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

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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.