TW202325853A - 治療性蛋白之生產 - Google Patents
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- TW202325853A TW202325853A TW111142592A TW111142592A TW202325853A TW 202325853 A TW202325853 A TW 202325853A TW 111142592 A TW111142592 A TW 111142592A TW 111142592 A TW111142592 A TW 111142592A TW 202325853 A TW202325853 A TW 202325853A
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Abstract
本揭露關於改善治療性蛋白的唾液酸含量之方法,該等方法包括在CHO細胞中表現α2,6-唾液醯基轉移酶-1(ST6)或ST6和β1,4半乳糖苷轉移酶1(B4GALT1)二者;以及在包含半乳糖和錳的培養基中培養該細胞。
Description
本揭露關於改善治療性蛋白的唾液酸含量之方法,該等方法包括在CHO細胞中表現α2,6-唾液醯基轉移酶-1(ST6)或ST6和β1,4半乳糖苷轉移酶1(B4GALT1)二者;以及在包含半乳糖和錳的培養基中培養該細胞。
由於需要高水平的表現和適當的翻譯後處理二者,治療性蛋白的藥物生物合成變得複雜,這涉及添加N-連接和O-連接的支鏈寡糖鏈。
在醣蛋白中,糖附接至天冬醯胺側鏈中的醯胺氮原子(稱為N-連接),或附接至絲胺酸或蘇胺酸側鏈中的氧原子(也稱為O-連接)。用於形成N-連接的碳水化合物的過程始於向內質網(ER)中的脂質連接的二元醇中添加14個單糖。在其形成後,然後在ER中被稱為「核心糖基化」的過程中藉由寡醣轉移酶(OST)複合物,將這種碳水化合物複合物轉移到蛋白質。寡醣轉移酶(OST)複合物係由核糖體結合蛋白I、II、OST48和DAD1組成的多蛋白單元(Kelleher和Gilmore, 1997, PNAS [美國國家科學院院刊] 94(10):4994-4999;Kelleher等人, 2003, Molecular Cell [分子細胞], 12(1):101-111;和Kelleher等人, 1992, Cell [細胞], 69(1):55-65)。
隨後,多肽被轉運到高爾基氏體複合體,在那裡添加O-連接糖鏈,並且以許多不同的方式修飾N-連接糖鏈。在高爾基氏體複合體的順式和中間間室中,可以藉由去除甘露糖(Man)殘基來修剪原始的14糖N-連接複合物,並且藉由添加N-乙醯葡萄糖胺(GlcNac)和/或岩藻糖(Fuc)殘基而將其延長。各種形式的N-連接碳水化合物共同具有由三個甘露糖和兩個N-乙醯葡萄糖胺殘基組成的五糖核心。最後,在反式高爾基氏體中,可以添加其他GlcNac殘基,隨後添加半乳糖(Gal)和末端唾液酸(Sial)。高爾基氏體複合體中的碳水化合物處理被稱為「末端糖基化」,從而差異於核心糖基化。
唾液酸係約30種天然存在的酸性單糖家族的總稱,它們通常是醣蛋白和醣脂上發現的碳水化合物的末端糖。重組醣蛋白的唾液酸化非常重要,並且可以賦予醣蛋白許多重要特性,包括電荷、免疫原性、對蛋白酶降解的抗性、血漿清除率和生物活性。
本申請基於以下發現:(a) 在中國倉鼠卵巢(CHO)細胞中,表現α2,6-唾液醯基轉移酶1(ST6)(SEQ ID NO: 1、3、5、7、9或11);或共表現ST6(SEQ ID NO: 1、3、5、7、9或11)和β1,4半乳糖苷轉移酶1(B4GALT1)(SEQ ID NO: 13、15、17、19、21、23、25或27),以及;視需要地 (b) 在包含半乳糖和錳的培養基中培養該等細胞可以增加所述CHO細胞產生的重組抗體中的唾液酸含量(特別是具有α2,6連接的唾液酸的N-乙醯神經胺酸(NANA)形式的增加)。
因此,在一個方面,本發明提供了一種增加由CHO細胞產生的治療性蛋白的唾液酸含量之方法,該方法包括在該CHO細胞中表現α2,6-唾液醯基轉移酶-1(ST6)(SEQ ID NO: 1、3、5、7、9或11)。
在另一個方面,本發明提供了一種增加由CHO細胞產生的治療性蛋白的唾液酸含量之方法,該方法包括在該CHO細胞中共表現α2,6-唾液醯基轉移酶-1(ST6)(SEQ ID NO: 1、3、5、7、9或11)和β1,4-半乳糖苷轉移酶1(B4GALT1)(SEQ ID NO: 13、15、17、19、21、23、25或27)。
在另一個方面,提供了一種增加由CHO細胞產生的治療性蛋白的唾液酸含量之方法,該方法包括:(a) 在該CHO細胞中表現α2,6-唾液醯基轉移酶1(ST6)(SEQ ID NO: 1、3、5、7、9或11);以及 (b) 在包含半乳糖和錳的培養基中培養該細胞,其中在細胞培養期間,與在不包含添加的錳和半乳糖的培養基中培養的CHO細胞相比,在包含半乳糖和錳的培養基中培養該細胞增加了由該細胞產生的蛋白的唾液酸化。
在另一個方面,提供了一種增加由CHO細胞產生的治療性蛋白的唾液酸含量之方法,該方法包括:(a) 在該CHO細胞中表現α2,6-唾液醯基轉移酶-1(ST6)(SEQ ID NO: 1、3、5、7、9或11)和β1,4-半乳糖苷轉移酶1(B4GALT1)(SEQ ID NO: 13、15、17、19、21、23、25或27);以及 (b) 在包含半乳糖和錳的培養基中培養該細胞,其中在細胞培養期間,與在不包含添加的錳和半乳糖的培養基中培養的CHO細胞相比,在包含半乳糖和錳的培養基中培養該細胞增加了由該細胞產生的蛋白的唾液酸化。
在一些實施方式中,在細胞培養期間,與在不包含添加的錳和半乳糖的培養基中培養的CHO細胞產生的蛋白相比,CHO細胞產生的蛋白的唾液酸化增加了至少10%或至少20%。
在一些實施方式中,本文所述方法包括在細胞培養的第3天向培養基中添加錳和半乳糖。在一些實施方式中,在第3天向培養基中添加至少100 ppb(十億分率)的錳和至少15 mM半乳糖。
在一些實施方式中,本文所述方法進一步包括在第6天向培養基中添加錳和半乳糖。在一些實施方式中,在第6天向培養基中添加至少100 ppb的錳和至少15 mM半乳糖。
在一些實施方式中,本文所述方法進一步包括在第8天向培養基中添加錳和半乳糖。
在一些實施方式中,該培養基進一步包含銅。
在一些實施方式中,本文所述方法進一步包括在培養期間累積添加約10 mM至約100 mM半乳糖。在一個實例中,本文所述方法包括在培養期間累積添加約45 mM半乳糖。
在一些實施方式中,本文所述方法進一步包括在培養期間累積添加約40 ppb至約400 ppb錳。在一個實例中,本文所述方法包括在培養期間累積添加約400 ppb錳。
在一些實施方式中,本文所述方法進一步包括在培養期間累積添加約0.01 mM至約0.5 mM銅。在一個實例中,本文所述方法包括在培養期間累積添加約0.1 mM銅。在一些實施方式中,如本文所述之治療性蛋白係分泌蛋白和重組蛋白。在一些實施方式中,該治療性蛋白係抗體或其抗原結合片段、抗體或抗體片段的衍生物、雙特異性T細胞接合物分子或融合多肽。在一些實施方式中,該抗體係抗IL12抗體、抗IL23抗體、或抗IL12/23抗體(例如,優特克單抗(ustekinumab)抗體,其包含根據SEQ ID NO: 29的重鏈序列和根據SEQ ID NO: 30的輕鏈序列)。
在一些實施方式中,本文所述之唾液酸係α2,6-唾液酸化聚糖。在一些實施方式中,可以藉由親水相互作用液相層析(HILIC)-質譜(MS)分析確認α2,6-唾液酸化聚糖的水平。在一些實施方式中,α2,6-唾液酸化聚糖的水平可以在延長的細胞培養持續時間產生的蛋白中保持恒定。在一些實施方式中,延長的細胞培養持續時間係約27個群體倍增(PDL)。
在一些實施方式中,根據本文所述方法產生的治療性蛋白(例如抗體)可以用於治療斑塊狀牛皮癬、牛皮癬性關節炎、克羅恩病或潰瘍性結腸炎。
相關申請
本申請要求於2021年11月9日提交的美國臨時申請案號63/277,501和於2022年3月31日提交的申請案號63/326,194的權益,兩者的內容藉由引用以其全文特此併入。
序列表
本申請連同序列表一起以電子格式提交。序列表以標題為A-2752-WO01-SEC_Final_SeqListing_10252022的文件提供,該文件創建於2022年10月25日,大小為49 KB。電子格式的序列表中的資訊藉由引用以其全文併入本文。
本申請基於以下發現:(a) 在中國倉鼠卵巢(CHO)細胞中,表現α2,6-唾液醯基轉移酶1(ST6)(SEQ ID NO: 1、3、5、7、9或11);或共表現ST6(SEQ ID NO: 1、3、5、7、9或11)和β1,4半乳糖苷轉移酶1(B4GALT1)(SEQ ID NO: 13、15、17、19、21、23、25或27),以及;視需要地 (b) 在包含半乳糖和錳的培養基中培養該細胞可以增加所述CHO細胞產生的重組抗體中的唾液酸含量(特別是具有α2,6連接的唾液酸的N-乙醯神經胺酸(NANA)形式的增加)。此外,本文已表明,向培養基中添加錳和半乳糖導致產生治療性蛋白(例如,抗原結合蛋白,例如抗體)的培養細胞對治療性蛋白的翻譯後處理的顯著改變。如本文所示,培養基中錳和半乳糖的組合減少了產生的低級唾液酸化醣蛋白的量,並且增加了回收的高級唾液酸化醣蛋白的量。
因此,在一個方面,本發明提供了一種增加由CHO細胞產生的治療性蛋白的唾液酸含量之方法,該方法包括在該CHO細胞中表現α2,6-唾液醯基轉移酶-1(ST6)(SEQ ID NO: 1、3、5、7、9或11)。
在另一個方面,本發明提供了一種增加由CHO細胞產生的治療性蛋白的唾液酸含量之方法,該方法包括在該CHO細胞中共表現α2,6-唾液醯基轉移酶-1(ST6)(SEQ ID NO: 1、3、5、7、9或11)和β1,4-半乳糖苷轉移酶1(B4GALT1)(SEQ ID NO: 13、15、17、19、21、23、25或27)。
在另一個方面,提供了一種增加由CHO細胞產生的治療性蛋白的唾液酸含量之方法,該方法包括:(a) 在該CHO細胞中表現α2,6-唾液醯基轉移酶1(ST6)(SEQ ID NO: 1、3、5、7、9或11);以及 (b) 在包含半乳糖和錳的培養基中培養該細胞,其中在細胞培養期間,與在不包含添加的錳和半乳糖的培養基中培養的CHO細胞相比,在包含半乳糖和錳的培養基中培養該細胞增加了由該細胞產生的蛋白的唾液酸化。
在另一個方面,提供了一種增加由CHO細胞產生的治療性蛋白的唾液酸含量之方法,該方法包括:(a) 在該CHO細胞中表現α2,6唾液醯基轉移酶1(ST6)(SEQ ID NO: 1、3、5、7、9或11)和β1,4半乳糖苷轉移酶1(B4GALT1)(SEQ ID NO: 13、15、17、19、21、23、25或27);以及 (b) 在包含半乳糖和錳的培養基中培養該細胞,其中在細胞培養期間,與在不包含添加的錳和半乳糖的培養基中培養的CHO細胞相比,在包含半乳糖和錳的培養基中培養該細胞增加了由該細胞產生的蛋白的唾液酸化。
定義
如本文所用,唾液酸化係將唾液酸殘基添加到蛋白質中,該蛋白質可為醣蛋白。
如本文所用,術語唾液酸涵蓋含有9個或更多碳原子的糖家族,包括羧基基團。下文示出了涵蓋所有已知天然形式的唾液酸的一般結構。
單個分子上不同位置處的R1基團可以彼此相同或不同。R1可為氫或乙醯基、乳酸基、甲基、硫酸根、磷酸根、脫水、唾液酸、岩藻糖、葡萄糖或半乳糖基團。R2可為N-乙醯基、N-羥乙醯基、胺基、羥基、N-羥乙醯基-O-乙醯基、或N-羥乙醯基-O-甲基基團。R3代表寡糖中的前一個糖殘基,唾液酸在醣蛋白的背景下附接至該寡糖。R3可為半乳糖(連接在其3、4或5的位置)、N-乙醯基-半乳糖(連接在其6的位置)、N-乙醯基-葡萄糖(連接在其4或6的位置)、唾液酸(連接在其8或9的位置)、或5-N-羥乙醯基-神經胺酸。Essentials of Glycobiology [糖生物學精要], 第15章, Varki等人編輯, Cold Spring Harbor Laboratory Press [冷泉港實驗室出版社], New York [紐約] (1999)。自然界中已經發現了超過40種形式的唾液酸。Essentials of Glycobiology [糖生物學精要], 第15章, Varki等人編輯, Cold Spring Harbor Laboratory Press [冷泉港實驗室出版社], New York [紐約] (1999)。唾液酸的常見形式係N-乙醯神經胺酸(NANA),其中R1在所有位置處都是氫,並且R2係N-乙醯基基團。
本文所用的術語「可操作地連接」或「功能性地連接」係指核酸序列在單個核酸片段上的締合,使得一者的功能受到另一者影響。例如,如果調控DNA序列和編碼DNA序列被定位為使得該調控DNA序列影響該編碼DNA序列的表現(即,編碼序列或功能性RNA處於在啟動子的轉錄控制之下),則該調控DNA序列被稱為與編碼RNA或多肽的DNA序列「可操作地連接」或「締合」。編碼序列可以在有義或反義方向上與調節序列可操作地連接。
術語「啟動子」係指一個核苷酸序列,其通常位於其編碼序列的上游(5'),藉由為RNA聚合酶和正確轉錄所需的其他因子提供識別位點來控制編碼序列的表現。「啟動子」包括最小啟動子,該最小啟動子係一個短DNA序列,在一些情況下由TATA盒和用於指定轉錄起始位點的其他序列組成,在該轉錄起始位點處添加調節元件以增強表現。「啟動子」還指包含最小啟動子和調節元件的核苷酸序列,並且其能夠控制編碼序列或功能性RNA的表現。這種類型的啟動子序列由近端元件和更遠端的上游元件組成,後者的元件通常被稱為強化子。因此,「強化子」係DNA序列,其可以刺激啟動子活性,並且可為啟動子的固有元件或插入以增強啟動子的水平或組織特異性的異源元件。它能夠在全部兩個方向(正常或翻轉)上運行,並且即使在從啟動子向上游或下游移動時也能夠發揮功能。強化子和其他上游啟動子元件二者都結合介導其作用的序列特異性DNA結合蛋白。啟動子可以整體來源於天然基因,或由來源於天然存在的不同啟動子的不同元件組成,或甚至由合成的DNA片段組成。
啟動子還可能含有參與蛋白因子的結合的DNA序列,該等蛋白因子控制轉錄起始對生理或發育條件的響應的有效性。「起始位點」係圍繞作為轉錄序列一部分的第一核苷酸的位置,其也被定義為位置+1。對於此位點,對該基因的所有其他序列及其控制區進行編號。下游序列(即3'方向上的另外的蛋白編碼序列)被命名為正,而上游序列(主要是5'方向上的控制區)被命名為負。
用於在細胞中重組表現酶和對哺乳動物細胞(包括CHO細胞)進行遺傳修飾的技術係熟悉該項技術者熟知的。典型地,此類技術涉及用包含相關序列的核酸構建體轉化細胞。例如,此類方法可以從標準手冊中獲知,例如Sambrook和Russel (2001) 「Molecular Cloning: A Laboratory Manual [分子選殖:實驗室手冊] (第3版)」, Cold Spring Harbor Laboratory [冷泉港實驗室], Cold Spring Harbor Laboratory Press [冷泉港實驗室出版社],或F.Ausubel等人編輯, 「Current protocols in molecular biology [分子生物學實驗指南]」, Green Publishing and Wiley Interscience [格林出版協會和威利跨學科出版社], New York [紐約] (1987)。用於真菌宿主細胞的轉化和遺傳修飾的方法描述於以下文獻中:例如,歐洲申請案號EP-A-0635574、國際專利公開案號WO 98/46772、國際專利公開案號WO 99/60102、國際專利公開案號WO 00/37671、國際專利公告號WO 90/14423、歐洲申請案號EP-A-0481008、歐洲申請案號EP-A-0635574和美國專利案號6,265,186,其揭露內容均藉由引用以其全文併入本文。
如本文所用,術語「抗體」係指具有常規免疫球蛋白形式、包含重鏈及輕鏈且包含可變區及恒定區的蛋白質。例如,抗體可為IgG,其係兩對相同多肽鏈的「Y形」結構,每對具有一條「輕」鏈(典型地具有約25 kDa的分子量)和一條「重」鏈(典型地具有約50-70 kDa的分子量)。抗體具有可變區和恒定區。在IgG形式中,可變區通常是約100個至110個或更多個胺基酸,包含三個互補決定區(CDR),主要負責抗原識別,並且與結合不同抗原的其他抗體差異很大。恒定區允許抗體募集免疫系統的細胞和分子。可變區由每條輕鏈和重鏈的N末端區域構成,而恒定區由每條重鏈和輕鏈的C末端部分構成。(Janeway等人, 「Structure of the Antibody Molecule and the Immunoglobulin Genes」[抗體分子和免疫球蛋白基因的結構], Immunobiology: The Immune System in Health and Disease [免疫生物學:健康與疾病的免疫系統], 第4版 Elsevier Science Ltd./Garland Publishing [愛思唯爾科學有限公司/加蘭出版社], (1999))。
