CN115190804A - 通过重定向内源性去泛素化酶稳定靶向蛋白质的组合物和方法 - Google Patents
通过重定向内源性去泛素化酶稳定靶向蛋白质的组合物和方法 Download PDFInfo
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Abstract
本公开尤其提供了二价纳米抗体分子和使用本文公开的二价纳米抗体分子在受试者中治疗或改善疾病例如长QT综合征或囊性纤维化的影响的方法。还提供了鉴定和制备靶向感兴趣的蛋白质的纳米抗体结合剂的方法。
Description
相关申请的交叉引用
本申请要求于2020年1月14日提交的美国临时专利申请序列号62/961,082的权益,该申请的全部内容通过引用并入本文。
技术领域
本公开尤其提供了二价纳米抗体分子和使用这种二价分子治疗或改善受试者中疾病例如长QT综合征或囊性纤维化的影响的方法。
通过引用序列表并入
本申请包含对与本申请同时提交的作为序列表文本文件“CU19201-seq.txt”的氨基酸和/或核苷酸序列的引用,文件大小未35KB,创建于2019年12月6日。根据37C.F.R.§1.52(e)(5),上述序列表通过引用整体并入本文。
政府资助
本发明是在国立卫生研究院授予的批准号HL 122421的政府支持下完成的。政府对本发明享有一定的权利。
背景技术
蛋白质稳定性对于细胞中所有蛋白质的正常功能是至关重要的。许多疾病过程源于一种或多种蛋白质的稳定性或表达的缺陷,其范围从使离子通道不稳定的遗传突变(即囊性纤维化,CFTR)到病毒介导的宿主防御的消除(即MHCI受体)和肿瘤细胞增殖中细胞周期抑制剂的降解(即p27、p21)。泛素是关键的翻译后修饰,其是蛋白质周转和降解的主要调节剂。然而,泛素信号传导的广泛生物学作用和混杂性在开发靶向该途径以选择性地稳定给定的感兴趣蛋白质的治疗剂中提供了显著的障碍。
泛素化由三种酶(E1、E2、E3)的逐步级联介导,导致76个残基的泛素与靶蛋白暴露的赖氨酸共价连接。泛素本身含有七个赖氨酸(K6、K11、K27、K29、K33、K48、K63),其与其N-末端(Met1)一起可以用作第二连接点,从而产生多种聚合物链,差异解释为分选、运输或降解信号。泛素化与遗传性疾病(囊性纤维化、心律失常、癫痫和神经性疼痛)、代谢调节(胆固醇稳态)、感染性疾病(病毒和细菌病原体劫持宿主系统)和癌症生物学(肿瘤抑制因子的降解、逃避免疫监视)有关。
去泛素化酶(DUB)是专门的异肽酶,其通过泛素链的修正和去除为泛素信号传导提供显著性。存在超过100种人DUB,包含6个不同的家族:1)泛素特异性蛋白酶(USP)家族,2)卵巢肿瘤蛋白酶(OUT)家族,3)泛素羧基末端水解酶(UCH)家族,4)Josephin结构域家族(Josephin),5)与含有泛素的新型DUB家族相互作用的基序(MINDY),和6)JAB1/MPN/Mov34金属酶结构域家族(JAMM)。每一类DUB具有其自身独特的催化性质,其中USP家族水解所有泛素链类型,与JAMM和OTU家族形成鲜明对比,其含有多种具有独特泛素连接偏好的酶组。最近,DUB作为药物靶标引起了人们的兴趣,多家公司都在寻求DUB抑制剂。然而,由于DUB调节途径中的混杂性,靶向DUB用于治疗具有挑战,其中单个DUB通常靶向多种蛋白质底物,并且特定底物可以通过多种DUB类型调节。
以离子通道/受体的异常运输、稳定性和功能障碍为特征的离子通道病构成了人类疾病中显著未满足的临床需求。遗传性离子通道病是罕见的疾病,其涵盖神经系统(癫痫、偏头痛、神经性疼痛)、心血管系统(长QT综合征、Brugada综合征)、呼吸系统(囊性纤维化)、内分泌系统(糖尿病、高胰岛素血症性低血糖症)和泌尿系统(Bartter综合征、尿崩症)中的广泛病症。尽管下一代基因组测序已经揭示了成千上万的通道突变(具有不同的潜在病理学机制)的快速扩展列表,但是这些罕见的疾病几乎完全是对症治疗的。例如,囊性纤维化(白种人中最常见的致死性遗传疾病)由于囊性纤维化跨膜传导调节因子(CFTR)(一种氯离子通道)的缺陷而产生。研究最多的突变(ΔF508)占所有病例的~85%,并导致通道错误折叠和泛素依赖性运输缺陷。在另一种破坏性疾病长QT综合征中,两个通道(KCNQ1、hERG)中超过500个突变涵盖了所有遗传病例的近90%。两个通道中的运输缺陷是大多数致病突变的机制基础。因此,理解功能丧失的根本原因对于采用个性化策略来治疗每种疾病中的根本功能缺陷是至关重要的。
发明概要
本公开提供了二价分子,其包含:a)去泛素化酶(DUB)结合剂;b)靶结合剂;和c)所述DUB结合剂和所述靶结合剂之间的可变接头,其中所述DUB结合剂选自细胞内抗体片段、scFv、纳米抗体、抗体模拟物、单体拟抗体(monobody)、DARPins、脂质运载蛋白和靶向序列。
本公开还提供了一种治疗或改善受试者疾病影响的方法,包括向受试者施用有效量的本文公开的二价分子。
本公开还提供了一种鉴定和制备靶向感兴趣的蛋白质的纳米抗体结合剂的方法,包括:a)构建表达合成纳米抗体的天然(naive)酵母文库;b)将天然酵母文库与感兴趣的蛋白质一起孵育;c)通过磁激活细胞分选(MACS)选择表达与感兴趣的蛋白质结合的纳米抗体的酵母细胞;d)扩增选定的细胞并构建富集酵母文库;e)将富集的酵母文库与感兴趣的蛋白质一起孵育;f)通过荧光激活细胞分选(FACS)选择表达与感兴趣的蛋白质结合的纳米抗体的酵母细胞;g)扩增选定的细胞并构建进一步富集酵母文库;h)重复步骤e)至g)两次;i)将选定的酵母细胞分选为单细胞并培养为单克隆菌落以进行结合验证和质粒分离。
附图的简要说明
申请文件包含至少一幅彩色附图。具有彩色附图的本专利或专利申请公开的副本将在请求并支付必要费用后由专利局提供。
以下附图构成本说明书的一部分,并且被包括在内以进一步说明本公开的某些方面。通过参考这些附图中的一个或多个并结合本文给出的具体实施方式的详细描述,可以更好地理解本公开。
图1A-1H显示enDUB逆转NEDD4L介导的KCNQ1泛素化。图1A是通过enDUB(nano,PDB:3K1K)靶向去泛素化的示意图。插图,OTUD1和enDUB-O1的模块结构域。在图1B中,左,用抗KCNQ1抗体作为探针从表达KCNQ1-YFP±NEDD4L,单独nano或enDUB-O1的HEK293细胞中进行KCNQ1的拉下实验(KCNQ1 pulldown)。右,在洗脱(stripping)之后印记之前,抗泛素标记KCNQ1拉下。图1C显示通过抗泛素与抗KCNQ1信号强度的比率计算的相对KCNQ1泛素化(n=4;平均值)。**p<0.002,使用Tukey’s多重比较检验的单因素方差分析。图1D提供了流式细胞图,其显示了表达BBS-KCNQ1-YFP的细胞中的表面(BTX647荧光)和总(YFP荧光)KCNQ1表达。基于对单色对照的分析,垂直和水平线分别代表YFP和BTX647阳性细胞的阈值。从YFP和CFP阳性细胞(n≥5000个细胞/实验;N=4;平均值±s.e.m)分析,图1E和1F显示了表面(图1E)和总KCNQ1表达(图1F)的流式细胞术实验的定量分析。数据以对照组(KCNQ1没有NEDD4L(虚线))的值进行标准化。*p<0.01,非配对双尾学生t检验。图1G显示了来自CHO细胞中全细胞膜片钳测量的KCNQ1电流的示例性家族。图1H提供了nano(黑色圆圈,n=9)、nano+NEDD4L(红色正方形,n=9)和enDUB-O1+NEDD4L(蓝色三角形,n=12)的群体I-V曲线。*p<0.01,与nano+NEDD4L相比、Tukey’s的多重比较测试的双因素方差分析。
图2A-2I显示了enDUB挽救了运输缺陷的突变LQT1通道。在图2A中,左,LQT1患者中沿KCNQ1的C端突变的示意图。右,从YFP和CFP阳性细胞(n≥5000个细胞/实验;N=3;平均值±s.e.m)分析,在单独nano(红色)或enDUB-O1(蓝色)的情况下,对LQT1突变通道的表面表达(BTX647)进行流式细胞实验的定量分析。数据以对照组WT KCNQ1的值(虚线)进行标准化。*p<0.05,非配对双尾学生t检验。右插图,用BTX647(品红色染料)染色,表达BBS标记的WTKCNQ1-YFP(上)或G589D-YFP+nano(中)或enDUB-O1(下)的活细胞的共聚焦图像。图2B显示了在CHO细胞中重构的WT和突变KCNQ1电流的示例性家族。图2C显示了WT+nano(黑色正方形,n=10)、R591H+nano(粉红色三角形,n=8)和R591H+nano+ML277(红色三角形,n=13)的群体I-V曲线。**p<0.001,使用Tukey’s多重比较检验的双因素方差分析。图2D显示了R591H+enDUB-O1(浅蓝色圆圈,n=9)和R591H+enDUB-O1+ML277(蓝色圆圈,n=9)的群体I-V曲线。WT KCNQ1和R591H+nano的数据复制自图2C(黑色和粉色线)。*p<0.01,**p<0.001,使用带有Tukey’s多重比较检验的双因素方差分析。图2E显示了表达WT KCNQ1-YFP(上)或G589D-YFP+nano(中)或enDUB-O1(下)的成年豚鼠心肌细胞的共聚焦图像。图2F显示了从表达WTKCNQ1-YFP(左;n=17)或G589D-YFP(右)+单独的nano(红色;n=16)或enDUB-O1(蓝色;n=14)的心肌细胞缓慢电压斜坡上升到+100mV的平均电流响应(平均值±s.e.m)。图2G显示了来自f中所示数据的单个细胞在+100mV时的Ipeak的定量分析(平均值±标准差)。*p<0.03,**p<0.002,使用Tukey’s多重比较检验的单因素方差分析。图2H显示了来自表达WTKCNQ1-YFP(左)或G589D-YFP(右)+单独nano(红色)或enDUB-O1(蓝色)的心肌细胞的代表性动作电位记录。图2I显示了90%复极化(APD90)时动作电位持续时间的定量分析(n=11–13;平均值±标准差)。**p<0.0002,使用Tukey’s多重比较检验的单因素方差分析。
图3A-3K显示了enDUB与Orkambi联合促进了突变CFTR通道的新挽救。图3A是BBS-CFTR-YFP通道中六个CF患者突变(II、VI类)的示意图。插图,USP21和enDUB-U21的模块化组件。从YFP和CFP阳性细胞(n≥5000个细胞/实验;N=3;平均值±s.e.m)分析,图3B显示了在单独的nano(黑色)或+鲁玛卡托(3μM)(红色)和单独的enDUB-O1(蓝色)或+鲁玛卡托(3μM)(绿色)的情况下,对CFTR突变通道的表面表达(BTX647)进行流式细胞实验的定量分析。数据以对照组WT CFTR的值(虚线)进行标准化。*p<0.02,**p<0.0001,双因素方差分析,然后是Dunnett’s检验。图3C显示了来自HEK293细胞中全细胞膜片钳测量的基础、毛喉素激活(10μM)和CFTRinh-172处理(10μM)的WT CFTR电流的示例性家族。图3D显示了基础(黑色方块,n=16)和毛喉素激活(红色方块,n=16)的WT CFTR电流的群体I-V曲线。图3E-3G显示了来自未转染细胞(图3E);和表达4326delTC细胞(图3F);表达N1303K CFTR突变体细胞(图3G)的基础和毛喉素激活的示例性家族。图3H显示了4236delTC突变通道在VX809处理(3μM)24小时和与nano(左)或enDUB-U21(右)共表达后的毛喉素激活、VX770增强(5μM)电流的示例性家族。图3I显示了来自表达nano的4326delTC突变体(黑色圆圈,n=17)与VX809处理的表达nano的4326delTC突变体(红色正方形,n=15)或enDUB-U21(绿色三角形,n=14)的毛喉素激活、VX770增强电流的群体I-V曲线。对于N1303K突变通道(n≥8),图3J和3K提供与图3H和3J相同的格式。**p<0.0001,使用Tukey’s多重比较检验的双因素方差分析。
图4A-4G显示了靶向CF的enDUB联合疗法在功能上挽救了FRT细胞中常见和罕见的运输缺陷型CFTR突变。图4A显示了改装自Liu等人,2017(PDB:5UAK)的全长CFTR通道的结构。NBD1以红色突出显示。在图4B中,顶部显示了通过酵母表面展示库进行纳米抗体选择的示意图。底部显示了用靶结合剂(红色)对酵母文库进行MACS/FACS富集后的示例性流式细胞图。在图4C中,顶部显示了在共表达Cerulean-nb.E3h(供体)和Venus-CFTR(受体)的HEK293细胞中进行FRET结合测定的示意图。底部显示了流式细胞FRET结合曲线,其中FRET供体效率作为游离受体的函数,Cerulean-nb.E3h(蓝色)和单独的Cerulean对照(黑色)(n≥10,000个细胞/实验;N=2)。图4D显示了在VX809处理24小时后稳定表达WT CFTR(左)或N1303K和共表达单一CFP(中)或enDUB-U21CF.E3h(右)的FRT细胞中毛喉素激活、VX770增强电流的示例性家族。图4E显示了与VX809处理的、毛喉素激活的和VX770增强的表达单独的CFP(绿色三角形,n=12)或enDUB-U21CF.E3h(蓝色三角形,n=10)的N1303K细胞相比,毛喉素激活的WT(黑色圆圈,n=7)和N1303K(红色正方形,n=8)细胞的群体I-V曲线。**p<0.0005,使用带有Tukey’s多重比较检验的双因素方差分析。图4F显示了在稳定表达WT CFTR(左)或和VX809处理24小时后F508del与nb.T2a(中)或enDUB-U21CF.E3h(右)共表达的FRT细胞中毛喉素激活、VX770增强电流的示例性家族。图4G显示了与VX809处理的、毛喉素激活的和VX770增强的表达单独的CFP(棕色菱形,n=8)、nb.T2a(绿色三角形,n=11)或enDUBU21CF.T2a(蓝色三角形,n=12)的N1303K细胞相比,毛喉素激活的WT(黑色圆圈,n=7)和F508del(红色正方形,n=8)细胞的群体I-V曲线。**p<0.005,使用Tukey’s多重比较检验的双因素方差分析。
