CN109071643B - 亲和力改造的血清蛋白载体结合结构域 - Google Patents
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Abstract
本公开内容涉及通过突变序列来调节特异于血清载体蛋白的结合结构域的半衰期的方法和经调节的特异于血清载体蛋白的结合结构域。
Description
本公开内容涉及通过突变序列来调节特异于血清载体蛋白的结合结构域的半衰期的方法和经调节的特异于血清载体蛋白的结合结构域。
背景
现已建立了通过靶向血清蛋白载体(例如人血清白蛋白(HSA)) 来增加生物药物的血清半衰期1,2。使用HSA是因为它的半衰期是19 天。它是血清中丰度最高的蛋白质(34-54g/L),并且广泛分布于组织中3。因此,它是对于结合易于获得且安全可靠的靶标,特别是因为占总量的一小部分的白蛋白被用于该方法。
在血清蛋白中,只有IgG具有相似的长半衰期(21天)。HSA 和IgG的长血清半衰期主要是由于新生儿Fc受体(FcRn)对细胞内溶酶体降解的保护作用4,5。FcRn将通过血管空间衬里的造血细胞和内皮细胞的血浆非特异性胞饮作用进入囊泡后HSA和IgG再循环回细胞表面。胞饮囊泡通过与早期内体融合而酸化,使得HSA和IgG 能够以pH依赖性方式结合FcRn。携带膜受体(包括FcRn)和结合的HSA和IgG的囊泡再循环回细胞表面,而剩余的未结合的物质被引导至溶酶体进行降解。HSA和IgG在中性pH下与FcRn结合较弱,因此当再循环的囊泡暴露于血液的中性pH时,它们会释放回循环系统中2。
可以通过两种方式之一开发HSA。一种方法是将治疗性蛋白质 (遗传上或化学上)直接偶联至HSA 6,7。第二种方法是使用白蛋白结合结构域。迄今使用的结合结构域的实例包括脂肪酸(肉豆蔻酸)8、有机分子(Albutag)9、合成肽10,11、细菌白蛋白结合结构域(AlbumodTm)12,13、单结构域抗体(NanobodyTm,AlbudAbTm)14-17和Fab18。
Nguyen等人,2006研究了与C末端白蛋白结合肽连接的Fab片段的半衰期。Nguyen得出以下结论:降低的对白蛋白的亲和力与降低的半衰期和更高的清除率相关。其中的图3表明该关系几乎是线性的。该文献还接着说,体内未结合的抗体级分的非常小的差异将对清除率产生深远的影响。
本发明人已经研究了包含特异于血清蛋白载体的VH和VL的结合结构域的亲和力与其体内半衰期之间的相关性。本发明人已经确定结合结构域的半衰期持续时间比白蛋白结合肽的响应更复杂,因为观察到的亲和力的大幅度降低通常转化为半衰期的适度减少,并且在一些情况下降低的亲和力能够导致半衰期延长,这是与直觉相反的。
发明概述
因此,提供了包含特异于血清载体蛋白的VH和VL的结合结构域,其中所述结构域通过轻链可变结构域(VL)、重链可变结构域(VH) 及其组合中的修饰而突变,并且突变的结合结构域的半衰期高于或低于未突变的结合结构域的半衰期,例如,条件是该突变不是由以下组成的突变:SEQ ID NO:1的I50A,T56A,T95A,V96A,P97A, G98A,Y99A,S100A,T100Aa,Y100Ca,I50A和T95A,I50A和 G98A,I50A和Y99A,T56A和T95A,T56A和G98A,以及T56A 和Y99A。
因此,提供了包含特异于血清载体蛋白的VH和VL的结合结构域,其中所述结合结构域通过选自以下的修饰突变:轻链可变结构域 (VL)中的一个或两个氨基酸取代、重链可变结构域(VH)中的一个或两个突变及其组合,并且突变的结合结构域的半衰期高于或低于未突变的结合结构域的半衰期,条件是突变不是由以下组成的突变: SEQ ID NO:1的I50A,T56A,T95A,V96A,P97A,G98A,Y99A, S100A,T100Aa,Y100Ca,I50A和T95A,I50A和G98A,I50A和 Y99A,T56A和T95A,T56A和G98A,以及T56A和Y99A。
在一个实施方案中,血清载体蛋白选自,例如甲状腺素结合蛋白、甲状腺素视黄质运载蛋白、α1-酸性糖蛋白、转铁蛋白、血纤蛋白原和白蛋白或其任何片段,例如白蛋白,特别是人血清白蛋白。
在一个实施方案中,结合结构域对白蛋白的结构域II具有特异性。
在一个实施方案中,突变是VL中的修饰,例如其中突变是VL 中的一个或两个氨基酸的取代,例如选自L1、L2、L3及其组合的CDR 中的修饰/取代,特别是其中CDR是L1。
在一个实施方案中,CDR L1中的突变氨基酸独立地选自位置26、 27、28、29、30、31、32、33、34和35,例如位置30。
在一个实施方案中,VL中相关位置的氨基酸被疏水残基取代,例如疏水残基选自丙氨酸、异亮氨酸、苯丙氨酸、缬氨酸、脯氨酸和甘氨酸,例如丙氨酸。
在一个实施方案中,突变由对VL的修饰组成。令人惊讶的是,本发明人已经确定能够进行VL中的修饰,其增加结合结构域的Kd 并降低结合结构域的亲和力但增加分子的半衰期。
在一个实施方案中,突变位于VH结构域中,例如突变是VH中的一个或两个氨基酸的取代,例如选自H1、H2、H3及其组合的CDR 中的突变,特别是其中CDR是H2和/或H3,更特别地其中CDR是 H2。
在一个实施方案中,CDR H2中的突变氨基酸独立地选自位置50、 51、52、53、54、55、56、57、58、59、60、61、62、63、64、65及其组合,例如,它们被疏水残基取代,疏水残基特别是独立地选自丙氨酸、异亮氨酸、苯丙氨酸、缬氨酸、脯氨酸和甘氨酸,例如丙氨酸。
在一个实施方案中,突变的氨基酸在CDR H3中,并且特别地,突变的氨基酸独立地选自位置95、96、97、98、99、100、101、102、 103、104、105、106或107,更特别地,残基101是经突变的。
在一个实施方案中,CDR H3中相关位置的氨基酸被疏水残基取代,例如独立地选自丙氨酸、异亮氨酸、苯丙氨酸、缬氨酸、脯氨酸和甘氨酸,例如丙氨酸。
在一个或多个实施方案中,一个或多个氨基酸取代是非保守氨基酸取代,例如其中非保守氨基酸选自天然氨基酸丙氨酸、缬氨酸、异亮氨酸、亮氨酸、甲硫氨酸、苯丙氨酸、酪氨酸、色氨酸、苏氨酸、天冬酰胺、谷氨酰胺、甘氨酸、脯氨酸、精氨酸、赖氨酸、天冬氨酸和谷氨酸。
在一个实施方案中,结合结构域包含CDR移植的可变结构域。
在一个实施方案中,结合结构域是人源化的,例如结合结构域在 VH和/或VL中包含人框架。
在一个实施方案中,VH框架是人(例如VH3,例如VH3 1-3 3-23) 并且包含1、2、3、4、5或6个氨基酸取代,例如其为供体残基的氨基酸。
在一个实施方案中,VH包含选自SEQ ID NO:2、3、4和5或其任一个的具有至少95、96、97、98或99%相似性或同一性的变体的序列,例如SEQ ID NO:2、3、4、5或6(特别是5或6)中所示的序列。
在一个实施方案中,VL框架是人的(例如Vκ1,例如2-1-(1)L5),例如包含1、2或3个氨基酸取代,例如其为供体残基的氨基酸。
在一个实施方案中,VL结构域包含选自SEQ ID NO:6、7、8 和9或其任一个的具有至少95、96、97、98或99%相似性或同一性的变体的序列。
在一个实施方案中,VH和VL序列选自组合SEQ ID NO:2和6、 2和7、2和8、2和9、3和6、3和7、3和8、3和9、4和6、4和7、 4和8、4和9、5和6、5和7、5和8以及5和9或其任一个的具有至少95、96、97、98或99%相似性或同一性的变体,特别是VL和 VH序列分别是SEQ ID NO:9和SEQ ID NO:3,或者VL和VH序列分别是SEQ ID NO:8和SEQ ID NO:4,或者VL和VH序列分别是SEQ ID NO:9和SEQ ID NO:5,或VL和VH序列是SEQ ID NO:9和SEQ ID NO:4。
在一个实施方案中,结合结构域是人的。
在一个实施方案中,结合配偶体的亲和力高,为5nM或更强,例如900、800、700、600、500、400、300、200、100、50或10pM或更强。
在一个实施方案中,提供了包含根据本发明的结合结构域的抗体分子,特别是多特异性抗体分子,例如双特异性抗体分子。
在一个实施方案中,提供了药物组合物,其包含根据本发明的结合结构域或本文所述的抗体分子。
