CN116948031A - 抗il-23r抗体及其用途 - Google Patents
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- CN116948031A CN116948031A CN202310571294.2A CN202310571294A CN116948031A CN 116948031 A CN116948031 A CN 116948031A CN 202310571294 A CN202310571294 A CN 202310571294A CN 116948031 A CN116948031 A CN 116948031A
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Abstract
本发明涉及白介素‑23受体的新型抗体或其抗体片段,所述抗体或其片段能够有效结合IL‑23R,阻断IL‑23R与人IL‑23α/IL‑12β异源二聚体配体的结合,具有良好的应用前景。所述抗体或其抗原结合片段包括重链可变区和轻链可变区,其中,重链可变区包括VH CDR1、VH CDR2和VH CDR3,轻链可变区包括VL CDR1、VL CDR2、VL CDR3,其中VH CDR1包含SEQ ID NO:3、13或23所示的氨基酸序列,VH CDR2包含SEQ ID NO:4、14或24所示的氨基酸序列,VH CDR3包含SEQ NO:5、15或25所示的氨基酸序列;VL CDR1包含SEQ ID NO:8、18或28所示的氨基酸序列,VL CDR2包含SEQ ID NO:9、19或29所示的氨基酸序列,VL CDR3包含SEQ ID NO:10、20或30所示氨基酸序列。
Description
本申请是申请号为202111193581.1、申请日为2021年10月13日、发明名称为“抗IL-23R抗体及其用途”的中国发明专利申请的分案申请。
技术领域
本发明属于生物医药领域,涉及抗体及其应用。
背景技术
白介素23(IL-23)是由两个蛋白亚基,即IL-23特有的p19亚基(IL-23α)和p40亚基(IL-12β,p40亚基与IL-12共同使用)构成的异二聚体细胞因子,一般由活化的巨噬细胞或者树突状细胞产生并作用于表达IL-23受体的Th17细胞促进其增殖和稳定性。
IL-23诱导CD4+T细胞产生IL-17的过程是由活化的Jak2、PI3K/Akt、STAT3和NF-κB介导的。Tyk2和STAT家族的其他成员如STAT1、STAT4、STAT5也参与此过程。IL-23受体由IL-12Rβ1亚基和IL-23R亚基组成,构成异二聚体,其中IL-12Rβ1结合Tyk2,IL-23R结合Jak2。IL-23与其受体复合物结合,激活其下游的Jak2和Tyk2后,引起受体复合物磷酸化和STATs(1,3,4,5)对接位点的形成。然后STATs发生聚合、磷酸化,向核内转移并活化相应基因。IL-12诱导的结合DNA的复合物中只包含STAT4,而IL-23诱导的结合DNA的复合物中包含STAT3、STAT1、STAT4,还可能包含STAT3/STAT4二聚体。在淋巴细胞中,IL-23对STAT3的磷酸化作用较强,STAT4磷酸化相对较弱。磷酸化的STAT3入核后,与IL-17A、IL-17F基因的启动子结合,直接参与启动其转录与合成;也可以与Th17细胞的特异性转录因子RORγt启动子结合,上调其表达,进而间接促进IL-17A、IL-17F的合成。
IL-23与多种自身免疫病的发生相关,如炎症性肠炎包括克罗恩病(CD)、或溃疡性结肠炎(UC)、银屑病(PS)、银屑病关节炎(PA)、系统性红斑狼疮(SLE)、类风湿性关节炎(RA)、强直性脊柱炎(AS)。
炎症性肠炎(IBD)是以克罗恩病(CD)、溃疡性结肠炎(UC)为代表的慢性复发性疾病,累及回肠、直肠、结肠的一种特发性肠道炎症性疾病,临床表现为腹泻、腹痛、甚至便血。近年研究人员通过免疫组化及实时定量PCR技术证实IL-23在IBD患者的炎性黏膜中呈高水平表达,其高表达可促使肠上皮内淋巴细胞(IEL)及NK细胞激活产生细胞毒性作用,同时刺激IBD病灶中部分亚群的T细胞分泌高水平的IFN-γ、TNF、IL-2和IL-17A等炎症因子,从而促使其分化为Th17细胞,加重炎症反应。
自上个世纪80年代初,T细胞异常在银屑病的发生发展中的作用逐渐受到了大家的关注。近年来人们对IL-23/Th17通路有了更深入的认识和了解。一般认为,银屑病患者真皮中的树突状细胞和巨噬细胞产生IL-23,诱导Th17细胞和γδT细胞活化,并释放IL-17A、IL-17F、IL-22、IL-6和肿瘤坏死因子-α(TNF-α)等炎性细胞因子。IL-17A、IL-17F及IL-22作用于角质形成细胞,导致表皮增生、棘层肥厚和角化过度等银屑病典型的病理改变。在皮肤炎症微环境下,角质形成细胞又可产生更多的IL-23和其他炎性因子、趋化因子,这样就形成IL-23/Th17正反馈循环,放大并加剧了银屑病慢性炎性病变过程为慢性皮肤角化疾病。
SLE患者中发现其症状与IL23R阳性T淋巴细胞存在正相关,另外在SLE患者血液中的IL-23与正常人相比异常升高。
类风湿性关节炎是一种关节滑膜细胞慢性炎症为主的自身免疫病。在机体中,NK细胞为重要免疫细胞,在特定情况下可参与免疫性疾病、超敏反应的发生。作为一种重要促炎因子,IL-23与NK细胞的功能、RA疾病活性、骨破坏等均存在密切相关性。
