Engineering Protein Scaffolds as Programmable Cancer Therapeutics

Kimmel, Blaise Russel

doi:https://doi.org/10.21985/n2-0xzx-2w59

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Engineering Protein Scaffolds as Programmable Cancer Therapeutics

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Biological therapeutics have revolutionized the way we treat cancer due to their ability to target tumors discriminately, leaving healthy cells unaffected. However, our inability to tailor the structure of biologics may hamper their optimization for efficacy. This lack of programmability contributes to factors such as immunogenic responses, inability to penetrate solid-tumors, and high dosage requirements. Our lab has developed a protein assembly method known as the ‘megamolecule’ approach that can address this limitation (Chapter 1) The megamolecule strategy provides precise control over the synthesis of protein scaffolds and enables the development of programmable therapeutics. Megamolecules represent next generation therapies whose structure can be tailored for optimum activity through controlling scaffold binding specificity, orientation, and protein stoichiometry. This dissertation introduces a new generation of the megamolecule assembly strategy through the development of a solid-phase megamolecule synthesis approach, the construction of programmable therapeutic megamolecules, expanding the number of orthogonal enzyme-inhibitor pairs for reactions, and the expansion of this synthesis strategy to construct tunable, dendritic scaffolds.First, this dissertation advances the megamolecule synthesis strategy through the development of a solid-phase strategy to efficiently assemble multi-protein scaffolds – known as megamolecules – without the need for protecting groups and with precisely-defined nanoscale architectures (Chapter 2). The megamolecules are assembled through sequential reactions of linkers that present irreversible inhibitors for enzymes and fusion proteins containing the enzyme domains. Here, a fusion protein containing an N’-terminal cutinase and a C’-terminal SnapTag domain react with an ethyl p-nitrophenyl phosphonate (pNPP) or a chloro-pyrimidine (CP) group, respectively, to give covalent products. Functionalized resin is leveraged in a series of high yielding reactions to form linear, branched, and dendritic protein scaffolds that are proteolytically released to give atomically precise, homogeneous scaffolds. The first demonstration of megamolecules as tunable therapeutics is detailed in Chapter 3 with applications in breast cancer research. Systemic chemotherapy delivers cytotoxic drugs to both cancer and non-cancer cells, leading to severe side-effects. Local intratumoral activation of a non-toxic prodrug increases the concentration of chemotherapeutic in the diseased tissue. To achieve local drug activation, we covalently assembled an antibody and enzyme to localize the drug-activating enzyme to cancer cells. We used the megamolecule approach to join the nanobody and enzyme proteins to create homogeneous megamolecules with variable numbers of nanobody and enzyme domains. Increasing the number of enzyme domains increased therapeutic efficacy. Megamolecules enable synthesis of homogeneous crosslinked proteins with enhanced activity through control of the number of protein domains. Chapter 4 focuses on the development of an enzyme-inhibitor chemistry using cellular retinoic acid binding protein II (CRABP2) for megamolecule assembly featuring one-pot reaction mixtures. I developed and optimized a 10-step convergent synthesis of a targeted covalent inhibitor that uses sulfur fluoride exchange click chemistry (SuFEx) to selectively inhibit the nucleophilic tyrosine residue in the active site of the monomeric CRABP2 domain. Further, this chapter highlights the integration of this enzyme-inhibitor pair into our assembly strategy to demonstrate exact control over the simultaneous placement of protein domains within several, unique homogeneous branched molecules. Monitoring the reaction progress over time allowed for modeling the reaction pathways and mechanism that formed the protein scaffold in the one-pot assembly mixture. The one-pot proteins scaffolds synthesized in this chapter were formed from up to six independent reactions using four molecules as starting reagents and were synthesized in mild conditions without the use of protecting groups. Chapter 5 first explores an evaluation of HaloTag as a surface chemistry and is followed by the optimization of inhibition reaction kinetics for protein scaffold synthesis. Next, the molecular toolbox is expanded through the synthesis of new chemical linkers and cloning of a fusion protein library for molecular targeting, fluorescent microscopy, and site-directed enzyme conversion. Finally, new megamolecule constructs are generated with the ability to create atomically-precise protein dendrimers with hypervalent targeting of cancerous tissue. In Chapter 6, I reflect on the work completed throughout this dissertation, including final insights, and potential research endeavors to advance the synthesis of programmable megamolecule therapeutics. One specific idea that arises from this dissertation involves marrying computational and experimental approaches to design megamolecule treatments for antibody-directed therapies in a microfluidic platform, known as a ‘body-on-a-chip’. This work could mimic a human vascular system by culturing solid tumors in microfluidic channels with synthetic blood will allow for the investigation of tumor conditions when influenced by the introduction of various chemical messengers and external drugs. Finally, several bodies of published and unpublished work that do not directly contribute to this dissertation, but were completed during the course of the PhD, are included in the Appendix chapters.

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