神經變性； (e) reducing neurodegeneration in α-Syn overexpressing cells;

(f)減少宿主中血清α-Syn含量； (f) reducing serum α-Syn content in the host;

(g)減少宿主腦中血清α-Syn含量； (g) reducing the serum α-Syn content in the host brain;

(h)減少宿主中的神經病變及回復運動功能；及其類似。 (h) Reduce neuropathy and restore motor function in the host; and similar.

上述方法包括給予一醫藥組成物至一所需個體，其中該醫學組成物包括一醫學上有效量之α-Syn胜肽免疫原結構。 The method includes administering a pharmaceutical composition to a desired individual, wherein the medical composition includes a medically effective amount of an α-Syn peptide immunogen structure.

具體實施例Specific embodiment

本發明包含，但不限於以下具體實施例：(1)一種α-突觸核蛋白(α-Syn)胜肽免疫原結構，包括：一B細胞抗原決定位，其包括約10至25個源自α-Syn C-端的胺基酸，其對應於全長α-Syn約第111個位置的甘氨酸(G111)至第135個位置的天門冬胺酸(D135)的序列SEQ ID NO：1；一T輔助抗原決定位，包括一擇自於SEQ ID NOs：70-98的胺基酸序列；以及一選擇性異源連接子，其擇自於一胺基酸、Lys-、Gly-、 Lys-Lys-Lys-、(α,ε-N)Lys及ε-N-Lys-Lys-Lys-Lys(SEQ ID NO：148)，其中B細胞抗原決定位共價直接或透過選擇性異源連接子連接T輔助抗原決定位。 The present invention includes, but is not limited to, the following specific examples: (1) an α-synuclein (α-Syn) peptide immunogen structure, including: a B-cell epitope, which includes about 10 to 25 sources The amino acid sequence from the C-terminus of α-Syn, which corresponds to the sequence of glycine (G111) to the 135th position of aspartic acid (D135) of the full-length α-Syn; SEQ ID NO: 1; The T helper epitope includes an amino acid sequence selected from SEQ ID NOs: 70-98; and a selective heterolinker selected from monoamino acids, Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N) Lys and ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148), in which the B cell epitope is covalently directly or through selective heterologous The linker connects the T helper epitope.

(2)如(1)之α-Syn胜肽免疫原結構，其中B細胞抗原決定位擇自於SEQ ID NOs：12-15、17及49-63所組成之群組。 (2) The α-Syn peptide immunogen structure according to (1), wherein the B-cell epitope is selected from the group consisting of SEQ ID NOs: 12-15, 17 and 49-63.

(3)如(1)之α-Syn胜肽免疫原結構，其中T輔助抗原決定位擇自於SEQ ID NOs：81、83及84所組成之群組。 (3) The α-Syn peptide immunogen structure according to (1), wherein the T helper epitope is selected from the group consisting of SEQ ID NOs: 81, 83, and 84.

(4)如(1)之α-Syn胜肽免疫原結構，其中選擇性異源連接子為(α,ε-N)Lys或ε-N-Lys-Lys-Lys-Lys(SEQ ID NO：148)。 (4) The α-Syn peptide immunogen structure according to (1), wherein the selective heterolinker is (α, ε-N) Lys or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148).

(5)如(1)之α-Syn胜肽免疫原結構，其中T輔助抗原決定位共價連接至B細胞抗原決定位的胺基端。 (5) The α-Syn peptide immunogen structure according to (1), wherein the T helper epitope is covalently linked to the amine end of the B cell epitope.

(6)如(1)之α-Syn胜肽免疫原結構，其中T輔助抗原決定位透過選擇性異源連接子共價連接B細胞抗原決定位的胺基端。 (6) The α-Syn peptide immunogen structure according to (1), wherein the T helper epitope is covalently connected to the amine base of the B cell epitope through a selective heterolinker.

(7)如(1)之α-Syn胜肽免疫原結構，包括下列結構(Th)m-(A)n-(α-Syn C-端片段)-X或(α-Syn C-端片段)-(A)n-(Th)m-X其中Th為T輔助抗原決定位；A為異源性連接子； (α-Syn C-端片段)為B細胞抗原決定位；X為一胺基酸的α-COOH或α-CONH₂；m為約1至約4；且n為1至約10。 (7) The α-Syn peptide immunogen structure according to (1), including the following structures (Th) m- (A) n- (α-Syn C-terminal fragment) -X or (α-Syn C-terminal fragment )-(A) n- (Th) mX where Th is the T helper epitope; A is a heterologous linker; (α-Syn C-terminal fragment) is the B cell epitope; X is a monoamino acid Α-COOH or α-CONH ₂ ; m is from about 1 to about 4; and n is from 1 to about 10.

(8)如(1)之α-Syn胜肽免疫原結構，包括一胺基酸序列，其擇自於SEQ ID NOs：107、108、111-113及115-147所組成之群組。 (8) The α-Syn peptide immunogen structure according to (1), including a monoamino acid sequence, selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147.

(9)如(1)之α-Syn胜肽免疫原結構，包括一胺基酸序列，其擇自於SEQ ID NOs：107、108及111-113所組成之群組。 (9) The α-Syn peptide immunogen structure according to (1), comprising a monoamino acid sequence selected from the group consisting of SEQ ID NOs: 107, 108, and 111-113.

(10)一種組成物，包括(1)之α-Syn胜肽免疫原結構。 (10) A composition comprising the α-Syn peptide immunogen structure of (1).

(11)一種組成物，包括一個以上之(1)的α-Syn胜肽免疫原結構。 (11) A composition comprising more than one α-Syn peptide immunogen structure according to (1).

(12)如(11)之組成物，其中α-Syn胜肽免疫原結構具有SEQ ID NOs：112及113胺基酸序列。 (12) The composition according to (11), wherein the α-Syn peptide immunogen structure has the amino acid sequences of SEQ ID NOs: 112 and 113.

(13)一種醫藥組成物，包括(1)之α-Syn胜肽免疫原結構及一藥學上可接受之載體及/或佐劑。 (13) A pharmaceutical composition comprising the α-Syn peptide immunogen structure of (1) and a pharmaceutically acceptable carrier and / or adjuvant.

(14)如(13)之醫藥組成物，其中a. α-Syn胜肽免疫原結構擇自於SEQ ID NOs：107、108、111-113及115-147所組成之群組；以及b. 該佐劑為礦物鹽類，其擇自於Al(OH)₃或AlPO₄所組成之群組。 (14) The pharmaceutical composition according to (13), wherein a. The α-Syn peptide immunogen structure is selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147; and b. The adjuvant is a mineral salt selected from the group consisting of Al (OH) ₃ or AlPO ₄ .

(15)如(13)之醫藥組成物，其中a. α-Syn胜肽免疫原結構擇自於SEQ ID NOs：107,108, 111-113,and 115-147所組成之群組；以及b. 該α-Syn胜肽免疫原結構與一CpG寡核苷酸(ODN)，以形成一穩定的免疫刺激複合物。 (15) The pharmaceutical composition according to (13), wherein a. The α-Syn peptide immunogen structure is selected from SEQ ID NOs: 107, 108, A group consisting of 111-113, and 115-147; and b. The α-Syn peptide immunogen structure and a CpG oligonucleotide (ODN) to form a stable immunostimulatory complex.

(16)一分離的抗體或其抗原決定位結合片段，其專一性地結合至(1)之α-Syn胜肽免疫原結構的B細胞抗原決定位。 (16) An isolated antibody or epitope-binding fragment thereof that specifically binds to the B-cell epitope of the α-Syn peptide immunogen structure of (1).

(17)如(16)之分離抗體或其抗原決定位結合片段，其結合至α-Syn胜肽免疫原結構。 (17) The isolated antibody or epitope-binding fragment thereof according to (16), which binds to an α-Syn peptide immunogen structure.

(18)一種分離抗體或其抗原決定位結合片段，其專一性地結合至(9)之α-Syn胜肽免疫原結構的B細胞抗原決定位。 (18) An isolated antibody or an epitope-binding fragment thereof that specifically binds to the B-cell epitope of the α-Syn peptide immunogen structure of (9).

(19)一種組成物，包括(16)之分離抗體或其抗原決定位結合片段。 (19) A composition comprising the isolated antibody of (16) or an epitope-binding fragment thereof.

(20)一種組成物，包括(18)之分離抗體或其抗原決定位結合片段。 (20) A composition comprising the isolated antibody of (18) or an epitope-binding fragment thereof.

(21)如(20)之組成物，包括a. 專一性結合至SEQ ID NO：112之B細胞抗原決定位之分離抗體或其抗原決定位結合片段；以及b. 專一性結合至SEQ ID NO：113之B細胞抗原決定位之分離抗體或其抗原決定位結合片段之組合。 (21) The composition of (20), comprising a. An isolated antibody or an epitope-binding fragment thereof that specifically binds to a B-cell epitope of SEQ ID NO: 112; and b. Specifically binds to SEQ ID NO : 113 B-cell epitope isolated antibody or combination of epitope-binding fragments thereof.

(22)一種製造辨識宿主中α-Syn的抗體，包括給予該宿主一組成物，其包括(1)之α-Syn胜肽免疫原結構及一載體及/或佐劑。 (22) An antibody for recognizing α-Syn in a host, comprising administering to the host a composition comprising the α-Syn peptide immunogen structure of (1) and a carrier and / or adjuvant.

(23)一種抑制動物中α-Syn聚集的方法，包括給予該動物一藥學上有效量之(1)的α-Syn胜肽免疫原結構。 (23) A method of inhibiting α-Syn aggregation in an animal, comprising administering the A pharmaceutically effective amount of (1) α-Syn peptide immunogen structure.

(24)一種降低動物中α-Syn聚集的方法，包括給予該動物一藥學上有效量之(1)的α-Syn胜肽免疫原結構。 (24) A method for reducing α-Syn aggregation in an animal, comprising administering to the animal a pharmaceutically effective amount of the α-Syn peptide immunogen structure of (1).

(25)一種辨識生物樣本中不同大小的α-Syn聚集體，包括：a.將該生物樣本在可與(16)之抗體或其抗原決定位結合片段結合的條件下與接觸；b.檢測與該生物樣品中α-Syn聚集體結合之抗體或其抗原決定位結合片段的量。 (25) An α-Syn aggregate of different sizes in a biological sample, including: a. Contacting the biological sample with the antibody or an epitope-binding fragment of (16) under conditions that are capable of being contacted; b. The amount of the antibody or epitope-binding fragment that binds to the α-Syn aggregate in the biological sample.

以下實施例提供詳細的實施方式。 The following examples provide detailed implementations.

【實施例】[Example]

實施例1. α-突觸核蛋白胜肽的合成及其配方的製備Example 1. Synthesis of α-synuclein peptide and preparation of its formulation

a. α-Syn C端片段的合成a. Synthesis of α-Syn C-terminal fragment

本實施例揭示開發α-Syn胜肽免疫原結構之α-Syn C端片段的合成方法。可進行用於血清學檢測、實驗室試驗和現場研究的小量合成，以及用於藥物組成物之工業/商業生產的大量(千克)合成。 This example discloses a synthetic method for developing an α-Syn C-terminal fragment of an α-Syn peptide immunogen structure. It can be used for small-scale synthesis for serological testing, laboratory tests and field research, as well as large-scale (kilogram) synthesis for industrial / commercial production of pharmaceutical compositions.

為了篩選和選擇有效α-Syn胜肽免疫原結構的最佳肽構建體，設計了具有長度為約10至40個氨基酸序列的大量α-Syn相關抗原性肽庫。 In order to screen and select the best peptide constructs for effective α-Syn peptide immunogen structure, a large library of α-Syn related antigenic peptides with a length of about 10 to 40 amino acid sequences was designed.

使用代表性的全長α-Syn(SEQ ID NO：1)及β-Syn(SEQ ID No：2)、α-Syn片段，如α-Syn_111-132、α-Syn_126-135等10-mer胜肽以各種血清試驗鑑定抗原決定位，如表1所示(SEQ ID NOs：1及3至69)。將所選擇的α-Syn片段合成連接至源自於麻疹病毒融合蛋白(MVF)、B型肝炎表面抗原蛋白(HBsAg)、流感、破傷風桿菌和艾伯斯坦-巴爾病毒(Epstein-Barr virus，EBV)之特別設計的輔助T細胞(Th)抗原決定位，以製作成α-Syn胜肽免疫原結構，如表2(SEQ ID NOs：70-98)所示。Th抗原決定位可使用單一序列(SEQ ID NOs：70-78及83-98)或合併序列庫(SEQ ID NOs：79-82)，以促進各α-Syn胜肽免疫原結構的免疫原性。 Use representative full-length α-Syn (SEQ ID NO: 1) and β-Syn (SEQ ID No: 2), α-Syn fragments, such as α-Syn _111-132 , α-Syn _126-135, etc. 10-mer The peptides identified epitopes in various serum tests, as shown in Table 1 (SEQ ID NOs: 1 and 3 to 69). The selected α-Syn fragment was synthetically linked to measles virus fusion protein (MVF), hepatitis B surface antigen protein (HBsAg), influenza, tetanus, and Epstein-Barr virus (EBV) ) Specially designed helper T cell (Th) epitopes to make an α-Syn peptide immunogen structure, as shown in Table 2 (SEQ ID NOs: 70-98). Th epitope can use a single sequence (SEQ ID NOs: 70-78 and 83-98) or a combined sequence library (SEQ ID NOs: 79-82) to promote the immunogenicity of each α-Syn peptide immunogen structure .

由超過100個胜肽結構選擇出的代表性α-Syn胜肽免疫原結構，如表3(SEQ ID NOs：99-147)所示。用於免疫原性研究或檢測及/或測量抗-α-Syn抗體相關血清學試驗的所有胜肽，使用F-moc化學以Applied BioSystems Models 430A、431及/或433的肽合成儀進行以小量合成。每個胜肽以在固相載體上獨立合成，其N端具有F-moc保護及三個官能氨基酸的支鏈保護基團。將胜肽由固相載體上切除，並以90%三氟乙酸(TFA)去除支鏈保護基團。以(MALDI-TOF)質譜法分析合成胜肽以確保氨基酸序列正確。各個合成胜肽也可以反相HPLC(RP-HPLC)確認合成結果及濃度。儘管嚴格控制合成程序(包括逐步監測耦合效率)，仍會因延長循環期間的意外事件，包括氨基酸***、缺失、置換和提前終止，而產生胜肽類似物。因此，胜肽合成一般會包括目標胜肽及多種胜肽類似物。儘管包含了這種非預期的肽類似物，但此最終合成胜肽仍適用於免疫應用，包括免疫檢測(抗體捕獲抗原)及醫藥組成物(胜肽免疫原)。一般來說，胜肽類似物，不論是刻意設計或是由合成過程中產生的混合副產物，只要以嚴謹QC程序監控製程與產品評估程序，能保證最終產品的再現性和功效，這些胜肽通常與所欲胜肽的效果相同。數百至數千克大量胜肽合成可利用客製化的自動胜肽合成儀UBI2003或其類似進行合成，濃度在15mmol至50mmol之間。對於臨床試驗之最終醫藥組合物中的活性成分，α-Syn胜肽結構體可利用RP-HPLC在低濃度梯度下進行純化，並以MALDI-TOF質譜法及胺基酸分析胜肽特徵，並以RP-HPLC分析純度和一致性。 Representative α-Syn peptide immunogen structures selected from more than 100 peptide structures are shown in Table 3 (SEQ ID NOs: 99-147). All peptides used for immunogenicity research or detection and / or measurement of anti-α-Syn antibody-related serological tests are performed using F-moc chemistry with Applied BioSystems Models 430A, 431, and / or 433 peptide synthesizers.量合成。 Volume synthesis. Each peptide is independently synthesized on a solid phase support, and its N-terminus has F-moc protection and three functional amino acid branch-chain protecting groups. The peptide was excised from the solid phase support and the branched protecting groups were removed with 90% trifluoroacetic acid (TFA). The synthetic peptide was analyzed by (MALDI-TOF) mass spectrometry to ensure that the amino acid sequence was correct. The synthesis results and concentration of each synthetic peptide can also be confirmed by reversed-phase HPLC (RP-HPLC). Despite strict control over synthetic procedures, including stepwise monitoring of coupling efficiency, peptide analogs can still be generated by unexpected events during extended cycles, including amino acid insertions, deletions, substitutions, and early termination. Therefore, peptide synthesis generally includes target peptides and various peptide analogs. Despite the inclusion of this unexpected peptide analog, this final peptide was synthesized Still suitable for immunological applications, including immunoassay (antibody capture antigen) and pharmaceutical composition (peptide immunogen). In general, peptide analogs, whether deliberately designed or mixed by-products generated during the synthesis process, as long as the manufacturing process and product evaluation procedures are monitored with rigorous QC procedures, the reproducibility and efficacy of the final product can be guaranteed. Usually the same effect as the desired peptide. Hundreds to several kilograms of large peptides can be synthesized using a customized automatic peptide synthesizer UBI2003 or similar, with a concentration between 15mmol and 50mmol. For the active ingredients in the final pharmaceutical composition of clinical trials, the α-Syn peptide structure can be purified by RP-HPLC at a low concentration gradient, and the characteristics of the peptide can be analyzed by MALDI-TOF mass spectrometry and amino acids, and Purity and consistency were analyzed by RP-HPLC.

b. 含有α-Syn胜肽免疫原結構之組成物的製備b. Preparation of composition containing α-Syn peptide immunogen structure

製備使用油包水乳化物和無機鹽懸浮液的配方。為了使醫藥組成物可應用於廣大族群，且預防也是給予的目的之一，安全性成為考量的另一個重要因素。僅管在人類臨床上許多醫藥組成物使用油包水乳化物，但基於安全性，明礬仍然是醫藥組合物的主要佐劑。因此，在臨床應用上，明礬或其礦物鹽ADJUPHOS(磷酸鋁)經常作為佐劑使用。 Formulations using water-in-oil emulsions and inorganic salt suspensions. In order to make the pharmaceutical composition applicable to a wide range of ethnic groups, and prevention is also one of the purposes of administration, safety has become another important factor to consider. Despite the use of water-in-oil emulsions in many pharmaceutical compositions in human clinical practice, alum remains the main adjuvant for pharmaceutical compositions based on safety. Therefore, in clinical applications, alum or its mineral salt ADJUPHOS (aluminum phosphate) is often used as an adjuvant.

以下所述特定配方含有所有類型的α-Syn胜肽免疫原結構。先於天竺鼠中評估超過100種α-Syn胜肽免疫原結構對於代表性α-Syn胜肽的相對免疫原性，且利用塗佈擇自SEQ ID NOs：1-153之不同胜肽的 96孔盤以ELISA分析各種同源肽之間的血清交叉反應性。 The specific formulations described below contain all types of α-Syn peptide immunogen structures. The relative immunogenicity of more than 100 α-Syn peptide immunogen structures to representative α-Syn peptides was evaluated in guinea pigs, and the application of different peptides selected from SEQ ID NOs: 1-153 was applied. 96-well plates were analyzed by ELISA for serum cross-reactivity between various homologous peptides.

α-Syn胜肽免疫原結構(i)以可用於人類之Seppic Montanide^TM ISA 51製備成油包水乳化物，或(ii)不同濃度的胜肽結構與ADJUPHOS(磷酸鋁)或ALHYDROGEL(明礬)混合。將α-Syn胜肽免疫原結構以20至800μg/mL的濃度溶於水中，並與Montanide^TM ISA 51(1：1體積比)，或礦物鹽或ALHYDROGEL(明礬)(1：1體積比)配製成油包水乳化物。將組成物在室溫維持約30分鐘，並在免疫前以震盪器混合10至15秒。特定組成物免疫動物2至3次，以肌內注射方式給予，在第0週及初始免疫後(wpi)3週(加強)進行進行免疫，選擇性地在5或6 wpi進行第2次加強免疫。利用經選擇的B抗原決定位胜肽測試經免疫的動物，以評估各α-Syn胜肽免疫原結構在配方中的免疫原性以及其與相關目標胜肽或蛋白質的交叉反應性。將於天竺鼠中初步篩選出可能具有免疫原性之α-Syn胜肽免疫原結構以油包水乳化物、礦物鹽及明礬配方形式進一步在靈長類動物中以特定的給藥程序進行試驗。 α-Syn peptide immunogen structure (i) Prepared as a water-in-oil emulsion with Seppic Montanide ^TM ISA 51, which can be used in humans, or (ii) peptide structures with different concentrations and ADJUPHOS (aluminum phosphate) or ALHYDROGEL (alum) mixing. The α-Syn peptide immunogen structure is dissolved in water at a concentration of 20 to 800 μg / mL, and is mixed with Montanide ^TM ISA 51 (1: 1 volume ratio), or mineral salt or ALHYDROGEL (alum) (1: 1 volume ratio) Formulated as a water-in-oil emulsion. The composition was maintained at room temperature for about 30 minutes and mixed with a shaker for 10 to 15 seconds before immunization. Animals were immunized with the specific composition 2 to 3 times and given by intramuscular injection. Immunization was performed at week 0 and 3 weeks (boost) after the initial immunization (wpi), and the second boost was optionally performed at 5 or 6 wpi Immunity. The immunized animals were tested using the selected B epitope peptides to evaluate the immunogenicity of each α-Syn peptide immunogen structure in the formulation and its cross-reactivity with the relevant target peptide or protein. The α-Syn peptide immunogen structure, which may be immunogenic, will be initially screened in guinea pigs. It will be further tested in primates with specific dosing procedures in the form of water-in-oil emulsions, mineral salts and alum formulations.

僅將最有希望的α-Syn胜肽免疫原結構體在被製成最終製劑前，以GLP規範進一步廣泛評估其免疫原性、持續時間、毒性及療效，以準備在突觸核蛋白病患者中提出新藥臨床申請和臨床試驗的進行。 Only the most promising α-Syn peptide immunogen structure is further evaluated for its immunogenicity, duration, toxicity, and efficacy according to GLP specifications before being prepared into a final preparation in preparation for patients with synucleinopathy In the clinical application of new drugs and clinical trials.

實施例2. 重組α-Syn蛋白的製備Example 2. Preparation of recombinant α- Syn protein

將α-Syn基因構築至pGEX-4T1載體的方式可參照Neurotoxicology and teratology 2004,26(3)：397-406。將目標序列(SEQ ID NOs：1)***pGEX-4T1載體中BamHI與XhoI之間的限制性內切位。以聚合酶鏈鎖反應(PCR)使用KAPA HiFi DNA聚合酶(Kapa Biosystems,Inc.,Woburn,MA,USA)複製此片段。引子序列如下：正向引子5’-cgggatccgatgtgtttatgaaaggtctgag-3’(SEQ ID NO：149)；反向引子5’-ggaattccgatgtgtttatgaaaggtctgag-3’(SEQ ID NO：150)。PCR的條件如下：94℃加熱1分鐘，94℃加熱15秒進行變性，於60℃下30秒進行接合，且於68℃下2分鐘進行延伸，進行30個循環，並於68℃下5分鐘進行結束反應。利用Q5 Site-Directed Mutagenesis套組(New England BioLabs,Beverly,MA,USA)完成A53T α-Syn的點突變。突變α-Syn的引子序列為：正向引子5’-tcatggtgtgaccaccgttgcag-3’(SEQ ID NO：151)；反向引子5’-accacgccttctttggttttg-3’(SEQ ID NO：152)。 For the method of constructing the α-Syn gene into the pGEX-4T1 vector, refer to Neurotoxicology and teratology 2004, 26 (3): 397-406. The target sequence (SEQ ID NOs: 1) was inserted into the restriction endonucleation site between BamHI and XhoI in the pGEX-4T1 vector. This fragment was replicated by polymerase chain reaction (PCR) using KAPA HiFi DNA polymerase (Kapa Biosystems, Inc., Woburn, MA, USA). The primer sequence is as follows: forward primer 5'-cgggatccgatgtgtttatgaaaggtctgag-3 '(SEQ ID NO: 149); reverse primer 5'-ggaattccgatgtgtttatgaaaggtctgag-3' (SEQ ID NO: 150). The PCR conditions were as follows: heating at 94 ° C for 1 minute, heating at 94 ° C for 15 seconds for denaturation, bonding at 60 ° C for 30 seconds, and extension at 68 ° C for 2 minutes, 30 cycles, and 5 minutes at 68 ° C. The end reaction is performed. A Q5 Site-Directed Mutagenesis kit (New England BioLabs, Beverly, MA, USA) was used to complete the A53T α-Syn point mutation. The primer sequence of the mutant α-Syn is: forward primer 5'-tcatggtgtgaccaccgttgcag-3 '(SEQ ID NO: 151); reverse primer 5'-accacgccttctttggttttg-3' (SEQ ID NO: 152).

將含有α-Syn的pGEX-4T1 GST載體轉染至E.coli BL21(DE3)進行蛋白質表現。將E.coli在37°C下培養於LB培養基中，並在OD600達到0.8時加入最終濃度4mM的β-d-1硫代半乳糖苷(IPTG)。培養4小時後，於4℃下，以5000xg離心20分鐘收集細胞。將所收集的細胞懸浮於PBS，置於冰上進行超音波破壞，再於4℃下，以5000xg離心20分鐘。將上清液置入穀胱甘肽瓊脂糖-4B管柱(GE Healthcare)，並以PBS進行平衡。以PBS清洗3次後，加入1mL的凝血酶(20U/mL溶於PBS)，於4℃靜置隔夜，以釋放融合蛋白的GST。將不含標誌的α-Syn洗堤出來，並以HiTrap Benzamidine FF管柱(GE Healthcare)去除凝血酶。將透析的α-Syn立即置於-80℃下冷凍。純化之α-Syn的分子量為14kDa MW，以10% SDS-PAGE分離後使用抗-α-Syn抗體(1：2000,Millipore,targeting α-Syn_111-131)進行西方墨點法分析。 The pGEX-4T1 GST vector containing α-Syn was transfected into E. coli BL21 (DE3) for protein expression. E. coli was cultured in LB medium at 37 ° C, and β-d-1 thiogalactoside (IPTG) was added at a final concentration of 4 mM when the OD600 reached 0.8. After 4 hours of incubation, cells were collected by centrifugation at 5000xg for 20 minutes at 4 ° C. The collected cells were suspended in PBS, placed on ice for ultrasonic destruction, and centrifuged at 5000xg for 20 minutes at 4 ° C. The supernatant was placed in a glutathione agarose-4B column (GE Healthcare) and equilibrated with PBS. After washing 3 times with PBS, 1 mL of thrombin (20 U / mL in PBS) was added and left at 4 ° C overnight to release the GST of the fusion protein. The unlabeled α-Syn was washed out and thrombin was removed on a HiTrap Benzamidine FF column (GE Healthcare). The dialyzed α-Syn was immediately frozen at -80 ° C. The purified α-Syn has a molecular weight of 14 kDa MW. After separation by 10% SDS-PAGE, anti-α-Syn antibody (1: 2000, Millipore, targeting α-Syn _111-131 ) was used for Western _blot analysis.