强直性脊柱炎(AS)是主要再脊柱和骶髂关节产生病变的慢性炎症性疾病。AS患者血液中的IL-23与正常人相比显著升高。
以上信息均表明IL-23介导的信号通路可作为自身免疫疾病的治疗靶点。开发出特异性阻断IL-23信号通路的单克隆抗体,则有望用于IL-23信号通路异常相关的各种自身免疫疾病的治疗、预防和诊断。
目前市场上多种靶向IL-23抗体药物处于研发或者临床阶段,其中靶向IL-23与IL-12共同亚基p40的抗体已有药物上市如ustekinumab,由于可以同时影响IL-23和IL-12两条信号通路,从而可以引发比较广泛的生物学效应,有可能引发较严重的副作用,如感染;靶向IL-23专有的p19亚基的抗体众多,如已经上市的Guselkumab(杨森)、Tildrakizumab(默沙东/太阳制药)、Risankizumab(艾伯维)以及其他众多在研的处于临床中或临床前的分子,赛道拥挤,市场同质化严重。
IL-23R作为IL-23的受体,为该信号通路的关键因子,开发其抗体具有以下几点优势:首先,IL-23R作为膜蛋白,其表达量远远低于IL-23配体,由此可见,低剂量的抗体便可以达到有效的阻断效果,因此可降低药物生产成本以及患者的经济负担;其次,IL-23R抗体在研药物很少,以默沙东公司于2008年申请专利(CN101675076 B)的分子为代表,其中鼠源m20D7性质最好并进行了人源化命名为Hu20D7。但是20D7分子由于性质不达预期,多年并未开展后续临床研究,因此本领域亟需开发性质更为优良的抗体。
发明内容
针对上述现有技术的需求,本发明提供一种新的抗IL-23R抗体,及其在疾病的治疗和诊断中的用途。
在第一个方面,本发明提供结合IL-23R或其片段的抗IL-23R抗体或其抗原结合片段,上述抗体或其抗原结合片段包括重链可变区和轻链可变区,其中,重链可变区包括VHCDR1、VH CDR2和VH CDR3,轻链可变区包括VL CDR1、VL CDR2和VL CDR3,其中VH CDR1包含SEQ ID NO:3、13或23所示的氨基酸序列或由其组成,VH CDR2包含SEQ ID NO:4、14或24所示的氨基酸序列或由其组成,VH CDR3包含SEQ NO:5、15或25所示的氨基酸序列或由其组成;VL CDR1包含SEQ ID NO:8、18或28所示的氨基酸序列或由其组成,VL CDR2包含SEQ IDNO:9、19或29所示的氨基酸序列或由其组成,VL CDR3包含SEQ ID NO:10、20或30所示氨基酸序列或由其组成。
在一种优选实施方式中,所述抗体或其抗原结合片段的重链可变区的VH CDR1、VHCDR2和VH CDR3的氨基酸序列分别如SEQ ID NO:3、4和5所示,轻链可变区的VL CDR1、VLCDR2和VL CDR3的氨基酸序列分别如SEQ ID NO:8、9和10所示。
在一种优选实施方式中,所述抗体或其抗原结合片段的重链可变区的VH CDR1、VHCDR2和VH CDR3的氨基酸序列分别如SEQ ID NO:13、14和15所示,轻链可变区的VL CDR1、VLCDR2和VL CDR3的氨基酸序列分别如SEQ ID NO:18、19和20所示;或者,
在一种优选实施方式中,所述抗体或其抗原结合片段的重链可变区的VH CDR1、VHCDR2和VH CDR3的氨基酸序列分别如SEQ ID NO:23、24和25所示,轻链可变区的VL CDR1、VLCDR2和VL CDR3的氨基酸序列分别如SEQ ID NO:28、29和30所示。
在进一步的实施方式中,所述重链可变区的氨基酸序列如SEQ ID NO:1、11或21所示,或与SEQ ID NO:1、11或21具有至少90%、至少95%、至少96%、至少97%、至少98%或至少99%序列同一性,所述轻链可变区的氨基酸序列如SEQ ID NO:6、16或26所示,或与SEQID NO:6、16或26具有至少90%、至少95%、至少96%、至少97%、至少98%或至少99%序列同一性。
在一些实施方式中,所述抗体或其抗原结合片段可进一步包含重链恒定区、轻链恒定区、Fc区或其组合。在一些优选的实施方式中,所述轻链恒定区是κ链恒定区。在一些优选实施方式中,所述抗体或其抗原结合片段是IgG1。
在进一步的实施方式中,所述抗体或其抗原结合片段包括重链和轻链,所述重链包含SEQ ID NO:34、35或36所示氨基酸序列或由其组成,所述轻链包含SEQ ID NO:31、32或33所示氨基酸序列或由其组成。
在第二个方面,提供一种核酸分子,包含编码本发明的抗体或其抗原结合片段的核苷酸。在一些实施方式中,所述核酸分子编码所述抗体或其抗原结合片段的重链可变区和/或轻链可变区。
在优选的实施方式中,所述核酸分子编码重链可变区,其核苷酸序列如SEQ IDNO:2、12或22所示,或与SEQ ID NO:2、12或22具有至少90%、至少95%、至少96%、至少97%、至少98%或至少99%序列一致性。在其它优选的实施方式中,所述核酸分子编码轻链可变区,其核苷酸序列如SEQ ID NO:7、17或27所示,或与SEQ ID NO:7、17或27具有至少90%、至少95%、至少96%、至少97%、至少98%或至少99%序列一致性。
在另一方面,本发明提供一种生物材料,为:
