CN110678196B - 抗hekr-k包膜抗体及其用途 - Google Patents
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- CN110678196B CN110678196B CN201880014059.2A CN201880014059A CN110678196B CN 110678196 B CN110678196 B CN 110678196B CN 201880014059 A CN201880014059 A CN 201880014059A CN 110678196 B CN110678196 B CN 110678196B
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Abstract
本发明涉及针对HERV‑K包膜的新型抗体及其在诊断和/或治疗中的用途,所述抗体靶向不受糖基化或天然构象影响的保守区。
Description
技术领域
本发明涉及抗HERV-K包膜的新型抗体及其在诊断和/或治疗中的用途,所述抗体靶向不受糖基化或天然构象影响的保守区。
背景技术
法国神经学家Jean-Martin Charcot首次描述了肌萎缩侧索硬化症(ALS),其名称反映了其中下行轴突显示脊髓侧索结构改变的皮质脊髓运动神经元的退化(侧脑硬化)和脊髓运动神经元死亡,伴随与肌肉萎缩相关的继发性去神经支配(肌萎缩)(Taylor,Brown和Cleveland 2016)。实际上,ALS是一种进行性且最终致命的神经退行性疾病,由大脑运动皮层、脑干和脊髓中涉及自主运动的计划、控制和执行的运动神经元退化引起。通常在诊断后3-5年发生致命结果(Taylor,Brown和Cleveland 2016)。ALS的患病率在10万例中大约达到5例,这反映了该疾病的快速致死率(Taylor,Brown和Cleveland 2016)。约10%的ALS病例似乎在家族中遗传传播(遗传性ALS),与特定基因组突变相关。例如,约20%的家族性ALS与超氧化物歧化酶(sod1)基因的突变相关(Vucic和Kiernan 2009;Rosen 1993)。其他非家族性病例被归类为散发性ALS(ALS病例的90%)(Lagier-Tourenne和Cleveland 2009),这意味着它没有家族史。
神经退行性疾病(如帕金森病、亨廷顿氏病、阿尔茨海默病、额颞叶变性(FTLD)和ALS)与中枢神经系统中神经元和神经胶质细胞内外错误折叠蛋白的积累有关(Polymenidou和Cleveland 2011)。这些错误折叠蛋白的聚集体是每种疾病的病理标志,并且可以在起始事件后通过朊病毒样机制在细胞之间传播。一种广泛持有的观点认为,这些聚集体在疾病的发生和进展中起着至关重要的作用,其中内源性蛋白质的错误折叠版本可能潜在地通过以下途径获得毒性:增加聚集体内必需细胞成分的疏水性和/或隔离、产生氧化性物种、抑制蛋白酶体和通过其他途径。另一种观点认为,大聚集体并不代表有毒形式,而是防御性细胞反应的最终产物,所述产物旨在保护细胞免受大多数技术仍然无法检测到的毒性较大的寡聚物的影响(Polymenidou和Cleveland,2011)。
来自ALS患者血清样品(人类免疫缺陷病毒(HIV)或人类T细胞白血病病毒(HTLV)外源病毒为血清阴性)的研究显示,50-60%ALS样品中的逆转录酶(RT)活性水平与HIV感染患者相当(MacGowan等2007;McCormick等2008;Andrews等2000;Steele等2005)。这与自从认识到鼠和人逆转录病毒都可以引起ALS样综合征起,几年来怀疑逆转录病毒参与的事实一致(McCormick等,2008)。可以通过抗逆转录病毒治疗缓解HIV阳性患者的ALS样疾病(Moulignier等,2001;von Giesen等,2002)。这对于HIV感染患者的ALS症状有效,并且可能代表ALS病例的特殊亚类。
在ALS患者的一级亲属的血清中也发现了增加的RT活性,这导致推测RT活性可能来自人内源性逆转录病毒(HERV)中的遗传活性拷贝,所述HERV占我们基因组的8%(Steele等,2005)。
尽管如此,ALS中RT活性的检测并未确定该酶的来源,但已显示HERV-K参与死后脑(Douville等,2011)。测序研究显示,与对照相比,ALS患者中的7q34和7q36.1染色体基因座(分别对应于HERV-K的HML-2和HML-3亚家族)更常表达(Douville和Nam 2014)。此外,最近观察到,与对照相比,ALS患者脑中的HERV-K gag-pol和env RNA表达显著升高(Li等,2015)。
HERV-K在人神经元培养物中的表达引起神经元细胞毒性,如转染整个HERV-K基因组或仅HERV-K-env基因后,观察到剂量依赖性方式的神经元数量减少和神经突收缩。这表明细胞内HERV-K-Env蛋白可能有助于神经毒性。这已经通过CRISP/casp9测定证实,所述测定通过VP64转录因子的LTR活化允许HERV-K表达增加两倍(Li等,2015)。HERV-K表达导致在皮质神经元中表达HERV-K-env基因的转基因小鼠体内运动皮质体积损失,其与免疫反应性无关,如用小胶质细胞的离子化钙结合适配子分子1(Iba-1)标记物所测量(Li等2015)。行为分析显示,HERV-K-env转基因小鼠在转杆实验(rotarod performance test)中走的距离较短,休息时间较长,并且掉得更快,显示后肢抓握增加的痉挛的证据。除了这些运动功能障碍外,转基因小鼠还发生了肢体和脊柱肌肉(包括用于呼吸的那些)的严重无力,导致10个月时的死亡率达到50%(Li等2015)。
有趣的是,HERV-K RT表达与ALS患者神经元中TDP-43水平升高相关,表明RT表达与该疾病特征性的其他异常细胞过程一起发生(Buratti和Baralie 2009;Geser等,2009;Douville等2011)。ALS中这种朊病毒样机制的证据现在涉及主要的错误折叠的蛋白质SOD1和TDP-43(Polymenidou和Cleveland 2011)。最近,Li等人证明TDP-43可以激活人神经元中的HERV-K-env表达,这与他们观察到TDP-43可以结合HERV-K长末端重复序列(LTR)的区域726-CCCTCTCCC-734一致(Li等2015)。他们还表明,内源性TDP-43沉默降低了HERV-K的表达。这些结果最近通过表明以下得到补充:正常TDP-43在体外对人星形胶质细胞和神经元中的HERV-K转录没有影响,而TDP-43在HERV-K启动子的U5区域具有结合位点。后一种结合通过炎症增强,例如存在肿瘤坏死因子(TNFα)或蛋白酶体抑制(Manghera,Ferguson-Parry和Douville 2016)。有趣的是,同样的研究表明,聚集形式的TDP-43的过表达增强了HERV-K病毒蛋白的表达和积累,而野生型(正常)TDP-43则没有(Manghera,Ferguson-Parry和Douville 2016)。此外,尽管证据表明ALS皮质神经元中的应激颗粒和自噬反应增强,但这些细胞未能清除过量的HERV-K蛋白积累。典型的大多数逆转录病毒限制因子,TDP-43启动子可能对干扰素和炎症相关的转录因子产生响应,因为它含有干扰素调节因子(IRF1、IRF3)和核因子-κB(NFκB)的结合位点(Douville等,2011)。
总之,这些研究结果表明,内源性逆转录病毒元件,特别是HERV-K参与了ALS的病理生理学,并且可能是TDP43与这种蛋白病之间缺失的连接(Alfahad和Nath 2013)。因此,ALS患者神经元内的HERV-K包膜蛋白表达可促成神经变性和疾病发病机制。
迄今为止,作为对症治疗,利鲁唑仍然是唯一相对有效的药物,并且仅将患者的平均存活期延长3-6个月(Hardiman,van den Berg和Kiernan 2011)。目前的治疗方案基于症状管理和保持生活质量,在多学科环境中提供。对于患有这种快速致命疾病的患者而言,发现有效治疗仍然是一项迫切需要(Hardiman,van den Berg和Kiernan 2011)。
因此,仍然存在对治疗ALS的有效治疗剂的未满足的需求。
发明简述
发明人开发了显示出乎意料的性质的抗HERV-K包膜蛋白的新抗体。
发明人已经表明,根据本发明的名为GN_mAb_Env_K01的抗体是鼠单克隆抗体(mAb),其选择性结合HERV-K-Env蛋白表面上的SLDKHKHKKLQSFYP(SEQ ID NO:9)线性表位。GN_mAb_Env_K01是IgG2b/κ鼠亚类的全长抗体。已经在ELISA和蛋白质印迹免疫测定中证实了GN_mAb_Env_K01的生物活性。令人惊讶地,GN_mAb_Env_K01不仅识别天然和变性的HERV-K-Env蛋白,而且也识别非糖基化和糖基化形式,而商业抗HERV-K-Env抗体不能检测糖基化形式。
此外且最意想不到的是,靶标表位似乎是高度保守的,具有在数据库中描述的HERV-K env基因的稳定氨基酸序列。这赋予该抗体独特的定位,无论其包膜蛋白的致病表达中涉及的HERV-K拷贝如何。这些意外的结果表明,GN_mAb_Env_K01是靶向HERV-K包膜蛋白的原创工具。他们还表明,即使对于技术人员,也不能通过给定的免疫方案预见或预测来选择和获得这种单克隆抗体。
因此,本发明涉及识别HERV-K包膜蛋白的抗体,其中所述抗体结合表位SLDKHKHKKLQSFYP(SEQ ID NO:9)。
本发明还涉及识别HERV-K包膜蛋白的抗体用于治疗的用途,其中所述抗体结合表位SLDKHKHKKLQSFYP(SEQ ID NO:9)。
本发明还涉及识别HERV-K包膜蛋白的抗体用于治疗肌萎缩侧索硬化(ALS),优选散发性ALS的方法中的用途,其中所述抗体结合表位SLDKHKHKKLQSFYP(SEQ ID NO:9)。
