CN115515975A - Fc变体及其制备 - Google Patents
Fc变体及其制备 Download PDFInfo
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- CN115515975A CN115515975A CN202180032434.8A CN202180032434A CN115515975A CN 115515975 A CN115515975 A CN 115515975A CN 202180032434 A CN202180032434 A CN 202180032434A CN 115515975 A CN115515975 A CN 115515975A
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Abstract
本发明涉及Fc变体蛋白及其制备。所述Fc变体对FcRn具有改变的结合亲和力。根据本发明制备的Fc变体可以用于制备FcRn拮抗剂组合物或者可以用于制备具有改变的效应子功能的含有Fc变体的药物或分子。
Description
发明领域
本发明涉及Fc变体蛋白及其制备。所述Fc变体对FcRn具有改变的结合亲和力。根据本发明制备的Fc变体可以用于制备FcRn拮抗剂组合物或者可以用于制备具有改变的效应子功能的含有药物或分子的Fc变体。
发明背景
新生Fc受体(FcRn)在结构上与由与可溶性轻链,β2-微球蛋白(β2m)非共价结合的I型跨膜重链组成的主要组织相容性复合物(MHC)I类异二聚体分子同源。β2m对于FcRn以及其它MHC I类同系物的正确折叠、运输和功能是必需的。FcRn重链含有三个可溶性细胞外结构域(α1、α2和α3)、单个跨膜螺旋和胞质尾区。与MHC I类分子不同,由于FcRn的顶面上破坏肽结合的点突变,因此FcRn不直接将抗原呈递给T细胞。FcRn保护IgG免受细胞内分解代谢的能力是与IgG的Fc部分特异性pH依赖性相互作用的结果。IgG在酸性(<6.5)而不是中性pH(>7)下以严格的pH依赖性方式结合FcRn,这是由IgG Fc的CH2-CH3结构域中的可滴定组氨酸残基与FcRn的α2结构域上的酸性残基之间的静电介导的。通过Fc与FcRnα2-结构域和β2m轻链N-末端内的残基之间的一系列疏水相互作用和氢键进一步稳定结合。由于IgG的同型二聚体性质,一个IgG分子可以同时结合两个FcRn分子,导致在pH6下FcRn和IgG之间由于增加的相互作用的亲合力而导致高亲和力相互作用。FcRn和IgG之间的2:1相互作用对于它们在表达FcRn的细胞中的有效结合、再循环和转胞吞作用是关键的,最终导致IgG的长期血清持久性的保存,使得保护性抗体分子不必由B细胞重复产生,从而提供针对感染的有效免疫。不幸的是,这种相同的相互作用也有助于那些对自身抗原具有反应性的IgG分子的致病性,从而导致许多自身免疫疾病。缺乏FcRn的小鼠被保护免于IgG介导的自身免疫疾病,这表明FcRn通过维持致病性IgG在循环中更长时间而至少部分地有助于自身免疫。因为FcRn有助于IgG的血清持久性,所以阻断IgG-FcRn相互作用的治疗剂代表IgG介导的自身免疫的潜在治疗方式。事实上,高剂量静脉内免疫球蛋白(IVIg)治疗剂是FDA批准的用于治疗IgG介导的自身免疫疾病,其中它部分地通过FcRn受体的饱和而提供治疗益处,从而增加致病性IgG的分解代谢。然而,IVIg治疗具有多种问题。例如,作为一种血液衍生的产品,其容易被人类病原体如HIV、HBV、HCV等污染,因此使慢性病症如自身免疫疾病的治疗对接受者患者非常危险。它也很昂贵,因为它需要从许多供血者的血浆中提取IgG。而且,因为它是人类供体依赖的产品,所以存在可扩展性的问题。因此,迫切需要新的重组方法以提供替代安全的、可扩展的和成本有效的治疗选择。
已经开发了基于单克隆抗体的拮抗剂来抑制内源性IgG-FcRn相互作用。一种方法是使用经由经典抗体:抗原结合机制起作用的针对FcRn的单克隆抗体。这种抗体的实例是M281和UCB7665,它们结合人FcRn以抑制IgG与FcRn的结合,从而加速内源性IgG的清除。用M281以剂量依赖性方式观察到范围为25-80%的内源性IgG的平均降低。此外,单剂量的30mg/kg或60mg/kg的M281分别将血清IgG维持在基线的50%或以下达18天和27天(1)。在健康对象中,在7mg/kg的UCB7665的单一IV剂量下,血清中的IgG浓度降低高达50%。在目前研究中观察到的血清IgG浓度的降低持续数周,在第7天至第10天达到最大降低,然后在第57天逐渐回到基线(2)。
降低血清中IgG水平的替代抗体工程方法是工程化IgG的Fc结构域,使得它在pH6和pH7.4下对FcRn均具有高亲和力。已经发现使用“Abdegs”(即,增强IgG降解的抗体)技术开发的这种分子在比IVIg低25至50倍的剂量下有效治疗鼠关节炎模型,表明这种IgG-FcRn相互作用的基于IgG Fc的有效拮抗剂是自身免疫中的替代治疗干预。这种分子Efgartigimod正由基于荷兰的生物技术公司arGEN-X进行临床开发。
最近,从噬菌体文库中鉴定了与IgG竞争结合FcRn的合成肽。在pH6和pH7.4下以亚纳摩尔亲和力结合FcRn的化学优化的肽二聚体(SYN1436)有效地增加小鼠中外源性IgG以及猴中内源性IgG的清除,但在IgG介导的自身免疫的治疗模型中尚未评价。可选地,在CH2-CH3结构域界面处反而结合Fc本身以阻断IgG-FcRn相互作用的另外分子包括通过噬菌体展示选择的13-氨基酸环肽(FcIII)、计算设计的IgG-Fc结合蛋白(FcBP6.1)和内源性Fc受体TRIM21。(3)因此,本发明提供了FcRn结合剂,其解决了各种未满足的需要,如下调FcRn活性、增加各种药物的循环半衰期、将药物和/或疫苗靶向特定细胞类型等。本发明的FcRn结合剂可用于治疗多种疾病。
存在另一类靶向免疫复合物(IC)形成和免疫复合物介导的FcγR介导的活化的分子。这类分子是重组Fc多聚体,如Pf-06755347(GL-2045)和CSL730(M230)。这些分子在结构上是复杂的,并可导致高度异质的群体。这些分子正被开发用于治疗自身免疫疾病。因此,靶向不同机制的分子正在进行早期临床试验,所述分子被开发为静脉内免疫球蛋白(IVIg)和皮下免疫球蛋白(SCIg)的替代物,以避免对供应人血浆和治疗所需的高达2g/kg体重的大剂量产品的依赖。在这种情况下,获得具有多种作用机制(如FcRn阻断、FcγR活化的抑制和/或完全补体活化的抑制)的组合作用的单一药物将是有利的。
在一个方面,本发明提供了Fc变体分子,其是FcRn结合剂以及FcγR结合剂,并且可以一起解决各种需要,如下调FcRn活性、增加多种药物的循环半衰期、IC抑制、由IC形成介导的细胞因子释放的抑制、由FcγR活化介导的吞噬作用的抑制。优选地,根据本发明制备的Fc变体分子以与IVIg的剂量相比更小的剂量提供一种或多种上述期望的效果。
发明内容
本发明提供了对FcRn具有改变的结合亲和力,优选对FcRn具有更高的结合亲和力的新的Fc变体。在一个方面,本发明的Fc变体对FcγR具有改变的结合亲和力,优选对FcγR具有更高的结合亲和力。优选地,所述Fc变体可用于开发具有FcRn拮抗剂功能或增加的循环半衰期,或用于靶向特定细胞和/或组织的药物组合物。本发明还提供了制备新的Fc变体的方法。根据本发明的Fc变体进一步用于制备用于治疗其中FcRn的活性是不利的疾病的药物,或用于增加药物的循环半衰期或用于将所述药物靶向某些细胞或组织。
附图简述
图1:其描述了用于文库生成以制备本发明的Fc变体的载体的图谱。
图2:其描述了用于生成本发明的Fc单体和Fc二聚体的载体的图谱。
图3:其描述了用于生成本发明的含有Fc单体的单克隆抗体的载体的图谱。
图4:其描述了测定Fc变体(Fc二聚体)和IVIg对野生型小鼠中总IgG血清水平的影响的实验结果。
图5:其描述了测定Fc变体(Fc二聚体)和IVIg对野生型小鼠中血清白蛋白水平的影响的实验结果。
图6:其描述了测定Fc变体对ADCC活性的影响的实验结果。
图7:其描述了在急性免疫血小板减少症(ITP)的小鼠模型中测定Fc变体对血小板计数的影响的实验结果。
定义
本文所用的术语“无岩藻糖基化的(afucosylated)”是指具有N-连接聚糖的糖基化蛋白,其缺少如US8067232中所述的核心岩藻糖分子,US8067232的内容通过引用以其整体并入本文。
本文所用的术语“氨基酸修饰”是多肽序列中的氨基酸取代、插入和/或缺失。
本文所用的术语“氨基酸取代”或“取代”是用另一个氨基酸代替亲本多肽序列中特定位置的氨基酸。例如,取代T307N是指变体多肽,在这种情况下是Fc变体,其中307位的苏氨酸被天冬酰胺代替。
根据本发明的术语“抗原结合分子”是指能够结合靶抗原并包含“FcRn结合结构域”的蛋白质。根据本发明的FcRn结合结构域存在于Fc蛋白中,优选存在于本发明的Fc变体中。根据本发明的优选的“抗原结合分子”可包括包含本发明Fc变体的抗体或肽体或融合蛋白。
本文所用的术语“抗体”包括完整抗体和任何抗原结合片段(即,“抗原结合部分”)。“抗体”是指包含通过二硫键相互连接的至少两条重(H)链和两条轻(L)链的糖蛋白,或其抗原结合部分。每条重链由重链可变区(本文缩写为VH)和重链恒定区(Fc)组成。重链恒定区由三个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(本文缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可进一步细分为称为互补决定区(CDR)的超变区,散布有称为框架区(FR)的更保守区域。每个VH和VL由从氨基末端到羧基末端按以下顺序排列的三个CDR和四个FR组成:FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白与宿主组织或因子的结合,所述宿主组织或因子包括免疫系统的各种细胞(例如效应细胞,如NK细胞、T细胞、巨噬细胞和树突细胞等)和经典补体系统的第一组分(C1q)。
术语“可操作地连接的”旨在意指将基因连接到载体中,使得载体内的转录和翻译控制序列发挥其调节这种基因的转录和翻译的预期功能。
术语“Ka”是两个分子之间相互作用的结合速率,而术语“Kd”是两个分子之间相互作用的解离速率。术语“KD”是从Kd与Ka的比率获得的亲和速率常数。它可以通过使用本领域众所周知的表面等离子体共振方法来测量。
本文所用的术语“单克隆抗体”或“单克隆抗体组合物”是指单分子组合物的抗体分子的制剂。单克隆抗体组合物显示对特定表位的单一结合特异性和亲和力。
术语“双特异性抗体”是指参与两种不同抗原决定簇或表位的高度特异性识别和结合的同质抗体群。
本文所用的术语“重组抗体”包括通过重组手段制备、表达、产生或分离的所有抗体。然而,在某些实施方案中,这种重组抗体可以通过体外诱变获得,因此本文所述的重组抗体的VH和VL区的氨基酸序列是在体内可能不天然存在于人抗体种系库中的序列。
本文所用的术语“效应子功能”是由抗体Fc区与Fc受体或配体相互作用产生的生化事件。效应子功能包括但不限于ADCC、ADCP和CDC。该术语还代表生理事件,如药物的循环半衰期或药物靶向特定细胞或组织类型。