图5A-5C显示了enDUB-O1需要催化活性和靶特异性用于KCNQ1通道的泛素依赖性挽救。在图5A中,(左)显示了实验策略的示意图;BBS-Q1-YFP与单独的纳米抗体(灰线)、NEDD4L+nano(红线)或NEDD4L+enDUB-O1(蓝线)共转染。(右)来自流式细胞仪分析的Alexa647荧光累积分布直方图。从YFP和CFP阳性细胞群生成的图(n≥5000个细胞/实验;N=2)。图5B显示了与图5A相同的实验,但使用了具有C320S的无催化活性的enDUB-O1*。图5C显示了与图5A相同的实验,但使用未标记的BBS-Q1与enDUB-O1共表达作为靶特异性的对照。
图6A-6B显示了与WT和V524G通道相比,G589D LQT1突变的泛素状态没有增强。图6A显示了用抗KCNQ1抗体作为探针从表达WT、G589D和V524G KCNQ1-YFP通道与单独的nano(左)或enDUB-O1(右)(两个独立实验的代表)的HEK293细胞KCNQ1拉下的蛋白质免疫印迹。图6B显示了在洗脱来自图6A的蛋白质印迹后KCNQ1拉下的抗泛素标记。
图7A-7E显示了enDUB处理挽救了总KCNQ1表达,但不能挽救N-末端和ERAD相关的LQT1突变的表面运输。图7A是沿KCNQ1的N端的两个ERAD相关LQT1患者突变的示意图。图7B显示了表达WT BBS-KCNQ1-YFP+纳米抗体(左,对照,黑色)和L114P突变体+nano(中间,红色)或enDUB-O1(右,蓝色)的细胞中总Q1表达(YFP荧光)的流式细胞术分析。图7C显示了图7B所示实验(左)和Y111CKCNQ1突变体的类似实验(右)的YFP荧光累积分布直方图。从YFP和CFP阳性细胞群体生成的图(n≥5000个细胞/实验;N=2)。图7D和7E显示了表面Q1表达的流式细胞分析和累积分布直方图(Alexa647荧光),使用与图7B和7C相同的格式。
图8A-8B显示了在N1303K CFTR突变通道的表面挽救中,enDUB-U21比enDUB-O1具有更大的功效。图8A显示了表达WT BBS-CFTR-YFP+nano(虚线)和与单独的nano(红色线)、enDUB-O1(青色线)和enDUB-U21(蓝色线)共表达的N1303K突变的细胞的流式细胞分析的Alexa647荧光的累积分布直方图。从YFP和CFP阳性细胞群体生成的图(n≥5000个细胞/实验;N=2)。图8B显示了与图8A相同的实验设计,但nano(绿色线)、enDUB-O1(青色线)和enDUB-U21(蓝线)与VX809孵育24小时。
图9A-9C显示了enDUB-U21需要催化活性和靶特异性用于CFTR突变体的泛素依赖性挽救。在图9A中,(左)显示了实验策略的示意图;WT BBS-CFTR-YFP+nano(虚线)或与nano共转染的N1303K突变体(红线)或与enDUB-U21共转染的N1303K突变体(蓝线)。流式细胞仪分析的Alexa647荧光的累积分布直方图(中)和定量分析(右)。从YFP和CFP阳性细胞群体生成的图(n≥5000个细胞/实验;N=3;平均值±s.e.m)。数据以对照组WT CFTR的值(虚线)进行标准化。图9B显示了与图9A相同的实验,但使用了具有C221S的无催化活性的enDUB-U21*。图9C显示了与图9A相同的实验,但使用靶向mCherry纳米抗体,m-enDUB-U21,作为靶特异性的对照。
图10A-10B显示了enDUB-U21与鲁玛卡托±依伐卡托联合增加了4326delTC CFTR突变通道的功能挽救。图10A显示了4236delTC突变通道在VX809处理(3μM)24小时后,与nano(左)或enDUB-U21(右)共表达的基础(顶部,黑色)、毛喉素激活(中间,红色)和VX770增强(底部,绿色)电流的示例性家族。图10B显示了来自与单独nano(左;n=15)或enDUB-U21(右;n=14)共表达的4326delTC突变体的基础(黑色方块)、毛喉素激活(红色圆圈)和VX770增强(绿色三角形)电流的群体I-V曲线。
图11A-11B显示了enDUB-U21与鲁玛卡托±依伐卡托联合增加了N1303K CFTR突变通道的功能挽救。图11A显示了N1303K突变通道在VX809处理(3μM)24小时后,与nano(左)或enDUBU21(右)共表达的基础(顶部,黑色)、毛喉素激活(中间,红色)和VX770增强(底部,绿色)电流的示例性家族。图11B显示了来自与单独nano(左;n=9)或enDUB-U21(右;n=11)共表达的N1303K突变体的基础(黑色方块)、毛喉素激活(红色圆圈)和VX770增强(绿色三角形)电流的群体I-V曲线。
图12A-12B显示了来自酵母表面展示纳米抗体文库的NBD1结合剂的开发。图12A显示了使用纯化的FLAG-NBD1的系列稀释液对9个纳米抗体克隆的在酵母上的结合亲和力测量。图12B显示了单独的WT CFTR表面密度(虚线)或与纳米抗体克隆共表达时的流式细胞表面标记测定和累积分布直方图。
图13A-13F显示了enDUB-U21CF.E3h与Orkambi联合在功能上挽救了HEK293细胞中不同的II类和VI类引起CF的突变。图13A是YFP传感器卤离子猝灭测定的示意图。图13B显示了在HEK293细胞中YFP猝灭的示例性轨迹,其中该细胞仅表达mCh(灰色)或mCh标记的4326delTC突变体(红色),以及4326delTC突变体用VX809(绿色)或VX809+enDUB-U21CF.E3h(蓝色)处理(添加毛喉素和VX770后)。图13C显示了碘流入率(n=9)的总结。**p<0.0001,与4326delTC相比,采用Tukey’s多重比较检验的单因素方差分析。对于N1303K突变通道(n=8),图13D和13E显示了与图13B和13C相同的格式。**p<0.0001,与N1303K相比,采用Tukey’s多重比较检验的单因素方差分析。图13F显示了来自mCh标记的WT CFTR通道(黑色圆圈,n=41)或4326delTC突变体用VX809处理并共表达单独的CFP(红色正方形,n=29)、nb.E3h(绿色三角形,n=9)或enDUB-U21CF.E3h(蓝色三角形,n=12)的基础(左)、毛喉素激活(中)和VX770增强(右)电流的群体I-V曲线。*p<0.02,**p<0.0001,采用Tukey’s多重比较检验的双因素方差分析。
图14A-14C显示了enDUB-U21CF.T2a与鲁玛卡托±依伐卡托联合挽救了HEK293细胞中F508del突变通道的运输和功能。图14A显示了流式细胞FRET结合曲线,FRET供体效率作为游离受体的函数,Cerulean-nb.T2a(绿色)和单独的Cerulean对照(黑色)(n≥10,000个细胞/实验;N=2)。图14B显示了从YFP和CFP阳性细胞分析(n≥5000个细胞/实验;N=4;平均值±s.e.m),在单独的CFP(红色)、enDUB-U21CF.E3h(橙色)、nb.T2a(绿色)或enDUB-U21CF.T2a(蓝色)存在的情况下,有或没有VX809处理(阴影或平坦),F508del突变通道的表面表达(BTX647)的流式细胞实验的定量分析。数据以WT CFTR对照组进行标准化,虚线代表只有VX809处理的F508del。p<0.05,与CFP+VX809相比,**p<0.0002,与全部相比,采用Tukey’s多重比较检验的单因素方差分析。图14C显示了来自mCh标记的WT CFTR通道(黑色圆圈,n=41)或VX809处理的与单独的CFP(红色正方形,n=8)、nb.T2a(绿色三角形,n=10)或enDUB-U21CF.T2a(蓝色三角形,n=9)的F508del突变体的基础(左)、毛喉素激活(中)和VX770增强(右)电流的群体I-V曲线。*p<0.05,**p<0.0001,采用Tukey’s多重比较检验的双因素方差分析。
图15A显示了囊性纤维化(CF)的潜在症状和当前治疗。图15B是详述泛素依赖性调节CFTR表面表达、稳定性和功能的示意图。正向运输路径以蓝色突出显示,反向运输路径以红色突出显示。
在图16A中,左,显示了示例性蛋白质靶标CFTR的结构。NBD1以红色突出显示。右,稳定酶DUB的结构。在图16B中,顶部,显示了通过酵母表面展示文库进行纳米抗体选择的示意图。底部,MACS/FACS富集靶结合剂后的流式细胞图(红色)。在图16C中,顶部,显示了基于纳米抗体的概念验证ReSTORx分子,ReSTORAb,由“活性”成分(DUB结合剂;蓝色)和“靶向”成分(NBD1结合剂;橙色)组成。底部,显示了每个组分的FRET分析和结合曲线。在图16D中,左,显示了CFTR表面标记测定和靶向CFTR的ReSTORAb共表达的示意图。右,显示来自ReSTORAb挽救突变通道的流式细胞图。图16E显示了与图16D相同的测定,其中USP2作为去泛素酶。图16F进一步显示了与图16D中相似的测定,其中存在鲁玛卡托(VX-809)。
图17显示了示例性的基于二价纳米抗体的ReSTORAb的示意图。
图18显示了基于二价纳米抗体的ReSTORAb能够挽救长QT综合征(LQTS)运输缺陷。
发明详述
本公开的一个实施方式是二价分子,其包含:a)去泛素化酶(DUB)结合剂;b)靶结合剂;和c)所述DUB结合剂和所述靶结合剂之间的可变接头,其中所述DUB结合剂选自细胞内抗体片段、scFv、纳米抗体、抗体模拟物、单体拟抗体、DARPins、脂质运载蛋白和靶向序列。
在一些实施方式中,DUB是内源性的。在一些实施方式中,DUB选自泛素特异性蛋白酶(USP)家族、卵巢肿瘤蛋白酶(OUT)家族、泛素羧基末端水解酶(UCH)家族、Josephin结构域家族(Josephin)、与含有泛素的新型DUB家族相互作用的基序(MINDY)和JAB1/MPN/Mov34金属酶结构域家族(JAMM)。在一些实施方式中,DUB是USP21或USP2。
在一些实施方式中,DUB结合剂选自细胞内抗体片段、scFv、纳米抗体、抗体模拟物、单体拟抗体、DARPins、脂质运载蛋白和靶向序列。在一些实施方式中,DUB结合剂是纳米抗体。在一些实施方式中,纳米抗体结合USP家族成员。在一些实施方式中,纳米抗体结合USP2。在一些实施方式中,纳米抗体结合USP21。在一些实施方式中,纳米体包含如SEQ IDNO:1至6中任一个所示的序列。在一些实施方式中,纳米体包含:a)如SEQ ID NO:7所示的互补决定区(CDR)1,如SEQ ID No:8所示的CDR2和如SEQ ID No:9所示的CDR3;b)如SEQ IDNo:10所示的CDR1、如SEQ ID No:11所示的CDR2和如SEQ ID No:12所示的CDR3;c)如SEQ IDNo:13所示的CDR1、如SEQ ID No:14所示的CDR2和如SEQ ID No:15所示的CDR3;d)如SEQ IDNo:16所示的CDR1、如SEQ ID No:17所示的CDR2和如SEQ ID No:18所示的CDR3;e)如SEQ IDNo:19所示的CDR1、如SEQ ID No:20所示的CDR2和如SEQ ID No:21所示的CDR3;或f)如SEQID No:22所示的CDR1、如SEQ ID No:23所示的CDR2和如SEQ ID No:24所示的CDR3。
在一些实施方式中,所述靶结合剂结合的靶标的异常泛素化引起疾病。在一些实施方式中,所述疾病是遗传性离子通道病。如本文所用,术语“遗传性离子通道病”是指罕见疾病,其涵盖神经系统、心血管系统、呼吸系统、内分泌系统和泌尿系统中的广泛病症。在本公开中,“遗传性离子通道病”包括但不限于:癫痫、偏头痛、神经性疼痛、心律失常、长QT综合征、Brugada综合征、囊性纤维化、糖尿病、高胰岛素血症性低血糖症、Bartter综合征和尿崩症。在一些实施方式中,所述疾病是长QT综合征。在一些实施方式中,所述疾病是囊性纤维化。
在一些实施方式中,靶结合剂结合的靶标是囊性纤维化跨膜传导调节因子(CFTR)。
在一些实施方式中,靶结合剂选自细胞内抗体片段、scFv、纳米抗体、抗体模拟物、单体拟抗体、DARPins、脂质运载蛋白和靶向序列。在一些实施方式中,靶结合剂是纳米抗体。在一些实施方式中,纳米抗体结合囊性纤维化跨膜传导调节因子(CFTR)的NBD1结构域。在一些实施方式中,纳米抗体包含如SEQ ID NO:25至38中任一个所示的序列。在一些实施方式中,纳米抗体包含:a)如SEQ ID NO:39所示的互补决定区(CDR)1、如SEQ ID No:40所示的CDR2和如SEQ ID No:41所示的CDR3;b)如SEQ ID No:42所示的CDR1、如SEQ ID No:43所示的CDR2和如SEQ ID No:44所示的CDR3;c)如SEQ ID No:45所示的CDR1、如SEQ ID No:46所示的CDR2和如SEQ ID No:47所示的CDR3;d)如SEQ ID No:48所示的CDR1、如SEQ ID No:49所示的CDR2和如SEQ ID No:50所示的CDR3;e)如SEQ ID No:51所示的CDR1、如SEQ IDNo:52所示的CDR2和如SEQ ID No:53所示的CDR3;f)如SEQ ID No:54所示的CDR1、如SEQ IDNo:55所示的CDR2和如SEQ ID No:56所示的CDR3;g)如SEQ ID No:57所示的CDR1、如SEQ IDNo:58所示的CDR2和如SEQ ID No:59所示的CDR3;h)如SEQ ID No:60所示的CDR1、如SEQ IDNo:61所示的CDR2和如SEQ ID No:62所示的CDR3;i)如SEQ ID No:63所示的CDR1、如SEQ IDNo:64所示的CDR2和如SEQ ID No:65所示的CDR3;j)如SEQ ID No:66所示的CDR1、如SEQ IDNo:67所示的CDR2和如SEQ ID No:68所示的CDR3;k)如SEQ ID No:69所示的CDR1、如SEQ IDNo:70所示的CDR2和如SEQ ID No:71所示的CDR3;l)如SEQ ID No:72所示的CDR1、如SEQ IDNo:73所示的CDR2和如SEQ ID No:74所示的CDR3;m)如SEQ ID No:75所示的CDR1、如SEQ IDNo:76所示的CDR2和如SEQ ID No:77所示的CDR3;或n)如SEQ ID No:78所示的CDR1、如SEQID No:79所示的CDR2和如SEQ ID No:80所示的CDR3。
