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Abstract

Immunotherapies offer new possibilities for treating pediatric cancers, but achieving treatment specificity remains challenging. Pediatric cancers have lower mutation rates than adult cancers, making it harder to identify selective therapeutic targets. A promising strategy involves focusing on cancer-specific proteolysis—a posttranslational modification that generates unique protein termini on cancer cells. These termini can serve as highly selective targets for N-recognin proteins such as ClpS. Furthermore, protein fragments released during this process may circulate in the bloodstream, serving as biomarkers for cancer-specific protein termini, thereby aiding in the development of precise therapies. However, targeting protein termini is difficult due to differences in epitopes recognized by antibodies or CAR-T cells and the complexity of detecting shed protein fragments in blood plasma, where protein abundances span >12 orders of magnitude. The low abundance of protein fragments shed by cancer cells further complicates detection. My research aimed to establish proof-of-concept methods to target cell surface protein termini and detect these fragments in blood plasma as potential biomarkers. My specific aims were: 1A) Optimize the expression and purification of ClpS and characterize its binding specificity to peptide termini. 1B) Develop tools for investigating ClpS binding to truncated proteins with diverse N-terminal sequences on Hela cells. 2) Establish a gel fractionation method based on molecular weight to enrich protein fragments. I successfully established the expression and automated purification of ClpS, validated its functionality in a biochemical binding assay, and demonstrated that electro-elution SDS-PAGE coupled with mass spectrometry can separate proteins based on molecular size and detect small proteins in plasma. This thesis provides foundational work towards establishing a new class of therapeutic targets and diagnostic markers. It addresses the critical challenge of preventing damage to healthy cells while targeting and destroying cancer cells. The project leverages the N-recognin protein class to target cancer-specific cell surface proteins and lays the foundation for a novel method to target and potentially kill cancer cells more selectively. Additionally, it develops a new methodology for the size-selective enrichment of protein fragments, with potential applications in liquid biopsy and immunotherapy selection to improve patient outcomes.