CN110760517B - 拮抗pd-1骆驼抗体类似物ap基因及蛋白和应用 - Google Patents
拮抗pd-1骆驼抗体类似物ap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骆驼抗体类似物AP基因及蛋白和应用,在分析PD‑L1与其抗体Avelumab结合模式后，确定了Avelumab与PD‑L1相互作用CDR区的关键氨基酸，并将这些参与结合的关键区域截取、移植到骆驼抗体的CDR区，构建了PD‑L1抗体Avelumab的骆驼抗体类似物AP基因及蛋白。AP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AP蛋白的功能活性。为开发有临床应用前景的免疫检测点抑制剂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AP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，PD-1能够与配体PD-L1、PD-L2相互作用，抑制T细胞增殖和功能。在正常机体中，PD-1作为一种T细胞活化后表达的负调节分子，能够结合相应配体PD-L1/2，调控T细胞活化和功能，抑制过度免疫反应，从而防止由此可能导致的组织损伤，对维持机体的免疫耐受有重要作用，是重要的免疫检测点(immune chekpoint)机制之一[1]。而在肿瘤和病毒感染时，靶细胞PD-L1和PD-L2表达上调，与T细胞表面的PD-1受体结合，传递负调控信号，能够抑制T细胞的活化、增殖及对靶细胞的杀伤，使肿瘤和感染细胞逃逸机体的免疫监控和免疫杀伤[2]。因此，以PD-1/PD-Ls信号通路分子为靶标，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。2017年，FDA批准了由默克/辉瑞公司开发的PD-L1抗体Avelumab(Bavencio)用于治疗Merkel细胞癌(成人或12岁以上儿童)和晚期或转移性尿路上皮癌，为众多癌症患者带来了新的希望[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(VHH)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。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[5]。纳米抗体结合抗原的互补决定区(complementary determine region，CDR)类似于人抗体分子中CDR区(见图1(自蛋白数据库Protein data bank database，5GRJ和1MEL))，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用于治疗获得性血栓性血小板减少性紫癜[7]，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 A H,Pauken K E.The diverse functions of the PD1 inhibitorypathway[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.Liu K,Tan S,Chai Y,et al.Structural basis of anti-PD-L1 monoclonalantibody avelumab for tumor therapy[J].Cell Research,2017；27(1):151-153.
4.Zhan M M,Hu X Q,Liu X X,et al.From monoclonal antibodies to smallmolecules:the development of inhibitors targeting the PD-1/PD-L1 pathway.DrugDiscovery Today,2016；21(6):1027-36
5.Desmyter A,Transue T R,Ghahroudi M A,et al.Crystal structure of acamel single-domain VH antibody fragment in complex with lysozyme.Nat StructBiol,1996；3(9):803-811.
6.Shi,D.,Zhou,S.,Liu,X.,Zhao,C.,Liu,H.,and Yao,X.Understanding thestructural and energetic basis of PD-1and monoclonal antibodies bound to PD-L1:A molecular modeling perspective.Biochim.Biophys.Acta Gen.Sub.2018；1862(3):576-588.
7.Poullin P,Bornet C,Veyradier A,Coppo P.Caplacizumab to treatimmune-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的骆驼抗体类似物AP基因。
本发明的另一个目的是提供拮抗PD-1的骆驼抗体类似物AP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骆驼抗体类似物AP基因，它具有序列表中SEQ ID No.1所示的核苷酸序列。
SEQ ID No.1:
GATGTTCAGCTGCAGGCAAGCGGTGGTGGTAGCGTTCAGGCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAGCGGTTATACCTTTAGCAGCTATATTATGATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAGCGTGAGGGTGTTGCGGCGATTTATCCGAGTGGTGGTATTACTTTTTATGCAGATTCAGTTAAAGGCCGTTTTACCATTAGCCAGGATAATGCGAAAAATACCGTTTATCTGCTGATGAATAGCCTGGAGCCTGAAGATACCGCCATTTATTATTGTGCAGCCATTAAACTGGGTACAGTGACAACCGTTGATAGCTGGGGACAGGGTACACAGGTGACCGTTTCCAGC。
拮抗PD-1骆驼抗体类似物AP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。
SEQ ID No.2：
DVQLQASGGGSVQAGGSLRLSCAASGYTFSSYIMMGWFRQAPGKEREGVAAIYPSGGITFYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAAIKLGTVTTVDSWGQGTQVTVSS。下划线标注的区域为Avelumab结合PD-L1的CDR三个关键区域序列。
