CN110982824B - 拮抗pd-1的抗体类似物bp基因及蛋白和应用 - Google Patents
拮抗pd-1的抗体类似物bp基因及蛋白和应用 Download PDFInfo
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Abstract
本发明公开了一种拮抗PD‑1的抗体类似物BP基因及蛋白和应用，基于抗体的结构，将PD‑L1抗体BMS‑963559重链的CDR区域的氨基酸序列替换到骆驼抗体的CDR区域，而得到的抗体类似物BP蛋白。该蛋白性质稳定，分子量小，能够在大肠杆菌中表达。进一步通过生物实验验证了BP蛋白的功能活性。为开发有临床应用前景的新药先导药物奠定了实验研究基础。
Description
技术领域
本发明属于生物医药领域，具体涉及一种拮抗PD-1骆驼抗体类似物BP基因及蛋白和应用。
背景技术
程序性死亡受体-1(programmed death-1，PD-1)是T细胞上的一种跨膜受体，主要在激活的T细胞和B细胞中表达，是激活型T细胞的一种表面受体，PD-1有两个配体，分别是PD-L1(B7-H1)和PD-L2(B7-DC)[1]。在正常情况下，PD-L1和PD-1的结合能够通过免疫负调控作用，维持外周淋巴细胞对自身抗原的免疫耐受，从而防止自身免疫性疾病的发生。但在肿瘤的发生发展中，肿瘤细胞高表达的PD-L1与PD-1结合后却能通过对淋巴细胞的抑制性作用，而促进肿瘤的免疫逃逸，这是导致肿瘤发生免疫逃逸的重要原因之一。因此，PD-1/PD-Ls负调控信号通路是重要的免疫检测点机制之一[2]。
目前，以PD-1/PD-Ls信号通路为靶标，研发针对PD-1或PD-Ls的阻断剂，即免疫检测点抑制剂(immune chekpoint inhibitors，ICIs)，能够增强T细胞对肿瘤细胞的杀伤。目前，已经有多个抗PD-1和PD-L1抗体药上市或者进入临床试验阶段。BMS-936559，一种阻断PD-L1的人IgG4抗体，已被证明在I期临床试验中对黑色素瘤，NSCLC和某些其他肿瘤有效[3]。
抗体分子是B淋巴细胞产生的一类大分子蛋白，它由两条重链和两条轻链组成，由于抗体具有高度的亲和性和特异性等特点。利用抗体分子作为药物治疗疾病已经在临床上广泛应用，其销售额每年都快速递增。全球抗体类药物销售额从2011年的不到500亿美元增加到2017年的1060亿美元。因此，抗体是重要的生物药物。尽管抗体分子有着较强的结合能力和选择性，但是由于抗体本身的一些性质，如相对分子质量大(约为1.5x105)、结构复杂、依赖二硫键的连结等，也导致了抗体分子的热稳定性差、制备过程复杂、进入实体瘤效率低等问题[4]。因此，抗体的临床应用仍然具有较大的局限性。然而，骆驼单链抗体是一种独特的仅由重链组成的小分子抗体。由来自骆驼科的仅有重链的抗体衍生的纳米抗体(VHH)具有分子量小，高溶解度，高稳定性的特点，与人VH3基因的同源性高，更适合作为移植骨架[5]。与人源VH比较发现，骆驼来源的VHH骨架区仅有十几个氨基酸不同，并且结构非常相似。纳米抗体结合抗原的互补决定区(complementary determine region，CDR)类似于抗体分子中CDR区(见图1)，可以被替换或突变，形成一个新的抗体[6]。在生产方面，骆驼来源抗体能够在大肠杆菌中高效、可溶性表达。综上所述，纳米抗体，具有比其他治疗药物具有多重的竞争优势。随着2018、2019年欧盟和FDA先后批准第一个纳米抗体，赛诺菲公司开发的纳米抗体药物Cablivi[7]，用于治疗获得性血栓性血小板减少性紫癜，基于骆驼抗体骨架蛋白设计的新型抗体类药物有望成为新一代生物药物，在临床上有广泛应用前景。
参考文献：
1.Sharpe AH，Pauken K E.The diverse functions of the PD 1inhibitorypathway[J].Nature Reviews Immunology，2018；18(3)：153-167.
