CN102264393B - 作为治疗和预防hcv感染药物的抗hcv单克隆抗体 - Google Patents
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Abstract
本发明涉及作为治疗和预防HCV感染药物的单克隆抗体e20或其功能性片段。e20抗体能够结合所有已知的HCV基因型,并展示了很强的对所述病毒特别是基因型Ia、Ib、2a和4的中和活性。本发明还记载了用于治疗或预防HCV感染的药物组合物,其包含单克隆抗体e20或其功能性片段,以及药学上可接受的辅料、载体或稀释剂。
Description
本发明涉及作为治疗和预防HCV感染药物的针对丙型肝炎病毒(HCV)E2糖蛋白的单克隆抗体。
HCV是一种具有外周衣壳(pericapsid)和单链RNA的属于黄病毒家族的病毒。基于在不同HCV分离株中所观察到的遗传差异,这类病毒被分为6种不同的基因型,每种均以数字标记。每种基因型依次包含若干亚型,每种亚型均以字母标记。不同HCV基因型的流行和分布在世界各地不相同。在欧洲,主要的基因型是1b,而在北美流行的是基因型1a。从临床来看,确定基因型很重要,因为这种特征有助于确定对基于α干扰素和利巴韦林组合治疗的潜在反应,这种组合治疗是目前最常用的治疗方式。事实上,基因型1和4对基于干扰素治疗的反应性弱于基因型2、3、5和6。
迄今为止,没有被证实对丙型肝炎病毒真正有效的疫苗和免疫疗法。尽管被赋予对不同病毒基因型的交叉反应性,HCV抗原结构的高变性至今仍阻碍可中和病毒的抗体的开发。因而需要具有这些特征的并由此在治疗和预防HCV感染中真正有效的抗HCV抗体。
现有技术中记载了针对HCV的抗体。例如,Burioni等,Hepatology第28卷第3期,1998年,记载了编码五种特异于HCV E2糖蛋白(HCV E2)的重组人抗体片段(Fab)的序列的克隆和鉴定,所述的抗体片段能够结合不同病毒基因型的糖蛋白(交叉反应性)。在这篇文章所记载的Fab中,有一种抗体片段名为e20。Burioni等,1998,cit,记载了e20在中和HCV E2结合中具有很高的最小活性(NOB活性)。然而,尽管具有高NOB活性,在国际专利申请WO03/064473中,e20抗体片段被描述为即使以高浓度(80μg/ml)也不能中和病毒感染(参见特别是WO 03/064473第16页第8-10行)。
本申请的发明人惊奇地发现,与现有技术、特别是WO 03/064473所声称的相反,e20片段能够在体外有效地中和不同HCV基因型的感染。这使得e20特别适合HCV中和以及清除HCV感染的细胞,并用作HCV感染免疫疗法和免疫保护的药剂。
这种结果的获得需要长期而复杂的实验工作,其在后续的实验部分详细说明。
在极端情况下,本申请发明人完成的实验可以证明e20抗体片段展现了以下预料不到的特征:
-其在属于基因型1b的HCV株导致的自然感染过程中产生,但能够结合所有已知HCV基因型的糖蛋白(特别是基因型1a、1b、2a、2b、3、4、5和6)并且因此是广泛地交叉反应性的;
-如通过基于HCV伪颗粒(HCVpp)的中和分析所测得的,其对HCV基因型1a、1b、2a和4具有特别高的中和能力;
-e20结合HCV E2糖蛋白所必需的某些氨基酸残基在HCV感染中也是必需的,这提示能够逃避e20结合的突变体同时拥有降低的复制能力。
上述特征显然有利于最终应用e20抗体片段作为HCV感染的免疫疗法和免疫保护的药剂。
因此,本发明的第一个目标是能够结合来自多种不同HCV基因型的HCVE2糖蛋白的单克隆抗体或其片段,其作为治疗或预防HCV感染的药剂,其特征在于所述的单克隆抗体或其片段包含至少一个包含氨基酸序列SEQ ID NO:1或与SEQ ID NO:1具有至少90%同一性的序列的重链可变区,以及至少一个包含氨基酸序列SEQ ID NO:2或与SEQ ID NO:2具有至少90%同一性的序列的轻链可变区。