實施例3. 血清學分析及試劑Example 3. Serological analysis and reagents

以下，揭示評估合成胜肽結構體及其製劑的功能性免疫原性的血清學測定與試劑。 In the following, serological assays and reagents for evaluating the functional immunogenicity of synthetic peptide structures and their preparations are disclosed.

a. 以胜肽ELISA分析抗體專一性a. Analysis of antibody specificity by peptide ELISA

以下實施例中用於評估免疫血清樣本的ELISA分析如下所述。96孔盤的孔洞分別以濃度2μg/mL(若無特別說明)，100μL溶於10mM NaHCO₃緩衝液(pH 9.5(若無特別說明))的目標胜肽α-Syn片段A85-A140、A91-A140、A101-A140、A111-A140、D121-A140、E126-A140、K97-D135、G101-D135、G111-D135、D121-D135、E123-D135、E126-D135、G101-132及G111-G132胜肽(SEQ ID NOs：4-17)以37℃，反應1小時以進行塗佈。 The ELISA analysis used to evaluate immune serum samples in the following examples is described below. The holes of the 96-well plate were at a concentration of 2 μg / mL (unless otherwise specified), 100 μL of target peptide α-Syn fragments A85-A140, A91- dissolved in 10 mM NaHCO ₃ buffer (pH 9.5 (unless specified)) A140, A101-A140, A111-A140, D121-A140, E126-A140, K97-D135, G101-D135, G111-D135, D121-D135, E123-D135, E126-D135, G101-132 and G111-G132 win The peptides (SEQ ID NOs: 4-17) were reacted at 37 ° C for 1 hour to perform coating.

b. 以Th胜肽ELISA評估抗體對Th胜肽的反應b. Evaluation of Th peptide response by Th peptide ELISA

塗佈胜肽(SEQ ID Nos：70-98)的孔洞與250μL，3重量%溶於PBS的明膠於37℃下反應1小時，以封阻非專一性蛋白結合位，接著以含有0.05體積%的TWEEN® 20清洗3次後，乾燥。將待測血清利用含20體積%山羊血清、1重量%明膠及0.05體積%TWEEN® 20的PBS以1：20比例稀釋(若無特別說明)。將100μL的稀釋樣本(如血清、血漿)添加至各孔洞中，於37℃反應60分鐘。接著，以0.05體積%溶於PBS的TWEEN® 20清洗孔洞6次，以去除未結合的抗體。辣根過氧化物酶(HRP)-共軛物種(如小鼠、天竺鼠或人類)專一性山羊抗-IgG為追蹤標誌，可與孔洞中的抗體/抗原複合體結合。將100μL稀釋之過氧化物酶標記的山羊抗-IgG添加至各個孔洞中，且於37℃反應30分鐘。以含0.05% TWEEN® 20的PBS清洗孔洞6次，以移除未結合的抗體，且與100μL含0.04重量%的3’,3’,5’,5’-四甲基聯苯胺(TMB)以及0.12體積%過氧化氫之檸檬酸鈉緩衝液反應15分鐘。此混合物可藉由產生有色物質來偵測過氧化物酶。加入100μL的1.0M H₂SO₄終止反應，偵測波長450nm的吸光值(A450)。測定10倍稀釋(稀釋比例為1：100至1：10,000)的血清力價並Log₁₀表示，製作A450線性回歸，以線性回歸及A450為0.5的閥值，計算各種經α-Syn胜肽免疫原免疫之動物的抗體力價。 The peptide-coated holes (SEQ ID Nos: 70-98) were reacted with 250 μL of 3% by weight gelatin in PBS at 37 ° C. for 1 hour to block non-specific protein binding sites, and then contained 0.05% by volume TWEEN® 20 is washed 3 times and dried. The serum to be tested was diluted 1:20 with PBS containing 20% by volume of goat serum, 1% by weight of gelatin and 0.05% by volume of TWEEN® 20 (unless otherwise specified). Add 100 μL of the diluted sample (such as serum and plasma) to each well and react at 37 ° C for 60 minutes. Next, wash the wells 6 times with TWEEN® 20 in 0.05% PBS to remove unbound antibodies. Horseradish peroxidase (HRP) -conjugated species (such as mouse, guinea pig, or human) specific goat anti-IgG is a tracking marker that can bind to the antibody / antigen complex in the hole. 100 μL of diluted peroxidase-labeled goat anti-IgG was added to each well, and reacted at 37 ° C. for 30 minutes. Wash the wells 6 times with PBS containing 0.05% TWEEN® 20 to remove unbound antibodies and 100 μL of 0.04% by weight of 3 ', 3', 5 ', 5'-tetramethylbenzidine (TMB) And 0.12% by volume sodium hydrogen citrate buffer for 15 minutes. This mixture can detect peroxidase by producing colored substances. The reaction was stopped by adding 100 μL of 1.0MH ₂ SO ₄ and the absorbance (A450) at 450 nm was detected. Measure the serum valence of 10-fold dilution (dilution ratio of 1: 100 to 1: 10,000) and express it as Log _10. Make A450 linear regression, and use linear regression and A450 threshold of 0.5 to calculate various α-Syn peptide immunity Antibody titer of the original immunized animal.

c. 以B細胞抗原決定位簇10-mer胜肽ELISA評估精確專一性及α-Syn片段的抗原決定位位置c. B-cell epitope 10-mer peptide ELISA to evaluate the specificity and epitope position of α-Syn fragment

利用抗原決定位作圖來測定免疫宿主中抗-α-Syn胜肽抗體的精確專一性。易言之，將96孔盤分別以每孔洞塗佈0.5μg/0.1mL的α-Syn 10-mer胜肽(SEQ ID NOs：18至69)，接著加入100μL血清樣本(1：100的比例以PBS稀釋)進行反應，並進行上述抗體ELISA分析步驟。也可利用相對應的α-Syn胜肽(SEQ ID No：99、102、108、110、112、113)或其片段，不含連接子及Th序列，或具有β-Syn(SEQ ID NO：153)胜肽進行額外的反應及專一性分析，以測試α-Syn胜肽免疫原的B細胞抗原決定位及相關的免疫血清抗-α-Syn抗體的專一性。 Epitope mapping was used to determine the precise specificity of anti-α-Syn peptide antibodies in immunized hosts. In other words, a 96-well plate was coated with 0.5 μg / 0.1 mL of α-Syn 10-mer peptide (SEQ ID NOs: 18 to 69) per hole, and then 100 μL of a serum sample (1: 100 ratio to PBS dilution) was performed, and the antibody ELISA analysis step was performed. The corresponding α-Syn peptide (SEQ ID No: 99, 102, 108, 110, 112, 113) or a fragment thereof can also be used, without the linker and Th sequence, or with β-Syn (SEQ ID NO: 153) The peptide performs additional responses and specificity analysis to test the specificity of the B-cell epitope of the α-Syn peptide immunogen and the associated immune serum anti-α-Syn antibody.

d. 免疫原性的評估d. Evaluation of immunogenicity

依照免疫程序收集動物免疫前及免疫血清樣本，以56℃加熱30分鐘以去活化血清補體因子。在給予醫藥組成物後，依程序獲得血液樣本，並分析特定目標位置的免疫原性。分析系列稀釋的血清，並以Log₁₀的倒數表示力價。以可引發針對目標抗原內抗原決定位特異性的高力價B細胞抗體反應，同時對用於增強B細胞反應之“輔助T細胞抗原決定位”保持在低、甚至可忽略的抗體反應性，來評估特定醫藥組成物的免疫原性。 Pre-immune and immune serum samples were collected according to the immunization program, and heated at 56 ° C for 30 minutes to deactivate serum complement factors. After administering the pharmaceutical composition, a blood sample is obtained in accordance with the procedure, and the immunogenicity of a specific target location is analyzed. Serially diluted sera were analyzed and force valence was expressed as the inverse of Log ₁₀ . With high-valence B-cell antibody response that can be specific for the epitope in the target antigen, while maintaining the "helper T-cell epitope" used to enhance the B-cell response at a low or even negligible antibody reactivity, To assess the immunogenicity of a particular pharmaceutical composition.

e. 小鼠免疫血清中α-Syn含量的免疫分析e. Immunoassay of α-Syn content in mouse immune serum

使用三明治ELISA(Cloud-clon,SEB222Mu)，以抗-α-Syn抗體作為捕捉抗體，以生物素標誌的抗體為偵測抗體，分析給予α-Syn胜肽免疫原小鼠血清中的α-Syn含量。易言之，將100ng/well溶於塗佈緩衝液(15mM Na₂CO₃，35mM NaHCO₃，pH 9.6)的抗體固定於96孔盤中，於4℃反應隔夜。以200μL/well的分析稀釋液(含0.5% BSA、0.05% TWEEN®-20、0.02% ProClin 300的PBS)在室溫下對塗佈的孔洞封阻1小時。使用含有純化重組α-Syn與5%小鼠血清之分析稀釋液獲得標準曲線(2倍系列稀釋，濃度156至1250ng/mL)。將50μL的稀釋血清(1：20)及標準物添加至經塗佈的孔洞。於室溫反應1小時。對所有的孔洞送氣，並以200μL/well的清洗緩衝液清洗6次。將被捕捉的人類α-Syn與100μL的偵測抗體溶液(50ng/ml溶於分析稀釋液之標誌生物素的HP6029)於室溫下反應1小時。接著，使用鏈黴親和素聚-HRP(1：10000稀釋，Thermo Pierce)與結合之生物素-HP6029反應1小時。對所有的孔洞送氣，以200μL/well的清洗緩衝液清洗6次，並加入100μL/well的1M H₂SO₄停止反應。使用SoftMax Pro軟體(Molecular Devices)製作標準曲線，並產生4參數邏輯曲線擬合，計算所有試驗樣品中的α-Syn濃度。 Using sandwich ELISA (Cloud-clon, SEB222Mu) with anti-α-Syn antibody as capture antibody and biotin-labeled antibody as detection antibody, α-Syn in the serum of mice administered with α-Syn peptide immunogen was analyzed content. In other words, 100 ng / well of an antibody dissolved in a coating buffer (15 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ , pH 9.6) was fixed in a 96-well plate and reacted at 4 ° C. overnight. 200 μL / well of analytical dilution (0.5% BSA, 0.05% TWEEN®-20, 0.02% ProClin 300 in PBS) was used to block the coated holes for 1 hour at room temperature. A standard curve (2-fold serial dilution, concentration 156 to 1250 ng / mL) was obtained using an analytical dilution containing purified recombinant α-Syn and 5% mouse serum. 50 μL of diluted serum (1:20) and standards were added to the coated wells. The reaction was carried out at room temperature for 1 hour. All holes were aerated and washed 6 times with 200 μL / well of washing buffer. The captured human α-Syn was reacted with 100 μL of a detection antibody solution (50 ng / ml of the biotin-labeled HP6029 in an analytical dilution) for 1 hour at room temperature. Next, streptavidin poly-HRP (1: 10000 dilution, Thermo Pierce) was reacted with the bound biotin-HP6029 for 1 hour. All holes were aerated, washed 6 times with 200 μL / well of washing buffer, and 100 μL / well of 1M H ₂ SO _{4 was added to} stop the reaction. A standard curve was prepared using SoftMax Pro software (Molecular Devices) and a 4-parameter logistic curve fit was generated to calculate the α-Syn concentration in all test samples.

f. α-Syn聚集體與重組α-Syn的製備f. Preparation of α-Syn aggregates and recombinant α-Syn

為了製備聚集的α-Syn，將純化的野生型或A53T的突變α-Syn[溶於100μL PBS/KCl聚集緩衝液 (2.5mM MgCl₂,50mM HEPES and 150mM KCl in 1 x PBS,pH 7.4)，濃度為0.1μg/μL]置於1.5mL微量離心管中，並以恆溫震盪器於37℃下反應7天。將所獲得之聚集α-Syn立即冷凍於-80℃，供後續使用。 To prepare aggregated α-Syn, purified wild-type or A53T mutant α-Syn [dissolved in 100 μL PBS / KCl aggregation buffer (2.5 mM MgCl ₂ , 50 mM HEPES and 150 mM KCl in 1 x PBS, pH 7.4), [Concentration: 0.1 [mu] g / [mu] L] was placed in a 1.5 mL microcentrifuge tube and reacted for 7 days at 37 [deg.] C. using a thermostatic shaker. The obtained aggregated α-Syn was immediately frozen at -80 ° C for subsequent use.

g. 抗-α-Syn抗體的純化g. Purification of anti-α-Syn antibodies

以含有不同序列(SEQ ID NOs：99-121)之α-Syn胜肽免疫原結構免疫天竺鼠，由注射後3至15週(WPI)所收集的血清利用親和性管柱(Thermo Scientific,Rockford)純化抗-α-Syn抗體。易言之，在緩衝液(0.1M磷酸與0.15M氯化鈉，pH 7.2)平衡後，將400μL的血清添加至Nab蛋白G Spin管柱中，上下混合10分鐘並以5,800 xg離心1分鐘。以結合緩衝液(400μL)清洗管柱3次。接著，加入洗湜緩衝液(400μL，0.1M甘胺酸，pH 2.0)至Spin管柱中，經5,800 x g離心1分鐘洗湜出抗體。將洗湜出的抗體與中和緩衝液(400μL,0.1M Tris pH 8.0)混合，以胎牛血清(BSA)作為標準，使用Nan-Drop偵測OD280以計算抗體濃度。 Guinea pigs were immunized with an α-Syn peptide immunogen structure containing different sequences (SEQ ID NOs: 99-121). Affinity columns (Thermo Scientific, Rockford) were used from serum collected 3 to 15 weeks after injection (WPI). Purified anti-α-Syn antibody. In other words, after equilibrating the buffer (0.1 M phosphoric acid and 0.15 M sodium chloride, pH 7.2), 400 μL of serum was added to a Nab Protein G Spin column, mixed up and down for 10 minutes, and centrifuged at 5,800 xg for 1 minute. To binding buffer (400 μ L) washed three times with the column. Next, wash buffer (400 μL, 0.1M glycine, pH 2.0) was added to the Spin column, and the antibodies were washed out by centrifugation at 5,800 xg for 1 minute. The washed out antibody was mixed with a neutralization buffer (400 μL, 0.1 M Tris pH 8.0), and fetal bovine serum (BSA) was used as a standard. The OD280 was detected using Nan-Drop to calculate the antibody concentration.

h. 不同α-Syn胜肽免疫原結構免疫之天竺鼠之抗-α-Syn抗體對於不同大小之α-Syn分子的專一性h. Specificity of anti-α-Syn antibodies of guinea pigs with different α-Syn peptide immunogen structural immunity against α-Syn molecules of different sizes

使用西方墨點法分析源自於經不同α-Syn胜肽免疫原結構免疫之天竺鼠之抗-α-Syn抗體對於不同大小之α-Syn分子的專一性。將20μM α-Syn以12% Tris-甘胺酸SDS-PAGE分離並在光誘導交聯(PICUP)處理前轉染至硝化纖維(NC)膜上。以1μg/mL源自天竺鼠之抗-α-Syn抗體處理硝化纖維膜，接著與共軛HRP之驢抗-天竺鼠抗體(706-035-148,Jackson)反應。以化學冷光試劑Western Lightning ECL Pro(PerkinElmer)顯影膜的墨點。由結果可知，單體α-Syn(Mw 14,460Da)的分子量為約14kDa，其二聚體、三聚體或寡聚體具有單體α-Syn數倍的分子量。使用可偵測各種寡聚體，如二聚體、三聚體或寡聚體的市售Syn211抗體(Abcam)作為陽性對照組。 Western blot analysis was used to analyze the specificity of anti-α-Syn antibodies derived from guinea pigs immunized with different α-Syn peptide immunogen structures for α-Syn molecules of different sizes. 20 μM α-Syn was separated with 12% Tris-glycine SDS-PAGE and transfected onto nitrocellulose (NC) membranes before photo-induced cross-linking (PICUP) treatment. Derived from 1 μg / mL The nitrocellulose membrane was treated with anti-α-Syn antibody of guinea pig, and then reacted with donkey anti-guinea pig antibody (706-035-148, Jackson) conjugated with HRP. The chemical dots of the film were developed with a chemical cold light reagent Western Lightning ECL Pro (PerkinElmer). From the results, it is found that the molecular weight of the monomer α-Syn (Mw 14,460 Da) is about 14 kDa, and the dimer, trimer or oligomer thereof has a molecular weight several times that of the monomer α-Syn. As a positive control group, a commercially available Syn211 antibody (Abcam) capable of detecting various oligomers such as dimer, trimer or oligomer was used.

i. 不同類澱粉蛋白的圓漬點墨法分析i. Analysis of different types of amyloids by circular staining method

Aβ_1-42、Tau及α-Syn之α-螺旋單體、β-折疊單體、β-折疊寡聚物及纖維狀β-折疊的製備如下所示。 The preparations of Aβ _1-42 , Tau and α-Syn α-helical monomers, β-sheet monomers, β-sheet oligomers, and fibrous β-sheets are shown below.

1. Aβ _1-42 α-螺旋單體：將20μg的Aβ_1-42 β-折疊單體(50μL)加至含有20%三氟乙酸及20%六氟異丙醇(10μL)，置於4℃反應24小時，以形成α-螺旋單體。 1. Aβ _1-42 α-helical monomer: Add 20 μg of Aβ _1-42 β-fold monomer (50 μL) to 20% trifluoroacetic acid and 20% hexafluoroisopropanol (10 μL), place in 4 It was reacted at 24 ° C for 24 hours to form an α-helical monomer.

2. Aβ _1-42 β-折疊單體：將60μg的Aβ_1-42置於120μL含5% TFA的1xPBS中，在37℃反應24小時，置於10kDa的過濾管柱(Millipore)以獲得β-折疊單體。 2. Aβ _1-42 β-folded monomer: Place 60 μg of Aβ _1-42 in 120 μL of 5% TFA in 1xPBS, react at 37 ° C for 24 hours, and place in a 10 kDa filter column (Millipore) to obtain β -Folding monomer.

3. Aβ _1-42 β-折疊寡聚物：將含有60μg Aβ_1-42的120μL 1xPBS在37℃反應3天，置於冰上以超音波震盪後，置於10與30kDa過濾管柱(Millipore)以獲得分子量小於35kDa的β-折疊纖維狀寡聚物。 3. Aβ _1-42 β-folded oligomer: 120 μL 1xPBS containing 60 μg Aβ _1-42 was reacted at 37 ° C. for 3 days, placed on ice for ultrasonic shock, and placed in 10 and 30 kDa filter columns (Millipore ) To obtain β-sheet fibrous oligomers with a molecular weight of less than 35 kDa.

4. Aβ1-42纖維狀β-折疊：將含有60μg Aβ_1-42的120μL 1xPBS在37℃反應3天，置於冰上以超音波震盪後，置於30kDa過濾管柱(Millipore)以獲得纖維狀β-折疊。 4. Aβ1-42 fibrous β-sheet: 120 μL 1xPBS containing 60 μg Aβ _1-42 was reacted at 37 ° C for 3 days, placed on ice for ultrasonic shock, and placed in a 30 kDa filter column (Millipore) to obtain fibers. Like β-sheet.

5. α-Syn α-螺旋單體：將40μg新鮮配製的α-Syn在4℃溶於100μL冰的1xPBS，且立刻置於10kDa過濾管柱(Millipore)以獲得α-螺旋單體。 5. α-Syn α-helical monomer: 40 μg of freshly prepared α-Syn was dissolved in 100 μL of ice 1xPBS at 4 ° C. and immediately placed in a 10 kDa filter column (Millipore) to obtain α-helical monomer.

6. α-Syn β-折疊單體：將100μL含有40μg α-Syn的PBS/KCl緩衝液於37℃反應24小時，並使用10kDa過濾管柱(Millipore)獲得β-折疊單體。 6. α-Syn β-folded monomer: 100 μL of PBS / KCl buffer containing 40 μg of α-Syn was reacted at 37 ° C. for 24 hours, and a β-folded monomer was obtained using a 10 kDa filter column (Millipore).

7. α-Syn β-折疊單體：將100μL含有40μg聚集之α-Syn的PBS/KCl緩衝液於37℃反應8天，置於冰上以超音波震盪後，置於30kDa與100kDa過濾管柱(Millipore)以獲得β-折疊單體。 7. α-Syn β-folded monomer: 100 μL of 40 μg aggregated α-Syn PBS / KCl buffer solution was reacted at 37 ° C. for 8 days, placed on ice for ultrasonic shock, and placed in 30 kDa and 100 kDa filter tubes. Column (Millipore) to obtain β-sheet monomer.

8. α-Syn纖維狀β-折疊：將100μL含有40μg聚集之α-Syn的PBS/KCl緩衝液於37℃反應8天，置於冰上以超音波震盪後，置於30kDa與100kDa過濾管柱(Millipore)以獲得β-折疊單體。 8. α-Syn fibrous β-sheet: 100 μL of PBS / KCl buffer solution containing 40 μg aggregated α-Syn is reacted at 37 ° C. for 8 days, placed on ice for ultrasonic shock, and placed in 30 kDa and 100 kDa filter tubes. Column (Millipore) to obtain β-sheet monomer.

9. Tau441 α-螺旋單體：將4℃，100μL含有60μg Tau的1xPBS置於100kDa過濾管柱(Millipore)以獲得α-螺旋單體。 9. Tau441 α-helical monomer: 100 μL of 1 × PBS containing 60 μg of Tau at 4 ° C. was placed in a 100 kDa filter column (Millipore) to obtain α-helical monomer.

10. Tau441 β-折疊單體：將100μL含60μg聚集Tau與10unit/mL肝素的1x PBS緩衝液於25℃反應48小時，在4℃下置於100kDa過濾管柱(Millipore)以獲得β-折疊單體。 10. Tau441 β-fold monomer: 100 μL of 60 μg aggregated Tau was reacted with 10 unit / mL heparin 1x PBS buffer at 25 ° C for 48 hours, and placed in a 100kDa filter column (Millipore) at 4 ° C to obtain β-fold monomer.

10. Tau441 β-折疊寡聚物：將100μL含有60μg聚集之Tau的1xPBS緩衝液於37℃反應48小時，置於100kDa與300kDa過濾管柱(Pall)以獲得β-折疊寡聚物。 10. Tau441 β-fold oligomer: 100 μL of 1 × PBS buffer containing 60 μg of aggregated Tau was reacted at 37 ° C. for 48 hours, and placed in 100 kDa and 300 kDa filter columns (Pall) to obtain β-fold oligomer.

11. Tau441纖維狀β-折疊：將100μL含有60μg聚集之Tau與10unit/mL肝素的1xPBS緩衝液於37℃反應6天，於4℃置於300kDa過濾管柱(Pall)以獲得纖維狀β-折疊。 11. Tau441 fibrous β-sheet: 100 μL of 60 μg aggregated Tau was reacted with 10 unit / mL heparin 1xPBS buffer at 37 ° C for 6 days, and placed in a 300kDa filter column (Pall) at 4 ° C to obtain fibrous β- fold.

此單體及寡聚物以硫黃素-T(ThT,Sigma)螢光或聚丙烯醯胺凝膠電泳(PAGE)確認。類澱粉蛋白的濃度以Nano-Drop測定，並以市售類澱粉Aβ_1-42為標準。將單體及寡聚物分別墨點至PVDF膜上，Aβ_1-42為3μg，α-Syn為4μg，且Tau為7μg。將PVDF膜與純化自天竺鼠的抗-α-Syn抗體(1：1000比例稀釋)作為一級抗體，接著與抗天竺鼠HRP-共軛二級抗體(1：5000；Vector Laboratories)雜交。將PVDF膜以Luminata Western HRP受質(Bio-Rad,Hercules,CA,USA)處理，並以ChemiDoc-It 810數位影像系統(UVP Inc.,Upland,CA,USA)偵測產生的訊號。 This monomer and oligomer were confirmed by thioflavin-T (ThT, Sigma) fluorescence or polyacrylamide gel electrophoresis (PAGE). The amyloid concentration was determined using Nano-Drop and was based on the commercially available amyloid Aβ _1-42 . Monomers and oligomers were respectively inked onto PVDF membranes, Aβ _1-42 was 3 μg, α-Syn was 4 μg, and Tau was 7 μg. The PVDF membrane was purified with guinea pig anti-α-Syn antibody (1: 1000 dilution) as a primary antibody, and then hybridized with an anti-guinea pig HRP-conjugated secondary antibody (1: 5000; Vector Laboratories). The PVDF membrane was treated with a Luminata Western HRP substrate (Bio-Rad, Hercules, CA, USA), and the generated signal was detected with a ChemiDoc-It 810 digital imaging system (UVP Inc., Upland, CA, USA).

j. 對經神經生長因子(NGF)處理後之過度表現α-Syn之PC12細胞中聚集的α-Syn結合特異性j. Specific α-Syn binding specificity in PC12 cells overexpressing α-Syn after treatment with nerve growth factor (NGF)

在8或9 WPI收集的天竺鼠的抗-α-Syn抗體與經NGF處理後之親源PC12細胞、空白對照組(mock-controlled)PC12細胞及過度表現α-Syn的PC12細胞進行免疫細胞化學(ICC)分析，以評估抗體的親和力。以DAPI(4',6-二脒基-2-苯基吲哚)複染細胞核。用螢光顯微鏡拍攝照片，且陽性染色的細胞數量與總細胞數量分別以“-”、“+”、“++”及“+++”表示“<1%”、“1-15%”、“16-50%”、“>50%”的比例。 The anti-α-Syn antibodies of guinea pigs collected at 8 or 9 WPI were subjected to immunocytochemistry with parental PC12 cells treated with NGF, mock-controlled PC12 cells, and PC12 cells overexpressing α-Syn ( ICC) analysis to assess antibody affinity force. Nuclei were counterstained with DAPI (4 ', 6-diamidino-2-phenylindole). Photographs were taken with a fluorescence microscope, and the number of positively stained cells and the total number of cells were expressed as "<1%", "1-15%" with "-", "+", "++", and "+++", respectively. , "16-50%", "> 50%".

實施例4. 免疫原性和有效性試驗中所使用的細胞及動物Example 4. Cells and animals used in immunogenicity and effectiveness tests

a. 過度表現α-Syn的PC12細胞：a. PC12 cells overexpressing α-Syn:

將編碼全長野生型α-Syn或A53T突變α-Syn的cDNA***pZD/XOL-L載體中，以構築pZD/XOL-L-α-Syn質體。依據操作說明書指示，利用Lipofectamine LTX轉染試劑(Invitrogen,Carlsbad,CA,USA)將質體轉染至PC12細胞。將2.5μL的轉染混合物、500μL的Opti-MEM培養基、2.5μL的PLUS試劑及8.75μL的lipofectamine LTX混合，並於室溫反應25分鐘。在將培養基置換為1.5mL的RMPI 1640生長培養基後，將500μL的轉染混合物直接添加至各孔洞中，並於37℃下反應1天。以PCR及西方墨點法確認轉染效率。 A cDNA encoding a full-length wild-type α-Syn or A53T mutant α-Syn was inserted into a pZD / XOL-L vector to construct a pZD / XOL-L-α-Syn plastid. According to the instruction manual, Lipofectamine LTX transfection reagent (Invitrogen, Carlsbad, CA, USA) was used to transfect plastids into PC12 cells. 2.5 μL of the transfection mixture, 500 μL of Opti-MEM medium, 2.5 μL of PLUS reagent, and 8.75 μL of lipofectamine LTX were mixed and reacted at room temperature for 25 minutes. After the medium was replaced with 1.5 mL of RMPI 1640 growth medium, 500 μL of the transfection mixture was directly added to each well, and reacted at 37 ° C. for 1 day. The transfection efficiency was confirmed by PCR and Western blot method.

b. 天竺鼠：b. Guinea pigs:

使用成熟、天然、成年雄性和雌性的Duncan-Hartley天竺鼠(300-350g/BW)中進行免疫原性試驗。每組至少有3隻天竺鼠。涉及Duncan-Hartley 天竺鼠(8-12週齡；Covance Research Laboratories,Denver,PA,USA)試驗的動物設施及UBI皆經IACUC同意核准。 Immunogenicity tests were performed using Duncan-Hartley guinea pigs (300-350 g / BW) in mature, natural, adult males and females. There are at least 3 guinea pigs in each group. Involving Duncan-Hartley Guinea pigs (8-12 weeks old; Covance Research Laboratories, Denver, PA, USA) were tested and approved by IACUC for animal facilities and UBI.

c.接種α-Syn纖維的帕金森氏症小鼠模型：c. Parkinson's mouse model inoculated with α-Syn fibers:

將FVB雌性小鼠(體重為25-30g)保持在12小時光照、12小時黑暗的週期，並依照AAALAC的指示飼養。將α-Syn胜肽(5mg/mL)於37℃下，在含有0.1% NaN₃的PBS/高KCl緩衝液中，搖晃7天，以製備α-Syn纖維。利用測量ThT螢光來監測纖維化，且當訊號超過原始α-Syn單體3倍時確認具纖維化。在接種至經異氟醚麻醉動物之單側黑質(前-後：-3.0mm；內側-外側：-1.3mm；背-腹：離囪穴及硬腦膜-4.7mm)及背側紋狀體(前-後：+0.2mm；內側-外側：-2mm；背-腹：離囪穴及硬腦膜-3.2mm)前，使用西方墨點法確認α-Syn的聚集。 FVB female mice (body weight 25-30 g) were kept in a 12-hour light, 12-hour dark cycle, and were reared according to the instructions of AAALAC. The α-Syn peptide (5 mg / mL) was shaken at 37 ° C. in PBS / high KCl buffer containing 0.1% NaN ₃ for 7 days to prepare α-Syn fibers. Fibrosis was monitored by measuring ThT fluorescence, and fibrosis was confirmed when the signal exceeded the original α-Syn monomer by three times. Unilateral substantia nigra (anterior-posterior: -3.0mm; medial-lateral: -1.3mm; dorsal-abdominal: away from the acupoint and dura mater -4.7mm) and dorsal striatum in isoflurane anesthetized animals Aggregation of α-Syn was confirmed in front of the body (anterior-rear: + 0.2mm; medial-lateral: -2mm; dorsal-abdominal: acupoint and dura mater -3.2mm) using Western blot method.

d. MPP+誘導的帕金森氏症小鼠模型：d. MPP + -induced Parkinson's disease mouse model:

Balb/c雌性小鼠(體重為18-20g)保持在12小時光照、12小時黑暗的週期，並依照AAALAC的指導方針飼養。將MPP+碘(Sigma,St.Luis,MO)溶於生理食鹽水中，並注射10μl含有18μg MPP+碘(0.8mg/kg)的溶液至麻醉動物的單側心室中。注射部位的立體座標為前囪穴-1.0mm，側邊1.0mm，深度2.0mm。 Balb / c female mice (18-20 g in weight) were maintained in a 12-hour light and 12-hour dark cycle and were reared according to AAALAC guidelines. MPP + iodine (Sigma, St. Luis, MO) was dissolved in physiological saline, and 10 μl of a solution containing 18 μg of MPP + iodine (0.8 mg / kg) was injected into the unilateral ventricle of the anesthetized animal. The three-dimensional coordinate of the injection site is the front pit -1.0mm, the side is 1.0mm, and the depth is 2.0mm.

實施例5. 含有α-Syn免疫原結構之多種成分的藥物組Example 5. Pharmaceutical group containing multiple components of the α-Syn immunogen structure 合物的設計原理、篩選、鑑定和優化Design principles, screening, identification and optimization of compounds

a. 設計歷程a. Design History

每種α-Syn胜肽免疫原結構或免疫治療產品基於特定疾病機制和目標蛋白皆有自身的設計重點和方法。模型設計的目標可包括疾病途徑的細胞蛋白或其中可能涉及來自病原體的多種蛋白質感染劑。從研發到商業化的過程非常長，通常需要十幾或數十年的時間才能完成。 Each α-Syn peptide immunogen structure or immunotherapy product has its own design focus and method based on specific disease mechanisms and target proteins. The target of the model design may include cellular proteins of the disease pathway or multiple protein infectious agents which may be involved from pathogens. The process from R & D to commercialization is very long and usually takes a dozen or decades to complete.

