CN114502591B - 靶向bcma的抗体、双特异性抗体及其用途 - Google Patents
靶向bcma的抗体、双特异性抗体及其用途 Download PDFInfo
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- C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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Abstract
提供了一种靶向BCMA的抗体、靶向BCMA和CD3的双特异性抗体及其应用。所述靶向BCMA的抗体包括两个重链可变区,所述重链可变区包含分别如SEQ ID NO:22、SEQ ID NO:33、SEQ ID NO:42的氨基酸序列所示的HCDR1、HCDR2、HCDR3;或如SEQ ID NO:26、SEQ ID NO:37、SEQ ID NO:41的氨基酸序列所示的HCDR1、HCDR2、HCDR3。该抗体有效地靶向人BCMA并结合食蟹猴BCMA;双特异性抗体有效地同时靶向人BCMA和人CD3、并能结合食蟹猴BCMA和食蟹猴CD3。
Description
本申请要求申请日为2019/9/30的中国专利申请2019109413110的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明涉及生物制药领域,尤其涉及一种靶向BCMA的抗体、双特异性抗体及其用途。
背景技术
BCMA(B-细胞成熟抗原,TNFRSF17,CD269)是一种属于TNF受体超家族的跨膜蛋白。BCMA是一种非糖基化跨膜蛋白,其参与B细胞成熟、生长和存活。BCMA是TNF超家族的两种配体的受体:高亲和力配体APRIL(增殖诱导配体)以及低亲和力配体BAFF(B细胞活化因子)。BCMA是一种高度分化的浆细胞选择性蛋白,其表达限于B-细胞谱系且主要存在于浆细胞和浆母细胞上,并在一定程度上存在于记忆B-细胞上,但是不存在于外周B-细胞上。BCMA在多发性骨髓瘤(MM)患者的恶性浆细胞中表达,支持多发性骨髓瘤细胞的生长和存活。
多发性骨髓瘤是继非霍奇金淋巴瘤的血液系统第二大恶性肿瘤,约占所有恶性肿瘤的1%,占血液系统恶性肿瘤的13%,占因恶性肿瘤死亡的2%。通常骨髓瘤细胞在骨髓内及骨骼海绵软组织内克隆性增生,引起溶骨性骨骼破坏,愈后不良多伴有贫血、肾衰竭和骨髓瘤细胞髓外浸润所导致的多种损害。截止2006年,MM的5年相对生产率约为34%,目前用于MM的所有疗法是非治愈性的。作为多发性骨髓瘤的一个新兴靶点,BCMA抗体可以通过多种机制作用于MM细胞,BCMA的研发方向主要集中在单克隆抗体、CAR-T疗法、ADC以及双特异性抗体疗法上。
现有技术抗体1(下文又称阳性对照1,在实施例中编号为PR000274)为葛兰素史克(GSK)公司的抗BCMA抗体CA8-J6M0 hIgG1,目前GSK基于抗BCMA抗体CA8-J6M0制备的抗体偶联药物belantamab mafodotin(CA8-J6M0-mcMMAF,GSK2857916)在多项临床试验中治疗不同类型的MM患者。此对照抗体的序列来源于(Tabs-Therapeutic Antibody Database)。来自一项小型临床研究的数据显示,在35例过度预治疗(大多数患者至少接受了5种疗法治疗失败)复发性或难治性(R/R)多发性骨髓瘤(MM)患者中,客观缓解率(0RR)达到了60%,中位无进展生存期(PFS)为12个月。安全性方面,最常见的副作用包括角膜事件、血小板减少和贫血,这些都与ADC中偶联的细胞毒性制剂有关。
目前,处于临床开发阶段的双特异性抗体有安进的AMG-420、再生元的REGN-5458、新基的CC-93269、强生JNJ-64007957、艾伯维的TNB383B。然而这些双特异性抗体还存在半衰期短、细胞因子释放综合征(CRS)等问题。现有技术抗体2(下文称阳性对照2,在实施例中编号为PR002199)来源于Teneobio公司的专利WO2018052503的抗BCMA(TNB308902)×CD3(TNB_F2B)双抗。Teneobio的TNB383B双特异性抗体于2019年进入临床一期用于MM。Teneobio的TNB383B采用了弱化的CD3抗体,具有减弱细胞因子释放的特点。但是TNB383B不结合食蟹猴的CD3和食蟹猴的BCMA,不能在食蟹猴进行毒理学评估。
因此,急需研发更加安全有效的靶向BCMA并能结合食蟹猴的BCMA的单克隆抗体,以及更加安全有效的同时靶向人的BCMA和CD3、并能结合食蟹猴的BCMA和CD3的双特异性抗体。
发明内容
为解决现有技术中缺乏安全有效的靶向人的BCMA并能结合食蟹猴的BCMA的单克隆抗体,以及同时靶向人的BCMA和CD3、并能结合食蟹猴的BCMA和CD3的双特异性抗体的缺陷的技术问题,本发明提供一种靶向BCMA的抗体、靶向BCMA和CD3的双特异性抗体及其应用。
为解决上述技术问题,本发明的第一方面的技术方案为:一种靶向BCMA的抗体,其包括两个重链可变区,所述重链可变区包含分别如SEQ ID NO:22、SEQ ID NO:33和SEQ IDNO:42的氨基酸序列所示的HCDR1、HCDR2和HCDR3;或,如SEQ ID NO:26、SEQ ID NO:37和SEQID NO:41的氨基酸序列所示的HCDR1、HCDR2和HCDR3。本发明的BCMA抗体具有与人BMCA和食蟹猴BCMA结合的活性。大小只有传统IgG抗体的一半,可以用于双特异性抗体的开发,并解决轻链错配和异源二聚化的问题。
在一较佳的具体实施例中,所述重链可变区的氨基酸序列如SEQ ID NO:59、SEQID NO:60所示或其突变;所述突变为在原氨基酸序列上的一个或多个氨基酸的添加、取代或删除,并保持或改善了所述第一蛋白功能区与所述BCMA抗原的结合。优选地,所述突变与如SEQ ID NO:59、SEQ ID NO:60所示的氨基酸序列有80%、85%、90%、95%、96%、97%、98%、99%或以上的序列同一性。
在一较佳的具体实施例中,所述靶向BCMA的抗体还包括Fc片段。由此,所述靶向BCMA的抗体包括两条重链。较佳地,所述Fc片段为hIgG1、hIgG2、hIgG3、hIgG4的Fc片段或其突变。
在一较佳的具体实施例中,所述重链的氨基酸序列如SEQ ID NO:1或SEQ ID NO:7或SEQ ID NO:8所示。