在另一方面,提供了治疗患者的方法,包括施用治疗有效量的根据本发明的结合结构域、本文所述的抗体分子或包含其任一种的药物组合物。
还提供了根据本发明的结合结构域、本文所述的抗体分子或包含其任一种的药物组合物,其用于治疗。
在一个实施方案中,提供了根据本发明的结合结构域、本文所述的抗体分子或包含其任一种的药物组合物,其用于治疗,特别是用于选自以下的治疗:感染(病毒、细菌、真菌和寄生虫感染),与感染有关的内毒素休克,关节炎如类风湿性关节炎,哮喘如严重哮喘,慢性阻塞性肺病(COPD),盆腔炎性疾病,阿尔茨海默病,炎症性肠病,克罗恩病,溃疡性结肠炎,阴茎纤维性海绵体炎,乳糜泻,胆囊疾病,藏毛病,腹膜炎,银屑病,血管炎,手术粘连,中风,I型糖尿病,莱姆病,脑膜脑炎,自身免疫性葡萄膜炎,免疫介导的中枢和外周神经系统炎症性疾病,如多发性硬化症,狼疮(如系统性红斑狼疮)和吉兰-巴雷综合征,特应性皮炎,自身免疫肝炎,纤维性肺泡炎,格雷夫斯病,IgA肾病,特发性血小板减少性紫癜,梅尼埃病,天疱疮,原发性胆汁性肝硬化,结节病,硬皮病,韦格纳肉芽肿病,其他自身免疫性疾病,胰腺炎,外伤(手术),移植物抗宿主病,移植排斥反应,心脏病包括缺血性疾病例如心肌梗塞以及动脉粥样硬化,血管内凝血,骨吸收,骨质疏松症,骨关节炎,牙周炎,胃酸过少和癌症,包括乳腺癌,肺癌,胃癌,卵巢癌,肝细胞癌,结肠癌,胰腺癌,食道癌,头颈癌,肾癌,特别是肾细胞癌,前列腺癌,肝癌,黑色素瘤,肉瘤,骨髓瘤,神经母细胞瘤,胎盘绒毛膜癌,宫颈癌和甲状腺癌,以及其转移性形式。
根据本公开内容的结合结构域、本文所述的抗体分子或包含其的药物组合物在制备药物中的用途,所述药物例如用于选自以下的治疗:感染(病毒、细菌、真菌和寄生虫感染),与感染有关的内毒素休克,关节炎如类风湿性关节炎,哮喘如严重哮喘,慢性阻塞性肺病 (COPD),盆腔炎性疾病,阿尔茨海默病,炎症性肠病,克罗恩病,溃疡性结肠炎,阴茎纤维性海绵体炎,乳糜泻,胆囊疾病,藏毛病,腹膜炎,银屑病,血管炎,手术粘连,中风,I型糖尿病,莱姆病,脑膜脑炎,自身免疫性葡萄膜炎,免疫介导的中枢和外周神经系统炎症性疾病,如多发性硬化症,狼疮(如系统性红斑狼疮)和格林-巴尔综合征,特应性皮炎,自身免疫肝炎,纤维性肺泡炎,格雷夫斯病, IgA肾病,特发性血小板减少性紫癜,梅尼埃病,天疱疮,原发性胆汁性肝硬化,结节病,硬皮病,韦格纳肉芽肿病,其他自身免疫性疾病,胰腺炎,外伤(手术),移植物抗宿主病,移植排斥反应,心脏病包括缺血性疾病例如心肌梗塞以及动脉粥样硬化,血管内凝血,骨吸收,骨质疏松症,骨关节炎,牙周炎,胃酸过少和癌症,包括乳腺癌,肺癌,胃癌,卵巢癌,肝细胞癌,结肠癌,胰腺癌,食道癌,头颈癌,肾癌,特别是肾细胞癌,前列腺癌,肝癌,黑色素瘤,肉瘤,骨髓瘤,神经母细胞瘤,胎盘绒毛膜癌,宫颈癌和甲状腺癌,以及其转移性形式。
在一个独立的方面,提供了选择血清蛋白载体结合结构域以提供定制半衰期的方法,包括以下步骤:提供一组特异于所述血清蛋白载体的VH/VL对,和
分析其体内半衰期,和
选择最紧密匹配人受试者中包含结构域的生物分子所需的半衰期的结构域。
在一个实施方案中,血清载体蛋白选自甲状腺素结合蛋白、甲状腺素视黄质运载蛋白、α1-酸性糖蛋白、转铁蛋白、血纤蛋白原和白蛋白或其任何片段,例如白蛋白,例如人血清白蛋白。
在一个方面,通过突变亲本抗体中的可变结构域来制备VH/VL 对的所述组。
在一个实施方案中,突变是对VL的至少一个修饰,例如其中VL 修饰选自轻链可变结构域(VL)中的一个或两个氨基酸取代,并且突变的结合结构域的半衰期高于或低于未突变的结合结构域的半衰期。
在一个实施方案中,VL中的突变是CDR L1、CDRL2和/或 CDRL3、特别是CDR L2或CDR L3中的突变。
在一个实施方案中,结合结构域中的突变由对VL结构域的一个修饰或多个修饰组成。
在一个实施方案中,突变是对VH的至少一个修饰。
在一个实施方案中,突变是重链可变结构域(VH)中的一个或两个突变,其中突变结合结构域的半衰期高于或低于未突变的结合结构域(在本文中也称为亲本抗体)的半衰期。
在一个实施方案中,突变位于CDR H1、CDRH2和/或CDRH3 中,特别是CDR H2或CDRH3中。
在一个实施方案中,根据本发明的方法还包括用替代性氨基酸残基替换VH和/或VL(来自所述组或针对所述组)中的组氨酸残基的步骤。
在一个实施方案中,该方法还包括在两个或更多个生物学相关的 pH(例如约pH5和pH7)下评估一种或多种结合结构域的性质的步骤。
在一个实施方案中,突变增加Kd的数值。
在一个实施方案中,提供了一种增加亲和力的方法。
在一个实施方案中,提供了一种降低亲和力的方法。
在一个实施方案中,使用具有抗体(例如亲本抗体)的血清载体蛋白的晶体结构来决定修饰/突变哪些残基。
在一个独立方面,本公开内容提供了以下段落中描述的方法:
1.一种选择血清蛋白质载体结合结构域以提供定制半衰期的方法,包括以下步骤:提供一组特异于所述血清蛋白质载体的VH/VL 对,和
分析其体内半衰期,和
选择最紧密匹配人受试者中包含结构域的生物分子所需的半衰期的结构域。
2.根据段落1的方法,其中血清载体蛋白选自甲状腺素结合蛋白、甲状腺素视黄质运载蛋白、α1-酸性糖蛋白、转铁蛋白、血纤蛋白原和白蛋白或其任何片段。
3.根据段落2的方法,其中血清蛋白质载体是白蛋白。
4.根据段落3的方法,其中白蛋白是人血清白蛋白。
5.根据段落1至4中任一段的方法,其中通过突变亲本抗体中的可变结构域来制备VH/VL对的所述组。
6.根据段落5的方法,其中所述突变是对VL的至少一个修饰。
7.根据段落6的方法,其中VL修饰选自轻链可变结构域(VL) 中的一个或两个氨基酸取代,并且突变的结合结构域的半衰期高于或低于未突变的结合结构域的半衰期。
8.根据段落5至7中任一段的方法,其中突变在CDR L1、CDRL2 和/或CDRL3中,特别是CDR L2或CDR L3中。
9.根据段落5至8中任一段的方法,其中所述突变由对VL结构域的一个修饰或多个修饰组成。
10.根据段落1至8中任一段的方法,其中所述突变是对VH的至少一个修饰。
11.根据段落9的方法,其中突变是重链可变结构域(VH)中的一个或两个突变及其组合,并且突变的结合结构域的半衰期高于或低于未突变的结合结构域(在本文中也称为亲本抗体)的半衰期。
12.根据段落10或11的方法,其中突变位于CDR H1、CDRH2 和/或CDRH3中,特别是CDR H2或CDR H3中。
13.根据段落1至12中任一段的方法,其进一步包括用替代性氨基酸残基取代所述组中使用的VH和/或VL中的组氨酸残基的步骤。
14.根据段落1-13中任一段的方法,其进一步包括在两个或更多个生物学相关pH,例如约pH5和pH7下评估一种或多种结合结构域的性质的步骤。
15.根据段落1至14中任一段的方法,其中突变增加Kd的数值。
16.根据段落1-15中任一段的方法,其中亲和力增加。
17.根据段落1-15中任一段的方法,其中亲和力降低。
发明详述
令人惊讶的是,本发明人已经发现VH和VL的序列能够被突变,并且相应的半衰期不一定对应于结合结构域的亲和力。特别地,能够对VL进行修饰,其中保持或降低亲和力并且增加半衰期。
这允许为分子设计和控制半衰期以提供与治疗适应症相关的半衰期。在一些实施方案中,可能需要长的半衰期。在一些实施方案中,适度/中等的半衰期可能是合适的。在其他实施方案中,相对短的半衰期可能是合适的。
本文所用的定制半衰期是这样的半衰期,其已通过修饰结合结构域而专门为该结合结构域进行了设计。
如本文所用的“结合结构域或位点”是与抗原接触的抗体部分。在一个实施方案中,结合结构域含有至少一个可变结构域或其衍生物,例如一对可变结构域或其衍生物,例如可变结构域的同源对或其衍生物。
在一个实施方案中,结合结构域包含6个CDR和框架,并且这些元件一起促成抗体或结合片段的结合相互作用的特异性。
可变区(在本文中也称为可变结构域)通常包含3个CDR和合适的框架。
如本文所用的术语“抗体”是指能够通过位于免疫球蛋白分子的可变区中的至少一个抗原识别位点(本文中也称为结合位点)特异性结合靶抗原(例如碳水化合物、多核苷酸、脂质、多肽、肽等)的免疫球蛋白分子。
如本文所用,“抗体分子”包括抗体及其结合片段。该术语还扩展至包含任何一种抗体分子的抗体形式。