2.Wykes M N，Lewin S R.Immune checkpoint blockade in infectiousdiseases[J].Nature Reviews Immunology，2018；18(2)：91-104.
3.Lee J Y，Lee H T，Shin W，et al.Structural basis of checkpointblockade by monoclonal antibodies in cancer immunotherapy[J].NatureCommunications，2017；27(1)：151-153.
4.Zhan MM，Hu X Q，Liu X X，et al.From monoclonal antibodies to smallmolecules：the development of inhibitors targeting the PD-1/PD-L1 pathway[J].Drug Discovery Today，2016；21(6)：1027-36.
5.Desmyter A，Transue TR，Ghahroudi M A，et al.Crystal structure of acamel single-domain VH antibody fragment in complex with lysozyme[J].NatStruct Biol，1996，3(9)：803-811.
6.Shi，D.，Zhou，S.，Liu，X.，Zhao，C.，Liu，H.，and Yao，X.(2018).Understandingthe structural and energetic basis of PD-1 and monoclonal antibodies bound toPD-L1：A molecular modeling perspective.Biochim.Biophys.Acta Gen.Sub.2018；1862(3)：576-588.
7.Poullin P，Bomet C，Veyradier A，Coppo P.Caplacizumab to treat immune-mediated thrombotic thrombocytopenic purpura.Drugs Today(Barc).2019；55(6)：367-376.
发明内容
本发明的目的是克服现有技术的不足，提供一种拮抗PD-1的抗体类似物BP基因。
本发明的另一个目的是提供拮抗PD-1的抗体类似物BP基因表达的蛋白。
本发明的第三个目的是提供上述基因和蛋白的应用。
本发明的技术方案为：
拮抗PD-1的抗体类似物BP基因，它具有序列表中SEQ ID No.1所示的核苷酸序列。
核苷酸序列(SEQ ID No.1)为：
5’-GATGTGCAGCTGCAAGCGAGCGGTGGCGGTAGCGTTCAAGCGGGCGGTAGCCTGCGTCTGAGCTGCGCGGCGAGCGGTTACACCATCGGTACCTATGCGATTAGCTGGTTCCGTCAAGCGCCGGGCAAGGAGCGTGAAGGCGTGGCGGGTATCATTCCGATCTTCGGCAAGGCGCACACCTACTATGCGGACAGCGTGAAAGGTCGTTTTACCATTAGCCAGGATAACGCGAAGAACACCGTTTACCTGCTGATGAACAGCCTGGAGCCGGAAGACACCGCGATCTACTATTGCGCGGCGAAATTCCACTTTGTTAGCGGCAGCCCGTTTGGTATGGATAGCTGGGGCCAGGGTACCCAAGTGACCGTTAGCAGC-3’。
拮抗PD-1的抗体类似物BP基因表达的蛋白，它具有序列表中SEQ ID No.2所示的氨基酸序列。
BP氨基酸序列(SEQ ID No.2)为：
DVQLQASGGGSVQAGGSLRLSCAASGYTIGTYAISWFRQAPGKEREGVAGIIPIFGKAHTYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAAKFHFVSGSPFGMDSWGQGTQVTVSS。下划线标注的区域为BMS-963559结合PD-L1的CDR三个关键区域序列。
