Silver, PamelaMitragotri, SamirJiang, Lauren Suyung2026-06-2420262026-06-242026Jiang, Lauren Suyung. 2026. Rationally-Engineered Protein Therapeutics for the Treatment of Ewing Sarcoma. Bachelors Thesis, Harvard University Engineering and Applied Sciences.32575917https://dash.harvard.edu/handle/1/42742211Ewing sarcoma (ES) is the second most common bone cancer in children and adolescents. Prognosis is generally dismal; the five-year post-relapse survival rate is 15-25%. No novel advances in its treatment have been made in the last 40 years beyond systemic chemotherapy. We designed and constructed two panels of rationally engineered fusion proteins to selectively kill ES cells. TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) and Pseudomonas exotoxin (PE) are cytotoxic proteins that have been of great interest in oncological research in the last several decades, but efforts have largely failed — TRAIL due to limited activity and PE toxicity (especially hepatotoxicity). We direct the apoptotic activity of TRAIL or PE to ES cells via fusion to antibody fragments that bind to Leucine-rich repeat and Immunoglobulin-like domain-containing protein 1 (LINGO-1/LERN-1), an ES-specific surface marker, so that cell killing is ES-preferential. The TRAIL constructs are attenuated via mutations that reduce systemic off-target effects and optimize affinity for true death receptors over decoys by one to two orders of magnitude. TRAIL cytotoxicity has been demonstrated in ES. The PE constructs incorporate complement factor binding loops that facilitate a novel resecretion mechanism to reduce hepatotoxicity, a very frequent key barrier to PE drug development. This complement binding system is modular and in principle adaptable to a broad range of recombinant protein drugs to increase therapeutic index and improve clinical feasibility. We demonstrated specific killing of ES cells by TRAIL constructs and explored the functionality of various design parameters. A second TRAIL panel is currently being designed. PE constructs remain in progress; they have been designed and ordered, but further purification, as well as functional testing is pending.application/pdfenEwing sarcomaImmunotoxinsProtein engineeringRational designBioengineeringThesis or Dissertation2026-06-240009-0003-4383-7943