为解决上述技术问题,本发明的第二方面的技术方案为:提供一种双特异性抗体,其包括靶向BCMA的第一蛋白功能区和靶向CD3的第二蛋白功能区,其中,所述第一蛋白功能区包括如本发明第一方面所述的靶向BCMA的抗体的两个重链可变区;较佳地,所述两个重链可变区之间以(G4S)n连接,所述n为非0自然数,优选1~20更优选3或4。本发明的BCMA×CD3双特异性抗体是一种特有的BCMA×CD3双特异性抗体,具有两个与BCMA结合的位点,从而提高了与肿瘤细胞BCMA结合的亲和力和特异性;双特异性抗体的两个蛋白功能区均具有良好的结合食蟹猴的活性。
在一较佳的具体实施例中,所述第二蛋白功能区包括含HCDR1、HCDR2和HCDR3的重链可变区和含LCDR1、LCDR2和LCDR3的轻链可变区,所述HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ ID NO:25、SEQ ID NO:38和SEQ ID NO:44所示,和/或,所述LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO:50、SEQ ID NO:53和SEQ ID NO:56所示。
较佳地,所述第二蛋白功能区的重链可变区的氨基酸序列如SEQ ID NO:63、SEQID NO:62所示或为其突变,轻链可变区的氨基酸序列如SEQ ID NO:66所示或为其突变,所述突变保持或改善了所述第二蛋白功能区与CD3的结合。本发明的双特异性抗体优化了CD3端的活性,减少细胞因子的释放,降低BCMA×CD3双特异性抗体的毒性。
在一较佳的具体实施例中,所述双特异性抗体还包括重链恒定区和轻链恒定区,优选为人重链恒定区和人轻链恒定区,所述人轻链恒定区优选人λ、κ轻链恒定区,所述人重链恒定区优选hIgG1、hIgG2、hIgG3、hIgG4的重链恒定区或其突变;
较佳地,所述人重链恒定区的突变选自以下组:
(1)L234A/L235A突变;
(2)knob突变T366W和hole突变T366S、L368A和Y407V;
(3)knob突变S354C和Hole突变Y349C;
更佳地,所述第一蛋白功能区包括氨基酸序列如SEQ ID NO:8或SEQ ID NO:7所示的重链,所述第二蛋白功能区包括氨基酸序列如SEQ ID NO:5或SEQ ID NO:4所示的重链和氨基酸序列如SEQ ID NO:47所示的轻链。本发明的双特异性抗体带有人Fc片段,保留了与FcRn的结合作用,从而具有较长的半衰期。
为解决上述技术问题,本发明的第三方面的技术方案为:提供一种编码根据如本发明第一方面所述的靶向BCMA的抗体或如本发明第二方面所述的双特异性抗体的分离的核酸。
为解决上述技术问题,本发明的第四方面的技术方案为:提供一种包含根据如本发明第三方面所述的分离的核酸的表达载体。
为解决上述技术问题,本发明的第五方面的技术方案为:提供一种宿主细胞,其包含如本发明第四方面所述的表达载体;优选地,所述宿主细胞是原核细胞或真核细胞。
为解决上述技术问题,本发明的第六方面的技术方案为:提供一种抗体的药物偶联物,其包含如本发明第一方面所述的靶向BCMA的抗体或如本发明第二方面所述的双特异性抗体,以及细胞毒性剂。
为解决上述技术问题,本发明的第七方面的技术方案为:提供一种药物组合物,其包含如本发明第一方面所述的靶向BCMA的抗体、如本发明第二方面所述的双特异性抗体或如本发明第六方面所述的抗体的药物偶联物,以及药学上可接受的载体。
为解决上述技术问题,本发明的第八方面的技术方案为:提供如本发明第一方面所述的靶向BCMA的抗体、本发明第二方面所述的双特异性抗体、本发明第六方面所述的抗体的药物偶联物或本发明第七方面所述的药物组合物在制备治疗和/或预防癌症的药物中的应用。
此外,为解决上述技术问题,本发明第八方面的技术方案为:提供一种药盒组合,其包括药盒A和药盒B;所述药盒A包含本发明第一方面所述的靶向BCMA的抗体、第二方面所述的双特异性抗体、第五方面所述的宿主细胞、第六方面所述的抗体药物偶联物或第七方面所述的药物组合物;所述药盒B包含其它抗体、双特异性抗体、宿主细胞或药物组合物,所述其它抗体、双特异性抗体、宿主细胞或药物组合物靶向CD3、BCMA或其它靶点。所述药盒A和药盒B的使用不分先后顺序,或先使用药盒A再使用药盒B,或先使用药盒B再使用药盒A。
本发明的第一方面所述的靶向BCMA的抗体、第二方面所述的双特异性抗体、第五方面所述的宿主细胞、第六方面所述的抗体药物偶联物或第七方面所述的药物组合物可施用于病人,用于治疗相关肿瘤。
在符合本领域常识的基础上,上述各优选条件,可任意组合,即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于:
1、本发明的BCMA抗体是一种全新的仅含“重链”的全人抗体,具有与人BMCA和食蟹猴BCMA结合的活性。该BCMA重链抗体的大小只有传统IgG抗体的一半,由于不含轻链的这一特点,使得该抗体可以用于双特异性抗体,并解决了轻链错配和异源二聚化的问题。
2、本发明的BCMA×CD3双特异性抗体:a.是一种特有的BCMA×CD3双特异性抗体,具有两个与BCMA结合的位点,从而提高了与肿瘤细胞BCMA结合的亲和力和特异性。b是一种带有人Fc片段的双特异性抗体结构,保留了Fc与FcRn的结合作用,从而具有较长的半衰期。c.优化了CD3端的活性,减少细胞因子的释放,降低BCMA×CD3双特异性抗体的毒性。d.BCMA端和CD3端抗体具有良好的结合食蟹猴的活性。
附图说明
图1为FACS检测抗人BCMA的HCAb单抗细胞水平的结合能力;
图2为ELISA检测抗人BCMA的HCAb单抗对人BCMA与配体BAFF蛋白结合的阻断;
图3为FACS检测BCMA×CD3双特异性抗体在高表达BCMA的NCI-H929细胞系上的体外结合;
图4为FACS检测BCMA×CD3双特异性抗体在过表达人和猕猴BCMA的HEK293T细胞株上的体外结合;
图5为FACS检测BCMA×CD3双体异性抗体与人和猕猴T细胞体外结合;
图6为BCMA×CD3双特异性抗体对BCMA高表达细胞系NCI-H929的体外杀伤实验以及细胞因子的释放;
图7为BCMA×CD3双特异性抗体对BCMA低表达细胞系RPIM8226的体外杀伤实验以及细胞因子的释放;
图8为FACS检测BCMA×CD3双体异性抗体在BCMA阴性细胞系HL-60上的结合;
图9为FACS方法检测BCMA×CD3双特异性抗体对BCMA阴性细胞系HL-60的非特异杀伤及细胞因子的释放;
图10为NCI-H929/人PBMC小鼠模型中BCMA×CD3双特异性抗体的抗肿瘤药效评估;
图11为BIACORE检测PR003178与人CD3e/g重组蛋白、食蟹猴CD3e/g重组蛋白及人BCMA重组蛋白、食蟹猴BCMA重组蛋白的亲和力。
具体实施方式
下面通过实施例的方式进一步说明本发明,但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
