CN103547595B - 人源化ctla‑4抗体 - Google Patents
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- CN103547595B CN103547595B CN201280022428.5A CN201280022428A CN103547595B CN 103547595 B CN103547595 B CN 103547595B CN 201280022428 A CN201280022428 A CN 201280022428A CN 103547595 B CN103547595 B CN 103547595B
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Abstract
本发明提供了一种抑制CTLA4结合人B7的CTLA4抗体,具体地,其抑制CTLA4结合人B7.1和/或人B7.2。提供了具有特异性可变区序列的特异性抗体以及含这种抗体的组合物用于治疗疾病。
Description
本发明涉及使用抗人CTLA4的新人源化抗体治疗和预防人类疾病,以及使用这些抗体治疗或预防人类疾病的方法。
背景技术
脊椎动物免疫系统需要多层次的分子和细胞相互作用以实现最佳的免疫应答。具体地,T淋巴细胞(T细胞)的激活是许多这种应答的重要组成部分。抗原呈递细胞(APC)可以通过将抗原经由主要组织相容性复合物(MHC)分子携带的肽呈递至TCR(T细胞受体)来激活T细胞。这种激活还需要APC的共刺激。将非特异性的共刺激信号递送至T细胞需要至少两个在APC上发现的同源B7家族成员B7-1(也称为B7、B7.1或CD80)和B7-2(也称为B7.2或CD86),两者都可在与T细胞上CD28抗原结合时递送共刺激信号,导致T细胞激活。CD28是免疫球蛋白(Ig)超家族的同源二聚体糖蛋白成员,其具有单个细胞外可变区,并且其存在于大多数成熟的人T细胞上。
被称为CTLA4(细胞毒性淋巴细胞相关抗原,也称为CD152)的CD28同系物,发现于1987年(Brunet et al.,(1987)Nature328:267-270),其与细胞毒性T细胞尤其相关。与CD28一样,CTLA4是Ig超家族的成员并且包含单个细胞外Ig结构域。然而,CTLA4的作用主要是抑制T细胞激活,这显示在患有大规模淋巴组织增生的CTLA4缺陷小鼠中(Chambers etal.,(1997)Immunity.7:8855-8959)。此外,表明了阻断CTLA4可在体外(Walunas et al.,(1994))Immunity.1:405-413)和体内(Kearney(1995)J.Immunol.155:1032-1036)增强T细胞应答,还可增加抗肿瘤免疫力(Leach(1996)Science.271:1734-1736)。因此,阻断CTLA4可提供其中免疫刺激可能有益的疾病尤其是人类疾病的新疗法,例如治疗癌症和传染性疾病的新疗法。
CTLA4功能的阻断剂的开发专注于使用单克隆抗体,尤其是来源于移入编码人免疫球蛋白的基因(并且宿主小鼠免疫球蛋白基因缺陷)的转基因小鼠的抗体。正在进行临床试验的这些抗体包括IgG1同种型的易普利姆玛(Ipilimumab)(Keler et al.,JImmunol171:6251-6259(2003))和IgG2同种型的Tremelimumab(Ribas et al.,Oncologist12:873-883(2005))。虽然通常报道免疫原性是低的(诱导针对注入的人单克隆抗体的抗体),但是值得注意的是这种人抗体由于体细胞突变和可变区序列中的重排(可能产生T细胞表位)而可在某些患者中诱导免疫原性,导致副作用和疗效缺乏。因此,需要改进的具有可能更低免疫原性的CTLA4单克隆抗体,以更有效地治疗人类疾病。
发明内容
本发明涉及特异性结合人CTLA4的新人源化抗体。本发明还提供了其中结合人CTLA4可抑制人CTLA4结合人B7的人源化抗体。本发明还提供了以至少10-8M的平衡解离常数(Kd)结合人CTLA4的人源化抗体。本发明还提供了特异性结合人CTLA4的人源化抗体,其可阻断人CTLA4结合人B7至少约10%、20%、30%、40%、50%、60%、70%、80%、90%、99%或100%。本发明还提供了特异性结合人CTLA4的人源化抗体,所述人源化抗体具有同种型IgG1、IgG2、IgG3或IgG4的抗体重链或具有突变的IgG恒定区,例如用以抑制结合Fc受体或用以抑制与补体的结合。本发明还提供了其中抗体轻链是κ轻链的人源化抗体。所述人源化抗体可由人IgG重链和人κ轻链核酸编码,所述IgG重链和人κ轻链核酸编码其可变区中如SEQ ID NO:31-SEQ ID NO:50列出的蛋白质序列。在本发明的优选实施方案中,所述人源化抗体包含来自SEQ ID NO:45和SEQ ID NO:49的可变区(另外也称为“VH5:VK4”)。
本发明还提供了特异性结合人CTLA4的人源化抗体,其中已经对所述抗体可变区进行了选择或修饰以排除一个或多个人CD4+T细胞表位。本发明还提供了特异性结合人CTLA4的人抗体,其中主要通过融合来自现有的人抗体可变区序列的序列片段形成所述抗体可变区。
本发明还提供了本发明的人源化抗体,其包含重链CDR1、CDR2和CDR3氨基酸序列(分别为“DYNMD”(SEQ ID No.9)、“NINPNSESTSYNQKFKG”(SEQ ID No.10)和“DGNRYDAWFAY”(SEQ ID No.11))以及轻链CDR1、CDR2和CDR3氨基酸序列(分别为“SASSSVTYMH”(SEQ IDNo.12)、“STSILAS”(SEQ ID No.13)和“QQRTSYPLT”(SEQ ID No.14))。
本发明还提供了本发明的人源化抗体,其包含重链CDR1、CDR2和CDR3氨基酸序列(分别为“SYWIN”(SEQ ID No.15)、“RIAPGSGTTYYNEVFKG”(SEQ ID No.16)和“GDYGSY”(SEQID No.17))以及轻链CDR1、CDR2和CDR3氨基酸序列(分别为“SASSSISYMH”(SEQ ID No.18)、“DTSKLAS”(SEQ ID No.19)和“HQRTSYPLT”(SEQ ID No.20))。