所述基因和所述蛋白在制备PD-1阻断剂中的应用。
本发明有益效果：
本发明是基于抗体的结构，将PD-L1抗体BMS-963559重链的CDR区域的氨基酸序列替换到骆驼抗体的CDR区域(见图1标注)，而得到的抗体类似物BP蛋白。该蛋白性质稳定，分子量小，能够在大肠杆菌中表达。进一步通过生物实验验证了BP蛋白的功能活性。为开发有临床应用前景的新药先导药物奠定了实验研究基础。
附图说明
图1.BMS-963559抗体重链与骆驼抗体示意图.(A)BMS-963559抗体重链；(B)骆驼抗体；
图2.BMS-963559/PD-L1的晶体结构；
图3BP基因的合成；
图4PD-1基因的合成；
图5PD-L1基因的合成；
图6.PD-1菌落PCR筛选；其中M，Marker；1，2PD-1阳性菌落；
图7.PD-L1菌落PCR筛选；其中M，Marker；1，2，PD-L1阳性菌落；
图8.蛋白SDS-PAGE电泳图；1.PD-1；2.PD-L1；3.BP：各条带分子量见左侧标注；
图9.BP显著结合PD-L1；
图10.BP抑制PD-1与PD-L1结合。
具体实施方式
下面结合附图和具体实施例进一步阐述本发明，应理解，这些实施例仅用于说明本发明而不用于限制本发明的保护范围。
1.计算机分析BMS-963559与PD-L1结合模式，确定植入骆驼抗体的序列
我们基于BMS-963559/PD-L1的晶体结构(自蛋白数据库Protein data bankdatabase，5GRJ)分析BMS-963559结合PD-L1的模式(图2)。复合物由BMS-963559重链(深灰)和PD-L1(浅灰)组成。基于这个3D结构，进一步通过分子对接(Molecular docking)、分子动力学(Molecular dynamics，MD)、自由能计算和丙氨酸突变等一系列方法分析BMS-963559结合PD-L1的模式。将BMS-963559结合PD-L1的CDR1区中的TYAIS替换骆驼抗体CDR1区的PYCMG序列，将BMS-963559结合PD-L1的CDR2中的GIIPIFGKAH序列替换骆驼抗体CDR2的AINMGGGI序列，将BMS-963559结合PD-L1的CDR3中的KFHFVSGSPFGM序列替换骆驼抗体CDR3的DSTIYASYYECGHGLSTGGYGY序列，构建能够结合PD-L1新的抗体类似物分子BP。
2.PD-1，PD-L1与BP基因的构建和表达
2.1 PD-1和PD-L1重组质粒的构建
PD-1、PD-L1 cDNA基因购买于北京义翘公司(图3、4)。含PD-1基因的载体经过限制性内切酶Nde I和HindIII酶切之后与同样经过Nde I和HindIII酶切的载体pET30a连接，含PD-L1基因的载体经过限制性内切酶Nde I和HindIII酶切之后与同样经过Nde I和HindIII酶切的载体pET30a连接。在无菌状态下，将连接产物转化入大肠杆菌DH5感受态细胞中。菌液均匀涂于含Kana固体培养基平板上，37℃培养12-20个小时，观察菌落生长情况并4℃保存平板。Kana固体培养基平板生长的克隆分别使用菌落PCR鉴定和质粒PCR鉴定(图6、7)。阳性克隆送公司测序鉴定。
2.2 BP重组质粒的构建
依据BMS-963559与PD-L1 CDR区结合的位置，确定替换骨架蛋白骆驼抗体CDR区序列，构建新的PD-L1结合蛋白BP。
核苷酸序列(SEQ ID No.1)为：
5’-GATGTGCAGCTGCAAGCGAGCGGTGGCGGTAGCGTTCAAGCGGGCGGTAGCCTGCGTCTGAGCTGCGCGGCGAGCGGTTACACCATCGGTACCTATGCGATTAGCTGGTTCCGTCAAGCGCCGGGCAAGGAGCGTGAAGGCGTGGCGGGTATCATTCCGATCTTCGGCAAGGCGCACACCTACTATGCGGACAGCGTGAAAGGTCGTTTTACCATTAGCCAGGATAACGCGAAGAACACCGTTTACCTGCTGATGAACAGCCTGGAGCCGGAAGACACCGCGATCTACTATTGCGCGGCGAAATTCCACTTTGTTAGCGGCAGCCCGTTTGGTATGGATAGCTGGGGCCAGGGTACCCAAGTGACCGTTAGCAGC-3’。