在一些实施方式中,接头是烷基、聚乙二醇(PEG)或其他类似分子,或点击接头。如本文所用,“烷基”可以是支链的或直链的、取代的或未取代的。选择烷基的长度以最大化或至少基本上不干扰DUB结合剂和靶结合剂的有效结合。例如,“烷基”可以是C1-C25,例如C1-C20,包括C1-C15、C1-C10和C1-C5。因此,烷基接头可以包括C1、C2、C3、C4、C5、C6、C7、C8、C9、C10、C11、C12、C13、C14、C15、C16、C17、C18、C19、C20、C21、C22、C23、C24、C25或更高碳链。如本文所用,“点击接头”是一类用于生物偶联的生物相容性小分子,允许将选择的底物与特定生物分子连接。它基于在Kolb等人,(2001)“Click Chemistry:Diverse ChemicalFunction from a Few Good Reactions”,Angewandie Chemie International Edition,40(11):2004-2021中充分描述的“点击(click)”化学。
本公开的另一个实施方式是治疗或改善受试者中疾病影响的方法,包括向受试者施用有效量的本文公开的二价分子。
在一些实施方式中,受试者是人。在一些实施方式中,所述疾病选自遗传性离子通道病、癌症、心血管病症、感染性疾病和代谢性疾病。在一些实施方式中,所述遗传性离子通道病选自癫痫、偏头痛、神经性疼痛、心律失常、长QT综合征、Brugada综合征、囊性纤维化、糖尿病、高胰岛素血症性低血糖症、Bartter综合征和尿崩症。在一些实施方式中,遗传性离子通道病是囊性纤维化。
如本文所用,术语“治疗(treat)”、“治疗(treating)”、“治疗(treatment)”及其语法变体意指使个体受试者经受方案(protocol)、方案(regimen)、方法(process)或疗法(remedy),其中期望在该受试者(例如患者)中获得生理反应或结果。然而,因为每个治疗的受试者可能对特定的治疗方案(protocol)、方案(regimen)、方法(process)或疗法(remedy)没有反应,所以治疗不需要在每个和每一个受试者或受试者群体(例如患者群体)中实现期望的生理反应或结果。因此,给定的受试者或受试者群体(例如患者群体)可能对治疗没有反应或反应不充分。
如本文所用,术语“改善(ameliorate)”、“改善(ameliorating)”及其语法变体意指降低受试者(优选人)的疾病症状的严重程度。
本文所用的,“施用(administration)”、“施用(administering)”及其变体是指将组合物,例如合成的膜-接受体复合物或试剂引入受试者,并且包括同时和顺序引入组合物或药剂。通过任何合适的途径将组合物或药剂引入受试者,包括口服、肺部、鼻内、肠胃外(静脉内、肌肉内、腹膜内或皮下)、直肠、淋巴管内或局部。施用包括自我施用和由他人施用。合适的施用途径允许组合物或药剂发挥其预期功能。例如,如果合适的途径是静脉内,则通过将组合物或药剂引入受试者的静脉来施用组合物。可以通过任何合适的途径进行施用。
如本文所用,“受试者(subject)”是哺乳动物,优选人。除了人之外,本公开范围内的哺乳动物的类别包括例如农场动物、家养动物、实验室动物等。农场动物的一些实例包括牛、猪、马、山羊等。家养动物的一些实例包括狗、猫等。实验室动物的一些实例包括灵长类动物、大鼠、小鼠、兔、豚鼠等。
本公开的另一个实施方式是鉴定和制备靶向感兴趣的蛋白质的纳米抗体结合剂的方法,包括:a)构建表达合成纳米抗体的天然酵母文库;b)将天然酵母文库与感兴趣的蛋白质一起孵育;c)通过磁激活细胞分选(MACS)选择表达与感兴趣的蛋白质结合的纳米抗体的酵母细胞;d)扩增选定的细胞并构建富集酵母文库;e)将富集的酵母文库与感兴趣的蛋白质一起孵育;f)通过荧光激活细胞分选(FACS)选择表达与感兴趣的蛋白质结合的纳米抗体的酵母细胞;g)扩增选定的细胞并构建进一步富集酵母文库;h)重复步骤e)至g)两次;i)将选择的酵母细胞分选为单细胞并培养为单克隆菌落以进行结合验证和质粒分离。
在一些实施方式中,感兴趣的蛋白质是囊性纤维化跨膜传导调节因子(CFTR)。在一些实施方案中,感兴趣的蛋白质是去泛素化酶(DUB)。
附加定义
术语“氨基酸”是指天然存在的和合成的氨基酸,以及功能类似于天然存在的氨基酸的氨基酸类似物和氨基酸模拟物。天然存在的氨基酸是由遗传密码编码的氨基酸,以及后来被修饰的氨基酸,例如羟脯氨酸、γ-羧基谷氨酸和O-磷酸丝氨酸。“氨基酸类似物”是指具有与天然存在的氨基酸相同的基本化学结构的化合物,例如与氢、羧基、氨基和R基团结合的碳,例如高丝氨酸,正亮氨酸、甲硫氨酸亚砜、甲硫氨酸甲基锍。此类类似物可能具有修饰的R基团(例如正亮氨酸)或修饰的肽骨架,但保留与天然存在的氨基酸相同的基本化学结构。亚氨基酸,例如脯氨酸,也在此处使用的“氨基酸”的范围内。“氨基酸模拟物”是指具有与氨基酸的一般化学结构不同的结构,但功能类似于天然存在的氨基酸的化合物。
如本文所用,术语“多肽”、“肽”和“蛋白质”在本文中可互换使用,是指氨基酸残基的聚合物。该术语适用于其中一个或多个氨基酸残基是相应天然存在的氨基酸的人工化学模拟物的氨基酸聚合物,以及天然存在的氨基酸聚合物、含有修饰残基的那些聚合物和非天然存在的氨基酸聚合物。
本文所用的“核酸”或“寡核苷酸”或“多核苷酸”是指共价连接在一起的至少两个核苷酸,核酸的许多变体可用于与给定核酸相同的目的,因此,核酸也包括基本上相同的核酸及其互补物。
核酸可以是单链或双链的,或者可以含有双链和单链序列的部分。核酸可以是DNA、基因组和cDNA、RNA或杂合体,其中核酸可以含有脱氧核糖核苷酸和核糖核苷酸的组合,以及碱基的组合,所述碱基包括尿嘧啶、腺嘌呤、胸腺嘧啶、胞嘧啶、鸟嘌呤、肌苷、黄嘌呤次黄嘌呤、异胞嘧啶和异鸟嘌呤。核酸可以合成为单链分子或使用合成基因在细胞(体外或体内)中表达。核酸可以通过化学合成方法或通过重组方法获得。
核酸还可以是RNA,例如mRNA、tRNA、短发夹RNA(shRNA)、短干扰RNA(siRNA)、双链RNA(dsRNA)、转录基因沉默RNA(ptgsRNA)、Piwi-相互作用RNA、pri-miRNA、pre-miRNA、micro-RNA(miRNA)或抗miRNA。
如本文所用,术语“抗体”涵盖天然或部分或完全合成产生的免疫球蛋白及其片段。该术语还涵盖具有与免疫球蛋白结合域同源的结合结构域的任何蛋白质。这些蛋白质可以来自天然来源,或部分或全部合成产生。“抗体”还包括多肽,其包含来自特异性结合和识别抗原的免疫球蛋白基因或其片段的框架区。术语抗体的使用意在包含完整抗体、多克隆、单克隆和重组抗体及其片段,并且进一步包含单链抗体、人源化抗体、鼠抗体、嵌合、小鼠-人、小鼠-灵长类、灵长类-人单克隆抗体、抗独特型抗体、抗体片段,例如scFv、(scFv)2、Fab、Fab'和F(ab')2、F(ab1)2、Fv、dAb和Fd片段、双抗体、纳米抗体和抗体相关多肽。抗体包含双特异性抗体和多特异性抗体,只要它们表现出所需的生物活性或功能即可。
本文使用的术语“抗原结合片段”是指完整免疫球蛋白的片段,以及多肽的任何部分,包括具有特异性结合抗原能力的抗原结合区。例如,抗原结合片段可以是F(ab')2片段、Fab'片段、Fab片段、Fv片段或scFv片段,但不限于此。Fab片段具有一个抗原结合位点,包含轻链和重链可变区、轻链恒定区和重链第一恒定区CH1。Fab'片段与Fab片段的不同之处在于Fab'片段另外包括重链的铰链区,包括在重链CH1区的C末端处的至少一个半胱氨酸残基。F(ab')2片段是由Fab'片段的半胱氨酸残基在铰链区通过二硫键连接而产生。Fv片段是仅具有重链可变区和轻链可变区的最小抗体片段,并且用于产生Fv片段的重组技术是本领域公知的。双链Fv片段可以具有重链可变区通过非共价键与轻链可变区连接的结构。单链Fv(scFv)片段通常可以具有二聚体结构,如在双链Fv片段中,其中重链可变区通过肽接头与轻链可变区共价结合,或者重链和轻链可变区在其C端直接相互连接。抗原结合片段可以使用蛋白酶获得(例如,将整个抗体用木瓜蛋白酶消化得到Fab片段,用胃蛋白酶消化得到F(ab')2片段),并且可以通过基因重组技术制备。dAb片段由VH结构域组成。单链抗体分子可以包含具有多个单独分子的聚合物,例如二聚体、三聚体或其他聚合物。
本文所用的“载体”是指能够指导所需蛋白质表达的装配体。载体必须包括与感兴趣的基因可操作地连接的转录启动子元件。载体可以由脱氧核糖核酸(“DNA”)、核糖核酸(“RNA”)或两者的组合(例如,DNA-RNA嵌合物)组成。任选地,载体可以包括聚腺苷酸化序列、一个或多个限制性位点以及一个或多个选择性标记,例如新霉素磷酸转移酶或潮霉素磷酸转移酶。另外,取决于所选择的宿主细胞和所采用的载体,其他遗传元件例如复制起点、另外的核酸限制性位点、增强子、赋予转录诱导性的序列和选择性标记也可以掺入本文所述的载体中。
如本文所用,术语“细胞”、“宿主细胞”或“重组宿主细胞”是指已被工程化以表达所需重组蛋白的宿主细胞。产生重组宿主细胞的方法是本领域熟知的。例如,参见Sambrook等人(MOLECULAR CLONING:A LABORATORY MANUAL(Sambrook等人编,Cold Spring HarborLaboratory Press,Cold Spring Harbor,1989),Ausubel等人(CURRENT PROTOCOLS INMOLECULAR BIOLOGY,Ausubel等人编,John Wiley&Sons,New York,1987)。在本公开中,用本文所述的载体转化宿主细胞。
本文所用的重组宿主细胞可以是用于重组蛋白生产的任何宿主细胞,包括但不限于细菌、酵母、昆虫和哺乳动物细胞系。
如本文所用,术语“增加”、“增强”、“刺激”和/或“诱导”(和类似术语)通常是指相对于自然、预期或平均或相对于对照条件,直接或间接改善或增加浓度、水平、功能、活性或行为的行为。
如本文所用,术语“抑制”、“压制”、“减少”、“干扰”和/或“降低”(和类似术语)通常是指相对于自然、预期或平均或相对于对照条件,直接或间接降低浓度、水平、功能、活性或行为的行为。
这里使用的术语仅用于描述特定实施方式的目的,而不是限制性的。如在说明书和所附权利要求书中所使用的,单数形式“一(a)”、“一(an)”和“该(the)”包括复数指示物。除非上下文另有明确规定。
对于本文中的数值范围的叙述,明确考虑了其间具有相同精度的每个中间数字。例如,对于6-9的范围,除了6和9之外还涵盖数字7和8,并且对于范围6.0-7.0,明确涵盖数字6.0、6.1、6.2、6.3、6.4、6.5、6.6、6.7、6.8、6.9和7.0。
提供以下实施例以进一步说明本公开的某些方面。这些实施例仅是说明性的,并不旨在以任何方式限制本公开的范围。
实施例
实施例1
靶向去泛素化可挽救运输缺陷型离子通道病
离子通道中的遗传性或从头突变是多种疾病(称为离子通道病)的基础,包括心律失常、癫痫和囊性纤维化(Kullmann,2010;Bohnen等人,2016;Cutting,2014)。通道向细胞表面的运输受损是许多不同离子通道病的基础(Curran和Mohler,2015年),这是一种共享机制,其代表了一个尚未开发的机会来制定治疗不同罕见疾病的共同策略。泛素化是一种普遍的翻译后修饰,它在离子通道中通过抑制正向运输、增强内吞作用和促进降解来限制其表面密度(Foot等人,2017;MacGurn等人,2012)。在这里,我们表明靶向去泛素化可以挽救由运输缺陷突变离子通道引起的不同疾病。我们开发了工程化去泛素酶(enDUB),其特点是纳米抗体与最小的去泛素酶催化成分融合,能够从目标通道中选择性地去除泛素链(表1)。这种靶向去泛素化方法成功地挽救了不同突变离子通道(KCNQ1和囊性纤维化跨膜调节因子(CFTR))的表面运输和功能电流,它们分别导致长QT综合征(LQT1)和囊性纤维化(CF)。在LQT1的豚鼠心室心肌细胞模型中,enDUB治疗挽救了缓慢延迟的整流K+电流和正常的动作电位持续时间。此外,当与FDA批准的疗法Orkambi联合使用时,以CFTR为靶点的enDUB在CF突变的功能挽救中表现出显著的协同作用。因此,我们引入靶向去泛素化作为一种强大的通用方法来分类和挽救多种疾病,其中离子通道向细胞表面的运输受损是主要机制。
离子通道的遗传性或从头突变导致的离子通道病是跨越神经系统(癫痫、偏头痛、神经性疼痛)(Kullmann,2010)、心血管系统(长QT综合征、Brugada综合征)(Bohnen等人,2016)、呼吸系统(囊性纤维化)(Cutting,2014年)、内分泌系统(糖尿病、高胰岛素血症性低血糖症)(Ashcroft和Rorsman,2013年)和泌尿系统(Bartter综合征、尿崩症)系统(Imbrici等人,2016年)的各种疾病的基础。数百个致病突变通常存在于单个离子通道中,对治疗提出了巨大的挑战。基于机制的方法来纠正通常可以跨不同离子通道适用的潜在异常将是有利的,但缺乏该方法(Imbrici等人,2016;Wulff等人,2019)。
