Adapting cell-based therapies for the treatment of pulmonary fibrosis

Project Summary Pulmonary fibrosis is a chronic and progressive disease characterized by the scarring and stiffening of lung tissue. Patients have poor quality of life, and usually die within 2-5 years. The only curative measure is lung transplantation, which is not an option for most patients. The primary catalyst of fibrosis, TGF-β, activates alveolar fibroblasts to become profibrotic fibroblasts, which deposit excess extracellular matrix protein and take-on an anti-apoptotic phenotype. TGF-β has been difficult to therapeutically target due to its universal expression and role in maintaining normal tissue homeostasis. Systemic inhibitors risk toxicity. This study aims to use chimeric antigen receptor (CAR)-T cell therapy combined with an inducible drug delivery circuit to target profibrotic fibroblasts. The inducible circuit using Synthetic Intramembrane Proteolysis Receptors (SNIPRs) drives a response element payload of potent TGF-β inhibitor TβRII-Fc. I am currently engineering CARs and SNIPRs that target membrane-bound extracellular proteins that are specifically and highly expressed on profibrotic fibroblasts, Lrrc15, TEM1, and Gremlin 1. The specificity of my targets limits unintended cell killing or inhibitor release. I anticipate that local TβRII-Fc release will reprogram and prevent the induction of profibrotic fibroblasts. For my thesis project, I will be testing the performance of engineered CARs and SNIPRs that target profibrotic fibroblasts and evaluating the efficacy of CAR-T SNIPR -> TβRII-Fc T cells in an in vivo model of pulmonary fibrosis. In Aim 1, I will take 41BBζ CAR and SNIPR construct backbones and individually clone-in short chain variable fragments (scFvs) with high binding affinities to my proteins of interest, Lrrc15, TEM1, or Gremlin 1. For each target, I will test CAR-T cell proliferation and killing ability, SNIPR response element activation, and the inhibition of TGF-β signaling (pSmad2/3) in vitro. I have bred mice that express membrane-bound GFP on profibrotic fibroblasts when treated with tamoxifen (Cthrc1-CreERT2-mGFP). The Lim Laboratory has already tested and optimized anti-GFP CARs and SNIPR circuits. In Aim 2, I will test the ability of anti-GFP SNIPR-TβRII-Fc CAR-T cells to target membrane-bound GFP on profibrotic fibroblasts and ameliorate pulmonary fibrosis in a bleomycin mouse model. Fibrosis will be measured by lung collagen content (hydroxyproline) and the amount of profibrotic fibroblasts present at endpoint. If successful, this project will broaden the applications of cell-based therapies beyond cancer to the treatment of pulmonary fibrosis and other complex diseases.