【作者】 胡沛臻；

【导师】 隋延仿；

【作者基本信息】 第四军医大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 恶性肿瘤严重地威胁着人类的健康,已成为人类主要死因之一。手术、化疗和放疗是治疗恶性肿瘤的三大传统疗法,其治疗效果并非尽如人意。随着免疫学、细胞生物学及分子生物学的飞速发展,肿瘤疫苗的研制与应用已成为治疗肿瘤复发、转移的有力手段。但是,现有肿瘤疫苗由于缺乏有效的抗原提呈,以及MHC限制性、肿瘤异质性、疫苗生物利用度差等问题,在走向临床运用的过程中还面临很多困难。黑色素瘤相关抗原MAGE（Melanoma associate antigen）属于肿瘤/睾丸抗原,其中MAGE1、MAGE3是肿瘤免疫治疗的理想靶分子,在多种肿瘤组织中表达而不在正常组织表达（除睾丸和胎盘）,被称为肿瘤特异性共有抗原。热休克蛋白是一种抗原递呈的辅助分子,可以增强肿瘤的免疫原性。在以往本室的研究中已证实MAGE1、MAGE3与HSP70的融合蛋白可以增强动物机体针对MAGE1和/或MAGE3的免疫反应,是一种优良的抗原物质,并构建了“恶性肿瘤基因工程纳米疫苗”。为了进一步提高该疫苗的抗肿瘤免疫活性,提高疫苗的生物利用度,也为了将该疫苗推向临床前研究,逐步进入成果的转化开发,本研究以肿瘤特异性抗原MAGE1、MAGE3与热休克蛋白HSP70融合蛋白等前期研究工作为基础,采用周期短、产率高且稳定可靠的中试发酵工艺,生产了足够后期临床前研究所用的重组蛋白,并对其进行酯化,再以纳米长循环脂质体作为载体,制备恶性肿瘤基因工程纳米脂质体疫苗,而后研究该疫苗对机体细胞免疫和体液免疫的作用,以及该疫苗在小鼠体内的吸收、分布、转化和排泄等过程的速度规律。1.鉴定重组工程菌pET28a-MAGE1/HSP70/MAGE3（MHM）/BL21（DE3）并检测其生物学性状的稳定性。结果显示含有该表达载体的工程菌经IPTG诱导后以包涵体形式表达重组蛋白,新生的蛋白条带能够和抗MAGE1单抗和抗MAGE3多抗特异性结合;对重组工程菌进行摇瓶培养,菌体经裂菌、包涵体的洗涤、溶解变性和透析复性,最后经亲和层析柱纯化获得纯度>80%的重组蛋白;该重组工程菌菌株连续传至50代后,进行质粒性状、扫描电镜观察、融合蛋白表达水平、细菌染色及各项生化反应检测,发现该菌株呈现典型的大肠杆菌形态,生物学形状稳定,可作为生产用菌种。2.为满足临床前各项实验的需要,将工程菌发酵和蛋白纯化工艺放大至中试规模。采用溶氧反馈-分批补料培养方式,对影响工程菌生长及目的蛋白表达的因素如发酵培养基、活化时间、诱导浓度及时间、pH值及分批补加营养物质等条件进行优化,在20L发酵罐中连续进行了三批工程菌发酵,工程菌生长正常,目的蛋白MHM和M3H的表达量均大于35%,保证了工程菌的稳定性;对实验室摸索的蛋白纯化工艺进行改进,连续纯化了三批M3H融合蛋白,纯度均在90%以上,生产规模达到中试要求;通过对融合蛋白的理化性质、纯度、生物学活性和残留杂质等几方面的控制,确认目的蛋白符合基因工程药物质量标准的要求。3.为有效提高脂质体包裹大分子蛋白的包封率,提高肿瘤抗原的生物利用度,运用化学合成方法将肿瘤特异性抗原融合蛋白与硬脂酸交联,使融合蛋白酯化后再进行纳米包裹,成功制备出平均粒径为87.9nm（CV=0.371）的衍生肿瘤抗原纳米脂质体疫苗,其药物包封率达到86%,显著高于单纯肿瘤抗原纳米脂质体（37%）,有效增强了抗原的生物利用率。该疫苗于4℃放置6月后无沉淀及分层,具备优良的理化性质。通过IFN-γELISPOT和LDH释放实验检测疫苗激活小鼠特异性细胞免疫反应的情况,发现该疫苗比游离蛋白疫苗能更好的诱导机体产生针对MAGE3的特异性CTL,对表达MAGE3的肿瘤细胞具有更强的杀伤作用。间接ELISA结果显示,该疫苗能够提高机体MAGE3的抗体水平,有效激活小鼠的体液免疫反应。4.在小鼠体内对该疫苗进行了药代动力学研究,首先制备¹²⁵I标记的衍生融合蛋白(¹²⁵I-SA-MH)和¹²⁵I标记的衍生融合蛋白脂质体(¹²⁵I-L-SA-MH),小鼠单次给药后,三氯醋酸（TCA）沉淀法测定血浆、组织、尿和粪的放射性含量,3p97软件拟合药物动力学模型,并计算相应参数。结果显示,¹²⁵I-SA-MH和¹²⁵I-L-SA-MH单次静脉注射后在小鼠体内的动力学过程符合两室模型。在相同剂量条件下,¹²⁵I-L-SA-MH分布相半衰期T1/2α和消除相半衰期T1/2β和AUC均有所增加;¹²⁵I-SA-MH的总体清除率比¹²⁵I-L-SA-MH大,证明脂质体包裹能保护药物,有一定的缓释作用,可进一步提高药物的生物利用度。体内分布试验结果表明,¹²⁵I-SA-MH进入血液后首先迅速聚集到肝脏,而¹²⁵I-L-SA-MH进入血液后则到脾脏发挥作用。排泄实验结果显示,¹²⁵I-SA-MH和¹²⁵I-L-SA-MH主要通过泌尿系统排泄。综上所述,我们研制的“恶性肿瘤基因工程纳米疫苗”能有效激发机体的细胞和体液免疫反应,杀伤肿瘤细胞;药代动力学显示该药物具有靶向性、缓释性及较高的生物利用率;同时还建立了稳定的中试发酵和蛋白纯化生产工艺,为进行临床前研究及药物开发奠定了基础。更多还原

【Abstract】 Neoplasm is a life-threatening disease and the leading cause of death. The traditional therapies such as surgery, radiotherapy and chemotherapy are far from satisfaction. With the progress in immunology, cellular biology and molecular biology, tumor vaccines are regarded as a promising method and play an important role in the prevention and treatment of tumor. However, there are still many difficulties before the tumor vaccines could be used clinically. The lack of tumor specific antigens, the shortage of effective antigen-presenting tools, the limitation of MHC, the polymorph of tumor cells and the low bioavailability are the major factors. The Melanoma Antigen-associated Gene （MAGE） belongs to Cancer/Testis antigen, which was the first reported example of tumor specific antigens expressed in most malignant tumors but not in normal tissue except