如本文所用的亲本抗体是指进行突变以改变半衰期之前的起始抗体。亲本抗体可以是人源化的(其可以包括掺入含有所谓的供体残基的回复突变)或经突变的,例如以从CDR中除去赖氨酸残基或类似的。然而,亲本抗体中存在的修饰不是为了改变/修饰半衰期。如本文所用的亲本抗体包括抗体结合片段。
如本文所用的“抗体片段”是指抗体结合片段,包括但不限于Fab,修饰的Fab,Fab',修饰的Fab',F(ab’)2,Fv,单结构域抗体,scFv,二、三或四价抗体,Bis-scFv,双抗体,三抗体,四抗体和任何上述的表位结合片段(参见例如Holliger and Hudson,2005,NatureBiotech. 23(9):1126-1136;Adair and Lawson,2005,Drug Design Reviews-Online 2(3),209-217)。用于产生和制造这些抗体片段的方法是本领域熟知的(参见例如Verma等,1998,Journal of Immunological Methods,216:165-181)。用于本公开内容的其他抗体片段包括国际专利申请WO05/003169、WO05/003170和WO05/003171中描述的Fab 和Fab'片段。多价抗体可包含多种特异性,例如双特异性的或可以是单特异性的(参见例如WO92/22853,WO05/113605,WO2009/040562, WO2010/035012,WO2015/197772)。
如本文所用的“结合片段”是指能够以足以表征该片段为特异于靶肽或抗原的亲和力结合该靶肽或抗原的片段。
本文所用的特异性(或特异于)是指相互作用中的配偶体仅彼此识别或与非配偶体相比对于彼此具有显著更高的亲和力,例如至少2、 3、4、5、6、7、8、9倍更高的亲和力,例如相较于结合的背景水平。
本文使用的配偶体是指抗原和抗体结合或配体和受体类型结合关系。
本文使用的至少一个修饰是指氨基酸的取代、添加或缺失,例如以改变序列的性质,例如改变疏水性或类似的。
抗体可变结构域中的残基通常根据Kabat等人,1987设计的系统编号。该系统在Kabat等人,1987,Sequences of Proteins of Immunological Interest,US Departmentof Health and Human Services,NIH,USA(以下称“Kabat等人(同上)”)中提出。除非另有说明,否则该编号系统用于本说明书中。
Kabat残基名称并不总是直接对应于氨基酸残基的线性编号。实际的线性氨基酸序列可含有比严格Kabat编号中更少或更多的氨基酸,其对应于基本可变结构域结构的结构组分(框架或互补决定区 (CDR))的缩短或在其中的插入。通过将抗体序列中的同源残基与“标准”Kabat编号序列比对,可以确定给定抗体的残基的正确Kabat 编号。
根据Kabat编号系统,重链可变结构域的CDR位于残基31-35 (CDR-H1)、残基50-65(CDR-H2)和残基95-102(CDR-H3)。然而,根据Chothia(Chothia,C.and Lesk,A.M.,J.Mol.Biol.,196, 901-917(1987)),相当于CDR-H1的环从残基26延伸至残基32。因此,除非另外指出,否则本文所用的“CDR-H1”意指残基26至35,如 Kabat编号系统和Chothia拓扑环定义的组合所述的。
如本文所用的术语“Fab片段”是指包含含有VL(可变轻)结构域和轻链恒定结构域(CL)的轻链片段和重链的VH(可变重)结构域和第一恒定结构域(CH1)的抗体片段。
如本文所用的Fab'片段是指进一步包含铰链区的Fab片段。
如本文所用,术语“单链Fv”或缩写为“scFv”是指包含连接(例如通过肽接头)以形成单个多肽链的VH和VL抗体结构域的抗体片段。在这种形式中省略了重链和轻链的恒定区。本文使用的单链Fv包括其二硫键稳定化形式,其中除肽接头外,在可变区之间存在二硫键。
二硫键稳定的scFv可以消除一些可变区域动态呼吸的倾向,这涉及可变区域分离和再次聚集在一起。
如本文所用的术语“单结构域抗体”是指由单个单体可变抗体结构域组成的抗体片段。单结构域抗体的实例包括VH或VL或VHH。
可以考虑所提出的抗体分子的功能,特别是可能需要的效应子功能而选择恒定区结构域(如果存在)。例如,恒定区结构域可以是人 IgA,IgD,IgE,IgG或IgM结构域。特别地,当抗体分子用于治疗用途并且需要抗体效应子功能时,可以使用人IgG恒定区结构域,尤其是IgG1和IgG3同种型。或者,当抗体分子用于治疗目的并且不需要抗体效应子功能时,可以使用IgG2和IgG4同种型。应当理解,也可以使用这些恒定区结构域的序列变体。例如,可以使用IgG4分子,其中241位的丝氨酸已经变为脯氨酸,如Angal等人,1993,MolecularImmunology,1993,30:105-108中所述。因此,在抗体是IgG4抗体的实施方案中,抗体可以包括突变S241P。
在一个实施方案中,抗体结合片段不包含Fc区。如本文所用,“不包含Fc区”是指较低的恒定结构域,例如CH2、CH3和CH4不存在。然而,可能存在恒定结构域诸如CH1,Cκ/Cλ。
本领域技术人员还将理解,抗体可经历多种翻译后修饰。这些修饰的类型和程度通常取决于用于表达抗体的宿主细胞系以及培养条件。此类修饰可包括糖基化、甲硫氨酸氧化、二酮哌嗪形成、天冬氨酸异构化和天冬酰胺脱酰胺的变化。常见的修饰是由于羧肽酶的作用而丧失羧基末端碱性残基(例如赖氨酸或精氨酸)(如Harris,RJ. Journal ofChromatography 705:129-134,1995中所述)。因此,可以不存在抗体重链的C末端赖氨酸。
在一个实施方案中,抗体重链包含CH1结构域,抗体轻链包含 CL结构域(κ或λ)。
在一个实施方案中,抗体重链包含CH1结构域、CH2结构域和 CH3结构域,并且抗体轻链包含CL结构域(κ或λ)。
如本文所用的“多特异性分子”是指具有特异性结合至少两种不同抗原(例如不同抗原)的能力的分子。在一个实施方案中,多特异性分子是双特异性、三特异性或四特异性分子,特别是双特异性分子。
合适的多特异性分子的实例是本领域已知的。
在一个实施方案中,多特异性形式包括本领域已知的那些和本文所述的那些,例如其中分子形式选自包括以下物质或由其组成的组:双抗体,scdiabody,triabody,三抗体,四抗体,串联scFv,FabFv, Fab'Fv,FabdsFv,Fab-scFv,diFab,diFab',串联scFv-Fc,scFv-Fc-scFv,scdiabody-Fc,scdiabody-CH3,Ig-scFv,scFv-Ig,V-Ig, Ig-V,Duobody和DVDIg,其将在下面更详细地讨论。
本文所用的分子在生物化学意义上用于指形成有机物质,特别是蛋白质物质的一组原子,其包括适合于在形成复合物后在适当条件下作为单一实体处理的复合物。
除非上下文另有说明,否则本文可互换使用分子和构建体。尽管构建体可以更频繁地用来指代多核苷酸分子,并且分子可以更频繁地用来指代主要包含氨基酸序列的实体。
可以由本公开内容的抗体分子中的结合结构域靶向的目标抗原也可以是任何医学相关的蛋白质,例如在疾病或感染期间上调的那些蛋白质,例如受体和/或其相应的配体。细胞表面蛋白的具体实例包括:粘附分子,整联蛋白如β1整联蛋白(例如VLA-4),E-选择蛋白,P 选择蛋白或L-选择蛋白,CD2,CD3,CD4,CD5,CD7,CD8,CD11a,CD11b,CD18,CD19,CD20,CD23,CD25,CD33,CD38,CD40, CDW52,CD69,CD134(OX40),ICOS,BCMP7,CD137,CD27L,CDCP1,DPCR1,DPCR1,dudulin2,FLJ20584,FLJ40787,HEK2, KIAA0634,KIAA0659,KIAA1246,KIAA1455,LTBP2,LTK, MAL2,MRP2,连接素样2,NKCC1,PTK7,RAIG1,TCAM1, SC6,BCMP101,BCMP84,BCMP11,DTD,癌胚抗原(CEA),人乳脂肪球蛋白(HMFG1和2),MHC I类和MHCII类抗原,和 VEGF,以及适当时其受体。