BP氨基酸序列(SEQ ID No.2)为：
DVQLQASGGGSVQAGGSLRLSCAASGYTIGTYAISWFRQAPGKEREGVAGIIPIFGKAHTYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAAKFHFVSGSPFGMDSWGQGTQVTVSS。
下划线标注的区域为BMS-963559结合PD-L1的CDR三个关键区域序列。
野生型骆驼抗体氨基酸序列(SEQ ID No.3)为：
DVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSS
2.3 PD-1，PD-L1和BP蛋白的诱导和表达
首先小样诱导筛选高表达的三个蛋白克隆。将测序正确的重组质粒pET30a-PD1、pET30a-PDL1和pET30a-BP转化表达宿主菌BL21。挑取转化平板的六个克隆，分别接种于2mLLB+Kana培养基中，37℃振荡过夜。按600μL菌液和400μL 50％甘油的比例保存菌种，剩余菌液按1∶1000加入IPTG，37℃振荡诱导过夜。SDS-PAGE电泳检测蛋白的表达，挑选高表达克隆。
表达和纯化BP蛋白是按照以下步骤进行的。取高表达菌种20μL接种于2mL LB+Kana培养基中，37℃培养过夜，转接于1800mL LB+Kana培养基中，37℃振荡培养至OD＝0.5，加0.5mM IPTG，37℃振荡诱导过夜。菌液8000rpm，4℃离心10min，得菌体用1×PBS洗涤一次，然后用80mL 1×PBS重悬，冷冻。反复冻融三次，冰浴中超声破菌。将破碎后的细胞12000rpm，4℃离心10min。得到的上清用4.5mm滤膜过滤，调pH为7.4。上清过Ni亲和层析柱，分别使用10、20、50、100、200和500mM的咪唑洗脱液洗脱结合蛋白。分别取各浓度梯度的洗脱液400μL，各加1mL无水酒精于-20℃浓缩2h，4℃，12000rpm离心，去上清，加40μL PBS缓冲溶液重悬，加10μL5×loading buffer混合均匀，沸水浴10min，冰浴2min，SDS-PAGE电泳观察并分析蛋白纯化结果(图8)。
3.ELISA实验验证BP的功能活性
按常规ELISA方法做BP与配体PD-L1蛋白结合的ELISA。其操作步骤如下：
a)包被：稀释制备50μg/ml的PD-L1底物包被液，将稀释的PD-L1按50μL/孔包被96孔板。只加包被液包被的孔做空白对照。4℃放置18h。
b)PBST洗板三次后，每孔加入150μL 5％的奶粉，室温封闭2h。
c)PBST洗板三次，将BP稀释至不同浓度(0、10、50、100、和200μg/mL)，每孔加入50μL。37℃，温育1h。
d)PBST洗板三次，加入用5％奶粉稀释的小鼠抗myc二抗(比例按说明书稀释)，每孔加入50μL。37℃，温育1h。
e)PBST洗板三次，加入用5％奶粉稀释的山羊抗小鼠3抗(比例按说明书稀释)，每孔加入50μL。37℃，温育1h。
f)终止反应，测450nm的OD值。
实验结果：
1.PD-1、PD-L1和BP基因合成：PD-1和PD-L1重组基因通过北京擎科生物技术有限公司测序。合成基因经测序，完全正确。BP的基因序列通过金斯瑞生物科技有限公司合成。合成基因经测序，完全正确(图3)。
2.PD-1、PD-L1和BP表达载体的构建与表达
将PD-1和PD-L1连接体系转化进入DH5α大肠杆菌中。PD-1和PD-L1菌落PCR结果显示1，2号菌种为阳性(图6，7)。对1号、2号菌种提取质粒做质粒PCR，结果分别有约1160bp和660bp目的条带，菌落PCR和质粒PCR结果显示pET30a-PD-1和PD-L1重组质粒正确。为进一步验证序列的准确性，将菌种送生物公司测序，测序结果进行序列比对，正确率100％，表明PD-1和PD-L1重组质粒构建成功。从宿主菌DH5a提取重组质粒，转化到表达宿主菌BL21，进行高表达菌株筛选，发现在IPTG浓度0.5mM，16℃慢速振摇的条件下诱导，能够诱导出目的蛋白PD-1，PD-L1。
