CN117417444A - 一种治疗纤维化疾病的人源化纳米抗体 - Google Patents
一种治疗纤维化疾病的人源化纳米抗体 Download PDFInfo
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Abstract
本发明提供了一种治疗纤维化疾病的人源化纳米抗体，属于纳米抗体领域。该人源化纳米抗体在保持与TGFβ亲和力的同时能阻断TGFβ与其受体的结合，并且最大程度的降低了异源性带来的免疫风险，更有利于穿透血脑屏障，在制备治疗纤维化疾病和晚期肿瘤的药物中具有广阔的应用前景。
Description
技术领域
本发明属于纳米抗体领域，具体涉及一种治疗纤维化疾病的人源化纳米抗体。
背景技术
纤维化疾病是以大量的成纤维细胞聚集、细胞外基质(ECG)沉积并伴有炎症和损伤所致组织结构破坏为特征的疾病。肺纤维化疾病包括特发性肺纤维化(IPF)、结节病、尘肺、过敏性肺炎、药物和放射线导致的纤维化，以及与胶原血管病有关的致纤维化肺泡炎等病因各异，范围广泛的疾病谱。其发病率和死亡率各不相同，严重性各异，但共同特点是缺少特异性治疗。目前对肺纤维化疾病的治疗仅限于非特异性抗炎、免疫抑制剂及糖皮质激素等，疗效尚不理想。
转化生长因子(TGF)β是一个分泌型的多肽信号分子超家族，具有转化作用，通过自分泌和旁分泌等不同的方式调节细胞的增殖、分化、移行，调节ECG的代谢，参与肺胚胎发育、组织损伤与修复。TGFβ具有刺激细胞从贴壁依赖性生长转变为非贴壁非依赖性生长的作用，对间充质起源的细胞起刺激作用，在细胞增殖、ECG的合成、血管形成等过程中具有重要作用。在多种纤维增生性疾病中，TGFβ表现出共同的促纤维化作用。抗TGFβ抗体能与TGFβ结合，阻碍TGFβ与其靶细胞表面的各型TGFβR结合而发挥其有效的抗纤维化作用。因此，开发能够抑制TGFβ与其受体结合的药物对抗纤维化治疗具有重要意义。
发明内容
本发明的目的在于提供一种抗TGFβ的人源化纳米抗体，以及该抗TGFβ的人源化纳米抗体在制备治疗纤维化疾病和晚期肿瘤的药物中的用途。
本发明提供了一种人源化纳米抗体，它包括互补决定区CDR1-CDR3，CDR1的氨基酸序列为FTFSGYA，CDR2的氨基酸序列为ISSSGGT，CDR3的氨基酸序列为ADLYYTYTY。
进一步地，所述人源化纳米抗体还包括与互补决定区CDR1-CDR3交替连接的四个框架区FR1-FR4，FR1的氨基酸序列为QVQLVESGGGLVQPGGSLRLSCAASG，FR2的氨基酸序列为LGWFRQAPGQEREAVAA，FR3的氨基酸序列为YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC，FR4的氨基酸序列为WGQGTLVTVSS。
进一步地，所述人源化纳米抗体的氨基酸序列为：
QVQLVESGGGLVQPGGSLRLSCAASGFTFSGYALGWFRQAPGQERE AVAAISSSGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ADLYYTYTYWGQGTLVTVSS。
本发明还提供了一种制备上述人源化纳米抗体的方法，所述方法包括以下步骤：
(1)将核苷酸序列如SEQ ID NO:9所示的核苷酸分子连接至表达载体中获得阳性质粒；
(2)将阳性质粒转化宿主细胞，诱导表达人源化纳米抗体。
本发明还提供了一种核苷酸分子，它的核苷酸序列如SEQ ID NO:9所示。
本发明还提供了一种表达载体，它包含核苷酸序列如SEQ ID NO:9所示的核苷酸分子。
本发明还提供了一种宿主细胞，它包含上述的表达载体。
本发明还提供了一种药物，它是以上述人源化纳米抗体为活性成分，加上要学上可接受的辅料制备而成的制剂。
本发明还提供了上述人源化纳米抗体在制备治疗纤维化疾病的药物中的用途；优选地，所述纤维化疾病为肺纤维化。
本领域技术人员公知的，肿瘤发展至晚期TGFβ会呈现出促进肿瘤恶化的趋势。抗TGFβ抗体可以治疗的晚期肿瘤包括：胃癌、胶质瘤、黑色素瘤、肾细胞癌、胰腺癌、乳腺癌、肺癌、前列腺癌、胆管癌、头颈部鳞状细胞癌、子宫颈癌等。
本发明还提供了上述人源化纳米抗体在制备治疗晚期肿瘤的药物中的用途。
进一步地，所述肿瘤为胃癌、胶质瘤、黑色素瘤、肾细胞癌、胰腺癌、乳腺癌、肺癌、前列腺癌、胆管癌、头颈部鳞状细胞癌或子宫颈癌。
本发明的抗TGFβ人源化纳米抗体在保持与TGFβ亲和力的同时能阻断TGFβ与其受体的结合，在制备治疗纤维化疾病和晚期肿瘤的药物中具有广阔的应用前景。
