TW201932488A - IgE受體之重組型胞外區域、包含其之藥學組成物以及用於製備其之方法 - Google Patents
IgE受體之重組型胞外區域、包含其之藥學組成物以及用於製備其之方法 Download PDFInfo
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- TW201932488A TW201932488A TW108100730A TW108100730A TW201932488A TW 201932488 A TW201932488 A TW 201932488A TW 108100730 A TW108100730 A TW 108100730A TW 108100730 A TW108100730 A TW 108100730A TW 201932488 A TW201932488 A TW 201932488A
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- glu
- polypeptide dimer
- ige
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- gly
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Abstract
本發明提供一種含有二個單體之多肽二聚蛋白,該單體各含有IgE Fc受體之α次單元的胞外區域(FcεRIa-ECD)。根據本發明之二聚蛋白的優點在於,相較於含有抗IgE抗體之習用治療劑,表現出極佳的IgE結合能力,以及由於無ADCC與CDC之功能,故表現出較少的其它副作用。因此,該二聚蛋白可應用於醫療產品,用於治療或預防IgE介導的過敏性疾病。
Description
發明領域
本發明有關經修飾的IgE Fc受體及其用途。
本發明有關經修飾的IgE Fc受體及其用途。
發明背景
過敏性疾病,如過敏性鼻炎、異位性皮膚炎及食物過敏,包括氣喘,在工業化和西化的現代社會中快速蔓延,且全身性過敏反應(anaphylaxis),一種嚴重的過敏疾病,之發生亦漸增。此等慢性的免疫疾病嚴重地妨害個人的生活品質,且社會經濟成本相應的飆升。因此,迫切需要採取措施克服這些疾病。
過敏性疾病,如過敏性鼻炎、異位性皮膚炎及食物過敏,包括氣喘,在工業化和西化的現代社會中快速蔓延,且全身性過敏反應(anaphylaxis),一種嚴重的過敏疾病,之發生亦漸增。此等慢性的免疫疾病嚴重地妨害個人的生活品質,且社會經濟成本相應的飆升。因此,迫切需要採取措施克服這些疾病。
大部分的過敏性疾病是因免疫球蛋白E (IgE)過度的免疫反應引起。IgE是一種抗體,其在一般情況下以極低的濃度存在於血清中。IgE通常也是由無害的抗原產生。有一種情況是IgE的數目在無任何特定刺激之情況下增加。此種情況可能引起過敏性疾病。數目異常增加的IgE會結合至表現在肥大細胞、嗜鹼性球等等表面上的高親和性IgE Fc受體(FceRIs)。此結合導致肥大細胞或嗜鹼性球釋出化學介導物,如組織胺、白三烯、前列腺素、 緩激肽及血小板活性因子。此等化學介導物之釋出導致過敏症狀。特別是,過敏性疾病可能因IgE與FceRI間之結合而表現出更惡化的症狀。已知FceRI表現細胞在過敏病患中會增加。
目前,已經有多方面用於治療過敏性疾病之方法被提出,例如避開過敏原、投與抗過敏藥物、調整體內IgE的合成及開發抗IgE抗體。然而,目前為止已知的治療方法具有許多的缺點,如無法治療過敏的根本病因、 藥效不足及嚴重副作用的發生。
此外,已研究出能夠以高親和力結合IgE及FceRIIb且抑制表現IgE之細胞的免疫球蛋白組成物(KR10-1783272B1)。此組成物據報導可用於治療IgE介導的病症,包括過敏及氣喘。此外,奧馬珠單抗(omalizumab) (商品名:Xolair),其會標靶IgE抗體之Fc部分,已開發用作為頑固性重症氣喘及頑固性蕁麻疹之治療劑。
然而,投與高劑量的奧馬珠單抗來維持治療效果,會導致高成本負擔以及諸如血管性水腫及全身性過敏反應之副作用(The Journal of Clinical Investigation Volume 99, Number 5, March 1997, 915-925)。除此之外,上市後的結果已經有過敏性肉芽腫血管炎及特發性血小板減少之報告。據此,越來越需要開發能夠有效治療過敏性疾病而沒有副作用的治療劑。
技術問題
本發明之一目的係提供一種用於治療IgE介導的過敏性疾病之多肽二聚蛋白。本發明之另一目的係提供一種編碼該蛋白之核酸分子、一種含有該核酸分子之表現載體及一種含有該表現載體之宿主細胞。本發明之又另一目的係提供一種用於製備該多肽二聚體之方法。
解決問題之方法
本發明之一目的係提供一種用於治療IgE介導的過敏性疾病之多肽二聚蛋白。本發明之另一目的係提供一種編碼該蛋白之核酸分子、一種含有該核酸分子之表現載體及一種含有該表現載體之宿主細胞。本發明之又另一目的係提供一種用於製備該多肽二聚體之方法。
解決問題之方法
為達到上述之目的,提供一種多肽二聚體,其包含二個單體,其各含有IgE Fc受體之a次單元的胞外區域。該單體含有一經修飾的Fc區,且該經修飾的Fc區與該IgE Fc受體之a次單元的胞外區域透過IgD抗體之鉸鏈(hinge)連接。在另一態樣中,提供一種用於治療或預防過敏性疾病之藥學組成物,其包含該多肽二聚體作為活性成份。
本發明之有利效果
本發明之有利效果
相較於習用的抗IgE抗體,根據本發明之多肽二聚蛋白不僅在體內具有極佳的安全性及持續性,且由於具有IgE結合能力比習用抗IgE抗體,奧馬珠單抗,大70倍,所以會與IgE強力結合,此容許延長投藥周期。此外,根據本發明之多肽二聚蛋白是一種通過採用經修飾的Fc獲得之物質,其僅以IgE作為單一標靶,而不會結合至Fc g受體,因此沒有抗體依賴性細胞毒性(ADCC)及補體依賴性細胞毒性(CDC)之功能。因此,不同於含有IgG1 Fc區之習用抗IgE抗體,該多肽二聚蛋白不會結合至Fcg受體,因此可抑制因結合至肥大細胞表面上之Fcg受體而引起之介導物的釋出,如此可使諸如因IgG1與肥大細胞上之Fcg受體III間之結合而導致全身性過敏反應之嚴重副作用的發生減低至最小限度。因此,根據本發明之多肽二聚蛋白可用作為取代含有習用抗IgE抗體之治療劑之新的藥學組成物。
本發明之詳細說明
本發明有關一種多肽二聚體,其包含二個單體,其各含有IgE Fc受體之a次單元的胞外區域(FceRIa-ECD),其中該單體含有一經修飾的Fc區,且該經修飾的Fc區與該FceRIa-ECD透過IgD抗體之鉸鏈連接。
本發明有關一種多肽二聚體,其包含二個單體,其各含有IgE Fc受體之a次單元的胞外區域(FceRIa-ECD),其中該單體含有一經修飾的Fc區,且該經修飾的Fc區與該FceRIa-ECD透過IgD抗體之鉸鏈連接。
本文中所使用之術語"IgE"意指稱作免疫球蛋白E之抗體蛋白。IgE對肥大細胞、血液嗜鹼性球等具親和力。此外,IgE抗體與其對應的抗原(過敏原)間之反應會引起發炎反應。此外,已知IgE是一種會因肥大細胞或嗜鹼性球發生突然的分泌而引起全身性過敏反應之抗體。
本文中所使用之術語"IgE Fc受體"亦稱作Fce受體,會結合IgE之Fc部分。受體有二種類型。具高IgE Fc親和力之受體稱作Fce受體I (FceRI)。具低IgE Fc親和力之受體稱作Fce受體II (FceRII)。FceRI在肥大細胞及嗜鹼性球中表現。在IgE抗體與FceRI之結合是經由多價抗原交聯之情況下,肥大細胞或嗜鹼性球會發生去顆粒反應,從而釋出各種化學傳遞物質,包括組織胺。此釋出會引起立即的過敏反應。
FceRI是一種由一個a鏈、一個b鏈及二個以雙硫鍵連接之g鏈構成的膜蛋白。此等鏈中,IgE結合的部分是a鏈(FceRIa)。FceRIa約60kDa大,且由存在於細胞膜內側之疏水性區域及存在於細胞膜外側之親水性區域構成。特別是,IgE會結合至a鏈的胞外區域。
具體地,IgE Fc受體之a次單元可具有NP_001992.1中所述之胺基酸序列。此外,IgE Fc受體之a次單元的胞外區域(FceRIa-ECD)可具有序列辨識編號1之胺基酸序列。在本說明書中,該IgE Fc受體之a次單元的胞外區域可為IgE Fc受體之a次單元的胞外區域的片段或變體,只要該片段或變體能夠結合IgE即可。
該變體可透過於野生型FceIa-ECD (胞外區域)中取代、刪除或加入一或多種蛋白製得,只要該方法不會改變FceRI之a鏈的功能即可。此各種蛋白或胜肽可與序列辨識編號1之胺基酸序列具有90%、91%、92%、93%、94%、95%、96%、97%、98%、99%或更高的一致性。此外,序列辨識編號1之FceRIa-ECD可由具有序列辨識編號5之多核苷酸編碼。
此外,本文中所使用之術語"經修飾的Fc區"意指其中抗體之Fc部分的一部分經過修飾之區。在此,術語"Fc區"意指含有免疫球蛋白之重鏈恆定區2 (CH2)及重鏈恆定區3 (CH3),但不含免疫球蛋白之重鏈與輕鏈之可變區與輕鏈恆定區1 (CH1)之部分。特別是,該經修飾的Fc區意指通過取代Fc區中之一些胺基酸或通過結合不同類型的Fc區而獲得之區。具體地,該經修飾的Fc區可具有序列辨識編號2之胺基酸序列。此外,具有序列辨識編號2之經修飾的Fc區,可由具有序列辨識編號6之序列的多核苷酸編碼。
此外,本發明之"經修飾的Fc區"可為具有天然形式的糖鏈、相對於天然形式糖鏈增加或相對於天然形式糖鏈減少之形式,或可為去除糖鏈之形式。免疫球蛋白Fc糖鏈可通過習用方法,如化學方法、酵素方法及使用微生物之基因工程方法修飾。
在此,本發明之"經修飾的Fc區"由於沒有FcgR或C1q之結合位置,可為無抗體依賴性細胞毒性(ADCC)及補體依賴性細胞毒性(CDC)功能之區。此外,該經修飾的Fc區及該FceRIa-ECD可透過IgD抗體之鉸鏈連接。IgD抗體之鉸鏈由64個胺基酸構成,且可選擇性地含有20至60個連續胺基酸、25至50個連續胺基酸或30至40個胺基酸。在一實施例中,IgD抗體之鉸鏈可由30或49個胺基酸構成,如下文所述。此外,該IgD抗體之鉸鏈可為通過修飾該鉸鏈區而獲得之變體,其中該鉸鏈可含有至少一個半胱胺酸。在此,為了使蛋白生產過程期間截短型的產生最小化,該鉸鏈變體可通過修飾IgD抗體中之一些鉸鏈序列獲得。
在一實施例中,該鉸鏈可含有下列序列:
Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa1 Xaa2 Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro (序列辨識編號17),其中Xaa1可為Lys或Gly,而Xaa2可為Glu、Gly或Ser。具體地,該鉸鏈可具有序列辨識編號3或序列辨識編號19之胺基酸序列,從而使蛋白生產過程期間截短型的產生最小化。
Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa1 Xaa2 Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro (序列辨識編號17),其中Xaa1可為Lys或Gly,而Xaa2可為Glu、Gly或Ser。具體地,該鉸鏈可具有序列辨識編號3或序列辨識編號19之胺基酸序列,從而使蛋白生產過程期間截短型的產生最小化。
在另一實施例中,該鉸鏈可含有下列序列:
Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa3 Xaa4 Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro (序列辨識編號18),其中Xaa3可為Lys或Gly,而Xaa4可為Glu、Gly或Ser。具體地,該鉸鏈可具有序列辨識編號4之胺基酸序列,從而使蛋白生產過程期間截短型的產生最小化。
Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa3 Xaa4 Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro (序列辨識編號18),其中Xaa3可為Lys或Gly,而Xaa4可為Glu、Gly或Ser。具體地,該鉸鏈可具有序列辨識編號4之胺基酸序列,從而使蛋白生產過程期間截短型的產生最小化。