本領域已經描述了抗體CDR的一般結構和特性。易言之,在抗體支架中,CDR嵌埋於重鏈及輕鏈可變區中的框架內,在這裡其構成主要負責抗原結合及識別的區域。可變區典型地包含至少三個重鏈CDR或輕鏈CDR(Kabat等人
, 1991, Sequences of Proteins of Immunological Interest [有免疫學意義的蛋白質序列], Public Health Service [公共衛生署] N.I.H., Bethesda [貝塞斯達], Md. [馬里蘭州];還參見Chothia和Lesk, 1987, J. Mol. Biol.[分子生物學雜誌] 196:901-917;Chothia等人, 1989, Nature [自然] 342: 877-883),位於框架區內(由Kabat等人, 1991指定框架區1-4、FR1、FR2、FR3和FR4;還參見Chothia及Lesk, 1987, 同上)。
抗體可以包含本領域已知的任何恒定區。人輕鏈分類為κ輕鏈及λ輕鏈。重鏈分類為μ、δ、γ、α或ε,並且將抗體的同種型分別定義為IgM、IgD、IgG、IgA和IgE。IgG具有若干個亞類,包括但不限於IgG1、IgG2、IgG3和IgG4。IgM具有亞類,包括但不限於IgM1和IgM2。本揭露之實施方式包括所有這種抗體類別或同種型。輕鏈恒定區可為例如κ型或λ型輕鏈恒定區,例如人κ型或λ型輕鏈恒定區。重鏈恒定區可為例如α型、δ型、ε型、γ型或μ型重鏈恒定區,例如人α型、δ型、ε型、γ型或μ型重鏈恒定區。因此,在示例性實施方式中,抗體係同種型IgA、IgD、IgE、IgG或IgM的抗體,包括IgG1、IgG2、IgG3或IgG4中之任一種。
抗體可為單株抗體或多株抗體。在一些實施方式中,抗體包含與由哺乳動物(例如,小鼠、兔、山羊、馬、雞、倉鼠、人等)產生的天然存在的抗體基本上相似的序列。在這方面,抗體可以被認為是哺乳動物抗體,例如小鼠抗體、兔抗體、山羊抗體、馬抗體、雞抗體、倉鼠抗體、人抗體等。在某些方面,抗體係人抗體。在某些方面,抗體係嵌合抗體或人源化抗體。術語「嵌合抗體」係指含有來自兩種或更多種不同抗體的結構域的抗體。嵌合抗體可以例如含有來自一個物種的恒定結構域和來自第二物種的可變結構域,或更一般地,可以含有來自至少兩個物種的胺基酸序列的區段。嵌合抗體還可以含有同一物種內的兩種或更多種不同抗體的結構域。術語「人源化」在關於抗體使用時係指至少具有來自經工程化以具有比原始來源抗體更類似於真人抗體的結構和免疫學功能的非人來源CDR區的抗體。例如,人源化可以涉及將來自非人抗體(諸如小鼠抗體)的CDR接枝到人抗體中。人源化還可以涉及所選擇的胺基酸取代以使非人序列更類似於人序列。
抗體可以藉由酶(例如像木瓜蛋白酶和胃蛋白酶)切割成片段。木瓜蛋白酶切割抗體以產生兩個Fab片段和單個Fc片段。胃蛋白酶切割抗體以產生F(ab’)
2片段和pFc’片段。在本揭露之示例性方面,治療性蛋白係抗原結合片段或抗體。如本文所用,術語「抗原結合抗體片段」係指能夠結合抗體的抗原,並且也被稱為「抗原結合片段」或「抗原結合部分」的抗體的一部分。在示例性情況下,抗原結合抗體片段為Fab片段或F(ab’)
2片段。
術語「ADCC」或「抗體依賴性細胞介導的細胞毒性」或「抗體依賴性細胞的細胞毒性」係指免疫系統的效應細胞(例如,自然殺傷細胞(NK細胞)、巨噬細胞、嗜中性球、嗜酸性球)主動裂解靶細胞(其膜表面抗原已被特異性抗體結合)的機制。ADCC係適應性免疫響應的一部分,當抗原特異性抗體 (1) 藉由其抗原結合區結合靶細胞上的膜表面抗原以及 (2) 藉由其Fc區結合效應細胞表面上的Fc受體時發生。抗體的Fc區與Fc受體的結合導致效應細胞釋放導致靶細胞死亡(例如,藉由細胞裂解或細胞去顆粒)的細胞毒性因子。
術語「ADCC活性」或「ADCC水平」係指ADCC被活化或刺激的程度。測量或確定抗體組成物的ADCC水平的方法,包括用於測量或確定ADCC水平的可商購測定和套組(kit),係本領域眾所周知的,如以下所述:Yamashita等人, Scientific Reports [科學報告] 6: 文章編號19772 (2016), doi:10.1038/srep19772); Kantakamalakul等人, 「A novel EGFP-CEM-NKr flow cytometric method for measuring antibody dependent cell mediated-cytotoxicity (ADCC) activity in HIV-1 infected individuals [用於測量HIV-1感染個體中抗體依賴性細胞介導的細胞毒性(ADCC)活性的新的EGFP-CEM-NKr流動式細胞分析術方法]」, J Immunol Methods [免疫學方法雜誌] 315 (第1-2期): 1-10; (2006);Gomez-Roman等人, 「A simplified method for the rapid fluorometric assessment of antibody-dependent cell-mediated cytotoxicity [快速螢光評估抗體依賴性細胞介導的細胞毒性的簡化方法]」, J Immunol Methods [免疫學方法雜誌] 308 (第1-2期): 53-67 (2006);Schnueriger等人, : Development of a quantitative, cell-line based assay to measure ADCC activity mediated by therapeutic antibodies [開發定量的用以測量治療性抗體介導的ADCC活性的基於細胞系的測定]」, Molec Immunology [分子免疫學] 38 (第12-13期): 1512-1517 (2011);以及Mata等人, 「Effects of cryopreservation on effector cells for antibody dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell activity in
51Cr-release and CD107a assays [在
51Cr-釋放和CD107a測定中低溫保存對抗體依賴性細胞介導的細胞毒性(ADCC)和自然殺傷(NK)細胞活性的效應細胞的影響]」, J Immunol Methods [免疫學方法雜誌] 406: 1-9 (2014);出於所有目的,所有該等均藉由引用併入本文。術語「ADCC測定」或「FcγR報告基因測定」係指可用於確定抗體的ADCC活性的測定、套組或方法。在本文所述方法中測量或確定抗體的ADCC活性的示例性方法包括實例2中描述的ADCC測定或可從普洛麥格公司(Promega)商購的ADCC報告基因測定(目錄號G7010和G7018)。在一些實施方式中,ADCC活性係使用包括以下一項或多項的鈣黃綠素釋放測定測量或確定的:表現FcγRIIa(158V)的NK92(M1)細胞作為效應細胞,HCC2218細胞或WIL2-S細胞作為用鈣黃綠素-AM標記的靶細胞。
如本文所用,除非上下文另外明確指示,否則單數形式「一個」、「一種」和「該」也包括複數指示物。因此,例如,對「一種試劑」的提及包括此類不同試劑中之一或多種,並且對「該方法」的提及包括提及熟悉該項技術者已知的可以修改或取代本文所述方法的等效步驟和方法。
除非另外指示,否則在一系列元素前面的術語「至少」應被理解為指該系列中之每一個元素。熟悉該項技術者將認識到,或能夠僅使用常規試驗確定本文所述之本發明的特定實施方式的許多等效內容。此類等效內容旨在由本發明所涵蓋。
術語「和/或」在本文使用時包括「和」、「或」和「由該等術語連接的要素的全部或任何其他組合」的含義。
如本文所用,術語「約」或「大約」意指在給定值或範圍的 ± 20%內、較佳的是在 ± 15%內、更較佳的是在 ± 10%內、並且最較佳的是在 ± 5%內。
貫穿本說明書及其後的申請專利範圍,除非上下文另有要求,否則詞語「包含(comprise)」以及變型如「包含(comprises)」和「包含(comprising)」將被理解成隱含包括所述整數或步驟或者整數或步驟的組,但不排除任何其他整數或步驟或者整數或步驟的組。當在本文中使用時,術語「包含/包括」可以用術語「含有」或「包括」來取代,或者有時在本文中使用時用術語「具有」取代。
當在本文中使用時,「由……組成」時,排除了在申請專利範圍元素中未指定的任何元素、步驟或成分。當在本文中使用時,「基本上由……組成」並不排除不實質性地影響申請專利範圍的基本和新穎特徵的材料或步驟。
在本文的每個例子中,術語「包含/包括」、「基本上由……組成」和「由……組成」中之任何一個可以用其他兩個術語中之任一個替代。
增加唾液酸含量之方法
本文所述方法包括在CHO細胞中表現α2,6-唾液醯基轉移酶1(ST6)或ST6和β1,4半乳糖苷轉移酶1(B4GALT1)二者以增加治療性蛋白(例如抗體)的唾液酸含量。
在一些實施方式中,該方法包括將編碼ST6的多核苷酸序列引入CHO細胞中。在一些實施方式中,該方法包括將編碼B4GALT1和ST6二者的多核苷酸序列引入CHO細胞中。下表1中列出了用於本發明的ST6和B4GALT1的多核苷酸序列和蛋白序列。
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表 1]
SEQ ID NO: 1 | 人ST6 ATGATTCACACCAACCTGAAGAAAAAGTTCAGCTGCTGCGTCCTGGTCTTTCTTCTGTTTGCAGTCATCTGTGTGTGGAAGGAAAAGAAGAAAGGGAGTTACTATGATTCCTTTAAATTGCAAACCAAGGAATTCCAGGTGTTAAAGAGTCTGGGGAAATTGGCCATGGGGTCTGATTCCCAGTCTGTATCCTCAAGCAGCACCCAGGACCCCCACAGGGGCCGCCAGACCCTCGGCAGTCTCAGAGGCCTAGCCAAGGCCAAACCAGAGGCCTCCTTCCAGGTGTGGAACAAGGACAGCTCTTCCAAAAACCTTATCCCTAGGCTGCAAAAGATCTGGAAGAATTACCTAAGCATGAACAAGTACAAAGTGTCCTACAAGGGGCCAGGACCAGGCATCAAGTTCAGTGCAGAGGCCCTGCGCTGCCACCTCCGGGACCATGTGAATGTATCCATGGTAGAGGTCACAGATTTTCCCTTCAATACCTCTGAATGGGAGGGTTATCTGCCCAAGGAGAGCATTAGGACCAAGGCTGGGCCTTGGGGCAGGTGTGCTGTTGTGTCGTCAGCGGGATCTCTGAAGTCCTCCCAACTAGGCAGAGAAATCGATGATCATGACGCAGTCCTGAGGTTTAATGGGGCACCCACAGCCAACTTCCAACAAGATGTGGGCACAAAAACTACCATTCGCCTGATGAACTCTCAGTTGGTTACCACAGAGAAGCGCTTCCTCAAAGACAGTTTGTACAATGAAGGAATCCTAATTGTATGGGACCCATCTGTATACCACTCAGATATCCCAAAGTGGTACCAGAATCCGGATTATAATTTCTTTAACAACTACAAGACTTATCGTAAGCTGCACCCCAATCAGCCCTTTTACATCCTCAAGCCCCAGATGCCTTGGGAGCTATGGGACATTCTTCAAGAAATCTCCCCAGAAGAGATTCAGCCAAACCCCCCATCCTCTGGGATGCTTGGTATCATCATCATGATGACGCTGTGTGACCAGGTGGATATTTATGAGTTCCTCCCATCCAAGCGCAAGACTGACGTGTGCTACTACTACCAGAAGTTCTTCGATAGTGCCTGCACGATGGGTGCCTACCACCCGCTGCTCTATGAGAAGAATTTGGTGAAGCATCTCAACCAGGGCACAGATGAGGACATCTACCTGCTTGGAAAAGCCACACTGCCTGGCTTCCGGACCATTCACTGCTAG |
SEQ ID NO: 2 | 人ST6 MIHTNLKKKFSCCVLVFLLFAVICVWKEKKKGSYYDSFKLQTKEFQVLKSLGKLAMGSDSQSVSSSSTQDPHRGRQTLGSLRGLAKAKPEASFQVWNKDSSSKNLIPRLQKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAVVSSAGSLKSSQLGREIDDHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC |
SEQ ID NO: 3 | 人ST6變體4 ATGATTCACACCAACCTGAAGAAAAAGTTCAGCTGCTGCGTCCTGGTCTTTCTTCTGTTTGCAGTCATCTGTGTGTGGAAGGAAAAGAAGAAAGGGAGTTACTATGATTCCTTTAAATTGCAAACCAAGGAATTCCAGGTGTTAAAGAGTCTGGGGAAATTGGCCATGGGGTCTGATTCCCAGTCTGTATCCTCAAGCAGCACCCAGGACCCCCACAGGGGCCGCCAGACCCTCGGCAGTCTCAGAGGCCTAGCCAAGGCCAAACCAGAGGCCTCCTTCCAGGTGTGGAACAAGGACAGCTCTTCCAAAAACCTTATCCCTAGGCTGCAAAAGATCTGGAAGAATTACCTAAGCATGAACAAGTACAAAGTGTCCTACAAGGGGCCAGGACCAGGCATCAAGTTCAGTGCAGAGGCCCTGCGCTGCCACCTCCGGGACCATGTGAATGTATCCATGGTAGAGGTCACAGATTTTCCCTTCAATACCTCTGAATGGGAGGGTTATCTGCCCAAGGAGAGCATTAGGACCAAGGCTGGGCCTTGGGGCAGGTGTGCTGTTGTGTCGTCAGCGGGATCTCTGAAGTCCTCCCAACTAGGCAGAGAAATCGATGATCATGACGCAGTCCTGAGGTTTAATGGGGCACCCACAGCCAACTTCCAACAAGATGTGGGCACAAAAACTACCATTCGCCTGATGAACTCTCAGTTGGTTACCACAGAGAAGCGCTTCCTCAAAGACAGTTTGTACAATGAAGGAATCCTAATTGTATGGGACCCATCTGTATACCACTCAGATATCCCAAAGTGGTACCAGAATCCGGATTATAATTTCTTTAACAACTACAAGACTTATCGTAAGCTGCACCCCAATCAGCCCTTTTACATCCTCAAGCCCCAGATGCCTTGGGAGCTATGGGACATTCTTCAAGAAATCTCCCCAGAAGAGATTCAGCCAAACCCCCCATCCTCTGGGATGCTTGGTATCATCATCATGATGACGCTGTGTGACCAGGTGGATATTTATGAGTTCCTCCCATCCAAGCGCAAGACTGACGTGTGCTACTACTACCAGAAGTTCTTCGATAGTGCCTGCACGATGGGTGCCTACCACCCGCTGCTCTATGAGAAGAATTTGGTGAAGCATCTCAACCAGGGCACAGATGAGGACATCTACCTGCTTGGAAAAGCCACACTGCCTGGCTTCCGGACCATTCACTGCTAA |