本文所用的术语“ADCC”或“抗体依赖性细胞介导的细胞毒性”是细胞介导的反应,其中表达FcγR的非特异性细胞毒性细胞识别靶细胞上的结合抗体并随后引起靶细胞的裂解。
本文所用的术语“ADCP”或“抗体依赖性细胞介导的吞噬作用”是细胞介导的反应,其中表达FcγR的非特异性细胞毒性细胞识别靶细胞上的结合抗体并随后引起靶细胞的吞噬作用。
本文所用的术语“效应细胞”是表达一种或多种Fc受体并介导一种或多种效应子功能的细胞。效应细胞包括但不限于单核细胞、巨噬细胞、嗜中性粒细胞、树突细胞、嗜酸性粒细胞、肥大细胞、血小板、B细胞、大颗粒淋巴细胞、朗格汉斯细胞、自然杀伤(NK)细胞和γδT细胞,并且可以来自任何有机体,包括但不限于人、小鼠、大鼠、兔和猴。
术语“Fc”片段,其名称反映了其容易结晶的能力。已经测定了人IgG Fc区的晶体结构(4)。在人IgG分子中,Fc区由木瓜蛋白酶切割Cys226的N-末端产生。Fc区是抗体的效应子功能的中心。
本文所用的术语“Fc蛋白”是指从恰好在木瓜蛋白酶切割位点上游的铰链区开始并终止于抗体的C-末端的单一免疫球蛋白重链的部分。因此,完整的Fc结构域包含铰链(例如,上、中和/或下铰链区)结构域、CH2结构域和CH3结构域的至少一部分。
本文所用的术语“含有Fc变体的蛋白”是指包含本发明的Fc变体的任何分子。优选地,含有Fc变体的蛋白包括抗体、融合蛋白和肽体。本文提及的抗体、融合蛋白和肽体包括任何批准的或正在进行临床试验的或正在进行临床前试验的抗体、融合蛋白和肽体。
本文所用的术语“含有Fc变体的分子”是指包含本发明的Fc变体的任何分子。它可以是融合产物,其中本发明的Fc变体与药物连接或缀合,其中药物可以是小肽或受体或毒素或任何化学分子。
本文所用的术语“Fc变体蛋白”或“Fc蛋白变体”或“Fc变体”是通过至少一个氨基酸修饰而不同于野生型Fc蛋白的Fc蛋白。Fc变体可以指Fc变体本身、包含Fc变体的组合物或编码Fc变体的氨基酸序列。优选地,Fc变体与亲本蛋白质相比具有至少一个氨基酸修饰,例如与亲本相比,约1至约10个氨基酸修饰,优选约1至约5个氨基酸修饰。
本文所用的术语“EU位置”是指参考文献5中所述的Fc区的EU编号惯例中的氨基酸位置。
本文所用的术语“CH1结构域”是指从约EU位置118-215延伸的免疫球蛋白重链的第一(最氨基末端)恒定区结构域。CH1结构域与免疫球蛋白重链分子的VH结构域和铰链区的氨基末端相邻,并且不形成免疫球蛋白重链的Fc区的一部分。
本文所用的术语“铰链区”是指将重链分子的CH1结构域与CH2结构域连接的部分。该铰链区包含约25个残基并且是柔性的,从而允许两个N-末端抗原结合区独立地移动。铰链区可以细分为三个不同的结构域:上铰链区、中铰链区和下铰链区(6)。本发明的Fc变体可包括铰链区的全部或一部分。
本文所用的术语“CH2结构域”是指重链免疫球蛋白分子的从约EU位置231-340延伸的部分。
本文所用的术语“CH3结构域”是指重链免疫球蛋白分子的从CH2结构域的N-末端延伸约110个残基,例如从约位置341-446延伸(EU编号系统)的部分。
本文所用的术语“Fcγ受体”或“FcγR”意指结合IgG抗体Fc区并由FcγR基因编码的蛋白质家族的任何成员。在人类中,该家族包括但不限于FcγRI(CD64),包括同种型FcγRIa、FcγRIb和FcγRIc;FcγRII(CD32),包括同种型FcγRIIa(包括同种异型H131和R131)、FcγRIIb(包括FcγRIIb-1和FcγRIIb-2)和FcγRIIc;和FcγRIII(CD16),包括同种型FcγRIIIa(包括同种异型V158和F158)和FcγRIIIb(包括同种异型FcγRIIIb-NA1和FcγRIIIb-NA2)(7),以及任何未发现的人FcγR或FcγR同种型或同种异型。FcγR可以来自任何有机体,包括但不限于人、小鼠、大鼠、兔和猴。小鼠FcγR包括但不限于FcγRI(CD64)、FcγRII(CD32)、FcγRIII(CD16)和FcγRIII-2(CD16-2),以及任何未发现的小鼠FcγR或FcγR同种型或同种异型。
本文所用的术语“FcRn”或“新生Fc受体”是结合IgG抗体Fc区域并至少部分由FcRn基因编码的蛋白质。FcRn可以来自任何有机体,包括但不限于人、小鼠、大鼠、兔和猴。如本领域已知的,功能性FcRn蛋白包含两条多肽,通常被称为重链和轻链。轻链是β-2-微球蛋白,重链由FcRn基因编码。除非本文另有说明,否则FcRn或FcRn蛋白是指FcRn重链与β-2-微球蛋白的复合物。
本文所用的术语“野生型Fc”是未修饰的Fc多肽,其随后被修饰以生成变体。野生型Fc可以是天然存在的多肽或天然存在的多肽的重组形式。野生型Fc可以指未修饰的Fc多肽本身、包含未修饰的Fc多肽的组合物或编码Fc多肽的氨基酸序列。
本文所用的术语“位置”是蛋白质序列中的位置。位置可以顺序地编号或根据建立的格式编号,例如在Kabat中的EU索引。例如,位置305是人抗体IgG1中的位置。
术语“患者”和“对象”可互换使用,并且以它们的常规意义使用,是指患有或易于患有可通过施用本发明的组合物来预防或治疗的病况的活有机体,并且包括人和非人动物两者。对象的实例包括但不限于人、黑猩猩及其它猿和猴物种;农场动物,如牛、绵羊、猪、山羊和马;家养哺乳动物,如狗和猫;实验动物,包括啮齿动物,如小鼠、大鼠和豚鼠;禽类,包括家禽、野禽和猎禽,如鸡、火鸡和其它鹑鸡类、鸭、鹅等。该术语不表示特定的年龄。因此,成年的、幼年的和新生个体都是感兴趣的。
本文所用的术语“残基”是蛋白质中的位置及其相关的氨基酸身份。例如,苏氨酸307(也称为Thr307,也称为T307)是人抗体IgG1中的残基。
本文所用的术语“免疫缀合物”或“缀合物”是指与细胞结合剂(即,如本文所述的抗体或Fc变体)连接并且由以下通式定义的化合物或其衍生物:A-L-D,其中A=本发明的细胞结合剂或抗体或Fc变体,L=接头以及D=药物(毒素或药物)。免疫缀合物也可以由通式以相反顺序定义:A-L-D。
术语“接头”是能够以稳定的共价方式将化合物(通常是药物,如美登木素生物碱(maytansinoid)或澳瑞他汀(auristatin))连接至细胞结合剂,如抗FcRn或其片段的任何化学部分。在化合物或抗体保持活性的条件下,接头可对酸诱导的切割、光诱导的切割、肽酶诱导的切割、酯酶诱导的切割和二硫键切割敏感或基本上抵抗酸诱导的切割、光诱导的切割、肽酶诱导的切割、酯酶诱导的切割和二硫键切割敏感。合适的接头是本领域众所周知的,并且包括例如二硫化物基团、硫醚基团、酸不稳定基团、光不稳定基团、肽酶不稳定基团和酯酶不稳定基团。接头还包括如本文所述和本领域已知的带电接头及其亲水形式。
术语“拮抗剂”是抑制或降低其结合的抗原如FcRn的生物活性的拮抗剂。在某些实施方案中,拮抗剂基本上或完全抑制抗原如FcRn的生物活性。理想地,生物活性降低10%、20%、30%、50%、70%、80%、90%、95%或甚至100%。
本文所用的术语“治疗”或“治疗剂”是指哺乳动物,特别是人中疾病的任何治疗。其包括:(a)预防疾病在可能易患所述疾病或处于患所述疾病风险但尚未诊断为患有所述疾病的对象中发生;(b)抑制疾病,即阻止其发展;以及(c)缓解疾病,即引起疾病的消退。
本文中的术语“野生型”或“WT”是在自然界中发现的氨基酸序列或核苷酸序列,包括等位基因变异。WT蛋白具有未经有意修饰的氨基酸序列或核苷酸序列。
术语“非天然编码的氨基酸”是指不是常见氨基酸或焦赖氨酸、吡咯啉-羧基-赖氨酸或硒代半胱氨酸之一的氨基酸。可以与术语“非天然编码的氨基酸”同义使用的其它术语是“非天然氨基酸(non-natural amino acid)”、“非天然氨基酸(unnatural aminoacid)”、“非天然存在的氨基酸”及其各种带连字符号和不带连字符的形式。术语“非天然编码的氨基酸”还包括但不限于通过修饰(例如翻译后修饰)天然编码的氨基酸(包括但不限于20种常见氨基酸或吡咯赖氨酸、吡咯啉-羧基-赖氨酸和硒代半胱氨酸)而出现,但本身不是通过翻译复合物天然掺入到生长多肽链中的氨基酸。这种非天然存在的氨基酸的实例包括但不限于N-乙酰氨基葡糖基-L-丝氨酸、N-乙酰氨基葡糖基-L-苏氨酸和O-磷酸酪氨酸。
表1:本申请中使用的氨基酸缩写
本专利申请中使用的其它缩写:
%:百分比
℃:摄氏度
μg:微克
μL:微升
A:腺嘌呤
ADCC:抗体依赖性细胞毒性
C:胞嘧啶
CDC:补体依赖性细胞毒性
CFU:菌落形成单位
CHO:中国仓鼠卵巢
DNA:脱氧核糖核酸
ELISA:酶联免疫吸附测定
EC50:任何药物的最大有效浓度的50%
EC75:任何药物的最大有效浓度的75%
FC:片段可结晶的
FcGRT:IgG受体和转运蛋白的Fc片段
FcRn:新生Fc受体
FcγR:Fcγ受体
G:鸟嘌呤
h:小时
H2SO4:硫酸
HRP:辣根过氧化物酶
IC:免疫复合物
IgG:免疫球蛋白G
IPTG:异丙基β-D-1-硫代半乳糖苷
ITP:急性免疫血小板减少症
IVIG:静脉内免疫球蛋白
Ka/kassoc:结合常数
Kd/kdissoc:解离常数
KD:平衡解离常数
M:摩尔
mg:毫克
MgCl2:氯化镁
min:分钟
mL:毫升
mM:毫摩尔
MOI:感染复数
NaCl:氯化钠
NaHCO3:碳酸氢钠
ng:纳克
nm:纳米
OD:光密度
OPD:邻苯二胺二盐酸盐
PBS:磷酸盐缓冲盐水
PBST:含有0.05%吐温的磷酸盐缓冲盐水
PEG:聚乙二醇
PFU:噬菌斑形成单位
rFcRn:重组新生Fc受体
rhFcRn:重组人新生Fc受体
rpm:每分钟转数
s:秒
SCIg:皮下免疫球蛋白
SEQ/seq:序列
SPR:表面等离子体共振
T:胸腺嘧啶
YT培养基:酵母提取物胰蛋白胨培养基本发明的实施方案
本发明的公开内容涉及可用于治疗目的的新型Fc变体蛋白。
在一个实施方案中,本发明的Fc变体蛋白或含有Fc变体的蛋白或含有Fc变体的分子相对于野生型Fc蛋白以高亲和力与人FcRn结合。在一个实施方案中,本发明的Fc变体对于FcRn的KD为10-8M或更低,更优选10-10M或更低。KD值是抗体对其靶抗原的结合亲和力的量度。
在一个实施方案中,相对于野生型IgG1 Fc区对Fcγ受体的亲和力,根据本发明的Fc变体对Fcγ受体具有改变(增加或降低)的亲和力。在某些实施方案中,相对于野生型IgG1 Fc区对FcγRIIIa(CD16a)的亲和力,Fc变体对FcγRIIIa(CD16a)具有增加的亲和力。
在一个实施方案中,本发明的Fc变体对于FcγRIIIa(CD16a),包括同种异型V158和F158的KD为10-7M或更低。在一个实施方案中,本发明的Fc变体抑制免疫复合物介导的FcγR活化,如通过ADCC的抑制所评估的。在一个实施方案中,本发明的Fc变体抑制由FcγR介导的吞噬作用。在一个实施方案中,本发明的Fc变体抑制由FcγR介导的细胞因子释放,如由IL-6或IL-8。
在一个实施方案中,Fc变体的氨基酸序列是IgGl、IgG2、IgG3、IgG4或IgG2/G4同种型,优选IgG1同种型。