一旦選擇目標分子，就需要進行廣泛的血清學試驗。目標分子內B細胞和T細胞抗原決定位的鑑定和分佈對於α-Syn胜肽免疫原結構的設計是重要的。一旦辨識出目標B細胞抗原決定位，即可進行小動物的連續先導免疫原性研究，以評估由設計胜肽藥物組成物引發抗體的功能特性。所有研究均在多個平行組別中進行，從免疫宿主收集血清進行分析。在人類藥物組合物的情況下，進行目標物種或非人靈長類動物的早期免疫原性研究，以進一步驗證免疫原性和設計方向。然後以各種混合物製備目標胜肽，當合併使用於製備各製劑設計時，評估各胜肽結構體間相互作用之功能特性的細微差異。在進一步評估之後，確定最終胜肽結構、胜肽組成物及其製劑，以及製劑的物理參數，即為最終產品開發過程。 Once the target molecule is selected, extensive serological testing is required. The identification and distribution of B-cell and T-cell epitopes within the target molecule is important for the design of the α-Syn peptide immunogen structure. Once the target B-cell epitope is identified, serial lead immunogenicity studies in small animals can be performed to assess the functional characteristics of antibodies raised by the designed peptide drug composition. All studies were performed in multiple parallel groups, and serum was collected from immunized hosts for analysis. In the case of human pharmaceutical compositions, early immunogenicity studies of target species or non-human primates are conducted to further verify immunogenicity and design direction. The target peptides are then prepared from various mixtures, and when used in combination to prepare each formulation design, the subtle differences in the functional characteristics of the interactions between the peptide structures are evaluated. After further evaluation, the final peptide structure, the peptide composition and its preparation, and the physical parameters of the preparation are determined, which is the final product development process.

b. 用於醫藥組成物之α-Syn衍生胜肽免疫原結構體的設計和驗證，可用於治療患有突觸核蛋白病的患者b. Design and verification of α-Syn-derived peptide immunogen structure for pharmaceutical composition, which can be used to treat patients with synucleinopathy

為了產生適於合併至醫藥組成物中的最有效胜肽結構體，從麻疹病毒融合(MVF)蛋白序列或B型肝炎表面抗原(HBsAg)蛋白進一步設計得到的各種病原體衍生的大量混雜T輔助細胞抗原決定位或人工輔助T輔助細胞抗原決定位進行天竺鼠免疫原性試驗。表3(SEQ ID NOs：99至121)中所示之α-Syn_126-140、α-Syn_121-140、α-Syn_111-140、α-Syn_101-140、α-Syn_91-140、α-Syn_85-140、α-Syn_121-135、α-Syn_111-135、α-Syn_101-135、α-Syn_97-135、α-Syn_123-135、α-Syn_126-135、α-Syn_111-132及α-Syn_101-132衍生胜肽結構透過εK及/或KKK連接子連接各種混雜的T輔助抗原決定位，進行代表性試驗。 In order to generate the most effective peptide structure suitable for incorporation into pharmaceutical compositions, a large number of promiscuous T helper cells derived from various pathogens were further designed from the measles virus fusion (MVF) protein sequence or hepatitis B surface antigen (HBsAg) protein. The epitope or artificial helper T helper epitope was used for guinea pig immunogenicity test. Α-Syn _126-140 , α-Syn _121-140 , α-Syn _111-140 , α-Syn _101-140 , α-Syn _91-140 , shown in Table 3 (SEQ ID NOs: 99 to 121) α-Syn _85-140 , α-Syn _121-135 , α-Syn _111-135 , α-Syn _101-135 , α-Syn _97-135 , α-Syn _123-135 , α-Syn _126-135 , α -Syn _111-132 and _α-Syn 101-132 Yansheng Sheng peptide structure is connected through a variety of mixed εK and / or linker KKK T helper epitopes bits, for a representative experiment.

i)選擇α-Syn C端部分作為胜肽免疫原設計的目標i) Selecting the C-terminal portion of α-Syn as a target for peptide immunogen design

α-Syn是一種內在的不正常蛋白質。其由140個胺基酸組成，且分為三個區域。N-末端區域(第1-60殘基)能夠形成雙極性螺旋結構，其為膜特徵及組合的典型構象。已知含有第61-95殘基的中心區域為非澱粉樣蛋白β部分(NAC)，其首先由AD老年斑中發現。此區域高度傾向形成富含β結構的構象及具有高度聚集性。此區域內不同類型的轉譯後修飾顯示出對調節α-Syn聚集的不同作用。具有第96-140殘基的C端區域富含脯胺酸和帶負電荷的殘基，此為內在無序蛋白質的共同特徵，用以維持溶解性。由於C端結構域具低疏水性和高淨負電荷，因此呈現不規則捲曲結構。體外試驗顯示，可透過降低pH值，中和負電荷來誘導α-Syn聚集。α-Syn具有極端的構象多樣性，其對應至膜結合、細胞質及澱粉樣蛋白聚集等狀態的不同條件並具有多種功能。經深思熟慮後，C-端不規擇捲曲及固有無序區域對保持蛋白質溶解性非常重要，其被選作為胜肽免疫原設計的目標，因為此區域最易受到抗體或其他物理因素調控，而非N-端雙極性螺旋和中央富含β-構型區域。 α-Syn is an inherently abnormal protein. It consists of 140 amino acids and is divided into three regions. The N-terminal region (residues 1-60) is capable of forming a bipolar helical structure, which is a typical conformation of membrane characteristics and combinations. The central region containing residues 61-95 is known as the non-amyloid beta portion (NAC), which was first found in AD senile plaques. This region is highly inclined to form a conformation rich in β structure and has a high degree of aggregation. Different types of post-translational modifications in this region show different effects on regulating α-Syn aggregation. The C-terminal region with residues 96-140 is rich in proline and negatively charged residues, which is a common feature of inherently disordered proteins to maintain solubility. Because the C-terminal domain has low hydrophobicity and high net negative charge, it exhibits an irregularly curled structure. In vitro experiments have shown that α-Syn aggregation can be induced by lowering the pH and neutralizing negative charges. α-Syn has extreme conformational diversity, which corresponds to different conditions of membrane binding, cytoplasm, and amyloid aggregation and has multiple functions. After careful consideration, the non-selective curl and inherent disordered regions of the C-terminus are important for maintaining protein solubility, and were selected as targets for peptide immunogen design because this region is most susceptible to regulation by antibodies or other physical factors, and Non-N-terminal bipolar helix and central beta-rich region.

ii)排除α-Syn B抗原決定位，鑑定自體Th抗原決定位ii) Exclude the α-Syn B epitope and identify the auto-Th epitope

初步免疫原性分析證實輔助T細胞抗原決定位結構特徵存在於α-Syn C-端，從α-Syn N-端進行序列刪除的α-Syn_126-140(SEQ ID NO：9)、α-Syn_121-140(SEQ ID NO：8)、α-Syn_111-140(SEQ ID NO：7)胜肽完全不具免疫原性，而α-Syn_101-140(SEQ ID NO：6)、α-Syn_91-140(SEQ ID NO：5)及α-Syn_85-140(SEQ ID NO：4)胜肽(表4)，在C-端序列內存在可能的自體Th結構，具有一定程度的免疫原性。在B抗原決定位中包含此序列，在加強免疫時，因自體T細胞的活化可能會誘發腦部發炎，如AN1792阿茲海默症疫苗。因此，本發明設計具有從G111胺基酸殘基起始的B細胞抗原決定位的α-Syn胜肽免疫原結構。 Preliminary immunogenicity analysis confirmed the presence of helper T cell epitope structural features at the C-terminus of α-Syn, α-Syn _126-140 (SEQ ID NO: 9), α- Syn _121-140 (SEQ ID NO: 8), α-Syn _111-140 (SEQ ID NO: 7) peptides are completely non-immunogenic, while α-Syn _101-140 (SEQ ID NO: 6), α- Syn _91-140 (SEQ ID NO: 5) and α-Syn _85-140 (SEQ ID NO: 4) peptides (Table 4) have a possible auto Th structure in the C-terminal sequence, which has a certain degree of Immunogenicity. This sequence is included in the B epitope. When boosting immunity, the activation of autologous T cells may induce brain inflammation, such as AN1792 Alzheimer's vaccine. Therefore, the present invention designs an α-Syn peptide immunogen structure having a B-cell epitope starting from a G111 amino acid residue.

iii)排列異源性T輔助抗原決定位及其包含在α-Syniii) Arrange heterologous T helper epitopes and their inclusion in α-Syn 胜肽免疫原結構中的設計可恢復和增強選定之α-Syn B抗原決定位胜肽的免疫原性Design in peptide immunogen structure restores and enhances the immunogenicity of selected α-Syn B epitope peptides

表2列出了共29種異源性Th抗原決定位(SEQ ID NOs：70-98)，已在小鼠、大鼠、天竺鼠、狒狒、獼猴等物種中測試其相對特性，發現可增強B細胞抗原決定位的免疫原性。如表5所示，源自於MvF蛋白的UBITh1(SEQ ID NO：83)及UBITh2(SEQ ID NO：84)T細胞抗原決定位可分別將非免疫原性的α-Syn_101-140(SEQ ID NO：6)胜肽轉變具有強至中等免疫原性。在ELISA試驗中，使用長的α-Syn胜肽A91-A140(SEQ ID NO：5)塗佈96孔盤，發現當UBITh3(SEQ ID NO：81)利用連接子共價連接至各種C-端的α-Syn胜肽(SEQ ID NO：4至9)時，具有類似的免疫增強特性，如表6中所示。 Table 2 lists a total of 29 heterologous Th epitopes (SEQ ID NOs: 70-98). Their relative properties have been tested in mice, rats, guinea pigs, baboons, macaques and other species, and they have been found to enhance B Cell epitope immunogenicity. As shown in Table 5, UBITh1 (SEQ ID NO: 83) and UBITh2 (SEQ ID NO: 84) T cell epitopes derived from the MvF protein can separate non-immunogenic α-Syn _101-140 (SEQ ID NO: 6) The peptide transition has strong to moderate immunogenicity. In an ELISA test, a long α-Syn peptide A91-A140 (SEQ ID NO: 5) was used to coat a 96-well plate, and it was found that when UBITh3 (SEQ ID NO: 81) was covalently linked to various C-terminal The α-Syn peptide (SEQ ID NOs: 4 to 9) has similar immune enhancing properties as shown in Table 6.

iv)C端的α-Syn胜肽免疫原結構與其相對應之α-Syn和β-Syn的抗體反應性的免疫原性評估iv) Immunogenicity assessment of the C-terminal α-Syn peptide immunogen structure and its corresponding α-Syn and β-Syn antibody reactivity

突觸核蛋白家族包括三種已知的蛋白質：α-Syn、β-Syn與γ-突觸核蛋白。所有突觸核蛋白具有共同的高度保守α-螺旋脂質結合區，其與可交換的載脂蛋白的A2類脂質結合結構域類似。β-Syn與α-Syn具高度同源性。β-Syn被建議可作為α-Syn聚集的抑製劑，其發生在神經退化性疾病中，如帕金森氏症。因此，β-Syn可以保護中樞神經系統不受α-Syn的神經毒性作用。所以較佳α-Syn胜肽免疫原結構所誘發的抗體，與α-突觸核蛋白反應，而非相對應的聚集保護性β-Syn。試驗6種胜肽免疫原結構，其全部以C-端A140結束，這些結構免疫血清所有的抗體與相對應大小的β-Syn具有顯著的交叉反應性，如表6所示。在仔細研究α-Syn和β-Syn(SEQ ID NO：1和2)之間的序列同源性時，發現兩種蛋白質之間，對應於C-端5個胺基酸YEPEA的序列是相同的。因此，本發明設計不包含此“YEPEA”5胺基酸序列的B抗原決定位。 The synuclein family includes three known proteins: α-Syn, β-Syn and γ-synuclein. All synucleins share a highly conserved alpha-helical lipid-binding region, which is similar to the A2 class lipid-binding domain of the exchangeable apolipoprotein. β-Syn is highly homologous to α-Syn. β-Syn is suggested as an inhibitor of α-Syn aggregation, which occurs in neurodegenerative diseases such as Parkinson's disease. Therefore, β-Syn can protect the central nervous system from the neurotoxic effects of α-Syn. Therefore, the resistance induced by the α-Syn peptide immunogen structure is preferred. Body, reacts with α-synuclein, rather than the corresponding aggregate-protective β-Syn. The six peptide immunogen structures were tested, all of which ended with C-terminal A140. All antibodies of these structural immune sera had significant cross-reactivity with the corresponding size of β-Syn, as shown in Table 6. When carefully studying the sequence homology between α-Syn and β-Syn (SEQ ID NOs: 1 and 2), it was found that the sequences corresponding to the 5 amino acids YEPEA at the C-terminus are the same between the two proteins of. Therefore, the present invention is designed to not contain the B epitope of this "YEPEA" 5 amino acid sequence.

仔細研究α-Syn和β-Syn(SEQ ID NO：1和2)間的序列同源性，發現對應於C-端的5個胺基酸“YEPEA”序列在此兩種蛋白質之間是相同的。因此，B抗原決定位的設計不含有此“YEPEA”胺基酸序列。由免疫研究的發現，將B抗原決定位的設計刪除YEPEA(Y136至A140)片段，如表6所示。在合併連接子序列上，例如，將人工T輔助胜肽UBITh1(SEQ ID NO：83)***不含有YEPEA序列之B細胞抗原決定位的α-Syn胜肽免疫原結構(SEQ ID NOs：107-114)，如表7所示，在評估長α-Syn胜肽K97-A140(SEQ ID NO：110)時，皆具高免疫原性。免疫血清不與β-Syn反應。因此，由表6及7的結果可知，免疫原結構的B抗原決定位設計被限定為α-Syn的G111至D135及其片段。 A careful study of the sequence homology between α-Syn and β-Syn (SEQ ID NOs: 1 and 2) revealed that the five amino acid "YEPEA" sequences corresponding to the C-terminus were identical between the two proteins . Therefore, the design of the B epitope does not contain this "YEPEA" amino acid sequence. Based on the findings of the immunological study, the design of the B epitope was deleted from the YEPEA (Y136 to A140) fragment, as shown in Table 6. On the merged linker sequence, for example, the artificial T helper peptide UBITh1 (SEQ ID NO: 83) is inserted into the α-Syn peptide immunogen structure (SEQ ID NOs: 107- 114), as shown in Table 7, when the long α-Syn peptide K97-A140 (SEQ ID NO: 110) was evaluated, all were highly immunogenic. Immune serum does not react with β-Syn. Therefore, from the results of Tables 6 and 7, it can be seen that the design of the B epitope of the immunogen structure is limited to G111 to D135 of α-Syn and fragments thereof.

v)由α-Syn肽免疫原結構所誘發的抗體僅與β-折疊單體、寡聚物或纖維狀反應，不與α-螺旋單體反應v) The antibodies induced by the α-Syn peptide immunogen structure only react with β-sheet monomers, oligomers or fibrils, and do not react with α-helical monomers

雖然本發明合理地設計α-Syn胜肽免疫原，但具有G111至D135序列或其片段之B抗原決定位的設計α-Syn肽免疫原所產生的抗體會與β-折疊的α-Syn單體、寡聚物或纖維狀具有特異性的反應，但不會與β-折疊的Aβ_1-42或Tau1-441反應，因此理想的α-Syn胜肽免疫原結構候選物，如代表性的α-Syn肽免疫原結構(SEQ ID Nos：112 and 113)所示，參照第8圖。 Although the present invention rationally designs the α-Syn peptide immunogen, the antibodies produced by the design of the α-Syn peptide immunogen with the B epitope of the G111 to D135 sequence or a fragment thereof will be single with β-sheet α-Syn Oligomers, oligomers, or fibrils have specific responses but do not react with β-folded Aβ _1-42 or Tau1-441, so ideal candidates for α-Syn peptide immunogen structure, such as representative The structure of the α-Syn peptide immunogen (SEQ ID Nos: 112 and 113) is shown in FIG. 8.

vi)使用具有不同混雜T輔助抗原決定位的α-Syn衍生胜肽免疫結構具寬廣的MHC覆蓋vi) Use of α-Syn-derived peptides with different promiscuous T helper epitopes with broad MHC coverage

在設計用於治療不同遺傳背景患者的藥物組成物時，設計覆蓋不同遺傳背景的最大族群是很重要的。因此，研究α-Syn衍生之胜肽免疫原結構對組成物的協同免疫原性。由於衍生自MVF或HBsAg的混雜T輔助抗原決定可提供最強的免疫原性，因此設計含有輔助T抗原決定位的胜肽結構組合，以進行研究。與單獨的胜肽結構相比，具相同B抗原決定位之2個胜肽免疫原結構的混合物可引發強大的免疫反應。 When designing pharmaceutical compositions for treating patients with different genetic backgrounds, it is important to design the largest populations that cover different genetic backgrounds. Therefore, the co-immunogenicity of the α-Syn-derived peptide immunogen structure to the composition was investigated. Since promiscuous T-helper epitopes derived from MVF or HBsAg provide the strongest immunogenicity, peptide peptide combinations containing helper T-epitopes were designed for research. Compared to a single peptide structure, a mixture of two peptide immunogen structures with the same B epitope can trigger a powerful immune response.

實施例6. α-Syn胜肽免疫原結構誘發僅針對目標B細胞抗原決定位的抗體反應Example 6. The α-Syn peptide immunogen structure elicits an antibody response against the epitope of the target B cell only

已知用於增強針對相標B細胞抗原決定位肽免疫反應的所有載體蛋白(例如匙孔血藍蛋白(KLH)或其他載體蛋白如白喉類毒素(DT)和破傷風類毒素(TT)蛋白)分別以化學共軛連接B細胞抗原決定位胜肽，而在宿主中所刺激產生的抗體超過90%為針對載體蛋白，且低於10%的抗體是針對目標B細胞抗原決定位。因此，評估本發明α-Syn胜肽免疫原構建體的特異性。將含有不同長度的B細胞抗原決定位的8種α-Syn胜肽免疫原結構(SEQ ID NO：107至114)，藉由連接子序列連接至異源性T細胞抗原決定位UBITh1(SEQ ID NO：83)，以評估免疫原性。將UBITh1(用於增強B抗原決定位免疫的T輔助胜肽)塗佈於孔盤上，且使用天竺鼠免疫血清測試與UBITh1胜肽的交叉反應性。相較於這些結構對於目標B抗原決定位的高免疫原性，如表6及7所示針對B抗原決定位的高力價抗體，絕大部分的免疫血清不會與UBITh1胜肽反應，如表8所示。 All carrier proteins (e.g. keyhole limpet hemocyanin (KLH) or other carrier proteins such as diphtheria toxoid (DT) and tetanus toxoid (TT) proteins) that are known to enhance the immune response against phase B epitope Chemically conjugated B-cell antigen-determining peptides, and more than 90% of the antibodies stimulated in the host are directed against carrier proteins. And less than 10% of the antibodies are directed against the target B cell epitope. Therefore, the specificity of the α-Syn peptide immunogen construct of the present invention was evaluated. Eight α-Syn peptide immunogen structures (SEQ ID NOs: 107 to 114) containing B cell epitopes of different lengths were linked to a heterologous T cell epitope UBITh1 (SEQ ID NO: 83) to evaluate immunogenicity. UBITh1 (T-helper peptide used to enhance B epitope immunity) was spread on well plates, and guinea pig immune serum was used to test cross-reactivity with UBITh1 peptide. Compared with the high immunogenicity of these structures for the target B epitope, as shown in Tables 6 and 7, the most potent antibodies against the B epitope, most of the immune serum will not react with the UBITh1 peptide, such as Table 8 shows.

綜上所述，將目標B細胞抗原決定位連接特別選擇的T輔助抗原決定位的簡單免疫原設計，可產生專一且針對α-Syn B細胞抗原決定位的免疫反應。對於藥物組合物的設計，其可產生更專一的免疫反應及更高的安全性。因此，本發明的α-Syn胜肽免疫原結構對其目標具有高度專一性且非常有效。 In summary, a simple immunogen design that links the target B-cell epitope with a specially selected T helper epitope can generate an immune response specific to the α-Syn B-cell epitope. For the design of the pharmaceutical composition, it can lead to a more specific immune response and higher safety. Therefore, the α-Syn peptide immunogen structure of the present invention is highly specific and very effective for its target.

實施例7. 以免疫血清(9 WPI)針對各種α-突觸核蛋白胜肽免疫原結構進行精確特異性分析的抗原決定位預測Example 7. Prediction of epitopes for precise and specific analysis of various α -synuclein peptide immunogen structures using immune serum (9 WPI)

在精確的抗原決定位預測研究(表9)中，為了確定α-Syn C-端區域內特定殘基的抗體結合位點，合成了52個重疊的10-mer(SEQ ID Nos：18-69)，其涵蓋α-Syn胺基酸序列(K80-A140)。陽性對照組使用(97-135，SEQ ID No：10)和(111-132，SEQ ID No：17)兩個更長的胜肽。將這些10-mer胜肽及兩個較長的胜肽分別塗佈至96孔盤上作為固相免疫吸附物。將混合的天竺鼠抗血清與樣品稀釋緩衝液以1：100稀釋，添加至塗佈2.0μg/mL 10-mer胜肽的孔盤中，在37℃下反應1小時。用緩衝液清洗孔洞後，加入辣根過氧化物酶共軛蛋白A/G反應30分鐘。再以PBS清洗，將反應物以ELISA平板測量儀偵測在450nm下的吸光值，每個樣品分析2次。抗血清與B抗原決定位免疫原結構的長α-Syn胜肽的結合代表最大結合性。 In an accurate epitope prediction study (Table 9), in order to determine antibody binding sites for specific residues in the C-terminal region of α-Syn, There are 52 overlapping 10-mers (SEQ ID Nos: 18-69), which cover the α-Syn amino acid sequence (K80-A140). The positive control group used (97-135, SEQ ID No: 10) and (111-132, SEQ ID No: 17) two longer peptides. These 10-mer peptides and two longer peptides were spread on a 96-well plate as solid-phase immunoadsorbants. The mixed guinea pig antisera and the sample dilution buffer were diluted 1: 100, added to a well plate coated with 2.0 μg / mL 10-mer peptide, and reacted at 37 ° C. for 1 hour. After the wells were washed with a buffer solution, horseradish peroxidase conjugated protein A / G was added and reacted for 30 minutes. After washing with PBS, the reaction was detected by an ELISA plate measuring instrument at 450 nm, and each sample was analyzed twice. The binding of the antiserum to the long alpha-Syn peptide of the B epitope immunogen structure represents maximum binding.

如表9所示，合併分別從6個α-Syn肽免疫原結構[(K97-D135,SEQ ID No：110)、(G111-D135,SEQ ID No：108)、(G111-G132,SEQ ID No：113)、(E126-D135,SEQ ID No：112)、(G101-A140,SEQ ID No：104)與(E126-A140,SEQ ID No：99)]所獲得的9 wpi天竺鼠免疫血清，以進行精確的抗原決定位預測。這些不同長度的6個B抗原決定位片段完全覆蓋了α-突觸核蛋白C-端區域的97-140序列。ELISA結果顯示所有6種免疫血清與代表性的α-Syn長胜肽(97-135，SEQ ID No：10)具有強烈的反應。對於10-mer的抗原決定位預測研究顯示，可覆蓋AA114至125(SEQ ID NO：52、53和54胜肽的114-123、115-124、116-125)區域周圍的免疫原性抗原決定位，且在C-端具有一高免疫原性區域，以胜肽131-140(SEQ ID NO：69)表示。有趣的是，大部分來自C-端α-Syn胜肽免疫原結構的免疫血清所刺激的抗體可辨識非線性構象的抗原決定位，除了位於具有EGYQDYEPEA序列(SEQ ID NO：69)之α-Syn C-端的抗原決定位外，也可與β-Syn蛋白產生交叉反應。 As shown in Table 9, the six α-Syn peptide immunogen structures were combined [(K97-D135, SEQ ID No: 110), (G111-D135, SEQ ID No: 108), (G111-G132, SEQ ID No. 113), (E126-D135, SEQ ID No: 112), (G101-A140, SEQ ID No: 104) and (E126-A140, SEQ ID No: 99)] obtained from 9 wpi guinea pig immune serum, For accurate epitope prediction. These 6 B epitope fragments of different lengths completely cover the 97-140 sequence of the C-terminal region of α-synuclein. ELISA results showed that all six immune sera had strong responses to the representative α-Syn long peptide (97-135, SEQ ID No: 10). The epitope prediction study for 10-mer showed that it can cover the area around AA114 to 125 (114-123, 115-124, 116-125 of SEQ ID NOs: 52, 53 and 54 peptides) The immunogenic epitope has a high immunogenic region at the C-terminus and is represented by peptide 131-140 (SEQ ID NO: 69). Interestingly, most antibodies stimulated by immune sera derived from the C-terminal α-Syn peptide immunogen structure can recognize epitopes in a non-linear conformation, except for those located in the α- In addition to the epitope of the C-terminus of Syn, it can also cross-react with β-Syn protein.

抗原決定位作圖發現不如預期，但與源自α-Syn 111-132(SEQ ID NO：113)及α-Syn 126-135(SEQ ID NO：112)之α-Syn胜肽免疫原結構的抗體完全相關，α-Syn胜肽免疫原結構來自於α-Syn的不規則捲曲區域，其與異源性Th抗原決定位連結，導致構象結構類似於α-Syn的變性β-折疊，且不與天然α-螺旋的α-Syn交叉反應。 The epitope mapping was found to be less than expected, but was similar to the structure of the α-Syn peptide immunogen derived from α-Syn 111-132 (SEQ ID NO: 113) and α-Syn 126-135 (SEQ ID NO: 112). Antibodies are completely related. The structure of the α-Syn peptide immunogen is derived from the irregular coiled region of α-Syn, which is linked to the heterologous Th epitope, resulting in a conformational structure similar to the degenerate β-fold of α-Syn, and does not Cross-reacts with α-Syn of natural α-helix.

實施例8. α-SYN胜肽免疫原結構及其製劑所誘發的抗體：抗-聚集和解聚集對重組α突觸核蛋白的影響Example 8. Antibodies induced by α- SYN peptide immunogen structure and its preparation: the effects of anti-aggregation and deaggregation on recombinant alpha synuclein

使用經重組α-Syn免疫之天竺鼠血清所純化的抗α-Syn抗體，評估α-Syn胜肽免疫原結構在in vitro抗-聚集試驗及解聚集試驗中的作用。 Anti-α-Syn antibodies purified from recombinant sera of immunized guinea pigs were used to evaluate the role of the α-Syn peptide immunogen structure in the in vitro anti-aggregation test and disaggregation test.

a. α-Syn聚集的抑制a. Inhibition of α-Syn aggregation

利用偵測thiofavin T定量α-Syn聚集的變化程度，初步篩選由不同α-Syn胜肽免疫原結構免疫之天竺鼠所純化的抗α-Syn抗體的抗-聚集能力，如實施例3所示。將製備於PBS的100μM重組α-Syn置於5μM 40μL PBS/KCl緩衝液(2.5mM MgCl₂，50mM HEPES和150mM KCl在1x PBS中，pH 7.4)中，在384孔盤中反應6天，以誘發聚集。將不同濃度(0.05、0.5或5μg/mL)從不同時間點收集之經不同α-Syn胜肽免疫原結構免疫天竺鼠之抗血清所純化的抗α-Syn抗體，分別添加到反應混合物中，評估抑制α-Syn聚集的效果。反應結束後，使用ThT分析聚集程度。將載體對照中的聚集程度設為100%，且在不含有α-Syn情況下所獲得的數值為0%，將各測試結果標準化。 The detection of thiofavin T was used to quantify the degree of change in α-Syn aggregation, and the anti-α-Syn antibodies purified from guinea pigs immunized with different α-Syn peptide immunogens were initially screened for anti-aggregation ability, as shown in Example 3. 100 μM recombinant α-Syn prepared in PBS was placed in 5 μM 40 μL PBS / KCl buffer (2.5 mM MgCl ₂ , 50 mM HEPES and 150 mM KCl in 1 × PBS, pH 7.4), and reacted in a 384-well plate for 6 days to Induced aggregation. Anti-α-Syn antibodies purified at different concentrations (0.05, 0.5, or 5 μg / mL) and purified from anti-sera from guinea pigs with different α-Syn peptide immunogen structures were collected from different time points and added to the reaction mixture for evaluation. Effect of inhibiting α-Syn aggregation. After the reaction was completed, the degree of aggregation was analyzed using ThT. The degree of aggregation in the vehicle control was set to 100%, and the value obtained without α-Syn was 0%, and the results of each test were standardized.

如表10所示，在9 WPI及以上所收集之經α-Syn_111-132、α-Syn_121-135或α-Syn_126-135引發的3種抗α-Syn抗體對於α-Syn聚集更具有效果與濃度依賴性抑制。在所有的抗α-Syn抗體中，以α-Syn_111-132(SEQ ID NO：113)、α-Syn_121-135(SEQ ID NO：107)、α-Syn_123-135(SEQ ID NO：111)或α-Syn_126-135(SEQ ID NO：112)(於9WPI收集)所誘發的抗體，相較於載體對照(100%)，其對α-Syn聚集物具有約40%的抑制作用(第1圖)。 As shown in Table 10, via the α-Syn 9 WPI collected above and _111-132, α-Syn _121-135 or 3 types of anti-α-Syn antibody α-Syn _126-135 induced aggregation for more α-Syn Has effect and concentration-dependent inhibition. Among all anti-α-Syn antibodies, α-Syn _111-132 (SEQ ID NO: 113), α-Syn _121-135 (SEQ ID NO: 107), α-Syn _123-135 (SEQ ID NO: 111) or α-Syn _126-135 (SEQ ID NO: 112) (collected at 9WPI), compared to the vehicle control (100%), it has about 40% inhibition of α-Syn aggregates (Figure 1).

b. 預形成α-Syn聚集體的解聚集b. Deaggregation of pre-formed α- Syn aggregates

從上述試驗中，經α-Syn胜肽免疫原結構免疫之天竺鼠抗血清所純化的抗α-Syn抗體具有抑制α-Syn聚集的作用。為了進一步評估由α-Syn胜肽免疫原結構引發之抗體在解聚集預形成之α-Syn聚集體中是否保持活性，使用從天竺鼠抗血清純化的抗-α-Syn 抗體進行體外解聚集試驗。 From the above test, the anti-α-Syn antibody purified from the guinea pig antiserum immunized with the α-Syn peptide immunogen has the effect of inhibiting the aggregation of α-Syn. To further evaluate whether antibodies raised by the α-Syn peptide immunogen structure remained active in the disaggregated pre-formed α-Syn aggregates, anti-α-Syn purified from guinea pig antisera was used The antibodies were subjected to an in vitro disaggregation test.

將α-Syn置於200μL的5μM PBS/KCl緩衝液中聚集3天。離心(13,000xg，4℃，30分鐘)後，收集α-Syn聚集體並進行ThT分析確認。將預形成的α-Syn聚集體在100μL PBS/KCl緩衝液中反應3天，此PBS/KCl緩衝液含有或不含從天竺鼠抗血清(5μg/mL)純化的抗-α-Syn抗體。反應後，在4℃下，以13,000 xg離心30分鐘收集聚集體，依實施例3所述，進行ThT定量分析。將載體對照組中自發性分解後的殘餘α-Syn聚集體標準化至100%。 Α-Syn was aggregated in 200 μL of 5 μM PBS / KCl buffer for 3 days. After centrifugation (13,000xg, 4 ° C, 30 minutes), α-Syn aggregates were collected and confirmed by ThT analysis. The pre-formed α-Syn aggregates were reacted in 100 μL of PBS / KCl buffer for 3 days. This PBS / KCl buffer contained or did not contain anti-α-Syn antibody purified from guinea pig antisera (5 μg / mL). After the reaction, aggregates were collected by centrifugation at 13,000 xg for 30 minutes at 4 ° C, and ThT quantitative analysis was performed as described in Example 3. Residual α-Syn aggregates after spontaneous decomposition in the vehicle control group were normalized to 100%.

對由α-Syn_111-152(SEQ ID NO：113)或α-Syn_126-135(SEQ ID NO：112)所誘發的2種抗-α-Syn抗體，以及α-Syn_111-132(SEQ ID NO：113)誘發及α-Syn_126-135(SEQ ID NO：112)誘發之抗-α-Syn抗體組合物進行in vitro解聚試驗。由結果可知，由α-Syn_126-135(SEQ ID NO：112)和α-Syn_111-132(SEQ ID NO：113)引發的抗-α-Syn抗體相較於載體對照組(100%)，對預形成α-Syn聚集體的解聚集作用為約50%，而其他抗-α-Syn抗體及免疫前動物中純化的抗體不具有此效果(第2圖)。 Against 2 anti-α-Syn antibodies _elicited by α-Syn _111-152 (SEQ ID NO: 113) or α-Syn _126-135 (SEQ ID NO: 112), and α-Syn _111-132 (SEQ The anti-α-Syn antibody composition induced by ID NO: 113) and α-Syn _126-135 (SEQ ID NO: 112) was subjected to an in vitro depolymerization test. From the results, it can be seen that the anti-α-Syn antibodies elicited by α-Syn _126-135 (SEQ ID NO: 112) and α-Syn _111-132 (SEQ ID NO: 113) were compared with the vehicle control group (100%). The de-aggregation effect on pre-formed α-Syn aggregates is about 50%, while other anti-α-Syn antibodies and purified antibodies in pre-immune animals do not have this effect (Figure 2).

實施例9. α-SYN胜肽免疫原結構及其製劑所誘發的抗體：α-SYN過度表現細胞中α-SYN聚集動力學的抗-聚集與解聚集效果Example 9. Anti-aggregation and deaggregation effects of α- SYN peptide immunogen structure and its preparation-induced antibodies: α- SYN overexpresses α- SYN aggregation kinetics in cells

已知在神經元分化過程中會加速α-Syn聚集。為了評估α-Syn胜肽免疫原結構在細胞條件下抑制α-Syn聚集或分離預形成α-Syn聚集體的效果，使用經NGF處理的神經元分化α-Syn過度表現之PC12細胞，進行抗聚集試驗與解聚集試驗，以評估經不同α-Syn胜肽免疫原結構免疫的天竺鼠抗血清所純化的抗α-Syn抗體。 Alpha-Syn aggregation is known to accelerate during neuronal differentiation. In order to evaluate the effect of α-Syn peptide immunogen structure on the inhibition of α-Syn aggregation or isolation of pre-formed α-Syn aggregates under cell conditions, NGF-treated neurons were used to differentiate PC12 cells with over-expressed α-Syn to perform anti- Aggregation test and disaggregation test to evaluate anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen structures.

a. α-Syn聚集的抑制a. Inhibition of α-Syn aggregation

將α-Syn過度表現之PC12細胞添加至聚-D-賴胺酸塗佈的96孔盤上，以神經生長因子(NGF)(100ng/mL)與經不同α-Syn胜肽免疫原結構(0或0.5μg/mL)免疫天竺鼠所純化之抗α-Syn抗體處理4天，確認抗-聚集活性。 Α-Syn over-expressed PC12 cells were added to poly-D-lysine-coated 96-well plates. Nerve growth factor (NGF) (100ng / mL) and different α-Syn peptide immunogen structures were used ( 0 or 0.5 μg / mL) treated with anti-α-Syn antibodies purified from guinea pigs for 4 days to confirm anti-aggregation activity.

