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

描述（由申请人提供）：晚期胰腺导管腺癌（PDAC）通常不能手术，并且对现有治疗只有短暂的反应。吲哚胺2,3-双加氧酶（IDO）在PDAC中的过度表达通过抑制抗肿瘤免疫来加速疾病进展。目前的IDO抑制剂不能充分逆转免疫抑制，而全身脱靶效应有助于其毒性。ShIDO-ST是一种新型的鼠伤寒沙门氏菌（ST）为基础的治疗方法，表达小发夹（sh）RNA特异性沉默肿瘤来源的IDO，降低毒性。ST作为shRNA递送载体，由于其运动性和对血管化差的缺氧组织的亲和力，对促结扎性PDAC组织具有良好的穿透性和抗转移功能。IDO沉默和大量ST肿瘤定植的结合通过募集和激活多形核中性粒细胞（PMN）增加肿瘤内局部活性氧，从而引起氧化应激诱导的肿瘤细胞、癌症干细胞（CSC）和血管间质凋亡，从而抑制PDAC的进展。PMN还具有抗转移功能，从而提供额外的防止肿瘤扩散的保护。建议的工作将详细分析影响shIDO-ST处理时PMN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阵列分析和流式细胞术对shIDO-ST处理小鼠的PDAC肿瘤进行评估，而shScr-ST不会沉默IDO。浸润PMN的极化和凋亡状态也将使用流式细胞术进行评估，因为IDO活性可以调节PMN的功能。ST和PMN均提供原发性和抗转移性保护，这一发现支持了shIDO-ST在更具侵袭性的PDAC模型中的评估。KrasG12D; Trp53R127H;Pdx1-Cre基因工程小鼠模型（KPC-GEMM）概括了人PDAC的结缔组织增生和转移特征。凭借我们与外科肿瘤学家的密切合作，独家获得新鲜的手术PDAC组织，将允许在异种移植模型中评估人类（Hu）shIDO-ST，为临床转化提供更大的支持。与索坦（tm）（辉瑞公司）联合治疗，可诱导PDAC血管塌陷，因为它增加了肿瘤的缺氧区域，以增加ST的定植和PMN的募集，因此将进行测试。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，增加血管通透性。通过对肿瘤体积、转移性负担、CSC标记物和细菌计数的横断面或纵向测量，将在转基因和异种移植环境中评估所提出的治疗方法对原发性和转移性负担、CSC频率和ST定植的影响。长期目标是将shIDO-ST发展成为一种适用于晚期无法手术的PDAC患者的有效治疗方法。

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.