CN111484553B - 一种靶向补体蛋白C5的pH依赖性抗体的筛选方法 - Google Patents
一种靶向补体蛋白C5的pH依赖性抗体的筛选方法 Download PDFInfo
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Abstract
本发明公开了一种靶向补体蛋白C5的pH依赖性抗体的筛选方法以及由此得到的抗体。本发明的筛选方法在生物分子相互作用分析系统上进行,包括设置实验组1、对照组1、实验组2和对照组2,使各组芯片如下结合抗原补体蛋白C5(A)、作为pH依赖性C5单克隆抗体的参考抗体(Ab1)、待测抗体(Ab2):实验组1 A‑Ab1‑Ab2;对照组1 A‑Ab1‑0;实验组2 A‑0‑Ab2;对照组2 A‑0‑0,通过检测结合信号并计算(实验组1‑对照组1)/(实验组2‑对照组2)的比值,来判断待测抗体是否为pH依赖性C5抗体。本发明的方法可缩短pH依赖性C5单抗开发周期,节省劳动量和时间成本;并且可将杂交瘤细胞培养上清作为待测样品直接进行鉴定,进一步提高抗体筛选效率。
Description
技术领域
本发明涉及生物医药领域,具体而言,本发明涉及一种靶向补体C5的pH依赖性抗体的筛选方法以及由其筛选得到的抗体。
背景技术
补体蛋白C5是补体系统的主要成分,是先天免疫系统的关键部分。C5是190kDa的糖蛋白,包含两个由二硫键连接的多肽链α和β,其分子质量分别是115kDa和75kDa。在C5 α-链N末端下游75个氨基酸的精氨酸残基处被C5转化酶切割,会产生7.4kDa C5a和180kDaC5b补体裂解产物。其中,C5a是一种过敏毒素,可通过嗜碱性细胞和巨细胞释放的组胺刺激血管舒张;C5b可在细胞表面依次与补体蛋白C6、C7、C8、C9一同形成膜攻击复合体(MAC)的初始组分,MAC在靶细胞表面的积累最终导致细胞胶体渗透性裂解,促成促炎环境和细胞损失。
针对补体蛋白C5,已有不同报道描述了抗C5的抗体,部分抗体已经被批准上市用于相关适应症的治疗,或处于临床开发中。亚力兄制药(Alexion)研发的单克隆抗体依库珠单抗(Eculizumab)于2007年由FDA获批上市。目前已获批的适应症有治疗阵发性睡眠性血红蛋白尿症、非典型溶血性尿毒综合症、重症型肌无力和视神经性脊髓炎。此外,针对2020年出现的新型冠状病毒肺炎(COVID-19),也开始应用依库珠单抗在美国开展了临床研究。但是,由于靶点介导的药物降解效应,该依库珠单抗在人体内的半衰期仅11.3天左右,且C5蛋白体内含量高,抗体用药量极大。
已知pH依赖性C5抗体可以延长半衰期,具体地,抗体与C5结合为pH敏感性的,抗体在细胞外中性条件下与C5结合,而被内皮细胞内吞后在酸性内体中与C5快速解离,避免了抗体被降解,从而再循环到细胞外继续结合C5,达到长效目的。
pH依赖性抗体的获得可以有两种方法。一种是抗体初步筛选时直接筛选pH依赖性抗体。天然的抗体中大约有5%左右的抗体具有pH依赖性;常用的筛选方法有膜干涉、ELISA法等,分别测定抗体在酸性和中性条件下的亲和力,两种条件下亲和力有差异的单抗为潜在的pH依赖性抗体。例如再生元制药公司(Regeneron)开发的靶向C5的单抗Pozelimab。但是,由于初步筛选的样品通常为杂交瘤上清,含有复杂成分的细胞培养基对数值有很大影响,且抗体含量未知,因此准确性低,假阴性率或假阳性率偏高,大大的增加了无效工作量及筛选的准确率。
pH依赖性抗体还可以通过对不具有pH依赖性的抗体进行工程改造获得。例如亚力兄制药开发了依库珠单抗的第二代Ravulizumab。具体策略为组氨酸扫描法:把轻链和重链CDR区的每个氨基酸逐个突变为组氨酸,筛选出中性条件下亲和力提高或不变的位点,然后进一步的组合突变。然而,整个改造流程完成下来,需要数月甚至更长时间,且工作量巨大。
Taku Fukuzawa等人报道了单抗SKY59,通过晶体结构分析发现,SKY59与C5结合的抗原表位位于C5蛋白β链MG1结构域(20-124氨基酸),其中His70、His72、His110三个组氨酸位点参与了C5抗原与SKY59抗体相互作用,组氨酸在酸性条件下发生质子化,从而使C5抗原与SKY59抗体的结合具有pH依赖性。