特別是,在具有序列辨識編號4之鉸鏈中,至少一個Thr可為糖基化的。具體地,在序列辨識編號18之胺基酸中,第13個、第14個、第18個或第19個Thr可為糖基化的。較佳地,全部四個胺基酸均為糖基化的。在此,糖基化可為O糖基化的。
此外,如上所述,本發明所提供之多肽二聚體可為其中二個單體彼此結合且各單體可通過IgE Fc受體之a次單元之胞外區域與經修飾的Fc區之間的結合而獲得之形式。該多肽二聚體可為其中相同的二個單體彼此通過位在鉸鏈處之半胱胺酸結合之形式。此外,該多肽二聚體可為其中二個不同的單體彼此結合之形式。例如,在該二個單體彼此不同之情況下,該多肽二聚體可為其中一個單體含有IgE Fc受體之a次單元的胞外區域,而另一個含有IgE Fc受體之a次單元的胞外區域之片段之形式。在此,該單體之實施例可具有序列辨識編號20、序列辨識編號21或序列辨識編號22之胺基酸序列。
此外,本發明所提供之多肽二聚體表現出比奧馬珠單抗,一種抗IgE抗體,高10至100倍、20至90倍、20至70倍、30至70倍或40至70倍之IgE結合能力,較佳地表現出比奧馬珠單抗高70倍之IgE結合能力。
在本發明之又另一態樣方面,提供一種編碼一單體之多核苷酸,其含有與經修飾的Fc區結合之IgE Fc受體的a次單元的胞外區域。
同時,該多核苷酸可額外地含有一訊息序列或一前導序列。本文中所使用之術語"訊息序列"意指編碼能指導目標蛋白的分泌之訊息肽的核苷酸。該訊息肽在宿主細胞中轉譯,然後切割。具體地,本發明之訊息序列是編碼能起動橫跨內質網(ER)膜之蛋白轉位的胺基酸序列之核苷酸。本發明之有用的訊息序列包括抗體輕鏈訊息序列,例如,抗體14.18 (Gillies et al., J. Immunol. Meth 1989. 125:191-202)、抗體重鏈訊息序列,例如MOPC141抗體重鏈訊息序列(Sakano et al., Nature, 1980. 286:676-683)及其它此技術領域中已知的訊息序列(見,列如,Watson et al., Nucleic Acid Research, 1984. 12:5145-5164)。
訊息序列之特徵是此技術領域熟知的。訊息序列典型地含有16至30個胺基酸殘基,且可含更多或更少的胺基酸殘基。典型的訊息肽由三個區構成,其等為鹼性N端區、中央疏水區及極性較高的C端區。中央疏水區含有4至12個疏水性殘基,其在未成熟的多肽轉位期間固定訊息序列通過膜脂質雙層。
起動之後,訊息序列會在內質網內腔中被稱作訊息肽酶之細胞酵素切斷。在此,訊息序列可為組織纖維蛋白溶解酶原活化因子(tPA)、單純皰疹病毒糖蛋白D (HSV gD)或生長激素之分泌訊息序列。較佳地,可使用高等真核細胞,包括哺乳動物等等使用之分泌訊息序列。此外,可以用在宿主細胞中具有高表現頻率之密碼子取代該分泌訊息序列,並使用之。
同時,訊息序列及經修飾的Fc區結合的IgE Fc受體之a次單元的胞外區域單體,可具有序列辨識編號11或序列辨識編號13之胺基酸序列。序列辨識編號11及序列辨識編號13之蛋白,可分別由具有序列辨識編號12及序列辨識編號14之序列之多核苷酸編碼。
在本發明之又另一態樣方面,提供一種加載編碼該單體之多核苷酸之表現載體。在此,該多核苷酸可具有序列辨識編號12或序列辨識編號14之序列。
本文中所使用之術語"載體",旨在被引入宿主細胞中且能夠與宿主細胞基因組重組並插入其中。選擇性地,載體是一種游離基因組,且被理解為含有可自動複製之核苷酸序列的核酸單元。載體包括線性核酸、質體、噬菌粒(phagemids)、黏接質體、RNA載體、病毒載體及其類似物。病毒載體之例子包括,但不限於,逆轉錄病毒、腺病毒及腺相關病毒。此外,質體可含一選擇性標記,如抗生素抗性基因,因此可在選擇性條件下培養攜帶該質體之宿主細胞。
本文中所使用之術語目標蛋白之"基因表現"或"表現",應理解為意指DNA序列之轉錄、mRNA轉錄本之轉譯及融合蛋白產物或其片段之分泌。可用的表現載體可為RcCMV (Invitrogen, Carlsbad)或其變體。表現載體可含有用於促進哺乳動物細胞中之目標基因的連續轉錄之人類巨大細胞病毒(CMV)啟動子,及用於增加轉錄後RNA之安定性位準之牛生長激素多腺苷酸化訊息序列。
在本發明之又另一態樣方面,提供一種其中引入了表現載體之宿主細胞。本文中所使用之術語"宿主細胞"意指可於其中引入重組表現載體之原核或真核細胞。本文中所使用之術語"轉導"、"轉型"及"轉染",意指使用此技術領域中已知的技術,將核酸(例如,載體)引入細胞中。
可用於本發明之較佳的宿主細胞包括永生融合瘤細胞、NS/0骨髓瘤細胞、293細胞、中國倉鼠卵巢細胞(CHO細胞)、HeLa細胞、人羊水來源細胞(CapT細胞)或COS細胞。較佳地,該宿主細胞可為CHO細胞。另一方面,該宿主細胞可為一種其中引入了該載體及加載唾液酸轉移酶基因之載體之宿主細胞。在此,該唾液酸轉移酶可為2,3-唾液酸轉移酶或2,6-唾液酸轉移酶。在此,該2,6-唾液酸轉移酶可具有序列辨識編號15之胺基酸序列。
在本發明之又另一態樣方面,提供一種用於治療或預防過敏性疾病之藥學組成物,其包含該多肽二聚體作為活性成份。
在本說明書中,術語"過敏性疾病"意指由肥大細胞活化作用,如肥大細胞去顆粒反應介導之過敏反應引起的病理症狀。此過敏性疾病包括食物過敏、異位性皮膚炎、氣喘、過敏性鼻炎、過敏性結膜炎、過敏性皮膚炎、過敏性接觸性皮膚炎、全身性過敏反應、蕁麻疹、瘙癢症、昆蟲過敏、慢性自發性蕁麻疹、藥物過敏等等。特別是,該過敏性疾病可為IgE介導的。
在本發明之用於治療或預防過敏性疾病之組成物中,可視用途、配方、摻合目的等等而包含任何數量的活性成份(有效量),只要該活性成份可表現出抗過敏活性即可。以該組成物之重量計,該活性成份一般的有效量測定在0.001重量%至20.0重量%之範圍內。在此,"有效量"意指活性成份能夠誘導抗過敏作用之數量。此一有效量可由熟悉此技術領域之人士實驗決定。
在此,該藥學組成物可進一步含有一藥學上可接受之載劑。對於該藥學上可接受之載劑,可使用任何載劑,只要該載劑為適合遞送給病患之無毒物質即可。可包含蒸餾水、酒精、脂肪、蠟及惰性固體作為載劑。在該藥學組成物中亦可包含藥學上可接受之佐劑(緩衝劑及分散劑)。
具體地,除了活性成份外,本發明之藥學組成物亦含有一藥學上可接受之載劑,且可視投藥途徑,以此技術領域中已知之慣用方法製成口服或非口服配方。在此,術語"藥學上可接受"意指具有不超過施用(處方)的受試者可接受之毒性且不會抑制活性成份之活性。
在本發明之藥學組成物是製成口服配方之情況,可依照此技術領域中已知之方法,將該藥學組成物與適合的載劑配在一起製成如粉劑、顆粒、錠劑、丸劑、糖衣錠、膠囊、液體、凝膠、糖漿、懸浮液及薄片之配方。在此,適合的藥學上可接受之載劑的例子可包括糖類,如乳糖、葡萄糖、蔗糖、右旋糖、山梨糖醇、甘露糖及木糖醇;澱粉類,如玉米澱粉、馬鈴薯澱粉及小麥澱粉;纖維素類,如纖維素、甲基纖維素、乙基纖維素、羧甲基纖維素鈉及羥丙基甲基纖維素;聚乙烯吡咯烷酮、水、羥基苯甲酸甲酯、羥基苯甲酸丙酯、硬脂酸鎂、礦物油、麥芽、明膠、滑石、多元醇、植物油等等。在製成製劑之情況,必要時,該製劑可透過包括稀釋劑和/或賦形劑,如填料、增量劑、粘合劑、潤濕劑、崩解劑及界面活性劑進行。
在本發明之藥學組成物是製成非口服配方之情況,可依照此技術領域已知之方法,將該藥學組成物與適合的載劑一起製成注射劑、穿皮藥物、鼻吸入劑及栓劑形式之製劑。在製成注射劑之情況,可使用無菌水、乙醇、多元醇,如甘油及丙二醇,或其混合物作為適合的載劑。至於該載劑,較佳地可使用等張溶液,如林格氏溶液、含三乙醇胺之磷酸鹽緩衝食鹽水(PBS)、注射用無菌水及5%右旋糖等等。
該藥學組成物之製備是此技術領域已知的,具體地可參考Remington's Pharmaceutical Sciences (第19版,1995)等等。該文獻視為本發明之一部分。
本發明之藥學組成物的較佳每日劑量範圍從0.01ug/kg至10g/kg,較佳地從0.01mg/kg至1g/kg,取決於患者的病況、體重、性別、年齡、疾病嚴重性或投藥途徑。一天可投藥一次或數次。此一劑量決不應被解釋為本發明之範圍的限制。
本發明之組成物可施用(處方)之受試者為哺乳動物及人,人是最適合的。除了該活性成份外,本發明之用於抗過敏的組成物可進一步包含安全性已經過認證且已知具有抗過敏活性之任何的化合物或天然萃出物,目的用於提升及強化抗過敏活性。
在本發明之另一態樣方面,提供一種用於改善及緩解過敏症狀之食品組成物,其包含該多肽二聚體作為活性成份。
在此,該多肽二聚體可結合至適當的遞送單元,供有效的遞送至腸子。本發明之食品組成物可製成任何形式,例如可製成飲料形式,如茶、果汁、碳酸飲料及離子飲料、加工乳製品,如牛奶及優格、保健功能性食品製劑,如錠劑、膠囊、丸劑、顆粒、液體、粉末、薄片、糊劑、糖漿、凝膠、膠凍及棒等等。此外,本發明之食品組成物可歸在任何合法的產品類別或功能分類,只要該食品組成物在製造及分銷時符合執法規定即可。例如,該食品組成物可為根據保健功能食品法之保健功能性食品,或屬於根據食品衛生法之食品法典(食品藥品管理局通報的食品標準和規範)中的每種食品類型之糖果甜點、豆類、茶、飲料、特殊用途食品等。至於其它可包含在本發明之食品組成物中之食品添加物,可參考根據食品衛生法之食品法典或食品添加物法典。
在本發明之又另一態樣方面,提供一種用於產生多肽二聚體之方法,其包含培養其中引入了編碼一單體及一唾液酸轉移酶基因之多核苷酸之宿主細胞的步驟;及回收多肽二聚體之步驟。
在此,編碼該單體之多核苷酸可以加載在一表現載體上之形式引入該宿主細胞中。此外,該唾液酸轉移酶基因可為加載在一載體上之形式引入該宿主細胞中。
首先,進行將加載編碼一單體之多核苷酸之載體及加載一唾液酸轉移酶基因之載體引入一宿主細胞中之步驟。在此,該唾液酸轉移酶可為2,3-唾液酸轉移酶或2,6-唾液酸轉移酶。
其次,進行培養該轉形細胞之步驟。
最後,進行回收多肽二聚體之步驟。在此,可從培養基或細胞萃出物中純化出該多肽二聚體。例如,在獲得該多肽二聚體分泌於其中之培養基的上清液後,使用市售蛋白濃縮濾器,如Amicon或Millipore Pellicon超過濾單元,濃縮該上清液。之後,使用此技術領域中已知之方法純化該濃縮液。例如,該純化可使用偶合至蛋白A之基質達成。
在本發明之又另一態樣方面,提供一種由上文所述用於產生二聚體之方法產生的多肽二聚體。
在此,該多肽二聚體具有高唾液酸含量,因此相對於理論pI值,具有非常高的酸性蛋白含量。
在本發明之又另一態樣方面,提供一種用於治療或預防過敏性疾病之藥學組成物,其包含由上文所述用於產生二聚體之方法產生的多肽二聚體作為活性成份。
在本發明之又另一態樣方面,提供一種改善或緩解過敏症狀之食品組成物,其包含由上文所述用於產生二聚體之方法產生的多肽二聚體作為活性成份。
在本發明之又另一態樣方面,提供一種用於治療或預防過敏性疾病之方法,其包含對一受試者投與含有二個單體之多肽二聚體之步驟,各單體含有IgE Fc受體之a次單元的胞外區域(FceRIa-ECD)。
該受試者可為哺乳動物,較佳地人。在此,投藥可以口服或非口服方式達成。在此,非口服投與可通過如皮下投與、靜脈投與、黏膜投與及肌肉投與之方法進行。
於下文中,將參考下列範例詳細說明本發明。然而,下列範例僅旨在說明本發明,本發明之範疇不是只受其限制。
範例1 ,含Fc eRI a-ECD 及經修飾的Fc 區之多肽的製備
範例1 ,含Fc eRI a-ECD 及經修飾的Fc 區之多肽的製備
IgE Fc受體之a次單元的胞外區域(FceRIa-ECD)之C端經修飾的多肽係依照美國專利案第7,867,491號中所揭示之方法製備。
首先,為了表現蛋白(FceRIaECD-Fc1)、蛋白(FceRIaECD-Fc2)及蛋白(FceR1aECD-Fc3),其中具有序列辨識編號1之胺基酸序列之FceRI的a鏈之胞外區域及序列辨識編號2之經修飾的免疫球蛋白Fc係分別透過序列辨識編號19之鉸鏈、序列辨識編號3之鉸鏈及序列辨識編號4之絞鏈連接,將連接編碼各蛋白之基因所獲得的盒選殖進入pAD15載體(Genexin, Inc.)中,建構FceRIaECD-Fc蛋白表現載體。之後,將各表現載體轉染進入CHO DG44細胞(來自Dr. Chasin, Columbia University, USA)中。
