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- All Subjects: Biology
- All Subjects: immunotherapy
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- Member of: Barrett, The Honors College Thesis/Creative Project Collection
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Description
Over the past decade, several high-value proteins have been produced using plant-based transient expression systems. However, these studies exposed some limitations that must be overcome to allow plant expression systems to reach their full potential. These limitations are the low level of recombinant protein accumulation achieved in some cases, and lack of efficient co-expression vectors for the production of multi-protein complexes. This study report that tobacco Extensin (Ext) gene 3' untranslated region (UTR) can be broadly used to enhance recombinant protein expression in plants. Extensin is the hydroxyproline-rich glycoprotein that constitutes the major protein component of cell walls. Using transient expression, it was found that the Ext 3' UTR increases recombinant protein expression up to 13.5- and 6-fold in non-replicating and replicating vector systems, respectively, compared to previously established terminators. Enhanced protein accumulation was correlated with increased mRNA levels associated with reduction in read-through transcription. Regions of Ext 3' UTR essential for maximum gene expression included a poly-purine sequence used as a major poly-adenylation site. Furthermore, modified bean yellow dwarf virus (BeYDV)-based vectors designed to allow co-expression of multiple recombinant genes were constructed and tested for their performance in driving transient expression in plants. Robust co-expression and assembly of heavy and light chains of the anti-Ebola virus monoclonal antibody 6D8, as well as E. coli heat-labile toxin (LT) were achieved with the modified vectors. The simultaneous co-expression of three fluoroproteins using the single replicon, triple cassette is demonstrated by confocal microscopy. In conclusion, this study provides an excellent tool for rapid, cost-effective, large-scale manufacturing of recombinant proteins for use in medicine and industry.
ContributorsRosenthal, Sun Hee (Author) / Mason, Hugh (Thesis advisor) / Mor, Tsafrir (Committee member) / Chang, Yung (Committee member) / Arntzen, Charles (Committee member) / Arizona State University (Publisher)
Created2012
Description
We, a team of students and faculty in the life sciences at Arizona State University (ASU), currently teach an Introduction to Biology course in a Level 5, or maximum-security unit with the support of the Arizona Department of Corrections and the Prison Education Program at ASU. This course aims to enhance current programs at the unit by offering inmates an opportunity to practice literacy and math skills, while also providing exposure to a new academic field (science, and specifically biology). Numerous studies, including a 2005 study from the Arizona Department of Corrections (ADC), have found that vocational programs, including prison education programs, reduce recidivism rates (ADC 2005, Esperian 2010, Jancic 1988, Steurer et al. 2001, Ubic 2002) and may provide additional benefits such as engagement with a world outside the justice system (Duguid 1992), the opportunity for inmates to revise personal patterns of rejecting education that they may regret, and the ability of inmate parents to deliberately set a good example for their children (Hall and Killacky 2008). Teaching in a maximum security prison unit poses special challenges, which include a prohibition on most outside materials (except paper), severe restrictions on student-teacher and student-student interactions, and the inability to perform any lab exercises except limited computer simulations. Lack of literature discussing theoretical and practical aspects of teaching science in such environment has prompted us to conduct an ongoing study to generate notes and recommendations from this class through the use of surveys, academic evaluation of students' work and ongoing feedback from both teachers and students to inform teaching practices in future science classes in high-security prison units.
ContributorsLarson, Anika Jade (Author) / Mor, Tsafrir (Thesis director) / Brownell, Sara (Committee member) / Lockard, Joe (Committee member) / Barrett, The Honors College (Contributor) / School of Politics and Global Studies (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Influenza is a deadly disease for which effective vaccines are sorely lacking. This is largely due to the phenomena of antigenic shift and drift in the influenza virus's surface proteins, hemagglutinin (HA) and neuraminidase (NA). The ectodomain of the matrix 2 protein (M2e) of influenza A, however, has demonstrated high levels of conservation. On its own it is poorly immunogenic and offers little protection against influenza infections, but by combining it with a potent adjuvant, this limitation may be overcome. Recombinant immune complexes, or antigens fused to antibodies that have been engineered to form incredibly immunogenic complexes with one another, were previously shown to be useful, immunogenic platforms for the presentation of various antigens and could provide the boost in immunogenicity that M2e needs to become a powerful universal influenza A vaccine. In this thesis, genetic constructs containing geminiviral replication proteins and coding for a consensus sequence of dimeric M2e fused to antibodies featuring complimentary epitopes and epitope tags were generated and used to transform Agrobacterium tumefaciens. The transformed bacteria was then used to cause Nicotiana benthamiana to transiently express M2e-RICs at very high levels, with enough RICs being gathered to evaluate their potency in future mouse trials. Future directions and areas for further research are discussed.