在另一个方面,本发明涉及药物组合物,其包含如上定义的抗体和药学上可接受的赋形剂。
在另一个方面,本发明还涉及用于检测生物样品中的HERV-K包膜蛋白的方法,包括将所述生物样品与如上定义的抗HERV-K包膜抗体接触的步骤。
在另一个方面,本发明还涉及用于诊断患者的ALS,特别是散发性ALS的方法,包括将从所述患者获得的生物样品与如上定义的抗HERV-K包膜抗体接触的步骤。
发明详述
定义
如本文所用,术语“治疗(treating)”或“治疗(treatment)”,如本文所用,意指逆转、减轻、抑制该术语所适用的疾病或病症的进展或预防该术语所适用的疾病或病症,或逆转、减轻、抑制该术语适用的疾病或病症的一种或多种症状的进展或预防该术语适用的疾病或病症的一种或多种症状。
如本文所用,术语“预防”是指在受试者中预防疾病或病症发生,所述受试者尚未呈现允许其临床诊断的临床症状、典型病变或生理功能障碍。
如本文所用,“抗体”或“免疫球蛋白”具有相同的含义,并且将在本发明中同等地使用。本文所用的术语“抗体”是指免疫球蛋白分子和免疫球蛋白分子的免疫活性部分,即包含特异性结合抗原的抗原结合位点的分子。因此,术语“抗体”不仅涵盖完整的抗体分子,还涵盖抗体片段,以及抗体衍生物。
如本文所用,表述“抗体片段”是指这种抗体的一部分模拟高变区,例如CDR(CDR-L1、CDR-L2、CDR-L3、CDR-H1、CDR-H2、CDR-H3)。根据本发明的抗体片段保留了所述抗体的结合亲和力和特异性。这种片段是所述抗体的功能等同物,它们与所述抗体结合基本相同的表位。抗体片段的实例包括但不限于重链、轻链、VL、VH、Fv、Fab、Fab'、F(ab)2和F(ab')2。
如本文所用,表述“抗体衍生物”是指与至少一个不同于天然序列的序列(例如,另一物种的接头序列......)融合的本发明抗体的片段,优选包括所述抗体的至少一个CDR,优选所述抗体的至少一个CDR3,所述衍生物具有与本发明抗体相当的对HERV-K Env的结合亲和力和特异性。根据本发明的衍生物保留了所述抗体的结合亲和力和特异性。这种衍生物是所述抗体的功能等同物,它们与所述抗体结合基本相同的表位。抗体衍生物的实例包括但不限于scFv、(scFv)2和双抗体。
在天然抗体中,两条重链(HC)通过二硫键彼此连接,并且每条重链通过二硫键与轻链(LC)连接。有两种类型的轻链,λ(1)和κ(k)。有五种主要的重链类型(或同种型),其决定了抗体分子的功能活性:IgM、IgD、IgG、IgA和IgE。每条链含有不同的序列结构域。
通常,轻链包括两个结构域,可变结构域(VL)和恒定结构域(CL)。重链包括四个结构域,可变结构域(VH)和三个恒定结构域(CH1、CH2和CH3,统称为CH)。轻链(VL)和重链(VH)的可变区决定了对抗原表位特异性的结合位点。轻链(CL)和重链(CH)的恒定区结构域赋予重要的生物学特性,例如抗体链结合、分泌、经胎盘移动、补体结合和与Fc受体(FcR)的结合。Fv片段是免疫球蛋白的Fab片段的N末端部分,并且为一条轻链和一条重链的可变部分。抗体的特异性在于抗体结合位点和抗原表位之间的结构互补性。抗体结合位点由主要来自高变或互补决定区(CDR)的残基组成。偶尔,来自非高变区或框架区(FR)的残基影响整个结构域结构,从而影响结合位点。互补决定区或CDR是指一起定义天然免疫球蛋白结合位点的天然Fv区的结合亲和力和特异性的氨基酸序列。免疫球蛋白的轻链和重链各自具有三个CDR,分别称为L-CDR1、L-CDR2、L-CDR3和H-CDR1、H-CDR2、H-CDR3。因此,抗原结合位点包括六个CDR,其包含来自每一个重链和轻链V区的CDR组。框架区(FR)是指插入CDR之间的氨基酸序列。
如本文所用,术语“嵌合抗体”是指包含以下的抗体:来自任何物种,优选小鼠的抗体的VH结构域和VL结构域,以及人抗体的CH结构域和CL结构域。
根据本发明,术语“人源化抗体”是指具有来自人抗体的可变区框架和恒定区的抗体,但保留来自任何物种,优选小鼠的抗体的CDR。
术语“Fab”表示具有约50,000的分子量和抗原结合活性的抗体片段,其中通过用蛋白酶(木瓜蛋白酶)处理IgG获得的片段中,约一半的H链的N末端侧和整个L链通过二硫键结合在一起。
如本文所用,术语“F(ab')2”是指具有约100,000的分子量和抗原结合活性的抗体片段,其中通过用蛋白酶(胃蛋白酶)处理IgG获得的片段中,其比通过铰链区的二硫键结合的Fab略大。
如本文所用,术语“Fab'”是指具有约50,000的分子量和抗原结合活性的抗体片段,其通过切割F(ab')2的铰链区的二硫键获得。
表述“单链Fv”或“scFv”是指多肽,其是共价连接的VH::VL异二聚体,并且通常由包括通过编码肽的接头连接的VH和VL编码基因的基因融合物表达。“dsFv”是通过二硫键稳定的VH::VL异二聚体。二价和多价抗体片段可以通过单价scFv的结合自发形成,或者可以通过肽接头偶联单价scFv产生,例如二价sc(Fv)2。
术语“双抗体”是指具有两个抗原结合位点的小抗体片段,该片段包含在同一多肽链(VH-VL)中与轻链可变结构域(VL)连接的重链可变结构域(VH)。通过使用太短而不允许同一链上的两个结构域之间配对的接头,结构域被迫与另一条链的互补结构域配对,产生两个抗原结合位点。
如本文所用,表述“本发明的抗体”是指抗(即,特异性结合)HERV-K包膜蛋白(HERV-K Env),优选抗K型人内源性逆转录病毒家族(HERV-K)的HERV-K包膜蛋白,更优选抗SEQ ID NO:9所示的表位的抗体。
如本文所用,本文所用的术语“生物样品”是指为了体外评价目的而获得的任何生物样品。在本发明中,样品或患者样品可包含任何体液或疾病特异性组织和病变,例如活组织检查。体液的实例包括血液、血清、血浆、乳头抽吸液、尿液、唾液、滑液和脑脊液(CSF)。
通常,本发明的抗体保护人神经元细胞免受神经元暴露于HERV_K Env蛋白而诱导的细胞毒性的危害。
更具体地,本发明的抗体优选表现出以下一种或多种功能特征:
-它保留暴露于HERV_K Env蛋白的神经元的神经元功能活性;
-它保留暴露于HERV_K Env蛋白的神经元的细胞活力;和/或
-它保留暴露于HERV_K Env蛋白的神经元的神经元形态。
如本发明的实施例2中所示,可以在体外评估根据本发明的抗体在针对细胞外HERV-K包膜细胞毒性方面对神经元功能活性的保护效果(参见点2.1.3)。特别地,这种效果可以通过以下来评估:用重组HERV-K Env蛋白处理后,记录人神经元在体外的自发电生理活性。因此,与未经重组HERV-K Env蛋白处理的人神经元相比,通常用本发明的抗体治疗使暴露于所述重组HERV-K Env蛋白的人神经元的自发活性恢复至少50%,特别是至少60%、至少70%、至少80%、至少90%或至少95%。
在一些实施方案中,本发明的抗体的保护作用可以通过用重组HERV-K Env蛋白处理后分析神经元培养物的活力来替代地或额外地在体外评估,如实施例2中所示(参见第2.1.1点)。典型地,在这种实施方案中,与未经本发明抗体处理的人神经元的活力相比,用本发明所述抗体处理使与重组HERV-K Env蛋白一起预孵育的人神经元的活力提高至少20%。
在一些实施方案中,本发明的抗体的保护作用可以通过评估用重组HERV-K Env蛋白处理后的培养物中的人神经元的神经元形态(例如,轴突长度)来替代地或额外地在体外评估,如实施例2中所示(参见第2.1.2点)。典型地,在这种实施方案中,与未经本发明抗体处理的人神经元的活力相比,用本发明所述抗体处理使与重组HERV-K Env蛋白一起预孵育的人神经元的轴突长度增加至少20%。
在一个实施方案中,本发明的抗体包含如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的6个CDR中的每一个。
在一个实施方案中,本发明的抗体包含:
-轻链,其中可变结构域包含以下3个CDR中的每一个:SEQ ID NO:1所示的CDR-L1、SEQ ID NO:2所示的CDR-L2和SEQ ID NO:3所示的CDR-L3;和
-重链,其中可变结构域包含以下3个CDR中的每一个:SEQ ID NO:4所示的CDR-H1、SEQ ID NO:5所示的CDR-H2和SEQ ID NO:6所示的CDR-H3。
如上所述的互补决定区(CDR)如表1所公开。
表1:根据本发明的抗体的CDR结构域
在一个实施方案中,本发明的抗体、片段或衍生物包含:
-如SEQ ID NO:7所示的轻链可变区(VL);和
-如SEQ ID NO:8所示的重链可变区(VH)。
如上所述的轻链和重链可变区如表2所公开。
表2:根据本发明的抗体的轻链和重链可变区
在一个实施方案中,本发明的抗体、片段或衍生物选自:Fv、Fab、F(ab')2、Fab'、dsFv、scFv、sc(Fv)2、双抗体和由抗体片段形成的多特异性抗体。
在优选的实施方案中,本发明的抗体是单克隆抗体。本发明的单克隆抗体是单价、二价、多价、单特异性、双特异性或多特异性的。在另一个实施方案中,抗HERV-K Env的抗体是结合片段或偶联物。例如,本发明的抗体可以与生长抑制剂、细胞毒性剂或前药活化酶偶联。
本发明抗体的另一种氨基酸修饰可用于改变抗体的原始糖基化模式。“改变”是指缺失抗体中发现的一个或多个碳水化合物部分,和/或添加抗体中不存在的一个或多个糖基化位点。抗体的糖基化通常是N-连接。“N-连接”是指碳水化合物部分与天冬酰胺残基的侧链连接。三肽序列天冬酰胺-X-丝氨酸和天冬酰胺-X-苏氨酸(其中X是除脯氨酸外的任何氨基酸)是碳水化合物部分与天冬酰胺侧链酶促连接的识别序列。因此,多肽中这些三肽序列中任一个的存在产生潜在的糖基化位点。通过改变氨基酸序列使其含有一个或多个上述三肽序列(对于N-连接的糖基化位点),可以方便地向抗体添加糖基化位点。