在本申请中,术语“抗体”通常是指包含结合抗原的部分的蛋白质,以及任选地允许结合抗原的部分采用促进抗体与抗原结合的构象的支架或骨架部分。可典型地包含抗体轻链可变区(VL)、抗体重链可变区(VH)或上述两者。例如本申请中的“重链抗体”不含VL区,仅含VH区。VH或VL区可进一步被区分为称为互补决定区(CDR)的高变区,它们散布在称为框架区(FR)的更保守的区域中。每个VH或VL可由三个CDR和四个FR区构成,它们从氨基端至羧基端可按以下顺序排列:FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的实例包括但不限于全长抗体、重链抗体(HCAb)、抗原结合片段(Fab,Fab’、F(ab)2、Fv片段、F(ab’)2、scFv、di-scFv和/或dAb)、免疫缀合物、多特异性抗体(例如双特异性抗体)、抗体片段、抗体衍生物、抗体类似物或融合蛋白等,只要它们显示出所需的抗原结合活性即可。
在本申请中,术语“可变”通常是指这样的事实,即抗体的可变结构域的序列的某些部分变化强烈,它形成各种特定抗体对其特定抗原的结合和特异性。然而,变异性并非均匀地分布在抗体的整个可变区中。它集中在轻链和重链可变区中的三个区段,称为CDR或高变区(HVR),FR为可变域中更高度保守的部分。天然重链和轻链的可变结构域各自包含四个FR区,大部分采用β-折叠构型,通过三个CDRs连接,形成环连接,并且在一些情况下形成β-折叠结构的一部分。每条链中的CDRs通过FR区紧密靠近在一起,并与来自另一条链的CDR一起形成抗体的抗原结合位点,恒定区不直接参与抗体与抗原的结合,但是它们表现出不同的效应功能,例如参与抗体的依赖于抗体的细胞毒性。
在本领域中,可以通过多种方法来定义抗体的CDR,例如基于序列可变性的Kabat定义规则(参见,Kabat等人,免疫学的蛋白质序列,第五版,美国国立卫生研究院,贝塞斯达,马里兰州(1991))和基于结构环区域位置的Chothia定义规则(参见,A1-Lazikani等人,JMol Biol 273:927-48,1997)。在本申请中,还使用包含了Kabat定义和Chothia定义的Combined定义规则确定可变结构域序列和全长抗体序列中的氨基酸残基(表1)。
表1本申请抗体CDR定义方法(可参见http://bioinf.org.uk/abs/)
其中,Laa-Lbb可以指从抗体轻链的N端开始,第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列;Haa-Hbb可以指从抗体重链的N端开始,第aa位(Chothia编码规则)至第bb位(Chothia编码规则)的氨基酸序列。例如,L24-L34可以指从抗体轻链N端开始,按照Chothia编码规则的从第24位至第34位的氨基酸序列;H26-H32可以指从抗体重链N端开始,按照Chothia编码规则的从第26位至第32位的氨基酸序列。
抗体Fc结构域介导的效应子功能如ADCC和CDC也有非常重要的生物学功能,不同的IgG亚型有着不同的ADCC或CDC功能,例如IgG1和IgG3有较强的ADCC和CDC作用,而IgG2和IgG4的作用相对较弱。另外,通过氨基酸突变或者修饰来改变Fc与Fc受体的结合能力也可以调节Fc原有的效应子功能。例如,IgG1中的“LALA”双突变体(L234A/L235A)能够显著降低与FcγRIIIA(CD16A)的亲和力,进而降低ADCC作用。另外,P329G突变能够显著降低与多种Fcγ受体的结合(参见,Schlothauer T,Herter S,Koller CEGrau-Richards S,SteinhartV,Spick C,Kubbies M,Klein C,P,/>E.Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effectorfunctions.Protein EngDes Sel.2016 Oct;29(10):457-466.doi:10.1093/protein/gzw040.Epub 2016 Aug 29.PubMed PMID:27578889)。在申请中,为了减少BCMA×CD3双特异性抗体与Fcγ受体的结合,这些抗体的Fc引入了“LALA”双突变体(L234A/L235A)或者“LALAPG”三突变体(L234A/L235A/P329G)。
实施例1.小鼠免疫和抗BCMA抗体分子的获得
可以利用BCMA抗原对实验动物进行免疫以获得针对BCMA特异性结合的抗体分子,该实验动物可以是小鼠、大鼠、兔、羊、骆驼等。通常,其得到的抗体分子是非人源的。在获得非人源抗体后,需要对这些分子利用抗体工程技术进行人源化改造,以降低免疫原性并提高成药性。然而,抗体的人源化过程有其技术复杂性,经过人源化改造的分子往往会降低对抗原的亲和力。另一方面,转基因技术的进步使得可以培育出基因工程化小鼠,其携带人免疫球蛋白免疫库并使其内源的鼠的免疫库缺失。这种转基因小鼠产生的抗体具有全人源的序列,因而无需再进一步做人源化改造,大大提高了治疗性抗体开发的效率。Harbour HCAb小鼠(Harbour Antibodies BV,WO 2002/085945 A3)是一种携带人免疫球蛋白免疫库的转基因小鼠,能够产生全新的仅“重链”抗体,该抗体的大小只有传统IgG抗体的一半。其产生的抗体仅具有人的抗体“重链”可变结构域和小鼠Fc恒定结构域。由于不含轻链的这一特点,该抗体几乎解决了轻链错配和异源二聚化的问题,使得这一技术平台能够开发出传统抗体平台难以实现的产品。
1.1用BCMA抗原免疫小鼠
用可溶的重组人BCMA-ECD-Fc融合蛋白对HarbourHCAb小鼠进行多轮免疫。抗原蛋白与免疫佐剂混合成免疫原试剂,然后通过皮下经腹股沟注射或通过腹腔注射。在每一轮免疫中,每只小鼠接受的总注射剂量是100微升。在首轮免疫中,每只小鼠接受用50微克抗原蛋白(重组人BCMA-ECD-Fc,ACRO,Cat.BC7-H82F0)与完全弗氏佐剂(Sigma,#F5881)以体积比1∶1混合配制的免疫原试剂的免疫。在随后的每轮增强免疫中,每只小鼠接受用25微克抗原蛋白与Sigma Adjuvant System佐剂(Sigma,#S6322)混合配制的免疫原试剂的免疫。每轮增强免疫的间隔时间至少为两周,通常不超过五轮增强免疫。免疫时间为第0、14、28、42、56、70天;并且在第49、77天,检测小鼠血清抗体滴度。在进行HCAb小鼠脾B细胞分离前5天,以每只小鼠25微克抗原蛋白的剂量进行最后一次增强免疫。
1.2获得HCAb单克隆和抗体序列
