PROJECT SUMMARY There is a steady rise in the clinical use of biologics, particularly monoclonal antibodies (mAbs). We present lasso peptides as a potential drug modality within the underleveraged region of chemical space between mAbs and small molecules. Lasso peptides represent a class of ribosomally-synthesized and post-translationally modified peptide (RiPP). Biosynthesis of lasso peptides involves a genetically encoded precursor peptide and maturation enzymes that catalyze a macrolactam ring and configure the peptide into a unique lariat knot. Lasso peptides recapitulate the desired properties of synthetic cyclic peptide but with key advantages: (i) lasso peptides are uniquely constrained, highly stable globular structures with the correct shape to engage peptide-binding targets, (ii) lasso peptides are entirely genetically encoded, straightforward generation of large and sequence- diverse libraries, (iii) enzymatic tolerance supports modification of the entire solvent-exposed surface, non- natural amino acids, and post-translational modifications, and (iv) the C-terminus provides a facile conjugation site for peptide display, and high-throughput screening. Due to their unique topology, lasso peptides cannot be chemical synthesized, thus we rely on the biosynthetic enzymes to thread the peptide. This proposal will design a biosynthesis-informed library of lasso peptides and as a proof-of-concept, use this library to select for lasso peptides that bind OX40. OX40 is an immunostimulatory member of the tumor necrosis factor receptors (TNFR), studies have found that the native OX40 ligand, OX40L, and agonist antibodies can activate OX40 to stimulate the proliferation and activation of T cells. There is a compelling need to validate immune targets so that therapeutics can be developed, complementing the success of anti-PD1 (ie Keytruda) and anti- TNFα (ie Humira) biologics. Our group has developed a high-throughput method with cell-free biosynthesis (CFB) to produce lasso peptide libraries. In Aim I we will combine CFB with mRNA display to identify library members that are successfully cyclized. Aim II will select for lasso peptides that bind OX40. We will assess structure-activity relationships by validating the three-dimensional structure of the OX40-binding variants and conduct binding affinity studies and in vitro signaling assays to evaluate their activity. Motifs related to OX40 binding and activity will be identified through structural and mutagenesis data. Improved understanding of receptor binding-activity relationship will significantly inform pharmaceutical efforts. This fellowship will provide the necessary training to prepare me for an academic career in mentoring researchers and teaching students. My goal is to develop my abilities to become a more comprehensive scientist through improving communication in both oral and written form, a strong collaborative work ethic, and mentorship of undergraduate and graduate students. Working wi...