ContributorsFavre, Brandon Chetan (Author) / Mason, Hugh (Thesis director) / Mor, Tsafrir (Committee member) / Diamos, Andrew (Committee member) / Department of Psychology (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The growing field of immunotherapy has generated numerous promising diseasetreatment platforms in recent years. By utilizing the innate capabilities of the immune system, these treatments have provided a unique, simplistic approach to targeting and eliminating cancer. Among these, the bispecific T cell engager (BiTEÒ) model has demonstrated potential as a treatment capable of bringing immune cells into contact with cancer cells of interest and initiating perforin/granzyme-mediated cell death of the tumor. While standard BiTE platforms rely on targeting a tumor-specific receptor via its complementary antibody, no such universal receptor has been reported for glioblastoma (GBM), the most common and aggressive primary brain tumor which boasts a median survival of only 15 months. In addition to its dismal prognosis, GBM deploys several immune-evasion tactics that further complicate treatment and make targeted therapy difficult. However, it has been reported that chlorotoxin, a 36-amino acid peptide found in the venom of Leiurus quinquestriatus, binds specifically to glioma cells while not binding healthy tissue in humans. This specificity positions chlorotoxin as a prime candidate to act as a GBM-targeting moiety as one half of an immunotherapeutic treatment platform resembling the BiTE design which I describe here. Named ACDClx∆15, this fusion protein tethers a truncated chlorotoxin molecule to the variable region of a monoclonal antibody targeted to CD3ε on both CD8+ and CD4+ T cells and is theorized to bring T cells into contact with GBM in order to stimulate an artificial immune response against the tumor. Here I describe the design and production of ACDClx∆15 and test its ability to bind and activate T lymphocytes against murine GBM in vitro. ACDClx∆15 was shown to bind both GBM and T cells without binding healthy cells in vitro but did not demonstrate the ability to activate T cells in the presence of GBM.
ContributorsSchaefer, Braeden Scott (Author) / Mor, Tsafrir (Thesis advisor) / Mason, Hugh (Committee member) / Blattman, Joseph (Committee member) / Arizona State University (Publisher)
Created2021
Description
Glioblastoma (GBM) is a highly invasive and deadly late stage tumor that develops from abnormal astrocytes in the brain. With few improvements in treatment over many decades, median patient survival is only 15 months and the 5-year survival rate hovers at 6%. Numerous challenges are encountered in the development of treatments for GBM. The blood-brain barrier (BBB) serves as a primary obstacle due to its innate ability to prevent unwanted molecules, such as most chemotherapeutics, from entering the brain tissue and reaching malignant cells. The GBM cells themselves serve as a second obstacle, having a high level of genetic and phenotypic heterogeneity. This characteristic improves the probability of a population of cells to have resistance to treatment, which ensures the survival of the tumor. Here, the development and testing of two different modes of therapy for treating GBM is described. These therapeutics were enhanced by pathogenic peptides known to improve entry into brain tissue or to bind GBM cells to overcome the BBB and/or tumor cell heterogeneity. The first therapeutic utilizes a small peptide, RVG-29, derived from the rabies virus glycoprotein to improve brain-specific delivery of nanoparticles encapsulated with a small molecule payload. RVG-29-targeted nanoparticles were observed to reach the brain of healthy mice in higher concentrations 2 hours following intravenous injection compared to control particles. However, targeted camptothecin-loaded nanoparticles were not capable of producing significant treatment benefits compared to non-targeted particles in an orthotopic mouse model of GBM. Peptide degradation following injection was shown to be a likely cause for reduced treatment benefit. The second therapeutic utilizes chlorotoxin, a non-toxic 36-amino acid peptide found in the venom of the deathstalker scorpion, expressed as a fusion to antibody fragments to enhance T cell recognition and killing of GBM. This candidate biologic, known as anti-CD3/chlorotoxin (ACDClx) is expressed as an insoluble protein in Nicotiana benthamiana and Escherichia coli and must be purified in denaturing and reducing conditions prior to being refolded. ACDClx was shown to selectively activate T cells only in the presence of GBM cells, providing evidence that further preclinical development of ACDClx as a GBM immunotherapy is warranted.
