CN114702589A - 治疗癌症的组合物和方法 - Google Patents
治疗癌症的组合物和方法 Download PDFInfo
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- CN114702589A CN114702589A CN202210457924.9A CN202210457924A CN114702589A CN 114702589 A CN114702589 A CN 114702589A CN 202210457924 A CN202210457924 A CN 202210457924A CN 114702589 A CN114702589 A CN 114702589A
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Abstract
本公开提供了一种对人Nectin‑4蛋白具有出色的结合亲和力的单域抗体。这些抗体特别适合被包含在双特异性抗体中,例如还靶向免疫细胞上的抗原的抗体。
Description
技术领域
本发明涉及一种用于治疗癌症的组合物及其制备方式,尤其涉及一种对人Nectin-4蛋白具有特异性的单域抗体。
背景技术
Nectin家族是由Nectin-1、-2、-3和-4四个成员组成的Ca2+非依赖性免疫球蛋白样分子。Nectin蛋白在细胞-细胞粘附中起作用。它们通过它们的细胞质尾部结合肌动蛋白丝(F-actin)结合蛋白afadin,并与肌动蛋白细胞骨架联合,与其他细胞粘附分子和细胞表面膜受体配合,调节许多其他细胞活动,如移动、分化、极化和病毒的进入。
Nectin-4,也称为脊髓灰质炎病毒受体相关蛋白4(PVRL4),是一种大小约52 kDa的I型单次跨膜蛋白。Nectin-4的胞外结构域有三个Ig样亚结构域,分别为V、C1和C2。
Nectin 1、2和3在成人组织中广泛表达,但Nectin-4在胚胎和胎盘中特异表达。然而,已经证明Nectin-4可以在各种癌细胞中表达,使其成为癌症治疗的合适靶点。
发明内容
在各种实施方式中,本公开提供了对人Nectin-4蛋白具有结合特异性的单域抗体。这些抗体可以与食蟹猴Nectin-4交叉反应。凭借出色的结合亲和力和小尺寸,这些抗体可以适当地用于生成双特异性抗体,例如也靶向免疫细胞的抗体。
因此,根据本公开的一个实施方式,提供了一种对人Nectin-4蛋白具有特异性的单域抗体或其抗原结合片段,其包含CDR1、CDR2和CDR3,其中所述CDR1、CDR2和CDR3分别包含抗体CMB7-1、CMB7-2、CMB7-3、CMB7-4或CMB7-5中任何一种的CDR1、CDR2和CDR3序列。这些示例性抗体具有如SEQ ID NO: 1-5所示的氨基酸序列。
在一些实施方式中,所述CDR1、CDR2和CDR3分别包含SEQ ID NO: 6-8、SEQ ID NO:9-11、SEQ ID NO: 12-14、SEQ ID NO: 15-17或SEQ ID NO: 18-20的氨基酸序列。
在一些实施方式中,所述单域抗体包含选自SEQ ID NO:1-5的氨基酸序列。
本公开还提供了一种双特异性抗体,其包含本文公开的单域抗体或其抗原结合片段和对不同于Nectin-4的抗原具有特异性的第二抗体或抗原结合片段。在一些实施方式中,所述抗原为人CD3。
在一些实施方式中,所述双特异性抗体包含四个单域抗体,每种单域抗体都融合至对人CD3具有特异性的全Fab抗体的重链可变区(VH)或轻链可变区(VL)。在一些实施方式中,每个单链结构域抗体都通过肽接头融合至VH或VL。
在一些实施方式中,所述肽接头具有长于7个氨基酸的长度。在一些实施方式中,肽接头具有短于50个氨基酸的长度。
本公开还提供了编码任何所述抗体或片段的多核苷酸。
本公开还提供了用所公开的抗体或片段治疗疾病(例如癌症)的方法。
附图说明
图1显示了证实抗体表达的SDS-PAGE图像。
图2显示了基于ELISA的抗体亲和力测试的结果。
图3显示了所有抗人Nectin-4 VHH抗体与食蟹猴Nectin-4交叉反应。
图4说明了测试的双特异性抗体的两种形式(形式A和形式B)。
图5显示了在Nectin-4表达细胞的存在下进行T细胞活化检测的结果。
图6-7显示了目标Nectin-4表达细胞的T细胞杀伤结果(图6: MCF-7细胞;图7: T-47D细胞)。
具体实施方式
定义
需要注意的是,术语“一种”实体是指所述实体中的一个或多个;例如,“一种抗体”被理解为代表一个或多个抗体。因此,术语“一”(或“一个”)、“一个或多个”和“至少一个”在本文中可以互换使用。
如本文所用,术语“多肽”旨在包含单数“多肽”以及复数“多肽”,并且是指由通过酰胺键(也称为肽键)线性连接的单体(氨基酸)组成的分子。术语“多肽”指两个或多个氨基酸的任何一条或多条链,而不是指产品的特定长度。因此,肽、二肽、三肽、寡肽、“蛋白质”、“氨基酸链”或用于指代两个或多个氨基酸链的任何其他术语包括在“多肽”的定义内,并且术语“多肽”可以用来代替、或与这些术语中的任何一个互换。术语“多肽”还意指多肽表达后修饰的产物,包括但不限于糖基化、乙酰化、磷酸化、酰胺化、通过已知的保护/阻断基团衍生、蛋白水解裂解或通过非天然存在的氨基酸修饰。多肽可以来自天然生物来源或通过重组技术生产,但不一定是从指定的核酸序列翻译而来。它可以以任何方式产生,包括通过化学合成。
本文中关于细胞、核酸(例如DNA或RNA)使用的术语“分离的”指分别与大分子天然来源中存在的其他DNA或RNA分离的分子。本文使用的术语“分离的”还指当通过重组DNA技术生产时基本上不含细胞材料、病毒材料或培养基的核酸或肽,或在化学合成时基本上不含化学前体或其他化学品的核酸或肽。此外,“分离的核酸”指的是不以片段形式天然出现且不会在天然状态下发现的核酸片段。术语“分离的”在本文中也用于指从其他细胞蛋白质或组织分离的细胞或多肽。分离的多肽意在包括纯化的和重组的多肽。
如本文所用,涉及多肽或多核苷酸的术语“重组”意指不天然存在的多肽或多核苷酸形式,其非限制性示例可通过组合通常不会同时出现的多核苷酸或多肽来创建。
“同源性”或“同一性”或“相似性”是指两个肽或两个核酸分子之间的序列相似性。同源性可以通过比较每个序列中的位置来确定,为了进行比较的目的,这些位置可能会被对齐。当比较序列中的一个位置被相同的碱基或氨基酸占据时,则分子在该位置是同源的。序列之间的同源程度是序列共享的匹配或同源位置数量的函数。“不相关的”或“非同源”序列与本公开的其中一个序列具有小于40%的同一性,但优选地小于25%的同一性。
多核苷酸或多核苷酸区域(或多肽或多肽区域)与另一个序列具有一定百分比(例如,60%、65%、70%、75%、80%、85%、90%、95%、98%或99%)的“序列同一性”,这意味着,当对齐时,在比较两个序列时,碱基(或氨基酸)的百分比相同。这种比对和同源性百分比或序列同一性可以使用本领域已知的软件程序来确定,例如Ausubel et al. eds. (2007) CurrentProtocols in Molecular Biology中所描述的那些。优选地,默认参数用于对齐。一种对齐程序是BLAST,使用默认参数。特别是,程序是BLASTN和BLASTP,使用以下默认参数:遗传代码=标准;过滤器=无;股=两者;截止值=60;期望值=10;矩阵=62;描述=50个序列;排序方式=高分;数据库=非冗余,GenBank+EMBL+DDBJ+PDB+GenBank-CDS-translations+SwissProtein+SPupdate+PIR。生物等效多核苷酸是指具有上述指定百分比同源性并编码具有相同或相似生物活性的多肽的多核苷酸。