(1)包含本发明的核酸分子的载体、宿主细胞或微生物等;或
(2)上述(1)的表达产物、悬浮液、上清液等。
本领域技术人员根据抗体的氨基酸序列能够容易地选择并制备包含所述抗体的编码序列的载体、宿主细胞或微生物,并能够知道如何培养这样的宿主细胞或微生物,从而获得相应的表达产物、悬浮液、上清液等,以获得相应的抗体。这都是本领域常规技术手段。
在另一方面,本发明提供一种组合物,其包含本发明的抗体或其抗原结合片段;优选地,所述组合物为药物组合物,进一步包含药学上可接受的载体。
在另一方面,提供制备本发明的抗体或其抗原结合片段的方法,包括:培养上述的宿主细胞使抗体或抗原结合片段表达,及自宿主细胞中分离所述抗体或抗原结合片段。
在另一方面,提供本发明的抗体或其抗原结合片段或本发明的核酸分子或本发明的生物材料或本发明的组合物在制备结合IL-23R蛋白的产品中的应用。在优选的实施方式中,所述IL-23R蛋白为表达于细胞膜表面的IL-23R蛋白。
在另一方面,提供一种阻断IL-23R与人IL-23α/IL-12β异源二聚体配体结合的方法,包括使用本发明的抗体或其抗原结合片段、或本发明的组合物。
在另一方面,提供本发明的抗体或其抗原结合片段或本发明的核酸分子或本发明的生物材料或本发明的组合物在制备治疗与自身免疫病相关的疾病的药物中的应用。在优选的实施方式中,所述疾病为炎症性肠炎包括克罗恩病、或溃疡性结肠炎、银屑病、银屑病关节炎、系统性红斑狼疮、类风湿性关节炎、强直性脊柱炎。
在另一方面,提供一种药物组合物,包括本发明抗体或其抗原结合片段以及另一种治疗剂。
在另一方面,提供一种联合用药方法,包括联合使用本发明抗体或其抗原结合片段以及另一种治疗剂。
所述另一种治疗剂包括但不限于其他抑制炎症的生物大分子药物如抗TNFα抗体、抗体IL-17抗体等。
本发明的抗IL-23R抗体是阻断性抗体,可阻断IL-23R与人IL-23α/IL-12β异源二聚体配体的结合,可以有效在HEK-Blue IL-23细胞(InvivoGen,hkb-il23)荧光素酶报告基因细胞株上抑制IL-23介导的STAT3的磷酸化激活,能够在过表达IL-23R/IL12β1的小鼠原B细胞株Baf3中(Baf3-IL23R/IL12Rβ)有效抑制STAT3磷酸化作用从而抑制IL-23介导的信号通路。本发明的抗IL-23R抗体有良好的应用潜质。
附图说明
图1显示了单克隆抗体与IL-23R蛋白的结合情况;
图2显示了单克隆抗体与293细胞膜表达的IL-23R的结合情况;
图3显示了单克隆抗体mAb#2C5、mAb5E2、mAb#7A12、mAb#5D4、mAb#9A10对IL-23与IL-23R结合的阻断情况;
图4显示了单克隆抗体mAb#2C5、mAb#7A12、mAb#5D4在HEK-Blue IL-23荧光素酶报告基因稳定细胞株中抑制IL-23介导的STAT3磷酸化激活的作用;
图5显示了单克隆抗体mAb#2C5、mAb#7A12、mAb#5D4在IL-23R/IL12β1过表达的Baf3稳定细胞株中抑制IL-23介导的信号通路激活作用。
具体实施方式
定义:
本发明中,术语“抗体”是指能够特异性识别抗原的免疫球蛋白,其涵盖多种抗体结构物,包括但不限于单克隆抗体、多克隆抗体、双特异抗体或抗体片段。
术语“可变区”指识别并特异性结合抗原表位的抗体重链或轻链的结构域。
CDR区或“互补决定区”指抗体可变区中在序列上高变并形成在结构上确定的环和/或含有抗原接触氨基酸残基的区域。
本发明的抗IL-23R抗体
本发明提供了对人IL-23R蛋白具有高亲和力的抗IL-23R抗体。抗体可有效抑制IL-23R与其配体IL-23的结合,从而阻断IL-23/IL-23R下游信号传导,从而实现从上游抑制炎症反应的效果。
如实施例所证明的,本发明提供的抗体具有极高的与IL-23R的亲和力以及对IL-23R/IL12Rβ1异源二聚体配体和IL-23结合的阻断作用。本发明的抗IL-23R抗体或其抗原结合片段包含取代、插入或缺失。本发明的抗IL-23R抗体包括对轻链可变区、重链可变区、轻链或重链的修饰,修饰后其氨基酸序列不同于衍生出该抗体的氨基酸序列。例如,衍生自同一指定蛋白质的氨基酸序列可以使与起始序列相似的,例如具有一定的百分比同一性,例如它可以与起始序列的百分比同一性是60%、65%、70%、75%、80%、85%、90%、95%、98%、99%。
在某些实施方案中,可在本文中所提供抗体的Fc区引入氨基酸修饰,所述氨基酸修饰可以是一个或多个,以此产生Fc变体。Fc变体可包含在一个或多个氨基酸位置处包含氨基酸修饰的人Fc区序列。
适用于本发明的“抗体及其抗原结合片段”包括但不限于单克隆、多克隆、单价、双特异性、多特异性、重组、异源、嵌合、人源化、去免疫原性的抗体,或Fab片段、Fab’片段、F(ab’)2片段、单链抗体、纳米抗体和上述任一种的表位结合片段。
在一些实施方案中,本发明的抗体可以是单特异性的、双特异性的或多特异性的。抗IL-23R抗体可以与另一种抗体或抗体片段链接以产生具有第二或者更多结合特异性的双特异性或多特异性抗体。
在某些实施方案中,抗体可经进一步修饰以添加功能性组分,适合抗体衍生作用的部分包括但不限于以下实例:PEG、葡聚糖、蛋白质、脂类、治疗剂或毒素。抗体可通过磷酸化、乙酰化、糖基化、聚乙二醇化、酰胺化或者其他蛋白链接等进行修饰。
抗体的治疗方法和用途
