CN113621066A - 一种抗pd-1抗体及其晶体制备方法 - Google Patents
一种抗pd-1抗体及其晶体制备方法 Download PDFInfo
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Abstract
本发明涉及对抗肿瘤介导的免疫抑制,具体提供了结合人类程序性细胞死亡1(PD‑1)的抗体,其能够特异性结合PD‑1分子,结合之后能够阻断PD‑1与选自PD‑L1,PD‑L2或其组合的结合,其可单独或与化学疗法和其他癌症治疗法组合用于治疗癌症,提供PD‑1单抗的晶体,以及产生此类晶体的方法,和包含此类抗体晶体的组合物在癌症治疗中的用途,本发明通过提供PD‑1单抗晶体的实施方案产生适合于X射线衍射的晶体。
Description
技术领域
本发明涉及对抗肿瘤介导的免疫抑制。更具体地,本发明涉及结合人类程序性细胞死亡1(PD-1),且可单独以及与化学疗法和其他癌症疗法组合用于治疗癌症的抗体。
背景技术
PD-1被公认为是免疫调节和外周耐受维持中的重要分子。PD-1在幼稚的T、B和NKT细胞上适度表达,并通过淋巴细胞、单核细胞和髓细胞上的T/B细胞受体信号传导上调。
PD-1/人类程序性细胞死亡1配体1(PD-L1)途径是一种此类免疫检查点。人类PD-1在T细胞上发现,且PD-L1和人类程序性细胞死亡1配体2(PD-L2)与PD-1的结合抑制T细胞增殖和细胞因子产生。因此,肿瘤细胞的PD-L1和PD-L2的产生可使得其逃避T细胞监督。在例如卵巢癌、肾癌、结肠直肠癌、胰腺癌、肝癌和黑色素瘤的大样品集中,PD-L1的表达显示为与不良的预后和总体生存期降低相关。
本领域需要拮抗PD-1活性的高效治疗性抗体,其可用于产生对肿瘤的强力免疫反应。
发明内容
本发明的目的是解决上述现有技术的不足,提供一种抗PD-1抗体及其晶体制备方法。
本发明是通过以下技术方案实现的:
一种抗PD-1抗体,所述抗体的重链可变区的CDR-H1为SEQ ID NO:1所示的氨基酸序列,CDR-H2为SEQ ID NO:2所示的氨基酸序列,和CDR-H3为SEQ ID NO:3所示的氨基酸序列;以及所述抗体的轻链可变区的CDR-L1为SEQ ID NO:4所示的氨基酸序列,CDR-L2为SEQID NO:5所示的氨基酸序列,和CDR-L3为SEQ ID NO:6所示的氨基酸序列。
优选的,所述抗体的重链氨基酸序列如SEQ ID NO:7所示。
优选的,所述抗体的轻链氨基酸序列如SEQ ID NO:8所示。
一种药物组合物,该组合物含有上述任一项所述的抗体和药物学上可接受的载体。
本发明还保护上述抗体在制药中的应用。
本发明还保护上述抗体在制备治疗癌症药物中的应用。
本发明还保护上述抗体的晶体制备方法,在抗PD-1B8抗体溶液中加入结晶缓冲液,用悬滴法,将由1:1的体积比例混合的抗PD-1B8抗体和结晶溶剂组成的液滴滴在硅化玻璃片上,同时将结晶池液加入到池中,达到平衡后,密封,在18℃获得抗PD-1B8抗体晶体;其中,所述结晶缓冲液的成分包括pH 8.0、100mM的Tris,150mM NaCl和1mM二硫苏糖醇;所述结晶溶剂为0.05M磷酸二氢钾、20%w/v聚乙二醇8,000;结晶池液包含:20%w/v聚乙二醇3350,pH 8.0、100mMTris,100mM CaCl2,1.26mM硫酸铵,pH 4.5的乙酸钠和0.2M NaCl。
本发明提供了抗PD-1抗体,即PD-1B8抗体。本发明还提供了此抗体表达载体的宿主细胞。
术语“治疗”是指减慢、中断、阻止、缓解、停止、减小或逆转现有症状、病症、病况或疾病的进展或严重度。除非另有指明,否则如本文中关于抗体对人类PD-1的亲和力所使用的“结合”意指KD为小于约1×10-6M,优选小于约1×10-9M,如通过本领域中已知的常用方法,包括基本上如本文所述通过在37℃下使用表面等离子共振(SPR)生物传感器所测定。
