双功能重组蛋白靶向免疫治疗肿瘤研究

发布时间：2018-06-03 23:02

本文选题：重组蛋白 + 表皮生长因子　；参考：《北京协和医学院》2015年博士论文

【摘要】：第一部分：可激活免疫细胞靶向EGFR高表达肿瘤细胞重组蛋白的设计、表达及对肿瘤的治疗作用肿瘤靶向治疗是当前癌症治疗的研究热点,并代表未来发展的方向。而肿瘤免疫治疗是继手术、放疗和化疗之后的第四种肿瘤治疗模式—肿瘤生物治疗,具有广阔的应用前景。有效地将两者结合起来是研发新型抗肿瘤药物的一大趋势。目前在抗肿瘤靶向药物的开发中,表皮生长因子受体(Epidermal growth factor receptor, EGFR)是公认的、选用最多的靶向治疗靶点。现在临床上主要有两大类EGFR靶向的抗肿瘤药物：一,小分子酪氨酸激酶抑制剂(small-molecule tyrosine kinase inhibitors, TKIs);二,单克隆抗体(monoclonal antibodies, mAbs)。但是在应用过程中发现有显著一部分患者对这两类药物均明显耐药,而最初敏感的患者在经过治疗后,转而发展为耐药,最终导致预后较差。通过分子水平的机制研究发现这种耐药多是由于EGFR激酶结构域存在突变,或其下游信号通路的持续活化造成。在这种情况下,肿瘤细胞不再依赖表皮生长因子(Epidermal growth factor, EGF)结合EGFR的启动激活信号,却可以持续地生长增殖,肿瘤细胞呈自主性生长状态。因此针对EGF-EGFR信号不敏感的肿瘤细胞,需要新的治疗方法。本研究旨在开发能够靶向免疫治疗该类肿瘤的重组蛋白类药物,即新型的Biosimih ar。为充分调动机体的免疫系统,将抗肿瘤免疫治疗机制运用至新型药物中,本研究将显性抗原肽构建至重组蛋白,组成一个EGFR靶向的双功能重组免疫调节蛋白,用于高表达EGFR肿瘤的治疗。本研究采用EGFR的天然配体EGF作为靶向结合载体,将其与来自李斯特菌溶细胞素O (Listeriolysin O, LLO)的3个显性T细胞表位融合,通过序列拼接构建了重组融合蛋白pLLO-hEGF。人EGF是一个由53个氨基酸残基组成的单链多肽,分子量小,与EGFR的亲和力高。而LLO是一个已经证实的强大的免疫原性分子,具有丰富的CD4+和CD8+T细胞抗原表位。本研究选用了经文献证实的2个CD4+和1个CD8+T细胞表位。所构建的重组蛋白pLLO-hEGF经生物信息学软件分析,理论分子量仅为16 kDa,性质稳定,血管穿透性将优于mAb。同时该重组蛋白应具有2种功能：既可以通过EGF靶向结合高表达EGFR肿瘤细胞,同时其LLO的显性抗原表位又可以充分活化机体的免疫细胞,使免疫细胞在肿瘤细胞部位聚集,从而加强攻击和杀伤肿瘤细胞,发挥靶向免疫治疗肿瘤的作用。我们首先采用生物工程的分子克隆技术以及蛋白表达纯化技术,成功克隆表达和获得了可溶性双功能重组蛋白pLLO-hEGF,4L重组菌液量可得(4.5-6)mL浓度为800 μg/mL左右(最大1.0 mg/m1)的纯化蛋白。接下来通过Western blot筛选了高表达EGFR的人肿瘤细胞系,从不同细胞系中各挑选出2种,包括人乳腺癌(MDA-MB-231、SK-BR-3)、人肺癌(A549、NCI-H157)和人结直肠癌(HCT116、HT-29),用于双功能重组蛋白pLLO-hEGF相关的功能研究。细胞免疫化学荧光染色实验结果显示了pLLO-hEGF可以很好地靶向结合这些肿瘤细胞表面。细胞增殖实验结果显示pLLO-hEGF对这些肿瘤细胞不产生促增殖作用。其免疫激活作用研究结果显示：pLLO-hEGF可使体外培养的人外周血单个核细胞(peripheral blood mononuclear cells, PBMCs)中 CD3+CD4+ T细胞明显增殖。单次实验结果显示pLLO-hEGF刺激14天时PBMCs中CD3+CD4+ T细胞百分比可增至54.5%,而对照组为44.8%。且刺激增殖活化的T淋巴细胞在体内外实验中均显示出显著的杀伤肿瘤细胞作用。体外对上述几种表达EGFR肿瘤细胞的杀伤率分别为：HCT116, (69.89 ±1.05)%; HT-29, (60.40±2.33)%; A549, (49.45±1.72)%; NCI-H157, (85.26±4.82)%；SK-BR-3,(47.90±1.39)%。体内移植瘤模型实验结果显示,将刺激增殖的T淋巴细胞注射裸小鼠体内,显著抑制了肿瘤的生长,平均瘤重(0.178/0.224 g)明显小于对照组(0.550 g)(*P0.05)。综上所述,我们的研究提供了一种研发新型抗肿瘤药物的新思路和新策略。此部分内容申请了国家发明专利(申请号：201410152549.2),目前在实审阶段,且相关内容已发表于Hum Vaccin Immunother (Human vaccines) (IF=3.643)和CellPhysiol Biochem杂志(IF=3.55)。第二部分：可激活免疫细胞靶向CD20阳性人淋巴瘤细胞重组蛋白的设计、表达及抗淋巴瘤作用的初步研究B细胞淋巴瘤是一种血液系统疾病,采用传统治疗手段临床疗效较差。随着对CD20分子结构特点的认识,抗CD20单克隆抗体的设计开发在B细胞淋巴瘤的临床治疗中取得重要进展。其中最具代表性的是1997年美国FDA批准上市的人鼠嵌合抗CD20单克隆抗体一Rituximab (C2B8,美罗华)。但人鼠嵌合抗体由于其分子量大,穿透力弱,且毒副作用大,明显限制了其临床效果。近年来,一些人源化抗体、小型或改型抗体、基因修饰抗体逐渐发展起来。其中单链抗体(single chain variable fragments, ScFv)由于其分子量小、穿透力强、能够较好地保持抗原亲和性等特点,成为应用生物工程方法进行肿瘤免疫治疗的一种重要手段。本研究同样利用LLO的显性抗原肽设计、构建了靶向CD20阳性B细胞淋巴瘤的双功能重组蛋白ScFv-pLLO。首先,我们设计了针对人CD20的ScFv序列,然后将LLO显性抗原肽与ScFv序列拼接,使构建的重组蛋白既可靶向结合CD20阳性B细胞淋巴瘤细胞,同时其LLO抗原表位又可增强肿瘤细胞的抗原性,从而充分激活机体的细胞免疫应答,发挥更长效的抗肿瘤免疫,达到靶向免疫治疗的效果。双功能重组蛋白ScFv-pLLO经原核克隆、表达和纯化,并初步分析了其抗B细胞淋巴瘤作用。生物信息学软件分析显示重组蛋白理论分子量为32 kDa,化学性质稳定。流式细胞术定量分析几种人淋巴瘤细胞系细胞表面CD20表达情况,结果显示Daudi细胞CD20阳性率在98%以上,Raji和NCI-BL2009细胞CD20阳性率分别为89.7%和92.2%,而RAMOS细胞CD20阳性率是52.3%。流式细胞术分析ScFv-pLLO靶向结合人淋巴瘤细胞的能力,结果显示ScFv-pLLO可不同程度的靶向结合CD20表达水平不同的人淋巴瘤细胞,其浓度为10μg/mL时,对Daudi和NCI-BL2009细胞的平均结合率分别为(93.25±3.45)%和(73.85±3.95)%。