Nanobody inhibitors of proton-sensing G protein-coupled receptors

项目总结/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的特征在于肿瘤微环境是密集纤维化的， 酸性的低pH的影响可以通过多种机制发生，包括激活质子敏感G 蛋白偶联受体，如GPR 65和GPR 68。GPR 65、GPR 68和GPR 132（另一种质子传感器 GPCR）在PDAC肿瘤中比在正常PDAC肿瘤中更高表达（数据来自癌症基因组图谱，TCGA）。 胰腺（GTEx数据库）。GPCR作为一类通常不靶向癌症，并且是一种未探索的基因。 这是治疗开发的高度关注领域，特别是对于癌症，如PDAC。 质子敏感GPCR可能是治疗PDAC的新靶点，并且GPR 65/GPR 68参与PDAC 为重点试点研究提供了一个很好的模型系统。它们在PDAC肿瘤中的显著上调 涉及肿瘤微环境（TME）中的不同细胞类型：免疫细胞中的GPR 65和癌症中的GPR 68。 相关成纤维细胞（CAFs）。预测GPR 65通过低pH促进的免疫抑制性增加而具有免疫抑制性。 环腺苷酸（cAMP）。GPR 68的低pH活化，通过患者来源的CAF中的cAMP起作用，增加生产 胶原蛋白、纤维化标记物和IL-6，其促进PDAC增殖。因此，GPR 68是以前的中心 PDAC细胞和CAF之间的未识别的正反馈回路。CAF表达的GPR 68的抑制 预测活性可钝化PDAC细胞增殖、纤维化并减小肿瘤大小。 我们建议使用一种新的方法来产生针对GPR 65和GPR 68的纳米抗体（Nbs）。 细胞外表面我们试图鉴定可以抑制低pH促进的信号传导和功能变化的Nbs， 包括来自患者来源的PDAC肿瘤的细胞。这样的Nbs还可以帮助检测PDAC肿瘤。 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， GPCR和受体，最小化（或消除）对纯化的受体蛋白作为抗原的需要。项目 具有两个目标：（1）获得细胞表面上对GPR 65和GPR 68特异性的Nbs组和（2）定义细胞表面上的GPR 65和GPR 68特异性的Nbs。 GPR 65和GPR 68靶向Nbs的特异性和生物活性，表征和验证它们 使用基于细胞的分析，将询问它们对它们在人类细胞中的信号传导和作用的影响。目标 是产生Nbs，其将抑制GPR 65和GPR 68的作用，作为最终产生Nbs的第一步。 Nbs针对所有4个质子感测GPCR。除了具有生物活性外，这种Nbs还可以帮助 在质子感应类GPCR的结构研究中，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.