CN117222665A - 具有改善的稳定性的抗tslp fab - Google Patents
具有改善的稳定性的抗tslp fab Download PDFInfo
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Abstract
本公开涉及一种具有改善的稳定性的抗TSLP Fab、编码所述Fab的核酸、包含所述核酸的宿主细胞和载体,以及使用所述Fab治疗TSLP相关疾患的方法。
Description
技术领域
本公开涉及一种具有改善的稳定性的抗TSLP Fab、编码所述Fab的核酸、包含所述核酸的宿主细胞和载体,以及使用所述Fab治疗疾病的方法。
背景技术
胸腺基质淋巴细胞生成素(TSLP)是一种细胞因子,它通过由IL-7Ra亚基和TSLP-R组成的异源二聚体受体传导信号,TSLP-R是一种独特的组分,与常见的y受体样链具有同源性(Pandey等人,Nat.Immunol.2000,1(1):59-64)。TSLP是由胸腺、肺、皮肤、肠道和扁桃体中的上皮细胞以及气道平滑肌细胞、肺成纤维细胞和基质细胞表达(Edwards,2008,Drugnews&perspectives 21,312-316;He和Geha,2010,Annals of the New York Academy ofSciences 1183,13-24;Reche等人,2001,Journal of immunology 167,336-343)。
这些细胞响应于促炎性刺激而产生TSLP,并且TSLP通过对包括树突状细胞(Soumelis等人,2002,Nature immunology 3,673-680)、单核细胞(Reche等人,2001,Journal of immunology 167,336-343)和肥大细胞(Allakhverdi等人,2007,The Journalof Experimental Medicine204,253-258)在内的多种先天性免疫细胞的活性来驱动过敏性炎症反应。已知TSLP-R和IL-7Ra表达量最高的细胞群是骨髓树突状细胞(Reche等人,2001,Journal of immunology 167,336-343)。
TSLP可促进幼稚T细胞的增殖,并驱动它们分化为高水平表达iL-4、IL-5和IL-13的Th2细胞(Omori和Ziegler,2007,Journal of immunology 178,1396-1404)。在哮喘性肺上皮细胞和慢性特应性皮炎病变中发现了高水平的TSLP表达,这表明TSLP在过敏性炎症中发挥作用(Ziegler和Artis,2010,Nature immunology 11,289-293)。最近的证据表明,TSLP与Thl7细胞分化和Thl7驱动的炎症过程有关(Hartgring等人,2011,Arthritis andrheumatism 63,1878-1887;Tanaka等人,2009,Clinical and experimental allergy:Journal of the British Society for Allergy and Clinical Immunology 39,89-100;Wu等人,2014,Journal of molecular and cellular cardiology 76,33-45)。慢性过敏性(特应性)哮喘通常以Th2型炎症为特征,而非过敏性哮喘性炎症则以中性粒细胞为主,具有混合Thl和Thl7细胞因子环境。哮喘中慢性炎症的后果包括支气管高反应性(BHR)、粘液过度分泌、气道壁重塑和气道狭窄(Lambrecht和Hammad,2014,Nature immunology 16,45-56)。经显示,TSLP参与过敏性哮喘反应的起始和维持/增强(Wang等人,2006,Immunity 24,827-838)。最近还发现,记忆T细胞对局部抗原激发的回忆反应也需要TSLP信号传导(Wang等人,2015,The Journal of allergy and clinical immunology 135,781-791e783)。
特折鲁单抗(Tezepelumab)是一种人免疫球蛋白G2(lgG2)单克隆抗体(mAb),该抗体与TSLP结合,从而阻止TSLP与TSLP受体复合物的相互作用。在轻度特应性哮喘患者中进行的概念验证研究表明,在吸入性过敏原激发后,特折鲁单抗抑制早期和晚期哮喘反应,并抑制Th2炎症的生物标志物。一项评价特折鲁单抗在患有重度不受控制哮喘的成人和青少年中的应用情况的研究(NCT03347279)近期刚结束,并满足其降低哮喘恶化年发作率(annualized asthma exacerbation rate,AERR)的主要终点[时间范围:基线至第52周]。
CSJ117是一种针对人胸腺基质淋巴细胞生成素(TSLP)的强效中和抗体片段,被配制成于硬胶囊中的PulmoSolTM工程化粉末形式以通过干粉吸入器递送到肺部(Gauvrea等人,ERS2020 56:增刊64,3690)。
因此,靶向TSLP以治疗哮喘等炎症性疾病已得到临床验证。鉴于大多数哮喘患者都习惯于通过吸入进行自我治疗,故通过吸入来施用TSLP拮抗剂的做法引起人们的兴趣。
本公开旨在以现有治疗选择为基础,特别是通过吸入这一新兴药物类别。
发明内容
本公开意外地发现,使特折鲁单抗的CH1结构域发生多个突变并将该分子转化为Fab将减少在加速稳定性条件(在1x PBS中,45℃,保持两周)下的聚集。在1x PBS中进行的加速稳定性研究有时被用作体内血清稳定性的粗略估计。
被施用于治疗的大多数抗体都具有超过14天(两周)的半衰期。在吸入递送方面,Fab可能比mAb更受青睐,因为它们的大小更小,并且气雾化后在肺部的生物分布更好。尽管是通过吸入施用,但血清稳定性仍然是肺部施用蛋白质类拮抗剂的一个相关因素,因为大部分活性剂最终都可能会分配入血清。因此,在用于治疗TSLP相关疾患时,示例性Fab的加速稳定性改善可能是有利的。
因此,在一方面,本公开提供一种Fab,该Fab包含:包含SEQ ID NO:1中所示氨基酸序列的重链,以及包含SEQ ID NO:2中所示氨基酸序列的轻链。
另一方面,本公开提供一种Fab,该Fab包含具有SEQ ID NO:1中所示氨基酸序列的重链,以及具有SEQ ID NO:2中所示氨基酸序列的轻链。
另一方面,本公开提供一种Fab,该Fab包含由SEQ ID NO:1中所示氨基酸序列组成的重链,以及由SEQ ID NO:2中所示氨基酸序列组成的轻链。
另一方面,本公开提供一种编码本文所描述的Fab的核酸。
另一方面,本公开提供一种包含本文所描述的核酸的载体。
另一方面,本公开提供一种包含本文所描述的核酸或载体的宿主细胞。
另一方面,本公开提供一种治疗方法,该方法包括向受试者施用治疗有效量的本文所描述的Fab。
另一方面,本公开提供一种用于疗法中的本文所描述的Fab。
另一方面,本公开提供本文所描述的Fab在疗法中的用途。
另一方面,本公开提供本文所描述的Fab在制造用于疗法的药剂中的用途。
附图说明
图1显示在4℃(A)和45℃(B)下用在1x PBS中约1mg/ml的特折鲁单抗进行的2周的加速稳定性测定。图中显示特折鲁单抗在各温度下培育规定时间后的HP-SEC迹线。表(C)说明特折鲁单抗存在下的单体损失率百分比和聚集体形成百分比,以及IgG1同型对照NIP228的结果。
图2显示在4℃(A)和45℃(B)下用在1x PBS中约0.8mg/ml的Fab1进行的2周加速稳定性测定。图中显示Fab1在各温度下培育规定时间后的HP-SEC迹线。表(C)说明Fab1存在下的单体损失率百分比和聚集体形成百分比,以及Fab同型对照R347的结果。
图3显示通过KinExA测量的Fab 1与人(hu)TSLP的结合。
图4显示通过KinExA测量的Fab1与食蟹猴(cyno)TSLP的结合。
图5显示使用HTRF测定测量的Fab 1与hu TSLP的竞争性结合。
图6显示Fab 1抑制由受TSLP激发的PBMC的CCL17释放。
具体实施方式
定义