免疫共沉淀实验结果进一步证明ScFv-pLLO可特异靶向结合人淋巴瘤细胞表面的CD20分子。细胞增殖实验结果显示ScFv-pLLO可抑制Raji细胞的生长,而对Daudi、RAMOS和NCI-BL2009淋巴瘤细胞的生长无影响。凋亡实验结果显示ScFv-pLLO可直接诱导Raji细胞凋亡,20μg/mL的ScFv-pLLO作用24 h后,可诱导24.6%的Raji细胞凋亡(早期+晚期),作用48 h后,Raji细胞总凋亡率可达47.0%。本研究的初步结果显示,双功能重组蛋白ScFv-pLLO可特异靶向结合CD20阳性B细胞淋巴瘤,对某些人淋巴瘤细胞系,如Raji细胞,可直接诱导其凋亡,结构和功能上类似“小型抗体”。同时,双功能重组蛋白ScFv-pLLO由于携带显性抗原肽,不同于纯粹的单克隆抗体,其靶向结合淋巴瘤细胞表面CD20分子后可增强肿瘤细胞激活免疫系统的能力,从而为我们下一步研究其对免疫细胞的激活作用以及激发长效抗肿瘤细胞免疫的功能奠定了基础。此部分内容已发表于《中国免疫学杂志》。第三部分：Leptin在结直肠癌细胞侵袭转移中的作用研究随着肿瘤分子生物学研究的深入,在肿瘤靶向治疗中,不断有新的候选抗肿瘤靶点出现。近几年,越来越多的研究发现Leptin介导的信号在肿瘤细胞的增殖、侵袭和肿瘤干细胞的诱导生成中发挥了重要作用,并被认为是潜在的有效抗肿瘤靶向治疗靶点。Leptin的中文名称为瘦素,是一种由167个氨基酸组成的非糖基化蛋白质类激素,主要由脂肪细胞分泌,参与机体的能量代谢。研究表明肿瘤组织也可表达Leptin及其受体(LEPR/Ob-R),其中较多数据显示结直肠癌的恶性程度与Leptin的表达量密切相关。一些实验结果显示高浓度的外源性Leptin可以促进人结直肠癌细胞的增殖,并增强其运动和侵袭能力。但目前较少关于肿瘤细胞产生的内源性Leptin在肿瘤细胞生长和侵袭力方面确切生物学作用的研究,而明确Leptin介导的信号通路对肿瘤细胞生物学行为的影响,有助于开发新型抗肿瘤靶向治疗药物。在研究中,我们首先分析了几种人结直肠癌细胞系中Leptin及其受体LEPR的表达情况,包括mRNA和蛋白水平。结果显示人结直肠癌细胞系均普遍表达Leptin和LEPR,包括细胞系HCT116、HT-29、COLO201、COLO205和SW480。以人结直肠癌细胞系HCT116和HT-29细胞为例,我们用siRNA有效敲低了肿瘤细胞中内源性Leptin的表达。细胞增殖实验结果显示,抑制内源性Leptin的表达对人结直肠癌细胞系HCT116和HT-29细胞的增殖无影响。然后我们通过Real-time PCR检测了一些与肿瘤细胞侵袭转移能力密切相关基因的表达情况,包括MMP1、MMP9、 TIMP-1、E-cadherin、Twist等。结果显示,抑制内源性Leptin表达,HCT116细胞中MMP1和MMP9的表达明显上调(*P0.05),TIMP-1、TIMP-2 和 E-cadherin的表达明显下调(*P0.05)；HT-29细胞中,MMP1和Vimentin的表达明显上调(*P0.05),TIMP-2的表达明显下调(*P0.05)。以HCT116为例,Western blot进一步检测蛋白表达水平变化,其结果与mRNA水平变化一致。此外,明胶酶谱实验结果显示,抑制内源性Leptin表达,HCT116细胞中MMP9的蛋白裂解活性增强。Transwell体外侵袭实验结果显示,与对照组穿膜细胞数(148±17)相比,HCT116细胞Leptin RNAi组穿出小室微孔膜的细胞数明显增多(512±23)(*P0.05),表明HCT116细胞的侵袭能力明显增强。本研究不同于以往的研究,重点分析了抑制人结直肠癌细胞内源性Leptin的表达,对肿瘤细胞增殖和侵袭能力的影响。研究发现,降低Leptin的表达对细胞增殖无影响,但却明显增强了细胞的侵袭能力。而以往的研究结果是提高Leptin的浓度会促进肿瘤细胞的增殖和侵袭。因此,进一步探明不同条件下Leptin信号通路在结直肠癌细胞中的具体生物学作用机制,可为确定此通路能否用于肿瘤靶向治疗靶点选择奠定基础。
[Abstract]:The first part: the design, expression, and therapeutic effect of the tumor targeting EGFR high expression of tumor cell recombinant protein, which is the hot spot of cancer therapy and the direction of the future development. Tumor immunotherapy is the fourth mode of tumor treatment following surgery, radiotherapy and chemotherapy. Epidermal growth factor receptor (EGFR) is the most widely accepted target for target therapy in the development of anti tumor targeting drugs. Two major EGFR targeting antitumor drugs: one, small molecule tyrosine kinase inhibitor (small-molecule tyrosine kinase inhibitors, TKIs); two, monoclonal antibodies (monoclonal antibodies, mAbs). But a significant number of patients were found to be significantly resistant to these two drugs during the application process, and the first sensitive patients were passing through After treatment, it turns to drug resistance and eventually leads to poor prognosis. Through molecular level mechanisms, it is found that the resistance is due to mutations in the EGFR kinase domain or the continuous activation of its downstream signal pathway. In this case, the tumor cells are no longer