在另一个实施方案中,本发明的一种或多种Fc变体具有修饰的或减少的效应子功能或没有效应子功能。在一个实施方案中,本发明的Fc变体或含有Fc变体的蛋白具有降低的引起ADCC和CDC的安全问题的可能性。在一个优选的实施方案中,本发明的Fc变体没有CDC活性。在一个优选的实施方案中,与野生型Fc的ADCC活性或IVIg的ADCC活性相比,本发明的Fc变体具有降低的ADCC活性。在一个实施方案中,本发明的Fc变体具有降低的ADCP活性或没有ADCP活性。在一个实施方案中,本发明的Fc变体不干扰FcRn与白蛋白的结合特性。
在一个实施方案中,本发明的Fc变体与来自人以外物种的FcRn交叉反应。
在一个实施方案中,本发明的Fc变体对人FcRn具有更高的结合特异性。
在一个实施方案中,本发明的Fc变体或含有Fc变体的蛋白或含有Fc变体的分子在对象中具有增加的半衰期。在某些实施方案中,聚乙二醇或人血清白蛋白可以与本发明的Fc变体连接以进一步增加所述Fc变体的半衰期。在替代实施方案中,本发明的Fc变体可以包括突变以增加其在对象中的半衰期。在另一个替代实施方案中,本发明的Fc变体可以以多聚体形式表达,以通过增加Fc变体的分子大小和通过更高亲合力的亲和力来增加半衰期。本文所用的术语“多聚体形式”是指包括一个以上蛋白单位的蛋白形式,在本文中优选Fc蛋白。例如,它可以是二聚体、三聚体、四聚体、五聚体、六聚体等。例如,单体Fc蛋白具有FcRn以及FcγR的两个结合位点。以类似的方式,根据本发明的Fc二聚体具有FcRn以及FcγR的四个结合位点。
在另一个实施方案中,本发明的Fc变体能够与猴FcRn结合,从而通过提供相关的动物药理学和毒理学模型而使得易于药物开发。
在某些实施方案中,Fc变体包含变体Fc区,其包含在EU位置297处的无岩藻糖基化的N-连接聚糖。
在一个实施方案中,Fc变体包含变体Fc区,其包含与野生型IgG相比更高的唾液酸化。
在一个实施方案中,本发明提供了包含Fc变体蛋白或含有Fc变体的蛋白或含有Fc变体的分子和可接受载体的组合物。
在另一个实施方案中,本发明的Fc变体蛋白或含有Fc变体的蛋白可用于治疗疾病,如感染、各种癌症、自身免疫病症等。
在某些实施方案中,Fc变体或含有Fc变体的蛋白或含有Fc变体的分子包含选自SEQ ID No.1至SEQ ID No.347中所示序列的氨基酸序列。本文表3中描述了SEQ ID No.1-347的序列。
在另一个实施方案中,本发明的Fc变体可用于制备全长抗体,其中抗体具有本发明的Fc变体代替常规Fc区。所述抗体可以是已经批准或发现的抗体的修饰形式,其中现有抗体的修饰形式具有本发明的Fc变体。它可以是针对任何靶标而开发的新抗体。
在一个单独的实施方案中,本发明的Fc变体可用于制备具有增加的半衰期的药物,其中本发明的Fc变体与循环中原始半衰期低的药物融合或连接或缀合。
在另一个实施方案中,本发明的Fc变体可用于增加ADC的半衰期并通过增加其通过FcRn受体的再循环来降低其脱靶毒性。
在一个实施方案中,本发明的Fc变体可以与任何药物融合以提高其半衰期和体内稳定性。
在一个实施方案中,本发明的Fc变体可用于代替白蛋白融合药物中的白蛋白以提高药物的药代动力学。
在一个实施方案中,本发明的Fc变体中的突变可用于提高完全抗体在pH6时对FcRn的亲和力,以通过减少分解代谢来增加其循环半衰期。
在一个实施方案中,含有Fc变体的抗体分子可用作清除剂以从循环中去除致病蛋白或毒素,如TNFα、VEGF等。
在一个实施方案中,含有Fc变体的抗体分子或蛋白可用作捕捉剂(trapper)以从环境中捕捉期望的蛋白、肽、碳水化合物或药物并带入表达FcRn的靶细胞内。
在一个实施方案中,本发明的Fc变体可用于开发与任何治疗性肽融合的单体Fc融合蛋白,以提高其在组织中的穿透性以及保留其FcRn结合活性。
在一个实施方案中,本发明的Fc变体可以与PEG融合以进一步提高其循环半衰期。
在一个实施方案中,本发明的Fc变体可用于经由非侵入性途径如肺部施用来递送药物。
在一个实施方案中,本发明的Fc变体阻断FcRn上的IgG结合位点,并与内源性IgG竞争FcRn,导致内源性IgG的更快清除。
在一个实施方案中,本发明的Fc变体可独立于其药代动力学半衰期而提高药物的效力/功效。
在一个实施方案中,与免疫原/抗原融合的本发明的Fc变体可以提高抗原向表达FcRn的APC的递送,导致提高的免疫应答。
在一个实施方案中,本发明的Fc变体可用于通过肠上皮在各种器官口服递送载有纳米颗粒的药物。
在一个实施方案中,与肽/免疫原融合的本发明的Fc变体可作为针对许多粘膜病原体的亚单位疫苗的一般递送途径用于鼻内免疫。
在一个实施方案中,与任何广泛中和抗体融合的本发明的Fc变体可用于延长这种抗体的半衰期,并且还可用于增强粘膜定位,该粘膜定位赋予针对病毒进入胃肠道或宫颈阴道的免疫保护。
发明详述
在一个实施方案中,本发明的Fc变体蛋白或含有Fc变体的蛋白或含有Fc变体的分子以高亲和力与人FcRn结合。本发明的Fc变体在pH6.0下与FcRn结合时的KD值比天然Fc低。本发明的Fc变体在pH6.0下对FcRn具有10-8M或更低,更优选10-10M或更低的KD。KD值是抗体对其靶抗原的结合亲和力的量度。
Fc变体的氨基酸序列
本发明的Fc变体包含Fc蛋白的CH2结构域或CH3结构域或CH2和CH3结构域的氨基酸取代。更优选地,本发明的Fc变体在选自以下的一个或多个残基处包含氨基酸取代:EU250、EU251、EU252、EU253、EU254、EU255、EU256、EU257、EU258、EU259、EU307、EU308、EU309、EU310、EU311、EU375、EU377、EU379、EU380、EU381、EU432、EU433、EU434和EU435。表2中显示了针对位置EU250、EU251、EU252、EU253、EU307、EU308、EU309、EU375、EU377、EU379、EU380、EU381、EU432、EU433、EU434、EU435、EU436和EU437的优选取代氨基酸。
表2:Fc变体中特定EU位置处的优选取代氨基酸
在优选的实施方案中,根据本发明的Fc变体的氨基酸取代选自T250Q、T250R、T250A、T250H、T250V、T250K、L251H、L251A、L251E、L251Q、L251R、L251Y、L251C、L251V、M252L、M252V、M252Q、M252Y、M252H、M252F、I253P、I253S、I253Q、I253R、I253T、I253N、I253Y、S254H、R255Q、T256Q、P257Q、E258Y、E258W、V259R、V259G、T307N、T307Y、T307Q、V308P、V308D、V308T、V308S、L309Y、L309S、L309T、L309Q、H310P、H310V、Q311H、Q311N、S375R、I377L、V379I、E380Q、W381Q、L432S、L432C、L432Q、L432Y、L432P、L432W、L432F、L432A、L432M、H433R、H433T、H433Q、H433P、H433K、H433F、H433S、H433N、H433M、N434W、N434Y、N434H、N434F、N434D、N434G、N434I、N434L、N434T、N434Q、H435Q、H435K、H435P、H435N、H435D、Y436F、T437N及其合适的组合。
本发明的Fc变体可以包括在野生型Fc中的两个或更多个位置的取代,其在本文中被称为取代的“组合”。野生型Fc的氨基酸序列如SEQ ID No.348中所提及的。Fc变体的氨基酸取代的优选组合显示在表3中。组合中存在的氨基酸取代由符号“/”分隔。
表3:Fc变体中特定EU位置处取代氨基酸的优选组合
在优选的实施方案中,根据本发明在Fc变体中的特定EU位置处取代氨基酸的组合选自以下示出的序列:SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ IDNO.5、SEQ ID NO.7、SEQ ID NO.9、SEQ ID NO.11、SEQ ID NO.14、SEQ ID NO.15、SEQ IDNO.18、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ IDNO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31、SEQ IDNO.32、SEQ ID NO.34、SEQ ID NO.35、SEQ ID NO.36、SEQ ID NO.38、SEQ ID NO.39、SEQ IDNO.40、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.45、SEQ IDNO.46、SEQ ID NO.47、SEQ ID NO.48、SEQ ID NO.49、SEQ ID NO.50、SEQ ID NO.51、SEQ IDNO.52、SEQ ID NO.53、SEQ ID NO.54、SEQ ID NO.55、SEQ ID NO.56、SEQ ID NO.57、SEQ IDNO.58、SEQ ID NO.59、SEQ ID NO.60、SEQ ID NO.61、SEQ ID NO.62、SEQ ID NO.63、SEQ IDNO.64、SEQ ID NO.65、SEQ ID NO.66、SEQ ID NO.67、SEQ ID NO.68、SEQ ID NO.69、SEQ IDNO.70、SEQ ID NO.71、SEQ ID NO.72、SEQ ID NO.73、SEQ ID NO.74、SEQ ID NO.75、SEQ IDNO.76、SEQ ID NO.77、SEQ ID NO.78、SEQ ID NO.79、SEQ ID NO.80、SEQ ID NO.81、SEQ IDNO.82、SEQ ID NO.83、SEQ ID NO.84、SEQ ID NO.85、SEQ ID NO.86、SEQ ID NO.87、SEQ IDNO.88、SEQ ID NO.89、SEQ ID NO.90、SEQ ID NO.91、SEQ ID NO.92、SEQ ID NO.93、SEQ IDNO.94、SEQ ID NO.95、SEQ ID NO.96、SEQ ID NO.97、SEQ ID NO.98、SEQ ID NO.99、SEQ IDNO.100、SEQ ID NO.101、SEQ ID NO.102、SEQ ID NO.103、SEQ ID NO.104、SEQ ID NO.105、SEQ ID NO.106、SEQ ID NO.107、SEQ ID NO.108、SEQ ID NO.109、SEQ ID NO.110、SEQ IDNO.111、SEQ ID NO.112、SEQ ID NO.113、SEQ ID NO.114、SEQ ID NO.115、SEQ ID NO.116、SEQ ID NO.117、SEQ ID NO.118、SEQ ID NO.119、SEQ ID NO.120、SEQ ID NO.121、SEQ IDNO.122、SEQ ID NO.123、SEQ ID NO.124、SEQ ID NO.125、SEQ ID NO.126、SEQ ID NO.127、SEQ ID NO.128、SEQ ID NO.129、SEQ ID NO.130、SEQ ID NO.131、SEQ ID NO.132、SEQ IDNO.133、SEQ ID NO.134、SEQ ID