本发明提供的抗IL-23R抗体或其抗原片段可用于诊断、预后、治疗或抑制炎症。本发明涉及向有需要的受试者施用本发明的抗IL-23R抗体或其片段而提供治疗受试者自身炎症性疾病的方法。本发明的治疗性化合物包括但不限于本发明所述抗体(包括本发明所述的变体和衍生物)和编码本发明的所述抗体(包括本发明所述的变体和衍生物)的核酸和多核苷酸。
本发明所述抗IL-23R抗体可以与另外一种治疗剂联合使用,所述另外一种治疗剂包括但不限于其他抑制炎症的生物大分子药物。
本发明的抗体(以及任何另外的治疗剂)可以通过任何合适的方式给药,包括但不限于腹腔内、静脉内、皮下、鼻内、肌肉内注射。抗体及其变体或组合物可以通过任何方便的途径实施,例如通过推注或输注,通过上皮或皮肤黏膜的吸收。本发明示例下的抗IL-23R抗体序列
表1:本发明抗体的VH、VH-CDR1、VH-CDR2、VH-CDR3、VL、VL-CDR1、VL-CDR2、VL-CDR3的氨基酸序列编号
表2:本发明的抗体VH、VL DNA序列编号
抗体编号 | VH序列编号 | VL序列编号 |
#7A12 | 2 | 7 |
#2C5 | 12 | 17 |
#5D4 | 22 | 27 |
实施例
下面将结合具体实施例来说明本发明内容。如无明确说明,以下方法中使用的试剂和仪器都是本领域常用试剂和仪器,可以通过商购方式获得;所使用的方法都是本领域常规方法,本领域技术人员根据实施例所描述的内容可以毫无疑义地施行所述方法并获得相应的结果。
实施例1抗IL-23抗体鼠单抗的制备
用商业化人IL-23R-ECD-Fc蛋白(Sino Biological,货号H02H)50μg对6-8周龄的BALB/c小鼠(商购)进行初次免疫。首次免疫后的第14天和第28天,用25μg IL-23R-ECD-Fc蛋白对免疫后的小鼠进行再次免疫。采用酶联免疫吸附反应(Elisa)法检测免疫后小鼠血清效价。将IL-23R-His(ACROBiosystem,H52H4)抗原以PBS稀释到0.5μg/ml,包被微孔板4℃过夜。然后用1%BSA-PBS封闭液37℃封闭1h;PBST洗板后,将来自小鼠的血清稀释液加入板中并37℃孵育1hr。洗板加入HRP标记的山羊抗鼠IgG(Sigma,A0170)(1:10000),37℃反应0.5h后洗板加入TMB溶液,室温避光反应5分钟,加入2N H2SO4终止反应,置于酶标仪上450nm波长检测吸光度。在免疫后第42天,用25μg IL-23R-ECD-Fc蛋白对有足够效价的小鼠进行加强免疫,所得小鼠用于融合。准备骨髓瘤细胞SP2/0(ATCC)悬液,用血球计数板(Qiujing,XB-K-25)计算SP2/0细胞总量。制备融合小鼠的脾细胞悬液,用血球计数板计数算出细胞总量。取骨髓瘤细胞和脾细胞按照1:3比例混合,利用电融合仪(BTX,ECM2001)进行细胞融合。将HAT培养基加入到融合后的50ml离心管中,混匀制成细胞悬液。然后将细胞悬液倒入培养皿中充分混匀,用多道移液器(Thermo,F1)将细胞悬液按照5个克隆/孔,100μl/孔的量铺入准备好的50块96孔板中。将融合后的细胞板放入5.5%二氧化碳培养箱(84-1A),37℃恒温培养7-10天。
用ELISA实验检测在杂交瘤上清中的抗IL-23R抗体。多个不同的杂交瘤被鉴定出来并用于进一步分析。
实施例2抗体与人IL-23R的结合检测
采用Elisa方法检测纯化后抗体与IL-23R蛋白的结合能力,具体方法如实施例1所述,其中鼠源抗体的起始浓度为740pM,用PBS进行3倍梯度稀释共得到10个梯度。以复孔对各抗体进行试验,通过曲线拟合分析EC50。结果,确认到命名为mAb#5D4、mAb#5E2、mAb#7A12、mAb#2C5、mAb#9A10的抗体对人IL-23R的高结合活性(图1)。
采用流式细胞术(FCM)分析以上鼠源单抗与外源过表达IL-23R的293细胞293-IL23 Res(Novoprotein,XCC05)膜上IL-23R的结合情况。将细胞与不同浓度的单抗(最高浓度为11.11nM,3倍梯度稀释,共9个浓度点)于4℃反应30分钟。洗涤细胞2次后,加入FITC标记的山羊抗鼠IgG(Sigma,A0170))(1:1000),4℃避光反应30分钟。洗涤细胞2次后,用流式细胞仪BD C6进行检测。以复孔对各抗体进行试验,通过曲线拟合分析EC50。结果显示以上抗体对膜表面的IL-23R均具有良好的结合活性(图2)。
实施例3抗体对IL-23R与IL-23配体结合的阻断作用
进一步检测这些抗体是否可以阻断IL-23R与IL-23的结合。将IL-23R(ACROBiosystem,ILR-H5254)稀释至1μg/mL,以100μL/孔加至酶标板中,4℃包被过夜。洗板后每孔加入240μL 5%BSA-PBST 37℃封闭1h。用稀释液(1×PBST)稀释生物素标记的IL-23α&IL-12β异源二聚体蛋白(ACROBiosystem,ILB-H82W6)至0.2μg/mL;稀释各抗体样本至66.7nM,后进行3倍梯度稀释,共获得10个浓度点。每孔加入梯度稀释的各抗体样本50μL以及IL-23α&IL-12β50μL,37℃水平放置1h洗板3遍,将HRP-链霉亲和素(Abcam,Ab7403)稀释后每孔加入100μL,37℃水平放置1h后洗涤3遍。每孔加入100μL TMB显色液,水平室温避光放置25min后加入100μL2N H2SO4终止反应;立即用酶标仪在波长450nm处测定吸光度;应用分析软件对读数进行数据分析。以复孔对各抗体进行试验,通过曲线拟合分析IC50。结果显示mAb#2C5、mAb#5D4、mAb#7A12具有相对明显的阻断作用,结果见图3。