dependent on the epidermal growth factor (Epidermal growth factor, EGF) binding EGFR. The activation signal can continue to grow and proliferate, and the tumor cells are autonomous growth. Therefore, new therapies are needed for tumor cells that are not sensitive to EGF-EGFR signals. This study aims to develop recombinant protein drugs that can target the immunotherapy of this type of tumor, that is, the new type of Biosimih ar. to fully mobilize the body. The Phytophthora system, which applies the antitumor immunotherapy mechanism to the new drug, constructs the dominant antigen peptide to the recombinant protein to form a EGFR targeted double functional recombinant immunoregulatory protein for the treatment of high expression of EGFR tumor. This study uses the natural ligand EGF of EGFR as a targeting carrier, and it is from Lester bacteria. The fusion of 3 dominant T cell epitopes of Listeriolysin O (LLO) O, the recombinant fusion protein pLLO-hEGF. human EGF is a single strand polypeptide composed of 53 amino acid residues. The molecular weight is small and the affinity to EGFR is high. LLO is a powerful immunogenic molecule that has been proved to be rich in CD4+. This study selected 2 CD4+ and 1 CD8+T cell epitopes confirmed by the literature. The constructed recombinant protein pLLO-hEGF was analyzed by bioinformatics software. The molecular weight of the recombinant protein was only 16 kDa, the properties were stable, the blood vessel penetration would be better than mAb. and the recombinant egg white should have the function of the EGF target node. At the same time, the high expression of EGFR tumor cells, and the dominant antigen epitopes of LLO can fully activate the immune cells in the body, so that the immune cells are gathered in the tumor cells, thus strengthening the attack and killing the tumor cells and playing the role of targeting the tumor in the target immunotherapy. The purified technique was successfully cloned and expressed and obtained the soluble bifunctional recombinant protein pLLO-hEGF, and the recombinant protein of 4L was obtained (4.5-6) with a concentration of about 800 mu g/mL (maximum 1 mg/m1). Then, the human tumor cell lines with high expression of EGFR were screened by Western blot, and 2 species were selected from different cell lines, including human breast. Cancer (MDA-MB-231, SK-BR-3), human lung cancer (A549, NCI-H157) and human colorectal cancer (HCT116, HT-29), used for the functional study of the bifunctional recombinant protein pLLO-hEGF related functions. The results of cell immunofluorescence staining showed that pLLO-hEGF could target the surface of these tumor cells well. The results of cell proliferation experiment showed that pLLO-hEGF against these cells. The tumor cells did not promote proliferation. The results of immunological activation showed that pLLO-hEGF could significantly increase the proliferation of CD3+CD4+ T cells in human peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMCs) cultured in vitro. The results of single experiment showed that the percentage of CD3+CD4+ T cells in PBMCs was increased at 14 days when pLLO-hEGF spiny was stimulated. To 54.5%, the control group was 44.8%. and stimulated the proliferation and activation of T lymphocytes to play a significant role in killing tumor cells in vitro and in vivo. In vitro, the killing rates of EGFR tumor cells were HCT116, (69.89 + 1.05)%; HT-29, (60.40 + 2.33)%; A549, (49.45 + 1.72)%; NCI-H157, (85.26 + 4.82)%; S K-BR-3, (47.90 + 1.39)%. In vivo transplantation tumor model experimental results showed that the proliferation of T lymphocytes injected into nude mice significantly inhibited the growth of tumor, and the average tumor weight (0.178/0.224 g) was significantly smaller than that of the control group (0.550 g) (*P0.05). In summary, our research provides a new idea for developing new antitumor drugs. New strategy. This part applies to the national invention patent (application number: 201410152549.2), currently in the actual trial stage, and the relevant content has been published in Hum Vaccin Immunother (Human vaccines) (IF=3.643) and CellPhysiol Biochem magazine (IF=3.55). The second part: can activate immune cells to target CD20 positive human lymphoma cell recombinant protein Preliminary study on the design, expression and anti lymphoma effect of B cell lymphoma is a blood system disease. The clinical effect of traditional treatment is poor. With the understanding of the structural characteristics of the CD20 molecular structure, the design and development of anti CD20 monoclonal antibodies have made important progress in the clinical treatment of B cell lymphoma. In 1997, the human mice approved by FDA in the United States were chimed with anti CD20 monoclonal antibody 1 Rituximab (C2B8). But the human chimeric antibody, because of its large molecular weight, weak penetration and toxic side effects, obviously restricts its clinical effect. In recent years, some people have derived antibodies, small or modified antibodies, and the gene modified antibodies have gradually developed. Single chain variable fragments (ScFv) is an important means for the application of bioengineering method for tumor immunotherapy due to its small molecular weight, strong penetration and good ability to maintain antigen affinity. This study also used the explicit antigen peptide of LLO to construct the targeted CD20 positive B cell lymph. The tumor's bifunctional recombinant protein ScFv-pLLO. first, we designed the ScFv sequence for human CD20, and then spliced the LLO dominant antigen peptide with the ScFv sequence, so that the recombinant protein can target both CD20 positive B cell lymphoma cells and the LLO antigen epitopes can enhance the antigenicity of the tumor cells, thus fully activating the body's fines. Cellular immune response, exerting a more effective anti-tumor immunity to achieve the