Browsing by Author "Thamm, Douglas H., advisor"

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Embargo
Development of a canine model to evaluate CAR-T cell subsets
(Colorado State University. Libraries, 2024) Brill, Samuel Austin, author; Thamm, Douglas H., advisor; Avery, Anne, committee member; Dow, Steven, committee member; Henao Tamayo, Marcela, committee member; Fry, Terry, committee member
Immunotherapy is a rapidly expanding therapeutic modality in the oncology clinic. Immunotherapy with chimeric antigen receptor (CAR) T cells has demonstrated success in human blood cancers, although relapse occurs in ~50% of patients. In the context of solid tumors, CAR-T cells show only transient effects. As a "living therapy", development of the next generation CAR-T cells requires a deeper understanding of how CAR biology, T cell biology, and tumor biology interact over the course of disease. CAR constructs are made up of an extracellular antigen binding domain fused with one or more intracellular signaling domains. The extracellular domain is frequently built from the fused heavy and light chains of an antibody, called a single chain fragmented variable (scFv) domain, and confers the CAR its tumor specificity. Intracellular signaling domains typically contain the zeta chain of CD3 along with one or more costimulatory domains, often CD28 and/or 4-1BB. Mechanisms of relapse and treatment failure for CAR-T cell therapy are related to loss of CAR-T targeting (e.g. target antigen loss) and dysfunction of CAR-T cells (e.g. immunosuppressive microenvironment). These mechanisms of resistance to CAR-T cell therapy have largely been resolved using in vitro and mouse models, where methods to overcome resistance have also been developed. While our mechanistic understanding of tumor and immune biology has been propelled to a place where beneficial therapies are being employed, there is a need for more clinically relevant models to help streamline clinical trials of promising next-generation therapies. Dogs provide an immune-intact animal model that can help address some fundamental questions of CAR-T cell biology in a clinically relevant animal model. Cancers in dogs share many pathobiological features to their human counterparts and receive similar treatments including chemotherapy, radiation, and surgery. Dogs' intact immune system, large size, and relatively outbred genetics provide an ideal environment to evaluate novel cellular therapies such as CAR T cell immunotherapies. This work sought to develop a model of CAR-T cell therapy in dogs to better support pre-clinical evaluation of novel CAR constructs and to better understand how CAR and T cell biology interact in the context of cancer. The first aim of this dissertation was to develop a canine CAR-T model for functional assessments of canine CAR-T cells. The second aim of this dissertation was to explore cell subsets using single cell sequencing. We first sought to establish a canine CAR-T cell in vitro model system. We hypothesized that the disialoganglioside GD2 would be a viable CAR tumor target in canine osteosarcoma (OS). The primary CAR used in these studies was a GD2 directed CAR which is currently undergoing human clinical trials. Using the same antibody clone from which the GD2 CAR is derived, we demonstrated that GD2 is expressed on a subset of canine OS and melanoma cell lines. We demonstrated that canine primary T cells are refractory towards lentiviral (HIV-1) based transduction, although they are susceptible to gammaretroviral (MSCV) transduction. Canine GD2+ cell lines were susceptible to GD2 CAR-T cells as assessed by IFN-g and IL-2 cytokine release, Incucyte live image microscopy, and luciferase killing assays. GD2 CARs with either CD28 or 4-1BB costimulatory domains were effective in killing GD2+ OS. The second aim of this proposal sought to elucidate and define canine immune cell subsets using single cell RNA sequencing (scRNASeq). We hypothesized that a phenotypically distinct population of CAR-T would preferentially expand and persist throughout CAR-T cell manufacturing and tumor engagement. To the aim of identifying specific immune subsets, we first sought to create an atlas of cells present in the canine lymph node (LN). In addition to peripheral blood and primary tumor lymphocytes, LNs are an additional key site for modulating cancer immunity. While the immune subsets in the peripheral blood and tumor have been described in dogs with OS, an atlas of canine LNs has not been made. We generated an immune atlas of cells with the LNs from healthy dogs and the regional LNs of OS bearing dogs. In addition to annotations of cell clusters present in l LNs, comparisons between dog populations revealed fewer B cells recovered in the OS group along with higher HLA-DQB2 gene expression across a variety of cell types. Taken together, these aims provide a basis for investigation of CAR-T cell therapy at single cell resolution. In the first aim, we developed canine GD2 directed CAR-T cells which could effectively kill GD2+ tumor in vitro. The second aim developed a workflow using scSeq to identify canine immune cell populations and provides a LN cell atlas to supplement those for the peripheral blood and tumor tissue. This platform will be used for translational oncology studies to assess novel CAR constructs, investigate CAR-T cell biology, and allow for optimization of next generation CAR-T cells.