根据C5抗原这一特性,可以考虑开发一种基于表位竞争实验的靶向C5抗体筛选方法,以SKY59或与其表位相同或相近的单克隆抗体作为参考抗体,筛选出靶向C5的具有pH依赖性的新抗体。
发明内容
本发明要解决的技术问题是,基于已知C5抗原MG1结构域具有三个暴露的组氨酸His70、His72、His110参与单抗SKY59的pH依赖性结合,以SKY59单抗或表位与其相同或相近的单克隆抗体作为参考抗体,利用生物膜干涉技术、表面等离子共振技术(SPR),来筛选靶向补体蛋白C5的pH依赖性单克隆抗体,从而缩短pH依赖性C5单抗开发周期,节省劳动量和时间成本;并且在筛选时,可将杂交瘤细胞培养上清作为待测样品,从而直接鉴定该上清中是否存在所述新抗体,进一步提高抗体筛选效率。
因此,本发明的目的在于提供一种靶向补体蛋白C5的pH依赖性抗体的筛选方法。本发明的目的还在于提供通过所述筛选方法得到的抗体。
本发明所述的“靶向补体蛋白C5的pH依赖性抗体”是指,与补体蛋白C5在酸性和中性条件下具有不同结合亲和力的抗体。一般而言,所述酸性条件是指pH 5.8,中性条件是指pH 7.4。
本发明的技术方案如下:
一方面,本发明提供一种靶向补体蛋白C5的pH依赖性抗体的筛选方法,所述筛选方法在生物分子相互作用分析系统上进行,包括以下步骤:
(1)设置实验组1、对照组1、实验组2和对照组2,各组芯片在缓冲液中平衡,然后浸入补体蛋白C5在缓冲液中的溶液以固化补体蛋白C5;
(2)使实验组1、对照组1的芯片再次在缓冲液中平衡,然后浸入参考抗体在缓冲液中的溶液以使补体蛋白C5结合所述参考抗体至饱和,再次在缓冲液中平衡,其中所述参考抗体为补体蛋白C5的pH依赖性抗体;之后使实验组1的芯片浸入包含待测抗体的待测样品,同时使对照组1的芯片浸入不包含待测抗体的对照溶液;
使实验组2、对照组2的芯片再次在缓冲液中平衡,然后使实验组2的芯片浸入所述待测样品,同时使对照组2的芯片浸入所述对照溶液;
(3)检测各实验组和对照组的结合信号,计算(实验组1-对照组1)/(实验组2-对照组2)的比值,比值为<30%指示待测抗体为潜在的靶向补体蛋白C5的pH依赖性抗体。
优选地,所述生物分子相互作用分析系统基于生物膜干涉技术或表面离子共振技术。
优选地,所述生物分子相互作用分析系统为fortebio或biacore生物分子相互作用分析系统。
优选地,所述补体蛋白C5为人补体蛋白C5。
优选地,所述参考抗体包含重链可变区(HCVR)和轻链可变区(LCVR),所述重链可变区和轻链可变区包含选自以下的重链CDR和轻链CDR的组合:
(1) 依次示于SEQ ID NO: 13、14、15的HCDR1(SSYYMA)、HCDR2(AIFTGSGAEYKAEWAKG)、HCDR3(DAGYDYPTHAMHY);和,依次示于SEQ ID NO: 16、17、18的LCDR1(RASQGISSSLA)、LCDR2(GASETES)、LCDR3(QNTKVGSSYGNT);
(2) 依次示于SEQ ID NO: 19、20、21的HCDR1(NNYLH)、HCDR2(WSYPENYDTKYNEKFKG)、HCDR3(SHFDGYLTGAMDY);和,依次示于SEQ ID NO: 22、23、24的LCDR1(RASKSVSKYLA)、LCDR2(SGSSLQF)、LCDR3(QQHNEYPWT);
(3) 依次示于SEQ ID NO: 25、26、27的HCDR1(SGYWN)、HCDR2(YIDYSGSTYYYLSLKS)、HCDR3(WGDYAAWFAY);和,依次示于SEQ ID NO: 28、29、30的LCDR1(KASQDVNTGVA)、LCDR2(WASTRHT)、LCDR3(QQHYSTPYT);和
(4) 依次示于SEQ ID NO: 31、32、33的HCDR1(DFYMH)、HCDR2(WSYPKNDNTKYNEKFKA)、HCDR3(SHFYGYLTGAMDH);和,依次示于SEQ ID NO: 34、35、36的LCDR1(RASENTYSYLA)、LCDR2(DAKTLAE)、LCDR3(QHHYGTPYT)。
进一步优选地,所述参考抗体包含选自以下的重链可变区和轻链可变区的组合:
