Cancer immunotherapies, or treatments that utilize a patient’s own immune system to fight cancer, have been met with a great deal of success. Although most current immunotherapies have focused on boosting the adaptive immune system, there has been a growing interest in targeting components of the innate immune system. An important member of the innate immune system is the natural killer (NK) cell. Like T cells, NK cells are cytotoxic effector cells with the ability to distinguish and eliminate virally infected or transformed cells. They play a key role in the anti-tumor response and are able to identify target cells by recognizing stress-induced ligands that are frequently overexpressed on cancer cells. NK cells are best known for their ability to perform antibody-dependent cellular cytotoxicity (ADCC), a mechanism that is used by several therapeutic antibodies to eradicate tumor cells. Whereas all T cells can be targeted through the T cell receptor (TCR)/CD3 complex, NK cells have multiple activating, costimulatory, and inhibitory receptors that can be targeted to modulate their activity. This dissertation provides a strategy used to identify antibodies that can target and stimulate NK cells and the development of bispecific antibodies to redirect NK cell-mediated cytotoxicity towards tumor cells of interest. Chapter 1 discusses the background of NK cells and their role in anti-tumor immunity. Chapter 2 describes a mass spectrometry-based screening approach to identify cell surface proteins on NK cells. Chapter 3 provides the method designed to identify antibodies that are able to stimulate NK cell-mediated cytotoxicity and the generation of bispecific NK engagers.

Allostery is an inherent feature of proteins, but it remains challenging to reveal the mechanisms by which allosteric signals propagate. A clearer understanding of this intrinsic circuitry would afford new opportunities to modulate protein function. Here, we have identified allosteric sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small-molecule fragment soaks. New modeling approaches reveal 'hidden' low-occupancy conformational states for protein and ligands. Our results converge on allosteric sites that are conformationally coupled to the active-site WPD loop and are hotspots for fragment binding. Targeting one of these sites with covalently tethered molecules or mutations allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure, revealed here by multitemperature crystallography, can elucidate allosteric mechanisms and open new doors for long-range control of protein function.

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl₂, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.