术语“等效核酸或多核苷酸”是指具有与所述核酸或其补体的核苷酸序列具有一定程度的同源性或序列同一性的核苷酸序列的核酸。双链核酸的同源物旨在包括与其补体具有一定程度的同源性核苷酸序列的核酸。一方面,核酸的同源物能够与核酸或其补体杂交。同样,“等效多肽”指与参考多肽的氨基酸序列具有一定程度的同源性或序列同一性的多肽。在一些方面,序列同一性为至少约70%、75%、80%、85%、90%、95%、98%或99%。在一些方面,与参考多肽或多核苷酸相比,等效多肽或多核苷酸具有一个、两个、三个、四个或五个添加、缺失、取代及其组合。在一些方面,等效序列保留参考序列的活性(例如表位结合)或结构(例如盐桥)。
杂交反应可以在不同的“严格”条件下进行。一般来说,低严格杂交反应在约40°C下,在约10 x SSC或具有同等离子强度/温度的溶液中进行。中严格杂交通常在约50°C下在约6 x SSC中进行,而高严格杂交反应通常在约60°C下在约1 x SSC中进行。杂交反应也可以在本领域技术人员熟知的“生理条件”下进行。生理条件的非限制性示例是细胞中通常存在的温度、离子强度、pH值和Mg2+浓度。
多核苷酸由四个核苷酸碱基的特定序列组成:腺嘌呤(A);胞嘧啶(C);鸟嘌呤(G);胸腺嘧啶;当多核苷酸是RNA时,尿嘧啶(U)代表胸腺嘧啶。因此,术语“多核苷酸序列”是多核苷酸分子的按字母顺序排列的表示。这种按字母顺序排列的表示可以输入具有中央处理单元的计算机中的数据库,并用于生物信息学应用,如功能基因组学和同源性搜索。术语“多态性”指的是一种以上的基因形式或其部分共存。基因的一部分中至少有两种不同形式,即两种不同的核苷酸序列,被称为“基因多态区”。多态区可以是单个核苷酸,其身份在不同的等位基因中不同。
术语“多核苷酸”和“寡核苷酸”可互换使用,是指任何长度的核苷酸的聚合形式,无论是脱氧核糖核苷酸或核糖核苷酸或其类似物。多核苷酸可以具有任何三维结构,并且可以执行任何已知或未知的功能。以下是多核苷酸的非限制性示例:基因或基因片段(例如探针、引物、EST或SAGE标签)、外显子、内含子、信使RNA(mRNA)、转移RNA、核糖体RNA、核酶、cDNA、dsRNA、siRNA、miRNA、重组多核苷酸、分支多核苷酸、质粒、载体、任何序列的分离DNA、任何序列的分离RNA、核酸探针和引物。多核苷酸可包含经修饰的核苷酸,例如甲基化核苷酸和核苷酸类似物。如果存在,可在多核苷酸组装之前或之后对核苷酸结构进行修饰。核苷酸序列可被非核苷酸成分打断。多核苷酸可以在聚合后进一步被修饰,例如通过与标记组分共轭。该术语还指双链和单链分子。除非另有说明或要求,否则本公开的任何多核苷酸实施方式均包含双链形式和已知或预测构成双链形式的两种互补单链形式中的每一种。
用于多核苷酸的术语“编码”是指多核苷酸,如果在其天然状态下或当由本领域技术人员熟知的方法操作时,其可被转录和/或翻译以产生所述多肽和/或其片段的mRNA,则被称为“编码”多肽。反义链是这种核酸的补体,编码序列可以从中推导出来。
如本文所用,“抗体”或“抗原结合多肽”是指特异性识别并结合抗原的多肽或多肽复合物。抗体可以是整个抗体和任何抗原结合片段或其单链。因此,术语“抗体”包括任何含有蛋白质或肽的分子,其包含具有与抗原结合的生物活性的免疫球蛋白分子的至少一部分。此类示例包括但不限于重链或轻链或其配体结合部分的互补决定区(CDR)、重链或轻链可变区、重链或轻链恒定区、框架(FR)区或其任何部分,或结合蛋白的至少一部分。
术语“抗体片段”或“抗原结合片段”,如本文所用,是抗体的一部分,例如F(ab’)2、F(ab)2、Fab’、Fab、Fv、scFv等。无论结构如何,抗体片段都与完整抗体所识别的相同抗原结合。术语“抗体片段”包括适体、镜像体和双体。术语“抗体片段”还包括通过结合特定抗原形成复合物而起到抗体作用的任何合成或基因工程蛋白质。
“单链可变片段”或“scFv”是指免疫球蛋白重链(VH)和轻链(VL)可变区的融合蛋白。在一些方面,这些区域与10至约25个氨基酸的短接头连接。所述接头可以富含甘氨酸以提高灵活性,也可以富含丝氨酸或苏氨酸以提高溶解度,并且可以连接VH的N-末端和VL的C-末端,反之亦然。尽管去除了恒定区并引入了接头,该蛋白仍保留了原始免疫球蛋白的特异性。本领域已知并描述了scFv分子,例如在US patent 5,892,019中。
术语抗体包括各种可以在生物化学上区分的广泛类别的多肽。本领域技术人员将理解,重链被分类为γ、μ、α、δ、ε,其中包括一些子类(例如,γ1-γ4)。正是这条链的性质决定了抗体的“类别”分别为IgG、IgM、IgA、IgG或IgE。免疫球蛋白亚类(同种型),例如IgG1、IgG2、IgG3、IgG4、IgG5等都有很好的特征,并且已知具有功能专一性。鉴于本公开,本领域技术人员容易识别出这些类别和同种型中的每一个的修改版本,并且因此在本公开的范围内。所有免疫球蛋白类别显然都在本公开的范围内,以下讨论通常针对免疫球蛋白分子的IgG类别。关于IgG,标准免疫球蛋白分子包含两条相同的分子量约为23000道尔顿的轻链多肽和两条相同的分子量为53000-70000道尔顿的重链多肽。这四条链通常通过二硫键以“Y”形结构连接,其中轻链从“Y”口开始包围重链,并继续通过可变区域。
“特异性结合”或“具有特异性”通常意味着抗体通过其抗原结合结构域与表位结合,并且这种结合需要抗原结合结构域与表位之间的一些互补性。根据这一定义,当抗体通过其抗原结合结构域与表位结合时,它比随机的、不相关的表位更容易“特异性结合”到所述表位。本文使用术语“特异性”来限定特定抗体与特定表位结合的相对亲和力。例如,抗体“A”可被认为比抗体“B”对给定表位具有更高的特异性,或者抗体“A”可能被认为与表位“C”结合的特异性比相关表位“D”更高。
如本文所用,术语“治疗”或“疗法”是指医疗性治疗和预防或预防性措施,其中目的是预防或减缓(减轻)不期望的生理变化或紊乱,例如癌症的进展。有益的或期望的临床结果包括但不限于症状缓解、疾病程度减轻、疾病状态稳定(即不恶化)、疾病进展延迟或减缓、疾病状态改善或减轻,以及缓解(部分或全部),无论可检测或不可检测。“治疗”还可以意味着与未接受治疗的预期生存期相比延长生存期。需要治疗的人包括已经患有该疾病或紊乱的人,以及容易患有该疾病或紊乱的人,或者需要预防该疾病或紊乱的人。
“对象”或“个体”或“动物”或“患者”或“哺乳动物”是指需要诊断、预后或治疗的任何对象,尤其是哺乳动物对象。哺乳动物对象包括人类、家畜、农场动物、动物园动物、运动动物或宠物动物,如狗、猫、豚鼠、兔子、大鼠、小鼠、马、牛、奶牛等。
如本文所用,诸如“对需要治疗的患者”或“需要治疗的对象”之类的短语包括将受益于施用了用于检测、诊断程序和/或治疗的本公开的抗体或组合物的对象,例如哺乳动物对象。
单域抗Nectin-4抗体
Nectin蛋白在细胞-细胞粘附中起重要作用。Nectin-4是一种大小约52 kDa的I型单次跨膜蛋白。与在成人组织中广泛表达的Nectin 1、2和3不同,Nectin-4在胚胎和胎盘中特异表达。然而,Nectin-4也可以在各种癌细胞中表达,使其成为癌症治疗的合适靶点。
本公开提供单域抗体形式的抗-Nectin-4抗体。单域抗体(sdAb),也称为纳米抗体,是由单个单体可变抗体域组成的抗体片段。最早的单域抗体是从骆驼科动物中发现的重链抗体改造而来的,也称为VHH片段。与常规抗体的VH一样,每个VHH包括三个CDR,CDR1、CDR2和CDR3。VHH可以进一步包括恒定域,例如CH2和CH3。
