CN108285485A - 一种抗pd-1的单域抗体及其应用 - Google Patents
一种抗pd-1的单域抗体及其应用 Download PDFInfo
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- CN108285485A CN108285485A CN201810016014.0A CN201810016014A CN108285485A CN 108285485 A CN108285485 A CN 108285485A CN 201810016014 A CN201810016014 A CN 201810016014A CN 108285485 A CN108285485 A CN 108285485A
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Abstract
本发明提供了一种新的抗PD‑1的单域抗体。本发明的抗体具有SEQ ID NO.1或SEQ ID NO.2或SEQ ID NO.3所示的氨基酸序列。本发明进一步提供了编码该抗PD‑1单域抗体的核苷酸序列、包含该核苷酸序列的表达载体和宿主细胞。本发明的抗PD‑1单域抗体可与PD‑1特异性结合，可以用于PD‑1的测定，以及研发PD‑1治疗性抗体药物。
Description
技术领域
本发明属于生物技术领域，涉及抗PD-1抗体，具体涉及新的抗PD-1的单域抗体及其氨基酸、核苷酸编码序列，本发明还涉及包含该核苷酸编码序列的表达载体和宿主细胞。
背景技术
PD-1(程序性死亡受体-1)是一种重要的免疫抑制分子，是CD28受体家族的成员。PD-1有两种细胞表面糖蛋白配体：PD-L1(程序性死亡受体配体-1，又称作CD274或者B7-H1)和PD-L2(程序性死亡受体配体-2，又称作CD273或者B7-DC)。
PD-1与PD-L1或PD-L2特异性相结合，通过招募Src癌基因同源的酪氨酸蛋白磷酸酶-2(The Src homology phosphotyrosylphosphatase 2，SHP-2)，进而去减弱抗原识别受体(BCR/TCR)的信号通路，负调控免疫应答过程，从而来调控机体内的免疫平衡。PD-1的两种配体PD-Ls广泛表达在多种细胞表面，因此PD-1/PD-Ls途径在抗病毒感染，免疫排斥和肿瘤免疫各个方面均发挥着重要作用。
多种肿瘤及肿瘤相关细胞通过产生大量的PD-Ls去抑制细胞毒性T细胞的功能，避免免疫系统的杀伤。PD-1/PD-Ls通路的阻断可加速肿瘤清除。阻断PD-1/PD-Ls通路还可以抑制癌细胞的转移。目前，已有PD-1单抗Nivolumab和Lambrolizumab上市，用于治疗非小细胞肺癌和黑色素瘤。但由于单抗分子自身具有分子量较大、组织穿透的能力较弱以及较难结合到位于细胞表面的受体和配体结合部位等缺点。此外较高的经济成本和靶向针对性差等因素使得单抗药物在临床治疗上仍有许多不足。
重链抗体(HCAbs)是发现于骆驼和软骨鱼等体内的一种天然缺失轻链，仅有重链的抗体。这类抗体的抗原结合位点仅由重链可变区单域结构域组成，称为单域重链抗体(variable domain ofthe heavy chain ofHCAbs，VHH)。由于天然缺失轻链，单域重链抗体靠仅有的3个互补决定区(CDRs)就具备了特异的抗原结合能力及高亲和力，而普通的抗体则需要6个CDRs才具备与抗原结合的能力。VHH是目前可以得到的具有完整功能的最小抗体分子片段。其可溶性较高，高表达于大肠杆菌细胞，易于制备和耦联于其他效应分子且成本较低。此外，在与抗原结合方面，单域抗体能结合到酶活中心、受体和配体结合间的裂隙等常规抗体所不易结合的位点。纳米抗体的这些优点使其在抗体结构研究、抗肿瘤治疗和新药研发等方面具有巨大潜力。
尽管已有多株抗PD-1单抗被用于临床实验以及PD-1治疗性药物，但抗PD-1的单域抗体在国内外还未被报道。
发明内容
本发明所要解决的技术问题是提供一种抗PD-1的单域抗体，同时提供了该单域抗体的编码序列，含有该编码序列的载体和宿主细胞。
本发明解决上述技术问题的技术方案如下：
本发明使用PD-1蛋白免疫双峰驼并利用噬菌体展示技术建立了PD-1单域抗体库，随机选择40个克隆进行测序，结果表明38个克隆具有编码重链抗体的可变区基因，计算得到库容为2.6×108的骆驼免疫单域抗体库，从中筛选出具有高特异性和抗原结合能力的单域抗体(VHH)。
一种抗PD-1的单域抗体，其特征在于所述单域抗体的氨基酸序列为以下序列之一：
SEQ ID NO.1：