应注意,术语“一个(种)(a)”或“一个(种)(an)”实体是指一个或多个该实体;例如,“一种抗TSLP Fab”应理解为表示一种或多种抗TSLP Fab。
术语“抗体”是以最广泛含义使用并涵盖各种抗体结构,包括但不限于单克隆抗体、多克隆抗体、多特异性抗体(例如双特异性抗体)和抗体片段,只要它们表现出期望的抗原结合活性。
“抗体片段”包括抗体的抗原结合部分,尤其包括Fab、Fab'、F(ab')2、Fv、结构域抗体(dAb)、互补决定区(CDR)片段、CDR移植抗体、单链抗体(scFv)、单链抗体片段、嵌合抗体、双功能抗体、三功能抗体、四功能抗体、微型抗体、线性抗体、螯合重组抗体、三抗体(tribady)或双抗体(bibody)、胞内抗体、纳米抗体(nanobody)、小模块免疫药物(smallmodular immunopharmaceutical,SMIP)、抗原结合结构域免疫球蛋白融合蛋白、单结构域抗体(包括骆驼化抗体)、含VHH的抗体或者其变体或衍生物、以及含有足以使多肽具有特异性抗原结合的免疫球蛋白的至少一部分的多肽,如一个、二个、三个、四个、五个或六个CDR序列,只要抗体保持所期望的生物活性即可。
"Fab"是指包含VH-CH1和VL-CL配对的抗体片段。所述术语涵盖包含非典型序列变体的Fab,所述非经典序列变体是例如在Fab内典型地与具有高序列变异性相关的序列区域之外的氨基酸取代、缺失或插入。例如,Fab变体包括在VH或VL构架区中或者CH1或CL结构域中包含非典型氨基酸或序列变化的Fab。这些变化可包括非典型半胱氨酸或其它可衍生化氨基酸的存在,所述氨基酸可用于将所述Fab变体与异源部分缀合。其它此类变化包括非典型多肽接头的存在,所述接头是在两个结构域之间共价桥接的多肽序列。例如,Fab变体可包含接头多肽,它将CH1结构域共价连接到VL结构域,或将CL结构域共价连接到VH结构域,由此使Fab能够以单一多肽链的形式表达。
"宿主细胞"是指带有载体的细胞,所述载体是使用DNA重组技术构建并编码至少一个异源基因。在描述从重组宿主分离抗TSLP Fab的过程时,除非另有明确规定,否则术语"细胞"和"细胞培养物"可以互换使用,表示抗TSLP Fab的来源。换句话说,从"细胞"中回收多肽可以是指从短暂离心的全细胞中回收,或是从含有培养基和悬浮细胞的细胞培养物中回收。
"分离的"是指呈自然界中未发现的形式的多肽、抗体、多核苷酸、载体、细胞或组合物。分离的多肽、抗体、多核苷酸、载体、细胞或组合物包括已被纯化达到不再呈它们在自然界中发现的形式的程度的那些。在一些方面,分离的抗体、多核苷酸、载体、细胞或组合物是大体上纯的。
“药物组合物”是指这样一种制剂,该制剂呈允许活性成分(例如本文所公开的抗TSLP Fab)的生物活性有效的形式,并且不含对将施用该组合物的受试者具有不可接受的毒性的其它组分。这种组合物可以是无菌的。
“多核苷酸”或“核酸”在本文中可互换使用,意思指任何长度的核苷酸聚合物,并且包括DNA和RNA。核苷酸可以是脱氧核糖核苷酸、核糖核苷酸、修饰过的核苷酸或碱基,和/或其类似物,或是可被DNA或RNA聚合酶并入聚合物中的任何底物。多核苷酸可包含修饰过的核苷酸,如甲基化核苷酸及其类似物。前述描述适用于本文所提到的所有多核苷酸,包括RNA和DNA。
“重组”多肽或蛋白质是指通过重组DNA技术产生的多肽或蛋白质。出于本发明的目的,在工程化宿主细胞中表达的重组产生的多肽和蛋白质,以及已通过任何适合技术分离、分级分离或者部分或大体上纯化的天然或重组多肽被认为是分离的。本文所公开的多肽可以使用本领域已知的方法重组产生。或者,本文所公开的蛋白质和肽可以是化学合成的。
“受试者”或“个体”或“动物”或“患者”或“哺乳动物”是指需要进行诊断、预后或治疗的任何受试者,特别是哺乳动物受试者,受试者被定义为“健康受试者”的情形除外。哺乳动物包括人类;家畜;农场动物;如狗、猫、豚鼠、兔、大鼠、小鼠、马、牛、奶牛等。优选地,受试者是人。
“治疗(Treat)”或“治疗(treatment)”既指治疗性治疗措施,也指防治性或预防性措施,其目的是预防或减慢(减轻)不希望出现的生理变化或紊乱。有益的或期望的临床结果包括但不限于症状减轻、疾病程度降低、疾病状态稳定(即,不恶化)、疾病进展延迟或减慢、疾病状态改善或缓和,以及缓解(部分或完全的),无论是可检测还是不可检测的。“治疗”还可指使存活期相较于未接受治疗时的预期存活期延长。需要治疗的那些包括已经患有疾患或病症的那些,以及易患疾患或病症的那些,或将预防疾患或病症的那些。
“治疗有效量”是指对“治疗”受试者或哺乳动物的疾病或病症有效的本文所公开的抗TSLP Fab或其它药物的量。
“TSLP”是指胸腺基质淋巴细胞生成素。TSLP是一种细胞因子,它通过由IL-7Ra亚基和TSLP-R组成的异源二聚体受体传导信号,TSLP-R是一种独特的组分,与常见的g受体样链具有同源性(Pandey等人,Nat.Immunol.2000,l(l):59-64)。TSLP是由胸腺、肺、皮肤、肠道和扁桃体中的上皮细胞以及气道平滑肌细胞、肺成纤维细胞和基质细胞表达(Edwards,2008,Drug news&perspectives 21,312-316;He和Geha,2010,Annals of the New YorkAcademy of Sciences 1183,13-24;Reche等人,2001,Journal of immunology 167,336-343)。这些细胞响应于促炎性刺激而产生TSLP,并且TSLP通过对包括树突状细胞(Soumelis等人,2002,Nature immunology 3,673-680)、单核细胞(Reche等人,2001,Journal ofimmunology 167,336-343)和肥大细胞(Allakhverdi等人,2007,The Journal ofExperimental Medicine 204,253-258)在内的多种先天性免疫细胞的活性来驱动过敏性炎症反应。已知TSLP-R和IL-7Ra表达量最高的细胞群体是骨髓树突状细胞(Reche等人,2001,Journal of immunology 167,336-343)。
TSLP可促进幼稚T细胞的增殖,并驱动它们分化为高水平表达IL-4、IL-5和IL-13的Th2细胞(Omori和Ziegler,2007,Journal of immunology 178,1396-1404)。在哮喘性肺上皮细胞和慢性特应性皮炎病变中发现了高水平的TSLP表达,这表明TSLP在过敏性炎症中发挥作用(Ziegler和Artis,2010,Nature immunology 11,289-293)。最近的证据表明,TSLP与Th17细胞分化和Th17驱动的炎症过程有关(Hartgring等人,201 1,Arthritis andrheumatism 63,1878-1887;Tanaka等人,2009,Clinical and experimental allergy:Journal of the British Society for Allergy and Clinical Immunology 39,89-100;Wu等人,2014,Journal of molecular and cellular cardiology 76,33-45)。慢性过敏性(特应性)哮喘通常以Th2型炎症为特征,而非过敏性哮喘性炎症则以中性粒细胞为主,具有混合Th1和Th17细胞因子环境。哮喘中慢性炎症的后果包括支气管高反应性(BHR)、粘液过度分泌、气道壁重塑和气道狭窄(Lambrecht和Hammad,2014,Nature immunology 16,45-56)。经显示,TSLP参与过敏性哮喘反应的起始和维持/增强(Wang等人,2006,Immunity 24,827-838)。最近还发现,记忆T细胞对局部抗原激发的回忆反应也需要TSLP信号传导(Wang等人,2015,The Journal of allergy and clinical immunology 135,781-791e783)。
