基于胞外诱捕网理念制备的CpG ODN胞外修饰肿瘤全细胞疫苗研究

Various whole-tumor cell vaccines have been developed and some of them have been tested in III clinical trials, but their clinic effectiveness is unsatisfactory for tumor patients. Therefore, more innovative strategies must be found to develop new whole-tumor cell vaccines. Toll-like receptors (TLRs) expressed on the phagocyte surfaces are the bridge between innate and adaptive immune responses. CpG ODN (DNA) is one of the ligands for TLR9 and an effective adjuvants used in many types of traditional and newly developed vaccines at present. Extracellular traps (ETs) are produced by several immune cells including neutrophils, eosinophils, mast cells, monocytes and macrophages. The release of ETs results in extrusion of a fibrous network of nuclear DNA, histones and a concentration of granular proteins. ETs have been implicated in a growing number of human diseases with beneficial roles in bacterial and fungal infections and deleterious roles in autoimmunity. In addition, previous studies have identified CD47, a cell-surface molecule, as a “marker of self” that prevents cells of the innate immune system from attacking tumors. CD47 acts by sending inhibitory signals through SIRPα, a receptor expressed on the surface of macrophages and other myeloid cells. In this sense, the CD47/SIRPα interaction serves as a myeloid-specific immune checkpoint, and blocking the interaction lowers the threshold for macrophage phagocytosis of cancer. Thus, in this study, we will use our proprietary technology to adhere the CpG ODN on the tumor cell surface (we called these tumor cells as ETs tumor cells), through which CpG ODN can act with TLR9 on DCs (including other APCs) and covered the CD47 on the tumor cells, leading to promote immune system to induce phagocytosis of tumor cells and presentation of the tumor cell antigens. After in vitro investigate the physico-chemical characteristics and functional activities of the ETs tumor cells, we will further use the ETs tumor cells as a whole-tumor cell vaccine to investigate their anti-tumor effects and potential side-effects in mouse tumor models. Moreover, we will study its immune mechanisms, including whether TLR9 and CD47/SIRPα pathways were involved in. Lastly, the immune mechanisms especially those that involved to antigenic cell death and micro-environment will also be investigated. Thus, our current study will potentially offer a new approach to develop an effective whole-tumor cell vaccine.

肿瘤全细胞疫苗进入临床试验后效果并不理想，需要寻找新的制备方法。Toll样受体(TLRs)是特异与非特异免疫反应的桥梁，CpG ODN(DNA)是TLR9 的配体和常用的免疫佐剂。免疫吞噬细胞通过一种以DNA为主要成分的胞外诱捕网(ETs)结构，将病原体或细胞包裹从而将包裹物清除并诱导免疫反应。研究还发现封闭肿瘤细胞CD47分子可以促使免疫细胞通过阻断SIRPα通路吞噬肿瘤细胞并诱导免疫反应。因此，本项目利用自主技术将CpG ODN粘附于肿瘤细胞表面(ETs化，同时封闭CD47)。在体外研究这种ETs化肿瘤细胞的理化特征和功能活性后将其用作全细胞疫苗，在肿瘤模型小鼠中了解其治疗肿瘤的效果和副作用，同时了解这种ETs化肿瘤细胞疫苗是否通过TLR9和CD47/SIRPα通路诱导特异的抗肿瘤免疫反应和肿瘤免疫原性死亡，是否改变免疫微环境等分子机理，为ETs化肿瘤细胞疫苗的转化应用提供理论依据。

利用肿瘤整个细胞加上基因重组修饰进而增强免疫功能是肿瘤疫苗一种新的策略，目前一种利用重组表达GM-CSF的全肿瘤疫苗已经进入临床试验，但总体效果欠佳，因此全肿瘤细胞疫苗应用仍然需要创新现有的制备模式。肿瘤细胞经过不断的基因突变选择，利用多种策略逃避免疫监视和清除，其中重要的一种策略就是不表达免疫识别所需要的分子模式，细菌则普遍存能被免疫识别的病原相关分子模式（PAMPs），因而能有效诱导免疫反应。Toll样受体(TLRs)是PAMP受体，是特异与非特异免疫反应的桥梁，CpG ODN(DNA)是TLR9 的配体和常用的免疫佐剂。另外，目前研究发现免疫吞噬细胞通过一种以DNA和瓜氨酸化组蛋白为主要成分的胞外诱捕网(ETs)结构，该结构能将病原体或细胞包裹从而将其清除并诱导免疫反应。研究还发现封闭肿瘤细胞免疫检查点分子CD47可以促使免疫细胞通过阻断SIRPα通路从而吞噬肿瘤细胞并诱导特异免疫反应。因此，本项目将CpG ODN和瓜氨酸化组蛋白粘附于肿瘤细胞表面（我们称ETs化，同时也可以封闭CD47)，我们将这种修饰的全肿瘤细胞作为疫苗并将其称为NET疫苗。本项目研究发现，NET疫苗可以明显促进DC成熟和交叉递呈肿瘤抗原，在多个小鼠肿瘤模型中有理想的治疗和抑制肿瘤转移的作用，并且没有明显的副作用。在本研究中，我们还改良了一种新的体内测定细胞免疫反应的技术同时结合传统技术检测肿瘤特异免疫功能，发现我们制备的NET疫苗能够诱导特异的细胞和体液免疫反应，这种NET全肿瘤细胞疫苗不但能够有效逆转肿瘤免疫抵制的内环境，还可以诱导包括铁死亡在内的多种细胞死亡。进一步的机理研究表明疫苗免疫后诱导产生的CD8阳性T淋巴分泌的细胞因子特别是INFγ是诱导肿瘤细胞特异性铁死亡的主要分子和机制，同时证明这种铁死亡和其能够逆转免疫抑制环境有关。项目研究不但创新性地提出了一种制备肿瘤细胞疫苗的新技术（同时在申请中国和美国技术专利），而且发现了疫苗（免疫治疗）逆转肿瘤免疫抑制微环境的一种新的分子机制，为这种细菌化肿瘤细胞疫苗的转化应用提供了理论依据。