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

项目总结/摘要 HPV相关的头颈部鳞状细胞癌造成了巨大的公共卫生负担， 对新的、广泛有效的疗法的巨大需求，这些疗法具有降低的治疗发病率和改善的长期 生存肿瘤特异性CD 8 + T细胞免疫的产生需要有效的抗原交叉呈递， 树突状细胞（DC），因为肿瘤细胞不能有效地呈递相关的CD 8 + T细胞表位。cDC 1已知 因为它们能够处理外源性抗原并有效地交叉呈递给CD 8 + T细胞。正因为如此， 增强cDC 1 s的创新策略可以稳健地诱导HPV特异性免疫， 治疗HPV相关HNC的潜力。FMS样酪氨酸激酶3配体（Flt 3L）是一种细胞因子， 将DC前体扩增并分化为鼠cDC 1，但Flt 3L的治疗潜力有限， 它的半衰期短，在体内的全球分布。我们已经通过产生Flt 3L的所述问题， 白蛋白（Alb）与Flt 3L的遗传融合体，称为白蛋白-Flt 3L（Alb-Flt 3L）。由于FcRn，Alb具有长半衰期- 介导的转细胞再循环，并表现出选择性运输到LN和TM。一旦cDC 1扩增， 需要一个强大的，组织局部的炎症源激活，或者他们将提出T细胞表位，而不 足够的共刺激，导致抑制。肠道细菌如沙门氏菌是一种理想的病原体， 肿瘤特异性cDC 1激活，因为它们可以为cDC 1提供许多病原体相关的分子模式 活化，并且已经描述了可能由于缺氧而有效地定殖肿瘤。关于细菌的一个警告 在癌症治疗中，必须考虑临床前模型的选择，因为定殖变化 自发性和可移植性肿瘤模型之间的差异很大。我们的初步数据表明，Alb-Flt 3L融合 与天然Flt 3L相比，该蛋白在LN和肿瘤中表现出增加的半衰期和选择性积累。白- Flt 3L能够在单次注射后在体内扩增交叉呈递的DC群体，并导致增强的免疫应答。 疫苗接种模型中的HPV特异性T细胞和原理验证实验中的肿瘤控制。沙门氏菌 在我们的实验中使用的菌株成功地定殖肿瘤，并可以有效地激活Alb-Flt 3L衍生的 区议会在该提议中，Alb-Flt 3L+沙门氏菌促进HPV特异性细胞毒性T细胞抗肿瘤的能力被证实。 通过扩增和激活交叉呈递的DC以及随后的肿瘤控制， 将研究自发的和可移植的HPV相关HNC模型。HPV特异性CD 8 + T细胞 将使用四聚体和TCR测序方法评价应答。这是一种机制， Flt 3L介导其免疫刺激功能也将使用适当的缺陷型小鼠进行研究。 模型关于沙门氏菌在自发肿瘤模型与可移植肿瘤模型中定植的研究将在 使用我们的高分辨率nanoPET/CT系统和适当的PET示踪剂进行研究。成功完成 这一建议将产生有价值的临床前和机制数据的治疗潜力的白蛋白- Flt 3L+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.