SEQ ID NO: 4 | 人ST6變體4 MIHTNLKKKFSCCVLVFLLFAVICVWKEKKKGSYYDSFKLQTKEFQVLKSLGKLAMGSDSQSVSSSSTQDPHRGRQTLGSLRGLAKAKPEASFQVWNKDSSSKNLIPRLQKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAVVSSAGSLKSSQLGREIDDHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC |
SEQ ID NO: 5 | 小鼠ST6變體1 ATGATTCATACCAACTTGAAGAGAAAGTTCAGCTGCTTTGTCCTGGTCTTTCTCCTGTTTGCCATCATCTGCGTGTGGAAGAAAGGGAGCGACTATGAGGCTCTTACATTGCAAGCCAAGGTATTCCAGATGCCGAAGAGCCAGGAGAAAGTGGCCGTGGGGCCTGCTCCCCAGGCTGTGTTCTCAAACAGCAAACAAGACCCTAAGGAAGGCGTTCAGATCCTCAGTTACCCCAGGGTCACAGCCAAGGTCAAGCCACAGCCCTCCTTGCAGGTGTGGGACAAGGACTCCACATACTCAAAACTTAACCCCAGGCTGCTGAAGATCTGGAGGAACTATCTGAACATGAATAAATATAAAGTGTCCTACAAGGGGCCGGGACCAGGAGTCAAGTTCAGCGTAGAGGCGCTGCGCTGCCACCTTCGAGACCACGTGAATGTGTCTATGATAGAGGCCACAGATTTTCCCTTCAACACCACTGAATGGGAGGGTTACCTGCCCAAGGAGAACTTCAGAACCAAGGCTGGGCCTTGGCATAAGTGTGCCGTCGTGTCTTCTGCAGGATCTCTGAAGAACTCCCAGCTGGGTCGAGAGATTGATAATCATGATGCGGTCCTGAGGTTTAATGGGGCACCTACAGACAACTTCCAACAGGATGTGGGCACAAAAACTACCATCCGCCTAGTGAACTCTCAGTTAGTCACCACAGAAAAGCGCTTCCTGAAGGACAGTTTGTACACCGAAGGAATCCTGATTCTGTGGGACCCATCTGTGTATCATGCAGACATTCCGCAGTGGTATCAGAAGCCAGACTACAACTTCTTCGAAACCTATAAGAGTTACCGAAGGCTTCACCCCAGCCAGCCTTTTTACATCCTCAAGCCCCAGATGCCATGGGAACTATGGGACATCATTCAGGAAATCTCTCCAGATCTGATTCAGCCGAATCCCCCATCCTCCGGCATGCTGGGTATCATCATTATGATGACGCTGTGTGACCAAGTTGATATTTACGAGTTCCTCCCATCCAAGCGCAAGACAGATGTGTGCTACTATCACCAGAAGTTCTTTGACAGCGCCTGCACGATGGGTGCCTACCATCCGCTCCTCTTCGAGAAGAATATGGTGAAGCATCTCAATGAGGGAACAGATGAAGACATTTATTTGTTTGGGAAAGCTACCCTGTCTGGCTTCCGGAACAATCGCTGTTGA |
SEQ ID NO: 6 | 小鼠ST6變體1 MIHTNLKRKFSCFVLVFLLFAIICVWKKGSDYEALTLQAKVFQMPKSQEKVAVGPAPQAVFSNSKQDPKEGVQILSYPRVTAKVKPQPSLQVWDKDSTYSKLNPRLLKIWRNYLNMNKYKVSYKGPGPGVKFSVEALRCHLRDHVNVSMIEATDFPFNTTEWEGYLPKENFRTKAGPWHKCAVVSSAGSLKNSQLGREIDNHDAVLRFNGAPTDNFQQDVGTKTTIRLVNSQLVTTEKRFLKDSLYTEGILILWDPSVYHADIPQWYQKPDYNFFETYKSYRRLHPSQPFYILKPQMPWELWDIIQEISPDLIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYHQKFFDSACTMGAYHPLLFEKNMVKHLNEGTDEDIYLFGKATLSGFRNNRC |
SEQ ID NO: 7 | 小鼠ST6變體2 ATGATTCATACCAACTTGAAGAGAAAGTTCAGCTGCTTTGTCCTGGTCTTTCTCCTGTTTGCCATCATCTGCGTGTGGAAGAAAGGGAGCGACTATGAGGCTCTTACATTGCAAGCCAAGGTATTCCAGATGCCGAAGAGCCAGGAGAAAGTGGCCGTGGGGCCTGCTCCCCAGGCTGTGTTCTCAAACAGCAAACAAGACCCTAAGGAAGGCGTTCAGATCCTCAGTTACCCCAGGGTCACAGCCAAGGTCAAGCCACAGCCCTCCTTGCAGGTGTGGGACAAGGACTCCACATACTCAAAACTTAACCCCAGGCTGCTGAAGATCTGGAGGAACTATCTGAACATGAATAAATATAAAGTGTCCTACAAGGGGCCGGGACCAGGAGTCAAGTTCAGCGTAGAGGCGCTGCGCTGCCACCTTCGAGACCACGTGAATGTGTCTATGATAGAGGCCACAGATTTTCCCTTCAACACCACTGAATGGGAGGGTTACCTGCCCAAGGAGAACTTCAGAACCAAGGCTGGGCCTTGGCATAAGTGTGCCGTCGTGTCTTCTGCAGGATCTCTGAAGAACTCCCAGCTGGGTCGAGAGATTGATAATCATGATGCGGTCCTGAGGTTTAATGGGGCACCTACAGACAACTTCCAACAGGATGTGGGCACAAAAACTACCATCCGCCTAGTGAACTCTCAGTTAGTCACCACAGAAAAGCGCTTCCTGAAGGACAGTTTGTACACCGAAGGAATCCTGATTCTGTGGGACCCATCTGTGTATCATGCAGACATTCCGCAGTGGTATCAGAAGCCAGACTACAACTTCTTCGAAACCTATAAGAGTTACCGAAGGCTTCACCCCAGCCAGCCTTTTTACATCCTCAAGCCCCAGATGCCATGGGAACTATGGGACATCATTCAGGAAATCTCTCCAGATCTGATTCAGCCGAATCCCCCATCCTCCGGCATGCTGGGTATCATCATTATGATGACGCTGTGTGACCAAGTTGATATTTACGAGTTCCTCCCATCCAAGCGCAAGACAGATGTGTGCTACTATCACCAGAAGTTCTTTGACAGCGCCTGCACGATGGGTGCCTACCATCCGCTCCTCTTCGAGAAGAATATGGTGAAGCATCTCAATGAGGGAACAGATGAAGACATTTATTTGTTTGGGAAAGCTACCCTGTCTGGCTTCCGGAACAATCGCTGTTGA |
SEQ ID NO: 8 | 小鼠ST6變體2 MIHTNLKRKFSCFVLVFLLFAIICVWKKGSDYEALTLQAKVFQMPKSQEKVAVGPAPQAVFSNSKQDPKEGVQILSYPRVTAKVKPQPSLQVWDKDSTYSKLNPRLLKIWRNYLNMNKYKVSYKGPGPGVKFSVEALRCHLRDHVNVSMIEATDFPFNTTEWEGYLPKENFRTKAGPWHKCAVVSSAGSLKNSQLGREIDNHDAVLRFNGAPTDNFQQDVGTKTTIRLVNSQLVTTEKRFLKDSLYTEGILILWDPSVYHADIPQWYQKPDYNFFETYKSYRRLHPSQPFYILKPQMPWELWDIIQEISPDLIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYHQKFFDSACTMGAYHPLLFEKNMVKHLNEGTDEDIYLFGKATLSGFRNNRC |
SEQ ID NO: 9 | 小鼠ST6變體3 ATGATTCATACCAACTTGAAGAGAAAGTTCAGCTGCTTTGTCCTGGTCTTTCTCCTGTTTGCCATCATCTGCGTGTGGAAGAAAGGGAGCGACTATGAGGCTCTTACATTGCAAGCCAAGGTATTCCAGATGCCGAAGAGCCAGGAGAAAGTGGCCGTGGGGCCTGCTCCCCAGGCTGTGTTCTCAAACAGCAAACAAGACCCTAAGGAAGGCGTTCAGATCCTCAGTTACCCCAGGGTCACAGCCAAGGTCAAGCCACAGCCCTCCTTGCAGGTGTGGGACAAGGACTCCACATACTCAAAACTTAACCCCAGGCTGCTGAAGATCTGGAGGAACTATCTGAACATGAATAAATATAAAGTGTCCTACAAGGGGCCGGGACCAGGAGTCAAGTTCAGCGTAGAGGCGCTGCGCTGCCACCTTCGAGACCACGTGAATGTGTCTATGATAGAGGCCACAGATTTTCCCTTCAACACCACTGAATGGGAGGGTTACCTGCCCAAGGAGAACTTCAGAACCAAGGCTGGGCCTTGGCATAAGTGTGCCGTCGTGTCTTCTGCAGGATCTCTGAAGAACTCCCAGCTGGGTCGAGAGATTGATAATCATGATGCGGTCCTGAGGTTTAATGGGGCACCTACAGACAACTTCCAACAGGATGTGGGCACAAAAACTACCATCCGCCTAGTGAACTCTCAGTTAGTCACCACAGAAAAGCGCTTCCTGAAGGACAGTTTGTACACCGAAGGAATCCTGATTCTGTGGGACCCATCTGTGTATCATGCAGACATTCCGCAGTGGTATCAGAAGCCAGACTACAACTTCTTCGAAACCTATAAGAGTTACCGAAGGCTTCACCCCAGCCAGCCTTTTTACATCCTCAAGCCCCAGATGCCATGGGAACTATGGGACATCATTCAGGAAATCTCTCCAGATCTGATTCAGCCGAATCCCCCATCCTCCGGCATGCTGGGTATCATCATTATGATGACGCTGTGTGACCAAGTTGATATTTACGAGTTCCTCCCATCCAAGCGCAAGACAGATGTGTGCTACTATCACCAGAAGTTCTTTGACAGCGCCTGCACGATGGGTGCCTACCATCCGCTCCTCTTCGAGAAGAATATGGTGAAGCATCTCAATGAGGGAACAGATGAAGACATTTATTTGTTTGGGAAAGCTACCCTGTCTGGCTTCCGGAACAATCGCTGTTGA |
SEQ ID NO: 10 | 小鼠ST6變體3 MIHTNLKRKFSCFVLVFLLFAIICVWKKGSDYEALTLQAKVFQMPKSQEKVAVGPAPQAVFSNSKQDPKEGVQILSYPRVTAKVKPQPSLQVWDKDSTYSKLNPRLLKIWRNYLNMNKYKVSYKGPGPGVKFSVEALRCHLRDHVNVSMIEATDFPFNTTEWEGYLPKENFRTKAGPWHKCAVVSSAGSLKNSQLGREIDNHDAVLRFNGAPTDNFQQDVGTKTTIRLVNSQLVTTEKRFLKDSLYTEGILILWDPSVYHADIPQWYQKPDYNFFETYKSYRRLHPSQPFYILKPQMPWELWDIIQEISPDLIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYHQKFFDSACTMGAYHPLLFEKNMVKHLNEGTDEDIYLFGKATLSGFRNNRC |
SEQ ID NO: 11 | 大鼠ST6變體3 ATGATTCATACCAACTTGAAGAAAAAGTTCAGCCTCTTCATCCTGGTCTTTCTCCTGTTCGCAGTCATCTGTGTTTGGAAGAAAGGGAGCGACTATGAGGCCCTTACACTGCAAGCCAAGGAATTCCAGATGCCCAAGAGCCAGGAGAAAGTGGCCATGGGGTCTGCTTCCCAGGTTGTGTTCTCAAACAGCAAGCAAGACCCTAAGGAAGACATTCCAATCCTCAGTTACCACAGGGTCACAGCCAAGGTCAAACCACAGCCTTCCTTCCAGGTGTGGGACAAGGACTCCACATACTCAAAACTTAACCCCAGGCTGCTGAAGATCTGGAGAAACTATCTGAACATGAACAAATATAAAGTATCCTACAAGGGACCGGGGCCAGGAGTCAAGTTCAGCGTAGAAGCACTGCGTTGCCACCTTCGAGACCATGTGAACGTGTCTATGATAGAGGCCACAGATTTTCCCTTCAACACCACTGAGTGGGAGGGTTACCTGCCCAAGGAGAACTTTAGAACCAAGGTTGGGCCTTGGCAAAGGTGTGCCGTCGTCTCTTCTGCAGGATCTCTGAAAAACTCCCAGCTTGGTCGAGAGATTGATAATCATGATGCAGTTCTGAGGTTTAATGGGGCCCCTACCGACAACTTCCAACAGGATGTGGGCTCAAAAACTACCATTCGCCTAATGAACTCTCAGTTAGTCACCACAGAAAAGCGCTTCCTCAAGGACAGTTTGTACACCGAAGGAATCCTAATTGTATGGGACCCATCCGTGTATCATGCAGATATCCCAAAGTGGTATCAGAAACCAGACTACAATTTCTTCGAAACCTATAAGAGTTACCGAAGGCTGAACCCCAGCCAGCCATTTTATATCCTCAAGCCCCAGATGCCATGGGAACTGTGGGACATCATTCAGGAAATCTCTGCAGATCTGATTCAGCCAAATCCCCCATCCTCCGGCATGCTGGGTATCATCATCATGATGACGCTGTGTGACCAGGTAGATATTTACGAGTTCCTCCCATCCAAGCGCAAGACGGACGTGTGCTATTATCACCAAAAGTTCTTTGACAGCGCTTGCACGATGGGTGCCTACCACCCGCTCCTCTTCGAGAAGAATATGGTGAAGCATCTCAATGAGGGAACAGATGAAGACATTTATTTGTTTGGGAAAGCCACCCTTTCTGGCTTCCGGAACATTCGTTGTTGA |
SEQ ID NO: 12 | 大鼠ST6變體3 MIHTNLKKKFSLFILVFLLFAVICVWKKGSDYEALTLQAKEFQMPKSQEKVAMGSASQVVFSNSKQDPKEDIPILSYHRVTAKVKPQPSFQVWDKDSTYSKLNPRLLKIWRNYLNMNKYKVSYKGPGPGVKFSVEALRCHLRDHVNVSMIEATDFPFNTTEWEGYLPKENFRTKVGPWQRCAVVSSAGSLKNSQLGREIDNHDAVLRFNGAPTDNFQQDVGSKTTIRLMNSQLVTTEKRFLKDSLYTEGILIVWDPSVYHADIPKWYQKPDYNFFETYKSYRRLNPSQPFYILKPQMPWELWDIIQEISADLIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYHQKFFDSACTMGAYHPLLFEKNMVKHLNEGTDEDIYLFGKATLSGFRNIRC |
