Many cancers have long been known to display an absolute requirement for the amino acid methionine (L-Met). Studies have shown that in the absence of L-Met, sensitive neoplasms experience cell cycle arrest and perish. Without the metabolic deviations that characterize L-Met auxotrophs, normal cells are able to grow on precursors such as homocysteine and tolerate periods of L-Met starvation. The differential requirement for this amino acid between normal and tumor cells has been exploited through enzymatic serum degradation of L-Met by a bacterial methionine-γ-lyase (MGL). Though MGL was able to deplete L-Met to therapeutically useful levels in animal models and exert a significant cytotoxic effect on malignant cell lines in vitro and on tumor xenografts in vivo, the clinical implementation of this enzyme is hampered by its short serum half-life and potential for catastrophic immune response. In the chapters that follow, we describe the engineering of a novel human methionine degrading enzyme (hMGL) that overcomes the limitations of the bacterial therapeutic. We have shown that hMGL is capable of degrading methionine at a therapeutically useful rate and inducing extensive cell killing in a variety of neoplasms. This enzyme is expected to have low immunogenicity in patients and a high therapeutic index. We have developed a high throughput screen for methionine degrading activity that we can utilize to further engineer the enzyme based on the results of additional preclinical development. We have found that hMGL is also capable of degrading cystine to operate as a dual amino acid depletion treatment that is expected to be more potent than methionine depletion alone. Due to the wide array of neoplasms sensitive to methionine and cystine starvation, the engineered enzyme holds a great deal of promise as a unique and powerful cancer therapeutic. / text

Affibody molecules are small protein scaffolds that have been engineered to bind to a variety of targets with diversetherapeutic and diagnostic applications. In this study, an array of affibody containing therapeutic constructs,targeting HER2 and HER3, and diagnostic anti-HER3 imaging agents have been purified in preparation for subsequentcancer cell assays and imaging studies in tumour-bearing mice respectively. Herein, the workflow for severalpurification techniques is delineated.

Recombinant proteins have many medical and industrial applications, but their use is complicated by commercial production and stability constraints. These issues are particularly challenging for recombinant proteins used in pharmaceutical therapeutics and clinical diagnostics. Expensive production and distribution limit the accessibility of therapeutics and diagnostics especially in the developing world. Additionally, clinical use of recombinant proteins face further challenges within biological systems including biological degradation and immunogenicity. To increase the accessibility of recombinant proteins, the cost and inefficiencies of protein manufacturing and distribution need to be significantly reduced. A powerful tool to aid in this endeavor is cell-free protein synthesis (CFPS) technology. CFPS is a versatile platform for recombinant protein production due to its open reaction environment, flexible reaction conditions, and rapid protein expression capabilities. These avoid the disadvantages of conventional manufacturing and present the capability of on-demand protein therapeutic production outside of centralized facilities. To improve the efficacy of recombinant proteins for medicinal use, protein engineering techniques such as PEGylation, or the conjugation of PEG polymers to protein surfaces, can be employed. PEGylation is widely used to enhance the pharmacokinetic properties of protein therapeutics. Deciphering optimal PEG conjugation sites is a continuing area of research that can be facilitated by CFPS systems that enable high-throughput, site-specific PEGylation. This dissertation presents advances in CFPS technology to promote increased accessibility and stability of life-saving therapeutics and diagnostics. The work presented here (1) improves on-demand therapeutic production capabilities by creating shelf-stable, endotoxin-free CFPS systems, (2) aids the rational design of next-generation PEGylated protein therapeutics through an in silico-in vitro CFPS screening platform, and (3) advances the development of portable clinical diagnostics for rapid and sustainable deployment at point-of-care through CFPS biosensor technology. The innovations of this dissertation are described in four publications. Specifically, an endotoxin-free CFPS system lyophilized with lyoprotectants is demonstrated that shows improved shelf-stability over standard lyophilized systems. A streamlined procedure for preparing endotoxin-free extract using auto-induction media is presented that significantly reduces CFPS preparation labor and time. A combinatorial screening approach is demonstrated in which coarse-grain molecular simulation informs PEGylation site selection as verified by CFPS experimental results. An inexpensive paper-based, saliva-activated CFPS biosensor platform is developed for the detection of SARS-CoV-2 sequences.

Hepatocellular carcinoma (HCC) is the third commonest cancer worldwide. However HCC is considered to be highly resistant to chemotherapy. Gene therapies aimed to regulate Bd-2 proteins may sensitize HCC cells to chemotherapy. Studies have demonstrated that Bid/tBid are crucial in hepatocyte apoptosis. Bid also plays important roles in the development and chemotherapeutic sensitivity of HCC. The objective of this study is to test effect of Ad/AFPtBid and chemotherapeutic agents in combination on an orthotopic HCC model. / In conclusion, (1) Ad/AFPtBid can specifically target and effectively suppress the AFP-producing HCC. (2) Ad/AFPtBid can significantly sensitize HCC to 5-FU, their combination can significantly increase the anti-tumor effectiveness. (3) Ad/AFPtBid shows little toxicity in vivo. (4) The complementary effect of tBid and 5-FU on different phases of the cell cycle may explain the better therapeutic result if both are used to treat HCC. (5) The elucidation of phase specific effect of tBid points to a possible therapeutic option that combines tBid with different phase specific agents to treat HCC. / It is well established that many apoptosis inducers act in a cell cycle-specific fashion. This leads us to hypothesize that tBid might have phase specific effect. So, we tested the susceptibility of Hep3B cells at 00/01, S or G2/M phases to tBid. The results revealed that tBid significantly reduced Hep3B cells in G0/G1 phase, increased cells in G2/M phase. On the contrary, 5-FU arrested Hep3B cells in G0/G1 phase, and significantly reduced cells in G2/M phase. The levels of cell cycle-related proteins were altered in line with the result of the cell cycle. This suggests Hep3B cells in G0/G1 phase may be more susceptible to tBid. The complementary effects tBid and 5-FU on different phases of the cell cycle may explain the better therapeutic result if both are used to treat HCC. / The mice bearing orthotopic HCC tumors were treated with Ad/AFPtBid alone or in combination with 5-FU/Dox. Serum AFP levels were measured to mornitor tumor progression. The mice were killed four weeks after treatment. Liver tissues were subjected to immunohistochemical staining of proliferation cell nuclear antigen (PCNA) and TUNEL staining. Another batch of mice was observed for survival rate over a six month period. In addition, possible side effects of Ad/AFPtBid were tested in BALB/c mice. Results demonstrated that Ad/AFPtBid significantly inhibited Hep3B tumor growth. The combination of Ad/AFPtBid with 5-FU was more effective in tumor regression than either agent alone. However, the combination of Ad/AFPtBid with Dox treatment failed to demonstrated better effect than Dox treatment alone because the mice that received Dox exhibited serious weight loss. Tumor tissues from Ad/AFPtBid alone or combination treatment groups showed a decrease in cells positive for PCNA, and an increase in apoptosis by TUNEL staining, indicating that Ad/AFPtBid induced tumor regression through its pro-apoptotic effect. Inflammatory cell infiltration was also increased. Furthermore, Ad/AFPtBid did not suppress the hepatic tumor formed by non-AFP producing SK-HEP-1 or DLD-1. Finally, Ad/AFPtBid and 5-FU in combination results in better survival rate. No acute toxic effect of Ad/AFPtBid was observed. / Ma, Shihong. / "December 2009." / Adviser: CHEN Gong George. / Source: Dissertation Abstracts International, Volume: 72-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 114-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.