MetAla Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala GlyGly Ser Leu Arg Leu Ser Cys Val Ala Ser Gln Tyr Thr TyrAsn Thr Val Gly TrpPhe Arg GlnAla Pro Gly Lys Glu Arg Glu Gly Val Ala Gly Ile TyrAsn Gly Gly AspGln Thr Tyr Tyr Ser Glu Ser Ala Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn AlaLys Arg Thr Val Tyr Leu Gln MetAsn Ser Leu Lys Pro GluAsp ThrAla Met Tyr TyrCys Ala Ala Gly Arg Leu Ile Val Ser Gly Arg Trp Ser Met Thr Lys Glu Glu TyrGlnTyrTrp Gly Gln Gly Thr GlnVal ThrVal Ser Ser
SEQ ID NO.2：
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly SerLeuArg Leu Ser Cys AlaAla Ser Gly Ser Thr Val ArgArg Arg Cys Met Gly Trp PheArg Gln Ala Pro Gly Lys Glu Arg Glu Glu Val Ala Ile Val Asp Asn Glu Gly IleGlu Gln Tyr Ala Asp Phe Val Lys GlyArg Phe Thr Ile SerArg Asp Asn Ala Lys AsnThr Leu Tyr Leu Gln MetAsn Ser Leu Lys Pro GluAsp ThrAla Met Tyr Tyr Cys AlaMetAla Pro Gly Tyr Thr Pro Thr Gly Cys Leu Val TyrAsn Thr Trp Gly Gln GlyThrGlnVal ThrVal Ser Ser
SEQ ID NO.3：
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly SerLeuArg Leu Ser Cys Ala Val Ser Gly Gly Thr ThrArgArgArg Cys Met Gly Trp PheArg Gln Ala Pro Gly Lys Glu Arg Glu Glu Val Ala Ser Tyr Asp Asn Gln Gly IleIle Lys Tyr Ala Asp Phe Val Lys Gly Arg Phe Thr Ile SerArg Asp Asn Ala LysAsn Thr Leu Tyr Leu Gln MetAsn Thr Leu Lys Pro GluAsp ThrAla Met Tyr Tyr CysAla MetAla Pro Gly TyrAsp AsnArg Gly Cys Leu Val TyrAsn Thr Trp Gly GlnGlyThr GlnVal ThrVal Ser Ser
进一步，本发明还提供了编码以上所述PD-1单域抗体的核苷酸序列，所述核苷酸序列为以下所述序列之一：
SEQ ID NO.4
SEQ ID NO.5
SEQ ID NO.6
进一步，本发明还提供了一种含有以上任一项所述的核苷酸序列的重组表达载体。
进一步，本发明还提供了一种含有以上任一所述的核苷酸序列的宿主细胞，其特征在于，所述的宿主细胞含有所述重组表达载体，且能够表达所述单域抗体。
本发明的有益效果是，本发明对得到的编码所述双峰驼单域抗体的基因进行了原核表达和纯化，通过特异性筛选，首次获得3个新的抗PD-1的单域抗体，本发明制备的基于双峰驼单域重链抗体的抗PD-1单域抗体具有分子量小、结构单一、穿透性好，特异性强，亲和力高等特点。
进一步，本发明提供了一种所述的单域抗体以及编码该单域抗体的所述核酸序列在制备PD-1治疗性药物中的应用。
采用上述进一步方案的有益效果是，本发明获得了PD-1特异性的单域抗体基因，使得所述单域抗体能在工程菌中大量表达，在PD-1治疗性抗体药物的制备中具有广阔应用前景提供可能。
附图说明
图1为本发明血清效价实验结果；
图2为本发明VHH噬菌体文库插入片段阳性率；
图3为本发明单域抗体VHH-B7、VHH-H5、VHH-H12重组蛋白表达情况以及纯化情况的SDS-PAGE电泳检测结果，其中泳道M为标记物；