for testis and placenta, and has been used as the ideal target in tumor immunotherapy. Heat shock protein （HSP） participates in processing and presentation of tumor antigen and plays an important role in promoting anti-tumor immunity. Our recent researches have demonstrated that the fusion protein of MAGE1 and MAGE3 to M. tuberculosis HSP70 can lead to the enhancement of tumor vaccines potency in tumor immunotherapy, and we have prepared nanoemulsion encapsulated tumor antigen protein vaccine. To improve its antitumor immunoactivity and bioavailability and meet the requirement of pre-clinical experiments, based on the MAGE1, MAGE3 and HSP70 fusion protein, a short cycle, high expression and stabilized fermentation process of recombinant fusion protein has been used, which would be the basis for further purification and large-scale production of recombinant fusion protein. To prepare nanoliposome encapsulated tumor specific antigen derivative vaccine, the purified recombinant fusion protein was esterified and then was wrapped up with long circulating nano-liposome. Its immunological mechanisms and the dynamic rules of ADME in mice were studied.1. The structure and stability of biological characteristics of recombinant bacterial strain pET28a-MAGE1/HSP70/MAGE3（MHM）/BL21（DE3） were studied. The recombinant fusion protein expressed in inclusion bodies induced with IPTG. The expressed protein, binding with anti-MAGE1 mAb as well as anti- MAGE3 polyclonal Ab specifically, is over 30% of total bacteria protein. Then the engineering bacteria were cultured in shake flask. The inclusion bodies were washed, denatured, renatured and purified by affinity chromatography. The purity of the product was over 80%. The recombinant strain was subcultured for 50 passages, and the expression level of MHM,property of plasmid,as well as morphology,cultivation characters and biochemical reactions of various passages were studied.pET28a-MHM/BL21（DE3） showed a typical morphology of E.coli, and the characteristics of various passages were not significantly different from that of primary strain.The strain is suitable for large-scale production. 2. To match the requirement of pre-clinical experiments, the production and purification procedure of recombinant fusion protein were scaled up to pilot-scale. The bacteria were cultured in 20L DO feed-back fed-batch culture system. The effects of the composition of the fermentation medium, activation time, induction time, the range of pH and fed-batch carbon sources on the expression level of MHM and cell output were analyzed.We performed a triplicate fermentation. The expression level of recombinant fusion protein MHM and M3H was higher than 35 % of the total protein in E.coli. Three batches M3H protein were purified to homogeneity and the products amounted to 600-700mg/batch. Quality control was established by the tests of characteristics, purity, bioactivity and impurity residue of the fusion protein.3. To improve the efficiency of envelopment of protein by liposomes and the bioavailability of tumor