CN106715472A - 用于监测组织蛋白酶s抑制的方法 - Google Patents
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Abstract
本发明涉及用于监测动物组织样品中的组织蛋白酶S抑制剂活性的方法，其包括：a)提供已对其施用组织蛋白酶S抑制剂的动物的组织样品，或提供在体外与组织蛋白酶S抑制剂接触过的动物组织样品，其中组织样品包含白细胞；b)通过流式细胞术测量步骤a)的组织样品的白细胞中的li p10肽水平；和c)使白细胞中的li p10肽水平与组织蛋白酶S抑制剂剂量关联，其中组织蛋白酶S抑制剂导致白细胞中li p10肽水平提高。
Description
本发明提供抗li p10肽的抗体及其在用于监测组织蛋白酶S抑制的方法中的用途。
组织蛋白酶S因其在蛋白水解消化不变链蛋白伴侣分子，从而控制抗原通过主要组织相容性复合体(MHC)II类分子呈递给CD4+ T细胞或通过CD1分子呈递给NK1.1+ T细胞中的重要功能而闻名。组织蛋白酶S似乎还参与通过MHC II类呈递给CD4+ T细胞的外源抗原的直接加工，或参与通过MHC I类分子交叉呈递给CD8+ T细胞。此外，组织蛋白酶S以其分泌形式涉及胞外基质降解，这可促成许多疾病的病理，包括关节炎、动脉粥样硬化和慢性阻塞性肺疾病。因此，对于开发用于多种适应症的新治疗剂，组织蛋白酶S的抑制是有前景的靶标。
MHC II类分子结合并展示抗原肽，作为抗原呈递细胞(APC)的细胞表面上的由CD4+辅助细胞识别的II类-肽复合体。导致形成II类-肽复合体并在细胞表面呈递抗原的分子机制始于内质网中的II类αβ异二聚体合成。在生物合成期间，这些αβ异二聚体与II型膜蛋白(不变链li的同种型)结合，形成αβli的九聚复合体。αβli复合体穿过高尔基体，递送至胞内区室。在这些酸性区室中，li被降解，并最终从αβli复合体释放，允许II类分子结合抗原肽。li的蛋白水解加工产生更短的片段。在APC中，半胱氨酸蛋白酶组织蛋白酶S在加工li中发挥关键作用。组织蛋白酶S降解来自II类-li复合体的的不变链li，产生II类-CLIP。因此，组织蛋白酶S的抑制导致CLIP肽减少，前体片段(即不变链li降解肽p10(li p10))累积。
EP 1 315 966公开了用于监测体内施用组织蛋白酶S抑制剂的作用的方法。该方法包括从用组织蛋白酶S抑制剂处理的个体的血样纯化白细胞，制备所纯化的白细胞的全细胞裂解物，并针对不变链li的li p10片段的存在分析全细胞裂解物。此方法需要约10-30ml血样，且耗费劳力(分离白细胞和制备全细胞裂解物)。li p10的检测通过ELISA或Western印迹进行。
本发明的目的是提供监测组织蛋白酶S抑制的测定，该测定克服现有技术方法的至少一些缺点。
在第一目的中，本发明提供用于监测动物组织样品中的组织蛋白酶S抑制剂活性的方法，其包括：
a)提供已对其施用组织蛋白酶S抑制剂的动物的组织样品，或提供在体外与组织蛋白酶S抑制剂接触过的动物组织样品，其中组织样品包含白细胞；
b)通过流式细胞术测量步骤a)的组织样品的白细胞中的li p10肽水平；和
c)使白细胞中的li p10肽水平与组织蛋白酶S抑制剂剂量关联，其中组织蛋白酶S抑制剂导致白细胞中li p10肽水平提高。
在第二目的中，本发明提供用于鉴定组织蛋白酶S抑制剂的方法，其包括：
a)用测试化合物处理非人动物；
b)提供步骤a)的动物的包含白细胞的组织样品；
c)测量步骤b)的组织样品的白细胞中的li p10肽水平；
其中与未经处理的动物的组织样品中的白细胞中li p10肽的水平相比，步骤a)的样品中的白细胞中li p10肽水平的提高指示组织蛋白酶S抑制剂。
在本发明方法的优选实施方案中，该组织样品是血液。
在本发明方法的另一优选实施方案中，该白细胞是抗原呈递细胞(APC)，优选地，该APC选自B细胞、单核细胞、巨噬细胞和树突细胞，更优选选自B细胞和单核细胞。