进一步优选的序列同一性百分比是至少91%、92%、93%、94%、95%、96%、97%、98%或99%,其中所述表述“至少”是指对于所列的每个百分比。
在本发明的一个实施方式中,所述的重链可变区由核苷酸序列SEQ ID NO:3编码,并且所述轻链可变区由SEQ ID NO:4编码。
术语“抗体”意指任意种类的全长免疫球蛋白及其包含轻链可变区与重链可变区的任意片段,例如Fab、F(ab’)2、CDR(互补决定区),或同时包含重链与轻链可变区或CDRs的单链抗体,或包含来源于免疫球蛋白重链可变区与轻链可变区的CDR片段的一个或更多个拷贝的骨架。这包括了命名为ScFvs、diabodies、VHHs或分离的轻链或重链的功能性抗体片段。表述“抗体”进一步包含可通过在轻链可变区重排方法中应用e20抗体片段可变区序列,以构建具有改善亲和力、稳定性和/或重组生产特性的VH/VL组合所产生的抗体。表述“抗体”还包括任意类型的融合特异性全长免疫球蛋白或免疫球蛋白片段的全长免疫球蛋白或免疫球蛋白片段,其可靶向所述e20抗体片段或免疫球蛋白至特定组织、细胞或可溶性蛋白结构。本发明的单克隆抗体优选是人的。
本发明的抗体可以是游离形式或缀合形式。缀合形式是与能够调节体内持续性、促进或限制体内分布、降低对蛋白水解剂的敏感性、降低抗原性、提高细胞毒能力和/或易于在体液和组织中检测的分子缀合的上述的抗体。适于缀合的分子的非限制例子包括人血清白蛋白,麦芽糖结合蛋白,谷胱甘肽-S-转移酶,噬菌体外壳p3或p8蛋白,肽,糖,PEG或PEG样分子,动物、植物或微生物源毒素,细胞因子,酶,化学发光化合物,生物发光化合物,金属原子,放射性同位素,荧光化合物,标签基团或磷酸化、糖基化、泛素化、苏素化或内肽酶解的底物。为便于缀合,抗体的C末端或N末端可以是修饰的,例如,通过插入额外的氨基酸残基,如一个或更多个可形成二硫桥的半胱氨酸残基。本发明应用的抗体还可以连接人红血球或其它细胞载体,连接特定制剂,或连接缓释系统例如但不限于脂质体、树形聚合物、微粒体、纳米颗粒、微胶囊、病毒载体等等。
本发明的另一目标是治疗或预防HCV感染的药物组合物,包含药学有效量的上述定义的单克隆抗体或其片段。
本发明的组合物可被施用给被HCV感染的或有感染风险的受试者。可以应用任何合适的施用途径,包括胃肠外的、口服、眼、局部(topical)、局部区域(loco-regional)、灌肠或气溶胶给药。胃肠外给药包括肌内注射、静脉注射、淋巴管内注射、皮下或皮内注射及输注。
本发明的组合物可被制备成适合所选择施用途径的任意药学形式,例如可注射溶液或悬浮液、输液、片剂、胶囊、霜剂、软膏、洗剂或栓剂形式。
本发明的组合物包含作为有效成分的上述抗体或其片段,以及本领域技术人员知晓的合适的药物辅料、载体或稀释剂。
本发明的进一步的目标是能够特异性结合上述抗体或其片段独特型的抗独特型抗体。本发明的抗独特型抗体可通过本领域技术人员本身已知的获得抗独特型抗体的传统方法获得。
本发明参照附图在后续仅通过例证的方式提供的实验部分进一步详述,其中:
图1显示了e20与不同基因型来源的HCV E2糖蛋白的结合。数据显示为阳性荧光细胞百分比。
图2显示了通过应用展示来源于基因型1a:UKN1A20.8(a)、E1E2基因型1b:UKN1B5.23(b)、E1E2基因型2a:UKN2A1.2(c)、E1E2基因型2b:UKN2B1.1(d)、E1E2基因型4:UKN4.21.16(e)的E1-E2糖蛋白的病毒伪颗粒的Fab e20中和活性。(f)通过应用展示来源于不同基因型(UKN1A20.8,UKN1B5.23,UKN2A1.2,UKN2B1.1;UKN3A13.6,UKN4.21.16,UKN5.15.11,UKN6.5.8)的E1-E2的病毒伪颗粒,15μg/ml Fab e20的中和活性。
图3显示了通过应用HCVcc系统(基因型2a)的e20及其它抗HCV抗体(e137,AP33)的中和活性。在e20和阴性对照Fab(c33-3)存在时的JFH-1传染性显示为标准化至甘油醛-3-磷酸脱氢酶RNA的病毒RNA的量,其通过定量反转录PCR确定。