本发明的抗TGFβ人源化纳米抗体分子量约15KDa，这种抗体是人源化抗体，其最大程度的降低了异源性带来的免疫风险，更有利于穿透血脑屏障，更易到达肿瘤内部发挥治疗效果。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1.人源化纳米抗体与TGFβ1亲和力检测。
图2.人源化纳米抗体与TGFβ2亲和力检测。
图3.人源化纳米抗体与TGFβ3亲和力检测。
具体实施方式
本发明所用原料与设备均为已知产品，通过购买市售产品所得。
实施例1：人源化纳米抗体的制备
1.构建表达载体
(1)骨架载体酶切：工具载体pcDNA3.1-x-IgG1进行BamH I/EcoR I酶切，37℃双酶切5h后采用PCR产物回收试剂盒(Cycle-Pure Kit PCR产物纯化试剂盒OMEGA D6492-01)回收载体。
(2)同源重组：将用于重组表达的核苷酸片段使用ddH2O稀释20倍，取1ul稀释后的样品与上述酶切回收的载体进行同源重组(重组酶，NovoRec Plus one step PCR CloningKit近岸蛋白货号NR005-01B)。
用于重组表达的核苷酸片段的序列为：
CCAGGCTACTTTATGCTTCGGCTCGTATGTTGTGTGGAATTGTGAGCGG
ATAACAATTGAATTCAGGAGGAATTTAAAATGAAAAAGACAGCTATCGC
GATTGCAGTGGCACTGGCTGGTTTCGCTACCGTGGCCCAGGCGGCCCA
GGTGCAGCTGGTTGAAAGTGGCGGTGGCCTGGTGCAGCCGGGTGGTTC
ACTGCGTCTGAGTTGTGCCGCAAGCGGTTTTACCTTTAGCGGTTATGCC
CTGGGCTGGTTTCGTCAGGCCCCTGGTCAGGAACGCGAAGCAGTTGCA
GCCATTAGTAGCAGCGGTGGTACATATTATGCAGATAGCGTTAAAGGCC
GCTTTACCATTAGCCGCGATAATAGCAAAAATACCCTGTATCTGCAGATG
AATAGTCTGCGCGCCGAAGATACCGCAGTTTATTATTGTGCAGATCTTTA
TTATACGTATACCTATTGGGGTCAGGGCACCCTGGTTACCGTTAGCAGTG
CGCACCACAGCGAAGACCCCCATGGCCAGGCCGGCCAGCACCATCACC
ATCACCATGGCGCATACCCGTACGACGTTCCGGACTACGCTTCTTAGGA
GGGTGGTGGCTCTGAGGGTGGCGGTTCTGAGGGTGGCGGCTCTGAGG
GAGGCGGTTCCGGTGGTGGCTCTGGTTCCGGTGATTTTGATTATGAAAA
GATGGCAAACGCTAATAAGGGGGCTATGACCGAAAATGCCGATGAAAA
CGTGCTACAGTCTGACGCTAAAGGCAAACTTGATTCTGTCGCTACTGAT
TACGGTGCTGCTATCGATGGTTTCATTGGTGACGTTTCCGGCCTTGCTAATGGTAATGGTGCTACTGGTGATTTTGCTGGCTCTAATTCC(SEQ ID NO:9)。
(3)大肠杆菌菌液PCR鉴定：从平板上挑取单克隆大肠杆菌菌落于200ul LB培养基37℃,220rpm培养3h，取1ul菌液作为模板，进行菌液PCR鉴定，选择鉴定的阳性克隆送去测序，将PCR产物电泳切角回收(Gel Extraction Kit胶回收试剂盒OMEGA，货号D2500-01)后，再次进行同源重组，菌液PCR鉴定后，选择鉴定的阳性克隆送去测序。
2.人源化纳米抗体Hek293F细胞表达与纯化
(1)抗体表达
将菌种接种到20ml含有氨苄青霉素的LB培养基中，37℃培养箱培养过夜，采用质粒提取试剂盒(Plasmid Miniprep Kit II，倍沃医学Cat:BW-PD1213)抽提质粒。传代Hek293细胞保持细胞良好生长状态，活率大于95％，转染时调整Hek293细胞密度为2.5×106cells/ml。取50ug表达质粒加入到1ml OPM培养基中混匀、取150ug PEI加入到1ml OPM培养基中混匀，上述两者混合后摇匀，室温静置30min后加入至50ml Hek293细胞中，于CO2摇床培养，第二天加入5％终体积的OPM培养基补料，继续培养至第7天，10000rmp离心20min收获细胞培养上清，用于蛋白纯化。
(2)抗体纯化
protein A重力柱纯化抗体蛋白：从冰箱中取出重力柱，用超纯水冲洗一个柱体积，0.1M NaOH冲洗一个柱体积，PBS缓冲液冲洗3个柱体积。