可溶性抗原包括白细胞介素,如IL-1,IL-2,IL-3,IL-4,IL-5, IL-6,IL-8,IL-10,IL-12,IL-13,IL-15,IL-16或IL-17,IL-21, IL-23,病毒抗原,例如呼吸道合胞病毒或巨细胞病毒抗原,免疫球蛋白,例如IgE,干扰素例如干扰素α、干扰素β或干扰素γ,肿瘤坏死因子-α,肿瘤坏死因子-β,集落刺激因子如G-CSF或GM-CSF,和血小板衍生的生长因子如PDGF-α和PDGF-β及适当时其受体。其他抗原包括细菌细胞表面抗原,细菌毒素,病毒例如流感,EBV,HepA、 B和C,生物恐怖主义药剂,放射性核素和重金属,以及蛇和蜘蛛毒液和毒素。
在本说明书的上下文中,“包含”应解释为“包括”。
包括某些元件的本发明的方面也旨在扩展到由相关元件组成或基本上由相关元件组成的替代实施方案。
在技术上适当的情况下,可以组合本公开内容的实施方案和描述。
本文描述的任何肯定实施方案或其组合可以是否定排除的基础,即否认声明。
实施例
图1.抗体CA645的人源化和亲和力降低。抗体CA645的重链和轻链序列与人种系受体框架序列VH3 1-3 3-23/JH4和Vκ1 2-1-(1) L5/Jκ4比对。兔残基为红色,人残基为黑色,CDR为蓝色(显示J 区CDR残基,但不显示受体V区CDR)。移植的VH(gH)和VL (gL)序列显示在它们相应的人受体种系框架下面。兔和人框架序列之间的框架序列差异用星号表示。保留在人源化移植物中的兔框架残基以粗体突出显示。
图2.在存在或不存在CA645 gL4gH5 Fab的情况下FcRn与HSA 和MSA的结合。在不存在Fab的情况下与HSA结合(红色圆圈),在Fab存在下与HSA结合(红色三角形),在不存在Fab的情况下与MSA结合(蓝色方块),在Fab存在下与MSA结合(蓝色三角形)。
图3.(A)与HSA复合的CA645 gL4gH5 Fab的晶体结构,(B) CA645 Fab-HSA与FcRn-HSA复合物(PDB代码4N0F)的晶体结构的的叠加。FcRn由以绿色显示的重链和以橙色显示的常见的β2-微球蛋白(β2M)组成,(C)CA645 Fab-HSA与如下物质的晶体结构的叠加:与以红色显示的肉豆蔻酸(PDB代码1BJ5)、以蓝色显示的布洛芬(PDB代码2BXG)和以洋红色显示的华法林(PDB代码2BXD) 复合的HSA。白蛋白中的七个脂肪酸(FA)结合位点也被标记。
图4.CA645-HSA与RbSA的叠加。位置(A)364、(B)320 和(C)358处白蛋白残基周围区域的特写视图。CA645重链显示为蓝色;CA645轻链显示银色;HSA以小麦色显示;RbSA以粉红色显示。冲突定义为来自不同残基的两个重原子彼此在以内并且用黑色圆圈表示。
图5.药代动力学。将CA645 Fab移植物以10mg/kg静脉内注射到小鼠中,并通过ELISA在不同时间点测定Fab的血清浓度。考虑第一时间点的最大浓度,将数据归一化。
图6.血液中游离CA645 Fab的百分比相对于MSA的亲和力。使用质量作用二次方程的解计算KD范围为1-106nM(蓝色菱形)的游离Fab%45。对于移植物gL4gH5、gL5gH5、gL4gH37和gL5gH47(针对MSA的亲和力分别为2.2、316、1146和62400nM)的游离Fab%显示为红色方块。
图7.本公开内容的序列
表1.抗人血清白蛋白(HSA)抗体的活性谱。就在存在或不存在 25μM白蛋白结合化合物(华法林、布洛芬、肉豆蔻酸和氯化铜)的情况下结合100ng/ml HSA和就结合100ng/ml大鼠血清白蛋白(RSA) 对B细胞上清液中分泌的抗HSA抗体进行荧光微量体积测定技术(FMAT)筛选。FL=荧光强度。通过表面等离子体共振(SPR)测定对于人和小鼠血清白蛋白(MSA)的抗HSA兔Fab片段的平衡结合常数(KD),以及对于HSA、MSA和RSA的等效人源化IgG抗体的平衡结合常数(KD)。
表2.CA645 gL4gH5 Fab对来自不同物种的血清白蛋白的亲和力。通过SPR测定的缔合(ka)和解离(kd)速率常数和平衡结合常数(KD)。
表3.X射线数据收集和精化统计。括号中的值是针对最高分辨率层的。
表4.CA645Fab移植物的结合动力学和药代动力学。通过SPR测定的缔合(ka)和解离(kd)速率常数和平衡结合常数(KD)。对3 只小鼠(M1-3)/组用每种CA645移植物以10mg/kg静脉内施用。显示了每组的平均值和标准偏差(SD)。*通过稳态测量。
表5显示了对各种移植物的亲和力。
表6.在pH5.0-7.0的范围内CA645 gL4gH5 Fab对HSA的亲和力
表7.(A)(B)(C)CA645Fab移植物的结合动力学。通过SPR 测定的缔合(ka)和解离(kd)速率常数和平衡结合常数(KD)。
抗体发现
用200μg HSA(Jackson ImmunoResearch)皮下免疫两只Half Lop兔。完全弗氏佐剂(Sigma Aldrich)与第一剂量共同施用,随后的剂量包括不完全弗氏佐剂。从兔血清中收获B细胞并培养7天以诱导克隆扩增和抗体分泌。使用荧光微量体积测定技术(FMAT)就与HSA的结合筛选上清液20-22。将上清液与包被有生物素化的山羊抗兔Fc和Alexa Fluor647Chrompure人白蛋白(Jackson ImmunoResearch)的链霉抗生物素蛋白珠(BangsLaboratories,Inc) 混合。在Applied Biosystems 8200细胞检测系统上读板。将具有最高荧光强度(FL)信号的48个孔转移到单个主板上,并如前所述重复筛选,但是进行另外两个筛选。在一个筛选中,将Alexa Fluor 647 Chrompure人白蛋白与25μM白蛋白结合剂(华法林、布洛芬、肉豆蔻酸和氯化铜(均来自Sigma Aldrich))溶液预孵育1小时。在第二个筛选中,用使用Alexa Fluor单克隆抗体标记试剂盒(Molecular Probes)标记的大鼠血清白蛋白(Sigma Aldrich)代替HSA。
通过荧光焦点法分离单个HSA特异性B细胞20-22。将来自阳性孔的B细胞与包被有生物素化-HSA(Jackson ImmunoResearch)和山羊抗兔Fc片段荧光素异硫氰酸盐缀合物(Chemicon)的链霉抗生物素蛋白珠(Bangs Laboratories,Inc)混合。在37℃孵育1小时后,由于B细胞周围存在荧光晕,可以鉴定抗原特异性B细胞。使用 Olympus IX70显微镜和Eppendorf显微操作器鉴定单个B细胞并将其转移至PCR管。通过RT-PCR扩增单细胞的重链和轻链免疫球蛋白可变(V)区基因,并分别克隆到含有兔重CH1和兔轻Cκ区的UCB 哺乳动物表达载体中。在HEK293细胞中瞬时表达后,在针对HSA 和MSA的SPR结合测定中进一步筛选抗HSA重组Fab。
人源化
通过计算机模拟将抗体V区的CDR移植到Vκ1和VH3人种系抗体V区框架上来白蛋白特异性抗体人源化。从供体移植到受体序列的 CDR如Kabat等人所定义32,除了CDR-H1(残基26-35)之外,其中使用了Kabat等人和环结构的组合定义23。当供体兔序列和受体人序列之间的框架残基在被认为对保留抗原结合重要的位置上不同时,则供体残基被包括在最初的保守移植物中21。保守的移植基因是由 Entelechon,GmbH化学合成的。将重链移植基因(gH1)克隆到两个UCB表达载体中,一个含有人γ1CH1结构域,另一个含有完整的人γ1恒定区。将轻链移植基因(gL1)克隆到含有人κ恒定区(Km3 同种异型)的UCB表达中。随后通过寡核苷酸定向诱变修饰这些构建体以产生重链和轻链移植物的许多不同变体。将重链和轻链载体共转染到HEK293细胞中,并使用SPR结合测定法筛选重组Fab或IgG 分子以测量对HSA、MSA、RSA、CSA、RbSA和BSA的亲和力。
抗体表达
使用293Fectin脂质转染(Life Technologies,目录号12347-019,根据制造商的说明书)在HEK-293细胞中或使用电穿孔在CHO-S XE 细胞(CHO-K1衍生细胞系33)中瞬时表达抗体。HEK-293细胞用于小规模表达(<100ml)以制备用于SPR分析的抗体。CHO-S XE细胞用于大规模表达(1升)以制备用于结晶学和体内药代动力学研究的抗体。
蛋白质纯化