(1) 包含示于SEQ ID NO: 1的氨基酸序列的重链可变区;和,包含示于SEQ IDNO: 2的氨基酸序列的轻链可变区;
(2) 包含示于SEQ ID NO: 3的氨基酸序列的重链可变区;和,包含示于SEQ IDNO: 4的氨基酸序列的轻链可变区;
(3) 包含示于SEQ ID NO: 5的氨基酸序列的重链可变区;和,包含示于SEQ IDNO: 6的氨基酸序列的轻链可变区;和
(4) 包含示于SEQ ID NO: 7的氨基酸序列的重链可变区;和,包含示于SEQ IDNO: 8的氨基酸序列的轻链可变区。
特别优选地,所述参考抗体为单克隆抗体或scFv、dsFv、(dsFv)2、Fab、Fab'、F(ab')2抗体;优选地,所述单克隆抗体为人或鼠的IgG型。
优选地,所述待测样品为待测抗体在缓冲液中的溶液或包含待测抗体的细胞培养液;所述对照溶液为缓冲液或细胞培养基。
优选地,所述待测样品为待测抗体在缓冲液中的溶液或包含待测抗体的单克隆杂交瘤细胞培养上清;所述对照溶液为缓冲液或单克隆杂交瘤细胞培养基。
优选地,在步骤(1)和步骤(2)中,所述缓冲液为磷酸盐缓冲液 + 0.1% BSA +0.02%吐温20 + 0.05% Priclin300,pH 7.4。
优选地,在步骤(1)和步骤(2)中,所述平衡为将芯片在所述缓冲液中放置60s。
优选地,在步骤(1)中,固化补体蛋白C5至信号高度2nm。
优选地,在步骤(2)中,使实验组1的芯片浸入待测样品300s,并且使对照组1的芯片浸入对照溶液300s。
优选地,在步骤(2)中,使实验组2的芯片浸入待测样品300s,并且使对照组2的芯片浸入对照溶液300s。
优选地,所述补体蛋白C5在缓冲液中的浓度为50nM。
优选地,所述参考抗体在缓冲液中的浓度为100nM。
优选地,所述待测抗体在缓冲液中的浓度为100nM。或者,所述待测样品为稀释或未稀释的单克隆杂交瘤细胞培养上清。根据本发明的具体实施方式,其中单克隆杂交瘤细胞是以人补体C5蛋白作为抗原免疫动物而得到的。
进一步地,本发明还包括对步骤(3)得到的抗体进行验证的步骤:
(4) 检测所述抗体在酸性和中性条件下与补体蛋白C5的结合亲和力,在二者有差异的情况下,确认所述抗体为靶向补体蛋白C5的pH依赖性抗体。
优选地,在步骤(4)中,所述酸性条件为pH 5.8,中性条件为pH 7.4。
另一方面,本发明还提供一种靶向补体蛋白C5的pH依赖性抗体或其抗原结合片段,所述抗体或其抗原结合片段包含重链可变区(HCVR)和轻链可变区(LCVR),所述重链可变区和轻链可变区包含选自以下的重链CDR和轻链CDR的组合:
(1) 依次示于SEQ ID NO: 19、20、21的HCDR1(NNYLH)、HCDR2(WSYPENYDTKYNEKFKG)、HCDR3(SHFDGYLTGAMDY);和,依次示于SEQ ID NO: 22、23、24的LCDR1(RASKSVSKYLA)、LCDR2(SGSSLQF)、LCDR3(QQHNEYPWT);
(2) 依次示于SEQ ID NO: 25、26、27的HCDR1(SGYWN)、HCDR2(YIDYSGSTYYYLSLKS)、HCDR3(WGDYAAWFAY);和,依次示于SEQ ID NO: 28、29、30的LCDR1(KASQDVNTGVA)、LCDR2(WASTRHT)、LCDR3(QQHYSTPYT);和
(3) 依次示于SEQ ID NO: 31、32、33的HCDR1(DFYMH)、HCDR2(WSYPKNDNTKYNEKFKA)、HCDR3(SHFYGYLTGAMDH);和,依次示于SEQ ID NO: 34、35、36的LCDR1(RASENTYSYLA)、LCDR2(DAKTLAE)、LCDR3(QHHYGTPYT)。
进一步优选地,所述抗体或其抗原结合片段包含选自以下的重链可变区和轻链可变区的组合:
(1) 包含示于SEQ ID NO: 3的氨基酸序列的重链可变区;和,包含示于SEQ IDNO: 4的氨基酸序列的轻链可变区;
(2) 包含示于SEQ ID NO: 5的氨基酸序列的重链可变区;和,包含示于SEQ IDNO: 6的氨基酸序列的轻链可变区;和
(3) 包含示于SEQ ID NO: 7的氨基酸序列的重链可变区;和,包含示于SEQ IDNO: 8的氨基酸序列的轻链可变区。