SEQ ID NO: 13 | 人B4GALT1 ATGAGGCTTCGGGAGCCGCTCCTGAGCGGCAGCGCCGCGATGCCAGGCGCGTCCCTACAGCGGGCCTGCCGCCTGCTCGTGGCCGTCTGCGCTCTGCACCTTGGCGTCACCCTCGTTTACTACCTGGCTGGCCGCGACCTGAGCCGCCTGCCCCAACTGGTCGGAGTCTCCACACCGCTGCAGGGCGGCTCGAACAGTGCAGCAGCTATAGGCCAGTCAAGCGGTGAACTGAGGACTGGGGGTGCCCGACCACCACCTCCACTCGGCGCTTCATCCCAACCACGTCCTGGGGGAGACTCCAGCCCAGTCGTGGATTCTGGCCCTGGCCCCGCTAGCAACTTGACCTCGGTCCCAGTGCCCCACACCACCGCACTGTCGCTGCCCGCCTGCCCTGAGGAGTCCCCGCTGCTTGTGGGCCCCATGCTGATTGAGTTTAACATGCCTGTGGACCTGGAGCTCGTGGCAAAGCAGAACCCAAATGTGAAGATGGGCGGCCGCTATGCCCCCAGGGACTGCGTCTCTCCTCACAAGGTGGCCATCATCATTCCATTCCGCAACCGGCAGGAGCACCTCAAGTACTGGCTATATTATTTGCACCCAGTCCTGCAGCGCCAGCAGCTGGACTATGGCATCTATGTTATCAACCAGGCGGGAGACACTATATTCAATCGTGCTAAGCTCCTCAATGTTGGCTTTCAAGAAGCCTTGAAGGACTATGACTACACCTGCTTTGTGTTTAGTGACGTGGACCTCATTCCAATGAATGACCATAATGCGTACAGGTGTTTTTCACAGCCACGGCACATTTCCGTTGCAATGGATAAGTTTGGATTCAGCCTACCTTATGTTCAGTATTTTGGAGGTGTCTCTGCTCTAAGTAAACAACAGTTTCTAACCATCAATGGATTTCCTAATAATTATTGGGGCTGGGGAGGAGAAGATGATGACATTTTTAACAGATTAGTTTTTAGAGGCATGTCTATATCTCGCCCAAATGCTGTGGTCGGGAGGTGTCGCATGATCCGCCACTCAAGAGACAAGAAAAATGAACCCAATCCTCAGAGGTTTGACCGAATTGCACACACAAAGGAGACAATGCTCTCTGATGGTTTGAACTCACTCACCTACCAGGTGCTGGATGTACAGAGATACCCATTGTATACCCAAATCACAGTGGACATCGGGACACCGAGCTAG |
SEQ ID NO: 14 | 人B4GALT1 MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS |
SEQ ID NO: 15 | 人B4GALT1變體2 ATGCCAGGCGCGTCCCTACAGCGGGCCTGCCGCCTGCTCGTGGCCGTCTGCGCTCTGCACCTTGGCGTCACCCTCGTTTACTACCTGGCTGGCCGCGACCTGAGCCGCCTGCCCCAACTGGTCGGAGTCTCCACACCGCTGCAGGGCGGCTCGAACAGTGCCGCCGCCATCGGGCAGTCCTCCGGGGAGCTCCGGACCGGAGGGGCCCGGCCGCCGCCTCCTCTAGGCGCCTCCTCCCAGCCGCGCCCGGGTGGCGACTCCAGCCCAGTCGTGGATTCTGGCCCTGGCCCCGCTAGCAACTTGACCTCGGTCCCAGTGCCCCACACCACCGCACTGTCGCTGCCCGCCTGCCCTGAGGAGTCCCCGCTGCTTGTGGGCCCCATGCTGATTGAGTTTAACATGCCTGTGGACCTGGAGCTCGTGGCAAAGCAGAACCCAAATGTGAAGATGGGCGGCCGCTATGCCCCCAGGGACTGCGTCTCTCCTCACAAGGTGGCCATCATCATTCCATTCCGCAACCGGCAGGAGCACCTCAAGTACTGGCTATATTATTTGCACCCAGTCCTGCAGCGCCAGCAGCTGGACTATGGCATCTATGTTATCAACCAGGCGGGAGACACTATATTCAATCGTGCTAAGCTCCTCAATGTTGGCTTTCAAGAAGCCTTGAAGGACTATGACTACACCTGCTTTGTGTTTAGTGACGTGGACCTCATTCCAATGAATGACCATAATGCGTACAGGTGTTTTTCACAGCCACGGCACATTTCCGTTGCAATGGATAAGTTTGGATTCAGCCTACCTTATGTTCAGTATTTTGGAGGTGTCTCTGCTCTAAGTAAACAACAGTTTCTAACCATCAATGGATTTCCTAATAATTATTGGGGCTGGGGAGGAGAAGATGATGACATTTTTAACAGATTAGTTTTTAGAGGCATGTCTATATCTCGCCCAAATGCTGTGGTCGGGAGGTGTCGCATGATCCGCCACTCAAGAGACAAGAAAAATGAACCCAATCCTCAGAGGTTTGACCGAATTGCACACACAAAGGAGACAATGCTCTCTGATGGTTTGAACTCACTCACCTACCAGGTGCTGGATGTACAGAGATACCCATTGTATACCCAAATCACAGTGGACATCGGGACACCGAGCTAG |
SEQ ID NO: 16 | 人B4GALT1變體2 MPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVS TPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS |
SEQ ID NO: 17 | 人B4GALT1變體3 ATGAGGCTTCGGGAGCCGCTCCTGAGCGGCAGCGCCGCGATGCCAGGCGCGTCCCTACAGCGGGCCTGCCGCCTGCTCGTGGCCGTCTGCGCTCTGCACCTTGGCGTCACCCTCGTTTACTACCTGGCTGGCCGCGACCTGAGCCGCCTGCCCCAACTGGTCGGAGTCTCCACACCGCTGCAGGGCGGCTCGAACAGTGCCGCCGCCATCGGGCAGTCCTCCGGGGAGCTCCGGACCGGAGGGGCCCGGCCGCCGCCTCCTCTAGGCGCCTCCTCCCAGCCGCGCCCGGGTGGCGACTCCAGCCCAGTCGTGGATTCTGGCCCTGGCCCCGCTAGCAACTTGACCTCGGTCCCAGTGCCCCACACCACCGCACTGTCGCTGCCCGCCTGCCCTGAGGAGTCCCCGCTGCTTGTGGGCCCCATGCTGATTGAGTTTAACATGCCTGTGGACCTGGAGCTCGTGGCAAAGCAGAACCCAAATGTGAAGATGGGCGGCCGCTATGCCCCCAGGGACTGCGTCTCTCCTCACAAGGTGGCCATCATCATTCCATTCCGCAACCGGCAGGAGCACCTCAAGTACTGGCTATATTATTTGCACCCAGTCCTGCAGCGCCAGCAGCTGGACTATGGCATCTATGTTATCAACCAGGCGGGAGACACTATATTCAATCGTGCTAAGCTCCTCAATGTTGGCTTTCAAGAAGCCTTGAAGGACTATGACTACACCTGCTTTGTGTTTAGTGACGTGGACCTCATTCCAATGAATGACCATAATGCGTACAGGTGTTTTTCACAGCCACGGCACATTTCCGTTGCAATGGATAAGTTTGGATTCAGATTAGTTTTTAGAGGCATGTCTATATCTCGCCCAAATGCTGTGGTCGGGAGGTGTCGCATGATCCGCCACTCAAGAGACAAGAAAAATGAACCCAATCCTCAGAGGTTTGACCGAATTGCACACACAAAGGAGACAATGCTCTCTGATGGTTTGAACTCACTCACCTACCAGGTGCTGGATGTACAGAGATACCCATTGTATACCCAAATCACAGTGGACATCGGGACACCGAGCTAG |
SEQ ID NO: 18 | 人B4GALT1變體3 MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS |
SEQ ID NO: 19 | 人B4GALT1變體4 ATGAGGCTTCGGGAGCCGCTCCTGAGCGGCAGCGCCGCGATGCCAGGCGCGTCCCTACAGCGGGCCTGCCGCCTGCTCGTGGCCGTCTGCGCTCTGCACCTTGGCGTCACCCTCGTTTACTACCTGGCTGGCCGCGACCTGAGCCGCCTGCCCCAACTGGTCGGAGTCTCCACACCGCTGCAGGGCGGCTCGAACAGTGCCGCCGCCATCGGGCAGTCCTCCGGGGAGCTCCGGACCGGAGGGGCCCGGCCGCCGCCTCCTCTAGGCGCCTCCTCCCAGCCGCGCCCGGGTGGCGACTCCAGCCCAGTCGTGGATTCTGGCCCTGGCCCCGCTAGCAACTTGACCTCGGTCCCAGTGCCCCACACCACCGCACTGTCGCTGCCCGCCTGCCCTGAGGAGTCCCCGCTGCTTGTGGGCCCCATGCTGATTGAGTTTAACATGCCTGTGGACCTGGAGCTCGTGGCAAAGCAGAACCCAAATGTGAAGATGGGCGGCCGCTATGCCCCCAGGGACTGCGTCTCTCCTCACAAGGTGGCCATCATCATTCCATTCCGCAACCGGCAGGAGCACCTCAAGTACTGGCTATATTATTTGCACCCAGTCCTGCAGCGCCAGCAGCTGGACTATGGCATCTATGTTATCAACCAGTATGAAAAAATAAGAAGACTCCTGTGGTGA |
SEQ ID NO: 20 | 人B4GALT1變體4 MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQYEKIRRLLW |
SEQ ID NO: 21 | CHO B4GALT1 ATGAGGTTTCTGAGGCCGGTCCTGGGCGGCAGCGCCGCGATGCCGGGCGCGACCCTGCAGCGGGCCTGCCGCCTGCTCGTGGCCGTCTGCGCGCTGCACCTCGGCGTCACCCTGGTCTATTACCTCTCGGGCCGCGACCTGAGCCGCCTGCCTCAGCTAGTCGGGGTTTCCTCTACACTTCGGAGCGGCACGATCGGCGCCACCGCCAACAAGCAGCCCCCCGGCGCGCGGCCGCCGCCTCCTGTGGGAGTCTCCTCGAAGCCTCGCCCGGGTCCCGACTCCAGCCCTGGTACGGCTTTTGACCCCGGCCTGAAGAGCAACTGGACTTCGGTCCTAGTGCCCCCTACCACTGCACTGTTGACGCTGCCCGCTTGCCCCGAGGAGTCCCCGCTGCTCGTTGGCCCCATGGTGATTGACTTTAATATTGCTGTGGACCTGGAGCTTTTGGCAAAGAAGAACCCAGAGATAAAGATGGGCGGTCGTTATTCCCCCAAGGACTGTATTTCTCCTCACAAGGTGGCCATCATTATCCCATTTCGCAACCGGCAGGAGCACCTCAAATATTGGCTGTATTATTTGCATCCGGTCCTACAGCGCCAGCAACTGGACTATGGTATCTACGTCATCAACCAGGCTGGAGACACCATGTTCAATCGAGCTAAGCTTCTCAACATAGGCTTCCAAGAGGCCTTGAAGGACCATGACTACAACTGCTTTGTGTTCAGTGATGTGGACCTTATTCCAATGGACGACCATAATGCCTACAGGTGTTTTTCACAGCCCCGGCACATTTCTGTTGCAATGGACAAGTTTGGGTTTAGCCTGCCTTATGTTCAGTACTTTGGAGGCGTTTCTGCCCTCAGTAAACAACAGTTTCTTGCCATCAATGGATTTCCTAATAATTATTGGGGCTGGGGAGGAGAAGATGATGACATTTTTAACAGAATAGTTCATAAGGGAATGTCTATATCACGCCCAAATGCTGTAGTTGGAAGATGTCGCATGATACGACACTCAAGAGACAAGAAAAATGAGCCCAATCCTCAGAGGTTTGACCGGATTGCTCACACAAAGGAAACTATGCGCTTTGATGGTTTGAACTCACTTACCTACCAGGTGTTGAACGTAGAGAGATACCCATTATATACCAAAATCACAGTGGACATCGGGACACCAAGATAGCATTGTGGAACAAATAACAGACCTGAAAATGGCCAGGGGCTCAGA |
SEQ ID NO: 22 | CHO B4GALT1 MRFLRPVLGGSAAMPGATLQRACRLLVAVCALHLGVTLVYYLSGRDLSRLPQLVGVSSTLRSGTIGATANKQPPGARPPPPVGVSSKPRPGPDSSPGTAFDPGLKSNWTSVLVPPTTALLTLPACPEESPLLVGPMVIDFNIAVDLELLAKKNPEIKMGGRYSPKDCISPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQAGDTMFNRAKLLNIGFQEALKDHDYNCFVFSDVDLIPMDDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLAINGFPNNYWGWGGEDDDIFNRIVHKGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMRFDGLNSLTYQVLNVERYPLYTKITVDIGTPR |
SEQ ID NO: 23 | 小鼠B4GALT1變體1 ATGAGGTTTCGTGAGCAGTTCCTGGGCGGCAGCGCCGCGATGCCGGGCGCGACCCTGCAGCGGGCCTGCCGCCTGCTCGTGGCCGTCTGCGCGCTGCACCTCGGCGTCACCCTCGTCTATTACCTCTCTGGCCGCGATCTGAGCCGCCTGCCCCAGTTGGTCGGAGTCTCCTCTACACTGCAGGGCGGCACGAACGGCGCCGCAGCCAGCAAGCAGCCCCCAGGAGAGCAGCGGCCGCGGGGTGCGCGGCCGCCGCCTCCTTTAGGCGTCTCCCCGAAGCCTCGCCCGGGTCTCGACTCCAGCCCTGGTGCAGCTTCTGGCCCCGGCTTGAAGAGCAACTTGTCTTCGTTGCCAGTGCCCACCACCACTGGACTGTTGTCGCTGCCAGCTTGCCCTGAGGAGTCCCCGCTGCTCGTTGGCCCCATGCTGATTGACTTTAATATTGCTGTGGATCTGGAGCTTTTGGCAAAGAAGAACCCAGAGATAAAGACGGGCGGCCGTTACTCCCCCAAGGACTGTGTCTCTCCTCACAAGGTGGCCATCATCATCCCATTCCGTAACCGGCAGGAGCATCTCAAATACTGGCTGTATTATTTGCATCCCATCCTTCAGCGCCAGCAACTCGACTATGGCATCTACGTCATCAATCAGGCTGGAGACACCATGTTCAATCGAGCTAAGCTGCTCAATATTGGCTTTCAAGAGGCCTTGAAGGACTATGATTACAACTGCTTTGTGTTCAGTGATGTGGACCTCATTCCGATGGACGACCGTAATGCCTACAGGTGTTTTTCGCAGCCACGGCACATTTCTGTTGCAATGGACAAGTTCGGGTTTAGCCTGCCATATGTTCAGTATTTTGGAGGTGTCTCTGCTCTCAGTAAACAACAGTTTCTTGCCATCAATGGATTCCCTAATAATTATTGGGGTTGGGGAGGAGAAGATGACGACATTTTTAACAGATTAGTTCATAAAGGCATGTCTATATCACGTCCAAATGCTGTAGTAGGGAGGTGTCGAATGATCCGGCATTCAAGAGACAAGAAAAATGAGCCCAATCCTCAGAGGTTTGACCGGATCGCACATACAAAGGAAACGATGCGCTTCGATGGTTTGAACTCACTTACCTACAAGGTGTTGGATGTACAGAGATACCCGTTATATACCCAAATCACAGTGGACATCGGGACACCGAGATAG |