当检测小鼠血清中BCMA特异的抗体滴度达到一定的水平后,将小鼠的脾细胞取出分离B细胞,用BD FACS AriaII Cell Sorter分选CD138阳性的浆细胞和BCMA抗原阳性的B细胞群。提取RNA,反转录cDNA后PCR扩增人VH基因。扩增的VH基因片段构建到编码人IgG1抗体重链Fc结构域序列的哺乳动物细胞表达质粒pCAG载体中,质粒转染哺乳动物宿主细胞(如人胚肾细胞HEK293)进行表达,表达的HCAb的抗体上清与重组人BCMA-Fc,Avitag重组蛋白(ACRO,Cat.BC7-H82F0)进行Mirrorball筛选,获得的Mirrorball阳性单克隆抗体用FACS进一步的鉴定,与过表达人BCMA的HEK293T细胞株(HEK293T/huBCMA,北京康源)、过表达食蟹猴BCMA的HEK293T细胞株(HEK293T/cynoBCMA,北京康源)和高表达人BCMA的细胞系NCI-H929细胞的结合能力。筛选获得的阳性克隆的克隆号HBM1005P63B7,下文编号为PR001046。利用常规的测序手段获得编码抗体分子可变结构域的核苷酸序列以及对应的氨基酸序列。在本实施例中,从免疫的Harbour HCAb小鼠得到的抗BCMA单克隆抗体分子可变结构域的序列是人源抗体序列。
表2列出了本实施例中BCMA抗体的重链可变结构域氨基酸序列、重链全长氨基酸序列,和根据Combined定义规则定义的CDR的氨基酸序列。本发明抗体根据其他规则定义的CDR的氨基酸序列请参见序列表。
表2抗BCMA的HCAb抗体相关氨基酸序列
1.3制备抗BCMA全人重组抗体
将编码抗体PR001046重链的质粒转染哺乳动物宿主细胞(如人胚肾细胞HEK293),利用常规的重组蛋白表达和纯化技术,可以得到纯化的抗BCMA重组重链抗体。具体说来,将HEK293细胞在FreeStyleTMF17 Expression Medium培养基(Thermo,A1383504)扩培。瞬时转染开始之前,调节细胞浓度至6×105细胞/ml,于37℃8%CO2摇床中培养24小时,细胞浓度在1.2×106细胞/ml。准备30ml培养的细胞,将上述编码PR001046重链的质粒30μg质粒溶解于1.5ml Opti-MEM减血清培养基(Thermo,31985088),再取1.5ml Opti-MEM溶入1mg/ml PEI(Polysciences,Inc,Cat#23966-2)120μl,静置5分钟。把PEI缓慢加入质粒中,室温孵育10分钟,边摇晃培养瓶边缓慢滴入质粒PEI混合溶液,于37℃8%CO2摇床中培养5天。5天后观测细胞活率。收集培养物,以3300G转速离心10分钟后取上清;然后将上清高速离心去除杂质。用PBS(pH7.4)平衡含有MabSelectTM(GE Healthcare Life Science,Cat#71-5020-91AE)的重力柱(Bio-Rad,#7311550),2-5倍柱体积冲洗。将上清样品过柱。用5-10倍柱体积的PBS冲洗柱子。再用pH3.5的0.1M甘氨酸洗脱目的蛋白,后用pH 8.0的Tris-HCl调节至中性,最后用超滤管(Millipore,UFC901024)浓缩换液至PBS缓冲液,得到纯化的抗BCMA重链抗体溶液。
下表为阳性对照1即全长抗体PR000274的轻、重链序列,轻、重链可变区序列以及根据Combined定义规则定义的CDR的氨基酸序列。
表3全人重组抗体相关氨基酸序列
实施例2.FACS检测抗人BCMA的HCAb单抗细胞水平的结合能力
本实施例是为了研究抗人BCMA的HCAb单抗体外结合人和食蟹猴BCMA的活性。采用过表达人BCMA的HEK293T细胞株(HEK293T/huBCMA,北京康源)、过表达食蟹猴BCMA的HEK293T细胞株(HEK293T/cynoBCMA,北京康源)和高表达人BCMA的细胞系NCI-H929进行细胞水平上的抗体结合实验。简言之,消化细胞HEK293T/huBCMA和HEK293T/cynoBCMA细胞,并用DMEM完全培养基重悬,收集NCI-H929细胞悬液。将3种细胞密度分别调整为1×106细胞/mL。以100μL细胞/孔接种于96孔V底板(Coming,Cat#:3894),随后加入100μL/孔,2倍于终浓度的3倍浓度梯度稀释的待测抗体。将细胞放置于4℃,避光孵育1小时。之后,加入100μL/孔预冷PBS漂洗细胞两次,于500g、4℃下离心5分钟,弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-HumanIgG,FcγFragment Specific,Jackson,Cat#:109-545-06,1∶500稀释),4℃,避光孵育30分钟。用100μL/孔预冷PBS洗涤细胞两次,于500g、4℃下离心5分钟,弃上清。最后,200μL/孔预冷PBS重悬细胞,使用BD FACS CANTOII读取荧光发光信号值。
如图1所示,抗人BCMA的HCAb单抗PR001046在过表达人BCMA的HEK293T细胞株(A)、过表达食蟹猴BCMA的HEK293T细胞株(B)和高表达人BCMA的细胞系NCI-H929(C)上的结合能力均优于阳性对照1(PR000274)。具体表现为,PR001046结合曲线的EC50均小于阳性对照1,具体最大荧光值和EC50参见图1中的(D)。
实施例3.ELISA检测抗人BCMA的HCAb单抗对人BCMA与配体BAFF蛋白结合的阻断
本实施例是为了评价抗人BCMA的HCAb单抗对人BCMA(ACRO,BCA-H522y-100μg)与配体BAFF蛋白(ACRO,BAF-H5248-50ug)结合的阻断能力。利用生物素化试剂盒(ThermoFisher,A39257,EZ-Link Sulfo-NHS-LC-Biotin),按照说明书要求对BAFF蛋白进行生物素化。采用1μg/mL人BCMA蛋白,FcTag(ACRO,Cat#:BC7-H5254)包板(Corning,Cat#:9018),过夜。PBST洗涤3次后加2%BSA室温封闭1小时。PBST洗涤3次后加入100μL/孔3倍浓度梯度稀释的待测抗体,起始浓度50nM,室温孵育1小时后,弃上清,加入100μL/孔0.5μg/mL生物素化的人BAFF蛋白(根据本领域常规方法制备),室温孵育1小时。弃上清,加入100μL/孔1∶4000稀释的链霉亲和素耦连的HRP(Bio-RAD,Cat#:1610380)。室温孵育1小时后,PBST洗涤3次。加入100μL/孔TBM显色,15分钟后加入终止液终止反应。使用Enspire(PerkinElmer)取OD450值。
如图2中(A)所示,抗人BCMA的HCAb单抗PR001046具有与阳性对照1抗体相当的阻断人BCMA与其配体BAFF蛋白结合的能力。具体IC50值参见图2中的(B)。
实施例4.CD3抗体的人源化改造