“载体”是指能够在宿主细胞中递送,并在一些方面表达一个或多个感兴趣基因或序列的构建体。载体的实例包括但不限于病毒载体;裸DNA或RNA表达载体;质粒、黏粒或噬菌体载体;与阳离子缩合剂相联的DNA或RNA表达载体;囊封于脂质体中的DNA或RNA表达载体;以及某些真核细胞,如生产者细胞。
抗TSLP Fab
本公开提供一种Fab,所述Fab包含:包含SEQ ID NO:1中所示氨基酸序列的重链和包含SEQ ID NO:2中所示氨基酸序列的轻链。
另一方面,本公开提供一种Fab,所述Fab包含:包含SEQ ID NO:1中所示氨基酸序列的重链和包含SEQ ID NO:2中所示氨基酸序列的轻链。
另一方面,本公开提供一种Fab,所述Fab包含:包含SEQ ID NO:1中所示氨基酸序列的重链和包含SEQ ID NO:2中所示氨基酸序列的轻链。
这些实例表明,与具有类似VH和VL结构域序列的全长抗体相比,具有这些序列特征的Fab在加速稳定性研究条件下具有改善的稳定性。具有改善的稳定性的示例性Fab在本文中称为Fab 1。
在一些实例中,本公开提供一种与Fab 1具有等效稳定性的Fab。在一些实例中,等效稳定性是指在加速稳定性研究条件下的等效稳定性。在一些实例中,加速稳定性研究的条件是1X PBS,45℃,保持两周。在一些实例中,在加速研究条件下Fab的浓度是约0.8mg/ml。
在一些实例中,Fab包含与SEQ ID NO:1的氨基酸序列具有至少80%、85%、90%或95%序列同一性的重链。在一些实例中,Fab包含与SEQ ID NO:1的氨基酸序列具有至少95%序列同一性的重链。在一些实例中,Fab包含与Fab 1等效的稳定性。
在一些实例中,Fab包含与SEQ ID NO:2的氨基酸序列具有至少80%、85%、90%或95%序列同一性的轻链。在一些实例中,Fab包含与SEQ ID NO:2的氨基酸序列具有至少95%序列同一性的轻链。在一些实例中,Fab包含与Fab 1等效的稳定性。
在一些实例中,Fab包含与SEQ ID NO:1的氨基酸序列具有至少80%、85%、90%或95%序列同一性的重链以及与SEQ ID NO:2的氨基酸序列具有至少80%、85%、90%或95%序列同一性的轻链。在一些实例中,Fab包含与Fab 1等效的稳定性。
在一些实例中,Fab包含与SEQ ID NO:1的氨基酸序列具有至少95%序列同一性的重链以及与SEQ ID NO:2的氨基酸序列具有至少95%序列同一性的轻链。在一些实例中,Fab包含与Fab 1等效的稳定性。
在一些实例中,本公开提供一种包含SEQ ID NO:1重链的抗原结合片段。
在一些实例中,本公开提供抗原结合片段,所述抗原结合片段包含与SEQ ID NO:1的氨基酸序列具有至少80%、85%、90%或95%序列同一性的重链。在一些实例中,所述抗原结合片段包含与SEQ ID NO:1的氨基酸序列具有至少95%序列同一性的重链。在一些实例中,所述抗原结合片段包含与Fab 1等效的稳定性。
在一些实例中,所述抗原结合片段包含与SEQ ID NO:2的氨基酸序列具有至少80%、85%、90%或95%序列同一性的轻链。在一些实例中,所述抗原结合片段包含与SEQID NO:2的氨基酸序列具有至少95%序列同一性的轻链。在一些实例中,所述抗原结合片段包含与Fab1等效的稳定性。
在一些实例中,所述抗原结合片段包含与SEQ ID NO:1的氨基酸序列具有至少80%、85%、90%或95%序列同一性的重链以及与SEQ ID NO:2的氨基酸序列具有至少80%、85%、90%或95%序列同一性的轻链。在一些实例中,所述抗原结合片段包含与Fab 1等效的稳定性。
在一些实例中,所述抗原结合片段包含与SEQ ID NO:1的氨基酸序列具有至少95%序列同一性的重链以及与SEQ ID NO:2的氨基酸序列具有至少95%序列同一性的轻链。在一些实例中,所述抗原结合片段包含与Fab 1等效的稳定性。
核苷酸
本公开还提供了编码本文所公开的Fab和抗原结合片段的核酸(或“多核苷酸”)。
本文所公开的多核苷酸还可包括另外的核酸,所述核酸编码例如信号肽以引导本文所描述的编码多肽的分泌。
所述多核苷酸可以通过本领域已知的任何方法产生或制造。例如,如果Fab的核苷酸序列已知,则可以由化学合成的寡核苷酸组装编码抗TSLP Fab的多核苷酸(例如Kutmeier等人,Bio Techniques17:242(1994)中所描述),简单地说,所述方法涉及合成含有编码抗TSLP Fab的序列的各部分的重叠寡核苷酸,退火并连接这些寡核苷酸,然后通过PCR扩增连接的寡核苷酸。
或者,可以从适合来源的核酸产生编码抗TSLP Fab的多核苷酸。如果无法得到含有编码Fab的核酸的克隆,但Fab的序列是已知的,则可通过化学合成或从适合的来源获得编码Fab的核酸,例如通过使用可与所述序列的3'端和5'端杂交的合成引物进行PCR扩增,或通过使用对感兴趣的特定序列具有特异性的寡核苷酸探针进行克隆。然后,由PCR产生的扩增的核酸可以使用本领域已知的任何方法克隆到可复制的克隆载体中。
在确定了Fab的核苷酸序列和相应氨基酸序列后,就可以使用本领域众所周知的核苷酸序列操作方法,例如重组DNA技术、定点诱变、PCR等,对其核苷酸序列进行操作(参见例如Sambrook等人(1990)Molecular Cloning,A Laboratory Manual(第2版;Cold SpringHarbor Laboratory,Cold Spring Harbor,N.Y.)和Ausubel等人编(1998)CurrentProtocols in Molecular Biology(John Wiley&Sons,NY)中所描述的技术,所述文献均以引用的方式整体并入本文中),以产生具有不同氨基酸序列的Fab,例如产生氨基酸替换、缺失和/或插入。
编码Fab的多核苷酸可以由任何多核糖核苷酸或多脱氧核糖核苷酸构成,所述多核糖核苷酸或多脱氧核糖核苷酸可以是未修饰的RNA或DNA,或是经过修饰的RNA或DNA。例如,编码抗TSLP Fab的多核苷酸可以由以下构成:单链和双链DNA;呈单链区和双链区的混合物形式的DNA;单链和双链RNA,以及呈单链区和双链区的混合物形式的RNA;包含DNA和RNA的杂交分子,所述DNA和RNA可以是单链的,或更典型地是双链的,或者是单链区和双链区的混合物。此外,编码所述Fab的多核苷酸还可以由包含RNA或DNA或者RNA和DNA两者的三链区构成。编码抗TSLP Fab的多核苷酸也可含有针对稳定性或其它原因进行修饰的一个或多个经过修饰的碱基或者DNA或RNA主链。“经过修饰”的碱基包括例如三苯甲基化碱基和不常见的碱基,如肌苷。DNA和RNA可以进行各种修饰;因此,“多核苷酸”包括化学、酶或代谢修饰的形式。
通过在免疫球蛋白的核苷酸序列中引入一个或多个核苷酸取代、添加或缺失,使得在编码的蛋白质中引入一个或多个氨基酸取代、添加或缺失,可以产生编码来源于免疫球蛋白(例如免疫球蛋白重链部分或轻链部分)的多肽的非天然变体的分离的多核苷酸。突变可通过标准技术引入,如定点诱变和PCR介导的诱变。优选地,在一个或多个非必需氨基酸残基处进行保守氨基酸取代。
制造方法
编码抗TSLP Fab或抗原结合片段的多核苷酸典型地被插入表达载体中,以便引入宿主细胞中,用于生产期望数量的Fab。因此,本文涵盖了包含编码本文所定义的Fab的多核苷酸的表达载体,以及包含所述表达载体的宿主细胞。
Fab或抗原结合片段的重组表达需要构建一种表达载体,该表达载体含有编码该Fab的多核苷酸。在获得了编码本公开的Fab的多核苷酸后,就可以通过DNA重组技术,使用本领域众所周知的技术产生用于产生Fab的载体。
编码Fab的DNA序列可以根据众所周知的方法,使用逆转录酶和DNA聚合酶同时或分别制备。PCR可以使用共同恒定区引物或根据已公布的DNA和氨基酸序列,使用更具特异性的引物来起始。PCR还可用于分离编码抗体可变轻链和重链的DNA克隆。在这种情况下,可以使用共同引物或较大的同源探针,如小鼠恒定区探针对文库进行筛选。
因此,本文描述通过表达含有编码Fab的核苷酸序列的多核苷酸来制备蛋白质的方法。可以使用本领域技术人员众所周知的方法构建表达载体,所述表达载体含有抗TSLPFab编码序列以及适当的转录和翻译控制信号。这些方法包括例如体外重组DNA技术、合成技术和体内基因重组。因此,本公开提供可复制的载体,所述载体包含可操作地连接至启动子的编码本公开的Fab的核苷酸序列。
