基于天然免疫通路的胃癌相关抗原合理化修饰及其抑瘤效应的机理研究

Gastric cancer is a kind of cancer with high incidence in China, which severely threatens people's life. In the past, surgery, chemotherapy, radiotherapy were frequently used in gastric cancer treatment. However, nowadays immunotherapy has drawn more and more scientific attention, for it has more advantages than traditional therapies. Tumor vaccine is an important portion of immunotherapy, which usually includes two components, tumor associated antigen (TAA) and immune activator. As for gastric cancer, MG7-Ag is the most widely investigated TAA. MG7-Ag epitope peptide is often used in the research of gastric cancer vaccine. Meanwhile, Toll like receptors (TLR) are a family of receptors found in recent years, which are able to activate immune response through innate immune pathway. TLRs have more than ten kinds of subtypes, while each subtype has its specific natural ligands. Among them, only TLR7 has small molecule ligands which can be synthesized in the lab. Based on the above discussion, we propose the project which is coupling small molecule TLR7 agonist (immune activator) and MG7-Ag epitope peptide (gastric cancer TAA), to produce super antigen (MG7-TLR7) with high sensitivity and specificity to overcome gastric cancer. Therefore, first we will synthesize TLR7 agonist and couple it to MG7-Ag epitope peptide. For this step, our lab already has the patent on the small molecule compound and coupling route. Second, we will use in vitro experiment to investigate MG7-TLR7 on the ability of inducing immune factors, such as INF-γ and IL-12. Third, we will use in vivo experiment to further investigate MG7-TLR7 on the ability of enhancing immune level and suppressing gastric cancer. Here, immune enhancement in mice depends on the activity of antigen-specific CD4, CD8 T cell and CTL cell. On the other hand, MG7-Ag epitope peptide is a heptapeptide containing only 7 amino acids, which theoretically speaking, can not give high antigenicity. Therefore, we expect to design and synthesize a series of heptapeptide polymer (such as dimer, trimer, tetramer, and so on) coupling with the same TLR7 agonist. We will use the same experimental methods as mentioned above to decide which polymer is the most desirable. In conclusion, it is anticipated that our project (MG7-TLR7) is the first time to couple small molecule TLR7 agonist and cancer antigen,and prove its antitumor effect and mechanism through innate immune pathway. We wish our results could firstly demonstrate the prospect of MG7-TLR7 on gastric cancer therapy.

胃癌是我国发病率较高的一种癌症，目前具有更多优势的免疫疗法已逐步取代传统疗法成为胃癌的研究热点。肿瘤疫苗通常由肿瘤相关抗原和免疫激活剂两部分组成。MG7-Ag是目前研究最多的胃癌相关抗原，胃癌疫苗研究中常采用其表位相似七肽。TLR则是近年来发现的一类在天然免疫通路中激活免疫应答的受体，包括十余种亚型，其中只有TLR7具有人工合成小分子配体。本课题将本实验室具有专利的TLR7小分子激动剂与胃癌相关抗原MG7表位七肽进行偶联，期望产生高活性的特异性超级抗原，并通过体外实验研究其诱导免疫因子的能力，通过动物实验研究其增强小鼠免疫功能（抗原特异性CD4、CD8 T细胞及CTL细胞活性等）及抗肿瘤的作用。另外由于MG7-Ag七肽本身抗原性较弱，我们设计七肽的一系列多聚体与TLR7激动剂偶联，通过活性实验确定最佳多聚体数目。预期本课题可以首次展示TLR7小分子激动剂偶联抗原作为疫苗在胃癌治疗中的前景。

胃癌是一种严重威胁着人类健康的常见病和多发病。胃癌本身易侵袭转移和复发的生物学特性，使得传统的手术、放疗和化疗的治疗效果受到局限，因此生物免疫治疗和胃癌疫苗已成为最新的研究热点。肿瘤疫苗通常由肿瘤相关抗原和免疫激活剂两部分组成。MG7-Ag是目前研究最多的胃癌相关抗原，胃癌疫苗研究中常采用其表位相似七肽。TLR则是近年来发现的一类在天然免疫通路中激活免疫应答的受体，包括十余种亚型，其中只有TLR7具有人工合成小分子配体。本项目主要研究成果是成功合成TLR7小分子激动剂和不同构型及聚合度的MG7表位相似多肽的化学偶联物（MG7-TLR7），并进行下列研究：1．不同构型及聚合度的MG7-Ag多肽多聚体与TLR7激动剂偶联后诱导产生免疫因子能力，总结出一定的构效关系。2．MG7-TLR7体内激发小鼠免疫能力，关键指标为诱发抗体产生、ADCC活性、CTL活性等。3．MG7-TLR7体内作为肿瘤疫苗的抗癌能力，并阐明其相关机制。具体研究内容概述如下：1．胃癌相关抗原MG7-Ag表位相似多肽多聚体：由于七肽从理论和经验上来讲属于弱抗原，存在免疫原性不足的缺点。因此，我们合成MG7-Ag七肽的二聚、三聚、四聚等多聚体，将这些聚合物分别和TLR7激动剂偶联，形成如下通式的抗原（MG7-TLR7），另外我们设计的聚合体包括直链状和分支状两种构型。2．TLR7激动剂和MG7-Ag的共价化学偶联。3．体内和体外研究评价TLR7激动剂和MG7-Ag偶联抗原（MG7-TLR7）作为肿瘤疫苗的抗癌能力，并从细胞和分子水平阐明具体的抗肿瘤机制。①体外实验验证并比较一系列MG7-TLR7诱导相关免疫因子（INF-γ、IL-12等）和免疫反应的能力。②动物实验水平上验证MG7-TLR7增强小鼠免疫功能。主要通过对小鼠进行多次免疫后，检验血清中抗MG7抗体的效价、小鼠脾脏CTL细胞的活性等。③动物水平上验证MG7-TLR7的抗癌能力。对上述免疫过的小鼠进行肿瘤激发实验，通过测定肿瘤大小和抑瘤率等指标反映MG7-TLR7作为肿瘤疫苗的效果，并且检测肿瘤激发后小鼠的免疫状态。本项目首次设计并合成TLR7小分子激动剂偶联胃癌相关抗原作为肿瘤疫苗，并进行初步的体内体外活性研究。此疫苗的良好效果极大的推进胃癌以及其他肿瘤疫苗等肿瘤生物治疗方法的发展和完善。