本发明的人源化抗体可以由上述CDR序列SEQ ID No.9至SEQ ID No.20和这些CDR序列的小变体(minor variants)组成,其中改变一个或多个氨基酸不会明显地改变与人CTLA4的结合。人源化抗体可通过将所述CDR序列与来自人可变区框架的序列连接在一起来产生,其中这种框架序列来源于单个或多个其他的人抗体可变区框架序列。通常,这种人可变区框架序列会包括造成所述人源化抗体与CTLA4的最佳结合或改善结合的一个或多个突变。在本发明的一个优选实施方案中,所述人源化抗体中的这种人可变区框架序列全部来源于其他的人抗体可变区中的序列,如EP1844074(Antitope Ltd)的方法所述。这些序列包含来自其他人抗体可变区的序列的连接在一起的片段,以及人恒定区。具体地,这种人源化抗体还含有来源于来自其他人抗体可变区的CDR序列、框架序列或部分框架/CDR序列的CDR序列以及人恒定区,从而产生其中可变区序列全部来源于其他人抗体可变区并且含有人恒定区的人源化抗体,从而产生“全人”抗体。
本发明还提供了特异性结合人CTLA4的人源化抗体,其中所述人源化抗体由哺乳动物细胞系,尤其是CHO或NS0细胞产生。本发明还提供了特异性结合人CTLA4的人源化抗体,其为Fab片段或单链Fv(scFv)。本发明还提供了多特异性抗体(将两种或多种不同的抗体分子连接在一起以给出两种或多种不同的特异性),对于抗体3B10,其包括至少一种来自序列SEQ ID NO:31至35(用于重链)和SEQ ID NO:36至40(用于轻链)的人源化抗体;或者对于抗体8H5,其包括至少一种来自序列SEQ ID NO:41至45(用于重链)和SEQ ID NO:46至50(用于轻链)的人源化抗体,所述人源化抗体均特异性结合人CTLA4。在一个优选的实施方案中,本发明提供了多特异性抗体,其具有由SEQ ID NO:45(用于重链)和SEQ ID NO:49(用于轻链)组成的可变区。可将包括在每种多特异性抗体内的不同抗体彼此共价或非共价连接。
本发明提供了一种药物组合物,其包含特异性结合人CTLA4的人源化抗体和可药用载体。所述药物组合物还可包含可有效诱导针对靶抗原的免疫应答的试剂,或者一种或多种化学治疗剂。
本发明提供了一种用于诱导、加强(augmenting)或延长患者中针对抗原的免疫应答的方法,其包括向所述患者给予有效剂量的特异性结合人CTLA4的人源化抗体,其中所述抗体阻断人CTLA4结合人B7。所述抗原可包括肿瘤抗原、病原体相关抗原、中枢神经系统(CNS)疾病相关抗原、血液系统疾病(包括高血压和动脉粥样硬化)相关抗原、炎性疾病(包括类风湿关节炎和自身免疫性疾病)相关抗原或变态反应相关抗原。肿瘤抗原可以是肿瘤的细胞表面上的一个或多个抗原、与所述肿瘤相互作用的一个或多个分子、来源于肿瘤抗原的肽的一个或多个MHC复合物,或与肿瘤不直接相关但针对其的免疫应答对所述肿瘤有副作用的抗原,例如肿瘤血管系统相关抗原。病原体可以是病毒、细菌、真菌或寄生物。CNS抗原包括与阿尔茨海默病中的斑块沉积相关的β淀粉状蛋白。血液系统抗原包括整联蛋白和粘附素,以及与动脉粥样硬化中的斑块沉积相关的抗原。炎性疾病抗原包括细胞因子和细胞因子受体。变态反应抗原包括与食物、植物、化学和环境变应原相关的抗原。本发明的方法还可包括向所述患者给予所述抗原或其片段或类似物,其中所述抗原与所述人源化抗体结合可诱导、加强或延长免疫应答。
本发明还提供了一种抑制患者中的免疫应答的方法,其包括向所述患者给予有效剂量的包含至少两个彼此相连接的抗人CTLA4的人源化抗体的多价制剂,导致例如调节性T细胞的诱导或CTLA4的下调。本发明还提供了一种抑制患者中的免疫应答的方法,其包括向所述患者给予有效剂量的多克隆制剂,所述多克隆制剂包含至少两个抗人CTLA4的人源化抗体。
本发明还提供了特异性结合人CTLA4的人源化单克隆抗体以及含有这类抗体的一个或结合物的组合物。本发明的一些人源化抗体特征在于以高亲和力结合人CTLA4,和/或在于阻断人CTLA4与其配体人B7-1和B7-2分子的相互作用。因此,本发明的这种人源化抗体可在体内和体外用作诊断或治疗试剂。
本发明的人源化抗体可包括多种抗体同种型或其混合物,例如IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgAsec、IgD、IgE或这些IgG的突变形式例如可减弱或消除与Fc受体的结合的突变。通常,它们包括IgG4(例如IgG4k)和IgG1(例如IgG1k)。所述人源化抗体可以是全长(例如IgG4或IgG1抗体)或者可以仅仅包括抗原结合部分(例如,Fab、F(ab')2、Fv或scFv片段)。
本发明的一些人源化CTLA4抗体可以通过一个或多个以下特性来表征:a)对人CTLA4的特异性(特异性结合人CTLA4);b)对人CTLA4的结合亲和力具有至少10-8M的平衡解离常数(Kd)。
在另一方面,本发明提供了编码本发明的人源化抗体或抗原结合部分的核酸分子。因此,本发明还涵盖了包括本发明的抗体编码核酸的重组表达载体和用这种载体转染的宿主细胞,以及通过培养这些宿主细胞制备本发明的抗体的方法。
可将本发明的人CTLA4的人源化单克隆抗体或其抗原结合部分(例如Fab)衍生化或连接至另一功能性分子,例如另个一肽或蛋白质(例如Fab'片段)。例如,可将本发明的人源化抗体的抗体或抗原结合部分功能化地连接(例如,通过化学偶联、基因融合、非共价连接或以其他方式)至一个或多个其他分子实体。例如,可将所述人源化CTLA4抗体或其抗原结合片段缀合至治疗部分,例如细胞毒类药、有酶活性毒素(enzymatically activetoxin)或其片段、放射性同位素、治疗性核酸或小分子抗癌药。还可将本发明的抗体缀合至细胞毒性药物,例如用细胞毒性试剂(例如131I)进行放射性标记,或者可以将其偶联至核糖体失活蛋白例如假单胞菌(Pseudomonas)外毒素(PE38片段、植物或细菌毒素例如蓖麻毒蛋白、蓖麻毒蛋白的α链、皂草素、美洲商陆抗病毒蛋白、白喉毒素、或假单胞菌外毒素A(Kreitman and Pastan(1998)Adv.Drug Delivery Rev.31:53.)。
在另一方面,本发明提供了组合物例如药物组合物和诊断组合物,其包含可药用载体和特异性结合人CTLA4的至少一种本发明的人源化单克隆抗体或其抗原结合部分。一些组合物还可包含本发明的人源化抗体或抗原结合部分的结合物。这类组合物还可包含与作为单独分子的一种或多种其他生物活性分子的结合物,例如至少一种本发明的人源化单克隆抗体与另一生物活性分子的结合物,或者可在同一分子内结合与一个或多个其他生物活性分子的多个结合物,例如作为双特异性或多特异性分子,为两个或多个本发明的人源化抗体的结合物或为与一个或多个其他生物活性分子的结合物。