本文所用的术语抗原呈递细胞(APC)指将抗原肽作为(MHC)II类-抗原肽复合体展示在其表面的细胞。APC包括树突细胞、单核细胞、巨噬细胞和B细胞。
在第三目的中，本发明提供抗li p10抗体，其包含：
包含含有氨基酸序列Seq.Id.No.4的CDR1、含有氨基酸序列Seq.Id.No.5的CDR2、含有氨基酸序列Seq.Id.No.6的CDR3的VH结构域，及包含含有氨基酸序列Seq.Id.No.7的CDR1、含有氨基酸序列Seq.Id.No.8的CDR2、含有氨基酸序列Seq.Id.No.9的CDR3的VL结构域。
在本发明的具体实施方案中，抗li p10抗体的VH结构域包含氨基酸序列Seq.Id.No.10，抗li p10抗体的VL结构域包含氨基酸序列Seq.Id.No.11.
本发明的抗li-p10特异性抗体在本文中称为源自家兔(Oryctologouscuniculus)的7B8新表位抗体。此抗体的特征在于Seq.Id.No.4-11中所公开的CDR和VH、VL序列。
术语组织蛋白酶S指木瓜蛋白酶超家族中的半胱氨酸蛋白酶。人组织蛋白酶S的氨基酸序列在Seq.Id.No.1中提供。
术语“不变链li”指主要组织相容性复合体或CD74的不变多肽。人不变链li的氨基酸序列在Seq.Id.No.2中提供。
术语li p10肽指II类结合li肽。人li p10的氨基酸序列在Seq.Id.No.3中提供。
本文所用的术语“MHC II多肽”指组成MHC II复合体的α链和β链。
本文所用的术语动物包括人类和非人动物，如猴、狗、猪、兔、豚鼠和啮齿动物。
本文在与本发明的测定结合描述的“测试化合物”或“候选药物化合物”的背景中使用术语“化合物”。因此，这些化合物包括合成来源或来自自然来源的有机或无机化合物。化合物包括无机或有机化合物，如多核苷酸、脂质或表征为相对低的分子量的激素类似物。其他生物聚合有机测试化合物包括含有约2至约40个氨基酸的肽和含有约40至约500个氨基酸的更大的多肽，如抗体或抗体缀合物。
本发明的发明人惊奇地发现，可以在白细胞中检测li p10肽。与现有技术中所述的方法相比，本发明的方法具有可以直接测定组织样品(优选血液)而不涉及分离白细胞的优势。在本发明的方法中，无需为了能够在本发明的方法中处理而从全血样品分离例如白细胞。如果在必须处理大量样品的临床环境中使用，则这具有重要意义。本发明的方法需要比现有技术方法小的组织样品，优选血样。
用于检测和/或测量生物样品中的多肽的方法为本领域公知，包括但不限于Western印迹、流式细胞术、ELISA或RIA、或多种蛋白质组学技术。可以为了检测多肽或肽片段的目的而例如通过用纯化的蛋白质免疫兔来产生识别li p10肽或其片段的单克隆或多克隆抗体，或者可以使用识别多肽或肽片段的已知抗体。例如，在Western印迹中，可以用能够结合变性蛋白质的抗体(如多克隆抗体)来检测LI P10肽或MHC II多肽。ELISA是测量多肽的方法的实例。此类蛋白质定量基于能够捕获特异性抗原的抗体和能够检测所捕获的抗原的第二抗体。用于制备和使用抗体的方法及上文提到的测定描述于Harlow,E.和Lane,D.Antibodies:A Laboratory Manual,(1988),Cold Spring Harbor Laboratory Press中。
用于检测li p10肽的优选方法是流式细胞术。流式细胞术方法描述于Handbookof Flow Cytometry Method,J.Paul Robinson(编辑)；Flow Cytometry-A BasicIntroduction,Michael G Ormerod(2008)和Current Protocols in Cytometry(2010),Wiley中。在此方法中，蛋白质不变性，而是保持其天然形式。
WO 2010/121918公开了可在本发明的方法中测试的组织蛋白酶S抑制剂。