“有效量”意指将引发研究人员、医师或其他临床医师所寻求的组织、系统、动物、哺乳动物或人类的生物或医学反应或对其的期望治疗效应的,本发明的抗体或包含本发明的抗体的药物组合物的量。抗体的有效量可根据诸如以下的因素而变化:个体的疾病状态、年龄、性别和体重以及抗体在该个体内引发期望反应的能力。有效量也是其中治疗有益效应胜过抗体的任何毒性或有害效应的量。
本发明的有益效果在于:
本发明提供一种抗体晶体的结晶条件以及其结构,该抗体具有高亲和力、更好的癌症治疗效果等优势。
附图说明
图1是人PD-1抗原SDS-PAGE电泳检测结果(其中,1,PD-1-Fc抗原融合蛋白;2,PD-1-Fc肠激酶酶切产物;3,PD-1胞外区抗原);
图2是鼠源抗PD-1单克隆抗体WB结果;
图3是抗体洗脱峰图;
图4是抗PD-1B8抗体纯度SDS-PAGE鉴定图;
图5是抗PD-1B8抗体的晶体照片;
图6是悬滴法示意图;
图7是抗PD-1B8抗体解析的晶体结构模型;
图8是抗PD-1B8抗体在小鼠给药后体重变化图;
图9是抗PD-1B8抗体在小鼠给药后肿瘤体积变化图;
图10是抗PD-1B8抗体在动物模型中的寿命对比图。
具体实施方式
以下通过实施例对本发明进行更具体的说明。但是本领域技术人员可以理解的是,以下的实施例仅是为了说明本发明的目的,而非用于限制本发明。
实施例1:PD-1抗原的制备
从南京金斯瑞公司合成人的PD-1的cDNA,Gene ID为5133,cDNA ID为NM_005018.2。在合成的胞外区PD-1基因后加上人源IgG1 Fc标签,并在两端引入Xba I,Bam HI两个限制性酶切位点连接到pTT5表达质粒,经测序验证正确。测序完的质粒转染Trans10(购自北京全式金生物技术有限公司),挑取单克隆,接种到1升LB液体培养基,至OD600为1时,离心收集菌体,用质粒大提试剂盒(购自Qiagen公司)提取质粒。
将测序鉴定正确的表达载体转染293F细胞(购自Invitrogen公司),37℃,5%CO2,130rpm/min培养7天后,离心收集上清。将上清于4000rpm离心10min,再用0.45μm滤膜过滤;滤液加入400mM NaCl;调整pH至8.0。样品经0.2μm滤膜再次过滤后,上样至已用PBS平衡好的5mLHiTrap Protein A柱;待样品上完后用PBS冲洗,流速5mL/min,紫外监测为水平。1M甘氨酸,pH 3.5洗脱,流速1mL/min,收集流出峰用Tris中和至pH 7.5。用超滤浓缩管浓缩洗脱峰换液至PBS中,由此得到PD-1-Fc抗原融合蛋白。将PD-1-Fc根据说明书用肠激酶(购自上海近岸蛋白科技有限公司)进行酶切,置于37℃12小时,酶切后产物过Protein A柱用于吸附切除的Fc标签,收集流出液,即为PD-1胞外区抗原。PD-1-Fc抗原融合蛋白和PD-1胞外区抗原SDS-PAGE电泳检测结果见图1。
实施例2:抗原免疫小鼠和杂交瘤筛选
本实验选用8周龄雌性BALB/c小鼠3只,采用腹腔注射法,用PD-1胞外区抗原与弗氏完全佐剂混合免疫小鼠,每周1次,共3次。最后一次免疫后一周测量小鼠血清效价,满足条件效价大于8K后加强免疫一次,结果显示3只小鼠全部满足效价,3天后处死小鼠,取小鼠脾脏,经碾磨后获得脾细胞群。小鼠血清效价ELISA检测结果见表1。
表1 20871小鼠免疫血清ELISA检测
利用流式细胞术(FACS)筛选出抗人PD-1抗体的B细胞,置于RPMI1640培养基中,加入骨髓瘤细胞(SP2/0)混匀,采用50%PEG溶液进行细胞融合。融合后的细胞经过适当稀释,分置于多块96孔培养板中培养,加入HAT选择性培养基,使未融合的B细胞和骨髓瘤细胞死亡,得到杂交瘤细胞。培养2周后收集96孔板细胞培养上清,与铺有PD-1抗原的96孔酶标板结合1小时,加入抗鼠/HRP二抗孵育1小时,最后加入TMB显色试剂反应10分钟,用酶标仪测定450nm处的光吸收值,选出与PD-1具有结合活性的杂交瘤细胞(初筛:12块96孔板,得到OD值≥0.5的孔数42个)。随后进行流式细胞术(FACS)筛选,选出具有PD-1/PD-L1阻断活性的杂交瘤细胞,在进行有限稀释法亚克隆,最终得到一株抗PD-1鼠源单克隆抗体。有限稀释法亚克隆结果见表2,亲和力鉴定结果见表3,免疫印迹结果见图2。