在另一个实施方案中,本发明的抗体是单克隆人源化抗体,更优选IgG4人源化单克隆抗体。
所述人源化抗体可以通过以下来产生:获得编码CDR结构域的核酸序列,将它们插入动物细胞的表达载体中,并通过将其导入动物细胞中来表达表达载体,所述动物细胞具有编码与人抗体相同的重链恒定区和与人抗体相同的轻链恒定区的基因。人源化抗体表达载体可以是其中编码抗体重链的基因和编码抗体轻链的基因存在于不同载体上的类型,或者是两个基因存在于同一载体上的类型(串联型)。鉴于构建人源化抗体表达载体的容易性,导入动物细胞的容易性,以及动物细胞中抗体H和L链的表达水平之间的平衡,更优选串联型人源化抗体表达载体。串联型人源化抗体表达载体的实例包括pKANTEX93、pEE18等。基于常规重组DNA和基因转染技术产生人源化抗体的方法是本领域熟知的。可以使用本领域已知的多种技术将抗体人源化,包括例如CDR移植、镶饰(veneering)或表面重塑(resurfacing)以及链替换。用于制备此类抗体的一般重组DNA技术也是已知的。
因此,本发明的一个实施方案涉及单克隆人源化抗体,其包含:
-轻链,其中可变结构域包含以下3个CDR中的每一个:SEQ ID NO:1所示的CDR-L1、SEQ ID NO:2所示的CDR-L2和SEQ ID NO:3所示的CDR-L3;和
-重链,其中可变结构域包含以下3个CDR中的每一个:SEQ ID NO:4所示的CDR-H1、SEQ ID NO:5所示的CDR-H2和SEQ ID NO:6所示的CDR-H3。
药物组合物
本发明的另一个目的涉及药物组合物,其包含有效剂量的抗HERV-K包膜蛋白(HERV-K Env)的抗体和药学上可接受的赋形剂。
如上所述的本发明的任何治疗剂可以与药学上可接受的赋形剂和任选的缓释基质(例如可生物降解的聚合物)组合以形成治疗组合物。
“药学上”或“药学上可接受的”是指当适当地施用于哺乳动物,尤其是人时,不产生有害的、过敏或其他不良反应的分子实体和组合物。药学上可接受的载体或赋形剂是指无毒的固体、半固体或液体填充剂、稀释剂、包封材料或任何类型的制剂助剂。
药物组合物的形式、施用途径、剂量和方案自然取决于待治疗的病症、疾病的严重程度、患者的年龄、体重和性别等。
本发明的药物组合物可以配制用于局部、口服、鼻内、眼内、静脉内、鞘内(直接在脑脊液中)、肌内或皮下施用等。
在一些实施方案中,配制用于鞘内施用的组合物可能是特别有利的。实际上,这种施用方式可以允许在特定治疗策略中短期施用非人抗体,例如小鼠抗体或嵌合抗体。实际上,CNS的“免疫-特权”生理学允许通过全身途径施用不可能的耐受性。因此,鞘内注射模式是常见的神经和神经外科手术。例如,利妥昔单抗是一种小鼠-人嵌合抗体,已用于多发性硬化和CNS淋巴瘤患者的鞘内注射(特别参见Bonnan M,Ferrari S,Bertandeau E,Demasles S,Krim E,Miquel M,Barroso B."Intrathecal rituximab therapy inmultiple sclerosis:review of evidence supporting the need for futuretrials.Curr Drug Targets".2014;15(13):1205-14.Topping J,Dobson R,Lapin S,Maslyanskiy A,Kropshofer H,Leppert D,Giovannoni G,Evdoshenko E."The effectsof intrathecal rituximab on biomarkers in multiple sclerosis".Mult SclerRelat Disord.2016 Mar;6:49-53.和Kadoch C,Li J,Wong VS,Chen L,Cha S,Munster P,Lowell CA,Shuman MA,Rubenstein JL."Complement activation and intraventricularrituximab distribution in recurrent central nervous system lymphoma".ClinCancer Res.2014 Feb 15;20(4):1029-41)。
优选地,药物组合物含有对于能够注射的制剂是药学上可接受的介质。这些可以特别是等渗的、无菌的、盐溶液(磷酸二氢钠或磷酸二钠、氯化钠、氯化钾、氯化钙或氯化镁等或这些盐的混合物),或干燥的,特别是冷冻干燥的组合物,其在根据情况添加无菌水或生理盐水时,允许构成可注射溶液。
用于施用的剂量可以根据各种参数进行调整,特别是根据所用的施用方式、相关病理学或所需治疗持续时间的函数进行调整。
为了制备药物组合物,可以将有效量的抗HERV-K包膜蛋白(HERV-K Env)的抗体溶解或分散在药学上可接受的载体或水性介质中。
适于注射使用的药物形式包括无菌水溶液或分散体;包括芝麻油、花生油或丙二醇水溶液的制剂;和用于临时制备无菌注射溶液或分散体的无菌粉末。在所有情况下,形式必须是无菌的并且必须是以易于注射的程度流动的。它必须在制造和储存条件下稳定,并且必须防止微生物如细菌和真菌的污染作用。
可以在与表面活性剂如羟丙基纤维素适当混合的水中制备作为游离碱或药理学上可接受的盐的活性化合物的溶液。也可以在甘油、液体聚乙二醇、其混合物和油中制备分散体。在通常的储存和使用条件下,这些制剂含有防腐剂以防止微生物的生长。
配制后,将以与剂量配方相容的方式并以治疗有效的量施用溶液。易于以多种剂型施用制剂,例如上述可注射溶液的类型,但也可使用药物释放胶囊等。
优选地,可以将本发明的抗HERV-K包膜蛋白(HERV-K Env)的抗体配制在将其溶解的缓冲液中,储存并向患者注射。优选地,所述缓冲液包含20mM组氨酸、5%蔗糖和0.01%聚山梨醇酯20,且pH为6.0。
对于在水溶液中的肠胃外给药,例如,可以适当地缓冲溶液,并且首先用足够的盐水或葡萄糖使液体稀释剂等渗。这些特定的水溶液特别适用于静脉内、肌肉内、皮下和腹膜内施用。就此而言,根据本公开内容,可以使用的无菌水性介质对于本领域技术人员而言是已知的。例如,可以将一个剂量溶解在1ml等渗NaCl溶液中,并且将其添加到1000ml的皮下注射液中或者在建议的输注部位注射(参见例如,“Remington's PharmaceuticalSciences”第15版,第1035页-1038和1570-1580)。取决于所治疗患者的病症,剂量必然发生一些变化。无论如何,负责施用的人将确定个体患者的适当剂量。
除了配制用于肠胃外施用(例如静脉内、鞘内或肌肉内注射)的化合物,其他药学上可接受的形式包括例如,用于口服施用的片剂或其他固体;时间释放胶囊;以及目前使用的任何其他形式。
本发明的诊断方法
另一方面,本发明还涉及检测生物样品中HERV-K包膜蛋白的方法,包括将所述生物样品与如上定义的抗HERV-K包膜抗体接触的步骤。
另一方面,本发明还涉及诊断患者ALS的方法,包括将从所述患者获得的生物样品与如上定义的抗HERV-K包膜抗体接触的步骤。
通常,生物样品可以是体液,例如脑脊液。
根据本发明的治疗方法和监测方法
在一个方面,本发明还涉及治疗患有ALS的患者的方法,包括向所述患者施用有效量的如上定义的识别HERV-K包膜蛋白的抗体。
优选地,患者患有ALS,特别是散发性ALS。
通常,所述抗体通过鞘内、静脉内或皮下施用。
本发明还涉及治疗患有ALS,特别是散发性ALS的患者的方法,包括以下步骤:
1)通过检测和/或定量生物样品中的HERV-K病毒来预测患者的预后;然后
2)如果所述步骤1)显示K型人内源性逆转录病毒(HERV)的表达,则本发明的方法包括向所述患者提供本发明的抗体的步骤。
本发明还涉及一种用于监测对患有ALS,特别是散发性ALS患者治疗的响应的方法,所述方法包括以下步骤:
a.用根据本发明的抗体治疗所述患者;然后
b.检测和/或定量所述患者的生物样品中的HERV-K。
根据本发明,在监测对患有ALS患者治疗的响应的情况下,生物样品可以是体液样品,例如血液、脑脊液、尿液或疾病特异性组织活检。
通常,检测和/或定量步骤可以根据本领域技术人员公知的常规技术进行。通常,所述步骤包括将患者的生物样品与选择性试剂如探针、引物、配体或抗体接触,从而检测最初在样品中存在的目标核酸或蛋白质。
在一个实施方案中,可以用如上定义的抗HERV-K抗体进行检测和/或定量步骤。
附图说明
图1:GN_mAb_Env_K01是IgG2b鼠抗体并具有κ轻链
通过ELISA对1:10稀释的上清液进行同种型分型,所述上清液来自通过抗鼠免疫球蛋白捕获的GN_mAb_Env_K01杂交瘤(单克隆期)。用各种抗小鼠轻(A)或重(B)链抗体检测,表明GN_mAb_Env_K01是IgG2b/κ鼠抗体。将结果绘制为两次重复的OD450mm值的平均值±SD。
图2:GN_mAb_En v_K01轻链和重链序列
提取杂交瘤细胞的RNA,并将其逆转录成cDNA,通过PCR扩增,然后用靶向编码鼠抗体重(A)和轻(B)链的cDNA的引物测序。
(A)来自κ轻链的CDR1(粗体)、CDR2(下划线)和CDR3(粗斜体)序列;
(B)来自重链的CDR4(粗体)、CDRS(下划线)和CDR6(粗体斜体)序列;恒定的鼠IgG2序列(灰色斜体)。
图3:GN_mAb_Env_K01结合HERV-K-Env的SLDKHKHKKLQSFYP表位
显示来自HERV-K-Env(MyBiosource)(右)的每种肽的强度谱(左)。这些具有一个残基偏移的15个氨基酸的重叠肽显示,GN_mAb_Env_K01结合线性SLDKHKHKKLQSFYP(SEQ IDNO:9)表位。结果表示为在所使用的CCD相机(与标准96孔板ELISA读数器类似)上获得的信号强度(mAU)。