出于本公开的目的,可以使用多种表达载体系统。例如,一类载体利用来源于动物病毒的DNA元件,所述动物病毒例如为牛乳头状瘤病毒、多瘤病毒、腺病毒、牛痘病毒、杆状病毒、逆转录病毒(RSV、MMTV或MOMLV)或SV40病毒。其它方法涉及使用具有内部核糖体结合位点的多顺反子系统。此外,还可以通过引入一种或多种允许选择转染后的宿主细胞的标记物来选择已将DNA整合到染色体中的细胞。所述标记物可提供对营养缺陷型宿主的原生营养、生物杀灭剂抗性(如抗生素)或对铜等重金属的抗性。选择性标记物基因可以直接连接至待表达的DNA序列,或是通过共转化引入同一个细胞中。要实现mRNA的最佳合成,可能还需要另外的元件。这些元件可包括信号序列、剪接信号以及转录启动子、增强子和终止信号。
能够在真核细胞中引起表达的任何表达载体都可用于本公开中。适合载体的实例包括但不限于质粒pcDNA3、pHCMV/Zeo、pCR3.1、pEF 1/His、pIND/GS、pRc/HCMV2、pSV40/Zeo2、pTRACER-HCMV、pUB6/V5-His、pVAXl和pZeoSV2(可从加利福尼亚州圣地亚哥(SanDiego,Calif.)的Invitrogen公司获得),以及质粒pCI(可从威斯康星州麦迪逊(Madison,Wis.)的Promega公司获得)。一般来说,筛选大量的转化后的细胞以获得能表达
更一般地说,在制备出编码Fab的载体或DNA序列后,就可以将表达载体引入适当的宿主细胞中。将质粒引入宿主细胞可通过本领域技术人员众所周知的各种技术实现。这些技术包括但不限于转染(包括电泳和电穿孔)、原生质体融合、磷酸钙沉淀、包膜DNA的细胞融合、显微注射和完整病毒感染。参见Ridgway(1988)“Mammalian Expression Vectors”in Vectors,Rodriguez和Denhardt编辑(Butterworths,Boston,Mass.),第24.2章,第470-472页。典型地,质粒是通过电穿孔引入宿主体内。使带有表达构建体的宿主细胞在适合产生抗TSLP Fab的条件下生长,并进行蛋白质合成测定。示例性测定技术包括酶联免疫吸附测定(ELISA)、放射免疫测定(RIA)或荧光激活细胞分选仪分析(FACS)、免疫组织化学等。
通过常规技术将表达载体转移到宿主细胞中,然后通过常规技术培养转染后的细胞,产生用于本文所描述的方法中的抗TSLP Fab。因此,本公开包括宿主细胞,所述宿主细胞含有编码本公开的Fab,例如重链或轻链,或者可变重链或可变轻链的多核苷酸,所述多核苷酸可操作地连接至异源启动子。
在一个实例中,提供一种包含所述宿主细胞的培养基。在一个实例中,提供一种包含所述培养基的发酵容器。
在一些实例中,培养基和发酵容器适于进行产生本文所定义的Fab的方法。
多种宿主表达载体系统可以用来表达本文所描述的抗TSLP Fab。这些宿主表达系统不仅表示可以产生且随后纯化感兴趣编码序列的媒介物,而且还表示可以在用适当核苷酸编码序列转化或转染时原位表达本公开的分子的细胞。这些包括但不限于:用含有编码序列的重组噬菌体DNA、质粒DNA或黏粒DNA表达载体转化的微生物,例如细菌(如大肠杆菌(E.coli)、枯草芽孢杆菌(B.subtilis));用含有编码序列的重组酵母表达载体转化的酵母(如酵母(Saccharomyces)、毕赤酵母(Pichia));用含有编码序列的重组病毒表达载体(如杆状病毒)感染的昆虫细胞系统;用重组病毒表达载体(如花椰菜花叶病毒(cauliflowermosaic virus),即CaMV;烟草花叶病毒(tobacco mosaic virus),即TMV)感染或用含有编码序列的重组质粒表达载体(例如Ti质粒)转化的植物细胞系统;或带有重组表达构建体的哺乳动物细胞系统(例如COS、CHO、BLK、293、3T3细胞),所述重组表达构建体含有来源于哺乳动物细胞基因组的启动子(例如金属硫蛋白启动子)或来源于哺乳动物病毒的启动子(例如腺病毒晚期启动子;牛痘病毒7.5K启动子)。
使用细菌细胞表达Fab,例如大肠杆菌,并且更优选真核细胞。例如,中国仓鼠卵巢细胞(CHO)等哺乳动物细胞联合来自人巨细胞病毒的主要立即早期基因启动子元件等载体是一种有效的抗体表达系统(Foecking等人,Gene 45:101(1986);Cockett等人,Bio/Technology8:2(1990))。
用于蛋白质表达的宿主细胞系通常是哺乳动物来源的;相信本领域技术人员有能力优先确定最适合表达期望基因产物的特定宿主细胞系。示例性宿主细胞系包括但不限于CHO(中国仓鼠卵巢)、DG44和DUXB13(中国仓鼠卵巢系,DHFR-)、HELA(人宫颈癌)、CVI(猴肾系)、COS(带有SV40T抗原的CVI衍生物)、VERO、BHK(幼仓鼠肾)、MDCK、293、WI38、R1610(中国仓鼠成纤维细胞)、BALBC/3T3(小鼠成纤维细胞)、HAK(仓鼠肾系)、SP2/0(小鼠骨髓瘤)、P3×63-Ag3.653(小鼠骨髓瘤)、BFA-lclBPT(牛内皮细胞)、RAJI(人淋巴细胞)和293(人肾)。宿主细胞系典型地可从商业服务机构、美国组织培养物保藏中心(American TissueCulture Collection)或发表的文献中获得。
此外,还可以选择一种宿主细胞株来调节插入序列的表达,或以期望的特定方式修饰和加工基因产物。蛋白质产物的这种修饰(例如糖基化)和加工(例如切割)对于蛋白质的功能非常重要。不同的宿主细胞在蛋白质和基因产物的翻译后加工和修饰方面具有各自特有的机制。可选择适当的细胞系或宿主系统以确保对所表达的外来蛋白进行正确的修饰和加工。为此,可以使用真核宿主细胞,这些细胞拥有适当加工主转录本、糖基化和磷酸化基因产物的细胞机制。
要长期、高产率生产重组蛋白,稳定表达是首选的。例如,可以对稳定表达抗TSLPFab的细胞系进行工程化。与使用含有病毒复制起点的表达载体不同,宿主细胞可以用受适当的表达控制元件(例如启动子、增强子、序列、转录终止子、多聚腺苷酸化位点等)和选择性标记物控制的DNA进行转化。引入外来DNA后,可使工程化的细胞在加富培养基中生长1-2天,然后转换成选择性培养基。重组质粒中的选择性标记物使细胞对选择具有抗性,并使细胞将质粒稳定地整合到其染色体中,并生长形成灶点(foci),进而可以克隆并扩增成细胞系。这种方法可有利地用于对稳定表达抗TSLP Fab的细胞系进行工程化。
可使用多种选择系统,包括但不限于单纯疱疹病毒胸苷激酶(Wigler等人,Cell13:223(1977))、次黄嘌呤-鸟嘌呤磷酸核糖转移酶(Szybalska和Szybalski,Proc.Natl.Acad.Sci.USA 48:202(1992))和腺嘌呤磷酸核糖转移酶(Lowy等人,Cell 22:817(1980))基因可分别用于tk-细胞、hgprt-细胞或aprt-细胞。此外,抗代谢物抗性也可用作以下基因的选择基础:dhfr,它赋予对甲氨蝶呤(methotrexate)的抗性(Wigler等人,Natl.Acad.Sci.USA 77:357(1980);O'Hare等人,Proc.Natl.Acad.Sci.USA 78:1521(1981));gpt,它赋予对霉酚酸的抗性(Mulligan和Berg,Proc.Natl.Acad.Sci.USA 78:2012(1981));neo,它赋予对氨基糖苷G-418的抗性(Clinical Pharmacy 12:488-505;Wu和Wu,Biotherapy 3:87-95(1991);Tolstoshev,Ann.Rev.Pharmacol.Toxicol.52:573-596(1993);Mulligan,Science 260:926-932(1993);以及Morgan and Anderson,Ann.Rev.Biochem.62:191-217(1993);TIB TECH 13(5):155-215(1993年5月);和hygro,它赋予对潮霉素(hygromycin)的抗性(Santerre等人,Gene 30:141(1984)。本领域中通常已知的可以使用的重组DNA技术方法描述于Ausubel等人(1993)Current Protocols inMolecular Biology(John Wiley&Sons,NY);Kriegler(1990)“Gene Transfer andExpression”in A Laboratory Manual(Stockton Press,NY);Dracopoli等人(编)(1994)Current Protocols in Human Genetics(John Wiley&Sons,NY)第12和13章;Colberre-Garapin等人(1981)J.Mol.Biol.150:1中,所述文献以引用的方式整体并入本文中。
