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Programmed cell death ligand-1 (PD-L1) is an overexpressed protein on many tumor cell types. PD-L1 is involved in normal immune regulation, playing an important role in self-tolerance and controlling autoimmunity. However, ligation of PD-L1 to PD-1 on activated T cells leads to tumor-mediated T cell suppression. Inhibiting the PD-1/PD-L1 pathway has emerged as an effective target for anti-tumor immunotherapies. Monoclonal antibodies (mAbs) targeting tumor-associated antigens such as PD-L1 have proven to be effective checkpoint blockades, improving therapeutic outcomes for cancer patients and receiving FDA approval as first line therapies for some cancers. A single chain variable fragment (scFv) is composed of the variable heavy and light chain regions of a mAb, connected by a flexible linker. We hypothesized that scFv proteins based on the published anti-PD-L1 monoclonal antibody sequences of atezolizumab and avelumab would bind to cell surface PD-L1. Four single chain variable fragments (scFvs) were constructed based on the sequences of these mAbs. PCR was used to assemble, construct, and amplify DNA fragments encoding the scFvs which were subsequently ligated into a eukaryotic expression vector. Mammalian cells were transfected with the scFv and scFv-IgG plasmids. The scFvs were tested for binding to PD-L1 on tumor cell lysates by western blot and to whole tumor cells by staining and flow cytometry analysis. DNA sequence analysis demonstrated that the scFv constructs were successfully amplified and cloned into the expression vectors and recombinant scFvs were produced. The binding capabilities of the scFvs constucts to PD-L1 protein were confirmed by western blot and flow cytometry analysis. This lead to the idea of constructing a CAR T cell engineered to target PD-L1, providing a possible adoptive T cell immunotherapy.
ContributorsPfeffer, Kirsten M. (Author) / Lake, Douglas (Thesis director) / Ho, Thai (Committee member) / Hastings, Karen (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Quiescin sulfhydryl oxidase 1 (QSOX1) is an enzyme that catalyzes disulfide bond formation by oxidizing two free sulfhydryl groups. QSOX1 consists of a thioredoxin (Trx) and an ERV (essential for respiration and viability)/ALR (augmenter of liver regeneration) domain which each contain CxxC motifs that work to bind to substrates and shuttle electrons to a flavin adenine dinucleotide (FAD) cofactor that accepts the electrons and reduces molecular oxygen to hydrogen peroxide. Investigation of the role of QSOX1 in cancer progression started when it was found at higher abundance in pancreatic ductal adenocarcinoma (PDA) patient plasma compared to healthy normal donor plasma. Increased expression in QSOX1 has been further identified in breast, lung, kidney, prostate, and other cancers. QSOX1 expression is associated with cell proliferation and invasion in vitro and tumor growth in vivo. Additionally, the enzymatic activity of QSOX1 in the extracellular matrix (ECM) is important for cell invasion in vitro. Small molecule inhibitors of QSOX1 have been shown to have antitumorigenic properties in vitro and in vivo. It was hypothesized that monoclonal antibodies (mAbs) against QSOX1 would inhibit cell invasion in vitro. In this work, mice were immunized with eukaryotic-derived rQSOX1 for generation of hybridomas. Hundreds of hybridoma clones were screened by enzyme-linked immunosorbent assay (ELISA) and a fluorescent QSOX1 activity assay. Multiple rounds of subcloning and screening identified 2F1.14 and 3A10.6 as mAbs of interest. The genes for the variable regions of the antibodies were rescued and sequenced. The sequences were aligned with the variable region sequences of another published αQSOX1 mAb scFv492.1. 2F1.14 inhibits the enzymatic activity of QSOX1 by binding to the active site of QSOX1, which was determined by epitope mapping against mutants of QSOX1 that contained mutations in the active site. 3A10.6 did not appear to inhibit the function of QSOX1 in the activity assay; however, it, along with 2F1.14, suppressed tumor invasion in a 3D invasion model. These findings support the developing idea that QSOX1 is a viable target for cancer treatment because targeted inhibition of QSOX1 extracellularly reduced invasive activity. The mAbs and rQSOX1 variants produced here can serve as tools in furthering the characterization of QSOX1 and its role in cancer.
