Blocking Latent TGF-β2 Activation as a Therapeutic Strategy for Renal Fibrosis

阻断潜在的 TGF-β2 激活作为肾纤维化的治疗策略

• 批准号: 10215977
• 负责人: Viet Quoc Le
• 金额: $ 15.53万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10215977
• 关键词: Acute Renal Failure with Renal Papillary Necrosis; Affinity; Antibodies; Aristolochic Acids; Binding; Binding Proteins; Biological Assay; Biology; Blocking Antibodies; Blood Platelets; Boston; Capital; Cardiovascular Diseases; Cell Cycle Arrest; Cell Line; Cells; Chronic Kidney Failure; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complement; Complement Factor B; Complex; Crystallization; Crystallography; DNA Insertion Elements; DNA Repair; Data; Deposition; Deuterium; Development; Diabetic Nephropathy; Disease model; Drug Kinetics; Drug Targeting; Education; Electron Microscopy; End stage renal failure; Endothelial Cells; Ensure; Environment; Epithelial Cells; Epitopes; Etiology; Exhibits; Expression Profiling; Extracellular Matrix; Failure; Fellowship; Fibroblasts; Fibrosis; Follow-Up Studies; Foundations; Functional disorder; Future; Genetic; Goals; Grant; Growth; Growth Factor; Hospitals; Human; Hydrogen; Immunofluorescence Microscopy; Immunoglobulin G; Infiltration; Inflammatory; Infrastructure; Injury to Kidney; Institutes; Institution; Integrin Inhibition; Integrins; Ischemia; Kidney; Kidney Diseases; Knock-in Mouse; LRRC32 gene; Libraries; Link; M cell; Mediating; Mentors; Mentorship; Modality; Modeling; Molecular; Molecular Biology; Molecular Conformation; Monitor; Mus; Mutation; Myofibroblast; National Institute of Diabetes and Digestive and Kidney Diseases; Negative Staining; Pathway interactions; Pediatric Hospitals; Pericytes; Pharmaceutical Preparations; Property; Protein Biochemistry; Proteins; Regulatory T-Lymphocyte; Renal function; Reperfusion Therapy; Reporter; Research; Research Personnel; Resolution; Resources; Role; Signal Transduction; Structure; Surface; System; Techniques; Testing; Therapeutic; Therapeutic antibodies; Training; Transforming Growth Factor Beta 2; Transforming Growth Factor beta Receptors; Transforming Growth Factors; Treatment Efficacy; Ureteral obstruction; Vision; Vocational Guidance; Woman; X-Ray Crystallography; Yeasts; antibody libraries; base; career; career development; cell type; diabetic rat; dimer; drug development; drug discovery; extracellular; global health; improved; in vitro activity; in vitro testing; in vivo; innovation; insertion/deletion mutation; insight; kidney fibrosis; lead candidate; medical schools; model building; mortality; mouse model; nanobodies; novel strategies; novel therapeutic intervention; novel therapeutics; prevent; programs; response; skills; structural biology; success; symposium; therapeutic development; therapeutic target