实施例4抗体阻断IL-23信号通路激活介导的STAT3磷酸化作用
根据实施例3的结果,选择能够明显阻断IL-23R与IL-23结合的mAb#2C5、mAb#5D4、mAb#7A12克隆进行STAT3磷酸化检测。将HEK-Blue IL-23细胞(InvivoGen,hkb-il23)收集后用实验培养基(DMEM,10%热灭活FBS,100U/mL青霉素,100μg/mL链霉素,100μg/mLNormocin)洗涤细胞2次,调整细胞密度至5×105个/mL;用实验培养基稀释HIS标签的IL-23α&IL-12β异源二聚体蛋白(ACROBiosystem,ILB-H52W5)至2ng/mL;mAb#2C5、mAb#5D4、mAb#7A12样品稀释至2666.67nM,再进行4倍梯度稀释,共获得9个浓度点。在96孔板中,每孔加入100μL细胞悬液,50μL各梯度稀释的抗体样品,置于37℃、5% CO2培养箱中孵育30min后每孔加入50μLIL-23α&IL-12β,置于37℃、5% CO2培养箱中培养24h。从每个测试孔中取20μLHEK-BlueTM IL-23细胞培养上清液转移至96孔板中,每孔加入180μLQUANTI-BlueTM溶液(InvivoGen,rep-qbs),在37℃下孵育2h。用酶标仪在波长630nm处测定吸光度并应用分析软件对读数进行数据分析,以复孔对各抗体进行试验,通过曲线拟合分析IC50。结果显示mAb#2C5、mAb#5D4、mAb#7A12均具有明显的阻断效果(图4)。
实施例5抗体在过表达人IL-23R/IL12Rβ1的小鼠原B细胞株Baf3(Baf3-IL-23R/IL12Rβ1)中抑制STAT5磷酸化作用
采用慢病毒的转染的方式构建Baf3-IL-23R/IL12Rβ1过表达细胞。取状态良好、处于对数期的293细胞(ATCC)分别进行IL-23R和IL12Rβ1慢病毒包装。首先第一天铺400万状态良好的293细胞于10厘米培养皿中,共铺两皿。次日将载有IL-23R的pLV-EF1a-IRES-Blastcidin(该质粒由中南大学湘雅医院肿瘤医学中心赠送)的质粒与PxpAx2(Addgene,#85132)、pCMV-VSV-G(Addgene,#8454)以1:4:3比例,总量为20μg添加到总体积为500μL的DMEM无血清培养基中,作为A液,震荡混匀后静置5分钟。类似地,将载有IL-12Rβ1的pLV-EF1a-IRES-puro质粒(Addgene,#85132)与PxpAx2(Addgene,#12259)、pCMV-VSV-G(Addgene,#8454)以1:4:3比例,总量为20μg添加到总体积为500μL的DMEM无血清培养基中,作为B液,震荡混匀静置5分钟。另外取120μl的Lipo2000加入到总体积1ml的DMEM无血清培养基中,作为C液,轻轻震荡混匀后静置5分钟。5分钟后分别将A液与500μl C液、B液与500μlC液混合,轻轻震荡混匀并静置20-30分钟后分别加入准备好的293细胞平皿中进行病毒组装。次日,将含有转染试剂的293上清更换为10ml DMEM完全培养基并继续培养,收集转染后72hrs的含有病毒的上清备用。当Baf3细胞(酶研生物,CC-Y2104)处于对数期且细胞密度控制在40%左右同时感染带有杀稻瘟菌素(Blastcidin)筛选标记的IL-23R慢病毒上清和带有嘌呤霉素(puromysine)筛选标记的IL12Rβ1慢病毒上清。转染8小时后更换为完全培养基进行培养。感染3-4天后加入1.5μg/ml的嘌呤霉素和15μg/ml的杀稻瘟菌素进行加压筛选获得IL-23R/IL12Rβ1过表达的Baf3。
利用IL-23R/IL12Rβ1过表达的小鼠原B细胞进一步检测候选分子对信号通路的阻断效果。在本实验中,mAb#2C5、mAb#7A12、mAb#5D4以2666.67nM为起始浓度,4倍梯度稀释,共8个浓度点;收集Baf3-IL-23R/IL12Rβ1细胞,用实验培养基(1640+10%FBS)洗涤细胞3次,调整细胞密度至5×106个/mL,40μL/孔接种于96孔板中。每孔加入梯度稀释好的样品20μL,置于37℃、5%CO2培养箱中孵育30min。后每孔加入20μL 40ng/ml的IL-23(ACROBiosystem,ILB-H52W5),置于37℃、5% CO2培养箱中培养4h。按照InstantOneELISAphospho-STAT3(Tyr705)试剂盒(Invitrogen,85-86102-11)说明书步骤,每孔添加20μL细胞裂解混合液(5×),室温300rpm振荡10min。然后取50μL/孔细胞裂解液转移至酶标板中,向每个测试孔中添加50μL试剂盒中的检测抗体,室温300rpm振荡1h。用洗液(1×)以200μL/孔洗板3次后向每个测定孔中添加100μL TMB显色液,室温避光300rpm振荡30min后加入100μL终止液。用酶标仪在波长450nm处测定吸光度并应用分析软件对读数进行数据分析。结果显示三个候选分子均表现出显著的信号通路激活抑制作用,结果如图5所示。
实施例6测序分析
