Nanobody inhibitors of proton-sensing G protein-coupled receptors

质子感应 G 蛋白偶联受体的纳米抗体抑制剂

• 批准号: 10216432
• 负责人: GEOFFREY A CHANG
• 金额: $ 15.78万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10216432
• 关键词: Acidity; Acidosis; Antibodies; Antigens; Area; Binding; Biochemical; Bioinformatics; Biological; Biological Assay; Biological Models; Biology; Biophysics; Blood Vessels; Cancer Etiology; Cell Proliferation; Cell surface; Cells; Cessation of life; Clinical; Collagen; Cyclic AMP; Data; Databases; Desmoplastic; Detection; Detergents; Development; Diagnostic; Directed Molecular Evolution; Disease; Extracellular Space; Family; Family member; Feedback; Fibroblasts; Fibrosis; Funding; Future; G-Protein-Coupled Receptors; GPR4 gene; GPR68 gene; Generations; Genotype-Tissue Expression Project; Goals; Human; Immune; Inflammatory; Interleukin-6; Malignant Neoplasms; Malignant neoplasm of pancreas; Mediating; Membrane; Molecular Conformation; Pancreas; Pancreatic Ductal Adenocarcinoma; Patients; Pharmaceutical Preparations; Phenotype; Pilot Projects; Production; Proteins; Protons; Reagent; Signal Transduction; Specificity; Structure; Surface; Surface Antigens; Techniques; The Cancer Genome Atlas; Therapeutic; Therapeutic Agents; Thromboplastin; Up-Regulation; base; cell type; extracellular; inhibitor/antagonist; interest; malignant phenotype; member; nanobodies; novel; novel strategies; novel therapeutics; pancreatic ductal adenocarcinoma cell; receptor; selective expression; therapeutic development; therapeutic target; therapeutically effective; therapy resistant; tool; tumor; tumor microenvironment

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.

项目摘要/摘要 胰腺导管腺癌（PDAC）很快成为美国癌症死亡的第二大原因， 总体生存率为9％。 PDAC的特征是肿瘤微环境，该环境密集纤维化，并且 酸性。低pH的影响可以通过多种机制发生，包括激活质子感应G 蛋白质偶联受体，例如GPR65和GPR68。 GPR65，GPR68和GPR132（另一种质子感应 GPCR）在PDAC肿瘤（来自癌症基因组图集，TCGA的数据）中比正常 胰腺（GTEX数据库）。 GPCR作为一类通常不是针对癌症的目标，并且是未开发的 对治疗性发育感兴趣的领域，尤其是对于PDAC等癌症的领域。 质子感应GPCR可能是治疗PDAC和GPR65/GPR68参与PDAC的新靶标 为重点的试点研究提供了出色的模型系统。它们在PDAC肿瘤中的突出上调 在肿瘤微环境（TME）中涉及不同的细胞类型：免疫细胞中的GPR65和癌症 - gpr68 相关的成纤维细胞（CAF）。预计GPR65通过低ph促进了免疫抑制作用。 循环放大器（营地）。 GPR68的低pH激活，在患者衍生的CAF中通过CAMP作用，增加了生产 胶原蛋白，纤维化标记和IL-6，可促进PDAC增殖。因此，gpr68是以前的枢纽 PDAC细胞和CAF之间未识别的正反馈回路。抑制CAF表达的GPR68 活性预计将钝性PDAC细胞增殖，纤维化和减少肿瘤大小。 我们建议使用一种新颖的方法来生成针对GPR65和GPR68的纳米型（NB） 细胞外表面。我们试图确定可以抑制低pH值促进的信号传导和功能变化的NB， 包括来自患者衍生PDAC肿瘤的细胞。这样的NB也可能有助于检测PDAC肿瘤 他们的酸性TME。如果成功的话，我们的方法可能会被推广，以找到其他NB拮抗剂 GPCR和受体，最小化（或消除）将纯化受体蛋白作为抗原的需求。项目 有两个目标：（1）在细胞表面获得一个特定于GPR65和GPR68的NB的面板，（2）定义 GPR65和GPR68靶向NB的特异性和生物活性，表征和验证它们 使用基于细胞的测定，将其对人类细胞中的信号传导和作用的影响进行质疑。目标 是生成NB，将抑制GPR65和GPR68的行为，作为最终产生的第一步 NBS均定向所有4个质子感应GPCR。除了进行生物学活动之外，此类NB还可以帮助 在GPCR的质子感应类别的结构研究中，并用作IDG生成的试剂。