靶向Clec9a+DC的胞内信号传导通路及其介导交叉递呈作用机制研究

To overcome off-target effect and to effectively stimulate specific CTL in vivo are the core problems in development of anti-tumor vaccine. Only dendritic cells (DC) can induce naïve T cells transforming CTL, so the choice of DC receptor and elucidate its mechanism are the key issues of anti-tumor vaccine. We previously reported that peptides targeting to Clec9a was obtained by phage display library, which coupled antigen can also target Clec9a+DC and stimulate specific anti-tumor immune response. However, the mechanism of intracellular SYK downstream signaling pathway and the mechanism of cross delivery are still unclear. Therefore, this study intends to verify the antigen presenting function by DC/OVA257-264/OT-1 model and study its signal pathway by SYK inhibitor and downstream key nodes.The cross-presentation pathway were determined by co-localization of peptide and endosome/lysosomal biomarkers. The guided protein of cross-presentation by Clec9a was obtained by Co-IP. The effects of anti-tumor immunotherapy were checked by using the antagonists of the key notes of SYK signal pathway or guided protein in vivo. To clarify the intracellular signal pathway and the cross-presentation mechanism of Clec9a on DC can provide a theoretical basis for developing anti-tumor targeted vaccine.

克服抗原脱靶效应及高效刺激机体产生特异性CTL是抗肿瘤疫苗研发的核心问题。只有树突状细胞（DC）才能诱导naïveT细胞形成CTL，因此选择DC表面特异性受体并阐明其作用机制是研究的关键。前期我们用噬菌体肽库获得DC表面特异性受体Clec9a的靶向肽，偶联抗原后能靶向Clec9a，进而刺激机体产生特异性抗肿瘤免疫应答。但目前抗原靶向Clec9a后，胞内SYK下游信号通路及引起交叉递呈作用机制仍不清楚。因此，本研究拟通过DC/OVA257-264/OT-1模型验证靶向肽偶联抗原递呈功能，用SYK下游关键节点抑制剂研究其信号通路；同时用靶向肽与内体/溶酶体标志蛋白共定位确定交叉递呈作用，并获得Clec9a的导向蛋白；利用SYK下游拮抗剂及导向蛋白抑制剂研究其抗肿瘤效果。本项目通过阐明Clec9a胞内信号通路及交叉递呈作用机制，为靶向Clec9a+DC疫苗的临床应用提供重要理论基础和技术支持。

通过本项目的支持，重点开发利用生物信息学方法在前期WH肽的基础上获得了一条亲和力高、特异性强的Clec9a靶向肽CBP-12。通过肽和蛋白相互作用模式分析，发现直接相互作用力的增加对于CBP-12亲和力提高很重要。通过体外吞噬实验表明CBP-12在体内外均能高效地被cDC1摄取。进一步通过交叉递呈实验发现CBP-12能显著增强DC交叉递呈抗原和引发CD8+ T细胞活化。CBP-12偶联抗原的多肽疫苗在无佐剂情况下引发强烈的抗原特异性CTL反应。在无佐剂的情况下，检测了CBP-12偶联抗原的多肽疫苗的抗肿瘤活性，结果表明无佐剂CBP-12多肽疫苗在两种肿瘤模型中均能显著抑制小鼠肿瘤生长，增加肿瘤组织浸润淋巴细胞，刺激抗原特异性CD8+T细胞活化。通过机制探究，发现无佐剂CBP-12多肽疫苗可能是通过激活cDC1中Syk-ITAM信号调控IL-21分泌。cDC1分泌的IL-21又能增强DC交叉递呈抗原和抗原特异性CD8+T细胞活化。最后，发现无佐剂CBP-12多肽疫苗与放疗在B16-OVA和TC-1肿瘤模型中具有显著的抗肿瘤协同作用，不仅能够抑制小鼠肿瘤生长、延长小鼠生存期，且能重塑肿瘤微环境。科学意义在于开发了一个无佐剂多肽疫苗载体（CBP-12），CBP-12可作为疫苗载体递送新抗原和其他药物到Clec9a+ DC，提高了其在肿瘤免疫应用的价值；并且阐明了抗原通过活化Syk-ITAM信号通路调控IL-21的分泌以增强了抗肿瘤作用。成果发表在Theranostics杂志上。另外发表4篇SCI论文，获批专利一项和4项软件著作权。