NO.135、SEQ ID NO.136、SEQ ID NO.137、SEQ ID NO.138、SEQ ID NO.139、SEQ ID NO.140、SEQ ID NO.141、SEQ ID NO.142、SEQ ID NO.143、SEQ IDNO.144、SEQ ID NO.145、SEQ ID NO.146、SEQ ID NO.147、SEQ ID NO.148、SEQ ID NO.149、SEQ ID NO.150、SEQ ID NO.151、SEQ ID NO.152、SEQ ID NO.153、SEQ ID NO.154、SEQ IDNO.155、SEQ ID NO.156、SEQ ID NO.157、SEQ ID NO.158、SEQ ID NO.159、SEQ ID NO.160、SEQ ID NO.161、SEQ ID NO.162、SEQ ID NO.163、SEQ ID NO.164、SEQ ID NO.165、SEQ IDNO.166、SEQ ID NO.167、SEQ ID NO.168、SEQ ID NO.169、SEQ ID NO.170、SEQ ID NO.171、SEQ ID NO.172、SEQ ID NO.173、SEQ ID NO.174、SEQ ID NO.175、SEQ ID NO.176、SEQ IDNO.177、SEQ ID NO.178、SEQ ID NO.179、SEQ ID NO.180、SEQ ID NO.181、SEQ ID NO.182、SEQ ID NO.183、SEQ ID NO.184、SEQ ID NO.185、SEQ ID NO.186、SEQ ID NO.187、SEQ IDNO.188、SEQ ID NO.189、SEQ ID NO.190、SEQ ID NO.191、SEQ ID NO.192、SEQ ID NO.193、SEQ ID NO.194、SEQ ID NO.195、SEQ ID NO.196、SEQ ID NO.197、SEQ ID NO.198、SEQ IDNO.199、SEQ ID NO.200、SEQ ID NO.201、SEQ ID NO.202、SEQ ID NO.203、SEQ ID NO.204、SEQ ID NO.205、SEQ ID NO.206、SEQ ID NO.207、SEQ ID NO.208、SEQ ID NO.209、SEQ IDNO.210、SEQ ID NO.211、SEQ ID NO.212、SEQ ID NO.213、SEQ ID NO.214、SEQ ID NO.215、SEQ ID NO.216、SEQ ID NO.217、SEQ ID NO.218、SEQ ID NO.219、SEQ ID NO.220、SEQ IDNO.221、SEQ ID NO.222、SEQ ID NO.223、SEQ ID NO.224、SEQ ID NO.225、SEQ ID NO.226、SEQ ID NO.227、SEQ ID NO.228、SEQ ID NO.229、SEQ ID NO.230、SEQ ID NO.231、SEQ IDNO.232、SEQ ID NO.233、SEQ ID NO.234、SEQ ID NO.235、SEQ ID NO.236、SEQ ID NO.237、SEQ ID NO.238、SEQ ID NO.239、SEQ ID NO.240、SEQ ID NO.241、SEQ ID NO.242、SEQ IDNO.243、SEQ ID NO.244、SEQ ID NO.245、SEQ ID NO.246、SEQ ID NO.247、SEQ ID NO.248、SEQ ID NO.249、SEQ ID NO.250、SEQ ID NO.251、SEQ ID NO.252、SEQ ID NO.253、SEQ IDNO.254、SEQ ID NO.255、SEQ ID NO.256、SEQ ID NO.257、SEQ ID NO.258、SEQ ID NO.259、SEQ ID NO.260、SEQ ID NO.261、SEQ ID NO.262、SEQ ID NO.263、SEQ ID NO.264、SEQ IDNO.265、SEQ ID NO.266、SEQ ID NO.267、SEQ ID NO.268、SEQ ID NO.269、SEQ ID NO.270、SEQ ID NO.271、SEQ ID NO.272、SEQ ID NO.273、SEQ ID NO.274、SEQ ID NO.275、SEQ IDNO.276、SEQ ID NO.277、SEQ ID NO.278、SEQ ID NO.279、SEQ ID NO.280、SEQ ID NO.281、SEQ ID NO.282、SEQ ID NO.283、SEQ ID NO.284、SEQ ID NO.285、SEQ ID NO.286、SEQ IDNO.287、SEQ ID NO.288、SEQ ID NO.289、SEQ ID NO.290、SEQ ID NO.291、SEQ ID NO.292、SEQ ID NO.293、SEQ ID NO.294、SEQ ID NO.295、SEQ ID NO.296、SEQ ID NO.297、SEQ IDNO.298、SEQ ID NO.299、SEQ ID NO.300、SEQ ID NO.301、SEQ ID NO.302、SEQ ID NO.303、SEQ ID NO.304、SEQ ID NO.305、SEQ ID NO.306、SEQ ID NO.307、SEQ ID NO.308、SEQ IDNO.309、SEQ ID NO.310、SEQ ID NO.311、SEQ ID NO.312、SEQ ID NO.313、SEQ ID NO.314、SEQ ID NO.315、SEQ ID NO.316、SEQ ID NO.317、SEQ ID NO.318、SEQ ID NO.319、SEQ IDNO.320、SEQ ID NO.321、SEQ ID NO.322、SEQ ID NO.323、SEQ ID NO.324、SEQ ID NO.325、SEQ ID NO.326、SEQ ID NO.327、SEQ ID NO.328、SEQ ID NO.329、SEQ ID NO.330、SEQ IDNO.331、SEQ ID NO.332、SEQ ID NO.333、SEQ ID NO.334、SEQ ID NO.335、SEQ ID NO.336、SEQ ID NO.337、SEQ ID NO.338、SEQ ID NO.339、SEQ ID NO.340、SEQ ID NO.341、SEQ IDNO.342、SEQ ID NO.343、SEQ ID NO.344、SEQ ID NO.345、SEQ ID NO.346和SEQ IDNO.347。
在一个优选的实施方案中,根据本发明在Fc变体中的特定EU位置处取代氨基酸的组合选自以下示出的序列:SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ IDNO.5、SEQ ID NO.7、SEQ ID NO.9、SEQ ID NO.11、SEQ ID NO.14、SEQ ID NO.15、SEQ IDNO.18、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ IDNO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31、SEQ IDNO.32、SEQ ID NO.34、SEQ ID NO.35、SEQ ID NO.36、SEQ ID NO.38、SEQ ID NO.39、SEQ IDNO.40、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.45、SEQ IDNO.46、SEQ ID NO.47、SEQ ID NO.48和SEQ ID NO.49。
在一个优选的实施方案中,根据本发明在Fc变体的特定EU位置处取代氨基酸的组合如SEQ ID NO.3、SEQ ID NO.5、SEQ ID NO.10、SEQ ID NO.20或SEQ ID NO.22中所示。
在更优选的实施方案中,本发明的Fc变体的氨基酸序列选自SEQ ID NO.22a、SEQID NO.22b、SEQ ID NO.22c、SEQ ID NO.22d、SEQ ID NO.22e及SEQ ID NO.22f和SEQ IDNO.22g。
如本文所述,Fc变体的残基、它们各自的氨基酸取代和特定残基的氨基酸取代的组合可以存在于IgG1、IgG2、IgG3、IgG4或IgG2/G4同种型,优选IgG1同种型的Fc蛋白中。IgG2、IgG4或IgG2/G4同种型的Fc变体构建体可以在Fc变体的铰链区中含有单个氨基酸取代(即,S228P),以减少两条Fc链之间二硫键的破坏。(8)根据本发明制备的Fc变体可以任选地被修饰以修饰功能性,例如以消除残留的效应子功能,如ADCC和CDC活性。优选地,本发明的Fc变体没有CDC活性。优选地,与野生型Fc的ADCC活性或IVIg的ADCC活性相比,本发明的Fc变体具有降低的ADCC活性。相对于野生型IgG1Fc区对所述Fcγ受体的亲和力,根据本发明的Fc变体对Fc受体具有改变(增加或降低)的亲和力。在一个实施方案中,相对于野生型IgG1Fc区对FcγRIIIa(CD16a)、FcγRIIa(CD32a)和FcγRI(CD64)的亲和力,Fc变体对所述FcγRIIIa(CD16a)、FcγRIIa(CD32a)和FcγRI(CD64)具有增加的亲和力。在一个实施方案中,本发明的Fc变体对于FcγRIIIa(CD16a),包括同种异型V158和F158的KD为10-7M或更低。KD值是抗体对其靶抗原的结合亲和力的量度。在一个实施方案中,本发明的Fc变体抑制免疫复合物介导的FcγR活化,如通过ADCC的抑制所评估的。在一个实施方案中,本发明的Fc变体抑制由FcγR介导的吞噬作用。在一个实施方案中,本发明的Fc变体抑制由FcγR介导的细胞因子释放,如IL-6或IL-8。本发明提供了能够以高亲和力与FcRn以及FcγR结合的Fc变体。因此,本发明的Fc变体具有单一药物的多种作用机制的组合作用,如FcRn阻断和FcγR活化的抑制,因此其导致本发明的Fc变体的强健功效。
在本文的实施例中,用本发明的非限制性Fc变体之一说明了组合效果。所述Fc变体具有表3中所述的SEQ ID No.22的取代。本发明的非限制性Fc变体的氨基酸序列提供于下表4中。在本文实施例给出的不同实验中使用了SEQ ID NO.22的特定变体SEQ IDNO.22b。
表4:Fc变体SEQ ID NO.22的氨基酸序列
在一个方面,本发明的Fc变体可以在一个或多个位置处包含非天然氨基酸。在肽中引入非天然氨基酸是本领域技术人员众所周知的。制备非天然存在的氨基酸并将其引入蛋白质的方法是已知的(美国专利号7,083,970和7,524,647)。在一个方面,根据本发明的Fc变体具有增加的FcRn结合和增加的半衰期,以及修饰的或降低的或没有ADCC和/或CDC活性。将本发明的Fc变体的ADCC和/或CDC活性与野生型Fc蛋白或IVIg的所述活性进行比较。在一个实施方案中,根据本发明的Fc变体具有选自P329G和/或M428L&N434S突变的氨基酸取代。