ContributorsKoelbel, Calvin John (Author) / Lake, Douglas (Thesis advisor) / Chen, Qiang "Shawn" (Committee member) / Ho, Thai (Committee member) / Arizona State University (Publisher)
Created2019
Description
Coccidioidomycosis (valley fever) is caused by inhalation of arthrospores from soil-dwelling fungi, Coccidioides immitis and C. posadasii. This dimorphic fungus and disease are endemic to the southwestern United States, central valley in California and Mexico. The Genome of Coccidioidies has been sequenced but proteomic studies are absent. To address this gap in knowledge, we generated proteome of Spherulin (lysate of Spherule phase) using LC-MS/MS and identified over 1300 proteins. We also investigated lectin reactivity to spherules in human lung tissue based on the hypothesis that coccidioidal glycosylation is different from mammalian glycosylation, and therefore certain lectins would have differential binding properties to fungal glycoproteins. Lectin-based immunohistochemistry using formalin-fixed paraffin-embedded human lung tissue from human coccidioidomycosis patients demonstrated that Griffonia simplificonia lectin II (GSL II) and succinylated wheat germ agglutinin (sWGA) bound specifically to endospores and spherules in infected lungs, but not to adjacent human tissue. GSL II and sWGA-lectin affinity chromatography using Spherulin, followed by LC-MS/MS was used to isolate and identify 195 proteins that bind to GSL-II lectin and 224 proteins that bind to sWGA lectin. This is the first report that GSL II and sWGA lectins bind specifically to Coccidioides endospores and spherules in infected human tissues. Our list of proteins from spherulin (whole and GSL-II and sWGA binding fraction) may also serve as a Coccidioidal Rosetta-Stone generated from mass spectra to identify proteins from 3 different databases: The Broad Institutes Coccidioides Genomes project, RefSeq and SwissProt. This also serves as a viable avenue for proteomics based diagnostic test development for valley fever. Using lectin chromatography and LC MS/MS, we identified over 100 proteins in plasma of two patients and six proteins in urine of one patient. We also identified over eighty fungal proteins isolated from spherules from biopsied infected lung tissue. This, to the best of our knowledge, is the first such example of detecting coccidioidal proteins in patient blood and urine and provides a foundation for development of a proteomics based diagnostic test as opposed to presently available but unreliable serologic diagnostic tests reliant on an antibody response in the host.
ContributorsKaushal, Setu (Author) / Lake, Douglas (Thesis advisor) / Magee, Dewey Mitchell (Committee member) / Chandler, Douglas (Committee member) / Rawls, Jeffery (Committee member) / Arizona State University (Publisher)
Created2015
Description
Duchenne muscular dystrophy (DMD) is a lethal, X-linked disease characterized by progressive muscle degeneration. The condition is driven by out-of-frame mutations in the dystrophin gene, and the absence of a functional dystrophin protein ultimately leads to instability of the sarcolemma, skeletal muscle necrosis, and atrophy. While the structural changes that occur in dystrophic muscle are well characterized, resulting changes in muscle-specific gene expression that take place in dystrophin’s absence remain largely uncharacterized, as they are potentially obscured by the characteristic chronic inflammation in dystrophin deficient muscle.
The conservation of the dystrophin gene across metazoans suggests that both vertebrate and invertebrate model systems can provide valuable contributions to the understanding of DMD initiation and progression. Specifically, the invertebrate C. elegans possesses a dystrophin protein ortholog, dys-1, and a mild inflammatory response that is inactive in the muscle, allowing for the characterization of transcriptome rearrangements affecting disease progression independently of inflammation. Furthermore, C. elegans do not possess a satellite cell equivalent, meaning muscle regeneration does not occur. This makes C. elegans unique in that they allow for the study of dystrophin deficiencies without muscle regeneration that may obscure detection of subtle but consequential changes in gene expression.
I hypothesize that gaining a comprehensive definition of both the structural and signaling roles of dystrophin in C. elegans will improve the community’s understanding of the progression of DMD as a whole. To address this hypothesis, I have performed a phylogenetic analysis on the conservation of each member of the dystrophin associated protein complex (DAPC) across 10 species, established an in vivo system to identify muscle-specific changes in gene expression in the dystrophin-deficient C. elegans, and performed a functional analysis to test the biological significance of changes in gene expression identified in my sequencing results. The results from this study indicate that in C. elegans, dystrophin may have a signaling role early in development, and its absence may activate compensatory mechanisms that counteract disease progression. Furthermore, these findings allow for the identification of transcriptome changes that potentially serve as both independent drivers of disease and potential therapeutic targets for the treatment of DMD.