对于体内方法,可向人受试者给予所述抗体或其抗原结合部分(或本发明的双特异性或多特异性分子),所述人受试者患有T细胞相关的疾病或患有可通过诱导、加强、延长或抑制免疫应答而改善或预防的疾病。
还可与其他已知疗法(例如抗癌疗法)结合给予本发明的人源化单克隆抗体组合物。因此,本发明提供了一种用于治疗受试者中的癌症的方法,其包括向所述受试者给予治疗有效量的人源化抗体的药物组合物以及药物载体。这种方法中的一些包括与疫苗结合。这种疫苗中的一些包括肿瘤细胞疫苗、GM-CSF-修饰的肿瘤细胞疫苗、核酸(例如DNA)疫苗和肿瘤相关抗原或负载抗原的树突细胞疫苗。
可将抗人CTLA4的人源化抗体用于需要刺激免疫应答或抑制免疫应答的治疗方法中。刺激使用可阻断人CTLA4结合人B7的抗体来实现,可通过刺激、加强和延长免疫应答治疗的疾病包括前列腺、肾、结肠、肺或乳腺的癌症;病原体感染;CNS相关疾病,例如包括阿尔兹海默病的淀粉样蛋白形成疾病;和有炎症或变应性成分(component)的疾病。免疫抑制还可使用抗人CTLA4的人源化抗体,例如通过诱导调节性T细胞(Coquerelle et al.,Gut2009;58:1363-1373)来实现。可治疗的疾病包括移植物抗宿主病、宿主抗移植物病、变态反应、自身免疫性疾病和其他炎性疾病。
在另一方面,本发明提供了一种使用本发明的抗体在体外或体内检测样品中人CTLA4抗原的存在的方法,例如用于诊断人CTLA4相关疾病。在一些方法中,这通过使要测试的样品和对照样品一起,与本发明的人源化单克隆抗体或其抗原结合部分(或双特异性或多特异性分子)在允许形成所述抗体和人CTLA4的复合物的条件下接触来实现。然后,检测测试样品中的复合物形成(例如通过ELISA),所述测试样品和对照样品之间复合物形成的任何统计学显著的增加都指示了所述测试样品中人CTLA4抗原的存在。
本领域的技术人员会理解,除了本文描述的那些之外,本发明的人源化抗体会具有另外的用途或组合物,在其中所述人源化抗体结合人CTLA4抗原的所有情况中,这种用途和组合物被认为在本发明的范围之内。本领域的技术人员会理解,可对本发明的人源化抗体的可变区序列(SEQ ID NO:31至SEQ ID NO:50)或本发明的人源化抗体的CDR(SEQ IDNO:9至SEQ ID NO:20)作出改变,所述改变不会显著改变本发明人源化抗体的特性,因此应认为这种变体在本发明的范围之内。此外,在所述人源化抗体的可变区或CDR序列内的这种改变应被认为是在本发明的范围之内,只要这种变体的可变区序列与本发明的人源化序列具有显著同源性。例如,可确定变体核酸在本发明的范围内,只要其包括含有SEQ ID NO:21至SEQ ID NO:30的序列或含有通过其在严格条件下与本发明核酸杂交的能力确定的与SEQID NO:21至SEQ ID NO:30基本相同的序列。在一个实施方案中,可通过核酸序列在严格条件下与本发明范围内核酸杂交(例如SEQ ID NO:21至SEQ ID NO:30)的能力确定其在本发明的范围内(例如与SEQ ID NO:21至SEQ ID NO:30基本相同)。术语“杂交”是指当特定核苷酸序列存在于复杂混合物(例如总细胞或文库DNA或RNA)中时,分子与该序列在严格杂交条件下的结合、形成双链体(duplex)或杂交,其中至少以约10倍背景检测到所述特定核苷酸序列。严格杂交条件被选择为例如比具体序列在确定离子强度pH下的热解链点(thermalmelting point,Tm)低5-10℃。
实施例
以下实施例不应被认为对本发明的范围进行限制。与下文的实施例有关的图和表如下:
图1-鼠抗体结合CTLA4-Fc。
图2-对于结合CTLA4-Fc,鼠抗体相对抗生物素化的B7.1的竞争ELISA。
图3-对于结合CTLA4-Fc,鼠抗体相对抗生物素化的B7.2的竞争ELISA。
图4-嵌合3B10和8H5人IgG1抗体结合CTLA4-Fc。
图5-对于结合CTLA4-Fc,嵌合3B10和8H5人IgG1抗体相对抗生物素化的B7.1的竞争ELISA。
图6-对于结合CTLA4-Fc,嵌合3B10和8H5人IgG1抗体相对抗生物素化的B7.2的竞争ELISA。
图7-在针对嵌合3B10和8H5人IgG1抗体的应答中人PBMC的T细胞增殖。
图8-pANT抗体表达载体图。
图9-3B10可变区(VH和VK)DNA序列。
图10-8H5VH和VK DNA序列。
图11-3B10VH和VK氨基酸序列。
图12-8H5VH和VK氨基酸序列。
图13-人源化3B10VH氨基酸序列。
图14-人源化3B10VK氨基酸序列。
图15-人源化8H5VH氨基酸序列。
图16-人源化8H5VK氨基酸序列。
图17-对于结合CTLA4-Fc,人源化8H5抗体相对抗生物素化的嵌合8H5人IgG1(=“h8H5亲本IgG1”)的竞争ELISA。
图18-在具有供体对的人混合淋巴细胞反应中由首要(lead)人源化VH5/VK4CTLA4和MDX0101引起的IFNχ分泌。
图19-在从第2天开始每周给予抗体剂量的人CTLA4敲入小鼠中MC38肿瘤的生长。
表1-用于扩增鼠cDNA可变区的引物序列。
表2-用于扩增用于克隆至pANT17和pANT13中的鼠可变区的引物序列。
除非另有说明,根据制造商的说明书使用实施例中提到的可商购试剂。在实施例和整个说明书中通过ECACC登录号识别的细胞的来源是英国索尔兹伯里的欧洲细胞培养物收集中心(European Collection of Cell Cultures,ECACC)。除非另有定义,本文使用的所有技术和科学术语与本发明所属领域的普通技术人员之一所通常理解的含义相同。下文描述了示例性的方法和材料,然而与本文描述的那些类似或等价的方法和材料也可用于实施或测试本发明。所述材料、方法和实施例仅是示例性的,并且在范围上不意欲是限制性的。
实施例1-产生小鼠单克隆抗体
包含融合至人IgG1恒定区的人CTLA4的细胞外结构域的重组CTLA4-融合蛋白购自R&D Systems(Oxford,UK)。细胞外CTLA4片段如下制备:用凝血因子Xa(Qiagen,Crawley,UK)对CTLA4-Fc融合蛋白进行蛋白水解切割,然后依次用凝血因子Xa去除树脂(Qiagen)除去所述蛋白酶,用蛋白A-琼脂糖除去所切割的Fc片段,以仅留下CTLA4细胞外结构域。
用含20μg CTLA4-Fc融合蛋白的200μl弗氏完全佐剂(Sigma-Aldrich,Dorset,UK)的1:1乳液皮下免疫雌性Balb/c小鼠。随后,大约每三周腹腔内注射含20μg CTLA4-Fc的弗氏完全佐剂(Sigma-Aldrich)的1:1乳液最多达3次而对免疫的小鼠进行加强。骨髓瘤融合前3天,显示出最高抗体滴度的两只小鼠接受了全抗原或CTLA4细胞外结构域的脾内加强免疫。
