一种基于细菌蛋白质N-糖基化系统的新型生物技术制备肿瘤糖抗原-蛋白缀合疫苗

Malignant neoplasm (also known as cancer) remains a leading threat to human health. The conventional therapies including radiation, chemotherapy and surgery cannot always cure cancer completely and the recurrence is often observed after successful early treatments. With the developments in the cancer-related research fields, cancer immunotherapy is now becoming an effective complementary therapy to the conventional ways. Cancer immunotherapy is to stimulate the patient's immune system to kill tumor cells either through immunization of the patient with a cancer vaccine in which case the immune system is trained to recognize tumor cells as targets to be destroyed or through the administration of therapeutic antibodies in which case the immune system is recruited by the antibodies to destroy tumor cells. Tumor-associated carbohydrate antigens (TACAs) are expressed aberrantly on the surface of tumor cells. In addition to acting as cancer biomarkers, TACAs can also play critical roles in the cancer pathologic process, making them obvious targets for the development of cancer vaccines. However, carbohydrates are T cell-independent type II antigens and by themselves cannot induce the activation of T cells. Without the involvement of T cells, the memory B cells and the IgM to IgG class switch cannot be induced. A major strategy to enhance the immunogenicity of carbohydrate antigens is to conjugate them to a carrier protein, forming conjugate vaccines. Currently, many researchers are focusing on the development of anti-cancer TACA-protein conjugate vaccines, and some such vaccines have showed great promise in clinical trials. The most widely used method for preparing such conjugate vaccines is the chemical conjugation that involves the preparation of both TACAs and carrier proteins and the subsequent ligation between two components. However, the chemical conjugation still has some drawbacks of which the fatal problems are the poor reproducibility of the conjugation reactions and non-site-specific coupling of TACAs which may cause heterogeneities and ambiguities of the vaccines in structure and composition. In the present study, we will establish a platform for the production of anti-cancer TACA-protein conjugate vaccines using engineered E. coli. We will first genetically engineer E. coli lipopolysaccharide biosynthetic pathway, and subsequently introduce both the genes that can drive the assembly of TACAs and the bacterial protein N-glycosylation system from C. jejuni that automatically ligates the pre-assembled TACA onto a carrier protein inside E. coli cells. Using this platform, we plan to produce conjugate vaccines toward seven important TACAs in one-step fermentation that will greatly reduce the production cost. Furthermore, to enhance the immunogenicity of the conjugate vaccines, we will also optimize the carbohydrate loading amount on the carrie protein and insert a MHC II-restricted T cell peptide in the region close to the glycosylation site.

肿瘤免疫疗法正逐步成为肿瘤治疗的有效辅助甚至替代手段，基于肿瘤疫苗的主动免疫治疗是免疫疗法的一种重要方式。肿瘤糖抗原因在肿瘤细胞表面异常表达并参与肿瘤的生长和转移，成为研制肿瘤疫苗的理想靶分子。然而糖抗原本身无法激活T细胞,将肿瘤糖抗原与具有较强免疫原性的蛋白载体缀合可激活T细胞并进一步刺激B细胞分泌IgG抗体。目前该策略已被广泛应用并取得了一些重要成果。化学合成是当前普遍使用的制备方法，但存在一些固有缺陷(产物结构不均一、过程复杂等)。本项目针对7种重要肿瘤糖抗原，建立一种基于原核蛋白N-糖基化系统的工程化大肠杆菌制备肿瘤糖抗原-蛋白缀合疫苗的技术体系。大肠杆菌的改造包括对其脂多糖合成路径的改造及之后引入肿瘤糖抗原合成基因和原核蛋白N-糖基化系统，在大肠杆菌体内实现肿瘤糖抗原与载体蛋白自动偶联。项目旨在利用这种新型生物技术制备肿瘤糖抗原-蛋白缀合疫苗，提高疫苗免疫活性并降低制备成本。

肿瘤相关糖抗原（Tumor-associated carbohydrate antigens, TACAs）是一类重要的肿瘤抗原。随着糖生物学、基因组学和生物信息学的迅速发展，越来越多的糖基转移酶基因被发现和鉴定。使部分肿瘤糖抗原可以在体外（in vitro）利用酶法进行合成。本项目将在大肠杆菌中引入了两套细菌蛋白N-糖基化系统，即寡糖转移酶PglB和最近新发现的N-葡萄糖转移酶(NGT)。前者可将与类脂链接的寡糖转移到新生肽链的N-糖基化位点，后者则利用UDP-Glc为供体对新生或者折叠后的蛋白进行糖基化。相对于PglB，NGT是目前唯一已知的以NDP-糖为供体，并在胞浆中可对新生或成熟蛋白进行糖基化修饰的N-糖基转移酶，因此具有很大的应用前景。目前，我们已经利用来自Actinobacillus pleuropneumoniae来源的NGT在胞外合成了携带多个N-Glc（多达10个）的均一化糖蛋白。的一个同时，我们克隆表达了所有成UDP-GlcNAc,UDP-GalNAc，GDP-Fucose，CMP-sialic acid等糖供体合成基因，以及LewisX，LewisY，Globo H，Gb5，GM2，GD2，GD3合成的全部基因，并在体外进行了活性鉴定。然而在胞内组装肿瘤相关糖抗原GM2时，我们发现同一启动子调控一组基因时，往往会照成下游尤其时末端基因表达严重衰减，导致GM2中间产物的大量积累（不均一化）。同时，能够适应肿瘤相关糖抗原的跨膜转运系统（Wzx）需要经过大量筛选，依然存在转运效率低下等问题。

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