ABSTRACT. Transforming growth factor beta2 (TGF-β2) is an important therapeutic target for renal fibrosis, the principal cause of end-stage renal failure in chronic kidney disease (CKD). TGF-β2 triggers renal fibrosis in vivo and, in response to kidney injury, is upregulated in renal myofibroblasts, pericytes, and proximal tubule epithelial cells—cell types that mediate kidney fibrosis. Earlier studies showed that an antibody to mature TGF-β2 arrested renal fibrosis in a rat model of diabetic kidney disease, but further therapeutic development was not followed up. In vivo, TGF-β2 exists mainly as a latent pro-complex (proTGF-β2) in which prodomains are noncovalently bound to the growth factor. Secreted proTGF-β2 is stored in different extracellular milieus where it undergoes activation, i.e. release of the growth factor (mature TGF-β2), to initiate signaling. Preliminary data point to αVβ6-dependent and -independent mechanisms of proTGF-β2 activation as different modalities that can be therapeutically targeted for renal fibrosis. Aim 1 of this proposal is to develop new antibodies that specifically target the prodomain and block proTGF-β2 activation as a novel therapeutic strategy for renal fibrosis. Antibodies will be selected from an innovative yeast display antibody library, screened for activation-blocking activity in vitro, and tested for therapeutic efficacy in mouse models of acute kidney injury. Aim 2 is to determine high-resolution crystal structures of proTGF-β2 to define the mechanism underlying latency and facilitate drug development by uncovering new strategies to prevent activation. The candidate has assembled an exceptional team of mentors and advisors with expertise in renal pathophysiology, drug discovery, and structural biology to ensure the success of the project. The team will provide career guidance and training in techniques essential for the candidate’s future independent career at the interface of structural biology, drug discovery, renal fibrosis, and CKD. The candidate will receive extensive training in 1) X-ray crystallography, 2) antibody discovery, 3) renal pathophysiology, 4) immunofluorescence microscopy, and 5) mouse models of acute kidney injury and renal fibrosis. These skills will extend the candidate’s already versatile foundation in genetics, molecular biology, protein biochemistry, and structural biology. Boston Children’s Hospital and surrounding institutions (e.g., Harvard Medical School) constitute a robust training environment with unparalleled intellectual capital and remarkable infrastructure, which include cutting-edge yeast display platforms for antibody discovery at the Institute for Protein Innovation and unparalleled resources and expertise in the Renal Division at Brigham and Women’s Hospital, that will enhance the candidate’s growth and support his proposed research. Career development will be accomplished through direct mentorship, education through fellowship training offices, and attendance of conferences. The results of this proposal will establish the foundation of the candidate’s future research programs as an independent investigator in renal biology. The candidate plans to apply for the NIDDK Small Grant Program if available to K awardees and an R01 to facilitate his transition to independence.

抽象的。转化生长因子β2（TGF-β2）是肾纤维化的重要治疗靶点， 慢性肾脏病（CKD）终末期肾衰竭的主要原因。 TGF-β2 触发体内肾纤维化 并且，作为对肾损伤的反应，肾肌成纤维细胞、周细胞和近端小管上皮细胞中的表达上调 细胞——介导肾纤维化的细胞类型。早期研究表明，成熟 TGF-β2 的抗体可抑制 糖尿病肾病大鼠模型中的肾纤维化，但没有跟进进一步的治疗开发。 在体内，TGF-β2 主要作为潜在的前复合物 (proTGF-β2) 存在，其中前结构域非共价结合 到生长因子。分泌的 proTGF-β2 储存在不同的细胞外环境中，并在那里进行激活， 即释放生长因子（成熟 TGF-β2）以启动信号传导。初步数据表明 αVβ6 依赖性 -proTGF-β2 激活的独立机制作为可以治疗的不同方式 针对肾纤维化。该提案的目标 1 是开发专门针对 前结构域并阻断 proTGF-β2 激活作为肾纤维化的新治疗策略。抗体将会 从创新的酵母展示抗体库中筛选，在体外筛选激活阻断活性，并且 测试了小鼠急性肾损伤模型的治疗效果。目标 2 是确定高分辨率 proTGF-β2 的晶体结构可定义潜伏期的潜在机制并促进药物开发 发现防止激活的新策略。候选人组建了一支出色的导师团队 以及具有肾脏病理生理学、药物发现和结构生物学专业知识的顾问，以确保 项目的成功。该团队将提供职业指导和必要的技术培训 候选人未来在结构生物学、药物发现、肾纤维化等领域的独立职业生涯 慢性肾病。候选人将接受以下方面的广泛培训：1) X 射线晶体学、2) 抗体发现、3) 肾病 病理生理学，4) 免疫荧光显微镜，5) 急性肾损伤和肾损伤的小鼠模型 纤维化。这些技能将扩展候选人在遗传学、分子生物学、 蛋白质生物化学和结构生物学。波士顿儿童医院及周边机构（例如， 哈佛医学院）构建了一个强大的培训环境，拥有无与伦比的智力资本和 卓越的基础设施，其中包括用于抗体发现的尖端酵母展示平台 布莱根大学蛋白质创新研究所以及肾脏科无与伦比的资源和专业知识 妇女医院，这将促进候选人的成长并支持他提出的研究。职业 发展将通过直接指导、通过奖学金培训办公室进行的教育以及 出席会议。该提案的结果将为候选人的未来奠定基础 作为肾脏生物学独立研究者的研究项目。候选人计划申请 NIDDK 小额赠款计划（如果适用于 K 获奖者和 R01），以促进他向独立过渡。