如图2所示,从CMB7-24到CMB7-28的所有已鉴定的VHH抗体均表现出与人Nectin-4蛋白的强结合。同时,所有这些抗体与相应的食蟹猴蛋白也有很强的亲和力(图3),使其可以在作为临床前模型的食蟹猴中进行测试。
其中一种抗体CMB7-24(VHH 37)以两种不同形式(图4)的双特异性抗体进行测试,其也靶向人CD3复合物。图5-7中的结果表明,B形式的双特异性抗体表现出优异的T细胞活化和T细胞杀伤活性。因此,这些VHH抗体可适当用于治疗癌症等疾病。
在一个实施方式中,提供了一种抗体或抗原结合片段,其包括CDR1、CDR2和CDR3,分别具有抗体CMB7-1、CMB7-2、CMB7-3、CMB7-4或CMB7-5的CDR1、CDR2和CDR3的氨基酸序列。这些抗体的序列在表1中提供,如SEQ ID NO: 1-5所示。
在一个实施方式中,提供了一种抗体或抗原结合片段,其包括分别具有SEQ IDNO: 6-8的氨基酸序列的CDR1、CDR2和CDR3。在一个实施方式中,提供了一种抗体或抗原结合片段,其包括分别具有SEQ ID NO: 9-11的氨基酸序列的CDR1、CDR2和CDR3。在一个实施方式中,提供了一种抗体或抗原结合片段,其包括分别具有SEQ ID NO: 12-14的氨基酸序列的CDR1、CDR2和CDR3。在一个实施方式中,提供了一种抗体或抗原结合片段,其包括分别具有SEQ ID NO: 15-17的氨基酸序列的CDR1、CDR2和CDR3。在一个实施方式中,提供了一种抗体或抗原结合片段,其包括分别具有SEQ ID NO: 18-20的氨基酸序列的CDR1、CDR2和CDR3。
在一个实施方式中,提供了一种抗体或抗原结合片段,其包括SEQ ID NO: 1-5中任一项的氨基酸序列,或与SEQ ID NO: 1-5中任一项具有至少85%、90%、95%、98%或99%序列同一性的氨基酸序列。在一些实施方式中,与SEQ ID NO: 1-24中的任一项具有至少85%、90%、95%、98%或99%序列同一性的氨基酸序列保留了相应参考抗体的CDR序列。
在一个实施方式中,提供了一种抗体或抗原结合片段,其包括SEQ ID NO: 1的氨基酸序列,或与SEQ ID NO: 1具有至少85%、90%、95%、98%或99%序列同一性的氨基酸序列。在一些实施方式中,与SEQ ID NO: 1具有至少85%、90%、95%、98%或99%序列同一性的氨基酸序列保留了相应参考抗体的CDR序列,例如SEQ ID NO: 6、7和8。
在一个实施方式中,提供了一种抗体或抗原结合片段,其包括SEQ ID NO: 2的氨基酸序列,或与SEQ ID NO: 2具有至少85%、90%、95%、98%或99%序列同一性的氨基酸序列。在一些实施方式中,与SEQ ID NO: 2具有至少85%、90%、95%、98%或99%序列同一性的氨基酸序列保留了相应参考抗体的CDR序列,例如SEQ ID NO: 9、10和11。
在一个实施方式中,提供了一种抗体或抗原结合片段,其包括SEQ ID NO:3的氨基酸序列,或与SEQ ID NO:3具有至少85%、90%、95%、98%或99%序列同一性的氨基酸序列。在一些实施方式中,与SEQ ID NO:3具有至少85%、90%、95%、98%或99%序列同一性的氨基酸序列保留了相应参考抗体的CDR序列,例如SEQ ID NO: 12、13和14。
在一个实施方式中,提供了一种抗体或抗原结合片段,其包括SEQ ID NO: 4的氨基酸序列,或与SEQ ID NO: 4具有至少85%、90%、95%、98%或99%序列同一性的氨基酸序列。在一些实施方式中,与SEQ ID NO: 4具有至少85%、90%、95%、98%或99%序列同一性的氨基酸序列保留了相应参考抗体的CDR序列,例如SEQ ID NO: 15、16和17。
在一个实施方式中,提供了一种抗体或抗原结合片段,其包括SEQ ID NO: 5的氨基酸序列,或与SEQ ID NO: 5具有至少85%、90%、95%、98%或99%序列同一性的氨基酸序列。在一些实施方式中,与SEQ ID NO:5具有至少85%、90%、95%、98%或99%序列同一性的氨基酸序列保留了相应参考抗体的CDR序列,例如SEQ ID NO: 18、19和20。
在一个实施方式中,提供了一种抗体或抗原结合片段,其包括SEQ ID NO: 1-5中任一项的氨基酸序列,可选地具有1、2、3、4或5个氨基酸的添加、缺失和/或取代。在一些实施方式中,所述取代是保守取代。在一些实施方式中,所述添加、缺失和/或取代在框架区内。
在一些实施方式中,所述抗体或片段进一步包括恒定结构域,例如CH2和/或CH3。在一些实施方式中,所述CH2和/或CH3来自人IgG1、IgG2、IgG3或IgG4序列。
在一些实施方式中,所述取代是保守取代。“保守氨基酸取代”是指用具有类似侧链的氨基酸残基取代氨基酸残基。本领域已经定义了具有类似侧链的氨基酸残基家族,包括碱性侧链(例如,赖氨酸、精氨酸、组氨酸)、酸性侧链(例如,天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如,甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸),非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸、色氨酸)、β支链侧链(例如苏氨酸、缬氨酸、异亮氨酸)和芳香侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此,免疫球蛋白多肽中的非必需氨基酸残基优选地被来自相同侧链家族的另一氨基酸残基取代。在另一种实施方式中,氨基酸串可被结构相似的串取代,其在侧链家族成员的顺序和/或组成上不同。
下表提供了保守氨基酸取代的非限制性示例,其中0或更高的相似性得分表示两种氨基酸之间的保守取代。
表A. 氨基酸相似性矩阵
表B. 保守氨基酸取代
氨基酸 | 取代为 |
丙氨酸 | D-Ala, Gly, Aib, β-Ala, L-Cys, D-Cys |
精氨酸 | D-Arg, Lys, D-Lys, Orn D-Orn |
天冬酰胺 | D-Asn, Asp, D-Asp, Glu, D-Glu Gln, D-Gln |
天冬氨酸 | D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln |
半胱氨酸 | D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr, L-Ser, D-Ser |
谷氨酰胺 | D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp |
谷氨酸 | D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln |
甘氨酸 | Ala, D-Ala, Pro, D-Pro, Aib, β-Ala |
异亮氨酸 | D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met |
亮氨酸 | Val, D-Val, Met, D-Met, D-Ile, D-Leu, Ile |
赖氨酸 | D-Lys, Arg, D-Arg, Orn, D-Orn |
蛋氨酸 | D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val |
