第一部分  防御素与人血清白蛋白的重组融合蛋白的制备及其抗胰腺癌活性研究
目的：研究表明，巨胞饮作用是细胞从细胞外获取营养和液体的一种细胞内运输形式。细胞膜内陷将细胞外颗粒包裹成小囊泡（胞饮囊泡），最终与溶酶体融合;然后囊泡内的物质被水解或分解。超过95%的胰腺导管癌具有KRAS突变，KRAS突变的胰腺癌细胞可以通过强烈的巨胞饮作用摄取白蛋白作为营养物质。此外，NF-kB在胰腺癌中过度表达。这些表明KRAS突变和NF-κB在胰腺癌中都起着至关重要的作用。防御素是先天免疫中的生物活性介质。除了具有抗菌和抗病毒活性外，防御素，特别是人β-防御素2(Human β defensin 2，HBD2)，还表现出抗肿瘤功效。研究表明，防御素可以抑制NF-κB。基于NF-κB抑制作用和KRAS突变驱动的巨胞饮作用双重靶向策略，我们设计并制备了一种重组融合蛋白DF2-HSA，它由两分子β-防御素2 (HBD2)和一分子人血清白蛋白(Human serum albumin，HSA)组成。在这种情况下，HBD2用作“弹头”药物，HSA用作定向递送药物至肿瘤部位的载体。我们将研究重组融合蛋白DF2-HSA对胰腺癌的抗肿瘤作用及其机制。
方法：采用毕赤酵母表达系统表达和分泌重组融合蛋白DF2-HSA，浓缩后通过Histrap柱纯化，脱盐柱脱盐来提高蛋白纯度。利用激光共聚焦实验和流式细胞术检测巨胞饮介导的胰腺癌细胞对重组蛋白DF2-HSA的摄取；利用CCK-8、克隆形成实验、愈合实验、Transwell侵袭实验、细胞成球实验检测重组蛋白DF2-HSA对胰腺癌增殖、迁移、侵袭、肿瘤细胞成球的影响；建立人胰腺癌MIA PaCa-2无胸腺裸鼠移植瘤模型检测重组蛋白在裸鼠体内的特异性生物分布及疗效评价。
结果:KRAS突变的胰腺癌细胞MIA PaCa-2比KRAS野生型的胰腺癌细胞BxPC-3对重组蛋白表现出更强的巨胞饮摄取能力；重组蛋白DF2-HSA的杀伤能力强于游离防御素HBD2和HSA。相比之下，KRAS突变的胰腺癌细胞MIA PaCa-2比KRAS野生型的胰腺癌细胞BxPC-3对DF2-HSA更敏感；重组蛋白DF2-HSA可以抑制胰腺癌细胞的迁移、侵袭；此外，DF2-HSA抑制肿瘤细胞球的形成并可消除细胞表面的丝状伪足，其效果与盐霉素诱导的效果相当。体内成像显示DF2-HSA可以快速汇聚于肿瘤部位并能滞留较长时间，而游离防御素HBD2则迅速消失；DF2-HSA显著抑制无胸腺裸鼠胰腺癌MIA PaCa-2异种移植瘤的生长；并且其与吉西他滨的联合用药取得了更高的抗肿瘤疗效。
结论：由人β防御素2和人血清白蛋白HSA构成的重组蛋白DF2-HSA，可以被KRAS突变的胰腺癌以巨胞饮作用大量摄取；同时下调NF-κB。重组融合蛋白DF2-HSA可以抑制胰腺癌细胞的增殖、迁移、侵袭，并减弱其干细胞特性；此外，DF2-HSA能特异性生物分布于肿瘤部位，并对无胸腺裸鼠的胰腺癌异种移植瘤发挥治疗作用。结果表明，使用HBD2作为弹头药物和人血清白蛋白作为递送载体是开发新的靶向癌症疗法的可行策略。重组融合蛋白DF2-HSA对胰腺癌的治疗有很大的潜力。
关键词：人血清白蛋白，防御素，巨胞饮，KRAS突变，NF-κB

 
第二部分  多特异性重组融合蛋白的构建及制备
癌症是一个重大的公共卫生问题，它可以发生在身体的各种器官中，并且可以转移到附近甚至更远的其它组织，是当前导致死亡的主要原因之一。现阶段针对肿瘤治疗常用的策略包括手术切除、放射疗法、化学疗法和激素疗法等，但这些治疗方法都有自身的不足。化疗是癌症治疗中常用的治疗手段，现有的抗肿瘤药物大多在抑制肿瘤的同时对人体正常组织有较大的毒性，药物不能有效地渗透到肿瘤组织内部，并且还会诱导肿瘤细胞产生耐药，耐药是癌症治疗失败的主要原因。因此，开发包含小分子药物和单克隆抗体的分子靶向治疗药物已成为高效抗肿瘤治疗的热点。抗体可通过与效应细胞结合或激活细胞信号通路诱导细胞毁灭。表皮生长因子受体（Epidermal growth factor receptor, EGFR）是受体酪氨酸激酶的一种，属于ErbB/HER家族成员，其在一些脑癌、肺癌、以及其他癌症中大量表达，并且在癌细胞的发展、高活跃性和失调等过程中至关重要。程序性细胞死亡蛋白1（Programmed cell death protein 1, PD-1）是一种在抗原刺激下T细胞表面产生的共抑制受体，程序性死亡配体1（Programmed death-ligand 1, PD-L1）是PD-1的其中一种配体，两者结合可抑制T细胞增殖和效应功能。CD3分子是T细胞膜上的重要生物标志物，它们参与T细胞抗原识别、信号转导和T细胞发育的调节，CD3在双特异性抗体领域是一个很有前景的肿瘤免疫治疗靶点。D3是人血清白蛋白的同源结构域III，在HSA与新生儿Fc受体（neonatal Fc receptor, FcRn）的结合中起重要作用，两者结合可延长HSA在血清中的半衰期。人β防御素2（HBD2）是一种抗菌肽，除了抗菌、抗病毒作用外，对肿瘤也有一定的毒性。因此，HBD2还可用于抗肿瘤治疗。本研究通过基因工程技术构建了三种重组蛋白Fv-PDL1-D3、Fv-CD3-D3、Fv-DF-D3，其中Fv为抗EGFR单链抗体，将不同特性的片段融合，得到多特异性重组蛋白。这些重组蛋白是使用毕赤酵母分泌表达系统表达的。表达载体成功构建后转化毕赤酵母GS115细胞，得到重组蛋白的表达菌株，Fv-DF-D3表达水平较低，需进一步对重组蛋白Fv-DF-D3的表达菌株经过G418高压筛选及诱导条件的优化。
关键词：多特异性，EGFR，PD-L1，CD3，D3

Part I  Preparation and study of the anti-pancreatic cancer activity of a recombinant fusion protein consisting of defensin and human serum albumin  