表2阳性克隆孔板位
序号 | 阳性克隆 | 96孔板位 | 384孔板位 | OD值 |
1 | 2G8-1N8 | 2G8 | 1N8 | 1.022 |
表3亲和力鉴定
实施例3:抗PD-1鼠源抗体可变区基因调取
选取抗PD-1杂交瘤克隆,采用Trizol方法抽提总RNA,利用抗体亚型(Isotype)特异性引物或者通用引物进行反转录PCR,分别扩增抗体轻链可变区(VL)和重链可变区(VH)基因,随后连接至克隆载体进行DNA测序分析。最终获得完整VL和VH的DNA序列,并翻译成相应的氨基酸序列。
实施例4:抗PD-1鼠源单抗可变区基因人源化改造
使用Ig Blast(http://www.ncbi.nlm.nih.gov/igblast)分析与小鼠PD-1抗体的基因同源性较高的人种系基因(germlinegene)。抗PD-1人源化抗体的重链氨基酸序列为SEQ ID NO:7;其中,重链可变区的CDR-H1、CDR-H2和CDR-H3氨基酸序列分别为SEQ ID NO:1-3。抗PD-1人源化抗体的轻链氨基酸序列为SEQ ID NO:8;其中,轻链可变区的CDR-L1、CDR-L2和CDR-L3氨基酸序列分别为SEQ ID NO:4-6。
实施例5:抗PD-1人源化抗体的亲和力成熟
针对抗PD-1人源化抗体的5个CDR区(L1,L3,H1,H2和H3)分别设计了抗体突变体库,突变位点覆盖了所有CDR区的非保守位点。采用SOE-PCR反应获得单链抗体(scFv)基因,经DNA凝胶回收和酶切之后,与酶切后的pCANTAB-5E噬菌体展示载体连接,电转化TG1感受态细菌,获得5个含有CDR突变的单链抗体库。通过感染M13KO7辅助噬菌体,制作重组噬菌体,共进行了三轮淘洗,保留并富集结合能力强的抗体突变体。每轮淘洗分别将重组噬菌体与生物素标记的重组人PD-1抗原结合2小时,再加入链亲和素磁珠结合30分钟,先后用2%TPBS、1%TPBS和PBS清洗5次,每次5分钟,淘洗之后立刻感染TG1细胞,用于下一轮重组噬菌体制备。挑选三轮淘洗后富集的TG1单克隆,制备重组噬菌体上清,与铺有1μg/mL PD-1抗原的96孔酶标板结合1小时,加入M13/HRP二抗孵育1小时,最后加入OPD进行显色反应10分钟,用酶标仪测定490nm处的光吸收值。对数据进行分析后,计算含有抗体突变体的相对亲和力,分别从L3,H1,H3突变体库中筛选得到3个,6个,5个亲和力有明显提高的克隆,最终从H3突变体库中选择1个亲和力最高的克隆B8开展下一步研究。
实施例6:抗PD-1B8抗体的制备
1.抗PD-1B8抗体的表达与纯化
1)收获上清:将实施例5中得到的B8转染HEK293细胞,转染后加新鲜培养基,得到250mL细胞培养液,1000rpm,10min离心,获得细胞表达上清。向上清中加入2M Tris,pH 8.0母液2.5mL,1.6g NaCl,搅拌均匀,使用0.22μm滤膜过滤。
2)Protein G亲和柱纯化:使用Buffer D平衡Protein G柱5-10个柱体积,样品上样,速度为3mL/min。完成上样后,使用80mL Buffer D清洗柱子。使用0.1M甘氨酸pH 2.7直接洗脱(图3),15mL一管收集洗脱液,收集管提前加入800μL 2M Tris pH 8.0母液和900μL2M NaCl母液,将洗脱液迅速混合均匀。
2.纯度鉴定
将收集洗脱的样品,制样,SDS-PAGE凝胶电泳,鉴定目标蛋白纯度(图4)。抗PD-1B8抗体纯度超过90%。浓缩样品至1-2mL,加入PBS,再浓缩,重复3次,测定浓度,将抗PD-1B8抗体样品浓度稀释至0.5mg/mL,200μL/管,13管,共1.3mg,液氮速冻,-80℃保存。
3.结晶
