Doctor of Philosophy, The Ohio State University, 2020, Chemistry

Cancer is one of the leading causes of death worldwide. The number of patients has been rapidly growing with 18.1 million new cases and 9.6 million deaths in 2018 alone. Part of the problem is that the targeting of current therapeutics against cancers is not adequate. Monoclonal antibodies (mAbs) have great potential for use in therapeutics with their strong affinity to target and have become one of the most successful strategies for cancer treatment in the last 20 years. However, mAbs often show immunogenic responses when injected into patients limiting their utility for therapeutic applications. In addition, their large and complex structure makes them difficult to study. This has led to a discovery of single-chain variable fragments (scFvs), the smallest functional unit of mAbs. Its small size allows bacterial expression making engineering easy, and further gains several pharmacokinetic advantages such as better tumor penetration, and reduced immunogenicity. Most importantly, the engineered scFvs can be easily put into different modalities such as mAb, ADC, multispecifics, and many others to tune its half-life and functions. However, scFvs have not made it into the clinic partly due to a decrease in stability and affinity. Despite the importance of such features in therapeutics, there has not been much study done on the compositional effect of the loop regions. There are two different kinds of loops in scFvs; a connecting loop (linker) for stability, and six CDR loops for affinity. In order to overcome the challenges with scFvs, the composition of these loops was explored to find factors contributing to their biophysical characteristics and ultimately to engineer the antibody 3E8 as cancer therapeutics with enhanced/novel properties. The model scFv, 3E8, has a high affinity to TAG-72, which is present in most adenocarcinomas but in healthy human tissue, and therefore makes it a great candidate for cancer therapeutics. Nearly all scFvs have been e (open full item for complete abstract)

Committee: Thomas Magliery (Advisor); Dalbey Ross (Committee Member); Jaroniec Christopher (Committee Member) Subjects: Biochemistry; Biomedical Research; Biophysics; Immunology; Medicine; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2018, Biochemistry Program, Ohio State

Proteins play a major role in virtually every biological process. Thus, proteins are an ideal platform for the next generation of therapeutics. Over the last few decades, technological and scientific advances in protein production and engineering have led to a new wave of protein-based biologics used in clinical settings. In this body of work, we have engineered both a protein-based cancer diagnostic and an immunotherapeutic. Antibody-based biologics are becoming one of the most widely approved drug platforms and owe their success to their versatility in binding targets, high stability, and low toxicity. The anti-TAG-72 cancer-targeting antibody, 3E8, is one such molecule that shows great potential as a diagnostic. We have designed and biophysically characterized a library of 3E8 single chain antibody fragments (scFV) with varying linker composition and length as well as domain orientations. In this library, we have found substantial variation in protein stability, binding affinity, and oligomeric states. Surprisingly, a drastic difference in the oligomeric state of these constructs was seen between conventional IMAC purification and Protein L purification. Therefore, the literature rules for scFV linker design must be updated to include the dependencies on purification method. A single antibody construct with optimal biophysical properties (3E8.G4S) was further characterized and subjected to in vivo pharmacokinetic studies. Due to its multimeric composition, 3E8.G4S showed a longer and more favorable clearance time compared to that of a fast clearing scFV. Xenograft mouse imaging and biodistribution studies revealed successful targeting of a colorectal tumor by 3E8.G4S with little accumulation in normal tissues. To determine the versatility of 3E8-based diagnostics and therapeutic agents, an expansive immunohistochemical analysis of TAG-72 expression was performed in over 1,500 tumors spanning 18 different cancer types. The results of this study showed enh (open full item for complete abstract)

Committee: Thomas Magliery (Advisor); Christopher Jaroniec (Committee Member); Edward Martin Jr. (Committee Member); Richard Swenson (Committee Member) Subjects: Biochemistry; Biomedical Research; Biophysics; Immunology; Medicine; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2010, Biomedical Engineering

ImmunoFET technology for protein sensing in highly ionic buffer solutions was long deemed infeasible. However, our research asserts that this is both theoretically and experimentally incorrect. In the theoretical assessment, the biochemical properties suggested in the erroneous model are not in accordance with established knowledge of antibody structure and function. Much is known about the biochemistry of antibodies and antibody fragments that are antithetical to the description of these biomolecules in previous immunoFET feasibility arguments. We use knowledge from years of immunological research to support our assertions and the experimental data supports this as well. Empirical evidence suggests that protein detection at physiological salt concentrations is not only plausible, but also indeed possible, with an immunoFET device. Detection of the monokine induced by interferon gamma (MIG) in physiologic salt conditions using an immunoFET device has vast implications for transplant medicine, specifically in allograft rejection. The optimization of a MIG sensing immunoFET has vast implications for immunoFET sensors in general and the application of a MIG detecting device to clinical problems. MIG is an early marker for transplant rejection and has great potential in preventing catastrophic graft failure. In this work, we highlight several aspects of the immunoFET device that can be optimized to improve device performance. ImmunoFETs can be optimized in the area of the device itself, the thin surface film, and the receptor. Each area has several subsections that are important in determining the best method to increase device sensitivity. The application of nanobiotechnology, namely protein engineering and interfacial design, is paramount to the implementation of such optimization strategies. This research looks to provide empirical and theoretical evidence to support immunoFET feasibility and elucidate strategies to improve the application of immunoFET technology.

Committee: Stephen Lee PhD (Advisor); Mark Ruegsegger PhD (Committee Member); Derek Hansford PhD (Committee Member); Jessica Winter PhD (Committee Member) Subjects: Biomedical Engineering; Biomedical Research