The conservation of the dystrophin gene across metazoans suggests that both vertebrate and invertebrate model systems can provide valuable contributions to the understanding of DMD initiation and progression. Specifically, the invertebrate C. elegans possesses a dystrophin protein ortholog, dys-1, and a mild inflammatory response that is inactive in the muscle, allowing for the characterization of transcriptome rearrangements affecting disease progression independently of inflammation. Furthermore, C. elegans do not possess a satellite cell equivalent, meaning muscle regeneration does not occur. This makes C. elegans unique in that they allow for the study of dystrophin deficiencies without muscle regeneration that may obscure detection of subtle but consequential changes in gene expression.
I hypothesize that gaining a comprehensive definition of both the structural and signaling roles of dystrophin in C. elegans will improve the community’s understanding of the progression of DMD as a whole. To address this hypothesis, I have performed a phylogenetic analysis on the conservation of each member of the dystrophin associated protein complex (DAPC) across 10 species, established an in vivo system to identify muscle-specific changes in gene expression in the dystrophin-deficient C. elegans, and performed a functional analysis to test the biological significance of changes in gene expression identified in my sequencing results. The results from this study indicate that in C. elegans, dystrophin may have a signaling role early in development, and its absence may activate compensatory mechanisms that counteract disease progression. Furthermore, these findings allow for the identification of transcriptome changes that potentially serve as both independent drivers of disease and potential therapeutic targets for the treatment of DMD.
ContributorsHrach, Heather (Author) / Mangone, Marco (Thesis advisor) / LaBaer, Joshua (Committee member) / Newbern, Jason (Committee member) / Rawls, Jeffery (Committee member) / Arizona State University (Publisher)
Created2020
Description
Quiescin Sulfhydryl Oxidase 1 (QSOX1) generates disulfide bonds in its client substrates via oxidation of free thiols. Localized to the Golgi and secreted, QSOX1 helps to fold proteins into their active form. Early work with QSOX1 in cancer began with the identification of a peptide from the long form of QSOX1 in plasma from patients with pancreatic ductal adenocarcinoma. Subsequent work confirmed the overexpression of QSOX1 in numerous cancers in addition to pancreatic, including those originating in the breast, lung, brain, and kidney. For my work, I decided to answer the question, “How does inhibition of QSOX1 effect the cancer phenotype?” To answer this I sought to fulfill the following goals A) determine the overexpression parameters of QSOX1 in cancer, B) identify QSOX1 small molecule inhibitors and their effect on the cancer phenotype, and C) determine potential biological effects of QSOX1 in cancer. Antibodies raised against rQSOX1 or a peptide from QSOX1-L were used to probe cancer cells of various origins for QSOX1 expression. High-throughput screening was utilized to identify 3-methoxy-n-[4(1pyrrolidinyl)phenyl]benzamide (SBI-183) as a lead inhibitor of QSOX1 enzymatic activity. Characterization of SBI-183 activity on various tumor cell lines revealed inhibition of viability and invasion in vitro, and inhibition of growth, invasion, and metastasis in vivo, a phenotype that was consistent with QSOX1 shKnockdown cells. Subsequent work identified 3,4,5-trimethoxy-N-[4-(1-pyrrolidinyl)phenyl]benzamide (SPX-009) as an SBI-183 analog with stronger inhibition of QSOX1 enzymatic activity, resulting in a more potent reduction in tumor invasion in vitro. Additional work with QSOX1 shKnockdown and Knockout (KO) cell lines confirmed current literature that QSOX1 is biologically active in modulation of the ECM. These results provide evidence for the master regulatory role of QSOX1 in cancer, making it an attractive chemotherapeutic target. Additionally, the small molecules identified here may prove to be useful probes in further elucidation of QSOX1 tumor biology and biomarker discovery.
ContributorsFifield, Amber (Author) / Lake, Douglas (Thesis advisor) / Ho, Thai (Committee member) / Rawls, Jeffery (Committee member) / Borges, Chad (Committee member) / Arizona State University (Publisher)
Created2020