可通过载体扩增来提高Fab的表达水平(相关评述,参见Bebbington和Hentschel(1987)“The Use of Vectors Based on Gene Amplification for the Expression ofCloned Genes in Mammalian Cells in DNA Cloning”(Academic Press,NY)第3卷)。当载体系统中表达抗TSLP Fab的标记物可扩增时,提高宿主细胞培养物中的抑制剂水平将增加标记物基因的拷贝数。由于扩增的区域与抗TSLP Fab基因相关联,故抗TSLP Fab的产量也会增加(Crouse等人,Mol.Cell.Biol.3:251(1983))。
体外生产允许扩大规模,生产出大量期望的多肽。用于在组织培养条件下进行哺乳动物细胞培养的技术是本领域已知的,并且包括均相悬浮培养,例如在气升式反应器或连续搅拌反应器中;或者固定或包埋式细胞培养,例如在中空纤维中、微胶囊中、琼脂糖微珠上或陶瓷筒上。如有必要和/或需要,可以例如在优先生物合成了合成铰链区多肽之后,或者在本文所描述的HIC层析步骤之前或之后,采用常用的层析方法,例如凝胶过滤、离子交换层析、DEAE-纤维素层析或(免疫)亲和层析等,对多肽溶液进行纯化。
编码本公开的抗TSLP Fab的基因也可以在非哺乳动物细胞中表达,例如昆虫、细菌或酵母或植物细胞。容易接受核酸的细菌包括以下细菌成员:肠杆菌科(enterobacteriaceae),例如大肠杆菌或沙门氏菌(Salmonella)菌株;芽孢杆菌科(Bacillaceae),例如枯草芽孢杆菌;肺炎球菌(Pneumococcus);链球菌(Streptococcus)和流感嗜血杆菌(Haemophilus influenzae)。还应理解,在细菌中表达时,异源多肽典型地会成为包涵体的一部分。异源多肽必须分离、纯化,然后组装成功能分子。如果需要四价形式的抗体,则亚基会自组装成四价抗体(WO 02/096948 A2)。
在细菌系统中,取决于所表达的抗TSLP Fab的预定用途,可以有利地选择多种表达载体。例如,当打算生产大量这种蛋白质,以产生Fab的药物组合物时,可能需要引导高水平表达易于纯化的融合蛋白产物的载体。这种载体包括但不限于大肠杆菌表达载体pUR278(Ruther等人,EMBO J.2:1791(1983)),其中的编码序列可独立地与lacZ编码区同框连接到载体中,从而产生融合蛋白;pIN载体(Inouye和Inouye,Nucleic Acids Res.iJ:3101-3109(1985);Van Heeke和Schuster,J.Biol.Chem.24:5503-5509(1989))等。pGEX载体还可用于以与谷胱甘肽S-转移酶(GST)的融合蛋白形式表达外来多肽。一般来说,这种融合蛋白是可溶的,并且可以通过吸附并结合到基质谷胱甘肽-琼脂糖珠粒上,随后在游离谷胱甘肽存在下进行洗脱,很容易地从裂解的细胞中纯化出来。pGEX载体被设计成包括凝血酶或Xa因子蛋白酶切割位点,由此使克隆的靶基因产物能从GST部分释放。
除原核生物外,还可使用真核微生物。在真核微生物中,最常用的是酿酒酵母(Saccharomyces cerevisiae),或是常见的面包酵母,但也可商购许多其它菌株,如巴斯德毕赤酵母(Pichia pas tons)。
在酵母中表达时,通常使用例如质粒YRp7(Stinchcomb等人,Nature 282:39(1979);Kingsman等人,Gene 7:141(1979);Tschemper等人,Gene 10:151(1980))。这种质粒已含有TRP1基因,该基因提供用于不能在色氨酸中生长的酵母突变株,例如ATCC编号44076或PEP4-1的选择标记物(Jones,Genetics 85:12(1977))。酵母宿主细胞基因组中特有的trpl损伤的存在则提供了用于检测由在无色氨酸存在下生长引起的转化的有效环境。
在昆虫系统中,苜蓿银纹夜蛾(Autographa californica)核多面体病毒(AcNPV)典型地被用作表达外来基因的载体。所述病毒在草地贪夜蛾(Spodoptera frugiperda)细胞中生长。Fab编码序列可单独克隆到病毒的非必要区(例如多面体蛋白基因)中,并置于AcNPV启动子(例如多面体蛋白启动子)的控制之下。
在重组表达出本公开的Fab后,就可采用本领域已知的任何纯化免疫球蛋白分子的方法进行纯化,例如层析法(例如离子交换层析法、亲和层析法,特别是利用蛋白质A后对特定抗原的亲和力,以及定尺寸柱层析法)、离心法、差异溶解度法或任何其它纯化蛋白质的标准技术。或者,美国专利申请公开号2002 0123057Al中公开了一种用于增加本公开抗体的亲和力的优选方法。
治疗方法
本公开还提供治疗方法,所述方法包括向有需要的受试者施用治疗有效量的本文所描述的抗TSLP Fab或药物组合物。在一些实例中,所述方法用于治疗TSLP相关疾患。
本公开还提供用于疗法中的本文所描述的抗TSLP Fab或药物组合物。在一些实例中,所述疗法是TSLP相关疾患的治疗。
本公开还提供抗TSLP Fab或药物组合物在制造用于治疗疾病的药剂中的用途。在一些实例中,所述疾病是TSLP相关疾患。
本公开还提供抗TSLP Fab或药物组合物在疗法中的用途。在一些实例中,所述疗法是TSLP相关疾患的治疗。
在一些实例中,TSLP相关疾患是TSLP相关炎症性疾患。在一些实例中,TSLP相关炎症性疾患选自哮喘、脓毒症、脓毒性休克、特应性皮炎、过敏性鼻炎、过敏性鼻窦炎、过敏性结膜炎、嗜酸性粒细胞性食管炎、类风湿性关节炎、慢性阻塞性肺病(COPD)、哮喘、COPD重叠综合征(ACOS)、慢性支气管炎、肺气肿、伴有或不伴有鼻息肉的慢性鼻窦炎、血管炎、GvHD、葡萄膜炎、慢性特发性荨麻疹、鼻窦炎或胰腺炎。
在一些实例中,TSLP相关炎症性疾患是哮喘。
哮喘是一种复杂的异质性气道炎症性疾病,其特征是症状多变且反复发作、可逆性气流阻塞和支气管痉挛。
哮喘的症状可包括喘鸣、咳嗽、胸闷和呼吸短促。暴露于过敏原或刺激物会引发症状。根据症状是由过敏原诱发(特应性)还是并非由过敏原诱发(非特应性),哮喘可分为特应性(外因性)和非特应性(内因性)两种。哮喘急性加重通常被称为“哮喘发作”。哮喘发作期间可能出现的其它病征包括使用辅助呼吸肌(颈部的胸锁乳突肌和斜角肌),可能出现奇脉(吸气时脉搏较弱,而呼气时脉搏较强),以及胸部过度扩张。缺氧可能导致皮肤和指甲呈蓝色。除了这些积极的治疗反应外,正在接受抗TSLP Fab治疗的受试者还可能会体验到一种或多种与疾病相关的这些症状的有益效果或改善。
临床反应可使用筛选技术,如磁共振成像(MRI)扫描、x射线成像、计算机断层(CT)扫描、流式细胞术或荧光激活细胞分选仪(FACS)分析、组织学、大体病理学和血液化学等筛查技术进行评估,包括但不限于可通过ELISA、RIA、层析法等方法检测的变化。
本文所公开的Fab可以与任何已知的炎症性疾病治疗方法结合使用,包括已知可用于或者已经用于或当前正用于治疗哮喘或COPD等炎症性疾病的任何药剂或药剂组合。可与本文所描述的Fab组合施用的示例性活性剂包括但不限于吸入性皮质类固醇(ICS)、支气管扩张剂(包括长效β激动剂(LABA)、长效抗毒蕈碱激动剂(LAMA)、短效β激动剂(SABA)和毒蕈碱β2激动剂(MABA))、抗组胺药、抗白三烯、PDE-4抑制剂、两面神激酶(janus kinase)抑制剂和磷酸肌醇3激酶抑制剂。