在此,在轉染進入細胞株之時,同時將通過選殖a-2,6-涶液酸轉移酶基因進入pCI Hygro載體(Invitrogen)所獲得之表現載體,轉染進入分開準備之能夠表現FceRIaECD-Fc2ST及FceRIaECD-Fc3ST蛋白(其中加入涶液酸)的細胞株中。
使用5-羥色胺(HT)-無10% dFBS培養基(Gibco, USA, 30067-334)、MEMa培養基(Gibco, 12561, USA, Cat No. 12561-049)及HT+培養基(Gibco, USA, 11067-030)進行HT選擇,作為主要篩選程序。之後,使用HT選擇的選殖株進行胺甲喋呤(MTX)擴增,以便放大使用二氫葉酸還原酶(DHFR)系統之產率。
在MTX擴增完成後,進行繼代培養約1至5次 以穩定細胞,供產率之評估。之後,進行MTX擴增的細胞之單位產率的評估。結果顯示於下表1中。
[表1]
[表1]
如表1所示,FceRIaECD-Fc3細胞株在2uM胺甲喋呤擴增後,表現出16.9ug/106
個細胞之產率。另一方面,與2,6-涶液酸轉移酶共轉染之FceRIaECD-Fc3細胞株(FceRIaECD-Fc3ST),在1uM胺甲喋呤擴增後,表現出17.5ug/106
個細胞之產率。此外,FceRIaECD-Fc2細胞株在0.5uM胺甲喋呤擴增下表現出20.9ug/106
個細胞之產率。此外,與2,6-涶液酸轉移酶共轉染之FceRIaECD-Fc2細胞株(FceRIaECD-Fc2ST),在1uM胺甲喋呤擴增後,表現出25.1ug/106
個細胞之產率。即,確定了與2,6-涶液酸轉移酶共轉染之FceRIaECD-Fc2細胞株,其已經過在1uM胺甲喋呤擴增條件下之選擇,表現出最好的產率。
範例2 ,Fc eRI aECD 融合蛋白之純化及其純度之鑑定
範例2 ,Fc eRI aECD 融合蛋白之純化及其純度之鑑定
針對以上範例1中選擇的細胞株,利用批次培養方法,以60ml的規模培養i) FceRIaECD-Fc3、ii) FceRIaECD-Fc3ST及iii) FceRIaECD-Fc2ST。使用蛋白A親和性管柱純化所產生的培養物,然後對純化的蛋白進行SDS-PAGE及粒徑篩析HPLC (SE-HPLC)之處理,以鑑定蛋白之純度。
如圖1所示,所有各別通過SE-HPLC方法純化之蛋白經鑑定均具有93%或更高之純度。此外,根據SDS-PAGE分析之結果,確定檢測到具有約150kDa及約75kDa大小之蛋白(圖1,第1至6道)。從這裡發現,Fc結合FceRIaECD形成二聚體。此外,在SDS-PAGE結果中沒有觀察到諸如截短型的雜質。特別是,即使在解凍/冷凍之製程後(圖1,第7至8道),全部的蛋白經鑑定均具有93%或更高的純度且沒有雜質。從這裡發現,相對於使用野生型IgD鉸鏈之FceRIaECD-Fc1,截短蛋白型之產量減少。
在此,在下列測試條件下進行Gel-IEF,以鑑定在引入涶液酸轉移酶後的蛋白中涶液酸含量之程度。從這裡確定了酸性蛋白之含量由於涶液酸含量之增加而增加。
[表2]
[表2]
為了確定純化產率之再現性,將FceRIaECD-Fc2ST細胞株以250ml之規模批次培養於1L燒瓶中,然後使用蛋白A親和性管柱純化。之後,使培養上清液及純化的產物在4%至15% TGXTM
凝膠(Bio-Rad Laboratories, Inc.)上,於Tris-甘胺酸SDS (TGS)緩衝液及200V條件下電泳30分鐘,然後進行SDS PAGE分析之處理。結果確定了不僅純化出具有非常高純度(98%或更高)的蛋白(即使僅通過第一步驟之純化),且在培養上清液中亦表現出非常高純度的蛋白。此指出,在開發已經於所討論的細胞系中表現之FceRIaECD-Fc蛋白成為醫療產品時,可簡化製程開發步驟,因此,極有可能顯著降低醫療產品的開發成本。
範例3 ,Fc eRI aECD 融合蛋白之IgE 結合能力的鑑定
範例3 ,Fc eRI aECD 融合蛋白之IgE 結合能力的鑑定
比較測量下列四種蛋白及市售抗IgE抗體,奧馬珠單抗(商品名:Xolair)之IgE結合能力:i) FceRIaECD-Fc2、ii) FceRIaECD-Fc2ST、iii) FceRIaECD-Fc3及iv) FceRIaECD-Fc3ST,其等已經過以上範例2中之方法純化。具體地,將IgE塗佈在蛋白GLC感測晶片(Bio-Rad Laboratories, Inc., Cat # 176-5011)之管道上,然後使奧馬珠單抗或不同濃度之各個FceR1aECD-Fc蛋白以30μl/分之速率流過以測量IgE之結合能力。
實驗之進行是使用25mM NaOH作為再生緩衝液,確定零基線,然後重複以上步驟。之後,使用蛋白結合分析器(ProteOn XPR36, Bio-Rad Laboratories, Inc., USA)確定結合曲線。結果示於表3及圖3與4中。
[表3]
[表3]
如表3所示,根據本發明之一實施例之多肽二聚體測得之結合速率(ka)值比奧馬珠單抗低1.5至2.0倍。即,發現其對IgE以外的物質之結合能力比奧馬珠單抗低1.5至2.0倍。此外,根據本發明之一實施例之多肽二聚體測得之解離速率(kd)值比奧馬珠單抗高40至106倍。此外,如圖3及4所示,能夠確定奧馬珠單抗在結合後經過一段時間之情況下會喪失其對IgE之結合,然而本發明之FceRIaECD融合蛋白之多肽二聚體一旦結合至IgE,該多肽二聚體不會與IgE分開。
即,可看出本發明之多肽二聚體不容易和IgE分開,且維持其結合狀態的能力比奥馬珠單抗好很多。結果可看出根據本發明之一實施例之多肽二聚體具有比奥馬珠單抗高22至69倍之平衡解離常數(KD <kd/ka>)值。從這裡可確定,相較於奥馬珠單抗,本發明之FceRIaECD融合蛋白之IgE結合能力顯著的增加。特別是可確定其中添加涶液酸之FceRIaECD-Fc2 (FceRIaECD-Fc2ST)表現出最高的IgE結合能力,其比奥馬珠單抗高69倍。
範例4 ,Fc eRI aECD 融合蛋白之IgG 受體結合能力的鑑定
範例4 ,Fc eRI aECD 融合蛋白之IgG 受體結合能力的鑑定
使用Octet RED384系統(ForteBio, CA, USA)鑑定IgETRAP
及奥馬珠單抗之IgG受體結合程度。將FcgRI、FcgRIIA、FcgRIIB、FcgRIIIA及FcgRIIIB重組蛋白(R & D Systems Inc., 5 μg/ml)固定在300mM醋酸鹽緩衝液(pH5)下之激活AR2G生物感測器上。使用含有0.1% Tween-20及1%牛血清之PBS作為電泳緩衝液。所有的實驗在30℃下,以1,000rpm速率之樣本盤振盪器進行。結果示於圖5A至5E中,定量奥馬珠單抗及IgETRAP
之IgG受體結合能力,並示於圖6中。
範例5 ,透過 b- 己糖胺酶分析法,鑑定Fc eRI aECD 融合蛋白在小鼠骨髓來源肥大細胞中之活性
範例5 ,透過 b- 己糖胺酶分析法,鑑定Fc eRI aECD 融合蛋白在小鼠骨髓來源肥大細胞中之活性
進行b-己糖胺酶分析來分析本發明之FceRIaECD融合蛋白的體外活性。具體地,將各個濃度之根據本發明之一實施例之FceRIaECD-Fc2融合蛋白與小鼠IgE (1ug/mL)混合,於室溫下(20℃)培育30分鐘,製備樣本。用Hank平衡鹽溶液(HBSS)緩衝液清洗培養供肥大細胞活化之小鼠骨髓來源肥大細胞,除去培養基,然後測量細胞數目。之後,調整細胞數目,以便以5×105
個細胞之數目注入40μL之HBSS緩衝液中。
之後,將50uL通過預培育製得的樣本溶液加至活化的肥大細胞中。然後,令產物在5% CO2
、37℃培育箱中培育30分鐘。各添加10μL外來抗原DNP (2,4-二硝基苯,100ng/mL)後,再次於5% CO2
、37℃下培育30分鐘,然後分開30μL的上清液。使30uL分開的上清液與30uL基質(4-硝基苯N-乙醯基-β-D-葡萄糖苷,5.84mM)充分混合, 然後於5% CO2
、37℃下培育20分鐘。之後,添加140μL作為終止溶液之0.1M碳酸鈉緩衝液(pH10),終止該反應。之後,測量在405nm下之吸收度,以鑑定活化的肥大細胞中因外來抗原而分泌的β-己糖胺酶之分泌量。結果示於圖7中。
如圖7所示,本發明之一實施例之多肽二聚體在一半濃度的小鼠IgE (0.5ug/mL)之情況下表現出約49.4%的肥大細胞抑制率,及在具有相同濃度的小鼠IgE (1ug/mL)之情況下表現出約99.4%的肥大細胞抑制率。即,可看出IgE誘導骨髓來源肥大細胞之活性大幅地受到本發明之FceRIa-ECD多肽二聚體的抑制。
範例6 ,使用β- 己糖胺酶分析法,比較Fc eRI aECD 融合蛋白與抗人IgE 抗體在Fc eRI 骨髓來源肥大細胞中之活性
範例6 ,使用β- 己糖胺酶分析法,比較Fc eRI aECD 融合蛋白與抗人IgE 抗體在Fc eRI 骨髓來源肥大細胞中之活性
進行β-己糖胺酶分析法,透過體外活性分析確定FceRIaECD融合蛋白相對於Xolair之優勢。製備各別藥物,FceRIaECD-Fc2ST (IgETRAP)及Xolair之各個濃度,然後與人IgE (1ug/mL)混合。之後,在室溫下培育30分鐘。在藥物之預培育期間,引入人FceRI基因及製備從小鼠骨髓衍生及分化而來之肥大細胞(其中已移除小鼠FceRI基因)。用HBSS緩衝液清洗製得的肥大細胞,然後將5×105
個細胞注入60μL之HBSS緩衝液中。於製得的肥大細胞中添加20μL之預培育的樣本,然後在5% CO2
、37℃培育箱中培育30分鐘。
隨後,在添加20uL之抗人IgE抗體(BioLegend, Cat No. 325502, 0.5 ug/mL)後,再次於5% CO2
、37℃培育箱中培育產物30分鐘。之後,在1,500rpm、4℃下離心後,分開30uL之上清液。使30uL分開的上清液與30uL基質(4-硝基苯N-乙醯基-β-葡萄糖苷,5.84mM)充分混合,然後於5% CO2
、37℃培育箱中培育25分鐘。之後,添加140uL之0.1M碳酸鈉緩衝液(pH 10)結束反應。
之後,測量在405nm下之吸收度,比較分泌的β-己糖胺酶之相對量,並確定依各藥物濃度之肥大細胞的抑制作用。結果示於圖8中。如圖8所示,FceRIaECD融合蛋白之IC50
經測量大約為11.16ng/mL,而Xolair蛋白之IC50
經測量大約為649.8ng/mL。因此,確定了FceRIaECD融合蛋白在肥大細胞活性上之抑制能力比Xolair大58倍。
範例7 ,Fc eRI aECD 融合蛋白之活體內分析( 食物過敏模型)
範例7 ,Fc eRI aECD 融合蛋白之活體內分析( 食物過敏模型)
以14天的間隔,經腹腔對Balb/c小鼠(Orientbio Inc.)投與50ug之卵白蛋白(OVA)及1mg之氫氧化鋁二次,用以誘發致敏。之後,在第28、30、32、34及36天經口投與50mg的OVA五次,用以誘發腸道食物過敏。
在經口投與OVA二次後,即在第31天,將小鼠分成三群,各有7隻小鼠。被分開的三群如下:第一群接受高濃度(200ug)之FceRIaECD-Fc2ST融合蛋白,第二群接受低濃度(20ug)之FceRIaECD-Fc2ST融合蛋白,而第三群沒有接受任何東西。在經口投與OVA的同時,鑑定是否因食物過敏誘導而發生腹瀉。在第37天犧牲小鼠,分析各群之小鼠的小腸中肥大細胞的數目、血液中IgE濃度及血液中肥大細胞去顆粒產生的酵素(肥大細胞蛋白酶-1 (MCPT-1))之濃度。
如圖9所示,相較於屬於沒有接受任何東西之小鼠群,確定了接受高濃度FceRIaECD-Fc2ST (其是多肽二聚體)之小鼠群,表現出呈濃度依賴性方式緩和食物過敏。
序列辨識編號1
VPQKPKVSLN PPWNRIFKGE NVTLTCNGNN FEVSSTKWF HNGSLSEETN SSLNIVNAKFEDSGEYKCQH QQVNESEPVY LEVFSDWLLL QASAEVVMEG QPLFLRCHGW RNWDVYKVIY
