【作者】 张海涛；

【导师】 郭长虹； 李会成；

【作者基本信息】 哈尔滨师范大学 ， 遗传学， 2012， 博士

【摘要】 人干扰素(Interferon)α2b是重要的肿瘤免疫治疗药物，在许多医院中作为一线的肿瘤治疗药物被广泛使用。己获美国FDA批准用于多种病毒性疾病和恶性肿瘤的治疗。然而，IFNα2b在临床治疗中需要大剂量长期给药，因此常常引发如急性和慢性毒性、神经系统损害和血液系统损害等明显的毒副作用，严重制约了其在临床上的广泛应用。人血管内皮抑素(Endostatin)是血管生成的抑制剂，在抑制肿瘤血管生成类药物中表现优秀，2009年被SFDA批准用于治疗肿瘤。经过大量的研究证实，人血管内皮抑素N末端的27个氨基酸是其核心功能区域。在内皮抑素N末端的27个氨基酸基础上进行氨基酸的替换和增补，引入串联的RGD(Arg-Gly-Asp)序列，形成内皮抑素多肽(29amino acid, EP29)，在保持内皮抑素抑制肿瘤血管生成活性的同时，RGD序列赋予内皮抑素27肽靶向肿瘤血管内皮细胞、抑制肿瘤细胞生长与转移和增强其抗肿瘤活性的效果。实验室研究证实，EP29体外抗肿瘤活性明显高于天然内皮抑素，体内抗肿瘤活性也略高于天然内皮抑素，病理切片研究显示肿瘤组织大面积坏死，肿瘤萎缩。因此，有望成为新一代抗肿瘤小分子药物。在本研究中，我们采用基因工程技术，将EP29融合在IFNα2b的C末端形成IFNα2b-内皮抑素29个氨基酸多肽(IEP29)融合蛋白，在大肠杆菌中进行表达。期望该融合蛋白通过EP29的RGD序列与肿瘤新生血管内皮细胞的整合素αvβ3/αvβ5特异结合，使IEP29在肿瘤组织的新生血管处富集，既能针对性地发挥EP29抑制肿瘤血管生成和抗肿瘤作用，又可以抑制整合素介导的新生血管形成，从而降低IFNα2b临床用药量，提高量效比。通过一系列的体内和体外实验分析证实，IEP29蛋白具有较高的生物活性，能够抑制肿瘤生长和肿瘤血管生成，同时具有良好的肿瘤靶向性。因此，符合最初的设计，具有较高的临床应用开发价值，有望成为新一代抗肿瘤药物。研究具体内容如下：1. IEP29融合蛋白上游研究本实验采用DNA重组技术，成功构建含IEP29融合基因的原核表达载体pET3a-IEP29，将表达载体转染BL21工程菌，经IPTG诱导后以包涵体形式表达重组蛋白，表达量占菌体总蛋白的10%左右，重组蛋白条带能够和抗IFNα2b单克隆抗体特异性结合。重组工程菌使用5L的NBS发酵罐培养，收集菌体经裂解、包涵体洗涤、溶解变性和透析复性，最后经亲和层析和离子交换柱纯化获得纯度大于95%的重组蛋白，内毒素检测完全符合药典标准。为适应临床前试验研究的需要，我们优化了发酵和纯化工艺，并将发酵和纯化工艺放大至中试规模。采用NBS5L发酵罐连续培养三批工程菌，每批次收获湿菌体约150g，IEP29蛋白表达量约占总蛋白量的10%。纯化三批次IEP29蛋白，纯度均达到95%以上，蛋白产量为每批30~50mg，生产规模基本达到中试要求。按照基因工程药物质量标准的要求对IEP29融合蛋白的理化性质、纯度、生物活性、内毒素含量和杂质残留等进行检测和控制。2. IEP29融合蛋白活性研究为了验证融合蛋白是否同时具有IFNα2b和EP29的生物活性，我们通过肿瘤细胞侵袭和肿瘤细胞生长抑制实验来验证融合蛋白体外活性。通过内皮细胞粘附实验来验证融合蛋白在体外与肿瘤特异的内皮细胞亲和能力。结果说明，在体外IEP29融合蛋白有效抑制肿瘤细胞繁殖和迁移，其能力明显高于IFNα2b和EP29，同时特异粘附于内皮细胞。3. IEP29融合蛋白抗肿瘤研究通过小鼠体内抑瘤实验、药物体内分布研究、肿瘤病理组织切片研究、鸡胚尿囊膜试验等，一方面研究和探讨IEP29融合蛋白在小鼠体内的抑瘤效果和间接证明药物的肿瘤靶向性。另一方面初步探讨融合蛋白抑制肿瘤生长的作用机理。结果显示IEP29的抗肿瘤效果比IFNα2b和EP29更加显著，有效抑制肿瘤血管生成，具有肿瘤靶向性。为了进一步研究IEP29是否保持着IFNα2b和EP29的生物学功能，我们进行了一系列的抗肿瘤研究，有助于揭示靶向性药物的作用途径，同时对融合蛋白药物的开发也具有指导意义。在抑瘤实验中，IEP29融合蛋白能明显减轻S180、H22和Lewis肿瘤细胞荷瘤裸鼠的瘤重，和对照组比具有显著性差别，并且具有明显的量效关系。IEP29融合蛋白组抑瘤率均明显高于同剂量的EP29组，且和同剂量IFNα2b组比较有显著性差别。体内分布试验显示，给药30min后IEP29在肿瘤组织的浓度是IFNα2b的5倍，给药1h后IEP29在肿瘤组织的浓度是IFNα2b的1.8倍，表明IEP29可以在小鼠肿瘤组织中有效聚集。在鸡胚绒毛尿囊膜实验中，EP29、IFNα2b和IEP29蛋白在不影响原有血管的前提下，均有一定程度的抑制新生血管的生长。EP29和IEP29抑制新生血管的能力明显强于IFNα2b。从结果可以看出对照组血管生长良好，毛细血管清晰可见，主血管粗壮，分支适中。IFNα2b组有部分毛细血管减少，但不明显。EP29组毛细血管数量部分减少，局部血管变模糊。而IEP29组的血管抑制较明显，许多毛细血管开始消失，大部分血管变模糊。综上所述，我们获得了IEP29蛋白，经过体内和体外实验研究证实IEP29融合蛋白与IFNα2b和EP29相比，是一种高效抗肿瘤新生血管形成和抑制肿瘤生长的药物，本研究结果为IEP29重组蛋白将来的工业化生产和临床研究奠定了基础。此外，建立了比较稳定的生产工艺流程，确定了优化的融合蛋白表达和纯化系统及质量控制标准。更多还原

【Abstract】 Interferon α2b is an important tumor immunotherapy drug, as first-line drugs arewidely used in cancer treatment. It has been approved by FDA for the treatment ofvarious viral diseases and cancer. However, IFNα2b in the clinical treatment oflong-term administration of large doses required, as so often lead to acute and chronictoxicity, damage to the nervous system and blood system and other significant sideeffects, has seriously hampered its wide application in clinical practice.Human endostatin is an inhibitor of angiogenesis, the inhibition of tumorangiogenesis drugs in outstanding performance in2009, approved by SFDA for thetreatment of tumors. After a number of studies confirm that human endostatinN-terminal27amino acids is the core functional areas. It