图4:在ELISA中通过GN_mAb_ENV_K01检测糖基化的HERV-K Env
在HEK细胞裂解物的ELISA中,将各种稀释度(1:25、1:50、1:100、1:200、1:400)的GN_mAb_Env_K01(A)或来自AMSBIO的抗-HERV-K-Env(B)(1μg/ml)用作一抗。与抗-HERV-K-Env(AMSBio)相反,GN_mAb_Env_K01识别糖基化的HERV-K-Env。将结果绘制为两次重复的OD450mm值的平均值±SD。
图5:在ELISA中通过GN_mAb_ENV_K01检测非糖基化的HERV-K Env
在来自大肠杆菌的1μg/ml his-SUMO-HERV-K-Env重组蛋白的ELISA中,将GN_mAb_Env_K01或来自AMSBIO的抗-HERV-K-Env(1μg/ml)用作一抗。两种抗-HERV-K-Env均识别非糖基化的HERV-K-Env蛋白。将结果绘制为两次重复的OD450mm值的平均值±SD。
图6:通过蛋白质印迹用GN_mAB_ENV_K01检测HERV-K Env
在蛋白质印迹中,将来自AMSBIO的抗HERV-K-Env(1μg/ml)(孔#1和2)或来自GN_mAB_ENV_K01杂交瘤的稀释的(1:5)上清液(孔#3和4)用作一抗。将0.2μg his-SUMO标记的HERV-K-Env加入孔#1和3,并将24.5μg从HERV-K-Env转染的HEK细胞提取的蛋白质加入孔#2和4。通过两种抗体在75kDa检测到非糖基化的his-SUMO标记的HERV-K-Env蛋白,以及高MW的多聚体和较低MW的切割片段。用GN_mAb_Env_K01仅检测到糖基化的HERV-K-Env(90kDa)。
MW:分子量。
图7:GN_mAb_Env_K01(GN K01)抗体特异性地保护人神经细胞免受细胞外HERV-K包膜蛋白诱导的细胞毒性的危害:细胞存活测定。
用分化培养基(参见实施例2)和终浓度为3μg/mL的IgG样品GN K01或对照非免疫IgG(Thermo Product#MA 1-10418)处理神经元培养物。预孵育60分钟后,加入重组HERV-KEnv蛋白(My BioSource,氨基酸90-632,Cat#MBS 1391552)至终浓度100nM。将一个GN K01Ig样品与HERV-K Env预孵育30分钟,然后一起加入人神经元。在不同时间点(在该实验中是Env处理后5天)用GE INCell Analyzer 2000 Biolmager观察神经元培养物,以获得每个孔的图像(每孔4个图像)。用GE Investigator高含量成像软件测定神经元细胞计数。直方图顶部的箭头显示GN_mAb_Env_K01(GN K01)抗体的结果。
图8:GN_mAb_Env_K01(GN K01)抗体特异性地保护人神经细胞免受细胞外HERV-K包膜蛋白诱导的细胞毒性的危害:轴突长度。
用分化培养基(如上所述)和终浓度为3μg/mL的IgG样品GN K01或对照非免疫IgG(Thermo Product#MA 1-10418)处理神经元培养物。预孵育60分钟后,加入重组HERV-K Env蛋白(My BioSource,氨基酸90-632,Cat#MBS 1391552)至终浓度100nM。在不同时间点(在该实验中是Env处理后5天)用GE INCell Analyzer 2000 Biolmager观察神经元培养物,以获得每个孔的图像(每孔4个图像)。用GE Investigator高含量成像软件测定平均轴突纤维长度。
图9:GN_mAb_Env_K01(GN K01)抗体特异性地保护人神经细胞免受细胞外HERV-K包膜蛋白诱导的细胞毒性的危害:神经元整体电生理活性。
在21天,在体外通过孔中发放率增加注意到的电生理活性显著增加,并通过每天记录所有孔中的自发电活性5分钟来监测。在这一点上,用分化培养基(如上所述)和终浓度为3μg/mL的IgG样品GN K01或对照非免疫IgG(Thermo Product#MA 1-10418)处理人神经元培养物。预孵育60分钟后,加入重组HERV-K Env蛋白(My BioSource)至终浓度100nM。从处理后24小时开始,之后每天记录自发电活性。在HERV-K Env暴露24小时后,测定每个处理组的平均放电率。
直方图顶部的箭头显示GN_mAb_Env_K01(GN K01)抗体的结果。直方图顶部的箭头显示GN_mAb_Env_K01(GN K01)抗体的结果。
具体实施方式
实施例1:GN_mAb_Env_K01抗体的开发和表征
1.材料和方法
1.1单克隆抗体开发
1.1.1免疫接种和免疫细胞回收
根据来自Biotem公司的保密RAD(快速抗体开发)方案,用来自Mybiosource提供的大肠杆菌的his-SUMO标记的HERV-K-Env蛋白(75kDa)(MBS 1391552)免疫接种三只雌性OF小鼠(Charles River)。
简言之,在第+10天(D+10),通过在重组HERV-K Env蛋白(MyBiosource,MBS1391552)或作为阴性对照的大肠杆菌裂解物上的直接ELISA分析来自免疫小鼠的血液样品。在D+13,处死免疫的小鼠并收集来自淋巴结的免疫细胞,用45ml Dulbecco改良的Eagle培养基(DMEM,SIGMA,D5671)洗涤三次,在244g离心7分钟后,重悬于20ml DMEM(SIGMA,D5671)。将来自免疫小鼠的免疫细胞(420×106个细胞)与指数生长期的骨髓瘤细胞(107×106个细胞)以1:3.9的比例混合。将细胞在244g离心7分钟,将沉淀重悬于1ml用作融合剂的聚乙二醇(PEG)(SIGMA,P7181)中。在洗涤步骤(包括以108g离心12分钟)后,将细胞重悬于10ml DMEM(SIGMA,D5671)、1x次黄嘌呤氨基蝶呤胸苷(HAT,SIGMA,H0262-10VL)、20%胎牛血清(FCS,PAA,A15)、4mM L-谷氨酰胺(SIGMA,G7513),并在244g离心7分钟。将细胞重悬浮于DMEM(SIGMA,D5671)、1X HAT(SIGMA,H0262-10VL)、20%FCS(PAA,A15-251)、4mM L-谷氨酰胺(SIGMA,G7513)中,并在室温下储存2小时。
1.1.2融合
在D-1,向免疫缺陷裸鼠(BIOTEM)注射5ml含有20%FCS(PAA,A 15-251)的DMEM(SIGMA,D5671),2+/-1分钟后,收集并在DMEM培养基(SIGMA,D5671)中培养来自腹膜液的巨噬细胞。
与P3X63Ag8骨髓瘤融合的来自用绵羊红细胞免疫接种的小鼠的BALB/c脾细胞已经通过BIOTEM选择、表征和储存。在D-10,将这些骨髓瘤解冻,并在DMEM(SIGMA,D5671)-8-氮鸟嘌呤(AZA,SIGMA,A5284)-10%FCS(PAA,A 15-251)中培养。
计数来自裸鼠的巨噬细胞,并将其以104个巨噬细胞/ml重悬于DMEM(SIGMA,D5671)、1X HAT(SIGMA,H0262-10VL)、20%FCS(PAA,A15-251)、4mM L-谷氨酰胺(SIGMA,G7513)、1%青霉素/链霉素(SIGMA,P0781)中。然后,将50μl用作生长因子的巨噬细胞悬浮液(相当于500个巨噬细胞)铺板在含有50μl杂交瘤细胞悬浮液的96孔板中。将这些细胞在37℃、5%CO2下培养21天。
1.1.3克隆
将杂交瘤细胞解冻,并在37℃、5%CO2下用DMEM(SIGMA,D5671)、HT(次黄嘌呤100μM、胸苷16μM-SIGMA H0137)、20%FCS(PAA,A15-251)、2%杂交瘤增强补充物(HES,SIGMA,H6020)、4mM L-谷氨酰胺(SIGMA,G7513)、1%青霉菌/链霉素(SIGMA,P0781)在24孔板中培养1周。克隆前一天,将杂交瘤细胞分开。
在D0,在培养基中以104、50、25、5和2.5个细胞/ml连续稀释后,将杂交瘤悬浮液以5、1和0.5个细胞/200μl铺板到96孔板的孔中。在D+6,用新鲜DMEM(SIGMA,D5671)、HT(次黄嘌呤100μM、胸苷16μM-SIGMA H0137)、20%FCS(PAA,A15-251)、2%HES(SIGMA,H6020)、1%青霉素/链霉素(SIGMA,P0781)替换含有细胞的孔中的100μl上清液(通过光学显微镜筛选进行选择)。
在对应于D+10的第一次ELISA筛选后,在24孔板中培养抗HERV-K-Env阳性杂交瘤(0.5ml/孔)。
在对应于D+14的第二次ELISA筛选后,在平板或培养瓶(Coming)中培养抗HERV-KEnv阳性杂交瘤,并将含有4-5×106个细胞的5个小瓶在-196℃下冷冻(液氮)在DMEM(SIGMA,D5671)、15%FCS(PAA,A 15-251)、4mM L-谷氨酰胺(SIGMA,G7513)、1%HES(SIGMA,H6020)、1%青霉素/链霉素(SIGMA,P0781)、20%二甲基亚砜(DMSO.Sigma,D2650)培养基中。
1.2抗HERV-K-Env ELISA
在室温下用50μl的1μg/ml HERV-K Env蛋白(Mybiosource,MBS 1391552)、大肠杆菌裂解物(XL 1-Blue MRF,Stratagene)、在1x磷酸盐缓冲盐水中的HEK细胞裂解物(PBS,BIOTEM)包被maxisorp锥形底96孔板(NUNC,449824)过夜。用[1X PBS+0.05%Tween20(VWR,28829.296)]洗涤缓冲液洗涤平板(300μl/孔)。在室温下用[1X PBS+0.05%Tween20+2.5%牛奶(Regilait)]封闭缓冲液封闭非特异性结合位点1小时。用[1X PBS+0.05%Tween20]洗涤缓冲液洗涤平板(300μl/孔)。