亲和层析用于从培养物上清液中纯化Fab蛋白。以使得柱接触时间为25分钟的流速使上清液通过HiTrap Protein G柱(GE Healthcare)。在使用pH7.4的PBS的洗涤步骤后,用pH2.7的0.1M 甘氨酸洗脱结合的物质,并用2m Tris-HCl(pH8.5)中和。合并含有 Fab的级分,通过280nm处的吸光度定量,并使用Amicon Ultra离心过滤器(Merck Millipore)浓缩。为了分离单体级分,使用在用pH7.4 的PBS平衡的HiLoad 16/60,Superdex 200柱(GEHealthcare)上的尺寸排阻色谱。合并含有单体Fab的级分,定量,浓缩并在4℃储存。
表面等离子体共振
通过使用CM5传感器芯片(GE Healthcare Bio-Sciences AB)和 HBS-EP(10mMHEPES,150mM NaCl,3mM EDTA,0.05%v/v P20, pH7.4)运行缓冲液在Biacore T200或Biacore 3000上进行的表面等离子体共振(SPR)测定抗体相互作用的结合亲和力和动力学参数。为了在pH 7.0、6.0、5.5和5.0下进行分析,使用40mM柠檬酸、80mM 磷酸钠50mM NaCl、3mM EDTA、0.05%v/v P20的运行缓冲液。通过改变柠檬酸与磷酸钠的比例来实现所需的pH。所有实验均在25℃进行。使用人F(ab’)2特异性或人Fc特异性山羊Fab(JacksonImmunoResearch)将抗体样品捕获至传感器芯片表面。通过标准胺偶联化学实现捕获抗体的共价固定,达到6000-7000响应单位(RU) 的水平。
人(Jackson ImmunoResearch,目录号009-000-051)、小鼠(Sigma Aldrich,目录号A3559)、大鼠(Sigma Aldrich,目录号A6414)、兔(Sigma Aldrich,目录号A0764)、牛(Sigma Aldrich,目录号 05470)和食蟹猴(Equitech-Bio,#CMSA-0050)白蛋白在捕获的抗体上以50nM至500μM的各种浓度滴定。每个测定循环包括首先使用 1分钟注射捕获抗体样品,然后进行由3分钟的白蛋白注射组成的缔合阶段,之后监测解离。在每个循环后,用40mMHCl的两次1分钟注射然后30秒的5mM NaOH再生捕获表面。使用的流速为10μl/min 用于捕获,30μl/min用于缔合和解离阶段,并且10μl/min用于再生。空白流动池用于参考扣除,并且包括缓冲液空白注射以扣除仪器噪声和漂移。通过使用Biacore T200Evaluation软件v2.0.1和 BIAEvaluation软件v4.1.1将所得传感图同时全局拟合至标准1:1结合模型来确定动力学参数,CA645gL5gH47除外,其在Prism中使用稳态亲和力模型拟合。
为了通过SPR测量CA645Fab对FcRn对白蛋白的结合效力的影响,将Biacore3000仪器与通过将HSA和MSA固定在分开的流动池上(分别达到270RU和247RU的水平)制备的CM5芯片一起使用。在运行缓冲液(100mM MES,150mM NaCl,0.05%v/v P20,pH 5.5) 中在50nM至50μM的范围内制备FcRn样品,并且它们还含有0或100nM CA645 Fab。每个测定循环以10μl/min的流速进行,并且包括 100nM CA645 Fab的5分钟注射以预饱和固定的白蛋白,然后在CA645 Fab的存在下进行上述制备的FcRn溶液之一的5分钟注射,或运行缓冲液的5分钟注射,然后在不存在CA645 Fab的情况下进行上述FcRn溶液之一的5分钟注射。在任一种情况下,在第二次注射结束后立即使用“共同注射”模式,进行分别包含缓冲液或100nM CA645Fab的第三次5分钟注射。包括使用用于参考扣除和空白循环 (其中用缓冲液代替FcRn)的空白流动池以扣除漂移和噪声。循环再生如上所述。将FcRn的空白校正的平台结合水平绘制在Prism中并拟合至稳态模型。
还使用固定化白蛋白芯片在Biacore3000上以反向形式研究了在 pH5.5下野生型和突变体CA645 Fab的结合动力学。在这种情况下,运行循环,其中注射5至5000nM范围的Fab溶液,使用5分钟的结合和解离阶段。也包括缓冲空白循环以校正漂移。
结晶学
为了制备复合物,将纯化的CA645Fab和不含脂肪酸的HSA (Sigma Aldrich,目录号A3782)以1:1的摩尔比混合,并在4℃孵育过夜。CA645Fab和复合物均通过尺寸排阻色谱法在用50mM NaCl、25mM Tris、5%(v/v)甘油平衡的HiLoad 16/60,Superdex 200柱(GEHealthcare)上纯化。合并含有CA645Fab或复合物的级分并分别浓缩至10mg/ml和70mg/ml。通过使用市售结晶筛(Qiagen) 的坐滴蒸气扩散法鉴定适合于晶体生长的条件。
为了产生衍射品质的晶体,使用悬滴蒸汽扩散法,其中将1μl蛋白质溶液与1μl储库溶液混合。对于CA645 Fab,储库含有500μl 2M DL-苹果酸。收获晶体并在液氮中快速冷冻,无需另外的冷冻保护剂。在Diamond Light Source,Oxford,UK的I04光束线上从单晶中收集针对的衍射数据,并使用MOSFLM和SCALA34-36处理。使用内部Fab结构的坐标作为搜索模型,通过使用相位器(Phaser)的分子置换来解析CA645Fab的结构37。对于复合物,储库含有pH 4.4 的500μl 0.1M柠檬酸、0.1M二磷酸氢钠、38%v/v乙醇和5%v/v聚乙二醇1000(PEG1000)。通过向液滴多次添加含有25%(v/v)PEG1000 的1μl储库缓冲液直至滴剂中PEG1000的浓度达到20%来将晶体冷冻保护。为了使晶体应力最小化,每次添加间隔至少1小时。收获晶体并在液氮中快速冷冻。在Diamond Light Source,Oxford,UK的I02 光束线上从单晶中收集针对/>的衍射数据,并使用XDS处理38。通过使用CA645Fab结构和HSA(PDB代码4G03)39的坐标作为搜索模型用相位器进行分子置换来解析复合物的结构。
使用CNS40,41和COOT42,通过模拟退火、能量最小化和手动重建的迭代循环来精细化两个初始结构。由于复合物的分辨率相当低,通过使用CNS的DEN功能在精细化期间约束模型。使用Molprobity 验证模型几何结构43。用Pymol生成分子可视化影像44。数据收集和精细化统计数据总结在表3中。
登录代码
CA645 Fab和CA645 Fab-HSA复合物的坐标和结构因子已经分别以登录代码X和Y保存在蛋白质数据库(PDB)中。
小鼠药代动力学
用抗体以10mg/kg静脉内给药于三只雄性BALB/c小鼠。在给药后最多100小时的几个时间点从尾静脉穿刺收集系列血液样品。为了获得血清,将血液样品在室温下以10,000rpm离心5分钟并通过 ELISA分析抗体浓度。抗Fab抗原抗体和抗人κ链-辣根过氧化物酶缀合物(Stratech)分别用作捕获抗体和二抗。纯化的Fab抗原样品用作标准品。使用TMB过氧化物酶溶液(Sigma-Aldrich)将板显色,并在450nm处读数(参考在630nm处)。使用Phoenix WinNonlin 6.2 软件从最终数据集计算药代动力学参数。
游离CA645 Fab的计算
为了确定在10mg/kg的剂量后在20g小鼠的2ml血液中未结合的 Fab(分子量=47907Da)的浓度,使用以下等式:31
游离Fab浓度x=(-b+SQRT(b2–4ac))/2a
其中:
b=(-(KA*[Tab])+1+(KA*[Tag]))
a=KA=1/KD
c=(-[Tab])
KD=Fab的亲和力
KA=缔合的平衡常数
[Tab]=Fab浓度(2087nM)
[Tag]=白蛋白浓度(600μM)
对于计算游离Fab的百分比:
游离Fab%=([游离Fab]/[Tab])*100
结果
针对跨物种的血清白蛋白的mAb的产生和表征
为了产生一组具有跨物种反应性的抗HSA抗体,用HSA免疫两只兔子。从血清中收获B细胞,培养,刺激以分泌IgG并使用荧光微量体积测定技术(FMAT)筛选以鉴定抗原特异性孔20-22。进一步的 FMAT筛选评估在已知的白蛋白结合化合物(华法林、布洛芬、肉豆蔻酸和氯化铜)存在或不存在时与RSA的结合和与HSA的结合。表 1中显示了五种排名靠前的抗体的数据。对于CA645,结合HSA的荧光强度信号最低。然而,CA645在化合物存在下确实保留了80%的结合活性,而CA646、CA647、CA648和CA649仅保留了40%。对于 CA645和CA646,与HSA和RSA的结合水平最紧密匹配,在5倍范围内。相反,CA647、CA648和CA649与RSA的结合水平比与HSA 的结合水平低9至18倍。