长QT综合征1型(LQT1)和囊性纤维化(CF)分别由KCNQ1(Kv7.1)(Bohnen等人,2016;Tester等人,2005)和囊性纤维化跨膜调节因子(CFTR)(Cutting,2014)通道中的功能丧失突变引起。LQT1增加了劳累引发心律失常和心源性猝死的风险,而CF患者表现出气道粘液清除受损,导致反复细菌感染、不受控制的炎症、肺损伤和预期寿命低。对于KCNQ1和CFTR,许多功能丧失突变的一个显著机制是通道向表面的运输受损(Wilson等人2005;Haardt等人,1999;Cheng等人,1990)。本公开利用这种共享机制来开发一种方法,该方法适合于治疗开发并且可以应用于多种离子通道。
因为泛素化/去泛素化是离子通道表面密度的主要决定因素(图1A),假设从突变通道中去除泛素将挽救运输缺陷的离子通道。由于泛素化是一种广泛存在的生理现象,因此目标是开发一种靶向去泛素化方法,以规避通常与靶向泛素/蛋白酶体系统相关的脱靶效应的问题(Nalepa等人,2006;Huang和Dixit,2016)。最初,我们使用了YFP标记的KCNQ1,这是一种已知在蛋白质和功能水平上被E3泛素连接酶NEDD4L下调的K+离子通道(Jespersen等人,2007)。我们通过将卵巢肿瘤去泛素酶1(OTUD1)(一种对K63多泛素链水解具有内在偏好的去泛素酶)(Mevissen等人,2013年)的最小催化单元与纳米抗体融合开发了YFP-靶向工程化去泛素酶(enDUB-O1),所述纳米抗体对GFP/YFP是特异性的,但对CFP18不是(图1A,插图)。我们在瞬时转染的HEK293细胞中使用生化和功能测定法测试了enDUB-O1的功效和选择性(图1B-1H)。
在表达KCNQ1-YFP和纳米抗体(nano)的对照细胞中的免疫沉淀实验显示了泛素化KCNQ1通道的强烈表达,反映了内源E3连接酶活性(图1B和1C)。与KCNQ1-YFP和抗GFP纳米抗体(nano)共表达的NEDD4L导致KCNQ1水平降低(图1B,左),但泛素信号增加(图1B和1C)。在NEDD4L存在的情况下,enDUB-O1挽救了KCNQ1的表达,并阻止了通道泛素化的增加(图1B和1C)。
进行流式细胞术测定以同时测量KCNQ1-YFP总表达和表面密度,并评估enDUB-O1(使用P2A自切割肽质粒与CFP以1:1的比例表达)拮抗NEDD4L对这两个指标的影响的能力。NEDD4L显著降低了KCNQ1的表面密度(通过荧光银环蛇毒素与细胞外表位标签的结合来评估)和总表达(通过YFP荧光来评估),并且这两种效果都被enDUB-O1逆转(图1D-1F)。催化死亡的enDUB-O1*没有挽救KCNQ1-YFP表面密度,证明DUB酶活性对于这种效果是必要的(图5B)。此外,enDUB-O1没有挽救缺乏YFP标签的KCNQ1通道的表面密度,证实了靶向enDUB方法的特异性(图5C)。
全细胞膜片钳电生理学用于确定enDUB-O1挽救的KCNQ1通道的功能。表达KCNQ1+nano的对照细胞显示出强大的KCNQ1电流,这些电流被NEDD4L的表达消除(图1G和1H);共表达enDUB-O1完全挽救了KCNQ1电流(图1G和1H),证实了用enDUB进行靶向去泛素化以特异性去泛素化和稳定细胞表面感兴趣的功能通道的功效。
下一个问题是,enDUB是否会挽救作为LQT1基础的运输缺陷型突变KCNQ1通道。我们使用流式细胞术来确定在KCNQ1的C-末端的14种不同LQT1突变(Tester等人,2005;Aromolaran等人,2014),以及先前描述的N末端内质网相关降解(ERAD)依赖性突变(L114P)(Peroz等人,2009)(图2A)对于通道表面密度的影响。除了L114P之外,与WT KCNQ1相比,14个C-末端突变中的9个显示出显著减少的表面运输(图2A,红色bar)。值得注意的是,6个突变通道的表面密度部分或完全被enDUB-O1共表达挽救(图2A,蓝色bar和插图)。响应突变通道沿着KCNQ1卷曲螺旋四聚结构域(螺旋D)聚集,定义了一个空间“热点”,适合于enDUB介导的运输挽救(图2A,紫色文本)。
在功能上,与WT KCNQ1相比,同源四聚体R591H通道显示出显著降低的电流,这与其受损的表面运输一致(图2B和2C)。KCNQ1激活剂ML277(Mattmann等人,2012)(1μM)的应用适度增加了R591H电流,这意味着表面有一小部分通道(图2B和2C)。共表达enDUB-O1显著地将R591H电流挽救至WT KCNQ1的约50%,鉴于表面密度的完全挽救(图2A),表明突变导致开放频率(probability)或电导的额外损害(图2B和2D)。引人注目的是,ML277显着增加了enDUB-O1挽救的R591H电流幅度,超过了WT KCNQ1水平(图2B和2D)。
LQT1通常以常染色体显性方式遗传,其中患者拥有一个WT和一个突变等位基因(Bohnen等人,2016)。因此,我们接下来试图在IKs对心脏动作电位复极化很重要的物种的心肌细胞中重述LQT1的异四聚体本质。我们使用腺病毒在分离的成年豚鼠心肌细胞中表达YFP标记的WT或LQT1突变体KCNQ1通道(图2E)。与表达WT KCNQ1的心肌细胞相比,具有G589D的心肌细胞通过缓慢的电压斜坡测量到+100mV显示出延迟外向电流减少(图2F和2G),动作电位持续时间(APD)显著延长(图2H和2I),这是LQTS的特征。值得注意的是,enDUB-O1治疗表达G589D的心肌细胞将IKs和APD恢复到WT KCNQ1水平(图2F-2I)。
值得注意的是,突变体KCNQ1对enDUB-O1介导的挽救的适应性与通道泛素化水平3/4不是简单地相关,例如,G589D的基线泛素信号低于用V524G观察到的泛素信号,所述V524G是不能被enDUB-O1挽救的突变(图6A-6B)。此外,enDUB-O1挽救了ERAD敏感的L114P和Y111C的总蛋白表达,但没有挽救其表面密度(图7A-7E),这表明有额外的机制可以防止错误折叠的蛋白质正向运输,而不管它们的泛素化状态如何。
为了确定enDUB是否可以类似地挽救不同离子通道病中的功能通道,其中运输受损是主要的根本原因,我们转向囊性纤维化(CF),这是一种由CFTR(Cl-离子通道)功能丧失突变引起的破坏性单基因疾病。超过2000种不同的CF突变已被映射到CFTR,其中许多因折叠/运输受损(II类)或质膜稳定性降低(VI类)导致降低通道表面密度(Veit等人,2016;Boeck和Amaral,2016)。从高通量筛选中发现的药理学伴侣(校正剂)和门控修饰剂(增强剂)导致了FDA批准的联合疗法,Orkambi,其由鲁玛卡托(lumacaftor)(VX809;校正剂)和依伐卡托(ivacaftor)(VX770;增强剂)组成,用于治疗纯合F508del突变(Wainwright等人,2015;Goor等人,2011;Goor等人,2009)。然而,Orkambi的临床疗效通常不是最理想的(用力呼气量变化约3%),并且大量CFTR突变对治疗无效,强调迫切需要开发补充疗法(Boeck和Amaral,2016;Farinha和Matos,2016)。
BBS标记的YFP-CFTR被设计成能够使用流式细胞术同时评估总通道表达和表面密度,并探测先前分类为II类(F508del、R560T、N1303K)或VI类(Q1412X、4279insA、4326delTC)的六种不同突变的影响(图3A)。与WT CFTR相比,所有六种突变都显著损害了通道表面密度(图3B,黑色bar)。将细胞与鲁玛卡托预孵育24小时并没有增加F508del和R560T的表面表达,但改善了剩余4个突变的运输(图3B,红色bar),提供了一个黄金标准校正基准,用于评估enDUB的功效。我们使用了第二个enDUB(enDUB-U21),其包含泛素特异性蛋白酶USP21的催化成分(图3A),其去除所有泛素连接类型(Faesen等人,2011)。在试点实验中,与enDUB-O1相比,enDUB-U21在挽救CFTR运输方面更有效(图8A-8B),导致我们采用该形式进行CFTR实验。与鲁玛卡托类似,enDUB-U21并没有显著挽救F508del和R560T表面密度;然而,它在纠正其他四个突变方面同等或更加有效,其中两个(N1303K和4279insA)被挽救至WT CFTR水平(图3B,蓝色bar)。DUB活性和CFTR靶向都是逆转运输缺陷所必需的,因为催化失活的enDUB-U21和mCh靶向的enDUB-U21没有改善YFP标记的N1303K的表面表达(图9A-9C)。最重要的是,共同应用鲁玛卡托和enDUB-U21产生了突变CFTR的表面密度的协同挽救,表明一种新的CF联合校正疗法(图3B;绿色bar)。
关键的下一步是确定enDUB-U21挽救的突变CFTR通道是否有功能。我们聚焦于N1303K和4326delTC,其分别代表II类和VI类突变。表达WT CFTR的HEK293细胞表现出强大的毛喉素激活的氯离子电流,这些电流被CFTR抑制剂阻断(图3C和3D),并且在未转染的细胞中未观察到(图3E)。相比之下,单独表达N1303K或4326delTC的细胞没有产生毛喉素诱导的电流(图3F和3G),这与其有限的表面运输和作为致病突变体的状态一致。在表达nano的细胞中,与鲁玛卡托预孵育产生了相对较小的毛喉素诱导的4326delTC(图10A-10B)和N1303K电流(图11A-11B),依伐卡托进一步提高了这些电流(图3H-3K)。令人兴奋的是,在相同条件下,共表达enDUB-U21的细胞产生了明显更大的毛喉素刺激的4326delTC或N1303K电流(图10A-10B和图11A-11B),依伐卡托进一步增强了这些电流(图3H-3K)。
虽然靶向GFP的enDUB为靶向去泛素化方法在挽救不同的运输缺陷型重组突变通道中提供了关键的概念验证功效,但关键的下一步是开发针对CFTR本身的纳米抗体,以实现靶向内源性通道。因此,我们使用纯化的CFTR的NBD1结构域(图4A)作为诱饵,使用酵母纳米抗体库表面展示方法(McMahon等人,2018)来鉴定结合剂(图4B)。在几轮磁激活细胞分选(MACS)和荧光激活细胞分选(FACS)选择之后,我们分离了14种独特的纳米抗体结合剂,这些结合剂对NBD1具有一系列如酵母结合试验所报告的亲和力(图12A;表1)。令人欣慰的是,当与WT CFTR共表达时,许多纳米抗体结合剂不会从本质上干扰通道的表面运输(图12B)。在初步研究中,我们使用卤离子敏感的YFP猝灭试验(Galietta等人,2001)来筛选不同的纳米抗体,其用于enDUB介导的HEK293细胞中CFTR碘化物电流的功能挽救(图13A)。我们选择了一种纳米抗体克隆nb.E3h,因为其在转化为enDUB(称为enDUB-U21CF.E3h)时在卤离子传感器和膜片钳测定(图13A-13F)中均具有出色的性能。通过流式细胞术荧光共振能量转移(flow-FRET)测定法证实了nb.E3h与细胞中全长CFTR的结合(图4C)。
为了测试靶向CF的enDUB在相关细胞环境中的功效,我们利用预测性体外CF模型(稳定表达突变CFTR通道的Fischer大鼠甲状腺(FRT)上皮细胞),该模型已用于产生临床试验之前的临床前数据(Goor等人,2011;Goor等人,2009;Yu等人,2012)并促进FDA药物Kalydeco(依伐卡托)的标签扩展(Ratner,2017;Durmowicz等人,2018)。与之前的研究结果一致(Han等人,2018;Goor等人,2014),与WT对照细胞相比,稳定表达N1303K通道的FRT细胞表现出很小的功能电流,并且对VX809+VX770处理无反应(图4D和4E)。值得注意的是,enDUB-U21CF.E3h与相同的CFTR调节剂联合产生了令人印象深刻的N1303K电流挽救,高达约WT细胞的40%(图4D和4E)。
F508del代表最常见的CF突变,在NBD1中具有苯丙氨酸缺失,从而导致CFTR折叠、组装和运输的热稳定性缺陷(Lukacs和Verkman,2012;Okiyoneda等人,2013;Okiyoneda等人,2010)。在HEK293细胞中,在VX809存在的情况下,enDUB-U21CF.E3h仅导致F508del表面表达的适度改善(图14A-14C)。假设具有双重功能的备用enDUB,所述双重功能为1)增强结合时的NBD1热稳定性,2)通过催化作用调节CFTR泛素状态,其将导致改善F508del挽救。值得注意的是,最近的一项研究开发了一种结合分离的wt和F508del NBD1的纳米抗体(nb.T2a),其在无细胞制剂中具有热稳定性(Sigoillot等人,2019);然而,未原位检测nb.T2a对全长F508del CFTR突变体的功能影响。我们通过使nb.T2a适应我们的enDUB-U21系统(enDUB-U21CF.T2a)来测试热稳定enDUB的潜在协同作用。尽管表达nb.T2a联合VX809导致F508del表面运输的适度增加,但我们的功能化enDUB-U21CF.T2a+VX809在HEK293细胞中表现出显著增强的表面挽救(图14A-14C)。此外,与单独的VX809±nb.T2a相比,enDUB-U21CF.T2a显著改善了F508del功能电流,HEK293膜片钳研究证实了这卓越的功能挽救(图14A-14C)。最后,在稳定表达F508del的FRT细胞中,enDUB-U21CF.T2a+VX809+VX770联合治疗导致功能性F508del挽救至WT水平的约45%,与单独使用VX809+VX770±nb.T2a治疗相比有显著改善(图4F和4G)。