将抗体样品稀释于[1X PBS+0.05%Tween20+0.5%BSA(VWR,1.12018.0100)稀释缓冲液中。在室温下孵育抗体样品或来自AMSBIO的纯化的抗-HERV-K-Env(1μg/ml)(50μl/孔)2小时。用[1X PBS+0.05%Tween20]洗涤缓冲液洗涤平板三次(300μl/孔),并在室温下用50μl/孔的多克隆过氧化物酶偶联的affini Pure F(ab)'2片段山羊抗小鼠IgG+IgM(Jackson,115-036-068)(1/10000,在1X PBS+0.05%Tween20+0.5%BSA中)孵育1小时。将平板洗涤三次,并在室温下用四甲基联苯胺(TMB,Eurobio,52-00-01)底物溶液(50μL/孔)显色10分钟。用0.1M H2SO4(Merck,1.12080.1000)封闭反应(50μl/孔)。使用光密度(OD)读数器(Dynex)测量450nm处的光密度(OD)。
1.3制备、纯化、透析
将GN_mAb_Env_K01杂交瘤细胞解冻,并首先在T75cm2组织培养瓶中培养,然后在T300cm2组织培养瓶(Corning)中培养。最后,在37℃、5%CO2下,将10-12×106个细胞在Hyperflask(Corning,10030)中的500ml DMEM(SIGMA,D5671)、15%FCS(PAA,A15-251)、4mML-谷氨酰胺(SIGMA,G7513)、1%HES(SIGMA H6020)、1%青霉素/链霉素(SIGMA,P0781)培养基中培养10+/-1天。
将培养上清液在244g离心7分钟,并通过11μm尼龙网过滤器(SIGMA,NY 1104700)过滤。用软化水洗涤蛋白A色谱柱(GE Healthcare,Mab Select Xtra)两次,并用5倍体积的1X PBS(Biotem)平衡。然后,加载0.5L不含细胞的培养上清液。用5倍体积的1X PBS洗涤柱子。在酸性pH下用3.5+/-0.5倍体积的乙酸(SIGMA,A6283)洗脱免疫球蛋白。用100μl1MTris pH 8.8缓冲液(Biotem)中和含有免疫球蛋白的洗脱级分,并在4℃下储存。
将IgG纯化的级分在0.5ml微透析胶囊(Roth,H448-1)上,用10kDaSnakeSkin透析管,22mm(Thermofischer,68100)在4℃下在1X PBS中透析2次,并通过于4℃在Vivaspin 20(30Kda)(Sartorius,ref)下离心浓缩。在0.22μm/>过滤器(Sartorius,ref)上过滤抗体,并通过分光光度法在280nm处测量蛋白质浓度。
1.4通过SDS PAGE凝胶电泳分析纯度
将先前在Laemmli缓冲液(Biotem)中稀释的抗体(5μl,0.2μg/μl)在95℃下加热5分钟,并在含有堆积凝胶的13.5%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)上用5%SDS-PAGE分离。凝胶在90V下运行30分钟,然后在120V下运行2小时。在搅拌下用着色溶液(Biotem)进行蛋白质检测1小时。在搅拌下用褪色溶液(Biotem)洗涤凝胶1小时。
1.5同种型分型
1.5.1通过ELISA进行同种型分型
在室温下用50μl的1μg/ml抗鼠免疫球蛋白(Climsciences,1010-01)包被maxisorp锥形底96孔板(NUNC,449824)过夜。用[1X PBS+0.05%Tween20]洗涤缓冲液洗涤平板(300μl/孔)。在室温下用[1X PBS+0.05%Tween20+2.5%牛奶(Regilait)]封闭缓冲液封闭非特异性结合位点1小时(300μl/孔)。用[1X PBS+0.05%Tween20]洗涤缓冲液洗涤平板(300μl/孔)。将杂交瘤上清液以1:10稀释于[1X PBS+0.05%Tween20+0.5%BSA]稀释缓冲液中。在室温下孵育样品(50μl/孔)2小时。用[1X PBS+0.05%Tween20]洗涤缓冲液洗涤平板一次(300μl/孔),并在室温下用50μl/孔的过氧化物酶偶联的山羊抗小鼠重链(IgA、IgGl、IgG2a、IgG2b、IgG3、IgM)(Clinisciences,5300-05)(1/2000,在1X PBS+0.05%Tween20+0.5%BSA中)孵育1小时。将平板洗涤一次,并在室温下用TMB(Eurobio,52-00-01)底物溶液(50μL/孔)显色10分钟。用0.1M H2SO4(Merck,1.12080.1000)封闭反应(50μl/孔)。使用光密度(OD)读数器(Dynex)测量450nm处的光密度(OD)。
1.5.2通过测流免疫测定进行同种型分型
用测流免疫测定(LFIA)(ThermoFisher,26179)表征轻链(λ或κ)。
1.6 HEK转染
用1μg表达HERV-K-Env(登录号AY037928.1)的质粒转染人胚胎细胞(1.106个细胞/mL)。将将转染细胞在37℃、8%CO2、120rpm震荡下培养。
1.7蛋白质印迹分析
将12.5ng/μl的重组HERV-K-Env蛋白(Mybiosource)和1.5μl/μl的来自HEK转染细胞的蛋白质裂解物以(1:1)稀释在2X Laemmli缓冲液(SIGMA,S3401)中,并在90℃加热5分钟。然后,在8-16%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE,Novex,EC60452BOX)上加载32μl样品。在1X Tris-甘氨酸SDS运行缓冲液(Novex,LC2675)中,以160mA运行凝胶1小时。在1X Tris-甘氨酸转移缓冲液(Novex,LC3675)中将蛋白质转移到0.2μm硝酸纤维素膜(Biorad,162-0146)上后,在室温下的旋转平台上用[1X PBS+5%牛奶(La Vie Claire)]封闭缓冲液将膜封闭1小时。将来自GN_mAb_Env_K01杂交瘤的细胞上清液以1:5稀释于[1XPBS+1%牛奶]抗体稀释液中,并用作一抗孵育1小时。然后将膜在[1XPBS+0.05%Tween20(SIGMA,P7949)]洗涤缓冲液中洗涤三次,每次5分钟,并用1:1000稀释的HRP-偶联的山羊抗鼠IgG抗体(Jackson,115035-146)孵育30分钟。将膜洗涤三次,并根据提供的方案用比色反应(Opti 4-CN,Biorad,170-8235)检测目标蛋白质。
1.8表位定位
在Pepscan Presto BV(Zuidersluisweg 2,8243RC Lelystad,The Netherlands)进行表位定位。
1.8.1肽文库合成
为了重建靶分子的表位,合成了肽文库。通过以下获得氨基官能化的聚丙烯支持物:用专有亲水聚合物制剂接枝,然后用二环己基碳二亚胺(DCC)和N-羟基苯并三唑(HOBt)与叔丁氧基羰基-六亚甲基二胺(BocHMDA)反应,然后使用三氟乙酸(TFA)裂解Boc-基团。通过定制改良的JANUS液体处理站(Perkin Elmer),使用标准的Fmoc-肽合成在氨基官能化的固体支持物上合成肽。使用Pepscan专有的在支架上化学连接肽(CLIPS)技术合成结构模拟物。CLIPS技术允许将肽构建成单环、双环、三环、片状折叠、螺旋状折叠及其组合。将CLIPS模板与半胱氨酸残基偶联。将肽中多个半胱氨酸的侧链与一个或两个CLIPS模板偶联。例如,将0.5mM的P2CLIPS(2,6-双(溴甲基)吡啶)溶液溶解于碳酸氢铵(20mM,pH7.8)/乙腈(1:3(v/v))中。将该溶液添加到肽阵列上。CLIPS模板将与存在于肽阵列的固相结合肽中的两个半胱氨酸的侧链结合(具有3μl孔的455孔板)。在完全覆盖在溶液中的情况下,将肽阵列在溶液中轻轻摇动30至60分钟。最后,用过量的H2O彻底洗涤肽阵列,并在含有1%SDS/0.1%β-巯基乙醇的PBS(pH7.2)破碎缓冲液中于70℃超声处理30分钟,然后在H2O中超声处理45分钟。以类似的方式制备携带肽的T3CLIPS,但现在使用三个半胱氨酸。
1.8.2 ELISA筛选
在基于Pepscan的ELISA中测试抗体与每种合成肽的结合。将肽阵列与一抗溶液(Pepscan缓冲液中1μg/ml的GN_mAb_Env_K01)在4℃孵育过夜。洗涤后,将肽阵列与兔抗小鼠IgG(H+L)HRP偶联抗体(Southern Biotech;表4)的1/1000稀释物在25℃孵育1小时。洗涤后,加入过氧化物酶底物2,2’-连氮基-二-3-乙基苯并噻唑啉磺酸盐(ABTS)和20μl/ml的3%H2O2。一小时后,测量显色。用电荷耦合设备(CCD)-相机和图像处理系统定量显色。从CCD相机获得的值范围为0至3000mAU,与标准96孔板ELISA读取器类似。
1.9测序
使用PureLink RNA Mini试剂盒(Life technologies,12183018A)从杂交瘤细胞中提取并纯化高质量的RNA,并在琼脂糖凝胶上进行控制。从纯化的总RNA开始,然后使用Superscript酶(Invitrogen,18064022)合成第一链互补DNA(cDNA),然后使用HotStarHiFidelity Polymerase试剂盒(Qiagen,202602)和简并引物(Biotem)进行聚合酶链式反应(PCR),所述简并引物特异性靶向编码鼠抗体重链和轻链的cDNA。在琼脂糖凝胶上控制PCR产物,并进行测序(双链测序)。使用专用生物信息学工具(Blast-ClustalW)分析所得序列的组装和质量控制,并将其翻译成肽序列。
1.10 Biotem缓冲液组成
