Cancer poses a significant burden on the global health system and represents a leading cause of death worldwide. For late-stage cancers, the traditional treatments of chemotherapy, radiation, and surgery are not always viable, and they can pose unnecessary health risks to the patients. New immunotherapies, such as adoptive cell transfer, are being developed and refined to treat such cancers. T cell immunotherapies in particular, where a patient’s T cell lymphocytes are isolated and amplified to be re-infused into the patient or where human cell lines are engineered to express T cell receptors for the recognition of common cancer antigens, are being expanded on because for some cancers, they could be the only option. Constructing an optimal pipeline for cloning and expression of antigen-specific TCRs has significant bearing on the efficacy of engineered cell lines for ACT. Adoptive T cell transfer, while making great strides, has to overcome a diverse T cell repertoire – cloning and expressing antigen-specific TCRs can mediate this understanding. Having identified the high frequency FluM1-specific TCR sequences in stimulated donor PBMCs, it was hypothesized that the antigen-specific TCR could be reconstructed via Gateway cloning methods and tested for expression and functionality. Establishing this pipeline would confirm an ability to properly pair and express the heterodimeric chains. In the context of downstream applications, neoantigens would be used to stimulate T cells, the α and β chains would be paired via single-cell or bulk methods, and instead of Gateway cloning, the CDR3 hypervariable regions α and β chains alone would be co-expressed using Golden Gate assembly methods.

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.

Inhibitor of growth factor 4 (ING4) is a tumor suppressor of which low expression has been associated with poor patient survival and aggressive tumor progression in breast cancer. ING4 is characterized as a transcription regulator of inflammatory genes. Among the ING4-regulated genes is CXCL10, a chemokine secreted by endothelial cells during normal inflammation response, which induces chemotactic migration of immune cells to the site. High expression of CXCL10 has been implicated in aggressive breast cancer, but the mechanism is not well understood. A potential signaling molecule downstream of Cxcl10 is Janus Kinase 2 (Jak2), a kinase activated in normal immune response. Deregulation of Jak2 is associated with metastasis, immune evasion, and tumor progression in breast cancer. Thus, we hypothesized that the Ing4/Cxcl10/Jak2 axis plays a key role in breast cancer progression. We first investigated whether Cxcl10 affected breast cancer cell migration. We also investigated whether Cxcl10-mediated migration is dependent on ING4 expression levels. We utilized genetically engineered MDAmb231 breast cancer cells with a CRISPR/Cas9 ING4-knockout construct or a viral ING4 overexpression construct. We performed Western blot analysis to confirm Ing4 expression. Cell migration was assessed using Boyden Chamber assay with or without exogenous Cxcl10 treatment. The results showed that in the presence of Cxcl10, ING4-deficient cells had a two-fold increase in migration as compared to the vector controls, suggesting Ing4 inhibits Cxcl10-induced migration. These findings support our hypothesis that ING4-deficient tumor cells have increased migration when Cxcl10 signaling is present in breast cancer. These results implicate Ing4 is a key regulator of a chemokine-induced tumor migration. Our future plan includes evaluation of Jak2 as an intermediate signaling molecule in Cxcl10/Ing4 pathway. Therapeutic implications of these findings are targeting Cxcl10 and/or Jak2 may be effective in treating ING4-deficient aggressive breast cancer.

Necrotic enteritis (NE) is caused by type A strains of the bacterium Clostridium perfringens, leading to an estimated 2 billion dollar global economic loss in the poultry industry annually. Traditionally, NE has been effectively controlled by antibiotics added to the diet of poultry. Concerns about increasing antibiotic resistance of poultry and human based pathogens have led to the consideration of alternative approaches for controlling disease, such as vaccination. NE causing strains of C. perfringens produce two major toxins, α-toxin and NetB. Immune responses against either toxin can provide partial protection against NE. We have developed a fusion protein combining a non-toxic carboxy-terminal domain of the α-toxin (PlcC) and an attenuated, mutant form of NetB (NetB-W262A) for use as a vaccine antigen to immunize poultry against NE. We utilized a DNA sequence that was codon-optimized for Nicotiana benthamiana to enable high levels of expression. The 6-His tagged PlcC-NetB fusion protein was synthesized in N. benthamiana using a geminiviral replicon transient expression system. The fusion protein was purified by metal affinity chromatography and used to immunize broiler birds. Immunized birds produced a strong serum IgY response against both the plant produced PlcC-NetB protein and against bacterially produced His-PlcC and His-NetB. However, the PlcC-NetB fusion had antibody titers four times that of the bacterially produced toxoids alone. Immunized birds were significantly protected against a subsequent in-feed challenge with virulent C. perfringens when treated with the fusion protein. These results indicate that a plant-produced PlcC-NetB is a promising vaccine candidate for controlling NE in poultry.