effect of targeted immunotherapy. Double functional recombinant protein ScFv-pLLO was expressed and purified by prokaryotic cloning, and its anti B cell lymphoma was preliminarily analyzed. Bioinformatics software analysis showed that the theory of recombinant protein was 32 kDa, chemical properties were stable. Flow cytometry was used. The quantitative analysis of the expression of CD20 on the cell surface of several human lymphoma cell lines showed that the positive rate of CD20 in Daudi cells was above 98%, and the positive rates of CD20 in Raji and NCI-BL2009 cells were 89.7% and 92.2% respectively, while the positive rate of RAMOS cell CD20 was the ability to analyze ScFv-pLLO targeted lymphoma cells by 52.3%. flow cytometry, and the results showed ScFv-. PLLO can target different levels of CD20 expression in human lymphoma cells with a concentration of 10 mu g/mL, the average binding rate of Daudi and NCI-BL2009 cells is (93.25 + 3.45)% and (73.85 + 3.95)%, respectively. The results of immunoprecipitation test further demonstrate that ScFv-pLLO can specifically target the CD20 molecules on the surface of human lymphoma cells. The results of cell proliferation test showed that ScFv-pLLO could inhibit the growth of Raji cells, but had no effect on the growth of Daudi, RAMOS and NCI-BL2009 lymphoma cells. Apoptosis experiment showed that ScFv-pLLO could induce apoptosis of Raji cells directly. After 24 h of ScFv-pLLO action of 20 u g/mL, apoptosis of 24.6% Raji cells (early + late) could be induced. After the action of 48 h The initial results of the total apoptosis rate of the cell up to 47.0%. show that the bifunctional recombinant protein ScFv-pLLO can specifically target the binding of CD20 positive B cell lymphoma. For some human lymphoma cell lines, such as Raji cells, the apoptosis can be directly induced and the structure and function are similar to "small anti body". At the same time, the dual function recombinant protein ScFv-pLLO is carried out. The dominant antigen peptide, which is different from the pure monoclonal antibody, can enhance the ability of the tumor cells to activate the immune system after binding to the CD20 molecules on the surface of the lymphoma cells. This provides a basis for our next step to study the activation of the immune cells and to stimulate the function of the long effective anti-tumor cell immunity. The third part of China's Journal of Immunology >. The third part: the role of Leptin in the invasion and metastasis of colorectal cancer cells, with the development of tumor molecular biology, new candidate anti-tumor targets are constantly emerging in tumor targeting therapy. In recent years, more and more studies have been conducted on the proliferation of Leptin mediated signals in tumor cells and the invasion of tumor cells in recent years. It has played an important role in the induction and formation of tumor stem cells. The Chinese name is leptin, which is