SEQ ID NO: 24 | 小鼠B4GALT1變體1 MRFREQFLGGSAAMPGATLQRACRLLVAVCALHLGVTLVYYLSGRDLSRLPQLVGVSSTLQGGTNGAAASKQPPGEQRPRGARPPPPLGVSPKPRPGLDSSPGAASGPGLKSNLSSLPVPTTTGLLSLPACPEESPLLVGPMLIDFNIAVDLELLAKKNPEIKTGGRYSPKDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPILQRQQLDYGIYVINQAGDTMFNRAKLLNIGFQEALKDYDYNCFVFSDVDLIPMDDRNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLAINGFPNNYWGWGGEDDDIFNRLVHKGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMRFDGLNSLTYKVLDVQRYPLYTQITVDIGTPR |
SEQ ID NO: 25 | 小鼠B4GALT1變體2 ATGCCGGGCGCGACCCTGCAGCGGGCCTGCCGCCTGCTCGTGGCCGTCTGCGCGCTGCACCTCGGCGTCACCCTCGTCTATTACCTCTCTGGCCGCGATCTGAGCCGCCTGCCCCAGTTGGTCGGAGTCTCCTCTACACTGCAGGGCGGCACGAACGGCGCCGCAGCCAGCAAGCAGCCCCCAGGAGAGCAGCGGCCGCGGGGTGCGCGGCCGCCGCCTCCTTTAGGCGTCTCCCCGAAGCCTCGCCCGGGTCTCGACTCCAGCCCTGGTGCAGCTTCTGGCCCCGGCTTGAAGAGCAACTTGTCTTCGTTGCCAGTGCCCACCACCACTGGACTGTTGTCGCTGCCAGCTTGCCCTGAGGAGTCCCCGCTGCTCGTTGGCCCCATGCTGATTGACTTTAATATTGCTGTGGATCTGGAGCTTTTGGCAAAGAAGAACCCAGAGATAAAGACGGGCGGCCGTTACTCCCCCAAGGACTGTGTCTCTCCTCACAAGGTGGCCATCATCATCCCATTCCGTAACCGGCAGGAGCATCTCAAATACTGGCTGTATTATTTGCATCCCATCCTTCAGCGCCAGCAACTCGACTATGGCATCTACGTCATCAATCAGGCTGGAGACACCATGTTCAATCGAGCTAAGCTGCTCAATATTGGCTTTCAAGAGGCCTTGAAGGACTATGATTACAACTGCTTTGTGTTCAGTGATGTGGACCTCATTCCGATGGACGACCGTAATGCCTACAGGTGTTTTTCGCAGCCACGGCACATTTCTGTTGCAATGGACAAGTTCGGGTTTAGCCTGCCATATGTTCAGTATTTTGGAGGTGTCTCTGCTCTCAGTAAACAACAGTTTCTTGCCATCAATGGATTCCCTAATAATTATTGGGGTTGGGGAGGAGAAGATGACGACATTTTTAACAGATTAGTTCATAAAGGCATGTCTATATCACGTCCAAATGCTGTAGTAGGGAGGTGTCGAATGATCCGGCATTCAAGAGACAAGAAAAATGAGCCCAATCCTCAGAGGTTTGACCGGATCGCACATACAAAGGAAACGATGCGCTTCGATGGTTTGAACTCACTTACCTACAAGGTGTTGGATGTACAGAGATACCCGTTATATACCCAAATCACAGTGGACATCGGGACACCGAGATAG |
SEQ ID NO: 26 | 小鼠B4GALT1變體2 MPGATLQRACRLLVAVCALHLGVTLVYYLSGRDLSRLPQLVGVSSTLQGGTNGAAASKQPPGEQRPRGARPPPPLGVSPKPRPGLDSSPGAASGPGLKSNLSSLPVPTTTGLLSLPACPEESPLLVGPMLIDFNIAVDLELLAKKNPEIKTGGRYSPKDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPILQRQQLDYGIYVINQAGDTMFNRAKLLNIGFQEALKDYDYNCFVFSDVDLIPMDDRNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLAINGFPNNYWGWGGEDDDIFNRLVHKGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMRFDGLNSLTYKVLDVQRYPLYTQITVDIGTPR |
SEQ ID NO: 27 | 大鼠B4GALT1 ATGAGGTTTCGTGAGCCGTTCCTGGGCGGCAGCGCCGCGATGCCGGGCGCGACCCTGCAGCGGGCCTGCCGCCTGCTCGTGGCGGTCTGCGCGCTGCACCTTGGCGTCACCCTGGTCTATTACCTCTCCGGTCGCGATCTGAGCCGCCTGCCCCAACTGGTCGGAGTCTCCTCTTCACTGCAAGGCGGCACGAACGGCGCCGCCGCCAGCAAGCAGCCCTCGGGAGAGCTCCGGCCCCGGGGCGCGCGGCCGCCGCCTCCTTTAGGCGTCTCCCCGAAGCCTCGCCCGGGTTCTGACTCCAGCCCTGATGCGGCTTCTGGCCCCGGCCTGAAGAGCAACTTGACTTCGGTGCCAATGCCCACCAGCACTGGATTGTTGACTCTGCCTGCTTGCCCTGAGGAGTCCCCGCTGCTCGTTGGCCCCATGGTGATTGACTTTAATATTCCTGTGGATCTGGAGCTTTTGGCAAAGAAGAACCCAGAGATAAAGATGGGCGGCCGTTACTTCCCCAAGGACTGTATCTCCCCTCACAAGGTGGCCATCATTATCCCATTCCGTAACCGGCAGGAGCACCTCAAATACTGGCTGTATTATTTGCATCCAGTCCTTCAGCGCCAGCAACTCGACTATGGCATCTACGTCATCAATCAGGCTGGAGACACCATGTTTAATCGAGCTAAGCTGCTCAACGTTGGCTTTCAAGAGGCCTTGAAAGACTATGACTACAACTGCTTTGTGTTCAGTGATGTGGACCTCATTCCAATGGATGACCATAATGCCTACAGGTGCTTTTCACAGCCACGGCATATTTCTGTCGCAATGGACAAGTTCGGGTTTAGCCTGCCTTACGTTCAGTATTTTGGAGGTGTCTCCGCTCTCAGTAAACAACAGTTCCTTACCATCAATGGATTTCCTAATAATTACTGGGGCTGGGGAGGAGAAGATGATGACATTTTTAACAGATTAGTTCATAAAGGCATGTCTATATCACGCCCAAATGCTGTGGTAGGCAGGTGTCGCATGATCCGGCACTCAAGAGACAAGAAAAATGAGCCCAACCCTCAGAGGTTTGACCGGATCGCACATACAAAGGAAACGATGCGCCTTGATGGTTTGAACTCACTTACCTACCAGGTGTTGGACATACAGAGATACCCGTTATATACCAAAATCACAGTGGACATCGGGACACCAAGATAG |
SEQ ID NO: 28 | 大鼠B4GALT1 MRFREPFLGGSAAMPGATLQRACRLLVAVCALHLGVTLVYYLSGRDLSRLPQLVGVSSSLQGGTNGAAASKQPSGELRPRGARPPPPLGVSPKPRPGSDSSPDAASGPGLKSNLTSVPMPTSTGLLTLPACPEESPLLVGPMVIDFNIPVDLELLAKKNPEIKMGGRYFPKDCISPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQLDYGIYVINQAGDTMFNRAKLLNVGFQEALKDYDYNCFVFSDVDLIPMDDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTINGFPNNYWGWGGEDDDIFNRLVHKGMSISRPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMRLDGLNSLTYQVLDIQRYPLYTKITVDIGTPR |
在一些實施方式中,對於包括將編碼B4GALT1和ST6的多核苷酸序列引入CHO細胞的方法,在同一載體中提供這種多核苷酸序列。在一些實施方式中,在兩個不同的載體中提供編碼B4GALT1的多核苷酸序列和編碼ST6的多核苷酸序列。
在一些實施方式中,將編碼ST6的多核苷酸序列和/或編碼B4GALT1的多核苷酸序列可操作地連接至啟動子。
本文所述方法進一步包括:在包含一定量的錳和半乳糖的培養基中培養CHO細胞,該細胞已經被修飾為1) 單獨表現ST6;或2) 共表現B4GALT1和ST6,該一定量的錳和半乳糖有效增加由在培養基中生長的細胞產生的治療性蛋白的唾液酸化。在一個實施方式中,所述量的錳和半乳糖對細胞無毒,即不會降低細胞活力、細胞生長、或蛋白質產生。在相關實施方式中,本揭露提供了一種培養基,該培養基包含一定量的錳和半乳糖,該一定量的錳和半乳糖有效增加由在該培養基中生長的細胞產生的治療性蛋白的唾液酸化。
在一些實施方式中,在細胞培養期間,與在不包含添加的錳和半乳糖的培養基中培養的CHO細胞產生的蛋白相比,使用本文所述方法由CHO細胞產生的治療性蛋白的唾液酸化增加了至少5%。在一些實施方式中,在細胞培養期間,與在不包含添加的錳和半乳糖的培養基中培養的CHO細胞產生的蛋白相比,由CHO細胞產生的治療性蛋白的唾液酸化增加了至少約5%、6%、7%、8%、9%、10%、11%、12%、13%、14%、15%、16%、17%、18%、19%、20%、21%、22%、23%、24%、25%、30%、35%、40%、50%、60%、70%或更多。
在一些實施方式中,在本文所述方法中使用的培養基可以具有從0 ppb至200 ppb(十億分率)範圍內(例如,液體配製物中的99 ppb)的痕量的錳,並且本文所述方法包括在細胞培養期間進一步用錳補充培養基。在一些實施方式中,培養基中的痕量的錳係99 ppb或100 ppb。在一些實施方式中,本文所述方法包括向培養基中添加從約40 ppb至約500 ppb或從約200 ppb至約400 ppb、或從約100 ppb至約300 ppb範圍內的量的錳。在其他示例性實施方式中,以所需範圍的下限的錳的濃度(例如,將補充錳的培養基添加到培養中的宿主細胞後的最終濃度)的範圍可為約40 ppb、50 ppb、60 ppb、70 ppb、80 ppb、90 ppb、100 ppb、120 ppb、140 ppb、160 ppb、180 ppb、200 ppb、220 ppb、240 ppb、260 ppb、280 ppb,300 ppb、320 ppb、340 ppb,360 ppb、380 ppb、400 ppb或更高;以範圍的上限的錳的濃度(例如,將補充錳的培養基添加到培養中的宿主細胞後的最終濃度)的範圍也可以高達約500 ppb、450 ppb、400 ppb、350 ppb、300 ppb、280 ppb、260 ppb、240 ppb、220 ppb、200 ppb、200 ppb、180 ppb、160 ppb、140 ppb、120 ppb、100 ppb、90 ppb、80 ppb、70 ppb、60 ppb、50 ppb或40 ppb。在一些實施方式中,培養基中的錳的濃度(例如,將補充錳的培養基添加到培養中的宿主細胞後的最終濃度)係約300 ppb。在一些實施方式中,培養基中的錳的濃度(例如,將補充錳的培養基添加到培養中的宿主細胞後的最終濃度)係約400 ppb。在一些實施方式中,將錳作為錳鹽(例如,硫酸錳(II),一水合物)添加到培養基中。
在一些實施方式中,在本文所述方法中使用的培養基在標準培養基組成物中可以具有從0 mM至50 mM範圍內的痕量的半乳糖,並且本文所述方法包括在細胞培養期間進一步添加一定量的半乳糖。在一些實施方式中,培養基在細胞培養之前不含任何半乳糖。在示例性實施方式中,該等方法包括向培養基中添加範圍從約10 mM至約100 mM、從約30 mM至約90 mM、或從約25 mM至約50 mM的量的半乳糖。在其他示例性實施方式中,以所需範圍的下限的半乳糖的濃度(例如,將補充半乳糖的培養基添加到培養中的宿主細胞後的最終濃度)的範圍可為約10 mM、15 mM、20 mM、25 mM、30 mM、35 mM、40 mM、45 mM、50 mM、55 mM、60 mM、65 mM、70 mM、75 mM、80 mM、85 mM、90 mM或更高。在一些實施方式中,以範圍的上限的半乳糖的濃度(例如,將補充半乳糖的培養基添加到培養中的宿主細胞後的最終濃度)的範圍也可以高達約100 mM、95 mM、90 mM、85 mM、80 mM、75 mM、70 mM、65 mM、60 mM、55 mM、50 mM、45 mM、40 mM、35 mM、30 mM、25 mM、20 mM、15 mM、或10 mM。在一些實施方式中,培養基中的半乳糖的濃度(例如,將補充半乳糖的培養基添加到培養中的宿主細胞後的最終濃度)係約45 mM或46 mM。在一些實施方式中,培養基中的錳的濃度(例如,將補充半乳糖的培養基添加到培養中的宿主細胞後的最終濃度)係約40 mM。在一些實施方式中,將半乳糖作為無水半乳糖添加到培養基中。
在一些實施方式中,本文所述方法包括在培養期間的一或多個時間點將錳和半乳糖添加到培養基中,作為營養料的一部分。例如,在一些實施方式中,該方法包括在培養期的第3天向培養基中添加錳和半乳糖。在一些實施方式中,該方法包括在培養期的第3天和第6天向培養基中添加錳和半乳糖。在一些實施方式中,該方法包括在培養期的第3天、第6天和第8天向培養基中添加錳和半乳糖。
在一些實施方式中,本文所述方法包括在培養期間的一或多個時間點將至少100 ppb錳和至少15 mM半乳糖添加到培養基中,作為營養料的一部分。例如,在一些實施方式中,在培養期的第3天將100 ppb錳和15 mM半乳糖添加到培養基中。在一些實施方式中,本文所述方法包括在培養期的第3天和第6天中之每一天將100 ppb錳和15 mM半乳糖添加到培養基中。在一些實施方式中,本文所述方法包括在培養期的第3天、第6天和第8天中之每一天將100 ppb錳和15 mM半乳糖添加到培養基中。
在一些實施方式中,在本文所述方法中使用的培養基可以具有從0 ppb至100 ppb範圍內(例如,28 ppb)的痕量的銅,並且本文所述方法包括在細胞培養期間進一步用銅補充培養基。在一些實施方式中,本文所述方法包括向培養基中添加銅,視需要地,以從約0.01 mM至約0.5 mM範圍的量。在示例性實施方式中,培養基中銅的濃度(例如,將補充半乳糖和錳的培養基添加到培養中的宿主細胞後的最終濃度)的範圍係從約0.01 mM至約0.1 mM或從約0.05 mM至約0.15 mM、或從約0.1 mM至約0.5 mM。在一些實施方式中,培養基中銅的濃度係0.1 mM。在一些實施方式中,可以使用五水硫酸銅(II)鹽製備銅儲備液。
培養基還可以包括本領域已知的任何其他必要或所需成分,例如碳水化合物,包括葡萄糖、必需和/或非必需胺基酸、脂質和脂質先質、核酸先質、維生素、無機鹽、痕量元素(包括稀有金屬)和/或細胞生長因子。培養基可為化學成分確定的,或者可以包括血清、植物水解物、或其他衍生物質。考慮了培養基可以基本上或完全不含動物組分。培養基可以基本上或完全不含血清、不含蛋白、不含生長因子和/或不含蛋白腖。基本上不含血清意味著培養基缺乏任何血清或含有微量的血清。基本上不含蛋白意味著培養基缺乏任何蛋白或含有微量的蛋白。基本上不含生長因子意味著培養基缺乏任何生長因子或含有微量的生長因子。基本上不含蛋白腖意味著培養基缺少任何蛋白腖或含有微量的蛋白腖。