总而言之,我们的数据揭示了靶向去泛素化可作为一种强有力的策略来挽救作为不同疾病基础的不同运输受损的离子通道。虽然高通量筛选能够鉴定药理学纠正剂(例如用于CFTR的鲁玛卡托),但这些通常仅对一个目标通道有效,并且它们的作用机制未知。另一方面,低温和非特异性化学伴侣(例如甘油)(Okiyoneda等人,2013;Delisle等人,2004)可以挽救不同的运输受损离子通道子集;然而,这种方法不适合治疗发展。有鉴于此,enDUB代表了一种令人兴奋的基于机制的新策略,其具有针对不同通道类型的适应性和靶向特异性,可用于定制治疗应用。除了膜蛋白之外,考虑活细胞中调节不同蛋白质靶标的多种泛素依赖性过程的机会是吸引人的(Nalepa等人,2006;Huang和Dixit,2016)。将这些见解转化为针对多种疾病的有效分子疗法是未来研究的一个令人兴奋的前景。
材料和方法
质粒载体的分子生物学和克隆
如前所述(Kanner等人,2017)生成了自定义的双顺反子CMV哺乳动物表达载体(nano-xx-P2A-CFP);我们通过PCR扩增了GFP纳米抗体(vhhGFP4)的编码序列(Rothbauer等人,2008),并使用NheI/AflII位点将其克隆到xx-P2A-CFP中。为了生成enDUB-O1构建体,我们使用由灵活的GSG接头分隔的AscI/AflII位点从OTUD1(Addgene#61405)中PCR扩增OTU结构域+UIM(残基287-481)。为了构建无催化活性的enDUB-O1*,我们通过定点突变在催化半胱氨酸残基[C320S]处引入点突变。如前所述(Kanner等人,2017)生成了第二个自定义的双顺反子载体(CFP-P2a-nano-xx)。为了生成enDUB-U21,我们从USP21(Addgene#22574)中PCR扩增了USP结构域(残基196-565),并使用AscI/NotI位点将该片段克隆至CFP-P2a-nano-xx中。为了构建无催化活性的enDUB-U21*,我们通过定点突变在催化半胱氨酸残基[C221S]处引入点突变。使用与上述类似的克隆策略,使用mCh纳米抗体LaM-4(Fridy等人,2014)生成了靶向mCh的enDUB-U21。
如前所述(Aromolaran等人,2014)制备KCNQ1构建体。简而言之,使用重叠延伸PCR将增强型黄色荧光蛋白(EYFP)融合到KCNQ1的C端。根据制造商的说明书使用QuikChangeLightning定点突变试剂盒(Stratagene)在KCNQ1的细胞外S1--S2环中的残基148-149之间引入了一个13个残基的银环蛇毒素结合位点(BBS;TGGCGGTACTACGAGAGCAGCCTGGAGCCCTACCCCGAC;SEQ ID No:81)(Aromolaran等人,2014;Sekine-Aizawa和Huganir,2004)。通过定点突变在KCNQ1的N端和C端之间引入LQT1突变。NEDD4L(PCI_NEDD4L;Addgene#27000)来自Joan Massague的馈赠(Gao等人,2009)。
CFTR构建体来自pAd.CB-CFTR(75468)。为了创建CFTRYFP,使用PCR扩增将EYFP融合到CFTR的N端。为了创建BBS-CFTR-YFP,使用重叠延伸PCR在CFTR的第四个细胞外环(ECL4)中的残基901-902之间引入BBS位点(Peters等人,2011)。通过定点突变在CFTR的NBD1、NBD2和C端引入CF患者特异性突变。使用了YFP卤离子传感器(EYFP H148Q/I152L)(Galietta等人,2001)(Addgene#25872)。为了生成靶向CF的enDUB,我们构建了一个模块化CFP-P2axx-U21载体,其在USP结构域上游具有扩展的(GGGGS)x5(GGGTG)连接子。然后使用BglII/AscI位点克隆选择的NBD1纳米抗体结合剂。
腺病毒载体的生成
如前所述(Aromolaran等人,2014),根据制造商的说明书使用pAdEasy系统(Stratagene)生成了腺病毒载体。含有nano-P2A-CFP、WT KCNQ1-YFP和G589D KCNQ1-YFP的cDNA的质粒穿梭载体(pShuttle CMV)经PmeI线性化并电转到用pAdEasy-1病毒质粒(Stratagene)预转化的BJ5183-AD-1电感受态细胞中。使用PacI限制性消化来鉴定重组成功的转化体。使用XL-10-Gold细菌扩增阳性重组体,并用PacI消化将重组腺病毒质粒DNA线性化。HEK细胞在60mm直径的培养皿中以70-80%的融合度培养,并使用PacI消化的线性化腺病毒DNA转染。监测转染板的细胞病变效应(CPE)和腺病毒空斑。收获细胞并进行三个连续的冻融循环,然后离心(2,500×g)以去除细胞碎片。使用上清液(2mL)感染90%融合度的HEK293细胞的10cm培养皿。然后在2-3天后观察CPE,使用细胞上清液再次感染新的HEK293细胞板。如前所述(Aromolaran等人,2014),进行病毒扩增和纯化。简而言之,使用如上所述获得的病毒上清液(1mL)感染在15cm培养皿(×8)上生长的融合HEK293细胞。48小时后,收获来自所有板的细胞,离心获得沉淀,并重悬于含有(以mM计)Tris·HCl 20、CaCl2 1和MgCl2 1(pH 8.0)的8mL缓冲液中。通过四个连续的冻融循环裂解细胞并通过离心沉淀细胞碎片。在经三种密度的CsCl(1.25、1.33和1.45g/mL)分层的氯化铯(CsCl)不连续梯度上纯化载有病毒的上清液。离心(50,000rpm;SW41Ti转子,Beckman-Coulter Optima L-100K超速离心机;1小时,4℃)后,移除1.33和1.45g/mL密度层间界面处的病毒带并用PBS透析(12h,4℃)。腺病毒载体等分试样在-80℃下冻存于10%甘油中直至使用。由VectorBiolabs(Malvern,PA)生成enDUB-O1-P2A-CFP。
细胞培养和转染
使用人胚肾(HEK293)细胞。通过MycoFluor支原体检测试剂盒(Invitrogen)测定,细胞不含支原体。低传代的HEK293细胞在37℃下在补充有8%胎牛血清(FBS)和100mg/mL青霉素-链霉素的DMEM中培养。使用磷酸钙沉淀法完成HEK293细胞转染。简而言之,将质粒DNA与62μL的2.5M CaCl2和无菌去离子水混合(最终体积为500μL)。将混合物逐滴添加,不断轻敲至500μL的2×Hepes缓冲盐溶液中,所述缓冲盐溶液含有(以mM计):Hepes 50、NaCl 280、Na2HPO4 1.5、pH 7.09。将得到的DNA-磷酸钙混合物在室温下孵育20分钟,然后滴加到HEK293细胞中(60–80%融合)。4-6小时后,用不含Ca2+的磷酸盐缓冲盐溶液洗涤细胞并维持在补充的DMEM中。
中国仓鼠卵巢(CHO)细胞获自ATCC,并在37℃下在补充有8%FBS和100mg/mL青霉素-链霉素的Kaighn's改良的Ham's F-12K(ATCC)中培养。根据制造商的说明书(Roche),使用X-tremeGENE HP(1:2,DNA/试剂比率)在35mm组织培养皿中用目的构建体瞬时转染CHO细胞——KCNQ1(0.5μg)和nano-P2A-CFP(0.5μg)或enDUBO1-P2A-CFP(0.5μg)。
使用稳定表达WT和突变CFTR通道的FRT上皮细胞(Han等人,2018)。FRT细胞保持在37℃下在补充有5%FBS、100mg/mL青霉素-链霉素、7.5%(w/v)碳酸氢钠和100μg/mL潮霉素(Invitrogen)的Ham’s F-12Coon’s改良型中(Sigma)。根据制造商的说明书(Thermo),使用Lipofectamine 3000完成FRT细胞的瞬时转染。
根据哥伦比亚大学动物管理委员会的指导方针进行成年豚鼠心肌细胞的分离。在分离之前,电镀皿(plating dish)预涂有15μg/mL层粘连蛋白(Gibco)。用5%异氟醚对成年Hartley豚鼠(Charles River)实施安乐死,切除心脏并首先在KH溶液中灌注分离心室肌细胞,所述KH溶液(mM):118NaCl、4.8KCl、1CaCl2 25HEPES、1.25K2HPO4、1.25MgSO4、11葡萄糖,0.02EGTA,pH 7.4,然后使用Langendorff灌注装置加入不含钙的KH溶液。在不含钙的KH缓冲液中,用0.3mg/mL的4型胶原酶(Worthington)、0.08mg/mL蛋白酶和0.05%BSA进行酶解消化6分钟。消化后,使用40mL高K+溶液进行心脏灌注,所述高K+溶液(mM):120谷氨酸钾、25KCl、10HEPES、1MgCl2和0.02EGTA,pH 7.4。随后将细胞分散在高K+溶液中。健康的棒状肌细胞在199培养基(Life Technologies)中培养,以促进对培养皿的附着,所述199培养基补充有(mM):10HEPES(Gibco)、1×MEM非必需氨基酸(Gibco)、2L-谷氨酰胺(Gibco)、20D-葡萄糖(Sigma Aldrich)、1%(v/v)青霉素-链霉素-谷氨酰胺(Fisher Scientific)、0.02mg/mL维生素B-12(Sigma Aldrich)和5%(v/v)FBS(Life Technologies)。5小时后,将培养基换成含有1%(v/v)血清的199培养基,但其他补充成分如上所述。培养物保持在37℃和5%CO2的加湿培养箱中。
总通道和表面通道的流式细胞术测定
如前所述(Kanner等人,2017;Kanner等人,2018),通过流式细胞术在活的、转染的HEK293细胞中测定细胞表面和总离子通道池。简而言之,转染后48小时,使用含有Ca2+和Mg2 +的冰冷PBS(以mM计:0.9CaCl2,0.49MgCl2,pH 7.4)轻轻洗涤在12孔板中培养的细胞,然后在封闭培养基(含有3%BSA的DMEM)中在4℃下孵育30分钟。然后在DMEM/3%BSA中将HEK293细胞与1μM Alexa Fluor 647偶联的a-银环蛇毒素(BTX647;Life Technologies)在摇床上在4℃下孵育1小时,然后用PBS(含有Ca2+和Mg2+)洗涤3次。在不含Ca2+的PBS中轻柔地收获细胞,并使用BD LSRII细胞分析仪(BD Biosciences,San Jose,CA,USA)通过流式细胞术进行测定。CFP和YFP标记的蛋白分别在405和488nm处被激发,Alexa Fluor 647在633nm处被激发。
FRET流式细胞术测定
如前所述(Lee等人,2016),通过流式细胞术在活的、转染的HEK293细胞中进行FRET结合测定。简而言之,细胞在转染后培养24小时,并在分析前与放线菌酮(100μM)一起孵育2-4小时,并与H89(30μM)一起孵育30分钟,以减少荧光蛋白成熟度和基础激酶活性的细胞变化。用冰冷的PBS(含有Ca2+和Mg2+)轻柔地洗涤细胞,在不含Ca2+的PBS中收获,并使用BD LSRII细胞分析仪(BD Biosciences,San Jose,CA,USA)通过流式细胞术进行测定。分别使用以下激光/滤光片组配置:BV421(Ex:405nm,Em:450/50)、FITC(Ex:488nm,Em:525/50)和BV520(Ex:405nm,Em:525/50)分析Cerulean(Cer)、Venus(Ven)和FRET信号。为实验准备了几个对照,包括用于背景扣除的未转染空白、用于光谱分离的单色Ven和Cer、用于浓度依赖性虚假FRET估计的一起共表达的Cer+Ven、以及用于FRET校准的一系列Cer-Ven二聚体。使用定制的Matlab软件分析FRET供体/受体效率并生成FRET结合曲线作为[受体]游离和[供体]游离的函数。
电生理学
对于钾通道测量,使用由PatchMaster软件(HEKA)控制的EPC-10膜片钳放大器(HEKA Electronics)在室温下在CHO细胞中记录全细胞膜电流。将粘附有CHO细胞的盖玻片放置在记录室(体积为0.7-1mL)的玻璃底部,所述记录室安装在倒置Nikon Eclipse Ti-U显微镜载物台上。微量移液器由1.5mm薄壁玻璃制成并经过火抛光。内部溶液包含(mM):133KCl、0.4GTP、10EGTA、1MgSO4、5K2ATP、0.5CaCl2和10HEPES(pH 7.2)。外部溶液包含(以mM计):147NaCl、4KCl、2CaCl2和10HEPES(pH 7.4)。当填充内部溶液时,移液器电阻通常为1.5MΩ。I-V曲线由一系列阶跃去极化(从-80mV的保持电位开始,以10mV阶跃从-40至+100mV)生成。电流在20kHz采样并在5kHz过滤。以10秒的重复间隔采集轨迹。
在感染48-72小时后进行心肌细胞的全细胞记录。如上所述使用内部和外部溶液。使用慢速电压斜坡步骤(从-80mv到+100mV超过2s)激发全细胞电流。通过短电流脉冲(150pA,10ms)的0.25Hz刺激获得电流钳下的动作电位记录。
对于CFTR通道测量,在室温下在HEK293和FRT细胞中进行全细胞记录。内部溶液包含(mM):113L-天冬氨酸、113CsOH、27CsCl、1NaCl、1MgCl2、1EGTA、10TES、3MgATP(pH 7.2)。外部包含(以mM计):145NaCl、4CsCl、1CaCl2、1MgCl2、10葡萄糖和10TES(pH 7.4)。I-V曲线由一系列阶跃去极化(从-40mV的保持电位开始,以20mV阶跃从-80至+80mV)生成。通过灌注10μM毛喉素激活CFTR电流。在使用鲁玛卡托(3μM)的实验中,在转染24小时后加入药物并在37℃下孵育。依伐卡托以5μM浓度急剧使用。电流在20kHz采样并在7kHz过滤。以10秒的重复间隔采集轨迹。
免疫沉淀和蛋白质印迹