图4为本发明Western Blot鉴定VHH-B7、VHH-H5、VHH-H12单域抗体的抗原特异性；
图5A、图5B和图5C为本发明单域抗体的抗原结合活性的ELISA检测结果，其中，图5A为VHH-B7的抗原结合活性，图5B为VHH-H5的抗原结合活性，图5C为VHH-H12的抗原结合活性；
图6A、图6B和图6C为本发明单域抗体在不同抗原包被量下的ELISA曲线，其中，图6A为VHH-B7的ELISA检测结果，图6B为VHH-H5的ELISA检测结果，图6C为VHH-H12的ELISA检测结果；
图7A、图7B和图7C为本发明单域抗体的模拟亲和力常数曲线，其中，图7A为VHH-B7的模拟亲和力常数曲线，图7B为VHH-H5的模拟亲和力常数曲线，图7C为VHH-H12的模拟亲和力常数曲线。
具体实施方式
以下结合附图及具体实施例对本发明作进一步的说明，所举实例只用于解释本发明，并非用于限定本发明的范围。
实施例1单克隆噬菌体抗体的筛选
利用噬菌体展示技术对双峰驼噬菌体抗体库进行多轮富集筛选，以PD-1蛋白为抗原进行了4轮“吸附-洗脱-扩增”的富集筛选。
具体方法如下：
1、PD-1蛋白免疫双峰驼及免疫响应测试
PD-1蛋白免疫新疆双峰驼：每次注射的蛋白量为600μg，初次免疫注射使用的佐剂为完全弗式佐剂，后续增强免疫改用不完全弗式佐剂，每次均与PD-1蛋白等体积充分混匀，每间隔14d免疫1次，共计6次，同时抽取免疫前后血液，制取血清存于-20℃。
免疫响应测试：测定骆驼血清抗PD-1蛋白的血清效价，ELISA板中包被PD-1抗原4℃过夜，包被的抗原浓度为2.5μg/ml，连续倍比稀释采集的免疫前和免疫后双峰驼的血清，将其作为一抗加入，羊抗驼IgG(H+L)-HRP为二抗(1∶3000)，TMB为底物，测定450nm吸光值。从1:2000开始倍比稀释骆驼血清至1：64000，结果如图1所示，抗体的滴度最少为1∶32000。
2、双峰驼外周血淋巴细胞总RNA的提取和VHH噬菌体展示文库的构建
采集骆驼的外周血并提取淋巴细胞，然后提取总RNA(TRIZOL法)，通过反转录得到的cDNA充当VHH基因扩增中的模板，用巢式聚合酶链反应扩增VHH片段。一轮PCR使用Call01-leader和Call02-CH2分别作为上游和下游引物进行扩增，反应结束后，琼脂糖凝胶电泳检测获得700bp大小的VHH片段，以回收的VHH片段为模板，使用PMCF和VHH-back分别作为上游和下游引物进行扩增，二轮扩增得到大小为450bp的VHH片段。将纯化得到的VHH基因片段与pMECS载体分别用QuickCutTM Not I和QuickCutTM PstI进行双酶切，用T4DNA连接酶连接，16℃恒温过夜，次日转化至TG1，得到抗体库。随机挑取了40个单克隆进行菌液PCR，如图2所示，其中36个表现为阳性，文库阳性率为90％，且基因序列均不同，文库多样性为100％，梯度稀释菌液后涂板，稀释梯度为10-5培养皿中生长的菌落数有260个，则噬菌体展示文库库容为2.6×108cfu/ml。引物Call01-leader、Call02-CH2、PMCF和VHH-back的序列分别为SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9和SEQ ID NO.10。
| 引物 | 引物序列 |
| Call01-leader | SEQ ID NO.7：gtcctggctgctcttctacaag |
| Call02-CH2 | SEQ ID NO.8：ggtacgtgctgttgaactgttcc |
| PMCF | SEQ ID NO.9：ctagtgcggccgctgaggagacggtgacctgggt |
| VHH-back | SEQ ID NO.10：gatgtgcagctgcaggagtctggtggagg |
3、单克隆噬菌体抗体的筛选
(1)噬菌体抗体库的扩增：
取200μl宿主菌液，接种到40ml 2×YTAG(50μg/ml氨苄青霉素，2％葡萄糖)培养基中，37℃震荡培养至至对数期，加4ml辅助噬菌体M13K07，37℃静置15min，然后放置于摇床，37℃，250r/min培养。转移菌液至50ml离心管，4℃，4000rpm，离心10min，弃去上清。然后用新鲜的2×YTAK(50μg/ml氨苄青霉素，50μg/ml卡那霉素)培养基重新悬浮沉淀，30℃，250r/min过夜培养。将过夜的菌液转移到50ml的离心管中，4000rpm，4℃，离心10min。吸取上清转移至新的50ml高速离心管，加入适宜5×PEG/NaCl(20％PEG/2.5M NaCl)冰浴30min以上。4℃，12000g，离心15min。除去上清，完全晾干后用PBS重新悬浮沉淀，转移到1.5ml的EP管中。4℃，12000g，离心2min，将上清转移新的1.5ml的EP管中，再次加入5×PEG/NaCl进行沉降，4℃，12000g，离心15min。尽可能吸取上清，用PBS重新悬浮沉淀。4℃，12000g，离心1min，上清即为扩增后的噬菌体抗体库，转上清到新的1.5ml EP管中，4℃保存。