抗CD3抗体是构建T细胞桥接器双特异性抗体(T-Cell Engager BispecificAntibody)的重要组成部分。针对已知的结合人CD3e(CD3 epsilon单亚基或者含有CD3e的复合物)的小鼠抗体SP34进行人源化改造。如WO2017084495A1(江苏恒瑞)或CN104136610B(中外制薬株式会社)中所述,并简述如下:
本实施例使用“CDR移植”的方法进行序列的人源化,即:将鼠抗的VH的CDR移植到人抗体VH的框架区,将鼠抗的VL的CDR移植到人抗体VL的框架区。人抗体VH或VL的框架区的序列可以来源于人的胚系基因序列或者经过V(D)J重排后的抗体序列或者人抗体特定VH或VL基因家族的一致性(consensus)序列。本实施例使用人的胚系基因序列提供的框架区序列作为人源化模板序列,即:人的胚系V基因片段提供框架区FR1,FR2,FR3的序列,人的胚系J基因片段提供框架区FR4的序列。最后以(人)FR1-(鼠)CDR1-(人)FR2-(鼠)CDR2-(人)FR3-(鼠)CDR3-(人)FR4的排列方式构建人源化可变区(VH或VL)序列。
本实施例使用人胚系V基因片段IGHV3-73*01或人胚系V基因片段IGHV3-23*01结合人胚系J基因片段IGHJ1*01的序列作为人源化模板提供框架区序列。并且在第30位、第73位、第76位、第78位、第93位或第94位(按照Chothia编码规则)引入一个或者多个位点的氨基酸突变,得到多个不同的VH变体序列。
本实施例使用人胚系V基因片段IGLV7-46*02结合人胚系J基因片段IGLJ2*01的序列或者人胚系V基因片段IGKV1-39*01结合人胚系J基因片段IGKJ4*01的序列作为人源化模板提供框架区序列。并且在第2位、第36位、第46位、第49位、第66位、第69位、第71位或第87位(按照Chothia编码规则)引入零个或者多个位点的氨基酸突变,得到多个不同的VL变体序列。下表4为其中一个CD3抗体的氨基酸序列,该抗体的轻链(LC)、重链(下文中,本发明CD3抗体的重链、重链可变区分别在双特异性抗体中简称HCl、VH1)与本发明BCMA抗体的重链(下文中,本发明BCMA抗体的重链、重链可变区分别在双特异性抗体中简称HC2、VH2)序列将构成双特异性抗体PR003178、PR002299(具体构建方法见实施例5)。
表4获得的CD3抗体氨基酸序列
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实施例5.BCMA×CD3双特异性抗体的制备
自实施例4之后所选的抗BCMA抗体和抗CD3细胞用于制备双特异性抗体。这种BCMA×CD3双特异性抗体的制备双抗可以同时结合两个靶点,其中一端可以识别肿瘤细胞表面特异表达的BCMA,而另一端可以结合T细胞上的CD3分子。当BCMA×CD3双抗分子结合到肿瘤细胞表面后,可以招募并激活肿瘤细胞附近的T细胞,从而杀死肿瘤细胞。
对于每一个双特异性抗体,涉及三条蛋白链,其分别包含相应抗BCMA抗体的重链,以及上述抗CD3抗体的重链和轻链。为了将具有错配的重链(例如抗CD3抗体的两条重链错配)的副产物形成最小化,使用了突变的异源二聚体Fc区,其携带“knob-hole”突变和改造的二硫键,如WO2009080251和WO2009080252中所述。BCMA×CD3双特异性抗体为IgG1,具有Fc突变L234A和L235A(根据EU索引编号)。
通过同时共转染三个不同的哺乳动物表达载体产生每一个双特异性抗体,分别编码:1)相应的BCMA抗体的重链,其在Fc区携带“Hole”突变以产生异源二聚体抗体,Fc的CH3携带L234A、L235A突变。2)相应的CD3抗体的重链,其在Fc区携带“knob”突变以产生异源二聚体抗体,Fc的CH3携带L234A、L235A突变。3)相应的CD3抗体的轻链。人IgG1Fc区的“knob”突变由以下组成:T366W,“Hole”突变由以下组成:T366S、L368A、Y407V。此外,可以包括“knob”Fc区的S354C和“Hole”Y349C形成一对二硫键以增加稳定性和异源二聚体抗体产量。下表5~7分别是本发明的双特异性抗体以及阳性对照2的序列信息。
表5 PR003178双特异性抗体BCMA链的序列信息
表6 PR002299双特异性抗体BCMA链的氨基酸序列信息
表7阳性对照2(PR002199)的氨基酸序列信息
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实施例6.FACS检测BCMA×CD3双特异性抗体在高表达BCMA的NCI-H929细胞系上的体外结合
为了研究BCMA×CD3双特异性抗体BCMA臂的结合人BCMA的活性。采用高表达BCMA的NCI-H929细胞系进行细胞上与人BCMA结合实验。简言之,收集NCI-H929细胞悬液。将细胞密度分别调整为1×106细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,Cat#:3894),随后加入100μL/孔,2倍于终浓度的3倍浓度梯度稀释的待测抗体。将细胞放置于4℃,避光孵育2小时。之后,加入100μL/孔预冷PBS漂洗细胞两次,于500g、4℃下离心5分钟,弃上清。再加入100μL/孔荧光二抗(Alexa Fluor 488-conjugated AffiniPure Goat Anti-HumanIgG,FcγFragment Specific,Jackson,Cat#:109-545-06,1∶500稀释),4℃,避光孵育1小时。用100μL/孔预冷PBS洗涤细胞两次,于500g,4℃下离心5分钟,弃上清。最后,200μL/孔预冷PBS重悬细胞,使用BDFACSCANTOII读取荧光发光信号值。
如图3中(A)和(B)所示,BCMA×CD3双特异性抗体PR003178与NCI-H929细胞系上人BCMA分子的结合优于阳性对照2,具体表现为,抗体PR003178结合曲线的EC50小于阳性对照2约8倍。同时,抗体PR002299与NCI-H929细胞系上人BCMA分子的结合从最大结合荧光值及结合曲线的EC50两方面均弱于抗体PR003178。
实施例7.FACS检测BCMA×CD3双特异性抗体在过表达人和食蟹猴BCMA的HEK293T细胞株上的体外结合