HEK293细胞用不含Ca2+的PBS洗涤一次,收获,并重悬于RIPA裂解缓冲液中,所述RIPA裂解缓冲液包含(以mM计)Tris(20,pH 7.4)、EDTA(1)、NaCl(150)、0.1%(w/v)SDS、1%Triton X-100、1%脱氧胆酸钠和补充有蛋白酶抑制剂混合物(10μL/mL,Sigma-Aldrich)、PMSF(1mM,Sigma-Aldrich)、Nethylmaleimide(2mM,Sigma-Aldrich)和PR-619去泛素酶抑制剂(50μM,LifeSensors)。通过在4℃下孵育1小时制备裂解物,偶尔涡旋,并通过离心(10,000×g,10分钟,4℃)清除。将上清液转移到新试管中,取出等分试样以定量由BCA蛋白估算试剂盒(Pierce Technologies)确定的总蛋白质浓度。裂解物通过与10μL Protein A/G琼脂糖珠(Rockland)在4℃下孵育40分钟进行预清除,然后与0.75μg的抗Q1(Alomone)在4℃下孵育1小时。向含有25μL Protein A/G琼脂糖珠的离心柱中加入等量的总蛋白量,在4℃下翻滚过夜。免疫沉淀物用RIPA缓冲液洗涤3次,用RIPA-500mM NaCl洗涤两次,以500×g旋转,用40μL加热的样品缓冲液(50mM Tris,10%(v/v)甘油,2%SDS,100mM DTT和0.2mg/mL溴酚蓝)洗脱,煮沸(55℃,15分钟)。蛋白质在4–12%Bis·Tris梯度预制凝胶(LifeTechnologies)上在Mops-SDS电泳缓冲液(Life Technologies)中以200V恒定电压约1小时进行分辨。我们在样品旁边上样了10μL PageRuler Plus预染蛋白分子量标准(10–250kDa,Thermo Fisher)。经电泳槽在转膜缓冲液(25mM Tris pH 8.3、192mM甘氨酸、15%(v/v)甲醇和0.1%SDS)中将蛋白条带转移至硝酸纤维素膜上。用Tris缓冲盐-吐温溶液(TBS-T)(25mM Tris pH 7.4、150mM NaCl和0.1%Tween-20)的5%脱脂牛奶(BioRad)在室温下封闭膜1小时,然后在4℃下与封闭溶液中的一抗(抗Q1,Alomone)一起孵育过夜。印迹用TBS-T洗涤3次,每次10分钟,然后与辣根过氧化物酶偶联的二抗在室温下孵育1小时。在TBS-T洗涤后,用化学发光检测试剂盒(Pierce Technologies)显色印迹,然后在凝胶成像仪上观察。然后用苛刻的膜洗脱缓冲液(2%SDS,62mM Tris pH 6.8,0.8%β-巯基乙醇)在50℃下洗脱膜30分钟,在流水下冲洗2分钟,并用TBST洗涤(3×,10分钟)。根据制造商的说明书,用0.5%戊二醛对膜进行预处理,并用抗泛素(VU1,LifeSensors)重新印迹。
用于纳米抗体结合剂的酵母表面展示
使用先前描述的酵母表面展示库方法(McMahon等人,2018)进行纳米抗体结合剂的分离。具有N端His×6-Smt3融合的人NBD1(残基387-646,Δ405-436)构建体获自亚利桑那州立大学质粒库(克隆:HsCD00287374)。使用Gibson组件在His×6-Smt3标签的下游立即插入一个FLAG标签。通过定制订单(Genscript)表达和His纯化蛋白质。使用SUMO蛋白酶试剂盒(Invitrogen)去除His×6-Smt 3标签,Ulp1蛋白酶在4℃下孵育过夜,随后进行亲和层析纯化(HisPur spin columns;Thermo)。将天然酵母文库(6×109酵母)在25℃下在含半乳糖的色氨酸缺陷型(Trp-)培养基中培养2-3天以诱导纳米抗体表达。将诱导的细胞洗涤并重悬于选择缓冲液(PBS、0.1%BSA、5mM麦芽糖)中。第一轮磁激活细胞分选(MACS)选择从预清除步骤开始,将酵母与抗FLAG M2-FITC偶联抗体(Sigma)和抗FITC微珠(Miltenyi)在4℃下孵育30分钟,然后通过LD柱(Miltenyi)移除抗体/微珠结合剂。然后通过在4℃下将预先清除的酵母与500nM His×6-Smt 3-FLAG-NBD1和抗FLAG M2-FITC孵育1小时,然后在选择缓冲液中洗涤,并与抗FITC在4℃下孵育20分钟,从而MACS-富集NBD1结合纳米抗体。标记的酵母通过LS柱(Miltenyi),用选择缓冲液洗涤3次,并通过移除MACS磁力架(Miltenyi)进行洗脱。富集的NBD1结合剂在含葡萄糖的Trp-培养基中于30℃培养过夜。通过如上所述在半乳糖Trp-培养基中孵育,用富集的NBD1文库(-1×108酵母)重复诱导纳米抗体表达。随后,通过荧光激活细胞分选(FACS)进行两轮阳性筛选,首先是用500nM His×6-Smt 3-FLAG-NBD1孵育诱导细胞(-5×106酵母,此处和然后),接下来是用500nM FLAG-NBD1移除任何Smt3结合剂。用第三轮阴性筛选FACS移除非特异性FITC偶联抗体结合剂,仅用抗FLAG FITC孵育细胞。最后,通过与100nM FLAG-NBD1孵育来选择高亲和力NBD1结合剂,FACS作为单细胞分选到96孔板中,并作为单克隆菌落生长,用于结合验证研究和质粒分离。通过使用FLAG-NBD1的连续稀释度(以nM计):5000、1000、500、100、50、10和1,来标记细胞(约105酵母),对独特的、经过验证的NBD1结合剂进行酵母上Kd测量。
YFP卤离子淬灭试验
YFP卤离子淬灭读板仪测定改装自先前的工作(Galietta等人,2001)。简而言之,将HEK293细胞分裂到24孔黑色培养板(VisiPlate;PerkinElmer)并与卤离子敏感的eYFP(H148Q/I152L)、单独的mCh或mCh标记的突变CFTR通道和单独的CFP或CFP-P2a靶向CF的enDUB-U21构建体共转染。2-3天后,用PBS(含Ca2+和Mg2+)洗涤细胞一次,并在37℃下在200μL PBS(含145mM NaCl)中孵育30分钟。使用SpectraMax M5读板仪(Molecular Devices)获取基线YFP读数(Ex:510nm,Em:538nm)。加入等量的含有碘化物的2×活化溶液以获得最终浓度(70mM NaI、10μM毛喉素、5μM VX770),每2秒拍摄一次记录YFP荧光的时间序列。测定在37℃下进行。
共聚焦显微镜
将细胞铺板到35mm MatTek培养皿上(MatTek Corporation)。心肌细胞在室温(RT)下用4%甲醛固定10分钟。如上所述,用BTX647对活HEK293细胞进行染色。使用具有40倍油浸物镜的Nikon A1RMP共聚焦显微镜捕获图像。
数据和统计分析
使用FlowJo、PulseFit(HEKA)、Microsoft Excel、Origin和GraphPad Prism软件离线分析数据。使用内置函数在Origin或GraphPad Prism中进行统计分析。使用带有Tukey’s多重比较检验的单因素方差分析或用于两组之间比较的双尾非配对t检验确定平均值之间的统计学显著差异(p<0.05)。除非另有说明,否则数据以平均值±s.e.m表示。
实施例2
RESTORx:基于靶向蛋白稳定化的下一代治疗模式
蛋白质稳定性是细胞中调控所有蛋白质的关键点。泛素化在细胞内蛋白质稳态中起主要作用,并且该过程的失调可导致许多疾病的发病机制。本公开聚焦于囊性纤维化(CF),一种作为主要适应症,具有高度未满足需求的罕见遗传性疾病。尽管绝大多数CF突变导致氯离子通道CFTR的稳定性缺陷,但目前的黄金标准治疗绝大多数是基于症状的:肺气道清除技术、粘液稀释剂的吸入和细菌感染的抗生素治疗(图15A-15B)。虽然这些治疗改善了预期寿命(~30-40岁),但仍然没有明确的治疗,并且CF患者继续经历快速恶化的生活质量。仅在最近才推动了药理学伴侣或“纠正剂”的开发,其看起来促进突变CFTR向细胞膜的运输;然而,迄今为止,此类治疗的临床功效相对不强,其中许多突变对治疗保持抗性。
本公开采用完全不同的小分子方法来挽救CFTR运输和稳定性(图16A)。特别地,目标是利用泛素化的强大但可逆的性质提出新的假设:我们是否可以将内源性去泛素化酶(DUB)募集到突变CFTR通道中,以便选择性调节泛素状态,增强通道稳定性,并恢复功能?我们将这种一般方法称为关于内源性DUB重定向的挽救和稳定化(Rescue&Stabilization onRedirection of Endogenous DUBs,ReSTORED),以及利用这种机制所得的分子,称为挽救和稳定化治疗(Rescue and Stabilization Therapeutics,ReSTORx)。基本上,我们的ReSTORx是由3个不同模块组成的异双功能分子:1)DUB结合分子,2)靶结合分子,和3)连接两者的可变接头。因此,我们的ReSTORx化合物充当分子桥,将内源性DUB活性连接到感兴趣的靶蛋白。为了测试这种新方法,我们首先使用酵母表面展示库开发了DUB和CFTR二者的基于纳米抗体的结合剂(图16B,表1)。得到ReSTORx分子,基于二价纳米抗体的ReSTORAb(图17),其能够结合活细胞内的两种蛋白质,并显著挽救突变CFTR表面运输至WT水平(图16C-16F)。此外,已经表明基于二价纳米抗体的ReSTORAb能够挽救LQTS运输缺陷(图18)。
表1.DUB和CFTR蛋白的基于纳米抗体的结合剂
ReSTORx技术作为首创的CFTR稳定剂出现,不同于市场上或在CF开发中的任何治疗剂,并且被合理设计用于靶向从突变通道去除泛素。其独特的作用机制促进与当前调节剂的协同功效,并挽救先前无响应的CFTR突变。此外,ReSTORx技术的模块性质表明高度适应性的蛋白质稳定平台。因此,“活性”DUB募集组分可以容易地适于与任何给定的靶标结合分子一起使用,具有改善目前市售药物的功效或使先前作为接合靶标而没有治疗效果的静止化合物有功能的潜力。
这种ReSTORx平台的潜在影响延伸到泛素治疗空间。泛素治疗剂中的竞争主要限于泛素蛋白酶体系统(UPS)的非选择性抑制剂。蛋白酶体抑制剂已经取得了巨大的商业成功,例如,首次上市的UPS调节剂(硼替佐米)仅在2014年就产生了30亿美元的收入;然而,由于这些药物靶向整个蛋白质降解途径,因此缺乏靶特异性限制了它们的使用并导致患者中的显著副作用。因此,焦点逐渐从蛋白酶体抑制剂转移到靶向UPS的特定组分(即E3泛素连接酶)。然而,这些泛素酶仍在许多不同底物的调节中遭受混杂性。相比之下,本文公开的ReSTORx分子在靶向方面具有特异性并且在作用方面具有普遍性,利用了对选择性UPS调节剂的巨大未满足的市场需求。这种全新的治疗方式可以进一步将适应症扩展到其他遗传性通道病和癌症治疗剂。
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如此描述的公开内容,显然相同内容可以以多种方式改变。此类变化不应被视为背离本公开的精神和范围,并且所有此类修改旨在包括在所附权利要求的范围内。
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
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Gly Gln Gly Thr Gln Val Thr Val Ser Ser
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Ile Phe Ser Tyr Gly
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Tyr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Arg Gly Ala Thr Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
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Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
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Val Val Gly Leu Arg Val Gln Tyr Gln Ala Tyr Leu Tyr Arg Tyr Trp
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Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 32
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Ser Arg Phe Gly
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Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
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Ala Ala Ile Ala Ser Gly Thr Thr Thr Tyr Tyr Ala Asp Ser Val Lys
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Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
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Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
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Gln Val Thr Val Ser Ser
115
<210> 33
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