將經處理的細胞裂解，以SDS-PAGE分離20μg細胞裂解物，並使用α-Syn抗體(BD)進行分析。將在高分子量區域中所檢測到的α-Syn訊號量化，並將其標準化為100%的載體對照組。參照第3圖，與載體對照組中α-Syn聚集體的量相比，在由α-Syn_111-132(SEQ ID NO：113)、α-Syn_121-135(SEQ ID NO：107)、α-Syn_123-135(SEQ ID NO：111)或α-Syn_126-135(SEQ ID NO：112)α-Syn胜肽免疫原結構所誘發的4種抗α-Syn抗體對α-Syn聚集體的抑制效果高達80-90%。 The treated cells were lysed, 20 μg of cell lysate was separated by SDS-PAGE, and analyzed using α-Syn antibody (BD). The α-Syn signal detected in the high molecular weight region was quantified and normalized to a 100% vehicle control group. Referring to FIG. 3, compared with the amount of α-Syn aggregates in the vehicle control group, the ratio of α-Syn _111-132 (SEQ ID NO: 113), α-Syn _121-135 (SEQ ID NO: 107), _Aggregation of 4 anti-α-Syn antibodies to α-Syn induced by α-Syn _123-135 (SEQ ID NO: 111) or α-Syn _126-135 (SEQ ID NO: 112) α-Syn peptide immunogen structure The inhibitory effect of the body is as high as 80-90%.

b. 預形成α-Syn聚集體的解聚集b. Deaggregation of pre-formed α-Syn aggregates

為了證明在預形成之α-Syn聚集體上的解聚集，使用NGF(100ng/mL)處理之α-Syn過度表現的PC12細胞3天，進行神經元分化造成α-Syn的聚集，再以經不同α-Syn胜肽免疫原結構(0或0.5μg/mL)免疫之天竺鼠所純化的抗-α-Syn抗體處理4天。 To demonstrate disaggregation on pre-formed α-Syn aggregates Set, PC12 cells overexpressed with α-Syn treated with NGF (100ng / mL) for 3 days, undergoing neuronal differentiation to cause aggregation of α-Syn, and then different α-Syn peptide immunogen structures (0 or 0.5 μg) / mL) treated with anti-α-Syn antibodies purified from guinea pigs for 4 days.

將經處理的細胞裂解，以SDS-PAGE分離20μg細胞裂解物，並使用α-Syn抗體(BD)進行分析。將在高分子量區域中所檢測到的α-Syn訊號量化，並將其標準化為100%的載體對照組。參照第3圖，與載體對照組中α-Syn聚集體的量相比，在由α-Syn_121-135(SEQ ID NO：107)、α-Syn_123-135(SEQ ID O：111)或α-Syn_126-135(SEQ ID NO：112)α-Syn胜肽免疫原結構所誘發的抗-α-Syn抗體對α-Syn聚集體的抑制效果為50-60%，而由α-Syn_111-132(SEQ ID NO：113)誘發的抗-α-Syn抗體可降低超過90%聚集α-Syn的量。 The treated cells were lysed, 20 μg of cell lysate was separated by SDS-PAGE, and analyzed using α-Syn antibody (BD). The α-Syn signal detected in the high molecular weight region was quantified and normalized to a 100% vehicle control group. Referring to FIG. 3, compared to the amount of α-Syn aggregates in the vehicle control group, the ratio of α-Syn _121-135 (SEQ ID NO: 107), α-Syn _123-135 (SEQ ID O: 111), or α-Syn _126-135 (SEQ ID NO: 112) The inhibitory effect of anti-α-Syn antibodies on α-Syn aggregates induced by α-Syn peptide immunogen structure is 50-60%, while α-Syn _111-132 (SEQ ID NO: 113) induced anti-α-Syn antibodies can reduce the amount of aggregated α-Syn by more than 90%.

實施例10. α-SYN胜肽免疫原結構及其製劑所誘發的抗體：對降低TNF-α和IL-6分泌的影響Example 10. Antibodies Induced by the Structure of α- SYN Peptide Immunogen and Its Preparation: Effects on Reducing TNF- α and IL-6 Secretion

據信，黑質神經元損傷會將聚集的α-Syn釋放到黑質中，活化小神經膠質細胞產生發炎介質，而導致PD中持續和進行性黑質神經變性。為了評估從不同α-Syn胜肽免疫原結構免疫天竺鼠所純化之抗-α-Syn抗體降低小膠質細胞活化的作用，在不同抗-α-Syn抗體存在或不存在的情況下，分析經α-Syn聚集體處理之小膠質細胞所釋放之發炎介質TNF-α(腫瘤壞死因子α)和IL-6(白細胞介素-6)的量。 It is believed that damage to substantia nigra neurons releases aggregated α-Syn into substantia nigra, activates microglia to produce inflammatory mediators, and results in continuous and progressive substantia nigra degeneration in PD. In order to evaluate the effect of anti-α-Syn antibodies purified from guinea pigs immunized with different α-Syn peptide immunogen structures on reducing microglial activation, in the presence or absence of different anti-α-Syn antibodies, -Syn Ag aggregate-treated microglia releases inflammatory mediator TNF-α (tumor Amount of necrosis factor alpha) and IL-6 (interleukin-6).

將老鼠BV2細胞或人類SVG p12細胞以5,000細胞/孔的量添加至含有1%FBS的RPMI 1640培養基中。將細胞以1μM α-Syn處理並在37℃，5%CO₂，潮濕的環境下處理24小時。之後，收集培養基、離心並分離上清液。分別使用小鼠IL-6或人類TNF-α小鼠ELISA套組(Thermofisher)，分析上清液中BV2細胞分泌的IL-6和SVG p12細胞分泌的TNF-α濃度，重複3次。將訊號標準化，使載體對照組設定為100%。 Mouse BV2 cells or human SVG p12 cells were added to RPMI 1640 medium containing 1% FBS in an amount of 5,000 cells / well. The cells were treated with 1 μM α-Syn and treated at 37 ° C., 5% CO ₂ , and humid environment for 24 hours. After that, the culture medium was collected, centrifuged, and the supernatant was separated. The mouse IL-6 or human TNF-α mouse ELISA kit (Thermofisher) was used to analyze the concentration of IL-6 secreted by BV2 cells and the TNF-α secreted by SVG p12 cells in the supernatant, and this was repeated 3 times. Normalize the signal so that the vehicle control group is set to 100%.

試驗結果顯示由α-Syn_111-132(SEQ ID NO：113)和α-Syn_123-135(SEQ ID NO：111)所誘發之抗α-Syn抗體，可降低30至50%因α-Syn聚集體所造成之SVG p12細胞的TNF-α釋放，而由α-Syn_123-135(SEQ ID NO：111)誘發的抗-α-Syn抗體可減少約30%SVGP12細胞釋放的IL-6(第4圖)。結果顯示，由α-Syn_123-135(SEQ ID NO：111)誘發的抗-α-Syn抗體，相較於其它抗-α-Syn抗體，可更有效地降低α-Syn聚集體所引起的小膠質細胞活化。 Test results showed that the _{α-Syn 111-132 (SEQ ID NO} : 113): the induced antibody anti-α-Syn, can be reduced by 30 to 50% α-Syn and _{α-Syn 123-135 (111 SEQ ID} NO) Aggregate-induced TNF-α release from SVG p12 cells, and anti-α-Syn antibodies induced by α-Syn _123-135 (SEQ ID NO: 111) reduced IL-6 release from SVGP12 cells by approximately 30% ( (Figure 4). The results show that the anti-α-Syn antibodies induced by α-Syn _123-135 (SEQ ID NO: 111) can reduce the effects caused by α-Syn aggregates more effectively than other anti-α-Syn antibodies. Microglia activation.

實施例11. α-SYN胜肽免疫原結構及其製劑所誘發的抗體：外源性α突觸核蛋白誘導之神經退化性的降低的影響Reduce the impact of exogenous [alpha] -synuclein-induced neurodegeneration of: 11. α -SYN peptide immunogen structure and formulation induced antibody Example

為了評估不同α-Syn胜肽免疫原結構免疫之天竺鼠抗血清所純化之抗-α-Syn抗體的神經保護作用，使用具有外源性預形成α-Syn聚集體之經NGF處理之神經元分化PC12細胞的體外神經變性模型。 In order to evaluate the neuroprotective effect of anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogens, An in vitro neurodegeneration model of PC12 cells differentiated with NGF-treated neurons with exogenous pre-formed α-Syn aggregates.

PC12細胞經NGF(100ng/mL)處理6天以誘導神經元分化。使用InCell高含量圖像分析系統(GE Healthcare)確認及分析神經元分化細胞的形態。評估反應神經營養作用的神經突起生長以及神經元分化細胞的數量。在標準化後，以百分比(平均值±SEM)顯示神經突生長和神經元分化細胞數量的程度。將經及未經NGF處理之PC12細胞的神經突長度分別設定為100%和0%。 PC12 cells were treated with NGF (100ng / mL) for 6 days to induce neuronal differentiation. The morphology of neuronal differentiated cells was confirmed and analyzed using the InCell high-content image analysis system (GE Healthcare). Assess the growth of neurites that respond to neurotrophic effects and the number of neuronal differentiated cells. After normalization, the degree of neurite growth and the number of neuronal differentiated cells is shown as a percentage (mean ± SEM). The neurite lengths of PC12 cells treated with and without NGF were set to 100% and 0%, respectively.

於神經元分化的PC12細胞中加入外源性預形成的α-Syn聚集物觀察神經變性。在預形成α-Syn聚集體的存在下，在神經元分化的PC12細胞中，神經突起長度縮短且細胞數目減少。此α-Syn聚集體驅動的神經變性與外源性α-Syn聚集體的添加量成正比，且可被薑黃素以濃度依賴性的方式阻斷，薑黃素已知可抵抗α-Syn聚集體的神經毒性，具有神經保護作用。市售的抗α-Syn抗體(BD bioscience)，不是由天竺鼠所純化的抗體，可緩解α-Syn聚集體所造成的神經退化性病變。採用此模型作為篩選平台，以鑑別具濃度依賴性恢復神經突起生長及神經元存活中之神經保護作用的抗-α-Syn抗體(表11和12)。 Neuron-differentiated PC12 cells were added with exogenous pre-formed α-Syn aggregates to observe neurodegeneration. In the presence of pre-formed α-Syn aggregates, in neuron-differentiated PC12 cells, the length of neurites was shortened and the number of cells was reduced. This α-Syn aggregate-driven neurodegeneration is proportional to the amount of exogenous α-Syn aggregates added, and can be blocked by curcumin in a concentration-dependent manner, which is known to resist α-Syn aggregates The neurotoxicity has neuroprotective effects. Commercially available anti-α-Syn antibodies (BD bioscience), not antibodies purified by guinea pigs, can alleviate neurodegenerative lesions caused by α-Syn aggregates. This model was used as a screening platform to identify anti-α-Syn antibodies with concentration-dependent restoration of neuroprotective effects in neurite growth and neuron survival (Tables 11 and 12).

由結果可知，超過一半經不同α-Syn胜肽免疫原結構免疫之天竺豚鼠抗血清所純化的抗-α-Syn抗體以濃度依賴性方式恢復神經突起生長(表11)，且幾乎所有的抗-α-Syn抗體可保護神經元分化的PC12細胞免於α-Syn聚集體觸發的神經元死亡(表12)。將兩種不同的參數放在一起，發現接近三分之一的抗-α-Syn抗體可影響神經突起長度和抵抗α-Syn聚集體神經毒性的細胞存活。觀察並量化以α-Syn_111-132(SEQ ID NO：113)、α-Syn_126-135(SEQ ID NO：112)誘發之抗-α-Syn抗體與天竺鼠的免疫前抗體，對於神經突起長度以及利用鈣螢光素AM(Life Technologies)(一種螢光活細胞標記染料)標定之細胞數量的影響。顯示在富含神經突起之神經元所分化的PC12細胞中，由α-Syn_111-132(SEQ ID NO：113)(第5B圖)和α-Syn_126-135(SEQ ID NO：112)(圖5C)誘發的抗體對於α-Syn聚集體所造成之神經突起縮短具有保護作用，但天竺鼠免疫前抗體則無(圖5A)。 It can be seen from the results that more than half of the anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen structures restored the growth of neurites in a concentration-dependent manner (Table 11), and almost all anti- -α-Syn antibody protects neuron-differentiated PC12 cells from neuronal death triggered by α-Syn aggregates (Table 12). Putting two different parameters together, it was found that nearly one-third of anti-α-Syn antibodies can affect neurite length and cell survival against neurotoxicity of α-Syn aggregates. To observe and quantify _{α-Syn 111-132 (SEQ ID NO} : 113), α-Syn 126-135 (SEQ ID NO: 112) -α-Syn preimmune anti-antibodies and the guinea pig induced antibodies, neurite length for And the effect of cell number calibration using calcein AM (Life Technologies), a fluorescent living cell marker dye. Shown in PC12 cells differentiated by neurite-rich neurons, α-Syn _111-132 (SEQ ID NO: 113) (Fig. 5B) and α-Syn _126-135 (SEQ ID NO: 112) ( Fig. 5C) The induced antibodies have a protective effect on the shortening of neurites caused by α-Syn aggregates, but the guinea pigs do not have pre-immune antibodies (Fig. 5A).

實施例12. α-SYN胜肽免疫原結構及其製劑所誘發的抗體：α-SYN過度表現細胞中神經退化性病變降低的影響Example 12. Antibodies Induced by the Structure of α- SYN Peptide Immunogens and Their Preparations: Effects of Reduced Neurodegenerative Lesions in α- SYN Overexpressing Cells

為了評估從不同α-Syn胜肽免疫原結構免疫之天竺鼠抗血清純化的抗α-Syn抗體的神經保護作用，使用α-Syn過度表現之野生型PC12細胞和α-Syn過度表現之A53T突變的PC12細胞作為體外神經退化性病變模型。 To evaluate the neuroprotective effect of anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogens, wild-type PC12 cells overexpressing α-Syn and A53T mutations overexpressing α-Syn PC12 cells serve as a model of neurodegenerative lesions in vitro.

在與NGF反應後，空白對照組細胞(轉染質質載體)產生長的神經突起延伸且細胞數增加，此結果類似親本野生型PC12細胞，然而相對於α-Syn過度表現之野生型PC12細胞和α-Syn過度表現之A53T突變的PC12細胞並未形成神經突起延伸或細胞數增加，證實在NGF處理後會伴隨α-Syn聚集的神經性病變。使用西方墨點法和ThT分析測定NGF處理之α-Syn過度表現之野生型PC12細胞的細胞裂解物，以了解其特徵。西方墨點法結果顯示，在NGF處理後，細胞裂解物中過度表現的α-Syn為寡聚物，ThT分析結果顯示，細胞裂解物中過度表現的α-Syn具有β-折疊結構(即增強的ThT螢光訊號)。與未經NGF處理之α-Syn過度表現之野生型PC12細胞的西方墨點法和ThT測定結果相比，經NGF誘導之神經元分化中過度表現的α-Syn由α-螺旋轉變為β-折疊，接著引發β-折疊α-Syn寡聚物的神經性病變。 After reacting with NGF, the blank control cells (transfected with cytoplasmic vector) produced long neurite extensions and increased the number of cells. This result is similar to the parent wild-type PC12 cells, but it is over-expressed compared to α-Syn wild-type PC12. Cells and A53T mutant PC12 cells with over-expression of α-Syn did not form neurite outgrowth or increase the number of cells, confirming that neuropathy accompanied by α-Syn aggregation after NGF treatment. Western blot methods and ThT analysis were used to determine cell lysates of wild-type PC12 cells overexpressed by NGF-treated α-Syn to understand their characteristics. Western blotting results show that after NGF treatment, the over-represented α-Syn in the cell lysate is an oligomer. ThT analysis results show that the over-represented α-Syn in the cell lysate has a β-sheet structure (that is, enhanced ThT fluorescent signal). Compared with Western blot and ThT measurements of wild-type PC12 cells overexpressing α-Syn without NGF treatment, the overexpression of α-Syn in the differentiation of neurons induced by NGF changed from α-helix to β- Folding, which in turn causes neuropathy of the β-sheet α-Syn oligomer.

此外，與野生型α-Syn過度表現的PC12細胞相比，過度表現A53T突變的α-Syn會導致更強的神經性退化反應，反映出神經突起長度減短與NGF處理之細胞數減少，其顯示A53T突變的α-Syn在α-Syn過度表現之PC12細胞中造成比野生型α-Syn更強的神經變性作用。 In addition, compared to wild-type α-Syn over-expressed PC12 cells, α-Syn over-expressing A53T mutations will lead to a stronger neurodegenerative response, reflecting a reduction in the length of neurites and a decrease in the number of cells treated with NGF. It was shown that A53T mutant α-Syn caused stronger neurodegenerative effects in wild-type α-Syn than in PC12 cells overexpressed by α-Syn.

由α-Syn_101-132(sEQ ID NO：114)、α-Syn_111-132(SEQ ID NO：113)、α-Syn_121-135(SEQ ID NO：107)、α-Syn_123-135(SEQ ID NO：111)或α-Syn_126-135(SEQ IID NO：112)所誘導的抗-α-Syn抗體，以及由α-Syn_111-132(SEQ ID NO：113)與α-Syn_126-135(SEQ IID NO：112)所誘導的抗-α-Syn抗體組成物，可利用α-Syn過度表現野生型PC12細胞的體外神經變性模型測定，以評估抗體對個體抵抗神經變性的保護作用。α-Syn過度表現之野生型PC12細胞經NGF處理3天造成神經元分化，然後再與抗-α-Syn抗體(最終濃度為5μg/mL)和NGF反應3天。反應結束後以顯微鏡觀察細胞，相較於載體對照組，顯示與選定的抗-α-Syn抗體反應後可恢復神經突起長度和增加細胞數。將經NGF處理6天之親本PC12細胞的偵測值標準化設定為100%，以定量神經突起長度和細胞數。由結果可知，與載體對照組相比，由α-Syn_101-132(SEQ ID NO：114)、α-Syn_111-132(SEQ ID NO：113)、或α-Syn_123-135(SEQ ID NO：111)所誘導的抗-α-Syn抗體，以及由α-Syn_111-132(SEQ ID NO：113)與α-Syn_126-135(SEQ ID NO：112)所誘導的抗-α-Syn抗體組成物具有顯著較多的細胞數，而α-Syn_101-132(SEQ ID NO：114)、α-Syn_111-132(SEQ ID NO：113)、α-Syn_123-135(SEQ ID NO：111)或α-Syn_126-135(SEQ ID NO：112)所誘導的抗-α-Syn抗體，以及由α-Syn_111-132(SEQ ID NO：115)及α-Syn_126-135(SEQ ID NO：114)所誘導的抗-α-Syn抗體組成物具有顯著較長的神經突起(第6A及6B圖)。 From α-Syn _101-132 (sEQ ID NO: 114), α-Syn _111-132 (SEQ ID NO: 113), α-Syn _121-135 (SEQ ID NO: 107), α-Syn _123-135 ( (SEQ ID NO: 111) or α-Syn _126-135 (SEQ IID NO: 112) induced anti-α-Syn antibody, and α-Syn _111-132 (SEQ ID NO: 113) and α-Syn _{126 -135} (SEQ IID NO: 112) -induced anti-α-Syn antibody composition can be measured using an in vitro neurodegeneration model of wild-type PC12 cells overexpressing α-Syn to assess the protective effect of antibodies on neurodegeneration in individuals . α-Syn over-expressed wild-type PC12 cells treated with NGF for 3 days caused neuronal differentiation, and then reacted with anti-α-Syn antibody (final concentration of 5 μg / mL) and NGF for 3 days. After the reaction, the cells were observed under a microscope. Compared with the vehicle control group, it showed that the reaction with the selected anti-α-Syn antibody could restore the neurite length and increase the number of cells. The detection value of parental PC12 cells treated with NGF for 6 days was standardized to 100% to quantify neurite length and cell number. From the results, compared with the vector control group, α-Syn _101-132 (SEQ ID NO: 114), α-Syn _111-132 (SEQ ID NO: 113), or α-Syn _123-135 (SEQ ID NO: 111) -induced anti-α-Syn antibodies, and anti-α- induced by α-Syn _111-132 (SEQ ID NO: 113) and α-Syn _126-135 (SEQ ID NO: 112) The Syn antibody composition has a significantly larger number of cells, while α-Syn _101-132 (SEQ ID NO: 114), α-Syn _111-132 (SEQ ID NO: 113), α-Syn _123-135 (SEQ ID NO: 111) or α-Syn _126-135 (SEQ ID NO: 112) induced anti-α-Syn antibody, and α-Syn _111-132 (SEQ ID NO: 115) and α-Syn _126-135 (SEQ ID NO: 114) The induced anti-α-Syn antibody composition has significantly longer neurites (Figures 6A and 6B).

實施例13. α-SYN胜肽免疫原結構及其製劑所誘發的抗體：對β-折疊寡聚物及纖維狀α突觸核蛋白的特異性Example 13. Antibodies induced by α- SYN peptide immunogen structure and its preparation: specificity for β -sheet oligomers and fibrous α- synuclein

為了分析由不同α-Syn胜肽免疫原結構免疫天竺鼠之抗血清所純化的抗-α-Syn抗體特異性，針對不同大小的α-Syn分子複合物，包括α-Syn、Aβ和tau蛋白等不同的類澱粉蛋白、與經NGF處理後在α-Syn過度表現的PC12細胞中聚集的α-Syn，進行了一系列體外試驗。 In order to analyze the specificity of anti-α-Syn antibodies purified from anti-sera from guinea pigs with different α-Syn peptide immunogen structures, specific α-Syn molecular complexes of different sizes, including α-Syn, Aβ, and tau proteins, etc. A series of in vitro experiments were performed with different amyloids and α-Syn aggregated in PC12 cells overexpressed by α-Syn after NGF treatment.

a. 對更大之α-Syn分子複合物的特異性a. Specificity for larger α- Syn molecular complexes

使用從用不同α-Syn胜肽免疫原結構免疫天竺鼠抗血清所純化之抗-α-Syn抗體作為一級抗體，對不同大小的α-Syn分子複合物進行西方墨點法試驗。結果顯示，除了較小的α-Syn單體之外，所有抗-α-Syn抗體與較大的α-Syn分子複合物，包括二聚體、三聚體、四聚體和寡聚體有強烈反應。與市售抗-α-Syn抗體相比，Syn211(Abcam)、α-Syn_111-132(SEQ ID NO：113)、α-Syn_121-135(SEQ ID NO：107)、α-Syn_123-135(SEQ ID NO：111)及α-Syn_126-135(SEQ ID NO：112)所誘導之抗-α-Syn抗體具有較高的較大α-Syn分子複合物(包括二聚體，三聚體，四聚體和寡聚體)與較小α-Syn單體的訊號比(第7A和7B圖)，表示抗-α-Syn對較大的α-Syn 分子複合物具有特異性。 Western blotting tests were performed on α-Syn molecular complexes of different sizes using anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen structures as primary antibodies. The results show that except for the smaller α-Syn monomers, all anti-α-Syn antibodies are complexed with larger α-Syn molecules, including dimers, trimers, tetramers, and oligomers. Strong response. Compared with commercially available anti-α-Syn antibodies, Syn211 (Abcam), α-Syn _111-132 (SEQ ID NO: 113), α-Syn _121-135 (SEQ ID NO: 107), α-Syn _123- Anti-α-Syn antibodies induced by ₁₃₅ (SEQ ID NO: 111) and α-Syn _126-135 (SEQ ID NO: 112) have higher and larger α-Syn molecular complexes (including dimers, The signal ratios of polymers, tetramers, and oligomers) to smaller α-Syn monomers (Figures 7A and 7B) indicate that anti-α-Syn is specific for larger α-Syn molecular complexes.

b. α-Syn在不同類澱粉蛋白中的特異性b. Specificity of α- Syn in different amyloids

如實施例3所述，製備不同種類(即α-螺旋單體、β-折疊單體、β-折疊低聚物與β-折疊纖維)的不同類澱粉蛋白(即α-Syn、Aβ1-42與Tau441)，使用由不同α-Syn胜肽免疫原結構免疫天竺鼠抗血清所純化的抗-α-Syn抗體作為一級抗體，進行圓漬點墨法。結果顯示，由α-Syn_126-135(SEQ ID NO：112)和α-Syn_111-132(SEQ ID NO：113)引發的抗-α-Syn抗體特異性地與所有β-折疊(單體、寡聚體和纖維狀物種)的α-Syn反應，而不是與α-螺旋單體反應(第8A、8B和8C圖)。此外，由α-Syn_126-135(SEQ ID NO：112)和α-Syn_111-132(SEQ ID NO：113)誘發的抗-α-Syn抗體與纖維狀β-折疊α-Syn和β-折疊α-Syn寡聚體的反應，比對與β-折疊α-Syn單體的反應強。相較之下，由α-Syn_126-135(SEQ ID NO：112)和α-Syn_111-132(SEQ ID NO：113)誘發的抗-α-Syn抗體不會與β-Syn或不同種類(α-螺旋單體、β-折疊單體、β-折疊寡聚體和β-折疊纖維)的類澱粉蛋白產生蛋白Aβ_1-42和Tau441的反應(第8A、8B和8C圖)。這些發現顯示由α-Syn_126-135(SEQ ID NO：112)和α-Syn_111-132(SEQ ID NO：113)誘發的抗-α-Syn抗體對β-折疊單體、β-折疊寡聚體和β-折疊纖維狀的α-Syn具有特異性。 As described in Example 3, different amyloids (ie α-Syn, Aβ1-42) of different species (ie α-helical monomers, β-fold monomers, β-fold oligomers, and β-fold fibers) were prepared. And Tau441), using anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogen structures as primary antibodies, and performing the round staining method. The results show that anti-α-Syn antibodies elicited by α-Syn _126-135 (SEQ ID NO: 112) and α-Syn _111-132 (SEQ ID NO: 113) specifically bind to all β-sheets (monomers). , Oligomers, and fibrous species) instead of α-Syn monomers (Figures 8A, 8B, and 8C). In addition, anti-α-Syn antibodies induced by α-Syn _126-135 (SEQ ID NO: 112) and α-Syn _111-132 (SEQ ID NO: 113) are associated with fibrous β-sheet α-Syn and β- The reaction of folded α-Syn oligomers is stronger than the reaction with β-folded α-Syn monomers. In contrast, anti-α-Syn antibodies _elicited by α-Syn _126-135 (SEQ ID NO: 112) and α-Syn _111-132 (SEQ ID NO: 113) do not differ from β-Syn or different species Reactions of amyloid-producing proteins Aβ _1-42 and Tau441 (α-helical monomers, β-fold monomers, β-fold oligomers, and β-fold fibers) (Figures 8A, 8B, and 8C). These findings show that anti-α-Syn antibodies against β-sheet monomers, β-sheet oligos induced by α-Syn _126-135 (SEQ ID NO: 112) and α-Syn _111-132 (SEQ ID NO: 113) Polymers and β-sheet fibrous α-Syn are specific.

c. 經NGF處理之α-Syn過度表現PC12細胞中聚集的α-Syn的結合特異性c. α- Syn treated with NGF overexpresses the binding specificity of α- Syn aggregated in PC12 cells

從不同α-Syn胜肽免疫原結構免疫之天竺鼠抗血清所純化的抗-α-Syn抗體在親代PC12細胞、空白對照PC12細胞、α-Syn過度表現野生型PC12細胞與α-Syn過度表現A53T突變PC12細胞中進行免疫細胞化學試驗(ICC)，以評估抗體對NGF處理後之聚集α-Syn的結合親和性，如實施例3所述。如第9圖的定量結果所示，由α-Syn_111-132(SEQ ID NO：113)、α-Syn_121-135(SEQ ID NO：107)或α-Syn_126-135(SEQ ID NO：112)誘發的抗-α-Syn抗體，比起在親代PC12細胞或NGF處理之空白對照組PC12細胞，在α-Syn過度表現野生型PC12細胞和α-Syn過度表現A53T突變PC12細胞中具有更強的反應性。由於NGF處理可誘導過度表現的α-Syn聚集物，發現由α-Syn_111-132(SEQ ID NO：113)、α-Syn_121-135(SEQ ID NO：107)或α-Syn_126-135(SEQ ID NO：112)引發的抗-α-Syn抗體對NGF處理之α-Syn過度表現野生型PC12細胞和α-Syn過度表現A53T突變PC12細胞中聚集的α-Syn具有特異性。 Anti-α-Syn antibodies purified from guinea pig antisera immunized with different α-Syn peptide immunogens were overexpressed in parental PC12 cells, blank control PC12 cells, α-Syn and wild-type PC12 cells and α-Syn. An A53T mutant PC12 cell was subjected to an immunocytochemical test (ICC) to evaluate the binding affinity of the antibody to aggregated α-Syn after NGF treatment, as described in Example 3. The quantitative results shown in FIG. 9, the _{α-Syn 111-132 (SEQ ID NO} : 113), α-Syn 121-135 (SEQ ID NO: 107) , or _{α-Syn 126-135 (SEQ ID NO} : 112) Induced anti-α-Syn antibodies have higher levels of α-Syn overexpressing wild-type PC12 cells and α-Syn overexpressing A53T mutant PC12 cells than parental PC12 cells or NGF-treated blank control PC12 cells. Greater reactivity. Since NGF treatment can induce over-expressing α-Syn aggregates, it was found that α-Syn _111-132 (SEQ ID NO: 113), α-Syn _121-135 (SEQ ID NO: 107), or α-Syn _126-135 (SEQ ID NO: 112) The elicited anti-α-Syn antibody is specific for α-Syn aggregated in NGF-treated α-Syn overexpressing wild-type PC12 cells and α-Syn overexpressing A53T mutant PC12 cells.

實施例14. 帕金森氏患者腦部免疫組織化學染色以評估α-SYN胜肽免疫原結構及其配方的組織特異性Example 14. Immunohistochemical staining of the brain of Parkinson's patients to evaluate the tissue specificity of the α- SYN peptide immunogen structure and its formulation

使用免疫前、以α-Syn_126-135(SEQ ID NO：112) 或α-Syn_111-132(SEQ ID NO：113)誘發的抗-α-Syn抗體、以及1：1比例混合2種抗體的抗-α-Syn抗體組合，於正常人組織中進行免疫組織病理學試驗，以監測特異性和不良的抗體自身反應。在給予經α-Syn_126-135(SEQ ID NO：112)或α-Syn_111-132(SEQ ID NO：113)免疫之天竺鼠的抗體及1：1比例混合2種抗體的組合物前，將人類組織切片(Pantomics)用二甲苯去除石蠟，在乙醇中脫水，接著以0.25%胰蛋白酶溶液與0.5% CaCl₂的PBS處理30分鐘，並在含1%過氧化氫的甲醇溶液中反應以阻斷內源性過氧化酶活性，再與含10%Block Ace(Sigma)的PBS反應。將組織切片以3-3'-二氨基聯苯胺(DAB)染色，並使用蘇木素進行複染色後以顯微鏡觀察。與市售抗-α-Syn抗體(BD，610708)的陽性反應相比，免疫α-Syn_126-135(SEQ ID NO：112)或α-Syn_111-132(SEQ ID NO：113)之天竺鼠的抗體及1：1比例混合2種抗體的組合物對正常人體組織呈陰性反應，此結果與天竺鼠免疫前抗體的結果一致(第10A圖)。 Anti-α-Syn antibody _elicited with α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113) before immunization, and the two antibodies were mixed in a 1: 1 ratio The anti-α-Syn antibody combination is used to perform immunohistopathological tests in normal human tissues to monitor specific and adverse antibody self-response. Before administering an antibody to a guinea pig immunized with α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113) and a composition in which the two antibodies are mixed in a ratio of 1: 1, Human tissue sections (Pantomics) were deparaffinized with xylene, dehydrated in ethanol, then treated with 0.25% trypsin solution and 0.5% CaCl ₂ in PBS for 30 minutes, and reacted in a methanol solution containing 1% hydrogen peroxide to prevent Endogenous peroxidase activity was cut off, and then reacted with 10% Block Ace (Sigma) in PBS. Tissue sections were stained with 3-3'-diaminobenzidine (DAB), counterstained with hematoxylin, and observed under a microscope. Compared with the positive response of commercially available anti-α-Syn antibody (BD, 610708), guinea pigs were immunized with α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113) The composition of the antibody and the 1: 1 ratio of the two antibodies was negative to normal human tissues. This result is consistent with the results of guinea pig pre-immune antibodies (Figure 10A).