苯丙氨酸 | D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp |
脯氨酸 | D-Pro |
丝氨酸 | D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-Cys |
苏氨酸 | D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Val, D-Val |
酪氨酸 | D-Tyr, Phe, D-Phe, His, D-His, Trp, D-Trp |
缬氨酸 | D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met |
在一些实施方式中,所述抗体或片段属于IgG1、IgG2、IgG3或IgG4类。在一些实施方式中,所述抗体或片段具有抗体依赖性细胞毒性(ADCC)活性。在一些实施方式中,所述抗体或片段不具有ADCC活性。
双特异性抗体
如上所述,这些新鉴定的抗-Nectin-4 VHH抗体适合被包含在双特异性抗体中。对两种形式(形式A和形式B)进行了测试,但形式A与细胞表面表达的Nectin-4没有充分结合。形式B相对于 Nectin-4是四价的,与细胞有很强的结合力。此外,当与T细胞(通过结合CD3靶向)和表达Nectin-4的肿瘤细胞孵育时,这些双特异性抗体表现出强烈的T细胞活化和T细胞介导的肿瘤细胞杀伤。
因此,根据本公开的一个实施方式,提供了一种双特异性抗体,其包括本公开的任何VHH抗体和结合另一抗原的第二抗体或抗原结合片段。在一些实施方式中,所述第二抗原是在免疫细胞上表达的蛋白质。
免疫细胞上可被靶向的蛋白质包括但不限于CD3、CD47、PD1、PD-L1、4-1BB、OX40、SIRPA、CD16、CD28、CTLA4和CD27。在一些实施方式中,所述免疫细胞表面蛋白为CD3。
如数据所示,与测试的其他形式相比,4:2 VHH:Fab形式(形式B)具有出色的治疗活性。4:2形式如图4B所示,其包括常规Fab形式的抗体(例如,抗CD3)和四个特异于Nectin-4的VHH单元。每个VHH通过肽接头融合到Fab可变区的N-末端,肽接头例如是GS(GGGGS)(SEQID NO: 21)、GS(GGGGS)3(SEQ ID NO: 22)和GS(GGGGS)6(SEQ ID NO: 23)。
对于某些VHH,一个有趣的发现是,肽接头的长度对双特异性抗体与细胞表面的Nectin-4结合的活性有显著影响。因此,在一些实施方式中,所述肽接头的长度可为至少2个氨基酸,或至少5、7、8、9、10、12、15、17、20、22或25个氨基酸。在一些实施方式中,长度不超过15、20、25、30、35、40、45或50个氨基酸。
示例性接头包括多个甘氨酸(G)和丝氨酸(S)。在一些实施方式中,所述接头包括至少50%、60%、70%或80%的甘氨酸。示例性接头序列包括但不限于GS(GGGGS)(SEQ ID NO:21)、GS(GGGGS)3(SEQ ID NO: 22)和GS(GGGGS)6(SEQ ID NO: 23)。
因此,在一个实施方式中,本公开提供了一种对免疫细胞(例如,靶向CD3)和Nectin-4具有特异性的双特异性抗体。在一些实施方式中,所述双特异性抗体包括对人CD3复合物具有特异性的常规抗体。在一些实施方式中,所述双特异性抗体包括多个(例如,2个和4个)靶向Nectin-4的VHH。
因此,在一个实施方式中,提供了一种包含第一部分和第二部分的双特异性抗体,其中第一部分包括两对VH和VL,每对都能够结合人CD3复合物,第二部分包括如本文所公开的四个单域抗体(VHH)片段,其中每个VHH片段通过肽接头融合到第一部分的每个VH和VL的N-端。
在一些实施方式中,所述双特异性抗体进一步包括恒定结构域,例如CH1和CL,以及CH2和/或CH3。在一些实施方式中,所述恒定区来自人IgG1、IgG2、IgG3或IgG4序列。
本领域的普通技术人员还将理解,本文所公开的抗体可以被修饰,以使其在氨基酸序列上与从其衍生的天然存在的结合多肽不同。例如,从指定蛋白质衍生的多肽或氨基酸序列可能是相似的,例如,与起始序列具有一定的百分比同一性,例如,它可能与起始序列60%、70%、75%、80%、85%、90%、95%、98%或99%相同。
在某些实施方式中,抗体包含通常不与抗体结合的氨基酸序列或一个或多个部分。下面更详细地描述示例性修饰。例如,本公开的抗体可包含灵活的接头序列,或可被修饰以添加功能部分(例如,PEG、药物、毒素或标记)。
本公开的抗体、其变体或衍生物(包括经修饰的衍生物),即通过将任何类型的分子共价连接到抗体,从而使共价连接不会阻止抗体结合到表位。例如,但不限于,抗体可以被修饰,例如通过糖基化、乙酰化、聚乙二醇化、磷酸化、磷酸化、酰胺化、通过已知保护/阻断基团衍生化、蛋白水解裂解、与细胞配体或其他蛋白质的连接等。可以通过已知的技术来进行许多化学修饰中的任何一种,包括但不限于特定化学裂解、乙酰化、甲酰化、衣霉素的代谢合成等。此外,抗体可能包含一个或多个非经典氨基酸。
在一些实施方式中,抗体可与治疗剂、前药、肽、蛋白质、酶、病毒、脂质、生物反应调节剂、药剂或PEG结合。
抗体可与治疗剂结合或融合,治疗剂可包括可检测标记,例如放射性标记、免疫调节剂、激素、酶、寡核苷酸、光活性治疗剂或诊断剂、细胞毒性剂(可能是药物或毒素)、超声增强剂、非放射性标记、其与本领域已知的其他此类试剂的组合。
抗体可以通过将其与化学发光化合物偶联而被可检测地标记。然后通过检测化学反应过程中出现的发光来确定化学发光标记的抗原结合多肽的存在。特别有用的化学发光标记化合物的示例包括鲁米诺、异鲁米诺、theromatic吖啶酯、咪唑、吖啶盐和草酸酯。
抗体也可以用荧光发射金属(如152Eu)或其他镧系元素标记。可以使用二乙烯三胺五乙酸(DTPA)或乙二胺四乙酸(EDTA)等金属螯合基团将这些金属连接到抗体上。将不同部分结合到抗体上的技术是众所周知的,例如,参见Arnon et al., “MonoclonalAntibodies For Immunotargeting Of Drugs In Cancer Therapy”, in MonoclonalAntibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R.Liss, Inc. (1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al., (eds.), Marcel Dekker,Inc., pp. 623- 53 (1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents InCancer Therapy: A Review”, in Monoclonal Antibodies ‘84: Biological AndClinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis,Results, And Future Prospective Of The Therapeutic Use Of RadiolabeledAntibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer DetectionAnd Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), andThorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-ToxinConjugates”, Immunol. Rev. (52:119-58 (1982))。