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115 120
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Ser Tyr Tyr Leu
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Ala Ala Ile Asn Arg Gly Ala Thr Thr Tyr Tyr Ala Asp Ser Val Lys
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Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
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Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
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Val Arg Ala Ile Gln Thr Ser Ser Glu Arg Arg Tyr Phe Thr Tyr Trp
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Gly Gln Gly Thr Gln Val Thr Val Ser Ser
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Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Ile Ser Leu Ala Arg
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Ala Gly Ile Thr Tyr Gly Thr Thr Thr Tyr Tyr Ala Asp Ser Val Lys
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Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
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Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
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Gly Gln Gly Thr Gln Val Thr Val Ser Ser
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<223> 纳米抗体
<400> 36
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Val Ser Tyr Ala Met
20 25 30
Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala
35 40 45
Ile Thr Leu Gly Ser Asn Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg
50 55 60
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met
65 70 75 80
Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Tyr
85 90 95
Arg Arg Tyr Gly Lys Thr Leu Tyr Leu Leu Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser
115
<210> 37
<211> 122
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 37
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Ile Ser Ser Asp Ala
20 25 30
Trp Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Ser Ile Ser Thr Gly Ala Thr Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Val Pro Arg Arg Arg Gly Tyr Tyr Thr Tyr Tyr Phe Arg Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 38
<211> 122
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 38
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ile Phe Gln Tyr Ala
20 25 30
Ser Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Gly Ile Ser Ala Gly Ala Thr Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Arg Trp Tyr Asp Leu Ser Gln Tyr Pro Arg Arg His His Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 39
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 39
Gly Thr Ile Ser Gly Ser Gly Ser Met
1 5
<210> 40
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 40
Glu Phe Val Ala Ala Ile Asn Val Gly Ser Asn Thr Tyr Tyr
1 5 10
<210> 41
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 41
Ala Val Arg Phe Gly Tyr Tyr Tyr Arg His Thr Tyr
1 5 10
<210> 42
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 42
Gly Ser Ile Phe Ser Arg Phe Tyr Met
1 5
<210> 43
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 43
Glu Phe Val Ala Gly Ile Ser Ala Gly Gly Thr Thr Tyr Tyr
1 5 10
<210> 44
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 44
Ala Val Val Ala Gly Arg Leu Leu Arg Tyr Arg Tyr
1 5 10
<210> 45
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 45
Gly Thr Ile Ser Tyr His Gly Thr Met
1 5
<210> 46
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 46
Glu Phe Val Ala Ala Ile Ala Arg Gly Gly Asn Thr Asn Tyr
1 5 10
<210> 47
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 47
Ala Ala Leu Leu Arg Arg Ser Gly Tyr Ile Thr Ser Ser Phe Leu Tyr
1 5 10 15
<210> 48
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 48
Gly Thr Ile Ser Arg Tyr Thr Thr Met
1 5
<210> 49
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 49
Glu Leu Val Ala Gly Ile Thr Pro Gly Gly Ser Thr Tyr Tyr
1 5 10
<210> 50
<211> 11
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 50
Ala Ala Arg Asp Tyr Trp Ala Lys Leu Ser Tyr
1 5 10
<210> 51
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 51
Gly Ser Ile Phe Ser Arg Thr Ser Met
1 5
<210> 52
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 52
Glu Leu Val Ala Gly Ile Thr Trp Gly Gly Asn Thr Tyr Tyr
1 5 10
<210> 53
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 53
Ala Val Leu Val Pro Ile Gly Arg Asp Val Lys Gly Tyr His Arg Tyr
1 5 10 15
<210> 54
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 54
Gly Thr Ile Phe Arg Tyr Ala Val Met
1 5
<210> 55
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 55
Glu Phe Val Ala Ala Ile Asn Ser Gly Thr Asn Thr Asn Tyr
1 5 10
<210> 56
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 56
Ala Ala Leu Tyr Arg Asn Pro Ala Phe Pro Ile Tyr Ala His Thr Tyr
1 5 10 15
<210> 57
<211> 10
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 57
Gly Thr Ile Phe Ser Tyr Gly Tyr Met Gly
1 5 10
<210> 58
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 58
Glu Phe Val Ala Gly Ile Ser Arg Gly Ala Thr Thr Asn Tyr
1 5 10
<210> 59
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 59
Ala Val Val Gly Leu Arg Val Gln Tyr Gln Ala Tyr Leu Tyr Arg Tyr
1 5 10 15
<210> 60
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 60
Gly Ser Ile Ser Arg Phe Gly Val Met
1 5
<210> 61
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 61
Glu Phe Val Ala Ala Ile Ala Ser Gly Thr Thr Thr Tyr Tyr
1 5 10
<210> 62
<211> 12
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 62
Ala Ala Arg Glu Tyr Gly Tyr Gly Gly His Leu Tyr
1 5 10
<210> 63
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 63
Gly Ser Ile Phe Tyr Tyr Ser Arg Met
1 5
<210> 64
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 64
Glu Leu Val Ala Gly Ile Gly Arg Gly Thr Thr Tyr Tyr
1 5 10
<210> 65
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 65
Ala Val Tyr Pro Asn Tyr Gln Trp Ala Tyr Ala Val Leu His Gly Tyr
1 5 10 15
<210> 66
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 66