术语“组合”是指呈一种剂量单位形式的固定组合,或是指组合施用,在组合施用情况下,抗TSLP Fab和组合搭配物(例如另一种药物,又称为“治疗剂”或“共用药剂(co-agent)”)可在同一时间独立地施用,或在时间间隔内分开施用,尤其是这些时间间隔允许组合搭配物显示出合作、例如协同作用的情形。各单独组分可以被包装于一个试剂盒中,或是分开包装。其中一种或两种组分(例如粉末或液体)可在施用前重构或稀释至期望剂量。如本文所使用,术语“共同施用”或“组合施用”等意图涵盖将选定的组合搭配物施用给单一有需要的受试者(例如患者),并且打算包括各药剂不一定通过相同施用途径或在相同时间施用的治疗方案。如本文所使用,术语“药物组合”是指由混合或组合多于一种治疗剂所得到的产品,并包括治疗剂的固定组合和非固定组合。术语“固定组合”意思指将多种治疗剂,例如抗TSLP Fab和组合搭配物,以单一实体或剂量形式同时施用给患者。术语“非固定组合”意思指将多种治疗剂,例如抗TSLP Fab和组合搭配物,以分开的实体形式同时、并行或依序且无具体时间限制地施用给患者,其中这种施用在患者体内提供治疗有效水平的所述两种化合物。后者还适用于鸡尾酒疗法,例如施用三种或更多种治疗剂。
术语“组合疗法”是指施用两种或更多种治疗剂以治疗本公开中所描述的治疗性疾患或病症。此类施用涵盖以大体上同时的方式,例如以具有固定活性成分比率的单一胶囊形式共同施用这些治疗剂。或者,此类施用涵盖每种活性成分以多个容器或分开的容器(例如片剂、胶囊、粉剂和液体)共同施用。粉剂和/或液体可在施用前重构或稀释至期望剂量。此外,此类施用还涵盖在大约相同时间或在不同时间依序使用每种类型的治疗剂。在任一情形中,治疗方案将在治疗本文所描述的疾患或病症中提供药物组合的有益作用。
组合物
本文所公开的医疗用途和方法中的抗TSLP Fab可以呈药物组合物形式施用给受试者。
在一些实例中,任何本文提及的‘一种/所述抗TSLP Fab’还可以指包含一种/所述抗TSLP Fab的药物组合物。
在一些实例中,抗TSLP Fab或其药物组合物可根据上述治疗方法/医疗用途,以足以产生治疗效果的量施用给人或其它动物。
在一些实例中,抗TSLP Fab或其药物组合物可以呈常规剂型施用给此类人或其它动物,所述剂型是根据已知技术,通过将抗TSLP Fab与常规药学上可接受的载体或稀释剂组合来制备。
本领域的技术人员将认识到,药学上可接受的载体或稀释剂的形式和性质是由打算与之组合的活性成分的量、施用途径和其它众所周知的变量决定。
在一些实例中,药物组合物被配制成包含药学上可接受的无毒无菌载体,如生理盐水、无毒缓冲剂、防腐剂等。在一些实例中,药物组合物可以包括无菌水性或非水性溶液、悬浮液和乳液。适合用于本文所公开的治疗方法中的配制物描述于Remington'sPharmaceutical Sciences(Mack Publishing Co.)第16版(1980)中。
在一些实例中,抗TSLP Fab或其药物组合物的施用途径可以是例如口服、肠胃外、通过吸入或表面施用。在一些实例中,如本文所使用,术语肠胃外包括例如静脉内、动脉内、腹膜内、肌肉内、皮下、直肠或阴道施用。
在一些实例中,抗TSLP Fab或其药物组合物可以通过鼻用气雾剂或吸入施用。
在一些实例中,本文所描述的用于制备所述药物组合物的组分可以包装成试剂盒形式并销售。在一些实例中,这种试剂盒具有标签或包装插页,说明相关药物组合物可用于治疗患有或易患某种疾病或病征的受试者。
实施例
实施例1
在4℃或45℃下,在1x PBS中对特折鲁单抗进行稳定性研究。将包含1mg/ml特折鲁单抗的溶液在每种条件下储存两周。
结果表明,在45℃下于D-PBS中放置2周后,观察到10%的单体损失率(图1)。通过定量HP-SEC层析图的曲线下面积(AUC)计算单体损失率%、聚集率%和断裂率%。根据洗脱体积,将峰指定为单体、聚集体或断裂产物。可以使用HP SEC分析软件提供的标准分析工具计算AUC。
构建一种Fab,它包含特折鲁单抗重链和轻链互补决定区(CDR),并在CH1结构域具有多个突变。这种Fab在本文中称为Fab 1。如以上关于特折鲁单抗所描述来分析Fab 1的稳定性。结果示于图2中。
意外地是,在加速稳定性研究条件(1x PBS,45℃)下,当以0.8mg/ml储存两周时,Fab 1显示出改善的稳定特性。
实施例2–Fab1以pM亲和力与人和食蟹猴TSLP结合
通过BIAcore确定Fab1与TSLP结合的亲和力
使用Biacore 8K SPR仪器(GE Healthcare,Little Chalfont,Bucks,UK)确定Fab1对重组哺乳动物细胞表达的人和食蟹猴TSLP的特异性和亲和力。
S系列C1生物传感芯片、胺偶联试剂盒、基于hepes缓冲盐水的缓冲液和再生缓冲液均获自GE Healthcare,并根据制造商的说明使用。使用冻干的链霉亲和素(streptavidin)制备链霉亲和素表面,所述链霉亲和素用D-PBS重构。简单地说,将链霉亲和素在10mM乙酸钠(pH 4.5)中稀释至4μg mL-1,并通过标准胺偶联方法共价固定至S系列C1生物传感芯片的三个流通池表面。获得有170个反应单元(RU)的最终链霉亲和素表面。另外,使用胺偶联试剂制备未固定链霉亲和素的对照空白表面,以充当每个流通池内的参照表面。接着,将N末端标记的生物素化TSLP(人和食蟹猴)滴定到每个链霉亲和素表面上,以使<100RU的Fab1能够饱和结合(Rmax)。低水平的分析物结合确保最大限度地减少质量传输诱导的假像,尤其是与在动力学测量步骤期间使用的相对较快的50μL min-1测定流速组合时。以50μL min-1测定流速注入单体化Fab1的稀释液(多循环动力学)(在HBS-EP+缓冲液中2倍稀释,范围在1.25与20nM之间),缔合2分钟并且解离10分钟。在整个实验中,在相同条件下进行多次单独缓冲液注射,以允许对最终传感图集进行双重参考处理。
通过流过两个30秒的10mM甘氨酸(pH 1.7)脉冲使芯片表面完全再生。使用1:1朗缪尔(Langmuir)模型确定结合亲和力和动力学。
表1中所示的结果展示,Fab1以相似亲力与固定的人和食蟹猴TSLP结合(2倍以内;分别为46pM和88pM)。
表1使用BIAcore确定的Fab1对人和食蟹猴TSLP的亲和力
分析物 | ka(M-1s-1) | kd(s-1) | KD(pM) |
人TSLP | 2.39E6 | 1.11E-4 | 46.3 |
食蟹猴TSLP | 1.75E6 | 1.55E-4 | 88.4 |
通过动力学排除测定(KinExA)确定结合亲和力。
另外,使用KinExA 3200仪器(Sapidyne Instruments,Boise,Idaho,USA)确定Fab1对人和食蟹猴TSLP的溶液相结合亲和力(KD),并使用4.1.11版KinExA Pro软件处理所得数据。KinExA方法已有评述(Darling和Brault,2004)。
将Fab 1分别与不同浓度的人和食蟹猴TSLP预混合,直至达到平衡(使用2倍系列稀释法分别制备至少12种浓度的人和食蟹猴TSLP)。接着,使用KinExA仪器,通过使用涂有人TSLP的珠粒捕获游离Fab,洗去未结合的物质,并使用商购的物种特异性抗体(AlexaFluor 647标记的小鼠抗人重链和轻链特异性抗体(Jackson Immunoresearch 209-605-088))荧光检测结合的Fab 1,由此测量游离Fab 1的量。通过全局1:1拟合将Fab 1对人TSLP的KD提取到三个数据集,这三个数据集由人TSLP滴定至1000pM(实心菱形)、500pM(倒置实心三角形)或40pM(空心正方形)固定的Fab1浓度溶液中得到(图3)。通过全局1:1拟合将Fab1对食蟹猴TSLP的KD提取到两个数据集,这两个数据集由食蟹猴TSLP滴定至1000pM(实心菱形)或40pM(空心正方形)固定的Fab1浓度溶液中得到(图4)。
将分别在人和食蟹猴TSLP浓度下检测到的游离Fab 1的量对TSLP的滴定浓度作图(分别为图3和图4)。使用KinExA软件计算平衡解离常数(KD)。表2中所示的结果展示,Fab1与人TSLP结合的亲和力是它在游离溶液中与食蟹猴TSLP结合的亲和力的1.7倍。