实验部分
克隆策略
丝状噬菌体表面展示的随机组合文库的制备代表了一种用于选择高亲和力人单克隆抗体的高度有效工具。实际上,基于噬菌体展示的选择步骤非常灵活,并且可被优化用以选择交叉反应的抗体。特别地,e20作为来源于一位58岁女性的B淋巴细胞库的IgG1 Fab片段克隆,该女性持续一直感染有基因型1b的HCV株。为选择交叉反应克隆,源自患者的文库经历对来源于不同基因型病毒分离株的重组HCV E2糖蛋白的淘选,所述不同基因型是1a。简要地,用这种方法可以获得在自然感染过程中产生的抗体,但其仍然能够结合从未被所选择研究患者的免疫系统遇到过的不同糖蛋白。
重链和轻链序列研究
e20重链及轻链基因的测序及其突变模式(表1)的研究表明,这种抗体来源于体细胞突变过程,这是一种为了提高抗体本身对抗原的亲和力,在抗体克隆中通过持续接触特异性抗原刺激的过程。
关于重链,e20具有与胚系基因低于85%的核苷酸序列同源性。突变模式显示了体细胞突变克隆的典型分布,在互补决定区(CDRs)具有特异性极化。对e20连接区域(产生CDR3的连接区域)的检查显示其由属于VH 1-69亚家族(一种在人抗HCV体液应答中高度表现的基因)的V基因、属于D2-21亚家族的D基因以及属于JH4亚家族的JH基因组成。
以类似的方式,e20轻链(独特型κ)具有与体细胞突变过程一致的突变百分比,正如CDRs中的极化所示。对连接区域的检查揭示了e20轻链CDR3来自κV3-15亚家族的κV基因以及κJ5亚家族的κJ基因的连接。
这些序列数据可以得出这样一个结论:e20不是一种人工抗体,而且正好相反,其实际存在于所选择研究的患者的抗体库中。
表1
b)
上面的表1显示了胚系以及e20重链a)与轻链b)中的基因V的突变模式。氨基酸和核苷酸突变百分比,经考虑轻链及重链的FR1、FR2和FR3,重链的CDR1和CDR2,以及轻链的CDR1、CDR2和CDR3,依据Kabat和Wu的比对方法计算。取代突变(R)与沉默突变(S)的比例也予以报道。
e20结合不同HCV基因型来源E2的评估
测试了Fab形式的e20片段识别1b(即已感染e20获取来源患者的株系的基因型)和1a(即用于克隆所述Fab的基因型)以外HCV基因型来源的E2糖蛋白的能力。从不同HCV基因型中获取可溶性E2形式所经历的困难,要求应用替代性基于FACS的方法。
简要地,293T人肾上皮细胞(HEK)在Dulbecco’s modified Eagle’s培养基(DMEM)中生长,加入10%胎牛血清、5%非必需氨基酸、200mM的谷氨酰胺、链霉素(100μg/ml)和盘尼西林(100U/ml)。一旦达到80%的融合,将2×106HEK细胞接种在10cm培养板中,24小时后应用磷酸钙转染流程以3μg的phCMV-7转染,phCMV-7是一种编码不同HCV基因型来源E1E2糖蛋白的表达载体。转染16小时后更换培养基,然后所述细胞在37℃孵育24小时。去掉培养基并以PBS洗涤细胞单层两次。加入5ml的解离缓冲液并将细胞在37℃孵育5分钟。以PBS洗涤细胞两次并以1000rpm离心5分钟;向每个培养板所获得的球团加入1.2ml固定试剂。细胞在室温下孵育15分钟。样品以5ml加入了2%胎牛血清的PBS(FPBS)洗涤,然后以1000rpm离心5分钟。
每个球团中加入100μl含有50μl Fab e20的通透剂,Fab e20终浓度为10μg/ml。非转染细胞也按照相似的流程,作为对照。室温孵育40分钟后,样品以5ml的FPBS洗涤,球团中加入50μl的FITC缀合的二抗。细胞在室温孵育20分钟并以5ml FPBS洗涤两次。最终,移除上清,在300μl FPBS中重悬所述球团,并以FACS分析所述细胞。结合活性以比没有Fab e20的细胞具有更高荧光水平的细胞百分数中获得的阳性荧光细胞百分数来表示。特异于非结构性HCV抗原(NS3)的重组人Fab(c33-3)在每个实验中作为阴性对照。
