Matching Items (6)
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- All Subjects: immunotherapy
- Creators: Blattman, Joseph
- Creators: Mor, Tsafrir
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
Since Metastatic Osteosarcoma is unresponsive to most of the current standards of care currently available, and yields a survival rate of 20%, it is pertinent that novel approaches to treating it be undertaken in scientific research. Past studies in our lab have used a The Immune Blockade Therapy, utilizing α-CTLA-4 and α-PD-L1 to treat mice with metastatic osteosarcoma; this resulted in 60% of mice achieving disease-free survival and protective immunity against metastatic osteosarcoma. 12 We originally wanted to see if the survival rate could be boosted by pairing the immune blockade therapy with another current, standard of care, radiation. We had found that there were certain, key features to experimental design that had to be maintained and explored further in order to raise survival rates, ultimately with the goal of reestablishing the 60% survival rate seen in mice treated with the immune blockade therapy. Our results show that mice with mature immune systems, which develop by 6-8 weeks, should be used in experiments testing an immune blockade, or other forms of immunotherapy, as they are capable of properly responding to treatment. Treatment as early as one day after should be maintained in future experiments looking at the immune blockade therapy for the treatment of metastatic osteosarcoma in mice. The immune blockade therapy, using α-PD-L1 and α-CTLA-4, seems to work synergistically with radiation, a current standard of care. The combination of these therapies could potentially boost the 60% survival rate, as previously seen in mice treated with α-PD-L1 and α-CTLA-4, to a higher percent by means of reducing tumor burden and prolonging length of life in metastatic osteosarcoma.
ContributorsLabban, Nicole (Author) / Blattman, Joseph (Thesis director) / Appel, Nicole (Committee member) / Barrett, The Honors College (Contributor)
Created2017-05
Description
The p53 gene functions as a tumor suppressor that inhibits proliferation, regulates apoptosis, DNA repair, and normal cell cycle arrest. Mutation of the p53 gene is linked to be prevalent in 50% of all human cancers. In this paper, we are exploring triple negative breast cancer and the effects of simvastatin on tumor growth and survival. Simvastatin is a drug that is primarily used to treat high cholesterol and heart disease. Simvastatin is unique because it is able to inhibit protein prenylation through regulation of the mevalonate pathway. This makes it a potential targeted drug for therapy against p53 mutant cancer. The mechanism behind this is hypothesized to be correlated to aberrant activation of the Ras pathway. The Ras subfamily functions to transcriptionally regulate cell growth and survival, and will therefore allow for a tumor to thrive if the pathway is continually and abnormally activated. The Ras protein has to be prenylated in order for activation of this pathway to occur, making statin drug treatment a viable option as a cancer treatment. This is because it acts as a regulator of the mevalonate pathway which is upstream of protein prenylation. It is thus vital to understand these pathways at both the gene and protein level in different p53 mutants to further understand if simvastatin is indeed a drug with anti-cancer properties and can be used to target cancers with p53 mutation. The goal of this project is to study the biochemistry behind the mutation of p53's sensitivity to statin. With this information we can create a possible signature for those who could benefit from Simvastatin drug treatment as a possible targeted treatment for p53 mutant cancers.
ContributorsGrewal, Harneet (Co-author) / Loo, Yi Jia Valerie (Co-author) / Anderson, Karen (Thesis director) / Blattman, Joseph (Committee member) / Ferdosi, Shayesteh (Committee member) / Department of Psychology (Contributor) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
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
Lung metastatic cancers represent a major challenge in both basic and clinical cancer research. The ability to treat lung metastases to date has been challenging, current treatment paradigms are a mix of classic radiotherapy, chemotherapies and tumor-targeted therapies, with no one treatment that is effective for all tumors. Oncolytic viruses (OVs) represent a new therapeutic modality for hard-to-treat tumors. However, major questions still exist in the field, especially around how to therapeutically arm and deliver OVs to sites of disseminated tumors. To address this need, oncolytic myxoma viruses (MYXV) that expresses TNF superfamily member transgenes (vMYX-hTNF or vMyx-mLIGHT) were tested in an immunocompetent syngeneic lung metastatic murine osteosarcoma model. Three versions of this model were used; 1-an early intervention model, 2-an established tumor model, defined by both average tumor burden and failure of anti-PD-L1 and vMyx-TNF monotherapies, and 3-a late-stage disease model, defined by the failure the combination of vMyx-hTNF/PBMCs and anti-PD-L1 therapy. These three models were designed to test different questions about therapeutic efficacy of armed MYXV and delivery of MYXV to lung metastases. In the early intervention model, vMyx-hTNF was found to be an effective therapy, especially when delivered by leukocyte carrier cells (either bone marrow or PBMCs). Next, the combination of immune checkpoint inhibitors, including anti-PD-L1, anti-PD-1 and anti-CTLA-4, with vMyx-TNF/PBMCs were found to increase efficacy in treated mice compared to monotherapies. The established model was used to test potential synergy of vMyx-hTNF with anti-PD-L1 therapy. This model was defined by the failure of the monotherapies, however, in combination, treated mice survived significantly longer, and had lower average tumor burden throughout. This model was also used to test tumor specific delivery using ex vivo loaded PBMCs as carrier cells. Using MYXV expressing Tdtomato, PBMCs were found to deliver MYXV to tumors more effectively than free virus. In the most stringent late-stage disease model, vMyx-mLIGHT/PBMCs and vMyx-mLIGHT/PBMCs plus anti-PD-1 were tested and found to be efficacious where combination vMyx-TNF/PBMCs plus PD-1 failed. These results taken together show that TNFSF arming of MYXV, especially when delivered by autologous PBMCs, represents a new potential treatment strategy for lung metastatic tumors.
ContributorsChristie, John Douglas (Author) / McFadden, Grant (Thesis advisor) / Blattman, Joseph (Committee member) / Jacobs, Bertram (Committee member) / Anderson, Karen (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