将离心后的细胞上清全部上样至重力柱，PBS缓冲液冲洗3个柱体积后，加入800ul的0.1M甘氨酸盐酸盐(Gly-HCl)洗脱，重复洗脱2次，收集目标蛋白(命名为RS98)，测定蛋白纯度不低于95％。
目标蛋白RS98的氨基酸序列如SEQ ID NO:8所示，它包括互补决定区CDR1-CDR3，互补决定区被四个框架区FR1、FR2、FR3和FR4所分隔。
表1.目标蛋白的氨基酸序列
以下通过实验例证明本发明人源化纳米抗体的活性。
实验例1：人源化纳米抗体与TGFβ1/2/3的亲和力检测
1.实验方法
TGFβ1/2/3抗原以1ug/ml浓度包被酶标板，人源化纳米抗体RS98从50ug/ml开始5倍比稀释后加入，采用anti-Fc二抗检测抗体与TGFβ1/2/3结合情况。以可同时靶向TGFβ1/2/3三种亚型的已知单克隆抗体Fresolimumab(非苏木单抗)作为对照抗体。
2.实验结果
亲和力ELISA试验结果如图1-3所示，可以看出，本发明RS98抗体能够同时靶向TGFβ1/2/3三种亚型。
实验例2：人源化纳米抗体对TGFβ1与TGFβR2阻断作用检测
1.实验方法
先用ELISA检测TGFβR2-FC-His与TGFβ1结合情况，选择合适浓度(该浓度处OD450以1左右为宜)的TGFβR2-FC-His作为与抗体竞争实验浓度。采用HRP标记的anti-His二抗检测，同时以50ul PBS和等体积的50ug/ml TGFβR2-FC-His做为对照，若实验组OD450明显弱于对照组，则可视为竞争。
2.实验结果
从竞争性ELISA试验结果可以看出，RS98抗体能够高效地阻断TGFβ1与其受体TGFβR2的结合(表2)，效果与照抗体Fresolimumab相当。
表2.RS98对TGFβ1与TGFβR2结合的阻断作用
| 实验组 | OD值 |
| RS98 | 0.2474 |
| Fresolimumab | 0.2915 |
| PBS | 0.4773 |
综上，本发明提供了一种治疗纤维化疾病的人源化纳米抗体，该人源化纳米抗体在保持与TGFβ亲和力的同时能阻断TGFβ与其受体的结合，并且最大程度的降低了异源性带来的免疫风险，更有利于穿透血脑屏障，在制备治疗纤维化疾病和晚期肿瘤的药物中具有广阔的应用前景。
Claims (10)
1.一种人源化纳米抗体，其特征在于，它包括互补决定区CDR1-CDR3，CDR1的氨基酸序列为FTFSGYA，CDR2的氨基酸序列为ISSSGGT，CDR3的氨基酸序列为ADLYYTYTY。
2.根据权利要求1所述的人源化纳米抗体，其特征在于，它还包括与互补决定区CDR1-CDR3交替连接的四个框架区FR1-FR4，FR1的氨基酸序列为QVQLVESGGGLVQPGGSLRLSCAASG，FR2的氨基酸序列为LGWFRQAPGQEREAVAA，FR3的氨基酸序列为YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC，FR4的氨基酸序列为WGQGTLVTVSS。
3.根据权利要求2所述的人源化纳米抗体，其特征在于，它的氨基酸序列为：
QVQLVESGGGLVQPGGSLRLSCAASGFTFSGYALGWFRQAPGQERE AVAAISSSGGTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC ADLYYTYTYWGQGTLVTVSS。
4.一种制备权利要求1-3任一项所述人源化纳米抗体的方法，其特征在于，所述方法包括以下步骤：
(1)将核苷酸序列如SEQ ID NO:9所示的核苷酸分子连接至表达载体中获得阳性质粒；
(2)将阳性质粒转化宿主细胞，诱导表达人源化纳米抗体。
5.一种核苷酸分子，其特征在于，它的核苷酸序列如SEQ ID NO:9所示。
6.一种表达载体，其特征在于，它包含权利要求5所述的核苷酸分子。
7.一种宿主细胞，其特征在于，它包含权利要求6所述的表达载体。
8.一种药物，其特征在于，它是以权利要求1-3任一项所述人源化纳米抗体为活性成分，加上要学上可接受的辅料制备而成的制剂。
9.权利要求1-3任一项所述人源化纳米抗体在制备治疗纤维化疾病的药物中的用途；优选地，所述纤维化疾病为肺纤维化。
10.权利要求1-3任一项所述人源化纳米抗体在制备治疗晚期肿瘤的药物中的用途；优选地，所述肿瘤为胃癌、胶质瘤、黑色素瘤、肾细胞癌、胰腺癌、乳腺癌、肺癌、前列腺癌、胆管癌、头颈部鳞状细胞癌或子宫颈癌。
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