表2使用KinExA确定的Fab1对人和食蟹猴TSLP的可溶相亲和力
配体 | 亲和力(KD)pM |
人TSLP | 8.0(95%置信区间6.27-10.01pM) |
食蟹猴TSLP | 13.6(95%置信区间9.07-19.22pM) |
实施例3–Fab1和特折鲁单抗以相似结合特征与TSLP结合
将Fab 1与人TSLP的结合特征直接与特折鲁单抗相比较。
使用基于均相荧光共振能量转移(FRET)均相时间分辨荧光(CisbioInternational)的TSLP:mAb结合测定来确定Fab 1的体外结合效力。使用链霉亲和素穴状化合物检测生物素化TSLP。简单地说,将未标记的Fab 1样品滴定到HTRF测定中,以与DyLight标记的特折鲁单抗竞争结合至生物素化的His-Avi人TSLP。还使用未标记的特折鲁单抗和DyLight标记的特折鲁单抗进行竞争测定,作为阳性对照。
结果表明,Fab 1与特折鲁单抗竞争结合至人TSLP,并以与特折鲁单抗类似的效力结合至人TSLP(IC50:Fab1-0.38nM;特折鲁单抗-0.23nM-图5)。
实施例4—在外周血单核细胞(PBMC)测定中Fab 1中和TSLP活性
接下来,在原代细胞测定中,通过测量在用Fab 1处理后TSLP诱导的CCL17从PBMC释放来确定Fab 1与TSLP的结合是否具有功能阻断活性。
根据英国剑桥(Cambridge,UK)的MedImmune建立的献血者程序,从健康供体获取血液。通过标准程序,使用ficoll梯度分离外周血单核细胞。简单地说,将20ml用PBS稀释的血液(10ml血液:30ml PBS)铺在15ml ficoll上。在室温下,将管以400g离心40分钟,无制动。收集PBMC层,并用50ml PBS洗涤细胞两次。使用血细胞计数器和锥虫蓝(trypan blue)排除死细胞来对PBMC计数,并将其再悬浮于培养基(含10%胎牛血清和1%青霉素/链霉素的RPMI)中,随后平板接种于96孔板中。在结合TSLP的抗体片段Fab1的存在下,用TSLP(0.5ng/ml)刺激细胞48小时。还使用结合TSLP的抗体特折鲁单抗进行测定,作为阳性对照。48小时后,取出上清液并使用R&D duoset ELISA,根据制造商的方案测定CCL17的产生。在三个独立实验中使用六名供体进行实验。
结果表明,Fab 1抑制PBMC产生CCL17,并且IC50是1.39nM(图6)。
序列
SEQ ID NO:1(Fab 1重链)
QMQLVESGGGVVQPGRSLRLSCAASGFTFRTYGMHWVRQAPGKGLEWVAVIWYDGSNKHYADSVKGRFTITRDNSKNTLNLQMNSLRAEDTAVYYCARAPQWELVHEAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDK
SEQ ID NO:2(Fab 1轻链)
SYVLTQPPSVSVAPGQTARITCGGNNLGSKSVHWYQQKPGQAPVLVVYDDSDRPSWIPERFSGSNSGNTATLTISRGEAGDEADYYCQVWDSSSDHVVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
序列表
<110> 免疫医疗有限公司(MEDIMMUNE LIMITED)
<120> 具有改善的稳定性的抗TSLP FAB
<130> 008231433
<150> EP 21169183.7
<151> 2021-04-19
<160> 2
<170> PatentIn 3.5版
<210> 1
<211> 227
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> FAB1重链
<400> 1
Gln Met Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Thr Tyr
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Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
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Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys His Tyr Ala Asp Ser Val
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Lys Gly Arg Phe Thr Ile Thr Arg Asp Asn Ser Lys Asn Thr Leu Asn
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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
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Ala Arg Ala Pro Gln Trp Glu Leu Val His Glu Ala Phe Asp Ile Trp
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Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
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Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
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Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
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Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
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Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
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Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
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His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser
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Cys Asp Lys
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<210> 2
<211> 214
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> FAB1轻链
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Ser Tyr Val Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln
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Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Leu Gly Ser Lys Ser Val