编码抗体的多核苷酸和制备抗体的方法
本公开还提供了编码本公开的抗体、其变体或衍生物的分离多核苷酸或核酸分子。本公开的多核苷酸可以在同一个多核苷酸分子或分离的多核苷酸分子上编码抗原结合多肽、其变体或衍生物的整个重链和轻链可变区。此外,本公开的多核苷酸可以在同一个多核苷酸分子或分离的多核苷酸分子上编码抗原结合多肽、其变体或衍生物的重链和轻链可变区的部分。
制备抗体的方法是本领域所熟知的,并在本文进行了描述。在某些实施方式中,本公开的抗原结合多肽的可变区和恒定区均为全人源的。可使用本领域所述和本文所述的技术制备全人源抗体。例如,针对特定抗原的全人源抗体可通过向转基因动物施用抗原来制备,该转基因动物已被修饰以产生此类抗体以响应抗原攻击,但其内源性位点已被无效。可用于制造此类抗体的示范性技术如U.S. 专利6,150,584; 6,458,592; 6,420,140中所述,其通过引用整体并入本文。
在某些实施方式中,制备的抗体不会在待治疗的动物(例如在人)中引发有害的免疫反应。在一种实施方式中,使用本领域公认的技术对本公开的抗原结合多肽、其变体或衍生物进行修饰以降低其免疫原性。例如,抗体可以是人源化的、灵长类化的、去免疫化的、或者可以制备嵌合抗体。这些类型的抗体源于非人抗体,通常是鼠类或灵长类抗体,其保留或基本上保留亲本抗体的抗原结合特性,但在人类中免疫原性较低。这可以通过多种方法实现,包括(a)将整个非人源可变结构域移植到人恒定区以产生嵌合抗体;(b)将一个或多个非人源互补决定区(CDR)的至少一部分移植到人源框架和恒定区中,并保留或不保留关键框架残基;或者(c)移植整个非人源可变结构域,但通过替换表面残基,用类似人源的部分“遮盖”它们。此类方法如Morrison et al., Proc. Natl. Acad. Sci. USA 57:6851-6855(1984); Morrison et al., Adv. Immunol. 44:65-92 (1988); Verhoeyen et al.,Science 239:1534-1536 (1988); Padlan, Molec. Immun. 25:489-498 (1991);Padlan, Molec. Immun. 31:169-217 (1994)及U.S. Pat. Nos.: 5,585,089, 5,693,761, 5,693,762和6,190,370中所述,其通过引用整体并入本文。
去免疫也可用于降低抗体的免疫原性。如本文所用,术语“去免疫”包括改变抗体以修饰T细胞表位(例如,参见国际申请公开号:WO/9852976 A1和WO/0034317 A2)。例如,分析来自起始抗体的可变重链和可变轻链序列,并创建每个V区的人源T细胞表位“图谱”,显示与互补决定区(CDR)和序列内其他关键残基相关的表位位置。分析T细胞表位图中的单个T细胞表位,以识别具有改变最终抗体活性的低风险的可选地氨基酸取代。设计了一系列可选地可变重序列和可变轻序列,包括氨基酸取代的组合,并且这些序列随后被并入一系列结合多肽中。通常,产生12到24种变异抗体,并测试其结合和/或功能。然后将包含修饰可变区和人源恒定区的完整重链和轻链基因克隆到表达载体中,并且将随后的质粒导入细胞系以产生完整的抗体。然后在适当的生化和生物试验中比较抗体,并确定最佳变体。
本公开的抗原结合多肽的结合特异性可通过体外试验来确定,例如免疫沉淀、放射免疫试验(RIA)或酶联免疫吸附试验(ELISA)等。
癌症治疗
如本文所述,本公开的抗体、变体或衍生物可用于某些治疗和诊断方法。
本公开进一步涉及基于抗体的疗法,其涉及将本公开的抗体施用于患者(例如动物、哺乳动物和人类),以治疗本文所述的一种或多种疾病或病症。本公开的治疗性化合物包括但不限于本公开的抗体(包括如本文所述的其变体和衍生物)和编码本公开抗体的核酸或多核苷酸(包括如本文所述的其变体和衍生物)。
本公开的抗体也可用于治疗或抑制癌症。在一些实施方式中,Nectin-4在肿瘤细胞中过度表达。因此,在一些实施方式中,提供了用于在有需要的患者中治疗癌症的方法。在一种实施方式中,该方法需要向患者施用有效量的本公开的抗体。在一些实施方式中,患者的至少一种癌细胞(例如基质细胞)表达、过度表达或被诱导表达肿瘤抗原。例如,可以通过施用肿瘤疫苗或放射疗法来实现基因表达的诱导。
可适当治疗的肿瘤包括膀胱癌、非小细胞肺癌、肾癌、乳腺癌、尿道癌、结直肠癌、头颈癌、鳞状细胞癌、默克尔细胞癌、胃肠道癌、胃癌、食管癌、卵巢癌、肾癌和小细胞肺癌。因此,目前的抗体可用于治疗任何一种或多种此类癌症。
可通过本公开的抗体或变体或其衍生物治疗、预防、诊断和/或预测的与细胞存活增加相关的其他疾病或病症包括但不限于恶性肿瘤和相关疾病的进展和/或转移,如白血病(包括急性白血病(例如,急性淋巴细胞白血病、急性髓细胞白血病(包括成髓细胞、早幼粒细胞、粒单核细胞、单核细胞和红细胞白血病))和慢性白血病(例如,慢性髓细胞(粒细胞)白血病和慢性淋巴细胞白血病)、真性红细胞增多症、淋巴瘤(例如,霍奇金病和非霍奇金病)、多发性骨髓瘤、华氏巨球蛋白血症、重链疾病和实体瘤,包括但不限于肉瘤和癌,例如纤维肉瘤、粘液肉瘤、脂肪肉瘤、软骨肉瘤、成骨肉瘤、脊索瘤、血管肉瘤、内皮肉瘤、淋巴管肉瘤、淋巴管内皮肉瘤、滑膜瘤、间皮瘤、尤文氏瘤、平滑肌肉瘤、横纹肌肉瘤、结肠癌、胰腺癌、乳腺癌、甲状腺癌、子宫内膜癌、黑色素瘤、前列腺癌、卵巢癌、前列腺癌、鳞状细胞癌、基底细胞癌、腺癌、汗腺癌、皮脂腺癌、乳头状癌、乳头状腺癌、囊腺癌、髓样癌、支气管癌、肾细胞癌、肝癌、胆管癌、绒毛膜癌、精原细胞瘤、胚胎癌、肾母细胞瘤、宫颈癌、睾丸肿瘤、肺癌、小细胞肺癌、膀胱癌、上皮癌、神经胶质瘤、星形细胞瘤、髓母细胞瘤、颅咽管瘤、室管膜瘤、松果体瘤、血管母细胞瘤、听神经瘤、少突胶质细胞瘤、血管瘤、黑色素瘤、神经母细胞瘤和视网膜母细胞瘤。
任何特定患者的特定剂量和治疗方案将取决于多种因素,包括使用的特定抗体、其变体或衍生物、患者的年龄、体重、一般健康状况、性别和饮食、以及给药时间、排泄率、药物组合和治疗的特定疾病的严重程度。医务人员对此类因素的判断属于本领域的普通技术。剂量还取决于待治疗的个体患者、给药途径、制剂类型、所用化合物的特性、疾病的严重程度和预期的效果。用量可通过本领域所熟知的药理学和药代动力学原理确定。
抗体、变体或衍生物的施用方法包括但不限于皮内、肌肉内、腹膜内、静脉内、皮下、鼻内、硬膜外和口服途径。抗原结合多肽或组合物可通过任何方便的途径施用,例如通过输液或快速浓注,通过上皮或粘膜皮肤内层(例如,口腔粘膜、直肠和肠粘膜等)吸收,并且可与其他生物活性剂一起施用。因此,含有本公开抗原结合多肽的药物组合物可口服、经直肠、肠胃外、池内(intracistemally)、阴道内、腹膜内、局部(如通过粉末、软膏、滴剂或透皮贴剂)、经颊或作为口腔或鼻腔喷雾剂施用。
如本文所用,术语“肠胃外”是指包括静脉内、肌肉内、腹膜内、胸骨内、皮下和关节内注射和输注的施用方式。
施用可以是系统性的或局部性的。此外,可能需要通过任何合适的途径将本公开的抗体引入中枢神经系统,包括心室内和鞘内注射;可以通过心室内导管促进心室内注射,例如连接到储液器,如Ommaya储液器。也可采用肺部施用,例如通过使用吸入器或雾化器,以及使用气溶胶制剂。