为了研究BCMA×CD3双特异性抗体BCMA臂的体外结合活性。采用过表达人BCMA的HEK293T细胞株(HEK293T/huBCMA)和过表达食蟹猴BCMA的HEK293T细胞株(HEK293T/cynoBCMA)进行细胞水平上的抗体结合实验。简言之,消化细胞HEK293T/huBCMA和HEK293T/cynoBCMA细胞,并用DMEM完全培养基重悬。将2种细胞密度分别调整为1×106细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,Cat#:3894),随后加入100μL/孔,2倍于终浓度的3倍浓度梯度稀释的待测抗体。将细胞放置于4℃,避光孵育1小时。之后,加入100μL/孔预冷PBS漂洗细胞两次,于500g、4℃下离心5分钟,弃上清。再加入100μL/孔荧光二抗(AlexaFluor 488-coniugated AffiniPure Goat Anti-Human IgG,FcγFragment Specific,Jackson,Cat#:109-545-06,1∶500稀释),4℃,避光孵育30分钟。用100μL/孔预冷PBS洗涤细胞两次,于500g、4℃下离心5分钟,弃上清。最后,200μL/孔预冷PBS重悬细胞,使用BD FACSCANTOII读取荧光发光信号值
如图4中(A)所示,BCMA×CD3双特异性抗体PR003178与HEK293T/huBCMA细胞上人BCMA的结合强于阳性对照2。具体表现为,抗体PR003178结合曲线的EC50小于阳性对照2约5倍,如图4中(C)所示。同时,如图4中(B)所示,PR003178能够与HEK293T/cynoBCMA细胞上食蟹猴BCMA交叉结合,而阳性对照2不具备交叉结合食蟹猴BCMA的能力。
实施例8.FACS检测BCMA×CD3双体异性抗体与人和食蟹猴T细胞体外结合
为了研究BCMA×CD3双特异性抗体CD3臂的体外结合活性。采用人和食蟹猴T细胞进行细胞水平上的抗体结合实验。简言之,将人和食蟹猴T细胞密度分别调整为1×106细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,Cat#:3894),随后加入100μL/孔,2倍于终浓度的3倍浓度梯度稀释的待测抗体。将细胞放置于4℃,避光孵育1小时。之后,加入100μL/孔预冷PBS漂洗细胞两次,于500g,4℃下离心5分钟,弃上清。再加入100μL/荧光二抗(Alexa Fluor 488-coniugated AffiniPure Goat Anti-Human IgG,FcγFragmentSpecific,Jackson,Cat#:109-545-06,1∶500稀释),4℃,避光孵育30分钟。用100μL/孔预冷PBS洗涤细胞两次,于500g,4℃下离心5分钟,弃上清。最后,200μL/孔预冷PBS重悬细胞,使用BD FACS CANTOII读取荧光发光信号值.
如图5中(A)所示,BCMA×CD3双特异性抗体PR003178与人T细胞上人CD3的结合强于阳性对照2。具体表现为,在相同抗体浓度情况下,PR003178结合的荧光值高于阳性对照2结合的荧光值。同时如图5中(B)所示,抗体PR003178能够与食蟹猴T细胞上食蟹猴CD3交叉结合,而阳性对照2不具备交叉结合食蟹猴CD3的能力。
实施例9.BCMA×CD3双特异性抗体对BCMA高表达细胞系NCI-H929的体外杀伤实验以及细胞因子的释放
为了研究BCMA×CD3双特异性抗体体外介导的靶细胞杀伤能力。采用人PBMC作为效应细胞,高表达BCMA的细胞系NCI-H929作为靶细胞进行体外杀伤实验以及细胞因子释放的检测。具体的,用RPMI1640/5%FBS培养基将PBMC的密度调整为1.1×106细胞/mL,NCI-H929的密度调整为0.11×106细胞/mL,以两种细胞悬液各90μL细胞/孔接种于96孔U底板(Corning,Cat#:3799),随后加入20μL/孔,10倍于终浓度的4倍浓度梯度稀释的待测抗体,其中抗体最高终浓度为30nM,每个抗体共10个浓度,最终效靶比为10∶1,设置两个重复。同时,在板内设置不同的对照组:ER(0):NCI-H929+PBMC+RPMI1640/5%FBS培养基;ESR:PBMC+RPMI1640/5%FBS培养基;TSR:NCI-H929+RPMI1640/5%FBS培养基;CMB:仅RPMI1640/5%FBS培养基;TMR:NCI-H929+RPMI1640/5%FBS培养基+裂解缓冲液(孵育24小时后加入);VCC:RPMI1640/5%FBS培养基+裂解缓冲液(孵育24小时后加入)。96孔板置于37℃二氧化碳培养箱孵育24小时。孵育完成后,取50μl上清液,加入96孔板(Corning,Cat#:3599),并加入50u1细胞毒性检测试剂(CytoTox非放射性细胞毒性检测试剂盒,Promega,USA,cat#G1780),室温孵育30分钟,加入终止溶液停止反应并用Enspire(Perkin Elmer)读取OD490值。OD490读值按照以下公式来计算杀伤效果,特异性杀伤%={(ER-CMB)-[(ESR-CMB)+(TSR-CMB)]}/(TMR-VCC)*100%。收集细胞培养上清,用于检测细胞因子IL-6和TNF-α的释放。ELISA检测方法参照IL-6(IL-6 Human Uncoated ELISA Kit,Thermo,Cat#:88-7066-88)和TNF-α(TNF alpha Human Uncoated ELISA Kit,Thermo,Cat#:88-7346-88)试剂盒操作说明。
如图6所示,BCMA×CD3双特异性抗体PR003178介导的PBMC对高表达BCMA的细胞系NCI-H929的杀伤优于阳性对照2(A)。具体表现为,PR003178在高浓度所达到杀伤百分比高于阳性对照2以及PR003178杀伤曲线的EC50小于阳性对照2约10倍(D)。同时,PR003178的杀伤体系中产生的细胞因子IL-6和TNF-α的释放均高于阳性对照2(B、C、E)。
实施例10.BCMA×CD3双特异性抗体对BCMA的低表达细胞系RPMI8226的体外杀伤实验以及细胞因子的释放
为了研究BCMA×CD3双特异性抗体体外介导的靶细胞杀伤能力。采用人PBMC作为效应细胞,低表达BCMA的细胞系RPMI8226(中科院CAS细胞库)作为靶细胞进行体外杀伤实验以及细胞因子释放的检测。具体的,用RPMI1640/5%FBS培养基将PBMC的密度调整为1.1×106细胞/mL,RPMI8226的密度调整为0.11×106细胞/mL,以两种细胞悬液各90μL细胞/孔接种于96孔U底板(Corning,Cat#:3799),随后加入20μL/孔,10倍于终浓度的4倍浓度梯度稀释的待测抗体,其中抗体最高终浓度为30nM,每个抗体共10个浓度,最终效靶比为10∶1,设置两个重复。同时,在板内设置不同的对照组:ER(0):RPMI8226+PBMC+RPMI1640/5%FBS培养基;ESR.PBMC+RPMI1640/5%FBS培养基;TSR.RPMI8226+RPMI1640/5%FBS培养基;CMB:仅RPMI1640/5%FBS培养基;TMR:RPMI8226+RPMI1640/5%FBS培养基+裂解缓冲液(孵育24小时后加入);VCC:RPMI1640/5%FBS培养基+裂解缓冲液(孵育24小时后加入)。