Open Access
Overactive NF-KB signaling as a druggable target and evaluation of parthenolide an NF-KB inhibitor in canine cancer
(Colorado State University. Libraries, 2022) Schlein, Lisa Janelle, author; Thamm, Douglas H., advisor; Avery, Paul, committee member; Duval, Dawn, committee member; MacNeill, Amy, committee member
This study provides a unique translational research opportunity to help both humans and dogs diagnosed with diseases that carry dismal prognoses in both species: histiocytic sarcoma (HS), hemangiosarcoma (HSA), and disseminated mastocytosis (MCT). Lymphoma is one of the most common cancer types affecting dogs and humans, and therefore, novel therapeutic approaches are always needed. For all of these cancer types, dogs and human cancers share common molecular abnormalities, consistent with a conserved pathogenesis between species. Relative to traditional murine models for human cancers, dogs are genetically diverse, large mammals with heterogeneous, spontaneous tumors. Dogs generally receive good medical care and share the environmental factors with humans, and accordingly, dogs with spontaneous tumors are an excellent model for human oncology generally. Additionally, although disseminated HS, MCT and visceral HSA are exceedingly rare diseases in humans, they are more common in some dog breeds, giving us the opportunity to study this disease in a larger population than would otherwise be available. Therapeutics evaluated in dogs with these diseases stand to benefit both canine and human patients. NF-kB proteins are a family of structurally related, eukaryotic transcription factors that have 400+ genetic targets, and are involved in many vital cellular processes, including innate immunity, inflammatory responses, development, cellular growth, and survival. Not surprisingly, overactivation of NF-kB is a feature of many chronic disease processes, including cardiac disease, neurodegenerative disease, immune-mediated disease, and cancer. While NF-kB overactivation has been documented extensively in human oncology, there is a relative paucity of data documenting the same phenomenon in veterinary medicine. As part of this study, large scale validation of NF-kB overactivation was performed in canine cancer via immunohistochemistry of 215 tumor samples (lymphoma, HS, HSA, and MCT). Antibodies were validated for use via western blot, immortalized cell pellets, and evaluation of normal canine tissues. In addition to validation of NF-kB overactivation, assays were performed to assess the therapeutic potential of parthenolide (PTL), a known, canonical NF-kB signaling inhibitor with additional mechanisms of antineoplastic activity, including alteration of cellular redox balance. Growth inhibition assays were performed with canine cell lines and primary lymphoma cells isolated from canine patients, using PTL alone or in combination with redox-perturbing standard-of-care therapeutics. Cell death was assessed using flow cytometry. Immunofluorescence was used to assess NF-kB localization, western blot was used to assess NF-kB activity with and without PTL, and canine cells were transfected with a reporter gene cassette containing the NFkB consensus sequence followed by firefly luciferase gene to study the effect of PTL on NF-kB-related luminescence. PTL's effects on glutathione and reactive oxygen species generation were assessed with a colorimetric assay and a fluorescent H2DCFDA assay, respectively. Genetic expression changes were assessed with RNA sequencing of HS cells, with and without PTL treatment. A mouse model of disseminated HS was created with NF-kB luminescent cells to study the effect of PTL on this disease in vivo. Many spontaneous canine tumor samples have nuclear p65 and p100/p52 IHC staining that is of greater magnitude than observed in comparable, normal cell populations, indicating the promise of PTL and other therapeutics that target aberrant NF-kB signaling. Canine cell lines and primary cells are sensitive to PTL and undergo dose-dependent apoptosis following exposure to drug. PTL exposure also leads to glutathione depletion, reactive oxygen species generation, and NF-kB inhibition in canine cells. Standard-of-care therapeutics broadly synergize with PTL. In two canine HS cell lines, genetic expression of NF-kB pathway signaling partners is downregulated with PTL therapy. Preliminary data suggest that PTL inhibits NF-kB activity of cells in a mouse model of disseminated HS. Overall, these data support further investigation of compounds that can antagonize canonical and alternative NF-kB pathway signaling, which are overactivated in canine lymphoma, HS, HSA, and MCT disease. PTL is one promising therapeutic that acts, in part, via canonical NF-kB antagonism in canine neoplasms. Further investigation of this compound in vivo is underway in a mouse model of disseminated HS, and if this study is successful, it will provide strong justification for clinical trials with this compound in dogs.