特别优选地,所述抗体为单克隆抗体或scFv、dsFv、(dsFv)2、Fab、Fab'、F(ab')2抗体;优选地,所述单克隆抗体为人或鼠的IgG抗体。
根据本发明的具体实施方式,本发明的筛选方法采用PALL生产的型号为OCTETRED 96的Fortebio OCTET Red生物分子相互作用仪和PALL生产的货号为18-5101的NTA芯片进行;用Fortebio的Data analysis 10.0处理数据。
相对于现有技术,本发明提供了一种靶向补体C5的pH依赖性单克隆抗体的筛选方法,该方法可以利用表位竞争实验,如生物膜干涉、表面等离子共振技术(SPR),初步筛选出与C5蛋白MG1结构域结合的抗体,所述抗体可以进一步验证是否为pH依赖性抗体,例如通过在不同pH条件下检测抗体与C5的结合情况来进行验证。实验证明,本发明的筛选方法以SKY59或与其表位相同的抗体D11、J11或C15作为参考抗体进行,能够有效地筛选出与抗原结合具有pH依赖性的C5抗体。本发明提供的筛选方法大大缩短了pH依赖性C5单抗开发周期,节省了劳动量和时间成本。
附图说明
以下结合附图来详细说明本发明的实施方案,其中:
图1为本发明筛选方法的示意图。
图2显示了杂交瘤细胞株上清的ELISA鉴定结果。
具体实施方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解,这些实施例仅用于说明本发明,其不以任何方式限制本发明的范围。
下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的药材原料、试剂材料等,如无特殊说明,均为市售购买产品。
(一)主要仪器与设备信息,见表1:
表1. 仪器与设备
(二)参考抗体与抗原信息,如下:
Chugai制药公司Crovalimab抗体(参见WO2016098356A1),简称SKY59。重链可变区示于SEQ ID NO: 1,轻链可变区示于SEQ ID NO: 2。构建成人IgG1形式。再将重轻链共转染到293细胞中,培养5-7天后收集上清,用Mabselect Sure柱纯化。用Kabat定义的CDR1、HCDR2、HCDR3依次示于SEQ ID NO: 13、14、15;LCDR1、LCDR2、LCDR3依次示于SEQ ID NO:16、17、18。
抗原重组人补体蛋白C5(Gln19-Cys1676),购自义翘神州(货号13416-H18H),该蛋白由人293细胞(HEK293)重组表达,C端含有组氨酸标签,N端含有Flag标签。
(三)缓冲液,如下:
缓冲液1:磷酸盐缓冲液(PBS)+0.1% BSA+0.02%吐温20(Tween 20)+0.05%Priclin 300,pH 7.4。
缓冲液2:磷酸盐缓冲液(PBS)+0.1% BSA+0.02%吐温20(Tween 20)+0.05%Priclin 300,pH 5.8。
(四)筛选方法,以参考抗体和待测抗体均为单克隆抗体为例:
将抗原补体蛋白C5固化在芯片上,然后依次与参考抗体和待测抗体或含有待测抗体的杂交瘤上清液相互作用,检测结合信号以判定待测抗体是否与参考抗体识别同一表位,由此筛选与参考抗体同为靶向补体蛋白C5的pH依赖性单克隆抗体的新抗体。示意图见图1。
设置以下实验组和对照组。A表示抗原,Ab1表示参考抗体,Ab2表示待测抗体或含有待测抗体的杂交瘤上清液,0表示不含有抗体的缓冲液1或杂交瘤细胞培养基。
A-Ab1-Ab2 实验组1
A-Ab1-0 对照组1
A-0-Ab2 实验组2
A-0-0 对照组2
(1)分别用缓冲液1配制抗体和抗原:
参考抗体配制成100nM;
待测抗体配制成100nM,作为待测样品;在待测样品为含待测抗体的杂交瘤上清液的情况下,直接使用;
抗原人C5蛋白配制成50nM。
(2)实验组1及其对照组1:
实验组1:在NTA芯片上偶联抗原A,先与Ab1反应,再与Ab2反应;对照组1:在NTA芯片上偶联抗原A,先与Ab1反应,再与缓冲液1或培养基反应。具体为:
1:NTA芯片先浸入缓冲液1中平衡60s,然后将芯片浸入抗原溶液固化抗原至信号高度2nm。
2:浸入缓冲液1中平衡60s。
3:浸入参考抗体溶液进行结合反应至饱和,即信号不再升高。