96孔板置于37℃二氧化碳培养箱孵育24小时。孵育完成后,取50μl上清液,加入96孔板(Corning,Cat#:3599),并加入50μl细胞毒性检测试剂(CytoTox非放射性细胞毒性检测试剂盒,Promega,USA,Cat#:G1780),室温孵育30分钟,加入终止溶液停止反应并用Enspire(PerkinElmer)读取OD490值。OD490读值按照以下公式来计算杀伤效果,特异性杀伤%={(ER-CMB)-[(ESR-CMB)+(TSR-CMB)]}/(TMR-VCC)*100%。收集细胞培养上清,用于检测细胞因子IL-6和TNF-α的释放。ELISA检测方法参照IL-6(IL-6 Human Uncoated ELISA Kit,Thermo,Cat#:88-7066-88)和TNF-α(TNF alpha Human Uncoated ELISA Kit,Thermo,Cat#:88-7346-88)试剂盒操作说明。
如图7所示,BCMA×CD3双特异性抗体PR003178介导的PBMC对低表达BCMA的细胞系的杀伤优于阳性对照2(A)。具体表现为,PR003178在高浓度所达到的靶细胞杀伤百分比高于阳性对照2(D)。同时,PR003178的杀伤体系中产生的细胞因子IL-6和TNF-α的释放均高于阳性对照2(B、C、E)。
实施例11.BCMA×CD3双特异性抗体对不表达BCMA细胞系HL-60的结合
为了研究BCMA×CD3双特异性抗体体外结合的特异性。采用不表达人BCMA的细胞系HL-60(CCL-240TM)进行细胞水平上的抗体结合实验。简言之,收集HL-60细胞悬液。将细胞密度分别调整为1×106细胞/mL。以100μL细胞/孔接种于96孔V底板(Corning,Cat#:3894),随后加入100μL/孔,2倍于终浓度的3倍浓度梯度稀释的待测抗体,抗体最高终浓度为300nM,每个抗体4个浓度。将细胞放置于4℃,避光孵育1小时。之后,加入100μL/孔预冷PBS漂洗细胞两次,于500g、4℃下离心5分钟,弃上清。再加入100μL/孔荧光二抗(AlexaFluor 488-conjugated AffiniPure Goat Anti-Human IgG,FcγFragment Specific,Jackson,Cat#:109-545-06,1∶500稀释),4℃,避光孵育30分钟。用100μL/孔预冷PBS洗涤细胞两次,于500g,4℃下离心5分钟,弃上清。最后,200μL/孔预冷PBS重悬细胞,使用BDFACSCANTOII读取荧光发光信号值。
如图8所示,在300nM、100nM、33.3nM和11.1nM下,BCMA×CD3双特异性抗体PR003178与阳性对照2均不能与BCMA阴性细胞系HL-60非特异结合。
实施例12.FACS方法检测BCMA×CD3双特异性抗体对BCMA阴性细胞系HL60的非特异杀伤及细胞因子的释放
为了研究BCMA×CD3双特异性抗体体外介导的靶细胞杀伤能力的特异性。采用人PBMC作为效应细胞,不表达BCMA的细胞系HL-60作为靶细胞进行体外杀伤实验以及细胞因子释放的检测。具体的,将靶细胞HL-60用0.5μM的CFSE染料标记。用RPMI1640/5%FBS培养基将3个供体的PBMC的密度调整为1.1×106细胞/mL,CFSE标记的HL-60细胞的密度调整为0.11×106细胞/mL,以两种细胞悬液各90μL细胞/孔接种于96孔U底板(Corning,Cat#:3799),随后加入20μL/孔,10倍于终浓度的待测抗体,其中抗体终浓度为100nM、30nM、10nM和3nM共4个浓度,最终效靶比为10∶1,设置两个重复。96孔板置于37℃二氧化碳培养箱孵育24小时。孵育完成后,收集细胞培养上清,用于检测细胞因子IL-6和TNF-α的释放。ELISA检测方法参照IL-6(IL-6 Human Uncoated ELISA Kit,Thermo,Cat#:88-7066-88)和TNF-α(TNF alpha Human Uncoated ELISA Kit,Thermo,Cat#:88-7346-88)试剂盒操作说明。将培养的细胞转至96孔V底板(Corning,Cat#:3894),以100μL/孔的7-AAD(BD,Cat#:559925,1∶100)染色体系进行染色,室温孵育30分钟,加入100μL/孔PBS终止反应。使用BD FACSCANTOII进行检测。按照以下公式来计算杀伤效果,特异性杀伤%=7-AAD+CFSE+百分比/(7-AAD+CFSE+百分比+7-AAD-CFSE+百分比)*100%。
如图9的A-E所示,在100nM、30nM、10nM和3nM下,BCMA×CD3双特异性抗体PR003178和阳性对照2不能介导的2个供体PBMC对BCMA阴性细胞系HL-60产生非特异杀伤(图9的A、D),该体系中也不能检测到非特异的细胞因子IL-6(图9的B、E)和TNF-α(图9的C、F)的释放。
实施例13.NCI-H929/人PBMC小鼠模型中BCMA×CD3双特异性抗体的抗肿瘤药效评估
为了评估BCMA×CD3双特异性抗体的体内抗肿瘤药效。采用6-8周雌性NCG小鼠,瘤细胞接种前3天,所有小鼠腹腔注射(i.p.)3×106人PBMC,然后于肿瘤细胞接种当天每只实验小鼠皮下接种5×106NCI-H929细胞,细胞重悬在PBS与Matrigel(1∶1)混合液中(0.1mL/只)。当肿瘤体积达到90mm3时对小鼠进行随机分组,每组6只小鼠。分组后将特定浓度经PBS稀释的药物以静脉注射(i.v.)、每周给药1次总共给药2次(QW*2)的方式进行给药,以PBS为空白对照组。在初次给药后第3天,7天,10天和14天对肿瘤体积和小鼠体重进行测量。肿瘤大小计算公式:肿瘤体积(mm3)=0.5×(肿瘤长径×肿瘤短径2)。
如图10中(A)所示,BCMA×CD3双特异性抗体PR003178在10微克/鼠和3微克/鼠均有抑制肿瘤生长的药效,10微克/鼠组的药效优于3微克/鼠组的药效。同时地,在10微克/鼠的给药剂量下PR003178的药效优于同等剂量的阳性对照2。如图10中(B)所示,受试各组小鼠体重变化均在正常范围内。
实施例14.BIACORE检测PR003178与人CD3e/g重组蛋白、食蟹猴CD3e/g重组蛋白及人BCMA重组蛋白、食蟹猴BCMA重组蛋白的亲和力