为了回收五种抗体的重链和轻链可变区,使用荧光焦点方法分离单个B细胞,然后进行RT-PCR。将可变区克隆到含有兔重CH1和轻链恒定区的表达载体中,然后对DNA进行测序。这表明抗体序列是独特的,除了具有相同重链序列的CA645和CA646以外。鉴于CA645 和CA646必须通过重链结合相同的表位,不清楚为什么CA646结合受配体存在的影响更大。还通过测序确定所有抗体的互补决定区 (CDR)缺乏组氨酸残基。这对于这些抗体的进一步进展是重要的,因为组氨酸残基在酸性pH下质子化并且这可能潜在地破坏抗原结合。
转染HEK293细胞后,通过表面等离振子共振(SPR)分析重组 Fab分子对HSA和小鼠血清白蛋白(MSA)的亲和力。CA645和CA646 对HSA的亲和力最强,分别为0.31nM和0.14nM,对MSA的亲和力分别为2.6nM和1.6nM(表1)。此外,它们对HSA和MSA的亲和力是所有抗体中最紧密匹配的。这与针对HSA和RSA的B细胞上清液筛选数据一致。
人源化和主要候选物的选择
通过将CDR移植到人Vκ1和VH3框架上以及反向突变在对于保留结合活性而言重要的位置上的框架残基来人源化所有五种抗体23。 CA645的人源化方案如图1所示。值得注意的是兔供体重链的框架三区域(残基66-94)短于人受体框架序列的相应区域。CA647和CA649 缩短了一个残基,而CA645和CA646(相同序列)和CA648缩短了两个残基。在所有情况下,在最初的保守性gL1gH1移植物中保留该缺口。
保守移植物表达为人IgG1抗体,并通过SPR分析其与HSA、 MSA和RSA的结合。如用重组亲本兔Fab所观察到的,人源化IgG 显示出与HSA和MSA结合的相同趋势(表1)。CA647、CA648和 CA649对HSA的亲和力与CA645和CA646的亲和力相似,但是通过比较它们显示出对MSA的亲和力降低6-10倍。与CA645和CA646 相比,CA648和CA649对RSA的亲和力也显示出5至10倍的降低。与CA645相比,CA646对HSA、MSA和RSA的亲和力稍强,但与 161mg/ml相比,35mg/ml的瞬时表达产率低4倍(表1)。基于在已知白蛋白结合剂存在下与HSA结合的近似最大保留、对于跨多种物种的白蛋白的一致结合活性和瞬时表达的良好产率,选择CA645作为进一步进展的主要候选物。通过用人受体残基取代兔供体残基并用相当的人残基填充重链框架3中的缺口,产生CA645 gL1gH1的进一步移植物变体。评估移植物变体对HSA的亲和力和瞬时表达产率(数据未显示)。选择的最终移植物配对是gL4和gH5(图1)。
CA645 gL4gH5 Fab对HSA、MSA、RSA和兔血清白蛋白(RbSA) 的亲和力分别显示为4.6、7.1、54和162nM(表2)。显著地,对于 CA645 gL4gH5 Fab在食蟹猴毒理学研究和疾病模型中的应用,对食蟹猴血清白蛋白(CSA)的亲和力为3.3nM,其与HSA的亲和力非常相似。此外,CA645 Fab不能与牛血清白蛋白结合。
为了确定CA645 gL4gH5 Fab是否可能在早期内体的酸性环境中保持与白蛋白结合并再循环到细胞表面,在pH 5.0-7.0下测量亲和力 (表S1)。在pH5.0、pH5.5、pH6.0和pH7.0下,CA645 gL4gH5 Fab 对HSA的亲和力为7.1、10.7、12.5和13.3nM,表明在该生理学相关的pH范围内结合基本上不受影响。
为了确定在CA645 gL4gH5 Fab存在下HSA是否可以与FcRn结合,使用SPR。在pH5.5下进行动力学测定以确保FcRn与白蛋白的最佳结合。将HSA或MSA直接结合到传感器芯片上,然后结合CA645 gL4gH5 Fab以使CA645白蛋白结合位点饱和,或将运行缓冲液注入流动池中。然后注射FcRn加CA645 Fab或单独的FcRn。CA645 Fab包含在与FcRn的共注射中以维持CA645结合位点饱和。图2显示扣除CA645的信号后FcRn与HSA和MSA结合的水平。FcRn与两种白蛋白的结合不受CA645 gL4gH5 Fab的存在的影响。
结晶学
为了鉴定CA645与HSA结合的位置,确定了CA645 Fab-HSA 复合物的晶体结构。将CA645 Fab-HSA复合蛋白制剂浓缩至70mg/ml24,并使用乙醇和PEG1000作为沉淀剂来结晶。为了帮助解析具有分子置换的复合物的结构,我们还确定了未结合的CA645 Fab 的结构。我们在它们各自晶体的不对称单元中观察到Fab和Fab-HSA 复合物二者的单拷贝(表3)。游离Fab的结构精细化至其中最终Rwork值为21.14%,并且Rfree值为25.13%。复合物的结构精细化至/>其中最终Rwork值为21.38%,Rfree值为25.23%。
CA645 Fab-HSA复合物的晶体结构显示CA645结合HSA的结构域II(图3A)。与HSA复合的FcRn的晶体结构(PDB代码4N0F)25的叠加显示CA645不阻断HSA与FcRn的结合(图3B)。HSA含有七个脂肪酸(FA)结合位点。位点FA7和FA3/FA4是两个主要的药物结合位点26。药物还在FA1、FA5和FA6位点结合但亲和力较弱。金属离子结合位点位于结构域I和II之间以及N末端的位点27。与华法林(PDB代码2BXD)28、布洛芬(PDB代码2BXG)28和肉豆蔻酸(PDB代码1BJ5)29复合的HSA的晶体结构与复合物的叠加显示 CA645接近FA6位点结合并且不阻塞主要药物(FA7和FA3/FA4)、脂肪酸或金属离子结合位点(图3C)。
CA645 gL4gH5 Fab与HSA的结合动力学与MSA、CSA、RSA 和RbSA(表2)的结合动力学的比较可以通过对晶体结构的仔细目视检查来解释。HSA上的表位由残基F206、G207、R209、C316、K317、 AEAKD 320-324、K351、E354、E358、K359、C361、A362和A364 形成。CA645对CSA(3.3nM)和MSA(7.1nM)的亲和力与对HSA 的亲和力(4.6nM)非常相似。这可能是由于在CSA和MSA中存在形成HSA中表位的相同残基。除了其为甘氨酸的位置364外,RSA 共有所有这些残基。位置364位于短环的尖端(位置362-365),其将两个α-螺旋(位置366-398和342-361)连接在一起(图4A)。该短环由CA645重链的CDR1和2结合。CA645对RSA的亲和力是对 HSA的亲和力的约十分之一。可能的是,与HSA相比,丙氨酸侧链的缺失增加环的柔性,并改变结合动力学。
除了位置320、358和364之外,RbSA共有所有HSA表位残基。RbSA的晶体结构(PDB代码3V09)30的叠加显示在位置320和358 处与CA645 Fab的明显冲突,以及在位置364处的潜在冲突。在RbSA 中,位置364是天冬氨酸,虽然没有明显的冲突,但这个位置是接触残基,因此可能影响CA645的结合。在HSA中,位置320是丙氨酸,并且它与CDRH2的F58形成疏水相互作用(图4B)。在RbSA中,位置320是谷氨酸,它与CDRH2残基W52和F58发生冲突。HSA 中的残基E358与CDRH3的S100和T100a形成氢键网络(图4C)。 RbSA中的位置358是赖氨酸,它与CDRH3的Y99发生冲突。与HSA 相比,CA645对RbSA的亲和力较弱,完全是由于缔合速率降低18 倍(表2)。这很可能是由于在位置320和358及可能364处较大侧链存在于RbSA中而引起的。
降低亲和力变体的药代动力学
为了研究CA645的半衰期与其对白蛋白的亲和力之间的相关性,我们产生了CA645gL4gH5 Fab的一组突变体,其具有宽范围的降低的亲和力,然后分析它们在小鼠中的药代动力学特性。使用CA645 gL4gH5 Fab-HSA复合物的晶体结构作为指导设计突变。将寡核苷酸定向诱变用于在重链的六个残基位置产生二十个变体,并在轻链的六个残基位置产生二十七个变体(表4,S2A、S2B和S2C)。通过以 10mg/kg单次静脉内注射CA645 gL4gH5 Fab和四种Fab变体 (gL5gH5、gL4gH37、gL5gH37和gL5gH47)的亚组来对BALB/c 小鼠给药。在103小时内对血清进行取样,并通过ELISA定量Fab 水平。
