Genetic Modulation of a Human Kinase Using Engineered Intracellular Antibody Fragments

Mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that regulates eukaryotic cell growth and metabolism in response to nutrient and growth factor cues. Dysregulated mTOR signaling is implicated in the progression of a wide range of human diseases, including cancer, neurodegeneration, metabolic disorders, and age-related conditions. Despite significant therapeutic potential, efforts to safely and effectively modulate aberrant mTOR activity with small molecules remain hindered by an incomplete understanding of how its substrate recruitment modalities, conformational dynamics, and subcellular spatial functions are coupled to distinct physiological outputs.

To address these limitations, this thesis describes an approach for modular genetic control of mTOR-mediated signal transduction. Herein, a series of synthetic antibody fragments targeting multiple epitopes and conformations of an mTOR substrate recruitment domain were generated using phage display. When genetically encoded as intracellular single-chain variable fragment "intrabodies" in living cells, these binders enabled programmable modulation of mTOR activity with conformational, spatial, and epitope-based precision. A combination of high-resolution crystallographic studies and cell-based functional assays provided key insights into FRB-mediated substrate docking, an allosteric mechanism governing mTOR complex 1 stability, the subcellular regulation of nuclear and cytoplasmic mTOR signaling, and an inhibitory binding site for unconventional modulation of mTOR function. In summary, this work integrates protein engineering, molecular structure, and synthetic biology approaches to establish engineered intracellular antibody fragments as essential tools for investigating the structural and spatial mechanisms driving therapeutically relevant protein kinase activity.