具有体内DC疫苗特性的细菌化肿瘤细胞疫苗抗肿瘤作用研究

Various whole-tumor cell vaccines have been developed and some of them have been tested in III clinical trials, but their clinic effectives are short of satisfactory for tumor patients. Therefore, it is necessary that more innovative strategies must be found to developed new whole-tumor cell vaccines. Sensing the presence of a pathogen is the first step for the immune system to mount an effective response to eliminate an invading organism and establish protective immunity. The understanding of innate immunity has led to the discovery of pattern recognition receptors being able to recognize pathogen-associated molecular patterns (PAMPs) of all classes of microorganisms. Thus, in this study, we will use our proprietary technology to adhere the whole proteins (antigens) of mycobacteria (BCG, a comment adjuvant in vaccination) on the tumor cell surface (we called this process as “bacterialized” tumor cells) in which tumor cells are artificially cover the PAMPs of mycobacteria on the tumor cell surface to promote immune system to induce recognize the tumor cell antigens. In addition, to amplify the immune response induced by the bacterialized tumor cell vaccine, we will transfect the bacterialized tumor cells with recombinant (a chemokine of DCs) adenovirus (we called it as “bacterialized MIP-3α tumor cell”), in which the bacterialized tumor cells will express and secrete MIP-3α. In this way, bacterialized MIP-3α tumor cells will attract DCs to the vaccine-injected site and promote antigen presentation and enhance the immune response by the attracted DCs. After in vitro investigate the physico-chemical characteristics and functional activity of the bacterialized MIP-3α tumor cells, we will further use the bacterialized MIP-3α tumor cells as whole-tumor cell vaccine to investigate their anti-tumor effects and potential side-effects in mouse tumor models. Lastly, we will study its immune mechanism, especially those that involved to antigenic cell death and micro-environment. Our current study will offer potentially offer a new approach to develop a effective whole-tumor cell vaccine.

全肿瘤细胞疫苗已经进入临床试验，但效果欠佳，需要创新现有的疫苗模式。肿瘤细胞缺少免疫识别的分子模式，细菌则普遍存在病原相关分子模式（PAMPs），因而能有效诱导免疫反应。为此，本项目拟利用我们自主技术将分枝杆菌蛋白（抗原）粘附于肿瘤细胞表面（将这一过程称为细菌化），同时将表达树突状细胞（DCs）趋化因子MIP-3α的重组腺病毒转染这种细菌化肿瘤细胞（称为细菌化MIP-3α肿瘤细胞），期望利用MIP-3α的活性在疫苗注射部位吸引DCs并特异呈递肿瘤抗原来增强这种全细胞疫苗的免疫反应。在体外研究这种细菌化MIP-3α肿瘤细胞的理化特征和功能活性后将其用作全细胞疫苗，在小鼠肿瘤模型中了解抗肿瘤作用效果和可能的副作用，同时利用免疫学、细胞学等现代生物技术了解这种疫苗是否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，为这种细菌化肿瘤细胞疫苗的转化应用提供理论依据。

目前，最成功有效的疫苗都是针对细菌的，这主要就是因为细菌自身存在PAMPs。MIP-3α是一种DC趋化因子，如果在肿瘤组织内表达就可认将DC吸引抵达肿瘤组织，如果能同时诱导肿瘤细胞免疫原性死亡则有可能诱导更好、更特异的免疫反应。为此，利用我们自主技术将分枝杆菌蛋白（抗原）粘附于肿瘤细胞表面（将这一过程称为细菌化），同时将表达MIP-3α的重组新城疫病毒转染这种细菌化肿瘤细胞（称为细菌化MIP-3α肿瘤细胞），期通过细菌抗原和PAMPs，同时利用MIP-3α的活性在疫苗注射部位吸引DCs并特异呈递肿瘤抗原等特性来增强这种全细胞疫苗的免疫反应。在本项目研究过程中，我们利用重组技术制备了重组MIP-3α新城疫病毒（NDV-MIP3α，原计划为腺病毒），体内外试验证明NDV-MIP3α保留有原野生病毒的所有溶瘤和诱导免疫原性死亡的活性，可以有效分泌目标活性蛋白MIP-3α，可以吸引DC抵达肿瘤组织，具有更好的诱导肿瘤特异性免疫反应，机制研究发现CD4和CD8细胞均参与免疫调节，可以改善肿瘤免疫抵制的内环境。与此同时，本项目还成功制备了多个细菌化肿瘤细胞模式疫苗，详细研究了这种疫苗抗原体内被处理和递呈的模式，发现可以明显促进DC成熟和抗原递呈，在多个小鼠肿瘤模型中有理想的治疗和抑制制肿瘤转移的作用，但没有明显的副作用。在本研究中，我们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发现，我们制备的细菌化肿瘤细胞疫苗能够诱导特异的细胞和体液免疫反应，同样能够有效逆转肿瘤免疫抵制的内环境。项目研究还创新性发现（此前没有报道），细菌化肿瘤细胞疫苗免疫后能通过CD8+细胞诱导肿瘤细胞细胞铁死亡，这种铁死亡和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理论依据。