使用免疫前、α-Syn_126-135(SEQ ID NO：112)或α-Syn_111-132(SEQ ID NO：113)所誘發的抗-α-Syn抗體以及1：1比例混合2種抗體的組合物，測試其與人類帕金森氏症腦組織的反應性。測定三個區域(即小腦、胼胝體及丘腦)(BioChain)的組織切片。由結果可知，與健康大腦切片中的陰性反應相比，α-Syn_126-135(SEQ ID NO：112)或α-Syn_111-132(SEQ ID NO：113)所誘發的抗 -α-Syn抗體以及1：1比例混合2種抗體的組合物對於PD大腦所有3個區域皆呈陽性反應(第10B及10C圖)。以顯微鏡觀察計數陽性染色，以定量PD大腦切片中對α-Syn聚集體的反應性。相較於健康大腦切片，α-Syn_126-135(SEQ ID NO：112)或α-Syn_111-132(SEQ ID NO：113)所誘發的抗-α-Syn抗體以及1：1比例混合2種抗體的組合物對於PD大腦切片有顯著陽性反應。在測定三種不同的抗-α-Syn抗體中，由α-Syn_111-132(SEQ ID NO：113)誘發的抗體對PD大腦切片中的α-Syn聚集體具有最強的免疫反應。 Pre-immune, α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113) induced anti-α-Syn antibody and a 1: 1 mixture of the two antibodies Composition to test its reactivity with human Parkinson's disease brain tissue. Tissue sections from three regions (ie, cerebellum, corpus callosum, and thalamus) (BioChain) were measured. From the results, compared with negative reactions in healthy brain sections, anti-α-Syn induced by α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113) The combination of the antibody and the 1: 1 ratio of the two antibodies was positive for all three regions of the PD brain (Figures 10B and 10C). Positive staining was counted under a microscope to quantify the reactivity to α-Syn aggregates in PD brain sections. Compared to healthy brain sections, α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113) induced anti-α-Syn antibodies and a 1: 1 ratio 2 A combination of these antibodies had a significant positive response to PD brain sections. Of the three different anti-α-Syn antibodies tested, antibodies induced by α-Syn _111-132 (SEQ ID NO: 113) had the strongest immune response to α-Syn aggregates in PD brain sections.

實施例15. α-SYN胜肽免疫原結構及其配方在動物模型中的效力證明Example 15. Proof of the efficacy of α- SYN peptide immunogen structure and its formulation in animal models

a. 免疫及血液/腦組織的收集a. Immune and blood / brain tissue collection

帕金森氏症(PD)小鼠模型的建立可參照實施例4。在注射MPP+後兩週，或接種α-Syn纖維後7週，將小鼠隨機分成三組，包括UBITh1-α-Syn_111-132(SEQ ID NO：113)胜肽、UBITh1-α-Syn_126-135(SEQ ID NO：112)胜肽及兩種胜肽的組合，以及佐劑組(組合物中所使用的佐劑和溶劑進行免疫(ISA 51 VG、CpG3、0.2% TWEEN®-80))。以肌內注射(IM)免疫3次，間隔3週，劑量為40μg。注射與採血時間表如表13所示。 For the establishment of a mouse model of Parkinson's disease (PD), refer to Example 4. Two weeks after MPP + injection or 7 weeks after α-Syn fiber inoculation, mice were randomly divided into three groups, including UBITh1-α-Syn _111-132 (SEQ ID NO: 113) peptide, UBITh1-α-Syn _{126 -135} (SEQ ID NO: 112) peptide, a combination of two peptides, and an adjuvant group (adjuvants and solvents used in the composition for immunization (ISA 51 VG, CpG3, 0.2% TWEEN®-80) ). Intramuscularly (IM) immunized 3 times, 3 weeks apart, a dose of 40 μ g. The injection and blood collection schedule is shown in Table 13.

在每個時間點，通過臉部靜脈採樣抽取200μL血液。從頜下靜脈滴下的血液收集至離心管中，並以300rpm離心10分鐘製備血清。動物犧牲後，收集腦組織樣品進行西方墨點法分析。 At each time point, the extraction face 200 μ L of blood through venous sampling. Blood dripped from the submandibular vein was collected into a centrifuge tube and centrifuged at 300 rpm for 10 minutes to prepare serum. After the animals were sacrificed, brain tissue samples were collected for Western blot analysis.

b. 接種α-Synb. Inoculation with α-Syn _{111-132111-132} (SEQ ID NO：113)及/或α-Syn(SEQ ID NO: 113) and / or α-Syn _{126-135126-135} (SEQ ID NO：112)胜肽免疫原結構之PD小鼠模式的反應(SEQ ID NO: 112) PD mouse model response of peptide immunogen structure

將各實驗組的混合血清樣本以含有1%BSA的PBST稀釋，接著添加至塗佈200μL全長α-Syn胜肽、含0.1M碳酸氫鈉的ELISA盤(Cloud-clone)(α-Syn濃度4.4μg/μL，pH9.6)中。在室溫下反應2小時後，以PBST清洗3次，加入100μL以1：3000比例經1%BSA稀釋的HRP共軛抗小鼠IgG抗體，於室溫再反應2小時。接著，以PBST清洗3次，與100μL的3,3,5,5-四甲基聯苯胺(TMB)在黑暗中反應10分鐘。加入100μL 2M H₂SO₄，反應15至30分鐘，使用SpectraMax i3x Multi-Mode Detection(Molecular Devices)測量波長450nm的吸光值(OD)。 The mixed serum samples of each experimental group were diluted with PBST containing 1% BSA, and then added to an ELISA disk (Cloud-clone) coated with 200 μL of full-length α-Syn peptide and containing 0.1 M sodium bicarbonate (α-Syn concentration 4.4 μg / μL, pH 9.6). After reacting at room temperature for 2 hours, washing with PBST three times, adding 100 μL of HRP conjugated anti-mouse IgG antibody diluted with 1% BSA at a ratio of 1: 3000, and reacting for 2 hours at room temperature. Next, it was washed 3 times with PBST, and reacted with 100 μL of 3,3,5,5-tetramethylbenzidine (TMB) in the dark for 10 minutes. Add 100 μL of 2M H ₂ SO ₄ , react for 15 to 30 minutes, and measure the absorbance (OD) at 450 nm using SpectraMax i3x Multi-Mode Detection (Molecular Devices).

在第二次免疫後，以α-Syn_111-132(SEQ ID NO：113)、α-Syn_126-135(SEQ ID NO：112)或2種胜肽免疫原結構之組合免疫MPP+誘導模型(第11A圖)或纖維狀α-Syn-模型(第11B圖)所產生之抗-α-Syn抗體的吸光值(OD值)大於3.0，並一直維持至15wpi或19wpi實驗結束，而注射佐劑的動物則無法偵測至抗-α-Syn的免疫反應。 After the second immunization, _{α-Syn 111-132 (SEQ ID NO} : 113), α-Syn 126-135 (SEQ ID NO: 112) or a combination of two structures immunogenic peptides immunity induced by MPP + model ( (Fig. 11A) or fibrous α-Syn-model (Fig. 11B) The absorbance (OD) of the anti-α-Syn antibody produced is greater than 3.0 and remains until the end of the 15wpi or 19wpi experiment, and the adjuvant is injected Animals cannot detect an anti-α-Syn immune response.

應注意的是，在α-Syn纖維模型中， α-Syn_111-132結構可誘發比α-Syn_126-135結構更強的免疫反應(第11B圖)，但在MPP+模型中則沒有此免疫原性的差異(第11A圖)。 It should be noted that in the α-Syn fiber model, the α-Syn _111-132 structure can induce a stronger immune response than the α-Syn _126-135 structure (Figure 11B), but it is not available in the MPP + model. Difference in originality (Figure 11A).

c. 血清中α-Syn量的降低c. Decreasing the amount of α-Syn in serum

使用可檢測實施例3中所述之α-螺旋和β-折疊α-Syn二者的ELISA試劑套組(SEB222Mu，USCN)，測定各實驗組動物血清的α-Syn量。 The amount of α-Syn in the serum of each experimental group was determined using an ELISA reagent kit (SEB222Mu, USCN) capable of detecting both α-helix and β-fold α-Syn described in Example 3.

使用α-Syn定量ELISA分析，與未免疫動物相比，免疫組抗-α-Syn抗體反應是否與週邊α-Syn量的減少有關。結果顯示，相較於注射佐劑的動物，用α-Syn_126-135(SEQ ID NO：112)、α-Syn_111-132(SEQ ID NO：113)或此兩種結構組合免疫MPP+誘導模型(第12A圖)或纖維狀α-Syn模型(第12B圖)皆可降低α-Syn的吸光值(OD)。 The α-Syn quantitative ELISA analysis was used to determine whether the anti-α-Syn antibody response of the immunized group was related to a decrease in the amount of peripheral α-Syn compared to the non-immunized animals. The results showed that compared with animals injected with adjuvant, the MPP + induction model was immunized with α-Syn _126-135 (SEQ ID NO: 112), α-Syn _111-132 (SEQ ID NO: 113), or a combination of these two structures. (Figure 12A) or the fibrous α-Syn model (Figure 12B) can reduce the absorbance (OD) of α-Syn.

結果顯示α-Syn胜肽免疫原結構可產生抗-α-Syn抗體反應，使週邊循環系統中的α-Syn量相對應減少。 The results show that the structure of the α-Syn peptide immunogen can produce anti-α-Syn antibody response, and the amount of α-Syn in the peripheral circulation system is correspondingly reduced.

d. 腦中α-Syn寡聚物的減少d. Reduced α-Syn oligomers in the brain

動物犧牲後，收集腦組織樣品進行西方墨點法分析。對於MPP+誘導的小鼠，將腦取出並均質化；對於接種α-Syn的纖維小鼠，先分離紋狀體和黑質區域，並進行均質。將裂解緩衝液(Amresco)和1x蛋白酶抑製劑(Roche)添加至均質物中以製備腦組織裂解物。以10% SDS-PAGE(十二烷基硫酸鈉-聚丙烯醯胺凝膠電泳)分離裂解物，轉染至PVDF膜上，與含5%牛奶的PBS反應隔夜。為了檢測多巴胺神經元的數量，將PVDF膜與抗-酪胺酸羥化酶抗體(1：1000稀釋，Abcam)反應，再與山羊抗-兔IgG(H+L)HRP共軛的二級抗體(1：5000稀釋，Jackson Immunoresearch)雜交。為了將數據視覺化，使用Luminata Western HRP基質，並使用ChemiDoc-It810數位圖像系統處理實驗結果。α-Syn寡聚物的定量以GAPDH的量標準化，並將未損傷組織的裂解物比例設定為100%，以進行比較。 After the animals were sacrificed, brain tissue samples were collected for Western blot analysis. For MPP + -induced mice, the brain was removed and homogenized; for fibrous mice inoculated with α-Syn, the striatum and substantia nigra regions were first separated and homogenized. Lysis buffer (Amresco) and 1x protease inhibitor (Roche) were added to the homogenate to prepare a brain tissue lysate. 10% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel Electrophoresis) The lysate was separated, transfected onto a PVDF membrane, and reacted with 5% milk in PBS overnight. In order to detect the number of dopamine neurons, PVDF membrane was reacted with anti-tyrosine hydroxylase antibody (1: 1000 dilution, Abcam), and then reacted with goat anti-rabbit IgG (H + L) HRP conjugated secondary antibody (1: 5000 dilution, Jackson Immunoresearch) hybridization. To visualize the data, the Luminata Western HRP matrix was used, and the experimental results were processed using the ChemiDoc-It810 digital imaging system. The quantification of the α-Syn oligomer was normalized to the amount of GAPDH, and the lysate ratio of the undamaged tissue was set to 100% for comparison.

在MPP⁺小鼠模型中，在經α-Syn_111-132胜肽免疫原結構免疫的動物中顯示α-Syn寡聚物減少(第13A圖)。同樣地，對接種α-Syn纖維小鼠中之黑質及同側紋狀體的裂解物進行西方墨點法分析(第14A與14D圖)，並對對側紋狀體進行西方墨點法分析(第14F圖)，結果顯示在經α-Syn_111-132(SEQ ID NO：113)和α-Syn_126-135(sEQ ID NO：112)結構處理後，佐劑對照小鼠中α-Syn的量增加2至3倍。西方墨點法分析結果的量化如第13B、14B、14C、14D及14G圖所示。 In the MPP ⁺ mouse model, α-Syn oligomers were shown to be reduced in animals immunized with α-Syn _111-132 peptide immunogen structurally (Figure 13A). Similarly, Western blot analysis was performed on lysates of nigra and ipsilateral striatum in mice inoculated with α-Syn fibers (Figures 14A and 14D), and Western blot analysis was performed on the contralateral striatum. analysis (of FIG. 14F), the results are shown by _{α-Syn 111-132 (SEQ ID NO} : 113) and _{α-Syn 126-135 (sEQ ID NO} : 112) after the configuration process, the adjuvant control mice α- The amount of Syn is increased by 2 to 3 times. The quantification of Western blot analysis results is shown in Figures 13B, 14B, 14C, 14D and 14G.

e. 神經病理的減少e. Reduction of neuropathology

分離注射α-Syn纖維小鼠的黑質區，並均質化。將裂解緩衝液(Amresco)和1x蛋白酶抑製劑(Roche)添加至均質物中以製備組織裂解物。以10% SDS-PAGE(十二烷基硫酸鈉-聚丙烯醯胺凝膠電泳)分離裂解物，轉染至PVDF膜上，與5%牛奶的PBS反應隔夜。為了檢測多巴胺神經元的量，將PVDF膜與抗-酪胺酸羥化酶抗體(1：1000稀釋，Abcam)反應，再與山羊抗-兔IgG(H+L)HRP共軛的二級抗體(1：5000稀釋，Jackson Immunoresearch)雜交。為了將數據視覺化，使用Luminata Western HRP基質，並以ChemiDoc-It810數位圖像系統接收所偵測到的數值。利用GAPDH標準化α-Syn寡聚物的量，並將未損傷組織裂解物的比例設定為100%，以進行比較。為了將數據視覺化，使用Luminata Western HRP基質，並使用ChemiDoc-It810數位圖像系統處理實驗結果。α-Syn表現量以GAPDH(甘油醛3-磷酸脫氫酶)的量為標準，且GAPDH也作為上樣對照組(protein loading control)。 The substantia nigra area of mice injected with α-Syn fibers was isolated and homogenized. Lysis buffer (Amresco) and 1x protease inhibitor (Roche) were added to the homogenate to prepare a tissue lysate. The lysate was separated by 10% SDS-PAGE (sodium lauryl sulfate-polyacrylamide gel electrophoresis), transfected onto a PVDF membrane, and reacted with 5% milk in PBS Overnight. In order to detect the amount of dopamine neurons, the PVDF membrane was reacted with anti-tyrosine hydroxylase antibody (1: 1000 dilution, Abcam), and then reacted with goat anti-rabbit IgG (H + L) HRP conjugated secondary antibody (1: 5000 dilution, Jackson Immunoresearch) hybridization. To visualize the data, a Luminata Western HRP matrix was used and the detected values were received with a ChemiDoc-It810 digital imaging system. GAPDH was used to normalize the amount of α-Syn oligomers, and the ratio of lysed tissue lysate was set to 100% for comparison. To visualize the data, the Luminata Western HRP matrix was used, and the experimental results were processed using the ChemiDoc-It810 digital imaging system. The expression of α-Syn is based on the amount of GAPDH (glyceraldehyde 3-phosphate dehydrogenase), and GAPDH is also used as a protein loading control.

結果證明用α-Syn_111-132結構體免疫將酪氨酸羥化酶的量恢復到與未損傷正常動物相當的程度(第14C-14D圖)，表示α-Syn胜肽免疫原結構對於接種聚集α-Syn小鼠所造成的神經毒性具有神經保護作用。 The results demonstrated that the amount of tyrosine hydroxylase was restored to a level equivalent to that of normal animals without damage by the immunization with the α-Syn _111-132 structure (Figures 14C-14D), indicating that the α-Syn peptide immunogen structure was suitable for vaccination. The neurotoxicity caused by the aggregation of α-Syn mice is neuroprotective.

f. 活動力的恢復f. Recovery of activity

CatWalk^TMXT(Noldus information Technology,Wageningen,Netherlands)是一種影像分析系統，可以動態的方式，基於位置、壓力和每個腳步的表面積等來衡量腳步的各種情況。在實驗前，所有小鼠都要以一致的方式訓練，一天至少跑三次。一次成功的跑步定義為一隻動物跑步過程沒有中斷或猶豫，且將訓練失敗的老鼠被排除。分析每隻小鼠5次跑步的平均值。由於α-Syn纖維接種在右腦，所以在跑步期間，左後腳站立時間可為參考參數。 CatWalk ^TM XT (Noldus information Technology, Wageningen, Netherlands) is an image analysis system that can dynamically measure the various conditions of the footsteps based on position, pressure, and the surface area of each footstep. Prior to the experiment, all mice were trained in a consistent manner and ran at least three times a day. A successful run was defined as an animal running without interruption or hesitation, and rats that failed training were excluded. The average of 5 runs per mouse was analyzed. Since α-Syn fiber is inoculated in the right brain, the standing time of the left hind foot during running can be used as a reference parameter.

在α-Syn纖維接種模型中，在經含有α-Syn_126-135(SEQ ID NO：112)或α-Syn_111-132(SEQ ID NO：113)的組成物接種後，發現左後肢站立時間有顯著差異(第15A圖)。此外，在以α-Syn_111-132(SEQ ID NO：113)(第15B和15C圖)處理後，α-Syn纖維接種模型和MPP+誘導模型的跑步時間有顯著差異。結果顯示在此兩種PD模型中，α-Syn_126-135(SEQ ID NO：112)或α-Syn_111-132(SEQ ID NO：113)的α-Syn胜肽免疫原結構的處理和運動功能有關聯性。 In the α-Syn fiber inoculation model, after inoculation with a composition containing α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113), the standing time of the left hind limb was found There are significant differences (Figure 15A). In addition, after treatment with α-Syn _111-132 (SEQ ID NO: 113) (Figures 15B and 15C), there was a significant difference in running time between the α-Syn fiber inoculation model and the MPP + induction model. The results show that in these two PD models, the processing and movement of the α-Syn peptide immunogen structure of α-Syn _126-135 (SEQ ID NO: 112) or α-Syn _111-132 (SEQ ID NO: 113) Functionality is related.

實施例16. α-syn胜肽免疫原結構所產生之抗體與神經變性疾病中不同種類α-syn的反應性Example 16. Reactivity of antibodies produced by α-syn peptide immunogen structure with different types of α-syn in neurodegenerative diseases

α-Syn造成帕金森氏症和其他突觸核蛋白疾病。α-Syn蛋白可形成具有不同大小與結構的不同類型聚集體，且對細胞會有不同的影響，因此各種疾病是由一或多種不同類型聚集體所造成。不同形狀的α-Syn聚集體可能會導致大腦中不同的損傷模式，甚至會導致不同的腦部疾病。本研究在評估由α-Syn胜肽免疫原結構所產生之抗體如何與神經退行性疾病中不同α-Syn間相互作用。 Alpha-Syn causes Parkinson's disease and other synuclein diseases. α-Syn protein can form different types of aggregates with different sizes and structures, and has different effects on cells. Therefore, various diseases are caused by one or more different types of aggregates. Different shapes of α-Syn aggregates may cause different damage patterns in the brain, and even different brain diseases. This study evaluates how antibodies produced by the α-Syn peptide immunogen structure interact with different α-Syns in neurodegenerative diseases.

本試驗與Ronald Melki博士合作。在實驗室中產生各種不同的α-Syn聚集體，包括(a)纖維狀-一種長、扭曲、鉸鍊狀的α-Syn蛋白質；(b)帶狀-一種更廣、更扁平的結構，以及(c)α-Syn寡聚物(O550)、多巴胺穩定(ODA)與戊二醛穩定(OGA)的寡聚物。 This trial is in collaboration with Dr. Ronald Melki. Produce a variety of different α-Syn aggregates in the laboratory, including (a) fibrous-a Long, twisted, hinged α-Syn protein; (b) ribbon-like, a broader, flatter structure, and (c) α-Syn oligomer (O550), dopamine stable (ODA), and glutaraldehyde Stable (OGA) oligomers.

測試本發明各種α-Syn胜肽免疫原結構於天竺鼠中所產生之抗體的相對親和力。將源自於PD-021514(α-Syn_85-140，wpi 08)、PD-021522(α-Syn_85-140，wpi 13)、PD-100806(α-Syn_126-135，wpi 09)、PRX002及一市售單株抗體Syn1(clone 42)的代表性樣本與不同的α-Syn胜肽聚集體(纖維狀、帶狀(ribbons)、纖維65、纖維91、纖維110、纖維聚集途徑上的纖維寡聚物(O550)、多巴胺穩定(ODA)與戊二醛穩定(OGA)的寡聚物、以及對照組單體)進行濾紙結合分析。 The relative affinities of antibodies produced by various α-Syn peptide immunogen structures of the present invention in guinea pigs were tested. Will be derived from PD-021514 (α-Syn _85-140 , wpi 08), PD-021522 (α-Syn _85-140 , wpi 13), PD-100806 (α-Syn _126-135 , wpi 09), PRX002 Representative samples of a commercially available monoclonal antibody Syn1 (clone 42) and different α-Syn peptide aggregates (fibrous, ribbons, fibers 65, fibers 91, fibers 110, fiber aggregation pathways) Fiber oligomers (O550), dopamine stabilized (ODA) and glutaraldehyde stabilized (OGA) oligomers, and control monomers were analyzed by filter paper binding.

材料與方法Materials and Methods

a. α-Syn聚集成纖維及帶狀a. α-Syn aggregated into fiber and ribbon

對於纖維的形成，將可溶性WT α-Syn置於緩衝液A(50mM Tris-HCl、pH 7.5、150mM KCl)中，於37℃下以600rpm持續震盪。在硫磺素T(15μM)存在下，以磁石(6×3mm)攪動(100rpm)，利用Cary Eclipse分光光度計(Varian Inc.,Palo Alto,CA,USA)觀察聚集情況，其中激發波長為440nm，發射波長為440nm和480nm，平均時間為1s。對於帶狀的形成，將WT α-Syn於4℃下在1,000倍體積的緩衝液B(5mM Tris-HCl pH 7.5)透析16小時，並於37℃下以600rpm持續震盪。透過分析440nm的分散光來觀察聚集情況。另外，在經35,000xg沉降後，以Hewlett Packard 8453二極管陣列分光光度計的280nm吸光值偵測上清液中的蛋白質量。使用Jeol 1400(Jeol Ltd.)TEM，將樣品吸附至塗覆碳的200目網格上，使用1%乙酸鈾醯進行負染色，分析寡聚物種類的特性。使用Gatan Orius CCD照相機(Gatan)記錄圖像。以下述方式評估α-Syn結合剛果紅的能力：將纖維狀與帶狀α-Syn與20mM溶於Tris緩衝液(pH7.5)中的100μM剛果紅(Sigma-Aldrich，St Louis，MO，USA)反應1小時。然後將聚合物使用TL100桌上型Beckman超速離心機(Beckman Instruments，Inc.，Fullerton，CA，USA)於20℃下以25,000g離心30分鐘。使用等體積的水清洗沉澱顆粒4次。重新懸浮顆粒後，將等份的樣本立即置於玻璃蓋玻片上並攝影，或進行乾燥。使用具有交叉偏光鏡(Leica Microsystems，Ltd.，Heerbrugg，瑞士)的Leica(MZ12.5)顯微鏡，通過偏光顯微鏡觀察明視野和交叉偏光下的樣本。 For fiber formation, soluble WT α-Syn was placed in Buffer A (50 mM Tris-HCl, pH 7.5, 150 mM KCl) and shaken continuously at 600 rpm at 37 ° C. In the presence of Thioflavin T (15 μM), agitate (100 rpm) with a magnet (6 × 3 mm) and observe the aggregation using a Cary Eclipse spectrophotometer (Varian Inc., Palo Alto, CA, USA), where the excitation wavelength is 440 nm, The emission wavelength is 440nm and 480nm, and the average time is 1s. For band formation, WT α-Syn was dialyzed at 1,000 times the volume of buffer B (5 mM Tris-HCl pH 7.5) at 4 ° C. for 16 hours, and continuously shaken at 600 rpm at 37 ° C. Observe aggregation by analyzing 440nm scattered light Happening. In addition, after sedimentation at 35,000xg, the amount of protein in the supernatant was detected using a 280 nm absorbance of a Hewlett Packard 8453 diode array spectrophotometer. A Jeol 1400 (Jeol Ltd.) TEM was used to adsorb the sample onto a 200-mesh grid coated with carbon, and negative staining was performed with 1% uranium acetate. Images were recorded using a Gatan Orius CCD camera (Gatan). The ability of α-Syn to bind Congo red was evaluated in the following manner: Fibrous and ribbon-shaped α-Syn and 20 μM Congo red (Sigma-Aldrich, St Louis, MO, USA) dissolved in Tris buffer (pH7.5) ) Reaction for 1 hour. The polymer was then centrifuged at 25,000 g for 30 minutes at 20 ° C using a TL100 desktop Beckman ultracentrifuge (Beckman Instruments, Inc., Fullerton, CA, USA). Wash the pellet 4 times with an equal volume of water. Immediately after resuspending the particles, place an aliquot of the sample on a glass coverslip and photograph or dry it. A Leica (MZ12.5) microscope with a cross polarizer (Leica Microsystems, Ltd., Heerbrugg, Switzerland) was used to observe the bright field and cross-polarized samples through a polarizing microscope.

b. 纖維狀和帶狀α-Syn濃度的測定b. Determination of fibrous and ribbon α-Syn concentration

使用超音波細胞粉碎儀UIS250v(250W,2.4kHz；Hielscher Ultrasonic,Teltow,Germany)，振幅為75%，脈衝為0.5秒，在冰上進行超音波震碎20分鐘，以降低纖維狀和帶狀α-Syn的長度。測量纖維狀和帶狀α-Syn的沉降速度。使用Sedfit軟體，以最小平方邊界模型ls-g *(s)分析沉降界面。產生沉降係數 50S至150S之帶狀α-Syn的顆粒分佈、沉降係數100S至1,000S之纖維狀α-Syn的顆粒分佈，分別以沉積係數為~90S及375S之纖維狀和帶狀α-Syn的物質為中心，相對於分子量為約11,500kDa，例如，約800個帶狀α-Syn分子(12,000kDa/14.5kDa)；約102,000kDa，例如約7,000個纖維狀α-Syn分子(10,2000,kDa/14.5kDa)。因此，在20μM的操作濃度下，所有的帶狀和纖維狀α-Syn分別為20μM/~800=~0.02μM、20μM/~7,000=~0.003μM，100%的α-Syn在穩定狀態下形成帶狀或纖維狀，樣本離心後100%的蛋白質存在於沉澱顆粒中。 Ultrasound cell pulverizer UIS250v (250W, 2.4kHz; Hielscher Ultrasonic, Teltow, Germany) with an amplitude of 75% and a pulse of 0.5 seconds. Ultrasonic crushing on ice for 20 minutes to reduce fibrous and band α -Syn length. The sedimentation rates of fibrous and banded α-Syn were measured. The Sedfit software was used to analyze the settlement interface with the least square boundary model ls-g * (s). Settlement coefficient The particle size distribution of banded α-Syn from 50S to 150S and the particle size distribution of fibrous α-Syn from 100S to 1,000S are based on fibrous and banded α-Syn with deposition coefficients of ~ 90S and 375S, respectively. Center, with a molecular weight of about 11,500 kDa, for example, about 800 band-shaped α-Syn molecules (12,000 kDa / 14.5 kDa); about 102,000 kDa, for example, about 7,000 fibrous α-Syn molecules (10, 2000, kDa / 14.5kDa). Therefore, at an operating concentration of 20 μM, all banded and fibrous α-Syns are 20 μM / ~ 800 = ~ 0.02 μM, 20 μM / ~ 7,000 = ~ 0.003 μM, and 100% α-Syn is formed in a stable state. Banded or fibrous, 100% of the protein is present in the pellet after centrifugation.

c. 包涵體對不同纖維狀和帶狀α-Syn的親和力評估c. Evaluation of the affinity of inclusion bodies for different fibrous and banded α-Syn

使用濾紙結合分析，以抗體為對照組，評估本發明α-Syn胜肽免疫原結構所產生之抗體對不同α-Syn聚集體的親和力。α-Syn聚集體(纖維狀、帶狀、纖維65、纖維91、纖維110、纖維聚集途徑上的纖維寡聚物(O550)、多巴胺穩定(ODA)與戊二醛穩定(OGA)的寡聚物)已揭示於Bousset L.et al.,2013 Nat Commun 4：2575；Makky A.et al.,2016 Sci Rep 6：37970；and Pieri L.et al,2016 Sci.Rep 6：24526。本實施例包括使用對照組單體α-Syn。 Using filter paper binding analysis and using antibodies as a control group, the affinity of the antibodies produced by the α-Syn peptide immunogen structure of the present invention to different α-Syn aggregates was evaluated. α-Syn aggregates (fibrous, ribbon-like, fiber 65, fiber 91, fiber 110, fiber oligomer (O550) on fiber aggregation pathway, dopamine-stabilized (ODA) and glutaraldehyde-stabilized (OGA) oligomerization Has been disclosed in Bousset L. et al., 2013 Nat Commun 4: 2575; Makky A. et al., 2016 Sci Rep 6: 37970; and Pieri L. et al, 2016 Sci. Rep 6: 24526. This example includes the use of the control monomer α-Syn.

使用轉漬過濾槽將將20pg-200ng纖維狀、寡聚物或單體α-Syn轉漬至硝酸纖維素膜上。將硝酸纖維素膜以脫脂奶粉封組，並與稀釋之PRX002、Syn1 抗體或本實施例測試GP抗體反應。在充份清洗後，使用二級抗-人類或抗-天竺鼠IgG-HRP分析一級抗體的結合特徵。同樣分析對照組的二級抗體。加入Super Signal ECL(Pierce #34096)，並以BioRad imager(Chemidoc MP imaging system/BioRad imagelab軟體)進行顯影。曝光時間及其動態如第16A-16H圖所示。在實驗組中，將DLB病人的腦部勻質物轉漬至膜上。 A 20 pg-200 ng fibrous, oligo or monomeric α-Syn was transferred to a nitrocellulose membrane using a transfer filter tank. The nitrocellulose membrane was sealed with skim milk powder, and reacted with diluted PRX002, Syn1 antibody or GP antibody tested in this example. After sufficient washing, the binding characteristics of the primary antibodies were analyzed using secondary anti-human or anti-guinea pig IgG-HRP. The secondary antibodies of the control group were also analyzed. Super Signal ECL (Pierce # 34096) was added and developed with BioRad imager (Chemidoc MP imaging system / BioRad imagelab software). Exposure time and its dynamics are shown in Figures 16A-16H . In the experimental group, the brain homogenate of DLB patients was transferred to the membrane.

d. 結果d. Results

使用濾紙結合分析，比較由免疫天竺鼠(GP)所獲得之抗體PD-021514(α-Syn_85-140，wpi 08)、PD-021522(α-Syn_85-140，wpi 13)、PD-100806(α-Syn_126-135，wpi 09)、PRX002及市售抗體Syn1(clone 42)對於不同α-Syn聚集物的親和力。所使用的α-Syn聚集物包括纖維狀、帶狀、纖維65、纖維91、纖維110、纖維聚集途徑上的纖維寡聚物(O550)、多巴胺穩定(ODA)與戊二醛穩定(OGA)的寡聚物、以及對照組單體。 Filter paper binding analysis was used to compare the antibodies PD-021514 (α-Syn _85-140 , wpi 08), PD-021522 (α-Syn _85-140 , wpi 13), PD-100806 ( _{Affinity of} α-Syn _126-135 , wpi 09), PRX002, and commercially available antibody Syn1 (clone 42) to different α-Syn aggregates. The α-Syn aggregates used include fibrous, ribbon, fiber 65, fiber 91, fiber 110, fiber oligomers on the fiber aggregation pathway (O550), dopamine stabilization (ODA) and glutaraldehyde stabilization (OGA) Oligomers, and control monomers.