CA645 gL5gH37显示SPR没有检测到与HSA的结合,并且其被快速清除,血清半衰期仅为0.48±0.06小时(表4)。这与在大鼠中观察到的抗TNF Fab的短半衰期(0.7小时)一致19。相比之下,gL4gH5 显示出显著延长的84±4.6小时的半衰期。具有最弱亲和力的变体是 gL5gH5(与gL4gH5的药代动力学特征没有差异)(图5)。gL5gH5 在轻链中含有单个突变W30A,并且其亲和力为453nM。该亲和力比 gL4gH5(1.23nM)弱368倍,但其半衰期(96.7±20.4h)与gL4gH5 相当。观察到gL4gH37的药代动力学特征的变化。它在重链中具有单突变F58E,并且其亲和力为955nM。该亲和力比gL4gH5低776倍,但半衰期仍延长至61±16.8小时。gL5gH47在轻链中含有一个突变 W30A,并在重链中含有一个突变T100aS,并且如通过稳态SPR测量具有52μM的亲和力。该亲和力比gL4gH5的亲和力弱42,276倍,但药代动力学特征与gL4gH37没有显著差异,并且半衰期增加至 26.3±3.1小时。
基于对HSA的亲和力设计和选择突变体,但药代动力学模型是鼠。因此,为了证实突变体对HSA的亲和力反映了它们在小鼠中对白蛋白的亲和力,重复SPR(表5)。对于HSA,gL4gH5和gL5gH5 的亲和力为1.8和254nM,并且类似地,对于MSA,亲和力为2.2和 316nM。这些数据符合先前确定的gL4gH5和gL5gH5对HSA的亲和力(分别为1.23和453nM)。
使用质量作用二次方程的解,我们可以估计每种变体的血液中游离Fab的百分比31。如果我们假设MSA(65.9kDa)的浓度是44g/L 和20g小鼠的血液的体积为2ml,然后对于10mg/kg的剂量,CA645Fab (47.9kDa)的浓度将是2087nM。图6显示了游离Fab的百分比相对于在1-106nM范围内的对白蛋白的亲和力的图。由于对MSA的亲和力为2.2nM,预计仅0.0003%的gL4gH5Fab在血液中未结合。gL5gH5 Fab对MSA的亲和力是316nM。它具有与gL4gH5相匹配的药代动力学特征和半衰期,并且被计算为具有0.05%的类似低水平的游离Fab。我们无法测量gL4gH37和gL5gH47 Fab对MSA的亲和力。然而,由于gL4gH5和gL5gH5的亲和力对MSA比对HSA弱1.2倍(表 5),因此预测gL4gH37和gL5gH47的亲和力将成比例地弱1.2倍是合理的。因此,使用1146nM和62.4μM的对MSA的预测亲和力,计算出分别0.17%的gL4gH37和8.57%的gL5gH47可能在血液中是游离的。
表2:CA645 gL4Gh5 Fab与HSA、MSA、CSA、RSA和RbSA的结合动力学
白蛋白 | kax104(1/Ms) | kdx10-4(1/s) | KDx10-9(M) |
人 | 9.0 | 4.1 | 4.6 |
小鼠 | 4.8 | 3.4 | 7.1 |
大鼠 | 2.4 | 13 | 54 |
食蟹猴 | 10 | 3.5 | 3.3 |
兔 | 0.2 | 2.9 | 162 |
牛 | - | - | 无结合 |
表3:X射线数据
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表5
表6
pH | KD(nM) |
5.0 | 7.1 |
5.5 | 10.7 |
6.0 | 12.5 |
7.0 | 13.3 |
表7A
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表7B
表7C
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序列表
<110> UCB生物制药私人有限公司
<120> 亲和力改造的血清蛋白载体结合结构域
<130> PF0083
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 118
<212> PRT
<213> Artificial Sequence
<220>
<223> CA645 VH
<400> 1
Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Asn Tyr Ala
20 25 30
Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly
35 40 45
Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Arg Pro Ser Thr Thr Val Asp Leu Glu Met Thr
65 70 75 80
Ser Leu Thr Thr Ala Asp Thr Ala Thr Tyr Phe Cys Ala Arg Thr Val
85 90 95
Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly Pro Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 2
<211> 119
<212> PRT
<213> Artificial Sequence
<220>
<223> CA645 VH gH1
<400> 2
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr
20 25 30
Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Ser Thr Thr Val Tyr Leu Gln Met
65 70 75 80
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr
85 90 95
Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 3
<211> 121
<212> PRT
<213> Artificial Sequence
<220>
<223> CA645 VH gH5
<400> 3
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr
20 25 30
Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 4
<211> 121
<212> PRT
<213> Artificial Sequence
<220>
<223> CA645 VH gH37
<400> 4
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr
20 25 30
Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Ile Ile Trp Ala Ser Gly Thr Thr Ala Tyr Ala Thr Trp Ala Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 5
<211> 121
<212> PRT
<213> Artificial Sequence
<220>
<223> CA645 VH gH47
<400> 5
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr
20 25 30
Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Arg Thr Val Pro Gly Tyr Ser Ala Ala Pro Tyr Phe Asp Leu Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 6
<211> 110
<212> PRT
<213> Artificial Sequence
<220>
<223> CA645 VL
<400> 6