将合成的BP基因直接转化进去BL21菌种进行表达，发现在IPTG浓度为0.0.5mM，葡-萄糖浓度为10g/L，16℃慢速振摇的条件下能够诱导出目的蛋白BP。
选取菌株进行大量表达，经金属Ni螯合琼脂糖凝胶亲和层析纯化，洗脱组分做SDS-PAGE电泳，发现PD-1，PD-L1和BP分别在100mM，100mM，250mM洗脱组分有目的条带，说明这些组分中有目的蛋白。将上述组分洗脱液在1×PBS中透析，收集。取透析后的目的蛋白进行SDS-PAGE电泳，发现，目的蛋白的大小和PD-1，PD-L1和BP蛋白的理论预测值44.36KD，27.43KD和16.80KD大小相符，并且具有较高纯度。
3.BP结合PD-L1并竞争抑制PD-1和PD-L1的结合
ELISA实验结果显示，BP明显结合PD-L1，结合作用与融合蛋白的浓度呈正相关，并且结合活性稍优于PD-1结合PD-L1，在200μg/ml时，结合强度能够达到PD-1结合PD-L1的115％。
竞争ELISA实验结果显示，在PD-1为50μg/ml时，BP对PD-1与PD-L1的结合有一定的抑制，并与BP的浓度呈正相关，在浓度是200μg/ml时，BP对PD-1与PD-L1结合的抑制率可达25.04％。阴性对照WFN没有抑制作用，说明BP抑制作用是特异性的。
关于附图的结果说明
图1 BMS-963559抗体重链与骆驼抗体结构示意图.(A)为BMS-963559抗体重链；(B)为骆驼抗体。
图2 BMS-963559抗体结合PD-L1的复合物晶体结构。
图3-图5BP，PD-1和PD-L1基因合成，编码BP的基因序列通过金斯瑞生物科技有限公司合成。合成基因经测序，完全正确。
图6 PD-1基因与Linker-Fc基因进行重叠PCR，合成带Fc标签的PD-1基因，然后经过限制性内切酶Nde I和HindIII酶切之后与同样经过Nde I和HindIII酶切的载体pET30a连接、转化入大肠杆菌DH5α感受态细胞中。PD-L1基因经过PCR后经过限制性内切酶Nde I和EcoR I酶切之后与同样经过Nde I和EcoR I酶切的载体pET30a连接、转化入大肠杆菌DH5α感受态细胞中。从转化平板中挑取菌落进行PCR鉴定，以含有重组质粒的菌落作为模板，前后引物作为上下游引物，1％琼脂糖凝胶电泳。能够看到目的条带。
图7选取菌落PCR阳性的菌株，提取质粒，以质粒为模板，前后引物作为两端引物，1％琼脂糖凝胶电泳。结果能够看到目的条带，大小正确。
小样诱导结果显示，PD-1和PD-L1都能在大肠杆菌中表达；
图8蛋白纯化使用金属Ni螯合琼脂糖凝胶亲和层析，用PBS过夜透析之后，聚丙烯酰胺凝胶电泳检测蛋白大小及纯度。
图9 ELISA结合实验结果表明：BP明显结合PD-L1，结合作用与融合蛋白的浓度呈正相关，并且稍优于PD-1结合PD-L1，在200g/ml时，结合强度能够达到PD-1结合PD-L1的115％。图10ELISA竞争实验结果表明：在PD-1为50μg/ml的浓度条件下，BP对PD-1与PD-L1的结合有一定的抑制，并与BP的浓度呈正相关，在浓度是200μg/ml时，BP对PD-1与PD-L1结合的抑制率可达25.04％。阴性对照WFN没有抑制作用，说明BP抑制作用是特异性的。抑制作用与融合蛋白的浓度呈正相关。
本发明并不局限于上述实施例，很多细节的变化是可能的，但这并不因此违背本发明的范围和精神。
<110> 天津大学
<120> 拮抗PD-1的抗体类似物BP基因及蛋白和应用
<160> 3
<170> PatentIn version 3.5
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Claims (3)
1.拮抗PD-1的抗体类似物BP基因，其特征在于，其核苷酸序列如序列表中SEQ ID No.1所示。
2.拮抗PD-1的抗体类似物BP基因表达的蛋白，其特征在于，其氨基酸序列如序列表中SEQ ID No.2所示。
3.权利要求1所述基因、权利要求2所述蛋白在制备PD-1阻断剂中的应用。
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