第16A-16H圖顯示相較於α-Syn單體，對照抗體PRX002對α-Syn具有稍微良好的親和性，而可辨識本發明α-Syn_126-135胜肽結構之PD-100806與PD-021514抗體對α-Syn纖維狀具有較高的親和性，而兩者對纖維狀α-Syn具有很好的結合性。Syn1單株抗體可結合纖維狀α-Syn以及寡聚物、單體α-Syn，彼此無太大差異。 Of FIG. 16A-16H show compared to monomeric α-Syn, a control antibody having a slightly PRX002 good affinity for α-Syn, but may recognize α-Syn PD-100806 peptide structure of the present invention for PD- _126-135 wins 021514 antibody has a high affinity for α-Syn fibrous, and the two have a good binding to fibrous α-Syn. Syn1 monoclonal antibodies can bind fibrous α-Syn as well as oligomers and monomer α-Syn without much difference from each other.

實施例17. 對帕金森氏症(PD)、多重系統退化症(MSA)和路易氏體型失智症(DLB)患者腦部α-SYN胜肽免疫原所形成之抗體的免疫組織化學研究Example 17. Immunohistochemical Study of Antibodies Formed by Alpha-SYN Peptide Immunogen in the Brain of Patients with Parkinson's Disease (PD), Multiple System Degeneration (MSA), and Lewy Body Dementia (DLB)

使用以本發明代表性α-Syn_126-135胜肽免疫原結構免疫天竺鼠所獲得之抗體進行免疫組織化學研究，以分析其與α-突觸核蛋白疾病患者大腦切片中α-Syn結合的能力。此研究與Roxana Carare教授合作。分析抗體與PD、LBD、及MSA患者大腦切片中α-Syn蛋白結合的能力。以健康組織作為陰性對照組。使用用於驗屍的市售單株抗體NCL-L-ASYN作為陽性對照組。本實驗提供了α-Syn_126-135胜肽免疫原結構抗體對PD、LBD和MSA患者大腦組織切片具有陽性免疫反應性的證據。在突觸核蛋白疾病患者大腦中特異性地看到結合，但在非病患大腦中則未發現結合，與市售診斷抗體相比，測試抗體的結合更為明顯。 Immunohistochemical studies were performed using antibodies obtained by immunizing guinea pigs with the representative α-Syn _126-135 peptide immunogen structure of the present invention to analyze their ability to bind to α-Syn in brain slices of patients with α-synuclein disease . This research is in collaboration with Professor Roxana Carare. The ability of antibodies to bind to α-Syn protein in brain slices from PD, LBD, and MSA patients was analyzed. Healthy tissue was used as the negative control group. A commercially available monoclonal antibody NCL-L-ASYN for autopsy was used as a positive control group. This experiment provides _evidence that α-Syn _126-135 peptide immunogen structural antibodies have positive immunoreactivity to brain tissue sections from patients with PD, LBD, and MSA. Binding was specifically seen in the brains of patients with synuclein disease, but was not found in the brains of non-patients, and the binding of test antibodies was more pronounced compared to commercially available diagnostic antibodies.

材料與方法Materials and Methods

a. 試劑與其供應商a. Reagents and their suppliers

將代表性α-Syn_126-135胜肽免疫結構免疫天竺鼠所獲得之抗體以1：100比例稀釋。PD062220-09-1-2-Syn、PD062205-09-1-2-Syn、PD100806-09-1-2-Syn抗體來自於聯腦科學公司(UNS)，NCL-L-ASYN抗體(小鼠單株抗體以1：100比例稀釋) 來自於Leica Biosystems，Santa Cruz Biotechnology，的HuD(E-I)(小鼠單株抗體以1：100比例稀釋)、Olig2抗體(兔子抗體以1：100比例稀釋)來自於Millipore，Alexa Flour 594抗體(山羊抗天竺鼠抗體以1：200比例稀釋)、Alexa Flour 488抗體(山羊抗天竺鼠抗體以1：200比例稀釋)與Alexa Flour 488抗體(山羊抗天竺鼠抗體以1：200比例稀釋)來自於Molecular Probes life technologies。 Antibodies obtained from a representative α-Syn _126-135 peptide immunized guinea pig were diluted at a ratio of 1: 100. PD062220-09-1-2-Syn, PD062205-09-1-2-Syn, PD100806-09-1-2-Syn antibodies were derived from United Brain Scientific Corporation (UNS), NCL-L-ASYN antibody (mouse single Strain antibody diluted 1: 100) HuD (EI) from Leica Biosystems, Santa Cruz Biotechnology, (mouse monoclonal antibody diluted 1: 100), Olig2 antibody (rabbit antibody diluted 1: 100) from In Millipore, Alexa Flour 594 antibody (goat anti-guinea pig antibody diluted 1: 200), Alexa Flour 488 antibody (goat anti-guinea pig antibody diluted 1: 200) and Alexa Flour 488 antibody (goat anti-guinea pig antibody 1: 200 (Proportional dilution) from Molecular Probes life technologies.

b. 人類腦組織b. Human brain tissue

本實施例使用來自UCL大腦銀行，厚度為μm的切片。所有樣本均依國家研究倫理服務委員會核准的方式進行收集和準備。 This example uses a slice from the UCL brain bank with a thickness of μm. All samples were collected and prepared in a manner approved by the National Research Ethics Services Committee.

由典型的α-Syn病理，包括多重系統退化症(MSA；n=3)、路易氏體癡呆症(DLB；n=3)和帕金森氏症(PD；n=3)受試者獲得組織(表15)。根據公佈的標準對受試者進行診斷**。 Tissue obtained from subjects with typical α-Syn pathology including multiple system degenerative disease (MSA; n = 3), Lewy body dementia (DLB; n = 3), and Parkinson's disease (PD; n = 3) (Table 15). Subjects were diagnosed according to published criteria **.

c. 人類突觸核蛋白疾病受試者的免疫組織化學分析c. Immunohistochemical analysis of human synuclein disease subjects

進行三種不同突觸核蛋白疾病(MSA，DLB和PD)患者的免疫組織化學(IHC)分析，以定量3種UNS抗體對α-Syn聚集體的特異性。比較UNS抗體(PD062220、PD062205及PD100806)與市售診斷抗體(NCL-L-ASYN)對於α-Syn聚集體的特異性。分析各患者4個大腦區域中抗體的特異性，(1)殼核(Putamen)、內囊(Internal Capsule)、島葉皮質(Insula Cortex)；(2) 中腦：黑質；(3)顳葉皮質(Temporal Cortex)：皮質灰質(Cortical Grey Matter)；以及(4)小腦(Cerebellum)：皮質下白質(Subcortical White Matter)；小腦白質(Cerebellar White Matter)。 Immunohistochemical (IHC) analysis was performed on patients with three different synuclein diseases (MSA, DLB, and PD) to quantify the specificity of the three UNS antibodies to α-Syn aggregates. The specificity of UNS antibodies (PD062220, PD062205, and PD100806) and commercially available diagnostic antibodies (NCL-L-ASYN) for α-Syn aggregates was compared. Analysis of antibody specificity in 4 brain regions of each patient, (1) Putamen, Internal Capsule, and Insula Cortex; (2) Midbrain: Substantia nigra; (3) Temporal Cortex: Cortical Grey Matter; and (4) Cerebellum: Subcortical White Matter; Cerebellar White Matter .

已知這些腦部區域在不同程度上以及各疾病類型的疾病進展階段皆會受到α-Syn聚集的影響。一般來說，在DLB、PD與MSA早期，基底核(basal ganglia)和中腦受到影響，且具有最高的累積負擔。顳葉皮質和小腦(cerebellum)在疾病後期受到影響，PD和DLB存在非常少的小腦聚集體。依據各IHC程序使用陰性對照組(未使用一級抗體)，確認二級抗體的非特異性結合。石蠟包埋的載玻片在60℃烤箱中脫蠟15-20分鐘，以二甲苯I & II各浸泡5分鐘。將組織置於100%至50% IMS的4倍稀釋液中再水化，每次5分鐘。以1xPBS中清洗組織3次，每次5分鐘，接著在100%甲酸中反應3分鐘以進行抗原修復。以1xPBS徹底清洗組織後，與3% H₂O₂反應10分鐘以去除內源性過氧化物酶活性。將組織冷凍，並進一步以1xPBS清洗3次(每次5分鐘)，用檸檬酸鹽緩衝液(15mM檸檬酸三鈉，TWEEN，pH6)在中等溫度下微波25分鐘，為確保每次進行相同的微波，每次使用3個容器及3個切片架。將切片冷凍並以1xPBS清洗3次(每次5分鐘)，以15%的山羊血清封阻非特異性結合位。組織在4℃下與一級抗體(與0.1%TBS/t以1：100比例稀釋)反應隔夜。以1xPBS清洗組織3次，每次5分鐘，並在室溫下與具有生物素的二級抗體反應1小時。使用前30分鐘，新鮮配製ABC溶液。以1xPBS清洗組織3次，每次5分鐘後，與ABC溶液於室溫下反應1小時。使用ImmPACTVIP peroxidase套組依操作說明製備VIP過氧化酶基質。於室溫下加入VIP過氧化酶基質反應7分鐘，並以dH₂O清洗。在將組織置於DPX前，先分別於IMS 50%、70%、95%、100%、100%及二甲苯I & II中脫水。對於雙重免疫螢光染色，在給予一級抗體前組織未經過3% H₂O₂處理。在給予第1種一級抗體與相同的二級抗體後，以15%山羊血清封阻組織30分鐘，並與第2種一級與二級抗體反應。在給予最終螢光標誌的二級抗體後，將組織與1% Sudan Black反應5分鐘，以去除自體螢光，以0.1%TBS/T清洗，並立即置於mowiol cituflour。將螢光染色的組織置於4℃直到完成圖像攝影。 It is known that these brain regions are affected by α-Syn aggregation to varying degrees and disease progression stages of each disease type. Generally, in the early stages of DLB, PD, and MSA, the basal ganglia and midbrain are affected and have the highest cumulative burden. The temporal lobe cortex and cerebellum are affected late in the disease, and there are very few cerebellar aggregates in PD and DLB. A negative control group was used in accordance with each IHC program (primary antibody was not used) to confirm non-specific binding of the secondary antibody. The paraffin-embedded glass slides were dewaxed in an oven at 60 ° C for 15-20 minutes, and immersed in xylene I & II for 5 minutes each. Tissues were rehydrated in 4-fold dilutions of 100% to 50% IMS for 5 minutes each. The tissues were washed 3 times in 1xPBS for 5 minutes each, and then reacted in 100% formic acid for 3 minutes for antigen retrieval. After thoroughly washing the tissue with 1xPBS, it was reacted with 3% H ₂ O ₂ for 10 minutes to remove endogenous peroxidase activity. The tissues were frozen and further washed 3 times (5 minutes each) with 1xPBS, and microwaved with citrate buffer (15 mM trisodium citrate, TWEEN, pH 6) at medium temperature for 25 minutes. Microwave, use 3 containers and 3 slicing racks each time. Sections were frozen and washed 3 times (5 minutes each) with 1xPBS, blocking non-specific binding sites with 15% goat serum. Tissues reacted with primary antibodies (diluted with 0.1% TBS / t at a ratio of 1: 100) at 4 ° C overnight. Tissues were washed 3 times in 1xPBS for 5 minutes each and reacted with a secondary antibody with biotin for 1 hour at room temperature. 30 minutes before use, freshly prepare ABC solution. The tissue was washed 3 times with 1xPBS, and after 5 minutes each time, it was reacted with the ABC solution at room temperature for 1 hour. ImmPACTVIP peroxidase kit was used to prepare VIP peroxidase substrate according to the instructions. VIP peroxidase substrate was added for 7 minutes at room temperature, and washed with dH ₂ O. Before placing the tissue in DPX, dehydrate it in IMS 50%, 70%, 95%, 100%, 100% and xylene I & II, respectively. For dual immunofluorescence staining, the tissues were not treated with 3% H ₂ O ₂ before the primary antibody was administered. After the first primary antibody and the same secondary antibody were administered, the tissue was blocked with 15% goat serum for 30 minutes, and reacted with the second primary and secondary antibodies. After giving the final fluorescently labeled secondary antibody, the tissue was reacted with 1% Sudan Black for 5 minutes to remove autofluorescence, washed with 0.1% TBS / T, and immediately placed in mowiol cituflour. The fluorescently stained tissue was placed at 4 ° C until image photography was completed.

d. 圖像分析及統計d. Image analysis and statistics

使用Olympus VS110高通量虛擬顯微系統或Olympus掃描切片虛擬顯微系統，以x20物鏡倍率掃描切片進行分析。使用Olympus VS軟體將每個受試者每個區域的相同位置由掃描圖像中擷取30張圖像(每張圖像500μm²)(第17A-17D、18A-18D、19A-19B、20A-20E、21A-21F、22A-22C、24A-24D及25A-25D圖)。分析每個腦部中總面積7.5mm²的區域。使用 ImageJ version Fiji windows-64軟體定量分析每個圖像的α-Syn免疫反應性。 The Olympus VS110 high-throughput virtual microscopy system or Olympus scanning microscopy virtual microscopy system was used to scan the sections at x20 objective magnification for analysis. Use Olympus VS software to capture 30 images (500 μm ² each) of the same position of each subject in each area from the scanned images (17A-17D, 18A-18D, 19A-19B, 20A -20E, 21A-21F, 22A-22C, 24A-24D and 25A-25D). An area with a total area of 7.5 mm ² in each brain was analyzed. ImageJ version Fiji windows-64 software was used to quantify the α-Syn immunoreactivity of each image.

為了分析每種抗體檢測到的α-Syn總量，以圖像總面積的百分比作為免疫反應性。為了分析背景染色的差異，選擇每個腦部區域調整α-Syn陽性免疫反應性的閥值。對於每個抗體和腦部區域的分析，計算α-Syn陽性聚集體覆蓋面積的平均百分比。 To analyze the total amount of α-Syn detected by each antibody, the percentage of the total area of the image was used as the immunoreactivity. In order to analyze the differences in background staining, each brain region was selected to adjust the threshold of α-Syn positive immunoreactivity. For the analysis of each antibody and brain region, the average percentage of α-Syn positive aggregate coverage area was calculated.

為了分析每種抗體對LBs或LNs的相對特異性，使用Fiji軟體，基於尺寸和圓度的參數來量化LBs的免疫反應性，以與LNs區分(參照第24A-24D、25A-25D、與26A-26B圖)。選擇具有不同形態LBs和LNs的腦部區域進行分析，以避免偽陽性，且腦部區域包括基底核的島葉皮質和顳皮質的灰質。LB免疫反應性以總α-Syn免疫反應性的百分比表示。 In order to analyze the relative specificity of each antibody to LBs or LNs, Fiji software was used to quantify the immunoreactivity of LBs based on parameters of size and roundness to distinguish them from LNs (refer to sections 24A-24D, 25A-25D, and 26A Figure -26B). Brain regions with different morphologies of LBs and LNs were selected for analysis to avoid false positives, and the brain regions included the basal nucleus of the insular cortex and the gray matter of the temporal cortex. LB immunoreactivity is expressed as a percentage of total α-Syn immunoreactivity.

使用GraphPad Prism v7.01軟體進行統計分析，並以平均值±SD表示(除非另外說明)。以單因素方差分析(ANOVA)分析結果，並以Dunnett校正進行事後分析(post hoc analys)。當p<0.05(*)時，視為具有顯著的差異。數字(n)指的是每個實驗使用的樣本數量。 Statistical analysis was performed using GraphPad Prism v7.01 software and expressed as mean ± SD (unless otherwise stated). Results were analyzed by one-way analysis of variance (ANOVA) and post hoc analys were analyzed by Dunnett's correction. When p <0.05 (*), it is considered to have a significant difference. The number (n) refers to the number of samples used in each experiment.

如先前所述，利用雙免疫螢光染色完成α-Syn在神經元或神經膠質中的定性分析。使用Leica SP8雷射掃描共軛焦顯微鏡觀察載玻片。在x40系列物鏡下獲得最大重疊影像。影像包括一系列z-方向之2種顏色重疊圖像，以顯示其相對位置。 Qualitative analysis of α-Syn in neurons or glial cells was performed using dual immunofluorescence staining as previously described. The glass was observed using a Leica SP8 laser scanning conjugate focal microscope. Get maximum overlapping images under x40 series objectives. The image includes a series of 2 colors in the z-direction Color overlay the image to show its relative position.

e. 以α-Syne. Take α-Syn _{126-135126-135} 抗體偵測α-Syn聚集體的不同樣態，並與NCL-L-ASYN抗體比較Antibodies detect different states of α-Syn aggregates and compare with NCL-L-ASYN antibodies

α-Syn聚集體的細胞類型和亞細胞位置，在不同的突觸核蛋白疾病之間有所不同。MSA的特徵為膠質胞質內涵物(GCI)，而在DLB和PD，α-Syn聚集發生於神經元細胞體(稱路易氏體(LB))和軸突過程(稱路易氏突起(LN))內。分析染色區域的百分比可定量每種抗體檢測到的總α-Syn聚集體。然而，這沒有考慮到聚集體的類型或亞細胞位置的差異。在PD和DLB的情況下，細胞體與神經元突內α-Syn聚集體的樣態不同，可使本發明UNS抗體對量化的這些不同類型α-Syn聚集體具相對敏感性。 The cell type and subcellular location of α-Syn aggregates varies between different synuclein diseases. MSA is characterized by glial cytoplasmic inclusions (GCI), while in DLB and PD, α-Syn aggregation occurs in neuronal cell bodies (called Lewy bodies (LB)) and axonal processes (called Lewy processes (LN) )Inside. Analysis of the percentage of stained areas quantifies the total α-Syn aggregates detected by each antibody. However, this does not take into account differences in aggregate types or subcellular locations. In the case of PD and DLB, the appearance of the α-Syn aggregates in the cell body and neuronal processes is different, which can make the UNS antibody of the present invention relatively sensitive to the quantification of these different types of α-Syn aggregates.

為了研究此現象，在DLB和PD情況下，分析每個抗體在細胞體內檢測到的聚集體比例。使用FIJI軟體，根據它們的大小和圓度選擇細胞體內的聚集體。計算細胞體聚集體的面積平均百分比作為總α-Syn，結果如第24A-24D與25A-25D所示。依據組織定性分析，整體與細胞體中α-Syn面積百分比的差異與α-Syn的軸突聚集體(LNs)有關。細胞體α-Syn比例的降低會反應出LN中的增加。由於顳葉皮質和島葉皮質同時表現出類似LB與LN的病理，因此在顳葉皮質和島葉皮質進行分析。LNs非常少且不均勻地分佈在殼核和內囊上，因此未選擇基底核進行分析。在中腦的黑質中觀察到類似的相關性(第26A與26B圖)，相較於DLB與PD中的NCL-L-ASYN抗體，本發明UNS抗體可檢測到更高量的LN。然而，由於LNs和LBs的複雜形態，無法用相同的方法可靠地區分和量化。 To investigate this phenomenon, in the case of DLB and PD, the proportion of aggregates detected by each antibody in the cell was analyzed. Using FIJI software, aggregates within cells were selected based on their size and roundness. The area average percentage of cell body aggregates was calculated as the total α-Syn, and the results are shown in Nos. 24A-24D and 25A-25D. According to the qualitative analysis of tissue, the difference between the percentage of α-Syn area in the whole and the cell body was related to the axon aggregates (LNs) of α-Syn. A decrease in the cell body α-Syn ratio reflects an increase in LN. Since the temporal lobe cortex and the insular cortex both show pathologies similar to LB and LN, analysis was performed in the temporal lobe cortex and insular cortex. LNs are very few and unevenly distributed on the putamen and inner capsule, so the basal nuclei were not selected for analysis. In the substantia nigra A similar correlation was observed (Figures 26A and 26B), and the UNS antibody of the present invention detected a higher amount of LN than the NCL-L-ASYN antibodies in DLB and PD. However, due to the complex morphology of LNs and LBs, they cannot be reliably distinguished and quantified in the same way.

第24A-24D圖的結果顯示每種抗體檢測到的總α-Syn量，與NCL-L-ASYN相比，UNS抗體在細胞體內檢測到的聚集體比例較低。表示細胞體內包涵體(inclusions)對LNs的比例降低，並且用UNS抗體檢測到更高比例的LNs。在UNS抗體中，PD062205抗體在DLB和PD中檢測島葉皮質高比例LNs的結果是一致的(第17A-17D與18A-18D圖)。相較之下，與NCL-L-ASYN相比，所有α-Syn_126-135抗體在DLB和PD患者的顳葉皮質灰質中可檢測到高比例的細胞體聚集(cell-body aggregates)(第25A-25B圖)。 The results of Figures 24A-24D show that the total amount of α-Syn detected by each antibody was lower than that of NCL-L-ASYN. This indicates that the ratio of inclusions to LNs in the body is reduced, and a higher proportion of LNs is detected with UNS antibodies. Among the UNS antibodies, the PD062205 antibody detected the high proportion of LNs in the insular cortex in DLB and PD (Figures 17A-17D and 18A-18D). In contrast, compared to NCL-L-ASYN, all α-Syn _126-135 antibodies detected a high proportion of cell-body aggregates in the gray matter of the temporal lobe of patients with DLB and PD (p. 25A-25B).

f. α-Syn的聚集體具細胞類型特異性f. α-Syn aggregates are cell type specific

含有α-Syn聚集體是突觸核蛋白疾病的特徵性致病標誌，包括MSA、DLB和PD。而α-Syn聚集是突觸核蛋白疾病中的主要致病蛋白，聚集和細胞的樣態會影響聚集的形成，且在特定疾病亞型之間不同。MSA、DLB和PD的臨床特徵顯示，在DLB和PD中神經元的細胞內與神經性過程(neritic processes)中有α-Syn的聚集，但在MSA中則主要在神經膠質細胞和少突膠質細胞(oligodendrocytes)內發現α-Syn的聚集。 Containing α-Syn aggregates are characteristic pathogenic markers of synuclein diseases, including MSA, DLB and PD. And α-Syn aggregation is the main pathogenic protein in synuclein diseases. Aggregation and cell appearance will affect the formation of aggregation, and it varies between specific disease subtypes. The clinical features of MSA, DLB, and PD show that α-Syn aggregates in the intracellular and neritic processes of neurons in DLB and PD. Aggregation of α-Syn was found in oligodendrocytes.

為了確定α-Syn_126-135抗體對細胞特異性α-Syn聚集體的選擇性，使用PD062205與針對神經元(HuD)或少突膠質細胞(Olig2)的標記進行雙重免疫螢光染色。 To determine the selectivity of the α-Syn _126-135 antibody for cell-specific α-Syn aggregates, dual immunofluorescence staining was performed using PD062205 with a marker against neurons (HuD) or oligodendrocytes (Olig2).

第27A-27C圖結果顯示，PD062205檢測出α-Syn位於PD和DLB中的基底核和中腦(高病理區域)的神經元細胞體內，但不是MSA。第28A-28C圖顯示，使用少突膠質細胞(Olig2)標記，在MSA發現α-Syn聚集於膠質細胞中，而未在PD或DL發現。此結果證明α-Syn_126-135抗體與突觸核蛋白疾病的臨床特徵一致，且證明這些抗體對α-Syn病理性聚集體的特異性。 Figures 27A-27C show that PD062205 detected α-Syn in neurons of the basal nucleus and midbrain (high pathological region) in PD and DLB, but not MSA. Figures 28A-28C show that using oligodendrocyte (Olig2) labeling, α-Syn was found to aggregate in glial cells in MSA, but not in PD or DL. This result demonstrates that the α-Syn _126-135 antibodies are consistent with the clinical characteristics of synuclein diseases and that these antibodies are specific for α-Syn pathological aggregates.

結果result

a. 用代表性的α-Syna. Use a representative α-Syn _{126-135126-135} 胜肽免疫原結構體免疫天竺鼠進行免疫治療所產生之抗體的定量分析Quantitative analysis of antibodies produced by immunizing guinea pigs with peptide immunogen structure

為了研究免疫治療的新型抗α-Syn抗體，以免疫組織化學(IHC)對三種突觸核蛋白疾病(MSA、DLB和PD)人類患者以各種抗體對α-Syn相對特異性的定量分析。 In order to study the novel anti-α-Syn antibodies for immunotherapy, immunohistochemistry (IHC) was used to quantitatively analyze the relative specificity of α-Syn in human patients with three synuclein diseases (MSA, DLB and PD) with various antibodies.

b. 以代表性α-Synb. Representative α-Syn _{126-135126-135} 胜肽免疫原結構免疫天竺鼠所獲得之抗體在結合α-Syn聚集體上比市售診斷抗體更敏感Antibodies derived from peptide immunogens structurally immunized guinea pigs are more sensitive to binding to α-Syn aggregates than commercially available diagnostic antibodies

為了研究本發明α-Syn_126-135抗體的相對抗原性，將各抗體檢測到的α-Syn量與市售突觸核蛋白疾病診斷抗體(NCL-L-ASYN)相比較。首先，分析第 17A-17D至22A-22C圖結果的整體，可發現與NCL-L-ASYN相比，α-Syn_126-135抗體檢測到的α-Syn平均百分比面積明顯較大。此趨勢在每個腦部區域和疾病類型中是一致的，且顯示本發明α-Syn_126-135抗體比起NCL-L-ASYN在結合聚集的α-Syn上更具有敏感性或選擇性。儘管本發明樣本量相對較小(n=3)，但數據仍可以看出明顯的趨勢。本發明α-Syn_126-135抗體對α-Syn的特異性可對照非病患與病患大腦中相同的腦區域來確認。第23A-23B圖的結果顯示每種抗體，包括NCL-L-ASYN皆都沒有任何陽性免疫染色。此數據表示本發明α-Syn_126-135抗體對致病形式的α-Syn具特異性。 In order to study the relative antigenicity of the α-Syn _126-135 antibody of the present invention, the amount of α-Syn detected by each antibody was compared with a commercially available synuclein disease diagnostic antibody (NCL-L-ASYN). First, by analyzing the overall results of the 17A-17D to 22A-22C plots, it can be found that compared with NCL-L-ASYN, the average percentage area of α-Syn detected by the α-Syn _126-135 antibody is significantly larger. This trend is consistent in each brain region and disease type, and shows that the α-Syn _126-135 antibody of the present invention is more sensitive or selective in binding to aggregated α-Syn than NCL-L-ASYN. Although the sample size of the present invention is relatively small (n = 3), the data still shows a clear trend. The specificity of the α-Syn _126-135 antibody of the present invention for α-Syn can be confirmed by comparing the same brain region in the non-patient and the patient's brain. The results in Figures 23A-23B show that each antibody, including NCL-L-ASYN, did not have any positive immunostaining. This data indicates that the alpha-Syn _126-135 antibodies of the invention are specific for the pathogenic form of alpha-Syn.

c. α-Sync. α-Syn _{126-135126-135} 抗體可檢測到較高量α-Syn，顯示其比市售抗體具有更高的敏感性和特異性Antibodies can detect higher amounts of α-Syn, showing higher sensitivity and specificity than commercially available antibodies

本發明α-Syn_126-135抗體比起NCL-L-ASYN可檢測到較高量的α-Syn，顯示本發明抗體更有利於免疫治療的應用，以促進α-Syn聚集體的清除。 The α-Syn _126-135 antibody of the present invention can detect a higher amount of α-Syn than NCL-L-ASYN, showing that the antibody of the present invention is more conducive to the application of immunotherapy to promote the elimination of α-Syn aggregates.

選擇作為免疫療法試劑之適當抗體的第一步是，確定抗體對具有初級α-Syn病理學之人腦組織中目標抗原(α-Syn)的選擇性。不同的突觸核蛋白疾病在α-Syn聚集體的機制、神經解剖學模式以及特定細胞類形聚集的損壞性(vulnerability)有所不同。 The first step in selecting an appropriate antibody as an immunotherapy agent is to determine the selectivity of the antibody for the target antigen (α-Syn) in human brain tissue with primary α-Syn pathology. Different synuclein diseases have different mechanisms of α-Syn aggregates, neuroanatomical patterns, and vulnerability of specific cell type aggregation.

為了研究一般將試劑作為突觸核蛋白疾病的免疫療法，可評估α-Syn_126-135抗體對不同突觸核蛋白疾病中不同神經病理學之α-Syn的選擇性是重要的。為此，選擇臨床確診的PD、DLB和MSA病例。PD和DLB是第二常見的癡呆型式，且主要是α-Syn在神經元(LB和LN)內的累積所引起。與PD相反，已知類澱粉蛋白-β和tau的病理會導致DLB2中的神經退化。在MSA中可見α-Syn聚集的不同模式，其中聚集體主要在神經膠質細胞而不是神經元中形成(第27A-27C與28A-28B圖)。此外，α-Syn病理的進展在不同疾病類型之間不同，早期病理常見於中腦和基底核區域。可藉由分析腦部中各抗體受不同階段疾病影響的抗原性，以分辨哪種抗體可更有效地治療疾病早期。 In order to study the immunotherapy using agents as synuclein diseases in general, it is important to evaluate the selectivity of α-Syn _126-135 antibodies for α-Syn of different neuropathologies in different synuclein diseases. To this end, clinically confirmed cases of PD, DLB, and MSA were selected. PD and DLB are the second most common form of dementia, and are mainly caused by the accumulation of α-Syn in neurons (LB and LN). In contrast to PD, the pathologies of amyloid-beta and tau are known to cause neurodegeneration in DLB2. Different patterns of α-Syn aggregation can be seen in MSA, where aggregates are formed mainly in glial cells rather than neurons (Figures 27A-27C and 28A-28B). In addition, the progression of α-Syn pathology varies between different disease types, with early pathology common in the midbrain and basal nucleus regions. By analyzing the antigenicity of each antibody in the brain affected by different stages of the disease, it can be distinguished which antibody can be more effective in treating the early stages of the disease.

d. α-Synd. α-Syn _{126-135126-135} 抗體(PD062220、PD062205和PD100806)可特異性結合PD、DLB和MSA(第17A-D至22A-22C圖)人腦組織中α-Syn病理聚集體，而在健康對照組未觀察到任何突觸核蛋白病理(圖23A-23B)Antibodies (PD062220, PD062205, and PD100806) specifically bind PD, DLB, and MSA (Figures 17A-D to 22A-22C) human α-Syn pathological aggregates, while no synapses were observed in healthy controls Riboprotein Pathology (Figures 23A-23B)

利用已公開的α-Syn_126-135抗體對α-Syn進行檢測，以臨床神經病理學描述相同細胞類型的特異性(第27A-27B與28A-28B圖)。重要的是，本發明α-Syn_126-135抗體對所有形式的人類α-Syn都沒有相同的抗原性。 Α-Syn was detected using the published α-Syn _126-135 antibody, and the specificity of the same cell type was described in clinical neuropathology (Figures 27A-27B and 28A-28B). Importantly, the α-Syn _126-135 antibodies of the invention do not have the same antigenicity to all forms of human α-Syn.