Gly Ser Ile Ser Tyr Tyr Leu Tyr Met
1 5
<210> 67
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 67
Glu Phe Val Ala Ala Ile Asn Arg Gly Ala Thr Thr Tyr Tyr
1 5 10
<210> 68
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 68
Ala Val Arg Ala Ile Gln Thr Ser Ser Glu Arg Arg Tyr Phe Thr Tyr
1 5 10 15
<210> 69
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 69
Gly Thr Ile Ser Leu Ala Arg Tyr Met
1 5
<210> 70
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 70
Glu Phe Val Ala Gly Ile Thr Tyr Gly Thr Thr Thr Tyr Tyr
1 5 10
<210> 71
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 71
Ala Ala Tyr Leu Arg Ser Thr Thr Ser Gly Tyr Leu Tyr His Arg Tyr
1 5 10 15
<210> 72
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 72
Gly Thr Val Ser Tyr Ala Met
1 5
<210> 73
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 73
Glu Phe Val Ala Ala Ile Thr Leu Gly Ser Asn Thr Asn Tyr
1 5 10
<210> 74
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 74
Ala Ala Tyr Arg Arg Tyr Gly Lys Thr Leu Tyr Leu Leu Tyr
1 5 10
<210> 75
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 75
Gly Thr Ile Ser Ser Asp Ala Trp Met
1 5
<210> 76
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 76
Glu Leu Val Ala Ser Ile Ser Thr Gly Ala Thr Thr Tyr Tyr
1 5 10
<210> 77
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 77
Ala Ala Val Pro Arg Arg Arg Gly Tyr Tyr Thr Tyr Tyr Phe Arg Tyr
1 5 10 15
<210> 78
<211> 9
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 78
Gly Tyr Ile Phe Gln Tyr Ala Ser Met
1 5
<210> 79
<211> 14
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 79
Glu Leu Val Ala Gly Ile Ser Ala Gly Ala Thr Thr Tyr Tyr
1 5 10
<210> 80
<211> 16
<212> PRT
<213> 人工序列
<220>
<223> 纳米抗体
<400> 80
Ala Ala Arg Trp Tyr Asp Leu Ser Gln Tyr Pro Arg Arg His His Tyr
1 5 10 15
<210> 81
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 银环蛇毒素结合位点
<400> 81
tggcggtact acgagagcag cctggagccc taccccgac 39
Claims (30)
1.二价分子,其包含:
a)去泛素酶(DUB)结合剂;
b)靶结合剂;和
c)所述DUB结合剂和所述靶结合剂之间的可变接头,
其中所述DUB结合剂选自细胞内抗体片段、scFv、纳米抗体、抗体模拟物、单体拟抗体、DARPins、脂质运载蛋白和靶向序列。
2.根据权利要求1所述的二价分子,其中所述DUB是内源性的。
3.根据权利要求1所述的二价分子,其中所述DUB选自泛素特异性蛋白酶(USP)家族、卵巢肿瘤蛋白酶(OUT)家族、泛素羧基末端水解酶(UCH)家族、Josephin结构域家族(Josephin)、与含有泛素的新型DUB家族相互作用的基序(MINDY)和JAB1/MPN/Mov34金属酶结构域家族(JAMM)。
4.根据权利要求1所述的二价分子,其中所述DUB是USP21或USP2。
5.根据权利要求1所述的二价分子,其中所述DUB结合剂是纳米抗体。
6.根据权利要求5所述的二价分子,其中所述纳米抗体结合USP家族成员。
7.根据权利要求5所述的二价分子,其中所述纳米抗体结合USP2。
8.根据权利要求5所述的二价分子,其中所述纳米抗体结合USP21。
9.根据权利要求8所述的二价分子,其中所述纳米抗体包含如SEQ ID NO:1至6中任一项所示的序列。
10.根据权利要求8所述的二价分子,其中所述纳米抗体包含:
a)如SEQ ID No:7所示的互补决定区(CDR)1、如SEQ ID No:8所示的CDR2和如SEQ IDNo:9所示的CDR3;
b)如SEQ ID No:10所示的CDR1、如SEQ ID No:11所示的CDR2和如SEQ ID No:12所示的CDR3;
c)如SEQ ID No:13所示的CDR1、如SEQ ID No:14所示的CDR2和如SEQ ID No:15所示的CDR3;
d)如SEQ ID No:16所示的CDR1、如SEQ ID No:17所示的CDR2和如SEQ ID No:18所示的CDR3;
e)如SEQ ID No:19所示的CDR1、如SEQ ID No:20所示的CDR2和如SEQ ID No:21所示的CDR3;或者
f)如SEQ ID No:22所示的CDR1、如SEQ ID No:23所示的CDR2和如SEQ ID No:24所示的CDR3。
11.根据权利要求1所述的二价分子,其中所述靶结合剂结合的靶标的异常泛素化引起疾病。
12.根据权利要求11所述的二价分子,其中所述疾病是遗传性离子通道病。
13.根据权利要求12所述的二价分子,其中所述遗传性离子通道病选自癫痫、偏头痛、神经性疼痛、心律失常、长QT综合征、Brugada综合征、囊性纤维化、糖尿病、高胰岛素血症性低血糖症、Bart ter综合征和尿崩症。
14.根据权利要求12所述的二价分子,其中所述疾病是长QT综合征。
15.根据权利要求12所述的二价分子,其中所述疾病是囊性纤维化。
16.根据权利要求1所述的二价分子,其中所述靶结合剂结合的靶标是囊性纤维化跨膜传导调节因子(CFTR)。
17.根据权利要求1所述的二价分子,其中所述靶结合剂选自细胞内抗体片段、scFv、纳米抗体、抗体模拟物、单体拟抗体、DARPins、脂质运载蛋白和靶向序列。
18.根据权利要求1所述的二价分子,其中所述靶结合剂是纳米抗体。
19.根据权利要求18所述的二价分子,其中所述纳米抗体结合囊性纤维化跨膜传导调节因子(CFTR)的NBD1结构域。
20.根据权利要求19所述的二价分子,其中所述纳米抗体包含如SEQ ID NO:25至38中任一项所示的序列。
21.根据权利要求19所述的二价分子,其中所述纳米抗体包含:
a)如SEQ ID No:39所示的互补决定区(CDR)1、如SEQ ID No:40所示的CDR2和如SEQ IDNo:41所示的CDR3;
b)如SEQ ID No:42所示的CDR1、如SEQ ID No:43所示的CDR2和如SEQ ID No:44所示的CDR3;
c)如SEQ ID No:45所示的CDR1、如SEQ ID No:46所示的CDR2和如SEQ ID No:47所示的CDR3;
d)如SEQ ID No:48所示的CDR1、如SEQ ID No:49所示的CDR2和如SEQ ID No:50所示的CDR3;
e)如SEQ ID No:51所示的CDR1、如SEQ ID No:52所示的CDR2和如SEQ ID No:53所示的CDR3;
f)如SEQ ID No:54所示的CDR1、如SEQ ID No:55所示的CDR2和如SEQ ID No:56所示的CDR3;
g)如SEQ ID No:57所示的CDR1、如SEQ ID No:58所示的CDR2和如SEQ ID No:59所示的CDR3;
h)如SEQ ID No:60所示的CDR1、如SEQ ID No:61所示的CDR2和如SEQ ID No:62所示的CDR3;
i)如SEQ ID No:63所示的CDR1、如SEQ ID No:64所示的CDR2和如SEQ ID No:65所示的CDR3;
j)如SEQ ID No:66所示的CDR1、如SEQ ID No:67所示的CDR2和如SEQ ID No:68所示的CDR3;
k)如SEQ ID No:69所示的CDR1、如SEQ ID No:70所示的CDR2和如SEQ ID No:71所示的CDR3;
l)如SEQ ID No:72所示的CDR1、如SEQ ID No:73所示的CDR2和如SEQ ID No:74所示的CDR3;
m)如SEQ ID No:75所示的CDR1、如SEQ ID No:76所示的CDR2和如SEQ ID No:77所示的CDR3;或者
n)如SEQ ID No:78所示的CDR1、如SEQ ID No:79所示的CDR2和如SEQ ID No:80所示的CDR3。
22.根据权利要求1所述的二价分子,其中所述接头是烷基、聚乙二醇(PEG)或点击接头。
23.一种治疗或改善受试者中疾病影响的方法,包括向受试者施用有效量的前述权利要求中任一项所述的二价分子。
24.根据权利要求23所述的方法,其特征在于,所述受试者是人。
25.根据权利要求23所述的方法,其中所述疾病选自遗传性离子通道病、癌症、心血管病症、感染性病和代谢性疾病。
26.根据权利要求25所述的方法,其中所述遗传性离子通道病选自癫痫、偏头痛、神经性疼痛、心律失常、长QT综合征、Brugada综合征、囊性纤维化、糖尿病、高胰岛素血症性低血糖症、Bartter综合征和糖尿病尿崩症。
27.根据权利要求25所述的方法,其中所述遗传性离子通道病是囊性纤维化。
28.一种鉴定和制备靶向感兴趣的蛋白质的纳米抗体结合剂的方法,包括:
a)构建表达合成纳米抗体的天然酵母文库;
b)将天然酵母文库与感兴趣的蛋白质一起温育;
c)通过磁激活细胞分选(MACS)选择表达与感兴趣的蛋白质结合的纳米抗体的酵母细胞;
d)扩增选定的细胞并构建富集酵母文库;
e)将富集的酵母文库与感兴趣的蛋白质一起孵育;
f)通过荧光激活细胞分选(FACS)选择表达与感兴趣的蛋白质结合的纳米抗体的酵母细胞;
g)扩增选定的细胞并构建进一步富集的酵母文库;
h)重复步骤e)至g)两次;和
i)将选定的酵母细胞分类为单细胞并培养为单克隆菌落以进行结合验证和质粒分离。
29.根据权利要求28所述的方法,其中所述感兴趣的蛋白质是囊性纤维化跨膜传导调节因子(CFTR)。
30.根据权利要求28所述的方法,其中所述感兴趣的蛋白质是去泛素酶(DUB)。
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JP2017516801A (ja) * | 2014-05-27 | 2017-06-22 | ファーマケア,インク. | 脱ユビキチン化酵素阻害剤の組成物と送達の方法 |
CN110740748A (zh) * | 2017-01-17 | 2020-01-31 | 芝加哥大学 | 肿瘤微环境中功能障碍的抗原特异性cd8+t细胞 |
CN110612294B (zh) * | 2017-01-31 | 2024-01-16 | 阿尔维纳斯运营股份有限公司 | 人小脑蛋白配体和包含其的双官能化合物 |
US11668021B2 (en) * | 2017-05-09 | 2023-06-06 | Yale University | Basehit, a high-throughput assay to identify proteins involved in host-microbe interaction |
CN111787940A (zh) * | 2017-11-06 | 2020-10-16 | 纽约市哥伦比亚大学理事会 | 使用工程化的去泛素化酶探测泛素依赖性细胞过程的组合物和方法 |
CN112585127A (zh) * | 2018-06-13 | 2021-03-30 | 安菲斯塔治疗有限责任公司 | 用于靶向UchL5的双功能分子 |
EP3927726A1 (en) * | 2019-02-21 | 2021-12-29 | Locki Therapeutics Limited | Survival-targeting chimeric (surtac) molecules |
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