可能需要将本公开的抗体多肽或组合物局部施用于需要治疗的区域;这可以通过,例如但不限于,在手术期间局部输注、局部施用,例如,与手术后的伤口敷料结合,通过注射、通过导管、通过栓剂、或通过植入物的方式,所述植入物是多孔的、非多孔的、或凝胶材料(包括膜,例如唾液酸膜、或纤维)。优选地,当施用本公开的蛋白质(包括抗体)时,必须注意使用蛋白质不吸收的材料。
组合物
本公开还提供了药物组合物。此类组合物包括有效量的抗体和可接受的载体。
在特定的实施方式中,术语“药学上可接的受”是指经联邦或州政府监管机构批准或在美国药典或其他公认的用于动物,尤其是用于人类的药典中列出。此外,“药学上可接受的载体”通常是无毒的固体、半固体或液体填充剂、稀释剂、封装材料或任何类型的辅助制剂。
术语“载体”是指与治疗剂一起施用的稀释剂、佐剂、赋形剂或载体。此类药物载体可以是无菌液体,例如水和油,包括石油、动物、植物或合成来源的那些,例如花生油、大豆油、矿物油、芝麻油等。当药物组合物经静脉施用时,水是优选的载体。盐水溶液、葡萄糖水溶液和甘油水溶液也可用作液体载体,特别是用于可注射溶液。合适的药物赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、大米、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石粉、氯化钠、脱脂奶粉、甘油、丙烯、乙二醇、水、乙醇等。如果需要,该组合物还可包含少量润湿剂或乳化剂,或pH缓冲剂,例如醋酸盐、柠檬酸盐或磷酸盐。抗菌剂,如苯甲醇或对羟基苯甲酸甲酯;抗氧化剂,如抗坏血酸或亚硫酸氢钠;螯合剂,如乙二胺四乙酸;此外,还设想了用于调节张力的试剂,例如氯化钠或葡萄糖。
这些组合物可以采取溶液、悬浮液、乳液、片剂、丸剂、胶囊、粉末、缓释制剂等形式。该组合物可用传统粘合剂和载体(如甘油三酯)制成栓剂。口服制剂可包括标准载体,例如药物级甘露醇、乳糖、淀粉、硬脂酸镁、糖精钠、纤维素、碳酸镁等。合适的药物载体的示例如E.W.Martin在Remington’s Pharmaceutical Sciences中所述,其通过引用并入本文。此类组合物将包含治疗有效量的抗原结合多肽(优选纯化形式)以及适量的载体,以便为患者提供适合施用的形式。制剂应适合施用模式。亲代制剂可以装在安瓿、一次性注射器或玻璃或塑料制成的多剂量瓶中。
在一个实施方式中,根据常规程序将该组合物配制成适合对人类静脉内施用的药物组合物。通常,用于静脉内给药的组合物是无菌等渗水缓冲液中的溶液。必要时,该组合物还可包括溶解剂和局部麻醉剂,例如利多卡因,以减轻注射部位的疼痛。通常,这些成分以单位剂型单独提供或混合在一起,例如,作为干燥的冻干粉末或无水浓缩物装在如安瓿或小袋的密封容器中,指示活性剂的数量。当通过输液施用该组合物,其可与含有无菌药物级水或生理盐水的输液瓶一起配药。当通过注射施用该组合物,可以提供一安瓿无菌注射用水或生理盐水,从而可以在施用前混合。
本发明的化合物可以配制成中性或盐形式。药学上可接受的盐包括与阴离子形成的盐,例如衍生自盐酸、磷酸、乙酸、草酸、酒石酸等的盐,以及与阳离子形成的盐,例如衍生自钠、钾、铵、钙、氢氧化铁、异丙胺、三乙胺、2-乙基氨基乙醇、组氨酸、普鲁卡因等的盐。
实施例
实施例1. 抗人Nectin-4单域抗体的制备
本实施例展示了如何通过羊驼免疫,然后构建和选择噬菌体库,产生抗人Nectin-4单域抗体。
以重组人Nectin-4/hFc融合蛋白作为免疫原产生抗人Nectin-4抗体。收集羊驼PBMC,通过RNA分离、PCR扩增和克隆到噬菌体展示载体中产生抗体cDNA库。然后对这些库进行一轮液相选择和一轮固相选择。
通过PCR从抗原阳性噬菌体中扩增结合物并测序。用SDS-PAGE确认表达的蛋白质(图1)。下表提供了独特的抗体及其CDR区域的序列。
表1. 抗体序列
表2. CDR序列
实施例 2. ELISA 结合测试
在该实例中,对抗体进行基于ELISA的结合测试。将相同浓度(0.5μg/mL)的人Nectin-4涂覆在96孔酶板上。封闭后加入2μg/mL的每种抗体和不同浓度(0.2μg/mL和1μg/mL)的山羊抗人Nectin-4抗体(作为对照)。洗去多余的样本后,加入偶联辣根过氧化物酶(HRP)的山羊抗人IgG Fc交叉吸附抗体(或兔抗山羊IgG抗体)。HRP能与底物3,3',5,5'-四甲基联苯胺(TMB)反应生成有色产物。CMB7与人Nectin-4的结合亲和力可通过读取反应溶液的OD450值来计算,因为反应溶液的吸光度与结合了抗原的抗体的含量呈正相关。因此,采用ELISA测试CMB7与人Nectin-4的结合亲和力。
试验步骤:
包被抗原制剂
在PBS中将包被抗原(人Nectin-4)稀释至工作浓度(0.5μg/mL)。立即以每孔100μL的稀释包被抗原包被96孔微孔板。密封微孔板并在4℃下孵育过夜。
封闭
吸出所有微孔并用300 µL/孔洗涤缓冲液通过微孔板洗涤机洗涤5次。在每个洗涤步骤中留出浸泡时间(约1分钟),可提高洗涤效果。然后用微孔板脱水机将微孔板脱水,以去除任何残留的缓冲液。用200µL的封闭缓冲液封闭微孔。在37℃的水浴中孵育1小时。
样品制备
重复洗涤/脱水。在试验缓冲液中将样品稀释至工作浓度(2μg/mL)。将100µL/孔的预稀释样品添加到适当的孔中。密封微孔板,在37℃的水浴中孵育1小时。
二级抗体孵育
重复洗涤/脱水。将二级抗体在试验缓冲液中以1:2000稀释。向每个孔中添加100µL/孔的稀释的二级抗体(1:2000)。将板在37℃的水浴中避光孵育1小时。
信号检测
重复洗涤/脱水。向每个孔中添加100µL/孔的基质溶液(TMB)。将板在室温下避光孵育3分钟。添加50µL/孔的终止溶液。在450 nm处读取板并分析数据。
结果:
ELISA测试结果如图2和表3所示。如结果所示,所有抗体均表现出与人Nectin-4蛋白的强结合亲和力。
表3. ELISA 亲和力测试结果
实施例3. 动力学结合测试
本实施例测量了抗体(与人Fc,VHH Fc融合)与人Nectin-4的动力学结合亲和力。
通过生物层干涉法检测抗体与人Nectin-4的动力学结合亲和力。将HFC(抗HIgGFC)探针(Probelife)在Crimson 96 MAX 96孔反应板(ET Healthcare,06-0098)中在30℃的动力学缓冲液(Probelife)中预湿5分钟。然后将探针浸入Greiner黑色微孔板的含有在动力学缓冲液中的4 μg/mL抗体的孔中。使用不同浓度梯度稀释(从200 nM开始,逐步稀释2倍,共5个浓度梯度)的分析物(人Nectin-4)进行5分钟的结合步骤,然后在动力学缓冲液中进行16分钟的解离步骤。通过减去参考样品对数据进行分析,并使用数据分析软件1.7.2.0609(Gator)对亲和常数的结构化数据方法的1:1 K结合模型进行拟合。
试验过程:
探针平衡
将板的温度设置为30℃,将采集速率设置为标准动力学(5.0 HZ),在Crimson 96MAX 96 孔反应板的第1列中每孔添加260μL/孔预湿缓冲液(K缓冲液),将探针置于缓冲液中,并将探针预湿5 min,1000 rpm。
基线1
在Greiner 96孔聚丙烯微孔板的第1列中,以1000 rpm的速度在200µL/孔K缓冲液中对6个探针进行基线试验2分钟。尽可能平衡,最终斜率最好不高于0.02 nm/min。
将抗体加载到探针上
用K缓冲液将抗体稀释至工作浓度(4μg/mL,200µL/孔),然后在Greiner 96孔聚丙烯微孔板的2/3/4/5/6列上以1000 rpm的速度加载到探针上5分钟。
基线2