Objective: Studies have shown that macropinocytosis is a form of intracellular transport for cells to obtain nutrients and fluid from outside the cell. The invagination of the cell membrane wraps extracellular particles into small vesicles (pinocytosis vesicles) which finally fuse with lysosomes; and then the substances inside the vesicles are hydrolyzed or decomposed. More than 95% of pancreatic ductal carcinomas have KRAS mutations; notably, KRAS-mutated pancreatic cancer cells could uptake albumin as a nutrient by intense macropinocytosis. Moreover, NF-κB over-expression occurs in pancreatic cancers. Those indicate that KRAS mutation and NF-κB both play crucial role in pancreatic cancer. Defensins are bioactive mediators in innate immunity. Besides having antibacterial and antiviral activity, the human beta-defensin 2 (hBD2), in particular, also exhibit antitumor efficacy. Studies have shown that defensin can inhibit NF-kB. Based on the proposed dual-targeting strategy that inhibits NF-κB and concomitantly interferes with the enhanced macropinocytosis driven by KRAS mutation, we designed and generated a recombinant fusion protein DF2-HSA which consists of two molecules of human beta-defensin 2 (HBD2) and a moiety of human serum albumin (HSA). In this case, HBD2 is utilized as “warhead” agent and HSA as carrier for tumor-site oriented delivery. The anti-tumor efficacy of the recombinant fusion protein DF2-HSA against pancreatic cancer and its mechanism were investigated.
Methods: The Pichia pastoris expression system was used to express and secrete the recombinant fusion protein DF2-HSA. After concentration, it was purified by Histrap column and desalted by desalting column to improve the protein purity. Macropinocytosis-mediated uptake of the recombinant fusion protein DF2-HSA by pancreatic cancer cells was detected with confocal laser experiments and flow cytometry. The effects of the recombinant fusion protein DF2-HSA on the proliferation, migration, invasion and tumor cell spheroidization of pancreatic cancer cells were determined by CCK-8 assay, clone formation assay, healing assay, Transwell invasion assay, and cell spheroidization assay. The model of human pancreatic cancer MIA PaCa-2 xenograft in athymic mice was employed for detecting the specific biodistribution and evaluating the therapeutic efficacy of the recombinant fusion protein.