ContributorsCook, Rebecca Leanne (Author) / Blattman, Joseph N (Thesis advisor) / Sirianni, Rachael W. (Thesis advisor) / Mor, Tsafrir (Committee member) / Anderson, Karen (Committee member) / Arizona State University (Publisher)
Created2019
Description
Fusion protein immunotherapies such as the bispecific T cell engager (BiTE) have displayed promising potential as cancer treatments capable of engaging the immune system against tumor cells. It has been shown that chlorotoxin, a 36-amino peptide found in the venom of the deathstalker scorpion (Leiurus quinquestriatus), binds specifically to glioblastoma (GBM) cells without binding healthy tissue, making it an ideal GBM cell binding moiety for a BiTE-like molecule. However, chlorotoxin’s four disulfide bonds pose a folding challenge outside of its natural context and impede production of the recombinant protein in various expression systems, including those relying on bacteria and plants. To overcome this difficulty, we have engineered a truncated chlorotoxin variant (Cltx∆15) that contains just two of the original eight cystine residues, thereby capable of forming only a single disulfide bond while maintaining its ability to bind GBM cells. We further created a BiTE (ACDClx∆15) which tethers Cltx∆15 to a single chain ⍺-CD3 antibody in order to bring T cells into contact with GBM cells. The gene for ACDClx∆15 was cloned into a pET-11a vector for expression in Escherichia coli and isolated from inclusion bodies before purification via affinity chromatography. Immunoblot analyses confirmed that ACDClx∆15 can be expressed in E. coli and purified with high yield and purity; moreover, flow cytometry indicated that ACDClx∆15 is capable of binding GBM cells. These data warrant further investigation into the ability of ACDClx∆15 to activate T cells against GBM cells.
ContributorsSchaefer, Braeden Scott (Author) / Mor, Tsafrir (Thesis director) / Mason, Hugh (Committee member) / Cook, Rebecca (Committee member) / School of Life Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
HIV continues to remain a global health issue, in particular in many low and middle-income countries. The World Health Organization (WHO) estimates that of the nearly 38 million HIV-1 positive individuals, 25% are unaware they are infected. Despite decades of research, a safe and effective preventative vaccine has yet to be produced. The HIV-1 envelope glycoprotein41 and the Gag structural protein have been identified to be particularly important in HIV-1 transcytosis and cytotoxic lymphocyte response, respectively. Enveloped virus-like particles (VLPs) consisting of Gag and a deconstructed form of glycoprotein (dgp41) comprising the membrane proximal external region (MPER), transmembrane domain and cytoplasmic tail may present a unique and safe way of presenting these proteins in a state mimicking their natural formation. Another form of presenting the immunogenic glycoprotein41, particularly the MPER component, is by presenting it onto the N-terminal of an IgG molecule, thereby creating an IgG fusion molecule. In our lab, both VLPs and IgG fusion molecules are highly expressed and purified within GnGn Nicotiana benthamiana. The results indicated that these recombinant proteins can be assembled properly within plants and can elicit an immune response in mice. This provides a preliminary step in using such Gag/dpg41 VLPs and RIC as present a safe, effective, and inexpensive HIV vaccine.
ContributorsGarcia, Izamar (Author) / Mor, Tsafrir (Thesis director) / Mason, Hugh (Committee member) / Kamzina, Aigerim (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05