considered as a potential target of effective antitumor targeting therapy.Leptin. It is a non glycosylated protein hormone composed of 167 amino acids, which is secreted mainly by fat cells and participates in the body's energy metabolism. Leptin and its receptor (LEPR/Ob-R) were also expressed, which showed that the malignancy of colorectal cancer was closely related to the expression of Leptin. Some experimental results showed that high concentrations of exogenous Leptin could promote the proliferation of human colorectal cancer cells and enhance their movement and emplacement ability. The exact biological role of endogenous Leptin in the growth and invasion of tumor cells, and the effect of Leptin mediated signaling pathway on the biological behavior of tumor cells is helpful for the development of new antitumor targeting drugs. In the study, we first analyzed the Leptin and its receptor LEPR in several human colorectal cancer cell lines. The expression, including mRNA and protein levels, showed that Leptin and LEPR were generally expressed in human colorectal cancer cell lines, including cell line HCT116, HT-29, COLO201, COLO205 and SW480. in colorectal cancer cell lines HCT116 and HT-29 cells, and we used siRNA to effectively reduce the expression of endogenous Leptin in tumor cells. Cell proliferation was true. The results showed that inhibition of the expression of endogenous Leptin had no effect on the proliferation of HCT116 and HT-29 cells in human colorectal cancer cell lines. Then we detected some genes closely related to the invasion and metastasis of tumor cells by Real-time PCR, including MMP1, MMP9, TIMP-1, E-cadherin, Twist and so on. The results showed that the inhibition of endogenous Le was inhibited. The expression of MMP1 and MMP9 in HCT116 cells was obviously up-regulated (*P0.05), and the expression of TIMP-1, TIMP-2 and E-cadherin was obviously down regulated (*P0.05), and the expression of MMP1 and Vimentin was obviously up regulated in HT-29 cells. In addition, the results of the gelatinase spectrum test showed that the expression of endogenous Leptin was inhibited and the protein lysis activity of MMP9 in HCT116 cells increased in HCT116 cells. The results of the invasion of.Transwell in vitro showed that the number of cells in the Leptin RNAi group of HCT116 cells increased significantly (51) compared with the control group (148 + 17) (51 2 + 23) (*P0.05) showed that the invasiveness of HCT116 cells was significantly enhanced. Different from previous studies, this study focused on the effects of inhibiting the expression of endogenous Leptin in human colorectal cancer cells and on the proliferation and invasiveness of tumor cells. Invasiveness. Previous studies have shown that increasing the concentration of Leptin can promote the proliferation and invasion of tumor cells.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R730.51

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