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表1-BIOTEM缓冲液组成2.结果
使用能够检测单克隆抗体的不同重链和轻链的特异性抗免疫球蛋白抗体,我们已经表明,通过抗-κ轻链抗体(图1A)和抗-IgG2b重链(图1B)检测到GN_mAb_Env_K01。
对GN_mAb_Env_K01重链和轻链进行测序,显示其三个CDR区(图2),并确认了IgG2b重链和κ轻链。
2.1 GN_mAb_Env_K01识别来自HERV-K-Env表面单元的线性表位SLDKHKHKKLQSFYP。
在高严格条件下,使用来自Mybiosource表位定位测试的截短HERV-K-Env蛋白序列(无信号肽和截短的跨膜结构域)的529个肽组,显示GN-mAb-Env_K01抗体与具有SLDKHKHKKLQSFYP核心序列的线性肽结合(图3,左图)。该表位包含在HERV-K-Env蛋白的细胞外结构域内(图3右图),对应于Dewannieux等(Dewannieux,Blaise和Heidmann 2005)描述的HERV-K-Env蛋白的298-312区域。在国家生物技术信息中心(NCBI)的Blastp中检索该表位,发现它是高度保守的表位,与对应于HERV-K-Env序列的前100个blast命中具有100%同源性(数据未显示)。
2.2 GN_mAb_Env_K01在天然条件下识别糖基化和非糖基化的HERV-K-Env蛋白。
我们进一步分析了GN_mAb_Env_K01识别糖基化HERV-K-Env的能力。为此目的,用编码HERV-K-Env蛋白的质粒转染人胚肾(HEK)细胞。尽管使用各种缓冲液进行了多次尝试,我们未能提取可溶性HERV-K-Env蛋白(数据未显示)。然而,通过ELISA,来自转染HEK细胞裂解物的糖基化HERV-K-Env蛋白的这种不溶性级分可以显示,GN_mAb_Env_K01特异性识别糖基化的HERV-K-Env抗原(图4,左图)。相反,来自AMSBIO的经典商业抗-HERV-K-Env mAb(HERM-1821-5,IgG2b)(图4,右图)未检测到糖基化的HERV-K-Env。
本结果表明,GN_mAb_Env_K01在ELISA中具有生物活性,并证实在天然条件下SLDKHKHKKLQSFYP表位是可接近的。
虽然GN_mAb_Env_K01和来自AMSBIO的抗HERV-K-Env mAb(HERM-1821-5,IgG2b)都识别来自大肠杆菌的天然的his-SUMO标记的重组HERV-K-Env(图5),但当以相同浓度(1μg/ml)测试两者时,与来自AMSBIO的抗-HERV-K-Env mAb对应物相比,GN_mAb_Env_K01似乎给出了高得多的检测(图5)。这证明了GN_mAb_Env_K01对HERV-K Env的亲和力更高。
如图6所示,GN_mAb_Env_K01和来自AMSBIO的抗HERV-K-Env mAb(HERM-1821-5,IgG2b)均识别来自大肠杆菌的变性的his-SUMO标记的重组HERV-K-Env,因为观察到75kDa信号。
重要的是,GN_mAb_Env_K01还检测来自HEK转染细胞的变性的HERV-K-Env糖基化蛋白(因为观察到90kDa的信号),而用来自AMSBIO的抗HERV-K-Env抗体未检测到信号。除了先前使用非变性蛋白的ELISA结果,GN_mAb_Env_K01在蛋白质印迹中具有生物学活性。因此,在变性条件下SLDKHKHKKLQSFYP表位也可接近。
4.结论
本报告表明,在小鼠免疫接种、抗体筛选和单克隆杂交瘤选择后,已开发出识别HERV-K-Env SLDKHKHKKLQSFYP表位的鼠单克隆抗体(命名为GN_mAb_Env-K01),并且表明其显示出意想不到的性质。
与来自AMSBIO的另一种抗HERV-K Env mAb(HERM-1821-5,IgG2b)的生物学比较证实,尽管它们具有相似的起源(鼠)、同种型(IgG2b,κ)和蛋白质靶标(HERV-W-Env蛋白),GN_mAb_EnvK-01是有利的,因为识别糖基化和非糖基化蛋白质,同时在天然和变性条件下都显示出高亲和力。此外,在来自数据库中描述的许多各种拷贝的HERV-K包膜序列中,GN_mAb_EnvK-01抗体靶向稳定且保守的表位。
因此,GN_mAb_EnvK-01是一种有用的工具,不仅用于免疫测定,而且用于针对例如在ALS中HERV-K Env蛋白作为治疗靶标的治疗目的。它在HERV-K拷贝中的稳定表位序列、它的高亲和力和它与天然糖基化形式的有效结合,满足了对例如ALS患者(其中不同HERV-K拷贝似乎显著表达)的有价值治疗的重要要求。
实施例2:GN_mAb_Env_K01(GN K01)抗体有效中和对人神经细胞的HERV-K包膜神经毒性
1.材料和方法
1.1人神经元细胞
如所述制备人神经干细胞(NSC)衍生的神经元培养物(Efthymiou,Shaltouki等,2014)。简言之,以7500-10,000个细胞/cm2将NSC分到涂有0.002%聚-L-鸟氨酸(Sigma,St.Louis,MO)和10μg/mL层粘连蛋白(Life Technologies)的96孔板中,并在铺板后24小时加入神经元分化培养基。分化培养基含有DMEM/F12和GlutaMax、1.8%牛血清白蛋白(BSA)、1x StemPro hESC补充剂(均来自Life Technologies)、10ng/mL脑源性神经营养因子(BDNF)和神经胶质细胞系衍生的神经营养因子(GDNF;R&D Systems,Minneapolis,MN),且细胞每隔一天接受新鲜培养基和生长因子。在体外第7-12天的神经元用于神经毒性测定。
1.2神经毒性试验
1.2.1神经形态和活力
如上所述,将稳定表达Td-番茄荧光蛋白以标记细胞和过程的人神经元培养物(每孔15-20,000个细胞)铺板到96孔板上,并在5%CO2、37℃下保持在加湿的组织培养箱中。用分化培养基(如上所述)和终浓度为3μg/mL的IgG样品GN K01或对照非免疫IgG(ThermoProduct#MA 1-10418)处理神经元培养物。预孵育60分钟后,加入重组HERV-K Env蛋白(MyBioSource,氨基酸90-632,Cat#MBS 1391552)至终浓度100nM。将一个GN K01 Ig样品与HERV-K Env预孵育30分钟,然后一起加入人神经元。在处理后24、48、72h的不同时间点,用GE INCell Analyzer 2000 Biolmager观察神经元培养物以获得每个孔的图像(每孔4个图像)。使用GE Investigator 1.93分析软件实现这些培养物的高含量成像/分析。定量每个样品的神经元活力、轴突长度和其他形态参数。用Graph Pad Prism 7.02描述数据。
1.2.2电生理分析
使用Axion Maestro微电极阵列(MEA)测定进行电生理分析。
使用48孔t-MEA平板铺板人神经元培养物用于分析。这些板每孔含有16个活性记录电极。在t-MEA板的每个孔施加200,000个神经元,并将培养物保持在5%CO2、37℃下保持在加湿的组织培养箱中。在体外通过孔中发放率增加注意到,电生理活性在21天显著增加,并通过每天记录所有孔中的自发电活性5分钟来监测。在这一点上,用分化培养基(如上所述)和终浓度为3μg/mL的IgG样品GN K01或对照非免疫IgG(Thermo Product#MA 1-10418)处理神经元培养物。预孵育60分钟后,加入重组HERV-K Env蛋白(My BioSource)至终浓度100nM。在这些条件下,首先将抗体加入到没有Env的细胞中,从而再现在其脑组织中扩散的治疗性抗体存在的治疗患者的状况。此后,加入活性Env蛋白(未与抗体预孵育,未被预先中和且不是作为无活性蛋白质添加),从而再现在细胞外空间中具有细胞外分泌的致病蛋白的表达。将一个GN K01 Ig样品与HERV-K Env预孵育30分钟,然后一起加入人神经元。从处理后24小时开始,之后每天记录自发电活性。用Axion Axis软件定量电活性。测定每次处理的参数,例如发放数、平均放电率和爆发次数。
2.结果
2.1细胞外HERV-K包膜蛋白对人神经元细胞有毒,其毒性受到GN_mAb_Env_K01(GNK01)抗体的特异性抑制
2.1.1神经元活力
用100nM重组HERV-K Env蛋白处理的人神经元培养物显示出显著的神经毒性,导致在接下来的几天内重要的神经元细胞损失。在暴露于HERV-K Env蛋白5天后,定量HERV-KEnv的作用。我们观察到,与Env处理的神经元相比,Env+GN K01 Ig(3μg/mL)处理的神经元具有增加的存活率。当在将HERV-K包膜蛋白加入培养基后5天进行分析时,用Env加3μg/mL非免疫IgG对照抗体处理的细胞表现出相似的毒性。用3μg/mL GN K01处理(在HERV-K Env之前,或用Env预孵育然后应用于神经元培养物)显示显著更有活力的神经元,从而证实GNK01抗体在中和HERV-K Env蛋白毒性方面的作用(图7)。
2.1.2神经元轴突长度
平行分析HERV-K Env对平均轴突长度的影响,与Env或Env+对照非免疫Ig处理的神经元相比,向暴露于Env的神经元添加GN K01(3μg/ml)显著增加了轴突长度。
2.1.3神经元功能活性