对抗体mAb#2C5、mAb#7A12、mAb#5D4进行测序分析,所得序列信息分别如下所示:
SEQ ID NO:1#7A12 VH氨基酸
QVSLKESGPGMLQPSQTLSLTCSFSGFSLTTFDMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPALKSRLTISKDTSKNQVFLKIANVDTADTATYYCARLQGSDFDYWGQGTTLIVSS
SEQ ID NO:2#7A12 VH DNA
CAGGTTAGTCTGAAAGAGTCTGGCCCTGGAATGTTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTTTCTGGGTTTTCACTGACCACTTTTGATATGGGTGTAGGCTGGATTCGTCAGCCTTCAGGGAAGGGACTGGAGTGGCTGGCACACATTTGGTGGGATGATGATAAATACTATAATCCGGCCCTGAAGAGTCGGCTCACAATCTCCAAGGATACCTCCAAAAACCAGGTATTCCTCAAGATCGCCAATGTGGACACTGCAGATACTGCCACATACTACTGTGCTCGATTACAGGGTTCGGACTTTGACTACTGGGGCCAAGGCACCACTCTCATAGTCTCCTCA
SEQ ID NO:3#7A12 VH-CDR1
TFDMGVG
SEQ ID NO:4#7A12 VH-CDR2
HIWWDDDKYYNPALKS
SEQ ID NO:5#7A12 VH-CDR3
LQGSDFDY
SEQ ID NO:6#7A12 VL
QLVLTQSSSASFSLGASAKLTCTLSSQHSTYTIEWYQQQPLKAPKYVMEVKKDGSHSTGDGIPDRFSGSSFGADRYLSISNIQPEDEAIYICGVGDTIKEQFLYVFGGGTKVTVL
SEQ ID NO:7#7A12 VL DNA
CAACTTGTGCTCACTCAATCATCTTCAGCCTCTTTCTCCCTGGGAGCCTCAGCAAAACTCACGTGCACCTTGAGTAGTCAGCACAGTACGTACACCATTGAATGGTATCAGCAACAGCCACTCAAGGCTCCTAAGTATGTGATGGAGGTTAAGAAAGATGGAAGCCACAGTACAGGTGATGGGATTCCTGATCGCTTCTCTGGATCCAGCTTTGGTGCTGATCGCTACCTTAGCATTTCCAACATCCAGCCTGAAGATGAAGCAATATACATCTGTGGTGTGGGTGATACAATTAAGGAACAATTTTTGTATGTTTTCGGCGGTGGAACCAAGGTCACTGTCCTA
SEQ ID NO:8#7A12 VL-CDR1
TLSSQHSTYTIE
SEQ ID NO:9#7A12 VL-CDR2
VKKDGSHSTGD
SEQ ID NO:10#7A12 VL-CDR3
GVGDTIKEQFLYV
SEQ ID NO:11#2C5 VH氨基酸
QVTLKESGPGILQPSQTLSLTCSFSGFSLTTFNVGIGWIRQPSGKGLEWLAHIWWDDD KYYNPALKTRLAISRDTSKNQVFLKIANVDTADTATYYCSRLEGNNFDYWGQGTTLTVSSSEQ ID NO:12#2C5 VH DNA
CAGGTTACTCTGAAGGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCCTTCTCTGGGTTTTCGCTTACCACTTTTAATGTGGGAATAGGCTGGATTCGTCAGCCTTCAGGGAAGGGTCTGGAGTGGCTGGCACACATTTGGTGGGATGATGATAAGTACTATAATCCAGCCCTGAAGACTCGGCTCGCTATCTCCAGGGATACCTCCAAAAACCAGGTTTTCCTCAAGATCGCCAATGTGGACACTGCAGATACTGCCACATACTACTGTTCTAGATTAGAGGGAAATAACTTCGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA
SEQ ID NO:13#2C5 VH-CDR1
TFNVGIG
SEQ ID NO:14#2C5 VH-CDR2
HIWWDDDKYYNPALKT
SEQ ID NO:15#2C5 VH-CDR3
LEGNNFDY
SEQ ID NO:16#2C5 VL氨基酸
QLVLTQSSSASFSLGASATLTCTLSSQHSTYTIEWYQQQPLKPPKYVMEIKKDGSHNT GDGIPDRFSGSSSGADRYLSISNIQPEDEAIYICGVGDTITEQFVYVFGGGTKVTVL
SEQ ID NO:17#2C5 VL DNA
CAACTTGTGCTCACTCAGTCATCTTCAGCCTCTTTCTCCCTGGGAGCCTCAGCAACACTCACGTGCACCTTGAGTAGTCAGCACAGTACATACACCATTGAATGGTATCAGCAACAGCCACTCAAGCCTCCTAAGTATGTGATGGAGATTAAGAAAGATGGAAGCCACAACACAGGTGATGGGATTCCTGATCGCTTCTCTGGATCCAGCTCTGGTGCTGATCGCTACCTTAGCATTTCCAACATCCAGCCTGAAGATGAAGCAATTTACATCTGTGGTGTGGGTGATACAATTACGGAACAATTTGTGTATGTTTTCGGCGGTGGAACCAAGGTCACTGTCCTA