YKDGEALKYW YENHNISITN ATVEDSGTYY CTGKVWQLDY ESEPLNITVI KAPREKYWLQ
序列辨識編號2
SHTQPLGVFL FPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPR
EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLP
PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSRLTV
DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS LSLGK
序列辨識編號3
RNTGRGGEEK KGSKEKEEQE ERETKTPECP
序列辨識編號4
AQPQAEGSLA KATTAPATTR NTGRGGEEKK GSKEKEEQEE RETKTPECP
序列辨識編號5
gtgccccaga agcccaaggt gagcctgaac cctccctgga acagaatctt caagggcgag
aacgtgaccc tgacctgcaa cggcaacaac ttcttcgagg tgagcagcac caagtggttc
cacaatggca gcctgagcga ggagaccaac agctccctga acatcgtgaa cgccaagttc
gaggacagcg gcgagtacaa gtgccagcac cagcaggtga acgagagcga gcccgtgtac
ctggaggtgt tcagcgactg gctgctgctg caggccagcg ccgaggtggt gatggagggc
cagcccctgt tcctgagatg ccacggctgg agaaactggg acgtgtacaa ggtgatctac
tacaaggatg gcgaggccct gaagtactgg tacgagaacc acaacatctc catcaccaac
gccaccgtgg aggacagcgg cacctactac tgcacaggca aggtgtggca gctggactac
gagagcgagc ccctgaacat caccgtgatc aaggctccca gagagaagta ctggctgcag
序列辨識編號6
tgcgtggtcg tggatgtgag ccaggaagat cccgaagtgc agttcaactg gtacgtggat
ggcgtggaag tgcacaacgc caagaccaag cccagagaag agcagttcaa ctccacctac
agagtggtga gcgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag
tgcaaggtgt ccaacaaagg cctgcccagc tccatcgaga agaccatcag caaagccaaa
ggccagccca gagaacccca ggtgtacacc ctgcctccca gccaggaaga gatgaccaag
aaccaggtgt ccctgacctg cctggtgaaa ggcttctacc ccagcgacat cgccgtggag
tgggaaagca acggccagcc cgagaacaat tacaagacaa cccctcccgt gctggatagc
gatggcagct tctttctgta cagcagactg accgtggaca agagcagatg gcaggaaggc
aacgtgttca gctgcagcgt gatgcacgaa gccctgcaca accactacac ccagaagagc
ctgtccctga gcctgggcaa g
序列辨識編號7
aggaacaccg gcagaggagg cgaggaaaag aaaggaagca aggagaagga ggagcaggag
gaaagagaaa ccaagacccc cgagtgcccc agccacaccc agcccctggg cgtgttcctg
ttccccccca agcccaagga caccctgatg atcagcagaa cccccgaggt gacc
序列辨識編號8
gcccagcccc aggccgaggg cagcctggct aaggccacca cagctcccgc caccaccagg
aacaccggca gaggaggcga ggaaaagaaa ggaagcaagg agaaggagga gcaggaggaa
agagaaacca agacccccga gtgccccagc cacacccagc ccctgggcgt gttcctgttc
ccccccaagc ccaaggacac cctgatgatc agcagaaccc ccgaggtgac c
序列辨識編號9
MDAMLRGLCC VLLLCGAVFV SPSHA
序列辨識編號10
atggacgcca tgctgagagg cctgtgctgt gtgctgctgc tgtgcggcgc cgtgttcgtg
tcccctagcc acgcc
序列辨識編號11
MDAMLRGLCC VLLLCGAVFV SPSHAVPQKP KVSLNPPWNR IFKGENVTLT CNGNNFFEVS
STKWFHNGSL SEETNSSLNI VNAKFEDSGE YKCQHQQVNE SEPVYLEVFS DWLLLQASAE
VVMEGQPLFL RCHGWRNWDV YKVIYYKDGE ALKYWYENHN ISITNATVED SGTYYCTGKV
WQLDYESEPL NITVIKAPRE KYWLQRNTGR GGEEKKGSKE KEEQEERETK TPECPSHTQP
LGVFLFPPKP KDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN
STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE
MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW
QEGNVFSCSV MHEALHNHYT QKSLSLSLGK
序列辨識編號12
atggacgcca tgctgagagg cctgtgctgt gtgctgctgc tgtgcggcgc cgtgttcgtg
tcccctagcc acgccgtgcc ccagaagccc aaggtgagcc tgaaccctcc ctggaacaga
atcttcaagg gcgagaacgt gaccctgacc tgcaacggca acaacttctt cgaggtgagc
agcaccaagt ggttccacaa tggcagcctg agcgaggaga ccaacagctc cctgaacatc
gtgaacgcca agttcgagga cagcggcgag tacaagtgcc agcaccagca ggtgaacgag
agcgagcccg tgtacctgga ggtgttcagc gactggctgc tgctgcaggc cagcgccgag
gtggtgatgg agggccagcc cctgttcctg agatgccacg gctggagaaa ctgggacgtg
tacaaggtga tctactacaa ggatggcgag gccctgaagt actggtacga gaaccacaac
atctccatca ccaacgccac cgtggaggac agcggcacct actactgcac aggcaaggtg
tggcagctgg actacgagag cgagcccctg aacatcaccg tgatcaaggc tcccagagag
aagtactggc tgcagaggaa caccggcaga ggaggcgagg aaaagaaagg aagcaaggag
aaggaggagc aggaggaaag agaaaccaag acccccgagt gccccagcca cacccagccc
ctgggcgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatcag cagaaccccc
gaggtgacct gcgtggtcgt ggatgtgagc caggaagatc ccgaagtgca gttcaactgg
tacgtggatg gcgtggaagt gcacaacgcc aagaccaagc ccagagaaga gcagttcaac
tccacctaca gagtggtgag cgtgctgacc gtgctgcacc aggactggct gaacggcaag
gagtacaagt gcaaggtgtc caacaaaggc ctgcccagct ccatcgagaa gaccatcagc
aaagccaaag gccagcccag agaaccccag gtgtacaccc tgcctcccag ccaggaagag
atgaccaaga accaggtgtc cctgacctgc ctggtgaaag gcttctaccc cagcgacatc
gccgtggagt gggaaagcaa cggccagccc gagaacaatt acaagacaac ccctcccgtg
ctggatagcg atggcagctt ctttctgtac agcagactga ccgtggacaa gagcagatgg
caggaaggca acgtgttcag ctgcagcgtg atgcacgaag ccctgcacaa ccactacacc
cagaagagcc tgtccctgag cctgggcaag
序列辨識編號13
MDAMLRGLCC VLLLCGAVFV SPSHAVPQKP KVSLNPPWNR IFKGENVTLT CNGNNFFEVS
STKWFHNGSL SEETNSSLNI VNAKFEDSGE YKCQHQQVNE SEPVYLEVFS DWLLLQASAE
VVMEGQPLFL RCHGWRNWDV YKVIYYKDGE ALKYWYENHN ISITNATVED SGTYYCTGKV
WQLDYESEPL NITVIKAPRE KYWLQAQPQA EGSLAKATTA PATTRNTGRG GEEKKGSKEK
EEQEERETKT PECPSHTQPL GVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY
VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK
AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL
DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK
序列辨識編號14
atggacgcca tgctgagagg cctgtgctgt gtgctgctgc tgtgcggcgc cgtgttcgtg
tcccctagcc acgccgtgcc ccagaagccc aaggtgagcc tgaaccctcc ctggaacaga
atcttcaagg gcgagaacgt gaccctgacc tgcaacggca acaacttctt cgaggtgagc
agcaccaagt ggttccacaa tggcagcctg agcgaggaga ccaacagctc cctgaacatc
gtgaacgcca agttcgagga cagcggcgag tacaagtgcc agcaccagca ggtgaacgag
agcgagcccg tgtacctgga ggtgttcagc gactggctgc tgctgcaggc cagcgccgag