上述基因、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Avelumab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)，而设计了骆驼抗体类似物AP基因和蛋白。并按照设计的AP核苷酸和氨基酸序列，制备了AP基因，构建了AP基因大肠杆菌的表达载体并在大肠杆菌中表达、纯化。AP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AP蛋白的功能活性。为开发有临床应用前景的免疫检测点抑制剂先导药物奠定了实验研究基础。
附图说明
图1.Avelumab重链与骆驼抗体示意图；(A)Avelumab重链；(B)骆驼抗体；
图2.Avelumab/PD-L1的晶体结构；
图3AP基因的合成；
图4PD-1基因；
图5PD-L1基因；
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.PD-1,PD-L1和AP小样诱导结果；
图9.蛋白SDS-PAGE电泳图；1.PD-1；2.PD-L1；3.AP；
图10.AP显著结合PD-L1；
图11.AP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。
野生型骆驼抗体氨基酸序列为SEQ ID No.3：
DVQLQASGGGSVQAGGSLRLSCAASGYTIGPYCMGWFRQAPGKEREGVAAINMGGGITYYADSVKGRFTISQDNAKNTVYLLMNSLEPEDTAIYYCAADSTIYASYYECGHGLSTGGYGYDSWGQGTQVTVSS。
实施方法：
1.计算机分析Avelumab与PD-L1结合模式，确定植入骆驼抗体的序列
我们基于Avelumab/PD-L1的晶体结构(自蛋白数据库Protein data bankdatabase，5GRJ)分析Avelumab结合PD-L1的模式(图2)。复合物由Avelumab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Avelumab结合PD-L1的模式。将Avelumab结合PD-L1的CDR1区中的FSSYIM替换骆驼抗体CDR1区的IGPYC序列,将Avelumab结合PD-L1的CDR2中的YPSGGITF序列替换骆驼抗体CDR2的NMGGGITY序列，将Avelumab结合PD-L1的CDR3中的IKLGTVTTV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。
2.PD-1,PD-L1与AP基因的构建和表达
2.1 PD-1和PD-L1重组质粒的构建
依据Avelumab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，设计Avelumab的骆驼抗体类似物AP。其核苷酸序列为SEQ ID No.1:
5’-GATGTTCAGCTGCAGGCAAGCGGTGGTGGTAGCGTTCAGGCGGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAGCGGTTATACCTTTAGCAGCTATATTATGATGGGTTGGTTTCGTCAGGCACCGGGTAAAGAGCGTGAGGGTGTTGCGGCGATTTATCCGAGTGGTGGTATTACTTTTTATGCAGATTCAGTTAAAGGCCGTTTTACCATTAGCCAGGATAATGCGAAAAATACCGTTTATCTGCTGATGAATAGCCTGGAGCCTGAAGATACCGCCATTTATTATTGTGCAGCCATTAAACTGGGTACAGTGACAACCGTTGATAGCTGGGGACAGGGTACACAGGTGACCGTTTCCAGC-3’。AP的基因通过北京擎科生物技术有限公司合成(图3)。
PD-1、PD-L1 cDNA基因购买于北京义翘公司(图4，图5)。PD-1基因与Linker-Fc基因进行重叠PCR，合成带Fc标签的PD-1-Fc基因,然后经过限制性内切酶NdeⅠ和HindⅢ酶切之后与同样经过NdeⅠ和HindⅢ酶切的载体pET30a连接、转化入大肠杆菌DH5α感受态细胞中。含PD-L1基因的载体经过限制性内切酶NdeⅠ和HindⅢ酶切之后与同样经过NdeⅠ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(附图6、7)。阳性克隆样品送北京擎科生物技术公司测序鉴定。
2.2 PD-1，PD-L1和AP蛋白的诱导和表达
首先小样诱导筛选高表达蛋白的三个克隆。将测序正确的重组质粒pET30a-PD1、pET30a-PDL1和pET30-AP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l：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AP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10μL 5×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。
3.ELISA实验验证AP的功能活性
按常规ELISA方法做AP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AP稀释至不同浓度(0、10、50、100、和200μg/mL)。PD-L1天然受体PD-1作为阳性对照，也按照AP的稀释梯度稀释。每孔分别加入不同浓度的AP和PD-1各50μL。37℃，温育1h。
d)PBST洗板三次，加入用5％奶粉稀释的二抗小鼠抗myc标签抗体(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％奶粉稀释的辣根过氧化物酶标记的三抗山羊抗小鼠抗体(比例按说明书稀释)，每孔加入50μL。37℃，温育1h。
f)洗板后，显色。
g)终止反应，测450nm的OD值。
实施结果：
1.PD-1和PD-L1重组质粒的构建
PD-1、PD-L1和AP基因合成：AP的基因序列通过北京擎科生物技术有限公司合成。合成基因经测序，完全正确(图3)。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，序列完全正确。
2.PD-1、PD-L1和AP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号菌种提取质粒并做质粒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(图8)。