使用Axion Maestro MEA系统进行电生理学研究以评估HERV-K Env处理是否导致自发电活性(是正常神经元活动的主要特征)的功能性改变。将人神经元培养物铺板到48孔MEA平板上21天后,将培养物在分化培养基(如上所述)和终浓度为3μg/ml的GN K01或对照非免疫IgG(Thermo Product#MA1-10418)中孵育。预孵育60分钟后,加入重组HERV-K Env蛋白(My BioSource,Cat#MBS 1391552)至终浓度100nM。将一个GN K01 Ig样品与HERV-K Env预孵育30分钟,然后一起加入人神经元。从处理后24小时开始记录自发电活性。HERV-K Env处理后24h,Env处理将发放数和平均放电率降低了40%。暴露于HERV-K Env加对照非免疫IgG处理的神经元显示出发放数和平均放电率下降约30%。最有趣的是,暴露于HERV-K Env加GN K01的神经元显示出的发放数和平均放电率与仅用对照培养基孵育的孔类似,从而显示在全局神经元功能活性上对HERV-K Env致病性的完全抑制(图9)。
3.结论
因此,GN K01抗体的功效是特异性的,其对神经元暴露于HERV K Env蛋白的致病后果的有益效果通过以下来证明:与用无关对照抗体或不用抗体的相同HERV-K Env暴露相比,其显著的(i)抑制细胞死亡,(ii)维持神经元细胞形态和轴突长度,和(iii)在存在致病性HERV-K Env蛋白的情况下通过电生理活性测量的神经元功能活性的完全恢复。
因此,(i)表明了在散发性ALS患者的脑实质内改变的神经元中特异性检测到HERV-K Env蛋白后,(ii)在通过在表达编码这种独特蛋白质的HERV-K env基因的转基因小鼠中再现的散发性ALS的临床和组织学体征证明了单独的HERV-K Env蛋白驱动致病性这一概念后(Li,Lee等,2015),GN K01抗体的特异性功效(如发明人在人神经元细胞和功能方面所证明的那样)证明了其在ALS中,特别是在散发性ALS中的治疗价值。
这种抗体的特殊活性似乎中和(Le:治疗)HERV-K包膜(Env)蛋白的致病作用,所述Env蛋白本身被证明与散发性ALS患者大脑中的ALS特异病征性神经元病变相关,并且当添加到培养的神经元中时,或当在具有与ALS相同临床体征的小鼠中作为单个转基因表达时,其再现相同的神经元改变。此外,在HERV-K Env致病蛋白存在下添加GN K01抗体显示出比在体外用HERV-K Env重组蛋白预孵育GN K01更显著的功效,显示其在生理条件下(从而在治疗应用中)的最佳功效。
根据先前的知识,HERV_K蛋白毒性不仅涉及转染的靶细胞中或转基因动物的神经元中的细胞内表达,还涉及对初始(非HERV-K转染或过表达)神经元致病的分泌的细胞外HERV-K蛋白。因此,本发明的抗体还保护人神经元细胞免受由神经元暴露于分泌的和/或细胞外HERV_K Env蛋白诱导的细胞毒性的旁分泌扩散的危害。隐含地,这也适用于在表达或过表达HERV-K env编码基因的神经元中产生的HERV_K Env蛋白的自分泌细胞毒性。
总之,根据本发明的特异性靶向HERV-K包膜蛋白的抗体可以中和其在ALS中,特别是在散发性ALS中,或在再现ALS特征的HERV-K env转基因小鼠模型中观察到的病理生理学特性。
参考文献
Alfahad,T.,and A.Nath.2013.'Retroviruses and amyotrophic lateralsclerosis',Antiviral Res,99:180-7.
Andrews,W.D.,P.W.Tuke,A.Al-Chalabi,P.Gaudin,S.Ijaz,M.J.Parton,andJ.A.Garson.2000.'Detection of reverse transcriptase activity in the serum ofpatients with motor neurone disease',J Med Virol,61:527-32.
Buratti,E.,and F.E.Baralle.2009.'The molecular links between TDP-43dysfunction and neurodegeneration',Adv Genet,66:1-34.
Dewannieux,M.,S.Blaise,and T.Heidmann.200S.'Identification of afunctional envelope protein from the HERV-K family of human endogenousretroviruses',J Virol,79:15573-7.
Douville,R.,J.Liu,J.Rothstein,and A.Nath.2011.'Identification ofactive loci of a human endogenous retrovirus in neurons of patients withamyotrophic lateral sclerosis',Ann Neurol,69:141-51.
Douville,R.N.,and A.Nath.2014.'Human endogenous retroviruses and thenervous system',Handb Clin Neurol,123:465-85.
Duperray,A.,D.Barbe,G.Raguenez,B.B.Weksler,I.A.Romero,P.O.Couraud,H.Perron,and P.N.Marche.2015.'Inflammatory response of endothelial cells to ahuman endogenous retrovirus associated with multiple sclerosis is mediated byTLR4',Int Immunol,27:545-53.
Efthymiou,A.,A.Shaltouki,J.P.Steiner,B.Jha,S.M.Heman-Ackah,A.Swistowski,X.Zeng,M.S.Rao and N.Malik(2014)."Functional screening assayswith neurons generated from pluripotent stem cell-derived neural stem cells."J Biomol Screen 19(1):32-43.
Geser,F.,M.Martinez-Lage,L.K.Kwong,V.M.Lee,and J.Q.Trojanowski.2009.'Amyotrophic lateral sclerosis,frontotemporal dementia and beyond:the TDP-43diseases',J Neurol,256:1205-14.
Hardiman,O.,L.H.van den Berg,and M.C.Kiernan.2011.'Clinical diagnosisand management of amyotrophic lateral sclerosis',Nat Rev Neurol,7:639-49.
Lagier-Tourenne,C,and D.W.Cleveland.2009.'Rethinking ALS:the FUSabout TDP-43',Cell,136:1001-4.
Li,W.,M.H.Lee,L.Henderson,R.Tyagi,M.Bachani,J.Steiner,E.Campanac,D.A.Hoffman,G.von Geldern,K.Johnson,D.Marie,H.D.Morris,M.Lentz,K.Pak,A.Mammen,L.Ostrow,J.Rothstein,and A.Nath.2015.'Human endogenous retrovirus-Kcontributes to motor neuron disease',Sci Transl Med,7:307ral 53.
MacGowan,D.J.,S.N.Scelsa,T.E.Imperato,K.N.Liu,P.Baron,andB.Polsky.2007.′A controlled study of reverse transcriptase in serum and CSFof HIV-negative patients with ALS',Neurology,68:1944-6.
Mallet,F.,O.Bouton,S.Prudhomme,V.Cheynet,G.Oriol,B.Bonnaud,G.Lucotte,L.Duret,and B.Mandrand.2004.The endogenous retroviral locus ERVWEl is abonafide gene involved in hominoid placental physiology*,Proc Natl Acad SetUSA,101:1731-6.
Manghera,M.,J.Ferguson-Parry,and R.N.Douville.2016.TDP-43 regulatesendogenous retrovirus-K viral protein accumulation',Neurobiol Dis,94:226-36.
McCormick,A.L.,R.H.Brown,Jr.,M.E.Cudkowicz,A.Al-Chalabi,andJ.A.Garson.2008.'Quantiflcation of reverse transcriptase in ALS andelimination of a novel retroviral candidate',Neurology,70:278-83.
Moulignier,A.,A.Moulonguet,G.Pialoux,and W.Rozenbaum.2001.'ReversibleALS-like disorder in HIV infection1,Neurology,57:995-1001.
