Nanobody inhibitors of proton-sensing G protein-coupled receptors

项目摘要/摘要 胰腺导管腺癌(PDAC)很快将成为美国癌症死亡的第二大原因， 总体存活率为9%。PDAC的特征是肿瘤微环境密集纤维化和 是酸性的。低pH值的影响可以通过多种机制发生，包括激活质子感应G 蛋白偶联受体，如GPR65和GPR68。GPR65、GPR68和GPR132(另一种质子传感 (来自癌症基因组图谱，TCGA的数据)在PDAC肿瘤中比在正常组织中更高表达 胰腺(GTEx数据库)。GPCR作为一个类别，通常不是针对癌症的，是一种未被探索的 对治疗开发高度感兴趣的领域，特别是对癌症，如PDAC。 质子传感GPCRs可能成为治疗PDAC的新靶点及GPR65/GPR68在PDAC中的参与 为重点先行研究提供了一个极好的模型系统。它们在PDAC肿瘤中的显著上调 在肿瘤微环境(TME)中涉及不同类型的细胞：免疫细胞中的GPR65和癌症中的GPR68- 相关成纤维细胞(CAF)。GPR65被预测为通过低pH促进的免疫抑制。 环磷酸腺苷(CAMP)。低pH激活GPR68，通过cAMP作用于患者来源的CAF，增加产量 胶原、纤维化标志物和促进PDAC增殖的IL-6的结合。因此，GPR68是以前的 PDAC细胞和CAF之间存在无法识别的正反馈环路。抑制CaF基因表达的GPR68 据预测，活性可以抑制PDAC细胞的增殖、纤维化和缩小肿瘤大小。 我们建议使用一种新的方法来生成针对GPR65和GPR68的纳米体(NBS) 胞外表面。我们试图确定可以抑制低pH促进的信号和功能变化的NBS， 包括来自患者来源的PDAC肿瘤的细胞。这样的NBS也可以帮助检测PDAC肿瘤 他们的酸性TME。如果成功，我们的方法可以推广到寻找潜在的NB拮抗剂 GPCRs和受体，最大限度地减少(或消除)作为抗原的纯化受体蛋白的需要。该项目 有两个目标：(1)获得细胞表面GPR65和GPR68特异的NBS面板和(2)定义 GPR65和GPR68靶向NBS的特异性和生物学活性及其表征和验证 使用基于细胞的分析来询问它们对它们在人类细胞中的信号和行为的影响。目标是 是产生NBS，它将抑制GPR65和GPR68的行动，作为最终产生 国家统计局针对所有4个质子传感GPCR。除了具有生物活性外，这样的NBS还可以帮助 在质子传感类GPCRs的结构研究中，并用作IDG生成的试剂。

PROJECT SUMMARY/ABSTRACT Pancreatic ductal adenocarcinoma (PDAC), soon to be the second leading cause of cancer deaths in the U.S, has an overall 9% survival. PDAC is characterized by a tumor microenvironment that is densely fibrotic and acidic. Effects of low pH can occur through a variety of mechanisms, including activation of proton-sensing G protein-coupled receptors, such as GPR65 and GPR68. GPR65, GPR68 and GPR132 (another proton-sensing GPCR) are more highly expressed in PDAC tumors (data from The Cancer Genome Atlas, TCGA) than in normal pancreas (GTEx database). GPCRs as a class are generally not targeted for cancers and are an unexplored area of high interest for therapeutic development, especially for cancers, such as PDAC. Proton-sensing GPCRs may be novel targets for treating PDAC and involvement of GPR65/GPR68 in PDAC provides an excellent model system for a focused pilot study. Their prominent up-regulation in PDAC tumors involves different cell types in the tumor microenvironment (TME): GPR65 in immune cells and GPR68 in cancer- associated fibroblasts (CAFs). GPR65 is predicted to be immunosuppressive via low-pH promoted increase in cyclic AMP (cAMP). Low pH activation of GPR68, acting via cAMP in patient-derived CAFs, increases production of collagens, fibrotic markers, and IL-6, which promotes PDAC proliferation. GPR68 is thus a hub of a previously unrecognized positive feedback loop between PDAC cells and CAFs. Inhibition of CAF-expressed GPR68 activity is predicted to blunt PDAC cell proliferation, fibrosis and reduce tumor size. We propose to use a novel approach to generate nanobodies (Nbs) directed at GPR65 and GPR68 on the extracellular surface. We seek to identify Nbs that can inhibit low pH-promoted signaling and functional changes, including in cells from patient-derived PDAC tumors. Such Nbs may also aid in the detection of PDAC tumors in their acidic TME. If successful, our approach could be generalized to find potential Nb antagonists to other GPCRs and receptors, minimizing (or eliminating) the need for purified receptor proteins as antigens. The project has two goals: (1) obtain a panel of Nbs specific to GPR65 and GPR68 on the cell surface and (2) define the specificity and biological activity of the GPR65 and GPR68-targeted Nbs, characterizing and validating them using cell-based assays that will interrogate their impact on their signaling and action in human cells. The goal is to generate Nbs that will inhibit the actions of GPR65 and GPR68 as a first step toward ultimately generating Nbs directed at all 4 of the proton-sensing GPCRs. In addition to having biological activities, such Nbs could aid in structural studies of the proton-sensing class of GPCRs and serve as IDG-generated reagents.