4:浸入缓冲液1中平衡60s。
5:实验组1芯片浸入待测样品300s。同时,对照组1浸入缓冲液1或杂交瘤细胞培养基300s。
(3)实验组2及其对照组2:
实验组2:在NTA芯片上偶联抗原A,先与缓冲液1反应,再与Ab2反应;对照组2:在NTA芯片上偶联抗原A,先与缓冲液1反应,再与缓冲液1或培养基反应。具体为:
1:NTA芯片先浸入缓冲液1中平衡60s(信号不再发生明显波动),然后将芯片浸入抗原溶液固化抗原至信号高度2nm。
2:浸入缓冲液1中平衡60s。
3:实验组2芯片浸入待测样品进行结合反应300s,对照组2芯片浸入缓冲液1或杂交瘤细胞培养基300s。
检测各实验组和对照组的结合信号,用Fortebio的Data analysis 10.0处理数据,计算(实验组1-对照组1)/(实验组2-对照组2)的比值,比值为<30%表明待测抗体与参考抗体完全竞争与该抗原的结合。在参考抗体为靶向补体蛋白C5的pH依赖性单克隆抗体时,比值为<30%表明待测抗体是潜在的靶向补体蛋白C5的pH依赖性单克隆抗体。可选地,通过在不同pH条件下检测抗体与补体蛋白C5结合亲和力的差异来进行验证该抗体是否为pH依赖性单克隆抗体。
实施例1 小鼠免疫和人C5抗体杂交瘤细胞株的筛选鉴定
参考Antibodies: A Laboratory Manual,Second Edition (Edward A.Greenfield 2012),以14天为间隔共计42天的过程免疫8周龄Balb/c小鼠。将人C5蛋白在完全或不完全弗氏佐剂中乳化,将其以单侧的方式注射于小鼠颈背部、尾根部、腹股沟3处皮下组织和腹膜腔内。在免疫第35天尾静脉采血,用ELISA方法检测抗体滴度后,取免疫小鼠脾细胞与骨髓瘤细胞融合。
取人C5蛋白免疫Balb/c小鼠脾脏细胞,与骨髓瘤细胞使用PEG或者电融合方法进行融合,将融合后的杂交瘤细胞以1x107个/孔密度接种于含HAT培养基中铺于384孔板中,培养5天后换含HT培养基继续培养2-3天筛选杂交瘤细胞。于384孔板中培养7-10天后,取细胞上清进行ELISA实验,筛选能够分泌抗人C5抗体的杂交瘤母克隆。
用ELISA法筛选人C5结合剂的方法如下:将人C5蛋白用PBS缓冲液稀释至1μg/ml,每孔100µl包被于96孔板(Microwell 96F 167008,Thermo)4℃孵育过夜;次日取出96孔板,用PBST(含0.5% Tween 20)洗板,每次浸润1 min后彻底甩干残余水分。样品孔中分别加入200µl的含2% BSA的PBST,置于37℃封闭1 h;然后用PBST洗板,并甩干孔中水分。分别向96孔板中加入待测样品100µl 4℃孵育过夜。取出96孔板后用PBST洗板后每孔加入1:20000稀释的二抗羊抗鼠偶联辣根过氧化物酶100µl,并置于37℃孵育1 h。再用PBST洗5次,每孔加入100µl 底物溶液(Substrate Solution, Invitrogen),于37℃ 孵育10 min;每孔加入2N硫酸50µl终止反应后于酶标仪(M5e,Molecular Device)450nm波长处检测吸光度。部分结果见图2,OD450值大于0.3为分泌C5抗体的阳性克隆。
随后将ELISA阳性的克隆从384孔板转到96孔板或其它型号孔板扩大培养,杂交瘤细胞培养上清可用于后续检测。
实施例2 用SKY59作为参考抗体筛选与其竞争表位的抗体
采用基于生物膜干涉原理的ForteBio OCTET Red生物分子相互作用仪筛选与SKY59表位竞争的鼠抗,以SKY59作为参考抗体,待测样品为实施例1筛选得到的小鼠杂交瘤上清。
配制缓冲液1(磷酸盐缓冲液(PBS)+0.1% BSA+0.02%吐温20(Tween 20)+0.05%Priclin300,pH7.4),用于稀释抗原和参考抗体。按照本发明上文所述的方法进行检测,比值60%-100%或甚至>100%为不竞争;30-60%为部分竞争;<30%为竞争;大于100%为不竞争。结果见表2,表明D11和J11克隆上清与SKY59竞争,C15克隆上清与SKY59部分竞争。
表2 Octet小鼠杂交瘤上清分泌抗体与SKY59的表位竞争
为了进一步确定C15、D11、J11与SKY59的表位竞争关系,从杂交瘤上清中纯化鼠抗,抗体编号同克隆编号。