(2)抗体库的富集
将定量后的PD-1蛋白稀释至10μg/ml，100μL/well加入到96孔酶标板，4℃孵育过夜。次日每孔加200μl 2％BSA进行封闭，37℃孵育1h。PBST(0.5％Tween20)洗涤三次后每孔加入100μl扩增分离得到的噬菌体，37℃，2h。然后分别用PBST洗6次，PBS洗2次洗涤后完全拍干液体，100μl/孔加Glycine-Hcl洗脱缓冲液，37℃温育10min，收集洗脱液，加入适量Tris-Hcl，使pH＝7.0左右。取洗脱下来的噬菌体，按10-1至10-7梯度稀释后等比例加到新鲜TG1菌液中，37℃感染约30min。取1ml洗脱液感染10ml生长处于对数期的TG1菌液，离心后重悬浮沉淀涂大板，次日刮取大板上的菌体，甘油保存用于下一轮筛选。亲和筛选共进行三轮。包被的抗原量每轮依次递减，二轮为5μg/ml，三轮为1μg/ml。
(3)ELISA法检测单克隆抗体与PD-1的结合
三轮富集后，在三轮输出板上挑去95个克隆进行ELISA的检测，在96孔深孔板中加入300ul 2×YTAG(50μg/ml Ampicillin，2％Glucose)，分别挑取第三轮筛选后输出平板上的单克隆接种到孔中；30℃，220rpm过夜培养作为母板。另取一个96孔深孔板，加入500μl 2×YTA，每孔接种20μl母板菌液，对应加入各孔中，37℃，220rpm，摇菌4h。加入0.01mmol/LIPTG，30℃，220rpm过夜诱导。
分别稀释PD-1抗原和BSA至终浓度为2μg/ml，分别包被96孔ELISA板，4℃孵育过夜。次日取出，PBST洗3次，然后每孔加入200μl 5％质量浓度的BSA封闭，37℃封闭2h，封闭期间1000g，4℃，10min离心过夜诱导的菌液，作为一抗备用。封闭结束后，PBST洗3次，每孔各加入30μl 3％浓度的BSA和90μl离心后的菌液上清作为一抗，37℃孵育2h。二抗采用HRP-Anti-HA-tag鼠单抗(1∶5000)。TMB作底物，450nm测吸光值。
95个克隆中进行ELISA检测，共有46个阳性克隆，可以与PD-1特异性结合。
实施例2阳性克隆NDA序列测定及单域抗体DNA序列分析
自46个阳性克隆中随机挑选30个克隆进行测序，27个测序成功。通过VBASE2比对，27个克隆共包含6个不同的序列。每种序列均有2次以上的重复，剔除结构上有残缺的序列后，选取了VHH-B7(重复4次)、VHH-H5(重复5次)及VHH-H12(重复2次)的克隆构建至pET22b载体并转化到BL21进行可溶性表达。VHH-B7、VHH-H5和VHH-H12的核苷酸序列分别为SEQ IDNO.4、SEQ ID NO.5和SEQ ID NO.6。
实施例3抗PD-1单域抗体的表达和纯化
分别提取pMECS-PD-1-VHH重组质粒和pET22b的质粒，使用Quick cut NotⅠ和Quick cut NocⅠ进行双酶切，切取凝胶，回收目的片断和酶切后载体，T4DNA连接酶连接目的基因片断和载体。转化连接产物至BL21(DE3)感受态，提取pET22b-VHH-B7，pET22b-VHH-H5和pET22b-VHH-H12的质粒，用QUICK Cut NotⅠ和QUICK Cut NocⅠ双酶切，跑1.5％琼脂糖凝胶鉴定条带完全正确，同时将转化子进行测序。选取阳性克隆培养，加入IPTG后，150r/min，16℃过夜诱导蛋白表达。将诱导后的菌液离心，超声后再12000rpm离心取上清，使用Ni柱亲和层析纯化His-VHH-B7-c-Myc、His-VHH-H5-c-Myc和His-VHH-H12-c-Myc重组抗体蛋白，以15％SDS-PAGE检测重组蛋白的表达情况，结果如图3显示，得到了较高纯度的3种单域抗体。
实施例4单域抗体的抗原特异性检测
Western Blot检测抗PD-1抗体与抗原的特异性结合，一抗为筛选出的纳米抗体，，二抗为鼠抗c-Myc-HRP单抗(1:2000)。阳性对照为鼠源抗PD-1单抗(1：1000)，阴性为无关蛋白CD-47。采用ECL显色，Las-4000显影。结果如图4所示，3种单域抗体均能特异性结合抗原PD-1。
实施例5ELISA检测单域抗体的抗原结合活性