SEQ ID NO:18#2C5 VL-CDR1
TLSSQHSTYTIE
SEQ ID NO:19#2C5 VL-CDR2
IKKDGSHNTGD
SEQ ID NO:20#2C5 CDR3
GVGDTITEQFVYV
SEQ ID NO:21#5D4 VH氨基酸
QVQLQQPGAELVMPGASVKLSCKASGYIFSSYWMHWVKQRPGQGLEWIGEIDPSDSYTNYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARSLYANDLLDNWGQGTTLTVSS
SEQ ID NO:22#5D4 VH DNA
CAGGTCCAACTGCAGCAGCCTGGGGCTGAACTTGTGATGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTATATTTTCTCCAGTTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAGAGATTGATCCTTCTGATAGTTATACTAACTACAATCAAAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGTCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCTCTCTATGCTAACGACCTCCTTGACAACTGGGGCCAAGGGACCACTCTCACAGTCTCCTCA
SEQ ID NO:23#5D4 VH-CDR1
SYWMH
SEQ ID NO:24#5D4 VH-CDR2
EIDPSDSYTNYNQKFKG
SEQ ID NO:25#5D4 VH-CDR3
SLYANDLLDN
SEQ ID NO:26#5D4 VL氨基酸
DILMTQSPASMSIPLGDTVSITCHASQGITSNIGWLQQKPGKSFKGLIYHGTNLEDGV PSRFSGSGSGADYSLTISSLESEDFADYYCVQYDQFPFTFGSGTKLEIK
SEQ ID NO:27#5D4 VL DNA
GACATCCTGATGACCCAATCTCCGGCCTCCATGTCTATACCTCTGGGAGACACAGTCAGCATCACTTGCCATGCAAGTCAGGGCATTACCAGTAATATAGGGTGGTTGCAGCAGAAACCAGGGAAATCATTTAAGGGCCTGATTTATCATGGAACCAACTTGGAAGATGGAGTTCCATCAAGGTTCAGTGGCAGTGGATCTGGAGCAGATTATTCTCTCACCATCAGCAGCCTGGAATCTGAAGATTTTGCAGACTATTACTGTGTACAGTATGATCAGTTTCCATTCACGTTCGGCTCGGGGACAAAGCTGGAAATAAAA
SEQ ID NO:28#5D4 VL-CDR1
HASQGITSNIG
SEQ ID NO:29#5D4 VL-CDR2
HGTNLED
SEQ ID NO:30#5D4 CDR3
VQYDQFPFT
SEQ ID NO:34#7A12重链全长
MGRLTSSFLLLIVPAYVLSQVSLKESGPGMLQPSQTLSLTCSFSGFSLTTFDMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPALKSRLTISKDTSKNQVFLKIANVDTADTATYYCARLQGSDFDYWGQGTTLIVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK
SEQ ID NO:31#7A12轻链全长
MAWTPLFFFFVLHCSGSFSQLVLTQSSSASFSLGASAKLTCTLSSQHSTYTIEWYQQQPLKAPKYVMEVKKDGSHSTGDGIPDRFSGSSFGADRYLSISNIQPEDEAIYICGVGDTIKEQFLYVFGGGTKVTVLRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC
SEQ ID NO:35#2C5重链全长
MGRLTSSFLLLIVPAYVLSQVTLKESGPGILQPSQTLSLTCSFSGFSLTTFNVGIGWIRQPSGKGLEWLAHIWWDDDKYYNPALKTRLAISRDTSKNQVFLKIANVDTADTATYYCSRLEGNNFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK
SEQ ID NO:32#2C5轻链全长
MAWTPLFFFFVLHCSGSFSQLVLTQSSSASFSLGASATLTCTLSSQHSTYTIEWYQQQPLKPPKYVMEIKKDGSHNTGDGIPDRFSGSSSGADRYLSISNIQPEDEAIYICGVGDTITEQFVYVFGGGTKVTVLRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC
SEQ ID NO:36#5D4重链全长
MGWSCFILFLVSTATGVHSQVQLQQPGAELVMPGASVKLSCKASGYIFSSYWMHWVKQRPGQGLEWIGEIDPSDSYTNYNQKFKGKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARSLYANDLLDNWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:33#5D4轻链全长
MVLAQFLAFLLLWFPGARCDILMTQSPASMSIPLGDTVSITCHASQGITSNIGWLQQKPGKSFKGLIYHGTNLEDGVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYDQFPFTFGSGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC
Claims (10)
1.一种抗IL-23R抗体或其抗原结合片段,所述抗体或其抗原结合片段包括重链可变区和轻链可变区,其中,重链可变区包括VH CDR1、VH CDR2和VH CDR3,轻链可变区包括VLCDR1、VL CDR2和VL CDR3,
所述抗体或其抗原结合片段的重链可变区的VH CDR1、VH CDR2和VH CDR3的氨基酸序列分别如SEQ ID NO:3、4和5所示,轻链可变区的VL CDR1、VL CDR2和VL CDR3的氨基酸序列分别如SEQ ID NO:8、9和10所示;或者,
所述抗体或其抗原结合片段的重链可变区的VH CDR1、VH CDR2和VH CDR3的氨基酸序列分别如SEQ ID NO:13、14和15所示,轻链可变区的VL CDR1、VL CDR2和VL CDR3的氨基酸序列分别如SEQ ID NO:18、19和20所示。
2.根据权利要求1所述的抗IL-23R抗体或其抗原结合片段,其中,所述重链可变区的氨基酸序列如SEQ ID NO:1或11所示,或与SEQ ID NO:1或11具有至少90%、至少95%、至少96%、至少97%、至少98%或至少99%序列同一性;所述轻链可变区的氨基酸序列如SEQ IDNO:6或16所示,或与SEQ ID NO:6或16具有至少90%、至少95%、至少96%、至少97%、至少98%或至少99%序列同一性。
3.根据权利要求1或2所述的抗IL-23R抗体或其抗原结合片段,其中,所述抗体或其抗原结合片段进一步包含重链恒定区和轻链恒定区;优选地,所述轻链恒定区是κ链恒定区;进一步优选地,所述重链恒定区是IgG1的重链恒定区;更优选地,所述抗体或其抗原结合片段包括重链和轻链,所述重链包含SEQ ID NO:34或35所示氨基酸序列或由其组成,所述轻链包含SEQ ID NO:31或32所示氨基酸序列或由其组成。
4.核酸分子,编码如权利要求1~3任一项所述的抗IL-23R抗体或其抗原结合片段;优选地,所述核酸分子编码所述抗体或其抗原结合片段的重链可变区和/或轻链可变区;进一步优选地,所述核酸分子编码重链可变区,其核苷酸序列如SEQ ID NO:2或12所示,或与SEQID NO:2或12具有至少90%、至少95%、至少96%、至少97%、至少98%或至少99%序列同一性;和/或,所述核酸分子编码轻链可变区,其核苷酸序列如SEQ ID NO:7或17所示,或与SEQID NO:7或17具有至少90%、至少95%、至少96%、至少97%、至少98%或至少99%序列同一性。
5.一种生物材料,为:
(1)包含如权利要求4所述的核酸分子的载体、宿主细胞或微生物等;或
(2)上述(1)的表达产物、悬浮液或上清液。
6.一种组合物,其包含如权利要求1~3任一项所述的抗IL-23R抗体或其抗原结合片段;优选地,所述组合物为药物组合物,进一步包含药学上可接受的载体。
7.制备如权利要求1~3任一项所述的抗IL-23R抗体或其抗原结合片段的方法,包括:培养权利要求5所述的宿主细胞使抗体或其抗原结合片段表达,及自宿主细胞中分离所述抗体或其抗原结合片段。
8.根据权利要求1~3任一项所述的抗IL-23R抗体或其抗原结合片段或权利要求4所述的核酸分子或权利要求5所述的生物材料或权利要求6所述的组合物在制备结合IL-23R蛋白的产品中的应用;优选地,所述IL-23R蛋白为表达于细胞膜表面的IL-23R蛋白。
9.根据权利要求1~3任一项所述的抗IL-23R抗体或其抗原结合片段或权利要求4所述的核酸分子或权利要求5所述的生物材料或权利要求6所述的组合物在制备治疗与自身免疫病相关的疾病的药物中的应用;优选地,所述与自身免疫病相关的疾病为炎症性肠炎包括克罗恩病、溃疡性结肠炎、银屑病、银屑病关节炎、系统性红斑狼疮、类风湿性关节炎或强直性脊柱炎。
10.一种药物组合物,包含如权利要求1~3任一项所述的抗IL-23R抗体或其抗原结合片段以及另一种治疗剂;优选地,所述另一种治疗剂为抑制炎症的生物大分子药物;更优选地,所述另一种治疗剂为抗TNFα抗体或抗体IL-17抗体。
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