on the basis of the replacementand addition, the introduction of series of RGD (Arg-Gly-Asp) sequence, formationendostatin peptide (29amino acid, EP29), while maintaining the endostatin inhibition oftumor angiogenesis activity and given by the endostatin27peptide-RGD targetingtumor vascular endothelial cells, inhibit tumor cell growth, metastasis and enhancing theeffectiveness of its anti-tumor activity. Laboratory studies have confirmed, EP29in vitroanti-tumor activity was significantly higher than the natural endostatin, in vivoanti-tumor activity of endostatin is also slightly higher than the natural, pathologicalstudies have shown that a large area of tumor necrosis and shrinkage. Therefore, it wasexpected to become a new generation of small molecule anti-cancer drugs.In this study, we used genetic engineering techniques to integrate the IFNα2b andEP29formation IFNα2b-endostatin29peptide (IEP29) fusion protein in E. coli forexpression. Expect that the RGD sequence of fusion protein can specific bindintegrin-αvβ3/αvβ5of endothelial cells within tumor angiogenesis. IEP29enriched attumor tissue to play targeted inhibition tumor angiogenesis of EP29and anti-tumor roleof IFNα2b, but also can inhibit integrin-mediated angiogenesis, thereby reducing theIFNα2b clinical dosage, increased dose-effect ratio. Through a series of in vivo and invitro analysis confirmed that, IEP29protein with high biological activity, can inhibittumor growth and tumor angiogenesis, but also has good tumor targeting. Therefore,according with the original design, with a high clinical application development value,is expected to become a new generation of anticancer drugs. Research details are as follows:1. IEP29fusion protein upstream researchIn this study, we successfully constructed IEP29prokaryotic expression vectorpET3a-IEP29by DNA recombinant technology, the vector was transfected into BL21engineering bacteria and induced by IPTG as inclusion bodies after expression, IEP29expression about10%of total bacterial protein. IEP29can be specific binding withmonoclonal antibodies of anti-IFNα2b. Re-engineering of bacteria using5L NBSfermentor cultivation, collection of bacteria by lysis, inclusion body wash, dissolve thedenaturation and refolding dialysis, and finally by affinity chromatography andion-exchange column purification the purity greater than95%of the recombinantprotein, in full compliance with USP standards for toxin detection.In order to meet the needs of pre-clinical study, we optimized the fermentation andpurification process to enlarge and pilot scale. Continuous fermentation using NBS5LFermentation batches, each batch of wet biomass harvest of about150g, IEP29proteinfusion protein is about the total amount of10%. Consecutive batches of IEP29purifiedfusion protein, more than95%purity, yield30~50mg/batch scale of production toachieve the basic test requirements. IEP29by physical and chemical properties, purity,biological activity, endotoxin content, residual impurities and other aspects of control,confirm IEP29protein gene engineering drugs meet quality standards.2. Activity analsis of IEP29proteinIn