将合成的AP基因表达载体直接转化进去BL21菌种进行表达，发现在IPTG浓度为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AP。
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AP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AP蛋白的理论预测值44.36KD，27.43KD和16.80KD大小一致，并且具有较高纯度。
3.APP结合PD-L1并竞争抑制PD-1和PD-L1的结合
ELISA结果显示，AP能够结合PD-L1，结合作用与蛋白的浓度呈正相关，并且结合强度优于PD-1与PD-L1的结合。在200μg/ml时，AP结合强度能够达到PD-1结合PD-L1的115％(图10)。阴性对照WFN没有结合，说明AP结合PD-L1是特异性的。
竞争ELISA实验结果显示，在PD-1为50μg/ml的浓度条件下，AP对PD-1与PD-L1的结合有一定的抑制，并与AP的浓度呈正相关，在浓度是200μg/ml时，AP对PD-1与PD-L1结合的抑制率可达24.15％。阴性对照WFN没有抑制作用，说明AP抑制作用是特异性的。
关于附图结果的说明
图1 Avelumab重链和骆驼抗体结构的示意图。
图2 Avelumab与其配体PD-L1相互作用的结构信息和关键区域的确定。
图3编码AP的基因通过北京擎科生物技术有限公司合成。合成基因经测序，完全正确。
图4PD-1基因由北京义翘公司合成。
图5PD-L1基因由北京义翘公司合成。
图6PD-1基因与Linker-Fc基因进行重叠PCR，合成带Fc标签的PD-1-Fc基因,然后经过限制性内切酶NdeⅠ和HindⅢ酶切之后与同样经过NdeⅠ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NdeⅠ和HindⅢ酶切之后与同样经过NdeⅠ和HindⅢ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。
图8小样诱导结果显示，PD-1、PD-L1和AP都能在大肠杆菌中表达。
泳道1和2是AP蛋白小样诱导结果，泳道3和4是PD蛋白小样诱导结果，泳道5，6和7是PD-L1蛋白小样诱导结果，目的条带用箭头标注。
图9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10 ELISA结合实验结果表明：AP结合PD-L1，结合作用与蛋白的浓度呈正相关，并且结合强度优于PD-1与PD-L1的结合，200μg/ml时结合强度能够达到PD-1结合PD-L1的115。阴性对照WFN没有结合，说明AP结合PD-L1是特异性的。
图11 ELISA竞争实验结果表明：在PD-1为50μg/ml的浓度条件下，AP对PD-1与PD-L1的结合有一定的抑制，并与AP的浓度呈正相关，在浓度是200μg/ml时，AP对PD-1与PD-L1结合的抑制率可达24.15％。阴性对照WFN没有抑制作用，说明AP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骆驼抗体类似物AP基因及蛋白和应用
<160> 3
<170> PatentIn version 3.5
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<211> 363
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<213> 人工序列
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gcaccgggta aagagcgtga gggtgttgcg gcgatttatc cgagtggtgg tattactttt 180
tatgcagatt cagttaaagg ccgttttacc attagccagg ataatgcgaa aaataccgtt 240
tatctgctga tgaatagcct ggagcctgaa gataccgcca tttattattg tgcagccatt 300
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agc 363
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Val Thr Val Ser Ser
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Claims (3)
1.拮抗PD-1骆驼抗体类似物AP基因，其特征在于，其核苷酸序列为序列表中SEQ IDNo.1所示。
2.拮抗PD-1骆驼抗体类似物AP基因表达的蛋白，其特征在于，其氨基酸序列为序列表中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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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016145085A2 (en) * | 2015-03-09 | 2016-09-15 | Celldex Therapeutics, Inc. | Cd27 agonists |
| WO2016205277A1 (en) * | 2015-06-16 | 2016-12-22 | Merck Patent Gmbh | Pd-l1 antagonist combination treatments |
| CN109310755A (zh) * | 2016-02-02 | 2019-02-05 | 卡德门企业有限公司 | Pd-l1和kdr的双特异性结合蛋白 |
| CN109942705A (zh) * | 2019-04-15 | 2019-06-28 | 石河子大学 | 一种mstn纳米抗体、构建方法及其应用 |
| CN110092826A (zh) * | 2019-02-28 | 2019-08-06 | 天津大学 | CTLA-4类似物CFN13及CFN13-Fc基因和蛋白 |
| CN110982824A (zh) * | 2019-10-09 | 2020-04-10 | 天津大学 | 拮抗pd-1的抗体类似物bp基因及蛋白和应用 |
| CN111278861A (zh) * | 2018-01-10 | 2020-06-12 | 江苏恒瑞医药股份有限公司 | Pd-l1抗体、其抗原结合片段及医药用途 |
| WO2020233539A1 (en) * | 2019-05-17 | 2020-11-26 | Nanjing GenScript Biotech Co., Ltd. | Anti-cd47/anti-pd-l1 multiple antigen binding proteins and methods of use thereof |