Ala Ala Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Ala Val Gly
1 5 10 15
Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Pro Ser Val Trp Ser Asn
20 25 30
Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu
35 40 45
Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Lys
50 55 60
Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp Val Gln
65 70 75 80
Cys Gly Asp Ala Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile
85 90 95
Ser Asp Thr Thr Phe Gly Gly Gly Thr Lys Val Val Val Lys
100 105 110
<210> 7
<211> 110
<212> PRT
<213> Artificial Sequence
<220>
<223> CA645 VL gL1
<400> 7
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn
20 25 30
Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Lys
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
65 70 75 80
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile
85 90 95
Ser Asp Thr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 8
<211> 110
<212> PRT
<213> Artificial Sequence
<220>
<223> CA645 VL gL4
<400> 8
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn
20 25 30
Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
65 70 75 80
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile
85 90 95
Ser Asp Thr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 9
<211> 110
<212> PRT
<213> Artificial Sequence
<220>
<223> CA645 VL gL5
<400> 9
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Ala Ser Asn
20 25 30
Phe Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Glu Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
65 70 75 80
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile
85 90 95
Ser Asp Thr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
Claims (8)
1.结合结构域,其包含特异于血清载体蛋白的VL和VH,
其中所述血清载体蛋白是白蛋白或其片段,并且
其中VL和VH序列分别是SEQ ID NO:9和SEQ ID NO:3。
2.权利要求1的结合结构域,其中所述血清载体蛋白是人血清白蛋白。
3.权利要求1或权利要求2的结合结构域,其中所述结合结构域结合白蛋白的结构域II。
4.抗体分子,其包含权利要求1~3中任一项的结合结构域。
5.药物组合物,其包含权利要求1~3中任一项的结合结构域或权利要求4的抗体分子。
6.权利要求1~3中任一项的结合结构域、权利要求4的抗体分子,或权利要求5的药物组合物,其用于治疗。
7.权利要求1~3中任一项的结合结构域、权利要求4的抗体分子或权利要求5的药物组合物,其用于治疗,其中所述治疗选自以下的治疗:感染,与感染有关的内毒素休克,关节炎,哮喘,慢性阻塞性肺病,盆腔炎性疾病,阿尔茨海默病,炎症性肠病,克罗恩病,溃疡性结肠炎,阴茎纤维性海绵体炎,乳糜泻,胆囊疾病,藏毛病,腹膜炎,银屑病,血管炎,手术粘连,中风,I型糖尿病,莱姆病,脑膜脑炎,自身免疫性葡萄膜炎,免疫介导的中枢和外周神经系统炎症性疾病,狼疮和吉兰-巴雷综合征,特应性皮炎,自身免疫性肝炎,纤维性肺泡炎,格雷夫斯病,IgA肾病,特发性血小板减少性紫癜,梅尼埃病,天疱疮,原发性胆汁性肝硬化,结节病,硬皮病,韦格纳肉芽肿病,自身免疫性疾病,胰腺炎,外伤,移植物抗宿主病,移植排斥反应,心脏病,血管内凝血,骨吸收,骨质疏松症,骨关节炎,牙周炎,胃酸过少和癌症。
8.权利要求1~3中任一项的结合结构域、权利要求4的抗体分子或权利要求5的药物组合物在制备药物中的用途,所述药物用于选自以下疾病的治疗:感染,与感染有关的内毒素休克,关节炎,哮喘,慢性阻塞性肺病,盆腔炎性疾病,阿尔茨海默病,炎症性肠病,克罗恩病,溃疡性结肠炎,阴茎纤维性海绵体炎,乳糜泻,胆囊疾病,藏毛病,腹膜炎,银屑病,血管炎,手术粘连,中风,I型糖尿病,莱姆病,脑膜脑炎,自身免疫性葡萄膜炎,免疫介导的中枢和外周神经系统炎症性疾病,狼疮和吉兰-巴雷综合征,特应性皮炎,自身免疫性肝炎,纤维性肺泡炎,格雷夫斯病,IgA肾病,特发性血小板减少性紫癜,梅尼埃病,天疱疮,原发性胆汁性肝硬化,结节病,硬皮病,韦格纳肉芽肿病,自身免疫性疾病,胰腺炎,外伤,移植物抗宿主病,移植排斥反应,心脏病,血管内凝血,骨吸收,骨质疏松症,骨关节炎,牙周炎,胃酸过少和癌症。
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GBGB1607636.6A GB201607636D0 (en) | 2016-05-01 | 2016-05-01 | Molecules & method |
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GBGB1607828.9A GB201607828D0 (en) | 2016-05-04 | 2016-05-04 | Method |
GB1607828.9 | 2016-05-04 | ||
PCT/EP2017/060266 WO2017191062A1 (en) | 2016-05-01 | 2017-04-28 | Affinity engineered serum protein carrier binding domain |
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US20190144529A1 (en) | 2019-05-16 |
JP2019516785A (ja) | 2019-06-20 |
CN109071643A (zh) | 2018-12-21 |
JP2022115961A (ja) | 2022-08-09 |
BR112018071288A2 (pt) | 2019-02-26 |
CA3022494A1 (en) | 2017-11-09 |
JP7133544B2 (ja) | 2022-09-08 |
EP3452506A1 (en) | 2019-03-13 |
US11466076B2 (en) | 2022-10-11 |
WO2017191062A1 (en) | 2017-11-09 |
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