利用ForteBio OCTET Red生物分子相互作用仪,用缓冲液1稀释抗原和各抗体。按照本发明上文所述的方法进行检测,比值60%-100%或甚至>100%为不竞争;30-60%为部分竞争;<30%为竞争。结果见表3,表明D11、C15和J11都与SKY59竞争。
表3 Octet检测纯化鼠抗与SKY59的表位竞争
实施例3 鼠抗序列鉴定及嵌合抗体构建
将筛选出的单克隆杂交瘤细胞在12或24孔板中扩大培养后,按照RNAfast200试剂盒(上海飞捷生物技术有限公司)说明书步骤提取细胞总RNA;利用5×PrimeScript RTMaster Mix(Takara)将杂交瘤细胞总RNA反转录成cDNA;使用简并引物和Extaq PCR试剂(Takara)扩增抗体轻链可变区IgVL(κ)和重链可变区VH序列;利用PCR clean-up Gelextraction试剂盒(Macherey-Nagel)纯化PCR扩增产物;按照pmd19T Simple Vector Kit试剂盒(Takara)说明书将扩增PCR产物连接至T载体并转化大肠杆菌感受态细胞,菌株扩增、抽提质粒后进行DNA测序获得单克隆抗体可变区序列。获取鼠源抗体的可变区序列并分析,见表4。
表4 部分鼠源抗体可变区序列以及CDR(用Kabat定义)
将鼠源抗体的完整轻、重链可变区克隆进入重组表达载体,重链恒定区均选用hIgG1亚型,轻链均为hIgG-Kappa亚型,得到的重组嵌合抗体命名为“鼠源抗体简称-xi”,进行抗体生产和纯化。
利用ForteBio OCTET Red生物分子相互作用仪,为了进一步确定几种嵌合抗体与SKY59的表位竞争关系。按照本发明上文所述的方法进行检测,比值60%-100%或甚至>100%为不竞争;30-60%为部分竞争;<30%为竞争。结果见表5,表明D11、C15和J11嵌合抗体都与SKY59竞争,K23、H20嵌合抗体与SKY59不竞争。
表5 Octet检测嵌合抗体与SKY59的表位竞争
实施例4 嵌合抗体在不同pH下与C5蛋白的结合和解离
为了验证筛选的C15、D11和J11几种嵌合抗体与C5的结合是否有pH依赖性,用Octet RED 96仪器分析嵌合抗体与人C5蛋白在不同pH条件下的结合和解离。
用缓冲液1稀释抗原和各抗体。使用抗人IgG Fc捕获(AHC)传感器,在pH7.4条件下,先结合浓度为100nM的SKY59,再结合100mM C5蛋白,再在pH7.4的缓冲液1中解离;传感器再生后,在pH7.4条件下,先结合浓度为100nM的SKY59,再结合100mM C5蛋白,再在pH5.8的缓冲液2(磷酸盐缓冲液(PBS)+0.1% BSA+0.02%吐温20(Tween 20)+0.05% Priclin 300,pH 5.8)中解离。采用相同方法检测本发明的嵌合抗体。
结果见表6,发现与SKY59表位竞争的C15、D11和J11嵌合抗体的解离常数Koff在pH5.8下均大于pH7.4时,其中C15和J11嵌合抗体的Koff5.8/Koff7.4远大于1,即在酸性条件下比中性条件下解离快很多,具有明显的pH依赖性结合;而与SKY59表位不竞争的K23和H20嵌合抗体在酸性和中性条件下的解离速率相近。
表6 Octet检测抗体在不同pH下与C5蛋白的结合和解离
实施例5 分别用C15、D11和J11嵌合抗体为参考抗体筛选与其表位竞争的C5抗体
采用基于生物膜干涉原理的ForteBio OCTET Red生物分子相互作用仪筛选与C15、D11和J11嵌合抗体表位竞争的抗体,以C15、D11或J11嵌合抗体作为参考抗体,待测样品为纯化的嵌合抗体。
配制缓冲液1(磷酸盐缓冲液(PBS)+0.1% BSA+0.02%吐温20(Tween 20)+0.05%Priclin300,pH7.4),用于稀释抗原和各抗体。按照本发明上文所述的方法进行检测,比值60%-100%或甚至>100%为不竞争;30-60%为部分竞争;<30%为竞争。结果见表7至表9,类似于SKY59为参考抗体的表位竞争筛选,C15、D11或J11为参考抗体时,均能有效筛选出pH依赖性的C5抗体。
表7 以C15-xiIgG为参考抗体,用Octet检测各抗体与C15的表位竞争
