Browsing by Subject "Enzyme engineering"

Now showing 1 - 5 of 5

Engineering a novel human methionine degrading enzyme as a broadly effective cancer therapeutic
(2014-08) Paley, Olga M.; Georgiou, George; Iverson, Brent; Alper, Hal S; Maynard, Jennifer; Johnson, Kenneth A
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.
Engineering and validation of a human cyst(e)ine degrading enzyme as a novel cancer therapeutic
(2017-05) Cramer, Shira Lyla; Georgiou, George; Stone, Everett; Alper, Hal; Maynard, Jennifer; Ehrlich, Lauren; DiGiovanni, John
Cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than do non-malignant cells because of genetic alterations and abnormal growth; as a result, maintenance of the antioxidant glutathione (GSH) is essential for their survival and proliferation. As a precursor for the biosynthesis of GSH, L-cysteine (L-Cys) availability is critical for maintaining the intracellular thiol redox potential and under conditions of elevated ROS, endogenous L-Cys production is insufficient for GSH synthesis. This necessitates the import of extracellular L-cyst(e)ine (predominantly in its disulfide form, L-cystine (CSSC)) to meet cellular antioxidant requirements. Since L-Cys is a non-essential amino acid in animals, eliminating L-Cys and CSSC uptake should selectively impact tumors that display increased ROS production and thus exhibit a higher demand for antioxidants, without causing an adverse effect on normal physiology. This can be accomplished by eliminating the extracellular pool of L-Cys and CSSC through the action of an enzyme that selectively converts these amino acids into non-toxic products. Unfortunately, no human enzyme displays sufficient catalytic properties towards both L-Cys and CSSC to be relevant for clinical applications. In the chapters that follow, we describe the engineering of a human L-Cys and CSSC degrading enzyme (cyst(e)inase) as a novel and potent therapeutic for tumors displaying elevated levels of ROS. We show that administration of cyst(e)inase mediates sustained depletion of the extracellular L-Cys and CSSC pool in mice and non-human primates at a therapeutically useful rate. In a wide variety of models, treatment with this enzyme suppresses tumor growth in mice, yet results in no apparent toxicities even after months of continuous treatment. Through additional engineering, we describe the isolation of novel enzymes that display the requisites for clinical development; including increases in soluble protein yields and catalytic activity towards both L-Cys and CSSC, which together translates to practical doses for a human therapeutic at reasonable manufacturing costs. Finally, upon further investigation into the therapeutic effect of cyst(e)inase, we show that cyst(e)inase treatment dramatically inhibits metastasis, as well as suggesting an important role of ROS regulation and the immune system in a syngeneic mouse model.
Enzymatic depletion of L-Met using an engineered human enzyme as a novel therapeutic strategy for melanoma
(2022-08-11) Wilder, Carly Strecker; DiGiovanni, John; Vasquez, Karen; Kidane, Dawit; Tiziani, Stefano; Georgiou, George
Metastatic melanoma is an aggressive form of cancer responsible for the majority of skin cancer related deaths. While treatment for metastatic melanoma has improved in recent years with the introduction of targeted therapies and immunotherapies, the five year survival rate for stage IV melanoma remains only 15-20%. To address the need for alternative options for melanoma treatment, we have evaluated the use of an engineered human enzyme called methionine-ɣ-lyase (hMGL). Many cancers including melanoma, have a high requirement for L-methionine (L-Met) in comparison with non-cancerous cells. The hMGL enzyme exploits this metabolic vulnerability by degrading extracellular L-Met resulting in cancer cell starvation. The goal of this project was to assess the efficacy and identify mechanisms of action of the hMGL enzyme in melanoma models. In vitro and in vivo methods were used to evaluate the efficacy of L-Met depletion using hMGL on melanoma skin cancer. Four melanoma cancer cell lines were used, three were derived from human melanomas while one was a commonly utilized mouse melanoma line. Cell viability, cell cycle, and cell death parameters were evaluated first to establish that melanoma is sensitive to hMGL treatment. Global omics data sets including RNA-seq and metabolomics were generated from in vitro samples to give some insight into potential mechanisms of action to be investigated further. Since L-Met is involved in many cellular processes, it is not surprising that multiple mechanisms were found to be perturbed with hMGL treatment. Upregulation of the uncharged tRNA amino acid sensing pathway and an increase in DNA replication stress and ROS were observed with hMGL treatment. Drugs to be used in combination with hMGL were identified based on mechanistic relevance and screened in vitro. Treatment with hMGL inhibited tumor growth in both human xenograft and mouse allograft orthotopic melanoma models. The results of this study provide rationale for further mechanistic evaluation and clinical development of hMGL for the treatment of melanoma skin cancer.