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His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Val Tyr
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Asp Asp Ser Asp Arg Pro Ser Trp Ile Pro Glu Arg Phe Ser Gly Ser
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Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Gly Glu Ala Gly
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Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Ser Ser Asp His
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Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys
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Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln
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Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly
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Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly
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Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala
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Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser
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Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val
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Ala Pro Thr Glu Cys Ser
210
Claims (16)
1.一种Fab,所述Fab包含:包含SEQ ID NO:1中所示氨基酸序列的重链和包含SEQ IDNO:2中所示氨基酸序列的轻链。
2.一种Fab,所述Fab包含具有SEQ ID NO:1中所示氨基酸序列的重链和具有SEQ IDNO:2中所示氨基酸序列的轻链。
3.一种Fab,所述Fab包含由SEQ ID NO:1中所示氨基酸序列组成的重链和由SEQ IDNO:2中所示氨基酸序列组成的轻链。
4.根据权利要求1至3中任一项所述的Fab,其中所述Fab是IgG1 Fab。
5.根据权利要求1至4中任一项所述的Fab,其中所述Fab在45℃下稳定保持至少两周的时间段。
6.根据权利要求5所述的Fab,其中所述Fab在1X PBS中以0.8mg/ml浓度稳定。
7.根据前述权利要求中任一项所述的Fab,其中所述Fab在45℃下于1X PBS中培育2周后显示小于10%的单体损失率,其中单体是由HP-SEC确定。
8.一种药物组合物,所述药物组合物包含权利要求1至7中任一项所述的Fab。
9.一种核酸,所述核酸编码权利要求1至7中任一项所述的Fab。
10.一种载体,所述载体包含权利要求9所述的核酸。
11.一种宿主细胞,所述宿主细胞包含权利要求9所述的核酸或权利要求10所述的载体。
12.一种产生权利要求1至7中任一项所述的Fab的方法,所述方法包括培养权利要求7所述的宿主细胞,表达所述Fab并纯化所述Fab。
13.一种能够由权利要求12所述的方法获得的Fab。
14.根据权利要求1至7中任一项所述的Fab,用于疗法中。
15.根据权利要求1至7中任一项所述的Fab,用于治疗TSLP相关疾患。
16.根据权利要求15所述使用的Fab,其中所述TSLP相关疾患是哮喘。
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CN1306272C (zh) | 2000-11-17 | 2007-03-21 | 罗切斯特大学 | 筛选编码抗原特异性免疫球蛋白分子或其抗原特异性片段的方法 |
WO2002096948A2 (en) | 2001-01-29 | 2002-12-05 | Idec Pharmaceuticals Corporation | Engineered tetravalent antibodies and methods of use |
JOP20190243A1 (ar) * | 2017-04-12 | 2019-10-13 | Medimmune Llc | علاج الربو بجسم مضاد لـ tslp |
CN113423733B (zh) * | 2019-09-04 | 2023-09-22 | 正大天晴药业集团股份有限公司 | 结合tslp的抗体及其用途 |
BR112022008031A2 (pt) * | 2019-10-28 | 2022-07-12 | Medimmune Ltd | Formulações de pó seco de anticorpos de ligação à linfopoietina estromal tímica (tslp) e métodos de uso das mesmas |
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2022
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- 2022-04-19 TW TW111114769A patent/TW202306982A/zh unknown
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- 2022-04-19 CA CA3216894A patent/CA3216894A1/en active Pending
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EP4326767A1 (en) | 2024-02-28 |
CA3216894A1 (en) | 2022-10-27 |
CL2023003082A1 (es) | 2024-04-19 |
WO2022223514A1 (en) | 2022-10-27 |
KR20230172508A (ko) | 2023-12-22 |
AU2022263281A9 (en) | 2023-11-16 |
AU2022263281A1 (en) | 2023-11-09 |
JP2024516962A (ja) | 2024-04-18 |
AR125379A1 (es) | 2023-07-12 |
TW202306982A (zh) | 2023-02-16 |
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