Fc变体的制备
本发明的Fc变体是在使用如本文实施例所述的定点诱变方法制备突变文库之后,使用噬菌体展示文库方法制备的。
编码Fc变体的核酸分子、载体和宿主细胞
在一个实施方案中,本发明提供了编码Fc变体或含有Fc变体的蛋白的核酸分子,以及包含这种核酸的表达载体和包含来自表达载体的编码Fc变体的这种核酸分子的宿主细胞。通过重组方法产生本发明的Fc变体或含有Fc变体的蛋白的合适载体是本领域技术人员已知的。在专利文献WO2007/017903、WO2012/046255(将其并入本文)中描述了这种已知载体的实例。根据本发明的宿主细胞是原核细胞或真核细胞,优选地,宿主细胞是哺乳动物细胞,更优选CHO细胞。
药物组合物
可以开发一种药物组合物,其含有本发明的Fc变体或含Fc变体的蛋白或含Fc变体的分子中的一种或组合与药学上可接受的载体一起配制。这种组合物可以包括本发明的(例如两种或更多种不同的)Fc变体或含Fc变体的蛋白或免疫缀合物或双特异性分子中的一种或组合。例如,本发明的药物组合物可以包含Fc变体或含Fc变体的蛋白和抗体(或免疫缀合物或双特异性的)(其结合靶抗原上的不同表位)的组合。
治疗用途
Fc变体或含Fc变体的蛋白或含Fc变体的分子可用于治疗涉及需要药物与FcRn结合的治疗方法的疾病。
在本发明的一个实施方案中,Fc变体或含Fc变体的蛋白可用于在患有疾病(如但不限于自身免疫疾病和炎症)的对象(包括人)体内抑制FcRn。
在某些实施方案中,Fc变体或含Fc变体的蛋白或含Fc变体的分子用于治疗选自以下的疾病:自身免疫性溶血性贫血、恶性贫血、特发性血小板减少性紫癜、Goodpasture综合征、大疱性类天疱疮、寻常型天疱疮、桥本氏甲状腺炎、胰岛素依赖型糖尿病(IDDM)、格雷夫斯病、重症肌无力、CIDP糖尿病、抗贫血药(β-地中海贫血)、造血剂、眼科药物、骨病、神经遗传病症、年龄相关性黄斑变性、糖尿病性视网膜病、黄斑疾病、非小细胞肺癌疗法、眼部遗传病症、血友病B、用于(凝血因子IX缺乏症)心血管疾病的药剂、2型糖尿病、免疫抑制剂、类风湿关节炎、移植排斥的治疗、脑癌、乳腺癌、结肠直肠癌、糖尿病性视网膜病、消化/胃肠癌、内分泌癌、女性生殖系统癌症、胃癌、泌尿生殖系统癌症、黄斑疾病、黑色素瘤、多发性骨髓瘤、骨髓增生异常综合征治疗、骨髓性白血病治疗、非霍奇金淋巴瘤治疗、非小细胞肺癌、卵巢癌、胰腺癌、前列腺癌疗法、肾癌疗法、视网膜病、再生障碍性贫血、止痛药、血液遗传病症、多系统遗传病症、骨关节炎、硬皮病、自身免疫疾病的治疗、痛风的治疗、荨麻疹、强直性脊柱炎、哮喘疗法、皮肤病药物、特发性炎性肌病、免疫抑制剂、炎性肠病、间质性肺病、多发性骨髓瘤疗法、多发性硬化症、肾炎、银屑病关节炎、系统性红斑狼疮、急性酒精性肝炎的药剂、阿尔茨海默氏痴呆、抗过敏药物/平喘药物、抗关节炎药物、抗银屑病药、乳腺癌疗法、癌症相关病症、皮肤病药物、心力衰竭疗法、淋巴瘤疗法、肾炎、神经病学药物(混杂的)、呼吸系统病症、先天性代谢异常的治疗。
在本文中,用以下非限制性实例说明本发明,这些实施例不应被解释为以任何方式限制本发明的范围。
实施例
提出以下实施例以向本领域普通技术人员提供如何实施本文要求保护的方法和Fc变体的公开内容和描述。它们仅仅是为了举例说明,而不旨在限制本公开内容的范围。本发明的其它Fc变体可以使用如提供的实施例中所述以及进行了一些修改的方法开发。这种修改对于本领域技术人员来说是已知的。
实施例1:噬菌体展示文库制备
(a)野生Fc质粒产生
为了产生文库,化学合成了含有带有重链CH2和CH3结构域(EU221-447)的铰链的部分序列以及EcoRI和BamHI的突出端(overhangs)的人IgG1Fc区,并将其克隆到pMA/pMK载体中。
用EcoRI和BamHI消化之后,从这些构建体中分离Fc基因(SEQ ID No.348)。克隆的Fc基因的氨基酸序列在本文中提供为SEQ ID No.:348.
修饰pSEX81(目录号:PR3005,Progen)噬菌粒载体,其中噬菌粒载体的pIII基因被截短,用EcoRI和BamHI消化,并将线性化的载体与消化的Fc基因连接。所得的修饰载体在本文中被称为pSEX83。pSEX83的载体图谱在本文中以图1给出。在琼脂糖凝胶上分析消化的Fc和pSEX83载体片段,并使用QIAquick凝胶提取试剂盒(Qiagen目录号28706)从凝胶中纯化。将Fc(插入物)和pSEX83(载体)的连接产物转化到电感受态大肠杆菌TG1细胞(目录号:60502-1,Lucigen)中。将转化的细胞铺板在含有氨苄青霉素(100μg/mL)的2xYT琼脂板上。通过EcoRI和BamHI限制性消化以及使用Sanger法的DNA测序分析克隆。
(b)用于亲和力成熟的二级文库的制备
按照两种不同的策略制备Fc变体噬菌体展示文库。
策略1:基于PCR引物的定点诱变
靶向Fc基因的CH2-CH3(EU250-259、EU307-311、EU375-381和EU432-437)结构域中的四个不同区域用于引入随机突变和制备噬菌体展示文库。
通过使用具有SapI限制性位点(表4)的相应引物对(RK173/RK174、RK175/RK176、RK177/RK178、RK179/RK180、RK181/RK182和RK183/RK184)的PCR引入选定区域中的突变。设计每个引物对,使它们以线性形式扩增完整的质粒,经SapI限制性消化,然后用T4 DNA连接酶连接,产生具有通过引物对引入的突变的环状质粒。每对用于制备单独的文库。另外,使用来自一个文库的DNA制备用于向其它区域添加突变的文库。
简而言之,使用表5中提及的引物对,将pSEX83载体中Fc基因的100ng模板DNA用于扩增。一旦使用随机引物扩增模板,用SapI消化产物以获得粘性末端。在PCR纯化之后,将消化的产物在16℃下连接过夜。然后将连接的DNA转化到新鲜制备的电感受态细胞(TG1)中。培养转化的细胞以生产噬菌体。如前所述(Brockmann等人,2011),对每个文库分别进行噬菌体生产。
表5:基于PCR引物的定点诱变中使用的引物列表
策略2:Kunkel诱变和sRCA
按照参考文献9,用sRCA进行Kunkel诱变。用携带Fc基因的质粒的噬菌体感染CJ236细胞的小培养物。感染的细胞在含有尿苷(6μg/mL)和羧苄青霉素抗生素(100μg/mL)的2xYT中生长。用VCS M13辅助噬菌体(Agilent technologies,USA)重复感染之后产生噬菌体,并通过用PEG6000(4%)和NaCl(500mM)沉淀来纯化。通过使用来自M13DNA微型试剂盒(OMEGA bio-tek,USA)的M13纯化试剂盒按照制造商的说明从纯化的噬菌体中提取单链尿苷化(uridylated)DNA(ss(U)DNA)。
将该ss(U)DNA用作Kunkel诱变中的模板,并将具有随机突变的不同引物用于制备文库。将靶向Fc的不同区域的引物(表6)分别与ss(U)DNA模板杂交并通过Kunkel诱变方法延伸。对产物进行UDG处理并通过RCA选择性地扩增(9)。用Hind III消化RCA产物,使用T4DNA连接酶环化并转化到SS320细胞中。如下所述,通过用辅助噬菌体重复感染,为每个诱变引物分别产生噬菌体(二级文库)。
表6:Kunkel诱变中使用的引物列表
实施例2:由PCR和Kunkel文库进行突变Fc噬菌体产生及其纯化
用来自上述文库的甘油储备液的细胞以OD600为0.06的初始浓度接种含有含羧苄青霉素或氨苄青霉素(100μL/mL)的2xYT培养基的烧瓶。使培养物在37℃下以250rpm摇动生长,直到它们达到高达约0.4-0.6的OD600。将辅助噬菌体VCSM13(Agilent,目录号200251)或M13KO7(GE healthcare,目录号27152401)以20的感染复数(MOI)加入到培养物中,并且首先在37℃下不摇动孵育40分钟,然后再在37℃下摇动孵育40分钟。然后将卡那霉素加入到培养基中并将培养物在26℃下以150rpm生长过夜。将过夜的噬菌体培养物以4000g离心并弃去细胞沉淀。将比例为1:5的PEG(20%)/NaCl(2.5M)溶液加入到上清液中以沉淀噬菌体。将重悬的溶液在冰上孵育20分钟,然后在4℃下以14,000g离心15分钟。弃去上清液,并将沉淀重悬在1mL的含有0.01%叠氮化钠的无菌PBS中。将噬菌体储存在4℃直至进一步使用。
实施例3:表达高亲和力Fc变体的噬菌体的生物淘选
筛选实施例2中制备的Fc突变体文库(1×1012PFU)对FcRn受体的高亲和力Fc结合剂。使用抗原固定的免疫管(Quidel,USA)进行pH 6.0下针对FcRn/FcGRT抗原(Sinobiological,目录号CT071-H27H)的第一轮淘洗,所述免疫管通过在4℃下用在碳酸盐缓冲液(0.1M,pH 9.6)中的5μg/mL FcRn蛋白溶液孵育过夜而制备。将免疫管用PBS洗涤3次,然后与PBS中的噬菌体一起在25℃下持续旋转孵育2小时。用含有吐温20(0.1%)的4mL PBS将管洗涤10次,随后用PBS洗涤10次。用甘氨酸-HCL pH2.1(0.1M)洗脱结合的噬菌体。通过感染TG1大肠杆菌细胞来拯救洗脱的噬菌体,铺板以及如实施例3所述产生噬菌体。
以将来自第一轮淘洗(1×1011CFU)和第二轮淘洗(1×1010CFU)的输出噬菌体分别用作第二轮和第三轮淘洗的输入噬菌体的方式,在FcRn抗原包被的免疫管中进行第二轮和第三轮淘洗。如前所述,在TG1细胞中感染第三轮淘洗之后的噬菌体,并分离噬菌粒DNA,用于在合适的表达载体中克隆Fc变体。
实施例4:筛选作为可溶性Fc蛋白的各Fc突变体克隆
克隆到表达载体和蛋白生产
将在3轮淘选后产生的来自富集文库的Fc变体基因克隆到表达载体pOPE101(羧苄青霉素抗性)(Progen,Germany)或其修饰形式pOPE102(卡那霉素抗性)中,使得各Fc突变体克隆可以以HIS标记的融合产物产生。用限制酶(EcoRI和BamHI)消化载体DNA(pOPE101或pOPE102)和噬菌粒DNA,以分别分离载体和Fc变体基因。连接限制性消化的载体和Fc基因并转化到TG1电感受态细胞中。将转化的细胞铺板到含有羧苄青霉素抗生素的2xYT琼脂板上。从2xYT琼脂板中挑选各克隆并在32℃下在含5mL含有羧苄青霉素或氨苄青霉素(100μL/mL)的2xYT培养基的15mL管中以200rpm培养过夜。第二天,将培养物以1:200的体积比重新接种到含有羧苄青霉素(100μg/mL)和葡萄糖(0.1%)的新鲜2xYT培养基中。使培养物生长直到OD600达到~0.6-0.8。之后,将1mM异丙基β-D-1-硫代半乳糖苷(IPTG)加入到培养物中并在30℃下以200rpm生长过夜。将过夜培养物以10,800g旋转15分钟,并除去上清液。将细胞沉淀重悬于1/20体积的原始培养物中,在含有2mg/mL溶菌酶、0.1%triton X和1U/100mLbenzonase的pH-7.4的1xPBS缓冲液中,并在37℃下孵育1h。将重悬的沉淀在10,800g离心15分钟,并收集上清液作为含有可溶性His-标记的Fc变体的细胞裂解物。将该细胞裂解物级分用于免疫测定中,以在定量周质级分中的可溶性Fc之后研究FcRn结合。