附图简述
图1：用新表位抗体检测B细胞和T细胞上的Ii p10表达。用递增浓度的组织蛋白酶S抑制剂1或溶媒(vehicle)孵育从健康供体(R213)的献血分离的PBMC 20小时。用不同的新表位抗体7B8(黑色)通过流式细胞术测定B细胞(CD20+)和T细胞(CD3+)上的Ii p10检测。
图2：用新表位抗体检测B细胞上的Ii p10表达。用递增浓度的组织蛋白酶S抑制剂1或溶媒孵育从健康供体(R172)的献血分离的PBMC 20小时。用两种不同的新表位抗体(即7B8(黑色)和13C4(灰色))通过流式细胞术测定B细胞(CD20+)和T细胞(CD3+)上的Ii p10检测。染色指数＝平均荧光强度(MFI)B细胞/MFIT细胞。
图3A：用组织蛋白酶S抑制剂处理时Ii p10累积在B细胞中。用递增浓度的组织蛋白酶S抑制剂1或溶媒孵育从11个健康供体(R39、R185、R198、R204、R209、R140、R154、R175、R213、R4、R214)的献血分离的PBMC 20小时。用7B8新表位抗体通过流式细胞术测定B细胞(CD20+)和T细胞(CD3+)上的Ii p10检测。染色指数＝MFIB细胞/MFIT细胞。
图3B：用组织蛋白酶S抑制剂处理时Ii p10累积在B细胞中。用递增浓度的组织蛋白酶S抑制剂2或溶媒孵育从11个健康供体(R39、R185、R198、R204、R209、R140、R154、R175、R213、R4、R214)的献血分离的PBMC 20小时。用7B8新表位抗体通过流式细胞术测定B细胞(CD20+)和T细胞(CD3+)上的Ii p10检测。染色指数＝MFIB细胞/MFIT细胞。
图4：组织蛋白酶S抑制剂IC50浓度。用递增浓度的组织蛋白酶S抑制剂1、组织蛋白酶S抑制剂2或溶媒孵育从11个健康供体(R39、R185、R198、R204、R209、R140、R154、R175、R213、R4、R214)的献血分离的PBMC 20小时。用7B8新表位抗体通过流式细胞术测定B细胞(CD20+)和T细胞(CD3+)上的Ii p10检测。用GraphPad Prism软件计算反应曲线和IC50值。
图5A-F：Ii p10新表位测定纵向变异性。在两个时间点从11个健康供体(R198、R209、R154、R213、R214)的献血分离PBMC，并用递增浓度的组织蛋白酶S抑制剂1或溶媒孵育20小时。用7B8新表位抗体通过流式细胞术测定B细胞(CD20+)和T细胞(CD3+)上的Ii p10检测。
图6A：通过新表位测定检测B细胞中的Ii p10累积。用7B8新表位抗体克隆通过流式细胞术测定B细胞(CD20+)上的Ii p10检测。用递增浓度的组织蛋白酶S抑制剂1或溶媒孵育从3份食蟹猴(Macaca fascicularis)献血分离的PBMC 20小时。通过用B细胞的平均荧光强度(MFI)除以前向散射低CD20阴性(FSCloCD20-)细胞的MFI来测定食蟹猴样品的p10染色指数。
图6B：组织蛋白酶S抑制剂单个口服剂量后通过新表位测定检测B细胞中的Ii p10累积。用7B8新表位抗体克隆通过流式细胞术测定B细胞(CD20+)上的Ii p10检测。50mg/kg的单个剂量后2小时和7小时从动物采血。在全血中进行Ii p10新表位测定。通过用B细胞的平均荧光强度(MFI)除以前向散射低CD20阴性(FSCloCD20-)细胞的MFI来测定食蟹猴样品的p10染色指数。
图7A-7D：从参与评价单剂量组织蛋白酶抑制剂1在健康男性和女性志愿者中的安全性、耐受性、药代和药效作用的单中心、随机化、双盲、安慰剂对照、单递增剂量研究的个体获得的样品上的p10定量。为了解释和限制临床前试验中观察到的预期的个体间变异的影响，利用了交错群组(“跳蛙”)设计。招募个体进入各八名个体的两个群组(群组A和B)。对于群组内的每个个体，研究为随机、安慰剂对照、四次治疗、四个时期、四因素交叉。图7A至7D(群组A)和图8A至8D(群组B)上各显示时间点0小时后立即应用的单剂量。