整个测试使用HBS-EP+(10mM HEPES,150mM NaCl,3mM EDTA和0.05%P20,pH7.4,GE Healthcare,Cat#BR-1006-69)作为运行缓冲液,系列S CM5(GE Healthcare,Cat#BR-1005-30)为实验芯片。人BCMA-Fc,Avitag重组蛋白(ACRO,Cat.BC7-H82F0)、食蟹猴BCMA-Fc,Avitag(ACRO,Cat.BCA-C82F4)购买自ACRO,人CD3e/g ECD-hFc重组蛋白(Lot.20190508002)、食蟹猴CD3e/g ECD-hFc重组蛋白及人(Lot.2018040001)购买自睿智化学。
14.1抗原的偶联
设置流速10μl/min,在两张CM5的4个通道上偶联4种抗原:1)设置注入时间300s,将50mM NHS和200mM EDC以1∶1体积比新鲜混合后注入4个通道;2)用pH4.5的醋酸钠(Cat#BR-1003-50)将人BCMA-Fc,Avitag重组蛋白、食蟹猴BCMA-Fc,Avitag、人CD3e/g ECD-hFc重组蛋白、食蟹猴CD3e/g ECD-hFc重组蛋白均稀释至1ug/ml,分别注入芯片一的2、3、4和芯片二的2通道;3)注入1M pH8.5乙醇胺300s,以封闭芯片表面剩余的活性羧基。封闭后继续用1×HBS-EP+缓冲液平衡仪器两小时,人BCMA-Fc,Avitag重组蛋白、食蟹猴BCMA-Fc,Avitag、人CD3e/g ECD-hFc重组蛋白和食蟹猴CD3e/g ECD-hFc重组蛋白最终偶联量分别为120RU、70RU、200RU和290RU。
14.2亲和力测定
设置多循环动力学模式,每个循环包括PR003178的结合以及芯片的再生。将PR003178两倍梯度稀释注入四个通道,流速为30μl/min,设置结合时间180s,解离时间400s或600s。最后以同样流速注入10mM甘氨酸-盐酸pH1.5(GE Life Sciences,Cat#BR-1003-54)60s,以再生芯片。
用Biacore T200分析软件对实验结果进行分析,1通道作为参比通道扣除,分析模型选用1∶1动力学拟合模型。
如图11中的(B)、(D)所示,BCMA×CD3双特异性抗体PR003178与人BCMA具有高亲和力结合,与人BCMA-Fc的亲和力约9.493E-11M,且具有很好的交叉结合人和食蟹猴BCMA的活性。如图11中的(A)、(C)所示,BCMA×CD3双特异性抗体PR003178与人CD3 e/g ECD-hFc的亲和力约2.541E-08 M,并且具有很好的交叉结合人和食蟹猴CD3 e/g ECD-hFc的活性。如图11中的(E)为BCMA×CD3双特异性抗体PR003178与BCMA和CD3 e/g ECD-hFc亲和力测定的具体数据,包括Ka(1/Ms),Kd(1/s),KD。
Claims (21)
1. 一种靶向BCMA的重链抗体,其特征在于,其包括重链可变区,所述重链可变区包含分别如SEQ ID NO: 22、SEQ ID NO: 33和SEQ ID NO: 42的氨基酸序列所示的HCDR1、HCDR2和HCDR3。
2. 如权利要求1所述的靶向BCMA的重链抗体,其特征在于,所述重链可变区的氨基酸序列如SEQ ID NO: 59所示。
3.如权利要求1所述的靶向BCMA的重链抗体,其特征在于,所述靶向BCMA的抗体还包括Fc片段。
4.如权利要求3所述的重链抗体,其特征在于,所述Fc片段为hIgG1、hIgG2、hIgG3、hIgG4的Fc片段或其突变体。
5. 如权利要求1-4中任一项所述的靶向BCMA的重链抗体,其特征在于,所述靶向BCMA的重链抗体的重链的氨基酸序列如SEQ ID NO: 1或SEQ ID NO: 8所示。
6.一种双特异性抗体,其包括靶向BCMA的第一蛋白功能区和靶向CD3的第二蛋白功能区,其特征在于,所述第一蛋白功能区包括如权利要求1-5中任一项所述的靶向BCMA的重链抗体的重链可变区的两个重复片段。
7.如权利要求6所述的双特异性抗体,其特征在于,所述重链可变区的两个重复片段之间以(G4S)n连接,所述n为非0自然数。
8.如权利要求7所述的双特异性抗体,其特征在于,所述n为1~20。
9.如权利要求8所述的双特异性抗体,其特征在于,所述n为3或4。
10. 如权利要求6所述的双特异性抗体,其特征在于,所述第二蛋白功能区包括重链可变区和轻链可变区,所述重链可变区的HCDR1、HCDR2和HCDR3的氨基酸序列分别如SEQ IDNO: 25、SEQ ID NO: 38和SEQ ID NO: 44所示,和,所述轻链可变区的LCDR1、LCDR2和LCDR3的氨基酸序列分别如SEQ ID NO: 50、SEQ ID NO: 53和SEQ ID NO: 56所示。
11. 如权利要求6所述的双特异性抗体,其特征在于,所述第二蛋白功能区的重链可变区的氨基酸序列如SEQ ID NO: 63所示,轻链可变区的氨基酸序列如SEQ ID NO: 66所示。
12.如权利要求6-11任一项所述的双特异性抗体,其特征在于,所述双特异性抗体还包括重链恒定区和轻链恒定区。
13.如权利要求12所述的双特异性抗体,其特征在于,所述重链恒定区和轻链恒定区为人重链恒定区和人轻链恒定区。
14.如权利要求13所述的双特异性抗体,其特征在于,所述人轻链恒定区为人λ、κ轻链恒定区,所述人重链恒定区为hIgG1、hIgG2、hIgG3、hIgG4的重链恒定区或其突变体;
所述人重链恒定区的突变体选自以下组:
(1) L234A/L235A突变;
(2) knob突变T366W和hole突变T366S、L368A和Y407V;
(3) knob突变S354C和Hole突变Y349C;
所述突变的位点根据EU索引编号。
15. 如权利要求14所述的双特异性抗体,其特征在于,所述第一蛋白功能区包括氨基酸序列如SEQ ID NO: 8所示的重链,所述第二蛋白功能区包括氨基酸序列如SEQ ID NO: 5所示的重链和氨基酸序列如SEQ ID NO: 47所示的轻链。
16.一种编码根据权利要求1-5中任一项所述的靶向BCMA的重链抗体或权利要求6-15中任一项所述的双特异性抗体的分离的核酸。
17.一种包含根据权利要求16所述的分离的核酸的表达载体。
18.一种宿主细胞,其包含根据权利要求17所述的表达载体。
19.如权利要求18所述的宿主细胞,其特征在于,所述宿主细胞是原核细胞或真核细胞。
20.一种药物组合物,其包含如权利要求1-5中任一项所述的靶向BCMA的重链抗体或权利要求6-15中任一项所述的双特异性抗体,以及药学上可接受的载体。
21.如权利要求1-5中任一项所述的靶向BCMA的重链抗体、权利要求6-15中任一项所述的双特异性抗体或权利要求20中所述的药物组合物在制备治疗和/或预防BCMA阳性的癌症的药物中的应用。
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