在Greiner 96孔聚丙烯微孔板的第8/9/10/11/12列中,以1000 rpm的速度在200µL/孔K缓冲液中对探针进行基线试验2分钟。为了从生物传感器中去除未结合的mAb,应尽量减少非特异性结合或减少缓冲效应的偏差。
结合
用K缓冲液将抗原(人Nectin-4)稀释至6种浓度(200、100、50、25、12.5、0 nM、200µL/孔),然后将这一系列抗原添加到Greiner 96孔聚丙烯微孔板第7列A行至F行的孔中。在第7列中,探针上的抗体在1000 rpm下与不同浓度梯度的抗原结合5分钟。
解离
探针在K缓冲液中解离16分钟,抗原在Greiner 96孔聚丙烯微孔板上从第8/9/10/11/12列的探针解离。
再生
探针运行再生程序(探针在Crimson 96 MAX 96 well反应板11列的R缓冲液中再生5s,然后在Crimson 96 MAX 96 well反应板12列的Q缓冲液中中和5s,该过程重复3次)。
结果:
下表4总结了测试结果。所有抗体都与人Nectin-14蛋白具有强大的亲和力。
表4. 抗-Nectin 4 VHH-Fc 的亲和力测量
抗体 | KD (M) | 响应 |
CMB7-24 | 5.89E-21 | 0.967 |
CMB7-25 | 5.80E-21 | 0.927 |
CMB7-26 | 4.18E-21 | 0.968 |
CMB7-27 | 5.00E-11 | 0.465 |
CMB7-28 | 5.96E-21 | 0.884 |
实施例 4. 与食蟹猴 Nectin-4 的交叉反应
本实施例测试了抗体与食蟹猴Nectin-4的结合亲和力。
通过ELISA测试抗体与食蟹猴Nectin-4的结合亲和力。将相同浓度(0.5μg/mL)的食蟹猴Nectin-4包被在96孔酶板上。封闭后加入稀释的不同浓度梯度的抗体(从2μg/mL开始,逐步3倍稀释,共7个浓度梯度)。在洗去过量的样本后,加入偶联了辣根过氧化物酶(HRP)的山羊抗人IgG Fc交叉吸附抗体。HRP能与底物3,3',5,5'-四甲基联苯胺(TMB)反应生成有色产物。CMB7与食蟹猴Nectin-4的结合亲和力可通过读取反应溶液的OD450值来计算,因为反应溶液的吸光度与结合了抗原的抗体的含量呈正相关。因此,采用ELISA测试CMB7与食蟹猴Nectin-4的结合亲和力。
试验过程:
包被抗原制剂
在PBS中将包被抗原(食蟹猴Nectin-4)稀释至工作浓度(0.5μg/mL)。立即以每孔100μL稀释包被抗原包被96孔微孔板。密封微孔板并在4℃下孵育过夜。
封闭
吸出所有微孔并用300 µL/孔洗涤缓冲液通过微孔板洗涤机洗涤5次。在每个洗涤步骤中留出浸泡时间(约1分钟),可提高洗涤效果。然后用微孔板脱水机将微孔板脱水,以去除任何残留的缓冲液。用200µL的封闭缓冲液封闭微孔。在37℃的水浴中孵育1小时。
样品准备
重复洗涤/脱水。在试验缓冲液中将样品稀释至工作浓度(2μg/mL),并进行3倍连续稀释,以制作总共7个点的曲线。将100µL/孔的预稀释样品添加到适当的孔中。密封微孔板,在37℃的水浴中孵育1小时。
二级抗体孵育
重复洗涤/脱水。将二级抗体在试验缓冲液中稀释至1:2000。向每个孔中添加100µL/孔的稀释的二级抗体(1:2000)。将板在37℃的水浴中避光孵育1小时。
信号检测
重复洗涤/脱水。向每个孔中添加100µL/孔的基质溶液(TMB)。将板在室温下避光孵育3分钟。添加50µL/孔的终止溶液。在450 nm处读取板并分析数据。
结果:
测试结果如图3所示,这表明所有测试的抗体都与食蟹猴Nectin-4发生交叉反应,其中大多数表现出强结合亲和力。这表明食蟹猴可以成为测试这些抗体的合适的临床前模型。
实施例5. 与人乳腺癌细胞结合的流式细胞术分析
本实施例通过荧光激活细胞分选仪(FACS)检测抗体与表达Nectin-4的人乳腺癌细胞的结合亲和力。
试验步骤:
细胞制备
1. 细胞达到80%汇合后,从100 mm培养皿中收集细胞。
2. 用2 mL PBS洗涤。加入1 mL 0.25%胰蛋白酶EDTA,在37℃下培养至细胞从板上解离。加入5 mL温培养基。收集细胞并转移至15 mL锥形管中。
4. 收集细胞并转移至15ml锥形管中。
5. 在室温下离心300 g 5分钟。
细胞铺板
用5mL PBS-0.2%BSA洗涤,在300g,4℃下离心5 min。重复两次。丢弃上清液,在2mLPBS-0.2%BSA中重新悬浮。计数细胞并向每个孔中添加1×105-2×105个细胞(100µL)。
一级抗体孵育
向每个孔中加入蛋白质(10 µg/mL或5 nM),并在4℃下孵育1小时。
洗涤
向每个孔中加入200µL冰冷的PBS-0.2% BSA,在300 g,4℃下离心5 min。重复3次。加入100µL PBS-0.2%BSA以重新悬浮细胞。
二级抗体孵育
在冰冷的PBS-0.2%BSA中向每个孔中加入Alexa Fluor 488山羊抗人IgG(H+L)(1:500稀释度),并在4℃下孵育1小时。
洗涤
向每个孔中加入200µL冰冷的PBS-0.2% BSA,在300 g,4℃下离心5 min。重复3次。加入200µL PBS-0.2%BSA以重新悬浮细胞。
应用于流式细胞术。
结果:
FACS结果显示,在每个测试浓度下,每个抗体都与人乳腺癌细胞系MCF-7结合。
实施例6. 抗-Nectin-4/抗-CD3双特异性抗体的制备
单域抗体(VHH)CMB7-24(“VHH 37”)用于构建还靶向人CD3的双特异性抗体。如图4所示,使用了两种不同形式的双特异性抗体。
图4A示出了A形式,其中单个VHH和来自抗CD3抗体的单个VH/VL对被融合到Fc片段。因此,A形式是不对称的,具有对CD3和Nectin-4的1:1的效价。
在图4B的形式(形式B)中,4个VHH中的每一个都融合到一个完全的抗CD3抗体的变体结构域的N-末端。因此,这种形式是对称的,具有对CD3和Nectin-4的2:4的效价。双特异性构型和每条链的结构如表5-6所示。
表5. 双特异性抗体和相关链的结构
抗体 | 形式 | 接头 (SEQ ID NO:) |
BJ182/12L1/183-37 | A | - |
BJ192-37/BJ196-37 | B | GS(GGGGS)<sub>1 </sub>(SEQ ID NO:21) |
BJ193-37/BJ197-37 | B | GS(GGGGS)<sub>3 </sub>(SEQ ID NO:22) |
BJ194-37/BJ198-37 | B | GS(GGGGS)<sub>6 </sub>(SEQ ID NO:23) |
链 | 结构 |
BJ192-37 | Anti-Nectin VHH-linker 1-Anti-CD3-Fc (PGLALA) |
BJ193-37 | Anti-Nectin VHH-linker 2-Anti-CD3-Fc (PGLALA) |
BJ194-37 | Anti-Nectin VHH-linker 3-Anti-CD3-Fc (PGLALA) |
BJ196-37 | Anti-Nectin VHH-linker 1-Anti-CD3-VL-CL |
BJ193-37 | Anti-Nectin VHH-linker 2-Anti-CD3-VL-CL |
BJ198-37 | Anti-Nectin VHH-linker 3-Anti-CD3-VL-CL |
合成了编码这些双特异性抗体的cDNA序列,并用于制备抗体。
实施例7. 双特异性抗体的T细胞活化
本实施例测试了双特异性抗体在存在表达Nectin-4的MCF-7细胞或T-47D细胞时活化T细胞的能力。