gtggtgatgg agggccagcc cctgttcctg agatgccacg gctggagaaa ctgggacgtg
tacaaggtga tctactacaa ggatggcgag gccctgaagt actggtacga gaaccacaac
atctccatca ccaacgccac cgtggaggac agcggcacct actactgcac aggcaaggtg
tggcagctgg actacgagag cgagcccctg aacatcaccg tgatcaaggc tcccagagag
aagtactggc tgcaggccca gccccaggcc gagggcagcc tggctaaggc caccacagct
cccgccacca ccaggaacac cggcagagga ggcgaggaaa agaaaggaag caaggagaag
gaggagcagg aggaaagaga aaccaagacc cccgagtgcc ccagccacac ccagcccctg
ggcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagcag aacccccgag
gtgacctgcg tggtcgtgga tgtgagccag gaagatcccg aagtgcagtt caactggtac
gtggatggcg tggaagtgca caacgccaag accaagccca gagaagagca gttcaactcc
acctacagag tggtgagcgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag
tacaagtgca aggtgtccaa caaaggcctg cccagctcca tcgagaagac catcagcaaa
gccaaaggcc agcccagaga accccaggtg tacaccctgc ctcccagcca ggaagagatg
accaagaacc aggtgtccct gacctgcctg gtgaaaggct tctaccccag cgacatcgcc
gtggagtggg aaagcaacgg ccagcccgag aacaattaca agacaacccc tcccgtgctg
gatagcgatg gcagcttctt tctgtacagc agactgaccg tggacaagag cagatggcag
gaaggcaacg tgttcagctg cagcgtgatg cacgaagccc tgcacaacca ctacacccag
aagagcctgt ccctgagcct gggcaag
序列辨識編號15
MIHTNLKKKF SCCVLVFLLF AVICVWKEKK KGSYYDSFKL QTKEFQVLKS LGKLAMGSDS
QSVSSSSTQD PHRGRQTLGS LRGLAKAKPE ASFQVWNKDS SSKNLIPRLQ KIWKNYLSMN
KYKVSYKGPG PGIKFSAEAL RCHLRDHVNV SMVEVTDFPF NTSEWEGYLP KESIRTKAGP
WGRCAVVSSA GSLKSSQLGR EIDDHDAVLR FNGAPTANFQ QDVGTKTTIR LMNSQLVTTE
KRFLKDSLYN EGILIVWDPS VYHSDIPKWY QNPDYNFFNN YKTYRKLHPN QPFYILKPQM
PWELWDILQE ISPEEIQPNP PSSGMLGIII MMTLCDQVDI YEFLPSKRKT DVCYYYQKFF
DSACTMGAYH PLLYEKNLVK HLNQGTDEDI YLLGKATLPG FRTIHC
序列辨識編號16
atgatccaca ccaacctgaa gaagaagttc agctgctgcg tgctggtgtt cctgctgttc
gccgtgatct gcgtgtggaa ggagaagaag aaaggcagct actacgacag cttcaagctg
cagaccaagg agttccaggt gctgaagagc ctgggcaagc tggccatggg cagcgacagc
cagagcgtgt ccagctcctc cacccaggat ccccacagag gcagacagac cctgggcagc
ctgagaggcc tggccaaggc caagcccgag gccagcttcc aggtgtggaa caaggacagc
agcagcaaga acctgatccc cagactgcag aagatctgga agaactacct gagcatgaac
aagtacaagg tgagctacaa aggacccgga cccggcatca agttcagcgc cgaggccctg
aggtgccacc tgagagacca cgtgaacgtg agcatggtgg aagtgaccga cttccccttc
aacaccagcg agtgggaagg ctacctgccc aaggagagca tcaggaccaa ggctggcccc
tggggcagat gcgccgtggt gagcagcgct ggcagcctga agagctccca gctgggcaga
gagatcgacg accacgatgc cgtgctgagg ttcaatggcg ctcccaccgc caacttccag
caggacgtgg gcaccaagac cacaatccgg ctgatgaaca gccagctggt gacaaccgag
aagcggttcc tgaaggacag cctgtacaac gagggcatcc tgatcgtgtg ggatcccagc
gtgtaccaca gcgacatccc caagtggtac cagaatcccg actacaactt cttcaacaac
tacaagacct atagaaagct gcaccccaac cagcccttct acatcctgaa gccccagatg
ccctgggagc tgtgggacat cctgcaggag atcagccctg aagagatcca gcccaaccct
ccctccagcg gcatgctggg cattatcatc atgatgaccc tgtgcgacca ggtggacatc
tacgagttcc tgcccagcaa gagaaagacc gacgtgtgct actactatca gaagttcttc
gacagcgcct gcaccatggg cgcctaccac cccctgctgt acgagaagaa cctggtgaag
cacctgaacc agggcaccga cgaggacatc tacctgctgg gcaaagccac cctgcccggc
ttcagaacca tccactgc
序列辨識編號17
RNTGRGGEEK KXXKEKEEQE ERETKTPECP
序列辨識編號18
AQPQAEGSLA KATTAPATTR NTGRGGEEKK XXKEKEEQEE RETKTPECP
序列辨識編號19
RNTGRGGEEK KKEKEKEEQE ERETKTPECP
序列辨識編號20
VPQKPKVSLN PPWNRIFKGE NVTLTCNGNN FFEVSSTKWF HNGSLSEETN SSLNIVNAKF
EDSGEYKCQH QQVNESEPVY LEVFSDWLLL QASAEVVMEG QPLFLRCHGW RNWDVYKVIY
YKDGEALKYW YENHNISITN ATVEDSGTYY CTGKVWQLDY ESEPLNITVI KAPREKYWLQ
RNTGRGGEEK KKEKEKEEQE ERETKTPECP SHTQPLGVFL FPPKPKDTLM ISRTPEVTCV
VVDVSQEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK
VSNKGLPSSI EKTISKAKGQ PREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE
SNGQPENNYK TTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS
LSLGK
序列辨識編號21
VPQKPKVSLN PPWNRIFKGE NVTLTCNGNN FFEVSSTKWF HNGSLSEETN SSLNIVNAKF
EDSGEYKCQH QQVNESEPVY LEVFSDWLLL QASAEVVMEG QPLFLRCHGW RNWDVYKVIY
YKDGEALKYW YENHNISITN ATVEDSGTYY CTGKVWQLDY ESEPLNITVI KAPREKYWLQ
RNTGRGGEEK KGSKEKEEQE ERETKTPECP SHTQPLGVFL FPPKPKDTLM ISRTPEVTCV
VVDVSQEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK
VSNKGLPSSI EKTISKAKGQ PREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE
SNGQPENNYK TTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS
LSLGK
序列辨識編號22
VPQKPKVSLN PPWNRIFKGE NVTLTCNGNN FFEVSSTKWF HNGSLSEETN SSLNIVNAKF
EDSGEYKCQH QQVNESEPVY LEVFSDWLLL QASAEVVMEG QPLFLRCHGW RNWDVYKVIY
YKDGEALKYW YENHNISITN ATVEDSGTYY CTGKVWQLDY ESEPLNITVI KAPREKYWLQ
AQPQAEGSLA KATTAPATTR NTGRGGEEKK GSKEKEEQEE RETKTPECPS HTQPLGVFLF
PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV
SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV
SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF
SCSVMHEALH NHYTQKSLSL SLGK
VPQKPKVSLN PPWNRIFKGE NVTLTCNGNN FEVSSTKWF HNGSLSEETN SSLNIVNAKFEDSGEYKCQH QQVNESEPVY LEVFSDWLLL QASAEVVMEG QPLFLRCHGW RNWDVYKVIY
YKDGEALKYW YENHNISITN ATVEDSGTYY CTGKVWQLDY ESEPLNITVI KAPREKYWLQ
序列辨識編號2
SHTQPLGVFL FPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV EVHNAKTKPR
EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKGLPSSI EKTISKAKGQ PREPQVYTLP
PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLYSRLTV
DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS LSLGK
序列辨識編號3
RNTGRGGEEK KGSKEKEEQE ERETKTPECP
序列辨識編號4
AQPQAEGSLA KATTAPATTR NTGRGGEEKK GSKEKEEQEE RETKTPECP
序列辨識編號5
gtgccccaga agcccaaggt gagcctgaac cctccctgga acagaatctt caagggcgag