進一步分析各抗體能力中PD062205和PD100806抗體的特異性，在基底核中檢測到比LNL-L-ASYN更大的LN比例(第24A-24D圖)。在中腦中也可觀察到相同的現象(第26A-26B圖)。總之，由 PD062205和PD100806抗體可檢測到較高的α-Syn百分比面積，此結果表示本發明α-Syn_126-135抗體檢測到的額外α-Syn部分，可歸因於這些抗體對LN的特異性增加。此結果有助於免疫治療，因為在疾病的早期，LN是基底節中α-Syn聚集的主要形式。其它臨床前開發階段中的突觸核蛋白病治療試劑不提供對LN的IHC的檢測。因此，相較於其它市售產品，本發明胜肽免疫原結構與此胜肽免疫原結構所產生的α-Syn_126-135抗體具有相同的性質與特徵。 Further analysis of the specificity of PD062205 and PD100806 antibodies in each antibody's ability, a greater proportion of LN than LNL-L-ASYN was detected in the basal nucleus (Figures 24A-24D). The same phenomenon was observed in the midbrain (Figures 26A-26B). In summary, higher percentage areas of α-Syn can be detected by PD062205 and PD100806 antibodies. This result indicates that the additional α-Syn portion detected by the α-Syn _126-135 antibodies of the present invention can be attributed to the specificity of these antibodies for LN Sex increased. This result is helpful for immunotherapy, because in the early stages of the disease, LN is the main form of α-Syn aggregation in the basal ganglia. Other synucleinopathy treatment agents in the preclinical development phase do not provide detection of IHC for LN. Therefore, compared with other commercially available products, the peptide immunogen structure of the present invention has the same properties and characteristics as the α-Syn _126-135 antibody produced by this peptide immunogen structure.

測量受影響腦部區域中α-Syn聚集體的平均量，以IHC分析本發明胜肽免疫原結構所產生之α-Syn_126-135抗體的靈敏度。本發明量化腦部樣本中α-Syn的平均百分比面積，證明比起市售抗體，α-Syn_126-135抗體對MSA、DLB和PD的疾病進展早期的α-Syn檢測非常靈敏。 The average amount of α-Syn aggregates in the affected brain region was measured, and the sensitivity of the α-Syn _126-135 antibody produced by the peptide immunogen structure of the present invention was analyzed by IHC. The invention quantifies the average percentage area of α-Syn in brain samples, and proves that compared to commercially available antibodies, α-Syn _126-135 antibodies are very sensitive to detection of α-Syn in the early stages of disease progression in MSA, DLB, and PD.

在本發明較高的靈敏度可歸因於本發明抗體對LN的比起診斷抗體NCL-L-ASYN有更高的特異性。此結果證明本發明α-Syn_126-135抗體可能是抗體輔助清除突觸核蛋白疾病中α-Syn聚集體的最有效候選者。 The higher sensitivity in the present invention can be attributed to the higher specificity of the antibody of the invention for LN compared to the diagnostic antibody NCL-L-ASYN. This result proves that the α-Syn _126-135 antibody of the present invention may be the most effective candidate for antibody-assisted clearance of α-Syn aggregates in synuclein diseases.

所有說明書中所揭示之發明技術特點可以任意方式組合。說明書中揭示之每一技術特點可以提供相同、等同或相似目的之其他方式替換。因此，除非另有特別說明，文中所有揭示之特點均只是等同或相似特點之一般系列之實例。 The technical features of the invention disclosed in all the specifications can be combined in any way. Each technical feature disclosed in the description can be replaced in other ways for the same, equivalent or similar purpose. Therefore, unless stated otherwise, all features disclosed herein are merely examples of a general series of equivalent or similar features.

由上述可知，熟習此技藝者能輕易地了解本發明之必要特徵，在不脫離其精神與範圍之下能就本發明做許多改變與調整以應用於不同用途與條件。 As can be seen from the above, those skilled in the art can easily understand the necessary features of the present invention, and without departing from the spirit and scope, can make many changes and adjustments to the present invention to apply to different uses and conditions.

<110> 美國聯合生物醫學公司王長怡 <110> United United Biomedical Corporation

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<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白81-90 <223> Alpha -synuclein 81-90

<400> 19

<400> 19

<210> 20 <210> 20

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白82-91 <223> Alpha -synuclein 82-91

<400> 20

<400> 20

<210> 21 <210> 21

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白83-92 <223> Alpha -synuclein 83-92

<400> 21

<400> 21

<210> 22 <210> 22

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白84-93 <223> Alpha -synuclein 84-93

<400> 22

<400> 22

<210> 23 <210> 23

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白85-94 <223> Alpha -synuclein 85-94

<400> 23

<400> 23

<210> 24 <210> 24

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白86-95 <223> Alpha -synuclein 86-95

<400> 24

<400> 24

<210> 25 <210> 25

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白87-96 <223> Alpha -synuclein 87-96

<400> 25

<400> 25

<210> 26 <210> 26

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白88-97 <223> Alpha -synuclein 88-97

<400> 26

<400> 26

<210> 27 <210> 27

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白89-98 <223> Alpha -synuclein 89-98

<400> 27

<400> 27

<210> 28 <210> 28

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白90-99 <223> Alpha -synuclein 90-99

<400> 28

<400> 28

<210> 29 <210> 29

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白91-100 <223> Alpha -synuclein 91-100

<400> 29

<400> 29

<210> 30 <210> 30

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白92-101 <223> Alpha -synuclein 92-101

<400> 30

<400> 30

<210> 31 <210> 31

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白93-102 <223> Alpha -synuclein 93-102

<400> 31

<400> 31

<210> 32 <210> 32

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白94-103 <223> Alpha -synuclein 94-103

<400> 32

<400> 32

<210> 33 <210> 33

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白95-104 <223> Alpha -synuclein 95-104

<400> 33

<400> 33

<210> 34 <210> 34

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白96-105 <223> Alpha -synuclein 96-105

<400> 34

<400> 34

<210> 35 <210> 35

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白97-106 <223> Alpha -synuclein 97-106

<400> 35

<400> 35

<210> 36 <210> 36

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白98-107 <223> Alpha -synuclein 98-107

<400> 36

<400> 36

<210> 37 <210> 37

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白99-108 <223> Alpha -synuclein 99-108

<400> 37

<400> 37

<210> 38 <210> 38

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白100-109 <223> Alpha -synuclein 100-109

<400> 38

<400> 38

<210> 39 <210> 39

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白101-110 <223> Alpha -synuclein 101-110

<400> 39

<400> 39

<210> 40 <210> 40

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白102-111 <223> Alpha -synuclein 102-111

<400> 40

<400> 40

<210> 41 <210> 41

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白103-112 <223> Alpha -synuclein 103-112

<400> 41

<400> 41

<210> 42 <210> 42

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白104-113 <223> Alpha -synuclein 104-113

<400> 42

<400> 42

<210> 43 <210> 43

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白105-114 <223> Alpha -synuclein 105-114

<400> 43

<400> 43

<210> 44 <210> 44

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白106-115 <223> Alpha -synuclein 106-115

<400> 44

<400> 44

<210> 45 <210> 45

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白107-116 <223> Alpha -synuclein 107-116

<400> 45

<400> 45

<210> 46 <210> 46

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白108-117 <223> Alpha -synuclein 108-117

<400> 46

<400> 46

<210> 47 <210> 47

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白109-118 <223> Alpha -synuclein 109-118

<400> 47

<400> 47

<210> 48 <210> 48

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白110-119 <223> Alpha -synuclein 110-119

<400> 48

<400> 48

<210> 49 <210> 49

<211> 10 <211> 10

<212> PRT <212> PRT

<213> Homo sapiens <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白111-120 <223> Alpha -synuclein 111-120

<400> 49

<400> 49

<210> 50 <210> 50

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白112-121 <223> Alpha -synuclein 112-121

<400> 50

<400> 50

<210> 51 <210> 51

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> alphal-Synuclein 113-122 <223> alphal-Synuclein 113-122

<400> 51

<400> 51

<210> 52 <210> 52

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白114-123 <223> Alpha -synuclein 114-123

<400> 52

<400> 52

<210> 53 <210> 53

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白115-124 <223> Alpha -synuclein 115-124

<400> 53

<400> 53

<210> 54 <210> 54

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白116-125 <223> Alpha -synuclein 116-125

<400> 54

<400> 54

<210> 55 <210> 55

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白117-126 <223> Alpha -synuclein 117-126

<400> 55

<400> 55

<210> 56 <210> 56

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白118-127 <223> Alpha -synuclein 118-127

<400> 56

<400> 56

<210> 57 <210> 57

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白119-128 <223> Alpha -synuclein 119-128

<400> 57

<400> 57

<210> 58 <210> 58

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白120-129 <223> Alpha -synuclein 120-129

<400> 58

<400> 58

<210> 59 <210> 59

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白121-130 <223> Alpha -synuclein 121-130

<400> 59

<400> 59

<210> 60 <210> 60

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白122-131 <223> Alpha -synuclein 122-131

<400> 60

<400> 60

<210> 61 <210> 61

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白123-132 <223> Alpha -synuclein 123-132

<400> 61

<400> 61

<210> 62 <210> 62

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白124-133 <223> Alpha -synuclein 124-133

<400> 62

<400> 62

<210> 63 <210> 63

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白125-134 <223> Alpha -synuclein 125-134

<400> 63

<400> 63

<210> 64 <210> 64

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白126-135 <223> Alpha -synuclein 126-135

<400> 64

<400> 64

<210> 65 <210> 65

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白127-136 <223> Alpha -synuclein 127-136

<400> 65

<400> 65

<210> 66 <210> 66

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白128-137 <223> Alpha -synuclein 128-137

<400> 66

<400> 66

<210> 67 <210> 67

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白129-138 <223> Alpha -synuclein 129-138

<400> 67

<400> 67

<210> 68 <210> 68

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白130-139 <223> Alpha -synuclein 130-139

<400> 68

<400> 68

<210> 69 <210> 69

<211> 10 <211> 10

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(10) <222> (1) .. (10)

<223> α-突觸核蛋白131-140 <223> Alpha -synuclein 131-140

<400> 69

<400> 69

<210> 70 <210> 70

<211> 17 <211> 17

<212> PRT <212> PRT

<213> 破傷風梭菌(Clostridium tetani) <213> Clostridium tetani

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(17) <222> (1) .. (17)

<223> 破傷風梭菌(Clostridium tetani)1 Th <223> Clostridium tetani 1 Th

<400> 70

<400> 70

<210> 71 <210> 71

<211> 15 <211> 15

<212> PRT <212> PRT

<213> 麻疹病毒(Measles virus) <213> Measles virus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(15) <222> (1) .. (15)

<223> MvF1 Th <223> MvF1 Th

<400> 71

<400> 71

<210> 72 <210> 72

<211> 24 <211> 24

<212> PRT <212> PRT

<213> 百日咳菌(Bordetella pertussis) <213> Bordetella pertussis

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(24) <222> (1) .. (24)

<223> 百日咳菌(Bordetella pertussis)Th <223> Bordetella pertussis Th

<400> 72

<400> 72

<210> 73 <210> 73

<211> 17 <211> 17

<212> PRT <212> PRT

<213> 破傷風梭菌(Clostridium tetani) <213> Clostridium tetani

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(17) <222> (1) .. (17)

<223> 破傷風梭菌(Clostridium tetani)2 Th <223> Clostridium tetani 2 Th

<400> 73

<400> 73

<210> 74 <210> 74

<211> 23 <211> 23

<212> PRT <212> PRT

<213> 白喉桿菌(diphtheria bacilli) <213> diphtheria bacilli

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(23) <222> (1) .. (23)

<223> 白喉(Diphtheria)Th <223> Diphtheria Th

<400> 74

<400> 74

<210> 75 <210> 75

<211> 21 <211> 21

<212> PRT <212> PRT

<213> 惡性瘧原蟲(Plasmodium falciparum) <213> Plasmodium falciparum

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(21) <222> (1) .. (21)

<223> 惡性瘧原蟲(Plasmodium falciparum)Th <223> Plasmodium falciparum Th

<400> 75

<400> 75

<210> 76 <210> 76

<211> 17 <211> 17

<212> PRT <212> PRT

<213> 曼氏血吸蟲(Schistosoma mansoni) <213> Schistosoma mansoni

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(17) <222> (1) .. (17)

<223> 曼氏血吸蟲(Schistosoma mansoni)Th <223> Schistosoma mansoni Th

<400> 76

<400> 76

<210> 77 <210> 77

<211> 25 <211> 25

<212> PRT <212> PRT

<213> 霍亂毒素(Cholera Toxin) <213> Cholera Toxin

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(25) <222> (1) .. (25)

<223> 霍亂毒素(Cholera Toxin)Th <223> Cholera Toxin Th

<400> 77

<400> 77

<210> 78 <210> 78

<211> 15 <211> 15

<212> PRT <212> PRT

<213> 麻疹病毒 <213> Measles virus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(15) <222> (1) .. (15)

<223> MvF 2 Th <223> MvF 2 Th

<400> 78

<400> 78

<210> 79 <210> 79

<211> 22 <211> 22

<212> PRT <212> PRT

<213> 麻疹病毒 <213> Measles virus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(22) <222> (1) .. (22)

<223> KKKMvF 3 Th <223> KKKMvF 3 Th

<220> <220>

<221> SITE <221> SITE

<222> (7)..(7) <222> (7) .. (7)

<223> S或T <223> S or T

<220> <220>

<221> SITE <221> SITE

<222> (10)..(10) <222> (10) .. (10)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (11)..(11) <222> (11) .. (11)

<223> G或T <223> G or T

<220> <220>

<221> SITE <221> SITE

<222> (15)..(15) <222> (15) .. (15)

<223> H或T <223> H or T

<220> <220>

<221> SITE <221> SITE

<222> (16)..(16) <222> (16) .. (16)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (19)..(19) <222> (19) .. (19)

<223> G或T <223> G or T

<400> 79

<400> 79

<210> 80 <210> 80

<211> 18 <211> 18

<212> PRT <212> PRT

<213> Hepatitis B virus <213> Hepatitis B virus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(18) <222> (1) .. (18)

<223> HBsAg 1 Th <223> HBsAg 1 Th

<220> <220>

<221> SITE <221> SITE

<222> (1)..(1) <222> (1) .. (1)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (2)..(2) <222> (2) .. (2)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (3)..(3) <222> (3) .. (3)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (4)..(4) <222> (4) .. (4)

<223> L或I或V或F <223> L or I or V or F

<220> <220>

<221> SITE <221> SITE

<222> (5)..(5) <222> (5) .. (5)

<223> F或K或R <223> F or K or R

<220> <220>

<221> SITE <221> SITE

<222> (6)..(6) <222> (6) .. (6)

<223> L或I或V或F <223> L or I or V or F

<220> <220>

<221> SITE <221> SITE

<222> (7)..(7) <222> (7) .. (7)

<223> L或I或V或F <223> L or I or V or F

<220> <220>

<221> SITE <221> SITE

<222> (9)..(9) <222> (9) .. (9)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (10)..(10) <222> (10) .. (10)

<223> L或I或V或F <223> L or I or V or F

<220> <220>

<221> SITE <221> SITE

<222> (11)..(11) <222> (11) .. (11)

<223> L或I或V或F <223> L or I or V or F

<220> <220>

<221> SITE <221> SITE

<222> (13)..(13) <222> (13) .. (13)

<223> L或I或V或F <223> L or I or V or F

<220> <220>

<221> SITE <221> SITE

<222> (15)..(15) <222> (15) .. (15)

<223> Q或L或I或V或F <223> Q or L or I or V or F

<220> <220>

<221> SITE <221> SITE

<222> (17)..(17) <222> (17) .. (17)

<223> L或I或V或F <223> L or I or V or F

<220> <220>

<221> SITE <221> SITE

<222> (18)..(18) <222> (18) .. (18)

<223> D或R <223> D or R

<400> 80

<400> 80

<210> 81 <210> 81

<211> 19 <211> 19

<212> PRT <212> PRT

<213> 麻疹病毒 <213> Measles virus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF 4 Th <223> MvF 4 Th

<220> <220>

<221> SITE <221> SITE

<222> (4)..(4) <222> (4) .. (4)

<223> S或T <223> S or T

<220> <220>

<221> SITE <221> SITE

<222> (7)..(7) <222> (7) .. (7)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (8)..(8) <222> (8) .. (8)

<223> G或T <223> G or T

<220> <220>

<221> SITE <221> SITE

<222> (12)..(12) <222> (12) .. (12)

<223> H或T <223> H or T

<220> <220>

<221> SITE <221> SITE

<222> (13)..(13) <222> (13) .. (13)

<223> K或R <223> K or R

<400> 81

<400> 81

<210> 82 <210> 82

<211> 18 <211> 18

<212> PRT <212> PRT

<213> Hepatitis B virus <213> Hepatitis B virus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(18) <222> (1) .. (18)

<223> HBsAg 2 Th <223> HBsAg 2 Th

<220> <220>

<221> SITE <221> SITE

<222> (4)..(4) <222> (4) .. (4)

<223> I或F <223> I or F

<220> <220>

<221> SITE <221> SITE

<222> (5)..(5) <222> (5) .. (5)

<223> I或F <223> I or F

<220> <220>

<221> SITE <221> SITE

<222> (6)..(6) <222> (6) .. (6)

<223> T或L <223> T or L

<220> <220>

<221> SITE <221> SITE

<222> (7)..(7) <222> (7) .. (7)

<223> I或L <223> I or L

<220> <220>

<221> SITE <221> SITE

<222> (11)..(11) <222> (11) .. (11)

<223> I或L <223> I or L

<220> <220>

<221> SITE <221> SITE

<222> (14)..(14) <222> (14) .. (14)

<223> P或I <223> P or I

<220> <220>

<221> SITE <221> SITE

<222> (15)..(15) <222> (15) .. (15)

<223> Q或T <223> Q or T

<220> <220>

<221> SITE <221> SITE

<222> (16)..(16) <222> (16) .. (16)

<223> S或T <223> S or T

<220> <220>

<221> SITE <221> SITE

<222> (17)..(17) <222> (17) .. (17)

<223> L或I <223> L or I

<400> 82

<400> 82

<210> 83 <210> 83

<211> 19 <211> 19

<212> PRT <212> PRT

<213> 麻疹病毒 <213> Measles virus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF 5 Th <223> MvF 5 Th

<400> 83

<400> 83

<210> 84 <210> 84

<211> 18 <211> 18

<212> PRT <212> PRT

<213> Hepatitis B virus <213> Hepatitis B virus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(18) <222> (1) .. (18)

<223> HBsAg 3 Th <223> HBsAg 3 Th

<400> 84

<400> 84

<210> 85 <210> 85

<211> 11 <211> 11

<212> PRT <212> PRT

<213> Influenza virus <213> Influenza virus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(11) <222> (1) .. (11)

<223> Influenza Matrix protein 1_1 Th <223> Influenza Matrix protein 1_1 Th

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(11) <222> (1) .. (11)

<223> Influenza Matrix protein 1_1 Th <223> Influenza Matrix protein 1_1 Th

<400> 85

<400> 85

<210> 86 <210> 86

<211> 15 <211> 15

<212> PRT <212> PRT

<213> Influenza virus <213> Influenza virus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(15) <222> (1) .. (15)

<223> Influenza Matrix protein 1_2 Th <223> Influenza Matrix protein 1_2 Th

<400> 86

<400> 86

<210> 87 <210> 87

<211> 9 <211> 9

<212> PRT <212> PRT

<213> Influenza virus <213> Influenza virus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(9) <222> (1) .. (9)

<223> Influenza Non-structural protein 1 Th <223> Influenza Non-structural protein 1 Th

<400> 87

<400> 87

<210> 88 <210> 88

<211> 19 <211> 19

<212> PRT <212> PRT

<213> Epstein-Barr virus <213> Epstein-Barr virus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> EBV BHRF1 Th <223> EBV BHRF1 Th

<400> 88

<400> 88

<210> 89 <210> 89

<211> 15 <211> 15

<212> PRT <212> PRT

<213> Clostridium tetani <213> Clostridium tetani

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(15) <222> (1) .. (15)

<223> Clostridium tetani TT1 Th <223> Clostridium tetani TT1 Th

<400> 89

<400> 89

<210> 90 <210> 90

<211> 20 <211> 20

<212> PRT <212> PRT

<213> Epstein-Barr virus <213> Epstein-Barr virus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(20) <222> (1) .. (20)

<223> EBV EBNA-1 Th <223> EBV EBNA-1 Th

<400> 90

<400> 90

<210> 91 <210> 91

<211> 21 <211> 21

<212> PRT <212> PRT

<213> Clostridium tetani <213> Clostridium tetani

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(21) <222> (1) .. (21)

<223> Clostridium tetani TT2 Th <223> Clostridium tetani TT2 Th

<400> 91

<400> 91

<210> 92 <210> 92

<211> 16 <211> 16

<212> PRT <212> PRT

<213> Clostridium tetani <213> Clostridium tetani

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(16) <222> (1) .. (16)

<223> Clostridium tetani TT3 Th <223> Clostridium tetani TT3 Th

<400> 92

<400> 92

<210> 93 <210> 93

<211> 16 <211> 16

<212> PRT <212> PRT

<213> Clostridium tetani <213> Clostridium tetani

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(16) <222> (1) .. (16)

<223> Clostridium tetani TT4 Th <223> Clostridium tetani TT4 Th

<400> 93

<400> 93

<210> 94 <210> 94

<211> 18 <211> 18

<212> PRT <212> PRT

<213> Epstein-Barr virus <213> Epstein-Barr virus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(18) <222> (1) .. (18)

<223> EBV CP Th <223> EBV CP Th

<400> 94

<400> 94

<210> 95 <210> 95

<211> 14 <211> 14

<212> PRT <212> PRT

<213> Human cytomegalovirus <213> Human cytomegalovirus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(14) <222> (1) .. (14)

<223> HCMV IE1 Th <223> HCMV IE1 Th

<400> 95

<400> 95

<210> 96 <210> 96

<211> 15 <211> 15

<212> PRT <212> PRT

<213> Epstein-Barr virus <213> Epstein-Barr virus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(15) <222> (1) .. (15)

<223> EBV GP340 Th <223> EBV GP340 Th

<400> 96

<400> 96

<210> 97 <210> 97

<211> 13 <211> 13

<212> PRT <212> PRT

<213> Epstein-Barr virus <213> Epstein-Barr virus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(13) <222> (1) .. (13)

<223> EBV BPLF1 Th <223> EBV BPLF1 Th

<400> 97

<400> 97

<210> 98 <210> 98

<211> 11 <211> 11

<212> PRT <212> PRT

<213> 艾伯斯坦-巴爾病毒(Epstein-Barr virus) <213> Epstein-Barr virus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(11) <222> (1) .. (11)

<223> EBV EBNA-2 Th <223> EBV EBNA-2 Th

<400> 98

<400> 98

<210> 99 <210> 99

<211> 38 <211> 38

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF 4 Th <223> MvF 4 Th

<220> <220>

<221> SITE <221> SITE

<222> (4)..(4) <222> (4) .. (4)

<223> S或T <223> S or T

<220> <220>

<221> SITE <221> SITE

<222> (7)..(7) <222> (7) .. (7)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (8)..(8) <222> (8) .. (8)

<223> G或T <223> G or T

<220> <220>

<221> SITE <221> SITE

<222> (12)..(12) <222> (12) .. (12)

<223> H或T <223> H or T

<220> <220>

<221> SITE <221> SITE

<222> (13)..(13) <222> (13) .. (13)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε-K <223> ε -K

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(23) <222> (20) .. (23)

<223> ε K-KKK為連接子 <223> ε K-KKK is the linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (24)..(38) <222> (24) .. (38)

<223> α-突觸核蛋白126-140 <223> Alpha -synuclein 126-140

<400> 99

<400> 99

<210> 100 <210> 100

<211> 43 <211> 43

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF 4 Th <223> MvF 4 Th

<220> <220>

<221> SITE <221> SITE

<222> (4)..(4) <222> (4) .. (4)

<223> S或T <223> S or T

<220> <220>

<221> SITE <221> SITE

<222> (7)..(7) <222> (7) .. (7)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (8)..(8) <222> (8) .. (8)

<223> G或T <223> G or T

<220> <220>

<221> SITE <221> SITE

<222> (12)..(12) <222> (12) .. (12)

<223> H或T <223> H or T

<220> <220>

<221> SITE <221> SITE

<222> (13)..(13) <222> (13) .. (13)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε-K <223> ε -K

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(23) <222> (20) .. (23)

<223> ε K-KKK為連接子 <223> ε K-KKK is the linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (24)..(43) <222> (24) .. (43)

<223> α-突觸核蛋白121-140 <223> Alpha -synuclein 121-140

<400> 100

<400> 100

<210> 101 <210> 101

<211> 53 <211> 53

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF 4 Th <223> MvF 4 Th

<220> <220>

<221> SITE <221> SITE

<222> (4)..(4) <222> (4) .. (4)

<223> S或T <223> S or T

<220> <220>

<221> SITE <221> SITE

<222> (7)..(7) <222> (7) .. (7)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (8)..(8) <222> (8) .. (8)

<223> G或T <223> G or T

<220> <220>

<221> SITE <221> SITE

<222> (12)..(12) <222> (12) .. (12)

<223> H或T <223> H or T

<220> <220>

<221> SITE <221> SITE

<222> (13)..(13) <222> (13) .. (13)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε-K <223> ε -K

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(23) <222> (20) .. (23)

<223> ε K-KKK為連接子 <223> ε K-KKK is the linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (24)..(53) <222> (24) .. (53)

<223> α-突觸核蛋白111-140 <223> Alpha -synuclein 111-140

<400> 101

<400> 101

<210> 102 <210> 102

<211> 63 <211> 63

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF 4 Th <223> MvF 4 Th

<220> <220>

<221> SITE <221> SITE

<222> (4)..(4) <222> (4) .. (4)

<223> S或T <223> S or T

<220> <220>

<221> SITE <221> SITE

<222> (7)..(7) <222> (7) .. (7)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (8)..(8) <222> (8) .. (8)

<223> G或T <223> G or T

<220> <220>

<221> SITE <221> SITE

<222> (12)..(12) <222> (12) .. (12)

<223> H或T <223> H or T

<220> <220>

<221> SITE <221> SITE

<222> (13)..(13) <222> (13) .. (13)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε-K <223> ε -K

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(23) <222> (20) .. (23)

<223> ε K-KKK為連接子 <223> ε K-KKK is the linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (24)..(63) <222> (24) .. (63)

<223> α-突觸核蛋白126-140 <223> Alpha -synuclein 126-140

<400> 102

<400> 102

<210> 103 <210> 103

<211> 63 <211> 63

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF5 Th <223> MvF5 Th

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε-K <223> ε -K

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(23) <222> (20) .. (23)

<223> ε K-KKK為連接子 <223> ε K-KKK is the linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (24)..(63) <222> (24) .. (63)

<223> α-突觸核蛋白101-140 <223> Alpha -synuclein 101-140

<400> 103

<400> 103

<210> 104 <210> 104

<211> 62 <211> 62

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(18) <222> (1) .. (18)

<223> HBsAg3 Th <223> HBsAg3 Th

<220> <220>

<221> SITE <221> SITE

<222> (19)..(19) <222> (19) .. (19)

<223> ε-K <223> ε -K

<220> <220>

<221> 胜肽 <221> Peptide

<222> (19)..(22) <222> (19) .. (22)

<223> ε K-KKK為連接子 <223> ε K-KKK is the linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (23)..(62) <222> (23) .. (62)

<223> α-突觸核蛋白101-140 <223> Alpha -synuclein 101-140

<400> 104

<400> 104

<210> 105 <210> 105

<211> 73 <211> 73

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF 4 Th <223> MvF 4 Th

<220> <220>

<221> SITE <221> SITE

<222> (4)..(4) <222> (4) .. (4)

<223> S或T <223> S or T

<220> <220>

<221> SITE <221> SITE

<222> (7)..(7) <222> (7) .. (7)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (8)..(8) <222> (8) .. (8)

<223> G或T <223> G or T

<220> <220>

<221> SITE <221> SITE

<222> (12)..(12) <222> (12) .. (12)

<223> H或T <223> H or T

<220> <220>

<221> SITE <221> SITE

<222> (13)..(13) <222> (13) .. (13)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε-K <223> ε -K

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(23) <222> (20) .. (23)

<223> ε K-KKK為連接子 <223> ε K-KKK is the linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (24)..(73) <222> (24) .. (73)

<223> α-突觸核蛋白91-140 <223> Alpha -synuclein 91-140

<400> 105

<400> 105

<210> 106 <210> 106

<211> 79 <211> 79

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF 4 Th <223> MvF 4 Th

<220> <220>

<221> SITE <221> SITE

<222> (4)..(4) <222> (4) .. (4)

<223> S或T <223> S or T

<220> <220>

<221> SITE <221> SITE

<222> (7)..(7) <222> (7) .. (7)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (8)..(8) <222> (8) .. (8)

<223> G或T <223> G or T

<220> <220>

<221> SITE <221> SITE

<222> (12)..(12) <222> (12) .. (12)

<223> H或T <223> H or T

<220> <220>

<221> SITE <221> SITE

<222> (13)..(13) <222> (13) .. (13)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε-K <223> ε -K

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(23) <222> (20) .. (23)

<223> ε K-KKK為連接子 <223> ε K-KKK is the linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (24)..(79) <222> (24) .. (79)

<223> α-突觸核蛋白85-140 <223> Alpha -synuclein 85-140

<400> 106

<400> 106

<210> 107 <210> 107

<211> 38 <211> 38

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF5 Th <223> MvF5 Th

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε-K <223> ε -K

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(23) <222> (20) .. (23)

<223> ε K-KKK為連接子 <223> ε K-KKK is the linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (24)..(38) <222> (24) .. (38)

<223> α-突觸核蛋白121-135 <223> Alpha -synuclein 121-135

<400> 107

<400> 107

<210> 108 <210> 108

<211> 48 <211> 48

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF5 Th <223> MvF5 Th

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε-K <223> ε -K

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(23) <222> (20) .. (23)

<223> ε K-KKK為連接子 <223> ε K-KKK is the linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (24)..(48) <222> (24) .. (48)

<223> α-突觸核蛋白111-135 <223> Alpha -synuclein 111-135

<400> 108

<400> 108

<210> 109 <210> 109

<211> 58 <211> 58

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF5 Th <223> MvF5 Th

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε-K <223> ε -K

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(23) <222> (20) .. (23)

<223> ε K-KKK為連接子 <223> ε K-KKK is the linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (24)..(58) <222> (24) .. (58)

<223> α-突觸核蛋白101-135 <223> Alpha -synuclein 101-135

<400> 109

<400> 109

<210> 110 <210> 110

<211> 62 <211> 62

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF5 Th <223> MvF5 Th

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε-K <223> ε -K

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(23) <222> (20) .. (23)

<223> ε K-KKK為連接子 <223> ε K-KKK is the linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (24)..(62) <222> (24) .. (62)