这种方法表明Fab e20能够识别所有表达的HCV E2基因型(1a、1b、2a、2b、3、4、5、6),其相对于用对照Fab获得的的具有更高的荧光细胞百分比。所述结果见图1。
e20结合来自CD81结合区内突变的HCV 1a的E2糖蛋白的评估
Fab e20同样针对保守区内突变的H77(基因型1a)来源的E1E2组进行测试,所述的保守区被描述为对CD81结合以及HCV伪颗粒(HCVpp)的传染性至关重要。该区域的每个保守位点突变为丙氨酸。所有这些替代导致下面所记载的HCVpp分析中传染性的丧失。Fab e20 HCV E2糖蛋白的结合被这些关键突变中的某一些所取消(表2)。
表2中记载的数据提示,e20结合的E2区域对于病毒感染是必需的。这些数据也确证了e20结合对AP33结合至关重要的区域,AP33是具有最大交叉反应的中和性抗HCV抗体,但考虑到以异源抗体的免疫疗法并非切实可行以及人源相似抗体的存在非常罕见(Tarr等,J Gen Virol.88:2991.2007),其不太适合作为设计疫苗药物的模板以及用于免疫疗法。更有趣的是,该分析清楚地表明所有不被e20识别的突变体不允许伪病毒模型中的靶细胞感染。
表2
在上文的表2显示了e20结合H77突变体(基因型1a)来源的E1E2。结合活性以相对于应用野生型H77蛋白检测结果的百分比表示。
来源于不同基因型的HCV伪颗粒上e20中和活性的评估
然后在基于HCV伪颗粒(HCVpp)的中和分析中检验Fab e20的中和活性。
简要地,293T人肾上皮细胞(HEK)以及Huh-7人肝细胞瘤细胞在加入10%胎牛血清、5%非必需氨基酸、200mM的谷氨酰胺、链霉素(100μg/ml)和盘尼西林(100U/ml)的DMEM中生长。一旦达到80%的融合,2×106HEK细胞接种在10cm培养板中,24小时后应用8μg小鼠白血病病毒载体(MLV)Gag-Pol、8μg编码萤光素酶的MLV转移载体以及3μg编码不同HCV基因型来源E1E2糖蛋白的全长phCMV-7a表达载体共转染。一天后,以5ml含有10mM HEPES的新鲜培养基替换转染培养基。细胞在37℃孵育24小时。靶细胞(Huh-7)以每孔2.5×104细胞接种于24孔板并在37℃孵育过夜。在替换培养基24小时后收集HCV伪颗粒(HCVpp),2000rpm离心10分钟并通过0.45μm孔径的膜过滤,并用于中和分析。
特别地,将60μl含有HCVpp的培养基与90μl不同浓度的Fab e20混合并在37℃孵育1小时。将这种混合液加入到Huh-7靶细胞,并且所述细胞在37℃孵育3小时。最终,移除接种物,每孔中加入1ml的新鲜培养基,细胞在37℃孵育4天。所述细胞以PBS洗涤两次然后以100μl裂解缓冲液(Promega)参照厂商说明裂解。将细胞裂解物转移至96孔板,并在每孔中加入100μl底物/缓冲液(Promega)。通过测量给定相对光单位(RLUs)下的发光活性(Chameleon platereader,Hidex)分析细胞的感染。中和活性通过所获得的发光与没有竞争性抗体的HCVpp孔中(阴性)所检测的相比较以感染百分比来确定。特异于非结构性HCV抗原(NS3)的重组人Fab(c33-3)在每个实验中作为阴性对照。
这种方法表明e20能够强有力地中和HCV基因型1a和4。E20显示了在7.5μg/ml浓度时对基因型1a 50%的中和活性,以及在15μg/ml浓度时对基因型475%的中和活性(图2a、2e和2f)。然而,这种抗体也能够强有力地中和HCV基因型1b和2a。更详细地,在15μg/ml时,e20对基因型1b和2a分别显示了40%的中和与75%的感染(图2b、2c和2f)。最后,e20能够中和较低度的具有基因型2b E1E2糖蛋白的HCVpps,在15μg/ml浓度显示了20%的抑制(图2d和2f)。
细胞培养物中生长的HCV株(基因型2a,JFH1株)的e20中和活性
