Novel immunotherapeutic regimen combining the dendritic cell expansion power of Albumin-Flt3L and inflammatory cues of Salmonella

项目摘要/摘要 HPV相关的头颈部鳞状细胞癌造成了压倒性的公共卫生负担 对新的、广泛有效的治疗方法的巨大需求，同时降低治疗发病率并改善长期疗效 生死存亡。肿瘤特异性CD8+T细胞免疫的产生需要强大的抗原交叉提呈，通过 树突状细胞(DC)，因为肿瘤细胞不能有效地提供相关的CD8+T细胞表位。CDC1s是已知的 因为它们有能力处理外源抗原，并有效地交叉呈现给CD8+T细胞。正因为如此， 增强cDC1s的创新策略可以有力地诱导HPV特异性免疫，并具有很好的治疗作用 治疗HPV相关性HNC的潜力。FMS样酪氨酸激酶3配体(Flt3L)是一种细胞因子， 将DC前体扩增并分化为小鼠CDC1s，但Flt3L的治疗潜力有限，因为 其短暂的半衰期和体内的全球分布。我们已经克服了Flt3L的上述问题，方法是生成 白蛋白(Alb)与Flt3L的基因融合，称为白蛋白-Flt3L(Alb-Flt3L)。由于FcRN，Alb有很长的半衰期- 介导的跨细胞循环，并显示有选择性地贩运到LN和TM。一旦CDC1扩张，它们就会 需要一个强大的、组织局部的炎症来源才能激活，否则他们将在没有 充分的共刺激，导致抑制。肠道细菌，如沙门氏菌，是治疗 肿瘤特异性的cDc1激活，因为它们可以为cDc1提供多种病原体相关的分子模式 激活，并已被描述为有效地定植肿瘤可能是由于缺氧。关于细菌的一个警告 癌症的治疗是必须考虑临床前模型的选择，因为定植性不同。 在自发性肿瘤模型和可移植肿瘤模型之间有很大的区别。我们的初步数据显示，Alb-Flt3L融合 与天然Flt3L相比，蛋白质在LN和肿瘤中的半衰期和选择性蓄积增加。白蛋白- Flt3L能够在体内一次注射后扩大交叉呈现的DC群体，并导致增强 在疫苗接种模型和肿瘤控制中的HPV特异性T细胞的原理验证实验。沙门氏菌 我们实验中使用的菌株成功地在肿瘤上定植，并能有效地激活Alb-Flt3L来源的 集散控制系统。在这项建议中，Alb-Flt3L+沙门氏菌促进HPV特异性细胞毒T细胞抗肿瘤的能力 免疫通过扩增和激活交叉呈递的DC和随后的肿瘤控制在我们的 自发性和可移植的HPV相关的HNC模型将被研究。HPV特异性CD8+T细胞 反应将使用四聚体和TCR测序方法进行评估。Alb-的作用机制 Flt3L介导的免疫刺激功能也将被用适当的缺陷小鼠来询问 模特们。关于沙门氏菌在自发性肿瘤模型和可移植肿瘤模型中定植的研究将是 使用我们的高分辨率纳米PET/CT系统和适当的PET示踪剂进行了研究。成功完成 将产生关于白蛋白治疗潜力的有价值的临床前和机制数据。 Flt3L+沙门氏菌作为治疗HPV相关HNC以及许多其他类型癌症的通用策略。

Project Summary/Abstract The overwhelming public health burden of HPV-associated head and neck squamous cell carcinoma has created great demand for novel, broadly effective therapies with reduced treatment morbidity and improved long-term survival. The generation of tumor-specific CD8+ T cell immunity requires potent antigen cross-presentation by dendritic cells (DCs) since tumor cells do not efficiently present relevant CD8+ T cell epitopes. cDC1s are known for their ability to process exogenous antigen and potently cross-present to CD8+ T cells. Because of this, innovative strategies to enhance cDC1s could robustly induce HPV-specific immunity and have great therapeutic potential in the treatment of HPV-associated HNC. FMS-like tyrosine kinase 3 ligand (Flt3L) is a cytokine that expands and differentiates DC precursors to murine cDC1s, but therapeutic potential of Flt3L is limited because of its short half-life and global distribution in vivo. We have overcome the described issues of Flt3L by generating a genetic fusion of Albumin (Alb) to Flt3L, named Albumin-Flt3L (Alb-Flt3L). Alb has a long half-life due to FcRn- mediated transcytolic recycling and exhibits selective trafficking to the LN and TM. Once cDC1s expand, they require a strong, tissue localized source of inflammation for activation, or they will present T cell epitopes without adequate costimulation, causing suppression. Enteric bacteria such as Salmonella serve as an ideal agent for tumor specific cDC1 activation as they can provide numerous pathogen-associated molecular patterns for cDC1 activation, and have been described to colonize the tumor efficiently likely due to hypoxia. One caveat of bacteria therapy in cancer is that consideration must be made regarding preclinical model selection, as colonization varies greatly between spontaneous and transplantable tumor models. Our preliminary data shows that Alb-Flt3L fusion protein exhibits increased half-life and selective accumulation in LN and tumor compared to native Flt3L. Alb- Flt3L is able to expand cross-presenting DC populations in vivo following a single injection and leads to enhanced HPV-specific T cells in a vaccination model and tumor control in a proof-of-principle experiment. The Salmonella strain used in our experiments successfully colonizes the tumor and can efficiently activate Alb-Flt3L-derived DCs. In this proposal, the ability of Alb-Flt3L + Salmonella to promote HPV-specific cytotoxic T cell antitumor immunity through the expansion and activation of cross-presenting DCs and subsequent tumor control in our spontaneous and transplantable HPV-associated HNC models will be investigated. HPV-specific CD8+ T cell responses will be evaluated using tetramer and TCR sequencing approaches. The mechanism by which Alb- Flt3L mediates its immunostimulatory function will also be interrogated using appropriate deficient mouse models. Studies regarding colonization of Salmonella in spontaneous vs transplantable tumor models will be investigated using our high-resolution nanoPET/CT system and appropriate PET tracers. Successful completion of this proposal will generate valuable preclinical and mechanistic data regarding the therapeutic potential of Alb- Flt3L + Salmonella as a universal strategy to treat HPV-associated HNC as well as many other types of cancer.