表8 以D11-xiIgG为参考抗体,用Octet检测各C5抗体与D11的表位竞争
表9 以J11-xiIgG为参考抗体,用Octet检测各C5抗体与J11的表位竞争
以上对本发明具体实施方式的描述并不限制本发明,本领域技术人员可以根据本发明作出各种改变或变形,只要不脱离本发明的精神,均应属于本发明所附权利要求的范围。
序列表
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<212> PRT
<213> 人工序列(artificial sequence)
<400> 46
Arg Ala Ser Gln Ser Val Ser Ser Ser Ser Tyr Ser Tyr Met His
1 5 10 15
<210> 47
<211> 7
<212> PRT
<213> 人工序列(artificial sequence)
<400> 47
Tyr Ala Ser Asn Leu Glu Ser
1 5
<210> 48
<211> 9
<212> PRT
<213> 人工序列(artificial sequence)
<400> 48
Gln His Ser Trp Glu Ile Pro Pro Thr
1 5
Claims (22)
1.一种靶向人补体蛋白C5的pH依赖性抗体的筛选方法,所述筛选方法在生物分子相互作用分析系统上进行,包括以下步骤:
(1)设置实验组1、对照组1、实验组2和对照组2,各组芯片在缓冲液中平衡,然后浸入人补体蛋白C5在缓冲液中的溶液以固化人补体蛋白C5;
(2)使实验组1、对照组1的芯片再次在缓冲液中平衡,然后浸入参考抗体在缓冲液中的溶液以使所述人补体蛋白C5结合所述参考抗体至饱和,再次在缓冲液中平衡,其中所述参考抗体为人补体蛋白C5的pH依赖性抗体;之后使实验组1的芯片浸入包含待测抗体的待测样品,同时使对照组1的芯片浸入不包含待测抗体的对照溶液;
使实验组2、对照组2的芯片再次在缓冲液中平衡,然后使实验组2的芯片浸入所述待测样品,同时使对照组2的芯片浸入所述对照溶液;
(3)检测各实验组和对照组的结合信号,计算(实验组1-对照组1)/(实验组2-对照组2)的比值,比值为<30%指示待测抗体为潜在的靶向人补体蛋白C5的pH依赖性抗体;
(4)检测步骤(3)得到的抗体在酸性和中性条件下与人补体蛋白C5的结合亲和力,在二者有差异的情况下,确认所述抗体为靶向人补体蛋白C5的pH依赖性抗体;
其中,所述生物分子相互作用分析系统基于生物膜干涉技术或表面离子共振技术。
2.根据权利要求1所述的筛选方法,其特征在于,所述生物分子相互作用分析系统为fortebio或biacore生物分子相互作用分析系统。
3.根据权利要求1所述的筛选方法,其特征在于,所述参考抗体包含重链可变区(HCVR)和轻链可变区(LCVR),所述重链可变区和轻链可变区包含选自以下的重链CDR和轻链CDR的组合:
(1)依次示于SEQ ID NO:13、14、15的HCDR1、HCDR2、HCDR3;和,依次示于SEQ ID NO:16、17、18的LCDR1、LCDR2、LCDR3;
(2)依次示于SEQ ID NO:19、20、21的HCDR1、HCDR2、HCDR3;和,依次示于SEQ ID NO:22、23、24的LCDR1、LCDR2、LCDR3;
(3)依次示于SEQ ID NO:25、26、27的HCDR1、HCDR2、HCDR3;和,依次示于SEQ ID NO:28、29、30的LCDR1、LCDR2、LCDR3;和
(4)依次示于SEQ ID NO:31、32、33的HCDR1、HCDR2、HCDR3;和,依次示于SEQ ID NO:34、35、36的LCDR1、LCDR2、LCDR3。
4.根据权利要求3所述的筛选方法,其特征在于,所述参考抗体包含选自以下的重链可变区和轻链可变区的组合:
(1)包含示于SEQ ID NO:1的氨基酸序列的重链可变区;和,包含示于SEQ ID NO:2的氨基酸序列的轻链可变区;
(2)包含示于SEQ ID NO:3的氨基酸序列的重链可变区;和,包含示于SEQ ID NO:4的氨基酸序列的轻链可变区;