Oluwole,S.O.,Y.Yao,S.Conradi,K.Kristensson,and H.Karlsson.2007.'Elevated levels of transcripts encoding a human retroviral envelope protein(syncytin)in muscles from patients with motor neuron disease',AmyotrophLateral Scler,8:67-72.
Perron,H.,J.P.Perm,F.Rieger,and P.M.Alliel.2000.'Particle-associatedretroviral RNA and tandem RGH/HERV-W copies on human chromosome 7q:possiblecomponents of a'chain-reaction'triggered by infectious agents in multiplesclerosis?',J Neurovirol,6 Suppl 2:S67-75.
Polymenidou,M.,and D.W.Cleveland.2011.The seeds of neurodegeneration:prion-like spreading in ALS',Cell,147:498-508.
Rolland,A.,E.Jouvin-Marche,C.Viret,M.Faure,H.Perron,andP.N.Marche.2006.The envelope protein of a human endogenous retrovirus-Wfamily activates innate immunity through CD14/TLR4 and promotes Thl-likeresponses',J Immunol,176:7636-44.
Rosen,D.R.1993.'Mutations in Cu/Zn superoxide dismutase gene areassociated with familial amyotrophic lateral sclerosis',Nature,364:362.
Steele,A.J.,A.Al-Chalabi,K.Ferrante,M.E.Cudkowicz,R.H.Brown,Jr.,andJ.A.Garson.2005.'Detection of serum reverse transcriptase activity inpatients with ALS and unaffected blood relatives'.Neurology,64:454-8.
Taylor,J.P.,R.H.Brown,Jr.,and D.W.Cleveland.2016.'Decoding ALS:fromgenes to mechanism',Nature,539:197-206.
Turner,G.,M.Barbulescu,M.Su,M.I.Jensen-Seaman,K.K.Kidd,andJ.Lenz.2001.'Insertional polymorphisms of full-length endogenous retrovirusesin humans',Curr Biol,11:1531-5.
von Giesen,H.J.,R.Kaiser,H.Koller,K.Wetzel,and G.Arendt.2002.'Reversible ALS-like disorder in HIV infection.An ALS-like syndrome with newHIV infection and complete response to antiretroviral therapy*,Neurology,59:474;author reply 74-5.
Vucic,S.,and M.C.Kieman.2009.Tathophysiology of neurodegeneration infamilial amyotrophic lateral sclerosis',CurrMol Med,9:255-72.
Claims (8)
1.识别K型人内源性逆转录病毒家族(HERV-K)包膜蛋白的抗体,其中所述抗体结合如SEQ ID NO:9所示的表位,其包含:
-包含轻链可变区(VL)的轻链,所述轻链可变区包含三个互补决定区(CDR-L1、CDR-L2和CDR-L3),和
-包含重链可变区(VH)的重链,所述重链可变区包含三个互补决定区(CDR-H1、CDR-H2和CDR-H3),
其中CDR-L1由如SEQ ID NO:1所示的氨基酸序列组成,CDR-L2由如SEQ ID NO:2所示的氨基酸序列组成,CDR-L3由如SEQ ID NO:3所示的氨基酸序列组成,CDR-H1由如SEQ ID NO:4所示的氨基酸序列组成,CDR-H2由如SEQ ID NO:5所示的氨基酸序列组成,和CDR-H3由如SEQ ID NO:6所示的氨基酸序列组成。
2.根据权利要求1所述的抗体,其中:
-轻链可变区(VL)如SEQ ID NO:7所示;和
-重链可变区(VH)如SEQ ID NO:8所示。
3.根据权利要求1或2所述的抗体,其中所述抗体是单克隆抗体。
4.根据权利要求3所述的抗体,其中所述单克隆抗体是鼠单克隆抗体、嵌合单克隆抗体或人源化单克隆抗体。
5.根据权利要求1-4任一项所述的抗体在制备用于治疗与K型人内源性逆转录病毒(HERV-K)相关的肌萎缩侧索硬化(ALS)的药物中的用途。
6.根据权利要求5所述的用途,其中所述ALS是散发性ALS。
7.药物组合物,其包含根据权利要求1-4任一项所述的抗体和药学上可接受的赋形剂。
8.根据权利要求1-4任一项所述的抗HERV-K包膜抗体在制备用于诊断患者的由K型人内源性逆转录病毒(HERV-K)引起的ALS的药物中的用途。
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994011514A1 (en) * | 1992-11-10 | 1994-05-26 | Asta Medica Aktiengesellschaft | Human endogenous retrovirus-k-envelope-gene encoded glycoprotein, and a monoclonal antibody for diagnostic and therapeutic purposes |
CN1926153A (zh) * | 2004-01-23 | 2007-03-07 | 拜奥默里克斯公司 | 治疗msrv/herv-w相关病理学的组合物 |
WO2013059426A1 (en) * | 2011-10-21 | 2013-04-25 | The Regents Of The University Of California | Human endogenous retrovirus peptides, antibodies to the peptides, and methods of use thereof |
CN103755805A (zh) * | 2014-02-11 | 2014-04-30 | 中国疾病预防控制中心病毒病预防控制所 | HIV-1Env特异性的全人单克隆抗体 |
CN104684927A (zh) * | 2012-10-02 | 2015-06-03 | 吉纳罗公司 | 用于治疗与herv-w包膜蛋白表达相关的疾病中的髓鞘再生阻断的化合物 |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20060160087A1 (en) | 2003-01-31 | 2006-07-20 | Mcgrath Michael | Monitoring and treatment of amyotrophic lateral sclerosis |
US20090297530A1 (en) * | 2006-05-22 | 2009-12-03 | Feng Wang-Johanning | Herv-k antigens, antibodies, and methods |
FR2912314B1 (fr) * | 2007-02-09 | 2012-08-03 | Geneuro Sa | Composition pharmaceutique comprenant des anticorps diriges contre l'enveloppe de herv-w. |
WO2014004385A2 (en) | 2012-06-25 | 2014-01-03 | Board Of Regents, The University Of Texas System | Anti-cancer vaccines |
EP3355924A1 (en) * | 2015-09-29 | 2018-08-08 | The U.S.A. as represented by the Secretary, Department of Health and Human Services | Methods of treating and preventing amyotrophic lateral sclerosis |
WO2018044970A1 (en) | 2016-08-31 | 2018-03-08 | University Of Rochester | Human monoclonal antibodies to human endogenous retrovirus k envelope (herv-k) and uses thereof |
EP3351265A1 (en) * | 2017-01-20 | 2018-07-25 | Geneuro SA | Anti-herv-k envelope antibody and uses thereof |
-
2017
- 2017-01-20 EP EP17305062.6A patent/EP3351265A1/en not_active Withdrawn
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2018
- 2018-01-19 AR ARP180100129A patent/AR110949A1/es unknown
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- 2018-01-22 TW TW107102219A patent/TWI793098B/zh active
-
2019
- 2019-07-09 IL IL267955A patent/IL267955A/en unknown
-
2020
- 2020-06-17 US US16/903,947 patent/US10981977B2/en active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994011514A1 (en) * | 1992-11-10 | 1994-05-26 | Asta Medica Aktiengesellschaft | Human endogenous retrovirus-k-envelope-gene encoded glycoprotein, and a monoclonal antibody for diagnostic and therapeutic purposes |
CN1926153A (zh) * | 2004-01-23 | 2007-03-07 | 拜奥默里克斯公司 | 治疗msrv/herv-w相关病理学的组合物 |
WO2013059426A1 (en) * | 2011-10-21 | 2013-04-25 | The Regents Of The University Of California | Human endogenous retrovirus peptides, antibodies to the peptides, and methods of use thereof |
CN104684927A (zh) * | 2012-10-02 | 2015-06-03 | 吉纳罗公司 | 用于治疗与herv-w包膜蛋白表达相关的疾病中的髓鞘再生阻断的化合物 |
CN103755805A (zh) * | 2014-02-11 | 2014-04-30 | 中国疾病预防控制中心病毒病预防控制所 | HIV-1Env特异性的全人单克隆抗体 |
Non-Patent Citations (4)
Title |
---|
Human endogenous retrovirus-K contributes to motor neuron disease;Wenxue Li等;《Sci Transl Med》;第7卷(第307期);全文 * |
The role of human endogenous retroviruses in brain development and function;Kristien Mortelmans等;《APMIS》;第124卷(第1-2期);第3、7页 * |
肌萎缩侧索硬化的药物治疗进展;冯新红;崔丽英;;中国神经免疫学和神经病学杂志;20100515(第03期);全文 * |
高效抗逆转录病毒疗法引起神经炎症的研究进展;舒建洪;杨意;;浙江理工大学学报(自然科学版);20161110(第06期);全文 * |
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AU2018210388B2 (en) | 2024-05-23 |
IL267955A (en) | 2019-09-26 |
TWI793098B (zh) | 2023-02-21 |
EP3351265A1 (en) | 2018-07-25 |
BR112019014773A8 (pt) | 2020-06-09 |
US20200308258A1 (en) | 2020-10-01 |
UA126677C2 (uk) | 2023-01-11 |
MX2019008648A (es) | 2019-12-02 |
AU2018210388A1 (en) | 2019-08-01 |
AR110949A1 (es) | 2019-05-22 |
KR20190119593A (ko) | 2019-10-22 |
EA201991736A1 (ru) | 2019-12-30 |
KR102599010B1 (ko) | 2023-11-06 |
WO2018136774A1 (en) | 2018-07-26 |
BR112019014773A2 (pt) | 2020-05-19 |
TW201831518A (zh) | 2018-09-01 |
JP2020505383A (ja) | 2020-02-20 |
CA3050286A1 (en) | 2018-07-26 |
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