Enzyme-mediated methylthioadenosine depletion as a novel immune checkpoint therapy
(2021-05) Gjuka, Donjeta; Georgiou, George; Stone, Everett Monroe, 1971-; Maynard, Jennifer; Jiang, Ning; Ehrlich, Lauren
Methylthioadenosine phosphorylase (MTAP) is an enzyme that is homozygously deleted in approximately 15% of all human cancer types. MTAP deletion has been correlated with cancer progression, poor patient survival, and resistance to immune checkpoint therapies. Frequent MTAP loss in cancer leads to the accumulation of its substrate 5’-deoxy-5’-methylthioadenosine (MTA), which exerts potent immunosuppressive effects in T cells. We hypothesize that accumulated MTA in the tumor microenvironment of MTAP-deficient tumors induces an immunomodulatory effect on surrounding T cells and engenders tumor tolerance. In this dissertation, we explore the therapeutic efficacy of enzyme-mediated MTA depletion in several MTAP-deficient tumor models. Our results indicate that treating MTAP-deficient tumors with MTA-degrading enzymes can drastically suppress tumor growth. Moreover, our in vivo studies and immunophenotyping experiments suggest that the therapeutic benefit of MTA-depletion is mediated by CD8 T cells that infiltrate the tumor microenvironment. Additionally, we tested the combined therapeutic effects of MTA depletion with immune checkpoint therapy (e.g., anti-PD1 or anti-CTLA4) and observed a potent synergistic effect. Furthermore, we investigate MTA’s mechanism of action on T cells. Due to MTA’s structural similarities with adenosine, we examined putative signaling of MTA through adenosine receptors. Our findings show that adenosine signaling is not the main pathway that MTA utilizes to suppress T cells. Instead, our proteomics analysis of T cells incubated with MTA suggest that MTA inhibits T cells, at least in part, via the differential expression of methyltransferase/demethylase enzymes, upregulation of proapoptotic proteins, and inhibition of proteins crucial for TCR activation and cytokine signaling. In this work, we also discuss the engineering and optimization of the MTAP enzyme for therapeutic use. Collectively, this dissertation elucidates the suppression mechanism of MTA on T cells and demonstrates the potential of utilizing MTA-depletion therapy as a biomarker-driven (namely, MTAP status) immunotherapeutic modality.
Novel high-throughput screening methods for the engineering of hydrolases
(2011-05) Gebhard, Mark Christopher; Georgiou, George; Alper, Hal; Ellington, Andrew D.; Iverson, Brent L.; Maynard, Jennifer A.
Enzyme engineering relies on changes in the amino acid sequence of an enzyme to give rise to improvements in catalytic activity, substrate specificity, thermostability, and enantioselectivity. However, beneficial amino acid substitutions in proteins are difficult to rationally predict. Large numbers of enzyme variants containing random amino acid substitutions are screened in a high throughput manner to isolate improved enzymes. Identifying improved enzymes from the resulting library of randomized variants is a current challenge in protein engineering. This work focuses on the development of high-throughput screens for a class of enzymes called hydrolases, and in particular, proteases and esterases. In the first part of this work, we have developed an assay for detecting protease activity in the cytoplasm of Escherichia coli by exploiting the SsrA protein degradation pathway and flow cytometry. In this method, a protease-cleavable linker is inserted between a fusion protein consisting of GFP and the SsrA degradation tag. The SsrA-tagged fusion protein is degraded in the cell unless a co-expressed protease cleaves the linker conferring higher cellular fluorescence. The assay can detect specific cleavage of substrates by TEV protease and human caspase-8. To apply the screen for protease engineering, we sought to evolve a TEV protease variant that has altered P1 specificity. However, in screening enzyme libraries, the clones we recovered were found to be false positives in that they did not express protease variants with the requisite specificities. These experiments provided valuable information on physiological and chemical parameters that can be employed to optimize the screen for directed evolution of novel protease activities. In the second part of this work, single bacterial cells, expressing an esterase in the periplasm, were compartmentalized in aqueous droplets of a water-in-oil emulsion also containing a fluorogenic ester substrate. The primary water-in-oil emulsion was then re-emulsified to form a water-in-oil-in-water double emulsion which was capable of being analyzed and sorted by flow cytometry. This method was used to enrich cells expressing an esterase with activity towards fluorescein dibutyrate from an excess of cells expressing an esterase with no activity. A 50-fold enrichment was achieved in one round of sorting, demonstrating the potential of this method for use as a high-throughput screen for esterase activity. This method is suitable for engineering esterases with novel catalytic specificities or higher stabilit