图5显示了所有双特异性抗体的T细胞活化结果。A形式(BJ182/12L1/BJ183-37)仅包含一个VHH,未显示出可观察到的T细胞活化活性。有趣的是,所有格式B的双特异性抗体均表现出剂量依赖性的强活性。。
实施例8. 双特异性抗体的细胞毒性活性
本研究的目的是检测抗Nectin-4抗体对MCF-7和T-47D细胞的细胞毒性。
通过基于图像的细胞杀伤试验检测抗Nectin-4抗体对MCF-7和T-47D细胞的细胞毒性。在这项研究中,MCF-7和T-47D细胞是靶细胞,原代人T细胞是效应细胞。从人PBMC细胞中分离出原代人T细胞,并将其冷冻在液氮中。靶细胞和效应细胞以1:2的比例(MCF-7/T-47D细胞为3x104,原代人T细胞为6x104)添加到含有100 nM抗Nectin-4抗体的96孔板的每个孔中。共培养40小时后扫描图像。
试验过程:
细胞培养
T细胞
将装有T细胞的小瓶在37℃的水浴中轻轻搅拌解冻。为了减少污染的可能性,使O形圈和盖子远离水。解冻必须迅速。内容物解冻后,立即将小瓶从水浴中取出,并通过浸入或喷洒70%乙醇进行净化。注:从这一步开始的所有步骤都应在严格的无菌条件下进行。将细胞转移到含有15 ml预热的生长培养基的更大的小瓶中。以400 g离心小瓶5分钟。去除含有冷冻保护剂的上清液,并用1 ml T细胞生长培养基重新悬浮细胞。将小瓶的内容物转移到含有15 ml T细胞生长培养基的T75细胞培养瓶中。将细胞置于37℃、5% CO2中。
MCF-7/T-47D细胞
将装有MCF-7/T-47D细胞的小瓶在37℃水浴中轻轻搅拌解冻。为了减少污染的可能性,使O形圈和盖子远离水。解冻必须迅速。内容物解冻后,立即将小瓶从水浴中取出,并通过浸入或喷洒70%乙醇进行净化。从这一步开始的所有步骤都应在严格的无菌条件下进行。将细胞转移到含有10 ml预热培养基的100 mm培养皿中。当细胞生长到80-90%时,去除细胞上清液,用PBS洗涤1-2次,并用1mL 0.25%胰蛋白酶EDTA(1X),酚红消化。用生长培养基终止消化,轻轻吹动细胞,将其完全去除。300 g离心5 min。去除上清液并加入1 ml培养基以吹走。将小瓶内容物转移到含有10 ml 生长培养基的100 mm 培养皿中。将培养物置于37℃、5% CO2中。
靶细胞扩散(第1天)
在测试培养基中制备靶细胞。向培养皿中加入200µL细胞悬浮液(每孔约3*104个细胞)。将细胞置于37℃、5% CO2中,使细胞粘附。
T 细胞制备(第 2 天)
在测试培养基中以 6*105/mL 准备足量T 细胞(每孔约6*104个细胞)。
抗体稀释(第 2 天)
在测试培养基中将BJ-009和12H3-1/12L1稀释至工作浓度(从10 nM开始,3倍稀释,6点)。为了达到10nM的工作浓度,应将抗体稀释至100 nM作为样品浓度。
将靶细胞与抗体和 T 细胞混合(第 2 天)
用测试培养基洗涤靶细胞2次。向每个孔中加入80µL测试培养基。向培养皿中加入20µL抗体溶液。向培养皿中加入100µL T细胞悬浮液(约6*104个细胞/孔)。
成像
设置活细胞成像仪(BioTek,Cytation5)。共培养40小时后扫描图片。
结果:
图6(MCF-7细胞)和图7(T-47D细胞)显示了所有双特异性抗体的T细胞杀伤结果。较大且较暗的颗粒表明靶细胞死亡。与实施例7中的T细胞活化结果一致,使用A形式的任何抗体的治疗不会导致T细胞杀伤,并且使用大多数B形式的双特异性抗体的治疗会导致靶细胞(MCF-7或T-47D细胞)的细胞死亡,证明了这些抗体的功效。
本公开的范围不受所述具体实施例的限制,所述具体实施例旨在作为本公开各个方面的单一说明,且功能等效的任何组合物或方法均包含在本公开的范围内。对于本领域技术人员而言,显而易见的是,在不脱离本公开的精神或范围的情况下,可以对本公开的方法和组合物进行各种修改和变化。因此,本公开旨在涵盖本公开的修改和变化,只要它们在所附权利要求书及其等效物的范围内。
本说明书中提及的所有出版物和专利申请均通过引用并入本文,其程度与每个被具体和单独指示为通过引用并入的出版物或专利申请相同。
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<110> 博际生物医药科技(杭州)有限公司
<120> 治疗癌症的组合物和方法
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Claims (14)
1.一种对人Nectin-4蛋白具有特异性的单域抗体或其抗原结合片段,其包含CDR1、CDR2和CDR3,其中所述CDR1、CDR2和CDR3分别包含由选自SEQ ID NO:1-5的氨基酸序列表示的单域抗体的CDR1、CDR2和CDR3序列。
2.根据权利要求1所述的单域抗体或其抗原结合片段,其中所述CDR1、CDR2和CDR3分别包含SEQ ID NO: 6-8、SEQ ID NO: 9-11、SEQ ID NO: 12-14、SEQ ID NO: 15-17或SEQ IDNO: 18-20的氨基酸序列。
3.根据权利要求1所述的单域抗体或其抗原结合片段,其包含选自SEQ ID NO: 1-5的氨基酸序列。
4.根据权利要求1所述的单域抗体或其抗原结合片段,其中所述CDR1、CDR2和CDR3分别包含SEQ ID NO: 6、7和8的氨基酸序列。
5.根据权利要求4所述的单域抗体或其抗原结合片段,其包含SEQ ID NO: 1的氨基酸序列。
6.一种双特异性抗体,包含权利要求1-5中任一项所述的单域抗体或其抗原结合片段和对不同于Nectin-4的抗原具有特异性的第二抗体或抗原结合片段。
7.根据权利要求6所述的双特异性抗体,其中所述抗原是人CD3。
8.根据权利要求7所述的双特异性抗体,其包含四个所述单域抗体,每个单域抗体都融合至对人CD3具有特异性的完全Fab抗体的重链可变区(VH)或轻链可变区(VL)。
9.根据权利要求8所述的双特异性抗体,其中每个单链结构域抗体都通过肽接头与VH或VL融合。
10.根据权利要求9所述的双特异性抗体,其中所述肽接头具有长于7个氨基酸的长度。
11.根据权利要求9或10所述的双特异性抗体,其中所述肽接头具有短于50个氨基酸的长度。
12.一种或多种编码权利要求1-11中任一项所述的抗体或片段的多核苷酸。
13.一种包含权利要求12所述的一种或多种多核苷酸的细胞。
14.一种治疗癌症的方法,包括向癌症患者施用有效量的权利要求1-11中任一项所述的抗体或片段。
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EP4438624A1 (en) * | 2023-03-27 | 2024-10-02 | LAVA Therapeutics N.V. | Antibodies that bind nectin-4 and gamma-delta t cell receptors |
WO2024200573A1 (en) * | 2023-03-27 | 2024-10-03 | LAVA Therapeutics N.V. | Nectin-4 binding agents and methods of use |
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CN114702589B (zh) | 2022-11-01 |
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