Monoclonal antibodies have created tremendous value for patients, with six of the top ten selling FDA approved drugs being monoclonal antibodies. Generally, monoclonal antibodies work by blocking protein function or inducing cell death via effector functions, such as antibody-dependent cellular toxicity. Though monoclonal antibodies have been greatly successful for various targets and diseases, some disease contexts could benefit from added functionality. For example, on-target, off-tumor toxicity is common when using a monoclonal antibody to target a disease protein that is also expressed on normal cells, as is the case with human epidermal growth factor receptor in breast cancer. Bispecific or bifunctional antibodies, which can recognize two different antigens or have dual functionalities, have the potential to offer greater selectivity, efficacy, and functionality compared to monoclonal antibodies. Various forms of bispecific antibodies exist to date, including bispecific IgGs, which add disease specificity by targeting a disease-specific protein and the target protein simultaneously, bispecific T cell engagers, which enable recruitment of cytotoxic T cells to tumor cells, and antibody-drug conjugates, which involve the tumor specific delivery of a highly cytotoxic small molecule payload using antibody-induced receptor internalization. Here, I describe three novel approaches using bifunctional antibody-based technologies. In Chapter 1, I describe the use of bispecific antibody-cytokine fusions, termed KineTACs, which can be applied as a novel strategy for the targeted degradation of cell surface and extracellular proteins. In Chapter 2, I highlight an orthogonal targeted degradation approach using antibody-drug conjugates in which the antibody recruits a cell surface E3 ligase, and the small molecule binds a cell surface multi-pass membrane protein, enabling its degradation. In Chapter 3, I describe the use of bispecific VH-Fab IgGs targeting different epitopes on the Spike protein as a novel anti-viral strategy against SARS-CoV-2 virus.

Bio-conjugation and protein engineering are two powerful biochemical methods to alter proteins to have enhanced functions. Bio-conjugation allows for proteins to be attached to other protein and non-protein molecules in ways that would never appear naturally in the biological world, bestowing new properties onto a protein. Protein engineering allows for manipulation of the linear amino-acid sequence of a protein to alter its biochemical properties. These types of substitutions can change the way a protein folds, how it interacts with partner proteins, and its own chemical reactivity. These methods are currently highly utilized in the development of novel therapeutics and research tools. For both methods, location is one of the most important considerations for successful applications. Years of bio-conjugation research has revealed that attachment at a controlled and known site improves the reproducibility and properties of a given conjugate. In protein engineering, not all sites on a protein have the same chemical environment and thus even the same amino acid at a different location may react differently. Thus, even with these powerful biochemical methods, careful research must be done to determine the optimal attachment method, mutation location, and site. However, the optimized conjugate or engineered protein has great potential as a novel therapeutic, diagnostic, or research tool. Here, I describe three approaches using bio-conjugation and protein engineering to develop novel and optimized protein conjugates or engineered protein fusions for various applications. In Chapter 1, I describe the use of a methionine-based conjugation method to attach cytotoxic cancer drugs on antibodies as selective drug delivery carriers. In Chapter 2, I highlight the development of rapid, facile protein biosensors to detect antibodies to COVID-19. In Chapter 3, I describe the development of an improved cell-surface biotinylation method utilizing novel enzyme conjugates.