图8A-8D：群组B数据，参见图7描述。
实验部分
组织蛋白酶S抑制剂体外测定
使用无菌聚苯乙烯24孔板
加入500μl完全RPMI+递增浓度(0至10μM)的组织蛋白酶S抑制剂向每个孔加入500μl PBMC悬液(2x106)
混合孔中内容物，在湿润培养箱中37℃、5％CO2孵育平板20小时
FACS染色
配制1x裂解/固定溶液
每2x106PBMC或200μl血液加入4mL预热的1x裂解/固定溶液37℃孵育10分钟，用PBS洗涤细胞1x(250xg，5分钟，RT)，弃上清每管加入2mL 1x透化溶液
RT孵育20分钟
用2ml Cell Stain Buffer(250xg，5分钟，RT)洗涤细胞2x，弃上清
在Cell Staining Buffer中加入10μg/mL未标记的抗p10抗体，RT，1小时
用2ml Cell Stain Buffer(250xg，5分钟，RT)洗涤细胞1x，弃上清
在Cell Staining中加入1:125PE缀合山羊抗兔IgG抗体，RT避光，30分钟
用2ml Cell Stain Buffer(250xg，5分钟，RT)洗涤细胞2x，弃上清
向每个管中加入抗体(竞争抗体)或抗体混合物(稀释于100μl/样品Cell StainBuffer)
+4℃避光孵育20分钟
用2ml Cell Stain Buffer(250xg，5分钟，RT)洗涤细胞1x，弃上清
重悬沉淀在200μl Cell Stain Buffer中，保持细胞避光，直至在FACS上分析
抗体：
材料和缓冲液
24孔板，BD，订购ID：351147。保存：室温，无菌，非组织培养处理聚苯乙烯。
10x BD Phosphoflow Lyse/Fix缓冲液裂解缓冲液(BD,订购ID:558049)。保存：+4℃。使用10x Phosflow Perm Buffer IV透化缓冲液(BD,订购ID:560746)前，用双蒸水稀释5x BD Phosphoflow Lyse/Fix缓冲液至1x，并预热至所需温度。保存于室温。用PBS稀释10xBD Phosphoflow Perm Buffer IV至1x。
1x Cell Satining Buffer(BioLegend,订购ID:420201。保存：+4℃，1x CellSatining Buffer为即用型。不含CaCl2且不含MgCl2的1x PBS(Gibco,订购ID:14190-094)。保存：室温。PBS为即用型。5ml聚苯乙烯圆底管(FACS管,BD,订购ID:352052)。保存：室温。
氨基酸序列
| 序列名称 | Seq.Id.No. |
| 人组织蛋白酶S | 1 |
| 人不变链li | 2 |
| 人li p10 | 3 |
| 抗li p10抗体7B8的VH CDR 1 | 4 |
| 抗li p10抗体7B8的VH CDR 2 | 5 |
| 抗li p10抗体7B8的VH CDR 3 | 6 |
| 抗li p10抗体7B8的VL CDR 1 | 7 |
| 抗li p10抗体7B8的VL CDR 2 | 8 |
| 抗li p10抗体7B8的VL CDR 3 | 9 |
| 抗li p10抗体7B8的VH链 | 10 |
| 抗li p10抗体7B8的VL链 | 11 |
Claims (12)
1.用于监测动物组织样品中的组织蛋白酶S抑制剂活性的方法，其包括：
a)提供已对其施用组织蛋白酶S抑制剂的动物的组织样品，或提供在体外与组织蛋白酶S抑制剂接触过的动物组织样品，其中组织样品包含白细胞；
b)通过流式细胞术测量步骤a)的组织样品的白细胞中的li p10肽水平；和
c)使白细胞中的li p10肽水平与组织蛋白酶S抑制剂剂量关联，其中组织蛋白酶S抑制剂导致白细胞中li p10肽水平提高。
2.用于鉴定组织蛋白酶S抑制剂的方法，其包括：