提取脾脏并进行均质化以产生单细胞悬液。使用聚乙二醇(PEG)将1×108个脾细胞与5×107个NS0小鼠骨髓瘤细胞(比例为2:1)进行融合。将融合的细胞重悬于200ml含杂交瘤细胞选择剂偶氮丝氨酸和次黄嘌呤的DMEM/20%FCS/5%BM Condimed H1(Roche,Burgess Hill,UK)——“HAZA培养基”中,并用移液管以200μl的体积移至10×96孔板中。将板在37℃下在5%CO2中孵育,每隔一日用新鲜的含2.5%BM Condimed H1的HAZA培养基替换每个培养孔的一半体积(100μl)。12天的孵育之后,将每个培养孔的100μl消耗后的培养基转移至96孔储藏板中,并使用下文所述的CTLA4-融合蛋白ELISA来测试所分泌的CTLA4-融合蛋白抗体的存在情况。通过转移至24孔板中的1ml“H-培养基”(DMEM/20%FCS/次黄嘌呤)并允许继续生长5-7天来扩大免疫阳性培养物。然后,将阳性培养物通过有限稀释进行亚克隆、扩大并通过CTLA4-融合蛋白ELISA进行测试。另外,通过下文所述的FACS测试阳性培养物。
对于有限稀释,使用血球细胞计数器并在含2.5%BM Condimed H1的培养基中对细胞进行系列稀释来确定细胞数量,直至细胞密度达到5-15个细胞/ml。对于每个杂交瘤,将200μl的细胞溶液用移液管移至48孔中,密度为1-3个细胞/孔。将培养物在37℃下在5%CO2中培养2周,在培养1周后加入一半体积的另外的培养基。通过ELISA测试培养基中对CTLA4-融合蛋白特异的抗体的存在情况。选出ELISA阳性克隆,并将其在DMEM/20%FCS/2.5%BMCondimed H1中扩大到10ml培养物。然后,将克隆冷冻在含10%DMSO的培养基中并保存在液氮中,并且还将其进一步扩大用于抗体纯化。将命名为3B10和8H5的两种杂交瘤细胞进行亚克隆,然后将亚克隆冷冻并用于进一步研究中的单克隆抗体产生。
为了鉴定分泌抗人CTLA4特异性小鼠抗体的杂交瘤细胞,将ELISA板(VWR,Lutterworth,UK)用100μl/孔的PBS中0.5μg/ml的重组CTLA4融合蛋白或人IgG1(Sigma-Aldrich,Poole,UK)在4℃下包被过夜。洗涤板,并将其用150μl/孔的含2%BSA的PBS封闭。将细胞培养上清液或纯化的抗体在PBS/2%BSA中稀释,向每个板加入100μl,然后在室温下孵育1小时。将板用PBS-吐温(0.05%)洗涤三次,并用100μl/孔的缀合辣根过氧化物酶的山羊抗小鼠Ig(Fab-特异性)(Sigma-Aldrich)孵育1小时。将板用PBS-吐温洗涤三次,然后加入SigmaFast OPD底物(Sigma-Aldrich)并在室温下在黑暗中孵育4分钟。通过加入50μl的3MHCl终止反应。使用Dynex酶标仪(Dynex,Worthing,UK)在490nm下读板。
使用快速ELISA小鼠抗体同种型鉴定试剂盒(Rapid ELISA Mouse AntibodyIsotyping Kit,Perbio,Cramlington,UK)对单克隆抗体进行同种型鉴定。在1ml蛋白A-琼脂糖柱(GE Healthcare,Little Chalfont,UK)上纯化抗体。纯化之前,将管道和蛋白A柱用0.4M NaOH去热原。将柱用20CV的1×PBS(pH7.4)重新平衡。收获杂交瘤细胞培养物上清液,使用10×PBS将其调至1×PBS(pH7.4)并过滤除菌。将过滤的上清液以0.5ml/分钟泵送通过柱。用1×PBS(pH7.4)洗涤柱,使用无菌0.1M柠檬酸钠(pH3)洗脱IgG,收集了0.9ml级分并用0.1ml的无菌1MTris-HCl(pH9)中和。在无菌条件下,将所述产品缓冲液交换为PBS(pH7.4)以除去任何的洗脱缓冲液并浓缩所述样品。浓缩之后,使用1.4的消光系数Ec(0.1%)通过OD280nm对抗体进行定量。纯化的抗体通过Novex NuPAGE电泳系统用4-12%NuPage凝胶(Invitrogen,Paisley,UK)和MES电泳缓冲液通过SDS-PAGE来分析。与4×NuPAGE样品缓冲液和β-巯基乙醇一起准备1μg抗体并加热。将所述凝胶用InstantBlue染色液(Expedeon,Cambridge,UK)染色并通过比较染色带与PageRulerTMPlus Prestained Protein Ladder(Fermentas,York,UK)来估计分子大小。对每个抗体确定两条带,不存在可检测到的污染物。
为了评估抗体与CTLA4的结合以及对CTLA4与CTLA4配体B7.1和B7.2之间相互作用的阻断,通过ELISA进行了竞争测定。使用Biotin TagTM微型生物素化试剂盒(Biotin TagTMMicro Biotinylation kit,Sigma–Aldrich)将配体B7.1-Ig和B7.2-Ig(R&D Systems)生物素化。将96孔MaxiSorp板(Nunc)用Dulbecco's PBS(PAA Laboratories,Yeovil,UK)中0.5μg/ml的重组人CTLA4-Ig(IgG1)(R&D Systems)(终体积80μl)在4℃下包被过夜。丢弃CTLA4-Ig并将板用Dulbecco’s PBS-2%BSA在室温下封闭1小时。将板用洗涤缓冲液(Dulbecco’s-PBS中0.05%吐温20)洗涤3次。将不同浓度的测试抗体与生物素化的-B7.1-Ig(终浓度0.36μg/ml)或生物素化的-B7.2-Ig(终浓度0.65μg/ml)预混合,然后加至所述CTLA4-Ig板(终体积80μl)。所有样品以两个重复进行测试。将板在室温下孵育1小时,并用洗涤缓冲液洗涤3次。加入80μl抗生物素蛋白链菌素HRP(Sigma-Aldrich)的1:500(1in500)稀释物,并在室温下孵育1小时。将板用洗涤缓冲液洗涤三次,加入80μl SigmaFast OPD底物(Sigma-Aldrich,Cat#P9187)并在室温下在黑暗中孵育4分钟。通过加入50μl的3M HCl终止反应。使用Dynex酶标仪在490nm下读板。基于与CTLA4的结合(图1),选择亚克隆8H5-1B1、3B10-4F7、7B9-1A3和2C7-1G10作为首要单克隆抗体的生产者。在这些首要者中,除了7B9-1A3以外都显示出与生物素化的B7.1(图2)和生物素化的B7.2(图3)竞争结合人CTLA4。