抗原PD-1包被浓度为2μg/ml。抗体稀释终浓度为：5μg/ml,2.5μg/ml,1.25μg/ml,0.625μg/ml,0.313μg/ml,0.156μg/ml，0.078μg/ml共7个梯度。阳性对照为鼠源抗PD-1单克隆抗体，浓度为2μg/ml，阴性抗原为BSA。单域抗体VHH-B7、VHH-H5和VHH-H12的ELISA结果分别如图5A、图5B和图5C所示，3个单域抗体在各浓度下均可与PD-1蛋白结合，且结合活性高于抗PD-1鼠单抗。
实施例6非竞争ELISA法测定单域抗体亲和常数
用浓度梯度分别为2μg/ml,1μg/ml,0.5μg/ml和0.25μg/ml的PD-1抗原包被ELISA板，将抗PD-1单域抗体浓度调至10-10mol/L水平，用3％BSA溶液倍比稀释1：2到1：128，100μl/孔加至相应的反应孔中作一抗，Anti-c-myc鼠单抗作二抗，三抗为羊抗鼠IgG(H+L)-HRP，用TMB显色，在450nm处测吸光值。抗体亲和常数计算：按抗原和抗体结合的S形曲线，得出半数吸光值所对应的纳米抗体的量浓度，代到KA＝(n-1)/(nAb'-Ab)公式中求解亲和力常数，公式中Ab和Ab'表示当抗原浓度为Ag和Ag'时，其半数吸光值所对应的纳米抗体的量浓度(mol/L)，且n＝Ag/Ag'。
单域抗体VHH-B7、VHH-H5和VHH-H12的ELISA结果分别如图6A、图6B和图6C所示，分别选抗原的两个浓度，对亲和常数测定的曲线模拟分别如图7A、图7B和图7C所示，求得相应的6个半数吸光值相对应的抗体的量浓度，代入公式KA＝(n-1)/(nAb'-Ab)计算亲和常数，B7,H5和H12这3种抗体的亲和力常数分别为KA＝2.31×1011L/mol，KA＝2.62×1011L/mol和KA＝4.79×1011L/mol。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
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Claims (6)
1.一种抗PD-1的单域抗体，其特征在于，所述单域抗体的氨基酸序列为SEQ ID NO.1或SEQ ID NO.2或SEQ ID NO.3。
2.一种编码权利要求1所述抗PD-1的单域抗体的核苷酸序列，其特征在于，所述的核苷酸序列为SEQ ID NO.4或SEQ ID NO.5或SEQ ID NO.6。
3.一种含有权利要求2所述的核苷酸序列的重组表达载体。
4.一种含有权利要求3所述的重组表达载体的宿主细胞，其特征在于，所述宿主细胞能够表达所述抗PD-1的单域抗体。
5.权利要求1所述的单域抗体在制备PD-1治疗性抗体药物中的应用。
6.权利要求2所述的核苷酸序列在制备PD-1治疗性抗体药物中的应用。
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| CN110003336B (zh) * | 2019-04-12 | 2023-05-12 | 深圳普瑞金生物药业股份有限公司 | Pd-1单域抗体、核苷酸序列及试剂盒 |
| WO2021043261A1 (zh) * | 2019-09-06 | 2021-03-11 | 北京拓界生物医药科技有限公司 | 抗pd-1单域抗体、其衍生蛋白及其医药用途 |
| CN114222759A (zh) * | 2019-09-06 | 2022-03-22 | 北京拓界生物医药科技有限公司 | 抗pd-1单域抗体、其衍生蛋白及其医药用途 |
| WO2022012639A1 (zh) * | 2020-07-16 | 2022-01-20 | 和铂医药(上海)有限责任公司 | Pd-1抗原结合蛋白及其应用 |
| WO2022141378A1 (zh) * | 2020-12-31 | 2022-07-07 | 浙江道尔生物科技有限公司 | 一种抗pd-1的单域抗体 |
| WO2022152222A1 (zh) * | 2021-01-14 | 2022-07-21 | 立凌生物制药(苏州)有限公司 | 靶向pd-1的单域抗体及其衍生物和用途 |
| WO2022188801A1 (zh) * | 2021-03-10 | 2022-09-15 | 北京拓界生物医药科技有限公司 | Pd-1结合蛋白及其医药用途 |
| WO2023116781A1 (zh) * | 2021-12-21 | 2023-06-29 | 博生吉医药科技(苏州)有限公司 | 一种新型pd1单域抗体的开发 |
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