a)使非人动物与测试化合物接触；
b)提供步骤a)的动物的包含白细胞的组织样品；
c)通过流式细胞术测量步骤b)的组织样品的白细胞中的li p10肽水平；
其中与未经处理的动物的组织样品中的白细胞中li p10肽的水平相比，步骤a)的样品中的白细胞中li p10肽水平的提高指示组织蛋白酶S抑制剂。
3.权利要求1或2的方法，其中组织样品是血液。
4.权利要求1至3的方法，其中白细胞是抗原呈递细胞(APC)，优选B细胞、单核细胞、巨噬细胞和树突细胞。
5.权利要求4的方法，其中APC选自B细胞和单核细胞。
6.权利要求1和3-5的方法，其中动物是人类。
7.权利要求2-6的方法，其中非人动物是啮齿动物，优选大鼠或小鼠。
8.权利要求1-7的方法，其中透化白细胞，以通过流式细胞术测量li p10肽。
9.权利要求1-8的方法，其中在流式细胞术中用抗li p10的抗体来测量白细胞中的lip10。
10.权利要求9的方法，其使用权利要求11或12的抗体。
11.抗li p10抗体，其包含VH结构域和VL结构域，所述VH结构域包含含有氨基酸序列Seq.Id.No.4的CDR1、含有氨基酸序列Seq.Id.No.5的CDR2、含有氨基酸序列Seq.Id.No.6的CDR3序列，所述VL结构域包含含有氨基酸序列Seq.Id.No.7的CDR1、含有氨基酸序列Seq.Id.No.8的CDR2、含有氨基酸序列Seq.Id.No.9的CDR3序列。
12.权利要求9的抗li p10抗体，其中VH结构域包含氨基酸序列Seq.Id.No.10，VL结构域包含氨基酸序列Seq.Id.No.11。
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| PCT/EP2015/071002 WO2016041915A1 (en) | 2014-09-18 | 2015-09-15 | Method for monitoring cathepsin s inhibition |
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| WO2002021129A1 (en) * | 2000-09-06 | 2002-03-14 | Ortho-Mcneil Pharmaceutical, Inc. | Method of monitoring the effect of cathepsin s inhibitors |
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| WO2012041824A1 (en) * | 2010-10-01 | 2012-04-05 | F. Hoffmann-La Roche Ag | Method for monitoring cathepsin s inhibition |
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| WO2002021129A1 (en) * | 2000-09-06 | 2002-03-14 | Ortho-Mcneil Pharmaceutical, Inc. | Method of monitoring the effect of cathepsin s inhibitors |
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| EP3194610A1 (en) | 2017-07-26 |
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| JP6826527B2 (ja) | 2021-02-03 |
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