为了确定所述首要单克隆抗体是否结合在T细胞表面上表达的CTLA4,进行了流式细胞分析。从人PBMC(外周血单核细胞)分离人外周T细胞,并对其进行刺激以增强CTLA4的表达。使用CD4+T细胞分离试剂盒(Miltenyi Biotec,Bisley,UK)从PBMC纯化CD4+细胞,铺板在24孔板(1×106个细胞/孔)中的AIM-V培养基(Invitrogen,Paisley,UK)中,在37℃下过夜孵育。用伊屋诺霉素(1μg/ml)和PMA((12-)十四酸佛波酯(-13-)乙酸盐)(50ng/ml)刺激细胞,并在37℃下孵育4小时。将细胞在AIM-V培养基中洗涤一次,在含2%甲醛的PBS中固定15分钟,以2×106个细胞/ml重悬于FACS缓冲液(含1%BSA、0.05%叠氮化钠和0.1%皂苷的D-PBS),在4℃下孵育30分钟。
将2×105个细胞用CTLA4-PE缀合抗体(BNI3)(Abcam,Cambridge,UK)的1:10稀释物(作为阳性对照)染色,或者用5μg/ml的各个CTLA4单克隆抗体和小鼠IgG-PE缀合抗体(Sigma)的1:50稀释液一起染色。还包括小鼠IgG(Sigma)作为首要单克隆抗体内存在的不同鼠同种型的单独对照。将细胞在4℃下染色1小时。还包括仅小鼠IgG-PE缀合抗体的对照。将细胞用FACS缓冲液洗涤两次,任选在4℃下在黑暗中用小鼠抗人CD4-FITC缀合抗体(Caltag,Buckingham,UK)的1:40稀释物或小鼠IgG2a-FITC缀合抗体(Caltag)染色1小时。用FACS缓冲液洗涤两次之后,将细胞重悬于FACS缓冲液并使用Beckton DickinsonFACSCalibur(Becton Dickinson,Oxford,UK)进行流式细胞术。通过分析相关同种型对照抗体来确定仪器设置。基于所观察到的与CTLA4的结合,单克隆抗体8H5和3B10被分别指定作为第一和第二首要单克隆抗体。
实施例2–可变区基因测序
对产生首要单克隆抗体8H5和3B10的亚克隆3B10-4F7、3B10-6E3、8H5-1A1和8H5-1B1进行可变区(V-区域)序列分析。使用RNAqueous-4PCR试剂盒(Ambion,Warrington,UK)从3×106-10×106个杂交瘤细胞提取总RNA,并将其用于合成cDNA。使用如表1所示的简并小鼠前导序列引物(Sigma)和独特恒定域引物(Sigma)通过PCR扩增鼠免疫球蛋白重链和κ轻链V-区域片段。将所得的PCR片段亚克隆至pGEM-T Easy I载体系统(Promega,Southampton,UK)中,并使用载体特异性引物M13Forward(Sigma)对插入片段进行测序。所有DNA测序由Geneservice Ltd,Cambridge,UK进行。获得了3B10(SEQ ID No 1和2)和8H5(SEQ ID No5和6)的独特V-区域核苷酸序列。
确定了如下的3B10和8H5高变区(CDR)序列;
SEQ ID No.93B10CDRH1DYNMD
SEQ ID No.103B10CDRH2NINPNSESTSYNQKFKG
SEQ ID No.113B10CDRH3DGNRYDAWFAY
SEQ ID No.123B10CDRL1SASSSVTYMH
SEQ ID No.133B10CDRL2STSILAS
SEQ ID No.143B10CDRL3QQRTSYPLT
SEQ ID No.158H5CDRH1SYWIN
SEQ ID No.168H5CDRH2RIAPGSGTTYYNEVFKG
SEQ ID No.178H5CDRH3GDYGSY
SEQ ID No.188H5CDRL1SASSSISYMH
SEQ ID No.198H5CDRL2DTSKLAS
SEQ ID No.208H5CDRL3HQRTSYPLT
实施例3–产生嵌合抗体
将首要3B10和8H5单克隆抗体的重链和轻链可变域序列进行PCR扩增并亚克隆至pANT抗体表达载体中(图8),重链和轻链V区域分别克隆至pANT17和pANT13中。将重链V-区域基因与人γ1重链基因(G1m3(G1m(f))同种异型)或人γ4重链基因在阅读框内经MluI和HindIII位点克隆至pANT17中,将轻链V-区域基因与人κ轻链恒定区基因(Km3同种异型)在阅读框内经BssHII和BamHI位点克隆至pANT13中。重链和轻链基因的转录都在CMV I/E启动子的控制下(US5168062和US5385839,爱荷华大学),pANT17质粒含有在SV40启动子控制下的突变型dhfr小基因(Simonsen&Levinson1983,PNAS80:2495-2499)和用于在真核细胞中进行选择的polyA序列。pANT17和pANT13都含有用于原核选择的β-内酰胺酶(ApR)基因和用于在原核细胞中增殖的pMB1复制起始点。所有质粒在大肠杆菌(E.coli)XL1-blue中增殖(Stratagene Cat.No.200130)。用于扩增所述V-区域基因以克隆至pANT表达载体的引物示于表2。
然后,将所述重链和轻链表达构建体通过基于磷酸钙的转染瞬时地共转染至HEK293细胞中,或者通过电穿孔稳定地转染至NS0细胞中。分泌的抗体通过蛋白A色谱法从细胞培养物上清液纯化。通过对CTLA4结合ELISA(图4)、相对于B7.1和B7.2的CTLA4竞争ELISA(图5和6)进行分析,以及通过如实施例1中的流式细胞术分析的与T细胞上表达的CTLA4的结合,3B10和8H5嵌合抗体都显示保留了初始单克隆抗体的CTLA4结合。
实施例4–T-细胞增殖测定
使用包被人CD2抗体、人CD3抗体和人CD28抗体的珠子(Miltenyi Biotec,Bisley,Surrey)激活PBMC(外周血单核细胞)。将5×105个细胞铺板在96-孔板每个孔中的AIM-V培养基中,并且以1珠子/细胞的比例向细胞加入珠子。将测试抗体或同种型对照抗体在AIM-V培养基中酌情稀释,并以50μl/孔加入到所述细胞中,得到200μl的终体积。还包括仅培养基(无抗体)的对照。将板在37℃下孵育4天,然后将细胞用培养基中的0.75μCi[3H]-胸苷(Perkin Elmer,Beaconsfield,UK)进行脉冲(pulse),并再孵育18小时,然后使用TomTec Mach III(Hamden CT,USA)细胞收集器在过滤垫(Perkin Elmer)上收集细胞。每个孔的每分钟计数(cpm)通过MeltilexTM(Perkin Elmer)闪烁计数法在1450MicrobetaWallac Trilux Liquid Scintillation Counter(Perkin Elmer)上在paralux低本底计数中确定。将每种抗体样品的每分钟计数相对于仅培养基对照进行归一化。在两个独立研究中,显示嵌合抗体可逆转CTLA4诱导的对T细胞增殖的抑制,与用初始单克隆抗体可观察到的一样(图7)。
