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

项目摘要 胰腺导管腺癌（PDAC）是一种高致死性恶性肿瘤， 7%左右。PDAC患者的预后不良是由于多种因素的综合作用，包括晚期诊断， 肿瘤灌注不良导致的低药物蓄积，以及免疫抑制的肿瘤微环境， 我是说。因此，尽管针对其他癌症类型的免疫疗法取得了重大进展， 例如黑色素瘤和非小细胞肺癌，PDAC免疫疗法的临床试验还没有 被证明是成功的。逆转局部免疫抑制的一种研究途径是抑制- 趋化因子受体CXCR 4与化疗剂和αPD-1化合物的组合。CXCR4 作为CXCL 12的受体，CXCL 12由癌症相关的成纤维细胞分泌并调节T细胞流入 转化为肿瘤最近，已经发现CXCR 7也作为CXCL 12的受体，除了CXCL 12以外，CXCR 7还作为CXCL 12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CXCR 7可能与PDAC的表达相关。 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进行感知和响应的兴趣 人体内的疾病，如癌症。细菌机器可以重新设计以感知特定的 微环境，启动定殖，并直接和特异性地触发治疗剂的产生。 这个微环境。特别是与PDAC相关的是，细菌在组织中移动而不受干扰的能力。 坚固的直接脉管连接为药物输送到灌注不良的肿瘤提供了很大的优势。 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， 我们将探讨CXCR 7在维持PDAC局部免疫抑制中的作用，并评估 靶向该途径作为PDAC治疗干预手段的临床前疗效。一是 检查在多种细胞类型中抑制CXCR 7及其通路组分的功能后果 在PDAC中，通过scRNAseq分析和多重分析阐明免疫抑制的分子机制。 PDAC外植体的免疫荧光。为了研究CXCR 7抑制在体内的作用，我们将施用 益生菌E.大肠杆菌Nissle 1917工程化以选择性地定殖肿瘤并产生CXCR 7抑制纳米抗体 直接在我们的PDAC临床前模型KPC小鼠的肿瘤内。肿瘤进展，免疫系统 在CXCR 7单药治疗和与传统疗法联合治疗后， 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肿瘤中的作用，以及验证解决PDAC治疗的两个挑战的细菌疗法; 药物可及性差和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.