aacgtgaccc tgacctgcaa cggcaacaac ttcttcgagg tgagcagcac caagtggttc
cacaatggca gcctgagcga ggagaccaac agctccctga acatcgtgaa cgccaagttc
gaggacagcg gcgagtacaa gtgccagcac cagcaggtga acgagagcga gcccgtgtac
ctggaggtgt tcagcgactg gctgctgctg caggccagcg ccgaggtggt gatggagggc
cagcccctgt tcctgagatg ccacggctgg agaaactggg acgtgtacaa ggtgatctac
tacaaggatg gcgaggccct gaagtactgg tacgagaacc acaacatctc catcaccaac
gccaccgtgg aggacagcgg cacctactac tgcacaggca aggtgtggca gctggactac
gagagcgagc ccctgaacat caccgtgatc aaggctccca gagagaagta ctggctgcag
序列辨識編號6
tgcgtggtcg tggatgtgag ccaggaagat cccgaagtgc agttcaactg gtacgtggat
ggcgtggaag tgcacaacgc caagaccaag cccagagaag agcagttcaa ctccacctac
agagtggtga gcgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag
tgcaaggtgt ccaacaaagg cctgcccagc tccatcgaga agaccatcag caaagccaaa
ggccagccca gagaacccca ggtgtacacc ctgcctccca gccaggaaga gatgaccaag
aaccaggtgt ccctgacctg cctggtgaaa ggcttctacc ccagcgacat cgccgtggag
tgggaaagca acggccagcc cgagaacaat tacaagacaa cccctcccgt gctggatagc
gatggcagct tctttctgta cagcagactg accgtggaca agagcagatg gcaggaaggc
aacgtgttca gctgcagcgt gatgcacgaa gccctgcaca accactacac ccagaagagc
ctgtccctga gcctgggcaa g
序列辨識編號7
aggaacaccg gcagaggagg cgaggaaaag aaaggaagca aggagaagga ggagcaggag
gaaagagaaa ccaagacccc cgagtgcccc agccacaccc agcccctggg cgtgttcctg
ttccccccca agcccaagga caccctgatg atcagcagaa cccccgaggt gacc
序列辨識編號8
gcccagcccc aggccgaggg cagcctggct aaggccacca cagctcccgc caccaccagg
aacaccggca gaggaggcga ggaaaagaaa ggaagcaagg agaaggagga gcaggaggaa
agagaaacca agacccccga gtgccccagc cacacccagc ccctgggcgt gttcctgttc
ccccccaagc ccaaggacac cctgatgatc agcagaaccc ccgaggtgac c
序列辨識編號9
MDAMLRGLCC VLLLCGAVFV SPSHA
序列辨識編號10
atggacgcca tgctgagagg cctgtgctgt gtgctgctgc tgtgcggcgc cgtgttcgtg
tcccctagcc acgcc
序列辨識編號11
MDAMLRGLCC VLLLCGAVFV SPSHAVPQKP KVSLNPPWNR IFKGENVTLT CNGNNFFEVS
STKWFHNGSL SEETNSSLNI VNAKFEDSGE YKCQHQQVNE SEPVYLEVFS DWLLLQASAE
VVMEGQPLFL RCHGWRNWDV YKVIYYKDGE ALKYWYENHN ISITNATVED SGTYYCTGKV
WQLDYESEPL NITVIKAPRE KYWLQRNTGR GGEEKKGSKE KEEQEERETK TPECPSHTQP
LGVFLFPPKP KDTLMISRTP EVTCVVVDVS QEDPEVQFNW YVDGVEVHNA KTKPREEQFN
STYRVVSVLT VLHQDWLNGK EYKCKVSNKG LPSSIEKTIS KAKGQPREPQ VYTLPPSQEE
MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SRLTVDKSRW
QEGNVFSCSV MHEALHNHYT QKSLSLSLGK
序列辨識編號12
atggacgcca tgctgagagg cctgtgctgt gtgctgctgc tgtgcggcgc cgtgttcgtg
tcccctagcc acgccgtgcc ccagaagccc aaggtgagcc tgaaccctcc ctggaacaga
atcttcaagg gcgagaacgt gaccctgacc tgcaacggca acaacttctt cgaggtgagc
agcaccaagt ggttccacaa tggcagcctg agcgaggaga ccaacagctc cctgaacatc
gtgaacgcca agttcgagga cagcggcgag tacaagtgcc agcaccagca ggtgaacgag
agcgagcccg tgtacctgga ggtgttcagc gactggctgc tgctgcaggc cagcgccgag
gtggtgatgg agggccagcc cctgttcctg agatgccacg gctggagaaa ctgggacgtg
tacaaggtga tctactacaa ggatggcgag gccctgaagt actggtacga gaaccacaac
atctccatca ccaacgccac cgtggaggac agcggcacct actactgcac aggcaaggtg
tggcagctgg actacgagag cgagcccctg aacatcaccg tgatcaaggc tcccagagag
aagtactggc tgcagaggaa caccggcaga ggaggcgagg aaaagaaagg aagcaaggag
aaggaggagc aggaggaaag agaaaccaag acccccgagt gccccagcca cacccagccc
ctgggcgtgt tcctgttccc ccccaagccc aaggacaccc tgatgatcag cagaaccccc
gaggtgacct gcgtggtcgt ggatgtgagc caggaagatc ccgaagtgca gttcaactgg
tacgtggatg gcgtggaagt gcacaacgcc aagaccaagc ccagagaaga gcagttcaac
tccacctaca gagtggtgag cgtgctgacc gtgctgcacc aggactggct gaacggcaag
gagtacaagt gcaaggtgtc caacaaaggc ctgcccagct ccatcgagaa gaccatcagc
aaagccaaag gccagcccag agaaccccag gtgtacaccc tgcctcccag ccaggaagag
atgaccaaga accaggtgtc cctgacctgc ctggtgaaag gcttctaccc cagcgacatc
gccgtggagt gggaaagcaa cggccagccc gagaacaatt acaagacaac ccctcccgtg
ctggatagcg atggcagctt ctttctgtac agcagactga ccgtggacaa gagcagatgg
caggaaggca acgtgttcag ctgcagcgtg atgcacgaag ccctgcacaa ccactacacc
cagaagagcc tgtccctgag cctgggcaag
序列辨識編號13
MDAMLRGLCC VLLLCGAVFV SPSHAVPQKP KVSLNPPWNR IFKGENVTLT CNGNNFFEVS
STKWFHNGSL SEETNSSLNI VNAKFEDSGE YKCQHQQVNE SEPVYLEVFS DWLLLQASAE
VVMEGQPLFL RCHGWRNWDV YKVIYYKDGE ALKYWYENHN ISITNATVED SGTYYCTGKV
WQLDYESEPL NITVIKAPRE KYWLQAQPQA EGSLAKATTA PATTRNTGRG GEEKKGSKEK
EEQEERETKT PECPSHTQPL GVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY
VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK
AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL
DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK
序列辨識編號14
atggacgcca tgctgagagg cctgtgctgt gtgctgctgc tgtgcggcgc cgtgttcgtg
tcccctagcc acgccgtgcc ccagaagccc aaggtgagcc tgaaccctcc ctggaacaga
atcttcaagg gcgagaacgt gaccctgacc tgcaacggca acaacttctt cgaggtgagc
agcaccaagt ggttccacaa tggcagcctg agcgaggaga ccaacagctc cctgaacatc
gtgaacgcca agttcgagga cagcggcgag tacaagtgcc agcaccagca ggtgaacgag
agcgagcccg tgtacctgga ggtgttcagc gactggctgc tgctgcaggc cagcgccgag
gtggtgatgg agggccagcc cctgttcctg agatgccacg gctggagaaa ctgggacgtg
tacaaggtga tctactacaa ggatggcgag gccctgaagt actggtacga gaaccacaac
atctccatca ccaacgccac cgtggaggac agcggcacct actactgcac aggcaaggtg
tggcagctgg actacgagag cgagcccctg aacatcaccg tgatcaaggc tcccagagag
aagtactggc tgcaggccca gccccaggcc gagggcagcc tggctaaggc caccacagct
cccgccacca ccaggaacac cggcagagga ggcgaggaaa agaaaggaag caaggagaag
gaggagcagg aggaaagaga aaccaagacc cccgagtgcc ccagccacac ccagcccctg
ggcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagcag aacccccgag