HCV e20的中和活性也通过应用HCVcc模型系统(HCV细胞培养物)测试,通过应用含有来源于HCV基因型2a(JFH1)和高生产性传染性病毒的整合入染色体的cDNA的稳定人源肝细胞系(图3)。这种系统可以通过应用传染性丙型肝炎病毒株评估中和活性。在这一系列实验中,不同浓度的Fab e20与含有HCVcc试验中生成的病毒的培养基一起孵育。3小时后,将混合物加入靶细胞(Huh7.5)。通过测量HCV阳性链RNA的水平评估传染性。Fab e20显示出很强的中和活性,因为在非常低的1μg/ml浓度,其能够完全消除HCV基因型2a的传染性。Fab e20中和活性与小鼠AP33 IgG单克隆抗体相当,该抗体是迄今所记载的最强的交叉中和抗体之一。
Claims (11)
1.作为治疗或预防HCV感染药物的能够结合来自多种不同HCV基因型的HCV E2糖蛋白的单克隆抗体或其片段,其特征在于,所述单克隆抗体或其片段含有至少一个包含氨基酸序列SEQ ID NO:1的重链可变区,以及至少一个包含氨基酸序列SEQ ID NO:2的轻链可变区。
2.权利要求1所述的单克隆抗体或其片段,其是全长免疫球蛋白或包含至少一个重链可变区和一个轻链可变区的免疫球蛋白片段。
3.权利要求2所述的单克隆抗体或其片段,其中所述片段选自Fab、F(ab’)2、CDR(互补决定区),或同时包含重链与轻链可变区或CDRs的单链抗体,或包含来源于免疫球蛋白重链可变区与轻链可变区的CDR片段的一个或更多个拷贝的骨架。
4.权利要求1-3任一项的单克隆抗体或其片段,其是游离形式的。
5.权利要求1-4任一项的单克隆抗体或其片段,其与能够调节体内持续性、促进或限制体内分布、降低对蛋白水解剂的敏感性、降低抗原性、提高细胞毒能力和/或易于在体液和组织中检测的分子缀合。
6.权利要求1-3任一项的单克隆抗体或其片段,其与能够靶向所述抗体至特定组织、细胞或可溶性蛋白结构的特异性全长免疫球蛋白或免疫球蛋白片段融合。
7.用于治疗或预防HCV感染的药物组合物,其包含药物有效量的权利要求1-6任一项的单克隆抗体或其片段。
8.权利要求7的药物组合物,其是适于胃肠外的、口服、眼、局部、局部区域、灌肠或气溶胶施用的药物剂型。
9.权利要求6或7的药物组合物,其是可注射溶液或悬浮液、输液、片剂、胶囊、霜剂、软膏、洗剂或栓剂形式。
10.权利要求1-6任一项的单克隆抗体或其片段用于制备治疗或预防HCV感染药物的用途。
11.能够特异性结合权利要求1-6任一项的抗体或其片段的独特型的抗独特型抗体。
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WO2010073204A1 (en) | 2010-07-01 |
BRPI0923569B1 (pt) | 2021-09-21 |
MX2011006722A (es) | 2011-07-20 |
US8623363B2 (en) | 2014-01-07 |
JP2012513457A (ja) | 2012-06-14 |
BRPI0923569A2 (pt) | 2016-01-26 |
KR20110096591A (ko) | 2011-08-30 |
RU2011130522A (ru) | 2013-01-27 |
RU2596409C2 (ru) | 2016-09-10 |
CN102264393A (zh) | 2011-11-30 |
ITTO20080964A1 (it) | 2010-06-23 |
KR101450955B1 (ko) | 2014-10-15 |
EP2376119A1 (en) | 2011-10-19 |
EP2376119B1 (en) | 2015-04-22 |
US20110256140A1 (en) | 2011-10-20 |
ES2541323T3 (es) | 2015-07-17 |
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