Bacterial Delivery of CXCR7 Nanobodies to Alleviate Immune Suppression in Pancreatic Cancer

项目摘要 胰腺导管腺癌(PDAC)是一种高度致命的恶性肿瘤，中位生存期为5年。 大约7%。PDAC患者预后不良是多种因素综合作用的结果，包括晚期诊断。 肿瘤血流不畅导致药物积聚低，以及免疫抑制的肿瘤微环境- 门槛。因此，尽管针对其他癌症类型的免疫治疗取得了重大进展 例如黑色素瘤和非小细胞肺癌，针对PDAC的免疫治疗的临床试验还没有 事实证明是成功的。逆转局部免疫抑制的一个研究途径是INHI-1。 趋化因子受体CXCR4与化疗药物和αPD-1化合物的联合应用。CXCR4 作为CXCL12的受体，CXCL12由癌症相关的成纤维细胞分泌，调节T细胞内流 变成了肿瘤。最近发现，CXCR7除了作为CXCL12的受体外，还可以作为CXCL12的受体。 其他几种趋化因子。鉴于其在PDAC间质中的表达升高，我们假设CXCR7可能 可作为逆转PDAC免疫抑制的候选治疗靶点。 合成生物学的最新进展激发了人们对编程细菌感知和反应的兴趣 人体内的疾病，如癌症。细菌机器可以被重新设计来感知一种特殊的 微环境，启动殖民，并直接和专门在 那个微环境。与PDAC特别相关的是细菌在组织中移动的能力，而不需要 强大的、直接的血管连接为药物输送到血流灌注不良的肿瘤提供了极大的优势。 利用我们在合成生物学、分子生物学、小鼠模型和临床前治疗方面的专业知识， 我们将询问CXCR7在维持PDAC局部免疫抑制中的作用并评估 以此通路为靶点作为PDAC治疗干预手段的临床前疗效。首先，我们将 在多种细胞类型中检测抑制CXCR7及其通路成分的功能后果 并通过scRNAseq分析和多重分析阐明其免疫抑制的分子机制 PDAC外植体的免疫荧光。为了研究CXCR7在体内的抑制作用，我们将给 设计用于选择性定植肿瘤并产生抑制CXCR7的纳米体的益生菌E.ColiNissle 1917 直接在我们的临床前PDAC模型的肿瘤内，KPC小鼠。肿瘤进展、免疫系统 CXCR7的激活和总存活率将在CXCR7单独治疗以及与传统药物联合治疗后进行评估。 化疗药物和其他免疫靶向药物。 总之，本文提出的实验将有助于我们理解局部免疫抑制。 在PDAC肿瘤内，以及验证细菌疗法，以解决PDAC治疗的两个挑战； 药物可及性差，免疫微环境受到高度抑制。

Project Summary Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy, with a median 5-year survival of around 7%. The poor prognosis of PDAC patients is due to a combination of factors, including late stage diag- nosis, low drug accumulation due to poorly perfused tumors, and an immunosuppressed tumor microenviron- ment. Consequently, while significant progress has been made in immunotherapy against other cancer types such as melanoma and non-small cell lung cancers, clinical trials of immunotherapy for PDAC have not yet proven to be successful. One avenue of investigation for reversing local immunosuppression has been the inhi- bition of chemokine receptor CXCR4 in combination with chemotherapeutic and αPD-1 compounds. CXCR4 serves as a receptor for CXCL12, which is secreted by cancer associated fibroblasts and regulates T-cell influx into tumors. Recently, it has been discovered the CXCR7 also serves as a receptor for CXCL12, in addition to several other chemokines. Given its elevated expression in PDAC stroma, we hypothesized that CXCR7 may serve as a candidate therapeutic target for reversing PDAC immunosuppression. Recent advances in synthetic biology have stimulated interest in programming bacteria to sense and respond to diseases in the human body such as cancer. Bacterial machinery can be re-engineered to sense a particular microenvironment, initiate colonization, and trigger the production of a therapeutic directly and specifically within that microenvironment. Particularly relevant for PDAC, the ability of bacteria to move through tissues without a robust, direct vasculature connection offers a great advantage for drug delivery to poorly perfused tumors. Leveraging our expertise in synthetic biology, molecular biology, mouse models, and preclinical therapeutics, we will interrogate the role of CXCR7 in the maintenance of local immune suppression in PDAC and evaluate the preclinical efficacy of targeting this pathway as a means of therapeutic intervention in PDAC. First, we will examine the functional consequences inhibiting CXCR7 and its pathway components in the multiple cell types in PDAC and elucidate the molecular mechanism of immune suppression via scRNAseq analysis and multiplex immunofluorescence of PDAC explants. To study the effects of CXCR7 inhibition in vivo, we will administer probiotic E. coli Nissle 1917 engineered to selectively colonize tumors and produce CXCR7 inhibiting nanobodies directly within the tumor of our preclinical model of PDAC, the KPC mouse. Tumor progression, immune system activation, and overall survival will be assessed following CXCR7 monotherapy and in combination with tradi- tional chemotherapeutics and other immune-targeted agents. In summary, the experiments proposed herein will aid our understanding of the local immune suppression within PDAC tumors as well as validate a bacterial therapy that addresses two challenges of PDAC treatment; poor drug accessibility and a highly suppressed immune microenvironment.