实施例5-产生人源化抗体
使用EP1844074(Antitope Ltd)中描述的方法产生人源化抗体。使用瑞士PDB产生小鼠V区域的结构模型并对其进行分析,以鉴定来自3B10和8H5V-区域的可能对所述抗体的CTLA4结合特性重要的重要氨基酸(“限制残基(constraining residues)”)。使用人V-区域序列的数据库以鉴定含有要用于设计所述人源化抗体的各个限制残基的人V-区域序列片段。通常,使用两个或多个可选择的V-区域序列片段以提供各个限制残基,获得8H5和3B10的人源化抗CTLA4V-区域序列的大量可能序列。然后,通过如Fothergill et al.(WO9859244,Eclagen Ltd)中所述的计算机分析来分析这些序列以预测非生殖细胞系MHCII类肽结合,还使用包括“The Immune Epitope Database and Analysis Resource”,http://www.immuneepitope.org/的数据库来分析已知CD4+T-细胞表位。去掉含有预测的非生殖细胞系MHC II类结合肽或含有与T细胞表位数据库明显匹配的序列的V-区域序列。这导致V-区域序列集变小。然后,将选择的V-区域序列片段组合结合以产生人源化重链和轻链可变区氨基酸序列。分别为8H5(分别为SEQ ID No41-45和46-50)和3B10(分别为SEQID No31-35和36-40)选择五条重链和五条轻链序列(分别命名为VH1-VH5,VK1-VK5)。
合成编码人源化变体V-区域的DNA并将其亚克隆至如实施例3所述的表达载体pANT17和pANT13中。将人源化VH和VK链的所有组合(即,对于8H5和3B10各自总计有25种配对)瞬时转染至HEK293中,还转染至NS0细胞中,并如实施例3所述通过蛋白A色谱法从培养物上清液纯化抗体。
实施例6-分析人源化抗体
在针对合适的亲本嵌合抗体的竞争ELISA中评估了来源于HEK和来源于NS0的8H5和3B10人源化变体与重组CTLA4的结合。使用Biotin TagTM微型生物素化试剂盒(Sigma–Aldrich)将亲本8H5和3B10嵌合抗体生物素化。将96孔MaxiSorp板(Nunc)用Dulbecco'sPBS中0.5μg/ml的重组人CTLA4-Ig(终体积100μl)在4℃下包被过夜。弃去CTLA4-Ig并将板用Dulbecco's PBS-2%BSA在室温下封闭1小时。将板用洗涤缓冲液(Dulbecco’s-PBS中0.05%吐温20)洗涤3次。将不同浓度的测试人源化抗体与生物素化的亲本嵌合抗体(终浓度0.02μg/ml)预混合,然后加入到所述CTLA4-Ig板中(终体积100μl)。所有样品进行两次重复测试。将板在室温下孵育1小时,并用洗涤缓冲液洗涤3次。加入100μl抗生物素蛋白链菌素HRP(Sigma-Aldrich)的1:500稀释液,并在室温下孵育1小时。将板用洗涤缓冲液洗涤三次,加入100μl SigmaFast OPD底物(Sigma-Aldrich,Cat#P9187)并在室温下在黑暗中孵育4分钟。通过加入50μl的3M HCl终止反应。使用Dynex酶标仪在490nm下读板。
所有首要8H5人源化变体都显示出与8H5嵌合抗体类似的竞争结合谱,但含κ链VK5的变体表现出与其他变体相比略减少的结合(图17)。类似地,所有的首要人源化3B10变体显示出与3B10嵌合抗体类似的竞争结合谱。此外,所有首要人源化8H5和3B10变体,当在相对于B7.1和B7.2的CTLA4竞争ELISA中测试时(实施例3),给出了图5和6所示的与所述嵌合抗体非常类似的竞争结合谱,其中在所述首要人源化变体的最大浓度下>90%的B7.1或B7.2结合被抑制。选择首要人源化变体VH5/VK4(分别为SEQ ID No45和39)作为用于进一步研究的首要抗体。
实施例7–产生scFv’s和Fab’s
将来自实施例6的人源化8H5和3B10变体转变为scFv’s并使用pCANTAB5E载体RPAS表达模块(Amersham Pharmacia Biotech,Little Chalfont,UK)克隆至如Benhar I.andReiter Y.,Current Protocols in Immunology,Unit10.19B,Wiley Online Library,May2002(http://www.currentprotocols.com/protocol/im1019b)所述的M13噬菌体展示载体中。使用引物扩增人源化VH和VK基因,所述引物提供末端SfiI和NotI限制位点、内部Gly4Ser接头和C末端his6标签。将所述scFv构建体作为SfiI-NotI片段插入至pCANTAB5E载体并转化至大肠杆菌HB2151中,产生输出至周质并部分输出至生长培养基的scFv。通过镍螯合物亲和色谱法使用HIS-Select HF Cartridges(Sigma-Aldrich)从生长培养基纯化scFv’s。在如实施例1所述的B7.1-Ig和B7.2-Ig竞争测定中测试了纯化的scFv’s,所有的人源化scFvs都显示出与CTLA4的竞争结合。还使用用于scFv’s的方法将实施例6的人源化8H5和3B10变体转变为Fab’s,另外将扩增的人源化VH和VK基因用CH1和Cκ恒定区基因进一步扩增以形成VH-CH1和VK-Cκ片段,并将其用引物进一步扩增以将这些片段与22个氨基酸的pelB前导序列(Lei S.P.,Lin H.C.,Wang S.S.,Callaway J.,and Wilcox G.,JBacteriol.169(1987)p4379–4383)在上游VH-CH1和下游VK-Cκ基因片段之间连接起来,产生双顺反子Fab基因。产生了来自人源化抗体变体的Fab’s,并按上文关于scFv’s的描述进行纯化,并在如实施例1所述的B7.1-Ig和B7.2-Ig竞争测定中进行测试。所有的人源化Fab’s显示出与CTLA4的竞争结合。
实施例8–分析CD4+T细胞应答
