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重组长效透明质酸酶的构建及其在皮下给药与肿瘤治疗中的应用研究
刘珊
学位类型博士
导师惠觅宙  
2016-07
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业生物化工
关键词长效透明质酸酶 表达与纯化 皮下给药 肿瘤微环境 尺寸效应
摘要

透明质酸酶通过降解胞外基质中透明质酸,有利于药物扩散,在皮下给药以及肿瘤治疗中均有应用。而在皮下给药中,大分子联合给药生物利用度仍与静脉给药存在较大差距。另一方面在透明质酸高表达的实体瘤中,其体内循环半衰期严重影响了体内药效的发挥。本论文针对这两大应用领域存在的问题,采用基因工程技术构建重组长效透明质酸酶,以期提升大分子皮下给药的生物利用度以及实现对体内肿瘤的药效提升效果。论文具体开展的研究工作如下:1. 采用分子克隆技术成功构建Igκ-rhPH20、Igκ-rhPH20-HSA与Igκ-rhPH20-Fc的融合基因。整合到表达载体之后将其成功转染到宿主细胞中,筛选克隆并进行表达量的比较。对于不同宿主细胞,CHO KSD细胞获得克隆的平均表达水平(18 U/24h·mL)高于CHO S细胞(12 U/24h·mL)。另外,构建带有IRES-eGFP的双顺反子表达载体,成功根据荧光强度通过流式分选术筛选高表达克隆,该法高效快速,获得克隆稳定性好。还考察了载体结构对蛋白表达量的影响,载体GC含量高,DNA对转录因子开放,蛋白表达量上调。2. 对三种重组透明质酸酶rhPH20、rhPH20-Fc以及rhPH20-HSA的克隆40mL批次培养基础表达量为402 U/mL、596U/mL以及737 U/mL。在5 L激流式生物反应式实现了培养工艺的优化及放大。确定了不同蛋白的层析柱选择以及层析条件,经纯化获得纯度大于95%的产品,61 kDa (rhPH20), 79 kDa (rhPH20-HSA) and 190 kDa (rhPH20-Fc)。比较三种重组蛋白比活性:rhPH20为106,724 U/mL;rhPH20-HSA为10,256U/mL,维持原来10%左右的活性,rhPH20-Fc为40,124 U/mL,能维持原来40%的活性。3.在皮下结构重建方面,重组透明质酸酶rhPH20与提纯的透明质酸酶产品在24h内皮肤结构恢复,rhPH20-Fc与rhPH20-HSA在小鼠皮下更稳定,造成皮下空洞恢复时间由延长到85~120 h,成功实现了皮下长效。透明质酸酶与蛋白药物联合给药,均能够增加AUC,增大Cmax,并且Tmax提前。对于Stelara,rhPH20-Fc将其生物利用度由rhPH20组的86%提升到93%;对于250 kDa的TFI大分子,rhPH20联用生物利用度提升至64%,而与长效rhPH20-Fc联用生物利用度提升至97%,接近静脉给药。4. 比较性研究透明质酸酶对不同尺寸肿瘤治疗药物的促进扩散作用以及药效提升效果。选取三种不同尺寸的典型抗肿瘤药物:大小约为1.5 × 1.0 × 0.7 nm的小分子化疗药物阿霉素(Dox)、直径约10~15 nm的曲妥珠单抗(Tmab)以及长120 nm×宽35 nm的热疗药物金纳米棒(GNR)。透明质酸酶对二维细胞水平药物量效关系没有影响,仅在胞外基质以及肿瘤微环境存在的三维细胞培养水平对三种药物的扩散、蓄积及生长抑制表现出不同程度的促进作用,其中对抗体扩散的增幅最大。重组透明质酸酶rhPH20-Fc在体内比原始rhPH20更稳定,rhPH20的循环半衰期仅为2.3 min,融合透明质酸酶rhPH20-Fc的循环半衰期提升至2.8 h,大幅提升血液循环时间能够增加透明质酸酶在肿瘤组织的截留。rhPH20-Fc促进瘤内药物扩散,利于药物在瘤内的蓄积,并且增强了化疗药物对肿瘤生长的抑制作用,针对三种药物不同治疗效果进行定量分析,得到对抗体分子的辅助治疗效果最好。