纯化抗PD-1B8抗体,通过分子筛凝胶层析,纯化获得抗PD-1B8抗体;使用Amicorn30KD(millipore公司)浓缩管超滤浓缩蛋白至10mg/mL,蛋白保存在结晶缓冲液中。使用悬滴法(如图6)筛选和优化蛋白晶体,池液体积为400mL,悬滴为1μL蛋白液和1μL结晶溶剂混合,悬滴法的晶体在结晶液滴中更容易被捞出。将由抗PD-1B8抗体和结晶溶剂的混合物(1:1的体积比例)组成的液滴滴在硅化玻璃片上,同时将结晶池液加入到池中。为了达到平衡,液滴开始挥发。随着液滴不断挥发,样品会相对过饱和,浓度会增加。随着不断挥发,样品和试剂的浓度都会增加。当液滴中的试剂浓度与池液的浓度大致相同时,即达到平衡。密封后,放置于18℃培养箱中;1周后观察晶体生长情况;获得抗PD-1B8抗体晶体,在普通显微镜下200倍观察(图5)。
其中,结晶缓冲液主要为100mMTris(pH 8.0)、150mM NaCl、1mM DTT,结晶溶剂为0.05M磷酸二氢钾、20%w/v聚乙二醇8,000。结晶池液包含以下混合物:20%PEG 3350、100mMTris(pH 8.0)、100mM CaCl2、1.26mM硫酸铵、乙酸钠(pH 4.5)和0.2M NaCl。
4.X-ray衍射
将晶体快速浸泡在含有20%甘油的结晶池液中,使用结晶LOOP迅速将晶体捞起,并在液态氮气中快速冷藏。晶体衍射数据是从中国上海同步辐射光源(SSRF)BL18 U光束线的收集,在100K的温度下,使用衍射波长为 衍射数据用HKL3000处理。相关统计数据汇总在表4中。
表4.数据采集和数据表格统计表
5.结构解析建模和验证
相位解析使用分子置换法,在分子置换中,使用FAB模板作为搜索模板;用分子置换法解析结构PDB 6TCN作为搜索模型;初始模型是使用PHENIX.autobuild搭建的。使用程序COOT进行模型的手动调整,并通过PHENIX.refinement和Refmac5对模型进行优化。使用PROCHECK检查结构的立体化学质量。大多数残基位于有利和允许的区域。细化导致模型具有出色的统计和立体化学角度(表5);使用蛋白质数据库ADIT服务器进行结构验证。中文简要方法总结见表6;所有结构图像均使用PyMOL分子图形系统(LLC)、UCSFChimera和BIOVIA Discovery Studio制备。解析结构如图7。
表5.数据收集和验证表格
表6.衍射和模型构建方法中文总结
实施例7:ELISA BINDING检测抗PD-1B8抗体与PD-1的结合能力
抗PD-1B8抗体由浓度3μg/mL起3倍梯度稀释,稀释12个梯度,2个平行实验。ELISA检测结果如表7所示,结果显示:抗PD-1B8抗体与PD-1具有较强的结合能力。
表7 ELISA检测抗PD-1B8抗体与PD-1的结合能力
实施例8:SPR/BLI Kon/Koff分析
在25℃进行作为配体的人类PD-1-Fc至传感器芯片表面上的固定。将本发明的抗体用作分析物,并注射至固定有人类PD-1-Fc的传感器芯片表面上。样品分析物在从其起始浓度(12.5μg/mL)起的2倍连续稀释液中运行,总计2种稀释样品。分析在37℃下进行。使用Biacore T200评估软件分析结合动力学。数据以空白流动室为基准,并将数据拟合至1:1结合模型。
上述实验,分析结果见表8:
表8抗PD-1B8抗体与PD-1的结合(SPR法)
抗PD-1B8抗体与PD-1的结合检测中,结果显示Ka=9.18×104M-1s-1,
Kd=7.12×10-4s-1,抗PD-1B8抗体以774pM的KD结合PD-1。
实施例9:动物实验
试验药物:PDAB(即PD-1B8)抗体来源于公司生产组,Nivolumab市场购买(来自百时美施贵宝)。
表9实验设计方案
组号 | 动物数 | 给药组 | 给药剂量(mg/kg) | 给药途径 | 给药频率 |
G1 | 5 | 生理盐水 | — | i.p | BIW*3 |
G2 | 5 | PDAB | 10 | i.p | BIW*3 |
G3 | 5 | PDAB | 3 | i.p | BIW*3 |
G4 | 5 | PDAB | 1 | i.p | BIW*3 |
G5 | 5 | BMS Opdivo | 10 | i.P | BIW*3 |