根据Addenbrooke’s Hospital Local Research Ethics Committee的批准,从获自UK National Blood Transfusion Service(Addenbrooke’s Hospital,Cambridge,UK)的健康社会供体血沉棕黄层(来自24小时内抽取的血液)分离PBMC。通过Lymphoprep(Axis-shield,Dundee,UK)密度离心从血沉棕黄层分离PBMC,并使用CD8+RosetteSepTM(StemCellTechnologies Inc,London,UK)耗尽CD8+T细胞。供体通过使用基于HLA SSP-PCR的组织分型试剂盒(Biotest,Solihull,UK)鉴定HLA-DR单元型来表征。还确定了针对对照抗原(包括回忆抗原破伤风毒素)的T细胞应答(KLH Pierce,Cramlingtom,UK和来源于A型流感病毒和爱泼斯坦巴尔病毒(Epstein Barr virus)的肽)。然后,将PBMC冷冻并保存在液氮中待用。
为了制备单核细胞衍生的树突细胞(DC),选择了50份不同的供体PBMC以提供与全世界人群频率类似的HLA-DR和HLA-DQ同种异型的频率分布。将PBMC在培养基中复苏,使用Miltenyi CD14微珠(Microbead)和LS柱(Miltenyi Biotech,Oxford,UK)分离CD14+细胞。将单核细胞重悬于补充有1000U/ml IL-4和1000U/ml GM-CSF的中(“DC培养基”)至4×106-6×106个PBMC/ml,然后分配到24孔板中(终培养体积2ml)。在第2天,通过更换一半体积的DC培养基来给细胞补料。到第3天时,单核细胞已经分化为半成熟DC,将所述半成熟DC与40μg/ml的测试人源化抗体或嵌合抗体、100μg/ml KLH或仅培养基预孵育。将半成熟DC与抗原孵育24小时,之后将所述细胞洗涤两次除去过量的测试抗体并重悬于补充有50ng/ml TNF-α(Peprotech,London,UK)的DC培养基。在第7天,通过更换一半体积的DC培养基(补充有50ng/ml TNFα)来为DC细胞补料,然后在第8天收集成熟DC。对收集的成熟DC进行计数并使用台盼蓝染色排除来评估生存力。然后,将所述DC进行γ-照射(4000拉德)并以2×105个细胞/ml重悬于AIM-V培养基,然后用于ELISpot和增殖测定。另外,在第8天,还制备了新鲜的CD4+T细胞。为了纯化CD4+T细胞,将PBMC在培养基中复苏,使用Miltenyi CD4微珠和LS柱(Miltenyi Biotech,Oxford,UK)分离CD4+细胞并将其以2×106个细胞/ml重悬于培养基中。
在第8天,建立了T细胞增殖测定,其中在96孔U型底板中,将1×105个自体CD4+T细胞加入到1×104个负载有人源化抗体或嵌合抗体的DC(比例为10:1)中,加入培养基至终体积为200μl/孔。在第14天,将测定板每孔用25μl AIMV中的1uCi[3H](PerkinElmer,Beaconsfield,UK)进行脉冲6小时,然后使用TomTec Mach III(Hamden CT,USA)细胞收集器在过滤垫(Perkin Elmer)上收集细胞。将所有的抗体制剂在一式六份的培养物中进行测试。每孔的每分钟计数(cpm)通过MeltilexTM(Perkin Elmer)闪烁计数法在1450Microbeta Wallac Trilux Liquid Scintillation Counter(Perkin Elmer)上在paralux低本底计数中确定。将每种抗体样品的每分钟计数按仅培养基的对照进行归一化。
对于ELISpot测定,将ELISpot板(Millipore,Watford,UK)用100μl/孔的PBS中的IL-2捕获抗体(R&D Systems,Abingdon,UK)包被。然后,将板在PBS中洗涤两次,在封闭缓冲液(PBS中1%BSA(Sigma))孵育过夜并在培养基中洗涤。在第8天,在96孔ELISpot板中,将1×105个自体CD4+T细胞加入到1×104个负载有抗原的DC(比例为10:1)中。将所有的抗体制剂在一式六份的培养物中进行测试。对于每种供体PBMC,还包括阴性对照(仅培养基)、无细胞对照和PHA(10μg/ml)阳性对照。
在继续孵育7天之后,将ELISpot板在dH2O和PBS中依次洗涤三次,然后加入100μl过滤的PBS/1%BSA中的生物素化检测抗体(R&DSystems,Abingdon,UK),进行显影。在37℃下孵育1.5小时后,将板再在PBS中洗涤三次,加入100μl过滤的PBS/1%BSA中的抗生物素蛋白链菌素-AP(R&D Systems),维持1小时(在室温下孵育)。弃去抗生物素蛋白链菌素-AP,并将板在PBS中洗涤四次。向每孔加入BCIP/NBT(R&D Systems),并在室温下孵育30分钟。通过用dH2O洗涤孔和孔背面(back)三次来终止斑点显影。将干燥的板在Immunoscan TMAnalyser上扫描,使用ImmunoscanTM第4版软件确定每孔斑点数(spw)。
对于增殖测定和IL-2ELISpot测定,结果表示为刺激指数(SI),其定义为测试抗体相对于仅培养基对照的cpm(增殖测定)或斑点(ELISpot测定)的比例,对于阳性T细胞应答使用等于或大于2的SI阙值(SI≥2.0)。数据显示,嵌合8H5抗体和嵌合3B10抗体在测试的50份供体PBMC中的8份或更多份中诱导T细胞应答(>=16%),而所述人源化8H5抗体或3B10抗体没有一种在50份供体中的多于2份供体中诱导T细胞应答(<=4%,平均数2%+-2%),这表明人源化过程有效去除了V-区域的T细胞应答。平行地,使用如实施例5所述的方法合成具有全人CTLA4抗体MDX010(易普利姆玛)(Keler et al.,ibid)和Tremelimumab(Ribas et al.,ibid)的V-区域序列的DNA,并用其产生这些抗体的重组IgG1/κ形式。然后,将这些抗体的来源于NS0的制剂用上述相同的50份供体PBMC在一式六份的培养物中针对CD4+辅助T细胞应答的诱导进行测试。对于易普利姆玛,在平均4份供体中检测到T细胞应答(8%+-2%),对于Tremelimumab的IgG1/κ形式,平均5份供体中检测到T细胞应答(10%+-2%),因此表明当在体外测试50份人全血样品中CD4+辅助T细胞应答的诱导时,仅本发明的人源化CTLA4抗体能够在<=4%的供体中给出CD4+T细胞应答。
实施例9–人混合的淋巴细胞细胞反应(MLR)模型