其他摘要

By degrading hyaluronan in extracellular matrix, hyaluronidase can facilitate therapeutic agents diffusion, and has been applied in subcutaneous administration and antitumor therapy. However, the bioavailabilities of SC-administered macro molecules would be lower than the IV route in general. On the other hand, the application in antitumor therapy was limited as the short in vivo circulation time. Focusing on the primary restrictions in these two areas, we constructed novel recombinant hyaluronidase and explored whether these reconstruction techniques could improve stability in the dermis and enhance antitumor efficacy in vivo. This thesis mainly included the following issues:1. The fused gene fragments of Igκ-rhPH20, Igκ-rhPH20-HSA and Igκ-rhPH20-Fc were successfully constructed and ligated to the cloning vector pMD18-T and expression vector pMH3, respectively. After gene transfection, high-level expression clones were chosen. Average expression levels of CHO KSD (18 U/24h·mL) derived clones were higher than CHO S (12 U/24h·mL) derived ones. The addition of IRES-eGFP to the expression vection facilitate clones screening by flow cytometer, which was high-efficient and convenient. The influence of expression vector skeleton was also explored, which indicated the higher GC content, the high expression level of protein, as the DNA was open for transcription factor. 2. The basic expression levels of batch culture in 40-mL flasks for each of the selected clones were 402, 596, and 737 U/mL for rhPH20, rhPH20-HSA and rhPH20-Fc, respectively. In this study, expression of the new protein was undertaken in CHO cells cultured in a 5-L disposable bioreactor. Purification was carried out by a series of chromatographic methods to obtain high-purity products of 61 kDa (rhPH20), 79 kDa (rhPH20-HSA) and 190 kDa (rhPH20-Fc). The specific activity of rhPH20-Fc was 35,600 U/mg, higher than that of rhPH20-HSA (10,000 U/mg).3. The chimeric proteins rhPH20-HSA and rhPH20-Fc performed fairly well as spreading factors in short-term trypan blue intradermal (ID) infusion in comparison with recombinant hyaluronidase (rhPH20). They extended the channel opening from 24 hours (rhPH20) to 85-120 hours in vivo. Co-administration of rhPH20-Fc with two biomacromolecular pharmaceuticals, Stelara (150 KDa) and TNFRII-Fc-IL1ra (TFI, 250 kDa), through an ID route increased the bioavailability from 86% to 93% and from 64% and 97%, respectively, compared with rhPH20. 4. Natural hyaluronidase PH20 and long-acting PH20-Fc were constructed and evaluated as diffusion promoter for typical therapeutics agents in different nanoscales: doxorubicin (~1.5 × 1.0 × 0.7 nm) in chemotherapy, trastuzumab (10~15 nm) in biotherapy, and gold nanorods (~100 × 35 nm) in thermotherapy. In traditional 2D culture, PH20 and PH20-Fc showed little influence, while in 3D culture, the promotion effect by hyaluronidase came out. Further in vivo evaluation revealed that the circulation time of PH20-Fc was significantly prolonged to 2.8 h, from 2.3 min of nature PH20. As a result, co-administration with PH20-Fc further facilitated drugs diffusion and accumulation, and inhibited tumor growth on the tumor xenografts. Meanwhile, we also noticed the PH20-Fc performances varied among these three therapeutic agents due to their different nanoscales. Trastuzumab with a best promotion on the penetration and accumulation benefited most from PH20-Fc. 

语种中文
文献类型学位论文
条目标识符http://ir.ipe.ac.cn/handle/122111/22916
专题研究所(批量导入)
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刘珊. 重组长效透明质酸酶的构建及其在皮下给药与肿瘤治疗中的应用研究[D]. 北京. 中国科学院研究生院,2016.
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