gtgacctgcg tggtcgtgga tgtgagccag gaagatcccg aagtgcagtt caactggtac
gtggatggcg tggaagtgca caacgccaag accaagccca gagaagagca gttcaactcc
acctacagag tggtgagcgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag
tacaagtgca aggtgtccaa caaaggcctg cccagctcca tcgagaagac catcagcaaa
gccaaaggcc agcccagaga accccaggtg tacaccctgc ctcccagcca ggaagagatg
accaagaacc aggtgtccct gacctgcctg gtgaaaggct tctaccccag cgacatcgcc
gtggagtggg aaagcaacgg ccagcccgag aacaattaca agacaacccc tcccgtgctg
gatagcgatg gcagcttctt tctgtacagc agactgaccg tggacaagag cagatggcag
gaaggcaacg tgttcagctg cagcgtgatg cacgaagccc tgcacaacca ctacacccag
aagagcctgt ccctgagcct gggcaag
序列辨識編號15
MIHTNLKKKF SCCVLVFLLF AVICVWKEKK KGSYYDSFKL QTKEFQVLKS LGKLAMGSDS
QSVSSSSTQD PHRGRQTLGS LRGLAKAKPE ASFQVWNKDS SSKNLIPRLQ KIWKNYLSMN
KYKVSYKGPG PGIKFSAEAL RCHLRDHVNV SMVEVTDFPF NTSEWEGYLP KESIRTKAGP
WGRCAVVSSA GSLKSSQLGR EIDDHDAVLR FNGAPTANFQ QDVGTKTTIR LMNSQLVTTE
KRFLKDSLYN EGILIVWDPS VYHSDIPKWY QNPDYNFFNN YKTYRKLHPN QPFYILKPQM
PWELWDILQE ISPEEIQPNP PSSGMLGIII MMTLCDQVDI YEFLPSKRKT DVCYYYQKFF
DSACTMGAYH PLLYEKNLVK HLNQGTDEDI YLLGKATLPG FRTIHC
序列辨識編號16
atgatccaca ccaacctgaa gaagaagttc agctgctgcg tgctggtgtt cctgctgttc
gccgtgatct gcgtgtggaa ggagaagaag aaaggcagct actacgacag cttcaagctg
cagaccaagg agttccaggt gctgaagagc ctgggcaagc tggccatggg cagcgacagc
cagagcgtgt ccagctcctc cacccaggat ccccacagag gcagacagac cctgggcagc
ctgagaggcc tggccaaggc caagcccgag gccagcttcc aggtgtggaa caaggacagc
agcagcaaga acctgatccc cagactgcag aagatctgga agaactacct gagcatgaac
aagtacaagg tgagctacaa aggacccgga cccggcatca agttcagcgc cgaggccctg
aggtgccacc tgagagacca cgtgaacgtg agcatggtgg aagtgaccga cttccccttc
aacaccagcg agtgggaagg ctacctgccc aaggagagca tcaggaccaa ggctggcccc
tggggcagat gcgccgtggt gagcagcgct ggcagcctga agagctccca gctgggcaga
gagatcgacg accacgatgc cgtgctgagg ttcaatggcg ctcccaccgc caacttccag
caggacgtgg gcaccaagac cacaatccgg ctgatgaaca gccagctggt gacaaccgag
aagcggttcc tgaaggacag cctgtacaac gagggcatcc tgatcgtgtg ggatcccagc
gtgtaccaca gcgacatccc caagtggtac cagaatcccg actacaactt cttcaacaac
tacaagacct atagaaagct gcaccccaac cagcccttct acatcctgaa gccccagatg
ccctgggagc tgtgggacat cctgcaggag atcagccctg aagagatcca gcccaaccct
ccctccagcg gcatgctggg cattatcatc atgatgaccc tgtgcgacca ggtggacatc
tacgagttcc tgcccagcaa gagaaagacc gacgtgtgct actactatca gaagttcttc
gacagcgcct gcaccatggg cgcctaccac cccctgctgt acgagaagaa cctggtgaag
cacctgaacc agggcaccga cgaggacatc tacctgctgg gcaaagccac cctgcccggc
ttcagaacca tccactgc
序列辨識編號17
RNTGRGGEEK KXXKEKEEQE ERETKTPECP
序列辨識編號18
AQPQAEGSLA KATTAPATTR NTGRGGEEKK XXKEKEEQEE RETKTPECP
序列辨識編號19
RNTGRGGEEK KKEKEKEEQE ERETKTPECP
序列辨識編號20
VPQKPKVSLN PPWNRIFKGE NVTLTCNGNN FFEVSSTKWF HNGSLSEETN SSLNIVNAKF
EDSGEYKCQH QQVNESEPVY LEVFSDWLLL QASAEVVMEG QPLFLRCHGW RNWDVYKVIY
YKDGEALKYW YENHNISITN ATVEDSGTYY CTGKVWQLDY ESEPLNITVI KAPREKYWLQ
RNTGRGGEEK KKEKEKEEQE ERETKTPECP SHTQPLGVFL FPPKPKDTLM ISRTPEVTCV
VVDVSQEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK
VSNKGLPSSI EKTISKAKGQ PREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE
SNGQPENNYK TTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS
LSLGK
序列辨識編號21
VPQKPKVSLN PPWNRIFKGE NVTLTCNGNN FFEVSSTKWF HNGSLSEETN SSLNIVNAKF
EDSGEYKCQH QQVNESEPVY LEVFSDWLLL QASAEVVMEG QPLFLRCHGW RNWDVYKVIY
YKDGEALKYW YENHNISITN ATVEDSGTYY CTGKVWQLDY ESEPLNITVI KAPREKYWLQ
RNTGRGGEEK KGSKEKEEQE ERETKTPECP SHTQPLGVFL FPPKPKDTLM ISRTPEVTCV
VVDVSQEDPE VQFNWYVDGV EVHNAKTKPR EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK
VSNKGLPSSI EKTISKAKGQ PREPQVYTLP PSQEEMTKNQ VSLTCLVKGF YPSDIAVEWE
SNGQPENNYK TTPPVLDSDG SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS
LSLGK
序列辨識編號22
VPQKPKVSLN PPWNRIFKGE NVTLTCNGNN FFEVSSTKWF HNGSLSEETN SSLNIVNAKF
EDSGEYKCQH QQVNESEPVY LEVFSDWLLL QASAEVVMEG QPLFLRCHGW RNWDVYKVIY
YKDGEALKYW YENHNISITN ATVEDSGTYY CTGKVWQLDY ESEPLNITVI KAPREKYWLQ
AQPQAEGSLA KATTAPATTR NTGRGGEEKK GSKEKEEQEE RETKTPECPS HTQPLGVFLF
PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV
SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP SQEEMTKNQV
SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF
SCSVMHEALH NHYTQKSLSL SLGK
圖1顯示各細胞株中所產生之蛋白的SDS-PAGE及凝膠等電聚焦(IEF)結果。在此可看出在還原及非還原二者條件下沒有產生截短型,及酸性蛋白之含量由於引入涶液酸轉移酶基因引起的涶液酸含量增加而增加。
圖2顯示根據本發明之實施例之多肽二聚蛋白於非還原及還原形式下之SDS-PAGE結果。特別是,可看出即使在對應於輸入的培養上清液中,該多肽二聚體均具有高純度。
圖3顯示奧馬珠單抗與IgE的結合能力之圖。該圖顯示固定奥馬珠單抗及分析其依處理的IgE濃度之結合能力所獲得的結果。
圖4描述顯示根據本發明之實施例之多肽二聚蛋白之IgE結合能力之圖。該圖顯示固定該二聚蛋白及分析其依處理的IgE濃度之結合能力所獲得的結果。
圖5描述通過生物膜干涉技術(BLI)分析鑑定本發明之實施例之多肽二聚蛋白(IgETRAP
)及奥馬珠單抗與IgG受體FcgRI (圖5A)、FcgRIIA (圖5B)、FcgRIIB (圖5C)、FcgRIIIA (圖5D)及FcgRIIIB (圖5E)之交互反應所獲得的結果。
圖6描述量化IgETRAP
與IgG受體及奥馬珠單抗與IgG受體之間之結合能力所獲得的圖。
圖7描述顯示根據本發明之實施例之多肽二聚蛋白(IgETRAP
)依其濃度在小鼠來源肥大細胞活性上的抑制能力之圖。
圖8描述顯示根據本發明之實施例之多肽二聚蛋白(IgETRAP
)及Xolair (奥馬珠單抗)依其等之濃度,在人FceRI表現小鼠來源肥大細胞活性上的抑制能力之比較圖。
圖9描述顯示根據本發明之實施例之多肽二聚蛋白在食物過敏模型中的投藥效果之圖。
Claims (19)
- 一種多肽二聚體,其包含: 二個單體,其各含有IgE Fc受體之a次單元的胞外區域(FceRIa-ECD), 其中該單體含有一經修飾的Fc區,及 該經修飾的Fc區與該FceRIa-ECD透過一鉸鏈(hinge)連接。
- 如請求項1之多肽二聚體,其中該IgE Fc受體之a次單元的胞外區域是序列辨識編號1之胺基酸序列或其片段。
- 如請求項1之多肽二聚體,其中該經修飾的Fc區是序列辨識編號2。
- 如請求項1之多肽二聚體,其中該鉸鏈是衍生自免疫球蛋白IgD之鉸鏈區或其變體。
- 如請求項4之多肽二聚體,其中該衍生自免疫球蛋白IgD之鉸鏈區或其變體含有至少一個半胱胺酸。
- 如請求項4之多肽二聚體,其中該衍生自免疫球蛋白IgD之鉸鏈區或其變體是Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa1 Xaa2 Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro (序列辨識編號17), Xaa1是Lys或Gly,及 Xaa2是Glu、Gly或Ser。