(3)包含示于SEQ ID NO:5的氨基酸序列的重链可变区;和,包含示于SEQ ID NO:6的氨基酸序列的轻链可变区;和
(4)包含示于SEQ ID NO:7的氨基酸序列的重链可变区;和,包含示于SEQ ID NO:8的氨基酸序列的轻链可变区。
5.根据权利要求4所述的筛选方法,其特征在于,所述参考抗体为单克隆抗体或scFv、dsFv、(dsFv)2、Fab、Fab'、F(ab')2抗体。
6.根据权利要求5所述的筛选方法,其特征在于,所述单克隆抗体为人或鼠的IgG型。
7.根据权利要求1所述的筛选方法,其特征在于,所述待测样品为待测抗体在缓冲液中的溶液或包含待测抗体的细胞培养液;所述对照溶液为缓冲液或细胞培养基。
8.根据权利要求1所述的筛选方法,其特征在于,所述待测样品为待测抗体在缓冲液中的溶液或包含待测抗体的单克隆杂交瘤细胞培养上清;所述对照溶液为缓冲液或单克隆杂交瘤细胞培养基。
9.根据权利要求1至8中任一项所述的筛选方法,其特征在于,在步骤(1)和步骤(2)中,所述缓冲液为磷酸盐缓冲液+0.1%BSA+0.02%吐温20+0.05%Priclin300,pH 7.4。
10.根据权利要求1至8中任一项所述的筛选方法,其特征在于,在步骤(1)和步骤(2)中,所述平衡为将芯片在所述缓冲液中放置60s。
11.根据权利要求1至8中任一项所述的筛选方法,其特征在于,在步骤(1)中,固化补体蛋白C5至信号高度2nm。
12.根据权利要求1至8中任一项所述的筛选方法,其特征在于,在步骤(2)中,使实验组1的芯片浸入待测样品300s,并且使对照组1的芯片浸入对照溶液300s。
13.根据权利要求1至8中任一项所述的筛选方法,其特征在于,在步骤(2)中,使实验组2的芯片浸入待测样品300s,并且使对照组2的芯片浸入对照溶液300s。
14.根据权利要求1至8中任一项所述的筛选方法,其特征在于,所述补体C5蛋白在缓冲液中的浓度为50nM。
15.根据权利要求1至8中任一项所述的筛选方法,其特征在于,所述参考抗体在缓冲液中的浓度为100nM。
16.根据权利要求1至8中任一项所述的筛选方法,其特征在于,所述待测抗体在缓冲液中的浓度为100nM;或者,所述待测样品为稀释或未稀释的单克隆杂交瘤细胞培养上清。
17.根据权利要求16所述的筛选方法,其特征在于,所述待测样品为以人补体蛋白C5作为抗原免疫动物而得到的单克隆杂交瘤细胞的培养上清。
18.根据权利要求1所述的筛选方法,其特征在于,所述酸性条件为pH5.8,中性条件为pH 7.4。
19.一种靶向人补体蛋白C5的pH依赖性抗体,所述抗体包含重链可变区(HCVR)和轻链可变区(LCVR),所述重链可变区和轻链可变区包含选自以下的重链CDR和轻链CDR的组合:
(1)依次示于SEQ ID NO:19、20、21的HCDR1、HCDR2、HCDR3;和,依次示于SEQ ID NO:22、23、24的LCDR1、LCDR2、LCDR3;
(2)依次示于SEQ ID NO:25、26、27的HCDR1、HCDR2、HCDR3;和,依次示于SEQ ID NO:28、29、30的LCDR1、LCDR2、LCDR3;和
(3)依次示于SEQ ID NO:31、32、33的HCDR1、HCDR2、HCDR3;和,依次示于SEQ ID NO:34、35、36的LCDR1、LCDR2、LCDR3。
20.根据权利要求19所述的抗体,其特征在于,所述抗体包含选自以下的重链可变区和轻链可变区的组合:
(1)包含示于SEQ ID NO:3的氨基酸序列的重链可变区;和,包含示于SEQ ID NO:4的氨基酸序列的轻链可变区;
(2)包含示于SEQ ID NO:5的氨基酸序列的重链可变区;和,包含示于SEQ ID NO:6的氨基酸序列的轻链可变区;和
(3)包含示于SEQ ID NO:7的氨基酸序列的重链可变区;和,包含示于SEQ ID NO:8的氨基酸序列的轻链可变区。
21.根据权利要求19或20所述的抗体,其特征在于,所述抗体为单克隆抗体或scFv、dsFv、(dsFv)2、Fab、Fab'、F(ab')2抗体。
22.根据权利要求21所述的抗体,其特征在于,所述单克隆抗体为人或鼠的IgG抗体。
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