<223> α-突觸核蛋白97-135 <223> Alpha -synuclein 97-135

<400> 110

<400> 110

<210> 111 <210> 111

<211> 36 <211> 36

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF5 Th <223> MvF5 Th

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε-K <223> ε -K

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(23) <222> (20) .. (23)

<223> ε K-KKK as a spacer <223> ε K-KKK as a spacer

<220> <220>

<221> 胜肽 <221> Peptide

<222> (24)..(36) <222> (24) .. (36)

<223> α-突觸核蛋白123-135 <223> Alpha -synuclein 123-135

<400> 111

<400> 111

<210> 112 <210> 112

<211> 33 <211> 33

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF5 Th <223> MvF5 Th

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε-K <223> ε -K

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(23) <222> (20) .. (23)

<223> ε K-KKK為連接子 <223> ε K-KKK is the linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (24)..(33) <222> (24) .. (33)

<223> α-突觸核蛋白126-135 <223> Alpha -synuclein 126-135

<400> 112

<400> 112

<210> 113 <210> 113

<211> 45 <211> 45

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF5 Th <223> MvF5 Th

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε-K <223> ε -K

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(23) <222> (20) .. (23)

<223> ε K-KKK為連接子 <223> ε K-KKK is the linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (24)..(45) <222> (24) .. (45)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 113

<400> 113

<210> 114 <210> 114

<211> 55 <211> 55

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF5 Th <223> MvF5 Th

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε-K <223> ε -K

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(23) <222> (20) .. (23)

<223> ε K-KKK為連接子 <223> ε K-KKK is the linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (24)..(55) <222> (24) .. (55)

<223> α-突觸核蛋白101-132 <223> Alpha -synuclein 101-132

<400> 114

<400> 114

<210> 115 <210> 115

<211> 45 <211> 45

<212> PRT <212> PRT

<213> 小家鼠(Mus musculus) <213> Mus musculus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF 5 Th <223> MvF 5 Th

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε-K <223> ε -K

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(23) <222> (20) .. (23)

<223> ε K-KKK為連接子 <223> ε K-KKK is the linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (24)..(45) <222> (24) .. (45)

<223> 小鼠α-突觸核蛋白111-132 <223> Mouse α -synuclein 111-132

<400> 115

<400> 115

<210> 116 <210> 116

<211> 33 <211> 33

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF 4 Th <223> MvF 4 Th

<220> <220>

<221> SITE <221> SITE

<222> (4)..(4) <222> (4) .. (4)

<223> S或T <223> S or T

<220> <220>

<221> SITE <221> SITE

<222> (7)..(7) <222> (7) .. (7)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (8)..(8) <222> (8) .. (8)

<223> G或T <223> G or T

<220> <220>

<221> SITE <221> SITE

<222> (12)..(12) <222> (12) .. (12)

<223> H或T <223> H or T

<220> <220>

<221> SITE <221> SITE

<222> (13)..(13) <222> (13) .. (13)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε-K <223> ε -K

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(23) <222> (20) .. (23)

<223> ε K-KKK為連接子 <223> ε K-KKK is the linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (24)..(33) <222> (24) .. (33)

<223> α-突觸核蛋白126-135 <223> Alpha -synuclein 126-135

<400> 116

<400> 116

<210> 117 <210> 117

<211> 45 <211> 45

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF 4 Th <223> MvF 4 Th

<220> <220>

<221> SITE <221> SITE

<222> (4)..(4) <222> (4) .. (4)

<223> S或T <223> S or T

<220> <220>

<221> SITE <221> SITE

<222> (7)..(7) <222> (7) .. (7)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (8)..(8) <222> (8) .. (8)

<223> G或T <223> G or T

<220> <220>

<221> SITE <221> SITE

<222> (12)..(12) <222> (12) .. (12)

<223> H或T <223> H or T

<220> <220>

<221> SITE <221> SITE

<222> (13)..(13) <222> (13) .. (13)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε-K <223> ε -K

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(23) <222> (20) .. (23)

<223> ε K-KKK為連接子 <223> ε K-KKK is the linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (24)..(45) <222> (24) .. (45)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 117

<400> 117

<210> 118 <210> 118

<211> 30 <211> 30

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF5 Th <223> MvF5 Th

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (21)..(30) <222> (21) .. (30)

<223> α-突觸核蛋白126-135 <223> Alpha -synuclein 126-135

<400> 118

<400> 118

<210> 119 <210> 119

<211> 42 <211> 42

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> MvF5 Th <223> MvF5 Th

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (21)..(42) <222> (21) .. (42)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 119

<400> 119

<210> 120 <210> 120

<211> 29 <211> 29

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(18) <222> (1) .. (18)

<223> HBsAg3 Th <223> HBsAg3 Th

<220> <220>

<221> SITE <221> SITE

<222> (19)..(19) <222> (19) .. (19)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(29) <222> (20) .. (29)

<223> α-突觸核蛋白126-135 <223> Alpha -synuclein 126-135

<400> 120

<400> 120

<210> 121 <210> 121

<211> 41 <211> 41

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(18) <222> (1) .. (18)

<223> HBsAg3 Th <223> HBsAg3 Th

<220> <220>

<221> SITE <221> SITE

<222> (19)..(19) <222> (19) .. (19)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(41) <222> (20) .. (41)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 121

<400> 121

<210> 122 <210> 122

<211> 40 <211> 40

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(17) <222> (1) .. (17)

<223> 破傷風梭菌(Clostridium tetani)1 Th <223> Clostridium tetani 1 Th

<220> <220>

<221> SITE <221> SITE

<222> (18)..(18) <222> (18) .. (18)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (19)..(40) <222> (19) .. (40)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 122

<400> 122

<210> 123 <210> 123

<211> 38 <211> 38

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(15) <222> (1) .. (15)

<223> MvF1 Th <223> MvF1 Th

<220> <220>

<221> SITE <221> SITE

<222> (16)..(16) <222> (16) .. (16)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (17)..(38) <222> (17) .. (38)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 123

<400> 123

<210> 124 <210> 124

<211> 47 <211> 47

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(24) <222> (1) .. (24)

<223> 百日咳菌(Bordetella pertussis)Th <223> Bordetella pertussis Th

<220> <220>

<221> SITE <221> SITE

<222> (25)..(25) <222> (25) .. (25)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (26)..(47) <222> (26) .. (47)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 124

<400> 124

<210> 125 <210> 125

<211> 40 <211> 40

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(17) <222> (1) .. (17)

<223> 破傷風梭菌(Clostridium tetani)2 Th <223> Clostridium tetani 2 Th

<220> <220>

<221> SITE <221> SITE

<222> (18)..(18) <222> (18) .. (18)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (19)..(40) <222> (19) .. (40)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 125

<400> 125

<210> 126 <210> 126

<211> 46 <211> 46

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(23) <222> (1) .. (23)

<223> 白喉(Diphtheria)Th <223> Diphtheria Th

<220> <220>

<221> SITE <221> SITE

<222> (24)..(24) <222> (24) .. (24)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (25)..(46) <222> (25) .. (46)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 126

<400> 126

<210> 127 <210> 127

<211> 44 <211> 44

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(21) <222> (1) .. (21)

<223> 惡性瘧原蟲(Plasmodium falciparum)Th <223> Plasmodium falciparum Th

<220> <220>

<221> SITE <221> SITE

<222> (22)..(22) <222> (22) .. (22)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (23)..(44) <222> (23) .. (44)

<223> α-突解核蛋白111-132 <223> Alpha -nucleolytic protein 111-132

<400> 127

<400> 127

<210> 128 <210> 128

<211> 40 <211> 40

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(17) <222> (1) .. (17)

<223> 曼森氏住血吸蟲(Schistosoma mansoni)Th <223> Schistosoma mansoni Th

<220> <220>

<221> SITE <221> SITE

<222> (18)..(18) <222> (18) .. (18)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (19)..(40) <222> (19) .. (40)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 128

<400> 128

<210> 129 <210> 129

<211> 48 <211> 48

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(25) <222> (1) .. (25)

<223> 霍亂毒素Th <223> Cholera toxin Th

<220> <220>

<221> SITE <221> SITE

<222> (26)..(26) <222> (26) .. (26)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (27)..(48) <222> (27) .. (48)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 129

<400> 129

<210> 130 <210> 130

<211> 38 <211> 38

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(15) <222> (1) .. (15)

<223> MvF2 Th <223> MvF2 Th

<220> <220>

<221> SITE <221> SITE

<222> (16)..(16) <222> (16) .. (16)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (17)..(38) <222> (17) .. (38)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 130

<400> 130

<210> 131 <210> 131

<211> 45 <211> 45

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(22) <222> (1) .. (22)

<223> KKKMvF3 Th <223> KKKMvF3 Th

<220> <220>

<221> SITE <221> SITE

<222> (7)..(7) <222> (7) .. (7)

<223> S或T <223> S or T

<220> <220>

<221> SITE <221> SITE

<222> (10)..(10) <222> (10) .. (10)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (11)..(11) <222> (11) .. (11)

<223> G或T <223> G or T

<220> <220>

<221> SITE <221> SITE

<222> (15)..(15) <222> (15) .. (15)

<223> H或T <223> H or T

<220> <220>

<221> SITE <221> SITE

<222> (16)..(16) <222> (16) .. (16)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (19)..(19) <222> (19) .. (19)

<223> G或T <223> G or T

<220> <220>

<221> SITE <221> SITE

<222> (23)..(23) <222> (23) .. (23)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (24)..(45) <222> (24) .. (45)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 131

<400> 131

<210> 132 <210> 132

<211> 41 <211> 41

<212> PRT <212> PRT

<213> B型肝炎病毒 <213> Hepatitis B virus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(18) <222> (1) .. (18)

<223> HBsAg 1 Th <223> HBsAg 1 Th

<220> <220>

<221> SITE <221> SITE

<222> (1)..(1) <222> (1) .. (1)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (2)..(2) <222> (2) .. (2)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (3)..(3) <222> (3) .. (3)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (4)..(4) <222> (4) .. (4)

<223> L或I或V或F <223> L or I or V or F

<220> <220>

<221> SITE <221> SITE

<222> (5)..(5) <222> (5) .. (5)

<223> F或K或R <223> F or K or R

<220> <220>

<221> SITE <221> SITE

<222> (6)..(6) <222> (6) .. (6)

<223> L或I或V或F <223> L or I or V or F

<220> <220>

<221> SITE <221> SITE

<222> (7)..(7) <222> (7) .. (7)

<223> L或I或V或F <223> L or I or V or F

<220> <220>

<221> SITE <221> SITE

<222> (9)..(9) <222> (9) .. (9)

<223> K或R <223> K or R

<220> <220>

<221> SITE <221> SITE

<222> (10)..(10) <222> (10) .. (10)

<223> L或I或V或F <223> L or I or V or F

<220> <220>

<221> SITE <221> SITE

<222> (11)..(11) <222> (11) .. (11)

<223> L或I或V或F <223> L or I or V or F

<220> <220>

<221> SITE <221> SITE

<222> (13)..(13) <222> (13) .. (13)

<223> L或I或V或F <223> L or I or V or F

<220> <220>

<221> SITE <221> SITE

<222> (15)..(15) <222> (15) .. (15)

<223> Q或L或I或V或F <223> Q or L or I or V or F

<220> <220>

<221> SITE <221> SITE

<222> (17)..(17) <222> (17) .. (17)

<223> L或I或V或F <223> L or I or V or F

<220> <220>

<221> SITE <221> SITE

<222> (18)..(18) <222> (18) .. (18)

<223> D或R <223> D or R

<220> <220>

<221> SITE <221> SITE

<222> (19)..(19) <222> (19) .. (19)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(41) <222> (20) .. (41)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 132

<400> 132

<210> 133 <210> 133

<211> 41 <211> 41

<212> PRT <212> PRT

<213> B型肝炎病毒 <213> Hepatitis B virus

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(18) <222> (1) .. (18)

<223> HBsAg 2 Th <223> HBsAg 2 Th

<220> <220>

<221> SITE <221> SITE

<222> (4)..(4) <222> (4) .. (4)

<223> I或F <223> I or F

<220> <220>

<221> SITE <221> SITE

<222> (5)..(5) <222> (5) .. (5)

<223> I或F <223> I or F

<220> <220>

<221> SITE <221> SITE

<222> (6)..(6) <222> (6) .. (6)

<223> T或L <223> T or L

<220> <220>

<221> SITE <221> SITE

<222> (7)..(7) <222> (7) .. (7)

<223> I或L <223> I or L

<220> <220>

<221> SITE <221> SITE

<222> (11)..(11) <222> (11) .. (11)

<223> I或L <223> I or L

<220> <220>

<221> SITE <221> SITE

<222> (14)..(14) <222> (14) .. (14)

<223> P或I <223> P or I

<220> <220>

<221> SITE <221> SITE

<222> (15)..(15) <222> (15) .. (15)

<223> Q或T <223> Q or T

<220> <220>

<221> SITE <221> SITE

<222> (16)..(16) <222> (16) .. (16)

<223> S或T <223> S or T

<220> <220>

<221> SITE <221> SITE

<222> (17)..(17) <222> (17) .. (17)

<223> L或I <223> L or I

<220> <220>

<221> SITE <221> SITE

<222> (19)..(19) <222> (19) .. (19)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(41) <222> (20) .. (41)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 133

<400> 133

<210> 134 <210> 134

<211> 34 <211> 34

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(11) <222> (1) .. (11)

<223> 流感病毒(Influenza)MP1_1 Th <223> Influenza MP1_1 Th

<220> <220>

<221> SITE <221> SITE

<222> (12)..(12) <222> (12) .. (12)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (13)..(34) <222> (13) .. (34)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 134

<400> 134

<210> 135 <210> 135

<211> 38 <211> 38

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(15) <222> (1) .. (15)

<223> 流感病毒(Influenza)MP1_2 Th <223> Influenza MP1_2 Th

<220> <220>

<221> SITE <221> SITE

<222> (16)..(16) <222> (16) .. (16)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (17)..(38) <222> (17) .. (38)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 135

<400> 135

<210> 136 <210> 136

<211> 32 <211> 32

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(9) <222> (1) .. (9)

<223> 流感病毒(Influenza)NSP1 Th <223> Influenza NSP1 Th

<220> <220>

<221> SITE <221> SITE

<222> (10)..(10) <222> (10) .. (10)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (11)..(32) <222> (11) .. (32)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 136

<400> 136

<210> 137 <210> 137

<211> 42 <211> 42

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(19) <222> (1) .. (19)

<223> EBV BHRF1 Th <223> EBV BHRF1 Th

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(20) <222> (20) .. (20)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> SITE <221> SITE

<222> (20)..(20) <222> (20) .. (20)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (21)..(42) <222> (21) .. (42)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 137

<400> 137

<210> 138 <210> 138

<211> 38 <211> 38

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(15) <222> (1) .. (15)

<223> 破傷風桿菌(Clostridium tetani)TT1 Th <223> Clostridium tetani TT1 Th

<220> <220>

<221> SITE <221> SITE

<222> (16)..(16) <222> (16) .. (16)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (17)..(38) <222> (17) .. (38)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 138

<400> 138

<210> 139 <210> 139

<211> 43 <211> 43

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(20) <222> (1) .. (20)

<223> EBV EBNA-1 Th <223> EBV EBNA-1 Th

<220> <220>

<221> SITE <221> SITE

<222> (21)..(21) <222> (21) .. (21)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (22)..(43) <222> (22) .. (43)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 139

<400> 139

<210> 140 <210> 140

<211> 44 <211> 44

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(21) <222> (1) .. (21)

<223> 破傷風桿菌(Clostridium tetani)TT2 Th <223> Clostridium tetani TT2 Th

<220> <220>

<221> SITE <221> SITE

<222> (22)..(22) <222> (22) .. (22)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (23)..(44) <222> (23) .. (44)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 140

<400> 140

<210> 141 <210> 141

<211> 39 <211> 39

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(16) <222> (1) .. (16)

<223> 破傷風桿菌(Clostridium tetani)TT3 Th <223> Clostridium tetani TT3 Th

<220> <220>

<221> SITE <221> SITE

<222> (17)..(17) <222> (17) .. (17)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (18)..(39) <222> (18) .. (39)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 141

<400> 141

<210> 142 <210> 142

<211> 39 <211> 39

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(16) <222> (1) .. (16)

<223> 破傷風桿菌(Clostridium tetani)TT4 Th <223> Clostridium tetani TT4 Th

<220> <220>

<221> SITE <221> SITE

<222> (17)..(17) <222> (17) .. (17)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (18)..(39) <222> (18) .. (39)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 142

<400> 142

<210> 143 <210> 143

<211> 41 <211> 41

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(18) <222> (1) .. (18)

<223> EBV CP Th <223> EBV CP Th

<220> <220>

<221> SITE <221> SITE

<222> (19)..(19) <222> (19) .. (19)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (20)..(41) <222> (20) .. (41)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 143

<400> 143

<210> 144 <210> 144

<211> 37 <211> 37

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(14) <222> (1) .. (14)

<223> HCMV IE1 Th <223> HCMV IE1 Th

<220> <220>

<221> SITE <221> SITE

<222> (15)..(15) <222> (15) .. (15)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (16)..(37) <222> (16) .. (37)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 144

<400> 144

<210> 145 <210> 145

<211> 38 <211> 38

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(15) <222> (1) .. (15)

<223> EBV GP340 Th <223> EBV GP340 Th

<220> <220>

<221> SITE <221> SITE

<222> (16)..(16) <222> (16) .. (16)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (17)..(38) <222> (17) .. (38)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 145

<400> 145

<210> 146 <210> 146

<211> 36 <211> 36

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(13) <222> (1) .. (13)

<223> EBV BPLF1 Th <223> EBV BPLF1 Th

<220> <220>

<221> SITE <221> SITE

<222> (14)..(14) <222> (14) .. (14)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (15)..(36) <222> (15) .. (36)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 146

<400> 146

<210> 147 <210> 147

<211> 34 <211> 34

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(11) <222> (1) .. (11)

<223> EBV EBNA-2 Th <223> EBV EBNA-2 Th

<220> <220>

<221> SITE <221> SITE

<222> (12)..(12) <222> (12) .. (12)

<223> ε K為連接子 <223> ε K is a linker

<220> <220>

<221> 胜肽 <221> Peptide

<222> (13)..(34) <222> (13) .. (34)

<223> α-突觸核蛋白111-132 <223> Alpha -synuclein 111-132

<400> 147

<400> 147

<210> 148 <210> 148

<211> 4 <211> 4

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(4) <222> (1) .. (4)

<223> ε K-KKK為連接子 <223> ε K-KKK is the linker

<220> <220>

<221> SITE <221> SITE

<222> (1)..(1) <222> (1) .. (1)

<223> ε-K <223> ε -K

<400> 148

<400> 148

<210> 149 <210> 149

<211> 31 <211> 31

<212> DNA <212> DNA

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 引子_結合位置 <221> Primer_binding position

<222> (1)..(31) <222> (1) .. (31)

<223> α-突觸核蛋白的正向引子序列 <223> Forward primer sequence of α -synuclein

<400> 149

<400> 149

<210> 150 <210> 150

<211> 31 <211> 31

<212> DNA <212> DNA

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 引子_結合位置 <221> Primer_binding position

<222> (1)..(31) <222> (1) .. (31)

<223> α-突觸核蛋白的反向引子 <223> Alpha -synuclein reverse primer

<400> 150

<400> 150

<210> 151 <210> 151

<211> 23 <211> 23

<212> DNA <212> DNA

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 引子_結合位置 <221> Primer_binding position

<222> (1)..(23) <222> (1) .. (23)

<223> 突變α-突觸核蛋白的引子序列 <223> Primer sequence of mutant α -synuclein

<400> 151

<400> 151

<210> 152 <210> 152

<211> 21 <211> 21

<212> DNA <212> DNA

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 引子_結合位置 <221> Primer_binding position

<222> (1)..(21) <222> (1) .. (21)

<223> 突變α-突觸核蛋白的反向引子 <223> Reverse primer for mutant α -synuclein

<400> 152

<400> 152

<210> 153 <210> 153

<211> 32 <211> 32

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> 胜肽 <221> Peptide

<222> (1)..(32) <222> (1) .. (32)

<223> β-突觸核蛋白103-134 <223> β -synuclein 103-134

<400> 153

<400> 153

Claims

一種α-突觸核蛋白(α-Syn)胜肽免疫原結構，包括：一B細胞抗原決定位，包括10至25個源自α-Syn之C-端片段的胺基酸，其對應於SEQ ID NO：1之第111個位置的甘胺酸(G111)至第135個位置的天門冬胺酸(D135)；一T輔助抗原決定位，包括一擇自由SEQ ID NOs：70-98所組成之群組的胺基酸序列；以及一選擇性異源性連接子，其擇自由一胺基酸、Lys-、Gly-、Lys-Lys-Lys-、(α,ε-N)Lys及ε-N-Lys-Lys-Lys-Lys(SEQ ID NO：148)所組成之群組，其中B細胞抗原決定位係直接或透過選擇性異源性連接子共價連接至T輔助抗原決定位。An α-synuclein (α-Syn) peptide immunogen structure, including: a B cell epitope, including 10 to 25 amino acids derived from the C-terminal fragment of α-Syn, which corresponds to SEQ ID NO: 1 Glycine (G111) from position 111 to 135 aspartic acid (D135); one T-helper epitope, including optional free SEQ ID NOs: 70-98 A group of amino acid sequences; and a selective heterologous linker selected from amino acids, Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N) Lys, and A group consisting of ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 148), in which the B cell epitope is covalently linked to the T helper epitope directly or through a selective heterologous linker .

如申請專利範圍第1項所述之α-Syn胜肽免疫原結構，其中該B細胞抗原決定位係擇自由SEQ ID NOs：12-15、17及49-63所組成之群組。The α-Syn peptide immunogen structure according to item 1 of the patent application range, wherein the B cell epitope is selected from the group consisting of SEQ ID NOs: 12-15, 17 and 49-63.

如申請專利範圍第1項所述之α-Syn胜肽免疫原結構，其中該T輔助抗原決定位係擇自由SEQ ID NOs：81、83及84所組成之群組。The α-Syn peptide immunogen structure according to item 1 of the patent application scope, wherein the T helper epitope is selected from the group consisting of SEQ ID NOs: 81, 83, and 84.

如申請專利範圍第1項所述之α-Syn胜肽免疫原結構，其中該選擇性異源性連接子為(α,ε-N)Lys或ε-N-Lys-Lys-Lys-Lys(SEQ ID NO：148)。The α-Syn peptide immunogen structure according to item 1 of the scope of the patent application, wherein the selective heterologous linker is (α, ε-N) Lys or ε-N-Lys-Lys-Lys-Lys ( (SEQ ID NO: 148).

如申請專利範圍第1項所述之α-Syn胜肽免疫原結構，其中該T輔助抗原決定位共價連接至B細胞抗原決定位的N端。The α-Syn peptide immunogen structure according to item 1 of the scope of the patent application, wherein the T helper epitope is covalently linked to the N-terminus of the B cell epitope.

如申請專利範圍第1項所述之α-Syn胜肽免疫原結構，其中該T輔助抗原決定位透過該選擇性異源性連接子共價連接至B細胞抗原決定位的N端。The α-Syn peptide immunogen structure according to item 1 of the patent application scope, wherein the T helper epitope is covalently connected to the N-terminus of the B cell epitope through the selective heterologous linker.

如申請專利範圍第1項所述之α-Syn胜肽免疫原結構，包括下式：(Th)_m-(A)_n-(α-Syn C-端片段)-X或(α-Syn C-端片段)-(A)_n-(Th)_m-X其中Th為該T輔助抗原決定位；A為該異源性連接子；(α-Syn C-端片段)為該B細胞抗原決定位；X為一胺基酸的α-COOH或α-CONH₂；m為1、2、3或4；以及n為1、2、3、4、5、6、7、8、9或10。The α-Syn peptide immunogen structure according to item 1 of the patent application scope, including the following formula: (Th) _m- (A) _n- (α-Syn C-terminal fragment) -X or (α-Syn C -Terminal fragment)-(A) _n- (Th) _m -X where Th is the T helper epitope; A is the heterologous linker; (α-Syn C-terminal fragment) is the B cell epitope Position; X is α-COOH or α-CONH ₂ of an amino acid; m is 1, 2, 3, or 4; and n is 1, ₂ , 3, 4, 5, 6, 7, 8, 9, or 10 .

如申請專利範圍第1項所述之α-Syn胜肽免疫原結構，包括一胺基酸序列，其擇自由SEQ ID NOs：107、108、111-113及115-147所組成之群組。The α-Syn peptide immunogen structure according to item 1 of the patent application scope includes a monoamino acid sequence, which can be selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147.

如申請專利範圍第1項所述之α-Syn胜肽免疫原結構，包括一胺基酸序列，其擇自由SEQ ID NOs：107、108及111-113所組成之群組。The α-Syn peptide immunogen structure described in item 1 of the patent application scope includes a monoamino acid sequence, which can be selected from the group consisting of SEQ ID NOs: 107, 108, and 111-113.

一種組成物，包括申請專利範圍第1項之α-Syn胜肽免疫原。A composition comprising the α-Syn peptide immunogen of the first patent application.

一種組成物，包括一個以上之申請專利範圍第1項的α-Syn胜肽免疫原。A composition comprising more than one α-Syn peptide immunogen in the first patent application.

如申請專利範圍第11項所述之組成物，其中該α-Syn胜肽免疫原結構具有SEQ ID NOs：112及113胺基酸序列。The composition according to item 11 of the scope of patent application, wherein the α-Syn peptide immunogen structure has SEQ ID NOs: 112 and 113 amino acid sequences.

一種醫藥組成物，包括該申請專利範圍第1項之α-Syn胜肽免疫原結構及一藥學上可接受之傳輸載體及/或佐劑。A medicinal composition includes the α-Syn peptide immunogen structure and the pharmaceutically acceptable delivery carrier and / or adjuvant in the first patent scope of the application.

如申請專利範圍第13項所述之醫藥組成物，其中：a.該α-Syn胜肽免疫原結構係擇自由SEQ ID NOs：107、108、111-113、及115-147所組成之群組；以及b.該佐劑為一鋁礦物鹽，其係擇自由Al(OH)₃或AlPO₄所組成之群組。The pharmaceutical composition according to item 13 of the patent application scope, wherein: a. The α-Syn peptide immunogen structure is selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147. group; and b. the mineral adjuvant is an aluminum salt, which lines the desired group consisting of Al (OH) ₃ or AlPO ₄ composed of.

如申請專利範圍第13項所述之醫藥組成物，其中a.該α-Syn胜肽免疫原結構係擇自由SEQ ID NOs：107、108、111-113及115-147所組成之群組；以及b.該α-Syn胜肽免疫原結構與一CpG寡核苷酸(ODN)混合以形成一穩定的免疫刺激複合物。The pharmaceutical composition according to item 13 of the patent application scope, wherein a. The α-Syn peptide immunogen structure is selected from the group consisting of SEQ ID NOs: 107, 108, 111-113, and 115-147; And b. The α-Syn peptide immunogen structure is mixed with a CpG oligonucleotide (ODN) to form a stable immunostimulatory complex.

一種分離抗體或其抗原決定位結合片段，其專一性地與申請專利範圍第1項之α-Syn胜肽免疫原結構的B細胞抗原決定位結合。An isolated antibody or epitope-binding fragment thereof specifically binds to the B-cell epitope of the α-Syn peptide immunogen structure of the first patent application.

如申請專利範圍第16項所述之分離抗體或其抗原決定位結合片段，其與該α-Syn胜肽免疫原結構結合。The isolated antibody or epitope-binding fragment thereof according to item 16 of the scope of the patent application, which binds to the α-Syn peptide immunogen structure.

一種分離抗體或其抗原決定位結合片段，其專一性地與申請專利範圍第9項之α-Syn胜肽免疫原結構之B細胞抗原決定位結合。An isolated antibody or an epitope-binding fragment thereof specifically binds to a B-cell epitope of an α-Syn peptide immunogen structure of the patent application No. 9.

一種組成物，包括申請專利範圍第16項之分離抗體或其抗原決定位結合片段。A composition comprising an isolated antibody or an epitope-binding fragment thereof according to item 16 of the patent application.

一種組成物，包括申請專利範圍第18項之分離抗體或其抗原決定位結合片段。A composition comprising an isolated antibody or an epitope-binding fragment thereof according to item 18 of the patent application.

如申請專利範圍第20項所述之組成物，包括下述之混合物：a.一與SEQ ID NO：112之B細胞抗原決定位專一性結合之分離抗體或其抗原決定位結合片段；以及b.一與SEQ ID NO：113之B細胞抗原決定位專一性結合之分離抗體或其抗原決定位結合片段。The composition according to item 20 of the scope of patent application, including the following mixture: a. An isolated antibody or an epitope-binding fragment thereof specifically binding to the B-cell epitope of SEQ ID NO: 112; and b An isolated antibody or epitope-binding fragment thereof that specifically binds to a B-cell epitope of SEQ ID NO: 113.

一種如申請專利範圍第1項所述之α-Syn胜肽免疫原結構的用途，其係用於製造辨識宿主中之α-Syn之抗體的醫藥組成物，其包括給予該宿主該醫藥組成物，其中該醫藥組成物包括申請專利範圍第1項之α-Syn胜肽免疫原以及一傳輸載體及/或佐劑。An application of the α-Syn peptide immunogen structure according to item 1 of the scope of patent application, which is a pharmaceutical composition for manufacturing an antibody that recognizes α-Syn in a host, and comprises administering the pharmaceutical composition to the host The pharmaceutical composition includes an alpha-Syn peptide immunogen and a delivery vehicle and / or an adjuvant in the first patent application.

一種如申請專利範圍第1項所述之α-Syn胜肽免疫原結構的用途，其係用於製造抑制一動物中α-Syn聚集的醫藥組成物，其包括給予該動物該醫藥組成物，其中該醫藥組成物包括一藥學上有效量之申請專利範圍第1項之α-Syn胜肽免疫原。An application of the α-Syn peptide immunogen structure as described in item 1 of the scope of patent application, which is used for manufacturing a pharmaceutical composition that inhibits the aggregation of α-Syn in an animal, and comprises administering the pharmaceutical composition to the animal The medicinal composition includes a pharmaceutically effective amount of an α-Syn peptide immunogen in the first patent application.

一種如申請專利範圍第1項所述之α-Syn胜肽免疫原結構的用途，其係用於製造降低一動物中α-Syn聚集之量的醫藥組成物，其包括給予該動物該醫藥組成物，其中該醫藥組成物包括一藥學上有效量之申請專利範圍第1項之α-Syn胜肽免疫原。An application of the α-Syn peptide immunogen structure as described in item 1 of the scope of the patent application, which is used for manufacturing a pharmaceutical composition for reducing the amount of α-Syn aggregation in an animal, which comprises administering the pharmaceutical composition to the animal The pharmaceutical composition includes a pharmaceutically effective amount of an α-Syn peptide immunogen in the first patent application.

一種辨識一生物樣本中不同大小之α-Syn聚集的方法，包括：a.將該生物樣本在允許抗體或其抗原決定位結合片段結合α-Syn聚集體的條件下，暴露於該申請專利範圍第16項之抗體或其抗原決定位結合片段；以及b.偵測在該生物樣本中與α-Syn聚集體結合之抗體或其抗原決定位結合片段的量。A method for identifying α-Syn aggregates of different sizes in a biological sample, including: a. Exposing the biological sample to the scope of the patent application under conditions that allow antibodies or epitope-binding fragments thereof to bind α-Syn aggregates The antibody or epitope-binding fragment thereof according to item 16; and b. Detecting the amount of the antibody or epitope-binding fragment that binds to the α-Syn aggregate in the biological sample.