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

摘要。转化生长因子β 2（TGF-β2）是肾纤维化的重要治疗靶点， 慢性肾脏病（CKD）中终末期肾衰竭的主要原因。TGF-β2在体内引发肾纤维化 在肾损伤时，在肾肌成纤维细胞、周细胞和近曲小管上皮细胞中上调 细胞-介导肾纤维化的细胞类型。早期的研究表明，成熟TGF-β2的抗体可以阻止 在糖尿病肾病的大鼠模型中发现了肾纤维化，但没有随访进一步的治疗开发。 在体内，TGF-β2主要以前体复合物（proTGF-β2）的形式存在 到生长因子。分泌的proTGF-β2储存在不同的细胞外环境中，在那里它经历活化， 即释放生长因子（成熟TGF-β2），启动信号传导。初步数据表明αVβ6依赖 proTGF-β2激活的独立和非独立机制作为不同的模式，可以治疗 治疗肾纤维化该提案的目的1是开发特异性靶向 前结构域和阻断proTGF-β2活化作为肾纤维化的新治疗策略。抗体将 选自创新的酵母展示抗体文库，筛选体外激活阻断活性，和 在急性肾损伤的小鼠模型中测试治疗功效。目标2：确定高分辨率 proTGF-β2的晶体结构，以确定潜在的机制，并通过以下方式促进药物开发： 发现防止激活的新策略候选人已经组建了一个杰出的导师团队 和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。该小组将提供职业指导和培训的基本技术， 候选人未来在结构生物学，药物发现，肾纤维化和 CKD。候选人将接受1）X射线晶体学，2）抗体发现，3）肾脏 病理生理学，4）免疫荧光显微镜，和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。波士顿儿童医院和周边机构（例如， 哈佛医学院）构成了一个强大的培训环境，拥有无与伦比的智力资本， 卓越的基础设施，其中包括尖端的酵母展示平台，用于在 研究所蛋白质创新和无与伦比的资源和专业知识，在肾脏部门在布里格姆和 妇女医院，这将促进候选人的成长和支持他提出的研究。职业生涯 发展将通过直接指导，通过研究金培训办公室进行教育， 出席会议。这一提议的结果将为候选人的未来奠定基础 作为肾脏生物学的独立研究者进行研究计划。候选人计划申请NIDDK 小额赠款计划，如果可用于K获奖者和R 01，以促进他过渡到独立。

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.