| WO2020249071A1 (en) * | 2019-06-12 | 2020-12-17 | Nanjing GenScript Biotech Co., Ltd. | Anti-pd-l1/anti-lag-3 multiple antigen binding proteins and methods of use thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103965295B (zh) * | 2014-04-18 | 2015-12-02 | 天津大学 | BLyS拮抗肽、含TA-Fc融合蛋白基因的质粒及TA-Fc融合蛋白 |
| MX2020008569A (es) * | 2018-02-16 | 2020-09-21 | Abbvie Inc | Inhibidores selectivos de bcl-2 en combinacion con un anticuerpo anti-pd-1 o anti-pd-l1 para el tratamiento de tipos de cancer. |
-
2019
- 2019-10-09 CN CN201910953260.3A patent/CN110760517B/zh not_active Expired - Fee Related
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016145085A2 (en) * | 2015-03-09 | 2016-09-15 | Celldex Therapeutics, Inc. | Cd27 agonists |
| WO2016205277A1 (en) * | 2015-06-16 | 2016-12-22 | Merck Patent Gmbh | Pd-l1 antagonist combination treatments |
| CN109310755A (zh) * | 2016-02-02 | 2019-02-05 | 卡德门企业有限公司 | Pd-l1和kdr的双特异性结合蛋白 |
| CN111278861A (zh) * | 2018-01-10 | 2020-06-12 | 江苏恒瑞医药股份有限公司 | Pd-l1抗体、其抗原结合片段及医药用途 |
| CN110092826A (zh) * | 2019-02-28 | 2019-08-06 | 天津大学 | CTLA-4类似物CFN13及CFN13-Fc基因和蛋白 |
| CN109942705A (zh) * | 2019-04-15 | 2019-06-28 | 石河子大学 | 一种mstn纳米抗体、构建方法及其应用 |
| WO2020233539A1 (en) * | 2019-05-17 | 2020-11-26 | Nanjing GenScript Biotech Co., Ltd. | Anti-cd47/anti-pd-l1 multiple antigen binding proteins and methods of use thereof |
| WO2020249071A1 (en) * | 2019-06-12 | 2020-12-17 | Nanjing GenScript Biotech Co., Ltd. | Anti-pd-l1/anti-lag-3 multiple antigen binding proteins and methods of use thereof |
| CN110982824A (zh) * | 2019-10-09 | 2020-04-10 | 天津大学 | 拮抗pd-1的抗体类似物bp基因及蛋白和应用 |
Non-Patent Citations (10)
| Title |
|---|
| A tumor-targeted immune checkpoint blocker;Zhang, YH等;《ROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA》;20190806;第116卷(第32期);第15889-15894页 * |
| Avelumab, an anti-PD-L1 antibody, in patients with locally advanced or metastatic breast cancer: a phase 1b JAVELIN Solid Tumor study;Dirix Luc Y等;《Breast cancer research and treatment》;20180228;第167卷(第3期);第671-686页 * |
| BCMA-Fc-Myc融合蛋白的构建、表达及活性鉴定;朱燕锋等;《生物技术》;20131015(第05期);全文 * |
| Chain H, Avelumab H Chain;Liu K等;《NCBI GenBank database》;20170118;Accession No：5GRJ_H * |
| Photothermally activatable PDA immune nanomedicine combined with PD-L1 checkpoint blockade for antimetastatic cancer photoimmunotherapy;Qianglan Lu等;《Journal of materials chemistry B》;20190410;第7卷(第15期);第2499-2511页 * |
| Soluble Expression of Small Antibody Fragments against PD-L1 Using Escherichia coli with High Yield and Purity;Sun-Hee Kim等;《applied sciences》;20210831;第11卷(第19期);全文 * |
| sTACI-Fc-Myc重组质粒的构建、原核表达及活性鉴定;白乌仁图雅等;《现代生物医学进展》;20150120(第02期);全文 * |
| Structural basis of anti-PD-L1 monoclonal antibody avelumab for tumor therapy;Kefang Liu等;《Cell research》;20160830;第27卷;第151-153页 * |
| 免疫检测点PD-1/PD-L1的机制研究及免疫治疗;胡耿维等;《国际肿瘤学杂志》;20190208;第46卷(第2期);第87-90页 * |
| 靶向肿瘤PD-1/PD-L1抗体药物治疗中出现疾病"超进展"现象及其合理应用研究进展;袁司辰等;《药学学报》;20190902;第54卷(第10期);第1735-1740页 * |
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