- 如請求項4之多肽二聚體,其中該衍生自免疫球蛋白IgD之鉸鏈區或其變體是Ala Gln Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala Pro Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Xaa3 Xaa4 Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro (序列辨識編號:18), Xaa1是Lys或Gly,及 Xaa2是Glu、Gly或Ser。
- 如請求項1之多肽二聚體,其中該鉸鏈具有選自於由序列辨識編號3、序列辨識編號4及序列辨識編號19所構成之群組之任一個胺基酸序列。
- 一種多核苷酸,其編碼如請求項1之單體,其含有IgE Fc受體之a次單元之胞外區域(FceRIa-ECD)。
- 一種表現載體,其加載了如請求項9之多核苷酸。
- 一種宿主細胞,其中引入了如請求項10之表現載體。
- 一種用於治療或預防過敏性疾病之藥學組成物,其包含如請求項1至8中任一項之多肽二聚體作為活性成份。
- 如請求項12之藥學組成物, 其中該過敏性疾病是選自於由下列所構成之群組之任一個:食物過敏、異位性皮膚炎、氣喘、過敏性鼻炎、過敏性結膜炎、過敏性皮膚炎、慢性自發性蕁麻疹及過敏性接觸性皮膚炎。
- 一種用於改善及緩解過敏症狀之食品組成物,其包含如請求項1至8中任一項之多肽二聚體作為活性成份。
- 一種用於產生多肽二聚體之方法,其包含: 培養其中引入了如請求項9之多核苷酸及涶液酸轉移酶之細胞的步驟;及 回收多肽二聚體之步驟。
- 一種於請求項15中獲得之多肽二聚體。
- 一種用於治療或預防過敏性疾病之藥學組成物,其包含如請求項16之多肽二聚體作為活性成份。
- 一種用於改善及緩解過敏症狀之食品組成物,其包含如請求項16之多肽二聚體作為活性成份。
- 一種用於治療或預防過敏性疾病之方法,其包含: 對一受試者投與如請求項1和/或請求項16之多肽二聚體之步驟。
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TW108100730A TWI713971B (zh) | 2018-01-08 | 2019-01-08 | IgE受體之重組型胞外區域、包含其之藥學組成物以及用於製備其之方法 |
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AU (1) | AU2019205743B2 (zh) |
BR (1) | BR112020013805A2 (zh) |
CA (1) | CA3086222A1 (zh) |
CL (1) | CL2020001798A1 (zh) |
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PE (1) | PE20210043A1 (zh) |
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SG (1) | SG11202005858WA (zh) |
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WO (1) | WO2019135668A1 (zh) |
Families Citing this family (6)
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PE20210043A1 (es) | 2018-01-08 | 2021-01-08 | Gi Innovation Inc | Dominio extracelular de subunidad alfa del receptor fc de ige, composicion farmaceutica que comprende el mismo y metodo para producir el mismo |
ES2969110T3 (es) * | 2018-01-12 | 2024-05-16 | Gi Innovation Inc | Composición que comprende probióticos y polipéptidos que tienen afinidad de unión por IgE y uso de la misma |
BR112022000246A2 (pt) * | 2019-07-08 | 2022-02-22 | Gi Innovation Inc | Dímero polipeptídico com elevado teor de ácido siálico, compreendendo o domínio extracelular da subunidade alfa do receptor fc de ige e composição farmacêutica compreendendo o mesmo |
US20230279124A1 (en) * | 2020-07-17 | 2023-09-07 | Gi Innovation, Inc. | Fusion protein comprising ige fc receptor alpha subunit extracellular domain and anti-il-4r antibody, and use thereof |
KR20220011931A (ko) * | 2020-07-22 | 2022-02-03 | (주)지아이이노베이션 | IgE Fc 수용체를 포함하는 융합단백질 및 이를 포함하는 개 알레르기성 질환 치료 용도 |
JP2024509937A (ja) | 2021-03-09 | 2024-03-05 | ジーアイ・イノベイション・インコーポレイテッド | Ige fc受容体のアルファサブユニットの細胞外ドメインを含む融合タンパク質の製剤 |
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US3067334A (en) | 1959-03-13 | 1962-12-04 | Garrett Corp | Driving and control means for a plurality of alternators |
DE60135158D1 (de) * | 2000-09-26 | 2008-09-11 | Genentech Inc | Antagonisten des ige-rezeptors |
US7829084B2 (en) * | 2001-01-17 | 2010-11-09 | Trubion Pharmaceuticals, Inc. | Binding constructs and methods for use thereof |
DK1896071T3 (en) * | 2005-06-30 | 2015-05-26 | Janssen Biotech Inc | Methods and compositions with increased therapeutic activity |
WO2008028068A2 (en) * | 2006-08-30 | 2008-03-06 | Genentech, Inc. | NON-HUMAN PRIMATE FCεR1α POLYPEPTIDES |
US7867491B2 (en) * | 2007-05-30 | 2011-01-11 | Genexine Co., Ltd. | Immunoglobulin fusion proteins |
DK2853545T3 (en) | 2008-09-17 | 2016-08-29 | Xencor Inc | Antibody specific for IgE |
US20110256641A1 (en) * | 2010-04-19 | 2011-10-20 | Michael Ling | Methods and Systems for Detecting Free IgE |
KR101825048B1 (ko) * | 2014-12-31 | 2018-02-05 | 주식회사 제넥신 | GLP 및 면역글로불린 하이브리드 Fc 융합 폴리펩타이드 및 이의 용도 |
TWI691512B (zh) * | 2015-02-20 | 2020-04-21 | 日商橘生藥品工業股份有限公司 | Fc融合高親和性IgE受體α鏈 |
US10195272B2 (en) * | 2015-03-02 | 2019-02-05 | The Nemours Foundation | Adoptive t-cell therapy using FcεRI-based chimeric antigen receptors for treating IgE-mediated allergic diseases |
KR101873201B1 (ko) * | 2015-06-11 | 2018-07-02 | 주식회사 제넥신 | 변형된 인터루킨-7 단백질 및 이의 용도 |
KR102038672B1 (ko) * | 2018-01-08 | 2019-10-30 | (주)지아이이노베이션 | IgE Fc 수용체의 알파 서브유닛의 세포외 도메인을 포함하는 약학적 조성물 |
PE20210043A1 (es) | 2018-01-08 | 2021-01-08 | Gi Innovation Inc | Dominio extracelular de subunidad alfa del receptor fc de ige, composicion farmaceutica que comprende el mismo y metodo para producir el mismo |
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BR112020013805A2 (pt) | 2020-12-01 |
EP3738977A4 (en) | 2021-12-22 |
SG11202005858WA (en) | 2020-07-29 |
PE20210043A1 (es) | 2021-01-08 |
CN111587251A (zh) | 2020-08-25 |
WO2019135668A1 (ko) | 2019-07-11 |
RU2020122056A (ru) | 2022-02-10 |
AU2019205743B2 (en) | 2023-11-16 |
JP7488303B2 (ja) | 2024-05-21 |
KR102038675B1 (ko) | 2019-10-30 |
KR20190084885A (ko) | 2019-07-17 |
JP2022176971A (ja) | 2022-11-30 |
JP2021509590A (ja) | 2021-04-01 |
JP7128291B2 (ja) | 2022-08-30 |
IL275591B2 (en) | 2023-06-01 |
EP3738977A1 (en) | 2020-11-18 |
PH12020551021A1 (en) | 2021-05-31 |
MX2020007030A (es) | 2020-12-03 |
CL2020001798A1 (es) | 2020-11-13 |
US20210070833A1 (en) | 2021-03-11 |
AU2019205743A1 (en) | 2020-06-25 |
TWI713971B (zh) | 2020-12-21 |
IL275591A (en) | 2020-08-31 |
CA3086222A1 (en) | 2019-07-11 |
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