Thesis advisor: Jianmin Gao / Covalent drugs present a unique situation in the clinical world. Formation of a covalent bond between a drug molecule and its target protein can lead to significant increases in a number of desirable traits such as residence time, potency, and efficacy of a drug. From a kinetic perspective, the formation of a covalent bond between a drug and its target functionally eliminates the dissociation rate (koff) of the compound, ensuring that the compound will stay engaged with its target. However, development of covalent drugs has been met with caution and concern, as an irreversible covalent bond forming on the wrong target can have disastrous results, so specificity is of the utmost importance. One option for increasing specificity is by linking a covalent binding electrophile, or warhead, to a peptide. Peptide-based therapeutics have already been shown to serve as effective protein-targeting modalities with high specificity, a specificity that would greatly benefit covalent drugs. Phage display is a powerful technique for the discovery of selective peptides which utilizes the screening of vast libraries of randomized peptides to identify strong binders. This technology has been used to discover a large number of protein-targeting peptides, but also a smaller number of cyclic, covalent binding peptides that function as enzymatic inhibitors. Herein, this study aimed to explore the idea of adding covalent-binding functionality to phage libraries in novel ways and expand upon the scope of proteins that can be targeted with phage libraries containing covalent libraries. We sought to develop a mechanistic and chemical understanding of the interactions between bacteriophage and chemical warheads to best understand both the limits and the potential of this technology. In order to best understand the relationship between chemical warhead and phage particle, a model system was developed based on the M13KE pIII protein. It was found that the extracellular N-terminal domains of this protein could be expressed and purified in low yields in bacterial cells and that these domains would behave similarly in solution as in the membrane of the M13KE bacteriophage. With this protein in hand, experiments previously performed using small, cysteine containing peptides, could be performed on a full protein to mimic the phage labeling environment. This protein was used to identify efficient cysteine crosslinkers, most notably dichloroacetone (DCA) and bis-chlorooxime (BCO). The pIII protein system was then used to study the viability of bifunctional warhead molecules containing a covalent warhead and a cysteine crosslinker. Based on preliminary analyses with the pIII protein, aryl sulfonyl fluoride was chosen as a novel warhead candidate that warranted further pursuit. Kinetic NMR studies verified that aryl sulfonyl fluoride was capable of forming covalent bonds with phenols under phage labeling conditions. Labeling experiments analyzed with LC/MS seemed to indicate a degradation of the warhead. However, as the source of the degradation was not able to be determined, it was decided that various affinity assays would be used to identify if phage could be labeled with an aryl sulfonyl fluoride-DCA conjugate. Both streptavidin-bead pulldown assays and ELISA assays were used, however both assays yielded results that could not conclusively verify the integrity of the warhead. During phage labeling experiments, a phenomenon was noted that phage titers after modification showed a 2-3 order of magnitude drop in phage count. Covalent modification of phage beyond what is intended could have troubling consequences for all covalent phage libraries, and so a more in-depth approach was taken to identify and better understand phage toxicity as it relates to covalent warheads. As a model, a well-studied diazaborine-mediated warhead with a slow dissociation rate was selected and used in a range of phage toxicity screenings. Despite statistical fluctuations between trials, toxicity screenings using this warhead served to highlight a unique concern for bifunctional covalent warheads. A concentration-dependent toxicity can be seen in phage incubated with bifunctional small molecules that is not present when incubated with the monofunctional equivalents. The presence of this toxicity even towards a phage with no free thiols highlights a unique challenge of off-target labeling within phage particles that, if solved, could provide the next significant step towards developing novel covalent phage libraries. / Thesis (MS) — Boston College, 2022. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Antibody-recruiting molecules (ARMs) are therapeutic tools that simultaneously bind a hapten-specific serum antibody and a cancer cell surface protein, resulting in the activation and recruitment of an immune cell to the cancer surface. However, ARM efficacy is limited by the ability of ARMs to form a quaternary complex with the immune cell receptor, antibody, and cancer cell surface. The Rullo lab has previously developed and characterized a covalent ARM (cARM) that irreversibly links the ARM to the antibody and simplifies the quaternary binding equilibria. cARMs have shown a marked increase in both target immune recognition and therapeutic efficacy. However, cARM efficacy is still limited by the affinity of the antibody for the immune receptor. We aim to investigate how direct covalent engagement of the immune receptor and elimination the antibody-immune receptor binding equilibria impacts immune activation and therapeutic efficacy. This thesis focuses on the chemical programming of macrophages through direct covalent immune receptor engagement. We have developed and characterized covalent immune programmers (CIPs), which are molecules that contain a macrophage targeting domain and a tumour targeting domain. The macrophage targeting domain binds the activating receptor CD64 on the macrophage surface and contains a chemical warhead that covalently labels the receptor once bound. The tumour targeting domain can promote macrophage tumour engagement resulting in tumoricidal function. Flow cytometry experiments have shown that CIPS are able to bind Fc receptors specifically and effectively on the surface of macrophages. Further, CIPs were able to induce macrophage activation and induce target specific phagocytosis. These experiments have also shown that direct engagement of the receptor by the CIP is more effective than antibody-mediated engagement, suggesting that overall immune complex stability affects immune cell activation. Taken together, these concepts can be used to guide future immunotherapeutic design. / Thesis / Master of Science (MSc)