Agrobacterium tumefaciens has the ability to transfer its tumor inducing (Ti) plasmid into plant cells. In the last decade, agroinfiltration of Nicotiana benthamiana plants has shown promising results for recombinant protein production. However, A. tumefaciens produce endotoxins in the form of lipopolysaccharides (LPS), a component of their outer membrane that can induce organ failure and septic shock. Therefore, we aimed to detoxify A. tumefaciens by modifying their Lipid A structure, the toxic region of LPS, via mutating the genes for lipid A biosynthesis. Two mutant strains of A. tumefaciens were infiltrated into N. benthamiana stems to test for tumor formation to ensure that the detoxifying process did not compromise the ability of gene transfer. Our results demonstrated that A. tumefaciens with both single and double mutations retained the ability to form tumors. Thus, these mutants can be utilized to generate engineered A. tumefaciens strains for the production of plant-based pharmaceuticals with low endotoxicity.

A chimeric, humanized monoclonal antibody that recognizes a highly conserved fusion loop found on flaviviruses was constructed with a geminiviral replicon and transiently expressed in Nicotiana benthamiana plants through Agrobacterium tumefaciens infiltration. Characterization and expression studies were then conducted to confirm correct assembly of the antibody. Once the antibody was purified, an ELISA was conducted to validate that the antibody was able to bind to the flavivirus fusion loop.

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.

This study focused on the connection between the EnvZ/OmpR two-component regulatory system and the iron homeostasis system in Escherichia coli, specifically how a mutant form of EnvZ11/OmpR is able to reduce the expression of fepA::lacZ, a reporter gene fusion in E. coli. FepA is one of several outer membrane siderophore receptors that allow extracellular siderophores bound to iron to enter the cells to power various biological processes. Previous studies have shown that in E. coli cells that expressed a mutant allele of envZ, called envZ11, which led to altered expression of various iron genes including down regulation of fepA::lacZ. The wild type EnvZ/OmpR system is not considered to regulate iron genes, but because these envz11 strains had downregulated fepA::lacZ, this study was undertaken to understand the connection and mechanisms of this downregulation. A large number of Lac+ revertants were obtained from the B32-2483 strain (envz11 and fepA::lacZ) and 7 Lac+ revertants that had reversion mutations not directly correcting the envZ11 allele were further characterized. With P1 phage transduction genetic mapping that involved moving a kanamycin resistance marker linked to fepA::lacZ, two Lac+ revertants were found to have their reversion mutations in the fepA promoter region, while the other five revertants had their mutations mapping outside the fepA region. These two revertants underwent DNA sequencing and found to carry two different single base pair mutations in two different locations of the fepA promoter region. Each one is in the Fur repressor binding region, but one also may have affected the Shine-Dalgarno region involved in translation initiation. All 7 reveratants underwent beta-galactosidase assays to measure fepA::lacZ expression. The two revertants that had mutations in the fepA promoter region had significantly increased fepA activity, with the revertant with the Shine-Dalgarno mutation having the most elevated fepA expression. The other 5 revertants that did not map in the fepA region had fepA expression elevated to the same level as that found in the wild type EnvZ/OmpR background. The data suggest that the negative effect of envZ11 can be overcome by multiple mechanisms, including directly correcting the envZ11 allele or changing the fepA promoter region.

Succinylcholine-induced apnea is a common problem in pre-hospital medicine that affects 1/1800 patients who undergo rapid sequence intubation. Succinylcholine is an anesthetic that mimics the neurotransmitter, acetylcholine. It binds to cholinergic receptors, blocking acetylcholine access, and causes paralysis for (normally) only a short time. Butyrylcholinesterase, which is responsible for succinylcholine hydrolysis, is deficient in a small percentage of the population. Previous studies have shown that wild-type butyrylcholinesterase (BChE) can be produced in transient-expression Nicotiana benthamiana, and can reverse the effects of succinylcholine induced apnea through enzyme replacement therapy. The wild type enzyme is also capable of irreversibly binding and inactivating organophosphorus nerve agents and pesticides, and has also exhibited cocaine hydrolase activity. Super cocaine-hydrolyzing BChE mutants, which exceed 2000 times the catalytic capability of the wild-type, have been optimized and expressed in N. benthamiana. The purpose of this study was to determine whether these mutants also hydrolyze succinylcholine with improved efficiency. Variant 3 and Variant 4 exhibited catalytic efficiencies of 2.08 x 106 M-1 min-1 and 3.48 x 106 M-1 min-1, respectively, against their preferred substrate, butyrylthiocholine, in the Ellman assay. The wild-type plant-expressed BChE did exhibit hydrolysis of succinylcholine, as we had previously determined; however, neither Variant 3 nor Variant 4 demonstrated the ability to hydrolyze succinylcholine in our particular assay. Therefore, N. benthamiana-expressed Variant 3 and Variant 4 may not succeed as a dual treatment against cocaine toxicity and prolonged succinylcholine-induces paralysis.