order to verify that both the biological activity of IFNα2b and EP29, the tumorcell growth inhibition and invasion assay were adopted. The experimental validation invitro fusion protein can effectively inhibit tumor cell invasion. Endothelial cell adhesionassay verificated the fusion protein in vitro effective adhesion endothelial cells andtumor-specific high affinity. Results indicate that IEP29in vitro inhibition of tumor cellproliferation and migration was significantly higher than IFNα2b and EP29, whilespecific adhesion to endothelial cells, statistically significant higher.3. IEP29fusion protein anti-cancer researchExperiments of drug biodistribution studies, tumor pathology biopsy study, chickchorioallantoic membrane test, etc shows the IEP29protein inhibitory effect of tumor.The other hand, some assaies study the mechanism of inhibition of tumor growth. Theresults showed that IEP29anti-tumor effective than IFNα2b and EP29. It is more significant and effective inhibition of tumor angiogenesis, targeting both the tumor. Tofurther study if the IEP29is maintained IFNα2b and EP29of the biological function, weconducted a series of studies, help to reveal the pathway-targeted drug, while the fusionprotein drug development is also instructive.In tumor inhibition assay, IEP29was significantly reduced tumor weight thattumor-bearing nude mice was injected tumor cells H22and Lewis’s, and the controlgroup was significant difference between the rate of tumor weight in a dose-dependentinhibition. IEP29group were significantly higher than the same dose of the EP29group,and the same dose IFNα2b significantly different. In vivo distribution studies show that30min after administration of tumor tissue the concentration of IEP29is IFNα2b5times,60min after administration of tumor tissue the concentration of IEP29is1.8times IFNα2b. IEP29can effectively gather in tumor tissue in mice.In the chick embryo chorioallantoic membrane experiments, EP29, IFNα2b andIEP29protein without affecting the blood vessels under the premise of the original,there is a certain degree of inhibition of the growth of new blood vessels. EP29andIEP29inhibition of angiogenesis was significantly stronger than IFNα2b. The resultscan be seen from the control group grew well in blood vessels, capillaries clearly visible,the main blood vessel stout, branch moderate. IFNα2b group shows part of the capillaryto reduce, but not obvious. EP29group significantly reduced the number of capillaries,blood vessels become thin and blurred. IEP29group was more obvious, a large numberof capillaries disappear, the main blood vessels as well as some disappear.In summary, we have established a relatively stable IEP29protein productionprocess, the establishment of an optimized expression and purification system andquality control standards. In vivo and in vitro experiments have confirmed that, IEP29and EP29fusion protein compared with IFNα2b, is a highly effective anti-tumorangiogenesis and inhibit tumor growth, results of this study shows IEP29is hopefulindustrial production and laid the foundation for clinical trials in the future.更多还原