G6 | 5 | BMS Opdivo | 3 | i.p | BIW*3 |
G7 | 5 | BMSOpdivo | 1 | i.p | BIW*3 |
备注:给药容积:10μL/g BIW:每周给药两次
表10药物配置
自中国科学院典型培养物保藏委员会细胞库购入MC38结肠直肠癌细胞,大量培养扩增后背部植入C57BL/6转人PD-1基因小鼠(2×106个细胞/小鼠),注射MC38细胞后,成瘤性及均一性较好,后期有个别组肿瘤出现溃烂。
至肿瘤体积均值约为123mm3,决定给药,入组后每组平均肿瘤体积在119mm3。3周后给药结束。各种指标详见表11。小鼠给药后体重变化和肿瘤体积变化如图8和图9。停药后继续饲养小鼠至81天时,处死小鼠,期间小鼠寿命对比如图10。
表11给药前后小鼠体征指标
注:TGI肿瘤生长抑制率40%,相对肿瘤增殖率T/C≤0.6
以上所述实施方式仅仅是对本发明的优选实施方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案作出的各种变形和改进,均应落入本发明的权利要求书确定的保护范围内。
SEQUENCE LISTING
<110> 安徽瀚海博兴生物技术有限公司
<120> 一种抗PD-1抗体及其晶体制备方法
<130> 2021
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<213> 人工序列
<400> 8
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Asn Asn Tyr
20 25 30
Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Ser Leu Ile
35 40 45
Tyr Arg Ala Ser Arg Leu Val Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Thr Leu
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
Claims (7)
1.一种抗PD-1抗体,其特征在于:所述抗体的重链可变区的CDR-H1为SEQ ID NO:1所示的氨基酸序列,CDR-H2为SEQ ID NO:2所示的氨基酸序列,和CDR-H3为SEQ ID NO:3所示的氨基酸序列;以及所述抗体的轻链可变区的CDR-L1为SEQ ID NO:4所示的氨基酸序列,CDR-L2为SEQ ID NO:5所示的氨基酸序列,和CDR-L3为SEQ ID NO:6所示的氨基酸序列。
2.根据权利要求1所述的一种抗PD-1抗体,其特征在于:所述抗体的重链氨基酸序列如SEQ ID NO:7所示。
3.根据权利要求1所述的一种抗PD-1抗体,其特征在于:所述抗体的轻链氨基酸序列如SEQ ID NO:8所示。
4.一种药物组合物,其特征在于:该组合物含有权利要求1~6任一项所述的抗体和药物学上可接受的载体。
5.权利要求1~3任一项所述的抗体在制药中的应用。
6.权利要求1~3任一项所述的抗体在制备治疗癌症药物中的应用。
7.权利要求1~3任一项所述的抗体的晶体制备方法,其特征在于:在抗PD-1B8抗体溶液中加入结晶缓冲液,用悬滴法,将由1:1的体积比例混合的抗PD-1B8抗体和结晶溶剂组成的液滴滴在硅化玻璃片上,同时将结晶池液加入到池中,达到平衡后,密封,在18℃获得抗PD-1B8抗体晶体;其中,所述结晶缓冲液的成分包括pH 8.0、100mM的Tris,150mM NaCl和1mM二硫苏糖醇;所述结晶溶剂为0.05M磷酸二氢钾、20%w/v聚乙二醇8,000;结晶池液包含:20%w/v聚乙二醇3350,pH 8.0、100mMTris,100mM CaCl2,1.26mM硫酸铵,pH 4.5的乙酸钠和0.2M NaCl。
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