培養基還可以包含在細胞培養期間耗盡的補充胺基酸,例如,天冬醯胺、天冬胺酸、半胱胺酸、胱胺酸、異白胺酸、白胺酸、色胺酸、和纈胺酸。胺基酸補充可以在初始生長培養基中和/或在快速生長階段期間或之後添加的培養基中。
該培養基可以包含脂質和/或脂質先質,例如膽鹼,乙醇胺,或磷酸乙醇胺,膽固醇,脂肪酸,例如油酸、亞麻油酸、亞麻酸、甲酯、D-α-生育酚(例如,乙酸形式)、硬脂酸、豆蔻酸、棕櫚酸、棕櫚油酸、或花生四烯酸。可獲得多種可商購的脂質混合物。
該培養基可以包含鐵補充物,該鐵補充物包含鐵和鐵結合的合成運輸分子。培養基可以包含無機化合物或痕量元素,以適當的鹽形式供應,例如鈉、鈣、鉀、鎂、銅、鐵、鋅、硒、鉬、釩、錳、鎳、矽、錫、鋁、鋇、鎘、鉻、鈷、鍺、鉀、銀、銣、鋯、氟化物、溴化物、碘化物、和氯化物。可獲得多種可商購的痕量元素的混合物。
培養基還可以視需要地包含非離子界面活性劑或界面活性劑,從而保護細胞免於混合或充氣。培養基還可以包含緩衝液,例如碳酸氫鈉、一元和二元磷酸鹽、HEPES、和/或Tris。
培養基還可以包含蛋白產生的誘導物,例如丁酸鈉、或咖啡因。其他已知的誘導物包括但不限於以下化合物:N-乙醯-L-半胱胺酸、放線菌素D、7-胺基-、巴佛洛黴素A1、灰色鏈黴菌、鈣磷酸蛋白C、枝狀枝孢、喜樹鹼、喜樹、CAPE、2-氯-2’-去氧腺苷、2-氯-2'-去氧腺苷5’-三磷酸鹽、四鋰鹽、環己醯亞胺、一水合環磷醯胺、環孢素、多孢木黴、柔紅黴素、鹽酸鹽、地塞米松、多柔比星、鹽酸鹽、(-)-表沒食子兒茶素沒食子酸酯、依託泊苷、磷酸依託泊苷、ET-18-OCH3、5-氟尿嘧啶、H-7、二鹽酸鹽、染料木素、4-羥基壬烯酸、維甲醯酚胺、羥基脲、IL-1β抑制劑、(±)-S-亞硝基-N-乙醯青黴胺、S-亞硝基麩胱甘肽、佛波醇-12-豆蔻酸-13-乙酸酯、嘌呤黴素、二鹽酸鹽、1-吡咯啶二硫代甲酸、銨鹽、槲皮素、二水合物、雷帕黴素、丁酸鈉、4-苯丁酸鈉,D-赤型神經胺、N-乙醯基-、D-赤型神經胺、N-辛醯基-、十字組孢鹼、鏈黴菌屬物種、舒林酸、毒胡蘿蔔素、TRAIL、大腸桿菌、曲古抑菌素A、鏈黴菌屬物種、(±)-維拉帕米、鹽酸鹽、藜蘆定鹼、維生素D3、和維生素E琥珀酸酯(VWR和Calbiochem公司)。
培養基視需要地排除A23187或其它消耗二價陽離子的化合物。
在細胞培養物中維持細胞
關於本揭露之治療性蛋白組成物的方法,可以藉由在細胞培養物中維持細胞來產生治療性蛋白組成物。可以根據適於重組糖基化蛋白質生產的任何一組條件維持細胞培養物。例如,在一些方面,將細胞培養物維持在特定pH、溫度、細胞密度、培養體積、溶解氧水平、壓力、莫耳滲透壓濃度等下。在示例性方面,將接種前的細胞培養物在CO
2培養箱中在標準加濕條件下在5% CO
2下振盪(例如,以70 rpm)。在示例性方面,在1.5 L培養基中以約1 x 10
6個細胞/mL的接種密度接種細胞培養物。
在示例性方面,本揭露之方法包括將細胞培養基中的細胞維持在約6.5至約7.2的pH下,例如,在各個方面,約6.5、約6.55、約6.6、約6.65、約6.7、約6.75、約6.8、約6.85、約6.86、約6.87、約6.88、約6.89、約6.90、約6.91、約6.92、約6.93、約6.94、約6.95、約6.96、約6.97、約6.98、約6.99、約7.00、約7.0、約7.02、約7.03、約7.04、約7.05、約7.06、約7.07、約7.08、約7.09、約7.1、約7.11、約7.12、約、約7.13、約7.14、約7.15、約7.16、約7.17、約7.18、約7.19或約7.2。
在一些實施方式中,該方法包括將細胞培養物維持在30°C和40°C之間的溫度下。在示例性實施方式中,溫度在約32°C至約38°C之間或在約35°C至約38°C之間。
在一些實施方式中,該方法包括細胞培養期間的一或多個溫度和/或pH變化。例如,可以使用該等變化來影響細胞的行為。更高的溫度典型地用於促進細胞生長,更低的溫度典型地用於減緩生長並且促進重組蛋白的產生。
在一些實施方式中,該方法包括將莫耳滲透壓濃度維持在約200 mOsm/kg至約500 mOsm/kg之間。在示例性方面,該方法包括將莫耳滲透壓濃度維持在約225 mOsm/kg至約400 mOsm/kg或約225 mOsm/kg至約375 mOsm/kg之間。在示例性方面,該方法包括將莫耳滲透壓濃度維持在約225 mOsm/kg至約350 mOsm/kg之間。在各個方面,將莫耳滲透壓濃度(mOsm/kg)維持在約200、約225、約250、約275、約300、約325、約350、約375、約400、約425、約450、約475、或約500。
在一些實施方式中,該方法包括在初始細胞培養期期間將細胞培養物的溶解氧(DO)水平維持在約20%至約60%氧飽和度下。在示例性情況下,該方法包括在初始細胞培養期期間將細胞培養物的DO水平維持在約30%至約50%(例如,約35%至約45%)氧飽和度下。在示例性情況下,該方法包括在初始細胞培養期期間將細胞培養物的DO水平維持在約20%、約25%、約30%、約35%、約40%、約45%、約50%、約55%、或約60%氧飽和度下。在示例性方面,DO水平係約35 mm Hg至約85 mmHg或約40 mm Hg至約80 mmHg或約45 mm Hg至約75 mm Hg。
在一些實施方式中,將細胞培養物維持在適於細胞生長的培養基中和/或為細胞培養物提供根據任何合適的進料方案的一或多種進料培養基。
在一些實施方式中,細胞培養的類型係饋料批式培養或連續灌注培養。然而,本揭露之方法有利地不限於任何特定類型的細胞培養。
美國專利案號5,459,031(將其藉由引用以其全文併入本文)中描述了用於控制重組醣蛋白的唾液酸化,特別是用於控制糖鏈中的N-羥乙醯神經胺酸(NGNA)水平的方法,並且此類方法可以與本文所述培養基和培養方法組合使用。該等方法涉及調整培養參數,包括二氧化碳水平,從而實現碳水化合物中所需的NGNA含量。
糖基化和唾液酸化的評估
CHO細胞中產生的重組醣蛋白可以表現出可變的糖基化和唾液酸化。高唾液酸化形式的醣蛋白分子可以經由陰離子交換層析從此類分子的更低唾液酸化(包括非唾液酸化)形式中分離出來。唾液酸係酸性的,並因此帶負電,被捕獲在柱上,使得高唾液酸化分子保留在柱上而更低唾液酸化形式流過。可以確定每種級分中醣蛋白的量(保留在柱上對比流過級分),並且將其與從細胞培養基中載入的醣蛋白的起始量進行比較。
在一些實施方式中,本文所述之唾液酸係α2,6-唾液酸化聚糖。在一些實施方式中,α2,6-唾液酸化聚糖的水平在延長的細胞培養持續時間產生的蛋白中保持恒定。在一些實施方式中,延長的細胞培養持續時間係約1個群體倍增(PDL)、2個PDL、3個PDL、4個PDL、5個PDL、6個PDL、7個PDL、8個PDL、9個PDL、10個PDL、11個PDL、12個PDL、13個PDL、14個PDL、15個PDL、16個PDL、17個PDL、18個PDL、19個PDL、20個PDL、21個PDL、22個PDL、23個PDL、24個PDL、25個PDL、26個PDL、27個PDL、28個PDL、29個PDL、30個PDL、31個PDL、32個PDL、33個PDL、34個PDL、35個PDL、36個PDL、37個PDL、38個PDL、39個PDL、40個PDL、41個PDL、42個PDL、43個PDL、44個PDL、45個PDL、46個PDL、47個PDL、48個PDL、49個PDL、50個PDL、55個PDL、60個PDL、65個PDL、70個PDL、75個PDL、80個PDL、85個PDL、90個PDL、95個PDL、或100個PDL。在一些實施方式中,延長的細胞培養持續時間係約27個PDL。
對於醣蛋白組成物,唾液酸化的增加或改善可以根據Elliott等人, Biochemistry [生物化學], 33(37):11237-45 (1994)(將其藉由引用以其全文併入本文)藉由陰離子交換層析進行確定。較高唾液酸化的蛋白預期會帶更多的負電荷,並且與柱結合更強,而較低唾液酸化的蛋白流過或容易洗脫。可以例如藉由ELISA確定兩種級分中每一種的醣蛋白分子的量(保留在樹脂上對比流過級分),並且將其與從細胞培養基中載入的此類分子的起始量進行比較。示例性ELISA套組可商購,並且包括R & D系統公司的IVD人EPO EIA套組。
層析如下進行。為了消除細胞和碎片,將經培養產生治療性蛋白的哺乳動物細胞的培養基以約1000 rpm離心並且通過0.45微米過濾器過濾。在可替代方法中,為了去除細胞和碎片,使用深度過濾將產生治療性蛋白的哺乳動物細胞的培養基進行過濾。然後對過濾的材料進行陰離子交換層析以預純化主要含有醣蛋白分子的四至七種最高度唾液酸化物質的級分。可以使用強離子交換樹脂,例如,像TRICORNTM Mono-Q 5/50 GL(安瑪西亞公司(Amersham_),部件號17-5166-01)或其他強陰離子交換樹脂,特別是具有季胺-CH
2-N+-(CH
3)
3作為樹脂官能基的那些樹脂。確切的程序將取決於特定醣蛋白分子可以包含的唾液酸殘基的理論最大數量。將用於從陰離子交換柱洗脫醣蛋白分子的緩衝液設計為:(1) 從柱上洗脫大多數或所有屬於以下物質的蛋白分子,該等物質的唾液酸化程度低於由大約前三分之一最高度唾液酸化物質組成的物質組(「高度唾液酸化」物質係指每個蛋白分子具有大於或等於3個末端唾液酸殘基的那些物質),(2) 然後洗脫屬於四至七種最高度唾液酸化物質的蛋白分子,以及(3) 最後從柱中去除更多高度帶電物質,該等高度帶電物質可能包含帶有硫酸化N-聚糖的糖型。因此,洗滌和洗脫緩衝液的確切組成可以根據醣蛋白分子上唾液酸殘基的理論最大數量進行調整。熟悉該項技術者可以基於常規經驗性柱參數的優化和在分析等電聚焦凝膠上測定從柱中脫離的材料來進行此類調整。
相對於對照(例如,由不含錳或痕量元素量的錳的培養基產生),從保留在樹脂上的池中回收的抗原結合蛋白的百分比增加(或在流過級分中觀察到的此類分子的百分比減少)表明唾液酸化增加,無論是藉由增加產生的唾液酸化分子的百分比還是藉由增加其唾液酸化的程度。
實際聚糖結構可以藉由本領域已知的任何技術來確定,包括碳水化合物的酶消化、凝集素免疫墨點、1D和2D 1H-NMR光譜、質譜技術(包括電灑游離串聯質譜(ESI MS)或基質輔助雷射脫附游離飛行時間質譜(MALDI-TOF MS))和/或酶釋放的N-聚糖的螢光標記,隨後藉由HPLC進行拆分並且與已知的N-聚糖對照樣本進行比較。
在以下實例中描述的用於確定具有被佔據的O-糖基化位點的醣蛋白的量的示例性技術涉及N-聚糖酶消化以去除N-連接的碳水化合物,隨後進行逆相HPLC以將醣蛋白組成物分離成兩個峰。可以藉由質譜確認峰鑒定為被佔據的O位點或未被佔據的O-位點。
N-位點分支和唾液酸化(包括產生的唾液酸化分子的百分比和唾液酸化分子的唾液酸化的程度)可以藉由分析醣蛋白的結構含量來確定,該分析藉由N-聚糖映射和酶定序進行,例如藉由用N-聚糖酶和神經胺酸酶進行消化,與MALDI-TOF質譜聯用以確定釋放的糖的大小。在以下實例中描述了示例性技術。
例如,附接至抗原結合蛋白的糖(為半乳糖)的百分比可以藉由神經胺酸酶加半乳醣苷酶消化隨後藉由HPLC分離或MALDI-TOF質譜確定釋放糖的大小來確定。在以下實例中描述了示例性技術。
治療性蛋白
在各個方面,治療性蛋白係抗體蛋白產物。如本文所用,術語「抗體蛋白產物」係指在各種情況下基於抗體的架構但在自然界中未發現的若干抗體替代物中之任一種。在一些方面,抗體蛋白產物的分子量在至少約12-150 kDa的範圍內。在某些方面,該抗體蛋白產物具有從單體(n = 1)到二聚體(n = 2)、到三聚體(n = 3)、到四聚體(n = 4)的價數(n)範圍,如果不是更高階的價數的話。在一些方面,抗體蛋白產物係基於完整抗體結構的那些和/或模擬保留完整抗原結合能力的抗體片段的那些,例如scFv、Fab和VHH/VH(下文討論)。保留其完整抗原結合位點的最小抗原結合抗體片段為Fv片段,其完全由可變(V)區組成。使用可溶性柔性胺基酸肽連接子將V區連接至scFv片段(可變單鏈片段)以使該分子穩定,或將恒定(C)結構域添加至V區以產生Fab片段[抗原結合片段]。scFv和Fab片段可以容易地在宿主細胞例如原核宿主細胞中產生。其他抗體蛋白產物包括經二硫鍵穩定的scFv(ds-scFv)、單鏈Fab(scFab)以及二聚體及集合體抗體形式,如雙抗體、三抗體和四抗體,或包含由與寡聚結構域連接的scFv組成的不同形式的迷你抗體(miniAb)。最小的片段係駱駝重鏈Ab的VHH/VH以及單結構域Ab(sdAb)。最常用於建造新型抗體形式的構件為單鏈可變(V)結構域抗體片段(scFv),其包含由具有約15個胺基酸殘基的肽連接子連接的來自重鏈和輕鏈的V結構域(VH結構域和VL結構域)。肽體或肽-Fc融合物係另一種抗體蛋白產物。肽體的結構由嫁接到Fc結構域上的生物活性肽組成。本領域已充分描述了肽體。參見,例如,Shimamoto等人, mAbs [單株抗體] 4(5): 586-591 (2012)。
其他抗體蛋白產物包括單鏈抗體(SCA)、雙抗體、三抗體、四抗體、雙特異性或三特異性抗體等。雙特異性抗體可分成五個主要類別:BsIgG、附加IgG、BsAb片段、雙特異性融合蛋白以及BsAb軛合物。參見,例如,Spiess等人, Molecular Immunology [分子免疫學] 67(2) 部分A: 97-106(2015)。
在示例性方面,治療性蛋白係雙特異性T細胞接合物(BiTE®)分子,該雙特異性T細胞接合物分子係人工雙特異性單株抗體。典型BiTE®分子係融合蛋白,包含不同抗體的兩個scFv。一個與CD3結合,而另一個與靶抗原結合。BiTE®分子係本領域已知的。參見,例如Huehls等人, Immuno Cell Biol [免疫細胞生物學] 93(3): 290-296 (2015);Rossi等人, MAbs [單株抗體] 6(2): 381-91 (2014);Ross等人, PLoS One [公共科學圖書館期刊] 12(8): e0183390。
在示例性方面,治療性蛋白係嵌合抗原受體(CAR)。嵌合抗原受體係由多個結構域構建的基因工程融合蛋白,典型地由免疫細胞表現的其他天然存在的分子構成。在若干個方面,CAR包含細胞外抗原結合結構域或抗原識別結構域、傳訊結構域和共刺激結構域。本領域描述了CAR。參見例如Maus等人, Clin Cancer Res [臨床癌症研究](2016) 22(8): 1875-1884;Dotti等人, Immuno Rev [免疫學評論] (2014) 257(1): 10.1111/imr.12131;Lee等人, Clin Cancer Res [臨床癌症研究] (2012): 18(10): 2780-2790;以及June和Sadelain, NEJM (2018) 379: 64-73。
抗體經常在恒定結構域區域被糖基化。已經檢測到N-糖基化位點,如下(基於從重鏈恒定區的N末端的編號):IgG1-N180;IgG2-N176;IgG3-N227;IgG4-N177;IgM-N46、N209、N272、N279、N439;IgA1-N144、N340;IgA2-N47、N131、N205、N327(Chandler等人, Molecular & Cellular Proteomics [分子與細胞蛋白質組學] 18: 686-703, 2019)。已經表明,人內源性IgG中的唾液酸的水平係大約11%-15%(Boune等人, Antibodies [抗體] 9: 22, 2020)。在各種實施方式中,唾液酸位於本文所述之N-糖基化位點上。在各種實施方式中,唾液酸位於IgG1的N180(如果從可變區編號,則為N297)上。在各種實施方式中,唾液酸位於Fab區中的殘基上。在各種實施方式中,本文的抗體組成物包含至少約20%的唾液酸化的抗體。在各種實施方式中,至少約21%、21%、22%、23%、24%、25%、26%、27%、28%、29%、30%、31%、32%、33%、34%、35%、36%、37%、38%、39%、40%、41%、42%、43%、44%、45%、46%、47%、48%、49%、50%、或更多的抗體包含至少一個唾液酸殘基。
示例性治療性蛋白包括但不限於CD蛋白、生長因子、生長因子受體蛋白
