IDO-silencing Salmonella therapy for the treatment of primary and metastatic PDAC

描述(申请人提供)：晚期胰腺导管腺癌(PDAC)通常不能手术，对现有的治疗方法只有短暂的反应。吲哚胺2，3-双加氧酶(IDO)在PDAC中的过表达，通过抑制抗肿瘤免疫，在加速疾病进展中起重要作用。目前的IDO抑制剂不能充分逆转免疫抑制，而全身性脱靶效应导致了它们的毒性。SHIDO-ST是一种新型的基于鼠伤寒沙门氏菌(ST)的治疗方法，它表达一个小的发夹(Sh)RNA来特异性地沉默肿瘤来源的IDO，毒性降低。ST作为shRNA传递载体，由于其对低血管、低氧组织的运动性和亲和力，对促结缔组织PDAC组织具有优越的穿透能力和抗转移功能。IDO沉默和大量的ST肿瘤定植相结合，通过中性粒细胞(PMN)的募集和激活增加了肿瘤内的局部活性氧物种，PMN可以导致氧化应激诱导的肿瘤细胞、肿瘤干细胞(CSC)和血管间质的凋亡，从而抑制PDAC的进展。众所周知，PMN还具有抗转移功能，从而提供额外的保护，防止肿瘤扩散。这项拟议的工作将详细分析影响SHIDO-ST治疗中性粒细胞募集和激活的信号通路。参与PMN募集的细胞因子将通过蛋白质阵列分析和流式细胞仪对接受Shido-ST治疗的小鼠的PDAC肿瘤与不抑制IDO的杂乱序列对照(shScr-ST)进行评估。由于IDO活性是调节PMN功能的已知因素，因此也将使用流式细胞术来评估浸润性PMN的极化和凋亡状态。ST和PMN都提供了初级和抗转移保护，这一发现支持在更具侵袭性的PDAC模型中评估Shido-ST。KrasG12D；Trp53R127H；Pdx1-Cre基因工程小鼠模型(KPC-GEMM)概括了人PDAC的结缔组织发育和转移特征。凭借我们与外科肿瘤学家的密切合作，独家获得新鲜的外科PDAC组织，将允许在异种移植模型中评估人类(HU)Shido-ST，为临床翻译提供更大的支持。与Sutent(Tm)(辉瑞公司)联合治疗导致PDAC血管崩溃的药物将接受测试，因为它增加了肿瘤的缺氧区，增强了ST的定植和PMN的募集。为了提高SHIDO-ST和Sutent对肿瘤的穿透性，我们建议使用聚乙二醇化的人重组透明质酸酶(PEGPH20，Halozyme Inc.)，它可以消耗PDAC组织中丰富的透明质酸，并增加血管通透性。在转基因和异种移植环境中，将通过横断面或纵向测量肿瘤体积、转移负荷、CSC标志物和细菌计数来评估所建议的治疗方法对原发和转移负荷、CSC频率和ST定植的影响。长期目标是将SHIDO-ST发展成为治疗晚期不可手术PDAC患者的一种合适、有效的治疗方法。

DESCRIPTION (provided by applicant): Advanced pancreatic ductal adenocarcinoma (PDAC) is often inoperable, and is only transiently responsive to existing therapies. Overexpression of indoleamine 2,3-dioxygenase (IDO) in PDAC plays a major role in accelerating disease progression by suppressing antitumor immunity. Current IDO inhibitors inadequately reverse immunosuppression while systemic off target effects contribute to their toxicity. ShIDO-ST is a novel Salmonella typhimurium (ST)-based therapy that expresses a small hairpin (sh)RNA to specifically silence tumor-derived IDO with decreased toxicity. ST as an shRNA delivery vehicle offers superior penetration against desmoplastic PDAC tissue and anti-metastatic function due to its motility and affinity for poorly vascularized, hypoxic tissue. he combination of IDO silencing and abundant ST tumor colonization increases intratumoral local reactive oxygen species through the recruitment and activation of polymorphonuclear neutrophils (PMN), which can cause oxidative stress-induced apoptosis of tumor cells, cancer stem cells (CSC), and vascular stroma to inhibit PDAC progression. PMN are also known to have anti-metastatic function, thus providing additional protection against tumor spread. The proposed work will provide detailed analysis of signaling pathways influencing PMN recruitment and activation upon shIDO-ST treatment. Cytokines involved in PMN recruitment will be evaluated using protein array analysis and flow cytometry of PDAC tumors from mice treated with shIDO-ST versus scrambled sequence control (shScr-ST), which does not silence IDO. The polarization and apoptotic state of infiltrating PMN will also be evaluated using flow cytometry, as IDO activity is known to regulate PMN function. The finding that both ST and PMN provide primary and anti-metastatic protection supports evaluation of shIDO-ST in more aggressive models of PDAC. The KrasG12D;Trp53R127H;Pdx1-Cre genetically engineered mouse model (KPC-GEMM) recapitulates desmoplasia and metastasis characteristic of human PDAC. Exclusive access to fresh surgical PDAC tissue, by virtue of our close collaboration with surgical oncologists, will allow for evaluation of human (Hu)shIDO-ST in xenograft models to provide greater support for clinical translation. Combination therapy with Sutent(tm) (Pfizer), which induces vascular collapse in PDAC, will be tested because it increases the hypoxic area of the tumor to augment colonization by ST and the recruitment of PMN. To improve penetration of tumors by shIDO-ST and Sutent, we propose using PEGylated human recombinant hyaluronidase (PEGPH20, Halozyme Inc.), which depletes hyaluronan abundant in PDAC tissue and increases vascular permeability. The effects of proposed treatments on primary and metastatic burden, CSC frequency, and ST colonization will be evaluated in transgenic and xenograft settings through cross sectional or longitudinal measurements of tumor volume, metastatic burden, CSC markers, and bacterial enumeration. The long-term objective is to develop shIDO-ST into a suitable, effective therapy for the treatment of patients with advanced inoperable PDAC.