Results: As shown, the macropinocytosis-mediated uptake of the recombinant fusion protein DF2-HSA in KRAS mutant pancreatic cancer MIA PaCa-2 cells was more intense than that in KRAS wild-type pancreatic cancer BxPC-3 cells. The cytotoxicity of the recombinant protein DF2-HSA was more potent than that of the free defensin HBD2 or HSA. In comparison, KRAS-mutated pancreatic cancer MIA PaCa-2 cells were more sensitive to DF2-HSA than KRAS wild-type pancreatic cancer BxPC-3 cells. DF2-HSA inhibited the migration, and invasion of pancreatic cancer cells. Additionally, DF2-HSA inhibited tumor cell spheroid formation and depleted filopodia on cell surface; notably, the effects were comparable to those induced by salinomycin. In vivo imaging showed that DF2-HSA rapidly accumulated at the tumor-site and retained for a long period of time, while the free defensin HBD2 was quickly diminished. DF2-HSA markedly suppressed the growth of pancreatic cancer MIA PaCa-2 xenograft in athymic mice; and its combination with gemcitabine achieved higher antitumor efficacy. 
Conclusion: The recombinant protein DF2-HSA composed of human beta-defensin 2 and human serum albumin (HSA) could be taken up by KRAS mutant pancreatic cancer cells in massive amounts by macropinocytosis; and concomitantly it down-regulates NF-κB. The recombinant fusion protein DF2-HSA can inhibit the proliferation, migration, and invasion capability and alleviate stemness in pancreatic cancer cells. Furthermore, DF2-HSA displays tumor-site specific biodistribution and exerts therapeutic efficacy against pancreatic cancer xenograft in athymic mice. The results show that using HBD2 as the “warhead” agent and human serum albumin as the delivery carrier is a feasible strategy for the development of new targeted cancer therapeutics. The recombinant fusion protein DF2-HSA is potentially useful for pancreatic cancer therapy. 
Keywords: human serum albumin, defensins, macropinocytosis, KRAS mutation, NF-κB

 
Part II  Construction and preparation of multi-specific recombinant fusion proteins
Cancer is a major public health concern, which occur in various organs of the body and can metastasize to other nearby and even remote tissues, and is currently one of the leading causes of death. At present, the commonly used strategies for the treatment of tumors include surgical resection, radiotherapy, chemotherapy and hormone therapy, etc., however, those treatment methods have shortcomings. Chemotherapy is a commonly used in cancer treatment, but most of the existing anti-tumor drugs have major toxicity to normal human tissues while suppressing tumors. Drugs cannot effectively penetrate into tumor tissues, and may induce tumor cells to develop drug resistance, which is the main reason for the failure of cancer treatment. Therefore, the development of molecularly targeted therapeutic drugs including small molecule drugs and monoclonal antibodies has become a hot spot for highly effective anti-tumor therapy. Antibodies induce cellular destruction by binding to effector cells or activating cell signaling pathways. Epidermal growth factor receptor (EGFR) is a kind of receptor tyrosine kinase and belongs to ErbB/HER family, which is abundantly expressed in some brain tumors, lung cancers, and other cancers. EGFR is critical for the development, hyperactivity and dysregulation of cancer cells. Moreover, programmed cell death protein 1 (PD-1) is a co-inhibitory receptor produced on the surface of T cells under antigen stimulation. Programmed death-ligand 1 (PD-L1) is one of the ligands of PD-1, and the combination of PD-1 and PD-L1 can inhibit T cell proliferation and effector function. CD3 molecule is an important biomarker on T cell membranes, involving in T cell antigen recognition, signal transduction and the regulation of T cell development. CD3 is a promising target for tumor immunotherapy, particularly in the generation of bispecific antibodies. D3 is the homology domain III of human serum albumin, which plays an important role in the binding of HSA to the neonatal Fc receptor (FcRn), and their combination can prolong the half-life of HSA in serum. Human beta defensin 2 (HBD2) is an antibacterial peptide; in addition to the antibacterial and antiviral effects, it displays cytotoxicity to cancer cells. Therefore, HBD2 can be used for antitumor therapy. In this study, three recombinant proteins including Fv-PDL1-D3, Fv-CD3-D3, and Fv-DF-D3 were constructed by genetic engineering technology, wherein Fv was anti-EGFR single chain antibody, and the fragments with different characteristics were fused to obtain multi-specific recombinant proteins. These recombinant proteins were expressed using the Pichia pastoris secretory expression system. After successful construction, the expression vectors were transformed into Pichia pastoris GS115 cells, and then the expression strains of the recombinant proteins were obtained. The expression level of Fv-DF-D3 was low, so the expression strain for the recombinant protein Fv-DF-D3 needs to be further screened by G418 high pressure and optimized the induction conditions.
Keywords: multi-specific, EGFR, PD-L1, CD3, D3