使用混合的淋巴细胞反应测定测量阻断CTLA4途径对IFN-γ分泌的效应,作为人T细胞激活的量度。将来自多个人供体的新鲜血液(获自UK National Blood TransfusionService,实施例8)用PBS/2%人血清进行1:1稀释,并以900g离心使其在Lymphoprep溶液(Nycomed)上分层。将PBMC从分界面移出,洗涤并重悬于AIM-V培养基(Invitrogen)。然后,将从不同的不匹配供体对产生的PBMC以1:1比例组合,并铺板于96孔板中,以提供每样品孔总共2.5×105个PBMC。加入PHA(植物凝集素,Sigma Aldrich)至终浓度为2μg/ml,以刺激T细胞群的增殖。加入首要VH5/VK4 CTLA4抗体,MDX010 CTLA4对照抗体(实施例8)或同种型对照IgG1抗体至终浓度150μg/ml。还使用5μg/mlCTLA4-Fc替代抗体作为对照,以证明对IFN-γ分泌的抑制。每孔总的终体积为150μl,对于每种供体组合,每种抗体测试五次。将96孔板在正常培养条件孵育72小时,然后取100μl上清液用于依照制造商推荐的方法通过ELISA(Thermo scientific,ESS0002)测量IFN-γ。根据图19中的数据,对于所有的供体组合,所述首要VH5/VK4抗体显示出比MDX010CTLA4对照抗体更高的T细胞激活,与MDX010相比VH5/VK4的T细胞激活的平均增加为>2倍。
实施例10-肿瘤动物模型
肿瘤动物模型被用于在体内分析人CTLA4抗体对肿瘤生长的抑制。在所述模型中,在人CTLA4敲入小鼠(OncoImmune,Inc.)中培养MC38鼠结肠肿瘤细胞(Corbett et al.,(1975)Cancer Res35:2434-2439,由OncoImmune,Inc.,Ann Arbor,USA提供)。
对CTLA4敲入小鼠(7-10周龄,各组中雄性和雌性相等分布)侧面皮下注射0.1ml体积的5×105个MC38肿瘤细胞。从给予肿瘤细胞之后的那天(“第2天”)起,每周以5mg/kg或10mg/kg剂量(剂量体积10ml/kg)注射所述首要VH5/VK4CTLA4抗体、MDX010(实施例8)或同种型匹配对照抗体。在实验过程中,每两周一次通过测径器测量取得肿瘤测量结果,肿瘤大小表示为立方体积(mm3)。跟踪动物直至肿瘤体积达到2000mm3,或直到注射肿瘤细胞后的第45天。实施例19示出的结果表明,与MDX010相比首要VH5/VK4CTLA4抗体提高了对肿瘤生长的抑制。
Claims (23)
1.一种抑制CTLA4结合人B7的抗CTLA4的人源化抗体,其中所述抗体包含可变区,所述可变区的CDR序列为:
(i)SEQ ID NO:9所示的CDRH1
(ii)SEQ ID NO:10所示的CDRH2
(iii)SEQ ID NO:11所示的CDRH3
(iv)SEQ ID NO:12所示的CDRL1
(v)SEQ ID NO:13所示的CDRL2;和
(vi)SEQ ID NO:14所示的CDRL3;
或者
(i)SEQ ID NO:15所示的CDRH1
(ii)SEQ ID NO:16所示的CDRH2
(iii)SEQ ID NO:17所示的CDRH3
(iv)SEQ ID NO:18所示的CDRL1;
(v)SEQ ID NO:19所示的CDRL2;和
(vi)SEQ ID NO:20所示的CDRL3。
2.权利要求1的抗CTLA4的抗体,其包含选自SEQ ID NO:31-35的可变区序列用于重链可变区,以及选自SEQ ID NO:36-40的可变区序列用于轻链可变区。
3.权利要求1的抗CTLA4的抗体,其包含选自SEQ ID NO:41-45的可变区序列用于重链可变区,以及选自SEQ ID NO:46-50的序列用于轻链可变区。
4.权利要求3的抗CTLA4的抗体,其包含SEQ ID NO:45用于重链可变区,以及SEQ IDNO:49用于轻链可变区。
5.权利要求1的抗CTLA4的抗体,当在具有人群HLA-DR同种异型分布的至少50份人血样中体外测试CD4+辅助T细胞应答的诱导时,所述抗CTLA4的抗体产生<=4%的T细胞应答。
6.权利要求1的抗体,其中所述可变区序列全部来源于人抗体可变区中的序列。
7.权利要求1的抗体,其由可变区以及同种型IgG1、IgG2、IgG3或IgG4的重链恒定区或突变的IgG恒定区和同种型κ的轻链恒定区组成。
8.权利要求7的抗体,其中所述恒定区为IgG1和κ。
9.权利要求7的抗体,其中所述恒定区为IgG4和κ。
10.权利要求1-9中任一项的抗体,其中所述抗体为scFv或Fab。
11.一种多特异性抗体,其包含一种或多种权利要求1-10的抗体。
12.一种多核苷酸,其编码权利要求1-11中任一项的抗体。
13.一种载体,其包含权利要求12的多核苷酸。
14.一种宿主细胞,其包含权利要求13的载体。
15.权利要求14的宿主细胞,其中所述宿主细胞是原核细胞或真核细胞。
16.权利要求15的宿主细胞,其中所述宿主细胞是哺乳动物细胞。
17.一种组合物,其包含权利要求1-11中任一项的抗CTLA4的抗体或权利要求12的多核苷酸。
18.权利要求1-11中任一项的抗体或权利要求12的多核苷酸用于制备在患者中诱导、加强或延长针对抗原的免疫应答的药物的用途。
19.权利要求1-11中任一项的抗体或权利要求12的多核苷酸用于制备治疗需要此治疗的受试者中的结肠癌的药物的用途。
20.权利要求18或19的用途,其中所述药物与有效量的化学治疗剂共同给药。
21.权利要求18或19的用途,其中所述药物与药物载体共同给药,所述药物载体包括疫苗。
22.一种使用权利要求1-11中任一项的抗体在体外检测样品中人CTLA4抗原的存在情况的方法,所述方法是用于非诊断目的。
23.权利要求1-11中任一项的抗体用于制备在体外或体内检测样品中人CTLA4抗原的存在情况的试剂的用途。
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RU2013145243A (ru) | 2015-04-20 |
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DK2683739T3 (en) | 2016-05-23 |
ZA201306589B (en) | 2014-05-28 |
RU2629768C2 (ru) | 2017-09-06 |
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CN103547595A (zh) | 2014-01-29 |
MX344971B (es) | 2017-01-12 |
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