antigen, the recombinant fusion protein was cross linked with stearic acid by chemical methods. The purified recombinant fusion protein was esterified and then was wrapped up with nano-liposome. Nanoliposome-encapsulated tumor specific antigen derivative vaccine （L-SA-MH） was prepared and its shape and size were detected by electron microscope and particle sizing system. The mean size of nano-liposomes was 87.9nm（CV=0.371）, the encapsulation rate was 86%, and L-SA-MH had the required stability after stored in 4℃for 6 months. The anti-tumor immunity was tested by Enzyme-linked immunospot assay （ELISPOT） and Cytotoxicity assays. ELISPOT and Cytotoxicity assays showed the immunization using L-SA-MH vaccine enhanced the frequency of splenocytes secretingγ-IFN significantly compared with immunization using free protein vaccine alone. Cytotoxicity assays showed the MAGE3 B16 lysis of CTLs from mice immunized with L-SA-MH vaccine was greater than that from mice immunized with free protein vaccine. The fusion protein MH, SA-MH, L-MH and L-SA-MH vaccine could induce higher titer of anti-MAGE3 antibody than control groups. These results showed that nanoliposome had novel characters, and nanoliposome-encapsulated tumor specific antigen derivative vaccine could greatly enhance the potency of MAGE3 protein vaccines, and generate specific anti-tumor immunity against MAGE3 expression tumors.4. To clarify the process and properties of ADME and supply the pharmacokinetic parameters for safe use of nano-liposome L-SA-MH in clinics, ¹²⁵I-SA-MH and ¹²⁵I-L-SA-MH were prepared. The radioactivity in plasma or tissues or urine or feces was determined following trichloroacetic （TCA） precipitation of after a single intravenous injection. The concentration-time curves of ¹²⁵I-SA-MH and ¹²⁵I-L-SA-MH were proved to comply with two-compartment model. At the same amount of dosage, the distribution half life T1/2α, the elimination half life T1/2βand the AUC for plasma were increased after a single i.v. with ¹²⁵I-L-SA-MH compared i.v. with ¹²⁵I-SA-MH, and the total body clearance rate of ¹²⁵I-SA-MH was higher than that of ¹²⁵I-L-SA-MH. So liposome can protect drug and prolong the half time of SA-MH in serum and tissues, which enhanced drug bioavailability. ¹²⁵I-L-SA-MH was aggregated in most tissues especially in spleen while 25I-SA-MH in liver. Urinary system excretion test proved it to be the major pathway of ¹²⁵I-SA-MH and ¹²⁵I-L-SA-MH elimination.In summary, we constructed nanoliposome-encapsulated tumor specific antigen derivative vaccine, and it can induce tumor cell specific cellular and humoral immune reaction effectively. The enhanced immunity resulted in potential therapeutic effects against MAGE3-expressing tumors. The pharmacokinetic data indicate that it is tumor-targeting, delayed released and has higher bioavailability. Moreover, we established a stable production procedure of recombinant fusion protein and set up a standard of quality control. The data from this study grounds the large scale production and pre-clinical experiments of vaccine.更多还原