周质级分中可溶性Fc的定量
如上所述单独培养克隆以产生可溶性Fc变体。通过将可溶性Fc固定在用小鼠抗人IgG抗体(Sigma,目录号I6760)预包被的maxisorp板上来定量细胞裂解物或周质级分中的总Fc。用在包被缓冲液(0.1M NaHCO3,pH 9.6)中浓度为2.5μg/mL(100μL/孔)的小鼠抗人IgG抗体进行过夜包被。在洗涤板两次之后,加入100μL细胞裂解物(1/20v/v在1xPBS中),并在25℃下孵育1h。已知浓度的连续稀释的野生Fc IgG1用作计算细胞裂解物中Fc蛋白的量的标准。将板用PBST(0.1%吐温20在1xPBS中)洗涤3次。将100μL(1:10000)的HRP缀合的山羊抗人IgG Fc,F(ab)’2片段(Invitrogen,目录号A24476)加入到板的每个孔中并孵育1h。然后将板用PBST洗涤5次,然后在37℃下加入底物邻苯二胺二盐酸盐(OPD)(100μL/孔)保持15分钟。使用1N H2SO4(100μL/孔)终止反应,并使用TECANM1000pro在450nm处测量光密度(OD)。然后使用参考标准计算细胞裂解物中Fc的浓度。
Fc变体的定性分析
将FcRn抗原包被在聚苯乙烯板(100ng/100μL/孔)上,在4℃下在包被缓冲液(0.1MNaHCO3,pH 9.6)中过夜。在将板洗涤两次之后,将稀释在100μL PBS pH6.0中的100ng Fc变体加入到每个孔中并且在25℃下孵育1h。用PBST(0.1%吐温20在1xPBS pH6.0中)将板洗涤4次,然后加入100μL(1:10000)的HRP缀合的山羊抗人IgG Fc,F(ab)’2片段(Invitrogen,目录号A24476),并在25℃下孵育1h。然后将板用PBST洗涤5次,然后在37℃下加入底物邻苯二胺二盐酸盐(OPD)(100μL/孔)保持15分钟。使用1N H2SO4(100μL/孔)终止反应,并使用TECANM1000pro在450nm处测量光密度。筛选出与野生Fc相比具有相对高OD信号的各克隆,用于进一步表征。
实施例5:Fc变体与重组人新生Fc受体(rhFcRn)结合的动力学速率常数的测定
通过使用ProteOnTM XPR36(Bio-Rad)的基于表面等离子体共振的测量来测定Fc变体与重组人新生Fc受体(rhFcRn)结合的动力学常数。按照制造商的说明,将rhFcRn受体(Sino Biologics)固定在GLC芯片上。将10mM磷酸盐缓冲盐水(PBS)(10mM磷酸盐缓冲液,pH6.0,150mM NaCl,含有0.005%吐温20)用作运行缓冲液以进行动力学测量。为了测量结合速率常数(Ka)和解离速率常数(Kd),在上述运行缓冲液中制备Fc变体的5个稀释液,并以100μL/min的流速注射,结合时间为180s,解离时间为600s。在25℃下在PBS(pH6.0,0.005%表面活性剂P20)中进行反应。在每次样品运行之后,使用两个脉冲的PBS(0.005%表面活性剂P20),pH7.4,然后使用一个脉冲的甘氨酸缓冲液,pH1.5,再生芯片表面。使用ProteOnTM系统中的数据拟合程序分析传感图(sensogram)形式的数据。rhFcRn与不同Fc变体结合的动力学常数显示在表7中。
表7:所选FcRn结合剂的动力学常数
SEQ ID No. | 样品代码 | K<sub>a</sub> | K<sub>d</sub> | K<sub>D</sub> |
3 | A4 | 1.12E+06 | 1.98E-02 | 1.08E-08 |
5 | D9-6 | 3.42E+05 | 2.93E-03 | 8.60E-09 |
6 | H11_4-2 | 1.06E+06 | 1.07E-02 | 1.02E-08 |
10 | B9-10 | 5.26E+06 | 1.56E-02 | 2.98E-09 |
19 | A9-10 | 4.21E+06 | 1.17E-02 | 2.79E-09 |
20 | F3-9 | 7.27E+06 | 2.64E-02 | 3.63E-09 |
22 | H11+A4 | 7.51E+06 | 2.16E-03 | 2.88E-10 |
实施例6:在不同pH条件下Fc变体与重组人新生Fc受体(rhFcRn)结合的动力学速率常数的测定
在该实验中,在两种不同的条件下,通过使用ProteOnTM XPR36(Bio-Rad)的基于表面等离子体共振的测量来测定Fc变体与重组人新生Fc受体(rhFcRn)结合的亲和常数。将10mM磷酸盐缓冲盐水(PBS)(10mM磷酸盐缓冲液,150mM NaCl,含有0.005%吐温20)用作运行缓冲液以进行动力学测量。为了测量亲和常数,制备Fc变体的5个稀释液,并以100μL/min的流速注射,结合时间为180s,解离时间为600s。所有反应在25℃下进行。分析样品在不同pH条件下的FcRn结合以检查pH对FcRn结合和分子与FcRn解离的影响。测量了具有SEQ IDNo.22所示序列的Fc变体与rhFcRn的亲和常数。用于相互作用的结合阶段的缓冲液是样品稀释缓冲液,以及用于相互作用的解离阶段的缓冲液是运行缓冲液。在条件1中,用于结合阶段的缓冲液是PBST pH6.0,而用于解离阶段的缓冲液是PBST pH7.4。在条件2中,用于结合阶段的缓冲液是PBST pH7.4,而用于解离阶段的缓冲液是PBST pH6.0。在CHO细胞系中产生用于进行该实验的Fc变体。在两种不同pH条件下rhFcRn与Fc变体结合的亲和常数显示在表8中。
表8:两种不同pH条件下FcRn结合剂的亲和常数
从表8可以看出,与在pH 7.4下相比,本发明的Fc变体可以在pH6.0下以更高的亲和力阻断FcRn。因此,本发明的Fc变体保留了FcRn相互作用的pH依赖性(在pH 6.0下比在接近中性的pH下的亲和力更高)特征。因此,根据本发明制备的Fc变体可以降低循环中的总血清IgG水平。
实施例7:Fc变体与不同物种的重组新生Fc受体(rFcRn)结合的动力学速率常数的测定
在该实验中,通过使用ProteOnTM XPR36(Bio-Rad)的基于表面等离子体共振的测量来测定本发明的Fc变体与不同物种的FcRn的结合。为了检查动力学速率常数,使用小鼠、猴和人的重组FcRn与具有SEQ ID No:22(H11+A4)所示序列的Fc变体进行实验。用于进行该实验的Fc变体是使用CHO细胞作为表达系统产生的。使用标准胺偶联化学并且基本上按照制造商的说明,将(不同物种的)FcRn固定在GLC传感器芯片表面上。将10mM磷酸盐缓冲盐水(PBS)(10mM磷酸盐缓冲液,pH6.0,150mM NaCl,0.005%吐温20)用作运行缓冲液以进行动力学测量。为了测量结合速率常数(kassoc)和解离速率常数(kdissoc),在上述运行缓冲液中制备Fc突变蛋白的5个稀释液,并以100μL/min的流速注射,结合时间为180s,解离时间为600s。所有反应在25℃下进行。在每个样品运行之后,使用pH 7.4的PBST再生芯片表面。使用ProteOnTM系统中的数据拟合程序分析传感器图形式的数据。
(不同物种的)FcRn与Fc变体结合的动力学常数显示在表9中。
表9:针对不同物种的FcRn的FcRn结合剂的动力学常数
FcRn的类型 | K<sub>a</sub> | K<sub>d</sub> | K<sub>D</sub> |
人FcRn | 4.10E+06 | 3.24E-03 | 7.91E-10 |
小鼠FcRn | 1.18E+06 | 7.24E-04 | 6.15E-10 |
猴FcRn | 4.42E+06 | 3.54E-03 | 8.01E-10 |
从表9可以看出,本发明的Fc变体不仅可以体外阻断人FcRn,而且可以体外阻断小鼠FcRn以及猴FcRn。与猴FcRn交叉反应性的有利特征意味着本发明的Fc变体可以在非人灵长类动物中测试,并且所得数据可以在预测本发明的Fc变体在人中的药代动力学、药效学和毒性中非常有用。
实施例8:Fc变体与Fcγ受体结合的动力学速率常数的测定
在该实验中,通过使用ProteOnTM XPR36(Bio-Rad)的基于表面等离子体共振的测量来测定Fc变体与重组Fcγ受体结合的动力学常数。对于该实验,分析FcγRIIIa(Phe)和FcγRIIIa(Val)Fcγ受体与本发明的Fc变体的结合,所述Fc变体具有SEQ ID No.22(H11+A4)所示的序列。使用标准胺偶联化学并且基本上按照制造商的说明,将所有重组人Fc受体直接固定在GLC传感器芯片表面上。将10mM磷酸盐缓冲盐水(PBS)(10mM磷酸盐缓冲液,pH7.4,150mM NaCl,含有0.005%吐温20)用作运行缓冲液,以进行所有Fc受体的动力学测量。为了测量结合速率常数(kassoc)和解离速率常数(kdissoc),在上述运行缓冲液中制备Fc变体的5个稀释液,并以50μL/min的流速注射。在下表中提及了每次相互作用时的结合时间和解离时间。所有反应在25℃下进行。使用ProteOnTM系统可获得的数据拟合程序分析传感器图形式的数据。
表10:Fc受体结合Fc变体的实验细节
受体 | 结合时间 | 解离时间 |
FcγRIIIa(Phe) | 240 | 600 |
FcγRIIIa(Val) | 240 | 600 |
用于进行该实验的Fc变体在CHO细胞系中产生。保持代表人IgG1对照的对照分子(缺少本发明的取代)以测定本发明Fc变体对Fcγ受体的亲和力水平。
表11:Fc变体对Fcγ受体的动力学常数
从表11可以看出,与代表IgG1对照的分子相比,本发明的Fc变体可以以高亲和力阻断FcγRIIIa(包括同种异型V158和F158)。本发明Fc变体阻断Fcγ受体的这种示出活性显示本发明Fc变体能够抑制免疫复合物介导的FcγR活化。
实施例9:具有SEQ ID No.22的Fc变体的单体和多聚体(二聚体)DGV-H11A4的构建
将具有SEQ ID NO.22(H11+A4;它也可以被称为H11A4)的氨基酸序列的Fc变体单体和具有SEQ ID NO.22(H11+A4;它可以被称为H11A4)的氨基酸序列的Fc二聚体的化学合成的基因获自德国Geneart的pMK载体,其中Fc变体单体的两条链与甘氨酸-丝氨酸接头(SEQ ID NO.A-GGGSGGGSGGGSGGGSSGGGSS)连接并且在第二条链的EU位置226和229处的半胱氨酸残基变为丝氨酸以防止自二聚化。通过用HindIII和EcoRI限制性消化从pMKGeneart构建体分离FC变体单体和二聚体基因。将~0.7kb的HindIII和EcoRI消化的Fc变体单体H11A4基因和~1.5kb的Fc变体二聚体分别连接到HindIII和EcoRI消化的PXC-17.4载体(Lonza)和PXC-18.4载体(Lonza)。将连接产物转化到大肠杆菌Top10F’中,并根据氨苄青霉素抗性对转化体进行评分。通过用HindIII和EcoRI限制性消化来分析克隆。用SalI和NotI消化来自每个PXC-17.4H11A4单体和二聚体以及pXC-18.4 H11A4单体和二聚体的阳性克隆。将消化的产物在1%琼脂糖凝胶上解析。通过连接来自pXCH11A4单体的~6.9kb片段和来自pXC-18.4 H11A4单体的~2.7kb片段来制备携带两个表达装配体的最终DGV H11A4单体。类似地,通过连接来自pXCH11A4二聚体的~7.7kb片段和来自pXC-18.4H11A4二聚体的~3.5kb片段来制备携带两个表达装配体的最终DGV H11A4二聚体。将连接产物转化到大肠杆菌Top 10F’中,并根据氨苄青霉素抗性对转化体进行评分。通过限制性消化表征和Sanger测序证实最终的DGV-H11A4单体和二聚体载体。将载体用PvuI线性化用于在CHO-GS细胞(Lonza)中转染。用于制备Fc单体和Fc二聚体的载体的图谱如本文图2所示。