(例如HER受體家族蛋白)、細胞黏附分子(例如LFA-I、MoI、pl50、95、VLA-4、ICAM-I、VCAM和αv/β3整合素)、激素(例如胰島素)、凝血因子、凝血相關蛋白、群落刺激因子及其受體,其他受體和受體相關蛋白或該等受體的配體、以及病毒抗原。
例如,示例性治療性蛋白包括CD蛋白中之任一種,例如CD1a、CD1b、CD1c、CD1d、CD2、CD3、CD4、CD5、CD6、CD7、CD8、CD9、CD10、CD11A、CD11B、CD11C、CDw12、CD13、CD14、CD15、CD15s、CD16、CDw17、CD18、CD19、CD20、CD21、CD22、CD23、CD24、CD25、CD26、CD27、CD28、CD29、CD30、CD31,CD32、CD33、CD34、CD35、CD36、CD37、CD38、CD39、CD40、CD41、CD42a、CD42b、CD42c、CD42d、CD43、CD44、CD45、CD45RO、CD45RA、CD45RB、CD46、CD47、CD48、CD49a、CD49b、CD49c、CD49d、CD49e、CD49f、CD50、CD51、CD52、CD53、CD54、CD55、CD56、CD57、CD58、CD59、CDw60、CD61、CD62E、CD62L、CD62P、CD63、CD64、CD65、CD66a、CD66b、CD66c、CD66d、CD66e、CD66f、CD68、CD69、CD70、CD71、CD72、CD73、CD74、CD75、CD76、CD79α、CD79β、CD80、CD81、CD82、CD83、CDw84、CD85、CD86、CD87、CD88、CD89、CD90、CD91、CDw92、CD93、CD94、CD95、CD96、CD97、CD98、CD99、CD100、CD101、CD102、CD103、CD104、CD105、CD106、CD107a、CD107b、CDw108、CD109、CD114、CD115、CD116、CD117、CD118、CD119、CD120a、CD120b、CD121a、CDw121b、CD122、CD123、CD124、CD125、CD126、CD127、CDw128、CD129、CD130、CDw131、CD132、CD134、CD135、CDw136、CDw137、CD138、CD139、CD140a、CD140b、CD141、CD142、CD143、CD144、CD145、CD146、CD147、CD148、CD150、CD151、CD152、CD153、CD154、CD155、CD156、CD157、CD158a、CD158b、CD161、CD162、CD163、CD164、CD165、CD166和CD182。
示例性生長因子包括例如血管內皮生長因子(「VEGF」)、生長激素、甲狀腺刺激素(TSH)、促濾泡素(FSH)、黃體生成激素(LH)、生長激素釋放因子(GHRF)、甲狀旁腺激素(PTH)、米勒管抑制物質(mullerian-inhibiting substance)(MIS)、人類巨噬細胞炎性蛋白(MIP-I-α)、促紅血球生成素(EPO)、神經生長因子(NGF)(例如NGF-β)、血小板源性生長因子(PDGF)、纖維母細胞生長因子(FGF)(包括例如aFGF和bFGF)、上皮生長因子(EGF)、轉化生長因子(TGF)(尤其包括TGF-α和TGF-β,包括TGF-βl、TGF-β2、TGF-β3、TGF-β4或TGF-β5)、胰島素樣生長因子-I和胰島素樣生長因子-II(IGF-I和IGF-II)、des(l-3)-IGF-I(腦IGF-I)和骨誘導因子。在一些方面,治療性蛋白係胰島素或胰島素相關蛋白,例如胰島素、胰島素A鏈、胰島素B鏈、胰島素原、和胰島素樣生長因子結合蛋白。示例性生長因子受體包括任何以上生長因子的任何受體。在各個方面,生長因子受體係HER受體家族蛋白(例如HER2、HER3、HER4和EGF受體)、VEGF受體、TSH受體、FSH受體、LH受體、GHRF受體、PTH受體、MIS受體、MIP-1-α受體、EPO受體、NGF受體、PDGF受體,FGF受體、EGF受體(EGFR)、TGF受體、或胰島素受體。
示例性凝血蛋白和凝血相關蛋白包括例如因子VIII、組織因子、von Willebrands因子、蛋白C、α-1-抗胰蛋白酶、纖維蛋白溶酶原活化劑(如尿激酶)和組織性纖維蛋白溶酶原活化劑(「t-PA」)、bombazine、凝血酶和血小板生成素;(vii) 其他血液和血清蛋白,包括但不限於白蛋白、IgE和血型抗原。群落刺激因子及其受體,尤其包括以下:M-CSF、GM-CSF和G-CSF及其受體,如CSF-1受體(c-fms)。受體和受體相關蛋白,包括例如flk2/flt3受體、肥胖(OB)受體、LDL受體、生長激素受體、血小板生成素受體(「TPO-R」、「c-mpl」)、升糖素受體、白血球介素受體、干擾素受體、T細胞受體、幹細胞因子受體(如c-Kit)和其他受體。受體配體,包括例如OX40L,它係OX40受體的配體。神經滋養因子,包括骨源性神經滋養因子(BDNF)和神經滋養蛋白-3、神經滋養蛋白-4、神經滋養蛋白-5或神經滋養蛋白-6(NT-3、NT-4、NT-5或NT-6)。鬆弛素A鏈、鬆弛素B鏈和鬆弛素原;干擾素和干擾素受體,包括例如干擾素-α、干擾素-β和干擾素-γ及其受體。白血球介素和白血球介素受體,尤其包括IL-I至IL-33和IL-I至IL-33受體,如IL-8受體等等。病毒抗原,包括AIDS包膜病毒抗原。脂蛋白、降鈣素、升糖素、心房鈉尿因子、肺表面活性物質、腫瘤壞死因子-α和腫瘤壞死因子-β、腦啡肽酶、RANTES(正常T細胞表現和分泌的活化調節蛋白)、小鼠促性腺激素相關肽、DNA酶、抑制素和活化素。整合素、蛋白A或D、類風濕因子、免疫毒素、骨形態發生蛋白(BMP)、超氧化物歧化酶、表面膜蛋白、衰變加速因子(DAF)、AIDS包膜、運輸蛋白、歸巢受體、位址素、調節蛋白、免疫黏附素、抗體。另外的示例性治療性蛋白包括例如肌生成抑制蛋白、TALL蛋白(包括TALL-I)、澱粉樣蛋白(包括但不限於澱粉樣β蛋白)、胸腺基質淋巴球生成素(「TSLP」)、RANK配體(「OPGL」)、c-kit、TNF受體(包括TNF受體1型)、TRAIL-R2、血管生成素和任何上述物質的生物活性片段或類似物或變體。
在示例性方面,該治療性蛋白係被稱為以下的藥劑中之任一種:Activase®(阿替普酶);阿利庫單抗、Aranesp®(達依泊汀-α)、Epogen®(依泊汀α或促紅血球生成素);Avonex®(干擾素β-Ia);Bexxar®(托西莫單抗);Betaseron®(干擾素-β);百庫珠單抗(抗PCSK9單株抗體,命名為L1L3,參閱US 8080243);Campath®(阿侖單抗);Dynepo®(依泊汀δ);Velcade®(硼替佐米);MLN0002(抗α4β7 mAb);MLN1202(抗CCR2趨化介素受體mAb);Enbrel®(依那西普);Eprex®(依泊汀α);Erbitux®(西妥昔單抗);依伏庫單抗;Genotropin®(生長激素);Herceptin®(曲妥珠單抗);Humatrope®(注射用生長激素[rDNA起源]);Humira®(阿達木單抗);Infergen®(干擾素Alfacon-1);Natrecor®(奈西立肽);Kineret®(阿那白滯素)、Leukine®(沙格司亭);LymphoCide®(依帕珠單抗);BenlystaTM(貝利單抗);Metalyse®(替奈普酶);Mircera®(甲氧基聚乙二醇-依泊汀β);MyIotarg®(吉妥珠單抗奧佐米星);Raptiva®(依法利珠單抗);Cimzia®(賽妥珠單抗);SolirisTM(依庫麗單抗);培克珠單抗(抗C5補體);MEDI-524(Numax®);Lucentis®(蘭尼單抗);依決洛單抗(Panorex®);Trabio®(樂地單抗(lerdelimumab));TheraCim hR3(尼妥珠單抗);奧密塔克(帕妥珠單抗,2C4);Osidem®(IDM-I);OvaRex®(B43.13);Nuvion®(維西珠單抗);莫星-坎妥珠單抗(huC242-DMl);NeoRecormon®(依泊汀β);Neumega®(奧普瑞白介素);Neulasta®(聚乙二醇化非格司亭、聚乙二醇化G-CSF、聚乙二醇化hu-Met-G-CSF);Neupogen®(非格司亭);Orthoclone OKT3®(鼠源單株抗體-CD3)、Procrit®(依泊汀α);Remicade®(英夫利昔單抗),Reopro®(阿昔單抗),Actemra®(抗IL6受體mAb),Avastin®(貝伐單抗),HuMax-CD4(紮木單抗(zanolimumab)),Rituxan®(利妥昔單抗);Tarceva®(埃羅替尼);Roferon-A®-(干擾素α-2a);Simulect®(巴厘昔單抗);Stelara®(優特克單抗);Prexige®(羅美昔布);Synagis®(帕利珠單抗);146B7-CHO(抗IL15抗體,參見US7153507),Tysabri®(那他珠單抗);Valortim®(MDX-1303,抗炭疽桿菌保護性抗原mAb);ABthraxTM;Vectibix®(帕尼單抗);Xolair®(奧馬珠單抗)、ETI211(抗MRSA mAb)、IL-I Trap(人IgGl的Fc部分和兩個IL-I受體組分(I型受體和受體輔助蛋白)的細胞外結構域)、VEGF Trap(與IgGl Fc融合的VEGFRl的Ig結構域)、Zenapax®(達克珠單抗);Zenapax®(達克珠單抗),Zevalin®(替坦替伊莫單抗(Ibritumomab tiuxetan)),Zetia(依澤替米貝),阿塞西普(TACI-Ig),抗α4β7 mAb(維多珠單抗);加利昔單抗(galiximab)(抗CD80單株抗體),抗CD23 mAb(魯昔單抗);BR2-Fc(huBR3/huFc融合蛋白,可溶性BAFF拮抗劑);Simponi®(戈利木單抗);馬帕木單抗(Mapatumumab)(人抗TRAIL受體-1 mAb);奧瑞單抗(Ocrelizumab)(抗CD20人mAb);HuMax-EGFR(紮魯木單抗(zalutumumab));M200(伏洛昔單抗(Volociximab),抗α5β1整合素mAb);MDX-010(艾匹利木單抗,抗CTLA-4 mAb和VEGFR-I(IMC-18F1);抗BR3 mAb;抗艱難梭菌毒素A和毒素B C mAb MDX-066(CDA-I)和MDX-1388);抗CD22 dsFv-PE38軛合物(CAT-3888和CAT-8015);抗CD25 mAb(HuMax-TAC);抗TSLP抗體;抗TSLP受體抗體(US 8101182);指定為A5的抗TSLP抗體(US7982016);(抗CD3 mAb(NI-0401);阿德木單抗(Adecatumumab)(MT201,抗EpCAM-CD326 mAb);MDX-060、SGN-30、SGN-35(抗CD30 mAb);MDX-1333(抗IFNAR);HuMax CD38(抗CD38 mAb);抗CD40L mAb;抗Cripto mAb;抗CTGF特發性肺纖維化I期纖維蛋白原(FG-3019);抗CTLA4 mAb;抗eotaxinl mAb(CAT-213);抗FGF8 mAb;抗神經節苷脂GD2 mAb;抗硬化蛋白抗體(參見,US 8715663或US 7592429)被指定為Ab-5的抗硬化蛋白抗體(US 8715663或US 7592429);抗神經節苷脂GM2 mAb;抗GDF-8人mAb(MYO-029);抗GM-CSF受體mAb(CAM-3001);抗HepC mAb(HuMax HepC);MEDI-545、MDX-1103(抗IFNα mAb);抗IGFIR mAb;抗IGF-IR mAb(HuMax-Inflam);抗IL12/IL23p40 mAb(布瑞吉努單抗);抗IL-23p19 mAb(LY2525623);抗IL13 mAb(CAT-354);抗IL-17 mAb (AIN457);抗IL2Ra mAb(HuMax-TAC);抗IL5受體mAb;抗整合素受體mAb(MDX-Ol8,CNTO 95);抗IPIO潰瘍性結腸炎 mAb(MDX-1100);抗LLY抗體;BMS-66513;抗甘露糖受體/hCGβ mAb(MDX-1307);抗間皮素dsFv-PE38軛合物(CAT-5001);抗PDlmAb(MDX-1 106(ONO-4538));抗PDGFRα抗體(IMC-3G3);抗TGFβ mAb(GC-1008);抗TRAIL受體-2人mAb(HGS-ETR2);抗TWEAK mAb;抗VEGFR/Flt-1 mAb;抗ZP3 mAb(HuMax-ZP3);NVS抗體#1;NVS抗體#2;或β澱粉樣蛋白單株抗體。
在一些實施方式中,該抗體係抗IL12抗體、抗IL-23抗體、或抗IL12/23抗體(例如,優特克單抗抗體,其包含根據SEQ ID NO: 29的重鏈序列和根據SEQ ID NO: 30的輕鏈序列)。
在一些實施方式中,治療性多肽係BiTE®分子。博納吐單抗(BLINCYTO®)係對CD19特異的BiTE®分子的實例。經修飾的BiTE®分子(如經修飾以延長其半衰期的那些)也可以用於所揭露的方法中。
在示例性方面,藉由基於細胞的定量測定確定抗體組成物的ADCC水平,該測定測量抗體組成物的抗體在表現抗體抗原並藉由抗體的Fc結構域與效應細胞上的Fc-γRIIIA受體銜接的細胞中以劑量依賴性方式介導細胞的細胞毒性的能力。在各種實施方式中,該方法包括使用帶有可檢測標記的靶細胞,當靶細胞被效應細胞裂解時,該等可檢測標記被釋放。從靶細胞釋放的可檢測標記的量係抗體組成物的ADCC活性的量度。在一些方面,將從靶細胞釋放的可檢測標記的量與基線進行比較。此外,ADCC水平可以報告為相對於對照% ADCC的% ADCC。在各個方面,% ADCC係相對% ADCC,其視需要地是相對於對照% ADCC。在各個方面,對照% ADCC係參考抗體的% ADCC。
在一些實施方式中,根據本文所述方法產生的治療性蛋白(例如抗體)可以用於治療患有以下疾病的患者:斑塊狀牛皮癬、牛皮癬性關節炎、克羅恩病和/或潰瘍性結腸炎。例如,患有中度至重度斑塊狀牛皮癬的患者可以用本文所述方法產生的抗IL12/23抗體進行治療。此外,患有中度至重度活動性克羅恩病或潰瘍性結腸炎的患者可以用本文所述方法產生的抗IL12/23抗體進行治療。此外,患有活動性牛皮癬性關節炎的患者可以單獨用本文所述方法產生的抗IL12/23抗體進行治療,或將其與胺甲喋呤組合進行治療。
如從引文的上下文中顯而易見的,所有已鑒定的專利和其他出版物均藉由引用以其全文或相關部分明確併入本文,其目的係描述和揭露例如在這樣的出版物中描述的可能與本文所述之資訊結合使用的方法。
給出以下實例僅用於說明本發明,而不以任何方式限制其範圍。
實例 實例 1 - ST6 或 ST6 和 B4GALT1 二者在 CHO 細胞中的過表現增加了治療性蛋白的唾液酸化
此實例表明,單獨ST6(α2,6-唾液醯基轉移酶-1)或ST6和β1,4-半乳糖苷轉移酶1(B4GALT1)二者過表現的CHO細胞增加了重組抗IL12/23抗體(即,包含根據SEQ ID NO: 29的重鏈序列和根據SEQ ID NO: 30的輕鏈序列的優特克單抗抗體)的唾液酸含量。
用ST6(SEQ ID NO: 1)或ST6和B4GALT1(SEQ ID NO: 13)二者轉染表現重組抗IL12/23抗體的十一(11)個不同擴增的CHO(中國倉鼠卵巢)CS9細胞系(例如,來源於DXB-11細胞的DHFR(二氫葉酸還原酶)缺陷細胞)池,並且產生33個工程池。由於重點係增加α2,6連接和N-乙醯神經胺酸百分比(%NANA),因此使用SNA凝集素藉由流動式細胞分析術評估α-2,6連接的表現。凝集素係與聚糖的特定二醇/糖部分結合的蛋白。從接骨木樹皮中分離的西洋接骨木凝集素(SNA1)優先與α-2,6連接中附接至末端半乳糖的唾液酸結合。分析表明,不同的工程池表現出3.3%-25%的SNA陽性群體,即α-2,6連接。使用FACS(螢光活化細胞分選)增濃SNA陽性群體,使用基於凝集素的細胞表面定性FACS測定進行分選。
藉由FACS和Beacon(BLI)平臺二者進行選殖。在FACS選殖期間,使用了使用SNA-FITC的表面染色方案。根據包括%SNA-FITC群體、Fortebio滴定量和測量ST6和B4GALT1的mRNA水平的Quantigene mRNA測定的標準,將來源於FACS的386個殖株減少為96個殖株。此外,基於陽性SNA珠測定、Spotlight的滴定量估計、以及擴增和從筆轉移時的生長,還獲得了12個Beacon衍生殖株。
將殖株傳代並且在第24天深孔板分批進料中進行篩選。篩選具有滴定量 > 0.5 mg/ml並且%SA水平在8%-73%之間的封閉物(如藉由快速聚糖法所測量的(Shah等人, 「Rapid Automated LC-MS/MS Glycan Analysis for Monoclonal Antibodies [單株抗體的快速自動LC-MS/MS聚糖分析]」, American Society of Mass Spectrometry (ASMS) conference [美國質譜學會(ASMS)研討會] (2016年6月4-9日))),並且選擇34個殖株進行進一步分析。使用親水相互作用液體分析這34個殖株的聚糖譜。層析(HILIC)。藉由消除高甘露糖(HM)> 10%的殖株,將34個殖株減少為16個。在十六個殖株中,基於池多樣性、低%HM和唾液酸百分比(%SA)的範圍,選擇八個殖株進行進一步篩選。八個殖株中的五個殖株的唾液酸百分比範圍為16.7%至27% SA。
實例 2 - ST6 或 ST6 和 B4GALT1 二者在 CHO 細胞中的過表現在整個生產過程中維持遺傳穩定性
在群體倍增水平(PDL)的限定持續時間和間隔內對以下3個殖株進行前主細胞庫(前MCB)階段穩定性研究:殖株1(ST6過表現)、殖株2(ST6和B4GALT1過表現)、和殖株3(ST6過表現)。此研究的主要目的係使用類比細胞庫和製造過程的方法,檢測生長速率、表現滴定量和產生的聚糖隨細胞年齡的變化。藉由監測高分子量(HMW)來評估另外的產品品質隨細胞年齡的變化。
前主細胞庫(前MCB)係最終重組殖株的最早測試/表徵的細胞庫,並且將其用於創建主細胞庫(MCB)。將前MCB用作起始材料以創建四個另外的測試「模擬」庫,其中每個庫代表定義的PDL積累,代表不同的製造。參見表2。出於此研究的目的,已經將「0 PDL」指定到前MCB的PDL。
[
表 2]
測試庫 | PDL | 代表 |
前MCB | 0 | |
模擬MCB | 15 | MCB |
模擬WCB | 30 | WCB |
模擬WCB + 20 PDL | 50 | WCB + LIVCA要求 |
模擬代表MCB的PDL水平的「模擬」細胞庫係在10-12個PDL時從前MCB創建的。然後藉由解凍一小瓶模擬MCB並且累積另外的約15-17個PDL來創建類比WCB。另外,將「模擬」WCB解凍並且培養以積累適量的PDL,以創建
「模擬」 WCB+20 PDL庫和
「模擬」 WCB+35 PDL庫。
靠前殖株(1號)在0個PDL、30個PDL和50個PDL的樣本中表現出穩定的生長和生產力,其中滴定量CV僅是9%(變異係數=標準偏差/平均值),而殖株2號和3號的%CV分別係13%和64%。在具有PDL的所有三個殖株中都可以看到可接受的HMW,但是殖株2的SEC譜顯示,具有PDL的%LMW增加,這與rCE SDS中LC後見到的額外峰有關。在聚糖方面,觀察到%唾液酸、%高甘露糖、%β半乳糖和%去岩藻糖(afucose)。所有三個殖株在全部PDL中均顯示出穩定的聚糖譜(參見圖1)。類似地,所有三個殖株經0、30和50個PDL維持高FITC訊息強度(10
4-10
5),其中%CV小於1%,這表明ST6酶介導的α-2,6連接NANA聚糖表現一致(參見圖1)。
模擬穩定性樣本的遺傳表徵包括使用qPCR技術對HC(重鏈)、LC(輕鏈)、ST6和B4GALT1進行拷貝數分析,使用和不使用嘌呤黴素選擇對殖株進行評估。殖株2號和殖株號顯示出所有4種屬性的一致表現,而殖株3號顯示出HC和LC的拷貝數損失,這與殖株3號在分批進料中觀察到的滴定量降低一致。
隨後,從MCB解凍到工作細胞庫(WCB)的創建再到體外細胞年齡極限(LIVCA)時結束生產(EOP),使用若干測定來確認基於殖株1號的抗體細胞系的遺傳穩定性。藉由反轉錄聚合酶鏈反應(RT-PCR)製備抗IL12/23抗體LC和HC的互補去氧核糖核酸(cDNA),並且隨後的序列分析表明,抗體LIVCA庫的核苷酸序列與典型生產過程中MCB、WCB和EOP中的核苷酸序列相同。獲得的序列也確認了與抗體的預期cDNA序列的匹配。使用數字微滴PCR(ddPCR)確定整合在細胞系中的抗體LC和HC基因的基因組DNA(gDNA)拷貝數。來自典型生產和LIVCA樣本的EOP拷貝數與MCB和WCB類似。每個細胞庫樣本的DNA墨點分析表明,編碼整合到宿主基因組DNA(gDNA)中的抗體LC和HC的產物基因係穩定的。觀察到每個抗體LC和抗體HC基因的穩定重排。來自典型生產過程的MCB、WCB、EOP和LIVCA樣本的DNA墨點整合位點分析顯示出類似的條帶模式,這表明整合位點的遺傳穩定性。RNA墨點分析表明,抗體LC和HC預期轉錄物在每個細胞庫樣本中都存在並且穩定。還進行了遺傳表徵測定,以表徵人ST6基因在細胞庫中的穩定性。抗體細胞庫中ST6基因的分析表明,LIVCA庫的核苷酸序列與來自典型生產過程的MCB、WCB和EOP中的核苷酸序列相同。ST6基因在所有細胞庫中也具有類似的拷貝數。另外,預期的轉錄物在每個細胞庫中都存在並且穩定。
來自該等測定的結果表明抗體細胞系ST6基因或ST6和B4GALT1二者維持遺傳穩定性。
實例 3 - 錳和半乳糖作為 CHO 細胞培養組分增加治療性蛋白的唾液酸化
以下實例描述了可用於在CHO細胞中產生重組抗IL12/23抗體的上游分批進料過程(藉由實例1中描述的方法)。
將作為單獨儲備液或作為基礎培養基和進料培養基的一部分(添加前半乳糖為零)添加的總半乳糖濃度與生產生物反應器pH相組合進行研究,以調節重組蛋白的唾液酸化。基於早期的研究,將100 ppb 錳(Mn)添加至基礎培養基和進料培養基(其具有約99 ppb的基線錳水平)中,從而在整個生產階段期間向反應器中總共添加400 ppb的Mn。在pH 6.95和7.10下研究了40 mM累積的半乳糖,並且觀察到pH升高增加了唾液酸化。在第3天、第6天和第8天的基礎培養基體積目標係1,175 L,並且進料培養基總體積係400 L。因此,在整個生產過程中,總共添加1) 2.115 g錳鹽(例如,硫酸錳(II),一水合物,以357 mg經由基礎培養基和以1758 mg經由進料進行添加),和2) 15.4 kg的無水半乳糖。
此外,在pH設定點7.10下研究了20 mM、40 mM和60 mM半乳糖。另外,還在pH 7.00下研究了40 mM半乳糖。在pH 7.00下進行46 mM半乳糖濃度的最終研究。基於實例中呈現的數據,最終約46.08 mM半乳糖和400ppb Mn在pH設定點7.00下導致唾液酸水平至少高於22%。
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[圖1A、圖1B、圖1C、圖1D和圖1E]顯示了3個殖株(殖株1-3)的細胞系穩定性,如藉由評估滴定量、目的基因的mRNA表現和聚糖譜在限定的持續時間和群體倍增水平(PDL)的間隔內的變化,對此進行評估。針對α2,6連接,在 (A) %唾液酸 (B) %高甘露糖 (C) %半乳糖基化 (D) %去岩藻糖基化和 (E) FITC-西洋接骨木(
Sambucus nigra)凝集素(SNA1)染色的條件下,使用親水相互作用液相層析(HILIC)-質譜(MS)分析,針對殖株1、2和3,跨越0個PDL、30個PDL和50個PDL,評估不同聚糖。殖株的每個數據點代表不同的PDL,並且誤差槓顯示重復數據之間的標準偏差。
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TW202325853A_111142592_SEQL.xml
Claims (28)
- 一種增加由中國倉鼠卵巢(CHO)細胞產生的治療性蛋白的唾液酸含量之方法,該方法包括在該CHO細胞中表現α2,6-唾液醯基轉移酶-1(ST6)(SEQ ID NO: 1、3、5、7、9或11)。
- 一種增加由中國倉鼠卵巢(CHO)細胞產生的治療性蛋白的唾液酸含量之方法,該方法包括在該CHO細胞中共表現α2,6-唾液醯基轉移酶-1(ST6)(SEQ ID NO: 1、3、5、7、9或11)和β1,4-半乳糖苷轉移酶1(B4GALT1)(SEQ ID NO: 13、15、17、19、21、23、25或27)。
- 如請求項1或2所述之方法,其中該唾液酸係α2,6-唾液酸化聚糖。
- 如請求項1-3中任一項所述之方法,其中該蛋白係抗體或其抗原結合片段、抗體或抗體片段的衍生物、雙特異性T細胞接合物分子或融合多肽。
- 如請求項4所述之方法,其中該抗體係抗IL12抗體、抗IL23抗體、或抗IL12/23抗體。
- 一種增加由中國倉鼠卵巢(CHO)細胞產生的治療性蛋白的唾液酸含量之方法,該方法包括: (a) 在該CHO細胞中表現α2,6-唾液醯基轉移酶1(ST6)(SEQ ID NO: 1、3、5、7、9或11);以及 (b) 在包含半乳糖和錳的培養基中培養該細胞,其中在細胞培養期間,與在不包含添加的錳和半乳糖的培養基中培養的CHO細胞相比,在該包含半乳糖和錳的培養基中培養該細胞增加了由該細胞產生的蛋白的唾液酸化。
- 一種增加由中國倉鼠卵巢(CHO)細胞產生的治療性蛋白的唾液酸含量之方法,該方法包括: (a) 在該CHO細胞中表現α2,6-唾液醯基轉移酶-1(ST6)(SEQ ID NO: 1、3、5、7、9或11)和β1,4-半乳糖苷轉移酶1(B4GALT1)(SEQ ID NO: 13、15、17、19、21、23、25或27);以及 (b) 在包含半乳糖和錳的培養基中培養該細胞,其中在細胞培養期間,與在不包含添加的錳和半乳糖的培養基中培養的CHO細胞相比,在該包含半乳糖和錳的培養基中培養該細胞增加了由該細胞產生的蛋白的唾液酸化。
- 如請求項6或7所述之方法,其中在細胞培養期間,與在不包含添加的錳和半乳糖的培養基中培養的CHO細胞產生的蛋白相比,該CHO細胞產生的蛋白的唾液酸化增加了至少10%。
- 如請求項6或7所述之方法,其中在細胞培養期間,與在不包含添加的錳和半乳糖的培養基中培養的CHO細胞產生的蛋白相比,該CHO細胞產生的蛋白的唾液酸化增加了至少20%。
- 如請求項6-9中任一項所述之方法,該方法包括在細胞培養的第3天向該培養基中添加錳和半乳糖。
- 如請求項10所述之方法,其中在第3天向該培養基中添加至少100 ppb(十億分率)的錳和至少15 mM半乳糖。
- 如請求項6-11中任一項所述之方法,該方法進一步包括在第6天向該培養基中添加錳和半乳糖。
- 如請求項12所述之方法,其中在第6天向該培養基中添加至少100 ppb的錳和至少15 mM半乳糖。
- 如請求項6-13中任一項所述之方法,該方法進一步包括在第8天向該培養基中添加錳和半乳糖。
- 如請求項6-14中任一項所述之方法,其中該培養基進一步包含銅。
- 如請求項6-15中任一項所述之方法,該方法包括在培養期間累積添加約10 mM至約100 mM半乳糖。
- 如請求項16所述之方法,該方法包括在培養期間累積添加約45 mM半乳糖。
- 如請求項6-17中任一項所述之方法,該方法包括在培養期間累積添加約40 ppb至約400 ppb錳。
- 如請求項6-18中任一項所述之方法,該方法包括在培養期間累積添加約400 ppb錳。
- 如請求項15-19中任一項所述之方法,該方法包括在培養期間累積添加約0.01 mM至約0.5 mM銅。
- 如請求項20所述之方法,該方法包括在培養期間累積添加約0.1 mM銅。
- 如請求項6-21中任一項所述之方法,其中該蛋白係分泌蛋白和重組蛋白。
- 如請求項6-22中任一項所述之方法,其中該蛋白係抗體或其抗原結合片段、抗體或抗體片段的衍生物、雙特異性T細胞接合物分子或融合多肽。
- 如請求項23所述之方法,其中該抗體係抗IL12抗體、抗IL23抗體、或抗IL12/23抗體。
- 如請求項6-24中任一項所述之方法,其中該唾液酸係α2,6-唾液酸化聚糖。
- 如請求項3或25所述之方法,其中藉由親水相互作用液相層析(HILIC)-質譜(MS)分析確認該α2,6-唾液酸化聚糖的水平。
- 如請求項3或25所述之方法,其中該α2,6-唾液酸化聚糖的水平在延長的細胞培養持續時間產生的蛋白中保持恒定。
- 如請求項27所述之方法,其中該延長的細胞培養持續時間係約27個群體倍增(PDL)。
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WO1990014423A1 (en) | 1989-05-18 | 1990-11-29 | The Infergene Company | Microorganism transformation |
CA2063592C (en) | 1989-07-07 | 2003-01-21 | Marco L. F. Giuseppin | Process for preparing a protein by a fungus transformed by multicopy integration of an expression vector |
DE4028800A1 (de) * | 1990-09-11 | 1992-03-12 | Behringwerke Ag | Gentechnische sialylierung von glykoproteinen |
DE69432543T2 (de) | 1993-07-23 | 2003-12-24 | Dsm Nv | Selektionmarker-genfreie rekombinante Stämme: Verfahren zur ihrer Herstellung und die Verwendung dieser Stämme |
US5459031A (en) | 1993-11-05 | 1995-10-17 | Amgen Inc. | Methods for controlling sialic acid derivatives in recombinant glycoproteins |
US6432672B1 (en) | 1997-04-11 | 2002-08-13 | Gerardus Cornelis Maria Selten | Gene conversion as a tool for the construction of recombinant industrial filamentous fungi |
US6265186B1 (en) | 1997-04-11 | 2001-07-24 | Dsm N.V. | Yeast cells comprising at least two copies of a desired gene integrated into the chromosomal genome at more than one non-ribosomal RNA encoding domain, particularly with Kluyveromyces |
WO1999060102A2 (en) | 1998-05-19 | 1999-11-25 | Dsm N.V. | Improved in vivo production of cephalosporins |
CN1331751A (zh) | 1998-12-22 | 2002-01-16 | Dsm公司 | 改进的头孢菌素的体内生产方法 |
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US8003108B2 (en) | 2005-05-03 | 2011-08-23 | Amgen Inc. | Sclerostin epitopes |
US7592429B2 (en) | 2005-05-03 | 2009-09-22 | Ucb Sa | Sclerostin-binding antibody |
EA201000006A1 (ru) | 2007-06-20 | 2010-10-29 | Айрм Ллк | Способы и композиции для лечения аллергических заболеваний |
US7982016B2 (en) | 2007-09-10 | 2011-07-19 | Amgen Inc. | Antigen binding proteins capable of binding thymic stromal lymphopoietin |
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