实施例10:用具有SEQ ID No.22的Fc变体构建全长抗体DGV-H11A4
通过将Fc区与不相关(对人/小鼠/NHP无交叉反应)轻链组合的不相关(对人/小鼠/NHP无交叉反应)重链可变结构域连接而将Fc变体H11A4制备为全长单克隆抗体分子。
扩增Fc变体H11A4区域以插入CH1结构域和5’端的ApaI限制性位点和3’端的EcoRI限制性位点。用ApaI和EcoRI消化该~1.0kb的插入物,以便于在具有重链可变区的框架内克隆。将MluI和ApaI消化的重链可变区,~0.5kb的基因和ApaI-EcoRI消化的H11A4 PCR片段连接到MluI和EcoRI消化的含有轻链基因的DGV载体上。将连接产物转化到大肠杆菌Top10F’细胞中,并根据氨苄青霉素抗性对转化体进行评分。通过限制性消化表征和Sanger测序证实最终的DGV-H11A4 mAb载体。将载体用PvuI线性化以在CHO-GS细胞(Lonza)中转染。用于产生包含Fc单体的全长抗体的载体的图谱如本文图3所示。
实施例11:细胞系表达Fc构建体的产生
该实施例描述了表达Fc变体的稳定转染细胞系的产生。将实施例9和10中所述的所有载体构建体用于转染。转染前用PvuI限制酶将质粒线性化。中国仓鼠卵巢(CHO)是用于表达重组蛋白的合适宿主之一,用于细胞系的产生。在转染前~24小时以50万/mL的密度接种CHO细胞以使细胞处于指数期。使用Neon Transfection System(Invitrogen)按照制造商的说明通过电穿孔技术进行转染。转染后,将细胞铺板在24孔细胞培养板中,所述培养板含有包含选择压力的1mL预热的ProCHO5无血清培养基(Lonza,Switzerland);并在湿润的培养箱中在37℃下在5% CO2存在下孵育。定期监测所有转染池的细胞数目并进行定期的培养基更换。一旦细胞从转染中恢复,将细胞进一步扩增至6孔培养板、T瓶和培养管(TPP)。
在培养管(TPP)中对所有Fc变体候选物的转染池进行补料分批培养以产生重组蛋白。将细胞以0.3×106个细胞/mL的密度接种在来自Hyclone,GE的ActiPro生产培养基中。将培养管在湿润的Kuhner摇床中在37℃的温度、5% CO2水平以及230RPM的摇动速度孵育。使用来自Hyclone,GE的化学确定的饲料在所有池的培养期间遵循固定的每日饲养方案。在培养72小时之后,开始进料并持续直至收获批料。
收获后,收集培养物上清液,通过蛋白A亲和层析纯化蛋白。如以上实施例中所述,对这些纯化的蛋白候选物进一步测试各种体外测定。已经观察到多聚体形式的Fc变体(SEQID No.22)和全长抗体形式的Fc变体具有与Fc单体相似的技术效果。因此,本发明的Fc变体可以在所有构建形式(单体、多聚体和全长抗体)中实现相同的技术效果。
实施例12:Fc变体对野生型C57BL/6小鼠中总血清IgG水平的影响
在本研究中,将根据实施例9制备的Fc二聚体与市售IVIg进行比较,以测定本发明的Fc变体对循环中总血清IgG水平的影响。将野生型C57BL/6小鼠基于其体重而随机化。在零时间点(给药前)进行血液收集。在1小时内,将50mg/kg Fc二聚体和1g/kg IVIg经由静脉内途径施用至各组研究动物(每组6只小鼠)。在时间点-5小时,24小时(第1天)、48小时(第2天)、72小时(第3天)、144小时(第6天)、216小时(第9天)、288小时(第12天)和312小时(第13天)收集血样。使用ELISA测定在提及的时间点收集的样品中内源性IgG水平。图形表示的结果显示在图4中。与对照相比,IVIg和Fc二聚体均显示出内源性小鼠IgG水平的降低,与IVIg相比,Fc二聚体显示出更大的IgG清除率直到第6天。与相对于给药前IgG的IVIg的~66%相比,用Fc二聚体观察到~47%的最低%IgG降低。在所提及的时间点也测定白蛋白水平。总的来说,在所有时间点和组别中没有观察到差异(图5)。根据本发明制备的FC二聚体不影响血清中的白蛋白水平。显然,根据本发明制备的Fc变体不干扰白蛋白在FcRn上的结合位点,并允许FcRn维持血清中的白蛋白水平。按照与本文实施例12所述相同的方法,对于含有本发明Fc变体的全长抗体以及具有SEQ ID No.22的本发明Fc变体的单体形式进行类似的实验。
实施例13:Fc变体对Fcγ受体依赖性ADCC活性的影响
将SKBR3细胞用钙黄绿素-AM染料染色,分别在10.39ng/mL和31.17ng/mL的EC50和EC75浓度的曲妥珠单抗下接种,并在37℃和5%CO2下孵育30分钟。为了确定根据实施例9制备的Fc二聚体是否能够阻断ADCC,将从100μg/mL开始连续稀释的Fc二聚体和靶细胞与效应细胞的比率为1:30的人PBMC加入到含有SKBR3细胞的孔中,并在37℃和5% CO2下孵育4小时。孵育后,将板在25℃下以2000rpm离心5分钟,收集100μL上清液,并将其转移到96孔黑色透明底板中。在494nm激发和515nm发射下在平板读数器中进行读数,截止值设置在515nm处。结果显示在图6中。从图6中可以看出,本发明的Fc二聚体可以降低曲妥珠单抗介导的ADCC活性,证实通过其与Fcγ受体的更强结合,可以防止曲妥珠单抗与PBMC中存在的NK细胞的结合。
实施例14:Fc变体在慢性特发性血小板减少性紫癜(ITP)动物模型中的作用
在本研究中,在急性免疫性血小板减少症的小鼠模型中测试H11+A4(SEQ IDNO.22)。具体地,将C57BL/6小鼠基于其体重而随机化,随后经由静脉内注射用两种不同剂量的H11+A4(即0.5mg/20gm小鼠&1mg/20gm小鼠)或用磷酸盐缓冲盐水(7只动物/组)处理。1小时后,经由静脉内途径用抗血小板抗体MWReg30(5μg/20gm小鼠)或用磷酸盐缓冲盐水处理小鼠。24小时之后,再次经由腹膜内途径用MWReg30(5μg/20gm小鼠)或用磷酸盐缓冲盐水处理小鼠。收集血样用于在第一次注射MWReg30之后72小时测定血小板计数。经由血液学分析仪从收集的样品中测定血小板计数并作图。结果显示在图7中。从图7中可以看出,用H11+A4预处理以剂量依赖性方式在两个剂量水平上降低了MWReg30(抗血小板抗体)诱导的血小板减少症&分子具有改善血小板减少性病况的显著潜力。
并入本专利申请的参考文献:
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通过引用并入
本文所提及的每个专利文献和科学文章的全部公开内容出于所有目的通过引用并入。
等同物
在不脱离本发明的精神或基本特征的情况下,本发明可以以其它具体形式实施。因此,上述实施方案在所有方面都被认为是说明性的,而不是限制本文描述的发明。因此,本发明的范围由所附权利要求书而不是由前面的描述来指示,并且在权利要求书的等同物的含义和范围内的所有改变都旨在包含在其中。
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225
Claims (21)
1.Fc变体,其包含在野生型Fc蛋白中的特定EU位置处取代氨基酸的组合,其中所述Fc变体包含选自以下的氨基酸序列:SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.7、SEQ ID NO.9、SEQ ID NO.11、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.18、SEQ ID NO.20、SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ IDNO.24、SEQ ID NO.25、SEQ ID NO.26、SEQ ID NO.27、SEQ ID NO.29、SEQ ID NO.30、SEQ IDNO.31、SEQ ID NO.32、SEQ ID NO.34、SEQ ID NO.35、SEQ ID NO.36、SEQ ID NO.38、SEQ IDNO.39、SEQ ID NO.40、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44、SEQ IDNO.45、SEQ ID NO.46、SEQ ID NO.47、SEQ ID NO.48、SEQ ID NO.49、SEQ ID NO.50、SEQ IDNO.51、SEQ ID NO.52、SEQ ID NO.53、SEQ ID NO.54、SEQ ID NO.55、SEQ ID NO.56、SEQ IDNO.57、SEQ ID NO.58、SEQ ID NO.59、SEQ ID NO.60、SEQ ID NO.61、SEQ ID NO.62、SEQ IDNO.63、SEQ ID NO.64、SEQ ID NO.65、SEQ ID NO.66、SEQ ID NO.67、SEQ ID NO.68、SEQ IDNO.69、SEQ ID NO.70、SEQ ID NO.71、SEQ ID NO.72、SEQ ID NO.73、SEQ ID NO.74、SEQ IDNO.75、SEQ ID NO.76、SEQ ID NO.77、SEQ ID NO.78、SEQ ID NO.79、SEQ ID NO.80、SEQ IDNO.81、SEQ ID NO.82、SEQ ID NO.83、SEQ ID NO.84、SEQ ID NO.85、SEQ ID NO.86、SEQ IDNO.87、SEQ ID NO.88、SEQ ID NO.89、SEQ ID NO.90、SEQ ID NO.91、SEQ ID NO.92、SEQ IDNO.93、SEQ ID NO.94、SEQ ID NO.95、SEQ ID NO.96、SEQ ID NO.97、SEQ ID NO.98、SEQ IDNO.99、SEQ ID NO.100、SEQ ID NO.101、SEQ ID NO.102、SEQ ID NO.103、SEQ ID NO.104、SEQ ID NO.105、SEQ ID NO.106、SEQ ID NO.107、SEQ ID NO.108、SEQ ID NO.109、SEQ IDNO.110、SEQ ID NO.111、SEQ ID NO.112、SEQ ID NO.113、SEQ ID NO.114、SEQ ID NO.115、SEQ ID NO.116、SEQ ID NO.117、SEQ ID NO.118、SEQ ID NO.119、SEQ ID NO.120、SEQ IDNO.121、SEQ ID NO.122、SEQ ID NO.123、SEQID NO.124、SEQ ID NO.125、SEQ ID NO.126、SEQ ID NO.127、SEQ ID NO.128、SEQ ID NO.129、SEQ ID NO.130、SEQ ID NO.131、SEQ IDNO.132、SEQ ID NO.133、SEQ ID NO.134、SEQ ID NO.135、SEQ ID NO.136、SEQ ID NO.137、SEQ ID NO.138、SEQ ID NO.139、SEQ ID NO.140、SEQ ID NO.141、SEQ ID NO.142、SEQ IDNO.143、SEQ ID NO.144、SEQ ID NO.145、SEQ ID NO.146、SEQ ID NO.147、SEQ ID NO.148、SEQ ID NO.149、SEQ ID NO.150、SEQ ID NO.151、SEQ ID NO.152、SEQ ID NO.153、SEQ IDNO.154、SEQ ID NO.155、SEQ ID NO.156、SEQ ID NO.157、SEQ ID NO.158、SEQ ID NO.159、SEQ ID NO.160、SEQ ID NO.161、SEQ ID NO.162、SEQ ID NO.163、SEQ ID NO.164、SEQ IDNO.165、SEQ ID NO.166、SEQ ID NO.167、SEQ ID NO.168、SEQ ID NO.169、SEQ ID NO.170、SEQ ID NO.171、SEQ ID NO.172、SEQ ID NO.173、SEQ ID NO.174、SEQ ID NO.175、SEQ IDNO.176、SEQ ID NO.177、SEQ ID NO.178、SEQ ID NO.179、SEQ ID NO.180、SEQ ID NO.181、SEQ ID NO.182、SEQ ID NO.183、SEQ ID NO.184、SEQ ID NO.185、SEQ ID NO.186、SEQ IDNO.187、SEQ ID NO.188、SEQ ID NO.189、SEQ ID NO.190、SEQ ID NO.191、SEQ ID NO.192、SEQ ID NO.193、SEQ ID NO.194、SEQ ID NO.195、SEQ ID NO.196、SEQ ID NO.197、SEQ IDNO.198、SEQ ID NO.199、SEQ ID NO.200、SEQ ID NO.201、SEQ ID NO.202、SEQ ID NO.203、SEQ ID NO.204、SEQ ID NO.205、SEQ ID NO.206、SEQ ID NO.207、SEQ ID NO.208、SEQ IDNO.209、SEQ ID NO.210、SEQ ID NO.211、SEQ ID NO.212、SEQ ID NO.213、SEQ ID NO.214、SEQ ID NO.215、SEQ ID NO.216、SEQ ID NO.217、SEQ ID NO.218、SEQ ID NO.219、SEQ IDNO.220、SEQ ID NO.221、SEQ ID NO.222、SEQ ID NO.223、SEQ ID NO.224、SEQ ID NO.225、SEQ ID NO.226、SEQ ID NO.227、SEQ ID NO.228、SEQ ID NO.229、SEQ ID NO.230、SEQ IDNO.231、SEQ ID NO.232、SEQ ID NO.233、SEQ ID NO.234、SEQ ID NO.235、SEQ ID NO.236、SEQ ID NO.237、SEQ ID NO.238、SEQ ID NO.239、SEQ ID NO.240、SEQ ID NO.241、SEQ IDNO.242、SEQ ID NO.243、SEQ ID NO.244、SEQ ID NO.245、SEQ ID NO.246、SEQ ID NO.247、SEQ ID NO.248、SEQ ID NO.249、SEQ ID NO.250、SEQ ID NO.251、SEQ ID NO.252、SEQ IDNO.253、SEQ ID NO.254、SEQ ID NO.255、SEQ ID NO.256、SEQ ID NO.257、SEQ ID NO.258、SEQ ID NO.259、SEQ ID NO.260、SEQ ID NO.261、SEQ ID NO.262、SEQ ID NO.263、SEQ IDNO.264、SEQ ID NO.265、SEQ ID NO.266、SEQ ID NO.267、SEQ ID NO.268、SEQ ID NO.269、SEQ ID NO.270、SEQ ID NO.271、SEQ ID NO.272、SEQ ID NO.273、SEQ ID NO.274、SEQ IDNO.275、SEQ ID NO.276、SEQ ID NO.277、SEQ ID NO.278、SEQ ID NO.279、SEQ ID NO.280、SEQ ID NO.281、SEQ ID NO.282、SEQ ID NO.283、SEQ ID NO.284、SEQ ID NO.285、SEQ IDNO.286、SEQ ID NO.287、SEQ ID NO.288、SEQ ID NO.289、SEQ ID NO.290、SEQ ID NO.291、SEQ ID NO.292、SEQ ID NO.293、SEQ ID NO.294、SEQ ID NO.295、SEQ ID NO.296、SEQ IDNO.297、SEQ ID NO.298、SEQ ID NO.299、SEQ ID NO.300、SEQ ID NO.301、SEQ ID NO.302、SEQ ID NO.303、SEQ ID NO.304、SEQ ID NO.305、SEQ ID NO.306、SEQ ID NO.307、SEQ IDNO.308、SEQ ID NO.309、SEQ ID NO.310、SEQ ID NO.311、SEQ ID NO.312、SEQ ID NO.313、SEQ ID NO.314、SEQ ID NO.315、SEQ ID NO.316、SEQ ID NO.317、SEQ ID NO.318、SEQ IDNO.319、SEQ ID NO.320、SEQ ID NO.321、SEQ ID NO.322、SEQ ID NO.323、SEQ ID NO.324、SEQ ID NO.325、SEQ ID NO.326、SEQ ID NO.327、SEQ ID NO.328、SEQ ID NO.329、SEQ IDNO.330、SEQ ID NO.331、SEQ ID NO.332、SEQ ID NO.333、SEQ ID NO.334、SEQ ID NO.335、SEQ ID NO.336、SEQ ID NO.337、SEQ ID NO.338、SEQ ID NO.339、SEQ ID NO.340、SEQ IDNO.341、SEQ ID NO.342、SEQ ID NO.343、SEQ ID NO.344、SEQ ID NO.345、SEQ ID NO.346和SEQ ID NO.347。
2.如权利要求1所述的Fc变体,其相对于野生型Fc蛋白以高亲和力与人FcRn结合。
3.如权利要求2所述的Fc变体,其在pH 6.0下对FcRn的结合亲和力与其在中性pH下的所述亲和力相比更高。
4.如权利要求2所述的Fc变体,其对FcRn具有10-8M或更小,更优选10-10M或更小的KD。
5.如权利要求2所述的Fc变体,其与来自除了人之外的物种的FcRn交叉反应。
6.如权利要求1所述的Fc变体,其存在于IgG1、IgG2、IgG3、IgG4或IgG2/G4同种型,优选IgG1同种型的Fc蛋白中。
7.如权利要求1所述的Fc变体,其以多聚体形式表达,以通过增加所述Fc变体的分子大小和通过更高亲合力的亲和力来增加半衰期。
8.如权利要求7所述的多聚体形式,其选自二聚体、三聚体、四聚体、五聚体和六聚体。
9.如权利要求1所述的Fc变体,其以全长抗体形式存在。
10.如权利要求1所述的Fc变体,相对于野生型IgGlFc区对Fcγ受体的亲和力,所述Fc变体对所述Fcγ受体的亲和力改变。
11.如权利要求10所述的Fc变体,相对于野生型IgGlFc区对包括同种异型V158和F158的FcγRIIIa的亲和力,所述Fc变体对FcγRIIIa(CD16a)的亲和力增加。
12.如权利要求11所述的Fc变体,其包括以下特征中的至少一个:
a)在对象中具有增加的半衰期;
b)相对于野生型Fc蛋白具有降低的/无ADCC活性;
c)抑制FcγR介导的吞噬作用,以及
d)抑制FcγR介导的细胞因子释放。
13.如权利要求1所述的Fc变体,其与药物或治疗性肽或聚乙二醇或免疫原或中和抗体融合。
14.如权利要求1所述的Fc变体,其在EU位置297处包含非岩藻糖基化的N-连接的聚糖。
15.如权利要求1所述的Fc变体,其包含选自以下示出的序列的氨基酸序列:SEQ IDNO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.7、SEQ ID NO.9、SEQ ID NO.11、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.18、SEQ ID NO.20、SEQ IDNO.21、SEQ ID NO.22、SEQ ID NO.23、SEQ ID NO.24、SEQ ID NO.25、SEQ ID NO.26、SEQ IDNO.27、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31、SEQ ID NO.32、SEQ ID NO.34、SEQ IDNO.35、SEQ ID NO.36、SEQ ID NO.38、SEQ ID NO.39、SEQ ID NO.40、SEQ ID NO.41、SEQ IDNO.42、SEQ ID NO.43、SEQ ID NO.44、SEQ ID NO.45、SEQ ID NO.46、SEQ ID NO.47、SEQ IDNO.48和SEQ ID NO.49。
16.如权利要求1所述的Fc变体,其包含选自以下示出的序列的氨基酸序列:SEQ IDNO.3、SEQ ID NO.5、SEQ ID NO.10、SEQ ID NO.20或SEQ ID NO.22。
17.如权利要求16所述的Fc变体,其中SEQ ID NO.22包含取代T307N、V308P、L309Y、H433R和N434W。
18.如权利要求17所述的Fc变体,其中SEQ ID NO.22具有选自以下的氨基酸序列:SEQID NO.22a、SEQ ID NO.22b、SEQ ID NO.22c、SEQ ID NO.22d、SEQ ID NO.22e和SEQ IDNO.22f及SEQ ID NO.22g。
19.组合物,其包含前述权利要求的任一项所述的Fc变体和可接受的载体。
20.如前述权利要求中任一项所述的Fc变体,其用于制备用于治疗其中FcRn的活性是不利的疾病的药物,或用于增加药物的循环半衰期,或用于将所述药物靶向某些细胞或组织。
21.如权利要求20所述的Fc变体,其中疾病选自感染病、癌症和自身免疫疾病。
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